Lipid composition

Abstract

A drug delivery system, in particular to lipid compositions shown comprising therapeutic and / or prophylactic agents, such as RNA, can be used to deliver therapeutic and / or prophylactic agents to mammalian cells or organs, for example to modulate polypeptide, protein or gene expression.

Description

A lipid composition

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202210948906.0 filed on August 9, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a drug delivery system, and in particular to a lipid composition and related products and their use in intramuscular injection. Background Art

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

[0005] Existing drug delivery systems based on lipids, such as liposomes, lipid nanoparticle (LNP) drug delivery systems etc. have been widely used in the past few years. But in actual use, it is found that there are many problems in these drug delivery systems based on lipids, such as the liposome production method is complicated and needs to use organic solvents, low drug encapsulation efficiency; For example, the LNP system of unmodified has poor stability in vivo and poor targeting. And the existing lipid delivery system is after delivering nucleic acid to organism, and the expression amount of nucleic acid in vivo is low, and in the freeze-dried storage process, there is stability problem, and the nucleic acid expression amount after freeze-dried redissolution is obviously reduced, affecting use. In order to improve stability, it is also usually necessary to add stabilizers such as N-(methoxy-poly(ethylene glycol)-oxycarbonyl)-distearoylphosphatidylethanolamine, so that the preparation process is complicated and cost is increased. So it is necessary to study a more efficient and stable lipid delivery system.

[0006] Many new and efficient lipid delivery systems have been developed. For example, CN110974954A provides a lipid nanoparticle that enhances the immune effect of nucleic acid vaccines, which has the advantages of high nucleic acid encapsulation efficiency and narrow particle size distribution. For example, CN110638759A also provides a formulation for in vitro transfection and in vivo delivery of mRNA, which can have lower toxicity and better immune effects of nucleic acids in vivo. Although research on lipid-based drug delivery systems has made significant progress, there is still a need for more efficient, stable, and targeted lipid delivery systems.

[0007] Summary of the Invention

[0008] On the one hand, the present invention provides a lipid composition, which comprises a therapeutic agent or a prophylactic agent and a lipid that encapsulates the therapeutic agent or the prophylactic agent, wherein the lipid that encapsulates the therapeutic agent or the prophylactic agent comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid; the composition further comprises a cationic polymer, wherein the cationic polymer is associated with the therapeutic agent or the prophylactic agent to form a complex, and together they are encapsulated in the lipid to form a lipid polymer complex.

[0009] In one embodiment, the therapeutic or prophylactic agent is a nucleic acid, such as RNA, particularly mRNA.

[0010] 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-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.

[0011] In one embodiment, the cationic lipid does not include T5'.

[0012] In a preferred embodiment, the lipid composition comprises

[0013] 10-70 mol% cationic lipid, 10-70 mol% phospholipid, 10-70 mol% steroid and 0.05-20 mol% polyethylene glycol-modified lipid;

[0014] Preferably comprising 35-50 mol% of cationic lipids, 10-30 mol% of phospholipids, 24-44 mol% of steroids and 1-1.5 mol% of polyethylene glycol-modified lipids; and / or

[0015] cationic lipids, DOPE, cholesterol, and DMG-PEG;

[0016] Preferably, it comprises 50 mol% cationic lipid, 10 mol% DOPE, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG or 40 mol% cationic lipid, 15 mol% DOPE, 43.5 mol% cholesterol and 1.5 mol% DMG-PEG.

[0017] In one embodiment, the therapeutic or prophylactic agent is a polynucleotide comprising a coding region, wherein the coding region encodes a modified spike protein, wherein the modified spike protein comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:12; and wherein the polynucleotide is RNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:3; or wherein the polynucleotide is DNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:5.

[0018] In some embodiments, the polynucleotide is RNA comprising the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:6; or the polynucleotide is DNA comprising the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:7.

[0019] 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.

[0020] In another aspect, the present invention also provides an injection, comprising the lipid composition of the present invention and a pharmaceutically acceptable excipient for injection.

[0021] 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 injection of the present invention in preparing a medicament for treating or preventing a disease or condition in a subject in need thereof.

[0022] On the other hand, the present invention also provides use of the lipid composition of the present invention, the pharmaceutical composition of the present invention, or the injection of the present invention in the preparation of a medicament for preventing and / or treating SARS-CoV-2 infection.

[0023] In another aspect, the present invention also provides a method for preventing or treating a disease or condition in a subject in need thereof, the method comprising:

[0024] The lipid composition, pharmaceutical composition or injection of the present invention is administered to a subject in need thereof. The lipid composition or pharmaceutical composition can be administered by injection, preferably intramuscular injection.

[0025] In one aspect, the present invention also provides a method for delivering a therapeutic agent or a prophylactic agent to a mammalian cell of a subject, the method comprising administering a lipid composition or a pharmaceutical composition of the present invention to the subject, wherein the administration comprises contacting the cell with the lipid composition, thereby delivering the therapeutic agent and / or prophylactic agent to the cell.

[0026] In yet another aspect, the present invention also provides a method for producing a target polypeptide in a subject's mammalian cell, the method comprising contacting the cell with a lipid composition or a pharmaceutical composition of the present invention, wherein the therapeutic agent or prophylactic agent is mRNA, and wherein the mRNA encodes the target polypeptide, whereby the mRNA can be translated in the cell to produce the target polypeptide.

[0027] In some embodiments, the lipid composition, the pharmaceutical composition, or the medicament is administered parenterally, orally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, or intravaginally; preferably, by intramuscular injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 shows the luciferase expression results of LPP preparations prepared with CDC formulation and BIC formulation in mouse liver and injection site.

[0029] Figures 2A-2B show the results of in vivo luciferase expression and in vitro physicochemical properties of SW0123.351-LPP after storage for different periods of time. Figure 2A shows the results of in vivo luciferase expression. Figure 2B shows the results of average particle size (Z-Average).

[0030] Figures 3A-3B show the in vivo luciferase expression and in vitro physicochemical properties of SW0123.351-LPP solutions after different freeze-thaw cycles. Figure 3A shows the in vivo luciferase expression results. Figure 3B shows the average particle size (Z-Average) test results.

[0031] FIG4 shows the immunogenicity test results of SW0123.351-LPP in mice before and after freeze-drying.

[0032] Figures 5A-5C show the particle size, encapsulation efficiency, and immunogenicity test results of SW0123.351-LPP lyophilized powder after storage at 25°C for different periods of time. Figure 5A shows the average particle size (Z-Average) test results. Figure 5B shows the encapsulation efficiency test results. Figure 5C shows the immunogenicity test results.

[0033] FIG6 shows the luciferase expression results of LNP or LPP preparations prepared with different formulations of cationic lipid M5 at the injection site of mice from 0 to 24 hours.

[0034] Figures 7A-7C show the luciferase expression results at the injection site or liver of mice 3 hours and 6 hours after administration of LNP or LPP formulations prepared with different cationic lipid formulations using M5. Figure 7A shows the test results 3 hours after administration. Figure 7B shows the test results 6 hours after administration. Figure 7C shows the luciferase expression ratio of mouse liver / injection site 6 hours after injection.

[0035] FIG8 shows the luciferase expression results at the injection site of mice 0-24 hours after LNP or LPP preparations prepared with cationic lipids of different formulations, SM-102, ALC-0315, SW-II-115, and SW-II-121.

[0036] Figure 9 shows the luciferase expression ratio of mouse liver / injection site of LNP or LPP preparations prepared with different formulations of cationic lipids including M5, SM-102, ALC-0315, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2, SW-II-140-2, and SW-II-122.

[0037] FIG10 shows the luciferase expression results of LNP preparations prepared with different formulations of cationic lipids including M5, MC3, and SW-II-121 at the injection site and liver of mice.

[0038] Figures 11A-11E show the luciferase expression results of LPP preparations prepared with different prescriptions of SW-II-121 as cationic lipid at the injection site and liver of mice from 0 to 24 hours. Figure 11A shows the expression of each lipid composition at the injection site (muscle tissue). Figure 11B shows the expression of different phospholipid and PEG combinations at the injection site (muscle tissue). Figure 11C shows the expression of each lipid composition in the liver. Figure 11D shows the expression of different phospholipid and PEG combinations in the liver. Figure 11E shows the ratio of luciferase expression in muscle / liver for each lipid composition.

[0039] Figure 12 shows the preparation and characterization of LPP / mRNA vaccines. (A) shows the formulations of four LPP formulations encapsulating luciferase mRNA; (B and C) show the mRNA expression efficiency of the four LPP formulations encapsulating luciferase mRNA at the injection site and liver 24 hours after injection; (D) shows images of LPP-B2 characterized by cryo-electron microscopy; (E) shows bioluminescent images of mouse luciferase expression 6 hours after injection of LPP / eGFP; (F) shows the results of eGFP expression in C2C12 cells and DC2.4 cells detected by flow cytometry after incubation with LPP / eGFP for 24 hours; (G) shows the results of eGFP expression in mouse lymph node cell subsets detected by flow cytometry 24 hours after injection of LPP / eGFP; (H) shows the results of DC maturation markers CD40, CD80, CD86, and MHC-II detected by flow cytometry 24 hours after injection of empty vector LPP or LPP / Luc. The data in (F, G, and H) are expressed as mean ± SEM (n = 5).

[0040] FIG13 shows the expression of eGFP in A20 cells, Jurkat T cells and HSKMC cells after incubation with LPP / eGFP for 24 hours detected by flow cytometry.

[0041] Figure 14 shows the bioluminescence of luciferase expression in mice 24 hours after LPP / eGFP injection. (A) Statistical results of luciferase expression in the liver and injection site of mice 24 hours after LPP / eGFP injection; (B) Bioluminescence images of luciferase expression at the local injection site and major organs (heart, liver, spleen, lungs, kidneys, brain, and lymph nodes) of mice.

[0042] Figure 15 shows the gating strategy for identifying lymph node cell populations.

[0043] Figure 16 shows the identification of CD11c in lymph nodes + Cell gating strategy.

[0044] Figure 17 shows LPP / mRNA in vitro antigen characterization and in vivo immunogenicity. (A) shows a schematic diagram of the mRNA encoding the modified spike protein; (B) shows the spike glycoprotein expression and deglycosylation analysis, with arrows indicating glycosylated or deglycosylated spike protein bands, and triangles indicating glycosylated or deglycosylated S1 protein bands; (C) shows the immunization schedule for C57BL / 6 mice (n=8); (D) shows the RBD-specific IgG titer in the serum of immunized mice detected by ELISA, with data expressed as geometric mean and 95% confidence interval; (E) shows the neutralizing antibody titer against the wild-type strain in the serum of immunized mice detected by pseudovirus neutralization assay, with data expressed as geometric mean and 95% confidence interval; (F) shows the IFN-γ-secreting T cells in the spleen cells of immunized mice detected by ELISpot. Cell frequency, data are expressed as mean ± SEM; (GJ) shows the cytokine pattern of vaccine-induced T cells detected by flow cytometry, and the data are expressed as mean ± SEM; (K) shows the vaccine-specific IgG1 and IgG2c titers and IgG2c / IgG1 ratio in mouse sera collected 2 weeks after vaccination detected by ELISA; (L) shows the immunization and challenge experimental strategy of BALB / c aged mice; (M) and (N) show the survival rate and body weight changes of mice after challenge, respectively, and the data are expressed as mean ± SEM (n = 8).

[0045] Figure 18 shows the expression of mRNA encoding modified spike protein on the cell surface of HEK-293 cells and DC2.4 cells detected by flow cytometry.

[0046] FIG19 shows the results of IL-4 secretion induced by LPP / mRNA vaccine detected by flow cytometry.

[0047] Figure 20 shows that LPP / mRNA induces strong immunogenicity in rhesus macaques. (A) shows the experimental strategy for immunization, challenge, sample collection and evaluation of rhesus macaques; (B) shows the RBD-specific IgG titer in the serum of immunized rhesus macaques detected by ELISA, and the data are expressed as geometric means with 95% confidence intervals; (C) shows the neutralizing antibody titer against wild-type strains in the serum of immunized rhesus macaques detected by pseudovirus neutralization assay, and the data are expressed as geometric means with 95% confidence intervals; (D) shows the neutralizing antibody titer against Delta and Omicron (BA.1) variants in the serum of immunized rhesus macaques detected by pseudovirus neutralization assay (E) shows the neutralizing antibody titer against the variant strain detected by live virus cytopathic effect (CPE) test, and the data are expressed as geometric mean and 95% confidence interval; (F) shows the frequency of IFN-γ-secreting T cells in immunized rhesus macaque PBMCs detected by ELISpot, and the data are expressed as mean ± SEM; (G) shows the frequency of spike protein-specific MBCs in rhesus macaque PBMCs detected by flow cytometry, and the data are expressed as mean ± SEM.

[0048] Figure 21 shows that LPP / mRNA protects rhesus macaques from SARS-CoV-2 infection and mortality. After viral challenge, viral RNA copies were detected in (A) nasal swabs, (B) oropharyngeal swabs, and (C) anal swabs by RT-PCR; (D) shows the number of viral RNA copies in lung tissue detected by RT-PCR. (E) shows histopathological changes in the lungs of rhesus macaques 7 days after challenge.

[0049] FIG22 (A and B) shows the changes in body weight and body temperature of rhesus monkeys in the vaccine group and the normal saline group 1-7 days after challenge, respectively.

[0050] Figure 23 shows that homologous or heterologous LPP / mRNA boosters enhance vaccine efficacy. (A) shows the experimental strategy for homologous boosting immunization; (B) shows the RBD-specific IgG titer in the serum of immunized mice on day 84 detected by ELISA, and the data are expressed as geometric means with 95% confidence intervals; (C) shows the neutralizing antibody titer against wild-type strains in the serum of immunized mice on day 84 detected by pseudovirus neutralization assay, and the data are expressed as geometric means with 95% confidence intervals; (D) shows the frequency of IFN-γ-secreting T cells in splenocytes of immunized mice on day 84 detected by ELISpot, and the data are expressed as mean ± SEM; (EG) shows the neutralizing antibody titer against Delta and Omicron (BA.1 and BA.4 / 5) mutant strains in the serum of immunized mice on day 84 detected by pseudovirus neutralization assay, and the data are expressed as geometric means with 95% confidence intervals; (H) shows the experimental strategy for heterologous boosting immunization; (I) shows the fold change of RBD-specific IgG in the serum of immunized mice on day 84 relative to that on day 28 as detected by ELISA, and the data are expressed as geometric means with 95% confidence intervals; (J) shows the fold change of neutralizing antibody titers against wild-type strains in the serum of immunized mice on day 84 relative to that on day 28 as detected by pseudovirus neutralization assay, and the data are expressed as geometric means with 95% confidence intervals; (K) shows the frequency of IFN-γ-secreting T cells in the spleen and lungs of immunized mice on day 84 as detected by ELISpot, and the data are expressed as mean ± SEM; (LN) shows the fold change of neutralizing antibody titers against Delta and Omicron (BA.1 and BA.4 / 5) mutant strains in the serum of immunized mice on day 84 relative to that on day 28 as detected by pseudovirus neutralization assay, and the data are expressed as geometric means with 95% confidence intervals.

[0051] Figure 24 shows the safety study of the LPP / mRNA vaccine in rhesus macaques. (A) shows the immunization, sample collection, and assessment strategies for rhesus macaques. (B, E) show the results of white blood cell (WBC), lymphocyte (LYMPH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) detection at specific time points. (F and G) show the changes in body temperature and weight gain in rhesus macaques throughout the study period, with data presented as mean ± SEM. (H) shows the histopathological changes at the injection site of rhesus macaques on days 30 and 59.

[0052] FIG25 shows the results of detection of cytokine IL-6 in rhesus monkeys before administration, 24 hours after administration on day 1 and day 29, and on day 56 after administration. Specific implementation plan

[0053] General Definitions and Terminology

[0054] 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.

[0055] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are those widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0056] As used herein, the expressions "comprise," "include," "contain," and "have" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0057] As used herein, the singular forms "a," "an," or "the" include plural referents unless the context indicates otherwise. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0058] Recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. Unless expressly indicated to the contrary, values ​​or ranges recited herein are modified by "about" to mean ±20%, ±10%, ±5%, or ±3% of the recited or claimed value or range.

[0059] Unless otherwise stated, all methods described herein can be performed in any suitable order.

[0060] As used herein, "nucleotides" include deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "ribonucleotides" are constituents of ribonucleic acid (RNA), consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotides" are constituents of deoxyribonucleic acid (DNA), also consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and are the main chemical components of chromosomes. "Nucleotides" are usually referred to by a single letter representing the base: "A (a)" refers to deoxyadenosine or adenylate containing adenine, "C (c)" refers to deoxycytidine or cytidine containing cytosine, "G (g)" refers to deoxyguanosine or guanylate containing guanine, "U (u)" refers to uridine or uridylate containing uracil, and "T (t)" refers to deoxythymidylate containing thymine.

[0061] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It will be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, with deoxythymidylic acid in the DNA coding strand sequence corresponding to uridine in the RNA sequence it encodes.

[0062] As used herein, the term "% identity" with respect to sequences refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared. The identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window." Optimal alignment can be performed manually, or by means of algorithms known in the art, including but not limited to the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2,482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48,443, the search for similarity method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88,2444, or using computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the publicly available BLASTN or BLASTP algorithms available on the National Center for Biotechnology Information (NCBI) website.

[0063] In some embodiments, the % homogeneity is given in the region of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100%. In some embodiments, the homogeneity is given to the whole length of the reference sequence. The comparison of sequence homogeneity can be determined with instruments known in the art, preferably using optimal sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0064] As used herein, "modified" refers to non-natural. For example, RNA can be modified RNA. That is, RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linking groups. "Modified" groups can also be referred to herein as "altered" groups. Groups can be modified or altered chemically, structurally, or functionally. For example, a modified nucleobase can include one or more non-naturally occurring substitutions.

[0065] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and / or polypeptides. The term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract). A vector that can be used to produce a transcript is also referred to as a "transcription vector," which contains regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription."

[0066] As used herein, the term "host cell" refers to a cell used to receive, maintain, replicate, or express a polynucleotide or vector.

[0067] As used herein, an "aliphatic" group is a non-aromatic group in which the carbon atoms are linked in a chain, and can be saturated or unsaturated.

[0068] As used herein, the term "alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising one or more carbon atoms. 12 Alkyl" or "C 1-12 "Alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising 1 to 12 carbon atoms. As used herein, the term "alkoxy" refers to an alkyl group as described herein, which is attached to the remainder of the molecule through an oxygen atom. The term "alkylene" refers to a divalent group formed by the corresponding alkyl group having lost one hydrogen atom. The term "C1-C 12 "Alkylene" or "C 1-12 "Alkylene" refers to an optionally substituted straight or branched chain alkylene group comprising 1 to 12 carbon atoms.

[0069] As used herein, the term "alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms and at least one double bond. 12 Alkenyl" or "C 2-12 "Alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising 2 to 12 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may comprise one, two, three, four or more carbon-carbon double bonds.

[0070] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0071] As used herein, the term "carbocycle" refers to a monocyclic or polycyclic non-aromatic system comprising one or more rings consisting of carbon atoms. 3-8 "Carbocycle" means a carbon ring comprising 3-8 carbon atoms. The carbocycle may include one or more carbon-carbon double or triple bonds. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and the like. As used herein, when a carbocycle is saturated (i.e., does not contain unsaturated bonds), it may also refer to the corresponding cycloalkyl group. Unless otherwise specifically stated, the carbocycles described herein refer to unsubstituted and substituted, i.e., optionally substituted, carbocycles.

[0072] As used herein, the term "heterocycle" refers to a monocyclic or polycyclic ring system comprising one or more rings and comprising at least one heteroatom. The heteroatom can be, for example, a nitrogen, oxygen, phosphorus or sulfur atom. The heterocycle can include one or more double bonds or triple bonds and can be non-aromatic. Examples of heterocycles include, but are not limited to, imidazolidinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl and piperidinyl. The heterocycle can comprise, for example, 3-10 atoms (non-hydrogen), i.e., 3-10 membered heterocycles (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 members), wherein one or more atoms are heteroatoms (e.g., N, O, S or P). When the heterocycle is saturated (i.e., without unsaturated bonds), it can also refer to the corresponding heterocycloalkyl. Unless otherwise specifically stated, the heterocycles described herein refer to both unsubstituted and substituted heterocyclic groups, i.e., optionally substituted heterocycles.

[0073] As used herein, the term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. 10 The alkylaryl group can have 6 to 10 carbon atoms, for example 6, 7, 8, 9, 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.

[0074] As used herein, the term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, with the remaining ring atoms being C, and having at least one aromatic ring. A heteroaryl group can have 5-10 ring atoms (5-10 membered heteroaryl), including 5-, 6-, 7-, 8-, 9-, or 10-membered, particularly 5- or 6-membered heteroaryl groups. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

[0075] As used herein, the term "interrupted by one or more groups" means that the one or more groups are present on the carbon chain, and the rest of the carbon chain is connected to both ends of the one or more groups.

[0076] Unless otherwise specifically stated, the groups described herein (e.g., any one of R1-R7, such as alkyl, alkylene, alkenyl, aryl, amino, etc.) may be optionally substituted. Optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chloro, bromo, fluoro, or iodo), carboxylic acids (e.g., -C(O)OH), alcohols (e.g., hydroxy, -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyls (e.g., -C(O)R, or represented by C=O), acyl halides (e.g., -C(O)X, wherein X is a halide selected from bromo, fluoro, chloro, and iodo), carbonates (e.g., -OC(O)OR), alkoxy groups (e.g., -OR), acetals (e.g., -C(OR)2R"", wherein each OR is the same or different alkoxy group and R"" is an alkyl or alkenyl group), phosphates (e.g., P(O)4 3- ), thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfinic acids (e.g., -S(O)OH), sulfonic acids (e.g., -S(O)2OH), thialdehydes (e.g., -C(S)H), sulfates (e.g., S(O)4 2- ), sulfonyl (e.g., -S(O)2-), amide (e.g., -C(O)NR2 or -N(R)C(O)R), azido (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR2, NRH, or -NH2), carbamoyl (e.g., -OC(O)NR2, -OC(O)NRH, or -OC(O)NH2), sulfonamide (e.g., -S(O)2NR2, -S(O)2NRH, -S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H, -N(H)S(O)2H), C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 aryl, 5-10 membered heteroaryl or 3-10 membered heterocycle. In any of the foregoing, each R can independently be a substituent as defined herein, such as alkyl, alkoxy, alkylene, halogen, carbocycle, heterocycle, aryl, heteroaryl, alkenyl. In some embodiments, the substituent itself can be further substituted with, for example, one, two, three, four, five or six substituents as defined herein. For example, an alkyl group can be further substituted with one, two, three, four, five or six substituents as described herein.

[0077] As used herein, the term "compound" is intended to include isotopic compounds of the depicted structure. "Isotopes" refer to atoms having the same atomic number but different mass numbers due to the number of neutrons in their nuclei, such as deuterium isotopes. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts, or complexes of the present invention can be prepared by combining with solvents or water molecules to form solvates and hydrates by conventional methods.

[0078] The term "optionally" or "optionally" (e.g., optionally substituted) means that the subsequently described event may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and that the description includes both substituted and unsubstituted alkyl radicals.

[0079] It should be understood that when chemical groups are written in a particular order, the reverse order is also encompassed unless otherwise stated. For example, in the formula -(R) where M1 is defined as -C(O)NH- i -(M1) k -(R) m -(i.e., -(R) i -C(O)-NH-(R) m -), unless otherwise stated, also encompasses compounds where M1 is -NHC(O)- (i.e., -(R) i -NHC(O)-(R) m -).

[0080] As used herein, the term "contact" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a lipid composition means that the mammalian cell and lipid nanoparticle are physically connected. The method of contacting a cell with an external entity in vivo and in vitro is well-known in the biological field. For example, contacting a lipid composition with a mammalian cell in a mammal can be carried out by different routes of administration (such as intravenous, intramuscular, intradermal and subcutaneous) and can relate to different amounts of lipid compositions. In addition, the lipid composition can contact more than a mammalian cell.

[0081] As used herein, the term "delivery" refers to providing an entity to a target. For example, delivering a therapeutic or prophylactic agent to a subject can involve administering a composition comprising the therapeutic or prophylactic agent to the subject.

[0082] As used herein, the term "subject" describes an organism to which the compositions of the present invention may be administered. Subjects to which these compositions may be administered include, but are not limited to, humans, other primates, and other mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, or rats. Preferably, the subject may be a mammal, particularly a human.

[0083] As used herein, "encapsulation efficiency" refers to the ratio of the amount of therapeutic or prophylactic agent that becomes part of the composition to the initial total amount of therapeutic or prophylactic agent used to prepare the composition. For example, if 97 mg of the therapeutic or prophylactic agent is encapsulated in the composition out of a total of 100 mg of therapeutic or prophylactic agent initially provided to the composition, then the encapsulation efficiency can be determined to be 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial encapsulation, sealing, surrounding, or packaging.

[0084] 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.

[0085] 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.

[0086] As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety into its salt form (e.g., by reacting a free basic group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium salts, and the like; and non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present invention include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally speaking, these salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent, or in a mixture of the two; non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0087] 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.

[0088] As used herein, "zeta potential" refers to the zeta potential of lipids in a lipid composition, and is an important indicator for characterizing the stability of a dispersion.

[0089] As used herein, "size" or "average size" in the context of a composition refers to the average diameter of the composition.

[0090] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, or reducing the occurrence of one or more symptoms or features of a particular infection, disease, disorder, or condition. "Preventing" refers to guarding against an underlying disease or preventing the worsening of symptoms or the development of a disease.

[0091] The term "prophylactically or therapeutically 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 prophylactically or therapeutically effective amount is affected by factors including, but not limited to, the rate of development and severity of the disease or symptom, the age, sex, weight, and physiological condition of the subject, the duration of treatment, and the specific route of administration. The prophylactically or therapeutically effective amount can be administered in one or more doses. The prophylactically or therapeutically effective amount can be achieved by continuous or intermittent administration.

[0092] Lipid composition

[0093] The present invention provides a lipid composition. The lipid composition is a lipid delivery vehicle, wherein the lipid can encapsulate a therapeutic agent or a preventive agent (such as a nucleotide) to form nanoparticles, thereby delivering the nanoparticles into an organism.

[0094] As used herein, the term "lipid" refers to an organic compound comprising a hydrophobic portion and optionally a hydrophilic portion. Lipids are generally poorly soluble in water but soluble in many organic solvents. Typically, amphipathic lipids comprising a hydrophobic portion and a hydrophilic portion can be organized into a lipid bilayer structure in an aqueous environment, for example, in the form of vesicles. Lipids may include, but are not limited to, fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, steroids, and cholesterol esters.

[0095] As used herein, "lipid nanoparticle" or "LNP" refers to a lipid vesicle having a uniform lipid core, which is a particle formed by lipids, the lipid components of which undergo intermolecular interactions to form a nanostructured entity. A therapeutic or prophylactic agent (such as a nucleic acid, e.g., mRNA) is encapsulated in the lipids.

[0096] 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 a therapeutic or prophylactic agent (such as a nucleic acid, such as mRNA) is contained in a polyplex, which itself is encapsulated in a biocompatible lipid bilayer shell to form the lipid nanoparticles of the present invention. In some embodiments, the lipid composition of the present invention is a lipid polyplex (LPP). In some embodiments, the composition of the present invention is a lipid polyplex (LPP) comprising RNA.

[0097] In some embodiments, the lipid encapsulating the therapeutic or preventive agent (such as a nucleic acid, e.g., mRNA) is selected from one or more of the following lipids: a cationic lipid, a phospholipid, a steroid, and / or a polyethylene glycol-modified lipid. In a preferred embodiment, the cationic lipid is an ionizable cationic lipid.

[0098] In one embodiment, the lipid composition 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-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2. In a preferred embodiment, the cationic lipid comprises M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2, or SW-II-140-2.

[0099] In one embodiment, the lipid composition comprises a phospholipid and / or a steroid. In one embodiment, the lipid composition comprises a phospholipid as described herein, wherein the phospholipid comprises 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl oil Acylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) or a combination thereof. In one embodiment, the lipid composition comprises a steroid as described herein, wherein the steroid comprises cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and derivatives thereof.In one embodiment, the lipid composition comprises a phospholipid as described herein and a steroid. In one embodiment, the lipid composition comprises DOPE. In one embodiment, the lipid composition comprises DSPC. In one embodiment, the lipid composition comprises cholesterol. In one embodiment, the lipid composition comprises DOPE and cholesterol. In one embodiment, the lipid composition comprises DSPC and cholesterol.

[0100] In one embodiment, the lipid composition comprises a cationic lipid M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, a phospholipid DOPE and cholesterol. In one embodiment, the lipid composition comprises a cationic lipid M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, a phospholipid DSPC and cholesterol.

[0101] 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, and DSPE-PEG, or a combination thereof. 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).

[0102] In one embodiment, the lipid composition comprises a cationic lipid, DOPE, cholesterol, and DSPE-PEG.

[0103] In one embodiment, the lipid composition comprises a cationic lipid, DSPC, cholesterol, and DSPE-PEG.

[0104] In one embodiment, the lipid composition comprises a cationic lipid, DSPC, cholesterol, and DMG-PEG.

[0105] In a preferred embodiment, the lipid composition comprises a cationic lipid, DOPE, cholesterol, and DMG-PEG.

[0106] In a preferred embodiment, the lipid composition comprises the cationic lipid M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, DOPE, cholesterol and DMG-PEG.

[0107] In some embodiments, the lipid composition of the present invention further comprises a cationic polymer, which is associated with the therapeutic agent or prophylactic agent (such as a nucleic acid, for example, mRNA) as a complex and is co-encapsulated in the lipid.

[0108] 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.

[0109] In one embodiment, the amount of lipid in the lipid composition is calculated as a molar percentage (mol %), which is determined based on the total moles of lipid in the composition. Unless otherwise specified, the sum of the amounts (mol %) of the individual lipids in the composition is 100 mol %, i.e., the sum of the amounts (mol %) of cationic lipids, phospholipids, steroids, and polyethylene glycol-modified lipids is 100 mol %.

[0110] In one embodiment, the amount of cationic lipid in the lipid composition is about 10-about 70 mol%. In some embodiments, the amount of cationic lipid in the lipid composition is about 20-about 60 mol%, about 30-about 50 mol%, about 35-about 50 mol%, about 35-about 45 mol%, about 38-about 45 mol%, about 40-about 45 mol%, about 40-about 50 mol% or about 45-about 50 mol%. For example, the amount of cationic lipid can be about 10, 15, 20, 25, 30, 35, 38, 40, 45, 50, 55, 60, 65, 70 mol%.

[0111] In one embodiment, the amount of phospholipids in the lipid composition is about 10-about 70 mol%. In one embodiment, the amount of phospholipids in the lipid composition is about 20-about 60 mol%, about 30-about 50 mol%, about 10-about 30 mol%, about 10-about 20 mol%, or about 10-about 15 mol%. For example, the amount of phospholipids can be about 10, 15, 20, 25, 30, 33.5, 35, 40, 45, 50, 53.5, 55, 60, 65 or 70 mol%.

[0112] In one embodiment, the amount of cholesterol in the lipid composition is about 10-about 70 mol%. In one embodiment, the amount of cholesterol in the lipid composition is about 20-about 60 mol%, about 24-44 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, 13.5, 15, 18.5, 18.75, 20, 23.5, 23.75, 24, 25, 28.5, 28.75, 29, 30, 33.75, 34, 35, 38.5, 38.75, 39, 40, 43, 43.5, 44, 45, 48.5, 50, 55, 60, 65 or 70 mol%.

[0113] In one embodiment, the amount of polyethylene glycol-modified lipids in the lipid composition is about 0.05-about 20 mol%. In one embodiment, the amount of polyethylene glycol-modified lipids in the lipid composition is about 0.5-about 15 mol%, about 1-about 10 mol%, about 5-about 15 mol%, about 1-about 5 mol%, about 1-about 1.5 mol%, about 1.5-about 3 mol%, or about 2-5 mol%. For example, the amount of polyethylene glycol-modified lipids can be about 0.05, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15 or 20 mol%.

[0114] In one embodiment, the lipid composition comprises 10-70 mol % of cationic lipids, 10-70 mol % of phospholipids, 10-70 mol % of steroids and 0.05-20 mol % of polyethylene glycol-modified lipids. In a preferred embodiment, the lipid composition comprises 35-50 mol % of cationic lipids, 10-30 mol % of phospholipids, 24-44 mol % of steroids and 1-1.5 mol % of polyethylene glycol-modified lipids.

[0115] In one embodiment, the LPP comprises a therapeutic or prophylactic agent of the present invention (e.g., a nucleic acid, e.g., mRNA) associated with a cationic polymer as a complex; and a lipid encapsulating the complex, wherein the lipid encapsulating the complex comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol-modified lipid. 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 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-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.

[0116] In one embodiment, the lipid encapsulation complex comprises 50 mol% M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 10 mol% DOPE, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG. In one embodiment, the lipid encapsulation complex comprises 40 mol% M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 15 mol% DOPE, 43.5 mol% cholesterol and 1.5 mol% DMG-PEG.

[0117] In one embodiment, the therapeutic or prophylactic agent is a polynucleotide comprising a coding region, wherein the coding region encodes a modified spike protein, wherein the modified spike protein comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:12; and wherein the polynucleotide is RNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:3; or wherein the polynucleotide is DNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:5.

[0118] In one embodiment, the polynucleotide is RNA comprising the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:6; or the polynucleotide is DNA comprising the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:7.

[0119] In one embodiment, the lipid encapsulating the complex comprises 40 mol% M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 15 mol% DOPE, 43.5 mol% cholesterol and 1.5 mol% DMG-PEG; the therapeutic or prophylactic agent is a polynucleotide comprising a coding region encoding a modified spike protein, wherein the modified spike protein comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12; and wherein the polynucleotide is RNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 3; or wherein the polynucleotide is DNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO: NO:5 or a nucleotide sequence having at least 85% identity with the nucleotide sequence of SEQ ID NO:5.

[0120] In one embodiment, the lipid encapsulating the complex comprises 40 mol% M5, 15 mol% DOPE, 43.5 mol% cholesterol and 1.5 mol% DMG-PEG; the therapeutic or prophylactic agent is a polynucleotide comprising a coding region encoding a modified spike protein, wherein the modified spike protein comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:12; and wherein the polynucleotide is RNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:3; or wherein the polynucleotide is DNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:5.

[0121] In one embodiment, the lipid encapsulating the complex comprises 40 mol% of M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 15 mol% of DOPE, 43.5 mol% of cholesterol and 1.5 mol% of DMG-PEG; the therapeutic or prophylactic agent is a polynucleotide, which is RNA comprising the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 6; or the polynucleotide is DNA comprising the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 7.

[0122] In one embodiment, the lipid encapsulating the complex comprises 40 mol% M5, 15 mol% DOPE, 43.5 mol% cholesterol and 1.5 mol% DMG-PEG; the therapeutic or prophylactic agent is a polynucleotide, which is RNA comprising the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 6; or the polynucleotide is DNA comprising the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 7.

[0123] Cationic lipids

[0124] 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.

[0125] Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), didecyldimethylammonium bromide (DDAB), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), dioctadecyldimethylammonium chloride ... dioctadecyldimethylammonium chloride (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium brom chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (D MRIE), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis, cis-9,1-(cis,cis-9,12-octadecadienoxy)propane (3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane, CLinDMA), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide bromide, DMORIE), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), dioctadecylamidoglycyl spermine spermine (DOGS), N4-cholesteryl-spermine, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate, DLin-MC3-DMA, a compound of formula (I), (II), (III) or (IV) as described herein, or a combination thereof.

[0126] In some embodiments, the cationic lipid is preferably an ionizable cationic lipid. Ionizable cationic lipids carry a net positive charge at, for example, acidic pH, but are neutral at higher pH (eg, physiological pH). Examples of ionizable cationic lipids include, but are not limited to, dioctadecylamidoglycyl spermine (DOGS), N4-cholesteryl-spermine, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), a compound of formula (I), (II), (III) or (IV) as described herein, or a combination thereof.

[0127] In one embodiment, the cationic lipid comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0128] in,

[0129] R1 and R2 are each independently selected from a bond, a C1-C 12 Alkyl and C2-C 12 alkenyl;

[0130] R3 and R4 are each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 aryl and 5-10 membered heteroaryl; and R3 and R4 are each independently optionally substituted by t R6, t being an integer selected from 1-5;

[0131] R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl;

[0132] M1 and M2 are each independently selected from a bond, H, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, a 3-10 membered heterocycle, -NR7-, or

[0133] R5 together with one of M1 and M2 together with the nitrogen atom to which they are attached forms a 3-10 membered heterocyclic ring, and the corresponding R1 / R3 or R2 / R4 are absent, said heterocyclic ring being optionally substituted by R7;

[0134] R5 is selected from C 3-8 Carbocyclic ring, -C 1-12 Alkylene-Q, Q is selected from H, -OR7, -SR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)S(O)2R7, -N(R7)C(S)R7, -N(R7)2, cyano, C 3-8 Carbocyclic ring, 3-10 membered heterocyclic ring, C6-C 10 Aryl, each of the above groups is optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O) substitution;

[0135] m and n are each independently an integer selected from 0-12;

[0136] The alkyl, alkenyl and alkylene groups are each optionally and independently interrupted by one or more groups selected from: -O-, -S-, -NR7-, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 carbocycle, and said alkyl, alkenyl and alkylene are each optionally substituted with one or more R7;

[0137] R7 are each independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C 3-8 Carbocyclic ring, each of the above groups is optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0138] In one embodiment, R1 and R2 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl, such as C1-C12 In another embodiment, one of R1 and R2 is a bond, and the other is independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl, such as C1-C 12 alkyl.

[0139] In one embodiment, R3 and R4 are each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 In another embodiment, R3 and R4 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl.

[0140] R3 and R4 can each be independently optionally substituted by t R6, where t is 1, 2, 3, 4, or 5. In one embodiment, R6 is each independently selected from C1-C 12 alkyl.

[0141] In yet another embodiment, at least one of R3 and R4 is C6-C 10 Aryl or 5-10 membered heteroaryl, such as C6-C 10 Aryl.

[0142] In one embodiment, R5 is selected from C 3-8 Carbocyclic ring, -C 1-12 Alkylene-Q. Q can be selected from H, -OR7, -SR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)S(O)2R7, -N(R7)C(S)R7, -N(R7)2, cyano, C 3-8 Carbocyclic ring, 3-10 membered heterocyclic ring, C6-C 10 The above groups, including those encompassing the options of Q, may each be optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0143] In yet another embodiment, R5 is selected from C 3-8 Carbocyclic ring, -C 1-12 Alkylene-Q, Q is selected from H, -OR7, -SR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)S(O)2R7, -N(R7)C(S)R7, -N(R7)2, cyano, C 3-8Carbocyclic ring, 3-10 membered heterocyclic ring, C6-C 10 The above groups, including those encompassing the options of Q, may each be optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0144] In the compound of formula (I), R7 can be independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C 3-8 Carbocyclic ring, preferably selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl and 5-10 membered heteroaryl. The above groups (when appropriate, such as H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclic ring, C 3-8 carbocyclic ring) are each optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0145] In one embodiment, each group described above, such as C 3-8 Carbocyclic ring, -C 1-12 Alkylene-Q, including -OR7, -SR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)S(O)2R7, -N(R7)C(S)R7, -N(R7)2, C 3-8 Carbocyclic ring, 3-10 membered heterocyclic ring, C6-C 10Aryl, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C 3-8 The carbocyclic rings etc. may each be optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0146] In one embodiment, the alkyl, alkenyl and alkylene groups (e.g., those mentioned in R1-R7) in the compounds of formula (I) may each be optionally and independently interrupted by one or more groups selected from the group consisting of: -O-, -S-, -NR7-, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 The alkyl, alkenyl and alkylene groups are each optionally substituted by one or more R7. That is, the chains (straight or branched) of the alkyl, alkenyl and alkylene groups may each optionally contain one or more groups selected from the following: -O-, -S-, -NR7-, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 Carbon ring.

[0147] R7 are each independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C 3-8 Carbocyclic ring; preferably, R7 is independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl and 5-10 membered heteroaryl. The above groups (when appropriate, such as H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclic ring, C 3-8 carbocyclic ring) are each optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0148] 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.

[0149] In one embodiment, M1 or M2 is a bond, the corresponding m or n is not 0, and the carbon chain preceding M1 or M2 is connected to the corresponding R1 or R2.

[0150] 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.

[0151] 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.

[0152] In one embodiment, M1 and M2 are each independently selected from -C(O)-, -OC(O)-, -C(O)O-. In another embodiment, M1 and M2 are each independently selected from -NR7-, and R7 is as described above.

[0153] In another embodiment, R5 and one of M1 and M2 together with the nitrogen atom to which they are attached form a 3-10 membered heterocyclic ring, and the corresponding R1 / R3 or R2 / R4 are absent, said heterocyclic ring being optionally substituted with R7, R7 being as described above.

[0154] In one embodiment, R5 is selected from -C 1-12 Alkylene-Q, Q is selected from H, -OR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)2, cyano, and R7 is as described above.

[0155] In a preferred embodiment, R1 and R2 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl;

[0156] wherein R3 and R4 are each independently selected from C1-C 12 Alkyl and C2-C12 and R3 and R4 are each independently optionally substituted by t R6, t is an integer selected from 1-5; R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl.

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

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

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

[0160] In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

[0161] In a preferred embodiment, the cationic lipid comprises M5 or SM-102.

[0162] In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

[0163] In a preferred embodiment, the cationic lipid comprises MC3.

[0164] In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

[0165] In a preferred embodiment, the cationic lipid comprises ALC-0315.

[0166] In one embodiment, the cationic lipid comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0167] R1 and R2 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl;

[0168] R3 and R4 are each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 Aryl and 5-10 membered heteroaryl;

[0169] Provided that at least one of R3 and R4 is C6-C 10 aryl or 5-10 membered heteroaryl, and R3 and R4 are each independently optionally substituted by t R6, t is an integer selected from 1-5; R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl;

[0170] M1 and M2 are each independently selected from -OC(O)-, -C(O)O-, -SC(S)-, and -C(S)S-;

[0171] R5 is selected from -C 1-12 Alkylene-Q, Q is selected from -OR7 and -SR7, R7 is independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl and 5-10 membered heteroaryl;

[0172] m and n are each independently an integer selected from 1-12.

[0173] In one embodiment, R2 is selected from C1-C 12 In another embodiment, R2 is selected from C1-C6 alkyl.

[0174] In one embodiment, one of R3 and R4 is C6-C 10 Aryl or 5-10 membered heteroaryl, the other is C1-C 12 Alkyl or C2-C 12 Alkenyl.

[0175] In one embodiment, R3 and R4 are each independently selected from C1-C 12 In another embodiment, one of R3 and R4 is phenyl and the other is C1-C 12 alkyl.

[0176] In another embodiment, R3 and R4 are each independently substituted with t R6, t being an integer selected from 1-5; for example 1, 2, 3, 4 or 5. Preferably, t is an integer from 1-3, for example 1, 2 or 3, in particular 1 or 2.

[0177] In one embodiment, R6 is independently selected from C1-C 12 Alkyl groups, such as C1-C 10 alkyl.

[0178] In one embodiment, t is 1 and R6 is substituted at the meta or para position relative to R1 or R2 on the phenyl ring.

[0179] In another embodiment, t is 2 and R6 is substituted at the meta and para positions on the phenyl ring relative to R1 or R2.

[0180] In one embodiment, R4 is substituted at the 1st or last position of R2. The 1st position refers to the position of the carbon atom in R2 that is directly connected to M2. The last position refers to the position of the carbon atom in R2 that is farthest from M2. In a specific embodiment, R4 is selected from C1-C 12 Alkyl, R3 is phenyl.

[0181] In one embodiment, R3 is substituted at the 1st position or the last position of R1. The 1st position refers to the position of the carbon atom in R1 that is directly connected to M1. The last position refers to the position of the carbon atom in R1 that is farthest from M1. In a specific embodiment, R3 is selected from C1-C 12 Alkyl, R4 is phenyl.

[0182] In one embodiment, M1 and M2 are each independently selected from -OC(O)- and -C(O)O-.

[0183] In one embodiment, R5 is selected from -C 1-5 Alkylene-Q, such as C1, C2, C3, C4 or C5 alkylene-Q. In exemplary embodiments, R5 is selected from -C 1-3 Alkylene-Q, for example C1, C2 or C3 alkylene-Q.

[0184] In another embodiment, Q is selected from -OH and -SH, in particular -OH.

[0185] 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.

[0186] In certain embodiments, the compound of formula (I) includes a compound represented by formula (II):

[0187] or a pharmaceutically acceptable salt thereof, wherein each group is as defined herein.

[0188] In one embodiment,

[0189] R1 is selected from C1-C6 alkyl;

[0190] R2 is selected from C1-C 10 alkyl;

[0191] R4 is selected from C1-C 10 alkyl;

[0192] M1 and M2 are each independently selected from -OC(O)- and -C(O)O-;

[0193] R5 is selected from -C 1-5 Alkylene-Q, Q is selected from -OR7 and -SR7, R7 is independently selected from H, C1-C 12 Alkyl and C2-C 12 alkenyl;

[0194] R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl, especially C1-C 12 alkyl;

[0195] m and n are each independently an integer selected from 2-9, such as 2, 3, 4, 5, 6, 7, 8 or 9;

[0196] t is an integer selected from 1-3.

[0197] In one embodiment, R5 is selected from -C 1-3 Alkylene-Q, Q is selected from -OH and -SH, in particular -OH.

[0198] In one embodiment, m and n are each independently an integer selected from 2-7, such as 2, 3, 4, 5, 6 or 7.

[0199] In some embodiments, t is 1 or 2.

[0200] In one embodiment, R4 is substituted at position 1 or the last position of R2. Position 1 refers to the position of the carbon atom in R2 that is directly connected to M2. The last position refers to the position of the carbon atom in R2 that is farthest from M2.

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

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

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

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

[0205] In one embodiment,

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

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

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

[0209] R5 is selected from -C 1-3 Alkylene-Q, Q is selected from -OH and -SH, in particular -OH;

[0210] t is 1 or 2;

[0211] R6 is selected from C1-C 12 Alkyl and C2-C 12 Alkenyl, especially C1-C 12 alkyl;

[0212] m and n are each independently an integer selected from 2-7, for example 2, 3, 4, 5, 6 or 7.

[0213] In one embodiment, R4 is substituted at the 1st or last position of R2. The last position refers to the position of the C atom in R2 that is directly connected to the The position of the furthest C atom in the part.

[0214] In one embodiment, t is 1 and R6 is substituted at the meta or para position relative to R1 on the phenyl ring.

[0215] In another embodiment, t is 2 and R6 is substituted at the meta and para positions on the phenyl ring relative to R1.

[0216] In certain embodiments, the compound of formula (I) includes a compound represented by formula (IV):

[0217] or a pharmaceutically acceptable salt thereof, wherein each group is as defined herein.

[0218] In one embodiment,

[0219] R1 is selected from C1-C6 alkyl;

[0220] R2 is selected from C1-C 10 alkyl;

[0221] R4 is selected from C1-C 10 alkyl;

[0222] t is 1 or 2;

[0223] R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl, especially C1-C 12 alkyl;

[0224] m and n are each independently an integer selected from 2-7, for example 2, 3, 4, 5, 6 or 7.

[0225] In one embodiment, R4 is substituted at the 1st or last position of R2. The last position refers to the position of the C atom in R2 that is directly connected to the The position of the furthest C atom in the part.

[0226] In one embodiment, t is 1 and R6 is substituted at the meta or para position relative to R1 on the phenyl ring.

[0227] In another embodiment, t is 2 and R6 is substituted at the meta and para positions on the phenyl ring relative to R1.

[0228] In a specific embodiment, the substituents (eg, R1-R7) in the lipid compounds of the present invention do not include alkenyl groups.

[0229] In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

[0230] In a preferred embodiment, the cationic lipid comprises the following lipid compounds: SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2, or SW-II-140-2.

[0231] In a preferred embodiment, the cationic lipid does not comprise T5',

[0232] In a preferred embodiment, the cationic lipid comprises the following lipid compound: M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.

[0233] phospholipids

[0234] The lipid composition of the present invention contains phospholipids, which can assist the cell penetration of the lipid composition.

[0235] Examples of phospholipids include, but are not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl oil Acylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof.

[0236] steroids

[0237] The lipid compositions of the present invention contain steroids, which may serve as structural components of the lipid compositions.

[0238] Examples of steroids include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and derivatives thereof.

[0239] PEG-modified lipids

[0240] 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, 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).

[0241] 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:

[0242] The average value of n is 44.

[0243] Cationic polymers

[0244] 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.

[0245] The term "protamine" refers to a low molecular weight basic protein rich in arginine, which exists in sperm cells of various animals (especially fish) and replaces histones to bind to DNA. In a preferred embodiment, the cationic polymer is protamine (e.g., protamine sulfate).

[0246] Physical and chemical properties

[0247] The physicochemical properties of lipid compositions can depend on their components. For example, a composition comprising cholesterol as a structural lipid can have physicochemical properties different from compositions comprising different structural lipids. Similarly, the physicochemical properties of a composition can depend on the absolute or relative amounts of its components. For example, a composition comprising a higher mole fraction phospholipid can have physicochemical properties different from compositions comprising a lower mole fraction phospholipid. Physicochemical properties can also vary depending on the method and conditions for preparing the composition.

[0248] The physicochemical properties of lipid compositions can be characterized by a variety of methods. For example, the morphology and size distribution of the composition can be checked using microscopy (e.g., transmission electron microscopy or scanning electron microscopy). Dynamic light scattering or potentiometric analysis (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to measure particle size. Multiple features of the composition, such as particle size, polydispersity index, and zeta potential, can also be measured using instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK).

[0249] The average size of the composition can be between tens of nanometers and hundreds of nanometers, for example, as measured by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 250 nm, such as about 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, or 300 nm. In some embodiments, the average size of the composition can be from about 50 nm to about 300 nm, from about 50 nm to about 290 nm, from about 50 nm to about 280 nm, from about 50 nm to about 270 nm, from about 50 nm to about 260 nm, from about 60 nm to about 300 nm, from about 60 nm to about 290 nm, from about 60 nm to about 280 nm, from about 60 nm to about 270 nm, from about 70 nm to about 300 nm, from about 70 nm to about 290 nm, from about 70 nm to about 280 nm, from about 70 nm to about 270 nm, from about 70 nm to about 260 nm, from about 80 nm to about 280 nm, from about 80 nm to about 270 nm, from about 80 nm to about 260 nm, from about 80 nm to about 250 nm, from about 90 nm to about 280 nm, from about 90 nm to about 270 nm, or from about 90 nm to about 260 nm. In certain embodiments, the average size of the lipid composition can be about 90nm to about 290nm or about 100nm to about 250nm. In a specific embodiment, the average size can be about 100nm. In other embodiments, the average size can be about 150nm. In other embodiments, the average size can be about 200nm.

[0250] In some embodiments, the lipid composition can be relatively homogeneous. Polydispersity index can be used to indicate the homogeneity of lipid composition, such as the particle size distribution of lipid composition. Smaller (e.g., less than 0.3) polydispersity index generally indicates a narrower particle size distribution. The polydispersity index of composition can be approximately 0 to approximately 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25. In some embodiments, the polydispersity index of lipid composition can be approximately 0.10 to approximately 0.20.

[0251] The zeta potential of a composition can be used to indicate the zeta potential of the composition. For example, the zeta potential can describe the surface charge of a composition. Compositions with relatively low charges, i.e., positively or negatively charged, are generally desirable because compositions with higher charges may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0252] The encapsulation efficiency of therapeutic or preventive agent describes the ratio of the amount of the therapeutic or preventive agent that is encapsulated in the composition or otherwise combined with the composition after preparation relative to the initial amount provided. Higher encapsulation efficiency is ideal (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of the therapeutic or preventive agent in the solution containing the composition before and after splitting the composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of the therapeutic or preventive agent (e.g., RNA) in the solution. For compositions as described herein, the encapsulation efficiency of the therapeutic or preventive agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0253] Pharmaceutical composition

[0254] The present invention also provides a pharmaceutical composition comprising the lipid composition of the present invention and a pharmaceutically acceptable carrier.

[0255] Pharmaceutically acceptable carriers may include, but are not limited to, 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), sorbitan, In some embodiments, the carrier may be a lipid composition comprising a carrier such as a lipophilic acid ...

[0256] The pharmaceutical composition provided herein can be in various dosage forms, including but not limited to solid, semisolid, liquid, powder or lyophilized form. For the pharmaceutical composition comprising lipid composition, preferred dosage form can generally be such as injection and lyophilized powder. Pharmaceutical composition can be prepared into various forms suitable for multiple routes of administration and methods. For example, pharmaceutical composition can be prepared into liquid dosage form (such as emulsion, microemulsion, nanoemulsion, solution, suspension, syrup and elixir), injectable form, solid dosage form (such as capsule, tablet, pill, powder and granule), dosage form (such as ointment, paste, emulsifiable paste, lotion, gel, powder, solution, spray, inhalant and patch), suspension, powder and other form for surface and / or transdermal administration.

[0257] The pharmaceutical composition of the present invention may be an injectable pharmaceutical composition, which may accordingly contain a pharmaceutically acceptable excipient for injection, preferably an excipient for intramuscular injection.

[0258] Therefore, the present invention also provides an injection comprising the lipid composition of the present invention and a pharmaceutically acceptable injection excipient. For example, a sterile injectable aqueous or oily suspension can be included. The sterile injectable formulation can be a sterile injectable solution, suspension, and / or emulsion in a non-toxic parenteral acceptable diluent and / or solvent, such as a solution in 1,3-butanediol.

[0259] The above-mentioned injection excipients can include one or more of water, carbohydrate solution, electrolyte solution, and amino acid solution. For example, the injection excipient can include one or more of 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. Sterile fixed oil can also be used as a solvent or suspension medium. For this purpose, any gentle fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injectable preparations.

[0260] Therapeutic / preventive agents

[0261] The lipid composition may comprise one or more therapeutic or prophylactic agents. The present invention provides methods for delivering a therapeutic or prophylactic agent to a mammalian cell or organ, producing a target polypeptide in a mammalian cell, and treating a disease or condition in a mammal in need thereof, comprising administering to the mammal a lipid composition comprising a therapeutic or prophylactic agent.

[0262] Therapeutic or prophylactic agents include biologically active substances and are alternatively referred to as "active agents." A therapeutic or prophylactic agent can be a substance that, upon delivery to a cell or organ, causes a desired change in that cell or organ, or in other body tissues or systems. Such substances can be used to treat one or more diseases, disorders, or conditions. In some embodiments, a therapeutic or prophylactic agent is a small molecule drug that can be used to treat a specific disease, disorder, or condition.Examples of drugs that can be used in the composition include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, arabinoside), anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines

[0013] These agents include, but are not limited to, illicit drugs (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma medications, vitamins, sedatives, and imaging agents.

[0263] In some embodiments, the therapeutic or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, or another therapeutic or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, daunorubic ... dione), mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologues thereof. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and preparations that can provide immunity for one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis and can include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines can also include compounds and preparations that guide the immune response for cancer cells and can include mRNA encoding tumor cell-derived antigens, epitopes and / or new epitopes. Compounds that cause immune response can include vaccines, corticosteroids (e.g., dexamethasone) and other substances. In some embodiments, vaccines and / or compounds that can cause immune response are administered intramuscularly by a composition including a compound according to formula (I), (II), (III) or (IV).Other therapeutic or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, razithromycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide,

[0014] Examples of the present invention include, but are not limited to, daunomycin (formerly daunomycin), bleomycin, mithramycin, and anthramycin (AMC), and antimitotic agents such as vincristine, vinblastine, paclitaxel, and maytansine.

[0264] In other embodiments, the therapeutic or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of the present invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0265] polynucleotides

[0266] In some embodiments, the therapeutic or prophylactic agent is a polynucleotide or nucleic acid (eg, ribonucleic acid or deoxyribonucleic acid).

[0267] In some embodiments, the therapeutic or preventive agents of the present invention are RNA. As used herein, the definition of "RNA" encompasses single-stranded, double-stranded, linear and circular RNA. The RNA of the present invention can be chemically synthesized, recombinantly produced and in vitro transcribed RNA. In one embodiment, the RNA of the present invention is used to express a polypeptide in a host cell.

[0268] In one embodiment, the therapeutic or preventive agent of the present invention is a single-stranded RNA. In one embodiment, the RNA of the present invention is an in vitro transcribed RNA (IVT-RNA). IVT-RNA can be obtained by in vitro transcription using a DNA template using RNA polymerase.

[0269] In some embodiments, the therapeutic or preventive agent of the present invention is a messenger RNA (mRNA). Generally speaking, an mRNA may comprise a 5'-UTR sequence, a coding sequence for a polypeptide, a 3'-UTR sequence, and an optional poly(A) sequence. The mRNA can be produced, for example, by in vitro transcription or chemical synthesis. In one embodiment, the mRNA of the present invention comprises (1) a 5'-UTR, (2) a coding sequence, (3) a 3'-UTR, and (4) an optional poly(A) sequence. In one embodiment, the mRNA of the present invention is a nucleoside-modified mRNA. In one embodiment, the mRNA of the present invention comprises an optional 5' cap.

[0270] 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.

[0271] As used herein, the term "poly(A) sequence" or "poly(A) tail" refers to a nucleotide sequence containing continuous or discontinuous adenylate nucleotides. The poly(A) sequence is typically located at the 3' end of the RNA, for example, at the 3' end (downstream) of the 3'-UTR. In some embodiments, the poly(A) sequence does not contain nucleotides other than adenylate nucleotides at its 3' end. The poly(A) sequence can be transcribed from the coding sequence of the DNA template by a DNA-dependent RNA polymerase during the preparation of the IVT-RNA, or can be attached to the free 3' end of the IVT-RNA, for example, at the 3' end of the 3'-UTR, by a DNA-independent RNA polymerase (poly(A) polymerase).

[0272] As used herein, the term "5' cap" generally refers to an N7-methylguanosine structure (also known as an "m7G cap," "m7Gppp-") attached to the 5' end of an mRNA via a 5' to 5' triphosphate bond. The 5' cap can be co-transcriptionally added to the RNA during in vitro transcription (e.g., using the anti-reverse cap analog "ARCA"), or it can be attached to the RNA post-transcriptionally using a capping enzyme.

[0273] In some embodiments, the therapeutic or preventive agent of the present invention is DNA. Such DNA can be, for example, a DNA template for in vitro transcription of the RNA of the present invention or a DNA vaccine for expressing a polypeptide antigen in a host cell. The DNA can be double-stranded, single-stranded, linear, or circular.

[0274] The DNA template can be provided in a suitable transcription vector. Generally speaking, the DNA template can be a double-stranded complex, which comprises a nucleotide sequence identical to the coding sequence described herein (coding strand) and a nucleotide sequence complementary to the coding sequence described herein (template strand). As known to those skilled in the art, the DNA template can include a promoter, 5'-UTR, coding sequence, 3'-UTR and an optional poly (A) sequence. The promoter can be an available promoter for a suitable RNA polymerase (particularly DNA-dependent RNA polymerase) known to those skilled in the art, including but not limited to promoters of SP6, T3 and T7 RNA polymerases. The 5'-UTR, coding sequence, 3'-UTR and poly (A) sequence in the DNA template are the corresponding sequences included in the RNA described herein or are complementary thereto. As the polynucleotide of a DNA vaccine, it can be provided in a plasmid vector (e.g., a circular plasmid vector).

[0275] In a preferred embodiment, the therapeutic or preventive agent of the present invention is an mRNA encoding a modified SARS-CoV-2 spike protein. Its exemplary nucleic acid sequence can be found in SEQ ID NO: 3, 5, 6 or 7.

[0276] Coronavirus

[0277] As used herein, "severe acute respiratory syndrome coronavirus 2," "new coronavirus," and "SARS-CoV-2" are used interchangeably. SARS-CoV-2 is known to be the pathogen that causes "coronavirus disease 2019 (COVID-19)."

[0278] SARS-CoV-2 is a positive-sense, single-stranded RNA ((+)ssRNA) enveloped virus belonging to the genus Beta of the Coronaviridae family. SARS-CoV-2 encodes four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). The spike (S) protein mediates the specific binding of the virus to host cells and the fusion of the viral envelope with the host cell membrane, making it a key molecule in viral infection of host cells.

[0279] As used herein, "SARS-CoV-2 spike protein," "SARS-CoV-2 S protein," or "S protein" refers to the spike protein of SARS-CoV-2. SARS-CoV-2 includes, but is not limited to, prototype strains and variants. Multiple SARS-CoV-2 variants have been discovered, including, but not limited to, Alpha (B.1.1.7 and Q lineages), Beta (B.1.351 and descendant lineages), Gamma (P.1 and descendant lineages), Delta (B.1.617.2 and AY lineages), and Omicron (lineages B.1.1.529 and BA lineages) variants. SARS-CoV-2 variants may have mutant SARS-CoV-2 S proteins. SARS-CoV-2 variants can be identified, for example, based on the sequence of the spike protein. For example, a mutant SARS-CoV-2 S protein may comprise mutations, such as amino acid deletions and / or substitutions, compared to the prototype SARS-CoV-2 S protein (e.g., SEQ ID NO: 11).

[0280] The SARS-CoV-2 S protein is synthesized as a glycoprotein with approximately 1273-1300 amino acids (an exemplary amino acid sequence is shown in SEQ ID NO: 11), which includes an N-terminal signal peptide, an S1 subunit, and an S2 subunit. The S1 subunit contains an N-terminal domain, a receptor binding domain (RBD), and subdomains 1 and 2 (SD1 / 2). The S2 subunit contains a fusion peptide (FP), heptad repeats HR1, HR2, a transmembrane domain, and a cytoplasmic domain. For a description of the SARS-CoV-2 S protein, see, for example, Huang Y et al., Acta Pharmacol Sin. 2020; 41(9): 1141-1149.

[0281] Studies have shown that the RBD of the S1 subunit recognizes target host cells by binding to the specific receptor angiotensin-converting enzyme 2 (ACE2), while the S2 subunit is responsible for membrane fusion. In its natural state, the S protein exists on the surface of the virus in a metastable pre-fusion trimer conformation. During infection, the RBD binds to the host cell receptor, and host proteases (such as furin) cut the S1 / S2 cleavage site of the S protein, destroying the stability of the pre-fusion trimer, causing the S1 subunit to fall off and the S2 subunit to transform into a stable post-fusion conformation. The Furin cleavage site is an exposed ring structure containing multiple arginine residues, which contains the amino acid motif Arg-X aa -X bb -Arg(where X aa is any amino acid; X bbis any amino acid, preferably Arg or Lys. In one embodiment, the amino acid sequence of the furin cleavage site is Arg-Arg-Ala-Arg ("RRAR"), corresponding to amino acids 682-685 in SEQ ID NO:11.

[0282] The modified spike protein encoded by the therapeutic or preventive agent of the present invention may include an inactivated Furin cleavage site. Specifically, the polypeptide antigen of the present invention includes an inactivated Furin cleavage site Gln-Ser-Ala-Gln (QSAQ), thereby having a higher expression level in the host cell and / or inducing a stronger immune response in the subject. As used herein, "inactivated Furin cleavage site" refers to an amino acid sequence that cannot be recognized and cut by Furin. As used herein, "active Furin cleavage site" or "Furin cleavage site" refers to an amino acid sequence that can be recognized and cut by Furin.

[0283] In one aspect, the therapeutic or prophylactic agent of the invention is a polynucleotide encoding a modified spike protein comprising an inactivated furin cleavage site, wherein the inactivated furin cleavage site has an amino acid sequence of QSAQ, and the polypeptide comprises:

[0284] The following amino acid substitutions are numbered according to SEQ ID NO: 11: D614G, K986P, V987P ("K986P / V987P", also referred to as "2P mutation").

[0285] In some embodiments, the polypeptide encoded by the polynucleotide of the present invention comprises the amino acid sequence of SEQ ID NO:12 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:12.

[0286] In some embodiments, the polynucleotide comprises a coding region that encodes a modified spike protein.

[0287] As used herein, "coding sequence" refers to a nucleotide sequence in a polynucleotide that can be used as a template for synthesizing a nucleotide sequence having a defined nucleotide sequence (e.g., tRNA and mRNA) or a defined amino acid sequence in a biological process. The coding sequence can be a DNA sequence or an RNA sequence. If the mRNA corresponding to the DNA sequence (including a coding strand identical to the mRNA sequence and a template strand complementary thereto) is translated into a polypeptide in a biological process, the DNA sequence or mRNA sequence can be considered to encode the polypeptide.

[0288] As used herein, "codon" refers to three consecutive nucleotide sequences (also known as triplet codes) in a polynucleotide that encode a specific amino acid. Synonymous codons (codons encoding the same amino acid) are used at different frequencies in different species, which is called "codon preference". It is generally believed that for a given species, the coding sequence using the codons of its preference can have higher translation efficiency and accuracy in the species expression system. Therefore, polynucleotides can be "codon optimized", that is, the codons in the polynucleotides are changed to reflect the codons of the host cell preference, and preferably the amino acid sequence encoded by them is not changed. It will be understood by those skilled in the art that due to the degeneracy of codons, the polynucleotides of the present invention may include such coding sequences, which are different from the coding sequences described herein (e.g., having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the coding sequences described herein) but encode the same amino acid sequence. In a specific embodiment, the RNA of the present invention comprises codons optimized for host (eg, subject, particularly human) cells, such that the polypeptide of the present invention is optimally expressed in the host (eg, subject, particularly human).

[0289] In one embodiment, the polynucleotides of the present invention comprise a coding sequence for a polypeptide as described herein. In one embodiment, the polynucleotides of the present invention comprise a nucleotide sequence complementary to a coding sequence for a polypeptide as described herein. In one embodiment, the coding sequence comprises a start codon at its 5' end and a stop codon at its 3' end. In one embodiment, the coding sequence comprises an open reading frame (ORF) as described herein.

[0290] In one embodiment, the coding sequence of the present invention encodes a polypeptide comprising:

[0291] (1) the amino acid sequence of SEQ ID NO: 12; or (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence in SEQ ID NO: 12.

[0292] In one embodiment, the coding sequence of the polypeptide described herein comprises a nucleotide sequence comprising: (1) the nucleotide sequence of SEQ ID NO:3; (2) a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:3; (3) the nucleotide sequence of SEQ ID NO:5; or (4) a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:5.

[0293] In some embodiments, the polynucleotides of the present invention are RNA. In some embodiments, the RNA of the present invention further comprises structural elements that contribute to improving the stability and / or translation efficiency of the RNA, including but not limited to a 5' cap, a 5'-UTR, a 3'-UTR, and a poly(A) sequence.

[0294] In some embodiments, the RNA of the present invention comprises a 5'-UTR. In a preferred embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 13. In a preferred embodiment, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the RNA of the present invention comprises a 5'-UTR and a 3'-UTR. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 13, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 14.

[0295] In some embodiments, the RNA of the present invention comprises a poly(A) sequence. In one embodiment, the poly(A) sequence comprises contiguous adenylate nucleotides. In one embodiment, the poly(A) sequence may comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100, and up to 120, 150, 180, 200, or 300 adenylate nucleotides. In one embodiment, the poly(A) sequence comprises at least 50 nucleotides. In one embodiment, the poly(A) sequence comprises at least 80 nucleotides. In one embodiment, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises approximately 70, 80, 90, 100, 120, or 150 nucleotides. In one embodiment, the contiguous adenylate sequence in the poly(A) sequence is interrupted by a sequence comprising U, C, or G nucleotides. In one embodiment, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 17.

[0296] In one embodiment, the RNA of the present invention comprises the nucleotide sequence of SEQ ID NO: 3. In one embodiment, the RNA of the present invention comprises the nucleotide sequence of SEQ ID NO: 6.

[0297] In one embodiment, the RNA of the invention (a) comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO:3 or 6; and (b) encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:12.

[0298] In some embodiments, the polynucleotides of the present invention are DNA. In some embodiments, the DNA of the present invention comprises a coding sequence for a polypeptide as described herein. In some embodiments, the DNA of the present invention comprises, from the 5' end to the 3' end, (1) a T7 promoter, (2) a 5'-UTR, (3) a coding sequence, (4) a 3'-UTR, and (5) an optional poly(A) sequence as described herein.

[0299] In some embodiments, the T7 promoter comprises the nucleotide sequence of SEQ ID NO:19.

[0300] In some embodiments, the DNA of the present invention comprises a 5'-UTR. In a preferred embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 15. In a preferred embodiment, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the RNA of the present invention comprises a 5'-UTR and a 3'-UTR. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 15, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 16.

[0301] In some embodiments, the DNA of the present invention comprises a poly(A) sequence. In one embodiment, the poly(A) sequence comprises continuous deoxyadenylic acid. In one embodiment, the poly(A) sequence may comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100, and up to 120, 150, 180, 200, or 300 deoxyadenylic acid. In one embodiment, the continuous adenylic acid sequence in the poly(A) sequence is interrupted by a sequence comprising T, C, or G nucleotides. In one embodiment, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 18.

[0302] In one embodiment, the DNA of the present invention comprises the nucleotide sequence of SEQ ID NO: 5. In one embodiment, the DNA of the present invention comprises the nucleotide sequence of SEQ ID NO: 7.

[0303] In one embodiment, the DNA of the invention (a) comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO: 5 or 7; and (b) encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12.

[0304] Modified nucleotides

[0305] 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-ur ... The invention also includes alkynyl-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. Modifications are made to the modified nucleotides described in PCT / CN2020 / 074825 and PCT / CN2020 / 106696.

[0306] In one embodiment, the RNA (e.g., mRNA) of the present invention is modified by including one or more modified nucleobases. In one embodiment, the modified nucleobases include modified cytosine, modified uracil, or a combination thereof. In one embodiment, the modified uracil is independently selected from pseudouracil, 1-methyl-pseudouracil, 5-methyl-uracil, or a combination thereof. In one embodiment, the modified cytosine is independently selected from 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof. In one embodiment, the proportion of modified nucleobases in the RNA of the present invention is 10%-100%, i.e., the RNA of the present invention can be modified by replacing 10%-100% of the nucleobases therein with modified nucleobases.

[0307] In some embodiments, the RNA (e.g., mRNA) of the present invention is modified by replacing one or more uracils with modified uracils. In one embodiment, the modified uracil comprises 1-methylpseudouracil, pseudouracil, 5-methyl-uracil, or a combination thereof. In one embodiment, the modified uracil comprises pseudouracil. In one embodiment, the modified uracil comprises 5-methyl-uracil. In one embodiment, the modified uracil comprises 1-methyl-pseudouracil.

[0308] In one embodiment, the RNA is modified by replacing at least one uracil with a modified uracil. In one embodiment, the RNA is modified by replacing all uracils with modified uracils. In one embodiment, the proportion of modified uracils in the RNA is 10%-100%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In one embodiment, the proportion of modified uracils in the RNA is 20%-100%. In one embodiment, 20%-100% of the uracils in the RNA are replaced by 1-methylpseudouracil. In a preferred embodiment, 100% of the uracils in the RNA are replaced by 1-methylpseudouracil.

[0309] 1-Methyl-pseudouridine has the following structure:

[0310] In a specific embodiment, the mRNA of the present invention comprises the nucleotide sequence of SEQ ID NO: 6, wherein 100% of uracil is replaced by 1-methylpseudouracil.

[0311] Application of lipid compositions and pharmaceutical compositions

[0312] Lipid composition, pharmaceutical composition and injection can be used for treating disease, disease or the patient's condition. Specifically, these lipid composition, pharmaceutical composition and injection can be used for treating disease, disease or the patient's condition characterized by loss or abnormal protein or polypeptide activity. For example, lipid composition and pharmaceutical composition comprising the mRNA of coding loss or abnormal polypeptide can be applied or delivered to cell. This mRNA is translated subsequently and can produce described polypeptide, thus reducing or eliminating the problem caused by the non-existence or abnormal activity of this polypeptide. Because translation can occur rapidly, these methods and lipid composition, pharmaceutical composition and injection can be used for treating acute disease, disease or the patient's condition such as sepsis, apoplexy and myocardial infarction. The therapeutic agent or preventive included in the lipid composition can also change the transcription rate of given mRNA, thus affecting gene expression.

[0313] Diseases, disorders or conditions characterized by malfunction or abnormal protein or polypeptide activity that can be used with lipid compositions, pharmaceutical compositions and injections include but are not limited to rare diseases, infectious diseases (in the form of vaccines and therapeutic agents), cancer and proliferative diseases, genetic diseases (such as cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases. There are various diseases, disorders or conditions that can be characterized by the loss of protein activity (or substantially reducing so that appropriate protein function cannot occur). These proteins may not exist, or they may be substantially non-functional. The specific example of a malfunctioning protein is a missense mutation variant of the cystic fibrosis transmembrane conductance regulatory protein (CFTR) gene, which produces a malfunctioning protein variant of the CFTR protein, thereby causing cystic fibrosis. The present invention provides a method for treating such diseases, disorders or conditions in a subject by administering a lipid composition, pharmaceutical composition or injection, wherein the lipid composition comprises RNA and a lipid component, wherein the lipid component comprises a cationic lipid, a phospholipid, a PEG lipid and a structured lipid, wherein the RNA may be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes the activity of an abnormal protein present in the subject's cells.

[0314] The methods provided herein relate to administering lipid compositions containing one or more therapeutic or prophylactic agents, pharmaceutical compositions or injections comprising these compositions. For features and embodiments of the present invention, the terms therapeutic agent and prophylactic agent can be used interchangeably herein. Lipid compositions and pharmaceutical compositions can be administered to a subject using any reasonable amount and any route of administration that is effective for preventing, treating, diagnosing, or treating a disease, disorder, or condition, or for any other purpose. The specific amount administered to a given subject can vary depending on the species, age, and general condition of the subject; the purpose of administration; the specific composition; the mode of administration, etc.

[0315] In some embodiments, lipid compositions and pharmaceutical compositions comprising therapeutic or preventive agents of the present invention can be administered to a subject by any method known to those skilled in the art, such as parenteral, oral, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous or intraperitoneal. Preferably, lipid compositions of the present invention are administered by intramuscular injection.

[0316] In one aspect, the lipid composition, pharmaceutical composition or injection provided by the present invention is used to prevent and / or treat SARS-CoV-2 infection.

[0317] In one aspect, the present invention also provides use of the lipid composition, pharmaceutical composition or injection of the present invention in the preparation of a medicament for preventing and / or treating SARS-CoV-2 infection.

[0318] In one aspect, the present invention also provides a method for preventing and / or treating SARS-CoV-2 infection in a subject, the method comprising administering a therapeutically effective amount of a lipid composition, pharmaceutical composition, or injection of the present invention. In one embodiment, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the mRNA of the present invention, particularly a pharmaceutical composition comprising LNP or LPP as described herein.

[0319] In one embodiment, the SARS-CoV-2 infection includes at least one infection caused by SARS-CoV-2. In one embodiment, the SARS-CoV-2 includes the SARS-CoV-2 prototype strain and BA.1, BA.2, Beta, and Delta variants.

[0320] In a specific embodiment, the present invention provides a method for preventing and / or treating an infection caused by at least one SARS-CoV-2 in a subject, the method comprising administering a prophylactically or therapeutically effective amount of a lipid composition, pharmaceutical composition, or injection of the present invention. In one embodiment, the SARS-CoV-2 is selected from the SARS-CoV-2 prototype strain and the Alpha, Beta, Omicron (BA.1), Omicron (BA.4 / 5), and Delta variants.

[0321] In some embodiments, the prophylactically or therapeutically effective amount is provided in one or more administrations. In some embodiments, the prophylactically or therapeutically effective amount is provided in two administrations. In some embodiments, the prophylactically or therapeutically effective amount is provided in three administrations.

[0322] In some embodiments, the lipid composition, pharmaceutical composition or injection of the present invention can be administered with a homologous booster strategy or a heterologous booster strategy. The term "homologous booster" refers to the interval vaccination using the same technical route vaccine. The term "heterologous booster" refers to the interval vaccination of different technical route vaccines. In one embodiment, the lipid composition, pharmaceutical composition or injection of the present invention are administered three times to provide a preventive or therapeutically effective amount. In one embodiment, the lipid composition, pharmaceutical composition or injection of the present invention are administered at intervals after administering an inactivated vaccine to provide a preventive or therapeutically effective amount. In one embodiment, the lipid composition, pharmaceutical composition or injection of the present invention are administered at intervals after administering two inactivated vaccines to provide a preventive or therapeutically effective amount. Beneficial effects

[0323] The lipid composition, pharmaceutical composition or injection provided by the present invention can exhibit excellent effects, such as but not limited to: (1) improving the expression efficiency of the contained mRNA; (2) having high stability under different storage conditions, and the expression efficiency of the contained mRNA and the in vitro physicochemical properties of the lipid composition are maintained stable; (3) having high stability after repeated freezing and thawing; (4) having high stability after lyophilization; (5) having good targeting and low hepatotoxicity; and (6) leading to effective APC uptake and DC maturation.

[0324] Furthermore, the lipid composition, pharmaceutical composition or injection comprising mRNA encoding the modified spike protein provided by the present invention can exhibit excellent effects, such as but not limited to: (1) inducing high levels of humoral immune response and cellular immune response in the subject; (2) protecting the subject from death and corresponding lesions caused by SARS-CoV-2 infection, and having a significant preventive effect on the replication of SARS-CoV-2 in the lungs; (3) homologous or heterologous booster immunization can enhance the immune response to mutant strains; and (4) having good safety.

[0325] Example

[0326] The present invention is further described with reference to the following examples. It should be understood that these examples are intended to be illustrative only and are not intended to limit the present invention. The following materials and instruments are commercially available or prepared according to methods known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures known in the art.

[0327] Experimental Materials

[0328] The cationic lipid according to formula (I) is synthesized by Si microorganisms or prepared by reference, such as CN110520409A, WO2018081480A1 or US11,246,933B1; phospholipids (DOPE) are purchased from CordenPharma; cholesterol is purchased from Sigma-Aldrich; mPEG2000-DMG (i.e., DMG-PEG 2000) is purchased from Avanti Polar Lipids, Inc.; PBS is purchased from Invitrogen; protamine sulfate is purchased from Beijing Silian Pharmaceutical Co., Ltd.; mPEG2000-DSPE is purchased from lipoid GmbH; DSPC is purchased from Avanti Polar Lipids, Inc.

[0329] Example 1 Synthesis of the compound according to formula (I)

[0330] General considerations

[0331] Unless otherwise noted, all solvents and reagents used were commercially available and used as received. 1 H NMR spectra were recorded in CDCl3 at 300 K using a Bruker Ultrashield 300 MHz instrument. Chemical shifts are relative to 1 H is reported as parts per million (ppm) relative to TMS (0.00). Silica gel chromatography was performed on an ISCO CombiFlash Rf+Lumen instrument using ISCO RediSep Rf Gold flash columns (particle size: 20-40 microns).

[0332] The procedure described below can be used to synthesize compounds SW-II-115 to SW-II-140-2.

[0333] The following abbreviations are used herein: THF: tetrahydrofuran MeCN: acetonitrile LAH: lithium aluminum hydride DCM: dichloromethane DMAP: 4-dimethylaminopyridine LDA: lithium diisopropylamide rt: room temperature DME: 1,2-dimethoxyethane n-BuLi: n-butyllithium CPME: cyclopentyl methyl ether EDCI: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide DIEA: N,N-diisopropylethylamine PE: petroleum ether EA: ethyl acetate

[0334] A. Compound SW-II-115

[0335] 1. Synthesis of Intermediate 3

[0336] To a DCM solution (100 mL) containing compound 1 (10 g, 45 mmol, 1 eq.) and compound 2 (7.8 g, 54 mmol, 1.2 eq.) was added EDCI (17.3 g, 90 mmol, 2 eq.) and DMAP (2.2 g, 18 mmol, 0.4 eq.), followed by DIEA (23.2 g, 180 mmol, 4 eq.). The reaction mixture was stirred at room temperature under N protection for 16 hours. TLC (petroleum ether: ethyl acetate = 30: 1) showed that compound 1 was consumed and the desired product was formed. The reaction mixture was diluted with DCM (20 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether: ethyl acetate (1: 0-20: 1) to give compound 3 (4.365 g, 28%) as a colorless oil.

[0337] 2. Synthesis of Intermediate 5

[0338] A solution of compound 3 (500 mg, 1.437 mmol, 1 eq.) and compound 4 (2.63 g, 43.103 mmol, 30 eq.) in EtOH was stirred at 60 ° C for 16 hours under N protection. TLC (DCM: MeOH = 10: 1) showed that compound 3 was consumed, and TLC (DCM / MeOH = 10 / 1) showed that a new main point was observed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with EtOAc (50 mL) and washed with H2O (3 × 50 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (1: 0-10: 1, v / v) to give compound 5 (264 mg, 56%) as a yellow oil.

[0339] 3. Synthesis of Intermediate 8

[0340] To compound 6 (500 mg, 1.712 mmol, 1 eq.) and compound 7 (1.113 g, 8.562 mmol, 5 eq.) in a dioxane / water (5 mL / 0.5 mL) solvent mixture were added Pd(dppf)Cl2 (112 mg, 0.171 mmol, 0.1 eq.) and potassium carbonate (709 mg, 5.136 mmol, 3 eq.). The mixture was stirred at 100°C overnight under N2. TLC (PE:EA = 15:1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1) to give compound 8 (455 mg, 88%) as a colorless oil.

[0341] 4. Synthesis of Intermediate 9

[0342] To a solution of compound 8 (455 mg, 1.497 mmol, 1 eq.) in THF (5 mL) was added LiAlH₄ (1.5 mL, 1.497 mmol, 1 M in THF, 1 eq.) at 0°C under N₂ protection. The mixture was stirred at room temperature under N₂ for 2 hours. TLC (PE:EtOAc=5:1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (1.5 mL) and treated with 2N HCl to adjust the pH between 6 and 7, extracted with EA, and washed with brine. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo to afford crude compound 9 (419 mg, >100%) as a colorless oil without further purification.

[0343] 5. Synthesis of Intermediate 10

[0344] To a solution of compound 1 (339 mg, 1.518 mmol, 1 eq.) and compound 9 (419 mg, 1.518 mmol, 1 eq.) in DCM (4 mL) were added EDCI (583 mg, 3.036 mmol, 2 eq.) and DMAP (74 mg, 0.607 mmol, 0.4 eq.), followed by DIEA (783 mg, 6.072 mmol, 4 eq.). The reaction mixture was stirred at room temperature under N protection for 16 hours. TLC (petroleum ether:ethyl acetate = 10:1) showed the formation of the desired product. The reaction mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether:ethyl acetate (1:0-10:1) to obtain compound 10 (443 mg, 60.7%) as a colorless oil.

[0345] 6. Synthesis of the final product SW-II-115

[0346] To a mixed solvent of CPME / CH3CN (3 mL / 3 mL) containing compound 10 (307 mg, 0.64 mmol, 1 eq.) and compound 5 (210 mg, 0.64 mmol, 1 eq.) were added K2CO3 (530 mg, 3.84 mmol, 6 eq.) and KI (212 mg, 1.28 mmol, 2 eq.). After the addition was complete, the mixture was stirred at 90°C overnight under N2. TLC (DCM:MeOH = 10:1) indicated the reaction was complete, with a new major spot observed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM:MeOH (1:0-10:1, v / v) to afford compound SW-II-115 (266 mg, 57%) as a yellow oil.

[0347] LCMS:Rt:1.293min;MS m / z(ELSD):730.5[M+H] + ;

[0348] HPLC: 99.472% purity, ELSD; RT = 4.895 min.

[0349] 1 H NMR (400MHz, CDCl3) δ7.21–6.99(m,3H),5.05(s,2H),4.05(t,J=6.8Hz,2H),3.58(t,J=5.3Hz,2H), 2.69–2.46(m,10H),2.31(dt,J=20.0,7.5Hz,4H),1.69–1.18(m,51H),0.89(dt,J=12.4,6.3Hz,9H).

[0350] 13C NMR (101MHz, CDCl3) δ173.90 (s), 173.68 (s), 140.80 (d, J = 13.0Hz), 133.31 (s), 129.25 (d, J = 16.2Hz) ,128.30(s),125.75(s),77.30(d,J=11.5Hz),77.04(s),76.72(s),66.22(s),64.43(s),58.12(s),55 .72(s),53.90(s),34.32(d,J=1.9Hz),32.69(s),32.48(s),31.81(d,J=11.2Hz),31.25(s),29.59–28 .91(m),28.66(s),27.17(s),26.64(s),25.94(s),24.91(d,J=5.1Hz),22.65(d,J=3.3Hz),14.10(s).

[0351] B. Compound SW-II-118

[0352] 1. Synthesis of Intermediate 3

[0353] A solution of compound 1 (1.22 g, 5.0 mmol, 1.0 eq.) and compound 2 (765 mg, 7.5 mmol, 1.5 eq.), Pd(PPh3)4 (tetrakistriphenylphosphine palladium, 289 mg, 0.25 mmol, 0.05 eq.), and K2CO3 (1.38 g, 10.0 mmol, 2.0 eq.) in toluene (10 ml) and H2O (1 ml) was stirred at 110°C under N2 protection for 1 hour. TLC (petroleum ether:ethyl acetate = 19:1) showed that compound 1 was consumed and a new spot was observed. The reaction mixture was diluted with DCM (50 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether:ethyl acetate (1:0-10:1) to give compound 3 (0.5 g, 45%) as a colorless oil.

[0354] 1 H NMR (400MHz, CDCl3) δ7.16 (dd, J=23.5, 8.1Hz, 4H), 4.14 (q, J=7.1Hz, 2H), 3.57 (s, 2H), 2.64–2.48 (m ,2H),1.66–1.51(m,2H),1.35(dd,J=15.0,7.4Hz,2H),1.25(t,J=7.1Hz,3H),0.92(t,J=7.3Hz,3H).

[0355] 2. Synthesis of Intermediate 4

[0356] LiAlH4 (193 mg, 5.09 mmol, 4.0 eq.) was added to a THF (10 mL) solution containing compound 3 (280 mg, 1.27 mmol, 1.0 eq.) at -78°C, and the reaction was allowed to react at 10°C for 3 hours. TLC showed that the reaction was well, and the reaction was concentrated and diluted with Na2SO4 (20 mL) and extracted with EA (30 mL×2). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give compound 4 (3.12 g, crude product) as a yellow oil.

[0357] 3. Synthesis of Intermediate 6

[0358] A solution of compound 4 (215 mg, 1.2 mmol, 1.0 eq.), compound 5 (404 mg, 1.8 mmol, 1.5 eq.), EDCI (1.15 g, 6.0 mmol, 5.0 eq.), DMAP (732 mg, 1.8 eq.), DIEA (1.29 g, 12.0 mmol, 10.0 eq.), and DIEA (1.29 g, 12.0 mmol, 10.0 eq.) in DCM (5 mL) was stirred at 10°C for 16 h under N₂ protection. TLC (DCM:MeOH = 10:1) indicated the reaction was complete and a new major spot was observed. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1, v / v) to afford compound 6 (145 mg, 31%) as a colorless oil.

[0359] 1 H NMR (400MHz, CDCl3) δ7.12(s,4H),4.27(t,J=7.1Hz,2H),3.52(t,J=6.7Hz,1H),3.40(t,J=6.8Hz,1H),2.90(t,J=7.1Hz,2H ),2.65–2.50(m,2H),2.28(t,J=7.5Hz,2H),1.93–1.70(m,2H),1.64–1.56(m,4H),1.44–1.27(m,8H),0.92(t,J=7.3Hz,3H).

[0360] 4. Synthesis of the final product SW-II-118

[0361] A mixture containing compound 6 (140 mg, 0.37 mmol, 1.0 eq.), compound 7 (243 mg, 0.55 mmol, 1.5 eq.), K2CO3 (153 mg, 1.11 mmol, 3.0 eq.) and KI (123 mg, 0.74 mmol, 2.0 eq.) was stirred in a mixed solvent of CPME (1 mL) and CH3CN (1 mL) under N2 at 90 ° C for 16 hours. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with NaHCO3 (30 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with DCM:MeOH (1: 0-10: 1, v / v) to give SW-II-118 (105 mg, 61%) as a yellow oil.

[0362] LCMS:Rt:1.946min;MS m / z(ELSD):744.4[M+H] + ;

[0363] HPLC: 99.64% purity, ELSD; RT = 5.875 min.

[0364] 1 H NMR (400MHz, CDCl3) δ7.11(s,4H),4.91–4.79(m,1H),4.26(t,J=7.2Hz,2H),3.80–3.68(m,2H),2.90(t,J=7.1Hz,4H),2.8 1–2.67(m,4H),2.62–2.52(m,2H),2.28(td,J=7.5,2.6Hz,4H),1.64–1.51(m,11H),1.38–1.17(m,42H),0.93–0.82(m,9H).

[0365] 13C NMR (101MHz, CDCl3) δ173.61 (d, J = 11.7Hz), 141.11 (s), 134.90 (s), 128.74 (s), 128.51 (s), 77.40 (s), 77.08(s),76.77(s),74.17(s),64.90(s),57.48(s),56.24(s),53.98(s),35.25(s),34.66(d,J=14.4 Hz),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).

[0366] C. Compound SW-II-120

[0367] 1. Synthesis of Intermediate 3

[0368] A solution containing compound 1 (1.22 g, 5.0 mmol, 1.0 eq.), compound 2 (1.30 mg, 10.0 mmol, 2.0 eq.), Pd(PPh3)4 (289 mg, 0.25 mmol, 0.05 eq.), and K2CO3 (1.38 g, 10.0 mmol, 2.0 eq.) in a mixture of toluene (10 ml) and H2O (1 ml) was stirred at 110°C under N2 protection for 1 hour. TLC (petroleum ether:ethyl acetate = 19:1) showed that compound 1 was consumed and a new spot was observed. The reaction mixture was diluted with DCM (50 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether:ethyl acetate (1:0-10:1) to give compound 3 (0.78 g, 62%) as a colorless oil.

[0369] 1 H NMR (400MHz, CDCl3) δ7.19(d,J=8.1Hz,2H),7.13(d,J=8.1Hz,2H),4.14(q,J=7.1Hz,2H),3.5 7(s,2H),2.62–2.51(m,2H),1.58(d,J=11.1Hz,2H),1.35–1.21(m,9H),0.88(t,J=6.7Hz,3H).

[0370] 2. Synthesis of Intermediate 4

[0371] LiAlH4 (477 mg, 12.56 mmol, 4.0 eq.) was added to a solution of compound 3 (780 mg, 3.14 mmol, 1.0 eq.) in THF (10 mL) at -78°C, and the reaction was stirred at 10°C for 3 hours. Thin-layer chromatography indicated that the reaction proceeded well. The reaction was concentrated, diluted with Na2SO4 (20 mL), and extracted with EA (30 mL x 2). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford compound 4 (640 mg, crude) as a colorless oil.

[0372] 3. Synthesis of Intermediate 6

[0373] A solution of compound 4 (640 mg, 3.10 mmol, 1.0 eq.), compound 5 (1.06 g, 4.70 mmol, 1.5 eq.), EDCI (2.98 g, 15.5 mmol, 5.0 eq.), DMAP (1.85 g, 15.0 eq.), and DIEA (4.0 g, 31.0 mmol, 10.0 eq.) in DCM (10 mL) was stirred at 10°C for 16 h under N₂ protection. TLC (DCM:MeOH = 10:1) indicated the reaction was complete and a new major spot was observed. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1, v / v) to afford compound 6 (465 mg, 36%) as a colorless oil.

[0374] 4. Synthesis of the final product SW-II-120

[0375] A mixture containing compound 6 (100 mg, 0.25 mmol, 1.0 eq.), compound 7 (161 mg, 0.36 mmol, 1.5 eq.), K2CO3 (104 mg, 0.75 mmol, 3.0 eq.) and KI (83 mg, 0.50 mmol, 2.0 eq.) was stirred at 90 ° C for 16 hours in CPME (1 mL) and CH3CN (1 mL) under N2. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with NaHCO3 (30 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM:MeOH (1: 0-10: 1, v / v) to give SW-II-120 (100 mg, 52%) as a yellow oil.

[0376] LCMS:Rt:2.500min;MS m / z(ELSD):772.4[M+H] + ;

[0377] HPLC: 99.70% purity, ELSD; RT = 8.675 min.

[0378] 1 H NMR(400MHz, CDCl3) δ7.07(d,J=8.9Hz,4H),4.89–4.73(m,1H),4.23(t,J=7.2Hz,2H),3.83–3.65(m,2H),2.87(t,J=7.2Hz,4H),2.82–2 .67(m,4H),2.61–2.45(m,2H),2.25(td,J=7.5,2.5Hz,4H),1.65–1.44(m,15H),1.27(dd,J=13.2,11.3Hz,42H),0.85(t,J=6.8Hz,9H).

[0379] 13 C NMR (101MHz, CDCl3) δ173.57 (d, J = 11.5Hz), 141.13 (s), 134.88 (s), 128.73 (s), 128.48 (s), 77.45 (s), 77.13(s),76.81(s),74.14(s),64.89(s),57.34(s),56.17(s),53.92(s),35.57(s),34.64(d,J=16.1H z),34.14(d,J=3.3Hz),31.79(d,J=13.4Hz),31.49(s),29.50(d,J=2.2Hz),29.23(s),29.10–28.71(m ),26.85(d,J=5.0Hz),25.49–25.38(m),25.13(d,J=35.4Hz),24.72(s),22.63(d,J=5.8Hz),14.11(s).

[0380] D. Compound SW-II-121

[0381] 1. Synthesis of Intermediate 3

[0382] To a DCM (20 mL) solution containing compound 1 (1.3 g, 5.86 mmol, 1.5 eq.) and compound 2 (1 g, 3.9 mmol, 1.0 eq.) was added EDCI (1.495 g, 7.8 mmol, 2.0 eq.), DMAP (0.19 g, 1.56 mmol, 0.4 eq.), and DIEA (2.57 mL, 15.6 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature under N2 for 16 hours. TLC (petroleum ether: ethyl acetate = 19:1) showed that compound 2 was consumed and the desired product was formed. The reaction mixture was diluted with DCM (20 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether: ethyl acetate (1:0-10:1) to give compound 3 (1.2 g, 66.9%) as a yellow oil.

[0383] 1 H NMR (400MHz, CDCl3) δ4.92–4.82(m,1H),3.42(t,J=6.8Hz,2H),2.31(t,J=7.5Hz,2H),1.95–1.82(m,2H),1.70–1.19(m,36H),0.90(t,J=6.8Hz,6H).

[0384] 2. Synthesis of Intermediate 5

[0385] A solution of EtOH (5 mL) containing compound 3 (5.2 g, 11.30 mmol, 1.0 eq.) and compound 4 (20.6 g, 339 mmol, 30 eq.) was stirred at 60 ° C for 16 hours under N protection. TLC (petroleum ether: ethyl acetate = 19: 1) showed that compound 3 was consumed and TLC (DCM / MeOH = 10 / 1) showed that a new main point was observed. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with H2O (3 × 50 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM: MeOH (1: 0-10: 1, v / v) to give compound 5 (3 g, 60%) as a yellow oil.

[0386] 3. Synthesis of Intermediate 8

[0387] To a mixed solution of compound 6 (1 g, 4.115 mmol, 1 eq.) and compound 7 (889 mg, 6.173 mmol, 1.5 eq.) in toluene / water (10 mL / 1 mL) were added Pd(pph) (238 mg, 0.206 mmol, 0.05 eq.) and KCO (1.7 g, 12.35 mmol, 3 eq.). The mixture was stirred at 110°C under N2 for 2 hours. TLC (PE:EA = 10:1) indicated the reaction was complete, with a new major spot observed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1) to afford compound 8 (714 mg, 66%) as a colorless oil.

[0388] 4. Synthesis of Intermediate 9

[0389] Under N protection, LiAlH (2.7 mL, 2.725 mmol, 1 M in THF, 1 eq.) was added to a mixture of compound 8 (714 mg, 2.725 mmol, 1 eq.) in THF (7 mL) at 0°C, and the mixture was stirred at room temperature for 2 hours. TLC (PE:EtOAc=10:1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (2.7 mL) and treated with 2N HCl to adjust the pH between 6 and 7, extracted with EA and washed with brine. The organic layer was dried over anhydrous NaSO, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluting with PE:EA (1:0-10:1) to give compound 9 (103 mg, 63%) as a colorless oil.

[0390] 5. Synthesis of Intermediate 11

[0391] To DCM (3 mL) containing compound 9 (300 mg, 1.364 mmol, 1 eq.) and compound 10 (363 mg, 1.64 mmol, 1.2 eq.) was added EDCI (524 mg, 2.728 mmol, 2 eq.), DMAP (67 mg, 0.546 mmol, 0.4 eq.), and DIEA (704 mg, 5.456 mmol, 4 eq.). The reaction mixture was stirred at room temperature under N2 for 16 hours. TLC (petroleum ether: ethyl acetate = 10:1) showed the formation of the desired product. The reaction mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether: ethyl acetate (1:0-10:1) to give compound 11 (169 mg, 29%) as a colorless oil.

[0392] 6. Synthesis of the final product SW-II-121

[0393] To a CPME / CH3CN (2 mL / 2 mL) mixed solvent containing compound 11 (169 mg, 0.399 mmol, 1 eq.) and compound 5 (176 mg, 0.399 mmol, 1 eq.) was added K2CO3 (330 mg, 2.394 mmol, 6 eq.) and KI (132 mg, 0.798 mmol, 2 eq.). After the addition was complete, the mixture was stirred at 90°C overnight under N2. TLC (DCM:MeOH = 10:1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM:MeOH (1:0-10:1, v / v) to afford compound SW-II-121 (145 mg, 46%) as a yellow oil.

[0394] LCMS:Rt:1.493min;MS m / z(ELSD):786.5[M+H] + ;

[0395] HPLC: 99.869% purity, ELSD; RT = 10.655 min.

[0396] 1 H NMR (400MHz, CDCl3) δ7.11 (s, 4H), 4.92–4.80 (m, 1H), 4.26 (t, J = 7.2Hz, 2H), 3.80 (s, 2H), 2.87 (dd, J = 26.6, 19.4Hz, 7H),2.62–2.51(m,2H),2.28(td,J=7.2,3.6Hz,4H),1.75–1.45(m,14H),1.42–1.09(m,45H),0.88(t,J=6.8Hz,9H).

[0397] 13C NMR (101MHz, CDCl3) δ173.61 (d, J = 12.3Hz), 141.20 (s), 134.90 (s), 128.75 (s), 128.51 (s),77.35(s),77.03(s),76.72(s),74.21(s),64.93(s),54.15(s),35.59(s),34.66( d,J=16.6Hz),34.16(d,J=3.0Hz),31.85(d,J=4.4Hz),31.55(s),29.64–29.15(m),29. 15–28.78(m),26.85(d,J=4.5Hz),25.33(s),24.95(s),24.72(s),22.68(s),14.12(s).

[0398] E. Compound SW-II-122

[0399] 1. Synthesis of compound 3

[0400] Compound 1 (1 g, 4.65 mmol, 1 eq.) and compound 2 (726 mg, 5.58 mmol, 1.2 eq.) were dissolved in toluene / water (10 / 1, 20 mL), and KCO (1.92 g, 13.9 mmol, 3 eq.) and Pd(pph) (269 mg, 0.23 mmol, 0.05 eq.) were added to the mixture. The reaction mixture was heated to 110°C under N2 and stirred for 2 hours. TLC (petroleum ether / ethyl acetate = 19 / 1) showed that compound 1 was consumed and a new major spot was observed. The reaction mixture was quenched with H2O (80 mL) and extracted with ethyl acetate (60 mL x 3). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 3 (800 mg, 78%) as a yellow oil.

[0401] 2. Synthesis of compound 4

[0402] Under nitrogen protection at 0°C, LiAlH4 (3.2 mL, 3.18 mmol, 1 eq.) was added to compound 3 (700 mg, 3.18 mmol, 1.0 eq.) dissolved in THF (14 mL). The reaction was allowed to warm to room temperature and stirred for 2 hours under nitrogen protection. TLC (PE / EtOAc = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (3.2 mL) and 1M HCl (3.2 mL), respectively. Water (6 mL) was added to the mixture and extracted with ethyl acetate (60 mL × 3). The organic layer was washed with brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with ethyl acetate / petroleum ether = 1 / 10 to give compound 4 (600 mg, 98%) as a yellow oil.

[0403] 3. Synthesis of Compound 6

[0404] Compound 4 (680 mg, 3.5 mmol, 1.0 eq.) and compound 5 (1.13 g, 5.1 mmol, 1.5 eq.) were dissolved in DCM (10 mL), and EDCI (1.20 g, 6.25 mmol, 2.0 eq.), DMAP (166 mg, 1.36 mmol, 0.4 eq.), and DIEA (1.78 g, 13.8 mmol, 4.0 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at room temperature overnight under nitrogen. TLC (DCM / MeOH = 30 / 1) showed that the starting material was consumed and a new spot had formed. The mixture was quenched with water (70 mL) and extracted with DCM (80 mL x 3). The combined organic layers were washed with brine (2 x 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with ethyl acetate / petroleum ether = 3 / 97 solution to give compound 6 (680 mg, 48.5%) as a yellow oil.

[0405] 4. Synthesis of SW-II-122

[0406] Compound 6 (108 mg, 0.27 mmol, 1.2 eq) and compound 7 (100 mg, 0.23 mmol, 1 eq.) were dissolved in CPME (2 mL) and CHCN (2 mL). Potassium carbonate (157 mg, 1.14 mmol, 5.0 eq) and potassium iodide (75 mg, 0.45 mmol, 2.0 eq) were added to the mixture. After the addition, the reaction mixture was stirred at 90°C under nitrogen for 16 hours. TLC (DCM / MeOH = 10 / 1) indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to obtain SW-II-122 (68 mg, 40%) as a colorless oil.

[0407] LCMS:Rt:1.487min;MS m / z(ELSD):758.5[M+H] + ;

[0408] HPLC: 97.3% purity, ELSD; RT = 7.622 min.

[0409] 1 H NMR (400MHz, CDCl3) δ7.32(d,J=26.4Hz,1H),7.17(dd,J=27.2,21.1Hz,3H),5.09(s,2H),4.91–4.79(m,1H),3.85(s,2H),2.98(s,2H),2.87(s,4H) ,2.65–2.54(m,2H),2.35(t,J=7.6Hz,2H),2.28(t,J=7.6Hz,2H),1.74–1 .57(m,9H),1.50(d,J=5.6Hz,4H),1.37–1.15(m,43H),0.94–0.80(m,9H).

[0410] 13C NMR (101MHz, CDCl3) δ173.55 (d, J = 2.4Hz), 143.35 (s), 135.92 (s), 128.67–128.19 (m), 125.47 (s), 77. 36(s),77.04(s),76.73(s),74.22(s),66.27(s),57.15(s),56.74(s),54.14(s),35.88(s),34.55(s) ,34.15(d,J=3.6Hz),31.79(d,J=15.2Hz),31.43(s),29.52(d,J=2.8Hz),29.25(s),28.92(dd,J=14.2 ,5.8Hz),26.77(d,J=4.8Hz),25.33(s),24.92(s),24.71(s),24.48(s),22.64(d,J=6.8Hz),14.12(s).

[0411] F. Compound SW-II-127

[0412] 1. Synthesis of compound 3

[0413] Compound 1 (1.3 g, 5.86 mmol, 1.5 eq.) and compound 2 (1 g, 3.9 mmol, 1.0 eq.) were dissolved in DCM (20 mL). EDCI (1.495 g, 7.8 mmol, 2.0 eq.) and DMAP (0.19 g, 1.56 mmol, 0.4 eq.) were added to the mixture, followed by DIEA (2.57 mL, 15.6 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature for 16 hours under nitrogen. TLC (petroleum ether / ethyl acetate = 19 / 1) showed that compound 2 was consumed and the desired product was formed. The reaction mixture was diluted with DCM (20 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 3 (1.2 g, 66.9%) as a yellow oil.

[0414] 1 H NMR (400MHz, CDCl3) δ4.92–4.82(m,1H),3.42(t,J=6.8Hz,2H),2.31(t,J=7.5Hz,2H),1.95–1.82(m,2H),1.70–1.19(m,36H),0.90(t,J=6.8Hz,6H).

[0415] 2. Synthesis of compound 5

[0416] Compound 3 (5.2 g, 11.30 mmol, 1.0 eq.) and compound 4 (20.6 g, 339 mmol, 30 eq.) were added to EtOH (5 mL), and the mixture was stirred at 60 ° C for 16 hours under nitrogen protection. TLC (petroleum ether / ethyl acetate = 19 / 1) showed that compound 3 was consumed and TLC (DCM / MeOH = 10 / 1) showed that a new main spot was observed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with EtOAc (50 mL) and washed with H2O (3 × 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM / MeOH (1 / 0-10: 1, v / v) to obtain compound 5 (3 g, 60%) as a yellow oil.

[0417] 1 H NMR (400MHz, CDCl3) δ4.95–4.75(m,1H),3.74–3.58(m,2H),2.87–2.74(m,2H),2.69–2.56(m,2H), 2.36(s,2H),2.28(t,J=7.5Hz,2H),1.65–1.42(m,8H),1.38–1.17(m,30H),0.88(t,J=6.8Hz,6H).

[0418] 3. Synthesis of Compound 8

[0419] Compound 7 (522 mg, 2.5 mmol, 1.2 eq.) and compound 6 (400 mg, 2.083 mmol, 1 eq.) were dissolved in DCM (4 mL). EDCI (800 mg, 4.166 mmol, 2 eq.), DMAP (102 mg, 0.833 mmol, 0.4 eq.), and DIEA (1.075 mg, 8.332 mmol, 4 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at room temperature overnight under nitrogen. TLC (PE:EA = 10:1) showed that the starting material was consumed and a new spot had formed. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-10 / 1), to afford compound 8 (454 mg, 57%) as a colorless oil.

[0420] 4. Synthesis of SW-II-127

[0421] Compound 8 (100 mg, 0.262 mmol, 1 eq.) and compound 5 (139 mg, 0.314 mmol, 1.2 eq.) were dissolved in CPME / CH3CN (1 mL / 1 mL). Potassium carbonate (217 mg, 1.572 mmol, 6 eq.) and potassium iodide (87 mg, 0.524 mmol, 2 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) showed that the reaction was complete and the desired product had formed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to obtain compound SW-II-127 (42.49 mg, 22%) as a yellow oil.

[0422] LCMS:Rt:1.323min; MS m / z(ELSD):744.5[M+H] + ;

[0423] HPLC: 99.742% purity, ELSD; RT = 7.339 min.

[0424] 1 H NMR(400MHz, CDCl3)δ7.25(s,2H),7.17(d,J=8.0Hz,2H),5.07(s,2H),4.91–4.82(m,1H),3.83(s,2H),2.90(d,J=44.8Hz,5H), 2.64–2.55(m,2H),2.35(t,J=7.4Hz,2H),2.28(t,J=7.5Hz,2H),1.76–1.46(m,14H),1.42–1.19(m,41H),0.88(t,J=6.8Hz,9H).

[0425] 13C NMR (101MHz, CDCl3) δ173.50 (d, J = 8.5Hz), 133.17 (s), 128.61 (s), 128.34 (s), 77.29 (d, J = 11 .4Hz),77.03(s),76.71(s),74.23(s),66.19(s),54.20(s),35.71(s),34.56(s),34.10(d,J= 8.8Hz),31.80(d,J=15.4Hz),31.43(s),29.53(d,J=2.5Hz),29.25(s),28.95(d,J=10.5Hz),2 8.63(s),26.71(d,J=18.2Hz),25.33(s),24.93(s),24.62(s),22.65(d,J=6.6Hz),14.13(s).

[0426] G. Compound SW-II-134-1

[0427] 1. Synthesis of compound 3

[0428] To a mixture of compound 1 (500 mg, 2.283 mmol, 1 eq.) and compound 2 (890 mg, 6.849 mmol, 3 eq.) in toluene / water (5 mL / 1 mL) were added palladium acetate (51 mg, 0.228 mmol, 0.1 eq.), Ruphos (213 mg, 0.457 mmol, 0.2 eq.), and potassium carbonate (945 mg, 6.849 mmol, 3 eq.). The mixture was stirred at 110°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (723 mg, 99.6%) as a colorless oil.

[0429] 2. Synthesis of compound 4

[0430] To a mixture of compound 3 (723 mg, 2.27 mmol, 1 eq.) in THF (8 mL) was added lithium aluminum hydride (2.3 mL, 2.27 mmol, 1 M in THF, 1 eq.) at 0°C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated that the reaction was complete and a new major spot was observed. The mixture was quenched with water (2.3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give compound 4 (381 mg, 58%) as a colorless oil without further purification.

[0431] 3. Synthesis of Compound 6

[0432] To a mixture of compound 4 (381 mg, 1.3 mmol, 1 eq.) and compound 5 (352 mg, 1.6 mmol, 1.2 eq.) in DCM (4 mL) was added EDCI (499 mg, 2.6 mmol, 2 eq.) and DMAP (63 mg, 0.52 mmol, 0.4 eq.), followed by DIEA (671 mg, 5.2 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 20 / 1) showed that the desired product had formed. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluted with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (272 mg, 44%) as a colorless oil.

[0433] 4. Synthesis of SW-II-134-1

[0434] To a mixture of compound 6 (150 mg, 0.303 mmol, 1 eq.) and compound 7 (110 mg, 0.333 mmol, 1.1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (251 mg, 1.818 mmol, 6 eq.) and potassium iodide (101 mg, 0.61 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C under nitrogen overnight. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give a yellow oily compound SW-II-134-1 (168 mg, 75%).

[0435] LCMS:Rt:1.276min;MS m / z(ELSD):744.4[M+H] + ;

[0436] HPLC: 98.481% purity, ELSD; RT = 10.724 min.

[0437] 1 H NMR(400MHz, CDCl3) δ7.06(d,J=7.6Hz,1H),7.01–6.93(m,2H),4.25(t,J=7.3Hz,2H),4.05(t,J=6.8Hz,2H),3.85–3.72(m,2H),2 .98–2.69(m,8H),2.62–2.48(m,4H),2.29(t,J=7.5Hz,4H),1.72–1.48(m,14H),1.45–1.17(m,36H),0.89(dt,J=11.9,6.0Hz,9H).

[0438] 13 CNMR (101MHz, CDCl3) δ173.78 (d, J = 16.7Hz), 140.72 (s), 138.81 (s), 134.91 (s), 129.70 (s), 129.22 (s), 126.19 (s),77.30(d,J=11.4Hz),77.03(s),76.72(s),65.02(s),64.49(s),57.42(s),56.36(s),54.08(s),34.76(s),3 4.22(d,J=4.2Hz),32.74(s),32.36(s),31.81(d,J=9.1Hz),31.35(d,J=5.3Hz),29.49(d,J=2.8Hz),29.24(d,J= 2.2Hz), 28.92(s), 28.66(s), 26.86(s), 25.93(s), 25.04(s), 24.78(d,J=6.6Hz), 22.65(d,J=2.6Hz), 14.10(s).

[0439] H. Compound SW-II-134-2

[0440] 1. Synthesis of compound 3

[0441] To a mixture of compound 1 (500 mg, 2.283 mmol, 1 eq.) and compound 2 (1.08 g, 6.849 mmol, 3 eq.) in toluene / water (5 mL / 1 mL) were added palladium acetate (51 mg, 0.228 mmol, 0.1 eq.), Ruphos (213 mg, 0.457 mmol, 0.2 eq.), and potassium carbonate (945 mg, 6.849 mmol, 3 eq.). The mixture was stirred at 110°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (854 mg, 100%) as a colorless oil.

[0442] 2. Synthesis of compound 4

[0443] To a mixture of compound 3 (854 mg, 2.28 mmol, 1 eq.) in THF (9 mL) at 0°C under nitrogen was added lithium aluminum hydride (2.3 mL, 2.28 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction, with a new major spot observed. The mixture was quenched with water (2.3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (724 mg, 92%) as a colorless oil without further purification.

[0444] 3. Synthesis of Compound 6

[0445] To a mixture of compound 4 (724 mg, 2.09 mmol, 1 eq.) and compound 5 (560 mg, 2.51 mmol, 1.2 eq.) in DCM (8 mL) were added EDCI (803 mg, 4.18 mmol, 2 eq.) and DMAP (102 mg, 0.84 mmol, 0.4 eq.), followed by DIEA (1.078 g, 8.36 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 20 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (473 mg, 41%) as a colorless oil.

[0446] 4. Synthesis of SW-II-134-2

[0447] To a mixture of compound 6 (150 mg, 0.27 mmol, 1 eq.) and compound 7 (108 mg, 0.33 mmol, 1.1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (225 mg, 1.63 mmol, 6 eq.) and potassium iodide (90 mg, 0.54 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C under nitrogen overnight. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give a yellow oily compound SW-II-134-2 (71.77 mg, 33%).

[0448] LCMS:Rt:1.527min;MS m / z(ELSD):800.4[M+H] + ;

[0449] HPLC: 97.311% purity, ELSD; RT = 9.025 min.

[0450] 1 H NMR (400MHz, CDCl3) δ7.06(d,J=7.6Hz,1H),6.96(d,J=9.6Hz,2H),4.25(t,J=7.3Hz,2H),4.05(t,J=6.8Hz,2H),3.80–3.66(m,2H),2.86(dd,J=12.8,5 .6Hz,4H),2.78–2.67(m,4H),2.60–2.52(m,4H),2.29(t,J=7.5Hz,4H),1.5 7(dt,J=15.8,7.3Hz,14H),1.30(d,J=20.3Hz,45H),0.88(t,J=6.7Hz,9H).

[0451] 13C NMR(101MHz, CDCl3)δ173.82(d,J=16.9Hz),140.73(s),138.82(s),134.91(s),129.71(s),129.23(s),126.19( s),77.36(s),77.14(d,J=20.4Hz),76.72(s),65.03(s),64.49(s),57.57(s),56.13(s),54.02(s),34.76(s),3 4.25(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).

[0452] 1. Compound SW-II-134-3

[0453] 1. Synthesis of compound 3

[0454] To a mixture of compound 1 (10 g, 45 mmol, 1 eq.) and compound 2 (7.8 g, 54 mmol, 1.2 eq.) in DCM (100 mL) were added EDCI (17.3 g, 90 mmol, 2 eq.) and DMAP (2.2 g, 18 mmol, 0.4 eq.), followed by DIEA (23.2 g, 180 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 30 / 1) showed that compound 1 was consumed and the desired product was formed. The reaction mixture was extracted with ethyl acetate (20 mL) and washed with water (40 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 3 (4.365 g, 28%) as a colorless oil.

[0455] 2. Synthesis of compound 5

[0456] A mixture of compound 3 (5 g, 14.38 mmol, 1 eq.) and compound 4 (8.8 g, 143.7 mmol, 10 eq.) in ethanol (2 mL) was stirred at 55 ° C under nitrogen for 16 hours. TLC (DCM / MeOH=10 / 1) showed that a new major spot was observed. The reaction mixture was extracted with ethyl acetate (50 mL) and washed with water (3×50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give compound 5 (1.008 g, 21%) as a yellow oil.

[0457] 3. Synthesis of Compound 8

[0458] To a mixture of compound 6 (500 mg, 2.283 mmol, 1 eq.) and compound 7 (699 mg, 6.849 mmol, 3 eq.) in toluene / water (5 mL / 1 mL) were added palladium acetate (51 mg, 0.228 mmol, 0.1 eq.), Ruphos (213 mg, 0.457 mmol, 0.2 eq.), and potassium carbonate (945 mg, 6.849 mmol, 3 eq.). The mixture was stirred at 110°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 8 (507 mg, 85%) as a colorless oil.

[0459] 4. Synthesis of Compound 9

[0460] To a mixture of compound 8 (507 mg, 1.935 mmol, 1 eq.) in THF (5 mL) at 0°C under nitrogen was added lithium aluminum hydride (2 mL, 1.935 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (2 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 9 (492 mg, >100%) as a colorless oil without further purification.

[0461] 5. Synthesis of Compound 10

[0462] To a mixture of compound 9 (492 mg, 2.103 mmol, 1 eq.) and compound 1 (563 mg, 2.523 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (808 mg, 4.206 mmol, 2 eq.) and DMAP (103 mg, 0.84 mmol, 0.4 eq.), followed by DIEA (1.085 g, 8.412 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 10 (329 mg, 36%) as a colorless oil.

[0463] 6. Synthesis of SW-II-134-3

[0464] To a mixture of compound 10 (150 mg, 0.34 mmol, 1 eq.) and compound 5 (134 mg, 0.41 mmol, 1.2 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (282 mg, 2.04 mmol, 6 eq.) and potassium iodide (113 mg, 0.68 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C under nitrogen overnight. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give SW-II-134-3 (63.59 mg, 25%) as a yellow oil.

[0465] LCMS:Rt:1.247min;MS m / z(ELSD):688.3[M+H] + ;

[0466] HPLC: 95.945% purity, ELSD; RT = 6.186 min.

[0467] 11H NMR (400 MHz, CDCl3) δ 7.07 (d, J = 7.6 Hz, 1H), 6.97 (dd, J = 9.9, 2.2 Hz, 2H), 4.26 (t, J = 7.2 Hz, 2H), 4.05 (t, J = 6.8 Hz, 2H), 2.88 (dd, J = 14.8, 7.6 Hz, 4H), 2.78–2.74 (m, 2H), 2.67–2.54 (m, 8H), 2.29 (t, J = 7.5 Hz, 4H), 1.68–1.47 (m, 15H), 1.37–1.22 (m, 27H), 0.98–0.86 (m, 9H).

[0468] 13 13C NMR (101 MHz, CDCl3) δ 173.86 (d, J = 17.1 Hz), 140.66 (s), 138.76 (s), 134.93 (s), 129.74 (s), 129.24 (s), 126.19 (s), 77.36 (s), 77.04 (s), 76.72 (s), 65.01 (s), 64.48 (s), 57.73 (s), 55.73 (s), 53.93 (s), 34.76 (s), 34.28 (d, J = 3.9 Hz), 33.54 (d, J = 4.5 Hz), 32.41 (s), 31.95 (d, J = 16.5 Hz), 29.49 (s), 29.15 (dd, J = 21.1, 2.4 Hz), 28.66 (s), 27.04 (s), 25.95 (d, J = 3.3 Hz), 24.85 (d, J = 6.6 Hz), 22.98–22.58 (m), 14.08 (d, J = 7.5 Hz).

[0469] J.SW-II-135-1

[0470] 1. Synthesis of Compound 3

[0471] Compound 1 (500 mg, 2.16 mmol, 1.0 eq.) and compound 2 (750 mg, 6.46 mmol, 3.0 eq.) were dissolved in toluene / H₂O (5 mL / 1 mL). To this mixture were added Ruphos (201 mg, 0.43 mmol, 0.2 eq.), Pd(OAc)₂ (48.5 mg, 0.22 mmol, 0.1 eq.), and Cs₂CO₃ (2.10 g, 6.46 mmol, 3.0 eq.). The reaction mixture was heated at reflux at 110°C under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 10 / 1) indicated the reaction was complete and the desired product had formed. The reaction mixture was washed with H₂O (40 mL) and extracted three times with EA (50 mL). The resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-30 / 1) to give compound 3 (540 mg, 82.44%) as a yellow oil.

[0472] 2. Synthesis of compound 4

[0473] To compound 3 (540 mg, 1.78 mmol, 1.0 eq.) dissolved in THF (5 mL) was added LiAlH4 (3.55 mL, 3.55 mmol, 2 eq. in 1 M THF) under nitrogen at 0°C. The reaction was allowed to warm to room temperature and stirred for 2 hours under nitrogen. TLC (PE / EtOAc = 10 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL), then adjusted to pH 6-7 with 1 M hydrochloric acid and extracted three times with ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-10 / 1) to give compound 4 (442 mg, 90.2%) as a colorless oil.

[0474] 3. Synthesis of Compound 6

[0475] Compound 4 (442 mg, 1.60 mmol, 1.0 eq.) and compound 5 (428.5 mg, 1.92 mmol, 1.2 eq.) were dissolved in DCM (5 mL), and EDCI (612 mg, 3.2 mmol, 2.0 eq.) and DMAP (78.2 mg, 0.64 mmol, 0.4 eq.) were added to the mixture, followed by DIEA (826 mg, 6.4 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature for 16 hours under nitrogen protection. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that compound 4 was consumed and the desired product was formed. The reaction mixture was washed with H2O (40 mL) and extracted 3 times with EA (50 mL). The resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 3 (342 mg, 44.5%) as a yellow oil.

[0476] 4. Synthesis of SW-II-135-1

[0477] Compound 6 (175 mg, 0.365 mmol, 1.2 eq.) and compound 7 (100 mg, 0.304 mmol, 1.0 eq.) were dissolved in CPME / CH3CN (1 mL / 1 mL). Potassium carbonate (210 mg, 1.52 mmol, 5.0 eq.) and potassium iodide (101 mg, 0.61 mmol, 2.0 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) showed that the reaction was complete and the desired product had formed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to obtain compound SW-II-135-1 (83.89 mg, 55.6%) as a yellow oil.

[0478] LCMS:Rt:1.356min; MS m / z(ELSD):730.5[M+H] + ;

[0479] HPLC: 100% purity at ELSD; RT=12.614min.

[0480] 1H NMR(400MHz, CDCl3)δ6.97(d,J=7.6Hz,1H),6.91–6.74(m,2H),4.76(s,1H),3 .99(dt,J=13.6,6.4Hz,4H),3.72–3.58(m,2H),2.85–2.73(m,2H),2.72–2.61( m,4H),2.59–2.41(m,6H),2.22(dd,J=13.2,7.2Hz,4H),1.93–1.79(m,2H),1. 62–1.41(m,14H),1.23(d,J=24.4Hz,32H),0.82(ddd,J=13.6,8.0,5.6Hz,9H).

[0481] 13 C NMR (101MHz, CDCl3) δ172.81 (d, J = 6.4Hz), 139.55 (s), 137.38 (s), 137.14 (s), 128.16 (d, J = 2.4Hz), 124.69 ( s),76.51(s),76.19(s),75.88(s),63.43(s),62.78(s),56.53(s),54.90(s),52.84(s),33.23(d,J=2.4Hz) ,31.73(s),31.28(s),30.91(dd,J=20.0,6.4Hz),30.10(d,J=3.2Hz),29.29(s),28.36(d,J=22.8Hz),28.23 (s),27.97(s),27.64(s),25.92(s),24.92(s),24.34(s),23.84(s),21.62(d,J=7.6Hz),13.08(d,J=4.7Hz).

[0482] K. Compound SW-II-135-2

[0483] 1. Synthesis of compound 3

[0484] Compound 1 (500 mg, 2.16 mmol, 1.0 eq.) and compound 2 (931 mg, 6.46 mmol, 3.0 eq.) were dissolved in toluene / H₂O (5 mL / 1 mL). To this mixture were added Ruphos (201 mg, 0.43 mmol, 0.2 eq.), Pd(OAc)₂ (48.5 mg, 0.22 mmol, 0.1 eq.), and Cs₂CO₃ (2.10 g, 6.46 mmol, 3.0 eq.). The reaction mixture was heated at reflux at 110°C under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 10 / 1) indicated the reaction was complete and the desired product had formed. The reaction mixture was washed with H₂O (40 mL) and extracted three times with EA (50 mL). The resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-30 / 1) to give compound 3 (651 mg, 84%) as a yellow oil.

[0485] 2. Synthesis of compound 4

[0486] To compound 3 (651 mg, 1.81 mmol, 1.0 eq.) dissolved in THF (7 mL) was added LiAlH4 (3.62 mL, 3.62 mmol, 1 M in THF, 2 eq.) under nitrogen at 0°C. The reaction was allowed to warm to room temperature and stirred for 2 hours under nitrogen. TLC (PE / EtOAc = 10 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL), then adjusted to pH 6-7 with 1 M hydrochloric acid and extracted three times with ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-10 / 1) to give compound 4 (571 mg, 95.2%) as a colorless oil.

[0487] 3. Synthesis of Compound 6

[0488] Compound 4 (571 mg, 1.72 mmol, 1.0 eq.) and compound 5 (459 mg, 2.06 mmol, 1.2 eq.) were dissolved in DCM (6 mL), and EDCI (657 mg, 3.44 mmol, 2.0 eq.) and DMAP (84 mg, 0.68 mmol, 0.4 eq.) were added to the mixture, followed by DIEA (887.5 mg, 6.88 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature for 16 hours under nitrogen protection. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that compound 4 was consumed and the desired product was formed. The reaction mixture was washed with H2O (50 mL) and extracted 3 times with EA (60 mL). The resulting organic phase was washed twice with brine (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 3 (245 mg, 26.5%) as a yellow oil.

[0489] 4. Synthesis of SW-II-135-2

[0490] Compound 6 (245 mg, 0.456 mmol, 1.5 eq.) and compound 7 (100 mg, 0.3 mmol, 1.0 eq.) were dissolved in CPME / CH3CN (1 mL / 1 mL). Potassium carbonate (210 mg, 1.52 mmol, 5.0 eq.) and potassium iodide (101 mg, 0.61 mmol, 2.0 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) showed that the reaction was complete and the desired product had formed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to obtain compound SW-II-135-2 (31.41 mg, 21.9%) as a yellow oil.

[0491] LCMS:Rt:1.608min; MS m / z(ELSD):786.4[M+H] + ;

[0492] HPLC: 95.16% purity, ELSD; RT = 7.919 min.

[0493] 1H NMR(400MHz, CDCl3)δ6.98(d,J=7.6Hz,1H),6.87(d,J=2.4Hz,2H),4.28–4.13(m,1H),4.04–3.95(m,4H),3.94–3.84(m,2H),3.14–2.89(m,6H) ,2.59–2.43(m,6H),2.23(dd,J=13.8,7.2Hz,4H),1.88–1.82(m,2H),1 .70(s,4H),1.57–1.46(m,10H),1.33–1.16(m,40H),0.90–0.72(m,9H).

[0494] 13 C NMR(100MHz, CDCl3)δ172.82(d,J=6.8Hz),139.61(s),137.29(d,J=16.4Hz),128.15(s),124.67(s),7 6.41(s),76.09(s),75.77(s),63.50(s),62.87(s),55.49(s),54.92(s),52.98(s),33.16(d,J=2.4Hz ),31.77(s),31.33(s),30.80(d,J=6.5Hz),30.42(d,J=3.6Hz),29.29(s),28.99–28.66(m),28.47(s) ,28.23(d,J=2.8Hz),28.06–27.45(m),25.58(s),24.91(s),23.71(s),22.79(s),21.66(s),13.10(s).

[0495] L. Compound SW-II-136-2

[0496] 1. Synthesis of compound 3

[0497] Compound 1 (3 g, 13.70 mmol, 1.0 eq.) and compound 2 (5.34 g, 41.09 mmol, 3.0 eq.) were dissolved in toluene / H₂O (30 mL / 3 mL). To this mixture were added Ruphos (1.28 g, 2.74 mmol, 0.2 eq.), Pd(OAc)₂ (308.3 mg, 1.37 mmol, 0.1 eq.), and K₂CO₃ (5.67 g, 41.10 mmol, 3.0 eq.). The reaction mixture was heated at reflux at 110°C under nitrogen for 16 hours. TLC (PE / EA = 10 / 1) indicated the reaction was complete and the desired product had formed. The reaction mixture was washed with H₂O (90 mL) and extracted three times with EA (110 mL). The resulting organic phase was washed twice with brine (40 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-30 / 1) to give compound 3 (1.98 g, 45.5%) as a yellow oil.

[0498] 2. Synthesis of compound 4

[0499] To compound 3 (1.98 g, 6.23 mmol, 1.0 eq.) dissolved in THF (20 mL) was added LiAlH₄ (1 M, 12.45 mL, 2.0 eq.) under nitrogen at 0°C. The reaction was allowed to warm to room temperature and stirred under nitrogen for 2 hours. TLC (PE / EtOAc = 10 / 1) indicated completion of the reaction, with a new major spot observed. The mixture was quenched with H₂O (70 mL), then adjusted to pH 6-7 with 1 M hydrochloric acid and extracted three times with EA (80 mL). The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-10 / 1) to afford compound 4 (1.28 g, 71.1%) as a colorless oil.

[0500] 3. Synthesis of Compound 7

[0501] To compound 4 (900 g, 3.1 mmol, 1.0 eq.) dissolved in DCM (9 mL) under nitrogen at 0°C were added DMSO (3.63 g, 51.72 mmol, 15 eq.), TEA (1.25 g, 12.4 mmol, 4.0 eq.), and PySO (1.27 g, 7.97 mmol, 2.57 eq.). The mixture was stirred at 0°C for 30 minutes, then warmed to room temperature and stirred under nitrogen for 90 minutes. Compound 6 (4.74 g, 13.62 mmol, 3.0 eq.) was then added to the mixture, and the reaction mixture was allowed to react at 25°C under nitrogen for 2 hours. TLC (PE / EA = 10 / 1) indicated the reaction was complete and the desired product had formed. The reaction mixture was washed with H2O (60 mL) and extracted three times with EA (70 mL). The resulting organic phase was washed twice with brine (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-10 / 1) to give compound 7 (345 mg, 27.9%) as a yellow oil.

[0502] 4. Synthesis of Compound 8

[0503] Compound 7 (340 mg, 0.95 mmol, 1.0 eq.) and Pd / C (100 mg) were added to MeOH (4 ml), and the reaction mixture was stirred at room temperature under a hydrogen atmosphere for 16 h. TLC (PE / EA = 10 / 1) showed complete consumption of the starting material and the formation of the desired product. The reaction mixture was filtered through Celite and washed with MeOH (40 mL x 2), dried over anhydrous NaSO, and the filtrate was concentrated under reduced pressure to obtain Compound 8 (298 mg, 88.2%) as a pale yellow oil.

[0504] 5. Synthesis of Compound 9

[0505] To compound 8 (298 mg, 0.83 mmol, 1.0 eq.) dissolved in THF (3 mL) was added LiAlH₄ (1 M, 1.66 mL, 2.0 eq.) under nitrogen at 0°C. The reaction was allowed to warm to room temperature and stirred under nitrogen for 2 hours. TLC (PE / EtOAc = 10 / 1) indicated completion of the reaction, with a new major spot observed. The mixture was quenched with H₂O (20 mL), then adjusted to pH 6-7 with 1 M hydrochloric acid and extracted three times with EA (30 mL). The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-10 / 1), to afford compound 9 (254 mg, 98.3%) as a colorless oil.

[0506] 6. Synthesis of Compound 11

[0507] Compound 9 (254 mg, 0.80 mmol, 1.0 eq.) and compound 10 (214 mg, 0.96 mmol, 1.2 eq.) were dissolved in DCM (3 mL). EDCI (305.6 mg, 1.6 mmol, 2.0 eq.) and DMAP (39 mg, 0.32 mmol, 0.4 eq.) were added to the mixture, followed by DIEA (412.8 mg, 3.2 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (PE / EA = 10 / 1) showed that compound 9 was consumed and the desired product was formed. The reaction mixture was adjusted to pH 4-6 with 1 M hydrochloric acid and extracted three times with EA (30 mL). The resulting organic phase was washed twice with brine (15 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using PE / EA (1 / 0-7 / 1) as eluent to give compound 11 (210 mg, 50.5%) as a yellow oil.

[0508] 7. Synthesis of SW-II-136-2

[0509] Compound 11 (200 mg, 0.38 mmol, 1.2 eq.) and compound 12 (105 mg, 0.32 mmol, 1.0 eq.) were dissolved in CPME / CHCN (1.5 mL / 1.5 mL). To this mixture were added KCO (220.2 mg, 1.60 mmol, 5.0 eq.) and KI (106 mg, 0.64 mmol, 2.0 eq.). After the addition, the reaction mixture was stirred at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated completion of the reaction and formation of the desired product. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to afford SW-II-136-2 (208 mg, 90.4%) as a yellow oil.

[0510] LCMS:Rt:2.146min; MS m / z(ELSD):773.3[M+H] + ;

[0511] HPLC: 99.49% purity, ELSD; RT = 8.055 min.

[0512] 1H NMR (400MHz, CDCl3) δ7.04(d,J=7.6Hz,1H),6.92(d,J=9.6Hz,2H),4.45(s,1H),4.06(dd,J=12.0,5.2Hz,4H),3.64(t,J=5.2Hz,2 H),2.72(t,J=5.2Hz,2H),2.65–2.50(m,10H),2.29(t,J=7.6Hz,4H),1.69–1.48(m,18H),1.41–1.24(m,36H),0.95–0.78(m,9H).

[0513] 13 C NMR (101MHz, CDCl3) δ173.86 (d, J = 2.8Hz), 140.48 (s), 139.24 (s), 138.01 (s), 129.13 (d, J = 14.8Hz), 125.67 (s),77.37(s),77.05(s),76.73(s),64.45(s),64.23(s),57.88(s),55.91(s),53.94(s),35.07(s),34.29(d ,J=3.2Hz),32.79(s),32.35(s),31.82(d,J=8.4Hz),31.38(s),29.50(d,J=2.4Hz),29.16(dd,J=18.0,2.0H z),28.66(s),28.35(s),27.78(s),27.08(s),26.02(d,J=17.2Hz),24.89(d,J=1.6Hz),22.65(s),14.10(s).

[0514] M. Compound SW-II-137-1

[0515] 1. Synthesis of compound 3

[0516] Compound 1 (500 mg, 1.95 mmol, 1.0 eq.) was dissolved in toluene (5.0 mL), followed by the addition of compound 2 (239 mg, 2.34 mmol, 1.2 eq.), Pd(PPh3)4 (225 mg, 0.19 mmol, 0.1 eq.), water (1 mL), and K2CO3 (808 g, 5.85 mmol, 3.0 eq.). The reaction was allowed to proceed at 110°C for 3 hours under nitrogen. TLC (PE / EA = 5 / 1) indicated complete reaction of the starting material and the formation of the desired product. H2O (70 mL) was added to the reaction mixture, and the mixture was extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford the compound (320 mg, 70%) as a colorless oil.

[0517] 2. Synthesis of compound 4

[0518] Compound 3 (300 mg, 1.28 mmol, 1.0 eq.) was dissolved in THF (4.0 mL) and LAH (97 mg, 2.56 mmol, 2.0 eq.) was added at 0°C under nitrogen. The reaction was then allowed to react at room temperature for 2 hours. TLC (PE / EA = 10 / 1) indicated complete reaction of the starting material and the formation of the desired product. The mixture was quenched with 1 M HCl (4 mL) and H₂O (10 mL), and extracted with EA (50 mL x 3). The organic phase was washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-10 / 1, v / v) as the eluent to afford Compound 4 (224 mg, 84.8%) as a yellow oil.

[0519] 3. Synthesis of Compound 6

[0520] Compound 4 (90 mg, 0.47 mmol, 1.0 eq.) was dissolved in DCM (3.0 mL), and compound 5 (127 mg, 0.56 mmol, 1.2 eq.), EDCI (180 mg, 0.94 mmol, 2.0 eq.), DIEA (242 mg, 1.88 mmol, 4.0 eq.), and DMAP (23 mg, 0.18 mmol, 0.4 eq.) were added. The mixture was then allowed to react overnight at room temperature under nitrogen. TLC (PE / EA = 20 / 1) indicated complete reaction of the starting material and formation of the desired product. The reaction was quenched with 1 M HCl solution, the pH adjusted to 4-6, and extracted with EA (40 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-20 / 1, v / v) as eluent to give compound 6 (90 mg, 48.6%) as a colorless oil.

[0521] 4. Synthesis of SW-II-137-1

[0522] Compound 6 (90 mg, 0.25 mmol, 1.0 eq.) was dissolved in MeCN (2 mL) and compound 7 (110 mg, 0.25 mmol, 1.0 eq.), KI (76 mg, 0.50 mmol, 2.0 eq.), CPME (2 mL), and KCO (157 mg, 1.25 mmol, 5.0 eq.) were added. The reaction was allowed to proceed at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete reaction of the starting material and formation of the desired product. The mixture was quenched with water (50 mL) and extracted with EA (40 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous NaSO, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using DCM / MeOH (1 / 0-10 / 1, v / v) as the eluent to afford the compound (98 mg, 52.12%, SW-II-137-1) as a yellow oil.

[0523] LCMS:Rt:1.596min; MS m / z(ELSD):758.4[M+H] + ;

[0524] HPLC: 98.02% purity, ELSD; RT = 5.993 min.

[0525] 1H NMR(400MHz, CDCl3) δ7.02(d,J=8.8Hz,4H),4.92–4.71(m,1H),4.01(t,J=6.4Hz,2H),3.78(s,1H),3.55(t,J=5.2Hz,2H),2.76–2.40(m,10H ),2.21(dd,J=15.6,7.7Hz,4H),1.95–1.80(m,2H),1.49(ddd,J=24.4,15.8,6.2Hz,15H),1.34–1.13(m,37H),0.82(dt,J=13.6,7.2Hz,9H).

[0526] 13 C NMR(101MHz, CDCl3)δ173.79(s),173.57(s),140.49(s),138.30(s),128.43(s),128.22(s),77.43(s), 77.11(s),76.79(s),74.11(s),63.66(s),57.96(s),55.75(s),53.90(s),35.22(s),34.63(s),34.20( d,J=11.6Hz),33.70(s),31.80(d,J=11.2Hz),30.30(s),29.51(d,J=2.8Hz),29.13(dd,J=9.6,6.8Hz), 27.12(d,J=2.8Hz),26.29(s),25.31(s),24.97(d,J=15.6Hz),22.66(s),22.37(s),14.02(d,J=15.2Hz) .

[0527] N. Compound SW-II-137-2

[0528] 1. Synthesis of compound 3

[0529] Compound 1 (500 mg, 2.06 mmol, 1.0 eq.), compound 2 (286 mg, 2.47 mmol, 1.2 eq.), Pd(PPh3)4 (119 mg, 0.1 mmol, 0.1 eq.), and K2CO3 (851 mg, 6.21 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL), and water (0.5 mL) was added. The mixture was then reacted at 110°C for 3 hours under nitrogen. TLC (PE / EA = 5 / 1) indicated complete reaction of the starting materials and the formation of the desired compound. The reaction was quenched with H2O (70 mL) and extracted with EA (80 mL x 3). The organic phase was washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA = 5 / 1, v / v as the eluent to afford Compound 3 (420 mg, 87.5%) as a colorless oil.

[0530] 2. Synthesis of compound 4

[0531] Compound 3 (420 mg, 1.78 mmol, 1.0 eq.) was dissolved in THF (3.0 mL), and LAH (1 M, 7 mL, 2.0 eq.) was added dropwise at 0°C under nitrogen. The reaction was then allowed to react at room temperature for 2 hours. TLC (PE / EA = 5 / 1) indicated complete reaction of the starting material and formation of the desired product. The reaction was quenched with HCl (1 M, 4 mL) and H₂O (10 mL), and extracted with EA (50 mL x 3). The organic phase was washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA = 5 / 1, v / v as the eluent to afford compound 4 (320 mg, 94%) as a colorless oil.

[0532] 3. Synthesis of Compound 6

[0533] Compound 4 (320 mg, 1.55 mmol, 1.0 eq.) was dissolved in DCM (4.0 mL), and compound 5 (416 mg, 1.86 mmol, 1.2 eq.), EDCI (594 mg, 3.11 mmol, 2.0 eq.), DIEA (802 mg, 6.21 mmol, 4.0 eq.), and DMAP (76 mg, 0.62 mmol, 0.4 eq.) were added. The mixture was then allowed to react overnight at room temperature under nitrogen. TLC (PE / EA = 20 / 1) indicated complete reaction of the starting materials and formation of the desired product. The reaction was quenched with 1 M HCl solution and adjusted to pH 4-6. The mixture was then extracted with DCM (60 mL x 3). The organic phase was washed with saturated brine (2 x 35 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA=5 / 1, v / v) to give compound 6 (300 mg, 47.17%) as a colorless oil.

[0534] 4. Synthesis of SW-II-137-2

[0535] Compound 6 (167 mg, 0.41 mmol, 1.2 eq.), compound 7 (150 mg, 0.34 mmol, 1.0 eq.), KI (113 mg, 0.68 mmol, 2.0 eq.), and CPME (2 mL) were dissolved in MeCN (2 mL), and KCO (235 mg, 1.70 mmol, 5.0 eq.) was added. The reaction was allowed to proceed at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete reaction of the starting materials and the formation of the desired product. The reaction was quenched with water (50 mL) and extracted with EA (60 mL x 3). The organic phase was dried over anhydrous NaSO, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using DCM / MeOH (1 / 0-10 / 1, v / v) as the eluent to afford the compound (105 mg, 40.3%, SW-II-137-2) as a pale yellow oil.

[0536] LCMS:Rt:1.660min;MS m / z(ELSD):772.4[M+H] + ;

[0537] HPLC: 98.38% purity, ELSD; RT = 8.743 min.

[0538] 1H NMR (400MHz, CDCl3) δ7.10 (d, J=8.8Hz, 4H), 5.04–4.74 (m, 1H), 4.08 (t, J=6 .4Hz,2H),3.58(t,J=5.2Hz,2H),2.65(dd,J=9.6,5.6Hz,4H),2.60–2.44(m, 6H),2.29(dd,J=16.4,7.6Hz,4H),2.01–1.88(m,2H),1.59(dt,J=9.2,7.2H z,6H),1.54–1.42(m,8H),1.39–1.11(m,41H),0.88(dt,J=11.8,6.0Hz,9H).

[0539] 13 C NMR(101MHz, CDCl3)δ173.86(s),173.63(s),140.59(s),138.34(s),128.45(s),128.24(s),77.36(s),77 .04(s),76.72(s),74.14(s),63.69(s),58.11(s),55.71(s),53.90(s),35.53(s),34.68(s),34.23(d,J=1 4.8Hz),31.82(d,J=11.6Hz),31.56(s),31.26(s),30.32(s),29.53(d,J=2.8Hz),29.19(dd,J=8.0,4.4Hz ), 27.20 (d, J = 2.4Hz), 26.64 (s), 25.33 (s), 25.02 (d, J = 15.6Hz), 22.62 (d, J = 11.6Hz), 14.08 (d, J = 8.0Hz).

[0540] O. Compound SW-II-137-3

[0541] 1. Synthesis of compound 3

[0542] To a mixture of compound 1 (11.8 g, 53 mmol, 1.2 eq.) and compound 2 (11.2 g, 44 mmol, 1 eq.) in DCM (110 mL) were added EDCI (16.9 g, 88 mmol, 2 eq.) and DMAP (2.1 g, 18 mmol, 0.4 eq.), followed by DIEA (22.7 g, 176 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 30 / 1) showed that compound 1 was consumed and the desired product was formed. The reaction mixture was extracted with ethyl acetate (200 mL) and washed with water (200 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 3 (7.391 g, 37%) as a colorless oil.

[0543] 2. Synthesis of compound 5

[0544] A mixture of compound 3 (7.391 mg, 16.07 mmol, 1 eq.) and compound 4 (29.4 g, 482.02 mmol, 30 eq.) in ethanol (2 mL) was stirred at 55 ° C under nitrogen for 16 hours. TLC (DCM / MeOH=10 / 1) showed that a new major spot was observed. The reaction mixture was extracted with ethyl acetate (100 mL) and washed with water (3×100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give compound 5 (3.695 g, 52%) as a yellow oil.

[0545] 3. Synthesis of Compound 8

[0546] To a mixture of compound 6 (1 g, 4.12 mmol, 1 eq.) and compound 7 (803 g, 6.17 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dtbpf)Cl2 (269 mg, 0.41 mmol, 0.1 eq.) and potassium carbonate (1.7 g, 12.36 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-20 / 1) to give compound 8 (568 mg, 56%) as a colorless oil.

[0547] 4. Synthesis of Compound 9

[0548] To a mixture of compound 8 (568 mg, 2.29 mmol, 1 eq.) in THF (6 mL) at 0°C under nitrogen was added lithium aluminum hydride (2.3 mL, 2.29 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (2.3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 9 (541 mg, >100%) as a colorless oil without further purification.

[0549] 5. Synthesis of Compound 10

[0550] To a mixture of compound 9 (441 mg, 2 mmol, 1 eq.) and compound 1 (536 mg, 2.4 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (768 mg, 4 mmol, 2 eq.) and DMAP (98 mg, 0.8 mmol, 0.4 eq.), followed by DIEA (1.032 g, 8 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 10 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluted with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 10 (372 mg, 44%) as a colorless oil.

[0551] 6. Synthesis of SW-II-137-3

[0552] To a mixture of compound 10 (150 mg, 0.353 mmol, 1 eq.) and compound 5 (156 mg, 0.353 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (244 mg, 1.765 mmol, 6 eq.) and potassium iodide (117 mg, 0.706 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C overnight under nitrogen. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give SW-II-137-3 (56.17 mg, 20%) as a yellow oil.

[0553] LCMS:Rt:1.550min;MS m / z(ELSD):786.4[M+H] + ;

[0554] HPLC: 98.597% purity, ELSD; RT = 13.153 min.

[0555] 1 H NMR(400MHz, CDCl3)δ7.09(s,4H),4.92–4.78(m,1H),4.08(t,J=6.6Hz,2H),3.62(t,J=5.2Hz,2H),2.78–2.50(m,10H), 2.35–2.22(m,4H),2.00–1.88(m,2H),1.57(ddd,J=28.9,13.5,4.5Hz,14H),1.38–1.20(m,42H),0.88(t,J=6.8Hz,9H).

[0556] 13C NMR(101MHz, CDCl3)δ173.83(s),173.60(s),140.60(s),138.33(s),128.44(s),128.24(s),77.36 (s),77.04(s),76.72(s),74.15(s),63.69(s),57.95(s),55.83(s),53.95(s),35.56(s),34.65(s) ),34.21(d,J=13.0Hz),31.81(d,J=12.3Hz),31.54(s),30.32(s),29.52(d,J=3.1Hz),29.34–28.9 4(m),27.13(d,J=2.5Hz),26.31(s),25.33(s),24.99(d,J=15.7Hz),22.64(d,J=5.7Hz),14.11(s).

[0557] P. Compound SW-II-138-1

[0558] 1. Synthesis of Compound 2

[0559] Compound 1 (4 g, 16.46 mmol, 1.0 eq.) was dissolved in MeOH (40 mL), cooled to 0°C, and SOCl2 (3.9 g, 32.92 mmol, 2.0 eq.) was added dropwise. The reaction was then allowed to react at room temperature for 1 hour. TLC (PE / EA = 5 / 1) showed that the starting material had been consumed and the desired product had formed. The system was directly evaporated to dryness under reduced pressure. NaHCO3 solution (70 mL) was added to the residue, and the mixture was extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford Compound 2 (4.1 mg, 95%) as a yellow oil.

[0560] 2. Synthesis of compound 4

[0561] Compound 2 (500 mg, 1.95 mmol, 1.0 eq.), compound 3 (239 mg, 2.34 mmol, 1.2 eq.), Pd(PPh3)4 (225 mg, 0.19 mmol, 0.1 eq.), and K2CO3 (808 g, 5.85 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL) was added. The mixture was then reacted at 110°C under nitrogen for 3 hours. TLC (PE / EA = 5 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with H2O (70 mL) and extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford compound 4 (320 mg, 70%) as a yellow oil.

[0562] 3. Synthesis of compound 5

[0563] Compound 4 (300 mg, 1.28 mmol, 1.0 eq.) was dissolved in THF (4.0 mL), and LAH (97 mg, 2.56 mmol, 2.0 eq.) was added at 0°C. The reaction was then allowed to react at room temperature under nitrogen for 2 hours. TLC (PE / EA = 5 / 1) indicated complete consumption of the starting material and the formation of the desired product. The reaction was quenched with 1 M HCl (4 mL) and H₂O (10 mL), and extracted with EA (50 mL x 3). The organic phase was washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford Compound 5 (224 mg, 84.8%) as a yellow oil.

[0564] 4. Synthesis of Compound 7

[0565] Compound 7 (224 mg, 1.09 mmol, 1.0 eq.) was dissolved in DCM (3.0 mL), and compound 6 (290 mg, 1.30 mmol, 1.2 eq.), EDCI (415 mg, 2.17 mmol, 2.0 eq.), DIEA (561 mg, 4.35 mmol, 4.0 eq.), and DMAP (53 mg, 0.43 mmol, 0.4 eq.) were added. The reaction was then allowed to proceed overnight at room temperature under nitrogen. TLC (PE / EA = 30 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with 1 M HCl solution and adjusted to pH 4-6, followed by extraction with DCM (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-30:1, v / v) as eluent to give compound 7 (208 mg, 46.7%) as a colorless oil.

[0566] 5. Synthesis of SW-II-138-1

[0567] Compound 10 (110 mg, 0.25 mmol, 1 eq.), compound 7 (153 mg, 0.37 mmol, 1.5 eq.), KI (83 mg, 0.50 mmol, 2.0 eq.), and CPME (2 mL) were dissolved in MeCN (2 mL) and KCO (172 mg, 1.25 mmol, 5.0 eq.) was added. The reaction was allowed to proceed overnight at 90°C under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction mixture was then dried under reduced pressure. The residue was purified on a silica gel column using DCM / MeOH (1 / 0-10:1, v / v) as the eluent to afford the compound (65 mg, 32%, SW-II-138-1) as a light yellow oil.

[0568] LCMS:Rt:1.684min;MS m / z(ELSD):772.4[M+H] + ;

[0569] HPLC: 96.56% purity, ELSD; RT = 6.346 min.

[0570] 11H NMR (400 MHz, CDCl3) δ 7.09 (s, 4H), 4.86 (s, 1H), 4.09 (d, J = 6.0 Hz, 2H), 3.97 (s, 2H), 3.07 (d, J = 38.8 Hz, 6H), 2.69–2.51 (m, 4H), 2.28 (td, J = 7.3, 3.6 Hz, 4H), 1.79 (s, 4H), 1.70–1.46 (m, 16H), 1.42–1.17 (m, 37H), 0.90 (dt, J = 13.6, 7.2 Hz, 9H).

[0571] 13 13C NMR (101 MHz, CDCl3) δ 173.80 (s), 173.53 (s), 140.32 (s), 139.13 (s), 128.28 (d, J = 13.6 Hz), 77.43 (s), 77.11 (s), 76.8 (s), 74.21 (s), 64.22 (s), 56.85 (s), 55.98 (s), 53.93 (s), 35.22 (s), 35.01 (s), 34.54 (s), 34.14 (d, J = 5.6 Hz), 33.71 (s), 31.85 (s), 29.50 (d, J = 2.8 Hz), 29.22 (s), 29.12–28.60 (m), 28.26 (s), 27.78 (s), 26.70 (d, J = 4.4 Hz), 25.31 (s), 24.82 (d, J = 17.6 Hz), 24.28 (s), 22.65 (s), 22.37 (s), 14.03 (d, J = 15.2 Hz).

[0572] QSW-II-138-2

[0573] 1. Synthesis of Compound 3

[0574] Compound 1 (500 mg, 1.95 mmol, 1.0 eq.), compound 2 (271 mg, 2.34 mmol, 1.2 eq.), Pd(PPh3)4 (225 mg, 0.20 mmol, 0.1 eq.), and K2CO3 (809 g, 5.86 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL). The mixture was then reacted at 110°C under nitrogen for 3 hours. TLC (PE / EA = 5 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with water (70 mL) and extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-30:1, v / v) as the eluent to afford compound 3 (320 mg, 70%) as a colorless oil.

[0575] 2. Synthesis of compound 4

[0576] Compound 3 (320 mg, 1.29 mmol, 1.0 eq.) was dissolved in THF (3.0 mL) and LAH (67 mg, 1.77 mmol, 2.0 eq.) was added at 0°C. The mixture was then reacted at room temperature under nitrogen for 2 hours. TLC (PE / EA = 5 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with 1 M HCl (2 mL) and H₂O (10 mL) and extracted with EA (50 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-30:1, v / v) as the eluent to afford compound 4 (180 mg, 64%) as a colorless oil.

[0577] 3. Synthesis of Compound 6

[0578] Compound 4 (180 mg, 0.82 mmol, 1.0 eq.) was dissolved in DCM (3.0 mL) and compound 5 (245 mg, 1.10 mmol, 1.2 eq.), EDCI (347 mg, 1.82 mmol, 2.0 eq.), DIEA (470 mg, 3.63 mmol, 4.0 eq.), and DMAP (45 mg, 0.36 mmol, 0.4 eq.) were added. The reaction was then allowed to proceed under nitrogen at room temperature overnight. TLC (PE / EA = 30 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with 1 M HCl solution and adjusted to pH 5-6, followed by extraction with DCM (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-30:1, v / v) as eluent to give compound 6 (220 mg, 63.6%) as a colorless oil.

[0579] 4. Synthesis of SW-II-138-2

[0580] Compound 6 (158 mg, 0.37 mmol, 1.5 eq.) and compound 7 (110 mg, 0.25 mmol, 1.0 eq.), KI (83 mg, 0.50 mmol, 2.0 eq.), and CPME (2 mL) were dissolved in MeCN (2 mL), and KCO (172 mg, 1.25 mmol, 5.0 eq.) was added. The reaction was then allowed to proceed at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction mixture was then evaporated to dryness under reduced pressure, and the residue was purified on a silica gel column using DCM / MeOH (1 / 0-10:1, v / v) as the eluent to afford the desired product (100 mg, 51%, SW-II-138-2) as a colorless oil.

[0581] LCMS:Rt:1.834min; MS m / z(ELSD):786.4[M+H] + ;

[0582] HPLC: 99.20% purity, ELSD; RT = 7.990 min.

[0583] 11H NMR (400 MHz, CDCl3) δ 7.00 (s, 4H), 4.88–4.73 (m, 2H), 4.00 (t, J = 5.6 Hz, 2H), 3.81–3.54 (m, 2H), 3.00–2.81 (m, 2H), 2.81–2.65 (m, 4H), 2.50 (dd, J = 16.4, 8.4 Hz, 4H), 2.20 (td, J = 7.6, 3.2 Hz, 4H), 1.56 (ddd, J = 18.4, 10.4, 5.2 Hz, 13H), 1.43 (d, J = 5.6 Hz, 4H), 1.34–1.07 (m, 40H), 0.81 (dt, J = 11.2, 5.6 Hz, 9H).

[0584] 13 13C NMR (101 MHz, CDCl3) δ 173.78 (s), 173.52 (s), 140.32 (s), 139.11 (s), 128.25 (d, J = 11.6 Hz), 77.49 (s), 77.17 (s), 76.85 (s), 74.14 (s), 64.17 (s), 57.25 (s), 55.82 (s), 53.85 (s), 35.50 (s), 35.01 (s), 34.56 (s), 34.14 (d, J = 7.2 Hz), 31.84 (s), 31.53 (s), 31.23 (s), 29.49 (d, J = 2.8 Hz), 29.21 (s), 28.94 (dd, J = 6.4, 4.4 Hz), 28.25 (s), 27.77 (s), 26.84 (d, J = 4.4 Hz), 25.30 (s), 25.25–24.59 (m), 22.59 (d, J = 11.2 Hz), 14.05 (d, J = 7.6 Hz).

[0585] R.SW-II-138-3

[0586] 1. Synthesis of Compound 3

[0587] Compound 1 (500 mg, 1.95 mmol, 1.0 eq.), compound 2 (305 mg, 2.34 mmol, 1.2 eq.), Pd(PPh3)4 (225 mg, 0.20 mmol, 0.1 eq.), and K2CO3 (809 g, 5.86 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL) was added. The reaction was then incubated at 110°C for 3 hours under nitrogen. TLC (PE / EA = 5:1) indicated complete consumption of the starting material and formation of the desired compound. The reaction was quenched with water (80 mL) and extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 40 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford Compound 3 (260 mg, 51.3%) as a colorless oil.

[0588] 2. Synthesis of compound 4

[0589] Compound 3 (260 mg, 0.99 mmol, 1.0 eq.) was dissolved in THF (4.0 mL), and LAH (75 mg, 1.98 mmol, 2.0 eq.) was added at 0°C. The reaction was then allowed to proceed at room temperature under nitrogen for 2 hours. TLC (PE / EA = 5 / 1) indicated complete reaction of the starting material and the formation of the desired compound. The reaction was quenched with 1 M HCl (4 mL) and H₂O (20 mL), and extracted with EA (50 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford compound 4 (230 mg, 98%) as a colorless oil.

[0590] 3. Synthesis of Compound 6

[0591] Compound 4 (240 mg, 1.03 mmol, 1.0 eq.) was dissolved in DCM (4.0 mL), and compound 5 (275 mg, 1.23 mmol, 1.2 eq.), EDCI (392 mg, 2.07 mmol, 2.0 eq.), DIEA (530 mg, 4.10 mmol, 4.0 eq.), and DMAP (50 mg, 0.41 mmol, 0.4 eq.) were added sequentially. The reaction was then allowed to proceed overnight at room temperature under nitrogen. TLC (PE / EA = 20 / 1) indicated complete consumption of the starting material and formation of the desired compound. The reaction was quenched with HCl (1 M) and the pH was adjusted to 5-6. The mixture was then extracted with DCM (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-20:1, v / v) as eluent to give compound 6 (180 mg, 40.9%) as a colorless oil.

[0592] 4. Synthesis of SW-II-138-3

[0593] Compound 6 (164 mg, 0.37 mmol, 1 eq.), compound 7 (110 mg, 0.24 mmol, 1.0 eq.), KI (83 mg, 0.49 mmol, 2.0 eq.), and CPME (2 mL) were dissolved in MeCN (2 mL) and KCO (172 mg, 1.24 mmol, 5.0 eq.) was added. The mixture was then reacted at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction mixture was then dried under reduced pressure. The residue was purified on a silica gel column using DCM / MeOH (1 / 0-10:1, v / v) as the eluent to afford the desired product (108 mg, 52.76%, SW-II-138-3) as a colorless oil.

[0594] LCMS:Rt:2.007min;MS m / z(ELSD):800.4[M+H] + ;

[0595] HPLC: 97.95% purity, ELSD; RT = 9.455 min.

[0596] 1H NMR (400MHz, CDCl3) δ7.08(s,4H),4.86(p,J=6.4Hz,1H),4.08(s,2H),3.60(t,J=5.2Hz,3H),2.76–2.42( m,10H),2.28(td,J=7.6,2.8Hz,4H),1.70–1.42(m,18H),1.28(d,J=20.0Hz,41H),0.88(t,J=6.8Hz,9H).

[0597] 13 C NMR(101MHz, CDCl3)δ173.85(s),173.59(s),140.39(s),139.15(s),128.27(d,J=12.0Hz),77.38(s),7 7.07(s),76.75(s),74.13(s),64.17(s),58.06(s),55.75(s),53.92(s),35.57(s),35.03(s),34.66(s) ,34.22(d,J=13.2Hz),31.81(d,J=12.4Hz),31.54(s),29.52(d,J=2.9Hz),29.34–28.95(m),28.29(s), 27.79 (s), 27.16 (d, J = 3.6Hz), 26.50 (s), 25.32 (s), 24.99 (d, J = 17.6Hz), 22.64 (d, J = 5.6Hz), 14.10 (s).

[0598] S. Compound SW-II-139-1

[0599] 1. Synthesis of compound 3

[0600] To a mixture of compound 1 (1 g, 4.37 mmol, 1 eq.) and compound 2 (852 g, 6.55 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dtbpf)Cl2 (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (691 mg, 68%) as a colorless oil.

[0601] 2. Synthesis of compound 4

[0602] To a mixture of compound 3 (691 mg, 2.95 mmol, 1 eq.) in THF (7 mL) at 0°C under nitrogen was added lithium aluminum hydride (3 mL, 2.95 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (547 mg, 90%) as a colorless oil without further purification.

[0603] 3. Synthesis of Compound 6

[0604] To a mixture of compound 4 (447 mg, 2.17 mmol, 1 eq.) and compound 5 (581 mg, 2.6 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (833 mg, 4.34 mmol, 2 eq.) and DMAP (106 mg, 0.87 mmol, 0.4 eq.), followed by DIEA (1.12 g, 8.68 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (455 mg, 51%) as a colorless oil.

[0605] 4. Synthesis of SW-II-139-1

[0606] To a mixture of compound 6 (150 mg, 0.365 mmol, 1 eq.) and compound 7 (161 mg, 0.365 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (252 mg, 1.825 mmol, 6 eq.) and potassium iodide (121 mg, 0.73 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C under nitrogen overnight. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give a yellow oily compound SW-II-139-1 (54.53 mg, 19%).

[0607] LCMS:Rt:1.521min;MS m / z(ELSD):772.4[M+H] + ;

[0608] HPLC: 99.637% purity, ELSD; RT = 12.347 min.

[0609] 1 H NMR (400MHz, CDCl3) δ7.20(t,J=7.7Hz,1H),7.03(t,J=6.8Hz,3H),4.94–4.78(m,1H),4.27(t,J=7.2Hz,2H),3.65(t,J=5.1Hz,2H),2.90(t,J=7.2H z,2H),2.73(t,J=4.9Hz,2H),2.67–2.41(m,6H),2.28(td,J=7.5,2.7Hz, 4H),1.67–1.45(m,14H),1.41–1.19(m,42H),0.88(dd,J=7.9,5.7Hz,9H).

[0610] 13C NMR (101MHz, CDCl3) δ173.65 (d, J = 11.3Hz), 143.17 (s), 137.67 (s), 129.04 (s), 128.34 (s), 126.61(s),126.11(s),77.30(d,J=11.6Hz),77.04(s),76.72(s),74.16(s),64.85( s),57.88(s),55.93(s),53.97(s),35.94(s),35.13(s),34.64(s),34.20(d,J=10.5 Hz),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).

[0611] T. Compound SW-II-139-2

[0612] 1. Synthesis of compound 3

[0613] To a mixture of compound 1 (1 g, 4.37 mmol, 1 eq.) and compound 2 (668 g, 6.55 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dtbpf)Cl2 (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (605 mg, 67%) as a colorless oil.

[0614] 2. Synthesis of compound 4

[0615] To a mixture of compound 3 (605 mg, 2.94 mmol, 1 eq.) in THF (7 mL) was added lithium aluminum hydride (3 mL, 2.94 mmol, 1 M in THF, 1 eq.) at 0°C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (534 mg, >100%) as a colorless oil without further purification.

[0616] 3. Synthesis of Compound 6

[0617] To a mixture of compound 4 (434 mg, 2.44 mmol, 1 eq.) and compound 5 (652 mg, 2.93 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (937 mg, 4.88 mmol, 2 eq.) and DMAP (119 mg, 0.976 mmol, 0.4 eq.), followed by DIEA (1.259 g, 9.76 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (355 mg, 38%) as a colorless oil.

[0618] 4. Synthesis of SW-II-139-2

[0619] To a mixture of compound 6 (122 mg, 0.319 mmol, 1 eq.) and compound 7 (140 mg, 0.319 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (220 mg, 1.595 mmol, 5 eq.) and potassium iodide (106 mg, 0.638 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C overnight under nitrogen. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give SW-II-139-2 (45.48 mg, 19%) as a yellow oil.

[0620] LCMS:Rt:1.346min; MS m / z(ELSD):744.3[M+H] + ;

[0621] HPLC: 97.994% purity, ELSD; RT = 11.235 min.

[0622] 1 H NMR (400MHz, CDCl3) δ7.20(t,J=7.8Hz,1H),7.03(t,J=7.6Hz,3H),4.91–4.81(m,1H),4.27(t,J=7.2Hz,2H),3.89–3.75(m ,2H),2.99–2.79(m,7H),2.64–2.48(m,2H),2.28(td,J=7.5,3.1Hz,4H),1.74–1.08(m,53H),0.90(dt,J=13.6,7.2Hz,9H).

[0623] 13 C NMR (101MHz, CDCl3) δ173.60 (d, J = 11.7Hz), 143.13 (s), 137.65 (s), 129.06 (s), 128.34 (s), 126.64 (s), 126.11 (s) ),77.30(d,J=11.4Hz),77.04(s),76.72(s),74.22(s),64.88(s),57.28(s),56.55(s),54.11(s),35.60(s),35.1 2(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).

[0624] U. Compound SW-II-140-1

[0625] 1. Synthesis of compound 3

[0626] To a mixture of compound 1 (1 g, 4.37 mmol, 1 eq.) and compound 2 (852 g, 6.55 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dppf)Cl2 (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (748 mg, 73%) as a colorless oil.

[0627] 2. Synthesis of compound 4

[0628] To a mixture of compound 3 (748 mg, 3.2 mmol, 1 eq.) in THF (8 mL) at 0°C under nitrogen was added lithium aluminum hydride (3.2 mL, 3.2 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (493 mg, 75%) as a colorless oil without further purification.

[0629] 3. Synthesis of Compound 6

[0630] To a mixture of compound 4 (393 mg, 1.91 mmol, 1 eq.) and compound 5 (511 mg, 2.29 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (733 mg, 3.82 mmol, 2 eq.) and DMAP (93 mg, 0.76 mmol, 0.4 eq.), followed by DIEA (986 mg, 7.64 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (327 mg, 42%) as a colorless oil.

[0631] 4. Synthesis of SW-II-140-1

[0632] To a mixture of compound 6 (150 mg, 0.365 mmol, 1 eq.) and compound 7 (161 mg, 0.365 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (302 mg, 2.19 mmol, 6 eq.) and potassium iodide (121 mg, 0.73 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C under nitrogen overnight. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give a yellow oily compound SW-II-140-1 (180 mg, 64%).

[0633] LCMS:Rt:1.568min;MS m / z(ELSD):772.4[M+H] + ;

[0634] HPLC: 98.053% purity, ELSD; RT = 8.702 min.

[0635] 1 H NMR (400MHz, CDCl3) δ7.23–7.05(m,4H),4.95–4.79(m,1H),4.25(t,J=7.4Hz,2H),3.62(t,J=4.8Hz,2H),2.96(dd,J=15.4,8.0 Hz,2H),2.74–2.49(m,8H),2.28(dd,J=14.2,7.2Hz,4H),1.67–1.44(m,14H),1.41–1.20(m,42H),0.90(dt,J=13.2,7.1Hz,9H).

[0636] 13C NMR (101MHz, CDCl3) δ173.68 (d, J = 10.2Hz), 141.26 (s), 135.23 (s), 129.73 (s), 129.37 (s), 126. 72(s),125.92(s),77.35(s),77.03(s),76.71(s),74.17(s),64.52(s),57.99(s),55.87(s),53 .94(s),34.66(s),34.21(d,J=11.5Hz),32.75(s),31.83(d,J=9.8Hz),31.32(s),29.65–28.88( m), 27.15 (d, J = 3.7Hz), 26.35 (s), 25.33 (s), 25.07 (s), 24.83 (s), 22.66 (d, J = 3.4Hz), 14.12 (s).

[0637] V.SW-II-140-2

[0638] 1. Synthesis of compound 3

[0639] To a mixture of compound 1 (1 g, 4.37 mmol, 1 eq.) and compound 2 (668 g, 6.55 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dppf)Cl2 (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (406 mg, 45%) as a colorless oil.

[0640] 2. Synthesis of compound 4

[0641] To a mixture of compound 3 (406 mg, 1.97 mmol, 1 eq.) in THF (5 mL) at 0°C under nitrogen was added lithium aluminum hydride (2 mL, 1.97 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (2 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (341 mg, 97%) as a colorless oil without further purification.

[0642] 3. Synthesis of Compound 6

[0643] To a mixture of compound 4 (241 mg, 1.35 mmol, 1 eq.) and compound 5 (361 mg, 1.62 mmol, 1.2 eq.) in DCM (3 mL) were added EDCI (518 mg, 2.7 mmol, 2 eq.) and DMAP (66 mg, 0.54 mmol, 0.4 eq.), followed by DIEA (697 mg, 5.4 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluted with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (185 mg, 32%) as a colorless oil.

[0644] 4. Synthesis of SW-II-140-2

[0645] To a mixture of compound 6 (185 mg, 0.483 mmol, 1 eq.) and compound 7 (213 mg, 0.483 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (400 mg, 2.898 mmol, 6 eq.) and potassium iodide (160 mg, 0.966 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C overnight under nitrogen. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give SW-II-140-2 (161 mg, 45%) as a yellow oil.

[0646] LCMS:Rt:1.696min;MS m / z(ELSD):744.3[M+H] + ;

[0647] HPLC: 94.658% purity, ELSD; RT = 5.938 min.

[0648] 1 H NMR (400MHz, CDCl3) δ7.22–7.03(m,4H),4.94–4.78(m,1H),4.25(t,J=7.3Hz,2H),3.70–3.54(m,2H),2.96(t,J =7.4Hz,2H),2.77–2.41(m,8H),2.28(dd,J=14.3,7.1Hz,4H),1.65–1.18(m,52H),0.91(dt,J=13.3,7.1Hz,9H).

[0649] 13 C NMR (101MHz, CDCl3) δ173.67 (d, J = 10.8Hz), 141.22 (s), 135.23 (s), 129.73 (s), 129.39 (s), 126.72 (s), 12 5.92(s),77.36(s),77.04(s),76.72(s),74.17(s),64.52(s),57.92(s),55.92(s),53.96(s),34.66(s),3 4.21(d,J=11.2Hz),33.51(s),32.44(s),31.83(d,J=9.3Hz),29.53(d,J=2.9Hz),29.14(dd,J=11.3,8.5H z), 27.12 (d, J = 4.1Hz), 26.23 (s), 25.33 (s), 25.06 (s), 24.82 (s), 22.73 (d, J = 9.9Hz), 14.08 (d, J = 8.8Hz).

[0650] Example 2 In vivo expression of LPP preparations prepared with CDC and BIC formulations

[0651] This example uses the CDC formulation (see, for example, Yang R et al. A core-shell structured COVID-19 mRNA vaccine with favorable biodistribution pattern and promising immunity. Signal Transduct Target Ther. 2021 May 31; 6(1): 213) and the BIC formulation (see below) to prepare an LPP formulation containing luciferase mRNA (Trilink Biotechnologies, SEQ ID NO: 1).

[0652] The preparation process of the LPP preparation prepared by the CDC prescription is as follows: Preparation of lipid mixture: dissolve M5:DOPE:mPEG2000-DSPE=49:49:2 in ethanol solution according to the weight ratio.

[0653] Preparation of mRNA aqueous solution: Dilute luciferase mRNA with 50 mM citric acid-sodium citrate buffer (pH 3-4) to a 0.35 mg / mL mRNA aqueous solution.

[0654] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.2 mg / mL.

[0655] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution composed of protamine and mRNA: volume = 4.0 mL; flow rate ratio = 3 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature.

[0656] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: volume = 4.0 mL, flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature to obtain LPP-mRNA solution.

[0657] Centrifugal ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration centrifugation to remove ethanol (centrifugal force 3400g, centrifugation time 60min, temperature 4°C) to obtain the LPP preparation prepared according to the CDC prescription.

[0658] The preparation process of LPP preparation prepared by BIC prescription is as follows: Preparation of lipid mixture: dissolve M5:DOPE:cholesterol:mPEG2000-DMG=40:15:43.5:1.5 in ethanol solution according to the molar ratio;

[0659] Preparation of mRNA aqueous solution: Dilute luciferase mRNA with 50 mM citric acid-sodium citrate buffer (pH 3-4) to a 0.35 mg / mL mRNA aqueous solution.

[0660] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.2 mg / mL.

[0661] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution composed of protamine and mRNA: volume = 4.0 mL; flow rate ratio = 3 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature.

[0662] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: volume = 4.0 mL, flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature to obtain LPP-mRNA solution.

[0663] Centrifugal ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration centrifugation to remove ethanol (centrifugal force 3400 g, centrifugation time 60 min, temperature 4°C) to obtain the LPP preparation prepared according to the BIC prescription.

[0664] Female BALB / c mice (Charles River Laboratories), 6-8 weeks old, were divided into three groups (CDC formulation, BIC formulation, and PBS, 3 mice per group). 10 μg of luciferase mRNA was administered via intramuscular injection (into the hind limb) in LPP solution prepared with the CDC formulation, LPP solution prepared with the BIC formulation, or control PBS. Six hours after administration, mice were intraperitoneally injected with 30 mg of D-luciferin substrate (Mao Kang Biotechnology). Bioluminescence was measured 15 minutes after substrate injection using a Xenogen IVIS-200 imaging system to assess in vivo expression of luciferase mRNA at the injection site and in the liver.

[0665] The experimental results are shown in Figure 1. The three columns in each group, from left to right, represent luciferase expression at the injection site, luciferase expression in the liver, and the luciferase expression ratio of the liver to the injection site. It was observed that the fluorescence of the LPP prepared with the BIC formulation at the injection site was higher than that of the LPP prepared with the CDC formulation, while the luciferase expression ratio of the liver to the injection site (%) was lower than that of the LPP prepared with the CDC formulation. This indicates that the expression efficiency of the LPP prepared with the BIC formulation was higher than that of the LPP prepared with the CDC formulation, and that it was less expressed in the liver, had better targeting, and had lower hepatotoxicity. Considering that the LPP in the BIC formulation is a four-lipid composition, while the LPP in the CDC formulation is a three-lipid composition, the following examples further investigate the four-lipid composition and optimize this lipid composition delivery system.

[0666] Example 3 Comparison of expression levels of LPP preparations with different lipid ratios

[0667] In this example, the formula in Table 1 was used to prepare four lipid LPP formulations SW0123.351-1LPP to SW0123.351-23LPP containing eGFP mRNA (Trilink Biotechnologies, SEQ ID NO: 2).

[0668] The preparation process of SW0123.351-1LPP is as follows: Preparation of lipid mixture: dissolve M5:DOPE:cholesterol:mPEG2000-DMG in ethanol solution in the proportion of 40:15:43.5:1.5.

[0669] Preparation of mRNA aqueous solution: Dilute luciferase mRNA with 50 mM citric acid-sodium citrate buffer (pH 3-4) to a 0.35 mg / mL mRNA aqueous solution.

[0670] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.2 mg / mL.

[0671] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution composed of protamine and mRNA: volume = 4.0 mL; flow rate ratio = 3 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature.

[0672] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: volume = 4.0 mL, flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature to obtain LPP-mRNA solution.

[0673] Centrifugal ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration centrifugation to remove ethanol (centrifugal force 3400g, centrifugation time 60min, temperature 4°C) to obtain an LPP preparation numbered SW0123.351-1LPP.

[0674] The preparation method of LPP preparations numbered SW0123.351-2LPP to SW0123.351-23LPP is the same as that of SW0123.351-1LPP, except for the lipid ratio (as shown in Table 1). 100 ng of SW0123.351-1LPP to SW0123.351-23LPP solution containing eGFP mRNA was taken, and PBS was used as a control to transfect dendritic cells (DC2.4 cells). 24 hours after administration, the cells were lysed and the expression of GFP protein was detected. The results are shown in Table 1. Among them, SW0123.351-1, SW0123.351-5, SW0123.351-6, SW0123.351- 9. SW0123.351-10, SW0123.351-12, SW0123.351-13, SW0123.351-18, SW0123.351-19, SW0123.351-20, and SW0123.351-21 all showed good in vitro transfection efficacy, while SW0123.351-2, SW0123.351-3, and SW0123.351-4 showed essentially no expression. This indicates that the ratio of M5, DOPE, cholesterol, and mPEG2000-DMG affects expression, with an M5:DOPE:cholesterol:mPEG2000-DMG molar ratio of 35-50:15-30:24-44:1-1.5 exhibiting an increased expression effect (indicated by expression levels exceeding the average GFP expression level).

[0675] In the following examples, SW0123.351-13LPP was used as SW0123.351-LPP for further study.

[0676] Table 1. Effects of different lipid ratios on expression levels

[0677] Example 4 Comparison of in vivo expression and in vitro physicochemical properties of SW0123.351-LPP under different storage environments

[0678] In this example, the preparation method provided in Example 3 was used to prepare a SW0123.351-LPP solution containing luciferase mRNA (SEQ ID NO: 1, 0.1 mg / ml).

[0679] Take an appropriate amount of SW0123.351-LPP solution containing luciferase mRNA and store it at 4°C. Take another appropriate amount of SW0123.351-LPP solution containing luciferase mRNA and store it at -20°C. This is recorded as day 0. Samples were taken at 4 weeks, 8 weeks, 12 weeks (i.e., day 28, day 56, and day 84) after storage to test the luciferase signal expression in vivo and the physicochemical properties in vitro. The specific detection methods are as follows:

[0680] In vivo luciferase signal expression detection: Female BALB / c mice (Charles River Laboratories) aged 6-8 weeks were divided into eight groups (3 mice in each of PBS and LPP-D0 groups, 4 mice in each of LPP-D28--20℃, LPP-D56--20℃, LPP-D84--20℃, LPP-D28-4℃, LPP-D56-4℃ and LPP-D84-4℃ groups) and were administered with control PBS, SW0123.351-LPP solution containing 10 μg of luciferase mRNA on day 0, SW0123.351-LPP solution stored at -20℃ on days 28, 56 and 84, and SW0123.351-LPP solution stored at 4℃ on days 28, 56 and 84, respectively, by intramuscular injection (hind limbs of mice). Six hours after administration, mice were intraperitoneally injected with 30 mg of D-luciferin substrate. Fifteen minutes after substrate injection, bioluminescence was measured using a Xenogen IVIS-200 imaging system to assess the expression of luciferase mRNA in mice.

[0681] Particle size detection: Take 50ul of LPP sample (2 replicates of SW0123.351-LPP solution on day 0, 6 replicates of SW0123.351-LPP solution on day 28, day 56, and day 84 stored at -20°C, and 6 replicates of SW0123.351-LPP solution on day 28, day 56, and day 84 stored at 4°C), and dilute with 950uL of purified water to obtain the diluted LPP sample, and place the sample in a dynamic light scattering laser particle size analyzer (Malvern, ZS-90) for detection.

[0682] The results of in vivo luciferase signal expression under different storage environments are shown in Figure 2A. After SW0123.351-LPP was stored at 4°C and -20°C for 12 weeks, the in vivo luciferase expression did not change significantly, indicating that it was very stable.

[0683] The in vitro physicochemical properties, i.e., average particle size (Z-Average), under different storage environments are shown in Figure 2B. After SW0123.351-LPP was stored at 4°C and -20°C for 12 weeks, the particle size of the lipid nanoparticles was very stable, remaining at around 150 nm without significant changes, indicating its stability.

[0684] The above experimental results show that the in vivo expression level and in vitro physicochemical properties of SW0123.351-LPP provided in this article can maintain a stable state for a long time under a refrigerated environment of 4°C and a frozen environment of -20°C, indicating the high stability of this four-lipid composition under different storage environments.

[0685] Example 5 Comparison of in vivo expression and in vitro physicochemical properties of SW0123.351-LPP after different freeze-thaw cycles

[0686] In this example, the preparation method provided in Example 3 was used to prepare a SW0123.351-LPP solution containing luciferase mRNA (SEQ ID NO: 1, 0.1 mg / ml).

[0687] Appropriate amounts of SW0123.351-LPP solutions containing luciferase mRNA were taken and stored at -20°C. They were freeze-thawed 2, 4, 6, 8, and 10 times, respectively, to detect luciferase signal expression in vivo and physicochemical properties in vitro. The unfreeze-thawed sample was designated as LPP-FT0, and the LPPs that were freeze-thawed 2, 4, 6, 8, and 10 times were designated as LPP-FT2, LPP-FT4, LPP-FT6, LPP-FT8, and LPP-FT10, respectively. The specific detection methods are as follows:

[0688] In vivo luciferase signaling assay: 6-8-week-old female BALB / c mice (Charles River Laboratories) were divided into seven groups (n=3 in each of the PBS and LPP-FT0 groups, and n=4 in each of the LPP-FT2, LPP-FT4, LPP-FT6, LPP-FT8, and LPP-FT10 groups). Control PBS and SW0123.351-LPP solutions stored at -20°C containing 10 μg of luciferase mRNA, which had been freeze-thawed 0, 2, 4, 6, 8, or 10 times, were administered via intramuscular injection (into the hind limb). Six hours after administration, mice were intraperitoneally injected with 30 mg of D-luciferin substrate. Bioluminescence was measured 15 minutes after substrate injection using a Xenogen IVIS-200 imaging system to assess in vivo luciferase mRNA expression.

[0689] Particle size detection: Take 50ul of LPP sample (2 replicates of LPP-FT0, 6 replicates each of LPP-FT2, LPP-FT4, LPP-FT6, LPP-FT8, and LPP-FT10) and dilute it with 950uL of purified water to obtain the diluted LPP sample. The sample was placed in a dynamic light scattering laser particle size analyzer (Malvern, ZS-90) for detection.

[0690] The results of in vivo luciferase signal expression after SW0123.351-LPP solution was treated with different freeze-thaw cycles are shown in Figure 3A. The in vivo luciferase signal expression of SW0123.351-LPP after being treated with 2, 4, 6, 8, and 10 freeze-thaw cycles did not change significantly and remained stable.

[0691] The in vitro physicochemical properties of the SW0123.351-LPP solution after different freeze-thaw cycles, namely the average particle size (Z-Average), are shown in Figure 3B. After SW0123.351-LPP was frozen and thawed 2, 4, 6, 8, and 10 times, the particle size of the lipid nanoparticles did not change significantly and remained at around 150 nm.

[0692] The above experimental results show that the in vivo expression level and in vitro physicochemical properties of SW0123.351-LPP provided in this article can maintain a stable state after multiple freeze-thaw cycles, indicating the high stability of this four-lipid composition under repeated freeze-thaw cycles.

[0693] Example 6 Immunogenicity of SW0123.351 in mice before and after freeze-drying

[0694] In this example, the preparation method provided in Example 3 was used to prepare SW0123.351-LPP containing COVID-19 mRNA (SEQ ID NO: 3), and freeze-dried to obtain SW0123.351-LPP lyophilized powder containing COVID-19 mRNA (0.1 mg / ml).

[0695] Female C57BL / 6 mice aged 6-8 weeks (Shanghai Lingchang Biotechnology Co., Ltd.) were divided into three groups (PBS group, freshly prepared SW0123.351-LPP solution group and reconstituted SW0123.351-LPP solution group, 5 mice in each group). An appropriate amount of SW0123.351-LPP lyophilized powder containing COVID-19 mRNA was reconstituted with purified water. C57BL / 6 mice were immunized by intramuscular injection (mouse hind limb) on the 1st and 14th days, and 10 μg of reconstituted SW0123.351-LPP solution containing COVID-19 mRNA and freshly prepared SW0123.351-LPP solution containing COVID-19 mRNA were administered, respectively. On the 14th day after the second immunization, the mice were bled from their orbits, and the S protein-specific binding antibodies in the serum were detected, with PBS as a control. The specific detection method is as follows:

[0696] Recombinant SARS-CoV-2 spike protein antigen (50 ng, Sino Biological) was diluted in carbonate buffer (0.1 M, pH 9.6) and coated onto EIA / RIA plates (Corning) at 4°C overnight. The plates were washed in PBST (0.05% Tween-20) and blocked with 10% goat serum (in PBS) at 37°C for 2 hours. The sample serum was serially diluted with 2% goat serum, starting at a titer of 50. The serum samples to be tested were then diluted in 2-fold dilutions. A negative serum control was included on each plate. Diluted serum samples were added (100 μl) to each well and incubated at 37°C (incubator) for 2 hours. The serum samples were discarded, and the wells were washed three times with 250 μl of PBST per well using a dispenser. 100 μl of a 10,000-fold diluted goat anti-mouse IgG HRP (Abcam) was added to each well and incubated at 37°C (incubator) for 1 hour. The secondary antibody was discarded, and the wells were washed five times with 300 μl of PBST per well using a dispenser. After washing, 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) (eBioscience) colorimetric solution was added to each well for 5 minutes in the dark. The reaction was terminated with 100 μl of 2N H2SO4 per well and the assay was performed. The OD values ​​were read at 450 nm using a microplate reader (BioTek). The endpoint titer was determined as the reciprocal of the last serum dilution. A sample OD450 nm greater than 2.1 times the OD450 nm of the negative control was considered positive.

[0697] The immunogenicity results are shown in Figure 4. The S protein-specific binding antibodies of SW0123.351-LPP before and after lyophilization were comparable, with no significant difference. This indicates the stability of the immunogenicity of this four-lipid composition after lyophilization.

[0698] Example 7 Stability Study of SW0123.351 Lyophilized Powder

[0699] In this example, the preparation method provided in Example 3 was used to prepare SW0123.351-LPP containing COVID-19 mRNA (SEQ ID NO: 3), and freeze-dried to obtain SW0123.351-LPP lyophilized powder containing COVID-19 mRNA (0.1 mg / ml).

[0700] Take an appropriate amount of SW0123.351-LPP lyophilized powder and store it at 25°C, marking it as day 0. Samples were taken and reconstituted at the first, second, and third month after storage, and the particle size, encapsulation efficiency, and immunogenicity of the reconstituted LPP solution were tested. The specific testing methods are as follows:

[0701] Particle size detection: Take 50ul of SW0123.351 lyophilized powder stored at 25℃ on day 0, 1 month, 2 months and 3 months, and dilute it with 950uL of purified water to obtain the diluted LPP sample. The sample is placed in a dynamic light scattering laser particle size analyzer (Malvern, ZS-90) for detection.

[0702] Encapsulation efficiency detection: using Quant-iT TM RiboGreen TM RNA reagent (Thermo Scientific) was used to detect the mRNA content in the LPP solution after reconstitution of SW0123.351 lyophilized powder stored at 25°C on day 0, month 1, month 2, and month 3. The LPP solution was first diluted with nuclease-free water and 1XTE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.00), then mixed with an equal volume of 2% Triton X-100 and incubated at room temperature for 1 hour to release the encapsulated mRNA. 100 μL of each sample was then transferred to a 96-well plate, and 100 μL of 200-fold diluted RiboGreen was added thereto. TMRNA reagent. The plate was shaken and incubated at room temperature for 10 minutes. Afterwards, the fluorescence value was read using a Bio-Tek Synergy I plate reader (BioTek). Standard samples were treated in the same manner. A calibration curve of fluorescence and mRNA concentration was drawn by linear regression, from which the mRNA concentration of the sample was calculated. The encapsulation efficiency of the LPP solution was defined as the percentage of encapsulated mRNA in the test sample to the total mRNA amount.

[0703] Immunogenicity test: Female C57BL / 6 mice aged 6-8 weeks (Shanghai Lingchang Biotechnology Co., Ltd.) were divided into four groups (SW0123.351 lyophilized powder stored at 25°C on day 0, month 1, month 2, and month 3 groups, with 5 mice in each group). C57BL / 6 mice were immunized by intramuscular injection (mouse hind limb) on days 1 and 14, and were administered with SW0123.351-LPP solution containing 10 μg of COVID-19 mRNA stored at 25°C on days 0, 1 month, 2 months, and 3 months after reconstitution. On the 14th day after the second immunization, the mice were bled from their orbits, and the S protein-specific binding antibodies in the serum were detected as described in Example 6, with PBS as a control.

[0704] The particle size and encapsulation efficiency test results for the lyophilized powder at 25°C storage on day 0, month 1, month 2, and month 3 are shown in Figures 5A and 5B, respectively. After 3 months of storage at 25°C, the in vitro physicochemical properties of the SW0123.351-LPP lyophilized powder remained stable, demonstrating its stability. This further demonstrates that the SW0123.351-LPP lyophilized powder provided herein maintains stable properties in all respects when stored at 25°C, demonstrating the stability of the in vitro physicochemical properties of this four-lipid composition lyophilized powder.

[0705] The immunogenicity results of the lyophilized powder stored at 25°C on the 0th day, 1st month, 2nd month and 3rd month are shown in Figure 5C. After SW0123.351-LPP lyophilized powder was stored at 25°C for 3 months, there was no significant change in its in vivo expression, indicating the stability of its immunogenicity, further illustrating the stability of the immunogenicity of this four-lipid composition lyophilized powder.

[0706] Example 8 Comparison of LNP and LPP preparations prepared from different formulas containing cationic lipid M5

[0707] 8.1. Preparation of LNP-mRNA and LPP-mRNA Formulations

[0708] 8.1.1 Preparation of Lipid Nanoparticle (LNP-mRNA) Formulations:

[0709] Preparation of mRNA aqueous solution: Dilute luciferase mRNA with 50 mM citric acid-sodium citrate buffer (pH 3-4) to a 0.35 mg / mL mRNA aqueous solution.

[0710] Preparation of lipid solution: According to the lipid types listed in Table 2, M5 (or MC3): phospholipid: cholesterol: PEG were dissolved in ethanol solution at a molar ratio of 50:10:38.5:1.5 to prepare a 10 mg / mL lipid solution.

[0711] Preparation of LNPs: Using microfluidic technology (Maianna (Shanghai) Technology Co., Ltd., model: Inano D), the lipid solution and the mRNA aqueous solution were mixed under the following conditions: volume = 4.0 mL; flow rate ratio = 3 (mRNA aqueous solution): 1 (lipid solution), total flow rate = 12 mL / min, to obtain an LNP-mRNA solution.

[0712] Centrifugal ultrafiltration: The LNP-mRNA solution was added to an ultrafiltration tube for centrifugal ultrafiltration concentration (centrifugal force 3400 g, centrifugation time 60 min, temperature 4°C) to obtain LNP-mRNA preparations numbered M5-A, M5-B, M5-C, M5-D and MC3.

[0713] 8.1.2 Preparation of Lipid Polyplex (LPP-mRNA) Preparation:

[0714] Preparation of mRNA aqueous solution: Dilute luciferase mRNA with 50 mM citric acid-sodium citrate buffer (pH 3-4) to a 0.35 mg / mL mRNA aqueous solution.

[0715] Preparation of lipid solution: According to the lipid types listed in Table 2, M5 (or MC3): phospholipid: cholesterol: PEG were dissolved in ethanol solution at a molar ratio of 50:10:38.5:1.5 to prepare a 10 mg / mL lipid solution.

[0716] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.2 mg / mL.

[0717] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution composed of protamine and mRNA: volume = 4.0 mL; flow rate ratio = 3 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature.

[0718] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: volume = 4.0 mL, flow rate ratio = 3 (mRNA aqueous solution): 1 (lipid solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature, to obtain LPP-mRNA solution.

[0719] Centrifugal ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration centrifugation to remove ethanol (centrifugal force 3400g, centrifugation time 60min, temperature 4°C) to obtain LPP-mRNA preparations numbered M5-A, M5-B, M5-C, M5-D and MC3.

[0720] 8.2. Physicochemical Properties of Different Phospholipid-PEG Combinations

[0721] This example uses LNP and LPP preparations of M5-A, M5-B, M5-C, M5-D, and MC3 prepared as in Examples 8.1.1 and 8.1.2 to test the physicochemical properties of different phospholipid and PEG combinations. The specific testing methods are as follows:

[0722] Detection of particle size and zeta potential: Take 50 μL of LPP or LNP sample and dilute it with 950 μL of purified water to obtain the diluted LPP or LNP sample, and place the sample in a dynamic light scattering laser particle size analyzer (Malvern, ZS-90) for detection.

[0723] Encapsulation efficiency detection: As described in Example 7, Quant-iT TM RiboGreen TM RNA reagent (Thermo Scientific) was used to detect the encapsulation efficiency of LPP solution or LNP solution.

[0724] Polydispersity index (PDI) measurement: The polydispersity index (PDI) of LPP or LNP solutions was determined using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK).

[0725] The experimental results, shown in Table 2, show that the particle size of the prepared LPP nanoparticles is larger than that of the LNP nanoparticles. The LPP formulations M5-B, M5-C, M5-D, and M5-E exhibit higher encapsulation efficiencies than the LNP. The polydispersity index (PDI) of the LNP formulations is smaller, but both LNP and LPP formulations have a value less than 0.3, indicating a narrow particle size distribution. Zeta potential is similar between the two formulations.

[0726] Table 2. Physicochemical properties of different phospholipids and PEG combinations

[0727] 8.3. Comparison of in vivo expression levels of preparations prepared with different formulations

[0728] This example uses LNP and LPP preparations of M5-A, M5-B, M5-C, M5-D, and MC3 prepared as in Examples 8.1.1 and 8.1.2 to test the expression of luciferase signals in mice using preparations prepared with different formulations. The specific detection method is as follows:

[0729] In vivo luciferase signal expression detection: Female Balb / c mice aged 6-8 weeks (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were divided into 10 groups (MC3-LNP, MC3-LPP, A-LNP, A-LPP, B-LNP, B-LPP, C-LNP, C-LPP, D-LNP and D-LPP groups, 3 mice in each group), and MC3-LNP, MC3-LPP, M5-A-LNP, M5-A-LPP, M5-B-LNP, M5-B-LPP, M5-C-LNP, M5-C-LPP, M5-D-LNP and M5-D-LPP solutions containing 10 μg of luciferase mRNA were administered by intramuscular injection (hind limbs of mice). At 3, 6, and 24 hours after administration, mice were intraperitoneally injected with 30 mg of D-luciferin substrate. Fifteen minutes after substrate injection, bioluminescence was measured using a Xenogen IVIS-200 imaging system to assess the expression of luciferase mRNA at the injection site and in the liver of the mice.

[0730] The total luciferase signal expression results of preparations prepared with different formulations at the injection site of mice from 0 to 24 hours are shown in Figure 6. The results of all lipid compositions show that under the same formulation, the fluorescence expression signal of LPP in mice is higher than that of LNP, indicating that under the same formulation, the expression level of LPP is higher than that of LNP, and the expression level of lipid composition B in the LPP delivery system is the highest, indicating that the optimal expression is achieved when the phospholipid is DOPE and the PEG type is PEG-DMG.

[0731] Luciferase expression in the liver and injection site of mice for formulations prepared with different formulations 3 hours after administration is shown in Figure 7A and Table 3. The three columns in each group, from left to right, represent luciferase expression at the injection site, luciferase expression in the liver, and the liver / injection site luciferase expression ratio. Similar expression in muscle tissue at the injection site was observed for the LPP and LNP formulations. M5-A / B / C / D-LPP expression at the injection site was higher than that of the M5-A / B / C / D-LNP group, and MC3-LPP expression was also higher than that of the MC3-LNP group. Furthermore, the liver / injection site luciferase expression ratio for the LPP group was lower than that for the LNP group. This suggests that 3 hours after intramuscular injection, LPP expression at the injection site was higher than that for LNP, and that LPP had better targeting, with a lower proportion of expression entering the liver, resulting in minimal liver impact and hepatotoxicity.

[0732] Luciferase expression in the liver and injection site of mice for the formulations prepared with different formulations 6 hours after administration is shown in Figure 7B and Table 4. The three columns in each group, from left to right, represent luciferase expression at the injection site, luciferase expression in the liver, and the liver / injection site luciferase expression ratio. M5-A / B / C / D-LPP expression at the injection site was observed to be 2-4 times higher than that of the M5-A / B / C / D-LNP group, and MC3-LPP expression was also significantly higher than that of the MC3-LNP group. Meanwhile, the liver / injection site luciferase expression ratio for the LPP group remained lower than that for the LNP group. This indicates that 6 hours after intramuscular injection, the expression difference between the LPP and LNP formulations further increased, with the LPP formulation expressing significantly higher levels at the injection site than the LNP formulation and maintaining excellent targeting. Among them, it was observed that under the same prescription, the liver / injection site luciferase expression ratios of M5-B-LNP and M5-B-LPP were lower, mainly expressed at the injection site and less expressed in the liver, further indicating that when the phospholipid is DOPE and the PEG type is PEG-DMG, the preparation will have better in vivo distribution and stronger targeting.

[0733] Six hours after administration, the ratio of luciferase expression in the liver to the injection site is shown in Figure 7C. The ratio of luciferase expression in the liver to the injection site for LPP was significantly higher than that for LNP. This further demonstrates that LPP formulations not only have higher in vivo expression efficiency but also have better targeting, less impact on the liver, and less hepatotoxicity compared to LNP formulations.

[0734] Table 3. Luciferase expression in muscle and liver of each preparation 3 hours after intramuscular administration

[0735] Table 4. Luciferase expression in muscle and liver of each preparation 6 hours after intramuscular administration

[0736] Example 9 Comparison of LNP and LPP preparations prepared from different formulations of cationic lipids SM102, ALC0315, SW-II-115 and SW-II-121

[0737] In this example, the preparation method provided in Example 2 was used to prepare LPP and LNP formulations containing luciferase mRNA (0.1 mg / ml) with the cationic lipids SM-102, ALC-0315, SW-II-115, and SW-II-121, respectively, as described in Table 5, to detect the expression of luciferase signals in mice using LNP and LPP formulations prepared with different formulations. The specific detection method is as follows:

[0738] In vivo luciferase signal expression detection: Female Balb / c mice aged 6-8 weeks (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were divided into 8 groups (ALC0315-LNP, ALC0315LPP, SM102-LNP, SM102-LPP, SW-II-115-LNP, SW-II-115-LPP, SW-II-121-LNP, SW-II-121-LPP groups, 3 mice in each group), and ALC0315-LNP, ALC0315LPP, SM102-LNP, SM102-LPP, SW-II-115-LNP, SW-II-115-LPP, SW-II-121-LNP, SW-II-121-LPP solutions containing 10 μg of luciferase mRNA were administered by intramuscular injection (hind limbs of mice). At 3, 6, and 24 hours after administration, mice were intraperitoneally injected with 30 mg of D-luciferin substrate. Fifteen minutes after substrate injection, bioluminescence was measured using a Xenogen IVIS-200 imaging system to assess luciferase mRNA expression in mice.

[0739] The total luciferase signal expression results of each group from 0 to 24 hours are shown in Figure 8. The results of all lipid compositions show that under the same prescription, the fluorescence expression signal of the LPP preparation in mice is higher than that of the LNP preparation, indicating that under the same prescription, although the cationic lipids are different, the expression level of the LPP preparation is higher than that of the LNP preparation, and has better expression efficiency.

[0740] Table 5. Different formulations of cationic lipids: SM102, ALC0315, SW-II-115, and SW-II-121

[0741] Example 10 Comparison of LNP and LPP preparations prepared from different formulas of cationic lipids M5, SM102, ALC0315, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 and SW-II-140-2

[0742] This example uses the preparation methods provided in Examples 8.1.1 and 8.1.2 to prepare LPP and LNP formulations containing luciferase mRNA (0.1 mg / ml) with cationic lipids M5, SM-102, ALC-0315, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2, and SW-II-140-2, respectively, to detect the expression of luciferase signals in mice using lipid compositions prepared with different formulations. The specific detection method is as follows:

[0743] In vivo luciferase signal expression detection: Female Balb / c mice aged 6-8 weeks (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were divided into 18 groups (M5-LNP, M5-LPP, SM102-LNP, SM102-LPP, ALC0315-LNP, ALC0315LPP, SW-II-121-LNP, SW-II-121-LPP, SW-II-122-LNP, SW-II-122-LPP, SW-II-134-3-LNP, SW-II-134-3-LPP, SW-II-138-2-LNP, SW-II-138-2-LPP, SW-II-139-2-LNP, SW-II-139-2-LPP, SW-II-140-2-LNP, SW-II- 140-2-LPP group, 3 mice in each group), and M5-LNP, M5-LPP, SM102-LNP, SM102-LPP, ALC0315-LNP, ALC0315LPP, SW-II-121-LNP, SW-II-121-LPP, SW-II-122-LNP, SW-II-122-LPP, SW-II-134-3-LNP, SW-II-134-3-LPP, SW-II-138-2-LNP, SW-II-138-2-LPP, SW-II-139-2-LNP, SW-II-139-2-LPP, SW-II-140-2-LNP, and SW-II-140-2-LPP solutions containing 10 μg of luciferase mRNA were administered by intramuscular injection (hind limb of mice). Six hours after administration, mice were intraperitoneally injected with 30 mg of D-luciferin substrate. Fifteen minutes after substrate injection, bioluminescence was measured using a Xenogen IVIS-200 imaging system to assess the expression of luciferase mRNA in the mouse liver and injection site (muscle).

[0744] Six hours after injection, the luciferase signal expression ratios in the liver and muscle of LNP and LPP preparations prepared with different cationic lipid formulations are shown in Figure 9 and Table 6. The two columns in each group, from left to right, represent the luciferase expression ratios in the liver and injection site for the LNP preparation and the luciferase expression ratios in the liver and injection site for the LPP preparation, respectively. It was observed that, under the same formulation, the LPP preparation exhibited a higher fluorescence expression signal in mice than the LNP preparation. This further demonstrates that, under the same formulation, despite the different cationic lipids, the expression levels of the LPP preparation were higher than those of the LNP preparation, indicating better expression efficiency.

[0745] Table 6. Luciferase expression ratios in liver / muscle of LNP and LPP preparations prepared with different cationic lipid formulations

[0746] Example 11 Comparison of preparations prepared with different formulations of cationic lipid SW-II-121

[0747] 11.1 Comparison of Different LNP Formulations Containing MC3, M5, and SW-II-121 Cationic Lipids

[0748] In this example, the preparation method provided in Example 8.1.1 was used to prepare LNP formulations containing luciferase mRNA (0.1 mg / ml) with the cationic lipids M5, MC3, and SW-II-121, as described in Table 7, to test the in vivo expression of the LNP formulations of different cationic lipids 6 hours after intramuscular administration. The specific detection method is as follows:

[0749] In vivo luciferase signaling assay: 6-8 week-old female Balb / c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were divided into three groups (M5-LNP, MC3-LNP, and SW-II-121-LNP, 3 mice per group). M5-LNP, MC3-LNP, and SW-II-121-LNP solutions containing 10 μg of luciferase mRNA were administered intramuscularly (into the hind limb). Six hours after administration, mice were intraperitoneally injected with 30 mg of D-luciferin substrate. Bioluminescence was measured 15 minutes after substrate injection using a Xenogen IVIS-200 imaging system to assess luciferase mRNA expression in the mouse liver and injection site (muscle).

[0750] The results are shown in Figure 10. The two columns in each group are, from left to right, luciferase expression at the injection site and luciferase expression in the liver. SW-II-121 was observed to express significantly better in vivo than M5-LNP and MC3-LNP 6 hours after intramuscular administration, indicating better expression efficiency. Therefore, SW-II-121 was selected as the preferred cationic lipid to further investigate the effect of lipid ratio on lipid composition.

[0751] Table 7. Different formulations of cationic lipids including M5, SM102, and SW-II-121

[0752] 11.2 Effects of Changes in Lipid Type and Lipid Ratio on In Vivo Expression

[0753] This example uses the preparation method described in Example 8.1.2 to prepare LPP formulations containing luciferase mRNA (0.1 mg / ml) with SW-II-121 as the cationic lipid, and other lipid types and lipid ratios as described in Table 9. This was done to examine the effects of varying lipid types and lipid ratios on in vivo expression. The specific testing method is as follows:

[0754] In vivo luciferase signaling assay: 6-8 week-old female Balb / c mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were divided into 12 groups (A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, and A12, 3 mice each). LPP solutions containing 10 μg of luciferase mRNA were administered intramuscularly (into the hind limb) to the mice. Twenty-four hours after administration, mice were intraperitoneally injected with 30 mg of D-luciferin substrate. Bioluminescence was measured 15 minutes after substrate injection using a Xenogen IVIS-200 imaging system to assess luciferase mRNA expression in the liver and injection site (muscle).

[0755] The expression of each formulation at the injection site (muscle tissue) of mice 0-24 hours after intramuscular injection is shown in Figures 11A and 11B and Table 9. When the phospholipid was DOPE and the PEG was PEG-DMG, fluorescence expression at the injection site of mice was the highest, significantly outperforming the other lipid combinations. Furthermore, the highest expression efficiency at the injection site was achieved when the lipid ratio was 50% SW-II-121, 10% DOPE, 38.5% cholesterol, and 1.5% PEG-DMG. The expression of each formulation in mouse liver 0-24 hours after intramuscular injection is shown in Figures 11C and 11D and Table 9. There were no significant differences among the four groups, but the magnitude of the ordinate indicates that expression of each formulation in the mouse liver was significantly lower than at the injection site.

[0756] As shown in Figure 11E and Table 9, when the phospholipid is DOPE and the PEG is PEG-DMG, the muscle / liver luciferase expression ratio is the highest, targeting is the best, and hepatotoxicity is minimized. In particular, when the lipid ratio is 50 mol% SW-II-121, 10 mol% DOPE, 38.5% cholesterol, and 1.5% PEG-DMG, the muscle / liver luciferase expression ratio is the highest and targeting is the best. This further illustrates that the preferred phospholipid is DOPE, the preferred PEG is PEG-DMG, and the preferred lipid composition comprises 50 mol% of the cationic lipid SW-II-121, 10 mol% of the phospholipid DOPE, 38.5 mol% of cholesterol, and 1.5 mol% of PEG-DMG.

[0757] Table 8. Different formulations of cationic lipids for SW-II-121

[0758] Table 9. Ratios of fluorescence expression in muscle / liver with different lipid compositions of SW-II-121 as the cationic lipid

[0759] Example 12 Preparation of Lipid Nanoparticle (LNP-mRNA) Preparation and Lipid Polyplex (LPP-mRNA) Preparation

[0760] 12.1 Experimental Materials

[0761] Lipids DOPE, DSPC, PEG-DMG, and PEG-DSPE were purchased from Avanti Polar Lipids (Birmingham, AL, USA). Cholesterol was provided by Shanghai AVT Pharmaceuticals.

[0762] 12.2 Preparation of LNP-mRNA Formulations

[0763] Preparation of mRNA aqueous solution: mRNA was diluted with 50 mM citric acid-sodium citrate buffer (pH 3-4) to a 0.35 mg / mL mRNA aqueous solution.

[0764] Preparation of lipid solution: SM-102 (or ALC-0315): phospholipid: cholesterol: PEG were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare a 10 mg / mL lipid solution.

[0765] Preparation of LNPs: Using microfluidic technology (Maianna (Shanghai) Technology Co., Ltd., model: Inano D), the lipid solution and the mRNA aqueous solution were mixed under the following conditions: volume = 4.0 mL; flow rate ratio = 3 (mRNA aqueous solution): 1 (lipid solution), total flow rate = 12 mL / min, to obtain an LNP-mRNA solution.

[0766] Centrifugal ultrafiltration: The LNP-mRNA solution was added to an ultrafiltration tube for centrifugal ultrafiltration concentration (centrifugal force 3400 g, centrifugation time 60 min, temperature 4°C) to obtain LNP-mRNA preparations numbered SM-102LNP and ALC0315-LNP.

[0767] 12.3 Preparation of LPP-mRNA Preparation

[0768] Preparation of mRNA aqueous solution: mRNA was diluted with 50 mM citric acid-sodium citrate buffer (pH 3-4) to a 0.35 mg / mL mRNA aqueous solution.

[0769] Preparation of lipid solution: According to the lipid types and molar ratios in the prescriptions listed in Tables 10 and 11, lipids were dissolved in ethanol solution to prepare a 10 mg / mL lipid solution.

[0770] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.2 mg / mL.

[0771] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution composed of protamine and mRNA: volume = 4.0 mL; flow rate ratio = 3 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature.

[0772] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: volume = 4.0 mL, flow rate ratio = 3 (mRNA aqueous solution): 1 (lipid solution), total flow rate = 12 mL / min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature to obtain LPP-mRNA solution.

[0773] Centrifugal ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration centrifugation to remove ethanol (centrifugal force 3400 g, centrifugation time 60 min, temperature 4°C) to obtain the corresponding LPP-mRNA preparation.

[0774] The particle size of LPP was measured using a dynamic light scattering instrument (Zetasizer Nano ZS90, Malvern), and the morphology of LPP was examined using a cryo-electron microscope (Glacios, Thermo Fisher Scientific).

[0775] Table 10. Different formulations of the four preparations

[0776] Table 11. Different formulations of seven preparations

[0777] Example 13 Optimized LPP formulation leads to efficient APC uptake and DC maturation

[0778] To minimize systemic exposure to the vaccine following local injection, this example employed the preparation method described in Example 12 to prepare LPP formulations containing different lipids and luciferase mRNA (encoding sequence as shown in SEQ ID NO: 1, 0.1 mg / ml) for intramuscular delivery, as described in Figure 12(A). These formulations contained different combinations of phospholipids (DOPE and DSPC) and PEG lipids (PEG-DMG and PEG-DSPE). The specific lipid ratios and compositions of the four different LPP formulations are shown in Table 10.

[0779] As shown in Figure 12(B), among the four intramuscularly injected LPP preparations, LPP-B preparation induced the strongest luciferase signal at the injection site and the least luciferase expression in the liver. Therefore, the LPP preparation was further optimized based on this lipid combination.

[0780] Using the preparation method provided in Example 12, seven LPP formulations LPP / Luc containing luciferase mRNA (0.1 mg / ml) with different lipid ratios as shown in Table 11 were prepared and characterized. As shown in Figure 12(C) and Table 11, LPP-B2 had a moderate particle size and good biodistribution (high local expression of mRNA and limited liver expression), so further research was conducted based on the LPP-B2 formulation. As shown in the cryo-electron microscopy (Cryo-TEM) image in Figure 12(D), LPP-B2 has a condensed core and a lipid shell encapsulating it, indicating that the LPP-B2 nanoparticles have a typical core-shell structure.

[0781] In vitro experiments confirmed the ability of LPP-B2 to transfect muscle cells and immune cells. The in vitro experimental procedures are shown below.

[0782] The preparation method provided in Example 12 was used to prepare an LPP preparation containing enhanced green fluorescent protein (eGFP) mRNA (coding sequence as shown in SEQ ID NO: 2, 0.1 mg / ml) based on the LPP-B2 formulation. The expression of GFP was observed by flow cytometry. Briefly, A20 (mouse B cell lymphoma cells, purchased from ATCC), DC2.4 (mouse bone marrow-derived dendritic cells, purchased from ATCC), C2C12 (mouse myoblasts, purchased from the Cell Bank of the Chinese Academy of Sciences), HSkMC (human skeletal muscle cells, purchased from ATCC) and Jurkat T cells (human T lymphocyte leukemia cells, purchased from the Cell Bank of the Chinese Academy of Sciences) were respectively cultured at 1×10 5 The cells were seeded at a density of 100 μg / well in complete medium in a 6-well plate. The cells were incubated with LPP / eGFP for 24 hours (transfection amount: 2.5 μg / well), and the percentage of GFP-positive cells was determined by flow cytometry (BD FACSCanto II, Becton Dickinson).

[0783] The results are shown in Figures 12(F) and 13. After LPP-B2 / eGFP treatment, nearly 90% of C2C12, HSkMC and Jurkat T cells synthesized GFP protein, and 60% of A20 cells and 30% of DC2.4 cells were GFP positive, indicating that the LPP-B2 prescription can effectively induce protein expression in muscle cells and immune cells.

[0784] To test the muscle-targeted delivery of LPP-B2, this example also compared the in vivo expression of luciferase delivered by LPP-B2 and LNP (SM-102LNP and ALC0315-LNP). The specific operation method is as follows.

[0785] In vivo luciferase signaling assay: 6-8 week-old female BALB / c mice (Charles River Laboratories) were divided into three groups (3 mice each: SM-102LNP, ALC0315-LNP, and LPP-B2). LPP-B2, SM-102LNP, and ALC0315-LNP formulations containing 10 μg of luciferase mRNA were administered intramuscularly (into the hind limb). Twenty-four hours after administration, mice were intraperitoneally injected with 30 mg of D-luciferin substrate (purchased from Mocon Biotech). Bioluminescence was measured 15 minutes after substrate injection using a Xenogen IVIS-200 imaging system to assess luciferase mRNA expression in the mice.

[0786] The results are shown in Figure 12 (E). Six hours after intramuscular injection, LPP-B2 was mainly expressed at the injection site, while it was almost not expressed in the liver. As shown in Figure 14 (A), 24 hours after intramuscular injection, the luciferase signal caused by LPP-B2 at the injection site was 4-6 times higher than that of commercial LNP, while the luciferase expression in the liver was 4-5 times lower. As shown in Figure 14 (B), further in vitro dissection confirmed that LPP-B2 mainly expressed protein at the local injection site, and no obvious luciferase signal was seen in major organs (heart, liver, spleen, lungs, kidneys, brain, lymph nodes). These results indicate that the intramuscularly targeted LPP-B2 delivery system may cause fewer systemic adverse events compared to commercial LNP.

[0787] This example further investigated the expression of enhanced green fluorescent protein (eGFP) mRNA (0.1 mg / ml) in different types of immune cells in lymph nodes using an LPP formulation based on the LPP-B2 formulation. The detection method is as follows.

[0788] To examine the mRNA expression of different cell types in lymph nodes, C57BL / 6 mice (Charles River Laboratories, n=5) were intramuscularly injected with control PBS or LPP formulation containing enhanced green fluorescent protein (eGFP) mRNA (0.1 mg / ml) based on the LPP-B2 formulation at a dose of 30 μg / mouse 24 hours before the assay. Flow cytometry (BD FACSCanto II, Becton Dickinson) was used to detect GFP-positive cells of different cell types. T cells, B cells, DCs (dendritic cells), macrophages, granulocytes, and monocytes were defined as CD45, CD47, and CD53, respectively. + CD3 + CD19 - 、CD45 + CD3 - CD19+ 、CD45 + CD3 - CD19 - CD11c + 、CD45 + CD11b + f / 4 / 80 + 、CD45 + CD11b + Ly6G + and CD45 + CD11b + Ly6C + Ly6G - (CD45, CD3, CD19, CD11c, CD11b, F4 / 80, and Ly6C detection antibodies were purchased from Biolegend, and Ly6G detection antibody was purchased from BD Biosciences.) The gating strategy for identifying cell populations is shown in FIG15 .

[0789] As shown in Figure 12(G), the eGFP expression rates of DCs and macrophages were approximately 7% and 8%, respectively. In contrast, T cells, B cells, granulocytes, and monocytes showed no positive signals. These results suggest that antigen-presenting cells are more efficient in LPP uptake and mRNA translation.

[0790] Furthermore, we examined the maturation markers of DCs after injection of LPP preparations based on the LPP-B2 formulation. The assay was performed as follows.

[0791] C57BL / 6 mice (Charles River Laboratories, n = 5) were injected intramuscularly with PBS, blank LPP (LPP without mRNA), or LPP / Luc, an LPP formulation based on the LPP-B2 formulation containing luciferase mRNA, at a dose of 10 μg mRNA / mouse. 24 h after injection, lymph nodes were harvested and mechanically disrupted using a 70 μm cell strainer. Cells were pelleted by centrifugation (2000 rpm, 5 min) and adjusted to a concentration of 1 × 10 cells / mouse using FACS buffer. 6 cells / tube. The resulting cells were treated with Fixable Viability Stain (BD) and incubated at room temperature for 10 minutes. The cells were then washed with FACS buffer and stained with CD11c, CD40, CD80, CD86, and IA / IE-specific antibodies (all purchased from Biolegend) at 4°C for 30 minutes. The cells were washed twice and then analyzed by flow cytometry using a BD FACSCanto II (Becton Dickinson). The gating strategy for identifying cell populations is shown in Figure 16.

[0792] The results are shown in Figure 12 (H). Compared with the PBS-treated group, 24 hours after LPP / Luc injection, the levels of DC cell maturation markers (CD40, CD80, CD86 and MHC-II) in mice were significantly upregulated, further demonstrating the auxiliary effect of LPP, which can lead to effective DC maturation.

[0793] Example 14 Optimized antigen design improves translation of spike protein in vitro

[0794] Improving the concentration and potency of the presented effective epitopes is the primary goal of vaccine antigen design. The antigens for the currently licensed SARS-CoV-2 mRNA vaccines were designed in early 2020. As the epidemic evolves and our understanding of the spike protein structure increases, the antigen design of the next generation of SARS-CoV-2 mRNA vaccines needs to be refined.

[0795] In this example, an mRNA encoding a full-length SARS-CoV-2 spike protein with three mutations was designed and prepared, and the sequence structure is shown in Figure 17(A). The 2P mutation (986-987aa) and the furin mutation (682-685aa) stabilize the full-length spike protein locked in the prefusion conformation, preventing the cleavage of the S1 / S2 subunits, thereby maintaining the integrity and conformation of the S protein. These designs have been shown to improve the immunogenicity of the S protein (WB Alsoussi, et al., SARS-CoV-2 Omicron boosting induces de novo B cell response in humans. Nature, 1–3 (2023) and J. Pallesen, et al., Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proceedings of the National Academy of Sciences. 114, E7348–E7357 (2017)). Since all variants of concern contain the D614G mutation (B. Korber, et al., Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell. 182, 812–827 (2020)), the D614G substitution was also introduced in the antigen design. This design further limits premature conversion to the post-fusion conformation and gives the variant a potentially broader neutralizing ability (P.-A. Koenig, et al., Spike D614G—A Candidate Vaccine Antigen Against Covid-19. New England Journal of Medicine. 384, 2349–2351 (2021)). In addition, the substitution of G614 supports the RBD domain in the “upper” position, increases the presentation of effective epitopes, and thus enhances immunogenicity. The expression of mRNA encoding the spike protein (S protein) was characterized by in vitro cell transfection. The methods for mRNA synthesis, in vitro transfection, and expression detection are as follows.

[0796] mRNA synthesis: As mentioned above, the designed candidate mRNA vaccine antigen is based on the spike protein of the wild-type strain (GenBank accession number: MN908947.3) and contains the D614G mutation, proline substitution (KV986-987PP) and furin cleavage site elimination (RRAR 682-685 The mRNA synthesis is based on the most widely used T7 RNA polymerase for in vitro transcription and co-transcriptional capping technology. First, the codon-optimized nucleotide sequence encoding the modified spike protein is cloned into the pUC plasmid and flanked by non-coding elements such as the T7 promoter sequence, the 5′-UTR from the human acot7 gene, the 3′-UTR from the mouse α-globin gene, and a 75nt poly(A) tail. The mRNA is synthesized using T7 RNA polymerase (Thermo Fisher Scientific) in Reagent AG (Trilink) was used for co-transcription capping reaction and in vitro transcription of RNA. 1-Methyl-pseudouridine-triphosphate was used instead of uridine triphosphate (UTP) in in vitro transcription, and the modification ratio of 1-methyl-pseudouridine was 100%. After treatment with DNase I, the RNA was purified by chromatography, filtered and dissolved in 1 mM sodium citrate buffer (pH 6.4). The RNA purity was detected by agarose gel electrophoresis and ion pair reverse liquid chromatography (IPRP-HPLC), and double-stranded RNA was detected by homogeneous time-resolved fluorescence (HTRF) technology. The mRNA was then aliquoted and stored at ≤-60°C until use. The mRNA encoding the natural spike protein was prepared according to the above method. The nucleic acid sequence encoding the natural spike protein and the nucleic acid sequence encoding the modified spike protein are shown in Table 12.

[0797] Table 12. Nucleic acid sequences encoding native spike proteins and nucleic acid sequences encoding modified spike proteins

[0798] In vitro transfection: To evaluate the in vitro expression of S protein, HEK293 cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) and DC2.4 cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were transfected at 1.2×10 6 or 8×10 5 Cells were seeded in 6-well plates at a rate of 10 cells / well. After 18 hours, cells were transiently transfected with 2 μg of mRNA encoding native spike protein or the modified spike protein mRNAs described above using Lipofectamine MassengerMAX transfection reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were harvested for western blot or flow cytometry analysis.

[0799] Western blot: mRNA-transfected cells were lysed and treated with or without PNGase F (NEB) at 37°C for 2 hours. The cells were then denatured by mixing with SDS loading buffer, followed by SDS-PAGE (polyacrylamide) gel electrophoresis and incubation with antibodies. S protein was detected using a polyclonal rabbit anti-SARS-CoV-2 RBD protein antibody (1:2000, Sinobologic) and a secondary rabbit anti-horseradish peroxidase (HRP)-conjugated antibody (Beyotime). β-actin was detected using a monoclonal mouse anti-Actin HRP-conjugated antibody (Cell Signaling Technology). Blots were developed with high-signal ECL Western Blotting Substrate (Tanon) and imaged with an ECL Imager (Azure) and Image Lab version 6.0 software.

[0800] Flow cytometry: mRNA-transfected cells were collected by direct pipetting (HEK293 cells) or 10 mM EDTA treatment (DC2.4 cells) and resuspended in cell staining buffer (biolegend). Cells were stained with Fixed Viability Stain (BD) to assess cell viability and blocked with FcR blocking reagent (Miltenyi). Cells were stained with hACE2-Fc (GenScript) staining buffer for 30 minutes at 4°C to detect cell surface spike expression. Subsequently, cells were rinsed twice with cell staining buffer and incubated with PE anti-human IgG Fc (Biolegend) in cell staining buffer at 4°C for 30 minutes. Samples were acquired using BD FACSDiva software version 8.0.1 on a FACSCanto II flow cytometer (BD Biosciences).

[0801] The Western blot results are shown in Figure 17 (B). Cells transfected with mRNA encoding the native spike protein showed two major bands (S and S1) (Figure 2B, lanes 1 and 2), indicating that most of the spike protein has been cleaved into S1 and S2 subunits. However, the modified spike protein expressed by the designed mRNA, although partially cleaved, mainly exists as a complete protein (Figure 2B, lanes 3 and 4). At the same time, it can be found that compared with the mRNA encoding the native spike protein, the designed mRNA expresses a higher level of modified spike protein (Figure 2B, lanes 1 and 3).

[0802] The flow cytometry results are shown in Figure 18 , and the modified spike protein has a significantly higher binding ability to hACE2-Fc than the natural spike protein.

[0803] All the above data indicate that the designed mRNA is translated efficiently in cells and expressed on the cell surface.

[0804] Example 15 LPP / mRNA vaccine induces strong humoral and cellular immune responses in mice

[0805] This example uses the preparation method provided in Example 12 to prepare an LPP formulation LPP / mRNA vaccine based on the LPP-B2 formulation containing the above-mentioned modified spike protein mRNA (0.1 mg / ml). In order to study the immunogenicity of the vaccine, as shown in Figure 17 (C), two batches of 6-8 week old C57 / BL6 mice (Charles River Laboratories, where n=8 for the first batch and n=5 for the second batch) were immunized by bilateral intramuscular injection on days 0 and 14, respectively, with control PBS and LPP / mRNA containing 1 μg, 5 μg, or 10 μg mRNA. The spleens of the first batch of subjects were collected on day 21, and the blood of the second batch of subjects was collected on day 28 to analyze the humoral immune response and cellular immune response levels. The specific detection method is as follows.

[0806] Enzyme-linked immunosorbent assay (ELISA) was used to detect RBD-specific binding antibodies: recombinant RBD protein (Genscript, 330-530aa) was coated in 0.05 M sodium carbonate buffer. High binding Elisa plates (Greiner) were set up at 4°C overnight. The plates were washed with PBS-T (0.05% Tween-20, pH 7.4) and blocked with 1% BSA in PBS-T. Serial two-fold dilutions of the heat-inactivated serum to be tested were added to each well. After three washes, the plates were incubated with a suitable HRP-conjugated secondary antibody (Abcam) and incubated with 3,3',5,5'-tetramethylbenzidine (TMB) (ebioscience) substrate. The absorbance was read at 450 / 610 nm using a plate reader (BioTek). The endpoint titer was determined as the reciprocal value of the last dilution of the serum to produce an absorbance greater than the critical value of 0.21.

[0807] Pseudovirus neutralization assay: The inhibition of virus entry was further tested by SARS-CoV-2 pseudovirus neutralization assay (pVNT). The neutralizing antibody titers of immunized mouse sera were determined according to the protocol of Nanjing Vazyme Biotechnology Co., Ltd. Briefly, SARS-CoV-2 pseudovirus was pre-incubated with serially diluted positive controls or serum in Opti-MEM at 37°C for 1 hour and then added to African green monkey kidney cells (Vero, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) in 96-well plates. After incubation for 24 hours, the cells were lysed and the cells were incubated with Britelite TMLuciferase signal was detected using the luciferase plus luminescent reporter gene assay kit (PerkinElmer) according to the manufacturer's protocol. Luminescence intensity was measured using a plate reader (BioTek). Neutralization titer (NT50) was defined as the reciprocal serum dilution required to inhibit luciferase activity by 50% compared to virus control wells.

[0808] Enzyme-linked immunospot assay (ELISpot): The IFNγ ELISpotPLUS kit (Mabtech) was used to detect the spike protein or RBD-specific T cell responses in mouse splenocytes according to the manufacturer's instructions. Briefly, 3 × 10 5 Splenocytes were stimulated with phytohemagglutinin (PHA) and ionomycin as a positive control, or with RPMI 1640 medium as a negative control. After incubation at 37°C, 5% CO₂ for 20 hours, the plates were sequentially treated with a biotinylated IFNγ detection antibody, streptavidin alkaline phosphatase (ALP), and color was developed using 5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium chloride-plus (BCIP / NBT-plus) substrate. When spots were intense enough to be observed with the naked eye, color development was stopped with deionized water. IFNγ spot-forming cells were counted using an ELISpot plate reader (ImmunoSpot S6 Core Analyzer (CTL)).

[0809] Intracellular cytokine staining: Single-cell suspensions of splenocytes were prepared by manually grinding whole mouse spleens and then filtered at 70 μm and 40 μm. The filtered splenocytes were resuspended in R10 medium (RPMI 1640 supplemented with penicillin-streptomycin antibiotics and 10% heat-inactivated fetal bovine serum), incubated at 37°C for 12 hours, and stimulated with a spike protein peptide pool (Genscript). A peptide pool with a final concentration of 2 μg / ml of each peptide was used. Cells were stimulated with phorbol methyl ester (PMA) (500 ng / ml, Dakewe) and ionomycin (10 μg / ml, Dakewe) as positive controls. After stimulation, the cells were washed with PBS and then stained with live / dead cell dye (Fixable Viability Stain, BD, 1:1000) for 5 minutes at room temperature. The cells were then washed in PBS and resuspended in a surface staining mixture containing the following antibodies: CD3-APC / Cyanine 7 (Biolgend, 1:50), CD4PerCP / Cyanine 5.5 (Biolgend, 1:50), CD8a APC (Biolgend, 1:50), FcR blocking reagent (Miltenyi, 1:50) in staining buffer. After incubation for 30 minutes, the cells were washed with PBS and then fixed with fixation buffer (BD) for 20 minutes. The cells were washed in perm / wash solution (BD) and intracellularly stained with a mixture of the following antibodies (4°C, 30 minutes): IFN-γ BV421 (Biolgend, 1:50), TNF-α PE / Cyanine 7 (Biolgend, 1:100), IL-4 PE (Biolgend, 1:100) in 1× perm / wash buffer (BD, diluted with sterile water). Finally, cells were washed with perm / wash solution and resuspended in PBS. Samples were acquired on a FACSCanto II flow cytometer (BD Biosciences) using BD FACSDiva software version 8.0.1.

[0810] The results of the binding antibody assay are shown in Figure 17(D). On day 28, the geometric mean titers (GMTs) of RBD-binding serum IgG in mice injected intramuscularly with LPP / mRNA formulations containing 1 μg, 5 μg, and 10 μg of mRNA were 39481, 51200, and 72408, respectively. This indicates that after the second immunization, LPP / mRNA induced high levels of RBD-specific binding antibodies in mice.

[0811] The results of the pseudovirus neutralization assay are shown in Figure 17(E). LPP / mRNA induced potent pseudovirus neutralization activity in mice, with neutralizing antibody geometric mean titers (GMTs) of 34,771 (1 μg), 53,538 (5 μg), and 55,882 (10 μg) on ​​day 28 after primary immunization (Figure 2E). The humoral immune response was not dose-dependent, indicating that mice receiving a 1 μg dose achieved maximal antibody production.

[0812] By intracellular cytokine staining and ELISpot detection, it was found that on the 21st day, a high proportion of IFN-γ (Th1 cytokine) and limited IL-4 (Th2 cytokine) secretion was detected in the spleen (as shown in Figure 17 (F) and Figure 19). Further, the cytokine pattern of vaccine-induced T cells was detected by intracellular cytokine staining on the 21st day after the primary immunization. The results are shown in Figure 17 (G and H), after restimulation with the spike protein peptide pool, LPP / mRNA-induced CD4+T cells showed a Th1-dominated response, especially at higher immunogen doses. In addition, as shown in Figure 17 (I and J), a strong CD8+T cell response to the spike protein peptide pool was observed.

[0813] Furthermore, the applicant also used ELISA to measure serum IgG1 and IgG2c antibody titers using IgG2c heavy chain (HRP) antibody (ab97255, abcam) and goat anti-mouse IgG1, human ads-HRP antibody (1070-05, SouthernBiotech) according to the manufacturer's instructions (for specific detection methods, see the above-mentioned ELISA detection method). The results are shown in Figure 17(K). A high IgG2c / IgG1 ratio indicates that the LPP / mRNA vaccine induces a Th1 humoral immune response.

[0814] Taken together, these data indicate that LPP / mRNA can induce both a dominant humoral immune response and a Th1-dominant cellular immune response.

[0815] Example 16 Protective efficacy of LPP / mRNA vaccine in elderly mice

[0816] A mouse-adapted SARS-CoV-2 strain was generated by in vivo passage and causes 100% lethality in elderly mice. This strain has been used to evaluate the protective efficacy of candidate vaccines (S.Sun, et al., Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2. Nature Communications. 12, 5654 (2021) and F.Yan, et al., Characterization of two heterogeneous lethal mouse-adapted SARS-CoV-2 variants recapitulating representative aspects of human COVID-19. Frontiers in Immunology. 13 (2022)).

[0817] In this example, as shown in FIG17(L), 8-9 month old BALB / c mice (Charles River Laboratories, n=8) were immunized intramuscularly with a control PBS, LPP / mRNA vaccine containing 1 μg or 10 μg mRNA on day 0 and day 14, respectively. Ten days after the booster vaccine injection (day 24), all mice were injected intranasally with the mouse-adapted SARS-CoV-2 strain C57MA14 (50× LD 50 ) were challenged with the drug (the challenge experiment was conducted at the Changchun Veterinary Research Institute of the Chinese Academy of Agricultural Sciences). The mice were observed daily after the challenge, and their body weight was recorded for 14 days.

[0818] The results are shown in Figure 17 (M and N). All mice vaccinated with LPP / mRNA vaccine survived the lethal dose challenge, exhibiting only transient and mild weight loss, followed by full recovery without any evidence of disease exacerbation. In contrast, the control group lost weight rapidly and died within 6 days of challenge. These results indicate that LPP / mRNA has a full protective effect against lethal infection of C57MA14.

[0819] Example 17 LPP / mRNA vaccine induces strong immunogenicity in rhesus monkeys

[0820] In order to evaluate the immunogenicity of LPP / mRNA in rhesus monkeys, as shown in Figure 20 (A), 6-year-old male rhesus monkeys were randomly divided into an LPP / mRNA group and a normal saline control group (n = 6 in each group). On day 0 and day 21, male rhesus monkeys were immunized by bilateral intramuscular injection with 500 μL of LPP / mRNA or normal saline containing 100 μg mRNA (rhesus monkeys were immunized at Sichuan Hengshu Biotechnology Co., Ltd.). Blood was collected on days 0, 14, 21, 28, and 35 for detection and analysis. For specific RBD-specific binding antibody detection methods and pseudovirus neutralization assays, see Example 15.

[0821] The results of the binding antibody test are shown in Figure 20 (B). As early as 14 days after the primary immunization, RBD-bound IgG could be detected by ELISA. The binding antibody level in rhesus macaques increased further on day 7 after the secondary immunization (day 28), with a peak GMT of 182,456. The results of the pseudovirus neutralization assay are shown in Figure 20 (C). A single injection of LPP / mRNA induced high levels of neutralizing antibodies in all vaccinated animals, with a 50% inhibition dilution reciprocal (ID) of 14 days after the primary immunization. 50 ) The geometric mean titer (GMT) was 234, which increased sharply to 8396 on the 7th day after the second vaccination.

[0822] This example also tested the neutralization effect of pseudoviruses of other strains (all purchased from Nanjing Novezan Biotechnology Co., Ltd.). As shown in Figure 20 (D) (data from day 28 and day 35, from left to right), the neutralization effect of the Delta strain was only slightly reduced (1.75-fold) compared to the wild-type strain. In contrast, the neutralization effect of the Omicron (BA.1) strain pseudovirus was significantly reduced (16.5-fold).

[0823] In addition, neutralization activity against the wild-type strain was assessed using a live virus cytopathic effect (CPE) assay. To test the breadth of the neutralization response, the serum was further tested against multiple variants (Alpha / Beta / Delta / Omicron) using the CPE assay. The assay method is described below.

[0824] In addition to the wild-type strain, four new variants of concern were also evaluated. The wild-type strain (GD108) and beta variant strain (B.1.351, GDPCC-nCOV84, CSTR.16698.06.NPRC 2.062100001), alpha variant strain (B.1.1.7, SARS-CoV-2 / C-Tan-BJ202101, CSTR.16698.06.NPRC 2.062100002), delta variant strain (B.1.617.2, CQ79, CSTR.16698.06.NPRC 6. CCPM-BV-049-2105-8), and omicron variant strain (B.1.1.529CCPM-BV-049-2112-18) are stored at the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences. Vero E6 cells were used for amplification and titer determination. Briefly, a Vero-E6 cell monolayer in a T225 culture flask was inoculated with the virus. 72 hours after inoculation, the virus-induced CPE was observed, and the supernatant was collected and stored at -80°C. The next day, after slow thawing at 4°C, the viral supernatant was centrifuged, concentrated by ultrafiltration, and eluted with 200 ml of PBS. The resulting virus was then inoculated with half the tissue culture infectious dose (TCID 50 ) titer determination. Rhesus macaque serum was collected 4 days before the challenge to evaluate its neutralization effect on SARS-CoV-2 infection. Briefly, Vero E6 cells (5×10 4 ) were inoculated into 96-well tissue culture plates and cultured overnight. 50 SARS-CoV-2 virus was preincubated with an equal volume of serially diluted serum from immunized rhesus macaques at 37°C for 1 hour. The mixture was then added to Vero E6 cell monolayers. Virus-induced CPE was recorded microscopically on day 3 post-infection, and neutralization titers were determined by calculating the reciprocal serum dilution that completely prevented CPE in 50% of the wells using the Reed-Meunch method.

[0825] Serum was collected three weeks after the second dose before the challenge (day 44). The results are shown in Figure 20 (E). For the wild-type strain, serum samples from all vaccinated animals continued to show excellent NAb titers up to this time point, with a geometric mean titer of NAb close to 2299, while no virus-specific neutralizing antibodies were detected in the control group. Compared with the wild-type strain, LPP / mRNA vaccination also resulted in excellent neutralizing activity against Alpha / Beta / Delta / Omicron variant strains, although the neutralizing activity decreased by 3.7 times, 19.8 times, 8.2 ti...

Claims

1. A lipid composition comprising a therapeutic agent or a prophylactic agent and a lipid that encapsulates the therapeutic agent or the prophylactic agent, wherein the lipid that encapsulates the therapeutic agent or the prophylactic agent comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol-modified lipid; the composition further comprises a cationic polymer, wherein the cationic polymer is associated with the therapeutic agent or the prophylactic agent to form a complex and is co-encapsulated in the lipid to form a lipid multimer complex.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

20. The lipid composition of claim 8, wherein the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof: Preferably, the cationic lipid is SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.

21. The lipid composition of claim 1, wherein the cationic lipid does not include a T5' 22. The lipid composition of any one of claims 3 to 21, wherein the phospholipids comprise 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl Phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof; DSPC, DOPE or a combination thereof is preferred.

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

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

25. The lipid composition of claim 24, comprising 10-70 mol% cationic lipid, 10-70 mol% phospholipid, 10-70 mol% steroid and 0.05-20 mol% polyethylene glycol-modified lipid; Preferably comprising 35-50 mol% of cationic lipids, 10-30 mol% of phospholipids, 24-44 mol% of steroids and 1-1.5 mol% of polyethylene glycol-modified lipids; and / or cationic lipids, DOPE, cholesterol, and DMG-PEG; Preferably, it contains 50 mol% cationic lipid, 10 mol% DOPE, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG or 40 mol% cationic lipid, 15 mol% DOPE, 43.5 mol% cholesterol and 1.5 mol% DMG-PEG.

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

27. The lipid composition of any one of claims 1-26, wherein the therapeutic or prophylactic agent is a polynucleotide comprising a coding region encoding a modified spike protein, wherein The modified spike protein comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12; and wherein the polynucleotide is RNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 3; or The polynucleotide is a DNA, wherein the coding region comprises the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO:

5.

28. The lipid composition of claim 27, wherein the polynucleotide further comprises a 5'-UTR; and / or the polynucleotide further comprises a 3'-UTR; Preferably, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 13; and / or the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 14; or The 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 15; and / or the 3'-UTR comprises the nucleotide sequence of SEQ ID NO:

16.

29. The lipid composition of claim 27 or 28, wherein the polynucleotide further comprises a poly(A) sequence; Preferably, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 17; or The poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:

18.

30. The lipid composition of any one of claims 27-29, wherein The polynucleotide is RNA, comprising the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least 85% identity with the nucleotide sequence of SEQ ID NO: 6; or The polynucleotide is DNA, which comprises the nucleotide sequence of SEQ ID NO: 7 or has a sequence similar to the nucleotide sequence of SEQ ID NO:

7. Nucleotide sequences that are at least 85% identical.

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

32. A pharmaceutical composition comprising the lipid composition of any one of claims 27-30, and optionally a pharmaceutically acceptable excipient.

33. Use of the lipid composition of any one of claims 1 to 26 or the pharmaceutical composition of claim 31 in the preparation of a medicament for treating or preventing a disease or condition in a subject in need thereof.

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

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

36. Use of the lipid composition of any one of claims 27 to 30 or the pharmaceutical composition of claim 32 in the preparation of a medicament for preventing and / or treating SARS-CoV-2 infection.

37. A method of delivering a therapeutic or prophylactic agent to a subject's mammalian cell, the method comprising administering to the subject a lipid composition of any one of claims 1-30 or a pharmaceutical composition of claim 31 or 32, the administration comprising contacting the cell with the lipid composition, thereby delivering the therapeutic and / or prophylactic agent to the cell.

38. A method for producing a target polypeptide in a subject's mammalian cell, the method comprising contacting the cell with a lipid composition of any one of claims 1-30 or a pharmaceutical composition of claim 31 or 32, wherein the therapeutic or prophylactic agent is mRNA, and wherein the mRNA encodes the target polypeptide, whereby the mRNA can be translated in the cell to produce the target polypeptide.

39. The lipid composition of any one of claims 1-30, the pharmaceutical composition of claim 31 or 32, the use of any one of claims 33-36, or the method of claim 37 or 38, wherein the lipid composition, pharmaceutical composition, or medicament is administered parenterally, orally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, or intravaginally; preferably by intramuscular injection.

40. An injection comprising the lipid composition according to any one of claims 1 to 30 and a pharmaceutically acceptable excipient for injection.

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