CN103497154A - Water-soluble perylene imide compounds, usage as DNA intercalator, and applications thereof in growth inhibition of cancer cells - Google Patents
Water-soluble perylene imide compounds, usage as DNA intercalator, and applications thereof in growth inhibition of cancer cells Download PDFInfo
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Abstract
The invention discloses water-soluble perylene imide compounds, a usage as a DNA intercalator, and applications thereof in the growth inhibition of cancer cells, and belongs to the technical field of chemical synthesis of biological drugs. Through inducing cation amino functional groups into perylene anhydride derivatives and a perylene mono-anhydride system, the water solubility of the perylene anhydride derivatives and the perylene mono-anhydride system is greatly improved; furthermore, the cation amino functional groups are in a photo-stability combination with perylene derivatives, and thus a series of novel water-soluble perylene imide compounds are obtained. The synthesized water-soluble perylene imide compounds can effectively be embedded between the base pairs of DNA double helix of nucleus and be used as a DNA intercalator. The synthesized water-soluble perylene imide compounds have a planar rigid structure, so that the compounds can be specifically enriched around fixed tissue cell nucleus, the compounds have the advantages of strong fluorescence and easiness in fluorescent imaging, and have a similar effect as the DAPI effect of commercial cell nucleus dye. The synthesized water-soluble perylene imide compounds can effectively inhibit the growth of cancer cells such as U2OS, HTC116, Hela, AGS, etc., and have a very prominent anti-tumor effect.
Description
Technical field
The invention belongs to the bio-pharmaceutical chemosynthesis technical field, particularly a kind of water-soluble perylene diimide compounds and as DNA intercalation agent and the application in anticancer growth.
Background technology
Perylene and derivative thereof have good light, heat, chemical stability, 100% fluorescence quantum yield almost, narrow fluorescence emission peak, can separate with cell background fluorescence (395-479nm) and excellent dyeing behavior, be widely used at present organic electro-optic device, laser dyes and biological fluorescent labeling field.
Intercalation agent (intercalator) refers to that the reversibility of polycyclic aromatic hydrocarbons small-molecule substance and DNA interacts.This quasi-molecule volume is little, in the electron deficiency state, and has certain planar rigidity, can be inserted between the base pair of two accumulations of DNA, and telescopiny causes extending spirally and unwinding of DNA.The more DNA intercalation agent of research at present has acridine, pyrido-carbazole class, benzoglyoxaline [1,2-C] quinoline, Anthraquinones etc., and all is used as antitumor research.Yet the perylene analog derivative with excellent planar rigidity knot structure also is not used as DNA intercalation agent and studies its antitumor performance.
Perylene diimide compounds has been applied to biological field in recent years, as cell specific marker, yet poor water-soluble its application in this field that directly affected, therefore improve the water-soluble of this compounds and in the aqueous solution, keep high-fluorescence quantum yield to it at biological field, especially DNA intercalation agent field is significant.
Summary of the invention
The object of the present invention is to provide that a class light, heat, chemical stability are good, good water solubility, structure have designability perylene diimide compounds, and be applied to biomedical sector, specifically studied as DNA intercalation agent and the application in the anticancer growth.
Technical scheme of the present invention is: at first Xuan Ze perylene Gan Huo perylene list acid anhydride, as fluorescent emission group, then reacts with the compound that contains amidine functional group, and preparation has amidine functional group and modifies perylene diimide derivative Ji perylene list imide derivative; Then there is tertiary amine functional group by Eschweiler – Clarke reaction preparation and modify perylene diimide derivative Ji perylene list imide derivative; End reaction obtains containing the different cationization perylene diimide derivatives such as tertiary ammonium salt or quaternary ammonium salt Ji perylene list imide derivative.Analyze Zhe Xie perylene diimide derivative and DNA interaction by ultra-violet absorption spectrum, fluorescence emission spectrum, circular dichroism spectrum (CD), confirm that these compounds have excellent DNA intercalation performance, can be used as the growth that DNA intercalation agent carrys out inhibition tumor cell simultaneously.Then, by cell and research and the sign of organizing level, prove the effect that the synthetic perylene diimide derivative of the present invention has nuclear specific enrichment effect and anticancer growth aspect.
The polyamine derivative of water-soluble perylene diimide compounds Shi Han perylene diimide Huo perylene list imide structure of the present invention, its general structure is:
Or
Or
Or
Wherein,
r
2=CH
3or CH
2cH
3, R
3=H or CH
3or CH
2cH
3.
The synthetic method of water-soluble perylene diimide compounds of the present invention, its concrete preparation process is:
(1) aminated compounds of 2mmol perylene acid anhydride and 6-12mmol is added in 3-6mL water and stirs, then be warming up to 100-120 ℃, back flow reaction 12-24h, after dropping to room temperature, with the 1-2wt%KOH aqueous solution and water washing, drying obtains product;
(2) product 0.6mmol step (1) obtained and the p-methyl benzenesulfonic acid of 5-9mmol filter after reacting 4-8h in 50-60 ℃ in 3-6mL water, filtrate is removed desolventizing through rotary evaporation, then with acetone, disperse, with ether sedimentation, wash, centrifugal, dry, obtain tertiary amine Yan perylene diimide compounds;
The product perhaps 0.6mmol step (1) obtained and the p-methyl benzenesulfonic acid methyl esters of 5-9mmol 50-60 ℃ react 18-24h after, add 3-5mL methyl alcohol filters in reaction mixture, by rotary evaporation, filtrate is concentrated, then carry out washing of precipitate with ether, after centrifugal, drying, obtain Ji An Yan perylene diimide compounds;
The described aminated compounds of step (1) is the tertiary amine compounds that contains an end amido, and its molecular formula general formula is NH
2(R
1nH)
0-5r
1n(R
2)
2, R wherein
1for the alkyl chain that contains 2-6 methylene radical, R
2for methyl or ethyl;
The above-mentioned tertiary amine Yan perylene diimide compounds made and Ji An Yan perylene diimide compounds are the water-soluble Xian perylene diimide compounds containing 2-12 functional amido.
The preferred N of the described aminated compounds of step (1), N-dimethyl-ethylenediamine or N, N-diethyl divinyl triamine.
The synthetic method of water-soluble perylene diimide compounds of the present invention, its concrete preparation process is:
1) adds perylene acid anhydride and polyamine compounds in the back flow reaction bottle for (1:40)-(1:60) in molar ratio, be heated to 98-110 ℃ of reaction 24-28h under nitrogen atmosphere, then be warmed up to 170-180 ℃ and continue reaction 2-5h, after dropping to room temperature, add ethanol that volume ratio is (1:2)-(1:4) and the mixed solvent of ether, filter, then by toluene, ether washing solid product, drying;
2) product 138mg step 1) obtained joins in the mixing solutions of 1-2mL, the formic acid solution that described mixing solutions is 80-86wt% and the formaldehyde solution of 25-35wt% (1.2:1)-(1.5:1) preparation by volume obtain, stir 0.5-1.5 hour post-heating under nitrogen atmosphere to 90-120 ℃ of reaction 12-18 hour, after dropping to room temperature, with the ether sedimentation washing, drying obtains the functionalized perylene diimide of tertiary amine compounds;
3) by 100mg step 2) the functionalized perylene diimide of the tertiary amine compounds that obtains is dissolved in the methyl alcohol or acetonitrile solvent of 2-4mL, then drip 0.2-0.4mL methyl iodide or 0.2-0.5mL iodoethane, be heated to 60-80 ℃ under nitrogen atmosphere, obtain Ji An Yan perylene diimide compounds;
The described polyamine compounds of step 1) is the aminated compounds that contains three end amidos, and its molecular formula general formula is NH
2(R
1nH)
0-5r
1n(R
1nH
2)
2, R wherein
1for the alkyl chain that contains 2-6 methylene radical;
The above-mentioned tertiary amine made functionalized perylene diimide compounds and Ji An Yan perylene diimide compounds are the water-soluble Zhiization perylene diimide compounds containing 6-16 functional amido.
The described polyamine compounds of step 1) is three (2-amino-ethyl) amine preferably.
The synthetic method of water-soluble perylene diimide compounds of the present invention, its concrete preparation process is:
(1) aminated compounds of 2mmol perylene list acid anhydride and 5-10mmol is added in 3-6mL water and stirs, then be warming up to 100-120 ℃, back flow reaction 12-24h, after dropping to room temperature, with the 1-2wt%KOH aqueous solution and water washing, drying obtains product;
(2) product 0.5mmol step (1) obtained and the p-methyl benzenesulfonic acid of 2.5-5mmol filter after reacting 4-8h in 50-60 ℃ in 2-4mL water, filtrate is removed desolventizing through rotary evaporation, with the ether sedimentation washing, centrifugal, dry, obtain tertiary amine Yan perylene list imide analog compounds;
The product perhaps 0.5mmol step (1) obtained and the p-methyl benzenesulfonic acid methyl esters of 2.5-5mmol filter after reacting 18-24h at 50-60 ℃, by rotary evaporation, filtrate is concentrated, then carry out washing of precipitate with ether, after centrifugal, drying, obtain Ji An Yan perylene list imide analog compounds;
The described aminated compounds of step (1) is the tertiary amine compounds that contains an end amido, and its molecular formula general formula is NH
2(R
1nH)
0-5r
1n(R
2)
2, R wherein
1for the alkyl chain that contains 2-6 methylene radical, R
2for methyl or ethyl;
The above-mentioned tertiary amine Yan perylene list imide analog compounds made and Ji An Yan perylene list imide analog compounds are the water-soluble Xian perylene diimide compounds containing 1-6 functional amido.
The preferred N of the described aminated compounds of step (1), N-dimethyl-ethylenediamine or N, N-diethyl divinyl triamine.
The synthetic method of water-soluble perylene diimide compounds of the present invention, its concrete preparation process is:
1) adds perylene list acid anhydride and polyamine compounds in the back flow reaction bottle for (1:40)-(1:60) in molar ratio, be heated to 98-110 ℃ of reaction 24-28h under nitrogen atmosphere, then be warmed up to 170-180 ℃ and continue reaction 2-5h, after dropping to room temperature, add ethanol that volume ratio is (1:2)-(1:4) and the mixed solvent of ether, filter, then by toluene, ether washing solid product, drying;
2) product 150mg step 1) obtained joins in the mixing solutions of 1.0-2mL, the formic acid solution that described mixing solutions is 80-86wt% and the formaldehyde solution of 25-35wt% (1.2:1)-(1.5:1) preparation by volume obtain, stir 0.5-1.5 hour post-heating under nitrogen atmosphere to 90-120 ℃ of reaction 12-18 hour, after dropping to room temperature, with the ether sedimentation washing, drying obtains the functionalized perylene of tertiary amine list imide analog compounds;
3) by 100mg step 2) the functionalized perylene of the tertiary amine list imide analog compounds that obtains is dissolved in 2-4mL methyl alcohol or acetonitrile solvent, then drip 0.2-0.4mL methyl iodide or 0.2-0.5mL iodoethane, be heated to 60-80 ℃ under nitrogen atmosphere, obtain Ji An Yan perylene list imide analog compounds;
The described polyamine compounds of step 1) is the aminated compounds that contains three end amidos, and its molecular formula general formula is NH
2(R
1nH)
0-5r
1n(R
1nH
2)
2, R wherein
1for the alkyl chain that contains 2-6 methylene radical;
The above-mentioned tertiary amine made functionalized perylene list imide analog compounds and Ji An Yan perylene list imide analog compounds are the water-soluble Zhiization perylene diimide compounds containing 3-8 functional amido.
The described polyamine compounds of step 1) is three (2-amino-ethyl) amine preferably.
Application using above-mentioned water-soluble perylene diimide compounds as DNA intercalation agent.
Application using above-mentioned water-soluble perylene diimide compounds as the nucleus dyestuff.
The application of growth medicine using above-mentioned water-soluble perylene diimide compounds as anticancer.
The present invention has following beneficial effect:
1. using perylene acid anhydride analog derivative and analogue thereof as fluorescent emission group, by different synthetic methods, synthesized a series of compounds of the water-soluble perylene diimide with different amido functional groups; This compounds has the characteristics such as good light, heat, chemical stability, fluorescence quantum yield are high, good water solubility, structure designability.
2. the synthetic water-soluble perylene diimide compounds of the present invention can effectively be embedded between nucleus DNA duplex base pair, can be used as DNA intercalation agent, has good actual application value.
3. the synthetic water-soluble perylene diimide compounds of the present invention has plane rigid structure, can specificly be enriched in the fixing organization nucleus, and fluorescence is strong, be easy to fluorescence imaging, can match in excellence or beauty with business-like nucleus dyestuff DAPI effect.
4. the synthetic water-soluble perylene diimide compounds of the present invention can effectively suppress the growth of cancer cells such as U2OS, HCT116, Hela and AGS, has obvious antitumous effect; Its scientific research in biological medicine, diagnosis, treatment field are had broad application prospects.
5. synthetic method of the present invention is simple, easily a large amount of synthetic, has good using value.
The accompanying drawing explanation
Fig. 1 water-soluble perylene diimide compounds P2 intercalation is to the molecular diagram between the DNA base pair, and the fluorescence imaging figure and the histogram that suppresses the HCT116 Growth of Cells that are applied to the enrichment of specific cell core.
Fig. 2 embodiment 1 becomes the reacting flow chart of water-soluble perylene diimide compounds with He in embodiment 2.
Fig. 3 embodiment 3 becomes the reacting flow chart of Shui Rong perylene list imide analog compounds with He in embodiment 4.
Fig. 4 water-soluble perylene diimide compounds P2, P3, P5 and P6(5.0 μ M) in phosphoric acid buffer (10mM, pH7.4) with (A1-A4) uv-absorbing of Ct-DNA effect and (B1-B4) fluorescence emission spectrogram.
Fig. 5 fixation of C t-DNA concentration is 80 μ M, in phosphoric acid buffer (10mM, pH7.4), adds water-soluble perylene diimide compounds P2, P3, P5 and P6(3 μ M) and (A-D) CD spectrogram of blank test.
Fig. 6 fruit bat sialisterium fixing organization is at water-soluble perylene diimide compounds P2, the fluorescence imaging under P3 and DAPI dye altogether.(A-B) P2 of 10 μ Μ, and P3 (inferior acid amides position connects two amido substituting groups, P2, P3 has the rigid plane skeleton construction) difference dye marker fruit bat sialisterium, 546nm session fluorescence imaging, specific being enriched on nucleus.(A '-B ') nucleus dyestuff DAPI dye marker drosophila larvae salivary organization, fluorescence imaging figure under the 405nm session.(A "-B ") P2, P3 nucleus fluorescence localization imaging effect overlaps fully with DAPI fluorescence imaging effect.
Fig. 7 fruit bat sialisterium fixing organization is at water-soluble perylene diimide compounds P5, the fluorescence imaging under P6 and DAPI dye altogether.(A-B) P5 of 10 μ Μ, and P6 (inferior acid amides position connects four amido substituting groups, P5, P6 has the rigid plane skeleton construction) difference dye marker fruit bat sialisterium, 546nm session fluorescence imaging, specific being enriched on nucleus.(A '-B ') nucleus dyestuff DAPI dye marker drosophila larvae salivary organization, fluorescence imaging figure under the 405nm session.(A "-B ") P5, P6 nucleus fluorescence localization imaging effect overlaps fully with DAPI fluorescence imaging effect.
Embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated.The invention is not restricted to these specific embodiments.
The sign of product structure is used: nuclear-magnetism
1h and
13c composes (Bruker400), mass spectrum AXIMA-CFRplus MALDI-TOFMS.
The product optical property characterizes to be used: uv-vis spectra (Cintra20, GBC, Australia), fluorescence spectrum (HoribaJobin YvonFluoroMax-4NIR, NJ, USA).
The Bioexperiment of product characterizes to be used: fluorescent microscope (AMGEVOSf1microscope), laser confocal microscope (LeicaTCSSP2AOBSconfocalmicroscope).
Embodiment 1:
1) 0.7846g perylene acid anhydride (2.0mmol) and 0.34mLN, N-dimethyl-ethylenediamine (6.0mmol) is suspended in 4mL water and stirs, then be warming up to 100 ℃ of back flow reaction 12h, after equitemperature drops to room temperature, with the 1wt%KOH aqueous solution and water washing, obtain red solid product P 1 after vacuum-drying, productive rate is 75%;
1H-NMR(400MHz,CF
3COOD),δppm:8.77(d,J=12.3Hz,8H),4.77(s,4H),3.75(s,4H),3.19(s,12H).
13C-NMR(100MHz,CF
3COOD),δppm:168.67,138.82,135.64,131.77,128.84,126.85,123.97,60.73,46.42,38.74.MS(MALDI-TOF,m/z):CalcdforC
32H
28N
4O
4,532.6;Found,533.6(M+H
+).
2) 0.32gP1(0.6mmol) with 1.02g p-methyl benzenesulfonic acid (5.4mmol), in the 3.5mL deionized water solution, in 50 ℃, filter after reacting 4h, filtrate is removed desolventizing through rotary evaporation, then with acetone, disperse, with ether sedimentation, wash, obtain red solid product P 2 after centrifugal, drying, productive rate is 85%.
1H-NMR(400MHz,CF
3COOD),δppm:8.76(s,8H),7.77(s,<2H),7.37(d,J=7.7Hz,4H),6.95(d,J=7.7Hz,4H),4.81(s,4H),3.83(s,4H),3.27(d,J=2.9Hz,12H),1.96(s,6H).
13C-NMR(100MHz,CF
3COOD),δppm:168.73,146.03,138.67,138.52,135.51,131.73,131.41,128.60,127.62,126.62,124.13,60.54,46.50,38.66,21.72.UV-Vis(H
2O,c=5.0×10
-6M):λ
max(ε)=498nm(2.45×10
4M
-1cm
-1);Emission(H
2O,λ
ex=498nm):λ
max=545nm.
P1(0.32g, 0.6mmol) react 20h with 2.5mL p-methyl benzenesulfonic acid methyl esters (5.4mmol) in 50 ℃, after equitemperature drops to room temperature, add 3mL methyl alcohol filters in reaction mixture, by rotary evaporation, filtrate is concentrated, then carry out washing of precipitate with ether, after centrifugal, drying, obtain red solid product P 3, productive rate is 80%.
1H-NMR(400MHz,CF
3COOD),δppm:8.99(dt,J=15.1,7.6Hz,8H),7.77(d,J=8.1Hz,4H),7.33(d,J=8.0Hz,4H),4.97(d,J=30.0Hz,4H),3.99(d,J=37.7Hz,4H),3.55(s,12H),3.37(s,6H),2.38(s,6H).UV-Vis(H
2O,c=10
-5M):λ
max=498nm;Emission(H
2O,λ
ex=498nm):λ
max=545nm。
P2 and P3 are the water-soluble Xian perylene diimide compounds containing 2 functional amidos.
The P2 structural formula is
The P3 structural formula is
Embodiment 2:
1) 0.81g perylene acid anhydride (2.1mmol) and 15mL tri-(2-amino-ethyl) amine (100.5mmol) are added in the back flow reaction bottle, wherein three (2-amino-ethyl) amine is also reaction solvent; Solution is heated to 100 ℃ of reactions 28 hours under nitrogen atmosphere, then be warmed up to 170 ℃ and continue reaction 4 hours, after equitemperature drops to room temperature, add ethanol that volume ratio is 1:3 and the mixed solvent of ether, filter, then with toluene, ether washing solid product, obtain red solid product P 4 after drying, productive rate is 90%.
1H-NMR(400MHz,CF
3COOD),δppm:8.82-8.98(m,8H),4.93(m,4H),4.40(m,4H),4.04-4.21(m,16H).UV-Vis(H
2O,c=10
-5M):λ
max=498nm;Emission(H
2O,λ
ex=498nm):λ
max=545nm.MS(MALDI-TOF,m/z):calcdforC
36H
40N
8O
4:648.75.Found:648.3.
2) 138mgP4 is joined in the mixing solutions that the formaldehyde solution of the formic acid solution of 0.64mL85wt% and 0.44mL30wt% forms, first under nitrogen atmosphere, in stirring at room, react 1 hour, then be heated to 120 ℃ of reactions 16 hours, after equitemperature drops to room temperature, with ether sedimentation washing 3 times, obtain red solid product P 5 after drying, productive rate is 88%; UV-Vis (H
2o, c=10
-5m): λ
max=498nm; Emission (H
2o, λ
ex=498nm): λ
max=545nm.MS (MALDI-TOF, m/z): calcdforC
44h
56n
8o
4: 760.97.Found:761.63.
3) above-mentioned 100mgP5 is dissolved in the 3mL methanol solvate, then drips the methyl iodide of 0.2mL, be heated to 60 ℃ under nitrogen atmosphere, obtain Ji An Yan perylene diimide analog derivative P6.
1H-NMR(400MHz,CF
3COOD),δppm:8.95(d,J=34.1Hz,8H),5.04(s,4H),4.51(s,20H),4.26(s,6H),3.56(s,36H).UV-Vis(H
2O,c=10
-5M):λ
max=498nm;Emission(H
2O,λ
ex=498nm):λ
max=545nm.
P5 and P6 are the water-soluble Zhiization perylene diimide compounds containing 6 functional amidos.
The P5 structural formula is
The P6 structural formula is
Embodiment 3:
(1) 0.644g perylene list acid anhydride (2.0mmol) and 0.34mLN, N-dimethyl-ethylenediamine (6.0mmol) adds in 3mL water and stirs, then be warming up to 110 ℃, back flow reaction 15h, after dropping to room temperature, with the 1wt%KOH aqueous solution and water washing, obtain garnet solid product M1 after vacuum-drying, productive rate is 79%;
(2) 0.196gM1(0.5mmol) with 0.51g p-methyl benzenesulfonic acid (2.7mmol), in the 2mL deionized water solution, in 55 ℃, filter after reacting 5h, filtrate is removed desolventizing through rotary evaporation, with ether sedimentation washing, obtain red solid product M2 after centrifugal, drying, productive rate is 88%;
M1(0.196g, 0.5mmol) react 19h with 1.25mL p-methyl benzenesulfonic acid methyl esters (2.7mmol) in 53 ℃, after equitemperature drops to room temperature, filter, by rotary evaporation, filtrate is concentrated, then carry out washing of precipitate with ether, after centrifugal, drying, obtain red solid product M3, productive rate is 86%.
M2 and M3 are the water-soluble Xian perylene diimide compounds containing 1 functional amido.
The M2 structural formula is
The M3 structural formula is
Embodiment 4:
1) 0.644g perylene list acid anhydride (2.0mmol) and 15mL tri-(2-amino-ethyl) amine (100.5mmol) are added in the back flow reaction bottle, solution is heated to 105 ℃ of reactions 25 hours under nitrogen atmosphere, then be warmed up to 172 ℃ and continue reaction 3 hours, after equitemperature drops to room temperature, add ethanol that volume ratio is 1:3 and the mixed solvent of ether, filter, then with toluene, ether washing solid product, obtain red solid product M4 after drying, productive rate is 86%.
2) 150mgM4 is joined in the mixing solutions that the formaldehyde solution of the formic acid solution of 0.72mL85wt% and 0.53mL30wt% forms, first under nitrogen atmosphere, in stirring at room, react 45 minutes, then be heated to 110 ℃ of reactions 15 hours, after equitemperature drops to room temperature, with ether sedimentation washing 3 times, obtain red solid product M5 after drying, productive rate is 93%;
3) above-mentioned 100mgM5 is dissolved in the 3mL methanol solvate, then drips the methyl iodide of 0.25mL, be heated to 64 ℃ under nitrogen atmosphere, obtain Ji An Yan perylene list imide derivatives M6.
M5 and M6 are the water-soluble Zhiization perylene diimide compounds containing 3 functional amidos.
The M5 structural formula is
The M6 structural formula is
By the water-soluble perylene diimide compounds P2 of embodiment 1 and 2 preparations, P3, P5 and P6 are studied as DNA intercalation agent.
1) ultra-violet absorption spectrum test: by the water-soluble perylene diimide compounds P2 of embodiment 1 and 2 preparations, P3, P5 and P6 use respectively phosphoric acid buffer (10mM, pH7.4) being formulated as the solution that concentration is 5 μ M, is 1:0 according to mol ratio, 1:1,5:2,1:3 adds Ct-DNA, after room temperature is placed 12h, sample is carried out to the ultra-violet absorption spectrum test.Experimental result shows P2, P3, and after adding Ct-DNA in P5 and P6 sample, the ultra-violet absorption spectrum strength reduction, and its maximum peak position is attended by Red Shift Phenomena.
2) fluorescence emission spectrum test: by above-mentioned steps 1) the water-soluble perylene diimide compounds sample solution of preparation carries out the fluorescence emission spectrum test.Experimental result shows, P2, and P3, after adding Ct-DNA in P5 and P6 sample, the fluorescence emission spectrum strength reduction.
3) CD spectrum test: fixation of C t-DNA concentration is 80 μ M, adds respectively P2 in phosphoric acid buffer (10mM, pH7.4), P3, P5 and P6 and blank sample, be formulated as the solution that concentration is 3 μ M, after room temperature is placed 1h, sample is carried out to the CD spectrum test.Experimental result shows, in Ct-DNA solution, adds P2, P3, and after P5 and P6, the positive signal CD peak intensity at 277nm place strengthens, and the negative signal CD peak intensity at 247nm place weakens.
Uv-absorbing, fluorescence emission spectrum and CD spectrum experiment result show this water-soluble perylene diimide compounds P2, P3, and P5 and P6 can be inserted between the DNA base pair as DNA intercalation agent.
By the water-soluble perylene diimide compounds P2 of embodiment 1 and 2 preparations, P3, P5 and P6 are as the research of nucleus dyestuff.
By P2, P3, P5 and P6(10 μ M) dyeing processes the fruit bat fixing organization, after cultivating together 1 hour, P2, P3, P5 and P6 can with nucleus in DNA interact, thereby the specific nucleus that is enriched in is and in full accord with commercialization dyestuff DAPI mark.
By the water-soluble perylene diimide compounds P2 of embodiment 1 and 2 preparations, P3, P5 and P6 are as the research of anticancer growth medicine.
With tetrazolium (microculturetetrozolium, MTT) reduction method research P2, P3, P5 and P6 are to the U2OS(human osteosarcoma), the HCT116(colorectal carcinoma), the Hela(cervical cancer) and the AGS(adenocarcinoma of stomach) cancer cells carries out vitro inhibition growth of cancer cells active testing.Experimental result is as shown in the table, shows that P2 and P3 have good inhibition to growth of cancer cells such as U2OS, HCT116, Hela and AGS.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. a water-soluble perylene diimide compounds, is characterized in that, this compounds is that its general structure is containing the polyamine derivative of perylene diimide or perylene list imide structure:
Or
Or
Or
Wherein,
r
2=CH
3or CH
2cH
3, R
3=H or CH
3or CH
2cH
3.
2. the synthetic method of water-soluble perylene diimide compounds according to claim 1, is characterized in that, its concrete preparation process is:
(1) aminated compounds of 2mmol perylene acid anhydride and 6-12mmol is added in 3-6mL water and stirs, then be warming up to 100-120 ℃, back flow reaction 12-24h, after dropping to room temperature, with the 1-2wt%KOH aqueous solution and water washing, drying obtains product;
(2) product 0.6mmol step (1) obtained and the p-methyl benzenesulfonic acid of 5-9mmol filter after reacting 4-8h in 50-60 ℃ in 3-6mL water, filtrate is removed desolventizing through rotary evaporation, then with acetone, disperse, with ether sedimentation, wash, centrifugal, dry, obtain tertiary amine Yan perylene diimide compounds;
The product perhaps 0.6mmol step (1) obtained and the p-methyl benzenesulfonic acid methyl esters of 5-9mmol 50-60 ℃ react 18-24h after, add 3-5mL methyl alcohol filters in reaction mixture, by rotary evaporation, filtrate is concentrated, then carry out washing of precipitate with ether, after centrifugal, drying, obtain Ji An Yan perylene diimide compounds;
The described aminated compounds of step (1) is the tertiary amine compounds that contains an end amido, and its molecular formula general formula is NH
2(R
1nH)
0-5r
1n(R
2)
2, R wherein
1for the alkyl chain that contains 2-6 methylene radical, R
2for methyl or ethyl;
The above-mentioned tertiary amine Yan perylene diimide compounds made and Ji An Yan perylene diimide compounds are the water-soluble Xian perylene diimide compounds containing 2-12 functional amido.
3. the synthetic method of water-soluble perylene diimide compounds according to claim 1, is characterized in that, its concrete preparation process is:
1) adds perylene acid anhydride and polyamine compounds in the back flow reaction bottle for (1:40)-(1:60) in molar ratio, be heated to 98-110 ℃ of reaction 24-28h under nitrogen atmosphere, then be warmed up to 170-180 ℃ and continue reaction 2-5h, after dropping to room temperature, add ethanol that volume ratio is (1:2)-(1:4) and the mixed solvent of ether, filter, then by toluene, ether washing solid product, drying;
2) product 138mg step 1) obtained joins in the mixing solutions of 1-2mL, the formic acid solution that described mixing solutions is 80-86wt% and the formaldehyde solution of 25-35wt% (1.2:1)-(1.5:1) preparation by volume obtain, stir 0.5-1.5 hour post-heating under nitrogen atmosphere to 90-120 ℃ of reaction 12-18 hour, after dropping to room temperature, with the ether sedimentation washing, drying obtains the functionalized perylene diimide of tertiary amine compounds;
3) by 100mg step 2) the functionalized perylene diimide of the tertiary amine compounds that obtains is dissolved in the methyl alcohol or acetonitrile solvent of 2-4mL, then drip 0.2-0.4mL methyl iodide or 0.2-0.5mL iodoethane, be heated to 60-80 ℃ under nitrogen atmosphere, obtain Ji An Yan perylene diimide compounds;
The described polyamine compounds of step 1) is the aminated compounds that contains three end amidos, and its molecular formula general formula is NH
2(R
1nH)
0-5r
1n(R
1nH
2)
2, R wherein
1for the alkyl chain that contains 2-6 methylene radical;
The above-mentioned tertiary amine made functionalized perylene diimide compounds and Ji An Yan perylene diimide compounds are the water-soluble Zhiization perylene diimide compounds containing 6-16 functional amido.
4. the synthetic method of water-soluble perylene diimide compounds according to claim 1, is characterized in that, its concrete preparation process is:
(1) aminated compounds of 2mmol perylene list acid anhydride and 5-10mmol is added in 3-6mL water and stirs, then be warming up to 100-120 ℃, back flow reaction 12-24h, after dropping to room temperature, with the 1-2wt%KOH aqueous solution and water washing, drying obtains product;
(2) product 0.5mmol step (1) obtained and the p-methyl benzenesulfonic acid of 2.5-5mmol filter after reacting 4-8h in 50-60 ℃ in 2-4mL water, filtrate is removed desolventizing through rotary evaporation, with the ether sedimentation washing, centrifugal, dry, obtain tertiary amine Yan perylene list imide analog compounds;
The product perhaps 0.5mmol step (1) obtained and the p-methyl benzenesulfonic acid methyl esters of 2.5-5mmol filter after reacting 18-24h at 50-60 ℃, by rotary evaporation, filtrate is concentrated, then carry out washing of precipitate with ether, after centrifugal, drying, obtain Ji An Yan perylene list imide analog compounds;
The described aminated compounds of step (1) is the tertiary amine compounds that contains an end amido, and its molecular formula general formula is NH
2(R
1nH)
0-5r
1n(R
2)
2, R wherein
1for the alkyl chain that contains 2-6 methylene radical, R
2for methyl or ethyl;
The above-mentioned tertiary amine Yan perylene list imide analog compounds made and Ji An Yan perylene list imide analog compounds are the water-soluble Xian perylene diimide compounds containing 1-6 functional amido.
5. the synthetic method of water-soluble perylene diimide compounds according to claim 1, is characterized in that, its concrete preparation process is:
1) adds perylene list acid anhydride and polyamine compounds in the back flow reaction bottle for (1:40)-(1:60) in molar ratio, be heated to 98-110 ℃ of reaction 24-28h under nitrogen atmosphere, then be warmed up to 170-180 ℃ and continue reaction 2-5h, after dropping to room temperature, add ethanol that volume ratio is (1:2)-(1:4) and the mixed solvent of ether, filter, then by toluene, ether washing solid product, drying;
2) product 150mg step 1) obtained joins in the mixing solutions of 1.0-2mL, the formic acid solution that described mixing solutions is 80-86wt% and the formaldehyde solution of 25-35wt% (1.2:1)-(1.5:1) preparation by volume obtain, stir 0.5-1.5 hour post-heating under nitrogen atmosphere to 90-120 ℃ of reaction 12-18 hour, after dropping to room temperature, with the ether sedimentation washing, drying obtains the functionalized perylene of tertiary amine list imide analog compounds;
3) by 100mg step 2) the functionalized perylene of the tertiary amine list imide analog compounds that obtains is dissolved in 2-4mL methyl alcohol or acetonitrile solvent, then drip 0.2-0.4mL methyl iodide or 0.2-0.5mL iodoethane, be heated to 60-80 ℃ under nitrogen atmosphere, obtain Ji An Yan perylene list imide analog compounds;
The described polyamine compounds of step 1) is the aminated compounds that contains three end amidos, and its molecular formula general formula is NH
2(R
1nH)
0-5r
1n(R
1nH
2)
2, R wherein
1for the alkyl chain that contains 2-6 methylene radical;
The above-mentioned tertiary amine made functionalized perylene list imide analog compounds and Ji An Yan perylene list imide analog compounds are the water-soluble Zhiization perylene diimide compounds containing 3-8 functional amido.
6. water-soluble perylene diimide compounds according to claim 1 is as the application of DNA intercalation agent.
7. water-soluble perylene diimide compounds according to claim 1 is as the application of nucleus dyestuff.
8. water-soluble perylene diimide compounds according to claim 1 is as the application of anticancer growth medicine.
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