WO2017193757A1 - 水溶性Epothilone衍生物及其制备方法 - Google Patents

水溶性Epothilone衍生物及其制备方法 Download PDF

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WO2017193757A1
WO2017193757A1 PCT/CN2017/080254 CN2017080254W WO2017193757A1 WO 2017193757 A1 WO2017193757 A1 WO 2017193757A1 CN 2017080254 W CN2017080254 W CN 2017080254W WO 2017193757 A1 WO2017193757 A1 WO 2017193757A1
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compound
formula
added
group
epothilone
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PCT/CN2017/080254
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English (en)
French (fr)
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汪海波
况洪福
张伟
郑晓鹤
朱天民
杨志清
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浙江海正药业股份有限公司
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Priority to CN201780027084.XA priority Critical patent/CN109415378B/zh
Publication of WO2017193757A1 publication Critical patent/WO2017193757A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides

Definitions

  • the invention belongs to the technical field of organic chemistry and medicinal chemistry, and in particular relates to the preparation of water-soluble Epothilone B derivatives and intermediates thereof, and their tumor suppressing activities.
  • Epothilones are a class of macrolide compounds with broad-spectrum anti-tumor activity, which show potent therapeutic effects on human tumors such as breast cancer, prostate cancer, lung cancer and colon cancer. Its anti-tumor mechanism binds to the ⁇ -subunit of ⁇ , ⁇ -tubulin heterodimer, promotes the polymerization of tubulin, inhibits its depolymerization, prevents cells from undergoing normal mitosis, and leads to tumor cell death. . Therefore, the anti-cancer mechanism of the Epothilones family of compounds is similar to that of paclitaxel, but at the same time the Epothilones family of compounds is superior to paclitaxel in many respects.
  • anticancer activity is higher than paclitaxel; it can inhibit tumor cells that are allergic to paclitaxel or resistant to paclitaxel; it has better activity against cancers with multiple drug resistance; cancer patients who have failed to cure a variety of drugs still show Therapeutic effect; not affected by cell detoxification protein, so there is no problem of drug resistance of paclitaxel.
  • the chemical structure is simpler than paclitaxel, the Epothilones family of compounds has better chemical modification and optimization potential.
  • Epothilone B is one of the most studied compounds and is considered to be one of the most promising compounds in cancer therapy.
  • the defects in the structure and properties of Epothilone B have limited its clinical application.
  • Epothilone B has serious toxic side effects such as neurotoxicity, liver toxicity and the like. As a safe drug for human body treatment, it is necessary to overcome the defects in the side effects of Epothilone B. More extensive structural optimization of Epothilone B in order to obtain target compounds with less toxic side effects and a larger therapeutic window.
  • Epothilone B has very little solubility in water, and it is necessary to add a co-solvent such as castor oil or ethanol for injection. These cosolvents increase the side effects of the drug in the human body to some extent. Therefore, improving the water solubility of Epothilone B is one of the problems to be solved.
  • Epothilone B macrolide structure is unstable, enters the body under the action of human esterolytic enzyme, macrolide lactone is opened and leads to inactivation.
  • Ixabepilone replaces the Epothilone B lactone bond with a lactam bond, which solves the defect of Epothilone B in vivo and retains strong anticancer activity in vivo.
  • ixabepilone still has defects such as toxic side effects and poor water solubility.
  • Epothilone B and its derivative Ixabepilone have further structural improvements to Epothilone B and Ixabepilone, in order to obtain Epothilone derivatives with less toxic side effects, better water solubility and stronger anticancer activity.
  • Epothilone B Since there is currently no effective Epothilone B rapid synthesis method, its structural transformation is very inadequate. Therefore, Epothilone B, which is obtained by microbial fermentation, has great potential for the development of new drugs for lead drug molecules.
  • PEG is a water-soluble, non-toxic, biocompatible polymer that is approved by the FDA for the modification of intravenous or oral drug molecules. And it has been successfully coupled with a variety of protein or peptide drugs to improve the therapeutic effect of these drugs. Because PEG can increase the volume of drug molecules, thereby reducing the renal clearance rate of the drug in the body; at the same time, PEG can protect the drug from degradation by the enzyme, thereby improving the pharmacokinetic properties of the drug. In addition, the complex produced by the coupling of the small molecule drug with PEG can play the role of sustained release in vivo, prolong the action time of the drug in the body and improve the therapeutic effect of the drug. The formation of a complex of PEG with a small molecule drug can also improve the water solubility of small molecule drugs.
  • a polypeptide is an endogenous substance of the human body. It is composed of multiple amino acids and has good water solubility. And a wide range of biological activities, through the combination of polypeptides and small molecule drugs on the one hand to improve the solubility of small molecule drugs, on the other hand to achieve in vivo targeted release or improve biological activity with the help of peptides.
  • Glutathione is a common endogenous polypeptide that is bound by glutamate, cysteine and glycine. Glutathione is a tripeptide containing a thiol group, which has antioxidant effects and integrated detoxification. Glutathione is also a nutrient for cell growth and is easily taken up by cells, and is particularly easily taken up by tumor cells that multiply rapidly.
  • the complex prepared by coupling glutathione with small molecule drugs is likely to have certain selectivity for rapidly growing tumor cells, and at the same time can reduce the toxicity of anti-tumor drugs to normal human cells.
  • glutathione is a water-soluble tripeptide that forms a complex with a small fraction of drugs, which can greatly improve the water solubility of small molecule drugs.
  • the present invention provides a method for preparing a water-soluble derivative of Epothilone B and Ixabepilone, and obtains a target compound which is excellent in pharmacological action, less toxic and side effect, and more water-soluble.
  • the present invention provides a water-soluble Epothilone derivative having small toxic and side effects, good water solubility and strong anticancer activity, and has the structure shown in Formula I:
  • X is O or NH
  • R 1 and R 2 are each independently H or COCH 2 -R 3 , but R 1 and R 2 may not be H at the same time;
  • R 3 is S-(CH 2 CH 2 O) n R 4 , NH-(CH 2 CH 2 O) n R 4 or a polypeptide group, wherein the polypeptide is preferably glutathione;
  • R 4 is hydrogen or an alkyl group, preferably a methyl group
  • n is greater than or equal to 1, preferably n is greater than or equal to 1, and n is less than or equal to 24, more preferably n is 3, 5, 7, 12 or 24.
  • an Epothilone derivative of Formula II is provided:
  • an Epothilone derivative of formula III is provided:
  • Another object of the invention is to provide an intermediate compound of formula IV for use in the preparation of a compound of formula (I):
  • X is O or NH
  • R 5 and R 6 are each independently H or COCH 2 -X 2 , but R 5 and R 6 may not be H at the same time;
  • X 2 is a halogen atom, preferably Br and I.
  • the present invention further provides a process for the preparation of a water-soluble Epothilone derivative, but is not limited to the specific examples provided in the present invention. It covers the preparation of NH-PEG, S-PEG, glutathionylation and polypeptideized water-soluble derivatives of other compounds of the Epothilones family. The preparation methods and steps of these derivatives are as follows:
  • step b) reacting a compound of the formula IV prepared in step a) with a PEG derivative or polypeptide having a -NH 2 or -SH group to give a compound of formula I:
  • X is O or NH
  • R 1 and R 2 are each independently H or COCH 2 -R 3 , but R 1 and R 2 may not be H at the same time;
  • R 3 is S-(CH 2 CH 2 O) n R 4 , NH-(CH 2 CH 2 O) n R 4 or a polypeptide group;
  • R 4 is independently hydrogen or alkyl
  • R 5 and R 6 are each independently H or COCH 2 -X 2 , but R 5 and R 6 may not be H at the same time;
  • X 2 is a halogen atom, preferably Br and I.
  • n is greater than or equal to 1, preferably n is greater than or equal to 1, and n is less than or equal to 24, more preferably n is 3, 5, 7, 12 or 24.
  • the compound of formula I is a compound of formula II or formula III,
  • the present invention provides the use of a water-soluble Epothilone derivative of the formula I, II, III for the preparation of a medicament for inhibiting the growth of tumor cells.
  • the present invention further provides the use of the water-soluble Epothilone derivative represented by Formulas I, II, and III for the preparation of a medicament for inhibiting human liver cancer cells.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of an Epothilone derivative of Formula I, Formula II, Formula III, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable Carrier, excipient or combination thereof.
  • Alkyl as a group or as part of a group refers to a straight or branched saturated aliphatic hydrocarbon group. It is preferably a C 1 -C 10 alkyl group, more preferably a C 1 -C 6 alkyl group.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, n-pentyl, 1, 1-di Methylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1 -ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethyl Butyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl Wait.
  • Polypeptide group as a group or part of a group refers to a polypeptide or protein group. Preference is given to polypeptide groups formed by the removal of H at the thiol group by a thiol-containing polypeptide or protein. Examples of polypeptide based include but not Limited to glutathione groups.
  • PEG derivative means a modified polyethylene glycol derivative. Preferred is a polyethylene glycol derivative modified with -NH 2 , -SH, COOH.
  • “Pharmaceutically acceptable salt” refers to certain salts of the above compounds which retain their original biological activity and which are suitable for pharmaceutical use.
  • the pharmaceutically acceptable salt of the compound of formula I may be a salt formed with a suitable acid, including inorganic and organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, Fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, malic acid, maleic acid, mandelic acid, methanesulfonic acid, nitric acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, P-toluenesulfonic acid and the like
  • Epothilone B (4.0 g, 7.9 mmol) and iodoacetic acid (1.2 g, 6.5 mmol) were dissolved in 20 mL of dichloromethane and stirred to a solid. The mixture was cooled to 0 to 5 ° C, DCC (1.55 g, 7.5 mmol) was added, and stirred at 0 to 5 ° C for 10-15 minutes, and DMAP (0.78 g, 6.4 mmol) was added to the reaction mixture. The reaction solution was warmed to room temperature and stirred for 1.5-2 hours.
  • Epothilone B (4.0 g, 7.9 mmol) and bromoacetic acid (2.4 g, 17.3 mmol) were dissolved in 20 mL of dichloromethane and stirred until a solid solid.
  • the mixture was cooled to 0 to 5 ° C, DCC (1.55 g, 7.5 mmol) was added, and stirred at 0 to 5 ° C for 10-15 minutes, and DMAP (0.78 g, 6.4 mmol) was added to the reaction mixture.
  • the reaction solution was warmed to room temperature and stirred for 12 hours.
  • the insoluble solid was suction filtered through a Buchner funnel, and the filtrate was concentrated to dryness, and the obtained residue was purified by silica gel column to afford compound VI-2 (5.09 g, 6.8 mmol).
  • reaction solution was again cooled to 0-5 ° C, iodoacetic acid (2.7 g, 14.6 mmol) and DCC (4.5 g, 14.3 mmol) were added, and the mixture was stirred at 0-5 ° C for 10-15 minutes, then the reaction mixture was warmed to room temperature. Stir until the reaction to ixabepilone is complete.
  • the insoluble solid was suction filtered through a Buchner funnel, and the filtrate was concentrated to dryness under reduced pressure. The obtained residue was purified by silica gel column to afford compound V-2 (1.35 g, 2.0 mmol) and compound VI-3 (4.0 g, 4.8 mmol) .
  • Epothilone B dihaloacetate (VI) (1 eq) and mPEG n -NH 2 or mPEG n -SH (m is methyl abbreviation) (2.1 eq) were dissolved in 10 volumes of DMF and 1.1 eq of K 2 was added. CO 3 , stir at room temperature for 3-16 hours. The reaction was quenched by the addition of 8 eq of ammonium formate and stirred at room temperature for 30 min. The reaction solution was concentrated to dryness under reduced pressure at 40 °C.
  • the syrup was dissolved in 20 volumes of dichloromethane, the dichloromethane layer was washed twice with 20 volumes of 10 g/L of formic acid solution, and the dichloromethane layer was washed twice with 20 volumes of saturated brine.
  • the methylene chloride layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure at 35 ° C.
  • Epothilone B dihaloacetate (VI-1) was added to a 50 mL reaction flask, 5 mL of DMF was added, 197 mg of mPEG 3 -NH 2 and 87.8 mg of K 2 CO 3 were added , and the mixture was stirred at room temperature for 3 hours. The reaction was quenched by the addition of 0.3 g of EtOAc. The syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine. The methylene chloride layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure at 35 ° C.
  • Epothilone B dihaloacetate (VI-1) was added to a 50 mL reaction flask, 5 mL of DMF was added, 423 mg of mPEG 7 -NH 2 and 87.8 mg of K 2 CO 3 were added , and the mixture was stirred at room temperature for 3 hours. The reaction was quenched by the addition of 0.3 g of EtOAc. The syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine. The methylene chloride layer was dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure at 35 ° C.
  • Epothilone B dihaloacetate (VI-1) was added to a 50 mL reaction flask, 5 mL of DMF was added, 1220 mg of mPEG 24 -NH 2 and 81.0 mg of K 2 CO 3 were added and stirred at room temperature for 16 hours. The reaction was quenched by the addition of 0.2 g of ammonium formic acid. The syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine. The methylene chloride layer was dried over anhydrous sodium sulfate, and concentrated to dryness.
  • the Ixabepilone 430mg bis haloacetate (VI-3) The reaction flask was added 50mL, was added 5mL DMF, was added 1167mg mPEG 24 -NH 2 and 77.6mg K 2 CO 3, stirred at room temperature for 16 hours. The reaction was quenched by the addition of 0.2 g of ammonium formic acid. The syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine. The methylene chloride layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure at 35 ° C.
  • Epothilone B dihaloacetate (VI-1) was added to a 50 mL reaction flask, 5 mL of DMF was added, 447 mg of mPEG 7 -SH and 90.7 mg of K 2 CO 3 were added , and the mixture was stirred at room temperature for 6 hours. The reaction was quenched by the addition of 0.3 g of EtOAc. The syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine. The methylene chloride layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure at 35 ° C.
  • ixabepilone dihaloacetate (VI-3) was added to a 50 mL reaction flask, 5 mL of DMF was added, 427 mg of mPEG 7- SH and 86.6 mg of K 2 CO 3 were added , and the mixture was stirred at room temperature for 6 hours. The reaction was quenched by the addition of 0.25 g of EtOAc. The syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine. The methylene chloride layer was dried over anhydrous sodium sulfate, and concentrated to dryness.
  • Epothilone B monohaloacetate (V) (1 eq) and mPEG n -NH 2 or mPEG n -SH (m is methyl abbreviation) (1.2 eq) were dissolved in 10 volumes of DMF and 0.55 eq of K 2 was added. CO 3 , stir at room temperature for 3-16 hours. The reaction was quenched by the addition of 4 eq of ammonium formate and stirred at room temperature for 30 min. The reaction solution was concentrated to dryness under reduced pressure at 40 °C.
  • the syrup was dissolved in 20 volumes of dichloromethane, the dichloromethane layer was washed twice with 20 volumes of 10 g/L of formic acid solution, and the dichloromethane layer was washed twice with 20 volumes of saturated brine.
  • the methylene chloride layer was dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure at 35 ° C.
  • Epothilone B monohalogenated acetate V-1
  • 5 mL of DMF was added
  • 50.3 mg of mPEG 3 -NH 2 and 25.7 mg of K 2 CO 3 were added
  • the reaction was quenched by the addition of 0.15 g of EtOAc.
  • the syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine.
  • the methylene chloride layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure at 35 ° C.
  • V-2 ixabepilone monohalogenated acetate
  • 5 mL of DMF was added
  • 145 mg of mPEG 7 -NH 2 and 27 mg of K 2 CO 3 were added
  • the reaction was quenched by the addition of 0.15 g of EtOAc.
  • the syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine.
  • the methylene chloride layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure at 35 ° C.
  • Epothilone B monohalogenated acetate V-1 was added to a 50 mL reaction flask, 5 mL of DMF was added, 304 mg of mPEG 12- NH 2 and 34.5 mg of K 2 CO 3 were added , and the mixture was stirred at room temperature for 15 hours. The reaction was quenched by the addition of 0.18 g of EtOAc. The syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine. The methylene chloride layer was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure at 35 ° C.
  • V-2 ixabepilone monohaloacetate
  • 5 mL of DMF was added
  • 583 mg of mPEG 24 -NH 2 and 34 mg of K 2 CO 3 were added
  • the reaction was quenched by the addition of 0.18 g of EtOAc.
  • the syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine.
  • the methylene chloride layer was dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure at 35 ° C.
  • V-2 ixabepilone monohaloacetate
  • 5 mL of DMF was added to a 50 mL reaction flask
  • 200 mg of mPEG 5 -NH 2 and 35 mg of K 2 CO 3 were added , and the mixture was stirred at room temperature for 6 hours.
  • the reaction was quenched by the addition of 0.18 g of EtOAc.
  • the syrup was dissolved in 10 mL of dichloromethane, and the dichloromethane layer was washed twice with 10 mL of 10 g/L of ammonium formate solution, and the dichloromethane layer was washed twice with 10 mL of brine.
  • the methylene chloride layer was dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure at 35 ° C.
  • the antitumor activity and toxicity of these compounds of the present invention can be confirmed by a standard pharmacological experimental procedure for measuring the growth of the compound of the present invention on human hepatoma cells HepG2, lung cancer cells NCI460, breast cancer cells MCF-7. Inhibition or lethality of human primary hepatocytes.
  • the procedure used and the results obtained were briefly described by taking the HepG2 inhibitory activity test of human hepatoma cells as an example.
  • HepG2 Human hepatoma cells HepG2 grow in the following media:
  • BRL minimum essential medium 500 ml
  • Earle salts 500 ml
  • test procedure is as follows:
  • the compounds of the invention tested were dissolved in 10 microliters of phosphate buffered saline (PBS) and incubated at 37 degrees Celsius for 48 hours.
  • PBS phosphate buffered saline
  • each well of a 96-well plate was labeled with 1 microcurie T Thymidine (New England Nuclear thymidine). 1 microcurie was added to 10 ⁇ l of PBS (on the day of sample collection). The 96-well plate was returned to the incubator for the last 6 hours of incubation.
  • T Thymidine New England Nuclear thymidine
  • the IC 50 in this table is the semi-inhibitory activity concentration of the compound on human hepatoma cells.
  • the solubility of the compound in the present invention is determined by referring to the method for determining the solubility in the Pharmacopoeia of the People's Republic of China (2010 edition). Briefly read as follows:
  • the solubility test results showed that the compounds of the present invention showed significant solubility improvement relative to the positive control Epothilone B (or ixabepilone). Among them, the solubility-reducing effect of the glutathione-modified Epothilone derivative is more pronounced, such as compounds 18, 19, 20, 21.
  • the glutathione-modified derivatives 19, 20, 21 have better growth inhibitory activity on human hepatoma cells than the original compound;
  • the toxicity of 20, 21 to human primary hepatocytes (normal hepatocytes) was reduced by at least 1000 times compared with the original compound; moreover, the water solubility of derivatives 19, 20, 21 was significantly improved.

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Abstract

提供水溶性的通式（I）所示的Epothilone衍生物、制备方法及其中间体，以及它们在制备抑制肿瘤细胞生长的药物中的应用。

Description

水溶性Epothilone衍生物及其制备方法
本申请要求于2016年5月10日提交中国专利局、申请号为201610307285.2、发明名称为“水溶性Epothilone衍生物及其制备方法”以及2016年11月14日提交中国专利局、申请号为201611019538.2、发明名称为“水溶性Epothilone衍生物及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于有机化学和药物化学技术领域，具体涉及水溶性Epothilone B衍生物和其中中间体的制备，以及它们的肿瘤抑制活性。
背景技术
Epothilones是一类大环内酯类化合物，具有广谱抗肿瘤活性，它对乳腺癌、前列腺癌、肺癌和结肠癌等人类肿瘤显示出强效的治疗作用。其抗肿瘤机制是与α,β-微管蛋白异二聚物的β亚单位结合，促进微管蛋白的聚合，抑制它的解聚，使细胞不能进行正常的有丝分裂，进而导致肿瘤细胞的死亡。所以，Epothilones家族化合物抗癌的作用机理与紫杉醇相似，但同时Epothilones家族化合物在许多方面优于紫杉醇。例如，抗癌活性比紫杉醇高；能抑制对紫杉醇过敏或对紫杉醇耐药的肿瘤细胞；对有多种抗药性的癌症有较好的活性；对一些多种药物治疗未果的癌症病人依然显示出治疗效果；不受细胞排毒蛋白的作用，故没有紫杉醇类的抗药性问题。此外，由于化学结构比紫杉醇简单，Epothilones家族化合物具有更好的化学修饰与优化潜力。
在Epothilones家族中，Epothilone B是被研究最多，也被认为在肿瘤治疗中最有前景的化合物之一，但Epothilone B本身结构、性质等多方面的缺陷导致其在临床上的应用受到局限。
(1)Epothilone B有严重的毒副作用，如神经毒性，肝脏毒性等。而作为用于人体治疗的安全性药物，要克服Epothilone B毒副作用方面的缺陷，需要 对Epothilone B进行更多更广的结构优化，以便得到毒副作用更小、治疗窗口更大的目标化合物。
(2)Epothilone B在水中的溶解度极小，用于注射剂需要加入助溶剂，如蓖麻油、乙醇等。这些助溶剂在一定程度上加大了药物在人体中的副作用。所以改善Epothilone B的水溶性是丞待解决的问题之一。
(3)Epothilone B大环内酯结构不稳定，进入体内在人体酯解酶的作用下，大环内酯开环而导致失活。伊沙匹隆(Ixabepilone)便是以内酰胺键代替Epothilone B内酯键的衍生物，解决了Epothilone B体内不稳定这一缺陷，保留了较强的体内抗癌活性。但伊沙匹隆仍然存在毒副作用、水溶性差等缺陷。
综上所述Epothilone B及其衍生物Ixabepilone的缺陷，对Epothilone B、Ixabepilone进行进一步的结构改进，以期得到毒副作用更小、水溶性更好、抗癌活性仍较强的Epothilone衍生物。
由于目前没有有效的Epothilone B快速合成方法，对其结构改造非常不充分。所以，以微生物发酵得到的Epothilone B为先导药物分子进行新药开发，具有极大的潜力。
Figure PCTCN2017080254-appb-000001
Epothilone B、Ixabepilone的结构
PEG是一种水溶性、无毒、生物相容性好的聚合物，经FDA批准可用于对静脉注射或口服药物分子进行修饰。并且已经成功将其同多种蛋白或多肽类药物偶联，改善这些药物的治疗效果。因为PEG能够增大药物分子的体积，从而降低了药物在体内的肾清除率；同时，PEG可以保护药物受到酶的影响而降解，从而改善药物的药代动力学性质。此外，小分子药物同PEG偶联产生的复合物能够起到体内缓释的作用，延长药物在体内的作用时间和改善药物的治疗效果。PEG同小分子药物形成复合物也可以改善小分子药物的水溶性。
多肽是人体一种内源性的物质，由多个氨基酸组成，其具有良好的水溶性 和广泛的生物活性，通过多肽与小分子药物的结合可一方面改进小分子药物的溶解性能，另一方面在多肽的协助下实现体内靶向释放或改善生物活性。谷胱甘肽是一个常见的内源性多肽，它由谷氨酸、半胱氨酸和甘氨酸结合。谷胱甘肽是含有巯基的的三肽，具有抗氧化作用和整合解毒作用。谷胱甘肽也是细胞生长的营养物质，易被细胞摄取，特别易被繁殖较快的肿瘤细胞摄取。利用谷胱甘肽同小分子药物偶联制备的复合物，极可能对快速繁殖的肿瘤细胞具有一定的选择性，同时在一定程度上可以降低抗肿瘤药物对人体正常细胞的毒性。而且，谷胱甘肽是一种水溶性三肽，与小分药物形成复合物，在很大程度上可以改善小分子药物的水溶性。
基于以上理念，本发明提供Epothilone B和Ixabepilone的水溶性衍生物的制备方法，得到了的药效优异、毒副作用更小、水溶性更好的目标化合物。
发明内容
为了克服现有技术的不足，本发明提供一种毒副作用小、水溶性好、抗癌活性较强的水溶性Epothilone衍生物，结构如式Ⅰ所示：
Figure PCTCN2017080254-appb-000002
其中，
X为O或者NH；
R1和R2各自独立地为H或COCH2-R3，但R1和R2不可同时为H；
R3为S-(CH2CH2O)nR4,NH-(CH2CH2O)nR4或者多肽基，其中，多肽优选谷胱甘肽；
R4为氢或烷基，优选甲基；
n大于或等于1，优选n大于或等于1，并且n小于或等于24，更优选n为3、5、7、12或24。
本发明的优选方案，提供式II所示的Epothilone衍生物：
Figure PCTCN2017080254-appb-000003
其中，X和R3如上所述。
本发明的另一个优选方案，提供式III所示的Epothilone衍生物：
Figure PCTCN2017080254-appb-000004
其中，X和R3如上所述。
具体地说，本发明所述式Ⅰ化合物选自：
Figure PCTCN2017080254-appb-000005
Figure PCTCN2017080254-appb-000006
Figure PCTCN2017080254-appb-000007
本发明的另一个目的是提供了制备式(I)化合物所使用的中间体式IV化合物：
Figure PCTCN2017080254-appb-000008
其中，
X为O或者NH；
R5和R6各自独立地为H或COCH2-X2，但R5和R6不可同时为H；
X2为卤素原子，优选Br和I。
本发明的优选方案，提供式V所示的中间体：
Figure PCTCN2017080254-appb-000009
其中，X，X2如上所述。
本发明的另一个优选方案，提供式VI所示的中间体：
Figure PCTCN2017080254-appb-000010
其中，X，X2如上所述。
本发明进一步提供了水溶性Epothilone衍生物的制备方法，但不仅限于本发明中所提供的具体事例。它涵盖Epothilones家族其他化合物的NH-PEG，S-PEG，谷胱甘肽化以及多肽化的水溶性衍生物的制备。这些衍生物的制备方法和步骤如下：
a)以Epothilone B或伊沙匹隆为起始物料，卤代乙酸为连接基，制备得到式IV所示的化合物：
Figure PCTCN2017080254-appb-000011
b)将步骤a)制备得到的式IV化合物和带有-NH2或-SH基团的PEG衍生物或多肽反应，得到式I所示的化合物：
Figure PCTCN2017080254-appb-000012
其中，
X为O或者NH；
R1和R2各自独立的是H或COCH2-R3，但R1和R2不可同时为H；
R3为S-(CH2CH2O)nR4，NH-(CH2CH2O)nR4或者多肽基；
R4独立的是氢或者烷基；
R5和R6各自独立地为H或COCH2-X2，但R5和R6不可同时为H；
X2为卤素原子，优选Br和I。
n大于或等于1，优选n大于或等于1，并且n小于或等于24，更优选n为3、5、7、12或24。
其中，式IV所示的化合物为式V或式VI所示的化合物，
Figure PCTCN2017080254-appb-000013
式I所示的化合物为式II或式III所示的化合物，
Figure PCTCN2017080254-appb-000014
其中，X，X2和R3如上述所定义。
本发明提供了式I、II、III所示的水溶性Epothilone衍生物在制备抑制肿瘤细胞生长的药物中的应用。
本发明进一步提供了式I、II、III所示的水溶性Epothilone衍生物在制备抑制人体肝癌细胞的药物中的应用。
更近一步，本发明提供一种药物组合物，所述的药物组合物含有有效剂量的式I、式II、式III所示的Epothilone衍生物或其药学上可接受的盐，及可药用的载体、赋形剂或它们的组合。
如无特别定义，本发明中所使用的术语具有本领域普遍所接受的含义，进一步地，本发明所使用的部分术语定义如下：
“烷基”当作一基团或一基团的一部分是指直链或者带有支链的饱和脂肪烃基团。优选为C1-C10烷基，更优选为C1-C6烷基。烷基基团的实施例包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。
“多肽基”当作一基团或一基团的一部分是指多肽或者蛋白基团。优先为含巯基的多肽或蛋白在巯基处脱H形成的多肽基。多肽基的实施例包括但不 限于谷胱甘肽基。
“PEG衍生物”是指经过修饰后的聚乙二醇衍生物。优先为经-NH2,-SH，COOH修饰后的聚乙二醇衍生物。
“药学上可接受的盐”是指上述化合物能保持原有生物活性并且适合于医药用途的某些盐类。式I化合物药学上可接受的盐可以为与合适的酸形成的盐，合适的酸包括无机酸和有机酸，例如乙酸、苯磺酸、苯甲酸、樟脑磺酸、柠檬酸、乙磺酸、富马酸、葡糖酸、谷氨酸、氢溴酸、盐酸、羟乙磺酸、乳酸、苹果酸、马来酸、扁桃酸、甲磺酸、硝酸、磷酸、琥珀酸、硫酸、酒石酸、对甲苯磺酸等。
具体实施方式
下面具体实施例可以使本专业技术人员全面理解本发明，但不以任何方式限制本发明。
在下列实例中，除非另有指明，所有温度为摄氏温度，除非另有指明，各种起始原料和试剂均来自市售。市售原料和试剂均不经进一步纯化直接使用，除非另有指明。
下面的实例仅仅是用来说明所发明的具体化合物的合成方法。但在合成方法上并没有任何限制。在下面未列出的化合物，也可以用与下面同样的合成路线与合成方法，选择适当的起始原材料、在有必要的地方稍加适当的常识性的反应条件调整即可加以制备。
实施例1
Epothilone B单卤代乙酸酯(V-1)和Epothilone B双卤代乙酸酯(VI-1)的制备
Figure PCTCN2017080254-appb-000015
将Epothilone B(4.0g,7.9mmol)和碘乙酸(1.2g，6.5mmol)溶于20mL二氯甲烷中，搅拌至固体溶清。混合液冷却至0-5℃，加入DCC(1.55g，7.5mmol)，0-5℃搅拌10-15分钟，向反应液中加入DMAP(0.78g，6.4mmol)。反应液升温至室温，搅拌1.5-2小时。再次将反应液冷却至0-5℃，加入碘乙酸(1.8g，9.7mmol)和DCC(1.97g，9.5mmol)，0-5℃保持搅拌10-15分钟后，将反应液升温至室温，搅拌直至Epothilone B反应完毕。布氏漏斗抽滤不溶性固体，滤液减压浓缩至干，所得浆状物用硅胶柱分离纯化，分别得到化合物V-1(1.05g,1.55mmol)和化合物VI-1(2.8g,3.3mmol)。
化合物V-1:
Figure PCTCN2017080254-appb-000016
1H-NMR(400MHz,CDCl3),δ7.01(s,1H),6.67(s,1H),5.47(s,1H),5.34(m,1H),3.72(m,2H),3.51(m,2H),2.85(t,1H),2.74(s,3H),2.55(m,2H),2.14(d,3H),1.94(m,4H),1.72(m,4H),1.60(m,5H),1.50(s,4H),1.31(m,4H),1.12(m,3H),0.97(d,3H),0.88(t,1H).ESI-MS:[M+H]+675,[M+Na]+697,C29H42INO7S。
化合物VI-1:
Figure PCTCN2017080254-appb-000017
1H-NMR(400MHz,CDCl3),δ7.04(s,1H),6.54(s,1H),5.67(t,1H),5.51(m,1H),5.21(m,1H),3.74(m,2H),3.62(d,1H),3.46(m,2H),2.81(m,1H),2.73(s,4H),2.72(s,1H),2.19(m,1H),2.09(s,3H),1.87(m,4H),1.48(s,3H),1.39(m,1H),1.28(m,7H),1.14(d,4H),1.08(m,6H).ESI-MS:[M+H]+843,[M+Na]+865,C31H43I2NO8S.
实施例2
将Epothilone B(4.0g,7.9mmol)和溴乙酸(2.4g，17.3mmol)溶于20mL二氯甲烷中，搅拌至固体溶清。混合液冷却至0-5℃，加入DCC(1.55g，7.5mmol)，0-5℃搅拌10-15分钟，向反应液中加入DMAP(0.78g，6.4mmol)。反应液升温至室温，搅拌12小时。布氏漏斗抽滤不溶性固体，滤液减压浓缩至干，所得浆状物用硅胶柱分离纯化，得到化合物VI-2(5.09g,6.8mmol)。
化合物VI-2:
Figure PCTCN2017080254-appb-000018
1H-NMR(400MHz,CDCl3),δ7.30(s,1H),6.56(s,1H),5.70(m,1H),5.50(m,1H),5.25(m,1H),4.86(s,1H),4.04(t,2H),3.99(d,1H),3.95(s,1H),3.83(d,1H),3.57(t,1H),2.89(m,1H),2.80(t,2H),2.71(s,3H),2.20(m,1H),2.07(d,2H),1.91(m,2H),1.74(t,3H),1.57(m,3H),1.49(m,2H),1.14(d,4H),1.07(m,7H),0.92(t,1H).13C-NMR(400MHZ,CDCl3),δ212.8,168.9,166.9,165.7,151.5,137.4,121.0,117.2,62.0,61.9,53.1,48.2,48.0,47.4,42.3,36.4,33.4,32.8,32.6,31.3,29.8,25.4,24.9,24.6,22.3,21.4,21.1,17.4,16.5,13.3,13.0.ESI-MS:[M+H]+750,[M+Na]+772.C31H43Br2NO8S.
实施例3
将伊沙匹隆(6.0g,11.9mmol)和碘乙酸(1.8g，9.8mmol)溶于30mL二氯甲烷中，搅拌至固体溶清。混合液冷却至0-5℃，加入DCC(2.3g，11.3mmol),0-5℃搅拌10-15分钟，向反应液中加入DMAP(1.2g，9.6mmol)。反应液升温至室温，搅拌1.5-2小时。再次将反应液冷却至0-5℃，加入碘乙酸(2.7g，14.6mmol)和DCC(4.5g，14.3mmol)，0-5℃保持搅拌10-15分钟后，将反应液升温至室温，搅拌直至伊沙匹隆反应完毕。布氏漏斗抽滤不溶性固体，滤液减压浓缩至干，所得浆状物用硅胶柱分离纯化，分别得到化合物V-2(1.35g,2.0mmol)和化合物VI-3(4.0g,4.8mmol)。
化合物V-2：
Figure PCTCN2017080254-appb-000019
1H-NMR(400MHz,CDCl3),δ6.97(s,1H),6.86(d,1H),6.54(s,1H),4.67(t,1H),4.25(s,1H),4.13(s,1H),4.02(s,1H),3.84(s,1H),3.75(s,2H),3.33-3.38(m,1H),2.83(t,1H),2.71(s,3H),2.68(d,1H),2.40-2.46(m,1H),2.35(d,1H),2.13(s,3H),1.94-1.98(m,2H),1.56-1.64(m,4H),1.37-1.42(m,6H),1.34(s,3H),1.28(d,3H),1.19(s,3H),0.99(d,3H).ESI-MS:[M+H]+674,C29H43IN2O6S。
化合物VI-3：
Figure PCTCN2017080254-appb-000020
1H-NMR(400MHz,CDCl3),δ6.97(s,1H),6.86(d,1H),6.54(s,1H),4.67(t,1H),4.25(s,1H),4.13(s,1H),4.02(s,1H),3.84(s,1H),3.74(s,2H),3.49(s,2H),3.33-3.38(m,1H),2.83(t,1H),2.71(s,3H),2.68(d,1H),2.40-2.46(m,1H),2.35(d,1H),2.13(s,3H),1.94-1.98(m,2H),1.56-1.64(m,4H),1.37-1.42(m,6H),1.34(s,3H),1.28(d,3H),1.19(s,3H),0.99(d,3H).ESI-MS:[M+H]+842, C31H44I2N2O7S.
PEG-Epothilone B的制备
Epothilone B双卤代乙酸酯(VI)(1eq)和mPEGn-NH2或mPEGn-SH(m为甲基缩写)(2.1eq)溶于10体积的DMF中，加入1.1eq的K2CO3，室温搅拌3-16小时。加入8eq的甲酸铵淬灭反应，室温搅拌30分钟。反应液40℃减压浓缩至干。浆状物用20体积二氯甲烷溶解，用20体积10g/L甲酸胺溶液分两次洗涤二氯甲烷层，20体积饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到式III化合物。
Figure PCTCN2017080254-appb-000021
实施例4
将500mg Epothilone B双卤代乙酸酯(VI-1)加入50mL反应瓶中，加入5mL DMF，加入197mg mPEG3-NH2和87.8mg K2CO3，室温搅拌3小时。加入0.3g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物1(409mg)。
化合物1：
Figure PCTCN2017080254-appb-000022
1H-NMR(400MHz,CDCl3),δ7.60(s,1H),7.18(s,1H),6.55(s,1H),5.71(t,1H),5.47(m,1H),5.29(t,1H),4.61(s,5H),3.66(m,6H),3.62(d,4H),3.58(m,5H),3.40(s,3H),3.38(d,7H),3.35(s,2H),2.85(m,3H),2.75(m,2H),2.72(s,1H),2.18(d,1H),2.07(s,3H),1.88(m,2H),1.73(t,1H),1.68(m,2H),1.53(d,3H),1.43(m,4H),1.30(s,4H),1.27(s,3H),1.11(d,4H),1.08(s,3H),1.05(d,4H),0.91(m,4H).13C-NMR(400MHz,CDCl3),δ213.2,172.0,171.4,169.1,151.4,137.0,121.3,117.3,78.3,70.3,70.2,70.1,70.0,69.9,69.7,62.1,62.0,58.5,53.1,50.0,49.8,48.8,48.6,48.4,48.3,48.2,48.0,47.7,42.1,36.9,34.9,33.6,33.0,32.7,31.8,30.2,29.5,25.5,24.8,22.3,21.9,18.4,17.1,14.1,14.0.ESI-MS:[M+H]+914,[M+Na]+936,C45H75N3O14S.
实施例5
将500mg Epothilone B双卤代乙酸酯(VI-1)加入50mL反应瓶中，加入5mL DMF，加入423mg mPEG7-NH2和87.8mg K2CO3，室温搅拌3小时。加入0.3g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物2(790mg)。
化合物2：
Figure PCTCN2017080254-appb-000023
1H-NMR(400MHz,CDCl3),δ7.34(s,1H),6.55(s,1H),5.76(m,1H),5.51(m,1H),5.28(t,1H),3.55(m,61H),3.37(t,7H),2.86(m,5H),2.72(s,3H),2.65(m,2H),2.15(m,1H),2.08(d,4H),1.91(m,1H),1.73(m,2H),1.54(t,2H),1.43(t,2H),1.27(m,8H),1.12(d,3H),1.07(d,6H).13C-NMR(400MHz,CDCl3),δ213.1,172.0,171.4,169.1,165.7,151.7,137.3,120.8,117.4,78.2,76.6,70.0,69.8,69.8,61.9,61.8,57.8,53.1,49.7,49.7,48.3,48.1,48.0,48.0,47.9,47.7,47.5,47.3,47.1,36.8,34.8,33.0,32.7,30.1,22.1,21.8,21.6,21.3,17.6,17.5,16.5,13.9,13.5,13.4.ESI-MS:[M+H]+1266,[M+Na]+1288,C61H107N3O22S.
实施例6
将408mg伊沙匹隆双卤代乙酸酯(VI-3)加入50mL反应瓶中，加入5mL DMF，加入344mg mPEG7-NH2和67.0mg K2CO3，室温搅拌4小时。加入0.2g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物3(586mg)。
化合物3：
Figure PCTCN2017080254-appb-000024
1H-NMR(400MHz,CDCl3),δ6.97(s,1H),6.86(d,1H),6.54(s,1H),5.52(m,1H),5.21(t,1H),3.56(m,61H),3.39(t,7H),2.86(m,5H),2.72(s,3H),2.60(m,2H),2.13(m,1H),2.08(d,4H),1.91(m,1H),1.73(m,2H),1.54(t,2H),1.43(t,2H),1.26(m,8H),1.12(d,3H),1.07(d,6H).13C-NMR(400MHz,CDCl3),δ220.7,172.0,171.4,170.4,164.8,152.3,137.5,119.2,116.1,78.2,76.6,70.0,69.8,69.8,61.9,61.8,57.8,53.1,49.7,49.7,48.3,48.1,48.0,48.0,47.9,47.7,47.5,47.3,47.1,36.8,34.8,33.0,32.7,30.7,22.6,21.9,21.6,21.5,19.6,17.5,16.9,13.9,13.5,13.4.ESI-MS:[M+H]+1265,C61H108N4O21S.
实施例7
将412mg Epothilone B双卤代乙酸酯(VI-1)加入50mL反应瓶中，加入5mL DMF，加入574mg mPEG12-NH2和74.5mg K2CO3，室温搅拌15小时。加入0.2g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物4(723mg)。
化合物4：
Figure PCTCN2017080254-appb-000025
1H-NMR(400MHz,CDCl3),δ6.69(s,1H),6.41(s,1H),5.38(t,1H),5.36(t,1H),5.15(t,1H),3.56(m,77H),3.44(m,10H),3.26(m,14H),2.74(m,3H),2.60(s,6H),2.52(m,1H),2.06(m,1H),1.99(s,3H),1.76(m,1H),1.62(m,1H),1.53(s,1H),1.34(m,5H),1.17(s,4H),1.07(s,4H),0.96(m,10H),0.79(m,1H).13C-NMR(400MHz,CDCl3),δ212.6,171.7,171.5,168.8,164.9,152.0,136.6,121.5,117.4,78.3,77.0,71.8,70.6,70.5,70.4,70.2,70.2,61.8,61.7,58.9,53.2,50.3,50.3,48.5,41.9,37.1,34.7,33.2,32.7,30.3,22.8,22.2,19.2,18.2,17.3,14.3,14.0.ESI-MS:[M+H]+1707,[M+Na]+1729,C81H147N3O32S.
实施例8
将450mg Epothilone B双卤代乙酸酯(VI-1)加入50mL反应瓶中，加入5mL DMF，加入1220mg mPEG24-NH2和81.0mg K2CO3，室温搅拌16小时。加入0.2g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物5(1350mg)。
化合物5：
Figure PCTCN2017080254-appb-000026
1H-NMR(400MHz,CDCl3),δ6.97(s,1H),6.86(d,1H),6.54(s,1H),5.48(s,1H),5.28(t,1H),3.66(m,170H),3.52(d,9H),2.83(m,6H),2.72(s,4H),2.65(m,2H),2.18(m,2H),2.09(s,4H),1.90(m,2H),1.72(m,3H),1.56(m,3H),1.44(m,3H),1.29(t,15H),1.09(m,13H),0.89(m,3H).13C-NMR(400MHz,CDCl3),δ220.7,171.9,171.4,170.4,164.8,152.3,137.5,119.2,116.1,78.2,76.7,71.6,71.1,69.6,61.9,61.8,57.8,53.0,49.6,49.6,48.3,48.1,47.9,47.7,47.5,47.3,47,0,42.0,36.7,34.9,33.0,32.7,31.7,30.0,29.4,22.2,21.8,21.3,17.7,17.6,16.6,13.5,13.4,13.2.ESI-MS:[M+H]+2763,[M+Na]+2785,C129H243N3O56S.
实施例9
将430mg伊沙匹隆双卤代乙酸酯(VI-3)加入50mL反应瓶中，加入5mL DMF，加入1167mg mPEG24-NH2和77.6mg K2CO3，室温搅拌16小时。加入0.2g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物6(1180mg)。
化合物6：
Figure PCTCN2017080254-appb-000027
1H-NMR(400MHz,CDCl3),δ7.34(s,1H),6.55(s,1H),5.79(d,1H),5.49(s,1H),5.28(t,1H),3.65(m,170H),3.56(d,9H),2.84(m,6H),2.72(s,4H),2.66(m, 2H),2.19(m,2H),2.10(s,4H),1.91(m,2H),1.70(m,3H),1.56(m,3H),1.44(m,3H),1.29(t,15H),1.10(m,13H),0.90(m,3H).13C-NMR(400MHz,CDCl3),δ213.1,171.9,171.4,169.0,165.7,151.7,137.3,120.8,117.5,78.8,76.8,71.6,71.2,69.5,61.7,61.8,57.7,53.0,49.6,49.3,48.5,48.1,47.9,47.7,47.5,47.3,47,0,42.0,36.7,34.9,33.0,32.7,31.7,30.0,29.4,22.8,21.9,21.5,17.9,17.6,16.6,13.5,13.8,13.6.ESI-MS:[M+H]+2762,[M+Na]+2784,C129H244N4O55S.
实施例10
将503mg Epothilone B双卤代乙酸酯(VI-1)加入50mL反应瓶中，加入5mL DMF，加入447mg mPEG7-SH和90.7mg K2CO3，室温搅拌6小时。加入0.3g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物7(720mg)。
化合物7：
Figure PCTCN2017080254-appb-000028
1H-NMR(400MHz,CDCl3),δ6.99(s,1H),6.44(s,1H),5.60(t,1H),5.40(d,1H),5.14(t,1H),3.63(t,3H),3.46(t,5H),3.39(t,2H),3.27(d,9H),3.14(d,1H),3.00(d,1H),2.77(t,2H),2.70(m,9H),2.09(d,2H),1.98(d,4H),1.78(m,1H),1.66(t,1H),1.58(d,1H),1.36(m,5H),1.18(t,11H),1.04(d,5H),0.98(t,7H),0.80(m,2H).13C-NMR(400MHz,CDCl3),δ212.0,169.9,169.5,168.5,164.6,151.9,136.5,121.6,117.3,78.1,70.9,70.4,70.3,70.2,70.1,61.5,58.8,53.6,53.2,42.0,36.9,34.8,33.7,33.5,33.1,32.6,31.8,31.8,31.7,30.7,30.1,29.5,22.5,22.4,22.3,22.1,19.1,18.1,17.3,14.3,14.0.ESI-MS:[M+H]+1301,[M+Na]+1323,C61H105NO22S3.
实施例11
将480mg伊沙匹隆双卤代乙酸酯(VI-3)加入50mL反应瓶中，加入5mL DMF，加入427mg mPEG7-SH和86.6mg K2CO3，室温搅拌6小时。加入0.25g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物8(635mg)。
化合物8：
Figure PCTCN2017080254-appb-000029
1H-NMR(400MHz,CDCl3),δ6.97(s,1H),6.86(d,1H),6.54(s,1H),5.42(d,1H),5.18(t,1H),3.64(t,3H),3.45(t,5H),3.39(t,2H),3.26(d,9H),3.15(d,1H),3.08(d,1H),2.77(t,2H),2.72(m,9H),2.09(d,2H),1.97(d,4H),1.77(m,1H),1.65(t,1H),1.56(d,1H),1.32(m,5H),1.17(t,11H),1.03(d,5H),0.98(t,7H),0.80(m,2H).13C-NMR(400MHz,CDCl3),δ220.7,170.4,169.9,169.5,164.8,152.3,137.5,119.2,116.1,78.3,70.8,70.5,70.2,70.1,70.0,61.6,58.9,53.4,53.2,42.3,36.8,34.8,33.6,33.4,33.1,32.6,31.8,31.7,31.6,30.6,30.1,29.8,22.8,22.5,22.3,22.1,19.1,18.1,17.3,14.3,14.2.ESI-MS:[M+H]+1300,[M+Na]+1322,C61H106N2O21S3。
Epothilone B单卤代乙酸酯(V)(1eq)和mPEGn-NH2或mPEGn-SH(m为甲基缩写)(1.2eq)溶于10体积的DMF中，加入0.55eq的K2CO3，室温搅拌3-16小时。加入4eq的甲酸铵淬灭反应，室温搅拌30分钟。反应液40℃减压浓缩至干。浆状物用20体积二氯甲烷溶解，用20体积10g/L甲酸胺溶液分两次洗涤二氯甲烷层，20体积饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到式II化合物。
Figure PCTCN2017080254-appb-000030
实施例12
将206mg Epothilone B单卤代乙酸酯(V-1)加入50mL反应瓶中，加入5mL DMF，加入50.3mg mPEG3-NH2和25.7mg K2CO3，室温搅拌3小时。加入0.15g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物9(159mg)。
化合物9：
Figure PCTCN2017080254-appb-000031
1H-NMR(400MHz,CDCl3),δ7.15(s,1H),6.62(s,1H),5.45(d,1H),5.31(m,1H),4.27(t,1H),3.67(s,6H),3.48(m,4H),3.40(s,3H),2.90(m,1H),2.83(s,2H),2.71(s,3H)2.54(d,2H),2.14(m,1H),2.08(s,3H),1.99(m,1H),1.85(m,1H),1.69(m,3H),1.57(t,3H),1.11(m,5H),1.08(s,4H),0.97(d,4H),0.91(m,2H).13C-NMR(400MHz,CDCl3),δ216.6,170.7,165.6,151.6,137.8,119.4,116.4,78.3,76.7,69.5,62.0,61.7,58.4,53.6,48.8,48.6,48.4,48.0,47.8,47.6,42.6,39.0,34.9,33.5,32.4,31.7,29.8,29.5,25.5,24.8,22.5,21.9,21.4,19.0,18.1,14.9.ESI-MS:[M+H]+711,[M+Na]+733,C36H58N2O10S.
实施例13
将248mg Epothilone B单卤代乙酸酯(V-1)加入50mL反应瓶中，加入5mL DMF，加入110.8mg mPEG5-NH2和27.9mg K2CO3，室温搅拌5小时。加入0.15g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和 食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物10(236mg)。
化合物10：
Figure PCTCN2017080254-appb-000032
1H-NMR(400MHz,CDCl3),δ7.10(s,1H),6.62(s,1H),5.44(d,1H),5.32(d,1H),4.24(m,1H),3.63(m,14H),3.55(m,2H),3.49(s,2H),3.45(t,1H),3.37(d,4H),2.89(m,1H),2.82(t,2H),2.70(s,3H),2.53(m,2H),2.89(m,1H),2.82(t,7H),2.70(s,3H),2.53(m,2H),2.12(m,1H),2.07(m,1H),1.97(m,1H),1.67(m,2H),1.54(d,2H),1.44(m,1H),1.34(s,3H),1.24(m,8H),1.10(d,4H),1.04(s,3H),0.95(d,3H),0.89(m,1H).13C-NMR(400MHz,CDCl3),δ216.6,172.1,170.7,165.5,151.6,137.7,119.3,116.4,78.5,70.2,69.9,69.6,61.9,58.6,53.5,50.0,49.4,48.7,48.4,48.3,48.1,47.9,42.7,39.0,34.9,33.6,32.4,31.6,29.8,29.5,25.6,24.9,22.2,21.5,19.3,18.4,17.3,15.3,15.1.ESI-MS:[M+H]+799,[M+Na]+821,C40H66N2O12S.
实施例14
将265mg Epothilone B单卤代乙酸酯(V-1)加入50mL反应瓶中，加入5mL DMF，加入160mg mPEG7-NH2和29.8mg K2CO3，室温搅拌6小时。加入0.15g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物11(260mg)。
化合物11：
Figure PCTCN2017080254-appb-000033
1H-NMR(400MHz,CDCl3),δ6.92(s,1H),6.56(s,1H),5.39(s,1H),5.30(d, 1H),4.07(m,1H),3.50(t,3H),3.41(s,3H),3.36(s,5H),3.30(s,4H),2.63(s,4H),2.42(m,3H),2.02(s,4H),1.90(m,4H),1.61(d,3H),1.49(t,3H),1.38(m,2H),1.29(s,4H),1.19(d,10H),1.08(t,3H),1.00(s,8H),0.85(d,4H),0.80(m,2H).13C-NMR(400MHz,CDCl3),δ216.5,171.9,170.5,165.0,152.0,136.8,119.6,116.3,73.0,71.8,70.5,70.4,70.2,61.2,60.9,59.0,52.7,50.6,49.4,48.8,48.7,43.3,38.7,34.7,33.9,31.8,31.2,30.6,29.6,29.5,29.3,25.6,24.9,22.9,22.8,22.6,21.0,20.7,19.0,17.7,17.6,15.8,15.4,14.1.ESI-MS:[M+H]+888,[M+Na]+910,C44H74N2O14S.
实施例15
将240mg伊沙匹隆单卤代乙酸酯(V-2)加入50mL反应瓶中，加入5mL DMF，加入145mg mPEG7-NH2和27mg K2CO3，室温搅拌6小时。加入0.15g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物12(230mg)。
化合物12：
Figure PCTCN2017080254-appb-000034
1H-NMR(400MHz,CDCl3),δ6.97(s,1H),6.86(d,1H),6.54(s,1H),4.67(t,1H),4.08(m,1H),3.52(t,3H),3.46(s,3H),3.38(s,5H),3.35(s,4H),2.68(s,4H),2.44(m,3H),2.06(s,4H),1.91(m,4H),1.60(d,3H),1.47(t,3H),1.36(m,2H),1.22(s,4H),1.16(d,10H),1.05(t,3H),0.98(s,8H),0.85(d,4H),0.80(m,2H).13C-NMR(400MHz,CDCl3),δ220.7,171.9,170.4,164.8,152.3,137.5,119.2,116.1,73.1,71.6,70.5,70.4,70.2,61.5,60.8,59.2,52.8,50.6,49.5,48.9,48.5,43.6,38.8,34.5,33.6,31.7,31.2,30.6,29.7,29.5,29.3,25.68,24.9,22.6,22.8,22.6,21.0,20.7,19.0,17.8,17.6,15.8,15.3,14.0.ESI-MS:[M+H]+887,[M+Na]+909,C44H75N3O13S.
实施例16
将305mg Epothilone B单卤代乙酸酯(V-1)加入50mL反应瓶中，加入5mL DMF，加入304mg mPEG12-NH2和34.5mg K2CO3，室温搅拌15小时。加入0.18g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物13(486mg)。
化合物13：
Figure PCTCN2017080254-appb-000035
1H-NMR(400MHz,CDCl3),δ7.04(s,1H),6.53(s,1H),5.28(m,2H),4.49(s,5H),4.16(d,1H),3.57(s,27H),3.42(s,6H),3.25(m,9H),2.78(d,3H),2.62(s,4H),2.44(s,2H),2.02(t,1H),1.89(m,3H),1.61(s,3H),1.47(s,3H),1.22(d,11H),0.95(m,12H).13C-NMR(400MHz,CDCl3),δ215.7,170.9,170.0,164.8,150.9,137.0,118.6,115.8,76.7,75.9,70.9,70.8,69.4,69.3,69.2,68.7,61.3,60.9,57.8,52.9,49.0,48.5,48.3,48.1,47.9,47.7,47.5,47.4,47.2,47.0,41.9,38.3,34.2,32.9,31.7,31.0,29.1,24.8,24.1,21.8,18.4,17.6,16.5,14.4,14.2.ESI-MS:[M+H]+1107,[M+Na]+1129,C54H94N2O19S.
实施例17
将310mg Epothilone B单卤代乙酸酯(V-1)加入50mL反应瓶中，加入5mL DMF，加入600mg mPEG24-NH2和35mg K2CO3，室温搅拌16小时。加入0.2g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物14(788mg)。
化合物14：
Figure PCTCN2017080254-appb-000036
1H-NMR(400MHz,CDCl3),δ6.94(s,1H),6.54(s,1H),5.40(s,1H),5.27(s,4H),4.05(s,1H),3.52(m,41H),3.29(s,8H),2.78(s,5H),2.63(s,6H),2.46(m,4H),2.05(s,6H),1.94(d,4H),1.62(s,4H),1.48(s,4H),1.48(m,3H),1.28(s,6H),1.19(d,20H),0.86(s,14H),0.80(d,9H).13C-NMR(400MHz,CDCl3),δ216.4,170.4,164.7,152.0,136.5,119.6,116.4,78.7,75.9,73.2,71.8,70.4,70.4,70.2,60.9,60.6,58.9,53.6,52.5,43.3,38.6,34.6,31.8,31.6,31.0,29.5,29.4,22.9,22.8,22.5,21.3,20.4,19.1,17.7,15.7,15.4,14.0.ESI-MS:[M+H]+1636,[M+Na]+1658,C78H142N2O31S.
实施例18
将301mg伊沙匹隆单卤代乙酸酯(V-2)加入50mL反应瓶中，加入5mL DMF，加入583mg mPEG24-NH2和34mg K2CO3，室温搅拌16小时。加入0.18g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物15(670mg)。
化合物15：
Figure PCTCN2017080254-appb-000037
1H-NMR(400MHz,CDCl3),δ6.97(s,1H),6.86(d,1H),6.54(s,1H),5.27(s,4H),4.05(s,1H),3.50(m,41H),3.28(s,8H),2.78(s,5H),2.60(s,6H),2.45(m,4H),2.07(s,6H),1.93(d,4H),1.60(s,4H),1.48(s,4H),1.47(m,3H),1.25(s,6H),1.19(d,20H),0.89(s,14H),0.80(d,9H).13C-NMR(400MHz,CDCl3),δ220.7,170.4,164.8,152.3,137.5,119.2,116.1,78.8,75.7,73.3,71.8,70.5,70.4,70.2,60.8,60.6,58.9,53.5,52.5,43.8,38.9,34.5,31.8,31.6,31.3,29.5,29.4,22.9, 22.8,22.6,21.3,20.4,19.1,17.8,15.5,15.4,14.1.ESI-MS:[M+H]+1635,[M+Na]+1657,C78H143N3O30S.
实施例19
将305mg Epothilone B单卤代乙酸酯(V-1)加入50mL反应瓶中，加入5mL DMF，加入195mg mPEG5-NH2和34.5mg K2CO3，室温搅拌6小时。加入0.15g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物16(381mg)。
化合物16：
Figure PCTCN2017080254-appb-000038
1H-NMR(400MHz,CDCl3),δ6.93(s,1H),6.55(s,1H),5.40(s,1H),5.32(d,1H),4.06(m,1H),3.63(t,2H),3.57(s,23H),3.47(t,3H),3.40(t,1H),3.36(s,1H),3.30(s,3H),3.24(s,2H),2.78(t,4H),2.69(s,4H),2,43(m,2H),2.03(s,3H),1.92(m,2H),1.61(t,2H),1.49(d,2H),1.39(m,1H),1.29(s,3H),1.02(t,7H),0.87(d,3H).13C-NMR(400MHz,CDCl3),δ216.5,170.5,170.0,164.9,152.0,136.7,119.6,116.3,79.2,76.0,73.2,71.8,70.5,70.5,70.4,70.4,70.2,61.1,60.8,58.9,52.6,50.2,43.5,38.6,32.8,33.9,31.9,31.7,31.1,29.6,29.5,22.9,21.2,20.6,19.1,17.6,15.8,15.4.ESI-MS:[M+H]+905,[M+Na]+927,C44H73NO14S2.
实施例20
将315mg伊沙匹隆单卤代乙酸酯(V-2)加入50mL反应瓶中，加入5mL DMF，加入200mg mPEG5-NH2和35mg K2CO3，室温搅拌6小时。加入0.18g甲酸铵淬灭反应，反应液于40℃减压浓缩至干。浆状物用10mL二氯甲烷溶解，用10mL 10g/L甲酸胺溶液分两次洗涤二氯甲烷层，10mL饱和食盐水分两次洗涤二氯甲烷层。二氯甲烷层用无水硫酸钠干燥，35℃减压浓缩至干，所得浆状物高效制备液相制备得到化合物17(401mg)。
化合物17：
Figure PCTCN2017080254-appb-000039
1H-NMR(400MHz,CDCl3),δ6.97(s,1H),6.86(d,1H),6.54(s,1H),5.32(d,1H),4.08(m,1H),3.65(t,2H),3.56(s,23H),3.48(t,3H),3.41(t,1H),3.35(s,1H),3.30(s,3H),3.22(s,2H),2.76(t,4H),2.68(s,4H),2,45(m,2H),2.01(s,3H),1.90(m,2H),1.62(t,2H),1.51(d,2H),1.38(m,1H),1.27(s,3H),1.02(t,7H),0.89(d,3H).13C-NMR(400MHz,CDCl3),δ220.7,170.4,170.0,164.8,152.3,137.5,119.2,116.1,79.5,76.2,73.5,71.6,70.6,70.5,70.4,70.4,70.2,61.0,60.4,58.8,52.6,50.1,43.8,38.6,32.8,33.7,31.8,31.6,31.1,29.6,29.5,22.9,21.2,20.6,19.1,17.6,15.7,15.4.ESI-MS:[M+H]+904,[M+Na]+926,C44H74N2O13S2.
实施例21
Glutathione-Epothilone的制备
Figure PCTCN2017080254-appb-000040
将化合物V-1(0.51g,0.76mmol)和谷胱甘肽(0.31g,1mmol)加至50mL反应瓶中，加入DMF/EtOH/H2O混合溶液16mL，加入碳酸K2CO3(56.6mg，0.41mmol)，反应混合室温搅拌约24h。加入NaHCO3(21.9mg)淬灭反应。反应液50℃减压浓缩至干，所得浆状物经乙醇打浆的白色固体化合物18(429mg)。
化合物18：
Figure PCTCN2017080254-appb-000041
1H-NMR(400MHz,D2O),δ7.25(s,1H),6.55(s,1H),5.48(s,1H),5.11(s,1H),4.69(m,2H),4.48(m,2H),3.82(m,7H),3.53(d,3H),3.26(m,3H),2.73(s,4H),2.66(d,6H),2.54(m,7H),1.98(s,4H),1.77(s,3H),1.51(s,4H),0.98(d,9H).ESI-MS:[M+H]+855,[M+Na]+877,C39H58N4O13S2.
实施例22
将化合物V-2(0.4g,0.59mmol)和谷胱甘肽(0.22g,0.72mmol)加至50mL反应瓶中，加入DMF/EtOH/H2O混合溶液16mL，加入碳酸K2CO3(45mg，0.33mmol)，反应混合室温搅拌约24h。加入NaHCO3(20mg)淬灭反应。反应液50℃减压浓缩至干，所得浆状物经乙醇打浆的白色固体化合物19(390mg)。
化合物19：
Figure PCTCN2017080254-appb-000042
1H-NMR(400MHz,D2O),δ6.99(s,1H),6.87(d,1H),6.60(s,1H),5.12(s,1H),4.68(m,2H),4.45(m,2H),3.80(m,7H),3.51(d,3H),3.25(m,3H),2.72(s,4H),2.65(d,6H),2.55(m,7H),1.96(s,4H),1.72(s,3H),1.50(s,4H),0.98(d,9H).ESI-MS:[M+H]+854,[M+Na]+876,C39H59N5O12S2.
实施例23
Figure PCTCN2017080254-appb-000043
将化合物VI-1(0.6g,0.71mmol)和谷胱甘肽(0.55g,1.79mmol)加至50mL反应瓶中，加入DMF/EtOH/H2O混合溶液16mL，加入碳酸K2CO3(100.5mg，0.73mmol)，反应混合室温搅拌约24h。加入NaHCO3(303mg)淬灭反应。反应液50℃减压浓缩至干，所得浆状物经乙醇打浆的白色固体化合物20(510mg)。
化合物20：
Figure PCTCN2017080254-appb-000044
1H-NMR(400MHz,D2O),δ7.33(s,1H),6.53(s,1H),5.69(s,1H),5.55(s,1H),5.43(s,2H),5.07(s,1H),4.54(m,3H),3.94(s,6H),3.79(d,3H),3.68(m,4H),3.46(m,2H),3.12(m,8H),2.88(m,3H),2.69(s,4H),2.53(d,6H),2.14(d,7H),1.96(s,4H),1.81(d,3H),1.42(s,5H),0.98(m,10H).ESI-MS:[M+H]+1202,[M+Na]+1224,C51H75N7O20S3.
实施例24
将化合物VI-3(0.5g,0.74mmol)和谷胱甘肽(0.48g,1.56mmol)加至50mL反应瓶中，加入DMF/EtOH/H2O混合溶液16mL，加入碳酸K2CO3(112.7mg，0.82mmol)，反应混合室温搅拌约24h。加入NaHCO3(300mg)淬灭反应。反应液50℃减压浓缩至干，所得浆状物经乙醇打浆的白色固体化合物21(538mg)。
化合物21：
Figure PCTCN2017080254-appb-000045
1H-NMR(400MHz,D2O),δ7.06(s,1H),6.80(s,1H),6.52(s,1H),5.50(s,1H),5.46(s,2H),5.02(s,1H),4.50(m,3H),3.95(s,6H),3.75(d,3H),3.68(m,4H),3.46(m,2H),3.11(m,8H),2.89(m,3H),2.65(s,4H),2.53(d,6H),2.13(d,7H),1.96(s,4H),1.810(d,3H),1.45(s,5H),0.98(m,9H).ESI-MS:[M+H]+1201,[M+Na]+1223,C51H76N8O19S3.
活性及毒性测试
本发明的这些化合物的抗肿瘤活性及毒性，可通过用标准的药理实验操作程序来证实，该实验可测量本发明的化合物对人体肝癌细胞HepG2、肺癌细胞NCI460、乳腺癌细胞MCF-7生长的抑制情况或对人原代肝细胞的致死情况。以人体肝癌细胞HepG2抑制活性测试为例简要地描述所用的操作程序和获得的结果。
人体肝癌细胞HepG2在以下介质中生长：
生长介质：带Earle盐类的BRL最小必需介质(500毫升)，再向该介质中分别加入如下试剂：
加5毫升BRL MEM非必需的氨基酸(10mM)；
加5毫升BRL青霉素-链霉素(10000国际单位/毫升，10000微克/毫升)；
加5毫升BRL丙酮酸钠溶液(100mM)；
加5毫升BRL L-谷氨酰胺(200mM)；
加50毫升BRL小牛血清(合格品)，如上所述制得的生长介质备用。
试验操作程序如下：
1、将细胞胰蛋白酶化并在96孔板上布板，使每个小孔中细胞浓度约为104/孔，并使之在200微升最终体积的介质中生长，布板在37摄氏度条件下允许粘附24小时；
2、小心地通过抽吸除去介质，不扰动单细胞层。在每个孔中加入200微升的新鲜介质，允许有足够多的孔被点样以便进行3个平行的试验。
3、被测试的本发明中的化合物被溶解到10微升磷酸盐缓冲溶液中(PBS)，并在37摄氏度孵育48小时。
4、在孵育最后6小时期间，96孔板的每个小孔用1微居里T胸腺嘧啶脱氧核苷标记(New England Nuclear thymidine)。1微居里被加进10微升PBS中(采集试样测试当天)。96孔板放回孵育器中孵育最后的6小时。
5、在不扰动单细胞层的前提下，通过抽吸除去待放射性的介质。然后往每个小孔中加入50微升BRL 10×胰蛋白酶，并继续在37摄氏度的条件下孵育10分钟或直到单细胞层从孔底(或孔壁)松脱。用Skatron 96孔板采集器把试样采集在玻璃纤维簇上。纤维簇在Wallac Betaptate计数器中计数。
表1 化合物1-21的体外抗肿瘤活性筛选结果
Figure PCTCN2017080254-appb-000046
Figure PCTCN2017080254-appb-000047
注：本表中IC50为化合物对人体肝癌细胞的半抑制活性浓度，IC50值越小，说明化合物对癌细胞的抑制活性越强；本表中LC50为化合物对人原代肝细胞的半致死浓度，即对人体肝细胞的毒性，LC50越大，化合物的毒性越小。
在这种标准的药理试验操作程序中得到的结果(表1)显示，与阳性对照Epothilone B(或伊沙匹隆)相比较，本发明中的这些化合物表现出对人体肝癌细胞HepG2的生长具有抑制作用。同时，与阳性对照Epothilone B(或伊沙匹隆)相比较，本发明中的这些化合物对人体正常肝细胞(原代肝细胞)的毒性明显下降。其中，以谷胱甘肽修饰的Epothilone类化合物19、20、21改善效果最为明显，化合物19、20、21对人体肝癌细胞的抑制活性优于原化合物Epothilone B和伊沙匹隆，但毒性明显降低。这一结果为本发明中的这些化合物在肝癌治疗中可能提供积极的信号。
水溶性测定
本发明中的化合物溶解度测定参照《中华人民共和国药典》(2010年版)凡例中有关溶解度测定的方法。简述如下：
称取样品，加至10mL容量瓶中，并加入水，定容至10mL，在25℃±2℃每隔5分钟强力振摇30秒钟，30分钟观察溶解情况，如看不见溶质颗粒或液滴时，即认为完全溶解。
本发明中的化合物溶解度测定结果仅用于初步判断化合物相对于阳性对照Epothilone B(或伊沙匹隆)溶解度的改善情况。
表2 化合物1-21水溶性试验结果
Figure PCTCN2017080254-appb-000048
Figure PCTCN2017080254-appb-000049
溶解度测试结果显示，相对于阳性对照Epothilone B(或伊沙匹隆)，本发明中的化合物均表现出明显的溶解度改善。其中，谷胱甘肽修饰的Epothilone所得的衍生物水中溶解性改善作用更为明显，如化合物18、19、20、21。
结论：通过对Epothilone B和伊沙匹隆的结构修饰，经谷胱甘肽修饰得到的衍生物19、20、21对人体肝癌细胞的生长抑制活性比原化合物更好；同时，衍生物19、20、21对人体原代肝细胞(正常肝细胞)的毒性相对于原化合物降低了至少1000倍；而且，衍生物19、20、21的水溶性得到了明显的改善。

Claims (13)

  1. 式Ⅰ化合物或其药学上可接受的盐：
    Figure PCTCN2017080254-appb-100001
    其中，
    X为O或者NH；
    R1和R2各自独立的是H或COCH2-R3，但R1和R2不可同时为H；
    R3为S-(CH2CH2O)nR4，NH-(CH2CH2O)nR4或者多肽基；
    R4独立的是氢或者烷基；
    n大于或等于1。
  2. 根据权利要求1所述的化合物，其中n为大于或等于1，并且n小于或等于24，优选n为3、5、7、12或24。
  3. 根据权利要求1所述的化合物，其中，当R1为COCH2-R3且R2为H，即式Ⅱ所示的化合物，或当R1及R2同时为COCH2-R3，即式III所示的化合物：
    Figure PCTCN2017080254-appb-100002
    其中，X和R3的定义如权利要求1所述。
  4. 根据权利要求1-3任一项所述的化合物，其中，所述多肽基为谷胱甘肽基。
  5. 根据权利要求1-3任一项所述的化合物，其中，R4为烷基，优选甲基。
  6. 根据权利要求1所述的化合物，其中所述的式Ⅰ化合物选自：
    Figure PCTCN2017080254-appb-100003
    Figure PCTCN2017080254-appb-100004
  7. 权利要求1-6中任一项所述的化合物的合成方法，其特征在于，
    a)以Epothilone B或伊沙匹隆为起始物料，卤代乙酸为连接基，制备得到式IV所示的化合物：
    Figure PCTCN2017080254-appb-100005
    b)将步骤a)制备得到的式IV化合物和带有-NH2或-SH基团的PEG衍生物或多肽反应，得到式I所示的化合物：
    Figure PCTCN2017080254-appb-100006
    其中，
    X为O或者NH；
    R1和R2各自独立的是H或COCH2-R3，但R1和R2不可同时为H；
    R3为S-(CH2CH2O)nR4，NH-(CH2CH2O)nR4或者多肽基；
    R4独立的是氢或者烷基；
    R5和R6各自独立地为H或COCH2-X2，但R5和R6不可同时为H；
    X2为卤素原子，优选Br和I。
    n大于或等于1，优选n大于或等于1，并且n小于或等于24，更优选n为3、5、7、12或24。
  8. 根据权利要求7所述的制备方法，其中，式IV所示的化合物为式V或式VI所示的化合物，
    Figure PCTCN2017080254-appb-100007
    Figure PCTCN2017080254-appb-100008
    其中，X，X2如权利要求7所定义。
  9. 根据权利要求7所述的制备方法，其中，式I所示的化合物为式II或式III所示的化合物，
    Figure PCTCN2017080254-appb-100009
    其中，X和R3的定义如权利要求7所定义。
  10. 根据权利要求7所述的合成方法，其中，所述多肽基为谷胱甘肽基。
  11. 权利要求1-6中任一项所述的化合物在制备抑制肿瘤细胞生长的药物中的应用，所述肿瘤细胞优选为人体肝癌细胞。
  12. 一种药物组合物，所述的药物组合物含有有效剂量的权利要求1-6中任一项所述的化合物或其药学可接受的盐，及可药用的载体、赋形剂或它们的组合。
  13. 式IV所示的化合物：
    Figure PCTCN2017080254-appb-100010
    其中，
    X为O或者NH；
    R5和R6各自独立地为H或COCH2-X2，但R5和R6不可同时为H；
    X2为卤素原子，优选Br和I；
    或式V所示的化合物：
    Figure PCTCN2017080254-appb-100011
    或式VI所示的化合物：
    Figure PCTCN2017080254-appb-100012
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