CN115873042B - Divalent ruthenium compound containing bioactive group and synthesis method and application thereof - Google Patents

Abstract

The invention discloses a divalent ruthenium compound containing a biological active group, and a synthesis method and application thereof, and particularly relates to the technical field of medicines. The method comprises the steps of taking 3, 5-dimethoxy benzaldehyde as a raw material, and synthesizing a resveratrol intermediate product through sodium borohydride reduction, halogenation, wittig, iron powder reduction and dehydration condensation reaction; coordination of the soluble ruthenium hydrate salt with the nitrogen-containing compound to form a ruthenium-containing intermediate; and splicing the ruthenium-containing intermediate product with the resveratrol intermediate product to obtain the divalent ruthenium compound containing the bioactive group. The synthetic method of the invention carries out comparative research on the reaction conditions involved in the synthetic route, improves the reaction yield, and the total yield of the whole synthetic route reaches 30-35%. The divalent ruthenium compound containing the bioactive group has obvious tumor cell killing capacity and strong effect. The tumor cell inhibition ability is controlled by illumination, which has remarkable significance for reducing toxic and side effects.

Description

Divalent ruthenium compound containing bioactive group and synthesis method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a divalent ruthenium compound containing a biological active group, a synthesis method and application thereof, and specifically relates to a resveratrol derivative modified Ru (II) compound photosensitizer, a synthesis method and application thereof.

Background

Resveratrol is widely regarded as a natural polyphenol anticancer substance because of the advantages of high stability, low price, easy obtainment and the like. Research shows that resveratrol can play an anti-tumor role by controlling malignant tumor transformation, regulating metabolism of cancer cells such as oxidation and reduction, and the like, controlling neovascularization of cancer cells, and the like through various small molecular mechanisms, has been widely applied to diagnosis and prevention of tumors, and has great significance in the research and development processes of anti-tumor. Wang Chunzhong and the like show that the veratrole has good anti-colon cancer effect and can become a new candidate drug or sensitizer for treating colon cancer. Li Bihui and other studies show that resveratrol can enable the growth period of SW579 cell division to lag the S phase by controlling the expression of p-AKT protein, so that the normal growth of SW579 cell of thyroid cancer can be obviously controlled. However, resveratrol has the problems of low activity, poor stability and the like.

Photosensitizers (PS) are capable of undergoing photochemical reactions under light conditions. The use of photosensitizers in specific lesion areas can make them function centrally in tumor cells and are not harmful per se. Metal-based photoactive drugs can play a role in three different therapies, which are: photodynamic therapy, photoactivation chemotherapy, and photothermal therapy. The photosensitizer is a single compound and has the characteristics of good light stability, small dark toxicity, large phototoxicity, fluorescence in a near infrared region (650-800 nm), easy chemical synthesis, easy absorption by body tissues and the like.

Photodynamic therapy (PDT) is a method in which, after activation by light of a specific wavelength, a photosensitizer enriched in a tumor transfers energy to a biological matrix surrounding the photosensitizer to produce active oxygen, such as singlet oxygen # ¹ O ₂ ) And further to methods of treatment for killing tumor cells. The way in which reactive oxygen species damage cancer cells is as follows: (1) itself toxic and directly damaging; (2) Blocking and disrupting blood vessels (3) associated with tumor tissue directly stimulates the autoimmune system. Photodynamic therapy is an efficient treatment method because photosensitizers catalyze chemical reactions, and one photosensitizer can form thousands of photosensitizers ¹ O ₂ A molecule. Photodynamic therapy is an anti-cancer therapy approved by the FDA immediately following surgery, radiation therapy, chemotherapy, and immune function therapy.

Currently, the effectiveness of photodynamic therapy to destroy cancer cells depends on the dose of light. However, in the process of absorbing light, the human tissue can have a weakening effect on the illumination intensity, so that the application range of the photodynamic therapy on cancer cells is limited, and the photodynamic therapy is limited to the growth part of the cancer cells which can be irradiated by light, and has no remarkable killing effect on infiltrated cancer cells, so that the photodynamic therapy is limited in the treatment of deep tissue diseases. Second, photodynamic therapy has poor therapeutic efficacy in hypoxic tumor microenvironments due to its oxygen demand characteristics. Finally, traditional photodynamic therapy has a short excitation wavelength, cannot penetrate thicker human tissues, and has difficulty in treating tumors located in deep human tissues.

Disclosure of Invention

Therefore, the invention provides a divalent ruthenium compound containing a bioactive group, and a synthesis method and application thereof, so as to solve the problems of low activity, poor stability, shorter excitation wavelength of photodynamic therapy, incapability of penetrating thicker human tissues and the like of the existing resveratrol.

Due to the defects of low activity, poor stability and the like of resveratrol, the invention tries to research a novel resveratrol derivative with good selectivity, high efficiency and low toxicity by changing the structure of the resveratrol. Based on the outstanding advantages of high targeting property, low side effect and low toxicity of photodynamic therapy in clinical application of malignant tumors, the invention develops the high-performance anti-tumor photosensitizer with higher tumor cell inhibition capability and longer excitation wavelength by changing the phenolic hydroxyl structure of resveratrol and combining Ru (II) compound as photosensitizer to induce singlet oxygen in tumor cells so as to express tumor inhibition capability. Through the design concept of pharmacophore split drug molecules, the compound is hybridized with resveratrol structure and the structure of Ru (II) compound is introduced, so that the compound with good pharmacological activity is obtained.

In order to achieve the above object, the present invention provides the following technical solutions:

according to a first aspect of the present invention there is provided a method of synthesis of a divalent ruthenium compound containing a biologically active group, the method comprising:

3, 5-dimethoxy benzaldehyde is used as a raw material, and a resveratrol intermediate product is synthesized through sodium borohydride reduction, halogenation, wittig, iron powder reduction and dehydration condensation reaction;

coordination of the water-soluble ruthenium salt with the nitrogen-containing compound to form a ruthenium-containing intermediate;

and splicing the ruthenium-containing intermediate product with the resveratrol intermediate product to obtain the divalent ruthenium compound containing the bioactive group.

The halogenation may be selected from chloro, bromo or iodo, as exemplified by PBr employed in the present invention ₃ The bromination is carried out, so that the reaction rate is higher;

the soluble ruthenium hydrate salt is a soluble ruthenium hydrate salt, and as an example, the invention adopts ruthenium trichloride hydrate;

the nitrogen-containing compound is a nitrogen-containing organic matter or a nitrogen-containing inorganic matter which can coordinate with the soluble ruthenium hydrate salt, preferably the nitrogen-containing organic matter, more preferably the dinitrogen-containing cyclic compound, and the invention adopts 2, 2-dipyridine or 1, 10-phenanthroline as an example.

Further, the synthetic route of the synthetic method is as follows:

wherein i is NaBH ₄ ，MeOH，DCM，0℃-r.t.，2h；

ii is PBr ₃ ，DCM，0℃-r.t.，30min；

iii is triphenylphosphine, toluene, 110 ℃ for 3h;

iv is NaH, DCM,0 ℃ -r.t., overnight;

v is NH ₄ Cl, reduced iron powder, etOH, H ₂ O, refluxing for 2h;

vi is N ₂ MeOH,70 ℃, reflux, overnight; vii, N ₂ Refluxing LiCl, DMF at 155 ℃ overnight;

viii is N ₂ MeOH,70 ℃, reflux overnight.

Intermediate 2-I-7 may be intermediate 2-I-7a or intermediate 2-I-7b;

the reaction of the hydrated ruthenium trichloride, the 2, 2-bipyridine and the lithium chloride is the intermediate product 2-I-7a;

the intermediate product 2-I-7b is obtained by the reaction of the hydrated ruthenium trichloride, the 1, 10-phenanthroline and the lithium chloride. Further, the intermediates 2-I-7 and 2-I-6a were synthesized as 2-1 and 2-2.

Further, the chemical formula of the compound 2-1 is [ C ₄₃ H ₃₈ N ₆ O ₃ Ru] ²⁺ ；

The structural formula is as follows:

the chemical formula of the 2-2 is [ C ₄₇ H ₄₀ N ₆ O ₃ Ru] ²⁺ ；

The structural formula is as follows:

further, the intermediate 2-I-7 synthesizes compound 2-3 and compound 2-4 with intermediate 2-I-6 b.

The chemical formula of the compound 2-3 is [ C ₄₇ H ₃₈ N ₆ O ₃ Ru] ²⁺ ；

The structural formula is as follows:

the chemical formula of the compounds 2-4 is [ C ₅₁ H ₄₀ N ₆ O ₃ Ru] ²⁺ ；

The structural formula is as follows:

further, the toluene in iii is redistilled to remove water from the solvent before use.

According to the second aspect of the invention, the divalent ruthenium compound containing the bioactive group is a resveratrol derivative modified Ru (II) compound photosensitizer, and comprises a compound 2-1, a compound 2-2, a compound 2-3 or a compound 2-4.

The anionic portion of the compound is a common anion; including but not limited to PF ₆ ^- ，Cl ^- 。

According to the application of the divalent ruthenium compound containing the biological active group in preparing antitumor drugs, the invention provides a divalent ruthenium compound containing the biological active group.

The invention has the following advantages:

the synthetic method of the invention carries out comparative research on the reaction conditions involved in the synthetic route, improves the reaction yield, and the total yield of the whole synthetic route reaches 30-35%.

The divalent ruthenium compounds 2-1 and 2-3 containing the bioactive groups have stronger absorption peaks at 450 nm; the divalent ruthenium compounds 2-2 and 2-4 containing biological active groups have stronger absorption peaks at 525 nm. Compounds 2-2 and 2-4 have a stronger conjugated structure, resulting in a red shift in their absorption peaks. Longer wavelength light can penetrate human tissue effectively, which will positively affect photodynamic therapy.

The divalent ruthenium compound containing the bioactive group has obvious tumor cell killing capacity and strong effect. The tumor cell inhibition ability is controlled by illumination, which has remarkable significance for reducing toxic and side effects.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.

The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum (DMSO-d 6) of a target compound 2-1 provided in example 1 of the present invention;

FIG. 2 is a high resolution mass spectrum of the target compound 2-1 provided in example 1 of the present invention;

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum (DMSO-d 6) of the target compound 2-2 provided in example 1 of the present invention;

FIG. 4 is a high resolution mass spectrum of the target compound 2-2 provided in example 1 of the present invention;

FIG. 5 shows the hydrogen nuclear magnetic resonance spectrum (DMSO-d 6) of the target compound 2-3 according to example 1 of the present invention;

FIG. 6 is a high resolution mass spectrum of the target compounds 2-3 provided in example 1 of the present invention;

FIG. 7 shows the hydrogen nuclear magnetic resonance spectrum (DMSO-d 6) of the target compound 2-4 according to example 1 of the present invention;

FIG. 8 is a high resolution mass spectrum of the target compounds 2-4 provided in example 1 of the present invention;

FIG. 9 is a graph showing the ultraviolet-visible absorption spectra of compounds 2-1 to 2-4 provided in Experimental example 1 of the present invention;

FIG. 10 shows the singlet oxygen generating ability of Compounds 2-1 to 2-4 under green light irradiation provided in Experimental example 1 of the present invention;

wherein, A-compound 2-1; B-Compound 2-2; C-Compound 2-3; D-Compound 2-4;

FIG. 11 is a graph showing the singlet oxygen generating ability of Experimental example 1 of the present invention under red light irradiation of Compounds 2-1 to 2-4;

wherein, A-compound 2-1; B-Compound 2-2; C-Compound 2-3; D-Compound 2-4;

FIG. 12 is a photograph of a sample of the living and dead cells of compound 2-4 treated with (A-C) in the dark and (D-F) in the irradiation at 610nm for 30min, which is provided in Experimental example 2 of the present invention;

wherein, the scale: 80. Mu.M;

FIG. 13 shows the results of uptake of Compounds 2-4 of Experimental example 3 of the present invention in Hela cells;

FIG. 14 is a photograph of Hela cells imaged after treatment with Compounds 2-4 and DCFH-DA according to Experimental example 3 of the present invention;

wherein (A-C) Hela cells are co-applied with the compound 2-4 and DCFH-DA without illumination; (D-F) Hela cells were co-plated with Compound 2-4 and DCFH-DA and irradiated with 610nm light for 30min;

scale 20 μm.

FIG. 15 shows agarose gel electrophoresis patterns of experimental example 3pBR322 plasmid DNA of the present invention after co-application with compounds 2-4;

FIG. 16 shows the effect of Compounds 2-4 of Experimental example 3 of the invention on the expression of proteins associated with the endogenous apoptotic pathway of Hela cells;

wherein, A-Western immunoblotting strip; b-relative gray scale analysis.

Detailed Description

Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The absolute ethyl alcohol, petroleum ether, ethyl acetate, toluene, N, N-dimethylformamide, methanol, methylene dichloride, concentrated sulfuric acid, concentrated nitric acid, ethylene glycol, sodium chloride, anhydrous sodium sulfate, sodium borohydride and the like used in the preparation are all purchased from national pharmaceutical group chemical company, and the purity is analytically pure. The consumables and reagents used in the cell experiments were purchased from Jiangsu Kaiki Biotechnology Co. The light sources used in the experiments were 520nm and 610nm LED arrays with an optical power density of 30.0mW/cm ² 。

Example 1

The embodiment provides a synthesis method of a divalent ruthenium compound containing a bioactive group, which comprises the following steps:

the synthetic route is as follows:

wherein i is NaBH ₄ MeOH, DCM,0 ℃ -r.t.,2h; ii is PBr ₃ ，DCM，0℃-r.t.，

30min; iii is triphenylphosphine, toluene, 110 ℃ for 3h; iv is NaH, DCM,0 ℃ -r.t., overnight; v is NH ₄ Cl, reduced iron powder, etOH, H ₂ O, refluxing for 2h; vi is N ₂ MeOH,70 ℃, reflux, overnight; vii, N ₂ Refluxing LiCl, DMF at 155 ℃ overnight; viii is N ₂ MeOH,70 ℃, reflux overnight.

Synthesis of intermediate 2-I-1: 3, 5-Dimethoxybenzaldehyde (4.11 g,24.74 mmol) was dissolved in absolute methanol (60 mL), and methylene chloride was added in an appropriate amount to aid dissolution. NaBH at 0 DEG C ₄ A solution of (2.178 g,74.22 mmol) in methanol was added dropwise to the reaction solution and stirred at room temperature for 2h. The reaction was observed by TLC plate. After the reaction of the compound was completed, ice water was added to quench the reaction, followed by evaporation of the solvent using a rotary evaporator. 100mL of water was added to the solid and extracted with dichloromethane (30 mL. Times.3) and the organic phase was collected. Subsequently, 100mL of a saturated saline solution was added to the organic phase, and the mixture was extracted with methylene chloride (30 mL). The organic phase was collected as anhydrous Na ₂ SO ₄ And (5) drying. The filtrate was collected after filtration and the solvent was distilled off to obtain 3.44g of white crystals with a yield of 82.6%. (yield η=82.6%, actual yield/theoretical yield, the same applies below)

Synthesis of intermediate 2-I-2: intermediate 2-I-1 (3.26 g,19.38 mmol) was dissolved in dry dichloromethane (50 mL) and PBr was taken up ₃ (5.52 mL,58.12 mmol) was dissolved in anhydrous dichloromethane (50 mL), and the PBr3 solution was added dropwise to the above reaction solution at 0deg.C. Followed by stirring at room temperature for 30min. The reaction was observed by TLC plate. After the reaction of the compound is completed, adding ice water to quench the reaction. 100mL of water was added, extracted with dichloromethane (30 mL. Times.3), and the organic phase was collected. Subsequently, 100mL of a saturated saline solution was added to the organic phase, and the mixture was extracted with methylene chloride (30 mL). The organic phase was collected with anhydrous Na ₂ SO ₄ Drying and filtering. After the solvent is distilled offSeparation was performed using a silica gel column (eluent: petroleum ether/dichloromethane=2:1, v/v). 3.83g of white crystals were obtained in 79.84% yield.

Synthesis of intermediate 2-I-3: intermediate 2-I-2 (3.19 g,13.80 mmol) and triphenylphosphine (4.70 g,17.94 mmol) were added sequentially to a 250mL round bottom flask containing 100mL toluene. Reflux at 110℃for 3h. After completion of the reaction, the reaction mixture was cooled to room temperature as observed by thin layer chromatography. The solid was filtered off and washed with toluene (30 mL). After drying in a vacuum oven, 6.72g of a white solid was obtained in 98.70% yield.

Synthesis of intermediate 2-I-4: intermediate 2-I-3 (4.20 g,8.51 mmol) was dissolved in anhydrous dichloromethane (50 mL), naH (1.02 g,42.50 mmol) was added at 0deg.C and stirred at the same temperature for 30min. 4-methoxy-3-nitrobenzaldehyde (1.54 g,8.50 mmol) was dissolved in anhydrous dichloromethane (20 mL) and added dropwise to the above reaction solution, and reacted overnight at room temperature. After the completion of the reaction was detected by thin layer chromatography spot plate, the reaction was quenched with ice water. To the reaction solution was added 100mL of water, extracted with methylene chloride (30 mL. Times.3), and the organic phase was collected. Subsequently, 100mL of a saturated saline solution was added to the organic phase, and the mixture was extracted with methylene chloride (30 mL). The organic phases were combined with anhydrous Na ₂ SO ₄ Drying and filtering. After filtration, the filtrate was collected and the solvent was distilled off, and separated using a silica gel column (eluent: petroleum ether/ethyl acetate=20:1, v/v). 1.56g of yellow oil was obtained in 58.02% yield.

Synthesis of intermediate 2-I-5: intermediate 2-I-4 (3.77 g,11.96 mmol) was dissolved in absolute ethanol (50 mL) and NH was taken up ₄ Cl (2.56 g,47.85 mmol) was dissolved in water (5 mL) and added to the above reaction solution followed by reduced iron powder (3.35 g,59.82 mmol). The mixture was refluxed at 80℃for 2h. After completion of the reaction was observed by TLC plate, the mixture was filtered through celite to remove the reduced iron powder. To the reaction mixture was added 100mL of water, extracted with ethyl acetate (30 mL. Times.3), and the organic phase was collected. Subsequently, 100mL of a saturated saline solution was added to the organic phase, followed by extraction with ethyl acetate (30 mL). The organic phase was collected with anhydrous Na ₂ SO ₄ Drying and filtering. Filtering, collecting filtrate, and evaporating to remove solventThe preparation gave 2.90g of a tan oil in 85.10% yield.

Synthesis of intermediates 2-I-6a and 2-I-6 b: intermediate 2-I-5 (0.40 g,1.40 mmol) and pyridine-2-carbaldehyde (0.22 g,1.40 mmol) or quinoline-2-carbaldehyde (0.22 g,1.40 mmol) were dissolved in dry methanol (15 mL), and refluxed overnight at 70℃to complete the reaction, followed by complete resolution of the solvent by TLC. Separation was performed using a silica gel column (eluent: petroleum ether/ethyl acetate=50:1, v/v).

For the synthesis of 2-I-6a, 0.42g of yellow oil was obtained in 80.15% yield.

For the synthesis of 2-I-6b, 0.38g of yellow oil was obtained in 64.08% yield.

Synthesis of intermediate 2-I-7a, b: ruthenium trichloride hydrate (1.14 g,5.00 mmol) and 2, 2-bipyridine (1.56 g,10.00 mmol) were added sequentially to the above solution, and the amount of DMF was increased appropriately. The mixture was refluxed overnight at 150 ℃ under nitrogen protection. After the reaction was completed, the solvent was distilled off by using a rotary evaporator, 150mL of acetone was added thereto, and the mixture was allowed to stand at-20℃for 24 hours or more. The reaction solution was filtered, and the obtained cake was washed with water. After drying, intermediate 2-I-7a is obtained. 0.28g of yellow-purple-black solid is obtained, and the yield is 11.67%.

Ruthenium trichloride hydrate (1.14 g,5.00 mmol) and 1, 10-phenanthroline (1.80 g,10.00 mmol) and lithium chloride (1.41 g,33.00 mmol) were added sequentially to the above solution, and the above reaction was performed sequentially. After drying, intermediate 2-I-7b is obtained. 1.30g of yellow-purple-black solid is obtained, and the yield is 52.00%.

Synthesis of target products 2-1 to 2-4:

synthesis of target products 2-1 and 2-2

2-I-6a (0.26 g,0.70 mmol) and intermediate 2-I-7a (0.34 g,0.70 mmol) or 2-I-7b (0.34 g,0.70 mmol) were added separately to anhydrous methanol (30 mL), refluxed overnight at 70℃and the reaction was observed by TLC plates, after completion of the reaction the solvent was removed by rotary evaporator and separated using a neutral alumina column (eluent: dichloromethane/methanol=100:1, v/v). The target products 2-1 and 2-2 are obtained respectively.

Synthesis of target product 2-1: 0.35g of a reddish brown solid was obtained, and the yield was 46.67%.

As shown in figures 1 and 2 of the drawings, ¹ h NMR (600 mhz, dmso) δ9.25 (s, 1H), 8.87 (d, j=8.1 hz, 2H), 8.63 (d, j=5.3 hz, 1H), 8.52 (d, j=8.1 hz, 1H), 8.45 (d, j=7.7 hz, 1H), 8.31 (d, j=8.1 hz, 1H), 8.21 (dtd, j=22.9, 15.3,7.6hz, 4H), 7.85 (dd, j=17.5, 6.6hz, 2H), 7.76 (dd, j=11.6, 5.4hz, 2H), 7.72-7.65 (m, 1H), 7.61 (dd, j=12.0, 5.8hz, 2H), 7.56-7.47 (m, 2H), 7.37-7.29 (m, 1H), 6.94 (dd, j=17.5, 6.6hz, 2H), 7.76 (dd, j=11.6.6.6.6 hz, 2H), 7.76 (dd, j=11.6.6, 5.4hz, 2H), 7.72-7.65 (m, 1H), 7.61 (dd, 3H), 6.56-7.7.29 (m, 1H), 6.94 (j=3.9, 3H), 3.7.7H (j=3H): [ C ₄₃ H ₃₈ N ₆ O ₃ Ru] ²⁺ ([M] ²⁺ ) 394.1019; 394.1090 was measured.

Synthesis of target product 2-2: 0.39g of a reddish brown solid was obtained in 49.36% yield.

As shown in figures 3 and 4 of the drawings, ¹ H NMR(600MHz,DMSO)δ9.68(s,1H),8.85(d,J＝8.2Hz,1H),8.80(d,J＝8.4Hz,1H),8.72(d,J＝8.1Hz,1H),8.62(d,J＝8.2Hz,1H),8.47(d,J＝7.6Hz,2H),8.34(d,J＝8.2Hz,1H),8.30–8.21(m,2H),8.20–8.15(d,1H),8.16–8.08(m,1H),8.02(d,J＝5.9Hz,1H),7.92(d,J＝5.3Hz,1H),7.87–7.81(m,1H),7.72(t,J＝7.6Hz,1H),7.65–7.56(m,2H),7.56–7.49(m,1H),7.39(t,J＝11.5,4.3Hz,2H),7.27(t,J＝6.9Hz,2H),6.95(d,J＝8.6,1.8Hz,1H)，6.57(s,1H),6.42(d,1H),6.39(s,1H),6.32(d,2H),6.06(s,1H),5.48(s,1H),3.69 (s, 9H). Calculated: [ C ₄₇ H ₄₀ N ₆ O ₃ Ru] ²⁺ ([M] ²⁺ ) 419.1097; 419.1017 was measured.

(II) Synthesis of target products 2-3 and 2-4

2-I-6b (0.30 g,0.70 mmol) and intermediate 2-I-7a (0.34 g,0.70 mmol) or 2-I-7b (0.34 g,0.70 mmol) were added separately to anhydrous methanol (30 mL) and the reaction was refluxed overnight at 70℃as described above, and the solvent was removed by TLC after completion of the reaction. Neutral alumina column separation was used (eluent: dichloromethane/methanol=100:1, v/v). The target products 2-3 and 2-4 are obtained respectively.

Synthesis of target product 2-3: 0.41g of a tan solid was obtained in 44.09% yield.

As shown in figures 5 and 6 of the drawings, ¹ h NMR (600 mhz, dmso) δ9.28 (s, 1H), 9.16 (t, j=6.7 hz, 1H), 8.93 (dd, j=8.3, 0.9hz, 1H), 8.90-8.85 (m, 1H), 8.76 (dd, j=8.2, 0.9hz, 1H), 8.48 (d, j=7.6 hz, 1H), 8.44 (d, j=8.9 hz, 1H), 8.40 (d, j=9.2 hz, 2H), 8.29 (d, j=4.4 hz, 1H), 8.24 (d, j=8.9 hz, 1H), 8.21-8.08 (m, 4H), 8.05 (t, j=7.0 hz, 1H), 8.00 (ddd, j=15.9, 8.5,4.4hz, 1H), 7.75 (dd, j=7.5, 9hz, 1H), 8.40 (d, j=9.9 hz, 1H), 8.29 (d, j=4.4 hz, 1H), 8.24 (d, j=8.4.9 hz, 1H), 8.21-8.08 (m, 4H), 8.05 (t, j=7.7.0 hz, 1H), 3.7.7.9 hz, 1H), 3.7.7 (3H): [ C ₄₇ H ₃₈ N ₆ O ₃ Ru] ²⁺ ([M] ²⁺ ) 418.1019; 418.1010 was measured.

Synthesis of target product 2-4: 0.32g of purplish red solid was obtained in 32.99% yield.

As shown in figures 7 and 8 of the drawings, ¹ h NMR (600 mhz, dmso) δ9.30 (s, 1H), 9.15 (d, j=4.8 hz, 1H), 8.94 (d, j=8.2 hz, 1H), 8.88 (d, j=8.3 hz, 1H), 8.77 (d, j=8.4 hz, 1H), 8.50 (d, j=7.7 hz, 1H), 8.43 (dd, j=25.8, 8.8hz, 4H), 8.29 (d, j=4.7 hz, 1H), 8.25 (d, j=8.8 hz, 1H), 8.19-8.08 (m, 4H), 8.06 (d, j=8.9 hz, 1H), 8.01 (dd, j=8.0, 5.1hz, 1H), 7.96 (s, 1H), 7.85-7.71 (m, 4H), 7.49 (dd, j=4.7.7 hz, 1H), 8.25 (d, j=8.8 hz, 1H), 8.19-8.8H), 8.19 (d, 4H), 8.19-8.08 (m, 4H), 8.06 (j=8.9 hz, 1H), 7.9 hz, 1H), 7.01 (d, j=3.3H), 3.3H (3H): [ C ₅₁ H ₄₀ N ₆ O ₃ Ru] ²⁺ ([M] ²⁺ )，443.1097；443.1029.

1. The synthesis of intermediate 2-I-1 is a reduction reaction of 3, 5-dimethoxybenzaldehyde in the presence of sodium borohydride. The reaction yield is higher. Before adding sodium borohydride to the reaction solution, sodium borohydride is dissolved by methanol and then is added dropwise to the reaction solution through a constant-pressure bottom solution funnel, so that a large number of bubbles are prevented. The purpose of the two washes during the post-treatment is to remove impurities from the residual reaction solvent.

2. Intermediates 2-I-2 to 2-I-6 showed by nuclear magnetic resonance spectroscopy that the resulting products were all in accordance with the design. Wherein, the intermediate 2-I-3 needs to be redistilled with toluene before the synthesis reaction, and the moisture in the solvent is removed.

3. The synthesis of the intermediate 2-I-4 is a Wittig reaction, and the key of the reaction is that the whole reaction is carried out under anhydrous and anaerobic conditions, particularly the control of the anhydrous conditions, so as to ensure the normal formation of phosphorus ylide. The phosphine ylide is directly reacted with aldehyde groups to form olefins (cis-trans mixture) by the action of NaH. The trans structure in the product needs to be removed by column chromatography.

4. The synthesis of the intermediate 2-I-5 is iron powder reduction reaction, the reaction liquid obtained after the filtration by diatomite can be washed by ethanol to remove NH in the reaction liquid ₄ Cl。

5. In consideration of poor stability of the intermediate 2-I-6, the intermediate 2-I-5 and pyridine-2-formaldehyde or quinoline-2-formaldehyde can be dissolved in absolute methanol and refluxed overnight, and then the next reaction is directly carried out, so that the deterioration of the product is prevented.

6. The synthesis of intermediates 2-I-7a and 2-I-7b was improved in combination with the laboratory practice by reference to previous reports. In order to prevent the zero-valent catalyst from being oxidized during the reaction, the invention uses an oil pump to pump out the oxygen in the reaction liquid and carries out N on the reaction liquid ₂ Bubbling and applying nitrogen protection to the reaction system.

7. The synthesis of the target products 2-1 to 2-4 is a coordination reaction, and the target products 2-1 to 2-4 are relatively stable, so that the target products can be separated by means of column chromatography, and the obtained yellow or red powder is confirmed to be the designed target product by nuclear magnetic resonance hydrogen spectrum and mass spectrum.

The reaction conditions involved in the synthetic route are compared, the reaction yield is improved, and the total yield of the whole synthetic route reaches 30-35%. The experiment shows that the structures of four end products from 2-1 to 2-4 and a plurality of key intermediate products are analyzed by nuclear magnetic resonance hydrogen spectrum ¹ H NMR) analysis method. And four of the end products 2-1 to 2-4 were characterized by Mass Spectrometry (MS) analysis.

Experimental example 1

The experiment provides the photophysical and chemical properties of the resveratrol derivative modified Ru (II) compound photosensitizer 2-1-2-4:

1. preparing a solution: compounds 2-1 to 2-4 were each prepared as a solution at a concentration of 1.00mM using chromatographically pure acetonitrile as a solvent, and then diluted with chromatographically pure acetonitrile to a diluted solution at a concentration of 10.0. Mu.M.

Determination of the spectra: for the ultraviolet visible absorption spectrum: 3.00mL of the above 10.0. Mu.M solutions of compounds 2-1 to 2-4 were each taken in a quartz cuvette, and absorbance at 180-800nm was collected on an ultraviolet-visible spectrophotometer. And normalized with absorbance at 180 nm.

As a result, as shown in FIG. 9, it can be seen from FIG. 9 that compounds 2-1 and 2-3 have strong absorption peaks at 450 nm; compounds 2-2 and 2-4 have strong absorption peaks at 525 nm. Compounds 2-2 and 2-4 have a stronger conjugated structure, resulting in a red shift in their absorption peaks. Because longer wavelength light can penetrate human tissue effectively, this will have a positive impact on photodynamic therapy.

Preparation of DPBF: under dark conditions, a concentrated solution of DPBF at a concentration of 6.00mM was prepared in methanol. Then diluted with methanol to a concentration of 60.0. Mu.M. All DPBF solutions were prepared as ready to use.

Preparation of compound solution: under dark conditions, 2-1 to 2-4 Ru (II) compounds were each formulated as a concentrated solution in dimethyl sulfoxide (DMSO) at a concentration of 1.00 mM. Then diluted with methanol to a concentration of 10.0. Mu.M.

Detection of singlet oxygen: under the condition of avoiding light, 1.50mL of each of the two solutions is respectively taken in a quartz cuvette and uniformly mixed. The ultraviolet-visible absorption spectrum is measured by irradiation with 520nm green light every 1 min. Compounds 2-1 to 2-4 were then each irradiated with 610nm red light and tested for photodynamic activity according to the test procedure described above.

The singlet oxygen generating capacity results under green light irradiation are shown in FIG. 10, and it can be seen that the peak drop rates of the 2-3 and 2-4 compounds under green light irradiation are faster and the photodynamic activities are stronger. Under the condition of 520nm illumination, after the total illumination time reaches 18min, the characteristic absorption peak of DPBF almost completely disappears.

The singlet oxygen generating capacity under red light irradiation is shown in FIG. 11, and the result shows that under red light irradiation, the photodynamic power of the quinoline compound is stronger than that of the pyridine compound, and the absorption of the two quinoline products of 2-2 and 2-4 to red light is better.

Experimental example 2

To evaluate the in vitro antitumor activity of the four target compounds 2-1 to 2-4 synthesized in example 1, this experiment was conducted by CCK-8 to obtain a series of ICs under different conditions ₅₀ Values.

1. CCK-8 experiment

1. Cell culture and administration

Placing tumor cells in a complete culture solution containing 10% foetal calf serum, and placing in a sterile condition with 95% relative humidity and 5% CO ₂ Incubate in incubator and passaged after digestion with 0.25% pancreatin and 0.02% EDTA. And freezing and preserving the cell strain growing in the logarithmic phase, and preparing for subsequent experiments.

Cells were individually inoculated in 96-well plates, placed in a constant temperature incubator overnight, and cultured to 80%. The original medium was discarded. To the cells, 100. Mu.L of medium containing 0,0.1,0.5,1, 10, 50, 100. Mu.M of the compound was added, respectively. Three duplicate wells were set for each concentration. Cells were placed in a 37℃incubator (containing 5% CO) ₂ ) And incubated for 72h. For the illumination experiments, a 520nm or 610nm LED array was used to illuminate the 96-well plate for 30min, and then incubation was continued for 72h.

2.IC ₅₀ Measurement of values

After 72h of incubation, 10.0. Mu.L of CCK-8 working solution was added to each well and shaken well and the resulting cells incubated continuously for 2h in an incubator at 37 ℃. Absorbance was measured at 450nm using a microplate reader and OD values were read. The obtained data is used for calculating IC through SPSS Statistics software ₅₀ Values.

3. Results

The results are shown in Table 1. In dark conditions, similar to the positive control compounds, all target compounds have lower tumor cell inhibition ability, which indicates that the target compounds have certain safety in dark conditions. Under the irradiation of 520nm green light, all four compounds 2-1 to 2-4 show stronger anti-tumor activity. Taken together, the activity is as great as 2-4 > 2-2 > 2-3 > 2-1. And under the irradiation of 610nm red light, the activity of all the compounds is obviously reduced compared with green light. The reason for this is that the series of target compounds synthesized by the present invention have a main light absorption band in the green region, and have weak absorption of red light, thus exhibiting low activity. Similarly, since the major absorption band of the positive control is located near 450nm, the compound exhibits lower photodynamic activity, both under red and green light irradiation. Under red light irradiation, the compounds 2-4 still show stronger anti-tumor activity, which shows that chemical modification plays a role in the direction of enlarging pi electron delocalization degree, which is beneficial to the development of photodynamic therapy to clinical application. Given that Hela is more sensitive to compounds 2-4, subsequent biological experiments will select this cell as an in vitro model.

TABLE 1 in vitro inhibitory Activity (IC) of Compounds 2-1 to 2-4 against tumor cells under different conditions ₅₀ Value, mu M)

2. Live dead cell imaging experiments

1. Cell culture: resuscitate the cryopreserved cells, passaged in 6-well plates, and inoculated in 5% CO at 37℃under sterile conditions ₂ Culturing in an incubator for 24 hours.

2. Administration and illumination: after removal of the medium, the cells were washed 3 times with sterilized PBS (0.01 mm, ph=7.4) to remove cell metabolites and impurities. To each cell culture dish was added a solution of PBS (0.01 mM, pH=7.4) containing 1998. Mu.L of fresh medium and 2.00. Mu.L of compound 2-4 at a concentration of 50.0. Mu.M (containing 10% dimethyl sulfoxide) to give a final concentration of 5. Mu.M. Cells were incubated under the above conditions for 24h. After 24h incubation of the cells, the dishes were removed. The light is tightly attached to the upper cover of the culture dish by a 610nm LED for 30min. After the illumination is finished, the cells are incubated for 1 hour.

3. Calcein and propidium iodide staining: after the cell incubation was completed, the medium was changed and the cells were washed with PBS (0.01 mm, ph=7.4). The cells were then incubated with calcein AM and propidium iodide at a concentration of 5.00 μm each for 30min. Cells were then washed with PBS (0.01 mm, ph=7.4) buffer;

4. confocal shooting: the dishes were placed on a confocal laser scanning microscope. For the green channel, the excitation wavelength is set to 488nm and the detection wavelength is set to (500-540) nm; for the red channel, 561nm laser is used as excitation light, and the detection wavelength is (600-640) nm.

5. Live dead cell imaging experimental results:

as can be seen from fig. 12, the dosing concentration was 5 μm and the incubation time was 24h. When no light is applied, most cells fluoresce green, indicating that most cells are viable at this time. In contrast, after irradiation with light at 610nm for 30min, most cells exhibited a dead state. The experiment clearly shows that the compounds 2-4 have obvious tumor cell killing capacity and strong effect. In addition, the tumor cell inhibition capacity is controlled by illumination, which has significant meaning for reducing toxic and side effects.

Experimental example 3

Because the in vitro inhibition activity of the compound 2-4 on cancer cells is strong, the cell strain is selected for relevant biological research in the embodiment so as to reveal the intrinsic mechanism of the compound 2-4 for inhibiting tumor cells.

Uptake of Ru (II) compounds by cells

The metal drug cell uptake experiments were performed using ICP-MS technology. Inoculating experimental cells into 6-hole culture plate, removing culture medium after cell density in culture plate reaches 90%, and replacing new culture medium, adding 5 μm concentration of compound 2-4 and positive control [ Ru (bpy) respectively ₃ ] ²⁺ . After incubating the cells in a constant temperature incubator for 12 hours, the cells were collected by centrifugation, digested with 200. Mu.L of 65% nitric acid at 65℃for 10 hours, and the total content of Ru in the cells was determined by inductively coupled plasma mass spectrometry (ICP-MS) experimental method.

As a result, as shown in FIG. 13, [ Ru (bpy) ] was selected ₃ ] ²⁺ Is a photosensitizer. The administration concentration is 5 mu M, and the incubation time is 24 hours; it can be seen that the pair positive control compound of three tumor cells [ Ru (bpy) ₃ ] ²⁺ Is weak. In sharp contrast, the uptake capacity of these three tumor cells for compounds 2-4 was significantly superior to that of the positive control compound. The authors speculate that this difference is due to the more lipophilic organic ligand of compounds 2-4Resulting, as a water-soluble compound, [ Ru (bpy) ₃ ] ²⁺ Is significantly weaker than the target compound. This phenomenon will certainly have a positive promoting effect on the enhancement of the antitumor activity.

(II) ROS imaging experiments

1. Cell culture: cells were seeded in glass-bottomed confocal dishes and incubated overnight in a thermostated incubator.

2. Application of the drug and illumination: the culture medium in the dish was removed and washed 3 times with sterilized PBS buffer (0.01 mm, ph=7.4) to remove cell metabolites and impurities. To each cell culture dish was added 999 μl of fresh medium and 1.00 μl of a solution of compound 2-4 in PBS buffer (0.01 mM, ph=7.4) at a concentration of 5.0mM (containing 10% dimethyl sulfoxide) to a final concentration of 50nM. Cells were incubated under the above conditions for 24h. After 4h incubation of the cells, the dishes were removed. The light is tightly attached to the upper cover of the culture dish by a 610nm LED for 2min.

dcfh-DA staining: the medium was discarded, 500. Mu.L of DCFH-DA working solution at a concentration of 5.00. Mu.M was added, and incubated for 30min. Finally, the working solution was aspirated and washed 3 times with PBS buffer (0.01 mm, ph=7.4).

4. Confocal shooting: the petri dish was placed on a confocal laser scanning microscope, and the detection wavelength was set to (500-600) nm with 448nm laser as excitation light.

As shown in FIG. 14, at a drug administration concentration of 5. Mu.M, incubation time of 24 hours, no green fluorescence was observed in HeLa cells after application of compounds 2 to 4 under dark conditions, indicating that there was no ROS present in excess in HeLa cells, and to some extent, safety of the compounds under dark conditions. And after 30 minutes of red light irradiation, confocal microscopy detected significant green fluorescence in the cells. This demonstrates that the compound is effective in elevating ROS levels in cells and that the behavior of inducing ROS can be controlled by light.

(III) agarose gel electrophoresis experiments

The intracellular ROS level is extracted to possibly cause DNA damage, and the DNA damage effect of the compounds 2-4 before and after illumination is explored by using a DNA agarose gel electrophoresis technology.

pBR322 plasmid DNA at a concentration of 0.100. Mu.g/. Mu.L was mixed with 5.00. Mu.L of the corresponding compound to give a concentration of 1:0. Mu.M; 2:1. Mu.M; 3:2. Mu.M; 4:5. Mu.M; 5:10. Mu.M; 6:20. Mu.M; 7:50. Mu.M; 8:80. Mu.M; 9:100. Mu.M; a concentration gradient of 10:200. Mu.M. For the illumination group, each sample was illuminated sequentially for 30min using a 610nm LED. All samples were then incubated at 37℃in a incubator protected from light for 24h. Subsequently, a gel was prepared and loaded, and 2.00. Mu.L of loadingbuffer was added to each sample to give a final concentration of 10% (v/v). The resulting samples were separated using agarose gel at 100V for 1h and the indicator sites were observed. The gel sample obtained was incubated in EB solution at a concentration of 0.750. Mu.g/mL for 30min. Finally, deionized water is used for washing off the EB solution on the gel surface, and a gel digital imaging system is used for photographing the obtained gel.

As shown in FIG. 15, in the dark, as the concentration of the compound increases, a large amount of open-loop DNA (Form II), most of which is supercoiled DNA (Form I), does not appear in the DNA electrophoresis pattern. This indicates that compounds 2-4 do not cause DNA damage in dark conditions. Correspondingly, after light irradiation at 610nm for 30min, the proportion of open-loop DNA in the pattern gradually increases with the increase of the drug concentration, and finally all the DNA is cut into open-loop forms. This demonstrates that compounds 2-4 are more damaging to DNA and are under the control of light.

(IV) Western blot (Westernblot) experiment

The effect of compounds 2-4 on the expression of proteins associated with the endogenous apoptotic pathway of HeLa cells was studied by western blots experiments.

The cells in the logarithmic growth phase were counted and inoculated into 6-well plates and cultured until the cells were about 85%. Cells in the wells were digested, collected by centrifugation into centrifuge tubes and washed with PBS, an appropriate amount of lysis solution was added dropwise to the cells, and the cells were lysed in an ice box for 2h. Centrifuging at 10000rpm for 20-30 min, and collecting supernatant for later use. 0.5mL of supernatant was placed in an EP tube and stored at-20℃taking care of handling on ice. Protein content was determined using BCA protein kit (Thermo, waltham, MA). After gel preparation, proteins were loaded at equal concentrations on 8-12% polyacrylamide gels and separated by electrophoresis. The proteins were then transferred to Immobilon-P membranes on a semi-dry transfer membrane apparatus. Soaking Yu Lichun in red dye liquor, and cutting proteins on the membrane according to the indication of a Marker. It was immersed in a skim milk dish of PBST and blocked by shaking on a decolorizing shaker for 1h. Followed by incubation overnight at 4℃with primary antibodies to beta-actin, caspase3, bax and Bcl-2. After the membranes were washed three times with PBST, they were incubated with IRDye 800-crosslinked secondary antibodies for 1h at 37 ℃. AB luminescence liquid (A: B=1:1) was prepared and added dropwise to the strip for about 10 s. Removing excessive luminous liquid, covering with film, covering film with film, pressing for 70s, and exposing in an exposure machine. The film was washed out under dark conditions and the strips were observed. The resulting bands were greyscale analyzed using image J software and statistically analyzed with Excel software.

In FIG. 16, caspase3 protein expression was slightly elevated in dark conditions, whereas pro-apoptotic protein Bax was not significantly affected and anti-apoptotic protein Bcl-2 expression was slightly inhibited compared to the blank. In the light condition, the disturbance of the protein expression is obviously enhanced. Caspase3 and Bax are significantly elevated, while the anti-apoptotic protein Bcl-2 is significantly inhibited. This suggests that under light conditions, the endogenous apoptotic pathways within Hela cells are opened.

The compounds 2-2 and 2-4 have stronger conjugated structures, so that the absorption peak of the compounds is red shifted, the damage range and depth of photodynamic therapy in phototherapy of deep infiltration tissue diseases are improved to a certain extent, and the photodynamic therapy is positively influenced. Under the irradiation of green light, the peak value of DPBF experiments of the compounds 2-3 and 2-4 is fast in descending speed and strong in photodynamic activity. The compounds 2-2 and 2-4 can effectively trigger singlet oxygen under the irradiation of 610nm visible light, and have strong inhibition capability on detected tumor cells.

The invention evaluates the antitumor activity of the 2-1 to 2-4 Ru (II) complexes by a CCK-8 method. Experimental results prove that the target compound has certain safety under dark conditions; under the irradiation of 520nm green light, the compound 2-4 shows stronger anti-tumor activity, and the activity strength sequence is 2-4 & gt2-2 & gt2-3 & gt2-1; and under the irradiation of 610nm red light, the activity of all the compounds is obviously reduced compared with green light. The reason for this is that the series of target compounds synthesized by this study have a main light absorption band in the green region and a weak absorption of red light, and thus exhibit a low activity. Under red light irradiation, compounds 2-4 still showed strong antitumor activity, indicating that chemical modification acts in a direction that enlarges pi electron delocalization. The research further verifies that the compounds 2-4 have obvious tumor cell killing capability through live dead cell imaging experiments, and the tumor cell inhibition capability is controlled by illumination, so that a basic condition is created for reducing toxic and side effects.

The invention proves that the uptake capacity of tumor cells to the compounds 2-4 is obviously better than that of positive control compounds [ Ru (bpy) through a metal medicine cell uptake experiment ₃ ] ²⁺ This will have a positive effect on the enhancement of its antitumor activity. Compounds 2-4 have organic ligands that are more lipophilic. As a water-soluble complex, [ Ru (bpy) ₃ ] ²⁺ Is significantly lower than the target compound. In ROS imaging experiments, compounds 2-4 are effective in elevating ROS levels in cells, and the behavior of eliciting ROS can be controlled by light. The invention uses DNA agarose gel electrophoresis technology to prove that the compound 2-4 can not cause DNA damage under dark condition, and the compound 2-4 has stronger damage capability to DNA under 610nm illumination and is controlled by illumination. Finally, the project finds that the expression of the cell endogenous apoptosis channel related protein is obviously disturbed under the illumination condition in the western blots experiment. Caspase3 and Bax are significantly elevated, while the anti-apoptotic protein Bcl-2 is significantly inhibited. This suggests that under light conditions, the endogenous apoptotic pathways within Hela cells are opened.

Therefore, the divalent ruthenium compound containing the bioactive group prepared by the invention has low toxicity, high inhibition on tumor cells and good application prospect for resisting tumors.

While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (4)

1. A method for synthesizing a divalent ruthenium compound containing a biologically active group, the method comprising:

3, 5-dimethoxy benzaldehyde is used as a raw material, and a resveratrol intermediate product is synthesized through sodium borohydride reduction, halogenation, wittig, iron powder reduction and dehydration condensation reaction;

coordination of the soluble ruthenium hydrate salt with the nitrogen-containing compound to form a ruthenium-containing intermediate;

splicing the ruthenium-containing intermediate product and the resveratrol intermediate product to obtain a divalent ruthenium compound 2-1, a divalent ruthenium compound 2-2, a divalent ruthenium compound 2-3 and a divalent ruthenium compound 2-4 which contain biological active groups;

the chemical formula of the compound 2-1 is [ C ₄₃ H ₃₈ N ₆ O ₃ Ru] ²⁺ ；

The structural formula is as follows:

the chemical formula of the compound 2-2 is [ C ₄₇ H ₄₀ N ₆ O ₃ Ru] ²⁺ ；

The structural formula is as follows:

the chemical formula of the compound 2-3 is [ C ₄₇ H ₃₈ N ₆ O ₃ Ru] ²⁺ ；

The structural formula is as follows:

the chemical formula of the compounds 2-4 is [ C ₅₁ H ₄₀ N ₆ O ₃ Ru] ²⁺ ；

The structural formula is as follows:

the synthetic route of the synthetic method is as follows:

2. the method for synthesizing a divalent ruthenium compound having a biologically active group according to claim 1, wherein i is NaBH ₄ MeOH, DCM; ii is PBr ₃ DCM; iii is triphenylphosphine, toluene; iv is NaH, DCM; v is NH ₄ Cl, reduced iron powder, etOH, H ₂ O; vi is N ₂ ，MeOH；vii、N ₂ LiCl, DMF; viii is N ₂ ，MeOH。

3. A divalent ruthenium compound containing a biologically active group, characterized in that the cationic part of the compound is a resveratrol derivative modified Ru (II) compound photosensitizer, comprising compound 2-1, compound 2-2, compound 2-3 or compound 2-4;

the chemical formula of the compound 2-1 is [ C ₄₃ H ₃₈ N ₆ O ₃ Ru] ²⁺ ；

The structural formula is as follows:

the chemical formula of the compound 2-2 is [ C ₄₇ H ₄₀ N ₆ O ₃ Ru] ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the The structural formula is as follows:

the chemical formula of the compound 2-3 is [ C ₄₇ H ₃₈ N ₆ O ₃ Ru] ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the The structural formula is as follows:

the chemical formula of the compounds 2-4 is [ C ₅₁ H ₄₀ N ₆ O ₃ Ru] ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the The structural formula is as follows:

4. the divalent ruthenium compound according to claim 3, wherein the anion portion of the compound is PF ₆ ^- ，Cl ^- 。