A method for the coordination modification of a metal complex photosensitizer and catecholamine and application thereof

Abstract

The application discloses a metal complex photosensitizer and a catecholamine coordination modification method and application thereof. The application utilizes a biological source catecholamine compound with redox activity (a characteristic structure unit can realize reversible conversion between phenol and quinone, and can generate a semiquinone free radical through disproportionation reaction, that is, QH2+Q ⇌ 2SQ·) to perform coordination modification on a metal complex photosensitizer, so that a new type of high-efficiency stable photosensitizer can be constructed. The application simultaneously solves three core pain points of existing photosensitizers, that is, low efficiency, instability and insecurity, pushes the metal complex photosensitizer to a brand-new performance platform, and is extremely likely to become a key breakthrough for promoting clinical transformation and application of the photosensitizer.

Description

Technical Field

[0001] This invention belongs to the technical field, specifically relating to a metal complex photosensitizer and its coordination modification method and application with catecholamines. Background Technology

[0002] Photodynamic therapy (PDT) is a clinically approved treatment modality that has evolved into an effective means of treating more than 20 diseases. However, despite decades of clinical application, PDT is rarely included in first-line treatment regimens, partly due to the lack of photosensitizers that simultaneously meet the stringent requirements for clinical translation. An ideal photosensitizer should possess the following characteristics: low dark toxicity and high reactive oxygen species (ROS) yield (including singlet oxygen). 1 O2, superoxide anion free radical O2 – • It possesses high photoresponsiveness, long-wavelength photoactivation ability, good water solubility, excellent structural stability, and potential for large-scale synthesis.

[0003] Photosensitizers are the core of photodynamic therapy (PDT). In the classical photodynamic mechanism, photosensitizers absorb light energy and are excited to a triplet excited state, which then sensitizes molecular oxygen to generate reactive oxygen species (such as singlet oxygen). The photophysical efficiency of this process is jointly regulated by the properties of the photosensitizer itself (such as triplet quantum yield, triplet lifetime, and absorption cross-section) and its biological performance (stability, pharmacokinetics, and toxicological characteristics).

[0004] First-generation porphyrin photosensitizers laid the clinical foundation for photothermolysis (PDT), but their absorption capacity in the visible light region was limited, their ROS yield was insufficient, they were prone to photodegradation, and their tissue penetration depth was insufficient. Although second-generation organic photosensitizers achieved a redshift in the absorption spectrum through structural modification, they still faced multiple trade-offs. For example, while expanding the π-conjugated system could improve singlet oxygen yield and extend the absorption wavelength, it often came at the cost of sacrificing water solubility and increasing dark toxicity.

[0005] Currently approved photosensitizers in clinical practice, such as verteporfen (VT), 5-aminolevulinic acid (5-ALA), methylene blue (MB), and hematoporphyrin monomethyl ether (Hmme), are mostly small organic molecules that do not contain heavy metals. However, these photosensitizers in the prior art have the following significant drawbacks: Insufficient efficiency: Due to the "heavy atom effect" caused by the lack of heavy metal atoms, its intersystem crossing efficiency is low and its ability to generate reactive oxygen species is limited, making it difficult to achieve ideal therapeutic effects, especially in scenarios such as low-dose drug administration, low light flux irradiation, or deep tumor treatment.

[0006] Poor photostability: Clinical phototherapy (PDT) typically lasts 30-60 minutes. Studies have found that the reactive oxygen species (ROS) production of most clinical photosensitizers begins to decline significantly within 5-10 minutes of light exposure. This is because they are prone to structural collapse and decomposition under light, making it impossible for them to maintain their effectiveness within the long therapeutic window.

[0007] Poor water solubility and complex synthesis and purification: Most clinically approved photosensitizers have the problem of strong hydrophobicity or poor water solubility, which seriously limits their biological application and bioavailability; at the same time, their synthesis and purification processes are often time-consuming and cumbersome.

[0008] Compared to organic photosensitizers, transition metal complexes have attracted significant attention due to their unique electronic structure. The "heavy atom effect" significantly promotes intersystem crossing, allowing their photoexcitation energies to efficiently reside in the triplet excited state. Furthermore, their structural diversity and synthetic tunability offer the possibility of precisely controlling photophysical, photochemical, and biological activities, thus making them a highly promising PDT (photodynamic therapy) drug platform. However, despite these theoretical advantages, no transition metal complex photosensitizers have yet been approved for clinical trials globally (TLD1433 is the only candidate drug to have entered the clinical trial stage). Existing transition metal complex photosensitizers face the following bottlenecks: Structural instability: Continuous light exposure can cause irreversible valence state increases in the metal center, leading to inactivation of the active center, damage to the coordination structure, and ultimately, photosensitizer failure. Safety concerns: Existing complexes mostly rely on precious metals such as ruthenium, iridium, platinum, and rhenium. While these impart a "heavy atom effect," they also result in high dark toxicity and uncertain in vivo metabolic behavior, severely hindering their clinical translation. This is one of the important reasons why no transition metal complex photosensitizers have been approved for clinical use globally.

[0009] Therefore, there is an urgent need for a modification strategy that can significantly improve the performance of metal complex photosensitizers, is simple to synthesize, and is inexpensive, so as to achieve gram-scale laboratory preparation. At the same time, the strategy needs to have good versatility and be scalable to different metal centers and ligand systems, so as to provide a general technical platform for solving the clinical translation bottleneck of metal complex photosensitizers. Summary of the Invention

[0010] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0011] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0012] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a metal complex photosensitizer.

[0013] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a metal complex photosensitizer, characterized in that its general structural formula is [M(L)]. n (A) i ] m+ Where M is a metal ion, L is an auxiliary ligand, A is a catecholamine ligand, n is the number of auxiliary ligands (1≤n≤6), i is the number of catecholamine ligands (1≤i≤6), and m is the charge of the complex.

[0014] As a preferred embodiment of the metal complex photosensitizer described in this invention, the metal ion is preferably a transition metal ion with a heavy atom effect, selected from ruthenium, osmium, iridium, platinum or other metal ions.

[0015] As a preferred embodiment of the metal complex photosensitizer of the present invention, the auxiliary ligand is selected from pyridine, bipyridine, phenanthroline, quinoline or its derivatives.

[0016] As a preferred embodiment of the metal complex photosensitizer of the present invention, the catecholamine ligand is a catecholamine ligand having a catechol structure and capable of reversible phenol-quinone transformation, selected from dopamine, adrenaline, noradrenaline, levodopa or its derivatives, and exists in the complex in one or more forms of a completely reduced state, a completely oxidized state or a semiquinone radical state.

[0017] As a preferred embodiment of the metal complex photosensitizer of the present invention, the catecholamine ligand can form stable free radicals and has reversible electron storage and release capabilities. During illumination, it can effectively inhibit the irreversible increase in valence state of the active metal center, stabilize the oxidation state of the metal center and the overall molecular structure.

[0018] Another object of the present invention is to overcome the shortcomings of the prior art and provide a method for modifying catecholamines in metal complex photosensitizers, comprising, Metal salts and auxiliary ligands are dissolved in organic solvents and refluxed under inert gas protection. After the reaction is completed, the metal complex precursor is obtained by precipitation, filtration and purification. The metal complex precursor and ion exchange resin were added to a mixed solvent consisting of acetone and deionized water. Dopamine ligand was added under an inert atmosphere and degassed, followed by heating to carry out the reaction. After the reaction was completed and cooled, acetic acid was added to terminate the reaction, and the metal complex photosensitizer was obtained by separation and purification.

[0019] In a preferred embodiment of the modification method described in this invention, the organic solvent is N,N-dimethylformamide or ethylene glycol, the ion exchange resin is Dowex 1×8, and the inert atmosphere is argon or nitrogen.

[0020] In a preferred embodiment of the modification method described in this invention, the degassing time is 10-20 minutes, the heating reaction temperature is 50-80°C, and the time is 8-16 hours.

[0021] As a preferred embodiment of the modification method described in this invention, the specific steps of separation and purification are as follows: the reaction solution is cooled to 0°C, acetic acid is added to terminate the reaction, the solution is filtered through diatomaceous earth, the filter cake is washed with acetone, the combined filtrates are concentrated and then purified by column chromatography.

[0022] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a metal complex photosensitizer in the preparation of photodynamic therapy drugs.

[0023] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a metal complex photosensitizer in the preparation of a drug for treating tumors.

[0024] This invention discovers that a novel class of highly efficient and stable photosensitizers can be constructed by coordinating photosensitizers with redox-active biogenic catecholamine compounds (whose characteristic structural units enable reversible transformation between phenol and quinone and can generate semiquinone radicals via disproportionation reactions, i.e., QH2 + Q⇌2SQ•). Taking dopamine as an example, it exists in three tautomer forms after coordination: a fully reduced state (phenolic hydroxyl form, QH2), a fully oxidized state (quinone form, Q), and a semiquinone radical state (SQ•). These three forms coexist in the coordination system, contributing synergistically or independently to performance enhancement. Therefore, this invention covers the application of these three forms in metal complexes.

[0025] The introduction of dopamine coordination produces the following three key effects: Electronic buffering effect: Dopamine ligands have reversible electron storage and release capabilities. During light irradiation, they can effectively inhibit the irreversible increase in valence state of active metal centers, stabilize the oxidation state of metal centers and the overall molecular structure, and enable the complex to maintain its initial configuration and continuously generate reactive oxygen species under long-term light irradiation. This significantly prolongs the reaction time and increases the ROS yield by orders of magnitude.

[0026] Heavy atom effect synergy and toxicity shielding: While retaining the heavy atom effect of the metal center and ensuring efficient intersystem crossing, the introduction of dopamine effectively reduces the biological exposure of metal ions and significantly reduces the dark toxicity of the complex.

[0027] Optimization of physicochemical properties: The introduction of dopamine increases the polarity of the molecule and the number of hydrogen bonding sites, significantly improving the water solubility of the complex. Simultaneously, by regulating the coordination environment, its light absorption performance is effectively enhanced (including an increase in the molar extinction coefficient and a red shift in the absorption spectrum), thus strengthening its photoresponsiveness. This improvement allows the complex to maintain highly effective photodynamic therapy even under harsh conditions such as low drug dosage, low light flux irradiation, and deep tumor treatment.

[0028] Beneficial effects of this invention: (1) Achieved an order-of-magnitude leap in photodynamic therapy performance: Compared to unmodified parent metal complexes and clinically approved photosensitizers, the photosensitizer described in this invention achieves... 1 The ability to generate O2 has been increased by orders of magnitude, thanks to the synergistic effect of dopamine molecules (including three forms: QH2, Q, and SQ•) and the metal center.

[0029] (2) Breakthrough in overcoming the technical bottleneck of poor photostability of existing photosensitizers: Addressing the common problem of photosensitizers (whether clinical organic molecules or unmodified metal complexes) being easily deactivated under light in existing technologies, this invention provides a revolutionary solution, specifically: Ultra-long action time: This invention endows the complex with "electron buffering" ability through the redox activity of dopamine molecules (dynamic balance of QH2, Q, SQ•), effectively inhibiting the irreversible increase of the valence state of the metal center and structural damage during light irradiation.

[0030] Structural integrity retention: Experiments have shown that the photosensitizer of this invention can maintain its initial configuration and generate efficiently even under prolonged light exposure. 1 O2 is used, while the activity of clinical photosensitizers begins to decline within 5-10 minutes. This means that the present invention can perfectly match the clinical treatment window of 30-60 minutes, achieving highly efficient treatment throughout the entire process.

[0031] (3) A universal metal complex photosensitizer optimization platform was constructed: The technical effects of this invention are not isolated cases, but have broad applicability. Multi-metal center and multi-ligand systems have verified that this strategy has achieved significant success in different metal centers such as ruthenium (Ru) and osmium (Os), and in different auxiliary ligand systems such as pyridine and quinoline, all showing a significant improvement in performance. This proves that the method is applicable to a variety of different metal-ligand combinations, and also shows that the coordination modification of dopamine is a general and scalable platform technology.

[0032] (4) Comprehensive improvement in the drug-likeness of photosensitizers: In addition to the core photodynamic properties, this invention has unexpectedly solved several auxiliary problems that limit the clinical translation of photosensitizers, specifically: Significantly improved water solubility: The introduction of dopamine molecules increases the polarity of the molecules and hydrogen bonding sites, which greatly improves the water solubility of metal complexes and solves the problem that traditional photosensitizers need to be administered with the help of organic solvents or carriers.

[0033] Significantly improved biocompatibility: By coordinating with metals, the exposure of bare metals in organisms is reduced, thereby lowering the risk of dark toxicity leakage of metal ions. Simultaneously, the combination of high efficiency and low toxicity results in an extremely high "phototoxicity index," significantly improving drug safety while ensuring efficacy.

[0034] Optimized optical response performance: The optical absorption band has been broadened and enhanced, enabling it to be excited by longer wavelengths, while utilizing the excitation light more effectively and reducing dependence on high-power lasers.

[0035] (5) The catecholamine molecular coordination modification strategy proposed in this invention solves the three major pain points of existing photosensitizers: "low efficiency, instability and unsafety". It pushes metal complex photosensitizers to a new performance platform and is very likely to become a key breakthrough in promoting the clinical translation and application of this type of photosensitizer. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 The Ru(biq)2(DA)Cl metal complex prepared in Example 1 of this invention 1 H NMR spectrum.

[0037] Figure 2 The Ru(biq)2(DA)Cl metal complex prepared in Example 1 of this invention 13 C10 NMR spectrum.

[0038] Figure 3 In Example 1 of this invention, Ru(biq)2(DA)Cl, Ru(biq)2Cl2, and the clinical photosensitizers Hmme, 5-ALA, VT, Ce6, and MB were tested under the same conditions (500 mW / cm²). 2 Comparison of in-situ EPR spectra.

[0039] Figure 4 The Ru(biq)2(DA)Cl, Ru(biq)2Cl2 and clinical photosensitizer in Example 1 of this invention are used in low laser power (50 mW / cm²) 2 ) in situ 1O2-EPR spectrum.

[0040] Figure 5 This refers to the in-situ in-situ induction of Ru(biq)2(DA)Cl, Ru(biq)2Cl2, and the clinical photosensitizer at a low sample concentration (100 ppm) in Example 1 of the present invention. 1 O2-EPR spectrum.

[0041] Figure 6 This is a comparison of the XPS spectra of Ru(biq)2(DA)Cl and Ru(biq)2Cl2 before and after illumination in Example 1 of the present invention.

[0042] Figure 7 This is the in-situ XAFS spectrum of Ru(biq)2(DA)Cl and Ru(biq)2Cl2 during the illumination process in Example 1 of the present invention.

[0043] Figure 8 This is an HPLC chromatogram of Ru(biq)2(DA)Cl before and after light irradiation in Example 1 of the present invention.

[0044] Figure 9 HPLC analysis of clinical photosensitizers Hmme, 5-ALA, VT, Ce6 and MB before and after light exposure.

[0045] Figure 10 The UV-Vis absorption spectrum of Ru(biq)₂(DA)Cl and the generation under 808 nm laser excitation 1 O2-EPR spectrum.

[0046] Figure 11 This is a comparative test of the water solubility of Ru(biq)2(DA)Cl in Example 1 of the present invention.

[0047] Figure 12 The results of the cytotoxicity evaluation of Ru(biq)2(DA)Cl, Ru(biq)2Cl2 and clinical photosensitizers Hmme, 5-ALA, VT, Ce6 and MB under dark and light conditions in Example 1 of this invention are presented.

[0048] Figure 13 This invention provides a comparison of the photodynamic therapeutic effects of Ru(biq)2(DA)Cl, Ru(biq)2Cl2, and clinical photosensitizers Hmme and Ce6 in an in situ deep pancreatic tumor model, as described in Example 1 of this invention.

[0049] Figure 14 The Ru(bpy)2(DA)Cl metal complex prepared in Example 2 of this invention 1 H NMR spectrum.

[0050] Figure 15The Ru(bpy)2(DA)Cl metal complex prepared in Example 2 of this invention 13 C10 NMR spectrum.

[0051] Figure 16 This is a comparison of the in-situ EPR spectra of Ru(bpy)2(DA)Cl and Ru(bpy)2Cl2 under the same conditions in Example 2 of the present invention.

[0052] Figure 17 The Os(bpy)2(DA)Cl metal complex prepared in Example 3 of this invention 1 H NMR spectrum.

[0053] Figure 18 The Os(bpy)2(DA)Cl metal complex prepared in Example 3 of this invention 13 C10 NMR spectrum.

[0054] Figure 19 This is a comparison of the in-situ EPR spectra of Os(bpy)2(DA)Cl and Os(bpy)2Cl2 under the same conditions in Example 3 of the present invention.

[0055] Figure 20 The Ir(phen)2(DA)Cl metal complex prepared in Example 3 of this invention 1 H NMR spectrum.

[0056] Figure 21 The Ir(phen)2(DA)Cl metal complex prepared in Example 3 of this invention 13 C10 NMR spectrum.

[0057] Figure 22 This is a comparison of the in-situ EPR spectra of Ir(phen)2(DA)Cl and Ir(phen)2Cl2 under the same conditions in Example 3 of the present invention. Detailed Implementation

[0058] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0059] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0060] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0061] The raw materials used in this invention are: ruthenium trichloride hydrate, 2,2′-biquinoline, 2,2′-bipyridine, dopamine hydrochloride, verteporfen, 5-aminolevulinic acid, hematoporphyrin monomethyl ether, dihydroporphyrin E6, methylene blue trihydrate, lithium chloride, and 1,2-divinylbenzene (Dowex 1×8) purchased from Sigma-Aldrich. Tetramethyl-4-piperidinone hydrochloride was purchased from Dojindo. In the embodiments of this invention, the solution concentration was 100~1000ppm.

[0062] Performance testing method in this embodiment of the invention: The NMR spectra were obtained using a Bruker Advance III 400 MHz NMR spectrometer.

[0063] X-ray photoelectron spectroscopy was performed using a Thermo Fisher K-Alpha spectrometer.

[0064] High performance liquid chromatography analysis was performed using a Waters 600 system.

[0065] Electron paramagnetic resonance spectra were acquired using a Bruker EMX nano spectrometer.

[0066] In-situ X-ray absorption fine structure spectrum of ruthenium K edge (22117.2 eV) was acquired by the BL14W1 beamline of Shanghai Synchrotron Radiation Facility (SSRF, China).

[0067] Bioluminescence imaging was performed using the Maestro in vivo optical imaging system.

[0068] Example 1 This embodiment provides a method for modifying catecholamines in ruthenium complex 1, comprising the following steps: (1) Ruthenium trichloride hydrate (RuCl3·H2O, 1.34 mmol), 2,2′-biquinoline ligand (3 mmol), and lithium chloride (LiCl, 4.2 mmol) were dissolved in 14 mL of N,N-dimethylformamide (DMF) and stirred until completely dissolved. The reaction system was degassed with argon for 5 minutes and then refluxed under argon protection for 6 hours. After the reaction was completed, the system was slowly cooled to room temperature and then added dropwise to 500 mL of vigorously stirred deionized water, resulting in the precipitation of a blue-green precipitate. The precipitate was collected by suction filtration, redissolved in dichloromethane, and filtered to remove insoluble impurities. The resulting green filtrate was washed with deionized water (20 mL × 5), the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to a small volume by rotary evaporation. Excess diethyl ether was added to the concentrate, resulting in the precipitation of a green precipitate, which was collected by suction filtration to obtain the crude product of the ruthenium complex precursor. The crude product was purified by column chromatography to obtain the target precursor Ru(biq)2(Cl)2.

[0069] (2) The above-mentioned ruthenium precursor (2.8 mmol) and Dowex 1×8 resin (20 g) were added to the reaction flask, followed by a mixed solvent of acetone (60 mL) and deionized water (60 mL). Under an argon atmosphere, dopamine (DA, 5.61 mmol) was added to the system, and degassing was continued for 15 minutes. The reaction system was magnetically stirred at 80 °C for 16 hours under argon protection. After the reaction was completed, it was cooled to 0 °C, and acetic acid (1.35 mL) was added to terminate the reaction. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with acetone. The filtrates were combined and concentrated by rotary evaporation to obtain a solid crude product. The crude product was purified by column chromatography to obtain the pure dopamine-modified ruthenium complex Ru(biq)2(DA)Cl.

[0070] The obtained product was subjected to performance testing, specifically as follows: Singlet oxygen yield determination: 2,2,6,6-Tetramethylpiperidine (TEMP) was used as the singlet oxygen yield. 1 O2) trapping agent, monitored in real time using in-situ electron paramagnetic resonance (EPR) technology. 1 O2 generation. Aqueous solutions of Ru(biq)₂Cl₂, Ru(biq)₂(DA)Cl, Hmme, 5-ALA, VT, Ce₆, and MB were prepared respectively. 40 µL of each sample solution was mixed with 2 µL of TEMP and subjected to illumination (650 nm, 500 mW / cm²). 2 In-situ EPR data acquisition was performed (60 min). Low laser intensity (50 mW / cm²) was used. 2 Under conditions of ) and low dose (0.2 mg / ml) 1 The same testing method was used to assess O2 generation capacity.

[0071] Structural stability assessment: High-performance liquid chromatography (HPLC) was used to monitor the structural changes of Ru(biq)₂(DA)Cl during illumination, with chromatograms collected every 20 min of illumination. The structural stability of reference standards Hmme, 5-ALA, VT, Ce₆, and MB was analyzed by HPLC before and after 30 min of illumination to assess their photoinduced structural changes.

[0072] Valence stability analysis of the metal center: X-ray photoelectron spectroscopy (XPS) was used to characterize the valence state changes of the Ru metal center in ruthenium complexes before and after irradiation. Simultaneously, in-situ X-ray absorption fine structure spectroscopy (in-situ XAFS) was used to track the dynamic valence state evolution of the Ru center during irradiation in real time, revealing the mechanism by which coordination modification affects the stability of the metal center.

[0073] Evaluation of photosensitivity in deep tissues: The photosensitivity of Ru(biq)₂Cl₂, Ru(biq)₂(DA)Cl, Hmme, 5-ALA, VT, Ce₆, and MB in deep tissues was evaluated using a 1% fat emulsion injection to simulate the light scattering characteristics of biological tissues. Sample solutions treated with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) were added to 96-well plates, and fat emulsion layers of varying thicknesses (0–8 mm) were placed beneath the wells to simulate tissue-like light scattering.

[0074] The well plate was placed below the fat emulsion layer and irradiated with a 650 nm laser (500 mW / cm²). 2 Excitation (3 min) was performed, followed by acquisition of fluorescence images for evaluation. 1 O2 generation capacity.

[0075] In vitro cytotoxicity evaluation: The cytotoxicity of Ru(biq)₂Cl₂, Ru(biq)₂(DA)Cl, Hmme, 5-ALA, VT, Ce₆, and MB against human pancreatic cancer cells was determined using the CCK-8 assay. Cells were seeded in 96-well plates and cultured overnight in DMEM medium to allow adherence. Subsequently, different concentrations of the test samples were added, and the cells were incubated in the dark for 12 h. The absorbance of each well was measured at 450 nm to assess dark toxicity. In the phototoxicity assay, after 12 h of incubation, cells were irradiated with a 650 nm laser (500 mW / cm²). 2 After incubation for 1 hour (5 min), the absorbance was measured at 450 nm.

[0076] Evaluation of in vivo antitumor efficacy: Healthy female BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. A pancreatic cancer tumor model was established through in situ inoculation. On day 7 post-inoculation, mice with similar tumor volumes were randomly divided into 6 groups (n=6 per group). Ru(biq)2Cl2, Ru(biq)2(DA)Cl, Hmme, and Ce6 were administered via tail vein injection (20 mg / kg), while the control and light-exposed control groups received an equal volume of PBS. Following administration, mice in the corresponding groups received laser therapy (650 nm, 500 mW / cm²). 2 Tumor volume changes were recorded every 2 days using a Maestro in vivo optical imaging system (5 min). On day 15 of treatment, mice were sacrificed, and tumor tissue was extracted for H&E staining and immune-related analysis.

[0077] Figure 1 The Ru(biq)2(DA)Cl metal complex prepared in Example 1 of this invention 1 1H NMR spectrum. The spectral signal is clearly assigned, with no obvious impurity peaks, indicating that the target complex was successfully synthesized and has high purity.

[0078] Figure 2 The Ru(biq)2(DA)Cl metal complex prepared in Example 1 of this invention 13 C10 NMR spectrum. The spectrum signal is intact, further confirming the successful synthesis of the target complex and its structural purity.

[0079] Figure 3 This is a comparison of the in-situ EPR spectra of Ru(biq)2(DA)Cl, Ru(biq)2Cl2, and clinical photosensitizers Hmme, 5-ALA, VT, Ce6, and MB under the same conditions in Example 1 of this invention. The results show that Ru(biq)2(DA)Cl modified with catecholamines maintains a stable and high singlet oxygen generation capacity under long-term light irradiation, which is significantly better than the unmodified complex and the clinical control photosensitizer.

[0080] Figure 4 In Example 1 of this invention, Ru(biq)₂(DA)Cl, Ru(biq)₂Cl₂, and the clinical photosensitizer were used in situ under low laser power. 1 O2-EPR spectrum. The results show that Ru(biq)2(DA)Cl still exhibits excellent photosensitivity under low laser power conditions, confirming its high photosensitivity efficiency.

[0081] Figure 5 This refers to the in-situ application of Ru(biq)2(DA)Cl, Ru(biq)2Cl2, and the clinical photosensitizer at low sample concentrations in Example 1 of the present invention. 1O2-EPR spectrum. The results show that Ru(biq)2(DA)Cl still exhibits excellent photosensitivity at low sample concentrations, confirming its high photosensitivity efficiency.

[0082] Figure 6 This is a comparison of the XPS spectra of Ru(biq)₂(DA)Cl and Ru(biq)₂Cl₂ before and after illumination in Example 1 of this invention. After illumination, the valence state of Ru in Ru(biq)₂(DA)Cl did not change significantly, while the valence state of Ru in Ru(biq)₂Cl₂ increased significantly, indicating that the modification with catecholamines effectively inhibited the light-induced oxidation of the metal center.

[0083] Figure 7 This is the in-situ XAFS spectrum of Ru(biq)₂(DA)Cl and Ru(biq)₂Cl₂ during the illumination process in Example 1 of this invention. Quantitative analysis further confirmed that the valence state of Ru in Ru(biq)₂(DA)Cl remained stable before and after illumination, while Ru(biq)₂Cl₂ underwent significant oxidation after illumination, resulting in an increase in its valence state.

[0084] Figure 8 The HPLC chromatograms of Ru(biq)2(DA)Cl before and after light irradiation in Example 1 of this invention show that the chromatographic retention time and absorption spectrum of the complex did not change significantly after light irradiation, indicating that its molecular structure remains highly stable under light conditions.

[0085] Figure 9 For the HPLC analysis of clinical photosensitizers Hmme, 5-ALA, VT, Ce6 and MB before and after light exposure and 1 Characterization by 1H NMR spectroscopy. The results showed that the above photosensitizer underwent significant structural damage after light irradiation, confirming its poor photostability.

[0086] Figure 10 The UV-Vis absorption spectrum of Ru(biq)₂(DA)Cl and the generation under 808 nm laser excitation 1 O2-EPR spectrum. The complex exhibits enhanced absorption in the near-infrared region and can effectively generate singlet oxygen under 808 nm excitation, indicating that dopamine modification effectively extends its long-wavelength absorption and photoresponse range.

[0087] Figure 11 This is a comparative test of the water solubility of Ru(biq)2(DA)Cl in Example 1 of the present invention. The results show that the water solubility of the dopamine-modified complex is significantly improved and is superior to that of existing clinically approved photosensitizers.

[0088] Figure 12This document presents the cytotoxicity evaluation results of Ru(biq)₂(DA)Cl, Ru(biq)₂Cl₂, and the clinical photosensitizers Hmme, 5-ALA, VT, Ce₆, and MB under dark and light conditions in Example 1 of this invention. The experiments show that Ru(biq)₂(DA)Cl exhibits extremely low dark toxicity under dark conditions, while demonstrating significant phototoxicity after light exposure, confirming its excellent photodynamic therapy potential and good biocompatibility.

[0089] Figure 13 This paper compares the photodynamic therapeutic effects of Ru(biq)2(DA)Cl, Ru(biq)2Cl2, and clinical photosensitizers Hmme, 5-ALA, VT, Ce6, and MB in an in situ deep pancreatic tumor model, as described in Example 1 of this invention. The results show that Ru(biq)2(DA)Cl exhibits significantly superior photodynamic therapeutic effects compared to existing clinical photosensitizers, effectively inhibiting the growth of deep tumors and confirming its feasibility for treating deep tumors.

[0090] Example 2 This embodiment provides a method for modifying catecholamines in ruthenium complex 2, comprising the following steps: (1) 2,2'-bipyridine (6 mmol), ruthenium trichloride trihydrate (3 mmol), and lithium chloride (30 mmol) were added to a reaction flask, followed by 45 mL of N,N-dimethylformamide. Under argon protection, the mixture was heated to reflux at 150 °C for 8 hours with magnetic stirring. After the reaction was completed, the mixture was cooled to room temperature, and 500 mL of acetone was added to the system. The mixture was then allowed to stand overnight at 0 °C. The mixture was filtered to obtain a black microcrystalline solid, which was washed three times each with water and diethyl ether to obtain a blackish-brown solid. After vacuum drying for 24 hours, the ruthenium precursor compound Ru(bpy)2Cl2 was obtained.

[0091] (2) The above precursor (2.07 mmol) and Dowex 1×8 resin (15 g) were added to a reaction flask, followed by acetone (30 mL) and an equal volume of deionized water. Under nitrogen protection, dopamine (4.14 mmol) was added to the solution, and the mixture was degassed for 10 minutes. The mixture was magnetically stirred under a nitrogen atmosphere and heated to 70 °C for 10 hours. After cooling to 0 °C, 1 mL of acetic acid was added to terminate the reaction. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with acetone. The resulting orange-purple filtrate was concentrated by rotary evaporation to obtain a purple solid product, namely the ruthenium complex Ru(bpy)2(DA)Cl.

[0092] The obtained product was subjected to performance testing, specifically as follows: Singlet oxygen yield determination: 2,2,6,6-Tetramethylpiperidine was used as a singlet oxygen scavenger, and in-situ electron paramagnetic resonance (EPR) technology was used for real-time monitoring. 1O2 generation. Aqueous solutions of Ru(bpy)₂Cl₂ and Ru(bpy)₂(DA)Cl were prepared separately. 40 µL of each sample solution was mixed with 2 µL of TEMP and subjected to illumination (650 nm, 500 mW / cm²). 2 In situ EPR testing was performed (60 min). 1 Quantitative comparison of O2 concentrations was obtained by fitting analysis of EPR signal intensity.

[0093] Figure 14 The Ru(bpy)2(DA)Cl metal complex prepared in Example 2 of this invention 1 1H NMR spectrum. The spectral signal is clearly assigned, with no obvious impurity peaks, indicating that the target complex was successfully synthesized and has high purity.

[0094] Figure 15 The Ru(bpy)2(DA)Cl metal complex prepared in Example 2 of this invention 13 C10 NMR spectrum. The spectrum signal is intact, further confirming the successful synthesis of the target complex and its structural purity.

[0095] Figure 16 This is a comparison of the in-situ EPR spectra of Ru(bpy)₂(DA)Cl and Ru(bpy)₂Cl₂ under the same conditions in Example 2 of this invention. The results show that Ru(bpy)₂(DA)Cl modified with catecholamines maintains a stable and high singlet oxygen generation capacity under long-term light irradiation, which is significantly better than the unmodified complex Ru(bpy)₂Cl₂.

[0096] Example 3 This embodiment provides a method for modifying catecholamines in osmium complexes, comprising the following steps: (1) 2,2'-Bipyridine (2.3 mmol) and ammonium hexachloroosmium tetroxide (2.27 mmol) were added to a reaction flask, followed by 25 mL of ethylene glycol. Under argon protection, the mixture was heated under reflux with magnetic stirring for 1 hour. After cooling to room temperature, an equal volume of saturated sodium dithionite aqueous solution was added, and the mixture was stirred at room temperature for 10 minutes. The dark purple solid was collected by centrifugation and washed three times each with water and ether. The resulting dark purple solid was dried under vacuum for 24 hours to obtain the osmium precursor compound Os(bpy)2Cl2.

[0097] (2) The above precursor (2.61 mmol) and Dowex 1×8 resin (20 g) were added to a reaction flask, followed by acetone (60 mL) and an equal volume of deionized water. Under nitrogen protection, dopamine (5.23 mmol) was added to the solution, and the mixture was degassed for 20 minutes. The mixture was magnetically stirred under a nitrogen atmosphere and heated to 50 °C for 8 hours. After cooling to 0 °C, 1.2 mL of acetic acid was added to terminate the reaction. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with acetone. The resulting orange-yellow filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain the osmium complex Os(bpy)2(DA)Cl.

[0098] The obtained product was subjected to performance testing, specifically as follows: Singlet oxygen yield determination: 2,2,6,6-Tetramethylpiperidine was used as a singlet oxygen scavenger, and in-situ electron paramagnetic resonance (EPR) technology was used for real-time monitoring. 1 O2 generation. Aqueous solutions of Os(bpy)₂Cl₂ and Os(bpy)₂(DA)Cl were prepared separately. 40 µL of each sample solution was mixed with 2 µL of TEMP and subjected to illumination (650 nm, 500 mW / cm²). 2 In situ EPR testing was performed (60 min). 1 Quantitative comparison of O2 concentrations was obtained by fitting analysis of EPR signal intensity.

[0099] Figure 17 The Os(bpy)2(DA)Cl metal complex prepared in Example 3 of this invention 1 1H NMR spectrum. The spectral signal is clearly assigned, with no obvious impurity peaks, indicating that the target complex was successfully synthesized and has high purity.

[0100] Figure 18 The Os(bpy)2(DA)Cl metal complex prepared in Example 3 of this invention 13 C10 NMR spectrum. The spectrum signal is intact, further confirming the successful synthesis of the target complex and its structural purity.

[0101] Figure 19 This is a comparison of the in-situ EPR spectra of Os(bpy)2(DA)Cl and Os(bpy)2Cl2 under the same conditions in Example 3 of the present invention. The results show that Os(bpy)2(DA)Cl modified with catecholamines still maintains a stable and high singlet oxygen generation capacity under long-term light irradiation, which is significantly better than the unmodified complex Os(bpy)2Cl2.

[0102] Example 4 This embodiment provides a method for modifying catecholamines in iridium complexes, comprising the following steps: (1) 1,10-phenanthroline (6 mmol) and iridium trichloride hydrate (3 mmol) were added to a reaction flask, followed by 40 mL of water + ethanol (1:1) solution. The mixture was heated under reflux at 50 °C for 1 hour with magnetic stirring. After the reaction was completed, it was cooled to room temperature. The mixture was filtered to obtain a black microcrystalline solid, which was the iridium precursor compound Ir(phen)2Cl2.

[0103] (2) The above precursor (1.13 mmol) and Dowex 1×8 resin (30 g) were added to a reaction flask, followed by acetone (12 mL) and an equal volume of deionized water. Under nitrogen protection, dopamine (2.26 mmol) was added to the solution, and the mixture was degassed for 15 minutes. The mixture was magnetically stirred under a nitrogen atmosphere and heated to 60 °C for 12 hours. After cooling to 0 °C, 0.4 mL of acetic acid was added to terminate the reaction. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with acetone. The resulting orange-yellow filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain the iridium complex Ir(phen)2(DA)Cl.

[0104] The obtained product was subjected to performance testing, specifically as follows: Singlet oxygen yield determination: 2,2,6,6-Tetramethylpiperidine was used as a singlet oxygen scavenger, and in-situ electron paramagnetic resonance (EPR) technology was used for real-time monitoring. 1 O2 generation. Ir(phen)₂Cl₂ and Ir(phen)₂(DA)Cl aqueous solutions were prepared separately. 40 µL of each sample solution was mixed with 2 µL of TEMP and subjected to illumination (650 nm, 500 mW / cm²). 2 In situ EPR testing was performed (60 min). 1 Quantitative comparison of O2 concentrations was obtained by fitting analysis of EPR signal intensity.

[0105] Figure 20 The Ir(phen)2(DA)Cl metal complex prepared in Example 3 of this invention 1 1H NMR spectrum. The spectral signal is clearly assigned, with no obvious impurity peaks, indicating that the target complex was successfully synthesized and has high purity.

[0106] Figure 21 The Ir(phen)2(DA)Cl metal complex prepared in Example 3 of this invention 13 C10 NMR spectrum. The spectrum signal is intact, further confirming the successful synthesis of the target complex and its structural purity.

[0107] Figure 22This is a comparison of the in-situ EPR spectra of Ir(phen)₂(DA)Cl and Ir(phen)₂Cl₂ under the same conditions in Example 3 of this invention. The results show that Ir(phen)₂(DA)Cl modified with catecholamines maintains a stable and high singlet oxygen generation capacity under long-term light irradiation, which is significantly better than the unmodified complex Os(bpy)₂Cl₂.

[0108] This invention provides a modification method that significantly improves the performance of metal complex photosensitizers, addressing the problems of unstable valence states and easily damaged coordination structures of existing metal complexes under continuous illumination. This extends the effective action time of the photosensitizer and overcomes its early inactivation within the clinical therapeutic window (30-60 minutes). The method significantly increases the reactive oxygen species yield of the photosensitizer by orders of magnitude, addressing the insufficient photodynamic efficiency of existing photosensitizers (including many clinically approved ones). Furthermore, this invention comprehensively optimizes the biological and physicochemical properties of the photosensitizer by introducing bio-derived ligands to effectively shield metal toxicity and reduce dark toxicity; improves water solubility; and increases the molar extinction coefficient in long-wavelength regions (e.g., 650 nm and 808 nm), enhancing photosensitivity and extending the absorption spectrum into the near-infrared region.

[0109] The modification strategy provided by this invention is simple to synthesize and low in cost, enabling gram-scale laboratory preparation. At the same time, this strategy has good versatility and can be extended to different metal centers and ligand systems, providing a general technical platform for solving the clinical translation bottleneck of metal complex photosensitizers.

[0110] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A metal complex photosensitizer, characterized in that: Its general structural formula is [M(L)] n (A) i ] m+ Where M is a metal ion, L is an auxiliary ligand, A is a catecholamine ligand, n is the number of auxiliary ligands (1≤n≤6), i is the number of catecholamine ligands (1≤i≤6), and m is the charge of the complex.

2. The metal complex photosensitizer as described in claim 1, characterized in that: The metal ion is selected from ruthenium, osmium, iridium, platinum or other metal ions; the auxiliary ligand is selected from pyridine, bipyridine, phenanthroline, quinoline or their derivatives.

3. The metal complex photosensitizer as described in claim 1, characterized in that: The catecholamine ligand is a catecholamine ligand with a catechol structure that can achieve reversible phenol-quinone conversion, selected from one or more of dopamine, adrenaline, noradrenaline, levodopa or their derivatives, and exists in the complex in one or more of the following forms: fully reduced, fully oxidized or semiquinone free radical.

4. The metal complex photosensitizer as described in claim 1, characterized in that: The catecholamine ligands can form stable free radicals and have reversible electron storage and release capabilities. During illumination, they can effectively inhibit the irreversible increase in valence state of active metal centers, stabilize the oxidation state of metal centers and the overall molecular structure.

5. A method for modifying the catecholamine in the metal complex photosensitizer according to any one of claims 1 to 4, characterized in that: include, Metal salts and auxiliary ligands are dissolved in organic solvents and refluxed under inert gas protection. After the reaction is completed, the metal complex precursor is obtained by precipitation, filtration and purification. The metal complex precursor and ion exchange resin were added to a mixed solvent consisting of acetone and deionized water. Dopamine ligand was added under an inert atmosphere and degassed, followed by heating to carry out the reaction. After the reaction was completed and cooled, acetic acid was added to terminate the reaction, and the metal complex photosensitizer was obtained by separation and purification.

6. The modification method as described in claim 5, characterized in that: The organic solvent is N,N-dimethylformamide or ethylene glycol, the ion exchange resin is Dowex 1×8, and the inert atmosphere is argon or nitrogen.

7. The modification method as described in claim 5, characterized in that: The degassing time is 10-20 minutes, and the heating reaction temperature is 50-80°C for 8-16 hours.

8. The modification method as described in claim 5, characterized in that: The specific steps of separation and purification are as follows: the reaction solution is cooled to 0°C, acetic acid is added to terminate the reaction, the solution is filtered through diatomaceous earth, the filter cake is washed with acetone, the combined filtrates are concentrated and then purified by column chromatography.

9. The use of the metal complex photosensitizer as described in any one of claims 1 to 4 in the preparation of photodynamic therapy drugs.

10. The use of the metal complex photosensitizer according to any one of claims 1 to 4 in the preparation of a medicament for treating tumors.

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