CN103497154A - Water-soluble perylene imide compounds, usage as DNA intercalator, and applications thereof in growth inhibition of cancer cells - Google Patents

Abstract

The invention discloses a water-soluble perylene imide compound and its application as a DNA intercalating agent and in inhibiting the growth of cancer cells, which belong to the technical field of chemical synthesis of biological medicines. The present invention introduces cationic amine functional groups into perylene anhydride derivatives and perylene monoanhydride systems, greatly improving their water solubility, and combining with the light stability of perylene derivatives, a series of new water-soluble Sexual perylene imide compounds. The water-soluble perylene imide compound synthesized by the invention can effectively intercalate between the double-helix base pairs of cell nucleus DNA, and can be used as a DNA intercalation agent. The water-soluble peryleneimide compound synthesized by the invention has a planar rigid structure, can be specifically enriched in fixed tissue nuclei, has strong fluorescence, is easy to perform fluorescence imaging, and is comparable in effect to the commercial nuclear dye DAPI. The water-soluble peryleneimide compound synthesized by the invention can effectively inhibit the growth of cancer cells such as U2OS, HCT116, Hela and AGS, and has obvious antitumor effect.

Description

A water-soluble perylene imide compound and its application as a DNA intercalation agent and in inhibiting the growth of cancer cells

technical field

The invention belongs to the technical field of biopharmaceutical chemical synthesis, and particularly relates to a water-soluble perylene imide compound and its application as a DNA intercalation agent and in inhibiting the growth of cancer cells.

Background technique

Perylene and its derivatives have good light, thermal and chemical stability, almost 100% fluorescence quantum yield, narrow fluorescence emission peak, can be separated from cell background fluorescence (395-479nm) and excellent dyeing performance. It has been widely used in the fields of organic optoelectronic devices, laser dyes and bioluminescent probes.

Intercalator refers to the reversible interaction between polycyclic aromatic hydrocarbon small molecule substances and DNA. This type of molecule is small in size, in an electron-deficient state, and has a certain planar rigidity. It can be inserted between two stacked base pairs of DNA. The insertion process leads to the helical extension and unzipping of DNA. At present, DNA intercalation agents that have been studied more include acridines, pyridocarbazoles, benzimidazol[1,2-C]quinolines, anthraquinones, etc., and all of them have been used in antitumor research. However, perylene derivatives with good planar rigid structures have not been used as DNA intercalators to study their antitumor properties.

In recent years, perylene imide compounds have been used in the biological field, such as cell-specific labeling, but poor water solubility directly affects its application in this field, so the water solubility of these compounds and the Maintaining a high fluorescence quantum yield is of great significance in the field of biology, especially in the field of DNA intercalators.

Contents of the invention

The object of the present invention is to provide a class of peryleneimide compounds with good light, heat and chemical stability, good water solubility and designable structure, and apply it to the field of biomedicine. agents and their use in inhibiting the growth of cancer cells.

The technical scheme of the present invention is: first select perylene anhydride or perylene monoimide as the fluorescent emitting group, and then react with a compound containing an amino functional group to prepare perylene imide derivatives and perylene monoimide derivatives modified with amino functional groups. Then, the peryleneimide derivatives and perylene monoimide derivatives with tertiary amine functional group modification are prepared by Eschweiler–Clarke reaction; the final reaction is to obtain different cationized peryleneimides containing tertiary amine salts or quaternary ammonium salts. Derivatives and perylene monoimide derivatives. The interaction between these peryleneimide derivatives and DNA was analyzed by ultraviolet absorption spectroscopy, fluorescence emission spectroscopy, and circular dichroism (CD), confirming that these compounds have excellent DNA intercalation properties, and can be used as DNA intercalation agents to inhibit tumor cells growth. Then, through cell and tissue level research and characterization, it is proved that the perylene imide derivative synthesized by the present invention has the specific enrichment effect of the nucleus and the effect of inhibiting the growth of cancer cells.

The water-soluble peryleneimide compound of the present invention is a polyamine derivative containing peryleneimide or perylene monoimide structure, and its general structural formula is:

or

or

or

R₂=CH₃或CH₂CH₃，R₃=H或CH₃或CH₂CH₃。in,

R ₂ =CH ₃ or CH ₂ CH ₃ , R ₃ =H or CH ₃ or CH ₂ CH ₃ .

The synthetic method of water-soluble perylene imide compound of the present invention, its specific preparation steps are:

(1) Add 2mmol of perylene anhydride and 6-12mmol of amine compound into 3-6mL of water and stir evenly, then raise the temperature to 100-120°C, reflux for 12-24h, after cooling down to room temperature, use 1-2wt% KOH aqueous solution Wash with water and dry to obtain the product;

(2) React 0.6mmol of the product obtained in step (1) with 5-9mmol of p-toluenesulfonic acid in 3-6mL of water at 50-60°C for 4-8h and then filter. Disperse with acetone, precipitate and wash with ether, centrifuge and dry to obtain tertiary ammonium salt perylene imide compounds;

Or react 0.6mmol of the product obtained in step (1) with 5-9mmol of methyl p-toluenesulfonate at 50-60°C for 18-24h, add 3-5mL of methanol to the reaction mixture solution and filter it, and use rotary evaporation The filtrate is concentrated, then precipitated and washed with ether, centrifuged and dried to obtain quaternary ammonium perylene imide compounds;

The amine compound described in step (1) is a tertiary amine compound containing one terminal amino group, and its molecular formula is NH ₂ (R ₁ NH) _0-5 R ₁ N(R ₂ ) ₂ , wherein R ₁ is An alkyl chain containing 2-6 methylene groups, R ₂ is methyl or ethyl;

The tertiary ammonium salt perylene imide compound and the quaternary ammonium salt perylene imide compound prepared above are water-soluble linear perylene imide compounds containing 2-12 functionalized amine groups.

The amine compound described in step (1) is preferably N,N-dimethylethylenediamine or N,N-diethyldivinyltriamine.

The synthetic method of water-soluble perylene imide compound of the present invention, its specific preparation steps are:

1) Add perylene anhydride and polyamines in a molar ratio of (1:40)-(1:60) into a reflux reaction flask, heat to 98-110°C for 24-28h under a nitrogen atmosphere, and then heat up to 170- Continue to react at 180°C for 2-5 hours, after cooling down to room temperature, add a mixed solvent of ethanol and ether with a volume ratio of (1:2)-(1:4), filter, then wash the solid product with toluene and ether, and dry;

2) Add 138mg of the product obtained in step 1) to 1-2mL of the mixed solution, the mixed solution is 80-86wt% formic acid solution and 25-35wt% formaldehyde solution in volume ratio (1.2:1)- (1.5:1) prepared, stirred under nitrogen atmosphere for 0.5-1.5 hours, then heated to 90-120 ° C for 12-18 hours, after cooling down to room temperature, washed with ether precipitation, dried to obtain tertiary amine functionalized peryleneimide Compounds;

3) Dissolve 100 mg of the tertiary amine-functionalized perylene imide compound obtained in step 2) in 2-4 mL of methanol or acetonitrile solvent, then add 0.2-0.4 mL of iodomethane or 0.2-0.5 mL of iodoethane dropwise, Heating to 60-80°C under a nitrogen atmosphere to obtain quaternary ammonium peryleneimide compounds;

The polyamine compound in step 1) is an amine compound containing three terminal amino groups, and its molecular formula is NH ₂ (R ₁ NH) _0-5 R ₁ N(R ₁ NH ₂ ) ₂ , where R ₁ is an alkyl chain containing 2-6 methylene groups;

The above prepared tertiary amine functionalized peryleneimide compounds and quaternary ammonium salt peryleneimide compounds are water-soluble branched peryleneimide compounds containing 6-16 functionalized amine groups.

The polyamine compound described in step 1) is preferably tris(2-aminoethyl)amine.

The synthetic method of water-soluble perylene imide compound of the present invention, its specific preparation steps are:

(1) Add 2mmol of perylene monoanhydride and 5-10mmol of amine compounds into 3-6mL of water and stir evenly, then heat up to 100-120°C, reflux for 12-24h, after cooling down to room temperature, use 1-2wt% KOH Washing with aqueous solution and water, and drying to obtain the product;

(2) React 0.5mmol of the product obtained in step (1) with 2.5-5mmol of p-toluenesulfonic acid in 2-4mL of water at 50-60°C for 4-8h, then filter, remove the solvent by rotary evaporation, and diethyl ether precipitation washing, centrifugation and drying to obtain tertiary ammonium salt perylene monoimide compounds;

Or react 0.5mmol of the product obtained in step (1) with 2.5-5mmol of methyl p-toluenesulfonate at 50-60°C for 18-24h, then filter, concentrate the filtrate by rotary evaporation, and then wash with ether for precipitation , centrifuged and dried to obtain quaternary ammonium perylene monoimide compounds;

The amine compound described in step (1) is a tertiary amine compound containing one terminal amino group, and its molecular formula is NH ₂ (R ₁ NH) _0-5 R ₁ N(R ₂ ) ₂ , wherein R ₁ is An alkyl chain containing 2-6 methylene groups, R ₂ is methyl or ethyl;

The tertiary ammonium salt perylene monoimide compound and the quaternary ammonium salt perylene monoimide compound prepared above are water-soluble linear perylene imide compounds containing 1-6 functionalized amine groups.

The amine compound described in step (1) is preferably N,N-dimethylethylenediamine or N,N-diethyldivinyltriamine.

The synthetic method of water-soluble perylene imide compound of the present invention, its specific preparation steps are:

1) Add perylene monoanhydride and polyamines in a molar ratio of (1:40)-(1:60) into the reflux reaction flask, heat to 98-110°C for 24-28h under nitrogen atmosphere, and then raise the temperature to 170°C Continue the reaction at -180°C for 2-5 hours, after cooling down to room temperature, add a mixed solvent of ethanol and ether with a volume ratio of (1:2)-(1:4), filter, then wash the solid product with toluene and ether, and dry;

2) Add 150mg of the product obtained in step 1) to 1.0-2mL of the mixed solution, the mixed solution is 80-86wt% formic acid solution and 25-35wt% formaldehyde solution in volume ratio (1.2:1)- (1.5:1) prepared, stirred under nitrogen atmosphere for 0.5-1.5 hours, then heated to 90-120 ° C for 12-18 hours, after cooling down to room temperature, washed with ether precipitation, dried to obtain tertiary amine functionalized perylene monoimide Amine compounds;

3) Dissolve 100 mg of the tertiary amine-functionalized perylene monoimide compound obtained in step 2) in 2-4 mL of methanol or acetonitrile solvent, then add 0.2-0.4 mL of iodomethane or 0.2-0.5 mL of iodoethane dropwise, Heating to 60-80°C under a nitrogen atmosphere to obtain quaternary ammonium salt perylene monoimide compounds;

The polyamine compound in step 1) is an amine compound containing three terminal amino groups, and its molecular formula is NH ₂ (R ₁ NH) _0-5 R ₁ N(R ₁ NH ₂ ) ₂ , where R ₁ is an alkyl chain containing 2-6 methylene groups;

The above prepared tertiary amine functionalized perylene monoimide compounds and quaternary ammonium salt perylene monoimide compounds are water-soluble branched perylene imide compounds containing 3-8 functionalized amine groups.

The polyamine compound described in step 1) is preferably tris(2-aminoethyl)amine.

The application of the above-mentioned water-soluble peryleneimide compound as a DNA intercalation agent.

The application of the above-mentioned water-soluble peryleneimide compound as a cell nucleus dye.

The application of the above-mentioned water-soluble perylene imide compound as a drug for inhibiting the growth of cancer cells.

The present invention has following beneficial effects:

1. Using perylene anhydride derivatives and their analogs as fluorescent emission groups, a series of water-soluble peryleneimide compounds with different amine functional groups were synthesized through different synthetic methods; these compounds have good Light, heat, chemical stability, high fluorescence quantum yield, good water solubility, and structural designability.

2. The water-soluble peryleneimide compound synthesized by the present invention can effectively intercalate between the base pairs of the DNA double helix in the cell nucleus, and can be used as a DNA intercalation agent, which has good practical application value.

3. The water-soluble peryleneimide compound synthesized by the present invention has a planar rigid structure, can be specifically enriched in fixed tissue nuclei, has strong fluorescence, is easy to perform fluorescence imaging, and is comparable to the effect of the commercial nuclear dye DAPI.

4. The water-soluble peryleneimide compound synthesized by the present invention can effectively inhibit the growth of cancer cells such as U2OS, HCT116, Hela and AGS, and has obvious anti-tumor effect; it has broad application in the fields of scientific research, diagnosis and treatment of biomedicine application prospects.

5. The synthesis method of the present invention is simple, easy to synthesize in large quantities, and has good application value.

Description of drawings

Figure 1: Molecular model diagram of intercalation of water-soluble peryleneimide compound P2 into DNA base pairs, as well as fluorescence imaging diagram applied to specific nucleus enrichment and histogram of inhibition of HCT116 cell growth.

The reaction flow chart of synthesizing water-soluble perylene imide compounds in Fig. 2 embodiment 1 and embodiment 2.

The reaction flow chart of synthesizing water-soluble perylene monoimide compounds in Fig. 3 embodiment 3 and embodiment 4.

Figure 4 (A1-A4) UV absorption and (B1- B4) Fluorescence emission spectrum diagram.

Figure 5 The fixed Ct-DNA concentration is 80μM, in phosphate buffer (10mM, pH7.4), adding water-soluble peryleneimide compounds P2, P3, P5 and P6 (3μM) and blank test (A-D) CD Spectrum.

Fig. 6 Fluorescence imaging of Drosophila salivary gland fixed tissue under the co-staining of water-soluble peryleneimide compounds P2, P3 and DAPI. (A-B) 10 μM of P2, P3 (two amine substituents connected to the imide site, P2, P3 have a rigid planar framework structure) were stained to mark Drosophila salivary glands, 546nm channel fluorescence imaging, specifically enriched in the nucleus . (A'-B') Drosophila larval salivary gland tissue stained with nuclear dye DAPI, fluorescence imaging under 405nm light channel. (A"-B") P2, P3 nuclei fluorescence localization imaging effect completely coincides with DAPI fluorescence imaging effect.

Fig. 7 Fluorescence imaging of Drosophila salivary gland fixed tissue under the co-staining of water-soluble peryleneimide compounds P5, P6 and DAPI. (A-B) 10 μM of P5, P6 (the imide site is connected with four amine substituents, P5, P6 have a rigid planar frame structure) were stained to mark the salivary glands of Drosophila, and the 546nm channel fluorescence imaging was specifically enriched on the nucleus . (A'-B') Drosophila larval salivary gland tissue stained with nuclear dye DAPI, fluorescence imaging under 405nm light channel. (A"-B") P5, P6 nuclei fluorescence localization imaging effect completely coincides with DAPI fluorescence imaging effect.

Detailed ways

The present invention will be further elaborated below in conjunction with specific embodiments. The present invention is not limited to these specific examples.

The characterization of the product structure used: nuclear magnetic ¹ H and ¹³ C spectrum (Bruker400), mass spectrometry AXIMA-CFRplusMALDI-TOFMS.

The optical properties of the product were characterized using: ultraviolet-visible spectroscopy (Cintra20, GBC, Australia), fluorescence spectroscopy (HoribaJobinYvonFluoroMax-4NIR, NJ, USA).

The biological experimental characterization of the product uses: fluorescence microscope (AMGEVOSf1microscope), laser confocal microscope (LeicaTCSSP2AOBSconfocalmicroscope).

Implementation example 1:

1) Suspend 0.7846g perylene anhydride (2.0mmol) and 0.34mL N,N-dimethylethylenediamine (6.0mmol) in 4mL water and stir evenly, then heat up to 100°C and reflux for 12h. After the temperature drops to room temperature, use Wash with 1wt% KOH aqueous solution and water, obtain red solid product P1 after vacuum drying, yield rate is 75%;

¹ H-NMR (400MHz, CF ₃ COOD), δppm: 8.77 (d, J=12.3Hz, 8H), 4.77 (s, 4H), 3.75 (s, 4H), 3.19 (s, 12H). ¹³ C- NMR (100MHz, CF ₃ COOD), 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 ₃₂ H ₂₈ N ₄ O ₄ ,532.6;Found,533.6(M+H ⁺ ).

2) 0.32g P1 (0.6mmol) and 1.02g p-toluenesulfonic acid (5.4mmol) were reacted in 3.5mL deionized aqueous solution at 50°C for 4h and then filtered. The precipitate was washed, centrifuged and dried to obtain the red solid product P2 with a yield of 85%.

¹ H-NMR(400MHz,CF ₃ COOD),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). ¹³ C-NMR(100MHz,CF ₃ COOD),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 ₂ O,c=5.0×10 ^-max6 M): _λ (ε)=498nm(2.45×10 ⁴ M ^-1 cm ^-1 );Emission(H ₂ O,λ _ex =498nm):λ _max =545nm.

P1 (0.32g, 0.6mmol) was reacted with 2.5mL methyl p-toluenesulfonate (5.4mmol) at 50°C for 20h. After the temperature dropped to room temperature, 3mL methanol was added to the reaction mixture and filtered. The filtrate was concentrated, then precipitated and washed with ether, centrifuged and dried to obtain the red solid product P3 with a yield of 80%.

¹ H-NMR(400MHz,CF ₃ COOD),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 ₂ O,c=10 ^-5 M):λ _max =498nm; Emission(H ₂ O,λ _ex =498nm):λ _max =545nm.

P2 and P3 are water-soluble linear perylene imide compounds containing two functionalized amine groups.

The structural formula of P2 is

The structural formula of P3 is

Implementation example 2:

1) Add 0.81g perylene anhydride (2.1mmol) and 15mL tris(2-aminoethyl)amine (100.5mmol) into the reflux reaction flask, in which tris(2-aminoethyl)amine is also the reaction solvent; Heat to 100°C for 28 hours under atmosphere, then raise the temperature to 170°C and continue to react for 4 hours. After the temperature drops to room temperature, add a mixed solvent of ethanol and ether with a volume ratio of 1:3, filter, and then wash with toluene and ether The solid product was dried to obtain the red solid product P4 with a yield of 90%.

¹ H-NMR (400MHz, CF ₃ COOD), δppm: 8.82-8.98(m, 8H), 4.93(m, 4H), 4.40(m, 4H), 4.04-4.21(m, 16H).UV-Vis( H ₂ O,c=10 ^-5 M):λ _max =498nm;Emission(H ₂ O,λ _ex =498nm):λ _max =545nm.MS(MALDI-TOF,m/z):calcdforC ₃₆ H ₄₀ N ₈ O ₄ :648.75. Found: 648.3.

2) Add 138mg of P4 to a mixed solution consisting of 0.64mL85wt% formic acid solution and 0.44mL30wt% formaldehyde solution, stir and react at room temperature under nitrogen atmosphere for 1 hour, then heat to 120°C for 16 hours, and wait for the temperature to drop to After room temperature, it was precipitated and washed with diethyl ether for 3 times, and the red solid product P5 was obtained after drying with a yield of 88%; UV-Vis (H ₂ O, c=10 ^-5 M): λ _max =498nm; Emission (H ₂ O ,λ _ex =498nm):λ _max =545nm.MS(MALDI-TOF,m/z):calcdforC ₄₄ H ₅₆ N ₈ O ₄ :760.97.Found:761.63.

3) The above 100mg of P5 was dissolved in 3mL of methanol solvent, then 0.2mL of iodomethane was added dropwise, and heated to 60°C under a nitrogen atmosphere to obtain the quaternary ammonium salt peryleneimide derivative P6. ¹ H-NMR (400MHz, CF ₃ COOD), 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 ₂ O,c=10 ^-5 M):λ _max =498nm;Emission(H ₂ O,λ _ex =498nm):λ _max =545nm.

P5 and P6 are water-soluble branched perylene imide compounds containing 6 functionalized amine groups.

The structural formula of P5 is

The structural formula of P6 is

Implementation example 3:

(1) Add 0.644g of perylene monoanhydride (2.0mmol) and 0.34mL of N,N-dimethylethylenediamine (6.0mmol) into 3mL of water and stir evenly, then heat up to 110°C, reflux for 15h, after cooling down to room temperature, use Washed with 1wt% KOH aqueous solution and water, and dried in vacuo to obtain dark red solid product M1 with a yield of 79%;

(2) React 0.196g M1 (0.5mmol) with 0.51g p-toluenesulfonic acid (2.7mmol) in 2mL deionized aqueous solution at 55°C for 5h, then filter, remove the solvent by rotary evaporation, wash with ether precipitation, centrifuge, After drying, red solid product M2 was obtained with a yield of 88%;

M1 (0.196g, 0.5mmol) was reacted with 1.25mL methyl p-toluenesulfonate (2.7mmol) at 53°C for 19h, and after the temperature dropped to room temperature, filtered, the filtrate was concentrated by rotary evaporation, and then carried out with diethyl ether. The precipitate was washed, centrifuged and dried to obtain the red solid product M3 with a yield of 86%.

M2 and M3 are water-soluble linear perylene imide compounds containing one amine group functionalization.

The structural formula of M2 is

The structural formula of M3 is

Implementation example 4:

1) Add 0.644g perylene monoanhydride (2.0mmol) and 15mL tris(2-aminoethyl)amine (100.5mmol) into the reflux reaction flask, heat the solution to 105°C for 25 hours under a nitrogen atmosphere, and then raise the temperature to 172 Continue to react at ℃ for 3 hours, wait for the temperature to drop to room temperature, add a mixed solvent of ethanol and ether with a volume ratio of 1:3, filter, then wash the solid product with toluene and ether, and obtain the red solid product M4 after drying. The yield is 86%.

2) Add 150mg M4 to the mixed solution consisting of 0.72mL85wt% formic acid solution and 0.53mL30wt% formaldehyde solution, stir and react at room temperature under nitrogen atmosphere for 45 minutes, then heat to 110°C for 15 hours, wait for the temperature to drop to After room temperature, it was precipitated and washed 3 times with ether, and after drying, the red solid product M5 was obtained with a yield of 93%;

3) The above 100mg M5 was dissolved in 3mL methanol solvent, then 0.25mL iodomethane was added dropwise, and heated to 64°C under a nitrogen atmosphere to obtain the quaternary ammonium salt perylene monoimide derivative M6.

M5 and M6 are water-soluble branched peryleneimide compounds containing three amine groups.

The structural formula of M5 is

The structural formula of M6 is

The water-soluble peryleneimide compounds P2, P3, P5 and P6 prepared in Examples 1 and 2 were used as DNA intercalators for research.

1) Ultraviolet absorption spectrum test: the water-soluble peryleneimide compounds P2, P3, P5 and P6 prepared in Examples 1 and 2 were respectively prepared into solutions with a concentration of 5 μM in phosphate buffer (10 mM, pH 7.4), Ct-DNA was added according to the molar ratio of 1:0, 1:1, 5:2, and 1:3, and after 12 hours at room temperature, the samples were tested by ultraviolet absorption spectrum. The experimental results showed that after adding Ct-DNA to P2, P3, P5 and P6 samples, the intensity of ultraviolet absorption spectrum was weakened, and the maximum peak position was accompanied by red shift.

2) Fluorescence emission spectrum test: The water-soluble peryleneimide compound sample solution prepared in the above step 1) was subjected to a fluorescence emission spectrum test. The experimental results showed that after adding Ct-DNA to P2, P3, P5 and P6 samples, the intensity of fluorescence emission spectrum was weakened.

3) CD spectrum test: fix the concentration of Ct-DNA at 80 μM, add P2, P3, P5 and P6 and blank samples respectively in phosphate buffer (10 mM, pH7.4), prepare a solution with a concentration of 3 μM, and place it at room temperature After 1 h, the CD spectrum test was carried out on the sample. The experimental results showed that after adding P2, P3, P5 and P6 to the Ct-DNA solution, the CD peak intensity of the positive signal at 277nm was enhanced, and the CD peak intensity of the negative signal at 247nm was weakened.

The experimental results of ultraviolet absorption, fluorescence emission spectrum and CD spectrum show that the water-soluble peryleneimide compounds P2, P3, P5 and P6 can be inserted between DNA base pairs as DNA intercalators.

The water-soluble peryleneimide compounds P2, P3, P5 and P6 prepared in Examples 1 and 2 were used as nuclear dyes.

Drosophila fixed tissues were stained with P2, P3, P5 and P6 (10 μM), and after 1 hour of incubation, P2, P3, P5 and P6 were able to interact with DNA in the nucleus, thereby specifically enriching in the nucleus, and It is completely consistent with the commercial dye DAPI labeling.

The water-soluble peryleneimide compounds P2, P3, P5 and P6 prepared in Examples 1 and 2 were used as drugs for inhibiting the growth of cancer cells.

Inhibition of P2, P3, P5 and P6 on U2OS (human osteosarcoma), HCT116 (colon cancer), Hela (cervical cancer) and AGS (gastric adenocarcinoma) cancer cells in vitro using tetrazolium salt (microculturetetrozolium, MTT) reduction method Cancer cell growth activity test. The experimental results are shown in the table below, showing that P2 and P3 have good inhibitory effects on the growth of cancer cells such as U2OS, HCT116, Hela and AGS.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (8)

1. a water-soluble perylene imide compound, is characterized in that, this class compound is the polyamine derivative containing perylene imide or perylene monoimide structure, and its general structural formula is:

or

or

or in,

R ₂ =CH ₃ or CH ₂ CH ₃ , R ₃ =H or CH ₃ or CH ₂ CH ₃ . 2. the synthetic method of water-soluble perylene imide compound according to claim 1, is characterized in that, its concrete preparation steps are: (1) Add 2mmol of perylene anhydride and 6-12mmol of amine compound into 3-6mL of water and stir evenly, then raise the temperature to 100-120°C, reflux for 12-24h, after cooling down to room temperature, use 1-2wt% KOH aqueous solution Wash with water and dry to obtain the product; (2) React 0.6mmol of the product obtained in step (1) with 5-9mmol of p-toluenesulfonic acid in 3-6mL of water at 50-60°C for 4-8h and then filter. Disperse with acetone, precipitate and wash with ether, centrifuge and dry to obtain tertiary ammonium salt perylene imide compounds; Or react 0.6mmol of the product obtained in step (1) with 5-9mmol of methyl p-toluenesulfonate at 50-60°C for 18-24h, add 3-5mL of methanol to the reaction mixture solution and filter it, and use rotary evaporation The filtrate is concentrated, then precipitated and washed with ether, centrifuged and dried to obtain quaternary ammonium perylene imide compounds; The amine compound described in step (1) is a tertiary amine compound containing one terminal amino group, and its molecular formula is NH ₂ (R ₁ NH) _0-5 R ₁ N(R ₂ ) ₂ , wherein R ₁ is An alkyl chain containing 2-6 methylene groups, R ₂ is methyl or ethyl; The tertiary ammonium salt perylene imide compound and the quaternary ammonium salt perylene imide compound prepared above are water-soluble linear perylene imide compounds containing 2-12 functionalized amine groups. 3. the synthetic method of water-soluble perylene imide compound according to claim 1, is characterized in that, its concrete preparation steps are: 1) Add perylene anhydride and polyamines in a molar ratio of (1:40)-(1:60) into a reflux reaction flask, heat to 98-110°C for 24-28h under a nitrogen atmosphere, and then heat up to 170- Continue to react at 180°C for 2-5 hours, after cooling down to room temperature, add a mixed solvent of ethanol and ether with a volume ratio of (1:2)-(1:4), filter, then wash the solid product with toluene and ether, and dry; 2) Add 138mg of the product obtained in step 1) to 1-2mL of the mixed solution, the mixed solution is 80-86wt% formic acid solution and 25-35wt% formaldehyde solution in volume ratio (1.2:1)- (1.5:1) prepared, stirred under nitrogen atmosphere for 0.5-1.5 hours, then heated to 90-120 ° C for 12-18 hours, after cooling down to room temperature, washed with ether precipitation, dried to obtain tertiary amine functionalized peryleneimide Compounds; 3) Dissolve 100 mg of the tertiary amine-functionalized perylene imide compound obtained in step 2) in 2-4 mL of methanol or acetonitrile solvent, then add 0.2-0.4 mL of iodomethane or 0.2-0.5 mL of iodoethane dropwise, Heating to 60-80°C under a nitrogen atmosphere to obtain quaternary ammonium peryleneimide compounds; The polyamine compound in step 1) is an amine compound containing three terminal amino groups, and its molecular formula is NH ₂ (R ₁ NH) _0-5 R ₁ N(R ₁ NH ₂ ) ₂ , where R ₁ is an alkyl chain containing 2-6 methylene groups; The above prepared tertiary amine functionalized peryleneimide compounds and quaternary ammonium salt peryleneimide compounds are water-soluble branched peryleneimide compounds containing 6-16 functionalized amine groups. 4. the synthetic method of water-soluble perylene imide compound according to claim 1, is characterized in that, its concrete preparation steps are: (1) Add 2mmol of perylene monoanhydride and 5-10mmol of amine compounds into 3-6mL of water and stir evenly, then heat up to 100-120°C, reflux for 12-24h, after cooling down to room temperature, use 1-2wt% KOH Washing with aqueous solution and water, and drying to obtain the product; (2) React 0.5mmol of the product obtained in step (1) with 2.5-5mmol of p-toluenesulfonic acid in 2-4mL of water at 50-60°C for 4-8h, then filter, remove the solvent by rotary evaporation, and diethyl ether precipitation washing, centrifugation and drying to obtain tertiary ammonium salt perylene monoimide compounds; Or react 0.5mmol of the product obtained in step (1) with 2.5-5mmol of methyl p-toluenesulfonate at 50-60°C for 18-24h, then filter, concentrate the filtrate by rotary evaporation, and then wash with ether for precipitation , centrifuged and dried to obtain quaternary ammonium perylene monoimide compounds; The amine compound described in step (1) is a tertiary amine compound containing one terminal amino group, and its molecular formula is NH ₂ (R ₁ NH) _0-5 R ₁ N(R ₂ ) ₂ , wherein R ₁ is An alkyl chain containing 2-6 methylene groups, R ₂ is methyl or ethyl; The tertiary ammonium salt perylene monoimide compound and the quaternary ammonium salt perylene monoimide compound prepared above are water-soluble linear perylene imide compounds containing 1-6 functionalized amine groups. 5. the synthetic method of water-soluble perylene imide compound according to claim 1, is characterized in that, its concrete preparation steps are: 1) Add perylene monoanhydride and polyamines in a molar ratio of (1:40)-(1:60) into the reflux reaction flask, heat to 98-110°C for 24-28h under nitrogen atmosphere, and then raise the temperature to 170°C Continue the reaction at -180°C for 2-5 hours, after cooling down to room temperature, add a mixed solvent of ethanol and ether with a volume ratio of (1:2)-(1:4), filter, then wash the solid product with toluene and ether, and dry; 2) Add 150mg of the product obtained in step 1) to 1.0-2mL of the mixed solution, the mixed solution is 80-86wt% formic acid solution and 25-35wt% formaldehyde solution in volume ratio (1.2:1)- (1.5:1) prepared, stirred under nitrogen atmosphere for 0.5-1.5 hours, then heated to 90-120 ° C for 12-18 hours, after cooling down to room temperature, washed with ether precipitation, dried to obtain tertiary amine functionalized perylene monoimide Amine compounds; 3) Dissolve 100 mg of the tertiary amine-functionalized perylene monoimide compound obtained in step 2) in 2-4 mL of methanol or acetonitrile solvent, then add 0.2-0.4 mL of iodomethane or 0.2-0.5 mL of iodoethane dropwise, Heating to 60-80°C under a nitrogen atmosphere to obtain quaternary ammonium salt perylene monoimide compounds; The polyamine compound in step 1) is an amine compound containing three terminal amino groups, and its molecular formula is NH ₂ (R ₁ NH) _0-5 R ₁ N(R ₁ NH ₂ ) ₂ , where R ₁ is an alkyl chain containing 2-6 methylene groups; The above prepared tertiary amine functionalized perylene monoimide compounds and quaternary ammonium salt perylene monoimide compounds are water-soluble branched perylene imide compounds containing 3-8 functionalized amine groups. 6. The application of the water-soluble peryleneimide compound according to claim 1 as a DNA intercalator. 7. The application of the water-soluble peryleneimide compound according to claim 1 as a nuclear dye. 8. The application of the water-soluble peryleneimide compound according to claim 1 as a drug for inhibiting the growth of cancer cells.

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