Photosensitizer for inducing tumor cell copper death under illumination as well as preparation method and application of photosensitizer
The copper complex photodynamic agent integrates photodynamic therapy with copper-induced cell death, addressing drug resistance and specificity issues by enhancing ROS generation and targeted copper release, achieving effective tumor treatment.
Patent Information
- Application Number
- CN202510481532.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-16
- Publication Date
- 2025-07-15
AI Technical Summary
The prior art is difficult to effectively induce copper death in tumor cells under conditions that ensure biosafety, and the combination of photodynamic therapy and copper death has problems of insufficient targeting and limited efficacy.
A copper complex photosensitizer is designed to bind to copper ions through specific molecular structures, activate the copper death mechanism by using near-infrared light, and combine with photodynamic therapy to achieve selective release of copper ions in tumor cells and efficiently generate superoxide anions, downregulate FDX1 protein expression and induce DLAT protein aggregation.
The precise induction of copper death in tumor cells was achieved, the scope of application of copper death in tumor treatment was expanded, the treatment effect was significantly improved and the toxicity was reduced. The IC50 was 226nM under light, and the toxicity was less than 2μM under dark conditions, and the safety was excellent.
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Figure CN120309640A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical fields of fine chemicals and cancer treatment, and particularly to a photosensitizer capable of inducing cuproptosis in tumor cells under light irradiation, a preparation method thereof, and applications thereof. Background Art
[0002] Current cancer chemotherapy highly relies on the apoptosis mechanism. However, tumor cells can rapidly develop drug resistance through the abnormally high expression of anti-apoptotic proteins (such as a 70% upregulation of Bcl-2), resulting in a clinical failure rate of over 30%. Similarly, approaches targeting other death pathways also face challenges: ferroptosis is vulnerable to the failure of tumor microenvironment regulation, and necroptosis is difficult to avoid the risk of systemic inflammation. Although the recently discovered mitochondria-dependent cuproptosis pathway provides a direction for breaking drug resistance, its practical application is limited by a double contradiction: First, cuproptosis itself is not target-specific, and it is impossible to inhibit tumors under the condition of ensuring biosafety (in a clinical test of metastatic prostate cancer by simultaneously orally administering disulfiram and copper gluconate in the first phase, although strong uptake of copper by the liver was observed through 64 CuCl2 PET, no significant therapeutic effect was observed in all test groups); Second, the highly expressed glutathione in tumor cells (average GSH concentration of 1 - 10 mM, more than four times higher than that of normal cells) can directly neutralize copper ions through chelation, blocking its ability to induce cuproptosis; To overcome these limitations, photodynamic therapy is highly expected due to its spatially selective killing and ROS-mediated GSH depletion capabilities. However, traditional photosensitizers (such as porphyrins) have fundamental defects - low reactive oxygen generation efficiency (O2 - · production rate < 5 μM / min under 660 nm light irradiation), limited excitation wavelength (< 600 nm resulting in insufficient tissue penetration depth), and the killing mode solely relying on ROS cannot avoid apoptosis drug resistance.
[0003] Although researchers have tried to combine photosensitizers with free copper ions to activate cuproptosis, the unsolved synergistic problems have led to their failure: On the one hand, the most widely used porphyrin photosensitizers will chelate with copper ions through their active centers, significantly quenching the photosensitive activity (the ROS production rate of the porphyrin-Cu 2+ complex is reduced by 80%); On the other hand, photosensitizers with too weak chelating ability for copper ions will cause disordered diffusion of copper ions, resulting in serious toxicity to the liver and kidneys, further exacerbating the risk-benefit imbalance of the treatment plan. The above dilemmas reveal a long-neglected technical forbidden zone - how to precisely activate copper ion-induced cell death while ensuring photosensitive function. The existing technology neither provides a feasible molecular design idea nor a process path for integrating the two, making the combination of photodynamic therapy and cuproptosis generally regarded as an "unattainable" exploration blind spot. Summary of the Invention
[0004] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a photosensitizer with the ability to induce cuproptosis in tumor cells under light irradiation and a preparation method thereof, aiming to expand the application scope of cuproptosis in tumor treatment.
[0005] The technical solution of the present invention is as follows:
[0006] On the one hand, the present invention protects a copper complex photosensitizer, the structural formula of which is shown in Formula I:
[0007]
[0008] Wherein, X is a nucleophilic group, Y⁻ is an anion, and the number of charges carried by the anion is equal to the number of charges carried by the cation in Formula I; n is an integer selected from 1 - 20; more preferably, n is an integer selected from 4 - 10; most preferably 6.
[0009] For the above-mentioned technical solution, further preferably, X and Y are each independently selected from one of BF₄, F, Cl, Br, I, NO₃, ClO₄, CH₃COO, CH₃SO₃ and CF₃SO₃; more preferably F, Cl, Br and I, and most preferably Cl.
[0010] The second aspect of the present invention is to protect the synthesis method of the ligand of the copper complex photosensitizer, which is prepared by dehydrating the reaction of compound NBS-Cₙ-NH₂ with a carboxylic acid derivative of 8-hydroxyquinoline.
[0011] The third aspect of the present invention is to protect the synthesis method of the copper complex photosensitizer, which is obtained by further reacting the prepared ligand of the copper complex photosensitizer with a copper salt.
[0012]
[0013] Wherein, X is a nucleophilic group, Y - is an anion, and the number of charges carried by the anion is equal to the number of charges carried by the cation in Formula I; n is an integer selected from 1 - 20.
[0014] For the above-mentioned technical solution, further preferably, the compound NBS-Cₙ-NH₂ and the carboxylic acid derivative of 8-hydroxyquinoline react and dehydrate under the catalysis of a coupling agent and a base to obtain the ligand part of the copper complex photosensitizer.
[0015] For the above-mentioned technical solution, further preferably, the molar ratio of the amounts used of the compound NBS-Cₙ-NH₂, the carboxylic acid derivative of 8-hydroxyquinoline, the coupling agent, and the base is 1:(1 - 2):1:(1.5 - 4).
[0016] For the technical solutions described above, more preferably, the carboxylic acid derivative of 8-hydroxyquinoline is selected from one of 8-hydroxyquinoline-2-carboxylic acid and 8-hydroxyquinoline-7-carboxylic acid.
[0017] For the technical solutions described above, more preferably, the coupling agent is HATU, the base is DIPEA, and the molar ratio of their dosages used is 1:(1 - 4).
[0018] For the technical solutions described above, more preferably, the molar ratio of the ligand to the copper salt is 1:(1 - 10).
[0019] For the technical solutions described above, more preferably, the copper salt is selected from one of copper chloride and copper acetate.
[0020] Application of the described copper complex photosensitizer in the preparation of anti-tumor drugs. Further, its application in inducing photodynamic anti-tumor drugs directed by cuproptosis covers the light-controlled targeted therapy of human solid tumors (such as colon cancer HCT116) and animal models (such as the 4T1 model of breast cancer). In particular, it is limited to the core mechanism of triggering the synchronous release of mitochondrial copper ions induced by the local superoxide anion burst through 660 ± 10 nm near-infrared light, down-regulating the FDX1 protein and inducing the aggregation of the DLAT protein, directly anchoring the "photodynamic-cuproptosis" synergistic therapy.
[0021] Application of the described copper complex photosensitizer in the preparation of anti-tumor drugs, and its synergistic therapy is achieved through the following parameter conditions: under the irradiation of 660 ± 10 nm near-infrared light (power density 20 ± 5 mW / cm 2 , irradiation time 5 - 10 minutes), triggering the re-release rate of GSH-complexed copper ions ≥ 50% (measured 58%), synchronously achieving a GSH depletion rate ≥ 60%, a down-regulation amplitude of the FDX1 protein expression ≥ 60% (measured 64%), and an increase amplitude of the DLAT protein aggregation ≥ 40%, and based on light control selectivity, expanding the therapeutic window to the half-maximal inhibitory concentration IC 50 ≤ 250 nM (measured 226 nM) under light conditions, and IC 50 ≥ 2 μM under dark conditions.
[0022] For the technical solutions described above, more preferably, the copper complex photosensitizer has the following physicochemical properties: characteristic absorption peaks at 280 ± 5 nm, 300 ± 5 nm, and 660 ± 5 nm in the ultraviolet-visible absorption spectrum, the peak of [M - Cl - + in the ESI-MS spectrum is 701.00 ± 0.05 Da, and the mass percentage of copper atoms in the elemental analysis spectrum is 8.61 ± 0.5%.
[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0024] Through innovative molecular design, the copper complex photosensitizer of the present invention deeply integrates photodynamic therapy with the cuproptosis mechanism for the first time, solving the pain points of limited efficacy of traditional photosensitizers and insufficient targeting of cuproptosis drugs. The specific advantages include:
[0025] Synergistic effect of dual mechanisms: Retaining the near-infrared light response ability with Nile blue as the parent body, superoxide anion radicals (O2 - are efficiently generated under 660 nm light irradiation (the production rate is 40% higher than that of traditional photosensitizers). At the same time, copper ions are significantly released by the oxidation of intracellular GSH by O2 - · (the verified copper ion release rate reaches 58%), breaking through the inhibitory effect of GSH on cuproptosis.
[0026] High killing efficiency at low doses: Experimental data show that the half-maximal inhibitory concentration (IC 50 ) of 4T1 cells under light irradiation is only 226 nM, which is 50% lower than that of traditional photosensitizers (such as 450 nM of Photofrin), and the toxicity under dark conditions is low (IC 50 > 2 μM), with excellent safety.
[0027] Precisely induce the cuproptosis pathway: After light irradiation, the expression of FDX1 protein is significantly down-regulated (the decrease rate reaches 64%), and the aggregation of DLAT protein is induced. Moreover, inhibiting the ferroptosis pathway has no effect on its efficacy, proving that cuproptosis is the main mechanism of action.
[0028] Advantages of structural optimization: The long-chain alkyl structure (such as n = 6) balances lipophilicity and tissue penetration, enabling rapid cell uptake (enrichment in 30 minutes) and long-term retention (the retention amount > 50% after 12 hours). BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 : 1H NMR spectrum of the ligand of copper complex photosensitizer I1.
[0030] Figure 2 : 13C NMR spectrum of the ligand of copper complex photosensitizer I1.
[0031] Figure 3 : High-resolution mass spectrum of the ligand of copper complex photosensitizer I1.
[0032] Figure 4 : Mass spectrum of copper complex photosensitizer I1.
[0033] Figure 5 : UV-visible absorption spectra of copper complex photosensitizer I1 and its ligand.
[0034] Figure 6: Superoxide anion generation curves of copper complex photosensitizer I1 and its ligand under different conditions.
[0035] Figure 7 : Fluorescence images of copper complex photosensitizer I1 incubated with 4T1 cells for different times.
[0036] Figure 8 : Cell viability of 4T1 cells treated with different concentrations of copper complex photosensitizer I1 and its ligand under dark and light conditions.
[0037] Figure 9 : Cell viability of A673 cells treated with different concentrations of copper chloride under dark and light conditions.
[0038] Figure 10 : Expression of FDX1 protein in HCT116 cells after different treatments under dark and light conditions.
[0039] Figure 11 : Expression of DLAT protein in 4T1 cells after different treatments under light conditions Detailed implementation manners
[0040] The present invention provides a copper complex photosensitizer and its preparation method and application. To make the invention purpose and technical solution of the present invention clearer and more definite, the following further explains and illustrates the present invention in detail. It should be clear that the examples of the specific implementation manners described herein are only for further explaining and illustrating the present invention, rather than limiting the present invention.
[0041] In the embodiments of the present invention, the chemical reagents, reaction equipment, etc. used, unless otherwise specified, are all conventional materials in the art, and can be directly purchased through commercial channels or prepared according to the industry standard methods; the process methods involved, if not described in detail, are all implemented according to the conventional operation specifications in the technical field to which they belong. The names and units in the embodiments of the present invention are all common names and units in the art. For example, μM is the concentration unit micromole per milliliter.
[0042] Example 1: The copper complex photosensitizer I1 is prepared in the following manner
[0043]
[0044] (1) 8-Hydroxyquinoline-2-carboxylic acid (25 mg, 0.13 mmol), HATU (41 mg, 0.1 mmol) and DIPEA (38 μL, 0.2 mmol) were dissolved in DMF (3 mL). After stirring for 20 minutes at room temperature under a N2 atmosphere, NBS-C6-NH2 (50 mg, 0.1 mmol) was added dropwise to the above mixed system. The mixture was stirred for 12 hours. The mixture was evaporated under reduced pressure. The resulting crude product was purified by silica gel column chromatography (CH2Cl2: methanol = 200:1, v / v) to obtain the ligand part (NQ) of the copper complex photosensitizer as a dark blue solid (50 mg, yield 78%).
[0045] 1 1H NMR (400 MHz, DMSO) δ 10.04 (s, 1H), 9.75 (s, 1H), 9.60 (s, 1H), 9.01 (d, J = 7.8 Hz, 1H), 8.51 (d, J = 8.5 Hz, 1H), 8.43 (d, J = 8.6 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.95 (t, J = 7.5 Hz, 1H), 7.88 (t, J = 7.3 Hz, 1H), 7.57 (s, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.45 - 7.39 (m, 3H), 7.13 (d, J = 8.3 Hz, 1H), 3.71 - 3.62 (m, 5H), 3.50 (s, 1H), 3.41 (d, J = 6.6 Hz, 2H), 1.81 (s, 2H), 1.66 (s, 2H), 1.49 (s, 4H), 1.24 - 1.22 (m, 6H). ESI-MS: m / z [M-Cl - + : 604.2745.
[0046] (4) CuCl2 (16 mg, 0.12 mmol) was dissolved in MeOH (10 ml), and then a solution of NQ (20 mg, 0.03 mmol) in MeOH (5 ml) was added dropwise. The mixture was stirred at 55 °C for 12 hours. Then it was evaporated under reduced pressure and washed with water and MeOH. The resulting I1 was a dark blue solid (20 mg, yield 80%).
[0047] ESI-MS: m / z [M-Cl - + : 701.00.
[0048] The above examples demonstrated that the target compound was successfully prepared through a specific synthetic route (such as HATU / DIPEA-catalyzed amidation reaction and copper salt complexation), with a yield of 80%, verifying the feasibility of the synthetic method.
[0049] By adopting a modular synthesis strategy, the efficient binding of photosensitizer and copper ions is achieved, providing a stable chemical basis for targeted induction of cuproptosis.
[0050] Example 2: UV-Visible Spectral Characterization of Copper Complex Photosensitizer I1 and Its Ligand
[0051] The preparation method of the ligand solution of copper complex photosensitizer I1 in this example:
[0052] Weigh the solid of copper complex photosensitizer I1 and its ligand with a six-digit electronic balance, prepare a stock solution with a concentration of 3 mM in DMSO, and then dilute it with methanol to a concentration of 15 μM for spectral testing.
[0053] As Figure 5 shown, the absorption of I1 at 250 nm shows a significant red shift compared with that of the ligand NQ, and the absorption of I1 at 400 - 500 nm is also higher than that of NQ, proving the complexation of NQ with Cu 2+ . The absorption of I1 and NQ completely coincides in the near-infrared region corresponding to the Nile blue structure, further indicating that the NBS part in the molecule does not participate in the coordination with Cu 2+ .
[0054] The above examples prove that I1 has a red shift in absorption at 250 nm, an enhanced absorption in the 400 - 500 nm band, and the absorption peak of the Nile blue structure remains unchanged, indicating that copper ions only coordinate with the quinoline carboxylic acid part, retaining the parent photosensitizing activity.
[0055] Through selective coordination design, both the photodynamic performance of the photosensitizer is maintained and the function of inducing cuproptosis is introduced, realizing the synergistic effect of the dual mechanisms.
[0056] Example 3: Evaluation of the Performance of Copper Complex Photosensitizer I1 and Its Ligand in Generating Superoxide Anions in Vitro
[0057] Dihydro rhodamine 123 (DHR123) is used as an indicator for O2 - ·. In the presence of O2 - ·, this indicator is converted into rhodamine 123. The concentrations of I1 and NQ in PBS are 10 μM, DHR123 is 1 μM, and vitamin C is 1 mM. The cuvette containing the test item is exposed to 660 nm monochromatic light (power density: 20 mW / cm -2 ) for different times (1 - 7 minutes). The fluorescence spectrometer records the fluorescence changes of the samples.
[0058] As Figure 6As shown, whether under illumination or not, the fluorescence intensity of DHR123 itself at 525 nm hardly changes under this test condition. After adding I1 or NQ, without illumination, the fluorescence intensity of DHR123 does not increase, the same as when using DHR123 alone, while illumination can cause the fluorescence intensity of DHR123 to gradually increase with the illumination time, and the fluorescence intensities of the two are almost the same. After adding Vc to the solution, even illumination will not cause the fluorescence of DHR123 at 525 nm to increase. The above characterizations indicate that a large amount of O2 can be generated by I1 during the PDT process -· , and the ability of I1 to generate O2 -· is the same as that of the ligand NQ, which also corroborates that the NBS structure in I1 does not participate in the coordination with Cu 2+ .
[0059] The above examples prove that both I1 and the ligand NQ can efficiently generate O2 under illumination - ·, and the level of reactive oxygen species is not affected by copper ion coordination, confirming that the photodynamic effect is not destroyed
[0060] The photosensitizer still maintains a strong ability to generate reactive oxygen species in the presence of copper ions, providing a necessary condition for simultaneously triggering photodynamic damage and cuproptosis
[0061] Example 4: Cellular uptake of copper complex photosensitizer I1
[0062] The specific implementation method of cell culture in this example is as follows
[0063] The cells used are: HCT116 cells (human colon cancer cells), 4T1 cells (mouse breast cancer cells). All the above cells are cultured in a DMEM medium containing 1% double antibody (penicillin-streptomycin mixture) and 10% fetal bovine serum in a cell culture incubator at 37 °C with 5% CO2 and 21% O2. When the cells grow to the logarithmic growth phase, the cells are digested with trypsin containing EDTA and then transferred to 96-well plates, six-well plates or confocal culture dishes for subsequent cell experiments
[0064] To clarify the cellular uptake behavior of I1, 4T1 cells were cultured in a 35 mm glass-bottom culture dish at 37 °C for 24 hours. Then, I1 with a concentration of 250 nM was added. Meanwhile, the intracellular fluorescence was tracked by an FV-3000 laser confocal scanning microscope using a 60× oil immersion objective lens at different times (30 minutes, 1 hour, 2 hours, and 12 hours). The emission wavelength was 680 to 720 nm, and the excitation wavelength was 640 nm
[0065] As Figure 7As shown, I1 can be effectively taken up by cells after incubation at 37 °C for 30 minutes, and part of the drug remains in the cells even after 12 hours. In subsequent cytotoxicity experiments and Western blot experiments, the copper complex photosensitizer was incubated with cells for 12 hours before light irradiation.
[0066] The above examples demonstrate that I1 is rapidly taken up by tumor cells within 30 minutes and remains in the cells after 12 hours, showing good cell targeting and retention.
[0067] Through structural optimization (such as the design of lipophilic segments), the enrichment efficiency of the photosensitizer in tumor cells is significantly improved, enhancing the therapeutic effect.
[0068] Example 5: Cytotoxicity evaluation of copper chloride, copper complex photosensitizer I1 and its ligand
[0069] Cells (4000 - 5000 cells per well) were evenly seeded into 96-well plates and incubated overnight. Then, the original medium was replaced with serum-free medium. After 4 to 6 hours, the cells were incubated with different concentrations of copper chloride, I1, ligand NQ or serum-free medium. After 12 hours, light irradiation was applied to the light irradiation group using a 660 nm laser. After another 12 hours, the medium in each well was replaced with 150 μL of fresh serum-free medium containing 0.5 mg / mL MTT. The plate was incubated for another 4 hours to allow living cells to reduce the yellow tetrazolium salt (MTT) to dark blue formazan crystals. Finally, 200 μL of DMSO was added to dissolve the formazan crystals. The plate was shaken for 5 minutes, and the absorbance of each well at 490 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader and the cell viability was calculated:
[0070]
[0071] Among them, the experimental group represents the cell culture groups treated with different concentrations of I1; the blank group represents the cell culture group with only medium added: the control group represents the cell culture group without I1 added: OD is the absorbance value measured at 490 nm of the DMSO solution dissolving the formazan crystals. Each experiment was performed in parallel 4 times.
[0072] As Figure 8 shown, both I1 and NQ showed significant cytotoxicity under light irradiation, and their half-maximal inhibitory concentrations (IC50) were 226 nM and 309 nM, respectively. Under dark conditions, cytotoxicity was only observed when the dosing concentrations of both were greater than 1 μM, and their IC50 values were both greater than 2 μM. This indicates that both the copper complex photosensitizer I1 and its ligand NQ have photodynamic therapy effects, and the phototoxicity of I1 is significantly greater than that of its ligand.
[0073] The above examples demonstrated that the half-maximal inhibitory concentration (IC 50= 226 nM) was significantly lower than that of ligand NQ (309 nM), and it had low toxicity under dark conditions (IC 50 > 2 μM), indicating that copper ions synergistically enhanced phototoxicity.
[0074] As Figure 9 shown, free copper chloride began to exhibit cytotoxicity only when the concentration was greater than 60 μM, while a copper concentration greater than 40 μM in the blood might cause severe liver and kidney damage, indicating that it was unrealistic to use copper chloride alone for tumor treatment.
[0075] The introduction of copper ions in photodynamic therapy not only did not interfere with the photodynamic effect, but instead significantly improved the anti-tumor efficiency through a dual mechanism (ROS + cuproptosis). Under the same cell survival rate, the copper concentration of NC was only 0.1% of that of copper chloride used alone, improving the biosafety of the therapy and breaking through the efficacy bottleneck of traditional photosensitizers.
[0076] Example 6: Evaluation of the cuproptosis induction ability of copper complex photosensitizer I1
[0077] The specific implementation method of cell culture in this example:
[0078] HCT116 / 4T1 cells were seeded into six-well plates and incubated overnight. Then, the original medium was replaced with serum-free medium. After 4 to 6 hours, the cells were co-incubated with I1 (250 nM), NQ (250 nM), ES + CuCl2 (Elesclomol, a reported copper death inducer drug, as the positive control group) (100 nM + 400 nM), NBS + CuCl2 (250 nM + 1 μM), HQC + CuCl2 (HQC is 8-hydroxyquinoline-2-carboxylic acid, administered at four times the concentration of I1 as the positive group) (1 μM + 4 μM), or serum-free medium (as the Control group). After 12 hours, light irradiation was applied to the light irradiation group with a laser at 660 nm. After another 12 hours, the tumor cells were washed with pre-cooled PBS and lysed on ice for 10 minutes with RIPA lysis buffer containing protease and phosphatase inhibitors. Then, cell debris in the resulting lysate was removed by centrifugation at 12,000 rpm for 10 minutes, and the supernatant was evaluated using a BCA kit to quantify the protein in it. The protein extract for FDX1 analysis was boiled in denaturing non-reducing gel sample loading buffer for 5 minutes. The protein extract for DLAT analysis was directly mixed with non-denaturing non-reducing gel sample loading buffer. Then, an equal amount of protein was added to each lane of the Hepes-Tris gel for electrophoresis and blotted onto a PVDF membrane. After blocking with protein-free rapid blocking buffer for two hours at room temperature, it was further incubated overnight at 4 °C in the presence of primary antibodies (FDX1, β-actin, DLAT, GAPDH) at a dilution of 1:2000. Then, the membrane was washed with TBST and incubated with HRP-conjugated goat anti-rabbit antibody at a dilution of 1:2000 for 2 hours at room temperature. Finally, the membrane was visualized using a chemiluminescence kit.
[0079] As Figure 10 shown, when HCT116 cells were treated with NQ and I1 without light irradiation, the expression level of FDX1 was almost the same as that of the Control. However, under light irradiation, the expression of FDX1 in the I1 group was significantly decreased, similar to the group co-administered with ES and CuCl2 as the positive control group. This indicates that I1 has the ability to induce copper death in tumor cells under light irradiation.
[0080] As Figure 11 shown, the degree of DLAT aggregation in 4T1 cells co-treated with NBS and copper chloride under light irradiation was almost the same as that of the Control. However, the degree of DLAT aggregation in the group co-administered with NQ and copper chloride or the I1 group under light irradiation was even higher than that of the positive group administered at four times the dose. This indicates that the copper death of tumor cells induced by light irradiation requires the participation of the copper ionophore 8-hydroxyquinoline.
[0081] The above embodiments prove that under light illumination, I1 can significantly reduce the expression of FDX1 protein (comparable to the positive control group ES+CuCl2) and induce the aggregation of DLAT protein, directly demonstrating its ability to induce cuproptosis.
[0082] In summary, the present invention synthesizes for the first time a small molecule drug that activates the cuproptosis mechanism using photodynamic therapy. By means of light-controlled targeting and ROS-mediated GSH depletion, the non-specific toxicity and drug resistance problems of cuproptosis drugs are overcome. The photosensitizer prepared in the present invention combines photodynamic therapy with the cuproptosis mechanism, solving the problems of traditional chemotherapy drug resistance and insufficient targeting of cuproptosis drugs. Through structural design innovation (selective coordination of copper ions, retention of the Nile blue parent body) and mechanism integration (photodynamic + cuproptosis), this patent solves the problems of poor targeting, GSH inhibition and drug resistance in the prior art, showing a significant creative breakthrough.
[0083] The above is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
Claims
1. A copper complex photosensitizer, characterized in that: Its structural formula is shown in Formula I: wherein X is a nucleophilic group, and Y - is an anion, and the number of charges carried by the anion is equal to the number of charges carried by the cation in Formula I; n is selected from integers from 1 to 20; Each of said X and Y is independently selected from one of BF4, F, Cl, Br, I, NO3, ClO4, CH3COO, CH3SO3 and CF3SO3.
2. The synthesis method of the ligand of the copper complex photosensitizer according to claim 1, characterized in that: It is prepared by the dehydration reaction of compound NBS-Cn-NH2 with a carboxylic acid derivative of 8-hydroxyquinoline; wherein, X is a nucleophilic group, and Y - is an anion, and the number of charges carried by the anion is equal to the number of charges carried by the cation in Formula I; 3. The synthesis method according to claim 2, characterized in that: The said compound NBS-Cn-NH2 is prepared by reacting with a carboxylic acid derivative of 8-hydroxyquinoline under the catalysis of a coupling agent and a base to dehydrate.
4. The synthesis method according to claim 3, characterized in that: The molar ratio of the amounts used among the said compound NBS-Cn-NH2, the carboxyl derivative of 8-hydroxyquinoline, the coupling agent and the base is 1:(1 - 2):1:(1.5 - 4).
5. The synthesis method according to claim 3, wherein: The said carboxylic acid derivative of 8-hydroxyquinoline is selected from one of 8-hydroxyquinoline-2-carboxylic acid and 8-hydroxyquinoline-7-carboxylic acid.
6. The synthesis method according to claim 3, characterized in that: The said coupling agent is HATU and the base is DIPEA.
7. The synthesis method of the copper complex photosensitizer according to claim 1, characterized in that: It is prepared by further reacting the ligand prepared by the method of claim 2 with a copper salt, and the molar ratio of the two is 1:(1 - 10).
8. The synthesis method according to claim 7, characterized in that: The said copper salt is selected from one of copper chloride and copper acetate.
9. Use of the copper complex photosensitizer according to claim 1 in the preparation of a tumor therapeutic drug.
10. The application according to claim 9, characterized in that: The said use is to trigger the synchronous release of mitochondrial copper ions induced by the burst of local superoxide anions through 660 nm near-infrared light, down-regulate the FDX1 protein and induce the aggregation of the DLAT protein.