A selenium-containing ruthenium complex with radiotherapy sensitization and reversal of radiotherapy tolerance, and its preparation method and application

Abstract

The present invention discloses a selenium-containing ruthenium complex with radiosensitization and reversal of radiotherapy tolerance, as well as a preparation method and application. The selenium-containing ruthenium complex has an inhibitory effect on cervical cancer cells, especially can significantly inhibit cervical cancer cells in combination with X-ray radiotherapy, and can significantly improve radiotherapy tolerance in combination with X-ray radiotherapy, thereby not only being expected to reduce the amount of the selenium-containing ruthenium complex, but also providing a new treatment approach for radiotherapy-tolerant human cervical cancer cells. The preparation method provided by the present invention has the following advantages: the raw materials are cheap and easy to obtain, the synthesis and purification steps are highly operable, and the prepared product has good repeatability and stability.

Description

Technical Field

[0001] The present invention relates to the field of complex preparation, and in particular to a selenium-containing ruthenium complex with radiotherapy sensitization and radiotherapy tolerance reversal functions, and a preparation method and application thereof. Background Art

[0002] In the past decade, radiotherapy has developed rapidly and is widely used in clinical tumor treatment. Studies have shown that about 70% of cancer patients have undergone radiotherapy. However, radiotherapy can easily lead to tumor cells becoming resistant to radiotherapy, which seriously hinders the treatment of the disease. Cervical cancer is a malignant tumor that is common in women. In the treatment of cervical cancer, chemotherapy and radiotherapy are often combined, that is, chemoradiotherapy. Unfortunately, cervical cancer is prone to developing resistance to both chemotherapy drugs and radiotherapy.

[0003] Radiotherapy can cause cancer cell DNA to break and die, and cancer cell DNA cannot repair its own damage. Cancer stem cells can promote cancer cell tolerance to radiotherapy by activating the cell's own DNA damage response system, but the mechanism is unclear. In the treatment of cervical cancer, it is very important to overcome the resistance of tumor cells to radiotherapy. The incidence of cancer is increasing day by day. Although people now have more and more treatment methods for cancer and the number of drugs available is also increasing, most cancers are still incurable and patients have a short survival period. Therefore, any new safe and effective cancer treatment method or a method that can improve the effectiveness of existing treatment methods will have practical significance for the health of cancer patients.

[0004] At present, anti-tumor drugs have been widely used in the treatment of cancer, but these drugs are selective for tumor cells and normal cells. While treating cancer cells, they also affect normal body functions. Therefore, improving drug bioavailability and reducing systemic toxicity are urgent issues to be solved in tumor chemotherapy. It is imperative to develop a tumor delivery system that is safe, low-toxic, tumor-specific, and can efficiently load anti-tumor drugs. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a selenium-containing ruthenium complex having the functions of radiotherapy sensitization and reversal of radiotherapy tolerance.

[0006] Another object of the present invention is to provide a method for preparing the selenium-containing ruthenium complex having the functions of radiotherapy sensitization and reversal of radiotherapy tolerance.

[0007] One object of the present invention is to provide the application of the above-mentioned selenium-containing ruthenium complex having the functions of radiotherapy sensitization and reversal of radiotherapy tolerance.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A selenium-containing ruthenium complex having the functions of radiosensitization and reversal of radiotherapy tolerance, the chemical formula of which is [Ru(POP)(PhenSeN2)Cl]Cl, and the structural formula of which is shown in Formula I:

[0010]

[0011] The method for preparing the selenium-containing ruthenium complex comprises the following steps:

[0012] (1) Synthesis of the precursor tris(triphenylphosphine)ruthenium(II) dichloride

[0013] Weigh ruthenium trichloride hydrate and triphenylphosphine, dissolve them in methanol, stir and reflux for reaction; after the reaction is completed, cool and filter, wash the obtained reddish brown solid with ether, and dry it to obtain tris(triphenylphosphine)ruthenium (II) dichloride;

[0014] (2) Synthesis of Ru(POP) precursor

[0015] Dissolve 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene and tris(triphenylphosphine)ruthenium(II) dichloride obtained in step (1) in acetone, stir and reflux for reaction, cool after the reaction is completed, filter to obtain an orange-yellow solid, wash with ether, then recrystallize and dry to obtain a product precursor Ru(POP);

[0016] (3) Synthesis of ligand o-phenanthroline selenadiazole

[0017] Dissolve 5,6-diamino-1,10-phenanthroline in glacial acetic acid to obtain solution A; dissolve selenium dioxide in distilled water to obtain solution B; mix solution A and solution B, stir to react, then add ammonia water, filter, wash the obtained precipitate with water, and dry; then dissolve it in an organic solvent, evaporate and crystallize naturally, collect the product pink needle-shaped crystals, and obtain the ligand o-phenanthroline selenadiazole;

[0018] (4) Synthesis of the complex [Ru(POP)(PhenSeN2)Cl]Cl

[0019] The Ru(POP) prepared in step (2) and the o-phenanthroline selenadiazole prepared in step (3) are mixed and dissolved in ethylene glycol ethyl ether, and stirred and refluxed for reaction; after the reaction is completed, the mixture is cooled, and any insoluble matter is filtered out, and then ether is added to precipitate a yellow solid, which is filtered and washed with ether to obtain an orange-yellow solid, and after recrystallization, a selenium-containing ruthenium complex having the function of radiotherapy sensitization and reversal of radiotherapy tolerance is obtained.

[0020] The ruthenium trichloride hydrate and the triphenylphosphine in step (1) are preferably mixed in a molar ratio of 1:4 to 7; more preferably in a molar ratio of 1:6.

[0021] The methanol described in step (1) is a reaction medium and does not participate in the reaction. The amount used is preferably sufficient to completely dissolve the reaction components, preferably calculated according to the ratio of (ruthenium trichloride hydrate + triphenylphosphine): methanol = 7g: 40-60mL; more preferably calculated according to the ratio of (ruthenium trichloride hydrate + triphenylphosphine): methanol = 7g: 50mL.

[0022] The temperature of the stirring reflux reaction in step (1) is preferably 60-90°C; more preferably 70-80°C; most preferably 75°C.

[0023] The stirring reflux reaction time in step (1) is 2 to 6 hours, more preferably 4 hours.

[0024] The cooling in step (1) is preferably natural cooling to room temperature.

[0025] The 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene described in step (2) and the tris(triphenylphosphine)ruthenium(II) dichloride are mixed in a molar ratio of 1 to 1.1:1, preferably in a molar ratio of 1:1.

[0026] The acetone described in step (2) is a reaction medium and does not participate in the reaction. The amount of acetone used is preferably sufficient to completely dissolve the reaction components. It is preferably calculated according to the ratio of (4,5-bis(diphenylphosphine)-9,9-dimethyloxanthene + tris(triphenylphosphine)dichloride ruthenium (II)): acetone = 0.72 g: 9-11 mL; more preferably, it is calculated according to the ratio of (4,5-bis(diphenylphosphine)-9,9-dimethyloxanthene + tris(triphenylphosphine)dichloride ruthenium (II)): acetone = 0.72 g: 10 mL.

[0027] The temperature of the stirring reflux reaction in step (2) is preferably 50-80°C; more preferably 65°C.

[0028] The stirring reflux reaction time in step (2) is preferably 1 to 3 hours, more preferably 2 hours.

[0029] The drying in step (2) is preferably carried out by air drying.

[0030] The specific steps of the recrystallization described in step (2) are to dissolve the solid with dichloromethane and then add ether for recrystallization.

[0031] The 5,6-diamino-1,10-phenanthroline and the selenium dioxide in step (3) are preferably mixed in a molar ratio of 1:1 to 1.1; more preferably in a molar ratio of 1:1.

[0032] The glacial acetic acid described in step (3) is a reaction medium and does not participate in the reaction. The amount of glacial acetic acid used is suitable for completely dissolving 5,6-diamino-1,10-phenanthroline; preferably, it is calculated according to the ratio of 5,6-diamino-1,10-phenanthroline: glacial acetic acid = 0.21 g: 4 to 6 mL; more preferably, it is calculated according to the ratio of 5,6-diamino-1,10-phenanthroline: glacial acetic acid = 0.21 g: 5 mL.

[0033] The distilled water described in step (3) is a reaction medium and does not participate in the reaction. The amount of distilled water used is preferably enough to completely dissolve the selenium dioxide; preferably, the ratio of selenium dioxide: distilled water = 0.11 g: 2-4 mL; more preferably, the ratio of selenium dioxide: distilled water = 0.11 g: 3 mL.

[0034] The stirring reaction time in step (3) is preferably 20 to 60 minutes, more preferably 30 minutes.

[0035] The amount of ammonia water used in step (3) is calculated according to the ratio of 5,6-diamino-1,10-phenanthroline: ammonia water = 0.21 g: 6-8 mL; preferably, it is calculated according to the ratio of 5,6-diamino-1,10-phenanthroline: ammonia water = 0.21 g: 7 mL.

[0036] The ammonia water described in step (3) has a concentration of 25% by mass.

[0037] The organic solution in step (3) is preferably a solution obtained by mixing dichloromethane and methanol; more preferably a solution obtained by mixing dichloromethane and methanol in a volume ratio of 1:1.

[0038] The Ru(POP) described in step (4) and the o-phenanthroline selenadiazole are mixed in a molar ratio of 1:1 to 1.1, preferably in a molar ratio of 1:1.

[0039] The ethylene glycol ether described in step (4) is a reaction medium and does not participate in the reaction. Its usage is preferably an amount capable of completely dissolving Ru(POP) and o-phenanthroline selenadiazole; preferably, it is calculated according to the ratio of (Ru(POP)+o-phenanthroline selenadiazole):ethylene glycol ether=0.128g:9-11mL; more preferably, it is calculated according to the ratio of (Ru(POP)+o-phenanthroline selenadiazole):ethylene glycol ether=0.128g:10mL.

[0040] The temperature of the stirring reflux reaction in step (4) is 70-120°C, preferably 100°C.

[0041] The stirring reflux reaction time in step (4) is 2 to 6 hours, preferably 4 hours.

[0042] The cooling in step (4) is preferably natural cooling.

[0043] The amount of ether then added to the ether described in step (4) is preferably equivalent to 4 to 6 times the volume of ethylene glycol ethyl ether and is calculated; more preferably equivalent to 5 times the volume of ethylene glycol ethyl ether and is calculated.

[0044] The specific steps of the recrystallization described in step (4) are to dissolve the solid with dichloromethane and then add ether for recrystallization.

[0045] The application of the selenium-containing ruthenium complex having the functions of radiosensitization and reversal of radiotherapy tolerance in non-therapeutic tumor radiotherapy research preferably comprises the following steps:

[0046] The selenium-ruthenium complex is dissolved in a solvent, then added to a cell suspension for incubation, and then placed under X-rays for irradiation. The results are then tested after continued cultivation.

[0047] The incubation time is 2 to 12 hours, preferably 6 hours.

[0048] The X-ray irradiation dose is 2 to 16 Gy, preferably 4 Gy.

[0049] The continued culture time is 66 to 80 hours, preferably 72 hours.

[0050] The cells include at least one of Caski human cervical squamous cell carcinoma, HeLa human cervical squamous carcinoma cells, SiHa human cervical squamous carcinoma cells, X-Hela X-ray resistant human cervical squamous carcinoma cells, and Ect1 / E6E7 human cervical immortalized squamous cells.

[0051] The application of the selenium-containing ruthenium complex having the function of radiosensitization and reversal of radiotherapy tolerance in the preparation of radiosensitization or reversal of radiotherapy tolerance drugs.

[0052] Compared with the prior art, the present invention has the following advantages and effects:

[0053] 1. The present invention provides a selenium-containing ruthenium complex with the functions of radiosensitization and reversal of radiotherapy tolerance. The central metal ruthenium itself, as a heavy metal element, has a high X-ray photon capture interface and Compton scattering effect, which can produce a dose enhancement effect, increase the radiation damage of sensitive target molecules such as DNA, and produce a radiosensitization effect on irradiated cells. It can combine with X-ray to produce reactive oxygen species (ROS) and increase the killing power to tumor cells.

[0054] 2. The selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl prepared by the present invention has an inhibitory effect on cervical cancer cells (including Caski human cervical squamous cell carcinoma, HeLa human cervical squamous cell carcinoma, SiHa human cervical squamous cell carcinoma), especially in combination with X-ray radiotherapy, it can significantly inhibit cervical cancer cells, and also has an inhibitory effect on radiotherapy-resistant HeLa human cervical cancer cells (X-HeLa), and combined with X-ray radiotherapy, it can significantly improve radiotherapy tolerance.

[0055] 3. The ligand of the selenium-containing ruthenium complex involved in the present invention belongs to the benzoselenadiazole compound, which is easy to coordinate with the metal, which makes it easier to synthesize; more importantly, the raw materials of the selenium-containing ruthenium complex involved in the present invention are cheap and easy to obtain, and the synthesis and purification steps are highly operational. The prepared product has good repeatability and stability, and the commercialization and application of the drug can be achieved by optimizing the process and appropriately expanding the synthesis scale.

[0056] 4. Existing tumor treatment methods mainly include surgery, chemotherapy and radiotherapy, each of which has its own advantages and disadvantages. However, compared with the high incidence and mortality of cancer, the effects of existing treatment methods are still unsatisfactory. The high-priced nature of metal complexes combined with X-rays has brought new hope to tumor treatment, with good safety and significant efficacy. In the future, clinical applications are expected to reduce the amount of metal complexes and improve the treatment effect. While improving the survival time and quality of life of patients, it greatly reduces the cost of treatment and medicine, which has great economic value and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a schematic diagram of the synthesis principle of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl.

[0058] Figure 2 This is a single crystal image of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl.

[0059] Figure 3 This is the CV diagram of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl.

[0060] Figure 4 This is the infrared spectrum of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl.

[0061] Figure 5 This is the ultraviolet spectrum of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl.

[0062] Figure 6This is the mass spectrum of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl.

[0063] Figure 7 This is a graph showing the in vitro antitumor activity of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl combined with X-ray.

[0064] Figure 8 IC of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl combined with X-ray 50 Result graph.

[0065] Fig. 9 This is the isobologram of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl combined with X-ray. DETAILED DESCRIPTION

[0066] The following is a preferred implementation of the embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the embodiment of the present invention. These improvements and modifications are also considered to be within the scope of protection of the embodiment of the present invention.

[0067] The embodiments of the present invention are not limited to the following specific embodiments, and can be appropriately modified and implemented without changing the scope of the main rights.

[0068] Example 1 Preparation of Selenium-Containing Ruthenium Complex [Ru(POP)(PhenSeN2)Cl]Cl

[0069] The synthesis principle of the selenium-containing ruthenium complex [Ru(POP)(PhenSeN2)Cl]Cl is as follows Figure 1 As shown, the specific steps are as follows:

[0070] 1 Synthesis of Selenium-containing Ruthenium Complex [Ru(POP)(PhenSeN2)Cl]Cl

[0071] 1.1 Synthesis of Ru(PPh3)3Cl2 Precursor

[0072]

[0073] 1.00 g of RuCl3·3H2O (ruthenium trichloride hydrate, 3.83 mmol) and 6.00 g of PPh3 (triphenylphosphine, 22.9 mmol) were weighed and dissolved in 50 mL of MeOH (methanol). The solution was reddish brown and stirred at 75°C for 4 h. After the reaction was completed, it was naturally cooled to room temperature and filtered to obtain a reddish brown solid. The solid was washed several times with ether and dried in air to obtain 3.59 g of a dark brown product, Ru(PPh3)3Cl2 (tri(triphenylphosphine)dichlorinated ruthenium (II)), which was 98% of the theoretical yield (3.67 g), i.e., the yield was 98%.

[0074] 1.2 Synthesis of Ru(POP) Precursor

[0075]

[0076] Weigh 0.27 g (0.5 mmol) of POP (4,5-bis(diphenylphosphino)-9,9-dimethyloxanthene) and 0.45 g (0.5 mmol) of Ru(PPh3)3Cl2 prepared in step 1.1, add 10 mL of acetone to dissolve, stir and reflux at 65°C for 2 h, cool naturally after the reaction, filter to obtain an orange-yellow solid, and wash with ether several times. Dissolve the precipitate with dichloromethane, add ether for recrystallization, and obtain a dark red solid. Dry in air to obtain 0.34 g of the product with a yield of 71.4%.

[0077] 1.3 Synthesis of ligand PhenSeN2 (o-phenanthroline selenadiazole)

[0078] Weigh 0.21g (1mmol) 5,6-diamino-1,10-phenanthroline and dissolve it in 5ml glacial acetic acid, 0.11g (1mmol) SeO2 and dissolve it in 3ml distilled water, mix the two, stir and react for 30min, then slowly add about 7mL of 25% ammonia water, filter, wash the precipitate with distilled water, and dry. Then dissolve it with dichloromethane-methanol (volume ratio 1:1) solution, evaporate and crystallize naturally, collect pink needle-shaped crystals PhenSeN20.21g, and the yield is 75%.

[0079] 1.4 Synthesis of the complex [Ru(POP)(PhenSeN2)Cl]Cl

[0080]

[0081] 0.10g (0.1mmol) Ru(POP) and 0.028g (0.1mmol) o-phenanthroline selenadiazole were mixed and dissolved in 10mL ethylene glycol ether, and stirred at 100℃ for 4h. After the reaction was completed, the mixture was cooled naturally. If there was any insoluble matter, it was filtered out, and then 50mL of ether was added. A yellow solid was precipitated, filtered, and washed with ether to obtain an orange-yellow solid. Recrystallization was performed with dichloromethane-ether to obtain about 0.068g of the product [Ru(POP)(PhenSeN2)Cl]Cl, with a yield of 65.6%.

[0082] Example 2: Characterization of the complex [Ru(POP)(PhenSeN2)Cl]Cl

[0083] 2 Chemical characterization experimental methods

[0084] 2.1 Crystal cultivation and determination

[0085] The crystal culture was carried out by the volatilization method. Take an appropriate amount of the complex [Ru(POP)(PhenSeN2)Cl]Cl (about 0.03-0.04mmol) and dissolve it in methanol (about 4-5ml). After filtering, the clear filtrate was transferred to several clean test tubes. The test tubes were placed in a clean wide-mouth bottle filled with an appropriate amount of ether, sealed, and left to stand until the crystals grew. The crystal parameters were measured by X-ray single crystal diffractometer. The single crystal structure was analyzed by olex2 software.

[0086] 2.2 Infrared spectroscopy

[0087] Take an appropriate amount of the complex [Ru(POP)(PhenSeN2)Cl]Cl to prepare the sample to be tested by potassium bromide tablet method, detect it by Fourier transform infrared spectrometer, scan in the range of 400-4400nm, and obtain the infrared spectrum data of the sample. Use OMNIC software to analyze its infrared spectrum.

[0088] 2.3 UV / visible spectroscopy

[0089] Using Cary5000 UV-visible spectrometer, methanol was used as solvent, and an appropriate amount of the complex [Ru(POP)(PhenSeN2)Cl]Cl was prepared into a solution with a concentration of about 0.1mM (M is the abbreviation of mol / L), and the UV spectra of the two complexes were measured. The sample was placed in a 10mm quartz cuvette for UV measurement, and the scanning wavelength range was set to 200-1000nm. The UV graph was analyzed using the instrument's own software.

[0090] 2.4 Mass spectrometry analysis

[0091] Take an appropriate amount of the complex [Ru(POP)(PhenSeN2)Cl]Cl and prepare it into a solution with chromatographic methanol. Use liquid chromatography-mass spectrometry to measure the mass spectrum data of the two complexes in positive ion mode, analyze the mass spectrum to obtain the chemical structure and stability data of the complex. Use ApoloMs or AgtQual software to analyze its mass spectrum.

[0092] 2.5 Magnetic resonance imaging

[0093] Take an appropriate amount of complex [Ru(POP)(PhenSeN2)Cl]Cl crystals and dissolve them in deuterated DMSO reagent, the concentration is about 10-20mg / mL, then add them into the NMR tube, and use 300 MHz NMR to detect the hydrogen spectrum. Use MestReNova software to analyze its NMR map.

[0094] 2.6 Electrochemical performance test

[0095] The cyclic voltammetry curve of the complex [Ru(POP)(PhenSeN2)Cl]Cl was determined by the CHI660E electrochemical workstation. A three-electrode system was used for detection, in which the working electrode was a glassy carbon electrode, the reference electrode was a saturated calomel electrode, and the auxiliary electrode was a platinum wire electrode. First, tetrabutylammonium hexafluorophosphate was used as the electrolyte, and a 0.1M tetrabutylammonium hexafluorophosphate (nBu4NPF6) acetonitrile solution was prepared, benzyl alcohol was used as the substrate, the concentration was 10mM, trifluoroacetic acid with a final concentration of 0.5M and H2O with a final concentration of 5M were added, and the cyclic voltammetry curve of 1mM complex [Ru(POP)(PhenSeN2)Cl]Cl in a system with a total volume of 5mL was determined. The scanning speed was 100mV / s. During the measurement, the voltage was scanned from 0V to the forward direction, and the voltage was gradually increased to 2V. The scan was repeated to finally obtain its cyclic voltammetry curve. Its CV graph was analyzed using CHI660E.

[0096] Example 3: Data Analysis

[0097] Figure 2 The crystal diagram of the complex [Ru(POP)(PhenSeN2)Cl]Cl. The oxygen atom and two phosphorus atoms on Ru(POP) coordinate with the metal ruthenium to form a pincer structure. O-phenanthroline selenadiazole is a bidentate heterocyclic compound ligand, which chelates with the central metal ruthenium through the two nitrogen atoms on o-phenanthroline. The remaining coordination site on the plane of ruthenium is combined with a chloride ion to form a six-coordinated octahedral configuration.

[0098] like Figure 3As shown, the current is strong. Comparing the two curves in the figure, it can be seen that the catalytic current of the electrolyte solution with the complex added is lower than that of the blank solution, indicating that the complex has a certain catalytic oxidation ability as a catalyst and has a certain catalytic research value. When the complex [Ru(POP)(PhenSeN2)Cl]Cl is subsequently added to the hydrogen peroxide reaction, it can catalyze the decomposition of hydrogen peroxide to produce oxygen.

[0099] like Figure 4 As shown in the infrared image, 3415.0cm -1 The broad absorption peak near 2969.4cm is produced by the resonance of CH on the aromatic ring or NH on the heterocyclic ring of 1,1-phenanthroline selenadiazole; -1 It is the CH stretching vibration of the benzene ring or the antisymmetric stretching vibration of the methyl group, 1635.3cm -1 、1479.8cm -1 、1434.3cm -1 It is the skeleton vibration of the benzene ring, 775.8cm -1 、742.9cm -1 It is the out-of-plane bending vibration of the CH of the aromatic ring. Therefore, it can be analyzed that the measured complex contains a benzene ring, and the benzene ring is a monosubstituted benzene ring and a 1,2,3-trisubstituted benzene ring. 699.2cm -1 It is the absorption peak produced by the N-Se-N vibration on the selenadiazole heterocyclic ring. 1397.2cm -1 It is the bending vibration of the methyl group. The [Ru(POP)(SeN2)Cl]Cl complex contains two methyl groups, and the infrared spectrum data is consistent with the structure of the complex.

[0100] like Figure 5 As shown in the ultraviolet spectrum, 434nm is the ultraviolet absorption of divalent ruthenium.

[0101] like Figure 6 As shown in the mass spectrum, the peak at m / z 1001.04 is the molecular ion peak of the complex [Ru(POP)(SeN2)Cl] + This may be due to the high ESI energy or the instability of chlorine in the complex. - The mass spectrum peak is very clear, and it can be clearly seen here that the complex [Ru(POP)(SeN2)Cl]Cl was successfully synthesized.

[0102] In summary, the complex [Ru(POP)(SeN2)Cl]Cl was successfully synthesized and characterized.

[0103] Example 4: Study on the antitumor activity of selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl in vitro

[0104] The method for measuring the growth inhibition of malignant tumor cells (Caski human cervical cancer cells, HeLa human cervical cancer cells, SiHa human cervical cancer cells, and normal cells (Ect1 / E6E7 human cervical immortalized squamous cells) by the ruthenium complex in Example 1 using the MTT method mainly comprises the following steps:

[0105] (1) dissolving the selenium-containing ruthenium complex in DMSO solvent, placing it on an ultrasonicator for 1 to 2 minutes to prepare a 10 mM solution, and diluting it with DMSO to obtain a 0.1 μM to 10 mM selenium-containing ruthenium complex gradient concentration solution for use;

[0106] (2) Different tumor cells and normal cells in the logarithmic growth phase were collected at a density of 2×10 4 Cells / mL were inoculated in a 96-well plate (100 μL cell suspension per well, culture medium was DMEM), and allowed to adhere to the wall and grow for 24 hours; then 100 μL of the gradient concentration solution of the selenium-ruthenium complex of different concentrations in step (1) was added respectively, and the culture was continued for 72 hours. After the cells showed a certain degree of damage, 30 μL of MTT solution (5 mg / mL, PBS solution) was added to each well and incubated for 3 hours. Then, the upper culture medium (DMEM high-glucose culture medium, Gibco) in the 96-well plate was removed, 150 μL DMSO was added, and the plate was gently shaken on a shaker for 15 minutes to fully dissolve the purple crystals in the 96-well plate. Then, the absorbance of each well at 570 nm was measured using a multifunctional microplate reader, and the cell survival rate was calculated, and a graph was drawn to obtain the half inhibitory concentration (IC 50 ). Cell survival rate (%) = (OD570 experimental group / OD570 control group) × 100%.

[0107] Test results: The in vitro antitumor activity of the selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl is shown in Table 1.

[0108] Table 1 In vitro anticancer activity of selenium-ruthenium complexes

[0109] cell <![CDATA[[Ru(POP)(SeN2)Cl]Cl IC 50 (μM)]]> Hela 3.55 SiH 0.82 Caski 5.32 Ect1 / E6E7 2.44

[0110] Safety factor = IC 50 (Ect1 / E6E7) / IC 50 (Hela) = 0.69

[0111] As can be seen from Table 1, the selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl exhibits good antitumor activity. However, it also has high toxicity to normal cells Ect1 / E6E7 cells (human cervical immortalized squamous cells), with an IC50 of 2.44. The above results show that the selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl can exert excellent antitumor activity in vitro, but has low selectivity and no selectivity for normal cells.

[0112] Example 5: Study on the antitumor activity of selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl combined with X-ray in vitro

[0113] The method for measuring the growth inhibition of the selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl in Example 1 in combination with X-ray on HeLa human cervical squamous cell carcinoma cells and X-HeLa cells (the cells were prepared according to Hela-R3 (fractionated dose of 6 Gy, irradiated 4 times) in the document "Huang Jingran et al. Biological characteristics of radiotherapy-resistant cervical cancer Hela cells. Journal of Sun Yat-sen University (Medical Science Edition), Vol. 33 (Issue 1): 16-22") by using the MTT method mainly comprises the following steps:

[0114] (1) dissolving the selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl in DMSO solvent, placing it on an ultrasonicator for 1 to 2 minutes to prepare a 10 mM solution, and diluting it with DMSO to obtain a 0.1 μM to 10 mM selenium-containing ruthenium complex gradient concentration solution for use;

[0115] (2) Take different tumor cells in the logarithmic growth phase and collect them at a density of 2×10 4 100 μL of cell suspension per well was inoculated into a 96-well plate (100 μL of cell suspension per well, culture medium was DMEM) and allowed to adhere to the wall and grow for 24 hours. Then, 100 μL of the gradient concentration solution of selenium-ruthenium complex in step (1) was added respectively. After incubation for 6 hours, the 96-well plate was irradiated with 0 / 2 / 4 Gy dose of X-ray and cultured for 72 hours. After the cells showed a certain degree of damage, 30 μL of MTT solution (5 mg / mL, PBS solution) was added to each well and incubated for 3 hours. Then, the upper culture medium (DMEM high glucose culture medium, Gibco) in the 96-well plate was removed, 150 μL of DMSO (dimethyl sulfoxide) was added, and the plate was gently shaken on a shaker for 15 minutes to fully dissolve the purple crystals in the 96-well plate. Then, the absorbance of each well at 570 nm was measured using a multifunctional microplate reader, and the cell survival rate was calculated. At the same time, a graph was drawn to obtain the half inhibitory concentration (IC 50 ). Cell survival rate (%) = (OD570 experimental group / OD570 control group) × 100%.

[0116] Test results: At the same X-ray dose, as the drug concentration increases, the cell survival rate decreases; at the same drug concentration, as the X-ray dose increases, the cell survival rate decreases. The generated curve is as follows Figure 7 As shown, radiotherapy greatly increases the lethality of drugs. Figure 8 It can be seen that when the drug is irradiated with X-ray (0Gy, 2Gy, 4Gy), the half-lethal concentrations of HeLa human cervical squamous cell carcinoma cells are 1.54μM, 0.98μM, 0.67mΜ, respectively, and the half-lethal concentrations of X-HeLa cells are 8.06μM, 6.19μM, 4.96mΜ, respectively. These data show that after the action of X-ray, the selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl has significantly enhanced lethality to HeLa human cervical squamous cell carcinoma cells and X-HeLa.

[0117] Example 6: Isobologram analysis of the radiotherapy synergistic effect of the selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl

[0118] The isobologram analysis of the synergistic sensitization effect of X-ray and selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl mainly includes the following steps:

[0119] (1) Take different tumor cells in the logarithmic growth phase and add them at a density of 2×10 4 Cells / mL were inoculated in a 96-well plate (100 μL cell suspension per well, culture medium was DMEM) and allowed to adhere to the wall and grow for 24 hours. They were then irradiated with different doses of X-ray (0 Gy, 2 Gy, 4 Gy, 8 Gy, 16 Gy). After continuing to culture for 72 hours, 30 μL of MTT solution (5 mg / mL, PBS solution) was added to each well and incubated for 3 hours. Next, the upper culture medium in the 96-well plate was removed, 150 μL DMSO was added, and the plate was gently shaken on a shaker for 15 minutes to fully dissolve the purple crystals in the 96-well plate. The absorbance of each well at 570 nm was then measured using a multifunctional microplate reader to calculate the cell survival rate and the IC value of radiotherapy alone for the cells. 50 The value is set to point B on the Y axis.

[0120] (2) According to Example 6, when the X-ray is 0 Gy, the IC of the cell is 50 The value is set to point A on the X-axis.

[0121] (3) Connecting point A on the X-axis and point B on the Y-axis yields an isoequivalent line. When the X-ray dose is 2 Gy or 4 Gy, the IC 50 Value point.

[0122] Test results: After combined radiotherapy and chemotherapy, the theoretical IC 50 add and experiment IC 50 When there is no significant difference in mix, it indicates that the type of interaction between the two is additive; if IC 50 mix is ​​significantly lower than IC 50 When add (below the addition line), it means that the two have a synergistic effect; otherwise, it is an antagonistic effect. Fig. 9 As shown in the figure, no matter whether the X-ray dose is 2Gy or 4Gy, it is below the isoequivalent line. That is, the selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl and X-ray are synergistic, which greatly reduces the dose of radiotherapy alone and chemotherapy alone, thereby improving the biosafety of the selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl as a radiotherapy sensitizer.

[0123] Example 7: DCF probe detection of intracellular reactive oxygen species (ROS) levels

[0124] ROS mainly include superoxide anions (O 2 ·-), hydrogen peroxide (H2O2), hydroxyl free radical (HO·), and a method for detecting the effect of a high-valent selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl on the level of reactive oxygen species (ROS) in HeLa human cervical squamous cell carcinoma cells using a DCF probe, mainly comprising the following steps:

[0125] (1) The selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl of Example 1 was dissolved in a DMSO solution and ultrasonicated for 1 to 2 minutes to prepare a 5 mM solution for later use;

[0126] (2) HeLa human cervical squamous cell carcinoma cells in the logarithmic growth phase were inoculated into a 96-well plate (density of 2×10 4 / mL, 100μL / well), after the cells adhered for 24h, 100μL of 15μM selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl was added and incubated for 6h, irradiated with 4Gy X-ray, and then the culture medium in each well was removed, and the DCF probe was added and cultured for 30min. The absorbance was detected on a multifunctional fluorescence microplate reader (excitation and emission wavelengths were 488nm and 525nm, respectively). At the same time, the DCF fluorescence intensity in the cells was monitored in real time using a fluorescence microscope.

[0127] Test results: An ideal radiosensitizer can increase the sensitivity of hypoxic tumor cells to X-rays, produce a large number of free radicals under X-ray irradiation, thereby increasing the content of intracellular reactive oxygen species (ROS), and ultimately leading to tumor cell death. The heavy metal effect of the selenium-ruthenium complex [Ru(POP)(SeN2)Cl]Cl can increase the absorption capacity of X-rays and produce a large amount of reactive oxygen.

[0128] Example 8: Effect of Selenium-containing Ruthenium Complex [Ru(POP)(SeN2)Cl]Cl on Tumor Cell Cycle Distribution

[0129] Flow cytometry was used to analyze the effect of selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl combined with X-ray on the cell cycle distribution of HeLa human cervical squamous cell carcinoma cells, which mainly included the following steps:

[0130] (1) The selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl obtained in Example 1 was dissolved in a DMSO solution and placed on an ultrasonicator for 1 to 2 minutes to prepare a 10 mM solution for later use;

[0131] (2) 5 mL of HeLa human cervical squamous cell carcinoma cells were inoculated into a 6 cm culture dish (cell density was 8×10 4 / mL), after the cells are completely attached, different concentrations of selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl are added. After incubation for 6 hours, the culture medium is irradiated with X-ray (4Gy), and then incubated in an incubator for 48 hours. The cells are collected and fixed overnight at -20℃ with pre-cooled 70% ethanol. The supernatant is removed and centrifuged, and the tube is inverted to dry. 500μL PI (propidium iodide) is added to stain for 30min in the dark, and the cell cycle is detected by Beckman flow cytometer to ensure that the number of cells is not less than 10,000. The cell cycle distribution is analyzed using MultiCycle software.

[0132] Test results: In order to explore the radiosensitization effect of selenium-containing ruthenium complex [Ru(POP)(SeN2)Cl]Cl, flow cytometry was used to analyze the cycle of the cell groups treated with radiotherapy alone, drug alone, and radiotherapy plus drug combination. The main manifestations of the cell groups treated with drug alone and radiotherapy plus drug combination were G2 / M phase arrest, which increased with the increase of the complex dose.

[0133] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A selenium-containing ruthenium complex having the function of radiosensitization and reversal of radiotherapy tolerance, characterized in that: The chemical formula is [Ru(POP)(PhenSeN2)Cl]Cl, and the structural formula is shown in Formula I:

2. The method for preparing the selenium-containing ruthenium complex according to claim 1, characterized in that The following steps are involved: (1) Synthesis of the precursor tris(triphenylphosphine)ruthenium(II) dichloride Weigh ruthenium trichloride hydrate and triphenylphosphine, dissolve them in methanol, stir and reflux for reaction; after the reaction is completed, cool and filter, wash the obtained reddish brown solid with ether, and dry it to obtain tris(triphenylphosphine)ruthenium (II) dichloride; (2) Synthesis of Ru(POP) precursor Dissolve 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene and tris(triphenylphosphine)ruthenium(II) dichloride obtained in step (1) in acetone, stir and reflux for reaction, cool after the reaction is completed, filter to obtain an orange-yellow solid, wash with ether, then recrystallize and dry to obtain a product precursor Ru(POP); (3) Synthesis of ligand o-phenanthroline selenadiazole Dissolve 5,6-diamino-1,10-phenanthroline in glacial acetic acid to obtain solution A; dissolve selenium dioxide in distilled water to obtain solution B; mix solution A and solution B, stir to react, then add ammonia water, filter, wash the obtained precipitate with water, and dry; Then it is dissolved with an organic solvent, evaporated and crystallized naturally, and the pink needle-shaped crystals of the product are collected to obtain the ligand o-phenanthroline selenadiazole; (4) Synthesis of the complex [Ru(POP)(PhenSeN2)Cl]Cl The Ru(POP) prepared in step (2) and the o-phenanthroline selenadiazole prepared in step (3) are mixed and dissolved in ethylene glycol ethyl ether, and stirred and refluxed for reaction; after the reaction is completed, the mixture is cooled, and any insoluble matter is filtered out, and then ether is added to precipitate a yellow solid, which is filtered and washed with ether to obtain an orange-yellow solid, and after recrystallization, a selenium-containing ruthenium complex having the function of radiotherapy sensitization and reversal of radiotherapy tolerance is obtained.

3. The method for preparing a selenium-containing ruthenium complex according to claim 2, characterized in that: The ruthenium trichloride hydrate described in step (1) and the triphenylphosphine described are mixed in a molar ratio of 1:4-7; The 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene described in step (2) and the tris(triphenylphosphine)ruthenium(II) dichloride are mixed in a molar ratio of 1 to 1.1:1; The 5,6-diamino-1,10-phenanthroline described in step (3) and the selenium dioxide are mixed in a molar ratio of 1:1 to 1.1; The amount of ammonia water used in step (3) is calculated according to the ratio of 5,6-diamino-1,10-phenanthroline: ammonia water = 0.21 g: 6-8 mL; The ammonia water in step (3) is an ammonia water with a concentration of 25% by mass; The organic solution described in step (3) is a solution obtained by mixing dichloromethane and methanol; The Ru(POP) described in step (4) and the o-phenanthroline selenadiazole are mixed in a molar ratio of 1:1 to 1.

1.

4. The method for preparing a selenium-containing ruthenium complex according to claim 2, characterized in that: The amount of methanol used in step (1) is calculated according to the ratio of (ruthenium trichloride hydrate + triphenylphosphine): methanol = 7 g: 40-60 mL; The amount of acetone used in step (2) is calculated according to the ratio of (4,5-bis(diphenylphosphine)-9,9-dimethylxanthene+tris(triphenylphosphine)ruthenium(II) dichloride):acetone=0.72g:9-11mL; The amount of glacial acetic acid used in step (3) is calculated according to the ratio of 5,6-diamino-1,10-phenanthroline: glacial acetic acid = 0.21 g: 4-6 mL; The amount of distilled water used in step (3) is calculated according to the ratio of selenium dioxide: distilled water = 0.11 g: 2-4 mL; The amount of ethylene glycol ethyl ether described in step (4) is calculated according to the ratio of (Ru(POP)+phenanthroline selenadiazole):ethylene glycol ethyl ether=0.128g:9-11mL; The amount of ether then added to the ether in step (4) is equivalent to 4 to 6 times the volume of ethylene glycol ether.

5. The method for preparing a selenium-containing ruthenium complex according to claim 2, characterized in that: The temperature of the stirring reflux reaction in step (1) is 60 to 90° C. The stirring reflux reaction time in step (1) is 2 to 6 hours; The cooling described in step (1) is natural cooling to room temperature; The temperature of the stirring reflux reaction in step (2) is 50-80°C; The stirring reflux reaction time in step (2) is 1 to 3 hours; The drying in step (2) is to place in air to dry; The specific steps of the recrystallization described in step (2) are to dissolve the solid with dichloromethane and then add ether for recrystallization; The stirring reaction time in step (3) is 20 to 60 minutes; The temperature of the stirring reflux reaction in step (4) is 70 to 120°C; The stirring reflux reaction time in step (4) is 2 to 6 hours; The cooling described in step (4) is natural cooling; The specific steps of the recrystallization described in step (4) are to dissolve the solid with dichloromethane and then add ether for recrystallization.

6. Use of the selenium-containing ruthenium complex having the function of radiosensitization and reversal of radiotherapy tolerance as claimed in claim 1 in the preparation of drugs for radiosensitization or reversal of radiotherapy tolerance.

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