Lipid compounds and compositions thereof

Abstract

The invention relates to a lipid compound and a composition thereof. In particular, the present invention relates to lipid compounds of formula (I), lipid nanoparticles comprising the same, and methods of preparation and use thereof in drug delivery. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and biotechnology, and relates to an active ingredient delivery system for a new type of lipid compounds. Background Art

[0002] Nucleic acid drugs are various oligoribonucleotides (RNA) or oligodeoxyribonucleotides (DNA) with different functions. They can regulate genes expressing related proteins based on the principle of base complementarity, and can directly act on pathogenic target genes or target mRNAs to play a role in treating diseases at the genetic level. The sequence design of nucleic acid drugs is very easy, and it can also avoid the limitation of non-drugable targets faced by traditional small molecule drugs and antibody drugs. In addition, they can play a regulatory role on intracellular and extracellular and cell membrane proteins. However, the development of nucleic acid drugs mainly faces three major problems: 1. Instability of nucleic acid molecules, especially RNA, in vivo; 2. Potential side effects; 3. Difficulties in drug delivery systems, etc. With the development of new technologies, some problems have been better solved, among which breakthroughs in chemical modification and delivery system technology have played a vital role in the development of nucleic acid drugs. Although chemical modification can improve the stability and immunogenicity of nucleic acid drugs, nucleic acid drugs need to enter cells to work. Because nucleic acid drugs have a large molecular weight and are usually negatively charged, the efficiency of cell uptake and endosome escape is relatively low, so they need to rely on the power of the delivery system.

[0003] Lipid nanoparticles (LNPs) are a new type of nucleic acid biomolecule delivery technology. LNPs are usually composed of four components: (1) ionizable lipids, which can self-assemble with mRNA into virus-sized particles and release mRNA from endosomes into the cytoplasm; (2) PEGylated lipids, which can increase the half-life of LNPs in the blood; (3) cholesterol, which can increase the stability of nanoparticles; and (4) phospholipids, which help form lipid bilayer structures. LNPs can protect mRNA from being decomposed by RNases and protect mRNA molecules from being recognized by TLRs, thereby avoiding overactivation of the innate immune system; and ionizable lipids can promote cellular uptake and help drug molecules escape from endosomes to achieve therapeutic effects.

[0004] At present, the LNP delivery system is still regarded as one of the key technologies to promote the entry of nucleic acid drug molecules into therapeutic applications. Lipid compounds with novel structures and high efficiency and stable delivery performance are still to be developed. Summary of the invention

[0005] Based on this, it is necessary to detect novel ionized lipid compounds in response to existing technical problems to improve delivery efficiency and stability.

[0006] The present invention provides a novel lipid compound, a synthesis method thereof, and a mixed encapsulation of drug molecules with a PEGylated lipid compound, a structural lipid cholesterol, and a natural phospholipid to form a nanoparticle delivery system, which can be used for in vitro cell delivery and in vivo organ-targeted cell delivery, as follows:

[0007] In one embodiment, the present invention discloses a lipid compound of formula (I), or a salt or isomer thereof:

[0008]

[0009] Where R1 is -C 1-6 Alkylene-X,

[0010] Where X is selected from amino, hydroxy, ethynyl, cyano, -C(O)(CH2) 1-3 NR a R b 、-C(O)O(CH2) 1-3 NR a R b 、-OC(O)(CH2) 1-3 NR a R b 、-C(O)NH(CH2) 1-3 NR a R b 、-NHC(O)(CH2) 1-3 NR a R b 、-NHC(O)CH(NR a R b )(CH2) 1- 3NR a R b , C 3-7 Cycloalkyl, 4-7 membered heterocyclic group, C 6-10 aryl or 5-10-membered heteroaryl, the above-mentioned cycloalkyl, heterocyclyl, aryl and heteroaryl groups are optionally substituted by the following groups;-(CH2) 1-3 OH, -(CH2) 1-3 NR a R b or -(CH2) 1-3 C(O)NR a R b ;

[0011] Or X is:

[0012]

[0013] R2 is C 3-20An alkylene group, wherein one or more methylene groups are each independently replaced by the following groups: -CH=CH-, -C≡C- or -O-;

[0014] R3 is C 5-20 An alkylene group, wherein one or more methylene groups are each independently replaced by the following groups: -CH=CH-, -C≡C- or -O-;

[0015] M is independently selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, or -OP(O)(OH)-O-;

[0016] R4 is

[0017] Wherein R is selected from H, R * or -(CH2) 1-5 -YR * ;

[0018] Y is selected from -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC (O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, or -OP(O)(OH)-O-;

[0019] R * Each independently selected from C 1-12 Alkyl, C 2-12 Alkenyl or C 2-12 Alkynyl;

[0020] R a , R b Independently selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl.

[0021] In one embodiment, R1 in formula (I) is -C 1-6 alkylene-X, wherein X is selected from amino, hydroxy, ethynyl or cyano;

[0022] R2 is C 3-20 Alkylene;

[0023] R3 is C 5-20 Alkylene;

[0024] M is independently selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH- or -NHC(S)-;

[0025] R4 is

[0026] Wherein R is selected from H, R * or -(CH2) 1-5 -YR * ;

[0027] Y is selected from -NH-, -C(O)-, -C(S)-, -O- or -S-;

[0028] R * Each independently selected from C 1-12 Alkyl, C 2-12 Alkenyl or C 2-12 Alkynyl.

[0029] In one embodiment of formula (I), R1 is -C 1-6 Alkylene -OH, preferably -C 2-6 Alkylene -OH, preferably -C 2-4 The alkylene-OH group is preferably a -C2 alkylene-OH group.

[0030] In one embodiment of formula (I), R2 is C 3-20 Alkylene, preferably C 3-11 Alkylene, preferably C 7-11 Alkylene, preferably C 5-11 Alkylene.

[0031] In one embodiment of formula (I), R3 is C 5-20 Alkylene, preferably C 7-20 Alkylene, preferably C 7-11 Alkylene, preferably C 5-11 The alkylene group is preferably a C7-9 alkylene group, preferably a C7 alkylene group.

[0032] In one embodiment of formula (I), each M is independently -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-, preferably -C(O)O- or -OC(O)-, preferably -C(O)O-.

[0033] In one embodiment of formula (I), each Y is independently selected from -O-.

[0034] In one embodiment of Formula (I), each R * Each independently selected from C 1-11 alkyl, wherein one or more methylene groups are each independently replaced by the following groups: -CH=CH-, -C≡C-, preferably C 1-8 Alkyl, preferably C 1-6 Alkyl group, preferably C6 alkyl group.

[0035] In one embodiment, the invention discloses a lipid compound of formula (II), or a salt or isomer thereof:

[0036]

[0037] Among them, R1, R2, R3, M and R* are the same as those defined in formula (I).

[0038] In one embodiment of formula (II), R1 is -C 2-6 Alkylene-OH;

[0039] M is selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C (O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, -OP(O)(OH)-O-;

[0040] R2 is C 3-20 Alkylene;

[0041] R3 is C 7-20 Alkylene;

[0042] R* is each independently selected from C 1-11 An alkyl group, wherein one or more methylene groups are each independently replaced by the following groups: -CH=CH- or -C≡C-.

[0043] In one embodiment of formula (II), R1 is -C 2-6 alkylene-OH; M is selected from -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-; R2 is C 3-11 Alkylene; R3 is C 7-11 Alkylene; R* are each independently selected from C 1-6 alkyl.

[0044] In one embodiment of formula (II), R1 is -C 2-4 alkylene-OH; M is selected from -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-; R2 is C 7-11 Alkylene; R3 is C 7-9 Alkylene; R* are each independently selected from C 1-6 alkyl.

[0045] In one embodiment of formula (II), R1 is -C2 alkylene-OH; M is -C(O)O- or -OC(O)-; R2 is C 7-11 Alkylene; R3 is C 7-9 Alkylene; R* are each independently selected from C 1-6 alkyl.

[0046] In one embodiment of formula (II), R1 is -C2 alkylene-OH; M is -C(O)O- or -OC(O)-; R2 is C 7-11 alkylene; R3 is a C7 alkylene; R* is each independently selected from C6 alkyl.

[0047] In one embodiment of formula (II), R1 is -C2 alkylene-OH. In one embodiment, R1 is -C3 alkylene-OH. In one embodiment, R1 is -C4 alkylene-OH. In one embodiment, R1 is -C5 alkylene-OH. In one embodiment, R1 is -C6 alkylene-OH.

[0048] In one embodiment of formula (II), M are each independently selected from -C(O)O-.

[0049] In one embodiment of formula (II), R2 is independently selected from C 3-11 Alkylene, and R3 is independently selected from C 7-11 In one embodiment, R2 is independently C3 alkylene. In one embodiment, R2 is independently C4 alkylene. In one embodiment, R2 is independently C5 alkylene. In one embodiment, R2 is independently C6 alkylene. In one embodiment, R2 is independently C7 alkylene. In one embodiment, R2 is independently C8 alkylene. In one embodiment, R2 is independently C9 alkylene. In one embodiment, R2 is independently C 10 alkylene. In one embodiment, R2 is independently C 11In one embodiment, R3 is independently C7 alkylene. In one embodiment, R3 is independently C8 alkylene. In one embodiment, R3 is independently C9 alkylene. In one embodiment, R3 is independently C 10 alkylene. In one embodiment, R3 is independently C 11 Alkylene.

[0050] In one embodiment of formula (II), R* is each independently selected from C 1-6 alkyl. In one embodiment, R* is each independently selected from C 4-6 alkyl. In one embodiment, R* is C6 alkyl.

[0051] In one embodiment, the present invention discloses a lipid compound of formula (III), or a salt or isomer thereof:

[0052]

[0053] Among them, R2, R3 and R* are the same as those defined in formula (I), and p is an integer of 2-6.

[0054] In one embodiment of formula (III), p is 2; R2 is C 7-11 Alkylene; R3 is C 7-9 Alkylene; R* are each independently selected from C 1-6 alkyl.

[0055] In one embodiment of formula (III), p is 2; R2 is C 7-11 alkylene; R3 is C7 alkylene; R* is each independently C6 alkyl.

[0056] In one embodiment of formula (III), R2 is independently selected from C 7-11 Alkylene, and R3 is independently selected from C 7-9 In one embodiment, R2 is independently C7 alkylene. In one embodiment, R2 is independently C8 alkylene. In one embodiment, R2 is independently C9 alkylene. In one embodiment, R2 is independently C 10 alkylene. In one embodiment, R2 is independently C 11 alkylene. In one embodiment, R3 is independently C7 alkylene. In one embodiment, R3 is independently C8 alkylene. In one embodiment, R3 is independently C9 alkylene.

[0057] In one embodiment of formula (III), R* is each independently selected from C 1-6 alkyl. In one embodiment, R* is each independently selected from C 4-6alkyl. In one embodiment, R* is C6 alkyl.

[0058] In one embodiment, the present invention discloses a lipid compound of formula (IV), or a salt or isomer thereof:

[0059]

[0060] Among them, R2, R3, M and R* are the same as those defined in formula (I), and p is an integer of 2-6.

[0061] In one embodiment of formula (IV), p is 2-4;

[0062] M is selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C (O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, or -OP(O)(OH)-O-;

[0063] R2 is C 3-20 Alkylene;

[0064] R3 is C 7-20 Alkylene;

[0065] R* is each independently selected from C 1-11 An alkyl group, wherein one or more methylene groups are each independently substituted with the following groups: -CH=CH- or -C≡C-.

[0066] In one embodiment of formula (IV), p is 2-4; M is selected from -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-; R2 and R3 are each independently selected from C 5-11 Alkylene; R* are each independently selected from C 1-6 alkyl.

[0067] In one embodiment of formula (IV), p is 2-4; M is -C(O)O- or -OC(O)-; R2 is C 7-11 Alkylene; R3 is C 7-9 Alkylene; R* are each independently selected from C 1-6 alkyl.

[0068] In one embodiment, the present invention discloses a composition comprising an ionizable lipid compound of the present invention. In one embodiment, the composition further comprises a phospholipid. In one embodiment, the phospholipid is selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-arachidonoyl-sn-glycero-3-phosphocholine, 1,2-bis-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (DPPG), At least one of phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0069] In one embodiment, the composition further comprises a PEGylated lipid compound. In one embodiment, the PEGylated lipid compound is selected from at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0070] In one embodiment, the composition further comprises a structured lipid. In one embodiment, the structured lipid is selected from at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol.

[0071] In one embodiment, the composition further comprises an active ingredient, and the active ingredient is selected from at least one of DNA, RNA, protein, and pharmaceutically active molecules. In one embodiment, the RNA is selected from at least one of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, and lncRNA. In one embodiment, the protein is selected from at least one of antibodies, enzymes, recombinant proteins, polypeptides, and short peptides.

[0072] In one embodiment of the composition of the invention, the ionizable lipid compound of the invention is 20%-80%, the PEGylated lipid compound is 1%-10%, the structured lipid is 10%-50% and the phospholipid is 5-30%, by mole.

[0073] In one embodiment, the invention discloses a lipid nanoparticle comprising the composition of the invention.

[0074] In one embodiment, the present invention discloses a method for producing lipid nanoparticles, comprising step (1): dissolving and mixing the ionizable lipid compound of the present invention with a polyethanolated lipid compound, a structural lipid and a phospholipid. Optionally, the method further comprises step (2): mixing with an active ingredient through a mixer to form lipid nanoparticles.

[0075] In one embodiment, the present invention discloses the use of the ionizable lipid compound of the present invention in preparing lipid nanoparticles. Optionally, the lipid nanoparticles are neutral and uncharged in a neutral medium, and are positively charged after protonation in an acidic medium.

[0076] In one embodiment, the invention discloses a pharmaceutical composition comprising the lipid nanoparticles of the invention and a pharmaceutically acceptable carrier.

[0077] In one embodiment, the present invention discloses the use of the lipid nanoparticles or pharmaceutical compositions of the present invention in the preparation of a drug. In one embodiment, the drug comprises an active ingredient, and the active ingredient is selected from at least one of DNA, RNA, protein, and pharmaceutically active molecules. In one embodiment, the RNA is selected from at least one of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, and lncRNA. In one embodiment, the protein is selected from at least one of antibodies, enzymes, recombinant proteins, polypeptides, and short peptides. In one embodiment, the drug is administered to humans by intravenous injection, intramuscular injection, subcutaneous injection, microneedle patch, oral administration, oral and nasal spray, and smearing.

[0078] In one embodiment, the chemical formula of the ionizable lipid compound of the present invention is as follows:

[0079]

[0080]

[0081]

[0082] Compared with the prior art, the ionizable lipids of the present application are different from:

[0083] 1. Different chemical structures: Two or one of the ester-containing fatty chains connected to the nitrogen atom (N) of the tertiary amine forms novel ether-containing fatty chains with saturated fatty chains or unsaturated fatty chains through the hydroxyl groups at the 1st or 3rd positions of glycerol, respectively, and the results show better ionizable lipid transfection efficiency;

[0084] 2. Simple synthesis and easy to obtain raw materials: the starting materials are glycerol, short fatty alcohols and fatty acids. The raw materials are cheap and easy to obtain, and easy to obtain. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figures 1A-1D The number of photons per unit area of ​​the legs and liver at different time points after administration of the compounds of the present invention (p / sec / cm) in each group of mice 2 )

[0086] Figure 2 Schematic diagram of IgG antibody after the first immunization of mice in each group (ns: p>0.05; ****: p<0.0001);

[0087] Figure 3 Schematic diagram of IgG antibody after 14 days of second immunization of mice in each group (ns: p>0.05; ***: p<0.001);

[0088] Figure 4The results of endocrine T lymphocytes in mice after vaccination of each group of mice were obtained.

[0089] definition:

[0090] 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 "Including 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 carbon atoms.

[0091] "XY-membered" means containing XY ring atoms. For example, "4-7-membered" means 4-7 ring atoms, such as 4 ring atoms, 5 ring atoms, 6 ring atoms or 7 ring atoms, and the ring atoms can be carbon atoms or heteroatoms, such as nitrogen, oxygen, sulfur, boron, phosphorus and silicon.

[0092] The term "alkyl" refers to a straight or branched chain saturated hydrocarbon group comprising one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms). 1-12 alkyl" refers to a linear or branched saturated hydrocarbon group containing 1 to 12 carbon atoms. "C 2-18 alkyl" refers to an optionally substituted linear or branched saturated hydrocarbon group including 2 to 18 carbon atoms. Unless otherwise specified, alkyl groups described herein refer to unsubstituted and substituted alkyl groups.

[0093] The term "alkenyl" or "alkenyl" refers to a straight or branched chain hydrocarbon group (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) containing two or more carbon atoms and at least one carbon-carbon double bond. Alkenyl groups can include one, two, three, four or more carbon-carbon double bonds. Specifically, "C 3-12 Alkenyl" refers to a linear or branched hydrocarbon group comprising 3 to 12 carbon atoms and at least one carbon-carbon double bond. "C 4-18Alkenyl" refers to a linear or branched chain hydrocarbon group comprising 4 to 18 carbon atoms and at least one carbon-carbon double bond. Unless otherwise specified, alkenyl groups described herein refer to unsubstituted and substituted alkenyl groups.

[0094] The term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0095] The term "haloalkyl" refers to that the above-mentioned "alkyl" is substituted by one or more halogen groups. Exemplary haloalkyl includes but is not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc.

[0096] The term "cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group consisting of ring carbon atoms and containing no ring heteroatoms. 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.

[0097] The term "heterocyclic group" refers to a group of a non-aromatic ring system having a ring heteroatom, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon. In a heterocyclic group containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as the valence permits. Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidine, oxetane, and thiidine. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-diketone. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, 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, hexahydrotriazinyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thianyl.

[0098] The term "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) group consisting of ring carbon atoms and no ring 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"; for example, naphthyl, eg, 1-naphthyl and 2-naphthyl).

[0099] The term "heteroaryl" refers to a group of a monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having 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 rings are fused to one or more cycloalkyl or heterocyclic 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. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl and thienyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl) and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups 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.

[0100] The term "isomer" refers to compounds having the same molecular formula, including enantiomers, diastereomers, stereoisomers, etc. Stereoisomers are particularly preferred in the present invention, and the term "stereoisomer" refers to isomers that differ only in the spatial arrangement of atoms.

[0101] In some cases, the compounds of the present invention can form salts, which are also within the scope of the present invention. The term "salt(s)" refers to acidic and / or basic salts formed with inorganic and / or organic acids and bases. Pharmaceutically acceptable salts are particularly preferred in the present invention.

[0102] The term "pharmaceutically acceptable salt" means those carboxylates, amino acid addition salts of the compounds of the 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 invention.

[0103] 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.

[0104] 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.

[0105] 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).

[0106] The term "acceptable carrier" refers to a suitable carrier that can be used to utilize the existing material for the purposes of the present invention without excessive toxicity, irritation, allergic response, etc., equivalent to a reasonable benefit / risk ratio.

[0107] The following abbreviations can be used in the Examples and throughout the specification:

[0108] AOP: 7-Azabenzotriazole-1-yloxytris(dimethylamino)phosphine hexafluorophosphate

[0109] BOP: benzotriazole-1-yloxytris(dimethylamino)phosphine hexafluorophosphate

[0110] BOP-Cl: Bis(2-oxo-3-oxazolidinyl)phosphoryl chloride

[0111] BDMP: 1-N-methylpyrrolinium-3-oxy-benzotriazole hexachloroante antimony salt

[0112] BMMP: 1-N-ethylpyrrolidinium-3-oxy-benzotriazole hexachloroante antimony salt

[0113] BOMI: 1-tert-butoxycarbonyl-5-methoxy-indole-2-ylboric acid

[0114] CDI: 1,1'-Carbonyldiimidazole

[0115] CDMT: 2-chloro-4,6-dimethoxy-1,3,5-triazine

[0116] DCC: dicyclohexylcarbodiimide

[0117] DCMT: 4,5-dichloro-2-(methylthio)pyrimidine

[0118] DECP: diethyl cyanophosphonate

[0119] DIC: N, N'-diisopropylcarbodiimide

[0120] DPPA: diphenylphosphoryl azide

[0121] DPP-Cl: diphenylphosphoryl chloride

[0122] EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide

[0123] EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0124] EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-di-hydroquinoline

[0125] EMPA: Ethyl methyl phosphinic anhydride

[0126] HATU: O-(7-azabenzotriazole-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate

[0127] HBTU: O-(benzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate

[0128] HCTU: O-(5-chlorobenzotriazole-1-yl)-bis(dimethylamino)carbium hexafluorophosphate

[0129] HAPyU: O-(7-azabenzotriazole-1-yl)-bis(tetrahydropyrrolyl)carbium hexafluorophosphate

[0130] HBPyU: O-(benzotriazole-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate

[0131] MPTA: thiodimethylphosphoryl azide

[0132] PyAOP: 7-Azabenzotriazole-1-yloxy-tris(tetrahydropyrrolyl)phosphonium hexafluorophosphate

[0133] PyBOP: benzotriazole-1-yloxy-tris(tetrahydropyrrolyl)phosphonium hexafluorophosphate

[0134] T3P: n-propylphosphonic anhydride

[0135] TBTU: O-(benzotriazole-1-yl)-bis(dimethylamino)carbium tetrafluoroborate

[0136] TNTU: O-(N-endo-5-norcinene-2,3-dicarbodiimide)-bis(dimethylamino)carbium tetrafluoroborate

[0137] TSTU: O-(N-succinimido)-bis(dimethylamino)carbium tetrafluoroborate

[0138] DMAP: 4-dimethylaminopyridine

[0139] HOAt: 1-Hydroxy-7-azabenzotriazole

[0140] HOBt: 1-Hydroxybenzotriazole

[0141] HOSu: N-hydroxysuccinimide

[0142] 4-PPY: 4-pyrrolidinylpyridine

[0143] 9-AJ: 9-azajulonidine

[0144] TMAJ: 1,1,7,7-tetramethyl-9-azazuronidine

[0145] NHPI: N-hydroxyphthalimide Specific implementation plan

[0146] Scheme 1. A compound of formula (I), or a salt or isomer thereof:

[0147]

[0148] in,

[0149] R1 is -C 1-6 Alkylene-X,

[0150] Where X is selected from amino, hydroxy, ethynyl, cyano, -C(O)(CH2) 1-3 NR a R b 、-C(O)O(CH2) 1-3 NR aR b 、-OC(O)(CH2) 1-3 NR a R b 、-C(O)NH(CH2) 1-3 NR a R b 、-NHC(O)(CH2) 1-3 NR a R b 、-NHC(O)CH(NR a R b )(CH2) 1- 3NR a R b , C 3-7 Cycloalkyl, 4-7 membered heterocyclic group, C 6-10 aryl or 5-10-membered heteroaryl, the above-mentioned cycloalkyl, heterocyclyl, aryl and heteroaryl groups are optionally substituted by the following groups;-(CH2) 1-3 OH, -(CH2) 1-3 NR a R b or -(CH2) 1-3 C(O)NR a R b ;

[0151] Or X is:

[0152]

[0153] R2 is C 3-20 An alkylene group, wherein one or more methylene groups are each independently replaced by the following groups: -CH=CH-, -C≡C- or -O-;

[0154] R3 is C 5-20 An alkylene group, wherein one or more methylene groups are each independently replaced by the following groups: -CH=CH-, -C≡C- or -O-;

[0155] M is independently selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, or -OP(O)(OH)-O-;

[0156] R4 is

[0157] Wherein R is selected from H, R* or -(CH2) 1-5 -YR * ;

[0158] Y is selected from -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC (O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, or -OP(O)(OH)-O-;

[0159] R * Each independently selected from C 1-12 Alkyl, C 2-12 Alkenyl or C 2-12 Alkynyl;

[0160] R a , R b Independently selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl.

[0161] Scheme 2. The compound of formula (I) of Scheme 1 or a salt or isomer thereof, wherein

[0162] R1 is -C 1-6 Alkylene-X,

[0163] Where X is selected from amino, hydroxy, ethynyl or cyano;

[0164] R2 is C 3-20 Alkylene;

[0165] R3 is C 5-20 Alkylene;

[0166] M is independently selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH- or -NHC(S)-;

[0167] R4 is

[0168] Wherein R is selected from H, R * or -(CH2) 1-5 -YR * ;

[0169] Y is selected from -NH-, -C(O)-, -C(S)-, -O- or -S-;

[0170] R * Each independently selected from C 1-12 Alkyl, C 2-12 Alkenyl or C 2-12 Alkynyl.

[0171] Scheme 3. The compound or salt or isomer thereof according to any one of Scheme 1-2, wherein R1 is -C 1-6 Alkylene -OH, preferably -C 2-6 Alkylene -OH, preferably -C 2-4 The alkylene-OH group is preferably a -C2 alkylene-OH group.

[0172] Scheme 4. The compound or salt or isomer thereof according to any one of Scheme 1-3, wherein R2 is C 3-20 Alkylene, preferably C 3-11 Alkylene, preferably C 5-11 Alkylene, preferably C 7-11 Alkylene.

[0173] Scheme 5. The compound or salt or isomer thereof according to any one of Scheme 1-4, wherein R3 is C 5-20 Alkylene, preferably C 7-20 Alkylene, preferably C 7-11 Alkylene, preferably C 5-11 Alkylene, preferably C 7-9 Alkylene, preferably C7 alkylene.

[0174] Scheme 6. A compound according to any one of Schemes 1 to 5, or a salt or isomer thereof, wherein each M is independently -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-, preferably -C(O)O- or -OC(O)-, preferably -C(O)O-.

[0175] Scheme 7. A compound or a salt or isomer thereof according to any one of Scheme 1-6, wherein each Y is independently selected from -O-.

[0176] Scheme 8. The compound or salt or isomer thereof according to any one of Scheme 1-7, wherein each R * Each independently selected from C 1-11 alkyl, wherein one or more methylene groups are each independently replaced by the following groups: -CH=CH-, -C≡C-, preferably C 1-8 Alkyl, preferably C 1-6 Alkyl group, preferably C6 alkyl group.

[0177] Scheme 9. The compound according to Scheme 1, or a salt or isomer thereof, wherein the compound has formula (II):

[0178]

[0179] in,

[0180] R1 is -C 2-6 Alkylene-OH;

[0181] M is selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C (O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, -OP(O)(OH)-O-;

[0182] R2 is C 3-20 Alkylene;

[0183] R3 is C 7-20 Alkylene;

[0184] R* is each independently selected from C 1-11 An alkyl group, wherein one or more methylene groups are each independently replaced by the following groups: -CH=CH- or -C≡C-.

[0185] Scheme 10. The compound of formula (II) or a salt or isomer thereof according to Scheme 9, wherein

[0186] R1 is -C 2-6 Alkylene-OH;

[0187] M is selected from -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-;

[0188] R2 is C 3-11 Alkylene;

[0189] R3 is C 7-11 Alkylene;

[0190] R* is each independently selected from C 1-6 alkyl.

[0191] Scheme 11. The compound of formula (II) or a salt or isomer thereof according to Scheme 9, wherein

[0192] R1 is -C 2-4 Alkylene-OH:

[0193] M is selected from -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-;

[0194] R2 is C 7-11 Alkylene;

[0195] R3 is C 7-9 Alkylene;

[0196] R* is each independently selected from C 1-6 alkyl.

[0197] Scheme 12. The compound of formula (II) or a salt or isomer thereof according to Scheme 9, wherein

[0198] R1 is -C2 alkylene-OH;

[0199] M is -C(O)O- or -OC(O)-;

[0200] R2 is C 7-11 Alkylene;

[0201] R3 is C 7-9 Alkylene:

[0202] R* is each independently selected from C 1-6 alkyl.

[0203] Scheme 13. The compound of formula (II) or a salt or isomer thereof according to Scheme 9, wherein

[0204] R1 is -C2 alkylene-OH;

[0205] M is -C(O)O- or -OC(O)-;

[0206] R2 is C 7-11 Alkylene;

[0207] R3 is C7 alkylene;

[0208] R* is each independently selected from C6 alkyl.

[0209] Scheme 14. The compound according to Scheme 1, or a salt or isomer thereof, wherein the compound has formula (III):

[0210]

[0211] in,

[0212] p is 2;

[0213] R2 is C 7-11 Alkylene;

[0214] R3 is C 7-9Alkylene;

[0215] R* is each independently selected from C 1-6 alkyl.

[0216] Scheme 15. The compound of formula (III) or a salt or isomer thereof according to Scheme 14, wherein

[0217] p is 2;

[0218] R2 is C 7-11 Alkylene;

[0219] R3 is C7 alkylene;

[0220] R* is each independently a C6 alkyl group.

[0221] Scheme 16. The compound according to Scheme 1, or a salt or isomer thereof, wherein the compound has formula (IV):

[0222]

[0223] in,

[0224] p is 2-4;

[0225] M is selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C (O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, or -OP(O)(OH)-O-;

[0226] R2 is C 3-20 Alkylene;

[0227] R3 is C 7-20 Alkylene;

[0228] R* is each independently selected from C 1-11 An alkyl group, wherein one or more methylene groups are each independently substituted with the following groups: -CH=CH- or -C≡C-.

[0229] Scheme 17. The compound of formula (IV) or a salt or isomer thereof according to Scheme 16, wherein

[0230] p is 2-4;

[0231] M is selected from -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-;

[0232] R2 and R3 are each independently selected from C 5-11 Alkylene;

[0233] R* is each independently selected from C 1-6 alkyl.

[0234] Scheme 18. The compound of formula (IV) or a salt or isomer thereof according to Scheme 16, wherein

[0235] p is 2-4;

[0236] M is -C(O)O- or -OC(O)-;

[0237] R2 is C 7-11 Alkylene;

[0238] R3 is C 7-9 Alkylene;

[0239] R* is each independently selected from C 1-6 alkyl.

[0240] Scheme 19. A compound or a salt or isomer thereof selected from any one of Al-A17:

[0241]

[0242]

[0243]

[0244] Scheme 20. A composition characterized in that it comprises the compound of any one of Scheme 1-19.

[0245] Scheme 21. The composition according to Scheme 20, characterized in that it further comprises phospholipids.

[0246] Scheme 22. The composition according to Scheme 21, characterized in that the phospholipid is selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl At least one of 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-oleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0247] Scheme 23. The composition according to any one of Scheme 20-22, characterized in that it further comprises a polyethanolated lipid compound.

[0248] Scheme 24. A composition according to Scheme 23, characterized in that the polyethanolylated lipid compound is selected from at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0249] Scheme 25. The composition according to any one of Scheme 20-24, characterized in that it further comprises structural lipids.

[0250] Scheme 26. A composition according to Scheme 25, characterized in that the structured lipid is selected from at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and α-tocopherol.

[0251] Scheme 27. A composition according to any one of Schemes 20-26, characterized in that the composition comprises 20%-80% of the compound according to any one of Schemes 1-19, 1%-10% of a pegylated lipid compound, 10%-50% of a structural lipid and 5-30% of a phospholipid, by mole.

[0252] Scheme 28. The composition according to Scheme 27, further comprising an active ingredient selected from at least one of DNA, RNA, protein, and pharmaceutically active molecules.

[0253] Scheme 29. The composition of Scheme 28, characterized in that the RNA is selected from at least one of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, and lncRNA.

[0254] Scheme 30. The composition of Scheme 28, the protein selected from at least one of an antibody, an enzyme, a recombinant protein, a polypeptide, and a short peptide.

[0255] Scheme 31. A lipid nanoparticle comprising the composition according to any one of Scheme 20-30.

[0256] Scheme 32. A method for preparing the lipid nanoparticles described in Scheme 31, comprising step (1): dissolving and mixing the compounds described in Schemes 1-19 optionally with polyethanolated lipid compounds, structured lipids and phospholipids.

[0257] Scheme 33. The method according to Scheme 32, further comprising the step (2): mixing with the active ingredient through a mixer to form lipid nanoparticles.

[0258] Scheme 34. Use of the compound of any one of Scheme 1-19 in the preparation of lipid nanoparticles.

[0259] Scheme 35. The use according to Scheme 34, characterized in that the lipid nanoparticles are neutrally uncharged in the neutral medium and are positively charged after protonation in the acidic medium.

[0260] Schemes 36. A pharmaceutical composition comprising the lipid nanoparticles of Schemes 31 and a pharmaceutically acceptable carrier.

[0261] Scheme 37. Use of the lipid nanoparticles of Scheme 31 or the pharmaceutical composition of Scheme 36 in the preparation of a drug.

[0262] Scheme 38. The use according to Scheme 37, characterized in that the drug comprises an active ingredient selected from at least one of DNA, RNA, protein, and drug-active molecules.

[0263] Scheme 39. The use according to Scheme 38, characterized in that the RNA is selected from at least one of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, and lncRNA.

[0264] Scheme 40. The use according to Scheme 38, characterized in that the protein is selected from at least one of an antibody, an enzyme, a recombinant protein, a polypeptide, and a short peptide.

[0265] Scheme 41. The use according to any one of Schemes 37-40, characterized in that the drug is administered to humans by intravenous injection, intramuscular injection, subcutaneous injection, microneedle patch, oral administration, oral and nasal spray, or application. DETAILED DESCRIPTION

[0267] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail by the following examples and in conjunction with the accompanying drawings. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0268] Synthesis of Example 1-A1

[0269] Synthesis route:

[0270]

[0271] Synthesis steps:

[0272] 1. Synthesis of ((2,3-bis(hexyloxy)propoxy)methyl)benzene

[0273] Add 50 ml of n-hexane, 1-benzyloxy-2,3-propanediol (10.0 g) and tetrabutylammonium bromide (0.88 g), stir for 10 min, add sodium hydroxide solution (18 mol / L) and bromohexane (36.2 g), heat and stir to react. After the reaction is complete, cool to room temperature, stand for stratification, collect the organic layer, wash and extract, and separate and purify the organic layer by column (n-heptane: ethyl acetate = 5%) to obtain ((2,3-bis(hexyloxy)propoxy)methyl)benzene.

[0274] Synthesis of 2,3-bis(hexyloxy)-1-propanol

[0275] Add ((2,3-bis(hexyloxy)propoxy)methyl)benzene (15.0 g), ethyl acetate (50 ml), glacial acetic acid (3.75 ml) and palladium hydroxide on carbon (20%, 1.0 g), and stir with hydrogenation at room temperature to react. After the reaction is complete, filter, collect the filtrate, wash the filtrate, extract the liquid, and collect the organic phase. Concentrate to dryness under reduced pressure to obtain the compound 2,3-bis(hexyloxy)-1-propanol.

[0276] 3. Synthesis of 2,3-bis(hexyloxy)propyl-8-bromooctanoate

[0277] 8-Bromooctanoic acid (10.3 g) was dissolved in dichloromethane (100 ml), and DCC (10.3 g), DMAP (6.1 g), and 2,3-bis(hexyloxy)-1-propanol (10 g) were added, and the mixture was stirred at room temperature for reaction. After the reaction was complete, the dichloromethane solvent was removed by concentration, and the liquid was separated by extraction. The organic phase was dried and concentrated to dryness, and the crude product was separated and purified by column (PE: EA = 20: 1) to obtain 2,3-bis(hexyloxy)propyl-8-bromooctanoate.

[0278] 4. Synthesis of 2,3-bis(hexyloxy)propyl-8-((2-hydroxyethyl)amino)octanoate

[0279] 2,3-Bis(hexyloxy)propyl-8-bromooctanoate (5.0 g) was dissolved in anhydrous ethanol (50 ml), and sodium carbonate (2.2 g), KI (0.17 g) and ethanolamine (20 g) were added, and the mixture was stirred at room temperature for reaction. After the reaction was complete, the solvent was removed by concentration, and the liquid was washed and extracted. The organic phase was dried, filtered and concentrated to dryness, and the crude product was separated and purified by column (MeOH:DCM=3%) to obtain 2,3-bis(hexyloxy)propyl-8-((2-hydroxyethyl)amino)octanoate.

[0280] 5. Synthesis of A1

[0281] 1,3-Bis(hexyloxy)-2-propanol-8-bromooctanoate (2.08 g) and 2,3-bis(hexyloxy)propyl-8-((2-hydroxyethyl)amino)octanoate (2.0 g) were dissolved in anhydrous ethanol (20 ml), and sodium carbonate (0.95 g) and KI (0.15 g) were added to react under reflux. After the reaction was completed, the solvent was removed by concentration, and the liquid was washed and extracted. The organic phase was dried, filtered and concentrated to dryness, and the crude product was separated and purified by column chromatography (MeOH:DCM=3%) to obtain an oily product A1 (2.1 g). 1 H NMR (500MHz, CDCl3) δ (ppm) = 5.05 (p, J = 5.1Hz, 1H), 4.15 (dd, J = 11.6, 4.1Hz, 1H), 4.03 (dd, J=11.6, 5.8Hz, 1H), 3.55 (dt, J=10.4, 5.4Hz, 1H), 3.49 (m, 8H), 3.43-3.30 (m, 8H), 2.53 (t, J=5.1Hz, 2H), 2.43-2.35 (m, 4H), 2.25 (t, J=7.5Hz, 4H), 1.60-1.52 (m, 4H), 1. 48(m, 8H), 1.38(m, 4H), 1.31-1.15(m, 36H), 0.82(t, J=6.8Hz, 12H).MS(ESI): m / z(M+H) + 832.10, (M+Na) + 853.2.

[0282] Synthesis of Example 2-A2

[0283] Synthesis route:

[0284]

[0285] A2 was prepared with reference to the method of Example 1 to obtain a product as an oil (4.01 g). 1 H NMR (500MHz, CDCl3) δ (ppm) = 4.15 (dd, J = 11.6, 4.1Hz, 2H), 4.02 (dd, J = 11.6, 5.8Hz, 2 H), 3.54 (m, 2H), 3.48 (t, J=6.6Hz, 6H), 3.42-3.34 (m, 8H), 2.54 (t, J=5.0Hz, 2H), 2.4 4-2.36(t, 4H), 2.25(t, J=7.5Hz, 4H), 1.59-1.52(m, 4H), 1.52-1.44(m, 8H), 1.39(dd , J=14.0, 7.3Hz, 4H), 1.32-1.17 (m, 36H), 0.82 (t, J=6.8Hz, 12H).MS (ESI): m / z (M+H)+ 831.92.

[0286] Synthesis of Example 3-A3

[0287] Synthesis route:

[0288]

[0289] A3 was prepared by the method of Reference Example 1 to obtain an oily product (0.3 g). 1H NMR (500 MHz, CDCl3) δ (ppm) = 5.36-5.21 (m, 4H), 4.15 (dd, J = 11.6, 4.1 Hz, 1H), 4.03 (dd, J = 11.6, 5.8 Hz, 1H), 3.76-3.65 (m, 2H), 3.58-3.52 (m, 1H), 3.52-3.31 ( m, 8H), 2.81 (s, 2H), 2.73-2.65 (m, 4H), 2.25 (t, J=7.5Hz, 2H), 1.98 (m, 4H), 1.6 1-1.44(m, 10H), 1.33-1.16(m, 34H), 0.82(t, J=8.0Hz, 9H).MS(ESI): m / z(M+H) + 695.40.

[0290] Synthesis of Example 4-A4

[0291] Synthesis route:

[0292]

[0293] A4 was prepared by the method of Reference Example 1 to obtain an oily product (1.65 g). 1H NMR (500 MHz, CDCl3) δ (ppm) = 4.28-4.18 (dd, 1H), 4.14-4.00 (dd, 1H), 3.75 (m 1H), 3.62 (dd, J = 11.3, 6.7 Hz, 1H), 3.58-3.49 (m, 4H), 3.49-3.40 (m, 4H), 2.61 (s, 2H), 2.52-2.44 (m, 4H), 2.28 (t, J = 14.3 Hz, 4H), 1.60-1.45 (m, 16H), 1.28 (m, 48H), 0.87 (t, J = 6.4 Hz, 16H). MS (ESI): m / z (M+H) + 827.10.

[0294] Synthesis of Example 5-A5

[0295] Synthesis route:

[0296]

[0297] A5 was prepared with reference to the method of Example 1 to obtain an oily product (2.15 g). 1 H NMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J = 11.6, 4.1Hz, 1H), 4.13-4.02 (m, 3H), 3. 64-3.58 (m, 1H), 3.57-3.50 (m, 4H), 3.45 (dt, J=13.3, 5.6Hz, 4H), 2.58 (t, J=5.2H z, 2H), 2.45 (dd, J=14.5, 7.0Hz, 4H), 2.30 (t, 4H), 1.68-1.51 (m, 10H), 1.45 (dt, J =15.1, 7.6Hz, 4H), 1.37-1.20 (m, 36H), 0.88 (t, J = 6.2Hz, 9H). MS (ESI): m / z (M+H) + 714.9, (M+Na) + 736.8.

[0298] Synthesis of Example 6-A6

[0299] Synthesis route:

[0300]

[0301] A6 was prepared with reference to the method of Example 1 to obtain an oily product (1.3 g). 1 H NMR (500MHz, CDCl3) δ (ppm) = 4.21 (dd, J = 11.6, 4.0Hz, 2H), 4.09 (dd, J = 11.5, 5.8Hz, 2H ), 3.73-3.66 (t, 4H), 3.61 (dd, J=9.9, 5.1Hz, 2H), 3.54 (t, J=6.6Hz, 4H), 3.50-3.39 (m , 8H), 2.60 (s, 2H), 2.46 (s, 10H), 2.31 (t, J=7.5Hz, 4H), 1.66-1.58 (m, 4H), 1.58-1.50 (m, 8H), 1.43 (s, 4H), 1.28 (m, 36H), 0.88 (t, J=6.7Hz, 12H). MS (ESI): m / z (M+H) + 901.4.

[0302] Synthesis of Example 7-A7

[0303] Synthesis route:

[0304]

[0305] A7 was prepared with reference to the method of Example 1 to obtain an oily product (1.05 g). 1 H NMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J = 11.6, 4.1Hz, 2H), 4.09 (dd, J = 11.6, 5.8Hz, 2H), 3.78 (t, J=5.0Hz, 2H), 3.64-3.57 (m, 2H), 3.55 (t, J=6.7Hz, 4H), 3.46 (m, 8H), 2 .67-2.61(t, 2H), 2.46-2.37(t, 4H), 2.31(t, J=7.5Hz, 4H), 1.71-1.58(m, 6H), 1.52 (dq, J=33.4, 6.8Hz, 12H), 1.28 (m, 36H), 0.88 (t, J=6.8Hz, 12H). MS (ESI): m / z (M+H) + 846.0, (M+Na) + 868.0.

[0306] Synthesis of Example 8-A8

[0307] Synthesis route:

[0308]

[0309] A8 was prepared with reference to the method of Example 1 to obtain an oily product (1.19 g). 1 HNMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J1 = 11.5Hz, J2 = 2.1Hz, 2H), 4.09 (dd, J1 =11.6Hz, J2=5.8Hz, 2H), 3.64-3.58(m, 2H), 3.55(m, 6H), 3.49-3.41(m, 8H), 2. 60 (t, J=5.0Hz, 2H), 2.47 (dd, J=14.3, 6.8Hz, 4H), 2.32 (dd, J=13.4, 7.2Hz, 4H) , 1.64-1.46 (m, 16H), 1.30 (m, 32H), 0.88 (t, J=6.8Hz, 12H).MS (ESI): m / z (M+H) + 803.11.

[0310] Synthesis of Example 9-A9

[0311] Synthesis route:

[0312]

[0313] A9 was prepared with reference to the method of Example 1 to obtain an oily product (1.5 g). 1HNMR (500MHz, CDCl3) δ (ppm) = 4.23 (dt, J = 9.2, 6.2Hz, 2H), 4.13-4.05 (m, 2H), 3.65-3.58 (m, 2H), 3.57-3.50 (m, 6H), 3.45 (dt, J = 13.2, 5.5Hz, 8H), 2 .58 (t, J=5.0Hz, 2H), 2.46 (dt, J=26.2, 7.2Hz, 4H), 2.38-2.28 (t, 4H), 1.7 7-1.47 (m, 14H), 1.30 (m, 30H), 0.88 (t, J=6.6Hz, 12H).MS (ESI): m / z (M+H) + 775.44.

[0314] Example 10-Synthesis of A10

[0315] Synthesis route:

[0316]

[0317] A10 was prepared with reference to the method of Example 1 to obtain an oily product (0.8 g). 1 H NMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J = 11.6, 4.0Hz, 2H), 4.09 (dd, J = 11.6, 5.8Hz, 2H), 3.64-3.57 (m, 4H), 3.57-3.50 (t, J = 6.6Hz, 4H), 3.50-3 .40 (m, 8H), 2.66 (s, 2H), 2.58-2.48 (m, 4H), 2.31 (t, J=7.4Hz, 4H), 1.65 -1.47(m, 16H), 1.31(m, 40H), 0.88(t, J=6.7Hz, 12H).MS(ESI): m / z(M+H) + 859.30.

[0318] Example 11-Synthesis of A11

[0319] Synthesis route:

[0320]

[0321] A11 was prepared with reference to the method of Example 1 to obtain an oily product (0.9 g). 1H NMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J = 11.6, 4.0Hz, 2H), 4.09 (dd, J = 11.6, 5.9Hz, 2H), 3.64-3.58 (m, 2H), 3.57-3.50 (t, J = 6.6Hz, 6H), 3.49-3.40 ( m, 8H), 2.60 (t, J=4.8Hz, 2H), 2.51-2.43 (t, 4H), 2.31 (t, J=7.5Hz, 4H), 1. 57-1.45 (m, 16H), 1.31 (m, 44H), 0.88 (t, J=6.8Hz, 12H).MS (ESI): m / z (M+H) + 887.62.

[0322] Example 12-Synthesis of A12

[0323]

[0324] A12 was prepared with reference to the method of Example 1 to obtain an oily product (0.3 g). 1 H NMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J = 11.6, 4.0Hz, 2H), 4.09 (dd, J = 11.5, 5.8Hz, 2H), 3.62 (m, 4H), 3.55 (t, J = 6.6Hz, 4H), 3.5 0-3.40 (m, 8H), 2.41 (s, 6H), 2.31 (t, J=7.5Hz, 4H), 1.65-1.40 (m, 20H), 1.31 (m, 38H), 0.88 (t, J=6.6Hz, 12H). MS (ESI): m / z (M+H) + 873.30.

[0325] Example 13-Synthesis of A13

[0326]

[0327] A13 was prepared with reference to the method of Example 1 to obtain an oily product (0.73 g). 1HNMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J = 11.6, 4.1Hz, 2H), 4.09 (dd, J = 11.6, 5.8Hz, 2H), 3.62 (m, 2H), 3.55 (t, J = 6.7Hz, 4H), 3.45 (dt, J = 13.3, 5.7Hz, 8H), 3.38 (m, 4H), 2.53 (s, 4H), 2.31 (t, J=7.5Hz, 4H), 1.69-1.50 (m, 20H), 1.37-1.23 (m, 36H), 0.88 (t, J=6.8Hz, 12H).MS (ESI): m / z (M+H) + 859.33.

[0328] Example 14-Synthesis of A14

[0329]

[0330] A14 was prepared with reference to the method of Example 1 to obtain an oily product (0.70 g). 1 HNMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J1 = 11.6Hz, J2 = 4.1Hz, 2H), 4.09 (dd, J1 = 11.6Hz, J2 = 5.8Hz, 2H), 3.62 (m, 4H), 3.55 (t, J = 6.7H z, 4H), 3.51-3.39 (m, 8H), 2.46 (s, 6H), 2.32 (t, J=7.5Hz, 4H), 1.58 (m, 20H), 1.32 (m, 40H), 0.88 (t, J=6.7Hz, 12H). MS (ESI): m / z (M+H) + 887.57.

[0331] Example 15-Synthesis of A15

[0332]

[0333] A15 was prepared with reference to the method of Example 1 to obtain an oily product (0.35 g). 1H NMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J = 11.6, 4.1Hz, 2H), 4.10 (dd, J = 11.6, 5.8Hz, 2H), 3.61 (m, 2H), 3.55 (m, 6H), 3.45 (dt, J=13.3, 5.2Hz, 8H), 2.60 (t, J =5.1Hz, 2H), 2.51 (t, J = 7.2Hz, 4H), 2.34 (t, J = 7.2Hz, 4H), 1.78 (m, 4H), 1.59 -1.50(m, 8H), 1.38-1.22(m, 24H), 0.88(t, J=6.8Hz, 12H).MS(ESI): m / z(M+H) + 718.70.

[0334] Example 16-Synthesis of A16

[0335]

[0336] A16 was prepared with reference to the method of Example 1 to obtain an oily product (0.23 g). 1 H NMR (500MHz, CDCl3) δ (ppm) = 4.22 (dd, J = 11.6, 4.1Hz, 2H), 4.09 (dd, J = 11.6, 5 .9Hz, 2H), 3.63-3.59(m, 2H), 3.55(t, J=6.7Hz, 4H), 3.49-3.41(m, 8H), 2.73( t, 2H), 2.61 (t, 4H), 2.31 (t, J=7.5Hz, 4H), 1.65-1.59 (m, 4H), 1.59-1.50 (m, 1 2H), 1.30 (dd, J=21.2, 8.1Hz, 52H), 0.88 (t, J=6.8Hz, 12H). MS (ESI): m / z (M+H) + 943.15.

[0337] Example 17-Synthesis of A17

[0338]

[0339] A17 was prepared with reference to the method of Example 1 to obtain an oily product (0.2 g). 1H NMR (500MHz, CDCl3) δ (ppm) = 5.80 (d, J = 8.2Hz, 1H), 4.25-4.15 (m, 2H), 4.13-4.06 (m, 1H), 3.68-3.58 (m, 3H), 3.57-3.49 (m, 4H), 3.45 (m, 10H), 2.72 (t, 2H), 2.60 (t, 4H), 2.31 (t, J=7.5Hz, 2H), 2.16 (t, J=7.5Hz, 2H), 1.58 (m, 16H), 1.30 (m, 36H), 0.88 (t, J=6.3Hz, 12H). MS (ESI): m / z (M+H) + 830.07.

[0340] Example 18 - Encapsulation efficiency

[0341] The lipid nanoparticles include (1) ionizable lipid compounds, which can be purchased commercially or homemade, such as MC3 (purchased from Avanti) and A1-A17 which are homemade; (2) phospholipids (such as DOPE or DSPC, purchased from Avanti); (3) PEGylated lipid compounds (such as PEG-DMG, purchased from Avanti or homemade); (4) structural lipids (such as cholesterol, purchased from Sigma-Aldrich); (5) active ingredients (such as Luciferase mRNA, siRNA, SARS-CoV-2S protein mRNA, Cas9 mRNA, etc.).

[0342] Preparation and encapsulation method: (1) ionizable lipids, phospholipids, pegylated lipids and structural lipids are dissolved and mixed in ethanol in order at 50%, 10%, 1.5% and 38.5% (by mole), respectively; (2) the lipid mixture and the active ingredient (mRNA) are uniformly mixed at a ratio of 1:3 using a microfluidic chip or a T-type mixer to obtain lipid nanoparticles.

[0343] The encapsulation rate reflects the degree of encapsulated substances. The higher the encapsulation rate, the less likely the encapsulated substance to be decomposed during the delivery process in vivo.

[0344] Table 1: Performance of ionizable lipids and their lipid nanoparticles

[0345]

[0346]

[0347] Example 19 - Transfection Efficiency

[0348] Various cationic lipid compounds and luciferase mRNA nanoparticles were encapsulated according to Example 18, and the fluorescence intensity or total photon number of luciferase mRNAs encapsulated by different LNPs were tested.

[0349] Experimental animals: SPF-grade BALB / c mice that passed the quarantine, female, 7-9 weeks old, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., were randomly divided into groups according to their weight, with 3 mice in each group. All animals were adaptively fed for more than 7 days before the experiment, and had free access to food and water during the experiment. The light was alternating between light and dark for 12 / 12h, the indoor temperature was 20-26℃, and the humidity was 40-70%.

[0350] Experimental method: Female BALB / c mice were given different LNP-encapsulated luciferase mRNA by intramuscular injection (tibialis anterior muscle of the right leg of mice); bioluminescence detection was performed using a small animal in vivo imaging system (brand: Bruker, model: XTREME) at 3, 6, 24, 48, and 72 hours after administration. The specific steps are as follows: Substrate preparation: Take an appropriate amount of substrate Luciferin (brand: Promega) and add physiological saline to prepare a 10 mg / mL solution, which is kept away from light for standby use. The test substance dose is 5 μg / mouse, 50 μL / leg. After the mice are given the substrate, they move freely for 5-10 minutes, and then they are placed in an anesthesia box and anesthetized with 2.5% isoflurane. The anesthetized mice are placed in the machine, the bioluminescence parameters are set and photos are taken. After collecting the image, the upper and lower limits of the image are adjusted according to the fluorescence intensity of different groups, and data collection (such as fluorescence intensity, average photon number and total photon number) and data processing are performed on the parts where the fluorescence is concentrated. Statistical analysis: The results of in vivo imaging are expressed as the mean of the fluorescence intensity or total photon number of different animals in the same test group, so as to determine the fluorescence intensity or total photon number of luciferase mRNA encapsulated by different LNPs. The data of luciferase expression induced by ionizable lipid nanoparticle preparations in mice 6 hours after injection are shown in Tables 2-1 and 2-2.

[0351] The fluorescence intensity and total photon count reflect the transfection efficiency of LNP. The higher the value, the higher the efficiency of LNP to deliver the encapsulated substance into the cells.

[0352] Table 2-1: Luciferase expression induced by ionized lipid nanoparticle preparations after 6 hours of injection

[0353]

[0354]

[0355] Table 2-2: Luciferase expression induced by ionized lipid nanoparticle preparations after 6 hours of injection

[0356]

[0357] Example 20 - Study on the immunogenicity of LNP-encapsulated mRNA

[0358] The S protein mRNA of the SARS-CoV-2BA.2.75.2 variant (sequence see SEQ ID NO.17 in 202310812939.7) was prepared by the T7 in vitro transcription method, and the mRNA vaccine was obtained by encapsulation with ionizable lipids A18 (self-made, refer to A18 in 202110617445.4), A2 and A11, respectively.

[0359] Table 3: mRNA encapsulation results

[0360]

[0361]

[0362] Table 4: Immunization schedule

[0363]

[0364] The three mRNA vaccine preparations obtained were used for the immunization test of BALB / c mice. The specific operation was as follows: 6-8 week old female BALB / c mice were selected, with 10 mice in each group, and the vaccine preparations were inoculated twice by intramuscular injection on Day 0 and Day 21, with an injection volume of 100 μL. The negative control group was given 100 μL PBS buffer.

[0365] 14 days after the second immunization, spleens of 4 mice were taken to isolate spleen lymphocytes, and T lymphocytes secreting IFN-γ and IL-4 in mice were detected by ELISPOT method. The specific operation of ELISPOT test was carried out according to the instructions of Mouse IL-4precoated ELISPOT kit (Da Kewei, 221 0402) and Mouse IFN-γprecoated ELISPOT kit (Da Kewei, 2210005).

[0366] 14 days and 35 days after the first immunization (i.e. 14 days after the second immunization), blood was collected from the orbits of the remaining 6 mice (to avoid hemolysis), and serum was collected for detection of BA.2.75.2 type Spike (S) protein-specific IgG antibodies by indirect ELISA. The specific operation of indirect ELISA for detecting BA.2.75.2 type S protein-specific IgG antibody titer is as follows:

[0367] Coating antigen: BA.2.75.2S protein (ACROBiosystems, Cat#SPN-C522r) was diluted to 2 ng / uL, 100 uL / well in coating buffer, and coated overnight at 4°C.

[0368] Wash the plate three times with 1× PBST, 5 min each time.

[0369] Blocked with 1% BSA blocking solution, 200uL / well, and allowed to stand at 37°C for 1 h.

[0370] Wash the plate three times with 1× PBST, 5 min each time.

[0371] The serum was diluted with dilution buffer, added at 100 ul / well, incubated at 37°C for 1 h, and a negative serum control and a blank control well without serum were set.

[0372] Wash the plate three times with 1× PBST, 5 min each time.

[0373] Anti-IgG secondary antibody was diluted 1:000, 100 ul / added, and incubated at 37°C for 1 h.

[0374] Wash the plate three times with 1× PBST, 5 min each time.

[0375] Add freshly configured TMB substrate chromogenic solution, 100 ul / well, and incubate for appropriate time at 37 °C.

[0376] Add stop solution 2mol / L sulfuric acid, 50ul / well.

[0377] The absorbance at OD450nm was measured by an enzyme-labeled instrument.

[0378] The results of IgG antibody titer are as follows Figure 2 , 3 As shown in the figure, the three mRNA vaccine preparations can induce humoral immune responses in BALB / c mice and produce S protein-specific IgG antibodies. At low and high doses, the antibody IgG level produced by L2 was basically the same as that of L1, regardless of the first or second immunization. The IgG antibody titer reached more than 4Log10 after the first immunization and 6Log10~7Log10 after the second immunization. The antibody IgG level produced by L3 was lower, but the IgG antibody titer reached more than 6Log10 after the second immunization. The ELISPOT results are shown in the figure. Figure 4As shown in the figure, after vaccination with low and high doses of vaccine preparations, the T lymphocytes secreting INF-γ in the L1, L2, and L3 groups increased significantly, and the frequency of T lymphocytes secreting INF-γ in the L3 group was the highest, followed by the L2 group, and the worst was the L1 group; after vaccination with low and high doses of vaccine preparations, the T lymphocytes secreting IL-4 in the L1, L2, and L3 groups increased. At low doses, the frequency of T lymphocytes secreting IL-4 in the L3 group was the highest, followed by the L2 group, and the worst was the L1 group; at high doses, there was no significant difference between the groups. In summary, compared with L1, L2 and L3, L3 can induce the strongest cellular immune response. The humoral immune response induced by L2 is equivalent to that of L1, but the cellular immune response is stronger.

[0379] Please refer to the contents of all references cited throughout the text (including literature references, issued patents, published patent applications and concurrently pending patent applications) hereby expressly incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly known to those of ordinary skill in the art.

[0380] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature having the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an example of a series of equivalent or similar features.

[0381] According to the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the appended claims.

Claims

1. A compound of formula (I), or a salt or isomer thereof: in, R1 is -C 1-6 Alkylene-X, Wherein X is selected from amino, hydroxyl, ethynyl, cyano, -C(O)(CH2) 1-3 NR a R b 、-C(O)O(CH2) 1-3 NR a R b 、-OC(O)(CH2) 1-3 NR a R b 、-C(O)NH(CH2) 1-3 NR a R b 、-NHC(O)(CH2) 1-3 NR a R b 、-NHC(O)CH(NR a R b )(CH2) 1-3 NR a R b , C 3-7 Cycloalkyl, 4-7 membered heterocyclic group, C 6-10 Aryl or 5-10 membered heteroaryl, the above cycloalkyl, heterocyclic, aryl and heteroaryl groups are optionally substituted by the following groups; -(CH2) 1-3 OH, -(CH2) 1-3 NR a R b or -(CH2) 1-3 C(O)NR a R b ; Or X is: R2 is C 3-20 Alkylene, wherein one or more methylene groups are each independently replaced by -CH=CH-, -C≡C- or -O-; R3 is C 5-20 Alkylene, wherein one or more methylene groups are each independently replaced by -CH=CH-, -C≡C- or -O-; M is independently selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, or -OP(O)(OH)-O-; R4 is Wherein R is selected from H, R * or -(CH2) 1-5 -YR * ; Y is selected from -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC (O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, or -OP(O)(OH)-O-; R * Each independently selected from C 1-12 Alkyl, C 2-12 Alkenyl or C 2-12 Alkynyl; R a , R b Independently selected from H, C 1-6 Alkyl or C 1-6 Halogenated alkyl.

2. The compound of formula (I) according to claim 1, or a salt or isomer thereof, wherein R1 is -C 1-6 Alkylene-X, wherein X is selected from amino, hydroxy, ethynyl or cyano; R2 is C 3-20 Alkylene; R3 is C 5-20 Alkylene; M is independently selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH- or -NHC(S)-; R4 is Wherein R is selected from H, R * or -(CH2) 1-5 -YR * ; Y is selected from -NH-, -C(O)-, -C(S)-, -O- or -S-; R * Each independently selected from C 1-12 Alkyl, C 2-12 Alkenyl or C 2-12 Alkynyl.

3. The compound according to any one of claims 1 to 2, or a salt or isomer thereof, wherein R1 is -C 1-6 Alkylene -OH, preferably -C 2-6 Alkylene -OH, preferably -C 2-4 The alkylene-OH group is preferably a -C2 alkylene-OH group.

4. The compound according to any one of claims 1 to 3, or a salt or isomer thereof, wherein R2 is C 3-20 Alkylene, preferably C 3-11 Alkylene, preferably C 5-11 Alkylene, preferably C 7-11 Alkylene.

5. The compound according to any one of claims 1 to 4, or a salt or isomer thereof, wherein R3 is C 5-20 Alkylene, preferably C 7-20 Alkylene, preferably C 7-11 Alkylene, preferably C 5-11 Alkylene, preferably C 7-9 Alkylene, preferably C7 alkylene.

6. The compound according to any one of claims 1 to 5, or a salt or isomer thereof, wherein each M is independently -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-, preferably -C(O)O- or -OC(O)-, preferably -C(O)O-.

7. The compound according to any one of claims 1 to 6, or a salt or isomer thereof, wherein each Y is independently selected from -O-.

8. The compound according to any one of claims 1 to 7, or a salt or isomer thereof, wherein each R * Each independently selected from C 1-11 Alkyl, wherein one or more methylene groups are independently replaced by the following groups: -CH=CH-, -C≡C-, preferably C 1-8 Alkyl, preferably C 1-6 Alkyl group, preferably C6 alkyl group.

9. The compound according to claim 1, or a salt or isomer thereof, wherein the compound has formula (II): in, R1 is -C 2-6 Alkylene-OH; M is selected from -CH2-, -CH=CH-, -C≡C-, -NH-, -C(O)-, -C(S)-, -O-, -S-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)-S-, -SC(O)-, -C(S)-O-, -OC(S)-, -C(S)-S-, -SC(S)-, -SS-, -C(S)NH-, -NHC(S)-, -OP(O)(OH)-O-; R2 is C 3-20 Alkylene; R3 is C 7-20 Alkylene; R* is each independently selected from C 1-11 Alkyl, wherein one or more methylene groups are each independently replaced by -CH=CH- or -C≡C-.

10. The compound of formula (II) or its salt or isomer according to claim 9, wherein R1 is -C 2-6 Alkylene-OH; M is selected from -C(O)O-, -OC(O)-, -C(O)NH- or -NHC(O)-; R2 is C 3-11 Alkylene; R3 is C 7-11 Alkylene; R* is independently selected from C 1-6 alkyl.

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