A lysosome-targeted ruthenium complex and preparation and application thereof

Abstract

This invention belongs to the interdisciplinary field of coordination chemistry with biology and medicine, and relates to a lysosome-targeting ruthenium complex, its preparation, and its applications. The ruthenium complex prepared by this invention has a novel structure, exhibiting lysosome-targeting and phosphorescent properties. It can not only achieve phosphorescent imaging of cells and tissues but also act as a photosensitizer, generating reactive oxygen species under light irradiation for photodynamic therapy of tumors. Under weakly acidic conditions, the phosphorescence of this complex is significantly enhanced, and its ability to generate singlet oxygen and catalyze NADH is also increased, thus demonstrating a more effective killing effect on lysosomes and tumors. The preparation method of this invention has advantages such as simple preparation process, high yield, and good reproducibility. The ruthenium complex prepared by this invention can be used for bioimaging and photodynamic therapy of tumors.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of coordination chemistry, biology, and medicine, and specifically relates to a lysosome-targeted ruthenium metal complex, its preparation method, and its application in bioimaging and tumor photodynamic therapy. Background Technology

[0002] Targeting organelles in tumor therapy has become a new paradigm for precision medicine, attracting significant attention. These strategies precisely deliver drugs to critical or vulnerable subcellular organelles, such as mitochondria, lysosomes, and the Golgi apparatus. By precisely disrupting the structure and function of subcellular organelles, therapeutic efficacy can be significantly improved, drug resistance prevented, drug dosage reduced, and side effects minimized. Among various subcellular organelles, lysosomes, as essential organelles in human and mammalian cells, play crucial roles in various physiological and signal transduction processes, such as intracellular transport, protein degradation, endocytosis, and cell death. Compared to normal cells, cancer cells possess more lysosomes and a lower acidity (pH 4.5–5.5). Furthermore, once lysosomes are damaged, protons and dozens of hydrolases within them are released into the cytoplasm, which can trigger programmed cell death by cleaving various intracellular substrates, such as cysteine ​​proteases and several members of the Bcl-2 protein family. These characteristics make lysosomes ideal targets for tumor diagnosis and treatment. Therefore, developing drugs that target lysosomes is a key objective.

[0003] Transition metal complexes have attracted increasing attention as probes or drugs targeting cell organelles. This is due to the rich tunable structures and excellent photophysical properties of metal complexes, providing a variety of options for designing unique drugs / probes. Among transition metal complexes, ruthenium complexes have been widely used in bioimaging, biosensing, and photoactivated tumor therapy. TLD1433, as an outstanding representative of ruthenium complexes, has shown promising results in recent clinical trials. Due to their high solubility and stability, tunable structure and redox potential, excellent photophysical properties, and sub-organelle targeting capabilities, Ru(II) complexes have become promising organelle-targeting metal drugs in cancer therapy.

[0004] Photodynamic therapy (PDT) has been widely used in preclinical and clinical studies due to its non-invasiveness, photospecific toxicity, and spatiotemporal controllability. Many ruthenium polypyridine complexes have been explored as photosensitizers (PSs) for PDT. Recently, Huaiyi Huang et al. developed an iridium complex-based photoredox catalyst (PC) for the treatment of hypoxic tumors (Nat. Chem. 2019, 11, 1041–1048). This strategy differs from traditional PSs, which directly transfer energy to surrounding oxygen molecules under light, generating highly toxic reactive oxygen species. Instead, it directly catalyzes the substrate (NADH) via an electron transfer pathway, independent of oxygen concentration. This catalytic process disrupts the mitochondrial electron transport chain (ETC) and disturbs cellular redox homeostasis, ultimately leading to the death of damaged cells.

[0005] Although various ruthenium complex photosensitizers have been designed and synthesized for preclinical and basic scientific research in photodynamic therapy (PDT) of tumors, many of these complexes exhibit high affinity for DNA, enabling them to target the cell nucleus. However, such nuclear-targeted drugs may pose potential genotoxicity problems. In contrast, lysosomal-targeting drug strategies are considered a relatively safe and effective approach. Nevertheless, the number of ruthenium complexes capable of specifically targeting lysosomes remains very limited. Furthermore, the hypoxic nature of the tumor microenvironment significantly inhibits the activity of photosensitizers, while both tumor cells and lysosomes exhibit typical weakly acidic characteristics, posing challenges to the development of new therapeutic strategies. This invention aims to prepare a lysosome-targeting ruthenium complex that achieves the treatment of hypoxic tumors through the oxygen concentration-independent photocatalytic NADH pathway, exhibiting higher PDT and photocatalytic activity under weakly acidic conditions. Therefore, this research will significantly increase the efficacy of tumor treatment and provide an important tool for developing new lysosome-targeted therapeutic strategies. Summary of the Invention

[0006] In view of this, the present invention provides a lysosome-targeted ruthenium metal complex, its preparation method and application.

[0007] The ruthenium complex provided by this invention is composed of a ruthenium complex cation unit as shown in Formula I or Formula II and a coordinating anion.

[0008]

[0009] The coordinating anion includes Cl. - ,Br - I - NO3 - or PF6 - PF6 is preferred. - .

[0010] The ruthenium complex cation shown in Formula I is protonated to the structure shown in Formula II under low pH conditions.

[0011] This invention also provides a method for preparing the lysosome-targeted ruthenium metal complex described above, comprising the following steps:

[0012] 1) Dissolve 1,10-phenanthroline-5,6-dione, p-hydroxybenzaldehyde and ammonium acetate in an organic solvent and react to prepare intermediate 1 as shown in formula I-1;

[0013]

[0014] 2) Intermediate 1 and bis(1,10-phenanthroline) ruthenium dichloride were subjected to a coordination reaction in a mixed solvent of dichloromethane and methanol to obtain a ruthenium complex containing the cation shown in Formula I, Cl. - It is a ruthenium complex with a coordinating anion;

[0015] 3) The cation shown in Formula I above, Cl - The ruthenium complex with the coordinating anion undergoes an ion exchange reaction to yield Br. - I - NO3 - or PF6 - A ruthenium complex with a coordinating anion and structural formula I as a cation;

[0016] 4) The obtained ruthenium metal complex is protonated under acidic conditions to obtain a ruthenium metal complex containing the ruthenium complex cation shown in Formula II.

[0017] In step 1), the molar ratio of 1,10-phenanthroline-5,6-dione, p-hydroxybenzaldehyde, and ammonium acetate is 1:1:10.

[0018] The organic solvent is glacial acetic acid;

[0019] The reaction temperature is 80-130℃, and the time is 12-24h;

[0020] In step 2), the ratio of the molar number of intermediate 1 to the sum of the molar numbers of intermediate 1 and bis(1,10-phenanthroline)ruthenium dichloride is 0.2-0.8:1;

[0021] In a mixed solvent of dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 1:1;

[0022] The coordination reaction was carried out under reflux for 12-24 hours.

[0023] In step 3), the ion replacement reaction is to replace Cl with Cl. - Replace with Br- I - NO3 - or PF6 - Reagents used for ion exchange reactions include NaBr, NaI, NaNO3, or ammonium hexafluorophosphate;

[0024] The ion exchange reaction is carried out at room temperature for 1-4 hours.

[0025] The ruthenium complex has lysosomal targeting properties.

[0026] The ruthenium complex can generate reactive oxygen species under light irradiation, and its ability to generate reactive oxygen species is even stronger under acidic conditions, thus exhibiting enhanced photodynamic effects.

[0027] The ruthenium complex exhibits photocatalytic redox ability independent of oxygen concentration, and its photocatalytic redox ability is even stronger under acidic conditions.

[0028] The application of the above-mentioned ruthenium complex in the preparation of optical imaging reagents and tumor photodynamic therapy reagents is also within the scope of protection of this invention.

[0029] The present invention also provides a photosensitizer comprising the above-mentioned ruthenium complex.

[0030] The use of the ruthenium complex and / or photosensitizer in the preparation of antitumor drugs; preferably, the antitumor drugs include drugs suitable for photodynamic therapy.

[0031] The tumor cells for which the anti-tumor drugs are applicable include breast cancer 4T1 cells and breast cancer EMT6 cells.

[0032] The antitumor drug targets the lysosomes of tumor cells.

[0033] The ruthenium complex, as a photosensitizer, exhibits pH-responsive phosphorescence. This photosensitizer demonstrates low in vitro cellular dark toxicity and high phototoxicity.

[0034] The ruthenium complex prepared in this invention has a novel structure and exhibits lysosomal targeting and phosphorescence properties. It can not only achieve phosphorescent imaging of cells and tissues but also act as a photosensitizer, generating reactive oxygen species under light irradiation for photodynamic therapy of tumors. Under weakly acidic conditions, the phosphorescence of this complex is significantly enhanced, and its ability to generate singlet oxygen and catalyze NADH is also increased, thus demonstrating a more effective killing effect on lysosomes and tumors. The preparation method of this invention has advantages such as simple preparation process, high yield, and good reproducibility. The ruthenium complex prepared in this invention can be used for bioimaging and photodynamic therapy of tumors. Attached Figure Description

[0035] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0036] Figure 1 This is a synthetic route diagram for preparing the ruthenium metal complex in Example 1 of the present invention;

[0037] Figure 2 This is a high-resolution mass spectrum of the ruthenium complex prepared in Example 1 of the present invention;

[0038] Figure 3 The 1H NMR spectrum of the ruthenium complex prepared in Example 1 of this invention;

[0039] Figure 4 The ultraviolet absorption and phosphorescence spectra of the ruthenium complex measured in Example 2 of this invention are shown below.

[0040] Figure 5 This is a laser confocal microscope image of the ruthenium complex in mouse breast cancer cells 4T1 after co-staining with mitochondrial probes MTR and LTR, respectively, in Example 3 of the present invention.

[0041] Figure 6 This is a diagram showing the effect of reactive oxygen generation of the ruthenium complex in two different pH aqueous solutions in Example 4 of the present invention;

[0042] Figure 7 This is a graph showing the photocatalytic oxidation of NADH by the ruthenium complex in Example 5 of this invention in two aqueous solutions with different pH values.

[0043] Figure 8 This is a small animal in vivo fluorescence imaging image of the ruthenium complex on mouse tumors in Example 6 of the present invention;

[0044] Figure 9 This is a real-time monitoring image of the destruction of lysosomes by the ruthenium complex under light in Example 7 of the present invention;

[0045] Figure 10 This is a diagram illustrating the tumor-killing effect of the ruthenium complex at the cellular level in Example 7 of the present invention.

[0046] Figure 11 This is a diagram illustrating the in vivo tumor-killing effect of the ruthenium complex in Example 8 of the present invention. Detailed Implementation

[0047] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0048] Example 1

[0049] 1,10-phenanthroline-5,6-dione (1.05 g), p-hydroxybenzaldehyde (0.61 g), and ammonium acetate (3.85 g) were added to the reaction vessel, followed by 60 mL of glacial acetic acid. The mixture was stirred at 120 °C for 24 hours under argon protection. After cooling to room temperature, the pH was adjusted to neutral using concentrated ammonia. A large amount of white solid appeared. The mixture was filtered, and the precipitate was collected to obtain intermediate 1. (2) Synthesis of the target product. Intermediate 1 (312 mg) and bis(1,10-o-phenanthroline) ruthenium dichloride (532 mg) were added to a CH2Cl2 / MeOH (1 / 1 v / v) mixed solvent and refluxed for 24 hours under argon protection. After the reaction was completed, the mixture was rotary evaporated to dryness, dissolved in a small amount of dichloromethane, and ammonium hexafluorophosphate was added. The mixture was reacted at room temperature for 1 hour, and the target product, the ruthenium metal complex, was obtained by column chromatography. (3) Characterization of the ruthenium metal complex. The obtained complex was dissolved in acetonitrile and characterized by electrospray ionization mass spectrometry. The results are as follows: Figure 2 As shown. (4) The obtained ruthenium complex was dissolved in DMSO-d6, and the proton spectrum was measured by nuclear magnetic resonance, as shown. Figure 3 As shown.

[0050] Example 2

[0051] The UV absorption and fluorescence emission properties of lysosomal-targeted ruthenium metal complexes. For example... Figure 4 As shown, the ruthenium complex exhibits strong absorption in the 260-360 nm range, with an absorption peak at 263 nm. It also shows a broad, moderately intense peak in the 360-550 nm range, centered around 450 nm, characteristic of typical MLCT absorption. For cell imaging, visible light excitation has lower phototoxicity, making it more suitable for applications in biological systems.

[0052] Example 3

[0053] Live-cell optical imaging of lysosome-targeted ruthenium complexes. Mouse breast cancer 4T1 cells in logarithmic growth phase were separated into two 35mm confocal culture dishes and incubated for 24 hours at 37°C in 1640 medium containing 10% fetal bovine serum. Then, 30 μM ruthenium complexes were added to each dish, and the cells were incubated for 4 hours. After washing with PBS, mitochondrial red fluorescent probe MTR and lysosomal red fluorescent probe LTR were added to the two confocal dishes, respectively, and incubated for 20 minutes. Confocal microscopy was then performed immediately. The results are shown below. Figure 5 As shown, the ruthenium complex only partially overlaps with the mitochondrial probe MTR, but almost completely overlaps with the lysosomal probe LTR, indicating that the ruthenium complex is located in the lysosome of the cell, demonstrating its lysosomal targeting.

[0054] Example 4

[0055] Determination of reactive oxygen species (ROS) generation by ruthenium metal complexes under light irradiation. To investigate the ability of ruthenium metal complexes to generate ROS under light irradiation, this invention utilizes the ROS probe 9,10-anthratridiyl-bis(methylene)dicarboxylic acid (ABDA) to detect ROS generation. Figure 6 As shown, when the ruthenium complex coexists with ABDA, the absorption peak of ABDA at 378 nm gradually decreases with increasing light exposure time, indicating that the ruthenium complex can generate reactive oxygen species under light conditions. Simultaneously, this invention selected two buffer solutions with different pH values, such as... Figure 6 As shown, under low pH conditions (pH=4.5), the absorption peak of ABDA decreases more significantly over time, indicating that the ruthenium complex has a stronger ability to generate reactive oxygen species under acidic conditions and has an enhanced photodynamic effect.

[0056] Example 5

[0057] Determination of photocatalytic oxidation of NADH by ruthenium metal complexes under illumination. Since tumor tissues are hypoxic, the efficacy of oxygen concentration-dependent photodynamic therapy is inevitably weakened. In recent years, oxygen concentration-independent photocatalytic redox mechanisms have been considered a novel and highly effective strategy to overcome tumor hypoxia limitations. The ruthenium metal complexes of this invention can undergo photocatalytic redox reactions with the mitochondrial coenzyme NADH, such as... Figure 7 As shown, under 450nm LED illumination, the characteristic absorption peak of NADH at 338nm gradually decreased over time in the ruthenium complex and NADH reaction under neutral conditions (pH = 7.40). Under weakly acidic conditions (pH = 4.53), the decrease in NADH absorption peak over time was more pronounced. This phenomenon is further illustrated by the characteristic absorption peak at 338nm and the decrease in reaction rate. The above experiments demonstrate that the ruthenium complex can undergo photocatalytic redox reactions. Furthermore, it shows that the ruthenium complex exhibits stronger photocatalytic redox activity under acidic conditions.

[0058] Example 6

[0059] In vivo fluorescence imaging of ruthenium complexes for mouse tumors. A 4T1 mouse tumor model was established, and two mice with tumors approximately 100 mm² were selected. 3 Tumor-bearing mice were used in two groups: one as a control group, receiving only saline solution, and the other as the experimental group, receiving 25 μL of a ruthenium complex injected into the tumor. Imaging was performed using a small animal in vivo fluorescence imaging system. Figure 8 As shown, the tumor sites in the control group showed almost no fluorescence, while obvious fluorescence signals were observed in the tumor sites of the experimental group mice, indicating that this ruthenium complex can be used for in vivo fluorescence imaging in mice.

[0060] Example 7

[0061] Ruthenium metalloproteinase complex (RuTM) disrupts lysosomes in tumor cells. Since this RuTM complex can target lysosomes, and in vitro experiments showed that it generates reactive oxygen species (ROS) under light conditions, we further investigated its lysosomal killing effect on tumor cells using acridine orange as an indicator. We incubated the RuTM complex in 4T1 cells for 3 hours, then added acridine orange and incubated for half an hour. After removing the culture medium, the cells were washed three times with PBS and imaged using laser confocal microscopy. Fluorescence in the green and red channels was collected, and the results are shown below. Figure 9 As shown. In the absence of laser irradiation (0 min), green fluorescence was almost distributed throughout the entire cell, while red fluorescence was only distributed in lysosomes. Subsequently, the cells were irradiated using a laser confocal microscope, with fluorescence signals collected every 40 seconds until the experiment ended after 200 seconds. Real-time monitoring showed that after laser irradiation, some lysosomal fluorescence signals disappeared and diffused, indicating that the integrity of the lysosomal membrane was disrupted. After the lysosomal membrane structure is disrupted, hydrolases in the lysosomes are released into the cytoplasm. Therefore, we detected cathepsin B using the catheps in Bsubstrate Magic Red MR-(RR)2 kit. Figure 10 As shown, cells in the ruthenium complex group emitted bright Magic Red fluorescence, which highly overlapped with the fluorescence of the ruthenium complex itself. In contrast, cells in the ruthenium complex + light-illuminated group showed significantly weakened fluorescence from Magic Red, with reduced overlap with the ruthenium complex. This result indicates that reactive oxygen species generated by the ruthenium complex under light irradiation disrupt the lysosomal membrane structure, leading to the release of hydrolases from the lysosomes into the cytoplasm, ultimately resulting in cell death.

[0062] Example 8

[0063] To investigate whether the ruthenium complex of this invention can achieve tumor-killing effects in vivo, we constructed a 4T1 tumor-bearing mouse model. Twenty female Bal lb / c mice were randomly selected, and the hair on the right posterior back of each mouse was removed using scissors and depilatory cream. The next day, each mouse was subcutaneously injected with 50 μL of approximately one million 4T1 tumor cells. Seven days later, when the tumor volume grew to approximately 100 cubic millimeters, the experiment proceeded to the next stage. These mice were randomly assigned to four different experimental groups: 1) a PBS control group only; 2) a light-exposed group only; 3) a ruthenium complex injection group only; and 4) a ruthenium complex injection followed by light-exposed group. Mice in the control group were injected with 25 μL of PBS into the tumor; mice in the light irradiation group were exposed to a 450 nm LED lamp in the tumor area for 1 hour; mice in the complex group were injected with 25 μL of ruthenium complex (dose 5 mg / kg) into the tumor; and mice in the complex + light irradiation group were treated with light after receiving the ruthenium complex injection. The experimental results showed that, as Figure 11 As shown, only the complex + light irradiation group exhibited a significant inhibitory effect on tumor growth; the tumors of 2 out of 5 mice almost completely regressed, and the tumors of the remaining 3 mice were also significantly inhibited. These findings indicate that this ruthenium complex possesses strong tumor-killing potential in photodynamic therapy and warrants further exploration in clinical studies.

[0064] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of ruthenium metal complexes in the preparation of optical imaging reagents and tumor photodynamic therapy reagents; The ruthenium complex exhibits pH-responsive phosphorescence and lysosomal targeting properties; the optical imaging reagent is used for optical imaging of 4T1 breast cancer cells; the tumor is breast cancer. The ruthenium metal complex is .

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