A tripolybipyridine ruthenium complex and its preparation method and application

Abstract

The present invention belongs to the technical field of drug preparation, and discloses a trimeric bipyridine ruthenium complex and a preparation method and application thereof. The trimeric bipyridine ruthenium complex has a structure shown in general formula I: #imgabs0# The trimeric bipyridine ruthenium complex provided by the present invention has low dark toxicity and can target tumor cell mitochondrial fluorescence imaging. At the same time, the compound of the present invention has a significant photodynamic therapy effect, can be used in the preparation of drugs for in vitro and in vivo tumor fluorescence imaging, and generates singlet oxygen after specific laser irradiation, thereby effectively killing tumor cells, and can be used for the preparation of tumor diagnosis and / or treatment drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and specifically relates to a tripolybipyridine ruthenium complex and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy refers to the use of non-toxic photosensitizers exposed to light of a specific wavelength. The photosensitizers transfer photon energy to produce reactive oxygen species, which kill cancer cells through reactive oxygen species. Photodynamic therapy is mainly divided into type I and type II photodynamic therapy. Among them, the most widely studied photodynamic therapy is type II photodynamic therapy, which mainly relies on the production of singlet oxygen to kill target cells. Singlet oxygen can efficiently oxidize unsaturated fatty acids, nucleic acids, proteins, and mitochondrial membranes, induce the expression of tumor cell apoptosis genes, or directly destroy the normal life activities of cancer cells to cause them to necrotize, thereby achieving the purpose of killing tumor cells.

[0003] Photodynamic therapy, as a new method for treating cancer, has the advantages of low side effects and high targeting. In the past few years, many different photosensitizers have been reported, such as porphyrin, fluoroboron dipyrrole dyes and cyanine dyes, but their photodynamic therapeutic effects are not significant. Metal compounds are another research direction for the development of new photosensitizers. Metal compound photosensitizers based on Pt(II), Pt(IV), Ru(II) and others have been reported, but there are still defects such as poor stability of the complex and weak photodynamic therapeutic activity, which deserves further research and development. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a triplylpyridine ruthenium complex and its preparation method and application, wherein the triplylpyridine ruthenium complex has a large Stokes shift, good photostability and singlet oxygen ( 1 O2) has the characteristics of high quantum yield and can be used in the preparation of tumor diagnostic drugs or drugs for photodynamic therapy of malignant tumors.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A trimeric bipyridine ruthenium complex, the complex having a structure shown in general formula I:

[0007]

[0008] Among them, R is selected from

[0009] Preferably, R is selected from

[0010] The present invention also provides a method for preparing a trimeric bipyridine ruthenium complex, comprising the following steps:

[0011] S1. The methyl group at the 4'-position of 4,4'-dimethyl-2,2'-bipyridine (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 various nitrogen heterocycles to obtain compound (5);

[0012] S2. Compound (5) and hydrated ruthenium trichloride (6) undergo coordination reaction in an ethylene glycol solution containing ammonium hexafluorophosphate to obtain tripyridine ruthenium complex I;

[0013] The synthetic route is as follows:

[0014]

[0015] The R is selected from One of them.

[0016] The present invention also provides the use of the above-mentioned ruthenium tris-bipyridine complex or a pharmaceutically acceptable salt thereof in the preparation of tumor diagnostic drugs.

[0017] Furthermore, the diagnostic drug is a drug that can target tumor cell mitochondria to achieve fluorescent imaging of tumor tissues or cells.

[0018] The present invention also provides the use of the above-mentioned ruthenium tris-bipyridine complex or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating malignant tumors.

[0019] Furthermore, the drug for treating malignant tumors is a drug for photodynamic therapy of malignant tumors.

[0020] Furthermore, the drug is a drug that can generate singlet oxygen after laser irradiation to effectively kill tumor cells.

[0021] Furthermore, the malignant tumor is one of breast cancer, colon cancer, lung cancer and cervical cancer.

[0022] Compared with the prior art, the triplylpyridyl ruthenium complex of the present invention has near-infrared fluorescence and two-photon fluorescence characteristics, low tissue background fluorescence interference, large Stokes shift, good photostability and singlet oxygen ( 1O2) quantum yield, can target tumor cell mitochondria, exert the characteristics of tumor cell fluorescence imaging, can accurately image and diagnose tumor cells in vivo or in vitro, and can be used in the preparation of tumor diagnostic drugs. At the same time, after laser irradiation, it effectively produces a high level of singlet oxygen, effectively kills a variety of tumor cells, and has the advantage of low dark toxicity, and has broad application prospects in the preparation of tumor diagnosis and / or therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The ultraviolet absorption spectra of the compounds obtained in Examples 1-5 of the present invention;

[0024] Figure 2 The fluorescence emission spectra of the compounds obtained in Examples 1-5 of the present invention;

[0025] Figure 3 The compound I1 obtained in Example 1 of the present invention is co-localized with the mitochondrial probe in 4T1 cells;

[0026] Figure 4 Compound I4 obtained in Example 4 of the present invention was co-localized with a mitochondrial probe in A549 cells. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1 Preparation of tris(4-methyl-2,2'-bipyridyl-4'-((4-methylpiperidine)-N-methylene))ruthenium di(hexafluorophosphate) salt (I1)

[0029] Step 1: Preparation of 4-methyl-2,2'-bipyridine-4'-carbaldehyde (Compound 2)

[0030] 4,4'-Dimethyl-2,2'-bipyridine (Compound 1) (10mmol, 1.84g) and SeO2 (15mmol, 0.56g) were dissolved in 1,4-dioxane (25mL), and the mixture was heated under nitrogen protection and refluxed at 110°C for 24h. The reaction was monitored by thin layer chromatography to see if it was complete. After the reaction was completed, the mixture was cooled to room temperature, the solvent was removed by vacuum vortexing, and the crude product was purified by column chromatography (ammonia MeOH: DCM = 1:25) to obtain compound 2 (1.247g) as a white solid with a yield of 63%. 1H 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.

[0031] Step 2: Preparation of 4-methyl-2,2'-bipyridine-4'-methanol (Compound 3)

[0032] Compound 2 (8 mmol, 1.585 g) was dissolved in CH3CH2OH (20 mL), stirred for 5 min, and then NaBH4 (16 mmol, 0.605 g) was added, and the mixture was stirred for 1 h. The reaction was monitored by thin layer chromatography to determine whether it was complete. After the reaction was complete, 1 to 2 drops of water were added to quench the reaction, and then the solvent was dried under reduced pressure, and saturated brine (30 mL) was added, and the mixture was extracted with DCM (20 mL×3). The organic layer was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure to obtain compound 3 (1.361 g) as a light yellow solid, with a yield of 86%. 1 HNMR(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.

[0033] Step 3: Preparation of 4-methyl-2,2'-bipyridyl-4'-bromomethyl (Compound 4)

[0034] Compound 3 (6mmol, 1.2g) 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 solution temperature was restored to room temperature, and then stirred for 10h. The reaction was monitored by thin layer chromatography to see if it was complete. After the reaction was completed, the pH was adjusted to 7 with 2mol / L NaHCO3 solution, and then extracted with DCM (20mL×3). The organic layer was dried with anhydrous Na2SO4 and the solvent was removed under reduced pressure to obtain compound 4 (1.258g) as a light yellow solid with a yield of 87%. 1 HNMR (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.

[0035] Step 4: Preparation of 4-methyl-2,2'-bipyridine-4'-((4-methylpiperidinyl)-N-methylene) (Compound 5a)

[0036] Compound 4 (4mmol, 1.048g), 4-methylpiperidine (4mmol, 0.372g) 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℃ 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 and concentrated under reduced pressure. The crude product was purified by column chromatography (ammonia MeOH: DCM = 1:15) to obtain the target compound 5a (0.933g) as a light yellow solid with a yield of 83%. 1H 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 / z calcd for C 18 H 23 N3[M+H] + ,282.1970;found,282.1974.

[0037] Step 5: Preparation of tris(4-methyl-2,2'-bipyridyl-4'-((4-methylpiperidinyl)-N-methylene))ruthenium di(hexafluorophosphate) salt (I1)

[0038] Ruthenium trichloride hydrate (0.5mmol, 0.103g) and compound 5a (1.5mmol, 0.421g) were dissolved in ethylene glycol (5mL), and then heated under nitrogen protection at 120°C for 6h. TCL was used to detect the reaction. After the reaction was completed, after cooling to room temperature, three distilled water (40mL) was added to the mixture, followed by ammonium hexafluorophosphate (4mmol, 0.744g) and stirred for 10min. After stirring, the solution was filtered out using filter paper, leaving the solid. The solid was washed with three distilled water (5mL×3) and chloroform (10mL×3), and then the solid was vacuum dried. The crude product was purified by column chromatography (ammonia MeOH:DCM=1:20) to obtain the target compound I1 (0.321g) as an orange-red solid with a yield of 52%. 1H NMR(400MHz,DMSO-d6)δ8.68(d,J=16.4Hz,6H),7.71–7.27(m,12H,12ArH),3.65(s,6H,3CH2),2.7 7(s,6H,2CH3),2.01(s,6H,2CH3),1.59(s,6H,2CH3),1.37–1.09(m,12H,6CH2),0.90(s,8H,4CH2). 13 C NMR (101MHz, DMSO) δ156.9,156.5,151.3,150.7,149.9,129.0,125.5,124.2,53.8,34.2,30.4,22.2,21.1.

[0039] Example 2 Preparation of tris(4-methyl-2,2'-bipyridine-4'-((4-piperidinylpiperidine)-N-methylene)ruthenium di(hexafluorophosphate) salt (I2)

[0040] Preparation of 4-methyl-2,2'-bipyridine-4'-((4-piperidinylpiperidine)-N-methylene) (Compound 5b)

[0041] Referring to the method described for compound 5a, 4-piperidinylpiperidine was used instead of 4-methylpiperidine to react with compound 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 C 22 H 30 N4[M+H]+ ,351.2549;found,351.2553.

[0042] Preparation of tris(4-methyl-2,2'-bipyridyl-4'-((4-piperidinylpiperidinyl)-N-methylene)ruthenium di(hexafluorophosphate) salt (I2)

[0043] Referring to the method described for compound I1, compound 5b was used instead of compound 5a to react with hydrated ruthenium trichloride to obtain the target compound I2 (0.396 g, 55%) as an orange-red solid. 1 HNMR(400MHz,DMSO-d6)δ8.98(d,J=8.6Hz,5H,5ArH),8.77–8.64(m,5H,5ArH),8.32(s,3H,3ArH),8.06(m,5H,5ArH),4.41–3.81(m,19H,5CH2,3CH3),3.7 0(s,4H,2CH2),3.18(s,2H,CH2),2.93(d,J=25.7Hz,6H,3CH2),2.52(d,J=7. 6Hz, 8H, 4CH2), 2.26–1.52 (m, 27H, 12CH2, 3CH), 1.33 (d, J=76.1Hz, 6H, 3CH2). 13 C NMR(101MHz,DMSO)δ152.88,152.70,148.16,147.59,136.96,136.45,134.93, 130.70,130.26,127.93,126.85,126.40,106.80,64.11,59.04,51.59,34.93.

[0044] Example 3 Preparation of tris(4-methyl-2,2'-bipyridyl-4'-((4-piperidinone)-N-methylene)ruthenium di(hexafluorophosphate) salt (I3)

[0045] Preparation of 4-methyl-2,2'-bipyridine-4'-((4-piperidinone)-N-methylene) (Compound 5c)

[0046] 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.

[0047] Preparation of tris(4-methyl-2,2'-bipyridyl-4'-((4-piperidinone)-N-methylene)ruthenium di(hexafluorophosphate) salt (Compound I3)

[0048] Referring to the method described for compound I1, compound 5c was used instead of compound 5a to react with hydrated ruthenium trichloride to obtain the target compound I3 (0.327 g, 53%) as an orange-red solid. 1 H NMR(400MHz, DMSO-d6)δ8.62(dd,J=20.9,5.3Hz,6H,6ArH),7.61–7.37(m,9H,9ArH),7.28( m,3H,3ArH),3.79(s,12H,6CH2),3.63(s,6H,3CH2),2.41(s,9H,3CH3),1.58(m,12H,6CH2). 13 C NMR (101MHz, DMSO) δ156.90,156.53,151.27,150.82,149.95,128.99,127.49,125.55,124.03,106.63,64.09,60.21,55.38,51.48,34.86,21.13.

[0049] Example 4 Preparation of tris(4-methyl-2,2'-bipyridyl-4'-((N-benzylpiperazine)-N-methylene)ruthenium di(hexafluorophosphate) salt (I4)

[0050] Preparation of 4-methyl-2,2'-bipyridine-4'-((N-benzylpiperazine)-N-methylene) (Compound 5d)

[0051] Referring to the method described for compound 5a, N-benzylpiperazine was used instead of 4-methylpiperidine to react with compound 4 to obtain compound 5d (1.060 g, 74%) as a yellow solid. 1 H 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.

[0052] Preparation of tris(4-methyl-2,2'-bipyridyl-4'-((N-benzylpiperazine)-N-methylene)ruthenium di(hexafluorophosphate) salt (I4)

[0053] Referring to the method described for compound I1, compound 5d was used instead of compound 5a to react with hydrated ruthenium trichloride to obtain compound I4 (0.373 g, 51%) as an orange-red solid. 1 H NMR (400MHz, DMSO-d6) δ8.67(d,J=16.5Hz,6H,6ArH),7.68–7.21(m,27H,27ArH),3.67(s,6H,3CH2),3.47(s,10H,5CH2),2.42(s,29H,3CH3,10CH2). 13C NMR (101MHz, DMSO) δ156.8,156.5,151.2,150.7,150.3,149.9,129.3,129.0,128.6,127.6,127.4,125.5,124.2,62.4,60.6,53.2,52.8,21.1.

[0054] Example 5 Preparation of tris(4-methyl-2,2'-bipyridine-4'-((N-phenylpiperazine)-N-methylene)ruthenium di(hexafluorophosphate) (I5) Preparation of 4-methyl-2,2'-bipyridine-4'-((N-phenylpiperazine)-N-methylene) (5e)

[0055] Referring to the method described for compound 5a, N-phenylpiperazine was used instead of 4-methylpiperidine to react with 4 to obtain compound 5e (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 calcd for C 22 H 24 N4[M+H] + ,345.2079;found,345.2072.

[0056] Preparation of tris(4-methyl-2,2'-bipyridyl-4'-((N-phenylpiperazine)-N-methylene)ruthenium di(hexafluorophosphate) salt (I5)

[0057] Referring to the method described for compound I1, compound 5e was used instead of compound 5a to react with hydrated ruthenium trichloride to obtain compound I5 (0.405 g, 57%) as an orange-red solid.1 H NMR(400MHz,DMSO-d6)δ8.74(d,J=7.1Hz,6H,6ArH),7.75–7.48(m,9H,9ArH),7.38–7.15(m,8H,8ArH),7 .00–6.72(m,8H,8ArH),3.76(s,4H,2CH2),3.34(s,15H,6CH2,CH3),2.55(d,J=36.7Hz,23H,3CH3,7CH2). 13 C NMR (101MHz, DMSO) δ156.9,156.5,151.3,150.7,150.2,149.9,129.4,129.0,127.7,125.6,124.3,119.4,115.8,60.6,53.2,48.6,21.1.

[0058] Test Example 1 Ultraviolet absorption and fluorescence emission spectra of the compounds prepared in Examples 1-5 of the present invention

[0059] (1) Ultraviolet absorption spectrum of the compound

[0060] The test compound was prepared into a 10 μM sample solution using methanol or ethanol 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 As shown, the absorption spectrum of the compound of the present invention has a very strong absorption peak in the range of 400-500 nm, which may be caused by the dπ-π* electronic transition (MLCT) between metal Ru and ligand phen.

[0061] (2) Fluorescence emission spectra of compounds

[0062] The test compound was prepared into a 10 μM sample solution using methanol or ethanol as solvent, and the fluorescence emission spectrum of the compound was recorded using a fluorescence spectrophotometer with an excitation wavelength of 450 nm. The wavelength range of the scanning emission spectrum was 470-900 nm, and the scanning speed was 1.0 nm / s. Figure 2 As shown, the maximum value of the fluorescence emission spectrum of the compound of the present invention corresponds to the wavelength in the range.

[0063] Test Example 2 Determination of Singlet Oxygen Quantum Yield of the Compounds of the Invention

[0064] We selected DPBF as the singlet oxygen scavenger and methanol as the solvent. The absorbance of DPBF at 412 nm of the test compound was adjusted to about 1.0; then, in order to reduce the singlet oxygen quenching of the control and the test compound itself, I1, I2, I3, I4, I5 and the control Ru(bpy)3 2+ The absorbance value at 520nm was adjusted to below 0.1. Then, the laser (40mW) at 520nm was irradiated for 5min. The absorption spectrum was recorded at different time periods during the 5min. The scanning wavelength range was 300-600nm and the scanning speed was 1.0nm / s. With the singlet oxygen quantum yield of [Ru(bpy)3] 2+ As a standard, the singlet oxygen quantum yield was calculated by the following formula:

[0065] Φ T =Φ X (S T F X / SxF T )

[0066] 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 UV absorption value of the compound at the irradiation wavelength; X and T represent the reference standard and the unknown sample, respectively. After calculation, the results are shown in Table 1. Most of the complexes of the present invention have a higher singlet oxygen quantum yield, which is significantly higher than the control Ru(bpy)3 2+ .

[0067] Table 1 Singlet oxygen quantum yield of some compounds of the present invention

[0068]

[0069] Test Example 3 Cytotoxicity Test of the Compounds of the Invention

[0070] The phototoxicity of the compounds of the present invention on human colon cancer cells HT29, human lung cancer cells A549, human breast cancer cells Mcf-7 and human cervical cancer cells Hela was evaluated by using the MTT colorimetric method in vitro toxicity test. 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 4Take 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 12.5 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.

[0071] 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.

[0072] According to the experimental results in Table 2, it was found that the compounds of the present invention had less dark toxicity to tumor cells. After specific laser irradiation, the cell death rate increased greatly, which indicated that the compounds of the present invention had significant photodynamic therapy effects on tumor cells.

[0073] Table 2 Survival rate of some compounds of the present invention on human cancer cells (12.5 μM)

[0074]

[0075] ND: Not tested.

[0076] Test Example 4: Cell fluorescence imaging experiment using confocal microscope

[0077] Confocal microscopy was used to perform cell fluorescence imaging and mitochondrial localization experiments. Lung cancer A549 cells or breast cancer 4T1 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 pH = 7.4 phosphate buffer solution, and then 1 μM of the mitochondrial dye MitoTracker red solution was added and incubated for another half an hour, and then washed three times with a pH = 7.4 phosphate buffer solution. 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 = 600-650nm.

[0078] The results showed that the compounds of the present invention can clearly image tumor cells and overlap well with the fluorescence imaging of mitochondrial probes, indicating that the compounds of the present invention can target mitochondria in tumor cells and have significant effects ( Figure 3 and Figure 4 ).in Figure 3 This is the co-localization imaging of the compound I1 of the present invention and the mitochondrial probe in 4T1 cells. Figure 4 This is the co-localization imaging of the compound I4 of the present invention and the mitochondrial probe in A549 cells.

Claims

1. A ruthenium tris(bipyridine) complex, characterized in that: The trimeric bipyridine ruthenium complex has a structure shown in general formula I: Among them, R is selected from One of them.

2. A method for preparing the ruthenium tris-bipyridine complex according to claim 1, characterized in that: The synthetic route of the preparation method is shown below: R is selected from One of; The preparation method comprises the following steps: S1. The methyl group at the 4'-position of 4,4'-dimethyl-2,2'-bipyridine 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 different substituted secondary amines to obtain compound 5; S2. Compound 5 and hydrated ruthenium trichloride 6 undergo coordination reaction in an ethylene glycol solution containing ammonium hexafluorophosphate to obtain tripyridine ruthenium complex I.

3. Use of the tris-bipyridine ruthenium complex or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of tumor diagnostic drugs, wherein the tumor is one of breast cancer, colon cancer, lung cancer and cervical cancer.

4. The use according to claim 3, characterized in that: The tumor diagnostic drug is a drug that can target tumor cell mitochondria to achieve tumor tissue or cell fluorescence imaging.

5. Use of the tris-bipyridine ruthenium complex or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for photodynamic therapy of malignant tumors, wherein the malignant tumor is one of breast cancer, colon cancer, lung cancer and cervical cancer.

6. The use according to claim 5, characterized in that: The drug is a drug that generates singlet oxygen after laser irradiation.

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