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

Abstract

Disclosed are a nitrogen-containing chain compound, a preparation method thereof, 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 can encapsulate a nucleic acid drug, and can be used for delivering a nucleic acid prophylactic and / or therapeutic agent to mammalian cells and organs and playing a role.

Description

Technical Field

[0001] This invention relates to a nitrogen-containing chain compound, its preparation method, compositions comprising the compound, and its applications. Background Technology

[0002] Nucleic acid drugs represent an important direction in current basic and applied research. They can be used for the prevention and / or treatment of viral and bacterial infectious diseases, tumors, metabolic diseases, etc., and their production costs are lower and the cycle shorter, facilitating the rapid development of personalized medicines. However, nucleic acids are negatively charged macromolecules, making them difficult to penetrate cell membranes, and they also exhibit poor stability. Developing various nucleic acid packaging and delivery systems can, to some extent, overcome the instability of nucleic acid drugs and improve their delivery efficiency.

[0003] Lipid nanoparticles have been shown to be effective carriers for delivering bioactive substances (such as small molecule drugs, proteins, and nucleic acids) into cells and / or intracellular compartments. Optimizing nucleic acid drug delivery systems by designing and optimizing the types and amounts of components within lipid nanoparticles is crucial for improving the efficacy of nucleic acid drugs for prevention and treatment, especially for lipid compounds and related methods and compositions for delivering RNA preventative agents and / or therapeutic agents. Summary of the Invention

[0004] This invention aims to provide a novel ionizable lipid compound for delivering nucleic acid drugs, increasing the variety of ionizable lipid compounds and the selection of delivery carriers for nucleic acid prophylaxis and / or therapeutic agents. To address the above technical problems, this invention provides a nitrogen-containing chain compound, its preparation method, compositions comprising the compound, and its applications. The compositions of this invention can be used for the efficient delivery of nucleic acid drugs.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides a nitrogen-containing chain compound as shown in Formula I, or a pharmaceutically acceptable salt thereof.

[0007]

[0008] Among them, A 1 for A 2 for

[0009] Y is a C1-C6 alkylene group;

[0010] m is an integer between 0 and 4;

[0011] n is an integer between 0 and 4;

[0012] R 1 H, unsubstituted, or with 1, 2, or 3 Rs1-1 Substituted C1-C6 alkyl or

[0013] Each R 1-1 Independently, it is amino, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy,

[0014] R 1-1-1 It is a C1-C6 alkyl group; R 1-1-2 It is a C1-C6 alkyl group; R 1-1-3 It is an alkyl group of C1-C6;

[0015] R 1-2 It is an alkyl group of C1-C6;

[0016] R 2 H, unsubstituted, or with 1, 2, or 3 Rs 2-1 Substituted C1-C6 alkyl or

[0017] Each R 2-1 Independently, it is amino, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy,

[0018] R 2-1-1 It is a C1-C6 alkyl group; R 2-1-2 It is a C1-C6 alkyl group; R 2-1-3 It is an alkyl group of C1-C6;

[0019] R 2-2 It is an alkyl group of C1-C6;

[0020] R 3 For not replaced or by 1, 2 or 3 R 3-1 Replacement C6-C 20 Alkyl, unsubstituted or with 1, 2 or 3 R 3-2 Replacement C6-C 20 alkenyl groups;

[0021] Each R 3-1 Independently C1-C6 alkyl, amino, hydroxyl, halogen, C1-C6 alkoxy, C2-C 10 alkenyl,

[0022] R 3-1-1 It is a C1-C6 alkyl group; R 3-1-2 It is a C1-C6 alkyl group; R 3-1-3 It is an alkyl group of C1-C6;

[0023] Each R 3-2 Independently C1-C6 alkyl, amino, hydroxyl, halogen, C1-C6 alkoxy, C2-C 10 alkenyl,

[0024] R 3-2-1 It is an alkyl group of C1-C6;

[0025] R 3-2-2 It is an alkyl group of C1-C6;

[0026] R 3-2-3 It is an alkyl group of C1-C6;

[0027] R 4 For not replaced or by 1, 2 or 3 R 4-1 Replacement C6-C 20 Alkyl, unsubstituted or with 1, 2 or 3 R 4-2 Replacement C6-C 20 alkenyl groups;

[0028] Each R 4-1 Independently C1-C6 alkyl, amino, hydroxyl, halogen, C1-C6 alkoxy, C2-C 10 alkenyl,

[0029] R 4-1-1 It is a C1-C6 alkyl group; R 4-1-2 It is a C1-C6 alkyl group; R 4-1-3 It is an alkyl group of C1-C6;

[0030] Each R 4-2 Independently C1-C6 alkyl, amino, hydroxyl, halogen, C1-C6 alkoxy, C2-C 10 alkenyl,

[0031] R 4-2-1 It is a C1-C6 alkyl group; R 4-2-2 It is a C1-C6 alkyl group; R 4-2-3 It is a C1-C6 alkyl group.

[0032] In a preferred embodiment, in the compound represented by Formula I or its pharmaceutically acceptable salt, certain groups may be defined as follows, and other groups may be defined as in any other embodiment (hereinafter referred to as "in a preferred embodiment"): m is 0 or 1.

[0033] In a preferred solution, n is either 0 or 1.

[0034] In a preferred embodiment, in Y, the C1-C6 alkylene group may be...

[0035] For example

[0036] In a certain preferred solution, R 1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl; for example, methyl.

[0037] In a certain preferred solution, R 1-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0038] In a certain preferred solution, R 1-1 In this context, the C1-C6 alkoxy groups can be C1-C4 alkoxy groups, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy.

[0039] In a certain preferred solution, R 1-1-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0040] In a certain preferred solution, R 1-1-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0041] In a certain preferred solution, R 1-1-3 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0042] In a certain preferred solution, R 1-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0043] In a certain preferred solution, R 2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl; for example, methyl.

[0044] In a certain preferred solution, R2-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0045] In a certain preferred solution, R 2-1 In this context, the C1-C6 alkoxy groups can be C1-C4 alkoxy groups, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy.

[0046] In a certain preferred solution, R 2-1-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0047] In a certain preferred solution, R 2-1-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0048] In a certain preferred solution, R 2-1-3 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0049] In a certain preferred solution, R 2-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0050] In a certain preferred solution, R 3 In the context, the C6-C 20 The alkyl group can be C 10 -C 18 Alkyl groups, and can also be C 15 -C 18 Alkyl groups, preferably straight-chain alkyl groups, for example

[0051] In a certain preferred solution, R 3 In the context, the C6-C 20 The alkenyl group can be C 10 -C 18 The alkenyl group, preferably C 15 -C 18 The alkenyl group, more preferably a straight-chain alkenyl group; the C6-C 20 The alkenyl group can contain 1 to 4 double bonds, for example

[0052] In a certain preferred solution, R 3-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0053] In a certain preferred solution, R 3-1 In this context, the C1-C6 alkoxy groups can be C1-C4 alkoxy groups, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy.

[0054] In a certain preferred solution, R 3-1 In this context, the halogen may be fluorine, chlorine, bromine, or iodine.

[0055] In a certain preferred solution, R 3-1 In the context, C2-C 10 The alkenyl group can be a C2-C6 alkenyl group, preferably a straight-chain alkenyl group.

[0056] In a certain preferred solution, R 3-1-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0057] In a certain preferred solution, R 3-1-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0058] In a certain preferred solution, R 3-1-3 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0059] In a certain preferred solution, R 3-2 In this context, the halogen may be fluorine, chlorine, bromine, or iodine.

[0060] In a certain preferred solution, R 3-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0061] In a certain preferred solution, R 3-2 In this context, the C1-C6 alkoxy groups can be C1-C4 alkoxy groups, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy.

[0062] In a certain preferred solution, R 3-2 In this context, the halogen may be fluorine, chlorine, bromine, or iodine.

[0063] In a certain preferred solution, R 3-2 In the context, C2-C 10 The alkenyl group can be a C2-C6 alkenyl group, preferably a straight-chain alkenyl group.

[0064] In a certain preferred solution, R 3-2-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0065] In a certain preferred solution, R 3-2-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0066] In a certain preferred solution, R 3-2-3 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0067] In a certain preferred solution, R 4 In the context, the C6-C 20 The alkyl group can be C 10 -C 18 Alkyl groups, and can also be C 15 -C 18 Alkyl groups, preferably straight-chain alkyl groups, for example

[0068] In a certain preferred solution, R 4 In the context, the C6-C 20 The alkenyl group can be C 10 -C 18 The alkenyl group, preferably C 15 -C 18 The alkenyl group, more preferably a straight-chain alkenyl group; the C6-C 20 The alkenyl group can contain 1 to 4 double bonds, for example

[0069] In a certain preferred solution, R 4-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0070] In a certain preferred solution, R 4-1 In this context, the C1-C6 alkoxy groups can be C1-C4 alkoxy groups, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy.

[0071] In a certain preferred solution, R 4-1 In this context, the halogen may be fluorine, chlorine, bromine, or iodine.

[0072] In a certain preferred solution, R 4-1 In the context, C2-C 10 The alkenyl group can be a C2-C6 alkenyl group, preferably a straight-chain alkenyl group.

[0073] In a certain preferred solution, R 4-1-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0074] In a certain preferred solution, R 4-1-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0075] In a certain preferred solution, R 4-1-3 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0076] In a certain preferred solution, R 4-2 In this context, the halogen may be fluorine, chlorine, bromine, or iodine.

[0077] In a certain preferred solution, R 4-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0078] In a certain preferred solution, R 4-2 In this context, the C1-C6 alkoxy groups can be C1-C4 alkoxy groups, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy.

[0079] In a certain preferred solution, R 4-2 In this context, the halogen may be fluorine, chlorine, bromine, or iodine.

[0080] In a certain preferred solution, R 4-2 In the context, C2-C 10 The alkenyl group can be a C2-C6 alkenyl group, preferably a straight-chain alkenyl group.

[0081] In a certain preferred solution, R 4-2-1 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0082] In a certain preferred solution, R 4-2-2 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0083] In a certain preferred solution, R 4-2-3 In this context, the C1-C6 alkyl group can be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0084] In a preferred embodiment, the nitrogen-containing chain compound as shown in Formula I is a compound as shown in Formulas Ia, Ib, Ic, Id, or Ie.

[0085]

[0086] In a certain preferred solution, A 1 for Where a is connected to CH2, b is connected to R 3 Connected.

[0087] In a certain preferred solution, A 2 for Where a is connected to CH2, b is connected to R 4 Connected.

[0088] In a certain optimal solution, m and n are the same.

[0089] In a certain preferred solution, R 1 and R 2 same.

[0090] In a certain preferred solution, R 3 and R 4 same.

[0091] In a certain preferred solution, R 1 It is a C1-C4 alkyl group, such as methyl.

[0092] In a certain preferred solution, R 2 It is a C1-C4 alkyl group, such as methyl.

[0093] In a certain preferred solution, R 3 C 10 -C 18 alkyl or C 10 -C 18 The alkenyl group, preferably C 15 -C 18 alkyl or C 15 -C 18 alkenyl groups, for example

[0094] In a certain preferred solution, R 4 C 10 -C 18 alkyl or C 10 -C 18 The alkenyl group, preferably C 15 -C 18 alkyl or C 15 -C 18 alkenyl groups, for example

[0095] In a preferred embodiment, Y is a C1-C4 alkylene group, for example...

[0096] In a certain preferred solution, R 1 It is a C1-C4 alkyl group; R 2 It is a C1-C4 alkyl group;

[0097] R 3 C 10 -C 18 alkyl or C 10 -C 18 alkenyl groups;

[0098] R 4 C 10 -C 18 alkyl or C 10 -C 18 alkenyl groups;

[0099] Y is a C1-C4 alkylene group.

[0100] In a certain preferred solution, R 1 It is a C1-C4 alkyl group; R 2 It is a C1-C4 alkyl group;

[0101] R 3 C 15 -C 18 alkyl or C 15 -C 18 alkenyl groups;

[0102] R 4 C 15 -C 18 alkyl or C 15 -C 18 alkenyl groups;

[0103] Y is a C1-C4 alkylene group.

[0104] In a preferred embodiment, the nitrogen-containing chain compound as shown in Formula I is any of the following compounds:

[0105]

[0106]

[0107]

[0108]

[0109] The present invention also provides a method for preparing a compound as shown in Formula I, comprising the following steps: in a solvent, in the presence of a base and a condensing agent, a compound as shown in Formula I-1 and a compound as shown in Formula I-2 undergo a condensation reaction as shown in the following formula;

[0110]

[0111] Among them, A 1 A 2 m, n, R 1 R 2 R 3 R 4 As mentioned before, Y.

[0112] In the condensation reaction, the molar ratio of the compound shown in Formula I-2 to the compound shown in Formula I-1 can be 1:(1-1.2), for example 1:1 or 1:1.08.

[0113] In the condensation reaction, the condensing agent is a conventional condensing agent in the art, preferably one or more of 1-hydroxybenzotriazole (HOBT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), such as "1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride" or 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0114] In the condensation reaction, the molar ratio of the compound as shown in Formula I-2 to the condensing agent is 1:(1-3), for example 1:2.56, 1:1.2 or 1:1.3.

[0115] In the condensation reaction, the base is a conventional base in the art, preferably an organic base, such as triethylamine or N,N-diisopropylethylamine (DIPEA).

[0116] In the condensation reaction, the molar ratio of the compound as shown in Formula I-2 to the base can be 1:(2-4), for example 1:3.

[0117] In the condensation reaction, the solvent is a conventional organic solvent in the art, preferably a halogenated hydrocarbon solvent, such as dichloromethane.

[0118] In the condensation reaction, the mass-to-volume ratio of the compound as shown in Formula I-2 to the solvent can be 30 mg / mL to 60 mg / mL, for example, 50 mg / mL or 40 mg / mL.

[0119] In the condensation reaction, the reaction temperature is a conventional reaction temperature in the art, such as room temperature.

[0120] In the condensation reaction, the reaction time is a conventional reaction time in the art, such as 10h or ≥12h.

[0121] The condensation reaction may also include post-processing, which may be a conventional post-processing step for organic reactions in the art, and may include one or more of the following steps: dilution, extraction, solution washing, water washing, drying, concentration, sample mixing and column chromatography.

[0122] The present invention also provides a lipid carrier comprising substance Z, said substance Z being a compound as described above as shown in Formula I or a pharmaceutically acceptable salt thereof.

[0123] In a preferred embodiment, the lipid carrier further includes a diluent. The diluent may be a phosphate buffer or a Tris buffer, etc.

[0124] In a preferred embodiment, the lipid carrier further includes phospholipids.

[0125] In a preferred embodiment, the phospholipid may be a conventional phospholipid in the art, which is an amphoteric accessory molecule that facilitates the fusion of lipid particles and cell membranes. The phospholipid may be a phospholipid molecule having an electrically polar end and a nonpolar end of a fatty acid chain, such as distearylphosphatidylcholine (DSPC), dimyristicoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoylphosphatidylcholine (DPPC), 1,2-distearylphosphatidylcholine (DSPC), docosanoylphosphatidylcholine (DUPC), or palmitoylphosphatidylcholine (POPC), etc.

[0126] In a preferred embodiment, the lipid carrier further includes PEG lipids (polyethylene glycol-modified lipids).

[0127] In a preferred embodiment, the PEG lipid may be a lipid molecule modified with a polyethylene glycol hydrophilic end. 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).

[0128] In a preferred embodiment, the lipid carrier further includes sterols.

[0129] In a preferred embodiment, the sterol may be a conventional sterol in the art, including animal, plant, or fungal sterols. The sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol, such as cholesterol.

[0130] In a preferred embodiment, the molar ratio of substance Z to sterol in the lipid carrier is 0.5 to 5:1, preferably 0.5 to 3:1, for example 0.6 to 2:1.

[0131] In a preferred embodiment, the molar ratio of substance Z to phospholipid in the lipid carrier is 1 to 15:1, preferably 2 to 8:1, for example 3 to 6:1.

[0132] In a preferred embodiment, the molar ratio of substance Z to PEG lipid in the lipid carrier is 20–130:1, preferably 20–80:1, for example 20–40:1.

[0133] In a preferred embodiment, the molar content of substance Z is approximately 30 mol% to 60 mol%.

[0134] In this invention, molar content refers to the percentage of a substance in the total mass of the lipid carrier, and the sum of the molar contents of all components in the lipid carrier does not exceed 100 mol%. In a preferred embodiment, the molar content of the phospholipid is approximately 0 mol% to 30 mol%.

[0135] In a preferred embodiment, the molar content of the sterol is approximately 15 mol% to 55 mol%.

[0136] In a preferred embodiment, the molar content of the PEG lipid is approximately 0 mol% to 10 mol%.

[0137] In a preferred embodiment, the lipid carrier is composed of the substance Z, the diluent, the phospholipid, the PEG lipid, and the sterol.

[0138] The present invention also provides a lipid nanoparticle comprising a therapeutic agent and / or a preventive agent as well as the aforementioned lipid carrier.

[0139] In a preferred embodiment, the therapeutic agent and / or prophylactic agent may be one or two or more nucleic acids. The nucleic acid may be a conventional nucleic acid in the art. The therapeutic agent and / or prophylactic agent may be 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.

[0140] In a preferred embodiment, the nitrogen-to-phosphorus ratio in the lipid nanoparticles can be 2:1 to 30:1, where the nitrogen-to-phosphorus ratio refers to the ratio of the number of moles of ionizable nitrogen atoms in one or more ionizable lipid compounds to the number of moles of phosphate ester groups in the RNA. Preferably, it is 2:1 to 20:1, for example 3:1 to 20:1, and also, for example, 3:1 to 16:1.

[0141] In a preferred embodiment, the mass ratio of the lipid carrier to the therapeutic agent and / or preventive agent in the lipid nanoparticles can be 3 to 80:1, preferably 10 to 50:1.

[0142] In a preferred embodiment, the particle size (average particle size) of the lipid nanoparticles may be 10–250 nm, for example 10–200 nm, 40–150 nm, or 50–250 nm, or for example 60–150 nm.

[0143] In a preferred embodiment, the lipid nanoparticles contain a lipid carrier that encapsulates the therapeutic agent and / or preventative agent.

[0144] The present invention also provides a composition comprising substance Z, said substance Z being a compound as described above as shown in Formula I or a pharmaceutically acceptable salt thereof.

[0145] In a preferred embodiment, the composition further includes one or more of a diluent, phospholipids, PEG lipids, sterols, and therapeutic and / or preventative agents.

[0146] In a preferred embodiment, the diluent, phospholipid, PEG lipid, sterol, and therapeutic and / or preventative agent in the composition are as described above.

[0147] In a preferred embodiment, in the composition, substance Z forms a lipid carrier as described above with one or more of the diluent, phospholipid, PEG lipid, and sterol.

[0148] In a preferred embodiment, the lipid carrier in the composition forms lipid nanoparticles with the therapeutic agent and / or preventive agent as described above.

[0149] In a preferred embodiment, the encapsulation rate of the therapeutic agent and / or preventive agent in the composition is at least 50%, preferably at least 70%.

[0150] In a preferred embodiment, the polydispersity index of the composition is not higher than 0.5, for example, not higher than 0.3.

[0151] Unless otherwise specified, the terms used in this invention have the following meanings:

[0152] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0153] The term "pharmaceutical acceptable" means that something is relatively non-toxic, safe, and suitable for patient use.

[0154] The term "pharmaceuticalally acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a 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, and ammonium salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, sulfate, and methanesulfonate salts. For details, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0155] In structural fragments This refers to the structural segment being connected to the rest of the molecule through this site. For example, It refers to cyclohexyl.

[0156] The "-" at the end of a group indicates that the group is attached to the rest of the molecule through that site. For example, CH3-C(=O)- refers to an acetyl group.

[0157] The term "alkyl" refers to a saturated monovalent hydrocarbon group that has a specified number of carbon atoms (e.g., C1 to C6), is straight-chain or branched. Alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0158] The term "alkylene" refers to a divalent group that is attached to the rest of the molecule by two single bonds, and the rest of the definition is the same as that of the term "alkyl".

[0159] The term "alkoxy" refers to the group R. X -O-,R X The definition is the same as the term "alkyl". Alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, etc.

[0160] The term "alkenyl" refers to a group having a specified number of carbon atoms (e.g., C2-C3). 20 A straight-chain or branched, unsaturated monovalent hydrocarbon group having one or more (e.g., 1, 2, or 3) carbon-carbon sp groups. 2 Double bond. Alkenyl groups include, but are not limited to: vinyl groups, wait.

[0161] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0162] Unless otherwise specified, room temperature in this invention refers to 20-30℃.

[0163] The reagents and raw materials used in this invention are all commercially available.

[0164] The significant advantages of this invention are as follows: It provides a nitrogen-containing chain compound as shown in Formula I, which has a novel structure and can be used to prepare lipid nanoparticles. Lipid nanoparticles containing the nitrogen-containing chain compound as shown in Formula I have a lower polydispersity index compared to other chain compounds containing two nitrogen atoms, enabling efficient mRNA transport. Attached Figure Description

[0165] Figure 1 The chemiluminescence intensity of 293FT cells after co-culturing with each Fluc-mRNA LNP for 24 h. Detailed Implementation

[0166] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0167] For experimental methods in the following examples where specific conditions are not specified, follow conventional methods and conditions, or select according to the product instructions.

[0168]

[0169] The reagents used in this embodiment are sourced as follows:

[0170] DCC: Purchased from Adamas Reagents Ltd., item number: 012041444, purity: RG, 99%;

[0171] DMAP: Purchased from Adamas Reagents Ltd., catalog number: 01271081, purity: RG, 99%;

[0172] DCM: Purchased from Shanghai Titan Technology Co., Ltd., item number: 01111853, purity: AR, ≥99.5%;

[0173] N-Boc serine: purchased from Bid Pharmaceutical Technology Co., Ltd., product number: BD227276, purity: 97%;

[0174] Oleoyl chloride: purchased from Anaiji Chemical, product number: W810227, purity: 89%;

[0175] TEA: Purchased from E. En Chemical Technology Co., Ltd., item number: R033479, purity: GC, 99.8%;

[0176] Hydrochloric acid-ethyl acetate solution: purchased from Nanjing Shengbicheng Chemical Technology Co., Ltd.;

[0177] 4-Dimethylaminobutyrate salt: purchased from Bid Pharmaceutical Technology Co., Ltd., product number: BD3025, purity: 97%;

[0178] Stearic acid: purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; item number: S817778, purity: 98%;

[0179] Trifluoroacetic acid: purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; item number: T818778, purity: AR, 99%;

[0180] EDCI: Purchased from Shanghai Myriel Biochemical Technology Co., Ltd.; Product No.: M38555, Purity: 98%;

[0181] HOBt: Purchased from Jiangsu Aikon Biomedical R&D Co., Ltd.; Product No.: AK-PC-03GAR4; Purity: 99%;

[0182] HATU: Purchased from Nanjing Peptide Biotechnology Co., Ltd.; Product No.: R40005;

[0183] DIPEA: Purchased from E. En Chemical Technology Co., Ltd., item number: R004066, purity: 99%;

[0184] 3-Dimethylaminopropionate salt: purchased from Bid Pharmaceutical Technology Co., Ltd., product number: BD158063, purity: 97%;

[0185] N,N-Dimethylglycine hydrochloride: purchased from Bid Pharmaceutical Technology Co., Ltd., product number: BD2382, purity: 98%.

[0186] Example 1: Preparation of compound GJX001

[0187] Step 1: Preparation of GJX001-1

[0188]

[0189] Material proportions:

[0190] Material Name molecular weight Feed ratio Feeding amount mmol N-Boc serine 191 1eq 3.8g 20 Oleyl chloride 300.91 2eq 12g 40 DMAP 122.17 0.25eq 610mg 5 TEA 101 2eq 4g 40 dichloromethane - - 100mL -

[0191] Operation process:

[0192] N-Boc serine, TEA, DMAP, and dichloromethane were added to a reaction flask, and the mixture was cooled to 0°C. Petroyl chloride was added dropwise, and the mixture was then brought to room temperature and stirred for 2 hours. TLC (PE:EA = 6:1) showed that the reaction was complete. Column chromatography purification yielded 12 g of a colorless oil, with a yield of 76%.

[0193] TLC case: PE:EA = 6:1, Rf = 0.3.

[0194] Step 2: Preparation of GJX001-2

[0195]

[0196] Material proportions:

[0197] Material Name molecular weight Feeding amount mmol GJX001-1 720 2.6g 3.6 Hydrochloric acid-ethyl acetate solution (2M) - 26mL -

[0198] Operation process:

[0199] GJX001-1 was added to the reaction flask, followed by a 2M hydrochloric acid-ethyl acetate solution. The mixture was stirred at room temperature for 2 hours. The reaction was completed by TLC (PE:EA = 4:1). The solvent was evaporated to dryness, yielding 2 g of a colorless oily substance, with a yield of 90%.

[0200] TLC case: PE:EA = 1:1, Rf = 0.2.

[0201] Step 3: Preparation of GJX001

[0202]

[0203] Material proportions:

[0204] Material Name molecular weight Feed ratio Feeding amount mmol GJX001-2 620 1.0eq 2g 3.2 4-Dimethylaminobutyrate 167.63 1.0eq 0.54g 3.2 EDCI 191.7 1.3eq 0.8g 4.1 HOBt 135 1.3eq 0.56g 4.1 Triethylamine 101 3.0eq 1g 9.6 DCM - - 40mL -

[0205] Operation process:

[0206] GJX001-2, 4-dimethylaminobutyrate, EDCI, HOBt, triethylamine, and DCM were added to the reaction flask, and the reaction was carried out at room temperature for at least 12 hours. The next day, TLC (PE:EA = 1:1) showed that the reaction was complete. The mixture was diluted with 60 mL of DCM, washed once with 100 mL of water, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The sample was then mixed and purified by column chromatography to give 1 g of a colorless oily substance, with a yield of 30%.

[0207] 1 HNMR (400MHz, CDCl3) δ: 6.03 (s, 1H), 5.46-5.30 (m, 4H), 4.49 (dd, 1H), 4.31-4.05 (m, 4H), 2.43 (t, 4H), 2.32 (d, 6H), 2.24-2.17 (m, 2H), 2.09-1.98 (m, 8H), 1.87 (dd, 7H), 1.62 (d, 5H), 1.47-1.19 (m, 42H), 0.90 (t, 6H).

[0208] LCMS: Rt: 0.304-2.599min; MS m / z(ESI): 733.6[M] + .

[0209] Example 2: Preparation of compound GJX002

[0210] Step 1: Preparation of GJX002-1

[0211]

[0212] Material proportions:

[0213] Material Name molecular weight Feed ratio Feeding amount mmol N-Boc serine 191 1eq 1g 5.23 stearic acid 284 2.5eq 3.72g 13.1 DCC 206.33 2.5eq 2.7g 13.1 DMAP 122.17 0.1eq 0.31g 1.31 dichloromethane - - 40mL -

[0214] Operation process:

[0215] N-Boc serine, stearic acid, DCC, DMAP, and dichloromethane were added to the reaction flask, and the mixture was stirred at room temperature for 2 hours. The reaction was completed by TLC (PE:EA = 4:1), filtered through a diatomaceous earth layer, and the filtrate was concentrated and mixed. Column chromatography was used for purification, yielding 3.1 g of a white solid, with a yield of 81.8%.

[0216] TLC case: PE:EA = 4:1, Rf = 0.4.

[0217] Step 2: Preparation of GJX002-2

[0218]

[0219] Material proportions:

[0220] Material Name molecular weight Feed ratio Feeding amount mmol GJX002-1 724 - 2.5g 3.45 Trifluoroacetic acid - - 10mL - dichloromethane - - 30mL -

[0221] Operation process:

[0222] GJX002-1 was added to a reaction flask, followed by a mixed solution of trifluoroacetic acid and dichloromethane. The mixture was stirred at room temperature for 2 hours. The reaction was completed by TLC (PE:EA = 4:1). The solution was diluted with 70 mL of dichloromethane, washed once with 100 mL of saturated sodium carbonate solution, and then once with 100 mL of water. The organic phase was dried over anhydrous sodium sulfate and concentrated to yield 2 g of a white solid, GJX002-2, with a yield of 93%.

[0223] TLC case: PE:EA = 1:1, Rf = 0.2.

[0224] Step 3: Preparation of GJX002

[0225]

[0226] Material proportions:

[0227] Material Name molecular weight Feed ratio Feeding amount mmol GJX002-2 624 1.0eq 2g 3.2 4-Dimethylaminobutyrate 167.63 1.0eq 0.54g 3.2 EDCI 191.7 1.3eq 0.8g 4.1 HOBt 135 1.3eq 0.56g 4.1 Triethylamine 101 3.0eq 1g 9.6 DCM - - 40mL -

[0228] Operation process:

[0229] GJX002-2, 4-dimethylaminobutyrate, EDCI, HOBt, triethylamine, and DCM were added to the reaction flask, and the reaction was carried out at room temperature for at least 12 hours. The next day, the reaction proceeded to TLC (PE:EA = 1:1) with the starting material GJX002-2. After complete reaction, 60 mL of DCM was added for dilution, followed by washing once with 100 mL of water. The organic phase was dried over anhydrous sodium sulfate, concentrated, and mixed. Column chromatography purification was performed (DCM:MeOH = 20:1) to obtain 700 mg of white solid, GJX002, with a yield of 30%.

[0230] 1 HNMR (400MHz, CDCl3) δ: 7.08 (d, 1H), 4.51-4.41 (m, 1H), 4.19 (dd, 1H), 4.08 (dd, 2H ), 2.30(q, 6H), 2.24(s, 6H), 1.78(dt, 4H), 1.6(p, 4H), 1.26(d, 56H), 0.88(t, 6H).

[0231] LCMS: Rt: 0.063-0.262min; MS m / z (ESI): 737.68[M]+.

[0232] Example 3: Preparation of compound GJX003

[0233] Step 1: Preparation of GJX003-1

[0234]

[0235] Material proportions:

[0236] Material Name molecular weight Feed ratio Feeding amount mmol A 280 2.1eq 6.2g 22.0 B 191 1eq 2g 10.5 DCC 206 2.1eq 4.6g 22.0 DMAP 122 0.2eq 0.26g 2.1 DCM - - 100mL -

[0237] Operation process:

[0238] A, B, DMAP, DCC, and DCM were added to the reaction flask and stirred at room temperature for 8 hours. TLC (PE:EA = 20:1) showed that starting material A reacted completely, and the mixture was filtered. Then, saturated sodium bicarbonate solution was added to the organic filtrate and stirred for 30 minutes. The mixture was separated, dried over anhydrous sodium sulfate, concentrated, and mixed. Column chromatography was used to purify the product, yielding 3.6 g of product, with a yield of 45.5%.

[0239] 1 HNMR (400MHz, CDCl3) δ: 5.45-5.27 (m, 8H), 4.78 (d, 1H), 4.18 (d, 3H), 4.07 (s, 2H), 2.78 (t, 4H ), 2.32(t, 4H), 2.06(q, 8H), 1.70-1.59(m, 4H), 1.46(s, 9H), 1.38-1.26(m, 29H), 0.90(t, 6H).

[0240] Step 2: Preparation of GJX003-2

[0241]

[0242] Material proportions:

[0243]

[0244] Operation process:

[0245] GJX003-1 was dissolved in ethyl acetate, and then EA / HCl was added. The mixture was stirred at room temperature for 2 hours. TLC (PE:EA = 1:1) showed that GJX003-1 reacted completely. The solution was evaporated to dryness without purification and proceeded directly to the next step.

[0246] 1 HNMR (400MHz, CDCl3) δ: 8.84 (s, 3H), 5.44-5.31 (m, 8H), 4.51-4.33 (m, 4H), 3.85-3.75 (m, 1H) , 2.79 (t, 4H), 2.46 (t, 4H), 2.08 (dt, 8H), 1.68-1.58 (m, 4H), 1.38-1.28 (m, 28H), 0.91 (t, 6H).

[0247] Step 3: Preparation of GJX003

[0248]

[0249] Material proportions:

[0250] Material Name molecular weight Feed ratio Feeding amount mmol GJX003-2 615.9 1.0eq 400mg 0.647 4-Dimethylaminobutyrate 167 1.1eq 120mg 0.712 HATU 378 1.2eq 293mg 0.777 DIPEA 129.9 3.0eq 252mg 1.94 DCM - - 10mL -

[0251] Operation process:

[0252] GJX003-2, 4-dimethylaminobutyrate, HATU, DIPEA, and DCM were added to a reaction flask and stirred at room temperature for 10 hours. TLC (DCM:CH3OH = 10:1) showed that GJX003-2 reacted completely. Water (20 mL) was added to the reaction solution, and the mixture was extracted three times with DCM. The organic phase was then washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated, and mixed. Column chromatography was used to purify the product, yielding 170 mg of product, with a yield of 36.0%.

[0253] 1 HNMR (400MHz, CDCl3) δ: 5.47-5.29 (m, 5H), 4.47-4.11 (m, 8H), 3.73-3.62 (m, 2H), 3.18-3.04 (m, 4H), 2.83 (s, 6H), 2 .77(d, 2H), 2.58(d, 2H), 2.42-2.30(m, 5H), 2.19-1.97(m, 9H), 1.45(d, 4H), 1.39-1.25(m, 31H), 0.93-0.85(m, 6H).

[0254] LCMS: Rt: 0.187min; MS m / z (ESI): 729.6[M]+.

[0255] Example 4: Preparation of compound GJX004

[0256]

[0257] Material proportions:

[0258]

[0259]

[0260] Operation process:

[0261] GJX002-2, 3-dimethylaminopropionate, EDCI, HOBt, triethylamine, and dichloromethane were added to the reaction flask and reacted at room temperature for at least 12 hours. The following day, the reaction proceeded to TLC (PE:EA = 1:1) with the starting material GJX002-2. After complete reaction, 60 mL of DCM was added for dilution, followed by washing once with 100 mL of water. The organic phase was dried over anhydrous sodium sulfate, concentrated, and mixed. Column chromatography purification was performed (DCM:MeOH = 20:1) to obtain 700 mg of a white solid, GJX004, with a yield of 30%.

[0262] 1 HNMR (500MHz, CDCl3) δ: 5.96 (d, 1H), 4.27-4.14 (m, 3H), 3.71-3.58 (m, 2H), 2.78 (t, 1H) , 2.34(t, 2H), 2.22-2.16(m, 2H), 1.61(dd, 6H), 1.37(s, 1H), 1.27(d, 62H), 0.88(t, 6H).

[0263] LCMS: Rt: 0.063-0.195min; MS m / z (ESI): 723.66[M]+.

[0264] Example 5: Preparation of compound GJX005

[0265]

[0266] Material proportions:

[0267]

[0268] Operation process:

[0269] GJX001-2, 3-dimethylaminopropionate, EDCI, HOBt, triethylamine, and dichloromethane were added to the reaction flask, and the reaction was carried out at room temperature for at least 12 hours. The next day, TLC (PE:EA = 1:1) showed that the starting material GJX001-2 had reacted completely. The mixture was diluted with 60 mL of DCM, washed once with 100 mL of water, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The sample was then mixed. Column chromatography purification (DCM:MeOH = 20:1) yielded 1 g of product GJX005, with a yield of 38%.

[0270] 1HNMR (400MHz, CDCl3) δ: 8.89 (d, 1H), 5.41-5.329 (m, 4H), 4.51-4.41 (m, 1H), 4.13 (ddd, 4H), 2.54-2.4 8(m, 2H), 2.38-2.28(m, 6H), 2.25(s, 6H), 2.08-1.95(m, 8H), 1.62(s, 4H), 1.28(d, 40H), 0.88(t, 6H).

[0271] LCMS: Rt: 0.500min; MS m / z (ESI): 719.6[M]+.

[0272] Example 6: Preparation of compound GJX006

[0273]

[0274] Material proportions:

[0275]

[0276] Operation process:

[0277] GJX003-2, 3-dimethylaminopropionate, HATU, DIPEA, and DCM were added to the reaction flask and stirred at room temperature for 10 hours. TLC (EA:PE = 1:1) showed that GJX003-2 reacted completely. Water (20 mL) was added to the reaction solution, and the mixture was extracted three times with DCM. The organic phase was then washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated, and mixed. Column chromatography was used for purification to obtain 70 mg of product, GJX006, with a yield of 14.4%.

[0278] 1 HNMR (400MHz, CDCl3) δ: 5.43-5.27 (m, 8H), 4.33 (s, 1H), 4.18 (d, 4H), 3.18 (q, 2H), 2.86 (s, 6H), 2.77(t, 4H), 2.34(t, 4H), 2.04(q, 8H), 1.60(s, 4H), 1.38-1.22(m, 32H), 0.89(t, 6H).

[0279] LCMS: Rt: 0.260min; MS m / z (ESI): 715.6[M]+.

[0280] Example 7 Preparation of compound GJX007

[0281]

[0282] Material proportions:

[0283] Material Name molecular weight Feed ratio Feeding amount mmol GJX002-2 624 1.0eq 2g 3.2 N,N-Dimethylglycine hydrochloride 139.58 1.0eq 0.45g 3.2 EDCI 191.7 1.3eq 0.8g 4.1 HOBt 135 1.3eq 0.56g 4.1 Triethylamine 101 3.0eq 1g 9.6 DCM - - 40mL -

[0284] Operation process:

[0285] GJX002-2, N,N-dimethylglycine hydrochloride, EDCI, HOBt, triethylamine, and DCM were added to the reaction flask, and the reaction was carried out at room temperature for at least 12 hours. The next day, the reaction proceeded to TLC (PE:EA = 1:1) with the starting material GJX002-2. After complete reaction, 60 mL of DCM was added for dilution, followed by washing once with 100 mL of water. The organic phase was dried over anhydrous sodium sulfate, concentrated, and mixed. Column chromatography purification was performed (DCM:MeOH = 20:1) to obtain 700 mg of a white solid, GJX007, with a yield of 30%.

[0286] 1 HNMR (500MHz, CDCl3) δ: 7.38 (d, 1H), 4.52-4.42 (m, 1H), 4.22-4.10 (m, 4H), 2.31 (t, 4H), 2.28 (s, 5H), 1.61 (td, 6H), 1.25 (s, 56H), 0.87 (t, 6H).

[0287] LCMS: Rt: 0.072-0.238min; MS m / z (ESI): 709.6[M]+.

[0288] Example 8: Preparation of compound GJX008

[0289]

[0290] Material proportions:

[0291]

[0292]

[0293] Operation process:

[0294] GJX001-2, N,N-dimethylglycine hydrochloride, EDCI, HOBt, triethylamine, and DCM were added to the reaction flask and reacted at room temperature for at least 12 hours. The next day, TLC (PE:EA = 1:1) showed that the starting material GJX001-2 had reacted completely. The mixture was diluted with 60 mL of DCM, washed once with 100 mL of water, and the organic phase was dried over anhydrous sodium sulfate and concentrated. Column chromatography purification (DCM:MeOH = 20:1) yielded 1 g of product GJX008, with a yield of 38%.

[0295] 1HNMR (500MHz, CDCl3) δ: 7.40 (d, 1H), 5.36 (dt, 4H), 4.49 (dt, 1H), 4.26-4.11 (m, 4H), 2 .97(s, 2H), 2.34(t, 4H), 2.30(s, 6H), 2.03(dt, 8H), 1.37-1.25(m, 44H), 0.90(t, 6H).

[0296] Example 9: Preparation of compound GJX009

[0297]

[0298] Material proportions:

[0299] Material Name molecular weight Feed ratio Feeding amount mmol GJX003-2 615.9 1.0eq 400mg 0.645 N,N-Dimethylglycine hydrochloride 139.6 1.2eq 98mg 0.70 HATU 378 1.3eq 319mg 0.845 DIPEA 129.9 3.0eq 252mg 1.94 DCM - - 10mL -

[0300] Operation process:

[0301] GJX003-2, N,N-dimethylglycine, HATU, DIPEA, and DCM were added to the reaction flask and stirred at room temperature for 10 hours. TLC (EA:PE = 1:1) showed that GJX003-2 reacted completely. Water (20 mL) was added to the reaction solution, and the mixture was extracted three times with DCM. The organic phase was then washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated, and mixed. Column chromatography was used for purification to obtain 120 mg of product, GJX009, with a yield of 26.5%.

[0302] 1 HNMR (400MHz, CDCl3) δ: 7.38 (d, 1H), 5.42-5.29 (m, 7H), 4.52-4.43 (m, 1H), 4.16 (qd, 4H), 2.95 (s , 2H), 2.77(t, 4H), 2.35-2.26(m, 9H), 2.05(q, 7H), 1.60(d, 8H), 1.38-1.26(m, 27H), 0.86(t, 6H).

[0303] LCMS: Rt: 0.546min; MS m / z (ESI): 701.6[M]+.

[0304] Example 10 Preparation and Detection of Lipid Nanoparticles (LNPs)

[0305] A series of ionizable lipid compounds, distearate phosphatidylcholine (DSPC, Nippon Seika Co., Ltd., catalog number: S01005), cholesterol (Nippon Seika Co., Ltd., catalog number: O01001), and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000, Guobang Pharmaceutical Co., Ltd., catalog number: O02005) were dissolved in ethanol solutions and then mixed according to a certain molar ratio to prepare a mixed lipid ethanol solution (total lipid concentration: 12.5 mM). Firefly luciferase (Fluc) mRNA was diluted in 50 mM citrate buffer (pH 4.0) to obtain an mRNA solution. Lipid nanoparticles were prepared by mixing the lipid ethanol solution and mRNA solution at a flow rate of 12 mL / min and a volume ratio of 1:3 using a microfluidic device, with an ionizable lipid to mRNA nitrogen-to-phosphorus ratio of 3–15:1. Ethanol was removed by dialyzing with 0.01M phosphate-buffered saline (PBS) for 12–24 h. Finally, the lipid nanoparticle solution was filtered through a 0.22 μm sterile filter and concentrated by ultrafiltration (Amicon-Ultra, MWCO 10 kDa) to obtain an LNP formulation encapsulated with Fluc mRNA using ionizable lipid / DSPC / cholesterol / DMG-PEG2000. D-Lin-MC3-DMA cationic lipid (manufacturer ApexBio, catalog number: A8791) was used as a positive control; its structure is as follows:

[0306] Unless otherwise specified, the molar ratio of ionizable lipids / DSPC / cholesterol / DMG-PEG2000 was 50:10:38.5:1.5. The particle size and polydispersity index (PDI) of each LNP were determined using Malvern Zetasizer Ultra (dynamic light scattering); the encapsulation efficiency of the LNPs was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, catalog number: R11490). The test results are shown in Table 1.

[0307] In this field, a PDI less than 0.3 indicates that the nanoparticles in the LNP formulation are relatively uniform in size; the encapsulation efficiency is used to indicate whether the LNP can effectively encapsulate mRNA, and an encapsulation efficiency greater than 70% indicates that the LNP can effectively encapsulate mRNA; a single and bright band in an agarose gel electrophoresis image indicates that the mRNA structure is intact. A PDI closer to 0 is better, and an encapsulation efficiency closer to 100% is better.

[0308] Table 1. Particle size, PDI, and encapsulation efficiency of each LNP

[0309]

[0310] As shown in Table 1, the particle size of each LNP is between 60 and 120 nm, the PDI is less than 0.3, and the encapsulation efficiency is higher than 80%.

[0311] Example 11: In vitro cell experiments of LNP formulation

[0312] 10,000 293FT cells per well were seeded in 96-well plates and cultured overnight until cell adhesion was achieved. The Fluc-mRNA LNP formulation prepared in Example 10 (150 ng mRNA / well) was added to the cell culture medium in the 96-well plates (replaced with antibiotic-free DMEM medium containing 10% fetal bovine serum (FBS)) (manufacturer: Gibco, catalog number: C11995500BT). After culturing for another 24 hours, the cell supernatant was discarded, and the cells were lysed with 100 μL of cell lysis buffer. D-fluorescein potassium salt (PerkinElmer, catalog number: 122799, final concentration 1 mM) and ATP (manufacturer: ApexBio, catalog number: C6931, final concentration 2 mM) were added. The chemiluminescence intensity was detected using a microplate reader. The test results are shown below. Figure 1 (Chemiluminescence intensity of 293FT cells after co-culturing with each Fluc-mRNA LNP for 24 h (n=2)).

[0313] Depend on Figure 1 It is evident that the Fluc-mRNA LNPs corresponding to D-Lin-MC3-DMA, GJX001, GJX003, GJX005, and GJX006 can effectively deliver Fluc mRNA into cells and promote its expression.

Claims

1. A nitrogen-containing chain compound or a pharmaceutically acceptable salt thereof, characterized in that, The nitrogen-containing chain compound is any of the following compounds: 、 、 、 。 2. 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 a condensing agent, the compound shown in Formula I-1 and the compound shown in Formula I-2 undergo a condensation reaction as shown in the following formula; ； R 1 It is methyl, R 2 Y is methyl. or ; A 1 for Where a is connected to CH2, and b is connected to R 3 Connected; A 2 for Where a is connected to CH2, and b is connected to R 4 Connected; m and n are both 1; R 3 and R 4 Same, R 3 for or ; R 4 for or .

3. The method for preparing the nitrogen-containing chain compound as shown in Formula I according to claim 2, 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 condensation reaction, the molar ratio of the compound shown in Formula I-2 to the compound shown in Formula I-1 is 1:(1-1.2). (2) In the condensation reaction, the condensing agent is one or more of 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; (3) In the condensation reaction, the molar ratio of the compound shown in Formula I-2 to the condensing agent is 1:(1-3). (4) In the condensation reaction, the base is triethylamine or N,N-diisopropylethylamine; (5) In the condensation reaction, the molar ratio of the compound shown in Formula I-2 to the base is 1:(2-4). (6) In the condensation reaction, the solvent is dichloromethane; (7) In the condensation reaction, the mass-to-volume ratio of the compound as shown in Formula I-2 to the solvent is 30 mg / mL to 60 mg / mL; (8) In the condensation reaction, the reaction temperature is room temperature; (9) In the condensation reaction, the reaction time is 10 h or ≥12 h; The condensation reaction described in (10) further includes post-treatment steps including one or more of dilution, extraction, solution washing, water washing, drying, concentration, mixing and column chromatography.

4. A lipid carrier, characterized in that, It includes substance Z, which is a nitrogen-containing chain compound as described in claim 1 or a pharmaceutically acceptable salt thereof.

5. The lipid carrier as described in claim 4, 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 further includes PEG lipids; The lipid carriers described in (4) also include sterols.

6. The lipid carrier as described in claim 5, 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 distearylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, palmitoylphosphatidylcholine, 1,2-distearylphosphatidylcholine, docosanoylphosphatidylcholine or palmitoylphosphatidylcholine. (3) 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; (4) The sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid and α-tocopherol; (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 30 mol% to 60 mol%; (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%.

7. The lipid carrier according to claim 6, 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 sterols are selected from cholesterol; (3) In the lipid carrier, the molar ratio of substance Z to sterol is 0.5~3:1; (4) In the lipid carrier, the molar ratio of substance Z to phospholipid is 2~8:1; (5) In the lipid carrier, the molar ratio of substance Z to PEG lipid is 20~80:

1.

8. The lipid carrier as described in claim 7, characterized in that, The lipid carrier satisfies one or more of the following conditions: (1) In the lipid carrier, the molar ratio of substance Z to sterol is 0.6~2:1; (2) In the lipid carrier, the molar ratio of substance Z to phospholipid is 3~6:1; (3) In the lipid carrier, the molar ratio of substance Z to PEG lipid is 20~40:

1.

9. The lipid carrier as described in claim 5, characterized in that, The lipid carrier is composed of substance Z, the diluent, the phospholipid, the PEG lipid, and the sterol.

10. 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 4-9.

11. The lipid nanoparticles according to claim 10, 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 to 30:1; (3) In the lipid nanoparticles, the mass ratio of the lipid carrier to the therapeutic agent and / or preventive agent is 3~80:1; (4) The particle size of the lipid nanoparticles is 10~250 nm; In the lipid nanoparticles of (5), the lipid carrier encapsulates the therapeutic agent and / or preventive agent.

12. The lipid nanoparticles according to claim 10, 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 to 20:1; (3) In the lipid nanoparticles, the mass ratio of the lipid carrier to the therapeutic agent and / or preventive agent is 10~50:1; (4) The lipid nanoparticles have a particle size of 10~200nm or 50~250nm.

13. The lipid nanoparticles according to claim 12, 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 to 20:1; (3) The lipid nanoparticles have a particle size of 40~150 nm.

14. The lipid nanoparticles according to claim 13, characterized in that, The lipid nanoparticles satisfy one or more of the following conditions: (1) The therapeutic agent and / or preventive agent is firefly luciferase mRNA; (2) The nitrogen-to-phosphorus ratio in the lipid nanoparticles is 3:1 to 16:1; (3) The size of the lipid nanoparticles is 60~150 nm.

15. The lipid nanoparticles as described in claim 10, characterized in that, The encapsulation rate of the therapeutic and / or preventive agents is at least 70%.

16. The lipid nanoparticles according to claim 10, characterized in that, The polydispersity index of the lipid nanoparticles is no higher than 0.3.

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