CN105541679B

CN105541679B - A kind of naphthoquinone compound of 2 pungent sulfoxide 1,4

Info

Publication number: CN105541679B
Application number: CN201610046339.4A
Authority: CN
Inventors: 金成浩; 臧延青; 罗英花; 孙虎男; 申贵男; 刘畅; 蒋雪园; 吴丹丹; 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: 2017-05-31
Anticipated expiration: 2036-01-25
Also published as: CN105541679A

Abstract

The invention discloses a kind of 2 pungent sulfoxide Isosorbide-5-Nitrae naphthoquinone compounds, the less problem of the research of 5,8 Isosorbide-5-Nitrae naphthoquinones for not containing any substitution base is solved.It is characterized in that the structural formula of the compound is：.The 2 pungent sulfoxide Isosorbide-5-Nitrae naphthoquinone compounds that the present invention is provided, the 5 of the compound, 8 do not contain any substitution base, 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 pungent sulfoxide -1,4- naphthoquinone compounds of 2-

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 is that the naphthoquinone compound of representative has anti-inflammatory, antibacterial, antiviral, anti-malarial, anti-swollen Various physiologically actives such as knurl.Particularly in anticancer research, report that it has suppression growth of tumour cell, inducing cell withers Die, suppress DNA topoisomerases, suppress various mechanism of action such as protein tyrosine kinase, anti-angiogenesis.Therefore, naphthoquinones class An always Many researchers class compound interested.

The 5,8- dihydroxy 1 of 2 substitutions or 6 substitutions is concentrated mainly on for the research of naphthoquinone compound in recent years, 4- naphthoquinones and 5,8- dimethoxy 1,4-naphthoquinone, and the research that 5,8 are not contained with the 1,4-naphthoquinone of any substitution base 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 is free of substituted base, and the naphthaquinone derivatives that 2 sulphur is oxidized to sulfoxide are necessary.

The content of the invention

In order in solving the problems, such as background technology, the present invention provides a kind of pungent sulfoxide -1,4-naphthoquinones of new compound 2-.

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

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

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

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

The amount of 1,4-naphthoquinone and methyl alcohol, methyl alcohol is added to meet fully dissolving 1,4-naphthoquinone in reaction vessel, the two 1- spicy thioalcohols are added after well mixed, 1- spicy thioalcohols are 1.5 with the mol ratio of 1,4-naphthoquinone:1, after reacting 3-5 hours at room temperature, To sodium dichromate and the concentrated sulfuric acid is added in reaction vessel, sodium dichromate is 1 with the mol ratio of 1,4-naphthoquinone: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, and obtains the pungent sulfydryl -1,4-naphthoquinones of 2-；

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

In reaction bulb, step is added（1）Pungent sulfydryl-the 1,4-naphthoquinones of product 2- and chloroform, the amount of chloroform meet fully molten The solution pungent sulfydryl -1,4-naphthoquinones of 2-, continuously add 3- chloroperoxybenzoic acids, 3- chloroperoxybenzoic acids and 2- fourths sulfydryl-Isosorbide-5-Nitrae - 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；Product is extracted through dichloromethane and saturated aqueous common salt, anhydrous Sodium sulphate is dried, filtering, is concentrated to dryness, and obtains the pungent sulfoxide -1,4-naphthoquinones of 2-.

Beneficial effects of the present invention：The present invention provides a kind of pungent sulfoxide -1,4-naphthoquinones of new compound 2-, the compound 5,8 do not contain any substitution base, 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 OSNQ to Hep3B cells cell.

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

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

Fig. 4 A are after processing Hep3B cells with OSNQ, using fluorescence microscope Apoptosis situation map.

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

Fig. 5 A are after processing Hep3B cells with OSNQ, using Apoptosis by Flow Cytometry situation map.

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

Fig. 6 A are after processing HepG2 cells with OSNQ, using fluorescence microscope Apoptosis situation map.

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

Fig. 7 A are after processing Huh7 cells with OSNQ, using fluorescence microscope Apoptosis situation map.

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

Specific embodiment

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

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

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

In 100 ml reaction bulbs, the mg of 1,4-naphthoquinone 158.15 (1 mmol) and the ml of methyl alcohol 30 are added, after being well mixed The μ l (1.5 mmol) of 1- spicy thioalcohols 266.6 are added, after reacting 4 hours at room temperature, to addition sodium dichromate 59.6 in mixture Mg (0.2 mmol) and the μ l of the concentrated sulfuric acid 40.8 (0.75 mmol), reaction terminate after 5-10 minutes.Through dichloromethane and saturation Saline solution is extracted, appropriate anhydrous sodium sulfate drying, filtering, is concentrated to dryness, and obtains crude product, is prepared through TLC, obtains pungent sulfydryl -1 of 2-, 4- naphthoquinones.

（2）The synthesis of the pungent sulfoxide -1,4- naphthoquinones of 2-（OSNQ）

In 50 ml reaction bulbs, the above-mentioned pungent mg of sulfydryl -1,4-naphthoquinone 302.43 (1mmol) of product 2- and chloroform are added 20 ml, are slowly added into 3- chloroperoxybenzoic acids（MCPBA）276.1 mg (1.2 mmol), react two hours 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, and obtains crude product, is prepared through TLC, obtains the pungent sulfoxide -1,4-naphthoquinones of 2-.

Experimental example

First, lethal effects of the OSNQ to cancer cell

Experimental technique：（MTT experiment）

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

2. cultured cells：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 that will be prepared takes final concentration of 0,1,3,10,20,30,40,50,60 respectively, 70,80, 100 μM for the treatment of Hep3B cells, the h of HepG2 and Huh7 cells 24；

5. color reaction：Add MTT solution per hole（5 mg/ml, are prepared, pH 7.4 with PBS）20 μl.Continue to be incubated 2-4 h Afterwards, careful suction 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, record result, 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 OSNQ 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 is gradually risen with the increase of drug concentration.

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

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

2nd, apoptotic effects of the OSNQ to cancer cell

Experimental technique：（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 thin with 10,000, every hole Born of the same parents are inoculated into 12 orifice plates, are 1 ml per pore volume；

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

3. drug-treated：The BSNQ that addition is prepared（IC₅₀: 1.40 μM）, process 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, gently mixes；

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

⑦（A）The change of the form and color of observation of cell under fluorescence microscope, green fluorescence is Annexin V- FITC staining positive cells, red fluorescence is propidium iodide positive cells.Only dyeed by green fluorescence, and small volume cell It is 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 OSNQ to human liver cancer cell using Flow Cytometry methods.

1st, Hep3B cells, apoptosis of the detection OSNQ to Hep3B cells cell are processed with OSNQ

Using above-mentioned experimental technique, the experimental result for obtaining 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 OSNQ treatment Hep3B cells, using fluorescence microscope Apoptosis situation map, shikonin and 5-FU is positive right According to group；Fig. 4 B are the quantitative analysis figures of Fig. 4 A.Fig. 5 A be with OSNQ process Hep3B cells after, it is thin using Flow cytometry Born of the same parents' apoptosis situation map；Fig. 5 B are the quantitative analysis figures of Fig. 5 A.

Interpretation of result

Use OSNQ（Final concentration of 1.40 μM）After the h for the treatment of Hep3B cells cell 0,3,6,12,24, Annexin is carried out 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.Result illustrates that OSNQ can effectively induce the apoptosis of Hep3B cells, and in Time Dependent Property.

Use OSNQ（Final concentration of 1.40 μM）After the h for the treatment of Hep3B cells cell 0,3,6,12,24, Annexin is carried out V-FITC and PI are marked, by 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, OSNQ can effectively induce the apoptosis of Hep3B, and in the time according to Lai Xing.

2nd, HepG2 cells, apoptosis of the detection OSNQ to human hepatoma HepG2 cell are processed with OSNQ

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

Interpretation of result

Use OSNQ（Final concentration of 1.40 μM）After treatment human hepatoma HepG2 cell 0,3,6,12,24 h, Annexin is carried out 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.OSNQ can effectively induce the apoptosis of HepG2 cells, and in time dependence.

3rd, Huh7 cells, apoptosis of the detection OSNQ to human liver cancer Huh7 cells are processed with OSNQ

Using above-mentioned experimental technique, the experimental result for obtaining is shown in that Fig. 7 A and Fig. 7 B, wherein Fig. 7 A are to process Huh7 with OSNQ After cell, using fluorescence microscope Apoptosis situation map, shikonin and 5-FU is positive controls；Fig. 7 B are Fig. 7 A Quantitative analysis figure.

Interpretation of result

Use OSNQ（Final concentration of 1.40 μM）After the h for the treatment of human liver cancer Huh7 cells 0,3,6,12,24, Annexin is carried out 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 to 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 sum, OSNQ（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 pungent sulfoxides of 2- -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：

。