A class of aminoamide lipid compounds and preparation methods and applications thereof

Abstract

The present invention relates to a class of aminoamide lipid compounds, compounds represented by chemical formula (I), or pharmaceutically acceptable salts and prodrugs thereof, and also discloses a preparation method of the compound and its use as a delivery therapeutic component in the preparation of drugs.

Description

Technical Field

[0001] The present invention relates to amide lipid compounds, in particular to aminoamide lipid compounds which can be used for delivering genes into cells, and preparation methods and uses thereof. Background Art

[0002] Gene therapy is a very promising treatment method. It uses artificial means to deliver genes with specific genetic information to target cells, and regulates and treats diseases caused by gene defects by inhibiting or expressing target proteins in target cells. However, naked nucleic acid drugs are easily degraded by nucleic acid degrading enzymes in the blood, and the purpose of treatment cannot be achieved. In addition, since nucleic acids are negatively charged and cell membranes are also negatively charged, it is difficult to enter cells directly. Therefore, in order for nucleic acid drugs to efficiently enter cells and exert their effects, they need to be achieved with the help of delivery vectors.

[0003] Lipid nanoparticles (LNPs) have shown great application potential as nucleic acid drug delivery carriers due to their easy preparation, convenient transportation and storage, and non-immunogenicity. Lipid molecules, as the core components of lipid nanoparticles, generally include two major categories: cationic lipid molecules and ionizable lipid molecules. Although cationic lipids can efficiently encapsulate nucleic acid drugs, they have toxic side effects when entering the blood due to their positive charge, which limits their further application. Ionizable lipid molecules are neutrally charged in the blood environment (pH 7.4). After endocytosis, LNP / nucleic acid drugs will be transported to lysosomal vesicles, and the LNP environment gradually changes from neutral to acidic. At this time, the ionizable lipid molecules are protonated and change from neutral to positively charged. The LNPs bilayer membrane is cleaved, so that the coated nucleic acid drugs are released into the cytoplasm, and then the drug effect is exerted. This is the lysosomal escape process of nucleic acid drugs. Existing lipid molecules generally have a relatively low lysosomal escape efficiency, and the efficiency of gene drug delivery is low. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides an aminoamide lipid compound that can be used to deliver genes into cells, and a preparation method and use thereof.

[0005] The technical solution adopted by the present invention to solve the technical problem is: an aminoamide lipid compound, or a pharmaceutically acceptable salt or prodrug thereof, having a structure shown in chemical formula (I):

[0006]

[0007] Among them, R 1is a straight chain alkane containing a secondary or tertiary amine group, or is a substituted or unsubstituted 4 to 10 membered heterocycle containing one or more heteroatoms independently selected from nitrogen, sulfur or oxygen;

[0008] The R 2 , R 3 and R 4 Each is independently selected from a substituted or unsubstituted C4-C20 alkyl group, a C4-C20 alkenyl group, and a C4-C20 ester group, and the substituent of the substituted C4-C20 alkyl group, the C4-C20 alkenyl group, and the C4-C20 ester group is a C1-C6 hydrocarbon group.

[0009] Preferably, the R 1 One selected from CN1, CN2, CN3, CN4, CN5, CN6, CN7, CN8, CN9, CN10, CN11, CN12, CN13, CN14, CN15, CN16, CN17, CN18;

[0010] The R 2 , R 3 , R 4 Each is independently selected from one of A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, and A22;

[0011] The CN1, CN2, CN3, CN4, CN5, CN6, CN7, CN8, CN9, CN10, CN11, CN12, CN13, CN14, CN15, CN16, CN17, and CN18 are as follows:

[0012]

[0013] The A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22 are as follows:

[0014] A5:-(CH 2 ) 4 CH 3 ; A6: -(CH 2 ) 5 CH 3 ; A7: -(CH 2 ) 6 CH 3 ; A8: -(CH 2 ) 7 CH 3 ;

[0015] A9:-(CH 2 ) 8 CH 3 ; A10: -(CH 2 ) 9 CH 3 ; A11: -(CH 2 ) 10 CH 3 ; A12: -(CH 2 ) 11 CH 3 ;

[0016] A13: -(CH 2 ) 12 CH 3 ; A14: -(CH 2 ) 13 CH 3 ; A15: -(CH 2 ) 14 CH 3 ; A16: -(CH 2 ) 15 CH 3 ;

[0017] A17:-(CH 2 ) 16 CH 3 ; A18: -(CH 2 ) 17 CH 3 ; A19: -(CH 2 ) 18 CH 3 ; A20: -(CH 2 ) 19 CH 3 ;

[0018]

[0019] More preferably, the aminoamide lipid compound of the present invention is selected from the following compounds:

[0020]

[0021] Among them, R 2 , R 3 and R 4 Each independently

[0022] A method for preparing an amide lipid compound or a pharmaceutically acceptable salt or prodrug thereof, comprising the following preparation steps:

[0023] First, compound R2 -CHO is dissolved in the solvent and compound R is added under stirring. 3 -NH 2 , heating reaction, to obtain imine intermediate R 2 -CH=NR 3 , and then to the imine intermediate R 2 -CH=NR 3 Compound R was added to the solution 4 -COOH and R 1 -NC, and stirred for reaction to obtain compound (I), the structural formula of which is as follows:

[0024]

[0025] in,

[0026] The R 1 is one selected from CN1, CN2, CN3, CN4, CN5, CN6, CN7, CN8, CN9, CN10, CN11, CN12, CN13, CN14, CN15, CN16, CN17, CN18;

[0027] The R 2 , R 3 , R 4 Each is independently selected from one of A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, and A22;

[0028] The CN1, CN2, CN3, CN4, CN5, CN6, CN7, CN8, CN9, CN10, CN11, CN12, CN13, CN14, CN15, CN16, CN17, and CN18 are as follows:

[0029]

[0030] The A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22 are as follows:

[0031] A5:-(CH 2 ) 4 CH 3 ; A6: -(CH 2 ) 5 CH 3 ; A7: -(CH 2 ) 6 CH 3 ; A8: -(CH2 ) 7 CH 3 ;

[0032] A9:-(CH 2 ) 8 CH 3 ; A10: -(CH 2 ) 9 CH 3 ; A11: -(CH 2 ) 10 CH 3 ; A12: -(CH 2 ) 11 CH 3 ;

[0033] A13: -(CH 2 ) 12 CH 3 ; A14: -(CH 2 ) 13 CH 3 ; A15: -(CH 2 ) 14 CH 3 ; A16: -(CH 2 ) 15 CH 3 ;

[0034] A17:-(CH 2 ) 16 CH 3 ; A18: -(CH 2 ) 17 CH 3 ; A19: -(CH 2 ) 18 CH 3 ; A20: -(CH 2 ) 19 CH 3 ;

[0035]

[0036] The present invention also provides a use of any of the above-mentioned aminoamide lipid compounds in the preparation of lipid nanoparticles.

[0037] Specifically, the steps for preparing the lipid nanoparticles are as follows:

[0038] The aminoamide lipid compound described in the present invention is mixed with cholesterol, phospholipids and polyethylene glycol-lipid molecules in a certain proportion and dissolved in anhydrous ethanol. The mixed solution is then quickly injected into a 3-fold volume of sodium citrate buffer solution and stirred for 1 minute to prepare a pre-LNP solution.

[0039] Preferably, the aminoamide lipid compound is selected from the following representative compounds:

[0040] Table 1 Representative compounds of this application

[0041]

[0042]

[0043] Preferably, the phospholipid is DOPE, and the polyethylene glycol-lipid molecule is DMG-PEG2000.

[0044] Preferably, the proportions of the aminoamide lipid compound, cholesterol, phospholipid and polyethylene glycol-lipid molecule are as follows: aminoamide lipid compound: cholesterol: phospholipid: polyethylene glycol-lipid molecule = 20-50%: 30-50%: 10-30%: 0.5-5%.

[0045] More preferably, the proportions of the aminoamide lipid compound, cholesterol, phospholipid and polyethylene glycol-lipid molecule are as follows: aminoamide lipid compound: cholesterol: phospholipid: polyethylene glycol-lipid molecule = 30%: 10%: 45%: 0.5%.

[0046] The present invention also provides a use of any of the aforementioned aminoamide 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 above-mentioned "substitution" is optional substitution, that is, one or more hydrogen atoms connected to an atom or group are independently unsubstituted, or are substituted by one or more substituents, and the substituents are independently selected from: -Cl, -Br, -I, -OH, -SH, -CN, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C6 Alkynyl, cycloalkyl (preferably C 3 -C 8 cycloalkyl), aryl, acyl, alkoxy, heterocyclic group (preferably 3-8 membered heterocyclic group), heteroaryl, heteroaryl C 1 -C 6 Alkyl, C 1 -C 6 Haloalkyl, -O(C 1 -C 6 )alkyl, -O(C 2 -C 6 ) alkenyl, C 1 -C 6 Alkyl-OH, C 1 -C 6 Alkyl -SH, -NH 2 , C 1 -C 6 Alkyl-NH 2 、-N(C 1 -C 6 alkyl) 2 、-NH(C 1 -C 6 Alkyl), -NO 2 、-COOH、-CO-O-(C 1 -C 6 alkyl), -CO(C 1 -C 6 alkyl), -CO(C 1 -C 6 Haloalkyl), -CO-O-(C 1 -C 6 Haloalkyl), -O-CO(C 1 -C 6 Alkyl), -O-CO(C 1 -C 6 haloalkyl), -CO-NH-(C 1 -C 6 Alkyl), -CO-N(C 1 -C 6 Alkyl)-(C 1 -C 6 Alkyl), -NH-CO-(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl)-CO-(C 1 -C 6 Alkyl), -S(O) 2 -C 1 -C 6 Alkyl, -S(O)-C1 -C 6 Alkyl, -S(O) 2 -C 1 -C 6 Haloalkyl, -S(O) 2 -NH 2 、-S(O) 2 -NH(C 1 -C 6 Alkyl), -NH-S(O) 2 (C 1 -C 6 Alkyl), -NH-S(O) 2 (C 1 -C 6 When an atom or group is substituted with multiple substituents, the multiple substituents may be the same or different.

[0050] The "hydrocarbon group" herein refers to the remaining group after an aliphatic hydrocarbon loses a hydrogen atom, including straight-chain or branched, saturated or unsaturated hydrocarbon groups, and the hydrocarbon groups include alkyl, alkenyl and alkynyl.

[0051] The term "acyl" refers to a hydrocarbon-carbonyl group, preferably a C 4 -C 18 Acyl.

[0052] The term "alkoxy" refers to an alkyl-oxy group, preferably a C 1 -C 18 Alkoxy.

[0053] The "heterocycle" herein refers to a saturated or unsaturated cyclic group containing a heteroatom selected from N, O, S, etc., and the heterocycle may be optionally substituted by one or more substituents.

[0054] Beneficial effects of the present invention:

[0055] The present invention provides a novel ionizable lipid molecule, wherein the aminoamide lipid compound uses an amide bond commonly seen in a living body to construct a skeleton of a lipid molecule, and the amide bond can be degraded by amidase in the human body, so that the lipid molecule has less toxic side effects. The aminoamide lipid compound contains a long-chain non-polar group and an ionizable amino portion, and has not only a hydrophobic characteristic, but also a hydrophilic characteristic, and can effectively deliver nucleic acids to the cytoplasm of cells. In particular, because the aminoamide lipid compound has three hydrophobic tails at the same time, its escape ability in lysosomes is increased, and the delivery efficiency of lipid nanoparticles is significantly enhanced.

[0056] In addition, compared with the multi-step synthesis of traditional lipid molecules, the synthesis of the aminoamide lipid compounds described in the present application adopts a "one-pot method", which simplifies the process operation, avoids the introduction of impurities and operational errors during material transfer and separation and purification, and greatly improves production efficiency. The synthesized aminoamide lipid compounds have good biocompatibility and can achieve safe and efficient intracellular delivery of a variety of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 is the compound Lp1 of Example 1 1 H-NMR spectrum.

[0058] Figure 2 Example 2 Compound Lp2 1 H-NMR spectrum.

[0059] Figure 3 Example 3 Compound Lp3 1 H-NMR spectrum.

[0060] Figure 4 is the compound Lp4 of Example 4 1 H-NMR spectrum. DETAILED DESCRIPTION

[0061] 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.

[0062] Example 1 Synthesis route of compound Lp1

[0063]

[0064] Take 4.2mmol of lauric aldehyde and dissolve it in 5mL of anhydrous methanol. Add 4.2mmoL of decaamine under stirring, heat to 50℃ and react for 5h to obtain imine. Weigh 4.2mmol of palmitic acid and isonitrile respectively and dissolve them in 5mL of anhydrous methanol, and add them to the flask in turn. React at 25℃ for 30h, and detect the progress of 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 oil with a yield of 13.8%, and mass spectrometry and nuclear magnetic resonance analysis are performed.

[0065] 1H NMR(400MHz,Chloroform-d)δ6.00(s,1H),3.72(t,J=7.1Hz,1H),3.16(t,J=5.1Hz,3H),2.61(t,J=4.9Hz,3H),2.51(s ,2H),2.38(s,3H),1.64(p,J=7.4Hz,2H),1.46(dp,J=13.7,6.4Hz,2H),1.27(d,J=5.6Hz,59H),0.90(t,J=6.7Hz,9H)( Figure 1 )。 ESI-MS (m / z): [M+H] + calcd.for C 46 H 90 N 4 O 3 ,747.7;found,747.7.

[0066] Example 2 Synthesis route of compound Lp2

[0067]

[0068] Take 4.2mmol of lauryl aldehyde and dissolve it in 5mL of anhydrous methanol. Add 4.2mmoL of dodecylamine under stirring, heat to 50℃ and react for 5h to obtain imine. Weigh 4.2mmol of stearic acid and isonitrile respectively and dissolve them in 5mL of anhydrous methanol, and add them to the flask in turn. React at 25℃ for 30h, and detect the progress of 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 oil with a yield of 15.1%, and mass spectrometry and nuclear magnetic resonance analysis are performed.

[0069] 1 H NMR(400MHz,Chloroform-d)δ5.99(s,1H),3.69(t,J=7.1Hz,1H),3.14(t,J=5.0Hz,4H),2.57(t,J=5.1Hz,4H),2.50(t,J=7.3 Hz,2H),2.37(s,3H),1.75(d,J=7.5Hz,2H),1.44(dh,J=11.5,6.0,5.4Hz,4H),1.26(d,J=5.5Hz,64H),0.90(t,J=6.7Hz,9H)( Figure 2 )。 ESI-MS (m / z): [M+H] + calcd.for C 50 H 98 N 4 O 3 ,803.7;found,803.7.

[0070] Example 3 Synthesis of Compound Lp3

[0071]

[0072] Take 4.2mmol of lauric aldehyde and dissolve it in 5mL of anhydrous methanol, add 4.2mmoL of dodecylamine under stirring, heat to 50℃ and react for 5h to obtain imine. Weigh 4.2mmol of dodecanoic acid and isonitrile respectively and dissolve them in 5mL of anhydrous methanol, and add them to the flask in turn, react at 25℃ for 30h, and detect the progress of the reaction by thin layer chromatography. After the reaction is completed, the crude product is separated by column chromatography, and the eluent is ethyl acetate / methanol = 10 / 1 to obtain a yellow oil with a yield of 19%, and mass spectrometry and nuclear magnetic resonance analysis are performed. 1 HNMR (400MHz, Chloroform-d): δ0.89(t,J=3.56Hz,9H),1.27(s,59H),1.6(t,J=6.4Hz,2H),1.78(t,J=7.12Hz,2H),2.29(t,J=9.16H z,1H),2.38(s,3H),2.51(t,J=10.04Hz,2H),2.62(t,J=10.48Hz,4H),3.15(t,J=12.6Hz,4H),3.71(t,J=14.84Hz,1H),6.00(s,1H)( Figure 3 )。 ESI-MS (m / z): [M+H] + calcd.for C 44 H 86 N 4 O 3 ,719.6;found,719.6.

[0073] Example 4 Synthesis of Compound Lp4

[0074]

[0075] Take 4.2mmol of lauric aldehyde and dissolve it in 5mL of anhydrous methanol. Add 4.2mmoL of n-hexylamine under stirring, heat to 50℃ and react for 5h to obtain imine. Weigh 4.2mmol of hexadecanoic acid and piperidinyl isonitrile respectively and dissolve them in 5mL of anhydrous methanol. Add them to the flask in turn, react at 25℃ for 30h, and detect the progress of 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 oil with a yield of 20.6%. It is also analyzed by mass spectrometry and nuclear magnetic resonance. 1H NMR(400MHz,Chloroform-d)δ8.10(s,1H),4.31–4.19(m,3H),4.15(s,3H),3.47(q,J=5.5Hz,4H),2.67(t,J=5.9Hz,3H),2.60(t,J=6.1Hz, 4H),2.25(t,J=7.7Hz,2H),1.69(q,J=5.8Hz,8H),1.58(d,J=7.1Hz,2H),1.52–1.46(m,4H),1.26(d,J=16.5Hz,50H),0.87(t,J=6.5Hz,9H)( Figure 4 )。 ESI-MS (m / z): [M+H] + calcd.for C 44 H 86 N 4 O 3 ,719.7;found,719.7.

[0076] Example 5 Synthesis of Compound Lp5

[0077]

[0078] Take 4.2mmol of lauric aldehyde and dissolve it in 5mL of anhydrous methanol, add 4.2mmoL of n-hexylamine under stirring, heat to 50℃ and react for 5h to obtain imine. Weigh 4.2mmol of hexadecanoic acid and morpholinyl isonitrile respectively and dissolve them in 5mL of anhydrous methanol, and add them to the flask in turn, react at 25℃ for 30h, and detect the progress of 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 oil with a yield of 19%, and mass spectrometry and nuclear magnetic resonance analysis are performed. 1 H NMR(400MHz,Chloroform-d)δ4.69(s,6H),4.13(s,12H),3.69(t,J=4.7Hz,28H),3.37(q,J=5.7Hz,13H),2.5 3(t,J=6.1Hz,12H),2.47(t,J=4.6Hz,28H),1.56(p,J=7.4Hz,3H),1.27–1.18(m,48H),0.83(t,J=6.6Hz,9H). ESI-MS(m / z):[M+H] + calcd.forC 44 H 86 N 4 O 3 ,721.7;found,721.7.

[0079] Example 6 Synthesis of Compound Lp6

[0080]

[0081] Take 4.2mmol of lauryl aldehyde and dissolve it in 5mL of anhydrous methanol. Add 4.2mmoL of dodecylamine under stirring, heat to 50℃ and react for 5h to obtain imine. Weigh 4.2mmol of oleic acid and isonitrile respectively and dissolve them in 5mL of anhydrous methanol, and add them to the flask in turn. React at 25℃ for 30h, and detect the progress of the reaction by thin layer chromatography. After the reaction is completed, the crude product is separated by column chromatography, and the eluent is ethyl acetate / methanol = 10 / 1 to obtain a yellow oil with a yield of 24.6%, and mass spectrometry and nuclear magnetic resonance analysis are performed. 1 HNMR(400MHz,Chloroform-d)δ5.38(s,2H),4.32(s,1H),3.69(s,3H),3.45(s,2H),2.54–2.44(m,4H),2.3 6(t,J=17.8Hz,6H), 2.05(t,J=19.0Hz,8H), 1.65(t,J=6.60Hz,3H), 1.29(s,53H), 0.91(t,J=3.64Hz,9H). ESI-MS(m / z):[M+H] + calcd.for C 48 H 92 N 4 O 3 ,773.7;found,773.7.

[0082] Example 7 Lipid Nanoparticles Prepared from Aminoamide Lipid Compounds

[0083] The aminoamide 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 MalvernZetasizer Nano ZS in a 173° backscattering detection mode. The test results are shown in Table 2. The particle size distribution of the prepared lipid nanoparticles is in the range of 100-200nm, which can meet the needs of in vivo delivery.

[0084] Example 8 Evaluation of the hemolytic performance of lipid nanoparticles prepared from aminoamide lipid compounds

[0085] First, a pre-LNP solution was prepared according to the method in Example 7. Then, D-PBS buffers of pH 7.4 and pH 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 pH 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. Centrifuge after 4 hours, take the supernatant, and measure the absorbance at a wavelength of 540nm. The obtained OD value is used to calculate the hemolysis rate of the sample by the following equation.

[0086] Hemolysis rate % = [(OD sample - OD negative) / (OD positive - OD negative)] × 100%

[0087] As shown in Table 2, the lipid nanoparticles prepared by the aminoamide lipid compound of the present invention have a low hemolytic activity (5%-15%) 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 (78%-96%), indicating that under acidic conditions, the nucleic acid coated by the lipid nanoparticles is easily released. Therefore, the aminoamide lipid compound of the present invention is conducive to further development and application.

[0088] Example 9 Evaluation of luciferase mRNA cell-level transfection performance of lipid nanoparticles prepared from aminoamide lipid compounds

[0089] Preparation method: First, prepare a pre-LNP solution according to the method in Example 7. Dissolve luciferase mRNA (FlucmRNA) in a DEPC aqueous solution, add an equal volume of 50% ethanol solution, and prepare an ethanol solution with a final concentration of 25%. Then, mix the pre-LNP solution with 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, which are dialyzed in PBS buffer for 2 hours.

[0090] According to the manufacturer's instructions, the Quant-it Ribogreen RNA quantitative assay kit was used to measure the loading rate of lipid nanoparticles. The test results are shown in Table 2, indicating that the carrier prepared by the aminoamide lipid compound of the present invention has a high encapsulation rate for nucleic acid molecules and can successfully transport nucleic acid molecules into cells for expression.

[0091] Table 2

[0092]

[0093] Hemolysis rate* indicates the ratio when the concentration is 100ug / ml.

[0094] Human embryonic kidney cell line Hek293t in logarithmic growth phase was cultured at 25×10 3 Each well was inoculated in a 48-well cell culture plate. After the cells adhered to the wall, lipid nanoparticles loaded with mRNA prepared by the representative aminoamide lipid compound of the present invention were added for cell transfection (200ng mRNA / well), and 3 replicates were made for each sample. After transfection for 6 hours, the culture medium was discarded, 50 μL / well of PLB lysate was added, and the cells were lysed for 20 minutes. After the cell culture plate was centrifuged, 10 μL of lysate was drawn to a 96-well black board, and the sample fluorescence intensity was immediately detected with an ELISA instrument after adding 50 μL / well luciferase substrate.

[0095] 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 multiple aminoamide lipid nanoparticles loaded with luciferase mRNA of the present invention is similar to the fluorescence intensity of the commercial SM-102 lipid nanoparticles loaded with luciferase mRNA, and even the fluorescence intensity of lipid molecules transfection of 1# (108%), 2# (112%), and 4# (129%) is better than that of the control SM-102 (100%). It shows that the mRNA encapsulated by the lipid nanoparticles prepared by the representative aminoamide lipid compounds of the present invention has good transfection activity, and the aminoamide lipid compounds of the present invention can effectively deliver nucleic acid molecules.

[0096] Table 3

[0097]

[0098]

[0099] 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. An aminoamide lipid compound or a pharmaceutically acceptable salt thereof, It is characterized in that Its structure is shown in formula (I): ; in, The R 1 is one selected from CN1, CN2, CN3, CN4, CN5, CN6, CN7, CN8, CN9, CN10, CN11, CN12, CN14, CN17, CN18; The R 2 , R 3 , R 4 Each is independently selected from one of A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, and A21; The CN1, CN2, CN3, CN4, CN5, CN6, CN7, CN8, CN9, CN10, CN11, CN12, CN14, CN17, and CN18 are as follows: ; The A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A21 are as follows: A6:-(CH 2 ) 5 CH 3 ;A7:-(CH 2 ) 6 CH 3 ;A8:-(CH 2 ) 7 CH 3 ; <h2 style=";text-align:left;direction:ltr">A9:-(CH<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> CH<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> A10:-(CH<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> CH<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> A11:-(CH<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> CH<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> A12:-(CH<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> CH<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> ; A13:-(CH 2 ) 12 CH 3 ;A14:-(CH 2 ) 13 CH 3 ;A15:-(CH 2 ) 14 CH 3 ;A16:-(CH 2 ) 15 CH 3 ; A17:-(CH 2 ) 16 CH 3 ;A18:-(CH 2 ) 17 CH 3 ; A21： 。 2. The aminoamide lipid compound according to claim 1 or a pharmaceutically acceptable salt thereof, in, The aminoamide lipid compound is selected from: ; Among them, R 2 , R 3 and R 4 As defined in claim 1.

3. A method for preparing an aminoamide lipid compound or a pharmaceutically acceptable salt thereof, It is characterized in that The steps include: First, compound R 2 -CHO is dissolved in the solvent and compound R is added under stirring. 3 -NH 2 , heating reaction, to obtain imine intermediate R 2 -CH=NR 3 , then, to the imine intermediate R 2 -CH=NR 3 Compound R was added to the solution 4 -COOH and R 1 -NC, and stirred for reaction to obtain compound (I), the structural formula of which is as follows: ; Among them, R 1 , R 2 , R 3 and R 4 As defined in claim 2.

4. Use of the aminoamide lipid compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 2 in the preparation of lipid nanoparticles.

5. The use according to claim 4, It is characterized in that The method comprises synthesizing aminoamide lipid compounds with cholesterol, phospholipids and polyethylene glycol-lipid molecules to obtain LNP liposome nanoparticles. The LNP can improve the translation expression level of the load-mRNA in cells.

6. The use according to claim 4, It is characterized in that The aminoamide lipid compound is selected from: ; Among them, R 2 , R 3 and R 4 As defined in claim 2.

7. Use of the aminoamide lipid compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 2 in the preparation of a medicament for gene therapy, gene vaccination or nucleic acid transfer.

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