WO2024027789A1 - 用于递送核酸的阳离子脂质化合物和组合物及用途 - Google Patents

用于递送核酸的阳离子脂质化合物和组合物及用途 Download PDF

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WO2024027789A1
WO2024027789A1 PCT/CN2023/110927 CN2023110927W WO2024027789A1 WO 2024027789 A1 WO2024027789 A1 WO 2024027789A1 CN 2023110927 W CN2023110927 W CN 2023110927W WO 2024027789 A1 WO2024027789 A1 WO 2024027789A1
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compound
cationic lipid
dcm
5min
liposome preparation
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PCT/CN2023/110927
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French (fr)
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胡勇
李亚霏
胡昭宇
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深圳瑞吉生物科技有限公司
武汉瑞佶生物科技有限公司
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Priority to US18/410,875 priority Critical patent/US20240180836A1/en
Publication of WO2024027789A1 publication Critical patent/WO2024027789A1/zh

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    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
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    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • 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
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    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
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    • C07C323/12—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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Definitions

  • the invention relates to the field of lipid delivery carriers. It is a type of cationic lipid compound that can form drug-carrying nano-lipid particles after being combined with other lipid components, thereby realizing the delivery of nucleic acids from outside the cell to the inside of the cell in vitro and in vivo.
  • the present invention relates to cationic lipid compounds and compositions and uses for the delivery of nucleic acids.
  • Nucleic acid drugs achieve the purpose of treating and preventing diseases by introducing exogenous genes into target cells or tissues to replace, compensate, block or modify specific genes. Its R&D and production process is relatively simple, and it has the advantages of short R&D cycle, high clinical development success rate, and better improvement plasticity. Nucleic acid vaccines have been one of the mainstays in preventing COVID-19 in recent years and have proven their huge potential in the market.
  • lipid nanoparticles which have the characteristics of improving the efficacy of gene drugs and targeted delivery. They can protect nucleic acids from rapid degradation in the body, extend circulation time, and enhance targeted delivery. It consists of 2 to 4 lipid components, including cationic lipid compounds, 0 to 2 auxiliary lipids and 0 to 1 PEG lipid. Among them, cationic lipid compounds play a key role in nucleic acid entrapment and release, so it is crucial to develop new, efficient, and low-toxic cationic lipid compounds.
  • the invention provides a class of sulfur-containing cationic lipid compounds that are easy to degrade and have rapid metabolism in the body, including pharmaceutically acceptable salts thereof and stereoisomers or tautomers thereof. Its primary use is in combination with other lipid components in specific ratios to form lipid nanoparticles for the delivery of prophylactic or therapeutic agents, such as therapeutic nucleic acids.
  • Another object of the present invention is to provide a method for synthesizing the lipid compound, which uses readily available raw materials, adopts a reaction route with mild conditions, has high product yield, has low requirements for equipment and equipment, and is simple to operate.
  • therapeutic nucleic acids include plasmid DNA, messenger RNA, antisense oligonucleotides (ASON), microRNA (miRNA), interfering RNA (micRNA), dicer substrate RNA, complementary DNA (cDNA).
  • ASON antisense oligonucleotides
  • miRNA microRNA
  • miRNA interfering RNA
  • cDNA complementary DNA
  • the present invention also provides formulation ratios and usage methods when such cationic lipid compounds are used in combination with other lipid components, as well as applications in cells and animal models.
  • a cationic lipid compound having the following structure of formula (I) is used:
  • G 1 and G 2 are each independently C 3 -C 10 alkylene
  • R 1 is C 2 -C 12 alkyl
  • R 2 is H or C 2 -C 12 alkyl
  • R 3 is C 2 -C 12 alkyl
  • R 4 is H or C 2 -C 12 alkyl
  • R 7 is C 2 -C 18 alkylene
  • R 8 and R 9 are C 1 -C 8 linear alkyl groups, or R 8 , R 9 and the N atom to which they are connected form a C 3 -C 10 heterocycloalkyl group.
  • G 1 and G 2 are C 3 -C 8 alkylene.
  • R 7 is C 2 -C 6 alkylene.
  • R 1 and R 3 are each independently a C 3 -C 9 alkyl group; R 2 and R 4 are H or a C 3 -C 9 alkyl group.
  • one and only one of R 2 and R 4 is H.
  • R 5 is -R 7 -OH
  • R 7 is C 2 -C 8 alkylene
  • R 5 is R 7 -N(CH 2 CH 3 )CH 2 CH 3 .
  • the -C(R 1 )R 2 or -C(R 3 )R 4 structure in the formula (I) structure each independently meets the following characteristics:
  • G 1 is C 5 -C 8 alkylene
  • G 2 is C 4 -C 8 alkylene
  • R 5 is -R 7 -OH, or -R 7 -NR 8 (R 9 );
  • R 7 is C 2 -C 6 alkylene
  • R 8 and R 9 are C 1 -C 8 linear alkyl groups, or R 8 , R 9 and the N atom to which they are connected form a C 3 -C 10 heterocycloalkyl group.
  • the cationic lipid compound has one of the structures shown in the following table:
  • the present invention also provides a liposome preparation comprising one or more cationic lipid compounds of the invention and preventive or therapeutic nucleic acids, wherein the liposome preparation is used to prevent or treat a certain disease.
  • the liposome formulation contains one or more components selected from the group consisting of neutral lipids, charged lipids, steroids, and polymer-conjugated lipids.
  • the therapeutic substances used in the present invention are therapeutic nucleic acids, including plasmid DNA, messenger RNA, antisense oligonucleotides (ASON), microRNA (miRNA), interfering RNA (micRNA), dicer substrate RNA, complementary DNA (cDNA ).
  • ASON antisense oligonucleotides
  • miRNA microRNA
  • micRNA interfering RNA
  • cDNA complementary DNA
  • Preferred are plasmid DNA, messenger RNA and antisense oligonucleotides.
  • the molar ratio of the nucleic acid to the cationic lipid compound is 20:1 to 1:1.
  • the molar ratio of the nucleic acid to the cationic lipid compound is 10:1 to 4:1.
  • the diameter of the liposome preparation is 50 nm to 300 nm.
  • the diameter of the liposome preparation is 50 nm to 150 nm, or 150 nm to 200 nm.
  • one or more other lipid components are also included, including but not limited to structural lipids, steroids, and polymer-conjugated lipids.
  • the steroid included is cholesterol.
  • the molar ratio of the cholesterol to the cationic lipid compound is 0:1 to 1.5:1.
  • the molar ratio of the cholesterol to the cationic lipid compound is 0.2:1 to 1.2:1.
  • polymer in the polymer-conjugated lipid is polyethylene glycol (PEG).
  • the molar ratio of the cationic lipid compound to the PEGylated lipid is 100:1 to 20:1.
  • the pegylated lipid is PEG 8 DAG, PEG 8 PE, PEG 8 SDAG, PEG 8 cer, PEG-DMG or ALC-0159.
  • the liposome preparation includes one or more structural lipids selected from DPPG, DSPC, DPPC, DMPC, DOPC, POPC, DOPE and DSPE.
  • the structural lipid is DSPC or DOPE.
  • the molar ratio of the structural lipid to the cationic lipid compound is 0:1 to 0.5:1.
  • the molar ratio of the structural lipid to the cationic lipid compound is 0:1 to 0.3:1.
  • the liposome preparation includes nucleic acid
  • the nucleic acid is selected from antisense RNA and/or messenger RNA.
  • the nucleic acid is messenger RNA.
  • the present invention also provides the use of the cationic lipid compound or the liposome preparation of the present invention in the preparation of a medicament for inducing protein expression in a subject.
  • the subject is a mammal.
  • the subject is a non-human primate.
  • the subject is a human.
  • the present invention provides cationic lipid compounds and liposome formulations and uses for delivering nucleic acids.
  • the technical solution of the present invention has the following advantages:
  • the cationic lipid compound of the present invention has a thioester bond.
  • the introduction of the thioester bond makes the compound easier to degrade, improves the clearance rate of the lipid compound in the body, and makes the carrier composed of the compound less toxic and leaving less residue in the body.
  • the preparation method of the lipid compound has the advantages of readily available raw materials, mild reaction conditions, high product yield, low requirements for equipment and equipment, and simple operation.
  • Figure 1 is a fluorescence brightness diagram of the compound of Example 14;
  • Figure 2 is a hematocrit diagram of Example 15
  • Figure 3 is a cationic lipid metabolism rate diagram of Example 16.
  • step 1
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (10g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 15ml/min), click Plate monitoring was used, and part of the pure product was evaporated to obtain compound 1-2 (5.5 g, 89% yield) as a colorless oily liquid.
  • HBTU benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DIEA N,N-diisopropylethylamine
  • 1-nonanethiol 1.6g
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (25g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min) to obtain free Compound 1-4 was an oily liquid (2.9 g, 89% yield).
  • step 1
  • HBTU benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DIEA N,N-diisopropylethylamine
  • 1-undecanethiol 3.5g
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (40g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 30ml/min) to obtain free Compound 2-2 was an oily liquid (5.0 g, 89% yield).
  • step 1
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (25g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min), click Plate monitoring was used, and part of the pure product was evaporated to obtain compound 8-2 (3.5 g, 84% yield) as a colorless oily liquid.
  • the organic phase was evaporated under reduced pressure, and an appropriate amount of silica gel was added to mix and purify (40g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 10ml/min) to obtain light yellow oily liquid compound 8-7 (1.1g, 81% yield).
  • HBTU benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DIEA N,N-diisopropylethylamine
  • 8-7 1.1g
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (25g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min) to obtain free Compound 8-8 was an oily liquid (1.4 g, 82% yield).
  • step 1
  • step 1
  • the organic phase was evaporated under reduced pressure, and an appropriate amount of silica gel was added to mix and purify (40g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 10ml/min) to obtain light yellow oily liquid compound 15-4 (1.06g, 83% yield).
  • HBTU benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DIEA N,N-diisopropylethylamine
  • 6-bromocaproic acid (1.07g
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (25g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min) to obtain free Compound 15-5 was an oily liquid (1.09 g, 79% yield).
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (25g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min), click Plate monitoring was used, and part of the pure product was evaporated to obtain compound 15-6 (3.96 g, 72% yield) as a colorless oily liquid.
  • step 1
  • step 1
  • step 1
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • DMAP 4-dimethylaminopyridine
  • n-heptanol 2.5g
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (25g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min), click Plate monitoring was used, and part of the pure product was evaporated to obtain compound 28-2 (3.1 g, 76% yield) as a colorless oily liquid.
  • step 1
  • step 1
  • HBTU benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DIEA N,N-diisopropylethylamine
  • 7-bromoheptanoic acid (1.03g
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (25g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min) to obtain free Compound 35-5 was an oily liquid (1.03 g, 61% yield).
  • step 1
  • Dissolve compound 40-1 (3.0g) in ethanol (50ml), stir in an ice bath, weigh sodium ethoxide (EtONa, 1.33g) and 1-bromoheptane (2.8g) in batches and add to the reaction system at room temperature. Stir for 6h. The reaction solution was quenched with saturated ammonium chloride solution and extracted with ethyl acetate.
  • the resulting organic phase was dried over anhydrous sodium sulfate, and an appropriate amount of silica gel was added to mix and purify (25 g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min), spot plate monitoring, evaporate part of the pure product to obtain a colorless oily liquid compound 40-2 (2.0 g, 47% yield).
  • Dissolve compound 40-4 (1.0g) in DCM (20ml), stir at room temperature, and weigh out 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.1g) ), 4-dimethylaminopyridine (DMAP, 535 mg) and 6-bromo-n-hexanol (873 mg) were added to the reaction system in batches, and stirred at room temperature for 2 h.
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • DMAP 4-dimethylaminopyridine
  • 6-bromo-n-hexanol 873 mg
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (25g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min), click Plate monitoring was used, and part of the pure product fraction was evaporated to obtain colorless oily liquid compound 40-5 (1.3 g, 76% yield).
  • Dissolve compound 40-7 (3.0g) in DCM (40ml), stir in an ice bath, weigh triphenylphosphine (2.76g) and carbon tetrabromide (8.71g) and add them to the reaction system in batches, and stir at room temperature for 2 hours. . There are new points with smaller polarity.
  • the reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (40g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 30ml/min) to obtain colorless oily liquid compound 40-8 (2.6g, 66% yield).
  • the organic phase was evaporated under reduced pressure, and an appropriate amount of silica gel was added to mix and purify (40g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 10ml/min) to obtain light yellow oily liquid compound 40-10 (950mg, 76% yield).
  • HBTU benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DIEA N,N-diisopropylethylamine
  • 7-bromocaproic acid 1.1g
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (25g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 20ml/min) to obtain free Compound 40-11 was an oily liquid (1.20 g, 80% yield).
  • step 1
  • Luciferase mRNA (Luc mRNA) is diluted in 10-100mM, pH 4.0 citric acid buffer; each lipid component (cationic lipid shown in the present invention: DSPC: cholesterol: PEG lipid) is divided according to the total concentration 10mg/mL dissolved in ethanol. Among them, the 1.6% PEG lipid group uses ALC-0159, and the 1.5% PEG lipid group uses DMG-PEG2000.
  • the detection results of the particle size, PDI and encapsulation efficiency of the mRNA-loaded LNP prepared in this example are shown in Table 1.
  • the results show that the nanoparticles formed by lipids and mRNA under this formula have a high encapsulation rate and a uniform particle size of about 100 nm, which is in line with the basic characteristics of nucleic acid delivery carriers.
  • Table 1 *DLin-MC3-DMA is the cationic lipid of the commercial nucleic acid delivery system Onpattro.
  • Example 14 Determination of the in vivo expression effect of luciferase mRNA delivered using nanolipid particle compositions
  • mice Female Balb/c mice aged 6 to 8 weeks were injected with LUC-mRNA 8 lipid nanoparticles containing 3ug of mRNA through the tail vein (the nucleotide sequence corresponding to LUC-mRNA is shown in SEQ ID NO: 1 of patent application 202210286081.0 ), the preparation method is the same as Example 13.
  • mice were intraperitoneally injected with 200ug of D-Luciferin Potassium Salt, and detected using the Qinxiang small animal imaging system. Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability. It emits biological light in the presence of the substrate fluorescein.
  • the basic characteristics of the mRNA used are the ARCA cap structure, the polyA tail length is 100-120nt, and pseudouracil is completely substituted.
  • Detection junction As shown in Figure 1, the nanolipid particle compositions No. 1-8 deliver mRNA to the liver at a higher level than MC3 lipid nanoparticles.
  • Example 15 Expression and effect determination of erythropoietin (EPO) mRNA delivered in mice using nanolipid particle compositions
  • EPO-mRNA 8 lipid nanoparticles into female Balb/c mice aged 6 to 8 weeks through the tail vein (for the nucleotide sequence corresponding to EPO-mRNA, see SEQ ID NO: 2 of patent application 202210286081.0), preparation method Same as Example 13, 48 hours after injection, blood was collected from the eye box of the mice and the hematocrit was measured.
  • hEPO is commonly used as a characterization gene for protein expression levels in mammalian blood, with expression levels proportional to hematocrit.
  • the basic characteristics of the mRNA used are the ARCA cap structure, the polyA tail length is 100-120nt, and pseudouracil is completely substituted.
  • the test results are shown in Figure 2. From the results, it can be seen that the nanolipid particle composition composed of a variety of patented cations has a better level of delivering mRNA than DLin-MC3-DMA.
  • Example 16 In vivo metabolism rates of different cationic lipids
  • mice Female Balb/c mice aged 6 to 8 weeks were injected with 5ug of EPO-mRNA 8 lipid nanoparticles through the tail vein.
  • the preparation method was the same as in Example 13. The injection was performed at different time points (after injection, 1h, 3h, 6h, 24h, 48h) were sacrificed. After the liver was broken, the lipids were extracted with chloroform three times. Finally, the extracts were combined, the chloroform was removed by rotary evaporation, methanol was added to dissolve, and the content of cationic lipids was analyzed by HPLC-CAD (Thermo Vanquish). .
  • the analytical column was Acclaim TM C18 column. Mobile phase A is 0.5% TEAA aqueous solution, phase B is 0.5% TEAA methanol solution, and the sample is eluted according to the gradient in Table 2. The results are shown in Figure 3.

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Abstract

公开了式(I)所示的用于递送核酸的阳离子脂质化合物和组合物及用途。还公开了以所述化合物为关键组分的纳米脂质颗粒在核酸递送方面的用途，包含递送载体的组分、制备方法和使用方法。

Description

用于递送核酸的阳离子脂质化合物和组合物及用途 技术领域
本发明涉及脂质递送载体领域，是一类阳离子脂质化合物，与其他脂质成分结合后能够形成可载药的纳米脂质颗粒，从而在体外和体内实现细胞外向细胞内递送核酸。具体的说，本发明涉及用于递送核酸的阳离子脂质化合物和组合物及用途。
背景技术
核酸药物通过将外源基因导入靶细胞或组织，替代、补偿、阻断或修正特定基因，以达到治疗和预防疾病的目的。其研发生产工艺相对简单，具有研发周期短、临床开发成功率高、改良可塑性更好等优势。核酸疫苗在近几年作为预防COVID-19的主力军之一，也已经证明了它在市场的巨大潜力。
但是裸mRNA在体内循环时间短、易被降解，且难以进入靶细胞或者靶组织。因此提高mRNA药物的体内递送效率，是提高该类产品有效性的关键方向之一。
目前，应用最广的核酸药物的递送载体是脂质纳米颗粒，它具有提高基因药物疗效以及靶向递送作用等特点，可以保护核酸在体内不被迅速降解，延长循环时间，增强靶向递送。它由2～4个脂质组分组成，包括阳离子脂质化合物、0～2种辅助脂质和0～1种PEG脂质构成。其中阳离子脂质化合物在核酸包载和释放中起关键作用，因此研发新型、高效、低毒的阳离子脂质化合物至关重要。
发明内容
本发明提供了一类易于降解、体内代谢速度快的含硫的阳离子脂质化合物，包括其药物可接受的盐和其立体异构体或互变异构体。其主要用途是与其他脂质组分以特定比例联合使用，以形成用于递送预防或治疗剂(如治疗性核酸)的脂质纳米颗粒。
本发明的另一目的是提供该类脂质化合物的合成方法，使用原料易得、采用条件温和的反应路线、产品产率高、仪器设备要求低且操作简单。
在一些实例中，治疗性核酸包括质粒DNA、信使RNA、反义寡核苷酸(ASON)、微小RNA(miRNA)、干扰RNA(micRNA)、dicer底物RNA、互补DNA(cDNA)。
同时本发明还提供了此类阳离子脂质化合物与其他脂质组分联合使用时的制剂配比以及使用方法，以及在细胞和动物模型中的应用。
在本发明的实施方案中，采用的是具有如下式(I)结构的阳离子脂质化合物：
或其药物可接受的盐、互变异构体或立体异构体，其中：
L1为-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-OC(＝O)O-、-O-、-S-、-S-S-、-C(＝O)S-、-SC(＝O)-、-N(R6)C(＝O)-、-C(＝O)N(R6)-、-N(R6)C(＝O)O-、-OC(＝O)N(R6)-、-SC(＝O)N(R6)-、-N(R6)C(＝O)S-、-C(＝S)-、-SC(＝S)-、和-C(＝S)S-中任一种，所述R6为H或C1 8C12烷基；
G1和G2各自独立地为C3-C10亚烷基；
R1为C2-C12烷基；
R2为H或C2-C12烷基；
R3为C2-C12烷基；
R4为H或C2-C12烷基；
R5为H、C1-C6烷基、-R7-OH、-R7-OC(＝O)CH3、-R7-NHC(＝O)-CH3、-R7-OCH3或-R7-NR8(R9)；
R7为C2-C18亚烷基；
R8、R9为C1-C8的直链烷基，或者R8、R9和其连接的N原子形成C3-C10杂环烷基。
根据本发明一些具体实施方案，其中，L1为-OC(＝O)-、-C(＝O)O-、-SC(＝O)-或-C(＝O)S-。
根据本发明一些具体实施方案，其中，G1和G2为C3-C8亚烷基。
根据本发明一些具体实施方案，其中，R7为C2-C6亚烷基。
根据本发明一些具体实施方案，其中，R1和R3各自独立地为C3-C9烷基；R2和R4为H或C3-C9烷基。
根据本发明一些具体实施方案，其中，R2和R4有且仅有一个为H。
根据本发明一些具体实施方案，其中，R5为-R7-OH，R7为C2-C8亚烷基。
根据本发明一些具体实施方案，其中，R5为R7-N(CH2CH3)CH2CH3。
根据本发明一些具体实施方案，其中，其中，所述式(I)结构中的-C(R1)R2或-C(R3)R4结构各自独立地符合如下特征：
根据本发明一些具体实施方案，其中，
L1为-OC(＝O)-或-SC(＝O)-；
G1为C5-C8亚烷基；
G2为C4-C8亚烷基；
-C(R1)R2结构选自
-C(R3)R4结构选自
R5为-R7-OH、或-R7-NR8(R9)；
R7为C2-C6亚烷基；
R8，R9为C1-C8的直链烷基，或者R8,R9和其连接的N原子形成C3-C10杂环烷基。
根据本发明一些具体实施方案，其中，所述阳离子脂质化合物具有以下表中所示的结构之一：


本发明还提供了包含一种或多种本发明的阳离子脂质化合物与预防性或治疗性核酸的脂质体制剂，其中，所述脂质体制剂用于预防或者治疗某种疾病。
该脂质体制剂包含选自中性脂质、带电脂质、类固醇和聚合物缀合的脂质的一种或多种组分。本发明所用到的治疗物为治疗性核酸，包含质粒DNA、信使RNA、反义寡核苷酸(ASON)、微小RNA(miRNA)、干扰RNA(micRNA)、dicer底物RNA、互补DNA(cDNA)。优选为质粒DNA、信使RNA和反义寡核苷酸。
根据本发明一些具体实施方案，其中，所述核酸与所述阳离子脂质化合物的摩尔比为20：1至1：1。
根据本发明一些具体实施方案，其中，所述核酸与所述阳离子脂质化合物的摩尔比 为10：1至4：1。
根据本发明一些具体实施方案，其中，该脂质体制剂的直径为50nm至300nm。
根据本发明一些具体实施方案，其中，该脂质体制剂的直径为50nm至150nm，或150nm至200nm。
根据本发明一些具体实施方案，其中，还包含一种或多种其他脂质组分，包括但不限于结构脂质、类固醇和聚合物缀合的脂质。
根据本发明一些具体实施方案，其中，所包含的类固醇为胆固醇。
根据本发明一些具体实施方案，其中，所述胆固醇与阳离子脂质化合物的摩尔比为0:1至1.5:1。
根据本发明一些具体实施方案，其中，所述胆固醇与阳离子脂质化合物的摩尔比为0.2:1至1.2:1。
根据本发明一些具体实施方案，其中，聚合物缀合的脂质中的聚合物为聚乙二醇(PEG)。
根据本发明一些具体实施方案，其中，所述阳离子脂质化合物与所述聚乙二醇化脂质的摩尔比为100:1至20:1。
根据本发明一些具体实施方案，其中，所述聚乙二醇化脂质为PEG8DAG、PEG8PE、PEG8SDAG、PEG8cer、PEG-DMG或ALC-0159。
根据本发明一些具体实施方案，其中，所述脂质体制剂包含选自DPPG、DSPC、DPPC、DMPC、DOPC、POPC、DOPE和DSPE中的一种或多种结构脂质。
根据本发明一些具体实施方案，其中，所述结构脂质为DSPC或DOPE。
根据本发明一些具体实施方案，其中，所述结构脂质与所述阳离子脂质化合物的摩尔比为0:1至0.5:1。
根据本发明一些具体实施方案，其中，所述结构脂质与所述阳离子脂质化合物的摩尔比为0:1至0.3:1。
根据本发明一些具体实施方案，其中，所述脂质体制剂包括核酸。
根据本发明一些具体实施方案，其中，所述核酸选自反义RNA和/或信使RNA。
根据本发明一些具体实施方案，其中，所述核酸为信使RNA。
本发明还提供了本发明所述的阳离子脂质化合物或所述的脂质体制剂在制备用于在对象中诱导蛋白质表达的药物中的用途。
根据本发明一些具体实施方案，其中，所述对象为哺乳动物。
根据本发明一些具体实施方案，其中，所述对象是非人灵长类动物。
根据本发明一些具体实施方案，其中，所述对象是人。
综上所述，本发明提供了用于递送核酸的阳离子脂质化合物和脂质体制剂及用途。本发明的技术方案具有如下优点：
本发明的阳离子脂质化合物具有硫酯键，硫酯键的引入使得该化合物更易降解，提高了该脂质化合物的体内清除速度，使得由该化合物构成的载体毒性更低、体内残留更少。且所述脂质化合物的制备方法具有使用原料易得、反应条件温和、产品产率高、仪器设备要求低且操作简单的优点。
附图说明
图1为实施例14的化合物荧光亮度图；
图2为实施例15的红细胞压积图；
图3为实施例16的阳离子脂质代谢速度图。
具体实施方式
以下结合附图及实施例详细说明本发明的技术方案，但本发明的保护范围包括但是不限于此。
实施例1
化合物1的合成
步骤1：
将化合物1-1(3.0g)溶于DCM(10ml)，室温搅拌，依次称取1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI，3.35g)，4-二甲氨基吡啶(DMAP，1.64g)和 9-十七醇(3.79g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与1-1标样对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(10g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速15ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物1-2(5.5g，89％收率)。
步骤2：
向化合物1-2(5.0g)和乙醇胺(1.0g)的乙腈溶液(100mL)溶液中加入碳酸钾(4.5g)。将混合物在70℃搅拌2小时。TLC显示化合物1-2完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物1-3(3.0g，62.6％收率)。
步骤3：
将化合物1-1(3.0g)溶于DCM(40ml)，室温搅拌，依次称取苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU，4.1g)，N,N-二异丙基乙胺(DIEA，2.3g)和1-壬硫醇(1.6g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与1-1标样对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)得到无色油状液体化合物1-4(2.9g，89％收率)。
步骤4：
将化合物1-3(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(170mg)，K2CO3(470mg)和化合物1-4(500mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比1-3极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(25g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物1(600mg，73％收率)。
1H NMR(400MHz,Chloroform-d)δ4.01–3.96(m,1H),2.85(d,J＝7.6Hz,2H),2.81–2.75(m,2H),2.79–2.58(m,6H),2.57–2.40(m,2H),2.31–2.27(m,2H),1.95–1.48(m,16H),1.25(s,46H),0.86(d,J＝7.2Hz,9H)。
实施例2
化合物2的合成
步骤1：
将化合物2-1(3.0g)溶于DCM(40ml)，室温搅拌，依次称取苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU，7.0g)，N,N-二异丙基乙胺(DIEA，4.0g)和1-十一硫醇(3.5g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与2-1标样对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速30ml/min)得到无色油状液体化合物2-2(5.0g，89％收率)。
步骤2：
将化合物1-3(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(170mg)，K2CO3(470mg)和化合物2-2(500mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比1-3极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(25g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物2(600mg，73％收率)。
1H NMR(400MHz,Chloroform-d)δ4.01–3.96(m,1H),2.92-2.88(m,2H),2.81–2.75(m,2H),2.79–2.58(m,6H),2.57–2.40(m,2H),2.31–2.27(m,2H),1.95–1.48(m,16H),1.25(s,46H),0.86(d,J＝7.2Hz,9H)。
实施例3
化合物8的合成
步骤1：
将化合物8-1(3.0g)溶于DCM(10ml)，室温搅拌，依次称取1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI，3.43g)，4-二甲氨基吡啶(DMAP，1.46g)和正庚醇(1.67g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与8-1标样对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物8-2(3.5g，84％收率)。
步骤2：
向化合物8-2(3.0g)和乙醇胺(780mg)的乙腈溶液(100mL)溶液中加入碳酸钾(3.6g)。将混合物在70℃搅拌3小时。TLC显示化合物8-2完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物8-3(1.7g，60％收率)。
步骤3：
将化合物8-4(3.0g)溶于DCM(40ml)，冰浴下搅拌，依次称取三苯基膦(2.45g)和四溴化碳(7.76g)分批加入反应体系，室温搅拌2h。有极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速30ml/min)得到无色油状液体化合物8-5(2.1g，84％收率)。
步骤4：
将化合物8-5(2.0g)溶于无水乙腈(50ml)，室温搅拌。然后称取硫代乙酸钾(1.43g)加入到上述反应体系中，在85℃下加热回流搅拌24h。将反应液减压浓缩后，所得固体用乙酸乙酯溶解后，加饱和食盐水洗涤三次，所得有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速10ml/min)得到粉色油状液体化合物8-6(1.6g，81％收率)。
步骤5：
将化合物8-6(1.5g)溶于甲醇(100ml)，室温搅拌。然后加入4.77mL甲醇钠的甲醇溶液(1M)到上述反应体系中，在氮气氛围下室温搅拌2h。反应液用安伯莱特离子交换树脂IR120中和后，过滤，洗涤，有机相浓缩。所得固体用乙酸乙酯溶解后，加饱和食盐水洗涤三次，所得有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速10ml/min)得到淡黄色油状液体化合物8-7(1.1g，81％收率)。
步骤6：
将化合物1-1(800mg)溶于DCM(40ml)，室温搅拌，依次称取苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU，1.6g)，N,N-二异丙基乙胺(DIEA，930mg)和8-7(1.1g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与1-1标样对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)得到无色油状液体化合物8-8(1.4g，82％收率)。
步骤7：
将化合物8-3(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(228mg)，K2CO3(420mg)和化合物8-8(870mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比8-3极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(25g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物8(600mg，54％收率)。
1H NMR(400MHz,Chloroform-d)δ3.60–3.55(m,1H),2.86–2.83(d,J＝7.6Hz,2H),2.81–2.77(m,2H),2.75–2.58(m,6H),2.49–2.44(m,2H),2.31–2.27(m,2H),1.95–1.48(m,16H),1.25(s,46H),0.86(d,J＝7.2Hz,9H)。
实施例4
化合物9的合成
步骤1：
向化合物1-4(2.0g)和乙醇胺(669mg)的乙腈溶液(100mL)溶液中加入碳酸钾(2.3g)。将混合物在70℃搅拌3小时。TLC显示化合物1-4完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物8-3(1.2g，63％收率)。
步骤2：
将化合物9-1(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(217mg)，K2CO3(400mg)和化合物8-8(830mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比9-1极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(15g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物2(700mg，65％收率)。
1H NMR(400MHz,Chloroform-d)δ4.01–3.96(m,1H),2.92-2.88(m,2H),2.81–2.75(m,2H),2.79–2.58(m,6H),2.57–2.40(m,2H),2.31–2.27(m,2H),1.95–1.48(m,14H),1.25(s,48H),0.86(d,J＝7.2Hz,9H)。
实施例5
化合物15的合成
步骤1：
将化合物15-1(3.0g)溶于DCM(40ml)，冰浴下搅拌，依次称取三苯基膦(2.76g)和四溴化碳(8.71g)分批加入反应体系，室温搅拌2h。有极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速30ml/min)得到无色油状液体化合物15-2(3.06g，80％收率)。
步骤2：
将化合物15-2(2.0g)溶于无水乙腈(50ml)，室温搅拌。然后称取硫代乙酸钾(1.57g)加入到上述反应体系中，在85℃下加热回流搅拌24h。将反应液减压浓缩后，所得固体用乙酸乙酯溶解后，加饱和食盐水洗涤三次，所得有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速10ml/min)得到粉色油状液体化合物15-3(1.49g，76％收率)。
步骤3：
将化合物15-3(1.5g)溶于甲醇(100ml)，室温搅拌。然后加入5.2mL甲醇钠的甲醇溶液(1M)到上述反应体系中，在氮气氛围下室温搅拌2h。反应液用安伯莱特离子交换树脂IR120中和后，过滤，洗涤，有机相浓缩。所得固体用乙酸乙酯溶解后，加饱和食盐水洗涤三次，所得有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速10ml/min)得到淡黄色油状液体化合物15-4(1.06g，83％收率)。
步骤4：
将化合物15-4(800mg)溶于DCM(40ml)，室温搅拌，依次称取苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU，1.79g)，N,N-二异丙基乙胺(DIEA，1.04g)和6-溴己酸(1.07g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与15-4标样对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)得到无色油状液体化合物15-5(1.09g，79％收率)。
步骤5：
将7-溴庚酸(3.0g)溶于DCM(10ml)，室温搅拌，依次称取1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI，3.15g)，4-二甲氨基吡啶(DMAP，1.6g)和化合物15-1(2.75g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与15-1标样对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物15-6(3.96g，72％收率)。
步骤6：
向化合物15-6(3.0g)和4-氨基-1-丁醇(1.78mg)的乙腈溶液(100mL)溶液中加入碳酸钾(2.77g)。将混合物在70℃搅拌3小时。TLC显示化合物15-6完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物15-7(1.59g，52％收率)。
步骤7：
将化合物15-7(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(175mg)，K2CO3(485mg)和化合物15-5(591mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比15-7极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(25g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物15(557mg，61％收率)。
1H NMR(400MHz,Chloroform-d)δ4.01–3.96(m,1H),2.85(d,J＝7.6Hz,4H),2.81–2.75(m,1H),2.70–2.58(m,6H),2.57–2.40(m,2H),1.95–1.48(m,16H),1.25(s,52H),0.86(d,J＝7.2Hz,12H)。
实施例6
化合物24的合成
步骤1：
向化合物1-4(2.0g)和1-(3-氨基丙基)哌啶(2.18g)的乙腈溶液(100mL)溶液中加入碳酸钾(2.12g)。将混合物在70℃搅拌3小时。TLC显示化合物1-4完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物24-1(1.42g，61％收率)。
步骤2：
将化合物24-1(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(176mg)，K2CO3(486mg)和化合物8-8(672mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比24-1极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(15g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物24(700mg，65％收率)。
1H NMR(400MHz,Chloroform-d)δ3.24(d,J＝3.2Hz,2H),3.01(d,J＝4.2Hz,2H),2.79–2.64(m,1H),2.53(d,J＝3.4Hz,2H),2.44(d,J＝3.2Hz,2H),2.33–2.21(m,10H),1.95–1.83(m,6H),1.64–1.51(m,16H),1.42–1.25(m,48H),0.91–0.88(m,9H)。
实施例7
化合物25的合成
步骤1：
向化合物15-6(3.0g)和3-二乙胺基丙胺(1.9g)的乙腈溶液(100mL)溶液中加入碳酸钾(2.97g)。将混合物在70℃搅拌3小时。TLC显示化合物15-6完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物25-1(1.8g，54％收率)。
步骤2：
将化合物25-1(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(160mg)，K2CO3(442mg)和化合物15-5(662mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比15-5极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(25g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物25(150mg，17％收率)。
1H NMR(400MHz,Chloroform-d)δ4.81–4.82(m,1H),3.89(d,J＝7.2Hz,4H),3.23–3.12(m,1H),3.02(d,J＝3.4Hz,2H),2.48(d,J＝7.2Hz,4H),2.36(d,J＝3.4Hz,2H),2.31(d,J＝3.2Hz,2H),2.07–1.86(m,4H),1.65–1.56(m,4H),1.53–1.43(m,6H),1.41–1.19(m,54H),1.04(d,J＝7.8Hz,6H),0.85(d,J＝12.4Hz,12H)。
实施例8
化合物28的合成
步骤1：
将化合物28-1(2.5g)溶于DCM(10ml)，室温搅拌，依次称取1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI，2.9g)，4-二甲氨基吡啶(DMAP，1.42g)和正庚醇(2.5g)分批加入反应体系，室温搅拌2h。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物28-2(3.1g，76％收率)。
步骤2：
向化合物28-2(3.0g)和乙醇胺(700mg)的乙腈溶液(100mL)溶液中加入碳酸钾(2.4g)。将混合物在70℃搅拌3小时。TLC显示化合物28-2完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物28-3(1.42g，75％收率)。
步骤3：
将化合物28-3(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(228mg)，K2CO3(630mg)和化合物8-8(1.1g)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比28-3极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(25g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物28(450mg，41％收率)。
1H NMR(400MHz,Chloroform-d)δ4.13(d,J＝3.2Hz,2H),3.45(d,J＝3.4Hz,2H),3.01(d,J＝7.2Hz,4H),2.87–2.76(m,1H),2.53(d,J＝3.2Hz,2H),2.40(d,J＝3.2Hz,2H),2.32(d,J＝3.2Hz,2H),1.98–1.88(m,4H),1.77–1.63(m,6H),1.54–1.44(m,6H),1.42–1.26(m,46H),0.88(d,J＝9.8Hz,9H)。
实施例9
化合物31的合成
步骤1：
将原料31-1(5g)溶于DCM(50ml)，滴加一滴DMF，用N2置换气体。然后称取SOCl2(4.14g)滴加到上述反应液中，室温搅拌3h。取少量反应液稀释与31-1标样对照点板(PE/EA＝10/1,磷钼酸)，观察到有比31-1极性小的新点，31-1剩余较少。反应液减压旋干后，用少量DCM溶解滴加到硫代乙酰胺(4.36g)的甲苯溶液(50mL)中，40℃加热搅拌3h后，往反应体系中滴加10％NaOH溶液(30ml)，40℃下加热搅拌过夜。取少量反应液稀释点板(PE/EA＝20/1,磷钼酸)，观察到些许拖尾的主点，且有紫外吸收。反应液用6M/L HCl调pH至3-5，用乙酸乙酯萃取反应液，有机相干燥后减压蒸发。加适量DCM和硅胶拌样过柱(PE,600ml)，点板和紫外灯检测，得到淡黄色油状液体31-2(3.02g，55％收率)。
步骤2：
向1,7-二溴庚烷(3.29g)的四氢呋喃溶液(40mL)中分批加入化合物31-2(2.0g)和碳酸钾(2.94g)。将混合物在45℃搅拌3小时。TLC显示化合物31-2完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物31-3(2.33g，60％收率)。
步骤3：
向化合物31-3(2.0g)和乙醇胺(1.0g)的乙腈溶液(40mL)溶液中加入碳酸钾(2.27g)。将混合物在70℃搅拌3小时。TLC显示化合物31-3完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、 纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物31-4(0.92g，49％收率)。
步骤4：
将化合物31-4(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(217mg)，K2CO3(600mg)和化合物8-8(829mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比31-4极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(25g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物31(647mg，60％收率)。
1H NMR(400MHz,Chloroform-d)δ3.63(d,J＝3.2Hz,2H),3.23(d,J＝3.4Hz,2H),3.06(d,J＝7.2Hz,4H),2.91–2.85(m,1H),2.57(d,J＝3.2Hz,2H),2.38(d,J＝7.6Hz,4H),2.07–1.86(m,6H),1.74–1.63(m,4H),1.59–1.46(m,4H),1.42–1.26(m,48H),0.87(d,J＝9.8Hz,9H)。
实施例10
化合物35的合成
步骤1：
将原料35-1(5g)溶于DCM(50ml)，滴加一滴DMF，用N2置换气体。然后称取 SOCl2(2.8g)滴加到上述反应液中，室温搅拌3h。取少量反应液稀释与35-1标样对照点板(PE/EA＝10/1,磷钼酸)，观察到有比35-1极性小的新点，35-1剩余较少。反应液减压旋干后，用少量DCM溶解滴加到硫代乙酰胺(2.2g)的甲苯溶液(50mL)中，40℃加热搅拌3h后，往反应体系中滴加10％NaOH溶液(30ml)，40℃下加热搅拌过夜。取少量反应液稀释点板(PE/EA＝20/1,磷钼酸)，观察到些许拖尾的主点，且有紫外吸收。反应液用6M/L HCl调pH至3-5，用乙酸乙酯萃取反应液，有机相干燥后减压蒸发。加适量DCM和硅胶拌样过柱(PE,600ml)，点板和紫外灯检测，得到淡黄色油状液体35-2(3.1g，58％收率)。
步骤2：
向1,6-二溴己烷(2.15g)的四氢呋喃溶液(40mL)中分批加入化合物35-2(2.0g)和碳酸钾(2.03g)。将混合物在45℃搅拌3小时。TLC显示化合物35-2完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物35-3(2.1g，65％收率)。
步骤3：
向化合物35-3(2.0g)和4-氨基-1-丁醇(1.15g)的乙腈溶液(40mL)溶液中加入碳酸钾(1.78g)。将混合物在70℃搅拌3小时。TLC显示化合物35-3完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物35-4(1.02g，50％收率)。
步骤4：
将化合物15-4(800mg)溶于DCM(40ml)，室温搅拌，依次称取苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU，1.81g)，N,N-二异丙基乙胺(DIEA，0.99g)和7-溴庚酸(1.03g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与35-4标样对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)得到无色油状液体化合物35-5(1.03g，61％收率)。
步骤5：
将化合物35-4(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(169mg)，K2CO3(467mg)和化合物35-5(589mg)分批加入到上述反应体系中，在85℃下加热 回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比35-4极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(25g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物35(620mg，68％收率)。
1H NMR(400MHz,Chloroform-d)δ3.51(d,J＝3.2Hz,2H),3.04(d,J＝9.8Hz,6H),2.86(d,J＝3.4Hz,2H),2.71–2.63(m,1H),2.46(d,J＝3.2Hz,2H),2.39–2.28(m,1H),2.01–1.86(m,6H),1.63–1.52(m,6H),1.42–1.26(m,56H),0.87(d,J＝12.8Hz,12H)。
实施例11
化合物40的合成
步骤1：
将化合物40-1(3.0g)溶于乙醇(50ml)，冰浴下搅拌，依次称取乙醇钠(EtONa，1.33g)，1-溴代庚烷(2.8g)分批加入反应体系，室温搅拌6h。反应液用饱和氯化铵溶液淬灭，乙酸乙酯萃取，所得有机相经无水硫酸钠干燥，加适量硅胶拌样、纯化(25 g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物40-2(2.0g，47％收率)。
步骤2：
向化合物40-2(2.0g)的DMSO溶液(30mL)溶液中加入氯化锂(2.1g)。将混合物在160℃搅拌10小时。反应液降至室温，反应液中加水淬灭，乙酸乙酯萃取，所得有机相经经饱和食盐水洗涤和无水硫酸钠干燥后，加适量硅胶拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物40-3(800mg，51％收率)。
步骤3：
将化合物40-3(800mg)溶于乙醇(10mL)，四氢呋喃(10ml)然后加入氢氧化锂(748mg)的水(10mL)溶液，室温下搅拌16h。加水和乙酸乙酯，水相用稀盐酸调节至pH＝2，乙酸乙酯萃取三遍，所得有机相经无水硫酸钠干燥，浓缩后得到无色油状液体化合物40-4(660mg，92％收率)。
步骤4：
将化合物40-4(1.0g)溶于DCM(20ml)，室温搅拌，依次称取1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI，1.1g)，4-二甲氨基吡啶(DMAP，535mg)和6-溴正己醇(873mg)分批加入反应体系，室温搅拌2h。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物40-5(1.3g，76％收率)。
步骤5：
向化合物40-5(1.2g)和4-氨基-1-丁醇(820mg)的乙腈溶液(100mL)溶液中加入碳酸钾(1.3g)。将混合物在70℃搅拌3小时。TLC显示化合物40-5完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物40-6(800mg，65％收率)。
步骤6：
将化合物40-7(3.0g)溶于DCM(40ml)，冰浴下搅拌，依次称取三苯基膦(2.76g)和四溴化碳(8.71g)分批加入反应体系，室温搅拌2h。有极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min， 5-5％5min，流速30ml/min)得到无色油状液体化合物40-8(2.6g，66％收率)。
步骤7：
将化合物40-8(2.0g)溶于无水乙腈(50ml)，室温搅拌。然后称取硫代乙酸钾(1.57g)加入到上述反应体系中，在85℃下加热回流搅拌24h。将反应液减压浓缩后，所得固体用乙酸乙酯溶解后，加饱和食盐水洗涤三次，所得有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速10ml/min)得到粉色油状液体化合物40-9(1.5g，76％收率)。
步骤8：
将化合物40-9(1.5g)溶于甲醇(100ml)，室温搅拌。然后加入6.0mL甲醇钠的甲醇溶液(1M)到上述反应体系中，在氮气氛围下室温搅拌2h。反应液用安伯莱特离子交换树脂IR120中和后，过滤，洗涤，有机相浓缩。所得固体用乙酸乙酯溶解后，加饱和食盐水洗涤三次，所得有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速10ml/min)得到淡黄色油状液体化合物40-10(950mg，76％收率)。
步骤9：
将化合物40-10(800mg)溶于DCM(40ml)，室温搅拌，依次称取苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU，1.79g)，N,N-二异丙基乙胺(DIEA，1.04g)和7-溴己酸(1.1g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与40-10标样对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速20ml/min)得到无色油状液体化合物40-11(1.20g，80％收率)。
步骤10：
将化合物40-6(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(188mg)，K2CO3(519mg)和40-11(817mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比40-6极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(25g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物40(600mg，66％收率)。
1H NMR(400MHz,Chloroform-d)δ4.01–3.96(m,1H),2.85(d,J＝7.6Hz,4H),2.81–2.75(m,1H),2.70–2.58(m,6H),2.57–2.40(m,2H),1.95–1.48(m,14H),1.25(s,46H),0.86(d,J＝7.2Hz,12H)。
实施例12
化合物43的合成
步骤1：
向化合物31-3(2.0g)和1-(3-氨基丙基)吡咯烷(2.11g)的乙腈溶液(100mL)溶液中加入碳酸钾(2.27g)，将混合物在70℃搅拌3小时。TLC显示化合物31-3完全消失，有一个极性变大的点生成。将反应液过滤，所得滤液浓缩后的粗产品，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物43-1(1.10g，49％收率)。
步骤2：
将化合物43-1(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(181mg)，K2CO3(502mg)和化合物8-8(695mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比43-1极性小的新点。反应液冷却至室温后减压蒸发，加适量DCM和硅胶拌样，纯化(15g正相柱，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)，浓缩得到淡黄色油状液体化合物43(300mg，31％收率)。
1H NMR(400MHz,Chloroform-d)δ3.24(d,J＝3.2Hz,2H),3.01(d,J＝4.2Hz,2H),2.79–2.64(m,1H),2.53(d,J＝3.4Hz,2H),2.44(d,J＝3.2Hz,2H),2.33–2.21(m,10H),1.95–1.83(m,6H),1.64–1.51(m,16H),1.42–1.25(m,46H),0.91–0.88(m,9H)。
实施例13纳米脂质颗粒制备以及性状表征
将荧光素酶mRNA(Luc mRNA)稀释于10-100mM，pH 4.0的柠檬酸缓冲液中；将各脂质组分(本发明所示阳离子脂质：DSPC：胆固醇：PEG脂质)按总浓度10mg/mL溶于乙醇。其中1.6％PEG脂质组使用的ALC-0159，1.5％PEG脂质组使用的DMG-PEG2000。
将3mL mRNA缓冲液和1mL脂质溶液分别装入两个5mL注射器，安装于微流控注射泵上，将芯片连接到注射器，设定注射泵流速，点击注射泵的开始按键，以流速比 3：1的方式注入芯片。观察芯片出口的产品颜色，弃去前5滴乳白色液滴(约为100μL)后，后端样品收集到EP管中。将收集到的产品放入透析袋中，隔10mM PBS(pH 7.4)透析6小时(截留分子量：100KDa)，随后超滤浓缩至理想浓度，再将脂质纳米颗粒经0.22μm无菌过滤器过滤，保存于4℃。
按照Ribogreen试剂盒说明，测试计算产品的包封率；于马尔文公司的Zetasizer nano仪器上使用标准检测方法进行粒径与多分散系数(PDI)检测、Zeta电位分析。
本实施例所制备得到的负载mRNA的LNP的粒径、PDI和包封率的检测结果如表1所示。结果表明，该配方下的脂质和mRNA形成的纳米颗粒包封率较高、粒径均一，为100nm左右，符合核酸递送载体的基本特征。
表1

*DLin-MC3-DMA为商业核酸递送系统Onpattro的阳离子脂质。
实施例14利用纳米脂质颗粒组合物递送荧光素酶mRNA在体内表达效果测定
在688周龄的雌性雌性Balb/c小鼠通过尾静脉注射含3ug mRNA的LUC-mRNA8脂质纳米颗粒(LUC-mRNA对应的核苷酸序列见专利申请202210286081.0的SEQ ID NO:1)，制备方法同实施例13。在特定时间点小鼠腹腔注射D-Luciferin Potassium Salt 200ug，使用勤翔小动物成像系统进行检测。Fluc通常用于哺乳动物细胞培养物中以测量基因表达和细胞活力。其在底物荧光素存在下发射出生物性光。所用到的mRNA的基本特征为ARCA帽结构，polyA尾长度为100-120nt，假尿嘧啶完全取代。检测结 果如图1，编号1-8的纳米脂质颗粒组合物递送mRNA至肝脏的水平优于MC3纳米脂质颗粒。
实施例15利用纳米脂质颗粒组合物递送促红细胞生成素(EPO)mRNA在小鼠内表达与效果测定
在688周龄的雌性Balb/c小鼠通过尾静脉注射10ug EPO-mRNA8脂质纳米颗粒(EPO-mRNA对应的核苷酸序列见专利申请202210286081.0的SEQ ID NO:2)，制备方法同实施例13，注射后48h对小鼠进行眼框取血，测定红细胞压积。hEPO通常用于哺乳动物血液中蛋白质表达水平的表征基因，其表达水平与红细胞压积成正比。所用到的mRNA的基本特征为ARCA帽结构，polyA尾长度为100-120nt，假尿嘧啶完全取代。检测结果如图2所示，由结果可知，多种本专利阳离子组成的纳米脂质颗粒组合物递送mRNA的水平优于DLin-MC3-DMA。
实施例16不同阳离子脂质的体内代谢速度
在688周龄的雌性Balb/c小鼠通过尾静脉注射5ug EPO-mRNA8脂质纳米颗粒，制备方法同实施例13，注射后按不同时间点(注射后、1h、3h、6h、24h、48h)处死，取肝脏破碎后，分三次用氯仿萃取其中脂质，最后合并萃取液，旋蒸去除氯仿，加入甲醇溶解，再用HPLC-CAD(Thermo Vanquish)分析其中阳离子脂质的含量。分析柱为AcclaimTM C18柱。流动相A相为0.5％TEAA水溶液，B相为0.5％TEAA甲醇溶液，样品按下表2的梯度洗脱。结果如图3。
表2
由结果可见，化合物9的体内代谢速度快与DLin-MC3-DMA，代表着它有更好的生物安全性。

Claims (27)

  1. 具有以下式(I)结构的用于递送核酸的阳离子脂质化合物：
    或其药物可接受的盐、互变异构体或立体异构体，其特征在于：
    L1为-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-OC(＝O)O-、-O-、-S-、-S-S-、-C(＝O)S-、-SC(＝O)-、-N(R6)C(＝O)-、-C(＝O)N(R6)-、-N(R6)C(＝O)O-、-OC(＝O)N(R6)-、-SC(＝O)N(R6)-、-N(R6)C(＝O)S-、-C(＝S)-、-SC(＝S)-、和-C(＝S)S-中任一种，所述R6为H或C1 8C12烷基；
    G1和G2各自独立地为C3-C10亚烷基；
    R1为C2-C12烷基；
    R2为H或C2-C12烷基；
    R3为C2-C12烷基；
    R4为H或C2-C12烷基；
    R5为H、C1-C6烷基、-R7-OH、-R7-OC(＝O)CH3、-R7-NHC(＝O)-CH3、-R7-OCH3或-R7-NR8(R9)；
    R7为C2-C18亚烷基；
    R8、R9为C1-C8的直链烷基，或者R8、R9和其连接的N原子形成C3-C10杂环烷基。
  2. 如权利要求1所述的阳离子脂质化合物，其特征在于，L1为-OC(＝O)-、-C(＝O)O-、-SC(＝O)-或-C(＝O)S-。
  3. 如权利要求2所述的阳离子脂质化合物，其特征在于，G1和G2为C3-C8亚烷基。
  4. 如权利要求3所述的阳离子脂质化合物，其特征在于，R7为C2-C6亚烷基。
  5. 如权利要求4所述的阳离子脂质化合物，其特征在于，R5为-R7-OH。
  6. 如权利要求5所述的阳离子脂质化合物，其特征在于，R1和R3各自独立地为C3-C9烷基；R2和R4为H或C3-C9烷基。
  7. 如权利要求6所述的阳离子脂质化合物，其特征在于，R2和R4仅有一个为H。
  8. 如权利要求1所述的阳离子脂质化合物，其特征在于，R5为-R7-N(CH2CH3)CH2CH3。
  9. 如权利要求5所述的阳离子脂质化合物，其特征在于，所述式(I)结构中的-C(R1)R2或-C(R3)R4结构各自独立地符合如下特征：
  10. 如权利要求1所述的阳离子脂质化合物，其特征在于，所述阳离子脂质化合物具有以下表中所示的结构之一：



  11. 包含权利要求1-10任意一项所述阳离子脂质化合物与预防性或治疗性核酸的脂质体制剂，其特征在于，所述制剂用于预防或者治疗某种疾病。
  12. 如权利要求11所述的脂质体制剂，其特征在于，所述核酸与所述化合物的摩尔比为20：1至1：1。
  13. 如权利要求12所述的脂质体制剂，其特征在于，所述核酸与所述化合物的摩尔比为10：1至4：1。
  14. 如权利要求11所述的脂质体制剂，其特征在于，该脂质体制剂的直径为50nm至300nm。
  15. 如权利要求14所述的脂质体制剂，其特征在于，该脂质体制剂的直径为50nm至150nm，或150nm至200nm。
  16. 如权利要求11所述的脂质体制剂，其特征在于，还包含一种或多种其他脂质组分，包括结构脂质、类固醇和聚合物缀合的脂质。
  17. 如权利要求16所述的脂质体制剂，其特征在于，所包含的类固醇为胆固醇。
  18. 如权利要求17所述的脂质体制剂，其特征在于，所述胆固醇与阳离子脂质化合物的摩尔比为(0-1.5)：1。
  19. 如权利要求11所述的脂质体制剂，其特征在于，聚合物缀合的脂质中的聚合物为聚乙二醇(PEG)。
  20. 如权利要求19所述的脂质体制剂，其特征在于，所述化合物与所述聚乙二醇缀合的脂质的摩尔比为100:1至20:1。
  21. 如权利要求19所述的脂质体制剂，其特征在于，所述聚乙二醇缀合的脂质为PEG8DAG、PEG8PE、PEG8SDAG、PEG8cer、PEG-DMG或ALC-0159。
  22. 如权利要求16所述的脂质体制剂，其特征在于，所述结构脂质选自DPPG、DSPC、DPPC、DMPC、DOPC、POPC、DOPE和DSPE中的一种或多种的组合。
  23. 如权利要求22所述的脂质体制剂，其特征在于，所述结构脂质与阳离子脂质化合物的摩尔比为(0-0.5)：1。
  24. 如权利要求11所述的脂质体制剂，其特征在于，所述核酸选自反义RNA和/或信使RNA。
  25. 如权利要求1-10任意一项所述的阳离子脂质化合物或权利要求11-24任意一项所述的脂质体制剂在制备用于在对象中诱导蛋白质表达的药物中的用途。
  26. 如权利要求25所述的用途，其特征在于，所述对象为哺乳动物。
  27. 如权利要求25所述的用途，其特征在于，所述对象是非人灵长类动物或人。
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