CN1771931B - Use of diterpene compound in preparing medicines with synergistic effect to chemical therapy medicines - Google Patents
Use of diterpene compound in preparing medicines with synergistic effect to chemical therapy medicines Download PDFInfo
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- CN1771931B CN1771931B CN 200410088934 CN200410088934A CN1771931B CN 1771931 B CN1771931 B CN 1771931B CN 200410088934 CN200410088934 CN 200410088934 CN 200410088934 A CN200410088934 A CN 200410088934A CN 1771931 B CN1771931 B CN 1771931B
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Abstract
The present invention relates to the pharmaceutical use of diterpene compound with the general expression as shown or its precursor medicine, and is especially the use of the diterpene compound or its precursor medicine in preparing medicine with synergistic effect on tumor chemotherapy medicine.
Description
Technical field
The present invention relates to the pharmaceutical applications of a kind of diterpene-kind compound or its prodrug, be specifically related to Lushanrubescensin A LuA Lushanrubescensin, Lushanrubescensin B LuB (-)-Lushanrubescensin B and Lushanrubescensin E LuE Lushanrubescensin E. and tumor chemotherapeutic drug had purposes in the synergistic medicine in preparation.
Background technology
The Lushan rubescensin is by extracting the diterpene-kind compound that obtains in the Lushan Rabdosia rubescens (Rabdosia rubescens Hara f.lushanensisGao et Li).Wherein Lushanrubescensin A LuA Lushanrubescensin (Lushanrubescensin A, LuA), Lushanrubescensin B LuB (-)-Lushanrubescensin B (Lushanrubescensin B, LuB) and Lushanrubescensin E LuE Lushanrubescensin E. (Lushanrubescensin E LuE) is its main active.The chemical constitution of three chemical compounds is as follows:
The Lushan Rabdosia rubescens is made into syrup clinically, is used for the treatment of advanced esophageal carcinoma, carcinoma of gastric cardia, has obtained certain curative effect (Chen Shaotang etc., combination of Chinese and Western medicine magazine, 1988,8 (1): 43-44).Pharmaceutical research shows, Lushanrubescensin A LuA Lushanrubescensin, second is plain and penta element all has certain anti-tumor activity, as to P388, and L1212, B16, ECA, the inhibitory action of mouse tumors such as S180 and ARS (Wang Qingrui etc., Henan Medical Univ.'s journal, 1987,22 (4): 372-376).We find under study for action, monomeric compounds such as Lushan rubescensin (total diterpene enriched substance) and Lushanrubescensin A LuA Lushanrubescensin, second element and penta element are to the tumor cell of various human, comprise adenocarcinoma of stomach (AGS, BGC-823), the esophageal carcinoma (Eca-109, TE-1), nonsmall-cell lung cancer (SPC-A-1, A549), breast carcinoma (Bcap-37) etc. all has significant cytotoxicity.
Chemotherapy is the abbreviation of " chemotherapy ".The notion of chemotherapy generally is understood that " chemotherapy of tumor " at present, promptly uses the antitumor chemicals, adopts the method for some measure and scheme treatment tumor.Yet common chemotherapeutics such as vincristine, cisplatin, methotrexate, cyclophosphamide etc. can produce toxic and side effects such as injection site pain, venous thrombosis, bone marrow depression, gastrointestinal reaction, peripheral nervous pathological changes; In addition, because the toxicity of chemotherapeutics, complication such as infecting also can appear in chemotherapy patients sometimes, hemorrhage.
For the toxic and side effects that reduces chemotherapeutics, improve curative effect, reduce tumor recurrence and avoid drug-fast generation, select different medication combined uses, become one of important means of chemotherapy of tumors.For example, clinically cisplatin and 5-Fu, bleomycin or epipodophyllotoxin etc. are united the use treatment esophageal carcinoma.
The curative effect of the curative effect that synergism (Synergistic Effect) is produced when being meant two medication combined use when two prescriptions solely use during greater than same dose.According to middle effect principle (Joseph R.Bertino, Ting-Chao Chou, Chemotherapy:Synergism and Antagonism, Encyclopedia ofCancer, 1996, Academic Press, Inc.), the action effect that two medicines are united when using can be judged by association index CI (Combination Index):
Wherein, D1, D2 are respectively Lushan rubescensin (plain or penta element of first element, second) and chemotherapeutics (as cisplatin) when using separately, the drug level when cell proliferation inhibition rate reaches X%; (Dx) 1, (Dx) 2 when reaching the same cell proliferation inhibition rate concentration of Lushan rubescensin in the mixture (first element, second is plain or penta element) and chemotherapeutics.
To two kinds of separate medicine α=0; And medicine α=1 independently not mutually
When CI＜1, be synergism; During CI=1, be summation action; CI〉1 o'clock, be antagonism
Lushan rubescensin and chemotherapy drugs in combination use to produce does not see bibliographical information to the Synergistic killing effect of tumor cell.
Summary of the invention
Appearance of the present invention is based on such one and is surprised to find: Lushanrubescensin A LuA Lushanrubescensin (LuA), Lushanrubescensin B LuB (-)-Lushanrubescensin B (LuB), Lushanrubescensin E LuE Lushanrubescensin E. (LuE) have synergism to chemotherapeutics, can obviously improve the killing activity of chemotherapeutics to tumor cell.
Therefore, the present invention relates to the diterpene-kind compound of following general formula (I) or its prodrug and tumor chemotherapeutic drug had purposes in the synergistic medicine in preparation:
R wherein
1, R
2, R
3, R
4, R
5Be selected from OC (O) CH independently of one another
3, OH or H.
Particularly, the present invention relates to the diterpene-kind compound of a class general formula (I), wherein R
1, R
2, R
3, and R
5Be OC (O) CH independently of one another
3, R
4Be OH;
The invention still further relates to the diterpene-kind compound of a class general formula (I), wherein R
1, R
2, R
3Be OC (O) CH independently of one another
3, and R
4And R
5Be OH independently of one another;
The invention still further relates to the diterpene-kind compound of a class general formula (I), wherein R
1, R
2, R
3Be OC (O) CH independently of one another
3, R
4Be OH, and R
5Be OH;
The invention still further relates to a class and have the diterpene-kind compound of the following general formula (II) of stereoselectivity (stereospecificity):
Particularly, the present invention relates to following three typical diterpene-kind compounds:
Can be used for chemotherapeutics of the present invention comprises, but be not limited to: cisplatin (cisplatin), mitomycin (mitomycin C), Irinotecan (Irinotecan), Docetaxel (docetaxel), paclitaxel (paclitaxel), table podophyllin (podophyllotoxin), vincristine (vincristine, VCR), plicamycin (plicamycin), daunorubicin (daunorubicin, DNR), D actinomycin D (dactinomycin, DACT), amycin (doxorubicin, adriamycin, ADM), 5-fluorouracil (5-Fluorouracil), hormone, hormone antagonist and cytokine (as interleukin-2 and β transforming growth factor).
The pharmaceutical composition of diterpene-kind compound, chemotherapeutics, pharmaceutically suitable carrier that comprises general formula of the present invention (I) is also within protection scope of the present invention.
The specific embodiment of the present invention will set forth with the lower part.Other characteristics of the present invention, purpose and advantage will be shown by following elaboration.
The specific embodiment
The inventor discovers, when the diterpene-kind compound of general formula of the present invention (I) or its prodrug and chemotherapeutics administration simultaneously, can obviously improve the anti-tumor activity of chemotherapeutics, has synergism.Thereby can reduce the consumption of chemotherapeutics, reduce its toxic and side effects.Therefore, the diterpene-kind compound of general formula of the present invention (I) or its prodrug can be used for the preparation tumor chemotherapeutic drug is had synergistic medicine.
Wherein some is that nature exists and can separates from plant and obtain the diterpene-kind compound of general formula of the present invention (I).For example, LuA, LuB and LuE can extract from the leaf of Lushan Rabdosia rubescens (Rabdosia rubescensHaraf.lushanensis Gao et Li) and obtain.Other chemical compounds of the present invention can make by the common synthetic technology of this area.Be used to separate or the chemicals of synthetic diterpene-kind compound of the present invention comprises solvent, reagent, catalyst, blocking group reagent, removes blocking group reagent.Described separation can also comprise adding or remove suitable blocking group finally to obtain the step of required diterpene-kind compound with synthetic.The synthetic chemistry that is used to prepare diterpene-kind compound of the present invention transforms and the method for radical protection (going to protect) is known to the ordinary skill of this area, can be referring to R.Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W.Greenand P.G.M.Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wileyand Sons (1999), L.Fieser and M.Fieser, Fieser and Fieser ' s Reagents forOrganic Synthesis, John Wiley and Sons (1994); And L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and follow-up works.
Diterpene-kind compound of the present invention can comprise one or more " asymmetric centers " (asymmetriccenters).Therefore, chemical compound of the present invention can racemic modification, various ways such as single enantiomer, single diastereomer, non-enantiomer mixture exist.Above-mentioned various isomer is all in protection scope of the present invention.
The prodrug of chemical compound of the present invention comprises that the carboxylate that contains this chemical compound (can be by the conventional method C of this area
1-4Pure and strong contraction get), the hydroxy ester that contains this chemical compound (can be by the conventional method C of this area
1-4Carboxylic acid, C
3-6Dicarboxylic acids or its anhydride, as maleic anhydride, fumaric acid anhydride etc. are concentrated to be made), the enamine that contains this chemical compound (can be by the conventional method C of this area
1-4Aldehydes or ketones concentrate make) or contain the acetal or ketal (the Albert S.Kearney Advanced Drug Reviews.19 (1996): 229-234) such as (can concentrate with chloromethyl methyl ether or chloromethyl ether by the conventional method of this area and make) of this chemical compound.
Can be used for chemotherapeutics of the present invention sets forth in " summary of the invention " part.Also can be referring to Isselbacher et al., Harrison ' s Principles of Internal Medicine13th, McGraw-Hill, 1994..These chemotherapeutics all can obtain or make via the known technology of this area by commercial sources.Select the standard of suitable chemotherapeutics to be based on, for example, tumor type, tumor marker and patient's age and general health situation.
Diterpene-kind compound of the present invention or its prodrug can with chemotherapeutics administration simultaneously or non-while administration; Can pass through intestinal or parenteral route administration.The intestinal canal administration preparation comprises pill, granule, capsule, suspension or solution.Non-intestinal drug delivery agent comprises injection, cream, unguentum, patch or spray.That the parenterai administration approach comprises is subcutaneous, in the Intradermal, tremulous pulse, vein, muscle, joint, synovial fluid, breastbone, sheath, intralesional, intracranial injection or instillation.Other route of administration can comprise part, rectum, per nasal, through cheek, vagina, Sublingual, mucosa, trachea or urethra.In addition, diterpene-kind compound of the present invention can also suck or implant and accumulate or mode administration such as acupuncture by aerosol.
The intestinal canal administration preparation of diterpene-kind compound of the present invention or its prodrug includes but not limited to capsule, tablet, Emulsion, aqueous suspension agent, colloidal solution, solution, microcapsule, pill, lozenge, granule, powder.The pharmaceutically suitable carrier that is usually used in tablet comprises lactose and corn starch.Usually also can add lubricants such as magnesium stearate.The pharmaceutically suitable carrier that is usually used in capsule comprises lactose and dried corn starch.When making oral aqueous suspension agent and/or Emulsion, diterpene-kind compound can suspend or be dissolved in the oil phase and with emulsifying agent or suspending agent and combine.If desired, also can add some sweeting agents and/or flavouring agent and/or toner.
Diterpene-kind compound of the present invention or its prodrug can be made into aseptic injection, as aseptic water or oil phase suspension.This suspension can use suitable dispersant or wetting agent (as Tween80) and suspending agent etc. to make by the conventional method of this area.But it can also be at the nontoxic diluent of intestinal external administration or aqueous solution or the suspension in the solvent, as the solution in 1,3 butylene glycol.Relevant available support or solvent comprise mannitol, water, ringer's solution, isotonic sodium chloride etc.In addition, aseptic fixedly oil (blandfixed oil) often is used as the media of solvent or suspending agent, thereby comprises that the multiple soft fixedly oil of synthetic glycerine monoesters or diglyceride all is suitable for.Fatty acid can be used for preparing described injection as octadecenic acid and glyceride ester derivatives thereof (as olive oil or Oleum Ricini, particularly its polyoxyethylene radical derivative) etc.Described oil solution or suspension also can comprise a kind of ethanol dilution agent of long-chain or dispersant or carboxymethyl cellulose or similar other dispersants, and this type of material is usually used in preparing pharmaceutical acceptable emulsion and/or suspending agent.Surfactant that some other preparation is commonly used such as Tweens or Spans and/or other similar emulsifying agents or bioavailability promoter etc. can be used for preparing this preparation too.
Diterpene-kind compound of the present invention or its prodrug can be made into suppository and pass through rectally, method is that diterpene-kind compound or its prodrug are mixed with the non-irritating excipient that suits, the latter is liquid under rectal temperature for solid at room temperature, thereby this suppository is dissolvable in water in the rectum and discharges active ingredient.This type of excipient includes but not limited to: cupu oil, Cera Flava and polyethylene.The local administration preparation of diterpene-kind compound of the present invention or its prodrug (as ointment) can be directly used in the affected part.This type of topical formulations contains active ingredient and pharmaceutically suitable carrier, and the latter includes but not limited to mineral oil, liquid petroleum, white oil, propylene glycol, polyoxyethylene or the polyoxy third desaturation compound, emulsifying is cured or water.In addition, diterpene-kind compound of the present invention or its prodrug also can be made into lotion or oil preparation.The carrier that is suitable for includes but not limited to: mineral oil, sorbic alcohol monostearate, polysorbate60, spermaceti ester, hexadecanol, 2-octadecanol, benzyl ethanol or water.Diterpene-kind compound of the present invention or its prodrug also can be made into enema etc. and are used for the rectum topical.The topical transdermal patch is also within protection scope of the present invention.But diterpene-kind compound of the present invention or its prodrug be per nasal spraying or inhalation also, promptly by the conventional method of this area, uses benzyl ethanol or other antiseptic, absorption enhancer, fluorocarbon and/or other solubilizing agents or dispersant to make saline solution.
Diterpene-kind compound of the present invention or its prodrug also can pass through drug delivery implant.Adopt the drug delivery implant mode can reach in the administration subject and continue, regularly discharge the effect of diterpene-kind compound of the present invention or its prodrug.In addition, drug delivery implant can also be at local organization and organ site-specific delivery of drugs (Negrin et al., Biomaterials22 (6): 563,2001) regularly release tech also can be used in the administration of diterpene-kind compound of the present invention or its prodrug, as delayed release capsule, slow release method and the preparation technique for packing (as polymer and liposome) etc. based on the polymer technology.
Patch is included within protection scope of the present invention equally.It comprises basic unit's (as polymer, cloth, yarn and binder) and pharmaceutical composition of the present invention.One side of basic unit can be provided with a protective layer to prevent the outflow of active ingredient.Described patch also can contain a binding agent that is used for fixing, and the latter can be a kind of natural or synthetic material, can temporarily adhere on the skin when it contacts with the administration subject's skin.Binding agent can be a waterproof.
" pharmaceutically suitable carrier " can not destroy the pharmaceutical active of diterpene-kind compound of the present invention or its prodrug, its effective dose simultaneously, and promptly can playing pharmaceutical carrier, to make the consumption of time spent nontoxic to human body." pharmaceutically suitable carrier " includes but not limited to: ion exchange material, aluminium oxide, aluminium stearate, lecithin, self-emulsifying drug delivery system (SEDDS) is as d-alpha-tocopherol cetomacrogol 1000 succinate, the surfactant that pharmaceutical preparatioies such as tween (Tweens) or other similar polymerisation mediums are used, serum albumin such as human serum albumin, buffer substance such as phosphate, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acid partial glycerol ester admixture, water, salt, electrolyte such as sulfate protamine, phosphoric acid hydrogen two is received, potassium hydrogen phosphate, sodium chloride, zinc salt, silica gel, magnesium silicate etc.Polyvidon, cellulosic material, polyvinyl alcohol, sodium carboxymethyl cellulose, polypropylene acid esters, ethylene-polyoxyethylene-block polymer and wool grease, cyclodextrin such as α-, β-and gamma-cyclodextrin or its all can be used for promoting the drug delivery of diterpene-kind compound of the present invention or its prodrug through hydroxyalkyl cyclodextrin such as the derivant of chemical modification such as 2-and 3-HP-or other soluble derivatives etc.
Diterpene-kind compound of the present invention or its prodrug and the synergistic effectiveness of tumor chemotherapeutic drug can be verified that the latter carries out its therapeutic effect of drug combination post-evaluation to the animal that suffers from tumor by suitable in vitro tests (in vitro assay) and in vivo test (in vivo assay).On this basis, can also determine appropriate dosage scope and route of administration.
The invention will be further described below in conjunction with specific embodiment.But and do not mean that the present invention only limits to this.
The separation and the evaluation of embodiment 1 Lushanrubescensin A LuA Lushanrubescensin, Lushanrubescensin B LuB (-)-Lushanrubescensin B, Lushanrubescensin E LuE Lushanrubescensin E.
With broken section of 200 Crewe mountain Rabdosia rubescens with 95% ethanol percolate extraction 3 times, each 3 days.3 times percolates are merged, and heating concentrates, and removes the aqueous solution of alcohol to proper volume fully, uses petroleum ether, ethyl acetate extraction then respectively, gets ethyl acetate layer.After ethyl acetate layer concentrated, through normal phase silicagel column (200～300 order), the wet method upper prop was with petroleum ether-acetone (gradient elution of 9:1 → 8:2 → 7:3), Fractional Collections.Petroleum ether-acetone 9:1 eluting position, 8:2 eluting position, 7:3 eluting position are respectively based on a material that strong uv absorption is arranged at the 254nm place.Petroleum ether-acetone 9:1 eluting position concentrates after the RP-18 column purification, and with 50% methanol-eluted fractions, being collected in 254nm place has strong uv absorption part, must chemical compound Lushanrubescensin A LuA Lushanrubescensin 37mg (productive rate: 0.02%) behind the concentrate drying.Petroleum ether-acetone 8:2 eluting position concentrates after the RP-18 column purification, and with 45% methanol-eluted fractions, being collected in 254nm place has strong uv absorption part, must chemical compound Lushanrubescensin B LuB (-)-Lushanrubescensin B 59mg (productive rate: 0.03%) behind the concentrate drying.Petroleum ether-acetone 7:3 eluting position concentrates after the RP-18 column purification, and with 40% methanol-eluted fractions, being collected in 254nm place has strong uv absorption part, must chemical compound Lushanrubescensin E LuE Lushanrubescensin E. 81mg (productive rate: 0.04%) behind the concentrate drying.
Lushanrubescensin A LuA Lushanrubescensin physicochemical constant and spectral data
M.p.=188～190 ℃
(c=0.99, pyridine)
UV?λ?max(EtOH)nm：239
IR?υ?max(KBr)cm-1：3440，1730，1703，1645，1248，1225
1H-NMR(C5D5N，400MHz)δ：5.56(1H，ddd，J＝12.5，4.0，2.6Hz，H2)，5.30(1H，d，J＝2.6Hz，H3)，2.64(1H，br，H5)，5.54(1H，dd，J＝3.4，1.7Hz，H6)，4.02(1H，d，J＝3.4Hz，H7)，2.40(1H，br，H9)，5.36(1H，d，J＝4.3Hz，H11)，3.00(1H，br，H13)，2.55(1H，d，J＝12.5Hz，H14a)，1.48(1H，dd，J＝12.5，3.6Hz，H14b)，6.05(1H，br，H17a)，5.35(1H，br，H17b)，1.04(3H，s，H18)，1.12(3H，s，H19)，1.49(3H，s，H20)
13C-NMR(C5D5N，100MHz)δ：40.8t(C1)，67.6d(C2)，77.6d(C3)，38.3s(C4)，42.0d(C5)，71.0d(C6)，73.1d(C7)，50.0s(C8)，55.0d(C9)，39.8s(C10)，68.3d(C11)，38.3t(C12)，37.3d(C13)34.5t(C14)，212.7s(C15)，150.2s(C16)，114.6t(C17)，28.0q(C18)，23.2q(C19)，20.6q(C20)，170.6s(-OAc)，170.4s(-OAc)，169.8s(-OAc)，169.0s(-OAc)，21.2q(-OAc)，20.9q(-OAc)，20.6q(-OAc)，20.6q(-OAc)
EIMS?m/z：535[M+1]+，474，414，354，312，294，279
Lushanrubescensin B LuB (-)-Lushanrubescensin B physicochemical constant and spectral data
M.p.=219～221 ℃
(c=0.1, methanol)
IR?υ?max(KBr)cm-1：3440，1740，1710，1646，1265～1220
1H-NMR(C5D5N，400MHz)δ：5.54(1H，ddd，J＝12.0，4.2，2.8Hz，H2)，5.20(1H，d，J＝2.8Hz，H3)，2.58(1H，br，H5)，5.45(1H，dd，J＝3.2，1.4Hz，H6)，4.00(1H，d，J＝3.2Hz，H7)，2.55(1H，br，H9)，4.34(1H，d，J＝4.3Hz，H11)，3.03(1H，br，H13)，2.58(1H，d，J＝12.6Hz，H14a)，1.46(1H，dd，J＝12.6，3.4Hz，H14b)，6.02(1H，br，H17a)，5.36(1H，br，H17b)，1.00(3H，s，H18)，1.13(3H，s，H19)，1.51(3H，s，H20)
13C-NMR(C5D5N，100MHz)δ：40.8t(C1)，67.9d(C2)，77.8d(C3)，38.3s(C4)，42.1d(C5)，71.5d(C6)，73.7d(C7)，49.9s(C8)，59.1d(C9)，39.4s(C10)，65.0d(C11)，40.8t(C12)，38.1d(C13)35.2t(C14)，213.6s(C15)，151.0s(C16)，112.9t(C17)，28.0q(C18)，23.3q(C19)，20.7q(C20)，170.6s(-OAc)，170.6s(-OAc)，169.9s(-OAc)，21.3q(-OAc)，21.3q(-OAc)，21.0q(-OAc)
EIMS?m/z：492[M]+，432，372，312，297，279，252，240
Lushanrubescensin E LuE Lushanrubescensin E. physicochemical constant and spectral data
M.p.=215～217 ℃
(c=0.1, methanol)
UV?λ?max(EtOH)nm：238
IR?υ?max(KBr)cm-1：3550～3370，1740～1720，1710，1650，1265～12251H-NMR(C5D5N，400MHz)δ：5.74(1H，d，J＝3.0Hz，H3)，5.35(1H，m，H6)，4.68～4.46(1H，m，H11)，3.08(1H，br，H13)，6.03(1H，br，H17a)，5.29(1H，br，H17b)，1.13(3H，s，H18)，1.10(3H，s，H19)，1.55(3H，s，H20)
13C-NMR(C5D5N，100MHz)δ：44.5t(C1)，63.8d(C2)，81.4d(C3)，38.5s(C4)，49.2d(C5)，69.0d(C6)，38.5d(C7)，48.8s(C8)，63.6d(C9)，39.7s(C10)，65.0d(C11)，41.3t(C12)，37.9d(C13)37.9t(C14)，208.0s(C15)，150.8s(C16)，111.4t(C17)，28.2q(C18)，22.9q(C19)，20.2q(C20)，170.8s(-OAc)，169.9s(-OAc)，21.5q(-OAc)，20.9q(-OAc)
EIMS?m/z：434[M]+，416，392，374，356，314，299，296，281，253，235
Cooperative effect when embodiment 2 detects Lushanrubescensin A LuA Lushanrubescensin, Lushanrubescensin B LuB (-)-Lushanrubescensin B and Lushanrubescensin E LuE Lushanrubescensin E. and cisplatin combined the use on cellular level.
Experiment material:
Human tumor cell line and cultivation thereof: the esophageal carcinoma (Eca-109, TE-1), adenocarcinoma of stomach (AGS and BGC-823), breast cancer cell (Bcap-37), pulmonary carcinoma (A549), adenocarcinoma of lung cells such as (SPC-A-1) be all available from biochemical cell research institute of the Chinese Academy of Sciences.Cell in the IMDM that contains 10% hyclone (Gibco) (Gibco) culture medium, at 37 ℃, 5%CO
2Cultivate under the condition.
Other reagent: MTT and DMSO are Sigma company product.
Sample: self-control Lushan Rabdosia rubescens monomeric compound all is dissolved among the DMSO, obtains the 10mg/ml stock solution.
Experimental technique:
Lushan Rabdosia rubescens monomeric compound records by mtt assay the cytotoxicity of above-mentioned tumor cell line.Concrete steps are as follows:
1. cell is suspended in the IMDM culture medium that contains 10% hyclone after trypsinization and washing, through the blue dyeing of Placenta Hominis exclusive method meter viable count, and regulates cell suspending liquid density to 1 * 10
5Cell/ml.
2. in flat 96 orifice plates, every hole adds 100 microlitre cells, and total cellular score is 1 * 10 in every hole
4In 37 ℃, overnight incubation in the 5%CO2 cell culture incubator.
3. be 1000 μ g/ml with 10mg/ml sample stock solution with the cell growth medium dilution, this concentration solution is carried out 1/3 gradient dilution, obtain 333.3,111.1,37.0,12.3,4.11 μ g/ml respectively.Every hole adds the sample after the 10 μ l dilution.The DMSO of respective amount is added in the cell of not dosing in contrast, what do not add medicine and DMSO contains cell hole as a setting.Make three parallel testings at every.
With the dosing cell at 37 ℃, 5%CO
2Insulation is 48 hours in the cell culture incubator.
5. add 10 μ l5mg/ml MTT solution in every hole, continue in incubator, to be incubated 3～4 hours.
6. with cell plates in 1000rpm centrifugal 15 minutes, vacuum was inhaled and is removed culture fluid, and it is centrifugal and inhale carefully and remove solution to add 150 μ l PBS washing back.
7. every hole adds 150 μ l DMSO, places on the shaking table and shakes 15 minutes with 150rpm, and the first hairpin crystal of generation is fully dissolved.Measure the 492nm absorbance value.
8. calculate the Lushan rubescensin according to absorbance value and handle back cell relative survival rate.Computing formula is as follows:
9.
10. by the IC of Xlfit (ID Business Solutions) computed in software Lushan rubescensin to each tumor cell
50
The synergism experiment of Lushan Rabdosia rubescens monomeric compound and cisplatin:
According to said medicine cytotoxicity analysis method, medicine to be measured and cisplatin are joined in the cell so that certain proportion is mixed, to replace independent medication.Other operations are identical.
Experimental result:
Table 1. Lushan rubescensin and cisplatin coupling suppress the association index CI of (or killing and wounding) to the propagation of tumor cell
Claims (30)
1. the chemical compound of following general formula (I) or its prodrug have purposes in the synergistic medicine in preparation to tumor chemotherapeutic drug:
R wherein
1, R
2, R
3, R
4, R
5Be selected from OC (O) CH independently of one another
3, OH or H.
2. the purposes of claim 1, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma, gastric cancer, breast carcinoma, hepatocarcinoma, carcinoma of prostate, colorectal cancer, cancer of pancreas, leukemia or melanoma.
3. the purposes of claim 2, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma or gastric cancer.
4. the purposes of claim 1, wherein said tumor chemotherapeutic drug is a cisplatin.
5. the purposes of claim 4, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma, gastric cancer, breast carcinoma, hepatocarcinoma, carcinoma of prostate, colorectal cancer, cancer of pancreas, leukemia or melanoma.
6. the purposes of claim 5, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma or gastric cancer.
7. the purposes of claim 6, wherein said chemical compound is selected from following general formula (II):
8. the purposes of claim 7, wherein R
1, R
2, R
3Be OC (O) CH independently of one another
3
9. the purposes of claim 1, wherein said chemical compound is selected from following general formula (II):
10. the purposes of claim 9, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma, gastric cancer, breast carcinoma, hepatocarcinoma, carcinoma of prostate, colorectal cancer, cancer of pancreas, leukemia or melanoma.
11. the purposes of claim 10, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma or gastric cancer.
12. the purposes of claim 11, wherein R
1, R
2, R
3Be OC (O) CH independently of one another
3
13. the purposes of claim 1, wherein R
1, R
2, R
3, R
5Be OC (O) CH independently of one another
3, and R
4Be OH.
14. the purposes of claim 13, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma, gastric cancer, breast carcinoma, hepatocarcinoma, carcinoma of prostate, colorectal cancer, cancer of pancreas, leukemia or melanoma.
15. the purposes of claim 14, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma or gastric cancer.
16. the purposes of claim 15, wherein said tumor chemotherapeutic drug are cisplatin.
17. the purposes of claim 15, wherein said chemical compound is:
18. the purposes of claim 17, wherein said tumor chemotherapeutic drug are cisplatin.
19. the purposes of claim 1, wherein R
1, R
2, R
3Be OC (O) CH independently of one another
3, and R
4, R
5Be OH independently of one another.
20. the purposes of claim 19, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma, gastric cancer, breast carcinoma, hepatocarcinoma, carcinoma of prostate, colorectal cancer, cancer of pancreas, leukemia or melanoma.
21. the purposes of claim 20, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma or gastric cancer.
22. the purposes of claim 21, wherein said tumor chemotherapeutic drug are cisplatin.
23. the purposes of claim 21, wherein said chemical compound is:
24. the purposes of claim 23, wherein said tumor chemotherapeutic drug are cisplatin.
25. the purposes of claim 1, wherein R
1, R
2, R
3Be OC (O) CH independently of one another
3, and R
4Be H, R
5Be OH.
26. the purposes of claim 25, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma, gastric cancer, breast carcinoma, hepatocarcinoma, carcinoma of prostate, colorectal cancer, cancer of pancreas, leukemia or melanoma.
27. the purposes of claim 26, wherein said tumor is selected from the esophageal carcinoma, pulmonary carcinoma or gastric cancer.
28. the purposes of claim 27, wherein said tumor chemotherapeutic drug are cisplatin.
29. the purposes of claim 27, wherein said chemical compound is:
30. the purposes of claim 29, wherein said tumor chemotherapeutic drug are cisplatin.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410088934 CN1771931B (en) | 2004-11-09 | 2004-11-09 | Use of diterpene compound in preparing medicines with synergistic effect to chemical therapy medicines |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410088934 CN1771931B (en) | 2004-11-09 | 2004-11-09 | Use of diterpene compound in preparing medicines with synergistic effect to chemical therapy medicines |
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| CN108210485A (en) * | 2018-04-08 | 2018-06-29 | 王长国 | A kind of TLR7 agonists are in enhancing CIK cell to the application in terms of tumor cell killing potential |
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2004
- 2004-11-09 CN CN 200410088934 patent/CN1771931B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 王庆端，王绵英.鲁山冬凌草甲素的抗肿瘤作用研究.河南医科大学学报22 4.1987,22(4),372-376. |
| 王庆端,王绵英.鲁山冬凌草甲素的抗肿瘤作用研究.河南医科大学学报22 4.1987,22(4),372-376. * |
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