Amino lipid compound, preparation method therefor, and application thereof

Abstract

The present invention discloses an amino lipid compound, a preparation method therefor, and an application thereof. The present invention further provides a lipid particle containing the amino lipid and a use of the lipid particle for the delivery of a bioactive agent into a cell. The present invention further provides a use of a lipid particle containing the amino lipid, as a drug.

Description

Aminolipid compounds, methods of making and using the same TECHNICAL FIELD

[0001] The present disclosure relates to aminolipid compounds, methods of making and lipid particles comprising the aminolipid compounds. The lipid particles can be used for delivering bioactive agents into cells. The present disclosure also relates to the use of the lipid particles containing aminolipids as a medicament. BACKGROUND

[0002] Gene therapy refers to the introduction of a desired genetic material into a specific tissue cell through a suitable carrier for proper expression, replacing or correcting the disorder of the self gene structure or function, killing the diseased cells or enhancing the ability of the body to remove the diseased cells, so as to achieve the purpose of treatment. However, nucleic acid substances are easily hydrolyzed by nucleases and have a large number of negative charges, which are not conducive to cell phagocytosis, so it is particularly important to research and develop a gene delivery system with high efficiency, safety and tissue specificity.

[0003] The gene carriers currently studied can be divided into viral carriers and non-viral carriers. Although the transfection efficiency of viral carriers is high, the toxicity, immunogenicity and serious safety problems limit their application in clinical treatment. Non-viral carriers have attracted more and more attention from experts and scholars due to their easy preparation, transportation and storage, and the advantages of safety, effectiveness and non-immunogenicity.

[0004] Non-viral carriers mainly include cationic polymers and cationic liposomes. Cationic polymers refer to artificially synthesized or naturally formed polymers containing cations. Polyethyleneimine (PEI) is the most common cationic polymer non-viral gene carrier. It can wrap the negatively charged gene and form a nanocomposite, which can enter the cell through endocytosis and escape from the lysosome to exhibit good transfection efficiency. However, PEI has high cytotoxicity, and the toxicity is proportional to the transfection efficiency. Although chitosan has good biological safety, its transfection efficiency is low. Cationic liposomes are artificially prepared phospholipid vesicles with double membranes. The main difference between these liposomes is the difference in their charges and fine structures. When the target DNA is mixed with such liposomes, the DNA will be condensed and form a stable liposome DNA complex (lipoplex). Since the liposome has properties similar to biological membranes, when the liposome DNA complex contacts the cell membrane, it enters the cell through endocytosis. In addition to high transfection efficiency, liposomes have no immunogenicity, low cytotoxicity, and are easy to prepare, so cationic liposomes have become one of the most commonly used carriers for gene transfection today.

[0005] Although cationic liposome particles have been reported more frequently, and commercial transfection reagents are available for selection. The subtle differences of the gene structure to be delivered require the matching of the charge and the subtle structure of the liposome particles, and it is difficult for one or several transfection reagents to be applicable to the delivery of all gene materials. The subtle differences of the cell structure to be transfected with the gene material also require different properties of the liposome particles to match them. Therefore, in view of the rapid development of gene therapy and the huge market demand, it is urgent to develop a small platform delivery system to meet the delivery needs of nucleic acid materials of different structures.

[0006] SUMMARY

[0007] One aspect of the present application provides an amine-based lipid compound, which is a compound represented by the following Formula I:

[0008]

[0009] wherein:

[0010] R 1 and R 2 are the same as or different from each other, and each is independently selected from the group consisting of C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 alkynyl, and C4-C 24 acyl, wherein the C6-C 24 alkyl, the C6-C 24 alkenyl, the C6-C 24 alkynyl, and the C4-C 24 acyl are optionally substituted with C1-C6alkyl;

[0011] X 1 , X 2 , and X 3 are the same as or different from each other, and each is independently selected from the group consisting of C, N, O, S, S=O, S(=O)2, and S-S;

[0012] when X 1 is C, m=2, and the two R 6 are the same as or different from each other; when X 1 is N, m=1; when X 1 is O, S, S=O, S(=O)2, or S-S, m=0;

[0013] when X 2 is C, n=2, and the two R 7 are the same as or different from each other; when X 2 is N, n=1; when X 2 is O, S, S=O, S(=O)2, or S-S, n=0;

[0014] when X 3 is C, p = 2, and the two R 8 are the same or different from each other; when X 3 is N, p = 1 ; when X 3 is O, S, S=O, S(=O)2, or S-S, p = 0;

[0015] R 3 and R 4 are the same or different from each other, and each is independently selected from the group consisting of C1-C 12 alkyl, C2-C 12 alkenyl, and C2-C 12 alkynyl, wherein said C1-C 12 alkyl, said C2-C 12 alkenyl, and said C2-C 12 alkynyl are optionally substituted with C1-C6hydrocarbyl, or R 3 and R 4 combine with each other to form an optionally substituted 4- to 10-membered heterocyclic ring comprising 1 to 6 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen;

[0016] R 5 is absent, or R 5 is hydrogen or C1-C 12 alkyl to provide a quaternary amine;

[0017] R 6 , R 7 , R 8 is hydrogen or C1-C 12 alkyl;

[0018] L is C1-C 12 alkylene, C2-C 12 alkenylene, or C 2- C 12 alkynylene, wherein said C1-C 12 alkylene, said C2-C 12 alkenylene, and said C2-C 12 alkynylene are optionally substituted with one or more substituents selected from the group consisting of hydrocarbyl, carboxyl, acyl, and alkoxy, or L is an optionally substituted 4- to 10-membered heterocyclic ring comprising a heteroatom selected from the group consisting of nitrogen, sulfur, and oxygen.

[0019] Another aspect of the present application provides a method of preparing said amine-based lipid compound.

[0020] Another aspect of the present application provides use of said amine-based lipid compound for the preparation of a lipid particle.

[0021] Another aspect of the present application provides a lipid particle comprising said amine-based lipid compound.

[0022] Another aspect of the application provides the use of the lipid particle in the preparation of a medicament BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 shows the humoral antibody titres generated by subcutaneous delivery of ovalbumin mRNA (OVA mRNA) using a representative amine-based lipid compound.

[0024] Figure 2 shows micrographs of HEK293 cells treated with a reference reagent (Lipofectamine 2000) and S8N12D6 according to Example 4, wherein Figure 2(a) shows a brightfield image of HEK293 cells treated with a reference reagent (Lipofectamine 2000), Figure 2(b) shows a brightfield image of HEK293 cells treated with S8N12D6 according to Example 4; Figure 2(c) shows a Hoechst staining image of HEK293 cell nuclei treated with a reference reagent (Lipofectamine 2000), Figure 2(d) shows a Hoechst staining image of HEK293 cell nuclei treated with S8N12D6 according to Example 4, Figure 2(e) shows a GFP image of HEK293 cells treated with a reference reagent (Lipofectamine 2000), Figure 2(f) shows a GFP image of HEK293 cells treated with S8N12D6 according to Example 4. DETAILED DESCRIPTION

[0025] Hereinafter, the application will be described in more detail.

[0026] The term "optionally substituted" as used herein means that one or more hydrogen atoms attached to an atom or group is independently either unsubstituted, or substituted with one or more, for example one, two, three or four, substituents independently selected from the group consisting of: deuterium (D), halogen, -OH, thiol, cyano, -CD3, C1-C6 alkyl (preferably C1-C3 alkyl), C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl (preferably C3-C8 cycloalkyl), aryl, heterocyclyl (preferably 3-8 membered heterocyclyl), heteroaryl, aryl C1-C6 alkyl-, heteroaryl C1-C6 alkyl, C1-C6 haloalkyl, -OC1-C6 alkyl (preferably -OC1-C3 alkyl), -OC2-C6 alkenyl, OC1-C6 alkylphenyl, C1-C6 alkyl-OH (preferably C1-C4 alkyl-OH), C1-C6 alkyl-SH, C1-C6 alkyl-O-C1-C6 alkyl, OC1-C6 haloalkyl, NH2, C1-C6 alkyl-NH2 (preferably C1-C3 alkyl-NH2), -N(C1-C6 alkyl)2 (preferably -N(C1-C3 alkyl)2), -NH(C1-C6 alkyl) (preferably -NH(C1-C3 alkyl)), -N(C1-C6 alkyl)(C1-C6 alkylphenyl), -NH(C1-C6 alkylphenyl), nitro, -C(O)-OH, -C(O)OC1-C6 alkyl (preferably -C(O)OC1-C3 alkyl), -CONRiRii (wherein Ri and Rii are H, D and C1-C6 alkyl, preferably C1-C3 alkyl), -NHC(O)(C1-C6 alkyl), -NHC(O)(phenyl), -N(C1-C6 alkyl)C(O)(C1-C6 alkyl), -N(C1-C6 alkyl)C(O)(phenyl), -C(O)C1-C6 alkyl, -C(O)heteroaryl (preferably -C(O)-5-7 membered heteroaryl), -C(O)C1-C6 alkylphenyl, -C(O)C1-C6 haloalkyl, -OC(O)C1-C6 alkyl (preferably -OC(O)C1-C3 alkyl), -S(O)2-C1-C6 alkyl, -S(O)-C1-C6 alkyl, -S(O)2-phenyl, -S(O)2-C1-C6 haloalkyl, -S(O)2NH2, -S(O)2NH(C1-C6 alkyl), -S(O)2NH(phenyl), -NHS(O)2(C1-C6 alkyl), -NHS(O)2(phenyl) and -NHS(O)2(C1-C6 haloalkyl).each of the alkyl, cycloalkyl, phenyl, aryl, heterocyclyl, and heteroaryl groups described therein is optionally further substituted with one or more substituents selected from the group consisting of halogen, -OH, -NH2, cycloalkyl, 3-8 membered heterocyclyl, C1-C4 alkyl, C1-C4 haloalkyl-, -OC1-C4 alkyl, -C1-C4 alkyl-OH, -C1-C4 alkyl-O-C1-C4 alkyl, -OC1-C4 haloalkyl, cyano, nitro, -C(O)-OH, -C(O)OC1-C6 alkyl, -CON(C1-C6 alkyl)2, -CONH(C1-C6 alkyl), -CONH2, -NHC(O)(C1-C6 alkyl), -NH(C1-C6 alkyl)C(O)(C1-C6 alkyl), -SO2(C1-C6 alkyl), -SO2(phenyl), -SO2(C1-C6 haloalkyl), -SO2NH2, -SO2NH(C1-C6 alkyl), -SO2NH(phenyl), -NHSO2(C1-C6 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C6 haloalkyl). When one atom or group is substituted with multiple substituents, the multiple substituents can be the same or different.

[0027] The term "hydrocarbyl" as used herein means a group remaining after a hydrogen atom has been removed from a hydrocarbon, including straight-chain or branched-chain, saturated or unsaturated hydrocarbyl groups, including alkyl, alkenyl, and alkynyl groups, preferably the hydrocarbyl group is a C1-C 10 hydrocarbyl group, a C1-C6 hydrocarbyl group, or a C1-C3 hydrocarbyl group.

[0028] The term "alkyl" as used herein means a C1-C 24 alkyl group, a C1-C 20 alkyl group, a C1-C 18 alkyl group, a C1-C 12 alkyl group, a C1-C6 alkyl group, a C3-C 24 alkyl group, a C3-C 20 alkyl group, a C3-C 18 alkyl group, a C3-C 12 alkyl group, a C3-C6 alkyl group, a C6-C 24 alkyl group, a C6-C 20 alkyl group, a C6-C 18 alkyl group, or a C6-C 12 alkyl group.

[0029] The term "alkenyl" as used herein means a C2-C 24 alkenyl group, a C2-C 20 alkenyl group, a C2-C 18 alkenyl group, a C2-C 12 alkenyl group, a C2-C6 alkenyl group, a C3-C 20 alkenyl group, a C3-C18 alkenyl, C3-C6 alkenyl, C6-C 12 alkenyl, C3-C6 alkenyl, C6-C 24 alkenyl, C6-C 20 alkenyl, C6-C 18 alkenyl or C6-C 12 alkenyl. Alkenyl can contain one or more (e.g. 2, 3 or 4) C=C double bonds.

[0030] The term "alkynyl" as used herein means a C2-C 24 alkynyl, C2-C 20 alkynyl, C2-C 18 alkynyl, C2-C 12 alkynyl, C2-C6 alkynyl, C3-C 20 alkynyl, C3-C 18 alkynyl, C3-C 12 alkynyl, C3-C6 alkynyl, C6-C 24 alkynyl, C6-C 20 alkynyl, C6-C 18 alkynyl or C6-C 12 alkynyl.

[0031] The term "acyl" as used herein means a hydrocarbyl-carbonyl group, preferably the acyl group is a C4-C 24 acyl, C6-C 18 acyl, C6-C 12 acyl, C6-C 10 acyl, C4-C6 acyl.

[0032] The term "alkoxy" as used herein means an alkyl-oxy group, preferably the alkoxy group is a C1-C 10 alkoxy, more preferably the alkoxy group is a C1-C6 alkoxy group, most preferably the alkoxy group is a C1-C3 alkoxy group.

[0033] The term "heterocycle" as used herein means a saturated or unsaturated cyclic group comprising a heteroatom selected from N, O, S, etc., preferably the heterocycle is an optionally substituted 4- to 10-membered heterocycle comprising 1 to 6 heteroatoms selected from N, O, S, or is an optionally substituted 4- to 6-membered saturated heterocycle comprising 1, 2, or 3 heteroatoms selected from N, O, S. Examples of heterocycles include, but are not limited to: azetidine, oxetanyl, tetrahydrofuran, pyrrolidine, imidazolidine, pyrazolidine, tetrahydropyran, piperidine, morpholine, thiomorpholine, piperazine, preferably pyrrolidine, piperidine, piperazine and morpholine. The heterocycle can be optionally substituted with one or more substituents, the nature of which is as defined above in relation to "optionally substituted".

[0034] One aspect of the present application provides an amine-based lipid compound, which is a compound represented by the following Formula I:

[0035]

[0036] wherein:

[0037] R 1 and R 2 are the same as or different from each other, and each is independently selected from the group consisting of C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 alkynyl, and C4-C 24 acyl, wherein the C6-C 24 alkyl, the C6-C 24 alkenyl, the C6-C 24 alkynyl, and the C4-C 24 acyl are optionally substituted with C1-C6alkyl;

[0038] Preferably, R 1 and R 2 are the same as or different from each other, and each is independently C6-C 18 alkyl or C6-C 18 alkenyl.

[0039] X 1 , X 2 , and X 3 are the same as or different from each other, and each is independently selected from the group consisting of C, N, O, S, S=O, S(=O)2, and S-S;

[0040] when X 1 is C, m = 2, and the two R 6 are the same as or different from each other; when X 1 is N, m = 1; when X 1 is O, S, S=O, S(=O)2, or S-S, m = 0;

[0041] when X 2 is C, n = 2, and the two R 7 are the same as or different from each other; when X 2 is N, n = 1; when X 2 is O, S, S=O, S(=O)2, or S-S, n = 0;

[0042] when X 3 is C, p = 2, and the two R 8 are the same as or different from each other; when X 3 is N, p = 1; when X 3is O, S, S=0, S(=0)2, or S-S, p = 0;

[0043] R 3 and R 4 are the same as or different from each other and each is independently selected from the group consisting of C1-C 12 alkyl, C2-C 12 alkenyl, and C2-C 12 alkynyl, wherein the C1-C 12 alkyl, the C2-C 12 alkenyl, and the C2-C 12 alkynyl are optionally substituted with C1-C6alkyl, or R 3 and R 4 are combined with each other to form an optionally substituted 4- to 10-membered heterocyclic ring comprising 1 to 6 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen;

[0044] R 5 is absent, or R 5 is hydrogen or C1-C 12 alkyl to provide a quaternary amine;

[0045] R 6 , R 7 , R 8 is hydrogen or C1-C 12 alkyl;

[0046] L is C1-C 12 alkylene, C2-C 12 alkenylene, or C 2- C 12 alkynylene, wherein the C1-C 12 alkylene, the C2-C 12 alkenylene, and the C2-C 12 alkynylene are optionally substituted with one or more substituents selected from the group consisting of alkyl, carboxyl, acyl, and alkoxy, or L is an optionally substituted 4- to 10-membered heterocyclic ring comprising a heteroatom selected from the group consisting of nitrogen, sulfur, and oxygen.

[0047] Preferably, L is C1-C8alkylene, C2-C6alkylene, C2-C4alkylene, or C3-C4alkylene, wherein the C1-C8alkylene, the C2-C6alkylene, the C2-C4alkylene, or the C3-C4alkylene is optionally substituted with C1-C6alkyl.

[0048] In some embodiments, the present application provides the amphilipidic lipid compound of Formula I, wherein X 1 is C, R 6 is H, m = 2; or X 1 is N, R 6 is H, m = 1; or X1 is O, S, S=0, S(=0)2, or S-S, m = 0; alternatively or further alternatively, X 2 is C, R 7 is H, n = 2; or X 2 is N, R 7 is H, n = 1; or X 2 is O, S, S=0, S(=0)2, or S-S, n = 0; alternatively or further alternatively, X 3 is C, R 8 is H, p = 2; or X 3 is N, R 8 is H, p = 1; or X 3 is O, S, S=0, S(=0)2, or S-S, p = 0.

[0049] In other embodiments, the present application provides the amine-based lipid compound of formula I, wherein X 1 and X 2 are the same or different from each other, and are each independently N or S; when X 1 is N, m = 1; when X 1 is S, m = 0; when X 2 is N, n = 1; when X 2 is S, n = 0; alternatively or further alternatively, X 3 is N and p = 1, or X 3 is O and p = 0; alternatively or further alternatively, R 5 is absent; alternatively or further alternatively, L is C1-C 12 alkylene, said C1-C 12 alkylene being optionally substituted with C1-C6alkyl, e.g., L is (CH2) q wherein q is an integer from 1 to 12, e.g., an integer from 1 to 8 or 1 to 6. In some such embodiments, when m = 1, R 6 is hydrogen; alternatively or further alternatively, when n = 1, R 7 is hydrogen; alternatively or further alternatively, R 8 is hydrogen. In other such embodiments, the present application provides the amine-based lipid compound of formula I, wherein: R 1 and R 2 are the same or different from each other, and are each independently C6-C 24 alkyl or C6-C 24 alkenyl (e.g., C 12 -C 18 alkenyl); X 1 is N, R 6 is H, m = 1; or X 1 is S, m = 0; X2 is N, R 7 is H, n = 1 ; or X 2 is S, n = 0; X 3 is N, R 8 is H, p = 1 ; R 3 and R 4 are the same as or different from each other and are each independently C 1- C 12 alkyl, wherein the C 1- C 12 alkyl is optionally substituted with C1-C6alkyl, or R 3 and R 4 are combined with each other to form an optionally substituted 4- to 10-membered heterocyclic ring comprising 1 to 6 heteroatoms selected from nitrogen, sulfur, and oxygen; and R 5 is absent.

[0050] In some embodiments, the present application provides an amine-based lipid compound of Formula I as described above, wherein L is C1-C4alkylene, wherein the C1-C4alkylene is optionally substituted with C1-C6alkyl. For example, L is C2-C4alkylene, such as (CH2) q wherein q is 1, 2, 3, or 4.

[0051] In some embodiments, the present application provides an amine-based lipid compound of Formula I as described above, wherein R 1 and R 2 are the same as or different from each other and are each independently C6-C 18 alkyl or C6-C 18 alkenyl. In some embodiments, R 1 and R 2 are the same as or different from each other and are each independently C6-C 18 alkyl. In some embodiments, one of R 1 and R 2 is C6-C 18 alkyl, and the other is C6-C 18 alkenyl

[0052] In some embodiments, the present application provides an amine-based lipid compound of Formula I as described above, wherein:

[0053] m = 0 or 1, n = 0 or 1, and

[0054] are the same as or different from each other and are each independently S6, S7, S8, S9, S10, S11, S12, S14, S15, S16, S18, S19, S20, N6, N7, N8, N9, N11, N12, N13, N15, N16, N18, and2 N 12 one of:

[0055] S6: CH3(CH2)5S-; S7: CH3(CH2)6S-; S8: CH3(CH2)7S-;

[0056] S9: CH3(CH2)8S-; S10: CH3(CH2)9S-; S11: CH3(CH2) 10 S-;

[0057] S12: CH3(CH2) 11 S-; S14: CH3(CH2) 13 S-; S15: CH3(CH2) 14 S-;

[0058] S16: CH3(CH2) 15 S-; S18: CH3(CH2) 17 S-;

[0059] S19: S20:

[0060] N6: CH3(CH2)5NH-; N7: CH3(CH2)6NH-; N8: CH3(CH2)7NH-;

[0061] N9: CH3(CH2)8NH-; N11: CH3(CH2) 10 NH-; N12: CH3(CH2) 11 NH-;

[0062] N13: CH3(CH2) 12 NH-; N15: CH3(CH2) 14 NH-; N16: CH3(CH2) 15 NH-;

[0063] N18: CH3(CH2) 17 NH-; 2 N 12 : (CH3(CH2) 11 )2N-.

[0064] In some embodiments, the present application provides an amine-based lipid compound of Formula I as described above, wherein:

[0065] p = 1, and

[0066] D1, D2, D3, D4, D5, D6, D7, D8, D9, and D10 is one selected from the group consisting of:

[0067] D1: D2: D3:

[0068] D4: D5: D6:

[0069] D7: D8: D9:

[0070] D10:

[0071] In some embodiments, the present application provides an amine- based lipid compound of Formula I, as described above, wherein:

[0072] X 3 is O;

[0073] p is 0;

[0074] R 5 is absent; and

[0075] D1, D2, D3, D4, D5, D6, D7, D8, D9, and D10 is one selected from the group consisting of:

[0076] D1: D2: D3:

[0077] D4: D5: D6: D7: D8: D9:

[0078] D10:

[0079] In other embodiments, the present application provides an amine- based lipid compound of Formula I, as described above, wherein the amine- based lipid compound is a compound represented by the following Formula I’:

[0080]

[0081] wherein:

[0082] X 1’ and X 2’ are identical or different from each other and are each independently NH, O or S, preferably NH or S;

[0083] R 1 , R 2 , R 3 , R 4 and q are defined as in their definition in the foregoing description of the amine-based lipid compound of the formula I.

[0084] In other embodiments, the present application provides the amine-based lipid compound of the formula I described hereinbefore, wherein the amine-based lipid compound is a compound represented by the following formula I":

[0085]

[0086] wherein:

[0087] R 1 , R 2 , R 3 , R 4 and q are defined as in their definition in the foregoing description of the amine-based lipid compound of the formula I.

[0088] Another aspect of the present application provides a method of preparing the amine-based lipid compound of the formula I described hereinbefore, said method comprising the following steps:

[0089] (1) subjecting cyanuric chloride to a first reaction with a compound represented by R 1 (R 6 ) m -X 1 H in the presence of a base as an acid-binding agent at a temperature of -40°C to 30°C to obtain a first intermediate of the formula I-1;

[0090]

[0091] (2) subjecting the first intermediate to a second reaction with a compound represented by R 2 (R 7 ) n -X 2 H in the presence of a base as an acid-binding agent at room temperature or under heating conditions, without isolating or (preferably) with isolating the first intermediate, to obtain a second intermediate of the formula I-2;

[0092]

[0093] (3) subjecting the second intermediate to a third reaction with a compound represented by HX 3 (R8 ) p -L-NR 3 R 4 R 5 under heating conditions to obtain said amine-based lipid compound of formula I;

[0094] wherein X 1 , X 2 , X 3 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , L, m, n and p are defined as in the description hereinabove regarding said amine-based lipid compound of formula I.

[0095] For example, in some embodiments, X 1 and X 2 are the same or different from each other and are each independently N or S. When X 1 is N, m = 1; when X 1 is S, m = 0. When X 2 is N, n = 1; when X 2 is S, n = 0. In some embodiments, X 3 is N and p = 1. In some embodiments, R 5 is absent. In some embodiments, L is a C1-C 12 alkylene group, said C1-C 12 alkylene group being optionally substituted with a C1-C6alkyl group, for example L is (CH2) q wherein q is an integer from 1 to 12, for example an integer from 1 to 6. In some embodiments, when m = 1, R 6 is hydrogen. In some embodiments, when n = 1, R 7 is hydrogen. In some embodiments, R 8 is hydrogen.

[0096] Preferably, in this step (3), said third reaction is carried out in the presence of a base as an acid-binding agent.

[0097] In some embodiments, the present application provides a method of preparing said amine-based lipid compound of formula I’, said method comprising the following steps:

[0098] (1) reacting cyanuric chloride with a compound of formula R 1 -X 1’H represents is subjected to a first reaction to obtain a first intermediate of formula I'-1;

[0099]

[0100] (2) in the case of separation or (preferably) without separation of the first intermediate, the first intermediate is subjected to a second reaction with a compound represented by H2N-(CH2) 2 -X 2’ H represents is subjected to a second reaction in the presence of a base as a deacidifying agent to obtain a second intermediate of formula I'-2;

[0101]

[0102] (3) in the case of separation or (preferably) without separation of the second intermediate, the second intermediate is subjected to a third reaction with a diamine represented by H2N-(CH2) q -NR 3 R 4 to obtain the amine-based lipid compound of formula I'.

[0103] wherein X 1’ , X 2’ , R 1 , R 2 , R 3 , R 4 and q are defined as their definitions in the description above about the amine-based lipid compound of formula I'.

[0104] Preferably, in this step (3), the third reaction is carried out in the presence of a base as a deacidifying agent.

[0105] In some embodiments, the present application provides a method for preparing the amine-based lipid compound of formula I" described above,

[0106]

[0107] The method comprises the following steps:

[0108] (1) at a temperature of -40°C to 30°C, a first reaction is carried out between cyanuric chloride and a compound represented by R 1 -SH in the presence of a base as a deacidifying agent to obtain a first intermediate of formula I"-1;

[0109] (2) in the case of separation or (preferably) without separation of the first intermediate, the first intermediate is subjected to a second reaction with a compound represented by R 2 -SH in the presence of a base as a deacidifying agent to obtain a second intermediate of formula I"-2;

[0110]

[0111] (3) in the presence of a base as an acid-binding agent, the second intermediate is reacted with an alcohol amine represented by HO-(CH2) q -NR 3 R 4 to obtain the amine-based lipid compound of Formula I”;

[0112] wherein R 1 , R 2 , R 3 , R 4 and q are defined the same as their definitions in the description above regarding the amine-based lipid compound of Formula I”.

[0113] In preferred embodiments of any of the methods described above, the first reaction is performed at a temperature lower than 20°C (e.g., lower than 15°C, lower than 10°C, lower than 5°C, lower than 0°C, lower than -10°C, or lower than -20°C, and there is no specific limitation on the lower limit of the reaction temperature) and higher than -40°C (e.g., higher than -35°C, or preferably equal to or higher than -30°C).

[0114] As used herein, the term “room temperature” is the usual temperature at atmospheric pressure, which can refer to a temperature range of 0°C to 30°C, 0°C to 25°C, 0°C to 20°C, 5°C to 30°C, 5°C to 20°C, 10°C to 30°C, 10°C to 25°C, 15°C to 30°C, or 15°C to 25°C.

[0115] As used herein, the term “heating conditions” specifically refers to heating to a temperature range of 50°C to 120°C, 50°C to 110°C, 50°C to 80°C, 60°C to 120°C, 60°C to 110°C, 60°C to 100°C, 70°C to 120°C, 70°C to 100°C, 70°C to 90°C.

[0116] As used herein, the term “base” can be specifically a base compound commonly used in the art, such as but not limited to: an organic base, such as triethylamine, DIPEA, pyridine, DMAP; or an inorganic base, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate.

[0117] Preferably, q is an integer of 1 to 8, preferably an integer of 1 to 6, more preferably q is an integer of 1 to 4, such as 1, 2, 3, or 4.

[0118] In some embodiments, the present application provides a method of preparing an amine-based lipid compound of Formula II, which can be performed by the following Reaction Scheme 1:

[0119] [Reaction Scheme 1]

[0120]

[0121] wherein R 1 , R 2 , R 3 , R 4 and X 2 are defined as in the description above regarding the process for the preparation of the amine-based lipid compound of the formula I.

[0122] In some embodiments, the present application provides a process for the preparation of the amine-based lipid compound of the formula III, which can be carried out by the following Reaction Scheme 2:

[0123] [Reaction Scheme 2]

[0124]

[0125] wherein R 1 , R 2 , R 3 , R 4 and X 2 are defined as in the description above regarding the process for the preparation of the amine-based lipid compound of the formula I.

[0126] In some embodiments, the present application provides a process for the preparation of the amine-based lipid compound of the formula IV, which can be carried out by the following Reaction Scheme 3:

[0127] [Reaction Scheme 3]

[0128]

[0129] wherein R 1 , R 2 , R 3 , R 4 , X 2 and q are defined as in the description above regarding the amine-based lipid compound of the formula I;

[0130] Preferably, X 2 is selected from the group consisting of O, S, S=0, S(=0)2and S-S, preferably O or S, more preferably S.

[0131] In some embodiments, the present application provides a process for the preparation of the amine-based lipid compound of the formula V, which can be carried out by the following Reaction Scheme 4:

[0132] [Reaction Scheme 4]

[0133]

[0134] wherein R 1 , R 2 , R 3 , R 4 , X 2 and q are defined as in the description above regarding the amphilipidic lipid compounds of the formula I;

[0135] Preferably, X 2 is selected from the group consisting of O, S, S=0, S(=0)2, and S-S, preferably O or S, more preferably S.

[0136] It can be seen that the method for preparing the amphilipidic lipid compounds according to the present application is very versatile and can be used for the rapid synthesis of a library of amphilipidic lipid compounds for rapid cell-based screening experiments in a very inexpensive way.

[0137] Another aspect of the present application provides the use of an amphilipidic lipid compound according to any one of the formulae I, I', II, III, IV, and V for the preparation of a lipid particle.

[0138] Another aspect of the present application provides a lipid particle comprising an amphilipidic lipid compound according to the present application as described above.

[0139] Due to the amphilipidic lipid compounds of the present application containing long non-polar residues, the resulting compounds all have hydrophobic characteristics and due to the amine group, at the same time, have hydrophilic characteristics. This amphoteric characteristic can be used for the formation of lipid particles, e.g. lipid nanoparticles, lipid bilayers, micelles, liposomes, etc.

[0140] Within the scope of the present application, the term "lipid particle" means a nanometer-sized substance made by placing an amphilipidic lipid compound into an aqueous solution. These particles are in particular lipid nanoparticles, lipid bilayer vesicles (liposomes), multilayer vesicles, or micelles.

[0141] In a preferred embodiment of the present application, the lipid particle is a liposome containing an amphilipidic lipid compound according to the present application as described above. Within the scope of the present application, a liposome is a microvesicle consisting of a bilayer of lipid amphiphilic (amphiphilic) molecules enclosing an aqueous compartment.

[0142] Liposome formation is not a spontaneous process. When lipids are placed in water, lipid vesicles are first formed, thus forming a bilayer or a series of bilayers, each separated by water molecules. Liposomes can be formed by treating the lipid vesicles with ultrasound in water.

[0143] Within the scope of the present application, the term "lipid bilayer" means a thin film formed by two layers of lipid molecules. The term "micelle" means an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle is shaped like a sphere with the hydrophilic heads in the outer layer and the hydrophobic tails in the inner layer.

[0144] Within the scope of the present application, the term "cell" means a general term and includes a culture of single cells, tissues, organs, insect cells, avian cells, fish cells, amphibian cells, mammalian cells, primary cells, continuous cell lines, stem cells, and / or genetically engineered cells (such as recombinant cells expressing a heterologous polypeptide or protein). Recombinant cells include, for example, cells expressing a heterologous polypeptide or protein (such as a growth factor or a blood factor).

[0145] In some preferred embodiments, the lipid particle or liposome further contains one or more of a helper lipid, a sterol, and a biologically active agent.

[0146] For example, in some embodiments, the lipid particle or liposome contains a helper lipid. In preferred embodiments, the helper lipid is a non-cationic lipid. In more preferred embodiments, the helper lipid is a non-cationic phospholipid. Within the scope of the present application, a non-cationic lipid can contain a cationic functional group (e.g., an ammonium group), but should contain an anionic functional group to at least neutralize the molecule. The overall of all functional groups in the lipid molecule should be non-cationic. Liposomes composed of a mixture of amine-based lipids according to the present application and non-cationic (neutral) phospholipids are most effective for delivering nucleic acids into cells. In even more preferred embodiments, the non-cationic lipid is DOPE, DSPC, or a combination thereof. In some preferred embodiments, the molar ratio of the amine-based lipid compound to the helper lipid in the lipid particle is about (2 to 10): 1, preferably about (3 to 8): 1, more preferably about (4 to 6): 1, such as about 4: 1, about 4.5: 1, or about 5: 1.

[0147] In some alternative or further embodiments, the lipid particle or liposome comprises a sterol. Sterols, such as cholesterol, are natural components in cell membranes. They can be used to stabilize the particle and help with integration with the cell membrane. The sterol can be one or more selected from the group consisting of cholesterol, sitosterol, stigmasterol, and ergosterol, preferably cholesterol. In some preferred embodiments, the molar ratio of the amine-based lipid compound to the sterol in the lipid particle is about (1 to 1.5): 1, preferably about (1 to 1.4): 1, such as about (1 to 1.3): 1.

[0148] In some alternative or further embodiments, the lipid particle or liposome contains a biologically active agent. Within the scope of the present application, a biologically active agent is a substance that has a biological effect when introduced into a cell or a host, for example, by stimulating an immune or inflammatory response, by exerting an enzymatic activity, or by complementing a mutation, etc. The biologically active agent is in particular a nucleic acid, a peptide, a protein, an antibody, and a small molecule. The term "lipid particle drug" can be used both when a liposome is used to enclose a drug within the lipid bilayer or in the internal aqueous space of the liposome.

[0149] In most preferred embodiments, the biologically active agent is a nucleic acid, including but not limited to, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA), and small nuclear RNA (snRNA). In other preferred embodiments, the biologically active agent is selected from the group consisting of an antineoplastic agent, an antibiotic, an immunomodulator, an anti-inflammatory agent, a central nervous system acting agent, an antigen or fragment thereof, a protein, a peptide, a polypeptide, a polypeptoid, a vaccine, and a small molecule, and mixtures thereof.

[0150] In other embodiments, the lipid particle or liposome further contains at least one polyethylene glycol (PEG)-lipid. PEG lipids help to protect the particle and its contents from degradation in vitro or in vivo. In addition, PEG forms a protective layer on the surface of the liposome and improves the circulation time in vivo. It can be used in liposome drug delivery (PEG-liposomes). Preferably, the PEG-lipid can be one or more selected from the group consisting of PEG1000-DMG, PEG5000-DMG, PEG2000-DMG, and PEG2000-DSPE, preferably PEG2000-DMG. In some preferred embodiments, the molar ratio of the amine-based lipid compound to the PEG-lipid in the lipid particle is about (9 to 42): 1, preferably about (12 to 38): 1, more preferably about (16 to 36): 1, such as about (18 to 34): 1.

[0151] The lipid particle or liposome according to the present application has excellent properties for encapsulating a biologically active agent. The lipid particle or liposome comprising a biologically active agent can be used to deliver any of a variety of therapeutic agents into a cell. The present application includes the use of a lipid particle (especially a liposome) as described above for delivering a biologically active agent into a cell. The present application also provides a method of delivering a biologically active agent into a cell, the method comprising contacting the cell with a lipid particle or liposome of the present application comprising the biologically active agent.

[0152] As shown above, the lipid particles or liposomes comprising the amine-based lipid compounds of the present application are suitable for delivering biologically active agents into cells. A variety of different amine-based lipid compounds synthesized by the general synthetic method can be screened for specific characteristics of the liposomes. Important characteristics are, for example, transfection efficiency, cytotoxicity, adherence of the agent to be delivered into the cell, stability of the liposome, size of the liposome, etc. The methods of the present application can form specifically adapted liposomes for a particular application.

[0153] For example, the lipid particles or liposomes can be used for transfecting a multi-cellular tissue or organ. Thus, the present application also provides a method of transfecting a cell, a multi-cellular tissue or an organ, the method comprising contacting the cell with the lipid particles or liposomes of the present application comprising the nucleic acid. This provides new possibilities for the therapeutic treatment of a patient.

[0154] According to the present application, the patient can be any mammal, preferably selected from the group consisting of mouse, rat, pig, cat, dog, horse, goat, cow and monkey and / or others. In further preferred embodiments, the patient is a human.

[0155] Another aspect of the present application also provides the use of the lipid particles or liposomes comprising the amine-based lipid compounds of any one of formulae I, I', I", II, III, IV and V as a medicament. Another aspect of the present application also provides the use of the lipid particles or liposomes comprising the amine-based lipid compounds of any one of formulae I, I', I", II, III, IV and V for the manufacture of a medicament. For example, in some embodiments, the lipid particles are used as a carrier for encapsulating a biologically active agent.

[0156] In particular, the lipid particles or liposomes, or the medicament can be administered to a patient for use in gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA. Specific applications include, but are not limited to:

[0157] (1) The lipid particles of the present application can deliver nucleic acids for gene therapy. By introducing foreign genes into target cells via the amine-based lipids of the present application, diseases caused by defective and abnormal genes can be corrected or compensated to achieve therapeutic purposes. This also includes the technical application of transgenesis, i.e. the insertion of foreign genes into appropriate recipient cells of a patient by gene transfer technology, so that the products produced by the foreign genes can treat certain diseases, such as common lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, prostate cancer, etc. Genetically edited nucleic acid substances can also be introduced for the treatment of various genetic diseases, such as hemophilia, thalassemia, Gaucher's disease, etc.

[0158] (2) The lipid particles of the present application can be used in vaccination. The lipid particles or liposomes of the present application can be used to deliver antigens or nucleic acids encoding antigens. The lipid particles of the present application can also be used to elicit an immune response against various antigens for the treatment and / or prevention of a variety of conditions, such as cancer, allergies, toxicity, and infection by pathogenic organisms (e.g., viruses, bacteria, fungi, and other disease-causing organisms).

[0159] The present application also provides a method of gene therapy, gene vaccination, antisense therapy, or therapy by interfering RNA, comprising administering to a patient in need thereof a lipid particle or liposome of the present application comprising a biologically active agent (e.g., a desired nucleic acid or antigen).

[0160] The lipid particles of the present application can be used in the preparation of a medicament for the transfer of nucleic acids. Preferably, the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA), and small nuclear RNA (snRNA).

[0161] Encapsulation of non-biologically active agents is also an object of the lipid particles or liposomes of the present application. Thus, the present application also provides a lipid particle or liposome comprising a non-biologically active agent. Examples of non-biologically active agents include, but are not limited to, antioxidants, pigments, dyes, fragrances, and flavoring agents, and the like, such as those used in cosmetics. It is contemplated that the lipid particles or liposomes according to the present application also have excellent performance in encapsulating non-biologically active agents.

[0162] The present application also includes the following embodiments:

[0163] 1. An amine-based lipid compound, which is a compound represented by the following Formula I:

[0164]

[0165] wherein, R 1 and R 2 are the same as or different from each other, and each is independently selected from the group consisting of C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 alkynyl, and C4-C 24 acyl, wherein the C6-C 24 alkyl, the C6-C 24 alkenyl, the C6-C 24 alkynyl, and the C4-C 24 acyl are optionally substituted with C1-C6 hydrocarbyl;

[0166] X 1 , X2 and X 3 are the same or different from each other and are each independently selected from the group consisting of C, N, O, S, S=0, S(=0)2, and S-S;

[0167] when X 1 is C, m = 2, and the two R 6 are the same or different from each other; when X 1 is N, m = 1 ; when X 1 is O, S, S=0, S(=0)2, or S-S, m = 0;

[0168] when X 2 is C, n = 2, and the two R 7 are the same or different from each other; when X 2 is N, n = 1 ; when X 2 is O, S, S=0, S(=0)2, or S-S, n = 0;

[0169] when X 3 is C, p = 2, and the two R 8 are the same or different from each other; when X 3 is N, p = 1 ; when X 3 is O, S, S=0, S(=0)2, or S-S, p = 0;

[0170] R 3 and R 4 are the same or different from each other and are each independently selected from the group consisting of C1-C 12 alkyl, C2-C 12 alkenyl, and C2-C 12 alkynyl, wherein the C1-C 12 alkyl, the C2-C 12 alkenyl, and the C2-C 12 alkynyl are optionally substituted with C1-C6hydrocarbyl, or R 3 and R 4 combine with each other to form an optionally substituted 4- to 10-membered heterocyclic ring comprising 1 to 6 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen;

[0171] R 5 is absent, or R 5 is hydrogen or C1-C 12 alkyl to provide a quaternary amine;

[0172] R 6 , R 7 , R 8 is hydrogen or C1-C 12 alkyl;

[0173] L is C1-C 12 alkylene, C2-C12 alkenylene or C 2- C 12 alkynylene, wherein said C1-C 12 alkylene, said C2-C 12 alkenylene and said C2-C 12 alkynylene are optionally substituted with one or more substituents selected from the group consisting of alkyl, carboxyl, acyl and alkoxy, or L is an optionally substituted 4- to 10-membered heterocyclic ring comprising a heteroatom selected from the group consisting of nitrogen, sulfur and oxygen.

[0174] 2. The aminolipid compound according to item 1, wherein:

[0175] X 1 is C, R 6 is H, m = 2; or X 1 is N, R 6 is H, m = 1 ; or X 1 is O, S, S=0, S(=0)2 or S-S, m = 0;

[0176] X 2 is C, R 7 is H, n = 2; or X 2 is N, R 7 is H, n = 1 ; or X 2 is O, S, S=0, S(=0)2 or S-S, n = 0;

[0177] X 3 is C, R 8 is H, p = 2; or X 3 is N, R 8 is H, p = 1 ; or X 3 is O, S, S=0, S(=0)2 or S-S, p = 0.

[0178] 3. The aminolipid compound according to item 1, wherein:

[0179] R 1 and R 2 are the same or different from each other and are each independently C6-C 24 alkyl;

[0180] X 1 is N, R 6 is H, m = 1 ; or X 1 is S, m = 0;

[0181] X 2 is N, R 7 is H, n = 1 ; or X 2 is S, n = 0;

[0182] X3 is N, R 8 is H, p = 1 ;

[0183] R 3 and R 4 are the same as or different from each other and are each independently C 1- C 12 alkyl, wherein the C 1- C 12 alkyl is optionally substituted with C1-C6alkyl, or R 3 and R 4 are combined with each other to form an optionally substituted 4- to 10-membered heterocyclic ring comprising 1 to 6 heteroatoms selected from nitrogen, sulfur, and oxygen;

[0184] R 5 is absent.

[0185] 4. The amine-based lipid compound according to any one of items 1 to 3, wherein L is C1-C4alkylene, wherein the C1-C4alkylene is optionally substituted with C1-C6alkyl.

[0186] 5. The amine-based lipid compound according to any one of items 1 to 3, wherein R 1 and R 2 are the same as or different from each other and are each independently C6-C 18 alkyl.

[0187] 6. The amine-based lipid compound according to item 1, wherein:

[0188] m = 0, n = 0, p = 0,

[0189] R 1 -X 1 - and R 2 -X 2 - are the same as or different from each other and are each independently one selected from S6, S7, S8, S9, S10, S11, S12, S14, S15, S16, S18, N6, N7, N8, N9, N11, N12, N13, N15, N16, N18, 2 N 12 as described above;

[0190] -X 3 -L-N(R 3 )(R 4 )(R 5 ) is one selected from D1, D2, D3, D4, D5, D6, D7, D8, D9, D10 as described above.

[0191] 7. A method for preparing the amine-based lipid compound according to any one of items 1 to 6, the method comprising the steps of:

[0192] (1) performing a first reaction of cyanuric chloride with a compound represented by R 1 -X 1 H in the presence of a base as an acid-binding agent at a temperature of -30°C to 30°C to obtain a first intermediate, wherein X 1 is NH or S;

[0193] (2) without isolating the first intermediate, performing a second reaction by adding a compound represented by R 2 -X 2 H in the presence of a base as an acid-binding agent at room temperature or under heating conditions to obtain a second intermediate, wherein X 2 is NH or S;

[0194] (3) without isolating the second intermediate, performing a third reaction by adding a diamine represented by H2N-(CH2) q -NR 3 R 4 at a heating temperature to obtain the amine-based lipid compound;

[0195] wherein q is an integer of 1 to 12, R 1 , R 2 , R 3 and R 4 are defined as the same as their definitions in any one of items 1 to 6.

[0196] 8. A method for preparing the amine-based lipid compound according to the present application, which can be performed by the following Reaction Scheme 1:

[0197] [Reaction Scheme 1]

[0198]

[0199] wherein R 1 , R 2 , R 3 , R 4 , X 2 are defined as the same as defined in the foregoing description about the method for preparing the amine-based lipid compound.

[0200] 9. A method for preparing the amine-based lipid compound according to the present application, which can be performed by the following Reaction Scheme 2:

[0201] [Reaction Scheme 2]

[0202]

[0203] wherein R 1 , R 2 , R 3 , R 4 , X 2 are defined as the same as defined in the previous description about the preparation method of the amine-based lipid compound.

[0204] 10. Use of the amine-based lipid compound according to any one of items 1 to 6 for the preparation of a lipid particle, wherein the lipid particle is a lipid nanoparticle, a liposome, a multilamellar vesicle or a micelle; more preferably, the lipid particle is a lipid nanoparticle.

[0205] 11. A lipid particle comprising the amine-based lipid compound according to any one of items 1 to 6; preferably, the lipid particle is a lipid nanoparticle, a liposome, a multilamellar vesicle or a micelle; more preferably, the lipid particle is a lipid nanoparticle.

[0206] 12. The lipid particle according to item 11, further containing one or more of a helper lipid, a sterol and a bioactive agent;

[0207] Preferably, the helper lipid is a non-cationic lipid, more preferably, the helper lipid is a non-cationic phospholipid, further preferably, the non-cationic lipid is DOPE;

[0208] Preferably, the sterol is cholesterol;

[0209] Preferably, the bioactive agent is one or more of a nucleic acid, an antitumor agent, an antibiotic, an immunomodulator, an anti-inflammatory agent, a central nervous system acting agent, an antigen or fragment thereof, a peptide, a protein, an antibody, a vaccine and a small molecule; more preferably, the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA).

[0210] 13. Use of the lipid particle according to item 11 or 12 in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA;

[0211] Preferably, the gene therapy is useful for the treatment of cancer and genetic diseases; more preferably, the cancer is one or more of lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphatic cancer, blood cancer or prostate cancer, and the genetic disease is one or more of hemophilia, thalassemia, Gaucher's disease;

[0212] Preferably, the genetic vaccination is used to treat cancer, allergy, toxicity and pathogen infection; more preferably, the pathogen is one or more of a virus, a bacterium or a fungus.

[0213] 14. Use of the lipid particle according to item 11 or 12 for the manufacture of a medicament for the transfer of a nucleic acid, preferably RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA). Advantages

[0214] The amine-based lipid compound of the present application has the characteristics of strong functional group diversity and large quantity, the preparation method of the amine-based lipid compound has the advantages of easy availability of raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirement for instruments and equipment and simple operation, the preparation method of the present application is very universal, can be used for rapid synthesis of an ionizable amine-based lipid compound library, and can be used for rapid cell-based screening experiments in a very inexpensive way.

[0215] The amine-based lipid compound provided by the present application has various types and combination modes of functional groups, and can be conveniently used for delivering different nucleic acid substances to different cells through routine screening for characteristics such as transfection efficiency, cytotoxicity, adhesion of a drug to be delivered into cells, stability of a lipid particle, size of the lipid particle, etc., thereby improving the targeting and effectiveness of the delivery system.

[0216] The amine-based lipid compound of the present application can be synthesized in high throughput and high efficiency by using the method of combinatorial chemistry, and the synthesis method is simple and has high yield.

[0217] The lipid particle or liposome containing the amine-based lipid compound of the present application has excellent performance of encapsulating a bioactive agent, and can be used for delivery of a bioactive agent, especially a poorly water-soluble drug or an easily decomposed or degraded active agent (such as a nucleic acid), thereby improving the bioavailability and effectiveness thereof.

[0218] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be described below in combination with specific examples. The described examples are part of the embodiments of the present application, rather than all the embodiments. Based on the examples in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

[0219] Examples

[0220] Unless otherwise specified, all chemical reagents used in the examples are of analytical grade and were purchased from TCI, Aladdin, or J&K, etc.

[0221] Example 1: Synthesis of amino lipid compounds

[0222] To systematically study the relationship between alkyl chain length, alkyl chain combination, ionizable head groups, and transfection capability, 11 straight-chain alkyl thiols with 6-18 carbon chains (S6-S18) and 11 alkylamines with 6-24 carbon chains (N6- 2 Ten diamines (D1-D6) with distinct structures (N12) were synthesized in high throughput using combinatorial chemistry. A library of 2530 compounds containing two alkyl chains and ionizable head groups was synthesized in the following synthetic routes:

[0223] (a) Triazine as a linking group

[0224]

[0225] (b) Typical examples

[0226]

[0227] (c) Composite blocks

[0228]

[0229] (d) Compound Library

[0230]

[0231] Where n3 = 1, 2, or 3; -NRR represents the dialkylamino or cycloamino moiety of the diamines D1 to D10 mentioned above; and R 1 and R 2 As defined above.

[0232] The synthesized compounds have one of the structures shown in Table I below:

[0233] Table I: Synthesized Compounds

[0234]

[0235] Sz independently represents groups selected from S6 to S12, S14 to S16 and S18 listed in Table II below;

[0236] Nx independently represents N6 to N9, N11 to N13, N15, N16, N18 and N19 listed in Table II below. 2 N 12 The groups; and

[0237] Dy represents each of the groups selected from D1 to D10 listed in Table II below:

[0238] Table II

[0239]

[0240]

[0241] For the synthesized compound, the measured mass spectrum ([M+H)) + The data is shown in Table III below:

[0242] Table III

[0243] D1D2D3D4D5D6D7D8D9D10S6N6383.4425.3409.3423.4438.4397.3411.4439.3452.44 23.4S6N7397.3439.3423.3437.4452.4411.3425.4453.3466.4437.5S6N8411.4453. 3437.4451.4466.6425.4439.4467.3480.6451.4S6N9425.3467.4451.3465.4480.44 39.3453.4481.3494.4465.4S6N11453.3495.3479.3493.4508.4467.3481.5509.452 2.4493.4S6N12467.4509.3493.4507.4522.4481.3495.4523.3536.4507.6S6N13481 .3523.5507.3521.5536.4495.3509.4537.5550.6521.4S6N15509.6551.3535.5549. 4564.4523.3537.5565.3578.4549.4S6N16523.3565.3549.3563.4578.6537.3551.4 579.5592.4563.4S6N18551.3593.3577.3591.4606.4565.3579.4607.3620.4591.6S6 2N 126 35.667 7.466 1.467 5.569 0.564 9.666 3.569 1.470 4.567 5.5 S7 N 639 7.343 9.642 3.743 7.445 2.441 1.342 5.445 3.346 6.443 7.6 S7 N 741 1.345 3.343 7.345 1.446 6.442 5.343 9.446 7.348 0.645 1.4 S7 N 842 5.546 7.345 1.346 5.448 0.643 9.345 3.548 1.349 4.446 5.4 S7 N 943 9.348 1.346 5.347 9.449 4.445 3.546 7.449 5.550 8.447 9.4 S7 N 114 67.550 9.349 3.750 7.752 2.448 1.349 5.452 3.353 6.450 7.4 S7 N 124 81.352 3.350 7.652 1.453 6.749 5.350 9.653 7.355 0.652 1.4 S7 N 134 95.653 7.652 1.353 5.455 0.450 963 5.323 5.451 3.564.453 5.4 S7 N 155 23.356 5.354 9.356 3.457 8.453 7.355 1.457 9.359 2.456 3.4 S7 N 165 37.357 9.356 3.357 7.459 2.455 1.356 5.459 3.360 6.457 7.4 S7 N 185 65.360 7.359 1.360 5.462 0.457 9.359 3.462 1.363 4.460 5.4 S7 2 N 126 49.469 1.467 5.468 9.570 4.566 3.467 7.570 5.471 8.568 9.5 S8 N 641 1.345 3.343 7.345 1.446 6.442 5.343 9.446 7.348 0.445 1.4 S8 N 742 5.346 7.345 1.346 5.448 0.643 9.345 3.448 1.349 4.446 5.4 S8 N 843 9.848 1.346 5.347 9.449 4.445 3.646 7.449 5.350 8.647 9.4 S8 N 945 3.349 5.847 9.849 3.650 8.446 7.348 1.550 9.552 2.449 3.6 S8 N 124 95.753 7.352 1.353 5.455 0.650 9.352 3.455 1.356 4.453 5.4

[0244] D1D2D3D4D5D6D7D8D9D10S8N13509.3551.3535.6549.4564.4523.3537.6565.3578.5549.6S8N15537.3579.3563.3577.4592.4551.3565.4593.3606.4577.4S8N16551.3593.3577.3591.4606.4565.3579.4607.3620.4591.4S8N18579.6621.3605.3619.4634.4593.3607.4635.3648.4619.4S8 2 N12663.4705.4689.4703.5718.5677.4691.5719.4732.5703.5S9N6425.3467.3451.3465.4480.7439.3453.4481.3494.4465.6S9N7439.3481.3465.3479.6494.4453.3467.4495. 3508.4479.4S9N8453.6495.3479.3493.4508.4467.3481.4509.6522.4493.4S9N9467.3509.3493.3507.4522.4481.3495.4523.3536.4507.4S9N11495.3537.3521.6535.4550.45 09.3523.4551.3564.4535.6S9N12509.3551.6535.3549.4564.4523.3537.4565.3578.4549.4S9N13523.6565.6549.3563.4578.4537.3551.4579.3592.4563.4S9N15551.3593.35 77.3591.4606.4565.3579.4607.3620.4591.4S9N16565.3607.3591.3605.4620.4579.3593.4621.3634.4605.4S9N18593.3635.3619.6633.4648.4607.3621.4649.3662.4633.4S9 2N12677.4719.4703.4717.5732.5691.4705.5733.4746.5717.5S10N6439.5481.6465.6479.4494.4453.5467.4495.3508.4479.4S10N7453.3495.3479.3493.5508.4467.3481.4509.3522.4493.4S10N8467.3509.3493.3507.7522.4481.3495.4523.3536.4507.4S10N9481.3523.3507.3521.4536.4495.5509.4537.3550.4521.4S10N11509.3551.3535.3549.4564.4523.3537.4565.3578.4549.4S10N12523.3565.3549.3563.4578.4537.3551.4579.3592.4563.4S10N13537.3579.3563.3577.4592.4551.3565.4593.3606.7577.7S10N15565.3607.3591.3605.4620.4579.3593.4621.3634.4605.4S10N16579.3621.3605.3619.4634.4593.3607.4635.6648.4619.4S10N18607.3649.3633.3647.4662.4621.3635.4663.3676.4647.4S10 2N12691.4733.4717.4731.5746.5705.4719.7747.4760.5731.5S11N6453.3495.3479.3493.4508.4467.3481.4509.3522.4493.4S11N7467.3509.3493.3507.4522.4481.3495.4523.3536.4507.4S11N8481.3523.3507.3521.4536.4495.3509.4537.3550.4521.4S11N9495.3537.3521.6535.4550.4509.3523.4551.3564.4535.4S11N11523.3565.3549.3563.4578.6537.3551.4579.3592.4563.4S11N12537.6579.3563.3577.4592.4551.3565.4593.3606.4577.6S11N13551.3593.3577.3591.4606.4565.3579.4607.3620.4591.4S11N15579.6621.3605.6619.4634.4593.3607.4635.3648.4619.4S11N16593.3635.3619.3633.4648.4607.3621.4649.3662.4633.4S11N18621.3663.3647.3661.4676.4635.3649.4677.3690.4661.4S11 2N12705.4747.4731.6745.7760.5719.4733.5761.4774.5745.5S12N6467.3509.3493.3507.4522.4481.3495.4523.3536.4507.4S12N7481.3523.3507.3521.4536.4495.3509.4537.3550.4521.4S12N8495.3537.3521.3535.4550.4509.3523.4551.3564.4535.4S12N9509.3551.3535.3549.4564.4523.3537.4565.3578.4549.4S12N11537.3579.3563.3577.5592.7551.3565.4593.3606.4577.4S12N12551.3593.3577.3591.4606.4565.3579.7607.3620.4591.4S12N13565.3607.3591.3605.4620.4579.3593.4621.3634.4605.4S12N15593.3635.3619.3633.4648.4607.3621.4649.3662.4633.4

[0245] D1D2D3D4D5D6D7D8D9D10S12N16607.3649.3633.3647.4662.4621.3635.4663.3676.4647.4S12N18635.3677.3661.3675.4690.4649.3663.4691.5704.4675.4S12 2N12719.5761.4745.4759.5774.5733.4747.5775.4788.5759.5S14N6495.3537.3521.3535.4550.4509.3523.4551.3564.5535.5S14N7509.3551.5535.3549.4564.4523.3537.4565.3578.4549.4S14N8523.5565.3549.3563.4578.4537.5551.4579.3592.4563.4S14N9537.3579.6563.3577.4592.55516565.4593.6606.6577.4S14N11565.3607.3591.6605.4620.4579.3593.4621.3634.4605.4S14N12579.5621.3605.3619.4634.4593.3607.4635.3648.4619.4S14N13593.3635.6619.3633.4648.4607.3621.4649.3662.4633.4S14N15621.3663.3647.3661.4676.4635.3649.4677.3690.4661.4S14N16635.6677.3661.3675.4690.4649.3663.4691.3704.4675.4S14N18663.3705.3689.3703.4718.4677.3691.4719.3732.4703.6S14 2N12747.4789.4773.4787.5802.5761.4775.6803.4816.5787.5S15N6509.3551.7535.3549.4564.4523.6537.4565.3578.6549.4S15N7523.7565.3549.3563.4578.4537.3551.45796592.4563.4S15N8537.3579.3563.3577.4592.4551.3565.4593.3606.4577.4S15N9551.3593.3577.7591.4606.4565.3579.4607.3620.4591.4S15N11579.7621.3605.3619.7634.6593.3607.7635.3648.8619.9S15N12593.3635.3619.3633.4648.4607.3621.4649.3662.4633.4S15N13607.5649.3633.3647.7662.4621.3635.4663.3676.4647.4S15N15635.3677.36615675.4690.4649.3663.4691.3704.4675.4S15N16649.3691.5675.3689.4704.7663.3677.4705.3718.4689.4S15N18677.3719.3703.3717.4732.4691.3705.4733.3746.4717.4S15 2N12761.4803.4787.4801.5816.5775.4789.5817.4830.5801.5S16N6523.3565.3549.3563.4578.4537.3551.4579.3592.4563.4S16N7537.3579.3563.7577.4592.4551.3565.4593.3606.4577.4S16N8551.6593.6577.3591.4606.4565.3579.4607.3620.4591.4S16N9565.3607.3591.5605.6620.4579.3593.4621.3634.4605.4S16N11593.3635.3619.3633.4648.7607.3621.4649.3662.4633.4S16N12607.3649.3633.3647.4662.4621.3635.4663.3676.4647.4S16N13621.3663.6647.3661.4676.4635.3649.4677.3690.4661.4S16N15649.3691.3675.6689.4704.4663.7677.4705.3718.4689.4S16N16663.3705.3689.3703.4718.4677.3691.4719.3732.4703.4S16N18691.3733.3717.3731.4746.4705.3719.4747.3760.4731.4S16 2N1 27 75.481 7.480 1.481 5.583 0.578 9.480 3.583 1.784 4.781 5.5 S18 N6 51.359 3.357 7.359 1.460 6.456 5.357 9.460 7.362 0.459 1.4 S18 N7 65.360 7.359 1.360 5.462 0.457 9.359 3.462 1.363 4.460 5.8 S18 N8 79.362 1.360 5.661 9.463 4.759 3.360 7.463 5.364 8.461 9.4 S18 N9 93.363 5.361 9.363 3.464 8.460 7.562 1.664 9.766 2.863 3.8 S18 N11 621.366 3.364 7.366 1.467 6.463 5.364 9.467 7.369 0.466 1.4 S18 N12 635.367 7.366 1.367 5.469 0.464 9.366 3.469 1.370 4.467 5.4 S18 N13 649.569 1.367 5.368 9.470 4.466 3.367 7.470 5.371 8.468 9.4 S18 N15 677.371 9.370 3.371 7.473 2.469 1.370 5.473 3.374 6.471 7.4 S18 N16 691.373 3.371 7.373 1.474 6.470 5.371 9.474 7.376 0.473 1.4 S18 N18 191.376 1.674 5.375 9.477 4.473 3.374 7.477 5.378 8.475 9.4

[0246] D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 S18 2N 128 0 3.484 5.482 9.784 3.785 8.581 7.483 1.585 9.487 2.584 3.5 S6 S640 0.344 2.342 6.344 0.445 5.741 4.342 8.445 6.346 9.444 0.4 S6 S741 4.345 6.344 0.345 4.446 9.442 8.344 2.447 0.348 3.445 4.4 S6 S842 8.347 0.345 4.346 8.448 3.444 2.345 6.448 4.349 7.446 8.4 S6 S944 2.348 4.346 8.348 2.449 7.445 6.347 0.449 8.351 1.448 2.4 S6 S104 56.349 8.348 2.349 6.451 1.447 0.348 4.451 2.352 5.449 6.4 S6 S114 70.351 2.349 6.352 1.452 5.448 4.348 4.352 6.553 9.451 0.4 S6 S124 84.652 6.651 0.352 4.453 9.449 8.351 2.454 0.355 3.452 4.4 S6 S145 12.355 4.353 8.555 2.456 7.452 6.354 0.456 8.358 1.455 2.4 S6 S155 26.556 8.355 2.356 6.458 1.454 0.455 4.458 2.359 5.456 6.4 S6 S165 40.358 2.356 6.358 0.459 5.455 4.356 8.459 6.360 9.458 0.4 S6 S185 68.361 0.359 4.360 8.462 3.458 2.359 6.462 4.363 7.460 8.4 S7 S742 8.547 0.345 4.346 8.448 3.444 2.345 6.448 4.349 7.446 8.4 S7 S844 2.348 4.346 8.348 2.449 7.445 6.347 0.449 8.351 1.448 2.4 S7 S945 6.349 8.348 2.349 6.451 1.447 0.348 4.451 2.352 5.449 6.4 S7 S104 70.351 2.549 6.351 0.452 5.448 4.349 8.452 6.353 9.451 0.4 S7 S114 84.352 6.351 0.352 4.453 9.449 8.351 2.454 0.355 3.452 4.4 S7 S124 98.754 0.352 4.353 8.455 3.451 2.352 6.455 4.356 7.453 8.4 S7 S145 26.356 8.355 2.3566.4581.4540.3554.4582.3595.4566.4S7S15540.3582.3566.3580.4595.4554.3568.4596.3609.4580.4S7S16554.6596.3580.3594.4609.4568.3582.4610.3623.4594.4S7S18582.3624.3608.3622.4637.4596.3610.4638.3651.4622.4S8S8456.8498.3482.3496.4511.4470.3484.4512.3525.4496.4S8S9470.3512.3496.3510.6525.6484.3498.6526.3539.4510.4S8S10484.7526.3510.3524.4539.4498.3512.4540.3553.8524.7S8S11498.3540.3524.3538.4553.4512.3526.4554.3567.4538.4S8S12512.3554.3538.3552.4567.4526.3540.4568.3581.4552.4S8S14540.3582.3566.3580.4595.4554.3568.4596.3609.4580.4S8S15554.3596.3580.3594.4609.4568.3582.4610.3623.4594.4S8S16568.3610.3594.3608.4623.4582.3596.4624.3637.4608.4S8S18596.3638.3622.3636.4651.4610.3624.4652.3665.4636.4S9S9484.3526.3510.3524.4539.4498.3512.4540.3553.4524.4S9S10498.3540.7524.6538.4553.4512.3526.4554.3567.4538.4S9S11512.3554.3538.3552.4567.4526.3540.4568.3581.4552.4S9S12526.3568.3552.3566.4581.4540.3554.4582.3595.4566.4S9S14554.3596.3580.3594.4609.4568.3582.4610.3623.4594.4S9S15568.3610.6594.3608.5623.4 582.55 96.66 24.36 37.46 08.4 S9S1 6582.36 24.36 08.36 22.46 37.45 96.36 10.46 38.76 51.46 22.5 S9S1 610.36 52.36 36.36 50.46 65.46 24.36 38.46 66.36 79.46 50.4 S10S1 051.35 54.35 38.35 52.45 67.45 26.35 40.45 68.35 81.45 52.4 S10S1 152.36 68.35 52.36 66.45 81.45 40.35 54.45 82.35 95.45 66.4 S10S1 254.0 58.2 36.66 58.0 45.95 54.36 68.45 96.36 60.9 45.80.4 S10S1 468.36 10.66 94.36 08.46 23.45 82.35 96.46 24.36 37.46 08.4 S10S1 558.2 24.36 08.36 22.46 37.45 96.36 10.46 38.76 51.46 22.4.

[0247] D1D2D3D4D5D6D7D8D9D10S11S12554.3596.3580.3594.4609.4568.3582.4610.3623.4594.4S11S14582.3624.5608.3622.4637.4596.3610.4638.3651.4622.4S11S15596.3638.3622.6636.4651.4610.3624.4652.3665.4636.4S11S16610.3652.3636.3650.7665.4624.3638.4666.3679.4650.4S11S18638.3680.3664.3678.4693.4652.3666.4694.3707.4678.4S12S12568.3610.5594.3608.4623.6582.7596.4624.3637.4608.4S12S14596.3638.3622.3636.4651.4610.3624.4652.3665.4636.4S12S15610.3652.3636.3650.4665.4624.3638.4666.3679.4650.4S12S16624.3666.3650.3664.4679.4638.3652.4680.3693.4664.4S12S18652.3694.3678.3692.4707.4666.3680.8708.9721.4692.4S14S14624.3666.3650.3664.4679.4638.3652.4680.3693.4664.4S14S15638.3680.6664.3678.4693.4652.5666.4694.3707.4678.4S14S16652.3694.3678.3692.4707.4666.3680.4708.3721.4692.4S14S18680.4722.3706.3720.8735.7694.3708.4736.3749.4720.4S15S15652.3694.5678.3692.4707.4666.3680.4708.3721.4692.4S15S16666.3708.3692.3706.4721.4680.3694.4722.3735.4706.4S15S18694.3736.3720.3734.4749.4708.3722.4750.3763.4734.4S16S16680.3722.3706.3720.4 736.3 750.3 734.3 748.4 763.4 722.3 736.4 764.3 777.4 748.4 718.4 763.4 764.3 780.5 776.4 N6 N6 666.4 408.4 392.4 406.8 421.5 380.4 394.5 422.4 435.5 406.5 N6 N6 380.4 422.4 406.4 420.5 435.5 394.4 408.5 436.4 449.5 420.5 N6 N6 394.4 436.4 420.4 434.5 449.6 408.4 422.5 436.4 447.5 434.5 N6 N6 408.4 450.4 434.4 448.5 463.5 422.4 436.5 464.4 477.5 448.5 N6 N6 436.4 478.4 462.4 476.4 491.5 450.4 464.5 492.4 492.4 476.4 N6 N6 450.4 492.4 476.4 490.5 505.5 464.4 478.5 506.4 519.5 490.5 N6 N6 464.4 506.4 490.4 504.5 519.6 478.4 492.5 520.4 533.5 504.5 N6 N6 492.4 534.4 518.4 532.5 547.5 520.4 534.5 548.4 561.5 532.5 N6 N6 506.4 548.4 532.4 546.5 561.2 520.4 534.5 562.4 575.5 546.5 N6 N6 334.4 576.4 560.4 574.5 589.5 548.4 562.4 590.4 603.5 574.5 N6. 2N12618.5660.5644.5658.6673.6632.5646.6674.5687.6658.6N7N7394.4436.4420.4434.5449.5408.4422.5450.4463.5434.5N7N8408.4450.4434.4448.5463.5 422.4436.5464.4477.5448.5N7N9422.4464.4448.7462.5477.5436.4450.5478.4491.5462.5N7N11450.4492.4476.4490.5505.5464.4478.5506.4519.5490.5N7N 12464.4506.4490.4504.5519.5478.4492.5520.4533.5504.5N7N13478.4520.4504.4518.5533.5492.4506.5534.4547.5518.5N7N15506.8548.4532.8546.5561.5 520.5534.5562.4575.5546.5N7N16520.4562.4546.4560.5575.5534.4548.5576.4589.5560.5N7N18548.4590.4574.4588.5603.5562.4576.5604.4617.5588.5N7 2 N12632.7674.7658.5672.6687.6646.5660.6688.5701.6672.6N8N8422.4464.8448.4462.5477.5436.4450.5478.4491.5462.5N8N9436.4478.4462.4476.5491.5450.4464.5492.4505.5476.5N8N11464.7506.4490.4504.5519. 5478.4492.5520.4533.5504.5N8N12478.5520.4504.4518.5533.8492.4506.5534.4547.5518.5N8N13492.4534.7518.4532.5547.5506.4520.5548.4561.5532.5N8N15520.4562.6546.4560.5575.5534.4548.5576.4589.5560.5

[0248] N8 N8 34.45 76.85 60.45 74.55 89.55 48.45 62.55 90.46 03.55 74.5 N8 N8 56 2.46 04.45 88.46 02.56 17.55 76.45 90.56 18.46 31.56 02.5 N8 2 N9 N9 45 0.44 92.44 76.44 90.55 05.54 64.44 78.55 06.45 19.54 90.5 N9 N9 14 78.45 20.45 04.45 18.53 33.49 20.45 06.55 34.45 47.55 18.5 N9 N9 12 92.45 34.45 18.75 32.54 47.50 06.45 20.55 48.45 61.53 32.5 N9 N9 13 06.45 48.45 32.45 46.55 61.52 20.45 34.55 62.45 75.54 46.5 N9 N9 15 34.45 76.45 60.45 74.95 89.54 48.45 62.55 90.46 03.55 74.5 N9 N9 16 48.45 90.45 74.45 88.45 60.35 62.95 76.56 04.45 17.55 88.5 N9 N9 18 76.86 18.45 02.46 16.56 31.50 04.46 32.56 64.45 96.16.5 2 N11 N11 06.45 48.45 32.45 46.55 61.52 20.45 34.65 62.45 75.54 46.5 N11 N11 12 20.45 62.45 46.45 60.55 75.53 34.45 48.55 76.45 89.55 60.5 N11 N11 13 34.45 76.45 60.45 74.55 89.54 48.45 62.55 90.46 03.55 74.5 N11 N11 15 62.45 04.45 88.45 02.56 17.55 76.45 90.56 18.46 31.56 02.5 N11 N11 16 76.46 18.45 02.46 16.56 31.50 04.46 32.56 64.45 96.16.5 N11 N11 18 04.46 46.46 30.46 44.56 59.50 18.46 32.56 60.46 73.56 44.52 N12688.5730.5714.5728.6743.6702.5716.6744.5757.6728.6N12N12534.4576.4560.4574.5589.5 548.4562.5590.4603.5574.5N12N13548.4590.4574.4588.5603.5562.4576.5604.4617.8588.5N12 N15576.4618.4602.4616.5631.5590.4604.5632.8645.5616.5N12N16590.4632.4616.4630.5645.5 604.4618.8646.4659.5630.8N12N18618.4660.4644.4658.5673.5632.4646.5674.4687.5658.5N12 2 N12702.5744.5728.8742.6757.6716.5730.8758.5771.6742.6N13N13562.4604.4588.4602.8617.5576.4590.5618.4631.5602.5N13N15590.4632.4616.4630.5645.5 604.4618.5646.4659.5630.5N13N16604.4646.4630.4644.5659.8618.4632.5660.4673.5644.5N13N18632.4674.4658.4672.5687.5646.4660.5688.4701.5672.5N13 2 N12716.5758.5742.5756.6771.6730.6744.6772.5785.6756.6N15N15618.4660.4644.4658.5673.5632.4646.5674.4687.5658.5N15 N16632.4674.4658.4672.5687.9646.4660.5688.4701.5672.5N15N18660.4702.4686.4700.5715.5674.4688.5716.4729.5700.5N15 2N12 44.5 78.5 70.5 84.6 79.6 75.8 72.6 80.0 58.3 78.4 N16 46.9 88.4 72.4 86.9 70.1 60.4 74.5 70.2 71.4 86.5 N16 87.4 71.6 70.0 29.4 72.8 42.4 74.2 57.6 71.6 42.5 N16 2 N12 86.5 28.5 81.2 26.5 82.6 84.1 68.4 20.5 81.4 64.2 85.5 26.6 2 N12 2 N12 2 N12 70.6 91.2 69.6 96.9 10.7 92.6 18.4 89.8 92.6 93.9 79.1 1.2

[0249] Example 1.1: Parallel synthesis and characterization of S8NxDy series of amine-based lipid compounds library

[0250]

[0251] wherein n3, -NRR, R 1 and R 2 are as defined above.

[0252]

[0253] In a 50 mL dry reaction bottle, add cyanuric chloride (2 mmol), THF (10 mL), and cool to -20 °C under stirring. Then add 1-octanethiol (2 mmol) and DIPEA (2.4 mmol) successively. A large amount of precipitate is immediately precipitated. After 5 min of reaction, TLC detection shows that the reaction is complete. The reaction solution is transferred to a 15 mL centrifuge tube, and then diluted to 15 mL and centrifuged (2000 r / min, 1 min). The Step I reaction solution (15 mL, 0.13 M) is obtained.

[0254] The Step I reaction solution was transferred to 11 1.5 mL reaction vials (1.15 mL, 0.15 mmol each) using a pipette. The corresponding reaction vials were each charged with the linear alkyl amine (0.15 mmol), a solution of DIPEA in THF (0.35 mL, 0.5 M), and the reaction was stirred at room temperature for 2 h, at which time TLC indicated no Step I starting material.

[0255] The Step II reaction solution was transferred to 10 1.5 mL EP tubes (0.1 mL, 0.01 mmol each) using a pipette. The corresponding EP tubes were each charged with a solution of the diamine in THF (0.12 mL, 0.012 mmol, 0.1 M), a solution of DIPEA in THF (0.02 mL, 0.1 M), and the reaction was stirred at 78 °C for 1 h in a heated Thermo-Shaker, at which time TLC indicated no Step II starting material. Upon completion of the reaction, the solvent was evaporated from the reaction tubes at room temperature to yield 110 amine-based lipid compounds S8NxDy. Ten representative compounds were selected for mass spectrometry, the results of which are shown in Table 1.

[0256] Table 1: MW / z values of representative compounds

[0257]

[0258]

[0259] Example 1.1.1: One-pot synthesis and characterization of representative amine-based lipid compound S8N11D6.

[0260]

[0261] A 20 mL dry reaction tube was charged with cyanuric chloride (1 mmol), THF (10 mL), and the reaction was cooled to -20 °C with a cold bath. 1-Octanethiol (1 mmol) and DIPEA (1.2 mmol) were added sequentially, and the reaction mixture immediately precipitated a large amount of solids. The reaction was stirred for 5 min, at which time TLC indicated completion. The reaction was warmed to room temperature, and undecyl amine (1 mmol) and DIPEA (1.2 mmol) were added. The reaction was stirred at room temperature for 2 h, at which time TLC indicated no Step I starting material. N,N-dimethylpropylenediamine (1.2 mmol) and DIPEA (2 mmol) were added, and the reaction was stirred at 78 °C for 1 h, at which time TLC indicated no Step II starting material. The reaction was purified using an automated purification system (normal-phase silica gel column, DCM:MeOH gradient from 0 to 10%) to yield 440.4 mg of a white solid in 89% yield over three steps and >99% purity. 1H NMR (400 MHz, CDC13) δ 3.54 (m, 2H), 3.34 (m, 2H), 3.01 (m, 4H), 2.80-2.73 (m, 6H), 2.07 (m, 2H), 1.67 (m, 2H), 1.53 (m, 2H), 1.38 (m, 2H), 1.28 (m, 2H), 1.24 (m, 24H), 0.88-0.84 (m, 6H). ESI-MS calculated for C27H55N6S+[M+H]+495.4, found 495.6.

[0262] Example 1.2: Parallel synthesis and characterization of S6SxDy series of amine-based lipid compound library

[0263]

[0264] wherein n3, -NRR, R 1 and R 2 are as defined above.

[0265]

[0266] In a 50 mL dry reaction flask was added cyanuric chloride (2 mmol), THF (10 mL), and the reaction flask was cooled to -20 °C under stirring. Then 1-hexanethiol (2 mmol) and DIPEA (2.4 mmol) were added successively. A large amount of precipitate was immediately formed. The reaction was monitored by TLC after 5 min. The reaction was completed. The reaction solution was transferred to a 15 mL centrifuge tube and diluted to 15 mL. The solution was centrifuged (2000 r / min, 1 min). The Step I reaction solution (15 mL, 0.13 M) was obtained.

[0267] The Step I reaction solution was transferred to 11 1.5 mL reaction flasks (1.15 mL, 0.15 mmol, in each flask) using a pipette. The corresponding reaction flasks were added with linear alkyl mercaptan (0.15 mmol) and a THF solution of DIPEA (0.35 mL, 0.5 M). The reaction was stirred at room temperature for 2 h. TLC detection showed no Step I starting material.

[0268] Each Step II reaction was transferred into 10 1.5 mL EP tubes (0.1 mL, 0.01 mmol each) using a pipette, and the corresponding EP tubes were each added with the diamine THF solution (0.12 mL, 0.012 mmol, 0.1 M), the DIPEA THF solution (0.02 mL, 0.1 M), and reacted in a heating type shaker (Thermo-Shaker) at 78 °C for 1 h, and no Step II starting material was detected by TLC. After the reaction was completed, the solvent in the reaction tube was evaporated at room temperature to obtain 110 amine-based lipid compounds S6SxDy. Ten representative compounds were selected for mass spectrometry detection, and the results are shown in Table 2 below.

[0269] Table 2: MW / z values of representative compounds

[0270]

[0271]

[0272] Example 1.2.1: One-pot synthesis and characterization of representative amine-based lipid compound S6S11D6

[0273]

[0274] In a 20 mL dry reaction tube, tricyanopyrazine (1 mmol) and THF (10 mL) were added, and the reaction solution was cooled to -20 °C under stirring. Then, 1-hexanethiol (1 mmol) and DIPEA (1.2 mmol) were added successively, and a large amount of precipitate was immediately precipitated from the reaction solution. After 5 min of reaction, the reaction was completed as detected by TLC. After the reaction solution was warmed to room temperature, undecyl mercaptan (1 mmol) and DIPEA (1.2 mmol) were added, and the reaction was stirred at room temperature for 2 h. No Step I starting material was detected by TLC. Then, N,N-dimethylpropylenediamine (1.2 mmol) and DIPEA (2 mmol) were added, and the reaction was warmed to 78 °C for 1 h. No Step II starting material was detected by TLC. Purification was performed using an automatic purification system (forward silica gel column, DCM:MeOH gradient elution from 0 to 10%), and 445.1 mg of white solid was obtained, with a yield of 92% and a purity of 99% for the three-step reaction. 1 H NMR (400 MHz, CDC13) δ 3.51 (m, 2H), 3.04-3.01 (m, 4H), 2.81-2.74 (m, 6H), 2.09 (m, 2H), 1.68 (m, 2H), 1.55 (m, 2H), 1.33 (m, 2H), 1.29 (m, 2H), 1.23 (m, 22H), 0.88-0.84 (m, 6H). ESI-MS calculated for C 25 H50 N5S2 + [M+H] + 484.4, found 484.4.

[0275] Example 1.3: Parallel synthesis and characterization of N6NxDy series of amphi-lipidic compounds library

[0276]

[0277] wherein n3, -NRR, R 1 and R 2 are as defined above.

[0278]

[0279] In a 50 mL dry reaction flask was added cyanuric chloride (2 mmol), THF (10 mL), after cooling to -20 °C by stirring in a cold bath, 1-hexylamine (2 mmol), DIPEA (2.4 mmol) were added successively, the reaction solution immediately precipitated a large amount of precipitate, after 5 min of reaction, TLC detection, the reaction was completed. The reaction solution was transferred to a 15 mL centrifuge tube, constant volume to 15 mL, then centrifuged in a centrifuge (2000 r / min, 1 min). Step I reaction solution (15 mL, 0.13 M) was obtained.

[0280] The Step I reaction solution was transferred to 11 1.5 mL reaction flasks with a pipette (1.15 mL each, 0.15 mmol), and the corresponding reaction flasks were each added with a linear alkylamine (0.15 mmol), a DIPEA THF solution (0.35 mL, 0.5 M), and stirred at room temperature for 2 h, and TLC detection showed no Step I starting material.

[0281] Each Step II reaction solution was transferred to 10 1.5 mL EP tubes with a pipette (0.1 mL each, 0.01 mmol), and the corresponding EP tubes were each added with a diamine THF solution (0.12 mL, 0.012 mmol, 0.1 M), a DIPEA THF solution (0.02 mL, 0.1 M), and reacted in a heated shaking reactor (Thermo-Shaker) at 78 °C for 1 h, and TLC detection showed no Step II starting material. After the reaction was completed, the solvent in the reaction tube was evaporated at room temperature, and 110 amphi-lipidic compounds N6NxDy were obtained. Ten representative compounds were selected for mass spectrometry detection, and the results are shown in Table 3.

[0282] Table 3: MW / z values of representative compounds

[0283]

[0284]

[0285] Example 1.3.1: One-pot synthesis and characterization of representative amine-based lipid compound N6N11D6

[0286]

[0287] In a 20 mL dry reaction tube, add cyanuric chloride (1 mmol), THF (10 mL), cool to -20 °C with stirring under a cold bath, then add 1-hexylamine (1 mmol), DIPEA (1.2 mmol), the reaction solution immediately precipitated a large amount of precipitate, TLC detection after 5 min of reaction, the reaction was completed. After the reaction solution was raised to room temperature, add undecylamine (1 mmol), DIPEA (1.2 mol), stirred at room temperature for 2 h, TLC detection no Step I raw material. Add N,N-dimethylpropylenediamine (1.2 mmol), DIPEA (2 mol) under stirring and heating to 78 °C for 1 h, TLC detection no Step II raw material. Purification using an automated purification system (normal silica gel column, DCM:MeOH from 0 to 10% gradient elution) to give white solid 395.7 mg, three-step reaction yield 88%, purity 99%. 1 H NMR (400 MHz, CDC13) δ 3.54 (m, 2H), 3.05-3.02 (m, 4H), 2.82-2.73 (m, 6H), 2.08 (m, 2H), 1.69 (m, 2H), 1.54 (m, 2H), 1.34 (m, 2H), 1.30 (m, 2H), 1.24 (m, 22H), 0.89-0.85 (m, 6H). ESI-MS calculated for C 25 H 52 N7 + [M+H] + 450.4, found 450.4.

[0288] Example 1.4: Parallel synthesis and characterization of S6SxOy series of amine-based lipid compound library

[0289]

[0290] wherein n4 = 1, 2, 3, or 4; -NR’R’ represents a dialkylamino or cyclic amino moiety of O1 to O10 above; and R 1 and R 2 are as defined above.

[0291]

[0292] In a 50 mL dry reaction flask, cyanuric chloride (2 mmol) and THF (10 mL) were added. After cooling to -20 °C with stirring in a cold bath, 1-hexamethylenetetramol (2 mmol) and DIPEA (2.4 mmol) were added sequentially. A large amount of precipitate immediately formed in the reaction solution. The reaction was stopped by TLC after 5 min. The reaction solution was transferred to a 15 mL centrifuge tube, and the volume was adjusted to 15 mL before centrifugation (2000 r / min, 1 min). Step I reaction solution (15 mL, 0.13 M) was obtained.

[0293] The reaction solution from Step I was transferred to 11 reaction flasks of 1.5 mL each (1.15 mL, 0.15 mmol each) using a pipette. A straight-chain hydrocarbon thiol (0.15 mmol) and a THF solution of DIPEA (0.35 mL, 0.5 M) were added to each flask. The mixture was stirred at room temperature for 2 h. TLC analysis showed no presence of the Step I starting material.

[0294] Each Step II reaction solution was transferred using a pipette into ten 1.5 mL EP tubes (0.1 mL, 0.01 mmol each). Hydroxylamine THF solution (0.12 mL, 0.012 mmol, 0.1 M) and DIPEA THF solution (0.02 mL, 0.1 M) were added to the corresponding EP tubes. The mixtures were reacted at room temperature for 2 h in a Thermo-Shaker reactor. TLC analysis showed no Step II starting material. After the reaction, the solvent in the reaction tubes was evaporated at room temperature to obtain 110 amino lipid compounds S6SxOy. Ten representative compounds were selected for mass spectrometry analysis, and the results are shown in Table 1.4 below.

[0295] Table 1.4: MW / z values ​​of representative compounds

[0296]

[0297]

[0298] Example 1.4.1: One-pot synthesis and characterization of the representative amino-lipid compound S19S9O8

[0299]

[0300] In a 20 mL dry reaction tube, add cyanuric chloride (1 mmol), THF (10 mL), cool to -20 °C with stirring under cold bath, then add 1-octyl mercaptan (1 mmol), DIPEA (1.2 mmol) successively, the reaction solution immediately precipitated a large amount of precipitate, TLC detection after 5 min reaction, reaction was completed. After the reaction solution was raised to room temperature, add 1-nonanethiol (1 mmol), DIPEA (1.2 mol), stirred at room temperature for 2 h, TLC detection no Step I raw materials. Add N-(2-hydroxyethyl)-pyrrolidine (1.2 mmol), DIPEA (2 mol) at room temperature for 2 h, TLC detection no Step II raw materials. Purification using an automatic purification system (forward silica gel column, DCM:MeOH from 0 to 10% gradient elution) to get white solid 546.2 mg, three-step reaction yield 86%, purity 99%. 1 H NMR (400 MHz, CDC13) δ 5.35 (m, 2H), 4.21 (m, 2H), 3.51 (m, 4H), 2.67-2.54 (m, 6H), 2.19 (m, 4H), 1.68 (m, 4H), 1.55-1.23 (m, 38H), 0.88-0.84 (m, 6H). ESI-MS calculated for C 36 H 67 N4OS2 + [M+H] + 635.5, found 635.7.

[0301] Example 2: Evaluation of the luciferase mRNA delivery performance of the lipid nanoparticles prepared from the amine-based lipid compound in vivo

[0302] 1. Preparation of lipid nanoparticles

[0303] Preparation method 1:

[0304] The amine-based lipid compound of the present application was mixed with DOPE, cholesterol, PEG2000-DMG at a molar ratio of 45:10:42.5:2.5 and dissolved in anhydrous ethanol to make the molar concentration of the amine-based lipid compound 0.001-0.01 mmol / L. Using a microsyringe pump, the resulting ethanol solution and a sodium acetate solution (50 mM, pH = 4.0) with luciferase mRNA (Fluc mRNA, TriLink) dissolved therein were mixed in a microfluidic chip at a volume ratio of 1:3 to prepare a crude solution of lipid nanoparticles, which was then dialyzed in a dialysis cassette (Fisher, MWCO 20,000) in 1X PBS at a controlled temperature of 4°C for 6 h, and filtered through a 0.22 μm microporous filter before use. The mass ratio of the amine-based lipid compound to Fluc mRNA was about 10:1. The resulting lipid nanoparticle (LNP) solution was administered to the test animals by subcutaneous administration.

[0305] Characterization of lipid nanoparticles:

[0306] Characterization of particle size: The particle size and PDI of the prepared lipid nanoparticles were determined by Nano-ZSZEN3600 (Malvern). 40 uL of the LNP solution was taken for particle size measurement, and the cycle was repeated three times, each cycle for 30 s.

[0307] Encapsulation efficiency detection: The LNP RNA concentration was detected using the RNA HS Assay kit. The theoretical RNA concentration was the total RNA amount inputted divided by the total volume of the final solution.

[0308]

[0309] Table 4: Characterization data of LNP prepared using a representative amine-based lipid compound using formulation method 1.

[0310]

[0311]

[0312]

[0313] Formulation method 2:

[0314] The preparation method was the same as formulation method 1, except that the amine-based lipid compound, DSPC, cholesterol and PEG2000-DMG were used at a molar ratio of 50:10:38.5:1.5. The resulting lipid nanoparticle (LNP) solution was administered to the test animals by tail vein and intramuscular injection.

[0315] Table 5: Characterization data of LNP prepared using a representative amine-based lipid compound using formulation method 2.​

[0316]

[0317] 2. Animal Experiments

[0318] Animal Preparation: 6-week-old female BALB / c mice with a body weight of about 20 g were selected and raised in a SPF level feeding room. Animal experiments were strictly conducted in accordance with the guidelines of the National Health Organization and the requirements of animal ethics.

[0319] In vivo delivery: 9 mice were randomly selected in each group, and the lipid nanoparticle solution was injected by subcutaneous, intramuscular and tail vein injection at a dosage of 0.5 mg / kg Fluc mRNA (3 mice for each administration method). After 12 hours, 200 μL of 10 mg / mL D-luciferin potassium salt (PerkinElmer) was injected into each mouse through the tail vein. After 10 minutes, the mice were placed under the in vivo imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed and recorded by taking pictures. The expression intensity of Fluc mRNA delivered by the representative amino lipid compounds through the three administration methods is shown in Tables 5-7. DLin-MC3 was used as a control.

[0320] Table 5: Expression intensity of Fluc mRNA delivered by subcutaneous administration of representative amino lipid compounds.

[0321] Amino lipid number Fluorescence intensity S16N9D32.7E+07 S15N11D51.9E+08 S12N13D68.6E+06 S16N7D91.4E+07 CD103.7E+08 S7S18D32.9E+08 S9S16D54.2E+07 S11S12D81.1E+07 S8S16D94.2E+06 S10S16D104.7E+07 S19S9O81.1E+07 S19S9O92.8E+07 DLin-MC3 (control) 3.1E+06

[0322] Table 6: Expression intensity of Fluc mRNA delivered by intramuscular administration of representative amino lipid compounds.

[0323] Fluorescence intensity S16N9D 34.3E+06 S15N11D 53.7E+06 S12N13D 61.2E+06 S16N7D 94.3E+07 S16N8D 103.9E+06 S7S18D 38.7E+07 S9S16D 55.2E+06 S11S12D 84.7E+06 S8S16D 98.2E+06 S10S16D 103.2E+06 S19S9O 89.1E+07 S19S9O 91.8E+07 DLin-MC3 (control) 2.7E+07

[0324] Table 7: Expression intensity of Fluc mRNA delivered by representative amine lipid compounds tail vein administration.

[0325] Fluorescence intensity S16N9D 35.3E+06 S15N11D 53.7E+06 S12N13D 63.9E+06 S16N7D 95.1E+07 S16N8D 103.2E+06 S7S18D 38.1E+07 S9S16D 57.5E+06 S11S12D 83.8E+06 S8S16D 96.5E+06 S10S16D 102.9E+06 S19S9O 81.1E+08 S19S9O 93.1E+07 DLin-MC3 (control) 2.7E+07

[0326] Example 3: Ovalbumin mRNA in vivo delivery and immune performance evaluation of lipid nanoparticles prepared by amine lipid compounds

[0327] Preparation method:

[0328] The amine lipid compound of the present application was mixed with DOPE, cholesterol, PEG2000-DMG at a molar ratio of 50:10:38.5:1.5 and dissolved in anhydrous ethanol to make the molar concentration of the amine lipid compound 0.001-0.01 mmol / L. Ovalbumin mRNA (OVA mRNA, TriLink) was dissolved in sodium acetate solution (50 mM, pH = 4.0). Using a microsyringe pump, the resulting ethanol solution and sodium acetate solution (50 mM, pH = 4.0) were mixed in a microfluidic chip at a volume ratio of 1:3 to prepare a crude solution of lipid nanoparticles, which was then dialyzed in a dialysis box (Fisher, MWCO 20,000) in 1X PBS at a controlled temperature of 4°C for 6 h, and filtered through a 0.22 μm microporous filter before use. The mass ratio of the amine lipid compound to ovalbumin mRNA (OVA mRNA) was about 10:1.

[0329] Animal preparation: Six-week-old female BALB / c mice weighing approximately 20g were selected and housed in an SPF-grade enclosure. Animal experiments were conducted strictly in accordance with national health agency guidelines and animal ethics requirements.

[0330] In vivo delivery: Three mice were randomly selected from each group and injected intramuscularly into the leg with a lipid nanoparticle solution at a dose of 0.5 mg / kg mRNA (Day 0). A booster injection of the same dose was administered 7 days later (Day 7). Blood was collected from the tail vein on day 21 for serological analysis. DLin-MC3 was used as a control (Figure 1).

[0331] Enzyme-linked immunosorbent assay (ELISA): Flat-bottomed 96-well plates (Nunc) were pre-coated with 50 mM carbonate buffer, with an OVA protein concentration of 0.5 μg protein per well (pH 9.6) and incubated overnight at 4°C, followed by blocking with 5% glycine. Serum obtained from animals that received a solution of lipid nanoparticles containing mRNA was diluted with PBS-0.05% Tween (PBS-T) at pH 7.4 from 10... 2 Dilute to 10 6 The HRP substrate was added to the wells and incubated at room temperature for 1 hour at 37°C. Horseradish peroxidase (HRP, Invitrogen)-conjugated goat anti-mouse IgG was labeled in PBS-T-1% BSA at a dilution of 1:10,000. After adding the HRP substrate, the absorbance was measured at 450 nm using a Bio-Rad ELISA reader. As shown in Figure 1, the IgG antibody titrations for S7S18D3, S16N7D9, S19S9O8, and S19S9O9 were significantly better than the control group.

[0332] Example 4: Preliminary screening of amino lipid compounds as DNA carriers

[0333] Cell line: HEK293 cells (ATCC CRL-1573) TM )

[0334] Culture medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum.

[0335] Screening method: 96-well plate cell transfection

[0336] Detection (readout): Percentage of GFP fluorescent cells relative to the total cell count (total cell count determined using the nuclear dye Hoechst – see Figure 2). Lipofectamine 2000 (Invitrogen) was used as a positive control according to the manufacturer's instructions.

[0337] Method: Samples were added using an 8-channel pipette. The concentrations shown are from a single well of a 96-well plate.

[0338] 1. The compound prepared in Example 1 (0.01 mmol) was dissolved in 1 mL of absolute ethanol, and after sonicating to dissolve, 10 μL of the resulting amine-based lipid ethanol solution and 5 μL of DOPE ethanol solution (0.01 M) were mixed, and then 35 μL of 0.2 M sodium acetate buffer (pH 5.6) was added to the resulting mixture, which was vortexed for 30 s at a constant speed to prepare 50 μL of a liposome solution. From the 50 μL of the liposome solution, 4 μL was added to 46 μL of 0.02 M sodium acetate buffer (pH 5.6), which was vortexed for 30 s at a constant speed to form a final liposome solution. From the final liposome solution, 5 μL was mixed with 75 ng of plasmid DNA (pSin-EGFP-IRES-Puro, Clontech) dissolved in 5 μL of 0.02 M sodium acetate buffer, and left to stand at room temperature for 30 min to form a lipid / DNA transfection complex.

[0339] 2. After 10 μL of the lipid / DNA transfection complex was incubated at room temperature for 30 min, 90 μL of freshly resuspended cells (3-5 x 10 4 cells) were added and mixed with a pipette. 100 μL of the cell + lipid / DNA complex was immediately transferred to separate wells of a 96-well culture plate, and placed in a 37°C incubator containing 5% CO2.

[0340] 3. Twenty to 24 hours after the initial transfection of the cells, Hoechst 33258 (Invitrogen) was added to the cells at a final concentration of 0.2 μg / ml, and incubated at 37°C in the dark for 15 min. The cells were then washed once with PBS solution, and incubated in culture medium for 20 to 24 hours.

[0341] 4. The cells were placed in a high-throughput confocal microscope (Molecular Devices ImageXpress), and 4 image fields of the cells were captured from each well. For each sample, 3 laser wavelength images were captured: a bright field image of the cells (Figures 2(a) and 2(b)), a Hoechst-stained image showing total nuclei (Figures 2(c) and 2(d)), and a GFP image showing cells successfully transfected with plasmid DNA and expressing GFP (Figures 2(e) and 2(f)). The obtained Hoechst-stained and GFP images were subjected to cell counting using MetaXpress software, and the number of cells expressing GFP was divided by the number of total nuclei, which was the absolute transfection efficiency of the cells. The absolute transfection efficiency was calculated as follows:

[0342]

[0343] Results: The absolute transfection efficiency of 2530 compounds on DNA of HEK293 cells is shown in Table 8.

[0344] Table 8: Absolute transfection efficiency of 2530 compounds on DNA of HEK293 cells.

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352] Example 5: Preliminary screening of amine-based lipid compounds as mRNA carriers

[0353] Cell line: HEK293 cells (ATCC CRL-1573 TM )

[0354] Culture medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum

[0355] Screening format: 96-well plate cell transfection

[0356] Detection (readout): Percentage of GFP fluorescent cells relative to total cell number (total cell number was determined using the nuclear stain Hoechst). Lipofectamine 2000 (Invitrogen) was used as a positive control.

[0357] Method: The experimental method was essentially the same as in Example 4, with 50 ng of transfected EGFP mRNA (TriLink) per well.

[0358] Results: The absolute transfection efficiency of 2530 compounds on mRNA of HEK293 cells is shown in Table 9.

[0359] Table 9: Absolute transfection efficiency of 2530 compounds on mRNA of HEK293 cells.

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367] Example 6: Transfection of Hela cell line with amine lipid compounds as DNA carriers

[0368] Cell line: Hela cells (ATCC 35241)

[0369] Culture medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum

[0370] Screening format: 96-well plate cell transfection

[0371] Detection (readout): Percentage of GFP fluorescent cells relative to total cell number (total cell number was determined using the nuclear stain Hoechst). Lipofectamine 2000 (Invitrogen) was used as a positive control according to the manufacturer's instructions.

[0372] Method: The experimental method was essentially the same as in Example 4.

[0373] Results: The absolute DNA transfection efficiency of 170 compounds on Hela cells is shown in Table 10.

[0374] Table 10: Absolute DNA transfection efficiency of 170 compounds on Hela cells.

[0375]

[0376] Example 7: Transfection of Hela cell line with amine lipid compounds as mRNA carriers

[0377] Cell line: Hela cells (ATCC 35241)

[0378] Culture medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum

[0379] Screening format: 96-well plate cell transfection

[0380] Detection (readout): Percentage of GFP fluorescent cells relative to total cell number (total cell number was determined using nuclear stain Hoechst). Commercially available lipofection reagent Lipofectamine 2000 (Invitrogen) was used as a positive control group according to the manufacturer's instruction.

[0381] Method: The experimental method was basically the same as that of Example 4, and the mass of transfected EGFP mRNA was 50 ng per well.

[0382] Results: The absolute mRNA transfection efficiency of 170 compounds on Hela cells is shown in Table 11.

[0383] Table 11: Absolute mRNA transfection efficiency of 170 compounds on Hela cells.

[0384]

[0385] Example 8: Transfection of amine lipid compound as DNA carrier on MCF7 cell line

[0386] Cell line: MCF7 cells (ATCC HTB-22)

[0387] Culture medium: DMEM supplemented with 10% fetal bovine serum

[0388] Screening format: 96-well plate cell transfection

[0389] Detection (readout): Percentage of GFP fluorescent cells relative to total cell number (total cell number was determined using nuclear stain Hoechst). Lipofectamine 2000 was used as a positive control group according to the manufacturer's instruction.

[0390] Method: The experimental method was basically the same as that of Example 4.

[0391] Results: The absolute DNA transfection efficiency of 102 compounds on MCF7 cells is shown in Table 12.

[0392] Table 12: Absolute DNA transfection efficiency of 102 compounds on MCF7 cells

[0393]

[0394]

[0395] Example 9: Transfection of amine lipid compound as mRNA carrier on MCF7 cell line

[0396] Cell line: MCF7 cells (ATCC HTB-22)

[0397] Culture medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum

[0398] Screening format: 96-well plate cell transfection

[0399] Detection (readout): Percentage of GFP fluorescent cells relative to total cell number (total cell number was determined using nuclear stain Hoechst). Lipofectamine 2000 (Invitrogen) was used as a positive control.

[0400] Method: The experimental method was essentially the same as in Example 4, with 50 ng of transfected EGFP mRNA (TriLink) per well.

[0401] Results: The mRNA absolute transfection efficiency of 102 compounds on MCF7 cells is shown in Table 13.

[0402] Table 13: mRNA absolute transfection efficiency of 102 compounds on MCF7 cells.

[0403]

[0404] Example 10: Amino lipid compounds as DNA carriers in screening of a hard-to-transfect cell line (embryonic stem cells)

[0405] Cell line: Embryonic stem cells (hESC2, cultured according to reported method Stem Cells, 2005, 23, 544-549)

[0406] Culture medium: mTeSRl (STEMCELL)

[0407] Screening format: 96-well plate cell transfection

[0408] Detection (readout): Percentage of GFP fluorescent cells relative to total cell number. Lipofectamine Stem was used as a positive control.

[0409] Method: 8-channel pipette was used for sample addition. The amounts shown are for a single well of a 96-well plate.

[0410] 1. Culture stem cells in Matrigel-treated 96-well plates to 3-5 x 10 4 cells per well.

[0411] 2. The compound prepared in Example 1 (0.01 mmol) was dissolved in 1 mL of absolute ethanol, after ultrasonic dissolution, 10 μL of the resulting amine-based lipid ethanol solution and 5 μL of DOPE ethanol solution (0.01 M) were mixed, then 35 μL of 0.2 M sodium acetate buffer (pH 5.6) was added to the mixture, and vortexed for 30 s at a constant speed. 4 μL of the resulting product was added to 46 μL of 0.02 M sodium acetate buffer (pH 5.6), and vortexed for 30 s at a constant speed to form a liposome solution. 5 μL of the liposome solution was mixed with 75 ng of plasmid DNA (pSin-EGFP-IRES-Puro) dissolved in 5 μL of 0.02 M sodium acetate buffer, and left at room temperature for 30 min to form a lipid / DNA transfection complex.

[0412] 3. After 10 μL of the lipid / DNA transfection complex was incubated at room temperature for 30 min, 90 μL of mTeSRl medium was added and mixed with a pipette. 100 μL of the mixture was added to separate wells of a 96-well culture plate, and placed in a 37 °C incubator containing 5% CO2.

[0413] 4. 48 hours after transfection of the cells, the cells were digested with 0.05% Trypsin-EDTA, and resuspended in PBS buffer and filtered. The cell suspension was placed in a flow cytometer, and the number of cells expressing GFP in not less than 2000 cells was measured to obtain the absolute efficiency of cell transfection. The cell transfection efficiency was compared with the positive control Lipofectamine Stem to obtain the relative efficiency of cell transfection.

[0414] Results: The absolute DNA transfection efficiency of 60 compounds on embryonic stem cells is shown in Table 14.

[0415] Table 14: Absolute DNA transfection efficiency of 60 compounds on embryonic stem cells.

[0416]

[0417] Example 11: Screening of amine-based lipid compounds as mRNA carriers in difficult-to-transfect cell lines (embryonic stem cells)

[0418] Cell line: embryonic stem cells (hESC2, cultured according to the reported method Stem Cells, 2005, 23, 544-549)

[0419] Culture medium: mTeSRl (STEMCELL)

[0420] Screening format: 96-well plate cell transfection

[0421] Detection (readout): Percentage of GFP fluorescent cells relative to total cell number. Lipofectamine Stem was used as a positive control group.

[0422] Method: The experimental method was basically the same as in Example 10, and the mass of transfected EGFP mRNA was 50 ng per well.

[0423] Results: The absolute mRNA transfection efficiency of 60 compounds on embryonic stem cells is shown in Table 15.

[0424] Table 15: Absolute mRNA transfection efficiency of 60 compounds on embryonic stem cells.

[0425]

[0426] Example 12: Screening of amine lipid compounds as DNA carriers in difficult-to-transfect cell lines (cardiomyocytes)

[0427] Cell line: Cardiomyocytes (differentiated from hESC according to the reported method, J. Mol. Cell. Cardiol. 2011, 51, 288-298)

[0428] Culture medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum

[0429] Screening format: 96-well plate cell transfection

[0430] Detection (readout): Percentage of GFP fluorescent cells relative to total cell number (total cell number was determined using the nuclear dye Hoechst). Lipofectamine Stem (Invitrogen) was used as a positive control group according to the manufacturer's instructions.

[0431] Method: The experimental method was basically the same as in Example 4.

[0432] Results: The absolute DNA transfection efficiency of 60 compounds on cardiomyocytes is shown in Table 16.

[0433] Table 16: Absolute DNA transfection efficiency of 60 compounds on cardiomyocytes.

[0434]

[0435] Example 13: Screening of amine lipid compounds as mRNA carriers in difficult-to-transfect cell lines (cardiomyocytes)

[0436] Cell line: Cardiomyocytes (differentiated from hESC according to the reported method, J. Mol. Cell. Cardiol. 2011, 51, 288-298)

[0437] Medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum

[0438] Screening format: 96-well plate cell transfection

[0439] Detection (readout): Percentage of GFP fluorescent cells relative to total cell number (total cell number was determined using the nuclear stain Hoechst). Lipofectamine Stem (Invitrogen) was used as a positive control according to the manufacturer's instructions.

[0440] Method: The experimental method was essentially the same as in Example 4, with the exception that the amount of transfected EGFP mRNA was 50 ng per well.

[0441] Results: The absolute mRNA transfection efficiency of 60 compounds on cardiomyocytes is shown in Table 17.

[0442] Table 17: Absolute mRNA transfection efficiency of 60 compounds on cardiomyocytes.

[0443]

[0444] List of abbreviations:

[0445] DMEM Dulbecco's Modified Eagle's Medium

[0446] DNA Deoxyribonucleic acid

[0447] DOPE Dioleoyl phosphatidylethanolamine

[0448] DSPC Distearoyl phosphatidylcholine

[0449] PEG2000-DMG (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000);

[0450] GFP Green fluorescent protein

[0451] EGFP Enhanced GFP

[0452] kD kiloDalton

[0453] RNA Ribonucleic acid

[0454] THF Tetrahydrofuran

[0455] DIPEA N,N-diisopropylethylamine

[0456] PBS phosphate buffered saline solution.

[0457] Various modifications to the application will be apparent to those skilled in the art from the foregoing description, which modifications are intended to fall within the scope of the appended claims. Each of the references cited in the present application, including all patents, patent applications, journal articles, books, and any other publications, are incorporated by reference herein in their entireties.

Claims

1. An amine lipid compound, wherein the amine lipid compound is a compound represented by the following formula I: in: R 1 and R 2 are the same or different from each other and are each independently selected from C 6 -C 24 Alkyl, C 6 -C 24 Alkenyl, C 6 -C 24 Alkynyl and C 4 -C 24 Acyl, wherein the C 6 -C 24 Alkyl, the C 6 -C 24 Alkenyl, the C 6 -C 24 Alkynyl and the C 4 -C 24 The acyl group is optionally replaced by C 1 -C 6 Hydrocarbyl substitution; X 1 , X 2 and X 3 are the same as or different from each other and are each independently selected from C, N, O, S, S=O, S(=O) 2 and SS; When X 1 When it is C, m=2, and two R 6 The same or different from each other; when X 1 When it is N, m=1; when X 1 It is O, S, S=O, S(=O) 2 Or SS, m = 0; When X 2 When it is C, n=2, and two R 7 The same or different from each other; when X 2 When it is N, n=1; when X 2 O, S, S=O, S(=O) 2 Or SS, n = 0; When X 3 When it is C, p = 2, and the two R 8 The same or different from each other; when X 3 When it is N, p = 1; when X 3 O, S, S=O, S(=O) 2 Or SS, p = 0; R 3 and R 4 are the same or different from each other and are each independently selected from C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl and C 2 -C 12 Alkynyl, wherein the C 1 -C 12 Alkyl, the C 2 -C 12 The alkenyl group and the C 2 -C 12 The alkynyl group is optionally substituted with C 1 -C 6 Hydrocarbyl substituted, or R 3 and R 4 combine with each other to form an optionally substituted 4- to 10-membered heterocyclic ring comprising 1 to 6 heteroatoms selected from nitrogen, sulfur and oxygen; R 5 Does not exist, or R 5 is hydrogen or C 1 -C 12 Alkyl to provide a quaternary amine; R 6 , R 7 , R 8 is hydrogen or C 1 -C 12 alkyl; L is C 1 -C 12 Alkylene, C 2 -C 12 Alkenylene or C 2- C 12 Alkyne, wherein the C 1 -C 12 Alkylene, the C 2 -C 12 The alkenylene and the C 2 -C 12 The alkynylene group is optionally substituted with one or more substituents selected from hydrocarbyl, carboxyl, acyl and alkoxy, or L is an optionally substituted 4- to 10-membered heterocyclic ring containing a heteroatom selected from nitrogen, sulfur and oxygen.

2. The amino lipid compound according to claim 1, in: X 1 C, R 6 is H, m = 2; or X 1 N, R 6 is H, m = 1; or X 1 O, S, S=O, S(=O) 2 or SS, m=0; and / or X 2 C, R 7 is H, n = 2; or X 2 N, R 7 is H, n = 1; or X 2 O, S, S=O, S(=O) 2 or SS, n=0; and / or X 3 C, R 8 is H, p = 2; or X 3 N, R 8 is H, p = 1; or X 3 O, S, S=O, S(=O) 2 Or SS, p=0.

3. The amino lipid compound according to claim 1, in: X 1 and X 2 are the same as or different from each other, and are each independently N or S; When X 1 When it is N, m=1; when X 1 When it is S, m=0; When X 2 When it is N, n=1; when X 2 When S, n=0; and / or X 3 N and p = 1, or X 3 is O and p=0; and / or R 5 does not exist; and / or L is C 1 -C 12 Alkylene, the C 1 -C 12 The alkylene group is optionally replaced by C 1 -C 6 Hydrocarbyl substituted, for example L is (CH 2 ) q , wherein q is an integer from 1 to 12, for example, an integer from 1 to 8 or 1 to 6.

4. The amino lipid compound according to claim 3, in: When m = 1, R 6 is hydrogen; and / or When n = 1, R 7 is hydrogen; and / or R 8 It's hydrogen.

5. The amino lipid compound according to claim 1, 3 or 4, in: R 1 and R 2 are the same or different from each other and are each independently C 6 -C 24 Alkyl or C 6 -C 24 alkenyl; X 1 N, R 6 is H, m = 1; or X 1 is S, m=0; X 2 N, R 7 is H, n = 1; or X 2 is S, n = 0; X 3 N, R 8 is H, p = 1; R 3 and R 4 are the same or different from each other and are each independently C 1 -C 12 Alkyl, wherein the C 1 -C 12 The alkyl group is optionally replaced by C 1 -C 6 Hydrocarbyl substituted, or R 3 and R 4 are combined with each other to form an optionally substituted 4 to 10 membered heterocyclic ring comprising 1 to 6 heteroatoms selected from nitrogen, sulfur and oxygen; and R 5 Does not exist.

6. The amino lipid compound according to any one of claims 1 to 5, in: L is C 1 -C 4 Alkylene, wherein the C 1 -C 4 The alkylene group is optionally replaced by C 1 -C 6 Hydrocarbyl substituted, for example L is C 2 -C 4 Alkylene, such as (CH 2 ) q , where q is 1, 2, 3, or 4.

7. The amino lipid compound according to any one of claims 1 to 6, in: R 1 and R 2 are the same or different from each other and are each independently C 6 -C 18 Alkyl or C 6 -C 18 Alkenyl.

8. The amino lipid compound according to any one of claims 1 and 3 to 7, in: m = 0 or 1, n = 0 or 1, and are the same as or different from each other and are each independently selected from the following S6, S7, S8, S9, S10, S11, S12, S14, S15, S16, S18, S19, S20, N6, N7, N8, N9, N11, N12, N13, N15, N16, N18 and 2 N 12 One of: S6:CH 3 (CH 2 ) 5 S-;S7:CH 3 (CH 2 ) 6 S-;S8:CH 3 (CH 2 ) 7 S-; S9:CH 3 (CH 2 ) 8 S-;S10:CH 3 (CH 2 ) 9 S-;S11:CH 3 (CH 2 ) 10 S-; S12:CH 3 (CH 2 ) 11 S-;S14:CH 3 (CH 2 ) 13 S-;S15:CH 3 (CH 2 ) 14 S-; S16:CH 3 (CH 2 ) 15 S-;S18:CH 3 (CH 2 ) 17 S-; S19： S20： N6:CH 3 (CH 2 ) 5 NH-;N7:CH 3 (CH 2 ) 6 NH-;N8:CH 3 (CH 2 ) 7 NH-; N9:CH 3 (CH 2 ) 8 NH-;N11:CH 3 (CH 2 ) 10 NH-;N12:CH 3 (CH 2 ) 11 NH-; N13:CH 3 (CH 2 ) 12 NH-;N15:CH 3 (CH 2 ) 14 NH-;N16:CH 3 (CH 2 ) 15 NH-; N18:CH 3 (CH 2 ) 17 NH-; 2 N 12 :(CH 3 (CH 2 ) 11 ) 2 N- 9. The amino lipid compound according to any one of claims 1 and 3 to 8, in: p = 1, and It is one selected from the following D1, D2, D3, D4, D5, D6, D7, D8, D9 and D10: D1： D2： D3： D4： D5： D6： D7： D8： D9： D10： 10. The amino lipid compound according to any one of claims 1 and 3 to 9, in: X 3 It is O; p is 0; R 5 does not exist; and One selected from the following O1, O2, O3, O4, O5, O6, O7, O8, O9 and O10: O1： O2： O3： O4： O5： O6： O7： O8： O9： O10： 11. A method for preparing an amino lipid compound according to any one of claims 1 to 10, wherein the method The following steps are involved: (1) at a temperature of -40°C to 30°C (preferably -30°C to 30°C), in the presence of a base as an acid-binding agent, cyanuric chloride is reacted with a 1 (R 6 ) m -X 1 The compound represented by H is subjected to a first reaction to obtain a first intermediate of formula I-1; (2) with or without isolating the first intermediate, at room temperature or under heating conditions, reacting the first intermediate with a product obtained by R 2 (R 7 ) n -X 2 The compound represented by H undergoes a second reaction in the presence of a base as an acid binding agent to obtain a second intermediate of formula I-2; (3) with or without isolating the second intermediate, under heating conditions, preferably in the presence of a base as an acid-binding agent, reacting the second intermediate with HX 3 (R 8 ) p -L-NR 3 R 4 R 5 The diamine represented by is subjected to a third reaction to obtain the amino lipid compound of formula I; Among them, X 1 , X 2 , X 3 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , L, m, n and p are defined as in any one of claims 1 to 10.

12. Use of the amino lipid compound according to any one of claims 1 to 10 for preparing lipid particles, wherein the lipid particles are lipid nanoparticles, liposomes, multilamellar vesicles or micelles; more preferably, the lipid particles are lipid nanoparticles.

13. A lipid particle comprising the amino lipid compound according to any one of claims 1 to 10; preferably, the lipid particle is a lipid nanoparticle, a liposome, a multilamellar vesicle or a micelle; more preferably, the lipid particle is a lipid nanoparticle.

14. lipid granules according to claim 13, wherein said lipid granules further contain one or more of helper lipids, sterols and bioactive agents; Preferably, the lipid particle further comprises polyethylene glycol lipid (PEG)-lipid; and / or Preferably, in the lipid particle, the molar ratio of the amino lipid compound to the helper lipid is about (2 to 10): 1, preferably about (3 to 8): 1, more preferably about (4 to 6): 1, for example about 4: 1, about 4.5: 1 or about 5: 1; and / or Preferably, in the lipid particles, the molar ratio of the amino lipid compound to the sterol is about (0.5 to 1.5):1, preferably about (1 to 1.4):1, for example about (1 to 1.3):1; and / or Preferably, in the lipid particle, the molar ratio of the amino lipid compound to the PEG-lipid is about (9 to 42): 1, preferably about (12 to 38): 1, more preferably about (16 to 36): 1, for example about (18 to 34): 1; and / or Preferably, the helper lipid is a non-cationic lipid, more preferably, the helper lipid is a non-cationic phospholipid, further preferably, the non-cationic lipid is DOPE, DSPC or a combination thereof; and / or Preferably, the sterol is one or more selected from cholesterol, sitosterol, stigmasterol and ergosterol, preferably cholesterol; and / or Preferably, the PEG-lipid is one or more selected from PEG1000-DMG, PEG5000-DMG, PEG2000-DMG and PEG2000-DSPE, preferably PEG2000-DMG; and / or Preferably, the bioactive agent is one or more of nucleic acids, antitumor agents, antibiotics, immunomodulators, anti-inflammatory agents, agents acting on the central nervous system, antigens or fragments thereof, peptides, proteins, antibodies, vaccines and small molecules; more preferably, the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotides, DNA, plasmids, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA).

14. Use of the lipid particles according to claim 12 or 13 in the preparation of a drug, wherein the drug is a drug for gene therapy, gene vaccination, antisense therapy or treatment by interfering RNA; Preferably, the lipid particles are used as carriers for encapsulating bioactive agents; Preferably, the gene therapy can be used for the treatment of cancer and genetic diseases; more preferably, the cancer is one or more of lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer or prostate cancer, and the genetic disease is one or more of hemophilia, thalassemia and Gaucher's disease; Preferably, the genetic vaccination is used to treat cancer, allergy, toxicity and pathogen infection; more preferably, the pathogen is one or more of a virus, a bacterium or a fungus.

15. Use of the lipid particles according to claim 12 or 13 in the preparation of a medicament for nucleic acid transfer, preferably, the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA).