WO2023134325A1 - 一种脂质化合物、包含其的组合物及应用 - Google Patents

一种脂质化合物、包含其的组合物及应用 Download PDF

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WO2023134325A1
WO2023134325A1 PCT/CN2022/136397 CN2022136397W WO2023134325A1 WO 2023134325 A1 WO2023134325 A1 WO 2023134325A1 CN 2022136397 W CN2022136397 W CN 2022136397W WO 2023134325 A1 WO2023134325 A1 WO 2023134325A1
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lipid
lipid compound
compound
pharmaceutically acceptable
nucleic acid
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French (fr)
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张元�
谷飞
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华南理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a lipid compound, a composition containing it and an application.
  • Nucleic acid drugs correct, knock out or compensate for gene defects or abnormalities by specifically up-regulating or down-regulating gene expression, and treat genetic diseases, cancer, infectious diseases, autoimmune diseases, cardiovascular diseases, various related gene The method of treating diseases has also been pushed to the clinic, bringing new hope for human medical treatment and health.
  • Common nucleic acid drugs mainly include plasmid DNA (plasmid DNA, pDNA), messenger RNA (Message RNA, mRNA), small interfering RNA (Small interfering RNA, siRNA) and antisense oligonucleotide (Antisense oligonucleotide).
  • siRNA is a double-stranded small molecule RNA, generally composed of 19 to 25 nucleotides.
  • siRNA can specifically recognize the target sequence, bind to the mRNA complementary to its sequence, and promote the degradation of mRNA, thereby inhibiting gene expression at the transcriptional level, inducing cell-specific gene deletion, efficiently silencing disease-causing genes, and blocking the occurrence of diseases .
  • RNA interference the idea of siRNA as a gene drug has attracted widespread attention once it was put forward, and has broad development prospects.
  • nucleic acid drugs Compared with traditional chemical drugs and antibody drugs, nucleic acid drugs have the characteristics of high efficacy, high specificity, low side effects, and low risk, and the development process is relatively simple.
  • various "stuck neck" technical problems still exist.
  • nucleic acid molecules such as RNA are sensitive to enzymes and are easily degraded by ubiquitous RNases, thereby losing their drug activity.
  • nucleic acid drugs enter the body they need to go through complex processes, such as cell uptake, endosome escape, etc., and are released to specific parts to exert their biological functions. Therefore, developing an efficient and safe delivery system is one of the primary tasks to overcome the development problems of nucleic acid drugs.
  • Non-viral vectors including inorganic materials, polymer molecules, liposomes, etc., have lower transfection efficiency than viral vectors.
  • Inorganic materials are difficult to metabolize in vivo, have poor biocompatibility, and have certain safety problems, while liposomes and polymer molecules have low biotoxicity.
  • liposomes and polymer molecules have low biotoxicity.
  • cationic liposomes have become one of the most widely used non-viral vectors and have good biological safety, at present, the transfection efficiency of cationic liposomes is still relatively low.
  • the present invention aims to solve the technical problems existing in the above-mentioned prior art.
  • the present invention proposes a lipid compound, a composition comprising it and applications.
  • the structure and reaction path of the lipid compound are simple, and the yield is high.
  • the composition constructed from the lipid compound can efficiently deliver active pharmaceutical ingredients to cells or tissues, and has broad application prospects.
  • the first aspect of the present invention provides a lipid compound, which is obtained after the hydrogen atoms on the organic amine nitrogen are replaced by R groups; the organic amine is selected from the structures shown below:
  • the R group has a structure shown in formula (I):
  • n is any integer between 6-16.
  • the R group is selected from the structures shown below:
  • the organic amine is selected from A1, A2, A7, A8, A12, A13.
  • the R 1 group is selected from C12, C16, C18U.
  • the structure of the lipid compound does not contain free amino groups.
  • the lipid compound is selected from the structures shown below:
  • Cationic lipids usually consist of an amino-containing hydrophilic head, a nonpolar hydrophobic tail, and a connecting chain that connects the head and tail.
  • the structure of the head, as well as the number, length and saturation of the tail all have a great influence on the transfection efficiency of cationic lipids.
  • the middle chain part is kept as a three-carbon chain structure substituted by hydroxyl groups, and the number of hydrophobic tails is adjusted to 2-6, and the hydrophobic tails are saturated or unsaturated with 8-18 carbon atoms. chain, a series of lipid compounds with strong transfection efficiency were obtained, which can be used for the in vivo delivery of pharmaceutical active ingredients.
  • the preparation method of the lipid compound comprises the following steps:
  • the molar ratio of the acid chloride to glycidol is 1:1.2-1.5.
  • the alcoholysis is carried out in the presence of an organic base, and the organic base is triethylamine.
  • the molar ratio of the organic base to the acid chloride is 1:1-1.2.
  • the temperature of the alcoholysis is 10-30°C.
  • the alcoholysis time is 12-36h.
  • the temperature of the reaction is 80-100°C.
  • the reaction time is 2-3d.
  • the second aspect of the present invention provides a composition comprising the above-mentioned lipid compound, or a pharmaceutically acceptable salt thereof.
  • the composition further includes other lipid compounds.
  • the other lipid compounds include at least one of cholesterol, phospholipids, and polymer-conjugated lipids.
  • the phospholipids include egg yolk lecithin, hydrogenated egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, sphingomyelin, phosphatidylethanolamine, dimyristoylphosphatidylcholine, dimyristyl At least one of acylphosphatidylglycerol, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, preferably DSPC.
  • DSPC distearoylphosphatidylcholine
  • the polymer-conjugated lipids include polyethylene glycol (PEG)-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine , at least one of PEG-modified diacylglycerol and PEG-modified dialkylglycerol, preferably PEG-modified phosphatidylethanolamine.
  • PEG polyethylene glycol
  • the molar ratio of the lipid compound, or a pharmaceutically acceptable salt thereof, to cholesterol is 1:0.01-9, for example, 1:0.1-9, 1:1-9, 1 :1-5, 1:1-2.
  • the molar ratio of the lipid compound, or a pharmaceutically acceptable salt thereof, to the phospholipid is 0.5-100:1, for example, 1-100:1, 1-10:1, 1 -5:1, 2-5:1, 3-5:1, 3-4:1.
  • the molar ratio of the lipid compound, or a pharmaceutically acceptable salt thereof, to the polymer-conjugated lipid is 0.1-100:1, for example, 1-100:1, 1- 50:1, 5-50:1, 10-50:1, 10-20:1, 15-20:1.
  • the other lipid compounds include cholesterol, phospholipids and polymer conjugated lipids
  • the lipid compound, or a pharmaceutically acceptable salt thereof cholesterol: phospholipid: polymer
  • the molar ratio of conjugated lipids is 10-100:1-90:1-90:1-90, for example, 10-50:20-80:1-20:1-10, 20-50:30-80: 1-20: 1-10, 30-50: 40-80: 1-20: 1-10, 30-40: 40-70: 1-20: 1-10, 30-40: 40-60: 5- 20: 1-10, 30-40: 40-60: 5-15: 1-10, 30-40: 40-60: 5-15: 1-5.
  • the composition is lipid nanoparticles, liposomes.
  • the lipid nanoparticles or liposomes of the present invention can be used to prepare cell transfection reagents, and have high transfection efficiency.
  • the composition further includes a pharmaceutically active ingredient.
  • the molar ratio of the lipid compound, or a pharmaceutically acceptable salt thereof, to the active pharmaceutical ingredient is 1-100:1.
  • the active pharmaceutical ingredient includes at least one of nucleic acid molecules, polypeptides, proteins and small molecule compounds.
  • the nucleic acid molecules include siRNA, mRNA, miRNA, antisense RNA, CRISPR guide RNAs, replicable RNA, cyclic dinucleotide (cyclic dinucleotide, CDN), poly IC, CpG ODN, At least one of plasmad DNA, preferably siRNA.
  • the protein includes at least one of cell colony-stimulating factor, interleukins, lymphotoxins, interferon proteins, and tumor necrosis factor.
  • the pharmaceutically active ingredient when the pharmaceutically active ingredient includes a nucleic acid molecule, the lipid compound, or a pharmaceutically acceptable salt thereof, has a nitrogen to phosphorus ratio (N/P ratio) of 1- 50:1, preferably 1-40:1, more preferably 4-32:1.
  • N/P ratio nitrogen to phosphorus ratio
  • composition of the present invention can carry nucleic acid molecules through the cell membrane, so it can be used as a transfection reagent, especially when transfected with siRNA, it can effectively inhibit the expression of target genes.
  • the preparation method of the composition loaded with active pharmaceutical ingredients comprises the following steps:
  • the third aspect of the present invention provides the application of the above-mentioned lipid compound, or a pharmaceutically acceptable salt thereof, or the above-mentioned composition in the preparation of nucleic acid drugs, gene vaccines, polypeptide or protein drugs, and small molecule drugs.
  • the nucleic acid drug of the present invention is a drug for treating related diseases caused by gene abnormalities, and the diseases include monogenic diseases such as methemoglobinemia and sickle cell anemia; polygenic diseases such as tumors and cardiovascular diseases , metabolic diseases, neurological and psychiatric diseases, immune diseases; and acquired genetic diseases such as AIDS.
  • monogenic diseases such as methemoglobinemia and sickle cell anemia
  • polygenic diseases such as tumors and cardiovascular diseases , metabolic diseases, neurological and psychiatric diseases, immune diseases
  • acquired genetic diseases such as AIDS.
  • the hydrophobic end of the lipid is composed of long carbon chain alkanes or alkenes, which is difficult to be degraded by enzymes and relatively difficult to metabolize in vivo; while the lipid compound of the present invention introduces a biodegradable ester bond into the hydrophobic end , can be degraded by esterase in the body, making lipid compounds easy to be eliminated by metabolism.
  • the protonatable lipid compound prepared by the present invention can be ionized into cations under acidic conditions, and can be combined with negatively charged pharmaceutical active ingredients through charge interaction, and can be further combined with other lipid compounds such as DSPC, cholesterol, Lipid nanoparticles composed of DSPE-PEG etc.
  • siRNA can be transfected into cells to specifically knock down the target gene and inhibit the expression of the target gene.
  • the data in the examples also show that the lipid compound prepared by the present invention has a high transfection efficiency.
  • the present invention has the advantages of readily available raw materials, simple reaction and high yield.
  • pharmaceutically acceptable salt in this application includes conventional salts formed with pharmaceutically acceptable inorganic or organic acids, or inorganic or organic bases.
  • composition includes a product comprising an effective amount of a compound of the invention, as well as any product resulting, directly or indirectly, from a combination of compounds of the invention.
  • Fig. 1 is a Fourier transform infrared scanning diagram of C12 of the present invention.
  • Fig. 2 is the proton nuclear magnetic resonance spectrum figure of C12 of the present invention.
  • Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram of C16 of the present invention.
  • Fig. 4 is the proton nuclear magnetic resonance spectrum of A1-C12 of the present invention.
  • Fig. 5 is the proton nuclear magnetic resonance spectrum of A2-C12 of the present invention.
  • Fig. 6 is the proton nuclear magnetic resonance spectrum of A2-C16 of the present invention.
  • Fig. 7 is the proton nuclear magnetic resonance spectrum of A2-C18U of the present invention.
  • Fig. 8 is the proton nuclear magnetic resonance spectrum of A13-C16 of the present invention.
  • Fig. 9 is the proton nuclear magnetic resonance spectrum of A13-C18U of the present invention.
  • Fig. 10 is the proton nuclear magnetic resonance spectrum of A12-C12 of the present invention.
  • Fig. 11 shows that firefly luciferase small interfering RNA (luciferase siRNA, siLuc) was loaded with lipid nanoparticles constructed by A1-C8, A1-C10, A1-C12, A1-C16, and A1-C18U of the present invention.
  • the expression level of luciferase (Luciferase); 4:1, 8:1, 16:1 represent the nitrogen-phosphorus ratio of the lipid compound to siRNA, that is, the ratio of the protonatable amino group on the lipid compound to the phosphate group on the nucleic acid. The molar ratio between.
  • Figure 12 shows the expression level of firefly luciferase (Luciferase) after the lipid nanoparticles constructed by A2-C8, A2-C10, A2-C12, A2-C14, A2-C16, and A2-C18U of the present invention are loaded with siLuc 4:1, 8:1, 16:1 represent the nitrogen-phosphorus ratio of the lipid compound to siRNA, that is, the molar ratio between the protonatable amino group on the lipid compound and the phosphate group on the nucleic acid.
  • siLuc 4:1 the nitrogen-phosphorus ratio of the lipid compound to siRNA, that is, the molar ratio between the protonatable amino group on the lipid compound and the phosphate group on the nucleic acid.
  • Fig. 13 is a heat map of the transfection efficiency of lipid nanoparticles constructed by different lipid compounds of the present invention under different ratios of nitrogen and phosphorus.
  • the ratios of nitrogen and phosphorus are 4:1, 8:1, and 16:1 respectively. Values in units indicate transfection efficiency.
  • Figure 14 shows the siLuc entrapped in lipid nanoparticles constructed by A5-C12, A6-C12, A7-C12, A8-C12, A9-C12, A10-C12, A11-C12, A12-C12, A13-C12 of the present invention
  • the expression level of firefly luciferase (Luciferase) 4:1, 8:1, 16:1, 32:1 represent the nitrogen-phosphorus ratio of the lipid compound to siRNA, that is, the protonatable amino group on the lipid compound and the The molar ratio between phosphate groups on a nucleic acid.
  • Figure 15 is a heat map of the transfection efficiency of lipid nanoparticles constructed by different lipid compounds of the present invention under different ratios of nitrogen and phosphorus, the ratios of nitrogen and phosphorus are respectively 4:1, 8:1, 16:1, 32:1, The value of each cell in the heatmap indicates the transfection efficiency.
  • Fig. 16 is after A12-C12, A13-C16, A7-C12 of the present invention are made into lipid nanoparticle, transfects green fluorescent protein particle DNA, the fluorescent microscope figure after 48h; Wherein, polyethyleneimine (PEI) is Commercial transfection reagents.
  • PEI polyethyleneimine
  • 17 is a histogram of transfection efficiency of firefly luciferase plasmid DNA with protonatable lipid compounds at different ratios of nitrogen and phosphorus 48 hours after transfection.
  • Glycidol and lauryl chloride were reacted at a molar ratio of 1.2:1.0.
  • the specific operation is as follows: dissolve glycidol in anhydrous dichloromethane, place in a 25 mL round-bottomed flask with stopper, add a catalytic amount of triethylamine (TEA), mix, seal, and pre-cool in an ice bath for 30 min. Under magnetic stirring, the methylene chloride solution of lauryl chloride was slowly added dropwise to the mixed solution of glycidol and TEA by using a constant pressure dropping funnel, and the dropping speed was controlled. After the dropping was completed, the reaction was carried out overnight at room temperature.
  • TEA triethylamine
  • Glycidol was reacted with hexadecanoyl chloride at a molar ratio of 1.2:1.0.
  • the specific operation is as follows: dissolving glycidol in dichloromethane-free solution, placing it in a 25 mL round-bottomed flask with a stopper, adding a catalytic amount of TEA, mixing, sealing, and pre-cooling in an ice bath for 30 minutes. Under magnetic stirring, the methylene chloride solution of hexadecanoyl chloride was slowly added dropwise to the mixed solution of glycidol and TEA by using a constant pressure dropping funnel, and the dropping speed was controlled. After the dropping was completed, the reaction was carried out overnight at room temperature.
  • Glycidol and oleoyl chloride were reacted at a molar ratio of 1.2:1.0.
  • the specific operation is to dissolve glycidol in anhydrous dichloromethane, place in a 25mL round-bottomed flask with a stopper, add a catalytic amount of TEA, mix, seal, and pre-cool in an ice bath for 30 minutes.
  • the dichloromethane solution of oleoyl chloride was slowly added dropwise to the mixed solution of glycidol and TEA by using a constant pressure dropping funnel, and the dropping speed was controlled. After the dropping was completed, the reaction was carried out overnight at room temperature. Wash twice with saturated sodium bicarbonate solution and once with saturated sodium chloride solution, take the organic phase, concentrate, dry over anhydrous magnesium sulfate for 30 min, and purify through a 200-300 mesh silica gel column to obtain the product C18U.
  • the intermediate C-12 (glycidyl dodecanoate) was reacted with the compound A1 at a molar ratio of 2.4:1.
  • the specific operation is: take the corresponding amount of intermediate C-12 and compound A1 into a 2mL glass bottle, put in a magnetic stirrer, and react at 90°C for 72h to obtain the product.
  • NMR characterization is shown in Figure 4. 1 H NMR (400MHz, CDCl 3 ): ⁇ 0.85-0.88(t, 6H), 1.24-1.26(m, 32H), 1.60-1.66(m, 6H), 2.14-2.19(t, 6H), 2.31- 2.43 (m, 12H), 4.08-4.19 (m, 8H).
  • the intermediate C-12 (glycidyl dodecanoate) was reacted with the compound A2 at a molar ratio of 2.4:1.
  • the specific operation is: take the corresponding amount of intermediate C-12 and compound A2 into a 2mL glass bottle, put in a magnetic stirrer, and react at 90°C for 72h to obtain the product.
  • NMR characterization is shown in Figure 5.
  • the intermediate C16 (glycidyl palmitate) was reacted with the compound A2 at a molar ratio of 2.4:1.
  • the specific operation is: take the corresponding amount of intermediate C16 and compound A2 into a 2mL glass bottle, put in a magnetic stirrer, and react at 90°C for 72h to obtain the product.
  • NMR characterization is shown in Figure 6. 1 H NMR (400MHz, CDCl 3 ): ⁇ 0.88-0.91(t, 6H), 1.27(m, 48H), 1.56-1.68(m, 10H), 2.32-2.52(m, 16H), 4.08-4.14( m, 8H).
  • the intermediate C18U (glycidyl palmitate) was reacted with the compound A2 at a molar ratio of 2.4:1.
  • the specific operation is: take the corresponding amount of intermediate C18U and compound A2 into a 2mL glass bottle, put in a magnetic stirrer, and react at 90°C for 72h to obtain the product.
  • NMR characterization is shown in Figure 7.
  • the intermediate C16 (glycidyl palmitate) was reacted with the compound A13 at a molar ratio of 5:1.
  • the specific operation is: take the corresponding amount of intermediate C16 and compound A13 into a 2mL glass bottle, put in a magnetic stirrer, and react at 90°C for 72h to obtain the product.
  • NMR characterization is shown in Figure 8. 1 H NMR (400 MHz, CDCl 3 ): ⁇ 0.87-0.98(t, 12H), 1.20-1.29(m, 96H), 1.58-1.66(t, 8H), 2.14-2.28(brs, 3H), 2.32 -2.47(m, 24H), 4.08-4.33(m, 12H).
  • the intermediate C18U was reacted with the compound A13 at a molar ratio of 5:1.
  • the specific steps are: take the corresponding amount of intermediate C-18U and compound A13 into a 2mL glass bottle, put in a magnetic stirrer, and react at 90°C for 72h to obtain the product.
  • NMR characterization is shown in Figure 9.
  • the intermediate C12 is reacted with the compound A12 according to the molar ratio.
  • the specific steps are: take the corresponding amount of intermediate C12 and compound A12 into a 2mL glass bottle, put in a magnetic stirrer, and react at 90°C for 72h to obtain the product.
  • NMR characterization is shown in Figure 10. 1 H NMR (400MHz, CDCl 3 ): ⁇ 0.88-0.92(t, 18H), 1.28-1.35(m, 104H), 1.63-1.65(m, 12H), 2.33-2.39(m, 30H), 4.08- 4.33 (m, 24H).
  • the reaction mechanism of the lipid compound of the present invention is: the ternary epoxy compound has very low ring tension because of its extremely low chemical bond strength, and the system energy is very high, so it is very easy to have a ring-opening reaction with the amino group with strong nucleophilicity, thereby obtaining the present invention.
  • Invented lipid compounds The reaction mechanism is very mature, and the reaction process is well known in the art. Therefore, the specific types and reaction degrees of the compounds generated by the above reaction mechanism are completely predictable.
  • the above are the reaction conditions and structural characterization of some compounds synthesized in the present invention.
  • the synthesis of the remaining compounds of the present invention is the same as the above-mentioned compounds, and their structural formulas and structural characterization data will not be described in detail here.
  • siLuc was transfected into cells capable of stably expressing firefly luciferase (Luciferase, Luc).
  • Luc firefly luciferase
  • B16F10-Luc cells were seeded in 96-well cell culture plates. The next day, cells were grown to about 80% for transfection.
  • N/P ratio nitrogen-to-phosphorus ratio
  • siLuc was transfected with Lipo2000 commercial transfection reagent. Transfection was performed according to the instruction manual of lipo2000. Take 50ng siLuc and add it to 5uL Opti-MEM, take 0.3 ⁇ L lipo2000 and place it in another 50 ⁇ L Opti-MEM, and finally add the siRNA Opti-MEM solution to the lipo2000 Opti-MEM solution, mix well, and incubate at room temperature for 15min. into 96-well cell culture plates. Before transfection, the culture medium in the culture plate was aspirated, and 80 ⁇ L of new medium was added, and the amount of siRNA added was 50 ng/well.
  • Negative control group only B16F10-Luc cells without transfection.
  • the cells were lysed, and the cell debris and contents were removed by centrifugation. The supernatant was taken, and the substrate of firefly luciferase was added to measure the expression of firefly luciferase, so as to compare the transfection efficiency of the synthetic lipid compound into siLuc .
  • the detection results are shown in Figure 11 and Figure 13, most of the synthetic lipid compounds have relatively strong transfection efficiency. Among them, the transfection efficiency of A1-C12 can reach about 95%.
  • lipid compounds A2-C8, A2-C10, A2-C12, A2-C14, A2-C16, and A2-C18U as gene carrier materials to transfect siLuc into B16F10-Luc cell line, the specific steps are as follows:
  • B16F10-Luc cells were seeded in 96-well cell culture plates. The next day, cells were grown to about 80% for transfection.
  • mice the prepared lipid compounds A2-C8, A2-C10, A2-C12, A2-C14, A2-C16, A2-C18U and DSPC, cholesterol, DSPE-PEG were dissolved in absolute ethanol to prepare into respective mother liquors, stored in a -20°C refrigerator, diluted as needed when used, and then mixed according to the molar ratio of 38:10:50:2 (lipid compound: DSPC: cholesterol: DSPE-PEG).
  • siLuc was transfected with Lipo 2000 transfection reagent. Transfection was performed according to the instruction manual of lipo2000. Add 50ng siLuc to 5uL Opti-MEM, take 0.3mL lipo2000 and place it in another 50 ⁇ L Opti-MEM, and finally add siRNA Opti-MEM solution to lipo2000 Opti-MEM solution, mix well, and incubate at room temperature for 15min. into 96-well cell culture plates. Before transfection, the culture solution in the culture plate was aspirated, and 80 ⁇ L of new medium was added, and the amount of siRNA added was 50 ng/well.
  • Negative control group only B16F10-Luc cells without transfection.
  • Lipid compounds A5-C12, A5-C16, A6-C12, A7-C12, A7-C16, A8-C12, A8-C16, A9-C12, A9-C16, A10-C16, A10-C12, A11 -C16, A11-C12, A12-C12, A12-C16, A13-C12, A13-C16 are used as gene carrier materials to transfect siLuc into B16F10-Luc cell line, the specific steps are as follows:
  • B16F10-Luc cells were seeded in 96-well cell culture plates. The next day, cells were grown to about 80% for transfection.
  • the ratio of nitrogen to phosphorus of lipid compound and siRNA is 4:1, 8:1, 16:1, 32:1.
  • siLuc was transfected with Lipo 2000 transfection reagent. Transfection was performed according to the instruction manual of lipo2000. Take 50ng siLuc and add it to 5uL Opti-MEM, take 0.3 ⁇ L lipo2000 and place it in another 50 ⁇ L Opti-MEM, and finally add the siRNA Opti-MEM solution to the lipo2000 Opti-MEM solution, mix well, and incubate at room temperature for 15min. into 96-well cell culture plates. Before transfection, the culture solution in the culture plate was aspirated, and 80 ⁇ L of new medium was added, and the amount of siRNA added was 50 ng/well.
  • Negative control group only B16F10-Luc cells without transfection.
  • lipid compounds A1-C12, A1-C16, A1-C18U, A2-C12, A2-C16, A2-C18U, A7-C12, A13-C16, A12-C16, A8-C12, A12-C12 as genes Carrier material
  • the plasmid DNA (pDNA-GFP-Luc) of green fluorescent protein and firefly luciferase was transfected into 293T cell line, the specific steps are as follows:
  • 293T cells were seeded in 96-well cell culture plates. The next day, cells were grown to about 80% for transfection.
  • the plasmid DNA (pDNA-GFP-Luc) expressing green fluorescent protein and firefly luciferase was dissolved in citrate
  • the volume of the citrate buffer is twice the volume of the ethanol-lipid mixture.
  • the culture medium in the culture plate was aspirated, 80 ⁇ L of new medium was added, and the amount of DNA added was 80 ng/well.
  • the nitrogen to phosphorus ratio of protonatable lipid compound to plasmid is 8:1, 16:1, 32:1.
  • Positive control group 293T cells were transfected with PEI commercialized transfection reagent. Transfection was performed according to the instructions of PEI transfection reagent. Take 80ng of DNA in 5uL ddH 2 O, mix well; take 0.1 ⁇ L PEI into 5 ⁇ L water, mix well, then add the diluted PEI to DNA aqueous solution, mix well, and incubate at room temperature for 15min before transfection. Before transfection, aspirate the original medium, add 80 ⁇ L of fresh medium, and the DNA transfection dose is 80ng/well.
  • Negative control group only 293T cells without transfection.

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Abstract

一种脂质化合物、包含其的组合物及其应用，该脂质化合物由有机胺和缩水甘油酯通过开环反应制成，结构中不含游离氨基。所述脂质化合物在酸性条件下可电离成阳离子化合物，与带负电的药物活性成分通过静电相互作用结合，从而组装成载药脂质纳米颗粒，进行药物活性成分的递送。所述脂质化合物结构简单，反应路径简单，产率高，构建得到的载药脂质纳米颗粒可用于制备核酸药物、基因疫苗、多肽或蛋白质药物、小分子药物，具备广泛的应用前景。

Description

一种脂质化合物、包含其的组合物及应用 技术领域
本发明属于生物技术领域，具体涉及一种脂质化合物、包含其的组合物及应用。
背景技术
核酸类药物通过特异性上调或下调基因的表达以纠正、敲除或补偿基因缺陷或异常的情况，治疗遗传性疾病、癌症、传染性疾病、自身免疫性疾病、心血管疾病，各种关于基因治疗疾病的方法也已经推向了临床，为人类的医疗和健康带来了新的希望。常见的核酸类药物主要有质粒DNA(plasmid DNA，pDNA)，信使RNA(Message RNA，mRNA)，小干扰RNA(Small interfering RNA，siRNA)以及反义核苷酸(Antisense oligonucleotide)。siRNA是一种双链小分子RNA，一般由19至25个核苷酸组成。siRNA能够特异性地识别靶序列，与其序列互补的mRNA结合，促使mRNA的降解，从而在转录水平上抑制基因的表达，诱使细胞特定基因缺失，高效率沉默致病基因，阻断疾病的发生。应用RNA干扰的原理，siRNA作为基因药物的设想一经提出便获得广泛关注，具有广阔的发展前景。
相对于传统化学药、抗体药物而言，核酸类药物具有高疗效、高特异性、低副作用、低风险的特点，且开发过程相对简单。但在核酸药物的发展过程中，各种“卡脖子”技术问题依旧存在。首先，RNA等核酸分子对酶敏感，极易被无处不在的RNA酶降解，从而失去药物活性作用。其次，核酸药物进入机体，需要经过复杂的过程，如细胞的摄取，内涵体逃脱等，释放到特定部位发挥其生物学功能。因此，研发高效安全的递送系统是攻克核酸药物发展难题的首要任务之一。
目前高效转染核酸药物的技术手段主要有两种：(1)病毒载体，病毒载体转染效率高，但却有潜在的危险性，并且受携带基因大小的限制，靶向性差；和(2)非病毒载体，包括无机材料，聚合物分子，脂质体等，转染效率较病毒载体低。无机材料在体内难以代谢，生物相容性差，存在一定的安全性问题，而脂质体和聚合物分子的生物毒性低。而相对脂质体而言，外源合成的聚合物分子易产生免疫原性，因而脂质体成为目前最理想的可用于核酸类药物递送的非病毒基因载体材料。此外，已有文献报道，尽管细胞对纳米颗粒具有良好的摄取，但是仅有2％的纳米颗粒能够逃脱出内涵体外，到达细胞质中发挥其生理功能。而阳离子脂质携带的正电荷可以与带负电的核酸分子或蛋白质分子通过静电相互作用形成脂质/药物复合物，然后通过细胞的内吞作用进入细胞质，转移到内涵体中，正电荷脂质可以与内涵体膜融合，将脂质纳米粒包被的药物等内容物释放到细胞质中，从而实现内涵体逃逸。虽然阳离子脂质体已经成为应用最为广泛的非病毒载体之一，具有良好的生物安全性，但是目前，阳离子脂质体的转染效率仍相对较低。
发明内容
本发明旨在解决上述现有技术中存在的技术问题。为此，本发明提出一种脂质化合物、包含其的组合物及应用。所述脂质化合物结构和反应路径简单，产率高，另外，由脂质化合物构建得到的组合物可高效地将药物活性成分递送至细胞或者组织，具有广泛的应用前景。
本发明的第一方面，提供一种脂质化合物，所述脂质化合物由有机胺氮上的氢原子均被R 1基团取代后得到；所述有机胺选自如下所示的结构：
Figure PCTCN2022136397-appb-000001
所述R 1基团具有式(I)所示的结构：
Figure PCTCN2022136397-appb-000002
其中，n为6-16之间的任意整数。
在本发明的一些实施方式中，所述R 1基团选自如下所示的结构：
Figure PCTCN2022136397-appb-000003
Figure PCTCN2022136397-appb-000004
在本发明的一些优选的实施方式中，所述有机胺选自A1、A2、A7、A8、A12、A13。
在本发明的一些优选的实施方式中，所述R 1基团选自C12、C16、C18U。
在本发明的一些实施方式中，所述脂质化合物的结构中不含有游离氨基。
在本发明的一些实施方式中，所述脂质化合物选自如下所示的结构：
Figure PCTCN2022136397-appb-000005
Figure PCTCN2022136397-appb-000006
阳离子脂质通常由含氨基的亲水头部，非极性疏水尾部以及起着连接头尾作用的连接链组成。头部的结构，以及尾部的数量、长短和饱和度等都对阳离子脂质的转染效率有极大影响。本发明通过选用具有不同结构的有机胺，保持中间链部分为羟基取代的三碳链结构，并调整疏水尾部的数量为2-6，疏水尾部为8-18个碳原子的饱和或不饱和长链，得到了一系列脂质化合物，这些脂质化合物具有较强的转染效率，可用于药物活性成分的体内递送。
在本发明的一些实施方式中，所述脂质化合物的制备方法包括以下步骤：
酰氯与缩水甘油经醇解得到缩水甘油酯，再将所述缩水甘油酯与有机胺反应，即得。
在本发明的一些实施方式中，所述酰氯与缩水甘油的摩尔比为1：1.2-1.5。
在本发明的一些实施方式中，所述醇解在有机碱存在的条件下进行，所述有机碱为三乙胺。
在本发明的一些实施方式中，所述有机碱与所述酰氯的摩尔比为1：1-1.2。
在本发明的一些实施方式中，所述醇解的温度为10-30℃。
在本发明的一些实施方式中，所述醇解的时间为12-36h。
在本发明的一些实施方式中，所述反应的温度为80-100℃。
在本发明的一些实施方式中，所述反应的时间为2-3d。
本发明的第二方面，提供一种组合物，所述组合物包括上述的脂质化合物，或其药学上可接受的盐。
在本发明的一些实施方式中，所述组合物还包括其他脂质化合物。
在本发明的一些实施方式中，所述其他脂质化合物包括胆固醇、磷脂和聚合物共轭脂质中的至少一种。
在本发明的一些实施方式中，所述磷脂包括蛋黄卵磷脂、氢化蛋黄卵磷脂、大豆卵磷脂、氢化大豆卵磷脂、鞘磷脂、磷脂酰乙醇胺、二肉豆蔻酰磷脂酰胆碱、二肉豆蔻酰磷脂酰甘油、二棕榈酰磷脂酰胆碱、二硬酯酰磷脂酰胆碱(DSPC)、二油酰磷脂酰胆碱、二月桂酰磷脂酰胆碱中的至少一种，优选DSPC。
在本发明的一些实施方式中，所述聚合物共轭脂质包括聚乙二醇(PEG)修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油、PEG修饰的二烷基甘油中的至少一种，优选PEG修饰的磷脂酰乙醇胺。
在本发明的一些实施方式中，所述脂质化合物，或其药学上可接受的盐与胆固醇的摩尔比为1：0.01-9，例如，1：0.1-9、1：1-9、1：1-5、1：1-2。
在本发明的一些实施方式中，所述脂质化合物，或其药学上可接受的盐与磷脂的摩尔比 为0.5-100：1，例如，1-100：1、1-10：1、1-5：1、2-5：1、3-5：1、3-4：1。
在本发明的一些实施方式中，所述脂质化合物，或其药学上可接受的盐与聚合物共轭脂质的摩尔比为0.1-100：1，例如，1-100：1、1-50：1、5-50：1、10-50：1、10-20：1、15-20：1。
在本发明的一些实施方式中，当所述其他脂质化合物包括胆固醇、磷脂和聚合物共轭脂质时，所述脂质化合物，或其药学上可接受的盐：胆固醇：磷脂：聚合物共轭脂质的摩尔比为10-100：1-90：1-90：1-90，例如，10-50：20-80：1-20：1-10、20-50：30-80：1-20：1-10、30-50：40-80：1-20：1-10、30-40：40-70：1-20：1-10、30-40：40-60：5-20：1-10、30-40：40-60：5-15：1-10、30-40：40-60：5-15：1-5。
在本发明的一些实施方式中，所述组合物为脂质纳米颗粒、脂质体。本发明所述的脂质纳米颗粒或脂质体可用于制备细胞转染试剂，具有很高的转染效率。
在本发明的一些实施方式中，所述组合物还包括药物活性成分。
在本发明的一些实施方式中，所述脂质化合物，或其药学上可接受的盐与药物活性成分的摩尔比为1-100：1。
在本发明的一些实施方式中，所述药物活性成分包括核酸分子、多肽、蛋白质和小分子化合物中的至少一种。
在本发明的一些实施方式中，所述核酸分子包括siRNA、mRNA、miRNA、antisense RNA、CRISPR guide RNAs、可复制性RNA、环二核苷酸(cyclic dinucleotide，CDN)、poly IC、CpG ODN、plasmid DNA中的至少一种，优选siRNA。
在本发明的一些实施方式中，所述蛋白质包括细胞集落刺激因子、白介素类、淋巴毒素、干扰素类蛋白、肿瘤坏死因子中的至少一种。
在本发明的一些实施方式中，当所述药物活性成分包括核酸分子时，所述脂质化合物，或其药学上可接受的盐与核酸分子的氮磷比(N/P ratio)为1-50：1，优选1-40：1、更优选4-32：1。
具体的，本发明的组合物可携带核酸分子透过细胞膜，因此可以作为转染试剂，尤其当转染siRNA后，能够有效抑制靶基因的表达。
在本发明的一些实施方式中，负载药物活性成分的组合物的制备方法包括以下步骤：
将所述脂质化合物的乙醇溶液与所述药物活性成分的缓冲盐溶液(pH＝4-6)混合；若存在其他脂质化合物，在所述混合过程中加入所述其他脂质化合物的乙醇溶液；室温孵育15-60min，水中透析，即得。
本发明的第三方面，提供上述脂质化合物，或其药学上可接受的盐，或上述组合物在制备核酸药物、基因疫苗、多肽或蛋白质药物、小分子药物中的应用。
本发明所述的核酸药物为用于治疗由基因异常引起的相关疾病的药物，所述疾病包括单基因疾病，例如高铁血红蛋白血症、镰刀红细胞贫血；多基因疾病，例如，肿瘤、心血管疾病、代谢性疾病、神经和精神类疾病、免疫性疾病；以及获得性基因病，例如，艾滋病。
根据本发明实施方式的脂质化合物或组合物，至少具备如下有益效果：
在现有的技术中，脂质的疏水端由长碳链烷烃或烯烃构成，难以被酶降解，体内代谢相对困难；而本发明的脂质化合物在疏水端中引入了生物可降解的酯键，在体内可被酯酶降解， 使得脂质化合物易于代谢清除。另外，本发明制备的可质子化的脂质化合物在酸性条件下可电离成阳离子，与带负电的药物活性成分通过电荷的相互作用进行结合，也可进一步与其它脂质化合物如DSPC、胆固醇、DSPE-PEG等组成脂质纳米颗粒，有效递送药物活性成分至细胞或组织。如，可将siRNA转染进入细胞内，特异性敲低目的基因，抑制目的基因的表达，实施例中的数据也表明本发明制备得到的脂质化合物具有很高的转染效率。除此之外，本发明具有原料易得、反应简单、产率高的优点。
本申请中术语：“药学上可接受的盐”包括与药学上可以接受的无机酸或者有机酸、或者无机碱或有机碱形成的常规盐。
“组合物”包括含有效量的本发明的化合物的产品，以及直接地或间接地由本申请化合物的组合产生的任何产品。
附图说明
下面结合附图和实施例对本发明作进一步的说明，其中：
图1为本发明C12的傅里叶红外扫描图。
图2为本发明C12的核磁共振氢谱图。
图3为本发明C16的核磁共振氢谱图。
图4为本发明A1-C12的核磁共振氢谱图。
图5为本发明A2-C12的核磁共振氢谱图。
图6为本发明A2-C16的核磁共振氢谱图。
图7为本发明A2-C18U的核磁共振氢谱图。
图8为本发明A13-C16的核磁共振氢谱图。
图9为本发明A13-C18U的核磁共振氢谱图。
图10为本发明A12-C12的核磁共振氢谱图。
图11为本发明A1-C8，A1-C10，A1-C12，A1-C16，A1-C18U所构建的脂质纳米颗粒包载萤火虫萤光素酶小干扰RNA(luciferase siRNA，siLuc)后，萤火虫荧光素酶(Luciferase)的表达量；4:1，8:1，16:1表示脂质化合物与siRNA的氮磷比例，即脂质化合物上可质子化的氨基与核酸上的磷酸基团之间的摩尔比。
图12为本发明A2-C8，A2-C10，A2-C12，A2-C14，A2-C16，A2-C18U所构建的脂质纳米颗粒包载siLuc后，萤火虫荧光素酶(Luciferase)的表达量；4:1，8:1，16:1表示脂质化合物与siRNA的氮磷比例，即脂质化合物上可质子化的氨基与核酸上的磷酸基团之间的摩尔比。
图13为本发明不同脂质化合物所构建的脂质纳米颗粒在不同氮磷比例下的转染效率热图，氮磷比例分别为4:1，8:1，16:1，热图中各单元的数值表示转染效率。
图14为本发明A5-C12，A6-C12，A7-C12，A8-C12，A9-C12，A10-C12，A11-C12，A12-C12，A13-C12所构建的脂质纳米颗粒包载siLuc后，萤火虫荧光素酶(Luciferase)的表达量；4:1，8:1，16:1，32:1表示脂质化合物与siRNA的氮磷比例，即脂质化合物上可质子化的氨基与核酸上的磷酸基团之间的摩尔比。
图15为本发明不同脂质化合物所构建的脂质纳米颗粒在不同氮磷比例下的转染效率热图，氮磷比例分别为4:1，8:1，16:1，32:1，热图中各单元的数值表示转染效率。
图16为本发明的A12-C12、A13-C16、A7-C12制成脂质纳米颗粒后，转染绿色荧光蛋白质粒DNA，48h后的荧光显微镜图；其中，聚乙烯亚胺(PEI)为商业化转染试剂。
图17为转染48h后，可质子化的脂质化合物在不同氮磷比例下转染萤火虫荧光素酶质粒DNA效率的柱状图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
实施例
实施例1中间体C12的合成
Figure PCTCN2022136397-appb-000007
将缩水甘油与十二酰氯按照摩尔比1.2:1.0的比例进行反应。具体操作为：将缩水甘油溶于无水二氯甲烷中，置于25mL具塞圆底烧瓶中，加入催化量的三乙胺(TEA)，混合，密闭，冰浴预冷30min。在磁力搅拌下，使用恒压滴液漏斗将十二酰氯的二氯甲烷溶液缓慢滴加到缩水甘油与TEA的混合溶液中，控制滴加速度，滴加完毕后，室温反应过夜。用饱和碳酸氢钠溶液洗涤两次，饱和氯化钠溶液洗涤1次，取有机相，浓缩，无水硫酸镁干燥30min，过200-300目硅胶柱纯化得产物C12。所得产物红外表征结构如图1，核磁表征如图2。
实施例2中间体C16的合成
Figure PCTCN2022136397-appb-000008
将缩水甘油与十六酰氯按照摩尔比1.2:1.0的比例进行反应。具体操作为：将缩水甘油溶于无二氯甲烷中，置于25mL具塞圆底烧瓶中，加入催化量的TEA，混合，密闭，冰浴预冷30min。在磁力搅拌下，使用恒压滴液漏斗将十六酰氯的二氯甲烷溶液缓慢滴加到缩水甘油与TEA的混合溶液中，控制滴加速度，滴加完毕后，室温反应过夜。用饱和碳酸氢钠溶液洗涤两次，饱和氯化钠溶液洗涤1次，取有机相，浓缩，无水硫酸镁干燥30min，过200-300目硅胶柱纯化得产物C16。所得产品核磁表征如图3。
实施例3中间体C18U的合成
Figure PCTCN2022136397-appb-000009
将缩水甘油与油酰氯按照摩尔比1.2:1.0的比例进行反应。具体操作为将缩水甘油溶于无水二氯甲烷中，置于25mL具塞圆底烧瓶中，加入催化量的TEA，混合，密闭，冰浴预冷30min。在磁力搅拌下，使用恒压滴液漏斗将油酰氯的二氯甲烷溶液缓慢滴加到缩水甘油与TEA的混合溶液中，控制滴加速度，滴加完毕后，室温反应过夜。用饱和碳酸氢钠溶液洗涤两次，饱 和氯化钠溶液洗涤1次，取有机相，浓缩，无水硫酸镁干燥30min，过200-300目硅胶柱纯化得产物C18U。
实施例4脂质化合物A1-C12的合成
Figure PCTCN2022136397-appb-000010
将中间体C-12(十二酸缩水甘油酯)与化合物A1按照摩尔比2.4:1进行反应。具体操作为：取相应量的中间体C-12和化合物A1置于2mL玻璃瓶中，放入磁力搅拌子，于90℃下反应72h后，即得。核磁表征如图4。 1H NMR(400MHz,CDCl 3)：δ0.85-0.88(t，6H)，1.24-1.26(m，32H)，1.60-1.66(m，6H)，2.14-2.19(t，6H)，2.31-2.43(m,12H)，4.08-4.19(m，8H)。
实施例5脂质化合物A2-C12的合成
Figure PCTCN2022136397-appb-000011
将中间体C-12(十二酸缩水甘油酯)与化合物A2按照摩尔比2.4:1进行反应。具体操作为：取相应量的中间体C-12和化合物A2置于2mL玻璃瓶中，放入磁力搅拌子，于90℃下反应72h，即得。核磁表征如图5。 1H NMR(400MHz,CDCl 3)：δ0.86-0.90(t，6H)，1.21-1.30(m，32H)，1.56-1.68(m，10H)，2.03-2.33(m，16H)，4.08-4.14(m，8H)。
实施例6脂质化合物A2-C16的合成
Figure PCTCN2022136397-appb-000012
将中间体C16(棕榈酸缩水甘油酯)与化合物A2按照摩尔比2.4:1进行反应。具体操作为：取相应量的中间体C16和化合物A2置于2mL玻璃瓶中，放入磁力搅拌子，于90℃下反应72h，即得。核磁表征如图6。 1H NMR(400MHz，CDCl 3)：δ0.88-0.91(t，6H)，1.27(m，48H)，1.56-1.68(m，10H)，2.32-2.52(m，16H)，4.08-4.14(m，8H)。
实施例7脂质化合物A2-C18U的合成
Figure PCTCN2022136397-appb-000013
将中间体C18U(棕榈酸缩水甘油酯)与化合物A2按照摩尔比2.4:1进行反应。具体操作为：取相应量的中间体C18U和化合物A2置于2mL玻璃瓶中，放入磁力搅拌子，于90℃下反应72h，即得。核磁表征如图7。 1H NMR(400 MHz，CDCl 3)：δ0.85-0.92(t，6H)，1.27-1.40(m，40H)，1.56-1.68(m，10H)，2.02-2.14(m，8H)，2.20-2.24(m，16H)，4.08-4.14(m，8H)，5.37-5.40(m，4H)。
实施例8脂质化合物A13-C16的合成
Figure PCTCN2022136397-appb-000014
将中间体C16(棕榈酸缩水甘油酯)与化合物A13按照摩尔比5:1进行反应。具体操作为：取相应量的中间体C16和化合物A13置于2mL玻璃瓶中，放入磁力搅拌子，于90℃下反应72h，即得。核磁表征如图8。 1H NMR(400 MHz，CDCl 3)：δ0.87-0.98(t，12H)，1.20-1.29(m，96H)，1.58-1.66(t，8H)，2.14-2.28(brs，3H)，2.32-2.47(m，24H)，4.08-4.33(m，12H)。
实施例9脂质化合物A13-C18U的合成
Figure PCTCN2022136397-appb-000015
将中间体C18U与化合物A13按照摩尔比5:1的比例进行反应。具体步骤为：取相应量的中间体C-18U和化合物A13置于2mL玻璃瓶中，放入磁力搅拌子，于90℃下反应72h，即得。核磁表征如图9。1H NMR(400 MHz，CDCl 3)：δ0.86-0.90(t，12H)，1.13-1.42(m，88H)，1.58-1.66(m，8H)，1.96-2.30(m，19H)，2.40-2.76(m，16H)，4.09-4.14(m，12H)，5.35(m，12H)。
实施例10脂质化合物A12-C12的合成
Figure PCTCN2022136397-appb-000016
将中间体C12与化合物A12按照摩尔比的比例进行反应。具体步骤为：取相应量的中间体C12和化合物A12置于2mL玻璃瓶中，放入磁力搅拌子，于90℃下反应72h，即得。核磁表征如图10。 1H NMR(400MHz，CDCl 3)：δ0.88-0.92(t，18H)，1.28-1.35(m，104H)，1.63-1.65(m，12H)，2.33-2.39(m，30H)，4.08-4.33(m，24H)。
本发明的脂质化合物的反应机理为：三元环氧化合物因其环张力很大，化学键强度极低，体系能量很高，极易与亲核性强的氨基发生开环反应，从而得到本发明的脂质化合物。该反应机理十分成熟，反应过程也为本领域熟知，因此利用上述反应机理生成的化合物，其具体类型以及反应程度都是完全可以预料的。上述为本发明合成的部分化合物的反应条件以及结构表征，本发明其余化合物的合成同上述化合物，其结构式及结构表征数据在此不再详述。
实施例11
分别用脂质化合物A1-C8，A1-C10，A1-C12，A1-C14，A1-C16，A1-C18U作为药物载体将siLuc转染至能够稳定表达萤火虫萤光素酶(Luciferase，Luc)的黑色素瘤(B16F10-Luc)细胞系中，具体步骤如下：
将B16F10-Luc细胞种于96孔细胞培养板中。第二天，细胞生长至80％左右，进行转染。
实验组：将制得的可质子化的脂质化合物A1-C8，A1-C10，A1-C12，A1-C14，A1-C16，A1-C18U以及二硬脂酰基磷脂酰胆碱(DSPC)，胆固醇，二硬脂酰磷脂酰乙酰胺-聚乙二醇(DSPE-PEG)分别溶于无水乙醇中，制成各自的母液，-20℃冰箱中保存，使用时根据需要进行稀释，然后按照摩尔比38:10:50:2(脂质化合物：DSPC：胆固醇：DSPE-PEG)的比例混合。siLuc溶于枸橼酸盐缓冲液(pH＝4)中，其中枸橼酸盐缓冲液的体积是上述乙醇脂质混合液体积的两倍。最后将含有siLuc的枸橼酸盐缓冲液(pH＝4)与上述乙醇脂质混合液快速混合充分，室温震荡孵育30min，自组装形成脂质纳米颗粒。将组装好的脂质纳米颗粒分别加入B16F10-Luc的96孔细胞培养板中进行转染。转染前将培养板中培养液吸除，加入新的培养基80μL，siRNA加入的量为50ng/孔。可质子化的脂质化合物与siRNA的氮磷比例(N/P ratio)为4:1，8:1，16:1。
阳性对照组：采用Lipo2000商业化转染试剂对siLuc进行转染。按照lipo2000的使用说明书进行转染。取50ng siLuc加入到5uL Opti-MEM中，取0.3μL lipo2000置于另一50μL的Opti-MEM中，最后将siRNA Opti-MEM溶液加入到lipo2000 Opti-MEM溶液中，混匀，室温孵育15min后，加入96孔细胞培养板中。转染前将培养板中培养液吸除，加入新的培养基 80μL，siRNA加入的量为50ng/孔。
阴性对照组：仅B16F10-Luc细胞，不进行转染。
转染24h后，裂解细胞，离心去除细胞碎片和内容物，取上清，加入萤火虫荧光素酶的底物，测萤火虫荧光素酶的表达量，从而比较合成的脂质化合物转染siLuc的效率。检测结果如图11，图13所示，合成的脂质化合物大部分有比较强的转染效率。其中A1-C12的转染效率可以达到95％左右。
实施例12
分别用脂质化合物A2-C8，A2-C10，A2-C12，A2-C14，A2-C16，A2-C18U作为基因载体材料将siLuc转染至B16F10-Luc细胞系中，具体步骤如下：
将B16F10-Luc细胞接种于96孔细胞培养板中。第二天，细胞生长至80％左右，进行转染。
实验组：将制得的脂质化合物A2-C8，A2-C10，A2-C12，A2-C14，A2-C16，A2-C18U以及DSPC，胆固醇，DSPE-PEG分别溶于无水乙醇中，制成各自的母液，-20℃冰箱中保存，使用时根据需要进行稀释，然后按照摩尔比38:10:50:2(脂质化合物：DSPC：胆固醇：DSPE-PEG)的比例混合。siLuc溶于枸橼酸盐缓冲液(pH＝4)中，其中枸橼酸盐缓冲液的体积是上述乙醇脂质混合液体积的两倍。将含有siLuc的枸橼酸盐缓冲液(pH＝4)与上述乙醇脂质混合液快速混合充分，室温震荡孵育30min，自组装形成脂质纳米颗粒。然后将组装好的脂质纳米颗粒分别加入B16F10-Luc细胞的96孔培养板中进行转染。转染前将培养板中培养液换液，加入新的培养基80μL，siRNA加入的量为50ng/孔。脂质化合物与siRNA的氮磷比为4:1，8:1，16:1。
阳性对照组：采用Lipo 2000转染试剂对siLuc进行转染。按照lipo2000的使用说明书进行转染。取50ng siLuc加入到5uL Opti-MEM中，取0.3mL lipo2000置于另一50μL的Opti-MEM中，最后将siRNA Opti-MEM溶液加入到lipo2000 Opti-MEM溶液中，混匀，室温孵育15min后，加入96孔细胞培养板中。转染前将培养板中培养液吸除，加入新的培养基80μL，siRNA加入的量为50ng/孔。
阴性对照组：仅B16F10-Luc细胞，不进行转染。
转染24h后，裂解细胞，离心去除细胞碎片和内容物，取上清，加入萤火虫荧光素酶的底物，测萤火虫荧光素酶的表达量，从而比较合成的脂质化合物转染siLuc的效率。检测结果如图12，图13所示，合成的脂质化合物大部分有比较强的转染效率，其中A2-C12，A2-C16的转染效率可以达到95％左右。
实施例13
分别用脂质化合物A5-C12，A5-C16，A6-C12，A7-C12，A7-C16，A8-C12，A8-C16，A9-C12，A9-C16，A10-C16，A10-C12，A11-C16，A11-C12，A12-C12，A12-C16，A13-C12，A13-C16作为基因载体材料将siLuc转染至B16F10-Luc细胞系中，具体步骤如下：
将B16F10-Luc细胞接种于96孔细胞培养板中。第二天，细胞生长至80％左右，进行转染。
实验组：将制得的可质子化的脂质化合物A5-C12，A5-C16，A6-C12，A7-C12，A7-C16，A8-C12，A8-C16，A9-C12，A9-C16，A10-C16，A10-C12，A11-C16，A11-C12，A12-C12， A12-C16，A13-C12，A13-C16以及DSPC，胆固醇，DSPE-PEG分别溶于无水乙醇中，制成各自的母液，-20℃冰箱中保存，使用时根据需要进行稀释。然后按照摩尔比38:10:50:2(脂质化合物：DSPC：胆固醇：DSPE-PEG)的比例混合。siLuc溶于枸橼酸盐缓冲液(pH＝4)中，其中枸橼酸盐缓冲液的体积是上述乙醇脂质混合液体积的两倍。将含有siLuc的枸橼酸盐缓冲液(pH＝4)与上述乙醇脂质混合液快速混合充分，室温震荡孵育30min，自组装形成脂质纳米颗粒。然后将组装好的脂质纳米颗粒分别加入B16F10-Luc细胞的培养板中进行转染。脂质化合物与siRNA的氮磷比例为4:1，8:1，16:1，32:1。
阳性对照组：采用Lipo 2000转染试剂对siLuc进行转染。按照lipo2000的使用说明书进行转染。取50ng siLuc加入到5uL Opti-MEM中，取0.3μL lipo2000置于另一50μL的Opti-MEM中，最后将siRNA Opti-MEM溶液加入到lipo2000 Opti-MEM溶液中，混匀，室温孵育15min后，加入96孔细胞培养板中。转染前将培养板中培养液吸除，加入新的培养基80μL，siRNA加入的量为50ng/孔。
阴性对照组：仅B16F10-Luc细胞，不进行转染。
转染24h后，裂解细胞，离心去除细胞碎片和内容物，取上清，加入萤火虫荧光素酶的底物，测萤火虫荧光素酶的表达量，从而比较合成的脂质化合物转染siLuc的效率。检测结果如图14，图15所示，合成的脂质化合物大部分有比较强的转染效率，A12-C12，A5-C12，A8-C12的转染效率可以达到90％以上，其中A12-C12几乎完全抑制萤火虫荧光素酶基因表达。
实施例14
分别用脂质化合物A1-C12，A1-C16，A1-C18U，A2-C12，A2-C16，A2-C18U，A7-C12，A13-C16，A12-C16，A8-C12，A12-C12作为基因载体材料将绿色荧光蛋白和萤火虫荧光素酶的质粒DNA(pDNA-GFP-Luc)转染至293T细胞系中，具体步骤如下：
将293T细胞接种于96孔细胞培养板中。第二天，细胞生长至80％左右，进行转染。
实验组：将制得的脂质化合物A1-C12，A1-C16，A1-C18U，A2-C12，A2-C16，A2-C18U，A7-C12，A13-C16，A12-C16，A8-C12，A12-C12以及DSPC，胆固醇，DSPE-PEG分别溶于无水乙醇中，制成各自的母液，-20℃冰箱中保存，使用时根据需要进行稀释。然后按照摩尔比38:10:50:2(脂质化合物：DSPC：胆固醇：DSPE-PEG)的比例混合；表达绿色荧光蛋白和萤火虫荧光素酶的质粒DNA(pDNA-GFP-Luc)溶于枸橼酸盐缓冲液(pH＝4)中，其中枸橼酸盐缓冲液的体积是上述乙醇脂质混合液体积的两倍。将含有质粒DNA的枸橼酸盐缓冲液(pH＝4)与上述乙醇脂质混合液快速混合充分，室温震荡孵育30min，自组装形成脂质纳米颗粒。然后将组装好的脂质纳米颗粒分别加入293T细胞的培养板中进行转染。转染前将培养板中培养液吸除，加入新的培养基80μL，DNA加入的量为80ng/孔。可质子化的脂质化合物与质粒的氮磷比例为8:1，16:1，32:1。
阳性对照组：采用PEI商业化转染试剂对293T细胞进行转染。按照PEI转染试剂说明书进行转染。取80ng DNA置于5uL的ddH 2O中，混匀；取0.1μL PEI至于5μL水中，混匀，随后将稀释后的PEI加入到DNA水溶液中，混匀，室温孵育15min后进行转染。转染前，吸去原培养基，加入80μL新鲜培养基，DNA转染剂量80ng/孔。
阴性对照组：仅293T细胞，不进行转染。
转染后分别于12h，24h，36h，48h，在荧光显微镜下观察绿色荧光蛋白的表达。转染48h 后，裂解细胞，离心去除细胞碎片和内容物，取上清，加入萤火虫荧光素酶的底物，测萤火虫荧光素酶的表达量，从而比较合成的脂质化合物转染质粒的效率。检测结果如图16，图17所示，其中A12-C12的转染效率与商业化试剂PEI相当。
上面结合附图对本发明实施例作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。

Claims (10)

  1. 一种脂质化合物，或其药学上可接受的盐，其特征在于，所述脂质化合物由有机胺氮上的氢原子均被R 1基团取代后得到；所述有机胺选自如下所示的结构：
    Figure PCTCN2022136397-appb-100001
    所述R 1基团具有式(I)所示的结构：
    Figure PCTCN2022136397-appb-100002
    其中，n选自6-16的整数。
  2. 根据权利要求1所述的脂质化合物，其特征在于，所述有机胺选自如下所示的结构：
    Figure PCTCN2022136397-appb-100003
    进一步，所述R 1基团选自如下所示的结构：
    Figure PCTCN2022136397-appb-100004
  3. 根据权利要求1所述的脂质化合物，其特征在于，所述脂质化合物选自如下所示的结构：
    Figure PCTCN2022136397-appb-100005
    Figure PCTCN2022136397-appb-100006
  4. 一种组合物，其特征在于，所述组合物包括权利要求1-3中任一项所述的脂质化合物，或其药学上可接受的盐；进一步，所述组合物还包括其他脂质化合物；更进一步，所述其他脂质化合物包括胆固醇、磷脂和聚合物共轭脂质中的至少一种。
  5. 根据权利要求4所述的组合物，其特征在于，所述磷脂包括蛋黄卵磷脂、氢化蛋黄卵磷脂、大豆卵磷脂、氢化大豆卵磷脂、鞘磷脂、磷脂酰乙醇胺、二肉豆蔻酰磷脂酰胆碱、二肉豆蔻酰磷脂酰甘油、二棕榈酰磷脂酰胆碱、二硬酯酰磷脂酰胆碱、二油酰磷脂酰胆碱、二月桂酰磷脂酰胆碱中的至少一种。
  6. 根据权利要求4所述的组合物，其特征在于，所述聚合物共轭脂质包括聚乙二醇修饰的 磷脂酰乙醇胺、聚乙二醇修饰的磷脂酸、聚乙二醇修饰的神经酰胺、聚乙二醇修饰的二烷基胺、聚乙二醇修饰的二酰基甘油、聚乙二醇修饰的二烷基甘油中的至少一种。
  7. 根据权利要求4所述的组合物，其特征在于，所述脂质化合物，或其药学上可接受的盐与胆固醇的摩尔比为1：1-9，优选1：1-5，更优选1：1-2；
    进一步，所述脂质化合物，或其药学上可接受的盐与磷脂的摩尔比为1-10：1，优选1-5：1，更优选3-5：1；
    更进一步，所述脂质化合物，或其药学上可接受的盐与聚合物共轭脂质的摩尔比为10-50：1，优选10-20：1，更优选15-20：1。
  8. 根据权利要求4所述的组合物，其特征在于，还包括药物活性成分；进一步，所述药物活性成分包括核酸分子、多肽、蛋白质和小分子化合物中的至少一种；更进一步，所述核酸分子包括siRNA、mRNA、miRNA、antisense RNA、CRISPR guide RNAs、可复制性RNA、环二核苷酸、poly IC、CpG ODN、plasmid DNA中的至少一种，优选siRNA。
  9. 根据权利要求8所述的组合物，其特征在于，当所述药物活性成分包括核酸分子时，所述脂质化合物，或其药学上可接受的盐与核酸分子的氮磷比为1-50：1，优选1-40：1，更优选4-32：1。
  10. 权利要求1-3中任一项所述的脂质化合物，或其药学上可接受的盐，或权利要求4-9中任一项所述的组合物在制备核酸药物、基因疫苗、多肽或蛋白质药物、小分子药物中的应用。
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