A metal platinum complex dual-targeted to mitochondria and nucleus, and its preparation method and application

Abstract

The present invention discloses a metal platinum complex with dual targeting of mitochondria and cell nuclei, as well as a preparation method and application thereof. The metal platinum complex with dual targeting of mitochondria and cell nuclei is obtained by combining cisplatin with a molecule containing a mitochondrial targeting group. The complex of the present invention can effectively inhibit the proliferation of human tumor cells, induce mitochondrial and DNA damage in tumor cells, and induce cancer cell death through mitochondrial autophagy and necrosis. In addition, the complex of the present invention can degrade mutant p53 protein, thereby eliminating the acquired function phenotype associated with mutant p53, including significantly inhibiting tumor growth, invasion and drug resistance, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to a metal platinum complex, in particular to a mitochondria and cell nucleus dual-targeting metal platinum complex, and also to a preparation method and application of the complex. Background Art

[0002] Subcellular organelles, such as the nucleus, mitochondria, and lysosomes, are crucial intracellular organs responsible for maintaining biological processes such as cell proliferation and differentiation, signal transduction, and energy metabolism. Mitochondria, known as the "energy factories" of the cell, also play a crucial role in many essential cellular processes. They synthesize ATP to support various cellular processes and participate in many other important functions, including initiation of apoptotic pathways, calcium metabolism, electron transport, and reactive oxygen species generation. The nucleus is the control center of the eukaryotic cell, hosting most of the genetic material and the various proteins involved in DNA replication and transcription. Many fundamental biological functions, such as gene expression, cell differentiation, cell growth, and apoptosis, occur near the nucleus. Due to its critical role in cell growth and proliferation, the nucleus has been implicated in carcinogenesis. Due to their roles in energy production and programmed cell death, these organelles have become major therapeutic targets in various diseases and conditions. Rationally designed organelle-targeting metal complexes can directly disrupt subcellular structures, further activating certain cell death pathways, thereby improving therapeutic efficacy and even overcoming drug resistance. In recent years, the design of anticancer metal complexes that specifically target organelles to achieve precise drug delivery and avoid possible toxic side effects of drugs has attracted widespread attention.

[0003] The discovery of cisplatin and its clinical success represent a milestone in the development of anti-tumor drugs. p53, as a key transcription factor, regulates the transcription of target genes and is a downstream effector molecule of intracellular DNA damage, playing a critical role in the mechanism of action of cisplatin. However, more than 50% of human cancers carry TP53 gene mutations, which lead to the production of dysfunctional mutant p53 proteins. These proteins are highly stable and accumulate in tumor cells, exhibiting oncogenic GOF (gain of function) phenotypes, such as promoting tumor proliferation, survival, and migration. Literature reports that changes in TP53 are associated with the development of sensitivity and resistance of cancer cells to cisplatin, and accurate p53 stability and activity can reduce the cisplatin resistance of tumor cells. Currently, no platinum-based metal anticancer agents can successfully treat mutant p53 tumors and reverse cisplatin resistance. Therefore, there is an urgent need to develop degrader materials to overcome cisplatin resistance. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a metal platinum complex that is dual-targeted to mitochondria and cell nuclei, and also to provide a preparation method and application of the above-mentioned complex.

[0005] Technical solution: The present invention discloses a metal platinum complex that is dual-targeted to mitochondria and cell nuclei, the structural formula of which is shown below:

[0006]

[0007] The preparation method of the above-mentioned complex comprises the following steps:

[0008] (1) 3-(9,9-dimethylacridin-10(9H)-yl)-5-(pyridin-4-yl)phenol, triphenylphosphine and K2CO3 were dissolved in acetone and stirred under argon atmosphere for reaction under reflux. After the reaction, the supernatant was collected by centrifugation, the solvent was evaporated and recrystallized to obtain a triphenylphosphine-modified ligand.

[0009]

[0010] (2) mixing the triphenylphosphine-modified ligand obtained in step (1), cisplatin, and DMF, stirring and reacting in the dark; after the reaction, removing the solvent by rotary evaporation, redissolving, and recrystallizing to obtain a metal platinum complex that dual-targets mitochondria and cell nuclei;

[0011]

[0012] Wherein, in step (1), the molar ratio of 3-(9,9-dimethylacridin-10(9H)-yl)-5-(pyridin-4-yl)phenol, triphenylphosphine and K2CO3 is 1:1.3:3.

[0013] The reflux reaction is carried out at a temperature of 45-50°C for 10-12 hours, preferably at 50°C for 12 hours.

[0014] The molar ratio of the triphenylphosphine-modified ligand to cisplatin is 1:1.

[0015] Wherein, in step (2), the reaction temperature is 50-55°C and the reaction time is 48-60h; preferably 55°C and 60h.

[0016] Wherein, in step (2), the re-dissolving is carried out by using hot methanol.

[0017] Wherein, in step (1) or (2), the recrystallization is performed by using ether and dichloromethane for at least 3 times.

[0018] The above-mentioned mitochondria- and nucleus-dual-targeted metal platinum complex can also be used in the preparation of anti-tumor drugs or anti-tumor drug components.

[0019] The tumor is a human lung cancer cell; the metal platinum complex inhibits the proliferation of tumor cells and induces mitochondrial dysfunction in cancer cells and inhibits DNA damage repair.

[0020] Principle of the Invention: The planar square geometry of the present invention's dual-targeted platinum complex for both mitochondria and the nucleus facilitates ligand replacement. Chlorine ligands in the complex are replaced by water molecules, forming a hydrate. The positive charge at the center of the complex then attracts it to negatively charged nuclear DNA, which in turn causes it to induce nuclear DNA damage. Furthermore, the platinum complex contains a mitochondrial-targeting ligand, enabling it to simultaneously target mitochondria and induce mitochondrial dysfunction, thereby inhibiting DNA damage repair and suppressing tumor cell proliferation.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The complex DP-Pt of the present invention can solve the problem that platinum-based metal anticancer agents cannot treat mutant p53 tumors; (2) The complex DP-Pt of the present invention exhibits high cytotoxicity to various tumor cells, and its IC value for human lung cancer resistant cells (A549R) is 50 The value is 6.3uM, which can effectively inhibit the proliferation of tumor cells; (3) After entering the cell, the complex DP-Pt of the present invention achieves dual targeting of mitochondria and cell nucleus, and achieves good anti-tumor activity by inducing mitochondrial dysfunction in cancer cells and inhibiting DNA damage repair; (4) The complex DP-Pt of the present invention can also induce mitochondrial autophagy and necroptosis in tumor cells, and has a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 1. The subcellular organelle distribution (A) and cellular uptake quantitative diagram (B) of the complex DP-Pt of the present invention;

[0023] Figure 2 Figure (A) and immunofluorescence image (B) of the degradation of Mutp53 protein by the complex DP-Pt of the present invention;

[0024] Figure 3 This is a graph showing the production of reactive oxygen species in A549R cells induced by the complex DP-Pt of the present invention;

[0025] Figure 4 This is a diagram showing the depolarization of A549R cell mitochondria induced by the complex DP-Pt of the present invention;

[0026] Figure 5 This is a diagram of the mitochondrial autophagy induced by the complex DP-Pt of the present invention in A549R cells;

[0027] Figure 6This is a diagram showing DNA damage and cell necrosis in A549R cells induced by the complex DP-Pt of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below with reference to the examples. The test materials used in the examples can all be purchased through conventional channels.

[0029] Example 1

[0030] The preparation method of the mitochondrial and nucleus dual-targeted metal platinum complex (DP-Pt) of the present invention comprises the following steps:

[0031] (1) Synthesis of compound DP-PPh3: DP-OH (3-(9,9-dimethylacridin-10(9H)-yl)-5-(pyridin-4-yl)phenol) (37.8 mg, 0.1 mmol), triphenylphosphine (62.2 mg, 0.13 mmol), and potassium carbonate (41.5 mg, 0.3 mmol) were added to a Shrek tube. 15 mL of acetone was used as a solvent, and the mixture was reacted under argon at 50°C overnight. After the reaction was completed, the supernatant was centrifuged and the solvent was evaporated. The product was recrystallized with ether and dichloromethane at least three times to obtain a yellow-brown oily product, named DP-PPh3, with a yield of about (62 mg, 80%). 1 H NMR(400MHz,Chloroform-d)δ(ppm):8.71-8.66(m,2H),7.94-7.85(m,7H),7.75(dtd,J=8.4,6.9 ,1.7Hz,4H),7.67(td,J=7.5,3.4Hz,5H),7.62-7.57(m,2H),7.49(dd,J=7.4,1.9Hz,2H),7.36(t ,J=1.9Hz,1H),7.23(d,J=1.7Hz,1H),6.98(dtd,J=15.5,7.3,1.7Hz,4H),6.35(dd,J=7.8,1.7Hz ,2H),4.26(t,J=5.7Hz,2H),4.03(s,2H),2.37-2.25(m,2H),1.91(q,J=7.7Hz,2H),1.72(s,6H).

[0032]

[0033] (2) Synthesis of complex DP-Pt: Dissolve silver nitrate in 3 mL of anhydrous DMF and add dropwise to a solution of cisplatin (30.0 mg, 0.1 mmol) dissolved in 6 mL of anhydrous DMF. The mixture was reacted at 45°C in the dark. After the reaction was completed overnight, centrifugation was performed to obtain 9 mL of a clear solution, which was stored in the dark and labeled as solution A. Dissolve DP-PPh3 (77.6 mg, 0.1 mmol) in 3 mL of anhydrous DMF. Then, dissolve an equimolar amount of silver nitrate (17.0 mg, 0.1 mmol) in 3 mL of DMF and add dropwise to the DP-PPh3 solution using a syringe. The mixture was reacted at 50°C in the dark under an argon atmosphere overnight to remove bromide ions. After the reaction was completed, the supernatant was taken and labeled as solution B. 3 mL of solution A prepared in the previous step was added to solution B. The mixture was reacted at 55°C in the dark for 60 h. The solvent was removed by rotary evaporation. The mixture was redissolved in hot methanol and recrystallized at least three times using ether and dichloromethane. An off-white product was obtained, named DP-Pt, with a yield of about (45.5 mg, 42%). 1 H NMR(400MHz,DMSO-d6)δ8.75(s,2H),8.06(s,3H),7.78(s,15H),7.55(s,5H),6.97(s,5H),6.21 (s,1H),4.79(s,5H),4.28(s,2H),4.19(d,J=8.7Hz,2H),1.96(s,2H),1.71(s,2H),1.64(s,6H).

[0034]

[0035] Comparative Example 1

[0036] A method for preparing a lysosome-targeted metal platinum complex (DM-Pt), comprising the following steps:

[0037] (1) Synthesis of compound DP-MOR: DP-OH (3-(9,9-dimethylacridin-10(9H)-yl)-5-(pyridin-4-yl)phenol) molecules, 4-(2-chloroethyl)morpholine, and K2CO3 were dissolved in acetone; the mixture was stirred and refluxed under an argon atmosphere; after the reaction, the supernatant was obtained by centrifugation, and the crude product was purified by silica gel column chromatography using CH2Cl2 / MeOH as eluent to obtain a gray-brown product, named DP-MOR; the yield was approximately (184.2 mg, 75%). 1H NMR (400MHz, DMSO-d6) δ8.63(d,J=5.5Hz,2H),7.83(d,J=6.2Hz,2H),7.64-7.38(m,4H),7.11-6.86( m,5H),6.27(d,J=8.1Hz,2H),4.25(t,J=5.7Hz,2H),3.57(t,J=4.7Hz,4H),2.73(s,2H),1.64(s,6H).

[0038] (2) Synthesis of complex DM-Pt: Dissolve silver nitrate in 3 mL of anhydrous DMF and add dropwise to a solution of cisplatin (30.0 mg, 0.1 mmol) dissolved in 6 mL of anhydrous DMF. The mixture was reacted at 45°C in the dark. After the reaction was complete, centrifuge to obtain 9 mL of a clear solution, which was stored in the dark and labeled as solution A. Dissolve DP-MOR (49.1 mg, 0.1 mmol) in 3 mL of anhydrous DMF. Then, dissolve an equimolar amount of silver nitrate (17.0 mg, 0.1 mmol) in 3 mL of DMF and add dropwise to the DP-MOR solution using a syringe. The mixture was reacted at 50°C in the dark under an argon atmosphere overnight to remove bromide ions. After the reaction was complete, the supernatant was collected and labeled as solution B. 3 mL of solution A prepared in the previous step was added to solution B. The mixture was reacted at 55°C in the dark for 60 h. The solvent was removed by rotary evaporation, redissolved in hot methanol, and recrystallized at least three times using ether and dichloromethane. A gray-brown product was obtained, named DM-Pt, with a yield of about (25.7 mg, 56%). 1 H NMR(400MHz, DMSO-d6)δ8.78(dd,J=38.6,6.1Hz,2H),8.09(d,J=22.5Hz,2H),7.82-7.43(m,6H),7.19(d,J=19.8Hz,2H), 6.95(dt,J=28.6,7.6Hz,5H),6.26-6.18(m,2H),4.66(s,2H),4.28(d,J=22.2Hz,2H),3.57(s,4H),1.64(d,J=6.4Hz,6H).

[0039] For the complex DP-Pt prepared in Example 1, an in vitro cytotoxicity experiment on tumor cells was conducted:

[0040] The cytotoxicity of DP-Pt, DM-Pt, Cis-Pt, DP-PPh3 and DP-MOR to different tumor cells was determined by MTT assay. 100 μL cell suspension was diluted to 8×10 4Cells were seeded at a density of 100 μL / mL in a 96-well plate. After overnight incubation in an incubator, a gradient of complex concentrations was added to the 96-well plate and the cells were incubated for 48 hours. Then, 20 μL of MTT was added to each well and incubated for an additional 4 hours. All culture medium was carefully removed, and 150 μL of DMSO was added. The absorbance was measured using a microplate reader, and cytotoxicity data was calculated using SPSS software.

[0041] Table 1. IC values ​​of complexes DP-Pt, DM-Pt, Cis-Pt, DP-PPh3 and DP-MOR 50 (μM) value

[0042]

[0043] The results showed that the complex DP-Pt had higher proliferation inhibitory activity against A549R cells than the compound DP-MOR, complexes DM-Pt and Cis-Pt, indicating that the modification of the mitochondrial targeting group significantly increased the cytotoxic activity against tumor cells; at the same time, the complex DP-Pt had higher selectivity for normal cells than DP-PPh3, and had higher biosafety.

[0044] Application of Quantitative Subcellular Organelle Distribution and Cellular Uptake of DP-Pt Complex:

[0045] Methods: The cellular uptake and organelle distribution of DP-Pt, DM-Pt, and Cis-Pt by A549R cells were determined by ICP-MS. A549R cells were seeded in 10 mL culture dishes and incubated overnight. The culture medium was removed and replaced with fresh medium containing 10 μM of the complexes. After 6 hours of incubation, the cells were harvested, and the nuclei, mitochondria, and cytoplasm were extracted and digested using a mitochondrial / nuclear isolation kit. The platinum content in the samples was then determined by ICP-MS.

[0046] Subcellular organelle localization of complexes in uptake Figure 1 As shown, the results show that the complex DP-Pt is mainly distributed in mitochondria and cell nucleus.

[0047] For the complex DP-Pt prepared in Example 1, an experiment was conducted to degrade Mutp53 protein:

[0048] Western blotting: A549R cells were seeded in 10 mL culture dishes and incubated overnight. The culture medium was removed and replaced with fresh culture medium containing 10 μM of the complexes DP-Pt and Cis-Pt, and the cells were incubated for 48 hours. Cells were harvested in a 15 mL centrifuge tube, washed twice with PBS, and then transferred to a 1.5 mL centrifuge tube and lysed with 200–400 μL of cell lysis buffer. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein were separated by electrophoresis on an SDS-PAGE gel and transferred to a PVDF membrane. After transfer, the membrane was blocked in skim milk powder for 1–2 hours and then incubated with the specific antibody at 4°C with shaking overnight. The membrane was washed with PBST and incubated with the corresponding secondary antibody for an additional 1 hour at room temperature. The membrane was then washed four times with PBST, and immunoblotting was performed using a chemiluminescence analyzer.

[0049] Or take immunofluorescence experiments: A549R cells were seeded in 35mm confocal culture dishes and incubated overnight, and then DP-Pt and Cis-Pt were added and incubated for 48h. After fixing with 4% paraformaldehyde for 30min, wash three times with PBS. LC3, γ-H2AX, and p53 antibodies prepared with skim milk powder were added to the culture dish and incubated at 4°C overnight. After completing the skim milk powder blocking and PBS washing steps, the corresponding secondary antibodies were added and incubated for 1h. Finally, the cell nuclei were stained with DAPI (300nM) for 15min. DAPI was aspirated and washed three times with PBS. After completion, the cells were immediately observed with a confocal microscope (A1, Nikon, Japan), λ ex =488nm and λ em =530±20nm.

[0050] The results of the complex DP-Pt degradation of Mutp53 protein are as follows Figure 2 As shown; the results showed that DP-Pt effectively degraded mutp53 protein, while cisplatin and DM-Pt had no effect on mutp53 protein. Figure 2 Middle B also shows that the immunofluorescence phenomenon is consistent with the above results.

[0051] Application of the complex DP-Pt of the present invention in inducing A549R cells to produce reactive oxygen species:

[0052] Methods: Changes in reactive oxygen species in tumor cells were detected by confocal microscopy. Confocal imaging: A549R cells were seeded in 35 mm confocal culture dishes and incubated overnight. DP-Pt and Cis-Pt were then added and incubated for 24 h. Subsequently, the cells were stained with 10 μM DCF-DA reactive oxygen species probe for 30 min and washed three times with PBS. After treatment, the cells were observed using a confocal microscope (A1, Nikon, Japan). ex =488nm and λ em=530±20nm.

[0053] The results of the changes in active oxygen in A549R cells induced by the complex DP-Pt are shown in Figure 2. Figure 3 The results showed that no significant green fluorescence was observed in A549R cells in the presence of the control. However, a significant increase in green fluorescence was observed after treatment with DP-Pt. Therefore, DP-Pt can significantly activate the overexpression of ROS.

[0054] Application of the complex DP-Pt of the present invention to induce mitochondrial depolarization in A549R cells:

[0055] Methods: Changes in mitochondrial membrane potential in tumor cells were detected by confocal microscopy. Confocal imaging: A549R cells were seeded in 35 mm confocal culture dishes and incubated overnight. DP-Pt and Cis-Pt were then added and incubated for 24 h. Subsequently, the cells were stained with 10 μM JC-1 working solution for 30 min and washed four times with 1× buffer. After treatment, the cells were observed using a confocal microscope (A1, Nikon, Japan). JC-1 Monomers: λ ex =490nm,λ em =520±20nm; JC-1Aggregates: λ ex =525nm,λ em =580±20nm.

[0056] The results of the changes in mitochondrial membrane potential of A549R cells induced by the complex DP-Pt are shown in Figure 4 The results showed that compared with the control example, after the complex treatment, the green fluorescence was significantly enhanced and the red fluorescence was significantly weakened, indicating that the complex DP-Pt can effectively induce a decrease in mitochondrial membrane potential.

[0057] Application of the complex DP-Pt of the present invention in inducing mitochondrial autophagy in A549R cells:

[0058] Methods: Confocal microscopy was used to assess mitochondrial-lysosomal colocalization. Confocal imaging: A549R cells were seeded in 35 mm confocal culture dishes and incubated overnight. 10 μM DP-Pt was then added for 48 hours. Subsequently, cells were incubated with 25 nM LysoBlue probe for 30 minutes and washed four times with PBS. Cells were then incubated with 500 nM MitoRed probe for 30 minutes and washed four times with PBS. After treatment, cells were imaged using a confocal microscope (A1, Nikon, Japan).

[0059] The results of the complex DP-Pt inducing mitochondrial lysosome fusion in A549R cells are shown in Figure 5The results showed that the co-labeling degree of mitochondrial red probe and lysosomal blue probe in A549R cells treated with DP-Pt was significantly higher than that in the control group, which confirmed the occurrence of mitophagy.

[0060] Application of the complex DP-Pt to induce DNA damage and necroptosis in A549R cells:

[0061] Methods: Western blotting (WB) was used to detect changes in the levels of relevant proteins. A549R cells were seeded in 10 mL culture dishes and incubated overnight. The culture medium was removed and replaced with fresh culture medium containing 10 μM of the complexes DP-Pt and Cis-Pt for 48 hours. Cells were harvested in a 15 mL centrifuge tube, washed twice with PBS, and then transferred to a 1.5 mL centrifuge tube and lysed with 200-400 μL of cell lysis buffer. After lysis, protein concentration was determined using a BCA protein assay kit. Equal amounts of protein were separated by electrophoresis on SDS-PAGE gels and transferred to PVDF membranes. After transfer, the membranes were blocked in skim milk powder for 1-2 hours and then incubated with specific antibodies at 4°C with shaking overnight. The membranes were washed with PBST and then incubated with the corresponding secondary antibodies for an additional 1 hour at room temperature. The membranes were then washed four times with PBST, and immunoblotting was performed using a chemiluminescence analyzer.

[0062] The experimental results of the complex DP-Pt inducing DNA damage and necroptosis-related protein expression in A549R cells are as follows Figure 6 The results showed that, compared with the control, DP-Pt treatment significantly upregulated the marker protein γ-H2AX, suggesting that its inhibitory activity against PARP-1 may be a primary factor in preventing DNA damage repair. RIP3, a marker of cell necrosis, was also upregulated in a concentration-dependent manner following DP-Pt treatment. These results suggest that DP-Pt induces DNA damage and necroptosis in A549R cells.

[0063] Therefore, the mitochondrial and nucleus dual-targeted metal platinum complex of the present invention can effectively inhibit the proliferation of human tumor cells, induce mitochondrial and DNA damage in tumor cells, and induce cancer cell death through mitochondrial autophagy and necrosis; in addition, the complex of the present invention can degrade mutant p53 protein, thereby eliminating the acquired function phenotype associated with mutant p53, including significantly inhibiting tumor growth, invasion and drug resistance, and has broad application prospects.

Claims

1. A metal platinum complex that dual-targets mitochondria and nuclei, characterized in that: The structural formula of the complex is shown below: .

2. A method for preparing the complex according to claim 1, characterized in that: The following steps are involved: (1) 3-(9,9-dimethylacridin-10(9H)-yl)-5-(pyridin-4-yl)phenol, (4-bromobutyl)triphenylphosphine bromide and K2CO3 were dissolved in acetone and stirred under argon atmosphere for reflux reaction. After the reaction, the supernatant was collected by centrifugation, the solvent was evaporated and recrystallized to obtain a triphenylphosphine-modified ligand. ； (2) mixing the triphenylphosphine-modified ligand obtained in step (1), cisplatin, and DMF, stirring and reacting in the dark; after the reaction, removing the solvent by rotary evaporation, redissolving, and recrystallizing to obtain a metal platinum complex that is dual-targeted to mitochondria and cell nuclei; 。 3. The preparation method according to claim 2, characterized in that In step (1), the molar ratio of 3-(9,9-dimethylacridin-10(9H)-yl)-5-(pyridin-4-yl)phenol, (4-bromobutyl)triphenylphosphine bromide and K2CO3 is 1:1.3:

3.

4. The preparation method according to claim 2, characterized in that In step (1), the reflux reaction is carried out at a temperature of 45-50° C. for 10-12 h.

5. The preparation method according to claim 2, characterized in that In step (2), the molar ratio of the triphenylphosphine-modified ligand to cisplatin is 1:

1.

6. The preparation method according to claim 2, characterized in that In step (2), the reaction temperature is 50-55°C and the reaction time is 48-60 h.

7. The preparation method according to claim 2, characterized in that In step (2), the re-dissolving is performed using hot methanol.

8. The preparation method according to claim 2, characterized in that In step (1) or (2), the recrystallization is performed by using ether and dichloromethane for at least 3 times.

9. Use of the mitochondria- and nucleus-dual-targeting metal platinum complex according to claim 1 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that The tumor is a human lung cancer cell.

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