WO2023216232A1 - 一种含有二硫键的脂质化合物及其组合物 - Google Patents

一种含有二硫键的脂质化合物及其组合物 Download PDF

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WO2023216232A1
WO2023216232A1 PCT/CN2022/092697 CN2022092697W WO2023216232A1 WO 2023216232 A1 WO2023216232 A1 WO 2023216232A1 CN 2022092697 W CN2022092697 W CN 2022092697W WO 2023216232 A1 WO2023216232 A1 WO 2023216232A1
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compound
lipid
polyethylene glycol
lipid compound
modified
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PCT/CN2022/092697
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French (fr)
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曾臣
申志高
汪子芊
王鹏
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南方科技大学
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    • 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—Liposomes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

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  • the present invention relates to the field of compounds, and in particular to a lipid compound containing disulfide bonds and a composition thereof.
  • the mRNA vaccine has topped the list of the "Top Ten Global Breakthrough Technologies" in 2021 due to the huge changes it has brought about in the medical field.
  • the approval of the COVID-19 mRNA vaccine has triggered the third revolution in biomedicine after small molecule drugs and protein drugs.
  • Therapeutics based on mRNA technology will likely be applied to important fields such as infectious disease prevention, tumor treatment, rare disease treatment, monoclonal antibodies and other drug replacement treatments at a faster rate.
  • mRNA is a negatively charged polymer and is extremely hydrophilic. It is difficult for it to pass through the cell membrane, which is also negatively charged, and it is difficult for it to enter the cell itself. It is also very unstable and easily It is degraded by ubiquitous nucleases and has a short circulation time in the blood; mRNA itself is immunogenic and is quickly recognized by the immune system after being injected into the human body, triggering an immune response and being eliminated.
  • the mRNA vaccine delivery system effectively solves this series of problems.
  • Lipid nanoparticles (LNP) technology has high delivery efficiency and good safety, and is currently the most popular delivery technology.
  • LNP is usually composed of cationic lipids or ionizable cationic lipids, auxiliary lipids, structural lipids and modified polyethylene glycol lipids. Therefore, there is a need to develop lipid compounds capable of delivering nucleic acid drugs and related methods and compositions to promote the delivery of various nucleic acid drugs in cells, tissues and in vivo.
  • a lipid compound comprising: a lipid compound represented by general formula (I) and/or (II), or a pharmaceutically acceptable compound of the lipid compound Salt, or solvate of said lipid compound:
  • Each L 1 , L 2 and L 3 is independently selected from an alkyl group with a length of 1 to 22 carbon atoms, the alkyl group is a straight chain or contains at least one branch, and the carbon atoms on the alkyl group are not Substituted, alternatively, at least one carbon atom on the alkyl group is optionally replaced with at least one selected from the group consisting of: -C(O)-, -NH-, -O-, - S-, C 2 -C 10 alkenyl, C 2 ⁇ C 10 alkynyl, C 3 ⁇ C 8 cycloalkyl and C 6 ⁇ C 10 arylene;
  • R 1 is selected from the following structures: -NH 2 , monosubstituted amine group, disubstituted amine group, substituted or unsubstituted nitrogen-containing saturated heterocycle, substituted or unsubstituted azaaromatic compound;
  • R 2 and R 3 are independently selected from the following structures: 1) hydrogen atoms; 2) an alkyl group containing 1 to 22 carbon atoms, the alkyl group being a straight chain or containing at least one branch, the alkyl group having The carbon atoms are unsubstituted, or at least one carbon atom on the alkyl group is optionally replaced with at least one selected from the group consisting of: -C(O)-, -NH-, - O-, -S-, C 2 -C 10 alkenyl, C 2 to C 10 alkynyl, C 3 to C 8 cycloalkyl and C 6 to C 10 arylene.
  • a nanoparticle preparation including the lipid compound described in the first aspect.
  • a composition comprising the lipid compound of the first aspect, the lipid compound being loaded with an active agent.
  • the lipid compound containing a disulfide bond and a composition thereof according to the above embodiment, is non-toxic, and the lipid nanoparticles formed therefrom can efficiently deliver active reagents (including but not limited to nucleic acids), and have high Encapsulation efficiency and stability.
  • Figure 1.1 is the particle size diagram of compound 1.
  • Figure 1.2 is the particle size diagram of compound 2.
  • Figure 1.3 is the particle size diagram of compound 3.
  • Figure 1.4 is the particle size diagram of compound 4.
  • Figure 2 shows the expression intensity of Fluc mRNA delivered by intramuscular injection.
  • Figure 3.1 shows the hydrogen spectrum of compound 1.
  • Figure 3.2 shows the carbon spectrum of compound 1.
  • Figure 4.1 shows the hydrogen spectrum of compound 2.
  • Figure 4.2 shows the carbon spectrum of compound 2.
  • Figure 5.1 shows the hydrogen spectrum of compound 3.
  • Figure 5.2 shows the carbon spectrum of compound 3.
  • Figure 6.1 shows the hydrogen spectrum of compound 4.
  • Figure 6.2 shows the carbon spectrum of compound 4.
  • Figure 7.1 shows the hydrogen spectrum of compound 5.
  • Figure 7.2 shows the carbon spectrum of compound 5.
  • Figure 8.1 shows the hydrogen spectrum of compound 6.
  • Figure 8.2 shows the carbon spectrum of compound 6.
  • Figure 9.1 shows the hydrogen spectrum of compound 7.
  • Figure 9.2 shows the carbon spectrum of compound 7.
  • Figure 10.1 shows the hydrogen spectrum of compound 8.
  • Figure 10.2 shows the carbon spectrum of compound 8.
  • Figure 11.1 shows the hydrogen spectrum of compound 9.
  • Figure 11.2 shows the carbon spectrum of compound 9.
  • Figure 12.1 shows the hydrogen spectrum of compound 10.
  • Figure 12.2 shows the carbon spectrum of compound 10.
  • Figure 13.1 is the hydrogen spectrum of compound 11.
  • Figure 13.2 shows the carbon spectrum of compound 11.
  • Figure 14.1 is the hydrogen spectrum of compound 12.
  • Figure 14.2 shows the carbon spectrum of compound 12.
  • Figure 15.1 is the hydrogen spectrum of compound 13.
  • Figure 15.2 shows the carbon spectrum of compound 13.
  • Figure 16.1 shows the hydrogen spectrum of compound 14.
  • Figure 16.2 shows the carbon spectrum of compound 14.
  • Figure 17.1 shows the hydrogen spectrum of compound 15.
  • Figure 17.2 shows the carbon spectrum of compound 15.
  • Figure 18.1 shows the hydrogen spectrum of compound 16.
  • Figure 18.2 shows the carbon spectrum of compound 16.
  • Figure 19.1 shows the hydrogen spectrum of compound 17.
  • Figure 19.2 shows the carbon spectrum of compound 17.
  • Figure 20.1 is the hydrogen spectrum of compound 18.
  • Figure 20.2 shows the carbon spectrum of compound 18.
  • Figure 21.1 is the hydrogen spectrum of compound 19.
  • Figure 21.2 shows the carbon spectrum of compound 19.
  • connection and “connection” mentioned in this application include direct and indirect connections (connections) unless otherwise specified.
  • Disulfide bonds play an important role in protein folding to form secondary structure, reducing protein structural entropy and increasing protein mechanical strength.
  • animal hair contains a lot of cysteine, which is used to form disulfide bonds.
  • cysteine content in membrane proteins is higher than that of other proteins.
  • transmembrane proteins and secreted proteins must have a signal sequence added to their end after assembly in the Golgi apparatus, and this sequence is connected to the protein through disulfide bonds. This shows that the disulfide bond is stable in vivo and has excellent biocompatibility.
  • the reaction of disulfide bonds is a very common bioorthogonal reaction.
  • disulfide bonds some compounds or drugs with specific properties can be connected to some biological macromolecules in order to study the mechanism of action of biological macromolecules or improve Performance of antibody drugs.
  • the ADC drugs Mylotarg and Besponsa use disulfide bonds as cleavable linkers to connect to small molecule toxic drugs.
  • the present invention introduces a disulfide bond structure into a cationic lipid compound and prepares LNP.
  • the LNP is stable in the blood. Once it enters cells through endocytosis, intracellular reduced glutathione (GSH) can react with cationic lipids through thiol-disulfide exchange, thereby destroying the structure of the LNP. Promote the release of mRNA.
  • GSH reduced glutathione
  • the disulfide bond-containing lipid compound of the present invention and the LNP composed of it can efficiently deliver nucleic acids in the body, and at the same time, due to their biodegradability, they will have better safety.
  • a lipid compound comprising: a lipid compound represented by general formula (I) and/or (II), or a pharmaceutically acceptable compound of the lipid compound Salt, or solvate of said lipid compound:
  • Each L 1 , L 2 and L 3 is independently selected from an alkyl group having a length of 1 to 22 carbon atoms, the carbon atoms on the alkyl group being unsubstituted, or at least one carbon atom on the alkyl group Optionally replaced by at least one selected from the group consisting of: -C(O)-, -NH-, -O-, -S-, C 2 -C 10 alkenyl, C 2 ⁇ C 10 alkynyl, C 3 ⁇ C 8 cycloalkyl and C 6 ⁇ C 10 arylene;
  • R 1 is selected from the following structures: -NH 2 , monosubstituted amine group, disubstituted amine group, substituted or unsubstituted nitrogen-containing saturated heterocycle, substituted or unsubstituted azaaromatic compound;
  • R 2 and R 3 are independently selected from the following structures: 1) hydrogen atoms; 2) an alkyl group containing 1 to 22 carbon atoms, the alkyl group being a straight chain or containing at least one branch, the alkyl group having The carbon atoms are unsubstituted, or at least one carbon atom on the alkyl group is optionally replaced with at least one selected from the group consisting of: -C(O)-, -NH-, - O-, -S-, C 2 -C 10 alkenyl, C 2 to C 10 alkynyl, C 3 to C 8 cycloalkyl and C 6 to C 10 arylene.
  • the lipid compound includes at least one of the following structures:
  • n is an integer from 1 to 9.
  • each R 2 and R 3 is a hydrogen atom.
  • each L 2 and L 3 are independently selected from a linear alkylene or alkenyl structure with a length of 8 to 22 carbon atoms.
  • each L 2 and L 3 are independently selected from the following structure:
  • R 1 can be selected from the following structures:
  • the nitrogen-containing aromatic compound includes but is not limited to at least one of imidazole, pyrazole, pyridine, and pyrrole.
  • the alkane chain -(CH 2 ) n - between R 1 and the ester bond can be a straight chain, and n is an integer from 0 to 6.
  • R1 is selected from any one of the following structures:
  • the alkane chain -(CH 2 ) n - between R 1 and the ester bond is selected from any one of the following structures:
  • the lipid compound is selected from at least one of the following structures:
  • a nanoparticle preparation comprising the lipid compound of any one of the first aspects.
  • the nanoparticle preparation further includes at least one of auxiliary lipids, structural lipids, and polyethylene glycol lipids.
  • the molar ratio of the lipid compound to the auxiliary lipid, structural lipid, and polyethylene glycol lipid is (40-60): (5-25): (25-50): (0.1 ⁇ 10).
  • the auxiliary lipid is an ionic lipid or a neutral lipid, which may contain cationic functional groups (for example, amine groups, quaternary ammonium groups) and/or anionic functional groups (for example, phosphate groups, carboxylic acid groups) group).
  • cationic functional groups for example, amine groups, quaternary ammonium groups
  • anionic functional groups for example, phosphate groups, carboxylic acid groups
  • the average particle size of the nanoparticle preparation is 30 nm to 200 nm.
  • the polydispersity index of the nanoparticle formulation is ⁇ 0.3.
  • the mass ratio of the carrier to the therapeutic or preventive agent is (3-50):1.
  • the auxiliary lipid includes but is not limited to at least one of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, glycolipids, and lipids.
  • the auxiliary lipids include but are not limited to dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), (2,3-dioleoyl-propyl) -At least one of trimethylamine (DOTAP) and (2,3-dioleoyl-propyl)-dimethylamine (DODAP).
  • DOPE dioleoylphosphatidylethanolamine
  • DSPC distearoylphosphatidylcholine
  • DOTAP 2,3-dioleoyl-propyl
  • DODAP 2,3-dioleoyl-propyl-dimethylamine
  • the structural lipids can be used to stabilize lipid nanoparticles and aid in fusion with cell membranes.
  • the structural lipids include, but are not limited to, cholesterol, vitamin D, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassisterol, tomatine, tomatine, and ursolic acid. , ⁇ -tocopherol, and at least one of corticosteroids.
  • the hydrophilic polyethylene glycol chains of the polyethylene glycol-modified lipid are distributed on the surface of the nanoparticles, preventing fusion between nanoparticles and reducing the immunogenicity of the particles to prevent them from being eliminated by the immune system in the body. , thereby prolonging the circulation time in the body.
  • the polyethylene glycol-modified lipids include, but are not limited to, polyethylene glycol (PEG)-modified phosphatidylethanolamine, polyethylene glycol-modified phosphatidic acid, and polyethylene glycol-modified ceramide. , at least one of polyethylene glycol-modified dialkylamine, polyethylene glycol-modified diacylglycerol, and polyethylene glycol-modified dialkylglycerol.
  • the polyethylene glycol-modified lipids include, but are not limited to, 1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol (PEG 2000-DMG) (CAS No.: 1397695-86-1).
  • the lipid nanoparticles are used to deliver active therapeutic agents.
  • a composition comprising the lipid compound of the first aspect, and the lipid nanoparticles formed by the lipid compound are loaded with an active agent.
  • the active agent can be bound to the surface of the lipid compound or to the interior of the lipid compound.
  • the composition comprises the nanoparticle formulation of the second aspect, the nanoparticle formulation is loaded with an active agent.
  • the active agent includes, but is not limited to, at least one of a preventive agent and a therapeutic agent.
  • the active reagents include, but are not limited to, nucleic acids, immunomodulators, antigens or fragments thereof, vaccines, anti-inflammatory agents, anti-tumor agents, antibiotics, agents that act on the central nervous system, proteins, peptides, and polypeptides. classes, small molecules, or mixtures thereof.
  • the nucleic acid includes, but is not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA ( At least one of snRNA), antisense oligonucleotide (ASO), DNA, and plasmid.
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • siRNA small interfering RNA
  • small nuclear RNA At least one of snRNA
  • ASO antisense oligonucleotide
  • the mass ratio of the lipid nanoparticle preparation to the active agent is (3-50):1.
  • Lipid nanoparticle preparations serve as carriers to load active reagents and deliver them to target locations in the human or animal body to prevent and/or treat corresponding diseases.
  • the present invention has a new chemical composition, including cationic or ionizable lipids, auxiliary lipids, sterols and polyethylene glycol or modified polyethylene glycol, with a molar ratio of 50 :10:38.5:1.5.
  • LNPs will be used to encapsulate nucleic acids, active therapeutic agents, and deliver nucleic acids into cells, including but not limited to messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), Small interfering RNA (siRNA), small nuclear RNA (snRNA), antisense oligonucleotide (ASO), DNA, plasmid.
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • siRNA small interfering RNA
  • snRNA small nuclear RNA
  • ASO antisense oligonucleotide
  • Active therapeutic agents may also be immunomodulators, antigens or fragments thereof, vaccines, anti-inflammatory agents, anti-tumor agents, antibiotics, agents acting on the central nervous system, proteins, peptides, polypeptides and small molecules, or mixtures thereof.
  • This novel LNP can be used to transfect multicellular tissues or organs, providing a new treatment method for patients.
  • the patient can be any mammal, preferably from humans, mice, rats, pigs, cats, dogs, and horses. , goats, cows and monkeys and/or others.
  • the present invention provides a disulfide bond-containing ionizable cationic lipid compound, compositions containing the same, and applications.
  • the present invention also provides in detail a method for preparing disulfide bond ionizable lipids and a preparation method for lipid nanoparticles containing disulfide bond ionizable lipids, and also provides how to introduce nucleic acids. Methods used for therapeutic purposes.
  • a novel disulfide bond ionizable cationic lipid is provided, which can form stable, functionally specific lipid nanoparticles with phospholipids, sterols and polyethylene glycol lipids.
  • LNP polyethylene glycol lipids.
  • RNA RNA, DNA, proteins, peptides or small molecule drugs.
  • the preparation method of the lipid and the use of its formula to encapsulate RNA as a therapeutic drug are also disclosed.
  • the present invention provides a novel disulfide bond-containing cationic lipid that can be used to deliver mRNA in cells, tissues and in vivo, as well as its preparation method and use.
  • the present invention enriches the types of cationic lipid compounds and provides more choices for the effective delivery of nucleic acid drugs, gene therapy, small molecule drugs, polypeptides or protein drugs, and is especially of great significance to the development and application of nucleic acid preventive agents and therapeutic agents. .
  • the present invention provides a lipid nanoparticle, which uses the newly invented disulfide bond lipid as a cationic lipid, and an auxiliary lipid, cholesterol and polyethylene glycol ester to form the lipid nanoparticle.
  • the lipid nanoparticles provided by the invention are non-toxic, can efficiently deliver nucleic acids, and have high encapsulation rate and stability.
  • dichloromethane (10 mL) and 2,3-bis((9Z,12Z)-octadecyl-9,12-dien-1-yl)disulfide)propane were added to a 25 mL round-bottomed flask.
  • Base-1-ol (554mg, 0.8mmol), 4-dimethylaminobutyric hydrochloride (0.96mmol, 161mg), N,N-diisopropylethylamine (104mg, 0.8mmol), 4-dimethyl Aminopyridine (10 mg, 0.08 mmol), stirred at 0°C for 20 minutes, then added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (192 mg, 1.25 mmol) at room temperature Stir for 12 hours. The solvent was removed under reduced pressure, and compound 1 (413 mg, 64%) was obtained as a light yellow oily liquid through column chromatography.
  • Figure 3.1 is the hydrogen spectrum of compound 1
  • Figure 3.2 is the carbon spectrum of compound 1.
  • Figure 4.1 is the hydrogen spectrum of compound 2
  • Figure 4.2 is the carbon spectrum of compound 2.
  • Figure 5.1 is the hydrogen spectrum of compound 3
  • Figure 5.2 is the carbon spectrum of compound 3.
  • Figure 6.1 is the hydrogen spectrum of compound 4
  • Figure 6.2 is the carbon spectrum of compound 4.
  • dichloromethane (10 mL), 2,3-bis((hexadecyl-1-yl)dithio)propyl-1-ol (600 mg, 0.8 mmol), 4 -Dimethylaminobutyric acid hydrochloride (0.96mmol, 161mg), N,N-diisopropylethylamine (104mg, 0.8mmol), 4-dimethylaminopyridine (10mg, 0.08mmol) were stirred at 0°C 20 minutes, then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (192 mg, 1.25 mmol) was added and stirred at room temperature for 12 hours. The solvent was removed under reduced pressure, and compound 5 (402 mg, 67%) was obtained as a light yellow oily liquid through column chromatography.
  • Figure 7.1 is the hydrogen spectrum of compound 5
  • Figure 7.2 is the carbon spectrum of compound 5.
  • dichloromethane (10 mL), 2,3-di(octadecyl-dithio)propyl-1-ol (554 mg, 0.8 mmol), and 4-dimethylaminobutylene were added to a 25 mL round-bottomed flask.
  • Figure 8.1 is the hydrogen spectrum of compound 6, and Figure 8.2 is the carbon spectrum of compound 6.
  • Figure 9.1 is the hydrogen spectrum of compound 7, and Figure 9.2 is the carbon spectrum of compound 7.
  • Figure 10.1 is the hydrogen spectrum of compound 8
  • Figure 10.2 is the carbon spectrum of compound 8.
  • Figure 11.1 is the hydrogen spectrum of compound 9, and Figure 11.2 is the carbon spectrum of compound 9.
  • Compound 10 was prepared according to the synthetic method of compound 1.
  • Figure 12.1 is the hydrogen spectrum of compound 10
  • Figure 12.2 is the carbon spectrum of compound 10.
  • Figure 13.1 is the hydrogen spectrum of compound 11, and Figure 13.2 is the carbon spectrum of compound 11.
  • Compound 12 was prepared according to the synthetic method of compound 1.
  • Figure 14.1 is the hydrogen spectrum of compound 12, and Figure 14.2 is the carbon spectrum of compound 12.
  • Figure 15.1 is the hydrogen spectrum of compound 13
  • Figure 15.2 is the carbon spectrum of compound 13.
  • Figure 16.1 is the hydrogen spectrum of compound 14, and Figure 16.2 is the carbon spectrum of compound 14.
  • Compound 15 was prepared according to the synthetic method of compound 1.
  • Figure 17.1 is the hydrogen spectrum of compound 15, and Figure 17.2 is the carbon spectrum of compound 15.
  • Compound 16 was prepared according to the synthetic method of compound 1.
  • Figure 18.1 is the hydrogen spectrum of compound 16, and Figure 18.2 is the carbon spectrum of compound 16.
  • Compound 17 was prepared according to the synthetic method of compound 1.
  • Figure 19.1 is the hydrogen spectrum of compound 17, and Figure 19.2 is the carbon spectrum of compound 17.
  • Figure 20.1 is the hydrogen spectrum of compound 18, and Figure 20.2 is the carbon spectrum of compound 18.
  • Figure 21.1 is the hydrogen spectrum of compound 19
  • Figure 21.2 is the carbon spectrum of compound 19.
  • Lipid nanoparticle size and polydispersity index (PDI) were determined by dynamic light scattering using the Malvern Zetasizer Nano ZS in 173° backscatter detection mode.
  • the encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA Quantification Assay Kit (ThermoFisher Scientific). The final particle size, PDI and encapsulation efficiency of lipid nanoparticles are shown in Table 1.
  • Figures 1.1, 1.2, 1.3, and 1.4 are particle size diagrams of compounds 1, 2, 3, and 4, respectively.
  • LNPs made from multiple cationic lipid compounds prepared in Examples 1 to 19 can encapsulate nucleic acid molecules, and the obtained mRNA-LNP has good uniformity and high encapsulation rate.
  • mice Six-week-old female BALB/c mice were selected, weighing about 20g, and kept in an SPF-level breeding room. Animal experiments were conducted in strict accordance with the guidelines of national health agencies and animal ethics requirements. Three mice were randomly selected and intramuscularly injected with lipid nanoparticles at a dosage of 0.5 mg/kg. After 12 hours, 200 ⁇ L of 10 mg/mL D-luciferin potassium salt was injected intramuscularly into each mouse. After 10 minutes, the mice were placed under the in vivo imaging system (PerkinElmer IVIS Spectrum) to observe each mouse. The total fluorescence intensity was taken and recorded. Imaging was subsequently performed at 12 hours. The expression intensity of Fluc mRNA delivered by intramuscular injection is shown in Figure 2 and Table 2. In Figure 2, from left to right, the expression intensity of Fluc mRNA in mice treated with compounds 3, 1, 2, and 4 is shown.
  • the LNP carriers prepared from multiple cationic lipid compounds of the present invention can deliver Fluc mRNA to cells, express luciferase at high levels and emit light.
  • LNP prepared using the cationic lipid of the present invention can successfully encapsulate nucleic acid molecules with good uniformity and high encapsulation rate, and can transport nucleic acids into animals for expression and translation.

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Abstract

一种脂质化合物及其组合物，脂质化合物包含：通式(I)和/或(II)所示的脂质化合物，或所述脂质化合物的药学上可接受的盐，或所述脂质化合物的溶剂合物，或该脂质化合物所形成的脂质纳米颗粒。该脂质化合物无毒，其形成的脂质纳米颗粒可高效递送核酸，且具有高的包封率和稳定性。

Description

一种含有二硫键的脂质化合物及其组合物 技术领域
本发明涉及化合物领域，具体涉及一种含有二硫键的脂质化合物及其组合物。
背景技术
mRNA疫苗以其在医学领域掀起的巨大变革而荣登2021年“全球十大突破性技术”名单榜首。新冠mRNA疫苗的获批引发了继小分子药物、蛋白类药物之后的生物医药第三次革命。以mRNA技术为基础的治疗手段，将可能以更快的速度被应用于传染病预防、肿瘤治疗、罕见病治疗、单抗和其他药物替代治疗等重要领域。
然而，核酸治疗剂仍面临若干挑战，mRNA是带负电的高分子，且亲水性极强，很难穿过同为带负电荷的细胞膜，自身很难进入细胞；mRNA也很不稳定，容易被无处不在的核酸酶降解，在血液中的循环时间很短；mRNA本身具有免疫原性，注射进人体后很快被免疫系统识别，进而引发免疫反应并被清除。而mRNA疫苗递送系统有效的解决这一系列难题。脂质纳米颗粒(Lipid nanoparticles,LNP)技术递送效率高，安全性好，是目前最热门的递送技术。LNP通常由阳离子脂质或者可离子化的阳离子脂质、辅助性脂质、结构脂质和修饰的聚乙二醇脂质组成。因此，需要开发能够递送核酸药物的脂质化合物以及相关的方法和组合物，以促进各类核酸药物在细胞、组织和体内的递送。
发明内容
根据第一方面，在一实施例中，提供一种脂质化合物，包含：通式(I)和/或(II)所示的脂质化合物，或所述脂质化合物的药学上可接受的盐，或所述脂质化合物的溶剂合物：
Figure PCTCN2022092697-appb-000001
其中：
每个L 1、L 2和L 3独立地选自长度为1～22个碳原子的烷基，所述烷基为直链或含有至少一个支链，所述烷基上的碳原子未被取代，或者，所述烷基上的至少一个碳原子任选地被选自以下基团所组成的组中的至少一个所替换：-C(O)-、-NH-、-O-、-S-、C 2-C 10烯基、C 2～C 10炔基、C 3～C 8环烷基和C 6～C 10亚芳基；
R 1选自以下结构：-NH 2、单取代的胺基、二取代的胺基、取代或非取代的含氮饱和杂环、取代或非取代的氮杂芳香化合物；
R 2和R 3独立地选自以下结构：1)氢原子；2)包含1～22个碳原子的烷基，所述烷基为直链或含有至少一个支链，所述烷基上的碳原子未被取代，或者，所述烷基上的至少一个碳原子任选地被选自以下基团所组成的组中的至少一个所替换：-C(O)-、-NH-、-O-、-S-、C 2-C 10烯基、C 2～C 10炔基、C 3～C 8环烷基和C 6～C 10亚芳基。
根据第二方面，在一实施例中，提供一种纳米颗粒制剂，包含第一方面所述脂质化合物。
根据第三方面，在一实施例中，提供一种组合物，所述组合物包含第一方面所述脂质化合物，所述脂质化合物负载有活性试剂。
依据上述实施例的一种含有二硫键的脂质化合物及其组合物，该脂质化合物无毒，其形成的脂质纳米颗粒可高效递送活性试剂(包括但不限于核酸)，且具有高的包封率和稳定性。
附图说明
图1.1为化合物1的粒径图。
图1.2为化合物2的粒径图。
图1.3为化合物3的粒径图。
图1.4为化合物4的粒径图。
图2为肌注给药方式递送Fluc mRNA的表达强度。
图3.1为化合物1的氢谱图。
图3.2为化合物1的碳谱图。
图4.1为化合物2的氢谱图。
图4.2为化合物2的碳谱图。
图5.1为化合物3的氢谱图。
图5.2为化合物3的碳谱图。
图6.1为化合物4的氢谱图。
图6.2为化合物4的碳谱图。
图7.1为化合物5的氢谱图。
图7.2为化合物5的碳谱图。
图8.1为化合物6的氢谱图。
图8.2为化合物6的碳谱图。
图9.1为化合物7的氢谱图。
图9.2为化合物7的碳谱图。
图10.1为化合物8的氢谱图。
图10.2为化合物8的碳谱图。
图11.1为化合物9的氢谱图。
图11.2为化合物9的碳谱图。
图12.1为化合物10的氢谱图。
图12.2为化合物10的碳谱图。
图13.1为化合物11的氢谱图。
图13.2为化合物11的碳谱图。
图14.1为化合物12的氢谱图。
图14.2为化合物12的碳谱图。
图15.1为化合物13的氢谱图。
图15.2为化合物13的碳谱图。
图16.1为化合物14的氢谱图。
图16.2为化合物14的碳谱图。
图17.1为化合物15的氢谱图。
图17.2为化合物15的碳谱图。
图18.1为化合物16的氢谱图。
图18.2为化合物16的碳谱图。
图19.1为化合物17的氢谱图。
图19.2为化合物17的碳谱图。
图20.1为化合物18的氢谱图。
图20.2为化合物18的碳谱图。
图21.1为化合物19的氢谱图。
图21.2为化合物19的碳谱图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本申请能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他材料、方法所替代。在某些情况下，本申请相关的一些操作并没有在说明书中显示或者描述，这是为了避免本申请的核心部分被过多的描述所淹没，而对于本领域技术人员而言，详细描述这些相关操作并不是必要的，他们根据说明书中的描述以及本领域的一般技术知识即可完整了解相关操作。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以参照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
本文中为部件所编序号本身，例如“第一”、“第二”等，仅用于区分所描述的对象，不具有任何顺序或技术含义。而本申请所说“连接”、“联接，”如无特别说明，均包括直接和间接连接(联接)。
二硫键在蛋白质折叠形成二级结构、降低蛋白质的结构熵及增加蛋白质机械强度方面作用很大。如动物毛发中就含有很多半胱氨酸用于形成二硫键，其次膜蛋白中的半胱氨酸含量较其他蛋白较高。几乎所有的跨膜蛋白和分泌蛋白，在高尔基体完成组装后，都要在其末端添加一段信号序列，该序列都是通过二硫键与蛋白相连。这说明二硫键在生物体内是稳定的，而且生物体相容性极好。同时，二硫键的反应是一种很常见的生物正交反应，通过二硫键能够将一些具有特定性质的化合物或药物连接到一些生物大分子上，以期研究生物大分子的作用机制或改善抗体药物的性能。例如ADC药物Mylotarg和Besponsa就使用了二硫键作为可裂解的连接子(linker)与小分子毒性药物相连。
在一实施例中，本发明将二硫键的结构引入的阳离子脂质化合物，并制得LNP。该LNP在血液中是稳定的，一旦通过內吞进入细胞中，细胞内的还原性谷胱甘肽(GSH)可以通过硫醇-二硫键交换与阳离子脂质反应，从而破坏LNP的结构，促进mRNA的释放。本发明的含二硫键的脂质化合物及其构成的LNP能够高效的在 体内递送核酸，同时由于其可生物降解性，因而将拥有更优的安全性。
根据第一方面，在一实施例中，提供一种脂质化合物，包含：通式(I)和/或(II)所示的脂质化合物，或所述脂质化合物的药学上可接受的盐，或所述脂质化合物的溶剂合物：
Figure PCTCN2022092697-appb-000002
其中：
每个L 1、L 2和L 3独立地选自长度为1～22个碳原子的烷基，所述烷基上的碳原子未被取代，或者，所述烷基上的至少一个碳原子任选地被选自以下基团所组成的组中的至少一个所替换：-C(O)-、-NH-、-O-、-S-、C 2-C 10烯基、C 2～C 10炔基、C 3～C 8环烷基和C 6～C 10亚芳基；
R 1选自以下结构：-NH 2、单取代的胺基、二取代的胺基、取代或非取代的含氮饱和杂环、取代或非取代的氮杂芳香化合物；
R 2和R 3独立地选自以下结构：1)氢原子；2)包含1～22个碳原子的烷基，所述烷基为直链或含有至少一个支链，所述烷基上的碳原子未被取代，或者，所述烷基上的至少一个碳原子任选地被选自以下基团所组成的组中的至少一个所替换：-C(O)-、-NH-、-O-、-S-、C 2-C 10烯基、C 2～C 10炔基、C 3～C 8环烷基和C 6～C 10亚芳基。
在一实施例中，所述脂质化合物包含如下结构中的至少一种：
Figure PCTCN2022092697-appb-000003
其中n为1～9的整数。
在一实施例中，每个R 2和R 3为氢原子。
在一实施例中，每个L 2和L 3独立地选自长度为8～22个碳原子的直链亚烷基或烯基结构。
在一实施例中，每个L 2和L 3独立地选自如下结构：
Figure PCTCN2022092697-appb-000004
在一实施例中，R 1可选自以下结构：
1)二取代的胺基；2)取代或非取代的含氮饱和四元环、五元环、六元环、七元环、八元环；3)取代或非取代的含氮芳香化合物。
在一实施例中，所述含氮芳香化合物包含但不限于咪唑、吡唑、吡啶、吡咯中的至少一种。
在一实施例中，R 1与酯键之间的烷烃链-(CH 2) n-可为直链，n为0～6的整数。
在一实施例中，R 1选自以下结构中的任意一种：
Figure PCTCN2022092697-appb-000005
在一实施例中，R 1与酯键之间的烷烃链-(CH 2) n-选自以下结构中的任意一种：
1)-(CH 2) 1-；2)-(CH 2) 2-；3)-(CH 2) 3-；4)-(CH 2) 4-；5)-(CH 2) 5-；6)-(CH 2) 6-；7)-(CH 2)CH(CH 3)(CH 2)-。
在一实施例中，所述脂质化合物选自以下结构中的至少一种：
Figure PCTCN2022092697-appb-000006
根据第二方面，在一实施例中，提供一种纳米颗粒制剂，包含第一方面任意一项的脂质化合物。
在一实施例中，所述纳米颗粒制剂还包含辅助型脂质、结构脂质、聚乙二醇脂质中的至少一种。
在一实施例中，所述脂质化合物与辅助型脂质、结构脂质、聚乙二醇脂的摩尔比为(40～60)：(5～25)：(25～50)：(0.1～10)。
在一实施例中，所述辅助型脂质为离子型脂质或中性脂质，可以含有阳离子官能团(例如，胺基、季铵基)和/或阴离子官能团(例如，磷酸基团、羧酸基团)。
在一实施例中，所述纳米颗粒制剂的平均粒径为30nm～200nm。
在一实施例中，所述纳米颗粒制剂的多分散指数≤0.3。
在一实施例中，所述载体与所述治疗或预防剂的质量比为(3～50)：1。
在一实施例中，所述辅助型脂质包括但不限于磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂、神经酰胺、糖脂、类脂中的至少一种。
在一实施例中，所述辅助型脂质包括但不限于二油酰基磷脂酰乙醇胺(DOPE)、二硬脂酰磷脂酰胆碱(DSPC)、(2,3-二油酰基-丙基)-三甲胺(DOTAP)、(2,3-二油酰基-丙基)-二甲胺(DODAP)中的至少一种。
在一实施例中，所述结构脂质可以用于稳定脂质纳米颗粒，并且帮助与细胞膜的融合。
在一实施例中，所述结构脂质包括但不限于胆固醇、维生素D、非甾醇、谷固醇、麦角固醇、菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、番茄碱、熊果酸、α-生育酚、皮质类固醇中的至少一种。
在一实施例中，所述聚乙二醇修饰的脂质的亲水聚乙二醇链分布于纳米颗粒颗粒表面，防止纳米颗粒间的融合以及降低颗粒的免疫原性防止被体内免疫系统消灭，从而延长在体内的循环时间。
在一实施例中，所述聚乙二醇修饰的脂质包括但不限于聚乙二醇(PEG)修饰的磷脂酰乙醇胺、聚乙二醇修饰的磷脂酸、聚乙二醇修饰的神经酰胺、聚乙二醇修饰的二烷基胺、聚乙二醇修饰的二酰基甘油、聚乙二醇修饰的二烷基甘油中的至少一种。
在一实施例中，所述聚乙二醇修饰的脂质包括但不限于1-(单甲氧基聚乙二醇)-2,3二肉豆寇酰基甘油(PEG 2000-DMG)(CAS号：1397695-86-1)。
在一实施例中，所述脂质纳米颗粒用于递送活性治疗剂。
根据第三方面，在一实施例中，提供一种组合物，所述组合物包含第一方面所述脂质化合物，所述脂质化合物形成的脂质纳米颗粒负载有活性试剂。活性试剂既可以结合于脂质化合物的表面，也可以结合于脂质化合物内部。
在一实施例中，所述组合物包含第二方面所述纳米颗粒制剂，所述纳米颗粒制剂负载有活性试剂。
在一实施例中，所述活性试剂包括但不限于预防剂、治疗剂中的至少一种。
在一实施例中，所述活性试剂包括但不限于核酸、免疫调节剂、抗原或其片段、疫苗、抗炎剂、抗肿瘤剂、抗生素、作用于中枢神经系统的药剂、蛋白、肽、多肽类、小分子，或其混合物。
在一实施例中，所述核酸包括但不限于信使RNA(mRNA)、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小干扰RNA(siRNA)、小的核RNA(snRNA)、反义寡核苷酸(ASO)、DNA、质粒中的至少一种。
在一实施例中，所述脂质纳米颗粒制剂与所述活性试剂的质量比为(3～50)：1。脂质纳米颗粒制剂作为载体，用于负载活性试剂，将其递送至人或动物体的目标位置，实现对相应疾病的预防和/或治疗。
在一实施例中，本发明具有新的化学组成，包含阳离子或可离子化的脂质、辅助型脂质、固醇类物质和聚乙二醇或修饰的聚乙二醇，摩尔比为50：10：38.5：1.5。这些LNP将用来包裹核酸这种活性治疗剂，把核酸递送到细胞中，核酸包括但不限于信使RNA(mRNA)、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小干扰RNA(siRNA)、小的核RNA(snRNA)、反义寡核苷酸(ASO)、DNA、质粒。活性治疗剂还可以是免疫调节剂、抗原或其片段、疫苗、抗炎剂、抗肿瘤剂、抗生素、作用于中枢神经系统的药剂、蛋白、肽、多肽类和小分子，或其混合物。可以将该新型LNP用于转染多细胞组织或器官，给病人提供了新的治疗处理的方法，病人可以是任何哺乳动物，优选自人、小鼠、大鼠、猪、猫、狗、马、山羊、牛和猴子和/或其他。
在一实施例中，本发明提供一种含二硫键的可离子化阳离子脂质化合物、包含其的组合物及应用。
在一实施例中，本发明也详细给出了二硫键可离子化脂质的制备方法以及含二硫键可离子化脂质的脂质纳米颗粒的制备方法，同样给出了如何引入核酸用于治疗目的的方法。
在一实施例中，提供一种新型的二硫键可离子化阳离子脂质，该脂质可与磷脂、固醇类物质和聚乙二醇脂质形成稳定、具有特定功能的脂质纳米颗粒(LNP)，用于一系列药物(RNA、DNA、蛋白、多肽或小分子药物)的递送。同时还公开了该脂质的制备方法，以及用其配方包载RNA作为治疗药物。
在一实施例中，本发明提供一种新型的可用于细胞、组织中和体内递送mRNA的含二硫键的阳离子脂质及其制备方法和用途。本发明丰富了阳离子脂质化合物种类，为核酸药物、基因疗法、小分子药物、多肽或蛋白质药物的有效递送提供更多的选择，尤其对核酸预防剂及治疗剂的发展和应用具有重要的意义。
在一实施例中，本发明提供了一种脂质纳米颗粒，以新发明的二硫键脂质为阳离子脂质，与辅助型脂质、胆固醇以及聚乙二醇酯组成脂质纳米颗粒的配方。本发明提供的脂质纳米颗粒无毒、可高效递送核酸，且具有高的包封率和稳定性。
实施例1：2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-4-(二甲氨基)丁酯(化合物1 C4S18A)的合成
Figure PCTCN2022092697-appb-000007
在100mL的圆底烧瓶中加入2,2'-二硫二吡啶(1.8g,7.2mmol)、甲醇(25mL)，随后缓慢滴加二巯基丙醇(372mg,3.0mmol)，室温下搅拌反应12小时，减压蒸干溶剂所得残留物柱层析(二氯甲烷/石油醚1/1then二氯甲烷)，得淡黄色液体2，3-二(吡啶二硫化)丙醇(723mg，70％)。在50mL圆底烧瓶中加入亚油基硫醇(2.3g，8.0mmol)、甲醇(20mL)、2，3-二(吡啶二硫化)丙醇(1.23g，3.6mmol)，室温搅拌12小时，减压蒸干柱层析得2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-1-醇(2.02g，82％)。随后在25mL的圆底烧瓶中加入二氯甲烷(10mL)、2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-1-醇(554mg，0.8mmol)、4-二甲氨基丁酸盐酸盐(0.96mmol，161mg)、N,N-二异丙基乙胺(104mg，0.8mmol)、4-二甲氨基吡啶(10mg，0.08mmol)，于0℃下搅拌下20分钟，随后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(192mg，1.25mmol)室温搅拌12小时。减压除去溶剂柱层析得淡黄色油状液体化合物1(413mg，64％)。
1H NMR(400MHz,CDCl 3)δ5.42–5.29(m,8H),4.36(d,J＝5.6Hz,2H),3.28(dq,J＝11.8,5.8Hz,1H),3.05(dd,J＝13.7,6.2Hz,1H),2.92(dt,J＝12.6,3.7Hz,1H),2.77(t,J＝6.4Hz,4H),2.70(t,J＝6.6Hz,4H),2.39(t,J＝7.4Hz,2H),2.34(t,J＝7.4Hz,2H),2.25(s,6H),2.05(dd,J＝13.7,6.8Hz,8H),1.86–1.77(m,2H),1.72–1.62(m,4H),1.42–1.24(m,32H),0.89(t,J＝6.7Hz,6H). 13C NMR(101MHz,CDCl 3)δ173.24,130.36,130.21,128.17,128.05,64.17,58.79,49.27,45.37,40.20,39.97,39.00,32.01,31.67,29.80,29.57,29.56,29.49,29.39,29.36,29.36,29.33,29.30,28.65,28.62,27.36,27.35,25.77,22.80,22.72,14.23。
图3.1为化合物1的氢谱图，图3.2为化合物1的碳谱图。
HR-MS(ESI)m/z calcd for C 45H 84O 2NS 4[M+H] +798.5379,found 798.5364。
实施例2：2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-3-(二甲氨基)丙酯(化合物2 C3S18A)的合成
Figure PCTCN2022092697-appb-000008
在25mL的圆底烧瓶中加入二氯甲烷(10mL)、2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-1-醇(554mg，0.8mmol)、3-二甲氨基丙酸(0.96mmol，112mg)、N,N-二异丙基乙胺(104mg，0.8mmol)、4-二甲氨基吡啶(10mg，0.08mmol)，于0℃下搅拌20分钟，随后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(192mg，1.25mmol)，室温搅拌12小时。减压除去溶剂柱层析得淡黄色油状液体化合物2(489mg，78％)。
1H NMR(400MHz,CDCl 3)δ＝5.47–5.26(m,8H),4.45–4.32(m,2H),3.33–3.24(m,1H),3.04(dd,J＝13.2,6.8Hz,1H),2.94(dd,J＝13.6,8.0Hz,1H),2.77(t,J＝6.3Hz,4H),2.70(t,J＝7.3Hz,4H),2.62(t,J＝7.0Hz,2H),2.51(t,J＝7.2Hz,2H),2.24(s,3H),2.04(dd,J＝13.5,6.7Hz,8H),1.73–1.61(m,8H),1.41–1.24(m,32H),0.89(t,J＝6.2Hz,6H). 13C NMR(101MHz,CDCl 3)δ172.22,130.36,130.22,128.17,128.05,64.32,54.83,49.29,45.41,40.21,39.97,39.01,32.98,31.68,29.81,29.58,29.50,29.40,29.37,29.35,29.31,28.67,28.63,27.41,27.37,27.36,25.78,22.74,14.25。
HR-MS(ESI)m/z calcd for C 44H 82O 2NS 4[M+H] +784.5223,found 784.5222。
图4.1为化合物2的氢谱图，图4.2为化合物2的碳谱图。
实施例3：2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-3-(吡咯烷-1-基)丙酯(化合物3 C3AS18A)的合成
Figure PCTCN2022092697-appb-000009
在25mL的圆底烧瓶中加入二氯甲烷(10mL)、2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-1-醇(554mg，0.8mmol)、3-(吡咯烷基-1-基)丙酸(0.96mmol，137mg)、N,N-二异丙基乙胺(104mg，0.8mmol)、4-二甲氨基吡啶(10mg，0.08mmol)，于0℃下搅拌20分钟，随后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(192mg，1.25mmol)，室温搅拌12小时。减压除去溶剂，柱层析，得淡黄色油状液体2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-3-(吡咯烷-1-基)丙酯(460mg，71％)。
1H NMR(400MHz,CDCl 3)δ5.42–5.29(m,8H),4.38(qd,J＝11.6,5.6Hz,2H),3.28(dq,J＝11.9,5.8Hz,1H),3.04(dd,J＝13.8,6.3Hz,1H),2.94(dd,J＝13.8,8.0Hz,1H),2.83–2.75(m,6H),2.70(t,J＝7.3Hz,4H),2.57(t,J＝7.5Hz,2H),2.53(dd,J＝9.2,3.9Hz,4H),2.05(q,J＝6.8Hz,8H),1.81–1.75(m,4H),1.72–1.62(m,4H),1.44–1.22(m,32H),0.89(t,J＝6.9Hz,6H). 13C NMR(101MHz,CDCl 3)δ172.22,130.35,130.21,128.16,128.04,64.24,54.14,51.46,49.32,40.16,39.96,39.00,34.23,31.68,29.80,29.57,29.50,29.40,29.37,29.34,29.31,28.66,28.63,27.37,27.35,25.78,23.63,22.73,14.24。
HR-MS(ESI)m/z calcd for C 46H 84O 2NS 4[M+H] +810.5379,found 810.5373。
图5.1为化合物3的氢谱图，图5.2为化合物3的碳谱图。
实施例4：2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-3-(4-甲基哌嗪-1-基)丙酯(化合物4 C3BS18A)的合成
Figure PCTCN2022092697-appb-000010
在25mL的圆底烧瓶中加入二氯甲烷(10mL)、2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-1-醇(554mg，0.8mmol)、3-(4-甲基哌嗪-1-基)丙酸(0.96mmol，165mg)、N,N-二异丙基乙胺(104mg，0.8mmol)、4-二甲氨基吡啶(10mg，0.08mmol)于0℃下搅拌20分钟，随后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(192mg，1.25mmol)室温搅拌12小时。减压除去溶剂柱层析得淡黄色油状液体化合物4(349mg，52％)。
1H NMR(500MHz,CDCl 3)δ5.41–5.30(m,8H),4.41(dd,J＝11.6,5.7Hz,1H),4.35(dd,J＝11.6,5.4Hz,1H),3.28(dq,J＝7.9,5.7Hz,1H),3.04(dd,J＝13.7,6.3Hz,1H),2.94(dd,J＝13.8,8.1Hz,1H),2.77(t,J＝6.7Hz,4H),2.73–2.65(m,7H),2.57–2.41(m,6H),2.28(s,3H),2.08–2.01(m,8H),1.80–1.71(m,3H),1.66(dt,J＝14.6,7.3Hz,4H),1.41–1.24(m,32H),0.89(t,J＝6.9Hz,6H). 1 3C NMR(101MHz,CDCl 3)δ172.19,130.36,130.20,128.16,128.03,64.18,55.13,53.58,52.94,49.29,46.10,40.11,39.95,38.98,32.43,31.67,29.80,29.57,29.49,29.40,29.37,29.36,29.33,29.31,28.66,28.63,27.36,27.35,25.77,22.73,14.24.
HR-MS(ESI)m/z calcd for C 47H 87O 2N 2S 4[M+H] +839.5645,found 839.5651。
图6.1为化合物4的氢谱图，图6.2为化合物4的碳谱图。
实施例5：2,3-二((十六烷基-1-基)二硫基)丙基-4-(二甲氨基)丁酯(化合物5 C4S16)的合成
Figure PCTCN2022092697-appb-000011
在50mL圆底烧瓶中加入亚油基硫醇(2.07g，8.0mmol)、甲醇(20mL)、2，3-二(吡啶二硫化)丙醇(1.23g，3.6mmol)，室温搅拌12小时，减压蒸干柱层析得2,3-二((十六烷基-1-基)二硫基)丙基-1-醇(1.74，76％)。随后在25mL的圆底烧瓶中加入二氯甲烷(10mL)、2,3-二((十六烷基-1-基)二硫基)丙基-1-醇(600mg，0.8mmol)、4-二甲氨基丁酸盐酸盐(0.96mmol，161mg)、N,N-二异丙基乙胺(104mg，0.8mmol)、4-二甲氨基吡啶(10mg，0.08mmol)于0℃下搅拌20分钟，随后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(192mg，1.25mmol)室温搅拌12小时。减压除去溶剂柱层析得淡黄色油状液体化合物5(402mg，67％)。
1H NMR(400MHz,CDCl 3)δ4.35(dd,J＝9.2,3.8Hz,2H),3.27(dq,J＝8.0,5.8Hz,1H),3.03(dd,J＝12.4,6.2Hz,1H),2.91(dd,J＝13.7,8.1Hz,1H),2.70(td,J＝7.8,2.1Hz,4H),2.42–2.32(m,4H),2.26(s,6H),1.82–1.77(m,2H),1.71–1.62(m,2H),1.39–1.22(m,52H),0.87(t,J＝6.8Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝173.19,64.16,58.75,49.24,45.32,40.17,39.96,39.03,32.07,31.97,29.84,29.81,29.76,29.68,29.67,29.51,29.39,29.39,29.34,29.31,28.66,28.64,22.84,22.72,14.27。
HR-MS(ESI)m/z calcd for C 41H 84O 2NS 4[M+H] +750.5379,found 750.5388。
图7.1为化合物5的氢谱图，图7.2为化合物5的碳谱图。
实施例6：2,3-二((十八烷基-二硫基)丙基)4-(二甲氨基)丁酯(化合物6 C4S18)的合成
Figure PCTCN2022092697-appb-000012
在50mL圆底烧瓶中加入十八烷基硫醇(1.29g，4.5mmol)、甲醇(20mL)、2，3-二(吡啶二硫化)丙醇(0.70g，2.06mmol)，室温搅拌12小时，减压蒸干柱层析得2,3-二(十八烷基-二硫基)丙基-1-醇(1.11g，78％)。随后在25mL的圆底烧瓶中加入二氯甲烷(10mL)、2,3-二(十八烷基-二硫基)丙基-1-醇(554mg，0.8mmol)、4-二甲氨基丁酸盐酸盐(0.96mmol，161mg)、N,N-二异丙基乙胺(104mg，0.8mmol)、4-二甲氨基吡啶(10mg，0.08mmol)于0℃下搅拌下20分钟，随后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(192mg，1.25mmol)室温搅拌12小时。减压除去溶剂柱层析得淡黄色油状液体化合物6(368mg，57％)。
1H NMR(400MHz,CDCl 3)δ＝4.35(dd,J＝5.6,1.2Hz,2H),3.27(dq,J＝8.0,5.8Hz,1H),3.04(dd,J＝13.7,6.2Hz,1H),2.91(dd,J＝13.8,8.1Hz,1H),2.69(td,J＝7.7,2.1Hz,4H),2.36(dt,J＝15.1,7.5Hz,4H),2.24(s,6H),1.85–1.77(m,2H),1.66(dq,J＝14.4,7.1Hz,4H),1.39–1.20(m,60H),0.86(t,J＝6.8Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝173.17,64.14,58.68,49.22,45.25,40.15,39.94,39.00,32.05,31.96,29.82,29.79,29.74,29.65,29.65,29.49,29.37,29.36,29.32,29.29,28.64,28.62,22.81,22.68,14.25。
HR-MS(ESI)m/z calcd for C 45H 92O 2NS 4[M+H] +806.6005,found 806.6009。
图8.1为化合物6的氢谱图，图8.2为化合物6的碳谱图。
实施例7：2,3-二((八烷基-二硫基)丙基)4-(二甲氨基)丁酯(化合物7 C4S8)
Figure PCTCN2022092697-appb-000013
化合物7参照化合物1的合成方法制备。
1H NMR(500MHz,CDCl 3)δ＝4.39–4.33(m,2H),3.28(dq,J＝8.0,5.8Hz,1H),3.04(dt,J＝13.7,4.3Hz,1H),2.95–2.88(m,1H),2.70(td,J＝7.6,2.6Hz,4H),2.39(td,J＝7.5,3.3Hz,4H),2.28(s,6H),1.87–1.81(m,2H),1.68(dt,J＝14.6,7.4Hz,4H),1.41–1.35(m,4H),1.32–1.24(m,16H),0.8 8(t,J＝6.8Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝173.02,64.07,58.51,49.10,45.05,40.04,39.85,38.90,31.82,31.80,29.20,29.19,28.53,28.51,22.67,22.41,14.13。
HR-MS(ESI)m/z calcd for C 25H 52O 2NS 4[M+H] +526.2875,found 526.2879。
图9.1为化合物7的氢谱图，图9.2为化合物7的碳谱图。
实施例8：2,3-二((十烷基-二硫基)丙基)4-(二甲氨基)丁酯(化合物8 C4S10)
Figure PCTCN2022092697-appb-000014
化合物8参照化合物1的合成方法制备。
1H NMR(500MHz,CDCl 3)δ＝4.39–4.33(m,2H),3.30–3.25(m,1H),3.05(dd,J＝13.8,6.2Hz,1H),2.92(dd,J＝13.8,8.1Hz,1H),2.72–2.68(m,4H),2.38(t,J＝7.5Hz,2H),2.30(t,J＝7.1Hz,2H),2.22(s,6H),1.84–1.77(m,2H),1.71–1.63(m,4H),1.41–1.35(m,4H),1.32–1.24(m,24H),0.88(t,J＝7.0Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝173.30,64.14,58.86,49.26,45.49,40.19,39.96,39.03,32.06,32.04,29.70,29.67,29.66,29.46,29.38,29.38,29.34,29.31,28.66,28.64,22.94,22.82,14.27。
HR-MS(ESI)m/z calcd for C 29H 60O 2NS 4[M+H] +582.3501,found 582.3503。
图10.1为化合物8的氢谱图，图10.2为化合物8的碳谱图。
实施例9：2,3-二((十二烷基-二硫基)丙基)4-(二甲氨基)丁酯(化合物9 C4S12)
Figure PCTCN2022092697-appb-000015
化合物9参照化合物1的合成方法制备。 1H NMR(400MHz,CDCl 3)δ＝4.36(d,J＝5.7Hz,2H),3.28(dq,J＝8.1,5.8Hz,1H),3.08–3.02(m,1H),2.91(dd,J＝13.8,8.2Hz,1H),2.74–2.67(m,4H),2.41(dd,J＝15.5,8.1Hz,4H),2.31(s,6H),1.91–1.82(m,2H),1.71–1.62(m,4H),1.41–1.34(m,4H),1.31–1.24(m,32H),0.87(t,J＝6.8Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝173.07,64.20,58.58,49.22,45.08,40.15,39.98,39.03,32.06,31.86,29.81,29.79,29.76,29.68,29.67,29.50,29.39,29.35,29.32,28.66,28.64,22.84,22.41,14.28。
HR-MS(ESI)m/z calcd for C 33H 68O 2NS 4[M+H] +638.4127,found 638.4132。
图11.1为化合物9的氢谱图，图11.2为化合物9的碳谱图。
实施例10：2,3-二((十四烷基-二硫基)丙基)4-(二甲氨基)丁酯(化合物10 C4S14)
Figure PCTCN2022092697-appb-000016
化合物10参照化合物1的合成方法制备。
1H NMR(400MHz,CDCl 3)δ＝4.39–4.33(m,2H),3.28(dq,J＝8.0,5.8Hz,1H),3.05(dd,J＝13.7,6.2Hz,1H),2.95–2.88(m,1H),2.70(td,J＝7.7,2.3Hz,4H),2.38(t,J＝7.5Hz,2H),2.30(d,J＝7.5Hz,2H),2.23(s,6H),1.85–1.76(m,2H),1.67(dq,J＝14.4,7.1Hz,4H),1.43–1.34(m,4H),1.31–1.22(m,40H),0.88(t,J＝6.8Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝173.30,64.15,58.85,49.26,45.46,40.19,39.97,39.04,32.07,29.85,29.84,29.81,29.77,29.68,29.68,29.52,29.40,29.39,29.35,29.32,28.67,28.65,22.91,22.85,14.28。
HR-MS(ESI)m/z calcd for C 37H 76O 2NS 4[M+H] +694.4759,found 694.4756。
图12.1为化合物10的氢谱图，图12.2为化合物10的碳谱图。
实施例11：2,3-二((9Z-十八烷基-9-烯-1-基)二硫基)丙基-4-(二甲氨基)丁酯(化合物11 C4S18B)
Figure PCTCN2022092697-appb-000017
化合物11参照化合物1的合成方法制备。
1H NMR(500MHz,CDCl 3)δ＝5.37–5.30(m,4H),4.36(dd,J＝5.6,2.0Hz,2H),3.27(dq,J＝7.8,5.8Hz,1H),3.04(dd,J＝13.7,6.2Hz,1H),2.91(dd,J＝13.8,8.1Hz,1H),2.70(td,J＝7.7,2.1Hz,4H),2.36(dd,J＝15.8,7.6Hz,4H),2.26(s,6H),2.03–1.96(m,8H),1.70–1.63(m,2H),1.70–1.62(m,4H),1.38–1.23(m,44H),0.87(t,J＝6.9Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝173.17,130.10,129.88,64.15,58.69,49.21,45.25,40.14,39.93,38.97,32.03,31.94,29.89,29.87,29.83,29.79,29.74,29.65,29.55,29.45,29.37,29.35,29.34,29.31,29.28,28.63,28.61,27.34,27.31,22.82,22.66,14.26.
HR-MS(ESI)m/z calcd for C 45H 88O 2NS 4[M+H] +802.5692,found 802.5699。
图13.1为化合物11的氢谱图，图13.2为化合物11的碳谱图。
实施例12：2,3-二((十四烷基-二硫基)丙基-4-(二甲氨基)丙酯(化合物12 C3S14)
Figure PCTCN2022092697-appb-000018
化合物12参照化合物1的合成方法制备。
1H NMR(500MHz,CDCl 3)δ＝4.42–4.34(m,2H),3.28(dq,J＝7.8,5.9Hz,1H),3.04(dd,J＝13.8,6.3Hz,1H),2.93(dd,J＝13.6,8.3Hz,1H),2.73–2.68(m,4H),2.65–2.60(m,2H),2.52(dd,J＝8.8,5.3Hz,2H),2.25(s,6H),1.71–1.63(m,4H),1.37(d,J＝4.8Hz,4H),1.30–1.24(m,40H),0.87(t,J＝6.9Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝172.19,64.31,54.75,49.25,45.34,40.17,39.97,39.03,32.88,32.07,29.84,29.83,29.81,29.76,29.68,29.67,29.51,29.40,29.39,29.35,29.32,28.67,28.64,22.84,14.28。
HR-MS(ESI)m/z calcd for C 36H 74O 2NS 4[M+H] +680.4597,found 680.4601。
图14.1为化合物12的氢谱图，图14.2为化合物12的碳谱图。
实施例13：2,3-二((十六烷基-二硫基)丙基)4-(二甲氨基)丙酯(化合物13 C3S16)
Figure PCTCN2022092697-appb-000019
化合物13参照化合物1的合成方法制备。
1H NMR(500MHz,CDCl 3)δ＝4.42–4.34(m,2H),3.28(dq,J＝7.8,5.8Hz,1H),3.05(dd,J＝13.7,6.3Hz,1H),2.98–2.90(m,1H),2.71(t,J＝6.8Hz,4H),2.63(dd,J＝10.9,3.9Hz,2H),2.51(dd,J＝8.8,5.3Hz,2H),2.24(s,6H),1.71–1.63(m,4H),1.40–1.35(m,4H),1.30–1.23(m,48H),0.88(t,J＝7.0Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝172.21,64.31,54.79,49.27,45.38,40.18,39.98,39.04,32.94,32.07,29.85,29.81,29.77,29.68,29.52,29.40,29.36,29.32,28.67,28.65,22.84,14.28。
HR-MS(ESI)m/z calcd for C 40H 82O 2NS 4[M+H] +736.5223,found 736.5231。
图15.1为化合物13的氢谱图，图15.2为化合物13的碳谱图。
实施例14：2,3-二((9Z-十八烷基-9-烯-1-基)二硫基)丙基-4-(二甲氨基)丙酯(化合物14 C3S18B)
Figure PCTCN2022092697-appb-000020
化合物14参照化合物1的合成方法制备。
1H NMR(400MHz,CDCl 3)δ＝5.39–5.30(m,4H),4.42–4.33(m,2H),3.28(dq,J＝12.0,5.9Hz,1H),3.04(dd,J＝13.7,6.3Hz,1H),2.94(dd,J＝13.8,8.0Hz,1H),2.70(t,J＝7.3Hz,4H),2.63(t,J＝6.9Hz,2H),2.51(t,J＝7.0Hz,2H),2.25(s,6H),2.06–1.94(m,8H),1.71–1.64(m,4H),1.40–1.22(m,44H),0.88(t,J＝6.8Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝172.20,130.13,129.91,64.31,54.79,49.26,45.38,40.18,39.96,39.00,32.93,32.06,29.92,29.90,29.86,29.81,29.68,29.57,29.48,29.40,29.38,29.37,29.34,29.31,28.66,28.64,27.37,27.34,22.84,14.29。
HR-MS(ESI)m/z calcd for C 44H 86O 2NS 4[M+H] +788.5536found 788.5533。
图16.1为化合物14的氢谱图，图16.2为化合物14的碳谱图。
实施例15：2,3-二((十四烷基-二硫基)丙基)4-(二甲氨基)戊酯(化合物15 C5S14)
Figure PCTCN2022092697-appb-000021
化合物15参照化合物1的合成方法制备。
1H NMR(500MHz,CDCl 3)δ＝4.37(d,J＝5.6Hz,2H),3.28(dq,J＝8.2,5.7Hz,1H),3.05(dd,J＝13.7,6.1Hz,1H),2.90(dd,J＝13.8,8.3Hz,1H),2.71(td,J＝7.5,2.8Hz,4H),2.65(d,J＝7.4Hz,2H),2.53(s,6H),2.40(t,J＝6.9Hz,2H),1.74–1.63(m,6H),1.41–1.35(m,4H),1.31–1.23(m,42H),0.88(t,J＝6.9Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝172.90,64.15,58.45,49.20,44.14,40.11,39.98,39.02,33.61,32.07,29.84,29.83,29.81,29.81,29.76,29.68,29.51,29.40,29.39,29.34,29.32,28.66,28.64,22.84,22.35,14.28。
HR-MS(ESI)m/z calcd for C 38H 78O 2NS 4[M+H] +708.4910,found 708.48980。
图17.1为化合物15的氢谱图，图17.2为化合物15的碳谱图。
实施例16：2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-2-(二甲氨基)乙酯(化合物17 C2S18A)
Figure PCTCN2022092697-appb-000022
化合物16参照化合物1的合成方法制备。
1H NMR(400MHz,CDCl 3)δ＝5.41–5.30(m,8H),4.45–4.38(m,2H),3.30(dq,J＝7.9,6.0Hz,1H),3.21(s,2H),3.08–3.02(m,1H),2.92(dd,J＝13.7,8.0Hz,1H),2.77(t,J＝6.7Hz,4H),2.70(t,J＝6.7Hz,4H),2.37(s,6H),2.07–2.02(m,8H),1.70–1.63(m,6H),1.39–1.25(m,30H),0.89(t,J＝6.9Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝170.47,130.35,130.20,128.16,128.03,64.32,60.28,49.14,45.41,40.20,39.95,39.00,31.67,29.79,29.57,29.56,29.50,29.39,29.37,29.35,29.33,29.31,28.65,28.62,27.36,27.35,25.77,22.73,14.25。
HR-MS(ESI)m/z calcd for C 43H 80O 2NS 4[M+H] +770.5066,found 770.5073。
图18.1为化合物16的氢谱图，图18.2为化合物16的碳谱图。
实施例17：2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-2-(1H咪唑-1-基)乙酯(化合物18 C2BS18A)
Figure PCTCN2022092697-appb-000023
化合物17参照化合物1的合成方法制备。
1H NMR(500MHz,CDCl 3)δ＝7.51(s,1H),7.10(s,1H),6.96(s,1H),5.42–5.29(m,8H),4.74(s,2H),4.49(qd,J＝11.6,5.6Hz,2H),3.29(dq,J＝8.6,5.7Hz,1H),3.02(dd,J＝13.8,5.8Hz,1H),2.83–2.75(m,5H),2.70(td,J＝7.5,2.6Hz,4H),2.05(q,J＝6.9Hz,8H),1.72–1.62(m,4H),1.40–1.25(m,32H),0.89(t,J＝6.9Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝167.18,138.07,130.36,130.18,129.97,128.17,128.02,120.11,65.50,48.74,48.09,40.00,39.78,38.92,31.67,29.78,29.56,29.55,29.49,29.38,29.35,29.34,29.29,28.62,28.59,27.34,25.77,22.72,14.24。
HR-MS(ESI)m/z calcd for C 44H 77O 2N 2S 4[M+H] +793.4862,found 793.48682。
图19.1为化合物17的氢谱图，图19.2为化合物17的碳谱图。
实施例18：2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-3-吗啉-丙酯(化合物19 C3CS18A)
Figure PCTCN2022092697-appb-000024
化合物18参照化合物1的合成方法制备。
1H NMR(400MHz,CDCl 3)δ＝5.44–5.27(m,8H),4.39(qd,J＝11.6,5.5Hz,2H),3.71–3.68(m,4H),3.28(dq,J＝8.1,5.7Hz,1H),3.05(dd,J＝13.7,6.1Hz,1H),2.93(dd,J＝13.7,8.2Hz,1H),2.77(t,J＝6.5Hz,4H),2.70(dd,J＝14.8,7.3Hz,6H),2.53(t,J＝7.1Hz,2H),2.49–2.46(m,2H),2.05(dd,J＝13.7,6.8Hz,8H),1.67(ddd,J＝12.9,10.6,5.6Hz,6H),1.32(td,J＝13.3,8.5Hz,32H),0.89(t,J＝6.8Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝171.96,130.23,130.07,128.04,127.91,66.93,64.05,53.92,53.40,49.16,39.99,39.84,38.88,32.12,31.55,29.67,29.44,29.37,29.27,29.23,29.21,29.19,28.53,28.50,27.22,25.65,22.60,14.11。
HR-MS(ESI)m/z calcd for C 46H 84O 3NS 4[M+H] +826.5329,found 826.5330。
图20.1为化合物18的氢谱图，图20.2为化合物18的碳谱图。
实施例19：2,3-二(((9Z,12Z)-十八烷基-9,12-二烯-1-基)二硫基)丙基-3-(1H咪唑-1-基)丙酯(化合物20 C3DS18A)
Figure PCTCN2022092697-appb-000025
化合物19参照化合物1的合成方法制备。
1H NMR(400MHz,CDCl 3)δ＝7.52(s,1H),7.05(s,1H),6.94(s,1H),5.43–5.28(m,8H),4.40(d,J＝5.6Hz,2H),4.27(t,J＝6.6Hz,2H),3.26(dq,J＝8.3,5.7Hz,1H),3.02(dd,J＝13.8,6.0Hz,1H),2.85–2.75(m,6H),2.69(td,J＝7.5,2.8Hz,3H),2.09–1.99(m,8H),1.65(dt,J＝14.6,7.3Hz,4H),1.41–1.24(m,34H),0.89(t,J＝6.8Hz,6H). 13C NMR(101MHz,CDCl 3)δ＝170.29,137.39,130.36,130.19,129.92,128.18,128.03,118.93,64.81,48.97,42.28,40.00,38.97,36.00,31.67,29.79,29.56,29.49,29.39,29.35,29.34,29.29,28.64,28.60,27.35,25.77,22.72,14.24。
HR-MS(ESI)m/z calcd for C 45H 79O 2N 2S 4[M+H] +807.5019,found 807.5018。
图21.1为化合物19的氢谱图，图21.2为化合物19的碳谱图。
纳米脂质颗粒(LNP制剂)的制备和表征
制备LNP制剂的方法：将各实施例制得的二硫键阳离子脂质与DSPC、胆固醇、PEG2000-DMG以50：10：38.5：1.5的摩尔比混合溶解在无水乙醇中，总的脂质浓度为10mM。萤光素酶mRNA(Fluc mRNA)溶解在醋酸钠水溶液(10mM，pH＝4.6)中，mRNA浓度为0.1mg/mL。使用微流控仪器，控制乙醇溶液与醋酸钠水溶液 的流速比为1：3，在微流控芯片中制得脂质纳米颗粒的溶液，在4℃下PBS稀释离心除去乙醇再稀释再离心重复三次，所得浓缩液用PBS稀释备用。用Malvern Zetasizer Nano ZS，以173°反向散射检测模式通过动态光散射测定脂质纳米颗粒的大小及多分散性指数(PDI)。使用Quant-it Ribogreen RNA定量测定试剂盒(ThermoFisher Scientific)确定脂质纳米颗粒的包封效率。最终所得脂质纳米颗粒粒径、PDI和包封率如表1所示。图1.1、1.2、1.3、1.4分别为化合物1、2、3、4的粒径图。
表1
LNP制剂 阳离子脂质 代号 Size(nm) PDI 包封率
1 化合物1 C4S18A 111.0 0.115 94.1％
2 化合物2 C3S18A 109.0 0.143 93.0％
3 化合物3 C3AS18A 108.6 0.150 95.3％
4 化合物4 C3BS18A 104.3 0.135 96.5％
5 化合物5 C4S16 105.9 0.123 96.6％
6 化合物6 C4S18 108.1 0.098 94.8％
7 化合物7 C4S8 82.5 0.325 91.8％
8 化合物8 C4S10 100.0 0.266 90.5％
9 化合物9 C4S12 52.0 0.251 89.2％
10 化合物10 C4S14 102.0 0.139 95.6％
11 化合物11 C4S18B 62.1 0.338 91.8％
12 化合物12 C3S14 89.6 0.297 90.1％
13 化合物13 C3S16 110.3 0.383 92.3％
14 化合物14 C3S18B 78.1 0.235 94.2％
15 化合物15 C5S14 66.9 0.358 91.4％
16 化合物16 C2S18A 119.6 0.264 91.2％
17 化合物17 C2BS18A 63.2 0.323 93.6％
18 化合物18 C3CS18A 82.8 0.325 92.7％
19 化合物19 C3DS18A 64.7 0.146 86.5％
从表1可以看出，实施例1～19制得的多个阳离子脂质化合物制成的LNP能够包裹核酸分子，获得的mRNA-LNP均一性好，包封率高。
动物实验
选取6周龄的雌性BALB/c小鼠，体重在20g左右，饲养环境为SPF级的饲养室，动物试验严格参照国家健康机构的指南以及动物伦理要求进行。随机选取3只小鼠，按0.5mg/kg的用量，使用肌肉注射脂质纳米颗粒。12小时后，分别往每只小鼠体内通过肌肉注射200μL 10mg/mL的D-荧光素钾盐，10分钟后，将小鼠放置于活体成像系统(PerkinElmer IVIS Spectrum)下，观察每只小鼠总的萤光强度，并拍照记录下来。随后在12小时成像。肌注给药方式递送Fluc mRNA的表达强度见图2和表2。图2中，从左往右，依次为化合物3、1、2、4处理组小鼠的Fluc mRNA的表达强度。
表2
LNP制剂 阳离子脂质 总发光强度(p/sec/cm 2/sr)
1 化合物1 1.98E+08
2 化合物2 3.41E+08
3 化合物3 1.41E+08
4 化合物4 2.51E+08
从图2和表2可以看出，本发明的多个阳离子脂质化合物制备的LNP载体均能将Fluc mRNA递送至细胞，并高水平表达萤光素酶并发光。
以上实验数据证明，使用本发明的阳离子脂质制备的LNP可成功包裹核酸分子，且均一性好，包封率，并能将核酸转运至动物体内进行表达翻译。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (10)

  1. 一种脂质化合物，其特征在于，包含：通式(I)和/或(II)所示的脂质化合物，或所述脂质化合物的药学上可接受的盐，或所述脂质化合物的溶剂合物：
    Figure PCTCN2022092697-appb-100001
    其中：
    每个L 1、L 2和L 3独立地选自长度为1～22个碳原子的烷基，所述烷基为直链或含有至少一个支链，所述烷基上的碳原子未被取代，或者，所述烷基上的至少一个碳原子任选地被选自以下基团所组成的组中的至少一个所替换：-C(O)-、-NH-、-O-、-S-、C 2-C 10烯基、C 2～C 10炔基、C 3～C 8环烷基和C 6～C 10亚芳基；
    R 1选自以下结构：-NH 2、单取代的胺基、二取代的胺基、取代或非取代的含氮饱和杂环、取代或非取代的氮杂芳香化合物；
    R 2和R 3独立地选自以下结构：1)氢原子；2)包含1～22个碳原子的烷基，所述烷基为直链或含有至少一个支链，所述烷基上的碳原子未被取代，或者，所述烷基上的至少一个碳原子任选地被选自以下基团所组成的组中的至少一个所替换：-C(O)-、-NH-、-O-、-S-、C 2-C 10烯基、C 2～C 10炔基、C 3～C 8环烷基和C 6～C 10亚芳基。
  2. 如权利要求1所述的脂质化合物，其特征在于，所述脂质化合物包含如下结构中的至少一种：
    Figure PCTCN2022092697-appb-100002
    其中n为1～9的整数，R 1、R 2、R 3、L 2和L 3如权利要求1定义。
  3. 如权利要求2所述的脂质化合物，其特征在于，每个R 2和R 3为氢原子；
    L 2和L 3独立地选自长度为8～22个碳原子的直链亚烷基或烯基结构；
    优选地，每个L 2和L 3独立地选自如下结构：
    Figure PCTCN2022092697-appb-100003
  4. 如权利要求1所述的脂质化合物，其特征在于，R 1选自以下结构：
    1)二取代的胺基；2)取代或非取代的含氮饱和四元环、五元环、六元环、七元环、八元环；3)取代或非取代的含氮芳香化合物；
    优选地，所述含氮芳香化合物包含咪唑、吡唑、吡啶、吡咯中的至少一种。
  5. 如权利要求2所述的脂质化合物，其特征在于，R 1与酯键之间的烷烃链-(CH 2) n-为直链，n为0～6的整数；
    优选地，R 1选自以下结构中的任意一种：
    Figure PCTCN2022092697-appb-100004
    优选地，R 1与酯键之间的烷烃链-(CH 2) n-选自以下结构中的任意一种：
    1)-(CH 2) 1-；2)-(CH 2) 2-；3)-(CH 2) 3-；4)-(CH 2) 4-；5)-(CH 2) 5-；6)-(CH 2) 6-；7)-(CH 2)CH(CH 3)(CH 2)-。
  6. 如权利要求1-5任意一项所述的脂质化合物，其特征在于，所述脂质化合物选自如下结构所示化合物中的至少一种；
    Figure PCTCN2022092697-appb-100005
  7. 一种纳米颗粒制剂，其特征在于，包含权利要求1～6任意一项所述脂质化合物。
  8. 如权利要求7所述的纳米颗粒制剂，其特征在于，所述纳米颗粒制剂还包含辅助型脂质、结构脂质、聚乙二醇修饰的脂质中的至少一种；
    优选地，所述脂质化合物与辅助型脂质、结构脂质、聚乙二醇修饰的脂质的摩尔比为(40～60)：(5～25)：(25～50)：(0.1～10)；
    优选地，所述辅助型脂质为离子型脂质或中性脂质；
    优选地，所述辅助脂质含有阳离子官能团和/或阴离子官能团；
    优选地，所述阳离子官能团包括胺基、季铵基；
    优选地，所述阴离子官能团包括磷酸基团、羧酸基团；
    优选地，纳米颗粒制剂的平均粒径为30nm～200nm；
    优选地，所述纳米颗粒制剂的多分散指数≤0.3；
    优选地，所述辅助型脂质包括磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂、神经酰胺、糖脂、类脂中的至少一种；
    优选地，所述辅助型脂质包括二油酰基磷脂酰乙醇胺、二硬脂酰磷脂酰胆碱、(2,3-二油酰基-丙基)-三甲胺、(2,3-二油酰基-丙基)-二甲胺中的至少一种；
    优选地，所述结构脂质包括胆固醇、维生素D、非甾醇、谷固醇、麦角固醇、菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、番茄碱、熊果酸、α-生育酚、皮质类固醇中的至少一种；
    优选地，所述聚乙二醇修饰的脂质的亲水聚乙二醇链分布于纳米颗粒制剂表面；
    优选地，所述聚乙二醇修饰的脂质包括聚乙二醇修饰的磷脂酰乙醇胺、聚乙二醇修饰的磷脂酸、聚乙二醇修饰的神经酰胺、聚乙二醇修饰的二烷基胺、聚乙二醇修饰的二酰基甘油、聚乙二醇修饰的二烷基甘油中的至少一种；
    优选地，所述聚乙二醇修饰的脂质包括1-(单甲氧基聚乙二醇)-2,3二肉豆寇酰基甘油。
  9. 一种组合物，其特征在于，所述组合物包含如权利要求1～6任意一项所述脂质化合物，所述脂质化合物形成的脂质纳米颗粒负载有活性试剂。
  10. 如权利要求9所述的组合物，其特征在于，所述组合物包含如权利要求7～8任意一项所述纳米颗粒制剂，所述纳米颗粒制剂负载有活性试剂；
    优选地，所述活性试剂包括预防剂、治疗剂中的至少一种；
    优选地，所述活性试剂包括核酸、免疫调节剂、抗原或其片段、疫苗、抗炎剂、抗肿瘤剂、抗生素、作用于中枢神经系统的药剂、蛋白、肽、多肽类、小分子药物，或其混合物；
    优选地，所述核酸包括信使RNA、核糖体RNA、微RNA、转移RNA、小干扰RNA、小的核RNA、反义寡核苷酸、DNA、质粒中的至少一种；
    优选地，所述脂质纳米颗粒制剂与所述活性试剂的质量比为(3～50)：1。
PCT/CN2022/092697 2022-05-13 2022-05-13 一种含有二硫键的脂质化合物及其组合物 WO2023216232A1 (zh)

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