Bipyridine ruthenium complex with photodynamic therapy effect and preparation method and application thereof

Abstract

The present invention belongs to the field of medicine and discloses a bipyridine ruthenium complex with photodynamic therapy effect, its preparation method and application. The present invention discloses a bipyridine ruthenium complex with photodynamic therapy (PDT) effect, its preparation method and application, and the complex has a structure shown in the general formula I: wherein R is selected from or Y ‑ Represents halide anions or hexafluorophosphate anions; the bipyridine ruthenium complex of the present invention not only has mitochondrial-targeted fluorescence imaging, but also produces high concentrations of reactive oxygen species after illumination, thereby killing tumor cells and having excellent photodynamic therapy effects to exert anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to a bipyridine ruthenium complex with photodynamic therapy effect and its preparation method and application. Specifically, it relates to a class of bipyridine ruthenium complexes with photodynamic therapy effect and pharmaceutically acceptable salts thereof, their preparation method and their medical uses, especially their application in the preparation of drugs for photodynamic therapy of malignant tumors. Background Art

[0002] Currently, several methods have been used to treat cancer, including surgical resection, chemotherapy, and radiotherapy. Compared with these traditional methods, photodynamic therapy, as a new non-invasive cancer treatment method, has shown advantages of non-invasiveness and precise treatment, attracting widespread attention.

[0003] Photosensitizers play a crucial role in photodynamic therapy, being activated by specific wavelengths of light to produce cytotoxic reactive oxygen species. Photosensitizers typically have low dark toxicity but can induce high cytotoxicity upon irradiation. The effective treatment area is closely related to the size of the irradiated area, allowing photodynamic therapy to precisely target tumor tissue while avoiding damage to healthy organs and tissues.

[0004] Metal complexes are considered promising new photosensitizers, exhibiting excellent photophysical properties. Ruthenium complexes have garnered significant interest in photodynamic therapy. Notably, the ruthenium complex TLD-1433 has completed a Phase II clinical trial as a photosensitizer for bladder cancer, significantly increasing interest in the development of new ruthenium complexes for photodynamic therapy. Summary of the Invention

[0005] The purpose of the present invention is to provide a bipyridine ruthenium complex with photodynamic therapy effect and its preparation method and application. The bipyridine ruthenium complex has excellent photophysical properties, can realize fluorescence imaging of tumor cells, target the mitochondria of tumor cells, and simultaneously exert photodynamic therapy effect on tumor cells.

[0006] The technical solution of the present invention is:

[0007] A bipyridine ruthenium complex having photodynamic therapy effect has the following structure of general formula I:

[0008]

[0009] Among them, R represents One of the following; Y - Represents a halide anion or a hexafluorophosphate anion.

[0010] Preferably, R represents One of the following; Y - represents halide anion or hexafluorophosphate anion.

[0011] Preferably, in the structure of Formula I, R and Y are selected from the following combinations:

[0012] Y=I;

[0013] or Y = PF6;

[0014] or Y = PF6;

[0015] or Y = PF6;

[0016] or Y = PF6;

[0017] or Y = PF6;

[0018] or Y = PF6;

[0019] The present invention also provides a method for preparing a bipyridine ruthenium complex having a photodynamic therapy effect, comprising the following steps:

[0020] S1. First, the methyl group at the 4'-position of compound 1 is oxidized to an aldehyde group using selenium dioxide to obtain compound 2. Compound 2 is then directly reduced with sodium borohydride to obtain compound 3. Compound 3 then undergoes a substitution reaction with phosphorus tribromide to obtain compound 4. Compound 4 reacts with various nitrogen heterocycles to obtain compound 5.

[0021] S2. Compound 6 undergoes coordination reaction with hydrated ruthenium trichloride to obtain compound 7;

[0022] S3. Compound 5 and compound 7 undergo coordination reaction in a mixed solution of water and methanol to form bipyridine ruthenium complex I;

[0023] The synthetic route of the preparation method is as follows:

[0024]

[0025] Among them, R represents One of the following; Y - Represents a halide anion or a hexafluorophosphate anion.

[0026] The present invention also provides the use of the above-mentioned bipyridine ruthenium complex in the preparation of medicines for treating malignant tumors.

[0027] Preferably, the drug is a drug for photodynamic therapy of malignant tumors.

[0028] Preferably, the malignant tumor is one of breast cancer, colon cancer, cervical cancer and lung cancer.

[0029] The present invention also provides the use of the above-mentioned bipyridine ruthenium complex in the preparation of a fluorescent imaging agent with mitochondrial targeting.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] Bipyridine is a bidentate ligand with a planar aromatic ring. It has good planarity and high designability. Through structural modification, it can optimize molecular optical and physical properties. This invention introduces nitrogen-containing saturated heterocycles with different hydrophilicities into the methyl group at the 4' position of 4,4'-dimethyl-2,2'-bipyridine to design and synthesize a series of bipyridine ruthenium complexes. These complexes not only have excellent photophysical properties, including near-infrared fluorescence and two-photon excitation characteristics, high fluorescence stability, and a large Stokes shift, which can reduce tissue background fluorescence interference, but also singlet oxygen ( 1 O2) quantum yield is high, the lipid-water partition coefficient of the complex is improved, and fluorescence imaging of tumor cells is achieved, tumor cell mitochondria are targeted and located, while a significant photodynamic therapy effect is exerted on multiple tumor cells. Under the action of photodynamic therapy, the mitochondria of tumor cells are damaged, and the photodynamic therapy can be used to prepare fluorescence imaging reagents with mitochondrial targeting or drugs for photodynamic therapy of malignant tumors, and is applied to fluorescence diagnosis and treatment research of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the ultraviolet absorption spectrum of the bipyridine ruthenium complex of the present invention;

[0033] Figure 2 is the fluorescence emission spectrum of the bipyridine ruthenium complex of the present invention;

[0034] Figure 3 The present invention is a bipyridine ruthenium complex I3 co-localized with a mitochondrial probe in A549 cells.

[0035] Figure 4 This is the co-localization imaging of the bipyridine ruthenium complex I6 of the present invention and the mitochondrial probe in Hela cells. DETAILED DESCRIPTION

[0036] In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative and are only used to specifically describe the present invention, and should not be understood as limiting the present invention.

[0037] Example 1 Preparation of 4-methyl-2,2'-bipyridine-4'-((4-methylpiperidine)-N-methylene)-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I1)

[0038] Preparation of 4-methyl-2,2'-bipyridine-4'-carboxaldehyde (Compound 2)

[0039] Compound 1 (10 mmol, 1.84 g) and SeO2 (15 mmol, 0.56 g) were dissolved in 1,4-dioxane (25 mL) under nitrogen atmosphere and heated under reflux at 110°C for 24 h. Completion of the reaction was monitored by thin-layer chromatography. After cooling to room temperature, the solvent was removed by vacuum evaporation. The crude product was purified by column chromatography (ammonia MeOH:DCM = 1:25) to afford compound 2 (1.247 g) as a white solid in a 63% yield. 1 H NMR (400MHz, CDCl3) δ10.18(s,1H,CH=O),8.40(d,J=5.1Hz,2H,2ArH),7.85(dd,J=5.0,1.6Hz,2H,2ArH),7.72(dd,J=4.9,1.6Hz,2H,2ArH),2.04(s,3H,CH3). 13 C NMR (101MHz, CDCl3) δ192.51,160.74,150.32,149.79,149.21,143.30,142.63,125.42,122.11,121.39,120.62,21.22.

[0040] Preparation of 4-methyl-2,2'-bipyridine-4'-methanol (Compound 3)

[0041] Compound 2 (8 mmol, 1.585 g) was dissolved in CH₃CH₂OH (20 mL) and stirred for 5 min. NaBH₄ (16 mmol, 0.605 g) was added and the mixture was stirred for 1 h. Completion of the reaction was monitored by thin-layer chromatography. Upon completion, 1-2 drops of water were added to quench the reaction. The solvent was then evaporated under reduced pressure, and saturated brine (30 mL) was added. The mixture was extracted with DCM (20 mL x 3). The organic layer was dried over anhydrous Na₂SO₄, and the solvent was evaporated under reduced pressure to afford compound 3 (1.361 g) as a light yellow solid in an 86% yield. 1HNMR(400MHz,CDCl3)δ8.52(m,2H,2ArH),8.28(s,1H,ArH),8.17(s,1H,ArH),7.20 (dd,J=52.7,5.0Hz,2H,2ArH),4.75(s,2H,CH2),4.02(s,1H,OH),2.42(s,3H,CH3). 13 C NMR (101MHz, CDCl3) δ155.97,155.75,151.70,149.14,148.73,148.59,124.86,122.37,121.19,118.73,63.26,21.22.

[0042] Preparation of 4-methyl-2,2'-bipyridyl-4'-bromomethyl (Compound 4)

[0043] Compound 3 (6 mmol, 1.2 g) was dissolved in DCM (20 mL) and stirred in an ice bath for 5 min. PBr (6 mmol, 1.624 g) was then slowly added dropwise. Stirring was continued for 10 min, and the reaction mixture was allowed to return to room temperature and stirred for 10 h. Completion of the reaction was monitored by thin-layer chromatography. After completion, the pH was adjusted to 7 with 2 mol / L NaHCO solution, followed by extraction with DCM (20 mL x 3). The organic layer was dried over anhydrous NaSO, and the solvent was removed by evaporation under reduced pressure to afford compound 4 (1.258 g) as a light yellow solid in 87% yield. 1 H NMR (400MHz, CDCl3) δ8.57(d,J=4.9Hz,1H,ArH),8.46(d,J=4.9Hz,1H,ArH),8.33(d,J=1.8Hz,1H,ArH),8.19– 8.13(m,1H,ArH),7.29–7.23(m,1H,ArH),7.07(dd,J=5.1,1.7Hz,1H,ArH),4.40(s,2H,CH2),2.36(s,3H,CH3). 13 C NMR (101MHz, CDCl3) δ156.9,155.3,149.6,149.0,148.2,147.1,125.0,123.5,122.0,120.9,30.7,21.2.

[0044] Preparation of 4-methyl-2,2'-bipyridine-4'-((4-methylpiperidine)-N-methylene) (Compound 5a)

[0045] Compound 4 (4 mmol, 1.048 g), 4-methylpiperidine (4 mmol, 0.372 g), and K2CO3 (4 mmol, 0.553 g) were dissolved in MeCN (20 mL). After stirring for 5 min, 1-2 drops of DMF were added dropwise. Under nitrogen, the mixture was heated at reflux at 85°C for 12 h. Completion of the reaction was monitored by thin-layer chromatography. Upon completion, saturated brine (20 mL) was added, followed by extraction with DCM (20 mL x 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (ammonia MeOH:DCM = 1:15) to afford the title compound 5a (0.933 g) as a light yellow solid in 83% yield. 1 H NMR (400MHz, CDCl3) δ8.61(d,J=5.0Hz,1H,ArH),8.54(d,J=4.9Hz,1H,ArH),8.29(d ,J=1.6Hz,1H,ArH),8.22(d,J=1.7Hz,1H,ArH),7.36(dd,J=5.0,1.7Hz,1H,ArH),7.1 3(dd,J=5.0,1.7Hz,1H,ArH),3.56(s,2H,CH2),2.85(m,2H,CH2),2.44(s,3H,CH3), 2.01(m,2H,CH2),1.60(m,2H,CH2),1.28(m,3H,CH2,CH),0.92(d,J=6.1Hz,3H,CH3). 13 C NMR(101MHz, CDCl3)δ156.1,156.0,149.3,149.1,148.9,148.1,124.6,123.9,122.0,121.5,62.4,54.1,34.2,30.6,21.9,21.2.HRMS(ESI)m / zcalcd for C 18 H 23 N3[M+H] + ,282.1970;found,282.1974.

[0046] Preparation of Bis(2,2'-bipyridyl)ruthenium dichloride (Compound 7)

[0047] 2,2'-Bipyridine (3 mmol, 0.468 g), LiCl (3 mmol, 0.127 g), and hydrated ruthenium trichloride (3 mmol, 0.622 g) were dissolved in anhydrous DMF (25 ml) and heated under reflux at 165°C for 8 h. Completion of the reaction was monitored by thin-layer chromatography. After cooling to room temperature, 150 ml of acetone was added to the reaction solution, which was then incubated at 0°C overnight. The solution was filtered, leaving a solid. The solid was washed with triple-distilled water (10 mL x 3) and dried under vacuum to afford compound 7 (1.161 g) as a purple-black solid in 80% yield. 1 H NMR (400MHz, DMSO-d6) δ9.93 (d, J=5.5Hz, 2H, 2ArH), 8.63 (dd, J=8.2, 1.3Hz, 2H, 2ArH), 8.49 (dd, J=8.2, 1.3H z,2H,2ArH),8.07(m,2H,2ArH),7.75(m,2H,2ArH),7.65(m,2H,2ArH),7.45(s,2H,2ArH),7.12(m,2H,2ArH). 13 C NMR (101MHz, DMSO) δ153.2,134.9,125.7,122.9,122.6.

[0048] 4-Methyl-2,2'-bipyridyl-4'-((4-methylpiperidinyl)-N-methylene)-bis(2,2'-bipyridyl)ruthenium di(hexafluorophosphate) salt (I1)

[0049] Compound 7 (1 mmol, 0.483 g) and compound 5a (1 mmol, 0.281 g) were dissolved in MeOH / H₂O (40 mL, v / v = 1 / 2) and heated under reflux at 95°C for 4.5 h. Completion of the reaction was monitored by thin-layer chromatography. After cooling to room temperature, ammonium hexafluorophosphate (3 mmol, 0.504 g) was added to the reaction solution and stirred for 30 min. The mixed solvent was evaporated under reduced pressure, followed by the addition of triple-distilled water (2 mL). The solid was mixed with water, and the solution was filtered through filter paper. The remaining solid was washed with triple-distilled water (5 mL × 3) and chloroform (10 mL × 3). The solid was dried under vacuum, and the crude product was purified by column chromatography (ammonia, MeOH:DCM = 1:25) to obtain the target compound I1 (0.719 g) as an orange-red solid in a 73% yield. 1H NMR (400MHz, DMSO-d6) δ8.84(d,J=8.2Hz,6H,6ArH),8.18(m,4H,4ArH),7.79–7.68(m,4H,4ArH),7.63–7.28(m,8H,8ArH),4.41(s ,1H,CH),3.87–3.37(m,2H,CH2),3.17–2.62(m,2H,2CH2),2.55(s,3H,2CH3),2.10–1.13(m,6H,2CH2),0.93(d,J=6.0Hz,3H,CH3). 13 C NMR (101MHz, DMSO) δ157.0,151.7,150.2,138.3,128.3,128.2,124.9,21.2.

[0050] Example 2 Preparation of 4-methyl-2,2'-bipyridine-4'-((4-piperidinylpiperidine)-N-methylene)-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I2)

[0051] Preparation of 4-methyl-2,2'-bipyridine-4'-((4-piperidinylpiperidine)-N-methylene) (Compound 5b)

[0052] Referring to the method described for compound 5a, 4-piperidinylpiperidine was used instead of 4-methylpiperidine to react with 4 to obtain compound 5b (1.120 g, 80%) as a light yellow solid. 1 H NMR (400MHz, CDCl3) δ8.53(d,J=5.0Hz,1H,ArH),8.46(d,J=5.0Hz,1H,ArH),8.22(d ,J=1.6Hz,1H,ArH),8.15(d,J=1.6Hz,1H,ArH),7.26(m,1H,ArH),7.05(m,1H,ArH), 3.48(s,2H,CH2),2.86(m,2H,CH2),2.42(m,4H,2CH2),2.36(s,3H,CH3),2.17(m,1H ,CH),1.94(m,2H,CH2),1.69(m,2H,CH2),1.60–1.47(m,6H,3CH2),1.35(m,2H,CH2). 13 C NMR (101MHz, CDCl3) δ156.2,156.0,149.2,149.1,148.9,148.1,124.6,123.8,1 22.0,121.3,62.6,61.9,53.6,50.2,27.9,26.4,24.8,21.1.HRMS(ESI)m / zcalcd for C22 H 30 N4[M+H] + ,351.2549;found,351.2553.

[0053] Preparation of 4-methyl-2,2'-bipyridine-4'-((4-piperidinylpiperidine)-N-methylene)-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) (I2) Referring to the method described for compound I1, compound 5b was used instead of compound 5a to react with compound 7 to obtain compound I2 (0.737 g, 70%) as an orange-red solid. 1 H NMR (400MHz, DMSO-d6) δ8.83(d,J=8.3Hz,5H,5ArH),8.70(d,J=11.1Hz,2H,2ArH),8. 17(s,4H,4ArH),7.75(d,J=6.7Hz,5H,5ArH),7.65(d,J=5.8Hz,1H,ArH),7.51(dd,J= 25.8,6.2Hz,7H,7ArH),7.38(d,J=5.9Hz,1H,ArH),3.71(s,2H,CH2),3.16(d,J=12.3 Hz,1H,CH),3.01–2.88(m,4H,2CH2),2.16–1.61(m,13H,CH3,5CH2),1.42(s,1H,CH). 13 C NMR(101MHz,DMSO)δ157.1,157.0,156.7,156.3,151.6,151.3,150.8,150.6,150.2,138.2 ,129.1,128.3,127.6,125.6,124.8,124.3,63.0,60.0,52.0,49.6,26.2,23.3,21.9,21.1.

[0054] Example 3 Preparation of 4-methyl-2,2'-bipyridine-4'-((4-piperidinone)-N-methylene)-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I3)

[0055] Preparation of 4-methyl-2,2'-bipyridine-4'-((4-piperidinone)-N-methylene) (Compound 5c)

[0056] Referring to the method described for compound 5a, 4-piperidone was used instead of 4-methylpiperidine to react with compound 4 to obtain compound 5c (0.865 g, 77%) as a light yellow solid. 1H NMR (400MHz, CDCl3) δ8.57(d,J=5.0Hz,1H,ArH),8.47(d,J=5.0Hz,1H,ArH),8.30(d,J=1.6Hz,1H,ArH),8.17(d,J=1.7Hz,1H,ArH),7.31 (dd,J=4.9,1.7Hz,1H,ArH),7.08(dd,J=5.0,1.7Hz,1H,ArH),3.64(s,2H,CH2),2.72(m,4H,2CH2),2.42(m,4H,2CH2),2.37(s,3H,CH3). 13 C NMR (101MHz, CDCl3) δ207.7,155.5,154.7,148.3,147.9,147.5,147.2,123.8,122.5,121.0,120.1,59.9,52.1,40.2,20.1.HRMS(ESI)m / z calcd for C 17 H 19 N3O[M+H] + ,282.1606;found,282.1610.

[0057] Preparation of 4-methyl-2,2'-bipyridyl-4'-((4-piperidinone)-N-methylene)-bis(2,2'-bipyridyl)ruthenium di(hexafluorophosphate) (I3)

[0058] Referring to the method described for compound I1, compound 5c was used instead of compound 5a to react with compound 7 to obtain compound I3 (0.659 g, 67%) as an orange-red solid. 1 H NMR (400MHz, DMSO-d6) δ8.76(d,J=8.2Hz,4H,4ArH),8.67(s,2H,2ArH),8.09(m,4H,4ArH),7.72–7.62(m,4H,4ArH),7.58(d,J=5.8Hz,1H, ArH),7.51–7.40(m,6H,6ArH),7.30(dd,J=5.9,1.7Hz,1H,ArH),3.79(s,2H,CH2),3.27(s,3H,CH3),2.71(m,4H,2CH2),2.33(m,4H,2CH2). 13C NMR(101MHz,DMSO)δ157.1,157.0,157.0,156.8,156.4,151.7,151.6,151.6,151.3,1 50.8,150.3,138.2,129.1,128.3,127.6,125.6,124.9,124.1,59.1,52.8,22.9,21.1.

[0059] Example 4 Preparation of 4-methyl-2,2'-bipyridine-4'-(morpholine-N-methylene)-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I4)

[0060] 4-Methyl-2,2'-bipyridine-4'-(morpholine-N-methylene) (Compound 5d)

[0061] Referring to the method described for compound 5a, morpholine was used instead of 4-methylpiperidine to react with compound 4 to obtain compound 5d (0.796 g, 74%) as a light yellow solid. 1 H NMR (400MHz, CDCl3) δ8.55(dd,J=5.0,0.7Hz,1H,ArH),8.47(d,J=5.0Hz,1H,ArH),8.26(d,J=1.4Hz,1H,ArH),8.19–8.13(m,1H,ArH),7.28(dd,J=5. 0,1.6Hz,1H,ArH),7.07(dd,J=5.3,1.7Hz,1H,ArH),3.69–3.61(m,4H,2CH2),3.51(s,2H,CH2),2.41(dd,J=5.7,3.6Hz,4H,2CH2),2.37(s,3H,CH3). 13 C NMR(101MHz, CDCl3)δ156.3,155.9,149.2,149.0,148.3,148.1,124.7,123.9,122.0,121.4,66.9,62.3,53.6,21.2.HRMS(ESI)m / z calcd for C 16 H 19 N3O[M+H] + ,270.1606;found,270.1605.

[0062] Preparation of 4-methyl-2,2'-bipyridyl-4'-(morpholino-N-methylene)-bis(2,2'-bipyridyl)ruthenium di(hexafluorophosphate) (I4)

[0063] Referring to the method described for compound I1, compound 5d was used instead of compound 5a to react with compound 7 to obtain compound I4 (0.632 g, 65%) as an orange-red solid. 1 H NMR (400MHz, DMSO-d6) δ8.88–8.79(m,4H,4ArH),8.73(dd,J=13.4,1.8Hz,2H,2ArH),8.17(s,4H,4ArH),7.78–7.70(m,4H,4ArH),7.65(d,J=5.8Hz,1H ,ArH),7.58–7.47(m,6H,6ArH),7.38(dd,J=5.9,1.8Hz,1H,ArH),3.69(s,2H,CH2),3.62(m,4H,2CH2),2.54(s,3H,CH3),2.43(d,J=4.0Hz,4H,2CH2). 13 C NMR(101MHz,DMSO)δ157.1,157.0,157.03,156.8,156.3,151.6,151.6,151.3,150.7,1 50.4,150.3,138.2,129.1,128.3,127.8,125.6,124.9,124.4,66.5,61.0,53.7,21.1.

[0064] Example 5 Preparation of 4-methyl-2,2'-bipyridine-4'-((1,1-dioxythiomorpholine)-N-methylene)-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I5)

[0065] 4-Methyl-2,2'-bipyridine-4'-((1,1-dioxythiomorpholinyl)-N-methylene) (Compound 5e)

[0066] Referring to the method described for compound 5a, 1,1-dithiomorpholine dioxide was used instead of 4-methylpiperidine to react with compound 4 to obtain compound 5e (0.989 g, 78%) as a light yellow solid. 1 H NMR (400MHz, CDCl3) δ8.57(d,J=4.9Hz,1H,ArH),8.47(d,J=5.0Hz,1H,ArH),8.28(d,J=1.6Hz,1H,ArH),8.17(d,J=1.6Hz,1H ,ArH),7.23(dd,J=4.9,1.6Hz,1H,ArH),7.14–7.07(m,1H,ArH),3.68(s,2H,CH2),3.08–2.91(m,8H,4CH2),2.38(s,3H,CH3). 13C NMR (101MHz, CDCl3) δ156.7,155.5,149.5,149.0,148.3,147.5,124.9,123.3,122.1,120.9,77.3,77.0,76.7,60.5,51.5,50.9,21.2.HRMS (ESI) m / z calcd for C 16 H 19 N3O2S[M+H]+,318.1276; found,318.1270.

[0067] 4-Methyl-2,2'-bipyridyl-4'-((1,1-dioxythiomorpholinyl)-N-methylene)-bis(2,2'-bipyridyl)ruthenium di(hexafluorophosphate) salt (I5)

[0068] Referring to the method described for compound I1, compound 5e was used instead of compound 5a to react with compound 7 to obtain compound I5 (0.724 g, 71%) as an orange-red solid. 1 H NMR(400MHz,DMSO-d6)δ8.84(d,J=8.2Hz,6H,6ArH),8.17(s,4H,4ArH),7.83–7.31(m,12H, 12ArH),3.93(s,2H,CH2),3.21(d,J=6.0Hz,4H,2CH2),2.98(s,4H,2CH2),2.55(s,3H,CH3). 13 C NMR (101MHz, DMSO) δ157.1,157.0,156.9,156.3,151.7,151.6,151.2,150.8,150. 5,150.3,138.2,129.1,128.3,127.4,125.6,124.8,123.7,58.0,50.9,50.6,21.2.

[0069] Example 6 Preparation of 4-methyl-2,2'-bipyridine-4'-((N-benzylpiperazine)-N-methylene)-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) (I6)

[0070] 4-Methyl-2,2'-bipyridine-4'-((N-benzylpiperazine)-N-methylene) (Compound 5f)

[0071] Referring to the method described for compound 5a, N-benzylpiperazine was used instead of 4-methylpiperidine to react with compound 4 to obtain compound 5f (1.060 g, 74%) as a yellow solid. 1H NMR (400MHz, CDCl3) δ8.60(d,J=5.0Hz,1H,ArH),8.54(d,J=5.0Hz,1H,ArH),8.30(d,J=1.6Hz,1H,ArH),8.22(d,J=1.7Hz,1H,ArH),7.3 6–7.31(m,4H,4ArH),7.25(m,2H,2ArH),7.13(dd,J=5.0,1.7Hz,1H,ArH),3.59(s,2H,CH2),3.54(s,2H,CH2),2.48(m,11H,CH3,4CH2). 13 C NMR (101MHz, CDCl3) δ156.2,155.9,149.1,148.9,148.6,148.1,137.7,129.3,129.0,128.3, 128.2,127.3,127.1,124.7,123.9,122.0,121.5,62.9,61.8,53.0,52.9,21.2.HRMS(ESI)m / z calcd for C 23 H 26 N4[M+H] + ,359.2236;found,359.2237.

[0072] 4-Methyl-2,2'-bipyridyl-4'-((N-benzylpiperazine)-N-methylene)-bis(2,2'-bipyridyl)ruthenium di(hexafluorophosphate) salt (I6)

[0073] Referring to the method described for compound I1, compound 5f was used instead of compound 5a to react with compound 7 to obtain compound I6 (0.722 g, 68%) as an orange-red solid. 1 H NMR (400MHz, DMSO-d6) δ8.77(dd,J=49.9,12.2Hz,6H,6ArH),8.16(t,J=7.8Hz,4H,4ArH),7.74(dt,J=10.2,6.3Hz,4H,4ArH),7.64(d,J=5.7Hz,1H,Ar H),7.51(dd,J=16.9,10.3Hz,7H,7ArH),7.30(d,J=16.1Hz,5H,CH3,CH2),3.69(s,2H,CH2),3.53–3.45(m,2H,CH2),2.48(d,J=20.3Hz,7H,CH3,2CH2). 13C NMR(101MHz,DMSO)δ157.0,156.7,156.3,151.63,150.7,150.2,138.2,129.3,128.6,128.3,124.9,52.8,21.1.HRMS(ESI)m / z calcd for C 43 H 42 N8Ru[M / 2] + ,386.1288;found,386.1303.

[0074] Example 7 Preparation of 4-methyl-2,2'-bipyridine-4'-((N-phenylpiperazine)-N-methylene)-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) (I7)

[0075] 4-Methyl-2,2'-bipyridine-4'-((N-phenylpiperazine)-N-methylene) (Compound 5g)

[0076] Referring to the method described for compound 5a, N-phenylpiperazine was used instead of 4-methylpiperidine to react with compound 4 to obtain compound 5g (0.977 g, 71%) as a yellow solid. 1 H NMR (400MHz, CDCl3) δ8.64(d,J=5.0Hz,1H,ArH),8.55(d,J=5.0Hz,1H,ArH),8.35(s,1H,ArH),8.24(s,1H,ArH),7.41(s,1H,ArH),7.31–7.22(m,2H,2ArH ),7.15(d,J=3.3Hz,1H,ArH),6.93(d,J=8.2Hz,2H,2ArH),6.86(m,1H,ArH), 3.67(s,2H,CH2),3.23(m,4H,2CH2),2.67(m,4H,2CH2),2.45(s,3H,CH3).13C NMR (101MHz, CDCl3) δ156.40,155.93,151.28,149.28,149.03,148.22,129.13,124.7 9,123.98,122.09,121.54,119.78,116.13,61.94,53.24,49.12,21.24.HRMS(ESI)m / z calcdfor C 22 H 24 N4[M+H] + ,345.2079;found,345.2072.

[0077] 4-Methyl-2,2'-bipyridyl-4'-((N-phenylpiperazine)-N-methylene)-bis(2,2'-bipyridyl)ruthenium di(hexafluorophosphate) salt (I7)

[0078] Referring to the method described for compound I1, compound 5g was used instead of compound 5a to react with compound 7 to obtain compound I7 (0.712 g, 65%) as an orange-red solid. 1 H NMR(400MHz, DMSO-d6)δ8.80(dd,J=36.8,7.2Hz,6H,6ArH),8.18(d,J=8.3Hz,4H,4ArH),7.82–7.65(m,5H,5ArH),7.55(d,J= 10.2Hz,6H,5ArH),7.41–7.15(m,3H,3ArH),7.00–6.61(m,3H,3ArH),3.77(s,2H,CH2),3.36(s,8H,4CH2),2.60(s,3H,CH3). 13 C NMR (101MHz, DMSO) δ157.0,156.8,156.4,151.6,151.4,150.7,150.3,138.2,129.4,129. 1,128.3,127.8,125.6,124.8,124.4,119.3,115.8,60.6,53.2,48.5,21.1.HRMS(ESI)m / z calcd for C 42 H 40 N8Ru[M / 2] + ,379.1210;found,379.1211.

[0079] Example 8 Measurement of the UV Absorption and Fluorescence Emission Spectra of the Bipyridine Ruthenium Complex of the Present Invention

[0080] 1. Ultraviolet absorption spectra of bipyridine ruthenium complexes

[0081] The bipyridine ruthenium complex prepared in the embodiment of the present invention was prepared into a 10 μM sample solution using methanol as solvent and recorded using a UV-visible spectrophotometer. The temperature when recording the UV absorption spectrum was room temperature, the wavelength range of the scanning absorption spectrum was 200-800 nm, and the scanning speed was 1.0 nm / s. Figure 1 The UV absorption spectra of the bipyridine ruthenium complexes in methanol solution are shown in FIG. As can be seen from the figure, the absorption spectrum of the bipyridine ruthenium complexes of the present invention has a strong absorption peak in the range of 400-500 nm, which may be caused by the dπ-π* electronic transition (MLCT) between the metal Ru and the ligand Biq.

[0082] 2. Fluorescence emission spectra of bipyridine ruthenium complexes

[0083] The bipyridine ruthenium complex prepared in the embodiment of the present invention was prepared into a 10 μM sample solution using methanol as solvent. The fluorescence emission spectrum of the ruthenium complex was recorded using a fluorescence spectrophotometer with an excitation wavelength of 452 nm. The temperature when recording the emission spectrum was room temperature. The wavelength range of the emission spectrum was 470-900 nm, and the scanning speed was 1.0 nm / s. Figure 2 The fluorescence emission spectra of the bipyridine ruthenium complexes in methanol solution are shown in FIG. As can be seen from the figure, the maximum wavelength of the fluorescence emission spectrum of the bipyridine ruthenium complexes of the present invention appears in the range of 600-700 nm.

[0084] Example 9 Measurement of Singlet Oxygen Quantum Yield of Bipyridine Ruthenium Complexes of the Present Invention

[0085] We selected DPBF as a singlet oxygen scavenger and methanol as the solvent. In methanol, the absorbance of DPBF at 412 nm was adjusted to about 1.0; then, in order to reduce the singlet oxygen quenching of the control and the test complex itself, I1, I2, I3, I4, I5, I6, I7 and the control [Ru(bpy)3] 2+ The absorbance at 520 nm was adjusted to below 0.1. Next, the light-transmitting side of the quartz cell was exposed to a 520 nm laser (40 mW) for a total of 5 minutes. The absorption spectra were recorded at different time intervals within this 5-minute period. The scanning wavelength range was 300-600 nm, and the scanning speed was 1.0 nm / s.

[0086] [Ru(bpy)3] with known singlet oxygen quantum yield 2+ As a standard, the singlet oxygen quantum yield was calculated by the following formula:

[0087] Φ T =Φ X (S T F X / SxF T )

[0088] Φ: singlet oxygen quantum yield; S: slope of the linear fit with irradiation time as x-axis and the UV absorbance of DPBF at 412 nm after irradiation time as y-axis; F: absorption correction factor of unknown sample and reference standard, F = 1-10 -OD OD represents the UV absorbance of the compound at the irradiation wavelength; X and T represent the reference standard and unknown sample, respectively. The results are shown in Table 1, which shows that most of the bipyridine ruthenium complexes of the present invention have high singlet oxygen quantum yields.

[0089] Table 1 Singlet oxygen quantum yield of the bipyridine ruthenium complex of the present invention

[0090]

[0091] Test Example 3 Cytotoxicity Test of the Compounds of the Invention

[0092] The phototoxicity of the bipyridine ruthenium complexes of the present invention on human colon cancer cells HT29, human lung cancer cells A549, and human breast cancer cells Mcf-7 was evaluated in vitro using the MTT colorimetric assay. In the dark toxicity test, a vial of cells in good exponential growth phase was first taken and digested with 0.25% trypsin to detach the adherent cells. The resulting mixture contained 2×10 4 ~4×10 4 Prepare a suspension of cells. Inoculate the cell suspension onto a 96-well plate, 180 μL per well, and incubate in a constant temperature CO2 incubator for 24 hours. Change the medium and add the test compound (dissolve the compound in DMSO and dilute it with PBS, the test compound concentration is 25 μM) at 20 μL per well in the dark. Continue incubating in the dark for 48 hours. Add MTT to a 96-well plate, 20 μL per well, and react in an incubator for 4 hours. Aspirate the supernatant, add 150 μL DMSO to each well, and shake on a plate shaker for 5 minutes. Measure the absorbance of each well at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) to calculate cell viability.

[0093] The phototoxicity test method is basically the same as the dark toxicity test method. The difference is that after adding the drug and incubating for 24 hours, the light condition is adopted, and the 520nm laser (15mW / cm 2 ) After 10 min of irradiation, fresh complete culture medium was replaced and the cells were cultured in the incubator for another 24 h. Then, 20 μL MTT solution was added to each well and the cell survival rate was calculated.

[0094] Experimental results show that the bipyridine ruthenium complexes of the present invention exhibit minimal dark toxicity to tumor cells. However, after specific laser irradiation, cell survival rates are significantly reduced, demonstrating that the bipyridine ruthenium complexes of the present invention can significantly inhibit tumor cell proliferation under light. Therefore, the bipyridine ruthenium complexes of the present invention exhibit significant photodynamic therapy effects on tumor cells (Table 2).

[0095] Table 2 Survival rate of some bipyridine ruthenium complexes of the present invention on human cancer cells (25 μM)

[0096]

[0097] ND: Not tested.

[0098] Test Example 4: Cell fluorescence imaging experiment using confocal microscopy

[0099] Cell fluorescence imaging and mitochondrial localization experiments were performed using a confocal microscope. Human lung cancer A549 cells or human cervical cancer Hela cells were cultured in DEME medium in a laser confocal microscope dish for 12-24 hours. The test compound was then added at 5-20 μM and incubated for half an hour in a cell culture incubator at 37°C and 5% CO2. The cells were then washed three times with a pH 7.4 phosphate buffer solution. A 1-5 μM solution of the mitochondrial stain MitoTracker Green was added and incubated for another half an hour. The cells were then washed three times with a pH 7.4 phosphate buffer solution. The cells were then placed on the stage of a confocal microscope for confocal fluorescence imaging. The excitation wavelength for the test compound was set to λex = 400-500 nm, and the emission wavelength was set to λem = 600-680 nm.

[0100] The results showed that the bipyridine ruthenium complex of the present invention can clearly image the fluorescence of human tumor cells, and the fluorescence imaging overlaps well with that of mitochondrial probes, indicating that the bipyridine ruthenium complex of the present invention can effectively target and localize in the mitochondria of tumor cells ( Figure 3 and Figure 4 ), the co-localization coefficients in the figure are 0.87 and 0.91 respectively. Figure 3 This is the co-localization imaging of the bipyridine ruthenium complex I3 of the present invention and the mitochondrial probe in A549 cells. Figure 4 This is the co-localization imaging of the compound I6 of the present invention and the mitochondrial probe in Hela cells.

Claims

1. A bipyridine ruthenium complex having photodynamic therapy effect, characterized in that: The bipyridine ruthenium complex has a structure shown in the general formula I: ， Wherein, R and Y are selected from the following combinations: R = ,Y = I; Or R = , Y = PF6; Or R = , Y = PF6; Or R = , Y = PF6; Or R = , Y = PF6; Or R = , Y = PF6.

2. A method for preparing a bipyridine ruthenium complex according to claim 1, characterized in that: The synthetic route of the preparation method is as follows: ； The preparation method comprises the following steps: S1. First, the methyl group at the 4'-position of compound 1 is oxidized to an aldehyde group using selenium dioxide to obtain compound 2. Compound 2 is then directly reduced with sodium borohydride to obtain compound 3. Compound 3 then undergoes a substitution reaction with phosphorus tribromide to obtain compound 4. Compound 4 reacts with various nitrogen heterocycles to obtain compound 5. S2. Compound 6 undergoes coordination reaction with hydrated ruthenium trichloride to obtain compound 7; S3. Compound 5 and compound 7 contain Y - A coordination reaction occurs in a mixed solution of water and methanol containing ions to form a bipyridine ruthenium complex I.

3. Use of the bipyridine ruthenium complex according to claim 1 in the preparation of a drug for treating malignant tumors.

4. The use according to claim 3, characterized in that The medicine is a medicine for photodynamic therapy of malignant tumors.

5. The use according to claim 3, characterized in that The malignant tumor is one of breast cancer, colon cancer, cervical cancer and lung cancer.

6. Use of the bipyridine ruthenium complex according to claim 1 in the preparation of a fluorescent imaging agent with mitochondrial targeting.

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