WO2017092230A1 - 双黄酮化合物及其治疗癌症和制备药物的用途 - Google Patents

双黄酮化合物及其治疗癌症和制备药物的用途 Download PDF

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WO2017092230A1
WO2017092230A1 PCT/CN2016/080931 CN2016080931W WO2017092230A1 WO 2017092230 A1 WO2017092230 A1 WO 2017092230A1 CN 2016080931 W CN2016080931 W CN 2016080931W WO 2017092230 A1 WO2017092230 A1 WO 2017092230A1
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cancer
compound
pharmaceutically acceptable
group
extract
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French (fr)
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林新华
姚宏
李少光
吴尤佳
眭玉霞
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福建医科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
    • 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
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • 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
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/40—Separation, e.g. from natural material; Purification

Definitions

  • the present invention relates to a biflavonoid compound, a pharmaceutical composition thereof and use thereof for the treatment of cancer.
  • the present invention provides a biflavonoid compound which is extracted and prepared from Asparagus officinalis, and is used for the treatment and pharmaceutical preparation of cancer.
  • the cypress is a dry whole grass of the Selaginella plant Selaginella doederleinii Hieron.
  • Shi Shangbai or its group can enhance the metabolism and reticuloendothelial system function of cancer patients, and study its active ingredients.
  • cypress has biflavonoids such as aceoflavone, heveaflavone and 7,4',7",4"'-tetra--0-methyl-furose flavonoids. (7,4',7",4"'-tetra-0-methyl-amentoflavone).
  • a method for preparing polysaccharide extract of cypress cypress and application thereof discloses a preparation method of polysaccharide extract of cypress, and application thereof.
  • the polysaccharide extract of the stone cypress is degreased, extracted, alcohol precipitated, deproteinized, monosaccharide and oligosaccharide, gel column purified, etc. The steps are implemented.
  • the polysaccharide extract has a polysaccharide content of 90% or more.
  • the polysaccharide extract has significant antioxidant activity and can be used in the preparation of an antitumor drug or pharmaceutical composition in vitro.
  • a stone cypress active component and its preparation method and use discloses an active component of the stone cypress and a preparation method and use thereof.
  • the method removes the dregs of the stone on the cypress, and extracts and concentrates with the ethanol solution to obtain a cypress leaching extract.
  • the stone leaching extract was reconstituted by water and then passed through a macroporous adsorption resin. The gradient elution with different polar solutions was used to collect the different polar eluates of Shishangbai. After concentration, they were screened by tumor cells in vitro to obtain anti-tumor activity groups. Minute.
  • the active component is enriched with more than 70% of the flavonoids of the stone.
  • the obtained active components of the stone cypress can be prepared into various dosage forms by a pharmaceutical method.
  • the invention provides a preparation method of the active component of the stone cypress, and proves that the invention has obvious inhibitory effects on tumor cell lines such as lung cancer, leukemia, colon cancer and nasopharyngeal carcinoma in vitro.
  • the active ingredient prepared by this method can be used in the preparation of an antitumor drug or a pharmaceutical composition.
  • the present invention provides, for the first time, specific active compounds in the anti-tumor component of the stone, and their use in the treatment of tumors or in the preparation of medicaments for the treatment of tumors.
  • R 1 , R 2 and R 3 are each independently selected from the group consisting of H, hydroxy, lower alkyl, lower alkenyl, lower alkoxy, halogen, amino, hydroxyalkyl, aminoalkyl, nitro, aryl and hetero Aryl.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the above formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, such as an aqueous carrier.
  • the pharmaceutical composition is for treating cancer.
  • the invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula I above, or a pharmaceutically acceptable salt thereof.
  • the aforementioned cancer includes, without limitation, skin cancer, lung cancer, Kaposi's sarcoma, testicular cancer, lymphoma, leukemia, esophageal cancer, gastric cancer, colon cancer, breast cancer, endometrial cancer, ovarian cancer, central nervous system cancer, liver cancer. And prostate cancer.
  • the cancer is lung cancer (including small cell lung cancer and non-small cell lung cancer, especially small cell lung cancer), gastric cancer, leukemia (including acute promyelocytic leukemia and chronic myeloid leukemia), nasopharyngeal carcinoma, and liver cancer.
  • alkyl or “lower alkyl” refers to a C 1 to C 8 alkyl group, e.g. a C 1 to C 3 alkyl group, which may be linear or branched and may be saturated or unsaturated of.
  • alkenyl or “lower alkenyl” refers to a C 2 to C 8 alkenyl, for example C 2 to C 4 alkenyl.
  • alkoxy refers to a straight-chain or branched, saturated or unsaturated, contain a C 1 to C 8 alkyl group, e.g. a C 1 to C 3 alkyl oxo - hydrocarbon chain, including methoxy , ethoxy, propoxy, isopropoxy, butoxy, and tert-butoxy.
  • halogen refers to any halogen group such as chlorine, fluorine, bromine, or iodine.
  • hydroxyalkyl refers to a C 1 to C 4 straight or branched hydroxy-substituted alkyl group, i.e., -CH 2 OH, -(CH 2 ) 2 OH, and the like.
  • aminoalkyl refers to a C 1 to C 4 straight or branched amino-substituted alkyl group, wherein the term “amino” refers to the group NR'R", wherein R' and R "are independently selected from H or lower alkyl as defined above, i.e., -NH 2, -NHCH 3, -N ( CH 3) 2 and the like.
  • aryl refers to a C 3 to C 10 ring aromatic group such as phenyl, naphthyl and the like, and includes a substituted aryl group such as a tolyl group.
  • heteroaryl refers to a 5- or 6-membered aromatic ring comprising 1, 2, 3 or 4 heteroatoms such as nitrogen, oxygen or sulfur, and with aryl, cycloalkyl, hetero An aryl or heterocycloalkyl ring fused to such a ring (for example, thereby providing a C 1 -C 13 heteroaryl group). Examples include, but are not limited to, pyridinyl, pyrazolyl, thienyl, and the like.
  • the heteroaryl group may be unsubstituted or may optionally include from 1 to 4 substituents such as independently selected from the group other than the "heteroaryl" as defined herein with respect to the R 1 group. Substituted by a substituent.
  • treatment refers to any type of treatment that can provide benefits to a patient suffering from a disease, including improving the condition of the patient (eg, an improvement in one or more symptoms), delaying the progression of the disease, preventing or delaying the onset of the disease. and many more.
  • pharmaceutically acceptable refers to a compound or component that is suitable for administration to a patient to obtain the treatments described herein, and that does not have undue adverse side effects in terms of the severity of the disease and the need for treatment. .
  • the invention is primarily concerned with the treatment of human subjects, but it can also be used for veterinary purposes for the treatment of other animal individuals (i.e., mammals, birds).
  • mammals including, but not limited to, dogs, cats, rabbits, horses, etc.
  • humans being particularly preferred.
  • the invention provides a compound of formula I or a pharmaceutically acceptable salt thereof,
  • R 1 , R 2 and R 3 are each independently selected from the group consisting of H, hydroxy, lower alkyl, lower alkenyl, lower alkoxy, halogen, amino, hydroxyalkyl, aminoalkyl, nitro, aryl and hetero Aryl.
  • one aspect of the invention provides a compound of the formula: or a pharmaceutically acceptable salt thereof:
  • the active compounds of the invention can be prepared by the methods described herein or variations thereof which will be apparent to those skilled in the art.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, as previously described, together with a pharmaceutically acceptable carrier.
  • the compound of the formula I or a pharmaceutically acceptable salt thereof is used as the active substance of the pharmaceutical composition.
  • active substance includes pharmaceutically acceptable salts of said compounds.
  • a pharmaceutically acceptable salt retains the biological activity of the desired parent compound and does not cause undesirable The toxicological role of the salt.
  • examples of such salts are (a) acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.; and acid addition salts with organic acids, The organic acid such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, citric acid, palmitic acid, alginic acid, poly Valley And naphthalenesulfonic acid, naphthalenesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, polygalacturonic acid, and the like; and (b) a) acid addition
  • the active substance used to prepare the compositions of the invention may alternatively be in the form of a free base which is pharmaceutically acceptable for the active substance. Because the free base of the compound is less water soluble than its salt, the use of the free base component provides a more sustained release of the active to the targeted area. The active substance that is not dissolved in the targeted area cannot be used to induce a physiological response, but it acts as a reservoir for bioavailable drugs that gradually dissolve into the solution.
  • the compounds of the invention are useful pharmaceutically active substances and can be applied in bulk form. However, it is more preferred to prepare these compounds as pharmaceutical preparations for administration. Any of a number of suitable pharmaceutical preparations may be employed as a carrier for administering a compound of the present invention.
  • the compounds of the invention may be formulated for administration in a variety of situations.
  • the compound of the present invention and a physiologically acceptable salt thereof, or an acidic derivative of the compound of the present invention or a physiologically acceptable salt thereof (hereinafter referred to as "active compound" are generally used. ) is mixed with a pharmaceutically acceptable carrier.
  • the carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation and must be innocuous to the patient.
  • the carrier may be solid or liquid or solid and liquid, and is preferably prepared with the compound in unit dosage formulations, such as tablets, which may contain from 0.5% to 95% by weight of active compound.
  • the preparation of the present invention may be mixed with one or more of the active ingredients, and the preparation may be composed of any of the basic components.
  • the well-known pharmaceutical techniques are used for the preparation, and may optionally comprise one or more additional ingredients.
  • Formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g. sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) and transdermal administration, but in any given The most appropriate route will depend on the nature and severity of the condition being treated and the nature of the particular active compound employed.
  • Formulations suitable for oral administration may be presented in discrete units such as capsules, cachets, lozenges, or tablets each containing a predetermined quantity of active ingredient; may be in powder or granule form; may be in aqueous or nonaqueous liquids In the form of a solution or suspension; or it may be in the form of an oil-in-water or water-in-oil emulsion.
  • Such formulations may be prepared by any suitable pharmaceutical method including the step of bringing into association the active compound with a suitable carrier (which may contain one or more additional ingredients as described above).
  • Formulations of the invention suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient to whom it is administered. These preparations can be administered by subcutaneous, intravenous, intramuscular, or intradermal injection. Such formulations are conveniently prepared by mixing the compound with water or a glycine buffer and allowing the resulting solution to be sterile and isotonic with blood.
  • Formulations suitable for rectal administration are preferably presented as unit dosage suppositories. These preparations can be prepared by mixing the active compound with one or more conventional solid carriers, such as cocoa butter, and then shaping the resulting mixture.
  • Formulations suitable for topical administration to the skin are preferably in the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers which may be used include petrolatum, lanolin, polyethylene glycol, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • Formulations suitable for transdermal administration may be in prolonged intimate contact with the epidermis of the recipient The existence of discrete patches exists. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3:318 (1986)) and generally take the form of an aqueous solution of the active compound which is optionally buffered.
  • the present invention also provides useful methods of treatment.
  • the invention provides methods of treating cancer in an individual in need thereof.
  • the methods of the invention are generally human subjects, but the methods of the invention can also be used in veterinary subjects for other individuals, preferably mammalian individuals.
  • the present invention provides a pharmaceutical preparation for any suitable route of administration comprising a compound of the formula herein or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier, said suitable route of administration being non-limiting This includes oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, intravenous, and transdermal administration.
  • the therapeutically effective dose of any particular compound will vary somewhat from compound to compound, patient to patient, and will depend on the condition and route of delivery of the patient.
  • the present invention also provides a process for the preparation of the above compound of the formula I of the present invention, which comprises the step of condensing and refluxing the whole grass with 70% ethanol to obtain an ethanol extract extract, suspending the extract with water, and then using an equal volume of petroleum ether.
  • Dichloromethane and ethyl acetate were separately extracted, and the ethyl acetate extract was concentrated to obtain an ethyl acetate fraction extract. Finally, the ethyl acetate fraction extract was separated by HPLC to obtain the compound of the formula I.
  • the dried whole grass of Shishangbai is condensed and refluxed with 70% ethanol, concentrated by low temperature rotary evaporation to obtain an ethanol extract extract, and double distilled water suspension is added, and an equal volume of petroleum ether, dichloromethane and acetic acid are used.
  • the esters were separately extracted successively, and the ethyl acetate extract was concentrated to obtain an ethyl acetate fraction extract, and finally the ethyl acetate fraction extract was separated by HPLC to obtain the compound.
  • Figure 2 High performance liquid chromatogram of the purity analysis of the compounds of the invention.
  • Figure 2 (A) is a high performance liquid chromatogram of 70% ethanol extract of Asparagus officinalis; (B) is a high performance liquid chromatogram of the ethyl acetate extract; (C) is an analytical chromatogram of each monomer component Overlay: 1 (I), 2 (II), 3 (III), 4 (IV), 5.
  • Figure 3 In vitro anti-tumor animal test of the compound of the present invention, showing changes in tumor volume in nude mice during continuous administration.
  • compound 5 high dose group 30 mg / kg
  • compound 5 medium dose group 15 mg / kg
  • compound 5 low dose group 5 mg / kg
  • doxorubicin group 2 mg / kg.
  • Figure 4 Data of in vitro anti-tumor animal test of the compound of the present invention, and data of tumor volume change in nude mice during continuous administration.
  • compound 5 high dose group 30 mg / kg
  • compound 5 medium dose group 15 mg / kg
  • compound 5 low dose group 5 mg / kg
  • doxorubicin Group 2 mg/kg.
  • the dried whole grass of Shishangbai was cut into small pieces of less than 5 mm, and then condensed and refluxed with 70% ethanol, and concentrated by rotary evaporation at a low temperature (40-50 ° C) to obtain an ethanol extract extract.
  • Add 10 times volume (m / v) double distilled water suspension extract separate with equal volume of petroleum ether, dichloromethane and ethyl acetate, and extract the ethyl acetate extract by low temperature rotary evaporation to obtain ethyl acetate paste.
  • the ethyl acetate fraction extract was taken and the following procedure was carried out to prepare the compound of the present invention.
  • the preparative column was an Agilent SB-C18 column (250 mm x 21.2 mm, 7 ⁇ m), mobile phase acetonitrile-water (44:56, v/v), flow rate 7 ml/min, detection wavelength 280 nm, room temperature, injection volume 100 ⁇ L.
  • Fig. 1 is a preparative liquid chromatogram
  • compound III, compound IV, compound 5, and component Fr. 4-1 were obtained.
  • Compound 5 was a pale yellow powder.
  • Fig. 2(A) is an analytical liquid chromatogram of the total ethanol extract
  • Fig. 2(B) is an analytical liquid chromatogram of the ethyl acetate extraction site
  • Fig. 2(C) is the separation.
  • HPLC conditions used are: high performance liquid chromatography (LC-20A, Shimadzu, Japan ), column was Ultimate TM XB-C18column (Welch Materials Inc.; 4.6mm ⁇ 250mm, 5.0 ⁇ m), a wavelength of 203nm and 254nm, The injection volume was 10 ⁇ L, the column temperature was 30 ° C, and the flow rate was 1 ml/min.
  • the solvent system for chromatographic analysis was acetonitrile-water. The gradient elution procedure was: 10–42% (v/v), 0–30 min; 42–60% (v/v), 30–60 min; 100% (v/v). ), 60–70min. The system is balanced for 10 minutes.
  • the purity of the compound 5 was 95% or more by the peak area normalization method.
  • the obtained compound 5 was analyzed by thin layer chromatography (TLC), which was a single circular point, and was eluted by HPLC in different elution systems to obtain a single peak.
  • TLC thin layer chromatography
  • the compounds were analyzed by various spectra (UV, IR, NMR, MS), and the data and identification results were as follows:
  • the compound 5 prepared above was used for evaluation of in vitro anticancer activity in the following human tumor cell lines.
  • Compound solution preparation Weigh a certain amount of compound 5, dissolve it with dimethyl sulfoxide (DMSO), and dilute it to the concentration of 500, 250, 125, 62.5, 31.25 ⁇ g/mL with the medium grown by each cell. 5 concentrations of test solution, positive control drug doxorubicin The preparation liquid was sequentially diluted into three concentrations of test medium at a concentration of 5, 2.5, and 1.25 ⁇ M in a medium in which each cell was grown.
  • DMSO dimethyl sulfoxide
  • human tumor cell lines including five adherent tumor cell lines: human gastric cancer cell line (MKN-45), non-small cell lung cancer (A549), nasopharyngeal carcinoma (CNE1, CNE2), large cell lung cancer (PC) -9); 2 kinds of tumor cells in suspension type: human acute promyelocytic leukemia cell line (HL60), human chronic myeloid leukemia cell line (K562). These cell lines were purchased from the Cell Bank of Shanghai Academy of Sciences and frozen by the College of Pharmacy, Fujian Medical University.
  • the frozen cells of the above seven tumor cell lines were taken out from the liquid nitrogen and resuscitated, and then cultured in a 25 cm2 culture flask, and cultured under the respective culture conditions of various cell lines (see Table 2). After cell growth was carried out 6-8 times, when the cell fusion rate at the bottom of the bottle was about 80%, it was a logarithmic growth phase cell, which was digested and passaged with 0.25% trypsin solution. The cell suspension was adjusted to various cell growth rates, and the cell suspension was separately adjusted to an appropriate concentration to be seeded in a 96-well plate.
  • Table 2 7 kinds of tumor cell lines and corresponding culture conditions
  • the seven tumor cell lines were intervened with the different concentrations of the biflavonol test solution prepared above for 72 h, and 10 ⁇ L of MTT solution (5 mg/mL) was added to each test well to continue the culture. After 4 h, the liquid in the culture well was removed, 200 ⁇ L of DMSO was added, and the wells were thoroughly shaken to sufficiently dissolve the bottom of the nail. The OD value of each well was measured at a wavelength of 492 nm by a microplate reader, and the inhibition rate was calculated. The inhibition of each tumor cell by each sample was performed in parallel three times.
  • Compound 5 has a good inhibitory effect on the selected seven human tumor cell lines, and its I C50 is in the range of 13.2 ⁇ 0.91 ⁇ 49.29 ⁇ 2.8 ⁇ g/mL, and the tumor inhibition spectrum is broad. Among them, Compound 5 has a significant inhibitory effect on A549.
  • the compound 5 prepared above was used for evaluation of in vitro anticancer activity in the following human tumor cell lines.
  • Human non-small cell lung cancer cell line A549 cells purchased from the Cell Bank of Shanghai Academy of Sciences, Fujian Medical University School of Pharmacy frozen.
  • Nude mice were housed in SPF-level laminar flow system in 6 cages, given standard food and drinking water to nude mice daily, and kept in the light and dark cycle test (12/12h), room temperature 25 ⁇ 2°C, humidity 50 ⁇ 10 %, replace the drinking water and litter after autoclaving every day. It was kept in the holding room for one week before the experiment.
  • Compound 5 is slightly soluble in water, and the aqueous solution (or physiological saline solution) is acidic.
  • the pH value is adjusted to the specified range of the injection solution (6.0-8.0)
  • the solubility of the sample increases, but it is still low, and it is easy to settle after standing. Therefore, choose a suitable cosolvent to increase its solubility.
  • test solution was prepared in the dark and is now ready for use.
  • the proportion of FBS in A549 culture medium was increased from 6% to 10%, and the cells proliferated rapidly.
  • the cell coverage in the culture flask reached about 80% (that is, the logarithmic growth phase)
  • the A549 cells were digested to prepare a cell suspension, and washed twice with autoclaved PBS to remove the residual culture solution. It was resuspended in PBS and the cell concentration was adjusted to 1 ⁇ 10 7 /mL.
  • a 1 mL (5 # needle) disposable syringe was used to inoculate a 1 ⁇ 10 7 /mL cell suspension into the ventral side of nude mice, and each nude mouse was injected with 0.2 mL of cells. The suspension was completed in 30 min. The remaining cell suspension was transferred to the culture medium to investigate whether the cells were contaminated during the modeling process to help infer the success of the modeling.
  • mice After tumor cell inoculation success, to be nude mice tumor masses grew to 50mm 3 -100mm 3, animals were randomized into 5 groups, namely the high dose group, middle dose group, low dose group, positive control group and negative control group , 6 in each group.
  • the high dose group, the middle dose group, and the low dose group tail vein Compound 5 was injected into the test solution at doses of 30 mg/kg, 15 mg/kg, and 5 mg/kg, respectively.
  • the positive control group and the negative control group were injected with doxorubicin (ADM, dose 2 mg/kg) and physiological saline, respectively.
  • the drug was administered once a day, and the administration volume was adjusted according to the body weight for 12 days; the positive control group was given a tail vein injection of doxorubicin injection.
  • the experiment was designed to be administered once every 3 days and 4 times in 12 days; the negative control group was injected with a physiological saline solution in the tail vein of the nude mice.
  • the life and mental state of the nude mice were observed every day, the body weight was weighed, and the tumor volume was measured.
  • the nude mice were separately treated, the tumor pieces were removed, and the tumor weights were weighed separately.
  • the food intake of the nude mice was normal, no discomfort was observed, and the body weight increased slightly.
  • the tumor growth rate of the control nude mice was significantly faster than that of the other three groups, and the inhibition of tumor growth in the four groups of nude mice with drug intervention was more obvious, and no obvious Significant side effects.
  • the tumor volume curve of the nude mice in the intervention process was plotted.
  • the ventral subcutaneous xenograft specimen which was dissected was a single tumor block and had no adhesion to the skin.
  • the appearance is red-white, irregular circular or elliptical, the capsule is intact, the surface is rich in blood vessels, the base and the ventral muscles are non-adhesive, the texture of the tissue is tough and hard, the cut surface is grayish white, and no obvious infiltration is observed locally.
  • the average tumor inhibition rate after drug intervention was 56.8 ⁇ 5.6% in the positive control group (doxorubicin group), 62.2 ⁇ 21.3% in the compound 5 high dose group, and 43.7 in the compound 5 medium dose group. ⁇ 19.8%, compound 5 low dose group 36.9 ⁇ 6.1%, see Table 4.
  • compound 5 can significantly inhibit the transplantation of A549 cells. Proliferation, reducing the rate of tumor growth, its high concentration (30mg / kg) inhibition is better than the positive control drug doxorubicin. Doxorubicin is one of the drugs commonly used in clinical chemotherapy for lung cancer. In the tumor-bearing nude mouse model, the doses of Compound 5 (15 mg/kg) and low doses (5 mg/kg) also showed good effects of inhibiting tumor cell proliferation.
  • compound 5 can effectively inhibit the growth of human non-small cell lung cancer A549 solid tumor in nude mice, and the toxicity is low, and it is well tolerated by nude mice. It is a high-efficiency, low-toxic anti-tumor active ingredient with development potential.
  • the compounds provided by the present invention have good antitumor effects in various human tumor cell lines as described above, including lung cancer (including non-small cell lung cancer and small cell lung cancer), gastric cancer, leukemia (including acute promyelocytic leukemia and Chronic myeloid leukemia), nasopharyngeal carcinoma.
  • lung cancer including non-small cell lung cancer and small cell lung cancer
  • gastric cancer including gastric cancer, leukemia (including acute promyelocytic leukemia and Chronic myeloid leukemia), nasopharyngeal carcinoma.
  • the unit "degree” of temperature appearing in this document refers to degrees Celsius, or °C.

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Abstract

本发明提供了具有式（I）的结构的化合物或其可药用的盐。本发明还提供了含有所述化合物的药物组合物，以及所述化合物用于治疗癌症的用途。

Description

双黄酮化合物及其治疗癌症和制备药物的用途
本申请要求了2015年12月3日提交的、申请号为201510882246.0、发明名称为“双黄酮化合物及其制备治疗癌症的药物的用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及双黄酮化合物、其药物组合物以及其用于癌症的治疗的用途。具体的，本发明提供了从石上柏提取和制备得到的双黄酮化合物，以及将其用于癌症的治疗和药物制备。
背景技术
石上柏是卷柏属(Selaginella)植物深绿卷柏(Selaginella doederleinii Hieron)的干燥全草。现代药理研究发现石上柏或其组方能增强癌症病人机体代谢和网状内皮系统功能，并对其活性成分进行了研究。已报道的研究发现石上柏含生物碱、植物甾醇、皂甙等，如大麦芽碱-O-α-L-吡喃鼠李糖甙(hordenine-O-α-L-rhamnopyranoside)，N-甲基酪胺-O-α-L-吡喃鼠李糖甙(N-methyltyramine-O-α-L-rhamnopyranoside)等。另外，还发现石上柏含有双黄酮化合物，如穗花杉双黄酮(amentoflavone)，橡胶树双黄酮(heveaflavone)和7，4′，7"，4"'-四-0-甲基-穗花杉双黄酮(7，4′，7"，4"'-tetra-0-methyl-amentoflavone)。
CN104523740A，“一种石上柏多糖提取物制备方法及其应用”中公开了一种石上柏多糖提取物制备方法及其应用。所述石上柏多糖提取物经脱脂、提取、醇沉、除蛋白、除单糖和寡糖、凝胶柱纯化等 步骤实现。该多糖提取物中多糖含量在90％以上。该多糖提取物具有显著的抗氧化活性，可以在制备体外抗肿瘤的药物或药物组合物中应用。
CN104546952A，“一种石上柏活性组分及其制备方法和用途”中公开了一种石上柏活性组分及其制备方法和用途。该方法将石上柏脱脂后的药渣，用乙醇溶液提取浓缩得到石上柏流浸膏。取石上柏流浸膏经水复溶后过大孔吸附树脂，用不同极性溶液梯度洗脱，收集得到石上柏不同极性洗脱液，浓缩后分别经过肿瘤细胞体外筛选得到抗肿瘤活性组分。该活性组分富集了石上柏70％以上黄酮类成分。得到的石上柏活性组分通过药剂学上的方法，可制成各种剂型。本发明提供的石上柏活性组分制备方法，并证明其体外对肺癌、白血病、结肠癌、鼻咽癌等肿瘤细胞株具有明显的抑制作用。该方法制备的活性组分可以在制备抗肿瘤药物或药物组合物中应用。
然而，现有技术对石上柏抗肿瘤组分中的具体活性化合物尚无报道，因此存在物质基础不明确，质量不可控等弊端，缺乏制备高含量总黄酮活性化合物的报道。更没有关于石上柏抗肿瘤组分中的具体活性化合物对于治疗肿瘤等疾病的作用的报道。
发明内容
本发明第一次提供了石上柏抗肿瘤组分中的具体活性化合物以及其在治疗肿瘤或制备治疗肿瘤的药物中的用途。
在本发明的其中一个方面，提供了具有式I的化合物或其可药用盐：
Figure PCTCN2016080931-appb-000001
其中:
R1、R2和R3各自独立地选自H、羟基、低级烷基、低级链烯基、低级烷氧基、卤素、氨基、羟基烷基、氨基烷基、硝基、芳基和杂芳基。
在本发明的其中一个方面，其中所述式I化合物为下式化合物：
Figure PCTCN2016080931-appb-000002
3-(4-(5,7-二羟基-4-氧-4H-苯并吡喃-2-基)苯氧基)-5,7-二羟基-2-(4-羟基苯基)-4H-苯并吡喃-4-酮
本发明另一方面，提供了一种药物组合物，其包含前述式I化合物或其可药用的盐和可药用的载体，例如水性载体。在本发明的其中又一个方面，所述药物组合物用于治疗癌症。
本发明另一方面，提供一种治疗癌症的方法，其包括给需要其的个体使用治疗有效量的上述式I化合物或其可药用的盐。
本发明另一方面，提供了上述式I化合物用于制备治疗癌症的药物的应用。
前述癌症非限制性地包括皮肤癌、肺癌、卡波济氏肉瘤、睾丸癌、淋巴瘤,白血病,食管癌、胃癌、结肠癌、乳癌、子宫内膜癌、卵巢癌、中枢神经系统癌、肝癌和前列腺癌。优选的，所述癌症为肺癌(包括小细胞肺癌和非小细胞肺癌，特别是小细胞肺癌)、胃癌、白血病(包括急性早幼粒细胞白血病和慢性粒细胞白血病)、鼻咽癌、肝癌。
本发明的详细描述
在下文对本发明进行更充分的说明，其进一步说明了这里所述的本发明。但是，本发明可以以不同的形式被具体实施并且不能认为本发明将仅限于这里所述的实施方案。提供这些实施方案只是为了这种公开将是彻底和完全的，并且将充分对本领域技术人员传达本发明的范围。
在这里本发明的说明书中所说的术语仅仅是为了对特定的实施方案进行描述，而并不是要用其对本发明进行限制。
这里所用的术语“烷基”或“低级烷基”指的是C1至C8烷基，例如C1至C3烷基，其可以是直链或支链的并且可以是饱和或不饱和的。
同样，这里所用的“链烯基”或“低级链烯基”指的是C2至C8链烯基，例如C2至C4链烯基。
这里所用的“烷氧基”指的是直链或支链、饱和或不饱和的包含C1至C8烷基，例如C1至C3烷基的氧代-烃链，包括甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、和叔-丁氧基。
这里所用的“卤素”指的是任何卤素基团如氯、氟、溴、或碘。
这里所用的术语“羟基烷基”指的是C1至C4直链或支链的羟基 -取代的烷基，即-CH2OH、-(CH2)2OH等等。
这里所用的术语“氨基烷基”指的是C1至C4直链或支链的氨基-取代的烷基，其中术语“氨基”指的是基团NR'R"，其中R'和R"独立地选自H或上面所定义的低级烷基，即-NH2、-NHCH3、-N(CH3)2等等。
这里所用的术语“芳基”指的是C3至C10环芳族基团如苯基、萘基等等，并且包括被取代的芳基如甲苯基。
这里所用的术语“杂芳基”指的是包括1、2、3或4个杂原子如氮、氧或硫的5-或6-员芳族环，和与芳基、环烷基、杂芳基或杂环烷基环稠合的该类环(例如，从而提供一种C1-C13杂芳基)。实例非限制性地包括吡啶基、吡唑基、噻吩基等。该杂芳基可以是未被取代的或者可以被任选地包括1至4个取代基如独立选择的得自这些除这里涉及R1基团时所定义的“杂芳基”外的基团的取代基所取代。
这里所用的“治疗”指的是可以为受疾病折磨的患者提供益处的任何类型的治疗，包括改善患者情况(例如,一种或多种症状的改善)、延迟疾病进程、阻止或延迟疾病发作等等。
这里所用的“可药用的”指的是适于给药于患者获得这里所述的治疗，并且同时就疾病的严重程度和治疗的必要性而言没有不适当的有害副作用的化合物或组分。
本发明主要与人类个体的治疗有关，但是其也可以用于兽医目的用于其它动物个体(即哺乳动物、鸟类)的治疗。优选哺乳动物(非限制性地包括狗、猫、兔、马等等)，特别优选人。
如上所示，本发明提供了具有式I的化合物或其可药用盐，
Figure PCTCN2016080931-appb-000003
其中:
R1、R2和R3各自独立地选自H、羟基、低级烷基、低级链烯基、低级烷氧基、卤素、氨基、羟基烷基、氨基烷基、硝基、芳基和杂芳基。
更具体的，本发明的其中一个方面提供了下式化合物或其可药用盐：
Figure PCTCN2016080931-appb-000004
本发明的活性化合物可以用这里所述的方法或其对本领域技术人员而言显而易见的变型来进行制备。
本发明提供了一种药物组合物，其包含如前所述的式I化合物或其可药用的盐，以及可药用的载体。其中，所述的式I化合物或其可药用的盐作为药物组合物的活性物质。
这里所用的术语“活性物质”包括所说化合物可药用的盐。可药用的盐是保留了所需母体化合物的生物学活性并且不会产生不希望 的毒理学作用的盐。该类盐的实例有(a)与无机酸形成的酸加成盐，所说的无机酸例如盐酸、氢溴酸、硫酸、磷酸、硝酸等等；和与有机酸形成的酸加成盐，所说的有机酸如例如乙酸、草酸、酒石酸、琥珀酸、马来酸、富马酸、葡萄糖酸、枸橼酸、苹果酸、抗坏血酸、苯甲酸、鞣酸、棕榈酸、藻酸、聚谷氨酸、萘磺酸、甲磺酸、对-甲苯磺酸、萘二磺酸、聚半乳糖醛酸等等；和(b)与元素的阴离子如氯、溴、和碘形成的盐。
用于制备本发明组合物的活性物质或者可以为活性物质可药用的游离碱形式。因为该化合物游离碱的水溶性低于其盐，所以使用游离碱组分提供了活性物质向靶向区域更持久的释放。存在于靶向区域的还没有溶解的活性物质不能用来诱导生理学响应，但是其可作为一种逐渐溶解进入溶液的可生物利用的药物的储库。
本发明的化合物是有用的药学活性物质并且可以以散装形式被应用。但是，更优选地将这些化合物制备成用于给药的药物制剂。可以用许多适宜药物制剂中的任何一种来作为将本发明化合物给药的载体。
本发明的化合物可以被制备成用于各种情况的治疗的给药形式。在本发明药物制剂的制造中，通常将本发明的化合物以及其生理学上可接受的盐、或者本发明化合物或其生理学上可接受的盐的酸性衍生物(在下文被称为“活性化合物”)与可药用的载体进行混合。当然，该载体在与该制剂中的任何其它成分可相容的意义上必需是可接受的并且必需对患者无害。该载体可以是固体或液体或者固体和液体，并且优选地与该化合物一起被制备成单位剂量制剂，例如片剂，其可包含0.5％至95％重量的活性化合物。本发明的制剂中可以混有一种或多种各活性成分，该制剂可以用任何基本由将这些组分混合所组成 的众所周知的药学技术来进行制备，并且可任选地包含一种或多种附加成分。
本发明的制剂包括这些适于口服、直肠、局部、颊(例如舌下)、胃肠外(例如皮下、肌内、真皮内、或静脉内)和经皮给药的制剂，但是在任何给定情况中最适宜的途径将取决于被治疗情况的性质和严重程度以及所用特定活性化合物的性质。
适于口服给药的制剂可以以离散单位如各自包含预定数量活性成分的胶囊、扁囊剂、锭剂、或片剂的形式存在；可以为粉末或颗粒形式；可以为位于水性或非水性液体中的溶液或混悬液形式；或者可以为水包油或油包水乳剂的形式。该类制剂可以用任何包括将活性化合物与适宜载体(如上所述那样，其可包含一种或多种附加成分)联系到一起的步骤的适宜药学方法来进行制备。
适于胃肠外给药的本发明的制剂方便地包括活性化合物的无菌水性制剂，该制剂优选地与其将施用的领受者的血液等渗。这些制剂可以通过皮下、静脉内、肌内、或真皮内注射来进行给药。该类制剂可以方便地通过将该化合物与水或甘氨酸缓冲剂进行混合并使所得的溶液无菌和与血液等渗来进行制备。
适于直肠给药的制剂优选地是以单位剂量栓剂的形式存在的。这些制剂可以通过将活性化合物与一种或多种常规固体载体，例如可可豆脂进行混合，然后将所得的混合物成型来进行制备。
适于局部给药于皮肤的制剂优选地采取软膏、乳膏、洗剂、糊剂、凝胶、喷雾、气雾剂、或油的形式。可以使用的载体包括凡士林、羊毛脂、聚乙二醇、醇类、经皮增强剂、以及其两种或多种的组合。
适于经皮给药的制剂可以以适于与领受者的表皮长期密切接触 的离散贴剂的形式存在。适于经皮给药的制剂还可以通过离子电渗疗法来进行传递(见，例如，Pharmaceutical Research 3:318(1986))并且一般采取任选地被缓冲的活性化合物水溶液形式。
除这里所述结构式的化合物外，本发明还提供了有用的治疗方法。例如，本发明提供了治疗需要其的个体的癌症的方法。
可以用本发明方法治疗的个体一般是人类个体，但是本发明的方法也可用于兽医目用于其它个体，优选哺乳动物个体。如上所示，本发明提供了在可药用的载体中包含这里所述结构式的化合物或其可药用盐的用于任何适宜给药途径的药物制剂，所说的适宜给药途径非限制性地包括口服、直肠、局部、颊、胃肠外、肌内、真皮内、静脉内、和经皮给药。
任何特定化合物的治疗有效剂量将随着化合物与化合物、患者与患者的不同而有一定的差异，并且将取决于该患者的情况和传递途径。
本发明还提供了制备上述本发明式I化合物的方法，其中包括将石上柏干燥全草用70％的乙醇冷凝回流提取得到乙醇提取物浸膏，用水混悬浸膏，然后用等体积石油醚、二氯甲烷和乙酸乙酯依次分别萃取，浓缩乙酸乙酯萃取液得到乙酸乙酯部位浸膏，最后通过HPLC对乙酸乙酯部位浸膏进行分离得到所述式I化合物。
在本发明的其中一个方面，提供了制备下式化合物的方法，
Figure PCTCN2016080931-appb-000005
包括，将石上柏干燥全草用70％的乙醇冷凝回流提取，低温旋转蒸发浓缩得到乙醇提取物浸膏，加双蒸水混悬浸膏，用等体积的石油醚、二氯甲烷和乙酸乙酯依次分别萃取，乙酸乙酯萃取液浓缩得到乙酸乙酯部位浸膏，最后通过HPLC对乙酸乙酯部位浸膏进行分离得到所述化合物。
附图说明
图1.本发明化合物制备液相色谱图
图2.本发明化合物纯度分析高效液相色谱图。其中图2(A)为石上柏70％乙醇提取物的高效液相色谱图；(B)为乙酸乙酯萃取部分的高效液相色谱图；(C)为各个单体成分的分析型色谱图叠加图：1(I)、2(II)、3(III)、4(IV)、5。
图3.本发明化合物体外抗肿瘤动物测试，连续给药过程裸鼠体内瘤体积大小变化情况图。其中，化合物5高剂量组:30mg/kg,化合物5中剂量组:15mg/kg,化合物5低剂量组:5mg/kg,多柔比星组:2mg/kg。
图4.本发明化合物体外抗肿瘤动物测试，连续给药过程裸鼠体内瘤体积大小变化数据图。其中，化合物5高剂量组:30mg/kg,化合物5中剂量组:15mg/kg,化合物5低剂量组:5mg/kg,多柔比星 组:2mg/kg。
具体实施方式
下面将结合实施例进一步说明本发明的实质内容和有益效果，该实施例仅用于说明本发明而非对本发明的限制。
实施例1化合物1的制备
1.石上柏样品处理
将石上柏干燥全草剪碎成小于5mm小段后，用70％的乙醇冷凝回流提取，低温(40-50℃)旋转蒸发浓缩得到乙醇提取物浸膏。加10倍体积(m/v)双蒸水混悬浸膏，用等体积的石油醚、二氯甲烷和乙酸乙酯依次分别萃取，乙酸乙酯萃取液低温旋转蒸发浓缩得到乙酸乙酯部位浸膏。取乙酸乙酯部位浸膏进行以下步骤制备本发明的化合物。
2.HPLC制备分离
制备柱为Agilent SB-C18柱(250mm×21.2mm,7μm)，流动相乙腈-水(44：56，v/v)，流速为7ml/min，检测波长280nm，室温，进样量为100μL。通过高效制备液相色谱分离，如图1(图1为制备液相色谱图)所示，得到化合物III、化合物IV、化合物5，以及组分Fr.4-1。
化合物5为淡黄色粉末。
3.HPLC分析制备得到的化合物5的纯度
如图2所示，图2(A)为乙醇总提取物的分析型液相色谱图，图2(B)为乙酸乙酯萃取部位的分析型液相色谱图，图2(C)为分离纯化得到的7个化合物的分析型色谱图叠加图。
采用的HPLC条件为：高效液相色谱仪(LC-20A，Shimadzu， Japan)，色谱柱为UltimateTM XB-C18column(Welch Materials Inc.；4.6mm×250mm，5.0μm)，波长为203nm和254nm，进样体积为10μL，柱温为30℃，流速为1ml/min。色谱分析的溶剂系统为乙腈-水，梯度洗脱程序为：10–42％(v/v)，0–30min；42–60％(v/v)，30–60min；100％(v/v)，60–70min。系统平衡10min。
采用峰面积归一化法计算，化合物5纯度达到95％以上。
4.化合物5的结构鉴定
将得到的化合物5用薄层色谱法(TLC)分析，均为一个圆形单点，同时用HPLC在不同洗脱体系中洗脱，得到的均为一个单峰。将化合物进行各种光谱(UV、IR、NMR、MS)分析，数据及鉴定结果如下：
质谱数据：(–)ESI-MS m/z 537.2[M–H]–，说明其MW＝538；分子式推断为：C30H18O10；不饱和度Ω＝22；
Figure PCTCN2016080931-appb-000006
：210nm、268nm、330nm，黄酮类化合物特征吸收峰；
Figure PCTCN2016080931-appb-000007
(cm-1)：3588(芳环中OH)、3423(芳环中C-H)、1654(芳环中C＝O)、1610(芳环中C＝C)、1500(芳环中C＝C)、1289(C-O)、1028(C-O)、838(芳基对位取代)。
1H NMR谱中芳香区有13个氢，13C NMR谱中有2个羰基碳(δ182.3、δ176.5)，28个芳香碳，提示该化合物结构为双黄酮类。1H NMR谱中，有2个羟基氢12.86(1H,s)、12.24(1H,s)。根据耦合常数推断8.01(2H,d,J＝8.4Hz)、7.86(2H,d,J＝8.8Hz)，7.26(2H,d,J＝8.8Hz)和6.91(2H,d,J＝8.8Hz)构成了2个AA′BB′系统。6.56(1H,s,J＝1.2Hz)，6.50(1H,s,J＝1.2Hz)，6.27(1H,s,J＝1.6Hz)和5.88(1H,s,J＝1.6Hz)构成2对间位耦合氢，此外尚有一个单氢6.85(1H,s)。13C NMR信号δ72.9、δ99.4、δ94.7、δ99.4可以推断出，两个黄酮母环A上的C6和C8均未被取代。
结合1H NMR谱和13C NMR谱全部数据(详见表1)，鉴定化合物5具有以下结构式：
Figure PCTCN2016080931-appb-000008
表1化合物5的核磁共振数据(DMSO-d6,400MHz)
Figure PCTCN2016080931-appb-000009
实施例2化合物5体外抗癌活性评价
将上述制备得到的化合物5用于在以下人肿瘤细胞株做体外抗癌活性评价。
1.化合物溶液配制：精密称取一定量化合物5，用二甲基亚砜(DMSO)溶解，用各个细胞生长的培养基依次稀释成浓度为500、250、125、62.5、31.25μg/mL的5个浓度的供试液，阳性对照药多柔比星 准备液以各个细胞生长的培养基分别依次稀释成浓度为5、2.5、1.25μM的3个浓度供试液。
2.细胞株和细胞培养
七种人肿瘤细胞株，包括5种为贴壁型肿瘤细胞株：人胃癌细胞株(MKN-45)、非小细胞肺癌(A549)、鼻咽癌(CNE1、CNE2)、大细胞肺癌(PC-9)；2种为悬浮型的肿瘤细胞：人急性早幼粒细胞白血病细胞株(HL60)、人慢性粒细胞白血病细胞株(K562)。这些细胞株均购自上海中科院细胞库，由福建医科大学药学院冻存。
上述七种肿瘤细胞株的冻存细胞从液氮中取出复苏后，分别培养于25cm2培养瓶中，用各种细胞株对应的培养条件培养(见表2)。待细胞生长传代6-8次，细胞在瓶底融合率为80％左右时，即为对数生长期细胞，用0.25％的胰酶溶液消化传代。细胞悬液按照各类细胞生长速度，分别调节细胞悬液至适当浓度接种于96孔板中。
表2 7种肿瘤细胞株及对应培养条件
Figure PCTCN2016080931-appb-000010
3.MTT法测定化合物5对七种细胞的抑制率
分别用以上制备的不同浓度双黄酮供试溶液干预所述七种肿瘤细胞株72h，每个实验孔加入MTT溶液(5mg/mL)10μL，继续培养 4h后，移去培养孔中液体后加入200μL的DMSO，并充分震荡使孔底甲瓒充分溶解。于酶标仪492nm波长下测定各个孔的OD值，计算其抑制率。每个样品对每种肿瘤细胞的抑制作用实验均平行三次。
测定结果分析，采用Origin7.5软件分析处理每个样品孔测得的OD值，计算人肿瘤细胞株的半数抑制率(IC50值，mean±S.D.)，结果见表3。
表3化合物5对七种人肿瘤细胞株的半数抑制率(mean±S.D.,n＝3)
Figure PCTCN2016080931-appb-000011
化合物5对所选的七种人肿瘤细胞株均有很好的抑制作用，其I C50在13.2±0.91～49.29±2.8μg/mL范围内，抑瘤谱广。其中，化合物5对A549抑制作用显著。
实施例3化合物5动物模型体内抗癌活性评价
将上述制备得到的化合物5用于在以下人肿瘤细胞株做体外抗癌活性评价。
1.细胞株和实验动物
人非小细胞肺癌细胞株A549细胞，购自上海中科院细胞库，由 福建医科大学药学院冻存。
3-4周龄BALB/c裸鼠，体重18±2g(雄性)，购自上海斯莱克实验动物有限责任公司，实验动物许可证号：SCXK(沪)2012-0002，动物合格证编号：2007000537438。
裸鼠每6只一笼饲养于SPF级层流系统中，每日给予裸鼠标准食物和饮水，保持光照和避光循环实验(12/12h)，室温25±2℃，湿度为50±10％，每天更换高压蒸汽灭菌后的饮用水和垫料。实验前在保持室饲养一周。
2.化合物5供试液配制
化合物5微溶于水，水溶液(或生理盐水溶液)呈酸性。当调节其pH值到注射液规定范围(6.0-8.0)，样品溶解度有所增大，但依然较低，静置容易沉降。故选择适当的助溶剂来增加其溶解度。通过对比了吐温-80(终浓度0.02-2％，v/v)、PEG-400(终浓度<40％，v/v)、1,2-丙二醇(终浓度<40％，v/v)、甘油(终浓度<30％，v/v)和CMC-Na(终浓度1-5‰，w/v)对样品在水溶液的助溶作用。最终发现，各类助溶剂在注射剂允许的添加浓度范围中，PEG-400和吐温-80对样品水溶液的助溶效果较好。由于二者助溶效果相当，考虑到含有吐温的剂型溶血现象比较普遍，故最终选择加入PEG-400作为助溶剂。通过筛选不同比例的PEG-400助溶效果，发现PEG-400为10％时助溶效果理想。故选择PEG-400添加量为10％。
精确称取6mg化合物5粉末，加入助溶剂PEG-400(100μL)涡旋、超声，充分溶解后，逐步加入少量的生理盐水，并调节pH为7.4左右，补足溶液至1mL。
3.阳性对照药的配制
精密称取一定量的注射用多柔比星粉末，加入生理盐水配成0.4mg/mL供试液。该供试液避光配制，现配现用。
4.人肿瘤细胞A549异体移植裸鼠模型建立
4.1细胞培养
人非小细胞肺癌A549细胞株复苏后，将A549培养液中的FBS比例从6％提高到10％，使细胞快速增殖。待培养瓶中细胞覆盖率达到80％左右(即对数成长期)时，将A549细胞消化配制成细胞悬液，用高压灭菌的PBS洗2遍，除去残余培养液。再用PBS重悬并将细胞浓度调整为1×107个/mL。
4.2裸鼠模型的建立
动物适应环境后，在无菌条件下，用1mL(5#针头)的一次性注射器吸取浓度为1×107个/mL细胞悬液接种于裸鼠腹侧，每只裸鼠注射0.2mL细胞悬液，整个实验在30mi n内完成。并将剩余细胞悬液移于培养液中培养，考察建模过程中细胞是否出现污染，以辅助推断建模是否成功。
4.3模型动物分组
肿瘤细胞接种成功后，待裸鼠体内的瘤块长至50mm3-100mm3，将动物随机分为5组，分别为高剂量组、中剂量组、低剂量组、阳性对照组和阴性对照组，每组6只。
5.化合物5的体内抗肿瘤活性研究
5.1动物分组干预
根据动物分组情况，将高剂量组、中剂量组、低剂量组尾静脉 注射化合物5供试液，注射剂量分别为30mg/kg、15mg/kg、5mg/kg，阳性对照组和阴性对照组分别注射多柔比星(ADM，剂量为2mg/kg)和生理盐水。每天给药一次，每次给药根据体重调节给药体积，连续给药12天；阳性对照组给予尾静脉注射多柔比星注射液。本实验设计为每3天给药一次，12天给药4次；阴性对照组裸鼠尾静脉注射生理盐水溶液。
5.2瘤体体积测量
给药过程中，每天观察裸鼠生活和精神状态，称其体重，并测量瘤体积大小。用游标卡尺测量实体瘤结节部位的长径L(mm)和短径W(mm)，瘤体体积(mm3)＝1/2×L(mm)×W2(mm2)。
最后一次给药24h后，将裸鼠分别处理，剥离瘤块，并分别称取瘤体重量。计算肿瘤抑制率，其中，肿瘤抑制率(％)＝(1-实验组瘤重/阴性对照组瘤重)×100％。
5.3药物干预后荷瘤小鼠处置
每次给药24h后称重，采用脱颈处死的方法，剖取瘤块称重拍照。
以上实验动物操作遵守《实验动物管理条例》(中华人民共和国国家科学技术委员会令第2号，1988)。
5.4统计学分析
实验所得数据均采用SPSS 13.0软件进行统计学处理，组间抑瘤率比较使用单因素方差分析(one-way ANOVA)或两样本比较U检验，结果以mean±SE表示，P<0.05为差异有显著性意义。
5组动物12天连续干预过程中，裸鼠摄食量正常，未见不适，体重略微增长。其中，对照组裸鼠体内瘤块生长速度明显快于其他3组，有药物干预的4组裸鼠体内瘤块生长的抑制较为明显，且未见明 显毒副反应。根据每天测量瘤体大小计算的瘤体积，绘制干预过程裸鼠体内瘤体积变化曲线图。
如图3所示，剖离出的腹侧皮下移植瘤标本为单个瘤块，与皮肤无粘连。外观红白色，呈不规则圆形或椭圆形，被膜完整，表面有丰富的血管分布，基底与腹侧肌肉无粘连，组织质地坚韧且硬，切面呈灰白色，局部未见明显浸润。
按照抑瘤率计算公式计算出药物干预后的平均肿瘤抑制率分别为：阳性对照组(多柔比星组)56.8±5.6％，化合物5高剂量组62.2±21.3％，化合物5中剂量组43.7±19.8％，化合物5低剂量组36.9±6.1％，见表4。
表4化合物5体内试验中各组动物给药剂量、给药前后体重、瘤重以及抑瘤率,(n＝6)
Figure PCTCN2016080931-appb-000012
连续给药过程裸鼠体内瘤体积大小变化情况见图3和图4。化合物5的各治疗组和阳性对照组与阴性对照组相比差异均有统计学意义(P<0.05)。
在荷瘤裸鼠模型中，化合物5能明显抑制A549细胞移植瘤的 增殖，降低肿瘤生长速度，其高浓度(30mg/kg)抑制程度要好于阳性对照药多柔比星。多柔比星是临床上常用的化疗肺癌的药物之一。在荷瘤裸鼠模型中，化合物5中剂量(15mg/kg)和低剂量(5mg/kg)也表现出良好的抑制肿瘤细胞增殖的效果。
可见，化合物5能在裸鼠体内有效抑制人非小细胞肺癌A549实体瘤的生长，且毒性低，裸鼠对其具良好耐受性。是具开发潜力的高效、低毒的抗肿瘤活性成分。
本发明提供的化合物在前述的多种人肿瘤细胞系都有良好的抗瘤效果，其包括了肺癌(包括非小细胞肺癌和小细胞肺癌)、胃癌、白血病(包括急性早幼粒细胞白血病和慢性粒细胞白血病)、鼻咽癌。
这些化合物在动物体内抗瘤实验的结果也表现出独特的令人感兴趣的结果。实验证明，本发明的化合物在动物体内能够有效地抑制各种癌症细胞，特别是肺癌，如非小细胞肺癌。
上面是对本发明进行的说明，不能将其看成是对本发明进行的限制。除非另外指出，本发明的实践将使用有机化学、聚合物化学、生物技术等的常规技术，显然除在上述说明和实施例中所特别描述之外，还可以别的方式实现本发明。其它在本发明范围内的方面与改进将对本发明所属领域的技术人员显而易见。根据本发明的教导，许多改变和变化是可行的，因此其在本发明的范围之内。
如无特别表示，本文中出现的温度的单位“度”是指摄氏度，即℃。

Claims (13)

  1. 一种具有式I的化合物或其可药用的盐：
    Figure PCTCN2016080931-appb-100001
    其中，R1、R2和R3各自独立地选自H、羟基、低级烷基、低级链烯基、低级烷氧基、卤素、氨基、羟基烷基、氨基烷基、硝基、芳基和杂芳基。
  2. 如权利要求1所述的化合物或其可药用的盐，其中所述化合物为
    Figure PCTCN2016080931-appb-100002
  3. 一种治疗癌症的药物组合物，其包含如权利要求1或2所述的化合物或其可药用的盐和可药用的载体。
  4. 如权利要求3所述的药物组合物，其中所说的癌症选自皮肤癌、肺癌、卡波济氏肉瘤、睾丸癌、淋巴瘤,白血病,食管癌、胃癌、结肠癌、乳癌、子宫内膜癌、卵巢癌、中枢神经系统癌、肝癌和前列 腺癌。
  5. 如权利要求4所述的药物组合物，其中所说的癌是肺癌，例如非小细胞肺癌。
  6. 一种治疗癌症的方法，其包括给需要其的个体使用治疗有效量的如权利要求1或2所述的化合物或其可药用的盐。
  7. 如权利要求6所述的方法，其中所说的癌症选自皮肤癌、肺癌、卡波济氏肉瘤、睾丸癌、淋巴瘤,白血病,食管癌、胃癌、结肠癌、乳癌、子宫内膜癌、卵巢癌、中枢神经系统癌、肝癌和前列腺癌。
  8. 如权利要求7所述的方法，其中所说的癌是肺癌，例如非小细胞肺癌。
  9. 如权利要求1或2所述的化合物或其可药用的盐在制备治疗癌症的药物中的用途。
  10. 如权利要求9所述的用途，其中所说的癌症选自皮肤癌、肺癌、卡波济氏肉瘤、睾丸癌、淋巴瘤,白血病,食管癌、胃癌、结肠癌、乳癌、子宫内膜癌、卵巢癌、中枢神经系统癌、肝癌和前列腺癌。
  11. 如权利要求10所述的用途，其中所说的癌是肺癌、胃癌、白血病、鼻咽癌或肝癌。
  12. 如权利要求11所述的用途，其中所说的癌是肺癌，例如非小细胞肺癌。
  13. 制备权利要求1或2所述的化合物的方法,其中包括将石上柏干燥全草用70％的乙醇冷凝回流提取得到乙醇提取物浸膏，用水混悬浸膏，然后用等体积石油醚、二氯甲烷和乙酸乙酯依次分别萃取，浓缩乙酸乙酯萃取液得到乙酸乙酯部位浸膏，最后通过HPLC对乙酸乙酯部位浸膏进行分离得到如权利要求1或2所述的式I化合物。
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