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WO2025108353A1 - 一种阳离子脂质化合物、包含其的组合物及应用 - Google Patents

一种阳离子脂质化合物、包含其的组合物及应用 Download PDF

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Publication number
WO2025108353A1
WO2025108353A1 PCT/CN2024/133412 CN2024133412W WO2025108353A1 WO 2025108353 A1 WO2025108353 A1 WO 2025108353A1 CN 2024133412 W CN2024133412 W CN 2024133412W WO 2025108353 A1 WO2025108353 A1 WO 2025108353A1
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compound
cationic lipid
lipid compound
liposome preparation
added
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English (en)
French (fr)
Inventor
胡勇
余鹏程
滕文琪
欧阳立鹏
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Shenzhen Rhegen Biotechnology Co Ltd
Wuhan Rhegen Biotechnology Co Ltd
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Shenzhen Rhegen Biotechnology Co Ltd
Wuhan Rhegen Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a cationic lipid compound, a composition containing the same and applications thereof.
  • RNA Nucleic acids
  • RNA Nucleic acids
  • naked RNA has a short circulation time in the body, low efficiency of cell internalization, and is easily cleared by the kidneys or degraded by RNase in the body, which greatly reduces the actual effect.
  • cationic lipid compounds and related preparations are one of the key technologies.
  • LNP lipid nanoparticles
  • LNP lipid nanoparticles
  • LNP is currently the mainstream delivery carrier. It can bind to mRNA to increase the in vivo circulation time of mRNA, control its escape in the body to increase its transfection rate, and increase protein expression.
  • LNP is generally composed of four components, including: (1) cationic lipids, which provide charge to encapsulate mRNA molecules; (2) supporting phospholipids, which provide bilayer support and help endosomal escape; (3) cholesterol, which enhances the stability of LNP and promotes membrane fusion; (4) PEG lipids, which reduce the particle size of LNP and increase the in vivo circulation time.
  • the present invention provides a new class of highly effective, low-toxic cationic compounds.
  • An object of the present invention is to provide a cationic lipid compound
  • Another object of the present invention is to provide a liposome preparation
  • Another object of the present invention is to provide uses of the cationic lipid compound.
  • the present invention provides a cationic lipid compound represented by the general formula (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:
  • Ring A is a substituted or unsubstituted 4-10 membered N-containing heterocyclic ring; the N-containing heterocyclic ring contains 0, 1 or 2 heteroatoms selected from N, O or S in addition to the N atom connected to L1 ; when substituted, the heterocyclic ring is substituted with 1, 2, 3, 4, 5 or 6 substituents selected from halogen, hydroxyl, cyano, nitro, carboxyl, C1-C5 alkyl or C1-C5 alkoxy;
  • L1 is a C1-C6 straight chain alkylene group
  • L2 and L3 are the same or different, and are each independently a C1-C12 alkylene group
  • R3 and R4 are the same or different, and are independently C4-C17 straight chain alkyl, C4-C17 straight chain alkenyl or C8-C22 branched non-cyclic alkyl.
  • Ring A is a substituted or unsubstituted 4-10 membered N-containing heterocycle; the N-containing heterocycle contains 1-3 N atoms.
  • Ring A is a substituted or unsubstituted 5-membered, 6-membered or 7-membered N-containing heterocycle.
  • Ring A is a substituted or unsubstituted 5-membered, 6-membered or 7-membered N-containing heterocycle, and the heteroatoms in the N-containing heterocycle are all N atoms.
  • L 1 is a C2-C4 straight chain alkylene group.
  • At least one of L 2 and L 3 is a C1-C6 alkylene group.
  • L 2 and L 3 are each independently a C1-C6 alkylene group.
  • ring A is methylpiperazinyl, hydropyrrolyl, piperidinyl or cycloheximide.
  • ring A is:
  • R 3 and R 4 are each independently:
  • R5 and R6 are the same or different, and are each independently a C3-C11 straight-chain alkyl group.
  • R5 and R6 should satisfy the above-defined range of C8-C22.
  • one of R5 and R6 is a C3 straight-chain alkyl group
  • the other should be at least a C5 straight-chain alkyl group.
  • R 5 and R 6 are the same or different, and are each independently a C4-C8 straight-chain alkyl group.
  • R 3 and R 4 are each independently:
  • the cationic lipid compound is selected from one or more of the following structures:
  • the present invention also provides a liposome preparation comprising the cationic lipid compound of any one of the present invention or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof and a preventive or therapeutic nucleic acid.
  • the molar ratio of the cationic lipid compound to the nucleic acid is 20:1 to 1:1.
  • the average particle size of the liposome preparation is 50 nm to 200 nm.
  • the liposome preparation further comprises one or more other lipid components, and the other lipid components include neutral lipids, steroids and polymer-conjugated lipids.
  • the steroid is ⁇ -sitosterol, soya bean sterol, ergosterol, cholesterol or dihydrocholesterol; cholesterol is preferred.
  • the molar ratio of the steroid to the cationic lipid compound is (0.5-1):1.
  • the polymer in the polymer-conjugated lipid is polyethylene glycol.
  • the molar ratio of the cationic lipid compound to the polymer-conjugated lipid is 100:1 to 20:1.
  • the polyethylene glycol-conjugated lipid is PEG2k-DSG, PEG2k-DMG, PEG2k-DPPE, PEG2k-DSPE, PEG2k-cer, PEG2k-DMG or ALC-0159; preferably PEG2k-DMG.
  • the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-sodium phosphoglycerol (DPPG-Na), sphingomyelin (SM), ceramide and one or more combinations of sterols.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DMPC 1,2-dimyristoyl-sn-glycer
  • the molar ratio of the cationic lipid compound to the neutral lipid is 2:1 to 8:1.
  • the nucleic acid is selected from mRNA, siRNA, miRNA or ASO; the nucleic acid is preferably mRNA.
  • the present invention also provides use of the cationic lipid compound of the present invention or its pharmaceutically acceptable salt, tautomer or stereoisomer or the liposome preparation of the present invention in the preparation of a medicament for inducing protein expression in a subject.
  • the present invention provides a cationic lipid compound, a composition comprising the same, and an application thereof.
  • the cationic lipid compound of the present invention has the following advantages:
  • FIG1 is a hydrogen spectrum of compound 1.
  • FIG2 is a hydrogen spectrum of compound 2.
  • FIG3 is a hydrogen spectrum of compound 3.
  • FIG4 is a hydrogen spectrum of compound 4.
  • FIG5 is a hydrogen spectrum of compound 5.
  • FIG6 is a hydrogen spectrum of compound 6.
  • FIG7 is a hydrogen spectrum of compound 7.
  • FIG8 is a hydrogen spectrum of compound 8.
  • FIG9 is a hydrogen spectrum of compound 9.
  • FIG10 is a hydrogen spectrum of compound 10.
  • FIG11 is a hydrogen spectrum of compound 11.
  • FIG12 is a hydrogen spectrum of compound 12.
  • FIG13 is a hydrogen spectrum of compound 13.
  • FIG14 is a hydrogen spectrum of compound 14.
  • FIG15 is a hydrogen spectrum of compound 15.
  • FIG. 16 is a graph showing the effect of the nanolipid particle composition in delivering erythropoietin (EPO) mRNA in mice.
  • EPO erythropoietin
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure.
  • An appropriate amount of silica gel was added for sample mixing and purification (80 g normal phase column, PE/EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 40 ml/min) was performed using a spot plate for monitoring. Part of the pure product fractions were evaporated to give a colorless oily liquid compound 1-2 (6.0 g, 54.5% yield).
  • Trifluoroacetic acid (15 ml) was added to a solution of compound 1-2 (6.0 g) in dichloromethane (50 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 1-2 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (100 ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (100 ml ⁇ 2) was used for extraction.
  • the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and added with appropriate amount of silica gel and DCM to mix the sample and purify (80 g normal phase column, PE/EA, 0-0% 5 min, 0-50% 20 min, 50-50% min, flow rate 50 ml/min) to obtain white solid compound 1-3 (4.5 g, 93% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure.
  • An appropriate amount of silica gel was added for sample mixing and purification (80 g normal phase column, PE/EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 40 ml/min), and the plate was monitored.
  • the pure product fraction was evaporated to obtain a colorless oily liquid compound 3-1 (8.0 g, 68.9% yield).
  • Trifluoroacetic acid (15 ml) was added to a dichloromethane solution (50 ml) of compound 3-1 (8.0 g). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 3-1 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (100 ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (100 ml ⁇ 2) was used for extraction.
  • the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and added with appropriate amount of silica gel and DCM to mix the sample and purify (80 g normal phase column, PE/EA, 0-0% 5 min, 0-40% 20 min, 40-40% min, flow rate 50 ml/min) to obtain a colorless oily liquid compound 3-2 (5.8 g, 89% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure.
  • An appropriate amount of silica gel was added for sample mixing and purification (25 g normal phase column, PE/EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml/min), and the plate was monitored.
  • the pure product fraction was evaporated to obtain a colorless oily liquid compound 4-1 (2.5 g, 59.4% yield).
  • Trifluoroacetic acid (10 ml) was added to a solution of compound 4-1 (2.5 g) in dichloromethane (20 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 4-1 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (50 ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (50 ml ⁇ 2) was extracted.
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure.
  • An appropriate amount of silica gel was added for sample mixing and purification (80 g normal phase column, PE/EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 40 ml/min), and the plate was monitored.
  • the pure product fraction was evaporated to obtain a colorless oily liquid compound 7-2 (8.5 g, 66.3% yield).
  • Trifluoroacetic acid (30 ml) was added to a dichloromethane solution (100 ml) of compound 7-2 (8.5 g). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 7-2 completely disappeared and a point with increased polarity was generated.
  • reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (150 ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (100 ml ⁇ 2) was extracted, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, added with appropriate amount of silica gel and DCM, mixed with sample, purified (120 g normal phase column, PE/EA, 0-0% 5 min, 0-50% 20 min, 50-50% min, flow rate 60 ml/min) to obtain white solid compound 7-3 (6.3 g, 89% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure.
  • An appropriate amount of silica gel was added for sample mixing and purification (25 g normal phase column, PE/EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml/min), and the plate was monitored.
  • the pure product fraction was evaporated to obtain a colorless oily liquid compound 8-2 (3.0 g, 64% yield).
  • Trifluoroacetic acid (10m) was added to a dichloromethane solution (30ml) of compound 8-2 (3.0g). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 8-2 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (150ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (100ml ⁇ 2) was used for extraction.
  • the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and added with appropriate amount of silica gel and DCM to mix the sample and purify (120g normal phase column, PE/EA, 0-0% 5min, 0-50% 20min, 50-50% min, flow rate 60ml/min) to obtain a light yellow oily compound 8-3 (1.9g, 84% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure, and an appropriate amount of silica gel was added to mix the sample and purify (25 g normal phase column, PE/EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml/min), and the plate was monitored.
  • the pure product fraction was evaporated to obtain a colorless oily liquid compound 8-5 (3.6 g, 72% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure, and an appropriate amount of silica gel was added to mix the sample and purify (40 g normal phase column, PE/EA, 0-0% 10 min, 0-6% 30 min, 6-6% 20 min, flow rate 30 ml/min), spot plate monitoring, and the pure product fraction was evaporated to obtain a white solid compound 9-1 (3.2 g, 57% yield).
  • Trifluoroacetic acid (10 ml) was added to a dichloromethane solution (30 ml) of compound 9-1 (3.2 g). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 9-1 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (150 ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (100 ml ⁇ 2) was extracted.
  • the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and added with appropriate amount of silica gel and DCM to mix the sample and purify (120 g normal phase column, PE/EA, 0-0% 5 min, 0-40% 20 min, 40-40% 15 min, flow rate 30 mL/min) to obtain colorless oily compound 9-2 (2.0 g, 90% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure, and an appropriate amount of silica gel was added to mix the sample and purify (25 g normal phase column, PE/EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml/min), and the plate was monitored.
  • the pure product fraction was evaporated to obtain a colorless oily liquid compound 9-4 (3.6 g, 70% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure, and an appropriate amount of silica gel was added to mix the sample and purify (40 g normal phase column, PE/EA, 0-0% 10 min, 0-6% 30 min, 6-6% 20 min, flow rate 30 ml/min), and the spot plate was monitored.
  • the pure product fraction was evaporated to obtain a white solid compound 11-2 (4.0 g, 76% yield).
  • Trifluoroacetic acid (10 ml) was added to a solution of compound 11-2 (4.0 g) in dichloromethane (30 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 11-2 completely disappeared and a point with increased polarity was generated.
  • reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (150 ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (100 ml ⁇ 2) was extracted, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, added with appropriate amount of silica gel and DCM, and the sample was mixed and purified (25 g normal phase column, PE/EA, 0-0% 5 min, 0-40% 20 min, 40-40% 15 min, flow rate 30 ml/min) to obtain colorless oily compound 11-3 (2.5 g, 87% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure, and an appropriate amount of silica gel was added to mix the sample and purify (25 g normal phase column, PE/EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml/min), and the plate was monitored.
  • the pure product fraction was evaporated to obtain a colorless oily liquid compound 11-5 (3.0 g, 78% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure.
  • An appropriate amount of silica gel was added for sample mixing and purification (40 g normal phase column, PE/EA, 0-0% in 10 min, 0-6% in 30 min, 6-6% in 20 min, flow rate 30 ml/min) was performed using a spot plate for monitoring.
  • the pure product fractions were evaporated to give a white solid compound 12-2 (4.3 g, 70.4% yield).
  • Trifluoroacetic acid (10 ml) was added to a solution of compound 12-2 (4.3 g) in dichloromethane (30 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 12-2 completely disappeared and a point with increased polarity was generated.
  • reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (150 ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (100 ml ⁇ 2) was extracted, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, added with appropriate amount of silica gel and DCM, and the sample was mixed and purified (25 g normal phase column, PE/EA, 0-0% 5 min, 0-40% 20 min, 40-40% 15 min, flow rate 30 mL/min) to obtain colorless oily compound 12-3 (2.9 g, 89% yield).
  • reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure.
  • An appropriate amount of silica gel was added for sample mixing and purification (40 g normal phase column, PE/EA, 0-0% in 10 min, 0-6% in 30 min, 6-6% in 20 min, flow rate of 30 ml/min) was performed using a spot plate for monitoring.
  • the pure product fractions were evaporated to give a white solid compound 13-1 (3.4 g, 69.9% yield).
  • Trifluoroacetic acid (10 ml) was added to a dichloromethane solution (30 ml) of compound 13-1 (3.4 g). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 13-1 completely disappeared and a point with increased polarity was generated.
  • reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (150 ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (100 ml ⁇ 2) was extracted, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, added with appropriate amount of silica gel and DCM, and the sample was mixed and purified (40 g normal phase column, PE/EA, 0-0% 5 min, 0-40% 20 min, 40-40% 15 min, flow rate 30 mL/min) to obtain colorless oily compound 13-2 (2.1 g, 89% yield).
  • the reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure.
  • An appropriate amount of silica gel was added for sample mixing and purification (80 g normal phase column, PE/EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 40 ml/min), and the plate was monitored.
  • the pure product fraction was evaporated to obtain a colorless oily liquid compound 14-1 (2.1 g, 69.8% yield).
  • Trifluoroacetic acid (5 ml) was added to a dichloromethane solution (20 ml) of compound 14-1 (2.1 g). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 14-1 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried, saturated sodium bicarbonate aqueous solution (100 ml) was added to quench the excess trifluoroacetic acid, and ethyl acetate (100 ml ⁇ 2) was used for extraction.
  • the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and added with appropriate amount of silica gel and DCM to mix the sample and purify (80 g normal phase column, PE/EA, 0-0% 5 min, 0-50% 20 min, 50-50% min, flow rate 50 ml/min) to obtain colorless oily compound 14-2 (2.1 g, 88.9% yield).
  • aqueous phase dilute the mRNA (LUC-mRNA, the nucleotide sequence corresponding to LUC-mRNA is shown in SEQ ID NO: 1 of patent application 202210286081.0) in citric acid-sodium citrate buffer at a final concentration of 0.144 mg/mL.
  • LUC-mRNA the nucleotide sequence corresponding to LUC-mRNA is shown in SEQ ID NO: 1 of patent application 202210286081.0
  • the encapsulation efficiency of the sample was determined using the Ribogreen kit according to the operating instructions, and the sample fluorescence was determined using an ELISA reader at an excitation light of 485 nm and an emission light of 535 nm, and the sample encapsulation efficiency was calculated using the sample fluorescence value.
  • mice Female Balb/c mice aged 6-8 weeks were injected with EPO-mRNA-lipid nanoparticles at 0.5 mg/kg through the tail vein (the nucleotide sequence corresponding to EPO-mRNA is shown in SEQ ID NO: 2 of patent application 202210286081.0). Five parallel mice were used for each formulation, and mouse blood was collected at specific time points (6h and 12h). The basic characteristics of the mRNA used are ARCA cap structure, polyA tail length of 100-120nt, and complete replacement of pseudouracil. The obtained blood was centrifuged at 5000g for 10 minutes at 4°C to separate the serum, and ELISA analysis was performed according to commercially available kits. The test results are detailed in Table 2 and Figure 16.
  • the cationic lipid compound control group SM102 of the present invention was purchased from Xiamen Sinobond (M2212000880009), and the structure is shown below:
  • control group T13 was prepared according to CN114773217A (Compound 35), and the structure is shown below:

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Abstract

本发明提供了一种阳离子脂质化合物、包含其的组合物及应用，所述阳离子脂质化合物如式(I)所示。本发明的提供了一种新的阳离子脂质化合物，丰富了目前阳离子脂质化合物的种类。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。

Description

一种阳离子脂质化合物、包含其的组合物及应用 技术领域
本发明涉及生物医药领域，具体的说，本发明涉及一种阳离子脂质化合物、包含其的组合物及应用。
背景技术
核酸(nucleic acids，RNA)通过体内表达蛋白而具有巨大的潜力，根据蛋白功能的不同，展现出预防与治疗的效果，但裸露的RNA在体内循环时间短，被细胞内化效率低，且极易被肾脏清除或者被体内RNase降解，实际效果大打折扣。为了更好的将核酸递送到靶点用以提升其预防和治疗作用，阳离子脂质化合物与相关制剂是关键技术之一。
LNP(lipid nanoparticles)作为目前主流递送载体，其能够与mRNA结合提高mRNA的体内循坏时间，控制体内逃逸来提高其转染率，增加蛋白表达发挥作用。LNP一般由四种成分组成，包括：(1)阳离子脂质，提供电荷包载mRNA分子；(2)支撑磷脂，提供双分子层支撑，有助于内体逃逸；(3)胆固醇，增强LNP的稳定性，促进膜融合；(4)PEG脂质，减小LNP的粒径，提升体内循环时间。
在体内，阳离子作为LNP关键的成分之一，能够对LNP制剂的无稳定性和作用效力产生巨大的影响，本发明提供一类新型高效、低毒的阳离子化合物。
发明内容
本发明的一个目的在于提供一种阳离子脂质化合物；
本发明的另一目的在于提供一种脂质体制剂；
本发明的再一目的在于提供所述阳离子脂质化合物的用途。
为达上述目的，一方面，本发明提供了一种通式(I)所示的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体：
其中，
环A为取代的或未取代的4-10元含N杂环；所述含N杂环除了与L1连接的N原子外，还含有0、1或2个选自N、O或S的杂原子；当被取代时，所述杂环被1、2、3、4、5或6个选自卤素、羟基、氰基、硝基、羧基、C1-C5烷基或C1-C5烷氧基的取代基所取代；
L1为C1-C6的直链亚烷基；
L2和L3相同或不同，其各自独立的分别为C1-C12的亚烷基；
G1和G2相同或不同，其各自独立的分别为-(C＝O)O-、-O(C＝O)-、-S(C＝O)-、-O(C＝S)-或-(C＝O)S-；
R3和R4相同或者不同，其各自独立的分别为C4-C17的直链烷基、C4-C17的直链烯基或C8-C22的支链非环状烷基。
根据本发明一些具体实施方案，其中，环A为取代的或未取代的4-10元含N杂环；所述含N杂环含有1-3个N原子。
根据本发明一些具体实施方案，其中，环A为取代的或未取代的5元、6元或7元含N杂环。
根据本发明一些具体实施方案，其中，环A为取代的或未取代的5元、6元或7元含N杂环，且所述含N杂环中的杂原子均为N原子。
根据本发明一些具体实施方案，其中，L1为C2-C4的直链亚烷基。
根据本发明一些具体实施方案，其中，L2和L3至少一个为C1-C6亚烷基。
根据本发明一些具体实施方案，其中，L2和L3各自独立的分别为C1-C6的亚烷基。
根据本发明一些具体实施方案，其中，环A为甲基哌嗪基、氢化吡咯基、哌啶基或环己亚胺基。
根据本发明一些具体实施方案，其中，环A为：
根据本发明一些具体实施方案，其中，G1和G2为-(C＝O)O-。
根据本发明一些具体实施方案，其中，G1和G2为-(C＝O)O-，且G1和G2通过其中的-O-分别与R3和R4连接。
根据本发明一些具体实施方案，其中，R3和R4各自独立的分别为：
R5和R6相同或者不同，其各自独立的分别为C3-C11的直链烷基。
其中可以理解的是，R5和R6的碳原子数之和应当满足前面限定的C8-C22的范围，譬如，当R5和R6之一为C3的直链烷基时，另一个最低应为C5的直链烷基。
根据本发明一些具体实施方案，其中，R5和R6相同或者不同，其各自独立的分别为C4-C8的直链烷基。
根据本发明一些具体实施方案，其中，R3和R4各自独立的分别为：
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选自如下结构中的一种或多种：

另一方面，本发明还提供了包含本发明任意一项所述阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体与预防性或治疗性核酸的脂质体制剂。
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核酸的摩尔比为20：1至1：1。
根据本发明一些具体实施方案，其中，所述脂质体制剂的平均粒径为50nm至200nm。
根据本发明一些具体实施方案，其中，所述脂质体制剂还包含一种或多种其他脂质组分，所述其他脂质组分包括中性脂质、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，所述类固醇为β-谷甾醇、野大豆甾醇、麦角甾醇、胆固醇或二氢胆固醇；其中优选胆固醇。
根据本发明一些具体实施方案，其中，所述类固醇与阳离子脂质化合物的摩尔比为(0.5～1)：1。
根据本发明一些具体实施方案，其中，所述聚合物缀合的脂质中的聚合物为聚乙二醇。
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合物缀合的脂质的摩尔比为100:1至20:1。
根据本发明一些具体实施方案，其中，所述聚乙二醇缀合的脂质为PEG2k-DSG、PEG2k-DMG、PEG2k-DPPE、PEG2k-DSPE、PEG2k-cer、PEG2k-DMG或ALC-0159；优选PEG2k-DMG。
根据本发明一些具体实施方案，其中，所述中性脂质选自1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二油酰基-sn-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰基-sn-磷酸甘油钠(DPPG-Na)、鞘磷脂(SM)、神经酰胺及甾醇中的一种或多种的组合。
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中性脂质的摩尔比为2:1至8:1。
根据本发明一些具体实施方案，其中，所述核酸选自mRNA、siRNA、miRNA或ASO；所述核酸优选mRNA。
再一方面，本发明还提供了本发明所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体或本发明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。
综上所述，本发明提供了一种阳离子脂质化合物、包含其的组合物及应用。本发明的阳离子脂质化合物具有如下优点：
本发明的提供了一种新的阳离子脂质化合物，丰富了目前阳离子脂质化合物的种类。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为化合物1的氢谱图。
图2为化合物2的氢谱图。
图3为化合物3的氢谱图。
图4为化合物4的氢谱图。
图5为化合物5的氢谱图。
图6为化合物6的氢谱图。
图7为化合物7的氢谱图。
图8为化合物8的氢谱图。
图9为化合物9的氢谱图。
图10为化合物10的氢谱图。
图11为化合物11的氢谱图。
图12为化合物12的氢谱图。
图13为化合物13的氢谱图。
图14为化合物14的氢谱图。
图15为化合物15的氢谱图。
图16为纳米脂质颗粒组合物递送促红细胞生成素(EPO)mRNA在小鼠内表达效果图。
具体实施方式
以下通过具体实施例详细说明本发明的实施过程和产生的有益效果，旨在帮助阅读者更好地理解本发明的实质和特点，不作为对本案可实施范围的限定。
实施例1
化合物1的合成路线如下：
步骤1：
将化合物1-1(5.0g)溶于DCM(200ml)，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，12.3g)，4-二甲氨基吡啶(DMAP，262mg)，三乙胺(6.51g)和正癸醇(6.79g)分批加入反应体系，室温搅拌16h。取少量反应液稀释与1-1标样对照点板(PE/EA＝10/1，磷钼酸和溴甲酚绿)，观察到极性变小的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(80g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速40ml/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(6.0g，54.5％收率)。
步骤2：
向化合物1-2(6.0g)的二氯甲烷溶液(50ml)溶液中加入三氟乙酸(15ml)。将混合物在室温下搅拌16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。将反应液旋干，加入饱和碳酸氢钠水溶液(100ml)淬灭多余的三氟乙酸，乙酸乙酯(100ml×2)萃取，有机相经无水硫酸钠干燥，过滤，浓缩后，加适量硅胶和DCM拌样、纯化(80g正相柱，PE/EA，0-0％5min，0-50％20min，50-50％min，流速50ml/min)得到白色固体化合物1-3(4.5g，93％收率)。
步骤3：
将化合物1-3(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(115mg)和三光气(86mg)，冰浴下搅拌0.5h。取少量反应液与1-3标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物1-(2-羟乙基)-4-甲基哌嗪(500mg)和吡啶(10mL)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为橙黄色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，在吡啶下方有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到淡黄色油状液体化合物1(200mg，47％收率)。化合物1的氢谱图见图1。
1H NMR(400MHz,Chloroform-d)δ4.23(t,J＝6.0Hz,2H),4.16–4.03(m,8H),2.75–2.30(m,10H),2.28(s,3H),1.67–1.57(m,8H),1.31–1.24(m,24H),0.88(t,J＝8.0Hz,6H).
实施例2
化合物2的合成路线如下：
步骤1：
将化合物1-3(300mg)溶于二氯甲烷(5ml)，冰浴条件下搅拌，依次加入超干吡啶(115mg)和三光气(86mg)，冰浴条件下搅拌0.5h。取少量反应液与1-3标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加二氯甲烷(5ml)溶解后，滴加入化合物1-(3-羟丙基)-4-甲基哌嗪(500mg)和吡啶(10ml)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为橙黄色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，在吡啶下方有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到淡黄色油状液体化合物2(214mg，47％收率)。化合物2的氢谱图见图2。
1H NMR(400MHz,Chloroform-d)δ4.15–4.10(m,6H),4.06(s,2H),2.68–2.30(m,8H),2.28(s,3H),1.83–1.76(m,2H),1.64–1.52(m,12H),1.36–1.13(m,24H),0.88(t,J＝6.8Hz,6H).
实施例3
化合物3的合成路线如下：
步骤1：
将化合物1-1(5.0g)溶于DCM(200ml)，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，12.3g)，4-二甲氨基吡啶(DMAP，262mg),三乙胺(6.51g)和6-十一烷醇(7.39g)分批加入反应体系，室温搅拌16h。取少量反应液稀释与1-1标样对照点板(PE/EA＝10/1，磷钼酸和溴甲酚绿)，观察到极性变小的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(80g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速40ml/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3-1(8.0g，68.9％收率)。
步骤2：
向化合物3-1(8.0g)的二氯甲烷溶液(50ml)溶液中加入三氟乙酸(15ml)。将混合物在室温下搅拌16小时。TLC显示化合物3-1完全消失，有一个极性变大的点生成。将反应液旋干，加入饱和碳酸氢钠水溶液(100ml)淬灭多余的三氟乙酸，乙酸乙酯(100ml×2)萃取，有机相经无水硫酸钠干燥，过滤，浓缩后，加适量硅胶和DCM拌样、纯化(80g正相柱，PE/EA，0-0％5min，0-40％20min，40-40％min，流速50ml/min)得到无色油状液体化合物3-2(5.8g，89％收率)。
步骤3：
将化合物3-2(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(115mg)和三光气(86mg)，冰浴下搅拌0.5h。取少量反应液与3-2标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物1-(2-羟乙基)-4-甲基哌嗪(500mg)和吡啶(10ml)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为橙黄色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，在吡啶下方有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到淡黄色油状液体化合物3(162mg，39％收率)。化合物3的氢谱图见图3。
1H NMR(400MHz,Chloroform-d)δ4.96–4.86(m,2H),4.21(t,J＝6.4Hz,2H),4.12(s,2H),4.04(s,2H),2.71–2.32(m,10H),2.28(s,3H),1.61–1.51(m,10H),1.30–1.24(m,22H),0.90–0.85(m,12H).
实施例4
化合物4的合成路线如下：
步骤1：
将化合物1-1(1.5g)溶于DCM(50ml)，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，3.70g)，4-二甲氨基吡啶(DMAP，79mg)，三乙胺(1.95g)和十五烷-8-醇(2.94g)分批加入反应体系，室温搅拌16h。取少量反应液稀释与1-1标样对照点板(PE/EA＝10/1，磷钼酸和溴甲酚绿)，观察到极性变小的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物4-1(2.5g，59.4％收率)。
步骤2：
向化合物4-1(2.5g)的二氯甲烷溶液(20ml)溶液中加入三氟乙酸(10ml)。将混合物在室温下搅拌16小时。TLC显示化合物4-1完全消失，有一个极性变大的点生成。将反应液旋干，加入饱和碳酸氢钠水溶液(50ml)淬灭多余的三氟乙酸，乙酸乙酯(50ml×2)萃取，有机相经无水硫酸钠干燥，过滤，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，0-50％20min，50-50％5min，流速30ml/min)得到无色油状液体化合物4-2(1.8g，85％收率)。
步骤3：
将化合物4-2(200mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(58mg)和三光气(43mg)，冰浴下搅拌0.5h。取少量反应液与4-2标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，将所得固体用超干二氯甲烷(5ml)溶解后，滴加入化合物1-(3-羟丙基)-4-甲基哌嗪(500mg)和吡啶(10ml)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为橙黄色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，在吡啶下方有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到淡黄色油状液体化合物1(130mg，51％收率)。化合物4的氢谱图见图4。
1H NMR(400MHz,Chloroform-d)δ4.95–4.86(m,2H),4.14(t,J＝8.0Hz 2H),4.11–4.03(m,4H),2.72–2.31(m,10H),2.30(s,3H),1.84–1.77(m,2H),1.56–1.48(m,8H),1.30–1.21(m,40H),0.90–0.85(t,J＝6.8Hz,12H).
实施例5
化合物5的合成路线如下：
步骤1：
将化合物4-2(200mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(58mg)和三光气(43mg)，冰浴下搅拌0.5h。取少量反应液与4-2标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，将所得固体用超干二氯甲烷(5ml)溶解后，滴加入化合物1-(2-羟乙基)-4-甲基哌嗪(500mg)和吡啶(10mL)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为橙黄色，TLC(DCM/MeOH/NH4OH＝10/1/0.1，磷钼酸)显示，在吡啶下方有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到淡黄色油状液体化合物5(130mg，50％收率)。化合物5的氢谱图见图5。
1H NMR(400MHz,Chloroform-d)δ4.79(s,2H),4.08(s,2H),3.87(s,4H),2.79(s,2H),2.54(s,4H),2.49(s,2H),2.44(s,2H),2.27(s,3H),1.68(d,J＝12.4Hz,4H),1.56(d,J＝12.4Hz,4H),1.37(d,J＝0.6Hz,8H),1.34(d,J＝1.2Hz,8H),1.31-1.29(m,24H),0.90(s,12H).
实施例6
化合物6的合成路线如下：
步骤1：
将化合物4-2(200mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(58mg)和三光气(43mg)，冰浴下搅拌0.5h。取少量反应液与1-3标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，将所得固体用超干二氯甲烷(5ml)溶解后，滴加入化合物3-(1-吡咯烷基)-1-丙醇(500mg)和吡啶(10ml)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为红色，TLC(DCM/MeOH/NH4OH＝10/1/0.1，磷钼酸)显示，在吡啶下方有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到淡黄色油状液体化合物6(140mg，54.7％收率)。化合物6的氢谱图见图6。
1H NMR(400MHz,Chloroform-d)δ4.79(s,2H),4.14(s,2H),3.87(s,4H),2.67(s,2H),2.56(s,4H),1.90(s,2H),1.75(s,4H),1.69(d,J＝12.4Hz,4H),1.56(d,J＝12.4Hz,4H),1.37(d,J＝0.6Hz,8H),1.33(d,J＝1.2Hz,8H),1.31(s,8H),1.28(d,J＝4.6Hz,16H),0.90(s,12H).
实施例7
化合物7的合成路线如下：
步骤1：
将化合物1-1(5.0g)溶于DCM(200ml)，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，12.3g)，4-二甲氨基吡啶(DMAP，262mg)，三乙胺(6.51g)和1-十三醇(8.59g)分批加入反应体系，室温搅拌16h。取少量反应液稀释与1-1标样对照点板(PE/EA＝10/1，磷钼酸和溴甲酚绿)，观察到极性变小的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(80g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速40ml/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7-2(8.5g，66.3％收率)。
步骤2：
向化合物7-2(8.5g)的二氯甲烷溶液(100ml)溶液中加入三氟乙酸(30ml)。将混合物在室温下搅拌16小时。TLC显示化合物7-2完全消失，有一个极性变大的点生成。将反应液旋干，加入饱和碳酸氢钠水溶液(150ml)淬灭多余的三氟乙酸，乙酸乙酯(100ml×2)萃取，有机相经无水硫酸钠干燥，过滤，浓缩后，加适量硅胶和DCM拌样、纯化(120g正相柱，PE/EA，0-0％5min，0-50％20min，50-50％min，流速60ml/min)得到白色固体化合物7-3(6.3g，89％收率)。
步骤3：
将化合物7-3(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(96mg)和三光气(72mg)，冰浴下搅拌0.5h。取少量反应液与7-3标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物3-(1-吡咯烷基)-1-丙醇(500mg)和吡啶(10mL)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为红色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，在吡啶下方有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到黄色油状液体化合物7(180mg，45.7％收率)。化合物7的氢谱图见图7。
1H NMR(400MHz,Chloroform-d)δ4.14(s,2H),4.12(s,4H),3.88(s,4H),2.67(s,2H),2.56(s,4H),1.90(s,2H),1.75(s,4H),1.66(s,4H),1.38(s,4H),1.33(d,J＝6.2Hz,8H),1.30–1.23(m,28H),0.90(s,6H).
实施例8
化合物8的合成路线如下：
步骤1：
将化合物8-1(2.0g)溶于DCM(30ml)，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，3.28g)，4-二甲氨基吡啶(DMAP，140mg),三乙胺(1.73g)和(8Z,11Z)-十七碳-8,11-二烯-1-醇(3.17g)分批加入反应体系，室温搅拌16h。取少量反应液稀释与8-1标样对照点板(PE/EA＝10/1，高锰酸钾)，观察到极性变小的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物8-2(3.0g，64％收率)。
步骤2：
向化合物8-2(3.0g)的二氯甲烷溶液(30ml)溶液中加入三氟乙酸(10m)。将混合物在室温下搅拌16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。将反应液旋干，加入饱和碳酸氢钠水溶液(150ml)淬灭多余的三氟乙酸，乙酸乙酯(100ml×2)萃取，有机相经无水硫酸钠干燥，过滤，浓缩后，加适量硅胶和DCM拌样、纯化(120g正相柱，PE/EA，0-0％5min，0-50％20min，50-50％min，流速60ml/min)得到淡黄色油状化合物8-3(1.9g，84％收率)。
步骤3：
将溴乙酸(2.0g)溶于DCM(40ml)，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，4.14g)，4-二甲氨基吡啶(DMAP，176mg),三乙胺(2.18g)和十五烷-8-醇(3.62g)分批加入反应体系，室温搅拌16h。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物8-5(3.6g，72％收率)。
步骤4：
将8-3(1.5g)溶于乙腈(10ml)，室温搅拌，依次称取碳酸钾(1.34g)和8-5(1.86g)分批加入反应体系，反应在70℃搅拌3h。反应液过滤，滤饼用乙酸乙酯洗涤，有机相加适量硅胶拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-30％20min，30-30％10min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物8-4(800mg，28.6％收率)。
步骤5：
将化合物8-4(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(83mg)和三光气(62mg)，冰浴下搅拌0.5h。取少量反应液与8-4标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物3-(1-吡咯烷基)-1-丙醇(500mg)和吡啶(10mL)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为红色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到黄色油状液体化合物8(160mg，42％收率)。化合物8的氢谱图见图8。
1H NMR(400MHz,Chloroform-d)δ5.35(d,J＝16.0Hz,4H),4.79(s,1H),4.13(d,J＝9.8Hz,4H),3.88(d,J＝4.4Hz,4H),2.80–2.64(m,4H),2.56(s,4H),2.08–2.00(dt,J＝9.0,1.0Hz,4H),1.90(s,2H),1.75(s,4H),1.72–1.63(m,4H),1.55(d,J＝12.6Hz,2H),1.38–1.25(m,34H),0.90(s,9H).
实施例9
化合物9的合成路线如下：
步骤1：
将化合物8-1(3.0g)溶于DCM(30ml)，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，4.92g)，4-二甲氨基吡啶(DMAP，210mg),三乙胺(2.60g)和十一烷醇(3.25g)分批加入反应体系，室温搅拌16h。取少量反应液稀释与8-1标样对照点板(PE/EA＝20/1，磷钼酸)，观察到极性接近的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％10min，0-6％30min，6-6％20min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到白色固体化合物9-1(3.2g，57％收率)。
步骤2：
向化合物9-1(3.2g)的二氯甲烷溶液(30mL)溶液中加入三氟乙酸(10ml)。将混合物在室温下搅拌16小时。TLC显示化合物9-1完全消失，有一个极性变大的点生成。将反应液旋干，加入饱和碳酸氢钠水溶液(150ml)淬灭多余的三氟乙酸，乙酸乙酯(100ml×2)萃取，有机相经无水硫酸钠干燥，过滤，浓缩后，加适量硅胶和DCM拌样、纯化(120g正相柱，PE/EA，0-0％5min，0-40％20min，40-40％15min，流速30mL/min)得到无色油状化合物9-2(2.0g，90％收率)。
步骤3：
将溴乙酸(2.0g)溶于DCM(40ml)，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，4.14g)，4-二甲氨基吡啶(DMAP，176mg),三乙胺(2.18g)和十五烷-7-醇(3.62g)分批加入反应体系，室温搅拌16h。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物9-4(3.6g，70％收率)。
步骤4：
将9-2(2.0g)溶于乙腈(20ml)，室温搅拌，依次称取碳酸钾(2.41g)和9-4(3.35g)分批加入反应体系，反应在70℃搅拌3h。反应液过滤，滤饼用乙酸乙酯洗涤，有机相加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-30％20min，30-30％20min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物9-3(900mg，20.7％收率)。
步骤5：
将化合物9-3(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(96mg)和三光气(72mg)，冰浴下搅拌0.5h。取少量反应液与9-3标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物1-(3-羟丙基)-4-甲基哌嗪(500mg)和吡啶(10ml)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为红色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到黄色油状液体化合物9(180mg，43.9％收率)。化合物9的氢谱图见图9。
1H NMR(400MHz,Chloroform-d)δ4.79(s,1H),4.14–4.11(m,4H),3.87(d,J＝4.4Hz,4H),2.66(d,J＝1.6Hz,2H),2.50–2.44(m,8H),2.27(s,3H),2.01–1.83(m,2H),1.73–1.63(m,4H),1.55(d,J＝12.4Hz,2H),1.40–1.36(m,6H),1.34(d,J＝1.0Hz,2H),1.34–1.30(m,10H),1.30–1.25(m,18H),0.90(s,9H).
实施例10
化合物10的合成
步骤1：
将化合物3-2(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(115mg)和三光气(86mg)，冰浴下搅拌0.5h。取少量反应液与3-2标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物N-(2-羟乙基)六亚甲二胺(500mg)和吡啶(10mL)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为橙黄色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，在吡啶下方有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到淡黄色油状液体化合物10(170mg，41％收率)。化合物10的氢谱图见图10。
1H NMR(400MHz,Chloroform-d)δ4.95–4.86(m,2H),4.27–4.22(m,2H),4.09(d,J＝12.0Hz,4H),2.78–2.70(m,2H),2.65–2.50(m,4H),1.80–1.30(m,16),1.28–1.10(m,24H),0.87(t,J＝4.0Hz,12H).
实施例11
化合物11的合成路线如下：
步骤1：
将化合物11-1(3.0g)溶于DCM(30ml)，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，3.40g)，4-二甲氨基吡啶(DMAP，180mg),三乙胺(2.24g)和5-十一醇(2.80g)分批加入反应体系，室温搅拌16h。取少量反应液稀释对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％10min，0-6％30min，6-6％20min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到白色固体化合物11-2(4.0g，76％收率)。
步骤2：
向化合物11-2(4.0g)的二氯甲烷溶液(30ml)溶液中加入三氟乙酸(10ml)。将混合物在室温下搅拌16小时。TLC显示化合物11-2完全消失，有一个极性变大的点生成。将反应液旋干，加入饱和碳酸氢钠水溶液(150ml)淬灭多余的三氟乙酸，乙酸乙酯(100ml×2)萃取，有机相经无水硫酸钠干燥，过滤，浓缩后，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-40％20min，40-40％15min，流速30ml/min)得到无色油状化合物11-3(2.5g，87％收率)。
步骤3：
将4-溴丁酸(2.0g)溶于DCM(40ml)，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，3.44g)，4-二甲氨基吡啶(DMAP，147mg),三乙胺(1.82g)和5-十一醇(2.27g)分批加入反应体系，室温搅拌16h。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物11-5(3.0g，78％收率)。
步骤4：
将11-3(2.0g)溶于乙腈(20ml)，室温搅拌，依次称取碳酸钾(2.15g)和11-5(2.75g)分批加入反应体系，反应在70℃搅拌3h。反应液过滤，滤饼用乙酸乙酯洗涤，有机相加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-40％20min，40-40％20min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物11-4(950mg，24.6％收率)。
步骤5：
将化合物11-4(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(96mg)和三光气(72mg)，冰浴下搅拌0.5h。取少量反应液与11-4标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物N-(2-羟乙基)-吡咯烷(500mg)和吡啶(10ml)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为红色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到黄色油状液体化合物11(160mg，41.5％收率)。化合物11的氢谱图见图11。
1H NMR(400MHz,Chloroform-d)δ4.77(s,2H),4.10(d,J＝0.8Hz,2H),3.27(d,J＝0.8Hz,4H),2.85–2.70(m,6H),2.40–2.29(m,4H),2.28–2.16(m,4H),1.76(d,J＝4.0Hz,4H),1.72–1.63(dd,J＝12.4,4.0Hz,4H),1.61–1.51(dd,J＝12.4,8.4Hz,4H),1.42–1.32(m,16H),1.30(d,J＝8.4Hz,8H),0.91(d,J＝10.6Hz,12H).
实施例12
化合物12的合成路线如下：
步骤1：
将化合物12-1(3.00g)溶于DCM(30ml)，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，4.24g)，4-二甲氨基吡啶(DMAP，180mg)，三乙胺(2.24g)和十五烷-7-醇(3.71g)分批加入反应体系，室温搅拌16h。取少量反应液稀释对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％10min，0-6％30min，6-6％20min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到白色固体化合物12-2(4.3g，70.4％收率)。
步骤2：
向化合物12-2(4.3g)的二氯甲烷溶液(30ml)溶液中加入三氟乙酸(10ml)。将混合物在室温下搅拌16小时。TLC显示化合物12-2完全消失，有一个极性变大的点生成。将反应液旋干，加入饱和碳酸氢钠水溶液(150ml)淬灭多余的三氟乙酸，乙酸乙酯(100ml×2)萃取，有机相经无水硫酸钠干燥，过滤，浓缩后，加适量硅胶和DCM拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-40％20min，40-40％15min，流速30mL/min)得到无色油状化合物12-3(2.9g，89％收率)。
步骤3：
将4-溴丁酸(2.0g)溶于DCM(40ml)，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，3.44g)，4-二甲氨基吡啶(DMAP，147mg),三乙胺(1.82g)和十五烷-7-醇(3.01g)分批加入反应体系，室温搅拌16h。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(25g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物12-5(3.3g，66.6％收率)。
步骤4：
将12-3(2.0g)溶于乙腈(20ml)，室温搅拌，依次称取碳酸钾(1.76g)和12-5(2.65g)分批加入反应体系，反应在70℃搅拌3h。反应液过滤，滤饼用乙酸乙酯洗涤，有机相加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-40％20min，40-40％20min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物12-4(800mg，22.6％收率)。
步骤5：
将化合物12-4(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(85mg)和三光气(64mg)，冰浴下搅拌0.5h。取少量反应液与12-4标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物1-(3-羟丙基)-4-甲基哌嗪(500mg)和吡啶(10mL)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为红色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到黄色油状液体化合物12(180mg，52.0％收率)。化合物12的氢谱图见图12。
1H NMR(400MHz,Chloroform-d)δ4.78(s,2H),4.15(d,J＝0.8Hz,2H),3.26(d,J＝0.8Hz,4H),2.65(d,J＝1.2Hz,2H),2.50(s,2H),2.47(s,2H),2.45(s,2H),2.44(s,2H),2.38–2.29(m,4H),2.28–2.16(m,7H),1.96–1.84(m,2H),1.73–1.51(m,8H),1.41–1.36(m,8H),1.34(d,J＝1.0Hz,4H),1.31(d,J＝6.8Hz,12H),1.30–1.25(dd,J＝4.9,1.0Hz,16H),0.90(s,12H).
实施例13
化合物13的合成路线如下：
步骤1：
将化合物4-(叔丁氧羰基氨基)丁酸(3.00g)溶于DCM(30ml)，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，3.40g)，4-二甲氨基吡啶(DMAP，180mg),三乙胺(2.24g)和1-壬醇(2.13g)分批加入反应体系，室温搅拌16h。取少量反应液稀释对照点板(PE/EA＝10/1，磷钼酸)，观察到极性变小的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％10min，0-6％30min，6-6％20min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到白色固体化合物13-1(3.4g，69.9％收率)。
步骤2：
向化合物13-1(3.4g)的二氯甲烷溶液(30ml)溶液中加入三氟乙酸(10ml)。将混合物在室温下搅拌16小时。TLC显示化合物13-1完全消失，有一个极性变大的点生成。将反应液旋干，加入饱和碳酸氢钠水溶液(150ml)淬灭多余的三氟乙酸，乙酸乙酯(100ml×2)萃取，有机相经无水硫酸钠干燥，过滤，浓缩后，加适量硅胶和DCM拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-40％20min，40-40％15min，流速30mL/min)得到无色油状化合物13-2(2.1g，89％收率)。
步骤3：
将13-2(2.0g)溶于乙腈(20ml)，室温搅拌，依次称取碳酸钾(1.76g)和12-5(2.65g)分批加入反应体系，反应在70℃搅拌3h。反应液过滤，滤饼用乙酸乙酯洗涤，有机相加适量硅胶拌样、纯化(40g正相柱，PE/EA，0-0％5min，0-40％20min，40-40％20min，流速30ml/min)，点板监测，将纯产物部分馏分蒸发，得到无色油状液体化合物13-3(800mg，22.6％收率)。
步骤4：
将化合物13-3(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(90mg)和三光气(68mg)，冰浴下搅拌0.5h。取少量反应液与13-3标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物1-(2-羟乙基)-4-甲基哌嗪(500mg)和吡啶(10mL)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为橙红色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到黄色油状液体化合物13(200mg，50.4％收率)。化合物13的氢谱图见图13。
1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.15–4.08(m,4H),3.27(d,J＝0.8Hz,4H),2.79(d,J＝1.6Hz,2H),2.56(s,2H),2.52(s,2H),2.49(s,2H),2.44(s,2H),2.40–2.31(m,4H),2.30–2.24(m,6H),2.22–2.20(m,1H),1.72–1.61(m,4H),1.55(d,J＝12.4Hz,2H),1.39(d,J＝2.4Hz,4H),1.36(d,J＝0.6Hz,2H),1.34(d,J＝1.0Hz,2H),1.33(d,J＝1.0Hz,4H),1.31(s,6H),1.30–1.27(m,14H),0.90(d,J＝10.6Hz,9H).
实施例14
化合物14的合成路线如下：
步骤1：
将化合物1-1(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，2.47g)，4-二甲氨基吡啶(DMAP，524mg),三乙胺(1.30g)和(8Z,11Z)-十七碳-8,11-二烯-1-醇(2.16g)分批加入反应体系，室温搅拌16h。取少量反应液稀释与1-1标样对照点板(PE/EA＝10/1，磷钼酸和溴甲酚绿)，观察到极性变小的新点。反应液加水淬灭，分液，有机相减压蒸发，加适量硅胶拌样、纯化(80g正相柱，PE/EA，0-0％5min，0-10％20min，10-10％10min，流速40ml/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14-1(2.1g，69.8％收率)。
步骤2：
向化合物14-1(2.1g)的二氯甲烷溶液(20ml)溶液中加入三氟乙酸(5ml)。将混合物在室温下搅拌16小时。TLC显示化合物14-1完全消失，有一个极性变大的点生成。将反应液旋干，加入饱和碳酸氢钠水溶液(100ml)淬灭多余的三氟乙酸，乙酸乙酯(100ml×2)萃取，有机相经无水硫酸钠干燥，过滤，浓缩后，加适量硅胶和DCM拌样、纯化(80g正相柱，PE/EA，0-0％5min，0-50％20min，50-50％min，流速50ml/min)得到无色油状化合物14-2(2.1g，88.9％收率)。
步骤3：
将化合物14-2(300mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(79mg)和三光气(60mg)，冰浴下搅拌0.5h。取少量反应液与14-2标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，所得固体加超干二氯甲烷(5ml)溶解后，滴加入化合物1-(2-羟乙基)-4-甲基哌嗪(500mg)和吡啶(10ml)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为橙黄色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到淡黄色油状液体化合物14(160mg，41.6％收率)。化合物14的氢谱图见图14。
1H NMR(400MHz,Chloroform-d)δ5.37(s,4H),5.33(s,4H),4.12(s,4H),4.08(s,2H),3.88(s,4H),2.79(s,2H),2.78–2.66(m,4H),2.54(s,4H),2.49(s,2H),2.44(s,2H),2.27(s,3H),2.07–2.01(dt,J＝9.0Hz,1.0Hz,8H),1.37(s,4H),1.35–1.30(m,20H),1.29(s,4H),0.90(s,6H).
实施例15
化合物15的合成路线如下：
步骤1：
将化合物4-2(200mg)溶于超干二氯甲烷(5ml)，在冰浴下搅拌，依次加入超干吡啶(58mg)和三光气(43mg)，冰浴下搅拌0.5h。取少量反应液与4-2标样对照点板(PE/EA＝1/1，磷钼酸)，观察到极性变小的新点。反应液减压蒸发，将所得固体用超干二氯甲烷(5ml)溶解后，滴加入化合物1-哌啶丙醇(500mg)和吡啶(10ml)的混合溶液中，滴加完成后，反应液在70℃下搅拌三小时，溶液为红色，TLC(DCM/MeOH/NH4OH＝10/1/0.1,磷钼酸)显示，在吡啶下方有一个新点生成，加适量硅胶和二氯甲烷拌样、纯化(10g正相柱，DCM:DCM/MeOH/NH4OH，0-0％5min，0-70％20min，70-70％10min，流速20ml/min)得到黄色油状液体化合物15(164mg，62.8％收率)。化合物15的氢谱图见图15。
1H NMR(400MHz,Chloroform-d)δ4.79(s,2H),4.14(s,2H),3.87(s,4H),2.66(s,2H),2.47(s,4H),1.90(s,2H),1.69(d,J＝12.4Hz,4H),1.60–1.52(d,J＝12.4Hz,8H),1.46(s,2H),1.37(d,J＝0.6Hz,8H),1.35–1.25(m,32H),0.90(s,12H).
实施例16
脂质纳米颗粒的制备及性状表征
配制水相：将mRNA(LUC-mRNA，LUC-mRNA对应的核苷酸序列见专利申请202210286081.0的SEQ ID NO:1)按照0.144mg/mL的终浓度稀释于柠檬酸-柠檬酸钠缓冲液中。
配制有机相：将上述阳离子脂质、DSPC、胆固醇和PEG2k-DMG按照总浓度10mg/mL溶于乙醇中(组分比例为阳离子脂质：DSPC：胆固醇：PEG2k-DMG＝50:10:38.5:1.5)。
将3ml水相缓冲液和1ml脂质有机相加入到15ml离心管中，分别连接于流控的A、B两端，将芯片安装到微流控设备中，设置一定的流速比例，先用纯水和纯乙醇进行预实验，待压力与流速均处于平稳时，加入料液，料液流经芯片同时观察芯片出口的样品颜色，弃去前、后3～5滴乳白色液滴(约100μL)，将中端样品收集到EP管中，随后将样品快速转移到透析袋中，在20mM Tris-HCl缓冲液中透析12-24h，透析结束后转移至4℃冰箱保存。
使用Ribogreen试剂盒，按照操作说明测定样品的包封率，使用酶标仪在激发光485nm，发射光535nm处测定样品荧光，通过样品荧光值计算样品包封率。
使用马尔文公司的Zetasizer nano仪器上使用标准检测方法进行粒径与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。
表1
实施例17
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
以0.5mg/kg对6-8周龄的雌性Balb/c小鼠通过尾静脉注射EPO-mRNA-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)，每组配方使用5只平行鼠，在特定时间点(6h和12h)分别采集小鼠血液。其中所用到的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。将所得的血液在4℃下5000g离心10min分离出血清，根据市售试剂盒进行ELISA分析，各项检测结果详见表2和图16。
表2
本发明所述阳离子脂质化合物对照组SM102购自厦门赛诺邦格(M2212000880009)，结构如下所示：
对照组T13按照CN114773217A制备(化合物35)，结构如下所示：

Claims (23)

  1. 一种通式(I)所示的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体：
    其中，
    环A为取代的或未取代的4-10元含N杂环；所述含N杂环除了与L1连接的N原子外，还含有0、1或2个选自N、O或S的杂原子；当被取代时，所述含N杂环被1、2、3、4、5或6个选自卤素、羟基、氰基、硝基、羧基、C1-C5烷基或C1-C5烷氧基的取代基所取代；
    L1为C1-C6的直链亚烷基；
    L2和L3相同或不同，其各自独立的分别为C1-C12的亚烷基；
    G1和G2相同或不同，其各自独立的分别为-(C＝O)O-、-O(C＝O)-、-S(C＝O)-、-O(C＝S)-或-(C＝O)S-；
    R3和R4相同或者不同，其各自独立的分别为C4-C17的直链烷基、C4-C17的直链烯基或C8-C22的支链非环状烷基。
  2. 根据权利要求1所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体，其中，环A为取代的或未取代的5元、6元或7元含N杂环；优选环A为取代的或未取代的5元、6元或7元含N杂环，且所述含N杂环中的杂原子均为N原子。
  3. 根据权利要求1所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体，其中，L1为C2-C4的直链亚烷基。
  4. 根据权利要求1所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体，其中，L2和L3至少一个为C1-C6亚烷基。
  5. 根据权利要求4所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体，其中，L2和L3各自独立的分别为C1-C6的亚烷基。
  6. 根据权利要求1所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体，其中，环A为甲基哌嗪基、氢化吡咯基、哌啶基或环己亚胺基。
  7. 根据权利要求1所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体，其中，G1和G2为-(C＝O)O-；优选的，G1和G2通过其中的-O-分别与R3和R4连接。
  8. 根据权利要求1所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体，其中，当R3和R4任一为C8-C22的支链非环状烷基时，R3和R4各自独立的分别为：
    R5和R6相同或者不同，其各自独立的分别为C3-C11的直链烷基。
  9. 根据权利要求1所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体，其中，R3和R4各自独立的分别为：
  10. 根据权利要求1所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体，其中，所述阳离子脂质化合物选自如下结构中的一种或多种：

  11. 包含权利要求1-10任意一项所述阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体与预防性或治疗性核酸的脂质体制剂。
  12. 根据权利要求11所述的脂质体制剂，其中，所述阳离子脂质化合物与所述核酸的摩尔比为20：1至1：1。
  13. 根据权利要求11所述的脂质体制剂，其中，所述脂质体制剂的平均粒径为50nm至200nm。
  14. 根据权利要求11所述的脂质体制剂，其中，所述脂质体制剂还包含一种或多种其他脂质组分，所述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. 根据权利要求14所述的脂质体制剂，其中，所述类固醇为β-谷甾醇、野大豆甾醇、麦角甾醇、胆固醇或二氢胆固醇；其中优选胆固醇。
  16. 根据权利要求14所述的脂质体制剂，其中，所述类固醇与阳离子脂质化合物的摩尔比为(0.5～1)：1。
  17. 根据权利要求14所述的脂质体制剂，其中，所述聚合物缀合的脂质中的聚合物为聚乙二醇。
  18. 根据权利要求14所述的脂质体制剂，其中，所述阳离子脂质化合物与所述聚合物缀合的脂质的摩尔比为100:1至20:1。
  19. 根据权利要求17所述的脂质体制剂，其中，所述聚乙二醇缀合的脂质为PEG2k-DSG、PEG2k-DMG、PEG2k-DPPE、PEG2k-DSPE、PEG2k-cer、PEG2k-DMG或ALC-0159；优选PEG2k-DMG。
  20. 根据权利要求14所述的脂质体制剂，其中，所述中性脂质选自1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-3-磷酸乙醇胺、1,2-二棕榈酰基-sn-磷酸甘油钠、鞘磷脂、神经酰胺及甾醇中的一种或多种的组合。
  21. 根据权利要求14所述的脂质体制剂，其中，所述阳离子脂质化合物与所述中性脂质的摩尔比为2:1至8:1。
  22. 根据权利要求11所述的脂质体制剂，其中，所述核酸选自mRNA、siRNA、miRNA或反义寡核苷酸；所述核酸优选mRNA。
  23. 权利要求1-10任意一项所述的阳离子脂质化合物或其药物可用的盐、互变异构体或立体异构体或权利要求11-22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。
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