CN107721893B - 2- naphthalene sulfydryl -5,8- dimethoxy -1,4- naphthoquinone compound and preparation method thereof and drug containing it - Google Patents

Abstract

2- naphthalene sulfydryl -5,8- dimethoxy -1,4- naphthoquinone compound and preparation method thereof and drug containing it；Belong to pharmaceutical technology field.The compounds of this invention structural formula are as follows:The method of the present invention: 5,8- dimethoxys -1,4-naphthoquinone (DMNQ) is dissolved in methanol, and 2- naphthalene sulfydryl is added, and stirs 4h, and the concentrated sulfuric acid and Na is added₂Cr₂O₇·2H₂O stirs 2min, with saturation NaCl and CHCl₃Extraction, anhydrous sodium sulfate dehydration, is concentrated and dried, column Chromatographic purification.The application of the compound of the present invention and pharmaceutical composition in the drug of preparation treatment gastric cancer.

Description

2- naphthalene sulfydryl -5,8- dimethoxy -1,4- naphthoquinone compound and preparation method thereof and contain Its drug

Technical field

The invention belongs to pharmaceutical technology fields；More particularly to a kind of 2- naphthalene sulfydryl -5,8- dimethoxy -1,4- naphthoquinones chemical combination Object, preparation method and using the compound as the pharmaceutical composition of active constituent.

Background technique

Traditional new drug development approach is to develop using effective components from natural materials as lead compound and study new drug 's.Asian puccoon is that medicinal history is long, the extensive traditional Chinese medicine of pharmacological action, and main pharmacodynamics ingredient alkannin right and wrong are often with there is hair Open up the lead compound of potentiality.Alkannin is that the naphthoquinone compound of representative has anti-inflammatory, antibacterial, antiviral, anti-malarial, resists and swell A variety of physiological activity such as tumor.Especially in anticancer research, reports that it has and inhibited growth of tumour cell, inducing cell withers It dies, inhibits DNA topoisomerase, inhibit protein tyrosine kinase, a variety of mechanism of action such as anti-angiogenesis.Therefore, naphthoquinones class The always interested a kind of compound of Many researchers.

The drug of clinical treatment gastric cancer is divided into fluorouracil drug (Tegafur, excellent fudding, 5- by the difference of the mechanism of action Fluorouracil etc.), cell cycle specific drugs (mitomycin, cis-platinum, adriamycin etc.), molecular targeted agents (toltrazuril list Anti-, Avastin, Cetuximab etc.).Currently, clinical treatment cancer is for the purpose of killing cancer cell, but chemotherapy does not have There is resolving ability, often while killing cancer cell, kill a large amount of normal cell, it is de- hair occur by patient after multiple chemotherapy Fall, lean body mass, functions of intestines and stomach disorder, nausea and vomiting, the symptoms such as low fever does not move back, these be clinically curing gastric cancer drug it is general Time defect.

Summary of the invention

The present invention provides a kind of 2- naphthalene sulfydryl -5,8- dimethoxy -1,4-naphthoquinone compound, preparation method and with this Compound is the pharmaceutical composition of active constituent.Present invention reduces the toxic side effect of compound itself, and irritation and toxicity It is low.Also there is certain targeting to cancer cell, it is lower to the lethal effect of normal cell.

In order to solve the above technical problems, 2- naphthalene sulfydryl -5,8- dimethoxy -1,4-naphthoquinone compound structure of the invention Formula are as follows:Preparation method carries out in the steps below:

5,8- dimethoxys -1,4-naphthoquinone (DMNQ) is dissolved in methanol, and 2- naphthalene sulfydryl is added, and stirs 4h, dense sulphur is added Acid and Na₂Cr₂O₇·2H₂O stirs 2min, with saturation NaCl and CHCl₃Extraction, anhydrous sodium sulfate dehydration, is concentrated and dried, column layer Analysis purification, obtains 2- naphthalene sulfydryl -5,8- dimethoxy -1,4-naphthoquinone.

It further limits, the molar ratio of 5, the 8- dimethoxy -1,4-naphthoquinone and 2- naphthalene sulfydryl is 1:1.65.

It further limits, the Na₂Cr₂O₇·2H₂The molar ratio of O and 5,8- dimethoxy -1,4- naphthoquinones is 0.76: 1。

It further limits, the concentrated sulfuric acid and 5,8- dimethoxy -1,4-naphthoquinone molar ratio is 0.23:1；It is described dense The concentration of sulfuric acid is 98% (quality).

Pharmaceutical composition of the invention contain therapeutically effective amount claim 1 compound and pharmaceutically acceptable load Body.

The application of the compound of the present invention and pharmaceutical composition in the drug of preparation treatment gastric cancer.

Pharmaceutically acceptable carrier described above refers to the pharmaceutical carrier of pharmaceutical field routine, such as: diluent, figuration Agent such as water etc., filler such as starch slurry, hydroxypropyl methylcellulose, povidone, syrup etc., wetting agent such as ethyl alcohol, water etc., disintegrating agent is such as Dried starch, Sodium Hydroxymethyl Stalcs, low-substituted hydroxypropyl cellulose, gas-producing disintegrant, crospovidone etc., sorbefacient such as sulphur Sour calcium, calcium monohydrogen phosphate, light magnesium oxide, calcium carbonate etc., absorption carrier such as chitosan etc., lubricant such as magnesium stearate, poly- diethyl Alcohol, talcum powder, hydrogenated vegetable oil, superfine silica gel powder etc., colorant such as titanium dioxide, sunset yellow, methylenum careuleum, medicinal iron oxide red Deng coating material such as acrylic resin, hydroxyl methylcellulose, povidone, cellacefate etc..In addition it can add in the composition Enter other auxiliary materials such as flavouring agent and sweetener etc..

Pharmaceutical composition of the invention can be applied to by way of oral, rectum or parenteral administration needs this treatment Patient.When for taking orally, it can be made into conventional solid pharmaceutical preparation such as tablet, capsule, pulvis, granule etc., liquid is made Preparation such as water or oil-suspending agent or other liquid preparations such as syrup, oral solution, elixir etc.；It, can be by it when for parenteral administration Solution, powder needle, water or the oleaginous suspension etc. of injection is made.Preferred form be tablet, coated tablet, capsule, granule, Oral solution and injection.

The various dosage forms of Pharmaceutical composition of the invention can be prepared according to the conventional production process of pharmaceutical field.Such as make This compound activity ingredient is mixed with one or more carriers, is then made into required dosage form.

Pharmaceutical composition of the invention preferably comprises the active constituent that molar ratio is 0.1%-99.5%, most preferably rubs You are than the active constituent for 0.5%-95%.

The amount of application of Pharmaceutical composition of the invention can according to route of administration, patient age, weight, body surface area, controlled The variation such as type and severity of the disease for the treatment of, daily dose can be 0.01-100mg/m²Body surface area, preferably 10-100 mg/m²Body surface area.It one or many can apply.

The present invention provides one kind new compound 2- naphthalene sulfydryl -5,8- dimethoxy -1,4-naphthoquinone, and the 5,8 of the compound Position is changed to methoxyl group by hydroxyl through methylating, and cytotoxicity is reduced, and by Michael addition reaction, by 2- naphthalene sulfydryl addition Onto 5,8- dimethoxy -1,4-naphthoquinone, to show more superior anticancer activity.The present invention is thin by up-regulation gastric cancer AGS The level of intracellular reactive oxygen species generation (ROS) lowers the phosphoric acid of ERK, Akt and STAT3 to raise the phosphorylation level of JNK and p38 Change level, gradually rises pro apoptotic protein Bax, cleaved-caspase-3 and cleaved-PARP expression quantity, anti-apoptotic egg White Bcl-xl expression quantity gradually decreases, and then induces human gastric cancer ags cell that apoptosis occurs.Meanwhile the present invention passes through up-regulation liver cancer The level of HepB reactive oxygen species (ROS) lowers the phosphorylation water of Akt and STAT3 to raise the phosphorylation level of p38 It is flat, pro apoptotic protein Bad and cleaved-PARP expression quantity is gradually risen, anti-apoptotic proteins Bcl-2 expression quantity gradually decreases, And then induce human liver cancer Hep3 apoptosis.

The invention has the advantages that

The compound of the present invention activity is high, using low concentration compound when can kill cancer cell well.

The compound of the present invention is lower to normal cell toxicity.

The compound of the present invention strong drug action.

The compound of the present invention is almost without by-product, product purity 98%.

The compound of the present invention production method is simpler, easy to operate.

The compound of the present invention cost is more honest and cleaner.

Detailed description of the invention

Fig. 1 is lethal effect of the NTDMNQ to people's AGS stomach cancer cell；

Fig. 2 is lethal effect of the NTDMNQ to people's MKN-28 stomach cancer cell；

Fig. 3 is lethal effect of the NTDMNQ to people's MKN-45 stomach cancer cell；

Fig. 4 is lethal effect of the NTDMNQ to people's NCI-N87 stomach cancer cell；

Fig. 5 is lethal effect of the NTDMNQ to people's SNU-216 stomach cancer cell；

Fig. 6 is lethal effect of the NTDMNQ to people's SNU-484 stomach cancer cell；

Fig. 7 is lethal effect of the NTDMNQ to people's YCC-1 stomach cancer cell；

Fig. 8 is lethal effect of the NTDMNQ to people's YCC-6 stomach cancer cell；

Fig. 9 is lethal effect of the NTDMNQ to people's YCC-16 stomach cancer cell；

Figure 10 A is after NTDMNQ handles ags cell, to utilize fluorescence microscope Apoptosis situation map；

Figure 10 B is the quantitative analysis figure of Figure 10 A；

Figure 11 A is after handling ags cell with NTDMNQ, to utilize Apoptosis by Flow Cytometry situation map；

Figure 11 B is the quantitative analysis figure of Figure 11 A；

Figure 12 A is to utilize the table of protein immunoblotting method detection apoptosis-related protein after handling ags cell with NTDMNQ Up to situation map；

Figure 12 B is the quantitative analysis figure of Figure 12 A albumen；

Figure 13 A is to utilize the table of protein immunoblotting method detection upstream GAP-associated protein GAP after handling ags cell with NTDMNQ Up to situation map；

Figure 13 B is the quantitative analysis figure of Figure 13 A albumen；

Figure 14 A is to utilize the variation of flow cytometry instrument detection intracellular ROS level after handling ags cell with NTDMNQ Situation map；

Figure 14 B is the quantitative analysis figure of Figure 14 A；

Figure 15 is lethal effect of the NTDMNQ to Hep3B cells cell；

Figure 16 is lethal effect of the NTDMNQ to human hepatoma HepG2 cell；

Figure 17 is lethal effect of the NTDMNQ to human liver cancer Huh7 cell；

Figure 18 A is after NTDMNQ handles HepG2 cell, to utilize fluorescence microscope Apoptosis situation map；

Figure 18 B is the quantitative analysis figure of Figure 18 A；

Figure 19 A is after handling HepG2 cell with NTDMNQ, to utilize Apoptosis by Flow Cytometry situation map；

Figure 19 B is the quantitative analysis figure of Figure 19 A；

Figure 20 A is to utilize protein immunoblotting method detection apoptosis-related protein after handling HepG2 cell with NTDMNQ Expression figure；

Figure 20 B is the quantitative analysis figure of Figure 20 A albumen；

Figure 21 A is to utilize protein immunoblotting method detection upstream GAP-associated protein GAP after handling HepG2 cell with NTDMNQ Expression figure；

Figure 21 B is the quantitative analysis figure of Figure 21 A albumen；

Figure 22 A is to utilize the change of flow cytometry instrument detection intracellular ROS level after handling HepG2 cell with NTDMNQ Change situation map；

Figure 22 B is the quantitative analysis figure of Figure 22 A.

Specific embodiment

With reference to the accompanying drawing and specific embodiment the present invention is described further:

Embodiment: the preparation of 2- naphthalene sulfydryl -5,8- dimethoxy -1,4- naphthoquinone compound in the present embodiment:

5,8- dimethoxy -1,4-naphthoquinone (1mmol) 218.21mg is added in 250ml round-bottomed flask, uses 60ml first Alcohol is dissolved, and substituent group 2- naphthalene sulfydryl (1.65mmol) 188.24 μ l is added, and is stirred 4h, is added the H of 98% (quality)₂SO₄ (0.23mmol)24.90μl、Na₂Cr₂O₇·2H₂27.45 μ l of O (0.76mmol) stirs 2min, uses saturation NaCl and CHCl₃ Extraction, anhydrous sodium sulfate dehydration, is concentrated and dried, and column chromatography purifies compound, (character of product: orange-yellow crystal).Product Structural formula:

Product purity 98%.

Using following verification experimental verification invention effects:

One, lethal effect of the NTDMNQ to cancer cell

Experimental method: (MTT experiment)

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

2. cultivating cell: 5%CO₂, 37 DEG C are incubated for for 24 hours, until cell monolayer is paved with bottom hole；

3. serum starvation: changing culture solution (culture solution containing 1%FBS) before dosing 2h；

Drug-treated: the NTDMNQ prepared is taken into final concentration of 0.1,0.3,1,3 and 30 μM of processing human gastric cancer respectively AGS, MKN-28, MKN-45, NCI-N87, SNU-216, SNU-484, YCC-1, YCC-6 and YCC-16 cell are for 24 hours；It takes respectively Final concentration of 0.01,0.03,0.1,0.03,1,3 and 10 μM of processing Hep3B cells, HepG2 and Huh7 cell are for 24 hours.

4. color reaction: every hole adds 20 μ l of MTT solution (5mg/ml is prepared with PBS, pH 7.4) to continue after being incubated for 2-4h, Careful inhale abandons culture supernatant in hole, is carefully washed 2 times with PBS, and then every hole adds 100 μ l dimethyl sulfoxides (DMSO), concussion 10min makes to crystallize abundant dissolution；

5. colorimetric: selection 490nm wavelength measures each hole absorbance value on enzyme-linked immunosorbent assay instrument, records as a result, with dense Degree is abscissa, and survival rate is that ordinate draws cell growth histogram, the result is shown in Figure 1-9, Figure 15-17 and table 1 and 2.

Interpretation of result:

It can be seen that NTDMNQ (IC in Fig. 1-9₅₀: 0.42 μM) all there is good killing energy to 9 kinds of stomach cancer cells of people Power；It can be seen that NTDMNQ (IC in Figure 15-17₅₀: 0.874 μM) all have well to liver cancer Hep3B, HepG2 and Huh7 simultaneously Killing ability, killing intensity gradually risen with the increase of drug concentration.

By the following table 1 and 2 it can also be seen that NTDMNQ (IC₅₀: 0.42 μM) all there is good kill to 9 kinds of stomach cancer cells of people Hurt ability；NTDMNQ (IC simultaneously₅₀: 0.874 μM) all there is good killing ability to liver cancer Hep3B, HepG2 and Huh7, Killing intensity gradually rises with the increase of drug concentration.

IC of 1 NTDMNQ of table to gastric carcinoma cells lethal effect₅₀Value

IC of 2 NTDMNQ of table to human liver cancer cell lethal effect₅₀Value

Apoptotic effect of the NTDMNQ to cancer cell

Experimental method: (experiment in vitro-Annexin-V decoration method)

1. inoculating cell: individual cells suspension is made into containing the culture solution of 10% tire calf serum, with 10,000, every hole For cell inoculation to 12 orifice plates, every pore volume is 1ml；

2. cultivating cell: 5%CO₂, 37 DEG C are incubated for for 24 hours, until cell monolayer is paved with bottom hole；

3. drug-treated: the NTDMNQ (IC prepared being added in gastric cancer ags cell₅₀: 0.42 μM), handle different time (3,6,12 and for 24 hours)；NTDMNQ (the IC prepared is added in hepatoma Hep G 2 cells₅₀: 0.874 μM), processing different time (3, 6,12 and for 24 hours)；

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

5. 10 μ, 1 propidium iodide (Propidium Iodide, PI) dyeing liquor is added, mix gently；

6. room temperature (20-25 DEG C) is protected from light incubation 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 are propidium iodide positive cells.It is only dyed by green fluorescence, and small volume is thin Born of the same parents are apoptotic cell；By red or green and red double dyes, and the biggish cell of volume is non-viable non-apoptotic cell；The cell not being colored For normal cell.200 cells of random observation acquire percentage shared by various cells, are averaged for each sample counting 3 times；

(B) simultaneously, also using Flow Cytometry methods detection NTDMNQ to human gastric cancer ags cell and liver cancer HepG2 cell Apoptosis.

1, human gastric cancer ags cell, apoptosis of the detection NTDMNQ to human gastric cancer ags cell are handled with NTDMNQ.

Using above-mentioned experimental method, obtain experimental result is shown in Figure 10 A, Figure 10 B, Figure 11 A and Figure 11 B, wherein Figure 10 A It is after handling ags cell with NTDMNQ, using fluorescence microscope Apoptosis situation map, 5-FU is positive controls；Figure 10B is the quantitative analysis figure of Figure 10 A.Figure 11 A is after handling ags cell with NTDMNQ, to be withered using Flow cytometry cell Die situation map；Figure 11 B is the quantitative analysis figure of Figure 11 A.

Interpretation of result

After with (final concentration of 0.42 μM) processing human gastric cancer ags cell 3,6,12 of NTDMNQ and for 24 hours, Annexin is carried out The bis- dye experiments of V-FITC/PI, well is in fluorescence microscope.It is from Figure 10 B as can be seen that continuous with drug exposure times Increase, Annexin V-FITC fluorescence intensity also gradually increases, and Apoptosis degree also dramatically increases.Especially it is when the time When for 24 hours, the fluorescence intensity highest of cell.As a result illustrate, NTDMNQ can effectively induce the apoptosis of AGS cell, and the time is presented Dependence.

After with (final concentration of 0.42 μM) processing human gastric cancer ags cell 3,6,12 of NTDMNQ and for 24 hours, Annexin is carried out V-FITC and PI are marked, and pass through Apoptosis by Flow Cytometry situation.From Figure 11 B as can be seen that with drug The processing time is continuously increased, and the degree of gastric cancer ags cell apoptosis is consequently increased, especially when drug exposure times reach for 24 hours When, the level of apoptosis of cell obviously increases.The result shows NTDMNQ can effectively induce the apoptosis of AG cell, and present Time dependence.

2, human hepatoma HepG2 cell, apoptosis of the detection NTDMNQ to human hepatoma HepG2 cell are handled with NTDMNQ

Using above-mentioned experimental method, obtain experimental result is shown in Figure 18 A, Figure 18 B, Figure 19 A and Figure 19 B, wherein 18A is After handling HepG2 cell with NTDMNQ, fluorescence microscope Apoptosis situation map is utilized；Figure 18 B is quantitative point of Figure 18 A Analysis figure.Figure 19 A is after handling HepG2 cell with NTDMNQ, to utilize Apoptosis by Flow Cytometry situation map；Figure 19 B is The quantitative analysis figure of Figure 19 A.

Interpretation of result

After with (final concentration of 0.874 μM) processing human hepatoma HepG2 cell 3,6,12 of NTDMNQ and for 24 hours, Annexin is carried out The bis- dye experiments of V-FITC/PI, and in fluorescence microscope.It is from Figure 18 B as can be seen that continuous with drug exposure times Increase, Annexin V-FITC fluorescence intensity also gradually increases, and Apoptosis degree also dramatically increases.Especially it is when the time When for 24 hours, the fluorescence intensity highest of cell.As a result illustrate, NTDMNQ can effectively induce the apoptosis of HepG2 cell, and be in the time Dependence.

After with (final concentration of 0.874 μM) processing human hepatoma HepG2 cell 3,6,12 of NTDMNQ and for 24 hours, Annexin is carried out V-FITC and PI are marked, and pass through Apoptosis by Flow Cytometry situation.From it can be seen that in 19B with drug The reason time is continuously increased, and the degree of hepatoma Hep G 2 cells apoptosis is consequently increased, especially when drug exposure times reach for 24 hours When, the level of apoptosis of cell obviously increases.The result shows NTDMNQ can effectively induce the apoptosis of HepG2 cell, and be in Existing time dependence.

Three, effect of the NTDMNQ to the apoptosis-related protein and stream signal access of cancer cell

Experimental method: (protein immunoblotting method)

1. inoculating cell: individual cells suspension is made into containing the culture solution of 10% tire calf serum, with 10,000, every hole For cell inoculation to 6 orifice plates, every pore volume is 2ml；

2. cultivating cell: 5%CO₂, 37 DEG C are incubated for for 24 hours, until cell monolayer is paved with bottom hole；

3. drug-treated: the NTDMNQ (IC prepared being added in gastric cancer ags cell₅₀: 0.42 μM), handle different time (3,6,12 and for 24 hours)；NTDMNQ (the IC prepared is added in hepatoma Hep G 2 cells₅₀: 0.874 μM), processing different time (3, 6,12 and for 24 hours)；

4. measuring OD value with ultraviolet specrophotometer, protein is quantified；

5. 8-12%SDS-PAGE electrophoresis, by Protein transfer to NC film, skimmed milk closes 1h, TBST solution washing 5 Time；

6. respectively with ERK, JNK, p38, AKT, Bcl-xl, Bad, PARP, Caspase-3, Bcl-2, Bax and α- Tubulin is combined, and 4 DEG C of shaking tables are incubated overnight；

7. TBST is washed 5 times, using HRP label goat anti-rabbit igg, HRP label mountain sheep anti-mouse igg as secondary antibody, it is incubated at room temperature 2h；

8. electrochemical luminescence reagent colour development is added, it is imaged using AI600 Multifunctional imaging instrument；

1, human gastric cancer ags cell, shadow of the detection NTDMNQ to human gastric cancer ags cell apoptosis-related protein are handled with NTDMNQ It rings

Using above-mentioned experimental method, obtain experimental result is shown in Figure 12 A, Figure 12 B, Figure 13 A and Figure 13 B, wherein Figure 12 A It is that after handling ags cell with NTDMNQ, the situation of change of apoptosis-related protein is detected by protein immunoblotting method；Figure 12 B It is the quantitative analysis figure of Figure 12 A.Figure 13 A is after handling ags cell with NTDMNQ, to detect upstream by protein immunoblotting method The situation of change of GAP-associated protein GAP；Figure 13 B is the quantitative analysis figure of Figure 13 A.

Interpretation of result

After with (final concentration of 0.42 μM) processing human gastric cancer ags cell 3,6,12 of NTDMNQ and for 24 hours, Western Immuno is carried out Blotting experiment, the imaging of AI600 Multifunctional imaging instrument.It can be seen that the continuous increasing with drug exposure times from Figure 12 B Add, Bax, cleaved-caspase-3 and cleaved-PARP expression quantity for promoting apoptosis increase, the Bcl-xl expression quantity of anti-apoptotic It reduces.As a result illustrate, NTDMNQ can effectively induce ags cell that apoptosis occurs.

After with (final concentration of 0.42 μM) processing human gastric cancer ags cell 3,6,12 of NTDMNQ and for 24 hours, Western Immuno is carried out Blotting experiment, the imaging of AI600 Multifunctional imaging instrument.It can be seen that the continuous increasing with drug exposure times from Figure 13 B Add, p-JNK and p-p38 expression quantity increases, p-ERK, p-STAT3 and p-AKT expression quantity reduces.As a result illustrate, NTDMNQ passes through Regulate and control MAPKs, AKT and STAT3 signal path come induce ags cell occur apoptosis.

2, human hepatoma HepG2 cell is handled with NTDMNQ, detects NTDMNQ to human hepatoma HepG2 cell's apoptosis-related protein Influence

Using above-mentioned experimental method, obtain experimental result is shown in Figure 20 A, Figure 20 B, Figure 21 A and Figure 21 B, wherein 20A is After handling HepG2 cell with NTDMNQ, the situation of change of apoptosis-related protein is detected by protein immunoblotting method；Figure 20 B It is the quantitative analysis figure of Figure 20 A.Figure 21 A is after handling HepG2 cell with NTDMNQ, to be detected by protein immunoblotting method Swim the situation of change of GAP-associated protein GAP；Figure 21 B is the quantitative analysis figure of Figure 22 A.

Interpretation of result

After with (final concentration of 0.874 μM) processing human hepatoma HepG2 cell 3,6,12 of NTDMNQ and for 24 hours, protein is carried out Immunoblotting experiments, the imaging of AI600 Multifunctional imaging instrument.It is from Figure 20 B as can be seen that continuous with drug exposure times Increase, Bad the and cleaved-PARP expression quantity for promoting apoptosis increases, and the Bcl-2 expression quantity of anti-apoptotic reduces.As a result illustrate, NTDMNQ can effectively induce HepG2 apoptosis.

After with (final concentration of 0.874 μM) processing human hepatoma HepG2 cell 3,6,12 of NTDMNQ and for 24 hours, protein is carried out Immunoblotting experiments, the imaging of AI600 Multifunctional imaging instrument.It is from Figure 21 B as can be seen that continuous with drug exposure times Increase, p-p38 expression quantity increases, p-STAT3 and p-AKT expression quantity reduces.As a result illustrate, NTDMNQ passes through regulation p38, AKT And STAT3 signal path induces HepG2 apoptosis.

Four, influence of the NTDMNQ to ROS level in cancer cell

Experimental method: (DCFH-DA dyeing)

1. inoculating cell: individual cells suspension is made into containing the culture solution of 10% tire calf serum, with 10,000, every hole For cell inoculation to 6 orifice plates, every pore volume is 2ml；

2. cultivating cell: 5%CO₂, 37 DEG C are incubated for for 24 hours, until cell monolayer is paved with bottom hole；

3. drug-treated: the NTDMNQ (IC prepared being added in gastric cancer ags cell₅₀: 0.42 μM), handle different time (3,6,12 and for 24 hours)；NTDMNQ (the IC prepared is added in hepatoma Hep G 2 cells₅₀: 0.874 μM), processing different time (3, 6,12 and for 24 hours)；

4. being resuspended with PBS, 100 μ l PBS are added, add 1 μ LDCFH-DA, 37 DEG C are protected from light incubation 15min；

5. 5000rpm is centrifuged, 500 μ L PBS are resuspended, and are blown mixed；

6. influence of the flow cytomery NTDMNQ to ROS level in gastric cancer ags cell and hepatoma Hep G 2 cells；

1, human gastric cancer ags cell is handled with NTDMNQ, detects the influence of ROS level in NTDMNQ gastric cancer ags cell

Using above-mentioned experimental method, obtain experimental result is shown in Figure 14 A and Figure 14 B, wherein Figure 14 A is at NTDMNQ After managing ags cell, pass through Flow cytometry intracellular ROS level situation of change；Figure 14 B is the quantitative analysis figure of Figure 14 A.

Interpretation of result

After with (final concentration of 0.42 μM) processing human gastric cancer ags cell 3,6,12 of NTDMNQ and for 24 hours, DCFH-DA dye is carried out Color experiment, uses Flow cytometry.As can be seen that being continuously increased with drug exposure times, ags cell from Figure 14 B Interior ROS is horizontal constantly to be increased.As a result illustrate, NTDMNQ can induce ags cell that apoptosis occurs by regulating and controlling the level of ROS.

2, human hepatoma HepG2 cell is handled with NTDMNQ, detects the influence of ROS level in NTDMNQ hepatoma Hep G 2 cells

Using above-mentioned experimental method, obtain experimental result is shown in Figure 22 A and Figure 22 B, wherein 22A is handled with NTDMNQ After HepG2 cell, pass through Flow cytometry intracellular ROS level situation of change；Figure 22 B is the quantitative analysis of Figure 22 A Figure.

Interpretation of result

After with (final concentration of 0.874 μM) processing human hepatoma HepG2 cell 3,6,12 of NTDMNQ and for 24 hours, DCFH-DA is carried out Coloration experiment uses flow cytomery.It can be seen that being continuously increased with drug exposure times from Figure 22 B, into the cell ROS is horizontal constantly to be increased.As a result illustrate, NTDMNQ can induce HepG2 apoptosis by regulating and controlling the level of ROS.

In conclusion NTDMNQ (final concentration of 0.42 μM) can regulate and control MAPKs, AKT by increasing the level of ROS Induce gastric cancer ags cell that apoptosis occurs with STAT3 signal path；NTDMNQ (final concentration of 0.874) can be by improving ROS Level come regulate and control AKT and STAT3 signal path induce human hepatoma HepG2 cell occur apoptosis.

Claims (8)

1. The structural formula of 1.2- naphthalene sulfydryl -5,8- dimethoxy -1,4- naphthoquinone compound are as follows:
2. 2. the preparation method of claim 1 compound, this method carry out in the steps below:

   5,8- dimethoxys -1,4-naphthoquinone (DMNQ) is dissolved in methanol, be added 2- naphthalene sulfydryl, stir 4h, be added the concentrated sulfuric acid and Na₂Cr₂O₇·2H₂O stirs 5min, with saturation NaCl and CHCl₃Extraction, anhydrous sodium sulfate dehydration, is concentrated and dried, and column chromatography mentions It is pure, obtain 2- naphthalene sulfydryl -5,8- dimethoxy -1,4-naphthoquinone compound.
3. 3. preparation method according to claim 2, it is characterised in that 5, the 8- dimethoxy -1,4-naphthoquinone and 2- naphthalene mercapto The molar ratio of base is 1:1.65.
4. 4. preparation method according to claim 2, it is characterised in that the Na₂Cr₂O₇·2H₂O and dimethoxy -1 5,8-, The molar ratio of 4- naphthoquinones is 0.76:1.
5. 5. preparation method according to claim 2, it is characterised in that the concentrated sulfuric acid and 5,8- dimethoxy -1,4-naphthoquinone Molar ratio be 0.23:1.
6. 6. preparation method according to claim 2, it is characterised in that the concentration of the concentrated sulfuric acid is 98% (quality).
7. 7. for treating the pharmaceutical composition of gastric cancer, wherein the compound of the claim 1 containing therapeutically effective amount and pharmaceutically may be used The carrier of receiving.
8. 8. application of the compound of claim 1 in the drug of preparation treatment gastric cancer.