Liposome compound and lipid nanoparticle composition containing same

Abstract

Provided in the present invention are an ionizable liposome compound and a drug delivery system containing the ionizable liposome. Specifically, provided in the present invention are an ionizable liposome having a structure of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, and a prodrug thereof. Lipid nanoparticles constructed by using the ionizable liposome can achieve safe and efficient delivery of nucleic acid drugs, small molecule drugs, peptide drugs, and protein drugs.

Description

Liposome compounds and lipid nanoparticle compositions comprising them Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a cationic liposome compound and lipid particles prepared from the cationic liposome, as well as compositions and uses thereof. Background Technology

[0002] With the development of molecular biology, it has been discovered that in addition to nucleic acid sequences that encode proteins, a large number of non-coding sequences also play important regulatory roles in human life activities, such as promoters, enhancers, and ribozymes. Utilizing the translational or regulatory functions of nucleic acid molecules to act as drugs for intervening in or treating diseases is known as nucleic acid drug. Nucleic acid drugs exert their effects through naturally occurring transcription and translation mechanisms within target cells, eliminating the need for complex protein engineering production. They offer advantages such as simple design, short development cycles, and a wealth of candidate targets. Nucleic acid drugs are mainly classified into two categories based on their structure: small nucleic acid drugs and mRNA drugs.

[0003] Nucleic acid drug delivery systems refer to the technological systems that effectively deliver and release nucleic acid drugs to target cells to exert therapeutic effects. The key lies in solving problems related to the stability, targeting, and delivery efficiency of nucleic acid drugs in vivo. Lipid nanoparticles (LNPs) are currently one of the most widely used systems for nucleic acid drug delivery. LNPs are composed of ionizable cationic phospholipids, neutral helper phospholipids, cholesterol, and polyethylene glycol-modified phospholipids, exhibiting good stability. LNPs deliver nucleic acid drugs into cells through endocytosis and protect them from nuclease degradation. Furthermore, LNPs can promote immune activation and response by adding adjuvants. The core barrier of LNP delivery systems lies in the material patents of related components. Studies have shown that a reasonable lipid ratio significantly affects the formation, stability, and encapsulation efficiency of nanoparticles; simultaneously, this ratio has a significant impact on cellular uptake efficiency, promoting endosome escape of nucleic acid drugs, and reducing adverse interactions with serum proteins.

[0004] Therefore, it is necessary to develop effective liposome compounds to form lipid nanoparticles for the efficient and safe delivery of therapeutic agents such as therapeutic nucleic acids. Summary of the Invention

[0005] The purpose of this invention is to provide a cationic liposome compound with low toxicity and high delivery efficiency.

[0006] In a first aspect of the invention, a liposomal compound of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, or a prodrug thereof, is provided:

[0007] in,

[0008] R1 is a C2-C8 alkylene group; Z1 is O, S, SS, or SSS;

[0009] T2 is Or H;

[0010] A is selected from: A nitrogen-containing heterocyclic group of 5-10 quinones; wherein the heterocyclic group is saturated or partially unsaturated;

[0011] G is selected from: -(CH2) m1 -C3-C10 cycloalkyl groups, -(CH2) m1 -5-12 membered heterocyclic group, -(CH2) m1 -5-10 heteroaryl groups, -(CH2) m1 -OC(O)C3-C10 cycloalkyl, -(CH2) m1 -OC(O)-5-12 membered heterocyclic group, -(CH2) m1 -OC(O)-5-10 heteroaryl, -(CH2) m1 -Phenyl; the cycloalkyl or heterocyclic group is a monocyclic, bicyclic, or tricyclic system; m1 is 0, 1, 2, 3, 4, or 5;

[0012] R7 is selected from: hydroxyl group, -(CH2) 1-6 -hydroxyl, C1-C6 alkoxy, C1-C6 alkyl, -(CH2) 0-6 -N(CH3)2; m is 0, 1, 2, 3, 4 or 5; or two R7 atoms located on the same ring atom together form -OCH2CH2O-;

[0013] L1, R2, Z2, and L2 are each independently -(L) n - Each L is independently selected from the following group: substituted or unsubstituted CH2, O, S, -CO-, -S(O)-, -S(O)2-, where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

[0014] and Each has ≤20 carbon atoms, preferably ≤15, and more preferably ≤10;

[0015] X1 and X2 are each independently selected from the following groups: covalent bond, -(C=O)O-, -O(C=O)-, -(S=O)O-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -O(C=S)-;

[0016] Y1 and Y2 are each independently selected from the group consisting of: covalent bond, CH or N; preferably, Y1 and Y2 are CH.

[0017] R3 and R5 are each independently selected from the following group: substituted or unsubstituted straight or branched C1-C 30 Alkylene, substituted or unsubstituted straight or branched C2-C 30 alkenyl, substituted or unsubstituted straight or branched C2-C 30 Ethyne group, -C0-C 10 Alkylene-C3-C 10 cycloalkyl-C1-C 20 Alkylene, -C0-C 10 imide-C3-C 10 cycloalkyl-C1-C 20 Alkylene, -C0-C 10 Alkylene-C3-C 10 cycloalkyl-C1-C 20 Alkylene;

[0018] R4 and R6 are each independently selected from the following group: H, substituted or unsubstituted straight or branched C1-C 30 Alkylene, substituted or unsubstituted straight or branched C2-C 30 alkenyl, substituted or unsubstituted straight or branched C2-C 30 Ethyne group, -C0-C 10 Alkylene-C3-C 10 cycloalkyl-C1-C 20 Alkylene, -C0-C 10 imide-C3-C 10 cycloalkyl-C1-C 20 Alkylene, -C0-C 10 Alkylene-C3-C 10 cycloalkyl-C1-C 20 Alkylene;

[0019] The substitution refers to having one or more substituents selected from the group consisting of: deuterium, halogen, amino, hydroxyl, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkoxy, and C1-C6 haloalkyl.

[0020] In another preferred embodiment, R1 is a C3-C8 alkylene group.

[0021] In another preferred embodiment, the liposome compound has the structure shown in formula (II):

[0022] Among them, G, X1, X2, Y1, Y2, Z1, Z2, L1, L2, R1, R2, R3, R4, R5, R6, R7, and m are as defined above.

[0023] In another preferred example, G is selected from the group: -(CH2) m1 -C4-C8 cycloalkyl, -(CH2) m1 -5-10 cyclic groups, -(CH2) m1 -5-10 membered heteroaryl.

[0024] In another preferred embodiment, m is 0, 1, 2, or 3.

[0025] In another preferred embodiment, R2 is a C3-C8 alkylene group; Z2 is O, S, SS, or SSS.

[0026] In another preferred embodiment, X1 and X2 are each independently -(C=O)O-; wherein the -(C=O)- segment is connected to Y1, and the -O- segment is connected to L1.

[0027] In another preferred embodiment, R7 is selected from the group consisting of H, hydroxyl, methyl, -CH2OH, -C2H4OH, methoxy, substituted or unsubstituted imidazole, or two R7s located on the same ring atom that together constitute -OCH2CH2O-.

[0028] In another preferred embodiment, A is selected from:

[0029] In another preferred embodiment, Selected from the following group:

[0030] In another preferred embodiment, L1 and R2 are each independently -(L). n - Each L is independently selected from the following group: substituted or unsubstituted CH2, O, S, where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0031] In another preferred embodiment, R1-Z1-L1 is selected from the group consisting of: -(CH2) a1 -O-(CH2) b1 -、-(CH2) a1 -O-(CH2) b1 -O-(CH2) c1 -、-(CH2) a1 -S-(CH2) b1 -、-(CH2) a1 -SS-(CH2) b1 -、-(CH2) a1 -SSS-(CH2) b1 -、-(CH2) a1 -S-(CH2) b1 -S-(CH2)c1 -、-(CH2) a1 -(CH2-CH2-O) b1 -(CH2) c1 -; a1 independently represents 2, 3, 4, 5, 6, 7, 8; b1 and c1 independently represent 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0032] In another preferred embodiment, a1 is independently 3, 4, 5, 6, 7, 8.

[0033] In another preferred embodiment, R2-Z2-L2 is selected from the group consisting of -(CH2). a2 -、-(CH2) a2 -O-(CH2) b2 -、-(CH2) a2 -O-(CH2) b2 -O-(CH2) c2 -、-(CH2) a2 -S-(CH2) b2 -、-(CH2) a2 -SS-(CH2) b2 -、-(CH2) a2 -SSS-(CH2) b2 -、-(CH2) a2 -S-(CH2) b2 -S-(CH2) c2 -、-(CH2) a2 -(CH2-CH2-O) b2 -(CH2) c2 -; a2, b2, c2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0034] In another preferred embodiment, R1-Z1-L1 has a structure selected from the group consisting of:

[0035] In another preferred embodiment, R2-Z2-L2 has a structure selected from the group consisting of:

[0036] In another preferred embodiment, Each person independently selects from the following groups:

[0037] In another preferred embodiment, the liposome compound is selected from the group consisting of:

[0038] A second aspect of the present invention provides a lipid nanoparticle (LNP) comprising a liposomal compound as described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0039] In another preferred embodiment, the lipid nanoparticles further comprise auxiliary lipids.

[0040] In another preferred embodiment, the content of the ionizable lipid in the lipid nanoparticles is 30-70% molar of the total lipid content, more preferably 40-60% molar.

[0041] In another preferred embodiment, the auxiliary lipids include auxiliary phospholipids, sterols, polymer-conjugated lipids, or combinations thereof.

[0042] In another preferred embodiment, the auxiliary lipid is a combination of auxiliary phospholipids, sterols, and polymer-conjugated lipids.

[0043] In another preferred embodiment, the auxiliary phospholipid is preferably selected from: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dioleoyl lecithin (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine, 1,2-Myristoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycerol-3-phospho-rac-(1-glycerol) sodium salt, 1,2-palmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoyllecithin, 1-palmitoyl-2-oleoylphosphatidylethanolamine, distearate phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylethanolamine, or combinations thereof.

[0044] In another preferred embodiment, the sterol includes cholesterol or a cholesterol derivative.

[0045] In another preferred embodiment, the polymer-conjugated lipid is a polyethylene glycol (PEG) lipid.

[0046] In another preferred embodiment, the PEGylated lipid is preferably selected from the group consisting of DMG-PEG2000, DSPE-PEG2000, DSG-PEG2000, DSPE-PEG-Mannose, DMG-PEG2000 (polypeptides, proteins, amino acids, vitamins, and other active substances) or combinations thereof.

[0047] In another preferred embodiment, the lipid nanoparticles comprise a liposomal compound, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of liposomal compound:DSPC:cholesterol:DMG-PEG2000 is (30-70):(5-30):(30-60):(1-5), preferably (40-60):(8-15):(35-45):(1-2).

[0048] In another preferred embodiment, the lipid nanoparticles further comprise bioactive substances encapsulated within the lipid nanoparticles.

[0049] In another preferred embodiment, the bioactive substance is selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0050] In another preferred embodiment, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, small RNA, ribosomal RNA, microRNA, and transfer RNA, preferably mRNA.

[0051] A third aspect of the present invention provides a lipid nanoparticle drug formulation, the lipid nanoparticle drug formulation comprising:

[0052] i) Lipid nanoparticles as described in the second aspect of the present invention;

[0053] ii) Bioactive substances encapsulated in the lipid nanoparticles; and

[0054] iii) Pharmaceutically acceptable carriers.

[0055] In another preferred embodiment, the bioactive substance is selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0056] In another preferred embodiment, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, small RNA, ribosomal RNA, microRNA, and transfer RNA, preferably mRNA.

[0057] In another preferred embodiment, the lipid nanoparticle drug formulation can be used for the treatment and / or prevention of tumors, infectious diseases, and rare diseases.

[0058] In another preferred embodiment, the dosage form of the lipid nanoparticle drug formulation is selected from the group consisting of: injections, lyophilized formulations, nebulized inhalers, and topical formulations.

[0059] A fourth aspect of the invention provides the use of the liposomal compound as described in the first aspect of the invention, the liposomal compound being used to prepare lipid nanoparticle pharmaceutical formulations for delivering bioactive substances to cells in a subject in need.

[0060] In another preferred embodiment, the bioactive substance is selected from the group consisting of DNA, RNA (mRNA, tRNA, rRNA, miRNA), natural and synthetic oligonucleotides (including antisense oligonucleotides (ASO), interfering RNA (RNAi), and small interfering RNA (siRNA)).

[0061] In another preferred embodiment, the bioactive substance is mRNA.

[0062] In another preferred embodiment, the mRNA is transfected into cells and expressed in the cells.

[0063] In another preferred embodiment, the cells are immune cells or tumor cells.

[0064] A fifth aspect of the present invention provides a method for treating a disease in a subject in need, comprising: administering a lipid nanoparticle pharmaceutical formulation according to a third aspect of the present invention to the subject.

[0065] In another preferred embodiment, the disease is selected from the group consisting of: tumors, infectious diseases, and rare diseases.

[0066] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0067] Figure 1 shows the in vivo fluorescence distribution of B003-LNP in mice in Example 70.

[0068] Figure 2 shows the fluorescence distribution of B003-LNP in various organs of mice in Example 70.

[0069] Figure 3 shows the percentage of fluorescence intensity of B003-LNP in various organs of mice in Example 70.

[0070] Figure 4 shows the expression results of hEPO in mice from serum samples in Example 70.

[0071] Figure 5 shows the mIFN-γ expression pattern of B003-LNP in mouse spleen cells in Example 70. Detailed Implementation

[0072] Through extensive and in-depth structure-activity relationship studies, the inventors unexpectedly discovered for the first time a class of cationic liposome compounds with a cyclic head and degradable heteroatoms in the side chains. Drug delivery systems using the ionizable lipid-encapsulated drug payloads (e.g., mRNA) described in this invention exhibit high delivery efficiency and low toxicity. While efficiently delivering drug payloads and increasing their expression levels, the safety of the drug delivery system is also improved, resulting in more prominent preventative and therapeutic effects. Based on this, the present invention was completed.

[0073] the term

[0074] In this invention, the term "halogen" refers to F, Cl, Br, or I.

[0075] In this invention, "C1-C6 alkyl" refers to a straight-chain or branched alkyl group comprising 1 to 6 (1, 2, 3, 4, 5 or 6) carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, neopentyl or similar groups.

[0076] In this invention, the term "C3-C8 cycloalkyl" refers to a cyclic alkyl group having 3-8 (1, 2, 3, 4, 5, 6, 7, or 8) carbon atoms on a ring, and non-limitingly includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term "C..." 3-6 "Cycloalkyl" has a similar meaning.

[0077] In this invention, the term "C1-C6 alkoxy" refers to a straight-chain or branched alkoxy group having 1-6 carbon atoms, and includes, without limitation, methoxy, ethoxy, propoxy, isopropoxy, and butoxy. Preferably, it is a C1-C4 alkoxy group.

[0078] In this invention, the term "haloalkyl" refers to a branched or straight-chain alkyl group substituted with one or more halogen atoms, up to the maximum permissible number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, monofluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

[0079] The term "alkylamino" refers to a secondary or tertiary alkylamine group, wherein the alkyl group is independently selected from alkyl groups as defined herein. The linkage site of an alkylamino group is on a nitrogen atom. Examples of alkylamino groups include ethylamine, dimethylamine, and methylpropylamino.

[0080] In this invention, the term "heterocyclic group" refers to a 4-10 membered heterocyclic group containing 1, 2, or 3 heteroatoms selected from N, O, and S. It may be used alone or in combination with other terms to refer to a saturated or partially saturated cyclic group consisting of 4-10 (4, 5, 6, 7, 8, 9, or 10) ring atoms, wherein 1, 2, or 3 of the ring atoms are heteroatoms independently selected from O, S, and N, and the remainder are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the carbon, nitrogen, and sulfur atoms may optionally be oxidized (i.e., C(=O), NO, SO, and SO2). It includes monocyclic, bicyclic, and tricyclic systems, wherein bicyclic and tricyclic systems include spirocyclic, fused, and bridged rings. Furthermore, heteroatoms may occupy the connection positions between the heterocyclic group and the rest of the molecule; heterocyclic groups include saturated and partially unsaturated heterocyclic groups. Non-limiting examples of heterocyclic groups include azirrobutyl, oxacyclobutyl, thioheterobutyl, pyrrolidinyl, pyrazolyl, imidazoalkyl, tetrahydrothiophene (including tetrahydrothiophene-2-yl and tetrahydrothiophene-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuranyl-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperidinyl and 2-piperidinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxyl, dithiaalkyl, isoxazolyl, isothiazolyl, 1,2-oxazinyl, 1,2-thiaazinyl, hexahydropyridazinyl, homopiperidinyl, and homopiperidinyl.

[0081] In this invention, the terms "aromatic ring" or "aryl" have the same meaning, and are preferably "C". 6- C 10 "Aromatic". The term "C" 6- C 10 "Aryl" refers to an aromatic cyclic group with 6-10 carbon atoms that does not contain heteroatoms on the ring, such as phenyl and naphthyl.

[0082] In this invention, the terms "aromatic heterocycle" or "heteroaryl" have the same meaning, referring to a heteroaromatic group containing one or more heteroatoms. For example, "5-10-membered heteroaryl" refers to an aromatic heterocycle containing 1-3 heteroatoms selected from oxygen, sulfur, and nitrogen, and 2-7 carbon atoms. Non-limiting examples include: furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0083] In this invention, the term "substitution" refers to the substitution of one or more hydrogen atoms on a specific group by a specific substituent. The specific substituent is the substituent described accordingly above, or the substituent appearing in the various embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substituted site of that group, and the substituents may be the same or different at each position. Those skilled in the art will understand that the combinations of substituents contemplated in this invention are those that are stable or chemically feasible. Such substituents include, but are not limited to: halogens, hydroxyl groups, carboxyl groups (-COOH), cyano groups (-CN), C1-C6 alkyl groups, C2-C6 alkenyl groups, C3-C8 cycloalkyl groups, 3- to 12-membered heterocyclic groups, aryl groups, heteroaryl groups, C1-C8 aldehyde groups, C2-C10 acyl groups, C2-C10 ester groups, amino groups, C1-C6 alkoxy groups, C1-C10 sulfonyl groups, etc.

[0084] In this invention, the terms 1-6 refer to 1, 2, 3, 4, 5, or 6. Other similar terms each have a similar meaning independently. The term "multiple" refers to 2-6 or more, such as 2, 3, 4, 5, or 6.

[0085] It should be understood that when a certain group exists simultaneously at multiple different positions in a compound, its definition at each position is independent of each other; they can be the same or different.

[0086] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures where enantiomers are enriched in diastereomers, all of which fall within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0087] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

[0088] Another preferred class of salts are salts formed by the compounds of the present invention with a base, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (such as lower alkanol ammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0089] Ionizable lipids

[0090] As used herein, the terms “cationic liposomes of the present invention”, “ionizable cationic liposomes of the present invention”, and “liposomal compounds of the present invention” are used interchangeably and refer to liposomal compounds having the structure of Formula I, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0091] Liposomes protonate to form cationic liposomes at low pH levels, and then revert to helper phospholipids at normal physiological pH levels. Helper phospholipids interact less with the anionic cell membranes of blood cells, improving the biocompatibility of lipid nanoparticles. When lipid nanoparticles are endocytosed by cells, the lower pH within the endosomes causes the lipids to protonate and become positively charged, leading to decreased membrane stability or even disruption, thus facilitating the escape of lipid nanoparticle endosomes. In summary, the pH-sensitive nature of lipids is beneficial for the in vivo delivery of lipid nanoparticles carrying bioactive components (such as mRNA molecules).

[0092] assist lipids

[0093] As used herein, the term "accessory lipid" refers to other types of lipids besides liposomal compounds in lipid nanoparticles, including accessory phospholipids, sterols, polymer-conjugated lipids, or combinations thereof. Accessory lipids are primarily used to improve the properties of lipid nanoparticles, such as stability, delivery efficiency, tolerability, and biocompatibility.

[0094] In some embodiments, the auxiliary phospholipids include (but are not limited to) 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dioleoyl lecithin (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine. Amines, 1,2-myristoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycerol-3-phospho-rac-(1-glycerol) sodium salt, 1,2-palmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoyllecithin, 1-palmitoyl-2-oleoylphosphatidylethanolamine, distearate phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylethanolamine, or combinations thereof.

[0095] In a preferred embodiment of the present invention, the auxiliary phospholipid is DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine, also known as distearylphosphatidylcholine). DSPC is a commonly used phosphatidylcholine. The tail group of DSPC is a saturated alkane chain, with a melting point of -54°C and a cylindrical shape. It forms a layered structure in lipid nanoparticles, making the structure of lipid nanoparticles more stable.

[0096] In a preferred embodiment of the present invention, the auxiliary phospholipid is DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, also known as dioleoylphosphatidylethanolamine). DOPE is a commonly used phosphatidylethanolamine. The tail group of DOPE consists of two unsaturated alkane chains, has a melting point of -30°C, and is conical in shape. In lipid nanoparticles, it easily forms an inverted hexagon, causing instability in the endosome membrane and facilitating the escape of lipid nanoparticle endosomes.

[0097] In some embodiments, the sterols include (but are not limited to) cholesterol or cholesterol derivatives. Cholesterol can modulate the integrity and stiffness of lipid membranes, enhancing the stability of lipid nanoparticles, while the morphology of cholesterol derivatives can affect the delivery efficiency and biodistribution of lipid nanoparticles, such as the chain length of the hydrophobic tail group of cholesterol analogs, the flexibility of the sterol ring, and the polarity of the hydroxyl group. Cholesterol also affects the morphology of lipid nanoparticles; cholesterol derivatives result in lipid nanoparticles with a multilayered polyhedral structure defined by lipids rather than a spherical shape. Simultaneously, cholesterol affects the selectivity of lipid nanoparticles for their target sites: lipid nanoparticles containing cholesterol oleate are more selective for hepatic endothelial cells than for hepatocytes; when containing cholesterol with oxidized tail groups, the content of lipid nanoparticles in hepatic endothelial cells and Kupffer cells is higher than that in hepatocytes.

[0098] In some embodiments, the polymer-conjugated lipids are polyethylene glycol (PEG)-conjugated lipids, also known as PEGylated lipids or PEGylated lipids. PEGylated lipids have multiple effects on the properties of lipid nanoparticles: the amount of PEGylated lipids affects the particle size and potential of lipid nanoparticles; it reduces particle aggregation and improves the stability of lipid nanoparticles; it reduces the renal and mononuclear phagocyte system (MPS)-mediated particle clearance rate and prolongs particle circulation time; the surface functional groups can be modified with ligands to improve targeted delivery capability. Molar mass and lipid length affect the properties of PEGylated lipids. DMG-PEG2000 and DSG-PEG2000 are both neutral phospholipids with saturated alkyl chain lengths of C14, C16, or C18, respectively. However, DMG-PEG2000 can separate from lipid nanoparticles more quickly, facilitating cellular uptake of nanoparticles and endosome escape. Therefore, the delivery efficiency of DMG-PEG2000 is superior to that of DSG-PEG2000.

[0099] In a preferred embodiment of the invention, the auxiliary lipid is a combination of DSPC, cholesterol, and DMG-PEG2000.

[0100] Lipid nanoparticles (LNP)

[0101] As used herein, the term "lipid nanoparticle" or "LNP" refers to particles with a diameter of approximately 5 to 500 nm. In some embodiments, the lipid nanoparticles contain one or more active agents (bioactive substances). In some embodiments, the lipid nanoparticles include nucleic acids. In some embodiments, the nucleic acids are condensed within the nanoparticles with cationic liposomes, polymers, or multivalent small molecules, and an external lipid coating that interacts with the biological environment. Nucleic acids are naturally rigid polymers and tend to have elongated configurations due to the repulsive forces between phosphate groups. In cells, to cope with volume constraints, DNA can package itself under appropriate solution conditions with the help of ions and other molecules. Typically, DNA condensation is defined as the collapse of an elongated DNA strand into a compact, ordered particle containing only one or a few molecules. By binding to phosphate groups, cationic lipids can concentrate DNA and cause it to pack tightly together by neutralizing the phosphate charge.

[0102] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance can be anionic compounds, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins such as intact or partially deproteinized viral particles (viral particles), and oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0103] In LNP vaccine products, the bioactive substance is typically contained within the LNP itself. In some embodiments, the bioactive substance includes nucleic acids. Typically, water-soluble nucleic acids are condensed within the particle with cationic lipids or polycationic polymers, and the particle surface is enriched with accessory phospholipids or PEG lipid derivatives. Additional ionizable cationic lipids may also be located on the surface; upon entering the lysosome, these ionizable cationic lipids become positively charged due to the acidic environment of the lysosome, interacting with the lysosomal membrane and facilitating endosome escape.

[0104] Regarding LNPs, ionizable lipids can have different properties or functions. Due to the pKa of the amino group, when the external pH is lower than the pKa of the lipid molecule, it can be protonated and become positively charged. Under these conditions, the lipid molecule can electrostatically bind to the phosphate group of nucleic acid, which leads to LNP formation and nucleic acid encapsulation, and the surface charge of LNPs in biological fluids (e.g., blood) at physiological pH is essentially neutral. High LNP surface charge is associated with toxicity, rapid clearance of circulating LNPs by fixed and free macrophages, hemolytic toxicity, including immune activation (Filion et al., Biochim Biophys Acta. 1997 Oct 23; 1329(2):345-56).

[0105] In some embodiments, the pKa can be high enough that ionizable cationic lipids can take a positively charged form at acidic endosomal pH. This allows the cationic lipids to bind to endogenous endosomal anionic lipids to promote membrane lysis of non-bilayer structures, such as the hexagonal HII phase, resulting in more efficient intracellular delivery. In some embodiments, the pKa ranges from 6.2 to 6.5. For example, the pKa can be about 6.2, about 6.3, about 6.4, or about 6.5. Unsaturated tails also contribute to the lipids' ability to take a non-bilayer form. (Jayaraman et al., Angew Chem Int Ed Engl. 20 Aug 2012; 51(34):8529-33).

[0106] The release of nucleic acids in LNP formulations, as well as other characteristics such as liposome clearance and circulating half-life, can be altered by the presence of polyethylene glycol and / or sterols (e.g., cholesterol) or other potential additives in the LNP, and by the overall chemical structure (including the pKa of any ionizable cationic lipids that are part of the formulation).

[0107] In one aspect of the invention, a lipid nanoparticle (LNP) is provided, the lipid nanoparticle comprising the liposomal compound described in the first aspect of the invention, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. Further, the lipid nanoparticle also comprises one or more auxiliary lipids, including auxiliary phospholipids, steroids, and polymer-conjugated lipids.

[0108] Lipid nanoparticle drug formulation

[0109] In another aspect of the invention, a lipid nanoparticle pharmaceutical formulation (or lipid nanoparticle pharmaceutical combination or LNP composition) is provided, the lipid nanoparticle pharmaceutical formulation comprising lipid nanoparticles as described in the third aspect of the invention, a bioactive substance encapsulated within the lipid nanoparticles, and a pharmaceutically acceptable carrier. The lipid nanoparticle pharmaceutical formulation is used to deliver the bioactive substance to cells in a subject in need.

[0110] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance can be anionic compounds, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins such as intact or partially deproteinized viral particles (viral particles), and oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0111] In some embodiments, the LNP composition comprises: nucleic acid; an ionizable cationic lipid having the structure shown in Formula (I); a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, or combinations thereof); a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol); and a polymer-conjugated lipid (e.g., DMG-PEG2000). In some embodiments, the LNP composition comprises: nucleic acid; an ionizable cationic lipid having the structure shown in Formula I, comprising 30-65% (molar percentage, the same below) of the total lipids of the composition; a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, or combinations thereof) comprising 5-30% of the total lipids of the composition; a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol) comprising 30-55% of the total lipids of the composition; and a polymer-conjugated lipid (e.g., DMG-PEG2000) comprising 1-5% of the total lipids of the composition.

[0112] In a preferred embodiment of the invention, the LNP composition comprises: nucleic acid, an ionizable cationic liposome having the structure shown in formula (I), a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, etc., or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000, etc.). In a more preferred embodiment of the invention, the LNP composition comprises: nucleic acid, an ionizable cationic lipid having the structure shown in formula (I), a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, etc., or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000, etc.).

[0113] As used herein, the terms “encapsulation” and “encapsulated” refer to mRNA, DNA, siRNA, or other nucleic acid drugs being contained within or bound to lipid nanoparticles. As used herein, the term “encapsulation” refers to complete or partial encapsulation. For example, mRNA may be selected to treat and / or prevent associated diseases when administered to a subject in need of a lipid nanoparticle composition comprising mRNA.

[0114] As used herein, the term “pharmaceuticalally acceptable carrier” includes, but is not limited to, any adjuvant, carrier, excipient, scintillation agent, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the Food and Drug Administration for use in humans or livestock.

[0115] The main advantages of this invention include:

[0116] (1) The present invention introduces heteroatoms in the middle of the branched chain of cationic lipid compounds, which can change the metabolic behavior of lipid molecules in the human body and further improve the biosafety of mRNA-LNP while reducing possible toxicity.

[0117] (2) The LNP prepared using the liposome compound of the present invention has high transfection efficiency.

[0118] (3) The lipid nanoparticles prepared by the cationic lipid compounds of the present invention can form a stable nanostructure and have a good size distribution.

[0119] (4) The lipid nanoparticles of the present invention have a stable nanostructure and can be stored at low temperature for a long time.

[0120] (5) The cationic lipid compounds involved in this invention have a simple and easy synthetic route, and the raw materials are cheap and readily available, which is conducive to industrial production.

[0121] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0122] The term "alkyl" refers to a saturated carbon chain having 1 to 20 carbon atoms, which may be straight or branched or a combination thereof, unless otherwise defined. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, etc.

[0123] Unless otherwise specified in the specification, alkyl groups may be optionally substituted. The term "unsaturated hydrocarbon group" means that the group contains at least one C=C double bond (alkenyl) or at least one C≡C triple bond (alkynyl). "Alkenyl", "alkenyl" and "alkynyl" are collectively referred to as "hydrocarbon group".

[0124] In the claims of this invention, when describing "C1-C30 hydrocarbon group", it refers to a group that can be an alkane group, alkenyl group, or alkyne group having 1-30 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms). "C1-C5 hydrocarbon group", "C4-C..." 30 "hydrocarbon group", "C2-C" 30 "Hydrocarbon group" has a similar meaning.

[0125] When describing "alkyl," it refers to a saturated hydrocarbon group having a given number of carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms). It can be straight-chain or branched, and is typically a chain group without cyclic structures. Alkyl groups satisfying the aforementioned number of carbon atoms are all within the scope of this term.

[0126] When describing "alkenyl," it refers to an olefinic group having a given number of carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms), which can be linear or branched, and is generally a chain group without a cyclic structure. Alkenyl groups satisfying the aforementioned number of carbon atoms are all within the scope of this term. In different embodiments of the invention, the alkenyl group can be a group formed from a mono-olefin or a polyolefin (e.g., a diene).

[0127] When describing "straight chain or branched chain C2-C" 30 When "alkenyl" is used, it refers to an olefinic group that can have a given number of carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms). It can be linear or branched, and is typically a chain group without a cyclic structure. Olefinic groups that satisfy the aforementioned number of carbon atoms are all within the scope of this term. In different embodiments of the present invention, the olefinic group can be a hydrocarbon group containing a single alkenyl group or a hydrocarbon group containing multiple alkenyl groups (e.g., a diene).

[0128] When describing "C1-C" 30 When "alkylene" is used, it refers to an alkylene group having 0-10 carbon atoms (such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms). The alkylene group can be a straight chain or a branched chain structure, and is usually a chain group without a cyclic structure.

[0129] When describing "C2-C" 30 When "alkynyl group" is used, it refers to a group that is an alkylene group having 0-30 carbon atoms (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms). The alkylene group can be straight-chain or branched, and is typically a chain group without a cyclic structure. In different embodiments of the present invention, the C2-C...30 The alkynyl group can be a hydrocarbon group containing a single alkynyl group or a hydrocarbon group containing multiple alkynyl groups (such as a dialkynyl group).

[0130] In this application, when the definition of a divalent group includes "none", it means that the adjacent structural segments of the divalent group are directly connected by chemical bonds.

[0131] Examples of specific preparation methods for cationic lipid compounds:

[0132] Compound Abbreviation Name Reference Table

[0133] Synthesis of intermediates:

[0134] Synthesis of Inter A

[0135] Step 1.

[0136] Nonanal (2.8 g, 19.7 mmol) was added to THF (50 ml), and a hexylmagnesium bromide solution in n-hexane (12 ml, 2 mol / L, 24 mmol) was added dropwise at -10 °C. The mixture was then stirred at room temperature for 2 hours. The reaction mixture was quenched with H₂O (50 ml), filtered, and the filtrate was extracted with EA (50 ml) and H₂O (50 ml). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 20) to give compound 2 (2.1 g, Y = 46%), a colorless oil.

[0137] Step 2.

[0138] Compound 2 (300 mg, 1.24 mmol), compound 3 (254 mg, 1.22 mmol), EDCI (440 mg, 2.31 mmol), DMAP (100 mg, 0.89 mmol), and DIEA (300 mg, 2.32 mmol) were added to DCM (10 ml), and the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was washed with H2O (10 ml), dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 20) to give inter A (280 mg, Y = 54%), a colorless oil.

[0139] Synthesis of Inter B:

[0140] Compound 3 (5.2 g, 36.1 mmol), compound 4 (6.3 g, 30.2 mmol), EDCI (8.11 g, 42.4 mmol), DMAP (0.7 g, 5.7 mmol), and DIEA (10 g, 77.5 mmol) were added to DCM (100 ml), and the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was washed with saturated sodium chloride aqueous solution (100 ml), dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 20) to give inter B (3.6 g, Y = 29%), a colorless oil.

[0141] Synthesis of Inter C

[0142] Step 1.

[0143] Decanal 5 (11.1 g, 71.1 mmol) was added to THF (100 mL), and a solution of hexyl magnesium bromide in n-hexane (42 mL, 2 mol / L in hexane, 84 mmol) was added dropwise at -40 °C. The mixture was then stirred at room temperature for 2 hours. The reaction mixture was quenched with H₂O (100 mL), filtered, and the filtrate was extracted with MTBE (100 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 10) to give compound 6 (7 g, Y = 40%), a colorless oil.

[0144] Step 2.

[0145] Compound 6 (4.8 g, 19.8 mmol), 8-bromooctanoic acid 7 (5.2 g, 23.4 mmol), EDCI (5.7 g, 29.8 mmol), DMAP (0.49 g, 4.01 mmol), and DIEA (5.2 g, 40.3 mmol) were added to DCM (100 ml), and the mixture was stirred at room temperature for 2 days. After the reaction was complete, the mixture was washed with H2O (100 ml), dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 20) to give inter C (3 g, Y = 33%), a colorless oil.

[0146] Synthesis of Inter D

[0147] 8-Decanol (5 g, 31.6 mmol), 8-bromooctanoic acid (8 g, 35.8 mmol), EDCI (8.5 g, 44.5 mmol), DMAP (0.7 g, 5.7 mmol), and DIEA (11.5 g, 89.1 mmol) were added to DCM (200 ml), and the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was washed with H2O (200 ml), dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 10) to give inter D (4 g, Y = 34%), a colorless oil.

[0148] Synthesis of Inter E:

[0149] Step 1.

[0150] Propylene glycol E-1 (10 g, 0.13 mol), compound E-2 (15 g, 0.12 mol), and NaOH (150 mg, 3.75 mmol) were stirred at room temperature for 3 days. The reaction solution was directly purified by silica gel column chromatography (EA / PE = 1 / 3) to give compound E-3 (10 g, Y = 37%), a colorless oil.

[0151] Step 2.

[0152] Compound E-3 (10 g, 0.049 mol), CBr4 (17.7 g, 0.054 mol), and PPh3 (13.3 g, 0.051 mol) were added to DCM (200 ml) at 0–20 °C, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was washed with MTBE (200 ml x 2), and the washes were combined and evaporated to dryness. The mixture was then purified by silica gel column chromatography (EA / PE = 1 / 10) to give compound E-4 (8.9 g, Y = 68%) as a colorless oil.

[0153] Step 3.

[0154] Compound E-4 (8.9 g, 0.033 mol) was added to TFA (100 ml), and the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was evaporated to dryness, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 1) to give compound E-5 (6.5 g, Y = 93%), a colorless oil.

[0155] Step 4.

[0156] Compound E-5 (6.5 g, 0.03 mol), compound E-6 (7.7 g, 0.033 mol), EDCI (7 g, 0.036 mol), DMAP (0.7 g, 5.7 mmol), and DIEA (11 g, 0.085 mol) were added to DCM (150 ml), and the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was washed with H2O (150 ml x 2), dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 20) to give inter E (6 g, Y = 47%), a colorless oil.

[0157] Synthesis of Inter F

[0158] Compound E-5 (6.5 g, 0.03 mol), compound F-1 (7.7 g, 0.033 mol), EDCI (7 g, 0.036 mol), DMAP (0.7 g, 5.7 mmol), and DIEA (11 g, 0.085 mol) were added to DCM (150 ml), and the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was washed with H2O (150 ml x 2), dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 20) to give inter F (6 g, Y = 47%), a colorless oil.

[0159] Synthesis of Inter G

[0160] Step 1.

[0161] Butanediol G-1 (12 g, 0.13 mol), compound G-2 (15 g, 0.12 mol), and NaOH (150 mg, 3.75 mmol) were stirred at room temperature for 3 days. The reaction solution was directly purified by silica gel column chromatography (EA / PE = 1 / 3) to give compound G-3 (10 g, Y = 45%), a colorless oil.

[0162] Step 2.

[0163] Compound G-3 (13 g, 0.059 mol), CBr4 (17.2 g, 0.052 mol), and PPh3 (23 g, 0.087 mol) were added to DCM (260 ml) at 0–20 °C, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was washed with MTBE (200 ml), the wash buffer was evaporated to dryness, and the solution was purified by silica gel column chromatography (EA / PE = 1 / 20) to give compound G-4 (6 g, Y = 36%) as a colorless oil.

[0164] Step 3.

[0165] Compound G-4 (6 g, 0.021 mol) was added to TFA (60 ml), and the mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was evaporated to dryness, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 1) to give compound G-5 (4.3 g, Y = 91%), a colorless oil.

[0166] Step 4.

[0167] Compound G-5 (6.5 g, 0.03 mol), compound G-6 (7.7 g, 0.033 mol), EDCI (7 g, 0.036 mol), DMAP (0.7 g, 5.7 mmol), and DIEA (11 g, 0.085 mol) were added to DCM (150 ml), and the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was washed with H2O (150 ml x 2), dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography (EA / PE = 1 / 20) to give inter G (6 g, Y = 47%), a colorless oil.

[0168] Synthesis of intermediate amine H:

[0169] Step 1.

[0170] Compound H-1 (3.6 g, 18.2 mmol), K₂O₅SO₄·2H₂O (480 mg, 1.3 mmol), and NMO (3.2 g, 27.3 mmol) were added to ACN (40 mL) and H₂O (12 mL), and the mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with EA (100 mL) and H₂O (100 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give compound H-2 (2 g, Y = 47%), a brown solid.

[0171] Step 2.

[0172] Compound H-2 (1 g, 4.3 mmol) was added to HCl (20 ml, 4 mol / L in dioxane) and H2O (2 ml), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solution was evaporated under reduced pressure to obtain amine H (1 g, crude product), a brown solid.

[0173] Synthesis of intermediate amine I:

[0174] Step 1.

[0175] Compound I-1 (0.9 g, 4.9 mmol), K₂O₅SO₄·2H₂O (480 mg, 1.3 mmol), and NMO (3.2 g, 27.3 mmol) were added to ACN (40 mL) and H₂O (12 mL), and the mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with EA (100 mL) and H₂O (100 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give compound I-2 (2 g, Y = 47%), a brown solid.

[0176] Step 2.

[0177] Compound I-2 (360 mg, 1.65 mmol) was added to HCl (4 mL, 4 mol / L in dioxane) and H2O (0.5 mL), and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the product was evaporated under reduced pressure to obtain amine I (300 mg, crude product), a brown solid.

[0178] Example 1. Synthesis of A001:

[0179] Intermediate E (422 mg, 1 mmol), 4-aminocyclohexanol (46 mg, 0.4 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 ml), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (20 ml) and EA (20 ml). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 10 / 1) to give compound A001 (209 mg, Y = 65%), a brown oil. LCMS: [M+1] + =797. 1 H NMR(300MHz, DMSO-d6)δ4.78(q,J=6.1Hz,2H),4.45(d,J=4.2Hz,1H),3.57-3.52(m,4H),3.33-3.29(m, 4H),2.47-2.23(m,9H),1.84-1.80(m,3H),1.47-1.29(m,15H),1.28-1.15(m,43H),0.90-0.77(m,12H).

[0180] Example 2. Synthesis of A002:

[0181] Intermediate E (422 mg, 1 mmol), 3-aminocyclobutanol (35 mg, 0.4 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (20 mL) and EA (20 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 10 / 1) to give compound A002 (167 mg, Y = 54%), a brown oil. LCMS: [M+1] + =769.

[0182] Example 3. Synthesis of A003:

[0183] Intermediate F (436 mg, 1 mmol), 3-aminocyclobutanol (44 mg, 0.5 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (10 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (20 mL) and EA (20 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 10 / 1) to give compound A003 (220 mg, Y = 55%), a brown oil. LCMS: [M+1] + =797. 1 H NMR(300MHz, DMSO-d6)δ4.94-4.69(m,3H),3.56(t,J=6.0Hz,4H),3.35-3.31(m,4H),2.46-2.41( m,3H),2.40-2.29(m,6H),1.52-1.30(m,15H),1.23-1.15(m,46H),0.85(td,J=6.3,2.5Hz,12H).

[0184] Example 4. Synthesis of A004:

[0185] Intermediate E (422 mg, 1 mmol), 3-aminocyclohexanol (46 mg, 0.4 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (20 mL) and EA (20 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 10 / 1) to give compound A004 (134 mg, Y = 42%), a brown oil. LCMS: [M+1] + =797.

[0186] Example 5. Synthesis of A005

[0187] Intermediate E (422 mg, 1 mmol), 2-aminocyclohexanol (46 mg, 0.4 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (20 mL) and EA (20 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 10 / 1) to give compound A005 (161 mg, Y = 50%), a brown oil. LCMS: [M+1] + =797.

[0188] Example 6. Synthesis of A006:

[0189] Intermediate E (422 mg, 1 mmol), cis-4-aminocyclohexylmethanol (51 mg, 0.4 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (20 mL) and EA (20 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 10 / 1) to give compound A006 (150 mg, Y = 46%), a brown oil. LCMS: [M+1] + =811. 1 H NMR(300MHz, DMSO-d6)δ4.78(q,J=6.1Hz,2H),4.32(t,J=5.3Hz,1H),3.55-3.51(m, 4H),3.31-3.21(m,6H),2.49-2.32(m,9H),1.23-1.16(m,60H),0.96-0.70(m,12H).

[0190] Example 7. Synthesis of A007:

[0191] Intermediate E (422 mg, 1 mmol), trans-4-aminocyclohexylmethanol (51 mg, 0.4 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (20 mL) and EA (20 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 10 / 1) to give compound A007 (200 mg, Y = 61%), a brown oil. LCMS: [M+1] + =811.

[0192] Example 8. Synthesis of A008:

[0193] Intermediate G (654 mg, 1.5 mmol), 4-aminocyclohexanol (60 mg, 0.52 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (10 mL) and EA (10 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 10 / 1) to give compound A008 (263 mg, Y = 61%), a brown oil. LCMS: [M+1] + =825. 1 H NMR(300MHz,DMSO-d6)δ4.78(q,J=6.4Hz,2H),3.64-3.45(m,4H),3.35-3.31(m,4H) ,2.41-2.16(m,9H),1.80(t,J=8.7Hz,2H),1.52-0.92(m,63H),0.92-0.54(m,12H).

[0194] Example 9. Synthesis of A009:

[0195] Intermediate G (654 mg, 1.5 mmol), 3-aminocyclobutanol (60 mg, 0.52 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (249 mg, 1.5 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (10 mL) and EA (10 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE = 10 / 1) to give compound A009 (320 mg, Y = 77%), a brown oil. LCMS: [M+1] + =797. 1 H NMR (300MHz, DMSO-d6) δ4.89-4.75(m,3H),3.71-3.60(m,1H),3.57(t,J=5.9Hz,4H),3.37-3.29(m,4H ),2.46-2.39(m,2H),2.29-2.25(m,7H),1.52-1.38(m,12H),1.37-1.20(m,48H),0.93-0.76(m,12H).

[0196] Example 10. Synthesis of A010:

[0197] 4-Aminocyclohexane-1,2-cis-diol hydrochloride (66 mg, 0.39 mmol), intermediate A10-1 (422 mg, 1 mmol), K2CO3 (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H2O (5 mL) and EA (5 mL). The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude product was purified by silica gel column chromatography (EA / MeOH = 10 / 1) to give compound A010 (123 mg, Y = 38%) as a brown oil. LCMS: [M+1] + =813.

[0198] Example 11. Synthesis of A011:

[0199] 4-Aminocyclopentane-1,2-cis-diol hydrochloride amine I (50 mg, 0.32 mmol), intermediate E (420 mg, 1 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (10 mL) and EA (10 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / MeOH = 10 / 1) to give compound A011 (103 mg, Y = 40%), a brown oil. LCMS: [M+1] + =799. 1 H NMR (300MHz, DMSO-d6) δ4.79(p,J=6.2Hz,2H),4.29(br,2H),3.85(br,1H),3.67(br,1H),3.56(t,J=6.0H z,4H),3.31-3.21(m,4H),2.48-2.27(m,9H),1.50-1.38(m,16H),1.23-1.15(m,40H),0.99-0.75(m,12H).

[0200] Example 12. Synthesis of A012:

[0201] 4-Aminocyclohexane-1,2-cis-diol hydrochloride amine H (80 mg, 0.48 mmol), intermediate F (654 mg, 1.5 mmol), K₂CO₃ (400 mg, 2.87 mmol), and KI (166 mg, 1 mmol) were added to ACN (5 mL), and the mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was extracted with H₂O (10 mL) and EA (10 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated, and the crude product was purified by silica gel column chromatography (EA / MeOH = 10 / 1) to give compound A012 (220 mg, Y = 54%), a brown oil. LCMS: [M+1] + =841. 1 H NMR(300MHz,DMSO-d6)δ4.79(p,J=6.2Hz,2H),4.31-4.25(m,1H),3.57(t,J=5.9Hz,4H),3.31-3.30(m, 4H),2.46-2.44(m,4H),2.35-2.32(m,5H),1.58-1.37(m,16H),1.36-1.21(m,47H),0.95-0.77(m,12H).

[0202] Example 12. Synthesis of A013

[0203] Step 1.

[0204] Add A13-1 (2.41 g, 10 mmol), A13-2 (2.70 g, 12 mmol), EDCI (2.41 g, 12 mmol), DMAP (240 mg, 2 mmol), DIEA (5.16 g, 40 mmol), and dichloromethane (30 mL) to a 40 mL flask. Stir at room temperature for 16 hours. Monitor the reaction by TLC. After the reaction is complete, add ammonium chloride aqueous solution (200 mL), extract with ethyl acetate (100 mL x 2), combine the organic phases, wash with brine (50 mL), dry the resulting organic phase with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–10%) to obtain a pale yellow liquid A13-3 (2.3 g, 5 mmol), yield 50%.

[0205] Step 2.

[0206] Add A13-3 (1.15 g, 2.5 mmol), A13-4 (115 mg, 1 mmol), potassium carbonate (828 mg, 6 mmol), potassium iodide (166 mg, 1 mmol), and acetonitrile (14 mL) to a 40 mL flask. Stir at 80 °C for 16 hours, and monitor the reaction by TLC. After cooling the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to give a pale yellow liquid A013 (160 mg, 0.18 mmol), in 18% yield. MS: [M+1] + =856.80.

[0207] Example 14. Synthesis of A014:

[0208] Add A13-3 (1.15 g, 2.5 mmol), A14-1 (87 mg, 1 mmol), potassium carbonate (828 mg, 6 mmol), potassium iodide (166 mg, 1 mmol), and acetonitrile (14 mL) to a 40 mL flask. Stir at 80 °C for 16 hours, and monitor the reaction by TLC. After cooling the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to give a pale yellow liquid A014 (500 mg, 0.6 mmol), in 60% yield. MS: [M+1] + =828.70.

[0209] Example 15. Synthesis of A015

[0210] Step 1.

[0211] Add A15-1 (2.5 g, 12 mmol), A15-2 (3.47 g, 15 mmol), EDCI (2.88 g, 15 mmol), DMAP (290 mg, 2.4 mmol), DIEA (6.2 g, 48 mmol), and dichloromethane (25 mL) to a 40 mL flask. Stir at room temperature for 16 hours. Monitor the reaction by TLC. After the reaction is complete, add ammonium chloride aqueous solution (200 mL), extract with ethyl acetate (100 mL x 2), combine the organic phases, wash with brine (50 mL), dry the resulting organic phase with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–10%) to obtain pale yellow liquid A15-3 (3 g, 7.4 mmol), yield 61%.

[0212] Step 2.

[0213] Add A15-3 (1.01 g, 2.5 mmol), A15-4 (115 mg, 1 mmol), potassium carbonate (828 mg, 6 mmol), potassium iodide (166 mg, 1 mmol), and acetonitrile (10 mL) to a 40 mL flask. Stir at 80 °C for 16 hours, and monitor the reaction by TLC. After cooling the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to give a pale yellow liquid A015 (180 mg, 0.23 mmol), in 23% yield. MS: [M+1] + =769.

[0214] Example 16. Synthesis of A016

[0215] Add A15-3 (1.01 g, 2.5 mmol), A14-1 (87 mg, 1 mmol), potassium carbonate (828 mg, 6 mmol), potassium iodide (166 mg, 1 mmol), and acetonitrile (10 mL) to a 40 mL flask. Stir at 80 °C for 16 hours, and monitor the reaction by TLC. After cooling the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to give a pale yellow liquid A016 (460 mg, 0.62 mmol), in 62% yield. MS: [M+1] + =741.

[0216] Example 17. Synthesis of A017

[0217] Add A15-3 (500 mg, 1.2 mmol), A17-1 (50 mg, 0.5 mmol), potassium carbonate (414 mg, 3 mmol), potassium iodide (83 mg, 0.5 mmol), and acetonitrile (5 mL) to a 40 mL flask. Stir at 80 °C for 16 hours, and monitor the reaction by TLC. After cooling the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to give a pale yellow liquid A017 (140 mg, 0.18 mmol), in a yield of 36%. MS: [M+1] + =755.

[0218] Example 18. Synthesis of A018

[0219] Add A15-3 (500 mg, 1.2 mmol), A18-1 (50 mg, 0.5 mmol), potassium carbonate (414 mg, 3 mmol), potassium iodide (83 mg, 0.5 mmol), and acetonitrile (5 mL) to a 40 mL flask. Stir at 80 °C for 16 hours, and monitor the reaction by TLC. After cooling the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to give a pale yellow liquid A018 (300 mg, 0.4 mmol), in 80% yield. MS: [M+1] + =755.

[0220] Example 19. Synthesis of A019

[0221] Add A13-3 (473 mg, 1.05 mmol), A19-1 (83 mg, 0.5 mmol), potassium carbonate (414 mg, 3 mmol), potassium iodide (83 mg, 0.5 mmol), and acetonitrile (5 mL) to a 40 mL flask. Stir at 80 °C for 16 hours, and monitor the reaction by TLC. After cooling the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to give a colorless liquid A019 (103 mg, 0.12 mmol), yield 23%. MS: [M+1] + =871.

[0222] Example 20. Synthesis of A020

[0223] Add A15-3 (500 mg, 1.2 mmol), A20-1 (83 mg, 0.5 mmol), potassium carbonate (414 mg, 3 mmol), potassium iodide (83 mg, 0.5 mmol), and acetonitrile (5 mL) to a 40 mL flask. Stir at 80 °C for 16 hours, and monitor the reaction by TLC. After cooling the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to obtain colorless oil A020 ​​(75 mg, 0.1 mmol), yield 20%. MS: [M+1] + =783.

[0224] Example 21. Synthesis of A021

[0225] Add A15-3 (500 mg, 1.2 mmol), amine H (83 mg, 0.5 mmol), potassium carbonate (414 mg, 3 mmol), potassium iodide (83 mg, 0.5 mmol), and acetonitrile (5 mL) to a 40 mL flask. Stir at 80 °C for 16 hours, and monitor the reaction by TLC. After cooling the reaction solution to room temperature, filter, concentrate the filtrate under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to obtain colorless oil A021 (358 mg, 0.45 mmol), yield 90%. MS: [M+1] + =785.

[0226] Example 22. Synthesis of B001:

[0227] Step 1.

[0228] Compound B1-1 (10.00 g, 39.0 mmol) was added dropwise to a solution of compound B1-2 (6.80 g, 50.7 mmol), DCC (8.05 g, 39.0 mmol), and DMAP (5.24 g, 42.9 mmol) in DCM (140 mL) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered, and the filter cake was washed twice with DCM (25 mL). The crude product obtained by vacuum distillation of the filtrate was purified by silica gel column chromatography (EA / PE, 0-13%) to give compound B1-3 (10.2 g, 70%) as a colorless oil.

[0229] Step 2.

[0230] At room temperature, pyridinium chlorochromate (7.5 g, 34.9 mmol) was added to a DCM (100 mL) solution of compound B1-3 (10.0 g, 26.8 mmol). The resulting reaction solution was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was filtered, and the filter cake was washed twice with DCM (25 mL). The crude product obtained by vacuum distillation was purified by silica gel column chromatography (EA / PE, 0-10%) to give compound B1-4 (8.4 g, 85%) as a colorless oil.

[0231] Step 3.

[0232] AcOH (42 mg, 0.70 mmol) was added dropwise to a DCM solution (12 mL) of compound B1-4 (589 mg, 1.59 mmol), compound 5 (100 mg, 0.64 mmol), and STAB (337 mg, 1.59 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-4%) to give B001 (200 mg, 36%) as a colorless oil. LCMS: [M+H] + =867; 1 H NMR (300MHz, DMSO-d6) δ = 4.12-4.02 (m, 4H), 3.76-3.62 (m, 4H), 3.50-3.33 (m, 8H), 2.40-2. 25(m,7H),1.83-1.64(m,10H),1.51-1.40(m,14H),1.28-1.18(m,40H),0.91-0.81(m,12H).

[0233] Example 23. Synthesis of B002:

[0234] AcOH (76 mg, 1.26 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (1.06 g, 2.87 mmol), compound B2-1 (100 mg, 1.15 mmol), and STAB (608 mg, 2.87 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give B002 (210 mg, 23%) as a colorless oil. LCMS: [M+H] + =797;1 H NMR (300MHz, DMSO-d6) δ = 4.88 (s, 1H), 4.06 (t, J = 6.3, 4H), 3.43-3.32 (m, 8H), 2.39-2.20 (m,6H),1.78(p,J=6.2,6H),1.60-1.33(m,14H),1.28-1.16(m,40H),0.89-0.78(m,12H).

[0235] Example 24. Synthesis of B003:

[0236] AcOH (58 mg, 0.96 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (804 mg, 2.17 mmol), compound B3-1 (100 mg, 0.87 mmol), and STAB (460 mg, 2.17 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give B003 (65 mg, 9%) as a colorless oil. LCMS: [M+H] + =825; 1 H NMR (300MHz, DMSO-d6) δ = 4.12-4.04 (m, 4H), 3.50-3.33 (m, 9H), 3.22-3.12 (m, 1H), 2.49-2. 19(m,6H),1.94-1.67(m,10H),1.62-1.32(m,14H),1.27-1.17(m,40H),0.89-0.80(m,12H).

[0237] Example 25. Synthesis of B004:

[0238] AcOH (36 mg, 0.60 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (500 mg, 1.35 mmol), compound B4-1 (70 mg, 0.54 mmol), and STAB (287 mg, 1.35 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give B004 (240 mg, 53%) as a colorless oil. LCMS: [M+H]+ =839; 1 H NMR (300MHz, DMSO-d6) δ = 4.47 (s, 1H), 4.07 (t, J = 6.4, 4H), 3.42 (dt, J = 7.0, 3.9, 8H), 3.28-3.08 (m, 4H), 3.07-2.96 (m, 1H), 2.33 -2.24(m,2H),1.78(q,J=6.3,6H),1.68-1.54(m,4H),1.54-1.33(m,12H),1.31-1.15(m,42H),1.12(s,3H),0.89-0.81(m,12H).

[0239] Example 26. Synthesis of B005

[0240] Step 1.

[0241] Add B5-1 (1.9 g, 7.4 mmol), B5-2 (2.23 g, 14.8 mmol), DCC (3.07 g, 14.8 mmol), DMAP (0.9 g, 25 mmol), and 60 mL of dichloromethane to a 250 mL reaction flask and stir at room temperature for 2 hours. Monitor the reaction by TLC until the starting material is completely converted. Filter the reaction mixture and collect the filtrate. The crude product obtained after evaporating the filtrate to dryness is purified by silica gel (PE / EA = 0-20%) to give compound B5-3 (1.9 g, 66%) as a colorless liquid.

[0242] Step 2.

[0243] At room temperature, pyridinium chlorochromate (1.33 g, 6.4 mmol) was added to a solution of compound B5-3 (1.9 g, 4.9 mmol) in dichloromethane (30 mL). The reaction mixture was stirred overnight at room temperature. TLC showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was collected. The crude product after evaporation was purified by silica gel (PE / EA = 0-15%) to give compound B5-4 (1.3 g, 69%) as a colorless liquid.

[0244] Step 3.

[0245] Compounds B5-4 (300 mg, 0.78 mmol), B5-5 (50 mg, 0.35 mmol), acetic acid (20 mg, 0.35 mmol), sodium borohydride acetate (164 mg, 0.78 mmol), and dichloromethane (30 mL) were added to a reaction flask and stirred overnight at room temperature. The reaction was monitored by TLC, and the starting material conversion was complete. The crude product after rotary evaporation was purified by silica gel (PE / EA = 0-50%) to give compound B005 (85 mg, 28%) as a colorless liquid. LCMS: [M+H]+ =857; 1 HNMR(300MHz,DMSO-d6)δ4.08(t,4H),3.51(s,1H),2.33-2.23(m,3H),1.88-1 .76(m,6H),1.39(m,3H),1.34(m,3H),1.30-1.15(m,52H),0.89-0.83(m,12H).

[0246] Example 27. Synthesis of B006

[0247] Step 1.

[0248] Compounds B6-2 (533 mg, 2.93 mmol), DCC (402 mg, 1.953 mmol), and DMAP (262 mg, 2.148 mmol) were added to a 20 mL solution of compound B6-1 (500 mg, 1.953 mmol) in dichloromethane at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give B6-3 (644 mg, 89.7%) as a pale yellow oil. LCMS: [M+H] + =421

[0249] Step 2.

[0250] IBX (550 mg, 1.96 mmol) was added to a DMSO (6 mL) solution of B6-3 (550 mg, 1.31 mmol) at room temperature. The resulting mixture was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give B6-4 (455 mg, 83.2%) as a pale yellow oil. LCMS: [M+H] + =419

[0251] Step 3.

[0252] Compound B6-5 (50 mg, 0.435 mmol), sodium borohydride acetate (231 mg, 1.087 mmol), and acetic acid (29 mg, 0.478 mmol) were added to a DCM (10 mL) solution of B6-4 (454 mg, 1.087 mmol) at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B006 (210 mg, 52.5%) as a pale yellow oil; LCMS: [M+H] + =921.

[0253] Example 28. Synthesis of B007:

[0254] AcOH (41 mg, 0.70 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (574 mg, 1.55 mmol), compound B7-1 (103 mg, 0.62 mmol), and STAB (459 mg, 2.17 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give B007 (140 mg, 27%) as a colorless oil. LCMS: [M+H] + =839; 1 H NMR (300MHz, DMSO-d6) δ = 4.46 (s, 1H), 4.11-4.06 (m, 4H), 3.56 (t, J = 6.3, 1H), 3.42 (t, J = 6.1, 8H), 3.24-2.8 9(m,6H),2.33-2.25(m,2H),1.92-1.74(m,8H),1.57-1.34(m,11H),1.31-1.18(m,46H),0.89-0.81(m,12H).

[0255] Example 29. Synthesis of B008:

[0256] AcOH (41 mg, 0.70 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (574 mg, 1.55 mmol), compound B8-1 (103 mg, 0.62 mmol), and STAB (459 mg, 2.17 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give B008 (220 mg, 42%) as a colorless oil. LCMS: [M+H] + =839; 1 H NMR (300MHz, DMSO-d6) δ = 4.44 (s, 1H), 4.07 (dt, J = 6.4, 4H), 3.83 (s, 2H), 3.41 (t, J = 6.1, 8H), 2.91 (q, J = 7. 7,1H),2.35-2.17(m,4H),1.78(h,J=6.4,8H),1.58-1.33(m,17H),1.30-1.16(m,42H),0.90-0.79(m,12H).

[0257] Example 30. Synthesis of B009:

[0258] AcOH (46 mg, 0.77 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (644 mg, 1.74 mmol), compound B9-1 (80 mg, 0.70 mmol), and STAB (515 mg, 2.43 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give B009 (150 mg, 26%) as a colorless oil. LCMS: [M+H] + =825; 1H NMR (300MHz, DMSO-d6) δ = 4.72 (s, 1H), 4.08 (t, J = 6.3, 4H), 3.56 (t, J = 6.3, 1H), 3.42 (t, J = 6.1, 8H), 3.24-2.80 (m ,5H),2.28(tt,J=8.7,5.4,2H),1.97-1.74(m,8H),1.66-1.33(m,14H),1.30-1.17(m,42H),0.90-0.80(m,12H).

[0259] Example 31. Synthesis of B010:

[0260] AcOH (46 mg, 0.77 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (644 mg, 1.74 mmol), compound B10-1 (80 mg, 0.70 mmol), and STAB (515 mg, 2.43 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give B010 (400 mg, 70%) as a colorless oil. LCMS: [M+H] + =825; 1 H NMR (300MHz, DMSO-d6) δ = 4.08 (t, J = 6.4, 4H), 3.66 (s, 1H), 3.44 (q, J = 7.6, 6.8, 8H), 3.29-2.76 (m, 5H), 2.29 (dt, J =9.7,5.7,2H),1.98-1.91(m,4H),1.87-1.59(m,8H),1.57-1.29(m,12H),1.28-1.18(m,40H),0.90-0.81(m,12H).

[0261] Example 32. Synthesis of B011:

[0262] AcOH (39 mg, 0.65 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (550 mg, 1.48 mmol), compound B11-1 (60 mg, 0.59 mmol), and STAB (440 mg, 2.08 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give B011 (180 mg, 38%) as a colorless oil. LCMS: [M+H] + =811; 1 H NMR (300MHz, DMSO-d6) δ=4.83-4.70(m,1H),4.07(t,J=6.5,4H),3.77(t,J=9.8,8.1,1H),3.56(t,J=6.3,1H),3.45-3.40(m,8H) ,3.18-2.94(m,4H),2.27(tt,J=9.0,4.3,2H),2.02-1.61(m,12H),1.60-1.31(m,10H),1.30-1.11(m,40H),0.93-0.79(m,12H).

[0263] Example 33. Synthesis of B012:

[0264] AcOH (39 mg, 0.65 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (550 mg, 1.48 mmol), compound B12-1 (60 mg, 0.59 mmol), and STAB (440 mg, 2.08 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give B012 (170 mg, 35%) as a colorless oil. LCMS: [M+H] + =811; 1H NMR (300MHz, DMSO-d6) δ = 4.06 (t, J = 6.9, 4H), 3.46-3.37 (m, 8H), 2.92 (q, J = 7.2, 2H), 2.57-2.51 (m, 4H) ),2.33-2.24(m,2H),1.94-1.59(m,10H),1.58-1.33(m,12H),1.28-1.17(m,40H),0.89-0.81(m,12H).

[0265] Example 34. Synthesis of B013

[0266] Step 1.

[0267] Add B13-1 (500 mg, 2.5 mmol), B13-2 (335 mg, 2.5 mmol), DCC (560 mg, 2.7 mmol), DMAP (332 mg, 2.7 mmol), and 60 mL of dichloromethane to a 100 mL reaction flask and stir at room temperature for 2 hours. Monitor the reaction by TLC until the starting material is completely converted. Filter the reaction solution and collect the filtrate. The crude product obtained after evaporating the filtrate to dryness is purified by silica gel (PE / EA = 0-20%) to give a colorless liquid compound B13-3 (400 mg, 50.6%).

[0268] Step 2.

[0269] At room temperature, pyridinium chlorochromate (288 mg, 1.4 mmol) was added to a solution of compound B13-3 (400 mg, 1.26 mmol) in dichloromethane (30 mL). The reaction mixture was stirred overnight at room temperature. TLC showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was collected. The crude product after evaporation was purified by silica gel (PE / EA = 0-15%) to give compound B13-4 (360 mg, 90.6%) as a colorless liquid.

[0270] Step 3.

[0271] Compounds B13-4 (150 mg, 0.447 mmol), B13-5 (224 mg, 0.477 mmol), one drop of acetic acid, sodium borohydride acetate (177 mg, 0.84 mmol), and dichloromethane (30 mL) were added to a reaction flask and stirred overnight at room temperature. The reaction was monitored by TLC, and the starting material conversion was complete. The crude product after rotary evaporation was purified by silica gel (MeOH / DCM = 0-50%) to give a colorless liquid compound B013 (160 mg, 43.7%). LCMS: [M+H]+=769; 1H NMR(300MHz,DMSO-d6)δ4.59(m,1H),4.19-3.95(m,4H),3.51(s,1H),3.40(s,8H) ),3.17(m,4H),2.27(m,4H),1.90-1.68(m,8H),1.24(m,50H),0.93-0.81(m,9H)

[0272] Example 35. Synthesis of B014:

[0273] Step 1.

[0274] In a 250 mL reaction flask, add B14-1 (500 mg, 2.7 mmol), B14-2 (473 mg, 3.53 mmol), DCC (560 mg, 2.7 mmol), DMAP (332 g, 2.7 mmol), and 60 mL of dichloromethane. Stir at room temperature for 2 hours. Monitor the reaction by TLC until the starting material is completely converted. Filter the reaction mixture and collect the filtrate. The crude product obtained after evaporating the filtrate to dryness is purified by silica gel (PE / EA = 0-20%) to give compound B14-3 (500 mg, 61%) as a colorless liquid.

[0275] Step 2.

[0276] At room temperature, pyridinium chlorochromate (379 mg, 1.83 mmol) was added to a solution of compound B14-3 (500 mg, 1.66 mmol) in dichloromethane (30 mL). The reaction mixture was stirred overnight at room temperature. TLC showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was collected. The crude product after evaporation was purified by silica gel (PE / EA = 0-15%) to give compound B14-4 (400 mg, 80.5%) as a colorless liquid.

[0277] Step 3.

[0278] Compounds B14-4 (100 mg, 0.335 mmol), B13-5 (157 mg, 0.335 mmol), one drop of acetic acid, sodium borohydride acetate (177 mg, 0.84 mmol), and dichloromethane (30 mL) were added to a reaction flask and stirred overnight at room temperature. The reaction was monitored by TLC until the starting material was completely converted. The crude product after rotary evaporation was purified by silica gel (PE / EA = 0-50%) to give compound B014 (100 mg, 39.7%) as a colorless liquid. LCMS: [M+H] + =753

[0279] Example 36. Synthesis of B015:

[0280] Step 1.

[0281] Compound B15-2 (393 mg, 2.93 mmol), EDCI.HCl (375 mg, 1.953 mmol), and DMAP (48 mg, 0.391 mmol) were added to a solution of compound B15-1 (500 mg, 1.953 mmol) in dichloromethane (10 mL) at room temperature. The resulting mixture was stirred at room temperature for 6 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with dichloromethane, and the organic phase was washed with 1N HCl aqueous solution and saturated brine. The solution was dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give B15-3 (528 mg, 72.7%) as a pale yellow oil. LCMS: [M+H] + =373

[0282] Step 2.

[0283] IBX (539 mg, 1.923 mmol) was added to a DMSO (10 mL) solution of B15-3 (477 mg, 1.282 mmol) at room temperature. The resulting mixture was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give B15-4 (352 mg, 74.3%) as a pale yellow oil. LCMS: [M+H] + =371

[0284] Step 3.

[0285] Compound B13-5 (304 mg, 0.649 mmol), sodium borohydride acetate (172 mg, 0.811 mmol), and acetic acid (32 mg, 0.541 mmol) were added to a solution of B15-4 (200 mg, 0.541 mmol) in 10 mL of DCM at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B015 (183 mg, 41.1%) as a pale yellow oil. LCMS: [M+H] + =825; 1 H NMR (300MHz, DMSO-d6) δ4.05(td,J=6.4,3.1Hz,1H),3.85(s,4H),3.37(t,J=6.0Hz,1H),3.16(s,8H),2.01(s,2H),1. 77(t,J=6.3Hz,1H),1.57(s,6H),1.33(s,15H),1.23(d,J=3.8Hz,10H),1.03(s,38H),0.90-0.80(m,3H),0.64(s,8H).

[0286] Example 37. Synthesis of B016:

[0287] Step 1.

[0288] Add B16-1 (500 mg, 2.5 mmol), B16-2 (372 mg, 2.77 mmol), DCC (560 mg, 2.7 mmol), DMAP (332 mg, 2.7 mmol), and 60 mL of dichloromethane to a 100 mL reaction flask and stir at room temperature for 2 hours. Monitor the reaction by TLC until the starting material is completely converted. Filter the reaction mixture and collect the filtrate. The crude product obtained after evaporating the filtrate to dryness is purified by silica gel (PE / EA = 0-20%) to give compound B16-3 (400 mg, 50.4%) as a colorless liquid.

[0289] Step 2.

[0290] At room temperature, PPh3 (334 mg, 1.27 mmol) and CBr4 (844 mg, 2.54 mmol) were added to a solution of compound B16-3 (400 mg, 1.27 mmol) in dichloromethane (30 mL). The reaction mixture was stirred overnight at room temperature. TLC showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was collected. The crude product after evaporation was purified by silica gel (PE / EA = 0-15%) to give compound B16-4 (400 mg, 83.3%) as a colorless liquid.

[0291] Step 3.

[0292] Compounds B16-4 (150 mg, 0.4 mmol), B13-5 (224 mg, 0.48 mmol), potassium carbonate (165 mg, 0.48 mmol), potassium iodide (66 mg, 0.4 mmol), and acetonitrile (30 mL) were added to a reaction flask and stirred overnight at 80 °C. The reaction was monitored by TLC, and the starting material conversion was complete. The crude product after rotary evaporation was purified by silica gel (methanol / dichloromethane = 0-10%) to give compound B016 (190 mg, 62%) as a colorless liquid. LCMS: [M+H] + =767; 1 HNMR(300MHz,DMSO-d6)δ4.59(m,1H),4.06(q,4H),3.81(s,1H),3.17(m,8H),2.9 2(s,4H),2.38-2.08(m,3H),1.99-1.63(m,16H),1.54-0.99(m,41H),0.85(t,9H).

[0293] Example 38. Synthesis of B017:

[0294] AcOH (35 mg, 0.59 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (494 mg, 1.33 mmol), compound B17-1 (82 mg, 0.53 mmol), and STAB (396 mg, 1.87 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give B017 (110 mg, 25%) as a colorless oil. LCMS: [M+H] + =827; 1H NMR (300MHz, DMSO-d6) δ = 4.54 (s, 2H), 4.07 (t, 4H), 3.95 (s, 2H), 3.74 (s, 1H), 3.43-3.39 (m, 8H), 3.06-2.60 ( m,4H),2.32-2.25(m,2H),1.79(p,J=6.3,10H),1.56-1.29(m,10H),1.27-1.17(m,40H),0.91-0.79(m,12H).

[0295] Example 39. Synthesis of B018:

[0296] AcOH (39 mg, 0.66 mmol) was added dropwise to a DCM solution (12 mL) of compound B1-4 (553 mg, 1.49 mmol), compound B18-1 (100 mg, 0.60 mmol), and STAB (443 mg, 2.09 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give B018 (75 mg, 15%) as a colorless oil. LCMS: [M+H] + =841; 1 H NMR (300MHz, DMSO-d6) δ = 4.04 (t, J = 6.7, 4H), 3.56 (t, J = 6.3, 2H), 3.45-3.39 (m, 8H), 2.93 (q, J = 7.3, 1H), 2.58-2.51 (m, 4 H),2.33-2.24(m,2H),1.85-1.72(m,4H),1.68-1.59(m,2H),1.58-1.30(m,14H),1.29-1.18(m,42H),0.89-0.79(m,12H).

[0297] Example 40. Synthesis of B019:

[0298] AcOH (40 mg, 0.67 mmol) was added dropwise to a DCM (12 mL) solution of compound B01-4 (563 mg, 1.52 mmol), compound B19-1 (70 mg, 0.61 mmol), and STAB (451 mg, 2.13 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give B019 (180 mg, 36%) as a colorless oil. LCMS: [M+H] + =825; 1 H NMR (300MHz, DMSO-d6) δ = 4.07 (dt, J = 11.1, 4.7, 4H), 3.57 (dt, J = 6.4, 1H), 3.41 (t, J = 6.1, 8H), 3.21-2.85 ( m,5H),2.32-2.24(m,2H),1.95-1.58(m,12H),1.56-1.34(m,12H),1.29-1.19(m,40H),0.90-0.80(m,12H).

[0299] Example 41. Synthesis of B020:

[0300] AcOH (40 mg, 0.67 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (563 mg, 1.52 mmol), compound B20-1 (70 mg, 0.61 mmol), and STAB (451 mg, 2.13 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give BO20 (140 mg, 28%) as a colorless oil. LCMS: [M+H] + =825; 1H NMR (300MHz, DMSO-d6) δ = 4.55 (s, 1H), 4.08 (dt, J = 6.4, 4H), 3.86-3.77 (m, 1H), 3.43 (td, J = 6.0, 3.4, 8H), 3.27-2.90 (m, 5H), 2 .28(tt,J=8.7,5.4,2H),2.05-1.91(m,4H),1.87-1.67(m,10H),1.55-1.34(m,10H),1.30-1.15(m,40H),0.93-0.78(m,12H).

[0301] Example 42. Synthesis of B021:

[0302] Step 1.

[0303] At room temperature, compound B21-2 (411 mg, 2.539 mmol), DCC (402 mg, 1.953 mmol), and DMAP (262 mg, 2.148 mmol) were added to a solution of compound B21-1 (500 mg, 1.953 mmol) in dichloromethane (20 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with dichloromethane, and the organic phase was washed with 1N HCl aqueous solution and saturated brine. The solution was dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give B21-3 (355 mg, 45.5%) as a pale yellow oil. LCMS: [M+H] + =401

[0304] Step 2.

[0305] IBX (315 mg, 1.125 mmol) was added to a DMSO (6 mL) solution of B21-3 (300 mg, 0.75 mmol) at room temperature. The resulting mixture was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give B21-4 (260 mg, 87.1%) as a pale yellow oil. LCMS: [M+H] + =399

[0306] Step 3.

[0307] Compound B21-5 (30 mg, 0.26 mmol), sodium borohydride acetate (193 mg, 0.91 mmol), and acetic acid (23 mg, 0.39 mmol) were added to a solution of B21-4 (258 mg, 0.65 mmol) in 10 mL of DCM at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B021 (76 mg, 33.2%) as a pale yellow oil. LCMS: [M+H] + =881; 1 H NMR (300MHz, DMSO-d6) δ4.05(t,J=6.5Hz,4H),3.38(t,J=6.7Hz,9H),2.42(s,3H),2.26(t,J=7 .3Hz,4H),1.80(s,8H),1.58-1.45(m,8H),1.29(s,16H),1.24(s,46H),0.85(t,J=6.4Hz,7H).

[0308] Example 43. Synthesis of B022 / B023:

[0309] Step 1.

[0310] AcOH (44 mg, 0.73 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (614 mg, 1.66 mmol), compound B22-1 (150 mg, 0.66 mmol), and STAB (492 mg, 2.32 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-3%) to give compound B22-2 (380 mg, 61%) as a colorless oil. LCMS: [M+H] + =936

[0311] Step 2.

[0312] At room temperature, trifluoroacetic acid (1 mL) was added to a solution of compound B22-2 (380 mg, 0.41 mmol) in dichloromethane (4 mL). The reaction mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete, and the reaction mixture was concentrated under vacuum to give a brownish-yellow oil. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-10%) to give a colorless oil of B022 (310 mg, 91%). LCMS: [M+H] + =836; 1 H NMR (300MHz, DMSO-d6) δ = 4.08 (dt, J = 6.2, 4H), 3.50–3.44 (m, 4H) 3.41 (t, J = 6.1, 8H), 3.01 (s, 3H), 2.32–2.23(m,2H),1.96–1.68(m,12H),1.56–1.35(m,8H),1.29–1.18(m,44H),0.92–0.77(m,12H).

[0313] Step 3.

[0314] AcOH (15 mg, 0.25 mmol) was added dropwise to a DCM solution of BO22 (190 mg, 2.17 mmol), paraformaldehyde (10 mg, 0.34 mmol), and STAB (121 mg, 0.57 mmol) in 6 mL at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (30 mL) was added and the mixture was extracted with DCM (40 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to obtain BO23 (150 mg, 78%) as a colorless oil. LCMS: [M+H] + =850

[0315] Example 44. Synthesis of B024:

[0316] Step 1.

[0317] At room temperature, compound B24-2 (739 mg, 5.36 mmol), EDCI HCl (1.03 g, 5.36 mmol), DIEA (1.8 mL, 10.71 mmol), and DMAP (87 mg, 0.714 mmol) were added to a solution of compound B24-1 (1 g, 3.57 mmol) in dichloromethane (20 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with dichloromethane, and the organic phase was washed with 1N HCl aqueous solution and saturated brine. The solution was dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 8:1) to give B24-3 (860 mg, 60.1%) as a pale yellow oil. LCMS: [M+H] + =401 / 403

[0318] Step 2.

[0319] Compound B13-5 (263 mg, 0.561 mmol), potassium carbonate (232 mg, 1.683 mmol), and potassium iodide (112 mg, 0.673 mmol) were added to a DCM (10 mL) solution of B24-3 (269 mg, 0.673 mmol) at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B024 (220 mg, 49.7%) as a pale yellow oil. LCMS: [M+H] + =791

[0320] Example 45. Synthesis of B025

[0321] Step 1.

[0322] Compound B25-2 (882 mg, 6.579 mmol), EDCI HCl (842 mg, 4.386 mmol), and DMAP (107 mg, 0.877 mmol) were added to a solution of compound B25-1 (1 g, 4.386 mmol) in dichloromethane (20 mL) at room temperature. The resulting mixture was stirred at room temperature for 6 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with dichloromethane, and the organic phase was washed with 1N HCl aqueous solution and saturated brine. The solution was dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give a pale yellow oily substance of B25-3 (1.17 g, 78%). LCMS: [M+H] + =345

[0323] Step 2.

[0324] IBX (733 mg, 2.616 mmol) was added to a DMSO (6 mL) solution of B25-3 (600 mg, 1.744 mmol) at room temperature. The resulting mixture was stirred at room temperature for 3 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give a pale yellow oily substance of B25-4 (510 mg, 85.6%). LCMS: [M+H] + =343

[0325] Step 3.

[0326] Compound B13-5 (200 mg, 0.426 mmol), sodium borohydride acetate (136 mg, 0.64 mmol), and acetic acid (26 mg, 0.426 mmol) were added to a solution of B25-4 (219 mg, 0.64 mmol) in 10 mL of DCM at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B025 (232 mg, 68.4%) as a pale yellow oil. LCMS: [M+H] + =797; 1H NMR(300MHz,DMSO-d6)δ4.06(td,J=6.4,3.5Hz,1H),3.49-3.39(m,72H),2.73(t,J=1.9Hz,1H),1.7 9(p,J=6.5Hz,2H),1.54-1.48(m,2H),1.56-1.36(m,1H),1.23(d,J=3.8Hz,12H),0.90-0.80(m,2H).

[0327] Example 46. Synthesis of B026:

[0328] Compound B26-1 (35 mg, 0.304 mmol), sodium borohydride acetate (226 mg, 1.064 mmol), and acetic acid (27 mg, 0.456 mmol) were added to a solution of B25-4 (260 mg, 0.76 mmol) in 10 mL of DCM at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B026 (50 mg, 21.5%) as a pale yellow oil. LCMS: [M+H] + =769

[0329] Example 47. Synthesis of B027:

[0330] Compound B27-1 (20 mg, 0.174 mmol), sodium borohydride acetate (129 mg, 0.609 mmol), and acetic acid (16 mg, 0.261 mmol) were added to an 8 mL solution of B15-4 (161 mg, 0.435 mmol) in DCM at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B027 (110 mg, 76.9%) as a pale yellow oil. LCMS: [M+H] + =825; 1H NMR (300MHz, DMSO-d6) δ4.05(t,J=6.4Hz,2H),3.94(s,3H),3.44-3.32(m,4H),3.28-3.11(m,6H),2.41-2.21(m,6H),2.09(s ,3H),1.74(dt,J=24.5,6.2Hz,1H),1.47(d,J=22.6Hz,6H),1.23(s,21H),1.13(s,38H),0.86(d,J=5.9Hz,2H),0.74(s,5H).

[0331] Example 48. Synthesis of B028:

[0332] AcOH (41 mg, 0.68 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (572 mg, 1.54 mmol), compound B28-1 (60 mg, 0.62 mmol), and STAB (458 mg, 2.16 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give B028 (160 mg, 32%) as a colorless oil. LCMS: [M+H] + =807; 1 H NMR (300MHz, DMSO-d6) δ = 7.72 (s, 1H), 7.19 (s, 1H), 4.06 (dt, J = 6.4, 4H), 3.96 (s, 2H), 3.43-3.37 (m, 8H), 2.88-2.75 (m, 4 H),2.33-2.22(m,2H),1.76(q,J=6.4,4H),1.56-1.36(m,8H),1.31-1.13(m,40H),1.06-0.97(m,4H),0.92-0.77(m,12H).

[0333] Example 49. Synthesis of B029:

[0334] AcOH (42 mg, 0.69 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (583 mg, 1.57 mmol), compound B29-1 (70 mg, 0.63 mmol), and STAB (467 mg, 2.20 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give B029 (130 mg, 25%) as a colorless oil. LCMS: [M+H] + =821; 1 H NMR (300MHz, DMSO-d6) δ = 7.53 (s, 1H), 6.74 (s, 1H), 4.04 (dt, J = 6.7, 2.1, 4H), 3.59 (s, 3H), 3.46 (s, 2H), 3.32-3.27 (m, 8H) ,2.39(t,J=7.0,4H),2.30-2.24(m,2H),1.74(p,J=6.3,4H),1.52-1.33(m,12H),1.28-1.17(m,40H),0.87-0.79(m,12H).

[0335] Example 50. Synthesis of B030:

[0336] AcOH (42 mg, 0.69 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (583 mg, 1.57 mmol), compound B30-1 (70 mg, 0.63 mmol), and STAB (467 mg, 2.20 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give BO30 (240 mg, 47%) as a colorless oil. LCMS: [M+H] + =821

[0337] Example 51. Synthesis of B031:

[0338] Step 1.

[0339] At room temperature, pyridinium chlorochromate (2.9 g, 13.5 mmol) was added to a solution of compound B31-1 (3 g, 11.3 mmol) in dichloromethane (80 mL). The reaction mixture was stirred overnight at room temperature. TLC showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was collected. The crude product was evaporated to dryness and purified by silica gel (PE / EA = 0-15%) to obtain product B31-2 (2.8 g, 98.5%) as a colorless liquid.

[0340] Step 2.

[0341] Compounds B31-2 (2.8 g, 10.6 mmol), B31-3 (1.5 g, 12.7 mmol), acetic acid (0.6 g, 10.6 mmol), sodium borohydride acetate (4.5 g, 21.18 mmol), and dichloromethane (100 mL) were added to a reaction flask and stirred overnight at room temperature. The reaction was monitored by TLC, and the starting material conversion was complete. The crude product after rotary evaporation was passed through silica gel (MeOH / DCM = 0-20%) to give compound B31-4 (1.5 g, 39%) as a colorless liquid. LCMS: [M+H] + =613

[0342] Step 3.

[0343] Compounds B31-4 (1 g, 2.75 mmol), B1-4 (1 g, 2.75 mmol), acetic acid (166 mg, 2.75 mmol), sodium borohydride acetate (870 mg, 4.1 mmol), and dichloromethane (30 mL) were added to a reaction flask and stirred overnight at room temperature. The reaction was monitored by TLC, and the starting material conversion was complete. The crude product after rotary evaporation was purified by silica gel (MeOH / DCM = 0-10%) to give compound B031 (500 mg, 28%) as a colorless liquid. LCMS: [M+H] + =719

[0344] Example 52. Synthesis of B032:

[0345] Compound B32-1 (60 mg, 0.522 mmol), sodium borohydride acetate (387 mg, 1.827 mmol), and acetic acid (47 mg, 0.783 mmol) were added to a 10 mL solution of B21-4 (519 mg, 1.305 mmol) in DCM at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B032 (356 mg, 77.6%) as a pale yellow oil. LCMS: [M+H] + =881

[0346] Example 53. Synthesis of B033

[0347] Step 1.

[0348] Add B33-1 (2 g, 10 mmol), B33-2 (1.6 g, 12 mmol), EDCI (2.1 g, 11 mmol), DMAP (250 mg, 2 mmol), DIEA (5 g, 40 mmol), and dichloromethane (20 mL) to a 40 mL flask. Stir at room temperature for 16 hours. Monitor the reaction by TLC. After the reaction is complete, add ammonium chloride aqueous solution (200 mL), extract with ethyl acetate (100 mL x 2), combine the organic phases, wash with brine (50 mL), dry the resulting organic phase with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–10%) to obtain pale yellow liquid B33-3 (2 g, 6.3 mmol), yield 63%. MS: [M+1] + =317.

[0349] Step 2.

[0350] Add B33-3 (2 g, 6.3 mmol), PCC (1.36 g, 6.3 mmol), and dichloromethane (20 mL) to a 40 mL flask. Stir at room temperature for 16 hours, monitoring the reaction by TLC. After cooling to room temperature, filter the solution. Concentrate the filtrate under reduced pressure to obtain the crude product, which is then purified by silica gel column chromatography (ethyl acetate, petroleum ether = 0–10%) to give a pale yellow liquid, B33-4 (1.3 g, 4 mmol), in 65% yield. MS: [M+1] + =315;

[0351] Step 3.

[0352] Add B33-4 (500 mg, 1.6 mmol), B33-5 (90 mg, 0.78 mmol), STAB (850 mg, 4 mmol), and dichloromethane (10 mL) to a 40 mL flask. Stir at room temperature for 16 hours, and monitor the reaction by TLC. Concentrate the reaction solution under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to give a pale yellow liquid B033 (300 mg, 0.42 mmol), in 54% yield. MS: [M+1] + =713;

[0353] Example 54. Synthesis of B034

[0354] Step 1.

[0355] Compound B34-2 (566 mg, 4.22 mmol), EDCI HCl (676 mg, 3.52 mmol), and DMAP (86 mg, 0.704 mmol) were added to a solution of compound B34-1 (1 g, 3.52 mmol) in dichloromethane (20 mL) at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with dichloromethane, and the organic phase was washed with 1N HCl aqueous solution and saturated brine. The solution was dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give B34-3 (774 mg, 55.3%) as a pale yellow oil. LCMS: [M+H] + =401

[0356] Step 2.

[0357] IBX (813 mg, 2.903 mmol) was added to an 8 mL solution of B34-3 (774 mg, 1.935 mmol) in DMSO at room temperature. The resulting mixture was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 8:1) to give B34-4 (593 mg, 77%) as a pale yellow oil. LCMS: [M+H] + =399

[0358] Step 3.

[0359] Compound B34-5 (30 mg, 0.261 mmol), sodium borohydride acetate (138 mg, 0.652 mmol), and acetic acid (23 mg, 0.391 mmol) were added to a DCM (10 mL) solution of B34-4 (260 mg, 0.652 mmol) at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B034 (102 mg, 44.4%) as a pale yellow oil. LCMS: [M+H] + =881

[0360] Example 55. Synthesis of B035:

[0361] Step 1.

[0362] Compound B35-2 (553 mg, 4.13 mmol), EDCI HCl (660 mg, 3.44 mmol), and DMAP (84 mg, 0.688 mmol) were added to a 20 mL solution of compound B35-1 (784 mg, 3.44 mmol) in dichloromethane at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with dichloromethane, and the organic phase was washed with 1N HCl aqueous solution and saturated brine. The solution was dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give a pale yellow oily substance of B35-3 (671 mg, 56.9%). LCMS: [M+H] + =345

[0363] Step 2.

[0364] IBX (820 mg, 2.93 mmol) was added to an 8 mL solution of B35-3 (671 mg, 1.95 mmol) in DMSO at room temperature. The resulting mixture was stirred at room temperature for 3 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product was purified by normal-phase silica gel column chromatography (PE / EA = 8:1) to give a pale yellow oily substance of B35-4 (600 mg, 89.9%). LCMS: [M+H] + =342.

[0365] Step 3.

[0366] Compound B35-5 (40 mg, 0.348 mmol), sodium borohydride acetate (184 mg, 0.87 mmol), and acetic acid (31 mg, 0.522 mmol) were added to a DCM (10 mL) solution of B35-4 (297 mg, 0.87 mmol) at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B035 (81 mg, 30.3%) as a pale yellow oil. LCMS: [M+H] + =769

[0367] Example 56. Synthesis of B036:

[0368] Step 1.

[0369] Compound B36-2 (786 mg, 5.87 mmol), EDCI HCl (751 mg, 3.91 mmol), and DMAP (95 mg, 0.782 mmol) were added to a solution of compound B36-1 (1 g, 3.91 mmol) in dichloromethane (20 mL) at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with dichloromethane, and the organic phase was washed with 1N HCl aqueous solution and saturated brine. The solution was dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give a pale yellow oily substance of B36-3 (1.02 g, 70.3%). LCMS: [M+H] + =373

[0370] Step 2.

[0371] IBX (1.15 g, 4.11 mmol) was added to an 8 mL solution of B36-3 (1.02 g, 2.74 mmol) in DMSO at room temperature. The resulting mixture was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 8:1) to give B36-4 (740 mg, 73.3%) as a pale yellow oil. LCMS: [M+H] + =371

[0372] Step 3.

[0373] Compound B36-5 (45 mg, 0.391 mmol), sodium borohydride acetate (207 mg, 0.978 mmol), and acetic acid (35 mg, 0.587 mmol) were added to a DCM (10 mL) solution of B62-4 (362 mg, 0.978 mmol) at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B036 (91 mg, 28.3%) as a pale yellow oil. LCMS: [M+H] + =825;

[0374] Example 57. Synthesis of B037:

[0375] Step 1.

[0376] At room temperature, pyridinium chlorochromate (2.9 g, 13.5 mmol) was added to a solution of compound B37-1 (3 g, 11.3 mmol) in dichloromethane (80 mL). The reaction mixture was stirred overnight at room temperature. TLC showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was collected. The crude product was evaporated to dryness and purified by silica gel (PE / EA = 0-15%) to obtain product B37-2 (2.8 g, 98.5%) as a colorless liquid.

[0377] Step 2.

[0378] Compounds B37-2 (2.8 g, 10.6 mmol), B37-3 (1.5 g, 12.7 mmol), acetic acid (0.6 g, 10.6 mmol), sodium borohydride acetate (4.5 g, 21.18 mmol), and dichloromethane (100 mL) were added to a reaction flask and stirred overnight at room temperature. The reaction was monitored by TLC, and the starting material conversion was complete. The crude product after rotary evaporation was passed through silica gel (MeOH / DCM = 0-20%) to give compound B37-4 (2.5 g, 65%) as a colorless liquid.

[0379] Step 3.

[0380] Compounds B1-4 (0.5 g, 1.38 mmol), B37-5 (0.5 g, 1.38 mmol), acetic acid (83 mg, 1.38 mmol), sodium borohydride acetate (440 mg, 2.1 mmol), and dichloromethane (30 mL) were added to a reaction flask and stirred overnight at room temperature. The reaction was monitored by TLC, and the starting material conversion was complete. The crude product after rotary evaporation was purified by silica gel (MeOH / DCM = 0-10%) to give compound B037 (150 mg, 15%) as a colorless liquid. LCMS: [M+H] + =719; 1 H NMR(300MHz,DMSO-d6)δ5.42-5.24(m,4H),4.69(s,1H),4.08(t,2H),3.03(s,5H),2.73(t,2H),2.38-2.21(m ,2H),2.07-1.98(m,4H),1.89(m,5H),1.78(m,3H),1.53-1.40(m,6H),1.35-1.16(m,44H),0.89-0.83(m,9H).

[0381] Example 58. Synthesis of B038:

[0382] Compounds B38-1 (300 mg, 0.71 mmol), B37-4 (315 mg, 0.85 mmol), potassium carbonate (295 mg, 2.14 mmol), potassium iodide (118 mg, 0.71 mmol), and acetonitrile (30 mL) were added to a reaction flask and stirred overnight at 80 °C. The reaction was monitored by TLC, and the starting material conversion was complete. The crude product after rotary evaporation was purified by silica gel (methanol / dichloromethane = 0-10%) to give compound B038 (160 mg, 31.9%) as a colorless liquid. LCMS: [M+H] + =719; 1 H NMR (300MHz, DMSO-d6) δ5.43-5.24(m,4H),4.79(p,1H),4.73-4.60(m,1H),3.59(t,J=6.03Hz,2H),3.39(t,3H),3.03(m,5H ),2.73(t,2H),2.20(m,2H),2.02(m,4H),1.88(m,4H),1.56-1.40(m,11H),1.32-1.26(m,19H),1.23(s,20H),0.86(t,9H).

[0383] Example 59. Synthesis of B039

[0384] Add B33-4 (250 mg, 0.8 mmol), B39-1 (45 mg, 0.39 mmol), STAB (425 mg, 2 mmol), and dichloromethane (5 mL) to a 40 mL flask. Stir at room temperature for 16 hours, and monitor the reaction by TLC. Concentrate the reaction solution under reduced pressure to obtain the crude product, and purify by silica gel column chromatography (ethyl acetate, petroleum ether = 0–100%) to give a pale yellow liquid B039 (150 mg, 0.21 mmol), in 54% yield. MS: [M+1] + =713.

[0385] Example 60. Synthesis of B040:

[0386] Compound B40-1 (30 mg, 0.261 mmol), sodium borohydride acetate (138 mg, 0.652 mmol), and acetic acid (23 mg, 0.391 mmol) were added to a solution of B34-4 (260 mg, 0.652 mmol) in 10 mL of DCM at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B040 (106 mg, 46.2%) as a pale yellow oil. LCMS: [M+H] + =881.

[0387] Example 61. Synthesis of B041:

[0388] Compound B41-1 (40 mg, 0.348 mmol), sodium borohydride acetate (184 mg, 0.87 mmol), and acetic acid (31 mg, 0.522 mmol) were added to a solution of B35-4 (297 mg, 0.87 mmol) in 10 mL of DCM at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B041 (115 mg, 43.1%) as a pale yellow oil. LCMS: [M+H] + =769

[0389] Example 62. Synthesis of B042:

[0390] Compound B42-1 (45 mg, 0.391 mmol), sodium borohydride acetate (207 mg, 0.978 mmol), and acetic acid (35 mg, 0.587 mmol) were added to a solution of B36-4 (362 mg, 0.978 mmol) in 10 mL of DCM at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B042 (97 mg, 30.1%) as a pale yellow oil. LCMS: [M+H] + =825

[0391] Example 63. Synthesis of B043:

[0392] Step 1.

[0393] Compound B43-1 (13.67 g, 53.31 mmol) was added dropwise to a solution of compound B43-2 (9.05 g, 85.30 mmol), DCC (11.00 g, 53.31 mmol), and DMAP (7.16 g, 58.64 mmol) in DCM (140 mL) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered, and the filter cake was washed twice with DCM (25 mL). The crude product obtained by vacuum distillation was purified by silica gel column chromatography (EA / PE, 0-15%) to give compound B43-3 (14 g, 76%) as a colorless oil.

[0394] Step 2.

[0395] At room temperature, pyridinium chlorochromate (10.6 g, 49.1 mmol) was added to a DCM (100 mL) solution of compound B43-3 (13.0 g, 37.7 mmol). The resulting reaction solution was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was filtered, and the filter cake was washed twice with DCM (25 mL). The crude product obtained by vacuum distillation was purified by silica gel column chromatography (EA / PE, 0-10%) to give compound B43-4 (9.8 g, 76%) as a colorless oil.

[0396] Step 3.

[0397] AcOH (42 mg, 0.70 mmol) was added dropwise to a DCM (12 mL) solution of compound B43-4 (545 mg, 1.59 mmol), compound B43-5 (100 mg, 0.64 mmol), and STAB (337 mg, 1.59 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (EA / PE, 0-45%) to give B043 (170 mg, 33%) as a colorless oil. LCMS: [M+H] + =811.

[0398] Example 64. Synthesis of B044:

[0399] AcOH (76 mg, 1.26 mmol) was added dropwise to a DCM (12 mL) solution of compound B43-4 (983 mg, 2.87 mmol), compound B44-1 (100 mg, 1.15 mmol), and STAB (608 mg, 2.87 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give B044 (190 mg, 22%) as a colorless oil. LCMS: [M+H] + =741; 1 H NMR (300MHz, DMSO-d6) δ = 4.89 (s, 1H), 4.21-4.06 (m, 4H), 3.62-3.50 (m, 4H), 3.50-3.36 (m, 4H), 2 .68-2.51(m,4H),2.42-2.21(m,4H),1.59-1.33(m,10H),1.28-1.18(m,40H),0.89-0.80(m,12H).

[0400] Example 65. Synthesis of B045:

[0401] AcOH (58 mg, 0.96 mmol) was added dropwise to a DCM solution (12 mL) of compound B43-4 (744 mg, 2.17 mmol), compound B45-1 (100 mg, 0.87 mmol), and STAB (460 mg, 2.17 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give B045 (75 mg, 11%) as a colorless oil. LCMS: [M+H] + =769; 1 H NMR (300MHz, DMSO-d6) δ = 4.12 (t, J = 4.7, 4H), 3.61-3.50 (m, 4H), 3.43-3.35 (m, 5H), 2.65-2.52 (m, 5H) ),2.31-2.23(m,2H),1.65-1.58(m,2H),1.57-1.28(m,14H),1.27-1.18(m,40H),0.89-0.81(m,12H).

[0402] Example 66. Synthesis of B046:

[0403] AcOH (36 mg, 0.60 mmol) was added dropwise to a DCM (12 mL) solution of compound B43-4 (464 mg, 1.35 mmol), compound B46-1 (70 mg, 0.54 mmol), and STAB (287 mg, 1.35 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-8%) to give B046 (40 mg, 9%) as a colorless oil. LCMS: [M+H] + =783;

[0404] Example 67. Synthesis of B047:

[0405] Step 1.

[0406] Compound B47-2 (1.57 g, 11.72 mmol), EDCI HCl (1.5 g, 7.81 mmol), and DMAP (191 mg, 1.562 mmol) were added to a solution of compound B47-1 (2 g, 7.81 mmol) in dichloromethane (40 mL) at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with water, extracted with dichloromethane, and the organic phase was washed with 1N HCl aqueous solution and saturated brine. The solution was dried over anhydrous sodium sulfate, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 5:1) to give a pale yellow oily substance of B47-3 (1.886 g, 65.3%). LCMS: [M+H] + =373

[0407] Step 2.

[0408] IBX (1.13 g, 4.032 mmol) was added to a DMSO (10 mL) solution of B47-3 (1 g, 2.688 mmol) at room temperature. The resulting mixture was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (PE / EA = 8:1) to give B47-4 (810 mg, 81.4%) as a pale yellow oil. LCMS: [M+H] + =371

[0409] Step 3.

[0410] Compound B47-5 (120 mg, 0.876 mmol), sodium borohydride acetate (464 mg, 2.189 mmol), and acetic acid (79 mg, 1.314 mmol) were added to a DCM (10 mL) solution of B47-4 (810 mg, 2.189 mmol) at room temperature. The resulting mixture was stirred at room temperature for 24 hours. The reaction was confirmed by LCMS. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and evaporated under vacuum. The crude product after evaporation was purified by normal-phase silica gel column chromatography (DCM / MeOH = 10:1) to give B047 (334 mg, 47.1%) as a pale yellow oil. LCMS: [M+H] + =811

[0411] Example 68. Synthesis of B048:

[0412] AcOH (39 mg, 0.65 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (551 mg, 1.49 mmol), compound B48-1 (80 mg, 0.59 mmol), and STAB (441 mg, 2.08 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-10%) to give B048 (180 mg, 38%) as a colorless oil. LCMS: [M+H] + =808; 1 H NMR (300MHz, DMSO-d6) δ = 4.07 (t, J = 6.5, 4H), 3.41-3.34 (m, 16H), 2.85 (s, 4H), 2.33-2.24 (m, 2H), 1.79 (p,J=6.3,4H),1.59(dd,J=13.4,6.8,4H),1.53-1.37(m,8H),1.30-1.12(m,40H),0.93-0.76(m,12H).

[0413] Synthesis of Example 69.B048:

[0414] AcOH (39 mg, 0.65 mmol) was added dropwise to a DCM (12 mL) solution of compound B1-4 (551 mg, 1.49 mmol), compound B49-1 (80 mg, 0.59 mmol), and STAB (441 mg, 2.08 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, 1N NaOH (60 mL) was added and the mixture was extracted with DCM (80 mL). The organic phase was washed once with water (60 mL) and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (MeOH / DCM, 0-6%) to give B049 (210 mg, 44%) as a colorless oil. LCMS: [M+H] + =808; 1 H NMR (300MHz, DMSO-d6) δ = 4.07 (t, J = 6.4, 4H), 3.43-3.42 (m, 8H), 3.26-2.71 (m, 10H), 2.27 (tt, J =8.8,4.3,2H),1.88-1.63(m,8H),1.61-1.32(m,10H),1.29-1.17(m,40H),0.89-0.79(m,12H).

[0415] Example 70. Biological Experiment

[0416] 1. Screening of cationic lipid activity

[0417] 1) Preparation of lipid nanoparticles

[0418] Cationic lipids (SM102 and newly synthesized liposomes), DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol at a designed formulation ratio (Lipid (cationic lipid compound) DSPC / Cholesterol (cholesterol) / DMG-PEG (conjugated lipid) 50 / 10 / 38.5 / 1.5 (molar ratio)) and thoroughly mixed. The above lipid solution was then mixed with a green fluorescent protein (GFP) mRNA solution to prepare LNPs in a Y-type microfluidic chip at an organic phase to aqueous phase volume ratio of 1:3 and a total flow rate of 20 ml / min.

[0419] 2) Cell viability screening

[0420] Cells of 293T (3E+5 cells), Jurkat (5E+5 cells), and THP-1 were seeded into 12-well plates. 50 μL and 25 μL of freshly prepared LNP were used to treat the cells. Cells were cultured at 37°C with 5% CO2 for 24 h, and the results were observed and photographed under a fluorescence microscope. The newly synthesized lipids were evaluated primarily based on fluorescence intensity and cytotoxicity. Fluorescence intensity and cytotoxicity were measured against SM102-LNP-treated cells. Lipid transfection efficiency was assessed at the cellular level using both fluorescence and cytotoxicity; cytotoxicity was evaluated using the cell state at 24 h. The results are shown in Table 1 below.

[0421] Table 1. Lipid-cell transfection efficiency Note: +++ indicates that the brightness of SM102-LNP transfection of 293T, JURKAT, and THP-1 cells was used as the comparison standard. The transfection rate of SM102 is approximately 100%. ++ indicates that the overall fluorescence brightness of the cell line with added SM102 is lower than that of the same cell line, or the transfection rate is lower but the brightness is higher. + indicates that there is fluorescence but the brightness is low, or a few fluorescent cells can be seen. ++++ indicates that the overall brightness of the cell line with added SM102 is higher than that of the same cell line, and the transfection is 100%. * indicates that cell morphology changes can be seen at a 50ul LNP gradient. ** indicates that cell morphology changes are more obvious at a 50ul LNP gradient, and the effect is smaller at 25ul. *** indicates that there are basically no viable cells at 50ul and 25ul LNP gradients. - indicates that fluorescence was not detected.

[0422] 2. Distribution of B003-LNP in mice

[0423] To investigate the distribution of B003-LNP in mice via tail vein or intramuscular injection, BALB / c mice were injected with 5 μg / 100 μL of B003-derived mRNA luciferase-LNP via tail vein or intramuscular injection. Six hours later, the mice were anesthetized (using a small animal gas anesthesia system) by injection of D-luciferin potassium salt and placed in a live imaging system (Perkin Elmer IVIS Lumina III) for 10 minutes. The fluorescence values ​​(Total Flux [p / s] / Avg Radiance [p / s / cm² / sr]) and distribution of the mice were obtained, as shown in Figure 1. The mice were then euthanized with carbon dioxide, and the heart, liver, spleen, lungs, kidneys, and lymph nodes were removed for live imaging, and the corresponding fluorescence values ​​(Total Flux [p / s] / Avg Radiance [p / s / cm² / sr]) were recorded, as shown in Figure 2. The proportion of fluorescence intensity among different organs was calculated and analyzed, as shown in Figure 3.

[0424] 3. Study on hEPO expression in mice by B003-LNP

[0425] To investigate hEPO expression in mice following tail vein or intramuscular injection of mRNA hEPO-LNP, mRNA hEPO-LNP was prepared using B003. Six- to eight-week-old C57L / B6 mice were administered the mRNA via both intramuscular and intravenous injection. Three mice were injected per group (10 μg / mouse). A negative control group and a positive control group (mRNA hEPO-LNP prepared using SM102) were established. Serum samples were collected 24 hours post-injection, and hEPO levels in all serum samples were quantified using an hEPO ELISA kit. The results, shown in Figure 4, indicate that mRNA hEPO-LNP prepared using B003 exhibited significantly higher hEPO expression levels compared to mRNA hEPO-LNP prepared using SM102.

[0426] 4. Study on the induction of humoral and cellular immunity by B003-LNP in mice

[0427] To investigate the OVA-binding antibody (humoral immunity) and cellular immunity (IFN-γ ELISPOT) induced by intramuscular injection of mRNA Ovabulin (OVA)-LNP prepared using B003 and B019, mRNA OVA-LNP was prepared using either B003 or B019. Six- to eight-week-old C57L / B6 mice were intramuscularly injected with mRNA OVA-LNP at doses of 2.5 μg / mouse or 10 μg / mouse, with three mice per group, administered twice weekly. A negative control group and a positive control group using mRNA OVA-LNP prepared using SM102 were also established. One week after discontinuation of administration, all mice were euthanized, serum was collected, and spleens were harvested. Anti-OVA antibodies were detected using ELISA at concentrations of 1 / 100, 1 / 1000, and 1 / 10000. Spleen cells were isolated and counted from the spleen. The diluted spleen cell suspension was then added to the wells of an ELISPOT plate, along with two OVA peptides, OVA 257-264aa and OVA 323-339aa, for stimulation. The plate was then incubated at 37°C in a 5% CO2 incubator for 24 hours. Finally, the ELISPOT plate was developed. After all operations were completed, the plate was read using an ELISPOT analyzer (CTL), and the mIFN-γ expression spots were counted. The results are shown in Figure 5. The liposomes of this invention can enhance the in vivo immunogenicity of the vaccine at a lower dose (2.5 μg).

[0428] 5. Characterization of representative LNP particle size properties

[0429] The physical properties of the prepared LNPs, including particle size, aggregation index (PDI), and zeta potential, were analyzed using a nanoparticle size and potential analyzer. Furthermore, the encapsulation efficiency of the LNPs was also determined using Quant-iT. TM RiboGreen RNA was analyzed, and representative results are shown in Table 2 below.

[0430] Table 2

[0431] 6. Stability of LNP formulations with different formulations

[0432] Taking sample 1 (B003 / DOPE / cholesterol / PEG2000-DMG = 50%:10%:38.5%:1.5%) as an example, the lipid nanoparticles prepared according to the formula were stored at 4°C. At different time points (0 days, 7 days, 22 days, 30 days, and 63 days), the particle size (Size) and PDI of mRNA-LNPs (lipid nanoparticles encapsulating mRNA) were characterized using a Malvern Zetasizer Nano ZS. The mRNA encapsulation efficiency was determined using a Ribogreen RNA quantification kit. The results are shown in Table 3 below.

[0433] Table 3

[0434] Taking sample 2 (B003 / DSPC / cholesterol / PEG2000-DMG = 50%:10%:38.5%:1.5%) as an example, the lipid nanoparticles prepared according to the formula were stored at 4°C. At different time points (0 days, 7 days, 22 days, 30 days, and 63 days), the particle size (Size) and PDI of mRNA-LNP (lipid nanoparticles encapsulating mRNA) were characterized using a Malvern Zetasizer Nano ZS. The encapsulation efficiency of mRNA was measured using a Ribogreen RNA quantification kit (Thermo Fisher). The results are shown in Table 4 below.

[0435] Table 4

[0436] Overall, samples 1 and 2 tended to be stable at 4°C.

[0437] 7. Security Assessment:

[0438] 1) Cellular level: 293T, Jurkat, and THP1 cells were treated with 1 μg of GFP mRNA-LNP prepared from B003 and B019 for 24 h. After trypan blue staining, cells and cell viability were counted using a Countess 3FL automated cell counter. Compared with untreated cells, B003 and B019 had no significant effect on cell growth and cell viability.

[0439] 2) Animal Level: Balb / c mice aged 6-8 weeks were intramuscularly injected twice weekly with 10 μg and 30 μg of luciferase mRNA LNP prepared from B003 and B019, for a total of six injections over three weeks. After treatment was discontinued, mice were observed for one month. No mouse deaths occurred during the entire experiment. Mice showed no significant changes in appearance, and their fur remained smooth. Body temperature and appetite were normal. Weight gain and defecation were normal, with no soft stools or diarrhea observed. Mice showed no signs of lethargy or other unusual symptoms. No redness, swelling, ulceration, or other abnormalities were observed at the injection sites.

[0440] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A liposomal compound of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, or a prodrug thereof: in, R1 is a C2-C8 alkylene group; Z1 is O, S, SS, or SSS; T2 is Or H; A is selected from: A nitrogen-containing heterocyclic group of 5-10 quinones; wherein the heterocyclic group is saturated or partially unsaturated; G is selected from: -(CH2) m1 -C3-C10 cycloalkyl groups, -(CH2) m1 -5-12 membered heterocyclic group, -(CH2) m1 -5-10 heteroaryl groups, -(CH2) m1 -OC(O)C3-C10 cycloalkyl, -(CH2) m1 -OC(O)-5-12 membered heterocyclic group, -(CH2) m1 -OC(O)-5-10 heteroaryl, -(CH2) m1 -Phenyl; the cycloalkyl or heterocyclic group is a monocyclic, bicyclic, or tricyclic system; m1 is 0, 1, 2, 3, 4, or 5; R7 is selected from: hydroxyl group, -(CH2) 1-6 -hydroxyl, C1-C6 alkoxy, C1-C6 alkyl, -(CH2) 0-6 -N(CH3)2; m is 0, 1, 2, 3, 4 or 5; or two R7 atoms located on the same ring atom together form -OCH2CH2O-; L1, R2, Z2, and L2 are each independently -(L) n - Each L is independently selected from the following group: substituted or unsubstituted CH2, O, S, -CO-, -S(O)-, -S(O)2-, where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and Each has ≤20 carbon atoms, preferably ≤15, and more preferably ≤10; X1 and X2 are each independently selected from the following groups: covalent bond, -(C=O)O-, -O(C=O)-, -(S=O)O-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -O(C=S)-; Y1 and Y2 are each independently selected from the group consisting of: covalent bond, CH or N; preferably, Y1 and Y2 are CH. R3 and R5 are each independently selected from the following group: substituted or unsubstituted straight or branched C1-C 30 Alkylene, substituted or unsubstituted straight or branched C2-C 30 alkenyl, substituted or unsubstituted straight or branched C2-C 30 Ethyne group, -C0-C 10 Alkylene-C3-C 10 cycloalkyl-C1-C 20 Alkylene, -C0-C 10 imide-C3-C 10 cycloalkyl-C1-C 20 Alkylene, -C0-C 10 Alkylene-C3-C 10 cycloalkyl-C1-C 20 Alkylene; R4 and R6 are each independently selected from the following group: H, substituted or unsubstituted straight or branched C1-C 30 Alkylene, substituted or unsubstituted straight or branched C2-C 30 alkenyl, substituted or unsubstituted straight or branched C2-C 30 Ethyne group, -C0-C 10 Alkylene-C3-C 10 cycloalkyl-C1-C 20 Alkylene, -C0-C 10 imide-C3-C 10 cycloalkyl-C1-C 20 Alkylene, -C0-C 10 Alkylene-C3-C 10 cycloalkyl-C1-C 20 Alkylene; The substitution refers to having one or more substituents selected from the group consisting of: deuterium, halogen, amino, hydroxyl, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkoxy, and C1-C6 haloalkyl.

2. The liposome compound according to claim 1, characterized in that, The liposome compound has the structure shown in formula (II): Wherein, G, X1, X2, Y1, Y2, Z1, Z2, L1, L2, R1, R2, R3, R4, R5, R6, R7, and m are as defined in claim 1.

3. The liposome compound according to claim 1, characterized in that, A is selected from:

4. The liposome compound according to claim 1, characterized in that, Selected from the following group:

5. The liposome compound according to claim 1, characterized in that, R1-Z1-L1 is selected from the following group: -(CH2) a1 -O-(CH2) b1 -、-(CH2) a1 -O-(CH2) b1 -O-(CH2) c1 -、-(CH2) a1 -S-(CH2) b1 -、-(CH2) a1 -SS-(CH2) b1 -、-(CH2) a1 -SSS-(CH2) b1 -、-(CH2) a1 -S-(CH2) b1 -S-(CH2) c1 -、-(CH2) a1 -(CH2-CH2-O) b1 -(CH2) c1 -; a1 independently represents 2, 3, 4, 5, 6, 7, 8; b1 and c1 independently represent 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

6. The liposome compound according to claim 1, characterized in that, R1-Z1-L1 has a structure selected from the following group: R2-Z2-L2 has a structure selected from the following group:

7. The liposome compound according to claim 1, characterized in that, Each person independently selects from the following groups:

8. The liposome compound according to claim 1, characterized in that, The liposome compounds are selected from the group consisting of:

9. A lipid nanoparticle (LNP), characterized in that, The lipid nanoparticles comprise the liposomal compound as described in any one of claims 1-9, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

10. A lipid nanoparticle drug formulation, characterized in that, The lipid nanoparticle drug formulation comprises: i) The lipid nanoparticles as described in claim 9; ii) Bioactive substances encapsulated in the lipid nanoparticles; and iii) Pharmaceutically acceptable carriers.

11. The use of the liposome compound as claimed in claim 1, characterized in that, The liposomal compound is used to prepare lipid nanoparticle drug formulations for delivering bioactive substances to cells in a subject in need.

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