WO2023193341A1 - 鞘脂类化合物、含有鞘脂类化合物的脂质体和应用 - Google Patents

鞘脂类化合物、含有鞘脂类化合物的脂质体和应用 Download PDF

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WO2023193341A1
WO2023193341A1 PCT/CN2022/098765 CN2022098765W WO2023193341A1 WO 2023193341 A1 WO2023193341 A1 WO 2023193341A1 CN 2022098765 W CN2022098765 W CN 2022098765W WO 2023193341 A1 WO2023193341 A1 WO 2023193341A1
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sirna
cationic liposome
compound
sphingolipid
group
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French (fr)
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姜志宏
白丽萍
许婷
周小波
郭勇
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澳门科技大学
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    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
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    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
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    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
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    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
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    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
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    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C07D317/14—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the invention belongs to the technical field of organic synthesis, and in particular relates to a sphingolipid compound, a liposome containing a sphingolipid compound and applications.
  • TNBC triple negative breast cancer
  • paclitaxel is the first-line drug for the clinical treatment of TNBC.
  • traditional paclitaxel dosage forms have poor targeting effect, low drug transport efficiency, and are prone to drug resistance.
  • HIF-1 hypoxia inducible factor-1
  • HIF-1 pumps drugs such as paclitaxel out of cells by upregulating multidrug resistance (MDR) and P-glycoprotein, reducing intracellular drug concentrations and weakening the ability to kill cells.
  • MDR multidrug resistance
  • P-glycoprotein reducing intracellular drug concentrations and weakening the ability to kill cells.
  • Small interfering RNA has high specificity and low toxicity to target cells, and can successfully silence malignant oncogenes.
  • the half-life of naked siRNA in the blood is less than 1 hour and can be rapidly degraded by nucleases in plasma or excreted by the kidneys.
  • naked siRNA has difficulty penetrating cell membranes due to its high molecular weight, hydrophilicity, and charge density. Therefore, various carriers that can carry genetic drugs have emerged, including micelles, liposomes, and inorganic nanoparticles. Cationic liposomes have been shown to have advantages over other nanocarriers, including greater stability in body fluids and tissues, better biodegradability, and the ability to release drugs over long periods of time.
  • cationic liposomes Although a variety of cationic liposomes have been reported at home and abroad, and there are also cationic liposomes on the market such as LipofectamineTM2000; Transfection Reagent, most cationic liposomes show high cytotoxicity and low transfection efficiency. , and can only be used to deliver genetic drugs.
  • the present invention aims to solve at least one of the above technical problems existing in the prior art. To this end, the present invention provides a sphingolipid compound.
  • the invention also provides a cationic liposome.
  • the invention also provides a cationic liposome pharmaceutical preparation.
  • the invention also provides an application of cationic liposome or cationic liposome pharmaceutical preparation.
  • a first aspect of the present invention provides sphingolipid compounds, the structural formula of the sphingolipid compound is as shown in formula (I):
  • R 1 and R 2 are independently selected from substituted or unsubstituted C 8-22 alkyl groups, substituted or unsubstituted C 8-20 alkenyl groups;
  • the R 3 is selected from The n is an integer from 1 to 5, and m is an integer from 1 to 3;
  • the A is Amide bond, ester bond,
  • the R 4 is selected from the group consisting of H, azaaromatic hydrocarbon group, a guanidine-containing substituted aliphatic or aromatic hydrocarbon group, a substituted or unsubstituted C 1 to 10 alkyl group, a heterocyclyl hydrocarbon group, and a C 2 to 10 alkyl acid;
  • the R 5 is selected from substituted or unsubstituted amino groups.
  • the invention uses a long-chain alkyl group as the hydrophobic tail, and bonds the amino acid, quaternary ammonium salt, tertiary amine group, secondary amine group or primary amine group through the connecting arm (covalent bond, amide bond, ester bond containing triazole) and other hydrophilic heads, to design and synthesize a series of new sphingolipid compounds. It was applied to prepare new cationic liposomes, and high transfection efficiency, good stability and low toxicity cationic liposomes were obtained.
  • said R1 is selected from Where x and y are integers from 8 to 20.
  • the R 2 is selected from
  • the R 4 is selected from H,
  • said R 5 is selected from
  • the sphingolipid compound is selected from one of the following structural formulas:
  • a second aspect of the present invention provides a cationic liposome, which includes the sphingolipid compound described in any one of the above, cholesterol, dioleoylphosphatidylethanolamine and methoxy-polyethylene glycol -Phospholipids.
  • the molecular weight of the methoxy-polyethylene glycol-phospholipid ranges from 750 to 10,000, and the preferred molecular weight is 2,000.
  • the molar ratio of the sphingolipid compound, cholesterol, dioleoylphosphatidylethanolamine and methoxy-polyethylene glycol-phospholipid is 1: (0.5 ⁇ 50): (0.5 ⁇ 50 ): (0.015 ⁇ 1).
  • the third aspect of the present invention provides a cationic liposome pharmaceutical preparation, which pharmaceutical preparation includes the above-mentioned cationic liposome and pharmaceutical active ingredients.
  • the molar ratio of the sphingolipid compound and the active pharmaceutical ingredient is (0.1-50):1.
  • the pharmaceutical active ingredient is one or more of anti-tumor compounds, anti-viral compounds, anti-inflammatory compounds or rheumatoid drugs.
  • the pharmaceutically active ingredient is an anti-tumor compound.
  • the anti-tumor compound is paclitaxel or a paclitaxel derivative thereof.
  • the cationic liposome pharmaceutical preparation further includes nucleic acid.
  • the amount of nucleic acid is an N/P ratio of 1:1 to 16:1, and the N/P ratio is the molar content of the ionizable nitrogen atom N in the cationic liposome and Ratio of molar content of P in nucleic acids.
  • the nucleic acid is selected from siRNA, miRNA, antagomir, plasmid DNA or mRNA.
  • the preparation method of the cationic liposome pharmaceutical preparation includes the following steps:
  • step S4 Subject the solution in step S3 to sonication and filtration at 0 to 5°C to prepare a cationic liposome drug preparation.
  • step S5 is also included: mix the cationic liposome drug preparation with the nucleic acid complex and 10% fetal bovine serum, and incubate the sample at room temperature to obtain the result.
  • the organic solvent includes chloroform and/or methanol.
  • the fourth aspect of the present invention provides the use of the above-mentioned cationic liposome or the cationic liposome pharmaceutical preparation in the preparation of drugs for the treatment of related diseases caused by abnormal gene expression, the diseases include malignant Tumors, cardiovascular diseases, rheumatoid diseases, infectious diseases or genetic diseases.
  • the malignant tumor is triple negative breast cancer.
  • the cationic liposomes of the present invention co-deliver the gene drug HIF-1 ⁇ siRNA and the chemotherapy drug paclitaxel and exhibit synergistic effects.
  • This cationic liposome can be used to construct a drug delivery system that co-loads HIF-1 ⁇ siRNA and paclitaxel to overcome the resistance of triple-negative breast cancer to paclitaxel and improve the anti-cancer effect.
  • Room temperature in the present invention refers to a temperature from 10°C to 40°C. In some embodiments, “room temperature” refers to a temperature from 20°C to 30°C; in other embodiments, “room temperature” refers to a temperature from 25°C to 30°C.
  • Substituted or unsubstituted C 8-20 alkyl group means an alkyl group with a total number of carbon atoms of 8-20, including straight-chain alkyl, branched-chain alkyl and cycloalkyl; and optionally C 8-20 alkyl At least one H in the group is substituted by the corresponding group defined herein, for example, by a hydroxyl, alkoxy or amino group.
  • Substituted or unsubstituted C 1-10 alkyl groups have similar definitions, but the difference is that the number of carbon atoms is different.
  • Substituted or unsubstituted C 8-20 alkenyl group means a straight-chain or branched-chain hydrocarbon group with one or more double bonds, and the total number of carbon atoms in the group is 8-20, and the double bonds in the group It can be at any position, and optionally at least one H in the C8-20 alkenyl group is substituted by the corresponding group defined herein, for example, substituted by a hydroxyl group, an alkoxy group, etc.
  • Alkylene chain is a straight or branched divalent hydrocarbon chain consisting only of carbon and hydrogen that connects the remainder of the molecule to the radical, and is saturated or unsaturated (i.e., contains one or more divalent bonds and/or triple bonds), and have one to twelve carbon atoms, such as methylene, ethylene, propylene, n-butylene, vinylene, propenylene, n-butenylene, Propargyl, n-butynyl, etc.
  • the alkylene chain is connected to the rest of the molecule by single or double bonds and to the radical by single or double bonds.
  • the point of attachment of the alkylene chain to the rest of the molecule, and of the alkylene chain to the group may be through one carbon or any two carbons within the chain. Unless otherwise specifically stated in this specification, the alkylene chain is optionally substituted.
  • Aryl means a carbocyclic ring system group containing hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring.
  • an aryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems.
  • Aryl groups include, but are not limited to, derivatives derived from benzacenaphthylene, acenaphthylene, acephenanthrene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, asymmetric indyne, symmetric indyne, indene, indene, naphthalene, Phenadene, phenanthrene, pleiadene, pyrene and benzophenanthrene.
  • the term "aryl” or the prefix “aryl-" is intended to include optionally substituted aryl groups.
  • “Azaareneyl” means a group of the formula -R b -R c , wherein R b is an alkylene chain and R c is one or more aryl groups as defined above, wherein at least one H in the aryl group is replaced by N atoms.
  • Heterocyclyl means a stable 3- to 18-membered non-aromatic ring having one to twelve ring carbon atoms (eg, two to twelve) and one to six ring heteroatoms selected from nitrogen, oxygen, and sulfur. group.
  • heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused, spirocyclic ("spiro-heterocyclyl”) and/or bridged the ring system; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl group are optionally oxidized; the nitrogen atoms are optionally quaternized; and the heterocyclyl group is partially or fully saturated.
  • Heterocyclylalkyl means a group of the formula -RbRe , wherein Rb is an alkylene chain and Re is heterocyclyl as defined above. Unless otherwise specifically stated in the specification, heterocyclylalkyl groups are optionally substituted.
  • C 2-10 alkyl acid means a carboxylic acid with a total number of carbon atoms of 2-10, and the alkyl group includes straight-chain alkyl, branched-chain alkyl and cycloalkyl.
  • Substituted or unsubstituted amino means that at least one H atom of the amino group is substituted by a corresponding group as defined herein, such as a C 1 -C 8 alkyl or C 3 -C 8 cycloalkyl substituent group.
  • Figure 1 is a cytotoxicity diagram of the sphingolipid compounds of Examples 1 to 4;
  • Figure 2 is a graph of cytotoxicity of the cationic liposomes of Example 11 and the cationic liposomes loaded with siRNA of Example 11;
  • Figure 3 is a storage stability chart of the cationic liposomes of Example 11;
  • Figure 4 is a diagram of the encapsulation efficiency of paclitaxel at different concentrations in the cationic liposome pharmaceutical preparation prepared in Example 12;
  • Figure 5 is a graph showing the influence of the molar ratio of sphingolipid compound-cholesterol-DOPE on the encapsulation rate of paclitaxel in the cationic liposome pharmaceutical preparation prepared in Example 13;
  • Figure 6 is a graph showing the influence of different DSPE-mPEG molar ratios on the encapsulation efficiency of paclitaxel
  • Figure 7 is a diagram of the encapsulation efficiency and drug loading capacity of the cationic liposome pharmaceutical preparation prepared in Example 15;
  • Figure 8 is an in vitro release diagram of PTX in the cationic liposome pharmaceutical preparation prepared in Example 15;
  • Figure 9 is a diagram showing the blocking effect of the cationic liposome pharmaceutical preparation prepared in Example 15 on siRNA-Luc;
  • Figure 10 is an analysis diagram of particle size and potential of paclitaxel-loaded cationic liposomes and siRNA complexes with different N/P ratios;
  • Figure 11 is a graph showing the stability of the cationic liposome drug preparation prepared in Example 15 and the siRNA complex in serum at different time points;
  • Figure 12 is a graph showing the silencing efficiency of firefly luciferase delivered by cationic liposomes prepared in Example 11, siRNA-Luc;
  • Figure 13 is a diagram showing the delivery effect of cationic liposomes prepared in Example 11 on Cy3-siRNA
  • Figure 14 is a diagram showing the effect of delivering Cy3-siRNA using cationic liposomes prepared in Example 11 for quantitative analysis using flow cytometry;
  • Figure 15 is a diagram showing the delivery effect of the cationic liposomes prepared in Example 11 on EGFP;
  • Figure 16 is an intracellular distribution diagram of the cationic liposome prepared in Example 11 co-loaded with coumarin C6 and Cy3-siRNA;
  • Figure 17 is a graph of HIF-1 ⁇ protein expression in MDA-MB-231 cells.
  • Figure 18 is a graph showing the 72h cytotoxic effects of the cationic liposome drug preparation and genetic drug HIF-1 ⁇ siRNA prepared in Example 15 on MDA-MB-231 cells;
  • Figure 19 is a graph showing the weight change trend of tumor-bearing mice.
  • Figure 20 is a trend chart of tumor volume changes in tumor-bearing mice
  • Figure 21 is a graph showing the change trend of tumor volume in tumor-bearing mice.
  • Figure 22 is a graph showing the statistical results of the tumor mass of each group in the model after the in vivo treatment experiment of the 4T1 tumor model.
  • the reagents, methods and equipment used in the present invention are all conventional reagents, methods and equipment in this technical field unless otherwise specified.
  • the dissolution-high-speed centrifugation method was used to determine the encapsulation efficiency of paclitaxel.
  • DLE Drug loading efficiency
  • EE encapsulation efficiency
  • W1 and W2 represent the total mass of encapsulated PTX in liposomes and the amount of PTX fed, respectively, and W is the total amount of lipid fed.
  • Embodiment 1 provides a sphingolipid compound a, with the following structural formula, and the preparation method is as follows:
  • Embodiment 2 provides a sphingolipid compound b, the structural formula is as follows, and the preparation method is as follows:
  • reaction mixture was stirred at 0°C for 30 minutes and then at room temperature for an additional hour.
  • the reaction was quenched with H 2 O and extracted with CH 2 Cl 2.
  • the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to obtain compound 10 as a white solid with a yield of 40%.
  • Embodiment 3 provides a sphingolipid compound c, the structural formula is as follows, and the preparation method is as follows:
  • Embodiment 4 provides a sphingolipid compound d, the structural formula is as follows, and the preparation method is as follows:
  • Embodiment 5 provides seven kinds of sphingolipid compounds e-k, the preparation method is as follows, and the structural formula is as follows:
  • Embodiment 6 provides seven kinds of sphingolipid compounds l-r.
  • the preparation method is as follows and the structural formula is as follows:
  • Embodiment 7 provides seven kinds of sphingolipid compounds s-y.
  • the preparation method is as follows and the structural formula is as follows:
  • Embodiment 8 provides a sphingolipid compound a1, the structural formula is as follows, and the preparation method is as follows:
  • Embodiment 9 provides a sphingolipid compound b1, the structural formula is as follows, and the preparation method is as follows:
  • Embodiment 10 provides a sphingolipid compound c1, the structural formula is as follows, and the preparation method is as follows:
  • the sphingolipid compounds (a, b, c and d) prepared in Examples 1 to 4 were respectively prepared into cationic liposomes (Sp1, Sp2, Sp3 and Sp4).
  • the preparation method is as follows:
  • sphingolipid compounds (a, b, c and d) with cholesterol, auxiliary phospholipid DOPE and DSPE-mPEG respectively in a round-bottomed flask so that the molar ratio is 1:1:1:0.06.
  • the sphingolipid compounds (a, b, c and d) prepared in Examples 1 to 4 were prepared into cationic Liposome drug preparation, the preparation steps are as follows:
  • the preparation method is the same as in Example 12.
  • the molar ratio of sphingolipid compounds (a, b, c and d) to paclitaxel is 8:1.
  • the difference is that the molar ratios of sphingolipid compounds (a, b, c and d), cholesterol and auxiliary phospholipid DOPE are 0.5:1:1, 1:1:1, 2:1:1 and 4:1 respectively: 1.
  • the preparation method is the same as in Example 12, but the difference is that the molar ratio of sphingolipid compounds (a, b, c and d) and DSPE-mPEG is 1:0.015 respectively. , 1:0.03, 1:0.06 and 1:0.12.
  • Embodiment 15 provides a cationic liposome pharmaceutical preparation (Sphy1), a cationic liposome pharmaceutical preparation (Sphy2), a cationic liposome pharmaceutical preparation (Sphy3) and a cationic liposome pharmaceutical preparation (Sphy4).
  • the preparation method is as follows:
  • sphingolipid compounds (a, b, c and d), cholesterol, auxiliary phospholipids DOPE and DSPE-mPEG (molecular weight 2000) with a molar ratio of 1:1:1:0.06 respectively in a round-bottomed flask, and then Add paclitaxel and dissolve it fully with 2 mL of chloroform and methanol.
  • the molar ratios of sphingolipid compounds (a, b, c and d) and paclitaxel are all 8:1 respectively.
  • Cytotoxicity test of sphingolipid compounds MTT method was used to detect the effects of sphingolipid compound a, sphingolipid compound b, sphingolipid compound c and sphingolipid compound d of Examples 1 to 4 on MDA-MB-231 cells.
  • MDA-MB-231 cells were seeded in 96-well plates at 4 ⁇ 10 3 /well/100 ⁇ L, and the culture medium was 1640 medium containing 10% FBS, and cultured at 37°C, 5% CO 2 saturated humidity Incubate in the box for 24 hours.
  • Cytotoxicity test of cationic liposomes and their complexes with siRNA-Luc MTT method was used to detect Sp1, Sp2, Sp3, Sp4, Sp1+siRNA-Luc complex, Sp2+siRNA-Luc complex prepared in Example 11, Effects of Sp3+siRNA-Luc complex, Sp4+siRNA-Luc complex and free siRNA-Luc on the viability of MDA-MB-231 cells.
  • the main purpose was to investigate whether cationic liposomes inhibit the growth of MDA-MB-231 cells during cell transfection carrying siRNA.
  • MDA-MB-231 cells were seeded in a 96-well plate at 4 ⁇ 10 3 /well/100 ⁇ L, and cultured in 1640 medium containing 10% fetal calf serum for 24 h in a 37°C, 5% CO 2 saturated humidity incubator.
  • the final concentration was 50 nM, and the same concentration of free siRNA and the same concentration gradient of liposomes, as well as Lipo2000 and Lipo 2000+siRNA-Luc complex were set as control groups.
  • a series of cationic liposomes prepared in Example 11 were tested for storage stability.
  • the freshly prepared cationic liposomes were sterile filtered and packed into sterilized EP tubes, and stored in a 4°C refrigerator for 28 days.
  • the storage system was: Sterile RNase-free water, take out samples on days 1, 4, 7, 14, and 28, measure their particle size with a laser particle size analyzer, and observe changes during storage; the results are shown in Figure 3.
  • Four blank cationic lipids prepared After 28 days of storage the particle size did not change significantly, and there was no obvious precipitation or flocculation in appearance, showing high stability.
  • Example 11 A series of cationic liposomes prepared in Example 11 and a series of cationic liposome pharmaceutical preparations prepared in Example 15 were tested for particle size and potential. The results are shown in Table 1. From the table, it can be seen that the four cationic lipids The bulk particle size does not exceed 100nm, and the polydispersity index (PDI) is less than 0.3. It shows that the sphingolipid compounds of Examples 1 to 4 can form liposomes in aqueous solutions with the assistance of cholesterol, auxiliary phospholipids DOPE and DSPE-mPEG, and their particle size is smaller, the particle size distribution is narrower, and the uniformity is relatively low. good.
  • PDI polydispersity index
  • all four liposomes can effectively encapsulate the chemotherapy drug paclitaxel, and the particle size of the liposome-paclitaxel complex has no obvious change and the uniformity is good.
  • the zeta potential of these liposomes and their complexes is greater than 30mV.
  • the high positive surface charge shows that the liposomes have the characteristics of being an anionic drug carrier and are relatively stable in solution and are not prone to aggregation.
  • a series of cationic liposome pharmaceutical preparations prepared in Example 12 were tested for the encapsulation efficiency of paclitaxel to study the effect of the content of paclitaxel on the encapsulation efficiency.
  • the results are shown in Figure 4.
  • the encapsulation rates of Sphy1, Sphy2, Sphy3 and Sphy4 were 83.6%, 78.5%, 85.4% and 73.2% respectively. After the amount of PTX continued to decrease, the encapsulation rate did not increase significantly.
  • a series of cationic liposome pharmaceutical preparations prepared in Example 13 were tested for the encapsulation efficiency of paclitaxel. The results are shown in Figure 5 and Table 2. As the proportion of sphingolipid compounds continues to increase, the encapsulation efficiency also continues to increase. When the ratio of sphingolipid compound-cholesterol-DOPE is 1:1:1, the encapsulation rates of Sphy1, Sphy2, Sphy3 and Sphy4 are 78.6%, 68.5%, 83.7% and 63.2% respectively. When the proportion of sphingolipid compounds is further increased, the encapsulation efficiency does not change significantly, but the positive charge density further increases, which has the risk of increasing cytotoxicity.
  • Example 14 A series of cationic liposome pharmaceutical preparations prepared in Example 14 were tested for the encapsulation efficiency of paclitaxel. From Figure 6 and Table 3, it can be seen that with the increase of DSPE-mPEG, the encapsulation efficiency of the four liposomes further increased. When the molar ratio of DSPE-mPEG content to sphingolipid was 0.06:1, the encapsulation efficiency reached the optimal level, which were 93.6%, 90.3%, 95.4%, and 89.7% respectively. At the same time, the particle size reaches the minimum and the potential is above 35mV, making it suitable as a cationic liposome material.
  • Example 15 A series of cationic liposome drug preparations in Example 15 were tested for encapsulation efficiency and drug loading capacity. The results are shown in Figure 7. The encapsulation rates of Sphy1, Sphy2, Sphy3 and Sphy4 were all above 90%, which were 93.6 respectively. %, 90.3%, 95.4% and 92.7%. The drug loading contents were 4.89%, 4.97%, 5.21%, and 4.85% respectively.
  • a series of cationic liposome drug preparations prepared in Example 15 were subjected to a PTX release test. As shown in Figure 8, Sphy1, Sphy2, Sphy3 and Sphy4 can all slowly release paclitaxel. When the pH is 7.4, compared with the control free paclitaxel, there is no obvious burst release phenomenon of paclitaxel liposomes in the first 12 hours, and it has good stability, which can avoid the toxic side effects caused by the burst release of nano-preparations and improve the efficiency of paclitaxel administration. safety.
  • a series of cationic liposome drug preparations in Example 15 were mixed and incubated with siRNA and the binding rate was determined: an agarose gel electrophoresis retardation test was used to determine the binding status of the cationic liposome drug preparation and siRNA-Luc complex.
  • siRNA-Luc (10mM)
  • the particle size under different N/P is Between 84 and 150 nm, as the proportion of siRNA-Luc increases, the particle size shows an increasing trend, indicating that liposomes and siRNA combine with each other.
  • N/P is 4:1, the potential of the four cationic liposome drug preparations changes from negative to positive, with the potential between 15-20mV, and the potential increases with the increase of liposomes. This result is consistent with the agarose gel electrophoresis result.
  • the positive charge of the liposome complex indicates that it can better bind to the negatively charged cell membrane, allowing gene drugs to better enter the cell.
  • a series of cationic liposome pharmaceutical preparations of Example 15 were mixed with siRNA and incubated to study serum stability: four cationic liposome pharmaceutical preparations of Example 15 were complexed with siRNA (siRNA-Luc) and 10% fetal bovine The serum was mixed, and the samples were incubated at 37°C with equal volumes and the same concentration of free siRNA as a control. Take samples at 0, 6, 12, 24 and 48 hours respectively. Before gel electrophoresis analysis, add 2% heparin sodium solution to the sample to extract siRNA, and immediately mix it with 2 ⁇ Loading Buffer at a volume of 1:1, and proceed. Analyzed by agarose gel electrophoresis and photographed by gel imaging system. The results are shown in Figure 11.
  • Free siRNA was basically completely degraded at 12 hours. Although siRNA wrapped in liposomes was degraded, its stability was significantly improved compared to free siRNA. Clear siRNA could still be seen at 48 hours. The strips prove that the four liposomes have a certain protective effect on siRNA in the presence of serum, preventing siRNA from being degraded too quickly by nucleases in serum.
  • MDA-MB-231-Luc cells in logarithmic growth phase were seeded in a 24-well plate, with 40,000 cells per well, and cultured 24h.
  • Sp1+siRNA-Luc of Example 11 Sp2+siRNA-Luc of Example 11, Sp3+siRNA-Luc of Example 11, Sp4+siRNA-Luc of Example 11, Lipo2000+siRNA-Luc ( N/P 1:1, 2:1, 4:1, 8:1) and free siRNA-Luc, the siRNA-Luc in each well was 50nM, transfected, and after incubation for 4 hours, the medium was changed and the culture was continued for 48 hours.
  • siRNA-Luc When the liposomes are in an excess state compared to siRNA-Luc, siRNA-Luc is firmly bound to the centers of multiple liposomes. On the contrary, it is not conducive to the escape of siRNA-Luc.
  • the silencing efficiencies of these four cationic liposomes on intracellular luciferase were 83%, 68%, 75% and 63% respectively; the silencing efficiency of Lipo2000 was 78%.
  • Cy3 was used to fluorescently label siRNA, and the delivery of siRNA by cationic liposomes was observed through confocal microscopy.
  • MDA-MB-231 cells in the logarithmic growth phase were digested with trypsin and then seeded into a 6-well plate at 1 ⁇ 10 5 cells per well.
  • the experimental groups are Sp1+Cy3-siRNA in Example 11, Sp2+Cy3-siRNA in Example 11, Sp3+Cy3-siRNA in Example 11, Sp4+Cy3-siRNA in Example 11, Lipo2000+Cy3-siRNA, Free Cy3-siRNA and Blank groups.
  • MDA-MB-231 cells in the logarithmic growth phase were trypsinized and seeded in a 6-well plate at 2 ⁇ 10 5 /well. Transfection was performed 24 hours after cell inoculation.
  • the experimental groups are Sp1+Cy3-siRNA of Example 11, Sp2+Cy3-siRNA of Example 11, Sp3+Cy3-siRNA of Example 11, Lipo2000+Cy3-siRNA, Free Cy3-siRNA and Blank group.
  • the final concentration of Cy3-siRNA in each group was 50 nM, and the N/P ratio was 4:1. After 4 hours, the liquid was discarded, 1640 medium containing 10% fetal calf serum was added, and culture was continued for 48 hours.
  • EGFP is a green fluorescent protein.
  • the delivery of EGFP plasmid DNA by cationic liposomes was observed through an inverted fluorescence microscope.
  • MDA-MB-231 cells in the logarithmic growth phase were digested with trypsin and then seeded into a 6-well plate at 1 ⁇ 10 5 cells per well.
  • the experimental groups are Sp1+EGFP of Example 11, Sp2+EGFP of Example 11, Sp3+EGFP of Example 11, Sp4+EGFP of Example 11, Free EGFP and Blank groups.
  • the final concentration of EGFP plasmid in each group was 7.58nM, and the cells were transfected 24 hours after inoculation. After 4 hours, the liquid was discarded, and 1640 medium containing 10% fetal calf serum was added. After continuing to culture for 48 hours, the expression of green fluorescent protein in the cells was observed using an inverted fluorescence microscope. The results are shown in Figure 15. It can be seen from the figure that when the N/P is 4:1, all four cationic liposomes can deliver the EGFP plasmid. Among them, the cationic liposome Sp1 has the highest green fluorescence intensity, showing a strong response to EGFP. Optimal delivery of plasmids.
  • MDA-MB-231 cells in the logarithmic growth phase were seeded at 1 ⁇ 10 5 cells per well on 6 cells covered with glass coverslips. well cell culture dish and cultured for 24 h.
  • coumarin C6 and PTX are both hydrophobic drugs, and coumarin itself can emit green fluorescence, so C6 was chosen to replace PTX, and coumarin was co-administered by laser. Focused microscopy was used to determine the cellular entry and intracellular distribution of siRNA and C6. The results are shown in Figure 16. The cell outline was obtained under bright field.
  • HIF-1 ⁇ gene silencing efficiency of HIF-1 ⁇ siRNA delivered by cationic liposomes logarithmic growth phase MDA-MB-231 cells were digested with trypsin and seeded in a 6-well plate at 2 ⁇ 10 5 /well. Transfection was performed 24 hours after cell seeding.
  • the experimental groups are Sp1+HIF-1 ⁇ siRNA in Example 11, Sp2+HIF-1 ⁇ siRNA in Example 11, Sp3+HIF-1 ⁇ siRNA in Example 11, Sp4+HIF-1 ⁇ siRNA in Example 11, Lipo2000+ HIF-1 ⁇ siRNA, Free HIF-1 ⁇ siRNA and Blank group.
  • the final concentration of HIF-1 ⁇ siRNA in each group was 50nM, and the N/P ratio was 4:1.
  • SRB method was used to detect the cationic lipids under normoxic (21% O 2 ) and hypoxic (1% O 2 ) conditions.
  • the experimental group settings are: the cationic liposome pharmaceutical preparations (Sphy1, Sphy2, Sphy3 and Sphy4) of Example 15, the cationic liposome pharmaceutical preparations (Sphy1, Sphy2, Sphy3 and Sphy4) of Example 15 and HIF-1 ⁇ siRNA respectively.
  • MDA-MB-231 cells were seeded in a 96-well plate at 4 ⁇ 10 3 /well/100 ⁇ L, and the culture medium was 1640 medium containing 10% FBS, and cultured for 24 h in a 37°C, 5% CO 2 saturated humidity incubator. . Different groups of drugs were added, and 3 duplicate wells were set for each concentration. After continuing to culture for 72 hours, 50 ⁇ L of pre-cooled TCA (trichloroacetic acid) solution (30%, w/v) at 4°C was added to each well to fix the cells. The final concentration of the TCA solution was 10%.
  • TCA trichloroacetic acid
  • the cationic liposome drug formulation group co-loaded with paclitaxel and HIF-1 ⁇ siRNA showed stronger tumor cytotoxicity under hypoxic conditions than the free paclitaxel group and the cationic liposome drug formulation group loaded with only paclitaxel. It shows that the cationic liposomes co-loaded with the chemotherapy drug paclitaxel and the genetic drug HIF-1 ⁇ siRNA have an obvious synergistic effect and can overcome the resistance of tumor cells to paclitaxel.
  • the cationic liposome drug preparation Sp1 has the best effect as a carrier to co-load paclitaxel and HIF-1 ⁇ siRNA, and has the potential to be used as a new cationic liposome co-delivery system to overcome the problem of drug resistance of paclitaxel in treating tumors.
  • mice set up physiological saline, Sp1 of Example 11, free HIF-1 ⁇ siRNA, Sp1+HIF-1 ⁇ siRNA of Example 11, free PTX, Sphy1 of Example 15, PTX+HIF-1 ⁇ There are 8 groups of siRNA and the Sphy1+HIF-1 ⁇ siRNA of Example 15. The mice are administered by tail vein injection once every two days with a disposable sterile insulin syringe. After 7 consecutive administrations, the administration is stopped. execution on the seventh day after cessation;
  • the mouse tumor growth trend was significantly slowed down, showing obvious tumor growth. Inhibitory activity ( * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, **** P ⁇ 0.0001).
  • the paclitaxel-loaded liposome Sphy1 and Sphy1+HIF-1 ⁇ siRNA groups of Example 15 also have stronger anti-tumor activity, showing significant differences ( # P ⁇ 0.05, ## P ⁇ 0.01, ### P ⁇ 0.001, #### P ⁇ 0.0001).
  • the Sphy1+HIF-1 ⁇ siRNA group showed stronger tumor inhibitory activity ( $ P ⁇ 0.05, $$ P ⁇ 0.01).
  • the tumor-bearing mice were sacrificed and the tumor tissues were separated and weighed.
  • the tumor weight of the cationic liposome drug preparation Sphy1+HIF-1 ⁇ siRNA group of Example 15 was the smallest, followed by the Sphy1 group of Example 15.

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Abstract

提供一种式(I)所示的鞘脂类化合物、含有鞘脂类化合物的脂质体和应用。所述鞘脂类化合物以长链烷基为疏水尾部，通过连接臂，键合亲水头部来制备，将其应用于阳离子脂质体，得到转染效率高，稳定性好的低毒性阳离子脂质体。 (AA), (BB), (CC)

Description

鞘脂类化合物、含有鞘脂类化合物的脂质体和应用 技术领域
本发明属于有机合成技术领域，尤其涉及一种鞘脂类化合物、含有鞘脂类化合物的脂质体和应用。
背景技术
乳腺癌，发病率位居女性恶性肿瘤的首位，其中三阴性乳腺癌(triple negative breast cancer；TNBC)因其复发率高、病死率高和侵袭性强等临床特点成为乳腺癌中恶性程度最高的类型。目前紫杉醇是临床上治疗TNBC的一线药物，但传统的紫杉醇剂型靶向作用差、药物转运效率低且易产生耐药。多项研究表明肿瘤的侵袭、增殖和耐药都和缺氧诱导因子(hypoxia inducible factor-1；HIF-1)有着密切的关系。低氧状态下，HIF-1通过上调多药耐药(multidrug resistance，MDR)与P-糖蛋白从而将紫杉醇等药物泵出细胞外，使细胞内药物浓度降低，杀伤细胞能力减弱。小干扰RNA(siRNA)对目的细胞具有高特异性和低毒性，可以成功沉默恶性致癌基因。然而，裸siRNA在血液中的半衰期不超过1小时，可被血浆中的核酸酶迅速降解或被肾排出。此外，裸siRNA由于其高分子量、亲水性和电荷密度，很难穿透细胞膜。因此，各种可以负载基因药物的载体应运而生，包括胶束、脂质体和无机纳米颗粒。阳离子脂质体已被证明比其他纳米载体具有优势，包括在体液和组织中具有更高的稳定性，更好的生物降解性，能够长时间释放药物。虽然国内外已经报道了多种阳离子脂质体，市面上也有出售的阳离子脂质体如LipofectamineTM2000；Transfection Reagent，但大多数阳离子脂质体表现出了较高的细胞毒性，较低的转染效率，且只能单一的用于递送基因药物。
因此，有必要提供一种新的鞘脂类化合物，克服三阴性乳腺癌对肿瘤化疗药(紫杉醇)的耐药性，提高抗癌药效；且所构建的给药系统具有转染效率高，安全性高，稳定性高的特点。
发明内容
本发明旨在至少解决现有技术中存在的上述技术问题之一。为此，本发明提供了一种鞘脂类化合物。
本发明还提供了一种阳离子脂质体。
本发明还提供了一种阳离子脂质体药物制剂。
本发明还提供了一种阳离子脂质体或阳离子脂质体药物制剂的应用。
本发明的第一方面提供了鞘脂类化合物，所述鞘脂类化合物的结构式如式(Ⅰ)所示：
Figure PCTCN2022098765-appb-000001
其中，所述R 1和R 2独立地选自取代或未取代的C 8～22的烷基、取代或未取代的C 8～20的烯基；
所述R 3选自
Figure PCTCN2022098765-appb-000002
所述n为1～5的整数，m为1-3的整数；
所述A为
Figure PCTCN2022098765-appb-000003
酰胺键、酯键、
Figure PCTCN2022098765-appb-000004
所述R 4选自H、氮杂芳烃基、含胍基取代的脂肪或芳烃基、取代或未取代的C 1～10的烷基、杂环基烃基、C 2～10的烷基酸；所述R 5选自取代或未取代的氨基。
本发明关于鞘脂类化合物的技术方案中的一个技术方案，至少具有以下有益效果：
本发明以长链烷基为疏水尾部，通过(含有三氮唑的共价键、酰胺键、酯键)连接臂，键合氨基酸、季铵盐、叔胺基、仲胺基或伯胺基等亲水头部，设计合成系列新型鞘脂类化合物。将其应用于制备新型阳离子脂质体，得到了转染效率高，稳定性好的低毒性阳离子脂质体。
根据本发明的一些实施方式，所述R 1选自
Figure PCTCN2022098765-appb-000005
其中x和y为8～20的整数。
根据本发明的一些实施方式，所述R 2选自
Figure PCTCN2022098765-appb-000006
Figure PCTCN2022098765-appb-000007
根据本发明的一些实施方式，所述R 4选自H、
Figure PCTCN2022098765-appb-000008
Figure PCTCN2022098765-appb-000009
根据本发明的一些实施方式，所述R 5选自
Figure PCTCN2022098765-appb-000010
Figure PCTCN2022098765-appb-000011
根据本发明的一些优选的实施方式，所述鞘脂类化合物选自如下结构式中的一种：
Figure PCTCN2022098765-appb-000012
Figure PCTCN2022098765-appb-000013
本发明的第二方面提供一种阳离子脂质体，所述阳离子脂质体包括上述任一项所述的鞘脂类化合物、胆固醇、二油酰磷脂酰乙醇胺和甲氧基-聚乙二醇-磷脂。
根据本发明的一些实施方式，所述甲氧基-聚乙二醇-磷脂的分子量范围为750～10000，优选的为分子量2000。
根据本发明的一些实施方式，所述鞘脂类化合物、胆固醇、二油酰磷脂酰乙醇胺和甲氧基-聚乙二醇-磷脂的摩尔比为1：(0.5～50)：(0.5～50)：(0.015～1)。
本发明的第三方面提供一种阳离子脂质体药物制剂，所述药物制剂包括上述所述的阳离子脂质体和药物活性成分，以所述阳离子脂质体中的鞘脂类化合物的摩尔量计算，所述鞘脂类化合物和药物活性成分的摩尔比为(0.1～50)：1。
根据本发明的一些实施方式，所述药物活性成分为抗肿瘤化合物、抗病毒化合物、抗炎化合物或类风湿药物中的一种或多种。
根据本发明的一些实施方式，所述药物活性成分为抗肿瘤化合物。
根据本发明的一些实施方式，所述抗肿瘤化合物为紫杉醇或其紫杉醇衍生物。
根据本发明的一些实施方式，所述阳离子脂质体药物制剂还包括核酸。
根据本发明的一些实施方式，所述核酸的量为N/P比1：1～16：1，所述的N/P比是阳离子脂质体中的可电离的氮原子N的摩尔含量和核酸中P的摩尔含量之比。
根据本发明的一些实施方式，所述核酸选自siRNA、miRNA、antagomir、质粒DNA或mRNA。
根据本发明的一些实施方式，所述阳离子脂质体药物制剂的制备方法，包括如下步骤：
S1、将鞘酯类化合物、胆固醇、二油酰磷脂酰乙醇胺、甲氧基-聚乙二醇-磷脂和药物活性成分加入有机溶剂进行溶解得到混合物；
S2、将所述混合物在30～50℃下减压蒸馏至形成脂膜，继续减压蒸馏0.5～2h；
S3、加入无酶水，进行超声至形成乳白色溶液，室温下继续搅拌；
S4、在0～5℃下，将步骤S3的溶液进行探头超声、过滤，即制得阳离子脂质体药物制剂。
根据本发明的一些实施方式，若要加入核酸，还包括步骤S5：将阳离子脂质体药物制剂与核酸复合物和10％胎牛血清混合，将样品置于室温下孵育，即得。
根据本发明的一些实施方式，所述有机溶剂包括氯仿和/或甲醇。
本发明的第四方面提供上述所述的阳离子脂质体、或所述的阳离子脂质体药物制剂在用于治疗由基因异常表达引起的相关疾病的药物制备中的用途，所述疾病包括恶性肿瘤、心血管疾病、类风湿、感染性疾病或遗传病。
根据本发明的一些实施方式，所述恶性肿瘤为三阴性乳腺癌。
本发明的阳离子脂质体共递送基因药物HIF-1α siRNA和化疗药物紫杉醇表现出协同作用。该阳离子脂质体可以应用于构建共同负载HIF-1α siRNA和紫杉醇的给药系统，克服三阴性乳腺癌对紫杉醇的耐药性，提高抗癌效果。
定义和一般术语
术语“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
本发明中“室温”指的是温度由10℃到40℃。在一些实施例中，“室温”指的是温度 由20℃到30℃；在另一些实施例中，“室温”指的是温度由25℃到30℃。
“取代或未取代的C 8～20的烷基”表示碳原子总数为8-20的烷基，包括直链烷基、支链烷基和环烷基；并且任选C 8-20的烷基中有至少一个H被本文定义的相应基团所取代，例如被羟基、烷氧基或氨基等基团取代。取代或未取代的C 1～10的烷基具有相似的定义，其区别在于碳原子数不相同。
“取代或未取代的C 8～20的烯基”表示具有一个或多个双键的直链或支链的烃基，且该基团的碳原子总数为8～20，基团中的双键可以在任意位置，并且任选C8-20的烯基中有至少一个H被本文定义的相应基团所取代，例如被羟基、烷氧基等基团取代。
“亚烃基链”仅由碳和氢组成的使分子的其余部分与基团连接的直链或支链的二价烃链，其为饱和的或不饱和的(即，含有一个或多个双键和/或叁键)，且具有一至十二个碳原子，例如亚甲基、亚乙基、亚丙基、亚正丁基、亚乙烯基、亚丙烯基、亚正丁烯基、亚丙炔基、亚正丁炔基等。亚烃基链通过单键或双键连接至分子的其余部分且通过单键或双键连接至基团。亚烃基链与分子的其余部分的连接点以及亚烃基链与基团的连接点可通过该链内的一个碳或任两个碳。除非本说明书中另有特定说明，否则亚烃基链是任选取代的。
“芳基”表示包含氢、6至18个碳原子和至少一个芳环的碳环环系统基团。出于本发明的目的，芳基可为单环、双环、三环或四环环系统，其可包括稠合的或桥联的环系统。芳基包括但不限于衍生自苯并苊、苊烯、醋菲烯、蒽、薁、苯、屈、荧蒽、芴、不对称引达省、对称引达省、茚满、茚、萘、非那烯、菲、七曜烯(pleiadene)、芘和苯并菲。除非本说明书中另有特定说明，否则术语“芳基”或前缀“芳-”(如在“芳烃基”中)意指包括任选取代的芳基。
“氮杂芳烃基”表示式-R b-R c的基团，其中R b为亚烃基链且R c为一个或多个如上文所定义的芳基，其中芳基中有至少一个H被N原子所取代。
“杂环基”表示是指具有一至十二个环碳原子(例如二至十二个)和一至六个选自氮、氧和硫的环杂原子的稳定的3元至18元非芳环基团。除非本说明书中另有特定说明，否则杂环基为单环、双环、三环或四环环系统，其可包括稠合、螺环(“螺-杂环基”)和/或桥联的环系统；且杂环基中的氮、碳或硫原子任选地被氧化；氮原子任选地被季铵化；且杂环基为部分或完全饱和的。
“杂环基烃基”表示式-R bR e的基团，其中R b为亚烃基链且R e为如上文所定义的杂环基。除非本说明书中另有特定说明，否则杂环基烃基是任选取代的。
“C 2～10的烷基酸”表示碳原子总数为2～10的羧酸，烷基包括直链烷基、支链烷基和环烷基。
“取代或未取代的氨基”表示氨基中至少一个H原子被本文所定义的相应基团所取代，例如C 1-C 8烷基或C 3-C 8环烷基取代基的基团取代。
附图说明
图1是实施例1～4的鞘脂类化合物的细胞毒性图；
图2是实施例11的阳离子脂质体以及实施例11的阳离子脂质体负载siRNA的细胞毒性图；
图3是实施例11的阳离子脂质体的存储稳定性图；
图4是实施例12制备的阳离子脂质体药物制剂中不同浓度的紫杉醇包封率图；
图5是实施例13制备的阳离子脂质体药物制剂中鞘脂类化合物-胆固醇-DOPE的摩尔比对紫杉醇包封率的影响图；
图6是不同DSPE-mPEG的摩尔比对紫杉醇包封率的影响图；
图7是实施例15制备的阳离子脂质体药物制剂的包封率和载药量图；
图8是实施例15制备的阳离子脂质体药物制剂中的PTX体外释放图；
图9是实施例15制备的阳离子脂质体药物制剂对siRNA-Luc的阻滞作用图；
图10是不同N/P比的负载紫杉醇的阳离子脂质体与siRNA复合物的粒径与电位分析图；
图11是实施例15制备的阳离子脂质体药物制剂与siRNA复合物血清中不同时间点的稳定性图；
图12是实施例11制备的阳离子脂质体递送siRNA-Luc对萤火虫荧光素酶的沉默效率图；
图13是实施例11制备的阳离子脂质体对Cy3-siRNA递送效果图；
图14是采用流式细胞仪定量分析实施例11制备的阳离子脂质体递送Cy3-siRNA效果图；
图15是实施例11制备的阳离子脂质体对EGFP的递送效果图；
图16是实施例11制备的阳离子脂质体共载香豆素C6和Cy3-siRNA的胞内分布图；
图17是MDA-MB-231细胞中HIF-1α蛋白表达量图；
图18是实施例15制备的阳离子脂质体药物制剂和基因药物HIF-1α siRNA对MDA-MB-231细胞的72h细胞毒性作用图；
图19是荷瘤小鼠体重变化趋势图；
图20是荷瘤小鼠肿瘤体积变化趋势图；
图21是荷瘤小鼠肿瘤体积变化趋势图；
图22是4T1肿瘤模型活体治疗实验结束后对模型中各组肿瘤质量的统计结果图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，但本发明的实施方式不限于此。
本发明所采用的试剂、方法和设备，如无特殊说明，均为本技术领域常规试剂、方法和设备。
本发明实施例涉及到的包封率和载药量的计算方法如下：
采用溶解-高速离心法测定紫杉醇包封率。分别吸取100μL Sphy1、Sphy2，Sphy3，Sphy4置于3500Da超速滤管中，加入0.4％吐温80-PBS(0.05mol/L，pH 7.4)溶液1.9mL，涡旋3min后，16000r/min的转速离心30min，取100μL上清液转移至1mL容量瓶中以无水甲醇定容，HPLC测定上清液中游离紫杉醇PTX的量；另分别取适量负载紫杉醇的脂质体至1mL容量 瓶中以无水甲醇破乳并定容，进样分析，HPLC测定PTX总量。
载药量(Drug loading efficiency,DLE)和包封率(Encapsulation efficiency,EE)用以下公式计算：
DLE＝W1/W×100％
EE＝W1/W2×100％
其中，W1和W2分别代表脂质体中被包封PTX的总质量和PTX的投料量，W为脂质总投料量。
实施例1
实施例1提供一种鞘脂类化合物a，结构式如下，制备方法如下：
Figure PCTCN2022098765-appb-000014
将化合物1植物鞘氨醇(2.83g,8.9mmol)和 tBoc 2O(2.23g,10.2mmol)置于反应瓶中，加入 tBuOH(60mL)溶液在室温下搅拌8h。待反应完全，减压浓缩反应溶液，以石油醚/乙酸乙酯(v/v，5:1)重结晶得到化合物2,产率为96％，白色固体。
称取产物2(2.55g,6.1mmol)和TsOH(116mg,0.61mmol)于反应瓶中，加入丙酮(20mL)溶液，室温下搅拌，待反应完全。减压浓缩反应溶液，柱层析分离得到化合物3，白色固体，产率89％。
将上述化合物3(2.09g,4.57mmol)置于反应瓶中，加入CH 2Cl 2(10mL)溶解，在0℃冰浴条件下缓慢滴加甲烷磺酰氯MsCl(0.728mL,9.45mmol)，最后加入三乙胺(1.48mL,10.65mmol)，将反应混合物在0℃下继续搅拌30分钟，然后在室温下搅拌一小时。反应液中加入H 2O进行淬灭，用CH 2Cl 2萃取(3×10mL)，收集有机相，无水Na 2SO 4干燥，减压下浓缩，柱层析分离得到化合物4，白色固体，产率为90％。
称取化合物4(1.89g,3.53mmol)于反应瓶中，加入DMF(10mL)溶解，后加入叠氮化钠(1.147g,28.67mmol)，反应混合物在65℃下搅拌过夜。反应完全后用H 2O猝灭，乙酸乙酯萃取(3×10mL)，收集有机相，无水Na 2SO 4干燥，减压下浓缩，柱层析分离得到化合物5，白色固体，产率为87％。
称取化合物5(1.8g,3.73mmol)用甲醇(10mL)溶解，再缓慢滴加浓盐酸(0.8mL)。将反应混合物在室温下搅拌过夜至反应完全，将反应溶液减压浓缩，得到粗品。继续加入油酸(1.32g,4.67mmol)，EDCI(893mg,4.66mmol)，和1-羟基苯并三唑HOBT(629mg,4.66mmol)，用二氯甲烷(15mL)溶解，后缓慢滴加三乙胺(0.723mL,5.20mmol)，将反应混合物在室温下搅拌16h，然后用二氯甲烷稀释。稀释后的溶液用饱和氯化铵水溶液洗涤。水层用二氯甲烷萃取。收集有机相，无水硫酸钠干燥，减压浓缩，柱层析得到化合物6，白色固体,产率为64％。
称取化合物6(0.1179g,0.19mmol)，抗坏血酸钠(94.9mg,0.48mmol),CuSO 4(6.7mg,0.04 mmol)和化合物7(98.3mg,0.26mmol)于圆底烧瓶中，后加入5.5mL的甲醇/水(10:1,v/v)，在室温下搅拌12h。反应液用乙酸乙酯萃取(3×10mL)，收集有机相，无水Na 2SO 4干燥，减压下浓缩，柱层析分离得到中间体8，白色固体，产率为65％。
称取化合物8(0.127g,0.13mmol)于反应瓶中，加入2mL甲醇溶解，缓慢滴加浓HCl，室温下搅拌至反应完全，反应液用水萃取(3×10mL)，收集水相。再向水相缓慢滴加饱和碳酸氢钠，调至PH＝7，水层用二氯甲烷萃取，收集有机相，无水硫酸钠干燥，减压浓缩，得到目标化合物a(80mg收率79％)。
目标化合物a的结构鉴定数据: 1H NMR(600MHz,CD 3OD)δ8.00(s,1H),5.32-5.35(m,2H),4.97-5.00(m,1H),4.69-4.74(m,1H),4.57-4.60(m,1H),4.48-4.52(m,1H),3.92-3.97(m,3H),2.95-2.98(m,2H),2.42-2.45(m,2H),2.00-2.05(m,4H),1.93-1.98(m,1H),1.87-1.91(m,2H),1.71-1.74(m,2H),1.62-1.67(m,3H),1.49-1.54(m,2H),1.29-1.34(m,45H),0.89(t,6H,J＝7.2Hz)； 13C NMR(150MHz,CD 3OD)δ175.2,170.2,145.9,131.0,130.8,126.0,74.0,72.4,54.3,54.1,40.4,35.4,33.12,33.11,30.82,30.73,30.68,30.66,30.53,30.50,30.46,30.40,30.39,30.31,28.2,26.2,26.0,23.8,23.0,14.51,14.50.HRMS(ESI):m/z calcd for C 45H 88N 7O 4([M+H] +):790.6892,found 790.6972。
其中，化合物1～8的结构式如下
Figure PCTCN2022098765-appb-000015
实施例2
实施例2提供一种鞘脂类化合物b，结构式如下，制备方法如下：
Figure PCTCN2022098765-appb-000016
将上述实施例1制备的化合物6(0.1845g,0.3mmol)和SnCl 2(0.569g,3.0mmol)的5mL乙醇溶液在室温下搅拌12h，二氯甲烷溶解，饱和NaCl溶液洗涤，收集有机相，无水硫酸钠 干燥，减压浓缩得到粗产品化合物9，无需进一步提纯。将上述粗品用无水CH 2Cl 2(7mL)溶解，在0℃搅拌，并缓慢加入三乙胺(0.4mL)和氯化乙酰氯(36μL)。将反应混合物在0℃下搅拌30分钟，然后在室温下下继续搅拌一小时。反应用H 2O猝灭，用CH 2Cl 2萃取，收集有机相，用无水硫酸钠干燥，减压浓缩，柱层析分离，得到化合物10，白色固体，产率为40％。
称取化合物10(41.6mg，0.12mmol)于反应瓶中，加入5mL乙酸乙酯溶解，后加入三甲胺(0.1mL,2M in THF)。在室温下搅拌24小时，产物沉淀为白色固体。将得到的悬浮液冷却到0℃，过滤，用冰冷的乙二醛洗涤，真空干燥，得到目标产物化合物b(45mg，收率65％)。
目标化合物b的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ8.73(t,1H,J＝6.0Hz,NH),7.52(d,1H,J＝8.4Hz,NH),5.32-5.34(m,2H),4.51(s,2H),4.17(s,1H),3.63-3.72(m,3H),3.48-3.52(m,1H),3.42(s,9H),2.19(t,2H,J＝7.8Hz),1.99-2.02(m,4H),1.42-1.58(m,8H),1.25-1.34(m,42H),0.88(t,6H,J＝7.2Hz)； 13C NMR(150MHz,CDCl 3)δ174.1,163.9,130.1,129.7,74.4,72.3,65.2,54.8,50.1,42.5,40.4,36.6,33.2,32.0,31.92,31.4,30.0,29.93,29.89,29.87,29.83,29.81,29.74,29.59,29.57,29.45,29.42,29.37,29.34,27.3,26.3,25.8,22.71,22.69,14.1.HRMS(ESI):m/z calcd for C 41H 82N 3O 4 +([M +]):680.6300,found 680.6286。
其中，化合物9和化合物10的结构式如下：
Figure PCTCN2022098765-appb-000017
实施例3
实施例3提供一种鞘脂类化合物c，结构式如下，制备方法如下：
Figure PCTCN2022098765-appb-000018
称取化合物10(139.6mg,0.23mmol),赖氨酸(79.7mg,0.23mmol),EDCI(51mg,0.27mmol)和HOBT(37mg,0.27mmol)于反应瓶中，加入2mL无水二氯甲烷溶解,后缓慢滴加三乙胺(0.06mL,0.43mmol)，室温下搅拌16h。反应加H 2O淬灭，反应液用二氯甲烷萃取(3×10mL)，收集有机相，无水Na 2SO 4干燥，减压下浓缩，柱层析分离得到化合物11，白色固体，产率为61％。
称取化合物11(0.127g,0.13mmol)于反应瓶中，加入2mL甲醇溶解，缓慢滴加浓HCl，室温下搅拌至反应完全，反应液用水萃取(3×10mL)，收集水相。再向水相缓慢滴加饱和碳酸氢钠，调至PH＝7，水层用二氯甲烷萃取，收集有机相，无水硫酸钠干燥，减压浓缩，得到的目标产物化合物c(40mg，收率75％)。
目标化合物c的结构鉴定数据: 1H NMR(600MHz,CD 3OD)δ7.00(s,1H),6.17(d,1H,J＝ 7.2Hz),5.33-5.37(m,2H),4.85-4.91(m,1H),4.04-4.14(m,6H),3.64-3.76(m,2H),3.46-3.48(m,1H),2.17-2.22(m,2H),2.00-2.01(m,4H),1.59-1.73(m,6H),1.25-1.30(m,50H),0.86(t,6H,J＝6.6Hz)； 13C NMR(150MHz,CD 3OD)δ174.23,168.52,167.88,130.03,129.72,78.33,72.85,71.58,52.05,50.23,42.42,41.14,41.07,40.83,40.27,36.78,36.69,33.95,31.94,31.92,29.78,29.74,29.71,29.67,29.65,29.59,29.54,29.48,29.37,29.34,29.33,29.26,29.21,29.17,28.86,27.24,27.20,25.63,25.45,22.70,14.13.HRMS(ESI):m/z calcd for C 42H 85N 4O 4([M+H] +)709.6565,found 709.6574。
其中，化合物11的结构式如下：
Figure PCTCN2022098765-appb-000019
实施例4
实施例4提供一种鞘脂类化合物d，结构式如下，制备方法如下：
Figure PCTCN2022098765-appb-000020
称取化合物1鞘氨醇(1.8g，3.73mmol)用甲醇(10mL)溶解，再缓慢滴加浓盐酸(0.8mL)。将反应混合物在室温下搅拌过夜至反应完全，将反应溶液减压浓缩，得到粗品。继续加入油酸(1.32g，4.67mmol)，EDCI(893mg,4.66mmol)，和HOBT(629mg，4.66mmol)，用二氯甲烷(15mL)溶解，后缓慢滴加三乙胺(0.723mL，5.20mmol)，将反应混合物在室温下搅拌16h，然后用二氯甲烷稀释。稀释后的溶液用饱和氯化铵水溶液洗涤。水层用二氯甲烷萃取。收集有机相，无水硫酸钠干燥，减压浓缩，柱层析分离得到化合物12，白色固体，产率为72％。
称取化合物12(2.55g,6.1mmol)和TsOH(116mg,0.61mmol)于反应瓶中，加入丙酮(20mL)溶液，室温下搅拌，待反应完全。减压浓缩反应溶液，柱层析分离得到目标化合物13，白色固体，产率89％。
称取化合物13(107.5mg,0.33mmol)于反应瓶中，加入2mL CH 2Cl 2溶解后在0℃下搅拌，继续加入Et 3N(0.05mL)和氯乙酰氯(20μL)。将反应混合物在0℃下搅拌30分钟，然后在室温下再搅拌一小时。反应用H 2O猝灭，反应液用二氯甲烷萃取(3×10mL)，收集有机相，无水Na 2SO 4干燥，减压浓缩，硅胶层析纯化，得到化合物14，白色固体，产率为67％，
称取化合物14(42.6mg，0.13mmol)于反应瓶中，加入5mL乙酸乙酯溶解，后加入三甲胺(0.1mL,2M in THF)。在室温下搅拌24小时，产物沉淀为白色固体。将得到的悬浮液冷却到0℃，过滤，用冰冷的乙二醛洗涤，真空干燥，得到目标化合物d(35mg，收率45％)。
目标化合物d的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.96(d,J＝8.4Hz,1H,-NH),5.33-5.36(m,2H),4.13-4.19(m,2H),4.09-4.11(m,1H),3.87-3.89(m,1H),3.66-3.68(m,1H),2.50(s,1H),2.17-2.20(m,2H),1.99-2.02(m,3H),1.53-1.64(m,6H),1.46(s,3H),1.33(s,3H),1.25-1.30(m,43H),0.86(t,J＝6.6Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:173.05,130.02,129.73,108.09,78.76,76.80,63.63,50.00,36.83,31.94,31.91,29.78,29.72,29.71,29.67,29.61,29.57,29.54,29.52,29.38,29.34,29.33,29.28,29.15,27.37,27.23,27.18,26.78,25.68,25.02,22.70,22.69,14.13.HRMS(ESI):m/z calcd for C 44H 85N 2O 5([M] +)721.6453,found 721.6474。
其中，化合物12，13和14的结构式如下：
Figure PCTCN2022098765-appb-000021
实施例5
实施例5提供七种鞘脂类化合物e-k，制备方法如下，结构式如下：
称取化合物14(78.9mg，0.12mmol)于反应瓶中，加入5mL无水二氯甲烷溶解，后加入三乙胺(33.4μL,0.24mmol)和一系列仲胺
Figure PCTCN2022098765-appb-000022
(0.24mmol)。在室温下搅拌24小时，TLC检测直至反应完全。柱层析分离得到一系列目标化合物e-k(收率29-91％)。
Figure PCTCN2022098765-appb-000023
目标化合物e的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.87(s,1H,-NH),5.32-5.36(m,2H),4.31-4.36(m,2H),4.20-4.22(m,1H),4.06-4.15(m,2H),3.18(s,2H),2.49(s,4H),2.12(t,J＝7.8Hz,2H),1.99-2.02(m,4H),1.61-1.62(m,7H),1.52-1.54(m,3H),1.43(s,3H,-C(CH 3) 2),1.25-1.31(m,47H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.57,170.55,130.08,129.74,108.20,78.78,77.63,64.65,60.38,54.34,48.03,36.85,31.94,31.91,29.78,29.76,29.72,29.69,29.68,29.63,29.59,29.57,29.54,29.38,29.34,29.33,29.30,29.20,29.15,28.98,27.42,27.37,27.23,27.19,26.72,25.73,25.68,25.38,25.02,23.77,22.70,22.69,14.13. HRMS(ESI):m/z calcd for C 46H 87N 2O 5([M+H] +)747.6537,found 747.6578。
Figure PCTCN2022098765-appb-000024
目标化合物f的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.55(d,J＝8.4Hz,1H,-NH),5.32-5.35(m,2H),4.27-4.34(m,3H),4.13-4.16(m,1H),4.02-4.04(m,1H),3.73-3.75(m,4H),3.20(s,2H),2.56-2.58(m,4H),2.12(t,J＝8.4Hz,2H),1.99-2.02(m,4H),1.54-1.62(m,4H),1.43(s,3H,-C(CH 3) 2),1.25-1.31(m,47H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.48,170.14,130.05,129.67,108.22,77.63,77.55,66.78,64.75,59.53,53.28,48.19,36.88,31.94,31.91,29.77,29.75,29.71,29.68,29.62,29.57,29.54,29.38,29.34,29.32,29.31,29.29,29.18,28.95,27.25,27.24,27.18,26.79,25.67,25.29,22.70,22.69,14.13.HRMS(ESI):m/z calcd for C 45H 85N 2O 6([M+H] +)749.6329,found 749.6370。
Figure PCTCN2022098765-appb-000025
目标化合物g的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.60(s,1H,-NH),5.32-5.37(m,2H),4.30-4.34(m,2H),4.19-4.21(m,1H),4.12-4.15(m,1H),4.05-4.07(m,1H),3.32(s,2H),2.51-2.53(m,4H),2.12-2.14(m,2H),1.98-2.02(m,4H),1.53-1.63(m,4H),1.43-1.47(m,6H),1.25-1.31(m,48H),0.86(t,J＝6.6Hz,12H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.45,171.79,130.02,129.70,108.16,77.58,77.44,64.26,56.42,55.06,48.23,36.87,31.94,31.91,29.78,29.75,29.71,29.68,29.63,29.57,29.55,29.54,29.38,29.34,29.33,29.30,29.18,28.99,27.35,27.23,27.19,26.73,25.64,25.33,22.70,22.69,20.69,14.12,11.76.HRMS(ESI):m/z calcd for C 47H 91N 2O 5([M+H] +)763.6850,found 763.6885。
Figure PCTCN2022098765-appb-000026
目标化合物h的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.61(s,1H,-NH),5.31-5.37(m,2H),4.30-4.34(m,2H),4.19-4.20(m,1H),4.12-4.15(m,1H),4.04-4.06(m,1H),3.32(s,2H),2.53-2.56(m,4H),2.12-2.15(m,2H),1.98-2.02(m,4H),1.53-1.61(m,4H),1.39-1.43(m,6H),1.25-1.31(m,52H),0.89(t,J＝7.2Hz,12H,-CH 2CH 3),0.86(t,J＝7.2Hz,12H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.44,171.74,130.02,129.70,108.16,77.58,77.42,64.26,55.02,54.15,48.23,36.87,31.94,31.91,29.78,29.76,29.71,29.69,29.68,29.63,29.58,29.55,29.54,29.38,29.34,29.33,29.31,29.19,28.99,27.36,27.23,27.19,26.74,25.64,25.34,22.70,22.69, 20.53,14.13,14.05.HRMS(ESI):m/z calcd for C 49H 95N 2O 5([M+H] +)791.7163,found 791.7201。
Figure PCTCN2022098765-appb-000027
目标化合物i的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.82(s,1H,-NH),5.31-5.37(m,2H),4.24-4.36(m,3H),4.06-4.15(m,2H),3.35(s,2H),2.65(s,4H),2.12-2.15(m,2H),1.98-2.02(m,4H),1.83(s,2H),1.53-1.63(m,6H),1.43(s,3H,-C(CH 3) 2),1.25-1.31(m,47H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:173.04,172.56,130.02,129.71,108.16,78.75,77.61,64.70,56.87,54.00,48.14,36.84,31.94,31.91,29.78,29.76,29.71,29.68,29.62,29.58,29.56,29.54,29.38,29.34,29.32,29.30,29.19,28.98,27.38,27.23,27.19,26.74,25.67,25.36,23.77,22.70,22.69,14.13.HRMS(ESI):m/z calcd for C 45H 85N 2O 5([M+H] +)733.6380,found 733.6425。
Figure PCTCN2022098765-appb-000028
目标化合物j的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.68(s,1H,-NH),5.30-5.37(m,2H),4.29-4.32(m,2H),4.25-4.27(m,1H),4.12-4.15(m,1H),4.06-4.07(m,1H),3.22(s,2H),2.50-2.62(m,8H),2.30(s,3H,-NCH 3),2.13(d,J＝7.8Hz,2H),1.98-2.02(m,4H),1.53-1.60(m,4H),1.43(s,3H,-C(CH 3) 2),1.25-1.31(m,47H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.52,170.35,130.03,129.69,108.18,77.59,77.39,64.71,59.31,54.83,52.83,48.12,45.95,36.84,31.94,31.91,29.78,29.76,29.72,29.68,29.62,29.59,29.54,29.38,29.34,29.32,29.30,29.19,28.97,27.35,27.24,27.19,26.76,25.67,25.34,22.70,22.69,14.13.HRMS(ESI):m/z calcd for C 46H 88N 3O 5([M+H] +)762.6646,found 762.6690。
Figure PCTCN2022098765-appb-000029
目标化合物k的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.67(s,1H,-NH),5.31-5.36(m,2H),4.31-4.33(m,2H),4.21-4.23(m,1H),4.12-4.15(m,1H),4.05-4.07(m,1H),3.32(s,2H),2.63-2.65(m,4H),2.12(d,J＝7.8Hz,2H),1.98-2.02(m,4H),1.53-1.59(m,4H),1.43(s,3H,-C(CH 3) 2),1.25-1.31(m,47H),1.04(t,J＝7.2Hz,6H,-CH 2CH 3),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.50,171.58,130.03,129.70,108.17,77.58,77.47,64.44,54.16,48.24,47.74,36.86,31.94,31.91,29.78,29.75,29.71,29.68,29.63,29.58,29.54,29.38,29.34,29.33,29.31,29.30,29.18,28.99,27.33,27.23,27.19,26.74,25.65,25.33,22.70,22.69,14.13,12.20.HRMS(ESI):m/z calcd for C 45H 87N 2O 5([M+H] +)735.6537,found 735.6574。
实施例6
实施例6提供七种鞘脂类化合物l-r，制备方法如下，结构式如下：
化合物13(622.0mg，1.0mmol)于反应瓶中，加入4-溴丁酸(1.25mmol)，二环己基碳二亚胺(257.5mg,1.25mmol)，4-二甲氨基吡啶(12.2mg,0.1mmol)后，用5mL无水二氯甲烷溶解，室温搅拌，TLC检测直至反应完全。将反应浓缩，后经柱层析分离得到中间体化合物15。
Figure PCTCN2022098765-appb-000030
称取化合物15(78.9mg，0.12mmol)于反应瓶中，加入5mL无水二氯甲烷溶解，后加入三乙胺(33.4μL,0.24mmol)和一系列仲胺
Figure PCTCN2022098765-appb-000031
(0.24mmol)。在室温下搅拌24小时，TLC检测直至反应完全。柱层析分离得到一系列目标化合物l-r(收率31-87％)。
Figure PCTCN2022098765-appb-000032
目标化合物l的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:6.71(s,1H,-NH),5.32-5.34(m,2H),4.36-4.38(m,1H),4.16-4.27(m,4H),2.93(s,2H),2.58-2.59(m,1H),2.49-2.52(m,1H),2.21-2.25(m,4H),1.98-2.02(m,8H),1.53-1.61(m,10H),1.42(s,3H,-C(CH 3) 2),1.24-1.32(m,47H),0.86(t,J＝6.6Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:173.00,171.87,129.97,129.77,107.99,77.88,76.01,65.05,56.84,53.64,47.84,36.68,31.94,31.91,31.32,29.80,29.79,29.73,29.71,29.68,29.66,29.64,29.54,29.41,29.38,29.35,29.33,29.26,28.79,27.82,27.24,26.85,25.83,25.66,22.69,14.13.HRMS(ESI):m/z calcd for C 48H 91N 2O 5([M+H] +)775.6850,found 775.6881。
Figure PCTCN2022098765-appb-000033
目标化合物m的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.54(d,J＝8.4Hz,1H,-NH),5.32-5.35(m,2H),4.25-4.32(m,2H),4.19-4.22(m,1H),4.12-4.15(m,1H),4.04-4.06(m,1H),3.69-3.70(m,4H),2.34-2.42(m,7H),2.12-2.15(m,2H),1.99-2.02(m,4H),1.79-1.82(m,2H),1.53-1.61(m,4H),1.43(s,3H,-C(CH 3) 2),1.25-1.32(m,47H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:173.50,172.48,130.05,129.67,108.18,77.57,77.55,66.95,64.42,57.94,53.60,48.28,36.89,31.94,29.77,29.75,29.71,29.69,29.68,29.62,29.57,29.55,29.54, 29.37,29.34,29.32,29.31,29.27,29.18,28.96,27.31,27.24,27.19,26.77,25.67,25.33,22.69,21.70,14.13.HRMS(ESI):m/z calcd for C 47H 89N 2O 6([M+H] +)777.6642,found 777.6688。
Figure PCTCN2022098765-appb-000034
目标化合物n的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.32-5.35(m,2H),4.24-4.28(m,3H),4.12-4.14(m,2H),2.50-2.60(m,4H),2.38-2.42(m,2H),2.15(t,J＝7.8Hz,2H),1.87-2.02(m,6H),1.53-1.60(m,8H),1.43(s,3H,-C(CH 3) 2),1.25-1.31(m,49H),0.89(t,J＝7.8Hz,6H,-CH 2CH 3),0.86(t,J＝6.6Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:173.14,172.65,130.00,129.72,108.09,77.71,77.57,64.53,55.41,52.85,48.08,36.78,31.94,31.92,31.67,29.78,29.72,29.70,29.68,29.64,29.61,29.59,29.54,29.38,29.35,29.33,29.21,28.91,27.53,27.24,27.21,26.79,25.71,25.47,22.70,14.13,11.73.HRMS(ESI):m/z calcd for C 49H 95N 2O 5([M+H] +)791.7163,found 791.7201。
Figure PCTCN2022098765-appb-000035
目标化合物o的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.59(s,1H,-NH),5.31-5.37(m,2H),4.27-4.31(m,2H),4.18-4.20(m,1H),4.12-4.15(m,1H),4.05-4.07(m,1H),2.32-2.39(m,8H),2.13-2.15(m,2H),1.98-2.02(m,4H),1.53-1.74(m,8H),1.43(s,3H,-C(CH 3) 2),1.36-1.39(m,4H),1.25-1.32(m,49H),0.89(t,J＝7.8Hz,6H,-CH 2CH 3),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:173.77,172.50,130.02,129.70,108.14,77.60,77.35,64.28,53.74,53.19,48.24,36.87,31.99,31.94,31.91,29.78,29.76,29.71,29.69,29.68,29.62,29.59,29.56,29.54,29.38,29.34,29.32,29.28,29.19,28.97,27.37,27.23,27.20,26.75,25.67,25.37,22.70,22.69,20.70,14.13,14.10.HRMS(ESI):m/z calcd for C 51H 99N 2O 5([M+H] +)819.7476,found 819.7512。
Figure PCTCN2022098765-appb-000036
目标化合物p的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:6.82(d,J＝7.8Hz,1H,-NH),5.32-5.34(m,2H),4.41-4.43(m,1H),4.27-4.29(m,2H),4.17-4.21(m,2H),3.16-3.19(m,2H),2.55-2.68(m,2H),2.16-2.26(m,8H),1.99-2.00(m,4H),1.51-1.61(m,8H),1.42(s,3H,-C(CH 3) 2),1.24-1.32(m,47H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:173.13,171.56,129.97,129.77,107.99,77.90,75.79,65.06,54.55,53.74,47.79,36.64,31.94,31.91,30.89,29.81,29.79,29.73,29.71,29.68,29.65,29.54,29.44,29.38,29.35,29.33,29.27,28.77,27.89,27.24,26.85,25.85,25.72,23.37,22.70,22.69,20.90,14.13.HRMS(ESI):m/z calcd for C 47H 89N 2O 5([M+H] +)761.6693,found 761.6732。
Figure PCTCN2022098765-appb-000037
目标化合物q的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.56(d,J＝8.4Hz,1H,-NH),5.32-5.35(m,2H),4.26-4.31(m,2H),4.18-4.20(m,1H),4.12-4.14(m,1H),4.04-4.06(m,1H),2.33-2.44(m,10H),2.27(s,3H,-NCH 3),2.12-2.15(m,2H),1.99-2.00(m,4H),1.78-1.82(m,2H),1.53-1.61(m,4H),1.43(s,3H,-C(CH 3) 2),1.25-1.31(m,49H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:173.52,172.48,130.03,129.68,108.16,77.58,77.47,64.36,57.53,55.13,53.09,48.26,46.05,36.88,32.07,31.94,31.91,29.77,29.75,29.71,29.68,29.67,29.62,29.58,29.55,29.54,29.37,29.34,29.32,29.31,29.27,29.18,28.97,27.34,27.23,27.19,26.75,25.66,25.34,22.70,22.12,14.13.HRMS(ESI):m/z calcd for C 48H 92N 3O 5([M+H] +)790.6959,found 790.6999。
Figure PCTCN2022098765-appb-000038
目标化合物r的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.32-5.34(m,2H),4.24-4.31(m,3H),4.14-4.18(m,2H),2.79-2.87(m,4H),2.44-2.50(m,2H),2.17-2.20(m,2H),1.98-2.02(m,6H),1.54-1.60(m,4H),1.43(s,3H,-C(CH 3) 2),1.24-1.31(m,55H),0.86(t,J＝6.6Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.82,172.44,129.99,129.75,108.05,77.80,76.47,64.81,51.35,47.95,46.54,36.71,31.94,31.92,31.39,29.79,29.73,29.71,29.68,29.65,29.62,29.55,29.38,29.35,29.33,29.24,28.84,27.68,27.24,27.22,26.83,25.76,25.56,22.70,14.13,9.75.HRMS(ESI):m/z calcd for C 47H 91N 2O 5([M+H] +)763.6850,found 763.6880。
实施例7
实施例7提供七种鞘脂类化合物s-y，制备方法如下，结构式如下：
化合物13(622.0mg，1.0mmol)于反应瓶中，3-溴丙酸(1.25mmol)，二环己基碳二亚胺(257.5mg,1.25mmol)，4-二甲氨基吡啶(12.2mg,0.1mmol)后，用5mL无水二氯甲烷溶解，室温搅拌，TLC检测直至反应完全。将反应浓缩，后经柱层析分离得到中间体化合物16。
Figure PCTCN2022098765-appb-000039
称取化合物16(78.9mg，0.12mmol)于反应瓶中，加入5mL无水二氯甲烷溶解，后加入三乙胺(33.4μL,0.24mmol)和一系列仲胺
Figure PCTCN2022098765-appb-000040
(0.24mmol)。在室温下 搅拌24小时，TLC检测直至反应完全。柱层析分离得到一系列目标化合物s-y(收率30-83％)。
Figure PCTCN2022098765-appb-000041
目标化合物s的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.98(s,1H,-NH),5.32-5.36(m,2H),4.48-4.50(m,1H),4.27-4.30(m,1H),4.16-4.18(m,1H),4.10-4.12(m,1H),4.04-4.07(m,1H),2.47-2.69(m,8H),2.10-2.14(m,2H),1.98-2.02(m,4H),1.46-1.61(m,10H),1.43(s,3H,-C(CH 3) 2),1.25-1.33(m,47H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.75,172.37,130.00,129.74,108.14,77.72,76.50,63.96,54.83,54.47,48.08,36.75,32.17,31.94,31.91,29.78,29.72,29.71,29.68,29.64,29.60,29.55,29.38,29.35,29.33,29.22,28.91,27.71,27.24,27.21,26.65,25.57,24.02,22.70,22.69,14.13.HRMS(ESI):m/z calcd for C 47H 89N 2O 5([M+H] +)761.6693,found 761.6730。
Figure PCTCN2022098765-appb-000042
目标化合物t的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.66(d,J＝9.6Hz,1H,-NH),5.31-5.36(m,2H),4.29-4.37(m,2H),4.11-4.23(m,2H),4.01-4.04(m,1H),3.68(t,J＝4.8Hz,4H),2.67(t,J＝7.2Hz,2H),2.46-2.53(m,6H),2.11-2.14(m,2H),1.99-2.02(m,4H),1.52-1.62(m,4H),1.44(s,3H,-C(CH 3) 2),1.25-1.32(m,47H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.55,172.29,130.04,129.68,108.19,77.62,77.23(overlapped with C of CDCl 3),66.84,64.33,54.22,53.49,48.22,36.86,32.02,31.94,31.91,29.77,29.75,29.71,29.69,29.67,29.62,29.58,29.56,29.53,29.37,29.33,29.30,29.18,28.94,27.43,27.23,27.19,26.74,25.61,25.40,22.70,14.12.HRMS(ESI):m/z calcd for C 46H 87N 2O 6([M+H] +)763.6486,found 763.6526。
Figure PCTCN2022098765-appb-000043
目标化合物u的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.74(s,1H,-NH),5.32-5.35(m,2H),4.29-4.34(m,2H),4.20-4.23(m,1H),4.10-4.13(m,1H),4.04-4.06(m,1H),2.77(d,J＝7.2Hz,2H),2.38-2.46(m,6H),2.10-2.13(m,2H),1.98-2.02(m,4H),1.60-1.64(m,6H),1.52-1.53(m,2H),1.43(s,3H,-C(CH 3) 2),1.25-1.32(m,47H),0.85-0.89(m,12H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.79,172.57,130.02,129.71,108.15,77.64,77.10,64.14,55.73,49.59,48.16,36.83,32.56,31.94,31.92,29.78,29.77,29.72,29.69,29.68,29.63,29.59,29.54,29.38,29.33,29.20,28.95,27.49,27.24,27.20,26.71,25.62,25.43,22.70,22.69,19.81,14.13,11.90.HRMS(ESI):m/z calcd for C 48H 93N 2O 5([M+H] +)777.7006,found 777.7042。
Figure PCTCN2022098765-appb-000044
目标化合物v的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.32-5.35(m,2H),4.29-4.37(m,2H),4.19-4.22(m,1H),4.05-4.12(m,2H),2.82(s,2H),2.48(s,4H),2.11-2.14(m,2H),1.98-2.02(m,4H),1.51-1.62(m,9H),1.43(s,3H,-C(CH 3) 2),1.25-1.33(m,52H),0.90(t,J＝7.2Hz,6H,-CH 2CH 3),0.86(t,J＝6.6Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.66,172.63,130.00,129.72,108.13,77.67,77.23(overlapped with C of CDCl 3),64.19,53.43,49.52,48.12,36.80,31.94,31.91,29.78,29.72,29.69,29.68,29.63,29.60,29.56,29.54,29.38,29.35,29.33,29.22,28.92,27.57,27.23,27.20,26.73,25.63,25.48,22.69,20.66,14.12,14.04.HRMS(ESI):m/z calcd for C 50H 97N 2O 5([M+H] +)805.7319,found 805.7355。
Figure PCTCN2022098765-appb-000045
目标化合物w的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:6.39(d,J＝9.0Hz,1H,-NH),5.32-5.35(m,2H),4.65-4.68(m,1H),4.26-4.29(m,1H),4.08-4.12(m,2H),3.99-4.02(m,1H),2.87(s,2H),2.54-2.66(m,6H),1.98-2.10(m,6H),1.48-1.61(m,6H),1.44(s,3H,-C(CH 3) 2),1.24-1.34(m,49H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.78,171.99,129.98,129.76,108.14,77.87,76.17,63.73,54.28,52.61,47.84,36.60,31.94,29.79,29.72,29.68,29.64,29.61,29.55,29.54,29.40,29.38,29.34,29.33,29.22,28.81,27.91,27.24,27.21,26.62,25.70,25.51,23.45,22.70,14.13.HRMS(ESI):m/z calcd for C 46H 87N 2O 5([M+H] +)747.6537,found 747.6578。
Figure PCTCN2022098765-appb-000046
目标化合物x的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.75(d,J＝9.6Hz,1H,-NH),5.32-5.36(m,2H),4.42-4.44(m,1H),4.27-4.30(m,1H),4.17-4.20(m,1H),4.11-4.13(m,1H),4.02-4.04(m,1H),2.68-2.71(m,2H),2.47-2.55(m,8H),2.28(s,3H,-NCH 3),2.10-2.13(m,2H),1.98-2.02(m,4H),1.51-1.61(m,6H),1.43(s,3H,-C(CH 3) 2),1.25-1.33(m,47H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.59,172.36,130.02,129.70,108.17,77.66,77.23(overlapped with C of CDCl 3),64.03,55.00,54.07,53.03,48.17,46.03,36.85,32.30,31.94,31.91,29.78,29.72,29.70,29.68,29.60,29.56,29.54,29.37,29.34,29.20,28.94,27.59,27.24,27.20,26.70,25.57,25.50,22.70,14.13.HRMS(ESI):m/z calcd for C 47H 90N 3O 5([M+H] +)776.6802,found 776.6848。
Figure PCTCN2022098765-appb-000047
目标化合物y的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ:5.32-5.35(m,2H),4.29-4.46(m,2H),4.05-4.20(m,3H),2.50-2.84(m,6H),2.10-2.13(m,2H),1.98-2.02(m,4H),1.50-1.60(m,8H),1.43(s,3H,-C(CH 3) 2),1.25-1.33(m,51H),0.86(t,J＝7.2Hz,6H,-CH 2CH 3)； 13C NMR(150MHz,CDCl 3)δ:172.68,172.40,130.01,129.74,108.13,78.75,77.71,64.16,48.41,48.11,46.44,36.77,32.33,31.94,29.78,29.72,29.67,29.63,29.59,29.54,29.38,29.34,29.33,29.21,28.89,27.65,27.25,27.21,26.70,25.60,25.54,22.70,14.12,10.86.HRMS(ESI):m/z calcd for C 47H 89N 2O 5([M+H] +)749.6693,found 749.6736。
实施例8
实施例8提供一种鞘脂类化合物a1，结构式如下，制备方法如下：
Figure PCTCN2022098765-appb-000048
将化合物鞘氨醇17(2.67g,8.9mmol)和 tBoc 2O(2.23g,10.2mmol)置于反应瓶中，加入 tBuOH(60mL)溶液在室温下搅拌8h。待反应完全，减压浓缩反应溶液，以石油醚/乙酸乙酯(v/v，5:1)重结晶得到化合物18,产率为93％，白色固体。
将上述化合物18(1.83g,4.57mmol)置于反应瓶中，加入CH 2Cl 2(10mL)溶解，在0℃冰浴条件下缓慢滴加甲烷磺酰氯MsCl(0.728mL,9.45mmol)，最后加入三乙胺(1.48mL,10.65mmol)，将反应混合物在0℃下继续搅拌30分钟，然后在室温下搅拌一小时。反应液中加入H 2O进行淬灭，用CH 2Cl 2萃取(3×10mL),收集有机相，无水Na 2SO 4干燥，减压下浓缩，柱层析分离得到化合物19，白色固体，产率为93％。
称取化合物19(1.68g,3.53mmol)于反应瓶中，加入DMF(10mL)溶解，后加入叠氮化钠(1.147g,28.67mmol)，反应混合物在65℃下搅拌过夜。反应完全后用H 2O猝灭，乙酸乙酯萃取(3×10mL)，收集有机相，无水Na 2SO 4干燥，减压下浓缩，柱层析分离得到化合物20，白色固体，产率为83％。
称取化合物20(1.32g,3.73mmol)用甲醇(10mL)溶解，再缓慢滴加浓盐酸(0.8mL)。将反应混合物在室温下搅拌过夜至反应完全，将反应溶液减压浓缩，得到粗品。继续加入油酸(1.32g,4.67mmol)，EDCI(893mg,4.66mmol)，和1-羟基苯并三唑HOBT(629mg,4.66mmol)，用二氯甲烷(15mL)溶解，后缓慢滴加三乙胺(0.723mL,5.20mmol)，将反应混合物在室温下搅拌16h，然后用二氯甲烷稀释。稀释后的溶液用饱和氯化铵水溶液洗涤。水层用二氯甲烷萃取。收集有机相，无水硫酸钠干燥，减压浓缩，柱层析得到化合物21，白色固体,产率为60％。
称取化合物21(0.069g,0.19mmol)，抗坏血酸钠(94.9mg,0.48mmol),CuSO 4(6.7mg,0.04 mmol)和化合物7(98.3mg,0.26mmol)于圆底烧瓶中，后加入5.5mL的甲醇/水(10:1,v/v)，在室温下搅拌12h。反应液用乙酸乙酯萃取(3×10mL)，收集有机相，无水Na 2SO 4干燥，减压下浓缩，柱层析分离得到中间体22，白色固体，产率为57％。
称取化合物22(0.098g,0.13mmol)于反应瓶中，加入2mL甲醇溶解，缓慢滴加浓HCl，室温下搅拌至反应完全，反应液用水萃取(3×10mL)，收集水相。再向水相缓慢滴加饱和碳酸氢钠，调至PH＝7，水层用二氯甲烷萃取，收集有机相，无水硫酸钠干燥，减压浓缩，得到目标化合物a1(76mg收率78％)。
目标化合物a1的结构鉴定数据: 1H NMR(600MHz,CD 3OD)δ7.59(s,1H),5.64-5.67(m,2H),5.32-5.35(m,2H),4.80-5.00(m,2H),4.46-4.52(m,2H),3.92-3.97(m,2H),2.95-2.98(m,1H),2.69(m,2H),1.94-2.16(m,8H),1.77(m,2H),1.50-1.53(m,6H),1.25-1.30(m,44H),0.88(t,6H,J＝7.2Hz)； 13C NMR(150MHz,CD 3OD)δ173.7,171.2,134.3,130.8,130.6,129.8,122.9,74.0,55.7,54.3,54.1,40.4,35.4,33.12,33.11,30.82,30.73,30.68,30.66,30.53,30.50,30.46,30.40,30.39,30.31,43.0,42.1,36.8,34.1,31.9,29.6,29.3,28.9,27.6,25.7,22.7,22.3,14.10.HRMS(ESI):m/z calcd for C 45H 85N 7O 3([M+H] +)772.6714,found 772.6709。
其中，化合物17～22的结构式如下
Figure PCTCN2022098765-appb-000049
实施例9
实施例9提供一种鞘脂类化合物b1，结构式如下，制备方法如下：
Figure PCTCN2022098765-appb-000050
将上述实施例8制备的化合物21(0.177g,0.3mmol)和SnCl 2(0.569g,3.0mmol)的5mL乙醇溶液在室温下搅拌12h，二氯甲烷溶解，饱和NaCl溶液洗涤，收集有机相，无水硫酸钠干燥，减压浓缩得到粗产品化合物23，无需进一步提纯。将上述粗品用无水CH 2Cl 2(7mL)溶解，在0℃搅拌，并缓慢加入三乙胺(0.4mL)和氯化乙酰氯(36μL)。将反应混合物在0℃下搅拌30分钟，然后在室温下下继续搅拌一小时。反应用H 2O猝灭，用CH 2Cl 2萃取，收集有机相，用无水硫酸钠干燥，减压浓缩，柱层析分离，得到化合物24，白色固体，产率为37％。
称取化合物24(76.6mg，0.12mmol)于反应瓶中，加入5mL乙酸乙酯溶解，后加入三甲胺(0.1mL,2M in THF)。在室温下搅拌24小时，产物沉淀为白色固体。将得到的悬浮液冷却 到0℃，过滤，用冰冷的乙二醛洗涤，真空干燥，得到目标产物化合物b1(40mg，收率65％)。
目标化合物b1的结构鉴定数据: 1H NMR(600MHz,CDCl 3)δ8.14(t,1H,J＝6.0Hz,NH),8.01(d,1H,J＝8.4Hz,NH),6.18(s,1H,-OH),5.64-5.67(m,2H),5.32-5.34(m,2H),4.80(m,1H),3.48-3.52(m,1H),3.30(s,9H),2.19(t,2H,J＝7.8Hz),1.94-2.16(m,8H),1.53(m,2H),1.26-1.33(m,42H),0.88(t,6H,J＝7.2Hz)； 13C NMR(150MHz,CDCl 3)δ173.8,170.9,134.3,130.6,129.8,73.6,72.2,58.7,53.8,38.4,36.8,34.0,31.9,29.9,29.7,29.6,29.3,29.0,28.6,27.7,25.6,22.7,14.1；HRMS(ESI):m/z calcd for C 41H 80N 3O 3 +([M] +)662.6194,found 662.6194。
其中，化合物23和化合物24的结构式如下：
Figure PCTCN2022098765-appb-000051
实施例10
实施例10提供一种鞘脂类化合物c1，结构式如下，制备方法如下：
Figure PCTCN2022098765-appb-000052
称取化合物23(129.4mg,0.23mmol),赖氨酸(79.7mg,0.23mmol),EDCI(51mg,0.27mmol)和HOBT(37mg,0.27mmol)于反应瓶中，加入2mL无水二氯甲烷溶解,后缓慢滴加三乙胺(0.06mL,0.43mmol)，室温下搅拌16h。反应加H 2O淬灭，反应液用二氯甲烷萃取(3×10mL)，收集有机相，无水Na 2SO 4干燥，减压下浓缩，柱层析分离得到化合物25，白色固体，产率为57％。
称取化合物25(89.6mg,0.13mmol)于反应瓶中，加入2mL甲醇溶解，缓慢滴加浓HCl，室温下搅拌至反应完全，反应液用水萃取(3×10mL)，收集水相。再向水相缓慢滴加饱和碳酸氢钠，调至PH＝7，水层用二氯甲烷萃取，收集有机相，无水硫酸钠干燥，减压浓缩，得到的目标产物化合物c1(37mg，收率72％)。
目标化合物c1的结构鉴定数据: 1H NMR(600MHz,CD 3OD)δ8.86(s,2H),8.35(s,1H),8.22(s,1H),6.10(s,1H),5.68-5.78(m,2H),5.31-5.39(m,2H),4.78-4.86(m,1H),4.28-4.55(m,1H),3.23-3.31(m,3H),2.62-2.70(m,2H),2.08-2.15(m,4H),2.00(t,J＝6.0Hz,2H),1.76-1.91(m,4H),1.58-1.65(m,6H),1.22-1.29(m,44H),0.81(m,6H)； 13C NMR(150MHz,CD 3OD)δ173.8,173.4,134.3,130.6,129.8,73.6,58.7,54.3,42.0,38.7,36.8,34.2,34.0,31.9,29.9,29.7,29.6,29.3,29.0,28.6,27.7,25.6,22.7,22.3,14.1.HRMS(ESI):m/z calcd for C 42H 83N 4O 3([M+H] +)691.6460,found 691.6394。
其中，化合物25的结构式如下：
Figure PCTCN2022098765-appb-000053
实施例11
将实施例1～4制备的鞘脂类化合物(a、b、c和d)分别制备成阳离子脂质体(Sp1、Sp2、Sp3和Sp4)，其制备方法如下：
(1)将鞘脂类化合物(a、b、c和d)分别与胆固醇、辅助磷脂DOPE以及DSPE-mPEG置于圆底烧瓶内，使其摩尔比为1:1:1:0.06。
(2)用2mL氯仿-甲醇(v/v 1:1)溶液充分溶解，置于旋转蒸发仪上，40℃水浴减压蒸馏至圆底烧瓶底部形成均匀的脂膜，继续减压浓缩1h，除尽残留有机试剂。
(3)然后加入2mL纯化水，置于超声仪上超声5min，形成乳白色溶液，常温下继续搅拌水化1h。
(4)冰浴下探头超声120次(工作3s，间歇2s)，0.22μm滤膜过滤，即分别制得阳离子脂质体(Sp1)、阳离子脂质体(Sp2)、阳离子脂质体(Sp3)和阳离子脂质体(Sp4)。
实施例12
研究鞘脂类化合物(a、b、c和d)与紫杉醇的比例对包封率的影响，将实施例1～4制备的鞘脂类化合物(a、b、c和d)分别制备成阳离子脂质体药物制剂，制备步骤如下：
1)分别称取摩尔比为1：1：1的鞘脂类化合物(a、b、c和d)、胆固醇、辅助磷脂DOPE于圆底烧瓶内，再加入紫杉醇，以氯仿和甲醇2mL使其充分溶解，其中，鞘脂类化合物(a、b、c和d)和紫杉醇的摩尔比分别均为1:1、2:1、4:1、8:1和16:1。
2)充分溶解后，置于旋转蒸发仪上，40℃减压蒸馏至圆底烧瓶底部形成均匀的脂膜，继续减压蒸馏1h，除尽残留有机试剂。
3)加入适量无酶水，置于超声仪上超声至形成均一的乳白色溶液，常温下继续搅拌水化1h。
4)取出磁子，将上述溶液从圆底烧瓶转移至5mL样品瓶中。冰浴下，探头超声120次。0.22μm滤膜过滤，即可制得一系列阳离子脂质体药物制剂。
实施例13
研究鞘脂类化合物(a、b、c和d)的含量对紫杉醇的包封率的影响，制备方法同实施例12，鞘脂类化合物(a、b、c和d)和紫杉醇的摩尔比为8:1。其区别在于，鞘脂类化合物(a、b、c和d)、胆固醇和辅助磷脂DOPE的摩尔比分别为0.5:1:1、1:1:1、2:1:1和4:1:1。
实施例14
研究DSPE-mPEG的含量对紫杉醇包封率的影响，制备方法同实施例12，其区别在于，鞘脂类化合物(a、b、c和d)和DSPE-mPEG的摩尔比分别为1:0.015、1:0.03、1:0.06和1:0.12。
实施例15
实施例15提供阳离子脂质体药物制剂(Sphy1)、阳离子脂质体药物制剂(Sphy2)、阳离子脂质体药物制剂(Sphy3)和阳离子脂质体药物制剂(Sphy4)，其制备方法如下：
1)分别称取摩尔比为1：1：1：0.06的鞘脂类化合物(a、b、c和d)、胆固醇、辅助磷脂DOPE和DSPE-mPEG(分子量2000)于圆底烧瓶内，再加入紫杉醇，以氯仿和甲醇2mL使其充分溶解，其中，鞘脂类化合物(a、b、c和d)和紫杉醇的摩尔比分别均为8:1。
2)充分溶解后，置于旋转蒸发仪上，40℃减压蒸馏至圆底烧瓶底部形成均匀的脂膜，继续减压蒸馏1h，除尽残留有机试剂。
3)加入适量无酶水，置于超声仪上超声至形成均一的乳白色溶液，常温下继续搅拌水化1h。
4)取出磁子，将上述溶液从圆底烧瓶转移至5mL样品瓶中。冰浴下，探头超声120次。0.22μm滤膜过滤，即制得一系列阳离子脂质体药物制剂。
性能测试
鞘脂类化合物的细胞毒性测试：采用MTT法检测实施例1～4的鞘脂类化合物a、鞘脂类化合物b、鞘脂类化合物c和鞘脂类化合物d对MDA-MB-231细胞的增殖的影响，以4×10 3/孔/100μL将MDA-MB-231细胞接种于96孔板中，培养基分别为含10％FBS 1640培养基，在37℃，5％CO 2饱和湿度培养箱内培养24h。加入不同浓度的鞘脂类化合物a、鞘脂类化合物b、鞘脂类化合物c和鞘脂类化合物d(80、40、20、10、5、2.5μM)，每个浓度设置3个复孔。继续培养72h后，每孔加入10μL MTT溶液，继续孵育4h。吸出上清液，每孔加入100μL二甲基亚砜，置于震荡仪上震荡10min，使甲瓒晶体充分溶解，酶标仪测定吸光值(λ＝570nm)，据此计算细胞存活率。结果如图1所示。在浓度为2.5-60μM的范围内，四种鞘脂类化合物均未表现出细胞毒性，可以作为安全的载体材料递送化疗药物和基因药物。
阳离子脂质体及其与siRNA-Luc复合物的细胞毒性试验：采用MTT法检测实施例11制备的Sp1、Sp2、Sp3、Sp4、Sp1+siRNA-Luc复合物、Sp2+siRNA-Luc复合物、Sp3+siRNA-Luc复合物、Sp4+siRNA-Luc复合物及free siRNA-Luc对MDA-MB-231细胞活力的影响。因主要为考察阳离子脂质体在载siRNA进行细胞转染过程中是否抑制MDA-MB-231细胞的生长。以4×10 3/孔/100μL将MDA-MB-231细胞接种于96孔板中，用含10％胎牛血清的1640培养基在37℃，5％CO 2饱和湿度培养箱内培养24h。配制N/P为8:1的Sp1、Sp2、Sp3、Sp4、Sp1+siRNA-Luc复合物、Sp2+siRNA-Luc复合物、Sp3+siRNA-Luc复合物、Sp4+siRNA-Luc复合物，siRNA终浓度为50nM，另设置相同浓度的游离siRNA与同等浓度梯度的脂质体、以及Lipo2000、Lipo 2000+siRNA-Luc复合物作为对照组。与细胞共孵育4h后，吸去培养基，加入新的完全培养基继续培养72h后，加MTT进行测定，并计算细胞存活率。细胞毒性如图2所示。四种阳离子脂质体及负载siRNA-Luc阳离子脂质体在N/P为8：1的浓度下与细胞进行孵育时，并未产生显著的细胞毒性，与市售转染试剂Lipofectamin 2000(Lipo 2000)相比，也具有相当的存活率。由试验结果可知，阳离子脂质体运载基因进行转染时具有较好的生物兼容性。
将实施例11制备的一系列阳离子脂质体进行储存稳定性测试，取新鲜制备的阳离子脂质体无菌过滤分装至灭菌EP管中，于4℃冰箱存储28天，其存储体系为无菌无RNA酶水，在1、4、7、14、28天取出样品，以激光粒度仪测定其粒径，观察存储期间变化；结果如图3所示，制备的四种空白阳离子脂质体存储28天后，粒径没有明显变化，外观无明显沉淀和絮凝，表现出较高的稳定性。
将实施例11制备的一系列阳离子脂质体和实施例15制备的一系列阳离子脂质体药物制剂进行粒径和电位检测，结果如表1所示，从表中可知，四种阳离子脂质体粒径均不超过100nm，聚分散指数(PDI)均小于0.3。说明实施例1～4的鞘脂类化合物在胆固醇，辅助磷脂DOPE以及DSPE-mPEG的辅助下，能在水溶液中形成脂质体，且其粒径较小，粒径分布较窄，均一性较好。并且四种脂质体均能有效的包载化疗药物紫杉醇，脂质体紫杉醇复合物粒径没有明显变化，且均一性较好。此外这些脂质体及其复合物Zeta电位均大于30mV，较高的表面正电荷说明了该脂质体具有作为阴离子药物载体的特性，且在溶液内较稳定，不易发生聚集。
表1阳离子脂质体和阳离子脂质体药物制剂的粒径、电位和聚分散指数表
Figure PCTCN2022098765-appb-000054
将实施例12制备的一系列阳离子脂质体药物制剂进行紫杉醇的包封率测试，研究紫杉醇的含量对包封率效果的影响，结果如图4所示，当PTX与鞘脂类化合物的摩尔比减小到1：8时，Sphy1、Sphy2、Sphy3和Sphy4的包封率分别为83.6％，78.5％，85.4％，73.2％，PTX用量继续减少后，包封率没有明显提高。
将实施例13制备的一系列阳离子脂质体药物制剂进行紫杉醇的包封率测试，结果如图5和表2所示，随着鞘脂类化合物的比例不断增大，包封率也不断增大，鞘脂类化合物-胆固醇-DOPE为1：1：1时，Sphy1、Sphy2、Sphy3和Sphy4的包封率分别为78.6％、68.5％、83.7％和63.2％。鞘脂类化合物的比例进一步增大后，包封率没有明显变化，但是正电荷密度进一步增大，具有增加细胞毒性的风险。
表2不同鞘脂类化合物-胆固醇-DOPE的摩尔比制备的阳离子脂质体药物制剂的表征
Figure PCTCN2022098765-appb-000055
Figure PCTCN2022098765-appb-000056
将实施例14制备的一系列阳离子脂质体药物制剂进行紫杉醇的包封率测试，由图6和表3可知，随着DSPE-mPEG的增加四种脂质体包封率均进一步增加，当DSPE-mPEG含量与鞘脂的摩尔比例为0.06：1时包封率达到最优，分别为93.6％、90.3％、95.4％、89.7％。同时粒径达到最小，电位均在35mV以上，适合作为阳离子脂质体材料。
表3不同DSPE-mPEG比例制备的阳离子脂质体的表征
Figure PCTCN2022098765-appb-000057
将实施例15的一系列阳离子脂质体药物制剂进行包封率和载药量测试，结果如图7所示，Sphy1、Sphy2、Sphy3和Sphy4的包封率均在90％以上，分别为93.6％、90.3％、95.4％和92.7％。载药量分别为4.89％、4.97％、5.21％、4.85％。
将实施例15制备的一系列阳离子脂质体药物制剂进行PTX释放测试，由图8可知，Sphy1、Sphy2、Sphy3和Sphy4均能够缓慢释放紫杉醇。在PH为7.4时，与对照的游离紫杉醇对比，前12h内紫杉醇脂质体没有明显的突释现象，具有良好的稳定性，可以避免由于纳米制剂突释带来的毒副作用，提高紫杉醇用药的安全性。
将实施例15一系列阳离子脂质体药物制剂与siRNA混合孵育并测定结合率：采用琼脂 糖凝胶电泳阻滞试验来判断阳离子脂质体药物制剂与siRNA-Luc复合物的结合情况。取siRNA-Luc(10mM)，按N/P 1:1、2:1、4:1、8:1和16:1，加入阳离子脂质体药物制剂，孵育20min。以同浓度的游离siRNA-Luc作为对照组。然后与DNA加样缓冲液1:1混匀，上样于1％的琼脂糖凝胶电泳上样槽内，控制电压100V，20min，取出凝胶，用凝胶成像系统拍照分析。结果如图9所示，游离siRNA可在电场作用下完全向正极迁移，随着N/P增加，从上样孔中迁移的siRNA亮度逐渐降低，表明siRNA与阳离子脂质体逐渐结合，当N/P为4:1时，Sphy1、Sphy2、Sphy3和Sphy4几乎将所有的siRNA-Luc阻滞，说明四种脂质体均对siRNA-Luc有较好的结合能力。使用玛尔文纳米粒度分析仪对不同N/P的阳离子脂质体与siRNA复合物进行平均粒径和Zeta电位的测定。如图10所示(其中A为Sp1与siRNA复合物、B为Sp2与siRNA复合物、C为Sp3与siRNA复合物、D为Sp4与siRNA复合物)，在不同N/P下的粒径在84～150nm之间，随着siRNA-Luc的比例增加，粒径呈增大趋势，说明脂质体与siRNA相互结合。当N/P为4：1时，四种阳离子脂质体药物制剂的电位由负转正，电位在15-20mV之间,并且电位随着脂质体的增加而增加。该结果与琼脂糖凝胶电泳结果一致，脂质体复合物的正电性预示了其可以较好地与负电性的细胞膜结合，使基因类药物能更好的进入细胞内。
将实施例15的一系列阳离子脂质体药物制剂与siRNA混合孵育并研究血清稳定性：取实施例15的四种阳离子脂质体药物制剂与siRNA(siRNA-Luc)复合物和10％胎牛血清混合，将样品置于37℃孵育，并以等体积和相同浓度的游离siRNA作为对照。分别取0、6、12、24和48h的样品，在凝胶电泳分析前，在样品中加入2％的肝素钠溶液以提取siRNA，立即与2×Loading Buffer以1：1体积混匀，进行琼脂糖凝胶电泳分析并通过凝胶成像系统拍照。结果如图11所示，游离siRNA在12h基本完全降解，而由脂质体包裹的siRNA虽有降解，但其稳定性相较于游离siRNA己明显提高，在48h时仍然可以看到清晰的siRNA条带，证明四种脂质体在血清存在的条件下均对siRNA有一定的保护作用，避免siRNA被血清中的核酸酶等过快降解。
研究了阳离子脂质体携载萤火虫荧光素酶siRNA对萤火虫荧光素酶的沉默效率：取生长对数期MDA-MB-231-Luc细胞接种于24孔板内，每孔4万个细胞，培养24h。设置实验组:实施例11的Sp1+siRNA-Luc、实施例11的Sp2+siRNA-Luc、实施例11的Sp3+siRNA-Luc、实施例11的Sp4+siRNA-Luc、Lipo2000+siRNA-Luc(N/P 1:1，2:1，4:1，8:1)和游离siRNA-Luc，每孔内siRNA-Luc为50nM，进行转染，孵育4h后，换液继续培养48h。吸去培养液，PBS洗涤一次，加入细胞裂解液，提取萤火虫荧光素酶，按萤火虫荧光素酶测定试剂盒测定，计算荧光素酶沉默效率。结果如图12所示。在孵育时间为48h时四种负载siRNA-Luc的脂质体表现出最强的沉默效率，且随着N/P的增加，沉默效果呈增加趋势，当N/P为4:1时沉默效果达到最佳，而后N/P再增加时，沉默效果却又有所下降。这样的现象可能是脂质体与siRNA-Luc的结合程度引起的，当脂质体相较siRNA-Luc而言出现过量状态时，将siRNA-Luc牢牢结合在多个脂质体的中心，反而不利于siRNA-Luc的逃逸。这四种阳离子脂质体对细胞内荧光素酶的沉默效率分别为83％、68％、75％和63％；Lipo2000沉默效率为78％。
研究了阳离子脂质体对Cy3-siRNA的递送效率、入胞分布及行为：采用Cy3对siRNA进行荧光标记，通过共聚焦显微镜来观察阳离子脂质体对siRNA的递送情况。将呈对数生长期的MDA-MB-231细胞用胰酶消化后，以每孔1×10 5细胞接种于6孔板中。实验分组分别为实施例11的Sp1+Cy3-siRNA、实施例11的Sp2+Cy3-siRNA、实施例11的Sp3+Cy3-siRNA、实施例11的Sp4+Cy3-siRNA、Lipo2000+Cy3-siRNA、Free Cy3-siRNA和Blank组。各组Cy3-siRNA终浓度为50nM，细胞接种24h后进行转染。4h后，弃去液体，加入含10％血清的1640培养基，继续培养48h后用激光共聚焦显微镜观察细胞内的荧光表达。结果如图13所示，从图中看出：在N/P为4：1时，阳离子脂质体Sp1红色荧光强度最高，表现出对Cy3-siRNA最优的递送效果，但市售阳性对照Lipofectamin 2000却表现出对Cy3-siRNA相对较弱的递送效果，同时阳离子脂质体Sp3也显著超过市售阳性对照Lipofectamin 2000，对siRNA具有优异的递送效果。该实验结果与萤火虫荧光素酶沉默效率基本一致。
将对数生长期MDA-MB-231细胞用胰酶消化，以2×10 5/孔接种于6孔板中。细胞接种24h后进行转染。实验分组分别为实施例11的Sp1+Cy3-siRNA、实施例11的Sp2+Cy3-siRNA、实施例11的Sp3+Cy3-siRNA、Lipo2000+Cy3-siRNA、Free Cy3-siRNA和Blank组。各组Cy3-siRNA终浓度为50nM，N/P比为4：1。4h后，弃去液体，加入含10％胎牛血清的1640培养基，继续培养48h。弃去培养液，PBS清洗两遍，加入胰酶消化后，收集细胞，用流式细胞仪进行荧光分析。从图14可以看出，使用流式细胞仪定量评估48h时阳离子脂质在MDA-MB-231细胞上对Cy3-siRNA的递送效率,从流式分析图可看出，游离的Cy3-siRNA很难进入细胞，Sp1+Cy3-siRNA和Sp3+Cy3-siRNA组荧光阳性比例可以分别达到86.05％和59.98％，均高于阳性对照Lipo2000+Cy3-siRNA(41.98％)。这些结果与上述共聚焦显微镜观察结果一致。
研究了阳离子脂质体对编码EGFP的质粒DNA(大约含有6300个碱基对)的递送效率：EGFP为绿色荧光蛋白，通过倒置荧光显微镜来观察阳离子脂质体对EGFP质粒DNA的递送情况。将呈对数生长期的MDA-MB-231细胞用胰酶消化后，以每孔1×10 5细胞接种于6孔板中。实验分组分别为实施例11的Sp1+EGFP、实施例11的Sp2+EGFP、实施例11的Sp3+EGFP、实施例11的Sp4+EGFP、Free EGFP和Blank组。各组EGFP质粒终浓度为7.58nM，细胞接种24h后进行转染。4h后，弃去液体，加入含10％胎牛血清的1640培养基，继续培养48h后用倒置荧光显微镜观察细胞内的绿色荧光蛋白的表达。结果如图15所示，从图中看出：在N/P为4：1时，四种阳离子脂质体均能递送EGFP质粒，其中阳离子脂质体Sp1绿色荧光强度最高，表现出对EGFP质粒最优的递送效果。
研究阳离子脂质体共载香豆素C6和Cy3-siRNA的胞内分布：取对数生长期MDA-MB-231细胞，以每孔1×10 5个接种于铺有玻璃盖玻片的6孔细胞培养皿内，培养24h。分别加入实施例11的Sp1+C6+Cy3-siRNA、实施例11的Sp2+C6+Cy3-siRNA、实施例11的Sp3+C6+Cy3-siRNA、实施例11的Sp4+C6+Cy3-siRNA、Free C6和C6+FreeCy3-siRNA，使Cy3-siRNA终浓度为50nM，孵育4h后，弃去培养液，PBS轻轻洗涤一次，加入含10％胎 牛血清的1640培养基，继续培养48h。取出后，吸去培养液，PBS洗涤两次，加入4％多聚甲醛固定，再加入10μg/mL DAPI进行细胞核染色，15min后吸去染料，PBS洗涤，制作细胞爬片。置于激光共聚焦显微镜下观察红色荧光(λ＝550nm)，绿色荧光(λ＝443nm)与蓝色荧光(λ＝364nm)，经图像迭合后，确定Cy3-siRNA和C6在细胞内的分布情况。为证明阳离子脂质体可以有效地将siRNA和紫杉醇(PTX)递送进细胞，香豆素C6与PTX均为疏水药物，且香豆素自身可以发出绿色荧光，因此选用C6替代PTX，通过激光共聚焦显微镜确定siRNA和C6的入胞量和胞内分布。结果如图16所示，明场下获得细胞轮廓，与细胞核，C6及Cy3通道重叠后，可发现红色荧光和绿色荧光在细胞内，且部分与蓝色荧光重叠，说明了Cy3-siRNA和C6在阳离子脂质体的递送下，进入了细胞，且有部分已经进入了细胞核内。同时也可以观察到阳离子脂质体Sp1共递送C6和Cy3-siRNA的荧光最强，与单独递送Cy3-siRNA结果基本一致，表现出最优的递送效果。
研究阳离子脂质体递送HIF-1α siRNA对HIF-1α基因沉默效率：将对数生长期MDA-MB-231细胞用胰酶消化，以2×10 5/孔接种于6孔板中。细胞接种24小时后进行转染。实验分组分别为实施例11的Sp1+HIF-1α siRNA、实施例11的Sp2+HIF-1α siRNA、实施例11的Sp3+HIF-1α siRNA、实施例11的Sp4+HIF-1α siRNA、Lipo2000+HIF-1α siRNA、Free HIF-1α siRNA和Blank组。各组HIF-1α siRNA终浓度为50nM，N/P比为4：1。继续在1％O 2条件下培养48h，PBS清洗后，用含蛋白酶抑制剂的1×RIPA裂解液对细胞进行裂解和收集。采用BCA检测试剂盒定量测定蛋白浓度。用8％SDS-PAGE分离总蛋白，随后300mA条件下湿转到PVDF膜。膜在封闭缓冲液(5％脱脂牛奶TBS+0.1％Tween 20)中室温封闭1h。与一抗在4℃下孵育过夜后，再将膜用IRDye 800cw标记的山羊抗小鼠IgG二抗在室温下避光孵育1h，扫描检测免疫反应条带。
由图17的结果可以看出，与Blank组相比，阳离子脂质体Sp1、Sp2、Sp3、Sp4和Lipo2000本身并没有敲低HIF-1α蛋白表达的作用。游离的HIF-1α siRNA组的HIF-1α蛋白表达量与Blank相比也没有降低，证明其很难进入细胞敲低HIF-1α蛋白表达量；而通过转染试剂递送的HIF-1α siRNA组，HIF-1α蛋白表达量明显被敲低，其中Sp1+HIF-1α siRNA组蛋白表达量降低最为显著，优于阳性对照Lipo2000。
研究阳离子脂质体共递送化疗药物紫杉醇和基因药物HIF-1α siRNA的体外抗肿瘤活性：采用SRB法分别检测常氧(21％O 2)和低氧(1％O 2)条件下，阳离子脂质体共递送化疗药物紫杉醇和基因药物HIF-1α siRNA对MDA-MB-231细胞的抗增殖作用。实验组设置为：实施例15的阳离子脂质体药物制剂(Sphy1、Sphy2、Sphy3和Sphy4)、实施例15的阳离子脂质体药物制剂(Sphy1、Sphy2、Sphy3和Sphy4)分别与HIF-1α siRNA的复合物、游离的HIF-1α siRNA、游离PTX、PTX与HIF-1α siRNA的复合物。以4×10 3/孔/100μL将MDA-MB-231细胞接种于96孔板中，培养基分别为含10％FBS 1640培养基，在37℃，5％CO 2饱和湿度培养箱内培养24h。加入不同组别的药物，每个浓度设置3个复孔。继续培养72h后，每孔加入50μL 4℃预冷的TCA(三氯乙酸)溶液(30％，w/v)固定细胞，TCA溶液的终浓度为10％。 静置5min移入4℃冰箱中固定1h，取出用去离子水冲洗5遍，室温晾干。待96孔板室温下晾干后，每孔加入0.4％(w/v)的SRB染液(1％的乙酸配制)70μL，染色30min后倒掉染液，用1％(v/v)乙酸冲洗4次，去除未结合的染料，室温晾干。用100μL三碱基溶液(10mM，pH＝10.5)溶解与细胞蛋白结合的染料，水平摇床上振荡20min，采用酶标仪515nm处测定光吸收值，据此计算细胞存活率。
结果如图18所示，在常氧条件下，负载紫杉醇的阳离子脂质体药物制剂组表现出比游离紫杉醇组更强的肿瘤细胞毒性，表明紫杉醇由阳离子脂质体负载后入胞率更高，提升了紫杉醇的药效。而在低氧条件下，游离紫杉醇组的肿瘤细胞毒性较常氧条件下降低，表明在低氧条件下肿瘤细胞对紫杉醇具有一定的耐药性。而共负载紫杉醇和HIF-1α siRNA的阳离子脂质体药物制剂组，在低氧条件下表现出比游离紫杉醇组和仅负载紫杉醇的阳离子脂质体药物制剂组更强的肿瘤细胞毒性。表明阳离子脂质体共同负载化疗药物紫杉醇和基因药物HIF-1α siRNA具有明显的协同作用，可以克服肿瘤细胞对紫杉醇的耐药性问题。其中阳离子脂质体药物制剂Sp1作为载体共同负载紫杉醇和HIF-1α siRNA的效果最好，有潜力作为新型阳离子脂质体共给药系统，克服紫杉醇治疗肿瘤的耐药性的问题。
研究阳离子脂质体共递送化疗药物紫杉醇和基因药物HIF-1α siRNA的体内抗肿瘤活性：
建立鼠源乳腺癌肿瘤模型：
标准细胞培养间进行4T1细胞培养，待细胞经几次传代且状态稳定时，用胰蛋白酶消化收集，用预冷的PBS重悬收集的细胞，以每只小鼠1×10 6个4T1(100μL)细胞接种在小鼠右侧腋下，待肿瘤体积约100mm 3(体积计算公式：体积＝长×宽×宽/2)时，随机分组后开展抗肿瘤药效研究。
抗肿瘤效率研究：
1)小鼠给药治疗处理：设置生理盐水、实施例11的Sp1、游离HIF-1α siRNA、实施例11的Sp1+HIF-1α siRNA、游离PTX、实施例15的Sphy1、PTX+HIF-1α siRNA和实施例15的Sphy1+HIF-1α siRNA共8组，每两天用一次性无菌胰岛素注射器对小鼠进行尾静脉注射给药一次，连续给药7次后停止给药，在给药停止后的第七天处死；
2)给药当天用游标卡尺对小鼠肿瘤的生长情况进行测量，绘制肿瘤的生长曲线，同时监测小鼠体重的变化情况，并作统计处理分析；
3)在小鼠处死后对其进行解剖，收集肿瘤组织，并对瘤块质量进行测量统计。
在荷瘤小鼠给药的过程中，对小鼠体重进行测量统计，并用游标卡尺实时测量小鼠肿瘤大小，如图19所示，各组小鼠给药期间均未出现死亡，体重没有明显差异，表明脂质体制剂具有很好的生物相容性。由图20可知，与生理盐水组相比，实施例11的Sp1，游离的HIF-1α siRNA组及Sp1+HIF-1α siRNA组表现出类似的肿瘤快速生长趋势，没有显著性差异。而游离PTX组和PTX+HIF-1α siRNA组，实施例15的Sphy1给药组，实施例15的Sphy1+HIF-1α siRNA给药组，小鼠肿瘤生长趋势均明显减缓，表现出明显的肿瘤抑制活性( *P<0.05， **P<0.01， ***P<0.001， ****P<0.0001)。如图21所示，与游离紫杉醇组相比，实施例15的负载 紫杉醇的脂质体Sphy1和Sphy1+HIF-1α siRNA组也具有更强的抗肿瘤活性，表现出显著性的差异性( #P<0.05， ##P<0.01， ###P<0.001， ####P<0.0001)。与实施例15的Sphy1组相比，Sphy1+HIF-1α siRNA组表现出更强的肿瘤抑制活性( $P<0.05， $$P<0.01)。
给药结束后，处死荷瘤小鼠并将肿瘤组织分离称重。如图22所示，实施例15的阳离子脂质体药物制剂Sphy1+HIF-1α siRNA组肿瘤重量最小，实施例15的Sphy1组次之。游离PTX组和PTX+HIF-1α siRNA组肿瘤重量比生理盐水组、实施例11的阳离子脂质体Sp1组、Free HIF-1α siRNA组及实施例11的阳离子脂质体Sp1+HIF-1α siRNA组小。这些结果表明阳离子脂质体共递送化疗药物紫杉醇和HIF-1α siRNA具有协同增效作用，克服了三阴性乳腺癌对紫杉醇的耐药性，具有高效的体内抗肿瘤作用。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (10)

  1. 一种鞘脂类化合物，其特征在于，所述鞘脂类化合物的结构式如式(Ⅰ)所示：
    Figure PCTCN2022098765-appb-100001
    其中，所述R 1和R 2独立地选自取代或未取代的C 8～22的烷基、取代或未取代的C 8～20的烯基；
    所述R 3选自
    Figure PCTCN2022098765-appb-100002
    所述n为1～5的整数，m为1-3的整数；
    所述A为
    Figure PCTCN2022098765-appb-100003
    酰胺键、酯键、
    Figure PCTCN2022098765-appb-100004
    所述R 4选自H、氮杂芳烃基、含胍基取代的脂肪或芳烃基、取代或未取代的C 1～10的烷基、杂环基烃基、C 2～10的烷基酸；所述R 5选自取代或未取代的氨基。
  2. 根据权利要求1所述的鞘脂类化合物，其特征在于，所述R 1选自
    Figure PCTCN2022098765-appb-100005
    Figure PCTCN2022098765-appb-100006
    其中x和y为8～20的整数。
  3. 根据权利要求1所述的鞘脂类化合物，其特征在于，所述R 4选自H、
    Figure PCTCN2022098765-appb-100007
    Figure PCTCN2022098765-appb-100008
    优选地，所述R 5选自
    Figure PCTCN2022098765-appb-100009
    Figure PCTCN2022098765-appb-100010
  4. 根据权利要求1所述的鞘脂类化合物，其特征在于，所述鞘脂类化合物选自如下结构 式中的一种：
    Figure PCTCN2022098765-appb-100011
    Figure PCTCN2022098765-appb-100012
    Figure PCTCN2022098765-appb-100013
  5. 一种阳离子脂质体，其特征在于，所述阳离子脂质体包括权利要求1～4任一项所述的鞘脂类化合物、胆固醇、二油酰磷脂酰乙醇胺和甲氧基-聚乙二醇-磷脂。
  6. 根据权利要求5所述的阳离子脂质体，其特征在于，所述鞘脂类化合物、胆固醇、二油酰磷脂酰乙醇胺和甲氧基-聚乙二醇-磷脂的摩尔比为1：(0.5～50)：(0.5～50)：(0.015～1)。
  7. 一种阳离子脂质体药物制剂，其特征在于，所述药物制剂包括如权利要求5或6所述的阳离子脂质体和药物活性成分，以所述阳离子脂质体中的鞘脂类化合物的摩尔量计算，所述鞘脂类化合物和药物活性成分的摩尔比为(0.1～50)：1。
  8. 根据权利要求7所述的阳离子脂质体药物制剂，其特征在于，所述药物活性成分为抗肿瘤化合物、抗病毒化合物、抗炎化合物或类风湿药物中的一种或多种。
  9. 根据权利要求7所述的阳离子脂质体药物制剂，其特征在于，所述阳离子脂质体药物制剂还包括核酸；
    优选地，所述核酸的量为N/P比为1：1～16：1，所述的N/P比是阳离子脂质体中的可电离的氮原子N的摩尔含量和核酸中P的摩尔含量之比；
    优选地，所述核酸选自siRNA、miRNA、antagomir、DNA质粒或mRNA。
  10. 权利要求5或6任一项所述的阳离子脂质体、权利要求7～9任一项所述的阳离子脂质体药物制剂在用于治疗由基因异常表达引起的相关疾病的药物制备中的用途，所述疾病包括恶性肿瘤、心血管疾病、类风湿、感染性疾病或遗传病；
    优选地，所述恶性肿瘤为三阴性乳腺癌。
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