PDI inhibitor binuclear ruthenium complex as well as preparation method and application thereof
By synthesizing the PDI inhibitor binuclear ruthenium complex, the problems of low activity and great toxicity and side effects of existing PDI inhibitors are solved, and efficient tumor cell inhibition and low toxicity are achieved, and there are broad clinical application prospects.
Patent Information
- Application Number
- CN202510372743.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-27
- Publication Date
- 2025-06-27
AI Technical Summary
The existing PDI inhibitors have low biological activity and high toxic and side effects, making them difficult to effectively apply in clinical treatment.
By synthesizing a PDI inhibitor binuclear ruthenium complex, using 2-hydrazine benzothiazole and 2-acetylpyridine as raw materials, a binuclear ruthenium complex with good PDI inhibition activity was prepared through reflux reaction and subsequent reaction.
The PDI inhibitor binuclear ruthenium complex showed significant tumor cell inhibitory activity, low IC50 value, and low toxicity, and can be more widely used in the field of tumor treatment, and its therapeutic effect is better than the existing anti-cancer drug cisplatin.
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Figure CN120209045A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and particularly to a binuclear ruthenium complex as a PDI inhibitor, its preparation method and application. Background Art
[0002] Protein disulfide isomerase (PDI) is an important enzyme that widely exists in cells and participates in biological processes such as protein folding, modification, and cell signal transduction. Recent studies have shown that PDI plays a key role in the occurrence, development, and drug resistance of various malignant tumors, and its abnormal expression is closely related to the proliferation, invasion, and metastasis of tumor cells. Therefore, the development of specific inhibitors against PDI has become one of the research hotspots in the field of tumor treatment.
[0003] Currently, although a variety of PDI inhibitors have been developed, most of them have some limitations. For example, some inhibitors have low biological activity and it is difficult to achieve an ideal therapeutic effect in actual application; while some other PDI inhibitors have acceptable activity, but they have high toxic and side effects, which seriously limits their application in clinical treatment.
[0004] Therefore, the development of a new type of PDI inhibitor with high inhibitory activity and low toxic and side effects has important clinical significance. Summary of the Invention
[0005] In view of this, the present invention provides a binuclear ruthenium complex as a PDI inhibitor, its preparation method and application to solve the problems of low activity and high toxic and side effects of existing PDI inhibitors.
[0006] To achieve the above object, the present invention adopts the following technical solutions:
[0007] The present invention provides a binuclear ruthenium complex as a PDI inhibitor, and the structural formula of the binuclear ruthenium complex as a PDI inhibitor is shown in Formula I:
[0008]
[0009] The present invention also provides a preparation method of the above binuclear ruthenium complex as a PDI inhibitor, including the following steps:
[0010] 1) Mix 2-hydrazinobenzothiazole, 2-acetylpyridine, a first organic solvent and a catalyst, and carry out a reflux reaction to obtain a Schiff base ligand;
[0011] 2) Mix the Schiff base ligand, dichloro bis(4-methylcumene)ruthenium(II) dimer, a second organic solvent, and ammonium hexafluorophosphate, and carry out a reaction to obtain a binuclear ruthenium complex of a PDI inhibitor;
[0012] The structural formula of the Schiff base ligand is as shown in Formula II:
[0013]
[0014] Preferably, in step 1), the molar volume ratio of 2-hydrazinobenzothiazole, 2-acetylpyridine, the first organic solvent, and the catalyst is 3-10 mmol: 3-10 mmol: 15-100 mL: 0.2-1 mL.
[0015] Preferably, in step 1), the temperature of the reflux reaction is 45-75 °C, and the time is 12-24 h.
[0016] Preferably, in step 1), the first organic solvent includes one or more of ethanol, methanol, and acetonitrile; the catalyst includes acetic acid and / or p-toluenesulfonic acid.
[0017] Preferably, in step 2), the molar volume ratio of the Schiff base ligand, dichloro bis(4-methylcumene)ruthenium(II) dimer, the second organic solvent, and ammonium hexafluorophosphate is 0.1-0.2 mmol: 0.05-0.1 mmol: 10-40 mL: 0.1-0.4 mmol.
[0018] Preferably, in step 2), the time of the reaction is 24-48 h.
[0019] Preferably, in step 2), the second organic solvent includes ethanol and / or methanol.
[0020] The present invention also provides an application of the binuclear ruthenium complex of a PDI inhibitor prepared by the preparation method of the above binuclear ruthenium complex of a PDI inhibitor in the preparation of an antitumor drug.
[0021] According to the above technical solutions, compared with the prior art, the beneficial effects of the present invention are as follows:
[0022] The present invention uses 2-acetylpyridine and 2-hydrazine benzothiazole as raw materials to synthesize Schiff base ligands, and then reacts the Schiff base ligands with dichlorobis(4-methylisopropylphenyl)ruthenium(II) dimer and ammonium hexafluorophosphate to synthesize a novel PDI inhibitor binuclear ruthenium complex, which has good protein disulfide isomerase (PDI) inhibitory activity, can be used as a therapeutic drug for malignant tumors and has a significant therapeutic effect. Moreover, the PDI inhibitor also has lower toxic side effects and can be more widely used in the field of tumor treatment. In addition, compared with the existing anticancer drug cisplatin, the PDI inhibitor prepared by the present invention has a better therapeutic effect and less toxicity.
[0023] The preparation method of the present invention is simple, has mild reaction conditions and low cost, and is conducive to realizing large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0025] Figure 1 This is a physical picture of the PDI inhibitor binuclear ruthenium complex prepared in Example 1;
[0026] Figure 2 The crystal structure of the binuclear ruthenium complex of the PDI inhibitor prepared in Example 1;
[0027] Figure 3 This is a graph showing the inhibitory activity of the PDI inhibitor binuclear ruthenium complex prepared in Example 1 against protein disulfide isomerase (PDI). DETAILED DESCRIPTION
[0028] The present invention provides a PDI inhibitor binuclear ruthenium complex, the structural formula of the PDI inhibitor binuclear ruthenium complex is shown in Formula I:
[0029]
[0030] The present invention also provides a method for preparing the above-mentioned PDI inhibitor binuclear ruthenium complex, comprising the following steps:
[0031] 1) mixing 2-hydrazinobenzothiazole, 2-acetylpyridine, a first organic solvent and a catalyst, and performing a reflux reaction to obtain a Schiff base ligand;
[0032] 2) Mix the Schiff base ligand, dichloro bis(4-methylcumyl)ruthenium(II) dimer, a second organic solvent, and ammonium hexafluorophosphate, and carry out a reaction to obtain a binuclear ruthenium complex of the PDI inhibitor;
[0033] The structural formula of the Schiff base ligand is as shown in Formula II:
[0034]
[0035] In the present invention, in step 1), the molar volume ratio of 2-hydrazinobenzothiazole, 2-acetylpyridine, the first organic solvent, and the catalyst is 3-10 mmol: 3-10 mmol: 15-100 mL: 0.2-1 mL, preferably 4-8 mmol: 4-8 mmol: 30-80 mL: 0.3-0.9 mL, more preferably 5-7 mmol: 5-7 mmol: 40-60 mL: 0.4-0.6 mL, and even more preferably 6 mmol: 6 mmol: 50 mL: 0.5 mL.
[0036] In the present invention, in step 1), the temperature of the reflux reaction is 45-75 °C, preferably 50-70 °C, more preferably 55-68 °C, and even more preferably 65 °C; the time of the reflux reaction is 12-24 h, preferably 15-23 h, more preferably 16-22 h, and even more preferably 20 h.
[0037] In the present invention, in step 1), the first organic solvent includes one or more of ethanol, methanol, and acetonitrile; the catalyst includes acetic acid and / or p-toluenesulfonic acid.
[0038] In the present invention, in step 1), the mixing preferably first dissolves 2-hydrazinobenzothiazole and 2-acetylpyridine in the first organic solvent, and then adds the catalyst.
[0039] In the present invention, after the reflux reaction in step 1), it preferably further includes operations of subjecting the Schiff base ligand to rotary evaporation under reduced pressure, washing, and drying in sequence.
[0040] In the present invention, the molar volume ratio of the Schiff base ligand, dichloro bis(4-methylcumyl) ruthenium (II) dimer, the second organic solvent, and ammonium hexafluorophosphate in step 2) is 0.1 - 0.2 mmol: 0.05 - 0.1 mmol: 10 - 40 mL: 0.1 - 0.4 mmol, preferably 0.12 - 0.18 mmol: 0.06 - 0.09 mmol: 12 - 35 mL: 0.12 - 0.36 mmol, more preferably 0.13 - 0.16 mmol: 0.07 - 0.08 mmol: 15 - 30 mL: 0.16 - 0.28 mmol, and even more preferably 0.15 mmol: 0.075 mmol: 25 mL: 0.2 mmol.
[0041] In the present invention, the reaction time in step 2) is 24 - 48 h, preferably 25 - 42 h, more preferably 30 - 40 h, and even more preferably 36 h.
[0042] In the present invention, the second organic solvent in step 2) includes ethanol and / or methanol.
[0043] In the present invention, after the reaction in step 2), operations such as filtration and crystallization are preferably included.
[0044] In the present invention, the crystallization operation is preferably to seal the filtered reaction solution in a beaker with plastic wrap, then punch holes in the plastic wrap, and place it in a fume hood to stand for crystallization; the number of the punched holes is preferably 10 - 15, more preferably 11 - 14, and even more preferably 13; the crystallization time is preferably 7 - 15 d, more preferably 8 - 12 d, and even more preferably 10 d.
[0045] In the present invention, the PDI inhibitor binuclear ruthenium complex is a black-purple crystal.
[0046] The present invention also provides an application of the PDI inhibitor binuclear ruthenium complex prepared by the preparation method of the above PDI inhibitor binuclear ruthenium complex in the preparation of anti-tumor drugs.
[0047] The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.
[0048] Example 1
[0049] 1) Dissolve 6 mmol of 2 - hydrazinylbenzothiazole and 6 mmol of 2 - acetylpyridine in 50 mL of ethanol, then add 0.5 mL of acetic acid, and reflux the reaction at 65 °C for 24 h to obtain a reaction solution. Perform rotary evaporation under reduced pressure on this reaction solution at a pressure of 20 mmHg to obtain a gray powder. Then wash this gray powder 3 times with methanol solution and dry it to obtain a Schiff base ligand with the structural formula as follows.
[0050] 2) Dissolve 0.15 mmol of the Schiff base ligand obtained in step 1) and 0.075 mmol of dichloro - bis(4 - methylisopropylphenyl)ruthenium(II) dimer in 25 mL of ethanol, then add 0.2 mmol of ammonium hexafluorophosphate (NH4PF6), and react at room temperature (25 °C) for 36 h. Filter the reaction solution obtained from the reaction through filter paper into a beaker, then seal the beaker with plastic wrap, make 12 small holes in the plastic wrap, and place it in a fume hood for crystallization. After 10 days, black - purple crystals precipitate at the bottom of the beaker, namely, the dinuclear ruthenium complex of the PDI inhibitor is obtained, and its structural formula is shown as formula Ⅰ below:
[0051]
[0052] After testing, the yield of the Schiff base ligand in this example is 91.9%, and the yield of the dinuclear ruthenium complex of the PDI inhibitor is 85.6%. And the elemental analysis of the dinuclear ruthenium complex of the PDI inhibitor prepared in this example is C 34 H 39 Cl2F6N4PRu2S, the theoretical values are C.42.82:H.4.12:N,5.87, and the measured values are C.42.84:H.4.11:N,5.85. In addition, select a black - purple crystal of the dinuclear ruthenium complex of the PDI inhibitor prepared in this example (the physical picture of the black - purple crystal is as Figure 1 shown), and use an Agilent Technologies Xcalibur&Gemini single - crystal diffractometer to obtain the diffraction data of this black - purple crystal. The specific data results are shown in Table 1.
[0053] Table 1 Data table of the dinuclear ruthenium complex of the PDI inhibitor
[0054]
[0055]
[0056] As can be seen from Table 1, the dinuclear ruthenium complex of the PDI inhibitor was successfully prepared in this example.
[0057] In addition, use OLEX2 software to analyze the crystal structure of the dinuclear ruthenium complex of the PDI inhibitor prepared in this example, and the results are asFigure 2 As shown in Figure 2 It can also be seen from
[0058] Example 2
[0059] 1) Dissolve 6 mmol of 2-hydrazinobenzothiazole and 6 mmol of 2-acetylpyridine in 50 mL of methanol, then add 1.5 mL of p-toluenesulfonic acid, and then carry out a reflux reaction at a temperature of 50 °C. After reacting for 24 h, a reaction solution is obtained. The reaction solution is subjected to rotary evaporation under reduced pressure at a pressure of 10 mmHg to obtain a gray powder. Then, the gray powder is washed 3 times with a methanol solution and then dried to obtain the Schiff base ligand shown in Formula II.
[0060] 2) Dissolve 0.15 mmol of the Schiff base ligand obtained in step 1) and 0.075 mmol of dichloro-bis(4-methylisopropylphenyl)ruthenium(II) dimer in 40 mL of methanol, then add 0.1 mmol of ammonium hexafluorophosphate (NH4PF6), and then react at room temperature (25 °C) for 24 h. The reaction solution obtained is filtered through filter paper into a beaker, and then the beaker is sealed with plastic wrap. After making 10 small holes in the plastic wrap, it is placed in a fume hood for crystallization. After 15 days, black-purple crystals precipitate at the bottom of the beaker, that is, the binuclear ruthenium complex of the PDI inhibitor shown in Formula I is obtained.
[0061] Example 3
[0062] 1) Dissolve 6 mmol of 2-hydrazinobenzothiazole and 6 mmol of 2-acetylpyridine in 90 mL of acetonitrile, then add 2 mL of acetic acid, and then carry out a reflux reaction at a temperature of 75 °C. After reacting for 15 h, a reaction solution is obtained. The reaction solution is subjected to rotary evaporation under reduced pressure at a pressure of 50 mmHg to obtain a gray powder. Then, the gray powder is washed 3 times with a methanol solution and then dried to obtain the Schiff base ligand shown in Formula II.
[0063] 2) Dissolve 0.15 mmol of the Schiff base ligand obtained in step 1) and 0.075 mmol of dichloro-bis(4-methylisopropylphenyl)ruthenium(II) dimer in 40 mL of ethanol, then add 0.1 mmol of ammonium hexafluorophosphate (NH4PF6), and then react at room temperature (25 °C) for 48 h. The reaction solution obtained is filtered through filter paper into a beaker, and then the beaker is sealed with plastic wrap. After making 15 small holes in the plastic wrap, it is placed in a fume hood for crystallization. After 7 days, black-purple crystals precipitate at the bottom of the beaker, that is, the binuclear ruthenium complex of the PDI inhibitor shown in Formula I is obtained.
[0064] Experimental Example 1
[0065] The tumor cell inhibitory proliferation test was carried out on the dinuclear ruthenium complex of the PDI inhibitor prepared in Example 1 by the thiazolyl blue (MTT) method. The specific test method is as follows:
[0066] First, prepare four tumor cell lines: human cervical cancer cell line Hela, human breast cancer cell line MCF-7, human glioma cell line T98G, and normal cell line WI-38. Among them, these four tumor cell lines were all purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection. Then, the above four logarithmic-phase tumor cell lines were respectively added to the wells of a 96-well plate at a concentration of 5×10 4 cells / mL at a volume of 180 μL. After that, they were continuously cultured overnight in DMEM medium under the conditions of 5% CO2 and a temperature of 37 °C until the cells adhered to the wall. Then, 20 μL of the dinuclear ruthenium complex of the PDI inhibitor prepared in Example 1 with mass concentrations of 1, 2, 5, 10, and 15 μmol / L was added to each well. After continuous culture for 48 h, 10 μL of thiazolyl blue (MTT) solution (mass concentration of 5 mg / mL) was added and incubated for 4 h. Then, all the liquid was slowly removed, and 100 μL of dimethyl sulfoxide was added and shaken for 10 min. The absorbance at a wavelength of 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the IC 50 value was calculated. At the same time, the clinical drug cisplatin (purchased from Beijing Innochem Science & Technology Co., Ltd.) was set as a control group and tested using the exactly same test method. The results are shown in Table 2. Among them, the smaller the IC 50 value, the better the inhibitory activity of the PDI inhibitor against cancer cells.
[0067] Table 2 IC 50 values of the dinuclear ruthenium complex of the PDI inhibitor against different tumor cell lines
[0068]
[0069] As can be seen from the results in Table 2, the dinuclear ruthenium complex of the PDI inhibitor prepared in Example 1 showed high inhibitory activity against all four tumor cell lines. Among them, the inhibitory activity against the human cervical cancer cell line Hela was the best, and the inhibitory effect was significantly better than that of cisplatin. This shows that the dinuclear ruthenium complex of the PDI inhibitor provided by the present invention can be used as a therapeutic drug for malignant tumors and has a significant therapeutic effect. In addition, compared with the existing anti-cancer drug cisplatin, the dinuclear ruthenium complex of the PDI inhibitor provided by the present invention has a certain selectivity and has a small inhibitory activity against the normal cell line WI-38, indicating that the dinuclear ruthenium complex has low toxicity.
[0070] Experimental Example 2
[0071] The inhibitory activity of the dinuclear ruthenium complex, a PDI inhibitor prepared in Example 1, against protein disulfide isomerase (PDI) was detected using the PROTEOSTAT PDI Detection Kit (purchased from Lawson Enzo Life Sciences, Switzerland). The reductase activity of the PDI inhibitor was detected by measuring the insulin reduction catalyzed by the PDI inhibitor. The specific detection method was as follows:
[0072] The dinuclear ruthenium complex, a PDI inhibitor prepared in Example 1, was added to the insulin PDI solution. Among them, three detection groups with different concentrations were set up, namely the dinuclear ruthenium complex (1 μm) group, the dinuclear ruthenium complex (5 μm) group, and the dinuclear ruthenium complex (10 μm) group. Then, DTT was added to each of the above three detection groups with different concentrations to initiate the reduction activity of the PDI inhibitor. After incubation for 30 min, a stop reagent mixture was added to stop the reaction. Then, the insulin precipitate was labeled with the fluorescent Proteostat PDI detection reagent, and the fluorescence intensity was measured at an excitation wavelength of 500 nm and an emission wavelength of 603 nm, and the IC 50 value was calculated. At the same time, the existing PDI inhibitor isoquercetin (10 μm) was set as a control group and detected using the exactly same detection method as above.
[0073] The results were as Figure 3 shown. It could be seen from Figure 3 that compared with the existing PDI inhibitor isoquercetin, the dinuclear ruthenium complex, a PDI inhibitor provided by the present invention, exhibited good inhibitory activity at low concentrations. At the same concentration, the activity of the dinuclear ruthenium complex, a PDI inhibitor, was 1.8 times that of the existing PDI inhibitor isoquercetin. This indicates that the dinuclear ruthenium complex, a PDI inhibitor provided by the present invention, has good PDI inhibitory activity and broad application prospects.
[0074] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A PDI inhibitor binuclear ruthenium complex, characterized in that The structural formula of the PDI inhibitor binuclear ruthenium complex is shown in Formula I:
2. A method for preparing a PDI inhibitor binuclear ruthenium complex as claimed in claim 1, characterized in that: The steps include: 1) mixing 2-hydrazinobenzothiazole, 2-acetylpyridine, a first organic solvent and a catalyst, and performing a reflux reaction to obtain a Schiff base ligand; 2) mixing the Schiff base ligand, dichlorobis(4-methylisopropylphenyl)ruthenium(II) dimer, a second organic solvent and ammonium hexafluorophosphate, and reacting the mixture to obtain a PDI inhibitor binuclear ruthenium complex; The structural formula of the Schiff base ligand is shown in Formula II:
3. The method for preparing a PDI inhibitor binuclear ruthenium complex according to claim 2, characterized in that: The molar volume ratio of 2-hydrazinobenzothiazole, 2-acetylpyridine, the first organic solvent and the catalyst in step 1) is 3-10 mmol: 3-10 mmol: 15-100 mL: 0.2-1 mL.
4. The method for preparing a PDI inhibitor binuclear ruthenium complex according to claim 3, characterized in that: The temperature of the reflux reaction in step 1) is 45-75° C. and the time is 12-24 hours.
5. The method for preparing a PDI inhibitor binuclear ruthenium complex according to any one of claims 2 to 4, characterized in that: In step 1), the first organic solvent includes one or more of ethanol, methanol and acetonitrile; The catalyst includes acetic acid and / or p-toluenesulfonic acid.
6. The method for preparing a PDI inhibitor binuclear ruthenium complex according to claim 5, characterized in that: The molar volume ratio of the Schiff base ligand, dichlorobis(4-methylisopropylphenyl)ruthenium(II) dimer, the second organic solvent and ammonium hexafluorophosphate in step 2) is 0.1-0.2 mmol: 0.05-0.1 mmol: 10-40 mL: 0.1-0.4 mmol.
7. The method for preparing a PDI inhibitor binuclear ruthenium complex according to claim 6, characterized in that: The reaction time in step 2) is 24 to 48 hours.
8. The method for preparing a PDI inhibitor binuclear ruthenium complex according to claim 7, characterized in that: In step 2), the second organic solvent comprises ethanol and / or methanol.
9. Use of the PDI inhibitor binuclear ruthenium complex prepared by the method for preparing a PDI inhibitor binuclear ruthenium complex according to any one of claims 2 to 8 in preparing drugs for treating tumors.