CN101879158A青蒿素衍生物的新应用---防治肥胖病的|New application of artemisinin derivant
By Roger,
Artemisinin Patent|青蒿素专利
Description The new application of artemisinin derivative Technical field The present invention relates to the new application of artemisinin derivative in pharmacy, belong to chemical biology and cytobiology field. Background technology Arteannuin is the sesquiterpene lactones medicine that contains peroxy-radical that extracts from Herba Artemisiae annuae.The discovery of artemisinin-based antimalarial drug thing is the another milestone after quinine on the global antimalarial agent development history, it is first phytochemistry medicine that China finds, also be China unique that approved by World Health Organization (WHO), can be by the Chinese medicine of synthetic drug research standard exploitation.Over more than 30 year, arteannuin distinctive molecular structure and outstanding biological activity are attracting the eyeball of scientists deeply.Along with the development of medical sci-tech, prove that through pharmacological action and clinical practice artemisinin derivative is widely used in treating malaria, cancer is in the diseases such as actute infection and hyperpyrexia disease and dermatosis.Wherein the more artemisinin derivative of clinical research has dihydroartemisinine, Artemether, arteether and artesunate etc.Have not yet to see the bibliographical information that relevant artemisinin derivative suppresses the adipose cell differentiation. Obesity is meant that body feed heat is more than the health consumption, cause the transition accumulation of fat and the excessive amplification of fatty tissue, make body weight more than 20% of body weight that is above standard, and the class disease weak with giddy, that Mental fatigue is breathed hard, be the risk factor of hypertension, diabetes, hyperlipidemia, coronary heart disease, cerebrovascular.Now, fat one of fast, the most serious public health problem of global rate of propagation that become.In recent years, along with the raising of China's living standards of the people, fat number has increased in urban population, and do not occur over just the old people, also occur in child, women, the number of each age group body obesity is all increasing, and fat-reducing has become people's much-talked-about topic.The cause of disease that causes obesity has a lot, and wherein main and h and E factor has substantial connection.Environmental factors is meant eats high fat, high heat food for a long time, adds physical exertion and reduces, and under the effect of factors such as mental maladjustment, causes that the neuroendocrine of body fat regulated and control network is regulated disorderly. Therefore, the generation of prevention and controlling obesity disease has become the problem of medically very paying close attention to.At present, slimming medicine commonly used roughly is divided three classes: one, the medicine of control appetite, and this type of medicine has many untoward reaction, as insomnia, xerostomia, constipation, cardiopalmus and hypertension; Two, increase the medicine of energy expenditure; Three, the medicine that suppresses intestinal absorption.Although there are many researchs to be deep into the genomic medicine level now, but still do not find at present a kind of real effectively, slimming medicine that can life-time service, work out diagnosis and the treatment that even more ideal effective method and preparation be used for obesity and still have profound significance. 3T3-L1 be one derived from 3T3 cell (the fibroblastic cell line of standard that builds on 1962), in biological study, be applied to studying the cell line of fatty tissue.The 3T3-L1 cell has similar fibroblastic form, but under the inducing of felicity condition, can break up and form the phenotype of adipose cell.Adipose cell form after the 3T3-L1 cell differentiation maturation, the synthetic and accumulation of the intracellular glycerol three esters fibroblast state preceding with respect to differentiation all improves greatly, and 3T3-L1 cell at this time presents a kind of adipose cell form of signet ring.These cells are all responsive to hormone and the medicine of regulating lipogenesis and fat acid decomposition, epinephrine for example, norepinephrine, insulin etc. Because these characteristics of 3T3-L1 cell, this cell line are widely used in the research of fat differentiation and metabolic disease mechanism, be the cell line model of internationally recognized external fat differentiation.The cascade signal of transcribing that this Study of model has been helped to illustrate a main regulation and control fat differentiation in the terminal differentiation of adipose cell is transduceed, and has comprised a series of transcription factor, modulin and effect protein. The oil red O stain technology is a kind of lipid staining commonly used, and its advantage is that staining procedure is easy, and coloration result is sure.Dyeing back fat is cerise, and it is blue that nucleus is, and a matter is colourless.The redness that oil red O dyes is directly proportional with the amount of the oil droplet that the interior accumulation of adipose cell forms, and the latter is directly proportional with the height of adipose cell differentiation degree, so by this technology, both can judge by qualitative observation, can also detection by quantitative the height of adipose cell differentiation degree relatively. Herba Artemisiae annuae extensively is present in all over the world, but the higher Herba Artemisiae annuae of artemislnin content exists only in east, China Chongqing, Fujian, Guangxi, some areas, Hainan, belongs to the exclusive drug resource of China.Nearly decades, the research of artemisinin derivative mainly concentrates on malaria and anti-tumor aspect, and the research that applies it to appetrol is but very rare. Summary of the invention The objective of the invention is promptly provides a kind of new application of artemisin derivant in order to improve the application of artemisinin derivative in frontier. Concrete technical scheme is as follows: The application of artemisinin derivative in the fat medicine of preparation control with following formula I, II, III, IV, V structure, Wherein: N=0~1; R 1Be selected from C 1~C 3Alkyl; R 2Be selected from the aromatic heterocycle that aryl or nitrogen atom replace. Preferably, described artemisinin derivative is to have in the following structural formula any Wherein, dihydroartemisinine, arteannuin benzoate have the effect of inhibition 3T3-L1 cell differentiation the strongest in all artemisinin derivatives that detected.Next is an artesunate, has the effect of the inhibition 3T3-L1 cell differentiation that is only second to the above two.These 3 chemical compounds can both suppress the differentiation of 3T3-L1 cell fully in the level of 10 μ M. The objective of the invention is to develop the effect of artemisinin derivative in slimming medicine, it is suppressed the cellular fat differentiation study in great detail. The present invention has the following advantages: (1) arteannuin and derivant thereof are traditional anti-malaria medicaments, be used for the treatment of malaria all the time, characteristics of the present invention are that the part derivant that proposes arteannuin first has the effect that suppresses the adipose cell differentiation, disease with them and metabolism class connects first, and proposes their first as the potential probability that is applied to the medicine of obesity and obesity-related disease such as cardiovascular disease.The metabolism class disease relevant with obesity is the significant threat of human health in current society, by the medicine of the biological medicine for preparing of described artemisine as the differentiation of lipotropism fat, will play a significant role in these treatment of diseases. (2) the present invention has compared the power that suppresses the adipose cell differentiation capability between the different artemisinin derivatives, has drawn preliminary structure activity relationship, provides foundation for further optimizing the active better artemisinin derivative of acquisition. Description of drawings Fig. 1. be the inhibitory action of arteannuin series derivates to the adipose cell differentiation, abscissa is a drug use dosage (concentration, represent with μ M), vertical coordinate is relative differentiation rate (dosing handle experimental group oil red dyeing OD value/break up fully matched group oil red dye OD value); Fig. 2. be the oil red dyeing general effect of 5 the most representative chemical compounds of the inhibition 3T3-L1 cell differentiation effect of choosing; Wherein, Fig. 2 .1 is the oil red dyeing general effect that chemical compound 2 Concentraton gradient influence the 3T3-L1 cell differentiation; Fig. 2 .2 is the oil red dyeing general effect that chemical compound 4 Concentraton gradient influence the 3T3-L1 cell differentiation; Fig. 2 .3 is the oil red dyeing general effect that chemical compound 3 Concentraton gradient influence the 3T3-L1 cell differentiation; Fig. 2 .4 is the oil red dyeing general effect that chemical compound 5 Concentraton gradient influence the 3T3-L1 cell differentiation; Fig. 2 .5 is the oil red dyeing general effect that chemical compound 9 Concentraton gradient influence the 3T3-L1 cell differentiation; Fig. 3 is the flow process stage sketch map of inducing the 3T3-L1 cell differentiation. The specific embodiment Artemisinin derivative with following formula I, II, III, IV, V structure of the present invention is mainly the chemical compound with following structural formula: (a) described chemical compound with formula I structure is: (b) described chemical compound with formula II structure is: (c) described chemical compound with formula III structure is: (d) described chemical compound with formula IV structure is: (e) described chemical compound with formula V structure is: For making the present invention easier to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are used for illustration purpose, and be not used in the restriction scope of the invention. Those skilled in the art can obtain described artemisinin derivative with described formula I, II, III, IV, V structure from following description or prior art. Embodiment 1: deoxidation arteannuin synthetic 200mg (0.7087mmol) arteannuin is dissolved in 10mL THF, behind the material dissolution, adds 547mg (20.2688mmol) Al powder and 7.23g (30.4032mmol) NiCl 26H 2O, afterreaction finished in 4 hours, was spin-dried for solvent, was spin-dried for thing with acetic acid ethyl dissolution, and sucking filtration is with ethyl acetate drip washing filtering residue.Filtrate is through anhydrous Na 2SO 4Drying concentrates back column chromatography (PE: EA=16: 1) obtain the 72mg target product, yield 38%. 1H?NMR(CDCl 3,400MHz):δ=5.68(s,1H),3.19-3.16(m,1H),2.02-1.58(m,6H),1.51(s,3H),1.28-1.03(m,11H). Embodiment 2: deoxidation dihydroartemisinine synthetic 500mg (1.7596mmol) dihydroartemisinine is dissolved in 5mL DCM, adds 45mg 10%Pd/C, under the hydrogen shield; 40 ℃ of reaction overnight finish, and remove by filter Pd/C, are spin-dried for solvent; concentrate back column chromatography (PE: EA=8: 1) obtain the 202mg target product, yield 43%. 1H?NMR(CDCl 3,400MHz):δ=5.34(s,1H),4.78(t,J=6.8Hz,1H),2.83(d,J=7.2Hz,1H),2.40-0.79(m,21H). Embodiment 3: dihydroartemisinine synthetic 10g (35.4321mmol) arteannuin is dissolved in 500mL methanol, stirs and is cooled to 0-5 ℃, adds 10g (265.7408mmol) NaBH in 1 hour in batches 4Keep this temperature to continue to stir after 1.5 hours, in reaction bulb, slowly drip 15.5mLHAc adjusting pH=7, separate out a large amount of white solids gradually.Be spin-dried for most of solvent, add 10mL cold water, stirred 15 minutes under the room temperature, sucking filtration is used cold H 2O: MeOH=2: 1 washing leaching cake, collect filter cake, obtain the 8.0g target product after the drying, yield 80%. 1H?NMR(CDCl 3,400MHz):δ=5.60(s,1H),5.28(s,1H),2.85(s,1H),2.61(s,1H),2.41-0.89(m,20H). Embodiment 4: β-Artemether synthetic 150mg (0.5275mmol) dihydroartemisinine is dissolved in 1.0mL MeOH and the 2mL DCM mixed solvent, is heated to 45 ℃, under the argon shield, injects 0.01mL (0.0559mmol) BF rapidly 3Et 2O, reaction is 1 hour under this temperature, and after reaction finished, with sodium acetate aqueous solution washing reaction liquid, organic layer was through anhydrous MgSO 4After the drying, concentrate the mixture that promptly obtains β-Artemether and α-Artemether, mixture is dissolved in an amount of normal hexane, preserved 36 hours, collect white crystal, and, obtain the pure β-Artemether of 107mg after the drying, yield 65% with cold normal hexane drip washing at-20 ℃. 1H?NMR(CDCl 3,400MHz):δ=5.38(s,1H),4.68(d,J=3.2Hz,1H),3.42(s,3H),2.64-0.85(m,21H). Embodiment 5: arteannuin benzoate synthetic Under the argon shield; 3.386g (11.9158mmol) dihydroartemisinine is dissolved in 40mL DCM, adds 6.1mL (76.2613mmol) pyridine, stirs and is cooled to 0 ℃; add 2.2mL (19.0653mmol) Benzenecarbonyl chloride.; keep reacting 15 minutes under this temperature, remove ice bath and rise to room temperature, continue reaction and finished in 16 hours; add 7% aqueous citric acid solution cessation reaction; be spin-dried for DCM, add acetic acid ethyl dissolution and be spin-dried for thing, use 7% aqueous citric acid solution, saturated NaHCO successively 3Solution, saturated NaCl solution washing organic layer, organic layer is through anhydrous Na 2SO 4Drying concentrates back column chromatography (PE: EA=16: 1) obtain the 4.07g target product, yield 88%. 1H?NMR(CDCl 3,400MHz):δ=8.12(d,J=7.2Hz,2H),7.59-7.55(m,1H),7.44(t,J=8.0Hz,2H),6.01(d,J=9.6Hz,1H),5.53(s,1H),2.76-0.92(m,21H). Embodiment 6: β-pi-allyl arteannuin synthetic Add a small amount of 4 in the reaction bulb The anhydrous ZnCl of MS, 105mg (0.7790mmol) 2, the Ar protection is injected anhydrous DCM of 3mL and 0.5mL (3.1160mmol) allyl trimethyl silane down, stirs and is cooled to 0 ℃. Claim 252mg (0.6492mmol) arteannuin benzoate to be dissolved in the anhydrous dichloroethanes of 3mL, solution is splashed in the above-mentioned reactant liquor, keep 0 ℃ of reaction 1 hour, remove ice bath, rose to room temperature reaction 3 hours, add an amount of ethyl acetate diluting reaction system, use 7% aqueous citric acid solution, saturated NaHCO successively 3Solution, saturated NaCl solution washing organic layer, organic layer is through anhydrous Na 2SO 4Drying concentrates back column chromatography (PE: EA=30: 1), obtain the 114mg target product, yield 66%. 1H?NMR(CDCl 3,400MHz):δ=5.92(m,1H),5.32(s,1H),5.14-5.04(m,2H),4.30(m,1H),2.69-0.87(m,23H). Embodiment 7: β-acetic acid arteannuin synthetic 100mg (0.3245mmol) β-pi-allyl arteannuin is dissolved in 4mL CCl 4-CH 3CN-H 2In O (V: V: V=1: 1: the 2) mixed solvent, add 347mg (1.6225mmol) NaIO successively 4And 2mg (0.0097mmol) RuCl 3, the ambient temperature overnight reaction.Be spin-dried for solvent, add acetic acid ethyl dissolution and be spin-dried for thing, use saturated NaHSO successively 3, saturated NaHCO 3Solution, saturated NaCl solution washing organic layer, organic layer is through anhydrous Na 2SO 4Drying concentrates back column chromatography (DCM: MeOH=30: 1), obtain the 92mg target product, yield 87%. 1H?NMR(CDCl 3,400MHz):δ=5.37(s,1H),4.86(m,1H),2.71-0.88(m,24H). Embodiment 8:D-Biotin modifies the synthetic of arteannuin Reagents?and?conditions:(a)Boc 2O,Et3N,THF,R.T.,15h;(b)D-biotin,EDCI,HOBt,(i-Pr) 2NEt,DMF,R.T.,10h;(c)TFA/CH 2Cl 2,0℃~R.T.,9h;(d)EDCI,HOBt,(i-Pr) 2NEt,DMF,R.T.,11h. 2 synthesize: 2.035g (13.7360mmol) be dissolved among the 18mL THF, add people 0.63mL (4.5767mmol) NEt 3, stir and be cooled to 0 ℃. 1g (4.5767mmol) Boc 2O is dissolved among the 9mL THF, behind the mix homogeneously, dropwise splashes in the above-mentioned reactant liquor, after dropwising, rises to room temperature reaction naturally 15 hours.After reaction finishes, be spin-dried for solvent, behind the thin up, with DCM extraction, organic layer is with saturated NaCl solution washing, anhydrous Na 2SO 4Drying concentrates back column chromatography (DCM: MeOH=5: 1), obtain the 906mg target product, yield 80%. 1H?NMR(CDCl 3,400MHz):δ=5.18(s,1H),3.56(s,4H),3.50-3.45(m,4H),3.30-3.25(m,2H),2.82(t,J=5.6Hz,2H),1.53(s,2H),1.38(s,9H). 3 synthesize: Under the argon shield, (1.3mmol, 1.3equiv.) (1.3mmol, 1.3equiv.) HOBt is dissolved in the dry DMF of 4mL, adds 0.23mL (1.4mmol, 1.4equiv.) DIPEA, stirring at room for EDCI, 176mg for 318mg (1.3mmol) D-Biotin, 249mg. 248mg (1.0mmol) 2 is dissolved among the dry DMF of 3mL, solution is injected above-mentioned reaction bulb, room temperature reaction 10 hours.After reaction finishes, in reaction bulb, add NH 4The Cl saturated solution, chloroform extraction, organic layer is through NH 4Cl saturated solution, water, saturated NaCl solution washing, anhydrous Na 2SO 4Drying concentrates back column chromatography (DCM: MeOH=20: 1), obtain the 358mg target product, yield 76%. 1H?NMR(CDCl 3,400MHz):δ=6.67(s,1H),6.15(s,1H),5.52(s,1H),5.15(s,1H),4.45-4.40(m,1H),4.25-4.20(m,1H),3.52(s,4H),3.49-3.45(m,4H),3.37-3.34(m,2H),3.23-3.21(m,2H),3.10-3.04(m,1H),2.14(t,J=7.6Hz,2H),1.70-1.50(4H),1.36(s,12H). 4 synthesize: 118mg 3 is dissolved in the 3mL dichloromethane, stirring is cooled to 0 ℃, slowly drip 1.0mL TFA, add the recession deicing and bathe, rose to room temperature reaction naturally 9 hours, after reaction finishes, directly distilling under reduced pressure removes and desolvates, vacuum drying obtains crude product (by 100% calculating, should obtain the 93mg crude product) 4, is directly used in next step reaction without separation. 1H?NMR(MeOD,400MHz):δ=4.52-4.49(m,1H),4.32-4.29(m,1H),3.72-3.65(m,6H),3.56(t,J=5.6Hz,2H),3.39-3.35(m,2H),3.22-3.20(m,1H),3.14-3.11(m,2H),2.95-2.91(dd,J=12.8Hz,4.8Hz,1H),2.71(d,J=12.8Hz,1H),2.22(t,J=7.6Hz,2H),1.71-1.60(m,4H),1.47-1.43(m,2H). D-Biotin modifies the synthetic of arteannuin: Under the argon shield, 74mg (0.2269mmol) carboxylic acid arteannuin, 57mg (0.2949mmol) EDCI, 40mg (0.2949mmol) HOBt are dissolved in the 3mL dry DMF, inject 0.24mL (1.4293mmol) DIPEA, stirring at room. Claim 93mg (0.2496mmol) 4 to be dissolved in the dry DMF of 2mL, solution is injected above-mentioned reactant liquor, the ambient temperature overnight reaction.After reaction finishes, add the DCM dilute reaction solution, use NH successively 4Cl saturated solution, water, saturated NaCl solution washing, anhydrous Na 2SO 4Drying concentrates back column chromatography (DCM: MeOH=10: 1), obtain the 89mg target product, yield 57%. 1H?NMR(CDCl 3,400MHz):δ=7.24(s,1H),δ6.85(s,1H),δ6.30(s,1H),δ5.41(s,1H),δ5.35(s,1H),δ4.75-4.71(m,1H),δ4.52-4.50(m,1H),δ4.32-4.29(m,1H),δ3.61-3.40(m,12H),δ3.15-3.16(m,1H),δ2.90-0.88(m,33H). Embodiment 9: Claim 50mg (0.1288mmol) arteannuin benzoate and an amount of 4 MS under the argon shield, injects 2mL and heavily steams the DCM dissolving in reaction bulb, is cooled to 0 ℃, injects 0.02mL (0.1546mmol) TMSCl, keeps this thermotonus to rise to the ambient temperature overnight reaction after 1 hour and finishes.Be spin-dried for solvent, add acetic acid ethyl dissolution and be spin-dried for thing, use saturated NaHCO successively 3Solution, saturated NaCl solution washing, organic layer is through anhydrous Na 2SO 4Drying concentrates back column chromatography (PE: EA=8: 1), obtain the 33mg target product, yield 96%. 1H?NMR(CDCl 3,400MHz):δ=6.19(s,1H),5.54(s,1H),2.44-0.99(m,20H). Embodiment 10: Under the argon shield, 50mg (0.1865mmol) deoxidation dihydroartemisinine is dissolved among the 3mL DCM, is cooled to 0 ℃, injects 0.03mL (0.3729mmol) chloroacetic chloride, rises to the ambient temperature overnight reaction gradually and finishes.Reactant liquor is through water washing, and organic layer is through anhydrous Na 2SO 4Drying, evaporating column chromatography (PE: EA=16: 1), obtain racemization deoxidation dihydroartemisinine 30mg, yield 64% as by-product. 1H?NMR(CDCl 3,400MHz):δ=6.04(s,1H),5.47(s,1H),2.04-1.21(m,20H). Embodiment 11: 100mg (0.3517mmol) dihydroartemisinine is dissolved in 5mL DCE, adding people 73mg (0.4220mmol) 2-quinoxaline carboxylic acid stirs moments later, add 5mL (0.0422mmol) DMAP, stirring is cooled to 0 ℃, adds 80mg (0.3868mmol) DCC, stirs and rises to room temperature after 5 minutes, stopped reaction after 16 hours, filtering reacting liquid, filtrate is through water, saturated NaCl solution washing, and organic layer is through anhydrous Na 2SO 4After the drying, the evaporating column chromatography obtains the 120mg target product, yield 78%. 1H?NMR(CDCl 3,400MHz):δ=9.57(s,1H),8.31(d,J=8.0Hz,1H),8.17(d,J=8.0Hz,1H),7.91-7.86(m,2H),6.03-6.00(m,2H),5.64(s,1H),2.90-0.95(m,21H). Embodiment 12: According to as described in example 12,100mg (0.3517mmol) dihydroartemisinine and the reaction of 63mg (0.4220mmol) terephthalaldehydic acid, column chromatography obtains the 116mg target product, yield 79%. 1H?NMR(CDCl 3,400MHz):δ=10.10(s,1H),8.27(d,J=8.0Hz,2H),7.96(d,J=8.0Hz,2H),6.03-6.00(m,1H),5.54(s,1H),2.90-0.95(m,21H). Embodiment 13: According to as described in example 12,100mg (0.3517mmol) dihydroartemisinine and 133mg (0.4220mmol) Boc-3-(2-naphthyl)-L-alanine reaction, column chromatography obtains the 151mg target product, yield 76%. 1H?NMR(CDCl 3,400MHz):δ=7.86-7.83(m,1H),7.80-7.74(m,3H),7.47-7.38(m,1H),6.05-6.00(m,1H),5.54(s,1H),2.83-0.85(m,34H). Embodiment 14: According to as described in example 12,100mg (0.3517mmol) dihydroartemisinine and the reaction of 76mg (0.4220mmol) paranitrophenylacetic acid, column chromatography obtains the 123mg target product, yield 78%. 1H?NMR(CDCl 3,400MHz):δ=8.18(d,J=8.0Hz,2H),7.48(d,J=12.0Hz,2H),6.03-6.00(m,1H),5.44(s,1H),3.82(s,2H),3.82(s,2H),2.65-0.68(m,23H). Embodiment 15: Under the argon shield; 100mg (0.3517mmol) dihydroartemisinine and 0.08mL (0.5627mmol) anisoyl chloride are dissolved among the 5mL DCM; be cooled to 0 ℃; inject 0.2mL (2.2509mmol) pyridine; stir after 15 minutes, rise to the ambient temperature overnight reaction and finish, react with 7% aqueous citric acid solution cancellation; ethyl acetate extraction is again through saturated NaHCO 3, saturated NaCl solution washing, organic layer is through anhydrous Na 2SO 4Drying concentrates the back column chromatography, obtains the 119mg target product, yield 81%. 1H?NMR(CDCl 3,400MHz):δ=8.06(d,J=12Hz,2H),6.91(d,J=12Hz,2H),5.98(m,1H),5.51(s,1H),3.864(s,3H),2.81-0.85(m,21H). Embodiment 16: Described according to example 15,100mg (0.3517mmol) dihydroartemisinine and the reaction of 104mg (0.5627mmol) paranitrobenzoyl chloride, column chromatography obtains the 81mg target product, yield 53%. 1H?NMR(CDCl 3,400MHz):δ=8.28(s,4H),6.05-6.00(m,1H),5.55(s,1H),2.83-0.85(m,22H). Embodiment 17: artesunate synthetic Under the room temperature condition, 250mg (0.9mmol) dihydroartemisinine is dissolved among the dry DCM of 25mL, adds 202mg (2mmol) succinic anhydride, be cooled to 0~5 ℃, add 107mg (0.9mmol) DMAP, kept this thermotonus 0.5 hour, rose to room temperature reaction naturally 1 hour.After reaction finishes, add 5mL water, transfer to pH=3 with 10% dilute hydrochloric acid, organic layer is used anhydrous MgSO after washing 4Drying, removal of solvent under reduced pressure obtains the 317mg target product, yield 94%. 1H?NMR(CDCl 3,400MHz):δ=5.74(d,J=8.0Hz,1H),5.39(s,1H),2.72-2.59(m,4H),2.53-2.48(m,1H),2.36-2.28.(m,1H),2.01-1.96(m,1H),1.87-1.82(m,1H),1.76-1.66(m,2H),1.60-1.55(m,1H),1.45-1.21(m,8H),1.01-0.95(m,1H),0.92(d,J=8.0Hz,3H),0.80(d,J=4.0Hz,3H). Embodiment 18: artemisinin derivative is to the influence of 3T3-L1 cell differentiation The cultivation of 3T3-L1 cell The 3T3-L1 cell is at 37 ℃, 5%CO 2Adherent growth in the culture environment is used the DMEM high glucose medium (HyClone) that adds 10%FBS (Hyclone hyclone).Can be according to cell growth condition, changed a not good liquor in 2~3 days.Continue if desired repeatedly to go down to posterity, and need still have good differentiation capability by cell after repeatedly go down to posterity, too full (<70%) that will avoid so making cell long generally longly will go down to posterity the branch cell to 60%~70%.Cell can be with go down to posterity branch dish of rare ratio by 1: 15, but usually with 1: 10 or the higher ratio branch dish that goes down to posterity, need be as the criterion with experiment. The culture medium preparation of 3T3-L1 cell induction differentiation Inducing culture is divided into two kinds, and MDI inducing culture and insulin culture medium are respectively applied for different differentiation phases, is to add that by the DMEM culture medium FBS, dexamethasone, insulin and the IBMX of appropriate amount are made into. IBMX solution: IBMX is dissolved in the KOH solution of 0.5N, the IBMX final concentration is 0.0115g/ml, 0.22 μ m syringe filters filtration sterilization ,-20 ℃ of preservations. The insulin storage solutions: insulin is dissolved among the HCl of 0.02M to final concentration 167uM (1mg/ml), 0.22 μ m syringe filters filtration sterilization ,-20 ℃ of long preservation, 4 ℃ of short-terms are preserved. Dexamethasone storage solutions: stored frozen liquid: the 10mM dexamethasone is dissolved in dehydrated alcohol ,-20 ℃ of preservations; The work storage liquid: stored frozen liquid is diluted to 1mM with PBS, and filtration sterilization is stored in 4 ℃. MDI inducing culture (now with the current, 10ml/10cm culture dish, 5ml/6cm culture dish): the DMEM high glucose medium that contains 10%FBS adds again: 1: 100 IBMX solution, 1: 1000 insulin storage liquid, 1: 1000 dexamethasone work storage liquid. Insulin culture medium (now with the current, 10ml/10cm culture dish, 5ml/6cm culture dish): the DMEM high glucose medium that contains 10%FBS adds again: 1: 1000 insulin storage liquid. Induce undifferentiated 3T3-L1 cell (fibroblast state) to break up to mature fat cell: As shown in Figure 3: In containing the DMEM high glucose medium of 10%FBS with 3T3-L1 before adipose cell be cultured to engagement state. 2. support 2 days (being differentiation the 0th day this moment) after engaging again with MDI inducing culture irritation cell (change original complete medium, add the MDI inducing culture of new preparation).Can notice that in following 2 days cellular morphology takes place by significant change (spindle more becomes). 3. add MDI and induce (breaking up the 2nd day) after 2 days, change original MDI inducing culture, add the insulin culture medium of new preparation.Culture medium can begin the thickness more that becomes, because the synthetic free fatty of cell is by a large amount of being secreted in the culture medium. 4. (broke up the 4th day) after two days, change culture medium into common complete medium (the DMEM high glucose medium that contains 10%FBS), changed a not good liquor (the DMEM high glucose medium that contains 10%FBS) in per afterwards 2 days.Usually reach in the 8th day in differentiation and break up state completely. Medicine numbering 1 to 9 structural formula of compound of representative respectively is followed successively by: The analysis of oil red O stain data and processing The preparation of oil red O stain agents useful for same: Oil red O storage liquid: oil red O (AMRESCO, FW 408.51, Em (513nm)) 0.7g is dissolved in the 200ml isopropyl alcohol, spends the night and puts upside down the vibration mixing, is stored in 4 ℃ behind the 0.2 μ m membrane filtration. Oil red O working solution: 6 parts of oil red O storage liquid add 4 parts of dH 2O, room temperature left standstill 20 minutes behind the mixing, 0.2 μ m membrane filtration. Oil red O stain: 1. inhale and go most culture medium. 2. add 10% formalin (being dissolved among the PBS), incubated at room 5 minutes. 3. inhale and remove 10% formalin, add the fresh formalin (undiluted 37% formalin) of same volume, hatched at least 1 hour, in addition more of a specified duration.Annotate: cell can keep several days in formalin, and then dyeing.With around the culture plate with sealing film phonograph seal preventing formaldehyde volatilization evaporate to dryness, and wrap up with lucifuge with aluminium-foil paper. 4. inhale with the lancet head and remove all formalin. 5. (be dissolved in dH with 60% isopropyl alcohol 2Among the O) the cleaning culture hole. 6. allow cell culture hole finish-drying. 7. add oil red O working solution, dye 10 minutes (adding the fashionable hole wall that keeps off). 8. remove all oil red dye liquors, add dH immediately 2O washes with water 4 times (can directly wash under water tap). 9. take pictures according to the experiment needs. 10. remove all water, allow the culture hole bone dry. 11. add 100% isopropyl alcohol eluting oil red O, hatch 10 minutes (can be more of a specified duration). 12. repeatedly pressure-vaccum contain oil red isopropyl alcohol several times, so guarantee that oil red all dissolves in the solution. 13. will contain in the hole that the isopropyl alcohol of oil red is transferred to EP pipe or elisa plate. 14. measure the OD value at 500nm wavelength place, the reading duration is 0.5 second. 15. blank uses 100% isopropyl alcohol, dyeing background contrast is used from according to the painted isopropyl alcohol of the empty culture hole of foster cell of above-mentioned steps with not ing. The consumption of all ingredients in table 1. oil red O stain Tissue Culture Plate Formalin 60% isopropyl alcohol Oil red O 100% isopropyl alcohol 24 orifice plates ??500μl ??500μl ??200μl ??750μl 12 orifice plates ??1ml ??1ml ??400μl ??1.5ml 6 orifice plates ??2.4ml ??2.4ml ??1ml ??3.6ml Artemisinin derivative is to the influence of the differentiation of 3T3-L1 cell Solid arteannuin (Fig. 1 .1,3-7 chemical compound) is dissolved among the DMSO, is mixed with the solution of a series of gradient concentrations, this Concentraton gradient comprises 5 level: 1mM, 5mM, 10mM, 30mM, 50mM in this experiment.Dihydroartemisinine (chemical compound 2) Concentraton gradient is: 0.1mM, 0.5mM, 1mM, 5mM, 10mM, 30mM, 50mM; The Concentraton gradient of artesunate (chemical compound 9) is: 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM, 10mM, 30mM, 50mM. As shown in Figure 3, according to above-mentioned described operating process of inducing undifferentiated 3T3-L1 cell (fibroblast state) to the mature fat cell differentiation, in differentiation the 0th day, to reaching engagement state but still undifferentiated 3T3-L1 cell adds the MDI culture medium of inducing differentiation.The processing that adds the arteannuin medicine is operating as, and adds the solution of the arteannuin series gradient concentration that is configured to as mentioned above in the MDI culture medium in advance according to 1/1000 ratio, culture medium is added the cell culture hole after the mixing again.1/1000 ratio makes the final concentration of chemical compound 1,3-7 medicine become 1 μ M, 5 μ M, 10 μ M, 30 μ M, 50 μ M, the actual drug dose that uses of this experiment just.Need to design a processing simultaneously, the DMSO of adding 1/1000 is as solvent control in culture medium, and just drug level is the processing of 0 μ M. Cultivated treated 3T3-L1 cell 2 days according to above-mentioned described cell culture condition, according to above-mentioned described operating process of inducing undifferentiated 3T3-L1 cell (fibroblast state) to the mature fat cell differentiation, added the insulin culture medium on the 2nd day in differentiation, and with same as mentioned above method and dosage, add the arteannuin (same cell culture hole adds same a kind of medicine of same concentration) of serial gradient concentration. According to foregoing description, the 3T3-L1 cell of finishing differentiation is carried out oil red O stain, take pictures, survey absorbance, collect data, figure is in arrangement, relatively, the results are shown in Figure 1. The data of these 9 each concentration of chemical compound all derive from the meansigma methods that the repeated experiments of 3 different batches obtains among Fig. 1, and error is by the SE value representation between 3 repeated experiments. After medicine adds, can suppress or promote or not appreciable impact 3T3-L1 cellular fat differentiation (for the arteannuin medicine series, what the present invention paid close attention to is to suppress).The present invention represents drug effect with the degree of cellular fat differentiation.The degree of cell differentiation can be with after the dyeing of oil red staining after experiment is finished, and surveying OD is worth with detection by quantitative.Differentiation degree is high more, and the oil droplet accumulation is many more, and oil red dyeing is dark more, and the OD value is high more. Analytical Chemical Experiment of the present invention uses 24 porocyte culture plates to do, and each 24 orifice plate can design several holes of not dosing and do contrast, just the contrast of the cell that can break up fully. Each medicine is to obtain (differentiation degree relatively) by corresponding OD value than the OD value of the contrast of differentiation fully on the last same 24 porocyte culture plates in the drug effect of specific concentrations.Like this, the data that draw of different culture plate just can compare mutually. As shown in Figure 1, if differentiation fully, the value that obtains (differentiation degree relatively) is exactly 1 (100%); Differentiation is suppressed, and value is just less than 1 (between 0~1).Differentiation inhibition degree is high more, is worth more for a short time, and drug effect is good more. The result as shown in Figure 1, arteannuin itself (Fig. 1 .1 chemical compound) suppresses the poor effect of 3T3-L1 cell differentiation, concentration is in the level of 50 μ M, the relative differentiation degree of 3T3-L1 cell can reach 93.3%; In the level of 10 μ M, differentiation degree can reach 94.8% relatively. The results are shown in Figure 1, Fig. 2 .1, the effect that dihydroartemisinine (Fig. 1 .2 chemical compound) suppresses the 3T3-L1 cell differentiation be that all are one of best in the artemisinin derivative of tested mistake, can suppress the differentiation of 3T3-L1 cell fully in the level of 10 μ M, and relative differentiation degree has only 24.5%. The results are shown in Figure 1, Fig. 2 .3, the effect that this chemical compound (Fig. 1 .3 chemical compound) suppresses the 3T3-L1 cell differentiation is general, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell has 62.5%. The results are shown in Figure 1, Fig. 2 .2, the effect that arteannuin benzoate (Fig. 1 .4 chemical compound) suppresses the 3T3-L1 cell differentiation is that all are one of best in the artemisinin derivative of tested mistake, can suppress the differentiation of 3T3-L1 cell fully in the level of 10 μ M, differentiation degree has only 27.3% relatively. The results are shown in Figure 1, Fig. 2 .4, the effect that pi-allyl arteannuin (Fig. 1 .5 chemical compound) suppresses the 3T3-L1 cell differentiation is general, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell has 58.1%. The results are shown in Figure 1, the effect of acetate arteannuin (Fig. 1 .6 chemical compound) inhibition 3T3-L1 cell differentiation is relatively poor, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell has 81.3%. The results are shown in Figure 1, arteannuin-biotin (Fig. 1 .7 chemical compound) suppresses the poor effect of 3T3-L1 cell differentiation, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell has 91.6%. The results are shown in Figure 1, deoxidation dihydroartemisinine (Fig. 1 .8 chemical compound) does not almost suppress the effect of 3T3-L1 cell differentiation, and in the level of 10 μ M, the relative differentiation degree of 3T3-L1 cell can reach 98.8%. The results are shown in Figure 1, Fig. 2 .5, the effect that artesunate (Fig. 1 .9 chemical compound) suppresses the 3T3-L1 cell differentiation be that all are one of best in the artemisinin derivative of tested mistake, can suppress the differentiation of 3T3-L1 cell fully in the level of 10 μ M, and relative differentiation degree has only 31.5%.
Claims (3) Hide Dependent 1. the application of artemisinin derivative in the fat medicine of preparation control that has following formula I, II, III, IV, V structure, Wherein: N=0~1; R 1Be selected from C 1~C 3Alkyl; R 2Be selected from the aromatic heterocycle that aryl or nitrogen atom replace. 2. application according to claim 1 is characterized in that: described artemisinin derivative is to have in the following structural formula any: 3. application according to claim 1 is characterized in that: described artemisinin derivative is to have in the following structural formula any: https://patents.google.com/patent/CN101879158A/en?oq=CN101879158A青蒿素衍生物的新应用---防治肥胖病的
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