WO2018090891A1 - 联吡啶类生物碱、其制备方法和用途 - Google Patents

联吡啶类生物碱、其制备方法和用途 Download PDF

Info

Publication number
WO2018090891A1
WO2018090891A1 PCT/CN2017/110799 CN2017110799W WO2018090891A1 WO 2018090891 A1 WO2018090891 A1 WO 2018090891A1 CN 2017110799 W CN2017110799 W CN 2017110799W WO 2018090891 A1 WO2018090891 A1 WO 2018090891A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
group
methanol
dichloromethane
optionally
Prior art date
Application number
PCT/CN2017/110799
Other languages
English (en)
French (fr)
Inventor
朱伟明
梅显贵
付鹏
王乂
刘培培
Original Assignee
中国海洋大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国海洋大学 filed Critical 中国海洋大学
Priority to US16/349,915 priority Critical patent/US20190322638A1/en
Publication of WO2018090891A1 publication Critical patent/WO2018090891A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/68—One oxygen atom attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/69—Two or more oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • C07D213/803—Processes of preparation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/02—Heterocyclic radicals containing only nitrogen as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin

Definitions

  • This invention relates to bipyridyl alkaloids, to processes for their preparation and to their use.
  • Chemotherapy is a treatment method that uses chemical drugs to kill tumor cells, inhibit the growth and reproduction of tumor cells, and promote the differentiation of tumor cells.
  • chemotherapeutic drugs kill tumor cells while normal cells and immune cells are also It is killed a lot, and its side effects and toxic effects are enormous.
  • Chemotherapy can cause liver and kidney function damage, gastrointestinal disorders, decreased immune function, hair loss, nausea and vomiting. Almost all chemotherapeutic drugs can cause liver damage. In mild cases, liver function abnormalities may occur. Patients may develop liver discomfort, which may lead to toxic hepatitis. Some chemotherapeutic drugs may cause kidney damage and kidney pain and kidney pain. District is not suitable. How to find a drug that does not harm normal cells that sustain life when it kills a specific tumor in a patient will greatly improve the survival rate and quality of life of chemotherapy patients.
  • the present inventors have been working to develop a bipyridine alkaloid having an antitumor use.
  • the present inventors have found that bipyridyl alkaloids have important prospects in the treatment of tumors.
  • the invention provides a compound of formula I, a pharmaceutically acceptable salt or prodrug thereof,
  • R 4 and R 6 are each independently selected from: -H, alkyl, alkanoyl;
  • R 5 is selected from the group consisting of: -H, -OH, -NH 2 , alkoxy;
  • R 2 is selected from the group consisting of: -H, an alkyl group, a sugar group;
  • R 3 is selected from the group consisting of: -H, -OH, -NH 2 , alkoxy;
  • X is selected from the group consisting of: -H, halogen, -NO 2 , -SO 2 R 7 , -CO R 8 ;
  • R 7 is selected from the group consisting of: -H, aryl
  • R 8 is an aryl group.
  • the alkyl group and the alkyl group of the alkanoyl group and the alkoxy group are each independently a linear or branched C 1 -C 16 alkyl group, optionally a linear or branched C 1 -C 10 alkyl group. , optionally a linear or branched C 1 -C 6 alkyl group, optionally a linear or branched C 1 -C 4 alkyl group;
  • the glycosyl group is selected from the group consisting of: glucosyl, rhamnosyl, isorhamnose, ribosyl, galactosyl, algose, fucosyl, idose, talose , 2,4-dimethoxyrhamnosyl, 2,4-dimethoxyglucosyl, 2,4-dimethoxyiso rhamnose, 2,4-dimethoxyribosyl, 2,4-Dimethoxygalactosyl, 2,4-dimethoxyallosyl, 2,4-dimethoxyfucosyl, 2,4-dimethoxyidurtyl , 2,4-dimethoxy talose;
  • the halogen is selected from the group consisting of: -F, -Cl, -Br, -I;
  • the aryl group is a C 6 -C 18 monocyclic or polycyclic aryl group, optionally a C 6 -C 14 monocyclic or polycyclic aryl group, optionally a C 6 -C 10 monocyclic or polycyclic aromatic group. base.
  • R 2 is selected from the group consisting of: -H, methyl, glucosyl, rhamnose, isorhamnose, ribosyl, galactosyl, alenose, fucosyl, idose, talose Base, 2,4-dimethoxyrhamnose, 2,4-dimethoxyglucosyl, 2,4-dimethoxyiso rhamnose, 2,4-dimethoxyribosyl , 2,4-dimethoxygalactosyl, 2,4-dimethoxyalose, 2,4-dimethoxyfucosyl, 2,4-dimethoxyidose a 2,4-dimethoxy talose group; optionally, R 2 is an L-rhamnosyl group; alternatively, R 2 is a methyl group;
  • R 3 is selected from the group consisting of: -H, -OH, -OCH 3 ; optionally, R 3 is -H;
  • X is selected from the group consisting of: -H, -F, -Cl, -Br, -I, -NO 2 , -SO 3 H, -SO 2 C 6 H 5 , -COC 6 H 5 ; optionally, X is -H ;
  • the compound of formula I is Compound 1, or Compound 2, or Compound 3, or Compound 4, or Compound 5, or Compound 6, or Compound 7:
  • the pharmaceutically acceptable salt includes a salt of an organic or inorganic acid
  • the pharmaceutically acceptable salt is a salt of the compound of formula I with a compound selected from the group consisting of: hydrochloric acid; sulfuric acid; phosphoric acid; formic acid; acetic acid; propionic acid; lactic acid; citric acid; tartaric acid; ; fumaric acid; maleic acid; mandelic acid; malic acid; camphorsulfonic acid;
  • the pharmaceutically acceptable prodrug comprises a prodrug of the compound of formula I bonded to a pharmaceutically acceptable carrier; optionally, the pharmaceutically acceptable carrier comprises: triglyceride phosphate, poly Ethylene glycol ester, polyethylene glycol amide, polyethylene glycol ether.
  • the present invention also provides a process for the preparation of a compound of the above formula I, a pharmaceutically acceptable salt or prodrug thereof, which comprises: to a fermenting medium of A. cyanogriseus WH1-2216-6 The precursor Y is added, and the A. cyanogriseus WH1-2216-6 is fermented and cultured, and the fermentation product is separated and purified to obtain a product; optionally, the separated and purified product is used as a raw material to prepare a semi-synthesis reaction. ,
  • the fermentation culture comprises: culturing A. cyanogriseus WH1-2216-6 in a seed culture medium, inoculating into a fermentation medium, culturing, fermenting, and preparing a fermentation product;
  • the seed culture medium comprises: a carbon source, a nitrogen source, and an aqueous solution containing sodium chloride; optionally, the seed culture medium comprises: peptone, glycerin, soybean powder, soluble starch, calcium carbonate, and chlorination.
  • the separating and purifying comprises: extracting the fermentation product with an organic solvent, concentrating the organic phase, adding an acidic solution, extracting with an organic solvent, removing the organic phase, adjusting the pH with an aqueous phase, and extracting with an organic solvent.
  • the organic phase is concentrated to obtain an alkaloid fraction, and the obtained alkaloid fraction is selected from the group consisting of gel column chromatography and silica gel column chromatography (optionally, vacuum gel column chromatography, reversed-phase decompression silica gel column, and flash silica gel column chromatography).
  • each of the extraction organic solvents is independently selected from the group consisting of ethyl acetate, dichloromethane, chloroform, petroleum ether; alternatively, the acidic solution is selected from hydrochloric acid, Sulfuric acid, nitric acid, phosphoric acid; optionally, the base is selected from the group consisting of: ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate; optionally, the eluent of the gel column chromatography is selected from the group consisting of methanol, a dichloromethane-methanol mixture; optionally, the eluent of the silica gel column chromatography is selected from the group consisting of petroleum ether, dichloromethane, methanol, dichloromethane-methanol mixture, water; optionally, the reversed phase Vacuum gel column
  • the de-agent is selected from the group consisting of methanol and water; optionally, the high
  • the de-agent is selected from petroleum ether, dichloromethane, dichloromethane-methanol mixture; alternatively, the eluent of the Sephadex LH-20 gel column chromatography is methanol, dichloromethane-methanol mixture;
  • the eluent of the flash silica gel column chromatography is dichloromethane or methanol; optionally, the eluent of the reversed-pressure vacuum silica gel column is selected from the group consisting of methanol and water; optionally, the high-efficiency liquid
  • the phase-prepared eluent is selected from the group consisting of a methanol-water mixture and an acetonitrile-water mixture;
  • the semi-synthesis reaction using the separated and purified product as a raw material comprises:
  • the isolated compound of CONH 2 is chemically reacted with a common chemical reagent or a halogenated hydrocarbon R 4 Cl, or formaldehyde, or chromic acid, or a halogenated hydrocarbon R 6 Cl, or acetic anhydride, or potassium hydroxide, respectively, through an alkyl group.
  • a reaction, or an oxidation reaction, or a reduction reaction, or a hydrolysis reaction to introduce a compound containing these groups;
  • R 2 is an alkyl group, and when the glycosyl group is used, the isolated compound having R 2 is -H is used as a raw material and a common halogenated hydrocarbon R 2 Cl or a sugar is chemically reacted, respectively, by alkylation reaction, glycosidation reaction Introducing a compound containing these groups;
  • the compound of formula I is compound 1, or compound 2, or compound 3, or compound 4, or compound 5, or compound 6, or compound 7;
  • the compounds 1 to 4 are obtained by the following methods:
  • the A. cyanogriseus WH1-2216-6 was cultured in a seed culture medium, inoculated into a fermentation medium to which 2-picolinic acid was added, and fermented by a shaker to obtain a fermented product, and the fermented product was organic.
  • step (b) The other alkaloids described in the step (a) are combined and then subjected to a vacuum gel column chromatography, followed by gradient elution with petroleum ether, dichloromethane, dichloromethane-methanol as eluent:
  • dichloromethane-methanol eluent comprises the following elution components: dichloromethane:methanol (v/v) is 100:1, 50:1, 30:1, 25:1, 15:1 , 10:1, 8:1, 5:1, 2:1, 1:1, and 0:1;
  • the compound 5 is obtained by culturing the A. cyanogriseus WH1-2216-6 in a seed medium and inoculating the fermentation to which 5-fluoropicolinic acid (precursor 9) is added.
  • the fermentation is carried out in a shaker to obtain a fermented product, the fermentation product is extracted with an organic solvent, and the organic phase is concentrated to obtain a crude extract, which is separated by a vacuum gel column chromatography, followed by petroleum ether, dichloromethane, The dichloromethane-methanol mixture was eluted with a gradient of eluent.
  • the obtained fractions were separated on a Sephadex LH-20 gel column and eluted with methanol.
  • the compound 6 is obtained by the following synthesis method: adding an aqueous formaldehyde solution, hydrochloric acid and water to the isolated compound 2, heating under reflux, and then cooling to room temperature, adding a saturated aqueous solution of NaHCO 3 thereto, and extracting with an organic solvent After concentrating under reduced pressure, the organic phase is separated by a silica gel column, eluting with eluent from ethyl acetate, dichloromethane, chloroform, petroleum ether, preferably with dichloromethane and methanol as eluent gradient Elution, to obtain a compound 6;; optionally, the concentration of the aqueous formaldehyde solution is 37% (v / v); optionally, the concentration of the hydrochloric acid is 10N;
  • the compound 7 was obtained by the following method: A. cyanogriseus WH1-2216-6 was cultured in a seed medium, inoculated into a fermentation medium to which 2-picolinic acid was added, and fermented by a shaker to obtain a fermentation. The fermented product is extracted with an organic solvent, and the organic phase is concentrated to obtain a crude extract.
  • the crude crude extract is subjected to silica gel column chromatography under reduced pressure, followed by petroleum ether, dichloromethane, and dichloromethane-methanol mixed solvent (v/ Gradient elution of v, 100:1, 50:1, 30:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1) as eluent
  • the organic solvent for extraction is selected from the group consisting of ethyl acetate, dichloromethane, chloroform, and petroleum ether.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one of the above compounds of formula I, a pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition described above characterized in that the pharmaceutical composition comprises a solid preparation and a liquid preparation; optionally, the pharmaceutical composition comprises an oral preparation, an injection preparation, a transdermal preparation; optionally, the pharmaceutical composition comprises a tablet, a capsule, a powder, a granule, a lozenge, a suppository, an oral solution, a sterile parenteral suspension, an injection; optionally, the injection includes a frozen Dry powder injection.
  • the above compound of formula I, a pharmaceutically acceptable salt or prodrug thereof, or the use of the above pharmaceutical composition for the preparation of an antitumor drug is a tumor cell proliferation inhibitor Or tumor cell killing agent; optionally, the anti-tumor drug does not include anti-human lung adenocarcinoma A549 drug, anti-human acute promyelocytic leukemia HL60 drug, anti-human chronic myeloid leukemia K562 drug, anti-human oral epidermoid carcinoma KB drug; optionally, the anti-tumor drug is: anti-human colon cancer cell line HCT-116 drug, anti-human breast cancer cell line MCF-7 drug, anti-hepatoma cell line HepG2 drug, anti-cervical cancer cell line Hela Drug, or, anti-human peripheral blood leukemia T cell line Jurkat drug.
  • the antitumor drug is a tumor cell proliferation inhibitor Or tumor cell killing agent
  • the anti-tumor drug does not include anti-human lung adenocarcinoma A549 drug
  • the invention also provides the use of a compound of formula I above, a pharmaceutically acceptable salt or prodrug thereof, as a probe for inhibiting cell proliferation.
  • the present invention also provides the use of the above pharmaceutical composition as a probe for inhibiting cell proliferation.
  • the invention also provides a probe kit comprising the above compound of formula I, a pharmaceutically acceptable salt or prodrug thereof.
  • the present invention also provides a probe kit comprising the above pharmaceutical composition.
  • the probe kit further comprises a biocompatible medium; optionally, the biocompatible medium is selected from at least one of methanol, water, and dimethyl sulfoxide.
  • A. cyanogriseus WH1-2216-6 for the preparation of the compound of the formula I of the present invention the species collection number: CCTCC M 209277, the preservation date: November 28, 2009, the preservation location: Wuhan, Wuhan, China , China Typical Culture Collection.
  • the actinomycete strain WH1-2216-6 was isolated from sea mud collected from Weihai, Shandongzhou. Details of the strain have been reported (Fu P. Wang S, Hong K, LiX, Liu P, Wang Y, Zhu W. Cytotoxic Bipyridines from the marine-derived actinomycete Actinoalloteichus cyanogriseus WH1-2216-6.J.Nat.Prod.2011, 74,1751-1756), identified as a strain of A. faecalis by polyphasic taxonomy, designated as Actinoalloteichuscyanogriseus WH1 -2216-6.
  • the method of preparing the compound of the present invention by fermenting microorganisms may employ any other microorganism capable of producing the compound of the present invention, as long as the microorganism capable of producing the compound of the present invention can be used as a production strain for the preparation of the compound of the present invention.
  • the compound of the present invention has good antitumor activity and has good selectivity, and has significant inhibitory effect on tumor cell proliferation, and has no obvious inhibitory effect on growth of normal cell strain.
  • Figure 1 is a photograph of colon cancer (HCT116) of each group in Test Example 2, wherein Group A is a negative control (saline) group, B is a docetaxel (5 mg/kg) group, and C is a compound 2 of the present invention ( 7.5 mg/kg) group.
  • HCT116 colon cancer
  • Fermentation culture A. cyanogriseus WH1-2216-6 was cultured in a seed culture medium for 5 days, and inoculated into a 500 mL flask containing 150 mL of fermentation medium to which 2-pyridine was added. Formic acid 0.4g. The shaker was cultured and fermented for 12 days to obtain 40 L of the fermentation product.
  • Fermentation culture of the precursor 5-fluoropicolinic acid A. cyanogriseus WH1-2216-6 was cultured in the seed medium for 5 days, and inoculated with 0.4 g of the precursor 5-fluoropicolinic acid. In 150 mL of fermentation medium, the mixture was shaken, fermented for 12 days, and 7 bottles were co-fermented to obtain 1.0 L of the fermentation product.
  • the fermentation product was extracted three times with an equal volume of ethyl acetate, and the organic phase was combined and concentrated to give a crude extract.
  • the crude extract was separated by vacuum silica gel column chromatography, followed by petroleum ether, dichloromethane, dichloromethane.
  • the methanol was used as the eluent for the gradient elution.
  • Reaction step Compound 2 (30.0 mg, 0.13 mmol) was placed in a 25 mL reaction flask, 2.28 mL of 37% formaldehyde aqueous solution, 0.116 mL of 10 N hydrochloric acid and 1.032 mL of water were added, followed by heating at 105 ° C for 30 minutes. cooled to room temperature, 10mL of saturated aqueous NaHCO 3 was added to quench the reaction.
  • the compound 7 was obtained by the following method: A. cyanogriseus WH1-2216-6 was cultured in a seed medium, inoculated into a fermentation medium to which 2-picolinic acid was added, and fermented by a shaker to obtain a fermentation. The fermented product is extracted with an organic solvent, and the organic phase is concentrated to obtain a crude extract.
  • the crude crude extract is subjected to silica gel column chromatography under reduced pressure, followed by petroleum ether, dichloromethane, and dichloromethane-methanol mixed solvent (v/ Gradient elution of v, 100:1, 50:1, 30:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1) as eluent
  • cyanogriseus WH1-2216-6 was cultured in a seed culture medium, inoculated into a fermentation medium to which benzoic acid was added, and fermented by a shaker to obtain a fermented product, and the fermented product was organic.
  • Compounds 7 and 8 were identified by comparison with standards by thin layer chromatography (TLC) and HPLC (Compound 7: Fu P, Zhu Y, Mei X, Wang Y, Jia H, Zhang C, Zhu W. Acyclic Congeners from Actinoalloteichus cyanogriseus Provide Insights into Cyclic Bipyridine Glycoside Formation. Org. Lett. 2014, 16, 4264-4267; Compound 8: Fu P, Wang S, Hong K, Li X, Liu P, Wang Y, Zhu W. Cytotoxic bipyridines from the Marine-derived actinomycete Actinoalloteichus cyanogriseus WH1-2216-6.J. Nat. Prod. 2011, 74, 1751-1756).
  • test sample is the compound 1-8 prepared in the above Preparation Example 1, and an appropriate amount of the sample is accurately weighed, and the solution of the desired concentration is prepared with methanol for the activity test.
  • Subculture of cell lines and cells 5 tumor cell lines were tested for activity: human colon cancer cell line (HCT-116 cells), human hepatoma cell line (HepG2 cells), human breast cancer cell line (MCF-7 cells) , cervical cancer cell line (Hela cell), human peripheral blood leukemia T cell line (Jurkat cell), and a human normal liver cell line (L-02 cell), of which MCF-7, HepG2, Hela, L-02 Cell lines were tested using the MTT model; HCT-116 cell lines were tested using the SRB model.
  • Various cells were subcultured with RPMI-1640 medium containing 10% FBS and incubated at 37 ° C in a 5% carbon dioxide incubator.
  • MTT method Dehydrogenase in living cell mitochondria can metabolize yellow-purified 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium blue-purple water-insoluble formazan, The amount of formazan can be determined by measuring the absorbance by a microplate reader. Since the amount of formazan is proportional to the number of living cells, the number of living cells can be determined from the absorbance to understand the ability of the drug to inhibit or kill tumor cells.
  • the cell line of the logarithmic growth phase was prepared, and the cell suspension with a density of 3 ⁇ 10 4 cells per ml was prepared by using fresh RPMI-1640 medium, and inoculated into a 96-well plate at 100 ⁇ L per well.
  • IR% (OD blank control - OD sample ) / OD blank control ⁇ 100%.
  • the tumor cells in the logarithmic growth phase (medium: RPMI-1640 medium containing 10% newborn fetal bovine serum (FBS); cell density 3 ⁇ 10 4 cells/mL) at 180 ⁇ L / wells were seeded in 96-well culture plates, adherently grown for 24 hours at 37 ° C, 5% CO 2 , and 20 ⁇ L/well of test samples were added, with four replicate wells per concentration.
  • the final screening concentration of the sample was set to 10 ⁇ M, and 5 to 7 concentration gradients were set by the double dilution method when testing the IC 50 ; the positive drug was doxorubicin 1 ⁇ M; the blank control was an equal amount of the corresponding concentration of the medium).
  • the tumor cells after dosing were further cultured at 37 ° C under 5% CO 2 for 72 hours, then the culture solution was decanted, and the cells were fixed with 10% cold trichloroacetic acid (TCA), left at 4 ° C for 1 hour, and then washed with distilled water. The air is naturally dry. Then, 100 ⁇ L/well of a 4 mg/ml solution of sulforaprofen B (SRB, Sigma) prepared from 1% glacial acetic acid was added, and the mixture was stained at room temperature for 15 minutes, and the supernatant was removed, washed 5 times with 1% acetic acid, and air-dried.
  • TCA cold trichloroacetic acid
  • IR% (OD blank control - OD sample ) / OD blank control ⁇ 100% formula.
  • IC 50 half-inhibitory concentration against cancer cells was determined.
  • the Selection Index is the ratio of the CC 50 value of the test sample to the growth inhibition of the normal cell line and the IC 50 value of the tumor cell proliferation inhibition, which can reflect the selectivity and safety of the sample.
  • selection index CC 50 (normal cell L-02 strain) / IC 50 (tumor cell line)
  • compound 8 (caerulomycin F) has no tumor cytotoxic activity against HCT-116 cell line, and the compound of the present invention has good antitumor activity and has little toxic side effect on normal human cells.
  • High safety factor, especially compound 2 has very good anti-HCT-116 tumor activity and selectivity.
  • selection index CC 50 (normal cell L-02 strain) / IC 50 (tumor cell line)
  • compound 8 (caerulomycin F) has no cytotoxic activity against MCF-7 tumor cell line, and the compound of the present invention has good antitumor activity and has little toxic side effect on normal human cells.
  • High safety factor, especially compound 2 has very good anti-MCF-7 tumor cell activity and selectivity.
  • selection index CC 50 (normal cell L-02 strain) / IC 50 (tumor cell line)
  • compound 8 (caerulomycin F) has no cytotoxic activity against HepG2 tumor cell lines, and the compound of the present invention has good antitumor activity, has little toxic side effect on normal human cells, and has high safety factor.
  • Compound 2 has very good anti-HepG2 tumor cell activity and selectivity.
  • selection index CC 50 (normal cell L-02 strain) / IC 50 (tumor cell line)
  • compound 8 (caerulomycin F) has no cytotoxic activity against Hela tumor cell lines, and the compound of the present invention has good antitumor activity, has little toxic side effect on normal human cells, and has high safety factor.
  • Compound 2 has very good anti-Hela tumor cell activity and selectivity.
  • selection index CC 50 (normal cell L-02 strain) / IC 50 (tumor cell line)
  • compound 8 (caerulomycin F) has no cytotoxic activity against Jurkat tumor cell line, and the compound of the present invention has good antitumor activity, has little toxic side effect on normal human cells, and has high safety factor.
  • Compound 2 has very good anti-Jurkat tumor cell activity and selectivity.
  • the compound of the formula I of the present invention has high selectivity against cancer cell proliferation, has low toxicity, and has high safety factor, especially compound 2, which has very good antitumor activity and selectivity.
  • mice were BALB/C, nu/nu nude mice, weighing 18-20 g, male. Purchased from Beijing Huakangkang Biotechnology Co., Ltd., license number: SCXK (Beijing)-2014-0004.
  • Test drug Compound 2 was dissolved in DMSO at 10 mg/mL.
  • the physiological saline dilution concentration was 0.75 mg/mL, and the administration volume was 0.1 mL/10 g body weight.
  • the cell suspension of human colon cancer cell line HCT-116 at a concentration of 30 million/mL was prepared in 5A medium containing 10% fetal bovine serum. After routine disinfection, 0.2 ml of each was inoculated into the right forelimb of the right forelimb of the mouse. The tumor is administered after the tumor has grown to an average volume of about 100 mm 3 .
  • Set positive drugs, compounds, and negative control groups were: docetaxel (positive drug) 5 mg/kg, and compound 2: 7.5 mg/kg.
  • Docetaxel was administered intravenously, administered every other day; Compound 2 was intraperitoneally injected daily; the negative control group was intraperitoneally injected with the same volume of normal saline.
  • the formula for calculating the tumor inhibition rate is as follows:
  • Tumor inhibition rate (average tumor weight of the control group - mean tumor weight of the treatment group) / average tumor weight of the control group ⁇ 100%
  • Spleen index 100 x spleen mass / body mass.
  • the average weight of the tumor in the negative control group was 1.0 g or more, which proved that the colon cancer (HCT-116) model was successfully constructed.
  • the positive drug was selected from the broad-spectrum anti-tumor drug docetaxel, which was used in clinical trials.
  • the polymethicone 5 mg/kg group significantly inhibited the growth of liver cancer (HCT-116) with an inhibition rate of 87.39%. P ⁇ 0.01), indicating that the results of the experiment were credible; however, the polymetheta group decreased the body weight and spleen index of mice, reflecting the side effects of inhibition of the immune system by docetaxel.
  • Compound 2 had a significant colon cancer (HCT-116) growth inhibition compared with the negative control group at a dose of 7.5 mg/kg for 14 days (Fig. 1, Table 10), and the inhibition rate was 42.46% (p ⁇ 0.05); however, the effect of Compound 2 on the spleen index of mice was not observed (Table 11), indicating that Compound 2 is a safe and effective antitumor drug.
  • Tumor weight Inhibition rate% Negative control (saline) 1.03 ⁇ 0.32 / Docetaxel (5mg/kg) 0.13 ⁇ 0.07 87.39** Compound 2 (7.5 mg/kg) 0.59 ⁇ 0.33 42.46*

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Dermatology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

联吡啶类生物碱、其制备方法和用途，所述联吡啶类生物碱结构如式I所示。所述联吡啶类生物碱具有肿瘤细胞增殖抑制活性，可作为肿瘤细胞增殖抑制剂或用于抗肿瘤药物开发。

Description

联吡啶类生物碱、其制备方法和用途 技术领域
本发明涉及联吡啶类生物碱、其制备方法和用途。
背景技术
《中国居民营养与慢性病状况报告(2015年)》指出，在2012年全国居民慢性病死亡率为533/10万，占总死亡人数的86.6％。心脑血管病、癌症和慢性呼吸系统疾病为主要死因，占总死亡的79.4％，其中癌症死亡率为144.3/10万(前五位分别是肺癌、肝癌、胃癌、食道癌、结直肠癌)。而2016年发表于CA：A Cancer_Journal_for_Clinicians一篇报告指出，在中国，癌症已成为诸多疾病死因之首，发病率和死亡率持续攀升，2015年中国预计有429.2万例新发肿瘤病例和281.4万例死亡病例，全球几乎22％的新发癌症病例和27％的癌症死亡病例在中国。癌症已成为非常重要的公共健康问题，中国人口基数众多也使得国人的数据对全球癌症防控具有重要意义(Chen W，Zheng R，Baade PD，et al.Cancer statistics in China，2015.Ca-Cancer.J.Clin.2016，66：115132.doi：10.3322/caac.21338)。2011年，Science特刊“向癌宣战四十年Cancer Crusade at 40”指出，癌症在今天仍然是威胁人类的主要疾病，从1971年到现在，美国癌症死亡率的依然维持较高水平，许多类型的癌症如前列腺癌等依旧不可治愈(Kiberstis P，Marshall E.Cancer crusade at 40.Celebrating an anniversary.Introduction.Science 2011，331：1539)。
随着科学技术和医疗水平的不断发展，人类探索出多种治疗肿瘤的方法，主要分为手术、化疗、放疗、中医药介入多学科综合治疗等(闫胜祥，苑华萍.试论肿瘤治疗方法.中国医师杂志.2002(增刊)，55)。其中，化疗是治疗癌症患者的重要手段之一。化疗是利用化学药物杀死肿瘤细胞、抑制肿瘤细胞的生长繁殖和促进肿瘤细胞的分化的一种治疗方式，但由于化疗选择性差，化疗药在杀死肿瘤细胞的同时，正常细胞和免疫细胞也被大量杀死，副作用和毒害作用极大。化疗可引起肝肾功能受损，胃肠功能紊乱，免疫功能下降，脱发，恶心呕吐等不良后果。几乎所有的化疗药物均可引起肝功能损害，轻者可出现肝功能异常，患者可出现肝区不适，甚者可导致中毒性肝炎；有些化疗药大剂量可引起肾功能损害而出现腰痛、肾区不适等。如何能发现一种药物，在有效杀死患者体内的特定肿瘤时不会伤及维持生命的正常细胞，将会大大提高化疗患者的生存率和生存质量。
发明内容
本发明人致力于开发具有抗肿瘤用途的联吡啶类生物碱。本发明人发现，联吡啶类生物碱在治疗肿瘤方面有着重要的前景。
为此，本发明提供一种式I化合物、其药学上可接受的盐或前药，
Figure PCTCN2017110799-appb-000001
其中：
R1选自：-CH＝N-O-R4，-COR5，-CH2OR6，-CN，-NH2；
R4和R6各自独立地选自：-H，烷基，烷酰基；
R5选自：-H，-OH，-NH2，烷氧基；
R2选自：-H，烷基，糖基；
R3选自：-H，-OH，-NH2，烷氧基；
X选自：-H，卤素，-NO2，-SO2R7，-CO R8；
R7选自：-H，芳基；
R8为芳基。
可选地，上述的式I化合物、其药学上可接受的盐或前药，其中：
所述烷基和烷酰基、烷氧基中的烷基各自独立地为直链或支链的C1～C16烷基，可选地为直链或支链的C1～C10烷基，可选地为直链或支链的C1～C6烷基，可选地为直链或支链的C1～C4烷基；
所述糖基选自：葡萄糖基，鼠李糖基，异鼠李糖基，核糖基，半乳糖基，阿洛糖基，岩藻糖基(fucosyl)，艾杜糖基，塔洛糖基，2，4-二甲氧基鼠李糖基，2，4-二甲氧基葡萄糖基，2，4-二甲氧基异鼠李糖基，2，4-二甲氧基核糖基，2，4-二甲氧基半乳糖基，2，4-二甲氧基阿洛糖基，2，4-二甲氧基岩藻糖基，2，4-二甲氧基艾杜糖基，2，4-二甲氧基塔洛糖基；
所述卤素选自：-F，-Cl，-Br，-I；
所述芳基为C6～C18单环或多环芳基，可选地为C6～C14单环或多环芳基，可选地为C6～C10单环或多环芳基。
可选地，上述的式I化合物、其药学上可接受的盐或前药，其中：
R1选自：-CH2OH，-CH＝NOH，-CN，-CONH2，-NH2，-CHO，-COOH；可选地，R1为-CH＝NOH；
R2选自：-H，甲基，葡萄糖基，鼠李糖基，异鼠李糖基，核糖基，半乳糖基，阿洛糖基，岩藻糖基，艾杜糖基，塔洛糖基，2，4-二甲氧基鼠李糖基，2，4-二甲氧基葡萄糖基，2，4-二甲氧基异鼠李糖基，2，4-二甲氧基核糖基，2，4-二甲氧基半乳糖基，2，4-二甲氧基阿洛糖基，2，4-二甲氧基岩藻糖基，2，4- 二甲氧基艾杜糖基，2，4-二甲氧基塔洛糖基；可选地，R2为L-鼠李糖基；可选地，R2为甲基；
R3选自：-H，-OH，-OCH3；可选地，R3为-H；
X选自：-H，-F，-Cl，-Br，-I，-NO2，-SO3H，-SO2C6H5，-COC6H5；可选地，X为-H；
可选地，所述式I化合物为化合物1，或化合物2，或化合物3，或化合物4，或化合物5，或化合物6，或化合物7：
Figure PCTCN2017110799-appb-000002
可选地，上述的式I化合物、其药学上可接受的盐或前药，其中：
所述药学上可接受的盐包括有机或无机酸的盐；
可选地，所述药学上可接受的盐为所述式I化合物与选自以下的化合物形成的盐：盐酸；硫酸；磷酸；甲酸；乙酸；丙酸；乳酸；柠檬酸；酒石酸；琥珀酸；富马酸；马来酸；杏仁酸；苹果酸；樟脑磺酸；
所述药学上可接受的前药包括所述式I化合物与药学上可接受的载体键合而成的前药；可选地，所述药学上可接受的载体包括：磷酸甘油三酯、聚乙二醇酯、聚乙二醇酰胺、聚乙二醇醚。
本发明还提供一种制备上述的式I化合物、其药学上可接受的盐或前药的方法，其特征在于，包括：向异壁放线菌A.cyanogriseus WH1-2216-6发酵培养基中加入前体物Y，发酵培养异壁放线菌A.cyanogriseus WH1-2216-6，将发酵产物进行分离纯化制得产物；任选地，以分离纯化的产物为原料，进行半合成反应制得，
Figure PCTCN2017110799-appb-000003
其中，X如上定义；
可选地，所述发酵培养包括：将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基进行培养，接种到发酵培养基中，培养，发酵，制得发酵产物；
可选地，所述种子培养基包括：碳源、氮源、含氯化钠水溶液；可选地，所述种子培养基包括：蛋白胨、甘油、黄豆粉、可溶性淀粉、碳酸钙、含氯化钠水溶液(优选天然海水)；可选地，所述种子培养基为：蛋白胨15重量份、甘油15重量份、黄豆粉5重量份、可溶性淀粉15重量份、CaCO32重量份、陈海水1000重量份，pH＝7.8；
可选地，所述发酵培养基包括：可溶性淀粉、甘油、蛋白胨、碳酸钙、大孔吸附树脂、海水；可选地，所述发酵培养基为：可溶性淀粉20重量份、甘油20重量份、蛋白胨20重量份、CaCO32重量份、XAD-16大孔吸附树脂重量份、陈海水1000重量份，pH＝7.5；
可选地，所述分离纯化包括：将所述发酵产物用有机溶剂萃取，有机相浓缩后，加入酸性溶液，有机溶剂萃取，除去有机相，水相加碱调节pH，再用有机溶剂萃取，浓缩有机相，得生物碱部分，所得生物碱部分用选自凝胶柱色谱法、硅胶柱色谱法(可选地为减压硅胶柱色谱、反相减压硅胶色谱柱、快速硅胶柱色谱分离)、半制备高效液相色谱法分离；可选地，各个萃取用有机溶剂各自独立的选自乙酸乙酯、二氯甲烷、氯仿、石油醚；可选地，所述酸性溶液选自盐酸、硫酸、硝酸、磷酸；可选地，所述碱选自：氨水、氢氧化钠、氢氧化钾、碳酸钠、碳酸钾；可选地，所述凝胶柱色谱的洗脱剂选自甲醇、二氯甲烷-甲醇混合液；可选地，所述硅胶柱色谱的洗脱剂选自石油醚、二氯甲烷、甲醇、二氯甲烷-甲醇混合液、水；可选地，所述反相减压硅胶色谱柱的洗脱剂选自甲醇、水；可选地，所述高效液相半制备的洗脱剂选自甲醇-水混合液、乙腈-水混合液；可选地，所述减压硅胶柱色谱的洗脱剂选自石油醚、二氯甲烷、二氯甲烷-甲醇混合液；可选地，所述Sephadex LH-20凝胶柱色谱的洗脱剂为甲醇、二氯甲烷-甲醇混合液；可选地，所述快速硅胶柱色谱的洗脱剂为二氯甲烷、甲醇；可选地，所述反相减压硅胶色谱柱的洗脱剂选自甲醇、水；可选地，所述高效液相半制备的洗脱剂选自甲醇-水混合液、乙腈-水混合液；
可选地，以分离纯化的产物为原料进行半合成反应包括：
(i)当R1选自：-CH＝N-O-R4，-COR5，-CH2OR6，-CN，-NH2时，以R1为-CH＝N-OH或-CH2OH或-CONH2的分离化合物为原料和常见化学试剂或卤代烃R4Cl，或甲醛，或铬酸，或卤代烃R6Cl，或乙酸酐，或氢氧化钾发生化学反应，分别通过烷基化反应，或氧化反应，或还原反应，或水解反应引入含有这些基团的化合物；
(ii)R2为烷基，糖基时，以R2为-H的分离化合物为原料和常见的卤代烃R2Cl，或糖发生化学反应，分别通过烷基化反应，糖苷化反应引入含有这些基团的化合物；
(iii)当R3为-NH2或烷氧基时，以R3为-OH的分离化合物为原料和常见的卤代烷烃，分别通过卤代反应，置换反应，烷基化反应在苯环上引入-NH2和烷氧基。
上述反应可以通过本领域已知的方法进行。
可选地，所述式I化合物为化合物1，或化合物2，或化合物3，或化合物4，或化合物5，或化合物6，或化合物7；
可选地，所述化合物1～4由以下方法得到：
(a)将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养，接种到加入了2-吡啶甲酸的发酵培养基中，摇床培养发酵，获得发酵物，发酵物用有机溶剂萃取，有机相浓缩得粗提物，向粗提物中加入HCl溶液，用有机溶剂萃取，用氨水调节水相至pH＝8.0，再用有机溶剂萃取，浓缩有机相得生物碱部分，所得生物碱部分用凝胶柱分离，所述凝胶柱的流动相为二氯甲烷∶甲醇＝1∶1(v/v)，得化合物2和其他生物碱部分；
(b)将步骤(a)所述其他生物碱部分合并后再经减压硅胶柱色谱分离，依次以石油醚、二氯甲烷、二氯甲烷-甲醇为洗脱液进行梯度洗脱：
其中，二氯甲烷-甲醇洗脱剂包括以下配比的洗脱组分∶二氯甲烷∶甲醇(v/v)为100∶1、50∶1、30∶1、25∶1、15∶1、10∶1、8∶1、5∶1、2∶1、1∶1和0∶1；
其中二氯甲烷洗脱组分和二氯甲烷-甲醇洗脱剂中二氯甲烷∶甲醇＝100∶1(v/v)洗脱组分合并后经葡聚糖凝胶Sephadex LH-20色谱柱分离、以二氯甲烷∶甲醇＝1∶1(v/v)洗脱，所得组分再经SephadexLH-20凝胶柱色谱分离，以甲醇洗脱得到化合物4；
其中二氯甲烷-甲醇洗脱剂中二氯甲烷∶甲醇＝8∶1(v/v)洗脱部分先后经Sephadex LH-20凝胶柱色谱分离、以甲醇洗脱；快速硅胶柱色谱分离，以二氯甲烷、甲醇梯度洗脱，得到5个组分，按流出顺序依次为组分1、2、3、4、5，组分2经半制备高效液相色谱分离纯化、以甲醇∶水＝50∶50(v/v)洗脱得到化合物1；组分3经半制备高效液相色谱分离纯化、以甲醇∶水＝30∶70(v/v)洗脱得到化合物3；
可选地，所述化合物5由以下方法得到：将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养，接种到加入了5-氟吡啶甲酸(前体物9)的发酵培养基中，摇床培养发酵，获得发酵物，发酵产物用有机溶剂萃取，有机相浓缩得粗提物，所述粗提物用减压硅胶柱色谱分离，依次以石油醚、二氯甲烷、二氯甲烷-甲醇混合液为洗脱液进行梯度洗脱，所得组分先后经Sephadex LH-20凝胶柱色谱以甲醇洗脱，快速硅胶柱色谱以二氯甲烷、甲醇梯度洗脱进行分离，所得组分浓缩后再经半制备高效液相色谱分离纯化、以甲醇∶水＝75∶25(v/v)，即得到化合物5；
Figure PCTCN2017110799-appb-000004
5-氟吡啶甲酸(前体物9)
可选地，所述化合物6由以下合成方法制得：向分离得到的化合物2中加入甲醛水溶液、盐酸和水，加热回流，然后冷却至室温，向其中加入饱和NaHCO3水溶液，用有机溶剂萃取，有机相减压浓缩后，经加压硅胶色谱柱分离，以选自乙酸乙酯、二氯甲烷、氯仿、石油醚的洗脱液洗脱，优选以二氯甲烷和甲醇为洗脱液梯度洗脱，得到化合物6；；可选的，所述甲醛水溶液的浓度为37％(v/v)；可选的，所述盐酸的浓度为10N；
所述化合物7由以下方法得到：将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养，接种到加入了2-吡啶甲酸的发酵培养基中，摇床培养发酵，获得发酵物，发酵物用有机溶剂萃取，有机相浓缩得粗提物，发酵粗提物经经减压硅胶柱色谱分离，依次以石油醚、二氯甲烷、以及二氯甲烷-甲醇混合溶剂(v/v，100∶1，50∶1，30∶1，25∶1，15∶1，10∶1，5∶1，2∶1，1∶1，0∶1)为洗脱液进行梯度洗脱，其二氯甲烷-甲醇洗脱剂中二氯甲烷∶甲醇＝2∶1(v/v)洗脱组分再经半制备高效液相色谱分离纯化、以甲醇∶水＝50∶50(v/v)洗脱得到化合物7；
可选地，所述萃取用有机溶剂选自乙酸乙酯、二氯甲烷、氯仿、石油醚。
本发明提供一种药物组合物，其特征在于，所述药物组合物包括上述的式I化合物、其药学上可接受的盐或前药中的至少一种和药学上可接受的辅料。
可选地，上述的药物组合物，其特征在于，所述药物组合物的剂型包括固态制剂和液态制剂；可选地，所述药物组合物的剂型包括口服制剂、注射制剂、经皮制剂；可选地，所述药物组合物的剂型包括片剂、胶囊剂、粉剂、颗粒剂、锭剂、栓剂、口服液、无菌胃肠外悬液、针剂；可选地，所述针剂包括冻干粉针剂。
可选地，上述的式I化合物、其药学上可接受的盐或前药，或者上述药物组合物在制备抗肿瘤药物中的用途；可选地，所述抗肿瘤药物为肿瘤细胞增殖抑制剂或肿瘤细胞杀伤剂；可选地，所述抗肿瘤药物不包括抗人肺腺癌A549药物、抗人急性早幼粒白血病HL60药物、抗人慢性髓性白血病K562药物、抗人口腔表皮样癌KB药物；可选地，所述抗肿瘤药物为：抗人结肠癌细胞系HCT-116药物，抗人乳腺癌细胞系MCF-7药物，抗肝癌细胞系HepG2药物，抗子宫颈癌细胞系Hela药物，或者，抗人外周血白血病T细胞系Jurkat药物。
本发明还提供上述的式I化合物、其药学上可接受的盐或前药作为抑制细胞增殖的探针中的用途。
本发明还提供上述药物组合物作为抑制细胞增殖的探针中的用途。
本发明还提供一种探针试剂盒，其特征在于，包括上述的式I化合物、其药学上可接受的盐或前药。
本发明还提供一种探针试剂盒，其特征在于，包括上述的药物组合物。
可选地，所述探针试剂盒还包含生物相容性介质；可选地，所述生物相容性介质选自甲醇、水、二甲基亚砜中的至少一种。
制备本发明式I化合物所用的异壁放线菌A.cyanogriseus WH1-2216-6，该菌种保藏编号：CCTCC M 209277，保藏日期：2009年11月28日，保藏地点：中国武汉，武汉大学，中国典型培养物保藏中心。
该放线菌菌株WH1-2216-6是从采自山东威海的海泥中分离得到，菌株详细信息已报道(Fu P.Wang S，Hong K，LiX，Liu P，Wang Y，Zhu W.Cytotoxic bipyridines from the marine-derived actinomycete Actinoalloteichus cyanogriseus WH1-2216-6.J.Nat.Prod.2011，74，1751-1756)，经多相分类学研究鉴定为异壁放线菌属菌株，定为Actinoalloteichuscyanogriseus WH1-2216-6。经发酵微生物制取本发明化合物的方法可采用其它任何能生产本发明化合物的微生物，只要能生产本发明化合物的微生物均可作为生产菌用于制备本发明所述化合物。
本发明化合物具有好的抗肿瘤活性并且具有好的选择性，对肿瘤细胞增殖有显著的抑制作用，同时对正常细胞株生长抑制作用不明显。
附图说明
图1是试验例2中各组小鼠结肠癌(HCT116)照片，其中A组为阴性对照(生理盐水)组，B为多西他赛(5mg/kg)组，C为本发明化合物2(7.5mg/kg)组。
具体实施方式
以下通过实施例对本发明进行示例性说明，但本发明的范围不限于这些实施例。
Figure PCTCN2017110799-appb-000005
【制备例】
(1)化合物1～4，7，8的制备
发酵培养：将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养5天，接种到装有150mL发酵培养基的500mL三角瓶中，所述发酵培养基中加入了2-吡啶甲酸0.4g。摇床培养，发酵12天，获得发酵产物40L。培养基的组成如下：种子培养基(蛋白胨15g、甘油15g、黄豆粉5g、可溶性淀粉15g、CaCO32g、陈海水1L、pH＝7.8)；发酵培养基(可溶性淀粉20g、甘油20g、蛋白胨20g、CaCO32g、XAD-16大孔吸附树脂50g、陈海水1L、pH＝7.5)。
分离精制：
(a)化合物2的获得：发酵产物用等体积乙酸乙酯萃取三次，合并乙酸乙酯萃取液有机相后，浓缩得粗提物，向每克粗提物中加入50mL的3％的HCl溶液，搅拌，静置，用等体积二氯甲烷萃取三次，用氨水调节水相至pH＝8.0，再用二氯甲烷萃取，浓缩有机相得35.0g生物碱部分，所述生物碱部分用凝胶柱分离2次(流动相为：二氯甲烷∶甲醇＝1∶1，v/v)，得化合物2和其它生物碱部分，经HPLC分析，化合物2的纯度达到99％以上。
(b)化合物1、3～4的获得：将上述(a)所得其它生物碱部分合并后再经减压硅胶柱色谱分离，依次以石油醚、二氯甲烷、以及二氯甲烷-甲醇混合溶剂(v/v，100∶1，50∶1，30∶1，25∶1，15∶1，10∶1，8∶1、5∶1，2∶1，1∶1，0∶1)为洗脱液进行梯度洗脱：
其二氯甲烷洗脱组分和二氯甲烷-甲醇洗脱剂中二氯甲烷∶甲醇＝100∶1(v/v)洗脱组分合并，浓缩后经葡聚糖凝胶Sephadex LH-20色谱柱分离、以二氯甲烷∶甲醇＝1∶1(v/v)洗脱，所得组分再经凝胶Sephadex LH-20柱色谱分离、以甲醇洗脱得到化合物4(70.0mg)；
其二氯甲烷-甲醇洗脱剂中二氯甲烷∶甲醇＝8∶1(v/v)洗脱部分先后经SephadexLH-20凝胶柱色谱(甲醇洗脱)、快速硅胶柱色谱(二氯甲烷、甲醇梯度洗脱)分离，得到5个组分，按流出顺序依次为组分1、2、3、4、5；组分2经半制备高效液相色谱分离纯化、以甲醇∶水＝50∶50(v/v)洗脱得到化合物1(4.5mg，保留时间tR 7.5min)；组分3经半制备高效液相色谱分离纯化、以甲醇∶水＝30∶70(v/v)洗脱得到化合物3(3.0mg，保留时间tR 13.7min)；
化合物1：淡紫色粉末，高分辨质谱HRESIMS m/z362.1343[M+H]+(calcd for C17H20N3O6，362.1347)；旋光值[α]D 15-50.6(c 0.1CH3OH)；紫外吸收光谱UV(CH3OH)λmax(logε)212(4.33)，240(4.17)，285(3.91)nm；红外光谱IR(KBr)vmax3547，3327，3325，2929，2376，2304，1679，1586，1564，1515，1455，1427，1383，1203，1136，1024，839，798，720cm-1；核磁1H NMR谱(500MHz，DMSO-d6)及13C NMR谱(125MHz，DMSO-d6)数据见表1。
化合物2：白色粉末，低分辨质谱ESIMS m/z 230.2[M+H]+；核磁1H NMR谱(500MHz，DMSO-d6)及13CNMR谱(125MHz，DMSO-d6)数据见表1。
化合物3：白色粉末，高分辨质谱HRESIMS m/z 216.0781[M+H]+(calcd for C11H10N3O2，216.0773)；UV(MeOH)λmax(logε)：240(2.91)，280(2.75)nm；红外光谱IR(KBr)vmax 3171，2953，1651， 1502，1378，1302，1016，980，797，738cm-1；核磁1H NMR谱(600MHz，DMSO-d6)及13C NMR谱(150MHz，DMSO-d6)数据见表2。
化合物4：黄色粉末，高分辨质谱ESIMS m/z 246.096[M+H]+；旋光值[α]D 15-5.2(c 0.2，MeOH)；紫外吸收光谱UV(MeOH)λmax(logε)245(4.22)，324(3.80)nm；红外光谱IR(KBr)vmax3563，3445，2927，1646，1566，1540，1515，1380，1261，1151，1050，991，865，800cm-1；核磁1H NMR谱(500MHz，DMSO-d6)及13C NMR谱(125MHz，DMSO-d6)数据见表2。
表1.化合物1和2的1H和13C NMR数据
Figure PCTCN2017110799-appb-000006
表2.化合物3和4的1H和13C NMR数据
Figure PCTCN2017110799-appb-000007
Figure PCTCN2017110799-appb-000008
(2)化合物5的制备
加前体物5-氟吡啶甲酸的发酵培养：将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养5天，接种到加有0.4g前体物5-氟吡啶甲酸的150mL发酵培养基中，摇床培养，发酵12天，共发酵7瓶，获得发酵产物1.0L。其培养基组成如下：种子培养基(蛋白胨15g、甘油15g、黄豆粉5g、可溶性淀粉15g、CaCO32g、陈海水1L、pH＝7.8)；发酵培养基(可溶性淀粉20g、甘油20g、蛋白胨20g、CaCO32g、XAD-16大孔吸附树脂50g、陈海水1L、pH＝7.5)。
分离精制：将发酵产物用等体积乙酸乙酯萃取三次，合并有机相，浓缩得粗提物，所得粗提物用减压硅胶柱色谱分离、依次以石油醚、二氯甲烷、二氯甲烷-甲醇为洗脱液进行梯度洗脱，组分浓缩后先后经Sephadex LH-20凝胶柱色谱(甲醇洗脱)和快速硅胶柱色谱(二氯甲烷、甲醇梯度洗脱)分离，所得组分再经半制备高效液相色谱分离、以甲醇∶水＝75∶25(v/v)洗脱，即得化合物5(10.0mg，保留时间tR 10.3min)。
化合物5，白色结晶性粉末，高分辨质谱HRESIMS m/z 248.0831[M+H]+(calcd for C12H11N3O2F，248.0830)；紫外吸收光谱UV(MeOH)λmax(logε)242(4.69)，280(4.30)nm；红外光谱IR(KBr)vmax3744，3674，3227，3110，2924，1738，1650，1583，1489，1464，1359，1239，1166，1112，1026，982，930，840，767，714cm-1；核磁1H NMR谱(500MHz，DMSO-d6)及13C NMR谱(125MHz，DMSO-d6)数据见表3。
(3)化合物6的制备：
反应步骤：取化合物2(30.0mg，0.13mmol)置于25mL反应瓶中，加入2.28mL37％甲醛水溶液，0.116mL浓度为10N的盐酸和1.032mL水，随后在105℃加热回流30分钟，反应液冷却至室温，向其中加入10mL饱和NaHCO3水溶液淬灭反应。
分离精制：将反应液用等量乙酸乙酯(10mL/每次)萃取三遍，乙酸乙酯有机层减压浓缩后，经加压硅胶色谱柱以二氯甲烷、甲醇梯度洗脱分离纯化得到化合物6(25.0mg，反应收率为89％)。
化合物6，无色针状晶体，高分辨质谱HRESIMS m/z 215.0817[M+H]+(calcd for C12H11N2O2，215.0815)；紫外吸收光谱UV(MeOH)λmax(logε)：215(4.43)nm；红外光谱IR(KBr)vmax 3407，3089，2919，2815，1715，1584，1374，1220，1046cm-1；核磁1H NMR谱(600MHz，CDCl3)及13C NMR谱(150MHz，CDCl3)数据见表3。
表3.化合物5和6的1H和13C NMR数据
Figure PCTCN2017110799-appb-000009
(4)化合物7和8的制备：
所述化合物7由以下方法得到：将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养，接种到加入了2-吡啶甲酸的发酵培养基中，摇床培养发酵，获得发酵物，发酵物用有机溶剂萃取，有机相浓缩得粗提物，发酵粗提物经经减压硅胶柱色谱分离，依次以石油醚、二氯甲烷、以及二氯甲烷-甲醇混合溶剂(v/v，100∶1，50∶1，30∶1，25∶1，15∶1，10∶1，5∶1，2∶1，1∶1，0∶1)为洗脱液进行梯度洗脱，其二氯甲烷-甲醇洗脱剂中二氯甲烷∶甲醇＝2∶1(v/v)洗脱组分再经半制备高效液相色谱分离纯化、以甲醇∶水＝50∶50(v/v)洗脱得到化合物7(2.3mg，保留时间tR 12.1min)；
化合物8的制备：将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养，接种到加入了苯甲酸的发酵培养基中，摇床培养发酵，获得发酵物，发酵物用有机溶剂萃取，有机相浓缩得粗提物，发酵粗提物经经减压硅胶柱色谱分离，依次以石油醚、二氯甲烷、以及二氯甲烷-甲醇混合溶剂(v/v，100∶1，50∶1，30∶1，25∶1，15∶1，10∶1，5∶1，2∶1，1∶1，0∶1)为洗脱液进行梯度洗脱，其二氯甲烷-甲醇洗脱剂中二氯甲烷∶甲醇＝30∶1(v/v)洗脱组分再经葡聚糖凝胶Sephadex LH-20色谱柱分离、以二氯甲烷∶甲醇＝1∶1(v/v)洗脱，所得组分再经半制备高效液相色谱分离纯化、以甲醇∶水＝60∶40(v/v)洗脱得到化合物8(3.5mg，保留时间tR 13.3min)；
化合物7和8的结构与标准品进行薄层色谱(TLC)和HPLC比较分析而鉴定(化合物7：Fu P，Zhu Y，Mei X，Wang Y，Jia H，Zhang C，Zhu W.Acyclic Congeners from Actinoalloteichus cyanogriseus Provide Insights into Cyclic Bipyridine Glycoside Formation.Org.Lett.2014，16，4264-4267；化合物8：Fu P，Wang S，Hong K，Li X，Liu P，Wang Y，Zhu W.Cytotoxic bipyridines from the marine-derived actinomycete Actinoalloteichus cyanogriseus WH1-2216-6.J.Nat.Prod.2011，74，1751-1756)。
【试验例1】体外抗肿瘤活性测试
1、实验样品及实验方法
被测样品溶液的配制：测试样品为上述制备例1中分离制备的化合物1～8准确称取适量样品，用甲醇配制成所需浓度的溶液，供活性测试。
细胞系及细胞的继代培养：活性测试采用5株肿瘤细胞系：人结肠癌细胞系(HCT-116细胞)、人肝癌细胞系(HepG2细胞)、人乳腺癌细胞系(MCF-7细胞)、子宫颈癌细胞系(Hela细胞)、人外周血白血病T细胞系(Jurkat细胞)，以及1株人正常肝细胞系(L-02细胞)，其中MCF-7、HepG2、Hela、L-02细胞系采用MTT模型测试；HCT-116细胞系采用SRB模型测试。各种细胞均用含10％FBS的RPMI-1640培养基，在37℃通入5％二氧化碳的培养箱中继代培养。
MTT法：活细胞线粒体中脱氢酶能够代谢还原黄色的溴化3-(4，5-二甲基噻唑)-2，5-二苯基四氮唑为蓝紫色的不溶于水的formazan，formazan的多少可通过酶标仪测定其吸收度求得。由于formazan的量与活细胞数成正比，可根据吸收度求出活细胞的数目，从而了解药物抑制或杀伤肿瘤细胞的能力。活性测试时，取对数生长期的测试细胞系，用新鲜的RPMI-1640培养基配制成密度为每毫升3×104个细胞的细胞悬液，按每孔100μL接种于96孔板中，在37℃下培养24小时后，每孔加入100μL不同浓度的样品溶液，继续培养72小时。然后加入20μL含MTT的IPMI-1640溶液(5mg/L)，继续培养4小时后，将培养液慢慢倾出再加入150μLDMSO溶解formazan，在540nm处测定其吸收度。按照下式计算每个浓度下的细胞增殖抑制率(IR％)：IR％＝(OD空白对照-OD样品)/OD空白对照×100％。求出对癌细胞的半数抑制浓度(IC50)以及对正常细胞的半数抑制浓度(CC50)。
SRB法：根据细胞生长速率，将处于对数生长期的肿瘤细胞(培养基：含10％新生胎牛血清(FBS)的RPMI-1640培养基；细胞密度3×104细胞/mL)以180μL/孔接种于96孔培养板，在37℃、5％CO2条件下贴壁生长24小时，再加测试样品20μL/孔，每个浓度设四复孔。(样品初筛终浓度设为10μM，测试IC50时用二倍稀释法设定5～7个浓度梯度；阳性药为阿霉素1μM；空白对照为加入等量的相应浓度的培养基)。加药后的肿瘤细胞在37℃、5％CO2条件下继续培养72小时，然后倾去培养液，用10％冷三氯醋酸(TCA)固定细胞，4℃放置1小时后用蒸馏水洗涤5次，空气中自然干燥。然后加入由1％冰醋酸配制的磺酰罗单明B(SRB，Sigma)4mg/ml溶液100μL/孔，室温中染色15分钟，去上清液，用1％醋酸洗涤5次，空气干燥。最后加入150μL/孔的Tris溶液，用酶标仪在540nm波长下测定吸收度OD值。按照IR％＝(OD空白对照-OD样品)/OD空白对照×100％式计算每个浓度下的细胞增殖抑制率(IR％)。求出对癌细胞的半数抑制浓度(IC50)。
选择指数(Selection Index，SI)为测试样品对正常细胞株生长抑制CC50值与对肿瘤细胞增殖抑制IC50值的比值，可反映样品的选择性、安全性。
计算公式：SI＝CC50(正常细胞L-02株)/IC50(肿瘤细胞株)
2、实验结果
表4.各化合物对正常细胞的毒性CC50(μM)
细胞系 化合物1 化合物2 化合物3 化合物4 化合物5 化合物6 化合物7 化合物8
L-02(CC50) 28.3 3.9 2.5 1.7 7.5 31.4 59.5 ＞50
表5.各化合物对HCT-116细胞株的细胞毒活性IC50(μM)和选择指数SI
细胞系 化合物1 化合物2 化合物3 化合物4 化合物6 化合物7 化合物8
HCT-116(IC50/SIa) 0.7/40.4 0.3/13.0 0.3/8.3 1.4/1.2 6.0/5.2 2.2/27.0 ＞50
aSI：选择指数＝CC50(正常细胞L-02株)/IC50(肿瘤细胞株)
由表5可以看出，对于HCT-116细胞株，化合物8(caerulomycin F)对HCT-116细胞系没有肿瘤细胞毒活性，本发明化合物具有好的抗肿瘤活性，对正常人体细胞毒副作用小，安全系数高，尤其是化合物2，具有非常好的抗HCT-116肿瘤活性和选择性。
表6.各化合物对MCF-7细胞株的细胞毒活性IC50(μM)和选择指数SI
细胞系 化合物2 化合物6 化合物8
MCF-7(IC50/SIa) 1.0/3.9 12.0/2.6 ＞50
aSI：选择指数＝CC50(正常细胞L-02株)/IC50(肿瘤细胞株)
由表6可以看出，对于MCF-7细胞株，化合物8(caerulomycin F)对MCF-7肿瘤细胞系没有细胞毒活性，本发明化合物具有好的抗肿瘤活性，对正常人体细胞毒副作用小，安全系数高，特别是化合物2，具有非常好的抗MCF-7肿瘤细胞活性和选择性。
表7.各化合物对HepG2细胞株的细胞毒活性IC50(μM)和选择指数SI
细胞系 化合物1 化合物2 化合物3 化合物7 化合物8
HepG2(IC50/SIa) 2.7/10.5 0.2/19.5 2.0/1.3 10.0/6.0 ＞50
aSI：选择指数＝CC50(正常细胞L-02株)/IC50(肿瘤细胞株)
由表7可以看出，对于HepG2细胞株，化合物8(caerulomycin F)对HepG2肿瘤细胞系没有细胞毒活性，本发明化合物具有好的抗肿瘤活性，对正常人体细胞毒副作用小，安全系数高，特别是化合物2，具有非常好的抗HepG2肿瘤细胞活性和选择性。
表8.各化合物对Hela细胞株的细胞毒活性IC50(μM)和选择指数SI
细胞系 化合物1 化合物2 化合物3 化合物5 化合物6 化合物7 化合物8
Hela(IC50/SIa) 9.0/3.1 0.1/39 1.9/1.3 4.4/1.7 19.3/1.6 2.5/23.8 ＞50
aSI：选择指数＝CC50(正常细胞L-02株)/IC50(肿瘤细胞株)
由表8可以看出，对于Hela细胞株，化合物8(caerulomycin F)对Hela肿瘤细胞系没有细胞毒活性，本发明化合物具有好的抗肿瘤活性，对正常人体细胞毒副作用小，安全系数高，特别是化合物2，具有非常好的抗Hela肿瘤细胞活性和选择性。
表9.各化合物对Jurkat细胞株的细胞毒活性IC50(μM)和选择指数SI
细胞系 化合物1 化合物2 化合物3 化合物6 化合物7 化合物8
Jurkat(IC50/SIa) 10.8/2.6 0.2/19.5 1.2/2.1 14.5/2.2 1.3/45.8 ＞50
aSI：选择指数＝CC50(正常细胞L-02株)/IC50(肿瘤细胞株)
由表9可以看出，对于Jurkat细胞株，化合物8(caerulomycin F)对Jurkat肿瘤细胞系没有细胞毒活性，本发明化合物具有好的抗肿瘤活性，对正常人体细胞毒副作用小，安全系数高，特别是化合物2，具有非常好的抗Jurkat肿瘤细胞活性和选择性。
由以上表4-9的结果还可以得知：(1)化合物3具有较好的体外抗肿瘤活性，说明3-甲氧基变为羟基后，肿瘤细胞毒活性仍然保持；(2)化合物4与化合物2相比，3-位羟基的引入降低肿瘤细胞株的活性、增加对正常细胞株的毒性；(3)化合物5与化合物2相比，5-氟原子的引入，降低了其对人肿瘤细胞和正常细胞的毒性；(4)化合物6与化合物2相比，4-肟基变为醛基后，其对肿瘤细胞和正常细胞的毒性均降低；(5)化合物1和化合物7的结果表明：3-羟基或甲氧基被糖基取代后，所得的氧苷衍生物，其肿瘤细胞毒活性保留，但毒性却显著降低；(6)将化合物2中的2-吡啶基换成苯基后，所得化合物8(caerulomycin F)对HCT-116、MCF-7、HepG2、Hela、Jurkat等肿瘤细胞系没有肿瘤细胞毒活性，表明：改变联吡啶醛肟结构后得到的化合物的活性降低或消失，进一步证明了本发明通式I化合物具有好的抗肿瘤活性。
综上可知，本发明式I化合物对癌细胞增殖有效抑制的同时具有较高的选择性，毒性较小，安全系数较高，特别是化合物2，具有非常好的抗肿瘤活性和选择性。
【试验例2】体内抗肿瘤活性测试
1、实验样品及实验方法
针对结肠癌HCT-116的裸鼠体内移植瘤，进行了化合物2的体内的抗肿瘤试验。
1、实验样品及实验方法
(1)实验样品
实验小鼠为BALB/C，nu/nu裸小鼠、体重18～20g、雄性。购自于北京华阜康生物科技股份有限公司，许可证号：SCXK(京)-2014-0004。
阳性药：多烯他赛(Docetaxel)用吐温DMSO为溶剂溶解配制成9mg/mL溶液备用。生理盐水稀释。
受试药：化合物2用DMSO溶解原液10mg/mL。生理盐水稀释浓度为0.75mg/mL，给药体积为0.1mL/10g体重。
(2)实验方法
用含10％胎牛血清的5A培养基配制浓度为3000万/mL的人结肠癌细胞株HCT-116的细胞悬液，常规消毒后按每只0.2ml接种于小鼠右前肢腋窝部皮下，待肿瘤生长至平均体积100mm3左右后给药。设置阳性药、化合物和阴性对照组。给药剂量为：多烯他赛(阳性药)5mg/kg，化合物2：7.5mg/kg。给药方式：多烯他赛为静脉注射，隔天给药；化合物2为腹腔注射，每天给药；阴性对照组为腹腔注射同等体积的生理盐水。给药14天。期间每日称重，统计小鼠体重变化趋势。最后一次给药后24h，处死小鼠，剥离其肿瘤组织，称取肿瘤质量，计算抑瘤率。抑瘤率计算公式如下：
抑瘤率＝(对照组平均瘤重-治疗组平均瘤重)/对照组平均瘤重×100％
同时摘取小鼠的脾脏，剥离被膜，分别记录小鼠脾脏的质量，计算脾脏指数。计算公式为：
脾脏指数＝100×脾脏质量/体质量。
统计学分析：应用SPSS 13.0统计软件进行数据处理，所得数据用X±S标示，各组数据运用t检验分析，P值比较组间差异。
2、实验结果
实验中给予化合物2后，对小鼠行为学观察表明，给药14天内未见明显的过敏等不良反应，对小鼠的体重也无不良影响。
该实验中，阴性对照组的肿瘤平均重量为1.0g以上，证明结肠癌(HCT-116)模型构建成功。阳性药选用临床应用的广谱抗肿瘤药物多烯他赛，实验中多烯他赛5mg/kg组与阴性对照组比较，能明显抑制肝癌(HCT-116)的生长，抑制率为87.39％(P＜0.01)，说明该实验结果可信；但多烯他赛组使小鼠体重和脾指数下降，体现了多烯他赛对免疫系统的抑制等毒副作用。
化合物2在剂量为7.5mg/kg时、给药14天后，与阴性对照组比有明显的结肠癌(HCT-116)生长抑制作用(图1、表10)，抑制率为42.46％(p＜0.05)；但未见化合物2对小鼠脾指数的影响(表11)，表明化合物2为安全有效的抗肿瘤药物。
表10.小鼠结肠癌(HCT-116)质量统计结果(
Figure PCTCN2017110799-appb-000010
单位：g)
  瘤重 抑制率％
阴性对照(生理盐水) 1.03±0.32 /
多烯他赛(5mg/kg) 0.13±0.07 87.39**
化合物2(7.5mg/kg) 0.59±0.33 42.46*
**p＜0.01；*p＜0.05代表各给药组与阴性对照组比较的结果
表11.小鼠脾指数(脾指数＝100×脾脏质量/体重质量)
  脾指数
阴性对照(生理盐水) 0.41±0.08
多烯他赛(10mg/kg) 0.25±0.11**
化合物2(7.5mg/kg) 0.45±0.09*
**p＜0.01；*p＜0.05代表各给药组与阴性对照组比较的结果

Claims (10)

  1. 一种式I化合物、其药学上可接受的盐或前药，
    Figure PCTCN2017110799-appb-100001
    其中：
    R1选自：-CH＝N-O-R4，-COR5，-CH2OR6，-CN，-NH2；
    R4和R6各自独立地选自：-H，烷基，烷酰基；
    R5选自：-H，-OH，-NH2，烷氧基；
    R2选自：-H，烷基，糖基；
    R3选自：-H，-OH，-NH2，烷氧基；
    X选自：-H，卤素，-NO2，-SO2R7，-COR8；
    R7选自：-H，芳基；
    R8为芳基。
  2. 根据权利要求1所述的式I化合物、其药学上可接受的盐或前药，其中：
    所述烷基和烷酰基、烷氧基中的烷基各自独立地为直链或支链的C1～C16烷基，可选地为直链或支链的C1～C10烷基，可选地为直链或支链的C1～C6烷基，可选地为直链或支链的C1～C4烷基；
    所述糖基选自：葡萄糖基，鼠李糖基，异鼠李糖基，核糖基，半乳糖基，阿洛糖基，岩藻糖基(fucosyl)，艾杜糖基，塔洛糖基，2,4-二甲氧基鼠李糖基，2,4-二甲氧基葡萄糖基，2,4-二甲氧基异鼠李糖基，2,4-二甲氧基核糖基，2,4-二甲氧基半乳糖基，2,4-二甲氧基阿洛糖基，2,4-二甲氧基岩藻糖基，2,4-二甲氧基艾杜糖基，2,4-二甲氧基塔洛糖基；
    所述卤素选自：-F，-Cl，-Br，-I；
    所述芳基为C6～C18单环或多环芳基，可选地为C6～C14单环或多环芳基，可选地为C6～C10单环或多环芳基。
  3. 根据权利要求1或2所述的式I化合物、其药学上可接受的盐或前药，其中：
    R1选自：-CH2OH，-CH＝NOH，-CN，-CONH2，-NH2，-CHO，-COOH；可选地，R1为-CH＝NOH；
    R2选自：-H，甲基，葡萄糖基，鼠李糖基，异鼠李糖基，核糖基，半乳糖基，阿洛糖基，岩藻糖基，艾杜糖基，塔洛糖基，2,4-二甲氧基鼠李糖基，2,4-二甲氧基葡萄糖基，2,4-二甲氧基异鼠李糖基，2,4-二甲氧基核糖基，2,4-二甲氧基半乳糖基，2,4-二甲氧基阿洛糖基，2,4-二甲氧基岩藻糖基，2,4-二甲氧基艾杜糖基，2,4-二甲氧基塔洛糖基；可选地，R2为L-鼠李糖基；可选地，R2为甲基；
    R3选自：-H，-OH，-OCH3；可选地，R3为-H；
    X选自：-H，-F，-Cl，-Br，-I，-NO2，-SO3H，-SO2C6H5，-COC6H5；可选地，X为-H；
    可选地，所述式I化合物为化合物1，或化合物2，或化合物3，或化合物4，或化合物5，或化合物6：
    Figure PCTCN2017110799-appb-100002
  4. 根据权利要求1～3中任一项所述的式I化合物、其药学上可接受的盐或前药，其中：
    所述药学上可接受的盐包括有机或无机酸的盐；
    可选地，所述药学上可接受的盐为所述式I化合物与选自以下的化合物形成的盐：盐酸；硫酸；磷酸；甲酸；乙酸；丙酸；乳酸；柠檬酸；酒石酸；琥珀酸；富马酸；马来酸；杏仁酸；苹果酸；樟脑磺酸；
    所述药学上可接受的前药包括所述式I化合物与药学上可接受的载体键合而成的前药；可选地，所述药学上可接受的载体包括：磷酸甘油三酯，聚乙二醇酯，聚乙二醇酰胺，聚乙二醇醚。
  5. 一种制备权利要求1～4中任一项所述的式I化合物、其药学上可接受的盐或前药的方法，其特征在于：
    所述式I化合物为化合物1，或化合物2，或化合物3，或化合物4，或化合物5，或化合物6，或化合物7；
    所述化合物1～4由以下方法得到：
    (a)将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养，接种到加入了2-吡啶甲酸的发酵培养基中，摇床培养发酵，获得发酵物，发酵物用有机溶剂萃取，有机相浓缩得粗提物，向粗提物中加入HCl溶液，用有机溶剂萃取，用氨水调节水相至pH＝8.0，再用有机溶剂萃取，浓缩有机相得生物碱部分，所得生物碱部分用凝胶柱分离，所述凝胶柱的流动相为二氯甲烷:甲醇＝1:1(v/v)，得化合物2和其他生物碱部分；
    (b)将步骤(a)所述其他生物碱部分合并后再经减压硅胶柱色谱分离，依次以石油醚、二氯甲烷、二氯甲烷-甲醇为洗脱液进行梯度洗脱：
    其中，二氯甲烷-甲醇洗脱剂包括以下配比的洗脱组分：二氯甲烷:甲醇(v/v)为100:1、50:1、30:1、25:1、15:1、10:1、8:1、5:1、2:1、1:1和0:1；
    其中二氯甲烷洗脱组分和二氯甲烷-甲醇洗脱剂中二氯甲烷:甲醇＝100:1(v/v)洗脱组分合并后经葡聚糖凝胶Sephadex LH-20色谱柱分离、以二氯甲烷:甲醇＝1:1(v/v)洗脱，所得组分再经Sephadex LH-20凝胶柱色谱分离，以甲醇洗脱得到化合物4；
    其中二氯甲烷-甲醇洗脱剂中二氯甲烷:甲醇＝8:1(v/v)洗脱部分先后经Sephadex LH-20凝胶柱色谱分离、以甲醇洗脱；快速硅胶柱色谱分离，以二氯甲烷、甲醇梯度洗脱，得到5个组分，按流出顺序依次为组分1、2、3、4、5，组分2经半制备高效液相色谱(HPLC)分离纯化、以甲醇:水＝50:50(v/v)洗脱得到化合物1；组分3经半制备高效液相色谱分离纯化、以甲醇:水＝30:70(v/v)洗脱得到化合物3；
    所述化合物5由以下方法得到：将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养，接种到加入了5-氟吡啶甲酸的发酵培养基中，摇床培养发酵，获得发酵物，发酵产物用有机溶剂萃取，有机相浓缩得粗提物，所述粗提物用减压硅胶柱色谱分离，依次以石油醚、二氯甲烷、二氯甲烷-甲醇混合液为洗脱液进行梯度洗脱，所得组分先后经Sephadex LH-20凝胶柱色谱以甲醇洗脱，快速硅胶柱色谱以二氯甲烷、甲醇梯度洗脱进行分离，所得组分浓缩后再经半制备高效液相色谱分离纯化、以甲醇:水＝75:25(v/v)，即得到化合物5；
    所述化合物6由以下合成方法制得：向分离得到的化合物2中加入甲醛水溶液、盐酸和水，加热回流，然后冷却至室温，向其中加入饱和NaHCO3水溶液，用有机溶剂萃取，有机相减压浓缩后，经加压硅胶色谱柱分离，以选自乙酸乙酯、二氯甲烷、氯仿、石油醚的洗脱液洗脱，优选以二氯甲烷和甲醇为洗脱液梯度洗脱，得到化合物6；；可选的，所述甲醛水溶液的浓度为37％(v/v)；可选的，所述盐酸的浓度为10N；
    所述化合物7由以下方法得到：将异壁放线菌A.cyanogriseus WH1-2216-6在种子培养基培养，接种到加入了2-吡啶甲酸的发酵培养基中，摇床培养发酵，获得发酵物，发酵物用有机溶剂萃取，有机相浓缩得粗提物，发酵粗提物经经减压硅胶柱色谱分离，依次以石油醚、二氯甲烷、以及二氯甲烷-甲醇混合溶剂(v/v，100:1，50:1，30:1，25:1，15:1，10:1，5:1，2:1，1:1，0:1)为洗脱液进行梯度洗脱，其二氯甲烷-甲醇洗脱剂中二氯甲烷:甲醇＝2:1(v/v)洗脱组分再经半制备高效液相色谱分离纯 化、以甲醇:水＝50:50(v/v)洗脱得到化合物7；
    可选地，所述萃取用有机溶剂选自乙酸乙酯、二氯甲烷、氯仿、石油醚。
  6. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1～4中任一项所述的式I化合物、其药学上可接受的盐或前药中的至少一种和药学上可接受的辅料。
  7. 根据权利要求6所述的药物组合物，其特征在于，所述药物组合物的剂型包括固态制剂和液态制剂；可选地，所述药物组合物的剂型包括口服制剂、注射制剂、经皮制剂；可选地，所述药物组合物的剂型包括片剂、胶囊剂、粉剂、颗粒剂、锭剂、栓剂、口服液、胃肠外悬液、针剂；可选地，所述针剂包括冻干粉针剂。
  8. 权利要求1～4中任一项所述的式I化合物、其药学上可接受的盐或前药，或者权利要求6或7所述药物组合物在制备抗肿瘤药物中的用途；可选地，所述抗肿瘤药物为肿瘤细胞增殖抑制剂或肿瘤细胞杀伤剂；可选地，所述抗肿瘤药物不包括抗人肺腺癌A549药物、抗人急性早幼粒白血病HL60药物、抗人慢性髓性白血病K562药物、抗人口腔表皮样癌KB药物；可选地，所述抗肿瘤药物为：抗人结肠癌细胞系HCT-116药物，抗人乳腺癌细胞系MCF-7药物，抗肝癌细胞系HepG2药物，抗子宫颈癌细胞系Hela药物，或者，抗人外周血白血病T细胞系Jurkat药物。
  9. 一种抑制细胞增殖的探针，其包括权利要求1～4中任一项所述的式I化合物、其药学上可接受的盐或前药，或者权利要求6或7所述药物组合物。
  10. 一种探针试剂盒，其特征在于，包括权利要求9所述的探针；可选地，所述探针试剂盒还包含生物相容性介质；可选地，所述生物相容性介质选自甲醇、水、二甲基亚砜中的至少一种。
PCT/CN2017/110799 2016-11-15 2017-11-14 联吡啶类生物碱、其制备方法和用途 WO2018090891A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/349,915 US20190322638A1 (en) 2016-11-15 2017-11-14 Dipyridyl alkaloid, preparation method therefor and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201611014546.8 2016-11-15
CN201611014546 2016-11-15

Publications (1)

Publication Number Publication Date
WO2018090891A1 true WO2018090891A1 (zh) 2018-05-24

Family

ID=62146077

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/110799 WO2018090891A1 (zh) 2016-11-15 2017-11-14 联吡啶类生物碱、其制备方法和用途

Country Status (3)

Country Link
US (1) US20190322638A1 (zh)
CN (1) CN108484699B (zh)
WO (1) WO2018090891A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114292266A (zh) * 2021-06-07 2022-04-08 曲阜师范大学 一种抗肿瘤化合物β-咔唑啉的提取方法及其应用
CN115304611A (zh) * 2021-12-09 2022-11-08 广西师范大学 两面针中两对二氢苯并菲啶生物碱类化合物及其提取方法和应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110015991A (zh) * 2019-05-22 2019-07-16 四川轻化工大学 一种联吡啶化合物及其制备方法和用途
CN113214157A (zh) * 2021-04-25 2021-08-06 广西壮族自治区花红药业集团股份公司 吡咯烷酮类化合物在制备治疗炎症性疾病的药物中的用途
CN115417837A (zh) * 2022-06-07 2022-12-02 新疆维吾尔自治区中药民族药研究所 神香草提取物中去甲戊烷型二萜成分及其制备方法和应用
CN115433185B (zh) * 2022-09-05 2023-06-06 西南大学 单萜吲哚类生物碱及其制备方法和应用
CN116514916B (zh) * 2023-06-26 2023-09-08 江西省药品检验检测研究院 一种吲哚生物碱化合物及其制备方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FU PENG: "Antibiotic Compounds from Four Marine-Derived Actinomycete Strains", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, MEDICINE AND HEALTH SCIENCES, 15 January 2014 (2014-01-15), ISSN: 1674-022X *
FU PENG: "Four Marine-Derived Actinomycete Strains", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, MEDICINE AND HEALTH SCIENCES, 15 January 2014 (2014-01-15), ISSN: 1674-022X *
FU, PENG ET AL.: "Acyclic Congeners from Actinoalloteichus cyanogriseus provide insights into cyclic bipyridine glycoside formation", ORGANIC LETTERS, vol. 16, no. §6, 15 August 2014 (2014-08-15), pages 4264 - 4267, XP055503092, ISSN: 1523-7060 *
JIA HAIJIAN: "Fermentation optimization for the production of caerulomycin A from marine-derived Actinoalloteichus cyanogriseus WH1-2216-6", CHINA MASTER'S THESES FULL-TEXT DATABASE, ENGINEERING TECHNOLOGY, 15 January 2014 (2014-01-15), pages 3 - 418-23 -24-26 -28-30, ISSN: 1674-0246 *
MCINNES, A.G. ET AL.: "Caerulomycins B and C, new 2, 2'-dipyridyl derivatives from Streptomyces caeruleus", CANADIAN JOURNAL OF CHEMISTRY, vol. 55, no. 24, 15 December 1977 (1977-12-15), pages 4159 - 4165, XP055503091 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114292266A (zh) * 2021-06-07 2022-04-08 曲阜师范大学 一种抗肿瘤化合物β-咔唑啉的提取方法及其应用
CN115304611A (zh) * 2021-12-09 2022-11-08 广西师范大学 两面针中两对二氢苯并菲啶生物碱类化合物及其提取方法和应用
CN115304611B (zh) * 2021-12-09 2023-06-02 广西师范大学 两面针中两对二氢苯并菲啶生物碱类化合物及其提取方法和应用

Also Published As

Publication number Publication date
CN108484699B (zh) 2021-11-19
CN108484699A (zh) 2018-09-04
US20190322638A1 (en) 2019-10-24

Similar Documents

Publication Publication Date Title
WO2018090891A1 (zh) 联吡啶类生物碱、其制备方法和用途
CN109134586B (zh) 雷公藤红素衍生物及其应用
WO2012149667A1 (zh) 一类强心苷类化合物及其抗肿瘤用途
CN109761948B (zh) 海绵来源杂萜类化合物Dysiarenone及其制备方法与应用
CN106008502B (zh) 马齿苋中骨架生物碱化合物及其提取分离方法
CN108623607B (zh) 5,5,6-多环含特特拉姆酸大环内酰胺类化合物及其制备方法和用途
CN103627772A (zh) 一种雷公藤内酯醇衍生物的制备方法及其产物和应用
WO2011131102A1 (zh) 含笑内酯的制备方法及其用途
CN110922440B (zh) 吲哚氮苷类化合物及提取方法及在制备防治神经系统疾病药物中的应用
CN109867649B (zh) 一种双黄酮类化合物及其制备方法与应用
CN103191143B (zh) 一种强心苷化合物的用途
US9163035B2 (en) Melonine bisindole compounds, pharmaceutical compositions, preparation method and use thereof
CN107286123B (zh) 一种二苯呋喃类化合物的制备方法与应用
CN112679481A (zh) 一种新化合物及其制备方法和镇痛用途
CN113278026A (zh) 一种具有抗肿瘤活性的苦木素类新化合物及其制备方法和应用
JP2004292426A (ja) 抗菌剤と抗ガン剤
CN110305094A (zh) 马齿苋中两种黄酮类化合物及其提取分离方法与用途
JP7393457B2 (ja) フランアスピドスペルミン二量体又はその薬学的に許容される塩及びその製造方法と適用、ならびに医薬組成物
CN115558012B (zh) 一种薯蓣皂苷元-丹参素衍生物及其自组装纳米颗粒和制备方法及用途
CN109867644B (zh) 一种苯醌类化合物及其制备方法与其在制备抗肿瘤药物中的应用
CN114805382B (zh) 一种倍半萜色酮类化合物及其分离和在制备抗胰腺癌药物中的应用
CN114621129B (zh) 抗肿瘤化合物及其制备方法和用途
CN114409670A (zh) 具有抗肿瘤活性苦木素类化合物及其制备方法与应用
JP5718450B2 (ja) 抗悪性腫瘍剤およびその製造方法と用途
AU2014414533B2 (en) 20(R)-ginsenoside Rg3 multiacylated derivative, preparation, and application thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17871185

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17871185

Country of ref document: EP

Kind code of ref document: A1