Ionizable lipid compounds

Abstract

The present invention provides a novel class of ionizable lipid compounds, or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof, pharmaceutical compositions thereof, and use thereof in the preparation of preventive or therapeutic nucleic acid vaccines.

Description

Technical Field

[0001] The present invention relates to a class of ionizable cationic lipid compounds, or pharmaceutically acceptable salts, isotope variants, tautomers or stereoisomers thereof, and preparation methods and applications thereof. Background Art

[0002] Nucleic acid vaccines are vaccines that directly introduce exogenous genes (DNA or RNA) encoding specific antigen proteins into host cells, express the antigen proteins in the host cells, and induce the host to produce an immune response to the antigen proteins to achieve the purpose of preventing and treating diseases.

[0003] The nucleic acid vaccine developed by Moderna and BioNTech uses lipid nanoparticles (LNP) as a delivery system. The main components of LNP include cationic lipid molecules (Cationic Lipid), cholesterol, neutral lipids and polyethylene glycol-conjugated lipids. Among them, cationic lipid molecules are the core of the LNP delivery system, and their molecular structure plays a decisive role in the delivery efficiency, targeting, and formulation stability of the entire liposome nanoparticle.

[0004] 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

[0005] 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 efficient nucleic acid delivery efficiency when injected into the body by intravenous administration, mainly delivered to the liver. However, nucleic acid vaccines can cause the body's lymphoid organs to produce an immune response by intramuscular injection. Due to different applications, whether the lipid compound of the present invention produces a good body immune response is the focus of the research of the present invention. Unexpectedly, the compounds of the present invention are used to prepare nucleic acid vaccines, and the body's immune response effect is better than ALC0315.

[0006] Specifically, one aspect of the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

[0007]

[0008] in,

[0009] R1 and R2 are independently selected from C 4-20 Alkyl, C 4-20Alkenyl 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"-;

[0010] R is independently selected from H, C 1-14 Alkyl, -L a -OR a and -L a -NR a R' a ;

[0011] R" is independently selected from H and C 1-20 alkyl;

[0012] 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*;

[0013] 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*;

[0014] a is independently selected from 0, 1, 2, 3, 4 and 5;

[0015] b and d are independently selected from 3, 4, 5, 6, 7, 8 and 9;

[0016] c and e are independently selected from 0, 1, 2, 3 and 4;

[0017] b+c=3, 4, 5, 6, 7, 8 or 9, d+e=3, 4, 5, 6, 7, 8 or 9;

[0018] The methylene group in the 1-6 Alkyl substitution;

[0019] 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*;

[0020] 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 ;

[0021] L a Independently selected from chemical bonds and C 1-6 Alkylene;

[0022] L b Independently selected from chemical bonds and C 1-6 Alkylene;

[0023] R a and R' a Independently selected from H, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl;

[0024] R b and R' b Independently selected from H, C 1-6 alkyl, 3- to 10-membered cycloalkyl, and 3- to 10-membered heterocyclyl.

[0025] The compounds of the present invention are ionizable lipid compounds.

[0026] In one embodiment, a is selected from 2, 3 and 4, more preferably 2 or 3, more preferably 2.

[0027] In one embodiment, b and d are independently 4 or 5.

[0028] In one embodiment, c and e are independently 0 or 1.

[0029] In one embodiment, b+c=4, 5 or 6.

[0030] In one embodiment, d+e=4, 5 or 6.

[0031] In one embodiment, b+c=5, d+e=5.

[0032] In one embodiment, R1 or R2 is independently selected from a linear alkyl group 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.

[0033] In one embodiment, 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 replaced by C 4-6 Alkyl substitution.

[0034] In one embodiment, 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.

[0035] In one embodiment, 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*.

[0036] In one embodiment, 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.

[0037] In one embodiment, R3 and R4 are independently selected from methyl.

[0038] In one embodiment, R5, R6, R7 and R8 are independently selected from C 1-6 Alkyl; preferably methyl or ethyl, more preferably methyl.

[0039] Another aspect of the present invention provides the following compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

[0040]

[0041] In one embodiment, the above-mentioned compound is used to prepare a nucleic acid vaccine, preferably to prepare an RNA vaccine, and more preferably to prepare an mRNA vaccine.

[0042] Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, and optionally a pharmaceutically acceptable excipient, such as a carrier, adjuvant or vehicle.

[0043] In one embodiment, the pharmaceutical composition is a nanoparticle composition comprising a compound of the invention.

[0044] In one embodiment, the nanoparticle composition comprises a lipid component and, optionally, an active ingredient.

[0045] In one embodiment, the active ingredient is a nucleic acid.

[0046] In one embodiment, the lipid component comprises the following components in molar percentages:

[0047] Ionizable cationic lipids 20 mol%-85 mol%;

[0048] Structural lipids 10 mol%-75 mol%;

[0049] Neutral lipids 1.0 mol%-30 mol%;

[0050] Polymer lipid 0.25mol%-10mol%.

[0051] The ionizable cationic lipid is the above-mentioned compound, or a pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer thereof.

[0052] In one embodiment, the molar percentage content of the ionizable cationic lipid is 42.5mol%-50mol%, 32.5mol%-50mol%, 25mol%-65mol%, 30mol%-60mol%, 30mol%-50mol%, 30mol%-55mol% or 40mol%-52.5mol%.

[0053] In one embodiment, the molar percentage content of the structured lipid is 30.6mol%-61mol%, 30.6mol%-51mol%, 25mol%-70mol%, 27.5mol%-66mol%, 30.5mol%-66mol%, 28mol%-64mol% or 28mol%-54mol%.

[0054] In one embodiment, the molar percentage content of the neutral lipid is 1 mol%-25mol%, 1.5mol%-20mol%, 1.5mol%-20mol%, 5mol%-20mol% or 5mol%-20mol%.

[0055] In one embodiment, 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%.

[0056] In one embodiment, the N:P molar ratio of N atoms in the ionizable cationic lipid to P atoms in the load molecule is 1-15:1, for example, 3-12:1, 3-7:1, 1-10:1, 3-6:1 or 3-5:1.

[0057] In one embodiment, the particle size of the particles 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.

[0058] In one embodiment, the neutral lipid is selected from one or more of DSPC, DMPC, DOPC, DPPC, POPC, DOPE, DMPE, POPE or DPPE.

[0059] In one embodiment, 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.

[0060] In one embodiment, the polymer lipid is a pegylated lipid.

[0061] In one embodiment, 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.

[0062] In one embodiment, 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.

[0063] Another aspect of the present invention provides the above-mentioned compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or the above-mentioned pharmaceutical composition for use in treating, diagnosing and / or preventing a disease.

[0064] Another aspect of the present invention provides use of the above-mentioned compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or the above-mentioned pharmaceutical composition in the preparation of a drug for treating, diagnosing or preventing a disease.

[0065] In one embodiment, the drug for treating, diagnosing or preventing a disease is selected from a therapeutic vaccine or a preventive vaccine.

[0066] In one embodiment, the vaccine is used to prevent / treat diseases caused by viral infection.

[0067] In one embodiment, the vaccine is used to prevent / treat a disease caused by a coronavirus infection, preferably a disease caused by SARS-CoV-2;

[0068] In one embodiment, the vaccine is used to prevent / treat diseases caused by influenza virus infection.

[0069] In one embodiment, the vaccine is used to prevent / treat a disease caused by herpes zoster virus infection.

[0070] 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 compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof.

[0071] In one embodiment, the amount of the pharmaceutical composition administered is an effective amount.

[0072] In one embodiment, the disease is a disease caused by a viral infection.

[0073] In one embodiment, the disease is a disease caused by coronavirus infection, preferably a disease caused by SARS-CoV-2.

[0074] In one embodiment, the disease is a disease caused by influenza virus infection.

[0075] In one embodiment, the disease is a disease caused by herpes zoster virus infection.

[0076] Another aspect of the present invention provides the use of the above-mentioned compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or the above-mentioned pharmaceutical composition in the preparation of a drug for delivering a load, wherein the load is selected from one or more of a therapeutic agent, a preventive agent or a diagnostic agent.

[0077] 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 compound, 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 preventive agent or a diagnostic agent.

[0078] In one embodiment, the therapeutic, prophylactic or diagnostic agent is a nucleic acid.

[0079] In one embodiment, the nucleic acid is selected from antisense oligonucleotides (ASOs).

[0080] In one embodiment, the nucleic acid is selected from one or more of RNA or DNA.

[0081] In one embodiment, 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), micro RNA (miRNA), small activating RNA (saRNA), polycoding nucleic acid (MCNA), polycoding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA) or ribozyme.

[0082] In one embodiment, the RNA is mRNA, more preferably modified mRNA.

[0083] definition

[0084] Chemical Definition

[0085] Definitions of specific functional groups and chemical terms are described in more detail below.

[0086] The term "substituted" or "substitution" means that any one or more hydrogen atoms on a particular atom are replaced with a substituent.

[0087] The term "chemical bond" refers to a single bond, a double bond or a triple bond, preferably a single bond.

[0088] 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.

[0089] “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 by one or more substituents, for example, by 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).

[0090] “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). The 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.

[0091] “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. 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 may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0092] 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.

[0093] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0094] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 "Alkyl" is substituted with one or more halogen groups. The haloalkyl group can be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0095] “C 3-10 "Cycloalkyl" or "3- to 10-membered cycloalkyl" refers to a non-aromatic cyclic hydrocarbon radical 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 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 as above, wherein substituents on any non-adjacent carbon atoms are linked to form a bridged ring, together forming a polycyclic alkane sharing two or more carbon atoms. Cycloalkyl also includes cycloalkyl rings as above, wherein Substituents on the same carbon atom are linked to form a ring, together forming a polycyclic alkane sharing one carbon atom. Exemplary cycloalkyls 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 by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0096] "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 heterocyclyls containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as valence permits. Heterocyclyl also includes ring systems in which the above heterocyclyl ring is fused to one or more cycloalkyl groups, wherein the point of attachment is on the heterocyclyl ring, or in which the above heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein 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 as above, wherein 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 ring, wherein the substituents on the same carbon atom are connected to form a ring, together forming a polycyclic heteroalkane sharing a carbon atom. Exemplary 3-membered heterocyclyls containing one heteroatom include, but are not limited to: aziridine, oxirane, thiorenyl. Exemplary 4-membered heterocyclyls containing one heteroatom include, but are not limited to: azetidine, oxetane and thiidine. 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 heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazine (triazinanyl). Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxanyl, and thianyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to, dihydroindole, isoindole, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinone, and the like.Exemplary 6-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclic groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Heterocyclic groups also include the above heterocyclic groups and a cycloalkyl, heterocyclic group, aryl or heteroaryl group sharing one or two atoms to form a bridged ring or a spirocyclic ring, and the shared atoms may be carbon or nitrogen atoms as long as the valence permits. Heterocyclic groups also include the above heterocyclic groups and heterocyclic groups may be optionally substituted by one or more substituents, for example, substituted by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0097] “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, aryl has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, aryl has ten ring carbon atoms ("C 10 Aryl also includes ring systems in which the above aryl ring 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, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0098] "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 may be a carbon or nitrogen atom as long as the valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the above-mentioned heteroaryl rings are 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, which 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, pyridyl or pyridone. 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, oxacycloheptatrienyl, and thiacycloheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolyl, isoquinolyl, 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.

[0099] "Optionally substituted..." means that the group may be substituted by a designated substituent or may be unsubstituted.

[0100] 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".

[0101] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups and the like as defined herein are optionally substituted groups.

[0102] "Nucleic acid" refers to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules and their hybrid molecules. 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). Nucleic acids can be further chemically modified, and the chemical modification is selected from one of pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, and 5-methylcytosine, or a combination thereof. The mRNA molecule contains a protein coding region and may further contain an expression regulatory sequence, and 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 (Poly A), and a miRNA binding site.

[0103] "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.

[0104] "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).

[0105] "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 cyclopentane pyrophenanthrene carbon skeleton. In a preferred embodiment, the steroid is selected from cholesterol, sitosterol, coprostanol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol or campesterol.

[0106] "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 compounds with uncharged, hydrophilic, steric barrier moieties coupled to lipids, can also be used.

[0107] "Lipid nanoparticles" refer to particles containing lipid components and having nanometer dimensions.

[0108] "Biodegradable groups" refer to functional groups containing biodegradable bonds, such as esters, disulfide bonds, and amides. Biodegradation can affect the process of removing compounds from the body. The direction of the biodegradable groups of the present invention is from the head to the tail in the ionizable lipid molecule.

[0109] Other definitions

[0110] 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, the latter being as just defined.

[0111] Herein, the 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 included. In addition, unless otherwise specified, all isomeric compounds included in the present invention may appear in the form of Z and E with carbon-carbon double bonds. Compounds that exist in different tautomeric forms, a compound is not limited to any specific tautomer, but is intended to cover all tautomeric forms.

[0112] The term "pharmaceutically acceptable salt" as used herein refers to those carboxylates, amino acid addition salts of the compounds of the present invention which 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.

[0113] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metals and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, etc. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine.

[0114] Base addition salts of acidic compounds may be prepared by contacting the free acid form with a sufficient amount of the desired base to form the salt in a conventional manner. The free acid may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but for purposes of the present invention, the salts are equivalent to their respective free acids.

[0115] Salts can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides prepared from inorganic acids, such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc. 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, etc. 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, etc. 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, etc. Pharmaceutically acceptable salts may include cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Also contemplated are salts of amino acids, such as argininate, gluconate, galacturonate, and the like (see, e.g., Berge SM et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

[0116] "Subjects" for administration 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.

[0117] "Disease," "disorder," and "condition" are used interchangeably herein.

[0118] As used herein, and unless otherwise indicated, the terms "treatment," "treating," "treating," "treating," and "treatment" include actions that occur while a subject has a particular disease, disorder, or condition, that reduce the severity of, or delay or slow the development of, the disease, disorder, or condition ("therapeutic treatment"), and also include actions that occur before a subject develops a particular disease, disorder, or condition ("prophylactic treatment").

[0119] Unless otherwise specified, a "prophylactically effective amount" of a pharmaceutical composition as used herein 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 that provides a prophylactic benefit in the process of preventing a disease, disorder, or condition when used alone or in combination with other agents. The term "prophylactically effective amount" may include an amount that improves overall prevention, or an amount that enhances the prophylactic effect of other prophylactic agents.

[0120] "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 other therapeutic agents in separate unit dosage forms, or can be administered simultaneously with other therapeutic agents in a single unit dosage form.

[0121] The term "vaccine" as used herein refers to a composition that can provide active acquired immunity and / or therapeutic effects (e.g., treatment) for a specific disease or pathogen. Vaccines generally contain one or more agents that can induce an immune response to a pathogen or disease (i.e., a target pathogen or disease) in an individual. The immunogenic preparation 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 likely to recognize and eliminate any pathogen when subsequently contacted. Vaccines can be preventive (e.g., preventing or improving the effects of any natural or pathogen future infection or the effects of cancer expected to occur in susceptible individuals) or therapeutic (e.g., treating cancer in individuals diagnosed with cancer). The administration of vaccines is called vaccination. In some instances, a vaccine composition can provide an individual with nucleic acid, such as mRNA encoding an antigenic molecule (e.g., a peptide). The nucleic acid delivered to an individual by a vaccine composition can be expressed as an antigenic molecule and allows the individual to obtain immunity to the antigenic molecule. In the case of vaccination against infectious diseases, the vaccine composition may provide mRNA encoding antigenic molecules associated with a specific pathogen, such as one or more peptides known to be expressed in a pathogen (e.g., a pathogenic bacterium or virus). In the case of a viral vaccine, the vaccine composition may 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 may have immunity to the viral peptides, specifically killing cells that express them.

[0122] The term "nucleic acid vaccine" used in this article is also called a gene vaccine, which is a vaccine that directly introduces genetic material (DNA or RNA) that determines the specific antigens of pathogens into human cells, allowing human cells to produce these antigens themselves and stimulate the body to produce an immune response to the antigen, thereby enabling the vaccine recipient to obtain corresponding immune protection. It also contains adjuvants (such as lipid nanoparticles).

[0123] The term "delivery system" used herein refers to a class of substances that can carry antigenic substances to the body's immune system and store and exert their antigenic effects therein for a long time. The vaccine delivery system described herein can be a liposome adjuvant vaccine delivery system or a nanoadjuvant vaccine delivery system.

[0124] 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).

[0125] The term "virus" is used according to its ordinary meaning in the biological arts 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), the envelope includes lipids and optionally host cell membrane components, and / or viral proteins.

[0126] The terms "viral infection" or "viral disease" refer to a disease or condition caused by a virus, including 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 infections, cytomegalovirus infections, respiratory viral infections (e.g., adenovirus infections, influenza, severe acute respiratory syndrome, coronavirus infections (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV, COVID-19, MERS)), gastrointestinal viral infections (e.g., norovirus infections, rotavirus infections, astrovirus infections), exanthematous viral infections (e.g., measles, herpes zoster, smallpox, rubella), viral hemorrhagic diseases (e.g., Ebola virus, Lassa fever, dengue fever, yellow fever), nervous system viral infections (e.g., West Nile virus infection, poliomyelitis, viral meningitis, viral encephalitis, Japanese encephalitis, rabies), and human papillomavirus infections.

[0127] 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.

[0128] In the context of a disease, the term "prevention" and its various grammatical variations "prevention" means causing the clinical symptoms of the disease to not occur in an individual who has not yet experienced or developed symptoms of the disease.

[0129] As used herein, and unless otherwise indicated, the terms "treatment," "treating," "treating," "treating," and "treatment" include actions that occur while a subject has a particular disease, disorder, or condition, that reduce the severity of, or delay or slow the development of, the disease, disorder, or condition ("therapeutic treatment"), and also include actions that occur before a subject develops a particular disease, disorder, or condition ("prophylactic treatment").

[0130] 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, the latter being as just defined.

[0131] "Subjects" for administration 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.

[0132] "Disease," "disorder," and "condition" are used interchangeably herein.

[0133] The term "effective amount" refers to the amount of a formulation according to the invention that is sufficient to achieve such treatment when administered to a patient for treating a state, disorder or condition, or sufficient to produce such an immune response when administered to a patient for producing 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.

[0134] 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 drug refers to an amount of a therapeutic agent that provides a prophylactic benefit in the process of preventing a disease, disorder, or condition when used alone or in combination with other agents. The term "prophylactically effective amount" may include an amount that improves overall prevention, or an amount that enhances the prophylactic effect of other prophylactic agents. Example

[0135] 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 used 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, if not specifically stated, can be obtained from commercial sources.

[0136] Table 1

[0137]

[0138]

[0139] Example 1: Synthesis of Compound 1

[0140]

[0141] In a 2L four-necked round-bottom flask, THF (160mL) and compound 1-1 (80g, 688.7mmol) were added at room temperature. The system was cooled to -40℃ and LDA (344.4mL, 688.7mmol) was added dropwise for 2h, and stirred at -40℃ for 1h. Then dibromobutane (206.70g, 957.3mmol) was added dropwise at -40℃ for 30 minutes, and finally DMPU (12.7g, 99.2mmol) was added dropwise at -40℃ for 2h, and -40℃ was kept warm for 1h. The mixture was stirred for 15h. The reaction was monitored by TLC, and the raw materials were consumed. The reaction was quenched with saturated NH4Cl (200mL) at 0℃, and water (160mL) and ethyl acetate (160mL) were added to dilute the system, extract, and separate. The aqueous phase was back-extracted once with ethyl acetate (200mL). 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 light yellow oily compound 1-2 (100 g).

[0142] In a 2L four-necked round-bottom flask, THF (1L) and compound 1-2 (98g, 391.91mmol) were added at room temperature. The system was cooled to -10°C and LiAlH4 (313.5mL, 313.5mmol) was added dropwise for 30 minutes. The system was stirred at -10°C for 30 minutes and the reaction was monitored by TLC. The raw material was completely converted. The reaction was quenched with Na2SO4.10H2O (125g) at -4°C, and the system was diluted with water (500mL) and ethyl acetate (5L), extracted, and separated. The aqueous phase was back-extracted once with ethyl acetate (300mL). The organic phases were combined and washed twice with saturated NaCl (500mL), and the organic phase was dried with anhydrous Na2SO4, filtered, and concentrated. The crude product was passed through a silica gel column to obtain a light yellow oil 1-3 (73g).

[0143] In a 250mL three-necked flask, DMSO (40mL) and NaH (3.83g, 95.7mmol) were added at room temperature. TosMIC (4.67g, 23.9mmol) was added in batches under nitrogen protection at 25°C, followed by TBAI (883.5mg, 2.39mmol), and the system was stirred at 25°C for 15 minutes. Finally, compound 1-3 (10g, 47.8mmol) was dissolved in DMSO (100mL) and added dropwise to the above system at 25°C, and the system was stirred at 25°C for an additional 1h. TLC monitored the reaction and the raw material was completely converted. The reaction was quenched with ice water (400mL) at 20°C, and the system was extracted with MTBE (2x500mL). The organic phases were combined and washed once with saturated NaCl (500mL), and the organic phases were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product (10g) was directly used for the next step.

[0144] In a 500mL three-necked flask, crude compound 1-4 (10g), CH3OH (100mL) and 12N HCl (5.3mL) were added at room temperature. The system was reacted at 80°C for 3h. The reaction was monitored by LCMS, and the raw material conversion was complete. The system was cooled to room temperature, pH=7 was adjusted with saturated sodium carbonate, and the methanol was removed by concentration under reduced pressure. The system was dissolved and diluted with water (100mL) and ethyl acetate (100mL), extracted, and separated. The aqueous phase was back-extracted once with ethyl acetate (100mL), the organic phases were combined and washed once with saturated NaCl (100mL), the organic phases were dried with anhydrous Na2SO4, filtered, and concentrated. The crude product was passed through a silica gel column to obtain compound 1-5 (4.6g).

[0145] 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 reacted at 20°C for 18 h. The reaction was monitored by TLC and LCMS, and there was a product signal. 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 organic phases were combined and washed once with saturated NaCl (50 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was passed through a silica gel column to obtain a light yellow compound 1-6 (1.71 g).

[0146] 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. The reaction was monitored by TLC, and the raw material conversion was complete. 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), and the organic phases were combined and washed once with saturated NaCl (20 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column to obtain a light yellow oil compound 1-8 (301.8 mg).

[0147] 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, and NaBH4 (17.2 mg, 0.45 mmol) was added at 0°C. The reaction was allowed to react at 20°C for 1 h. The reaction was monitored by TLC, and the reaction of the raw material was complete. 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), and the organic phases were combined and washed once with saturated NaCl (20 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product (273.8 mg) was directly used for the next step.

[0148] 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 monitored the reaction and the raw material conversion was complete. 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), and the organic phases were combined and washed once with saturated NaCl (25 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column to obtain a light yellow oily compound 1 (239.6 mg).

[0149] 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] + .

[0150] Example 2: Synthesis of Compound 2

[0151]

[0152] Add the raw material 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) into an 8 mL sealed tube. Stir at room temperature for 18 hours. TLC monitoring shows that there is no raw material. Add silica gel and stir the sample. Purify by column chromatography to obtain 196 mg of light yellow oil 2-2.

[0153] Add the raw material compound 2-2 (196 mg, 0.29 mmol), MeOH (4 mL) and THF (1 mL) into a 25 mL three-necked flask. Cool to 0 °C and add NaBH4 (10.92 mg, 0.29 mmol). Stir at room temperature for 1 hour. No raw material was found under TLC monitoring. Add saturated aqueous ammonium chloride solution (10 mL) and extract with MTBE (1 x 20 mL). Wash the organic phase with saturated brine (1 x 20 mL), dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain 140 mg of product 2-3, which was directly used in the next step.

[0154] Add the raw material 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) into an 8 ml sealed tube. React at room temperature for 5 hours. TLC monitoring shows no raw material. Add silica gel and stir the sample. Purify by column chromatography to obtain 105.9 mg of compound 2.

[0155] 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] + .

[0156] Example 3: Synthesis of Compound 3

[0157]

[0158] In 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 and monitor by TLC. Directly mix the sample and purify by column chromatography to obtain 165.6 mg of yellow oil 3-2.

[0159] Compound 3-2 (165.6 mg, 0.25 mmol), MeOH (4 mL) and THF (1 mL) were added to a 25 mL three-necked flask. The temperature was lowered to 0°C and NaBH4 (9.62 mg, 0.25 mmol) was added, followed by stirring at room temperature for 1 hour and monitoring by TLC. After the reaction of the raw materials was complete, 10 mL of saturated NH4Cl aqueous solution was added, EA was extracted (1 x 20 mL), and the organic phase was washed with saturated brine (1 x 10 mL). Drying over anhydrous Na2SO4 and vacuum concentration were performed to obtain 166 mg of crude compound 3-3, which was used directly in the next step.

[0160] In 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 and monitor by TLC. Directly mix the sample and column chromatography pure DCM / MeOH (50:1) to obtain 142.4 mg of light yellow oily compound 3.

[0161] 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] + .

[0162] Example 4: Synthesis of Compound 4

[0163]

[0164] The tetrahydrofuran (800 mL) solution of compound 4-1 (100 g, 979 mmol) was cooled to -40 °C, and LDA (2M, 490 mL) was slowly added to the solution. After the addition was completed, stirring was continued for 1 hour. At the same temperature, a tetrahydrofuran (100 mL) solution of 4-2 (315 g, 1.37 mol) was added to the reaction system, and the reaction system was stirred overnight. The reaction system was quenched with saturated aqueous ammonium 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 a crude product. The crude product was separated and purified by silica gel column to obtain compound 4-3 (115 g).

[0165] 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) were cooled to 0 ° C, sodium hydride (20.7 g, 517 mmol) was slowly added in batches, and the reaction was allowed to proceed overnight at room temperature. The reaction system was quenched with saturated sodium chloride aqueous 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 directly used in the next step without separation and purification.

[0166] Add 330 mL of concentrated hydrochloric acid to a solution of compound 4-4 (110 g, 205 mmol) in dichloromethane (880 mL), react at room temperature for 2 hours, and monitor the substrate reaction completely by TLC. The reaction system is quenched with a saturated aqueous ammonium chloride solution, extracted with ethyl acetate, and the organic phases are combined and dried over anhydrous sodium sulfate. Filter and concentrate the filtrate to dryness to obtain a crude product. Separate and purify on a silica gel column to obtain compound 4-5 (30.0 g, 80.9 mmol, yield 39.4%).

[0167] TMSOK (11.0 g, 86.4 mmol) was added to a tetrahydrofuran (35.0 mL) solution of compound 4-5 (8.0 g, 21.6 mmol) at room temperature, and the reaction system was heated to 70°C for stirring. TLC monitored the complete consumption of the reaction raw materials. The reaction solution was cooled to room temperature, and the organic solvent was removed by rotary evaporation. 20 mL of water was added to the crude product and extracted with dichloromethane. The aqueous phase was collected, and the pH value of the solution was adjusted to less than 5 with 1M hydrochloric acid, and extracted with dichloromethane. The organic phases were combined and dried with anhydrous sodium sulfate, and the filtrate was collected by filtration and concentrated to obtain compound 4-6 (7.0 g).

[0168] 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), and then the temperature was raised to 60°C for 4 hours, cooled to room temperature, the reaction system was quenched with a saturated sodium chloride aqueous solution, 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 a crude product, which was purified by silica gel column to obtain compound 4-8 (682 mg).

[0169] Add 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) to a 20 mL sealed tube. React at room temperature overnight. LCMS monitors the product signal. Add 10 mL of water to quench, extract with CH2Cl2 (1x10 mL), wash the organic phase with saturated brine (1x10 mL), dry with Na2SO4, filter and spin dry, and purify by column to obtain compound 4-10 (543 mg).

[0170] Under nitrogen protection, compound 4-10 (543 mg, 0.83 mmol) and MeOH (10 mL) were added to a 50 mL three-necked flask, and NaBH4 (157.8 mg, 4.17 mmol) was added in batches at 0°C. The reaction was allowed to react for 2 h at room temperature. 20 mL of water was added dropwise at 0°C to quench the reaction, and the reaction was extracted with EtOAc (1 x 20 mL). The organic phase was washed with saturated brine (1 x 20 mL), dried with Na2SO4, filtered and dried to obtain compound 4-11 (480 mg).

[0171] Add 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) to an 8 mL sealed tube and react overnight at room temperature. LCMS monitors for product signals. Add 10 mL of water to quench, extract with DCM (2 x 20 mL). Wash the organic phase with brine (1 x 20 mL), dry with Na2SO4, filter and spin dry, and purify with Prep-TLC (DCM / MeOH 10:1) to obtain 4 (118.3 mg).

[0172] 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] + .

[0173] Example 5: Synthesis of Compound 5

[0174] Compound 5 was prepared according to the synthetic method of Example 70 of CN115850104A.

[0175]

[0176] Pharmacological experiments

[0177] Experimental Example 1: Nanoparticle Preparation

[0178] The materials used for lipid nanoparticle assembly 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, which is 1,2-distearoyl-SN-glycero-3-phosphocholine (Distearoylphosphatidylcholine, purchased from AVT) or DOPE, which is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Dioleoyl Phosphoethanolamine, purchased from AVT); (4) polyethylene glycol lipid compounds: such as DMG-PEG2000, which is dimyristoylglycerol-polyethylene glycol 2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, purchased from AVT) or ALC0159 (purchased from Sinobond); (5) Nucleic acid fragment active ingredient: novel coronavirus spike protein mRNA (self-made, the coding sequence is the same as WO2021154763A1 (Table 1) SARS-CoV-2S Protein Variant 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.

[0179] Preparation method of lipid nanoparticles: (1) Dissolve an ionizable lipid compound, cholesterol, phospholipid and pegylated lipid in ethanol in sequence according to a preferred formula. The optimized formulas of compounds 1-5 of the present invention are shown in Table 3. The preferred formula of ALC0315 is shown 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 is dissolved in 25 mM sodium acetate solution (pH = 4.5); (3) an automated high-throughput microfluidic system is used to mix the organic phase containing the lipid mixture and the aqueous phase containing the mRNA component at a flow rate ratio of 1:1 to 1:4, and the mixing speed is between 10 mL / min and 18 mL / min; (4) the prepared lipid nanoparticles are diluted with phosphate buffered saline, and the nanoparticle solution is 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 are sterilized by filtration through a 0.2 μm sterile filter membrane and stored at low temperature in a sealed glass bottle.

[0180] Methods for preparing lipid nanoparticles include, but are not limited to, microfluidic mixing systems and also include T-type mixers and ethanol injection methods.

[0181] Table 2

[0182]

[0183]

[0184] Table 3

[0185]

[0186] Experimental Example 2: Characterization of Physical Properties of Lipid Nanoparticles

[0187] 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 TM The 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.

[0188] Experimental Example 3: Mouse muscle immunization

[0189] The adaptive immune effect of lipid nanoparticles encapsulating mRNA encoding viral antigen proteins in mice was evaluated. The experimental mice were SPF-grade BALB / c mice, female, 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 were free to eat and drink, with 12 / 12h light and dark alternation, indoor temperature of 20-26°C, and humidity of 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 mRNA encoding viral antigen proteins were injected into the immunization group mice by intramuscular administration 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.

[0190] Table 4

[0191] Serial number Group dose Immunization interval 1 Novel coronavirus spike protein group 0.025mg / kg mRNA 14 days 2 Varicella-zoster virus envelope glycoprotein E group 0.05mg / kg mRNA 28 days

[0192] Experimental Example 4: Evaluation of Humoral Immunity Effect

[0193] 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.

[0194] 1. Detection of novel coronavirus-specific binding antibodies

[0195] 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 filter 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 a washing solution (1×TBS containing 0.2% Tween-20, purchased from Solebao) was added to each well for washing. After repeating the washing for 6 times, a blocking solution (1×TBS containing 2% BSA, purchased from Solebao) was added to each well, and the plate was incubated at 37°C for blocking; the plate washing operation was repeated after blocking for 2 hours; the immune mouse serum was diluted 10 times with an antibody diluent (washing solution containing 0.5% BSA) to obtain 10 -1 Up to 10 -6 Serum with different dilutions was added to the well plate and incubated in a 37°C incubator; the plate was washed repeatedly after 2 hours of incubation; horseradish peroxidase-labeled goat anti-mouse IgG (purchased from Bio-Time) was diluted 250 times with antibody diluent (washing solution containing 0.5% BSA) to obtain secondary antibody diluent, which was added to the well plate and incubated in a 37°C incubator; the plate was washed repeatedly after 1 hour of incubation; TMB substrate was added and incubated for 20 minutes at room temperature in the dark for color development; finally, stop solution (purchased from Solebol) was added to terminate the reaction and the OD value of A450 was detected on an enzyme reader.

[0196] Determination of serum binding antibody IgG titer: a certain dilution serum OD / negative control OD ≥ 2.1, the next dilution multiple OD / negative control OD < 2.1, the dilution multiple is the antibody titer corresponding to the serum sample (if the negative control OD < 0.05, it is calculated as 0.05). The titer of the novel coronavirus spike protein binding antibody in the serum of mice immunized with lipid nanoparticles containing novel coronavirus spike protein mRNA is shown in Table 5.

[0197] 2. Evaluation of the Neutralizing Effect of SARS-CoV-2-Specific Antibodies

[0198] The antibody neutralizing effect of mouse serum was evaluated using the 2019-nCoV spike protein pseudovirus.

[0199] The mouse immune serum was serially diluted 3-fold using DMEM complete medium (purchased from Gibco) to obtain 6 different serum dilutions, which were then mixed with 650 TCID 50 Pseudovirus (purchased from Yunling Biotechnology Co., Ltd.) was incubated at 37°C. A cell control group without pseudovirus and a pseudovirus control group without serum samples were set up. After incubation for one hour, 2×10 4 Huh 7 cells (liver cancer cells) were cultured at 37°C and 5% CO2. Since the pseudovirus enters the cells, it will express firefly luciferase. After 24 hours, it reacts with the luminescent substrate and performs luminescence detection. The percentage of pseudovirus inhibition is calculated by comparing the luminescence value with the pseudovirus control group. The dilution factor of serum when the pseudovirus is 50% inhibited is calculated by the Reed-Muench method, that is, the half neutralization dilution (NTD) 50 ). NT 50 Illustration of the neutralizing activity of serum antibodies against pseudoviruses. Neutralizing activity of serum from mice immunized with lipid nanoparticles loaded with mRNA of the spike protein of the novel coronavirus NT 50 See Table 5 for details.

[0200] It can be seen from the data in Table 5 that the humoral immune effect of the novel coronavirus vaccine prepared by the compound of the present invention is significantly better than that of the control ALC0315 in terms of serum IgG titer and serum neutralization ability. When the degradable group of the compound of the present invention (corresponding to the general formula groups M1 and M2) is -OC(O)-, the serum neutralization ability is better than that of the compound containing the group -C(O)O-.

[0201] Table 5

[0202]

[0203] 3. Detection of specific binding antibodies to varicella-zoster virus envelope glycoprotein E

[0204] 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 filter membrane) to form a protein coating solution, which was added to a 96-well plate and sealed and incubated at 4°C overnight; after overnight coating, the 96-well plate was poured to remove the protein coating solution, and a washing solution (1×TBS containing 0.2% Tween-20, purchased from Solebol) was added to each well for washing, and the plate was washed 6 times, and a blocking solution (1×TBS containing 2% BSA, purchased from Solebol) was added to each well, and the plate was incubated at 37°C for blocking; the plate washing operation was repeated after blocking for 2 hours; the immune mouse serum was diluted 10 times with an antibody diluent (washing solution containing 0.5% BSA) to obtain 10 -1 Up to 10 -6 Serum with 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 (H+L) (purchased from Beyotime, A0216) 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 (purchased from Tiangen) was added, and the reaction was incubated at room temperature, away from light, for 20 minutes for color development; finally, the stop solution (purchased from Solebo) was added to terminate the reaction and the OD value of A450 was detected on the microplate reader. The titer of serum binding antibody IgG was judged as follows: a certain dilution serum OD / negative control OD ≥ 2.1, and the next dilution multiple OD / negative control OD < 2.1, and the dilution multiple was the antibody titer corresponding to the serum sample (if the negative control OD < 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.

[0205] The data in Table 6 show that the nucleic acid vaccine encoding varicella-zoster virus prepared by the compounds of the present invention produces a strong humoral immune response.

[0206] Table 6

[0207]

[0208] Experimental Example 5: Evaluation of Cellular Immunity Effect

[0209] 1. Novel coronavirus-specific cellular immunity

[0210] On the 14th day after booster immunization, the spleens of immunized mice and mice in the negative control group were dissected, and spleen cells were isolated to prepare single cell suspensions. Mouse spleen cells were added with novel coronavirus spike protein peptides (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 spot (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.

[0211] 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 significantly better cellular immune effect than the control compound ALC0315.

[0212] Table 7

[0213]

[0214] 2. Varicella-zoster virus-specific cellular immunity

[0215] On the 14th day after booster immunization, spleens of immunized mice and mice in the negative control group were dissected, and 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 by enzyme-linked immunosorbent spot (ELISpot) method. The ELISpot detection kit was purchased from MABTECH. The specific implementation method refers to the instructions of the kit. 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 envelope glycoprotein E of varicella-zoster virus is shown in Table 8.

[0216] 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.

[0217] Table 8

[0218]

[0219] Experimental Example 6: In vitro safety assessment

[0220] 1. hERG test

[0221] The SyncroPatch 384i / 384 automated patch clamp system was used to evaluate the potential inhibitory effects of ionizable lipid compounds on human hERG channels. CHO cells stably overexpressing the hERG gene were used for evaluation, and cisapride was 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 50 ). The evaluation was completed by Kanglong Chemical. The test results are detailed in Table 9.

[0222] 2. Mini-Ames test for Salmonella typhimurium and Escherichia coli

[0223] 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 tested to evaluate the potential mutagenicity of the test substances. The test doses of ionizable lipid compounds were 1.5, 4, 10, 25, 64, 160, 400 and 1000 μg / well. Negative / solvent controls (methanol) and positive controls were also tested. The test was completed by Kanglong Chemical. The test results are detailed in Table 9.

[0224] The data in Table 9 show that the compounds of the present invention have substantially no potassium ion channel inhibitory effect and toxicity and have good safety.

[0225] Table 9

[0226]

[0227] Although the present invention has been comprehensively described through its embodiments, it is worth noting that various changes and modifications are obvious to those skilled in the art. Such changes and modifications should be included in the scope of the appended claims of the present invention.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof: (I) in, R1 and R2 are independently selected from C 4-20 Alkyl, the C 4-20 The alkyl group is optionally substituted with one or more R; R is independently selected from H and C 1-14 alkyl; R3 and R4 are independently selected from H and C 1-6 alkyl; a is selected from 2, 3 or 4; b and d are independently 4 or 5; c and e are independently 1; b+c=5 or 6, d+e=5 or 6; The methylene group in the 1-6 Alkyl substitution; R5, R6, R7 and R8 are independently selected from methyl or ethyl.

2. The compound according to claim 1, wherein a is 2 or 3.

3. The compound according to claim 2, wherein a is 2.

4. The compound according to any one of claims 1 to 3, wherein b and d are independently 4; b+c=5, d+e=5.

5. The compound according to any one of claims 1 to 3, wherein R1 and R2 are independently selected from a linear alkyl group 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.

6. The compound according to claim 5, wherein R1 and R2 are each independently a straight chain alkyl group having a total length of 9 carbon atoms, and one of R1 and R2 is replaced by C 4-6 Alkyl substitution.

7. The compound according to any one of claims 1 to 3, wherein R3 and R4 are methyl groups.

8. The compound according to any one of claims 1 to 3, wherein R5, R6, R7 and R8 are methyl groups.

9. The following compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof: 。 10. A pharmaceutical composition comprising the compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, and optionally a pharmaceutically acceptable excipient.

11. The pharmaceutical composition according to claim 10, wherein 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; The ionizable cationic lipid is a compound of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof.

12. The pharmaceutical composition according to claim 11, wherein 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.

13. The pharmaceutical composition according to claim 11, wherein 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.

14. The pharmaceutical composition according to claim 11, wherein The neutral lipid is selected from one or more of DSPC, DMPC, DOPC, DPPC, POPC, DOPE, DMPE, POPE or DPPE.

15. The pharmaceutical composition according to claim 11, wherein The structural lipid is selected from one or more of cholesterol, sitosterol, coprosterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol or campesterol.

16. The pharmaceutical composition according to claim 11, wherein 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.

17. Use of a compound as described in any one of claims 1 to 9, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or a pharmaceutical composition as described in any one of claims 10 to 16 in the preparation of a drug for delivering a load, wherein the load is selected from one or more of a therapeutic agent, a prophylactic agent or a diagnostic agent.

18. The use according to claim 17, wherein The therapeutic, prophylactic or diagnostic agent is a nucleic acid.

19. The use according to claim 18, wherein The nucleic acid is selected from ASO.

20. The use according to claim 18, wherein The nucleic acid is selected from one or more of RNA or DNA.

21. The use according to claim 20, wherein The RNA is selected from one or more of small interfering RNA, short hairpin RNA, antisense RNA, messenger RNA, modified messenger RNA, long non-coding RNA, micro RNA, small activating RNA, poly-encoding nucleic acid, polymer-encoding nucleic acid, guide RNA, CRISPR RNA or ribozyme.

22. The use according to claim 21, wherein The RNA is mRNA.

23. The use according to claim 22, wherein The RNA is a modified mRNA.

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