CN104610412B

CN104610412B - Anticancer compound, its preparation method and prepare the purposes of cancer therapy drug

Info

Publication number: CN104610412B
Application number: CN201510060052.2A
Authority: CN
Inventors: 朱海亮; 严晓强; 杨永安; 俞海荣; 钟飞
Original assignee: JIANGSU NAIQUE BIOLOGICAL ENGINEERING TECHNOLOGY Co Ltd
Current assignee: JIANGSU NAIQUE BIOLOGICAL ENGINEERING TECHNOLOGY Co Ltd
Priority date: 2015-02-04
Filing date: 2015-02-04
Publication date: 2016-07-06
Anticipated expiration: 2035-02-04
Also published as: CN104610412A

Abstract

The invention discloses a kind of structure such as anticancer compound shown in Formula VIII, its preparation method and prepare the purposes of cancer therapy drug,Wherein, R₁Selected from H, F, R₂Selected from H, F, R₃Selected from H, F.The present invention has the advantages such as better biological activity, higher selectivity, less toxicity and the shorter longevity of residure.

Description

Anticancer compound, its preparation method and prepare the purposes of cancer therapy drug

Technical field

The invention belongs to medicinal chemistry art, especially a kind of anticancer compound, its preparation method and prepare the purposes of cancer therapy drug.

Background technology

The steroidal saponin constituents obtained through extraction separation and purification from Chinese medicine Caulis Marsdeniae Tenacissimae, its aglycon is by unique steroidal saponin structure of 21 C atomic buildings, because of referred to herein as C₂₁Steroidal saponin.Now there are some researches show that there is C₂₁The pharmacologically active such as the compound of steroidal saponin parent nucleus has immunomodulating, relieving asthma, index active component as medicine has been obtained for wide application clinically clinically.

Inventor it have been investigated that: there is C₂₁The compound of steroidal saponin parent nucleus has good antineoplastic activity, kinds of tumors is had good inhibiting effect, as hepatocarcinoma, pulmonary carcinoma, esophageal carcinoma etc. are respectively provided with good inhibitory activity.At present to this type of C₂₁It also not deeply, is mainly easily separated purification and analysis detection research, carries out structural modification research for its parent nucleus and carry out pharmacology activity research for its architectural feature according to its physicochemical property by the research of steroidal saponin.

How to make it have better biological activity, higher selectivity, less toxicity are current important research content.

Summary of the invention

Goal of the invention a: purpose is to provide a kind of antitumoral compounds, at least to solve the subproblem that prior art exists.Further objective is that the preparation method that a kind of antitumoral compounds is provided.Further object is to provide the application in preparing antitumor drug of a kind of antitumoral compounds.

Technical scheme: a kind of structure is the compound shown in Formula VIII such as,

Wherein, R₁Selected from H, F, R₂Selected from H, F, R₃Selected from H, F.

A kind of preparation method of the structure such as compound shown in Formula VIII,

Wherein, R₁Selected from H, F, R₂Selected from H, F, R₃Selected from H, F；

Comprise the steps:

Step 1: prepare hydrazino-benzoic acid methyl ester, and be the structure such as compound shown in Formula II by structure converting compounds shown in formula I；

Step 2: add Azimethylene. ether mixed liquor, boron trifluoride-ether complex and the structure such as compound shown in Formula II in reactor, be obtained by reacting the structure such as compound shown in formula III；

Step 3: add the structure such as chemical combination shown in formula III, KOH aqueous solution, the structure such as compound shown in Formulas I V and ethanol in reactor, obtain structure compound shown as a formula V after reaction；

Step 4: by structure compound dehydrated alcohol shown as a formula V, hydrazino-benzoic acid methyl ester and glacial acetic acid are joined in reaction vessel, be obtained by reacting the structure such as compound shown in Formula IV；

Step 5: joined in the hydroxylamine solution of activation by the structure such as compound shown in Formula IV, is obtained by reacting the structure such as compound shown in Formula VII；

Step 6: add the structure such as compound shown in Formula VII, dichloromethane in reactor, and drip Boron tribromide, be obtained by reacting target product；

Formulas I V～VII, R₁Selected from H, F, R₂Selected from H, F, R₃Selected from H, F.

In a preferred embodiment, described step 1 is further:

At 0 ± 5 DEG C, successively in reactor add absolute methanol, thionyl chloride, to hydrazino-benzoic acid, after stirring 30～40min, continue stirring 3～5h at normal temperatures, filter the solid of gained successively with distilled water, ethanol, distilled water wash, dry, the solid product obtained is dissolved in dehydrated alcohol, recrystallization purifies, it is thus achieved that to hydrazino-benzoic acid methyl ester.

In room temperature (herein, room temperature refers to 15 DEG C～25 DEG C) under stirring, in reactor, add structure compound shown in formula I, aqueous hydrochloric acid solution, dehydrated alcohol successively, after stirring 2～3h, back flow reaction 7-9h, TLC follows the tracks of reaction, and after reaction terminates, the solid being filtrated to get is successively with aqueous hydrochloric acid solution, distilled water, ethanol, distilled water wash, dry, the solid crude product obtained is dissolved in dehydrated alcohol, and recrystallization purifies, and obtains the structure such as compound shown in Formula II.

It is furthermore preferred that structure compound 10mmol shown in formula I is dissolved in dehydrated alcohol 50ml. drip the hydrochloric acid 5ml of 1mol/L.

In a preferred embodiment, described step 2 is further:

Under ice-water bath stirring action, Azimethylene. ether mixed liquor, boron trifluoride-ether complex and the structure such as compound shown in formula III is added successively, after reaction 20～40min in reactor, back flow reaction 5～7h again, TLC follows the tracks of reaction, after reaction terminates, is filtrated to get solid successively with aqueous hydrochloric acid solution, distilled water, ethanol, distilled water wash, dry, the solid product obtained is dissolved in dehydrated alcohol, and recrystallization purifies, and obtains the structure such as compound shown in formula III.

It is furthermore preferred that each reactant molar ratio is in above-mentioned steps: structure is the compound shown in formula III such as: Azimethylene.: Boron tribromide=1:1.2～1.5:1.5～2.0.It is further preferred that ratio is 1:1.2:1.5,1:1.5:1.5,1:1.2:2.0.

In a preferred embodiment, described step 3 is further:

Under stirring at room temperature, the structure such as compound shown in formula III, KOH aqueous solution, the structure such as compound shown in Formulas I V, ethanol is added successively in reactor, after continuing stirring reaction 3～6h, pH is regulated with hcl acidifying, the solid being filtrated to get with distilled water, ethanol, distilled water wash, dries to obtain structure compound shown as a formula V successively.

It is furthermore preferred that each reactant molar ratio in above-mentioned steps: structure is the compound shown in Formulas I V such as: structure is the compound shown in formula III such as: Boron tribromide=1:1:1～1.5.

In a preferred embodiment, described step 4 is further:

Successively by structure compound, dehydrated alcohol shown as a formula V, hydrazino-benzoic acid methyl ester and glacial acetic acid are joined in reactor, after reaction 20～40min is stirred at room temperature, have fraction solids insoluble；Then back flow reaction 5～8h, TLC follows the tracks of reaction, after reaction terminates, filtering, solid is successively with aqueous hydrochloric acid solution, distilled water, ethanol, distilled water wash, dry, the solid product obtained is dissolved in dehydrated alcohol, and recrystallization purifies, and obtains the structure such as compound shown in Formula IV.

It is furthermore preferred that each reactant molar ratio: structure compound shown as a formula V: to Hydrazinobenzenesulfonamide: acetic acid=1:1.2～1.5:0.5.It is further preferred that ratio is: 1:1.2:0.5,1:1.5:0.5.

In a preferred embodiment, described step 5 is further:

Oxammonium hydrochloride., Feldalat NM are added in absolute methanol, after heated and stirred 20～40min, filters out solid, obtain the hydroxylamine solution of activation, then be added thereto to the structure such as compound shown in Formula IV, back flow reaction 3～5h, TLC follows the tracks of reaction, after reaction terminates, filter, the solid distilled water wash obtained, last vacuum drying, the solid obtained is dissolved in dehydrated alcohol, and recrystallization purifies, and obtains the structure such as compound shown in Formula VII.

It is furthermore preferred that each reactant molar ratio: VI: hydrochloric acid hydroxyl is pressed: Feldalat NM=1:4～5:4～5.It is further preferred that ratio is 1:4:4,1:5:4.

In a preferred embodiment, described step 6 is further:

Under stirring at-20 ± 5 DEG C, in reactor, add the structure such as compound shown in Formula VII, dichloromethane successively, and progressively drip Boron tribromide, stirring 20～40min, room temperature continues reaction 8～12h, TLC and follows the tracks of reaction, after reaction terminates, filter, solid distilled water wash, last vacuum drying, the solid obtained is dissolved in dehydrated alcohol, recrystallization purifies, and obtains lenticular target compound.

It is furthermore preferred that each reactant molar ratio is in above-mentioned steps: VII: Boron tribromide=1:0.5

This method is transformed for saponin aglycon, has effectively integrated hydroxamic acid skeleton, pyrazoline skeleton, assay reproducibility is strong, good stability, and the required condition of experiment reaction is simpler, and experimental situation is gentle, productivity is better, can produce in a large number in less input situation.

A kind of structure is the application in preparing cancer therapy drug of the compound shown in Formula VIII such as,

A kind of anticancer pharmaceutical composition, the such as compound shown in Formula VIII and medically acceptable carrier including structure.

Beneficial effect: the present invention has the advantages such as better biological activity, higher selectivity and less toxicity.Human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and hepatoma carcinoma cell (HepG2) there are is obvious inhibitory action, people's renal epithelial cell (293T) is shown quite simultaneously or be better than the cytotoxicity of positive control medicine Celecoxib.

Detailed description of the invention

The implementation process concrete in conjunction with certain describes the present invention.The structural formula of each reactant, intermediate product and target product is such as shown in literary composition:

The present invention is made up of the following step:

Step 1. is at 0 ± 5 DEG C, successively in round-bottomed flask add absolute methanol, thionyl chloride, to hydrazino-benzoic acid 1, after stirring about 1h, room temperature continues stirring 3-5h, filter, the solid obtained is successively with distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, dry, the solid crude product obtained is dissolved in dehydrated alcohol recrystallization and obtains carboxylate to hydrazino-benzoic acid methyl ester 2.

Step 2. under stirring at room temperature, adds TenacissosideA1 (a kind of C successively in round-bottomed flask₂₁Steroidal saponin) 3, dilute hydrochloric acid, dehydrated alcohol, stirring 1h after, back flow reaction 7-9h.TLC follows the tracks of reaction (developing solvent V_AcOEt:V_PE=1:2), after reaction terminates, filter, the solid obtained is successively with dilute hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, dry, the solid crude product obtained is dissolved in dehydrated alcohol recrystallization and obtains the lenticular C of partial reduction₂₁Steroidal saponin aglycon 4.

Step 3., under ice-water bath stirring action, adds Azimethylene. ether mixed liquor, boron trifluoride-ether complex, C successively in round-bottomed flask₂₁Steroidal saponin aglycon 4, after reaction about 0.5h, then back flow reaction 5-7h.TLC follows the tracks of reaction (developing solvent V_AcOEt:V_PE=1:2), after reaction terminates, filter, obtain solid successively with dilute hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, dry, the solid crude product obtained is dissolved in dehydrated alcohol recrystallization and obtains lenticular intermedium 5.

Step 4. is under stirring at room temperature, intermedium 5, KOH aqueous solution, the benzaldehyde of different substituents, ethanol is added successively in round-bottomed flask, after continuing stirring reaction 4h, dilute hydrochloric acid acidifying regulates pH, filter, the solid obtained with distilled water (3 × 100mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, dries to obtain intermediate 6-9 successively.

Step 5., successively by different compound 6-9, dehydrated alcohol, join in round-bottomed flask to hydrazino-benzoic acid methyl ester 2, glacial acetic acid, after reaction 1h is stirred at room temperature, still has fraction solids insoluble；Then back flow reaction 5h, TLC follow the tracks of reaction (developing solvent V_AcOEt:V_PE=1:2), after reaction terminates, filter, solid is successively with dilute hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, dry, the solid crude product obtained is dissolved in dehydrated alcohol recrystallization and obtains lenticular intermedium 10-13.

Equimolar hydrochloric acid hydroxyl is pressed by step 6., Feldalat NM adds in absolute methanol, after heated and stirred 0.5h, filters out solid, obtains the hydroxylamine solution of activation, at the crystal intermedium 10-13, the back flow reaction 3-5h that are being added thereto to correspondence.TLC follows the tracks of reaction (developing solvent V_AcOEt:V_PE=1:2), after reaction terminates, filter, solid distilled water wash, last vacuum drying, the solid obtained is dissolved in dehydrated alcohol, recrystallization purifies, and obtains lenticular target compound 14-17.

Step 7. adds the intermedium 14-17 of correspondence, dichloromethane successively in round-bottomed flask, and progressively drips Boron tribromide under stirring at-20 ± 5 DEG C, and after stirring about 1h, room temperature continues reaction about 12h.TLC follows the tracks of reaction (developing solvent V_AcOEt:V_{Normal hexane}=1:2), after reaction terminates, filter, solid distilled water wash, last vacuum drying, the solid obtained is dissolved in dehydrated alcohol, recrystallization purifies, and obtains lenticular target compound 18-21.

Embodiment one:

4-(5-phenyl-3-((3aR, 4aS, 8S, 10aS, 11S, 12S, 12aS)-(8,11,12-trihydroxy)-(10a, 12a-dimethyl)-(1,2-Pentamethylene. base)-1,10a-b-epoxy n-tetradecane base)-(4,5-pyrazoline)) preparation of benzoyl hydroxamic acid (compound 18)

Under stirring at-20 DEG C, corresponding intermedium 14 (10.0mmol), the dichloromethane (25mL) that step 6 obtains is added successively in the round-bottomed flask of 50mL, and progressively drip after Boron tribromide (5.0mmol) continues stirring reaction 1h, reaction flask is transferred to room temperature, continues reaction 12h.TLC follows the tracks of reaction (developing solvent V_AcOEt:V_{Normal hexane}=1:2), after reaction terminates, filter, solid distilled water wash, last vacuum drying, the solid obtained is dissolved in dehydrated alcohol, recrystallization purifies, and obtains lenticular target compound 12.

Obtain white crystal, productivity 53.7%.M.p.198～200 DEG C；¹HNMR(DMSO-d₆, 300MHz) and δ: 10.42 (s, 1H, OH), 8.01 (d, J=8.1Hz, 2H, ArH), 7.75 (d, J=7.7Hz, 2H, ArH), 7.70 (s, 1H, NH), 7.36～7.25 (m, 5H, ArH), 5.37 (s, 2H, OH), 5.19 (t, J=7.3Hz, 1H, CH), 4.49 (s, 1H, OH), 3.58～3.42 (m, 2H, CH), 3.34 (d, J=6.8Hz, 1H, CH), 2.95～2.73 (m, 2H, CH₂), 2.01 (t, J=7.3Hz, 1H, CH), 1.79～1.21 (m, 15H, CHandCH₂),1.14(dd,J₁=7.1Hz, J₂=7.8Hz, 1H, CH), 0.89 (s, 3H, CH₃),0.84(s,3H,CH₃).ESI-MS:602.7[M+H]⁺.Anal.CalcdforC₃₅H₄₃N₃O₆:C,H,N.

Embodiment two:

4-(5-(4-fluoro-phenyl)-3-((3aR, 4aS, 8S, 10aS, 11S, 12S, 12aS)-(8,11,12-trihydroxy)-(10a, 12a-dimethyl)-(1,2-Pentamethylene. base)-1,10a-b-epoxy n-tetradecane base)-(4,5-pyrazoline)) preparation of benzoyl hydroxamic acid (compound 19)

Preparation method reference example one.Obtain white crystal, productivity 51.5%.M.p.219～220 DEG C；¹HNMR(DMSO-d₆, 300MHz) and δ: 10.48 (s, 1H, OH), 8.03 (d, J=8.3Hz, 2H, ArH), 7.73 (d, J=8.1Hz, 2H, ArH), 7.65 (s, 1H, NH), 7.35～7.18 (m, 4H, ArH), 5.35 (s, 2H, OH), 5.19 (t, J=7.1Hz, 1H, CH), 4.49 (s, 1H, OH), 3.56～3.41 (m, 2H, CH), 3.32 (d, J=6.8Hz, 1H, CH), 2.97～2.71 (m, 2H, CH₂), 2.04 (t, J=7.0Hz, 1H, CH), 1.81～1.20 (m, 15H, CHandCH₂),1.14(dd,J₁=7.0Hz, J₂=7.6Hz, 1H, CH), 0.89 (s, 3H, CH₃),0.78(s,3H,CH₃).ESI-MS:620.7[M+H]⁺.Anal.CalcdforC₃₅H₄₂FN₃O₆:C,H,N.

Embodiment three:

4-(5-(2-fluoro-phenyl)-3-((3aR, 4aS, 8S, 10aS, 11S, 12S, 12aS)-(8,11,12-trihydroxy)-(10a, 12a-dimethyl)-(1,2-Pentamethylene. base)-1,10a-b-epoxy n-tetradecane base)-(4,5-pyrazoline)) preparation of benzoyl hydroxamic acid (compound 20)

Preparation method reference example one.Obtain white crystal, productivity 44.9%.M.p.212～213 DEG C；¹HNMR(DMSO-d₆, 300MHz) and δ: 10.56 (s, 1H, OH), 8.03 (d, J=8.1Hz, 2H, ArH), 7.74～7.55 (m, 5H, ArHandNH), 7.29～7.23 (m, 1H, ArH), 7.15～7.07 (m, 1H, ArH), 5.37 (s, 2H, OH), 5.19 (t, J=7.3Hz, 1H, CH), 4.48 (s, 1H, OH), 3.55～3.42 (m, 2H, CH), 3.34 (d, J=6.8Hz, 1H, CH), 2.95～2.73 (m, 2H, CH₂), 2.01 (t, J=6.9Hz, 1H, CH), 1.79～1.21 (m, 15H, CHandCH₂),1.14 (dd,J₁=7.1Hz, J₂=7.9Hz, 1H, CH), 0.84 (s, 3H, CH₃),0.81(s,3H,CH₃).ESI-MS:620.7[M+H]⁺.Anal.CalcdforC₃₅H₄₂FN₃O₆:C,H,N.

Embodiment four:

4-(5-(3-fluoro-phenyl)-3-((3aR, 4aS, 8S, 10aS, 11S, 12S, 12aS)-(8,11,12-trihydroxy)-(10a, 12a-dimethyl)-(1,2-Pentamethylene. base)-1,10a-b-epoxy n-tetradecane base)-(4,5-pyrazoline)) preparation of benzoyl hydroxamic acid (compound 21)

Preparation method reference example one.Obtain white crystal, productivity 37.8%.M.p.215～216 DEG C；¹HNMR(DMSO-d₆null,300MHz)δ:10.49(s,1H,OH),7.99(d,J=8.1Hz,2H,ArH),7.72(d,J=7.3Hz,2H,ArH),7.66(s,1H,NH),7.39～7.31 (m,1H,ArH),7.15～7.07 (m,2H,ArH),6.97(d,J=3.9Hz,1H,ArH),5.35(s,2H,OH),5.16(t,J=7.2Hz,1H,CH),4.49(s,1H,OH),3.54～3.41 (m,2H,CH),3.34(d,J=6.5Hz,1H,CH),,2.95～2.73 (m,2H,CH₂), 2.01 (t, J=7.3Hz, 1H, CH), 1.75～1.20 (m, 15H, CH₂andCH),1.13(dd,J₁=6.6Hz, J₂=7.2Hz, 1H, CH), 0.85 (s, 3H, CH₃),0.83(s,3H,CH₃).ESI-MS:620.7[M+H]⁺.Anal.CalcdforC₃₅H₄₂FN₃O₆:C,H,N.

Embodiment five:

MTT [3-(4,5)-bis-methyl-2-thiazoles-(2,5)-phenyl bromination tetrazole is blue] method is adopted to measure pyrazoline hydroxamic acid C₂₁The steroidal saponin aglycone derivative half-inhibition concentration (IC to human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and hepatoma carcinoma cell (HepG2)₅₀)。

(1) preparation of culture fluid (/L): 1. suspension cell: DMEM cultivates one bag of powder (10.4g), new-born calf serum 100mL, penicillin solution (2 × 10^-5U/mL) 0.5mL, Streptomycin Solution (2 × 10^-5U/mL) 0.5mL, after adding tri-distilled water dissolving, with the NaHCO of 5.6%₃Solution adjusts pH value to 7.2-7.4, is finally settled to 1000mL.Filtration sterilization.2. attached cell: ibid, adds NaHCO₃2.00g, HEPES2.38g.

(2) preparation of D-Hanks buffer (every liter): NaCl8.00g, KCl0.40g, Na₂HPO₄·12H₂O0.06g, KH₂PO₄0.06g, NaHCO₃0.35g.Autoclaving.

(3) preparation of trypsin solution: utilizing D-Hanks buffer to be made into concentration is 0.5% trypsin solution.Filtration sterilization.

(4) preparation of medicinal liquid is tested: dissolved by a small amount of tri-distilled water of test sample and be made into storing solution, i.e. 10 times of preparation storing solutions of empirically maximum concentration.Different according to compound dissolution, available tri-distilled water directly dissolves, or with a small amount of DMSO hydrotropy, then add tri-distilled water dissolving.Storing solution is stored in-20 DEG C of refrigerators standby.

(5) cultivation of human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and hepatoma carcinoma cell (HepG2): for adherent growth cell, cellar culture (containing 10% calf serum, 100U/mL streptomycin), is placed in 37 DEG C, 5%CO in DMEM culture fluid₂Incubator is cultivated, goes down to posterity once every 3-4d.Being transferred in centrifuge tube by culture fluid in former bottle when going down to posterity, 1000rpm is centrifuged 5min, discards original fluid, add equivalent fresh medium, piping and druming uniformly, pipettes appropriate to fresh cultured bottle, is supplemented with fresh medium to original volume (culture fluid volume is about the 1/10 of culture bottle capacity).

(6) cell incubation: the tumor cell of trophophase of taking the logarithm, tune concentration of cell suspension is 1-1.5 × 10⁵Individual/mL.In 96 well culture plates, every hole adds cell suspension 100 μ L, puts 37 DEG C, 5%CO₂Incubator is cultivated 24h.After cultivating 24h, add medicinal liquid by design respectively.

(7) dosing: being added separately in each hole by test medicinal liquid according to the Concentraton gradient of ultimate density, each concentration sets 6 parallel holes.Experiment is divided into drug test group (being separately added into the test medicine of variable concentrations), matched group (only adding culture fluid and cell, be not added with test medicine) and blank group (only adding culture fluid, be not added with cell and test medicine).96 orifice plates after dosing are placed in 37 DEG C, 5%CO₂Incubator is cultivated 48h.The activity of positive control medicine measures according to the method for test sample.

(8) mensuration of survivaling cell: in 96 orifice plates after having cultivated 48h, every hole adds MTT40 μ L (being made into 4mg/mL with D-Hanks buffer).After placing 4h at 37 DEG C, remove supernatant.Every hole adds 150 μ LDMSO, and vibrate 5min, makes formazan crystallization dissolve.Finally, automatic microplate reader is utilized to detect the optical density (OD value) in each hole at 570nm wavelength place.

Half-inhibition concentration (IC₅₀) it is defined as the drug level when the tumor cell survival of 50%.According to the optical density (OD value) measured, make the standard curve of inhibitory rate of cell growth, standard curve is tried to achieve the drug level of its correspondence.

The IC recorded₅₀It is shown in Table 1.

The listed novel pyrazoline hydroxamic acid C of table 1 present invention₂₁The steroidal saponin aglycone derivative suppression IC to tumor cell₅₀Value (μm ol/mL)

^a6 parallel tests, experimental result is averaged, and error is between 5-10%.

The pyrazoline hydroxamic acid C of the present invention₂₁Human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and hepatoma carcinoma cell (HepG2) are had obvious inhibitory action by steroidal saponin aglycone derivative, the inhibitory activity of contrast TenacissosideA is significantly improved, and contrast positive control drug then shows quite or is better than the inhibitory activity of positive control medicine.Therefore the pyrazoline hydroxamic acid C of the present invention₂₁Steroidal saponin aglycone derivative can apply to prepare antitumor drug.

Embodiment six:

The present invention tests the newly synthesized compound 18-21 cytotoxicity to people's renal epithelial cell (293T), and cytotoxicity result is table 2 such as, using Celecoxib as positive control.Concentration (the CC when toxicity of each compound suppresses T cell survival rate to 50%₅₀) represent.

Experimental technique:

(1) cultivate people's renal epithelial cell (293T) until reaching its logarithmic growth end of term cell and tending to merging, digest cell dispersion with cell dissociation buffer, be configured to 1 with cell culture fluid

×10⁴The cell suspension of individual/mL.Take 96 well culture plates, every hole adds the cell suspension of 100 μ L.Horizontally rotating culture plate gently makes cell be evenly dispersed in the surface in ware hole.

(2) it is placed in containing 5%CO₂In cell culture incubator, at 37 ± 2 DEG C of temperature, cultivate 24h.Discarding original fluid, every hole adds the blank liquid of 100 μ L, negative controls, positive control solution, the test specimen lixiviating solution of 100% and 50% concentration.Often organize and at least set 8 holes.Note: lixiviate stock solution or make the serial lixiviate diluent of diluent with culture medium.When adopting 0.9% sodium chloride injection lixiviate, use 2 times of culture medium of concentration when diluting lixiviate.

(3) it is placed in containing 5%CO₂In incubator, cultivate at 37 ± 2 DEG C of temperature.Cultivate 48h.

(4) after date between each cultivation, every hole adds MTT solution 20 μ L, is placed in containing 5%CO₂In incubator, at 37 ± 2 DEG C of temperature, cultivate 5h.

(5) discarding liquid in hole, every hole is separately added into 200 μ LDMSO, and culture plate is placed 10min, and level is rocked and made solution colour in hole uniform.

(6) measuring absorbance by microplate reader, wavelength adopts 570nm.

The CC recorded₅₀It is shown in Table 2.

The listed novel pyrazoline hydroxamic acid C of table 2 present invention₂₁The steroidal saponin aglycone derivative suppression CC to 293T cell₅₀Value (μm ol/mL)

^a6 parallel tests, experimental result is averaged, and error is between 5-10%

From the above: the pyrazoline hydroxamic acid C of the present invention₂₁Steroidal saponin aglycone derivative, to people's renal epithelial cell (293T) being shown quite or being better than the cytotoxicity of positive control medicine, is compared with TenacissosideA, and cytotoxicity also almost maintains an equal level.Therefore the pyrazoline hydroxamic acid C of the present invention₂₁Steroidal saponin aglycone derivative can apply to prepare antitumor drug.

Inventor thinks: hydroxamic acid structural formula is R-CO-NHOH, is a kind of organic sequestering agent, energy and Cu²⁺、Zn²⁺、Co²⁺、Ni²⁺、Fe³⁺Ions etc. form stable metallo-chelate, and it has significant complexing power.Pyrazoline is particularly important nitrogenous five member ring heterocyclic compound, and it has the anti-tumor activity of excellence.

In a word, the present invention will have outstanding bioactive hydroxamic acid skeleton and be incorporated in pyrazoline derivant, in combination with the C with activity very well₂₁Steroidal saponin aglycon skeleton, design has synthesized a series of pyrazoline hydroxamic acid C₂₁Steroidal saponin aglycone derivative, finds through experiment, it has the advantages such as better biological activity, higher selectivity and less toxicity.

The preferred embodiment of the present invention described in detail above; but, the present invention is not limited to the detail in above-mentioned embodiment, in the technology concept of the present invention; technical scheme can being carried out multiple equivalents, these equivalents belong to protection scope of the present invention.It is further to note that each the concrete technical characteristic described in above-mentioned detailed description of the invention, in reconcilable situation, it is possible to be combined by any suitable mode.In order to avoid unnecessary repetition, various possible compound modes are no longer illustrated by the present invention separately.Additionally, can also carry out combination in any between the various different embodiment of the present invention, as long as it is without prejudice to the thought of the present invention, it should be considered as content disclosed in this invention equally.

Claims

1. the preparation method of the structure such as compound shown in Formula VIII, it is characterised in that

Comprise the steps:

Step 3: add the structure such as compound shown in formula III, KOH aqueous solution, the structure such as compound shown in Formulas I V and ethanol in reactor, obtain structure compound shown as a formula V after reaction；

Step 5: pressed by hydrochloric acid hydroxyl, Feldalat NM adds in absolute methanol, after heated and stirred 20～40min, filter out solid, obtain the hydroxylamine solution of activation, the structure such as compound shown in Formula IV is joined in the hydroxylamine solution of activation, is obtained by reacting the structure such as compound shown in Formula VII；

2. the preparation method of compound as claimed in claim 1, it is characterised in that described step 1 is further:

At 0 ± 5 DEG C, successively in reactor add absolute methanol, thionyl chloride, to hydrazino-benzoic acid, after stirring 30～40min, continue stirring 3-5h at normal temperatures, filter the solid of gained successively with distilled water, ethanol, distilled water wash, dry, the solid product obtained is dissolved in dehydrated alcohol, recrystallization purifies, it is thus achieved that to hydrazino-benzoic acid methyl ester

Under stirring at room temperature, structure compound shown in formula I, aqueous hydrochloric acid solution, dehydrated alcohol is added successively in reactor, after stirring 2～3h, back flow reaction 7～9h, TLC follow the tracks of reaction, after reaction terminates, the solid being filtrated to get is successively with aqueous hydrochloric acid solution, distilled water, ethanol, distilled water wash, dry, and the solid crude product obtained is dissolved in dehydrated alcohol, recrystallization purifies, and obtains the structure such as compound shown in Formula II.

3. the preparation method of compound as claimed in claim 1, it is characterised in that described step 2 is further:

Under ice-water bath stirring action, Azimethylene. ether mixed liquor, boron trifluoride-ether complex and the structure such as compound shown in Formula II is added successively, after reaction 20～40min in reactor, back flow reaction 5-7h, TLC follows the tracks of reaction, after reaction terminates, is filtrated to get solid successively with aqueous hydrochloric acid solution, distilled water, ethanol, distilled water wash, dry, the solid product obtained is dissolved in dehydrated alcohol, and recrystallization purifies, and obtains the structure such as compound shown in formula III.

4. the preparation method of compound as claimed in claim 1, it is characterised in that described step 3 is further:

5. the preparation method of compound as claimed in claim 1, it is characterised in that described step 4 is further:

Successively by structure compound, dehydrated alcohol shown as a formula V, hydrazino-benzoic acid methyl ester and glacial acetic acid are joined in reactor, after reaction 20～40min is stirred at room temperature, have fraction solids insoluble；Back flow reaction 5～8h, TLC follow the tracks of reaction, after reaction terminates, filtering, solid is successively with aqueous hydrochloric acid solution, distilled water, ethanol, distilled water wash, dry, the solid product obtained is dissolved in dehydrated alcohol, and recrystallization purifies, and obtains the structure such as compound shown in Formula IV.

6. the preparation method of compound as claimed in claim 1, it is characterised in that described step 5 is further:

7. the preparation method of compound as claimed in claim 1, it is characterised in that described step 6 is further: