Cyclo-metal ruthenium (II) complexes containing polyadenosine diphosphate ribopolymerase-1 inhibitors and methods of making and using the same

Abstract

The application discloses a cyclometalated ruthenium (II) complex containing a poly-adenosine diphosphate ribose polymerase-1 inhibitor and a preparation method and application thereof, the complex is any one of Ru1, Ru2 and Ru3, and the cationic structure of the Ru1, Ru2 and Ru3 is shown in formula I. The compound disclosed by the application is a kind of bifunctional anticancer drug with PARP-1 inhibition and DNA damage, has novel structure, good antitumor activity, and has good application prospect in the field of antitumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and in particular relates to a cyclometalated ruthenium (II) complex containing a poly(ADP-ribose) polymerase-1 inhibitor, a preparation method thereof, and an application thereof. Background Art

[0002] The platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin are first-line treatment options for a variety of localized and metastatic cancers. These drugs enter cells via passive diffusion or transporter-mediated uptake, are activated by ligand exchange, and form covalent platinum-DNA adducts, inhibiting transcription and inducing apoptotic cell death in cancer cells. These platinum-based drugs comprise approximately 50% of total chemotherapy regimens and, as such, are included in the World Health Organization (WHO) Essential Medicines List. Despite their remarkable clinical success, major drawbacks, such as inherent and acquired cancer cell resistance and dose-limiting toxicities, severely limit the practical therapeutic efficacy of platinum-based chemotherapy. To address these issues, efforts are underway to develop anticancer compounds with distinct mechanisms of action from platinum-based drugs.

[0003] In recent years, research on ruthenium complexes as anticancer drugs has continued to grow, and several promising ruthenium (Ru) drug candidates have been discovered as potential alternatives to platinum drugs. The Ru(III) coordination compounds NAMI-A and KP1019, developed by Allesio and Keppler, respectively, have entered clinical trials and are activated by reduction in the tumor microenvironment. The teams of Dyson and Sadler have developed piano-stool Ru(II)-aromatic organometallic complexes, such as RM175 and RAPTA-C, which have excellent anti-metastatic, anti-angiogenic, and anti-cancer activities. In particular, this class of compounds exerts its activity through a novel mechanism of action and is able to overcome platinum resistance. In addition, Ru(II) polypyridine complexes have been widely used in fields such as DNA binding, cell imaging, and anti-cancer due to their facile synthesis, tunable photophysical / chemical properties, and good water solubility.

[0004] Poly(ADP-ribose) polymerases (PARPs) can transfer single or multiple ADP-ribose segments to target proteins, recruiting DNA repair-related proteins, thereby mediating DNA single-strand break repair, DNA double-strand break repair, and homologous recombination repair, making PARPs an important target for cancer treatment. PARP inhibitors (PARPis) inhibit DNA repair mechanisms and promote tumor cell apoptosis, proving to be a major breakthrough in clinical chemotherapy. Several PARPis, including Olaparib, Niraparib, and Fluazolepali, have been reported to be effective in the treatment of BRCA1 / 2-mutated breast cancer.

[0005] Ruthenium(II) exhibits similar ligand exchange kinetics to platinum(II), and the octahedral geometry of ruthenium complexes offers unique potential for binding to nucleic acids. Along with platinum-based anticancer drugs, DNA may remain the most commonly targeted biomolecule for metallodrugs. Small molecule drugs can bind to DNA through electrostatic attraction, either through large or small grooves, or by intercalation, where the flat portion of the molecule slides between stacked base pairs, causing DNA damage. However, DNA repair pathways significantly limit the therapeutic efficacy of DNA-targeted metallodrugs, resulting in reduced activity and the development of drug resistance. To address these issues, the limitations of single-agent combinations of drugs with specific functions have been explored. This approach is typically achieved by administering two or more drugs separately or via delivery vehicles such as liposomes or nanoparticles. However, this strategy often faces significant challenges, such as uncertain drug dosing, undesirable drug-drug interactions, and unpredictable immune responses to liposomes or nanoparticles. An alternative strategy is to combine the metallodrug with the pharmacological moiety of a bioactive ligand into a single entity to achieve a diverse spectrum of bioactivity and improve the properties of both components. Summary of the Invention

[0006] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a new cyclometallated ruthenium (II) complex containing a bioactive pharmacophoric poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) inhibitor to obtain a highly active cyclometallated ruthenium complex.

[0007] The present invention also provides a method for preparing a polypyridyl ruthenium (II) complex containing a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor.

[0008] The present invention also provides an application of a polypyridyl ruthenium (II) complex containing a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor in anti-tumor and PARP-1 inhibition.

[0009] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a polypyridyl ruthenium (II) complex containing a poly(ADP-ribose) polymerase-1 inhibitor or a pharmaceutically acceptable salt thereof, wherein the complex is any one of Ru1, Ru2 and Ru3, and the structures of Ru1, Ru2 and Ru3 are shown in Formula I:

[0010]

[0011] Wherein, the X - For halide ions, trifluoromethanesulfonate, perchlorate, BF4 - or PF6 - .

[0012] The method for preparing the polypyridyl ruthenium (II) complex containing a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor of the present invention comprises the following steps:

[0013] (1) Synthesis of product Ru1: Under an inert gas atmosphere, cis-[(bpy)2RuCl2] and AgNO3 are mixed in an organic solvent, stirred at high temperature, and the sediment is separated by filtration. The ligand and NEt3 are added to the filtrate; the solution is heated to reflux for reaction, and then salts with different counter anions are added to the reaction solution. The reaction solution is stirred at room temperature, the solvent is removed under reduced pressure, and Ru1 is purified by chromatography.

[0014] (2) Synthesis of the product Ru2: Under an inert gas atmosphere, cis-[(phen)2RuCl2] and AgNO3 are mixed in an organic solvent, stirred at high temperature to react, the sediment is separated by filtration, and the ligand and NEt3 are added to the filtrate; the solution is heated to reflux for reaction, and then salts with different counter anions are added to the reaction solution, stirred at room temperature, the solvent is removed under reduced pressure, and Ru2 is purified by chromatography.

[0015] (3) Synthesis of the product Ru3: Under an inert gas atmosphere, cis-[(bpy)(dppn)RuCl2] and AgNO3 are mixed in an organic solvent aqueous solution, stirred at high temperature to react, the precipitated material is filtered and separated, and the ligand and NEt3 are added to the filtrate; the solution is heated to reflux for reaction, and then salts with different counter anions are added to the reaction solution, stirred at room temperature, the solvent is removed under reduced pressure, and Ru3 is purified by chromatography.

[0016] The ligand is a S^N ligand, which is a potent PRAP-1 inhibitor 2-(2,3-Dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide.

[0017] Wherein, in said step (1), the molar ratio of cis-[(bpy)2RuCl2], AgNO3, ligand, NEt3 and salts of different counter anions is 1:2:1:1:(2-32).

[0018] Preferably, the molar ratio of cis-[(bpy)2RuCl2], AgNO3, ligand, NEt3 and salts of different counter anions in step (1) is 1:2:1:1:8.

[0019] Wherein, in said step (2), the molar ratio of cis-[(phen)2RuCl2], AgNO3, ligand, NEt3 and salts of different counter anions is 1:2:1:1:(2-32).

[0020] Preferably, the molar ratio of cis-[(phen)2RuCl2], AgNO3, ligand, NEt3 and salts of different counter anions in step (2) is 1:2:1:1:8.

[0021] Wherein, in step (3), the molar ratio of cis-[(bpy)(dppn)RuCl2], AgNO3, ligand, NEt3 and salts of different counter anions is 1:2:1:1:(2-32).

[0022] Preferably, the molar ratio of cis-[(bpy)(dppn)RuCl2], AgNO3, ligand, NEt3 and salts of different counter anions in step (3) is 1:2:1:1:8.

[0023] Preferably, the salts of different counter anions in steps (1) to (3) are ammonium hexafluorosulfate, sodium trifluoromethanesulfonate, sodium perchlorate or sodium tetrafluoroborate.

[0024] Preferably, the preparation method of the optimized complex Ru1-Ru3 of the present invention comprises the following steps:

[0025] Synthesis of Complex Ru1: Under a nitrogen atmosphere, cis-[(bpy)2RuCl2] (0.20 mmol) and AgNO3 (0.40 mmol) were mixed in ethanol (30 mL) and stirred at 70°C for 3 hours. The insoluble AgCl was removed by filtration, and the S^N ligand 2-(2,3-Dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (0.20 mmol) and NEt3 (0.20 mmol) were added to the filtrate. The solution was heated at reflux for 4 hours, followed by the addition of NH4PF6 (1.6 mmol). After 2 hours, the solvent was removed under reduced pressure, and the product was purified by Al2O3 column chromatography using CH2Cl2 / MeOH (30:1) as the eluent to obtain a brownish-black solid.

[0026] Synthesis of the Ru2 complex: Under a nitrogen atmosphere, cis-[(phen)2RuCl2] (0.20 mmol) and AgNO3 (0.40 mmol) were mixed in ethanol (30 mL) and stirred at 70°C for 3 hours. The insoluble AgCl was removed by filtration, and the S^N ligand 2-(2,3-Dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (0.20 mmol) and NEt3 (0.20 mmol) were added to the filtrate. The solution was heated at reflux for 4 hours, followed by the addition of NH4PF6 (1.6 mmol). After 2 hours, the solvent was removed under reduced pressure, and the product was purified by Al2O3 column chromatography using CH2Cl2 / MeOH (30:1) as the eluent to yield a brownish-black solid.

[0027] Synthesis of the Ru3 complex: Under a nitrogen atmosphere, cis-[(bpy)(dppn)RuCl2] (0.20 mmol) and AgNO3 (0.40 mmol) were mixed in ethanol / water (15 mL / 15 mL) and stirred at 70°C for 3 hours. The insoluble AgCl was removed by filtration, and the S^N ligand 2-(2,3-Dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (0.20 mmol) and NEt3 (0.20 mmol) were added to the filtrate. The solution was heated at reflux for 4 hours, followed by the addition of NH4PF6 (1.6 mmol). After 2 hours, the solvent was removed under reduced pressure, and the product was purified by Al2O3 column chromatography using CH2Cl2 / MeOH (30:1) as the eluent to obtain a brownish-black solid.

[0028] The specific synthetic route is as follows:

[0029]

[0030] The invention provides an application of the polypyridyl ruthenium (II) complex containing a poly(ADP-ribose) polymerase-1 inhibitor or a pharmaceutically acceptable salt thereof in the preparation of cancer treatment drugs and kinase inhibitors.

[0031] Wherein, the cancer is breast cancer or cervical cancer.

[0032] Wherein, the kinase is poly ADP-ribose polymerase-1 (PARP-1).

[0033] The pharmaceutical composition for treating cancer or inhibiting kinases of the present invention contains the cyclometallated ruthenium (II) complex containing a poly(ADP-ribose) polymerase-1 inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0034] This study, for the first time, synthesized three cyclometallated Ru(II) complexes with π-conjugated ligands of varying sizes by coordinating a cyclometallated Ru(II) subunit with a PARP-1 inhibitor (S^N ligand). Their anti-tumor and PARP-1 inhibitory activities were evaluated. Furthermore, the introduction of N^N ligands of varying planar sizes into the structures of these cyclometallated Ru(II) complexes was found to induce varying degrees of DNA damage.

[0035] The present invention is the first to improve the biological activity of the ruthenium complex by introducing a bioactive PARP-1 pharmacophore into the cyclometallated ruthenium unit, and for the first time coordinates a PARP-1 inhibitor to the cyclometallated ruthenium unit. Experimental results show that the ruthenium (II) complex provided by the present invention not only exhibits retained inhibition of PARP-1, but also exhibits potent inhibition against three types of cancer cells. The compound provided by the present invention is the first cyclometallated ruthenium complex with the dual effects of PARP-1 inhibition and DNA damage, and has great application and development value. At the same time, the use of ionic Ru complexes is higher in water solubility and better stability than organic small molecules.

[0036] This invention proposes a novel cyclometallated ruthenium complex that can both damage DNA and inhibit PARP-1 expression. This combination of a DNA-damaging drug and a kinase inhibitor enhances cell viability and is less likely to induce drug resistance than single-acting drugs. This is the first cyclometallated ruthenium complex to demonstrate both PARP-1 inhibition and DNA-damaging properties.

[0037] In fact, the present invention synthesizes a cyclometallated ruthenium complex with a completely new structure. The raw material used in the ruthenium complex is inherently inactive, but through a specific method, the pharmacophore is added to exhibit significant anticancer effects. The present invention coordinates the ruthenium complex with a specific S^N ligand to synthesize a cyclometallated ruthenium complex containing a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor. This complex not only inhibits PARP-1 but also has DNA-damaging effects, demonstrating significant activity against tumor cells. The cyclometallated ruthenium complex synthesized by the present invention combines these two effects: increasing cell sensitivity to chemotherapy and enhancing cytotoxicity, resulting in a synergistic anticancer effect.

[0038] The precursor ruthenium complex of the present invention has basically no activity against tumor cells. After being connected to the pharmacophore (i.e., the S^N ligand of the present invention), it shows a significant anti-cancer effect, and the effect is significantly improved compared with the ligand, further achieving synergistic enhancement, and also retains the PARP-1 inhibitory effect of the ligand.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0040] 1. The present invention provides a new cyclometallated ruthenium complex with a simple preparation method, mild reaction conditions, easy industrial mass production, stable structure, higher water solubility than organic small molecules, high reproducibility and high anticancer activity, making it a potential candidate for anticancer drugs.

[0041] 2. The cyclometal ruthenium (II) complex containing a poly(ADP-ribose) polymerase-1 inhibitor described in the present invention contains a metal ligand ion with a positive charge, which can increase the water solubility of such complexes. The octahedral geometry of the metal ruthenium complex provides many opportunities for structural modification, and different active molecules can be introduced for different substrates to achieve the purpose of increasing anti-cancer activity.

[0042] 3. The compounds provided herein incorporate ligands with different π-conjugated systems into their structures, resulting in complexes with varying degrees of DNA damage. Furthermore, the incorporation of S₆N ligands endows these complexes with the potential for PARP-1 inhibition, demonstrating enhanced PARP-1 inhibition compared to other ruthenium complexes. In summary, DNA damage and PARP-1 inhibition jointly contribute to these complexes' cytotoxicity, making them promising for development.

[0043] 4. The compounds provided by the present invention can effectively inhibit the growth of various tumor cells including breast cancer cells or cervical cancer cells; in particular, the IC of compound Ru3 against breast cancer cancer cells MDA-MB-231 and MCF-7 is 50 The IC50 values ​​for Hela cervical cancer cells were as low as 5.67±1.02μM and 4.33±0.91μM, respectively, and the IC50 value for Hela cervical cancer cells was as low as 5.27±0.56μM. The compounds provided by the present invention can be used to prepare drugs for the prevention and / or treatment of various tumors such as breast cancer, liver cancer, lung adenocarcinoma, esophageal cancer, and prostate cancer, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the H NMR spectrum of the complex Ru1;

[0045] Figure 2 This is the high-resolution mass spectrum of the complex Ru1;

[0046] Figure 3 is the H NMR spectrum of the complex Ru2;

[0047] Figure 4 This is the high-resolution mass spectrum of the complex Ru2;

[0048] Figure 5 is the H NMR spectrum of the complex Ru3;

[0049] Figure 6 This is the high-resolution mass spectrum of the complex Ru3;

[0050] Figure 7 This is the confocal imaging image of the comet experiment;

[0051] Figure 8 is the expression level of PARP-1 in MCF-7 cells. DETAILED DESCRIPTION

[0052] The present invention will be further described below with reference to the accompanying drawings and examples.

[0053] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0054] The S^N ligand has been reported as a potent PRAP-1 inhibitor: 2-(2,3-Dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide, synthesized according to the method in the literature J.Med.Chem.2019,62,3051-3067.

[0055] cis-[(bpy)2RuCl2] was synthesized according to the method described in Inorg.Chem. 2013, 52, 12492-12501.

[0056] cis-[(phen)2RuCl2] was synthesized according to the method described in Inorg.Chem.2017, 56, 7519-7532.

[0057] cis-[(bpy)(dppn)RuCl2] was synthesized according to the method in Inorg.Chem.2020, 59, 11193-11204.

[0058] Example 1

[0059] Under a nitrogen atmosphere, cis-[(bpy)2RuCl2] (0.20 mmol) and AgNO3 (0.40 mmol) were mixed in ethanol (30 mL) and stirred at 70°C for 3 hours. The insoluble AgCl was removed by filtration, and the S^N ligand 2-(2,3-Dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (0.20 mmol) and NEt3 (0.20 mmol) were added to the filtrate. The solution was heated at reflux for 4 hours, and then NH4PF6 (1.6 mmol) was added to the reaction solution. After stirring at room temperature for 2 hours, the solvent was removed under reduced pressure, and Ru1 was purified by Al2O3 column chromatography using CH2Cl2 / MeOH (30:1) as the eluent to obtain a brown-black solid, Ru1.

[0060] H NMR spectrum and mass spectrum Figure 1 and Figure 2 As shown: 1 H NMR (600MHz, DMSO-d6) δ9.85 (dd, J=5.6, 0.6Hz, 1H), 8.77 (d, J=8.0Hz, 2H), 8.72 (d, J=8.1Hz,1H),8.61(t,J=5.9Hz,2H),8.19–8.14(m,2H),8.08(s,2H),7.92–7.83(m, 4H),7.75(d,J=5.2Hz,1H),7.69(s,1H),7.49(d,J=5.2Hz,1H),7.38–7.36(m,1H),7 .24–7.22(m,1H),6.86(s,1H),3.86(s,3H),3.67(s,3H)ppm.ESI-MS(m / z),[M-PF6] + .Calcd for C 30 H 26 Br2RuN7O2S:809.92585.Found:809.93012.

[0061] Example 2

[0062] Under a nitrogen atmosphere, cis-[(phen)2RuCl2] (0.20 mmol) and AgNO3 (0.40 mmol) were mixed in ethanol (30 mL) and stirred at 70°C for 3 hours. The insoluble AgCl was removed by filtration, and the S^N ligand 2-(2,3-Dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (0.20 mmol) and NEt3 (0.20 mmol) were added to the filtrate. The solution was heated at reflux for 4 hours, and then NH4PF6 (1.6 mmol) was added to the reaction solution. After stirring at room temperature for 2 hours, the solvent was removed under reduced pressure, and Ru2 was purified by Al2O3 column chromatography using CH2Cl2 / MeOH (30:1) as the eluent to obtain a brown-black solid.

[0063] H NMR spectrum and mass spectrum Figure 3 and Figure 4 As shown: 1 H NMR (600MHz, DMSO-d6) δ10.19(dd,J=5.2,1.1Hz,1H),9.07(dd,J=5.2,1.0Hz,1H),8.83(dd,J=3.5,1.1Hz,1H),8.82( dd,J=3.4,1.0Hz,1H),8.49(dd,J=8.1,0.8Hz,1H),8.43(dd,J=8.2,0.9Hz,1H),8.38(d,J=1.4Hz,1H),8.37(d,J=1.4 Hz,1H),8.31–8.22(m,4H),8.14(s,2H),8.00(dd,J=5.3,0.9Hz,1H),7.66(s,1H),7.63(dd,J=5.3,1.0Hz,1H),7.56( dd,J=8.1,5.3Hz,1H),7.41(dd,J=8.1,5.3Hz,1H),6.73(s,1H),3.84(s,3H),3.63(s,3H)ppm.ESI-MS(m / z),[M-PF6] + .Calcd forC 34 H 26 Br2RuN7O2S:857.92585.Found:857.93022.

[0064] Example 3

[0065] Under a nitrogen atmosphere, cis-[(bpy)(dppn)RuCl2] (0.20 mmol) and AgNO3 (0.40 mmol) were mixed in ethanol / water (15 mL / 15 mL) and stirred at 70°C for 3 hours. The insoluble AgCl was removed by filtration, and the S^N ligand 2-(2,3-Dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (0.20 mmol) and NEt3 (0.20 mmol) were added to the filtrate. The solution was heated at reflux for 4 hours, and then NH4PF6 (1.6 mmol) was added to the reaction solution. After stirring at room temperature for 2 hours, the solvent was removed under reduced pressure, and Ru3 was purified by Al2O3 column chromatography using CH2Cl2 / MeOH (30:1) as the eluent to obtain a brown-black solid.

[0066] H NMR spectrum and mass spectrum Figure 5 and Figure 6 As shown: 1 H NMR(600MHz,DMSO-d6)δ9.96(dd,J=5.4,1.3Hz,1H),9.59(d,J=7.9Hz,1H),9.52(dd,J=8.0,1.0Hz,1H),9.21(s,1H),9.17 (s,1H),8.84(d,J=8.3Hz,1H),8.72(d,J=8.3Hz,1H),8.64(s,1H),8.61(d,J=5.2Hz,1H),8.46–8.39(m,2H),8.30(dd,J=8. 1,5.4Hz,1H),8.25–8.21(m,1H),8.19(dd,J=5.3,1.2Hz,1H),7.97(dd,J=8.1,5.4Hz,1H),7.96–7.92(m,1H),7.85–7.81(m ,1H),7.80–7.73(m,3H),7.26(s,2H),7.23–7.19(m,1H),6.74(s,1H),3.83(s,3H),3.62(s,3H)ppm.ESI-MS(m / z),[M-PF6] + .Calcd for C 42 H 30 Br2RuN9O2S:985.96330.Found:985.96788.

[0067] Example 4

[0068] Detection of the inhibitory activity of the complex Ru1-Ru3 on PARP-1 enzyme

[0069] Experimental Methods: The inhibitory activity of Ru1-Ru3, S^N ligands, cis-[(bpy)2RuCl2], and cis-[(phen)2RuCl2] against PARP-1 was determined using a universal PARP colorimetric assay kit (Trevigen, Cat#4677-096-K). Each well of a 96-well plate was pre-coated with 100 μL of histone (Trevigen, Cat#4677-096-K) (20 μg / mL) diluted in PBS buffer (10 mM NaH2PO4, 10 mM Na2HPO4, 150 mM NaCl, pH 7.4) and incubated overnight at 37°C. After incubation, the cells were washed three times with 200 μL of PBST buffer (1× PBS containing 0.05% (v / v) Tween 20), blocked with 200 μL of buffer (1× PBST buffer containing 5% (v / v) skim milk) at room temperature for 60 minutes, and then washed three times with 200 μL of PBST buffer as described above. + Activator oligonucleotide (8 μM) and activator oligonucleotide (100 μg / mL) were added to 70 μL of reaction buffer (50 mM Tris-HCl, 2 mM MgCl2, pH 8.0), followed by the addition of 10 μL of the compound at various concentrations. The test compound was diluted with 10% (v / v) DMSO and serially diluted 10 times, starting at 1 μM. 20 μL of PARP-1 (10 ng / well) was added and incubated at 37°C for 1 hour. The reaction mixture was discarded, the wells were rinsed three times with 200 μL of PBST buffer, and the wells were gently tapped onto a clean tissue as above. 50 μL of streptavidin-HRP was added to each well and incubated at room temperature for 30 minutes. The wells were rinsed three times with 200 μL of PBST buffer and gently tapped onto a clean tissue as above. Finally, 100 μL of ECL solution was added and incubated at room temperature for 15 minutes. Luminescence signals were measured using a multi-well spectrophotometer (Molecular Devices SpectraMax M5 microplate reader). Luminescence data were analyzed using GraphPad Prism software. In the absence of compound, the luminescence (L t ) was defined as 100% activity. In the absence of PARP-1, the luminescence (Lb) in each data set was defined as 0% activity. The percentage activity in the presence of each compound was calculated as follows: % activity = [(LL b ) / (L t -L b )]×100, where L is the luminescence when the compound exists, L b is the luminescence in the absence of PARP, L tThe inhibition rate was calculated according to the following formula: % inhibition = 100 - % activity. The results are shown in Table 1.

[0070] Table 1. Inhibition of PARP-1 by compounds

[0071]

[0072]

[0073] According to the data in Table 1, S^N ligand showed strong PARP-1 inhibitory activity, IC 50 The value was 36 nM, which is similar to the results reported previously [J.Med.Chem.2019,62,3051-3067]. Ru1-Ru3 retained the PARP-1 inhibitory activity of S^N ligand to varying degrees, IC 50 As expected, the Ru(II) precursors cis-[(bpy)2RuCl2] and cis-[(phen)2RuCl2] showed negligible PARP-1 inhibitory activity, with IC 50 The value exceeded 1000nM.

[0074] Example 5

[0075] In vitro cytotoxic activity test.

[0076] In order to evaluate the in vitro antitumor activity of the complexes Ru1-Ru3 on human breast cancer cells (MDA-MB-231 cells and MCF-7 cells) and human cervical cancer cells (HeLa cells), the MTT colorimetric assay was used. 4 Cells were seeded in 96-well plates at a concentration of 100 cells / mL and cultured overnight. The cells were exposed to different concentrations of the compound and incubated at 37°C for 48 hours. Then, MTT solution at a concentration of 5 mg / mL was added to each well and incubated for another 4 hours. Insoluble crystals were dissolved with dimethyl sulfoxide (DMSO) and the absorbance (OD value) was measured at 490 nm using a microplate reader. The formula for calculating cell viability is as follows:

[0077] Survival rate (%) = (OD 实验组 ) / (OD 对照组 )*100%

[0078] The half inhibitory concentration (IC 50 The values ​​are shown in Table 2.

[0079] Table 2. IC values ​​of Ru1-Ru3 and L1 for inhibiting the growth of different cell lines 50 value.

[0080]

[0081]

[0082] As shown in Table 2, the IC values ​​of Ru1-Ru3 for MDA-MB-231, HeLa, and MCF-7 cells were 50 The values ​​were 5.67-25.66 μM, 5.27-19.81 μM and 4.33-14.54 μM, respectively. Compared with cisplatin, Ru1-Ru3 showed comparable or stronger cytotoxicity in the three cancer cell lines. It is worth noting that the cytotoxic activity of Ru1-Ru3 was much higher than that of the Ru(II) precursor or S^N ligand, which indicates that the coordination of the Ru(II) part with the thiosemicarbazide analogue plays a decisive role in exerting the anticancer effect. It is worth noting that among the Ru(II) complexes, Ru3 showed the strongest inhibitory effect on the three cancer cell lines, especially on MCF-7 cells. In addition, the IC value of Ru3 against the normal human liver cell line (L02) was 0. 50 The value is high, about twice that of cisplatin, which is beneficial for chemotherapy. At the same time, with the increase of auxiliary ligand π-conjugated system (N^N ligand), the anticancer activity of Ru1-Ru3 is enhanced.

[0083] Example 6

[0084] DNA damage research

[0085] Comet assay was used to detect DNA damage. 5 10 μM Ru1, Ru2 and Ru3 were added to the culture medium respectively and incubated at 37°C for 24 hours. MCF-7 cells were collected by trypsin digestion after 24 hours. 100 μL of 1% normal melting point agarose was dissolved in PBS and gently dropped onto a completely frosted microslide. The slide was immediately covered with a coverslip and placed at 4°C for 15 minutes. After the gel solidified, the coverslip was removed. 10 μL of cell suspension (10 5cells / mL) were mixed with 75 μL of 0.75% low-melting point agarose (Beijing Biolab Technology Co., Ltd., kfs210) at 60-80°C. A total of 85 μL of the mixture was quickly dropped onto the gel, immediately covered with a coverslip, and placed at 4°C for 15 minutes. After the gel solidified, the coverslip was removed again. The third layer was coated with 75 μL of 0.75% low-melting point agarose and placed at 4°C for 20 minutes. After the agarose solidified, the cover layer was removed and the slide was immersed in ice-cold lysis solution (9 mL Lysis Buffer / 1 mL DMSO) and placed in a 4°C refrigerator for 1 hour. All of the above operations were performed under low light conditions to avoid additional DNA damage. After removing the slide from the lysis solution, it was placed horizontally in the electrophoresis chamber. The slides were covered with electrophoresis buffer (300 mM NaOH, 1.2 mM EDTA) by approximately 0.25 cm, and the DNA was allowed to lyse in the electrophoresis solution for 35 minutes. Electrophoresis was then performed at 25 V and 300 mA for 25 minutes. After electrophoresis, the slides were removed and washed three times in neutralization buffer (0.4 mM Tris, HCl, pH 7.5). MCF-7 cells were stained with 25 μL of EB (20.0 mg / mL) in the dark for 25 minutes. The slides were washed in chilled distilled water for 15 minutes to neutralize excess alkali, air-dried, and comets were imaged using an Olympus FV1000 confocal microscope (Olympus, Japan).

[0086] DNA damage results in Figure 7 As shown in the figure, after incubating MCF-7 cells with Ru1-Ru3 (10 μM) for 24 hours, the length of the tail DNA increased from 2.8% to 15.1%, 22.0%, and 32.5%, respectively, indicating that these complexes can induce severe DNA damage and lead to cell death. Furthermore, the larger the planar area of ​​the N^N ligand, the more severe the DNA damage. Therefore, the synthetic compounds of the present invention can not only cause DNA damage but also inhibit the activity of PARP-1, thereby preventing DNA damage from being repaired, improving activity, and preventing cells from developing drug resistance.

[0087] Example 7

[0088] Immunoblot analysis

[0089] MCF-7 cells (1×10 6) were cultured in a 10 cm dish for 24 h, and Ru1-Ru3 complexes (10 μM) were added and cultured for 12 h. After the culture was completed, the cells were washed twice with PBS, lysed and scraped. The lysate was collected by centrifugation and the protein concentration was determined by the BSA method. Afterwards, the extracted protein was added to 5× SDS protein loading buffer in proportion and mixed, and the protein was denatured by boiling at high temperature for 10 min. SDS polyacrylamide gel electrophoresis was used, and the protein was then transferred to a polyvinylidene fluoride membrane at 120 V for 90 minutes. Subsequently, the membrane was blocked with 5% (w / V) skim milk powder in Tris-buffered saline / 0.1% Tween 20 (TBST) buffer at room temperature for 2 hours and washed twice with TBST. Incubated with PARP-1 and β-actin primary antibodies at 4°C for 12 h, then washed 3 times with TBST, and incubated with the corresponding secondary antibodies at room temperature for 2 h. After washing three times, the membrane was observed with chemiluminescence reagent (ECL), and the labeled proteins were detected using an automatic chemiluminescence imaging analysis system (Tanon-5200, Shanghai, China).

[0090] Western Blot analysis was used to detect the expression of PARP-1 in MCF-7 cells treated with Ru1-Ru3 and cis-[(phen)2RuCl2] at the same concentration (10 μM). Figure 8 As can be seen, PARP-1 expression in MCF-7 cells treated with cis-[(phen)2RuCl2] remains unchanged, while Ru1-Ru3 can effectively induce the cleavage of the PARP-1 enzyme, which is consistent with the results of enzyme activity assays. Once cleaved, PARP-1 loses its activity, thereby inhibiting DNA repair. Therefore, enzyme inhibition may be one possible cause of the blockage of DNA damage repair. This experiment effectively demonstrates that the compounds of the present invention can cause the cleavage of PARP-1, thereby preventing DNA damage repair, enhancing activity, reducing the risk of drug resistance, and increasing sensitivity to chemotherapeutic drugs.

Claims

1. A cyclometallated ruthenium (II) complex or a pharmaceutically acceptable salt thereof, characterized in that: The complex is any one of Ru1, Ru2 and Ru3, and the structures of Ru1, Ru2 and Ru3 are shown in Formula I: ； Wherein, the X - For halide ions, trifluoromethanesulfonate, perchlorate, BF4 - or PF6 - .

2. A method for preparing the cyclometallated ruthenium (II) complex according to claim 1, characterized in that: The steps include: (1) Synthesis of product Ru1: Under an inert gas atmosphere, cis -[(bpy)2RuCl2] and AgNO3 are mixed in an organic solvent and stirred at high temperature to react. The sediment is separated by filtration, and the ligand and NEt3 are added to the filtrate; the solution is heated to reflux for reaction, and then salts with different counter anions are added to the reaction solution. The mixture is stirred at room temperature, the solvent is removed under reduced pressure, and Ru1 is purified by chromatography; (2) Synthesis of Ru2: In an inert gas atmosphere, cis -[(phen)2RuCl2] and AgNO3 are mixed in an organic solvent and stirred at high temperature to react. The sediment is separated by filtration, and the ligand and NEt3 are added to the filtrate; the solution is heated to reflux for reaction, and then salts with different counter anions are added to the reaction solution. The mixture is stirred at room temperature, the solvent is removed under reduced pressure, and Ru2 is purified by chromatography; (3) Synthesis of Ru3: In an inert gas atmosphere, cis -[(bpy)(dppn)RuCl2] and AgNO3 are mixed in an organic solvent and reacted at high temperature with stirring. The precipitated material is separated by filtration, and the ligand and NEt3 are added to the filtrate. The solution is heated to reflux for reaction, and then salts with different counter anions are added to the reaction solution. The solution is stirred at room temperature, the solvent is removed under reduced pressure, and Ru3 is purified by chromatography. Wherein, the ligand is S^N and the ligand structure is: 。 3. The preparation method according to claim 2, characterized in that In the step (1) cis The molar ratio of -[(bpy)2RuCl2], AgNO3, ligand, NEt3 and salts of different counter anions is 1:2:1:1:(2-32).

4. The preparation method according to claim 2, characterized in that In the step (2) cis The molar ratio of -[(phen)2RuCl2], AgNO3, ligand, NEt3 and salts of different counter anions is 1:2:1:1:(2-32).

5. The preparation method according to claim 2, characterized in that In the step (3) cis The molar ratio of -[(bpy)(dppn)RuCl2], AgNO3, ligand, NEt3 and salts of different counter anions is 1:2:1:1:(2-32).

6. Use of the cyclometallated ruthenium (II) complex or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of cancer treatment drugs and kinase inhibitors.

7. The use according to claim 6, characterized in that The cancer is breast cancer or cervical cancer.

8. The use according to claim 6, characterized in that The kinase is poly ADP-ribose polymerase-1 (PARP-1).

9. A pharmaceutical composition for treating cancer or inhibiting kinases, comprising the cyclometallated ruthenium (II) complex or a pharmaceutically acceptable salt thereof as claimed in claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.

Images