Imidazole derivative compound, composition containing imidazole derivative compound and application of imidazole derivative compound

Abstract

The invention discloses an imidazole derivative compound, a composition containing the imidazole derivative compound and application of the imidazole derivative compound. Specifically, the invention provides a compound as shown in a formula I or pharmaceutically acceptable salts thereof, the novel imidazole derivative compound can be used for preparing a lipid carrier, and has one or more of the following advantages: high transfection efficiency, high encapsulation efficiency, good stability, good biocompatibility and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmacy and biomaterials, and particularly relates to an imidazole derivative compound and a composition and application thereof. Background Art

[0002] Nucleic acid therapy is a research hotspot in disease treatment. Clinically, gene therapy is primarily used for genetic diseases, cancer, and infectious diseases. Genes are negatively charged, hydrophilic macromolecules that face a series of physiological barriers in the body. A safe and effective nucleic acid delivery system is crucial for its effectiveness.

[0003] Nucleic acid therapy vectors are primarily categorized into viral and non-viral vectors. Viral vectors, such as adenovirus, retrovirus, adeno-associated virus, and herpes simplex virus, offer high transfection efficiency, but they also suffer from limitations such as limited packaging capacity, complex production processes, broad tropism, cytotoxicity, immunogenicity, and tumorigenicity, limiting their widespread application. Non-viral vectors, such as lipid nanoparticles, cationic polymers, and cationic liposomes, have attracted significant attention in recent years due to their advantages, including high safety, low immunogenicity, low toxicity, high payload, and ease of synthesis.

[0004] Lipid nanoparticles (LNPs) are commonly used as nucleic acid delivery vehicles, but current LNPs prepared with ionizable lipids, such as ALC-0315 and SM-102, still have several drawbacks: ① Lipid nanoparticles have a high liver tropism, making them susceptible to off-target transfection into the liver after local administration, leading to hepatotoxicity. ② Endosomal escape of nucleic acids is low: After LNPs enter cells, only 1%–2% of the nucleic acids are released into the cytoplasm for protein translation, while the remaining nucleic acids reside in intracellular vesicles and are degraded within lysosomes as endosomes mature. ③ Lipid nanoparticles have low thermal stability. Currently available LNP formulations require ultra-low temperature storage and transportation. The mRNA-1273 vaccine must be stored at -20°C to -15°C for six months and at 2°C to 8°C for 30 days; the BNT162b2 COVID-19 vaccine must be stored at -80°C to -60°C for six months and at 2°C to 8°C for five days. This not only affects the effectiveness of mRNA but also increases application costs, significantly limiting the development and application of LNPs. Therefore, the development of safe and efficient new delivery vectors will help promote the promotion and application of gene therapy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the types of lipid nanoparticle delivery vectors in the existing technology are relatively single. To this end, the present invention provides an imidazole derivative compound and a composition and application containing the same. This type of novel imidazole derivative compound can be used to prepare lipid nanoparticle delivery vectors and has one or more of the following advantages: for example, high transfection efficiency, high encapsulation rate, good stability, good cell compatibility, etc.

[0006] The present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0007]

[0008] in:

[0009] L is -(CR a R b ) n1 , one or more CRs in the L a R b The groups are independently optionally replaced by R 1-1 Instead, n1 is an integer from 1 to 10;

[0010] R a and R b are independently H or deuterium;

[0011] R 1-1 are independently -NR c -, O, S, or

[0012] R c H, C 5-30 Alkyl, C 5-30 Alkenyl or C 5-30 Alkynyl, the C 5-30 Alkyl, C 5-30 Alkenyl and C 5-30 One or more methylene units in the alkynyl group are independently optionally replaced by R 1-2 Replacement;

[0013] X is CH or N;

[0014] R 1 and R 2 Independently C 5-30 Alkyl, C 5-30 Alkenyl or C 5-30 Alkynyl, the C 5-30 Alkyl, C 5-30 Alkenyl and C 5-30 One or more methylene units in the alkynyl group are independently optionally replaced by R 1-3 Replacement;

[0015] R 1-2 and R 1-3 are independently O, C(O),

[0016] In one embodiment, certain groups in the compound represented by Formula I have the following definitions, and the definitions of the groups not mentioned are as described in any embodiment of the present invention (hereinafter referred to as one embodiment):

[0017] In one embodiment, L is -(CR a R b ) n1 、-(CR a R b ) n2 -NR c -(CR a R b ) n3 -or-(CR a R b ) n2 -NR c -(CR a R b ) n4 -OP(O)(OH)-O-(CR a R b ) n5 -, wherein n1 is an integer from 1 to 10, and n2, n3, n4 and n5 are independently 1, 2, 3 or 4 (n2+n4+n5≤9);

[0018] Preferably, L is -(CH2) n1 、-(CH2) n2 -NR c -(CH2) n3 -or-(CH2) n2 -NH-(CH2) n4 -OP(O)(OH)-O-(CH2) n5 -

[0019] (e.g. “-O-(CH2) n5 -" end is connected to X), wherein n1, n2, n3, n4 and n5 are independently 1, 2, 3 or 4 (n2+n4+n5≤9);

[0020] More preferably, L is -(CH2) n1 , n1 is 1, 2, 3 or 4.

[0021] In one scheme, R a and R b For H.

[0022] In one scheme, R 1-2 is C(O).

[0023] In one scheme, R c H or C 5-30 Alkenyl, the C 5-30 One or more methylene units in the alkenyl group are independently optionally replaced by R 1-2 Alternatively, preferably, R care independently H or C 5-30 Alkenyl, the C 5-30 A methylene unit in the alkenyl group is independently replaced by R 1-2 Alternatively; more preferably, R c H or -C(O)-C 10-25 Alkenyl.

[0024] In one embodiment, L is a methylene group, Preferably, a "1" bit in L is connected to X.

[0025] In one embodiment, X is N.

[0026] In one scheme, R 1 and R 2 Independently C 5-30 Alkyl or C 5-30 Alkenyl, the C 5-30 Alkyl and C 5-30 One or more methylene units in the alkenyl group are independently optionally replaced by R 1-3 Alternatively, preferably, R 1 and R 2 Independently C 5-30 Alkyl and C 5-30 Alkenyl, the C 5-30 Alkyl and C 5-30 A methylene unit in the alkenyl group is independently replaced by R 1-3 Alternative.

[0027] In one scheme, R 1-3 Independently

[0028] In one scheme, R 1 and R 2 are independently -(CH2) t1 -OC(O)-C 10-25 Alkyl, -(CH2) t1 -C(O)OC 10-25 Alkyl, -(CH2) t1 -OC(O)-C 10-25 Alkenyl or -(CH2) t1 -C(O)OC 10-25 alkenyl, t1 is independently 0, 1, 2, 3 or 4;

[0029] Preferably, the -(CH2) t1 -OC(O)-C 10-25 Alkyl and -(CH2) t1 -C(O)OC 10-25 C in the alkyl group 10-25Alkyl is C 10-25 Branched alkyl; said -(CH2) t1 -OC(O)-C 10-25 Alkenyl and -(CH2) t1 -C(O)OC 10-25 C in alkenyl 10-25 Alkenyl is C 10-25 Straight chain alkenyl, the C 10-25 The number of olefinic bonds in a straight-chain alkenyl group is 1, 2 or 3;

[0030] More preferably, R 1 and R 2 Independently t1 is independently 0, 1, 2, 3 or 4, R 1-A and R 1-B Independently C 4-10 an alkyl group, such as n-hexyl or n-octyl;

[0031] Optimally, R 1 and R 2 Independently

[0032] In one scheme, R 1 and R 2 same.

[0033] In one scheme, R c 、R 1 and R 2 same.

[0034] In one scheme, R 1 and R 2 Independently

[0035]

[0036] In a certain embodiment, in the compound shown in formula I,

[0037] L is -(CH2) n1 , n1 is 1, 2, 3 or 4;

[0038] X is N;

[0039] R 1 and R 2 Independently R 1-A and R 1-B Independently C 4-10 Alkyl, t1 is independently 0, 1, 2, 3 or 4.

[0040] In one embodiment, the compound represented by formula I is any one of the following compounds:

[0041]

[0042]

[0043] The present invention provides a compound as shown in Formula II or an acceptable salt thereof:

[0044]

[0045] R z is an amino protecting group, such as tert-butyloxycarbonyl (Boc), L, X, R 1 and R 2 The definition of is as described in any of the previous schemes. In one scheme, the compound shown in formula II is any of the following compounds:

[0046]

[0047]

[0048] The present invention provides a use of the aforementioned compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a lipid carrier.

[0049] In one embodiment, the lipid carrier is a delivery vehicle for RNA (ribonucleic acid).

[0050] In one embodiment, the RNA is one or more of small interfering RNA (siRNA), self-replicating RNA (samRNA), microRNA (miRNA), circular RNA (circRNA) and messenger RNA (mRNA), such as messenger RNA (mRNA), and luciferase mRNA (Fluci mRNA).

[0051] In one embodiment, the lipid carrier is an (empty) lipid nanoparticle (ie, does not contain a drug).

[0052] In one embodiment, the lipid nanoparticles further comprise sterol and PEG lipid, and preferably, further comprise phospholipid.

[0053] In one embodiment, the lipid nanoparticles further include a solvent. The solvent is a conventional buffer solution in the art, such as a citrate buffer, a phosphate buffer, or an acetate buffer, such as a phosphate buffer solution. The concentrations of the components in the lipid nanoparticles are conventional concentrations in the art.

[0054] In one embodiment, the phospholipid is a neutral phospholipid, such as a neutral glycerophospholipid, and for example, the neutral glycerophospholipid is selected from one or more of distearoylphosphatidylcholine (DSPC), dimyristoylphosphocholine (DMPC), dioleoylphosphocholine (DOPC), palmitoylphosphocholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylglycerol (DSPG), dieucoylphosphatidylcholine (DEPC), palmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), heneicosanoylphosphocholine (DUPC) and palmitoylphosphocholine (POPC), and for example, distearoylphosphatidylcholine (DSPC).

[0055] In one embodiment, the sterol is an animal sterol, a plant sterol or a fungal sterol, for example, the sterol is selected from one or more of cholesterol, β-sitosterol, ergosterol, campesterol, brassicasterol and stigmasterol, such as cholesterol.

[0056] In a certain embodiment, 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, such as PEG-modified phosphatidylethanolamine or PEG-modified dialkylamine, and also such as DMG-PEG2000, DSPE-PEG2000 or ALC-0159.

[0057] In one embodiment, the lipid nanoparticles include (35-85) mol% of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, (0-30) mol% of phospholipids, (10-60) mol% of sterols and (0.1-5) mol% of PEG lipids, wherein the mol% of each component refers to: the total molar number of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, phospholipids, sterols and PEG lipids is 100%, and the molar percentage of each component accounts for the total molar number.

[0058] In one embodiment, the molar percentage of the compound of Formula I or a pharmaceutically acceptable salt thereof is (35-75) mol%, for example (45-50) mol%.

[0059] In one embodiment, the molar percentage of the compound of Formula I or a pharmaceutically acceptable salt thereof is 39.4 mol%, 44.1 mol%, 45.6 mol%, 46.4 mol%, 48.9 mol%, 51.4 mol%, 52.6 mol%, 54.4 mol%, 55.7 mol%, 59.5 mol%, 61.8 mol%, 62.7 mol%, 63.2 mol%, 65.1 mol%, 71.1 mol% or 73.7 mol%,

[0060] In one embodiment, the molar percentage of the phospholipid is (0-25) mol%, for example (10-15) mol%.

[0061] In one embodiment, the mole percentage of the phospholipid is 0, 6.4 mol%, 8.1 mol%, 10.8 mol%, 11.9 mol%, 12.2 mol%, 13.1 mol%, 14.3 mol%, 15.4 mol%, 15.7 mol%, 16.6 mol% or 19.1 mol%.

[0062] In one embodiment, the mole percentage of the sterol is (20-60) mol%, for example (35-40) mol%.

[0063] In one embodiment, the mole percentage of the sterol is 20.8 mol%, 21.2 mol%, 23.7 mol%, 24.9 mol%, 25.8 mol%, 28.4 mol%, 29.2 mol%, 32.0 mol%, 35.3 mol%, 36.3 mol%, 37.3 mol%, 38.5 mol%, 40.5 mol%, 41.2 mol%, 43.9 mol%, 45.2 mol%, 52.3 mol% or 53.2 mol%.

[0064] In one embodiment, the molar percentage of the PEG lipid is (0.2-4) mol%, such as (0.1-0.5) mol%.

[0065] In one embodiment, the mole percentage of the PEG lipid is 0.3 mol%, 0.4 mol%, 0.5 mol%, 1.1 mol%, 1.2 mol%, 1.4 mol%, 1.6 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.4 mol% or 3.0 mol%.

[0066] In one embodiment, the lipid nanoparticles include (45-50) mol% of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, (10-15) mol% of phospholipids, (35-40) mol% of sterols and (0.1-0.5) mol% of PEG lipids, or include a solvent, (45-50) mol% of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, (10-15) mol% of phospholipids, (35-40) mol% of sterols and (0.1-0.5) mol% of PEG lipids.

[0067] In one embodiment, the lipid nanoparticles are composed of 48.9 mol% of compound A33-D268, 12.2 mol% of distearoylphosphatidylcholine, 38.5 mol% of cholesterol and 0.3 mol% of DMG-PEG2000.

[0068] In one embodiment, the lipid nanoparticles are composed of phosphate buffer solution, 48.9 mol% of compound A33-D268, 12.2 mol% of distearoylphosphatidylcholine, 38.5 mol% of cholesterol and 0.3 mol% of DMG-PEG2000.

[0069] The present invention provides a lipid carrier, comprising a compound as shown in Formula I or a pharmaceutically acceptable salt thereof as described in any embodiment of the present invention. The definition of the lipid carrier can also be as described in any of the above embodiments.

[0070] The present invention provides a lipid medicine comprising:

[0071] (1) a lipid carrier as described in any embodiment of the present invention; and

[0072] (2) The RNA according to any embodiment of the present invention.

[0073] The concentration of the RNA in the lipid drug is a conventional concentration in the art. In a certain embodiment, the mass concentration of the RNA is (0.001-1) μg / μl, for example (0.0025-0.05) μg / μl, for example (0.005-0.015) μg / μl, and for example 0.01 μg / μl.

[0074] In the lipid drug, the total molar concentration of each of the components of the lipid carrier (the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, phospholipids, sterols and PEG lipids) is a conventional molar concentration in the art. In a certain embodiment, in the lipid nanoparticles, the total molar number of each of the components (the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, phospholipids, sterols and PEG lipids) is (0.01-10) mmol / L, for example (0.1-1) mmol / L, for example 0.05 mmol / L, 0.1 mmol / L, 0.2 mmol / L, 0.4 mmol / L or 0.8 mmol / L.

[0075] In one embodiment, in the lipid drug, the weight ratio of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof to the RNA is (2-40):1, for example (3-35):1, and for example (25-35):1.

[0076] In one embodiment, in the lipid drug, the weight ratio of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof to the RNA is 10:1, 20:1 or 30:1.

[0077] In one embodiment, the average particle size of the lipid drug is 50-400 nm, such as 70-150 nm, and further such as 80-110 nm.

[0078] In one embodiment, the polydispersity index of the lipid drug is 0.05-0.3, such as 0.09-0.3.

[0079] In a certain embodiment, the encapsulation efficiency of the lipid drug is greater than 50%, such as greater than 70% or greater than 80%, and further such as 75%-85%.

[0080] In a certain embodiment, the potential of the lipid drug is (0.5-6.5) ζ / mV, such as (1-3) ζ / mV, and another example is 1.70 ζ / mV.

[0081] In one embodiment, the pKa of the lipid drug is 5-9, such as 7-7.5, such as 7-7.2.

[0082] It will be understood by those skilled in the art that the structural formulas used in the present invention to describe groups are based on the conventions used in the art. It means that the corresponding group is connected to other fragments and groups in the compound through this site.

[0083] As used herein, a substituent group flanked by a single dash "-" indicates that the substituent is chemically bonded to the parent moiety.

[0084] The term "plurality" refers to 2, 3, 4 or 5, preferably 2 or 3.

[0085] The term "pharmaceutically acceptable" means that salts, solvents, excipients, etc. are generally non-toxic, safe, and suitable for use by patients. The "patient" is preferably a mammal, more preferably a human.

[0086] The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention prepared with relatively non-toxic, pharmaceutically acceptable acids or bases.

[0087] When any variable (such as R f ) appears multiple times in the definition of a compound, the definition of each position of the variable is independent of the definition of the other positions, and their meanings are independent of each other and do not affect each other. Therefore, if a group is replaced by 1, 2 or 3 R f group substituted, that is, the group may be replaced by up to 3 R f Replace, the position R f Definition and other positions R f In addition, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0088] The term "alkyl" refers to a straight or branched chain saturated alkyl group having the specified number of carbon atoms. 1-30 Alkyl, C 5-30 、C 1-6 、C 1-10 Alkyl, examples of which include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and similar alkyl groups.

[0089] The term "alkenyl" refers to a group having at least 2 carbon atoms (e.g., C 2-30 ), linear or branched, unsaturated monovalent hydrocarbon group containing one or more carbon-carbon double bonds and no carbon-carbon triple bonds, wherein the one or more carbon-carbon double bonds may be at the end of the group or may be located inside the group. Examples of alkenyl groups include

[0090] The term "alkynyl" refers to a group having at least 2 carbon atoms (e.g., C 2-30 ), a linear or branched unsaturated monovalent hydrocarbon group containing one or more carbon-carbon triple bonds and no carbon-carbon double bonds, wherein the one or more carbon-carbon triple bonds may be at the end of the group or may be located inside the group.

[0091] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0092] The reagents and raw materials used in the present invention are commercially available.

[0093] The positive progress of the present invention is that the novel imidazole derivative compound provided in this application can be used to prepare lipid nanoparticle delivery carriers and has one or more of the following advantages: for example, high transfection efficiency, high encapsulation rate, good stability and good biocompatibility, etc., and has good application development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 Transmission electron microscopy image of A33-D268 / mRNA delivery system particles. DETAILED DESCRIPTION

[0095] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0096] Example 1: Synthesis of 2-N-tert-Butyloxycarbonyl-(4-Formyl)-1H-Imidazole (AM)

[0097] Synthesis route of compound AM:

[0098]

[0099] Pyruvic acid dimethyl acetal (20 g, 0.169 mol) was dissolved in 400 mL of carbon tetrachloride. The reaction solution was cooled to 0°C, and Br2 (32.45 g, 0.202 mol) was slowly added dropwise to the above system. The reaction was allowed to react at room temperature under nitrogen for 12 hours, then cooled to 0°C and 100 mL of saturated sodium bicarbonate solution was added dropwise to the system. The organic phase was separated using a separatory funnel, washed three times with saturated sodium chloride solution (3 × 100 mL), and then dried over anhydrous sodium sulfate. The organic solvent was evaporated to obtain the intermediate 3,3-dimethoxy-2-oxobromopropane, which was used directly in the next reaction without purification.

[0100] Boc-guanidine (10 g, 63 mmol) and the intermediate 3,3-dimethoxy-2-oxobromopropane (18.5 g, 76 mmol) were dissolved in 200 mL of tetrahydrofuran. Tetraisopropyl titanate (5.96 g, 21 mmol) was added to the reaction solution and stirred at room temperature. After 48 hours, the mixture was filtered and the organic solvent was evaporated to obtain a crude product of N-tert-butoxycarbonyl-2-amino-4(dimethoxymethyl)-imidazole. The crude product was purified by column chromatography (eluent: dichloromethane / methanol / triethylamine = 3 / 1 / 0.5) to obtain 13.68 g of a white solid with a yield of 84.5%. 1H-NMR (600MHz, DMSO-d6): δ=1.54(9H,s,-CH3), 3.19(6H,s,-CH2O-), 5.12(1H,s,-CH-), 6.46(2H,s,-NH2), 6.70(1H,s,=CH-)ppm.

[0101] The intermediate N-tert-butoxycarbonyl-2-amino-4-(dimethoxymethyl)-imidazole (5 g, 19.45 mmol) was dissolved in 50 mL of tetrahydrofuran under N protection, and then diisopropylethylenediamine (3.01 g, 23.34 mmol) and di-tert-butyl dicarbonate (5.08 g, 23.34 mmol) were added to the reaction system. After stirring at room temperature for 12 h, the organic solvent was removed by evaporation under reduced pressure, redissolved with dichloromethane, washed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (eluent: dichloromethane / ethyl acetate = 5 / 1) to obtain 6.42 g of N-tert-butoxycarbonyl-((2-N-tert-butoxycarbonyl)-4-(dimethoxymethyl))-imidazole as a white solid in a yield of 92.1%. 1 H-NMR (600MHz, CDCl3): δ=1.51(9H,s,-CH3), 1.61(9H,s,-CH3), 3.37(6H,s,-CH3O-), 5.38(1H,s,-CH-), 7.05(1H,s,=CH), 9.01(1H,s,-NH-)ppm.

[0102] The compound N-tert-butoxycarbonyl-((2-N-tert-butoxycarbonyl)-4-(dimethoxymethyl))-imidazole (2.0 g, 5.6 mmol) was dissolved in 20 mL of CH2Cl2. The reaction system was cooled to 0°C, pyridine hydrochloride was added to the reaction solution, and the mixture was stirred at room temperature overnight. The reaction progress was monitored by TLC. After the reaction was completed, 10 mL of water was poured into the solution, and the mixture was washed three times with saturated sodium chloride solution (3×10 mL). The organic phase was dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain 0.99 g of 2-N-tert-butoxycarbonyl-(4-formyl)-1H-imidazole as a white solid with a yield of 84.0%. 1 H-NMR (600MHz, DMSO-d6): δ=1.50(9H,s,-CH3), 7.69(1H,s,=CH-), 9.59(1H,s,-CHO-), 10.73(1H,s,-NH-)ppm.

[0103] Example 2: Synthesis of 2-amino-4-(dioleoylphosphatidylethanolaminemethyl)-1H-imidazole (AM-DOPE)

[0104] Synthesis route of compound AM-DOPE:

[0105]

[0106] AM (10 mg, 0.047 mmol) and DOPE (1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine, 15 mg, 0.020 mmol) were dissolved in 1.5 ml of methanol and added to a 10 ml flask, stirring thoroughly. 3 μl of glacial acetic acid was then added while stirring, and the mixture was heated under reflux at 60°C for 4 h. Sodium triacetoxyborohydride (60 mg, 0.28 mmol) was then added in small portions to the reaction mixture. The reaction was continued at 60°C for 4 h before stopping and cooling to room temperature. The methanol in the reaction mixture was evaporated, dissolved in an appropriate amount of dichloromethane, and purified by preparative thin-layer chromatography (methanol:dichloromethane = 1:10) to obtain undecapped AM-DOPE. This was dissolved in 1.5 ml of dichloromethane, 20% trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2-3 h. After the reaction, the dichloromethane and trifluoroacetic acid were dried in a rotary evaporation cycle. An appropriate amount of anhydrous ethanol was then added, and the pH was adjusted to approximately 8-9 with aqueous ammonia. An appropriate amount of anhydrous magnesium sulfate was added to remove water. The reaction was filtered and the ethanol was dried in a rotary evaporation cycle to obtain 2-amino-4-(dioleoylphosphatidylethanolaminemethyl)-1H-imidazole (AM-DOPE). The yield was 32.2%. 1 H-NMR (400MHz, CDCl3): δ=7.28(1H,d,-NC=CH-), 5.35(4H,s,-HC=CH-), 5.23(1H,s,-C(-OOC-)-), 4.34-4.13(4H,s,-P-OCH2-), 3.96(2H,s,-CH2-OP-), 3.74(2H,s,=C-CH2-NH-), 2.98(2H,s,-NHCH2-), 2.31(4H,s,-OOC-CH2-), 2 .02(8H,s,-CH2-C=C-), 1.60-1.29(44H,s,d,-CH2-), 0.90(6H,s,-CH3)ppm. ESI-MS: m / z=839.58.

[0107] Example 3: Synthesis of 2-N-tert-Butoxycarbonyl-4-[2-amino-3,4-dihydroxypropyl]-1H-imidazole (AA)

[0108] Synthesis route of compound AA:

[0109]

[0110] AM (40 mg, 0.19 mmol) and (±)-3-amino-1,2-propanediol (17 mg, 0.19 mmol) were dissolved in 1.5 ml of methanol in a flask. 3 μl of glacial acetic acid was added with stirring, and the mixture was heated at reflux at 60°C for 4 h. Sodium triacetoxyborohydride (80 mg, 0.38 mmol) was then added in small portions to the reaction mixture. After heating at reflux at 60°C for another 4 h, the reaction was stopped and cooled to room temperature. An appropriate amount of methanol was added to bring the reaction volume to approximately 3 ml. Preparative liquid phase purification (mobile phase: 5%-40% acetonitrile / water) was performed, and the fraction with a retention time of approximately 20 min was collected and freeze-dried to obtain 2-N-tert-butyloxycarbonyl-4-[2-amino-3,4-dihydroxypropyl]-1H-imidazole in a yield of 13.1%. 1 H-NMR (400MHz, D2O): δ=6.65(1H,s,-NC=CH-), 3.68, 3.37(2H,ddd,dd,-CH2OH), 3.53(2H,d,= C-CH2-NH-), 3.46(1H,m,-CH(OH)-), 2.55, 2.46(2H,m,dd,-NH-CH2-), 1.36(9H,d,-CH3)ppm.

[0111] Example 4: Synthesis of 2-amino-4-[2-oleoyl-3,4-dioleoylpropyl]-1H-imidazole (AA-OA3)

[0112] Synthesis route of compound AA-OA3:

[0113]

[0114] Oleic acid (60 mg, 0.21 mmol) was dissolved in 700 μl of N,N-dimethylformamide, and 4-dimethylaminopyridine (4 mg, 0.033 mmol) was dissolved in 300 μl of N,N-dimethylformamide. These were added to a flask. N,N'-dicyclohexylcarbodiimide (50 mg, 0.24 mmol) dissolved in 500 μl of N,N-dimethylformamide was slowly added dropwise under an ice bath. After reacting at room temperature for 30 min, AA (16.5 mg, 0.058 mmol) dissolved in 1.5 ml of N,N-dimethylformamide was added. The reaction was allowed to proceed at room temperature for 12 h. After the reaction, the N,N-dimethylformamide was spin-dried to dryness, and the product was then dissolved in 3 ml of dichloromethane. The insoluble solid was filtered off and purified by preparative thin-layer chromatography (developing solvent: ethyl acetate:petroleum ether = 1:2) to obtain AA-OA3, which was not de-Bocated. Dissolve the product in 2 ml of dichloromethane, add 20% trifluoroacetic acid, and stir at room temperature for 2-3 hours. After the reaction, remove the dichloromethane and trifluoroacetic acid by rotary evaporation. Dissolve the product in anhydrous ethanol, adjust the pH of the solution to 8-9 with aqueous ammonia, remove the water by adding an appropriate amount of anhydrous magnesium sulfate, filter, and remove the ethanol by rotary evaporation to obtain 2-amino-4-[2-oleoyl-3,4-dioleoylpropyl]-1H-imidazole (AA-OA3) in a 9.5% yield. ESI-MS: m / z = 1079.76.

[0115] Example 5: Synthesis of 2-N-tert-Butyloxycarbonyl-4-(3-[bis(2-hydroxyethyl)amino]propylaminomethyl)-1H-imidazole (A4)

[0116] Synthesis route:

[0117]

[0118] AM (40 mg, 0.19 mmol) and N-(3-aminopropyl)diethanolamine (60 mg, 0.38 mmol) were dissolved in 1.5 ml of methanol in a flask. 3 μl of glacial acetic acid was added while stirring, and the mixture was heated at 60°C under reflux for 4 h. Sodium triacetoxyborohydride (80 mg, 0.038 mmol) was then added in small portions to the reaction mixture. After heating and reflux at 60°C for another 4 h, the reaction was stopped and cooled to room temperature. An appropriate amount of methanol was added to bring the reaction volume to approximately 3 ml. The mixture was purified by preparative liquid chromatography (5%-40% acetonitrile / water). The fraction with a retention time of approximately 23 min was collected and freeze-dried to yield 2-N-tert-butyloxycarbonyl-4-(3-[bis(2-hydroxyethyl)amino]propylaminomethyl)-1H-imidazole (A4) in an 8.2% yield. 1H-NMR (400MHz, D2O): δ=7.19 (1H,s,-NC=CH-), 4.06 (6H,d,=C-CH2-NH-,-CH2OH), 3.05(8H,s,-NH-CH2-,N(CH2-)3), 2.07(2H,s,-C-CH2-C-), 1.86(9H,m,-CH3)ppm.

[0119] Example 6: Synthesis of 2-amino-4-(3-[bis(2-oleoylethyl)amino]propyloleoylmethyl)-1H-imidazole (A4-OA3)

[0120] Synthesis route of compound A4-OA3:

[0121]

[0122] Oleic acid (50 mg, 0.18 mmol) was dissolved in 700 μl of N,N-dimethylformamide, and 4-dimethylaminopyridine (3.3 mg, 0.027 mmol) was dissolved in 300 μl of N,N-dimethylformamide. The mixture was added to an ane. N,N'-dicyclohexylcarbodiimide (42 mg, 0.20 mmol) dissolved in 500 μl of N,N-dimethylformamide was slowly added dropwise under an ice bath. After reacting at room temperature for 30 min, A4 (11 mg, 0.031 mmol) dissolved in 1 ml of N,N-dimethylformamide was added. The mixture was allowed to react at room temperature for 12 h. After the reaction, the N,N-dimethylformamide was spin-dried and then dissolved in 3 ml of dichloromethane. The insoluble solid was filtered off and the mixture was purified by preparative thin-layer chromatography (ethyl acetate:petroleum ether = 1:2). The undeBoc-degraded A4-OA3 was dissolved in an appropriate amount of dichloromethane and 20% trifluoroacetic acid was added. The mixture was stirred at room temperature for 2-3 hours. After the reaction, the dichloromethane and trifluoroacetic acid were evaporated. The solution was then dissolved in anhydrous ethanol and the pH was adjusted to 8-9 with aqueous ammonia. An appropriate amount of anhydrous magnesium sulfate was added to remove water. The solution was filtered and the ethanol was evaporated to obtain 2-amino-4-(3-[bis(2-oleic acid ethyl ester)amino]propyloleoylmethyl)-1H-imidazole (A4-OA3) in an approximately 9.1% yield. ESI-MS: A4-OA3 m / z = 1150.82.

[0123] Example 7: Synthesis of 2-amino-4-(3-[bis(2-linoleic acid ethyl ester)amino]propyllinoleoylmethyl)-1H-imidazole (A4-Lin3)

[0124] Synthesis route of compound A4-Lin3:

[0125]

[0126] Linoleic acid (50 mg, 0.18 mmol) was dissolved in 700 μl of N,N-dimethylformamide, and 4-dimethylaminopyridine (3.3 mg, 0.027 mmol) was dissolved in 300 μl of N,N-dimethylformamide. The mixture was added to an ane. N,N'-dicyclohexylcarbodiimide (42 mg, 0.20 mmol) dissolved in 500 μl of N,N-dimethylformamide was slowly added dropwise under an ice bath. After reacting at room temperature for 30 min, A4 (11 mg, 0.031 mmol) dissolved in 1 ml of N,N-dimethylformamide was added. The mixture was allowed to react at room temperature for 12 h. After the reaction, the N,N-dimethylformamide was spin-dried and then dissolved in 3 ml of dichloromethane. The insoluble solid was filtered off and the mixture was purified by preparative thin-layer chromatography (ethyl acetate:petroleum ether = 1:2). The undeBoc-degraded A4-Lin3 was dissolved in an appropriate amount of dichloromethane and added with 20% trifluoroacetic acid. The mixture was stirred at room temperature for 2-3 hours. Upon completion of the reaction, the dichloromethane and trifluoroacetic acid were evaporated, and the solution was then dissolved in anhydrous ethanol. The pH of the solution was adjusted to 8-9 with aqueous ammonia. An appropriate amount of anhydrous magnesium sulfate was added to remove water. The solution was filtered and the ethanol was evaporated to obtain 2-amino-4-(3-[bis(2-linoleic acid ethyl ester)amino]propyllinoleoylmethyl)-1H-imidazole (A4-Lin3) in an approximately 9.1% yield. ESI-MS: A4-Lin3 m / z = 1144.79.

[0127] Example 8: Synthesis of 2-N-tert-Butyloxycarbonyl-4-[bis(3-hydroxypropyl)aminomethyl]-1H-imidazole (A33)

[0128] Synthesis route of compound A33:

[0129]

[0130] AM (25 mg, 0.12 mmol) and 3,3'-iminobis-1-propanol (80 mg, 0.60 mmol) were dissolved in 1.5 ml of methanol in a flask. 3 μl of glacial acetic acid was added while stirring, and the mixture was heated at 60°C under reflux for 4 h. Sodium triacetoxyborohydride (80 mg, 0.038 mmol) was then added in small portions to the reaction mixture. After heating and reflux at 60°C for another 4 h, the reaction was stopped and cooled to room temperature. An appropriate amount of methanol was added to bring the reaction volume to approximately 3 ml. Preparative liquid phase purification (mobile phase: 5%-40% acetonitrile / water) was performed, and the fraction with a retention time of approximately 25 min was collected and freeze-dried to obtain 2-N-tert-butyloxycarbonyl-4-[bis(3-hydroxypropyl)aminomethyl]-1H-imidazole (A33) in a yield of 17.9%. 1H-NMR (400MHz, CDCl3): δ=10.29(1H,s,-NH-C=), 10.19(1H,s,Boc-NH-), 6.61(1H,s,-CH=), 3.72(4H, t,-CH2OH), 3.08(2H,s,=C-CH2N-), 2.65(4H,t,-N-CH2-), 1.75(4H,p,-CH2-), 1.51(9H,s,-Boc)ppm.

[0131] Example 9: Synthesis of 2-amino-4-[bis(3-oleoylpropyl)aminomethyl]-1H-imidazole (A33-DOA)

[0132] Synthesis route of compound A33-DOA:

[0133]

[0134] Dissolve oleic acid (80 mg, 0.28 mmol) in 700 μl of N,N-dimethylformamide, and 4-dimethylaminopyridine (5 mg, 0.041 mmol) in 300 μl of N,N-dimethylformamide. Add the resulting mixture to a flask and slowly add N,N'-dicyclohexylcarbodiimide (70 mg, 0.34 mmol) dissolved in 500 μl of N,N-dimethylformamide dropwise with stirring on ice. After reacting at room temperature for 30 minutes, add A33 (15 mg, 0.046 mmol) dissolved in 1 ml of N,N-dimethylformamide. Allow to react at room temperature for 12 hours. After the reaction, spin-dry the N,N-dimethylformamide, dissolve the mixture in 2 ml of dichloromethane, filter out the insoluble matter, and purify the mixture by preparative thin-layer chromatography (developing solvent: methanol:dichloromethane = 1:20). This yields A33-DOA, which does not undergo Boc removal. Dissolve the product in an appropriate amount of dichloromethane, add 20% trifluoroacetic acid, and stir at room temperature for 2-3 hours. After the reaction, remove the dichloromethane and trifluoroacetic acid by rotary evaporation. Dissolve the product in anhydrous ethanol, adjust the pH of the solution to 8-9 with aqueous ammonia, remove the water by adding an appropriate amount of anhydrous magnesium sulfate, filter, and remove the ethanol by rotary evaporation to obtain 2-amino-4-[bis(3-oleoylpropyl)aminomethyl]-1H-imidazole (A33-DOA) in an approximately 20% yield. A33-DOAm / z = 757.73.

[0135] Example 10: Synthesis of 2-amino-4-[bis(3-linoleic acid propyl)aminomethyl]-1H-imidazole (A33-DLin)

[0136] Synthesis routes of compounds and A33-DLin:

[0137]

[0138] Linoleic acid (80 mg, 0.29 mmol) was dissolved in 700 μl of N,N-dimethylformamide, and 4-dimethylaminopyridine (5 mg, 0.041 mmol) was dissolved in 300 μl of N,N-dimethylformamide. The mixture was added to a flask and stirred in an ice bath. N,N'-dicyclohexylcarbodiimide (70 mg, 0.34 mmol) dissolved in 500 μl of N,N-dimethylformamide was slowly added dropwise. After reacting at room temperature for 30 min, A33 (15 mg, 0.046 mmol) dissolved in 1 ml of N,N-dimethylformamide was added. The reaction was allowed to proceed at room temperature for 12 h. After the reaction, the N,N-dimethylformamide was dried and dissolved in 2 ml of dichloromethane. The insoluble matter was filtered off and the product was purified by preparative thin-layer chromatography (developing solvent: methanol:dichloromethane = 1:20). This afforded the undecapped A33-DLin. Dissolve the product in an appropriate amount of dichloromethane, add 20% trifluoroacetic acid, and stir at room temperature for 2-3 hours. After the reaction, remove the dichloromethane and trifluoroacetic acid by rotary evaporation. Dissolve the product in anhydrous ethanol, adjust the pH of the solution to 8-9 with aqueous ammonia, add an appropriate amount of anhydrous magnesium sulfate to remove water, filter, and rotary evaporation to obtain 2-amino-4-[bis(3-linoleic acid propyl ester)aminomethyl]-1H-imidazole (A33-DLin). The yield of A33-DLin is approximately 20%. 1 H-NMR (400MHz, CDCl3): δ=7.29(1H,m,-NC=CH-), 5.38(8H,s,-HC=CH-), 4.16(4H,s,-CH2OOC-), 3.13(2H,s,=C-CH2N-), 2 .79(6H,s,-CH2N-), 2.32(4H,s,-OOCCH2-), 2.07(12H,m,-CH2-C=C-), 1.62-1.28(32H,s,-CH2-), 0.90(6H,s,-CH3)ppm. ESI-MS: A33-DLin m / z=753.87.

[0139] Example 11: Synthesis of 2-amino-4-[bis(3-hexyldecanoate propyl)aminomethyl]-1H-imidazole (A33-D268)

[0140] Synthesis route of compound A33-D268:

[0141]

[0142] Dissolve 2-hexyldecanoic acid (80 mg, 0.31 mmol) in 700 μl of N,N-dimethylformamide and 4-dimethylaminopyridine (5 mg, 0.041 mmol) in 300 μl of N,N-dimethylformamide. Add the resulting mixture to a flask, then add N,N-diisopropylethylamine (120 mg, 0.93 mmol) dropwise with stirring. Next, slowly add N,N'-dicyclohexylcarbodiimide (70 mg, 0.34 mmol) dissolved in 500 μl of N,N-dimethylformamide dropwise with stirring in an ice bath. After reacting at room temperature for 30 minutes, add A33 (10 mg, 0.030 mmol) dissolved in 1 ml of N,N-dimethylformamide. Allow to react at room temperature for 18 hours. After the reaction, the N,N-dimethylformamide was dried by spin-drying, and the product was dissolved in 2 ml of dichloromethane. Insoluble matter was filtered off and purified by preparative thin-layer chromatography (methanol:dichloromethane = 1:20) to obtain A33-D268 without Boc removal. Dissolve the product in an appropriate amount of dichloromethane, add 20% trifluoroacetic acid, and stir at room temperature for 2-3 hours. After the reaction, the dichloromethane and trifluoroacetic acid were dried by spin-drying, and the product was dissolved in anhydrous ethanol. Ammonia water was used to adjust the pH of the solution to 8-9, and an appropriate amount of anhydrous magnesium sulfate was added to remove water. The product was filtered and the ethanol was dried by spin-drying to obtain 2-amino-4-[bis(3-hexyldecanoate propyl ester)aminomethyl]-1H imidazole (A33-D268) with a yield of approximately 16%. A33-D268 1 H-NMR (400MHz, CD3OD): δ=7.05(1H,s,-C=CH-), 4.16(4H,t,-CH2OOC-), 3.45(2H,m,=C-CH2N-), 2. 35(4H,tt,-CH2N-), 2.07(2H,tt,-CHCOO-), 1.53-1.28(52H,m,s,-CH2-), 0.89(12H,m,-CH3)ppm. ESI-MS: A33-D268 m / z=705.66.

[0143] Example 12: Synthesis of 2-amino-4-[bis(3-octyldecanoate propyl)aminomethyl]-1H-imidazole (A33-D288)

[0144] Synthesis route of compound A33-D288:

[0145]

[0146] Dissolve 2-octyldecanoic acid (80 mg, 0.28 mmol) in 700 μl of N,N-dimethylformamide and 4-dimethylaminopyridine (5 mg, 0.041 mmol) in 300 μl of N,N-dimethylformamide. Add the resulting mixture to a flask, then add N,N-diisopropylethylamine (120 mg, 0.93 mmol) dropwise with stirring. Next, slowly add N,N'-dicyclohexylcarbodiimide (70 mg, 0.34 mmol) dissolved in 500 μl of N,N-dimethylformamide dropwise with stirring in an ice bath. After reacting at room temperature for 30 minutes, add A33 (10 mg, 0.030 mmol) dissolved in 1 ml of N,N-dimethylformamide. Allow to react at room temperature for 18 hours. After the reaction, the N,N-dimethylformamide was dried by spin-drying, and the product was dissolved in 2 ml of dichloromethane. Insoluble matter was filtered off and purified by preparative thin-layer chromatography (methanol:dichloromethane = 1:20) to obtain A33-D288 without Boc removal. Dissolve the product in an appropriate amount of dichloromethane, add 20% trifluoroacetic acid, and stir at room temperature for 2-3 hours. After the reaction, the dichloromethane and trifluoroacetic acid were dried by spin-drying, and the product was dissolved in anhydrous ethanol. Ammonia water was used to adjust the pH of the solution to 8-9, and an appropriate amount of anhydrous magnesium sulfate was added to remove water. The product was filtered and the ethanol was dried by spin-drying to obtain 2-amino-4-[bis(3-octyldecanoate propyl)aminomethyl]-1H imidazole (A33-D288) with a yield of approximately 16%. A33-D288 1 H-NMR (400MHz, CDCl3): δ=7.30(1H,d,-C=CH-), 4.17(4H,d,-CH2OOC-), 3.68(2H,s,=C-CH2N-), 2.74(4H,s,-CH2N-), 2.38(2H,s,-CHCOO-), 1.65-1.29(60H,s,-CH2-), 0.91(12H,s,-CH3)ppm. ESI-MS: A33-D288 m / z=761.71.

[0147] Example 13: Preparation and Characterization of AM-LIPID / mRNA Lipid Nanoparticles

[0148] The above-synthesized AM-LIPID: AM-DOPE (Example 2), AA-OA3 (Example 4), A4-OA3 (Example 6), A4-Lin3 (Example 7), A33-DOA (Example 9), A33-Dlin (Example 10), A33-D268 (Example 11) and A33-D288 (Example 12) were dissolved in anhydrous ethanol with DSPC, cholesterol and ALC-0159 at a ratio of AM-LIPID:DSPC:Chol:ALC-0159 = 63.2:11.9:23.7:1.2 to prepare a mixed lipid solution with a total lipid concentration of 6240 nmol / ml. A positive control, ALC-0315, was dissolved in anhydrous ethanol with DSPC, cholesterol, and ALC-0159 at a ratio of 46.3:9.4:42.7:1.6 to create a mixed lipid solution with a total lipid concentration of 6240 nmol / ml. AM-LIPID / mRNA was prepared with a 20:1 weight ratio of ionizable lipid (i.e., the compound described in the above examples) to mRNA in 50 mM citrate buffer at pH 3, corresponding to Fluci mRNA (luciferase mRNA). A positive control, Fluci mRNA, was prepared at a 14:1 weight ratio of ALC-0315 to mRNA. Lipid nanoparticles were prepared using microfluidics at a total flow rate of 4 ml / min and a flow ratio of 1:3 alcohol phase to aqueous phase. The preparation volume was approximately 400 μl. Then ultrafiltration was performed using a 100 kDa ultrafiltration tube, and the lipid nanoparticles were diluted 10 times with 1×PBS (i.e., 0.01 M), and ultrafiltration was performed at a speed of 1700 g for 10 min. This was repeated three times to obtain lipid nanoparticles with a buffer system of 1×PBS. TM RiboGreen TM The LNP encapsulation efficiency was determined using a kit. Particle size and zeta potential were measured using a Malvern NanoZS particle size analyzer. The results are shown in Table 1.

[0149] Table 1

[0150] Size / nm PDI EE / % AM-DOPE 135.3 0.142 38.5 AA-OA3 129.9 0.219 39.5 A4-OA3 146.9 0.215 11.2 A4-Lin3 159.5 0.222 8.7 A33-DLin 124.0 0.208 61.1 A33-D268 131.3 0.126 73.8 A33-D288 98.4 0.238 62.6

[0151] Example 14: In vivo transfection effect of AM-LIPID / mRNA lipid nanoparticles

[0152] Following the method described in Example 13, each group of prepared lipid nanoparticles was prepared into a 50 μl formulation containing 0.5 μg of mRNA in 1×PBS and injected intramuscularly into the hind legs of ICR mice (Bikai, male, 20-25 g, grouped in groups of 3). Six hours after injection, each ICR mouse received an intraperitoneal injection of 100 μl of 15 mg / ml D-luciferin. Ten minutes later, luciferase expression in the hind leg muscles of the mice was imaged using a small animal in vivo imaging device. The results are shown in Table 2.

[0153] Table 2

[0154] AM-LIPID Total Flux[p / s] AM-DOPE 2.697e+06 AA-OA3 1.236e+06 A4-OA3 1.356e+05 A4-Lin3 1.045e+05 A33-DOA 1.624e+05 A33-DLin 4.256e+07 A33-D268 9.276e+07 A33-D288 4.345e+07

[0155] Example 15: Optimization of A33-D268 / mRNA lipid nanoparticle formulation

[0156] Based on the results of Example 14, the A33-D268 / mRNA lipid nanoparticles with the best in vivo transfection effect were selected for formulation optimization. The ratios of A33-D268, phospholipids, cholesterol, and PEG-lipids, as well as the types of phospholipids and PEG-lipids, and the weight ratio of A33-D268 to mRNA were optimized. The multi-factor formulation was optimized using the deterministic screening in the experimental design optimization method. JMP 17 Pro software was used for the deterministic screening experimental design, and the ratios of A33-D268, phospholipids, cholesterol, DMG-PEG2000, the weight ratio of A33-D268 to mRNA, and the types of PEG-lipids: DMG-PEG2000 or DSPE-PEG2000 were optimized. The experimental groups are shown in Table 3.

[0157] Table 3

[0158]

[0159]

[0160] Wright ratio represents the weight ratio of A33-D268 / mRNA.

[0161] Each group prepared lipid nanoparticles and characterized them according to the method described in Example 13. The results are shown in Table 4.

[0162] Table 4

[0163]

[0164]

[0165] Following the method described in Example 14, lipid nanoparticles were prepared in 1×PBS to prepare 50 μl of formulations containing 0.5 μg of mRNA. The formulations were injected intramuscularly into the hind legs of mice. Luciferase expression in the hind leg muscles of the mice was imaged using in vivo imaging at 3, 6, 9, 15, and 24 hours after injection. The AUC for expression was calculated semi-quantitatively for each formulation over 24 hours. The results are shown in Table 5.

[0166] Table 5

[0167]

[0168]

[0169] Example 16: Optimization of the preparation process of A33-D268 / mRNA lipid nanoparticles

[0170] Based on the results of Example 15, the preferred formulation of A33-D268 / mRNA lipid nanoparticles was determined, as shown in Table 6 (i.e., formulation 7 in Table 3).

[0171] Table 6

[0172]

[0173] To further optimize the efficacy of this set of lipid nanoparticles, the preparation method described in Example 13 was used as a foundation to optimize several key factors affecting lipid nanoparticle preparation and formation: microfluidic total flow rate (TFF), ultrafiltration speed, dilution ratio, and the mass ratio of A33-D268 to mRNA. The remaining preparation methods remained unchanged. A single-factor experiment was used, with particle size and encapsulation efficiency as evaluation indicators, to optimize each factor.

[0174] (1) Optimization of ultrafiltration speed and dilution ratio

[0175] The experimental settings for ultrafiltration speed and dilution ratio are shown in Table 7, the raw material prescription is shown in Table 6, and other conditions are the same as the preparation method in Example 13. The particle size results are shown in Table 8.

[0176] Table 7

[0177]

[0178] Table 8

[0179]

[0180]

[0181] (II) Optimization of the mass ratio of A33-D268 to mRNA

[0182] The experimental setup for the mass ratio of A33-D268 to mRNA is shown in Table 9A. The other raw material prescriptions are shown in Table 6. The ultrafiltration speed / G was 1200, the dilution ratio was 20:1, and other conditions were the same as the preparation method in Example 13. The test results are shown in Table 9B.

[0183] Table 9A

[0184]

[0185] Table 9B

[0186] Weight Ratio Size / nm PDI EE / % 10 76.4 0.162 75.5 20 84.7 0.106 70.7 30 75.7 0.135 53.3

[0187] (III) Optimization of microfluidic total flow rate (TFF)

[0188] The microfluidic total flow rate experimental set is shown in Table 10, the raw material prescription is shown in Table 6, the ultrafiltration speed / G is 1200, the dilution ratio is 20:1, the mass ratio is 20:1, and the other conditions are the same as the preparation method in Example 13. The test results are shown in Table 11.

[0189] Table 10

[0190]

[0191] Table 11

[0192] TFF Size / nm PDI EE / % 4 83.3 0.134 71.7 8 78.3 0.186 74.5 12 75.7 0.205 76.5

[0193] In summary, the formulation of A33-D268 / mRNA lipid nanoparticles was determined as shown in Table 6, with an ultrafiltration speed / G of 1200, a dilution ratio of 20:1, a total microfluidic flow rate of 12 ml / min, and other conditions were the same as in Example 13.

[0194] Example 17: Characterization of optimized A33-D268 / mRNA lipid nanoparticles

[0195] Positive control ALC-0315 / mRNA lipid nanoparticles were prepared according to the method described in Example 13. A33-D268 / mRNA lipid nanoparticles were prepared according to the preferred formulation and optimized preparation process for A33-D268 / mRNA lipid nanoparticles determined in Example 16. The particle size, potential, and encapsulation efficiency of the optimized A33-D268 / mRNA lipid nanoparticles were measured. The results are shown in Table 12.

[0196] Table 12

[0197] Size / nm 94.95 PDI 0.09 ζ / mV 1.70 EE / % 81.9％

[0198] 15 μL of A33-D268 / mRNA and 15 μL of uranium acetate were dropped onto two pieces of sealing film, and a copper mesh was floated on the droplets. After 90 seconds, the excess LNP liquid on the edge of the copper mesh was removed with filter paper. The copper mesh was then floated on the same side of the uranium acetate droplet. After 90 seconds, the excess uranium acetate liquid was removed with filter paper. After drying at room temperature, the morphology of A33-D268 / mRNA was observed using a transmission electron microscope. Figure 1 Transmission electron microscopy images showed that the A33-D268 / mRNA delivery system particles were uniform and densely spherical.

[0199] Dissolve 1.78g of sodium chloride, 424mg of trisodium phosphate, 1.13g of diammonium hydrogen citrate, and 308mg of ammonium acetate in 200mL of pure water. A series of solutions with a pH range of 2 to 12 were prepared with 50mg / mL hydrochloric acid and 50mg / mL sodium hydroxide solutions, with pH increments of 0.5. Dissolve 0.5mg of TNS in 10mL of pure water to prepare a TNS stock solution. Add 100μL of this pH gradient solution to a black 96-well plate. Add 2.5μL of AM-LNP / mRNA or ALC-0315LNP / mRNA to each well, followed by 4.0μL of the TNS stock solution for a final TNS concentration of 6μM. Incubate at room temperature in the dark for 5 minutes. Fluorescence values ​​for each well were measured using a microplate reader (Ex / Em = 322 / 431). Fluorescence intensity was normalized according to the following formula. The normalized fluorescence intensity was curve fitted using Graphpadprism software, and the apparent pKa of LNP / mRNA was the pH value corresponding to half the maximum normalized fluorescence intensity. The results are shown in Table 13.

[0200]

[0201] Table 13

[0202] LNP pKa A33-D268 7.06 ALC-0315 7.15

[0203] The physiological pH in the body is 7.35-7.45, so AM-LNP / mRNA and ALC-0315LNP / mRNA are both electrically neutral after local injection and have good biocompatibility.

[0204] The preferred A33-D268 / mRNA lipid nanoparticles and ALC-0315 LNP / mRNA lipid nanoparticles were placed at 4°C. The changes in LNP particle size and PDI were observed on days 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28, starting with the evening of preparation. The results are shown in Table 14.

[0205] Table 14

[0206]

[0207]

[0208] Under 4°C storage conditions, the particle sizes of A33-D268 / mRNA and ALC-0315 / mRNA remained stable, both exceeding 100 nm and with a PDI of no more than 0.200. This indicates that A33-D268 / mRNA has good storage stability at 4°C.

[0209] Example 18: Serum stability of optimized A33-D268 / mRNA lipid nanoparticles

[0210] The transfection efficiency of the preferred A33-D268 / mRNA and ALC-0315LNP / mRNA in HEK293t cells under different serum concentrations was measured in Example 17. HEK293t cells were counted and diluted to 8×104 cells / mL using DMEM (complete medium) supplemented with 10% FBS and 1% penicillin-streptomycin solution. The cell suspension was added to a 96-well plate at a rate of 150 μL per well. After the cells were cultured until adherent, the original medium was removed and AM-LNP / mRNA or ALC-0315LNP / mRNA was added at a dose of 0.5 μg mRNA / well (50 μL of lipid nanoparticles contained 0.5 μg mRNA). DMEM supplemented with 10%, 20%, 40%, 60%, and 80% FBS was added to a total of 150 μL, respectively. The cells were cultured at 37°C and 5% CO2 for 12 hours. Remove the original culture medium from each group, add 100μL 1×PBS to each well for rinsing, then add 100μL cell lysis buffer, place on ice for 10 minutes, and pipette to fully lyse the cells. Take the lysate from each group and centrifuge at 15000g for 2 minutes at 4°C, and collect the supernatant. Determine the total protein concentration in the supernatant using the BCA method. Take 20μL of the supernatant from each group and add it to a black 96-well plate. Add 100μL of firefly luciferase detection reagent to the sample well every 10 seconds, and measure the relative light unit (RLU) using a microplate reader. The final cell transfection efficiency of each group of LNP is expressed as relative mg total protein (RelativeRLU / %) based on 10% FBS. The results are shown in Table 15.

[0211] Table 15

[0212]

[0213] The transfection efficiency of A33-D268 / mRNA slowly decreased with increasing FBS concentration. At 80% FBS, the transfection efficiency dropped to 2.18×106 RLU / mg protein, but remained at 55.7% of that at 10% FBS. The transfection efficiency of ALC-0315 mRNA also decreased with increasing FBS concentration, but at a faster rate than that of AM-LNP / mRNA, reaching only 30.2% of that at 10% FBS at 80% FBS. These results demonstrate that the preferred AM-LNP / mRNA delivery system exhibits excellent serum stability and outperforms the positive control.

[0214] Example 19: Optimized cytocompatibility of A33-D268 / mRNA lipid nanoparticles

[0215] The cell compatibility of the preferred A33-D268 / mRNA and ALC-0315 LNP / mRNA lipid nanoparticles in Example 17 was determined. Mouse myoblasts C2C12 and mouse normal liver cells AML12 were plated and cultured as described in Example 18. The cell compatibility of the LNP lipid carriers was investigated using the CCK-8 assay. After cell culture adherence, the original culture medium was removed and complete culture medium was added to a final concentration (total lipid concentration) of 0.8 mM, 0.4 mM, 0.2 mM, 0.1 mM, or 0.05 mM of ALC-0315 / mRNA or A33-D268 / mRNA, respectively. A negative control group containing cells and complete culture medium and a blank group containing no cells and complete culture medium were also set up. After 6 h of incubation at 37°C and 5% CO2, the cells were replaced with fresh complete culture medium and cultured for 48 h. Remove the original culture medium and add 100 μL of complete culture medium containing 10% CCK-8 reagent to each well. Incubate at 37°C, 5% CO₂ for 1 hour. Measure the absorbance of each well at 450 nm using a microplate reader. Calculate cell viability using the following formula. The results are shown in Tables 16 and 17.

[0216]

[0217] Table 16

[0218]

[0219] Table 17

[0220]

[0221] As shown in Table 16, when the total lipid concentration of A33-D268 / mRNA reached 0.8 mM, which is four times the normal dosing concentration, the C2C12 cell viability still reached 95.1%. When the total lipid concentration of ALC-0315 / mRNA was 0.8 mM, the C2C12 cell viability was 94.1%. Both A33-D268 / mRNA and ALC-0315 / mRNA showed good compatibility with mouse muscle cells.

[0222] As shown in Table 17, A33-D268 / mRNA maintained near 100% AML12 cell viability within the experimentally determined range of 0.05mM to 0.8mM total lipid concentration. For ALC-0315 / mRNA, while AML12 cell viability remained above 90% within the established range, it decreased by approximately 5% at a total lipid concentration of 0.1mM, half the normal dosing concentration. Furthermore, at a concentration of 0.2mM, the normal dosing concentration, AML12 cell viability decreased by approximately 10%. In comparison, A33-D268 / mRNA exhibited superior compatibility with liver cells to ALC-0315 / mRNA.

[0223] Example 20: In vivo delivery effect of optimized A33-D268 / mRNA lipid nanoparticles

[0224] A33-D268 / mRNA lipid nanoparticles were prepared according to the optimized formulation and preparation method described in Example 17. Positive control ALC-0315 / mRNA lipid nanoparticles were prepared according to the method described in Example 13. In vivo transfection experiments in mice were conducted according to the method described in Example 14. The results are shown in Table 18.

[0225] Table 18

[0226] LNP <![CDATA[Liver / AUC 0-24hr (P / S * hr)]]> <![CDATA[Muscle / AUC 0-24hr (P / S*hr)]]> A33-D268 <![CDATA[4.60×10 8 ]]> <![CDATA[6.21×10 9 ]]> ALC-0315 <![CDATA[1.35×10 10 ]]> <![CDATA[7.38×10 9 ]]>

[0227] The mouse muscle transfection effect of A33-D268 / mRNA was able to reach a level comparable to that of the positive control ALC-0315 / mRNA, but A33-D268 / mRNA showed lower liver expression after local injection, indicating that A33-D268 / mRNA has lower off-target effects and better selective expression.

Claims

1. A compound as shown in Formula I or a pharmaceutically acceptable salt thereof: in: L is -(CR a R b ) n1 , one or more CRs in the L a R b The groups are independently optionally replaced by R 1-1 Instead, n1 is an integer from 1 to 10; R a and R b are independently H or deuterium; R 1-1 are independently -NR c -, O, S, or R c H, C 5-30 Alkyl, C 5-30 Alkenyl or C 5-30 Alkynyl, the C 5-30 Alkyl, C 5-30 Alkenyl and C 5-30 One or more methylene units in the alkynyl group are independently optionally replaced by R 1-2 Replacement; X is CH or N; R 1 and R 2 Independently C 5-30 Alkyl, C 5-30 Alkenyl or C 5-30 Alkynyl, the C 5-30 Alkyl, C 5-30 Alkenyl and C 5-30 One or more methylene units in the alkynyl group are independently optionally replaced by R 1-3 Replacement; R 1-2 and R 1-3 are independently O, C(O), 2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein: It meets one or more of the following conditions: (1) L is -(CR a R b ) n1 、-(CR a R b ) n2 -NR c -(CR a R b ) n3 -or-(CR a R b ) n2 -NR c -(CR a R b ) n4 -OP(O)(OH)-O-(CR a R b ) n5 -, wherein n1 is an integer from 1 to 10, and n2, n3, n4 and n5 are independently 1, 2, 3 or 4; (2)R a and R b is H; (3)R c H or C 5-30 Alkenyl, the C 5-30 One or more methylene units in the alkenyl group are independently optionally replaced by R 1-2 Alternatively, preferably, R c are independently H or C 5-30 Alkenyl, the C 5-30 A methylene unit in the alkenyl group is independently replaced by R 1-2 Alternatively; more preferably, R c H or -C(O)-C 10-25 alkenyl; (4) X is N; (5)R 1 and R 2 Independently C 5-30 Alkyl or C 5-30 Alkenyl, the C 5-30 Alkyl and C 5-30 One or more methylene units in the alkenyl group are independently optionally replaced by R 1-3 Alternatively, preferably, R 1 and R 2 Independently C 5-30 Alkyl or C 5-30 Alkenyl, the C 5-30 Alkyl and C 5-30 A methylene unit in the alkenyl group is independently replaced by R 1-3 Replacement; (6)R 1-3 Independently (7)R 1 and R 2 same; (8)R c 、R 1 and R 2 same.

3. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein: It meets one or more of the following conditions: (1) L is -(CH2) n1 、-(CH2) n2 -NR c -(CH2) n3 -or-(CH2) n2 -NH-(CH2) n4 -OP(O)(OH)-O-(CH2) n5 -, wherein n1, n2, n3, n4 and n5 are independently 1, 2, 3 or 4; preferably, L is -(CH2) n1 , n1 is 1, 2, 3 or 4; (2)R 1 and R 2 are independently -(CH2) t1 -OC(O)-C 10-25 Alkyl, -(CH2) t1 -C(O)OC 10-25 Alkyl, -(CH2) t1 -OC(O)-C 10-25 Alkenyl or -(CH2) t1 -C(O)OC 10-25 alkenyl, t1 is independently 0, 1, 2, 3 or 4; Preferably, the -(CH2) t1 -OC(O)-C 10-25 Alkyl and -(CH2) t1 -C(O)OC 10-25 C in the alkyl group 10-25 Alkyl is C 10-25 Branched alkyl; said -(CH2) t1 -OC(O)-C 10-25 Alkenyl and -(CH2) t1 -C(O)OC 10-25 C in alkenyl 10-25 Alkenyl is C 10-25 Straight chain alkenyl, the C 10-25 The number of olefinic bonds in a straight-chain alkenyl group is 1, 2 or 3; More preferably, R 1 and R 2 Independently t1 is independently 0, 1, 2, 3 or 4, R 1-A and R 1-B Independently C 4-10 Alkyl groups, such as n-hexyl or n-octyl; Optimally, R 1 and R 2 Independently More preferably, the compound as shown in Formula I satisfies one or both of the following conditions: (1) L is a methylene group, Preferably, the "1" bit in L is connected to X; (2)R 1 and R 2 Independently 4. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein: In the compound shown in formula I: L is -(CH2) n1 , n1 is 1, 2, 3 or 4; X is N; R 1 and R 2 Independently R 1-A and R 1-B Independently C 4-10 Alkyl, t1 is independently 0, 1, 2, 3 or 4.

5. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The compound shown in formula I is any of the following compounds:

6. A compound as shown in Formula II or an acceptable salt thereof: R z is an amino protecting group, such as tert-butyloxycarbonyl, L, X, R 1 and R 2 The definition is as described in any one of claims 1 to 5; preferably, the compound represented by formula II is any one of the following compounds:

7. Use of a compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in the preparation of a lipid carrier.

8. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to claim 7 in the preparation of a lipid carrier, wherein: It meets one or more of the following conditions: (1) The lipid carrier is a delivery vector for RNA; preferably, the RNA is one or more of small interfering RNA, self-replicating RNA, microRNA, circular RNA, and messenger RNA, such as messenger RNA, and also such as luciferase mRNA; (2) The lipid carrier is a lipid nanoparticle; preferably, the lipid nanoparticle further comprises a sterol and a PEG lipid, more preferably, further comprises a phospholipid; or, the lipid nanoparticle further comprises a solvent; Further preferably, the lipid nanoparticles comprise (35-85) mol% of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, (0-30) mol% of phospholipids, (10-60) mol% of sterols and (0.1-5) mol% of PEG lipids, wherein the mol% of each component refers to: the total molar number of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, phospholipids, sterols and PEG lipids is 100%, and the molar percentage of each component accounts for the total molar number.

9. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to claim 8 in the preparation of a lipid carrier, wherein: The lipid nanoparticles meet one or more of the following conditions: (1) The solvent is a citrate buffer, a phosphate buffer or an acetate buffer, such as a phosphate buffer solution; (2) The total molar number of each of the components is (0.01-10) mmol / L, for example (0.1-1) mmol / L; (3) The molar percentage of the compound of Formula I or a pharmaceutically acceptable salt thereof is (35-75) mol%, for example (45-50) mol%; (4) The phospholipid is a neutral phospholipid, such as a neutral glycerophospholipid, and for example, the neutral glycerophospholipid is selected from one or more of distearoylphosphatidylcholine, dimyristoylphosphocholine, dioleoylphosphocholine, palmitoylphosphocholine, dioleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, distearoylphosphatidylglycerol, dieruoylphosphatidylcholine, palmitoylphosphatidylglycerol, dipalmitoylphosphatidic acid, heneicosanoylphosphocholine and palmitoylphosphocholine, and for example, distearoylphosphatidylcholine; (5) The molar percentage of the phospholipid is (0-25) mol%, for example (10-15) mol%; (6) The sterol is an animal sterol, a plant sterol or a fungal sterol, for example, the sterol is selected from one or more of cholesterol, β-sitosterol, ergosterol, campesterol, brassicasterol and stigmasterol, for example, cholesterol; (6) The mole percentage of the sterol is (20-60) mol%, for example (35-40) mol%; (7) 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, such as PEG-modified phosphatidylethanolamine or PEG-modified dialkylamine, and also such as DMG-PEG2000, DSPE-PEG2000, or ALC-0159; (8) The molar percentage of the PEG lipid is (0.2-4) mol%, for example (0.1-0.5) mol%; Preferably, the lipid nanoparticles comprise (45-50) mol% of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, (10-15) mol% of phospholipids, (35-40) mol% of sterols and (0.1-0.5) mol% of PEG lipids, or comprise a solvent, (45-50) mol% of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, (10-15) mol% of phospholipids, (35-40) mol% of sterols and (0.1-0.5) mol% of PEG lipids; More preferably, the lipid nanoparticles are composed of 48.9 mol% of compound A33-D268, 12.2 mol% of distearoylphosphatidylcholine, 38.5 mol% of cholesterol and 0.3 mol% of DMG-PEG2000, or are composed of phosphate buffer solution, 48.9 mol% of compound A33-D268, 12.2 mol% of distearoylphosphatidylcholine, 38.5 mol% of cholesterol and 0.3 mol% of DMG-PEG2000.

10. A lipid carrier comprising a compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the lipid carrier may also be defined as described in any one of claims 8 or 9.

11. A lipid drug comprising: (1) The lipid carrier according to claim 10; and (2) The RNA according to claim 8.

12. The lipid drug according to claim 11, wherein It meets one or more of the following conditions: (1) The mass concentration of the RNA is (0.001-1) μg / μl, for example (0.0025-0.05) μg / μl, and for example (0.005-0.015) μg / μl; (2) The total molar amount of the compound of Formula I or a pharmaceutically acceptable salt thereof, phospholipid, sterol, and PEG lipid is (0.01-10) mmol / L, for example (0.1-1) mmol / L; (3) The weight ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the RNA is (2-40):1, for example (3-35):1, and for example (25-35):1; (4) The average particle size of the lipid drug is 50-400 nm, for example 70-150 nm, and for example 80-110 nm; (5) The polydispersity index of the lipid drug is 0.05-0.3, for example, 0.09-0.3; (6) The encapsulation efficiency of the lipid drug is greater than 50%, for example, greater than 70% or greater than 80%, and further for example, 75% to 85%; (7) The potential of the lipid drug is (0.5-6.5) ζ / mV, for example (1-3) ζ / mV; (8) The pKa of the lipid drug is 5-9, such as 7-7.5, and another example is 7-7.2.

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