Ionizable lipid compound and use thereof in delivering nucleic acid vaccine

Abstract

The present invention belongs to the technical field of biomedicine, and specifically relates to an ionizable lipid compound and a use thereof in delivering a nucleic acid vaccine. Provided is the ionizable lipid compound represented by formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof. The definition of each group in the formula is described in detail.

Description

Ionizable lipid compounds and their use in delivering nucleic acid vaccines

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application CN202311848564.6 filed on December 29, 2023 and Chinese patent application CN202311848734.0 filed on December 29, 2023; the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biomedicine technology, and particularly relates to ionizable lipid compounds and their use in delivering nucleic acid vaccines. Background Art

[0004] Nucleic acid vaccines are vaccines that directly introduce exogenous genes (DNA or RNA) encoding specific antigenic proteins into host cells, express the antigenic proteins in the host cells, and induce the host to produce an immune response to the antigenic proteins to achieve the purpose of preventing and treating diseases.

[0005] The nucleic acid vaccine developed by Moderna and BioNTech uses lipid nanoparticles (LNPs) as a delivery system. The main components of LNPs include cationic lipid molecules (Cationic Lipid), cholesterol, neutral lipids, and polyethylene glycol-conjugated lipids. Among them, the cationic lipid molecule is the core of the LNP delivery system, and its molecular structure plays a decisive role in the delivery efficiency, targeting, and formulation stability of the entire liposome nanoparticle.

[0006] Since the delivery of different types of nucleic acid substances and the specific delivery of different targets have different requirements for the delivery system, in order to meet the different needs of gene therapy, new lipid molecules need to be further developed. Summary of the Invention

[0007] The cationic lipid molecule ALC0315 used in BioNTech's nucleic acid vaccine is currently recognized as one of the cationic lipid compounds with the best nucleic acid delivery effect, which can cause the body to produce an efficient immune response. The inventors have developed a class of cationic lipid compounds containing geminal dimethyl groups (see CN115850104A), which have a high nucleic acid delivery efficiency when injected into the body by intravenous administration, mainly delivered to the liver. However, nucleic acid vaccines can produce an immune response in the body's lymphoid organs through intramuscular injection. Due to different applications, whether the lipid compound of the present invention produces a good immune response in the body is a key research issue of the present invention. Unexpectedly, the compound of the present invention is used to prepare a nucleic acid vaccine, and the body's immune response effect is better than ALC0315.

[0008] Specifically, one aspect of the present invention provides an ionizable lipid compound having a structure of formula (I) or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

[0009] in,

[0010] M1 and M2 are independently selected from -C(O)O-, -OC(O)-, -OC(O)O-, -SC(O)-, -C(O)S-, -C(O)NR a -and-NR a C(O)-;

[0011] a is independently selected from 0, 1, 2, 3, 4, and 5;

[0012] b and d are independently selected from 3, 4, 5, 6, 7, 8 and 9;

[0013] c and e are independently selected from 0, 1, 2, 3 and 4;

[0014] b+c=3, 4, 5, 6, 7, 8, or 9, d+e=3, 4, 5, 6, 7, 8, or 9;

[0015] The methylene group in the 1-6 Alkyl substitution;

[0016] R1 and R2 are independently selected from C 4-20 Alkyl, C 4-20 Alkenyl and C 4-20 Alkynyl, the C 4-20 Alkyl, C 4-20 Alkenyl or C 4-20 Alkynyl is optionally substituted with one or more R, said C 4-20 Alkyl, C 4-20 Alkenyl or C 4-20 One or more methylene units in the alkynyl group are optionally and independently replaced by -NR"-;

[0017] R is independently selected from H, C 1-14 Alkyl, -L a -OR a and -L a -NR a R' a ;

[0018] R" is independently selected from H and C 1-20 alkyl;

[0019] R3 and R4 are independently selected from H, C 1-6 Alkyl, C 1-6haloalkyl, 3 to 10-membered cycloalkyl and 3 to 10-membered heterocyclic group, the C 1-6 Alkyl, C 1-6 haloalkyl, 3- to 10-membered cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with 1, 2, 3, 4, or 5 R*;

[0020] or R3, R4 and the N atom to which they are attached together form a 3- to 10-membered heterocyclic group, wherein the 3- to 10-membered heterocyclic group is optionally substituted by 1, 2, 3, 4 or 5 R*;

[0021] R5, R6, R7 and R8 are independently selected from C 1-6 Alkyl, the C 1-6 Alkyl is optionally substituted with 1, 2, 3, 4 or 5 R*;

[0022] R* is independently selected at each occurrence from H, halogen, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, -L b -OR b or -L b -NR b R' b ;

[0023] L a independently selected from chemical bonds and C 1-6 alkylene;

[0024] L b independently selected from chemical bonds and C 1-6 alkylene;

[0025] R a and R' a Independently selected from H, C 1-6 alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl;

[0026] R b and R' b Independently selected from H, C 1-6 alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl.

[0027] In some embodiments, M1 and M2 are independently selected from -C(O)O-, -OC(O)-, -OC(O)O-, -SC(O)-, and -C(O)S-.

[0028] In some embodiments, M1 and M2 are independently selected from -C(O)O- or -OC(O)-.

[0029] In some embodiments, M1 and M2 are both selected from -C(O)O-.

[0030] In some embodiments, M1 and M2 are both selected from -OC(O)-.

[0031] Another aspect of the present invention provides an ionizable lipid compound having a structure represented by formula (II) or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

[0032] in,

[0033] R1 and R2 are independently selected from C 4-20 Alkyl, C 4-20 Alkenyl and C 4-20 Alkynyl, the C 4-20 Alkyl, C 4-20 Alkenyl or C 4-20 Alkynyl is optionally substituted with one or more R, said C 4-20 Alkyl, C 4-20 Alkenyl or C 4-20 One or more methylene units in the alkynyl group are optionally and independently replaced by -NR"-;

[0034] R is independently selected from H, C 1-14 Alkyl, -L a -OR a and -L a -NR a R' a ;

[0035] R" is independently selected from H and C 1-20 alkyl;

[0036] R3 and R4 are independently selected from H, C 1-6 Alkyl, C 1-6 haloalkyl, 3 to 10-membered cycloalkyl and 3 to 10-membered heterocyclic group, the C 1-6 Alkyl, C 1-6 haloalkyl, 3- to 10-membered cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with 1, 2, 3, 4, or 5 R*;

[0037] or R3, R4 and the N atom to which they are attached together form a 3- to 10-membered heterocyclic group, wherein the 3- to 10-membered heterocyclic group is optionally substituted by 1, 2, 3, 4 or 5 R*;

[0038] a is independently selected from 0, 1, 2, 3, 4, and 5;

[0039] b and d are independently selected from 3, 4, 5, 6, 7, 8 and 9;

[0040] c and e are independently selected from 0, 1, 2, 3 and 4;

[0041] b+c=3, 4, 5, 6, 7, 8, or 9, d+e=3, 4, 5, 6, 7, 8, or 9;

[0042] The methylene group in the 1-6 Alkyl substitution;

[0043] R5, R6, R7 and R8 are independently selected from C 1-6 Alkyl, the C 1-6 Alkyl is optionally substituted with 1, 2, 3, 4 or 5 R*;

[0044] R* is independently selected at each occurrence from H, halogen, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, -L b -OR b or -L b -NR b R' b ;

[0045] L a independently selected from chemical bonds and C 1-6 alkylene;

[0046] L b independently selected from chemical bonds and C 1-6 alkylene;

[0047] R a and R' a Independently selected from H, C 1-6 alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl;

[0048] R b and R' b Independently selected from H, C 1-6 alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl.

[0049] The compounds of the present invention are ionizable lipid compounds.

[0050] In some embodiments, a is selected from 2, 3 or 4, more preferably 2 or 3, and more preferably 2.

[0051] In some embodiments, b and d are independently 4 or 5.

[0052] In some embodiments, c and e are independently 0 or 1;

[0053] In some embodiments, b+c=4, 5, or 6, and d+e=4, 5, or 6.

[0054] In some embodiments, b+c=5, d+e=5.

[0055] In some embodiments, R1 and R2 are independently selected from linear alkyl groups having a total length of 6, 7, 8, 9 or 10 carbon atoms, wherein 1, 2 or 3 methylene groups in the linear alkyl group are optionally and independently replaced by C 1-8 Alkyl substitution.

[0056] In some embodiments, R1 or R2 are each independently a straight chain alkyl group having a total length of 9 carbon atoms, and one of R1 or R2 is replaced by C 4-6 Alkyl substitution.

[0057] In some embodiments, R1 and R2 are each independently a straight chain alkyl group having a total length of 9 carbon atoms, and one of R1 or R2 is substituted with n-butyl, n-pentyl, or n-hexyl.

[0058] In some embodiments, R3 and R4 are independently selected from C 1-6 Alkyl, the C 1-6 The alkyl group is optionally substituted with 1, 2, or 3 R*.

[0059] In some embodiments, R* is independently selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl and -OR a ; Among them, R a Independently selected from H and C 1-6 alkyl.

[0060] In some embodiments, R3 and R4 are independently selected from methyl.

[0061] In some embodiments, R5, R6, R7 and R8 are independently selected from C 1-6 Alkyl group; preferably methyl or ethyl, more preferably methyl.

[0062] In another aspect of the present invention, the ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein the ionizable lipid compound is selected from the following structures:

[0063] In another aspect of the present invention, the ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein the ionizable lipid compound is selected from the following structures:

[0064] Another aspect of the present invention provides an ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein the ionizable lipid compound is selected from the following compounds:

[0065] Another aspect of the present invention provides a pharmaceutical composition comprising the aforementioned ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, and optionally a pharmaceutically acceptable excipient.

[0066] In some embodiments, excipients include but are not limited to carriers, adjuvants, or vehicles.

[0067] In some embodiments, the pharmaceutical composition is a nanoparticle composition comprising a lipid component and, optionally, an active ingredient.

[0068] In some embodiments, the active ingredient is a nucleic acid.

[0069] In some embodiments, the lipid component comprises the following components in molar percentages:

[0070] Ionizable cationic lipids 20 mol%-85 mol%;

[0071] Structural lipids 10 mol%-75 mol%;

[0072] Neutral lipids 1.0 mol%-30 mol%;

[0073] Polymer lipid 0.25mol%-10mol%.

[0074] In some embodiments, the ionizable cationic lipid is an ionizable lipid compound as described above, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof.

[0075] In some embodiments, the lipid component comprises the following components in molar percentages:

[0076] Ionizable cationic lipids 30 mol%-50 mol%;

[0077] Structural lipids 30 mol%-60 mol%;

[0078] Neutral lipids 10 mol%-30 mol%;

[0079] Polymer lipid 0.25mol%-5mol%.

[0080] In some embodiments, the lipid component comprises the following components in molar percentages:

[0081] Ionizable cationic lipids 35 mol%-45 mol%;

[0082] Structural lipids 40 mol%-50 mol%;

[0083] Neutral lipids 10 mol%-20 mol%;

[0084] Polymer lipid 0.5mol%-2mol%;

[0085] In some embodiments, the molar percentage content of the ionizable cationic lipid is 42.5 mol%-50 mol%, 32.5 mol%-50 mol%, 25 mol%-65 mol%, 30 mol%-60 mol%, 30 mol%-50 mol%, 30 mol%-55 mol% or 40 mol%-52.5 mol%.

[0086] In some embodiments, the molar percentage content of the structured lipid is 30.6 mol%-61 mol%, 30.6 mol%-51 mol%, 25 mol%-70 mol%, 27.5 mol%-66 mol%, 30.5 mol%-66 mol%, 28 mol%-64 mol% or 28 mol%-54 mol%.

[0087] In some embodiments, the molar percentage content of neutral lipids is 1 mol%-25 mol%, 1.5 mol%-20 mol%, 1.5 mol%-20 mol%, 5 mol%-20 mol%, or 5 mol%-20 mol%.

[0088] In some embodiments, the molar percentage content of the polymer lipid is 1 mol%-5 mol%, 1 mol%-2 mol%, 0.5 mol%-8 mol%, 1 mol%-5 mol%, 1 mol%-3 mol% or 1 mol%-3 mol%.

[0089] In some embodiments, the N:P molar ratio of N atoms in the ionizable cationic lipid to P atoms in the charge molecule is 1-15:1, for example, 3-12:1, 3-7:1, 1-10:1, 3-6:1 or 3-5:1.

[0090] In some embodiments, the particle size is 65-200 nm, for example, 65-180 nm, 70-170 nm, 70-130 nm, 70-180 nm, 80-180 nm, 90-180 nm, or 100-135 nm.

[0091] In some embodiments, the neutral lipid is selected from one or more of DSPC, DMPC, DOPC, DPPC, POPC, DOPE, DMPE, POPE, or DPPE.

[0092] In some embodiments, the structured lipid is selected from one or more of cholesterol, sitosterol, coproposterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol, or campesterol.

[0093] In some embodiments, the polymer lipid is a PEGylated lipid.

[0094] In some embodiments, the PEGylated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0095] In some embodiments, the PEGylated lipid is selected from one or more of DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, and DSPE-PEG5000.

[0096] Another aspect of the present invention provides the use of the ionizable lipid compound as described above or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or the pharmaceutical composition as described above in the preparation of a drug for delivering a load or a drug for treating, diagnosing or preventing a disease, wherein the load is selected from one or more therapeutic agents, preventive agents or diagnostic agents.

[0097] Another aspect of the present invention provides the use of the ionizable lipid compound as described above or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or the pharmaceutical composition as described above in the preparation of a drug for delivering a load, wherein the load is selected from one or more therapeutic agents, prophylactic agents or diagnostic agents.

[0098] Another aspect of the present invention provides the use of the aforementioned ionizable lipid compound or its pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer, or the aforementioned pharmaceutical composition in the preparation of nucleic acid vaccines or the delivery of nucleic acid vaccines.

[0099] Another aspect of the present invention provides the use of the aforementioned ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or the aforementioned pharmaceutical composition in the preparation of a nucleic acid vaccine.

[0100] Another aspect of the present invention provides use of the aforementioned ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or the aforementioned pharmaceutical composition in delivering nucleic acid vaccines.

[0101] In some embodiments, the nucleic acid vaccine comprises a lipid component and a nucleic acid.

[0102] In some embodiments, the nucleic acid is selected from one or more of RNA or DNA.

[0103] In some embodiments, the nucleic acid is selected from antisense oligonucleotides (ASOs).

[0104] In some embodiments, the RNA is selected from one or more of small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long noncoding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polycoding nucleic acid (MCNA), polycoding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA) or ribozyme.

[0105] In some embodiments, the RNA is mRNA, more preferably modified mRNA.

[0106] In some embodiments, the therapeutic, prophylactic, or diagnostic agent is a nucleic acid.

[0107] In some embodiments, the nucleic acid is selected from one or more of RNA or DNA.

[0108] In some embodiments, the nucleic acid is selected from antisense oligonucleotides (ASOs).

[0109] In some embodiments, the RNA is selected from one or more of small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long noncoding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), polycoding nucleic acid (MCNA), polycoding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA) or ribozyme.

[0110] In some embodiments, the RNA is mRNA, more preferably modified mRNA.

[0111] In some embodiments, the drug for treating, diagnosing or preventing a disease is selected from a therapeutic vaccine or a preventive vaccine.

[0112] In some embodiments, the vaccine is used to prevent / treat diseases caused by viral infection.

[0113] In some embodiments, the vaccine is used to prevent / treat diseases caused by coronavirus infection, more preferably diseases caused by SARS-CoV-2.

[0114] In some embodiments, the vaccine is used to prevent / treat diseases caused by influenza virus infection.

[0115] In some embodiments, the vaccine is used to prevent / treat diseases caused by herpes zoster virus infection.

[0116] In some embodiments, the use elicits an immune response in a subject or vaccinates a subject against a viral infection.

[0117] In some embodiments, the use elicits an immune response in the subject.

[0118] In some embodiments, the use elicits an antiviral immune response in the subject.

[0119] In some embodiments, the use is for vaccinating a subject against a viral infection.

[0120] Another aspect of the present invention provides a method for preparing a nucleic acid vaccine, which comprises the following steps: mixing the ionizable lipid compound as described above or its pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer with a structural lipid, a neutral lipid, a polymer lipid and a solvent, and then mixing it with a solution in which mRNA encoding an antigen protein is dissolved.

[0121] In some embodiments, the antigenic protein is a viral antigenic protein.

[0122] In some embodiments, the antigenic protein is the novel coronavirus spike protein or the varicella-zoster virus envelope glycoprotein E.

[0123] In some embodiments, the solvent is an organic solvent, preferably an alcohol solvent, more preferably ethanol.

[0124] In some embodiments, a sodium acetate solution is used to dissolve the mRNA encoding the antigen protein, preferably a 20-30 mmol / L sodium acetate solution.

[0125] In some embodiments, the vaccine is administered intramuscularly.

[0126] In some embodiments, the vaccine is targeted to the liver.

[0127] In another aspect of the present invention, a method for delivering a nucleic acid in a subject is provided, comprising administering to the subject a pharmaceutical composition comprising the aforementioned ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof.

[0128] Another aspect of the present invention provides a method for delivering a load in a subject, comprising administering to the subject a pharmaceutical composition comprising the above-mentioned ionizable lipid compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein the load is selected from one or more therapeutic agents, prophylactic agents or diagnostic agents.

[0129] In some embodiments, the cargo of the present invention is selected from one or more therapeutic agents, prophylactic agents, or diagnostic agents;

[0130] In some embodiments, the therapeutic, prophylactic, or diagnostic agent of the present invention is selected from a vaccine;

[0131] In some embodiments, the therapeutic, prophylactic, or diagnostic agent of the present invention is selected from nucleic acids;

[0132] In some embodiments, the therapeutic, prophylactic, or diagnostic agent of the present invention is selected from nucleic acid vaccines;

[0133] In some embodiments, the therapeutic, prophylactic, or diagnostic agent of the present invention is selected from a therapeutic vaccine or a prophylactic vaccine;

[0134] In some embodiments, the therapeutic agent, prophylactic agent or diagnostic agent of the present invention is selected from a therapeutic nucleic acid vaccine or a prophylactic nucleic acid vaccine.

[0135] Another aspect of the present invention provides a method for treating, diagnosing or preventing a disease in a subject, comprising administering to the subject a pharmaceutical composition comprising the above-mentioned ionizable lipid compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof.

[0136] In some embodiments, the amount of the pharmaceutical composition administered is an effective amount.

[0137] Another aspect of the present invention provides a method for treating, diagnosing or preventing a disease in a subject, comprising administering to the subject the nucleic acid vaccine obtained by the preparation method as described above.

[0138] In some embodiments, the amount of the nucleic acid vaccine administered is a preventive / therapeutic effective amount.

[0139] In some embodiments, the method elicits an immune response in the subject.

[0140] In some embodiments, the method elicits an anti-viral immune response in the subject.

[0141] In some embodiments, the method vaccinates the subject against a viral infection.

[0142] In some embodiments, the disease is a disease caused by a viral infection.

[0143] In some embodiments, the disease is a disease caused by coronavirus infection, preferably a disease caused by SARS-CoV-2.

[0144] In some embodiments, the disease is a disease caused by influenza virus infection.

[0145] In some embodiments, the disease is caused by herpes zoster virus infection.

[0146] definition

[0147] Chemical definition

[0148] Definitions of specific functional groups and chemical terms are described in more detail below.

[0149] The term "substituted" or "substituted" means that any one or more hydrogen atoms on a particular atom are replaced with a substituent.

[0150] The term "chemical bond" refers to a single bond, a double bond or a triple bond, preferably a single bond.

[0151] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0152] “C 4-20 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 4 to 20 carbon atoms, further including C 9-15 Alkyl. C 1-6Examples of alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6), etc. The term “C 1-6 "Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3), or i-Bu(-CH2CH(CH3)2).

[0153] “C 4-20 "Alkenyl" refers to a straight or branched chain hydrocarbon group having 4 to 20 carbon atoms and at least one carbon-carbon double bond. 2-6 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6 "Alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkenyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0154] “C 4-20 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 4 to 20 carbon atoms, at least one carbon-carbon triple bond and optionally one or more carbon-carbon double bonds. The term "C 2-6 "Alkynyl" also includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups can be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0155] The term "the total length of variable A and variable B is x carbon atoms" means that the sum of the number of carbon atoms in the main chain of the group represented by variable A and the number of carbon atoms in the main chain of the group represented by variable B is x.

[0156] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0157] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 "alkyl" is substituted with one or more halo groups. A haloalkyl group can be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0158] “C 3-10 "Cycloalkyl" or "3- to 10-membered cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms, optionally containing 1, 2, or 3 double or triple bonds. Cycloalkyl also includes ring systems in which the above-mentioned cycloalkyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such cases, the number of carbons continues to refer to the number of carbons in the cycloalkyl system. Cycloalkyl also includes cycloalkyl rings in which the substituents on any non-adjacent carbon atoms are linked to form a bridged ring, together forming a polycycloalkane sharing two or more carbon atoms. Cycloalkyl also includes cycloalkyl rings in which Substituents on the same carbon atom are linked to form a ring to form a polycycloalkane sharing one carbon atom. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), and the like. The cycloalkyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0159] "3-10 membered heterocyclyl" or "3- to 10-membered heterocyclyl" refers to a saturated or unsaturated radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, and optionally containing 1, 2, or 3 double or triple bonds. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. Heterocyclyl also includes ring systems in which the above-mentioned heterocyclyl ring is fused to one or more cycloalkyl groups, where the point of attachment is on the heterocyclyl ring, or ring systems in which the above-mentioned heterocyclyl ring is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the heterocyclyl ring; and in such cases, the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. Heterocyclyl also includes heterocyclyl rings in which the substituents on any non-adjacent carbon or nitrogen atoms are linked to form a bridged ring, together forming a polycyclic heteroalkane sharing two or more carbon or nitrogen atoms. Heterocyclyl also includes the above-mentioned heterocyclyl rings, wherein the substituents on the same carbon atom are linked to form a ring, together forming a polycyclic heteroalkane sharing one carbon atom. Exemplary 3-membered heterocyclyls containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclyls containing one heteroatom include, but are not limited to, azetidinyl, oxetane, and thietidinyl. Exemplary 5-membered heterocyclyls containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyls containing two heteroatoms include, but are not limited to, pyrazolidinyl, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyls containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyls containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyls containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyls containing three heteroatoms include, but are not limited to, hexahydrotriazinyl. Exemplary 7-membered heterocyclyls containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 5-membered heterocyclyls fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyls) include, but are not limited to, dihydroindolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.Exemplary 6-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Heterocyclyl groups also include those in which the aforementioned heterocyclyl groups share one or two atoms with a cycloalkyl, heterocyclyl, aryl, or heteroaryl group to form a bridged or spirocyclic ring. The shared atoms may be carbon or nitrogen atoms as long as valence permits. Heterocyclyl groups also include those in which the aforementioned heterocyclyl and heterocyclyl groups may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0160] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, an aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms ("C 10 Aryl also includes ring systems in which an aryl ring as described above is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the aryl ring, in which case the number of carbon atoms continues to refer to the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0161] "5-14 membered heteroaryl" or "5 to 14 membered heteroaryl" refers to a group of a 5-14 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom as long as the valence permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the above-mentioned heteroaryl ring is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-10 membered heteroaryl is preferred, which is a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryls are particularly preferred and are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryls containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryls containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryls containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryls containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryls containing one heteroatom include, but are not limited to, pyridinyl or pyridonyl. Exemplary 6-membered heteroaryls containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azacycloheptatrienyl, oxepinyl, and thieptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indanyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. A heteroaryl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0162] "Optionally substituted..." means that the group may be substituted with a designated substituent or may be unsubstituted.

[0163] The divalent groups formed by removing another hydrogen from the above-defined alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups are collectively referred to as "subunits". The ring-forming groups such as cycloalkyl, heterocyclyl, aryl and heteroaryl groups are collectively referred to as "cyclyls".

[0164] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like are defined herein as optionally substituted groups.

[0165] "Nucleic acid" refers to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules and hybrid molecules thereof. Examples of nucleic acid molecules include, but are not limited to, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), and antisense oligonucleotides (ASOs). The nucleic acid may be further chemically modified, and the chemical modification is selected from one or a combination of pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, and 5-methylcytosine. The mRNA molecule contains a protein coding region and may further contain an expression regulatory sequence. Typical expression regulatory sequences include, but are not limited to, a 5' cap (5' cap), a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), a polyadenylic acid sequence (PolyA), and a miRNA binding site.

[0166] "Cationic lipid" refers to a lipid molecule that is capable of being positively charged under physiological pH conditions. In some embodiments, the cationic lipid is an amino lipid.

[0167] "Neutral lipids" refer to lipid molecules that are uncharged under specific pH conditions, such as physiological pH conditions. Examples of neutral lipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0168] "Structured lipids" refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids, such as steroids. Steroids are compounds with a cyclopentanylphenyl carbon skeleton. In a preferred embodiment, the steroid is selected from cholesterol, sitosterol, coprosterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol, or campesterol.

[0169] "Polymer lipid" refers to a molecule containing a polymer portion and a lipid portion. In some embodiments, the polymer lipid is a polyethylene glycol (PEG) lipid. Other lipids that can reduce aggregation, such as lipid-coupled compounds with uncharged, hydrophilic, steric barrier moieties, can also be used.

[0170] "Lipid nanoparticles" refer to particles containing lipid components and having nanometer dimensions.

[0171] "Biodegradable groups" refer to functional groups containing biodegradable bonds, such as esters, disulfide bonds, and amides. Biodegradation can affect the elimination of compounds from the body. The biodegradable groups of the present invention are oriented from the head to the tail of the ionizable lipid molecule.

[0172] Other definitions

[0173] The term "treat" as used herein relates to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such a disorder or condition. The noun "treat" as used herein relates to the action of the verb treat, which is as just defined.

[0174] Herein, compounds are named using standard nomenclature. For compounds with asymmetric centers, it should be understood that (unless otherwise specified) all optical isomers and mixtures thereof are encompassed. In addition, unless otherwise specified, all isomeric compounds encompassed by the present invention may occur in both Z and E forms with carbon-carbon double bonds. Compounds that exist in different tautomeric forms are not limited to any particular tautomer, but are intended to encompass all tautomeric forms.

[0175] As used herein, the term "pharmaceutically acceptable salt" refers to those carboxylate salts, amino acid addition salts of the compounds of the present invention that are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

[0176] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations include sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines include N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine.

[0177] Base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of this invention, the salts are equivalent to their respective free acids.

[0178] Salts can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides prepared from inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, methanesulfonate, glucoheptonate, lactobionate, laurylsulfonate, and isethionate, and the like. Salts can also be prepared from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Representative salts include acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Pharmaceutically acceptable salts may include cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Salts of amino acids, such as arginate, gluconate, galacturonate, and the like are also contemplated (see, e.g., Berge SM et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

[0179] The term "vaccine" refers to a composition that can provide active acquired immunity and / or therapeutic effects (such as treatment) for a specific disease or pathogen. Vaccines generally contain one or more medicaments that can induce an immune response against a pathogen or disease (i.e., a target pathogen or disease) in an individual. The immunogenic formulation stimulates the body's immune system to recognize the agent as a threat or indication of the presence of a target pathogen or disease, thereby inducing immune memory so that the immune system is more easily identified and eliminates any pathogen when subsequently contacted. Vaccines can be preventive (such as preventing or improving the effects of any natural or future infection of pathogens or the effects of cancer expected to occur in susceptible individuals) or therapeutic (such as treating cancer in individuals diagnosed with cancer). The administration of a vaccine is called vaccination. In some instances, a vaccine composition can provide nucleic acid, such as mRNA encoding an antigenic molecule (such as a peptide), to an individual. The nucleic acid delivered to the individual by the vaccine composition can be expressed as an antigenic molecule and allows the individual to obtain immunity against the antigenic molecule. In the case of vaccination against infectious diseases, the vaccine composition can provide mRNA encoding antigenic molecules associated with specific pathogens, such as one or more peptides known to be expressed in pathogens (e.g., pathogenic bacteria or viruses). In the case of viral vaccines, the vaccine composition can provide mRNA encoding certain viral peptides that are characteristic of the virus for which immunity is sought, such as peptides that are essentially exclusively or highly expressed on the surface of the virus (e.g., capsid proteins). After vaccination with the viral vaccine composition, an individual can have immunity against the viral peptides, specifically killing cells that express them.

[0180] The term "nucleic acid vaccine" used in this article is also called gene vaccine, which is to directly introduce the genetic genetic material (DNA or RNA) that determines the specific antigens of the pathogen into human cells, allowing the human cells to produce these antigens themselves and stimulate the body to produce an immune response to the antigen, so that the vaccine recipient obtains corresponding immune protection. It also contains adjuvants (such as lipid nanoparticles).

[0181] The term "delivery system" as used herein refers to a class of substances that can carry antigenic substances to the body's immune system, where they are stored and exert their antigenic effects for a long time. The vaccine delivery system described herein can be a liposome adjuvant vaccine delivery system or a nanoadjuvant vaccine delivery system.

[0182] The term "adjuvant" as used herein refers to a pharmaceutically acceptable substance that enhances the immune response to an antigen when co-administered with an antigen or before, during, or after the antigen is administered to a subject, including but not limited to: nucleic acid adjuvants (such as nucleic acid vectors), plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharide, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), and emulsion adjuvants (such as Freund's adjuvant).

[0183] The term "virus" is used according to its ordinary meaning in the field of biology to refer to a virus that includes the viral genome (e.g., DNA, RNA, single-stranded, double-stranded), a proteinaceous protective capsid (e.g., capsid protein) and associated proteins, and in the case of enveloped viruses (e.g., herpes viruses), an envelope including lipids and optionally host cell membrane components, and / or viral proteins.

[0184] The terms "viral infection" or "viral disease" refer to a disease or condition caused by a virus, including both symptomatic and asymptomatic infections. Non-limiting examples of viral infections include hepatoviral diseases (e.g., hepatitis A, B, C, D, E), herpesvirus infections (e.g., HSV-1, HSV-2, herpes zoster), flavivirus infections, Zika virus infection, cytomegalovirus infection, respiratory viral infections (e.g., adenovirus infection, influenza, severe acute respiratory syndrome, coronavirus infection (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV, COVID-19, MERS)), gastrointestinal viral infections (e.g., norovirus infection, rotavirus infection, astrovirus infection), exanthematous viral infections (e.g., measles, herpes zoster, smallpox, rubella), viral hemorrhagic diseases (e.g., Ebola virus, Lassa fever, dengue fever, yellow fever), neuroviral infections (e.g., West Nile virus infection, poliomyelitis, viral meningitis, viral encephalitis, Japanese encephalitis, rabies), and human papillomavirus infection.

[0185] SARS-CoV-2 belongs to the betacoronavirus family, whose members include two other zoonotic viruses that have caused severe disease outbreaks in the new millennium: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). The term "SARS-CoV" refers to the SARS coronavirus. The term "SARS-CoV" includes any coronavirus, such as SARS-CoV-2, SARS-CoV-1, and MERS-CoV.

[0186] The term "immunization" refers to administering a composition (e.g., a lipid nanoparticle composition) to a subject in an amount sufficient to induce a desired immune response (e.g., a humoral immune response or a cellular immune response to a nucleic acid vaccine) after one or more administration steps. Immunization can include administering (e.g., injecting) a composition from one to ten or more times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations. The first administration may not induce a detectable immune response because each subsequent administration will typically enhance the immune response generated by the previous administration. As used herein, the term "immunization" includes post-exposure prophylaxis.

[0187] The term "immune response" refers to the initiation of activity of one or more immune cell types in a subject. An immune response includes, for example, T cell and B cell responses.

[0188] The term "humoral immune response" refers to an immune response that produces plasma or serum antibodies (eg, IgG).

[0189] In the context of a disease, the term "prevent" and its various grammatical conjugations "prevention" means preventing the clinical symptoms of the disease from occurring in an individual who has not already experienced or developed symptoms of the disease.

[0190] As used herein, and unless otherwise indicated, the term "treating" includes actions that occur while a subject has a particular disease, disorder, or condition that reduces the severity of, or delays or slows the development of, the disease, disorder, or condition ("therapeutic treatment"), as well as actions that occur before a subject develops a particular disease, disorder, or condition ("prophylactic treatment").

[0191] The term "treat" as used herein relates to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such a disorder or condition. The noun "treat" as used herein relates to the action of the verb treat, which is as just defined.

[0192] "Subjects" to be administered include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0193] "Disease," "disorder," and "condition" are used interchangeably herein.

[0194] The term "effective amount" refers to an amount of a formulation according to the present invention that, when administered to a patient for treating a state, disorder or condition, is sufficient to achieve such treatment or that, when administered to a patient for generating an immune response, is sufficient to generate such an immune response. The "effective amount" will vary depending on the active ingredient, the state, disorder or condition to be treated and its severity, and the age, weight, physical condition and responsiveness of the subject to be treated.

[0195] Unless otherwise specified, a "prophylactically effective amount" is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or an amount to prevent the recurrence of a disease, disorder, or condition. A prophylactically effective amount of a pharmaceutical composition refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a disease, disorder, or condition. The term "prophylactically effective amount" can include an amount that improves overall prevention or enhances the prophylactic effect of other prophylactic agents.

[0196] "Combination" and related terms refer to the simultaneous or sequential administration of a pharmaceutical composition of the present invention and other therapeutic agents. For example, a pharmaceutical composition of the present invention can be administered simultaneously or sequentially with the other therapeutic agents in separate unit dosage forms, or can be administered simultaneously with the other therapeutic agents in a single unit dosage form. Example

[0197] In order to make the technical solution of the present invention clearer and more specific, the present invention is further described in detail by the following examples. The following examples are only used to illustrate the specific embodiments of the present invention so that those skilled in the art can understand the present invention, but are not intended to limit the scope of protection of the present invention. In the specific embodiments of the present invention, the technical means or methods not specifically described are conventional technical means or methods in the art. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0198] Table 1

[0199] Example 1: Synthesis of Compound 1

[0200] To a 2 L four-necked round-bottom flask, THF (160 mL) and compound 1-1 (80 g, 688.7 mmol) were added at room temperature. The system was cooled to -40°C and LDA (344.4 mL, 688.7 mmol) was added dropwise over 2 hours. The mixture was stirred at -40°C for 1 hour. Dibromobutane (206.70 g, 957.3 mmol) was then added dropwise at -40°C over 30 minutes. Finally, DMPU (12.7 g, 99.2 mmol) was added dropwise at -40°C over 2 hours. The mixture was kept at -40°C for 1 hour. The mixture was stirred with the mixture warming to room temperature for 15 hours. The reaction was monitored by TLC to confirm the complete consumption of the starting material. The reaction was quenched with saturated NH4Cl (200 mL) at 0°C, diluted with water (160 mL) and ethyl acetate (160 mL), and the mixture was extracted and separated. The aqueous phase was back-extracted once more with ethyl acetate (200 mL). The organic phases were combined and washed twice with saturated NaCl (300 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column to obtain compound 1-2 (100 g) as a light yellow oil.

[0201] To a 2-L four-necked round-bottom flask, THF (1 L) and compound 1-2 (98 g, 391.91 mmol) were added at room temperature. The system was cooled to -10°C and LiAlH₄ (313.5 mL, 313.5 mmol) was added dropwise over 30 minutes. The system was stirred at -10°C for 30 minutes, and the reaction was monitored by TLC, indicating complete conversion of the starting material. The reaction was quenched with Na₂SO₄.10H₂O (125 g) at -4°C, diluted with water (500 mL) and ethyl acetate (5 L), extracted, and separated. The aqueous phase was back-extracted once with ethyl acetate (300 mL). The combined organic phases were washed twice with saturated NaCl (500 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was passed through a silica gel column to afford 1-3 (73 g) as a pale yellow oil.

[0202] To a 250 mL three-necked flask, DMSO (40 mL) and NaH (3.83 g, 95.7 mmol) were added at room temperature. TosMIC (4.67 g, 23.9 mmol) was added portionwise at 25°C under nitrogen, followed by TBAI (883.5 mg, 2.39 mmol). The system was stirred at 25°C for 15 minutes. Finally, compound 1-3 (10 g, 47.8 mmol) was dissolved in DMSO (100 mL) and added dropwise at 25°C. The system was stirred at 25°C for an additional hour. TLC confirmed complete conversion of the starting material. The reaction was quenched with ice water (400 mL) at 20°C and extracted with MTBE (2 x 500 mL). The combined organic phases were washed once with saturated NaCl (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product (10 g) was used directly in the next step.

[0203] Crude compound 1-4 (10 g), CH3OH (100 mL), and 12N HCl (5.3 mL) were added to a 500 mL three-necked flask at room temperature. The system was reacted at 80°C for 3 h. LCMS monitoring of the reaction showed complete conversion of the starting material. The system was cooled to room temperature, adjusted to pH = 7 with saturated sodium carbonate, and concentrated under reduced pressure to remove methanol. The system was dissolved and diluted with water (100 mL) and ethyl acetate (100 mL), extracted, and separated. The aqueous phase was back-extracted once with ethyl acetate (100 mL), and the combined organic phases were washed once with saturated NaCl (100 mL). The organic phases were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was passed through a silica gel column to obtain compound 1-5 (4.6 g).

[0204] Compound 1-5 (2.5 g, 8.73 mmol), n-decanoic acid (1.50 g, 8.73 mmol), DMAP (53.3 mg, 0.431 mmol), and DCM (25 mL) were added to a 100 mL three-necked flask at room temperature. EDCI (1.67 g, 8.73 mmol) was added at 0°C, and the system was allowed to react at 20°C for 18 h. The reaction was monitored by TLC and LCMS, indicating the presence of product. The reaction system was quenched with saturated NH4Cl (25 mL), extracted, and separated. The aqueous phase was back-extracted once with DCM (25 mL), and the combined organic phases were washed once with saturated NaCl (50 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column to afford a pale yellow compound 1-6 (1.71 g).

[0205] Compound 1-6 (240 mg, 0.55 mmol), compound 1-7 (145.2 mg, 0.60 mmol), EDCI (156.6 mg, 0.82 mmol), DMAP (13.3 mg, 0.11 mmol), and DCM (3 mL) were added to an 8 mL sealed tube at room temperature. The system was reacted at 20°C for 18 h. TLC monitoring confirmed complete conversion of the starting material. The reaction system was quenched with saturated NH4Cl (10 mL), diluted with DCM (10 mL), extracted, and separated. The aqueous phase was back-extracted once with DCM (15 mL). The combined organic phases were washed once with saturated NaCl (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified on a silica gel column to afford compound 1-8 (301.8 mg) as a pale yellow oil.

[0206] Compound 1-8 (301.8 mg, 0.45 mmol), methanol (4 mL), and THF (1 mL) were added to a 25 mL three-necked flask at room temperature. NaBH₄ (17.2 mg, 0.45 mmol) was then added at 0°C. The reaction was allowed to react at 20°C for 1 h. TLC confirmed complete reaction of the starting material. The reaction system was quenched with water (20 mL), diluted with ethyl acetate (20 mL), extracted, and separated. The aqueous phase was back-extracted once with ethyl acetate (20 mL). The combined organic phases were washed once with saturated NaCl (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product (273.8 mg) was directly used in the next step.

[0207] Compound 1-9 (273.8 mg, 0.41 mmol), 4-dimethylaminobutyric acid (137.6 mg, 0.82 mmol), EDCI (236.0 mg, 1.23 mmol), DMAP (50.1 mg, 0.41 mmol), and DCM (3 mL) were added to an 8 mL sealed tube at room temperature. The system was reacted at 20°C for 2 h. TLC confirmed complete conversion of the starting material. The reaction system was quenched with saturated NH4Cl (10 mL), diluted with DCM (10 mL), extracted, and separated. The aqueous phase was back-extracted once with DCM (10 mL). The combined organic phases were washed once with saturated NaCl (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified on a silica gel column to afford Compound 1 (239.6 mg) as a pale yellow oil.

[0208] 1 H NMR (300MHz, CDCl3) δ: 4.85 (p, J=6.2Hz, 1H), 3.77 (s, 4H), 2.31 (ddd, J=14.8, 7.4, 4.3Hz, 12H),1.65-1.49(m,11H),1.36-1.20(m,52H),0.89(s,15H),0.88(t,J=6.8Hz,7H); ESI-MS m / z:780.65[M+H] + .

[0209] Example 2: Synthesis of Compound 2

[0210] To an 8 mL sealed tube, add the starting compound 1-6 (150 mg, 0.34 mmol), compound 2-1 (96.01 mg, 0.374 mmol), EDCI (97.87 mg, 0.51 mmol), DMAP (8.32 mg, 0.068 mmol), and DCM (3 mL). Stir at room temperature for 18 hours. TLC monitoring indicated the absence of starting material. Silica gel was added and the sample was stirred. Purification by column chromatography afforded 196 mg of 2-2 as a light yellow oil.

[0211] To a 25 mL three-necked flask, add the starting compound 2-2 (196 mg, 0.29 mmol), MeOH (4 mL), and THF (1 mL). Cool to 0°C, and add NaBH4 (10.92 mg, 0.29 mmol). Stir at room temperature for 1 hour. TLC monitoring indicates no starting material. Add saturated aqueous ammonium chloride (10 mL), and extract with MTBE (1 x 20 mL). The organic phase is washed with saturated brine (1 x 20 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to yield 140 mg of product 2-3, which is used directly in the next reaction.

[0212] To an 8 mL sealed tube, add the starting compound 2-3 (140 mg, 0.21 mmol), 4-dimethylaminobutyric acid (41.35 mg, 0.25 mmol), EDCI (59.10 mg, 0.31 mmol), DMAP (5.02 mg, 0.041 mmol), and DCM (2 mL). The reaction was allowed to react at room temperature for 5 hours. TLC monitoring indicated the absence of starting material. Silica gel was added and the sample was stirred. Purification by column chromatography afforded 105.9 mg of compound 2.

[0213] 1 H NMR (300MHz, CDCl3) δ: 4.85 (q, J = 6.3Hz, 1H), 3.77 (s, 4H), 2.45-2.22 (m, 13H), 1.84 (p, J = 7.4Hz, 2H), 1.61 (d, J = 7 .2Hz,4H),1.52(d,J=6.1Hz,5H),1.27(qd,J=6.8,4.5,3.4Hz,46H),0.89(s,17H),0.88(d,J=13.3Hz,3H); ESI-MS m / z:794.70[M+H] + .

[0214] Example 3: Synthesis of Compound 3

[0215] To an 8 mL sealed tube, add compound 1-6 (150 mg, 0.34 mmol), compound 3-1 (85.50 mg, 0.374 mmol), EDCI (97.87 mg, 0.510 mmol), DMAP (8.32 mg, 0.068 mmol), and DCM (2 mL). Stir at room temperature for 18 hours with TLC monitoring. The sample was directly mixed and purified by column chromatography to obtain 165.6 mg of 3-2 as a yellow oil.

[0216] To a 25 mL three-necked flask, add compound 3-2 (165.6 mg, 0.25 mmol), MeOH (4 mL), and THF (1 mL). Cool to 0°C and add NaBH4 (9.62 mg, 0.25 mmol). Stir at room temperature for 1 hour, monitoring by TLC. After the reaction is complete, add 10 mL of saturated aqueous NH4Cl solution. Extract with EA (1 x 20 mL), and wash the organic phase with saturated brine (1 x 10 mL). Dry over anhydrous Na2SO4 and concentrate in vacuo to yield 166 mg of crude compound 3-3, which is used directly in the next step.

[0217] To an 8 mL sealed tube, add compound 3-3 (166 mg, 0.25 mmol), 4-dimethylaminobutyric acid (51.13 mg, 0.31 mmol), EDCI (73.09 mg, 0.38 mmol), DMAP (6.21 mg, 0.051 mmol), and DCM (2 mL). Stir at room temperature for 5 hours with TLC monitoring. The sample was directly mixed and purified by column chromatography using DCM / MeOH (50:1) to afford 142.4 mg of compound 3 as a light yellow oil.

[0218] 1 H NMR (300MHz, CDCl3) δ: 4.87 (q, J = 6.2Hz, 1H), 3.77 (s, 4H), 2.39-2.26 (m, 12H), 1.84 (q, J = 7.4Hz ,2H),1.68-1.54(m,5H),1.52(d,J=6.2Hz,5H),1.34-1.19(m,47H),0.91-0.86(m,21H); ESI-MS m / z:766.55[M+H] + .

[0219] Example 4: Synthesis of Compound 4

[0220] A solution of compound 4-1 (100 g, 979 mmol) in tetrahydrofuran (800 mL) was cooled to -40°C. LDA (2 M, 490 mL) was slowly added dropwise to the solution. Stirring was continued for 1 hour. A solution of 4-2 (315 g, 1.37 mol) in tetrahydrofuran (100 mL) was added dropwise to the reaction system at the same temperature. The reaction system was stirred overnight. The reaction system was quenched with saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain compound 4-3 (115 g).

[0221] A solution of compound 4-3 (100 g, 398 mmol), TosMIC (38.9 g, 199 mmol), and TBAI (14.7 g, 39.8 mmol) in tetrahydrofuran (800 mL) was cooled to 0°C. Sodium hydride (20.7 g, 517 mmol) was slowly added portionwise and allowed to react overnight at room temperature. The reaction system was quenched with saturated sodium chloride solution, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain 115 g of crude product 4-4, which was used directly in the next step without further purification.

[0222] To a solution of compound 4-4 (110 g, 205 mmol) in dichloromethane (880 mL) was added 330 mL of concentrated hydrochloric acid and allowed to react at room temperature for 2 hours. TLC confirmed the complete reaction. The reaction system was quenched with saturated aqueous ammonium chloride and extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain the crude product. Compound 4-5 (30.0 g, 80.9 mmol, 39.4% yield) was obtained by silica gel column separation and purification.

[0223] TMSOK (11.0 g, 86.4 mmol) was added to a solution of compound 4-5 (8.0 g, 21.6 mmol) in tetrahydrofuran (35.0 mL) at room temperature. The reaction system was heated to 70°C with stirring. TLC confirmed complete consumption of the starting material. The reaction solution was cooled to room temperature, and the organic solvent was removed by rotary evaporation. The crude product was added with 20 mL of water and extracted with dichloromethane. The aqueous phase was collected, the pH of the solution was adjusted to less than 5 with 1 M hydrochloric acid, and the solution was extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was collected by filtration and concentrated to obtain compound 4-6 (7.0 g).

[0224] Potassium carbonate (1.55 g, 11.2 mmol) was added to a DMF solution of compound 4-6 (959 mg, 2.8 mmol) and 1-bromononane (638 mg, 3.08 mmol). The reaction was then heated to 60°C for 4 hours, cooled to room temperature, quenched with saturated sodium chloride solution, extracted with ethyl acetate, and the organic phases combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain the crude product, which was purified on a silica gel column to obtain compound 4-8 (682 mg).

[0225] To a 20 mL sealed tube, compound 4-8 (600 mg, 1.28 mmol), compound 4-9 (329.3 mg, 1.54 mmol), EDCI (294.5 mg, 1.54 mmol), DMAP (156.4 mg, 1.28 mmol), and DCM (5 mL) were added. The reaction was allowed to react overnight at room temperature. LCMS monitoring indicated a product signal. The mixture was quenched with 10 mL of water and extracted with CH2Cl2 (1 x 10 mL). The organic phase was washed with saturated brine (1 x 10 mL), dried over Na2SO4, filtered, and purified by column chromatography to afford compound 4-10 (543 mg).

[0226] Under nitrogen, compound 4-10 (543 mg, 0.83 mmol) and MeOH (10 mL) were added to a 50 mL three-necked flask. NaBH4 (157.8 mg, 4.17 mmol) was added portionwise at 0°C and allowed to react for 2 h at room temperature. The reaction was quenched by the addition of 20 mL of water dropwise at 0°C and extracted with EtOAc (1 x 20 mL). The organic phase was washed with saturated brine (1 x 20 mL), dried over Na2SO4, filtered, and spun down to afford compound 4-11 (480 mg).

[0227] To an 8 mL sealed tube, 4-11 (480 mg, 0.74 mmol), SM4 (289.23 mg, 2.21 mmol), EDCI (422.68 mg, 2.21 mmol), DMAP (89.8 mg, 0.74 mmol), and DCM (3 mL) were added and allowed to react overnight at room temperature. LCMS monitoring indicated product signal. The mixture was quenched with 10 mL of water and extracted with DCM (2 x 20 mL). The organic phase was washed with brine (1 x 20 mL), dried over Na2SO4, filtered, and purified by Prep-TLC (DCM / MeOH 10:1) to afford 4 (118.3 mg).

[0228] 1 H NMR (300MHz, CDCl3) δ: 4.86 (t, J = 6.2 Hz, 1H), 4.06 (td, J = 6.6, 2.5 Hz, 4H), 2.40 (t, J = 7.0 Hz, 2H), 2.00 (s ,2H),1.66-1.56(m,5H),1.50(s,9H),1.27(d,J=12.8Hz,45H),1.17(s,13H),0.96-0.86(m,9H); ESI-MS m / z:766.80[M+H] + .

[0229] Example 5: Synthesis of Compound 5

[0230] Compound 5 was prepared according to the synthetic method of Example 70 of CN115850104A.

[0231] Pharmacological experiments

[0232] Experimental Example 1: Nanoparticle Preparation

[0233] The materials used for the assembly of lipid nanoparticles are: (1) ionizable lipid compounds, such as Example compounds 1-5 or ALC0315 (purchased from AVT); (2) structural lipids, such as cholesterol (purchased from Sigma-Aldrich); (3) phospholipids, such as DSPC (1,2-distearoylphosphatidylcholine, purchased from AVT) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, purchased from AVT); (4) polyethylene glycol lipid compounds, such as DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol); glycol-2000 (purchased from AVT) or ALC0159 (purchased from Sinobond); (5) nucleic acid fragment active ingredient: novel coronavirus spike protein mRNA (self-made, coding sequence identical to SARS-CoV-2 S Protein Variant 1 in WO2021154763A1 (Table 1)) or varicella-zoster virus envelope glycoprotein E mRNA (self-made, coding sequence Uniprot: Q9J3M8). The names and structural formulas of lipid nanoparticle assembly materials are detailed in Table 2.

[0234] Preparation method of lipid nanoparticles: (1) Ionizable lipid compound, cholesterol, phospholipid and PEGylated lipid are dissolved and mixed in ethanol in sequence according to the preferred formula. The optimized formulas of Example compounds 1-5 of the present invention are shown in Table 3. The preferred formula of ALC0315 can be found in the literature (mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability, International Journal of Pharmaceutics, 2021), the LNP formula is detailed in Table 3; (2) the mRNA active ingredient was dissolved in 25 mM sodium acetate solution (pH = 4.5); (3) an automated high-throughput microfluidic system was used to mix the organic phase containing the lipid mixture and the aqueous phase containing the mRNA component at a flow rate ratio ranging from 1:1 to 1:4, and the mixing speed was between 10 mL / min and 18 mL / min; (4) the prepared lipid nanoparticles were diluted with phosphate buffered saline solution, and the nanoparticle solution was ultrafiltered to the original preparation volume using an ultrafiltration tube with a molecular weight cutoff of 30 kDa (purchased from Millipore); (5) the obtained nanoparticles were sterilized by filtration through a 0.2 μm sterile filter membrane and stored at low temperature in a sealed glass bottle.

[0235] Methods for preparing lipid nanoparticles include, but are not limited to, microfluidic mixing systems and also include T-type mixers and ethanol injection methods.

[0236] Table 2

[0237] Table 3

[0238] Experimental Example 2: Characterization of physical properties of lipid nanoparticles

[0239] The particle size and particle size dispersion index (PDI) of the prepared lipid nanoparticles were measured using Zetasizer Pro (purchased from Malvern Instruments Ltd) and DynaPro NanoStar (purchased from Wyatt) dynamic light scattering instruments. The degree of RNA encapsulation of lipid nanoparticles is characterized by the encapsulation efficiency (Encapsulation Efficiency%), which reflects the degree of binding between lipid nanoparticles and RNA fragments. This coefficient is measured by Quant-it TMThe lipid nanoparticle sample was diluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 7.5), and a portion of the sample solution was added with 0.5% Triton X-100 and allowed to stand at 37°C for 30 minutes. Immediately after the reaction, the fluorescence value was read on a Varioskan LUX multifunctional microplate reader (purchased from Thermofisher) at an absorption wavelength of 485 nm and an emission wavelength of 528 nm to obtain the encapsulation efficiency value.

[0240] Experimental Example 3: Mouse Muscle Immunization

[0241] The adaptive immune effect of lipid nanoparticles encapsulating viral antigen protein mRNA in mice was evaluated. The experimental mice were SPF-grade BALB / c female mice, 6-8 weeks old, weighing 18-22g, purchased from Beijing Sibeifu Biotechnology Co., Ltd. All animals were adaptively raised for more than 7 days before the experiment. During the experiment, they had free access to food and water, and the light was alternating between 12 / 12h of light and dark. The indoor temperature was 20-26°C and the humidity was 40-70%. The mice were randomly divided into an immunization group and a negative control group (DPBS, pH=7.4), with 5 mice in each group. The prepared lipid nanoparticles encapsulating viral antigen protein mRNA were injected intramuscularly into the immunization group mice according to the immunization dose in Table 4. Two immunizations were performed, the primary immunization and the booster immunization, with an interval of several days between the two immunizations.

[0242] Table 4

[0243] Experimental Example 4: Evaluation of humoral immunity effect

[0244] On the 10th day after each immunization, blood was collected from the mice through their eye sockets to obtain sufficient serum to evaluate the humoral immune response produced by lipid nanoparticles encapsulating mRNA encoding viral antigen proteins in mice.

[0245] 1. Detection of novel coronavirus-specific binding antibodies

[0246] The novel coronavirus spike protein (purchased from Sino Biological) was diluted to 2 ng / μl with carbonate buffer (50 mM, pH 9.6, filtered with a 0.22 μm membrane) to form a protein coating solution, which was added to a 96-well plate and sealed at 4°C overnight. After overnight coating, the 96-well plate was poured to remove the protein coating solution, and washing solution (1×TBS containing 0.2% Tween-20, purchased from Solebol) was added to each well for washing. After 6 washes, blocking solution (1×TBS containing 2% BSA, purchased from Solebol) was added to each well and incubated at 37°C for blocking. After blocking for 2 hours, the washing operation was repeated. The immune mouse serum was diluted 10-fold with antibody diluent (washing solution containing 0.5% BSA) to obtain 10 -1 to 10 -6 Serum of different dilutions was added to the well plate and incubated in a 37°C incubator; after incubation for 2 hours, the plate was washed repeatedly; horseradish peroxidase-labeled goat anti-mouse IgG (purchased from Beyotime) was diluted 250 times with antibody diluent (washing solution containing 0.5% BSA) to obtain secondary antibody diluent and added to the well plate for incubation at 37°C; after incubation for 1 hour, the plate was washed repeatedly; TMB substrate was added and incubated for 20 minutes at room temperature in the dark for color development; finally, stop solution (purchased from Solebow) was added to terminate the reaction and the A450 OD value was detected on a microplate reader.

[0247] Serum binding antibody IgG titer determination: OD of a certain serum dilution / negative control OD ≥ 2.1, and OD of the next dilution / negative control OD < 2.1. This dilution factor is the corresponding antibody titer for that serum sample (if the negative control OD is < 0.05, it is calculated as 0.05). The SARS-CoV-2 spike protein binding antibody titer of sera from mice immunized with lipid nanoparticles loaded with SARS-CoV-2 spike protein mRNA is detailed in Table 5.

[0248] 2. Evaluation of the Neutralizing Effect of Specific Antibodies Against the Novel Coronavirus

[0249] The neutralizing effect of antibodies in mouse serum was evaluated using a pseudovirus containing the novel coronavirus spike protein.

[0250] The mouse immune serum was serially diluted 3-fold with DMEM complete medium (purchased from Gibco) to obtain 6 different dilutions of serum, which were then mixed with 650 TCID 50 Pseudovirus (purchased from Yunling Biotechnology Co., Ltd.) was incubated at 37°C. At the same time, a cell control group without pseudovirus and a pseudovirus control group without serum samples were set up. After incubation for one hour, 2×10 4Huh7 cells (hepatocellular carcinoma cells) were cultured at 37°C in 5% CO2. Since the pseudovirus enters the cells, it expresses firefly luciferase. After 24 hours, the cells react with the luminescent substrate and luminescence is detected. The percentage of pseudovirus inhibition is calculated by comparing the luminescence value with that of the pseudovirus control group. The dilution of serum that inhibits 50% of the pseudovirus is calculated using the Reed–Muench method, i.e., the half-neutralization dilution (NTD). 50 ). NT 50 The neutralization activity of serum antibodies against pseudoviruses is shown in Figure NT. 50 See Table 5 for details.

[0251] The data in Table 5 show that the humoral immune response of the novel coronavirus vaccine prepared with the compounds of the present invention is significantly superior to that of the control, ALC0315, in terms of both serum IgG titer and serum neutralization capacity. When the degradable group of the compounds of the present invention (corresponding to the general formula groups M1 and M2) is -OC(O)-, the serum neutralization capacity is superior to that of compounds containing the group -C(O)O-.

[0252] Table 5

[0253] 3. Varicella-zoster virus envelope glycoprotein E specific binding antibody detection

[0254] Recombinant varicella-zoster virus (Oka strain vaccine) envelope glycoprotein E (purchased from Nearshore Protein, DRA224) was diluted to 1 ng / μl with carbonate buffer (50 mM, pH 9.6, filtered with a 0.22 μm membrane) to form a protein coating solution, which was added to a 96-well plate, sealed, and incubated at 4°C overnight. After overnight coating, the 96-well plate was poured to remove the protein coating solution, and washing solution (1×TBS containing 0.2% Tween-20, purchased from Solebol) was added to each well for washing. After washing the plate six times, blocking solution (1×TBS containing 2% BSA, purchased from Solebol) was added to each well and incubated at 37°C for blocking. After blocking for 2 hours, the plate washing operation was repeated. The immune mouse serum was serially diluted 10-fold with antibody diluent (washing solution containing 0.5% BSA) to obtain 10 -1 to 10 -6Serum at different dilutions was added to the plate and incubated at 37°C. After a 2-hour incubation, the plate was washed repeatedly. A secondary antibody dilution solution containing horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (purchased from Beyotime, A0216) was diluted 250-fold in antibody diluent (washing buffer containing 0.5% BSA) and added to the plate for incubation at 37°C. After a 1-hour incubation, the plate was washed repeatedly. TMB substrate (purchased from Tiangen) was added and incubated at room temperature, protected from light, for 20 minutes for color development. Finally, the reaction was terminated with stop solution (purchased from Solebol) and the A450 OD value was measured on a microplate reader. The titer of serum-bound IgG antibodies was determined as follows: if the OD of a given serum dilution was divided by the OD of the negative control ≥ 2.1, and the OD of the next dilution was less than 2.1, the titer corresponding to that serum sample was calculated (if the OD of the negative control was less than 0.05, it was calculated as 0.05). The titers of varicella-zoster virus envelope glycoprotein E binding antibodies in the serum of mice immunized with lipid nanoparticles loaded with varicella-zoster virus envelope glycoprotein E mRNA are shown in Table 6.

[0255] The data in Table 6 show that the nucleic acid vaccine encoding varicella-zoster virus prepared by the compound of the present invention produces a strong humoral immune response.

[0256] Table 6

[0257] Experimental Example 5: Evaluation of Cellular Immunity Effect

[0258] 1. Novel coronavirus-specific cellular immunity

[0259] On the 14th day after booster immunization, the spleens of the immunized mice and the negative control group were dissected, and the spleen cells were isolated to prepare single-cell suspensions. Mouse spleen cells were added with the novel coronavirus spike protein peptide (purchased from GenScript, 2 μg / ml / peptide) and specifically stimulated under cell culture conditions of 37°C and 5% CO2. After 18 hours of stimulation, the secretion of cytokines IFN-γ and IL-2 was detected using an enzyme-linked immunosorbent assay (ELISpot) detection kit (purchased from MABTECH). After the experiment, the color spot plate was naturally dried, and the spots were counted using the fully automatic AID iSpot ELISPOT plate reader, and various parameters of the spots were recorded. The secretion of IFN-γ and IL-2 specific to the novel coronavirus spike protein is detailed in Table 7.

[0260] It can be seen from the data in Table 7 that the novel coronavirus vaccine prepared by the compound of the present invention has a cellular immune effect significantly better than the control compound ALC0315.

[0261] Table 7

[0262] 2. Varicella-zoster virus-specific cellular immunity

[0263] On the 14th day after the booster immunization, the spleens of the immunized mice and the negative control group were dissected, and the spleen cells were isolated to prepare single-cell suspensions. Mouse spleen cells were added with a mixed overlapping peptide library of varicella-zoster virus (Oka strain vaccine) envelope glycoprotein E (purchased from GenScript, 2 μg / ml / peptide) and specifically stimulated under cell culture conditions of 37°C and 5% CO2. After 18 hours of stimulation, the secretion of cytokines IFN-γ and IL-2 was detected using the enzyme-linked immunosorbent assay (ELISpot) method. The ELISpot detection kit was purchased from MABTECH. The specific implementation method was referred to the kit instructions. After the experiment, the color spot plate was naturally dried, and the spots were counted using the fully automatic AID iSpot ELISPOT plate reader, and various parameters of the spots were recorded. The secretion of IFN-γ and IL-2 specific to the varicella-zoster virus envelope glycoprotein E is detailed in Table 8.

[0264] The data in Table 8 show that the varicella-zoster virus nucleic acid vaccine prepared by the compound of the present invention produces a strong cellular immune response.

[0265] Table 8

[0266] Experimental Example 6: In vitro safety assessment

[0267] 1. hERG test

[0268] The potential inhibitory effects of ionizable lipid compounds on human hERG channels were evaluated using the SyncroPatch 384i / 384 automated patch clamp system. CHO cells stably overexpressing the hERG gene were used for evaluation, with cisapride used as a positive control. Five concentrations of ionizable lipid compounds were set at 30, 10, 3.33, 1.11, and 0.37 μM, and the 50% inhibitory concentration (IC) was calculated. 50 The evaluation was completed by Pharmaron. The test results are detailed in Table 9.

[0269] 2. Mini-Ames test for Salmonella typhimurium and Escherichia coli

[0270] The ability of ionizable lipid compounds to induce reverse mutations in histidine-auxotrophic Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and tryptophan-auxotrophic Escherichia coli WP2 uvrA (pKM101) was assessed to evaluate the mutagenic potential of the test compounds. Ionizable lipid compounds were tested at doses of 1.5, 4, 10, 25, 64, 160, 400, and 1000 μg / well. Negative / solvent controls (methanol) and positive controls were also tested. Testing was performed by Pharmaron Chemicals. The test results are detailed in Table 9.

[0271] The data in Table 9 show that the compounds of the present invention have substantially no potassium channel inhibitory effect and toxicity and have good safety.

[0272] Table 9

[0273] Although the present invention has been fully described through its embodiments, it is noteworthy that various changes and modifications are obvious to those skilled in the art. Such changes and modifications should be included within the scope of the appended claims of the present invention.

Claims

1. An ionizable lipid compound having the structure of formula (I) or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof: in, M1 and M2 are independently selected from -C(O)O-, -OC(O)-, -OC(O)O-, -SC(O)-, -C(O)S-, -C(O)NR a -and-NR a C(O)-; a is independently selected from 0, 1, 2, 3, 4 and 5; b and d are independently selected from 3, 4, 5, 6, 7, 8 and 9; c and e are independently selected from 0, 1, 2, 3 and 4; b+c=3, 4, 5, 6, 7, 8 or 9, d+e=3, 4, 5, 6, 7, 8 or 9; The methylene group in the 1-6 Alkyl substitution; R1 and R2 are independently selected from C 4-20 Alkyl, C 4-20 Alkenyl and C 4-20 Alkynyl, the C 4-20 Alkyl, C 4-20 Alkenyl or C 4-20 Alkynyl is optionally substituted with one or more R, said C 4-20 Alkyl, C 4-20 Alkenyl or C 4-20 One or more methylene units in the alkynyl group are optionally and independently replaced by -NR"-; R is independently selected from H, C 1-14 Alkyl, -L a -OR a and -L a -NR a R' a ; R" is independently selected from H and C 1-20 alkyl; R3 and R4 are independently selected from H, C 1-6 Alkyl, C 1-6 haloalkyl, 3 to 10-membered cycloalkyl and 3 to 10-membered heterocyclic group, the C 1-6 Alkyl, C 1-6 haloalkyl, 3 to 10 membered cycloalkyl or 3 to 10 membered heterocyclyl is optionally substituted with 1, 2, 3, 4 or 5 R*; or R3, R4 and the N atom to which they are attached together form a 3- to 10-membered heterocyclic group, wherein the 3- to 10-membered heterocyclic group is optionally substituted by 1, 2, 3, 4 or 5 R*; R5, R6, R7 and R8 are independently selected from C 1-6 Alkyl, the C 1-6 Alkyl is optionally substituted with 1, 2, 3, 4 or 5 R*; R* is independently selected at each occurrence from H, halogen, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, -L b -OR b or -L b -NR b R' b ; L a Independently selected from chemical bonds and C 1-6 Alkylene; L b Independently selected from chemical bonds and C 1-6 Alkylene; R a and R' a Independently selected from H, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl; R b and R' b Independently selected from H, C 1-6 alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl.

2. The ionizable lipid compound according to claim 1 or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, characterized in that: in, M1 and M2 are independently selected from -C(O)O-, -OC(O)-, -OC(O)O-, -SC(O)- and -C(O)S-; Preferably, M1 and M2 are independently selected from -C(O)O- or -OC(O)-; more preferably, both M1 and M2 are selected from -C(O)O-; more preferably, both M1 and M2 are selected from -OC(O)-; Preferably, a is selected from 2, 3 or 4, more preferably 2 or 3, more preferably 2; Preferably, b and d are independently 4 or 5; c and e are independently 0 or 1; Preferably, b+c=4, 5 or 6, d+e=4, 5 or 6; more preferably, b+c=5, d+e=5.

3. An ionizable lipid compound having a structure represented by formula (II) or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof: in, R1 and R2 are independently selected from C 4-20 Alkyl, C 4-20 Alkenyl and C 4-20 Alkynyl, the C 4-20 Alkyl, C 4-20 Alkenyl or C 4-20 Alkynyl is optionally substituted with one or more R, said C 4-20 Alkyl, C 4-20 Alkenyl or C 4-20 One or more methylene units in the alkynyl group are optionally and independently replaced by -NR"-; R is independently selected from H, C 1-14 Alkyl, -L a -OR a and -L a -NR a R' a ; R" is independently selected from H and C 1-20 alkyl; R3 and R4 are independently selected from H, C 1-6 Alkyl, C 1-6 haloalkyl, 3 to 10-membered cycloalkyl and 3 to 10-membered heterocyclic group, the C 1-6 Alkyl, C 1-6 haloalkyl, 3 to 10 membered cycloalkyl or 3 to 10 membered heterocyclyl is optionally substituted with 1, 2, 3, 4 or 5 R*; or R3, R4 and the N atom to which they are attached together form a 3- to 10-membered heterocyclic group, wherein the 3- to 10-membered heterocyclic group is optionally substituted by 1, 2, 3, 4 or 5 R*; a is independently selected from 0, 1, 2, 3, 4 and 5; b and d are independently selected from 3, 4, 5, 6, 7, 8 and 9; c and e are independently selected from 0, 1, 2, 3 and 4; b+c=3, 4, 5, 6, 7, 8 or 9, d+e=3, 4, 5, 6, 7, 8 or 9; The methylene group in the 1-6 Alkyl substitution; R5, R6, R7 and R8 are independently selected from C 1-6 Alkyl, the C 1-6 Alkyl is optionally substituted with 1, 2, 3, 4 or 5 R*; R* is independently selected at each occurrence from H, halogen, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, -L b -OR b or -L b -NR b R' b ; L a Independently selected from chemical bonds and C 1-6 Alkylene; L b Independently selected from chemical bonds and C 1-6 Alkylene; R a and R' a Independently selected from H, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl; R b and R' b Independently selected from H, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl; Preferably, a is selected from 2, 3 or 4, more preferably 2 or 3, more preferably 2; Preferably, b and d are independently 4 or 5; c and e are independently 0 or 1; Preferably, b+c=4, 5 or 6, d+e=4, 5 or 6; more preferably, b+c=5, d+e=5.

4. The ionizable lipid compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, It is characterized in that, wherein R1 and R2 are independently selected from a straight-chain alkyl group with a total length of 6, 7, 8, 9 or 10 carbon atoms, and 1, 2 or 3 methylene groups in the straight-chain alkyl group are optionally and independently replaced by C 1-8 Alkyl substitution; Preferably, R1 or R2 are each independently a straight chain alkyl group having a total length of 9 carbon atoms, and one of R1 or R2 is replaced by C 4-6 Alkyl substitution.

5. The ionizable lipid compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, It is characterized in that, wherein R3 and R4 are independently selected from C 1-6 Alkyl, the C 1-6 Alkyl is optionally substituted with 1, 2 or 3 R*; R* is independently selected from H, C 1-6 Alkyl, C 1-6 Haloalkyl and -OR a ; Among them, R a Independently selected from H and C 1-6 alkyl; Preferably, R3 and R4 are independently selected from methyl.

6. The ionizable lipid compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, Characterized in that, wherein R5, R6, R7 and R8 are independently selected from C 1-6 Alkyl; preferably methyl or ethyl, more preferably methyl.

7. An ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein the ionizable lipid compound is selected from the following compounds:

8. A pharmaceutical composition comprising the ionizable lipid compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, and optionally a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, characterized in that in, The pharmaceutical composition is a nanoparticle composition: it comprises a lipid component and optionally an active ingredient; The lipid component comprises: 20 mol%-85 mol% of ionizable cationic lipids, 10 mol%-75 mol% of structural lipids, 1.0 mol%-30 mol% of neutral lipids, and 0.25 mol%-10 mol% of polymer lipids; wherein the ionizable cationic lipid is an ionizable lipid compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof; Preferably, the lipid component comprises: 30 mol%-50 mol% of ionizable cationic lipids, 30 mol%-60 mol% of structural lipids, 10 mol%-30 mol% of neutral lipids, and 0.25 mol%-5 mol% of polymer lipids; Preferably, the lipid component comprises: 35 mol%-45 mol% of ionizable cationic lipids, 40 mol%-50 mol% of structural lipids, 10 mol%-20 mol% of neutral lipids, and 0.5 mol%-2 mol% of polymer lipids; Preferably, the neutral lipid is selected from one or more of DSPC, DMPC, DOPC, DPPC, POPC, DOPE, DMPE, POPE or DPPE; Preferably, the structured lipid is selected from one or more of cholesterol, sitosterol, coprosterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol or campesterol; Preferably, the polymer lipid is a PEGylated lipid, and the PEGylated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

10. Use of the ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof according to any one of claims 1 to 8, or the pharmaceutical composition according to claim 9 in the preparation of a nucleic acid vaccine or the delivery of a nucleic acid vaccine; Preferably, the nucleic acid vaccine comprises a lipid component and a nucleic acid; Preferably, the nucleic acid is selected from one or more of RNA or DNA; Preferably, the nucleic acid is selected from ASO; Preferably, the RNA is selected from one or more of small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), micro RNA (miRNA), small activating RNA (saRNA), polycoding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA) or ribozyme, preferably mRNA, more preferably modified mRNA.

11. Use of the ionizable lipid compound or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof according to any one of claims 1 to 8, or the pharmaceutical composition according to claim 9 in the preparation of a drug for delivering a load or a drug for treating, diagnosing or preventing a disease, wherein the load is selected from one or more of a therapeutic agent, a preventive agent or a diagnostic agent; Preferably, the therapeutic, prophylactic or diagnostic agent is a nucleic acid; Preferably, the therapeutic, prophylactic or diagnostic agent is a vaccine; Preferably, the therapeutic agent, prophylactic agent or diagnostic agent is a nucleic acid vaccine; Preferably, the therapeutic, prophylactic or diagnostic agent is selected from a therapeutic vaccine or a prophylactic vaccine; Preferably, the therapeutic agent, prophylactic agent or diagnostic agent is selected from a therapeutic nucleic acid vaccine or a prophylactic nucleic acid vaccine. Preferably, the nucleic acid is selected from ASO; Preferably, the nucleic acid is selected from one or more of RNA or DNA; Preferably, the RNA is selected from one or more of small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), micro RNA (miRNA), small activating RNA (saRNA), polycoding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA) or ribozyme, preferably mRNA, more preferably modified mRNA.

12. The use according to claim 10 or 11, characterized in that in, The vaccine is selected from a therapeutic vaccine or a preventive vaccine; Preferably, the vaccine is used to prevent / treat diseases caused by viral infection; Preferably, the vaccine is used to prevent / treat diseases caused by coronavirus infection, more preferably diseases caused by SARS-CoV-2; Preferably, the vaccine is used to prevent / treat diseases caused by influenza virus infection; Preferably, the vaccine is used to prevent / treat diseases caused by herpes zoster virus infection.

13. The use according to any one of claims 10 to 12, wherein The use is to elicit an immune response in a subject or to vaccinate a subject against a viral infection.

14. A method for preparing a nucleic acid vaccine, comprising the following steps: The ionizable lipid compound or pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof according to any one of claims 1 to 8 is mixed with a structural lipid, a neutral lipid, a polymer lipid and a solvent, and then mixed with a solution in which mRNA encoding an antigen protein is dissolved; Preferably, the antigen protein is a viral antigen protein; Preferably, the antigen protein is a novel coronavirus spike protein or varicella-zoster virus envelope glycoprotein E; Preferably, the vaccine is a formulation for intramuscular injection.

15. A method of delivering a nucleic acid in a subject, comprising administering to the subject a pharmaceutical composition comprising the ionizable lipid compound of any one of claims 3 to 8, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof.

16. A method of delivering a load in a subject, comprising administering to the subject a pharmaceutical composition comprising the ionizable lipid compound of any one of claims 3 to 8, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein the load is selected from one or more of a therapeutic agent, a prophylactic agent or a diagnostic agent; Preferably, the therapeutic, prophylactic or diagnostic agent is selected from a vaccine; Preferably, the therapeutic, prophylactic or diagnostic agent is selected from nucleic acids; Preferably, the therapeutic, prophylactic or diagnostic agent is selected from a nucleic acid vaccine; Preferably, the therapeutic, prophylactic or diagnostic agent is selected from a therapeutic vaccine or a prophylactic vaccine; Preferably, the therapeutic agent, prophylactic agent or diagnostic agent is selected from a therapeutic nucleic acid vaccine or a prophylactic nucleic acid vaccine.

17. A method for treating, diagnosing or preventing a disease in a subject, comprising administering to the subject a pharmaceutical composition comprising the ionizable lipid compound of any one of claims 3 to 8 or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof.

18. A method for treating, diagnosing or preventing a disease in a subject, comprising administering to the subject the nucleic acid vaccine obtained by the preparation method of claim 14.

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