WO2023246074A1 - 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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WO2023246074A1
WO2023246074A1 PCT/CN2022/143764 CN2022143764W WO2023246074A1 WO 2023246074 A1 WO2023246074 A1 WO 2023246074A1 CN 2022143764 W CN2022143764 W CN 2022143764W WO 2023246074 A1 WO2023246074 A1 WO 2023246074A1
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compound
cationic lipid
independently
dcm
liposome preparation
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PCT/CN2022/143764
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English (en)
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 EP23826394.1A priority Critical patent/EP4378922A1/en
Priority to CN202380011678.7A priority patent/CN117529468A/zh
Priority to PCT/CN2023/101290 priority patent/WO2023246747A1/zh
Publication of WO2023246074A1 publication Critical patent/WO2023246074A1/zh
Priority to US18/625,544 priority patent/US20240360072A1/en

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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 present invention provides a class of sulfur-containing cationic lipid compounds, 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:
  • R 2 and R 3 are independently substituted or unsubstituted C 1 -C 18 linear alkylene or -R 9 -L 3 -R 10 -; the R 9 and R 10 are independently substituted or unsubstituted C 1 -C 10 linear alkylene group, L 3 is O or S;
  • R 4 , R 5 , R 6 and R 7 are independently hydrogen, or a substituted or unsubstituted C 1 -C 30 aliphatic hydrocarbon group, or -R 11 -L 4 -R 12 ; the R 11 and R 12 Each occurrence is independently a substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, and L 4 is O or S;
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 contain at least one O or S;
  • R 13 is C 1 -C 12 linear alkyl or branched alkyl
  • R 14 and R 15 are each independently H or C 1 -C 12 linear alkyl group, or R 14 and R 15 and the N atom to which they are connected form a C 3 -C 10 heterocycloalkyl group.
  • R 8 is H or C 1 -C 8 alkyl.
  • R 1 is -R 13 -OH or -R 13 -N(R 14 )(R 15 ), R 13 is C 1 -C 12 linear alkyl; R 14 and R 15 are each independently C 1 -C 12 linear alkyl;
  • R 2 is a C 1 -C 18 linear alkylene group
  • R 4 and R 5 are each independently a C 1 -C 30 aliphatic hydrocarbon group
  • R 3 is a C 1 -C 18 linear alkylene group
  • R 6 is methyl or ethyl
  • R 7 is -R 11 -L 4 -R 12 ;
  • R 11 and R 12 are independently substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon groups each time they appear, and L 4 is O or S.
  • R 13 is a C 1 -C 8 linear alkyl group or a branched alkyl group
  • R 14 and R 15 are each independently H or a C 1 -C 5 linear alkyl group
  • R 14 and R 15 and the N atom to which they are connected form a C 3 -C 8 heterocycloalkyl group.
  • R 2 and R 3 are independently substituted or unsubstituted C 1 -C 18 linear alkyl groups.
  • R 2 and R 3 are independently substituted or unsubstituted C 1 -C 12 linear alkyl.
  • R 4 , R 5 , R 6 and R 7 are independently hydrogen, or a substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, or -R 11 -L 4 - R 12 ; R 11 and R 12 are independently substituted or unsubstituted C 1 -C 10 aliphatic hydrocarbon groups each time they appear, L 4 is O or S; at least R 4 , R 5 , R 6 and R 7 Contains one O or S, and at most two are hydrogen.
  • R 4 , R 5 , R 6 and R 7 in the structure of formula (I) are each independently H or the following alkyl chain, or each is independently the following An ether or thioether formed by replacing any carbon atom in the alkyl chain with O or S:
  • R 1 is -R 13 -OH or -R 13 -N(R 14 )(R 15 ), and R 13 is a C 1 -C 5 linear alkyl group, preferably C 2 -C 4 linear alkyl; R 14 and R 15 are each independently C 1 -C 12 linear alkyl, preferably each independently is C 1 -C 3 linear alkyl;
  • R 2 is a C 2 -C 12 linear alkylene group, preferably a C 5 -C 9 linear alkyl group, and more preferably a C 5 -C 7 linear alkyl group;
  • R 4 and R 5 are each independently a C 3 -C 13 aliphatic hydrocarbon group, preferably a C 6 -C 10 linear alkyl group, and more preferably a C 6 -C 8 linear alkyl group;
  • R 3 is a C 2 -C 10 linear alkylene group, preferably a C 3 -C 7 linear alkyl group, and more preferably a C 5 -C 7 linear alkyl group;
  • R 6 is methyl or ethyl
  • R 7 is -R 11 -L 4 -R 12 ;
  • R 11 is C 1 -C 10 alkyl, preferably C 1 -C 3 alkyl;
  • R 12 is C 3 -C 13 alkyl, preferably C 6 -C 10 alkyl, more preferably C 6 -C 8 alkyl;
  • L 4 is O or S.
  • 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 neutral lipids, steroids, and polymer-conjugated lipids.
  • the steroid included is cholesterol.
  • the molar ratio of the cholesterol to the cationic lipid compound is (0-1.5):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-DAG, PEG-PE, PEG-SDAG, PEG-cer, PEG-DMG or ALC-0159.
  • the liposome preparation includes one or more neutral lipids selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.
  • the neutral lipid is DSPC or DOPE.
  • the molar ratio of the neutral lipid to the cationic lipid compound is (0-0.5):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 an ether bond or a thioether bond.
  • the introduction of the ether bond or the thioether 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. , less residue in the body.
  • the in vivo transfection efficiency of the selected cationic compounds was better than that of some commercial transfection cationic lipid compounds.
  • the preparation method of the amino 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 shows the relative fluorescence intensity of in vivo imaging of intramuscularly injected mice in Example 20;
  • Figure 2 is the relative fluorescence intensity of in vivo imaging of mice delivered by lung aerosol in Example 21;
  • Figure 3 is the neutralizing antibody titer of Example 22
  • Figure 4 is a liver and kidney function evaluation chart of Example 25.
  • 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) to obtain free Compound 2-4 was an oily liquid (2.8 g, 87% 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 (10g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 15ml/min) to obtain free Compound 3-4 was an oily liquid (2.6 g, 83% 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 (10g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 15ml/min) to obtain free Compound 4-4 was an oily liquid (2.6 g, 84% yield).
  • step 1
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • DMAP 4-dimethylaminopyridine
  • 5-bromo-1-pentanol 1.5g
  • 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) to obtain free Compound 6-4 was an oily liquid (2.5 g, 82% yield).
  • step 1
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • DMAP 4-dimethylaminopyridine
  • 5-bromo-1-pentanol 1.5g
  • 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) to obtain free Compound 7-4 was an oily liquid (2.5 g, 74% yield).
  • step 1
  • 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) to obtain free Compound 17-4 was an oily liquid (2.8 g, 74% yield).
  • reaction mixture was concentrated to obtain a crude product, which was purified by column chromatography (silica gel column, the eluent was petroleum ether solution containing 0-1% EA (volume percentage)), and the pure product fraction was evaporated to obtain compound 44 -6 (2.4g, 74% yield).
  • step 1
  • 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 29-3 (1.45 g, 95% yield) as a colorless oily liquid.
  • step 1
  • reaction mixture was concentrated to obtain a crude product, which was purified by column chromatography (silica gel column, the eluent was petroleum ether solution containing 0-1% EA (volume percent)), and the pure product fraction was evaporated to obtain compound 30 -3 (1.54g, 57% 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 (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 fraction was evaporated to obtain compound 35-3 (1.30 g, 79.2% yield) as a colorless oily liquid.
  • 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 36-3 (1.20 g, 74.7% yield) as a colorless oily liquid.
  • step 1
  • Dissolve compound 41-2 1000mg) in DCM (10ml), stir at room temperature, and weigh out 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.05g) in sequence.
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • 4-dimethylaminopyridine DMAP, 560 mg
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (15g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 15ml/min) to obtain free Compound 41-3 was an oily liquid (1.5 g, 77% yield).
  • step 1
  • reaction mixture was concentrated to obtain a crude product, which was purified by column chromatography (silica gel column, the eluent was petroleum ether solution containing 0-1% EA (volume percentage)), and the pure product fraction was evaporated to obtain compound 44 -3 (1.65g, 62% yield).
  • reaction mixture was concentrated to obtain a crude product, which was purified by column chromatography (silica gel column, the eluent was petroleum ether solution containing 0-1% EA (volume percentage)), and the pure product fraction was evaporated to obtain compound 44 -6 (2.40g, 74% yield).
  • step 1
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • DMAP 4-dimethylaminopyridine
  • 6-bromo-n-hexanol 792 mg
  • 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 46-4 (1.6 g, 84% yield) as a colorless oily liquid.
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • DMAP 4-dimethylaminopyridine
  • 6-bromo-n-hexanol (1.41 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 15ml/min), click Plate monitoring was used, and part of the pure product was evaporated to obtain compound 46-7 (2.8 g, 86% yield) as a colorless oily liquid.
  • step 1
  • step 1
  • reaction solution was filtered, and the obtained crude product after the filtrate was concentrated was added with an appropriate amount of silica gel and DCM to mix and purify (25g normal phase column, 0.1% NH 3 H 2 O, MeOH/DCM, 0-0% 5min, 0-10% 20min, 10-10% 5min, flow rate 20ml/min) to obtain colorless oily liquid compound 55-1 (1.3g, 32% yield).
  • reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added to mix and purify (15g normal phase column, PE/EA, 0-0% 5min, 0-5% 20min, 5-5% 5min, flow rate 15mL/min) to obtain free Compound 55-4 was an oily liquid (1.27 g, 70% yield).
  • step 1
  • Luciferase 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 (DMG-PEG2000)) is divided into moles. Ratio 50:10:38.5:1.5 is soluble in ethanol.
  • 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.
  • *DLin-MC3-DMA is the cationic lipid of the commercial nucleic acid delivery system Onpattro.
  • Example 20 Determination of the expression effect of luciferase mRNA delivered in vivo using tail vein injection of nanolipid particle composition
  • mice BALB/c mice aged 6-8 weeks were injected with LUC-mRNA-lipid nanoparticles containing 5 ⁇ g of mRNA through the tail vein (for the nucleotide sequence corresponding to LUC-mRNA, see SEQ ID NO: 1 of the patent publication CN114380724A), The preparation method is the same as Example 19.
  • mice were injected with 100 ⁇ g of D-Luciferin Potassium Salt into the tail vein, and detected using the PerkinElmer small animal imaging system. Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability, and it emits biological fluorescence 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.
  • the test results are shown in Figure 1, in which the nanolipid particle composition composed of the compounds designed in the present invention is mostly equivalent to or better than DLin-MC3-DMA in delivering mRNA to the liver; some compounds are better than Lipid M.
  • Example 21 Determination of the in vivo expression effect of luciferase mRNA delivered by pulmonary aerosolization using nanolipid particle compositions
  • LUC-mRNA-lipid nanoparticles containing 5 ⁇ g of mRNA were delivered to the lungs of BALB/c mice aged 6-8 weeks through aerosolization.
  • the preparation method was the same as in Example 19.
  • mice were injected with 100 ⁇ g of D-Luciferin Potassium Salt into the tail vein, and detected using the PerkinElmer small animal imaging system.
  • the test results are shown in Figure 2, in which the nanolipid particle composition composed of compounds 7 and 35 delivers better fluorescence levels of mRNA expression than Lipid M and SM-102.
  • 6-week-old BALB/c mice were used to intramuscularly inject the mRNA COVID-19 vaccine (Omicron antigen mRNA) delivered by different nanolipid particle compositions on days 0 and 14.
  • the corresponding nucleotide sequence can be found in the SEQ ID NO of the patent publication CN114380724A. :6)
  • Immunize collect blood on day 28 (day 14 after secondary immunization), measure the neutralizing antibody titer by enzyme-linked immunosorbent assay, and evaluate the effect of the mRNA new coronavirus vaccine delivered by different nanolipid particle compositions on SARS-CoV Protective effect against infection by -2 viral strains.
  • the results are shown in Figure 3. From the results, it can be seen that the neutralizing antibody titer caused by the delivery of COVID-19 mRNA by the nanolipid particle composition composed of Compound 35 is 1.4 million, while that of Lipid M is about 930,000.
  • the above compounds have high encapsulation efficiency, uniform particle size, and the efficiency of delivering Luciferase mRNA in vivo is much higher than that of the commercial lipid DLin-MC3-DMA.
  • EPO blood-stimulating erythropoietin
  • the preparation method of nanoparticles is the same as in Example 19.
  • the nucleotide sequence corresponding to EPO mRNA is shown in CN114380724B, SEQ ID NO: 2.
  • 6-8 week old female Balb/c mice were injected with 20 ⁇ g EPO-mRNA-lipid nanoparticles through the tail vein.
  • blood was taken from the eye box of the mice, and the serum was centrifuged to separate, and ELISA was used.
  • 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 preparation and protein detection results are shown in Table 4.
  • the above compounds have high encapsulation efficiency, uniform particle size, and the protein translation efficiency of EPO mRNA delivered in vivo is much higher than that of the commercial lipid DLin-MC3-DMA.
  • Example 25 the nanoparticles prepared in Example 25 were used.
  • Female SD rats weighing 200 to 250 g were used and injected into the tail vein at a dose of 5 mg/kg.
  • the mice in the control group were injected with a corresponding volume of physiological saline.
  • the mice in the DLin-MC3-DMA group died within 18 hours after injection, and the weight, eating, and activity status of the remaining mice showed no abnormalities during the observation period. This shows that the toxic and side effects of DLin-MC3-DMA lipid are stronger.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • BUN urea nitrogen
  • SCR creatinine

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Abstract

本发明提供了用于递送核酸的阳离子脂质化合物和组合物及用途。所述化合物如下式(I)所示。本发明还提供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：
Figure PCTCN2022143764-appb-000001
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：
L 1、L 2为连接键或二价连接基，所述二价连接基各自独立地选自-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-OC(＝O)O-、-O-、-S-、-S-S-、-C(＝O)S-、-SC(＝O)-、-N(R 8)C(＝O)-、-C(＝O)N(R 8)-、-N(R 8)C(＝O)O-、-OC(＝O)N(R 8)-、-SC(＝O)N(R 8)-、-N(R 8)C(＝O)S-、-C(＝S)-、-SC(＝S)-、和-C(＝S)S-中任一种，所述R 8为H或C 1-C 12烷基；
R 2和R 3独立地为取代或未取代的C 1-C 18直链亚烷基或者-R 9-L 3-R 10-；所述R 9和R 10独立地为取代或未取代的C 1-C 10直链亚烷基，L 3为O或者S；
R 4，R 5，R 6和R 7独立地为氢、或者是取代或未取代的C 1-C 30脂肪烃基，或者是-R 11-L 4-R 12；所述R 11和R 12每次出现时独立地为取代或未取代的C 1-C 18脂肪烃基，L 4为O或者S；
上述R 2，R 3，R 4，R 5，R 6和R 7中至少含有一个O或者S；
R 1为H、-R 13、-OR 13、-R 13-OH、-R 13-OR 14、-R 13-OC(＝O)R 14、-R 13-NHC(＝O)-R 14、-R 13-OCH 3或-R 13-N(R 14)(R 15)；R 13为C 1-C 12直链烷基或支链烷基，R 14和R 15各自独立的分别为H或C 1-C 12直链烷基，或者，R 14与R 15和其连接的N原子形成C 3-C 10杂环烷基。
根据本发明一些具体实施方案，其中，R 8为H或C 1-C 8烷基。
根据本发明一些具体实施方案，其中，R 1为-R 13-OH或-R 13-N(R 14)(R 15)，R 13为C 1-C 12直链烷基；R 14和R 15各自独立的分别为C 1-C 12直链烷基；
R 2为C 1-C 18直链亚烷基；
L 1为-C(＝O)S-、-SC(＝O)-、-OC(＝O)-或-C(＝O)O-；
R 4和R 5各自独立的为C 1-C 30脂肪烃基；
R 3为C 1-C 18直链亚烷基；
L 2为-C(＝O)S-、-SC(＝O)-、-OC(＝O)-或-C(＝O)O-；
R 6为甲基或者乙基；
R 7为-R 11-L 4-R 12；所述R 11和R 12每次出现时独立地为取代或未取代的C 1-C 18脂肪烃基，L 4为O或者S。
根据本发明一些具体实施方案，其中，R 13为C 1-C 8直链烷基或支链烷基，R 14和R 15各自独立的分别为H或C 1-C 5直链烷基，或者，R 14与R 15和其连接的N原子形成C 3-C 8杂环烷基。
根据本发明一些具体实施方案，其中，R 2和R 3独立地为取代或未取代的C 1-C 18直链烷基。
根据本发明一些具体实施方案，其中，R 2和R 3独立地为取代或未取代的C 1-C 12直链烷基。
根据本发明一些具体实施方案，其中，R 4，R 5，R 6和R 7独立地为氢、或者是取代或未取代的C 1-C 18脂肪烃基，或者是-R 11-L 4-R 12；所述R 11和R 12每次出现时独立地为取代或未取代的C 1-C 10脂肪烃基，L 4为O或者S；R 4，R 5，R 6和R 7中至少含有一个O或者S，至多有两个为氢。
根据本发明一些具体实施方案，其中，所述式(I)结构中的R 4，R 5，R 6和R 7结构各自独立地为H或下述烷基链，或各自独立地为下述烷基链上任意一个碳原子被O或者S替代而形成的醚或者硫醚：
Figure PCTCN2022143764-appb-000002
根据本发明一些具体实施方案，其中，R 1为-R 13-OH或-R 13-N(R 14)(R 15)，R 13为C 1-C 5直链烷基，优选为C 2-C 4直链烷基；R 14和R 15各自独立的分别为C 1-C 12直链烷基，优选各自独立的分别为C 1-C 3直链烷基；
R 2为C 2-C 12直链亚烷基，优选为C 5-C 9直链烷基，更优选为C 5-C 7直链烷基；
L 1为-C(＝O)S-、-SC(＝O)-、-OC(＝O)-或-C(＝O)O-；
R 4和R 5各自独立的为C 3-C 13脂肪烃基，优选为C 6-C 10直链烷基，更优选为C 6-C 8直链烷基；
R 3为C 2-C 10直链亚烷基，优选为C 3-C 7直链烷基，更优选为C 5-C 7直链烷基；
L 2为-C(＝O)S-、-SC(＝O)-、-OC(＝O)-或-C(＝O)O-；
R 6为甲基或者乙基；
R 7为-R 11-L 4-R 12；所述R 11为C 1-C 10烷基，优选为C 1-C 3烷基；R 12为C 3-C 13烷基，优选 为C 6-C 10烷基，更优选为C 6-C 8烷基；L 4为O或者S。
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：
Figure PCTCN2022143764-appb-000003
Figure PCTCN2022143764-appb-000004
Figure PCTCN2022143764-appb-000005
Figure PCTCN2022143764-appb-000006
Figure PCTCN2022143764-appb-000007
Figure PCTCN2022143764-appb-000008
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，包括但不限于中性脂质、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(0-1.5)：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PEG-DAG、PEG-PE、PEG-SDAG、PEG-cer、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DSPC、DPPC、DMPC、DOPC、POPC、DOPE和SM中的一种或多种中性脂质。
根据本发明一些具体实施方案，其中，所述中性脂质为DSPC或DOPE。
根据本发明一些具体实施方案，其中，所述中性脂质与所述阳离子脂质化合物的摩尔比为(0-0.5)：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醚键或者硫醚键，醚键或者硫醚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。而且经过结构优化后，筛选出的阳离子化合物的体内转染效率优于部分商 业转染阳离子脂质化合物。且所述氨基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20的肌肉注射小鼠活体成像相对荧光强度；
图2为实施例21的肺部雾化递送小鼠活体成像相对荧光强度；
图3为实施例22的中和抗体滴度；
图4为实施例25的肝肾功能评价图。
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2的合成
Figure PCTCN2022143764-appb-000009
步骤1：
向化合物2-1(3.00g)的叔丁醇(20mL)溶液中依次加入1-癸醇(3.23g)和碳酸铯(11.1g)。溶液在室温下搅拌4小时后，点板(石油醚：乙酸乙酯＝10：1)显示有新点生成。将反应液过滤，所得滤液浓缩后的粗产品通过柱色谱法(硅胶柱，洗脱液为含0-10％乙酸乙酯(体积百分比)的石油醚溶液)纯化得到化合物2-2(3.93g，69％收率)。
步骤2：
向化合物2-2(3.00g)的THF(20mL)和水的溶液中加入氢氧化锂(860mg)，将混合物在60℃搅拌16小时。TLC显示有极性变大的点生成。将反应混合物浓缩去除四氢呋喃，加水稀释后，用乙酸乙酯(30mL)萃取1次，将水相用稀盐酸调节pH＝2，用乙酸乙酯(30 mL)萃取2次，合并有机层，浓缩得到化合物2-3(2.10g，95％收率)。
步骤3：
将化合物2-3(2.0g)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，2.0g)，4-二甲氨基吡啶(DMAP，1.3g)和5-溴-1-戊醇(1.5g)分批加入反应体系，室温搅拌3h。取少量反应液稀释与2-3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)得到无色油状液体化合物2-4(2.8g，87％收率)。
步骤4：
将化合物2-5(5.0g)溶于二氯甲烷(70ml)，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，4.48g)，4-二甲氨基吡啶(DMAP，3.57g)和8-溴辛酸(4.78g)分批加入反应体系，室温搅拌3h。取少量反应液稀释与2-5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(60g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2-6(8.0g，88.9％收率)。下述实施例1-6，8-10，11-13，16-18的2-6化合物均采用此方法合成。
步骤5：
向化合物2-6(8.0g)和乙醇胺(1.59g)的乙腈溶液(50mL)溶液中加入碳酸钾(7.19g)。将混合物在70℃搅拌2小时。TLC显示化合物2-6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2-7(4.2g，54.9％收率)。
步骤6：
将化合物2-4(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(191mg)，K 2CO 3(527mg)和化合物2-7(673mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。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2-7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2(700mg，73％收率)。
1H NMR(400MHz,Chloroform-d)δ4.80(s,1H),4.16–4.01(d,J＝3.2Hz,2H),3.75–3.45(m,6H),2.78–2.68(dd,J＝8.2,5.8Hz,2H),2.61–2.51(m,2H),2.50–2.45(m,4H),2.31–2.16(m,2H),1.70–1.69(s,1H),1.68–1.66(s,1H),1.58–1.57(s,2H),1.57–1.55(d,J＝3.4 Hz,2H),1.55–1.51(m,6H),1.50–1.48(s,2H),1.38–1.35(d,J＝1.0Hz,4H),1.35–1.30(m,20H),1.30–1.27(m,20H),1.19–1.17(m,3H),0.91–0.88(m,9H)。
实施例2
化合物3的合成
Figure PCTCN2022143764-appb-000010
步骤1：
向化合物3-1(3.00g)的叔丁醇(20mL)溶液中依次加入1-癸醇(4.45g)和碳酸铯(15.3g)。混合物在室温下搅拌4小时后，点板(石油醚：乙酸乙酯＝10：1)显示有新点生成。将反应液过滤，所得滤液浓缩后的粗产品，将粗产品通过柱色谱法(硅胶柱，洗脱液为含0-10％乙酸乙酯(体积百分比)的石油醚溶液)纯化得到化合物3-2(3.1g，46％收率)。
步骤2：
向化合物3-2(3.00g)的THF(20mL)和水的溶液中加入氢氧化锂(860mg)将混合物在60℃搅拌16小时。TLC(石油醚：乙酸乙酯＝10：1)显示有极性变大的点生成。将反应混合物浓缩去除四氢呋喃，加水稀释后，用乙酸乙酯(30mL)萃取1次，将水相用稀盐酸调节pH＝2，用乙酸乙酯(30mL)萃取2次，合并有机层，浓缩得到化合物3-3(2.50g，92％收率)。
步骤3：
将化合物3-3(2.0g)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，2.0g)，4-二甲氨基吡啶(DMAP，1.3g)和5-溴-1-戊醇(1.5g)分批加入反应体系，室温搅拌3h。取少量反应液稀释与3-3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)得到无色油状液体化合物3-4(2.6g，83％收率)。
步骤4：
将化合物3-4(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(191mg)，K 2CO 3(527mg)和化合物2-7(673mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。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2-7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3(750mg，80％收率)。
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.14–4.02(d,J＝2.6Hz,2H),3.73–3.46(m,6H),2.61(s,1H),2.59–2.50(m,2H),2.49–2.45(m,4H),2.29–2.18(m,2H),1.71–1.63(m,4H),1.60–1.50(m,12H),1.49(s,2H),1.38–1.36(d,J＝0.6Hz,4H),1.35–1.30(m,20H),1.30–1.26(m,20H),0.92(s,3H),0.91–0.87(s,9H)。
实施例3
化合物4的合成
Figure PCTCN2022143764-appb-000011
步骤1：
向化合物4-1(3.00g)的叔丁醇(20mL)溶液中依次加入1-癸醇(4.6g)和碳酸铯(16.1g)。混合物在室温下搅拌4小时后，点板(石油醚：乙酸乙酯＝10：1)显示有新点生成。将反应液过滤，所得滤液浓缩后的粗产品，将粗产品通过柱色谱法(硅胶柱，洗脱液为含0-10％乙酸乙酯(体积百分比)的石油醚溶液)纯化得到化合物4-2(3.0g，47.3％收率)。
步骤2：
向化合物4-2(3.00g)的THF(20mL)和水的溶液中加入氢氧化锂(860mg)将混合物在60℃搅拌16小时。TLC(石油醚：乙酸乙酯＝10：1)显示有极性变大的点生成。将反应混合物浓缩去除四氢呋喃，加水稀释后，用乙酸乙酯(30mL)萃取1次，将水相用稀盐酸 调节pH＝2，用乙酸乙酯(30mL)萃取2次，合并有机层，浓缩得到化合物4-3(2.50g，92％收率)。
步骤3：
将化合物4-3(2.0g)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，2.1g)，4-二甲氨基吡啶(DMAP，1.4g)和5-溴-1-戊醇(1.6g)分批加入反应体系，室温搅拌3h。取少量反应液稀释与3-3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)得到无色油状液体化合物4-4(2.6g，84％收率)。
步骤4：
将化合物4-4(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(191mg)，K 2CO 3(527mg)和化合物2-7(673mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。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2-7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％(甲醇在DCM/MeOH溶液中的体积百分含量，后同)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4(610mg，65.7％收率)。
1H NMR(400MHz,Chloroform-d)δ4.80–4.74(s,1H),4.15–4.01(d,J＝2.6Hz,2H),3.73–3.44(m,6H),2.56–2.50(m,3H),2.49–2.45(m,4H),2.29–2.18(m,2H),1.71–1.47(m,18H),1.41–1.26(m,46H),0.96–0.92(m,3H),0.99(s,9H)。
实施例4
化合物6的合成
Figure PCTCN2022143764-appb-000012
步骤1：
向化合物6-1(19.0g)的叔丁醇(20mL)溶液中依次加入1-癸醇(3.0g)和碳酸铯(12.4g)。混合物在室温下搅拌4小时后，点板(石油醚：乙酸乙酯＝10：1)显示有新点生成。将反应液过滤，所得滤液浓缩后的粗产品，将粗产品通过柱色谱法(硅胶柱，洗脱液为含0-10％乙酸乙酯(体积百分比)的石油醚溶液)纯化得到化合物6-2(2.1g，43％收率)。
步骤2：
向化合物6-2(2.1g)的THF(20mL)和水(10mL)的溶液中加入氢氧化锂(584mg) 将混合物在60℃搅拌16小时。TLC(石油醚：乙酸乙酯＝10：1)显示有极性变大的点生成。将反应混合物浓缩去除四氢呋喃，加水稀释后，用乙酸乙酯(30mL)萃取1次，水相用稀盐酸调节pH＝2，用乙酸乙酯(30mL)萃取2次，合并有机相，浓缩得到化合物6-3(1.6g，85％收率)。
步骤3：
将化合物6-3(2.0g)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，2.0g)，4-二甲氨基吡啶(DMAP，1.3g)和5-溴-1-戊醇(1.5g)分批加入反应体系，室温搅拌3h。取少量反应液稀释与6-3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)得到无色油状液体化合物6-4(2.5g，82％收率)。
步骤4：
将化合物6-4(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(191mg)，K 2CO 3(527mg)和化合物2-7(673mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。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2-7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6(742mg，75％收率)。
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.11(s,2H),3.68–3.57(d,J＝5.0Hz,4H),3.55–3.46(s,2H),2.61–2.43(m,8H),2.28–2.17(s,2H),1.73–1.63(d,J＝3.9Hz,4H),1.60–1.46(m,12H),1.39–1.23(m,40H),0.96–0.84(s,9H)。
实施例5
化合物7的合成
Figure PCTCN2022143764-appb-000013
步骤1：
向化合物2-1(3.00g)的叔丁醇(20mL)溶液中依次加入辛醇(3.1g)和碳酸铯(11.0g)。溶液在室温下搅拌4小时后，点板(石油醚：乙酸乙酯＝10：1)显示有新点生成。将反应液过滤，所得滤液浓缩后的粗产品通过柱色谱法(硅胶柱，洗脱液为含0-10％乙酸乙酯(体积百分比)的石油醚溶液)纯化得到化合物7-2(3.6g，69％收率)。
步骤2：
向化合物7-2(3.00g)的THF(20mL)和水的溶液中加入氢氧化锂(860mg)将混合 物在60℃搅拌16小时。TLC显示有极性变大的点生成。将反应混合物浓缩去除四氢呋喃，加水稀释后，用乙酸乙酯(30mL)萃取1次，将水相用稀盐酸调节pH＝2，用乙酸乙酯(30mL)萃取2次，合并有机层，浓缩得到化合物7-3(2.0g，94％收率)。
步骤3：
将化合物7-3(2.0g)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，2.0g)，4-二甲氨基吡啶(DMAP，1.3g)和5-溴-1-戊醇(1.5g)分批加入反应体系，室温搅拌3h。取少量反应液稀释与7-3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)得到无色油状液体化合物7-4(2.5g，74％收率)。
步骤4：
将化合物7-4(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(191mg)，K 2CO 3(527mg)和化合物2-7(673mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。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2-7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7(750mg，75％收率)。
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.09(d,J＝2.2Hz,2H),3.78–3.37(m,6H),2.75(s,1H),2.55(d,J＝3.8Hz,2H),2.49–2.43(m,4H),2.30–2.16(m,2H),1.71–1.65(m,2H),1.64(s,2H),1.60–1.50(m,10H),1.49(s,2H),1.37(d,J＝0.6Hz,4H),1.35–1.30(m,20H),1.30–1.26(m,16H),1.22–1.14(m,3H),0.99(s,9H)。
实施例6
化合物14的合成
Figure PCTCN2022143764-appb-000014
步骤1：
向化合物14-1(3.0g)和三乙胺(5.3g)的二氯甲烷(50mL)溶液中在冰浴条件下加入对甲苯磺酰氯(7.4g)。将混合物室温下搅拌3小时后，将反应混合物用DCM(30mL)稀释，并用稀盐酸和盐水(100mL)洗涤。合并有机层经Na 2SO 4干燥，并真空除去溶剂，得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-10％EA(体积百分比)的正己烷溶液)纯化得到化合物14-2(6.0g，86％收率)。
步骤2：
冰浴条件下，向叔丁基二甲基羟乙氧基硅烷(2.0g)的DMF(20mL)溶液中加入NaH(680mg,60％)，将混合物0℃下搅拌半小时后，将14-2缓缓加入溶液中，溶液在80℃下搅拌两小时。待溶液降至室温后，加入饱和氯化铵溶液淬灭，乙酸乙酯萃取，合并有机层经Na 2SO 4干燥，并真空除去溶剂，得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-10％EA(体积百分比)的正己烷溶液)纯化得到化合物14-3(2.5g，80％收率)。
步骤3：
冰浴条件下，向14-3(2.5g)的无水四氢呋喃(20mL)溶液中加入1M TBAF溶液，将溶液升至室温，在室温下搅拌两小时，加入饱和氯化铵溶液，加水稀释后，乙酸乙酯萃取，合并有机层经Na 2SO 4干燥，并真空除去溶剂，得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-60％EA(体积百分比)的正己烷溶液)纯化得到化合物14-4(1.4g，96％收率)。
步骤4：
向化合物14-4(1.4g)的DCM(20mL)溶液中依次加入4-二甲氨基吡啶(DMAP，1.07g)，7-溴庚酸(2.01g)和1-乙基-(3-二甲基氨基丙基)(EDCl，2.01g)，反应混合物25℃搅拌12小时，将反应混合物用DCM(30mL)稀释，并用饱和NaHCO 3(100mL)和盐水(100mL)洗涤。合并有机层经Na 2SO 4干燥，并真空除去溶剂，得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-1％EA(体积百分比)的正己烷溶液)纯化，并将纯产物馏分蒸发，得到化合物14-5(2.10g，68％收率)。
步骤5：
将化合物14-5(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(191mg)，K 2CO 3(527mg)和化合物2-7(673mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。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2-7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14(820mg，81％收率)。
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.21(s,2H),3.64(d,J＝5.0Hz,2H),3.60(s,2H),3.53(s,2H),2.55(s,2H),2.48(s,4H),2.24(s,4H),1.71–1.65(m,4H),1.58(d,J＝6.4Hz,4H),1.53(s,4H),1.51(s,2H),1.38–1.35(m,6H),1.35–1.33(m,8H),1.32(d,J＝1.0Hz,4H),1.32–1.31(m,10H),1.29(s,4H),1.29(s,2H),1.29–1.27(m,8H),0.97–0.82(m,9H).
实施例7
化合物17的合成
Figure PCTCN2022143764-appb-000015
步骤1：
向化合物2-1(5.00g)的叔丁醇(40mL)溶液中依次加入正己醇(2.90g)和碳酸铯(27.8g)。溶液在室温下搅拌4小时后，点板(石油醚：乙酸乙酯＝10：1)显示有新点生成。将反应液过滤，所得滤液浓缩后的粗产品通过柱色谱法(硅胶柱，洗脱液为含0-10％乙酸乙酯(体积百分比)的石油醚溶液)纯化得到化合物17-2(3.14g，39.8％收率)。
步骤2：
向化合物17-2(3.00g)的THF(20mL)和水(20mL)的溶液中加入氢氧化锂(1.03g)将混合物在60℃搅拌16小时。TLC显示有极性变大的点生成。将反应混合物浓缩去除四氢呋喃，加水稀释后，用乙酸乙酯(30mL)萃取1次，将水相用稀盐酸调节pH＝2，用 乙酸乙酯(30mL)萃取2次，合并有机层，浓缩得到化合物17-3(1.80g，88.7％收率)。
步骤3：
将化合物17-3(2.0g)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，3.1g)，4-二甲氨基吡啶(DMAP，2.0g)和7-溴-1-庚醇(2.3g)分批加入反应体系，室温搅拌3h。取少量反应液稀释与2-3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)得到无色油状液体化合物17-4(2.8g，74％收率)。
步骤4：
向化合物44-5(2.00g)的DCM(15mL)溶液中依次加入4-二甲氨基吡啶(DMAP，200mg)和7-溴-1-庚醇(1.51g)。混合物在25℃搅拌5分钟后，加入1-乙基-(3-二甲基氨基丙基)(EDCl，1.62g)，反应混合物25℃搅拌1小时。TLC显示起始化合物44-5完全消失。将反应混合物浓缩得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-1％EA(体积百分比)的石油醚溶液)纯化，并将纯产物馏分蒸发，得到化合物44-6(2.4g，74％收率)。
步骤5：
向化合物44-6(2.0g)和乙醇胺(530mg)的乙腈溶液(50mL)溶液中加入碳酸钾(1.80g)。将混合物在70℃搅拌3小时。TLC显示化合物44-6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17-5(1.0g，53.8％收率)。
步骤6：
将化合物17-4(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(191mg)，K 2CO 3(527mg)和化合物17-5(673mg)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17-5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17(725mg，70％收率)。
1H NMR(400MHz,Chloroform-d)δ4.15–4.03(m,4H),3.56–3.45(m,6H),2.75(s,1H),2.55(d,J＝3.8Hz,2H),2.48(d,J＝1.0Hz,4H),2.29(s,1H),1.65(s,4H),1.50–1.46(m,10H),1.40–1.38(s,4H),1.36(d,J＝0.6Hz,2H),1.34(d,J＝0.6Hz,4H),1.32(d,J＝1.0Hz,10H),1.32–1.30(m,10H),1.28(d,J＝1.2Hz,12H),1.18(s,3H),0.94–0.84(m,9H).
实施例8
化合物28的合成
Figure PCTCN2022143764-appb-000016
步骤1：
向化合物2-6(2.0g)和N,N-二乙基乙二胺(755mg)的乙腈溶液(50mL)溶液中加入碳酸钾(1.2g)。将混合物在70℃搅拌3小时。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，DCM/MeOH，0.1％氨水，0-0％10min，0-7.5％20min，7.5-7.5％5min，流速25ml/min)得到无色油状液体化合物28-1(900mg，30％收率)。
步骤2：
将化合物28-1(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(146mg)，K 2CO 3(406mg)和化合物7-4(425mg)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)，观察到有新点生成。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7(94mg，11％收率)。
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.09(d,J＝2.2Hz,2H),3.73–3.60(m,4H),2.75(s,1H),2.69–2.57(m,8H),2.51–2.40(m,4H),2.31–2.17(m,2H),1.72–1.65(m,4H),1.60–1.50(m,10H),1.48(s,2H),1.36(d,J＝0.6Hz,4H),1.35–1.30(m,20H),1.30–1.26(m,16H),1.19(s,3H),1.00(s,6H),0.90(s,9H)。
实施例9
化合物29的合成
Figure PCTCN2022143764-appb-000017
步骤1：
将化合物1-癸硫醇(2.28g)和KOH(2.20g)溶于乙醇(20ml)，室温搅拌。然后称取化合物29-1(2.00g)分批加入到上述反应体系中，室温下搅拌过夜。TLC(PE/EA＝3/1， 磷钼酸)有新点生成。反应混合液加200ml水，滴加浓HCl调pH至3附近。用600ml乙酸乙酯萃取上述混合液，有机相Na 2SO 4干燥后减压蒸发。加适量DCM和硅胶拌样，纯化(30g正相柱，PE/EA，0-0％10min，0-2％20min，2-2％5min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到白色固体29-2(950mg，30％收率)。
步骤2：
将化合物29-2(950mg)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，924mg)，4-二甲氨基吡啶(DMAP，95mg)和5-溴-1-戊醇(708mg)分批加入反应体系，室温搅拌3h。取少量反应液稀释与29-2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29-3(1.45g，95％收率)。
步骤3：
将化合物29-3(750mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(170mg)，K 2CO 3(350mg)和化合物2-7(559mg)分批加入到上述反应体系中，在85℃下加热回流搅拌2h。取少量反应液稀释与2-7标样对照点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比2-7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)，点板监测，将纯产物部分馏分蒸发，得到淡黄色油状液体化合物29(850mg)。
1H NMR(400MHz,cdcl3)δ4.86(p,J＝6.4Hz,1H),4.09(t,J＝6.8Hz,2H),3.56(t,J＝5.4Hz,2H),2.77(t,J＝7.4Hz,2H),2.61(dt,J＝11.8,6.4Hz,4H),2.51(dd,J＝14.4,6.8Hz,6H),2.28(t,J＝7.6Hz,2H),1.70–1.55(m,6H),1.50(dd,J＝16.6,10.9Hz,8H),1.40–1.21(m,46H),0.88(t,J＝6.8Hz,9H)。
实施例10
化合物30的合成
Figure PCTCN2022143764-appb-000018
步骤1：
向化合物30-1(2.00g)的DMF(15mL)溶液中依次加入1-癸硫醇(2.10g)和氢氧化钠(1.20g)，反应混合物在70℃搅拌3小时。TLC显示起始化合物30-1完全消失。将反应液倒入H 2O(50mL)，加EA(20mL)萃取1次，水相用2M稀盐酸调节pH为3，加EA(20mL)萃取3次，合并有机相，用无水硫酸钠干燥。抽滤，浓缩得到化合物30-2(1.70g，54％收率)。
步骤2：
向化合物30-2(1.70g)的DCM(15mL)溶液中依次加入4-二甲氨基吡啶(DMAP，160mg)和5-溴戊醇(1.31g)。混合物在25℃搅拌5分钟后，加入1-乙基-(3-二甲基氨基丙基)(EDCl，1.56g)，反应混合物25℃搅拌2小时。TLC显示起始化合物30-2完全消失。将反应混合物浓缩得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-1％EA(体积百分比)的石油醚溶液)纯化，并将纯产物馏分蒸发，得到化合物30-3(1.54g，57％收率)。
步骤3：
向化合物30-3(1.5g)的乙醇(15mL)溶液中加入乙醇胺(783mg)，混合物在70℃搅拌12小时。TLC显示起始化合物30-3有少量剩余。将反应混合物浓缩得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-10％CH 3OH(体积百分比)的二氯甲烷溶液，甲醇中加1％氨水)纯化，并将纯产物馏分蒸发，得到化合物30-4(824mg，58％收率)。
步骤4：
向化合物30-4(724mg)的DMF(7mL)溶液中依次加入七烷-9-基8-溴辛酸酯(1.03g)，NaI(278mg)和K 2CO 3(770mg)，反应混合物在50℃反应12小时。TLC显示起始化合物30-4有少量剩余。将反应液倒入H 2O(50mL)，加EA(20mL)萃取3次，合并有机相，有机相用饱和食盐(20mL)洗2次，有机相用无水硫酸钠干燥。抽滤，浓缩得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-10％CH 3OH(体积百分比)的二氯甲烷溶液，甲醇中加1％氨水)纯化，并将纯产物馏分蒸发，得到化合物30(1.02g，71％收率)。
1H NMR(400MHz,Chloroform-d)δ4.86(dd,J＝12.4,6.0Hz,1H),4.09(t,J＝8.0Hz,2H),3.54(t,J＝6.0Hz,2H),2.83(d,J＝7.0Hz,1H),2.73–2.38(m,12H),2.27(t,J＝7.2Hz,3H),1.68–1.18(m,61H),0.87(t,J＝6.8Hz,9H)。
实施例11
化合物35的合成
Figure PCTCN2022143764-appb-000019
步骤1：
向化合物35-1(2.00g)的DMF(15mL)溶液中依次加入1-辛硫醇(2.10g)和氢氧化钠(1.20g)，反应混合物在70℃搅拌3小时。TLC显示起始化合物35-1完全消失。将反应液倒入H 2O(50mL)，加EA(20mL)萃取1次，水相用2M稀盐酸调节pH为3，加EA(20mL)萃取3次，合并有机相，用无水硫酸钠干燥。抽滤，浓缩得到化合物35-2(1.80g，56.6％收率)。
步骤2：
将化合物35-2(1000mg)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，924mg)，4-二甲氨基吡啶(DMAP，95mg)和5-溴-1-戊醇(708mg)分批加入反应体系，室温搅拌3h。取少量反应液稀释与35-2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35-3(1.30g，79.2％收率)。
步骤3：
将化合物35-3(750mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(170mg)，K 2CO 3(350mg)和化合物2-7(559mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。取少量反应液稀释与35-3标样对照点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比35-3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)，点板监测，将纯产物部分馏分蒸发，得到淡黄色油状液体化合物35(830mg，56.9％收率)。
1H NMR(400MHz,Chloroform-d)δ4.86(dd,J＝12.4,6.0Hz,1H),4.09(t,J＝8.0Hz,2H),3.54(t,J＝6.0Hz,2H),2.83(d,J＝7.0Hz,1H),2.73–2.38(m,12H),2.27(t,J＝7.2Hz,3H),1.68–1.18(m,61H),0.87(t,J＝6.8Hz,9H)。
实施例12
化合物36的合成
Figure PCTCN2022143764-appb-000020
步骤1：
将化合物1-辛硫醇(2.50g)和NaOH(2.20g)溶于乙醇(20ml)，室温搅拌。然后称取化合物36-1(2.00g)分批加入到上述反应体系中，室温搅拌过夜。TLC(PE/EA＝3/1，磷钼酸)监测有新点生成。反应混合液加200ml水，滴加浓HCl调pH至3附近。用600ml乙酸乙酯萃取上述混合液，有机相Na 2SO 4干燥后减压蒸发。加适量DCM和硅胶拌样，纯化(30g正相柱，PE/EA，0-0％10min，0-2％20min，2-2％5min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到白色固体36-2(1.09g，40.2％收率)。
步骤2：
将化合物36-2(1000mg)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，924mg)，4-二甲氨基吡啶(DMAP，95mg)和5-溴-1-戊醇(708mg)分批加入反应体系，室温搅拌3h。取少量反应液稀释与36-2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36-3(1.20g， 74.7％收率)。
步骤3：
将化合物36-3(750mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(170mg)，K 2CO 3(350mg)和化合物2-7(559mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。取少量反应液稀释与2-7标样对照点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比2-7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)，点板监测，将纯产物部分馏分蒸发，得到淡黄色油状液体化合物35(830mg，56.9％收率)。
1H NMR(400MHz,Chloroform-d)δ4.86(dd,J＝12.4,6.0Hz,1H),4.09(t,J＝8.0Hz,2H),3.54(t,J＝6.0Hz,2H),2.83(d,J＝7.0Hz,1H),2.73–2.38(m,14H),2.27(t,J＝7.2Hz,2H),1.68–1.18(m,57H),0.87(t,J＝6.8Hz,9H).
实施例13
化合物41的合成
Figure PCTCN2022143764-appb-000021
步骤1：
向化合物41-1(2.00g)的DMF(15mL)溶液中依次加入1-癸硫醇(2.30g)和氢氧化钠(1.20g)，反应混合物在40℃搅拌4小时。TLC显示起始化合物41-1完全消失。将反应液倒入H 2O(50mL)，加乙酸乙酯(20mL)萃取1次，水相用2M稀盐酸调节pH为3，加乙酸乙酯(20mL)萃取3次，合并有机相，用无水硫酸钠干燥。抽滤，浓缩得到化合物41-2(1.80g，56.6％收率)。
步骤2：
将化合物41-2(1000mg)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，1.05g)，4-二甲氨基吡啶(DMAP，560mg)和 8-溴辛酸(1.02g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与41-2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(15g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速15ml/min)得到无色油状液体化合物41-3(1.5g，77％收率)。
步骤3：
将化合物41-3(700mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(170mg)，K 2CO 3(350mg)和化合物2-7(559mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。取少量反应液稀释与2-7标样对照点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比2-7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)，得到淡黄色油状液体化合物41(600mg，44.8％收率)。
1H NMR(400MHz,Chloroform-d)δ4.86(p,J＝6.4Hz,1H),4.09(t,J＝6.8Hz,2H),3.56(t,J＝5.4Hz,2H),2.77(t,J＝7.4Hz,2H),2.61(dt,J＝11.8,6.4Hz,4H),2.51(dd,J＝14.4,6.8Hz,6H),2.28(t,J＝7.6Hz,2H),1.70–1.55(m,6H),1.50(dd,J＝16.6,10.8Hz,8H),1.40–1.21(m,50H),0.88(t,J＝6.8Hz,9H)。
实施例14
化合物44的合成
Figure PCTCN2022143764-appb-000022
Figure PCTCN2022143764-appb-000023
步骤1：
向化合物44-1(2.00g)的DMF(15mL)溶液中依次加入1-辛硫醇(2.63g)和氢氧化钠(1.44g)，反应混合物在70℃搅拌3小时。TLC显示起始化合物44-1完全消失。将反应液倒入H 2O(50mL)，加EA(20mL)萃取1次，水相用2M稀盐酸调节pH为3，加EA(20mL)萃取3次，合并有机相，用无水硫酸钠干燥。抽滤，浓缩得到化合物44-2(1.63g，53％收率)。
步骤2：
向化合物44-2(1.63g)的DCM(15mL)溶液中依次加入4-二甲氨基吡啶(DMAP，172mg)和5-溴戊醇(1.41g)。混合物在25℃搅拌5分钟后，加入1-乙基-(3-二甲基氨基丙基)(EDCl，1.75g)，反应混合物25℃搅拌1小时。TLC显示起始化合物44-2完全消失。将反应混合物浓缩得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-1％EA(体积百分比)的石油醚溶液)纯化，并将纯产物馏分蒸发，得到化合物44-3(1.65g，62％收率)。
步骤3：
向化合物44-3(1.5g)的乙腈(15mL)溶液中加入乙醇胺(960mg)和碳酸钾(1.15g),溶液在70℃搅拌12小时。TLC显示起始化合物44-3有少量剩余。将反应混合物浓缩得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-10％CH 3OH(体积百分比)的二氯甲烷溶液，甲醇中加1％氨水)纯化，并将纯产物馏分蒸发，得到化合物44-4(800mg，56％收率)。
步骤4：
向化合物44-5(2.00g)的DCM(15mL)溶液中依次加入4-二甲氨基吡啶(DMAP，200mg)和7-溴-1-庚醇(1.51g)。混合物在25℃搅拌5分钟后，加入1-乙基-(3-二甲基氨基丙基)(EDCl，1.62g)，反应混合物25℃搅拌1小时。TLC显示起始化合物44-5完全消失。将 反应混合物浓缩得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-1％EA(体积百分比)的石油醚溶液)纯化，并将纯产物馏分蒸发，得到化合物44-6(2.40g，74％收率)。
步骤5：
向化合物44-4(800mg)的DMF(7mL)溶液中依次加入44-6(1.12g)，NaI(332mg)和K 2CO 3(918mg)，反应混合物在50℃反应12小时。TLC显示起始化合物44-4有少量剩余。将反应液倒入H 2O(50mL)，加EA(20mL)萃取3次，合并有机相，有机相用饱和食盐(20mL)洗2次，有机相用无水硫酸钠干燥。抽滤，浓缩得到粗产物，将粗产物通过柱色谱法(硅胶柱，洗脱液为含0-10％CH 3OH(体积百分比)的二氯甲烷溶液，甲醇中加1％氨水)纯化，并将纯产物馏分蒸发，得到化合物44(950mg，58％收率)。
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.09(d,J＝3.2Hz,2H),3.72–3.55(m,2H),3.05–2.86(m,2H),2.84(s,1H),2.59–2.41(m,8H),2.31–2.17(m,2H),1.72–1.64(m,4H),1.62–1.60(s,2H),1.59–1.51(m,8H),1.55(s,2H),1.39–1.35(m,6H),1.33(d,J＝3.0Hz,6H),1.31(d,J＝3.0Hz,12H),1.31–1.28(m,4H),1.30-1.26(m,12H),1.24(s,3H),0.94–0.82(m,9H)。
实施例15
化合物46的合成
Figure PCTCN2022143764-appb-000024
步骤1：
将化合物1-庚硫醇(2.1g)和NaOH(1.0g)溶于DMF(20ml)，室温搅拌。然后称取化合物46-1(2.00g)分批加入到上述反应体系中，60℃下搅拌过夜。TLC(PE/EA＝3/1，磷钼酸)有新点生成。反应混合液加200ml水，滴加浓HCl调pH至3附近。用600ml乙酸乙酯萃取上述混合液，有机相Na 2SO 4干燥后减压蒸发。加适量DCM和硅胶拌样，纯化(30g正相柱，PE/EA，0-0％10min，0-2％20min，2-2％5min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体46-2(1.5g，62％收率)。
步骤2：
向化合物46-2(1.5g)的THF(20mL)和水的溶液中加入氢氧化锂(360mg)将混合物在60℃搅拌16小时。TLC显示有极性变大的点生成。将反应混合物浓缩去除四氢呋喃，加水稀释后，用乙酸乙酯(30mL)萃取1次，将水相用稀盐酸调节pH＝2，用乙酸乙酯(30mL)萃取2次，合并有机层，浓缩得到化合物46-3(1.2g，88％收率)。
步骤3：
将化合物46-3(1.2g)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，1.01g)，4-二甲氨基吡啶(DMAP，534mg)和6-溴正己醇(792mg)分批加入反应体系，室温搅拌3h。取少量反应液稀释与46-3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46-4(1.6g，84％收率)。
步骤4：
向化合物46-4(1.6g)和乙醇胺(447mg)的乙腈溶液(50mL)溶液中加入碳酸钾(1.52g)。将混合物在85℃下回流2小时。TLC显示化合物46-4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46-5(870mg，57％收率)。
步骤5：
将化合物46-6(2.0g)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，1.79g)，4-二甲氨基吡啶(DMAP，950mg)和6-溴正己醇(1.41mg)分批加入反应体系，室温搅拌3h。取少量反应液稀释与46-6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46-7(2.8g，86％收率)。
步骤6：
将化合物46-5(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(180mg)，K 2CO 3(497mg)和化合物46-7(502mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。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46-7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46(700mg，77％收率)。
1H NMR(400MHz,Chloroform-d)δ4.10(m,4H),3.64(m,3H),2.61–2.42(m,8H),2.29(s,1H),1.98–1.90(m,2H),1.67–1.61(m,6H),1.59–1.45(m,8H),1.43(s,4H),1.41–1.37(m,4H),1.37–1.33(m,10H),1.33–1.30(m,14H),1.30–1.26(m,12H),0.95–0.83(m,12H)。
实施例16
化合物48的合成
Figure PCTCN2022143764-appb-000025
步骤1：
向化合物2-6(8.0g)和丙醇胺(1.9g)的乙腈溶液(50mL)溶液中加入碳酸钾(7.19g)。将混合物在85℃下回流2小时。TLC显示化合物2-6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(40g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48-1(5.0g，63.3％收率)。
步骤2：
将化合物35-3(750mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(170mg)，K 2CO 3(350mg)和化合物48-1(620mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。取少量反应液稀释与48-1标样对照点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有比48-1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(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)，点板监测，将纯产物部分馏分蒸发，得到淡黄色油状液体化合物48(650mg，69％收率)。
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.09(d,J＝3.2Hz,2H),3.65–3.56(m,2H),3.00–2.88(m,3H),2.56–2.40(m,8H),2.31–2.17(m,2H),1.73–1.63(m,6H),1.62(d,J＝1.0Hz,2H),1.59–1.50(m,8H),1.58(s,2H),1.40–1.35(m,6H),1.34–1.30(m,18H),1.30–1.27(m,16H),1.24(s,3H),0.95–0.82(m,9H)。
实施例17
化合物55的合成
Figure PCTCN2022143764-appb-000026
步骤1：
向化合物2-6(4.0g)和N,N-二甲基乙二胺(1.53g)的乙腈溶液(50mL)溶液中加入碳酸钾(3.59g)。将混合物在85℃下搅拌回流3小时。TLC显示化合物2-6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，0.1％NH 3H 2O,MeOH/DCM，0-0％5min，0-10％20min，10-10％5min，流速20ml/min)得到无色油状液体化合物55-1(1.3g，32％收率)。
步骤2：
向溴乙醇(2.00g)的DMF(15mL)溶液中依次加入化合物55-2(1.93g)和氢氧化钠(1.20g)，反应混合物在40℃搅拌4小时。TLC显示起始化合物55-2完全消失。将反应液倒入H 2O(50mL)，加乙酸乙酯(20mL)萃取1次，水相用2M稀盐酸调节pH为3，加乙酸乙酯(20 mL)萃取3次，合并有机相，用无水硫酸钠干燥。抽滤，浓缩得到化合物55-3(1.56g，62.3％收率)。
步骤3：
将化合物55-3(872mg)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，1.05g)，4-二甲氨基吡啶(DMAP，560mg)和8-溴辛酸(1.02g)分批加入反应体系，室温搅拌2h。取少量反应液稀释与55-3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(15g正相柱，PE/EA，0-0％5min，0-5％20min，5-5％5min，流速15mL/min)得到无色油状液体化合物55-4(1.27g，70％收率)。
步骤4：
将化合物55-1(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(146mg)，K 2CO 3(406mg)和化合物55-4(393mg)分批加入到上述反应体系中，在85℃下加热回流搅拌3h。取少量反应液稀释点板(DCM/MeOH＝10/1，1d氨水，磷钼酸)，观察到有新点生成。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55(108mg，14％收率)。
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.23(s,2H),2.80(s,2H),2.60(s,2H),2.57(s,2H),2.52(s,2H),2.45(s,4H),2.30(s,6H),2.27(s,2H),2.24(s,2H),1.71–1.66(m,2H),1.62(s,2H),1.58–1.57(d,J＝6.8Hz,4H),1.54(d,J＝6.8Hz,2H),1.53–1.50(m,4H),1.37(d,J＝1.0Hz,6H),1.35–1.30(m,24H),1.30–1.25(m,16H),0.96–0.81(m,9H)。
实施例18
化合物57的合成
Figure PCTCN2022143764-appb-000027
步骤1：
将化合物55-1(500mg)溶于乙腈(10ml)，室温搅拌。然后依次称取NaI(146mg)，K 2CO 3(406mg)和化合物29-3(422mg)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)，观察到有新点生成。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57(103mg，12％收率)
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.12(s,2H),2.96–2.77(m,2H),2.62–2.56(m,6H),2.50(s,2H),2.45(d,J＝2.2Hz,4H),2.30(s,6H),2.24(s,2H),1.71–1.66(m, 2H),1.65(s,2H),1.62(s,2H),1.58(s,2H),1.57–1.50(m,6H),1.49(s,2H),1.38(d,J＝0.8Hz,6H),1.34–1.30(m,18H),1.30–1.26(m,20H),0.94–0.82(m,9H)。
实施例19
将荧光素酶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(DMG-PEG2000))按照摩尔比50：10：38.5：1.5溶于乙醇。
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、不同阳离子脂质制备的纳米颗粒表征结果
编号 阳离子脂质 包封率(％) 粒径(nm) PDI Zeta电位(mV)
1 化合物2 87.8 97.3 0.12 0.49
2 化合物3 87.5 113.9 0.10 1.95
3 化合物4 83.7 94.4 0.21 0.39
4 化合物6 63.9 95.6 0.17 0.79
5 化合物7 96.1 78.9 0.17 -3.77
6 化合物14 95.7 119.0 0.22 -3.27
7 化合物17 92.5 90.9 0.10 -0.15
8 化合物28 95.9 127.6 0.04 5.29
9 化合物29 95.6 105.5 0.03 -1.31
10 化合物30 95.7 121.2 0.11 -0.26
11 化合物35 90.4 104.5 0.11 -2.10
12 化合物36 93.9 98.4 0.11 0.55
13 化合物41 94.9 123.4 0.17 -7.02
14 化合物44 96.4 104.1 0.21 -2.82
15 化合物46 95.6 101.3 0.11 -4.12
16 化合物48 92.5 94.2 0.10 -0.14
17 化合物55 91.1 102.3 0.13 7.37
18 化合物57 94.1 122.8 0.24 7.09
19 DLin-MC3-DMA* 87.6 102.3 0.19 -0.03
20 Lipid M** 98.5 145.3 0.14 -0.53
*DLin-MC3-DMA为商业核酸递送系统Onpattro的阳离子脂质。
**表1的Lipid M出自《Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines》Mol Ther Nucleic Acids 2019 Vol.15Pages 1-11，为SM-102的同类脂质。
实施例20利用纳米脂质颗粒组合物尾静脉注射递送荧光素酶mRNA在体内表达效果测定
在6-8周龄的BALB/c小鼠通过尾静脉注射含5μg mRNA的LUC-mRNA-脂质纳米颗粒(LUC-mRNA对应的核苷酸序列见专利公开CN114380724A的SEQ ID NO:1)，制备方法同实施例19。在特定时间点小鼠尾静脉注射D-Luciferin Potassium Salt 100μg，使用PerkinElmer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荧光。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，其中本发明设计的化合物组成的纳米脂质颗粒组合物递送mRNA至肝脏的水平大部分等效或者优于DLin-MC3-DMA；部分化合物优于Lipid M。
实施例21利用纳米脂质颗粒组合物肺部雾化递送荧光素酶mRNA在体内表达效果测定
6-8周龄的BALB/c小鼠通过雾化方式，进行肺部递送入含有5μg mRNA的LUC-mRNA-脂质纳米颗粒，制备方法同实施例19。在特定时间点小鼠尾静脉注射D-Luciferin Potassium Salt 100μg，使用PerkinElmer小动物成像系统进行检测。检测结果如图2，其中化合物7，35组成的纳米脂质颗粒组合物递送mRNA表达的荧光水平优于Lipid M和SM-102。
实施例22利用纳米脂质颗粒组合物递送新型冠状病毒mRNA疫苗
使用6周BALB/c小鼠，于0、14天肌肉注射给与不同纳米脂质颗粒组合物递送的mRNA新冠肺炎疫苗(Omicron抗原mRNA，对应的核苷酸序列见专利公开CN114380724A的SEQ ID NO:6)进行免疫，第28天(二次免疫后第14天)采血，通过酶联免疫吸附测定中和抗体滴度，评价不同纳米脂质颗粒组合物递送的mRNA新冠肺炎疫苗对SARS-CoV-2病毒毒株感染的保护效果。结果如图3所示，由结果可知，化合物35组成的纳米脂质颗粒组合物递送新冠mRNA的引起的中和抗体滴度为140万，而Lipid M的为93万左右。
实施例23不同脂质组分和比例的纳米脂质颗粒组合物表征
为研究不同辅助脂质和脂质比例下的成药性，对化合物35展开了实验设计。尝试将不同结构脂质(DOPE、DSPC)、不同脂质比例(阳离子45～55％，PEG脂质1.5～2.5％，结构脂质8～22％，胆固醇20.5～45.5％)、不同氮磷比(5～10)的脂质混合物，和Luc mRNA通过微流控法(方法同实施例19)混合制备纳米颗粒。结果见表2所示。
表2、不同脂质比例(摩尔比例)制备的纳米颗粒表征结果
Figure PCTCN2022143764-appb-000028
Figure PCTCN2022143764-appb-000029
从表2结果可知，在该范围内的脂质混合物和mRNA形成的纳米颗粒粒径均一、包封率高、成药性好。
实施例24不同阳离子递送荧光素酶mRNA在体内表达效果研究
我们根据实施例19和实施例20的方法验证了更多阳离子脂质递送荧光素酶mRNA的效果。以下表3为表征结果。
表3
编号 阳离子脂质 包封率(％) 粒径(nm) PDI 相对荧光亮度(％)
1 化合物7 96.8 82.9 0.15 754±32
2 化合物16 95.6 90.9 0.20 574±102
3 化合物17 97.6 68.2 0.11 686±45
4 化合物23 97.0 75.4 0.10 764±39
5 化合物35 96.2 80.4 0.14 962±78
6 化合物44 97.1 71.1 0.10 843±52
7 化合物45 96.8 80.9 0.15 559±33
8 化合物51 98.0 91.0 0.11 734±51
9 DLin-MC3-DMA 0.2 101.5 0.16 100
注：因实验批次不同，故本表3包封率、粒径DPI数据与表1有所不同。
上表可见，以上化合物包封率高，粒径均一，体内递送Luciferase mRNA效率远高于商业脂质DLin-MC3-DMA。
实施例25不同阳离子脂质蛋白表达效率研究
本实施例中，对比了不同阳离子脂质体内转染促血红细胞生成素(EPO)的效果。纳米颗粒制备方式同实施例19，使用EPO mRNA对应的核苷酸序列见CN114380724B，SEQ ID NO:2。制备后用6-8周龄的雌性Balb/c小鼠，通过尾静脉注射20μg EPO-mRNA-脂质纳米颗粒，6小时后对小鼠进行眼框取血，离心分离血清后，使用ELISA法测定EPO蛋白表达量。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。制备及蛋白检测结果如表4所示。
表4
编号 阳离子脂质 包封率(％) 粒径(nm) PDI EPO浓度(pg/mL)
1 化合物7 97.8 88.8 0.09 4.21×10 7
2 化合物16 96.5 75.4 0.12 3.01×10 7
3 化合物17 96.9 74.0 0.09 3.25×10 7
4 化合物23 96.5 81.7 0.11 4.11×10 7
5 化合物35 96.1 79.1 0.12 5.73×10 7
6 化合物44 95.5 82.5 0.17 4.89×10 7
7 化合物45 93.9 81.0 0.15 3.46×10 7
8 化合物51 95.0 75.3 0.14 6.68×10 7
9 DLin-MC3-DMA 90.1 97.6 0.19 4.37×10 6
注：因实验批次不同，故本表4中包封率、粒径DPI数据与表1、表3有所不同。
上表可见，以上化合物包封率高，粒径均一，体内递送EPO mRNA的蛋白翻译效率远高于商业脂质DLin-MC3-DMA。
实施例26不同阳离子脂质异常毒性研究
本实验中使用了实施例25中制备的纳米颗粒，使用体重200～250g的雌性SD大鼠，按5mg/kg的剂量进行尾静脉注射，对照组小鼠注射对应体积的生理盐水。DLin-MC3-DMA组小鼠注射后18h内死亡，其余小鼠的体重、进食、活动状态在观察期内无异常表现。说明DLin-MC3-DMA脂质的毒副作用更强。注射后120h采血，使用全自动生化仪检测谷丙转氨酶(ALT)，谷草转氨酶(AST)、尿素氮(BUN)、肌酐(SCR)作为肝肾功能评价指标。图4的结果表明，除35号(化合物35)和51号(化合物51)脂质的BUN数值有轻微下降，其余脂质纳米颗粒其余指标未发生明显波动，说明该类阳离子脂质未影响肝肾功能，毒副作用低，适用于蛋白替代管线使用。

Claims (25)

  1. 具有以下结构式(I)结构的用于递送核酸的阳离子脂质化合物：
    Figure PCTCN2022143764-appb-100001
    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：
    L 1、L 2为连接键或二价连接基，所述二价连接基各自独立地选自-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-OC(＝O)O-、-O-、-S-、-S-S-、-C(＝O)S-、-SC(＝O)-、-N(R 8)C(＝O)-、-C(＝O)N(R 8)-、-N(R 8)C(＝O)O-、-OC(＝O)N(R 8)-、-SC(＝O)N(R 8)-、-N(R 8)C(＝O)S-、-C(＝S)-、-SC(＝S)-、和-C(＝S)S-中任一种，所述R 8为H或C 1-C 12烷基；
    R 2和R 3独立地为取代或未取代的C 1-C 18直链亚烷基或者-R 9-L 3-R 10-；所述R 9和R 10独立地为取代或未取代的C 1-C 10直链亚烷基，L 3为O或者S；
    R 4，R 5，R 6和R 7独立地为氢、或者是取代或未取代的C 1-C 30脂肪烃基，或者是-R 11-L 4-R 12；所述R 11和R 12每次出现时独立地为取代或未取代的C 1-C 18脂肪烃基，L 4为O或者S；
    上述R 2，R 3，R 4，R 5，R 6和R 7中至少含有一个O或者S；
    R 1为H、-R 13、-OR 13、-R 13-OH、-R 13-OR 14、-R 13-OC(＝O)R 14、-R 13-NHC(＝O)-R 14、-R 13-OCH 3或-R 13-N(R 14)(R 15)；R 13为C 1-C 12直链烷基或支链烷基，R 14和R 15各自独立的分别为H或C 1-C 12直链烷基，或者，R 14与R 15和其连接的N原子形成C 3-C 10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，R 8为H或C 1-C 8烷基。
  3. 如权利要求2所述的阳离子脂质化合物，其特征在于，R 13为C 1-C 8直链烷基或支链烷基，R 14和R 15各自独立的分别为H或C 1-C 5直链烷基，或者，R 14与R 15和其连接的N原子形成C 3-C 8杂环烷基。
  4. 如权利要求3所述的阳离子脂质化合物，其特征在于，R 2和R 3独立地为取代或未取代的C 1-C 18直链烷基。
  5. 如权利要求4所述的阳离子脂质化合物，其特征在于，R 2和R 3独立地为取代或未取代的C 1-C 12直链烷基。
  6. 如权利要求5所述的阳离子脂质化合物，其特征在于，R 4，R 5，R 6和R 7独立地为氢、或者是取代或未取代的C 1-C 18脂肪烃基，或者是-R 11-L 4-R 12；所述R 11和R 12每次出现时独 立地为取代或未取代的C 1-C 10脂肪烃基，L 4为O或者S；R 4，R 5，R 6和R 7中至少含有一个O或者S，至多有两个为氢。
  7. 如权利要求6所述的阳离子脂质化合物，所述式结构中的R 4，R 5，R 6和R 7结构各自独立地为H或下述烷基链，或各自独立地为下述烷基链上任意一个碳原子被O或者S替代而形成的醚或者硫醚：
    Figure PCTCN2022143764-appb-100002
  8. 如权利要求5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：
    Figure PCTCN2022143764-appb-100003
    Figure PCTCN2022143764-appb-100004
    Figure PCTCN2022143764-appb-100005
    Figure PCTCN2022143764-appb-100006
    Figure PCTCN2022143764-appb-100007
    Figure PCTCN2022143764-appb-100008
    Figure PCTCN2022143764-appb-100009
  9. 包含权利要求1-8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。
  10. 如权利要求9所述的脂质体制剂，其特征在于，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。
  11. 如权利要求10所述的脂质体制剂，其特征在于，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。
  12. 如权利要求9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。
  13. 如权利要求12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。
  14. 如权利要求9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，包括中性脂质、类固醇和聚合物缀合的脂质。
  15. 如权利要求14所述的脂质体制剂，其特征在于，所包含的类固醇为胆固醇。
  16. 如权利要求15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。
  17. 如权利要求9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)。
  18. 如权利要求17所述的脂质体制剂，其特征在于，所述阳离子脂质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。
  19. 如权利要求17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PEG-DAG、PEG-PE、PEG-SDAG、PEG-cer、PEG-DMG或ALC-0159。
  20. 如权利要求14所述的脂质体制剂，其特征在于，所述中性脂质选自DSPC、DPPC、DMPC、DOPC、POPC、DOPE和SM中的一种或多种的组合。
  21. 如权利要求20所述的脂质体制剂，其特征在于，所述中性脂质与阳离子脂质化合物的摩尔比为(0-0.5)：1。
  22. 如权利要求9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。
  23. 如权利要求1-8任意一项所述的阳离子脂质化合物或权利要求9-22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。
  24. 如权利要求23所述的用途，其特征在于，所述对象为哺乳动物。
  25. 如权利要求23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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