CN115873042B - A divalent ruthenium compound containing a biologically active group and its synthesis method and application - 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

A divalent ruthenium compound containing a biologically active group and its synthesis method and application

Technical field

The invention relates to the field of medical technology, specifically to a divalent ruthenium compound containing a biologically active group and its synthesis method and application, specifically to a resveratrol derivative-modified Ru(II) compound photosensitizer and its synthesis. Methods and Applications.

Background technique

As a natural polyphenol anti-cancer substance, resveratrol has attracted widespread attention due to its advantages such as high stability, low price, and easy availability. Research shows that resveratrol can exert anti-tumor effects through various small molecule mechanisms, such as controlling the transformation of malignant tumors, regulating the oxidation and reduction of cancer cells and other metabolic functions, and controlling the formation of new brain blood vessels in cancer cells. It has been widely used in The diagnosis, prevention and treatment of tumors are of great significance in the process of anti-tumor research and development. Wang Chunzhong and others have shown through experimental results that resveratrol has good anti-colon cancer effects and may become a new candidate drug or sensitizer for colon cancer treatment. Li Bihui et al. have found that resveratrol can lag the division and growth phase of SW579 cells in the S phase by controlling the expression of p-AKT protein, thereby significantly controlling the normal growth of thyroid cancer SW579 cells. However, resveratrol has problems such as low activity and poor stability.

Photosensitizer (PS) can undergo photochemical reactions under light conditions. The use of photosensitizers in specific disease areas allows them to concentrate on tumor cells without causing any harm. Metal-based photosensitizing drugs can play a role in three different therapies: photodynamic therapy, photoactivated chemotherapy and photothermal therapy. The photosensitizer is a single compound with good light stability, low dark toxicity, high phototoxicity, fluorescence in the near-infrared region (650-800nm), easy chemical synthesis, and easy absorption by body tissues.

In photodynamic therapy (PDT), after being activated by light of a specific wavelength, the photosensitizer concentrated in the tumor transfers energy to the biological matrix surrounding the photosensitizer to generate reactive oxygen species, such as singlet oxygen ( ¹ O ₂ ), and then Treatments that kill tumor cells. The ways in which reactive oxygen species damage cancer cells are as follows: (1) It is toxic and can cause direct damage; (2) It blocks and destroys blood vessels related to tumor tissue; (3) It directly stimulates its own tumor immune system. Photodynamic therapy is an efficient treatment method because the photosensitizer plays a catalytic effect in the chemical reaction _, and one photosensitizer can form thousands of ^1O2 molecules. Photodynamic therapy is an anti-cancer therapy approved by the FDA following surgery, radiotherapy, chemotherapy and immune function therapy.

Currently, the effectiveness of photodynamic therapy in destroying cancer cells depends on the dose of light. However, during the process of absorbing light, human tissue will have a weakening effect on the light intensity. As a result, the scope of photodynamic therapy's effect on cancer cells is limited. It is limited to the growth parts of cancer cells that can be irradiated by light. It has no significant killing effect on infiltrating cancer cells. , which limits the use of photodynamic therapy in the treatment of deep tissue diseases. Secondly, due to its requirement for oxygen, photodynamic therapy is not effective in hypoxic tumor microenvironments. Finally, traditional photodynamic therapy has a shorter excitation wavelength and cannot penetrate thicker human tissues, thus facing difficulties in treating tumors located in deep tissues of the human body.

Contents of the invention

To this end, the present invention provides a divalent ruthenium compound containing a bioactive group and its synthesis method and application to solve the problem that existing resveratrol has low activity, poor stability, short photodynamic therapy excitation wavelength, and inability to penetrate Problems such as penetrating thick human tissue.

Since resveratrol has shortcomings such as low activity and poor stability, the present invention attempts to develop new resveratrol derivatives with good selectivity, high efficiency and low toxicity by changing its structure. Based on the outstanding advantages of photodynamic therapy in clinical applications of malignant tumors, such as high targeting, low side effects, and low toxicity, the present invention changes the phenolic hydroxyl structure of resveratrol and combines it with Ru(II) compounds as photosensitizers in tumor cells. Initiate singlet oxygen internally to demonstrate tumor inhibition ability, and develop high-performance anti-tumor photosensitizers with high tumor cell inhibition ability and longer excitation wavelength. Through the pharmacophore assembly drug molecule design concept, the structure of resveratrol will be hybridized and the structure of Ru(II) compounds will be introduced, in order to obtain compounds with good pharmacological activity.

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

According to the first aspect of the present invention, a method for synthesizing a divalent ruthenium compound containing a biologically active group is provided, which method includes:

Using 3,5-dimethoxybenzaldehyde as raw material, the resveratrol intermediate product was synthesized through sodium borohydride reduction, halogenation, Wittig, iron powder reduction, and dehydration condensation reactions;

The ruthenium-containing intermediate product is generated by coordinating the hydrated soluble ruthenium salt with the nitrogen-containing compound;

The ruthenium-containing intermediate product and the resveratrol intermediate product are spliced to obtain a divalent ruthenium compound containing a bioactive group.

Halogenation can be chlorine, bromide or iodination. As an example, the present invention uses PBr ₃ for bromination, and the reaction rate is higher;

The soluble hydrated ruthenium salt is a soluble hydrated ruthenium salt. As an example, the present invention uses hydrated ruthenium trichloride;

The nitrogen-containing compound is a nitrogen-containing organic substance or a nitrogen-containing inorganic substance that can be coordinated with the soluble hydrated ruthenium salt, preferably a nitrogen-containing organic substance, and more preferably a dinitrogen-containing cyclic compound. As an example, the present invention uses 2,2-bis Pyridine or 1,10-phenanthroline.

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

Among them, i is NaBH ₄ , MeOH, DCM, 0℃-rt, 2h;

ii is PBr ₃ , DCM, 0℃-rt, 30min;

iii is triphenylphosphonium, toluene, 110℃, 3h;

iv is NaH, DCM, 0℃-r.t., overnight;

v is NH ₄ Cl, reduced iron powder, EtOH, H ₂ O, reflux, 2h;

vi is N ₂ , MeOH, 70°C, reflux, overnight; vii, N ₂ , LiCl, DMF, 155°C, reflux, overnight;

viii is N ₂ , MeOH, 70°C, reflux overnight.

Intermediate product 2-I-7 can be intermediate product 2-I-7a or intermediate product 2-I-7b;

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

The intermediate product 2-I-7b is obtained from the reaction of hydrated ruthenium trichloride, 1,10-phenanthroline and lithium chloride. Further, the intermediate product 2-I-7 and 2-I-6a are used to synthesize 2-1 and 2-2.

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

The structural formula is:

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

The structural formula is:

Further, the intermediate product 2-I-7 and the intermediate product 2-I-6b are used to synthesize compound 2-3 and compound 2-4.

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

The structural formula is:

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

The structural formula is:

Further, the toluene in iii is redistilled before use to remove the moisture in the solvent.

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

The anion part of the compound is a commonly used anion; including but not limited to PF ₆ ^- and Cl ^- .

According to the third aspect of the present invention, the application of a divalent ruthenium compound containing a biologically active group in the preparation of anti-tumor drugs is provided.

The invention has the following advantages:

The synthesis method of the present invention conducts comparative research on the reaction conditions involved in the synthesis route, improves the reaction yield, and the total yield of the entire synthesis route reaches 30 to 35%.

The divalent ruthenium compounds 2-1 and 2-3 containing bioactive groups obtained by the present invention have strong absorption peaks at 450 nm; the divalent ruthenium compounds 2-2 and 2-4 containing bioactive groups have There is a strong absorption peak at 525nm. Compounds 2-2 and 2-4 have stronger conjugated structures, resulting in a red shift of their absorption peaks. Longer wavelength light can effectively penetrate human tissue, which will have a positive impact on photodynamic therapy.

The divalent ruthenium compound containing a bioactive group of the present invention has obvious tumor cell killing ability and strong effect. Its ability to inhibit tumor cells is controlled by light, which is of significant significance in reducing its toxic and side effects.

Description of the drawings

In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the drawings that need to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only exemplary. For those of ordinary skill in the art, other implementation drawings can be obtained based on the extension of the provided drawings without exerting creative efforts.

The structures, proportions, sizes, etc. shown in this specification are only used to coordinate with the contents disclosed in the specification for the understanding and reading of people familiar with this technology. They are not used to limit the conditions under which the invention can be implemented, and therefore do not have any technical Any structural modification, change in proportion or size adjustment shall still fall within the scope of the technical content disclosed in the present invention without affecting the effectiveness and purpose achieved by the present invention. within the scope that can be covered.

Figure 1 is the proton nuclear magnetic resonance spectrum (DMSO-d6) of the target compound 2-1 provided in Example 1 of the present invention;

Figure 2 is a high-resolution mass spectrum of the target compound 2-1 provided in Example 1 of the present invention;

Figure 3 is the proton nuclear magnetic resonance spectrum (DMSO-d6) of the target compound 2-2 provided in Example 1 of the present invention;

Figure 4 is a high-resolution mass spectrum of the target compound 2-2 provided in Example 1 of the present invention;

Figure 5 is a proton nuclear magnetic resonance spectrum (DMSO-d6) of the target compound 2-3 provided in Example 1 of the present invention;

Figure 6 is a high-resolution mass spectrum of target compound 2-3 provided in Example 1 of the present invention;

Figure 7 is a proton nuclear magnetic resonance spectrum (DMSO-d6) of the target compound 2-4 provided in Example 1 of the present invention;

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

Figure 9 is the ultraviolet-visible absorption spectra of compounds 2-1 to 2-4 provided in Experimental Example 1 of the present invention;

Figure 10 shows the singlet oxygen generation ability of compounds 2-1 to 2-4 under green light irradiation in Experimental Example 1 of the present invention;

Among them, A-compound 2-1; B-compound 2-2; C-compound 2-3; D-compound 2-4;

Figure 11 shows the singlet oxygen generation ability of compounds 2-1 to 2-4 under red light irradiation in Experimental Example 1 of the present invention;

Among them, A-compound 2-1; B-compound 2-2; C-compound 2-3; D-compound 2-4;

Figure 12 is Experimental Example 2 of the present invention providing (A-C) dark conditions and (D-F) 610nm irradiation conditions for 30 minutes, living and dead cell staining pictures of HeLa cells treated with compound 2-4;

Among them, scale bar: 80μM;

Figure 13 shows the uptake results of compounds 2-4 in Experimental Example 3 of the present invention in HeLa cells;

Figure 14 is an imaging picture of HeLa cells after treatment with Compound 2-4 and DCFH-DA in Experimental Example 3 of the present invention;

Among them, (A-C) Hela cells were co-deposited with compound 2-4 and DCFH-DA, without adding light; (D-F) Hela cells were co-deposited with compound 2-4 and DCFH-DA and irradiated with 610nm light for 30 minutes;

Scale bar 20μm.

Figure 15 is an agarose gel electrophoresis pattern after co-deposition of pBR322 plasmid DNA and compound 2-4 in Experimental Example 3 of the present invention;

Figure 16 shows the effect of compounds 2-4 in Experimental Example 3 of the present invention on the expression of proteins related to the endogenous apoptosis pathway in Hela cells;

Among them, A-protein immunoblotting band; B-relative grayscale analysis.

Detailed ways

The following specific embodiments are used to illustrate the implementation of the present invention. Persons familiar with this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only part of the embodiments of the present invention. , not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Absolute ethanol, petroleum ether, ethyl acetate, toluene, N,N-dimethylformamide, methanol, dichloromethane, concentrated sulfuric acid, concentrated nitric acid, ethylene glycol, sodium chloride, anhydrous sulfuric acid used in preparation Sodium, sodium borohydride, etc. were purchased from Sinopharm Chemical Co., Ltd., and the purity was of analytical grade. The consumables and reagents used in cell experiments were purchased from Jiangsu Kaiji Biotechnology Co., Ltd. The light sources used in the experiment are 520nm and 610nm LED arrays, and the optical power density is 30.0mW/cm ² .

Example 1

This embodiment provides a method for synthesizing divalent ruthenium compounds containing biologically active groups:

The synthesis route is as follows:

Among them, i is NaBH ₄ , MeOH, DCM, 0℃-rt, 2h; ii is PBr ₃ , DCM, 0℃-rt,

30min; iii is triphenylphosphonium, toluene, 110℃, 3h; iv is NaH, DCM, 0℃-rt, overnight; v is NH ₄ Cl, reduced iron powder, EtOH, H ₂ O, reflux, 2h; vi is N ₂ , MeOH, 70°C, reflux, overnight; vii, N ₂ , LiCl, DMF, 155°C, reflux, overnight; viii is N ₂ , MeOH, 70°C, reflux, overnight.

Synthesis of intermediate 2-I-1: Dissolve 3,5-dimethoxybenzaldehyde (4.11g, 24.74mmol) in anhydrous methanol (60mL), and add an appropriate amount of dichloromethane to help dissolve. A methanol solution of NaBH ₄ (2.808g, 74.22mmol) was added dropwise to the reaction solution at 0°C, and stirred at room temperature for 2 h. The reaction was observed by TLC spot plate. After the compound reaction is complete, ice water is added to quench the reaction, and then the solvent is evaporated using a rotary evaporator. Add 100 mL of water to the solid, extract with dichloromethane (30 mL × 3), and collect the organic phase. Then, 100 mL of saturated aqueous sodium chloride solution was added to the organic phase, and the mixture was extracted with dichloromethane (30 mL). The organic phase was collected and dried over _{anhydrous Na2SO4} . After filtration, the filtrate was collected and the solvent was evaporated to obtain 3.44g of white crystals with a yield of 82.6%. (Yield eta = 82.6%, actual output/theoretical output, the same below)

Synthesis of intermediate 2-I-2: Dissolve intermediate 2-I-1 (3.26g, 19.38mmol) in anhydrous dichloromethane (50mL), and dissolve PBr ₃ (5.52mL, 58.12mmol) in anhydrous dichloromethane (50mL). into water dichloromethane (50 mL), and add PBr3 solution dropwise to the above reaction solution at 0°C. Then stir at room temperature for 30 min. The reaction was observed by TLC spot plate. After the compound reaction is complete, ice water is added to quench the reaction. Add 100 mL of water, extract with dichloromethane (30 mL × 3), and collect the organic phase. Then, 100 mL of saturated aqueous sodium chloride solution was added to the organic phase, and the mixture was extracted with dichloromethane (30 mL). The organic phase was collected, dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was evaporated and separated using a silica gel chromatography column (eluent: petroleum ether/dichloromethane=2:1, v/v). 3.83g of white crystals were obtained, with a yield of 79.84%.

Synthesis of intermediate 2-I-3: Intermediate 2-I-2 (3.19g, 13.80mmol) and triphenylphosphorus (4.70g, 17.94mmol) were added in sequence to a 250mL round-bottomed flask containing 100mL toluene. Reflux at 110°C for 3 hours. Observe through thin layer chromatography spot plate. After the compound reaction is complete, the reaction solution is cooled to room temperature. The solid was filtered off and washed with toluene (30 mL). After drying in a vacuum drying oven, 6.72g of white solid was obtained with a yield of 98.70%.

Synthesis of intermediate 2-I-4: Dissolve intermediate 2-I-3 (4.20g, 8.51mmol) in anhydrous dichloromethane (50mL), add NaH (1.02g, 42.50mmol) at 0°C ) and stir at the same temperature for 30 minutes. 4-Methoxy-3-nitrobenzaldehyde (1.54g, 8.50mmol) was dissolved in anhydrous dichloromethane (20mL) and added dropwise to the above reaction solution, and the reaction was carried out at room temperature overnight. After the completion of the reaction was detected by thin layer chromatography spot plate, ice water was added to quench the reaction. Add 100 mL of water to the reaction solution, extract with dichloromethane (30 mL × 3), and collect the organic phase. Then, 100 mL of saturated aqueous sodium chloride solution was added to the organic phase, and the mixture was extracted with dichloromethane (30 mL). The organic phases were combined, dried over anhydrous Na ₂ SO ₄ and filtered. After filtration, the filtrate was collected and the solvent was evaporated, and separated using a silica gel chromatography column (eluent: petroleum ether/ethyl acetate = 20:1, v/v). 1.56g of yellow oil was obtained, with a yield of 58.02%.

Synthesis of intermediate 2-I-5: Intermediate 2-I-4 (3.77g, 11.96mmol) was dissolved in absolute ethanol (50mL), and NH ₄ Cl (2.56g, 47.85mmol) was dissolved in water ( 5mL) and added to the above reaction solution, and then added reduced iron powder (3.35g, 59.82mmol). The mixture was refluxed at 80°C for 2 h. After the reaction was completed by observing the TLC spot plate, the mixture was filtered through diatomaceous earth to remove the reduced iron powder. 100 mL of water was added to the reaction solution, extracted with ethyl acetate (30 mL × 3), and the organic phase was collected. Then, 100 mL of saturated aqueous sodium chloride solution was added to the organic phase, and the mixture was extracted with ethyl acetate (30 mL). The organic phase was collected, dried over anhydrous Na ₂ SO ₄ and filtered. After filtration, the filtrate was collected and the solvent was evaporated to obtain 2.90 g of brown oil, with a yield of 85.10%.

Synthesis of intermediates 2-I-6a and 2-I-6b: combine intermediate 2-I-5 (0.40g, 1.40mmol) and pyridine-2-carboxaldehyde (0.22g, 1.40mmol) or quinoline-2- Formaldehyde (0.22g, 1.40mmol) was dissolved in anhydrous methanol (15mL), and the mixture was refluxed at 70°C overnight. The solvent was removed after the completion of the reaction was detected by TLC. Separate using a silica gel chromatography column (eluent: petroleum ether/ethyl acetate = 50:1, v/v).

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

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

Synthesis of intermediates 2-I-7a, b: combine hydrated ruthenium trichloride (1.14g, 5.00mmol) and 2,2-bipyridine (1.56g, 10.00mmol) and lithium chloride (1.41g, 33.00mmol) Add it to the above solution in sequence, and increase the amount of DMF appropriately. The mixture was refluxed at 150°C overnight under nitrogen protection. After the reaction is complete, use a rotary evaporator to evaporate the solvent, add 150 mL acetone, and let stand at -20°C for more than 24 hours. The reaction solution was filtered, and the obtained filter cake was washed with water. After drying, intermediate product 2-I-7a was obtained. 0.28g of yellow-purple black solid was obtained, with a yield of 11.67%.

Add hydrated ruthenium trichloride (1.14g, 5.00mmol), 1,10-phenanthroline (1.80g, 10.00mmol) and lithium chloride (1.41g, 33.00mmol) to the above solution in sequence, and follow the above reaction in sequence. conduct. After drying, intermediate product 2-I-7b was obtained. 1.30g of yellow-purple black solid was obtained, with a yield of 52.00%.

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

(1) Synthesis of target products 2-1 and 2-2

2-I-6a (0.26g, 0.70mmol) and intermediate 2-I-7a (0.34g, 0.70mmol) or 2-I-7b (0.34g, 0.70mmol) were added to anhydrous methanol (30mL) respectively. , reflux overnight at 70°C, observe the reaction through TLC spot plate, after the reaction is complete, spin off the solvent on a rotary evaporator, and separate using a neutral alumina chromatography column (eluent: dichloromethane/methanol=100:1 ,v/v). The target products 2-1 and 2-2 were obtained respectively.

For the synthesis of target product 2-1: 0.35g of brown-red solid was obtained with a yield of 46.67%.

As shown in Figure 1 and Figure 2, ¹ H NMR (600MHz, 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＝8.1Hz,1H),8.45(d,J＝7.7Hz,1H),8.31(d,J＝8.1Hz,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＝8.6,1.5Hz,1H),6.59(dd,J＝19.9,5.1Hz,1H),6.48 –6.39(m,3H),6.30(d,J＝1.9Hz,2H),6.06(d,J＝12.3Hz,1H),3.77–3.57(m,9H). Calculated value: [C ₄₃ H ₃₈ N ₆ O ₃ Ru] ²⁺ ([M] ²⁺ ), 394.1019; found 394.1090.

For the synthesis of target product 2-2: 0.39g of brown-red solid was obtained with a yield of 49.36%.

As shown in Figure 3 and Figure 4, 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 value: [C ₄₇ H ₄₀ N ₆ O ₃ Ru] ²⁺ ([M] ²⁺ ) , 419.1097; measured 419.1017.

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

2-I-6b (0.30g, 0.70mmol) and intermediate 2-I-7a (0.34g, 0.70mmol) or 2-I-7b (0.34g, 0.70mmol) were added to anhydrous methanol (30mL) respectively. , as in the above reaction, reflux overnight at 70°C, detect the completion of the reaction by TLC and then spin off the solvent. Separate using a neutral alumina chromatography column (eluent: dichloromethane/methanol=100:1, v/v). Target products 2-3 and 2-4 were obtained respectively.

For the synthesis of target product 2-3: 0.41g of brown solid was obtained, with a yield of 44.09%.

As shown in Figure 5 and Figure 6, ¹ H NMR (600MHz, DMSO) δ9.28 (s, 1H), 9.16 (t, J = 6.7Hz, 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＝7.6Hz,1H),8.44(d,J＝8.9Hz,1H),8.40 (d,J＝9.2Hz,2H),8.29(d,J＝4.4Hz,1H),8.24(d,J＝8.9Hz,1H),8.21–8.08(m,4H),8.05(t,J＝ 7.0Hz,1H),8.00(ddd,J＝15.9,8.5,4.4Hz,1H),7.75(ddd,J＝13.5,11.7,5.4Hz,3H),7.53–7.45(m,2H),6.64(dd ,J＝8.6,1.9Hz,1H),6.41–6.21(m,4H),6.15(t,J＝8.3Hz,2H),3.87–3.49(m,9H). Calculated value: [C ₄₇ H ₃₈ N ₆ O ₃ Ru] ²⁺ ([M] ²⁺ ), 418.1019; measured 418.1010.

For the synthesis of target product 2-4: 0.32g of purple-red solid was obtained, with a yield of 32.99%.

As shown in Figure 7 and Figure 8, ¹ H NMR (600MHz, DMSO) δ9.30 (s, 1H), 9.15 (d, J = 4.8Hz, 1H), 8.94 (d, J = 8.2Hz, 1H), 8.88(d,J＝8.3Hz,1H),8.77(d,J＝8.4Hz,1H),8.50(d,J＝7.7Hz,1H),8.43(dd,J＝25.8,8.8Hz,4H), 8.29(d,J＝4.7Hz,1H),8.25(d,J＝8.8Hz,1H),8.19–8.08(m,4H),8.06(d,J＝8.9Hz,1H),8.01(dd,J ＝8.0,5.1Hz,1H),7.96(s,1H),7.85–7.71(m,4H),7.49(dd,J＝12.7,5.3Hz,2H),6.63(d,J＝8.4Hz,1H) ,6.38(s,1H),6.33(d,J＝8.3Hz,1H),6.28(d,J＝12.2Hz,1H),6.16(s,2H),3.78–3.55(m,9H). Calculated value : [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, the sodium borohydride should be dissolved in methanol and then added dropwise to the reaction solution through a constant pressure bottom funnel to prevent the generation of a large number of bubbles. The purpose of the two washings during post-treatment is to remove residual impurities in the reaction solvent.

2. The NMR spectra of intermediates 2-I-2 to 2-I-6 show that the obtained products are consistent with the design. Among them, before the synthesis reaction of intermediate 2-I-3, toluene needs to be redistilled to remove the moisture in the solvent.

3. The synthesis of intermediate 2-I-4 is a Wittig reaction. The key to the reaction is that the entire reaction should be carried out under anhydrous and oxygen-free conditions, especially the control of anhydrous conditions to ensure the normal formation of phosphorus ylide. The phosphorus ylide generated by the action of NaH reacts directly with the aldehyde group to generate an alkene (cis-trans mixture). The trans structure in the product needs to be removed by column chromatography.

4. The synthesis of intermediate 2-I-5 is an iron powder reduction reaction. The reaction liquid obtained after filtering through diatomaceous earth can be washed with ethanol to remove NH ₄ Cl in the reaction liquid.

5. Considering that the stability of intermediate 2-I-6 itself is poor, intermediate 2-I-5 and pyridine-2-carbaldehyde or quinoline-2-carbaldehyde can be dissolved in anhydrous methanol and refluxed overnight, and then directly Carry out the next step of the reaction to prevent the product from deteriorating.

6. The synthesis of intermediates 2-I-7a and 2-I-7b was based on previous reports and improved based on the actual situation of this laboratory. In order to prevent the zero-valent catalyst from being oxidized during the reaction, the present invention uses an oil pump to remove oxygen from the reaction liquid, bubbles _N2 into the reaction liquid, and applies nitrogen protection to the reaction system.

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

The reaction conditions involved in the synthesis route were comparatively studied and the reaction yield was improved. The total yield of the entire synthesis route reached 30-35%. In this experiment, the structures of four final products from 2-1 to 2-4 and multiple key intermediates were characterized by hydrogen nuclear magnetic resonance spectroscopy ( ¹ H NMR) analysis. And the four final products 2-1 to 2-4 were characterized by mass spectrometry (MS) analysis method.

Experimental example 1

This experiment provides the photophysical and chemical properties of Ru(II) compound photosensitizers 2-1~2-4 modified with resveratrol derivatives:

1. Preparation of solution: Prepare compounds 2-1 to 2-4 using chromatographically pure acetonitrile as the solvent to prepare a solution with a concentration of 1.00mM, and then dilute it with chromatographically pure acetonitrile to a dilute solution with a concentration of 10.0 μM.

Measurement of the spectrum: For UV-visible absorption spectrum: Take 3.00 mL of the above 10.0 μM solution of compounds 2-1 to 2-4 in a quartz cuvette, and collect the absorbance at 180-800 nm on a UV-visible spectrophotometer. And normalized to the absorbance at 180nm.

The results are shown in Figure 9. It can be seen from Figure 9 that compounds 2-1 and 2-3 have strong absorption peaks at 450nm; compounds 2-2 and 2-4 have strong absorption peaks at 525nm. absorption peak. Compounds 2-2 and 2-4 have stronger conjugated structures, resulting in a red shift of their absorption peaks. Because longer wavelength light can effectively penetrate human tissue, this will have a positive impact on photodynamic therapy.

2. Preparation of DPBF: Under dark conditions, use methanol to prepare a concentrated solution of DPBF with a concentration of 6.00mM. Then use methanol to dilute to a concentration of 60.0 μM. All DPBF solutions are ready-to-use.

Preparation of compound solutions: Under light-proof conditions, prepare four Ru(II) compounds from 2-1 to 2-4 with dimethyl sulfoxide (DMSO) to prepare concentrated solutions with a concentration of 1.00mM. Then dilute with methanol to a concentration of 10.0 μM.

Detection of singlet oxygen: Under light-proof conditions, take 1.50mL of each of the above two solutions into a quartz cuvette and mix evenly. Irradiate with 520nm green light, and measure the UV-visible absorption spectrum every 1 minute. Then compounds 2-1 to 2-4 were irradiated under 610 nm red light respectively, and their photodynamic activity was detected according to the above test procedures.

The results of singlet oxygen generation ability under green light irradiation are shown in Figure 10. It can be seen that under green light irradiation, the peak value of 2-3 and 2-4 compounds decreases faster and the photodynamic activity is stronger. Under the condition of 520nm illumination, after the total illumination time reaches 18 minutes, the characteristic absorption peak of DPBF almost completely disappears.

The singlet oxygen generation ability under red light irradiation is shown in Figure 11. It was found that under red light irradiation, the photodynamic force of quinoline compounds was stronger than that of pyridine compounds. The two quinoline products 2-2 and 2-4 were sensitive to red light. Better absorption.

Experimental example 2

In order to evaluate the in vitro anti-tumor activity of the four target compounds 2-1 to 2-4 synthesized in Example 1, this experimental example obtained the IC ₅₀ values of a series of compounds under different conditions through the CCK-8 experiment.

1. CCK-8 experiment

1. Cell culture and drug administration

Tumor cells were placed in complete culture medium containing 10% fetal calf serum, cultured in a sterile, 95% relative humidity, 5% _CO2 incubator, and digested with 0.25% trypsin and 0.02% EDTA. pass on. The cell lines growing in the logarithmic phase were frozen and stored to prepare for subsequent experiments.

The cells were seeded in 96-well plates, placed in a constant temperature incubator overnight, and cultured to 80%. Discard the original culture medium. Add 100 μL of culture medium containing 0, 0.1, 0.5, 1, 10, 50, and 100 μM compounds to the cells. Three replicate wells were set up for each concentration. Cells were incubated in a 37°C incubator (containing 5% CO ₂ ) for 72 h. For illumination experiments, use a 520nm or 610nm LED array to illuminate the 96-well plate for 30 minutes, and then continue to culture for 72 hours.

2. Determination of IC ₅₀ value

After 72 h of culture, 10.0 μL of CCK-8 working solution was added to each well and shaken evenly, and the resulting cells were continuously incubated in a 37°C incubator for 2 h. Use a microplate reader to test the absorbance at 450nm and read the OD value. The obtained data were used to calculate IC ₅₀ values using SPSS Statistics software.

3.Results

The results are shown in Table 1. Under dark conditions, similar to the positive control compounds, all target compounds have low tumor cell inhibitory abilities, indicating that the target compounds have certain safety under dark conditions. Under the irradiation of 520nm green light, the four compounds 2-1 to 2-4 all showed strong anti-tumor activity. Taken together, the order of activity is 2-4>2-2>2-3>2-1. Under the irradiation of 610nm red light, the activity of all compounds showed a significant decrease compared with that of green light. The reason is that the main light absorption band of a series of target compounds synthesized by the present invention is located in the green light region, and the absorption of red light is weak, so it exhibits low activity. Similarly, since the main absorption band of the positive control is located near 450 nm, the compound exhibits relatively low photodynamic activity regardless of whether it is illuminated by red light or green light. Under red light irradiation, compounds 2-4 still showed strong anti-tumor activity, which shows that chemical modifications aimed at expanding the degree of π electron delocalization play a role, which will be beneficial to the development of photodynamic therapy for clinical application. Considering that Hela is relatively sensitive to compounds 2-4, this cell will be selected as an in vitro model in subsequent biological experiments.

Table 1 In vitro inhibitory activity of compounds 2-1 to 2-4 on tumor cells under different conditions (IC ₅₀ value, μM)

2. Live and dead cell imaging experiments

1. Cell culture: Resuscitate frozen cells, passage and inoculate them into 6-well plates, and culture them in a sterile, 37°C, 5% _CO2 incubator for 24 hours.

2. Administration and illumination: After removing the culture medium, wash 3 times with sterilized PBS (0.01mM, pH=7.4) to remove cell metabolites and impurities. To each cell culture dish, add 1998 μL of fresh medium and 2.00 μL of a 50.0 μM solution of compound 2-4 in PBS (0.01 mM, pH = 7.4) (containing 10% dimethyl sulfoxide) to the final concentration. is 5μM. Cells were incubated under the above conditions for 24 h. After the cells were incubated for 24 h, the culture dish was removed. Use a 610nm LED to illuminate the culture dish for 30 minutes. After the illumination was completed, the cells were incubated for 1 h.

3. Calcein and propidium iodide staining: After the cells are incubated, replace the culture medium and wash the cells with PBS (0.01mM, pH=7.4). The cells were then incubated with calcein AM and propidium iodide, each at a concentration of 5.00 μM, for 30 min. Then use PBS (0.01mM, pH=7.4) buffer to wash the cells;

4. Confocal photography: Place the culture dish on the laser scanning confocal microscope. For the green channel, the excitation light wavelength is set to 488nm, and the detection wavelength is set to (500-540) nm; for the red channel, the 561nm laser is used as the excitation light, and the detection wavelength is (600-640) nm.

5. Live and dead cell imaging experiment results:

As can be seen from Figure 12, the dosage concentration is 5 μM and the incubation time is 24 h. When no light is applied, the vast majority of cells emit green fluorescence, indicating that most cells are alive at this time. On the contrary, after 30 minutes of 610nm light irradiation, most cells appeared dead. This experiment clearly demonstrates that compounds 2-4 have obvious tumor cell killing ability and strong effects. In addition, its tumor cell inhibition ability is controlled by light, which is of significant significance in reducing its toxic and side effects.

Experimental example 3

Since Compound 2-4 has strong inhibitory activity against cancer cells in vitro, this cell line was selected for relevant biological research in this example, in order to reveal the intrinsic mechanism of Compound 2-4 inhibiting tumor cells.

(1) Cellular uptake of Ru(II) compounds

Cellular uptake experiments of metal drugs were performed using ICP-MS technology. The experimental cells were seeded in a 6-well culture plate. After the cell density in the culture plate reached 90%, the medium was removed and replaced with new medium. Compound 2-4 at a concentration of 5 μM and a positive control were added respectively [Ru(bpy ) ₃ ] ²⁺ . After culturing and incubating the cells in a constant temperature incubator for 12 hours, the cells were collected by centrifugation, digested with 200 μL of 65% nitric acid at 65°C for 10 hours, and the total Ru content in the cells was measured using the inductively coupled plasma mass spectrometry (ICP-MS) experimental method.

The results are shown in Figure 13. [Ru(bpy) ₃ ] ²⁺ was selected as the photosensitizer. The dosing concentration was 5 μM and the incubation time was 24 hours; it can be found that the three types of tumor cells have weak uptake ability of the positive control compound [Ru(bpy) ₃ ] ²⁺ . In stark contrast, the ability of these three tumor cells to uptake compounds 2-4 was significantly higher than that of the positive control compounds. The author speculates that this difference is caused by the fact that compound 2-4 has a highly lipophilic organic ligand, and as a water-soluble compound, the cell accumulation ability of [Ru(bpy) ₃ ] ²⁺ is obviously weaker than that of the target compound . This phenomenon will undoubtedly play a positive role in improving its anti-tumor activity.

(2) ROS imaging experiment

1. Cell culture: Seed cells in a glass-bottomed confocal culture dish and incubate overnight in a constant-temperature incubator.

2. Application of drugs and illumination: Remove the culture medium in the culture dish and wash 3 times with sterile PBS buffer (0.01mM, pH=7.4) to remove cell metabolites and impurities. Add 999 μL of fresh culture medium and 1.00 μL of a 5.0 mM compound 2-4 in PBS buffer (0.01 mM, pH = 7.4) solution (containing 10% dimethyl sulfoxide) to each cell culture dish and allow it to The final concentration is 50 nM. Cells were incubated under the above conditions for 24 h. After the cells were incubated for 4 hours, the culture dish was removed. Use a 610nm LED to illuminate the culture dish for 2 minutes.

3. DCFH-DA staining: Discard the culture medium, add 500 μL of DCFH-DA working solution with a concentration of 5.00 μM, and incubate for 30 minutes. Finally, the working solution was aspirated and washed 3 times with PBS buffer (0.01mM, pH=7.4).

4. Confocal photography: Place the culture dish on a laser scanning confocal microscope, use 448nm laser as the excitation light, and set the detection wavelength to (500-600) nm.

As shown in Figure 14, the dosage concentration was 5 μM and the incubation time was 24 h. After applying compound 2-4 under dark conditions, no green fluorescence was observed in Hela cells, which indicates that there is no excess ROS in Hela cells. The extent also indicates that the compound is safe under dark conditions. After 30 minutes of red light irradiation, a confocal microscope detected obvious green fluorescence in the cells. This shows that the compound can effectively increase ROS levels in cells, and the behavior of inducing ROS can be controlled by light.

(3) Agarose gel electrophoresis experiment

The extraction of intracellular ROS levels may cause DNA damage. DNA agarose gel electrophoresis technology was used to explore the DNA damage effect of compound 2-4 before and after illumination.

Mix pBR322 plasmid DNA with a concentration of 0.100 μg/μL and 5.00 μL of the corresponding compound evenly. The dosage concentrations are 1:0 μM; 2:1 μM; 3:2 μM; 4:5 μM; 5:10 μM; 6:20 μM; 7: Concentration gradient of 50μM; 8:80μM; 9:100μM; 10:200μM. For the illumination group, each sample was illuminated with 610nm LED for 30 minutes. All samples were then placed in a 37°C incubator and incubated in the dark for 24 hours. Subsequently, the gel was prepared, samples were loaded, and 2.00 μL loading buffer was added to each sample to a final concentration of 10% (v/v). The obtained sample was separated using agarose gel at a voltage of 100V for 1 h, and the position of the indicator was observed. The obtained gel sample was placed in an EB solution with a concentration of 0.750 μg/mL and incubated for 30 min. Finally, deionized water was used to wash away the EB solution on the gel surface, and the resulting gel was photographed using a gel digital imaging system.

As shown in Figure 15, under dark conditions, as the compound concentration increases, a large amount of open circular DNA (Form II) does not appear in the DNA electrophoresis pattern, and most of it is supercoiled DNA (Form I). This shows that compounds 2-4 do not cause DNA damage under dark conditions. Correspondingly, after irradiation with 610nm light for 30 minutes, as the drug concentration increased, the proportion of open-circular DNA in the pattern gradually became higher, and eventually all DNA was cleaved into open-circular form. This shows that compound 2-4 has a strong ability to damage DNA and is controlled by light.

(4) Western blot experiment

The effects of compounds 2-4 on the expression of proteins related to the endogenous apoptosis pathway in HeLa cells were studied through westernblots experiments.

Count the cells in the logarithmic growth phase, seed the cells in a 6-well plate, and culture until the cells grow to about 85%. Digest the cells in the wells, collect them into a centrifuge tube by centrifugation, and wash with PBS. Add an appropriate amount of lysis solution to the cells, and lyse the cells in an ice box for 2 hours. Centrifuge at 10,000 rpm for 20 to 30 minutes, and take the supernatant for later use. Take 0.5 mL of the supernatant and place it in an EP tube and store it at -20°C. Be careful to operate on ice. Protein content was determined using the BCA protein kit (Thermo, Waltham, MA). After gel preparation, the proteins were loaded onto an 8-12% polyacrylamide gel at equal concentrations and separated by electrophoresis. The protein was then transferred to Immobilon-P membrane on a semi-dry transfer membrane apparatus. Soak in Ponceau red staining solution and cut the protein on the membrane according to the instructions of the Marker. Immerse it in a PBST skimmed milk dish and shake on a destaining shaker for 1 hour. This was followed by incubation with primary antibodies for β-actin, caspase3, Bax and Bcl-2 overnight at 4°C. After the above membrane was washed three times with PBST, it was incubated with the secondary antibody cross-linked with IRDye800 for 1 hour at 37°C. Prepare AB luminescent liquid (A:B=1:1), drop it on the strip, and let it act for about 10 seconds. After removing the excess luminescent liquid, cover it with a film, put the film on the film, press it for 70 seconds, and place it in the exposure machine for exposure. Develop the film in the dark and observe the bands. The obtained bands were grayscale analyzed using image J software, and statistical analysis was performed using Excel software.

In Figure 16, compared with the blank control group, the expression of caspase3 protein was slightly increased under dark conditions, the pro-apoptotic protein Bax was not significantly affected, and the expression of the anti-apoptotic protein Bcl-2 was slightly inhibited. Under light conditions, the perturbation of the above protein expression was significantly enhanced. Caspase3 and Bax were significantly elevated, while the anti-apoptotic protein Bcl-2 was significantly inhibited. This shows that under light conditions, the endogenous apoptosis pathway in Hela cells is turned on.

Compounds 2-2 and 2-4 of the present invention have stronger conjugated structures, resulting in a red shift of their absorption peaks, which to a certain extent improves the damage range and depth of photodynamic therapy for deeply infiltrating tissue diseases. This Will have a positive impact on photodynamic therapy. Under green light irradiation, the peak value of the DPBF experiment of compounds 2-3 and 2-4 decreased faster, and the photodynamic activity was stronger. Compounds 2-2 and 2-4 can effectively induce singlet oxygen under the irradiation of 610nm visible light, and show strong inhibitory ability against the detected tumor cells.

The present invention evaluates the anti-tumor activity of four Ru(II) complexes from 2-1 to 2-4 through the CCK-8 method. The experimental results confirmed that the target compound has a certain degree of safety under dark conditions; under the irradiation of 520nm green light, compound 2-4 showed strong anti-tumor activity, and the order of activity strength was 2-4>2-2 >2-3>2-1; and under the irradiation of 610nm red light, the activity of all compounds showed a significant decrease compared with green light. The reason is that the main light absorption band of a series of target compounds synthesized in this study is located in the green light region, and the absorption of red light is weak, so it shows low activity. Under red light irradiation, compounds 2-4 still showed strong anti-tumor activity, which shows that chemical modifications aimed at expanding the degree of π electron delocalization play a role. This study further verified through live-dead cell imaging experiments that compound 2-4 has obvious tumor cell killing ability, and its tumor cell inhibition ability is controlled by light, which creates basic conditions for reducing its toxic and side effects.

Through metal drug cell uptake experiments, the present invention proves that the uptake ability of compound 2-4 by tumor cells is significantly greater than that of the positive control compound [Ru(bpy) ₃ ] ²⁺ , which will play a positive role in improving its anti-tumor activity. Compound 2-4 has a highly lipophilic organic ligand. As a water-soluble complex, the cell accumulation ability of [Ru(bpy) ₃ ] ²⁺ is significantly lower than that of the target compound. In ROS imaging experiments, compounds 2-4 can effectively increase ROS levels in cells, and the behavior of inducing ROS can be controlled by light. The present invention uses DNA agarose gel electrophoresis technology to confirm that compound 2-4 does not cause DNA damage under dark conditions, but under 610 nm light, compound 2-4 has a strong ability to damage DNA and is controlled by light. Finally, in the westernblots experiment, this project found that under light conditions, the expression of proteins related to the endogenous apoptosis pathway of cells was significantly perturbed. Caspase3 and Bax were significantly elevated, while the anti-apoptotic protein Bcl-2 was significantly inhibited. This shows that under light conditions, the endogenous apoptosis pathway in Hela cells is turned on.

It can be seen that the divalent ruthenium compound containing a bioactive group prepared by the present invention has low toxicity, high inhibitory effect on tumor cells, and has good anti-tumor application prospects.

Although the present invention has been described in detail with general descriptions and specific examples 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, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

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 ^- 。