An ionizable lipid compound, its preparation method, and its application in a nucleic acid drug delivery system.

Abstract

This invention belongs to the field of nucleic acid drug delivery carrier technology, specifically providing a novel ionizable lipid compound, its preparation method, lipid nanoparticles composed thereof, and their applications. The novel ionizable lipid compound has a structure as shown in Formula III. The ionizable lipid compound provided by this invention exhibits strong designability and high in vitro transfection efficiency. The lipid nanoparticle delivery system composed of it demonstrates high transfection efficiency, good delivery efficiency, low toxicity, high stability, and good safety, and can serve as a novel method for nucleic acid drug delivery, which is of great significance for promoting the development of nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drug delivery carrier technology, specifically providing a novel ionizable lipid compound, its preparation method, and its application in a nucleic acid drug delivery system. Background Technology

[0002] The efficient targeted delivery of bioactive substances, such as small molecule drugs, peptides, proteins, and nucleic acids, especially nucleic acids, remains a persistent medical challenge. Nucleic acid therapeutics face significant challenges due to their low endocytic efficiency and high sensitivity to degradation of certain nucleic acid molecules, including RNA, thus requiring the use of vectors to encapsulate them and deliver them to target cells.

[0003] Lipid nanoparticles (LNPs) have been proven to be effective nonviral gene vectors for delivering bioactive substances, particularly polynucleotides such as small interfering RNA (siRNA), messenger RNA (mRNA), and guide RNA (gRNA), into cells. Therefore, LNPs have become a research hotspot in the field of nonviral gene vectors in recent years. In 2018, the FDA approved LNP delivery of patisiran (onpattro) for the treatment of hereditary transthyretin amyloidosis. Since then, research using LNP technology to deliver nucleic acid drugs has experienced explosive growth. Particularly in late 2020, the FDA approved Moderna's and BioNTech & Pfizer's COVID-19 vaccines, both of which utilized LNP technology to deliver mRNA drugs, thus achieving prevention against the COVID-19 virus.

[0004] LNPs typically consist of four types of lipid compounds: ionized lipids, neutral lipids, sterols, and amphiphilic lipids. Among these, the choice of ionized lipid compounds has the greatest impact on LNPs. Ionized lipid compounds often have a hydrophilic end with an amino group, which can bind to hydrogen ions and become positively charged in an acidic environment. Nucleic acids, on the other hand, have a large number of phosphate groups and are therefore negatively charged. Thus, through electrostatic adsorption, nucleic acids can be encapsulated in lipid nanoparticles. Therefore, ionized lipids are a key factor determining the efficiency of nucleic acid delivery and transfection.

[0005] Currently, there are very few ionizable lipid compounds used clinically, only three: DLin-MC3-DMA (Alnylam's RNAi drug Onpattro), SM-102 (Moderna's COVID-19 vaccine mRNA-1273), and ALC-0315 (Pfizer's vaccine BNT162b2). Lipid nanoparticles prepared with DLin-MC3-DMA are mainly used for siRNA delivery, while lipid nanoparticles composed of SM-102 and ALC-0315 are mainly used for mRNA delivery. However, the transfection efficacy of these ionizable lipid compounds has not been very good.

[0006] A search of existing technologies revealed that CN114262275A, CN114191561A, CN113372226A, and CN114213295A disclose different ionizable lipid compounds. While these compounds enrich the variety of ionizable lipid compounds and exhibit significant advantages over viral vectors and other types of non-viral vectors in terms of encapsulation efficiency, nucleic acid expression, and cytotoxicity, the number of commercially available ionizable lipid molecules remains limited. Furthermore, many of these compounds tend to distribute easily into the liver, increasing its metabolic burden and potentially causing toxic side effects. Therefore, exploring more ionizable lipid compounds suitable for nucleic acid drug applications and developing nucleic acid drug delivery vectors that truly balance high transfection efficiency, high expression efficacy, and low toxicity is of great significance for the development and application of nucleic acid prophylaxis and therapeutic agents. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a novel ionizable lipid compound, its preparation method, lipid nanoparticles composed thereof, and their applications. The novel ionizable lipid compound exhibits strong designability and high in vivo and in vitro transfection efficiency. The lipid nanodelivery system composed of it can be used for in vivo and in vitro delivery of nucleic acid drugs, achieving high-efficiency expression of nucleic acid drugs while maintaining low expression levels in the liver, thereby reducing toxicity.

[0008] Based on the aforementioned objectives, the inventors, aiming to improve the transfection efficiency of lipid nanoparticle delivery systems and reduce liver toxicity, conducted repeated research on novel ionizable lipid compounds that constitute lipid nanoparticles, ultimately completing this invention.

[0009] To achieve the objectives of this invention, the following implementation scheme is provided:

[0010] A novel ionizable lipid compound, the general structural formula of which is shown in Formula III:

[0011]

[0012] in,

[0013] i and j are each independently selected as integers between 0 and 20; preferably, i and j are each independently selected as integers between 4 and 15; more preferably, i and j are each independently selected as integers between 5 and 10.

[0014] p and q are each independently selected as integers between 0 and 20; preferably, p is selected as an integer between 4 and 15, and q is selected as an integer between 0 and 12; more preferably, p is selected as an integer between 6 and 12, and q is selected as an integer between 0 and 7.

[0015] m and n are each independently selected as integers between 1 and 15; preferably, m and n are each independently selected as integers between 2 and 10; more preferably, m and n are each independently selected as integers between 4 and 8.

[0016] K is chosen as an integer between 1 and 6, preferably an integer between 2 and 4.

[0017] R1 and R2 are each independently carbonyl or oxygen, and R1 is different from R2.

[0018] R is an aromatic C1-C3 alkyl group substituted with or unsubstituted with at least one substituent, wherein the substituent is a C1-C6 alkyl group, an aromatic group, a C3-C6 cycloalkyl group, a C2-C6 alkenyl group, or a halogen. Preferably, R is an aromatic C1-C3 alkyl group substituted with at least two identical or different substituents, wherein the substituent is an aromatic group.

[0019] Preferably, the structural formula of the novel ionizable lipid compound is shown in Formula IV or Formula V:

[0020]

[0021] or

[0022]

[0023] The definition of R is the same as above.

[0024] Preferably, as an example, the novel ionizable lipid compound is selected from structures shown in the following formula:

[0025]

[0026]

[0027]

[0028] Furthermore, this invention provides a novel method for preparing an ionizable lipid compound, characterized in that, under the action of an amine organic base, a compound of formula I and a compound of formula II are reacted to obtain an ionizable lipid compound of formula III, as shown in the following reaction formula:

[0029]

[0030] The definitions of i, j, p, q, m, n, k, R1, R2, and R are the same as those above.

[0031] Preferably, the method for preparing the novel ionizable lipid compound is characterized in that, under the action of an amine organic base, a compound of formula I-1 is reacted with a compound of formula II to obtain an ionizable lipid compound of formula IV, as shown in the following reaction formula:

[0032]

[0033] The definition of R is the same as above.

[0034] Preferably, the method for preparing the novel ionizable lipid compound is characterized in that, under the action of an amine organic base, the compound of formula I-2 is reacted with the compound of formula II to obtain the ionizable lipid compound of formula V, as shown in the following reaction formula:

[0035]

[0036] The definition of R is the same as above.

[0037] Preferably, the method for preparing a novel ionizable lipid compound further includes using one or more of dimethylaminopyridine (DMAP) and DMAP analogs as a catalyst.

[0038] Preferably, the DMAP analog is 4-pyrrolidinylpyridine (4-PPY) or 9-azajulonidine.

[0039] Preferably, the catalyst is DMAP.

[0040] Preferably, the amine organic base is selected from triethylamine, diisopropylethylamine, diethylamine, ethylenediamine, ammonia, pyridine, and combinations of two or more thereof, with triethylamine being more preferred.

[0041] Preferably, the molar ratio of the amine organic base to the compound of formula I is (1.0-2.0):1, and more preferably 1.5:1.

[0042] Preferably, the molar ratio of the catalyst to the compound of formula I is (0.1-0.5):1, and more preferably 0.2:1.

[0043] Preferably, the molar ratio of the compound of formula II to the compound of formula I is (1-2):1, and more preferably 1.2:1.

[0044] Preferably, the reaction solvent is selected from dimethyl sulfoxide, diethyl ether, n-hexane, dichloromethane, chloroform, tetrahydrofuran, toluene, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, acetone, ethyl acetate, and combinations of two or more thereof, and more preferably N,N-dimethylformamide.

[0045] Preferably, the reaction temperature is controlled at 30-60℃, and more preferably at 45-55℃.

[0046] Preferably, the reaction time is controlled within 4-12 hours, and more preferably within 8-10 hours.

[0047] Furthermore, the present invention provides a method for detecting ionizable lipid compound III:

[0048] High-performance liquid chromatography (HPLC) was performed using a YMC Bio C4 150*4.6mm*3μm column, with 0.1% trifluoroacetic acid as mobile phase A and acetonitrile as mobile phase B. The flow rate was 1.5 mL / min, the detection wavelength was 190 nm, the column temperature was 40℃, and a CAD detector (nominal value 50, peak width 0.1 min, acquisition frequency 10 Hz, filtration constant 3.6 s) was used. Gradient elution was performed according to Table 1 below:

[0049] Table 1

[0050]

[0051] Using the high-performance gas chromatography method provided above, with ionizable lipid III compounds as the test sample, the content of ionizable lipid III compounds and the content of various impurities can be detected.

[0052] Furthermore, the present invention also provides a liposome, which is at least one of (a) or (b) below:

[0053] (a) Made from the above-mentioned ionizable lipid compounds;

[0054] (b) Made from the above-described ionizable lipid compounds and co-lipids, wherein the co-lipids include phospholipids and / or structural lipids and / or polyethanol-modified lipids.

[0055] Furthermore, the present invention also provides a use of the above-described ionizable lipid compound or the above-described liposomes, comprising at least one of the following 1)-3),

[0056] 1) In vitro cell transfection with therapeutic or preventative agents;

[0057] 2) Preparation of in vivo delivery agents for treatment or prevention;

[0058] 3) Prepare the transfection kit;

[0059] The therapeutic or preventive agent is selected from nucleic acid drugs; the nucleic acid drug is selected from at least one of DNA drugs and RNA drugs, preferably RNA drugs; the RNA drug is selected from at least one of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, and lncRNA, preferably mRNA.

[0060] Furthermore, the present invention also provides a lipid nanoparticle comprising the following (1) and (2):

[0061] (1) Treatment or preventative agent;

[0062] (2) Liposomes used to deliver therapeutic or preventative agents;

[0063] The definition of the therapeutic or preventive agent is the same as above; the liposome is at least one of the following (a) or (b):

[0064] (a) Made from the above-mentioned ionizable lipid compounds;

[0065] (b) Made from the above-mentioned ionizable lipid compound and cofactor, wherein the cofactor includes phospholipids and / or structural lipids and / or polyethanolized lipids; wherein the molar ratio of the ionizable lipid compound, phospholipids, structural lipids and polyethanolized lipids is (10-100):(0-50):(0-50):(0-50), more preferably (30-80):(2-20):(30-50):(0.5-5), more preferably (40-60):(5-15):(35-45):(0.5-2), and even more preferably 50:10:38.5:1.5.

[0066] Furthermore, the present invention also provides a method for preparing the above-mentioned lipid nanoparticles, the method comprising the following steps:

[0067] - Dissolve liposomes in an organic solvent to obtain an organic phase solution;

[0068] - Add the treatment or preventative agent to the buffer solution to obtain an aqueous solution;

[0069] -Lipid nanoparticles are obtained by mixing organic phase solution and aqueous phase solution.

[0070] Preferably, the organic solvent is DMSO (dimethyl sulfoxide), acetonitrile, DMF (N,N-dimethylformamide), N,N-dimethylacetamide, methanol, ethanol, propanol, tert-butanol, N-methylpyrrolidone, and combinations of two or more thereof, with ethanol being the most preferred.

[0071] Preferably, the buffer solution is a sodium acetate buffer solution or a sodium citrate buffer solution.

[0072] Preferably, the concentration of the buffer solution is 10-40 mM and the pH of the buffer solution is 3-7; more preferably, the concentration of the buffer solution is 20-30 mM and the pH of the buffer solution is 4-6.

[0073] Preferably, the volume ratio of the aqueous phase solution to the organic phase solution is (1-6):1, more preferably 3:1.

[0074] Preferably, the mass ratio of liposomes to therapeutic or preventive agents in the lipid nanoparticles is (1-30):1, more preferably 20:1.

[0075] The beneficial effects of this invention are as follows:

[0076] This invention provides a novel ionizable lipid compound, its preparation method, lipid nanoparticles composed thereof, and their applications. Compared to the commercially available SM-102 lipid compound, the lipid nanoparticles composed of the ionizable lipid compound obtained by this invention exhibit a higher level of in vitro transfection efficiency. Furthermore, the ionizable lipid compound prepared by this invention achieves high yield and high purity, with a yield exceeding 90% and a purity exceeding 92%. The preparation method is characterized by mild reaction conditions, simplicity, and ease of operation, making it more suitable for industrial-scale production. Therefore, this invention can serve as a novel method for delivering nucleic acid drugs, which is of significant importance for promoting the development of nucleic acid drugs. Attached Figure Description

[0077] Figure 1 The HNMR spectrum of the compound of formula PL-0271 prepared in Example 1 is shown.

[0078] Figure 2 The HPLC chromatogram of the compound of formula PL-0271 prepared in Example 1 is shown.

[0079] Figure 3 The transfection efficiency of lipid nanoparticles in serum-free systems in Examples 22-32 is shown.

[0080] Figure 4The transfection efficiency of lipid nanoparticles in serum-containing systems in Examples 22-32 is shown. Detailed Implementation

[0081] the term

[0082] In this invention, "C1-C6 alkyl" refers to a saturated hydrocarbon group, which is a hydrocarbon group formed by removing one hydrogen atom from an alkane molecule. It is preferably a straight-chain or branched alkyl group with 1-10 carbon atoms that is substituted or unsubstituted, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, etc., but is not limited to these.

[0083] In this specification, "aromatic group" refers to a group that is aromatic, including monocyclic, bicyclic, and polycyclic aromatic groups, such as benzene, naphthalene, anthracene, and pyrene. The aromatic group may be substituted at one or more ring positions by one or more substituents, such as halogens, azides, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, mercapto, imino, amide, phosphonate, phosphonite, carbonyl, carboxyl, silyl, aldehyde, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclic group, aromatic moiety or heteroaromatic moiety, fluoroalkyl (such as trifluoromethyl), oxy, etc., but is not limited thereto. The term "aromatic group" also includes polycyclic systems having two or more rings, wherein two or more carbons are shared by two adjacent rings (the rings are "fused rings"), wherein at least one of the rings is an aromatic hydrocarbon, and the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic groups, but are not limited thereto.

[0084] In this specification, "C2-C6 alkenyl" refers to a straight-chain or branched hydrocarbon with optional substitutions comprising 2-6 carbon atoms and at least one carbon-carbon double bond. Alkenyl groups may include one, two, three, or more carbon-carbon double bonds, but are not limited thereto.

[0085] In this specification, "C3-C6 cycloalkyl" refers to a non-aromatic carbon ring having 3-6 carbon atoms and may or may not include any double or triple bonds. Unless otherwise specified, the carbon ring refers to both unsubstituted and substituted carbon ring groups, i.e., optionally substituted carbon rings. Examples include cyclopropyl, cyclopentyl, cyclohexyl, etc., but are not limited to these.

[0086] In this specification, "halogen atom" refers to a fluorine atom, chlorine atom, bromine atom, or iodine atom.

[0087] In this specification, "phospholipids" are selected from 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine, 1,2-dimyristoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-diundecanoyl-sn-glycerol-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, and 1,2-diundecanoyl-sn-glycerol-3-phosphocholine. Acyl-sn-glycerol-3-phosphate choline, 1,20-di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate choline 3-Phosphoethanolamine, 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylphosphatidylglycerol The sphingoethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine are all selected from the following, preferably 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC).

[0088] In this specification, "structural lipids" are selected from any one of cholesterol, β-sitosterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, lycopene, ursolic acid, and α-tocopherol, with cholesterol being preferred.

[0089] In this specification, "polyethanol-modified lipids (PEG lipids)" are selected from any one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, preferably PEG2000-DMG.

[0090] In this specification, the structural formula of "4-pyrrolidinylpyridine (4-PPY)" is shown below:

[0091]

[0092] The structural formula of "9-azajulonidine" is shown below in this specification:

[0093]

[0094] To better understand the technical solution of the present invention, the technical solution of the present invention will be further described below with reference to specific embodiments. The embodiments are only for the purpose of helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0095] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0096] According to the preparation method of ionizable lipid compounds provided by the present invention, the compounds shown in Table 2 below can be obtained:

[0097] Table 2

[0098]

[0099]

[0100] The specific preparation methods of the products in the table above are shown in the following examples, in which compound PL-0271 is used as a representative compound.

[0101] Examples 1-5: Preparation of ionizable lipid compound PL-0271

[0102] At room temperature, 2g of compound I-1, 0.87g of triphenylchloromethane, 0.43g of TEA, 0.07g of DMAP, and 10ml of organic solvent were added sequentially to a 50ml single-necked flask. The solution was heated to 50℃ and stirred for 8-10 hours. After the reaction was complete, the mixture was cooled to room temperature, and 20ml of ethyl acetate and 20ml of H2O were added. The mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain compound PL-0271. The ¹H NMR spectrum of compound PL-0271 prepared in Example 1 is attached. Figure 1 As shown, 1HNMR(400MHz,Chloroform-d)δ7.53–7.36(m,6H),7.36–7.25(m,6H),7.25–7.18(m,3 H), 4.87 (t, J = 6.2Hz, 1H), 4.05 (t, J = 6.8Hz, 2H), 3.18 (t, J = 5.3Hz, 2H), 2.71 (t, J = 6. 2Hz,2H),2.54–2.34(m,2H),2.26(t,J=7.5Hz,4H),1.70–1.55(m,6H),1.50(q,J=6.1 Hz, 6H), 1.42 (q, J=5.8, 3.9Hz, 4H), 1.36–1.15 (m, 48H), 0.88 (td, J=6.9, 2.7Hz, 9H).

[0103] The organic solvents selected are shown in Table 3 below:

[0104] Table 3

[0105] Example organic solvents Yield yield purity 1 N,N-Dimethylformamide 2.45g 91.3％ 92.8％ 2 Tetrahydrofuran 1.89g 70.6％ 80.1％ 3 dichloromethane 2.21g 82.3％ 86.9％ 4 Acetonitrile 1.86g 69.4％ 78.4％ 5 Dimethyl sulfoxide 2.00g 74.6％ 83.5％

[0106] The HPLC chromatogram of the compound of formula PL-0271 prepared in Example 1 is attached. Figure 2 As shown, the data analysis table corresponding to this spectrum is shown in Table 4 below:

[0107] Table 4

[0108]

[0109] Examples 6-9: Preparation of ionizable lipid compounds of formula PL-0271

[0110] At room temperature, 2g of compound I-1, 0.87g of triphenylchloromethane, an amine organic base, 0.07g of DMAP, and 10ml of N,N-dimethylformamide were added sequentially to a 50ml single-necked flask. The solution was heated to 50℃ and stirred for 8-10h. After the reaction was completed, the solution was cooled to room temperature, 20ml of ethyl acetate and 20ml of H2O were added, and the mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain compound PL-0271.

[0111] The selected amine organic bases are shown in Table 5 below:

[0112] Table 5

[0113]

[0114] Examples 10-12: Preparation of ionizable lipid compounds of formula PL-0271

[0115] At room temperature, 2g of compound I-1, 0.87g of triphenylchloromethane, 0.43g of TEA, catalyst, and 10ml of N,N-dimethylformamide were added sequentially to a 50ml single-necked flask. The solution was heated to 50℃ and stirred for 8-10h. After the reaction was completed, the solution was cooled to room temperature, 20ml of ethyl acetate and 20ml of H2O were added, and the mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain compound PL-0271.

[0116] The catalysts selected are shown in Table 6 below:

[0117] Table 6

[0118] Example catalyst Yield yield purity 10 none 1.60g 58.9％ 60.1％ 11 0.08g 4-pyrrolidinylpyridine 2.14g 79.6％ 82.3％ 12 0.1g 9-azajulonidine 2.02g 75.4％ 84.9％

[0119] Example 13, Preparation of ionizable lipid compound PL-0285

[0120] At room temperature, 2 g of compound I-1, 1.04 g of 4-methoxytriphenylchloromethane, 0.43 g of TEA, 0.07 g of DMAP, and 10 ml of N,N-dimethylformamide were added sequentially to a 50 ml single-necked flask. The solution was heated to 50 °C and stirred for 8-10 h. After the reaction was completed, the mixture was cooled to room temperature, 20 ml of ethyl acetate and 20 ml of H2O were added, and the mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain 2.31 g of compound PL-0285, with a yield of 83.6% and a purity of 91.8%.

[0121] Example 14, Preparation of ionizable lipid compound PL-0294

[0122] At room temperature, 2 g of compound I-1, 1.14 g of 4,4'-dimethoxytriphenylmethyl chloride, 0.43 g of TEA, 0.07 g of DMAP, and 10 ml of N,N-dimethylformamide were added sequentially to a 50 ml single-necked flask. The solution was heated to 50 °C and stirred for 8-10 h. After the reaction was completed, the mixture was cooled to room temperature, 20 ml of ethyl acetate and 20 ml of H2O were added, and the mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain 2.24 g of compound PL-0294, with a yield of 78.6% and a purity of 90.9%.

[0123] Example 15, Preparation of ionizable lipid compound PL-0301

[0124] At room temperature, 2 g of compound I-1, 1.25 g of 4,4',4'-trimethoxytriphenylmethyl chloride, 0.43 g of TEA, 0.07 g of DMAP, and 10 ml of N,N-dimethylformamide were added sequentially to a 50 ml single-necked flask. The solution was heated to 50 °C and stirred for 8-10 h. After the reaction was completed, the mixture was cooled to room temperature, 20 ml of ethyl acetate and 20 ml of H2O were added, and the mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain 2.40 g of compound PL-0301, with a yield of 81.9% and a purity of 91.4%.

[0125] Example 16, Preparation of ionizable lipid compound PL-1017

[0126] At room temperature, 2 g of compound I-1, 0.73 g of 2-methyldiphenylmethyl chloride, 0.43 g of TEA, 0.07 g of DMAP, and 10 ml of N,N-dimethylformamide were added sequentially to a 50 ml single-necked flask. The solution was heated to 50 °C and stirred for 8-10 h. After the reaction was completed, the mixture was cooled to room temperature, 20 ml of ethyl acetate and 20 ml of H2O were added, and the mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain 1.63 g of compound PL-1017, with a yield of 64.9% and a purity of 90.1%.

[0127] Example 17, Preparation of ionizable lipid compound PL-1128

[0128] At room temperature, 2 g of compound I-1, 0.43 g of benzyl chloride, 0.43 g of TEA, 0.07 g of DMAP, and 10 ml of N,N-dimethylformamide were added sequentially to a 50 ml single-necked flask. The solution was heated to 50 °C and stirred for 8-10 h. After the reaction was completed, the mixture was cooled to room temperature, 20 ml of ethyl acetate and 20 ml of H2O were added, and the mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain 1.54 g of compound PL-1128, with a yield of 68.2% and a purity of 89.6%.

[0129] Example 18, Preparation of ionizable lipid compound PL-2009

[0130] At room temperature, 2.16 g of compound I-2, 0.87 g of triphenylchloromethane, 0.43 g of TEA, 0.07 g of DMAP, and 10 ml of N,N-dimethylformamide were added sequentially to a 50 ml single-necked flask. The solution was heated to 50 °C and stirred for 8-10 h. After the reaction was completed, the mixture was cooled to room temperature, and 20 ml of ethyl acetate and 20 ml of H2O were added. The mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain 2.57 g of compound PL-2009, with a yield of 90.4% and a purity of 92.3%.

[0131] Example 19, Preparation of ionizable lipid compound PL-2016

[0132] At room temperature, 2.16 g of compound I-2, 1.14 g of 4,4'-dimethoxytriphenylmethyl chloride, 0.43 g of TEA, 0.07 g of DMAP, and 10 ml of N,N-dimethylformamide were added sequentially to a 50 ml single-necked flask. The solution was heated to 50 °C and stirred for 8-10 h. After the reaction was completed, the mixture was cooled to room temperature, and 20 ml of ethyl acetate and 20 ml of H2O were added. The mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain 2.63 g of compound PL-2016, with a yield of 87.4% and a purity of 91.5%.

[0133] Example 20: Preparation of ionizable lipid compound PL-2105

[0134] At room temperature, 2.16 g of compound I-2, 0.73 g of 2-methyldiphenylmethyl chloride, 0.43 g of TEA, 0.07 g of DMAP, and 10 ml of N,N-dimethylformamide were added sequentially to a 50 ml single-necked flask. The solution was heated to 50 °C and stirred for 8-10 h. After the reaction was completed, the mixture was cooled to room temperature, and 20 ml of ethyl acetate and 20 ml of H2O were added. The mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain 2.00 g of compound PL-2105, with a yield of 75.1% and a purity of 90.6%.

[0135] Example 21, Preparation of ionizable lipid compound PL-2118

[0136] At room temperature, 2.16 g of compound I-2, 0.43 g of benzyl chloride, 0.43 g of TEA, 0.07 g of DMAP, and 10 ml of N,N-dimethylformamide were added sequentially to a 50 ml single-necked flask. The solution was heated to 50 °C and stirred for 8-10 h. After the reaction was completed, the mixture was cooled to room temperature, and 20 ml of ethyl acetate and 20 ml of H2O were added. The mixture was allowed to stand and separated. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain 2.13 g of compound PL-2118, with a yield of 88.4% and a purity of 91.7%.

[0137] Preparation of lipid nanoparticles in Examples 22-32

[0138] According to the preparation method of lipid nanoparticles provided by the present invention, the ionizable lipid compounds PL-0271, PL-0285, PL-0294, PL-0301, PL-1017, PL-1128, PL-2009, PL-2016, PL-2105, PL-2118 and SM-102 synthesized in the present invention are combined with a therapeutic or preventive agent (luciferase mRNA) to prepare different lipid nanoparticles, as detailed in Table 7. The specific preparation method includes:

[0139] The Luciferase mRNA stock solution was diluted to a concentration of 0.0111 mg / mL using 25 mM sodium acetate buffer (pH 5.0) as the aqueous phase. An organic phase was prepared by mixing four lipids—ionizable lipid compounds, DSPC, Cholesterol, and PEG2000-DMG—in a molar ratio of 50:10:38.5:1.5 and a liposome to mRNA mass ratio of 20:1. LNPs were then prepared using an aqueous phase to organic phase volume ratio of 3:1.

[0140] Table 7

[0141]

[0142] Example 1: Physicochemical Properties of Lipid Nanoparticles

[0143] The size and polydispersity index (PDI) of the lipid nanoparticles in Examples 22-32 were determined using a Malvern particle size analyzer, and the results are shown in Table 8 below. The encapsulation efficiency of the lipid nanoparticles was determined using a microplate reader and a Quant-it Ribogreen RNA quantification kit, and the results are shown in Table 8 below.

[0144] Table 8

[0145] Example Size (nm) PDI Encapsulation efficiency (%) 22 116.4 0.07 94.69 23 140.8 0.26 48.32 24 123.5 0.12 81.78 25 117.6 0.10 93.51 26 134.2 0.15 74.85 27 126.3 0.23 50.98 28 118.1 0.11 92.16 29 145.1 0.15 80.04 30 120.7 0.09 87.13 31 132.7 0.18 78.14 32 115.2 0.10 82.64

[0146] Experiment Example 2: Cell Transfection Experiment with Lipid Nanoparticles

[0147] Measurement of transfection fluorescence value:

[0148] 200 μL of DC2.4 cells in logarithmic growth phase (density 1.25 × 10⁻⁶) were collected. 6Cells (13,000 cells / well) were seeded in 96-well plates and cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin (PS) solution at 37°C and 5% CO2 for 24 h. The original culture medium was discarded, and fresh DMEM complete medium (containing 10% FBS and 1% PS) and serum-free DMEM medium (containing 0% FBS and 1% PS) were added to the wells. 15 μL of mRNA-LNP (containing 25 ng / well) and 60 μL (containing 100 ng / well) were added to the wells, respectively, and the cells were transfected at 37°C and 5% CO2 for 24 h. The expression of firefly luciferase in DC2.4 cells 24 h after mRNA-LNP transfection was detected using a microplate reader, and the transfection value was determined. Cell Culture Lysis 5×Reagent was diluted 5-fold with ultrapure water to prepare 1×CellCultureLysis and brought to room temperature. Lysis solution of lyophilized Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer to prepare Luciferase Assay Reagent. The original culture medium was aspirated from each well of a 96-well plate, and 100 μL of 1×CellCultureLysis cell lysis buffer was added. After lysis for 5 min, the expression of firefly luciferase was detected using a microplate reader.

[0149] BCA method for determining total protein expression:

[0150] Dilute 0.5 mg / mL protein standard solution with ultrapure water to prepare standards with protein concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL. Prepare BCA working solution according to (Reagent A: Reagent B = 50:1). Add 20 μL of standard solution and 100 μL of BCA working solution to a 96-well plate, incubate at 37°C for 30 min, and measure the absorbance at 562 nm using a microplate reader. Plot a standard curve for the protein standards based on the concentrations and absorbance values. Add 4 μL of sample cell lysis buffer, 16 μL of ultrapure water, and 100 μL of BCA working solution to a 96-well plate, incubate at 37°C for 30 min, measure the absorbance at 562 nm using a microplate reader, and calculate the total protein concentration and total protein mass using the protein standard curve.

[0151] Transfection efficiency = Transfection fluorescence value / Total protein mass × 100%.

[0152] Examples 22-32: Transfection efficiency of lipid nanoparticles in serum-free systems are shown in the appendix. Figure 3 As shown; the transfection efficiency in the serum-containing system is shown in the appendix. Figure 4 As shown.

Claims

1. An ionizable lipid compound, characterized in that, It has the following structural formula: ， ， ， ， ， ， ， 。 2. A method for preparing the ionizable lipid compound of claim 1, characterized in that, Under the action of an amine organic base, the reaction of compound I-1 with compound II yields an ionizable lipid compound of compound IV; or, under the action of an amine organic base, the reaction of compound I-2 with compound II yields an ionizable lipid compound of compound V, as shown in the following reaction formulas. ； ； Where R is , , , or One of them.

3. The preparation method according to claim 2, characterized in that, Further, it includes using one or more compositions of DMAP and its analogues as a catalyst, wherein the DMAP analogue is 4-pyrrolidinylpyridine or 9-azajulonidine.

4. The preparation method according to claim 3, characterized in that, The catalyst is DMAP.

5. The preparation method according to claim 3, characterized in that, The amine organic base is selected from triethylamine, diisopropylethylamine, diethylamine, ethylenediamine, ammonia, pyridine, and combinations of two or more thereof; the molar ratio of the amine organic base to the compound of formula I-1 or formula I-2 is (1.0-2.0):1; the molar ratio of the catalyst to the compound of formula I-1 or formula I-2 is (0.1-0.5):1; the molar ratio of the compound of formula II to the compound of formula I or formula I-2 is (1-2):

1.

6. The preparation method according to claim 5, characterized in that, The amine organic base mentioned is triethylamine.

7. The preparation method according to claim 5, characterized in that, The molar ratio of the amine organic base to the compound of formula I-1 or formula I-2 is 1.5:

1.

8. The preparation method according to claim 5, characterized in that, The molar ratio of the catalyst to the compound of formula I-1 or formula I-2 is 0.2:

1.

9. The preparation method according to claim 5, characterized in that, The molar ratio of the compound of formula II to the compound of formula I-1 or formula I-2 is 1.2:

1.

10. The preparation method according to claim 2, characterized in that, The reaction solvent is selected from dimethyl sulfoxide, diethyl ether, n-hexane, dichloromethane, chloroform, tetrahydrofuran, toluene, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, acetone, ethyl acetate, and combinations of two or more of them; the reaction temperature is controlled at 30-60℃; and the reaction time is controlled at 4-12h.

11. The preparation method according to claim 10, characterized in that, The reaction solvent is N,N-dimethylformamide.

12. The preparation method according to claim 10, characterized in that, The reaction temperature is controlled at 45-55℃.

13. The preparation method according to claim 10, characterized in that, The reaction time should be controlled within 8-10 hours.

14. A liposome, characterized in that, It is at least one of the following (a) or (b): (a) Made from the ionizable lipid compound of claim 1; (b) Made from the ionizable lipid compound of claim 1 and the co-lipid, wherein the co-lipid comprises phospholipids and / or structural lipids and / or polyethanol-modified lipids.

15. Use of the ionizable lipid compound of claim 1 or the liposome of claim 14, characterized in that, Includes at least one of the following 1)-2), 1) Preparation of in vivo delivery agents for therapeutic or preventative agents; 2) Prepare the transfection kit; The therapeutic or preventive agent is selected from nucleic acid drugs; the nucleic acid drug is selected from at least one of DNA drugs and RNA drugs; the RNA drug is selected from at least one of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, and lncRNA.

16. The use according to claim 15, characterized in that, The nucleic acid drug is an RNA drug.

17. The use according to claim 15 or 16, characterized in that, The RNA drug is mRNA.

18. A lipid nanoparticle, characterized in that, Including the following (1) and (2), (1) Treatment or preventative agent; (2) Liposomes used to deliver therapeutic or preventative agents; The definition of the therapeutic or preventive agent is the same as that in claim 15; the liposome is at least one of the following (a) or (b): (a) Made from the ionizable lipid compound of claim 1; (b) Made from the ionizable lipid compound and cofactor of claim 1, wherein the cofactor comprises phospholipids, structural lipids and polyethanol-modified lipids; wherein the molar ratio of the ionizable lipid compound, phospholipids, structural lipids and polyethanol-modified lipids is (10-100):(0-50):(0-50):(0-50).

19. The lipid nanoparticles according to claim 18, characterized in that, The molar ratio of the ionizable lipid compound, phospholipid, structural lipid and polyethanol-modified lipid is (30-80):(2-20):(30-50):(0.5-5).

20. The lipid nanoparticles according to claim 19, characterized in that, The molar ratio of the ionizable lipid compound, phospholipid, structural lipid and polyethanol-modified lipid is (40-60):(5-15):(35-45):(0.5-2).

21. The lipid nanoparticles according to claim 20, characterized in that, The molar ratio of the ionizable lipid compound, phospholipid, structural lipid, and polyethanol-modified lipid is 50:10:38.5:1.

5.

22. A method for preparing the lipid nanoparticles of claim 18, characterized in that, Includes the following steps: - Dissolve liposomes in an organic solvent to obtain an organic phase solution; - Add the treatment or preventative agent to the buffer solution to obtain an aqueous solution; -Lipid nanoparticles are obtained by mixing organic phase solution and aqueous phase solution.

23. The preparation method according to claim 22, wherein the organic solvent is DMSO, acetonitrile, DMF, N,N-dimethylacetamide, methanol, ethanol, propanol, tert-butanol, N-methylpyrrolidone, and a combination of two or more thereof; the buffer solution is sodium acetate buffer solution or sodium citrate buffer solution; the concentration of the buffer solution is 10-40 mM, and the pH of the buffer solution is 3-7; the volume ratio of the aqueous phase solution to the organic phase solution is (1-6):1; and the mass ratio of liposomes to therapeutic or preventive agents in the lipid nanoparticles is (1-30):

1.

24. The preparation method according to claim 23, characterized in that, The organic solvent is ethanol.

25. The preparation method according to claim 23, characterized in that, The concentration of the buffer solution is 20-30 mM, and the pH is 4-6.

26. The preparation method according to claim 23, characterized in that, The volume ratio of the aqueous phase solution to the organic phase solution is 3:

1.

27. The preparation method according to claim 23, characterized in that, The mass ratio of liposomes to therapeutic or preventative agents in lipid nanoparticles is 20:

1.

28. A method for analyzing ionizable lipid compounds according to claim 1, characterized in that, The determination was performed using high-performance liquid chromatography (HPLC). A YMC Bio C4 150*4.6mm*3um column was used, with 0.1% trifluoroacetic acid as mobile phase A and acetonitrile as mobile phase B. The flow rate was 1.5 mL / min, the detection wavelength was 190 nm, the column temperature was 40℃, a CAD detector with a nominal value of 50 was used, the peak width was 0.1 min, the acquisition frequency was 10 Hz, and the filtration constant was 3.6 s. Gradient elution was performed according to Table 1 below. Table 1 。

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