WO2019170092A1 - 含酮羰基的疏水性抗肿瘤药物及其缀合物、含有缀合物的纳米制剂及其制备方法及应用 - Google Patents

含酮羰基的疏水性抗肿瘤药物及其缀合物、含有缀合物的纳米制剂及其制备方法及应用 Download PDF

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WO2019170092A1
WO2019170092A1 PCT/CN2019/077038 CN2019077038W WO2019170092A1 WO 2019170092 A1 WO2019170092 A1 WO 2019170092A1 CN 2019077038 W CN2019077038 W CN 2019077038W WO 2019170092 A1 WO2019170092 A1 WO 2019170092A1
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isocyanate
group
conjugate
docetaxel
ketone carbonyl
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French (fr)
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颜德岳
耿仲毅
王瑶
朱新远
黄卫
周永丰
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江苏吉贝尔药业股份有限公司
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Priority to US16/975,996 priority Critical patent/US11833128B2/en
Priority to EP19764534.4A priority patent/EP3763391A4/en
Publication of WO2019170092A1 publication Critical patent/WO2019170092A1/zh

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Definitions

  • the invention belongs to the technical field of biomedicine, and particularly relates to a hydrophobic antitumor drug comprising a ketone carbonyl group, a polyethylene glycol and/or a lactose hydrazide terminated by a hydrazide, and a hydrophobic antitumor drug with a ketone carbonyl group obtained by dehydration condensation reaction.
  • Some hydrophobic antitumor drugs such as paclitaxel and docetaxel have good therapeutic effects on breast cancer, ovarian cancer, etc., but due to their poor water solubility, currently used paclitaxel injection (Taxol) and docetaxel Injection (Taxoter) is a solution of paclitaxel and docetaxel in a mixture of Cremophor EL or Tween 80 and absolute ethanol. Use saline or 5% glucose before use. Dilute to the appropriate concentration. However, injections containing Cremophor EL or Tween 80 can cause serious side effects to patients, posing a safety hazard for clinical applications. Therefore, the development of safe, non-toxic paclitaxel and docetaxel dosage forms has been a hot spot in cancer treatment research.
  • amphiphilic conjugate prodrugs are typically designed to introduce water-soluble or targeted groups by ester linkage at the hydroxyl position.
  • the resulting product is hydrolyzed by an ester bond to release a prodrug, which in turn exerts an anti-tumor effect, but it is important that the rate of hydrolysis of the ester bond greatly affects its anti-tumor activity.
  • the present invention provides a condensate obtained by dehydration condensation reaction of a hydrazide-terminated polyethylene glycol and/or a lactobionyl group with a hydrophobic antitumor drug into which a ketone carbonyl group is introduced, and a pH-responsive nano preparation containing the conjugate.
  • the present invention also provides a preparation method of a nano-preparation comprising the above conjugate and use thereof in the preparation of an antitumor drug. Further, the present invention provides a hydrophobic antitumor drug comprising a ketone carbonyl group, and a docetaxel derivative containing a ketone carbonyl group.
  • a first aspect of the present invention provides a conjugate obtained by dehydration condensation reaction of a hydrazide-terminated polyethylene glycol and/or a lactosyl hydrazide with a hydrophobic antitumor drug into which a ketone carbonyl group is introduced.
  • the dehydration condensation reaction takes place between a ketone carbonyl group and a hydrazide group.
  • the conjugate is an amphiphilic pH responsive conjugate.
  • the hydrophobic antitumor drug into which a ketone carbonyl group is introduced is obtained by reacting an isocyanate group in a compound containing an isocyanate group and a ketone carbonyl group with a hydroxyl group in a hydrophobic antitumor drug containing a hydroxyl group.
  • the isocyanate-containing and ketocarbonyl-containing compounds include: p-isocyanate benzaldehyde, meta-isocyanate benzaldehyde, o-isocyanate benzaldehyde, p-isocyanate acetophenone, meta-isocyanate acetophenone, o-isocyanate acetophenone, p-isocyanate-based diphenyl. Ketone, meta-isocyanate benzophenone, o-isocyanate benzophenone.
  • the hydrophobic antitumor drug containing a hydroxyl group includes at least any one of paclitaxel, docetaxel, a paclitaxel derivative, and a docetaxel derivative.
  • the hydrazide-terminated polyethylene glycol has a number average molecular weight of 148 to 100,000 and 1 ⁇ PDI ⁇ 2.
  • the hydrazide-terminated polyethylene glycol has a chemical structure as shown in Formula I below:
  • R is H, -CH3 or is selected from the group consisting of biotin, folic acid, arginine-glycine-aspartic acid (RGD), fluorouridine, cytarabine, gemcitabine, isatoribine, and tresal Any of a group derived from a derivative group of hydroxy, ureido, mitoxantrone, aramid, streptozotocin, pingyangmycin, and bleomycin.
  • lactosylhydrazide has the chemical structure shown in the following formula II:
  • the paclitaxel derivative includes 10-deacetylpaclitaxel having the structure shown in Formula III, or cephalosporin having the structure shown in Formula IV:
  • R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 are each independently hydrogen, deuterium or fluorine, provided that at least one of them is deuterium or fluorine; preferably, wherein R1, R2, R3, One or more of R4, R5, R6, R7, R8, and R9 are ⁇ , and R10 is ⁇ .
  • the conjugate preferably includes a structure represented by the formula (1) to formula (7).
  • R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are each independently hydrogen, hydrazine or fluorine, provided that at least one of them is hydrazine or fluorine; preferably, wherein R1 One or more of R2, R3, R4, R5, R6, R7, R8, and R9 are ⁇ , and R10 is ⁇ .
  • a second aspect of the invention provides a nano-formulation comprising nanoparticles comprising the aforementioned conjugate.
  • the nanoparticles have a particle size of less than 300 nm, preferably 20 to 200 nm.
  • the nanoformulation is a pH responsive nanoformulation.
  • a third aspect of the invention provides a method for preparing the nano preparation, which comprises the following steps:
  • Step (1) dehydrating condensation reaction of a hydrazide-terminated polyethylene glycol and/or lactosyl hydrazide with a hydrophobic antitumor drug introduced into a ketone carbonyl group to obtain a conjugate;
  • Step (2) dissolving the conjugate in an organic solvent to form a solution, adding the solution to water at room temperature, and removing the organic solvent to obtain an aqueous solution containing the nanoparticles.
  • an organic solution containing a hydrazide-terminated polyethylene glycol and/or a lactobionyl group is added to an organic solution of a hydrophobic antitumor drug introduced into a ketone carbonyl group, and the reaction is stirred.
  • the organic solvent in steps (1) and (2) is at least one selected from the group consisting of N,N'-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, acetonitrile, methanol, and dioxane.
  • a fourth aspect of the invention provides the use of the conjugate or nanoformulation for the preparation of an anti-tumor drug.
  • a fifth aspect of the present invention provides a hydrophobic antitumor drug comprising a ketone carbonyl group which is obtained by reacting an isocyanate group in a compound containing an isocyanate group and a ketone carbonyl group with a hydroxyl group in a hydrophobic antitumor drug containing a hydroxyl group.
  • the hydrophobic antitumor drug containing a hydroxyl group is selected from at least any one of paclitaxel, docetaxel, a paclitaxel derivative, and a docetaxel derivative.
  • the isocyanate-containing and ketocarbonyl-containing compounds include: p-isocyanate benzaldehyde, meta-isocyanate benzaldehyde, o-isocyanate benzaldehyde, p-isocyanate acetophenone, meta-isocyanate acetophenone, o-isocyanate acetophenone, p-isocyanate-based diphenyl. Ketone, meta-isocyanate benzophenone, o-isocyanate benzophenone.
  • the paclitaxel derivative includes 10-deacetylpaclitaxel having the structure shown in Formula III, or cephalosporin having the structure shown in Formula IV:
  • the derivative of docetaxel comprises the structure shown in formula V:
  • R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 are each independently hydrogen, deuterium or fluorine, provided that at least one of them is deuterium or fluorine; preferably, wherein R1, R2, R3, One or more of R4, R5, R6, R7, R8, and R9 are deuterium, and R10 is deuterium.
  • a sixth aspect of the present invention provides a ketone carbonyl-containing docetaxel derivative obtained by reacting an isocyanate group in a compound containing an isocyanate group and a ketone carbonyl group with a hydroxyl group in docetaxel or deuterated docetaxel;
  • the isocyanate-containing and ketocarbonyl-containing compounds include: p-isocyanate benzaldehyde, meta-isocyanate benzaldehyde, o-isocyanate benzaldehyde, p-isocyanate acetophenone, meta-isocyanate acetophenone, o-isocyanate acetophenone, p-isocyanate-based diphenyl.
  • the deuterated docetaxel has the structure represented by Formula V
  • the hydrophobic antitumor drug and the pH-responsive nano-injection of the ketone carbonyl group of the present invention have better anti-cancer activity and higher tumor inhibition rate in the treatment of malignant tumors than the currently used docetaxel preparations.
  • the amphiphilic pH-responsive conjugate of the present invention has a well-defined structure, and can be self-assembled to form nanoparticles in water for transport, thereby avoiding the side effects caused by reintroduction of other carriers. Through the high permeability and retention (EPR) effects of tumor tissue, pH-responsive nanoparticles can be passively targeted into tumor tissue.
  • the pH-responsive nanoparticle can rapidly release hydrophobic anti-tumor drug molecules, avoiding the attenuation or even loss of anti-tumor activity caused by the hysteresis of release.
  • Example 1 is a 1 H NMR spectrum of an amphiphilic pH-responsive monomethylated PEG-dene paclitaxel conjugate prepared in Example 1;
  • Example 2 is a graph showing hydrodynamic diameter data of nanoparticles prepared in Example 1;
  • Example 3 is a transmission electron micrograph of the nanoparticles prepared in Example 1;
  • Figure 4 shows the changes in body weight of mice with treatment time
  • Figure 5 shows the relative tumor volume as a function of treatment time
  • Figure 6 is a graph showing the relative tumor inhibition rate as a function of treatment time
  • Figure 7 is a photograph of a tumor of each group after the end of treatment
  • Figure 8 shows the tumor volume of human lung cancer A549 nude mice transplanted with treatment time
  • Figure 9 shows the changes in body weight of nude mice bearing human lung cancer with treatment time.
  • the conjugate of the present invention is obtained by dehydration condensation reaction of a hydrazide-terminated polyethylene glycol and/or lactosyl hydrazide with a hydrophobic antitumor drug into which a ketone carbonyl group is introduced.
  • the hydrophobic antitumor drug into which a ketone carbonyl group is introduced is obtained by reacting an isocyanate group in a compound containing an isocyanate group and a ketone carbonyl group with a hydroxyl group on a hydrophobic antitumor drug having a hydroxyl group.
  • the hydroxyl group-containing hydrophobic antitumor drug may be a hydroxyl group-containing paclitaxel drug and a derivative thereof, and may, for example, include at least any one selected from the group consisting of paclitaxel, docetaxel, and derivatives thereof.
  • the derivative includes an optical isomer, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug, a polymorph, a stereoisomer, a geometric isomer or a tautomer thereof, of the above compound, ⁇ .
  • the paclitaxel, docetaxel, and derivatives thereof are not only mainly structurally similar, but also have the same mechanism of action, so that the conjugate and the nano preparation can be formed by the method of the present invention to improve the pharmacological effect.
  • the hydrazide-terminated polyethylene glycol used in the present invention has the chemical structure shown in the following formula I:
  • R may be H, -CH 3 , or a derivative group of a hydrophilic molecule or a hydrophilic drug, such as biotin, folic acid, arginine-glycine-aspartate (RGD), fluorouridine, Among the derivatizing groups of cytarabine, gemcitabine, isatoribine, trisacitabine, hydroxyurea, mitoxantrone, aramid, streptozotocin, pingyangmycin, bleomycin, etc. Any one of the groups.
  • the hydrazide-terminated polyethylene glycol used in the present invention has a number average molecular weight of 148 to 100,000 and 1 ⁇ PDI ⁇ 2.
  • lactosyl hydrazide used in the present invention has a chemical structure represented by the following formula II:
  • paclitaxel derivatives of the present invention including 10-deacetylpaclitaxel and cephalosporin, each having the chemical structure shown in the following formula III and IV:
  • R1, R2, R3, R4, R5, R6, R7, R8 and R9 are each independently hydrogen, deuterium or fluorine, and R10 is deuterium.
  • R1, R2, R3, R4, R5, R6, R7, R8, R9 is hydrazine; more preferably, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 is ⁇ , or wherein R1, R2, R3, R4, R5, R6, R7, R8, and R9 are all fluorine, and R10 is ⁇ .
  • the conjugate of the present invention has amphiphilic properties due to a hydrophobic antitumor drug having a ketone carbonyl group introduced at one end and a hydrophilic compound at the other end.
  • the amphiphilic structure allows the conjugate to undergo hydrophobic association (primary) action in water and spontaneously assemble into nanomicelles.
  • the conjugate of the present invention is dissolved in an organic solvent to form a solution, and the solution is added to water at room temperature to remove an organic solvent to obtain an aqueous solution containing nanoparticles.
  • the conjugate is dissolved in an organic solvent at a concentration of 2 ng/ml to 70 mg/ml, preferably at a concentration of 3 ⁇ g/ml to 35 mg/ml, and the ratio of the obtained solution to water (volume ratio) may be 1:1. ⁇ 1:50, preferably 1:2 to 1:10.
  • the method of removing the organic solvent may be a method commonly used in the prior art, such as distillation under reduced pressure or the like.
  • the hydrophobic antitumor drug having a ketone carbonyl group is contained in an amount of from 0.8 ng/ml to 20 mg/ml, preferably from 1 ⁇ g/ml to 10 mg/ml.
  • the content of the hydrophobic antitumor drug into which the carbonyl group is introduced in the nanoformulation can be adjusted depending on the specific application.
  • the therapeutic dose of the nanoformulations described herein can be determined based on the particular therapeutic use, the type and extent of the disease, the health of the patient, and the judgment of the physician. Some typical dosages range from 40 to 300 mg/kg body weight per day, preferably 200 mg/kg body weight per day.
  • Polyethylene glycol monomethyl ether (M n 2000): purchased from Sigma-Aldrich
  • Lactose acid purity: 97%, purchased from Adamas
  • Docetaxel purity: 98%, purchased from Jiangsu Taxus Pharmaceutical Co., Ltd.
  • Paclitaxel Purity: 98%, purchased from Jiangsu Taxus Pharmaceutical Co., Ltd.
  • the polyethylene glycol monomethyl ether (20 g, 10 mmol) was completely dissolved in toluene (200 mL), the air in the reaction flask was removed by nitrogen, and a solution of potassium tert-butoxide (4.12 g, 36 mmol) in t-butanol (60 mL) was gradually added dropwise. ). After reacting for half an hour, ethyl bromoacetate (6.4 mL, 48 mmol) was added dropwise for half an hour, and the reaction solution was allowed to react at room temperature for 24 hours. After completion of the reaction, the reaction solution was filtered through a Buchner funnel, and the filtrate was concentrated and precipitated three times in ice diethyl ether. The white precipitate was dried under vacuum at 35 ° C for 24 hours to obtain ethyl acetate-terminated polyethylene glycol monomethyl ether. The rate is 90%.
  • ethyl acetate-terminated polyethylene glycol monomethyl ether (10.00 g, 4.8 mmol) was dissolved in methanol (100 mL), and a solution of hydrazine hydrate (30 mL) in methanol (40 mL) was gradually added dropwise. After reacting for 24 hours, the reaction mixture was filtered, and the filtrate was evaporated. After concentration of the filtrate, it was precipitated in iced diethyl ether, and the white precipitate was dried under vacuum at 35 ° C for 24 hours to finally obtain a hydrazide-terminated monomethylated polyethylene glycol in a yield of 90%.
  • methyl ester-terminated monohydroxy polyethylene glycol (5.00 g, 5 mmol) was dissolved in methanol (50 mL), and a solution of hydrazine hydrate (15 mL) in methanol (20 mL) was gradually added dropwise. After reacting for 24 hours, the reaction mixture was filtered, and the filtrate was evaporated. After concentration of the filtrate, it was precipitated in iced diethyl ether, and the white precipitate was dried under vacuum at 35 ° C for 24 hours to finally obtain a hydrazide-terminated monohydroxy polyethylene glycol in a yield of 90%.
  • Lactic acid (5.0000 g, 13.96 mmol) was dissolved in anhydrous methanol (70.0 mL) and refluxed at 75 °C until the lactose acid was completely converted to lactone lactone.
  • Lactone lactone (3.0000 g, 8.82 mmol) was dissolved in anhydrous methanol (40.0 mL) at 25 °C. Subsequently, hydrazine hydrate (2.2270 g, 44.10 mmol) was added dropwise to the reaction solution, and reacted at 25 ° C for 1 h. A white precipitate is produced. Distillation under reduced pressure gave a white solid, succinylhydrazide, yield 76.2%.
  • DTX-TES-AI (2 g) was dissolved in 16 ml of 5% HCl/methanol, and the reaction was started at 26 ° C. After 30 min, 50 ml of ethyl acetate was added, followed by washing three times with water, and ethyl acetate was evaporated under reduced pressure. The resulting mixture was eluted with a gradient of ethyl acetate and petroleum ether, and purified by column chromatography to give the desired carbonyl-introduced hydrophobic antitumor drug (DTX-AI) as a white powdery solid, yield 88%.
  • DTX-AI desired carbonyl-introduced hydrophobic antitumor drug
  • paclitaxel 6 g was dissolved in a mixed solution of 110 ml of anhydrous dichloromethane and pyridine. Under ice-cooling conditions, 40 ml of dimethyl t-butyl chlorosilane (TBSCl) was added dropwise to the PTX solution at a dropping rate of 10 ml/10 min, and a total of four drops were added. After the dropwise addition was completed, stirring was continued for 40 minutes under ice bath conditions. Subsequently, the reaction system was placed in a 35 ° C oil bath to continue the reaction for 24 h. After the reaction was completed, water was slowly added to cause no bubble generation. It was then washed 3 times with water, and dichloromethane was distilled off under reduced pressure. The resulting mixture was purified by column chromatography eluting with EtOAc EtOAc EtOAc.
  • PTX-TBS (2.06g), 4-dimethylaminopyridine (25mg), isocyanate acetophenone (AI) (1.63g) was dissolved in 100ml of anhydrous N, N-dimethylformamide, slowly stirred until The solid was completely dissolved. After the dissolution was completed, the reaction system was placed in a 56 ° C oil bath for 12 h. After completion of the reaction, N,N-dimethylformamide was removed by distillation under reduced pressure. The mixture was purified by column chromatography eluting EtOAc EtOAc EtOAc
  • PTX-TBS-AI (2 g) was dissolved in 16 ml of 5% HCl/methanol, and the reaction was started at 26 ° C. After 30 min, 50 ml of ethyl acetate was added, followed by washing three times with water, and ethyl acetate was evaporated under reduced pressure. The obtained mixture was eluted with a gradient of ethyl acetate and petroleum ether, and the silica gel column was used to obtain the desired ketone carbonyl-containing hydrophobic antitumor drug (PTX-AI) as a white powdery solid in a yield of 88%.
  • PTX-AI ketone carbonyl-containing hydrophobic antitumor drug
  • the hydrazide-terminated monomethylated PEG (6 g) obtained in Preparation Example 1 was dissolved in 150 ml of anhydrous methanol.
  • the ketokecarbonyl-derived docetaxel derivative (968.39 mg) obtained in Preparation Example 4 was dissolved in 20 ml of anhydrous methanol, and the resulting solution was added to the above solution containing hydrazide-terminated monomethylated PEG in nitrogen.
  • the reaction was stirred at room temperature for 48 hours under protection. Methanol was distilled off under reduced pressure, and the mixture was eluted with EtOAc and EtOAc (EtOAc) %.
  • the amphiphilic conjugate prepared above was dissolved in tetrahydrofuran, and added to water at room temperature to remove tetrahydrofuran to obtain an aqueous solution of an amphiphilic conjugate of nanoparticles in which the concentration of the docetaxel derivative was 1 mg/ Ml.
  • the nanoparticle (Nano mPEG-DTX-AI) containing the pH-responsive amphiphilic conjugate prepared in this example has an average particle size of about 170 nm.
  • the hydrodynamic diameter data of the nanoparticles obtained in this example are shown in Fig. 2, and the transmission electron microscope photograph is shown in Fig. 3.
  • the hydrazide-terminated monomethylated PEG (6 g) obtained in Preparation Example 1 was dissolved in 150 ml of anhydrous methanol.
  • the ketocarbonyl-introduced paclitaxel derivative (1015.06 mg) obtained in Preparation 5 was dissolved in 20 ml of anhydrous methanol, and the resulting solution was added to the above solution containing hydrazide-terminated monomethylated PEG under room temperature under nitrogen atmosphere. The reaction was stirred for 48 hours. Methanol was distilled off under reduced pressure, and the mixture was purified eluting with EtOAc EtOAc EtOAc (EtOAc)
  • the amphiphilic conjugate prepared above was dissolved in tetrahydrofuran, and added to water at room temperature to remove tetrahydrofuran to obtain an aqueous nanoparticle solution of the amphiphilic conjugate, wherein the concentration of the paclitaxel derivative was 1 mg/ml.
  • the nanoparticle (Nano mPEG-DTX-AI) containing the pH-responsive amphiphilic conjugate prepared in this example has an average particle size of about 200 nm.
  • the hydrazide-terminated monomethylated PEG (6 g) obtained in Preparation Example 1 was dissolved in 150 ml of anhydrous methanol to form a solution.
  • the ketokecarbonyl-derived deuterated docetaxel derivative (968.39 mg) obtained in a similar manner to Preparation Example 4 was dissolved in 20 ml of anhydrous methanol, and the resulting solution was added to the above-mentioned hydrazide-terminated monomethylated PEG. In the solution, the reaction was stirred at room temperature for 48 hours under a nitrogen atmosphere. Methanol was distilled off under reduced pressure, and the mixture was purified eluting with EtOAc EtOAc EtOAc EtOAc EtOAc
  • R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are all ruthenium.
  • the amphiphilic conjugate prepared above is dissolved in tetrahydrofuran, and added to water at room temperature to remove tetrahydrofuran to obtain an aqueous solution of the amphiphilic conjugate nanoparticle, wherein the concentration of the deuterated docetaxel derivative is 1 mg/ml.
  • the nanoparticles containing the pH-responsive amphiphilic conjugate prepared in this example have an average particle size of about 200 nm.
  • the hydrazide-terminated monomethylated PEG (6 g) obtained in Preparation Example 1 was dissolved in 150 ml of anhydrous methanol to form a solution.
  • the 10-deacetylpaclitaxel derivative (973.03 mg) obtained by a similar method to Preparation Example 4 was dissolved in 20 ml of anhydrous methanol, and the resulting solution was added to the above-mentioned hydrazide-terminated monomethylated PEG. In the solution, the reaction was stirred at room temperature for 48 hours under a nitrogen atmosphere. Methanol was distilled off under reduced pressure, and the mixture was purified eluting with EtOAc EtOAc EtOAc EtOAc EtOAc
  • the amphiphilic conjugate prepared above is dissolved in tetrahydrofuran, and added to water at room temperature to remove tetrahydrofuran to obtain an aqueous solution of the amphiphilic conjugate nanoparticle, wherein the concentration of the 10-deacetyl paclitaxel derivative is 1 mg/ml.
  • the nanoparticles containing the pH-responsive amphiphilic conjugate prepared in this example have an average particle size of about 200 nm.
  • the hydrazide-terminated monomethylated PEG (6 g) obtained in Preparation Example 1 was dissolved in 150 ml of anhydrous methanol to form a solution.
  • the ketone carbonyl-containing cephalosporin derivative (992.39 mg) obtained in a similar manner to Preparation Example 4 was dissolved in 20 ml of anhydrous methanol, and the resulting solution was added to the above hydrazide-terminated monomethylated PEG. In the solution, the reaction was stirred at room temperature for 48 hours under a nitrogen atmosphere. Methanol was distilled off under reduced pressure, and the mixture was purified eluting with EtOAc EtOAc EtOAc EtOAc EtOAc
  • amphiphilic conjugate prepared above is dissolved in tetrahydrofuran, and added to water at room temperature to remove tetrahydrofuran to obtain an aqueous solution of an amphiphilic conjugate nanoparticle, wherein the concentration of the cephalosporin derivative is 1 mg/ml.
  • the nanoparticles containing the pH-responsive amphiphilic conjugate prepared in this example have an average particle size of about 200 nm.
  • the lactosyl hydrazide (6 g) obtained in Preparation Example 3 was dissolved in 150 ml of anhydrous methanol.
  • the ketokecarbonyl-derived docetaxel derivative (1015.06 mg) obtained in Preparation Example 4 was dissolved in 20 ml of anhydrous methanol, and the resulting solution was added to the above lactobionate-containing solution, and the reaction was stirred at room temperature under a nitrogen atmosphere. hour. Methanol was distilled off under reduced pressure, and the mixture was purified eluting with EtOAc EtOAc EtOAc
  • the amphiphilic conjugate prepared above was dissolved in tetrahydrofuran, and added to water at room temperature to remove tetrahydrofuran to obtain an aqueous solution of an amphiphilic conjugate of nanoparticles in which the concentration of the docetaxel derivative was 1 mg/ Ml.
  • the nanoparticles containing the pH-responsive amphiphilic conjugate prepared in this example have an average particle size of about 200 nm.
  • the lactosyl hydrazide (6 g) obtained in Preparation Example 3 was dissolved in 150 ml of anhydrous methanol.
  • the ketone carbonyl-incorporated paclitaxel derivative (1015.06 mg) obtained in Preparation Example 5 was dissolved in 20 ml of anhydrous methanol, and the resulting solution was added to the above-mentioned lactobionate-containing solution, and the reaction was stirred at room temperature for 48 hours under a nitrogen atmosphere. Methanol was distilled off under reduced pressure, and the mixture was purified eluting with EtOAc (EtOAc)
  • the amphiphilic conjugate prepared above was dissolved in tetrahydrofuran, and added to water at room temperature to remove tetrahydrofuran to obtain an aqueous nanoparticle solution of the amphiphilic conjugate, wherein the concentration of the paclitaxel derivative was 1 mg/ml.
  • the nanoparticles containing the pH-responsive amphiphilic conjugate prepared in this example have an average particle size of about 200 nm.
  • Test method for hydrodynamic diameter of nanoparticles in the present invention is the same as Test method for hydrodynamic diameter of nanoparticles in the present invention.
  • the nano-micelle average particle size (Z-Average) and particle size distribution (PDI) were measured by Dynamic Light Scattering (DLS) at a test temperature of 25 ° C, a laser wavelength of 633 nm, and a probe angle of 173 °. Each sample was equilibrated for 2 min and tested 3 times. Experimental data was analyzed by software Dispersion Technology software version 5.32.
  • TEM testing was performed on the JEM-2010/INCA OXFORD model with an electron microscope acceleration voltage of 200 kV.
  • the sample preparation method was as follows: The assembly solution was dropped onto a copper mesh with a carbon film and allowed to air dry at room temperature. Care should be taken to avoid contamination of impurities such as dust in the air during the drying process.
  • Hela tumor-bearing mice were divided into four groups, one in group of five.
  • the first, second and third groups were the treatment group, that is, for each Hela tumor-bearing mouse, the tumor was treated by injecting the drug through the tail vein separately.
  • the first group was treated with docetaxel injection (25 mg DTX/kg)
  • the second group was treated with the antitumor drug DTX-AI obtained in Preparation Example 4 (40 mg DTX/kg)
  • the third group was used in Example 1.
  • the resulting pH-responsive nanoformulation was administered for treatment (200 mg DTX/kg).
  • the fourth group was a blank control group.
  • the first, second and third groups of mice were administered once every seven days for a total of three injections. Each tumor-bearing mouse in each group was labeled and recorded for body weight and tumor volume throughout the course of treatment.
  • the animals were sacrificed on day 45, the tumor pieces were dissected, and the tumor weight was weighed. The body weight of the animals after tumor removal was calculated.
  • Figure 4 shows the changes in body weight over time in mice.
  • the body weight of the mice in the second and third groups was basically the same as that in the blank control group, indicating that the DTX-AI and the nano-mPEG-DTX-AI were relatively safe in vivo.
  • Figure 5 shows the relative tumor volume as a function of time.
  • the second and third groups showed a situation in which the relative tumor volume was greatly reduced as compared with the blank control group and the first group.
  • the relative tumor volume of the first group continued to increase with the passage of time, and the antitumor effect gradually weakened and rebounded.
  • the relative tumor volume of the second and third groups showed a decreasing trend with time, that is, after stopping the drug, the antitumor effect continued to increase.
  • relative tumor inhibition rate % (1-T RTV / C RTV ) * 100%, (where T RTV represents the RTV of the treatment group; C RTV represents the blank control group RTV), The result is shown in Figure 6.
  • the treatment group showed a trend of increasing relative tumor inhibition rate.
  • the relative tumor inhibition rates of the second group and the third group showed a substantial increase compared with the first group, and continued to remain above 90% after 21 days (after stopping the administration).
  • the relative tumor inhibition rate of the first group was 67.62%
  • the relative tumor inhibition rate of the second group was 93.5%
  • the relative tumor inhibition rate of the third group was 95.61%.
  • Figure 7 shows photographs of tumors of each group after the end of treatment. It can be visually seen from the photograph that the tumor size of the second group and the third group is significantly smaller than that of the first group and the blank control group.
  • a well-developed lung cancer A549 solid tumor was cut into uniform small pieces of about 3 mm under sterile conditions. Each mouse was inoculated with a trocar under the right iliac crest. The average tumor volume on the 14th day after inoculation was about 210-250 mm 3 . According to the tumor size group, the average volume of each group of tumors was basically the same.
  • the antitumor drug DTX-AI obtained in Preparation Example 4 was prepared as a lyophilized powder needle, and the dose was as shown in Table 1 below.
  • the animals were sacrificed on day d43 (56 days after inoculation), the tumor pieces were dissected, the tumor weight was weighed, and the body weight of the animals after tumor removal was calculated.
  • DTX-AI lyophilized powder 4 doses 80mg / kg, 60mg / kg, 40mg / kg, 20mg / kg, once a week intravenous injection, a total of 4 times, from the last dose
  • the tumor inhibition rate of human lung cancer A549 at 21 days was 99.27%, 99.50%, 99.28%, and 62.38%, respectively.
  • the dosage of docetaxel injection was 40mg/kg, 20mg/kg, and the tail vein was injected once a week for 4 times.
  • the tumor inhibition rate of human lung cancer A549 was 99.24% and 84.50% from the last 21 days.
  • Animals in the DTX-AI lyophilized powder did not die in the 4 dose groups. Animals in the docetaxel 20 mg/kg group did not die, and in the 40 mg/kg group, one animal died on day d33.
  • DTX-AI lyophilized powder 20mg/kg had no effect on animal body weight after administration; DTX-AI lyophilized powder 40mg/kg, 60mg/kg gave a slight decrease in body weight after administration; DTX-AI The body weight of the animals after lyophilized powder 80 mg/kg was reduced. After docetaxel 20mg/kg, the body weight of the animals decreased, and gradually recovered after stopping the drug; the docetaxel 40mg/kg increased the body weight with the increase of the number of administrations, and remained low in weight after stopping the drug. The degree of weight loss after administration of docetaxel 40 mg/kg was significantly higher than that of DTX-AI lyophilized powder after administration. Although the body weight of the DTX-AI lyophilized powder after administration of 80 mg/kg was also decreased, the degree of reduction was also lower than that of the docetaxel 40 mg/kg administration group.
  • the tumor growth began to slow down after the first dose of DTX-AI lyophilized powder in four dose groups.
  • the tumor inhibition effect increased, 40 mg/kg, 60 mg/kg, 80 mg/kg.
  • the prolongation of withdrawal time the anti-tumor effect was maintained at 98-99% inhibition rate. After stopping the drug, no animal tumor rebounded.
  • hydrophobic antitumor drug and the pH-responsive nano-implant introduced with the ketone carbonyl group have better anti-cancer activity and higher tumor inhibition rate in the treatment of malignant tumor than the currently used docetaxel preparation. It has higher safety and has potential clinical application value.

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Abstract

本发明提供一种含酮羰基的疏水性抗肿瘤药物及其缀合物、含有缀合物的纳米制剂及其制备方法及应用。所述缀合物是由酰肼封端的聚乙二醇和/或乳糖酰肼与引入酮羰基的疏水性抗肿瘤药物经脱水缩合反应而得到的两亲性pH响应缀合物。所述引入酮羰基的疏水性抗肿瘤药物由含有异氰酸酯基和酮羰基的化合物中的异氰酸酯基团与含有羟基的疏水性抗肿瘤药物上的羟基反应而得到。所述含有羟基的疏水性抗肿瘤药物包括紫杉醇、多烯紫杉醇、紫杉醇衍生物及多烯紫杉醇衍生物中的至少任一种。所述含酮羰基的疏水性抗肿瘤药物及其缀合物、纳米制剂与多烯紫杉醇相比具有更高的抗肿瘤活性。

Description

含酮羰基的疏水性抗肿瘤药物及其缀合物、含有缀合物的纳米制剂及其制备方法及应用 技术领域
本发明属于生物医药技术领域，具体涉及包含酮羰基的疏水性抗肿瘤药物、由酰肼封端的聚乙二醇和/或乳糖酰肼与引入酮羰基的疏水性抗肿瘤药物经脱水缩合反应而得到的缀合物，含有该缀合物的纳米制剂及其制备方法和在制备抗肿瘤药物中的应用。
背景技术
一些疏水性抗肿瘤药物，例如紫杉醇和多烯紫杉醇，对乳腺癌、卵巢癌等具有良好的治疗效果，但由于其水溶性太差，目前临床上使用的紫杉醇注射液(Taxol)和多烯紫杉醇注射液(Taxoter)是将紫杉醇和多烯紫杉醇溶解在由聚氧乙烯蓖麻油(Cremophor EL)或吐温80(Tween 80)与无水乙醇混合的溶液中，使用前用生理盐水或5％葡萄糖稀释到适当浓度。但含有Cremophor EL或Tween 80的注射液会给病人带来严重的毒副作用，给临床应用带来安全隐患，因此开发安全、无毒的紫杉醇和多烯紫杉醇剂型一直是肿瘤治疗研究的热点。
近年来，随着纳米科技的发展，人们开发了各种各样的载体对紫杉醇和多烯紫杉醇进行包埋，以便其体内输送，如脂质体(Pharmaceutical Research,1994,11(6),889-896.)、聚合物胶束(Journal of Controlled Release,2005,109(1-3),158-168.)、纳米颗粒(Small,2009,5,1706-1721.)等。除了利用物理包埋的方法进行药物体内运输外，目前还有大量的研究工作尝试通过化学键合的方式在疏水性紫杉醇和多烯紫杉醇药物上连接一个亲水性基团，形成两亲性缀合物前药，继而通过各种胶束制备方式使其在水相中形成胶束。这样既能增加药物的水溶性，还能通过纳米颗粒的形成减少小分子药物在体内运输过程中的损失。
根据紫杉醇和多烯紫杉醇的结构特点，目前报道的两亲性缀合物前药的设计通常是在羟基位，通过酯键的方式引入水溶性或者靶向性基团。所得产物通过酯键水解释放出前药，继而发挥抗肿瘤效果，但重要的是酯键的水解速率会大大影响其抗肿瘤活性。Greenwald用低分子量的聚氧化乙烯(PEG,M n＝350,750,2000,5000)与紫杉醇的7-OH键合，修饰紫杉醇以形成药物前体，其产物的溶解度比紫杉醇增大30000倍，但抑瘤活性却几乎完全丧失(Journal of Organic Chemistry 1995,60(2),331-336)。若釆用高分子量PEG(40000)与紫杉醇的2'-OH反应，所得的产物容易水解释放出紫杉醇，其体外抗种瘤活性与紫杉醇原药相当，体内对白血病肿瘤P388的抑瘤效果略高于紫杉醇原药。然而高分子量载体一方面会显著降低药物前体的载药率，另一方面注射大量的非活性载体材料进入体内可能会带来毒副反应。因此，研制出抗肿瘤活性强、安全性好的新型紫杉醇、多烯紫杉醇及其衍生物的药物前体为临床肿瘤治疗所亟需。
发明内容
发明要解决的问题
本发明提供了由酰肼封端的聚乙二醇和/或乳糖酰肼与引入酮羰基的疏水性抗肿瘤药物经脱水缩合反应而得到的缀合物、含有该缀合物的pH响应性纳米制剂，以解决现有疏水性抗肿瘤药物例如紫杉醇、多烯紫杉醇及其衍生物在体内输送所存在的技术问题，并且提高疏水性抗肿瘤药物例如紫杉醇、多烯紫杉醇及其衍生物的体内抗肿瘤活性。本发明还提供了包含上述缀合物的纳米制剂的制备方法和在制备抗肿瘤药物中的应用。此外，本发明还提供了包含酮羰基的疏水性抗肿瘤药物，以及含有酮羰基的多烯紫杉醇衍生物。
用于解决问题的方案
本发明的第一方面提供一种缀合物，其由酰肼封端的聚乙二醇和/或乳糖酰肼与引入酮羰基的疏水性抗肿瘤药物经脱水缩合反应而得到。所述脱水缩合反应发生在酮羰基和酰肼基之间。所述缀合物为两亲性pH响应性缀合物。
所述引入酮羰基的疏水性抗肿瘤药物由含有异氰酸酯基和酮羰基的化合物中的异氰酸酯基与含有羟基的疏水性抗肿瘤药物中的羟基反应而得到。
所述含有异氰酸酯基和酮羰基的化合物包括：对异氰酸酯苯甲醛、间异氰酸酯苯甲醛、邻异氰酸酯苯甲醛、对异氰酸酯苯乙酮、间异氰酸酯苯乙酮、邻异氰酸酯苯乙酮、对异氰酸酯基二苯酮、间异氰酸酯基二苯酮、邻异氰酸酯基二苯酮。
所述含有羟基的疏水性抗肿瘤药物包括紫杉醇、多烯紫杉醇、紫杉醇衍生物及多烯紫杉醇衍生物中的至少任一种。
所述酰肼封端的聚乙二醇的数均分子量为148～100000，1<PDI<2。
所述酰肼封端的聚乙二醇具有如下式Ⅰ所示的化学结构：
Figure PCTCN2019077038-appb-000001
其中，R为H、-CH3或者选自生物素、叶酸、精氨酸-甘氨酸-天冬氨酸(RGD)、氟脲苷、阿糖胞苷、吉西他滨、艾沙托立宾、曲沙他滨、羟基脲、米托蒽醌、阿美蒽醌、链脲菌素、平阳霉素、博来霉素的衍生基团中的任一种基团。
所述乳糖酰肼具有如下式Ⅱ所示的化学结构：
Figure PCTCN2019077038-appb-000002
所述紫杉醇的衍生物，包括具有式Ⅲ所示结构的10-去乙酰紫杉醇，或者具有式Ⅳ所示结构的三尖杉宁碱:
Figure PCTCN2019077038-appb-000003
所述多烯紫杉醇的衍生物具有式Ⅴ所示结构：
Figure PCTCN2019077038-appb-000004
其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10各自独立地为氢、氘或氟，条件是其中至少有一个为氘或氟；优选，其中R1、R2、R3、R4、 R5、R6、R7、R8、R9中一个或多个为氘，且R10为氘。
所述缀合物，优选包括具有式(1)-式(7)所示结构。
Figure PCTCN2019077038-appb-000005
Figure PCTCN2019077038-appb-000006
式(7)中，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10各自独立地为氢、氘或氟，条件是其中至少有一个为氘或氟；优选，其中R1、R2、R3、R4、R5、R6、R7、R8、R9中一个或多个为氘，且R10为氘。
本发明的第二方面提供一种纳米制剂，其包含纳米颗粒，所述纳米颗粒中包含前述的缀合物。所述纳米颗粒的粒径小于300纳米，优选为20～200 纳米。所述纳米制剂为pH响应性纳米制剂。
本发明的第三方面提供所述纳米制剂的制备方法，其特征在于，包括以下步骤：
步骤(1)：将酰肼封端的聚乙二醇和/或乳糖酰肼与引入酮羰基的疏水性抗肿瘤药物进行脱水缩合反应，得到缀合物；
步骤(2)：将所述缀合物溶解在有机溶剂中形成溶液，在室温下在所述溶液加入水中，除去有机溶剂后，得到包含纳米颗粒的水溶液。
优选地，在步骤(1)中，将含酰肼封端的聚乙二醇和/或乳糖酰肼的有机溶液加入引入酮羰基的疏水性抗肿瘤药物的有机溶液中，搅拌反应。
优选地，步骤(1)和(2)中的有机溶剂选自N,N′-二甲基甲酰胺、二甲基亚砜、四氢呋喃、乙腈、甲醇、二氧六环中的至少一种。
本发明的第四方面提供所述缀合物或纳米制剂在制备抗肿瘤药物中的应用。
本发明的第五方面提供一种包含酮羰基的疏水性抗肿瘤药物，其由含有异氰酸酯基和酮羰基的化合物中的异氰酸酯基与含有羟基的疏水性抗肿瘤药物中的羟基反应而得到。
所述含有羟基的疏水性抗肿瘤药物选自紫杉醇、多烯紫杉醇、紫杉醇衍生物及多烯紫杉醇衍生物中的至少任一种。
所述含有异氰酸酯基和酮羰基的化合物包括：对异氰酸酯苯甲醛、间异氰酸酯苯甲醛、邻异氰酸酯苯甲醛、对异氰酸酯苯乙酮、间异氰酸酯苯乙酮、邻异氰酸酯苯乙酮、对异氰酸酯基二苯酮、间异氰酸酯基二苯酮、邻异氰酸 酯基二苯酮。
所述紫杉醇的衍生物，包括具有式Ⅲ所示结构的10-去乙酰紫杉醇，或者具有式Ⅳ所示结构的三尖杉宁碱:
Figure PCTCN2019077038-appb-000007
所述多烯紫杉醇的衍生物包括式Ⅴ所示结构：
Figure PCTCN2019077038-appb-000008
其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10各自独立地为氢、氘或氟，条件是其中至少有一个为氘或氟；优选，其中R1、R2、R3、R4、R5、R6、R7、R8、R9中一个或多个为氘，且R10为氘。
本发明的第六方面提供一种含有酮羰基的多烯紫杉醇衍生物，其由含有异氰酸酯基和酮羰基的化合物中的异氰酸酯基与多烯紫杉醇或氘代多烯紫杉醇中的羟基反应而得到；所述含有异氰酸酯基和酮羰基的 化合物包括：对异氰酸酯苯甲醛、间异氰酸酯苯甲醛、邻异氰酸酯苯甲醛、对异氰酸酯苯乙酮、间异氰酸酯苯乙酮、邻异氰酸酯苯乙酮、对异氰酸酯基二苯酮、间异氰酸酯基二苯酮、邻异氰酸酯基二苯酮。
所述氘代多烯紫杉醇具有式Ⅴ所示结构
发明的效果
本发明的引入酮羰基的疏水性抗肿瘤药物及pH响应纳米制剂与目前临床上使用的多烯紫杉醇制剂相比，在治疗恶性肿瘤过程中，抗癌活性更好，对肿瘤抑制率更高，具有潜在的临床应用价值。本发明的两亲性pH响应缀合物的结构明确，在水中可以自组装形成纳米颗粒进行输送，避免了再引入其它载体带来的毒副作用。通过肿瘤组织的高通透性和滞留(EPR)效应，pH响应纳米颗粒可以被动靶向到肿瘤组织中。由于肿瘤组织的微酸环境，pH响应纳米颗粒能够快速释放出疏水性抗肿瘤药物分子，避免了释放的滞后所引起的抗肿瘤活性的减弱甚至丧失。
附图说明
图1为实施例1制备的两亲性pH响应单甲基化PEG-多烯紫杉醇缀合物的 1H NMR谱图；
图2为实施例1制备得到纳米颗粒的流体力学直径数据图；
图3为实施例1制备纳米颗粒的透射电镜照片；
图4为小鼠体重随治疗时间的变化情况；
图5为相对肿瘤体积随治疗时间的变化情况；
图6为相对肿瘤抑制率随治疗时间的变化情况；
图7为治疗结束后各组的肿瘤照片；
图8为人体肺癌A549裸鼠移植瘤肿瘤体积随治疗时间的变化情况；
图9为荷人体肺癌A549裸鼠体重随治疗时间的变化情况。
具体实施方式
本发明的缀合物，是由酰肼封端的聚乙二醇和/或乳糖酰肼与引入酮羰基的疏水性抗肿瘤药物经脱水缩合反应而得到。
所述引入酮羰基的疏水性抗肿瘤药物，是由含有异氰酸酯基和酮羰基的化合物中的异氰酸酯基团与含有羟基的疏水性抗肿瘤药物上的羟基反应而得到。所述含羟基的疏水性抗肿瘤药物可以为含羟基的紫杉醇类药物及其衍生物，例如可以包括选自紫杉醇、多烯紫杉醇及它们的衍生物的至少任一种。
所述衍生物包括上述化合物的旋光异构体、药学可接受的盐、溶剂化物、水合物、前药、多晶型物、其立体异构体、几何异构体或互变异构体、氘代物等。
所述紫杉醇、多烯紫杉醇及它们的衍生物，不仅主要结构类似，而且其作用机理也基本相同，因此可以通过本发明的方法形成缀合物以及纳米制剂从而改善其药效。
本发明中所使用的酰肼封端的聚乙二醇，其具有如下式Ⅰ所示的化学结构:
Figure PCTCN2019077038-appb-000009
R可为H、-CH 3，也可为亲水性分子或亲水性药物的衍生基团，如生物素、叶酸、精氨酸-甘氨酸-天冬氨酸(RGD)、氟脲苷、阿糖胞苷、吉西他滨、艾沙托立宾、曲沙他滨、羟基脲、米托蒽醌、阿美蒽醌、链脲菌素、平阳霉素、博来霉素等的衍生基团中的任一种基团。
本发明所使用的酰肼封端的聚乙二醇的数均分子量为148～100000,1<PDI<2。
酰肼封端的聚乙二醇的制备方法参见本申请的制备例。
本发明中所使用的乳糖酰肼，其具有如下式Ⅱ所示的化学结构:
Figure PCTCN2019077038-appb-000010
乳糖酰肼的制备方法参见本申请的制备例。
本发明中所述紫杉醇的衍生物，包括10-去乙酰紫杉醇及三尖杉宁碱，其分别具有如下式Ⅲ及式Ⅳ所示的化学结构：
Figure PCTCN2019077038-appb-000011
所述多烯紫杉醇的衍生物，包括多烯紫杉醇的氘代衍生物，其具有如下式Ⅴ所示的化学结构：
Figure PCTCN2019077038-appb-000012
式Ⅴ中R1、R2、R3、R4、R5、R6、R7、R8、R9各自独立地为氢、氘或氟，R10为氘。优选，其中R1、R2、R3、R4、R5、R6、R7、R8、R9中一个或多个为氘；更优选，其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10均为氘，或者，其中R1、R2、R3、R4、R5、R6、R7、R8、R9均为氟，R10为氘。
上述紫杉醇、10-去乙酰紫杉醇、三尖杉宁碱或多烯紫杉醇的衍生物，包括其旋光异构体、溶剂化物、多晶型物、其立体异构体、几何异构体或互变异构体。
本发明的缀合物由于一端是引入酮羰基的疏水性的抗肿瘤药物，另一端是亲水性的化合物，因此具有两亲性。两亲性的结构使得缀合物在水中受到疏水缔合(主要)的作用，自发地组装成纳米胶束。
将本发明所述的缀合物溶解在有机溶剂中形成溶液，在室温下将所述溶液加入水中，除去有机溶剂后，得到包含纳米颗粒的水溶液。其中，所述缀合物以2ng/ml～70mg/ml的浓度、优选以3μg/ml～35mg/ml的浓度溶于有机溶剂中，所得溶液与水的比例(体积比)可以为1:1～1:50，优选1:2～1:10。除去有机溶剂的方法可以为现有技术中常用的方法，例如减压蒸馏等。在包含本发明所述缀合物的纳米制剂中，所述引入酮羰基的疏水性抗肿瘤药物的含量为0.8ng/ml～20mg/ml，优选为1μg/ml～10mg/ml。所述纳米制剂中的引入 羰基的疏水性抗肿瘤药物的含量可根据具体的应用进行调节。
本申请所述纳米制剂的治疗剂量可根据具体的治疗用途、疾病的种类和发展程度、患者的健康状况以及医师的判断而决定。某些典型的剂量范围为40～300mg/kg体重/日，优选为200mg/kg体重/日。
下面给出制备例以及实施例并结合附图对本发明的技术方案作进一步说明，但是以下制备例和实施例只是本发明的示例性说明，不能理解为对本发明保护范围的限制，该领域的技术熟练人员根据上述本发明的内容，对本发明作一些非本质性的改进和调整仍属于本发明的保护范围。
除非另有说明，所使用原料均为商购原料。
关于部分原料的说明：
聚乙二醇单甲醚(M n＝2000)：，购自Sigma-Aldrich公司
羧基封端的单羟基聚乙二醇(M n＝2000)，购自Sigma-Aldrich公司
乳糖酸：纯度：97％，购自Adamas公司
多烯紫杉醇：纯度：98％，购自江苏红豆杉药业有限公司
紫杉醇：纯度：98％，购自江苏红豆杉药业有限公司
<合成酰肼封端的聚乙二醇的制备例>
制备例1
将聚乙二醇单甲醚(20g,10mmol)完全溶于甲苯(200mL)中，通氮气除去反应瓶中空气，逐步滴加叔丁醇钾(4.12g,36mmol)的叔丁醇溶液(60mL)。反应半小时后，滴加溴代乙酸乙酯(6.4mL,48mmol)半个小时，反应溶液在室温下反应24小时。反应结束后，反应液用布氏漏斗过滤，滤液浓缩，并在冰乙醚中沉淀三次，白色沉淀物在35℃真空干燥24小时，最终得到乙酸乙酯封端的聚乙二醇单甲醚，产率90％。
1H NMR(400MHz,DMSO-d 6,20℃):δ＝1.18(t,J＝7.04Hz,3H；CH 3),3.22(s,3H；OCH 3),3.31-3.66(m,180H；OCH 2CH 2),4.22(m,2H； OCH 2CH 2OCO),4.70(s,2H；OCOOCH 2).
随后，将乙酸乙酯封端的聚乙二醇单甲醚(10.00g,4.8mmol)溶于甲醇(100mL)中，逐步滴加水合肼(30mL)的甲醇溶液(40mL)。反应24小时后，反应液过滤，滤液浓缩，用二氯甲烷萃取三次，无水硫酸镁干燥，布氏漏斗抽滤。滤液浓缩后，在冰乙醚中沉淀，白色沉淀物在35℃真空干燥24小时，最终得到酰肼封端的单甲基化聚乙二醇，产率90％。
1H NMR(400MHz,DMSO-d 6,20℃):δ＝3.22(s,3H；OCH 3),3.30-3.68(m,180H；OCH 2CH 2),3.87(s,2H；OCH 2CH 2OCO),8.87(s,1H；CONHNH 2).
制备例2
将羧基封端的单羟基聚乙二醇(10g,5mmol)，4-二甲氨基吡啶(DMAP)(61.1mg,0.5mmol)完全溶于甲醇(100mL)中，在冰浴的条件下，缓慢滴加二环己基碳二亚胺(DCC)(2.1g,10mmol)的甲醇(20ml)溶液，滴加完毕后，反应溶液在室温下反应48小时。反应结束后，反应液用布氏漏斗过滤，滤液浓缩，并在冰乙醚中沉淀三次，白色沉淀物在35℃真空干燥24小时，最终得到甲酯封端的单羟基聚乙二醇，产率76％。
随后，将甲酯封端的单羟基聚乙二醇(5.00g,5mmol)溶于甲醇(50mL)中，逐步滴加水合肼(15mL)的甲醇溶液(20mL)。反应24小时后，反应液过滤，滤液浓缩，用二氯甲烷萃取三次，无水硫酸镁干燥，布氏漏斗抽滤。滤液浓缩后，在冰乙醚中沉淀，白色沉淀物在35℃真空干燥24小时，最终得到酰肼封端的单羟基聚乙二醇，产率90％。
<合成乳糖酰肼的制备例>
制备例3
乳糖酸(5.0000g,13.96mmol)溶于无水甲醇(70.0mL)中，在75℃条件下回流直至乳糖酸完全转化成乳糖内酯。
1H NMR(400MHz,DMSO-d6)δ(ppm):5.33–4.01(br,OH),4.34–4.13(m, 2H,CH),4.02–3.86(m,1H,CH),3.75–3.25(m,10H,CH&CH2). 13C NMR(100MHz,DMSO-d6)δ(ppm):173.44,105.42,84.19,76.01,73.66,71.83,71.63,71.41,70.94,68.55,62.64,60.79.经HRMS:(ESI)[M-H] -检测C 12H 19O 11分子量为339.0927.
在25℃条件下，将乳糖内酯(3.0000g,8.82mmol)溶于无水甲醇中(40.0mL)中。随后，将水合肼(2.2270g,44.10mmol)逐滴加入反应液中，25℃条件下反应1h。有白色沉淀产生。减压蒸馏得到白色固体即乳糖酰肼，产率76.2％。
1H NMR(400MHz,DMSO-d6)δ(ppm):8.91–8.50(s,1H,NH),5.23–4.03(br,OH),4.32–4.17(d,J＝4.27Hz,1H,CH),4.19–4.09(d,J＝4.16Hz,1H,CH),4.04–3.93(m,1H,CH),3.73–3.60(m,2H,CH),3.60–3.46(m,5H,CH&CH2),3.45–3.24(m,3H,CH),3.18–3.16(s,2H,NH 2). 13C NMR(100MHz,DMSO-d6)δ(ppm):171.46,104.95,82.96,76.15,73.67,72.05,71.86,71.56,70.92,68.74,62.77,61.15.经HRMS:(ESI)[M+H] +检测C 12H 20O 11分子量为339.0927.
<合成引入酮羰基的疏水性抗肿瘤药物的制备例>
制备例4
将6g多烯紫杉醇(DTX)溶解在110ml无水二氯甲烷与吡啶的混合溶液中。在冰浴的条件下，向DTX溶液中滴加40ml三乙基氯硅烷(TESCl)，滴加速度为10ml/10min,共滴加四次。滴加完毕后，在冰浴的条件下继续搅拌40min。随后，将反应体系置于35℃油浴中继续反应24h。反应结束后，缓慢加入水致无气泡产生。随后用水洗涤3次，减压蒸馏除去二氯甲烷。所得混合物用乙酸乙酯和石油醚梯度洗脱，经硅胶柱分离得到白色粉末状固体三乙基硅烷保护的多烯紫杉醇(DTX-TES)，产率98％。
1H NMR(400MHz,DMSO-d6)δ8.01–7.94(m,2H),7.72–7.66(m,1H), 7.59(t,J＝7.6Hz,2H),7.48(d,J＝9.9Hz,1H),7.38–7.30(m,4H),7.16(tt,J＝6.0,2.5Hz,1H),5.86–5.74(m,1H),5.39(d,J＝7.1Hz,1H),4.92(tt,J＝12.6,5.0Hz,4H),4.50(s,1H),4.45(d,J＝7.1Hz,1H),4.29(dd,J＝10.5,6.6Hz,1H),4.03(s,2H),3.65(d,J＝7.1Hz,1H),2.34(s,4H),1.91(dd,J＝15.3,9.2Hz,1H),1.71–1.57(m,4H),1.53(s,3H),1.34(s,9H),0.96(d,J＝6.4Hz,6H),0.87(td,J＝7.9,2.2Hz,18H),0.60–0.39(m,12H).
将DTX-TES(2.06g)，4-二甲氨基吡啶(25mg),对异氰酸酯苯乙酮(AI)(1.63g)溶于100ml无水N，N-二甲基甲酰胺中，缓慢搅拌至固体完全溶解，溶解完毕后，将反应体系放入56℃油浴中，反应12h。反应结束后，减压蒸馏除去N，N-二甲基甲酰胺。所得混合物用乙酸乙酯和石油醚梯度洗脱，经柱层析分离得到白色粉末状固体(DTX-TES-AI)，产率98％。
1H NMR(400MHz,DMSO-d6)δ10.24(s,1H),8.03–7.93(m,2H),7.94–7.85(m,2H),7.75–7.65(m,1H),7.66–7.51(m,5H),7.41–7.28(m,4H),7.17(tt,J＝5.8,3.0Hz,1H),6.26(s,1H),5.78(t,J＝9.1Hz,1H),5.44(d,J＝7.1Hz,1H),4.95(d,J＝9.1Hz,1H),4.89(dd,J＝9.9,7.1Hz,1H),4.66(s,1H),4.49–4.33(m,2H),4.09–3.94(m,3H),3.62(d,J＝7.0Hz,1H),2.49(s,3H),2.36(d,J＝5.4Hz,3H),1.96(s,2H),1.79(s,3H),1.75–1.60(m,1H),1.54(s,3H),1.33(s,9H),1.12(d,J＝5.1Hz,3H),0.98(s,3H),0.85(td,J＝7.9,6.2Hz,18H),0.52(hept,J＝7.9Hz,12H).
将DTX-TES-AI(2g)溶于16ml 5％HCl/甲醇中，26℃下开始反应,30min后加入50ml乙酸乙酯，随后用水洗涤3次，减压蒸馏除去乙酸乙酯。所得混合物用乙酸乙酯和石油醚梯度洗脱，经柱层析分离得到目标得到的引入羰基的疏水性抗肿瘤药物(DTX-AI)，为白色粉末状固体，产率88％。
1H NMR(400MHz,DMSO-d6)δ10.21(s,1H),8.02–7.94(m,2H),7.91(d,J＝8.7Hz,2H),7.69(t,J＝7.4Hz,1H),7.66–7.56(m,4H),7.43(d,J＝9.4 Hz,1H),7.36(t,J＝7.6Hz,2H),7.29(d,J＝7.6Hz,2H),7.19(t,J＝7.2Hz,1H),6.31(s,1H),5.87(td,J＝9.6,8.6,5.5Hz,2H),5.45(d,J＝7.1Hz,1H),4.98(d,J＝6.9Hz,1H),4.95–4.84(m,2H),4.67(s,1H),4.34(t,J＝7.2Hz,1H),4.14(dt,J＝11.1,7.0Hz,1H),4.06–3.96(m,2H),3.63(d,J＝7.0Hz,1H),2.50(s,3H),2.38–2.27(m,1H),2.22(s,3H),1.95–1.75(m,4H),1.64(t,J＝12.5Hz,1H),1.52(s,3H),1.33(s,9H),1.14(d,J＝10.9Hz,3H),1.04(s,3H).
制备例5
将6g紫杉醇(PTX)溶解在110ml无水二氯甲烷与吡啶的混合溶液中。在冰浴的条件下，向PTX溶液中滴加40ml二甲基叔丁基氯硅烷(TBSCl)，滴加速度为10ml/10min,共滴加四次。滴加完毕后，在冰浴的条件下继续搅拌40min。随后，将反应体系置于35℃油浴中继续反应24h。反应结束后，缓慢加入水致无气泡产生。随后用水洗涤3次，减压蒸馏除去二氯甲烷。所得混合物用乙酸乙酯和石油醚梯度洗脱，经柱层析分离得到白色粉末状固体二甲基叔丁基硅烷保护的紫杉醇(PTX-TBS)，产率98％。
1H NMR(400MHz,DMSO-d6)δ8.81(d,J＝9.4Hz,1H),8.01–7.92(m,2H),7.87–7.79(m,2H),7.70(t,J＝7.4Hz,1H),7.58(t,J＝7.6Hz,2H),7.55–7.42(m,5H),7.38(t,J＝7.5Hz,2H),7.19(t,J＝7.3Hz,1H),6.24(s,1H),5.83(t,J＝9.1Hz,1H),5.52(t,J＝9.1Hz,1H),5.40(d,J＝7.2Hz,1H),4.94(t,J＝8.3Hz,2H),4.76(d,J＝8.7Hz,1H),4.68(s,1H),4.05(dq,J＝27.1,8.2,7.1Hz,3H),3.62(d,J＝7.1Hz,1H),2.45(s,3H),2.34(d,J＝9.4,8.6Hz,1H),2.07(s,3H),1.94(q,J＝10.3,7.2Hz,1H),1.63(t,J＝12.2Hz,2H),1.52(d,J＝17.6Hz,6H),0.98(d,J＝15.6Hz,6H),0.78(s,9H),0.03(d,J＝16.1Hz,6H).
将PTX-TBS(2.06g)，4-二甲氨基吡啶(25mg),对异氰酸酯苯乙酮(AI)(1.63g)溶于100ml无水N，N-二甲基甲酰胺中，缓慢搅拌至固体完全溶解，溶解完毕后，将反应体系放入56℃油浴中，反应12h。反应结束后，减 压蒸馏除去N，N-二甲基甲酰胺。所得混合物用乙酸乙酯和石油醚梯度洗脱，经柱层析分离得到白色粉末状固体(PTX-TBS-AI)，产率98％。
1H NMR(400MHz,DMSO-d6)δ9.87(s,1H),8.84(d,J＝9.4Hz,1H),8.03–7.94(m,2H),7.91–7.80(m,4H),7.72(t,J＝7.3Hz,1H),7.62(t,J＝7.6Hz,2H),7.59–7.37(m,8H),7.19(t,J＝7.4Hz,1H),6.29(s,1H),5.86(t,J＝9.1Hz,1H),5.58–5.38(m,3H),5.00(d,J＝9.4Hz,1H),4.80–4.67(m,2H),4.14–3.95(m,3H),3.79(d,J＝7.0Hz,1H),2.49(s,3H),2.43(s,2H),1.97(s,3H),1.91(dd,J＝15.2,9.6Hz,2H),1.69(d,J＝15.6Hz,7H),1.00(d,J＝6.5Hz,6H),0.79(s,9H),0.05(d,J＝23.3Hz,6H).
将PTX-TBS-AI(2g)溶于16ml 5％HCl/甲醇中，26℃下开始反应,30min后加入50ml乙酸乙酯，随后用水洗涤3次，减压蒸馏除去乙酸乙酯。所得混合物用乙酸乙酯和石油醚梯度洗脱，经硅胶柱分离得到目标得到的引入酮羰基的疏水性抗肿瘤药物(PTX-AI)，为白色粉末状固体，产率88％。
1H NMR(400MHz,DMSO-d6)δ8.81(d,J＝9.4Hz,1H),8.01–7.92(m,2H),7.87–7.79(m,2H),7.70(t,J＝7.4Hz,1H),7.58(t,J＝7.6Hz,2H),7.55–7.42(m,5H),7.38(t,J＝7.5Hz,2H),7.19(t,J＝7.3Hz,1H),6.29(s,1H),5.86(t,J＝9.1Hz,1H),5.58–5.38(m,3H),5.00(d,J＝9.4Hz,1H),4.80–4.67(m,2H),4.14–3.95(m,3H),3.79(d,J＝7.0Hz,1H),2.49(s,3H),2.43(s,2H),1.97(s,3H),1.91(dd,J＝15.2,9.6Hz,2H),1.69(d,J＝15.6Hz,7H),1.00(d,J＝6.5Hz,6H).
<合成pH响应性缀合物的实施例>
实施例1
将制备例1得到的酰肼封端的单甲基化PEG(6g)溶解在150毫升无水甲醇中。将制备例4得到的引入酮羰基的多烯紫杉醇衍生物(968.39mg) 溶于20毫升无水甲醇中，将所得溶液加入至上述含酰肼封端的单甲基化PEG的溶液中，在氮气保护下室温搅拌反应48小时。减压蒸馏除去甲醇，混合物用乙腈和水梯度洗脱，经反相柱分离得到白色粉末状产物单甲基化PEG-多烯紫杉醇衍生物缀合物(mPEG-DTX-AI)，产率90％。
本实施例合成的mPEG-DTX-AI的化学结构如式(1)所示。本实施例制得的缀合物的 1H NMR谱图如图1所示，测试溶剂为DMSO-d6。谱图1中各质子峰的归属如下： 1H NMR(400MHz,DMSO-d6)δ10.51(s,1H),10.06–9.87(m,2H),7.97(d,J＝7.6Hz,2H),7.74(d,J＝8.4Hz,1H),7.69(d,J＝7.9Hz,2H),7.60(t,J＝7.6Hz,2H),7.52(d,J＝8.3Hz,2H),7.41(d,J＝9.3Hz,1H),7.35(t,J＝7.5Hz,2H),7.29(d,J＝7.6Hz,2H),7.19(t,J＝7.3Hz,1H),6.30(s,1H),5.87(d,J＝7.7Hz,2H),5.44(d,J＝7.0Hz,1H),4.99–4.83(m,3H),4.65(s,1H),4.51(s,1H),4.34(t,J＝7.2Hz,1H),4.11(d,J＝14.4Hz,2H),4.01(s,2H),3.64(t,J＝4.9Hz,2H),3.49(s,195H),3.22(s,3H),2.32(s,1H),2.22(s,3H),2.17(s,3H),1.87(s,4H),1.63(t,J＝12.7Hz,1H),1.51(s,3H),1.33(s,9H),1.12(s,3H),1.03(s,3H).
将上述制得的两亲性缀合物溶解在四氢呋喃中，在室温下将其加入水中，除去四氢呋喃，得到两亲性缀合物的纳米颗粒水溶液，其中多烯紫杉醇衍生物的浓度为1mg/ml。本实施例制备的含有pH响应两亲性缀合物的纳米颗粒(Nano mPEG-DTX-AI)的粒径平均尺寸在170纳米左右。本实施例得到的纳米颗粒的流体力学直径数据如图2所示，透射电镜照片如图3所示。
实施例2
将制备例1得到的酰肼封端的单甲基化PEG(6g)溶解在150毫升无水甲醇中。将制备5得到的引入酮羰基的紫杉醇衍生物(1015.06mg)溶于20毫升无水甲醇中，将所得溶液加入至上述含酰肼封端的单甲基化PEG的溶液中，在氮气保护下室温搅拌反应48小时。减压蒸馏除去甲醇，混合物 用乙腈和水梯度洗脱，经反相柱分离得到白色粉末状产物单甲基化PEG-紫杉醇衍生物缀合物(mPEG-DTX-AI)，产率89％。
本实施例合成的两亲性pH响应单甲基化PEG-紫杉醇衍生物缀合物的化学结构如式(2)所示。
将上述制得的两亲性缀合物溶解在四氢呋喃中，在室温下将其加入水中，除去四氢呋喃，得到两亲性缀合物的纳米颗粒水溶液，其中紫杉醇衍生物的浓度为1mg/ml。本实施例制备的含有pH响应两亲性缀合物的纳米颗粒(Nano mPEG-DTX-AI)的粒径平均尺寸在200纳米左右。
实施例3
将制备例1得到的酰肼封端的单甲基化PEG(6g)溶解在150毫升无水甲醇中形成溶液。将与制备例4类似方法得到的引入酮羰基的氘代多烯紫杉醇衍生物(968.39mg)溶于20毫升无水甲醇中，将所得溶液加入至上述含酰肼封端的单甲基化PEG的溶液中，在氮气保护下室温搅拌反应48小时。减压蒸馏除去甲醇，混合物用乙腈和水梯度洗脱，经反相柱分离得到白色粉末状产物单甲基化PEG-氘代多烯紫杉醇衍生物缀合物，产率90％。
本实施例合成的两亲性pH响应单甲基化PEG-氘代多烯紫杉醇衍生物缀合物的化学结构如式(7)所示:
Figure PCTCN2019077038-appb-000013
其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10均为氘。
将上述制得的两亲性缀合物溶解在四氢呋喃中，在室温下将其加入水中，除去四氢呋喃，得到两亲性缀合物的纳米颗粒水溶液，其中氘代多烯紫杉醇衍生物的浓度为1mg/ml。本实施例制备的含有pH响应两亲性缀合物的纳米颗粒的粒径平均尺寸在200纳米左右。
实施例4
将制备例1得到的酰肼封端的单甲基化PEG(6g)溶解在150毫升无水甲醇中形成溶液。将与制备例4类似方法得到的引入酮羰基的10-去乙酰紫杉醇衍生物(973.03mg)溶于20毫升无水甲醇中，将所得溶液加入至上述含酰肼封端的单甲基化PEG的溶液中，在氮气保护下室温搅拌反应48小时。减压蒸馏除去甲醇，混合物用乙腈和水梯度洗脱，经反相柱分离得到白色粉末状产物单甲基化PEG-10-去乙酰紫杉醇衍生物缀合物，产率85％。
本实施例合成的两亲性pH响应单甲基化PEG-10-去乙酰紫杉醇衍生物缀合物的化学结构如式(3)所示:
Figure PCTCN2019077038-appb-000014
将上述制得的两亲性缀合物溶解在四氢呋喃中，在室温下将其加入水中，除去四氢呋喃，得到两亲性缀合物的纳米颗粒水溶液，其中10-去乙酰紫杉醇衍生物的浓度为1mg/ml。本实施例制备的含有pH响应两亲性缀合 物的纳米颗粒的粒径平均尺寸在200纳米左右。
实施例5
将制备例1得到的酰肼封端的单甲基化PEG(6g)溶解在150毫升无水甲醇中形成溶液。将与制备例4类似方法得到的引入酮羰基的三尖杉宁碱衍生物(992.39mg)溶于20毫升无水甲醇中，将所得溶液加入至上述含酰肼封端的单甲基化PEG的溶液中，在氮气保护下室温搅拌反应48小时。减压蒸馏除去甲醇，混合物用乙腈和水梯度洗脱，经反相柱分离得到白色粉末状产物单甲基化PEG-三尖杉宁碱衍生物缀合物，产率90％。
本实施例合成的两亲性pH响应单甲基化PEG-三尖杉宁碱衍生物缀合物的化学结构如式(4)所示:
Figure PCTCN2019077038-appb-000015
将上述制得的两亲性缀合物溶解在四氢呋喃中，在室温下将其加入水中，除去四氢呋喃，得到两亲性缀合物的纳米颗粒水溶液，其中三尖杉宁碱衍生物的浓度为1mg/ml。本实施例制备的含有pH响应两亲性缀合物的纳米颗粒的粒径平均尺寸在200纳米左右。
实施例6
将制备例3得到的乳糖酰肼(6g)溶解在150毫升无水甲醇中。将制备例4得到的引入酮羰基的多烯紫杉醇衍生物(1015.06mg)溶于20毫升无水 甲醇中，将所得溶液加入至上述含乳糖酰肼的溶液中，在氮气保护下室温搅拌反应48小时。减压蒸馏除去甲醇，混合物用乙腈和水梯度洗脱，经反相柱分离得到白色粉末状产物乳糖-多烯紫杉醇衍生物缀合物，产率87％。
本实施例合成的两亲性pH响应乳糖-多烯紫杉醇衍生物缀合物的化学结构如式(5)所示。
将上述制得的两亲性缀合物溶解在四氢呋喃中，在室温下将其加入水中，除去四氢呋喃，得到两亲性缀合物的纳米颗粒水溶液，其中多烯紫杉醇衍生物的浓度为1mg/ml。本实施例制备的含有pH响应两亲性缀合物的纳米颗粒的粒径平均尺寸在200纳米左右。
实施例7
将制备例3得到的乳糖酰肼(6g)溶解在150毫升无水甲醇中。将制备例5得到的引入酮羰基的紫杉醇衍生物(1015.06mg)溶于20毫升无水甲醇中，将所得溶液加入至上述含乳糖酰肼的溶液中，在氮气保护下室温搅拌反应48小时。减压蒸馏除去甲醇，混合物用乙腈和水梯度洗脱，经反相柱分离得到白色粉末状产物乳糖-紫杉醇衍生物缀合物，产率85％。
本实施例合成的两亲性pH响应乳糖-紫杉醇衍生物缀合物的化学结构如式(6)所示。
将上述制得的两亲性缀合物溶解在四氢呋喃中，在室温下将其加入水中，除去四氢呋喃，得到两亲性缀合物的纳米颗粒水溶液，其中紫杉醇衍生物的浓度为1mg/ml。本实施例制备的含有pH响应两亲性缀合物的纳米颗粒的粒径平均尺寸在200纳米左右。
本发明中纳米颗粒的流体力学直径的测试方法：
纳米胶束平均粒後(Z-Average)和粒径：分布(PDI)由动态光散射仪(Dynamic light scattering,DLS)测定，测试温度25℃，激光波长633nm，探头角度为173°。每个样品平衡时间为2min，测试3次。实验数据通过软 件Dispersion Technology software version 5.32来分析。
本发明中纳米颗粒的透射电镜照片的测试方法：
透射电镜(TEM)测试在JEM-2010/INCA OXFORD型上完成,电镜加速电压为200kV。样品制备方法如下：将组装体溶液滴加到附有碳膜的铜网上,室温下自然晾干。干燥过程中注意避免空气中的灰尘等杂质的污染。
药效实验：
实验1：pH响应纳米制剂对体内肿瘤的影响实验
将Hela荷瘤小鼠分为四组，5只一组。第一、二及三组为治疗组，即对于每一只Hela荷瘤小鼠，通过尾静脉处分别注射药物进行肿瘤治疗。对第一组用多烯紫杉醇注射液进行治疗(25mg DTX/kg)，第二组用制备例4得到的抗肿瘤药物DTX-AI进行治疗(40mg DTX/kg)及第三组用实施例1得到的pH响应纳米制剂进行给药治疗(200mg DTX/kg)。第四组为空白对照组。
对第一、二及第三组小鼠每七天进行一次给药治疗，共注射3次。每组的每只荷瘤小鼠都进行标记，记录其在整个治疗过程中的体重及肿瘤的体积。肿瘤的体积通过以下公式计算：V(mm 3)＝1/2×长(mm)×宽(mm) 2。相对肿瘤体积(relative tumor volume，RTV)的计算公式为：RTV＝V t/V 0。其中V 0为分笼给药时(即d 0)测量所得肿瘤体积，V t为每一次测量时的肿瘤体积。d45天处死动物，解剖取瘤块，称瘤重，计算去瘤后动物体重。
图4示出了小鼠体重随时间的变化情况。如图4所示，第二及三组的小鼠体重与空白对照组基本保持一致，说明DTX-AI及纳米mPEG-DTX-AI的体内安全性比较高。
图5示出了相对肿瘤体积随时间的变化情况。如图5所示，与空白对照组及第一组相比，第二及三组显示出相对肿瘤体积大幅度降低的态势。另外，在21天以后(停止给药后)后，第一组的相对肿瘤体积随着时间的推移， 持续增加，抑瘤作用逐渐减弱，出现反弹的现象。然而第二及三组的相对肿瘤体积随着时间的进行依然呈现降低的趋势，即停药后，抑瘤作用继续增强。
此外，还对相对肿瘤的抑制率进行了计算和分析。相对肿瘤抑制率％的计算公式为：相对肿瘤抑制率％＝(1-T RTV/C RTV)*100％，(其中，T RTV表示治疗组的RTV；C RTV表示空白对照组RTV)，其结果如图6所示。
治疗组均显示出相对肿瘤抑制率持续增长的趋势。与第一组相比，第二组与第三组的相对肿瘤抑制率呈现大幅度增长，并且在21天以后(停止给药后)持续保持90％以上。距离末次给药30天时，第一组的相对肿瘤抑制率为67.62％，第二组的相对肿瘤抑制率达到93.5％，第三组的相对肿瘤抑制率达到95.61％。
图7示出了治疗结束后各组的肿瘤照片。从照片中可以直观的看出第二组与第三组的肿瘤尺寸明显小于第一组和空白对照组。
实验2：引入酮羰基的疏水性抗肿瘤药物(DTX-AI)的肿瘤抑制作用
取生长良好的肺癌A549实体瘤，无菌条件下切割成约3mm大小的均匀小块，用套管针每只小鼠右腋皮下接种一块，接种后14日平均肿瘤体积约210-250mm 3，根据肿瘤大小分组，每组肿瘤平均体积基本一致。用制备例4得到的抗肿瘤药物DTX-AI制备成冻干粉针开始给药，给药剂量见下表1。每周2次用电子数显卡尺测瘤块的长径(a)、短径(b)，肿瘤体积(tumor volume,TV)计算公式为：TV＝1/2×a×b 2，相对肿瘤体积(relative tumor volume,RTV)计算公式为：RTV＝Vt/Vo，Vo为分笼时(即d1)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，T/C(％)＝T RTV/C RTV×100％，或相对肿瘤增殖抑制率(％)：(1-T/C)×100％。并进行t检验。d43天(接种后56天)处死动物，解剖取瘤块，称瘤重，计算去瘤后动物体重。
表1：
Figure PCTCN2019077038-appb-000016
试验结果，如表1和图8，DTX-AI冻干粉4个剂量80mg/kg、60mg/kg、40mg/kg、20mg/kg，每周尾静脉注射一次，共4次，距离末次给药21天时对人体肺癌A549的抑瘤率分别为99.27％、99.50％、99.28％、62.38％。多西他赛注射液剂量40mg/kg，20mg/kg，每周尾静脉注射一次，共4次，距离末次给药21天时对人体肺癌A549的抑瘤率为99.24％、84.50％。
DTX-AI冻干粉4个剂量组动物无死亡。多西他赛20mg/kg组动物无死亡，40mg/kg组在d33天死亡1只动物。
如表1和图9，DTX-AI冻干粉20mg/kg给药后对动物体重基本无影响；DTX-AI冻干粉40mg/kg、60mg/kg给药后体重稍有降低；DTX-AI冻干粉80mg/kg给药后动物体重有降低。多西他赛20mg/kg给药后，动物体重有降低，停药后逐渐恢复；多西他赛40mg/kg随着给药次数增加动物体重下降严重，停药后也维持在低体重状态。多西他赛40mg/kg给药后的体重降低程度显著高于DTX-AI冻干粉给药后的体重降低程度。虽然DTX-AI冻干粉80mg/kg给药后动物的体重也有降低，但降低程度也低于多西他赛40mg/kg给药组的体重降低程度。
如图8所示，DTX-AI冻干粉4个剂量组首次给药后肿瘤生长就开始减慢，随着给药次数增加，抑瘤作用增强，40mg/kg，60mg/kg，80mg/kg组随着停药时间的延长，抑瘤作用维持在98-99％抑瘤率，停药后，无动物的肿 瘤反弹。
以上数据说明引入酮羰基的疏水性抗肿瘤药物及pH响应纳米制剂与目前临床上使用的多烯紫杉醇制剂相比，在治疗恶性肿瘤过程中，抗癌活性更好，对肿瘤抑制率更高，且具有更高的安全性，具有潜在的临床应用价值。
以上公开的仅为本申请的几个具体实施例，但本申请并非局限于此，任何本领域的技术人员能思之的变化，都应落在本申请的保护范围内。

Claims (25)

  1. 一种缀合物，其特征在于，其由酰肼封端的聚乙二醇和/或乳糖酰肼与引入酮羰基的疏水性抗肿瘤药物经脱水缩合反应而得到。
  2. 根据权利要求1所述的缀合物，其特征在于，所述引入酮羰基的疏水性抗肿瘤药物由含有异氰酸酯基和酮羰基的化合物中的异氰酸酯基与含有羟基的疏水性抗肿瘤药物中的羟基反应而得到。
  3. 根据权利要求2所述的缀合物，其特征在于，所述含有异氰酸酯基和酮羰基的化合物包括：对异氰酸酯苯甲醛、间异氰酸酯苯甲醛、邻异氰酸酯苯甲醛、对异氰酸酯苯乙酮、间异氰酸酯苯乙酮、邻异氰酸酯苯乙酮、对异氰酸酯基二苯酮、间异氰酸酯基二苯酮、邻异氰酸酯基二苯酮。
  4. 根据权利要求2或3所述的缀合物，其特征在于，所述含有羟基的疏水性抗肿瘤药物包括紫杉醇、多烯紫杉醇、紫杉醇衍生物及多烯紫杉醇衍生物中的至少任一种。
  5. 根据权利要求1～4任一项所述的缀合物，其特征在于，所述酰肼封端的聚乙二醇的数均分子量为148～100000，1<PDI<2。
  6. 根据权利要求1～5任一项所述的缀合物，其特征在于，所述酰肼封端的聚乙二醇具有如下式Ⅰ所示的化学结构：
    Figure PCTCN2019077038-appb-100001
    其中，R为H、-CH 3或者选自生物素、叶酸、精氨酸-甘氨酸-天冬氨酸(RGD)、氟脲苷、阿糖胞苷、吉西他滨、艾沙托立宾、曲沙他滨、羟基脲、米托蒽醌、阿美蒽醌、链脲菌素、平阳霉素、博来霉素的衍生基团中的任一 种基团。
  7. 根据权利要求1～4任一项所述的缀合物，其特征在于，所述乳糖酰肼具有如下式Ⅱ所示的化学结构：
    Figure PCTCN2019077038-appb-100002
  8. 根据权利要求1～7任一项所述的缀合物，其特征在于，所述脱水缩合反应发生在酮羰基和酰肼基之间。
  9. 根据权利要求4～8任一项所述的缀合物，其特征在于，
    所述紫杉醇的衍生物，包括具有式Ⅲ所示结构的10-去乙酰紫杉醇，或者具有式Ⅳ所示结构的三尖杉宁碱:
    Figure PCTCN2019077038-appb-100003
    Figure PCTCN2019077038-appb-100004
    所述多烯紫杉醇的衍生物具有式Ⅴ所示结构：
    Figure PCTCN2019077038-appb-100005
    其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10各自独立地为氢、氘或氟，条件是其中至少有一个为氘或氟；优选，其中R1、R2、R3、R4、R5、R6、R7、R8、R9中一个或多个为氘，且R10为氘。
  10. 根据权利要求1～8任一项所述的缀合物，其特征在于，其包括具有式(1)-式(7)所示结构的缀合物:
    Figure PCTCN2019077038-appb-100006
    Figure PCTCN2019077038-appb-100007
    式(7)中，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10各自独立地为氢、氘或氟，条件是其中至少有一个为氘或氟；优选，其中R1、R2、R3、R4、R5、R6、R7、R8、R9中一个或多个为氘，且R10为氘。
  11. 根据权利要求1～10任一项所述的缀合物，其特征在于，所述缀合物为两亲性pH响应性缀合物。
  12. 一种纳米制剂，其特征在于，包含纳米颗粒，所述纳米颗粒中包含权利要求1～11中任一项所述的缀合物。
  13. 根据权利要求12所述的纳米制剂，其特征在于，所述纳米颗粒的粒径小于300纳米，优选为20～200纳米。
  14. 根据权利要求12或13所述的纳米制剂，其特征在于，所述纳米制剂为pH响应性纳米制剂。
  15. 根据权利要求12～14中任一项所述的纳米制剂的制备方法，其特征在于，包括以下步骤：
    步骤(1)：将酰肼封端的聚乙二醇和/或乳糖酰肼与引入酮羰基的疏水性 抗肿瘤药物进行脱水缩合反应，得到缀合物；
    步骤(2)：将所述缀合物溶解在有机溶剂中形成溶液，在室温下在所述溶液加入水中，除去有机溶剂后，得到包含纳米颗粒的水溶液。
  16. 根据权利要求15所述的纳米制剂的制备方法，其特征在于，在步骤(1)中，将含酰肼封端的聚乙二醇和/或乳糖酰肼的有机溶液加入引入酮羰基的疏水性抗肿瘤药物的有机溶液中，搅拌反应。
  17. 根据权利要求15或16所述的纳米制剂的制备方法，其特征在于，步骤(1)和(2)中的有机溶剂选自N,N′-二甲基甲酰胺、二甲基亚砜、四氢呋喃、乙腈、甲醇、二氧六环中的至少一种。
  18. 根据权利要求1～11所述的缀合物在制备抗肿瘤药物中的应用。
  19. 根据权利要求12～14所述的纳米制剂在制备抗肿瘤药物中的应用。
  20. 一种包含酮羰基的疏水性抗肿瘤药物，其特征在于，其由含有异氰酸酯基和酮羰基的化合物中的异氰酸酯基与含有羟基的疏水性抗肿瘤药物中的羟基反应而得到。
  21. 根据权利要求20所述的药物，其特征在于，所述含有异氰酸酯基和酮羰基的化合物包括：对异氰酸酯苯甲醛、间异氰酸酯苯甲醛、邻异氰酸酯苯甲醛、对异氰酸酯苯乙酮、间异氰酸酯苯乙酮、邻异氰酸酯苯乙酮、对异氰酸酯基二苯酮、间异氰酸酯基二苯酮、邻异氰酸酯基二苯酮。
  22. 根据权利要求20或21所述的药物，其特征在于，所述含有羟基的疏水性抗肿瘤药物选自紫杉醇、多烯紫杉醇、紫杉醇衍生物及多烯紫杉醇衍生物中的至少任一种。
  23. 根据权利要求22所述的药物，其特征在于，所述紫杉醇的衍生物，包括具有式Ⅲ所示结构的10-去乙酰紫杉醇，或者具有式Ⅳ所示结构的三尖杉宁碱:
    Figure PCTCN2019077038-appb-100008
    所述多烯紫杉醇的衍生物包括式Ⅴ所示结构：
    Figure PCTCN2019077038-appb-100009
    其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10各自独立地为氢、氘或氟，条件是其中至少有一个为氘或氟；优选，其中R1、R2、R3、R4、R5、R6、R7、R8、R9中一个或多个为氘，且R10为氘。
  24. 一种含有酮羰基的多烯紫杉醇衍生物，其特征在于，由含有异氰酸 酯基和酮羰基的化合物中的异氰酸酯基与多烯紫杉醇或氘代多烯紫杉醇中的羟基反应而得到；所述含有异氰酸酯基和酮羰基的化合物包括：对异氰酸酯苯甲醛、间异氰酸酯苯甲醛、邻异氰酸酯苯甲醛、对异氰酸酯苯乙酮、间异氰酸酯苯乙酮、邻异氰酸酯苯乙酮、对异氰酸酯基二苯酮、间异氰酸酯基二苯酮、邻异氰酸酯基二苯酮。
  25. 根据权利要求24所述的含有酮羰基的多烯紫杉醇衍生物，其特征在于，所述氘代多烯紫杉醇具有式Ⅴ所示结构，
    Figure PCTCN2019077038-appb-100010
    其中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10各自独立地为氢、氘或氟，条件是其中至少有一个为氘或氟；优选，其中R1、R2、R3、R4、R5、R6、R7、R8、R9中一个或多个为氘，且R10为氘。
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