Bipyridine / phenanthroline ruthenium complex and its preparation method and application

Abstract

The invention belongs to the field of medicines, and discloses a bipyridine / phenanthroline ruthenium complex and a preparation method and application thereof. The bipyridine / phenanthroline ruthenium complex has a structure shown in general formula I: #imgabs0# wherein #imgabs1# is selected from #imgabs2##imgabs3#, and Y represents a halide anion or a hexafluorophosphate anion. The novel bipyridine / phenanthroline ruthenium complex has mitochondrial targeting, generates a high concentration of active oxygen after being irradiated with light, thereby killing tumor cells, and has excellent photodynamic therapy effect and anti-tumor proliferation activity.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and in particular relates to a bipyridine / phenanthroline ruthenium complex and a preparation method and application thereof. Background Art

[0002] Cancer is one of the malignant diseases with high morbidity and mortality. Photodynamic therapy is a new technology that uses the photodynamic effect for disease diagnosis and treatment, and has gradually developed into a promising treatment option for localized cancer and non-malignant diseases.

[0003] In general, photosensitizers themselves have low toxicity, but after being irradiated with light of a specific wavelength, they produce cytotoxic reactive oxygen species. Highly reactive reactive oxygen species rapidly destroy nearby biomolecules, including proteins, lipids or nucleic acids, through oxidation, ultimately leading to cell death. In recent years, many different photosensitizers have been reported, such as porphyrins, fluoroboron dipyrroles and cyanines, but their photodynamic therapy effects are not significant. Metal complexes are another research direction for the development of new photosensitizers. Metal complex photosensitizers based on Pt(II), Pt(IV), Ru(II), etc. have been reported.

[0004] Ruthenium metal complexes have various applications in the medical field due to their excellent physical and chemical properties, such as cell imaging, fluorescent probes, antibacterial, and tumor treatment. In addition, studies have shown that bidentate ligands have good chelating ability for transition metals, and the complexes have high stability. Therefore, designing new ruthenium complexes containing bidentate ligands with different structures may improve the optical properties of the complexes, such as improving the fluorescence imaging effect and increasing the singlet oxygen yield. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a bipyridine / phenanthroline ruthenium complex and a preparation method and application thereof. The bipyridine / phenanthroline ruthenium complex can realize fluorescent imaging of tumor cells and can target and locate in the mitochondria of tumor cells, exerting a photodynamic therapy effect on a variety of tumor cells, and can be used in the preparation of malignant tumor diagnostic reagents or malignant tumor therapeutic drugs.

[0006] The present invention provides a bipyridine / phenanthroline ruthenium complex or a pharmaceutically acceptable salt thereof. The bipyridine / phenanthroline ruthenium complex has a structure shown in the following general formula I:

[0007]

[0008] Among them, R is selected from One of them; Y- represents a halide anion or a hexafluorophosphate anion.

[0009] Furthermore, R is selected from One of them.

[0010] Furthermore, R and Y are selected from the following combinations:

[0011] Y=I;

[0012] or 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 / phenanthroline ruthenium complex, and the synthesis route of the preparation method is shown in the following formula:

[0020]

[0021] Said Selected from Y- represents halide anion or hexafluorophosphate anion;

[0022] The preparation method comprises the following steps:

[0023] S1. The methyl group at the 5-position of compound 1 is oxidized to an aldehyde group by selenium dioxide to obtain compound 2, and then compound 2 is directly reduced by sodium borohydride to obtain compound 3, and compound 3 is then substituted with phosphorus tribromide to obtain compound 4, and compound 4 is reacted with various nitrogen heterocycles to obtain compound 5;

[0024] S2. Compound 6 undergoes coordination reaction with hydrated ruthenium trichloride to obtain compound 7;

[0025] S3. Compound 5 and compound 7 undergo coordination reaction in a mixed solution of water and methanol to generate bipyridine / phenanthroline ruthenium complex I.

[0026] The present invention also provides an application of the above-mentioned bipyridine / phenanthroline ruthenium complex or a pharmaceutically acceptable salt thereof in the preparation of a malignant tumor diagnostic reagent or a malignant tumor therapeutic drug.

[0027] Furthermore, the malignant tumor diagnostic reagent is a malignant tumor fluorescence imaging reagent.

[0028] Furthermore, the malignant tumor fluorescent imaging agent is a near-infrared fluorescent imaging agent with mitochondrial targeting.

[0029] Furthermore, the drug for treating malignant tumors is a drug used for photodynamic therapy of malignant tumors.

[0030] Furthermore, the malignant tumor includes one of breast cancer, colon cancer, lung cancer and cervical cancer.

[0031] Compared with the prior art, the present invention takes into account that phenanthroline is a bidentate ligand with a planar aromatic ring, good planarity and high designability. By introducing nitrogen-containing saturated heterocycles with different hydrophilicities into the methyl group at the 5-position of 5-methyl-1,10-phenanthroline, a series of novel bipyridine / phenanthroline ruthenium complexes are designed and synthesized, which have near-infrared fluorescence and two-photon excitation characteristics, large Stokes shift, low tissue background fluorescence interference, good photostability and singlet oxygen ( 1 O2) has the advantages of high quantum yield, which not only improves the lipid-water partition coefficient of the complex and realizes fluorescent imaging of tumor cells, but also can target and locate in the mitochondria of tumor cells, exerting the photodynamic therapy effect on a variety of tumor cells, and can be used in the preparation of malignant tumor diagnostic reagents or malignant tumor therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The ultraviolet absorption spectrum of the bipyridine / phenanthroline ruthenium complex provided by the present invention;

[0033] Figure 2 The fluorescence emission spectrum of the bipyridine / phenanthroline ruthenium complex provided by the present invention;

[0034] Figure 3 This is an imaging diagram of the co-localization of compound I3 provided in an embodiment of the present invention with a mitochondrial probe in Hela cells;

[0035] Figure 4 This is an imaging diagram of the co-localization of compound I8 provided in an example of the present invention with a mitochondrial probe in A549 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 5-(piperidinyl-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I1)

[0038] (1) Preparation of 5-formaldehyde-1,10-phenanthroline (2)

[0039] Compound 1 (10 mmol, 1.940 g) and SeO2 (30 mmol, 1.111 g) were dissolved in 1,3-dichlorobenzene (10 mL), and the mixture was refluxed at 165°C for 3 h under nitrogen protection. The reaction was monitored by thin layer chromatography to see if it was complete. After the reaction was completed, the reaction solution was cooled to room temperature, 1,3-dichlorobenzene was first sucked out with a plastic pipette, and the remaining black solid was washed with methanol (10 mL × 3) and the solid was filtered out. The filtrate was mixed with the previous 1,3-dichlorobenzene, and the organic layer was dried with anhydrous Na2SO4. The methanol was removed by decompression, and the crude product was purified by column chromatography (ammonia methanol: DCM = 1:15) to obtain a white solid, namely compound 2 (1.873 g), with a yield of 90%. 1 H NMR (400MHz, DMSO-d6) δ10.32(s,1H,CH),9.57(dd,J=8.5,1.8Hz,1H,ArH),9.18(dd,J=4.4,1 .8Hz,1H,ArH),9.10(dd,J=4.3,1.8Hz,1H,ArH),8.62–8.53(m,2H,2ArH),7.77(m,2H,2ArH). 13 C NMR (101MHz, DMSO-d6) δ193.5,153.2,150.7,147.6,145.8,140.4,138.4,133.8,130.1,127.2,125.5,124.5.

[0040] (2) Preparation of 5-hydroxymethyl-1,10-phenanthroline (3)

[0041] Compound 2 (8 mmol, 1.664 g) was dissolved in CH3CH2OH (20 mL), stirred for 5 min, and then NaBH4 (16 mmol, 0.605 g) was added and reacted for 1 h. The reaction was monitored by thin layer chromatography. After the reaction was completed, 1 to 2 drops of water were added to quench the reaction, and then the solvent was removed by vortexing under reduced pressure. Saturated brine (30 mL) was added and extracted with DCM (20 mL×3). The organic layer was dried over anhydrous Na2SO4, and the organic solvent was removed by vortexing under reduced pressure to obtain compound 3 (1.215 g) as a light yellow solid with a yield of 73%. 1H NMR (400MHz, DMSO-d6) δ9.01 (m, 2H, 2ArH), 8.52 (dd, J=8.4, 1.7Hz, 1H, ArH), 8.39 (dd, J=8.1, 1.8Hz,1H,ArH),7.90(s,1H,ArH),7.69(m,2H,2ArH),5.58(m,1H,OH),5.04–4.94(m,2H,CH2). 13 C NMR (101MHz, DMSO-d6) δ193.5,153.2,150.7,147.60,145.8,140.4,138.4,133.8,130.1,127.2,125.5,124.5.

[0042] (3) Preparation of 5-bromomethyl-1,10-phenanthroline (4)

[0043] Compound 3 (6mmol, 1.260g) was dissolved in DCM (20mL), stirred in an ice bath for 5min, and then PBr3 (6mmol, 1.624g) was slowly added dropwise. After stirring for 10min, the reaction liquid temperature was restored to room temperature and reacted for 12h. The reaction was monitored by thin layer chromatography to see if it was complete. After the reaction was complete, the pH was adjusted to 7 with 2mol / L NaHCO3, and then extracted with DCM (20mL×3). The organic layer was dried with anhydrous Na2SO4, and the organic solvent was removed under reduced pressure to obtain compound 4 (1.306g) as a light yellow solid with a yield of 80%. 1 HNMR(400MHz,CD3OD)δ9.00(m,2H,2ArH),8.64(dd,J=8.4,1.7Hz,1H,ArH),8.34–8.28(m,1H,ArH),7 .95(s,1H,ArH),7.74(dd,J=8.0,4.1Hz,1H,ArH),7.65(dd,J=8.1,4.4Hz,1H,ArH),5.02(s,2H,CH2). 13 C NMR (101MHz, CD3OD) δ149.9,149.5,145.2,144.9,137.0,133.6,133.1,128.2,127.5,126.9,123.8,123.3,29.5.

[0044] (4) Preparation of 5-(piperidinyl-N-methylene)-1,10-phenanthroline (5a)

[0045] Compound 4 (4mmol, 1.088g), piperidine (4mmol, 0.340g) and K2CO3 (4mmol, 0.553g) were dissolved in MeCN (20mL), stirred for 5min, and then 1-2 drops of DMF were added dropwise. The mixture was refluxed at 85°C for 12h under nitrogen protection. The reaction was monitored by thin layer chromatography. After the reaction was completed, saturated brine (20mL) was added, and then extracted with DCM (20mL×3). The organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography (ammonia methanol: DCM=1:20) to obtain a light yellow solid, namely compound 5a (0.798g), with a yield of 72%. 1 H NMR (400MHz, CDCl3) δ9.07(m,2H,2ArH),8.71(dd,J=8.4,1.7Hz,1H,ArH),8.10(dd,J=8.1,1.7Hz,1H,ArH),7.5 9(s,1H,ArH),7.52(m,2H,2ArH),3.79(s,2H,CH2),2.41–2.35(m,2H,CH2),1.41(m,6H,3CH2),1.17(s,2H,CH2). 13 C NMR (101MHz, CDCl3) δ149.9,149.8,146.4,146.1,135.6,133.7,133.6,128.6,127.9,126.5,123.0,122.5,61.8,54.7,26.0,24.3.HRMS (ESI) m / z calcd for C 18 H 19 N3[M+H] + ,278.1657;found,278.1670.

[0046] (5) Preparation of bis(2,2'-bipyridine)ruthenium dichloride (7)

[0047] Compound 6 (3mmol, 0.468g), LiCl (3mmol, 0.127g) and hydrated ruthenium trichloride (3mmol, 0.622g) were dissolved in anhydrous DMF (25ml) and heated to reflux at 165℃ for 8h. The reaction was monitored by thin layer chromatography. After the reaction was completed, it was cooled to room temperature, 150ml of acetone was added to the reaction solution, and then placed at 0℃ overnight. The solution was filtered off, leaving the solid, which was washed with triple distilled water (10mL×3) and vacuum dried to obtain compound 7 (1.161g) as a purple-black solid with a yield of 80%. 1H 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-d6) δ153.2,134.9,125.7,122.9,122.6.

[0048] (6) Preparation of 5-(piperidinyl-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I1)

[0049] Compound 7 (2mmol, 0.967g) and compound 5a (2mmol, 0.554g) were dissolved in methanol / H2O (40mL, v / v=1 / 2) and heated under reflux at 95°C for 4.5h. The reaction was monitored by thin layer chromatography. After the reaction was completed, the mixture was cooled to room temperature, and ammonium hexafluorophosphate (10mmol, 1.630g) was added to the reaction solution. The mixture was stirred for 30min, and the mixed solvent was dried under reduced pressure. Then, triple distilled water (2mL) was added, and the solid was mixed with water. The solution was filtered out with filter paper, leaving the solid. The solid was washed with triple distilled water (5mL×3) and chloroform (10mL×3), and the solid was vacuum dried. The crude product was purified by column chromatography (ammonia methanol: DCM=1:20) to obtain the target complex I1 as an orange-red solid with a yield of 63%. 1 H NMR (400MHz, CD3OD) δ9.06 (dd, J=8.5, 1.3Hz, 1H, ArH), 8.74–8.65 (m, 4H, 4ArH), 8.63 (dd, J=8. 3,1.3Hz,1H,ArH),8.22–8.10(m,5H,5ArH),8.09–7.99(m,2H,ArH),7.95–7.90(m,2H,2ArH),7. 84–7.77(m,2H,2ArH),7.66–7.59(m,2H,2ArH),7.53(d,J=1.7Hz,2H,2ArH),7.31(m,2H,2ArH), 4.17–3.95(m,2H,CH2),2.59(s,4H,2CH2),1.62–1.55(m,4H,2CH2),1.49(d,J=5.6Hz,2H,CH2). 13C NMR (101MHz, CD3OD) δ157.3,157.3,157.1,151.7,151.6,151.5,151.4,151.3,151.2,147.9,147.3,137.8,137.6,136. 5,136.3,134.9,131.0,130.3,129.4,128.0,127.5,127.5,127.4,126.1,125.5,124.2,124.1,60.2,54.4,25.5,23.9.

[0050] Example 2: Preparation of 5-((4-methylpiperidinyl)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I2)

[0051] (1) Preparation of 5-((4-methylpiperidinyl)-N-methylene)-1,10-phenanthroline (5b)

[0052] Referring to the method described for compound 5a, 4-methylpiperidine was used instead of piperidine to react with compound 4 to obtain compound 5b (0.861 g, 74%) as a light yellow solid. 1 H NMR (400MHz, CDCl3) δ9.12–9.03(m,2H,2ArH),8.71(dd,J=8.3,1.8Hz,1H,ArH),8.10(dd,J=8.0,1.8Hz,1H,ArH),7.59(s,1H,ArH),7.53(m,2H ,2ArH),3.81(s,2H,CH2),2.88–2.75(m,2H,CH2),1.98(m,2H,CH2),1.37–1.26(m,1H,CH),1.22–1.03(m,4H,2CH2),0.82(d,J=6.5Hz,3H,CH3). 13 C NMR (101MHz, CDCl3) δ149.9,149.8,146.4,146.1,135.6,133.7,133.6,128. 6,127.9,126.5,123.04,122.6,61.5,54.1,34.3,30.8,21.8.HRMS(ESI)m / z calcd for C 19 H 21 N3[M+H] + ,292.1814;found,292.1823.

[0053] 5-((4-Methylpiperidinyl)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridyl)ruthenium di(hexafluorophosphate) salt (I2)

[0054] (2) Preparation of 5-((4-methylpiperidinyl)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I2)

[0055] Referring to the method described for complex I1, compound 5b was used instead of compound 5a to react with compound 7 to obtain complex I2 as an orange-red solid (yield: 61%). 1 H NMR (400MHz, DMSO-d6) δ8.86(dd,J=20.0,8.1Hz,6H,6ArH),8.17(dd,J=39.8,7.9Hz,7H,7ArH),7.90(d,J=43.8Hz,4H,4ArH),7.63–7.53(m,4 H,4ArH),7.36(s,2H,2ArH),3.20(s,2H,2ArH),2.62(d,J=89.1Hz,2H,CH2),1.87(m,4H,2CH2),1.49–1.01(m,3H,CH2,CH),0.92(s,3H,CH3). 13 C NMR(101MHz,DMSO-d6)δ157.2,157.0,152.4,152.2,151.9,151.8,147.7,147.1,138.4,138.2,137.0,136.6 ,135.0,130.7,130.2,128.3,128.2,127.9,126.9,126.4,124.9,124.8,59.9,54.2,53.9,34.4,30.7,22.2.

[0056] Example 3: Preparation of 5-((4-piperidone)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I3)

[0057] (1) Preparation of 5-((4-piperidone)-N-methylene)-1,10-phenanthroline (5c)

[0058] Referring to the method described for compound 5a, 4-piperidone was used instead of piperidine to react with compound 4 to obtain compound 5c (0.815 g, 70%) as a light yellow solid, except that in the preparation of the complex. 1H NMR (400MHz, CDCl3) δ9.10(m,2H,2ArH),8.72(dd,J=8.4,1.7Hz,1H,ArH),8.12(dd,J=8.1,1.7Hz,1H ,ArH),7.62(s,1H,ArH),7.56(m,2H,2ArH),3.98(s,2H,CH2),2.78(m,4H,2CH2),2.37(m,4H,2CH2). 13 C NMR (101MHz, CDCl3) δ208.7,150.3,150.1,146.5,146.2,135.7,133.5,132.7,128.2,127.7,127.0,123.2,122.7,60.2,53.0,41.2.HRMS (ESI) m / z calcd for C 18 H 17 N3O[M+H] + ,292.1450;found,292.1456.

[0059] (2) Preparation of 5-((4-piperidone)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I3)

[0060] Referring to the method described for complex I1, compound 5c was used instead of compound 5a to react with compound 7 to obtain complex I3 as an orange-red solid (yield: 67%). 1 H NMR(400MHz,DMSO-d6)δ9.08(d,J=8.5Hz,1H,ArH),8.91–8.75(m,5H,5ArH),8.42–8.33(m,1H,ArH),8.26–8.09(m,6H,6ArH), 7.97–7.82(m,4H,4ArH),7.59(m,4H,4ArH),7.46–7.33(m,2H,2ArH),4.27(m,2H,CH2),2.93(s,4H,2CH2),2.42(s,4H,2CH2). 13 C NMR(101MHz,DMSO-d6)δ157.2,157.0,152.5,151.9,151.8,147.8,138.4,138.3,1 37.1,135.0,130.6,130.2,128.3,128.2,127.0,126.5,124.9,124.8,57.7,52.9.

[0061] Example 4: Preparation of 5-((4-hydroxypiperidinyl)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I4)

[0062] (1) Preparation of 5-((4-hydroxypiperidinyl)-N-methylene)-1,10-phenanthroline (5d)

[0063] Referring to the method described for compound 5a, 4-hydroxypiperidine was used instead of piperidine to react with compound 4 to obtain compound 5d (0.966 g, 75%) as a light yellow solid. 1 H NMR (400MHz, CDCl3) δ9.07(m,2H,2ArH),8.69(dd,J=8.4,1.7Hz,1H,ArH),8.10(dd,J=8.1,1.8Hz,1H,ArH),7.61–7.50(m,3H,CH=C,2ArH) ,3.84(s,2H,CH2),3.67(m,1H,CH),2.75(d,J=11.8Hz,2H,CH2),2.18(m,2H,CH2),1.97–1.70(m,3H,OH,CH2),1.50(d,J=9.4Hz,2H,CH2). 13 C NMR(101MHz, CDCl3)δ150.0,149.9,146.4,146.1,135.7,133.7,128.4,127.9,126.6,123.1,122.6,61.0,51.2,34.4.HRMS(ESI)m / zcalcd for C 18 H 19 N3O[M+H] + ,294.1606;found,294.1618.

[0064] (2) Preparation of 5-((4-hydroxypiperidinyl)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I4)

[0065] Referring to the method described for complex I1, compound 5d was used instead of compound 5a to react with compound 7 to obtain complex I4 as an orange-red solid (yield: 64%). 1H NMR(400MHz, DMSO-d6)δ9.07(s,1H,ArH),8.78(dd,J=18.2,8.2Hz,6H,6ArH),8.21–7.96(m,6H,6ArH),7.76(m,4H,4ArH),7.5 0(m,4H,4ArH),7.28(m,2H,ArH),4.96(s,1H,OH),2.73(d,J=65.1Hz,3H,CH2,CH),2.43(s,2H,CH2),1.98–1.07(m,6H,3CH2). 13 C NMR (101MHz, DMSO-d6) δ157.27,157.05,151.92,147.77,138.43,138.32,128.34,128.25,124.95,124.85.

[0066] Example 5: Preparation of 5-((4-piperidinylpiperidine)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I5)

[0067] (1) Preparation of 5-((4-piperidinylpiperidine)-N-methylene)-1,10-phenanthroline (5e)

[0068] Referring to the method described for compound 5a, 4-piperidinylpiperidine was used instead of piperidine to react with compound 4 to obtain compound 5e as a light yellow solid (yield: 74%). 1 H NMR (400MHz, CDCl3) δ9.16(m,2H,2ArH),8.78(dd,J=8.4,1.7Hz,1H,ArH),8.18(dd,J=8.1,1.8Hz,1H,ArH),7.67(s,1H,ArH),7.61(m,2H,2ArH),3. 90(s,2H,CH2),3.00(m,2H,CH2),2.52(m,4H,2CH2),2.31(m,1H,CH),2.08 (m,2H,CH2),1.80(m,2H,CH2),1.64–1.49(m,6H,3CH3),1.43(m,2H,CH2). 13 C NMR (101MHz, CDCl3) δ150.0,149.9,146.4,146.1,135.6,133.7,133.4,128.4,12 7.9,126.5,123.0,122.6,62.7,61.1,53.5,50.2,27.9,26.0,24.5.HRMS(ESI)m / z calcd for C 23 H 28N4[M+H] + ,361.2392;found,361.2398.

[0069] (2) Preparation of 5-((4-piperidinylpiperidine)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I5)

[0070] Referring to the method described for complex I1, compound 5e was used instead of compound 5a to react with compound 7 to obtain complex I5 as an orange-red solid (yield: 71%). 1 H NMR(400MHz,DMSO-d6)δ8.96–8.74(m,6H,6ArH),8.23–8.01(m,7H,2ArH),7.85–7.72(m,4H,4ArH),7.59–7.39(m,4H,4ArH ),7.34–7.19(m,2H,2ArH),3.14(s,2H,CH2),2.43(s,6H,3CH2),2.31–2.00(m,3H,CH2,CH),1.66(d,J=67.8Hz,10H,5CH2). 13 C NMR(101MHz,DMSO-d6)δ157.2,157.0,152.4,152.3,151.9,151.8,147.8,147.2,138.4,138.2,137.0,136.0,1 35.0,130.6,130.2,128.3,128.2,127.0,126.4,124.9,124.8,63.1,58.8,52.3,51.9,49.6,26.2,23.4,21.9.

[0071] Example 6: Preparation of 5-((N-methylpiperazine)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I6)

[0072] (1) Preparation of 5-((N-methylpiperazine)-N-methylene)-1,10-phenanthroline (5f)

[0073] Referring to the method described for compound 5a, N-methylpiperazine was used instead of piperidine to react with compound 4 to obtain compound 5f (0.840 g, 72%) as a light yellow oil. 1H NMR (400MHz, CDCl3) δ9.09 (m, 2H, 2ArH), 8.70 (dd, J=8.3, 1.8Hz, 1H, ArH), 8.12 (dd, J=8.1, 1.8Hz, 1 H,ArH),7.62(s,1H,ArH),7.55(m,2H,2ArH),3.88(s,2H,CH2),2.38(s,8H,4CH2),2.22(s,3H,CH3). 13 C NMR (101MHz, CDCl3) δ150.1,149.9,146.4,146.2,135.6,133.6,132.9,128.4,127.8,126.9,123.1,122.6,61.1,55.0,53.0,45.9.HRMS (ESI) m / z calcd for C 18 H 20 N4[M+H] + ,293.1766;found,293.1763.

[0074] (2) Preparation of 5-((N-methylpiperazine)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I6)

[0075] Referring to the method described for complex I1, compound 5f was used instead of compound 5a to react with compound 7 to obtain complex I6 as an orange-red solid (yield: 67%). 1 H NMR (400MHz, DMSO-d6) δ8.97(d,J=8.5Hz,1H,ArH),8.90–8.79(m,4H,4ArH),8.76–8.67(m,1H,ArH),8.31(s,1H,ArH),8.22(m,2H,2ArH),8.10(m,4H ,4ArH),7.90–7.81(m,4H,4ArH),7.62–7.52(m,4H,4ArH),7.36(m,2H,2Ar H),4.25–4.07(m,2H,CH2),3.10(d,J=58.7Hz,8H,4CH2),2.78(s,3H,CH3). 13 C NMR(101MHz,DMSO-d6)δ157.2,157.0,152.4,151.8,138.3,137.0,135.2, 130.5,130.2,128.62,128.3,127.0,126.4,124.9,58.2,53.1,50.0,42.7.

[0076] Example 7: Preparation of 5-((N-hydroxyethylpiperazine)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I7)

[0077] (1) Preparation of 5-((N-hydroxyethylpiperazine)-N-methylene)-1,10-phenanthroline (5 g)

[0078] Referring to the method described for compound 5a, N-hydroxyethylpiperazine was used instead of piperidine to react with compound 4 to obtain compound 5g (0.992 g, 77%) as a light yellow oil. 1 H NMR (400MHz, CDCl3) δ9.17(m,2H,2ArH),8.77(dd,J=8.4,1.7Hz,1H,ArH),8.21(dd,J=8.1,1.8Hz,1H,ArH ),7.70(s,1H,ArH),7.63(m,2H,2ArH),3.96(s,2H,CH2),3.62(m,2H,CH2,OH),2.59–2.43(m,10H,5CH2). 13 C NMR (101MHz, CDCl3) δ150.1,149.9,146.4,146.2,135.7,133.7,132.9,128 .4,127.8,126.9,123.1,122.6,61.1,59.2,57.7,53.2,52.8.HRMS(ESI)m / z calcd for C 19 H 22 N4O[M+H] + ,323.1872;found,323.1874.

[0079] (2) Preparation of 5-((N-hydroxyethylpiperazine)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I7)

[0080] Referring to the method described for complex I1, compound 5g was used instead of compound 5a to react with compound 7 to obtain complex I7 as an orange-red solid (yield: 69%). 1 H NMR(400MHz,DMSO-d6)δ9.06–8.71(m,6H,6ArH),8.38–8.08(m,7H,7ArH),7.86(m,4H,4ArH),7.57(m,4H,4ArH),7 .37(m,2H,2ArH),4.17–4.06(m,1H,CH),3.55(s,2H,CH2),3.44(s,2H,CH2),2.60(s,6H,3CH2),1.23(s,4H,2CH2).13 C NMR(101MHz,DMSO-d6)δ157.2,157.0,152.4,151.8,147.8,147.2,138.4,137 .0,135.0,130.7,130.2,128.2,127.0,126.4,124.9,55.3,29.5,22.5,14.4.

[0081] Example 8: Preparation of 5-((N-benzylpiperazine)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I8)

[0082] (1) Preparation of 5-((N-benzylpiperazine)-N-methylene)-1,10-phenanthroline (5h)

[0083] Referring to the method described for compound 5a, N-benzylpiperazine was used instead of piperidine to react with compound 4 to obtain compound 5h (1.059 g, 72%) as a light yellow solid. 1 H NMR (400MHz, CDCl3) δ9.17(m,2H,2ArH),8.77(dd,J=8.4,1.8Hz,1H,ArH),8.20(dd,J=8.1,1.8Hz,1H,ArH),7.70(s,1H, ArH),7.63(m,2H,2ArH),7.35–7.29(m,5H,5ArH),3.96(s,2H,CH2),3.53(s,2H,CH2),2.65(dd,J=6.2,4.7Hz,8H,4CH2). 13 C NMR (101MHz, CDCl3) δ150.1,150.0,146.4,146.2,135.7,133.7,133.0,129.3,1 29.0,128.4,128.2,127.9,127.2,126.9,123.1,122.6,62.9,61.1,53.0,53.0.

[0084] (2) Preparation of 5-((N-benzylpiperazine)-N-methylene)-1,10-phenanthroline-bis(2,2'-bipyridine)ruthenium di(hexafluorophosphate) salt (I8)

[0085] Referring to the method described for complex I1, compound 5h was used instead of compound 5a to react with compound 7 to obtain complex I8 as an orange-red solid (yield: 62%). 1H NMR (400MHz, DMSO-d6) δ8.99 (d, J=8.5Hz, 1H, 2ArH), 8.91–8.81 (m, 4H, 4ArH), 8.73(d,J=8.2Hz,1H,ArH),8.31(s,1H,ArH),8.22(m,3H,3ArH),8.15–8.06(m ,4H,4ArH),7.90–7.80(m,4H,4ArH),7.63–7.53(m,4H,4ArH),7.45(s,4H,2CH 2),7.37(m,2H,CH2),4.35–3.90(m,4H,2CH2),2.82(d,J=185.9Hz,8H,4CH2). 13 C NMR (101MHz, DMSO-d6) δ157.2,152.4,151.8,147.8,147.3,138.3,137.0,135.0,130.6,130.2,129.2,128.3,127.0,126.5,124.9,65.3,60.2.

[0086] Example 9: Measurement of UV absorption and fluorescence emission spectra of the novel bipyridine / phenanthroline ruthenium complex of the present invention

[0087] (1) Ultraviolet absorption spectrum of bipyridine / phenanthroline ruthenium complex

[0088] Using methanol as solvent, the compounds of the present invention were prepared into 1-10 μM sample solutions, 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. The UV absorption spectra of the compounds in methanol solutions were characterized as follows: Figure 1 As shown. Figure 1 It can be found that the absorption spectrum of the bipyridine / phenanthroline ruthenium complex of the present invention has a very strong absorption peak in the range of 400-500nm, which may be caused by the dπ-π* electronic transition (MLCT) between the metal Ru and the ligand biq.

[0089] (2) Fluorescence emission spectra of bipyridine / phenanthroline ruthenium complexes

[0090] The compound of the present invention was prepared into a 10 μM sample solution using methanol as solvent, and 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 scanning emission spectrum was 470-900 nm, and the scanning speed was 1.0 nm / s. The fluorescence emission spectra of the compounds in methanol solution were characterized as follows: Figure 2As shown in the figure, it can be found that the maximum wavelength of the fluorescence emission spectrum of the novel bipyridine / phenanthroline ruthenium complex of the present invention appears in the range of 600-680nm.

[0091] Example 10: Determination of the singlet oxygen quantum yield of the novel bipyridine / phenanthroline ruthenium complex of the present invention

[0092] 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 compound to be tested, I1-I8 and the control [Ru(bpy)3] 2+ The absorbance values ​​at 520 nm were adjusted to below 0.1. Then, the transparent side of the quartz cell was exposed to a laser (40 mW) with a wavelength of 520 nm for a total of 5 minutes. The absorption spectrum was recorded at different time periods during the 5 minutes. The scanning wavelength range was 300-600 nm, and the scanning speed was 1.0 nm / s.

[0093] With the known singlet oxygen quantum yield of [Ru(bpy)3] 2+ As a standard, the singlet oxygen quantum yield was calculated by the following formula:

[0094] Φ T =Φ X (S T F X / SxF T )

[0095] Φ: singlet oxygen quantum yield; S: the slope of the straight line with irradiation time as x-axis and the UV absorbance of DPBF at 412nm after the corresponding irradiation time as y-axis; F: absorption correction factor of unknown sample and reference standard, F = 1-10 -OD OD represents the ultraviolet absorption value of the compound under the irradiation wavelength; X and T represent the reference standard and the unknown sample, respectively. The results are shown in Table 1. We found from Table 1 that most of the novel bipyridine / phenanthroline ruthenium complexes of the present invention have a high singlet oxygen quantum yield.

[0096] Table 1 Singlet oxygen quantum yield of the novel bipyridine / phenanthroline ruthenium complex of the present invention

[0097]

[0098] Example 11: Cytotoxicity test of the compounds of the present invention

[0099] The phototoxicity of the compounds of the present invention on human colon cancer cells HT29, human lung cancer cells A549, and human breast cancer cells Mcf-7 was evaluated by using the MTT colorimetric method in vitro. In the dark toxicity test, a bottle of cells in good exponential growth phase was first taken, and 0.25% trypsin was added to digest the cells to make them fall off. The mixture was prepared into a solution containing 2×10 4 ~4×10 4 Cell suspension. Take the cell suspension and inoculate it on a 96-well plate, 180 μL per well, and culture it in a constant temperature CO2 incubator for 24 hours. Change the liquid, add the test compound (the compound is dissolved in DMSO and diluted with PBS, the test compound concentration is 20 μM) in the dark, 20 μL per well, and continue to culture in the dark for 48 hours. Add MTT to the 96-well plate, 20 μL per well, and react in the incubator for 4 hours. Aspirate the supernatant, add 150 μL DMSO to each well, and shake on a plate shaker for 5 minutes. Use an enzyme-linked immunosorbent assay to measure the absorbance of each well at a wavelength of 570 nm to calculate the cell survival rate.

[0100] 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 520nm laser (15mW / cm 2 ) After irradiation for 10 min, replace with fresh complete culture medium and continue culturing in the incubator for 24 h. Then, add 20 μL of MTT solution to each well and calculate the cell survival rate.

[0101] The experimental results show that the compounds of the present invention have less dark toxicity to tumor cells. However, after specific laser irradiation, the cell inhibition rate is greatly increased, which indicates that the compounds of the present invention have significant photodynamic therapy effects on tumor cells (Table 2).

[0102] Table 2 Survival rate of some compounds of the present invention on human cancer cells (20 μM)

[0103]

[0104] ND: Not tested.

[0105] Example 12: Cell fluorescence imaging experiment using confocal microscopy

[0106] Confocal microscopy was used to perform cell fluorescence imaging and mitochondrial localization experiments. Human lung cancer cells A549 or human cervical cancer Hela cells were cultured in a laser confocal dish with DEME culture medium for 12 to 24 hours, and 5 to 20 μM of the test compound was added. The cells were placed in a cell culture incubator at 37°C and 5% CO2 for half an hour. Then, the cells were washed three times with a phosphate buffer solution of pH = 7.4, and then 1 to 5 μM of the mitochondrial dye MitoTracker red solution was added and incubated for another half an hour, and then washed three times with a phosphate buffer solution of pH = 7.4. The incubated cells were placed on the stage of a confocal microscope for confocal fluorescence imaging, and the excitation wavelength of the test compound was set to: λex = 400-500nm, and the emission wavelength λem = 600-680nm.

[0107] The results showed that the compounds of the present invention can clearly image the fluorescence of human tumor cells and overlap well with the fluorescence imaging of mitochondrial probes, indicating that the compounds of the present invention can effectively target and localize in mitochondria in tumor cells ( Figure 3 and Figure 4 ).in Figure 3 This is the co-localization imaging of the compound I3 of the present invention and the mitochondrial probe in Hela cells. Figure 4 This is the co-localization imaging of the compound I8 of the present invention and the mitochondrial probe in A549 cells.

Claims

1. A bipyridine / phenanthroline ruthenium complex or a pharmaceutically acceptable salt thereof, characterized in that: The bipyridine / phenanthroline ruthenium complex has a structure shown in the following general formula I: Among them, R is selected from One of the following; Y - It stands for halide or hexafluorophosphate ion.

2. The bipyridine / phenanthroline ruthenium complex or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R is selected from One of them.

3. The bipyridine / phenanthroline ruthenium complex or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R and Y are selected from the following combinations: Y=I; or Y=I; or Y = PF6; or Y = PF6; or Y = PF6; or Y = PF6; or Y = PF6; or Y=PF6.

4. A method for preparing a bipyridine / phenanthroline ruthenium complex, characterized in that: The synthetic route of the preparation method is shown below: Said Selected from Y - represents halide anion or hexafluorophosphate anion; The preparation method comprises the following steps: S1. The methyl group at the 5-position of compound 1 is oxidized to an aldehyde group by selenium dioxide to obtain compound 2, and then compound 2 is directly reduced by sodium borohydride to obtain compound 3, and compound 3 is then subjected to a substitution reaction with phosphorus tribromide to obtain compound 4, and compound 4 is reacted with a nitrogen heterocycle to obtain compound 5; S2. Compound 6 undergoes coordination reaction with hydrated ruthenium trichloride to obtain compound 7; S3. Compound 5 and compound 7 undergo coordination reaction in a mixed solution of water and methanol to generate bipyridine / phenanthroline ruthenium complex I.

5. Use of the bipyridine / phenanthroline ruthenium complex or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a malignant tumor diagnostic reagent or a malignant tumor therapeutic drug, wherein the malignant tumor is one of breast cancer, colon cancer and lung cancer.

6. The use according to claim 5, characterized in that: The malignant tumor diagnostic reagent is a malignant tumor fluorescence imaging reagent.

7. The use according to claim 6, characterized in that: The malignant tumor fluorescent imaging reagent is a near-infrared fluorescent imaging reagent with mitochondrial targeting.

8. The use according to claim 5, characterized in that: The malignant tumor therapeutic drug is a drug used for photodynamic therapy of malignant tumors.

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