CN104829685A - Furan skeleton-containing dihydropyrazolsulfanilamide C21 steroid sapogenin derivative, and preparation method and application thereof - Google Patents

Furan skeleton-containing dihydropyrazolsulfanilamide C21 steroid sapogenin derivative, and preparation method and application thereof Download PDF

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CN104829685A
CN104829685A CN201510219525.9A CN201510219525A CN104829685A CN 104829685 A CN104829685 A CN 104829685A CN 201510219525 A CN201510219525 A CN 201510219525A CN 104829685 A CN104829685 A CN 104829685A
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formula
reaction
compound shown
successively
pyrazoline
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杨永安
魏元刚
金显友
钟慧
朱海亮
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JIANGSU NAIQUE BIOLOGICAL ENGINEERING TECHNOLOGY Co Ltd
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JIANGSU NAIQUE BIOLOGICAL ENGINEERING TECHNOLOGY Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/001—Oxiranes
    • C07J71/0021—Oxiranes at position 14(15)

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Abstract

The invention discloses a furan skeleton-containing dihydropyrazolsulfanilamide C21 steroid sapogenin derivative, and a preparation method and an application thereof. The structure of the furan skeleton-containing dihydropyrazolsulfanilamide C21 steroid sapogenin derivative is represented by formula VII shown in the specification; and in the formula VII, R1 is selected from H and CH3, and R2 is selected from H and F. The derivative has an obvious inhibition effect on human breast cancer cells (MCF-7), cervical carcinoma cells (HeLa), lung cancer cells (A549) and liver cancer cells (HepG2), has same or better cytotoxicity on human renal epithelial cells (293T) than positive control drug Celecoxib, and can be used to prepare antitumor drugs.

Description

Containing the pyrazoline sulfanilamide (SN) C of furans skeleton 21steroidal saponin aglycone derivative, its preparation method and application
Technical field
The invention belongs to medicinal chemistry art, particularly relate to the pyrazoline sulfanilamide (SN) C containing furans skeleton 21steroidal saponin aglycone derivative, 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 sulfanilamide (SN) C containing furans skeleton 21steroidal saponin aglycone derivative, 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; 3rd object, is to provide this steroidal saponin aglycone derivative and is preparing the application in cancer therapy drug.
Technical scheme: a kind of pyrazoline sulfanilamide (SN) C containing furans skeleton provided by the invention 21steroidal saponin aglycone derivative, its structure such as formula shown in VII,
Wherein, R 1be selected from H, CH 3; R 2be selected from H, F.
A kind of pyrazoline sulfanilamide (SN) C prepared containing furans skeleton that the present invention provides simultaneously 21the method of steroidal saponin aglycone derivative, the described pyrazoline sulfanilamide (SN) C containing furans skeleton 21the structure of steroidal saponin aglycone derivative such as formula shown in VII,
Wherein, R 1be selected from H, CH 3; R 2be selected from H, F;
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 Hydrazinobenzenesulfonamide and acetic acid, extract structure after reaction such as formula the compound shown in VI;
Step 5. adds structure successively such as formula the compound shown in VI and methylene dichloride, and drips boron tribromide under stirring at-25 ~-15 DEG C in reaction vessel, and obtained structure is such as formula the compound shown in VII;
In formula IV ~ formula VI, R 1be selected from H, CH 3; R 2be selected from H, F.
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 0.5 ~ 1.5h, 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, cold ethanol and distilled water wash successively, dry, and the solid product of acquisition is dissolved in dehydrated alcohol, recrystallization, obtains structure such as formula the compound shown in II.Further preferred, churning time is 0.55,0.8,1,1.2h, 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.Preferably, TLC is adopted to follow the tracks of developping agent V when reacting acOEt: V pE=1:2.
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, 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.Preferably, TLC is adopted to follow the tracks of developping agent V when reacting acOEt: V pE=1:2.
Described step 3 is further:
Under stirring at room temperature, the compound of structure as shown in formula III, the KOH aqueous solution, structure is added successively such as formula the compound shown in IV and ethanol in reaction vessel, continue stirring reaction 3 ~ 6 hours, pH to 6 ~ 7 are regulated with dilute hydrochloric acid, filter, the solid obtained is used successively distilled water, cold ethanol and distilled water wash, dry, obtain 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 Hydrazinobenzenesulfonamide and 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, cold ethanol and distilled water wash successively, dry, by solid product solution dehydrated alcohol, recrystallization, 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.Preferably, reactant mol ratio for: structure is such as formula the compound shown in V: to Hydrazinobenzenesulfonamide: acetic acid=1:1.2 ~ 1.5:0.5.Preferred mol ratio is 1:1.2:0.5,1:1.5:0.5.Preferably, TLC is adopted to follow the tracks of developping agent V when reacting acOEt: V pE=1:2.
Described step 5 is further:
Under stirring at-25 ~-15 DEG C, in reaction vessel, add structure successively such as formula the compound shown in VI and methylene dichloride, and drip boron tribromide, stir 0.5 ~ 1h, at room temperature continue reaction 11 ~ 13h, 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 such as formula the target compound shown in VII.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: VI: boron tribromide=1:2 ~ 3.Preferred mol ratio is 1:2.Preferably, TLC is adopted to follow the tracks of developping agent V when reacting acOEt: V normal hexane=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 sulfanilamide (SN) C containing furans skeleton provided by the invention 21steroidal saponin aglycone derivative is preparing the application in cancer therapy drug, its structure such as formula shown in VII,
Wherein, R 1be selected from H, CH 3; R 2be selected from H, F.
A kind of cancer therapy drug that the present invention provides simultaneously, comprises structure such as formula the compound shown in VII and medically acceptable carrier,
Wherein, R 1be selected from H, CH 3; R 2be selected from H, F.
Beneficial effect: 1, the pyrazoline sulfanilamide (SN) C containing furans skeleton provided by the present invention 21steroidal saponin aglycone derivative novel structure, compared with related compound in prior art, has better biological activity, higher selectivity, lower toxicity and the shorter longevity of residure.2, synthetic route provided by the present invention be easy to realize and reproducible.3, compound provided by the invention can be applied to antitumor drug, it has obvious restraining effect to human breast cancer cell (MCF-7), cervical cancer cell (HeLa), lung carcinoma cell (A549) and liver cancer cell (HepG2), show quite people's renal epithelial cell (293T) simultaneously or be better than the cytotoxicity of positive control medicine Celecoxib, toxicity is less.Therefore the pyrazoline sulfanilamide (SN) C containing furans skeleton provided by the 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 sulfanilamide (SN) C containing furans skeleton 21the synthesis of steroidal saponin aglycone derivative, it comprises the following steps:
Step 1. under stirring at room temperature, adds Tenacissoside A (a kind of C successively in round-bottomed flask 21steroidal saponin) 1, dilute hydrochloric acid, dehydrated alcohol, stir after 1h, then 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 2.
Step 2., under ice-water bath stirring action, adds diazomethane ether mixed solution (50mL), boron trifluoride-ether complex, C successively in 100mL round-bottomed flask 21steroidal saponin aglycon 2, 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 3.
Step 3. under stirring at room temperature, intermediate 3, the KOH aqueous solution, the furans-2-formaldehyde of different substituents, ethanol is added successively in round-bottomed flask, after continuing stirring reaction 4h, dilute hydrochloric acid acidifying regulates pH to 6 ~ 7, filter, the solid obtained uses distilled water (3 × 100mL), cold ethanol (3 × 50mL), distilled water (3 × 100mL) to wash successively, dry intermediate 4-6.
Step 4., successively by different compound 4-6, dehydrated alcohol, join in round-bottomed flask to Hydrazinobenzenesulfonamide, 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 7-9 by the solid crude product obtained.
Step 5. adds corresponding intermediate 7-9, methylene dichloride successively, and progressively drips boron tribromide under stirring at-25 ~-15 DEG C in round-bottomed flask, and after stirring reaction 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 10-12.
Embodiment one: 4-(5-(2-furans)-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 benzsulfamide (compound 10)
Under stirring at-20 DEG C, corresponding intermediate 7 (10.0mmol), methylene dichloride (25mL) that step 4 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 VAcOEt:V normal hexane=1:2), after reaction terminates, filters, 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 48.9%.m.p.196～197℃；1H NMR(DMSO-d6,300MHz)δ:8.01(d,J＝7.4Hz,2H,ArH),7.74(d,J＝7.9Hz,2H,ArH),7.56(d,J＝6.8Hz,1H,CH),6.89(s,2H,NH2),6.40(dd,J1＝4.2,J2＝3.8Hz,2H,CH),5.60(t,J＝4.9Hz,1H,CH),5.39(s,2H,OH),4.48(s,1H,OH),3.54(dd,J1＝4.7,J2＝4.2Hz,1H,CH),3.44(t,J＝8.1Hz,1H,CH),3.34(d,J＝6.8Hz,1H,CH),2.79(dd,J1＝4.8,J2＝5.1Hz,2H,CH2),2.01(t,J＝7.1Hz,1H,CH),1.77～1.26(m,15H,CH and CH2),1.14(dd,J1＝7.2Hz,J2＝7.1Hz,1H,CH),0.89(s,3H,CH3),0.84(s,3H,CH3).ESI-MS:612.8[M+H]+.Anal.Calcd for C32H41N3O7S:C,H,N.
Embodiment two: 4-(5-(2-(5-methyl-ribofuranosyl))-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 benzsulfamide (compound 11)
Preparation method is with embodiment one (adopting the furans-2-formaldehyde with embodiment one different substituents).Obtain white crystal, productive rate 44.2%.m.p.200～201℃；1H NMR(DMSO-d6,300MHz)δ:8.03(d,J＝7.5Hz,2H,ArH),7.73(d,J＝7.6Hz,2H,ArH),6.88(s,2H,NH2),6.17(d,J＝5.2Hz,1H,CH),6.02(d,J＝5.6Hz,1H,CH),5.58(t,J＝4.4Hz,1H,CH),5.39(s,2H,OH),4.48(s,1H,OH),3.54(dd,J1＝4.7,J2＝4.2Hz,1H,CH),3.44(t,J＝8.1Hz,1H,CH),3.34(d,J＝6.8Hz,1H,CH),2.79(dd,J1＝4.8,J2＝5.1Hz,2H,CH2),2.20(s,3H,CH3),2.01(t,J＝7.1Hz,1H,CH),1.72～1.31(m,15H,CH and CH2),1.13(dd,J1＝7.2Hz,J2＝7.1Hz,1H,CH),0.84(s,3H,CH3),0.80(s,3H,CH3).ESI-MS:626.8[M+H]+.Anal.Calcd for C33H43N3O7S:C,H,N.
Embodiment three: 4-(5-(2-(the fluoro-furans of 5-))-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 benzsulfamide (compound 12)
Preparation method is with embodiment one (adopting the furans-2-formaldehyde with embodiment one different substituents).Obtain white crystal, productive rate 53.1%.m.p.215～216℃；1H NMR(DMSO-d6,300MHz)δ:7.94(d,J＝7.1Hz,2H,ArH),7.77(d,J＝7.9Hz,2H,ArH),7.68(d,J＝5.1Hz,1H,CH),6.87(s,2H,NH2),6.50(t,J＝5.5Hz,1H,CH),5.58(t,J＝5.3Hz,1H,CH),5.34(s,2H,OH),4.44(s,1H,OH),3.58(dd,J1＝4.5,J2＝4.1Hz,1H,CH),3.44(t,J＝8.1Hz,1H,CH),3.34(d,J＝6.8Hz,1H,CH),2.79(dd,J1＝3.7,J2＝3.4Hz,2H,CH2),2.01(t,J＝7.1Hz,1H,CH),1.79～1.21(m,15H,CH2 and CH),1.14(dd,J1＝7.2Hz,J2＝7.1Hz,1H,CH),0.89(s,3H,CH3),0.84(s,3H,CH3).ESI-MS:630.7[M+H]+.Anal.Calcd for C32H40FN3O7S:C,H,N.
Embodiment four: containing the pyrazoline sulfanilamide (SN) C of furans 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 sulfanilamide (SN) C containing furans 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 sulfanilamide (SN) C containing furans 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 sulfanilamide (SN) C of furans skeleton 21steroidal saponin aglycone derivative anti tumor activity in vitro is about Cytotoxic research
The present invention tests new synthetic compound 10-12 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 sulfanilamide (SN) C21 steroidal saponin aglycone derivative containing furans 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 finds with experiment after deliberation: furans is oxygen containing heterogeneous ring compound, and many furfuran compounds are all the natural components in plant, and its isolated effective constituent from plant demonstrates has good biological activity.Sulfa drugs is widely used in antibacterial, hypotensive, diuresis etc., but such medicine is fungistat, and without germicidal action, easily produce resistance and often use can produce many untoward reactions, thus its range of application is greatly limited.Because it easily develops immunity to drugs, use range reduces gradually.But sulfa drugs has multiple biological activity, research finds that its derivative has otherwise activity except antibacterial, is wherein anti-tumor activity the most significantly.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 sulfanilamide (SN) 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 furans skeleton and sulfanilamide (SN) group is incorporated in pyrazoline derivative, combine simultaneously and have C active very well 21steroidal saponin aglycon skeleton, a series of pyrazoline sulfanilamide (SN) C containing furans 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 (9)

1. the pyrazoline sulfanilamide (SN) C containing furans skeleton 21steroidal saponin aglycone derivative, is characterized in that, its structure such as formula shown in VII,
Wherein, R 1be selected from H, CH 3; R 2be selected from H, F.
2. prepare the pyrazoline sulfanilamide (SN) C containing furans skeleton for one kind 21the method of steroidal saponin aglycone derivative, is characterized in that, the described pyrazoline sulfanilamide (SN) C containing furans skeleton 21the structure of steroidal saponin aglycone derivative such as formula shown in VII,
Wherein, R 1be selected from H, CH 3; R 2be selected from H, F;
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 Hydrazinobenzenesulfonamide and acetic acid, extract structure after reaction such as formula the compound shown in VI;
Step 5. adds structure successively such as formula the compound shown in VI and methylene dichloride, and drips boron tribromide under stirring at-25 ~-15 DEG C in reaction vessel, and obtained structure is such as formula the compound shown in VII;
In formula IV ~ formula VI, R 1be selected from H, CH 3; R 2be selected from H, F.
3. preparation as claimed in claim 2 is containing the pyrazoline sulfanilamide (SN) C of furans skeleton 21the method of steroidal saponin aglycone derivative, 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 0.5 ~ 1.5h, 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, cold ethanol and distilled water wash successively, dry, and the solid product of acquisition is dissolved in dehydrated alcohol, recrystallization, obtains structure such as formula the compound shown in II.
4. preparation as claimed in claim 2 is containing the pyrazoline sulfanilamide (SN) C of furans skeleton 21the method of steroidal saponin aglycone derivative, 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, obtains the compound of structure as shown in formula III.
5. preparation as claimed in claim 2 is containing the pyrazoline sulfanilamide (SN) C of furans skeleton 21the method of steroidal saponin aglycone derivative, 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, structure is added successively such as formula the compound shown in IV and ethanol in reaction vessel, continue stirring reaction 3 ~ 6 hours, pH is regulated to be 6 ~ 7, filter, the solid obtained is used successively distilled water, cold ethanol and distilled water wash, dry, obtain structure such as formula the compound shown in V.
6. preparation as claimed in claim 2 is containing the pyrazoline sulfanilamide (SN) C of furans skeleton 21the method of steroidal saponin aglycone derivative, 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 Hydrazinobenzenesulfonamide and 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, cold ethanol and distilled water wash successively, dry, by solid product solution dehydrated alcohol, recrystallization, obtains structure such as formula the compound shown in VI.
7. preparation as claimed in claim 2 is containing the pyrazoline sulfanilamide (SN) C of furans skeleton 21the method of steroidal saponin aglycone derivative, is characterized in that, described step 5 is further:
Under stirring at-25 ~-15 DEG C, in reaction vessel, add structure successively such as formula the compound shown in VI and methylene dichloride, and drip boron tribromide, stir 0.5 ~ 1h, at room temperature continue reaction 11 ~ 13h, 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 such as formula the target compound shown in VII.
8. containing the pyrazoline sulfanilamide (SN) C of furans skeleton 21steroidal saponin aglycone derivative, preparing the application in cancer therapy drug, is characterized in that, its structure such as formula shown in VII,
Wherein, R 1be selected from H, CH 3; R 2be selected from H, F.
9. a cancer therapy drug, is characterized in that, comprises structure such as formula the compound shown in VII and medically acceptable carrier,
Wherein, R 1be selected from H, CH 3; R 2be selected from H, F.
CN201510219525.9A 2015-04-30 2015-04-30 Furan skeleton-containing dihydropyrazolsulfanilamide C21 steroid sapogenin derivative, and preparation method and application thereof Pending CN104829685A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104530021A (en) * 2015-02-04 2015-04-22 江苏耐雀生物工程技术有限公司 Compounds and preparation method thereof, application of compounds in preparing antineoplastic drugs and antineoplastic drugs prepared from compounds

Patent Citations (1)

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CN104530021A (en) * 2015-02-04 2015-04-22 江苏耐雀生物工程技术有限公司 Compounds and preparation method thereof, application of compounds in preparing antineoplastic drugs and antineoplastic drugs prepared from compounds

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Application publication date: 20150812