CN104558093B - C21steroid saponin aglycone derivative, its preparation method and the application in preparing antineoplastic thereof - Google Patents
C21steroid saponin aglycone derivative, its preparation method and the application in preparing antineoplastic thereof Download PDFInfo
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Abstract
The invention discloses a kind of C21Steroid saponin aglycone derivative, its preparation method and the application in preparing antineoplastic thereof, described C21The structure of steroid saponin aglycone derivative is as shown in Formula IX, and the present invention has the advantage such as more preferable biologically active, higher selective and lower toxicity.Human breast cancer cell, cervical cancer cell, lung carcinoma cell and HCC there are is obvious inhibitory action,Wherein, R1Selected from H, F, R2Selected from H, F, R3Selected from H, F.
Description
Technical field
The invention belongs to medicinal chemistry art, especially a kind of C21Steroid saponin aglycone derivative, its preparation method and the application in preparing antineoplastic thereof.
Background technology
Can obtain steroid saponin constituents through extraction separation and purification from Chinese medicine CAULIS MARSDENIAE TENACISSIMAE, its aglycon is by unique steroid saponin structure of 21 C atomic buildings, because of referred to herein as C21Steroid saponin.Now there are some researches show that there is C21The compound of steroid saponin parent nucleus has immunological regulation, the pharmacologically active such as relieving asthma, and the most the index active component as medicine has been obtained for wide application clinically.
Inventor it has been investigated that: there is C21The compound of steroid saponin parent nucleus has good antineoplastic activity, and kinds of tumors is had good inhibiting effect, as liver cancer, lung cancer, cancer of the esophagus etc. are respectively provided with preferable inhibitory activity.At present to this type of C21It the most not deeply, is mainly carried out isolated and purified according to its physicochemical property and analyzes detection research, carry out structural modification research for its parent nucleus and carry out pharmacology activity research for its architectural feature by the research of steroid saponin.
How to make it have more preferable biologically active, higher selectivity, lower toxicity are current important research contents.
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 antineoplastic of a kind of antitumoral compounds.
Technical scheme: a kind of C21Steroid saponin aglycone derivative, structure as shown in Formula IX,
Wherein, R1Selected from H, F, R2Selected from H, F, R3Selected from H, F.
A kind of C21The preparation method of steroid saponin aglycone derivative,
Described C21The structure of steroid saponin aglycone derivative as shown in Formula IX,
Wherein, R1Selected from H, F, R2Selected from H, F, R3Selected from H, F；
Comprise the steps:
Step 1, preparation, to hydrazino-benzoic acid methyl esters, add structure compound, aqueous hydrochloric acid solution and absolute ethyl alcohol shown in formula I in reactor, prepare structure compound as shown in Formula II after reaction；
Step 2, in reactor, add diazomethane ether mixed liquor, boron trifluoride-ether complex and structure compound as shown in Formula II, after reaction, obtain structure compound as shown in formula III；
Step 3, in reactor, add structure compound as shown in formula III, the KOH aqueous solution, the structure compound as shown in formula IV and ethanol, prepared structure compound shown as a formula V；
Step 4, by structure compound, absolute ethyl alcohol shown as a formula V, hydrazino-benzoic acid methyl esters and glacial acetic acid are joined in reactor, reaction obtains structure compound as shown in Formula IV；
Step 5, structure compound as shown in Formula IV, KOH are joined in the mixed solution of methyl alcohol, THF, after reaction, obtain structure compound as shown in Formula VII；
Step 6, structure compound as shown in Formula VII and N methyl piperazine being joined in dichloromethane, mixing is dissolved, the dropping dichloromethane mixed solution containing DCC/DMAP, and reaction obtains structure compound as shown in Formula VIII.
Step 7, in reactor, add structure compound as shown in Formula VIII, dichloromethane, and drip Boron tribromide, produce target compound；
In formula IV～VIII, R1Selected from H, F, R2Selected from H, F, R3Selected from H, F.
In a preferred embodiment, it is characterised in that described step 1 is further:
At 0 ± 5 DEG C, absolute methanol, thionyl chloride and to hydrazino-benzoic acid is added successively in reactor, after stirring 30～40min, normal temperature continues stirring 3-5h, filters, and the solid obtained is successively with distilled water, ethanol and distilled water washing, it is dried, the solid product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains carboxylate to hydrazino-benzoic acid methyl esters；
Under stirring at room temperature, structure compound shown in formula I, aqueous hydrochloric acid solution, absolute ethyl alcohol, after stirring 2～3h, back flow reaction 7-9h is added successively in reactor, TLC follows the tracks of reaction, after reaction terminates, filtering, the solid obtained is successively with aqueous hydrochloric acid solution, distilled water, cold ethanol, distilled water washing, it is dried, the solid crude product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, and obtains the structure of the lenticular compound as shown in Formula II.
It is furthermore preferred that I 10mmol to be dissolved in the hydrochloric acid 5ml of absolute ethyl alcohol 50ml. dropping 1mol/L.
In a preferred embodiment, described step 2 is further:
Under ice-water bath stirring action, diazomethane ether mixed liquor, boron trifluoride-ether complex, structure compound as shown in Formula II is added successively in reactor, after reaction 20～40min, then back flow reaction 5～7h, TLC follows the tracks of reaction, after reaction terminates, filter, obtain solid successively with aqueous hydrochloric acid solution, distilled water, ethanol, distilled water washing, it is dried, the solid crude product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains structure compound as shown in formula III.
It is furthermore preferred that each reactant molar ratio: II: diazomethane: Boron tribromide=1:1.2～1.5:1.5～2.0.It is further preferred that ratio is 1:1.2:2.0,1:1.4:1.5.
In a preferred embodiment, described step 3 is further:
In room temperature (herein, room temperature refers to 15 DEG C～25 DEG C) stirring under, structure compound as shown in formula III, the KOH aqueous solution, the structure compound as shown in formula IV and ethanol is added successively in reactor, after continuing stirring reaction 3～6h, it is acidified with hydrochloric acid, regulates pH, filter, the solid obtained is successively with distilled water, cold ethanol, distilled water washing, dried structure compound shown as a formula V.
It is furthermore preferred that each reactant molar ratio: IV:III: Boron tribromide=1:1:1～1.5.It is further preferred that ratio is 1:1:1.5,1:1:1.
In a preferred embodiment, described step 4 is further:
Successively by structure compound, absolute ethyl alcohol shown as a formula V, hydrazino-benzoic acid methyl esters and glacial acetic acid are joined in reactor, after reaction 20～40min is stirred at room temperature, still have fraction solids insoluble；Back flow reaction 5～8h, TLC follows the tracks of reaction, after reaction terminates, filtering, solid with aqueous hydrochloric acid solution, distilled water, cold ethanol, distilled water washing, is dried successively, the solid product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains structure compound as shown in Formula IV.
It is furthermore preferred that mol ratio: 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: structure compound as shown in Formula IV and KOH are joined in the mixed solution of methyl alcohol, THF water, back flow reaction 4～8h, TLC follows the tracks of reaction, after reaction terminates, is acidified with hydrochloric acid, regulation pH is to acid, being poured into by solution in saturated aqueous common salt, filter, solid distilled water washs, finally it is vacuum dried, the solid obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains structure compound as shown in Formula VII.
It is furthermore preferred that volume ratio: methyl alcohol: THF: water=1:1:1.The structure of 10mmol compound as shown in Formula IV is dissolved in mol ratio: VI:KOH=1:2 in 50ml methyl alcohol THF water mixed liquid.
In a preferred embodiment, described step 6 is further:
Structure compound as shown in Formula VII and N methyl piperazine are joined in dichloromethane, mixing is dissolved, the dropping dichloromethane mixed solution containing DCC/DMAP under 0 ± 5 DEG C of DEG C of stirring, reaction 20～40min, then 6～10h are reacted under 35 ± 5 DEG C of stirrings, TLC follows the tracks of reaction, after reaction terminates, reactant liquor is poured in saturated aqueous common salt, filtering, solid with aqueous hydrochloric acid solution, distilled water, cold ethanol, distilled water washing, is dried successively, the solid product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies and obtains structure compound as shown in Formula VIII.
It is furthermore preferred that in above-mentioned steps, the mol ratio of each reactant: VII:N-methyl piperazine: DCC:DMAP=1:1:1.2:1.44.
In a preferred embodiment, described step 7 is further:
Under stirring at-20 ± 5 DEG C, in reactor, add structure compound as shown in Formula VIII, dichloromethane successively, and drip Boron tribromide, stirring 20～40min, room temperature continues reaction 8～12h, and TLC follows the tracks of reaction, after reaction terminates, filter, solid distilled water washs, and is finally vacuum dried, the solid obtained is dissolved in absolute ethyl alcohol, recrystallization purifies, and obtains the target compound of lenticular.
It is furthermore preferred that in above-mentioned steps, the mol ratio of each reactant: VIII: Boron tribromide=1:0.5.
This method is transformed for saponin(e aglycon, has effectively integrated piperazine 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 preferable, can produce in a large number in the case of less input.
A kind of C21The application in preparing antineoplastic of the steroid saponin aglycone derivative,
Described C21The structure of steroid saponin aglycone derivative as shown in Formula IX,
Wherein, R1Selected from H, F, R2Selected from H, F, R3Selected from H, F.
Beneficial effect: the present invention has the advantage such as more preferable biologically active, higher selective and lower toxicity.Human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and HCC (HepG2) are had 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
Technical scheme is as follows:
The pyrazoline piperazine C that one class is novel21The synthesis of steroid saponin aglycone derivative, is characterized in that it has a below formula:
Pyrazoline piperazine C of the present invention21The synthesis of steroid saponin aglycone derivative, comprises the following steps:
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, normal temperature continues stirring 3-5h, filter, the solid obtained with distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, is dried, the solid crude product obtained is dissolved in absolute ethyl alcohol and is recrystallized to give carboxylate to hydrazino-benzoic acid methyl esters 2 successively.
Step 2. under stirring at room temperature, adds Tenacissoside A (a kind of C successively in round-bottomed flask21Steroid saponin) 3, aqueous hydrochloric acid solution, absolute ethyl alcohol, stirring 1h after, back flow reaction 7-9h.TLC follows the tracks of reaction (solvent VAcOEt:VPE=1:2), after reaction terminates, filter, the solid obtained is successively with watery hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, it is dried, the solid crude product obtained is dissolved in absolute ethyl alcohol and is recrystallized to give the lenticular C of partial reduction21Steroid saponin aglycon 4.
Step 3., under ice-water bath stirring action, adds diazomethane ether mixed liquor, boron trifluoride-ether complex, C successively in round-bottomed flask21Steroid saponin aglycon 4, after reaction about 0.5h, then back flow reaction 5-7h.TLC follows the tracks of reaction (solvent VAcOEt:VPE=1:2), after reaction terminates, filter, obtain solid successively with watery hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, it is dried, the solid crude product obtained is dissolved in absolute ethyl alcohol and is recrystallized to give lenticular intermediate 5.
Step 4. is under stirring at room temperature, intermediate 5, the KOH aqueous solution, the benzaldehyde of different substituents, ethanol is added successively in round-bottomed flask, after continuing stirring reaction 4h, watery hydrochloric acid acidifying regulation pH, filter, the solid obtained with distilled water (3 × 100mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, is dried to obtain intermediate 6-9 successively.
Step 5., successively by different compound 6-9, absolute ethyl alcohol, join in round-bottomed flask to hydrazino-benzoic acid methyl esters 2, glacial acetic acid, after reaction 1h is stirred at room temperature, still has fraction solids insoluble；Then back flow reaction 5h.TLC follows the tracks of reaction (solvent VAcOEt:VPE=1:2), after reaction terminates, filter, solid is successively with watery hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, it is dried, the solid crude product obtained is dissolved in absolute ethyl alcohol and is recrystallized to give lenticular intermediate 10-13.
Compound 10-13, KOH are joined in the mixed solution of methyl alcohol, THF water by step 6., about back flow reaction 5h after.TLC follows the tracks of reaction (solvent VAcOEt:VPE=1:2), after reaction terminates, solution, to acid, is poured in saturated aqueous common salt by watery hydrochloric acid acidifying regulation pH, filtering, solid distilled water washs, and is finally vacuum dried, the solid obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains lenticular compound 14-17.
Step 7. joins compound 14-17 and N methyl piperazine in dichloromethane, and mixing is dissolved, the dropping dichloromethane mixed solution containing DCC/DMAP under about 0 DEG C stirring, after reaction about 0.5h, then reacts about 8h under 35 ± 5 DEG C of stirrings.TLC follows the tracks of reaction (solvent VAcOEt:VPE=1:2), after reaction terminates, reactant liquor is poured in saturated aqueous common salt, filter, solid is successively with watery hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) washing, it is dried, the solid crude product obtained is dissolved in absolute ethyl alcohol and is recrystallized to give lenticular intermediate 18-21.
Under step 8. stirs at-20 ± 5 DEG C, adding the intermediate 18-21 of correspondence, dichloromethane successively, and progressively drip Boron tribromide in round-bottomed flask, after stirring about 1h, room temperature continues reaction about 12h.TLC follows the tracks of reaction (solvent VAcOEt:VN-hexane=1:2), after reaction terminates, filter, solid distilled water washs, and is finally vacuum dried, and the solid obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains lenticular target compound 22-25.
Embodiment one:
N-methyl-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 piperazine (compound 22)
Under stirring at-20 DEG C, corresponding intermediate 18 (10.0mmol), the dichloromethane (25mL) that step 7 obtains is added successively in the round-bottomed flask of 50mL, and after progressively dripping Boron tribromide (5.0mmol) continuation stirring reaction 1h, reaction flask is transferred to room temperature, continues reaction 12h.TLC follows the tracks of reaction (solvent VAcOEt:VN-hexane=1:2), after reaction terminates, filter, solid distilled water washs, and is finally vacuum dried, and the solid obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains lenticular target compound 22.
Obtain white crystal, productivity 47.3%.M.p.221～223 DEG C；1H NMR(DMSO-d6, 300MHz) and δ: 7.98 (d, J=8.1Hz, 2H, ArH), 7.68 (d, J=7.7Hz, 2H, ArH), 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, 6H, CH and CH2), 3.34 (d, J=6.8Hz, 1H, CH), 2.95～2.73 (m, 2H, CH2), 2.31 (t, J=7.7Hz, 4H, CH2),2.18(s,3H,CH3), 2.01 (t, J=5.8Hz, 1H, CH), 1.79～1.21 (m, 15H, CH and CH2),1.14(dd,J1=7.1Hz, J2=7.8Hz, 1H, CH), 0.89 (s, 3H, CH3),0.84(s,3H,CH3).ESI-MS:669.9[M+H]+.Anal.Calcd for C40H52N4O5:C,H,N.
Embodiment two:
N-methyl-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 piperazine (compound 23)
Preparation method reference example one.Obtain white crystal, productivity 45.5%.M.p.226～227 DEG C；1H NMR(DMSO-d6, 300MHz) and δ: 8.03 (d, J=8.3Hz, 2H, ArH), 7.73 (d, J=8.1Hz, 2H, ArH), 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, 6H, CH and CH2), 3.32 (d, J=6.8Hz, 1H, CH), 2.97～2.71 (m, 2H, CH2), 2.28 (t, J=7.7Hz, 4H, CH2),2.15(s,3H,CH3), 2.04 (t, J=5.7Hz, 1H, CH), 1.81～1.20 (m, 15H, CH and CH2),1.14(dd,J1=7.0Hz, J2=7.6Hz, 1H, CH), 0.89 (s, 3H, CH3),0.78(s,3H,CH3).ESI-MS:687.9[M+H]+.Anal.Calcd for C40H51FN4O5:C,H,N.
Embodiment three:
N-methyl-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 piperazine (compound 24).
Preparation method reference example one.Obtain white crystal, productivity 44.9%.M.p.212～213 DEG C；1H NMR(DMSO-d6, 300MHz) and δ: 10.56 (s, 1H, OH), 8.03 (d, J=8.1Hz, 2H, ArH), 7.74～7.55 (m, 5H, ArH and NH), 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, 6H, CH and CH2), 3.34 (d, J=6.8Hz, 1H, CH), 2.95～2.73 (m, 2H, CH2), 2.32 (t, J=7.7Hz, 4H, CH2), 2.01 (t, J=5.4Hz, 1H, CH), 1.79～1.21 (m, 15H, CH and CH2),1.14(dd,J1=7.1Hz, J2=7.9Hz, 1H, CH), 0.84 (s, 3H, CH3),0.81(s,3H,CH3).ESI-MS:687.9[M+H]+.Anal.Calcd for C40H51FN4O5:C,H,N.
Embodiment four:
N-methyl-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 piperazine (compound 25).
Preparation method reference example one.Obtain white crystal, productivity 37.8%.M.p.215～216 DEG C；1H NMR(DMSO-d6, 300MHz) and 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, 6H, CH and CH2), 3.34 (d, J=6.5Hz, 1H, CH), 2.95～2.73 (m, 2H, CH2), 2.30 (t, J=7.7Hz, 4H, CH2), 2.01 (t, J=5.5Hz, 1H, CH), 1.75～1.20 (m, 15H, CH and CH2),1.13(dd,J1=6.6Hz, J2=7.2Hz, 1H, CH), 0.85 (s, 3H, CH3),0.83(s,3H,CH3).ESI-MS:687.9[M+H]+.Anal.Calcd for C40H51FN4O5:C,H,N.
Embodiment five:
MTT [3-(4,5)-bis-methyl-2-thiazoles-(2,5)-phenyl bromination tetrazole is blue] method is used to measure pyrazoline piperazine C21Steroid saponin aglycone derivative is to human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and the half-inhibition concentration (IC of HCC (HepG2)50)。
(1) preparation of nutrient solution (/L): 1. suspension cell: DMEM cultivates one bag of powder (10.4g), NBCS 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, HEPES 2.38g.
(2) preparation of D-Hanks buffer solution (every liter): NaCl 8.00g, KCl 0.40g, Na2HPO4·12H2O 0.06g, KH2PO40.06g, NaHCO30.35g.Autoclaving.
(3) preparation of trypsin solution: utilizing D-Hanks buffer solution to be made into concentration is 0.5% trypsin solution.Filtration sterilization.
(4) preparation of liquid is tested: test sample dissolved with a small amount of tri-distilled water and be made into storing solution, the most empirically 10 times of preparation storing solutions of maximum concentration.Different according to compound dissolubility, 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) human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and the cultivation of HCC (HepG2): for adherent growth cell, cellar culture (containing 10% calf serum, 100U/mL streptomysin), is placed in 37 DEG C, 5%CO in DMEM nutrient solution2Incubator is cultivated, passes on once every 3～4d.Being transferred in centrifuge tube by nutrient solution in former bottle when passing on, 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 (nutrient solution volume is about the 1/10 of blake bottle capacity).
(6) cell incubation: the tumour cell in growth period 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 liquid by design respectively.
(7) dosing: being added separately in each hole according to the concentration gradient of ultimate density by test liquid, each concentration sets 6 parallel holes.Experiment is divided into drug test group (being separately added into the test medicine of variable concentrations), control group (only adding nutrient solution and cell, be not added with testing medicine) and blank group (only adding nutrient solution, 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 MTT 40 μ L (being made into 4mg/mL with D-Hanks buffer solution).After placing 4h at 37 DEG C, remove supernatant.Every hole adds 150 μ L DMSO, and vibrate 5min, makes formazan crystallization dissolve.Finally, automatic ELIASA is utilized to detect the optical density (OD value) in each hole at 570nm wavelength.
Half-inhibition concentration (IC50) it is defined as the drug concentration when the tumor cell survival of 50%.According to the optical density (OD value) measured, make the calibration curve of inhibitory rate of cell growth, calibration curve is tried to achieve the drug concentration of its correspondence.
The IC recorded50It is shown in Table 1.
Pyrazoline piperazine C novel listed by table 1 present invention21The steroid saponin aglycone derivative suppression IC to tumour cell50Value (μm ol/mL)
a6 parallel tests, experimental result averages, and error is between 5-10%
From the above: the pyrazoline piperazine C of the present invention21Steroid saponin aglycone derivative has obvious inhibitory action to human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and HCC (HepG2), especially best to A549 cell, the inhibitory activity of contrast Tenacissoside A 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 piperazine C of the present invention21Steroid saponin aglycone derivative can apply to prepare antineoplastic.
Embodiment six:
The present invention tests the newly synthesized compound 18-21 cytotoxicity to people's renal epithelial cell (293T), and cytotoxicity result such as table 2, using Celecoxib and Tenacissoside A as positive control.The toxicity suppression T cell survival rate of each compound is to concentration (CC when 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 the cell suspension of 1 × 104/mL with cell culture fluid.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 in 5%CO2 cell culture incubator, at a temperature of 37 ± 2 DEG C, cultivates 24h.Discarding original fluid, every hole adds the blank liquid of 100 μ L, negative controls, positive control solution, the test specimen leaching liquor of 100% and 50% concentration.Often organize and at least set 8 holes.Note: extract stoste or make the series extraction dilution of diluent with culture medium.When using 0.9% sodium chloride injection extraction, use, when dilution extraction, the 2 times of culture mediums concentrated.
(3) it is placed in containing in 5%CO2 incubator, cultivates at a temperature of 37 ± 2 DEG C.Cultivate 48h.
(4) after date between each cultivation, every hole adds MTT solution 20 μ L, is placed in containing in 5%CO2 incubator, cultivates 5h at a temperature of 37 ± 2 DEG C.
(5) discarding liquid in hole, every hole is separately added into 200 μ L DMSO, and culture plate is placed 10min, and it is uniform that level rocks solution colour in making hole.
(6) measuring absorbance with ELIASA, wavelength uses 570nm.
The CC recorded50It is shown in Table 2.
Pyrazoline piperazine C novel listed by table 2 present invention21The steroid saponin aglycone derivative suppression CC to 293T cell50Value (μm ol/mL)
a6 parallel tests, experimental result averages, and error is between 5-10%.
The pyrazoline piperazine C of the present invention21Steroid saponin aglycone derivative is to showing quite people's renal epithelial cell (293T) or being better than the cytotoxicity of positive control medicine, and compared with Tenacissoside A, cytotoxicity the most almost maintains an equal level.Therefore the pyrazoline piperazine C of the present invention21Steroid saponin aglycone derivative can apply to prepare antineoplastic.
Inventor thinks: hexahydropyrazine is the hexa-member heterocycle in molecule containing 2 nitrogen-atoms, and its enthalpy of formation is high, Heat stability is good, is the ideal building blocks of azotic heterocyclic compound, has good nitrogen balance symmetrical structure.Introduce piperazine ring in the present invention, as the synergist groups of medicine, the pharmacokinetic property of medicine can be improved, improve the biologically active of medicine.And it also has that onset of action is fast, side reaction is little, toxicity is low, without the feature such as additive.Pyrazoles is the heterocyclic compound that a class is important, has efficient, low toxicity, and the character of the multi-faceted conversion of its ring substituents.
In a word, piperazine skeleton is incorporated in pyrazoline derivative by the present invention, in combination with the C with activity very well21Steroid saponin aglycon skeleton, design has synthesized a series of pyrazoline piperazine C21Steroid saponin aglycone derivative, according to experimental result, it has the advantage such as more preferable biologically active, higher selective and lower 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 be 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 the case of reconcilable, can be combined by any suitable means.In order to avoid unnecessary repetition, various possible combinations are illustrated by the present invention the most separately.Additionally, can also be combined 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 (8)
1. a C21The preparation method of steroid saponin aglycone derivative,
Described C21The structure of steroid saponin aglycone derivative as shown in Formula IX,
Wherein, R1Selected from H, F, R2Selected from H, F, R3Selected from H, F；
It is characterized in that, comprise the steps:
Step 1, preparation, to hydrazino-benzoic acid methyl esters, add structure compound, aqueous hydrochloric acid solution and absolute ethyl alcohol shown in formula I in reactor, prepare structure compound as shown in Formula II after reaction；
Step 2, in reactor, add diazomethane ether mixed liquor, boron trifluoride-ether complex and structure compound as shown in Formula II, after reaction, obtain structure compound as shown in formula III；
Step 3, in reactor, add structure compound as shown in formula III, the KOH aqueous solution, the structure compound as shown in formula IV and ethanol, prepared structure compound shown as a formula V；
Step 4, by structure compound, absolute ethyl alcohol shown as a formula V, hydrazino-benzoic acid methyl esters and glacial acetic acid are joined in reactor, reaction obtains structure compound as shown in Formula IV；
Step 5, structure compound as shown in Formula IV, KOH are joined in the mixed solution of methyl alcohol, THF, after reaction, obtain structure compound as shown in Formula VII；
Step 6, structure compound as shown in Formula VII and N methyl piperazine being joined in dichloromethane, mixing is dissolved, the dropping dichloromethane mixed solution containing DCC/DMAP, and reaction obtains structure compound as shown in Formula VIII；
Step 7, in reactor, add structure compound as shown in Formula VIII, dichloromethane, and drip Boron tribromide, produce target compound；
In formula IV～VIII, R1Selected from H, F, R2Selected from H, F, R3Selected from H, F.
2. C as claimed in claim 121The preparation method of steroid saponin aglycone derivative, it is characterised in that described step 1 is further:
At 0 ± 5 DEG C, absolute methanol, thionyl chloride and to hydrazino-benzoic acid is added successively in reactor, after stirring 30～40min, continuing stirring 3-5h at normal temperatures, filter, the solid obtained is successively with distilled water, ethanol and distilled water washing, it is dried, the solid product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains carboxylate to hydrazino-benzoic acid methyl esters；
Under stirring at room temperature, structure compound shown in formula I, aqueous hydrochloric acid solution, absolute ethyl alcohol, after stirring 2-3h, back flow reaction 7-9h is added successively in reactor, TLC follows the tracks of reaction, after reaction terminates, filtering, the solid obtained is successively with aqueous hydrochloric acid solution, distilled water, cold ethanol, distilled water washing, it is dried, the solid crude product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, and obtains the structure of the lenticular compound as shown in Formula II.
3. C as claimed in claim 121The preparation method of steroid saponin aglycone derivative, it is characterised in that described step 2 is further:
Under ice-water bath stirring action, diazomethane ether mixed liquor, boron trifluoride-ether complex, structure compound as shown in Formula II, after reaction 20～40min, back flow reaction 5-7h is added successively in reactor, TLC follows the tracks of reaction, after reaction terminates, filter, obtain solid successively with aqueous hydrochloric acid solution, distilled water, ethanol, distilled water washing, it is dried, the solid crude product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains structure compound as shown in formula III.
4. C as claimed in claim 121The preparation method of steroid saponin aglycone derivative, it is characterised in that described step 3 is further:
Under stirring at room temperature, structure compound as shown in formula III, the KOH aqueous solution, the structure compound as shown in formula IV and ethanol is added successively in reactor, after continuing stirring reaction 3～6h, it is acidified with hydrochloric acid, regulation pH, filtering, the solid obtained is successively with distilled water, cold ethanol, distilled water washing, dried structure compound shown as a formula V.
5. C as claimed in claim 121The preparation method of steroid saponin aglycone derivative, it is characterised in that described step 4 is further:
Successively by structure compound, absolute ethyl alcohol shown as a formula V, hydrazino-benzoic acid methyl esters 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 follows the tracks of reaction, after reaction terminates, filtering, solid with aqueous hydrochloric acid solution, distilled water, cold ethanol, distilled water washing, is dried successively, the solid product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies, obtains structure compound as shown in Formula IV.
6. C as claimed in claim 121The preparation method of steroid saponin aglycone derivative, it is characterized in that, described step 5 is further: structure compound as shown in Formula IV and KOH are joined in the mixed solution of methyl alcohol, THF water, back flow reaction 4～8h, TLC follows the tracks of reaction, after reaction terminates, being acidified with hydrochloric acid, solution, to acid, is poured in saturated aqueous common salt by regulation pH, filter, solid distilled water washs, and is finally vacuum dried, the solid obtained is dissolved in absolute ethyl alcohol, recrystallization purifies, and obtains structure compound as shown in Formula VII.
7. C as claimed in claim 121The preparation method of steroid saponin aglycone derivative, it is characterised in that described step 6 is further:
Structure compound as shown in Formula VII and N methyl piperazine are joined in dichloromethane, mixing is dissolved, the dropping dichloromethane mixed solution containing DCC/DMAP under 0 ± 5 DEG C of stirring, reaction 20～40min, then 6～10h are reacted under 35 ± 5 DEG C of stirrings, TLC follows the tracks of reaction, after reaction terminates, reactant liquor is poured in saturated aqueous common salt, filtering, solid with aqueous hydrochloric acid solution, distilled water, cold ethanol, distilled water washing, is dried successively, the solid product obtained is dissolved in absolute ethyl alcohol, and recrystallization purifies and obtains structure compound as shown in Formula VIII.
8. C as claimed in claim 121The preparation method of steroid saponin aglycone derivative, it is characterised in that described step 7 is further:
Under stirring at-20 ± 5 DEG C, in reactor, add structure compound as shown in Formula VIII, dichloromethane successively, and drip Boron tribromide, stirring 20～40min, room temperature continues reaction 8～12h, and TLC follows the tracks of reaction, after reaction terminates, filter, solid distilled water washs, and is finally vacuum dried, the solid obtained is dissolved in absolute ethyl alcohol, recrystallization purifies, and obtains the target compound of lenticular.
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| CN104829682A (en) * | 2015-04-29 | 2015-08-12 | 江苏耐雀生物工程技术有限公司 | Furan-skeleton-containing 2H-pyrazole piperazine C21 steroidal saponin aglycone derivatives, preparation method, and applications thereof |
| CN104877002A (en) * | 2015-04-29 | 2015-09-02 | 江苏耐雀生物工程技术有限公司 | C21 steroid sapogenin derivative of dihydro parazole piperazidine, and preparation method and application thereof |
| CN104829683A (en) * | 2015-04-30 | 2015-08-12 | 江苏耐雀生物工程技术有限公司 | Indole skeleton-containing dihydropyrazolpiperazine C21 steroid sapogenin derivative, and preparation method and application thereof |
| CN104861026A (en) * | 2015-04-30 | 2015-08-26 | 江苏耐雀生物工程技术有限公司 | Furan skeleton included 2H-pyrazole hydroxamic acid C21 steroid saponin aglycone derivative and preparation method and application thereof |
| CN104804062A (en) * | 2015-04-30 | 2015-07-29 | 江苏耐雀生物工程技术有限公司 | Pyrazoline piperazine steroid saponin aglycone derivative containing naphthalene skeleton, and preparation method and application of derivative |
| CN104829534A (en) * | 2015-05-20 | 2015-08-12 | 南京大学 | Preparation method of dihydro-pyrazole morpholine derivatives containing naphthalene nucleus frameworks and application of dihydro-pyrazole morpholine derivatives to preparation of antitumor drugs |
| CN105037265A (en) * | 2015-05-20 | 2015-11-11 | 南京大学 | Preparation method of quinolinone derivative containing chalcone framework, and application of the quinolinone derivative in anti-cancer medicines |
| CN104961683A (en) * | 2015-07-10 | 2015-10-07 | 南京大学 | Preparation method of dihydropyrazol piperazine derivatives containing naphthalene ring skeleton and application in anti-cancer drugs |
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