Nitrogen-containing chain compounds, methods of preparation, compositions containing the same, and uses

Abstract

The present application discloses a nitrogen-containing chain compound, a preparation method, a composition containing the same and applications. The present application provides a nitrogen-containing chain compound as shown in formula I or a pharmaceutically acceptable salt thereof. The nitrogen-containing chain compound as shown in formula I of the present application can be used for preparing a lipid carrier. The lipid carrier prepared by the present application can encapsulate nucleic acid drugs, and can be used for delivering nucleic acid prophylactic and / or therapeutic agents to mammalian cells and organs and playing a role.

Description

[0001] The present application claims priority to Chinese Patent Application No. 2022106524310, filed on June 6, 2022, Chinese Patent Application No. 2023102724825, filed on March 17, 2023, and Chinese Patent Application No. 2023106034922, filed on May 25, 2023, the contents of which are incorporated herein in their entirety. TECHNICAL FIELD

[0002] The present application relates to a nitrogen-containing chain compound, a preparation method, a composition comprising the same, and applications. BACKGROUND

[0003] Nucleic acid drugs are an important direction of current basic and applied research. Nucleic acid drugs can be used for the prevention and / or treatment of viral and bacterial infectious diseases, tumors, metabolic diseases, etc., and have lower production costs and shorter production cycles, which are beneficial to the rapid development of personalized drugs. However, nucleic acids are large molecules with negative charges, which are difficult to penetrate the cell membrane. At the same time, nucleic acids have poor stability. The development of various nucleic acid packaging and delivery systems can to some extent overcome the instability of nucleic acid drugs and improve their delivery efficiency.

[0004] Lipid nanoparticles have been shown to be useful as carriers for delivering biologically active substances, such as small molecule drugs, proteins, and nucleic acids, into cells and / or intracellular compartments. By designing and optimizing the types and amounts of each component in the lipid nanoparticle, it is important to optimize the nucleic acid drug delivery system for improving the efficacy of nucleic acid drug prevention and treatment, especially lipid compounds that can be used to deliver RNA prophylactic and / or therapeutic agents, and related methods and compositions. SUMMARY

[0005] The present application aims to provide a new ionizable lipid compound that can be used to deliver nucleic acid drugs, increasing the types of ionizable lipid compounds and the selection of nucleic acid prophylactic and / or therapeutic delivery carriers. To solve the above technical problems, the present application provides a nitrogen-containing chain compound, a preparation method, a composition comprising the same, and applications. The composition of the present application can be used for efficient delivery of nucleic acid drugs. The technical solutions of the present application are as follows:

[0006] The present application provides a nitrogen-containing chain compound as shown in Formula I, or a pharmaceutically acceptable salt thereof,

[0007]

[0008] wherein Z and W are independently C3-C 10 alkylene;

[0009] Y and Q are independently

[0010] A is C2-C6alkylene, each R A-1 and R A-2 is independently C2-C6alkylene;

[0011] M is C1-C6alkylene;

[0012] R 1 and R 2 is independently C6-C 20 alkyl;

[0013] R 5 is unsubstituted or substituted with 1, 2, or 3 R 5-1 C2-C 10 alkyl;

[0014] each R 5-1 is independently hydroxyl or each R 5-1-1 is independently C6-C 20 alkyl;

[0015] R 6 is unsubstituted or substituted with 1, 2, or 3 R 6-1 C2-C 10 alkyl;

[0016] each R 6-1 is independently hydroxyl or each R 6-1-1 is independently C6-C 20 alkyl.

[0017] The present application provides a nitrogen-containing chain compound as shown in formula I or a pharmaceutically acceptable salt thereof,

[0018]

[0019] wherein Z and W are independently C3-C 10 alkylene;

[0020] Y and Q are independently

[0021] A is C2-C6alkylene, each R A-1 and R A-2 is independently C2-C6alkylene;

[0022] M is C1-C6alkylene;

[0023] R 1 and R 2 is independently C6-C 20alkyl;

[0024] R 5 is unsubstituted or substituted with one, two, or three R 5-1 substituted C2-C 10 alkyl;

[0025] each R 5-1 is independently hydroxyl or R 5-1-1 is independently C6-C 20 alkyl;

[0026] R 6 is unsubstituted or substituted with one, two, or three R 6-1 substituted C2-C 10 alkyl;

[0027] each R 6-1 is independently hydroxyl or

[0028] R 6-1-1 is independently C6-C 20 alkyl.

[0029] The present application provides a nitrogen-containing chain compound as shown in formula I or a pharmaceutically acceptable salt thereof,

[0030]

[0031] wherein Z and W are independently C4-C 10 alkylene;

[0032] Y and Q are independently

[0033] A is C2-C6alkylene,

[0034] each R A-1 and R A-2 are independently C2-C6alkylene;

[0035] M is C1-C6alkylene;

[0036] R 1 and R 2 are independently C6-C 20 alkyl;

[0037] R 5 is unsubstituted or substituted with one, two, or three R 5-1 substituted C2-C 10 alkyl;

[0038] each R 5-1 is independently hydroxyl or

[0039] R 5-1-1 independently C6-C 20 alkyl;

[0040] R 6 is C2-C 6-1 alkyl, unsubstituted or substituted by one, two or three R 10

[0041] each R 6-1 is independently hydroxy or

[0042] R 6-1-1 is independently C6-C 20 alkyl.

[0043] In a certain preferred embodiment, in the nitrogen-containing chain compound of the formula I or a pharmaceutically acceptable salt thereof, the definition of certain groups can be as described below and the definition of other groups can be as described in any of the other embodiments (hereinafter referred to as "in a certain preferred embodiment"): In Z, the C4-C 10 alkylene can be C5-C8alkylene, preferably straight-chain alkane, for example

[0044] In a certain preferred embodiment, in Z, the C3-C 10 alkylene can be C3-C8alkylene, preferably straight-chain alkane, for example

[0045] In a certain preferred embodiment, in W, the C4-C 10 alkylene can be C4-C 10 alkylene, and also C5-C8alkylene, preferably straight-chain alkane, for example

[0046] In a certain preferred embodiment, in W, the C3-C 10 alkylene can be C3-C8alkylene, preferably straight-chain alkane, for example

[0047] In a certain preferred embodiment, in A, the C2-C6alkylene can be for example

[0048] In a certain preferred embodiment, in A, the C2-C6alkylene can be for example ​

[0049] In a preferred embodiment, R A-1 In a preferred embodiment, the C2-C6alkylene group can be For example

[0050] In a preferred embodiment, R A-2 In a preferred embodiment, the C2-C6alkylene group can be For example In a preferred embodiment, the C1-C6alkylene group can be

[0051] For example In a preferred embodiment, R 1 In a preferred embodiment, the C6-C 20 alkyl group can be C 10 -C 18 For example

[0052] In a preferred embodiment, R 1 In a preferred embodiment, the C6-C 20 alkyl group can be C 10 -C 19 For example

[0053] In a preferred embodiment, R 2 In a preferred embodiment, the C6-C 20 alkyl group can be C 10 -C 18 For example

[0054] In a preferred embodiment, R 2 In a preferred embodiment, the C6-C 20 alkyl group can be C 10 -C 19 For example

[0055] In a preferred embodiment, R 5 In a preferred embodiment, the C2-C 10 alkyl group can be C2-C8alkyl, for example

[0056] Also for example

[0057] In a preferred embodiment, R 5-1-1 In a preferred embodiment, the C6-C 20 In a preferred embodiment, the C6-C 11 In a preferred embodiment, the C6-C 18 For example, the C6-C

[0058] In a preferred embodiment, R 6 In a preferred embodiment, the C2-C 10 In a preferred embodiment, the C2-C

[0059] For example, the C2-C

[0060] In a preferred embodiment, R 6-1-1 In a preferred embodiment, the C6-C 20 In a preferred embodiment, the C6-C 11 In a preferred embodiment, the C6-C 18 For example, the C6-C

[0061] In a preferred embodiment, the nitrogen-containing chain compound of Formula I is a nitrogen-containing chain compound of Formula I-a

[0062]

[0063] In a preferred embodiment, Y is wherein a is attached to R 2 and b is attached to Z.

[0064] In a preferred embodiment, Q is wherein a is attached to R 1 and b is attached to W.

[0065] In a preferred embodiment, Q and Y are the same.

[0066] In a preferred embodiment, Z and W are the same.

[0067] In a preferred embodiment, R 1 and R 2 are the same.

[0068] In a preferred embodiment, R 5 and R 6 are the same.

[0069] In a preferred embodiment, Z and W are independently C5-C8alkylene.

[0070] In a preferred embodiment, Z and W are independently C3-C8alkylene.

[0071] In a preferred embodiment, A is C2-C6alkylene or In a preferred embodiment, R A-1 and R A-2 are independently C2-C4alkylene.

[0072] In a preferred embodiment, M is methylene.

[0073] In a preferred embodiment, R 1 and R 2 are independently C 10 -C 18 , for example C 10 -C 12 , and also for example

[0074]

[0075] In a preferred embodiment, R 1 and R 2 are independently C 10 -C 20 alkyl, preferably

[0076] more preferably

[0077]

[0078] In a preferred embodiment, R 5 is C2-C8alkyl substituted with 1, 2, or 3 R 5-1 .

[0079] In a preferred embodiment, R 5-1-1 is C 10 -C 18 alkyl, for example C 14 -C 18 alkyl, and also for example In a preferred embodiment, R 6 is C2-C8alkyl substituted with 1, 2, or 3 R 6-1 .

[0080] In a preferred embodiment, R 6-1-1 is C 10 -C 18 alkyl, for example C 14 -C 18 alkyl, and also for example In a preferred embodiment, Y is wherein a is attached to R 2 b is attached to Z; Q is wherein a is attached to R 1 b is attached to W;

[0081] Z and W are independently C5-C8 alkylene;

[0082] A is C2-C6 alkylene or R A-1 and R A-2 are independently C2-C4 alkylene;

[0083] M is methylene;

[0084] R 1 and R 2 are independently C 10 -C 20 alkyl;

[0085] R 5 is C2-C8 alkyl substituted with 1, 2, or 3 R 5-1 ; and

[0086] R 5-1-1 is C 10 -C 18 alkyl;

[0087] R 6 is C2-C8 alkyl substituted with 1, 2, or 3 R 6-1 ; and

[0088] R 6-1-1 is C 10 -C 18 alkyl.

[0089] In a certain preferred embodiment, Y is wherein a is attached to R 2 b is attached to Z; Q is wherein a is attached to R 1 b is attached to W;

[0090] Z and W are independently C5-C8 alkylene;

[0091] A is C2-C6 alkylene or R A-1 and R A-2 are independently C2-C4 alkylene;

[0092] M is methylene;

[0093] R 1 and R 2 are independently C 10 -C18 ;

[0094] R 5 For 1, 2 or 3 R 5-1 Substituted C2-C8 alkyl groups;

[0095] R 5-1-1 C 10 -C 18 Alkyl groups;

[0096] R 6 For 1, 2 or 3 R 6-1 Substituted C2-C8 alkyl groups;

[0097] R 6-1-1 C 10 -C 18 Alkyl groups.

[0098] In a certain preferred solution, Q and Y are the same;

[0099] Z and W are the same;

[0100] R 1 and R 2 same;

[0101] R 5 and R 6 same;

[0102] Z and W are independently C5-C8 alkylene groups;

[0103] A is a C2-C6 alkylene group or R A-1 and R A-2 Independently a C2-C4 alkylene group;

[0104] M stands for methylene;

[0105] R 1 and R 2 Independently R 5 For 1, 2 or 3 R 5-1 Substituted C2-C8 alkyl groups;

[0106] R 5-1-1 C 14 -C 18 Alkyl groups;

[0107] R 6 For 1, 2 or 3 R 6-1 Substituted C2-C8 alkyl groups;

[0108] R 6-1-1 C 14 -C18 Alkyl groups.

[0109] In a preferred embodiment, the nitrogen-containing chain compound as shown in Formula I is a compound with bilateral symmetry.

[0110] In a certain preferred solution, Z can be: In a certain preferred solution, Z can be:

[0111] In a certain preferred solution, W can be In a certain preferred solution, W can be

[0112] In a certain preferred solution, R 1 Can be In a certain preferred solution, R 1 Can be

[0113] In a certain preferred solution, R 2 Can be In a certain preferred solution, R 2 Can be

[0114] In a certain preferred solution, R 5 Can be

[0115] In a certain preferred solution, R 6 Can be

[0116] In a certain preferred solution, A can be:

[0117] In a certain preferred solution, A can be:

[0118] In a preferred embodiment, the nitrogen-containing chain compound as shown in Formula I is any of the following compounds:

[0119]

[0120]

[0121]

[0122] The present application also provides a preparation method of the nitrogen-containing chain compound as shown in Formula I, which comprises the following steps: in a solvent, a compound as shown in Formula I-1 is coupled with a compound as shown in Formula I-2 in the presence of a base and an iodine salt, i.e.

[0123]

[0124] X is halogen, A is C2-C6 alkylene, Y, Q, Z, W, R 5 , R 6 , R 1 and R 2 are the same as described above, and Y is the same as Q, R 1 is the same as R 2 , and Z is the same as W.

[0125] In the coupling reaction, the halogen can be fluorine, chlorine, bromine or iodine, for example, bromine.

[0126] In the coupling reaction, the molar ratio of the compound as shown in Formula I-2 to the compound as shown in Formula I-2 can be 1:(1-3), for example, 1:2.6.

[0127] In the coupling reaction, the base can be a conventional base in the art. The base can be an alkaline carbonate (the cation in the salt is an alkali metal ion, and the anion is carbonate), for example, K2CO3.

[0128] In the coupling reaction, the molar ratio of the compound as shown in Formula I-2 to the base can be 1:(1-5); for example, 1:3.5.

[0129] In the coupling reaction, the solvent can be a conventional solvent in the art, and the solvent can be an ether solvent or / and a nitrile solvent. The ether solvent can be methyl tert-butyl ether. The nitrile solvent can be acetonitrile. The volume ratio of the nitrile solvent to the ether solvent can be 1:1.

[0130] In the coupling reaction, the mass-volume ratio of the compound as shown in Formula I-2 to the solvent can be 10-50 mg / mL; for example, 16 mg / mL.

[0131] In the coupling reaction, the iodine salt can be a conventional iodine salt in the art. The iodine salt can be an alkaline iodine salt, for example, KI.

[0132] In the coupling reaction, the molar ratio of the compound as shown in Formula I-2 to the iodine salt can be 1:(1-2); for example, 1:1.2.

[0133] In the coupling reaction, the reaction temperature of the coupling reaction can be a conventional reaction temperature in the art, and is preferably 50-100℃, for example, 80℃.

[0134] The present application also provides a lipid carrier comprising a substance Z, which is a compound of formula I or a pharmaceutically acceptable salt thereof as described above.

[0135] In a preferred embodiment, the lipid carrier further comprises a diluent. The diluent can be a phosphate buffer or a Tris buffer, etc.

[0136] In a preferred embodiment, the lipid carrier further comprises a phospholipid.

[0137] In a preferred embodiment, the phospholipid can be a conventional phospholipid in the art, which is an amphipathic auxiliary molecule, which helps the fusion of lipid particles and cell membranes. The phospholipid can be a phospholipid molecule having a charged polar end and a non-polar end of a fatty chain, such as distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoylphosphatidylcholine (DPPC), 1,2-distearoylphosphatidylcholine (DSPC), heneicosanoylphosphatidylcholine (DUPC), or palmitoylphosphatidylcholine (POPC), etc.

[0138] In a preferred embodiment, the lipid carrier further comprises a PEG lipid (polyethylene glycol modified lipid).

[0139] In a preferred embodiment, the PEG lipid can be a lipid molecule modified with a hydrophilic end of polyethylene glycol. The PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, such as PEG-modified dimyristoylglycerol (DMG-PEG2000), etc.

[0140] In a preferred embodiment, the lipid carrier further comprises a sterol.

[0141] In a preferred embodiment, the sterol can be a conventional sterol in the art, which includes animal, plant or fungal sterol. The sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and a-tocopherol, such as cholesterol, etc.

[0142] In a preferred embodiment, in the lipid carrier, the molar ratio of the substance Z to the sterol is 0.5-5:1, preferably 0.5-3:1, for example 0.6-2:1.

[0143] In a preferred embodiment, the molar ratio of the substance Z to the sterol in the lipid carrier is 0.5-5:1, preferably 0.5-3:1, such as 0.68:1, 0.69:1, 0.71 :1, 0.74:1, 0.76:1, 0.77:1, 0.79:1, 0.83:1, 0.84:1, 0.85:1, 0.86:1, 0.88:1, 0.89:1, 0.9:1, 0.91 :1, 0.94:1, 0.99:1, 1.04:1, 1.07:1, or 1.28:1.

[0144] In a preferred embodiment, the molar ratio of the substance Z to the sterol in the lipid carrier is 0.5-5:1, preferably 0.5-3:1, such as 0.6-2:1 ; such as 0.66:1, 0.68:1, 0.69:1, 0.70:1, 0.71 :1, 0.72:1, 0.74:1, 0.76:1, 0.77:1, 0.79:1, 0.82:1, 0.83:1, 0.84:1, 0.85:1, 0.86:1, 0.87:1, 0.88:1, 0.89:1, 0.9:1, 0.91 :1, 0.92:1, 0.93:1, 0.94:1, 0.97:1, 0.99:1, 1.04:1, 1.07:1, 1.1 :1, 1.16:1, 1.23:1, 1.28:1, 1.30:1, 1.32:1,

[0145] 1.41 :1, 1.52:1, 1.58:1, 1.64:1, 1.65:1, 1.74:1, 1.79:1, or 1.96:1.

[0146] In a preferred embodiment, the molar ratio of the substance Z to the phospholipid in the lipid carrier is 1 -15:1, preferably 2-8:1, such as 3-6:1.

[0147] In a preferred embodiment, the molar ratio of the substance Z to the phospholipid in the lipid carrier is 1 -25:1, preferably 2-25:1, such as 22.5:1, 20:1, 17.5:1, 15:1, 1 1.25:1, 10:1, 8.75:1, 7.5:1, 6.67:1, 5:1, 4.75:1, 4.5:1, 4:1, 3.9:1, 3.6:1, 3.3:1, 3:1, 2.86:1, 2.5:1, or 2.2:1.

[0148] In a preferred embodiment, the molar ratio of the substance Z to the PEG lipid in the lipid carrier is 20-130:1, preferably 20-80:1, such as 20-40:1.

[0149] In a preferred embodiment, the molar ratio of the substance Z to the PEG lipid in the lipid carrier is 16-130: 1, preferably 16-80: 1, for example 16-40: 1; for example 16: 1, 18: 1, 18.8: 1, 20: 1, 21.9: 1, 22.5: 1, 25: 1, 26.9: 1, 27.5: 1, 28.1: 1, 29.6: 1, 30: 1, 31.25: 1, 33.3: 1 or 37.5: 1.

[0150] In a preferred embodiment, the molar ratio of the substance Z to the PEG lipid in the lipid carrier is 16-130: 1, preferably 16-80: 1, for example 16-40: 1; for example 16: 1, 18: 1, 18.8: 1, 20: 1, 21.9: 1, 22.5: 1, 25: 1, 26.9: 1, 27.5: 1, 28.1: 1, 29.6: 1, 30: 1, 31.25: 1, 33.3: 1 or 37.5: 1.

[0151] In a preferred embodiment, the molar content of the substance Z is about 30 mol% to 60 mol%.

[0152] In a preferred embodiment, the molar content of the substance Z is about 30 mol% to 60 mol%; preferably 40 mol% to 55 mol%; for example 40 mol%, 43 mol%, 45 mol%, 47.4 mol%, 50 mol%, 50 mol% or 55 mol%.

[0153] In the present application, the molar content means the percentage of a substance in the total mass of the substances in the lipid carrier, the sum of the molar content of each component in the lipid carrier does not exceed 100 mol%. In a preferred embodiment, the molar content of the phospholipid is about 0 mol% to 30 mol%.

[0154] In a preferred embodiment, the molar content of the phospholipid is about 0 mol% to 30 mol%; preferably 0 mol% to 18 mol%; for example 0 mol%, 2 mol%, 4 mol%, 6 mol%, 8 mol%, 10 mol%, 11 mol%, 12 mol%, 14 mol%, 16 mol% or 18 mol%.

[0155] In a preferred embodiment, the molar content of the sterol is about 15 mol% to 55 mol%.

[0156] In a preferred embodiment, the molar content of the sterol is about 15 mol% to 60 mol%, preferably 40.4% mol% to 58.4 mol%, for example 42.4 mol%, 44.4 mol%, 46.4 mol%, 48.4 mol%, 50.4 mol%, 52.4 mol% or 56.4 mol%.

[0157] In a preferred embodiment, the molar content of the sterol is about 15 mol% to 60 mol%, preferably 40.4% mol% to 58.4 mol%, for example 40.4 mol%, 41 mol%, 42.4 mol%, 43 mol%, 43.4 mol%, 44.4 mol%, 46.4 mol%, 47.4 mol%, 48 mol%, 48.4 mol%, 49 mol%, 49.4 mol%, 49.5 mol%, 50 mol%, 50.4 mol%, 50.5 mol%, 51 mol%, 51.4 mol%, 51.5 mol%, 52 mol%, 52.25 mol%, 52.5 mol%, 52.75 mol%, 53 mol%, 53.4 mol%, 54 mol%, 54.25 mol%, 54.4 mol%, 54.5 mol%, 54.75 mol%, 55 mol%, 56 mol%, 56.4 mol%, 56.5 mol%, 57 mol%, 57.5 mol%, 58 mol%, or 58.4 mol%.

[0158] In a preferred embodiment, when the lipid carrier does not comprise a phospholipid, the molar content of the sterol in the lipid carrier is about 15 mol% to 60 mol%, preferably 40.4% mol% to 58.4 mol%, for example 43 mol%, 43.4 mol%, 44.4 mol%, 46.4 mol%, 47.4 mol%, 48 mol%, 48.4 mol%, 49 mol%, 49.4 mol%, 49.5 mol%, 50 mol%, 50.4 mol%, 50.5 mol%, 51 mol%, 51.4 mol%, 51.5 mol%, 52 mol%, 52.4 mol%, 52.25 mol%, 52.5 mol%, 52.75 mol%, 53 mol%, 53.4 mol%, 54 mol%, 54.25 mol%, 54.4 mol%, 54.5 mol%, 54.75 mol%, 55 mol%, 56 mol%, 56.4 mol%, 56.5 mol%, 57 mol%, 57.5 mol%, 58 mol%, or 58.4 mol%; for example 52.5 mol% to 54.5 mol%, for example 53 mol% to 54.5 mol%.

[0159] In a preferred embodiment, the molar content of the PEG lipid is about 0 mol% to 10 mol%.

[0160] In a preferred embodiment, the molar content of the PEG lipid is about 0 mol% to 10 mol%, for example 1.5 mol% to 2.5 mol%; for example 1.6 mol% or 2 mol%.

[0161] In a preferred embodiment, the molar content of the PEG lipid is about 0 mol% to 10 mol%, which can be 0.5 mol% to 2.5 mol%, and further can be 0.5 mol% to 1.5 mol% or 1.5 mol% to 2.5 mol%, for example 1.6 mol% or 2 mol%.

[0162] In a preferred embodiment, the molar content of the PEG lipid is about 0 mol% to 10 mol%, for example 0.5 mol% to 2.5 mol%, in particular, for example 0.25 mol%, 0.5 mol%, 0.75 mol%, 1 mol%, 1.5 mol%, 1.6 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol% or 5 mol%; further 0.5 mol% to 2 mol%; and further can be 0.5 mol% to 1.5 mol% or 1.5 mol% to 2.5 mol%; and further can be 1.6 mol% or 2 mol%.

[0163] In a preferred embodiment, the molar content of the PEG lipid is about 0 mol% to 10 mol%, in particular, for example 0.25 mol%, 0.5 mol%, 0.75 mol%, 1 mol%, 1.5 mol%, 1.6 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol% or 5 mol%, when the lipid carrier does not comprise a phospholipid, or the content of the phospholipid is 4 mol% or less; for example 0.25 mol% to 3 mol%, further 0.5 mol% to 2.5 mol%, and further 0.5 mol% to 2 mol%.

[0164] In a preferred embodiment, the lipid carrier consists of the substance Z, the diluent, the phospholipid, the PEG lipid and the sterol.

[0165] In a preferred embodiment, the lipid carrier consists of the substance Z, the phospholipid, the PEG lipid and the sterol.

[0166] In a preferred embodiment, the lipid carrier consists of the substance Z, the diluent, the PEG lipid and the sterol.

[0167] In a preferred embodiment, the lipid carrier consists of the substance Z, the PEG lipid and the sterol.

[0168] In a preferred embodiment, the lipid carrier does not comprise a phospholipid.

[0169] In a preferred embodiment, when the lipid carrier does not comprise a phospholipid, the molar ratio of the substance Z to the sterol in the lipid carrier can be 0.6-2: 1; preferably 0.68: 1, 0.69: 1, 0.7: 1, 0.77: 1, 0.85: 1, 0.86: 1, 1.04: 1, or 1.28: 1.

[0170] In a preferred embodiment, when the lipid carrier does not comprise a phospholipid, the molar ratio of the substance Z to the sterol in the lipid carrier can be 0.6-2: 1; preferably 0.68: 1, 0.69: 1, 0.7: 1, 0.77: 1, 0.85: 1, 0.86: 1, 1.04: 1, or 1.28: 1.

[0171] In a preferred embodiment, when the lipid carrier does not comprise a phospholipid, the molar ratio of the substance Z to the PEG lipid in the lipid carrier can be 16-35: 1; preferably 16: 1, 18: 1, 20: 1, 22.5: 1, 25: 1, 27.5: 1, or 28.1: 1.

[0172] In a preferred embodiment, when the lipid carrier does not comprise a phospholipid, the molar ratio of the substance Z to the PEG lipid in the lipid carrier can be 16-35: 1; again for example 16: 1, 18: 1, 20: 1, 21.9: 1, 22.5: 1, 25: 1, 26.9: 1, 27.5: 1, 28.1: 1, 29.6: 1, or 30: 1.

[0173] The present application also provides a lipid nanoparticle comprising a therapeutic and / or prophylactic agent and the aforementioned lipid carrier.

[0174] In a preferred embodiment, the therapeutic and / or prophylactic agent can be one or more than two nucleic acids. The nucleic acid can be a conventional nucleic acid in the art. The therapeutic and / or prophylactic agent can be a single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), asymmetric double-stranded small interfering RNA (aiRNA), microRNA (miRNA), small hairpin RNA (shRNA), circular RNA (circRNA), transfer RNA (tRNA), messenger RNA (mRNA), and other forms of nucleic acid molecules known in the art, preferably mRNA, such as firefly luciferase (Fluc) mRNA or SARS-CoV-2 Spike mRNA.

[0175] In a preferred embodiment, the nitrogen to phosphorus ratio in the lipid nanoparticle can be 2:1-30:1, the nitrogen to phosphorus ratio of the composition refers to the ratio of the moles of ionizable nitrogen atoms in the ionizable lipid compound(s) to the moles of phosphate groups in the RNA. Preferably 2:1-20:1, such as 3:1-20:1, further such as 3:1-16:1.

[0176] In a preferred embodiment, the mass ratio of the lipid carrier to the therapeutic and / or prophylactic agent in the lipid nanoparticle can be 3-80:1, preferably 6-60:1.

[0177] In a preferred embodiment, the particle size (average particle size) of the lipid nanoparticle can be 10-200 nm, preferably 40-150 nm, such as 60-150 nm.

[0178] In a preferred embodiment, the particle size (average particle size) of the lipid nanoparticle can be 10-200 nm, preferably 40-150 nm, such as 60-150 nm; further such as 50-150 nm.

[0179] In a preferred embodiment, the lipid carrier encapsulates the therapeutic and / or prophylactic agent in the lipid nanoparticle.

[0180] The present application also provides a composition comprising a substance Z, the substance Z being a compound as shown in formula I or a pharmaceutically acceptable salt thereof as described previously.

[0181] In a preferred embodiment, the composition further comprises one or more of a diluent, a phospholipid, a PEG lipid, a sterol, and a therapeutic and / or prophylactic agent.

[0182] In a preferred embodiment, the diluent, the phospholipid, the PEG lipid, the sterol, and the therapeutic and / or prophylactic agent in the composition are as described previously.

[0183] In a preferred embodiment, in the composition, the substance Z forms a lipid carrier as described above with one or more of the diluent, the phospholipid, the PEG lipid and the sterol.

[0184] In a preferred embodiment, in the composition, the lipid carrier forms a lipid nanoparticle as described above with the therapeutic and / or prophylactic agent. In a preferred embodiment, in the composition, the encapsulation efficiency of the therapeutic and / or prophylactic agent is at least 50%, preferably at least 70%.

[0185] In a preferred embodiment, in the composition, the polydispersity index of the composition is not higher than 0.5, for example not higher than 0.3.

[0186] The terms used in the present application have the following meanings, unless otherwise specified:

[0187] The term "one or more" means 1, 2 or 3.

[0188] The term "halogen" means fluorine, chlorine, bromine or iodine.

[0189] The term "pharmaceutically acceptable" means relatively non-toxic, safe, suitable for use in patients.

[0190] The term "pharmaceutically acceptable salt" means a salt of a compound with a pharmaceutically acceptable acid or base. When a compound contains relatively acidic functionalities, base addition salts can be obtained by contacting the compound with a sufficient amount of the pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to sodium, potassium, calcium, aluminum, magnesium, bismuth, ammonium, and quaternary ammonium salts. When a compound contains relatively basic functionalities, acid addition salts can be obtained by contacting the compound with a sufficient amount of the pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to hydrochloride, sulfate, methanesulfonate, and the like. See, e.g., Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0191] In a structural fragment, means that the structural fragment is connected to the rest of the molecule through the site. For example, means cyclohexyl.

[0192] The "-" at the end of a group means that the group is connected to the rest of the molecule through the site. For example, CH3-C(=O)- means acetyl.

[0193] The term "alkyl" refers to a straight or branched chain, saturated, monovalent hydrocarbon radical having the indicated number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, and the like.

[0194] The term "alkylene" refers to a divalent radical, which is attached to the rest of the molecule by two single bonds, having the remaining definition of the term "alkyl".

[0195] The term "alkoxy" refers to the group R X -O-, R X The term "alkyl" refers to a straight or branched chain, saturated, monovalent hydrocarbon radical having the indicated number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, and the like.

[0196] The above preferred conditions can be combined in any manner without violating the common knowledge in the art, thus obtaining various preferred embodiments of the present application

[0197] The reagents and raw materials used in the present application are commercially available.

[0198] The positive progress effect of the present application is that the present application provides a nitrogen-containing chain compound as shown in Formula I, which has a novel structure and can be used for preparing lipid nanoparticles. The lipid nanoparticles containing the nitrogen-containing chain compound as shown in Formula I have a low polydispersity index and can efficiently deliver mRNA. When the nitrogen-containing chain compound of the present application is used for preparing LNP preparations, the LNP preparations still have good properties and delivery capacity even if the phospholipid component is as low as 4 mol% or less. Further, when the nitrogen-containing chain compound of the present application is used for preparing LNP preparations, the LNP preparations prepared under the condition of low PEG lipid still have good properties and delivery capacity, thereby reducing the risk of affecting the efficacy and safety due to high PEG lipid. BRIEF DESCRIPTION OF DRAWINGS

[0199] Figure 1 The nucleic acid gel electrophoresis result diagram of each LNP preparation prepared in Example 1;

[0200] Figure 2 The chemiluminescence intensity measured after the 293FT cells in Example 2 were co-cultured with the LNP preparations prepared in Example 1 for 18-24 hours;

[0201] Figure 3 and Figure 4A The total in vivo bioluminescence measured at different times after the mice in Example 3 were intravenously administered with the LNP preparations LQ104-1 to LQ104-8 prepared in Example 1;

[0202] Figure 4BIn vivo total antibody titers measured after intramuscular injection of LNP formulations LQ104-9, LQ104-10, or LQ107 in mice in Example 3;

[0203] Figures 5A to 5D In vivo total bioluminescence or total luminescence at the site of administration measured at various times after intravenous administration ( Figure 5A , Figure 5B ) or intramuscular administration ( Figure 5C , Figure 5D ) of each LNP formulation in Example 4 to mice;

[0204] Figures 6A to 6C In vivo total bioluminescence measured at various times after intravenous administration of each LNP formulation in Example 5 to mice;

[0205] Figure 7A In vivo total bioluminescence measured at various times after intravenous administration of each LNP formulation in Example 6 to mice; Figure 7B

[0206] In vivo total bioluminescence measured at various times after intravenous administration of each LNP formulation in Example 7 to mice; Figure 8

[0207] In vivo total bioluminescence measured at various times after intravenous administration of each LNP formulation in Example 8 to mice; Figure 9A Figure 9B In vivo total bioluminescence measured at various times after intravenous administration of each LNP formulation in Example 10 to mice;

[0208] Figures 10A to 10C In vivo total bioluminescence measured at various times after intravenous administration of each LNP formulation in Example 11 to mice;

[0209] Figures 11A to 11C In vivo total bioluminescence or total luminescence at the site of administration measured at various times after intravenous administration (

[0210] , Figures 12A to 12D ) or intramuscular administration ( Figure 12A , Figure 12B ) of each LNP formulation in Example 12 to mice. Figure 12C Figure 12D In vivo total bioluminescence measured at various times after intravenous administration of each LNP formulation in Example 13 to mice.

[0211] DETAILED DESCRIPTION Figure 13A Figure 13B In vivo total bioluminescence measured at various times after intravenous administration of each LNP formulation in Example 13 to mice. DETAILED DESCRIPTION

[0212] ​The application will be further described in the following by way of examples without limiting the application to the examples described. The experimental methods in the following examples, if not specified, are selected according to the conventional methods and conditions, or according to the commercial instruction.

[0213] Preparation of compound LQ104

[0214]

[0215] Preparation of LQ104

[0216] Material ratio:

[0217]

[0218]

[0219] Operation process:

[0220] LQ001-1, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI, methyl tert-butyl ether and acetonitrile were added into a reaction bottle, heated to 80°C and stirred for 12h. TLC (DCM:MeOH=10:1) showed that the reaction was complete.

[0221] Post-treatment:

[0222] The reaction solution was filtered and rotary evaporated, and 1.1g of colorless oil was obtained after column chromatography purification.

[0223] 1 HNMR (400MHz, CDCI3) δ: 4.86 (p, 2H), 3.65-3.59 (m, 4H), 2.68-2.59 (m, 8H), 2.57-2.49 (m, 4H), 2.27 (t, 4H), 1.61 (p, 5H), 1.49 (dt, 12H), 1.28 (d, 61H), 0.87 (t, 12H).

[0224] Preparation of compound LQ107

[0225] Reaction formula:

[0226]

[0227] Material ratio:

[0228] Material Name Molecular Weight Charge Ratio Charge Amount mmol LQ107-1 709 2.1 eq 1g 1.41 Malonic acid 104 1 eq 70 mg 0.67 DCC 206 2.5 eq 350 mg 1.68 DMAP 122 0.2 eq 20 mg 0.14 DCM - - 20ml -

[0229] Operation process:

[0230] Into a 1 L reaction flask was added LQ107-1, malonic acid, DCC, DMAP and DCM. The reaction was stirred at room temperature for 12 h. TLC (DCM:MeOH = 20:1) showed the reaction was complete.

[0231] Work-up:

[0232] The reaction solution was filtered through celite and rotary evaporated. Purification by column chromatography gave 700 mg of colorless oil in 70% yield.

[0233] 1 HNMR (400 MHz, CDC13) δ: 4.86 (p, 2H), 4.07 (dt, 8H), 2.66 (t, 4H), 2.49-2.38 (m, 8H), 2.28 (q, 8H), 2.05 (s, 5H), 1.62 (q, 17H), 1.83-1.36 (m, 17H), 1.27 (d, 90H), 0.87 (t, 18H).

[0234] Preparation of compound LQ104-E15b-1

[0235] Preparation of E15b-1

[0236] Reaction scheme:

[0237]

[0238] Material ratio:

[0239] Material Name Molecular Weight Charge Ratio Charge Amount mmol 8-Pentadecanol 228.42 1 eq 11.4g 50 6-Bromohexanoic acid 195.06 1.1 eq 10.7g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0240] Operation process:

[0241] Into a 1 L reaction flask was added 6-bromohexanoic acid, DCC, DMAP and DCM. Then 8-pentadecanol was added. The reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction was complete (product rf value was 0.6).

[0242] Work-up:

[0243] The reaction solution was filtered and rotary evaporated. Purification by column chromatography gave 15 g of colorless oil.

[0244] Preparation of LQ104-E15b-1

[0245] Reaction scheme:

[0246]

[0247] Material ratio:

[0248]

[0249] Operation process:

[0250] To a reaction flask was added E15b-1, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, heated to 80 °C and stirred for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction to be complete (product rf = 0.5).

[0251] Work-up:

[0252] The reaction solution was filtered and rotary evaporated, and 800 mg of colorless oil was obtained after purification by column chromatography.

[0253] 1 H NMR (600 MHz, CDC13) δ: 4.86 (p, J = 6.2 Hz, 2H), 3.62 (t, J = 4.9 Hz, 4H), 2.67 - 2.59 (m, 8H), 2.55 (t, J = 8.0 Hz, 4H), 2.28 (t, J = 7.5 Hz, 4H), 1.64 (p, J = 7.5 Hz, 4H), 1.50 (tt, J = 8.5, 4.5 Hz, 12H), 1.34 - 1.20 (m, 46H), 0.87 (t, J = 7.0 Hz, 12H).

[0254] Preparation of compound LQ104-E15b-2

[0255] Preparation of E15b-2

[0256] Reaction scheme:

[0257]

[0258] Material ratio:

[0259] Material Name Molecular Weight Charge Ratio Charge Amount mmol 9-Heptadecanol 256.47 1 eq 12.8g 50 5-Bromopentanoic acid 181 1.1 eq 9.96g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0260] Operation process:

[0261] To a 1 L reaction flask was added 5-bromopentanoic acid, DCC, DMAP and DCM, and then 9-heptadecanol was added. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction to be complete (product rf = 0.6).

[0262] Work-up:

[0263] The reaction solution was filtered and rotary evaporated, and 13.8 g of colorless oil was obtained after purification by column chromatography.

[0264] Preparation of LQ104-E15b-2

[0265] Reaction scheme:

[0266]

[0267] Material ratio:

[0268]

[0269]

[0270] Operation process:

[0271] Into a 1L reaction flask was added E15b-2, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, heated to 80°C and stirred for 12h. TLC (DCM:MeOH = 10:1) showed the reaction was complete (product rf value was 0.5).

[0272] Post-processing:

[0273] The reaction solution was filtered and rotary evaporated, and 760mg of colorless oil was obtained after column chromatography purification.

[0274] 1 H NMR (600 MHz, CDCl3) δ: 4.85 (p, J = 6.2 Hz, 2H), 3.70 (t, J = 4.9 Hz, 4H), 2.85-2.69 (m, 12H), 2.32 (t, J = 7.0 Hz, 4H), 1.65-1.54 (m, 9H), 1.49 (q, J = 6.4 Hz, 8H), 1.25 (d, J = 9.9 Hz, 50H), 0.87 (t, J = 7.0 Hz, 12H).

[0275] Preparation of compound LQ104-E15b-3

[0276] Preparation of E15b-3

[0277] Reaction formula:

[0278]

[0279] Material ratio:

[0280] Material Name Molecular Weight Charge Ratio Charge Amount mmol 10-Nonadecanol 284.5 1 eq 14.2g 50 4-Bromobutanoic acid 167 1.1 eq 9.2g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0281] Operation process:

[0282] Into a 1L reaction flask was added 4-bromobutyric acid, DCC, DMAP and acetonitrile, then 10-nonadecanol was added, and after completion, it was stirred at room temperature for 12h. TLC (PE:EA = 20:1) showed that the reaction was complete (product rf value was 0.6).

[0283] Post-processing:

[0284] The reaction solution was filtered and rotary evaporated, and 760mg of colorless oil was obtained after column chromatography purification.

[0285] Preparation of LQ104-E15b-3

[0286] Reaction Scheme:

[0287]

[0288] Material ratio:

[0289] Material Name Molecular Weight Charge Ratio Charge Amount mmol E15b-3 433.5 2.5 eq 2.17g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 [K2CO3] 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 Acetonitrile 40ml

[0290] Operation process:

[0291] Into a 1 L reaction flask was added E15b-3, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, and heated to 80 °C and stirred for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction was complete (product rf value was 0.5).

[0292] Post-treatment:

[0293] After the reaction solution was filtered and rotary evaporated, 820 mg of colorless oil was obtained after column chromatography purification.

[0294] 1 H NMR (600 MHz, CDC13) δ: 4.85 (p, J = 6.2 Hz, 2H), 3.69 (t, J = 4.8 Hz, 4H), 2.76 (s, 8H), 2.66 (t, J = 8.2 Hz, 4H), 2.27 (t, J = 7.5 Hz, 4H), 1.61 (p, J = 7.3 Hz, 4H), 1.50 (dq, J = 12.3, 6.4, 5.3 Hz, 12H), 1.35 - 1.20 (m, 54H), 0.87 (t, J = 7.0 Hz, 12H).

[0295] Preparation of compound LQ104-E16b-1

[0296] Preparation of E16b-1

[0297] Reaction Scheme:

[0298]

[0299] Material ratio:

[0300] Material Name Molecular Weight Charge Ratio Charge Amount mmol 8-Pentadecanol 228.42 1 eq 11.4g 50 7-Bromoheptanoic acid 209 1.1 eq 11.5g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0301] Operation process:

[0302] Into a 1 L reaction flask was added 7-bromoheptanoic acid, DCC, DMAP and DCM, and then 8-pentadecanol was added. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction was complete (product rf value was 0.6).

[0303] Work-up:

[0304] The reaction solution was filtered and rotary evaporated. After column chromatography purification, 14.5 g of colorless oil was obtained.

[0305] Preparation of LQ104-E16b-1

[0306] Reaction Scheme:

[0307]

[0308] Material ratio:

[0309] Material Name Molecular Weight Charge Ratio Charge Amount mmol E16b-1 419.5 2.5 eq 2.1g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 K2CO3 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 Acetonitrile 40ml

[0310] Operation process:

[0311] Into a reaction flask was added E16b-1, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile. The mixture was heated to 80 °C and stirred for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction was complete (product rf value was 0.5).

[0312] Work-up:

[0313] The reaction solution was filtered and rotary evaporated. After column chromatography purification, 730 mg of colorless oil was obtained.

[0314] 1 H NMR (600 MHz, CDC13) δ: 4.85 (p, J = 6.2 Hz, 2H), 3.61 (t, J = 4.9 Hz, 4H), 2.67 - 2.60 (m, 8H), 2.54 (t, J = 7.9 Hz, 4H), 2.27 (t, J = 7.5 Hz, 4H), 1.61 (p, J = 7.5 Hz, 4H), 1.50 (qd, J = 7.6, 3.4 Hz, 12H), 1.37 - 1.20 (m, 50H), 0.87 (t, J = 7.0 Hz, 12H).

[0315] Preparation of compound LQ104-E16b-2 in Preparation Example 7

[0316] Preparation of E16b-2

[0317] Reaction Scheme:

[0318]

[0319] Material ratio:

[0320]

[0321]

[0322] Procedure:

[0323] Into a 1 L reaction flask was added 6-bromohexanoic acid, DCC, DMAP and DCM, followed by 9-heptadecanol. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction to be complete (product rf value 0.6).

[0324] Work-up:

[0325] The reaction mixture was filtered and dried in vacuo. Purification by column chromatography gave 14.4 g of colorless oil.

[0326] Preparation of LQ104-E16b-2

[0327] Reaction Scheme:

[0328]

[0329] Material ratio:

[0330] Material Name Molecular Weight Charge Ratio Charge Amount mmol E16b-2 433.5 2.5 eq 2.17g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 [K2CO3] 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 Acetonitrile 40ml

[0331] Procedure:

[0332] Into a reaction flask was added E16b-2, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile. The reaction was stirred at 80 °C for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction to be complete (product rf value 0.5).

[0333] Work-up:

[0334] The reaction mixture was filtered and dried in vacuo. Purification by column chromatography gave 770 mg of colorless oil.

[0335] 1 H NMR (600 MHz, CDC13) δ: 4.85 (p, J = 6.2 Hz, 2H), 3.65 (t, J = 4.8 Hz, 4H), 2.73 - 2.65 (m, 8H), 2.61 (t, J = 8.1 Hz, 4H), 2.28 (t, J = 7.4 Hz, 4H), 1.64 (p, J = 7.5 Hz, 4H), 1.51 (dq, J = 19.0, 7.3, 6.8 Hz, 12H), 1.35 - 1.20 (m, 54H), 0.87 (t, J = 6.9 Hz, 12H).

[0336] Preparation of compound LQ104-E16b-3 according to Preparation Example 8

[0337] Preparation of E16b-3

[0338] Reaction Scheme:

[0339]

[0340] Material ratio:

[0341] Material Name Molecular Weight Charge Ratio Charge Amount mmol 7-Pentadecanol 228.42 1 eq 11.4g 50 6-Bromohexanoic acid 195.06 1.1 eq 10.7g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0342] Operation process:

[0343] Into a 1 L reaction flask was added 6-bromohexanoic acid, DCC, DMAP and DCM, then 7-pentadecanol was added. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction to be complete (product rf value 0.6).

[0344] Work-up:

[0345] The reaction solution was filtered and rotary evaporated. Purification by column chromatography gave 13.3 g of colorless oil.

[0346] Preparation of LQ104-E16b-3

[0347] Reaction scheme:

[0348]

[0349] Material ratio:

[0350] Material Name Molecular Weight Charge Ratio Charge Amount mmol E16b-3 405.5 2.5 eq 2g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 K2CO3 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 Acetonitrile 40ml

[0351] Operation process:

[0352] Into a reaction flask was added E16b-3, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile. The reaction was stirred at 80 °C for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction to be complete (product rf value 0.5).

[0353] Work-up:

[0354] The reaction solution was filtered and rotary evaporated. Purification by column chromatography gave 660 mg of colorless oil.

[0355] 1 H NMR (600 MHz, CDC13) δ: 4.85 (p, J = 6.3 Hz, 2H), 3.65 (t, J = 4.8 Hz, 4H), 2.69 (d, J = 5.2 Hz, 8H), 2.62 (d, J = 8.2 Hz, 4H), 2.28 (t, J = 7.4 Hz, 4H), 1.64 (p, J = 7.6 Hz, 4H), 1.51 (dq, J = 18.9, 6.9, 6.0 Hz, 12H), 1.35 - 1.18 (m, 46H), 0.87 (t, J = 6.9 Hz, 12H).

[0356] Preparation of compound LQ104-E16b-3R

[0357] Preparation of E16b-3R

[0358] Reaction scheme:

[0359]

[0360] Material ratio:

[0361] Material Name Molecular Weight Charge Ratio Charge Amount mmol 5-Bromo-1-pentanol 167 1 eq 8.35g 50 2-Hexylundecanoic acid 256.43 1.1 eq 14.1g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0362] Operation process:

[0363] Into a 1 L reaction flask was added 2-hexyl undecanoic acid, DCC, DMAP and DCM, then 5-bromo-1-pentanol was added. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed that the reaction was complete (product rf value was 0.6).

[0364] Work-up:

[0365] The reaction solution was filtered and rotary evaporated, and 14.2 g of colorless oil was obtained after column chromatography purification.

[0366] Preparation of LQ104-E16b-3R

[0367] Reaction scheme:

[0368]

[0369] Material ratio:

[0370]

[0371]

[0372] Operation process:

[0373] Into a reaction flask was added E16b-3R, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, and the reaction was stirred at 80°C for 12 h. TLC (DCM:MeOH = 10:1) showed that the reaction was complete (product rf value was 0.5).

[0374] Work-up:

[0375] The reaction solution was filtered and rotary evaporated, and 840 mg of colorless oil was obtained after column chromatography purification.

[0376] 1H NMR (600 MHz, CDC13) δ: 4.06 (t, J = 6.6 Hz, 4H), 3.70 (t, J = 4.8 Hz, 4H), 2.73 (d, J = 51.1 Hz, 12H), 2.30 (tt, J = 8.8, 5.3 Hz, 2H), 1.66 (p, J = 6.9 Hz, 4H), 1.57 (p, J = 7.9 Hz, 8H), 1.46 - 1.39 (m, 4H), 1.35 (p, J = 7.5 Hz, 4H), 1.32 - 1.19 (m, 42H), 0.87 (t, J = 6.9 Hz, 12H).

[0377] Preparation of compound LQ104-E17b-1

[0378] Preparation of E17b-1

[0379] Reaction scheme:

[0380]

[0381] Material ratio:

[0382] Material Name Molecular Weight Charge Ratio Charge Amount mmol 8-Pentadecanol 228.42 1 eq 11.4g 50 8-Bromooctanoic acid 223 1.1 eq 12.3g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0383] Operation process:

[0384] Into a 1 L reaction flask was added 8-bromooctanoic acid, DCC, DMAP and DCM, then 8-pentadecanol was added. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed that the reaction was complete (product rf value was 0.6).

[0385] Work-up:

[0386] The reaction solution was filtered and rotary evaporated, and 14.8 g of colorless oil was obtained after column chromatography purification.

[0387] Preparation of LQ104-E17b-1

[0388] Reaction scheme:

[0389]

[0390] Material ratio:

[0391] Material Name Molecular Weight Charge Ratio Charge Amount mmol E17b-1 433.5 2.5 eq 2.17g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 K2CO3 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 Acetonitrile 40ml

[0392] Operation process:

[0393] Into a reaction flask was added E17b-1, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, and the reaction was stirred at 80 °C for 12 h. TLC (DCM:MeOH = 10:1) showed that the reaction was complete (product rf value was 0.5).

[0394] Work-up:

[0395] The reaction solution was filtered and rotary evaporated, and 750 mg of colorless oil was obtained after column chromatography purification.

[0396] 1 H NMR (600 MHz, CDC13) δ: 4.86 (p, J = 6.2 Hz, 2H), 3.64 (t, J = 4.8 Hz, 4H), 2.68 (d, J = 6.8 Hz, 8H), 2.58 (t, J = 8.0 Hz, 4H), 2.27 (t, J = 7.5 Hz, 4H), 1.61 (p, J = 7.2 Hz, 4H), 1.49 (qd, J = 7.7, 5.2, 4.2 Hz, 12H), 1.36 - 1.20 (m, 54H), 0.87 (t, J = 7.0 Hz, 12H).

[0397] Preparation of compound LQ104-E17b-2

[0398] Preparation of E17b-2

[0399] Reaction scheme:

[0400]

[0401] Material ratio:

[0402] Material Name Molecular Weight Charge Ratio Charge Amount mmol 9-Heptadecanol 256.47 1 eq 12.8g 50 7-Bromoheptanoic acid 209 1.1 eq 11.5g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0403] Operation process:

[0404] Into a 1 L reaction flask was added 7-bromoheptanoic acid, DCC, DMAP and DCM, then 9-heptadecanol was added. After stirring at room temperature for 12 h, TLC (PE:EA = 20:1) showed that the reaction was complete (product rf value was 0.6).

[0405] Work-up:

[0406] The reaction solution was filtered and rotary evaporated, and 750 mg of colorless oil was obtained after column chromatography purification.

[0407] Preparation of LQ104-E17b-2

[0408] Reaction scheme:

[0409]

[0410] Material ratio:

[0411] Material Name Molecular Weight Charge Ratio Charge Amount mmol E17b-2 447.5 2.5 eq 2.24g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 K2CO3 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 acetonitrile 40ml

[0412] Operation process:

[0413] To a reaction flask was added E17b-2, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, heated to 80 °C and stirred for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction to be complete (product rf = 0.5).

[0414] Work-up:

[0415] The reaction mixture was filtered and dried in vacuo, and purified by column chromatography to give 680 mg of colorless oil.

[0416] 1 H NMR (600 MHz, CDC13) δ: 4.85 (p, J = 6.2 Hz, 2H), 3.67 (t, J = 4.9 Hz, 4H), 2.73 (s, 8H), 2.64 (t, J = 8.0 Hz, 4H), 2.27 (t, J = 7.5 Hz, 4H), 1.61 (p, J = 7.4 Hz, 4H), 1.51 (dd, J = 13.6, 7.0 Hz, 12H), 1.38 - 1.19 (m, 58H), 0.87 (t, J = 7.0 Hz, 12H).

[0417] Preparation of compound LQ104-E17b-3

[0418] Preparation of E17b-3

[0419] Reaction scheme:

[0420]

[0421] Material ratio:

[0422] Material name Molecular weight Charge ratio Charge amount mmol 7-pentadecanol 228.42 1 eq 11.4g 50 7-bromoheptanoic acid 209 1.1 eq 11.5g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0423] Operation process:

[0424] To a 1 L reaction flask was added 7-bromohexanoic acid, DCC, DMAP and DCM, then 7-pentadecanol was added, and the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction to be complete (product rf = 0.6).

[0425] Work-up:

[0426] The reaction mixture was filtered and dried in vacuo, and purified by column chromatography to give 12.8 g of colorless oil.

[0427] Preparation of LQ104-E17b-3

[0428] Reaction scheme:

[0429]

[0430] Material ratio:

[0431] Material name Molecular weight Charge ratio Charge amount mmol E17b-3 419.5 2.5 eq 2.1g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 K2CO3 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 acetonitrile 40ml

[0432] Operation process:

[0433] Into a 1 L reaction flask was added E17b-3, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, heated to 80 °C and stirred for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction was complete (product rf value was 0.5).

[0434] Post-processing:

[0435] The reaction solution was filtered and rotary evaporated, and 590 mg of colorless oil was obtained after column chromatography purification.

[0436] 1 H NMR (600 MHz, CDC13) δ: 4.86 (p, J = 6.3 Hz, 2H), 3.63 (t, J = 4.8 Hz, 4H), 2.66 (dd, J = 9.9, 5.1 Hz, 8H), 2.56 (t, J = 8.0 Hz, 4H), 2.27 (t, J = 7.4 Hz, 4H), 1.62 (p, J = 7.5 Hz, 4H), 1.49 (dd, J = 10.3, 4.9 Hz, 12H), 1.38 - 1.20 (m, 50H), 0.87 (t, J = 7.0 Hz, 12H).

[0437] Preparation of compound LQ104-E17b-3R

[0438] Preparation of E17b-3R

[0439] Reaction formula:

[0440]

[0441] Material ratio:

[0442] Material name Molecular weight Charge ratio Charge amount mmol 6-bromo-1-hexanol 181 1 eq 9.05g 50 2-hexylundecanoic acid 256.43 1.1 eq 14.1g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0443] Operation process:

[0444] Into a 1 L reaction flask was added 2-hexyl undecanoic acid, DCC, DMAP and DCM, then 6-bromo-1-hexanol was added, and after the addition was completed, it was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed that the reaction was complete (product rf value was 0.6).

[0445] Post-processing:

[0446] The reaction solution was filtered and rotary evaporated, and 14.5 g of colorless oil was obtained after column chromatography purification.

[0447] Preparation of LQ104-E17b-3R

[0448] Reaction Scheme:

[0449]

[0450] Material ratio:

[0451] Material name Molecular weight Charge ratio Charge amount mmol E17b-3R 419.5 2.5 eq 2.1g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 K2CO3 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 acetonitrile 40ml

[0452] Operation process:

[0453] Into a 1 L reaction flask was added E17b-3R, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, and heated to 80 °C and stirred for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction was complete (product rf value was 0.5).

[0454] Post-treatment:

[0455] After the reaction solution was filtered and rotary evaporated, 770 mg of colorless oil was obtained after column chromatography purification.

[0456] 1 H NMR (600 MHz, CDC13) δ: 4.05 (t, J = 6.6 Hz, 4H), 3.66 (t, J = 4.8 Hz, 4H), 2.66 (d, J = 52.0 Hz, 12H), 2.30 (tt, J = 8.9, 5.3 Hz, 2H), 1.66 - 1.48 (m, 12H), 1.46 - 1.20 (m, 54H), 0.87 (td, J = 7.0, 1.5 Hz, 12H).

[0457] Preparation of compound LQ104-E17b-4

[0458] Preparation of E17b-4

[0459] Reaction Scheme:

[0460]

[0461] Material ratio:

[0462] Material name Molecular weight Charge ratio Charge amount mmol 8-heptadecanol 256.47 1 eq 12.8g 50 6-bromohexanoic acid 195.06 1.1 eq 10.7g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0463] Operation process:

[0464] Into a 1 L reaction flask was added 6-bromohexanoic acid, DCC, DMAP and acetonitrile, and then 8-heptadecanol was added, and after completion of the addition, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction was complete (product rf value was 0.6).

[0465] Post-treatment:

[0466] The reaction solution was filtered and rotary evaporated, and 15.2 g of colorless oil was obtained after column chromatography purification.

[0467] Preparation of LQ104-E17b-4

[0468] Reaction formula:

[0469]

[0470] Material ratio:

[0471] Material name Molecular weight Charge ratio Charge amount mmol E17b-4 419.5 2.5 eq 2.1g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 K2CO3 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 acetonitrile 40ml

[0472] Operation process:

[0473] The reaction bottle was added with E17b-4, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, heated to 80°C and stirred for 12 h. TLC (DCM:MeOH=10:1) showed that the reaction was complete (the product rf value was 0.5).

[0474] Post-treatment:

[0475] The reaction solution was filtered and rotary evaporated, and 840 mg of colorless oil was obtained after column chromatography purification.

[0476] 1 H NMR (600 MHz, CDC13) δ: 4.85 (p, J = 6.3 Hz, 2H), 3.67 (t, J = 4.8 Hz, 4H), 2.74 (s, 8H), 2.66 (t, J = 8.0 Hz, 4H), 2.29 (t, J = 7.4 Hz, 4H), 1.64 (p, J = 7.5 Hz, 4H), 1.52 (dq, J = 26.5, 6.9, 6.0 Hz, 12H), 1.37 - 1.19 (m, 54H), 0.87 (t, J = 6.9 Hz, 12H).

[0477] Preparation of compound LQ104-E18b-2 in Preparation Example 15

[0478] Preparation of E18b-2

[0479] Reaction formula:

[0480]

[0481] Material ratio:

[0482] Material name Molecular weight Charge ratio Charge amount mmol 7-pentadecanol 228.42 1 eq 11.4g 50 8-bromooctanoic acid 223 1.1 eq 12.3g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0483] Operation process:

[0484] Into a 1 L reaction flask was added 8-bromooctanoic acid, DCC, DMAP and DCM, followed by 7-pentadecanol. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction to be complete (product rf value 0.6).

[0485] Work-up:

[0486] The reaction mixture was filtered and spun dry. Purification by column chromatography gave 14.6 g of colorless oil.

[0487] Preparation of LQ104-E18b-2

[0488] Reaction Scheme:

[0489]

[0490] Material Ratio:

[0491]

[0492]

[0493] Operation Process:

[0494] Into a 1 L reaction flask was added 8-bromooctanoic acid, DCC, DMAP and DCM, followed by 7-pentadecanol. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction to be complete (product rf value 0.6).

[0495] Work-up:

[0496] The reaction mixture was filtered and spun dry. Purification by column chromatography gave 750 mg of colorless oil.

[0497] 1 H NMR (600 MHz, CDC13) δ: 4.86 (p, J = 6.3 Hz, 2H), 3.62 (t, J = 4.9 Hz, 4H), 2.68 - 2.60 (m, 8H), 2.55 (t, J = 8.0 Hz, 4H), 2.27 (t, J = 7.5 Hz, 4H), 1.61 (t, J = 7.4 Hz, 4H), 1.49 (p, J = 7.7, 6.7 Hz, 12H), 1.35 - 1.21 (m, 54H), 0.87 (t, J = 7.0 Hz, 12H).

[0498] Preparation of Compound LQ104-E18b-2R

[0499] Preparation of E18b-2R

[0500] Reaction Scheme:

[0501] Preparation of E18b-2R

[0502] Material ratio:

[0503] Material name Molecular weight Charge ratio Charge amount mmol 7-bromo-1-heptanol 195 1 eq 9.75g 50 2-hexylundecanoic acid 256.43 1.1 eq 14.1g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0504] Operation process:

[0505] Into a 1 L reaction flask was added 2-hexyl undecanoic acid, DCC, DMAP and DCM, then 7-bromo-1-heptanol was added. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction to be complete (product rf value 0.6).

[0506] Work-up:

[0507] The reaction solution was filtered and spun dry. After column chromatography purification, 14.1 g of colorless oil was obtained.

[0508] Preparation of LQ104-E18b-2R

[0509] Reaction formula:

[0510]

[0511] Material ratio:

[0512] Material name Molecular weight Charge ratio Charge amount mmol E18b-2R 433.5 2.5 eq 2.17g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 [K2CO3] 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 acetonitrile 40ml

[0513] Operation process:

[0514] Into a reaction flask was added E18b-2R, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile. The reaction was stirred at 80 °C for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction to be complete (product rf value 0.5).

[0515] Work-up:

[0516] The reaction solution was filtered and spun dry. After column chromatography purification, 690 mg of colorless oil was obtained.

[0517] 1 H NMR (600 MHz, CDC13) δ: 4.05 (t, J = 6.7 Hz, 4H), 3.77 (t, J = 4.8 Hz, 4H), 2.96 - 2.87 (m, 8H), 2.81 (t, J = 8.2 Hz, 4H), 2.30 (tt, J = 8.9, 5.3 Hz, 2H), 1.65 - 1.52 (m, 12H), 1.46 - 1.38 (m, 4H), 1.37 - 1.19 (m, 54H), 0.87 (td, J = 7.1, 1.5 Hz, 12H).

[0518] Preparation of compound LQ104-E18b-3

[0519] Preparation of E18b-3

[0520] Reaction scheme:

[0521]

[0522] Material ratio:

[0523] Material name Molecular weight Charge ratio Charge amount mmol 8-heptadecanol 256.47 1 eq 12.8g 50 7-bromoheptanoic acid 209 1.1 eq 11.5g 55 DCC 206 2 eq 20.6g 100 DMAP 122 0.1 eq 610 mg 5 DCM 300ml

[0524] Operation process:

[0525] Into a 1 L reaction flask was added 7-bromoheptanoic acid, DCC, DMAP and DCM, then 8-heptadecanol was added. After the addition was complete, the reaction was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) showed the reaction was complete (product rf value was 0.6).

[0526] Work-up:

[0527] The reaction solution was filtered and rotary evaporated, and then purified by column chromatography to obtain 13.7 g of colorless oil.

[0528] Preparation of LQ104-E18b-3

[0529] Reaction scheme:

[0530]

[0531] Material ratio:

[0532] Material name Molecular weight Charge ratio Charge amount mmol E18b-3 447.5 2.5 eq 2.24g 5 N,N'-Bis(2-hydroxyethyl)ethylenediamine 148 1 eq 300 mg 2 K2CO3 138 4 eq 1.1g 8 KI 166 2 eq 664 mg 4 acetonitrile 40ml

[0533] Operation process:

[0534] Into a reaction flask was added E18b-3, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI and acetonitrile, and the reaction was stirred at 80 °C for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction was complete (product rf value was 0.5).

[0535] Work-up:

[0536] The reaction solution was filtered and rotary evaporated, and then purified by column chromatography to obtain 790 mg of colorless oil.

[0537] 1H NMR (600 MHz, CDC13) δ: 4.86 (p, J = 6.2 Hz, 2H), 3.61 (t, J = 4.8 Hz, 4H), 2.67 - 2.60 (m, 8H), 2.54 (t, J = 8.0 Hz, 4H), 2.27 (t, J = 7.5 Hz, 4H), 1.62 (p, J = 7.5 Hz, 4H), 1.49 (tt, J = 7.6, 4.6 Hz, 12H), 1.37 - 1.20 (m, 58H), 0.87 (t, J = 6.9 Hz, 12H).

[0538] The compound LQ104-H3 was prepared by Preparation Example 18

[0539] The preparation route of LQ104-H3 is as follows:

[0540]

[0541] Material ratio:

[0542] Material name Molecular weight Charge amount Mmol LQ001-1 461.5 2g 4.3 N,N'-Di(2-hydroxyethyl)-1,3-propanediamine 235.15 510 mg 2.15 K2CO3 138 2.1g 15 KI 166 860 mg 5.2 acetonitrile - 30ml -

[0543] Operation process:

[0544] Into a 1 L reaction flask was added LQ001-1, N,N'-di(2-hydroxyethyl)-1,3- propanediamine, K2CO3, KI, and acetonitrile, heated to 80°C, and stirred for 12 hours. TLC (DCM:MeOH = 10:1) showed that the reaction was complete.

[0545] Post-treatment:

[0546] After the reaction solution was filtered and rotary evaporated, 850 mg of colorless oil was obtained after column chromatography purification, which was the compound LQ104-H3, with a yield of 43%.

[0547] Mass spectrometry analysis:

[0548] In the ESI-MS positive ion mass spectrum, there were strong ion peaks at m / z 924 and 925, which matched the molecular weight of 923.5 of the compound.

[0549] 1 H NMR (400 MHz, CDC13) δ: 4.86 (p, 2H), 3.65-3.59 (m, 4H), 2.68-2.59 (m, 8H), 2.57-2.49 (m, 4H), 2.27 (t, 4H), 1.61 (p, 7H), 1.49 (dt, 12H), 1.28 (d, 61H), 0.87 (t, 12H).

[0550] The reagent sources of the foregoing preparation examples of the present application are as follows:

[0551] LQ001-1: self-made;

[0552] N,N'-bis(2-hydroxyethyl)ethylenediamine: purchased from Adamas Reagent Co., Ltd., item number: 013455310, purity: RG, 98%;

[0553] K2CO3: purchased from Shanghai Yinn Chemical Technology Co., Ltd., item number: RH425011, purity: AR, 99%;

[0554] KI: purchased from Shanghai Yinn Chemical Technology Co., Ltd., item number: RH432132, purity: AR, 99%;

[0555] Acetonitrile: purchased from Shanghai Titan Science and Technology Co., Ltd., item number: 01111797, purity: AR, ≥99.0%;

[0556] Methyl tert-butyl ether: purchased from Shanghai Titan Science and Technology Co., Ltd., item number: 01030342, purity: AR, ≥99.0%;

[0557] Malonic acid: purchased from Adamas Reagent Co., Ltd., item number: 01022573, purity: RG, 99%;

[0558] DCC: dicyclohexyl carbodiimide, purchased from Adamas Reagent Co., Ltd., item number: 012041444, purity: RG, 99%;

[0559] DMAP: 4-dimethylaminopyridine, purchased from Adamas Reagent Co., Ltd., item number: 01271081, purity: RG, 99%;

[0560] DCM: dichloromethane, purchased from Shanghai Titan Science and Technology Co., Ltd., item number: 01111853, purity: AR, ≥99.5%;

[0561] Diatomaceous earth: purchased from Shanghai Titan Science and Technology Co., Ltd., item number: 01589000, purity: extra pure, ≥89.0%, 200 mesh;

[0562] 8-pentadecanol: self-made;

[0563] 6-bromohexanoic acid: purchased from Adamas Reagent Co., Ltd., item number: 01073739, purity: RG, 98%+;

[0564] N,N'-bis(2-hydroxyethyl)ethylenediamine: purchased from Adamas Reagent Co., Ltd., item number: 013455310, purity: RG, 98%;

[0565] 9-heptadecanol: purchased from Dalian Ruiyin Technology Co., Ltd., purity: 98%;

[0566] 5-Bromopentanoic acid: purchased from Bide Pharmatech, Catalog No. BD9634, purity: 98%;

[0567] 10-Nonadecanol: made in-house

[0568] 4-Bromobutyric acid: purchased from Shanghai Titan Technology Co., Ltd., Catalog No. 011016520, purity: RG, 99%+;

[0569] 7-Bromoheptanoic acid: purchased from Shanghai Titan Technology Co., Ltd., Catalog No. 012345536, purity: RG, 98%;

[0570] 7-Pentadecanol: made in-house

[0571] 5-Bromo-1-pentanol: purchased from Bide Pharmatech, purity: 98%;

[0572] 2-Hexylundecanoic acid: purchased from Bide Pharmatech, Catalog No. BD75392, purity: 98%;

[0573] 8-Bromo-octanoic acid: purchased from Jiangsu Aikang, purity: 98%;

[0574] 6-Bromo-1-hexanol: purchased from Adamas Reagent Co., Ltd., Catalog No. 01074359, purity: RG, 98%;

[0575] 8-Heptadecanol: made in-house

[0576] 7-Bromo-1-heptanol: purchased from Adamas Reagent Co., Ltd., Catalog No. 01001821, purity: RG, 98%;

[0577] N,N'-Bis(2-hydroxyethyl)-1,3-propanediamine (purchased from Beijing Weisai Chemical, purity: 95%).

[0578] Example 1

[0579] Example 1 was used to verify whether the Lipid Nanoparticle (LNP) formulations prepared from the ionizable lipid compounds disclosed in this application can effectively encapsulate mRNA and maintain the structural integrity of mRNA. The ionizable lipid compounds prepared in Preparation Example 1 and Preparation Example 2, distearoylphosphatidylcholine (DSPC, purchased from NOF Corporation, Lot No. S01005), cholesterol (purchased from NOF Corporation, Lot No. O01001), and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000, purchased from Genview Biotech, Lot No. O02005) were dissolved in ethanol (manufacturer: Nanjing Chemical Reagent Co., Ltd., purity 99.6%) solution, respectively, and then mixed according to a certain molar ratio to prepare a mixed lipid ethanol solution, wherein the total concentration of lipids was 12.5 mM (the unit of measurement "M" appearing in this application means mol / L). The self-prepared firefly luciferase (Fluc) mRNA or self-prepared SARS-CoV-2 Spike protein (Spike) mRNA (SARS-CoV-2 Spike protein mRNA is described in Tan, S. et al., bioRxiv 2022.05.10.491301.) was diluted in 50 mM citrate buffer at pH 4.0 to obtain an mRNA solution. Lipid nanoparticles were prepared by using a microfluidic device, controlling the flow rate at 12 mL / min, and controlling the volume ratio of the mixed lipid ethanol solution and the mRNA solution prepared in the previous step at 1:3, according to the nitrogen-phosphorus ratio of ionizable lipids to mRNA at 3-15:1. Ethanol was removed by dialysis against 0.01 M phosphate buffered saline (PBS) for 12 to 24 hours. Finally, the LNP solution was filtered through a sterile filter with a pore size of 0.22 μm (manufacturer: Millex, Lot No. SLGPR33RB), and concentrated by ultrafiltration (manufacturer: Amicon-Ultra, molecular weight cut-off: 10 KDa) to obtain LNP formulations encapsulating Fluc mRNA or Spike mRNA with the ionizable lipids described in this application and DSPC, cholesterol, and DMG-PEG2000. The molar ratio of ionizable lipid compounds to DSPC, cholesterol, and DMG-PEG2000, and the nitrogen-phosphorus ratio of ionizable lipids to mRNA are shown in Table 1. The particle size and polydispersity index (PDI) of each LNP formulation were determined by dynamic light scattering using a Malvern Zetasizer Ultra instrument (manufacturer: Malvern); the encapsulation efficiency of LNP was determined using the Quant-it Ribogreen RNA quantitation assay kit (manufacturer: ThermoFisher Scientific, Lot No. R11490); and the mRNA integrity was investigated by nucleic acid gel electrophoresis (electrophoresis instrument, manufacturer: Shanghai Tian Neng), and the test results are shown in Table 1 andFigure 1 . Figure 1 The figure is the gel electrophoresis of nucleic acid of each LNP preparation of this example. The gel is 1% agarose gel (manufacturer: Biowest; item number: BY-R0100), and the test condition is 160V electrophoresis for 20 minutes.

[0580] Table 1

[0581]

[0582]

[0583] In the art, PDI less than 0.3 indicates that the size of nanoparticles in the LNP preparation is relatively uniform; the encapsulation efficiency is used to indicate whether the LNP can effectively encapsulate mRNA, and the encapsulation efficiency higher than 70% indicates that the LNP can effectively encapsulate mRNA; the single and bright band in the agarose gel electrophoresis indicates that the structure of mRNA is complete. Among them, the more PDI tends to 0, the better, and the more encapsulation efficiency tends to 100%, the better. From Table 1 and Figure 1 It can be seen that the particle size of each LNP of the present application is between 70-120 nm, PDI is less than 0.3, and the encapsulation efficiency is higher than 80%; specifically, the encapsulation efficiency of LQ104 series is stable at more than 90%, and the encapsulation efficiency of LNP preparation prepared from LQ107 is 83.7%. It can be seen that the LNP preparation prepared according to the molar ratio and nitrogen-phosphorus ratio shown in Table 1 can effectively encapsulate mRNA and maintain the structure of mRNA complete. And from Table 1 and the experimental data not exhaustively listed in the present application, it can be seen that the performance of LQ104 series is better than that of LQ107 series. In addition, according to the experimental data verified but not exhaustively listed in the present application, no matter what mRNA is encapsulated, even if the data obtained by the above in vitro experiment is slightly different, but it has no effect on the above conclusion.

[0584] Example 2

[0585] In this embodiment, we verified the in vitro cell delivery and expression of the LNP formulation of this application through in vitro cell experiments. 10,000 293FT cells per well were seeded in 96-well plates and cultured overnight until cell adhesion. The LNP formulations LQ104-1 to LQ104-8 from Example 1, containing 100 ng of mRNA per well, were added to the cell culture medium of the 96-well plates. Before adding the LNP formulations, the cell culture medium was replaced with antibiotic-free DMEM medium containing 10% fetal bovine serum (manufacturer: Gibco, catalog number: C11995500BT), and cultured for another 24 hours. Then, the cell culture medium was discarded, and cells were lysed at a dose of 100 μL / well using cell lysis buffer supplemented with D-luciferin potassium (manufacturer: PerkinElmer, catalog number: 122799, final concentration 1 mM) and ATP (manufacturer: ApexBio, catalog number: C6931, final concentration 2 mM). The chemiluminescence intensity was detected using an enzyme-linked immunosorbent assay (ELISA) reader (manufacturer: Thermo Scientific). The test results are shown below. Figure 2 .exist Figure 2 In this study, PBS served as a negative control, and the corresponding chemiluminescence intensity readings for this group can be considered as background readings. Higher chemiluminescence intensity readings indicate higher expression levels. Figure 2 As can be seen, compared with the PBS group, the readings of the LQ104-1 to LQ104-8 groups increased significantly, indicating that each LNP can effectively deliver Fluc mRNA into the cell and express it.

[0586] Example 3

[0587] In this example, LQ104-1 to LQ104-8 (encapsulating Fluc mRNA) of Example 1 were injected into 6 to 8-week-old female Balb / C mice (Vital River) via tail vein at a dose of 5 pg per mouse (n = 3, i.e. 3 mice were used for injection and test in each group, and the data presented are the average of the measurements of each group), and at specific time points after administration (6 hours, 24 hours, 48 hours in this example), D-luciferin potassium salt was injected intraperitoneally, and then the luminescence was detected by IVIS Spectrum small animal live imaging instrument (manufacturer: PerkinElmer), and the total luminescence intensity of the luminescence site (e.g. liver and other sites) in the mouse was counted, and the higher the luminescence intensity, the higher the expression of luciferase, i.e. the better the expression of the corresponding LNP preparation in the mouse. The total luminescence intensity is measured by bioluminescence imaging, and the luminescence intensity data of the luminescence site is obtained after 6 to 15 minutes (min) of intraperitoneal injection of D-luciferin potassium salt. The total luminescence intensity of the luminescence site is counted by Living Image software (manufacturer: PerkinElmer), and further, the area under the curve (AUC, unit: p / s*hour) is calculated by GraphPad software, and the AUC in each example of the present application is the area under the curve of the connecting line of the total luminescence intensity measurement points from 4 hours or 6 hours after administration (the peak value of 3-6 hours after administration is the same by using the experimental method of the present application) to 48 hours after administration. The test results are shown in Figure 3 and 4A and Table 2.

[0588] Table 2 Figure 4A AUC data of

[0589] Group AUC (p / s*hour) LQ104-1 2.95E+10 LQ104-2 1.83E+10 LQ104-3 4.81E+10 LQ104-4 1.01E+11 LQ104-5 5.58E+10 LQ104-6 3.98E+10 LQ104-7 5.97E+10 LQ104-8 3.96E+10

[0590] Note: E+10 is a scientific notation, which represents 10 to the power of 10; for example, E+11 represents 10 to the power of 11, and so on.

[0591] Generally, the total luminescence intensity of the mouse without administration treatment is in the order of 10 5 As can be seen from Figure 3 and 4A and Table 2, LQ104-1 to LQ104-8 have strong expression in mice. Further, in each example of the present application, we determine the LNP preparation with better expression by observing the area under the curve (AUC) of the connecting line of the measurement points, and the larger the area under the curve, the better the expression effect.

[0592] LQ104-9, LQ104-10, LQ107 (encapsulating Spike mRNA) were injected intramuscularly into 6-8 weeks old female Balb / C mice at a dose of 2 pg per mouse, and the same LNP formulation was injected again at day 21 after the first injection. The whole blood of mice was collected at a specific time point (in this example, day 7 after the second injection). The collected blood was centrifuged at 4°C, 2000xg for 10 min to separate the serum from the whole blood. Then, the serum was inactivated at 56°C for 30 min and stored at -80°C for analysis. The total antibody titer in the serum was determined by enzyme-linked immunosorbent assay (ELISA). Specifically, SARS-CoV-2 (2019-nCoV) Spike S1+S2 ECD-His Recombinant Protein (manufacturer: Sino Biological, catalog number: 40589-V08B1) was used to coat the antigen, SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody Mouse Mab (manufacturer: Sino Biological, catalog number: 40591-MM43) was used as a control, 2% bovine serum albumin (BSA) was used for blocking, Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, catalog number: 115-035-003) was used as the secondary antibody, and TMB (Invitrogen, catalog number: 00-4201-56) was used for color development according to the instructions. The Spike antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) analysis (the data was determined in the serum of mice at day 7 after the second injection, n = 8), and the test results are shown in Figure 4.

[0593] As can be seen from Figure 4, the total antibody titer of Spike protein in animals injected with LQ104-9 and LQ104-10 increased significantly compared with animals injected with PBS (statistical analysis by ANOVA, **p < 0.01, ***p < 0.001, compared with the group of animals injected with PBS), indicating that the mRNA delivered by the carrier was effectively expressed after administration and induced an immune response. The total antibody titer of the group of animals injected with LQ107 did not increase.

[0594] Example 4

[0595] The ionizable lipids prepared in Preparation Examples 3 to 17 were selected to prepare LNP formulations (encapsulating Fluc mRNA) according to the same method as in Example 1, according to the molar ratio and nitrogen-phosphorus ratio shown in Table 3. The particle size, PDI and surface potential of each LNP formulation were determined using a Malvern Zetasizer Ultra; the encapsulation efficiency of the LNP was determined using a Quant-it Ribogreen RNA quantification assay kit (manufacturer: ThermoFisher Scientific, item number: R11490); and the pKa of the LNP was measured using a 6-(p-Toluidino)-2-naphthalene sulfonic acid sodium salt (TNS, purchased from Nanjing Xizhe Biotechnology Co., Ltd., item number: XZ0743) dye binding test.

[0596] Table 3

[0597]

[0598]

[0599]

[0600] As can be seen from Table 3, the particle size of the LNP formulations prepared from each of the ionizable lipids described in Preparation Examples 3 to 17 is between 50-100 nm; the PDI is less than 0.3, specifically between 0.044 and 0.098; and the encapsulation efficiency is higher than 96%. This shows that the LNP formulations in this example can effectively encapsulate mRNA; the surface potential is weakly negative, and the pKa is between 6-7.3, which is comparable to the range recognized in the art.

[0601] Further, according to the mouse in vivo test method of Reference Example 3, each group of LNP formulations in this example was injected into 6 to 8 week old female Balb / C mice through the tail vein or intramuscular injection of the lower limbs at a dose of 5 μg per mouse, and the total luminescence intensity at the liver site or the lower limb administration site was counted, and the test results are shown in Table 4A to Table 4D. Figures 5A to 5D and Table 4A to Table 4D. Among them Figure 5A and Figure 5B show the luminescence counting results at the liver site of the mice after intravenous injection administration, Figure 5C and Figure 5D show the luminescence counting results at the lower limb administration site of the mice after intramuscular injection administration. From Figures 5A to 5DAs can be seen from Table 4A to Table 4D, the LNP formulations tested in this example all have strong expression in mice, indicating that the ionizable lipids described in Preparation 3 to Preparation 17 can effectively deliver mRNA to the body and express. We also tested other administration routes, such as intraperitoneal injection and subcutaneous injection, and the results showed that the LNP formulations in this example can express under various administration routes.

[0602] Table 4A Figure 5A AUC data of

[0603] Group AUC (p / s*hour) LQ104-E15b-1 2.26E+10 LQ104-E16b-1 2.74E+10 LQ104-E16b-2 8.98E+10 LQ104-E16b-3 1.95E+10 LQ104-E16b-3R 1.47E+10

[0604] Table 4B Figure 5B AUC data of

[0605]

[0606] Table 4C Figure 5C AUC data of

[0607] Group AUC (p / s*hr) LQ104-E15b-1 5.58E+09 LQ104-E16b-1 2.30E+09 LQ104-E16b-2 3.50E+09 LQ104-E16b-3 2.10E+09 LQ104-E16b-3R 2.19E+09

[0608] Table 4D Figure 5D AUC data of

[0609]

[0610] Example 5

[0611] In this example, LQ104-E16b-2 obtained from Preparation 7, LQ104-E17b-4 obtained from Preparation 14 and LQ104-E18b-3 obtained from Preparation 17 were each divided into 8 groups, and LNP formulations (loaded with Fluc mRNA) were prepared according to the formulation components and nitrogen to phosphorus ratios described in the following Table 5. Their particle size, PDI and encapsulation efficiency were determined, and the results are shown in Table 5.

[0612] Table 5

[0613]

[0614]

[0615]

[0616] As can be seen from Table 5, the particle size of the LNP formulations prepared from compound LQ104-E16b-2, LQ104-E17b-4 or LQ104-E18b-3 according to the above components and nitrogen to phosphorus ratios is between 60-90 nm; the PDI is less than 0.3, and most of them are below 0.1; the encapsulation efficiency is higher than 90%, and most of them are between 97%-99%.

[0617] Similarly, referring to the in vivo test method of mice in Example 3, all the LNP formulations prepared in this example were injected into 6-8 week old female Balb / C mice through the tail vein at a dose of 5 μg per mouse, and the total luminescence intensity of the liver site of the mice in vivo was counted, and the test results are shown in Table 6A and Table 6B. It can be seen that the LNP formulations in this example have strong expression in mice. Figures 6A-6C and Table 6A to Table 6B. It can be seen that the LNP formulations in this example have strong expression in mice.

[0618] Table 6A Figure 6A AUC data

[0619]

[0620] Table 6B Figure 6B AUC data

[0621] Grouping AUC (p / s*hr) LQ104-E18b-3(f3) 4.14E+10 LQ104-E18b-3(f4) 3.17E+10 LQ104-E18b-3(f7) 5.92E+10 LQ104-E18b-3(f8) 7.00E+10

[0622] Example 6

[0623] In this example, LQ104-E16b-2 obtained by Preparation Example 7 was selected as an ionizable lipid, and 21 kinds of LNP formulations (encapsulating Fluc mRNA) were prepared according to the molar ratio and nitrogen-phosphorus ratio in Table 7 by the same method as in Example 1, and their particle size, PDI and encapsulation efficiency were determined. This example is mainly used to verify the preferred component content of DSPC and cholesterol in the LNP formulation. Among them, the molar percentage content of LQ104-E16b-2 and DMG-PEG is kept unchanged, and the performance of each LNP formulation prepared by changing the molar percentage content of DSPC and cholesterol is verified. The total molar number of the four components is 100%.

[0624] Table 7

[0625]

[0626]

[0627]

[0628] As can be seen from Table 7, the particle size of each group of LNP formulations prepared from LQ104-E16b-2 is between 60-110 nm; the PDI is less than 0.3, specifically between 0.053 and 0.166; the encapsulation efficiency is higher than 90%, and is higher than 96.8%.

[0629] Similarly, referring to the in vivo test method of mice in Example 3, all the LNP formulations prepared in this example were injected into 6-8 week old female Balb / C mice through the tail vein at a dose of 5 μg per mouse, and the total luminescence intensity of the liver site of the mice in vivo was counted, and the test results are shown in Table 6A and Table 6B. It can be seen that the LNP formulations in this example have strong expression in mice.Figure 7A and Figure 7B and Table 8A to Table 8B. It can be seen that the LNP formulations tested in this example have strong expression in mice. Among them, LQ104-E16b-2(DS-f1) to LQ104-E16b-2(DS-f10) have higher total luminescence intensity, indicating that the LNP formulations with molar percentage of DSPC between 0% and 18% have better in vivo delivery ability.

[0630] Table 8A Figure 7A AUC data of

[0631] Grouping AUC (p / s*hr) LQ104-E16b-2(DS-f1) 4.74E+11 LQ104-E16b-2(DS-f2) 3.97E+11 LQ104-E16b-2(DS-f3) 2.13E+11 LQ104-E16b-2(DS-f4) 1.63E+11 LQ104-E16b-2(DS-f5) 1.19E+11 LQ104-E16b-2(DS-f6) 9.89E+10 LQ104-E16b-2(DS-f7) 9.79E+10 LQ104-E16b-2(DS-f8) 6.45E+10 LQ104-E16b-2(DS-f9) 5.09E+10 LQ104-E16b-2(DS-f10) 4.34E+10 LQ104-E16b-2(DS-f11) 1.83E+10 LQ104-E16b-2(DS-f12) 1.09E+10

[0632] Table 8B Figure 7B AUC data of

[0633] Grouping AUC (p / s*hr) LQ104-E16b-2(DS-f13) 1.11E+10 LQ104-E16b-2(DS-f14) 7.24E+09 LQ104-E16b-2(DS-f15) 3.74E+09 LQ104-E16b-2(DS-f16) 3.88E+09 LQ104-E16b-2(DS-f17) 1.29E+10 LQ104-E16b-2(DS-f18) 1.91E+10 LQ104-E16b-2(DS-f19) 1.73E+10 LQ104-E16b-2(DS-f20) 2.95E+10 LQ104-E16b-2(DS-f21) 1.34E+10

[0634] Example 7

[0635] Different from Example 6, in this example, the phospholipid DSPC in the LNP formulation components is replaced with DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamin, dioleoyl phosphatidyl ethanolamine), and the LNP formulations (encapsulating Fluc mRNA) are prepared according to the same method as Example 1 to test the particle size, PDI and encapsulation efficiency of the LNP formulations prepared by changing the component from 0% to 22% using DOPE as the phospholipid, as shown in Table 9.

[0636] Table 9

[0637]

[0638]

[0639] As can be seen from Table 9, the LNP formulations prepared in this example have a particle size of 70-100 nm, a PDI of less than 0.3, and an encapsulation efficiency of more than 96%.

[0640] According to the mouse in vivo test method of Reference Example 3, each LNP formulation prepared in this example is injected into 6 to 8 week old female Balb / C mice through the tail vein at a dose of 5 μg per mouse, and the total luminescence intensity of the mouse in vivo liver site is counted, and the test results are shown in Figure 8 and Table 10. It can be seen that each LNP formulation prepared in this example has strong expression in mice. In addition, we tested a variety of phospholipid molecules, and the prepared LNP formulations can achieve in vivo delivery and expression.

[0641] Table 10 Figure 8 AUC data of

[0642] Grouping AUC (p / s*hr) LQ104-E16b-2(DO-f1) 7.17E+11 LQ104-E16b-2(DO-f2) 7.82E+11 LQ104-E16b-2(DO-f3) 3.15E+11 LQ104-E16b-2(DO-f4) 2.50E+11 LQ104-E16b-2(DO-f5) 1.68E+11 LQ104-E16b-2(DO-f6) 1.17E+11 LQ104-E16b-2(DO-f7) 7.54E+10 LQ104-E16b-2(DO-f8) 1.13E+11 LQ104-E16b-2(DO-f9) 7.77E+10 LQ104-E16b-2(DO-f10) 4.35E+10 LQ104-E16b-2(DO-f11) 4.06E+10 LQ104-E16b-2(DO-f12) 2.78E+10

[0643] Example 8

[0644] This example selected LQ104-E16b-2 obtained from Preparation Example 7 as ionizable lipid, prepared LNP formulations (encapsulating Fluc mRNA) according to the molar ratio and nitrogen-phosphorus ratio in Table 11, and in the manner of Reference Example 1. Different from Example 1, the mRNA in this example was diluted in 25 mM sodium acetate solution with pH 5.0, and 20 mM Tris-acetate solution with pH 7.5 was used for dialysis. The particle size, PDI and encapsulation efficiency of all LNP formulations in this example were determined. This example was mainly used to verify the preferred component content of ionizable lipids in LNP formulations. We fixed the molar percentage content of DSPC at 0%, 2%, 4%, 10%, respectively, and simultaneously screened the content range of ionizable lipids of several specific components (cholesterol was used in this example to make up the rest after the other three components were determined) in combination with 1.6% molar percentage content of PEG lipid.

[0645] Table 11

[0646]

[0647]

[0648]

[0649] As can be seen from Table 11, the particle size of the LNP formulations prepared in this example was between 55-120 nm, the PDI was less than 0.3, and the encapsulation efficiency was higher than 90%.

[0650] According to the in vivo test method of Reference Example 3, all LNP reagents prepared in this example were injected into 6-8 week old female Balb / C mice through the tail vein at a dose of 5 μg per mouse, and the total luminescence intensity of the liver site of the mice in vivo was counted, and the test results are shown in Figure 9A and Figure 9B and Table 12A to Table 12B. It can be seen that all LNP formulations prepared in this example have strong expression in mice in vivo. In combination with our screening test of the component content of the series of ionizable lipids, the results show that the ionizable lipid in the range of 40%-55% corresponds to the better in vivo delivery effect of the LNP formulation.

[0651] Table 12A Figure 9A AUC data)

[0652] Grouping AUC (p / s*hr) LQ104-E16b-2(ION-f1) 1.57E+11 LQ104-E16b-2(ION-f2) 3.62E+11 LQ104-E16b-2(ION-f3) 6.47E+11 LQ104-E16b-2(ION-f4) 4.67E+10 LQ104-E16b-2(ION-f5) LQ104-E16b-2(ION-f6) LQ104-E16b-2(ION-f7) LQ104-E16b-2(ION-f8) LQ104-E16b-2(ION-f9) LQ104-E16b-2(ION-f10) LQ104-E16b-2(ION-f11) LQ104-E16b-2(ION-f12) Figure 9B Grouping AUC (p / s*hr) LQ104-E16b-2(ION-f13) LQ104-E16b-2(ION-f14) LQ104-E16b-2(ION-f15) LQ104-E16b-2(ION-f16) LQ104-E16b-2(ION-f17) LQ104-E16b-2(ION-f18) LQ104-E16b-2(ION-f19) LQ104-E16b-2(ION-f20) LQ104-E16b-2(ION-f21) Figure 10 2.12E+11 LQ104-E16b-2 (ION-f6) 4.38E+11 LQ104-E16b-2 (ION-f7) 5.05E+11 LQ104-E16b-2 (ION-f8) 5.82E+11 LQ104-E16b-2 (ION-f9) 2.28E+11 LQ104-E16b-2 (ION-f10) 5.75E+10

[0653] Table 12B Figure 9BAUC data)

[0654] Group AUC (p / s*hr) LQ104-E16b-2 (ION-f11) 7.23E+10 LQ104-E16b-2 (ION-f12) 1.23E+11 LQ104-E16b-2 (ION-f13) 2.05E+11 LQ104-E16b-2 (ION-f14) 3.05E+11 LQ104-E16b-2 (ION-f15) 1.05E+11 LQ104-E16b-2 (ION-f16) 2.03E+10 LQ104-E16b-2 (ION-f17) 3.15E+10 LQ104-E16b-2 (ION-f18) 7.36E+10 LQ104-E16b-2 (ION-f19) 5.00E+10 LQ104-E16b-2 (ION-f20) 7.84E+10

[0655] Example 9

[0656] The LNP formulations LQ104-E16b-2(DS-f1) to LQ104-E16b-2(DS-f7) prepared in Example 6 were stored at an environment of 4℃ for 14 days, and their particle sizes and PDIs were determined, with the results shown in Table 13.

[0657] Table 13

[0658]

[0659] As can be seen from Table 13, the LNP formulations LQ104-E16b-2 prepared according to different DSPC contents had a particle size variation of less than 5 nm after being placed at 4℃ for 14 days, indicating that the samples had good stability. Similarly, we selected other LNP formulations of the present application, and also obtained a comparable effect.

[0660] In addition, the LNP formulations LQ104-E16b-2(ION-f3), LQ104-E16b-2(ION-f6), LQ104-E16b-2(ION-f7), LQ104-E16b-2(ION-f8), LQ104-E16b-2(ION-f13), LQ104-E16b-2(ION-f14), and LQ104-E16b-2(ION-f20) prepared in Example 8 were each divided into 0.3 mL and stored at -80℃. It should be noted that each experiment of the present application requires the addition of sucrose as a protective agent at a final concentration of 8% when the sample is frozen, and the freezing time is more than 6 h to ensure that the sample can be completely frozen; when the sample is thawed, the sample is placed at 4℃ for no less than 90 min, and through observation, this condition can ensure that the LNP formulation is completely thawed. All samples were frozen and thawed 5 times, and their particle sizes and PDIs were determined. The results are shown in Table 14.

[0661] Table 14

[0662]

[0663]

[0664] As can be seen from Table 14, except for the LQ104-E16b-2(ION-f13) group, the particle size of the LNP formulations of the other groups changed little after being frozen and thawed 5 times at -80℃, with a variation of less than 10 nm, which preliminarily indicated that the samples had good stability.

[0665] Example 10

[0666] In the same manner as Example 8, LNP formulations (encapsulating Fluc mRNA) were prepared according to the molar ratios and N:P ratios in Table 15. In the same manner as Example 8, the mRNA solution of this example was diluted in 25 mM, pH 5.0 sodium acetate solution, and the solution was 20 mM, pH 7.5 Tris-acetate solution during dialysis. This example was mainly used to verify the performance of the LNP formulations of the present application prepared with three components (ionizable lipid, cholesterol and DMG-PEG) and four components (ionizable lipid, phospholipid, cholesterol and DMG-PEG), and to investigate the preferred component content of DMG-PEG in the LNP formulation. In this example, we selected the molar percentage content of DMG-PEG in the range of 1.5%-5% for experiment. The particle size, PDI and encapsulation efficiency of each LNP were determined, and the results are shown in Table 15.

[0667] Table 15

[0668]

[0669]

[0670]

[0671] As can be seen from Table 15, the particle size of the LQ104 series prepared with three components or four components is between 60-115 nm, the PDI is less than 0.3, and the encapsulation efficiency is higher than 96%.

[0672] In the same manner as Example 3, the LNP formulations of this example were injected into 6-8 week old female Balb / C mice through the tail vein at a dose of 5 μg per mouse, and the total luminescence intensity of the liver of the mice in vivo was counted, and the test results are shown in Figure 10A , Figure 10B and Figure 10C and Tables 16A to 16C. It can be seen that all the LNP formulations of this example have strong expression in mice in vivo. Moreover, the experiments have shown that when the molar percentage content of PEG lipid is between 1.5%-2.5%, the expression effect is better; more preferably, the molar percentage content of PEG lipid is 1.5%-2.0%. In addition, we tested a variety of PEG lipids, and the prepared LNP formulations can all achieve in vivo delivery and expression.

[0673] Table 16A Figure 10A AUC data of

[0674] Group AUC (p / s*hr) LQ104-E16b-2 (PEG-f1) 7.15E+11 LQ104-E16b-2 (PEG-f2) 4.36E+11 LQ104-E16b-2 (PEG-f3) 1.58E+11 LQ104-E16b-2 (PEG-f4) 1.15E+11 LQ104-E16b-2 (PEG-f5) 4.81E+10 LQ104-E16b-2 (PEG-f6) 2.49E+10 LQ104-E16b-2 (PEG-f7) 1.27E+10

[0675] Table 16B Figure 10B AUC data of

[0676] Group AUC (p / s*hr) LQ104-E16b-2 (PEG-f8) 5.97E+11 LQ104-E16b-2 (PEG-f9) 7.88E+11 LQ104-E16b-2 (PEG-f10) 2.53E+11 LQ104-E16b-2 (PEG-f11) 6.88E+10 LQ104-E16b-2 (PEG-f12) 5.90E+10 LQ104-E16b-2 (PEG-f13) 9.23E+09 LQ104-E16b-2 (PEG-f14) 6.51E+09

[0677] Table 16C Figure 10C AUC data of Table 16C

[0678] Group AUC (p / s*hr) LQ104-E16b-2 (PEG-f15) 4.62E+11 LQ104-E16b-2 (PEG-f16) 2.30E+11 LQ104-E16b-2 (PEG-f17) 1.35E+11 LQ104-E16b-2 (PEG-f18) 1.04E+11 LQ104-E16b-2 (PEG-f19) 1.78E+10 LQ104-E16b-2 (PEG-f20) 1.23E+10 LQ104-E16b-2 (PEG-f21) 9.78E+09

[0679] Example 11

[0680] In the same manner as in Reference Example 8, LNP formulations (encapsulating Fluc mRNA) were prepared according to the molar ratios and nitrogen to phosphorus ratios in Table 17. This example was mainly used to verify the performance of LNP formulations prepared with different nitrogen to phosphorus ratios. We prepared LNP formulations with nitrogen to phosphorus ratios of 4: 1 to 8: 1 based on the preferable ranges of the mole percentages of ionizable lipid and DSPC verified in the previous examples. The particle size, PDI and encapsulation efficiency of each LNP formulation were determined, and the results are shown in Table 17.

[0681] Table 17

[0682]

[0683]

[0684] As can be seen from Table 17, the particle size of the LNP formulations of this example was between 60-120 nm, the PDI was less than 0.3, and the encapsulation efficiency was higher than 96%.

[0685] In the same manner as in Reference Example 3, each group of LNP formulations of this example was injected into 6 to 8-week-old female Balb / C mice through the tail vein at a dose of 5 pg per mouse, and the total luminescence intensity of the liver of the mice in vivo was counted, and the test results are shown in Figure 11A , Figure 11B and Figure 11C and Tables 18A to 18C. It can be seen that when the nitrogen to phosphorus ratio is in the range of 4: 1 to 8: 1, the LNP formulations have strong expression in mice.

[0686] Table 18A Figure 11A AUC data of Table 18A

[0687] Group AUC (p / s*hr) LQ104-E16b-2 (NP-f1) 1.63E+11 LQ104-E16b-2 (NP-f2) 1.63E+11 LQ104-E16b-2 (NP-f3) 1.70E+11 LQ104-E16b-2 (NP-f4) 1.63E+11

[0688] Table 18B Figure 11B AUC data of Table 18B

[0689]

[0690]

[0691] Table 18C Figure 11C AUC data of Table 18C

[0692] Group AUC (p / s*hr) LQ104-E16b-2 (NP-f9) 1.65E+11 LQ104-E16b-2 (NP-f10) 1.95E+11 LQ104-E16b-2 (NP-fll) 1.22E+11 LQ104-E16b-2 (NP-f12 1.58E+11

[0693] Example 12

[0694] In the same manner as in Reference Example 8, the LNP formulations (encapsulating Fluc mRNA) were prepared according to the molar ratios and N:P ratios in Table 19. This example selected the ionizable lipids prepared in Preparation Examples 3 to 17, and was mainly used to verify the performance of the three-component (ionizable lipid, cholesterol and DMG-PEG) LNP formulations in this application. The particle size, PDI and encapsulation efficiency of each LNP were determined, and the results are shown in Table 19.

[0695] Table 19

[0696]

[0697]

[0698]

[0699] As can be seen from Table 19, the particle sizes of the LNP formulations in this example were all between 60-130 nm, the PDIs were all less than 0.3, and the encapsulation efficiencies were all higher than 90%.

[0700] In the same manner as in Reference Example 3, each LNP formulation in this example was injected into 6 to 8-week-old female Balb / C mice at a dose of 5 μg per mouse by tail vein injection or intramuscular injection in the lower limbs, and the total luminescence intensity at the liver site (mainly expressed region when administered by tail vein injection) and the lower limb administration site (mainly expressed region when administered by intramuscular injection in the lower limbs) was counted, and the test results are shown in Figures 12A to 12D and Tables 20A to 20D. Among them Figure 12A and Figure 12B show the luminescence counting results at the liver site of the mice after intravenous injection, Figure 12C and Figure 12D show the luminescence counting results at the lower limb administration site of the mice after intramuscular injection. As can be seen from Figures 12A to 12D all the LNP formulations in this example had strong expression in mice.

[0701] Table 20A Figure 12A AUC data of

[0702]

[0703]

[0704] Table 20B Figure 12B AUC data of

[0705] Group AUC (p / s*hr) LQ104-E16b-l (f2) 1.22E+11 LQ104-E16b-2 (f2) 3.50E+11 LQ104-E16b-2 (f3) 3.69E+11 LQ104-E16b-2 (f4) 3.67E+11 LQ104-E16b-2 (f5) 1.01E+11 LQ104-E16b-3 (f2) 6.34E+10

[0706] Table 20C (AUC data of the LNP formulations of Example 12) Figure 12C

[0707] Group AUC (p / s*hr) LQ104 (fl) 6.83E+09 LQ104 (f2) 9.21E+09 LQ104-E15b-l (f2) 1.01E+10 LQ104-E15b-2 (f2) 1.11E+10 LQ104-E15b-3 (f2) 1.24E+10 LQ104-E17b-l (f2) 6.86E+09 LQ104-E17b-2 (f2) 7.77E+09 LQ104-E17b-3 (f2) 9.34E+09 LQ104-E17b-4 (f2) 8.06E+09 LQ104-E18b-2 (f2) 8.96E+09 LQ104-E18b-3 (f2) 8.19E+09

[0708] Table 20D (AUC data of the LNP formulations of Example 13) Figure 12D

[0709]

[0710] Example 13

[0711] This example selected LQ104-E16b-2 obtained by Preparation Example 7 as an ionizable lipid, and prepared 16 kinds of LNP formulations (encapsulating Fluc mRNA) according to the molar ratio and nitrogen-phosphorus ratio in Table 21 in the manner of Reference Example 8, and determined their particle size, PDI and encapsulation efficiency.

[0712] Table 21

[0713]

[0714]

[0715]

[0716] As can be seen from Table 21, when DMG-PEG2000 is between 0.5 mol% and 3 mol%, the particle size of each group of LNP formulations prepared from LQ104-E16b-2 is between 70 and 150 nm; the PDI is less than 0.3; the encapsulation efficiency is higher than 90%, and is higher than 95.6%.

[0717] Similarly, according to the mouse in vivo test method of Reference Example 3, all the LNP formulations prepared in this example were injected into 6-8 week old female Balb / C mice through the tail vein at a dose of 5 μg per mouse, and the total luminescence intensity of the liver region was counted in the same manner as Example 3, and the test results are shown in Tables 22 and 23 and Figure 13A and 13B ​​It can be seen that the LNP formulations tested in this example have strong expression in mice. Among them, the total luminescence intensity of LQ104-E16b-2(PEG-f1) to LQ104-E16b-2(PEG-f6) in the 0% DSPC group is higher, indicating that the molar percentage content of PEG lipid in this group is between 0.25% and 2%, and the in vivo delivery ability of the corresponding LNP formulation is better; the total luminescence intensity of LQ104-E16b-2(PEG-f9) to LQ104-E16b-2(PEG-f15) in the 2% DSPC group is higher, indicating that the molar percentage content of PEG lipid in this group is between 0.25% and 2.5%, and the in vivo delivery ability of the corresponding LNP formulation is better.

[0718] Table 22 Figure 13A AUC data of

[0719] Group AUC (p / s*hr) LQ104-E16b-2 (PEG-fl) 5.71E+11 LQ104-E16b-2 (PEG-f2) 4.63E+11 LQ104-E16b-2 (PEG-f3) 7.64E+11 LQ104-E16b-2 (PEG-f4) 8.18E+11 LQ104-E16b-2 (PEG-f5) 4.43E+11 LQ104-E16b-2 (PEG-f6) 3.92E+11 LQ104-E16b-2 (PEG-f7) 1.94E+11 LQ104-E16b-2 (PEG-f8) 4.74E+10

[0720] Table 23 Figure 13B AUC data of

[0721]

[0722]

[0723] In the prior art, it is difficult to achieve good delivery effect when the content of phospholipid in the LNP formulation is below 4 mol%. However, in the LNP formulation prepared by using the ionizable lipid compound provided in the present application, even if the phospholipid component is as low as below 4 mol%, for example, 0 mol% to 2 mol%, as shown in this example, the prepared LNP formulation still has good properties and delivery ability. At the same time, without increasing the content of PEG lipid, for example, in the content range of 0.5 mol% to 2.5 mol%, the prepared LNP formulation still has good properties and delivery ability, thereby reducing the risk of affecting the efficacy and safety due to the increase of PEG lipid.

Claims

1. A nitrogen-containing chain compound as shown in Formula I, or a pharmaceutically acceptable salt thereof, characterized in that, ； Where Z and W are independently C4-C 10 Alkylene; Y and Q independently ; A is a C2-C6 alkylene group. , or ; Each R A-1 and R A-2 Independently a C2-C6 alkylene group; M is a C1-C6 alkylene group; R 1 and R 2 Independently for C6-C 20 Alkyl groups; R 5 For not replaced or by 1, 2 or 3 R 5-1 Replacement C2-C 10 Alkyl groups; Each R 5-1 Independently hydroxyl or ; R 5-1-1 Independently for C6-C 20 Alkyl groups; R 6 For not replaced or by 1, 2 or 3 R 6-1 Replacement C2-C 10 Alkyl groups; Each R 6-1 Independently hydroxyl or ; R 6-1-1 Independently for C6-C 20 Alkyl groups.

2. The nitrogen-containing chain compound of formula I as claimed in claim 1, characterized in that, The nitrogen-containing chain compound as shown in Formula I satisfies one or more of the following conditions: (1) In Z, the C4-C 10 The alkylene group is a C5-C8 alkylene group; (2) In W, the C4-C 10 The alkylene group is a C5-C8 alkylene group; (3) In A, the C2-C6 alkylene group is , , , , , , , , or ; (4) R A-1 In this context, the C2-C6 alkylene group is... , , , , , , , , or ; (5) R A-2 In this context, the C2-C6 alkylene group is... , , , , , , , , or ; (6) In M, the C1-C6 alkylene groups are , , , , , , , , , or ; (7) R 1 In the context, the C6-C 20 The alkyl group is C 10 -C 18 ; (8) R 2 In the context, the C6-C 20 The alkyl group is C 10 -C 18 ; (9) R 5 In the context, C2-C 10 The alkyl group is a C2-C8 alkyl group; (10) R 5-1-1 In the context, the C6-C 20 The alkyl group is C 11 -C 18 ; (11) R 6 In the context, C2-C 10 The alkyl group is a C2-C8 alkyl group; (12) R 6-1-1 In the context, the C6-C 20 The alkyl group is C 11 -C 18 ; The nitrogen-containing chain compound shown in formula I and (13) are nitrogen-containing chain compounds shown in formula Ia. .

3. The nitrogen-containing chain compound of formula I as described in claim 1, characterized in that, The nitrogen-containing chain compound as shown in Formula I satisfies one or more of the following conditions: (1) In Z, the C4-C 10 The alkylene groups are straight-chain alkanes; (2) In W, the C4-C 10 The alkylene groups are straight-chain alkanes; (3) In A, the C2-C6 alkylene group is ; (4) R A-1 In this context, the C2-C6 alkylene group is... ; (5) R A-2 In this context, the C2-C6 alkylene group is... ; (6) In M, the C1-C6 alkylene groups are ; (7) R 1 In the context, the C6-C 20 The alkyl group is or ; (8) R 2 In the context, the C6-C 20 The alkyl group is or ; (9) R 5 In the context, C2-C 10 The alkyl group is , , , , , , , , or ; (10) R 5-1-1 In the context, the C6-C 20 The alkyl group is or ; (11) R 6 In the context, C2-C 10 The alkyl group is , , , , , , , , or ; and (12)R 6-1-1 In the context, the C6-C 20 The alkyl group is or .

4. The nitrogen-containing chain compound of formula I as claimed in claim 1, characterized in that, The nitrogen-containing chain compound as shown in Formula I satisfies one or more of the following conditions: (1) In Z, the C4-C 10 The alkylene group is or ; (2) In W, the C4-C 10 The alkylene group is or ; (3) R 5 In the context, C2-C 10 The alkyl group is , or ; and (4)R 6 In the context, C2-C 10 The alkyl group is , or .

5. The nitrogen-containing chain compound of formula I as claimed in claim 1, characterized in that, The nitrogen-containing chain compound as shown in Formula I satisfies one or more of the following conditions: (1) Z and W are the same; (2) R 1 and R 2 same; (3) R 5 and R 6 same; (4) Q and Y are the same.

6. The nitrogen-containing chain compound of formula I as claimed in claim 1, characterized in that, The nitrogen-containing chain compound as shown in Formula I satisfies one or more of the following conditions: (1) Z and W are independently C5-C8 alkylene groups; (2) A is a C2-C6 alkylene group or ; (3) R A-1 and R A-2 Independently a C2-C4 alkylene group; (4) M is methylene; (5) R 1 and R 2 Independently for C 10 -C 18 ; (6) R 5 For 1, 2 or 3 R 5-1 Substituted C2-C8 alkyl groups; (7) R 5-1-1 C 10 -C 18 Alkyl groups; (8) R 6 For 1, 2 or 3 R 6-1 Substituted C2-C8 alkyl groups; (9) R 6-1-1 C 10 -C 18 Alkyl groups; (10) The nitrogen-containing chain compound shown in Formula I is a compound with bilateral symmetry; (11) Y is , where a and R 2 Connected, b is connected to Z; And (12)Q is , where a and R 1 Connected, b is connected to W.

7. The nitrogen-containing chain compound of formula I as claimed in claim 1, characterized in that, The nitrogen-containing chain compound as shown in Formula I satisfies one or more of the following conditions: (1) R 1 and R 2 Independently for C 10 -C 12 ; (2) R 5-1-1 C 14 -C 18 Alkyl groups; and (3)R 6-1-1 C 14 -C 18 Alkyl groups.

8. The nitrogen-containing chain compound of formula I as claimed in claim 1, characterized in that, The nitrogen-containing chain compound as shown in Formula I satisfies one or more of the following conditions: (1) R 1 and R 2 Independently ; (2) R 5-1-1 for ; and (3)R 6-1-1 for .

9. The nitrogen-containing chain compound of formula I as claimed in claim 1, characterized in that, The nitrogen-containing chain compound as shown in Formula I is Scheme 1 or Scheme 2: Option 1; Y is , where a and R 2 Connected, b is connected to Z; Q is , where a and R 1 Connected, b is connected to W; Z and W are independently C5-C8 alkylene groups; A is a C2-C6 alkylene group or ; R A-1 and R A-2 Independently a C2-C4 alkylene group; M stands for methylene; R 1 and R 2 Independently for C 10 -C 18 ; R 5 For 1, 2 or 3 R 5-1 Substituted C2-C8 alkyl groups; R 5-1-1 C 10 -C 18 Alkyl groups; R 6 For 1, 2 or 3 R 6-1 Substituted C2-C8 alkyl groups; R 6-1-1 C 10 -C 18 Alkyl groups; Option 2; Q and Y are the same; Z and W are the same; R 1 and R 2 same; R 5 and R 6 same; Z and W are independently C5-C8 alkylene groups; A is a C2-C6 alkylene group or ; R A-1 and R A-2 Independently a C2-C4 alkylene group; M stands for methylene; R 1 and R 2 Independently ; R 5 For 1, 2 or 3 R 5-1 Substituted C2-C8 alkyl groups; R 5-1-1 C 14 -C 18 Alkyl groups; R 6 For 1, 2 or 3 R 6-1 Substituted C2-C8 alkyl groups; R 6-1-1 C 14 -C 18 Alkyl groups.

10. Any of the following compounds or their pharmaceutically acceptable salts: , , , or .

11. A method for preparing a nitrogen-containing chain compound as shown in Formula I, characterized in that, It includes the following steps: in a solvent, in the presence of a base and an iodide salt, the compound shown in Formula I-1 and the compound shown in Formula I-2 undergo a coupling reaction as shown in the following formula; ； X is a halogen, A is a C2-C6 alkylene group, and Y, Q, Z, W, and R are also present. 5 R 6 R 1 and R 2 As described in any one of claims 1-10; and Y is the same as Q, R 1 With R 2 They are the same; Z and W are identical.

12. The preparation method according to claim 11, characterized in that, The method for preparing the nitrogen-containing chain compound as shown in Formula I satisfies one or more of the following conditions: (1) In the coupling reaction, the halogen is fluorine, chlorine, bromine or iodine; (2) In the coupling reaction, the molar ratio of the compound shown in Formula I-2 to the compound shown in Formula I-2 is 1:(1-3). (3) In the coupling reaction, the base is a basic carbonate; (4) In the coupling reaction, the molar ratio of the compound shown in Formula I-2 to the base is 1:(1-5). (5) In the coupling reaction, the solvent is an ether solvent and / or a nitrile solvent; (6) In the coupling reaction, the mass-to-volume ratio of the compound as shown in Formula I-2 to the solvent is 10-50 mg / mL; (7) In the coupling reaction, the iodine salt is a basic iodine salt; (8) In the coupling reaction, the molar ratio of the compound shown in Formula I-2 to the iodide salt is 1:(1-2). In the coupling reaction described in (9), the reaction temperature of the coupling reaction is 50-100℃.

13. The preparation method according to claim 12, characterized in that, The method for preparing the nitrogen-containing chain compound as shown in Formula I satisfies one or more of the following conditions: (1) In the coupling reaction, the halogen is bromine; (2) In the coupling reaction, the molar ratio of the compound shown in Formula I-2 to the compound shown in Formula I-2 is 1:2.6; (3) In the coupling reaction, the base is K2CO3; (4) In the coupling reaction, the molar ratio of the compound as shown in Formula I-2 to the base is 1:3.5; (5) In the coupling reaction, the ether solvent is methyl tert-butyl ether; (6) In the coupling reaction, the nitrile solvent is acetonitrile; (7) In the coupling reaction, the mass-to-volume ratio of the compound as shown in Formula I-2 to the solvent is 16 mg / mL; (7) In the coupling reaction, the iodine salt is KI; (8) In the coupling reaction, the molar ratio of the compound as shown in Formula I-2 to the iodide salt is 1:1.2; In the coupling reaction described in (9), the reaction temperature of the coupling reaction is 80°C.

14. The preparation method according to claim 12, characterized in that, The volume ratio of the nitrile solvent to the ether solvent is 1:

1.

15. A lipid carrier, characterized in that, It includes substance Z, which is selected from one or more of the compounds as described in any one of claims 1-10 or their pharmaceutically acceptable salts.

16. The lipid carrier according to claim 15, characterized in that, The lipid carrier satisfies one or more of the following conditions: (1) The lipid carrier further includes a diluent; (2) The lipid carrier also includes phospholipids; (3) The lipid carrier also includes PEG lipids; The lipid carriers described in (4) also include sterols.

17. The lipid carrier according to claim 16, characterized in that, The lipid carrier satisfies one or more of the following conditions: (1) The diluent is phosphate buffer or Tris buffer; (2) The phospholipid is a phospholipid molecule with an electric polar end and a nonpolar end of an adipose chain; (3) The PEG lipid is a lipid molecule modified with a hydrophilic end of polyethylene glycol; (4) The sterols include animal, plant or fungal sterols; (5) In the lipid carrier, the molar ratio of substance Z to sterol is 0.5-5:1; (6) In the lipid carrier, the molar ratio of substance Z to phospholipid is 1~15:1; (7) In the lipid carrier, the molar ratio of substance Z to PEG lipid is 20~130:1; (8) The molar content of substance Z is 30mol% to 60mol% (9) The molar content of the phospholipid is from 0 mol% to 30 mol%; (10) The molar content of the sterol is from 15 mol% to 55 mol%; The molar content of the PEG lipids described in (11) is from 0 mol% to 10 mol%.

18. The lipid carrier as described in claim 16, characterized in that, The lipid carrier satisfies one or more of the following conditions: (1) The phospholipid is distearylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, palmitoylphosphatidylcholine, 1,2-distearylphosphatidylcholine, docosanoylphosphatidylcholine or palmitoylphosphatidylcholine. (2) The PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol; (3) The sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid and α-tocopherol; (4) In the lipid carrier, the molar ratio of substance Z to sterol is 0.5-3:1; (5) In the lipid carrier, the molar ratio of substance Z to phospholipid is 2~8:1; In the lipid carrier described in (6), the molar ratio of substance Z to PEG lipid is 20~80:

1.

19. The lipid carrier as described in claim 16, characterized in that, The lipid carrier satisfies one or more of the following conditions: (1) The PEG lipid is a PEG-modified myristoyl glycerol; (2) The sterol mentioned is cholesterol; (3) In the lipid carrier, the molar ratio of substance Z to sterol is 0.6-2:1; (4) In the lipid carrier, the molar ratio of substance Z to phospholipid is 3~6:1; In the lipid carrier described in (5), the molar ratio of substance Z to PEG lipid is 20-40:

1.

20. A lipid nanoparticle, characterized in that, It includes therapeutic agents and / or preventive agents, as well as lipid carriers as described in any one of claims 15-19.

21. The lipid nanoparticles according to claim 20, characterized in that, The lipid nanoparticles satisfy one or more of the following conditions: (1) The therapeutic agent and / or preventive agent is one or two or more nucleic acids; (2) The nitrogen-to-phosphorus ratio in the lipid nanoparticles is 2:1-30:1; (3) The lipid nanoparticles have a particle size of 10-200 nm; (4) In the lipid nanoparticles, the mass ratio of the lipid carrier to the therapeutic agent and / or preventive agent is 3-80:1; In the lipid nanoparticles of (5), the lipid carrier encapsulates the therapeutic agent and / or preventive agent.

22. The lipid nanoparticles according to claim 20, characterized in that, The lipid nanoparticles satisfy one or more of the following conditions: (1) The therapeutic agent and / or preventive agent is single-stranded deoxyribonucleic acid, double-stranded DNA, small interfering RNA, asymmetric double-stranded small interfering RNA, microRNA, small hairpin RNA, circular RNA, transfer RNA or messenger RNA; (2) The nitrogen-to-phosphorus ratio in the lipid nanoparticles is 2:1-20:1; (3) The lipid nanoparticles have a particle size of 40-150 nm; In the lipid nanoparticles described in (4), the mass ratio of the lipid carrier to the therapeutic agent and / or preventive agent is 6-60:

1.

23. The lipid nanoparticles as described in claim 20, characterized in that, The lipid nanoparticles satisfy one or more of the following conditions: (1) The therapeutic agent and / or preventive agent is mRNA; (2) The nitrogen-to-phosphorus ratio in the lipid nanoparticles is 3:1-20:1; The lipid nanoparticles described in (3) have a particle size of 60-150 nm.

24. The lipid nanoparticles according to claim 20, characterized in that, The lipid nanoparticles satisfy one or two of the following conditions: (1) The therapeutic agent and / or preventive agent is firefly luciferase mRNA or SARS-CoV-2 spike protein mRNA; The nitrogen-to-phosphorus ratio in the lipid nanoparticles described in (2) is 3:1-16:

1.

25. A composition, characterized in that, It includes substance Z, which is a compound as described in any one of claims 1-10 or a pharmaceutically acceptable salt thereof.

26. The composition of claim 25, characterized in that, The composition further includes one or more of a diluent, phospholipids, PEG lipids, sterols, and therapeutic and / or preventative agents.

27. The composition of claim 26, characterized in that, In the composition, the diluent, phospholipid, PEG lipid, and sterol are as described in any one of claims 16-19; and / or the therapeutic and / or preventative agent are as described in any one of claims 21-24.

28. The composition of claim 26, characterized in that, In the composition, substance Z forms a lipid carrier as described in any one of the diluent, phospholipid, PEG lipid and sterol as described in any one of claims 15-19; Or, in the composition, the encapsulation rate of the therapeutic agent and / or preventive agent is at least 50%.

29. The composition according to claim 26, characterized in that, In the composition, the encapsulation rate of the therapeutic agent and / or preventive agent is at least 70%.

30. The composition of claim 26, characterized in that, In the composition, the lipid carrier forms lipid nanoparticles as described in any one of claims 20-24 with the therapeutic agent and / or preventive agent; and or, in the composition, the polydispersity index of the composition is not higher than 0.

5.

31. The composition according to claim 26, characterized in that, In the composition, the polydispersity index is not higher than 0.3.

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