WO2015078344A1 - 鬼臼毒素衍生物、其制备方法、药物组合物及应用 - Google Patents

鬼臼毒素衍生物、其制备方法、药物组合物及应用 Download PDF

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WO2015078344A1
WO2015078344A1 PCT/CN2014/092098 CN2014092098W WO2015078344A1 WO 2015078344 A1 WO2015078344 A1 WO 2015078344A1 CN 2014092098 W CN2014092098 W CN 2014092098W WO 2015078344 A1 WO2015078344 A1 WO 2015078344A1
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formula
compound
group
salt
cancer
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PCT/CN2014/092098
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English (en)
French (fr)
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肖旭华
孙亚飞
沈舜义
刘全海
肖璘
樊钱永
张志宏
任岩松
姚利霞
张锴婷
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上海医药工业研究院
中国医药工业研究总院
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Publication of WO2015078344A1 publication Critical patent/WO2015078344A1/zh

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    • 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
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention relates to a podophyllotoxin derivative, a process for its preparation, a pharmaceutical composition and use thereof.
  • Etoposide (VP-16) and teniposide (VM-26) are podophyllotoxin derivatives for the treatment of tumors in clinical practice. Etoposide is often used in combination with cisplatin in the treatment of lung cancer and testis. Cancer, good curative effect, also used for the treatment of lymphoma. Teniposide mainly treats He Jiejin and non-Hodgkin's lymphoma and has obvious effects. It also has therapeutic effects on brain tumors and childhood lymphocytic leukemia. However, the above antitumor drugs have a narrow spectrum of tumor inhibition, accompanied by severe myelosuppression and gastrointestinal side effects.
  • Podophyllotoxin derivatives generally have a good proliferation inhibitory effect on human oral epithelial carcinoma (KB) cells and on mouse lymphocytic leukemia cells (L1210), but not on human lung cancer (non-small cell lung cancer cell line A549) cells.
  • the inhibition of proliferation is very poor. Therefore, there is a need in the art to find a podophyllotoxin derivative with a broad antitumor spectrum to effectively solve the defects of narrow anti-tumor spectrum and large side effects of podophyllotoxin compounds.
  • the technical problem to be solved by the present invention is to solve the problem that the existing podophyllotoxin derivatives have a narrow tumor inhibition spectrum, severe bone marrow suppression and gastrointestinal side effects, etc., and provide a completely different technology from the prior art.
  • the podophyllotoxin derivatives of the invention have good tumor cell inhibitory activity, and some compounds have superior antitumor activity to etoposide, and some compounds have better inhibitory activity against human lung cancer cells (non-small cell lung cancer cell line A549).
  • the present invention provides a podophyllotoxin derivative represented by the following formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof;
  • X is oxygen or nitrogen; R 1 is mono- or poly-substituted, and the substitution position is arbitrary; R 1 is halogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, amino, nitro and One or more of the hydroxyl groups; R 2 is hydrogen or a C 1 -C 4 alkyl group; when X is O and R 1 is monosubstituted, R 1 is not 2-chloro, 6-chloro or 5- Bromine; when X is N and R 1 is monosubstituted, R 1 is not 2-chloro or 6-chloro; when X is N and R 1 is disubstituted, R 1 is not 4-chloro and 6- Methyl; when R 2 is hydrogen, the carbon marked with * is the ⁇ configuration chiral carbon; when R 2 is C 1 - C 4 alkyl, the carbon marked with * is the ⁇ configuration chiral carbon .
  • the halogen is preferably fluorine, chlorine, bromine or iodine.
  • the C 1 -C 4 alkyl group is preferably a methyl group, an ethyl group, a n-propyl group, an isopropyl group or a butyl group.
  • the butyl group is n-butyl, isobutyl or tert-butyl.
  • the C 1 -C 4 alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group or a butoxy group.
  • the propoxy group is preferably n-propoxy or isopropoxy.
  • the butoxy group is preferably n-butoxy, isobutoxy or tert-butoxy.
  • the substituent represented by R 1 is preferably one or more of the 2-position, 5-position and 6-position of the carbon atom of the pyridine ring.
  • R 1 is preferably one of a halogen, a C 1 -C 4 alkyl group, a C 1 -C 4 alkoxy group, an amino group and a hydroxyl group.
  • R 1 is preferably one or more of the 2-position, 5-position and 6-position of the carbon atom of the pyridine ring.
  • R 1 is preferably one or more of a halogen, a C 1 -C 4 alkoxy group and an amino group; a substituent represented by R 1 Preferably one or more of the 2, 5 and 6 positions of the carbon atom of the pyridine ring.
  • R 1 is a halogen and/or an amino group
  • the substituent represented by R 1 is preferably one or more of the 2-position, 5-position and 6-position of the carbon atom of the pyridine ring.
  • R 1 is a C 1 -C 4 alkoxy group
  • the substituent represented by R 1 is preferably at the 6-position of the carbon atom of the pyridine ring.
  • R 1 is preferably a halogen, a C 1 -C 4 alkyl group or a C 1 -C 4 alkoxy group.
  • R 1 is preferably one or more of the 2-position, 5-position and 6-position of the carbon atom of the pyridine ring.
  • R 1 is preferably one or more of a halogen, a C 1 -C 4 alkoxy group and an amino group.
  • R 1 is a halogen and/or an amino group
  • the substituent represented by R 1 is preferably one or more of the 2, 5 and 6 positions of the carbon atom of the pyridine ring; more preferably, the carbon of the pyridine ring 2 bits of the atom.
  • R 1 is a C 1 -C 4 alkoxy group
  • the substituent represented by R 1 is preferably at the 2-position of the carbon atom of the pyridine ring.
  • R 1 is preferably one of a halogen, a C 1 -C 4 alkyl group, a C 1 -C 4 alkoxy group, an amino group and a hydroxyl group.
  • R 1 is preferably one or more of the 2-position, 5-position and 6-position of the carbon atom of the pyridine ring.
  • R 1 is preferably one of halogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy and amino or A variety.
  • R 1 is a C 1 -C 4 alkyl group and/or an amino group
  • the substituent represented by R 1 is preferably one or more of the 2, 5 and 6 positions of the carbon atom of the pyridine ring; Preferably, it is at the 2 and/or 6 position of the carbon atom of the pyridine ring.
  • R 1 is a C 1 -C 4 alkoxy group
  • the substituent represented by R 1 is preferably at the 6-position of the carbon atom of the pyridine ring.
  • R 1 is a halogen
  • R 1 is preferably a monosubstituted group; and the substituent represented by R 1 is preferably at the 5-position of the carbon atom of the pyridine ring.
  • R 1 is preferably an amino group, and the amino group is preferably at the 2-position of the carbon atom of the pyridine ring.
  • the podophyllotoxin derivative of the formula I, a stereoisomer thereof, a tautomer thereof, a pharmaceutically acceptable salt, prodrug or solvate thereof, is preferably any of the following compounds:
  • the present invention also provides the podophyllotoxin derivative represented by the above formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof for use in the treatment of cancer.
  • the application of the drug is preferably lung cancer, oral cancer, liver cancer or leukemia, especially lung cancer.
  • the lung cancer is preferably human non-small cell lung cancer.
  • the oral cancer is preferably human oral epithelial cancer.
  • the tumor cells of the cancer are preferably human non-small cell lung cancer cells, human oral cancer epithelial cells, human liver cancer cells or mouse lymphoid leukemia cells.
  • the human non-small cell lung cancer cell is preferably a human non-small cell lung cancer cell line A549.
  • the human oral cancer cell is preferably a human oral cancer epithelial cell line KB.
  • the human liver cancer cell is preferably a human liver cancer cell line HepG2.
  • the mouse lymphocytic leukemia cell is preferably a mouse lymphocytic leukemia cell line L1210.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the podophyllotoxin derivative of the above formula I, a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a prodrug and a solvate thereof.
  • compositions can be formulated into various types of dosage unit dosage forms, such as: aqueous dispersions, liquids, mashes, syrups, elixirs, syrups, suspensions, aerosols, controlled release.
  • Agent fast solvent, effervescent agent, Lyophilizates, tablets, powders, pills, sugar-coated, capsules, delayed release agents, extended release agents, pulsed release agents, multiparticulates or immediate release agents.
  • the invention also provides a preparation method of the podophyllotoxin derivative represented by the formula I, which comprises the following steps: in an organic solvent, in the presence of a base, under the action of a condensing agent, The compound shown or a salt thereof is reacted with a compound of the formula B as shown below;
  • R is a hydroxyl group (-OH) or an amino group (-NH 2 ); *, R 1 and R 2 are as defined above.
  • the method and conditions of the reaction can be conventional methods and conditions for such reactions in the art.
  • the following methods and conditions are particularly preferred: a compound such as the formula A or a salt thereof is reacted with a compound of the formula B in the presence of a base in the presence of a base under the action of a condensing agent and a catalyst.
  • the salt of the compound of the formula A is a salt of a compound of the formula A and a salt conventionally used in the art.
  • the salt of the compound of formula A is preferably the hydrochloride salt of a compound of formula A.
  • the organic solvent is preferably a halogenated hydrocarbon solvent and/or an amide solvent.
  • the halogenated hydrocarbon solvent is preferably dichloromethane.
  • the amide solvent is preferably N,N-dimethylformamide (DMF).
  • the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-dicyclohexylcarbodiimide (DCC).
  • diethyl cyanophosphate (DEPC) or the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), one or more of N,N'-dicyclohexylcarbodiimide (DCC) and diethyl cyanophosphate (DEPC), with 1-hydroxybenzotriazole (HOBt) mixture.
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • DCC N,N'-dicyclohexylcarbodiimide
  • DEPC diethyl cyanophosphate
  • the mixture is preferably a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt), wherein The molar ratio of -(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) to 1-hydroxybenzotriazole (HOBt) is preferably 1:1.
  • the base is preferably an organic base, and the organic base is preferably triethylamine.
  • the catalyst is preferably 4-dimethylaminopyridine (DMAP).
  • the base is preferably from 1:1 to 3:1 in molar ratio to the compound of the formula A or a salt thereof.
  • the compound of the formula A or a salt thereof, as compared with the compound of the formula B, preferably has a molar ratio of from 1:1 to 1:1.5.
  • the molar ratio of the condensing agent to the compound of the formula A or a salt thereof is preferably from 1:1 to 3:1.
  • the catalyst preferably has a molar ratio of 0.05:1 to 0.1:1 to the compound of the formula A or a salt thereof.
  • the mass ratio of the organic solvent to the compound of the formula A or a salt thereof is preferably from 1 mL/g to 10 mL/g.
  • the temperature of the reaction is preferably from 0 to 30 °C.
  • the progress of the reaction can be carried out by conventional testing methods in the art (such as TLC, Monitoring by HPLC or NMR), the reaction time is preferably from 1 to 24 hours.
  • the compound represented by the formula A is esterified with the compound of the formula B;
  • the organic solvent is preferably a halogenated hydrocarbon solvent;
  • the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and / Or N,N'-dicyclohexylcarbodiimide (DCC); alternatively, the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and/or diethyl cyanophosphate (DEPC), a mixture with 1-hydroxybenzotriazole (HOBt).
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • DEPC diethyl cyanophosphate
  • the mixture is preferably a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt), wherein
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • HOBt 1-hydroxybenzotriazole
  • the molar ratio of -(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) to 1-hydroxybenzotriazole (HOBt) is preferably 1:1.
  • the organic solvent is preferably a halogenated hydrocarbon solvent and/or an amide solvent;
  • the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbamate Imine hydrochloride (EDCI) and/or diethyl cyanophosphate (DEPC), more preferably diethyl cyanophosphate (DEPC); alternatively, the condensing agent is preferably 1-(3-di) Mixture of methylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and/or diethyl cyanophosphate (DEPC) with 1-hydroxybenzotriazole (HOBt).
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • DEPC diethyl cyanophosphate
  • the mixture is preferably a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt), wherein
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • HOBt 1-hydroxybenzotriazole
  • the molar ratio of -(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) to 1-hydroxybenzotriazole (HOBt) is preferably 1:1.
  • a post-treatment operation may also be included.
  • the method and conditions for the post-treatment may be post-treatment conventional methods and conditions in the art, and preferably include the following steps: when the organic solvent is a halogenated hydrocarbon solvent, after the reaction is completed, the reaction solution is Washing with a saturated aqueous solution of ammonium hydrogencarbonate, washing with water, drying (for example, drying over anhydrous sodium sulfate), solvent removal, separation by column chromatography (eluent is preferably dichloromethane / ethyl acetate, dichloromethane / acetone , dichloromethane / methanol, cyclohexane / ethyl acetate, petroleum ether / ethyl acetate), when the organic solvent is an amide solvent, after the end of the reaction, extraction (such as ethyl acetate) The acid is washed (for example, a 5% aqueous solution of hydro
  • the room temperature is referred to as 10 to 30 °C.
  • therapeutically effective amount refers to an amount of a compound that is sufficient to effectively treat a disease or condition described herein when administered to a subject. While the amount of the compound that constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, and the age of the subject to be treated, it can be determined in a conventional manner by those skilled in the art.
  • the reagents and starting materials used in the present invention are commercially available.
  • the podophyllotoxin derivatives of the invention have good tumor cell inhibitory activity, and some compounds have superior antitumor activity to etoposide, and some compounds have better inhibitory activity against human lung cancer cells (non-small cell lung cancer cell line A549).
  • This provides a new research and development direction for the development of highly effective and low-toxic podophyllotoxin derivatives, and the preparation method of the podophyllotoxin derivatives of the present invention is simple and has a good market development prospect.
  • the room temperature in the following examples all means 10 to 35 ° C; the ice bath means 0 ° C.
  • 6-Methylnicotinic acid (196 mg, 1.6 mmol), HOBt (253 mg, 1.5 mmol) EDCI (305 mg, 1.5 mmol) was taken in 6 mL dichloromethane and stirred for 2 h, 4 ⁇ -amino-4'- The hydrochloride of the methyl epipodophyllotoxin (540 mg, 1.25 mmol) was added to the reaction solution, 0.5 mL of triethylamine was added dropwise, and the reaction was continued for 2 hours. The reaction was complete by TLC and the reaction was stopped. After work-up: EtOAc (EtOAc/EtOAc m. Purification gave 212 mg of a white solid. Yield: 38%.
  • 6-methylnicotinic acid (184 mg, 1.6 mmol), HOBt (270 mg, 1.6 mmol) EDCI (380 mg, 1.6 mmol) was taken in 6 mL dichloromethane and stirred for 2 h, 4'-demethyl Toxin (500 mg, 1.25 mmol) was added to the reaction solution, 0.5 mL of triethylamine was added dropwise, and the reaction was continued for 2 hours. The reaction was complete by TLC and the reaction was stopped. After work-up: the mixture was washed with EtOAc EtOAc EtOAc. A white solid 197 mg was obtained in a yield: 38%.
  • 6-methoxynicotinic acid (765 mg, 5 mmol), HOBt (1.043 g, 7.5 mmol), EDCI (1.437 g, 7.5 mmol) was taken in 7 mL of dichloromethane at 0 ° C and stirred for 2 h.
  • Demethylpodopoxin (1.2 g, 3 mmol) was added to the reaction solution, and 0.5 mL of triethylamine was added dropwise, and the reaction was continued for 3 hours to stop the reaction. After work-up: the mixture was washed with EtOAc EtOAc EtOAc. A white solid 203 mg was obtained in a yield: 38%.
  • 6-methoxynicotinic acid 230 mg, 1.5 mmol
  • HOBt 202 mg, 1.5 mmol
  • EDCI 288 mg, 1.5 mmol
  • the reaction solution was added, 0.5 mL of triethylamine was added dropwise, and the reaction was continued for 2 hours. The reaction was completely detected by TLC, and the reaction was stopped. After work-up: EtOAc (aq.) Purification gave 312 mg of a white solid. Yield: 57%.
  • 6-methylnicotinic acid (202 mg, 1.5 mmol), DCC (309 mg, 1.5 mmol) DMAP (catalytic amount) was added to 6 mL of anhydrous tetrahydrofuran (THF), stirred for 15 min, and the podophyllotoxin (414 mg, 1 mmol) was added.
  • THF anhydrous tetrahydrofuran
  • the reaction solution was added, and 2 mL of dichloromethane (increasing the amount of podophyllotoxin dissolved) was added, and the reaction was continued for 24 hours.
  • the reaction was stopped by TLC, and the reaction was stopped. After work-up: suction filtration, the solvent was evaporated to dryness crystals crystals crystals crystals
  • 2-hydroxynicotinic acid EDCI: HOBt (1.5: 1.5: 1.5) was added to dichloromethane (volume mass ratio of DCM to podophyllotoxin was 5:1) at room temperature with 417 mg of 2-hydroxynicotinic acid. After stirring for 2 hours, podophyllotoxin (1 eq) and triethylamine (2 eq) were added to the reaction mixture, and when the reaction was stopped by TLC, the reaction was stopped. The reaction mixture was washed with saturated sodium bicarbonate, water and dried over anhydrous sodium sulfate. The eluent was dichloromethane and ethyl acetate to give white title compound (yield:
  • 6-hydroxynicotinic acid 4'-demethylepipodophyllotoxin: DMAP: triethylamine (1.5:1:0.1:2) was added to dichloromethane at room temperature.
  • the volume-to-mass ratio of methyl chloride to 4'-demethylepipodophyllotoxin is 5:1), and two drops of triethylamine are added dropwise, stirred for 0.5 h, and EDCI (380, 1.25 mmol) is added to the reaction solution, 4.5. After h, TLC detected the reaction completely and stopped the reaction.
  • Post-treatment washed with a saturated aqueous solution of sodium bicarbonate, water and dried over anhydrous sodium sulfate.
  • the eluent was dichloromethane and methanol to give a white title compound, 170 mg, yield: 33.7%.
  • Post-treatment washed with a saturated aqueous solution of sodium bicarbonate, water and dried over anhydrous sodium sulfate.
  • the eluent was dichloromethane and methanol to give a white title compound (200 mg, yield: 77%).
  • 2-aminonicotinic acid at room temperature, 2-aminonicotinic acid: HOBt: EDCI is added to the appropriate amount of dichloromethane (DCM and 4 ⁇ -amino-4-deoxy-4'- go according to a feed ratio of 1.5:1.5:1.5
  • the volume-to-mass ratio of methyl epipodophyllotoxin hydrochloride was 10:1), and stirring for 2 hours, 1 eq of 4 ⁇ -amino-4'-demethylepipodophyllotoxin hydrochloride was added to the reaction solution, and 2 eq was added dropwise.
  • Triethylamine the reaction is continued, and the reaction is stopped when the TLC detects that the reaction is complete or the reaction is no longer carried out.
  • Work-up Wash with saturated aqueous sodium hydrogencarbonate, EtOAc (EtOAc m. 1:3, v/v), purified to give the objective compound 100 mg, yield: 19.2%.
  • Test compound Compounds 1 to 20 prepared in the above respective examples.
  • Control compound etoposide (VP-16), Cisplatin (CDDP).
  • Cell line human non-small cell lung cancer (A549) cell line, human oral cancer epithelial (KB) cell line, human liver cancer (HepG2) cell line and mouse lymphocytic leukemia (L1210) cell line.
  • Cell culture A549 cell line, HepG2 cell line, KB cell line and L1210 cell line were taken out from liquid nitrogen, rapidly thawed in a 37 ° C water bath, and the cells were transferred into a 10 mL sterile centrifuge tube in an aseptic table. 6mLDMEM cell culture medium, 1000 rpm / separation heart for 5 min. Discard the supernatant, add 5 ⁇ 6mLDMEM cell culture medium to the pellet, pipette it to suspend it, transfer it to the cell culture flask, place it in the cell culture incubator at 37 °C, and take the cells from the incubator the next day, discard the cells. The DMEM cell culture medium in the bottle was added with 5-6 mL.
  • the cells were taken out from the incubator the next day, the DMEM cell culture medium in the cell bottle was discarded, and 5-6 mLDMEM cell culture medium was added, and the cells were placed in a 37 °C cell culture incubator.
  • remove the cells from the incubator discard the DMEM cell culture medium in the cell vial, add PBS (pH 7.4) 2 to 3 mL, shake and wash, pour off the PBS solution and repeat the washing.
  • Sample preparation Samples (Compounds 1 to 20) were dissolved in dimethyl sulfoxide to obtain a solution having a concentration of 10 mg/mL. Further, PBS was used for gradient dilution to obtain diluted samples having concentrations of 1000 ⁇ g/mL, 100 ⁇ g/mL, 10 ⁇ g/mL, 1 ⁇ g/mL, 0.1 ⁇ g/mL, and 0.01 ⁇ g/mL, respectively.
  • Cisplatin was dissolved in PBS to give a solution having a concentration of 1 mg/mL. Further, PBS was used for gradient dilution to obtain diluted reference materials having concentrations of 1000 ⁇ g/mL, 100 ⁇ g/mL, 10 ⁇ g/mL, 1 ⁇ g/mL, 0.1 ⁇ g/mL, and 0.01 ⁇ g/mL, respectively.
  • the diluted sample was added to a flat-bottom 96-well plate at 10 uL per well, and two parallel tests were performed for each point.
  • the DMSO was diluted as a gradient and added to the plate as a control.
  • the cells are trypsinized and washed, suspended in DMEM medium containing 10% calf serum, counted by the trypan blue staining method, and the cell suspension is adjusted.
  • the liquid density was 2 x 10 5 cells/mL.
  • the cell activity showed that the antitumor activity of the amide compound was better than that of the ester compound as a whole.
  • the 6-position substitution activity is better than the 2-position substitution activity;
  • the substitution can increase the inhibitory activity of the compound on tumor cells, and when the 2, 5 and 6 positions of the pyridine ring have a halogen substituent (for example, the compound 8 of the present invention), the inhibitory activity against tumor cells is good.
  • the 2-position substitution activity is better than the 6-position substitution activity.
  • the 6-position substitution activity is better than the 2-position substitution activity.
  • the substituent on the pyridine ring is monosubstituted and F, the inhibitory activity against tumor cells is better, but when the 2,6 position of the pyridine ring is Cl, and 5 is F, the compound has poor antitumor activity ( For example, the compound of the invention 16).
  • the inhibitory activity against the four tumor cells is good regardless of the amide or the ester compound, especially the inhibitory activity against the A549 cell line, and the IC50 values are all below 10 ⁇ g/mL.
  • the 6-position of the pyridine ring is methyl or methoxy substitution activity is better than the same group substitution activity at the 2-position; the 2-position substitution, the methyl activity is higher than the methoxy activity, and it is presumed that the activity is substituted at this position.
  • the 2-position substitution the methyl activity is higher than the methoxy activity, and it is presumed that the activity is substituted at this position.
  • the size of the group is related to the size of the group.

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Abstract

本发明公开了一种通式I所示的鬼臼毒素衍生物、其制备方法、药物组合物及应用。本发明的通式I所示鬼臼毒素衍生物中，X为氧或氮；R1为单取代或多取代，取代位置任意，为卤素、C1~C4的烷基、C1~C4的烷氧基、氨基、硝基和羟基中的一种或多种；R2为氢或C1~C4的烷基。本发明的鬼臼毒素衍生物具有良好的肿瘤细胞抑制活性，一些化合物的抗肿瘤活性明显优于依托泊苷，部分化合物对人肺癌细胞，尤其是非小细胞肺癌细胞株A549具有较好的抑制活性，这为开发广谱、高效低毒的鬼臼毒素类衍生物提供了新的研发方向，并且本发明的鬼臼毒素衍生物的制备方法简单，具有良好的市场开发前景。

Description

鬼臼毒素衍生物、其制备方法、药物组合物及应用
本申请要求申请日为2013年11月26日的中国专利申请CN201310610906.0的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种鬼臼毒素衍生物、其制备方法、药物组合物及应用。
背景技术
依托波苷(Etoposide，VP-16)和替尼泊苷(VM-26)是临床上治疗肿瘤的鬼臼毒素类衍生物，依托泊苷在临床上常与顺铂联合用于治疗肺癌及睾丸癌，疗效较好，也用于淋巴瘤的治疗。替尼泊苷主要治疗何杰金和非何杰金淋巴瘤且具有明显效应，对脑瘤，儿童淋巴细胞性白血病也有治疗作用。但是，上述抗肿瘤药物的抑瘤谱比较窄，同时伴有严重的骨髓抑制及胃肠道副反应等。鬼臼毒素类衍生物一般对人口腔上皮癌(KB)细胞以及对小鼠淋巴细胞白血病细胞(L1210)具有很好的增殖抑制作用，而对人肺癌(非小细胞肺癌细胞株A549)细胞没有增殖抑制作用或很差，因此，本领域亟需寻找一种抗瘤谱广的鬼臼毒素类衍生物，以有效解决鬼臼毒素类化合物抗瘤谱窄，毒副作用大等缺陷。
发明内容
本发明所要解决的技术问题是为了解决现有的鬼臼毒素衍生物的抑瘤谱比较窄，有严重的骨髓抑制及胃肠道副反应等，而提供了一种与现有技术完全不同的鬼臼毒素衍生物、其制备方法、药物组合物及应用。本发明的鬼臼毒素衍生物具有良好的肿瘤细胞抑制活性，一些化合物的抗肿瘤活性明显优于依托泊苷，部分化合物对人肺癌细胞(非小细胞肺癌细胞株A549)具有较好的抑制活性，这为扩大鬼臼毒素类化合物的抗瘤谱，并开发高效低毒的鬼臼毒素类衍生物提供了新的研发方向，并且本发明的鬼臼毒素衍生物的制备方法简单，具有良好的市场开发前景。
本发明提供了下列通式I所示鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物；
Figure PCTCN2014092098-appb-000001
其中，X为氧或氮；R1为单取代或多取代，取代位置任意，R1为卤素、C1～C4的烷基、C1～C4的烷氧基、氨基、硝基和羟基中的一种或多种；R2为氢或C1～C4的烷基；当X为O，且R1为单取代时，R1不为2-氯，6-氯或5-溴；当X为N，且R1为单取代时，R1不为2-氯或6-氯；当X为N，且R1为双取代时，R1不为4-氯和6-甲基；当R2为氢时，用*号标注的碳为β构型手性碳；当R2为C1～C4的烷基时，用*标注的碳为α构型手性碳。
R1中，所述的卤素较佳地为氟、氯、溴或碘。R1或R2中，所述的C1～C4的烷基较佳地为甲基、乙基、正丙基、异丙基或丁基。所述的丁基为正丁基、异丁基或叔丁基。R1中，所述的C1～C4的烷氧基较佳地为甲氧基、乙氧基、丙氧基或丁氧基。所述的丙氧基较佳地为正丙氧基或异丙氧基。所述的丁氧基较佳地为正丁氧基、异丁氧基或叔丁氧基。
通式I化合物中，R1表示的取代基较佳地位于吡啶环的碳原子的2位、5位和6位中的一个或多个。
通式I化合物中，当X为氧，R2为氢时，R1较佳地为卤素、C1～C4的烷基、C1～C4的烷氧基、氨基和羟基中的一种或多种；R1表示的取代基较佳地位于吡啶环的碳原子的2位、5位和6位中的一个或多个。
通式I化合物中，当X为氧，R2为氢时，R1较佳地为卤素、C1～C4的烷氧基和氨基中的一种或多种；R1表示的取代基较佳地位于吡啶环的碳原子的2位、5位和6位中的一个或多个。当R1为卤素和/或氨基时，R1表示的取代基较佳地位于吡啶环的碳原子的2位、5位和6位中的一个或多个。当R1为C1～C4的烷氧基时，R1表示的取代基较佳地位于吡啶环的碳原子的6位。
通式I化合物中，当X为氧，R2为C1～C4的烷基时，R1较佳地为卤素、C1～C4的烷基、C1～C4的烷氧基、氨基和羟基中的一种或多种；R1表示的取代基较佳地位于吡啶环的碳原子的2位、5位和6位中的一个或多个。
通式I化合物中，当X为氧，R2为C1～C4的烷基时，R1较佳地为卤素、C1～C4的烷氧基和氨基中的一种或多种。当R1为卤素和/或氨基时，R1表示的取代基较佳地位于吡啶环的碳原子的2位、5位和6位中的一个或多个；更佳地位于吡啶环的碳原子的2位。 当R1为C1～C4的烷氧基时，R1表示的取代基较佳地位于吡啶环的碳原子的2位。
通式I化合物中，当X为氮，R2为氢时，R1较佳地为卤素、C1～C4的烷基、C1～C4的烷氧基、氨基和羟基中的一种或多种；R1表示的取代基较佳地位于吡啶环的碳原子的2位、5位和6位中的一个或多个。
通式I化合物中，当X为氮，R2为氢时，R1较佳地为卤素、C1～C4的烷基、C1～C4的烷氧基和氨基中的一种或多种。当R1为C1～C4的烷基和/或氨基时，R1表示的取代基较佳地位于吡啶环的碳原子的2位、5位和6位中的一个或多个；更佳地位于吡啶环的碳原子的2位和/或6位。当R1为C1～C4的烷氧基时，R1表示的取代基较佳地位于吡啶环的碳原子的6位。当R1为卤素时，R1较佳地为单取代；R1表示的取代基较佳地位于吡啶环的碳原子的5位。
通式I化合物中，当X为氧或氮，R2为氢时，R1较佳地为氨基，氨基较佳地位于吡啶环的碳原子的2位。
所述的通式I所示鬼臼毒素衍生物、其立体异构体、互变异构体、其药学上可接受的盐、前药或溶剂化物，最佳地，为如下任一化合物：
Figure PCTCN2014092098-appb-000002
Figure PCTCN2014092098-appb-000003
本发明还提供了所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物在制备用于治疗癌症的药物中的应用。所述的癌症较佳地为肺癌、口腔癌、肝癌或白血病，尤其是肺癌。所述的肺癌较佳地为人非小细胞肺癌。所述的口腔癌较佳地为人口腔上皮癌。所述的癌症的肿瘤细胞较佳地为人非小细胞肺癌细胞、人口腔癌上皮细胞、人肝癌细胞或小鼠淋巴白血病细胞。所述的人非小细胞肺癌细胞较佳地为人非小细胞肺癌细胞株A549。所述的人口腔癌细胞较佳地为人口腔癌上皮细胞株KB。所述的人肝癌细胞较佳地为人肝癌细胞株HepG2。所述的小鼠淋巴白血病细胞较佳地为小鼠淋巴细胞白血病细胞株L1210。
本发明还提供了一种药物组合物，其包含上述通式I所示鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药和溶剂化物中的一种或多种，以及药学上可接受的赋形剂。
根据治疗目的，可将上述药物组合物制成各种类型的给药单位剂型，例如：水性分散剂、液体、啫哩、糖浆、西也剂、药浆、悬浮液、气雾剂、控释剂、速溶剂、泡腾剂、 冻干剂、片剂、粉末、药丸、糖衣完、胶囊、延迟释放剂、延长释放剂、脉冲控释剂、多微粒剂或立即释放剂。
本发明还提供了一种所述的通式I所示鬼臼毒素衍生物的制备方法，其包含下列步骤：有机溶剂中，碱的存在下，在缩合剂的作用下，将如式A所示的化合物或其盐，与如式B所示的化合物进行如下所示的反应；
Figure PCTCN2014092098-appb-000004
其中，R为羟基(-OH)或氨基(-NH2)；*、R1和R2的定义均同前所述。
其中，所述的反应的方法和条件可为本领域此类反应常规的方法和条件。本发明特别优选下列方法和条件：有机溶剂中，碱的存在下，在缩合剂和催化剂的作用下，将如式A所示的化合物或其盐，与如式B所示的化合物进行反应。其中，所述的如式A所示的化合物的盐是指如式A所示的化合物与本领域常规的酸形成的盐。所述的如式A所示的化合物的盐较佳地为如式A所示的化合物的盐酸盐。所述的有机溶剂较佳地为卤代烃类溶剂和/或酰胺类溶剂。所述的卤代烃类溶剂较佳地为二氯甲烷。所述的酰胺类溶剂较佳地为N,N-二甲基甲酰胺(DMF)。所述的缩合剂较佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)、N,N’-二环己基碳二酰亚胺(DCC)和氰基磷酸二乙酯(DEPC)中的一种或多种，或者，所述的缩合剂较佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)、N,N’-二环己基碳二酰亚胺(DCC)和氰基磷酸二乙酯(DEPC)中的一种或多种，与1-羟基苯并三氮唑(HOBt)的混合物。所述的混合物较佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)与1-羟基苯并三氮唑(HOBt)的混合物，其中，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)与1-羟基苯并三氮唑(HOBt)的摩尔比较佳地为1:1。所述的碱较佳地为有机碱，所述的有机碱较佳地为三乙胺。所述的催化剂较佳地为4-二甲氨基吡啶(DMAP)。所述的碱，与如式A所示的化合物或其盐的摩尔比较佳地为1:1～3:1。所述的如式A所示的化合物或其盐，与如式B所示的化合物的摩尔比较佳地为1:1～1:1.5。所述的缩合剂，与如式A所示的化合物或其盐的摩尔比较佳地为1:1～3:1。所述的催化剂，与如式A所示的化合物或其盐的摩尔比较佳地为0.05:1～0.1:1。所述的有机溶剂与如式A所示的化合物或其盐的质量体积比较佳地为1mL/g～10mL/g。所述的反应的温度较佳地为0～30℃。所述的反应的进程可以采用本领域中的常规测试方法(如TLC、 HPLC或NMR)进行监控，反应时间较佳地为1～24小时。
所述的通式I所示的鬼臼毒素衍生物的制备方法中，当X为O时，较佳地，将如式A所示的化合物与如式B所示的化合物进行酯化反应；所述的有机溶剂较佳地为卤代烃类溶剂；所述的缩合剂较佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)和/或N,N’-二环己基碳二酰亚胺(DCC)；或者，所述的缩合剂较佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)和/或氰基磷酸二乙酯(DEPC)，与1-羟基苯并三氮唑(HOBt)的混合物。所述的混合物较佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)与1-羟基苯并三氮唑(HOBt)的混合物，其中，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)与1-羟基苯并三氮唑(HOBt)的摩尔比较佳地为1:1。
所述的通式I所示的鬼臼毒素衍生物的制备方法中，当X为N时，较佳地，将如式A所示的化合物的盐与如式B所示的化合物进行酰胺化反应；所述的有机溶剂较佳地为卤代烃类溶剂和/或酰胺类溶剂；所述的缩合剂较佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)和/或氰基磷酸二乙酯(DEPC)，更佳地为氰基磷酸二乙酯(DEPC)；或者，所述的缩合剂较佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)和/或氰基磷酸二乙酯(DEPC)，与1-羟基苯并三氮唑(HOBt)的混合物。所述的混合物较佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)与1-羟基苯并三氮唑(HOBt)的混合物，其中，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)与1-羟基苯并三氮唑(HOBt)的摩尔比较佳地为1:1。
所述的反应结束后，较佳地，还可包括后处理的操作。所述的后处理的方法和条件可为本领域后处理常规的方法和条件，较佳地包含下列步骤：当所述的有机溶剂为卤代烃类溶剂时，上述反应结束后，将反应液用饱和碳酸氢铵水溶液洗，水洗，干燥(例如用无水硫酸钠干燥)，除去溶剂，经柱层析分离(洗脱剂较佳地为二氯甲烷/乙酸乙酯、二氯甲烷/丙酮、二氯甲烷/甲醇、环己烷/乙酸乙酯、石油醚/乙酸乙酯)，即可；当所述的有机溶剂为酰胺类溶剂时，上述反应结束后，萃取(如乙酸乙酯)，酸洗(例如质量分数为5％的盐酸水溶液)，饱和碳酸氢钠水溶液洗，水洗，得到有机相后，除去有机溶剂，即可。本发明中，所述的通式I所示的鬼臼毒素衍生物的药学上可接受的盐的制备方法和条件还可按照本领域成盐反应的常规方法和条件制备。
本发明中，所述的室温均是指10～30℃。
本发明中，“治疗有效量”是指在给予受试者时足以有效治疗本文所述的疾病或病症的化合物的量。虽然构成“治疗有效量”的化合物的量将根据化合物、病症及其严重度、以及欲治疗受试者的年龄而变化，但可由本领域技术人员以常规方式确定。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的鬼臼毒素衍生物具有良好的肿瘤细胞抑制活性，一些化合物的抗肿瘤活性明显优于依托泊苷，部分化合物对人肺癌细胞(非小细胞肺癌细胞株A549)具有较好的抑制活性，这为开发高效低毒的鬼臼毒素类衍生物提供了新的研发方向，并且本发明的鬼臼毒素衍生物的制备方法简单，具有良好的市场开发前景。
具体实施方式
下述实施例中的室温均是指10～35℃；冰浴均是指0℃。
实施例1
4β-氯代乙酰胺基-4-脱氧-4’-去甲基表鬼臼毒素的制备
室温下，将4’-去甲基表鬼臼毒素(10g，25mmol)加入至氯乙腈(15mL，250mmol)中，滴加0.15mL 98％浓硫酸，搅拌，可看到不溶物溶解并迅速有白色固体生成，加入50mL异丙醇稀释固体，抽滤，滤饼继续用100mL异丙醇分洗2次，后用水洗至中性。放置真空干燥箱干燥40℃干燥4h，得白色固体粉末10g，收率：84％。
ESI-MS：476(M+H+)，493(M+H3O+).
1HNMR(DMSO)：8.60(1H,dd,NH),8.20(1H,d,OH重水交换),6.89(1H,d,5H),6.52(1H,d,8H),6.23(2H,s,H-2’,6’),6.00(2H,d,-OCH2O),5.19(1H,dd,H-4),4.5(1H,d,H-1),4.34(1H,t,H-11α),3.79(1H,t,H-11β),3.89(6H,S,2’,6’-O-CH3),3.21(1H,dd,s,H-1),3.15(1H,m,3H).
实施例2
4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐的制备
将4β-氯代乙酰基-4’-去甲基表鬼臼毒素(10g，21mmol)加入至50mL冰醋酸中，升温至80℃，加入硫脲(2.4g，31mmol)，约10min内可看到反应液有浑浊变为澄清，维持在80℃搅拌2.5h，有白色固体生成，至沉淀不再生成，停止反应，继续加入50mL冰醋酸，稀释固体，抽滤，滤饼用无水乙醚150mL洗涤三次，转移至真空干燥箱中干燥，4β-氨基-4’-去甲基表鬼臼毒素盐酸盐6.85g收率：84％。
ESI-MS：400(M+H+).
1HNMR(DMSO)：8.60(2H,s,-NH2重水交换消失),8.20(1H,s,OH重水交换消失), 7.20(1H,d,5H),6.80(1H,d,8H),6.19(2H,s,H-2’,6’),6.00(2H,d,-OCH2O),4.78(1H,d,H-4),4.57(1H,d,H-1),4.34(2H,m,H-11α,β),3.60(6H,s,2’,6’-O-CH3),3.08(1H,m,H-3).
实施例3化合物1的制备
室温下，将2-甲氧基烟酸(229mg，1.5mmol)，HOBt(204mg，1.5mmol)EDCI(396mg，2mmol)投入6mL二氯甲烷中，搅拌2h，将4β-氨基-4’-去甲基表鬼臼毒素的盐酸盐(435mg，1mmol)加入反应液，滴加0.5mL三乙胺，继续反应2h，TLC检测反应完全，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/乙酸乙酯梯度洗脱)纯化，得到白色固体212mg，收率：38％。
ESI-MS：535(M+H+)557(M+Na+).
1HNMR(DMSO)：8.6(1H,d,4-吡啶-H),8.25(2H,d,N-H,-OH重氢交换消失),8.00(1H,d,6-吡啶-H),7.08(1H,dd,3-吡啶-H),6.89(1H,d,H-5),6.52(1H,d,H-8),6.23(2H,s,H-2’,6’),6.00(2H,d,-OCH2O-),5.49(1H,dd,H-4),4.5(1H,d,H-1),4.54(1H,t,H-11α),3.99(1H,t,H-11β),3.99(6H,s,3’,5’-O-CH3),3.80(3H,s,2-吡啶-OCH3),3.36(2H,d,s,H-1,NH),3.15(1H,m,H-3).
实施例4化合物2的制备
室温下，将6-甲基烟酸(196mg，1.6mmol)，HOBt(253mg，1.5mmol)EDCI(305mg，1.5mmol)投入6mL二氯甲烷中，搅拌2h，将4β-氨基-4’-去甲基表鬼臼毒素的盐酸盐(540mg，1.25mmol)加入反应液，滴加0.5mL三乙胺，继续反应2h，TLC检测反应完全，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/乙酸乙酯梯度洗脱)纯化，得到白色固体212mg，收率：38％。
ESI-MS：519(M+H+)，541(M+Na+).
1HNMR(DMSO):8.99(1H,s,2-吡啶-H),8.80(1H,d,-NH),8.20(1H,s,OH,重水交换),8.19(1H,dd,4-吡啶-H),7.35(1H,d,5-吡啶-H),6.89(1H,s,H-5),6.52(1H,s,H-8),6.23(2H,s,H-2’,6’),6.00(2H,d,-OCH2O-),5.45(1H,dd,H-4),4.79-4.70(1H,m,11a-H),4.52(1H,d,H-1),4.45(1H,t,H-11α),3.80(1H,t,H-11β),3.69(6H,s,3’,5’-OCH3),3.5(1H,dd,2-H),3.15(1H,m,H-3),2.60(3H,s,6-吡啶-CH3).
实施例5化合物3的制备
室温下，将2-甲基烟酸(275mg，1.6mmol)，HOBt(270mg，1.6mmol)EDCI(380mg，1.6mmol)投入6mL二氯甲烷中，搅拌2h，将4β-氨基-4’-去甲基表鬼臼毒素的盐酸盐 (540mg，1.25mmol)加入反应液，滴加0.5mL三乙胺，继续反应2h，TLC检测反应完全，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/乙酸乙酯梯度洗脱)纯化，得到白色固体212mg，收率：33％。
ESI-MS：519(M+H+)，541(M+Na+).
1HNMR(DMSO):8.60(1H,d,4-吡啶-H),7.61(1H,d,6-吡啶-H),7.19(1H,m,OH,重水交换5-吡啶-H,6.82(1H,s,H-5),6.57(1H,s,H-8),6.25(2H,s,H-2’,6’),6.00(2H,d,-OCH2O-),5.45(1H,dd,H-4),4.60(1H,d,H-1),4.58(1H,d,H-11α),4.00(1H,t,H-11β),3.80(6H,s,3’,5’-OCH3),3.20(1H,dd,2-H),3.85(1H,m,H-3),2.65(3H,s,2-吡啶-CH3).
实施例6化合物4的制备
室温下，将2-甲基烟酸(184mg，1.6mmol)，HOBt(270mg，1.6mmol)EDCI(380mg，1.6mmol)投入6mL二氯甲烷中，搅拌2h，将4β-氨基-4’-去甲基表鬼臼毒素的盐酸盐(540mg，1.25mmol)加入反应液，滴加0.5mL三乙胺，继续反应2h，TLC检测反应完全，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/丙酮梯度洗脱)纯化，得到白色固体212mg，收率：38％。
ESI-MS：535(M+H+)，557(M+Na+).
1HNMR(DMSO)：8.79(1H,s,2-吡啶-H),8.75(1H,d,-NH),8.20(1H,s,OH,重水交换),8.19(1H,dd,4-吡啶-H),6.98(1H,d,5-吡啶-H),6.97(1H,s,H-5),6.52(1H,s,H-8),6.23(2H,s,H-2’,6’),6.00(2H,d,-OCH2O-),5.45(1H,dd,H-4),4.52(1H,d,H-1),4.35(1H,t,H-11α),3.90(6-吡啶-OCH3),3.80(1H,t,H-11β),3.69(6H,s,3′,5′-OCH3),3.08(1H,m,H-3).
实施例7化合物5的制备
室温下，将2-甲氧基烟酸(202mg，1.5mmol)，DCC(309mg，1.5mmol)，DMAP(催化量)投入6mL二氯甲烷中，搅拌15min，将4’-去甲基表鬼臼毒素(414mg，1mmol)加入反应液，继续反应24h，TLC检测反应不再进行，停止反应。后处理：抽滤，滤液减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/乙酸乙酯洗脱)纯化，得到白色固体139mg，收率：26％。
ESI-MS：536(M+H+)，558(M+Na+).
1HNMR(CDCl3)：8.25(1H,dd,4-吡啶-H),8.00(1H,d,6-吡啶-H),7.00(1H,s,H-5),6.99(1H,dd,5-吡啶-H),6.52(1H,s,H-8),6.30(1H,d,H-4),6.23(2H,s,H-2’,6’),6.00(2H, d,-OCH2O-),5.50(1H,s,-OH),4.56(1H,d,H-1),4.30(1H,t,H-11α),4.02(1H,t,H-11β),3.90(3H,s,2-吡啶-OCH3),3.69(6H,s,3’,5’-OCH3),3.30(1H,dd,2),3.00(1H,m,H-3).
实施例8化合物6的制备
室温下，将6-甲基烟酸(184mg，1.6mmol)，HOBt(270mg，1.6mmol)EDCI(380mg，1.6mmol)投入6mL二氯甲烷中，搅拌2h，将4’-去甲基表鬼臼毒素(500mg，1.25mmol)加入反应液，滴加0.5mL三乙胺，继续反应2h，TLC检测反应完全，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/丙酮梯度洗脱)纯化，得到白色固体197mg，收率：38％。
ESI-MS：520(M+H+)，542(M+Na+).
1HNMR(CDCl3)：9.20(1H,s,2-吡啶-H),8.25(1H,dd,4-吡啶-H),7.23(1H,d,5-吡啶-H),6.99(1H,s,H-5),6.58(1H,s,H-8),6.28(2H,s,H-2’,6’),5.99(2H,d,-OCH2O-),4.80(1H,d,H-4),4.64(1H,d,H-1),4.38(2H,m,11-α,β-H),3.64(6H,s,3’,5’-OCH3),3.23(1H,dd,2-H),2.82(1H,m,H-3),2.60(3H,s,6-吡啶-CH3).
实施例9化合物7的制备
室温下，将2-甲基烟酸(184mg，1.6mmol)，HOBt(270mg，1.6mmol)EDCI(380mg，1.6mmol)投入6mL二氯甲烷中，搅拌2h，将4’-去甲基表鬼臼毒素(500mg，1.25mmol)加入反应液，滴加0.5mL三乙胺，继续反应2h，TLC检测反应完全，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/丙酮梯度洗脱)纯化，得到白色固体223mg，收率：47％。
ESI-MS：552(M+Na+).
1HNMR(DMSO)：8.69(1H,d,4-吡啶-H),8.25(1H,d,6-吡啶-H),7.48(1H,dd,5-吡啶-H),7.00(1H,s,H-5),6.60(1H,s,H-8),6.23(2H,s,H-2’,6’),6.00(2H,d,-OCH2O-),5.69(1H,s,-OH,重水交换消失),4.58(1H,d,H-1),4.69(1H,d,H-4),4.48(1H,t,H-11α),4.23(1H,t,H-11β),3.69(9H,s,3’,5’-O-CH3),3.20(2H,d,H-1),2.89(1H,m,H-3),3.71(3H,s,2-吡啶-CH3).
实施例10化合物8的制备
室温下，将2,6-二氯-5-氟-烟酸(420mg，2mmol)，4’-去甲基表鬼臼毒素(400mg，1mmol)，DMAP(催化量)投入4mL二氯甲烷中，并滴入两滴三乙胺，搅拌0.5h，将EDCI(380，1.25mmol)加入反应液，4.5h后，TLC检测反应完全，停止反应。后处理： 依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/甲醇梯度洗脱)纯化，得到白色固体211mg，收率：36％。
ESI-MS：592(M+H+).
1HNMR(DMSO-d6)：8.79(1H,s,2-吡啶-H),6.97(1H,s,H-5),6.52(1H,s,H-8),6.39(2H,s,H-2′,6′),6.00(2H,d,-OCH2O-),5.45(1H,dd,-OH,重水交换消失),4.80(4H,dd,H-4),4.40(1H,d,H-1),4.20(1H,t,H-11α),3.80(1H,t,H-11β),3.69(6H,s,3′,5′-OCH3),3.38(1H,dd,2-H),3.08(1H,m,H-3).
实施例11化合物9的制备
在0℃下，将6-甲氧基烟酸(765mg，5mmol)，HOBt(1.043g，7.5mmol)，EDCI(1.437g，7.5mmol)投入7mL二氯甲烷中，搅拌2h，将4’-去甲基表鬼臼毒素(1.2g，3mmol)加入反应液，滴加0.5mL三乙胺，继续反应3h，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/丙酮梯度洗脱)纯化，得到白色固体203mg，收率：38％。
ESI-MS：536(M+H+)，558(M+Na+).
1HNMR(DMSO-d6)：8.90(1H,s,2-吡啶-H),8.25(1H,dd,4-吡啶-H),8.20(1H,s,OH,重水交换),7.00(1H,s,H-5),6.98(1H,d,5-吡啶-H),6.52(1H,s,H-8),6.35(1H,d,H-4),6.23(2H,s,H-2′,6′),6.00(2H,d,-OCH2O-),5.45(1H,dd,H-4),4.62(1H,d,H-1),4.35(1H,t,H-11α),3.90(1H,t,H-11β),3.80(3H,s,6-吡啶-OCH3),3.62(6H,s,3′,5′-OCH3),3.58(1H,dd,2-H),3.10(1H,m,H-3).
实施例12化合物10的制备
使用鬼臼毒素(76mg，0.18mmol)和2-甲氧基烟酸(22mg，0.27mmol)室温下，将2-甲氧基烟酸：EDCI：HOBt(1.5:1.5:1.5)加入至二氯甲烷(二氯甲烷与鬼臼毒素的体积质量比为5:1)中，搅拌2h后，向反应液中加入鬼臼毒素(1eq)和三乙胺(2eq)，TLC检测反应不再进行时，停止反应。反应液用饱和碳酸氢钠、水洗，无水硫酸钠干燥，旋蒸除去溶剂，所得固体柱层析分离纯化。洗脱剂为二氯甲烷和丙酮，制得目标化合物60mg，收率：63％。
ESI-MS：550(M+H+)，572(M+Na+)，397，282，209(碎片峰).
1HNMR(DMSO-d6)：8.42(1H,d,4-吡啶-H),8.25(1H,d,6-吡啶-H),7.08(1H,dd,5-吡啶-H),6.60(1H,s,5H),6.60(1H,s,8H),6.23(2H,s,H-2′,6′),6.00(2H,d,-OCH2O-),5.49(1H,dd,H-4),4.58(1H,d,H-1),4.54(1H,t,H-11α),4.23(1H,t,H-11β),3.99(3H,s,2- 吡啶-OCH3),3.60(9H,s,3’,4’,5’-O-CH3),3.36(1H,d,2-H),3.15(1H,m,H-3).
实施例13化合物11的制备
室温下，将6-甲氧基烟酸(230mg，1.5mmol)，HOBt(202mg，1.5mmol)EDCI(288mg，1.5mmol)投入6mL二氯甲烷中，搅拌2h，将鬼臼毒素(460mg，1mmol)加入反应液，滴加0.5mL三乙胺，继续反应2h，TLC检测反应完全，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：环己烷/乙酸乙酯梯度洗脱)纯化，得到白色固体312mg，收率：57％。
MS：550(M+H+)，572(M+Na+).
1HNMR(CDCl3)：8.82(1H,s,2-吡啶-H),8.19(1H,d,4-吡啶-H),6.83(1H,s,H-5),6.80(1H,d,5-吡啶-H),6.60(1H,s,H-8),6.43(2H,s,H-2′,6′),6.14(1H,dd,H-4),6.00(2H,d,-OCH2O-),4.62(1H,d,H-1),4.54(1H,t,H-11α),4.23(1H,t,H-11β),3.99(3H,s,2-吡啶-OCH3),3.78(9H,S,3’,4’,5’-O-CH3),3.00(2H,m,H-2,H-3).
实施例14化合物12的制备
室温下，将6-甲基烟酸(202mg，1.5mmol)，DCC(309mg，1.5mmol)DMAP(催化量)投入6mL无水四氢呋喃(THF)中，搅拌15min，将鬼臼毒素(414mg，1mmol)加入反应液，并补加2mL二氯甲烷(增加鬼臼毒素的溶解量)，继续反应24h，TLC检测反应不再进行，停止反应。后处理：抽滤，滤液减压蒸除溶剂，所得粗品经柱层析纯化(洗脱剂：二氯甲烷/乙酸乙酯洗脱)纯化，得到白色固体277mg，收率：57％。
MS：534(M+H+).
1HNMR(CDCl3)：9.19(1H,s,2-吡啶-H),8.25(1H,dd,4-吡啶-H),7.29(1H,d,5-吡啶-H),7.00(1H,s,H-5),6.52(1H,s,H-8),6.23(2H,s,H-2′,6′),6.12(1H,d,H-4),6.00(2H,d,-OCH2O-),4.62(1H,d,H-1),4.35(1H,t,H-11α),3.90(1H,t,H-11β),3.80(3H,s,4’-OCH3),3.62(6H,s,3′,5′,-OCH3),3.00(2H,dd,2-H,H-3),2.69(3H,s,6吡啶-CH3).
实施例15化合物13的制备
以2-羟基烟酸417mg，室温下，将2-羟基烟酸：EDCI：HOBt(1.5:1.5:1.5)加入至二氯甲烷(DCM与鬼臼毒素的体积质量比为5:1)中，搅拌2h后，向反应液中加入鬼臼毒素(1eq)和三乙胺(2eq)，TLC检测反应不再进行时，停止反应。反应液用饱和碳酸氢钠、水洗，无水硫酸钠干燥，旋蒸除去溶剂，所得固体柱层析分离纯化。洗脱剂为二氯甲烷和乙酸乙酯，得到白色目标化合物800mg，收率：72％。
MS：558(M+Na+)，574(M+K+).
1HNMR(DMSO-d6)：12(1H,s,2-吡啶-OH),8.19(1H,d,4-吡啶-H),7.68(1H,d,6-吡啶 -H),7.00(1H,s,H-5),6.58(1H,s,H-8),6.40(2H,s,H-2′,6′),6.23(1H,t,5-吡啶-H),6.04(1H,d,H-4),6.00(2H,d,-OCH2O-),4.62(1H,d,H-1),4.35(1H,t,H-11α),3.90(1H,t,H-11β),3.68(9H,s,3’,4’,5’-OCH3),3.40(1H,dd,2-H),2.89(1H,m,H-3).
实施例16化合物14的制备
使用208mg的6-羟基烟酸，室温下，6-羟基烟酸：4’-去甲基表鬼臼毒素：DMAP：三乙胺(1.5：1：0.1：2)投入至二氯甲烷(二氯甲烷与4’-去甲基表鬼臼毒素的体积质量比为5:1)中，并滴入两滴三乙胺，搅拌0.5h，将EDCI(380，1.25mmol)加入反应液，4.5h后，TLC检测反应完全，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化。洗脱剂为二氯甲烷和甲醇，得到白色目标化合物170mg，收率：33.7％。
ESI-MS：522(M+H+)，544(M+Na+).
1H-NMR(DMSO-d6)：12.17(1H,s,OH),8.18(1H,s,2-吡啶-H),7.86(1H,d,4-吡啶-H),6.96(1H,s,5H),6.55(1H,s,8H),6.42(1H,d,5-吡啶-H),6.36(2H,s,H-2′,6′),6.00(2H,d,-OCH2O-),5.43(1H,s,H-4),4.76(1H,s,H-1),4.62(1H,s,H-2),4.19-4.39(2H,t,11-α,β-H),3.60(9H,s,3′,5′-O-CH3),2.83(1H,m,H-3).
实施例17化合物15的制备
138mg 2-氨基烟酸，室温下，2-胺基烟酸：4’-去甲基表鬼臼毒素：DMAP：三乙胺(1.5：1：0.1：2)投入至二氯甲烷(二氯甲烷与4’-去甲基表鬼臼毒素的体积质量比为5:1)中，并滴入两滴三乙胺，搅拌0.5h，将EDCI(380，1.25mmol)加入反应液，4.5h后，TLC检测反应完全，停止反应。后处理：依次用饱和碳酸氢钠水溶液、水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化。洗脱剂为二氯甲烷和甲醇，得到白色目标化合物200mg，收率：77％。
ESI-MS：521(M+H+)，543(M+Na+).
1H-NMR(CDCl3)：8.35(1H,dd,6-吡啶-H),8.25(1H,dd,4-吡啶-H),6.83(1H,s,H-5),6.72(1H,m,5-吡啶-H),6.58(1H,s,H-8),6.37(3H,s,-NH2和H-2′,6′),5.99(2H,d,-OCH2O-),4.80(1H,d,J＝3.2HZ,H-4),4.33(2H,m,11-α,β-H),3.68(6H,s,3′,5′-OCH3),3.31(1H,dd,2-H),2.82(1H,m,H-3).
实施例18化合物16的制备
以104mg的2,6-二氯-5-氟烟酸，冰浴下，将2,6-二氯-5-氟烟酸：4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(1:1)投入N,N-二甲基甲酰胺(DMF)中(DMF与4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐的体积质量比为1:1)，冰浴下搅拌2h后，依次加入氰 基磷酸二乙酯(DEPC,1eq)和三乙胺(Et3N，2eq)，在冰浴下(0℃)继续反应，TLC检测反应完全后，向反应液中加入5倍体积水，并用乙酸乙酯萃取，依次用5％的盐酸水溶液，饱和碳酸氢钠水溶液和水洗涤乙酸乙酯层，除去乙酸乙酯，得目标化合物粗品，得到白色固体105mg，收率：35.5％。
1H-NMR(400MHz,DMSO-d6)δ9.09(d,J＝7.9Hz,1H),8.42(d,J＝7.8Hz,1H),8.18(s,1H),6.92(s,1H),6.54(s,1H),6.27(s,2H),6.01(d,J＝8.9Hz,2H),5.34(dd,J＝7.8,4.2Hz,1H),4.51(d,J＝4.8Hz,1H),4.39(t,J＝7.9Hz,1H),4.05(t,J＝9.5Hz,1H),3.65(s,6H),3.14(dd,J＝14.4,4.9Hz,1H),3.10-2.99(m,1H).
实施例19化合物17的制备
以2-氨基烟酸，室温下，2-氨基烟酸：HOBt：EDCI按照1.5:1.5:1.5的投料比加入适量的二氯甲烷中(DCM与4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐的体积质量比为10:1)，搅拌2h，将1eq的4β-氨基-4’-去甲基表鬼臼毒素盐酸盐加入反应液，滴加2eq的三乙胺，继续反应，TLC检测反应完全或反应不再进行时，停止反应。后处理：用饱和碳酸氢钠水溶液洗，水洗，无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析纯化，二氯甲烷/乙酸乙酯梯度洗脱(3:1～1:3，v/v)，纯化，得目标化合物100mg，收率：19.2％。
ESI-MS：520(M+H+)，542(M+Na+).
1H-NMR(CDCl3)：8.19(1H,dd,N-H),7.55(1H,dd,吡啶-H),6.80(1H,s,H-5),6.57(2H,m,吡啶-H，H-8),6.40(2H,S,-NH2),6.32(2H,s,H-2′,6′),6.00(2H,d,OCH2O),6.23(2H,s,H-2′,6′),6.00(2H,d,-OCH2O-),5.38(1H,dd,H-4),4.48(1H,m,H-1),4.46(1H,t,H-11α),3.87(1H,t,H-11β),3.78(6H,s,3′,5′-OCH3),3.08(2H,m,H-3，H-2).
实施例20化合物18的制备
2-羟基-5-溴烟酸(56mg，0.25mmol)，冰浴下，将2-羟基-5-溴烟酸：4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(1:1)加入至适量的N,N-二甲基甲酰胺(DMF)中(DMF与4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐的体积质量比为1:1)，冰浴下搅拌2h后，依次加入氰基磷酸二乙酯(DEPC,1eq)和三乙胺(Et3N，2eq)，在冰浴下(0℃)继续反应，TLC检测反应完全后，向反应液中加入5倍体积水，并用乙酸乙酯萃取，依次用5％的盐酸水溶液，饱和碳酸氢钠水溶液和水洗涤乙酸乙酯层，除去乙酸乙酯，得到白色固体68mg，收率：45％。
ESI-MS：600(M+H+)，601(M+H+,同位素峰).
1H-NMR(400MHz,DMSO-d6)δ11.35(s,1H),8.18(s,1H),8.07(t,J＝8.7Hz,1H), 7.90(d,J＝3.1Hz,1H),6.84(s,1H),6.53(s,1H),6.25(d,J＝23.5Hz,2H),5.99(d,J＝12.0Hz,2H),5.34(dd,J＝7.8,4.4Hz,1H),4.57(d,J＝4.9Hz,1H),4.39-4.28(m,1H),3.72(dd,J＝18.1,8.2Hz,2H),3.65(s,6H),3.08-2.88(m,3H).
实施例21化合物19的制备
用71mg5-氟烟酸，冰浴下，将5-氟烟酸：4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(1:1)加入至适量的N,N-二甲基甲酰胺(DMF)中(DMF与4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐的体积质量比为2:1)，冰浴下搅拌2h后，依次加入氰基磷酸二乙酯(DEPC,1eq)和三乙胺(Et3N，2eq)，在冰浴下(0℃)继续反应，TLC检测反应完全后，向反应液中加入5倍体积水，并用乙酸乙酯萃取，依次用5％的盐酸水溶液，饱和碳酸氢钠水溶液和水洗涤乙酸乙酯层，除去乙酸乙酯，得到白色固体154mg，收率：59％，熔点188-189℃。
ESI-MS：545(M+Na+).
1H-NMR(400MHz,DMSO-d6)δ8.95(s,1H),8.93(d,J＝1.7Hz,1H),8.73(d,J＝2.7Hz,1H),8.22–8.15(m,2H),6.88(s,1H),6.58(s,1H),6.30(s,2H),6.00(d,J＝12.0Hz,2H),5.44(dd,J＝7.9,4.7Hz,1H),4.54(d,J＝5.2Hz,1H),4.35(t,J＝8.0Hz,1H),3.85(dd,J＝10.8,8.7Hz,1H),3.66(s,5H),3.38(dd,J＝14.4,5.3Hz,1H),3.10–2.99(m,1H).
实施例22化合物20的制备
用52mg 5-溴烟酸，冰浴下，将5-溴烟酸：4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(1:1)加入至适量的N,N-二甲基甲酰胺(DMF)中(DMF与4β-氨基-4-脱氧-4’-去甲基表鬼臼毒素盐酸盐的体积质量比为5:1)，冰浴下搅拌2h后，依次加入氰基磷酸二乙酯(DEPC,1eq)和三乙胺(Et3N，2eq)，在冰浴下(0℃)继续反应，TLC检测反应完全后，向反应液中加入5倍体积水，并用乙酸乙酯萃取，依次用5％的盐酸水溶液，饱和碳酸氢钠水溶液和水洗涤乙酸乙酯层，除去乙酸乙酯，得到白色固体70mg，收率：48％，熔点：188-181℃。
ESI-MS：524(M+H+)，546(M+Na+).
1H-NMR(400MHz,DMSO-d6)δ9.10–8.89(m,2H),8.84(s,1H),8.51(s,1H),8.17(s,1H),6.88(s,1H),6.57(s,1H),6.29(s,2H),5.99(d,J＝11.7Hz,2H),5.42(s,1H),4.54(s,1H),4.33(s,1H),3.91-3.80(m,1H),3.66(s,6H),3.45-3.33(m,1H),3.12-2.95(m,1H).
效果实施例
本发明中的部分化合物抑制肿瘤细胞的活性实验
一、实验材料
1、供试化合物：上述各实施例所制备的化合物1～20。
2、对照化合物：依托泊苷(etoposide，VP-16)，顺铂(Cisplatin,CDDP)。
3、细胞株：人非小细胞肺癌(A549)细胞株，人口腔癌上皮(KB)细胞株，人肝癌(HepG2)细胞株和小鼠淋巴细胞白血病(L1210)细胞株。
二、试验方法
(1)细胞培养：将A549细胞株，HepG2细胞株、KB细胞株和L1210细胞株从液氮中取出，在37℃水浴中迅速解冻，细胞在无菌操作台中移入10mL无菌离心管中加入6mLDMEM细胞培养基，1000转/分离心5min。弃去上清液，沉淀中加入5～6mLDMEM细胞培养基，滴管吹打使其悬浮后移入细胞培养瓶中，置37℃细胞培养箱内，次日，自培养箱中取出细胞，弃去细胞瓶中的DMEM细胞培养基，加入5～6mL，次日自培养箱中取出细胞，弃去细胞瓶中DMEM细胞培养基，加入5～6mLDMEM细胞培养基，置37℃细胞培养箱内。隔日，自培养箱中取出细胞，弃去细胞瓶中DMEM细胞培养基，加入PBS(PH7.4)2～3mL晃动清洗，倒掉PBS溶液后再重复一次清洗。在培养瓶中加入3～5滴0.25％胰蛋白酶溶液晃动均匀，加盖置于37℃细胞培养箱内3分钟左右，于显微镜下观察发现细胞自培养瓶壁上脱离，加DMEM细胞培养基2mL，滴管吹打使细胞完全脱离瓶壁后，分别移入2个干净培养瓶中，加入DMEM细胞培养基5～6mL吹打均匀，置于37℃细胞培养箱内。在整个培养过程中，细胞不允许生长过密。
(2)样品制备：将样品(化合物1～20)溶解于二甲亚砜中，得到浓度为10mg/mL的溶液。再用PBS作梯度稀释，得到浓度分别为1000μg/mL、100μg/mL、10μg/mL、1μg/mL、0.1μg/mL、0.01μg/mL的稀释样品。
对照品制备：将顺铂溶解于PBS中，得到浓度为1mg/mL的溶液。再用PBS作梯度稀释，得到浓度分别为1000μg/mL、100μg/mL、10μg/mL、1μg/mL、0.1μg/mL、0.01μg/mL的稀释对照品。
(3)将稀释好的样品加入平底96孔板中，每孔10uL，每点作两个平行测试。将DMSO相应作梯度稀释后加入板中，作为对照。
(4)取处于对数生长期的细胞，细胞经胰酶消化并洗涤后悬浮于含10％小牛血清的DMEM培养基中，经苔盼蓝染色排除法计活细胞数，并调节细胞悬浮液密度至2×105细胞/mL。
(5)在平底96孔板中，每孔加入90微升细胞，于37℃、5％CO2细胞培养箱中培养过夜。
(6)将加入细胞的平底96孔板在37℃、5％CO2细胞培养箱中培养48小时。
(7)每孔中加入20uL5mg/mLMTT溶液，继续在培养箱中保温3～4小时。
(8)每孔加入100uL溶解液，继续在培养箱中保温过夜，使生成的甲臢晶体充分溶解。测定492nm光吸收值。
(9)根据光吸收值计算化合物处理后细胞相对存活率。计算公式如下：
Figure PCTCN2014092098-appb-000005
(10)通过软件计算化合物对各肿瘤细胞的IC50。
三、实验结果
本发明的鬼臼毒素衍生物对各细胞株的体外增殖抑制作用的药理数据如表1所示。
表1
Figure PCTCN2014092098-appb-000006
Figure PCTCN2014092098-appb-000007
细胞活性显示：整体上说，酰胺类化合物的抗肿瘤活性比酯类化合物的抗肿瘤活性好。
酯类化合物中，对于鬼臼毒素类衍生物来说，当吡啶环上的取代基为C1～C4的甲氧基时，6位取代活性较2-位取代活性好；吡啶环上卤素取代能够增加化合物对肿瘤细胞的抑制活性，并且吡啶环的2、5和6位同时有卤素取代基时(例如本发明化合物8)，对肿瘤细胞的抑制活性较好。对于表鬼臼毒素类化合物来说，当吡啶环上的取代基为C1～C4的甲氧基时，2位取代活性较6位取代活性好。
酰胺类化合物中，当吡啶环上的取代基单取代，且为甲氧基时，6位取代活性较2位取代活性好。当吡啶环上的取代基为单取代且为F时，对肿瘤细胞的抑制活性较好，但是，当吡啶环的2,6位为Cl，且5为F时，化合物抗肿瘤活性较差(例如本发明化合物16)。
吡啶环的2-氨基取代时，无论是酰胺或者是酯类化合物，对四种肿瘤细胞的抑制活性均较好，尤其是对A549细胞株的抑制活性，IC50值均在10μg/mL以下。
吡啶环的6-位为甲基还是甲氧基取代活性均较2-位同样的基团取代活性要好；2-位取代，甲基活性高于甲氧基活性，推测此位置取代时，活性与基团大小有关。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (20)

  1. 一种通式I所示鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物；
    Figure PCTCN2014092098-appb-100001
    其中，用*号标注的碳为α构型手性碳或β构型手性碳；
    X为氧或氮；R1为单取代或多取代，取代位置任意，R1为卤素、C1～C4的烷基、C1～C4的烷氧基、氨基、硝基和羟基中的一种或多种；R2为氢或C1～C4的烷基；
    当X为O，且R1为单取代时，R1不为2-氯，6-氯或5-溴；
    当X为N，且R1为单取代时，R1不为2-氯或6-氯；当X为N，且R1为双取代时，R1不为4-氯和6-甲基；
    当R2为氢时，用*号标注的碳为β构型手性碳；当R2为C1～C4的烷基时，用*标注的碳为α构型手性碳。
  2. 如权利要求1所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物，其特征在于，
    所述的卤素为氟、氯、溴或碘；和/或，R1或R2中，所述的C1～C4的烷基为甲基、乙基、正丙基、异丙基或丁基；和/或，所述的C1～C4的烷氧基为甲氧基、乙氧基、丙氧基或丁氧基。
  3. 如权利要求1或2所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物，其特征在于，R1表示的取代基位于吡啶环的碳原子的2位、5位和6位中的一个或多个。
  4. 如权利要求1～3中至少一项所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物，其特征在于，
    当X为氧，R2为氢时，R1为卤素、C1～C4的烷基、C1～C4的烷氧基、氨基和羟基中的一种或多种；R1表示的取代基位于吡啶环的碳原子的2位、5位和6位中的一个或多个；
    当X为氧，R2为C1～C4的烷基时，R1为卤素、C1～C4的烷基、C1～C4的烷氧基、氨基和羟基中的一种或多种；R1表示的取代基位于吡啶环的碳原子的2位、5位和6位中的一个或多个；
    当X为氮，R2为氢时，R1为卤素、C1～C4的烷基、C1～C4的烷氧基、氨基和羟基中的一种或多种；R1表示的取代基位于吡啶环的碳原子的2位、5位和6位中的一个或多个。
  5. 如权利要求1～4中至少一项所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物，其特征在于，
    当X为氧，R2为氢时，R1为卤素、C1～C4的烷氧基和氨基中的一种或多种；当R1为卤素和/或氨基时，R1表示的取代基位于吡啶环的碳原子的2位、5位和6位中的一个或多个；当R1为C1～C4的烷氧基时，R1表示的取代基位于吡啶环的碳原子的6位；
    当X为氧，R2为C1～C4的烷基时，R1为卤素、C1～C4的烷氧基和氨基中的一种或多种；当R1为卤素和/或氨基时，R1表示的取代基位于吡啶环的碳原子的2位、5位和6位中的一个或多个；当R1为C1～C4的烷氧基时，R1表示的取代基位于吡啶环的碳原子的2位；
    当X为氮，R2为氢时，R1为卤素、C1～C4的烷基、C1～C4的烷氧基和氨基中的一种或多种；当R1为C1～C4的烷基和/或氨基时，R1表示的取代基位于吡啶环的碳原子的2位、5位和6位中的一个或多个；当R1为C1～C4的烷氧基时，R1表示的取代基位于吡啶环的碳原子的6位；当R1为卤素时，R1为单取代；R1表示的取代基位于吡啶环的碳原子的5位。
  6. 如权利要求1～5中至少一项所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物，其特征在于，X为氧或氮，R2为氢，R1为氨基。
  7. 如权利要求1～6中至少一项所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物，其特征在于，其为如下任一化合物：
    Figure PCTCN2014092098-appb-100002
    Figure PCTCN2014092098-appb-100003
  8. 一种如权利要求1～7中至少一项所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物在制备用于治疗癌症药物中的应用。
  9. 如权利要求8所述的应用，其特征在于，所述的癌症为肺癌、口腔癌、肝癌或白血病。
  10. 如权利要求9所述的应用，其特征在于，所述的肺癌为人非小细胞肺癌；和/或，所述的口腔癌为人口腔上皮癌。
  11. 如权利要求8～10中至少一项所述的应用，其特征在于，所述的癌症的肿瘤细胞为人非小细胞肺癌细胞、人口腔癌上皮细胞、人肝癌细胞或小鼠淋巴白血病细胞。
  12. 如权利要求11所述的应用，其特征在于，所述的人非小细胞肺癌细胞为人非小细胞肺癌细胞株A549；和/或，所述的人口腔癌细胞为人口腔癌上皮细胞株KB；和/或，所述的人肝癌细胞为人肝癌细胞株HepG2；和/或，所述的小鼠淋巴白血病细胞为小鼠淋巴细胞白血病细胞株L1210。
  13. 一种治疗癌症的方法，其特征在于，通过给予治疗有效量如权利要求1～7中至少一项所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物。
  14. 一种用于治疗癌症的药物组合物，其特征在于，其包含如权利要求1～7中至少一项所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物中的一种或多种，以及药学上可接受的赋形剂，所述的癌症如权利要求8～12中至少一项所述。
  15. 一种药物组合物，其特征在于，其包含如权利要求1～7中至少一项所述的通式I所示的鬼臼毒素衍生物、其立体异构体、互变异构体、药学上可接受的盐、前药或溶剂化物中的一种或多种，以及药学上可接受的赋形剂。
  16. 如权利要求1～7中至少一项所述的通式I所示鬼臼毒素衍生物的制备方法，其特征在于，其包含下列步骤：有机溶剂中，碱的存在下，在缩合剂的作用下，将如式A所示的化合物或其盐，与如式B所示的化合物进行如下所示的反应；
    Figure PCTCN2014092098-appb-100004
    其中，R为羟基或氨基；*、R1和R2的定义如权利要求1～6任一项所述。
  17. 如权利要求16所述的制备方法，其特征在于，有机溶剂中，碱的存在下，在缩合剂和催化剂的作用下，将如式A所示的化合物或其盐，与如式B所示的化合物进行反应。
  18. 如权利要求17所述的制备方法，其特征在于，
    所述的如式A所示的化合物的盐为如式A所示的化合物的盐酸盐；
    和/或，所述的有机溶剂为卤代烃类溶剂和/或酰胺类溶剂；
    和/或，所述的缩合剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸、N,N’-二环己基碳二酰亚胺和氰基磷酸二乙酯中的一种或多种；或者，所述的缩合剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸、N,N’-二环己基碳二酰亚胺和氰基磷酸二乙酯中的一种或多种，与1-羟基苯并三氮唑的混合物；
    和/或，所述的碱为有机碱；
    和/或，所述的催化剂为4-二甲氨基吡啶；
    和/或，所述的碱，与如式A所示的化合物或其盐的摩尔比为1:1～3:1；
    和/或，所述的如式A所示的化合物或其盐，与如式B所示的化合物的摩尔比为1:1～1:1.5；
    和/或，所述的缩合剂，与如式A所示的化合物或其盐的摩尔比为1:1～3:1；
    和/或，所述的催化剂，与如式A所示的化合物或其盐的摩尔比为0.05:1～0.1:1；
    和/或，所述的有机溶剂，与如式A所示的化合物或其盐的质量体积比为1mL/g～10mL/g；
    和/或，所述的反应的温度为0～30℃。
  19. 如权利要求18所述的制备方法，其特征在于，所述的混合物为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸与1-羟基苯并三氮唑的混合物；其中，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸与1-羟基苯并三氮唑的摩尔比为1:1。
  20. 如权利要求19所述的制备方法，其特征在于，
    当X为O时，将如式A所示的化合物与如式B所示的化合物进行酯化反应；所述的有机溶剂为卤代烃类溶剂；所述的缩合剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸和/或N,N’-二环己基碳二酰亚胺；或者，所述的缩合剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸和/或N,N’-二环己基碳二酰亚胺，与1-羟基苯并三氮唑的混合物；
    当X为N时，将如式A所示的化合物的盐与如式B所示的化合物进行酰胺化反应；所述的有机溶剂为卤代烃类溶剂和/或酰胺类溶剂；所述的缩合剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸和/或氰基磷酸二乙酯；或者，所述的缩合剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸和/或氰基磷酸二乙酯，与1-羟基苯并三氮唑的混合物。
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