Preparation and application of phosphoramidate lipid compound

Abstract

The invention relates to an aminophospholipid lipid molecule compound, a compound as shown in a chemical formula (I), or pharmaceutically acceptable salt, prodrug and # imgabs0 # thereof. The invention also discloses a preparation method of the compound and application of the compound as a delivery treatment component in preparation of drugs.

Description

Technical Field

[0001] The present invention relates to amino lipid compounds, in particular to a class of aminophosphoryl lipid compounds which can be used for delivering genes into cells, and a preparation method and use thereof. Background Art

[0002] In gene therapy, nucleic acid drugs are delivered to target cells to express or inhibit the production of target proteins in target cells, thereby regulating and treating diseases caused by gene defects, which has broad application prospects. However, naked nucleic acid drugs are easily degraded by nucleic acid degrading enzymes in the blood and have no therapeutic effect. In addition, since nucleic acids and cell membranes are negatively charged, it is difficult for them to enter cells directly. In order for nucleic acid drugs to enter cells efficiently, they need to be delivered with the help of delivery tools.

[0003] As a carrier for the delivery of nucleic acid drugs, lipid nanoparticles (LNPs) have great application potential due to their quick preparation, convenient transportation and storage, and non-immunogenicity. Its core lipid components generally include two major categories: cationic lipid molecules and ionizable lipid molecules. However, the toxic side effects of cationic lipids limit their further application. Ionizable lipid molecules are electrically neutral in the blood. After endocytosis, LNPs / nucleic acid drugs will be transported to lysosomal vesicles, and the internal environment will change from neutral to acidic. At this time, the ionizable lipid molecules are protonated by the acidic environment and become positively charged, causing the LNPs bilayer membrane to cleave, so that the coated nucleic acid drug is released into the cytoplasm, and then exerts its effect. Existing lipid molecules generally have a relatively low lysosomal escape efficiency, and the efficiency of gene drug delivery is low. In addition, the biological toxicity of existing lipid molecules is relatively large, and the biological safety needs to be further improved.

[0004] Phospholipids are a class of natural lipid components, which are key components of biological membranes and organelles. They have good biocompatibility, contribute to the encapsulation of nucleic acids, and play a powerful role in the membrane fusion step, increasing the efficiency of escape in vivo. However, natural phospholipids are mainly found in animals and plants, and exist in a bound state with proteins, sugars, fatty acids, mycophenolic acid, vitamins and other substances. They are an important class of oil accompaniments, and it is difficult to separate a single phospholipid compound. In addition, natural phospholipids lack structural flexibility, that is, the types and quantities of phospholipids are limited. Therefore, it is of great significance to develop a multi-tailed ionizable phospholipid compound. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides an aminophosphoester lipid compound that can be used to deliver genes to cells, and a preparation method and use thereof, which not only enriches the diversity of phospholipid structures, but also can achieve targeting needs during nucleic acid delivery.

[0006] The technical solution adopted by the present invention to solve the technical problem is: the aminophosphoric acid ester lipid compound or its pharmaceutically acceptable salt, prodrug has a structure shown in chemical formula (I):

[0007]

[0008] Wherein, n and m are each an integer from 1 to 8;

[0009] The R 1 , R 2 Each is independently selected from substituted or unsubstituted C6-C18 alkyl, C6-C18 alkenyl, C6-C18 ester group, and the substituent of the substituted C6-C18 alkyl, C6-C18 alkenyl, C6-C18 ester group is a C1-C6 hydrocarbon group;

[0010] The R 3 -H or substituted or unsubstituted C1-C18 alkyl, C1-C18 alkenyl, C1-C18 ester, the substituent of the substituted C1-C18 alkyl, C1-C18 alkenyl, C1-C18 ester is C1-C6 hydrocarbon group;

[0011] The R 4 is a substituted or unsubstituted C6-C18 alkyl, C6-C18 alkenyl, or C6-C18 ester group, wherein the substituent of the substituted C6-C18 alkyl, C6-C18 alkenyl, or C6-C18 ester group is a C1-C6 hydrocarbon group;

[0012] The R 5 -H or -CH2CH(OH)-R 4 .

[0013] Preferably, n and m are integers of 2-6 respectively.

[0014] Preferably, the R 1 , R 2 Each is independently selected from substituted or unsubstituted C13-C18 alkyl, C13-C18 alkenyl, C13-C18 ester group, and the substituent of the substituted C13-C18 alkyl, C13-C18 alkenyl, C13-C18 ester group is a C1-C6 hydrocarbon group;

[0015] The R 3 is -H or a substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 ester group, and the substituent of the substituted C1-C18 alkyl, C1-C18 alkenyl, or C1-C18 ester group is a C1-C6 hydrocarbon group;

[0016] The R 4It is a substituted or unsubstituted C10-C18 alkyl, C10-C18 alkenyl or C10-C18 ester group, and the substituent of the substituted C6-C18 alkyl, C6-C18 alkenyl or C6-C18 ester group is a C1-C6 hydrocarbon group.

[0017] More preferably, the R 1 , R 2 Each independently selected from

[0018] The R 3 Select from -H or

[0019] The R 4 Selected from

[0020] A method for preparing a phosphoramidate lipid compound or a pharmaceutically acceptable salt or prodrug thereof, comprising the following preparation steps:

[0021] S1:

[0022]

[0023] S2:

[0024]

[0025] S3:

[0026]

[0027] Among them, the R 1 , R 2 Each is independently selected from substituted or unsubstituted C13-C18 alkyl, C13-C18 alkenyl, C13-C18 ester group, and the substituent of the substituted C13-C18 alkyl, C13-C18 alkenyl, C13-C18 ester group is a C1-C6 hydrocarbon group;

[0028] The R 3 is -H or a substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 ester group, and the substituent of the substituted C1-C18 alkyl, C1-C18 alkenyl, or C1-C18 ester group is a C1-C6 hydrocarbon group;

[0029] The R 4 is a substituted or unsubstituted C10-C18 alkyl, C10-C18 alkenyl, or C10-C18 ester group, and the substituent of the substituted C6-C18 alkyl, C6-C18 alkenyl, or C6-C18 ester group is a C1-C6 hydrocarbon group;

[0030] The R 5Selected from -H or -CH2CH(OH)-

[0031] Preferably, the R 1 , R 2 Each independently selected from

[0032] The R 3 Select from -H or

[0033] The R 4 Selected from

[0034] More preferably, the above-mentioned phosphoramidate lipid compound is selected from the following representative compounds:

[0035] Table 1: Representative compounds of this application

[0036]

[0037]

[0038]

[0039] The present invention also provides a use of any of the aforementioned phosphoramidate lipid compounds or pharmaceutically acceptable salts thereof in the preparation of lipid nanoparticles.

[0040] Specifically, the steps for preparing the lipid nanoparticles are as follows:

[0041] The aminophosphoryl lipid compound, auxiliary lipid molecule, cholesterol and PEG2000-DMG described in the present invention are mixed and dissolved in anhydrous ethanol in a certain proportion; the mixed solution is then quickly injected into 3 times the volume of sodium citrate buffer solution and stirred for 1 minute to prepare a pre-LNP solution.

[0042] Preferably, the auxiliary lipid molecule is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), dimyristoylphosphatidylcholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), diethyl pyrocarbonate (DEPC), 1,2-diacyl-sn-glycero-3-phosphocholine (HSPC) or 2-oleoyl-1-palmitoyltinglycero-3-phosphocholine (POPC).

[0043] More preferably, the helper lipid molecule is DOPE, DSPC or DOPC.

[0044] Preferably, the proportions of the aminophosphoric acid ester lipid compound, auxiliary lipid molecule, cholesterol and PEG2000-DMG are as follows: aminophosphoric acid ester lipid compound: auxiliary lipid molecule: cholesterol: PEG2000-DMG = 20%-60%: 10%-30%: 30%-50%: 0.2%-5%.

[0045] More preferably, the proportions of the aminophosphoric acid ester lipid compound, auxiliary lipid molecule, cholesterol and PEG2000-DMG are as follows: aminophosphoric acid ester lipid compound: auxiliary lipid molecule: cholesterol = 50%: 10%: 38.5%: 1.5%.

[0046] The present invention also provides a use of any of the above-mentioned phosphoamidate lipid compounds or a pharmaceutically acceptable salt thereof in the preparation of a drug for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

[0047] Wherein, the nucleic acid is RNA or DNA.

[0048] Preferably, the nucleic acid is an antisense oligonucleotide; the RNA is any one of mRNA, rRNA, miRNA, tRNA, siRNA, and snRNA; and the DNA is a plasmid.

[0049] The beneficial effects of the present invention are:

[0050] (1) The present invention provides an aminophosphoryl lipid compound, which contains a hydrophobic long chain of non-polar groups and a hydrophilic hydroxyl portion, and can effectively encapsulate nucleic acid drugs and deliver them into cells.

[0051] (2) The present invention uses a multi-tail phosphate structure as the core lipid component and introduces an ionizable amino part, which not only enriches the diversity of phospholipid structures, but also can create specific chemical structures according to targeting needs, thereby increasing the escape ability of phosphate in lysosomes and significantly enhancing the delivery efficiency of lipid nanoparticles.

[0052] (3) The phosphate structure has certain biocompatibility and high biosafety.

[0053] (4) The aminophosphoryl lipid compound can use unsaturated fatty acids as the hydrophobic end. Since unsaturated fatty acids have a lower phase transition temperature, they can promote the phase transition of lipid particles in the endosomes, destroy the stability of the endosomes, and further increase the escape performance of nucleic acid drugs from the endosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 For Example 1P4 1 H-NMR spectrum.

[0055] Figure 2 For Example 1P4 31 P-NMR spectrum.

[0056] Figure 3 For Example 2P5 1 H-NMR spectrum.

[0057] Figure 4 For Example 2P5 31 P-NMR spectrum.

[0058] Figure 5 For Example 3P6 1 H-NMR spectrum.

[0059] Figure 6 For Example 3P6 31 P-NMR spectrum. DETAILED DESCRIPTION

[0060] In order to more clearly illustrate the purpose, technical solutions and advantages of the embodiments of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments for a clear and complete description. The described embodiments are partial embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without paying creativity also belong to the protection scope of the present invention.

[0061] The synthetic route is as follows:

[0062] S1:

[0063]

[0064] S2:

[0065]

[0066] S3:

[0067]

[0068] R 1 =R 2 =-(CH2) 12 Taking CH3 as an example, the synthesis method of this type of lipid molecule is introduced.

[0069] Synthesis steps of intermediate (1):

[0070] Add 9.1mmol 3-(benzyloxy)propane-1,2-diyl tetracosanoate, 100mL dichloromethane, 1.0g 10% Pd / C to a 200mL single-necked flask equipped with a magnetic electron, then replace the air with nitrogen, then replace the nitrogen with hydrogen, heat to 30°C and stir for 2h, and detect the reaction by thin layer chromatography. After the reaction is completed, filter to remove the catalyst, and then concentrate the mother liquor to obtain a white solid with a yield of 99%, and perform mass spectrometry and nuclear magnetic resonance analysis. 1 H NMR(400MHz,Chloroform-d)δ5.08(p,J=5.0Hz,1H),4.35–4.20(m,2H),3.73(d,J=4.9Hz,2 H),2.38–2.28(m,4H),2.06(s,1H),1.67–1.58(m,4H),1.25(s,40H),0.88(t,J=6.7Hz,6H).

[0071] ESI-MS(m / z):[M+H] + calcd.for C 31 H 60 O5,512.4; found,512.4.

[0072] The synthesis steps of compound (2):

[0073] Add 3.2mmol 3-hydroxypropane-1,2-tetracosanoate, 10mL anhydrous tetrahydrofuran, 0.32mmol DMAP, 4.8mmol triethylamine to a 25mL single-necked flask equipped with a magnetic electron. When the temperature drops to 0°C, replace the air with nitrogen, then dilute 4.8mmol 2-chloro-2-oxo-1,3,2-dioxaphospholane with 4mL tetrahydrofuran, and add it dropwise to the single-necked flask through a constant pressure dropping funnel. After the addition is complete, slowly heat to 25°C and stir for 6h. Detect the reaction by thin layer chromatography. After the reaction is completed, filter to remove the precipitate, concentrate the solution, and separate the crude product by column chromatography. The eluent is petroleum ether / ethyl acetate = 2 / 1 to obtain a white solid with a yield of 85%, and perform mass spectrometry and nuclear magnetic resonance analysis.

[0074] 1 H NMR(400MHz,Chloroform-d)δ5.29–5.19(m,1H),4.45–4.04(m,7H),3.82(q,J=4.2Hz,1H) ,2.32(dt,J=15.0,7.6Hz,4H),1.62(q,J=7.1Hz,4H),1.25(s,40H),0.88(t,J=6.8Hz,6H).

[0075] ESI-MS(m / z):[M+H] + calcd.for C 33 H 63 O8P,618.4;found,618.4.

[0076] Synthesis steps of intermediate (3):

[0077] When m = 4, R 3 For -H, R 4 When it is -CH2(CH2)8CH3, add 3mmol N-tert-butyloxycarbonyl-1,4-butanediamine and 6mmol epoxydodecane to a 25mL single-necked flask equipped with a magnetic electron, heat to 90℃ and stir for 48h, and detect the reaction by thin layer chromatography. After the reaction is completed, the crude product is separated by column chromatography, and the eluent is dichloromethane / methanol = 20 / 1 to obtain a yellow oily liquid with a yield of 95.1%, and mass spectrometry and nuclear magnetic resonance analysis are performed.

[0078] 1H NMR (400MHz, DMSO-d6) δ6.74(s,1H),4.25(s,1H),4.20(s,1H),3.54–3.35(m,2H),2.89(q,J=6.2Hz, 2H),2.50–2.16(m,6H),1.45–1.35(m,13H),1.35–1.29(m,4H),1.24(s,32H),0.85(t,J=6.9Hz,6H).

[0079] ESI-MS(m / z):[M+H] + calcd.for C 33 H 68 N2O4,556.5;found,556.5.

[0080] The synthesis steps of compound (4):

[0081] 0.8 mmol (4-(bis(2-hydroxydodecyl)amino)butyl)carbamic acid tert-butyl ester, 8 mL dichloromethane, 4 mL trifluoroacetic acid were added to a 25 mL single-necked flask equipped with a magnetic electron, and stirred for 4 h at 25 ° C. The reaction was detected by thin layer chromatography. After the reaction was completed, the crude product was washed with a saturated sodium carbonate aqueous solution until neutral, dried over anhydrous sodium sulfate, and the mother liquor was concentrated. The crude product was separated by column chromatography, and the eluent was dichloromethane / methanol = 10 / 1 to obtain a yellow oily liquid with a yield of 98%, and mass spectrometry and nuclear magnetic resonance analysis were performed.

[0082] 1 H NMR (400MHz, DMSO-d6) δ7.84(s,2H),4.40(s,2H),3.48(td,J=8.0,7.5,4.0Hz,2H),2.77(dq,J=7.1,4.5,2.5Hz,2H),2.49–2.21( m,6H),1.55(dq,J=13.8,6.8Hz,2H),1.46(q,J=7.0Hz,2H),1.37(s,4H),1.24(s,32H),0.85(t,J=6.6Hz,6H).ESI-MS(m / z):[M+H] + calcd.for C 28 H 60 N2O2,456.5; found,456.5.

[0083] Example 1 Aminophosphoryl lipid molecule P4:C 14 N4E 12 The synthetic route

[0084] When m = 4, R 3 For -H, R4 When the compound (4) is -CH2(CH2)8CH3, the compound (4) is synthesized according to step S2 to be 1,1'-((2-aminoethyl)azo)bis(dodecan-2-ol), and the compound (2) reacts with the compound (4) to generate the compound C. 14 N4E 12 .

[0085]

[0086] 800 mg (2), 590 mg 1,1'-((2-aminoethyl)azo)bis(dodecan-2-ol) and 2.0 mL anhydrous acetonitrile were added to a 25 mL single-necked flask equipped with a magnetic rod, and the mixture was heated to 75°C and stirred for 12 h. The reaction was detected by thin layer chromatography. After the reaction was completed, the mother liquor was concentrated, and the crude product was separated by column chromatography with the eluent being dichloromethane / methanol = 15 / 1 to obtain a yellow oily liquid with a yield of 48.6%. The mixture was then subjected to mass spectrometry and nuclear magnetic resonance analysis.

[0087] 1 H NMR(400MHz,Chloroform-d)δ5.08(p,J=5.1Hz,1H),4.39–4.05(m,8H),3.76–3.62(m,2H),3.39(s,1H), 2.50(t,J=8.2Hz,2H),2.44–2.18(m,10H),1.61(q,J=7.0Hz,8H),1.26(s,76H),0.87(t,J=6.8Hz,12H).

[0088] ESI-MS(m / z):[M+H] + calcd.for C 61 H 123 N2O 10 P,1074.9;found,1074.9.

[0089] Example 2 Synthesis route of aminophosphoryl lipid molecule P5: C14N4E14

[0090] When m = 4, R 3 For -H, R 4 -CH2(CH2) 10 When CH3 is present, compound (4) is synthesized according to step S2 to be 1,1'-((2-aminoethyl)azo)bis(tetradecane-2-ol), and compound (2) reacts with compound (4) to generate compound C 14 N4E 14 .

[0091]

[0092] 600 mg (2), 498 mg 1,1'-((2-aminoethyl)azo)bis(tetradecane-2-ol) and 647 μL anhydrous acetonitrile were added to a 25 mL single-necked flask equipped with a magnetic rod, and the mixture was heated to 75°C and stirred for 12 h. The reaction was detected by thin layer chromatography. After the reaction was completed, the mother liquor was concentrated, and the crude product was separated by column chromatography with the eluent being dichloromethane / methanol = 15 / 1 to obtain a yellow oily liquid with a yield of 51.3%. The mixture was then subjected to mass spectrometry and nuclear magnetic resonance analysis.

[0093] 1 H NMR(400MHz,Chloroform-d)δ5.08(p,J=5.1Hz,1H),4.40–4.05(m,8H),3.76–3.65(m,2H),3.39(s,1H), 2.50(t,J=8.2Hz,2H),2.44–2.19(m,10H),1.61(q,J=7.0Hz,8H),1.25(s,84H),0.87(t,J=6.8Hz,12H).

[0094] ESI-MS(m / z):[M+H] + calcd.for C 65 H 131 N2O 10 P,1131.0;found,1131.0.

[0095] Example 3 Aminophosphoryl lipid molecule P6:C 14 N4E 16 The synthetic route

[0096] When m = 4, R 3 For -H, R 4 -CH2(CH2) 12 When CH3 is present, compound (4) is synthesized according to step S2 to be 1,1'-((2-aminoethyl)azo)bis(hexadecane-2-ol), and compound (2) reacts with compound (4) to generate compound C 14 N4E 16 .

[0097]

[0098] 600 mg (2), 735 mg 1,1'-((2-aminoethyl)azo)bis(hexadecane-2-ol) and 2 mL anhydrous acetonitrile were added to a 25 mL single-necked flask equipped with a magnetic rod, and the mixture was heated to 75°C and stirred for 12 h. The reaction was detected by thin layer chromatography. After the reaction was completed, the mother liquor was concentrated, and the crude product was separated by column chromatography with the eluent being dichloromethane / methanol = 15 / 1 to obtain a yellow oily liquid with a yield of 52.4%. The mixture was then subjected to mass spectrometry and nuclear magnetic resonance analysis.

[0099] 1 H NMR(400MHz,Chloroform-d)δ5.06(p,J=5.1Hz,1H),4.38–4.05(m,8H),3.76–3.63(m,2H),3.38(s,1H), 2.52(t,J=8.2Hz,2H),2.43–2.19(m,10H),1.61(q,J=7.0Hz,8H),1.24(s,92H),0.86(t,J=6.8Hz,12H).

[0100] ESI-MS(m / z):[M+H] + calcd.for C 69 H 139 N2O 10 P,1187.0;found,1187.0.

[0101] Example 4 Aminophosphoryl lipid molecule P20:C 14 N4O 14 The synthetic route

[0102] When m = 4, R 3 For -H, R 4 -CH2(CH2) 10 When CH3 is used, the molar ratio of N-tert-butyloxycarbonyl-1,4-butanediamine to epoxydodecane is 1:1, and compound (4) is synthesized according to step S2 to be 1-((4-aminobutyl)amino)tetradecane-2-ol. Compound (2) reacts with compound (4) to generate compound C. 14 N4O 14 .

[0103]

[0104] 800 mg (2), 388 mg 1-((4-aminobutyl)amino)tetradecan-2-ol and 1.95 mL anhydrous acetonitrile were added to a 25 mL single-necked flask equipped with a magnetic rod, and the mixture was heated to 75°C and stirred for 10 h. The reaction was detected by thin layer chromatography. After the reaction was completed, the mother liquor was concentrated, and the crude product was separated by column chromatography with the eluent being dichloromethane / methanol = 15 / 1 to obtain a yellow oily liquid with a yield of 48.6%. The mixture was then subjected to mass spectrometry and nuclear magnetic resonance analysis.

[0105] 1H NMR(400MHz,Chloroform-d)δ5.22(p,J=10.7,5.3Hz,1H),4.40–4.11(m,6H),4.02(p,J=7.5,5.5Hz,1H),3.21–3.14(m,2H),3 .07–2.93(m,4H),2.28(q,J=7.9Hz,6H),1.68(dq,J=11.0,6.7Hz,4H),1.57(dq,J=7.7,3.5Hz,6H),1.24(s,60H),0.86(t,9H).

[0106] ESI-MS(m / z):[M+H] + calcd.for C 51 H 103 N2O9P,918.7;found,918.7.

[0107] Example 5 Lipid Nanoparticles Prepared from Phosphoramidate Lipid Compounds

[0108] The aminophosphoryl lipid compound described in the present invention is mixed and dissolved in anhydrous ethanol with DOPE, cholesterol, and PEG2000-DMG in a preferred molar ratio of 50:10:38.5:1.5. The solution is then quickly injected into 3 volumes of a sodium citrate buffer solution with a pH of 4 and stirred for 1 minute to prepare a pre-LNP solution. The size and polydispersity index PDI of the lipid nanoparticles are determined by dynamic light scattering using a Malvern Zetasizer NanoZS in a 173° backscattering detection mode. The particle size distribution of the prepared lipid nanoparticles is in the range of 80-200nm, which can meet the needs of in vivo delivery.

[0109] Example 6 Evaluation of the hemolytic properties of lipid nanoparticles prepared from aminophosphoryl lipid compounds

[0110] First, a pre-LNP solution was prepared according to the method in Example 5. Then, D-PBS buffers of pH 7.4 and 5.5 were prepared, and the dialyzed pre-LNP was diluted to 12.5, 25, 50, 100, 200 and 400 μg / mL, respectively. Fresh blood from mice was taken, and after centrifugation to remove the supernatant, the blood cells were washed several times with D-PBS of pH 7.4 until the supernatant was colorless and transparent. The red blood cells were then resuspended with buffers of pH 7.4 and 5.5, respectively. According to V 红细胞悬液 :V LNP =1:4, add cell suspensions of different pH to LNP solutions of different concentrations of corresponding pH, mix gently and incubate together. Centrifuge after 4 hours, take the supernatant and measure the absorbance at a wavelength of 540nm.

[0111] Hemolysis rate % = [(OD sample - OD negative) / (OD positive - OD negative)] x 100%.

[0112] As shown in Table 2, the lipid nanoparticles prepared by the aminophosphoric acid ester lipid compound of the present invention have low hemolytic activity (7%-23%) at pH 7.4, indicating that in the human blood circulation system, the lipid nanoparticles are not easy to cause human red blood cells to rupture and dissolve and cause harm to the human body; and at pH 5.5, the hemolysis rate is high (75%-95%), indicating that under acidic conditions, the nucleic acid coated by the lipid nanoparticles is easily released. Therefore, the aminophosphoric acid ester lipid compound of the present invention is conducive to further development and application.

[0113] Example 7 Evaluation of luciferase mRNA cell-level transfection performance of lipid nanoparticles prepared from aminophosphoryl lipid compounds

[0114] Preparation method: First, prepare a pre-LNP solution according to the method in Example 5. Dissolve luciferase mRNA (FlucmRNA) in DEPC aqueous solution, add an equal volume of 50% ethanol solution to prepare an ethanol solution with a final concentration of 25%. Mix the pre-LNP solution and the mRNA ethanol solution at a volume ratio of 1.5:1. Incubate at 50°C for 20 minutes to obtain lipid nanoparticles encapsulating mRNA, and dialyze in PBS buffer for 2 hours.

[0115] According to the manufacturer's instructions, the encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantitative assay kit. The test results are shown in Table 2, indicating that the carrier prepared by the aminophospholipid lipid compound of the present invention has a high encapsulation efficiency for nucleic acid molecules and can successfully transport nucleic acid molecules into cells for expression.

[0116] Table 2

[0117]

[0118]

[0119]

[0120] Note: Hemolysis rate* indicates the ratio when the concentration is 100ug / ml

[0121] Human bronchial epithelial cell line HBE in logarithmic growth phase was cultured at 25×10 3Each well was inoculated in a 48-well cell culture plate. After the cells adhered to the wall, lipid nanoparticles loaded with mRNA were added for cell transfection (200ng mRNA / well), and 3 replicates were made for each sample. After 6 hours of transfection, the culture medium was discarded, 50μL / well of PLB lysis solution was added, and the cells were lysed for 20 minutes. After the cell culture plate was centrifuged, 10μL of lysis solution was aspirated to a 96-well black board, and 50μL / well of luciferase substrate was added, and the luminescence intensity of the sample was immediately detected with an ELISA reader.

[0122] The fluorescence intensity of each group of samples is related to the efficiency of Luc-mRNA transfection with fluorescent protein. As shown in Table 3, the commercial SM-102 ionizable ionic lipid with higher transfection efficiency on the market is used as the control group (100%). It can be seen that after cell transfection, the fluorescence intensity of the aminophospholipid lipid nanoparticles loaded with luciferase mRNA of the present invention is significantly higher than that of the commercial SM-102 lipid nanoparticles loaded with luciferase mRNA, and the average fluorescence intensity of compound P5 can reach up to 4586, indicating that the aminophospholipid liposomes of the present invention have quite reliable commercial transformation potential.

[0123] Table 3

[0124]

[0125]

[0126]

[0127]

[0128] Experiments have shown that the phosphoramidate lipid compounds of the present invention can deliver nucleic acid molecules, small molecule compounds, polypeptides or proteins, etc. The vector prepared using the lipid compounds of the present invention has a high encapsulation rate for nucleic acid molecules and can successfully transport nucleic acid molecules into cells for expression.

[0129] The above detailed description of the present invention is intended to enable those familiar with the technology in this field to understand the contents of the present invention and implement them. It does not limit the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit of the present invention are covered by the scope of protection of the present invention.

Claims

1. A phosphoramidate lipid compound or a pharmaceutically acceptable salt thereof, characterized in that: Its structure is shown in formula (I): Wherein, n and m are each an integer from 1 to 8; The R 1 , R 2 Each is independently selected from substituted or unsubstituted C6-C18 alkyl, C6-C18 alkenyl, C6-C18 ester group, and the substituent of the substituted C6-C18 alkyl, C6-C18 alkenyl, C6-C18 ester group is a C1-C6 hydrocarbon group; The R 3 -H or substituted or unsubstituted C1-C18 alkyl, C1-C18 alkenyl, C1-C18 ester, the substituent of the substituted C1-C18 alkyl, C1-C18 alkenyl, C1-C18 ester is C1-C6 hydrocarbon group; The R 4 is a substituted or unsubstituted C6-C18 alkyl, C6-C18 alkenyl, or C6-C18 ester group, wherein the substituent of the substituted C6-C18 alkyl, C6-C18 alkenyl, or C6-C18 ester group is a C1-C6 hydrocarbon group; The R 5 -H or -CH2CH(OH)-R 4 .

2. The phosphoramidate lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The R 1 , R 2 Each is independently selected from substituted or unsubstituted C13-C18 alkyl, C13-C18 alkenyl, C13-C18 ester group, and the substituent of the substituted C13-C18 alkyl, C13-C18 alkenyl, C13-C18 ester group is a C1-C6 hydrocarbon group; The R 3 is -H or a substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 ester group, and the substituent of the substituted C1-C18 alkyl, C1-C18 alkenyl, or C1-C18 ester group is a C1-C6 hydrocarbon group; The R 4 It is a substituted or unsubstituted C10-C18 alkyl, C10-C18 alkenyl or C10-C18 ester group, and the substituent of the substituted C6-C18 alkyl, C6-C18 alkenyl or C6-C18 ester group is a C1-C6 hydrocarbon group.

3. The phosphoramidate lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The R 1 , R 2 Each independently selected from The R 3 Select from -H or The R 4 Selected from 4. The phosphoramidate lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The phosphoramidate lipid compound is selected from:

5. A method for preparing a phosphoramidate lipid compound or a pharmaceutically acceptable salt thereof, comprising the following steps: S1： S2： S3： in, R 1 , R 2 , R 3 , R 4 and R 5 As defined in claim 2.

6. Use of the aminophosphoryl lipid compound or a pharmaceutically acceptable salt thereof as claimed in claims 1 to 4 in the preparation of lipid nanoparticles.

7. The use according to claim 6, characterized in that: The phosphoramidate lipid compound is selected from:

8. The use according to claim 6, characterized in that: The lipid nanoparticles are synthesized by phosphoramidate lipid compounds, auxiliary phospholipids, cholesterol and polyethylene glycol. The lipid nanoparticles can improve the translation expression level of the nucleic acid of the load in the cell.

9. The use according to claim 8, characterized in that: The auxiliary phospholipids are DOPE, DSPC and DOPC.

10. Use of the phosphoramidate lipid compound or a pharmaceutically acceptable salt thereof as claimed in claims 1 to 4 in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy, interfering RNA or nucleic acid transfer.

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