WO2015161745A1 - 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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WO2015161745A1
WO2015161745A1 PCT/CN2015/076505 CN2015076505W WO2015161745A1 WO 2015161745 A1 WO2015161745 A1 WO 2015161745A1 CN 2015076505 W CN2015076505 W CN 2015076505W WO 2015161745 A1 WO2015161745 A1 WO 2015161745A1
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substituted
group
unsubstituted
formula
heteroaryl
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French (fr)
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肖旭华
孙亚飞
姚利霞
沈舜义
刘全海
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中国医药工业研究总院
上海医药工业研究院
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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings

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.
  • the above antitumor drugs have a narrow spectrum of tumor inhibition, accompanied by severe myelosuppression and gastrointestinal side effects. Therefore, there is a need in the art for a podophyllotoxin derivative having a broad antitumor spectrum, no obvious myelosuppression and gastrointestinal side effects, and is used as an antitumor drug in clinical practice.
  • 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 and the pharmaceutical compositions thereof have good tumor cell inhibitory activity, and the preparation method and post-treatment thereof are simple and easy, and have good market development prospects.
  • the present invention provides a podophyllotoxin derivative of the formula I:
  • the substituent described in the group or the substituted C 2 -C 10 heterocycloalkyl group is substituted by one or more of the following substituents: hal
  • the "substituted or unsubstituted C 5 - C 10 aryl group” is preferably a substituted or unsubstituted naphthyl group.
  • the naphthyl group is preferably
  • the substituted or unsubstituted heteroaryl preferably C 2 ⁇ C 10 means that the heteroatom is N, O or S, the number of hetero atoms, 1 to 4 substituted or unsubstituted C 2 ⁇ C 10 Heteroaryl.
  • the hetero atom is N, O or S, and the substituted or unsubstituted C 2 -C 10 heteroaryl group having 1 to 4 hetero atoms preferably means the hetero atom is N or S, and the number of hetero atoms It is 1 or 2 substituted or unsubstituted C 4 -C 10 heteroaryl groups.
  • the substituted or unsubstituted C 4 -C 10 heteroaryl group is preferably a substituted or unsubstituted pyrrolyl group, a substituted or unsubstituted thienyl group, a substituted or unsubstituted quinolyl group, a substituted or unsubstituted group.
  • the pyrrolyl group is preferably The thiophene group is preferably The substituted thienyl group is preferably The quinolyl group is preferably The imidazolyl group is preferably The pyrazolyl group is preferably The benzopyrazolyl group is preferably The sulfhydryl group is preferably The substituted fluorenyl group is preferably
  • the substituted or unsubstituted heterocycloalkyl C 2 ⁇ C 10 is preferably a hetero atom means N, O or S, the number of hetero atoms, 1 to 4 substituted or unsubstituted C 2 ⁇ C 10 heterocycloalkyl.
  • the hetero atom is N, O or S, and the substituted or unsubstituted C 2 -C 10 heterocycloalkyl group having 1 to 4 hetero atoms preferably means that the hetero atom is N and the number of hetero atoms is One substituted or unsubstituted C 4 -C 8 heterocycloalkyl group.
  • the substituted or unsubstituted C 4 -C 8 heterocycloalkyl group is preferably a substituted or unsubstituted tetrahydropyrrolyl group (for example ).
  • the substituted tetrahydropyrrolyl group is preferably
  • R is a substituted or unsubstituted C 5 -C 10 aryl group, a substituted or unsubstituted C 2 -C. a heteroaryl group of 10 or a substituted C 2 -C 10 heterocycloalkyl group, n being 0, 1, 2 or 3.
  • R is a substituted or unsubstituted C 5 -C 10 aryl group
  • X is oxygen
  • R is a substituted or unsubstituted C 2 -C 10 heteroaryl group
  • the heteroaryl group of 2 to C 10 is preferably a substituted or unsubstituted pyrrolyl group, a substituted or unsubstituted thienyl group, a substituted or unsubstituted imidazolyl group, a substituted or unsubstituted pyrazolyl group, a substituted or unsubstituted one.
  • R is a substituted thienyl group, and the substitution is substituted by a C 1 -C 4 alkyl group, X is a nitrogen.
  • R is a substituted C 2 -C 10 heterocycloalkyl group, the substitution means a hydroxyl group and Replaced.
  • R is a substituted or unsubstituted C 2 -C 10 heteroaryl group, or a substituted or unsubstituted C. a heterocycloalkyl group of 2 to C 10 ; n is 0, 1, 2 or 3.
  • R is a substituted or unsubstituted C 5 -C 10 aryl group, or a substituted or unsubstituted C 2 group. a heteroaryl group of -C 10 wherein n is 0, 1, 2 or 3.
  • R is a substituted or unsubstituted C 5 -C 10 aryl group, n is 1, and X is oxygen; when R is a substituted or unsubstituted C 2 -C 10 heteroaryl group, the substitution or The unsubstituted C 2 -C 10 heteroaryl group is preferably a substituted or unsubstituted pyrrolyl group, a substituted or unsubstituted thienyl group, a substituted or unsubstituted imidazolyl group or a substituted or unsubstituted anthracenyl group.
  • R When R is a substituted thienyl group and the substitution is substituted by a C 1 -C 4 alkyl group, X is oxygen.
  • R When R is a substituted or unsubstituted fluorenyl group, when n is 1, 2 or 3, X is O; when the thiol group is When n is 0, X is N.
  • R When R is a substituted indenyl group, the substitution is substituted by a halogen, when n is 0, X is O; when R is a substituted indenyl group, the substitution is a C 1 -C 4 alkane Substituted by an oxy group, when n is 0, X is N.
  • R is a substituted or unsubstituted C 2 -C 10 heteroaryl group, and n is 0, 1, 2 Or 3.
  • R is a substituted or unsubstituted C 2 -C 10 heteroaryl group, and n is 0, 1, 2 Or 3.
  • the substituted or unsubstituted C 2 -C 10 heteroaryl group is a substituted or unsubstituted pyrrolyl group, a substituted or unsubstituted thienyl group or a fluorenyl group
  • X is N;
  • the substitution is replaced by a halogen, and when n is 0, X is O.
  • R is a substituted or unsubstituted C 2 -C 10 heteroaryl group, and n is 0 or 1;
  • the substituted or unsubstituted C 2 -C 10 heteroaryl group is preferably a substituted or unsubstituted pyrrolyl group, a substituted or unsubstituted thienyl group, a substituted or unsubstituted imidazolyl group or a substituted or unsubstituted anthracene.
  • the podophyllotoxin derivative represented by the above formula I preferably, is any of the following compounds:
  • 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 and the compound of formula III are subjected to a condensation reaction as shown below;
  • R 1 is a hydroxyl group (-OH) or an amino group (-NH 2 ); and X, R and n are as defined above.
  • the method and conditions of the condensation reaction can be conventional methods and conditions for such reactions in the art.
  • the following methods and conditions are particularly preferred: the compound of the formula II and the compound of the formula III are subjected to the condensation reaction in the presence of a base in the presence of a base under the action of a condensing agent and a catalyst.
  • the compound represented by Formula II may also participate in the reaction in the form of a hydrochloride.
  • the organic solvent is preferably a halogenated hydrocarbon solvent and/or an amide solvent.
  • the halogenated hydrocarbon solvent is preferably a chlorinated hydrocarbon solvent.
  • the chlorinated 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), 1-hydroxybenzotriazole (HOBt), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylhexafluorophosphate (HATU), N,N'-dicyclohexylcarbodiimide (DCC And one or more of diethyl cyanophosphate (DEPC), more preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and/or 1-Hydroxybenzotriazole (HOBt).
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • HOBt 1-hydroxybenzotriazole
  • the molar ratio of EDCI to HOBt is preferably 1:1.
  • the base is preferably an organic base.
  • the organic base is preferably triethylamine.
  • the catalyst is preferably 4-dimethylaminopyridine (DMAP).
  • DMAP 4-dimethylaminopyridine
  • the amount of the catalyst used may be a conventional amount of such a reaction catalyst in the art.
  • the molar ratio of the catalyst to the compound of the formula II is from 0.05:1 to 0.5:1.
  • the molar ratio of the organic base to the compound of formula II is preferably from 1:1 to 3:1.
  • the molar ratio of the condensing agent to the compound of formula II is preferably from 1:1 to 4:1.
  • the molar ratio of the compound of the formula II to the compound of the formula III is preferably from 1:1 to 1:3.
  • the mass ratio of the organic solvent to the compound of the formula II is preferably from 1 mL/g to 10 mL/g.
  • the temperature of the condensation reaction is preferably from 0 ° C to 30 ° C.
  • the progress of the condensation reaction can be monitored by conventional test methods in the art (e.g., TLC, HPLC or NMR), and the reaction time is preferably from 1 to 24 hours.
  • a post-treatment step may also be included.
  • the methods and conditions of the post-treatment steps can be post-processing conventional methods and conditions in the art, preferably including the following methods:
  • Method 1 After the completion of the above reaction, the reaction is quenched with water, extracted with an ester solvent (for example, ethyl acetate) or a halogenated hydrocarbon solvent (for example, dichloromethane) (3 times), and the organic layer is saturated aqueous sodium hydrogen carbonate, Washed with water, or washed with saturated sodium hydrogencarbonate, water, brine (saturated aqueous sodium chloride); the organic layer is dried (for example, dried with anhydrous sodium sulfate), filtered, and the solvent is removed (for example, under reduced pressure), and the crude product is subjected to column chromatography.
  • an ester solvent for example, ethyl acetate
  • a halogenated hydrocarbon solvent for example, dichloromethane
  • Method 2 After the completion of the above reaction, when solids are formed in the reaction solution, the mixture is filtered, washed (for example, DCM), and dried to obtain.
  • Method 3 When DEPC is used as the condensing agent, after the above reaction is completed, the reaction liquid is mixed with water, and extracted with an ester solvent (for example, ethyl acetate) or a halogenated hydrocarbon solvent (for example, dichloromethane), and the organic layer is sequentially The mixture was washed with a 5% aqueous solution of hydrochloric acid and a saturated aqueous solution of sodium hydrogencarbonate, and the organic layer was adjusted to pH 7-8 with 1% sodium hydroxide solution, washed with water three times, dried over anhydrous sodium sulfate, decolorized with activated carbon, and ethyl acetate was removed.
  • an ester solvent for example, ethyl acetate
  • a halogenated hydrocarbon solvent for example, dichloromethane
  • the target compound is obtained; or the crude product obtained is purified by column chromatography (petroleum ether/ethyl acetate system, dichloromethane/ethyl acetate system or dichloromethane/methanol system (3:1 to 1:3, v/v) )) to obtain the target compound.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the podophyllotoxin derivative of the above formula I and a pharmaceutically acceptable excipient.
  • compositions may be formulated into various types of dosage unit dosage forms, for example, aqueous fractions, depending on the purpose of the treatment.
  • the invention also provides the use of the podophyllotoxin derivative of the formula I described in the manufacture of a medicament for the treatment of cancer.
  • the cancer is preferably lung cancer, oral cancer, liver cancer or leukemia.
  • 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.
  • room temperature means 0 to 30 °C.
  • the ice bath means -5 to 0 °C.
  • the reagents and starting materials used in the present invention are commercially available.
  • the podophyllotoxin derivative of the invention has good tumor cell inhibitory activity, and the preparation method and post-treatment thereof are simple and easy, and have good market development prospects.
  • Fig. 1 is a photographic diagram showing the growth inhibition of ICR mouse S180 xenograft tumor by intravenous administration of Compound I-4 in Effect Example 2.
  • the feed ratio is generally referred to as a molar ratio unless otherwise specified.
  • Room temperature means 0 to 30 °C.
  • the ice bath means -5 to 0 °C.
  • Compound III Compound II (4'-desmethylepipodophyllotoxin DMEP): DMAP (1.5 eq: 1 eq: 0.05 eq) was added to an appropriate amount of dichloromethane at room temperature, and added dropwise with triethylamine (1 eq). After 15 min, DCC (1.5 eq) was added to the reaction solution, and the reaction was continued for 3-5 h. During the reaction, solids formed, and the reaction was stopped by TLC, and the reaction was stopped. Post-treatment: suction filtration (or filtration), the filter cake is washed with an appropriate amount of solvent (for example, dichloromethane), and dried to obtain the target compound.
  • solvent for example, dichloromethane
  • EA Ethyl acetate
  • the compound 4 ⁇ -(1-naphthoyloxy)-4-deoxy- was prepared by using 1-naphthoic acid (258 mg, 1.5 mmol) as raw material, 2 mL of DCM at 30 ° C, and the rest of the operation and feeding ratio were compared with reference to the second method. 4'-demethylepipodophyllotoxin, after the reaction was completed, the obtained crude product was separated by column chromatography eluting with petroleum ether and ethyl acetate.
  • N-Boc-L-hydroxyproline (395 mg, 1.725 mmol) was added to 3 mL of N,N-dimethylformamide (DMF), and the rest of the operation and charge ratio were compared with the third method to prepare I-22 to obtain white. Solid 333 mg, yield: 47.39%.
  • the cytotoxic effect of the podophyllotoxin derivatives of the present invention on human tumor cell lines was determined by MTT assay in vitro.
  • Cell lines human lung cancer (A549), human liver cancer (HepG2), human oral cancer (KB), and mouse leukemia cells (L1210), and the cell lines were purchased from the Institute of Cell Sciences of the Chinese Academy of Sciences.
  • DMEM cell culture medium containing 10% inactivated newborn calf serum (Shanghai Saida Biopharmaceutical Co., Ltd.); L-glutamyl ammonia (imported sub-package, SANGON); sodium pyruvate; ⁇ 10 5 UL -1 penicillin, 100 mg.L -1 streptomycin; sterile filtration, storage at 4 °C.
  • Trypsin purchased from Invitrogen, stored at -20 °C.
  • PBS Phosphate buffer
  • MTT (AMRESCO) solution A 5 mg/mL solution was prepared with PBS.
  • the cytotoxicity of the podophyllotoxin to the above tumor cell lines was measured by the MTT method. Specific steps are as follows:
  • Cell culture 1 The cells 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, and 6 mL of DMEM cell culture medium was added, 1000 rpm/separation of heart 5 minute. The supernatant was discarded, 5-6 mL of DMEM cell culture medium was added to the pellet, and the tube was dropped by a dropper, and then transferred to a cell culture flask, and placed in a 37 ° C cell culture incubator. On the 2nd day, the cells were removed from the incubator and the DMEM cells in the cell vials were discarded.
  • Sample preparation The sample was 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.
  • Control preparation Etoposide injection was diluted with PBS to obtain diluted control products at 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 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 numbers I-24, 1-2 to I-7 and VP16 in Table 1.1 are the results of the activity measured by the inventor Sun Yafei before the filing date of the Chinese patent application CN201410172493.7, and the numbers I-1, I in Table 1.2.
  • the activity results of -8 to I-23, I-25 to I-37, and VP16 were measured by the inventor Yao Lixia after the filing date of the Chinese patent application CN201410172493.7.
  • I-4 The anti-tumor effect of I-4 was studied by using ICR mouse S180 xenografts as a model (S180 cells were purchased from the Institute of Cell Sciences, Chinese Academy of Sciences).
  • Compound I-4 was prepared by first dissolving the sample with 0.5% DMSO, then dissolving with 4% Tween-80, and diluting to the desired concentration with physiological saline.
  • Positive control Etoposide, diluted with physiological saline during preparation.
  • mice 60 ICR mice, female, weighing 18-20g, were provided by Xibikai Experimental Animal Co., Ltd., certificate: SCXK (Shanghai) 2008-0016.
  • Tumor strain S180 ascites tumor, ICR mice taken from S180 ascites tumor.
  • mice S180 ascites tumors, were harvested in the vigorous growth stage. Ascites was aspirated under sterile conditions, diluted 1:6 with normal saline, and subcutaneously inoculated with 0.2 mL/mouse (60 inoculation). The mice were randomly divided into 6 groups on the next day, 10 in each group.
  • I-410 mg/kg iv group continuous administration for 4 days, iv means intravenous injection
  • I-45 mg/kg iv group continuous administration for 4 days
  • I-42.5 mg/kg iv group Continuous administration for 4 days
  • I-41.25 mg/kg iv group continuous administration for 4 days
  • etoposide 5 mg/kg iv administration for 7 days.
  • mice The administration of ICR mice began on the second day after inoculation.
  • the dosage and administration schedule are shown in Table 2. Animals that survived the end of treatment were euthanized, and the tumor pieces were weighed to calculate the tumor inhibition rate.
  • the ICR mice in the I-410 mg/kg iv group and the I-45 mg/kg iv group were given a weight loss of >20% after 4 days of administration, so the ICT mice of the I-4 group from the 5th day. The administration was stopped, and the positive control group was continued to be administered to the 7th day with etoposide 5 mg/kg.
  • I-410 mg/kg iv group ICR mice died on the 6th day after administration, and all died at the end of treatment, while I-45 mg/kg iv group ICR mice began to recover weight, and the other groups of ICR mice did not die. .
  • the structure-activity relationship of the podophyllotoxin derivative represented by the general formula I of the present invention is analyzed: when X is N, the compound of the formula I is represented by Ia; when X is 0
  • the compound of formula I is shown as Ib:
  • the antitumor activity ratio R is a small molecule such as a pyrrolyl group or a thienyl group.
  • the activity at the time of the group is low.
  • the number of carbon atoms of the R group is equivalent, for example, when R is a naphthyl group and a quinolyl group, when R is a quinolyl group, the antitumor activity of the compound is better than R being a naphthyl group, indicating that R is a heteroaryl group.
  • the antitumor activity is better than that of the aryl group.
  • R is a small molecular group, such as pyrrole or thiophene
  • the obtained compound has an inhibitory effect on cell lines such as HepG2, KB and L1210, and is VP-16.
  • A549 cells with weaker inhibitory effects also have an inhibitory effect.
  • R is a thiol group: when the 1R group is linked to 4-amino-4'-demethylepipodophyllotoxin at the ⁇ and ⁇ positions, it has an effect on the inhibitory activity of tumor cells.
  • I-25 inhibited the tumor cell line KB and A549 cell lines, and inhibited HepG2 and L1210 cells.
  • I-33 was superior to I-25, that is, ⁇ -substitution was better than ⁇ -position.
  • the introduction of an electron-donating substituent on the 2R group has an effect on the activity; when I-25 is compared with I-27, I-29, and I-31, the five methoxy groups on the anthracene ring and the halogen are substituted for four tumors.
  • the inhibition of cell lines was slightly reduced.
  • the substitution of methoxy at position 5 enhanced the inhibition of HepG2 and L1210 cell lines.
  • the introduction of halogen reduced the inhibitory activity of four cell lines; 3 the length of the linker affected antitumor activity.
  • I-33 has a reduced inhibition of four cell lines by carbon chain growth. All the obtained compounds had inhibitory effects on cell lines such as HepG2, KB and L1210, and also inhibited VP-16-inactivated A549 cells.
  • R is a thiol group: when the 1R group is linked to 4-hydroxy-4'-demethylepipodophyllotoxin at the ⁇ and ⁇ positions, it has an effect on the inhibitory activity of tumor cells.
  • the inhibitory effect of I-24 on the four tumor cell lines tested was better than I-32, that is, the ⁇ -position substitution was better than the ⁇ -position; the electron-donating substituent pair activity was introduced on the 2R group. influential.
  • I-26, I-28 and I-30 the inhibition of four tumor cell lines by 5-position methoxy group and halogen substitution on the anthracene ring did not improve much.
  • the methoxy substitution enhanced the inhibition of the L1210 cell line, and the introduction of chlorine enhanced the inhibitory activity against HepG2, KB and L1210; 3 the length of the linker affected the antitumor activity.
  • I-32 enhanced the inhibition of HepG2, A549 and KB cells by carbon chain growth, but decreased the inhibition of L1210 cell line. All the obtained compounds had inhibitory effects on cell lines such as HepG2, KB and L1210, and also inhibited VP-16-inactivated A549 cells.
  • the podophyllotoxin derivative represented by Formula Ib has better killing power against some cell lines, such as HepG2 and KB cell lines, but its anti-tumor spectrum is more general.
  • the podophyllotoxin derivative shown by Ia is narrow.
  • the podophyllotoxin derivative represented by the formula I-a obtained by the present invention has a broad antitumor spectrum, and when R contains a hetero atom, it is advantageous for enhancing the antitumor activity of the compound.

Abstract

本发明公开了一种鬼臼毒素衍生物、其制备方法、药物组合物及应用。本发明公开的制备方法，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II所示的化合物与如式III所示的化合物进行缩合反应。本发明公开的药物组合物，其包含通式I所示鬼臼毒素衍生物以及药学上可接受的赋形剂。本发明还公开了通式I所示的鬼臼毒素衍生物在制备用于治疗癌症的药物中的应用。本发明的鬼臼毒素衍生物具有良好的肿瘤细胞抑制活性，其制备方法和后处理简单易行，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2014年4月25日的中国专利申请CN201410172493.7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。因此，本领域亟需一种抗瘤谱广、无明显骨髓抑制及胃肠道副反应的鬼臼毒素类衍生物，作为抗肿瘤药物应用于临床。
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及其药物组合物具有良好的肿瘤细胞抑制活性，其制备方法和后处理简单易行，具有良好的市场开发前景。
本发明提供了一种通式I所示的鬼臼毒素衍生物：
Figure PCTCN2015076505-appb-000001
其中，X为氧或氮；R为取代或未取代的C5～C10的芳基、取代或未取代的C2～C10的杂芳基，或取代或未取代的C2～C10的杂环烷基；n为0、1、2、3或4；当n为0时，R不为取代或未取代的苯基或取代或未取代的吡啶基，当n＝1，X为N时，R不为取代或 未取代的吲哚基或取代或未取代的噻吩基；所述的取代的C5～C10的芳基、所述的取代的C2～C10的杂芳基或所述的取代的C2～C10的杂环烷基中所述的取代是指被下列一个或多个取代基所取代：卤素(优选氟、氯、溴或碘)、硝基、氨基、羟基、C1～C4的烷基(优选甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基)或
Figure PCTCN2015076505-appb-000002
其中，Ra为C1～C4的烷基(优选甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基)，当取代基为多个时，所述的取代基相同或不同。
所述的“取代或未取代的C5～C10的芳基”较佳地为取代或未取代的萘基。所述的萘基较佳地为
Figure PCTCN2015076505-appb-000003
所述的取代或未取代的C2～C10的杂芳基较佳地是指杂原子为N、O或S，杂原子数为1～4个的取代或未取代的C2～C10的杂芳基。所述的杂原子为N、O或S，杂原子数为1～4个的取代或未取代的C2～C10的杂芳基较佳地是指杂原子为N或S，杂原子数为1或2个的取代或未取代的C4～C10的杂芳基。所述的取代或未取代的C4～C10的杂芳基较佳地为取代或未取代的吡咯基、取代或未取代的噻吩基、取代或未取代的喹啉基、取代或未取代的咪唑基、取代或未取代的吡唑基、取代或未取代的苯并吡唑基或取代或未取代的吲哚基。其中，所述的吡咯基较佳地为
Figure PCTCN2015076505-appb-000004
所述的噻吩基较佳地为
Figure PCTCN2015076505-appb-000005
所述的取代的噻吩基较佳地为
Figure PCTCN2015076505-appb-000006
所述的喹啉基较佳地为
Figure PCTCN2015076505-appb-000007
所述的咪唑基较佳地为
Figure PCTCN2015076505-appb-000008
所述的吡唑基较佳地为
Figure PCTCN2015076505-appb-000009
所述的苯并吡唑基较佳地为
Figure PCTCN2015076505-appb-000010
所述的吲哚基较佳地为
Figure PCTCN2015076505-appb-000011
所述的取代的吲哚基较佳地为
Figure PCTCN2015076505-appb-000012
所述的取代或未取代的C2～C10的杂环烷基较佳地是指杂原子为N、O或S，杂原子 数为1～4个的取代或未取代的C2～C10的杂环烷基。所述的杂原子为N、O或S，杂原子数为1～4个的取代或未取代的C2～C10的杂环烷基较佳地是指杂原子为N，杂原子数为1个的取代或未取代的C4～C8的杂环烷基。所述的取代或未取代的C4～C8的杂环烷基较佳地为取代或未取代的四氢吡咯基(例如
Figure PCTCN2015076505-appb-000013
)。其中，所述的取代的四氢吡咯基较佳地为
Figure PCTCN2015076505-appb-000014
本发明一方面，所述的通式I所示的鬼臼毒素衍生物中，较佳地，R为取代或未取代的C5～C10的芳基、取代或未取代的C2～C10的杂芳基或取代的C2～C10的杂环烷基，n为0、1、2或3。当R为取代或未取代的C5～C10的芳基时，X为氧；当R为取代或未取代的C2～C10的杂芳基时，所述的取代或未取代的C2～C10的杂芳基较佳地为取代或未取代的吡咯基、取代或未取代的噻吩基、取代或未取代的咪唑基、取代或未取代的吡唑基、取代或未取代的苯并吡唑基或取代或未取代的吲哚基；当R为取代的噻吩基，且所述的取代为被C1～C4的烷基所取代时，X为氮。当R为取代的C2～C10的杂环烷基时，所述的取代是指被羟基和
Figure PCTCN2015076505-appb-000015
所取代。
本发明另一方面，所述的通式I所示的鬼臼毒素衍生物中，较佳地，R为取代或未取代的C2～C10的杂芳基，或取代或未取代的C2～C10的杂环烷基；n为0、1、2或3。
本发明另一方面，所述的通式I所示的鬼臼毒素衍生物中，较佳地，R为取代或未取代的C5～C10的芳基，或取代或未取代的C2～C10的杂芳基，n为0、1、2或3。当R为取代或未取代的C5～C10的芳基时，n为1，X为氧；当R为取代或未取代的C2～C10的杂芳基时，所述的取代或未取代的C2～C10的杂芳基较佳地为取代或未取代的吡咯基、取代或未取代的噻吩基、取代或未取代的咪唑基或取代或未取代的吲哚基。当R为取代的噻吩基，且所述的取代为被C1～C4的烷基所取代时，X为氧。当R为取代或未取代的吲哚基，n为1、2或3时，X为O；当所述的吲哚基为
Figure PCTCN2015076505-appb-000016
时，n为0，X为N。当R为取代的吲哚基，所述的取代为被卤素所取代，n为0时，X为O；当R为取代的吲哚基，所述的取代为被C1～C4的烷氧基所取代，n为0时，X为N。
本发明另一方面，所述的通式I所示的鬼臼毒素衍生物中，较佳地，R为取代或未取代的C2～C10的杂芳基，n为0、1、2或3。较佳地，当所述的取代或未取代的C2～C10的杂芳基为取代或未取代的吡咯基、取代或未取代的噻吩基或吲哚基时，X为N；当R为取代的吲哚基，所述的取代为被卤素所取代，n为0时，X为O。本发明另一方面，所述的通式I所示的鬼臼毒素衍生物中，较佳地，R为取代或未取代的C2～C10的杂芳基，n为0或1；所述的取代或未取代的C2～C10的杂芳基较佳地为取代或未取代的吡咯基、取代或未取代的噻吩基、取代或未取代的咪唑基或取代或未取代的吲哚基；当所述的取代或未取代的C2～C10的杂芳基为取代或未取代的吡咯基、取代或未取代的噻吩基或吲哚基时，X为N；当R为取代的吲哚基，所述的取代为被卤素所取代时，X为O；当R为取代的吲哚基，所述的取代为被C1～C4的烷氧基所取代时，X为N。
所述的通式I所示的鬼臼毒素衍生物，最佳地，其为如下任一化合物：
Figure PCTCN2015076505-appb-000017
Figure PCTCN2015076505-appb-000018
Figure PCTCN2015076505-appb-000019
本发明还提供了一种所述的通式I所示的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II所示的化合物与如式III所示的化合物进行如下所示的缩合反应，即可；
Figure PCTCN2015076505-appb-000020
其中，R1为羟基(-OH)或氨基(-NH2)；X、R和n的定义均同前所述。
所述的缩合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II所示的化合物与如式III所示的化合物进行所述的缩合反应。其中，所述的如式II所示的化合物也可以盐酸盐的形式参与到反应中。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氯代烃类溶剂。所述的氯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)、1-羟基苯并三氮唑(HOBt)、 2-(7-氮杂苯并三氮唑)-N,N,N’,N’-四甲基六氟磷酸酯(HATU)、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，更佳地为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸(EDCI)和/或1-羟基苯并三氮唑(HOBt)。当所述的缩合剂为EDCI和HOBt时，所述的EDCI和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)。所述的催化剂的用量可为本领域此类反应催化剂的常规用量，较佳地，所述的催化剂与如式II所示的化合物的摩尔比为0.05:1～0.5:1。所述的有机碱与如式II所示的化合物的摩尔比较佳地为1:1～3:1。所述的缩合剂与如式II所示的化合物的摩尔比较佳地为1:1～4:1。所述的如式II所示的化合物与如式III所示的化合物的摩尔比较佳地为1:1～1:3。所述的有机溶剂与如式II所示的化合物的质量体积比较佳地为1mL/g～10mL/g。所述的缩合反应的温度较佳地为0℃～30℃。所述的缩合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。
所述的缩合反应结束后，较佳地，还可包括后处理步骤。所述的后处理步骤的方法和条件可为本领域后处理常规的方法和条件，较佳地包含下列方法：
方法一：上述反应结束后，加水淬灭反应，采用酯类溶剂(例如乙酸乙酯)或卤代烃类溶剂(例如二氯甲烷)萃取(3次)，有机层用饱和碳酸氢钠水溶液、水洗，或者饱和碳酸氢钠、水、盐水(饱和氯化钠水溶液)洗；有机层干燥(例如用无水硫酸钠干燥)，过滤，除去溶剂(例如减压蒸馏)，所得粗品经柱层析(石油醚/乙酸乙酯体系、二氯甲烷/乙酸乙酯体系或者二氯甲烷/甲醇体系(3:1～1:3，v/v))，即得目标化合物，或者所得粗品经重结晶后，即得目标化合物。
方法二：上述反应结束后，当反应液中有固体生成时，过滤，洗涤(例如DCM)滤饼，干燥，即得。
方法三：当采用DEPC作为缩合剂时，上述反应结束后，将反应液与水混合，采用酯类溶剂(例如乙酸乙酯)或卤代烃类溶剂(例如二氯甲烷)萃取，有机层依次用5％的盐酸水溶液，饱和碳酸氢钠水溶液，水洗，有机层用1％氢氧化钠溶液调pH至7-8，水洗3次，无水硫酸钠干燥，活性炭脱色，除去乙酸乙酯后，即得目标化合物；或所得粗品经柱层析分离纯化(石油醚/乙酸乙酯体系、二氯甲烷/乙酸乙酯体系或者二氯甲烷/甲醇体系(3:1～1:3，v/v))即得目标化合物。
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。
本发明还提供了所述的通式I所示的鬼臼毒素衍生物在制备用于治疗癌症的药物中的应用。所述的癌症较佳的为肺癌、口腔癌、肝癌或白血病。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。
本发明中，室温是指0～30℃。冰浴是指-5～0℃。
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，其制备方法和后处理简单易行，具有良好的市场开发前景。
附图说明
图1为效果实施例2中的化合物I-4静脉给药对ICR小鼠S180移植瘤瘤生长抑制的照片图。
具体实施方式
下述实施例中，如未作特别说明，其投料比一般是指摩尔比。室温是指0～30℃。冰浴是指-5～0℃。
①关键中间体的合成：
实施例1
4β-氨基-4’-去甲基表鬼臼毒素的合成4β-氯代乙酰胺基-4’-去甲基表鬼臼毒素的合成
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0.15mL98％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-d6)：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),4.19(2H,s CH2),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’-去甲基表鬼臼毒素的制备
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-d6)：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).
②目标化合物的合成
方法一：
室温下，将化合物III：化合物II(4’-去甲基表鬼臼毒素DMEP)：DMAP(1.5eq：1eq：0.05eq)投入适量二氯甲烷中，并滴入三乙胺(1eq)搅拌15min，加入DCC(1.5eq)至反应液中，继续反应3-5h，反应过程中有固体生成，TLC检测反应不再进行，停止反应。后处理：抽滤(或者过滤)，滤饼用适量溶剂洗涤(例如二氯甲烷)，干燥，即得目标化合物。
方法二：
室温下，化合物III：化合物II(4’-去甲基表鬼臼毒素DMEP)：DMAP(2eq：1eq：0.1eq)投入适量二氯甲烷中，并滴入三乙胺(2eq)，搅拌0.5h，将EDCI(2eq)加入反应液中，4.5h后，TLC检测反应完全，停止反应。后处理：加水淬灭反应。二氯甲烷(DCM)抽提3次，合并二氯甲烷层，依次用饱和碳酸氢钠水溶液、水洗涤，有机层用无水硫酸钠干燥，过滤，减压蒸除溶剂，所得粗品经柱层析(石油醚/乙酸乙酯)纯化后即得目标化合物，或者所得粗品经重结晶(甲醇)后即得目标化合物。
方法三：
室温下，将化合物III：EDCI：HOBt(1.5:1.5:1.5)加入至适量二氯甲烷中，搅拌0.5h后，向反应液中加入4’-去甲基表鬼臼毒素(DMEP，1eq)和三乙胺(2eq)，TLC检测反应不再进行时，加水淬灭反应。二氯甲烷(DCM)抽提3次，合并二氯甲烷层，用饱和NaHCO3溶液、水、盐水洗涤有机层。有机层用无水硫酸钠干燥过夜，过滤除去无水硫酸钠，滤液减压蒸干，所得固体经柱层析，以DCM/EA为洗脱剂梯度洗脱得到目标化合物。
方法四：
室温下，将化合物III：EDCI：DMAP(1.5:1.5:0.1)加入至适量的二氯甲烷中，搅拌0.5h后，向反应液中加入4’-去甲基表鬼臼毒素(DMEP，1eq)和三乙胺(2eq)，TLC检测反应不再进行时，加水淬灭反应。二氯甲烷(DCM)抽提3次，合并二氯甲烷层，用饱和NaHCO3溶液、水、盐水洗涤有机层。有机层用无水硫酸钠干燥过夜，过滤除去无水硫酸钠，滤液减压蒸干，所得固体经柱层析，以DCM/EA为洗脱剂梯度洗脱得到目标化合物。
方法五：
室温下，将化合物III：2-(7-氮杂苯并三氮唑)-N,N,N’,N’-四甲基六氟磷酸酯(HATU)(1.5:1.4)加入至适量的N,N-二甲基甲酰胺(DMF)中，搅拌5～15min后，加入三乙胺(Et3N,3eq)，反应液变黄。继续搅拌30min加入4β-氨基4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(leq)。0.5～3h后TLC检测原料点消失，停止反应。加水淬灭反应。乙酸乙酯(EA)抽提3次，合并乙酸乙酯层，用饱和NaHCO3溶液、5％的盐酸水溶液、水、盐水洗涤有机层。有机层用无水硫酸钠干燥过夜，过滤除去无水硫酸钠，滤液减压蒸干，所得固体经柱层析，以DCM/EA为洗脱剂梯度洗脱得到目标化合物。
方法六：
室温下，将化合物III：EDCI：DMAP(1.5eq：1.5eq：0.1eq)加入至适量的干燥的二氯甲烷中，搅拌0.5h后，向反应液中加入4’-去甲基表鬼臼毒素(DMEP，1eq)和三乙胺(2eq)，TLC检测反应不再进行时，加水淬灭反应。用适量的二氯甲烷抽提3次，合并二氯甲烷层，用饱和NaHCO3溶液、水、盐水(饱和氯化钠水溶液)洗涤有机层。有机层用无水硫酸钠干燥过夜，过滤除去无水硫酸钠，滤液减压蒸干，所得固体经柱层析，以二氯甲烷/乙酸乙酯(DCM/EA)为洗脱剂梯度洗脱得到目标化合物。
方法七：
室温下，将化合物III：EDCI：HOBt(1.5eq：1.5eq：1.5eq)加入至干燥的二氯甲烷 中，搅拌0.5h后，向反应液中加入4’-去甲基表鬼臼毒素(DMEP，1eq)和三乙胺(2eq)，TLC检测反应不再进行时，加水淬灭反应。用适量的二氯甲烷抽提3次，合并二氯甲烷层，用饱和NaHCO3溶液、水、盐水(饱和氯化钠水溶液)洗涤有机层。有机层用无水硫酸钠干燥过夜，过滤除去无水硫酸钠，滤液减压蒸干，所得固体经柱层析，以二氯甲烷/乙酸乙酯(DCM/EA)为洗脱剂梯度洗脱得到目标化合物。
方法八：
室温下，将化合物III：EDCI：HOBt(1.5eq：1.5eq：1.5eq)加入至适量的干燥的N,N-二甲基甲酰胺中，搅拌0.5h后，向反应液中加入4β-氨基4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(1eq)和三乙胺(2eq)，TLC检测反应不再进行时，加水淬灭反应。用适量乙酸乙酯抽提3次，合并乙酸乙酯层，用饱和NaHCO3溶液、水、盐水(饱和氯化钠水溶液)洗涤有机层。有机层用无水硫酸钠干燥过夜，过滤除去无水硫酸钠，滤液减压蒸干，所得固体经柱层析，以二氯甲烷/乙酸乙酯(DCM/EA)为洗脱剂梯度洗脱得到目标化合物。
方法A：
室温下，化合物III，HOBt和EDCI按照1.5:1.5:1.5的投料比加入适量的二氯甲烷中，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2h，TLC检测反应完全，停止反应。后处理：加水淬灭反应。乙酸乙酯(EA)抽提3次，合并乙酸乙酯层，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。
方法B：
冰浴下，将化合物II(4’-去甲基表鬼臼毒素DMEP)和化合物III(1:1)，适量的N,N-二甲基甲酰胺(DMF)中，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)，在冰浴下继续反应3h，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，有机层用1％氢氧化钠溶液调pH至7-8，水洗3次，无水硫酸钠干燥，活性炭脱色，除去乙酸乙酯后，即得目标化合物；或所得粗品经柱层析分离纯化即得目标化合物。
实施例3
4β-(2-吡咯甲酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-1)的制备
DCM 5mL，2-吡咯甲酸(206mg,1.875mmol)，DMEP(500mg,1.25mmol)，其余操作和 投料比参照方法四，制备I-1，得到白色固体128mg，收率：20.8％。
ESI-MS：516(M+Na+)
1H NMR(400MHz,DMSO-d6)δ11.93(s,1H),7.08(d,J＝0.8Hz,1H),6.95(s,1H),6.92(d,J＝8.4Hz,1H),6.54(s,1H),6.36(s,2H),6.23(s,1H),5.98(d,J＝2.4Hz,2H),5.49(d,J＝6Hz,1H),4.78(dd,3.6Hz,4Hz,1H),4.61(d,J＝5.6Hz,1H),4.37(t,J＝15.6Hz,1H),4.21(m,1H),3.60(s,6H),3.39–3.30(m,2H),2.87–2.81(m,1H).
实施例4
4β-(1-萘甲酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-2)的制备
以1-萘甲酸(258mg,1.5mmol)为原料，2mL的DCM，30℃条件下，其余操作和投料比参照方法二，制备化合物4β-(1-萘甲酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素，反应结束后，所得粗品经柱层析分离(石油醚与乙酸乙酯梯度洗脱)得目标化合物277mg，收率50％。
EI-MS：577(M+Na+).
1H NMR(400MHz,DMSO-d6)δ6.98-7.98(6H,萘环氢),6.69(s,1H),6.49(s,2H),6.27(s,2H),6.96(d,J＝17.2Hz,2H),5.18(dd,J＝8.0,4.7Hz,1H),4.52(d,J＝5.2Hz,1H),4.38(t,J＝7.9Hz,1H),3.74(s,6H).
实施例5
4β-(1-萘乙酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-3)的制备
1-萘乙酸(1.16g,6mmol)，4’-去甲基表鬼臼毒素(800mg,2mmol)和EDCI(1.12g,6mmol)，1.2mL的DCM，15℃条件下，其余操作和投料比参照方法二，制备化合物4β-(1-萘乙酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素，反应结束后，所得粗品经柱层析分离，石油醚与乙酸乙酯梯度洗脱，得目标化合物158mg，收率14％。
ESI-MS：591(M+Na+).
1H NMR(400MHz,DMSO-d6)δ7.465-8.297(7H,萘环氢),6.94(s,1H),6.51(s,1H),6.31(s,2H),5.42(s,J＝6Hz,1H),4.75(d,J＝4.4Hz,1H),4.33(t,J＝7.9Hz,1H),4.19(t,J＝12.0Hz,1H),3.57(s,6H),2.74-2.83(m,1H).
实施例6
4β-(2-吡咯酰胺)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-4)的制备
以2-吡咯羧酸(166mg,1.5mmol)和4β-氨基4-脱氧-4’-去甲基表鬼臼毒素(65mg,1.5mmol)为原料，0.5mL的DCM，0℃条件下，其余操作和投料比参照方法B，得白色固体563mg，收率76.3％。
ESI-MS：493(M+H+)/515(M+Na+).
1H-NMR:1H NMR(400MHz,DMSO-d6)：δ11.47(s,1H),8.19(d,J＝8.6Hz,2H),6.88(d,J＝8.0Hz,2H),6.82(s,1H),6.57(s,1H),6.27(s,2H),6.08(s,1H),5.99(d,J＝11.4Hz,2H),5.44(dd,J＝8.3,4.8Hz,1H),4.54(d,J＝5.2Hz,1H),4.35(t,J＝8.0Hz,1H),3.81-3.72(m,1H),3.66(s,6H),3.40(dd,J＝14.4,5.2Hz,1H),3.10-2.95(m,1H).
实施例7
4β-(1-萘酰胺)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-5)的制备
DCM 3mL，以245mg的2-萘酸，DECI作为缩合剂，其余操作和投料比参照方法一，得白色固体380mg，收率：69％。
ESI-MS：554(M+H+)/576(M+Na+).
1H NMR(400MHz,DMSO-d6)δ7.5-9.0(8H,H-萘环和NH)，7.01(IH,s,H-5),6.91(s,1H),6.51(s,1H),6.32(s,2H),6.00(d,J＝11.8Hz,2H),5.53(dd,J＝8.1,4.8Hz,1H),4.51(d,J＝5.2Hz,1H),3.97(t,J＝8.1Hz,1H),3.89-3.80(m,1H),3.60(s,6H),3.36(dd,J＝14.4,5.2Hz,1H),3.14-3.03(m,1H).
实施例8
4β-(2-萘乙酰胺)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-6)的制备
DCM 3mL，以279mg 2-萘乙酸为原料，采用DECP作为缩合剂，其余操作和投料比参照方法一，得白色固体400mg，收率：70.5％。
ESI-MS：567(M+H+)/590(M+Na+).
1H NMR(400MHz,DMSO-d6)δ7.5-9.0(8H,H-萘环和NH),6.73(s,IH),6.55(s,1H),6.25(s,2H),6.00(d,J＝11.8Hz,2H),5.20(dd,J＝8.1,4.8Hz,1H),4.54(d,J＝5.2Hz,1H),4.18(t,J＝8.1Hz,1H),3.97(s,2H),3.66-3.71(m,7H),2.93(m,1H).
实施例9
4β-(5-喹啉酰胺)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-7)的制备
DCM 2mL，以173mg的6-喹啉羧酸，以DEPC为缩合剂，其余操作和投料比参照方法二，所得粗品用20mL无水甲醇重结晶，得白色固体150mg，收率：27％。
ESI-MS：555(M+H+)/577(M+Na+).
1H NMR(400MHz,DMSO-d6)δ8.99(dd,J＝4.2,1.7Hz,1H),9.02-8.88(m,2H),8.93(d,J＝8.2Hz,1H),8.58(d,J＝1.8Hz,1H),8.58(d,J＝1.8Hz,1H),8.47(d,J＝8.4Hz,1H),8.47(d,J＝8.4Hz,1H),8.25-8.17(m,2H),8.08(d,J＝8.8Hz,1H),7.61(dd,J＝8.3,4.2Hz,1H),6.91(s,1H),6.60(s,1H),6.32(s,2H),6.00(d,J＝11.8Hz,2H),5.53(dd,J＝8.1,4.8Hz, 1H),4.56(d,J＝5.2Hz,1H),4.41(t,J＝8.1Hz,1H),3.89-3.80(m,1H),3.67(s,5H),3.49(dd,J＝14.4,5.2Hz,1H),3.14-3.03(m,1H).
实施例10
4β-(2-噻吩甲酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-8)的制备
DCM 5mL，2-噻吩甲酸(240mg,1.875mmol)，DMEP(500mg,1.25mmol)，其余操作和投料比参照方法四，制备I-8，得到白色固体128mg，收率：20％。
ESI-MS：533(M+Na+).
1H NMR(400MHz,DMSO-d6)δ8.05(dd,J＝5.0,1.2Hz,1H),8.01–7.68(m,1H),7.38–7.15(m,1H),6.97(s,1H),6.57(s,1H),6.38(s,2H),6.01(d,J＝3.5Hz,2H),5.48(d,J＝5.9Hz,1H),4.77(dd,J＝5.7,3.6Hz,1H),4.63(d,J＝5.4Hz,1H),4.55–4.29(m,1H),4.21(dt,J＝23.1,11.6Hz,1H),3.62(s,6H),3.38–3.31(m,1H),3.05–2.64(m,1H).
实施例11
4β-(2-噻吩甲酰胺基)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-9)的制备
DMF 3mL，2-噻吩甲酸(147mg,1.15mmol)，4β-氨基4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(500mg,1.15mmol)，其余操作和投料比参照方法B，制备I-9，所得粗品经柱层析(洗脱剂：二氯甲烷/乙酸乙酯梯度洗脱)纯化，得到黄色固体160mg，收率：27.4％。
ESI-MS：532(M+Na+).
1H NMR(400MHz,DMSO-d6)δ8.69(d,J＝8.4Hz,1H),8.21(s,1H),7.84(d,J＝2.4Hz,1H),7.75(m,1H),7.14(m,1H),6.83(d,J＝2Hz,1H),6.55(d,J＝2.4Hz,1H),6.28(s,2H),5.98(d,J＝10.4Hz,2H),5.41(s,1H),4.53(d,J＝2.8Hz,1H),4.36(t,J＝8Hz,1H),3.76(t,J＝8.8Hz,1H),3.65(s,6H),3.40-3.37(m,1H),3.04-3.01(m,1H).
实施例12
4β-(3-噻吩甲酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-10)的制备
DCM 3mL，3-噻吩甲酸(240mg,1.875mmol)，DMEP(500mg,1.25mmol)，其余操作和投料比参照方法四，制备I-10，得到白色固体400mg，收率：62.74％。
ESI-MS：533(M+Na+).
1H NMR(400MHz,DMSO-d6)δ8.51(d,J＝1.9Hz,1H),7.71(dd,J＝4.9,3.0Hz,1H),7.54(d,J＝4.9Hz,1H),6.97(s,1H),6.57(s,1H),6.37(s,2H),6.01(d,J＝3.7Hz,2H),5.48(d,J＝5.9Hz,1H),4.77(dd,J＝5.4,3.7Hz,1H),4.63(d,J＝5.4Hz,1H),4.38(t,J＝7.9Hz,1H),4.22(dd,J＝10.4,8.6Hz,1H),3.61(s,6H),3.32(d,J＝5.6Hz,1H),2.90–2.78(m,1H).
实施例13
4β-(3-噻吩甲酰胺基)-4-脱氧-4’-去甲基表鬼臼毒素(化合物I-11)的制备
DMF 4mL，3-噻吩甲酸(147mg,1.15mmol)，4β-氨基4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(500mg,1.15mmol)其余操作和投料比参照方法B，制备I-11，所得粗品经柱层析(洗脱剂：二氯甲烷/乙酸乙酯梯度洗脱)纯化，得到黄色固体220mg，收率：37.58％。
ESI-MS：532(M+Na+).
1H NMR(400MHz,DMSO-d6)δ8.54(d,J＝8.3Hz,1H),8.26(s,1H),8.24(s,1H),7.60-7.54(m,2H),6.83(s,1H),6.57(s,1H),6.28(s,2H),5.99(d,J＝11.9Hz,2H),5.42(dd,J＝8.1,4.8Hz,1H),4.54(d,J＝5.2Hz,1H),4.35(t,J＝8.0Hz,1H),3.74(dd,J＝10.5,9.0Hz,1H),3.65(s,6H),3.41(dd,J＝14.4,5.2Hz,1H),3.08-2.97(m,1H).
实施例14
4β-(5-甲基-2-噻吩甲酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(I-12)的制备
DCM 4mL，5-甲基-2-噻吩甲酸(267mg,1.875mmol)，DMEP(500mg,1.25mmol)，其余操作和投料比参照方法四，制备I-12，得到白色固体460mg，收率：70.23％。
ESI-MS：547(M+Na+)/563(M+K+).
1H NMR(400MHz,DMSO-d6)δ7.76(d,J＝3.7Hz,1H),6.99(dd,J＝3.7,0.8Hz,1H),6.96(s,1H),6.56(s,1H),6.36(s,2H),6.01(d,J＝3.4Hz,2H),5.48(d,J＝5.9Hz,1H),4.76(dd,J＝5.7,3.6Hz,1H),4.62(d,J＝5.4Hz,1H),4.37(t,J＝7.9Hz,1H),4.21(dd,J＝10.6,8.4Hz,1H),3.61(s,6H),3.36-3.31(m,1H),2.89-2.77(m,1H),2.54(s,3H).
实施例15
4β-(5-甲基-2-噻吩甲酰胺基)-4-脱氧-4’-去甲基表鬼臼毒素(I-13)的制备
DMF 3mL，5-甲基-2-噻吩甲酸(164mg,1.15mmol)，4β-氨基4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(500mg,1.15mmol)，其余操作和投料比参照方法B，制备I-13，所得粗品经柱层析(洗脱剂：二氯甲烷/乙酸乙酯梯度洗脱)纯化，得到白色固体255mg，收率：42.43％。
ESI-MS：524(M+H+)/546(M+Na+).
1H NMR(400MHz,DMSO-d6)δ8.60(d,J＝8.3Hz,1H),8.26(s,1H),7.66(d,J＝3.7Hz,1H),6.83(d,J＝3.1Hz,2H),6.57(s,1H),6.28(s,2H),5.99(d,J＝11.2Hz,2H),5.38(dd,J＝8.2,4.8Hz,1H),4.53(d,J＝5.2Hz,1H),4.34(t,J＝8.0Hz,1H),3.73(dd,J＝10.6,8.9Hz,1H),3.65(s,6H),3.40(dd,J＝14.4,5.3Hz,1H),3.08-2.96(m,1H),2.46(s,3H).
实施例16
4β-(5-氯-2-噻吩甲酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(I-14)的制备
DCM 5mL，5-氯-2-噻吩甲酸(305mg,1.875mmol)，DMEP(500mg,1.25mmol)，其余操作和投料比参照方法四，制备I-14，得到白色固体553mg，收率81.32％。
ESI-MS：567(M+Na+).
1H NMR(400MHz,DMSO-d6)δ7.85(d,J＝4.1Hz,1H),7.34(d,J＝4.1Hz,1H),6.96(s,1H),6.56(s,1H),6.37(s,2H),6.01(d,J＝2.9Hz,2H),5.48(d,J＝5.9Hz,1H),4.76(dd,J＝5.7,3.6Hz,1H),4.63(d,J＝5.4Hz,1H),4.37(t,J＝7.9Hz,1H),4.21(dd,J＝10.6,8.4Hz,1H),3.62(s,6H),3.36-3.32(m,1H),2.89-2.78(m,1H).
实施例17
4β-(5-氯-2-噻吩甲酰胺基)-4-脱氧-4’-去甲基表鬼臼毒素(I-15)的制备
DMF 2mL，5-氯-2-噻吩甲酸(187mg,1.15mmol)，4β-氨基4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(500mg,1.15mmol)，其余操作和投料比参照方法B，制备I-15，所得粗品(白色固体)经柱层析(洗脱剂：二氯甲烷/乙酸乙酯梯度洗脱)纯化，得到白色固体275mg，收率：44.07％。
ESI-MS：566(M+Na+)/582(M+K+).
1H NMR(400MHz,DMSO-d6)δ8.82(d,J＝8.2Hz,1H),8.26(s,1H),7.75(d,J＝4.1Hz,1H),7.17(d,J＝4.0Hz,1H),6.85(s,1H),6.57(s,1H),6.28(s,2H),6.00(d,J＝11.1Hz,2H),5.37(dd,J＝8.0,4.7Hz,1H),4.54(d,J＝5.1Hz,1H),4.34(t,J＝8.0Hz,1H),3.74(dd,J＝10.4,9.1Hz,1H),3.65(s,6H),3.38(s,1H),3.03(ddd,J＝18.8,13.1,7.6Hz,1H).
实施例18
4β-(咪唑-2-甲酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(I-16)的制备
将咪唑-2-甲酸(504mg,4.5mmol)，EDCI(573mg,3mmol)，HOBt(405mg,3mmol)加入3mL二氯甲烷中，室温下搅拌0.5h后加入DMEP(600mg,1.5mmol)和DMAP(55mg,0.3mmol)，TLC检测反应完全后，后处理方法与方法三相同，得到白色固体114mg，收率：15.4％。
ESI-MS：517(M+Na+).
1H NMR(400MHz,DMSO-d6)δ13.47(s,1H),13.21(s,1H),8.18(s,1H),7.43(d,J＝16.4Hz,1H),7.23(d,J＝21.2Hz,2H),7.03(d,J＝28Hz,2H),6.60(d,J＝14.8Hz,1H),6.38(s,2H),6.35(d,J＝3.6Hz,1H),6.00(dd,J＝11.2Hz,9.2Hz,1H),5.43(d,J＝6Hz,1H),4.77(t,J＝3.6Hz,1H),4.63(d,J＝5.2Hz,2H),4.39(m,2H),4.21(t,J＝2Hz,1H),4.00(t,J＝2Hz,1H),3.36-3.23(m,1H).
实施例19
4β-(咪唑-2-甲酰胺基)-4-脱氧-4’-去甲基表鬼臼毒素(I-17)的制备
DMF 3mL，咪唑-2-甲酸(168mg,1.5mmol)，其余操作和投料比参照方法五，制备I-17，得到白色固体130mg，收率：26.34％。
ESI-MS：516(M+Na+).
1H NMR(400MHz,DMSO-d6)δ13.11(s,1H),8.66(d,J＝8.8Hz,1H),8.24(s,1H),7.31(s,1H),7.04(s,1H),6.82(s,1H),6.55(s,1H),6.27(s,2H),5.99(d,J＝11.9Hz,2H),5.42(dd,J＝8.7,4.9Hz,1H),4.48(d,J＝5.2Hz,1H),4.35(t,J＝8.0Hz,1H),3.79(dd,J＝10.7,8.7Hz,1H),3.71-3.59(m,7H),3.10-2.96(m,1H).
实施例20
4β-(吡唑-3-甲酰胺基)-4-脱氧-4’-去甲基表鬼臼毒素(I-18)的制备
DMF 4mL，吡唑-3-甲酸(168mg,1.5mmol)，其余操作和投料比参照方法五，制备I-18，得到白色固体80mg，收率：16.23％。
ESI-MS：516(M+Na+).
1H NMR(400MHz,DMSO-d6)δ13.26(s,1H),8.38(d,J＝8.1Hz,1H),8.25(s,1H),7.83(s,1H),6.81(s,1H),6.72(s,1H),6.55(s,1H),6.27(s,2H),5.99(d,J＝12.3Hz,2H),5.43(dd,J＝8.5,4.9Hz,1H),4.48(d,J＝3.9Hz,1H),4.35(t,J＝8.0Hz,1H),3.75(dd,J＝10.7,8.8Hz,1H),3.68-3.54(m,7H),3.02(s,1H).
实施例21
4β-(吲唑-3-甲酰胺基)-4-脱氧-4’-去甲基表鬼臼毒素(I-19)的制备
DMF 5mL，吡唑-3-甲酸(186mg,1.15mmol)，其余操作和投料比参照方法B，制备I-19，所得粗品经柱层析(洗脱剂：二氯甲烷/乙酸乙酯梯度洗脱)纯化，得到白色固体100mg，收率：16.02％。
ESI-MS：566(M+Na+).
1H NMR(400MHz,DMSO-d6)δ13.61(s,1H),8.65(d,J＝8.7Hz,1H),8.25(s,1H),8.21(d,J＝8.1Hz,1H),7.63(d,J＝8.4Hz,1H),7.43(t,J＝7.6Hz,1H),7.27(t,J＝7.5Hz,1H),6.88(s,1H),6.57(s,1H),6.29(s,2H),5.99(d,J＝13.1Hz,2H),5.53(dd,J＝8.6,4.9Hz,1H),4.50(d,J＝5.1Hz,1H),4.41(t,J＝8.0Hz,1H),3.88-3.80(m,1H),3.69(d,J＝5.2Hz,1H),3.66(s,6H),3.11-3.00(m,1H).
实施例22
4β-(N-Boc-DL-脯氨甲酰胺基)-4-脱氧-4’-去甲基表鬼臼毒素(I-20)制备
将N-Boc-DL-脯氨酸(560mg,2.6mmol)，HATU(532mg,1.4mmol)加入至5mL N,N-二 甲基甲酰胺(DMF)中，室温搅拌0.5h后加入三乙胺(0.35mL,2.5mmol)，反应液变黄，加入4β-氨基4-脱氧-4’-去甲基表鬼臼毒素盐酸盐(435mg,1.0mmol)。后处理方法与方法五相同。经柱层析(洗脱剂：二氯甲烷/乙酸乙酯梯度洗脱)纯化，得到白色固体160mg，收率：26.85％。
ESI-MS：619(M+Na+).
1H NMR(400MHz,DMSO-d6)δ8.33(d,J＝8.3Hz,1H),8.29-8.18(m,1H),6.73(dd,J＝14.7,5.8Hz,1H),6.58-6.49(m,1H),6.25(s,2H),5.98(dd,J＝18.1,4.6Hz,2H),5.25-5.17(m,1H),5.11(dd,J＝12.1,7.5Hz,1H),4.49(t,J＝7.5Hz,1H),4.27(dt,J＝33.2,7.8Hz,1H),4.02(ddt,J＝29.6,19.8,10.1Hz,1H),3.81-3.69(m,1H),3.63(s,5H),3.31-3.14(m,2H),2.95(s,1H),2.15(d,J＝8.1Hz,1H),1.79(dd,J＝15.2,9.3Hz,3H),1.41-1.29(m,8H).
实施例23
4β-(N-Boc-L-羟脯氨酸-2-甲酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(I-21)制备
将N-Boc-L-羟脯氨酸(300mg,1.3mmol)，EDCI(597mg,3.125mmol)，DMAP(61mg,0.5mmol)加入3mL二氯甲烷中，室温下搅拌0.5h后加入DMEP(400mg,1.0mmol)，TLC检测反应完全后，后处理方法与方法三相同。得到白色固体255mg，收率：41.6％。
ESI-MS：636(M+Na+).
1H NMR(400MHz,CDCl3)δ6.74(s,1H),6.49(s,1H),6.29(s,2H),5.95(d,J＝11.6Hz,2H),5.63(s,1H),5.19(d,J＝8Hz,1Hz),4.54(t,10.4Hz,3H),4.35(m,2H),3.76(s,6H),3.48(s,2H),2.93(s,2H),1.99(m,4H),1.43(s,9H).
实施例24
4β-(N-Boc-L-羟脯氨甲酰胺基)-4-脱氧-4’-去甲基表鬼臼毒素(I-22)制备
将N-Boc-L-羟脯氨酸(395mg,1.725mmol)投入至3mL N,N-二甲基甲酰胺(DMF)中，其余操作和投料比参照方法三，制备I-22，得到白色固体333mg，收率：47.39％。
ESI-MS：635(M+Na+).
1H NMR(400MHz,DMSO-d6)δ6.74(s,1H),6.49(s,1H),6.29(s,2H),5.95(d,J＝11.6Hz,2H),5.63(s,1H),5.19(d,J＝8Hz,1H),4.54(t,J＝13.2Hz,2H),4.37(m,2H),3.80(d,J＝19.2Hz,8H),3.49(s,2H),2.93(s,2H),2.70(s,1H),2.42(s,1H),1.43(s,10H).
实施例25
4β-(2-噻吩乙酰氧基)-4-脱氧-4’-去甲基表鬼臼毒素(I-23)的制备
DCM 3mL，2-噻吩乙酸(266mg,1.875mmol)，DMEP(500mg,1.25mmol)，其余操作和投料比参照方法四，制备I-23，得到白色固体360mg，收率54.96％。
ESI-MS：547(M+Na+).
1H NMR(400MHz,DMSO-d6)δ7.43(dd,J＝5.1,1.2Hz,1H),7.05(dd,J＝3.3,0.7Hz,1H),6.99(dd,J＝5.1,3.5Hz,1H),6.95(s,1H),6.54(s,1H),6.32(s,2H),6.00(d,J＝1.4Hz,2H),5.47(d,J＝5.9Hz,1H),4.75(dd,J＝5.8,3.6Hz,1H),4.60(d,J＝5.4Hz,1H),4.36(t,J＝7.9Hz,1H),4.23-4.15(m,3H),3.60(s,6H),3.34-3.29(m,1H),2.87-2.75(m,1H).
实施例26
4β-(吲哚-2-乙酰氧基)-4-脱氧-4′-去甲基表鬼臼毒素(I-24)的制备
DCM 3mL，吲哚-2-甲酸(364mg,2.25mmol)按照方法七所示投料和后处理，得到白色固体120mg，收率为21.4％。
ESI-MS：566(M+Na+).
1H NMR(400MHz,DMSO-d6)δ11.98(s,1H),7.71(d,J＝8Hz,1H),7.50(d,J＝8.4Hz,1H),7.30(t,J＝7.2Hz,2H),7.11(t,J＝8Hz,1H),6.97(s,1H),6.58(s,1H),6.41(s,2H),6.01(d,J＝2.8Hz,2H),5.72(s,1H),5.44(d,J＝5.6Hz,1H),4.78(t,J＝3.6Hz,1H),4.65(d,J＝5.6Hz,1H),4.39(t,J＝8Hz,1H),4,23(t,J＝10.8Hz,1H),3.64(s,6H),3.36(dd,J＝5.6Hz,5.6Hz,1H).
实施例27
4β-(吲哚-2-乙酰胺基)-4-脱氧-4′-去甲基表鬼臼毒素(I-25)的制备
DMF 3mL，吲哚-2-甲酸(242mg,1.5mmol)按照方法八所示投料和后处理，得到白色固体175mg，收率为32.3％。
ESI-MS：565(M+Na+).
1H NMR(400MHz,DMSO-d6)δ11.54(s,1H),8.65(d,J＝8.4Hz),8.17-7.88(m,1H),7.60(d,J＝8Hz,1H),7.45(d,8.4Hz,1H),7.22(t,J＝5.6Hz,1H),7.19(d,J＝1.2Hz,1H),7.03(t,J＝8Hz,1H),6.86(s,1H),6.58(s,1H),6.30(s,1H),6.00(d,J＝12Hz,1H),5.72(d,J＝0.4Hz,1H),5.50(dd,J＝4.8Hz,4.8Hz,1H),4.56(d,J＝5.2Hz,1H),4.38(t,J＝8Hz,1H),4.04(dd,J＝6.8Hz,1H),3.66(s,3H),3.47(dd,J＝5.2Hz,5.2Hz,1H),3.07(m,1H),2.89(s,2H),2.73(s,1H),1.98(d,J＝9.2Hz,1H),1.17(m,1H).
实施例28
4β-(5-氟吲哚-2-乙酰氧基)-4-脱氧-4′-去甲基表鬼臼毒素(I-26)制备
DCM 4mL，5-氟吲哚-2-甲酸(215mg,1.2mmol)按照方法七所示投料和后处理，得到白色固体220mg，收率为27％。
ESI-MS：584(M+Na+).
1H NMR(400MHz,DMSO-d6)δ12.12(s,1H),7.48(m,2H),7.30(d,J＝1.2Hz,1H),7.17(m, 1H),6.97(s,1H),6.57(s,1H),6.40(s,2H),6.01(d,J＝2.4Hz,2H),4.78(d,J＝3.2Hz,1H),4.64(d,J＝5.6Hz,1H),4.39(t,J＝8Hz,1H),4.23(t,J＝6.4Hz,1H),4.04(dd,J＝7.2Hz,7.2Hz,1H),3.64(m,6H),2.86-2.83(m,1H).
实施例29
4β-(5-氟吲哚-2-乙酰胺基)-4-脱氧-4′-去甲基表鬼臼毒素(I-27)制备
DMF 4mL，5-氟吲哚-2-甲酸(269mg,1.5mmol)按照方法八所示投料和后处理，得到白色固体170mg，收率为30.4％。
ESI-MS：583(M+Na+).
1H NMR(400MHz,DMSO-d6)δ11.67(s,1H),8.70(d,J＝8.4Hz,1H),8.16(s,1H),7.44(m,2H),7.22(d,J＝2.4Hz,1H),7.05(m,1H),6.86(s,1H),6.58(s,1H),6.30(s,2H),6.00(s,1H),5.97(s,1H),5.72(s,1H),5.50(m,1H),4.56(d,J＝5.2Hz,1H),4.37(t,J＝8.4Hz,1H),3.81(m,1H),3.66(s,6H),3.47-3.42(m,1H).
实施例30
4β-(5-氯吲哚-2-乙酰氧基)-4-脱氧-4′-去甲基表鬼臼毒素(I-28)制备
DCM 5mL，5-氯吲哚-2-甲酸(235mg,1.2mmol)按照方法七所示投料和后处理，得到白色固体116mg,收率为20％。
ESI-MS：600(M+Na+).
1H NMR(400MHz,DMSO-d6)δ12.21(s,1H),7.77(d,J＝1.6Hz,1H),7.51(d,J＝8.8Hz,1H),7.30(m,2H),6.97(s,1H),6.57(s,1H),6.40(s,2H),6.01(d,J＝2.4Hz,2H),5.72(s,1H),5.43(d,J＝4.8Hz,1H),4.78(s,1H),4.64(d,J＝5.2Hz,1H),4.39(t,J＝7.6Hz,1H),4.23(t,J＝8.4Hz,1H),3.64(s,6H),2.87-2.83(m,1H).
实施例31
4β-(5-氯吲哚-2-乙酰胺基)-4-脱氧-4′-去甲基表鬼臼毒素(I-29)制备
DMF 5mL，5-氯吲哚-2-甲酸(293mg,1.5mmol)按照方法八所示投料和后处理，得到白色固体214mg，收率为37.1％。
ESI-MS：599(M+Na+).
1H NMR(400MHz,DMSO-d6)δ11.77(s,1H),8,74(d,J＝8.4Hz,1H),8.16(s,1H),7.95(s,1H),7.67(s,1H),7,45(d,J＝8.8Hz,1H),7.20(m,2H),6.86(s,1H),6.58(s,1H),6.30(s,2H),6.00(d,J＝11.6Hz,2H),5.51-5.48(m,1H),4.56(d,J＝5.2Hz,1H),4.37(t,J＝8Hz,1H),3.80(t,J＝11.2Hz,1H),3.66(s,6H),3.47-3.42(m,1H).
实施例32
4β-(5-甲氧基吲哚-2-乙酰氧基)-4-脱氧-4′-去甲基表鬼臼毒素(I-30)制备
DCM 3mL，5-甲氧基吲哚-2-甲酸(288g,1.5mmol)按照方法七所示投料和后处理，得到淡黄色固体240mg，收率为41.9％。
ESI-MS：596(M+Na+).
1H NMR(400MHz,DMSO-d6)δ11.84(s,1H),7.39(d,J＝9.2Hz,1H),7.21(s,1H),7.14(d,J＝2Hz,1H),6.91(m,2H),6.57(s,1H),6.39(s,2H),6.01(d,J＝3.2Hz,2H),5.43(d,J＝6.4Hz,1H),4.77(t,J＝5.2Hz,1H),4.64(d,J＝5.2Hz,1H),4.38(t,J＝8Hz,1H),4.22(t,J＝10.8Hz,1H),3.77(s,3H),3.63(s,6H),2.86-2.83(m,1H).
实施例33
4β-(5-甲氧基吲哚-2-乙酰胺基)-4-脱氧-4′-去甲基表鬼臼毒素(I-31)制备
DMF 3mL，5-甲氧基吲哚-2-甲酸(288g,1.5mmol)按照方法八所示投料和后处理，得到淡黄色固体232mg，收率为40.6％。
ESI-MS：595(M+Na+).
1H NMR(400MHz,DMSO-d6)δ11.39(s,1H),8.60(d,J＝8.4Hz,1H),8.15(s,1H),7.34(d,J＝8.8.Hz,1H),7.13(s,1H),7.05(s,1H),6.87-6.84(m,2H),6.58(s,1H),6.31(s,2H),6.00(d,J＝11.6Hz,2H),5.72(s,1H),5.52-5.48(m,1H),4.56(d,J＝4.8Hz,1H),4.37(t,J＝8.4Hz,1H),3.80(t,J＝10.4Hz,1H),3.75(s,3H),3.66(s,6H),3.45(dd,J＝5.2Hz,5.2Hz,1H),3.07-3.05(m,1H).
实施例34
4β-(吲哚-3-甲酰氧基)-4-脱氧-4′-去甲基表鬼臼毒素(I-32)的制备
DCM 4mL，吲哚-3-甲酸(242mg,1.5mmol)按照方法七所示投料和后处理，得到白色固体150mg，收率为27.6％。
ESI-MS：566(M+Na+)，582(M+K+).
1H NMR(400MHz,DMSO-d6)δ11.61(s,1H),8.27(s,1H),8.20–8.14(m,2H),7.44(d,J＝7.5Hz,1H),7.19–7.09(m,2H),6.88(s,1H),6.59(s,1H),6.31(s,2H),6.00(d,J＝11.7Hz,2H),5.52(dd,J＝8.3,4.8Hz,1H),4.56(d,J＝5.1Hz,1H),4.40(t,J＝8.1Hz,1H),3.89–3.79(m,1H),3.67(s,6H),3.45(dd,J＝14.4,5.2Hz,1H),3.04(dd,J＝25.2,7.3Hz,1H).
实施例35
4β-(吲哚-3-甲酰胺基)-4-脱氧-4′-去甲基表鬼臼毒素(II-33)的制备
DMF 3mL，吲哚-3-甲酸(242mg,1.5mmol)按照酰胺类合成方法八所示投料和后处理，得到白色固体95mg，收率为17.5％。
ESI-MS：565(M+Na+).
1H NMR(400MHz,DMSO-d6)δ11.61(s,1H),8.31(s,1H),8.23–8.10(m,3H),7.44(d,J＝7.6Hz,1H),7.15(t,J＝14.9Hz,2H),6.88(s,1H),6.58(s,1H),6.31(s,2H),5.99(d,J＝11.7Hz,2H),5.53(s,1H),4.56(s,1H),4.42(s,1H),4.05(dd,J＝18.9,11.9Hz,1H),3.83(t,J＝9.5Hz,1H),3.66(s,6H),3.03(d,J＝8.0Hz,1H).
实施例36
4β-(吲哚-3-乙酰氧基)-4-脱氧-4′-去甲基表鬼臼毒素(I-34)的制备
DCM 5mL，吲哚-3-甲酸(242mg,1.5mmol)按照方法七所示投料和后处理，得到白色固体150mg，收率为27.6％。
ESI-MS：566(M+Na+)，582(M+K+).
1H NMR(400MHz,DMSO-d6)δ10.94(s,1H),7.55(d,J＝7.8Hz,1H),7.37(d,J＝8.1Hz,1H),7.32(s,1H),7.10(t,J＝7.5Hz,1H),7.01(t,J＝7.1Hz,1H),6.95(s,1H),6.53(s,1H),6.32(s,2H),5.99(s,2H),5.49(d,J＝5.9Hz,1H),4.77–4.73(m,1H),4.59(d,J＝5.4Hz,1H),4.41–4.32(m,1H),4.20(dd,J＝10.6,8.4Hz,1H),3.98(s,2H),3.62(s,6H),3.32(dd,J＝14.3,5.4Hz,1H),2.88–2.74(m,1H).
实施例37
4β-(吲哚-3-丁酰氧基)-4-脱氧-4′-去甲基表鬼臼毒素(I-35)的制备
DCM 3mL，吲哚-3-丁酸(302mg,1.49mmol)按照方法六所示投料和后处理，得到白色固体466mg，收率为63.8％，纯度：92.26％。
ESI-MS：608(M+Na+)，624(M+K+).
1H-NMR(400MHz,DMSO-d6)δ10.72(s,1H),7.45(d,J＝7.8Hz,1H),7.32(d,J＝8.1Hz,1H),7.06(dd,J＝14.6,4.7Hz,2H),6.95(t,J＝7.4Hz,1H),6.91(s,1H),6.56(s,1H),6.23(s,2H),6.09(d,J＝3.5Hz,1H),6.01(d,J＝17.2Hz,2H),4.56(d,J＝5.2Hz,1H),4.31(t,J＝7.9Hz,1H),3.84(t,J＝12.0Hz,1H),3.64(s,6H),3.21(d,J＝5.2Hz,1H),3.08-2.97(m,1H),2.70(t,J＝7.4Hz,2H),2.43(dd,J＝14.7,7.4Hz,2H),1.96-1.87(m,2H).
实施例38
4β-(吲哚-3-丁酰胺基)-4-脱氧-4′-去甲基表鬼臼毒素(I-36)的制备
DMF 3mL，吲哚-3-丁酸(302mg,1.49mmol)按照方法八所示投料和后处理，得到白色固体88mg，收率为15.0％，纯度：90.50％。
ESI-MS：607(M+Na+).
1H NMR(400MHz,CDCl3)δ8.06(s,1H),7.58(d,J＝7.6Hz,1H),7.36(d,J＝8Hz,1H), 7.19(t,J＝3.2Hz,1H),7.10(t,J＝4Hz,1H),6.98(s,1H),6.72(s,1H),6.50(s,1H),6.29(s,2H),5.97(d,J＝4.8Hz,2H),5.61(d,J＝6.8Hz,1H),5.21(t,J＝6.4Hz,1H),4.51(d,J＝4.4Hz,1H),4.37(t,J＝8.4Hz,1H),3.75(m,6H),2.91(m,3H),2.69(m,1H),2.31(t,J＝7.2Hz,2H),2.11(m,2H),1.36(m,2H).
效果实施例1化合物抑制肿瘤细胞的活性实验
1、实验目的
在体外用MTT法实验测定本发明的鬼臼类衍生物对人体肿瘤细胞株的细胞毒作用
2、仪器设备
(1)CO2细胞培养箱，PHARMA SCIENTIFIC。
(2)酶联免疫检测仪，Labsystems,wellscan,MK.2。
(3)垂直单面双人超净工作台，苏州净化设备厂。
(4)倒置生物显微镜，37XB/37×BTV，上海光学仪器六厂。
3、实验材料及配制
(1)细胞株：人肺癌(A549)、人肝癌(HepG2)、人口腔癌(KB)、小鼠白血病细胞(L1210)，细胞株均购自中科院细胞所。
(2)DMEM细胞培养基(GIBCO)：含10％灭活新生小牛血清(上海赛达生物药业有限公司)；L-谷氨酰氨(进口分装，SANGON)；丙酮酸钠；1×105U.L-1青霉素，100mg.L-1链霉素；无菌过滤，4℃保存。
(3)0.25％胰蛋白酶溶液(Trypsin)：购自Invitrogen公司，-20℃保存。
(4)磷酸缓冲液(PBS)：NaCl8g，KCl0.2g，Na2HPO41.15g，KH2PO40.2g，溶于1L双蒸水，121℃高压消毒20min，4℃保存。
(5)MTT(AMRESCO)溶液：用PBS配成5mg/mL溶液。
(6)溶解液：每100mL去离子双蒸水含SDS10g，异丁醇5mL，浓硫酸0.12mL。
(7)样品：本发明鬼臼毒素衍生物。
(8)对照品：依托泊苷注射液，购自江苏恒瑞医药股份有限公司。
4、实验方法
鬼臼类化合物对上述肿瘤细胞株的细胞毒性通过MTT法测得。具体步骤如下：
(1)细胞培养：①将细胞从液氮中取出，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6mL DMEM细胞培养基，1000转/分离心5分钟。弃去上清液，沉淀中加入5-6mL DMEM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。②次日，自培养箱中取出细胞，弃去细胞瓶中DMEM细胞 培养基〔除K562(人慢性髓原白血病细胞株和L1210(小鼠淋巴白血病细胞株)等悬浮细胞〕，加入5-6mL DMEM细胞培养基，置37℃细胞培养箱内。③隔日，自培养箱中取出细胞，弃去细胞瓶中DMEM细胞培养基〔除K562(人慢性髓原白血病细胞株)和L1210(小鼠淋巴白血病细胞株)等悬浮细胞〕，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。K562(人慢性髓原白血病细胞株)和L1210(小鼠淋巴白血病细胞株)等悬浮细胞取出1-2mL悬浮液，分别移入2个干净培养瓶中，加入DMEM细胞培养基5-6mL，置于37℃细胞培养箱内。④隔日，重复步骤③的操作。在整个培养过程中，贴壁细胞不允许生长过密，悬浮细胞始终保持对数生长期。
(2)样品制备：将样品溶解于二甲亚砜中，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：将依托泊苷注射液用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)将稀释好的样品和对照品加入平底96孔板中，每孔10μL，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90μL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20μL 5mg/mLMTT溶液，继续在培养箱中保温3～4小时。
(8)每孔加入100μL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 PCTCN2015076505-appb-000021
(10)通过软件计算化合物对各肿瘤细胞的IC50。
其中，表1.1中编号I-24、1-2～I-7和VP16是在中国专利申请CN201410172493.7的申请日之前由发明人孙亚飞所测活性结果，而表1.2中编号I-1、I-8～I-23、I-25～I-37和VP16在中国专利申请CN201410172493.7的申请日之后由发明人姚利霞所测活性结果。
表1.1 MTT法所测活性结果
Figure PCTCN2015076505-appb-000022
表1.2 MTT法所测活性结果
Figure PCTCN2015076505-appb-000023
Figure PCTCN2015076505-appb-000024
效果实施例2化合物I-4对ICR小鼠S180移植瘤的药效学研究
1、试验目的
以ICR小鼠S180移植瘤为模型(S180细胞购自中科院细胞所)对I-4的抗肿瘤作用进行研究。
2、试验内容
试验样品
样品：化合物I-4，白色粉末状。
阳性对照：注射用依托泊苷，江苏恒瑞医药股份有限公司，产品批号：10110731，5mL/0.1g/瓶。
3、配制方法
样品：化合物I-4，配制时先加0.5％DMSO溶解样品，再用4％的吐温-80助溶，再用生理盐水稀释至所需浓度。
阳性对照：依托泊苷，配制时用生理盐水稀释。
4、动物和瘤株
ICR小鼠60只，雌性，体重18～20g，由西必凯实验动物有限责任公司提供，合格证：SCXK(沪)2008-0016。
瘤株：S180腹水瘤，取自S180腹水瘤的ICR小鼠。
5、试验方法
取生长旺盛期的ICR小鼠S180腹水瘤2只，无菌条件下抽取腹水，用生理盐水1:6稀释，按0.2mL/只给小鼠腋皮下接种(共接种60只)。次日将小鼠随机均分为6组，每组10只。
分别为空白溶媒组、I-410mg/kg iv组(连续给药4天，iv表示静脉注射法)、I-45mg/kgiv组(连续给药4天)、I-42.5mg/kg iv组(连续给药4天)、I-41.25mg/kg iv组(连续给药4天)、依托泊苷5mg/kg iv(连续给药7天)。
ICR小鼠接种后第二天开始给药，给药剂量及给药方案见表2。治疗结束后存活下来的动物被执行安乐死，取瘤块称重，计算抑瘤率。
Figure PCTCN2015076505-appb-000025
表2 ICR小鼠给药剂量及给药方式
Figure PCTCN2015076505-appb-000026
6、结果
化合物I-4静脉给药对ICR小鼠S180移植瘤的抑瘤率见表3。
表3 化合物I-4静脉给药对ICR小鼠S180移植瘤的抑瘤率
Figure PCTCN2015076505-appb-000027
Figure PCTCN2015076505-appb-000028
I-410mg/kg iv组及I-45mg/kg iv组的ICR小鼠给药4天后与空白对照组相比体重减轻>20％，因此从第5天起I-4的各组ICR小鼠均停止给药，阳性对照组依托泊苷5mg/kg继续给药至第7天。I-410mg/kg iv组ICR小鼠于给药后第6天开始死亡，至治疗结束全部死亡，而I-45mg/kg iv组ICR小鼠体重开始恢复，其余各组ICR小鼠均无死亡。对上述各组ICR小鼠进行解剖，化合物I-4静脉给药对各组ICR小鼠S180移植瘤瘤生长抑制的照片图见图1，其中，A排表示I-45mg/kg iv组10只ICR小鼠中S180移植瘤、B排表示I-42.5mg/kg iv组10只ICR小鼠中S180移植瘤、C排表示I-41.25mg/kg iv组10只ICR小鼠中S180移植瘤、D排表示依托泊苷5mg/kg iv组10只ICR小鼠中S180移植瘤、E排表示空白溶媒组。从图1中可以看出：化合物I-45mg/kg iv组ICR小鼠给药后，S180移植瘤均未继续生长，相对于空白溶媒组对抑制S180移植瘤生长的有明显的抑制作用，并且比依托泊苷5mg/kg iv抑制效果好。
根据本发明的效果实施例，对于本发明的通式I所示的鬼臼毒素衍生物的构效关系进行分析：当X为N时，通式I化合物为I-a所示；当X为O时，通式I化合物为I-b所示：
Figure PCTCN2015076505-appb-000029
在通式I-a所示的鬼臼毒素衍生物中，当R为较大基团时：如萘基、喹啉基或吲哚基等，抗肿瘤活性比R为吡咯基、噻吩基等小分子基团时的活性低。当R基团的碳原子数相当的情况下，例如R为萘基和喹啉基时，当R为喹啉基时，化合物抗肿瘤活性较R为萘基好，说明R为杂芳基时，抗肿瘤活性较芳基好。当R为小分子基团时，例如吡咯或噻吩，所得到的化合物除对HepG2、KB和L1210等细胞株均有抑制作用，并且对VP-16 抑制作用较弱的A549细胞株也有抑制作用。
对于R为吲哚基的情况：①R基团以α和β位连接4-氨基-4′-去甲基表鬼臼毒素时，对肿瘤细胞的抑制活性有影响。I-25和I-33相比，对所测肿瘤细胞系KB和A549细胞系抑制作用相当，对HepG2和L1210细胞的抑制作用I-33优于I-25，即β位取代优于α位；②R基团上引入供电子取代基对活性有影响；I-25和I-27、I-29、I-31相比，吲哚环上5位甲氧基、卤素取代时对四种肿瘤细胞系的抑制作用略有降低，5位为甲氧基取代时增强了对HepG2和L1210细胞系的抑制作用，引入卤素降低了四种细胞系的抑制活性；③连接链长度影响抗肿瘤活性。I-33和I-16相比，碳链增长对四种细胞系的抑制作用都有所降低。得到的所有化合物除对HepG2、KB和L1210等细胞株均有抑制作用，并且对VP-16无效的A549细胞株也有抑制作用。
在通式I-b所示的鬼臼毒素衍生物中，当R为较大基团时：如萘基，其对HepG2和KB细胞株的活性较好，并且增加碳链的长度(即n的取值较大时)，抗肿瘤活性有所升高。
对于R为吲哚基的情况：①R基团以α和β位连接4-羟基-4′-去甲基表鬼臼毒素时，对肿瘤细胞的抑制活性有影响。I-24和I-32相比，对所测四种肿瘤细胞系的抑制作用I-24优于I-32，即α位取代优于β位；②R基团上引入供电子取代基对活性有影响。I-24和I-26、I-28、I-30相比，吲哚环上5位甲氧基、卤素取代时对四种肿瘤细胞系的抑制作用并未有很大改善，5位为甲氧基取代时增强了对L1210细胞系的抑制作用，引入氯增强了对HepG2、KB和L1210的抑制活性；③连接链长度影响抗肿瘤活性。I-32和I-34、I-35相比，碳链增长对HepG2、A549和KB细胞的抑制作用增强，但对L1210细胞系的抑制作用降低。得到的所有化合物除对HepG2、KB和L1210等细胞株均有抑制作用，并且对VP-16无效的A549细胞株也有抑制作用。
综上所述，当R为较大基团时，通式I-b所示的鬼臼毒素衍生物对部分细胞株的杀伤力较好，如HepG2、KB细胞株，但其抗肿瘤谱较通式I-a所示的鬼臼毒素衍生物窄。本发明得到的通式I-a所示的鬼臼毒素衍生物抗肿瘤谱宽，当R中含有杂原子时，有利于提高化合物的抗肿瘤活性。
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所示的鬼臼毒素衍生物：
    Figure PCTCN2015076505-appb-100001
    其中，X为氧或氮；R为取代或未取代的C5～C10的芳基、取代或未取代的C2～C10的杂芳基，或取代或未取代的C2～C10的杂环烷基；n为0、1、2、3或4；当n为0时，R不为取代或未取代的苯基或取代或未取代的吡啶基，当n＝1，X为N时，R不为取代或未取代的吲哚基或取代或未取代的噻吩基；所述的取代的C5～C10的芳基、所述的取代的C2～C10的杂芳基或所述的取代的C2～C10的杂环烷基中所述的取代是指被下列一个或多个取代基所取代：卤素、硝基、氨基、羟基、C1～C4的烷基或
    Figure PCTCN2015076505-appb-100002
    其中，Ra为C1～C4的烷基，当取代基为多个时，所述的取代基相同或不同。
  2. 如权利要求1所述的通式I所示的鬼臼毒素衍生物，其特征在于，
    当所述的取代的C5～C10的芳基、所述的取代的C2～C10的杂芳基或所述的取代的C2～C10的杂环烷基中所述的取代为被卤素所取代时，所述的卤素为氟、氯、溴或碘；
    当所述的取代的C5～C10的芳基、所述的取代的C2～C10的杂芳基或所述的取代的C2～C10的杂环烷基中所述的取代为被C1～C4的烷基所取代时，所述的C1～C4的烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；
    当所述的取代的C5～C10的芳基、所述的取代的C2～C10的杂芳基或所述的取代的C2～C10的杂环烷基中所述的取代为被
    Figure PCTCN2015076505-appb-100003
    所取代时，Ra中，所述的C1～C4的烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基。
  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，其特征在于，
    所述的取代或未取代的C5～C10的芳基为取代或未取代的萘基；
    和/或，所述的取代或未取代的C2～C10的杂芳基是指杂原子为N、O或S，杂原子数 为1～4个的取代或未取代的C2～C10的杂芳基；
    和/或，所述的取代或未取代的C2～C10的杂环烷基是指杂原子为N、O或S，杂原子数为1～4个的取代或未取代的C2～C10的杂环烷基。
  4. 如权利要求3所述的通式I所示的鬼臼毒素衍生物，其特征在于，
    所述的萘基为
    Figure PCTCN2015076505-appb-100004
    和/或，所述的杂原子为N、O或S，杂原子数为1～4个的取代或未取代的C2～C10的杂芳基是指杂原子为N或S，杂原子数为1或2个的取代或未取代的C4～C10的杂芳基；
    和/或，所述的杂原子为N、O或S，杂原子数为1～4个的取代或未取代的C2～C10的杂环烷基是指杂原子为N，杂原子数为1个的取代或未取代的C4～C8的杂环烷基。
  5. 如权利要求4所述的通式I所示的鬼臼毒素衍生物，其特征在于，
    所述的取代或未取代的C4～C10的杂芳基为取代或未取代的吡咯基、取代或未取代的噻吩基、取代或未取代的喹啉基、取代或未取代的咪唑基、取代或未取代的吡唑基、取代或未取代的苯并吡唑基或取代或未取代的吲哚基；
    和/或，所述的取代或未取代的C4～C8的杂环烷基为取代或未取代的四氢吡咯基。
  6. 如权利要求5所述的通式I所示的鬼臼毒素衍生物，其特征在于，
    所述的吡咯基为
    Figure PCTCN2015076505-appb-100005
    和/或，所述的噻吩基为
    Figure PCTCN2015076505-appb-100006
    和/或，所述的取代的噻吩基为
    Figure PCTCN2015076505-appb-100007
    和/或，所述的喹啉基为
    Figure PCTCN2015076505-appb-100008
    和/或，所述的咪唑基为
    Figure PCTCN2015076505-appb-100009
    和/或，所述的吡唑基为
    Figure PCTCN2015076505-appb-100010
    和/或，所述的苯并吡唑基为
    Figure PCTCN2015076505-appb-100011
    和/或，所述的吲哚基为
    Figure PCTCN2015076505-appb-100012
    所述的取代的吲哚基为
    Figure PCTCN2015076505-appb-100013
    和/或，所述的取代的四氢吡咯 基为
    Figure PCTCN2015076505-appb-100014
  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，其特征在于，所述的通式I所示的鬼臼毒素衍生物中，R为取代或未取代的C5～C10的芳基、取代或未取代的C2～C10的杂芳基或取代的C2～C10的杂环烷基，n为0、1、2或3；当R为取代或未取代的C5～C10的芳基时，X为氧；当R为取代或未取代的C2～C10的杂芳基时，所述的取代或未取代的C2～C10的杂芳基为取代或未取代的吡咯基、取代或未取代的噻吩基、取代或未取代的咪唑基、取代或未取代的吡唑基、取代或未取代的苯并吡唑基或取代或未取代的吲哚基；当R为取代的噻吩基，且所述的取代为被C1～C4的烷基所取代时，X为氮；当R为取代的C2～C10的杂环烷基时，所述的取代是指被羟基和
    Figure PCTCN2015076505-appb-100015
    所取代。
  8. 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，其特征在于，所述的通式I所示的鬼臼毒素衍生物中，R为取代或未取代的C2～C10的杂芳基，或取代或未取代的C2～C10的杂环烷基；n为0、1、2或3。
  9. 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，其特征在于，所述的通式I所示的鬼臼毒素衍生物中，R为取代或未取代的C5～C10的芳基，或取代或未取代的C2～C10的杂芳基，n为0、1、2或3；当R为取代或未取代的C5～C10的芳基时，n为1，X为氧；当R为取代或未取代的C2～C10的杂芳基时，所述的取代或未取代的C2～C10的杂芳基为取代或未取代的吡咯基、取代或未取代的噻吩基、取代或未取代的咪唑基或取代或未取代的吲哚基；当R为取代的噻吩基，且所述的取代为被C1～C4的烷基所取代时，X为氧；当R为取代或未取代的吲哚基，n为1、2或3时，X为O；当所述的吲哚基为
    Figure PCTCN2015076505-appb-100016
    时，n为0，X为N；当R为取代的吲哚基，所述的取代为被卤素所取代，n为0时，X为O；当R为取代的吲哚基，所述的取代为被C1～C4的烷氧基所取代，n为0时，X为N。
  10. 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，其特征在于，所述的通式I所示的鬼臼毒素衍生物中，R为取代或未取代的C2～C10的杂芳基，n为0、1、2或3；当所述的取代或未取代的C2～C10的杂芳基为取代或未取代的吡咯基或吲哚基时，X为N；当R为取代的吲哚基，所述的取代为被卤素所取代，n为0时，X为O。
  11. 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，其特征在于，所述的通式I所示的鬼臼毒素衍生物中，R为取代或未取代的C2～C10的杂芳基，n为0或1；所述的取代或未取代的C2～C10的杂芳基为取代或未取代的吡咯基、取代或未取代的噻吩基、取代或未取代的咪唑基或取代或未取代的吲哚基；当所述的取代或未取代的C2～C10的杂芳基为取代或未取代的吡咯基或者取代或未取代的噻吩基时，X为N；当R为取代的吲哚基，所述的取代为被卤素所取代时，X为O；当R为取代的吲哚基，所述的取代为被C1～C4的烷氧基所取代时，X为N。
  12. 如权利要求1～11中至少一项所述的通式I所示的鬼臼毒素衍生物，其特征在于，其为如下任一化合物：
    Figure PCTCN2015076505-appb-100017
    Figure PCTCN2015076505-appb-100018
    Figure PCTCN2015076505-appb-100019
  13. 一种如权利要求1～12中至少一项所述的通式I所示的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II所示的化合物与如式III所示的化合物进行如下所示的缩合反应，即可；
    Figure PCTCN2015076505-appb-100020
    R1为羟基或氨基；X、R和n的定义均如权利要求1～12任一项所述。
  14. 如权利要求13所述的制备方法，其特征在于，所述的缩合反应的方法包括下列步骤：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II所示的化合物与如式III所示的化合物进行所述的缩合反应。
  15. 如权利要求14所述的制备方法，其特征在于，所述的如式II所示的化合物以盐酸盐的形式参与到反应中；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、1-羟基苯并三氮唑、2-(7-氮杂苯并三氮唑)-N,N,N’,N’-四甲基六氟磷酸酯、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；所述的碱为有机碱；所述的催化剂为4-二甲氨基吡啶；所述的催化剂与如式II 所示的化合物的摩尔比为0.05:1～0.5:1；所述的有机碱与如式II所示的化合物的摩尔比为1:1～3:1；所述的缩合剂与如式II所示的化合物的摩尔比为1:1～4:1；所述的如式II所示的化合物与如式III所示的化合物的摩尔比为1:1～1:3；所述的有机溶剂与如式II所示的化合物的质量体积比为1mL/g～10mL/g；所述的缩合反应的温度为0℃～30℃；所述的缩合反应的时间为1～24小时。
  16. 一种药物组合物，其特征在于，所述的药物组合物包含如权利要求1～12任一项所述的通式I所示鬼臼毒素衍生物以及药学上可接受的赋形剂。
  17. 一种如权利要求1～12中至少一项所述的通式I所示的鬼臼毒素衍生物在制备用于治疗癌症的药物中的应用。
  18. 如权利要求17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。
  19. 如权利要求18所述的应用，其特征在于，所述的肺癌为人非小细胞肺癌；所述的口腔癌为人口腔上皮癌；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。
  20. 如权利要求19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；所述的人口腔癌细胞为人口腔癌上皮细胞株KB；所述的人肝癌细胞为人肝癌细胞株HepG2；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。
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