CN101575335B - Dehydrogenated silibinin substituted by meta-chlorobenzene formoxyl, preparation method and pharmaceutical applications thereof - Google Patents
Dehydrogenated silibinin substituted by meta-chlorobenzene formoxyl, preparation method and pharmaceutical applications thereof Download PDFInfo
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Abstract
The invention relates to a dehydrogenated silibinin substituted by meta-chlorobenzene formoxyl, a preparation method and pharmaceutical applications thereof, in particular to a preparation method and applications of 2,3-dehydrogenated silibinin substituted by 23-site meta-chlorobenzene formoxyl. The compound has applications of inhibiting activity of production of lipid superoxide induced by freeradical and can be expectedly used for preparing medicine for prevention and treatment of acute and chronic liver damage, hepatitis and cerebral atherosclerosis which are caused by the lipid superoxide. The compound further shows very strong resistance for PC12 cell damage caused by free radical and can be expectedly developed into medicine for protecting cranial nerve from oxidation and preventing and treating senile dementia. Besides, the compound has strong xanthine oxidase activity inhibition and can be expectedly developed into medicine for preventing and treating diseases caused by xanthine oxidase.
Description
Technical field
The present invention relates to medical technical field.Particularly; the present invention relates to 2 of one 23 interdigit chlorobenzene formacyl replacement; the preparation method and the medicinal use thereof of 3-dehydro-silibinin ester derivative; this compound has the activity that the lipid peroxide that suppresses free yl induction generates, and can expect to develop into that the control free radical causes or associated disease comprises the pharmaceutical use of diseases such as inflammation, autoimmune disorder, radiotherapy sequela, tumour, myocardial ischemia, myocardial hypertrophy, aging, transformation reactions, atherosclerosis.This compound also shows very strong rat suprarenal gland pheochromocytoma (PC12) the cell injury effect that Green Tea Extract is caused; promptly the PC12 cell to the simulation cranial nerve cell has the anti-oxidative damage provide protection; illustrating that it is anti-oxidant to the anti-oxidant and cranial nerve of protection brain cell tissue, prevent and treat senile dementia has active effect, can expect the purposes that is used to prepare such medicine.In addition, this compound has potent inhibition xanthine oxidase activity, can expect to develop into the medicine that disease that control causes by XOD comprises goat.
Background technology
In numerous Green Tea Extract natural products, silymarin is one of medicine of a few widespread use clinically.This medical instrument of the clinical trial certificate of three more than ten years has definite curative effect and hypotoxicity (to consult people such as FloraK., Am.J.Gastroenterol, 1998,93,139-143; People such as Saller R., Drugs, 2001,61 (14), 2035-2063).Silibinin content is maximum in the silymarin, and activity is also the highest relatively, so the most extensive about the pharmacodynamic study of silibinin.In national drug standards silymarin one hurdle that State Food and Drug Administration works out, require the moisture silibin that flies to calculate, must not be less than 68% with silibinin.This natural drug effect mainly contain following some: (one) Green Tea Extract activity: silymarin has provide protection for the hepatic injury that is caused by tetracol phenixin, GalN, alcohols and other hepatotoxin.People such as nineteen ninety Lotteron have reported in the Mouse Liver microsome, silymarin can reduce external lipid peroxidation that is caused by the tetracol phenixin metabolism and the peroxidation that is caused separately by reduced coenzyme, and these show that all silymarin is the chain interruption antioxidant or is free-radical scavengers.(2) protection liver plasma membrane: keep flowability of cell membranes by the anti peroxidation of lipid reaction, the protection liver plasma membrane.Can also block combining of special acceptor on mycotoxins phalloidin and α-amanitine etc. and the liver cell, suppress it, interrupt its liver sausage circulation, thereby the enhance hepatocyte film be for the resistibility of multiple damage factor hepatocellular attack and transmembrane transport.(3) promote hepatocellular reparation and regeneration: silibinin can combine with estradiol receptor after entering cell, and make it to activate, activated receptors can enhance hepatocyte nuclear RNA polysaccharase 1 activity, rna transcription is strengthened, promote enzyme and proteinic synthetic, and promote the synthetic of DNA indirectly, help hepatocellular reparation and regeneration.(4) antitumor action: various active oxygens can form 8-hydroxyl guanine by the oxidation guanine, cause dna damage, and then cause tumour, and silibinin has also shown the effect of prevention and treatment tumour as an effective Green Tea Extract material.
The silibinin compounds has definite anti-oxidant activity, but because the market that the some shortcomings on its water-soluble and bioavailability have limited this medicine.So seek the new derivative of silibinin class, make it can have pharmacologically active higher or that upgrade, in the hope of obtaining the new drug of independent intellectual property right, real genus is necessary.
Goat is common multiple disease, and modern medical theory confirms that the paathogenic factor of goat is relevant with oxyradical.XOD (EC1.2.3.2, abbreviate XO as) be the metabolic important enzyme of nucleic acid in vivo, energy catalysis xanthoglobulin and xanthine generate uric acid and superoxide anion, if body can not in time be removed the uric acid of generation, uric acid crystal salt is deposited at joint, will cause gout.Produce too much superoxide anion and also can cause many tissue damageds, it is significant to finding new XO inhibitor and its antioxygenation to the effect of XO therefore to measure compound.From natural product with and derivative filter out the vital task that the xanthine oxidase inhibitor lead compound also is an exploitation treatment goat new drug.In recent years, international the world of medicine has dropped into great amount of manpower and material resources, financial resources continuously, strives to find the antioxidant lead compound that antioxygenation is remarkable, have potent neuroprotective.
In sum; the present invention designs and synthesizes out 2 of one 23 interdigit chlorobenzene formacyl replacement; 3-dehydro-silibinin ester; tested that its lipid peroxide that suppresses free yl induction generates active and to the rat adrenal chromaffin knurl PC12 cell injury effect that Green Tea Extract causes also tested the restraining effect of this compound for XOD.Test result shows: this compound has significant pharmacodynamics activity to above-mentioned model, is expected to further develop the lead compound and the medicine that become the above-mentioned relative disease of treatment.Finished the present invention thus.
Summary of the invention
The purpose of this invention is to provide 2 of the 23 interdigit chlorobenzene formacyls replacement shown in a kind of formula (I); 3-dehydro-silibinin ester is compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3; 5; 7-trihydroxy--4-oxo-chromene-2)-2; 3-dihydro-1,4-benzodioxane-2]-methyl ester or its pharmacologically acceptable salt be used to prepare the purposes of control and anti-oxidant machine-processed relative disease and goat medicine.
Particularly, one of purpose of the present invention has provided a kind of m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester or its pharmacologically acceptable salt be used to the purposes for preparing control brain cell tissue oxidative damage or cranial nerve oxidative damage, prevent and treat the medicine or the pharmaceutical composition of degenerative brain disorder; It is used to prepare the disease that is caused by XOD and comprises the medicine of diseases such as goat or the purposes of pharmaceutical composition.
A further object of the present invention has provided the purposes that formula (I) compound or pharmaceutically acceptable salt thereof is used to prepare the medicine of relevant physiological change of control liver cephalopin peroxidation or disease, generates the purposes of the medicine of the acute and chronic liver injury class disease, inflammatory disease and the autoimmune disorder that is caused by lipid peroxidation that cause, radiotherapy sequela, tumour, myocardial ischemia, myocardial hypertrophy, aging, transformation reactions, atherosclerosis in particular for the lipid peroxide of preparation control free yl induction.
Another purpose of the present invention provided a kind of be used to prevent and treat by oxyradical cause or brain cell tissue oxidative damage, cranial nerve oxidative damage, the senile dementia relevant with oxyradical; and the relevant physiological change of control liver cephalopin peroxidation or the medicine or the pharmaceutical composition of disease; it contains 2 of the 23 interdigit chlorobenzene formacyls replacement shown in the formula as activeconstituents (I) for the treatment of significant quantity, 3-dehydro-silibinin ester or its pharmacologically acceptable salt and pharmaceutically acceptable auxiliaries.
A further object of the present invention provides medicine or the pharmaceutical composition that disease that a kind of control causes by XOD comprises goat; wherein contain 2 of the 23 interdigit chlorobenzene formacyls replacement shown in the formula (I) for the treatment of significant quantity, 3-dehydro-silibinin ester or its pharmacologically acceptable salt and pharmaceutical excipient.
The present invention also provides the method for a kind of preparation formula (I) compound, it is characterized in that: 2 of silibinin dehydration back formation, the 3-dehydro-silibinin is in the presence of condensation catalyst and phosphonium salt, carry out dehydration condensation with m-chlorobenzoic acid and get, the preferred diethyl azodiformate of condensation catalyst (DEAD) herein, the phosphonium salt triphenylphosphine.
Usefulness of the present invention is: the present invention adopts synthetic method to prepare 2 of 23 interdigit chlorobenzene formacyls replacements; 3-dehydro-silibinin ester; and disclosed it with pharmacological evaluation and have control liver, cerebral tissue resistance of oxidation, thereby can expect the pharmaceutical applications that develops into the antioxidant medicine and prevent and treat the senile dementia disease drug.Formula (I) compound is that parent preparation gets by natural drug, and method is simple, step is short, and productive rate is higher, and cost is low, pollutes for a short time, is suitable for industrialization and meets the related request of energy-saving and emission-reduction.In addition; the present invention also discloses 2 of this 23 interdigit chlorobenzene formacyl replacement; 3-dehydro-silibinin ester for above-mentioned common disease, frequently-occurring disease provide new basic substance, has certain social benefit and economic benefit in the potentiality to be exploited of preparation treatment goat pharmaceutical field.
Embodiment
In order to understand essence of the present invention better, at first use the process of the formal specification compound of embodiment below, embodiment has provided part physics and the chemistry and the Wave Spectrum data of compound.Mandatory declaration, embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Embodiment 1: the preparation of formula (I) compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester
1.1 2, the preparation of 3-dehydro-silibinin:
Add 1.02 gram silibinins in the exsiccant reaction flask, 10 gram Potassium ethanoates add 50 milliliters of Glacial acetic acid again, add 50 milliliters in the Glacial acetic acid that is dissolved with 1 gram iodine under the ice bath again.Reflux 5 hours.The cooling back adds 50 milliliters of frozen water, ethyl acetate extraction (20 milliliters * 4 times), and the extracting solution organic layer merges, washing, anhydrous sodium sulfate drying.Filter, filtrate concentrates, and gets the reddish-brown solid, adds 50 milliliters of ethanolic solns that contain 6 milliliters of hydrochloric acid.Reflux 3 hours.Yellow solid is separated out in cooling, and the vinyl acetic monomer recrystallization gets crude product 2, and 3-dehydro-silibinin yellow powder 0.95 gram is directly used in next step reaction.
1.2 the preparation of formula (I) compound:
In the exsiccant reaction flask, add 0.95 gram 2,3-dehydro-silibinin crude product, 0.62 gram m-chlorobenzoic acid, 1.57 gram triphenylphosphines add 1.04 gram (0.8 milliliter) DEAD, stirring at room 6 hours with 20 milliliters of anhydrous tetrahydro furan dissolving backs.Remove solvent under reduced pressure, add minimum of chloroform, the elimination insolubles, mother liquor concentrates the back with 6 grams, 200～300 order silica gel mixed samples, with 40 grams, 200～300 order silica gel column chromatographies, sherwood oil/vinyl acetic monomer=2: 1 wash-outs, thin layer TLC detects merging, and purifying obtains formula (I) compound 1.08 grams.
R
f(petrol ether/ethyl acetate=1: 1): 0.31; Proton nmr spectra
1HNMR (400MHz, deuterated acetone) δ: 4.63 (multiplet, 1H), 4.85 (multiplet, 2H), 5.02 (multiplets, 1H), 6.28 (bimodal, J=2.0Hz, 1H), 6.47～6.58 (multiplet, 3H, aromatic nucleus Ar-H), 6.62 (bimodal, J=2.0Hz, 1H), 6.81 is (bimodal, J=8.4Hz, 1H), 7.28～7.89 (multiplet, 4H, aromatic nucleus Ar-H), 7.96 (bimodal, J=2.0Hz, 1H), 8.01 (double doublet, J=8.4,2.0Hz, 1H), 11.1 5 (unimodal, 1H, 5-OH); Electrospray ionization mass spectrum ESI-MS:617 (M-1)
+
Formula (I) compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester has multiple important biological, the present invention tests the anti-oxidant activity of this compound by the pharmacodynamics test model, finds the activity that its lipid peroxide with definite vitro inhibition free yl induction generates.Showing that this compound can be expected is used to prepare prevention or treatment acute and chronic liver injury class disease and is caused or other physiological changes relevant with lipid peroxide or the medicine of disease by lipid peroxide.In addition, this compound also shows the very strong PC12 cell injury effect that Green Tea Extract is caused, promptly the PC12 cell to the simulation cranial nerve cell has the anti-oxidative damage provide protection.Illustrate that it is anti-oxidant to the protection cranial nerve, prevent and treat senile dementia active effect is arranged, can be used to prepare the purposes that the protection cranial nerve is anti-oxidant, prevent and treat the senile dementia disease drug.Moreover this compound also demonstrates potent inhibition xanthine oxidase activity, can expect to develop into the medicine that disease that control causes by XOD comprises goat.
Formula of the present invention (I) compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester or its pharmacologically acceptable salt can combine with auxiliary material or carrier pharmaceutically commonly used, prepare medicine or pharmaceutical composition with anti-oxidant activity, can be used for treating the disease that produces free radical in some pathological processes, as inflammatory disease, autoimmune disorder, the radiotherapy sequela, tumour, myocardial ischemia, myocardial hypertrophy, old and feeble, transformation reactions, diseases such as atherosclerosis.Above-mentioned various kinds of drug or pharmaceutical composition can adopt formulations such as injection, tablet, capsule, aerosol, suppository, film, pill, externally-applied liniment.
Formula of the present invention (I) compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester or its pharmacologically acceptable salt can also unite use with the Green Tea Extract oxidant drug that has now gone on the market such as superoxide-dismutase (SOD) etc., prepare and have the active antioxidant compositions of infringement that causes of defence free radical, be used for the treatment of above-mentioned disease.Its also can with other protect cranial nerve anti-oxidant, prevent and treat the medication combined use of senile dementia, obtain anti-ageing year dementia pharmaceutical composition.Aforementioned pharmaceutical compositions can adopt formulations such as injection, tablet, capsule, aerosol, suppository, film, pill, externally-applied liniment.
In addition, formula of the present invention (I) compound or pharmaceutically acceptable salt thereof can combine with auxiliary material or carrier pharmaceutically commonly used, prepares the medicine that suppresses XOD, treatment gout class diseases.Formula of the present invention (I) compound or pharmaceutically acceptable salt thereof can also be united use with treatment goat medicine that has now gone on the market such as xanthine oxidase inhibitor allopurinol etc., prepares the pharmaceutical composition with treatment goat, is used for the clinical treatment of goat.Above-mentioned various kinds of drug composition can adopt formulations such as injection, tablet, capsule, aerosol, suppository, film, pill, externally-applied liniment.
Formulation described in the present invention such as tablet, capsule, injection, aerosol, suppository, film, pill and externally-applied liniment and various slowly-releasing, controlled release form and nanometer formulation can get via the routine preparation according to the pharmaceutics general knowledge of now having generally acknowledged, and the medicine of preparing on these knowledge and technical foundation that contains claim compound of the present invention or other formulations of pharmaceutical composition are all in protection scope of the present invention.
In order to understand essence of the present invention better, use form enumerative (I) compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3 of pharmacology related embodiment below respectively, 5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester suppresses activity test that the lipid peroxide of free yl induction generates, to oxydol H
2O
2Due to the PC12 cell injury provide protection test, with and to the result of the restraining effect test of XOD, illustrate its in pharmacy field purposes and be used to prepare the basis of anti-oxidation medicine and goat medicine of being correlated with.Following pharmacology related embodiment has provided the part activity data of compound.Same mandatory declaration, these embodiment that the present invention enumerates are used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Embodiment 2: formula (I) compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester is to oxydol H
2O
2Due to the provide protection activity test of PC12 cell injury
2.1 experiment material and sample
2.1.1 cell: rat suprarenal gland pheochromocytoma (PC12) cell is available from Shanghai cell institute of the Chinese Academy of Sciences.
2.1.2 experiment reagent:
2.1.2.1 hydrogen peroxide (hydrogen peroxide, H
2O
2), nitro tetrazole orchid (nitroblue tetrazolium, NBT), luxuriant and rich with fragrance alloxazine (ferrozine) is available from Sigma company;
2.1.2.2 Quercetin (quercetin) provides (purity is 99%) by pharmaceutical college of Zhejiang University Chinese medicine and laboratory, natural drug research department; Silibinin (Silybin) is available from Panjin sky, Liaoning source pharmaceutcal corporation, Ltd, and HPLC detects purity 98%.
2.1.2.3Tris alkali, the DMEM substratum is available from Gibco company;
2.1.2.4 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is available from Amresco company for 3-;
2.1.2.5 calf serum is available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.;
2.1.2.6 penicillin and Streptomycin sulphate are produced by Shijiazhuang Pharmaceutical Group Co Ltd;
2.1.2.7 other reagent is homemade analytical pure, available from Hangzhou Huadong Medicine reagent company limited.
2.1.3 instrument:
2.1.3.1 microplate reader: Synergy-HT type, BIO-TEK company;
2.1.3.2 vertical automatic electric heating pressure steam sterilizer: LDZX-40BI type, Shen, Shanghai peace medical apparatus plant;
2.1.3.3 ultraviolet spectrophotometer: UV-1201 type, Beijing Rayleigh Analytical Instrument Co.,Ltd;
2.1.3.4 ultrapure water system: UPWS-I-60D type, Hangzhou Yongjieda Cleaning Technology Co.,Ltd;
2.1.3.5 sterilizing filter: Sterifil500 type, Millipore company;
2.1.3.6 gas bath constant temperature oscillator: THZ-C, Jintan City, Jiangsu Province Medical Instruments factory;
2.1.3.7CO
2Cell culture incubator: MMM, German company;
2.1.3.8 inverted microscope: XD-2 type, Chongqing Optical ﹠ Electrical Instrument Co., Ltd..
2.2 experimental principle:
Oxydol H
2O
2It is a kind of precursor of main living radical, it can cause the apoptosis of central nervous system cell, PC12 cell (rat suprarenal gland pheochromocyte oncocyte) can be simulated cranial nerve cell, therefore use it always and be used as studying the model that concerns between medicine and the neurocyte, with the survival rate of MTT survey cell, if testing compound has removing by H
2O
2The effect of the free radical that causes, protection cell anti-oxidative damage, then its cell survival rate is just high, and corresponding OD value is also high, on the contrary cell survival rate and OD value are all low.Experiment among the present invention adopted to Tang Xican institute reported method (Xiaoqiu Xiao etc., NeurosciLetter, 1999,275:73-76) improved the back in order to measure the provide protection of compound to the PC12 cell.
2.3 cell cultures: the PC12 cell is with containing 10% calf serum, and the DMEM culture medium culturing of 100U/ ml penicillin and 100U/ milliliter Streptomycin sulphate is in 37 ℃, 5% carbonic acid gas CO
2Hatch the ordinary method cultivation of going down to posterity in the cell culture incubator.
2.4 experimental technique:
2.4.1 Cytotoxic mensuration: at first measured the effect of testing compound to PC12 cell proliferation with the mtt assay of improvement.The PC12 cell digests with the pancreas enzyme-EDTA Digestive system, collecting cell, counting, be diluted to 8000/milliliter density with the DMEM substratum that contains 10% calf serum, be inoculated in then in the 96 porocyte plates, continue in the cell culture incubator to cultivate, after 24 hours, the dimethyl sulfoxide solution to be measured that to newly join respectively joins in each hole with concentration gradient, makes the ultimate density of hole Chinese style (I) compound be respectively 16,8 and 4 mcg/ml.After 72 hours, add the normal saline solution of 10 microlitre MTT (5 mg/ml), continue at 37 ℃ 5%CO
2Cultivated 3 hours in the incubator of damp atmosphere, discard stoste, add 150 microlitre methyl-sulphoxides in every hole, the MTT crystal Jia Za that the vibration dissolving generates, with microplate reader colorimetric under the 570nm wavelength, the inhibitory rate of cell growth calculation formula is as follows:
% inhibitory rate of cell growth=(OD
Solvent control-OD
Sample)/OD
Solvent control* 100%.
2.4.2 formula (I) compound is to H
2O
2Due to the mensuration of provide protection of PC12 cell injury
PC12 cell DMEM culture medium culturing contains 10% bovine serum in the substratum, 100U/ ml penicillin and 100U/ milliliter Streptomycin sulphate.Cell is added in 96 orifice plates with the density in 8000 in every hole, at 37 ℃, and 50%CO
2Cultivated 36 hours in the incubator of damp atmosphere.With the metamorphosis of inverted microscope observation of cell and the survival rate of measuring cell with mtt assay.Cell joins the dimethyl sulfoxide solution of formula (I) compound of newly joining in each hole with concentration gradient after 36 hours hatch.Act on the H that adding is newly joined after 2 hours
2O
2(final concentration is 600 micromoles per liter) effect 3 hours, the microscopic examination record discards original fluid, add new nutrient solution 100 microlitres, add MTT 10 microlitres then, after 3 hours, the careful suction removed nutrient solution, adds 150 microlitre DMSO Rong Xie Jia Za, in 570nm place reading.Adopt Quercetin and the positive control drug of silibinin.
2.4.3 test-results: see Table one.
2.5 conclusion: test-results shows, formula (I) compound has the very potent PC12 cell injury effect that Green Tea Extract is caused, this compound with oxidation resistance damaging action all is better than positive control medicine Quercetin under all high, medium and low concentration situations of test.2 of this 23 interdigit chlorobenzene formacyl replacement is described, 3-dehydro-silibinin ester belongs to the antioxidant of the PC12 cytosis with potent protection simulation cranial nerve cell.
Measure compound to H
2O
2Due to the provide protection of PC12 cell injury can be used as the foundation of the mechanism of action of its protection maincenter cranial nerve cell of preliminary discussion.So show the fact according to this compound, can expect that its treatment to senile dementia has very positive effect to the potent provide protection of PC12 cell due to the hydrogen peroxide damage.
Embodiment 3: formula (I) compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester suppresses the activity test that the lipid peroxide of free yl induction generates
3.1 experimental principle: lipid peroxidation is the product that free radical acts on polyunsaturated fatty acid, and the generation positive correlation of content and free radical because body has the provide protection of oxidation resistant enzyme system and non-enzyme system, constantly produces free radical and constantly is eliminated again.With advancing age, intravital antioxidant constantly descends, and the ability of removing free radical weakens gradually, and lipid peroxidation then strengthens, and lipid peroxidation product increases.Therefore, the working sample lipoid peroxidization resistant is one of important indicator of anti-oxidant, the antiaging agent of screening.This research vitamins C-Fe
2+The system induction rat liver homogenate produces lipid peroxidation product mda MDA, and MDA can react with thiobarbituricacid TBA, and product has maximum absorption at the 532nm place, and the variation of measuring by MDA reflects the restraining effect of compound to the liver homogenate lipid peroxidation indirectly.
3.2 experiment material and sample
3.2.1 experimental animal: SD (sprague-dawley) rat is available from Zhejiang University's Experimental Animal Center; Conformity certification number: SCXK (Zhejiang 2007-0029).
3.2.2 experiment reagent:
3.2.2.1 ferrous sulfate FeSO
4Solution: homemade FeSO
4Analyze pure reagent, available from East China, Hangzhou reagent company, autogamy as requested before the use;
3.2.2.2 vitamin V c solution: Domestic V c analyzes pure reagent, available from East China, Hangzhou reagent company, and autogamy as requested before the use;
3.2.2.3 trichloroacetic acid solution: homemade CCl
3CO
2H analyzes pure reagent, available from Hangzhou Huadong Medicine reagent company limited, and autogamy as requested before the use;
3.2.2.4 potassium primary phosphate, dipotassium hydrogen phosphate, thiobarbituricacid and other reagent are homemade analytical pure (Hangzhou Huadong Medicine reagent company limited);
3.2.2.5 Quercetin (quercetin) provides (purity is 99%) by pharmaceutical college of Zhejiang University Chinese medicine and laboratory, natural drug research department; Silibinin (Silybin) is available from Panjin sky, Liaoning source pharmaceutcal corporation, Ltd, and HPLC detects purity 98%.。
3.3 experiment material and sample
3.3.1 microplate reader: Synergy-HT type, BIO-TEK company;
3.3.2 vertical automatic electric heating pressure steam sterilizer: LDZX-40BI type, Shen, Shanghai peace medical apparatus plant;
3.3.3 ultraviolet spectrophotometer: UV-1201 type, Beijing Rayleigh Analytical Instrument Co.,Ltd;
3.3.4 ultrapure water system: UPWS-I-60D type, Hangzhou Yongjieda Cleaning Technology Co.,Ltd;
3.3.5 constant water bath box: Shanghai gloomy letter test science instrument company;
3.3.6 gas bath constant temperature oscillator: THZ-C, Jintan City, Jiangsu Province Medical Instruments factory;
3.3.7 desk type high speed refrigerated centrifuge: TGL-16G, last Hai'an booth instrument plant.
3.4 experimental technique: the lipid peroxide generation that suppresses free yl induction is to detect by the mouse hepatomicrosome.
3.4.1 the preparation of hepatomicrosome: get 5 sacrificed by decapitation of SD experimental rat, take out liver rapidly, prepare hepatomicrosome with ultracentrifugation.
3.4.2 sample is to the restraining effect of mouse liver lipid peroxidation: containing FeSO
4Add in the 200 mcg/ml microsomes in 1 milliliter of damping fluid of (4 micromoles per liter) and Vc (50 micromoles per liter), to induce the generation lipid peroxidation, the testing compound sample that adds different concns simultaneously, hatch for 37 ℃ and add 1 milliliter of Tricholroacetic Acid (20%) stopped reaction adding, 1.5 milliliters of thiobarbituricacids (0.76%) after 30 minutes, 100 ℃ of boiling water baths were handled 20 minutes, centrifugal removal albumen precipitation is measured the supernatant absorbancy under the 532nm wavelength.Lipid peroxidation represents with the mda that generates in the reaction, experiment with Quercetin and silibinin as the positive control medicine.As the blank group, what add same volume DMSO is the solvent control group with damping fluid surrogate response liquid.Sample is as follows to the calculation formula of rat liver microsomal lipid peroxidation inhibiting rate:
Inhibiting rate (%)=(OD
Sample hose-OD
Blank pipe)/(OD
Standard pipe-OD
Blank pipe) * 100%.
3.5 test-results: test-results sees Table two.
Table two. formula (I) compound is to the restraining effect of rat liver microsomal lipid peroxidation
Test-results shows, compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester has the lipid peroxide generative capacity of stronger inhibition free yl induction, its half-inhibition concentration IC
50Be 10.46 mcg/ml.
3.6 conclusion: formula (I) compound belongs to and has more potent protection cell antioxidant; though its anti peroxidation of lipid ability is inferior to positive control medicine Quercetin; but far exceed 3 times more than of reference substance silibinins; point out 2 of this 23 interdigit chlorobenzene formacyl replacement; 3-dehydro-silibinin ester has certain liver protecting effect and can expect the effect that is used to prepare prevention or treatment acute and chronic liver injury class disease; be worth further Application and Development, or unite use with other related drugs.
Embodiment 4: formula (I) compound m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester is to the restraining effect test of XOD XO
4.1 experiment material and sample
4.1.1 experimental animal: SD (sprague-dawley) rat is buied in Zhejiang University's Experimental Animal Center; Conformity certification number: SCXK (Zhejiang 2007-0029).
4.1.2 experiment reagent:
4.1.2.1 phenazine methosulfate (phenazine methosulfate, PMS), nitro tetrazole orchid (nitrobluetetrazolium, NBT), xanthine, luxuriant and rich with fragrance alloxazine (ferrozine) be available from Sigma company;
4.1.2.2Triton X-100 is available from Gibco company;
4.1.2.3 positive control medicine: allopurinol, Taizhou Mei Tong pharmaceutcal corporation, Ltd;
4.1.2.4 potassium primary phosphate, dipotassium hydrogen phosphate and other reagent are homemade analytical pure (East China, Hangzhou reagent company).
4.1.3 instrument:
4.1.3.1 microplate reader: Synergy-HT type, BIO-TEK company;
4.1.3.2 vertical automatic electric heating pressure steam sterilizer: LDZX-40BI type, Shen, Shanghai peace medical apparatus plant;
4.1.3.3 ultraviolet spectrophotometer: UV-1201 type, Beijing Rayleigh Analytical Instrument Co.,Ltd;
4.1.3.4 ultrapure water system: UPWS-I-60D type, Hangzhou Yongjieda Cleaning Technology Co.,Ltd;
4.1.3.5 sterilizing filter: Sterifil500 type, Millipore company;
4.1.3.6 gas bath constant temperature oscillator: THZ-C, Jintan City, Jiangsu Province Medical Instruments factory;
4.1.3.7 glass homogenizer: homemade.
4.2 experimental technique:
4.2.1 get the SD rat, sacrificed by decapitation, take out rapidly liver to the phosphoric acid buffer of precooling (100 mmoles/liter, pH 8.75) in, remove blood vessel, shred the back and dilute the back in directly homogenate on ice with phosphoric acid buffer 1: 1 (w/v), in-20 ℃ of preservations, with phosphoric acid buffer (1: 7) dilution, centrifugal under 4 ℃ of conditions (8000rpm/ minute) gets supernatant as proenzyme with preceding.
4.2.2 formula (I) compound is measured the restraining effect of XO and is adopted enzyme linked immunological ELISA method: add substrate xanthine (0.1 mg/ml in the sample well, 600 microlitres), enzyme (30 microlitre), formula (I) compound dissolves with methyl-sulphoxide, dilute with phosphoric acid buffer, every hole 30 microlitres, making its final concentration is 40 mcg/ml, 20 mcg/ml and 10 mcg/ml, add blue tetrazole of nitro and phenazine-methionine ester (30 microlitre), add Triton X-100 (0.4% at last, 10 microlitres), water bath heat preservation is 2 hours in 37 ℃, colorimetric estimation under the 550nm wavelength.Formula (I) compound is calculated for blank and contrast OD value by sample OD value the XOD inhibiting rate.XOD inhibiting rate (%)=[(OD
Mark Accurate pipe-OD
Blank pipe)-(OD
Sample hose-OD
The sample blank pipe)]/(OD
Standard pipe-OD
Blank pipe) * 100%.
4.3 experimental result:
Formula (I) compound is 35.21% to the XOD inhibiting rate when 40 mcg/ml, and inhibiting rate is 89.2% during positive control allopurinol 40 mcg/ml, half-inhibition concentration IC
50=3.34 * 10
-5Mol.
4.4 conclusion: formula (I) compound has certain inhibition activity to XOD, illustrates that this compound has to further develop the potential that becomes the xanthine of determined curative effect enzyme inhibitors.
Claims (5)
1. have that 23 interdigit chlorobenzene formacyls of structure shown in the formula (I) replace 2,3-dehydro-silibinin or its pharmacologically acceptable salt; The name of formula (I) compound is called: m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(3,5,7-trihydroxy--4-oxo-chromene-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester;
Formula (I).
2. method for preparing claim 1 formula shown (I) compound is characterized in that: silibinin dehydration back form 2, the 3-dehydro-silibinin carries out dehydration condensation with m-chlorobenzoic acid and gets in the presence of condensation catalyst and phosphonium salt.
3. according to the method for claim 2, it is characterized in that described condensation catalyst is meant diethyl azodiformate, phosphonium salt is meant triphenylphosphine.
According to 23 interdigit chlorobenzene formacyls shown in the formula (I) of claim 1 replace 2, the application in preparation xanthine oxidase inhibitor medicine of 3-dehydro-silibinin or its pharmacologically acceptable salt.
5. application according to claim 4 is characterized in that 2 of the 23 interdigit chlorobenzene formacyls replacement shown in the formula (I) according to claim 1, the application in preparation treatment goat medicine of 3-dehydro-silibinin or its pharmacologically acceptable salt.
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| CN1990484A (en) * | 2005-12-26 | 2007-07-04 | 浙江海正天华新药研发有限公司 | Silybin esters derivatives and preparation and use thereof |
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| GB2261434A (en) * | 1991-11-15 | 1993-05-19 | Inverni Della Beffa Spa | New radical scavenging and antipoperoxidant flavanolignans their preparation and formulations for therapeutic use |
| CN1990484A (en) * | 2005-12-26 | 2007-07-04 | 浙江海正天华新药研发有限公司 | Silybin esters derivatives and preparation and use thereof |
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