CN105541676B

CN105541676B - A kind of naphthoquinone compound of 2 fourth sulfoxide 1,4

Info

Publication number: CN105541676B
Application number: CN201610046370.8A
Authority: CN
Inventors: 金成浩; 罗英花; 孙虎男; 臧延青; 申贵男; 刘畅; 吴丹丹; 蒋雪园; 孟令旗; 王浩; 徐婉婷
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2016-01-25
Filing date: 2016-01-25
Publication date: 2017-08-04
Anticipated expiration: 2036-01-25
Also published as: CN105541676A

Abstract

The invention discloses a kind of 2 fourth sulfoxide Isosorbide-5-Nitrae naphthoquinone compound, solve 5,8 Isosorbide-5-Nitrae naphthoquinones for not containing any substituent research it is less the problem of.The structural formula of the compound is：.The 2 fourth sulfoxide Isosorbide-5-Nitrae naphthoquinone compounds that the present invention is provided, the 5 of the compound, 8 do not contain any substituent, and 2 are replaced by sulfydryl, and 2 sulphur are oxidized to sulfoxide so that naphthoquinone compound has more superior active anticancer.

Description

A kind of 2- fourths sulfoxide -1,4- naphthoquinone compounds

Technical field

The present invention relates to a kind of new compound.

Background technology

Traditional new drug development approach is to develop and study new drug using effective components from natural materials as lead compound 's.Asian puccoon is that medicinal history is long, the extensive traditional Chinese medicine of pharmacological action, and its main pharmacodynamics composition alkannin is that have very much hair Open up the lead compound of potentiality.Alkannin has anti-inflammatory for the naphthoquinone compound of representative, antibacterial, antiviral, anti-malarial, anti-swollen A variety of physiologically actives such as knurl.Particularly in anticancer research, report that it has and suppressed growth of tumour cell, inducing cell withers Die, suppression DNA topoisomerases, suppression protein tyrosine kinase, a variety of mechanism of action such as anti-angiogenesis.Therefore, naphthoquinones class An always Many researchers class compound interested.

Research for naphthoquinone compound in recent years is concentrated mainly on the 5,8- dihydroxy 1 of 2 substitutions or 6 substitutions, 4- naphthoquinones and 5,8- dimethoxy 1,4-naphthoquinone, and the research to 5,8 1,4-naphthoquinones for not containing any substituent is fewer.

Previous research report display：The naphthaquinone derivatives of 2 sulfydryl substitutions have preferable active anticancer.And 2 sulfydryls The compound of the sulfoxide series active anticancer more superior than the series display of unoxidized sulfydryl in substituted naphthaquinone derivatives.Cause This research and design 5,8 does not contain substituent, and the naphthaquinone derivatives that 2 sulphur are oxidized to sulfoxide are necessary.

The content of the invention

The problem of in order to solve in background technology, the present invention provides a kind of new compound, 2- fourths sulfoxide -1,4-naphthoquinone.

In order to realize foregoing invention purpose, the technical solution adopted by the present invention is：A kind of 2- fourths sulfoxide -1,4- naphthoquinones chemical combination Thing,

It is characterized in that the structural formula of the compound is：。

The preparation method of above-mentioned 2- fourths sulfoxide -1,4- naphthoquinone compounds is：

（1）The synthesis of 2- fourth sulfydryl -1,4- naphthoquinones

1,4-naphthoquinone and methanol are added in reaction vessel, the amount of methanol meets fully dissolving 1,4-naphthoquinone, the two The mol ratio of addition 1- butyl mercaptan after well mixed, 1- butyl mercaptan and 1,4-naphthoquinone is 1.5:1, after reacting 3-5 hours at room temperature, The mol ratio of addition sodium dichromate and the concentrated sulfuric acid into reaction vessel, sodium dichromate and 1,4-naphthoquinone is 1:5, the concentrated sulfuric acid and Isosorbide-5-Nitrae- The mol ratio of naphthoquinones is 3:4, continue to react 5-10 minutes；Then extracted with dichloromethane and saturated aqueous common salt, anhydrous sodium sulfate Dry, filtering is concentrated to dryness, obtains 2- fourths sulfydryl -1,4-naphthoquinone；

（2）The synthesis of 2- fourth sulfoxide -1,4- naphthoquinones

In reaction bulb, step is added（1）Product 2- fourths sulfydryl -1,4-naphthoquinone and chloroform, the amount of chloroform meet fully molten 2- fourths sulfydryl -1,4-naphthoquinone is solved, 3- chloroperoxybenzoic acids, 3- chloroperoxybenzoic acids and 2- fourths sulfydryl-Isosorbide-5-Nitrae-is continuously added The mol ratio of naphthoquinones is 1.2:Reacted 1.5-2.5 hours at a temperature of 1,0 DEG C, 5% NaHCO is added after reaction completely₃Solution is neutralized Terminating reaction after superfluous 3- chloroperoxybenzoic acids in reaction；Reaction product is extracted through dichloromethane and saturated aqueous common salt, anhydrous Sodium sulphate is dried, filtering, is concentrated to dryness, is obtained 2- fourths sulfoxide -1,4-naphthoquinone.

Beneficial effects of the present invention：The present invention provides a kind of new 2- fourths sulfoxide -1,4-naphthoquinone compound, the compound 5,8 do not contain any substituent, and 2 are replaced by sulfydryl, and 2 sulphur are oxidized to sulfoxide so that naphthoquinone compound has There is more superior active anticancer.

Brief description of the drawings

Fig. 1 is lethal effects of the BSNQ to Hep3B cells cell.

Fig. 2 is lethal effects of the BSNQ to human hepatoma HepG2 cell.

Fig. 3 is lethal effects of the BSNQ to human liver cancer Huh7 cells.

Fig. 4 A are, with after BSNQ processing Hep3B cells, to utilize fluorescence microscope Apoptosis situation map.

Fig. 4 B are Fig. 4 A quantitative analysis figures.

Fig. 5 A are, with after BSNQ processing Hep3B cells, to utilize Apoptosis by Flow Cytometry situation map.

Fig. 5 B are Fig. 5 A quantitative analysis figures.

Fig. 6 A are, with after BSNQ processing HepG2 cells, to utilize fluorescence microscope Apoptosis situation map.

Fig. 6 B are Fig. 6 A quantitative analysis figures.

Fig. 7 A are, with after BSNQ processing Huh7 cells, to utilize fluorescence microscope Apoptosis situation map.

Fig. 7 B are Fig. 7 A quantitative analysis figures.

Embodiment

Below in conjunction with the accompanying drawings and specific embodiment the present invention is described further：

Embodiment：The preparation of 2- fourth sulfoxide -1,4- naphthoquinones

（1）The synthesis of 2- fourth sulfydryl -1,4- naphthoquinones

In 100 ml reaction bulbs, the mg of 1,4-naphthoquinone 158.15 (1 mmol) and the ml of methanol 30 is added, after being well mixed The μ l (1.5 mmol) of 1- butyl mercaptan 166 are added, after reacting 4 hours at room temperature, the mg of sodium dichromate 59.6 is added into mixture (0.2 mmol) and the μ l of the concentrated sulfuric acid 40.8 (0.75 mmol), reaction terminates after 5-10 minutes.Through dichloromethane and saturated common salt Water is extracted, appropriate anhydrous sodium sulfate drying, filtering, is concentrated to dryness, is obtained crude product, prepared through TLC, obtains 2- fourths sulfydryl-Isosorbide-5-Nitrae-naphthalene Quinone.

（2）The synthesis of 2- fourth sulfoxide -1,4- naphthoquinones（BSNQ）

In 50 ml reaction bulbs, above-mentioned product 2- fourths sulfydryl-mg of 1,4-naphthoquinone 246.32 (1mmol) and chloroform are added 20 ml, are slowly added into 3- chloroperoxybenzoic acids（MCPBA）276.1 mg (1.2 mmol), two hours are reacted at a temperature of 0 DEG C, 5% NaHCO is added after reaction completely₃Solution, terminating reaction.Extracted through dichloromethane and saturated aqueous common salt, anhydrous sodium sulfate is done Dry, filtering is concentrated to dryness, obtains crude product, prepared through TLC, obtains 2- fourths sulfoxide -1,4-naphthoquinone.

Experimental example

First, lethal effects of the BSNQ to cancer cell

Experimental method：（MTT experiment）

1. inoculating cell：Individual cells suspension is made into the nutrient solution containing 10% tire calf serum, it is 10000 thin with every hole Born of the same parents are inoculated into 96 orifice plates, are 200 μ l per pore volume；

2. cell is cultivated：5% CO₂, 37 DEG C are incubated 24 h, and bottom hole is paved with to cell monolayer；

3. serum starvation：Nutrient solution is changed before the h of dosing 2（Nutrient solution containing 1% FBS）；

4. drug-treated：The BSNQ prepared is taken final concentration of 0,1,3,10,20,30,40,50,60 respectively, 70,80, 100 μM of processing Hep3B cells, the h of HepG2 and Huh7 cells 24；

5. color reaction：Add MTT solution per hole（5 mg/ml, are prepared with PBS, pH 7.4）20 μl.Continue to be incubated 2-4 h Afterwards, careful inhale abandons culture supernatant in hole, is carefully washed with PBS 2 times, then adds 100 μ l dimethyl sulfoxide (DMSO)s per hole（DMSO）, Concussion 10 minutes, makes crystallization fully dissolve；

6. colorimetric：490 nm wavelength are selected, each hole absorbance value is determined on enzyme-linked immunosorbent assay instrument, result is recorded, with Time is abscissa, and light absorption value is that ordinate draws cell growth block diagram, as a result sees Fig. 1-Fig. 3 and table 1.

Interpretation of result：

It can be seen that BSNQ in Fig. 1-Fig. 3（IC₅₀: 1.40 μM）All have to Hep3B cells, HepG2 and Huh7 There is good killing ability, it kills intensity and gradually risen with the increase of drug concentration.

By table 1 below it can also be seen that BSNQ（IC₅₀: 1.40 μM）All have to Hep3B cells, HepG2 and Huh7 good Good killing ability, it kills intensity and gradually risen with the increase of drug concentration.

ICs of the BSNQ of table 1 to human liver cancer cell lethal effect₅₀Value

2nd, apoptotic effects of the BSNQ to cancer cell

Experimental method：（Experiment in vitro-Annexin-V decoration methods）

1. inoculating cell：Individual cells suspension is made into the nutrient solution containing 10% tire calf serum, it is 10,000 thin with every hole Born of the same parents are inoculated into 12 orifice plates, are 1 ml per pore volume；

2. cell is cultivated：5% CO2,37 DEG C of 24 h of incubation, bottom hole is paved with to cell monolayer；

3. drug-treated：Add the BSNQ prepared（IC₅₀: 1.40 μM）, handle different time（0,3,6,12,24 h）；

4. washed with PBS 2 times, add 195 μ l Annexin V-FITC combination liquid, add 5 μ l Annexin V- FITC is gently mixed；

5. 10 μ l propidium iodides are added（Propidium Iodide, PI）Dyeing liquor, is gently mixed；

6. room temperature（20-25℃）Lucifuge is incubated 15 minutes；

⑦（A）In the change of the form and color of fluorescence microscopy Microscopic observation cell, green fluorescence is Annexin V- FITC staining positive cells, red fluorescence is propidium iodide positive cells.Only dyed by green fluorescence, and the cell of small volume For apoptotic cell；By red or green and red double dyes, and the larger cell of volume is non-viable non-apoptotic cell；The cell not being colored is Normal cell.200 cells of random observation, try to achieve the percentage shared by various cells, and each sample counting is averaged for 3 times；

（B）Meanwhile, also detect apoptosis of the BSNQ to human liver cancer cell using Flow Cytometry methods.

1st, Hep3B cells, apoptosis of the detection BSNQ to Hep3B cells cell are handled with BSNQ

Using above-mentioned experimental method, obtained experimental result is shown in Fig. 4 A, Fig. 4 B, Fig. 5 A and Fig. 5 B, and wherein Fig. 4 A are to use After BSNQ processing Hep3B cells, using fluorescence microscope Apoptosis situation map, shikonin and 5-FU are positive right According to group；Fig. 4 B are Fig. 4 A quantitative analysis figures.Fig. 5 A are to be handled with BSNQ after Hep3B cells, thin using Flow cytometry Born of the same parents' apoptosis situation map；Fig. 5 B are Fig. 5 A quantitative analysis figures.

Interpretation of result

Use BSNQ（Final concentration of 1.40 μM）Handle after the h of Hep3B cells cell 0,3,6,12,24, carry out Annexin The double dye experiments of V-FITC/PI, and in fluorescence microscope.As can be seen that continuous with drug exposure times from Fig. 4 B Increase, Annexin V-FITC fluorescence intensities also gradually strengthen, and its Apoptosis degree is also dramatically increased.It is 24 especially when the time During h, the fluorescence intensity highest of cell.As a result illustrate, BSNQ can effectively induce the apoptosis of Hep3B cells, and in Time Dependent Property.

Use BSNQ（Final concentration of 1.40 μM）Handle after the h of Hep3B cells cell 0,3,6,12,24, carry out Annexin V-FITC and PI are marked, and pass through Apoptosis by Flow Cytometry situation.As can be seen that at medicine from Fig. 5 B The reason time is continuously increased, and the degree of liver cancer Hep3B Apoptosis is consequently increased, especially when drug exposure times reach 24 h When, the level of apoptosis of cell substantially increases.The result shows, BSNQ can effectively induce Hep3B apoptosis, and in the time according to Lai Xing.

2nd, HepG2 cells, apoptosis of the detection BSNQ to human hepatoma HepG2 cell are handled with BSNQ

Using above-mentioned experimental method, obtained experimental result is shown in that Fig. 6 A and Fig. 6 B, wherein Fig. 6 A are with BSNQ processing After HepG2 cells, using fluorescence microscope Apoptosis situation map, hikonin and 5-FU are positive controls；Fig. 6 B are The quantitative analysis figure of 6A figures.

Interpretation of result

Use BSNQ（Final concentration of 1.40 μM）Handle after the h of human hepatoma HepG2 cell 0,3,6,12,24, carry out Annexin The double dye experiments of V-FITC/PI, and in fluorescence microscope.As can be seen that continuous with drug exposure times from Fig. 6 B Increase, Annexin V-FITC fluorescence intensities also gradually strengthen, and its Apoptosis degree is also dramatically increased.It is 24 especially when the time During h, the fluorescence intensity highest of cell.BSNQ can effectively induce the apoptosis of HepG2 cells, and in time dependence.

3rd, Huh7 cells, apoptosis of the detection BSNQ to human liver cancer Huh7 cells are handled with BSNQ

Using above-mentioned experimental method, obtained experimental result is shown in that Fig. 7 A and Fig. 7 B, wherein Fig. 7 A are to handle Huh7 with BSNQ After cell, using fluorescence microscope Apoptosis situation map, shikonin and 5-FU are positive controls；Fig. 7 B are Fig. 7 A Quantitative analysis figure.

Interpretation of result

Use BSNQ（Final concentration of 1.40 μM）Handle after the h of human liver cancer Huh7 cells 0,3,6,12,24, carry out Annexin The double dye experiments of V-FITC/PI, and in fluorescence microscopy Microscopic observation.As can be seen that from Fig. 7 B with drug exposure times not Disconnected increase, Annexin V-FITC fluorescence intensities also gradually strengthen, and its Apoptosis degree is also dramatically increased.Especially it is when the time During 24 h, the fluorescence intensity highest of cell.

In summary, BSNQ（IC₅₀: 1.40 μM）Hep3B cells, the apoptosis of HepG2 and Huh7 cells can be induced, Its cancer cell-apoptosis ability is increased over time and gradually risen.

Claims

1. a kind of 2- fourths sulfoxide -1,4-naphthoquinone compound is used to prepare the application in the medicine for the treatment of liver cancer, the knot of the compound Structure formula is：

。