CN104804063A - Pyrazoline hydroxamic acid steroid saponin aglycone derivative containing thiophene skeleton, as well as preparation method and application of derivative - Google Patents
Pyrazoline hydroxamic acid steroid saponin aglycone derivative containing thiophene skeleton, as well as preparation method and application of derivative Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
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- C07J71/0005—Oxygen-containing hetero ring
- C07J71/001—Oxiranes
Abstract
The invention discloses a pyrazoline hydroxamic acid steroid saponin aglycone derivative containing a thiophene skeleton, as well as a preparation method and an application of the derivative. The pyrazoline hydroxamic acid steroid saponin aglycone derivative containing the thiophene skeleton has a structural formula as shown as Formula VIII as shown in the specification, wherein R1 is selected from H and CH3; and R2 is selected from H and CH3. The derivative has an obvious inhibiting effect on human breast cancer cells (MCF-7), cervical cancer cells (HeLa), lung cancer cells (A549) and liver cancer cells (HepG2), shows equivalent or better cytotoxicity than a positive control drug Celecoxib for human kidney epithelial cells (293T), and can be applied to preparation of an anti-tumor drug.
Description
Technical field
The invention belongs to medicinal chemistry art, particularly relate to containing the pyrazoline hydroxamic acid steroidal saponin aglycone derivative of thiophene skeleton, its preparation method and application.
Background technology
C21 steroidal saponin is through the steroidal saponin constituents that extraction separation and purification obtains from Chinese medicine Caulis Marsdeniae Tenacissimae, and its aglycon is by unique steroidal saponin structure of 21 C atomic buildings, is therefore called C21 steroidal saponin.The pharmacologically active such as the compound with C21 steroidal saponin parent nucleus has immunomodulatory, relieving asthma, has obtained clinically as the index activeconstituents of medicine clinically and has applied more widely.
Further investigate discovery through applicant, the compound with C21 steroidal saponin parent nucleus may have good antineoplastic activity.The current research contents to this type of C21 steroidal saponin mainly contains: carry out separation and purification and analyzing and testing research according to its physico-chemical property to it, carry out structural modification research for its parent nucleus, carry out pharmacology activity research etc. for its constitutional features.
But also there is biological activity and the shortcoming such as selectivity is lower, toxicity is higher in existing product.
Summary of the invention
Goal of the invention a: object is to provide a kind of pyrazoline hydroxamic acid steroidal saponin aglycone derivative containing thiophene skeleton, to solve the problems referred to above that prior art exists.Further object is to provide the preparation method of this steroidal saponin aglycone derivative.
Technical scheme: a kind of pyrazoline hydroxamic acid steroidal saponin aglycone derivative containing thiophene skeleton, its structure such as formula shown in VIII,
Wherein, R
1be selected from H, CH
3; R
2be selected from H, CH
3.
Prepare a method for the pyrazoline hydroxamic acid steroidal saponin aglycone derivative containing thiophene skeleton, the structure of the described pyrazoline hydroxamic acid steroidal saponin aglycone derivative containing thiophene skeleton such as formula shown in VIII,
Wherein, R
1be selected from H, CH
3; R
2be selected from H, CH
3;
Preparation method comprises the steps:
Step 1. under stirring at room temperature, adds structure successively such as formula the compound shown in I, aqueous hydrochloric acid and dehydrated alcohol in reaction vessel, fully after reaction, extracts structure such as formula the compound shown in II;
Step 2., under ice-water bath stirs, add diazomethane ether mixed solution, boron trifluoride-ether complex successively, and structure is such as formula the compound shown in II in reaction vessel, extracts the compound of structure as shown in formula III;
Step 3. under stirring at room temperature, adds the compound of structure as shown in formula III, the KOH aqueous solution and structure successively such as formula the compound shown in IV, produces structure such as formula the compound shown in V in reaction vessel;
Step 4. successively by structure such as formula the compound shown in V, dehydrated alcohol, add in reaction vessel to hydrazino-benzoic acid methyl esters and Glacial acetic acid, extract structure after reaction such as formula the compound shown in VI;
Structure joins in the hydroxylamine solution of activation such as formula the compound shown in VI by step 5., fully reacts, and obtained structure is such as formula the compound shown in VII;
Step 6. adds structure successively such as formula the compound shown in VII and methylene dichloride, and drips boron tribromide under stirring at-20 ± 5 DEG C in reaction vessel, and obtained structure is such as formula the compound shown in VIII;
In formula IV ~ formula VII, R
1be selected from H, CH
3; R
2be selected from H, CH
3.
In a further embodiment, described step 1 is further:
Under stirring at room temperature, structure is added successively such as formula the compound shown in I, aqueous hydrochloric acid and dehydrated alcohol in reaction vessel, after stirring 1 ± 0.5h, back flow reaction 7 ~ 9h, TLC follow the tracks of reaction, reaction terminates rear filtering reacting liquid, the solid obtained uses aqueous hydrochloric acid, distilled water, ethanol and distilled water wash successively, dry, and the solid product of acquisition is dissolved in dehydrated alcohol, recrystallization is purified, and obtains structure such as formula the compound shown in II.
Further preferred, churning time is 0.55,0.8,0.85,1h, reflux time is 7.6,7.8,8.3,8.8h, the mol ratio of reactant is I:HCl=1:8 ~ 11, and preferred mol ratio is: 1:9.3.
Preferred described step 2 is further:
Under ice-water bath stirs, diazomethane ether mixed solution, boron trifluoride-ether complex is added successively in reaction vessel, and structure is such as formula the compound shown in II, after reaction 20 ~ 40min, back flow reaction 5-7h, TLC follows the tracks of reaction, filter after reaction and the solid product obtained is used aqueous hydrochloric acid, distilled water, cold ethanol and distilled water wash successively, dry, the solid product obtained is dissolved in dehydrated alcohol, recrystallization is purified, and namely obtains the compound of structure as shown in formula III.
Further preferred, the reaction times is 22,23,26,36min, reflux time is 5.6,5.8,6.2,6.8h, the mol ratio of reactant is II: diazomethane: boron trifluoride=1:1.2 ~ 1.3:1.2 ~ 1.3.Preferred mol ratio is: 1:1.25:1.25.
Described step 3 is further:
Under stirring at room temperature, the compound of structure as shown in formula III, the KOH aqueous solution and structure is added successively such as formula the compound shown in IV in reaction vessel, continue stirring reaction 3 ~ 6 hours, use salt acid for adjusting pH, filter, the solid obtained is used distilled water, ethanol and distilled water wash successively, produces structure such as formula the compound shown in V.
Further preferred, churning time is 3.5,5.3,5.6,5.7h, the mol ratio of reactant is III:IV is 1:1.
Described step 4 is further:
Successively by structure such as formula the compound shown in V, dehydrated alcohol, add in reaction vessel to hydrazino-benzoic acid methyl esters and Glacial acetic acid, stirring at room temperature reaction 0.5-1h, back flow reaction 5-6h, TLC follow the tracks of reaction, and reaction terminates rear filtration, obtain solid product, use solid product described in aqueous hydrochloric acid, distilled water, ethanol and distilled water wash successively, dry, by solid product solution dehydrated alcohol, recrystallization is purified, and obtains structure such as formula the compound shown in VI.
Further preferred, churning time is 35,45,48,55min, reflux time is 5.3,5.5,5.8h, the mol ratio of reactant is: V: to hydrazino-benzoic acid methyl esters: Glacial acetic acid=1:1.1 ~ 1.3:0.8 ~ 1.Preferred mol ratio is: 1:1.2:0.88.
Described step 5 is further:
By equimolar hydrochloric acid hydroxyl by adding in anhydrous methanol with sodium methylate, after heated and stirred 20-40min, filters solid, obtains the hydroxylamine solution activated, is adding structure wherein such as formula the compound shown in VI, back flow reaction 3-5h, TLC follows the tracks of reaction, and reaction terminates rear filtration and the solid distilled water wash that will obtain, then solid product is dissolved in dehydrated alcohol, recrystallization is purified, and obtained structure is such as formula the compound shown in VII.
Further preferred, stir 20,30,40min, back flow reaction 3.2,4.2,4.8h, the mol ratio of reactant is: VI: hydrochloric acid hydroxyl is pressed: sodium methylate 1:4 ~ 5:4 ~ 5.
Described step 6 is further:
Under stirring at-20 ± 5 DEG C, in reaction vessel, add structure successively such as formula the compound shown in VII and methylene dichloride, and drip boron tribromide, stir 0.5-1h, at room temperature continue reaction 12 ± 1h, TLC follows the tracks of reaction, and reaction terminates rear filtration, will filter the solid distilled water wash obtained, vacuum-drying, solid is dissolved in dehydrated alcohol, and recrystallization is purified, and obtains target compound.
Further preferred, churning time is 0.7,0.8,0.9h, the reaction times is 11.5,11.8,12.2h, the mol ratio of reactant is: VII: boron tribromide=1:2 ~ 3.Preferred mol ratio is: 1:2.
Present method is transformed for saponin(e aglycon, has effectively integrated above-mentioned each skeleton structure, and assay reproducibility is strong, good stability, and the required condition of experiment reaction is comparatively simple, and experimental situation is gentle, and productive rate is better, can produce in a large number in less input situation.
Pyrazoline hydroxamic acid steroidal saponin aglycone derivative containing thiophene skeleton is preparing the application in cancer therapy drug, its structure such as formula shown in VIII,
Wherein, R
1be selected from H, CH
3; R
2be selected from H, CH
3.
A kind of cancer therapy drug, comprises structure such as formula the compound shown in VIII and medically acceptable carrier,
Wherein, R
1be selected from H, CH
3; R
2be selected from H, CH
3.
Beneficial effect: the present invention has obvious restraining effect to human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and liver cancer cell (HepG2), shows quite people's renal epithelial cell (293T) simultaneously or is better than the cytotoxicity of positive control medicine Celecoxib.Therefore the pyrazoline hydroxamic acid C containing thiophene skeleton of the present invention
21steroidal saponin aglycone derivative can be applied to prepares antitumor drug.
Embodiment
In certain specific embodiment, the structural formula of preparation process of the present invention and associated products is as described below:
A kind of above-mentioned pyrazoline hydroxamic acid C containing thiophene skeleton
21the synthesis of steroidal saponin aglycone derivative, it comprises the following steps:
Step 1. is at 0 ± 5 DEG C, add in round-bottomed flask successively anhydrous methanol, sulfur oxychloride, to hydrazino-benzoic acid 1, after stirring about 1h, normal temperature continues to stir 3-5h, filter, the solid obtained uses distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) to wash successively, dry, the solid crude product obtained is dissolved in dehydrated alcohol recrystallization and obtains carboxylate to hydrazino-benzoic acid methyl esters 2.
Step 2. under stirring at room temperature, adds Tenacissoside A (a kind of C successively in round-bottomed flask
21steroidal saponin) 3, dilute hydrochloric acid, dehydrated alcohol, stir after 1h, back flow reaction 7-9h.TLC follows the tracks of reaction (developping agent V
acOEt: V
pE=1:2), after reaction terminates, filter, the solid obtained uses dilute hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) to wash successively, drying, is dissolved in the lenticular C that dehydrated alcohol recrystallization obtains partial reduction by the solid crude product obtained
21steroidal saponin aglycon 4.
Step 3., under ice-water bath stirring action, adds diazomethane ether mixed solution, boron trifluoride-ether complex, C successively in round-bottomed flask
21steroidal saponin aglycon 4, after reaction about 0.5h, then back flow reaction 5-7h.TLC follows the tracks of reaction (developping agent V
acOEt: V
pE=1:2), after reaction terminates, filter, obtaining solid uses dilute hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) to wash successively, drying, is dissolved in dehydrated alcohol recrystallization by the solid crude product obtained and obtains lenticular intermediate 5.
Step 4. under stirring at room temperature, intermediate 5, the KOH aqueous solution, the thiophene-2-formaldehyde 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 uses distilled water (3 × 100mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) to wash successively, dry intermediate 6-8.
Step 5., successively by different compound 6-8, dehydrated alcohol, join in round-bottomed flask to hydrazino-benzoic acid methyl esters 2, Glacial acetic acid, after stirring at room temperature reaction 1h, still has fraction solids insoluble; Then back flow reaction 5h, TLC follow the tracks of reaction (developping agent V
acOEt: V
pE=1:2), after reaction terminates, filter, solid uses dilute hydrochloric acid (3 × 100mL), distilled water (3 × 150mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) to wash successively, drying, is dissolved in dehydrated alcohol recrystallization and obtains lenticular intermediate 9-11 by the solid crude product obtained.
Equimolar hydrochloric acid hydroxyl adds in anhydrous methanol by, sodium methylate by step 6., and after heated and stirred 0.5h, filters solid, obtains the hydroxylamine solution activated, and is adding corresponding crystal intermediate 9-11 wherein, back flow reaction 3-5h.TLC follows the tracks of reaction (developping agent V
acOEt: V
pE=1:2), after reaction terminates, filter, solid distilled water wash, last vacuum-drying, is dissolved in dehydrated alcohol by the solid obtained, and recrystallization is purified, and obtains lenticular target compound 12-14.
Step 7. adds corresponding intermediate 12-14, methylene dichloride successively, and progressively drips boron tribromide under stirring at-20 ± 5 DEG C in round-bottomed flask, and after stirring about 1h, room temperature continues reaction about 12h.TLC follows the tracks of reaction (developping agent V
acOEt: V
normal hexane=1:2), after reaction terminates, filter, solid distilled water wash, last vacuum-drying, is dissolved in dehydrated alcohol by the solid obtained, and recrystallization is purified, and obtains lenticular target compound 15-17.
Embodiment one:
4-(5-(2-thiophene)-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 15)
Under stirring at-20 DEG C, corresponding intermediate 12 (10.0mmol), methylene dichloride (25mL) that step 6 obtains is added successively in the round-bottomed flask of 50mL, and progressively drip after boron tribromide (5mmol) continues stirring reaction 1h, reaction flask is transferred to room temperature, continues reaction 12h.TLC follows the tracks of reaction (developping agent V
acOEt: V
normal hexane=1:2), after reaction terminates, filter, solid distilled water wash, last vacuum-drying, the solid obtained is dissolved in the purification of dehydrated alcohol recrystallization and obtains lenticular target compound.
Obtain white crystal, productive rate 44.8%.m.p.197～198℃；
1H NMR(DMSO-d
6,300MHz)δ:10.54(s,1H,OH),8.04(d,J＝7.8Hz,2H,ArH),7.76(d,J＝7.9Hz,2H,ArH),7.71(s,1H,NH),7.34(d,J＝6.8Hz,1H,CH),7.01～6.82(m,2H,CH),5.32(s,2H,OH),4.48(s,1H,OH),3.90(t,J＝7.3Hz,1H,CH),3.53(dd,J
1＝4.7,J
2＝4.8Hz,1H,CH),3.41(t,J＝8.1Hz,1H,CH),3.33(d,J＝6.8Hz,1H,CH),2.79(dd,J
1＝4.7,J
2＝3.4Hz,2H,CH
2),2.01(t,J＝7.1Hz,1H,CH),1.79～1.21(m,15H,CH and CH
2),1.14(dd,J
1＝7.2Hz,J
2＝7.1Hz,1H,CH),0.89(s,3H,CH
3),0.84(s,3H,CH
3).ESI-MS:608.8[M+H]
+.Anal.Calcd for C
33H
41N
3O
6S:C,H,N.
Embodiment two:
4-(5-(2-(5-methyl-thiophene))-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 16)
Obtain white crystal, productive rate 43.9%.m.p.199～201℃；
1H NMR(DMSO-d
6,300MHz)δ:10.49(s,1H,OH),8.01(d,J＝7.9Hz,2H,ArH),7.73(d,J＝7.3Hz,2H,ArH),7.68(s,1H,NH),6.80(d,J＝6.7Hz,1H,CH),6.55(d,J＝5.8Hz,1H,CH),5.34(s,2H,OH),4.55(s,1H,OH),3.88(t,J＝6.6Hz,1H,CH),3.54(dd,J
1＝4.7,J
2＝4.8Hz,1H,CH),3.44(t,J＝8.1Hz,1H,CH),3.34(d,J＝6.8Hz,1H,CH),2.79(dd,J
1＝3.7,J
2＝3.4Hz,2H,CH
2),2.37(s,3H,CH
3),2.01(t,J＝7.1Hz,1H,CH),1.85～1.23(m,15H,CH and CH
2),1.14(dd,J
1＝7.2Hz,J
2＝7.1Hz,1H,CH),0.89(s,3H,CH
3),0.84(s,3H,CH
3).ESI-MS:622.8[M+H]
+.Anal.Calcd forC
34H
43N
3O
6S:C,H,N.
Embodiment three:
4-(5-(2-(3-methyl-thiophene))-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 17)
Preparation method's reference example one.Obtain white crystal, productive rate 55.5%.m.p.251～253℃；
1H NMR(DMSO-d
6,300MHz)δ:10.52(s,1H,OH),8.05(d,J＝7.8Hz,2H,ArH),7.73(d,J＝7.9Hz,2H,ArH),7.67(s,1H,NH),7.40(d,J＝5.4Hz,1H,CH),6.94(d,J＝5.9Hz,1H,CH),5.35(s,2H,OH),4.49(s,1H,OH),3.86(t,J＝6.7Hz,1H,CH),3.54(dd,J
1＝4.7,J
2＝4.8Hz,1H,CH),3.44(t,J＝8.1Hz,1H,CH),3.34(d,J＝6.8Hz,1H,CH),2.79(dd,J
1＝3.7,J
2＝3.4Hz,2H,CH
2),2.28(s,3H,CH
3),2.01(t,J＝7.1Hz,1H,CH),1.79～1.20(m,15H,CH and CH
2),1.14(dd,J
1＝7.2Hz,J
2＝7.1Hz,1H,CH),0.89(s,3H,CH
3),0.84(s,3H,CH
3).ESI-MS:622.8[M+H]
+.Anal.Calcd for C
34H
43N
3O
6S:C,H,N.
Embodiment four:
Containing the pyrazoline hydroxamic acid C of thiophene skeleton
21steroidal saponin aglycone derivative anti tumor activity in vitro is about the research of anti-tumour cell proliferative
Employing MTT [3-(4,5)-bis-methyl-2-thiazole-(2,5)-phenyl bromination tetrazole is blue] method measures the pyrazoline hydroxamic acid C containing thiophene skeleton
21steroidal saponin aglycone derivative is to the half-inhibition concentration (IC of human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and liver cancer cell (HepG2)
50).
(1) preparation of nutrient solution (/L): 1. suspension cell: DMEM cultivates one bag, 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 adjust pH, to 7.2-7.4, is finally settled to 1000mL.Filtration sterilization.2. attached cell: the same, then add NaHCO
32.00g, HEPES 2.38g.
(2) preparation of D-Hanks damping fluid (often liter): NaCl 8.00g, KCl 0.40g, Na
2hPO
412H
2o 0.06g, KH
2pO
40.06g, NaHCO
30.35g.Autoclaving.
(3) preparation of trypsin solution: utilizing D-Hanks damping fluid to be made into concentration is 0.5% trypsin solution.Filtration sterilization.
(4) preparation of 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 adds tri-distilled water and dissolve.Storing solution is stored in-20 DEG C of refrigerators for subsequent use.
(5) cultivation of human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and liver cancer cell (HepG2): be adherent growth cell, cellar culture is (containing 10% calf serum, 100U/mL Streptomycin sulphate) in DMEM nutrient solution, is placed in 37 DEG C, 5%CO
2cultivate in incubator, go down to posterity once every 3-4d.When going down to posterity, nutrient solution in former bottle is transferred in centrifuge tube, the centrifugal 5min of 1000rpm, discard original fluid, add equivalent fresh medium, piping and druming evenly, pipette appropriate in fresh culture bottle, then supplement fresh medium to original volume (nutrient solution volume is about 1/10 of culturing bottle capacity).
(6) cell incubation: the tumour cell in vegetative period of taking the logarithm, tune concentration of cell suspension is 1-1.5 × 10
5individual/mL.In 96 well culture plates, every hole adds cell suspension 100 μ L, puts 37 DEG C, 5%CO
224h is cultivated in incubator.After cultivating 24h, add liquid by design respectively.
(7) dosing: join in each hole by test liquid respectively according to the concentration gradient of ultimate density, each concentration establishes 6 parallel holes.Experiment is divided into drug test group (adding the test medicine of different concns respectively), control group (only add nutrient solution and cell, do not add test medicine) and blank group (only add nutrient solution, do not add cell and test medicine).96 orifice plates after dosing are placed in 37 DEG C, 5%CO
248h is cultivated in incubator.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 damping fluid).After placing 4h at 37 DEG C, remove supernatant liquor.Every hole adds 150 μ L DMSO, and vibration 5min, makes formazan dissolving crystallized.Finally, automatic microplate reader is utilized to detect the optical density(OD) (OD value) in each hole at 570nm wavelength place.
Half-inhibition concentration (IC
50) be defined as drug level when the tumor cell survival of 50%.According to the optical density(OD) (OD value) measured, make the typical curve of inhibitory rate of cell growth, typical curve is tried to achieve the drug level of its correspondence.
The IC recorded
50be shown in Table 1.
The listed pyrazoline hydroxamic acid C containing thiophene skeleton of table 1 the present invention
21steroidal saponin aglycone derivative is to the suppression IC of tumour cell
50value (μm ol/mL)
a6 parallel tests, experimental result is averaged, and error is between 5-10%
From above-mentioned experiment: the present invention has obvious restraining effect to human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and liver cancer cell (HepG2), compared with control group, activity significantly improves.
Embodiment five:
Containing the pyrazoline hydroxamic acid C of thiophene skeleton
21steroidal saponin aglycone derivative anti tumor activity in vitro is about Cytotoxic research
The present invention tests new synthetic compound 15-17 to the cytotoxicity of people's renal epithelial cell (293T), cytotoxicity result as table 2, using Celecoxib as positive control.The toxicity suppressor T cell survival rate of each compound to 50% time concentration (CC
50) represent.
Experimental technique:
(1) cultivator renal epithelial cell (293T) is tending towards fusion until reach its logarithmic growth end of term cell, digests cell dispersion, be mixed with 1 × 10 with cell culture fluid with cell dissociation buffer
4the cell suspension of individual/mL.Get 96 well culture plates, in every hole, add the cell suspension of 100 μ L.Horizontally rotating culture plate gently makes cell be evenly dispersed in the surface in ware hole.
(2) be placed in containing 5%CO
2in cell culture incubator, at 37 ± 2 DEG C of temperature, cultivate 24h.Discard original fluid, every hole adds the blank liquid of 100 μ L, negative controls, positive control solution, the test sample vat liquor of 100% and 50% concentration.Often organize and at least establish 8 holes.Note: lixiviate stoste or make the serial lixiviate diluent of thinner with substratum.When adopting 0.9% sodium chloride injection lixiviate, use 2 times of concentrated substratum when diluting lixiviate.
(3) be placed in containing 5%CO
2in 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%CO
2in incubator, at 37 ± 2 DEG C of temperature, cultivate 5h.
(5) discard liquid in hole, every hole adds 200 μ L DMSO respectively, and culture plate is placed 10min, and level is rocked and made solution colour in hole even.
(6) measure absorbancy by microplate reader, wavelength adopts 570nm.
The CC recorded
50be shown in Table 2.
The listed pyrazoline hydroxamic acid C21 steroidal saponin aglycone derivative containing thiophene skeleton of table 2 the present invention is to the suppression CC of 293T cell
50value (μm ol/mL)
a6 parallel tests, experimental result is averaged, and error is between 5-10%
From above-mentioned experiment: the present invention shows quite people's renal epithelial cell (293T) or is better than the cytotoxicity of positive control medicine, may be used for producing antitumor drug.
In a word, applicant thinks after deliberation afterwards: thiophene is sulfur heterocyclic ring compound, has cellular cytoxicity activity when being exposed to long wave ultraviolet light.Photochemistry Study shows, the phototoxicity of thiophene is mainly because create single oxygen of toxicity by II type photodynamic processes.Hydroxamic acid also claims hydroxamic acid, has two kinds of tautomers, i.e. hydroximic acid and hydroxamic acid, is a kind of organic sequestering agent, energy and Cu
2+, Zn
2+, Co
2+, Ni
2+, Fe
3+ions etc. form stable metallo-chelate.Pyrazoles is the important heterogeneous ring compound of a class, has efficient, low toxicity, and the character of the multi-faceted conversion of its ring substituents, and is used widely in pharmaceutical field.Pyrazoline is very important nitrogenous five member ring heterocyclic compound, and it has a lot of excellent biological activity, is a structural presence subunit with various pharmacological property, there is the active compound guide of general medicinal organism activity.In addition, because mostly pyrazoline is the conformation of the replacement on ring and molecule can be caused to have larger polytropy, thus, have chirality better biological activity potential quality.
The present invention is carrying out on the basis of separation and purification research to the chemical composition of Chinese medicine Caulis Marsdeniae Tenacissimae, is separated to obtain to have C
21the compound Caulis Marsdeniae Tenacissimae glycosides A (Tenacissoside A) of steroidal saponin parent nucleus, molecular formula is C
48h
74o
19, molecular weight is 954, and purity is 99.2%; With Caulis Marsdeniae Tenacissimae glycosides A for initial compounds, structural modification research is carried out to it, obtain the pyrazoline hydroxamic acid C of a class novelty
21steroidal saponin aglycone derivative, and by pharmacological research, result shows that this compounds has significant anti-tumor activity.
Based on this, the present invention will have outstanding bioactive thiophene skeleton and Hydroxamic acid is incorporated in pyrazoline derivative, combines simultaneously and has C active very well
21steroidal saponin aglycon skeleton, a series of pyrazoline hydroxamic acid C containing thiophene skeleton of design and synthesis
21steroidal saponin aglycone derivative, experimental result shows that this compound has better biological activity, higher selectivity, lower toxicity and the shorter longevity of residure etc.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple equivalents to technical scheme of the present invention, these equivalents all belong to protection scope of the present invention.It should be noted that in addition, each the concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode.In order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1., containing the pyrazoline hydroxamic acid steroidal saponin aglycone derivative of thiophene skeleton, it is characterized in that, its structure such as formula shown in VIII,
Wherein, R
1be selected from H, CH
3; R
2be selected from H, CH
3.
2. prepare a method for the pyrazoline hydroxamic acid steroidal saponin aglycone derivative containing thiophene skeleton, it is characterized in that, the structure of the described pyrazoline hydroxamic acid steroidal saponin aglycone derivative containing thiophene skeleton such as formula shown in VIII,
Wherein, R
1be selected from H, CH
3; R
2be selected from H, CH
3;
Preparation method comprises the steps:
Step 1. under stirring at room temperature, adds structure successively such as formula the compound shown in I, aqueous hydrochloric acid and dehydrated alcohol in reaction vessel, fully after reaction, extracts structure such as formula the compound shown in II;
Step 2., under ice-water bath stirs, add diazomethane ether mixed solution, boron trifluoride-ether complex successively, and structure is such as formula the compound shown in II in reaction vessel, extracts the compound of structure as shown in formula III;
Step 3. under stirring at room temperature, adds the compound of structure as shown in formula III, the KOH aqueous solution and structure successively such as formula the compound shown in IV, produces structure such as formula the compound shown in V in reaction vessel;
Step 4. successively by structure such as formula the compound shown in V, dehydrated alcohol, add in reaction vessel to hydrazino-benzoic acid methyl esters and Glacial acetic acid, extract structure after reaction such as formula the compound shown in VI;
Structure joins in the hydroxylamine solution of activation such as formula the compound shown in VI by step 5., fully reacts, and obtained structure is such as formula the compound shown in VII;
Step 6. adds structure successively such as formula the compound shown in VII and methylene dichloride, and drips boron tribromide under stirring at-20 ± 5 DEG C in reaction vessel, and obtained structure is such as formula the compound shown in VIII;
In formula IV ~ formula VII, R
1be selected from H, CH
3; R
2be selected from H, CH
3.
3. preparation as claimed in claim 2 is containing the method for the pyrazoline hydroxamic acid steroidal saponin aglycone derivative of thiophene skeleton, and it is characterized in that, described step 1 is further:
Under stirring at room temperature, structure is added successively such as formula the compound shown in I, aqueous hydrochloric acid and dehydrated alcohol in reaction vessel, after stirring 1 ± 0.5h, back flow reaction 7 ~ 9h, TLC follow the tracks of reaction, reaction terminates rear filtering reacting liquid, the solid obtained uses aqueous hydrochloric acid, distilled water, ethanol and distilled water wash successively, dry, and the solid product of acquisition is dissolved in dehydrated alcohol, recrystallization is purified, and obtains structure such as formula the compound shown in II.
4. preparation as claimed in claim 2 is containing the method for the pyrazoline hydroxamic acid steroidal saponin aglycone derivative of thiophene skeleton, and it is characterized in that, described step 2 is further:
Under ice-water bath stirs, diazomethane ether mixed solution, boron trifluoride-ether complex is added successively in reaction vessel, and structure is such as formula the compound shown in II, after reaction 20 ~ 40min, back flow reaction 5-7h, TLC follows the tracks of reaction, filter after reaction and the solid product obtained is used aqueous hydrochloric acid, distilled water, cold ethanol and distilled water wash successively, dry, the solid product obtained is dissolved in dehydrated alcohol, recrystallization is purified, and namely obtains the compound of structure as shown in formula III.
5. preparation as claimed in claim 2 is containing the method for the pyrazoline hydroxamic acid steroidal saponin aglycone derivative of thiophene skeleton, and it is characterized in that, described step 3 is further:
Under stirring at room temperature, the compound of structure as shown in formula III, the KOH aqueous solution and structure is added successively such as formula the compound shown in IV in reaction vessel, continue stirring reaction 3 ~ 6 hours, use salt acid for adjusting pH, filter, the solid obtained is used distilled water, ethanol and distilled water wash successively, produces structure such as formula the compound shown in V.
6. preparation as claimed in claim 2 is containing the method for the pyrazoline hydroxamic acid steroidal saponin aglycone derivative of thiophene skeleton, and it is characterized in that, described step 4 is further:
Successively by structure such as formula the compound shown in V, dehydrated alcohol, add in reaction vessel to hydrazino-benzoic acid methyl esters and Glacial acetic acid, stirring at room temperature reaction 0.5-1h, back flow reaction 5-6h, TLC follow the tracks of reaction, and reaction terminates rear filtration, obtain solid product, use solid product described in aqueous hydrochloric acid, distilled water, ethanol and distilled water wash successively, dry, by solid product solution dehydrated alcohol, recrystallization is purified, and obtains structure such as formula the compound shown in VI.
7. preparation as claimed in claim 2 is containing the method for the pyrazoline hydroxamic acid steroidal saponin aglycone derivative of thiophene skeleton, and it is characterized in that, described step 5 is further:
By equimolar hydrochloric acid hydroxyl by adding in anhydrous methanol with sodium methylate, after heated and stirred 20-40min, filters solid, obtains the hydroxylamine solution activated, is adding structure wherein such as formula the compound shown in VI, back flow reaction 3-5h, TLC follows the tracks of reaction, and reaction terminates rear filtration and the solid distilled water wash that will obtain, then solid product is dissolved in dehydrated alcohol, recrystallization is purified, and obtained structure is such as formula the compound shown in VII.
8. preparation as claimed in claim 2 is containing the method for the pyrazoline hydroxamic acid steroidal saponin aglycone derivative of thiophene skeleton, and it is characterized in that, described step 6 is further:
Under stirring at-20 ± 5 DEG C, in reaction vessel, add structure successively such as formula the compound shown in VII and methylene dichloride, and drip boron tribromide, stir 0.5-1h, at room temperature continue reaction 12 ± 1h, TLC follows the tracks of reaction, and reaction terminates rear filtration, will filter the solid distilled water wash obtained, vacuum-drying, solid is dissolved in dehydrated alcohol, and recrystallization is purified, and obtains target compound.
9. containing the pyrazoline hydroxamic acid steroidal saponin aglycone derivative of thiophene skeleton preparing the application in cancer therapy drug, it is characterized in that, its structure such as formula shown in VIII,
Wherein, R
1be selected from H, CH
3; R
2be selected from H, CH
3.
10. a cancer therapy drug, is characterized in that, comprises structure such as formula the compound shown in VIII and medically acceptable carrier,
Wherein, R
1be selected from H, CH
3; R
2be selected from H, CH
3.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104558094A (en) * | 2015-02-04 | 2015-04-29 | 江苏耐雀生物工程技术有限公司 | Sapogenin derivative, preparation method of derivative and application thereof in preparation of antitumor drugs |
Non-Patent Citations (2)
| Title |
|---|
| ANABEL ROMERO-LÓPEZ, ET AL.: "Synthesis of steroidal derivatives containing substituted, fused and spiro pyrazolines", 《STEROIDS》 * |
| SHRAVANKUMAR KANKALA, ET AL.: "Synthesis and anti-cancer evaluation of steroidal diglycoside–pyrazoline hybrids", 《RSC ADVANCES》 * |
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