Specific tetradentate copper chelating agents as anticancer drugs

Abstract

To provide a method for treating cancer and preventing cancer metastasis using a copper chelator.SOLUTION: There is provide a method for treating cancer and preventing cancer metastasis, comprising administering an effective amount of a copper chelator represented by a compound TDMQ20.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to the technical field of anti-cancer drugs. More specifically, the present invention discloses the use of tetraaza-monoketone (TDMQ)-based copper chelating agents as anti-cancer agents.

Background Art

[0002] Cancer is the second leading cause of death worldwide after cardiovascular disease. Over the years, various cancer treatment methods such as surgery, radiotherapy, chemotherapy, gene therapy, and immunotherapy have been developed. Due to the complexity of cancer and the ability of cancer cells to develop drug resistance, the development of new treatment methods in all these various treatment fields, including chemotherapy, is absolutely necessary (V. Schirrmacher, Intern. J. Oncology, 2019, 54, 407-419; N. Vasan et al., Nature, 2019, 575, 299-309; K. Bukowski et al., Intern. J. Mol. Sci., 2020, 21, 3233).

[0003] It has long been known that the metal ion content of cancer cells may differ from that of normal cells, but the classes of metal ligands have not been widely studied among the various approaches developed based on chemotherapy. For example, the copper ion concentrations in metastatic cancer and malignant glioma were 1.5 times and 1.3 times higher, respectively, than in the control group (D. Yoshida et al., J. Neurooncol., 1993, 16, 109-115). Furthermore, it has been demonstrated that angiogenesis is a copper-dependent process (E. Urso et al., J. Vasc. Res., 2015, 52, 172-196; L. Chen et al., Signal Transduct. Target. Ther., 2022, 7, 378). Copper is involved in the proliferation and spread (metastasis) of various cancer cells (S. Ishida et al., PNAS, 2013, 110, 19507-19512; G. Fnu et al., Front. Oncol. 2021, 11, 765329), and copper depletion has been shown to inhibit angiogenesis in various cancer cells and xenograft systems (L. Finney et al., Clin. Exp. Pharmacol. Physiol. 2009, 36, 88-94). Although copper chelating agents, such as tetrathiomolybdate, D-penicillamine, or trientine, have been used in xenograft mice (Q. Pan et al., Cancer Res., 2002, 62, 4854-4859) and in several anti-cancer clinical trials, they have not been successful in practice, mainly due to the lack of metal specificity and potential toxicity of these ligands (L. Finney et al., Clin. Exp. Pharmacol. Physiol., 2009, 36, 88-94; C. Santini et al., Chem. Rev., 2014, 114, 815-862; J. Yoshii et al., Int. J. Cancer, 2001, 94, 768-773; S. Brem et al., Neurooncol. July 2005, 246-253).

[0004] Therefore, in this invention, an effective and selective copper chelating agent is used as an anticancer agent. [Overview of the project]

[0005] The copper-specific tetradentate ligands, initially developed in this invention and named TDMQ, are potential therapeutic agents for Alzheimer's disease (AD), as it has been demonstrated that copper homeostasis is disrupted in the brains of Alzheimer's patients (Y. Liu et al. Acc. Chem. Res., 2019, 52, 2026-2035). In this invention, we investigated using these ligands to reduce the likelihood of cancer cells coming into contact with copper ions (because these metal ions are involved in tumor growth and tumor angiogenesis) (Ishida et al. and Fnu et al.; cited in full). Our research group holds patents for the TDMQ chelating agent series (Y. Liu et al.; China (GDUT-CNRS), May 27, 2016, Application No. 201610369550.X; Patent No. US10807957B2 (October 20, 2020); Canada, Patent No. 3025406 (June 1, 2021); Japan, Patent No. 6889825 (May 26, 2021); Europe, Patent No. EP3466931B1).

[0006] The chemical synthesis of TDMQ ligands is reported and published in the above patent (W. Zhang et al., ChemMedChem, 2018, 13, 864-704). Among various TDMQ ligands (see structures above), TDMQ20, which exhibited pharmacological properties suitable for the mouse AD model, was selected as the best drug candidate (J. Zhao et al., ACS Chem. Neurosci., 2021, 12, 140-149). JPEG0007900791000001.jpg38170 General formula of reported TDMQ ligand

[0007] In the case of TDMQ20, R = 5,7-dichloro-, n = m = 2.

[0008] This invention discloses the cytotoxicity and antiproliferative activity of TDMQ20 against several cancer cell lines.

[0009] Furthermore, the present invention discloses compounds and methods for treating cancer or inhibiting cancer metastasis.

[0010] In consideration of the above medical need for more effective compounds applicable to the treatment of cancer, the present invention relates to a series of highly selective copper chelating agents named TDMQ. It exhibits cytotoxic activity against several cancer cell lines in vitro.

[0011] In particular, this invention provides evidence demonstrating that TDMQ20 exhibits varying degrees of cytotoxicity against five test cancer cell lines. This copper chelator is especially effective against non-small cell lung cancer A549, cervical cancer HeLa cells, and liver cancer HepG2, at concentrations lower than those of the positive drug 5-FU. Furthermore, TDMQ20 exhibits dose-dependent cytotoxic effects on cancer cells.

[0012] Furthermore, the chelating agents disclosed in this invention exhibit significantly superior selectivity for cancer cells and non-cancerous HaCaT cells compared to 5-FU.

[0013] Studies investigating the mechanism of action of TDMQ ligands on HeLa cancer cell lines revealed that TDMQ20 induces ROS (reactive oxygen species) generation, mitochondrial damage, and cancer cell apoptosis. TDMQ20 can also suppress cancer cell migration.

[0014] As can be seen from the results above, TDMQ20 has higher anticancer activity and higher safety compared to 5-fluorouracil (5-FU). 5-fluorouracil is currently used in the clinical treatment of various cancers, including breast cancer, colon cancer, rectal cancer, and gastric cancer. [Brief explanation of the drawing]

[0015] [Figure 1]This shows the effect of the compound TDMQ20 on the colony-forming ability of HeLa cells 14 days after treatment. (A) Representative images of colonies formed 14 days after treatment of HeLa cells with increased concentrations of TDMQ20. (B) Quantitative analysis of clonal formation tests (**p<0.01, ***p<0.0001). "Control" refers to untreated cells. [Figure 2] This study demonstrates the inhibitory effect of the compound TDMQ20 (14.5 M concentration) on the migration ability of HeLa cells after 24 hours of treatment in a cell scratch assay. (A) Representative image of scratches formed in HeLa cells after treatment with TDMQ20. (B) Quantitative analysis of scratch closure after treatment with TDMQ20 (****: p<0.0001 compared to the control group). "Control" indicates untreated cells. Scale: 250 μM. [Figure 3] This shows the pro-apoptotic activity of TDMQ20 on HeLa cells after 48 hours of incubation. (A) Quantitative analysis of necrotic cells (Q1 quadrant), early apoptotic cells (Q2 quadrant), late apoptotic cells (Q3 quadrant), and non-apoptotic cells (Q4 quadrant), detected using annexin V / propidium iodide (PI) staining and flow cytometry. "Control" indicates untreated cells. (B) The histogram shows the percentage of apoptotic cells (Q2+Q3) in the flow cytometry figure, compared to the control group, with "ns" indicating p>0.05 and ***:p<0.0001. [Figure 4] HeLa cells showed TDMQ20-induced ROS production, which was detected by DCF fluorescence after 48 hours of incubation (***p<0.001, ***p<0.0001). [Figure 5] (A) HeLa cells treated with various concentrations of TDMQ20 and stained with JC-1 exhibited red / green fluorescence, and the mitochondrial membrane potential (ΔΨm) was indirectly measured (detected by flow cytometry). (B) In the histogram, the data from A are shown as the mean ± SD of three independent experiments. **** indicates p<0.0001 compared to the control group.

Mode for Carrying Out the Invention

[0016] Hereinafter, the technical means of the present invention will be clearly and completely described by way of examples of the present invention. Clearly, the following examples are only some examples of the present invention, not all examples. Based on the examples of the present invention, all other examples obtained by those skilled in the art without creative efforts should be included in the protection scope of the present invention.

[0017] The test methods used in the examples of the present invention are all ordinary methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0018] TDMQ refers to a series of compounds represented by formula (I).

[0019] The term "copper chelating agent of tetracoordinate monoquinoline" refers to TDMQ. Based on previous research, TDMQ can be applied to the treatment of Alzheimer's disease as a "copper chelating agent".

[0020] 5-FU represents 5-fluorouracil and is currently clinically used as an anticancer agent.

[0021] Cancer mainly refers to various malignant tumors. Tumors include, but are not limited to, malignant melanoma, lung cancer, breast cancer, cervical cancer, colon cancer, melanoma, cutaneous squamous cell carcinoma, liver cancer, osteosarcoma, prostate cancer, uveal melanoma. The structural formula of compound TDMQ20 is as follows. JPEG0007900791000002.jpg40170 <00?0095> Example 1: Pharmacological Activity of TDMQ Ligand Against Various Cancer Cell Lines Cytotoxicity Against Cancer Cells, Selectivity Against Non-Cancer Cells The cytotoxicity of several compounds derived from the TDMQ series, particularly TDMQ20, was evaluated using several human cancer cell lines. The clinically used anticancer drug 5-fluorouracil (5-FU) was used as a control.

[0023] The target human cancer cell lines are: 1) metastatic melanoma A375, 2) non-small cell lung adenocarcinoma A549, 3) melanoma COLO-829, 4) cervical cancer HeLa, and 5) hepatocellular carcinoma HepG2. The human immortalized keratinocyte cell line HaCaT was used as a non-cancer reference cell line.

[0024] Cell activity was detected using the MTT assay. In this assay, the added colorless thiazole blue is reduced by mitochondrial succinate dehydrogenase to produce a violet formazan, which is quantified by ultraviolet-visible spectroscopy at 570 nm.

[0025] Cell activity is dependent on the concentration of TDMC20, and the anticancer activity of TDMC20 is determined after incubation for 48 hours in each cell line (IC). 50 The drug concentration was quantified as the concentration that inhibits the proliferation of ) by 50%.

[0026] The pharmacological results are shown in Table 1.

[0027] Table 1: Results of measuring the viability of several cell lines treated with TDMQ20 for 48 hours using the MTT assay. JPEG0007900791000003.jpg23170 The results are the mean ± SD (μM) of at least three independent experiments.

[0028] TDMQ20 showed higher cytotoxicity (lower IC) than 5-FU against all cancer cells tested. 50 It showed a value of cytotoxicity, particularly against non-small cell lung cancer A549, cervical cancer HeLa cells, and liver cancer HepG2. 50 The value is in the range of 14-16 μM, IC 50The values ​​were lower than those of the control drug 5-FU. TDMQ20 showed cytotoxicity (IC) against the non-cancer cell line HaCaT. 50 TDMQ20 (at 41 μM) showed lower cytotoxicity than the control drug 5-FU. The selectivity (SI) of TDMQ20 for A549 cells, HeLa cells, and HepG2 cell lines was in the range of 2.5 to 2.8 for non-cancer cells HaCaT [SI=IC1]. 50 (HaCaT) / IC 50 (Cancer cells). Its safety was higher than that of 5-FU.

[0029] These results indicate that TDMQ20 exhibits dose-dependent cytotoxicity against several human cancer cell lines. Cytotoxicity was higher than that of the control drug 5-FU (four times higher than toxicity against lung adenocarcinoma A549 cells). Against non-cancerous HaCaT cells, TDMQ20 exhibits selective cytotoxicity against cancer cells (SI ≥ 2.5).

[0030] Antiproliferative activity of TDMQ20 against HeLa cells Cell cloning experiment: HeLa cells were exposed to various concentrations of TDMQ20, and cell proliferation was evaluated on day 14. The number of HeLa cells treated with TDMQ20 decreased sharply as the drug concentration increased from 1.5 to 12 μM. At a concentration of TDMQ20 = 12 μM, no HeLa cells were detected.

[0031] Figure 1 shows the colony-forming ability of HeLa cells treated with TDMQ20.

[0032] Cell scratch assay: The migratory ability of HeLa cells in the presence of TDMQ20 was evaluated using a wound healing assay monitored with an optical microscope. After treatment with the drug for 24 hours, the cell migration rate was measured by the width of the scratch formed on the cell monolayer. This method simulated cell migration during the in vitro scratch healing process. In cancer cells, cell migration is involved in several processes, including tumor invasion, angiogenesis, and metastasis (X. Wang et al., BMC Pharmacol. Toxicol. 2019, 20, 4).

[0033] In vitro, 14.5 μM TDMQ20 significantly inhibited HeLa cell migration, blocking scratches that occurred in the cell monolayer, demonstrating the anti-migration effect of this drug.

[0034] The antiproliferative activity of TDMQ20 in cell cloning experiments and scratch assays demonstrates that TDMQ20 can suppress tumor growth and metastasis.

[0035] Example 2: Mechanism of inhibition of HeLa cancer cell lines by TDMQ ligand Promotion of TDMQ20 in cellular apoptosis The pro-apoptotic activity of TDMQ20 in HeLa cells was evaluated after incubation with the drug for 48 hours using the annexin V-FITC / PI method (FITC and PI represent fluorescein-5-isothiocyanate and propidium iodide, respectively). After staining, very few viable cells or no fluorescence (annexin V- / PI-), early apoptotic cells showed green fluorescence (annexin V+ / PI-), and late apoptotic and necrotic cells showed red and green fluorescence (annexin V+ / PI+). Cells were detected by flow cytometry. Data were collected using IDEAS software and analyzed using FlowJo vX software. The results are shown in Figure 3. When HeLa cells were treated with TDMQ20 at concentrations of 14.5 μM, 7.3 μM, and 3.7 μM, the overall percentage of apoptotic cells, including early apoptosis (quadrant Q2), late apoptosis (quadrant Q3), and necrosis (quadrant Q1), was 51%, 15%, and 9%, respectively. Therefore, TDMQ20 induced apoptosis in HeLa cells in a dose-dependent manner.

[0036] Excessive production of reactive oxygen species (ROS) due to induction of TDMQ20 Cells continuously produce reactive oxygen species (ROS) during aerobic metabolic processes. ROS production plays an important protective and functional role in the immune system. Cells have a robust antioxidant defense system to counteract ROS overproduction. Oxidative damage is thought to play a significant role in many human diseases, including cancer, and oxidative damage occurs when ROS production exceeds the cell's natural antioxidant defenses.

[0037] The most direct method for measuring the production of reactive oxygen species within cells is to use the cell-permeable fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA). In the presence of ROS (mainly H2O2), H2DCF is rapidly oxidized to 2,7-dichlorodihydrofluorescein (DCF). This DCF is highly fluorescent, with excitation and emission wavelengths of 498 nm and 522 nm, respectively. Cells stained by flow cytometry were analyzed using FlowJo vX software (E. Eruslanov et al., Methods Mol. Biol. 2010, 594, 57-52).

[0038] The results are shown in Figure 4. After incubation with the drug for 48 hours, DCF fluorescence in HeLa cells incubated with TDMQ20 at 3.7 μM, 7.3 μM, and 14.5 μM was 125%, 138%, and 150%, respectively, compared to control cells. Therefore, TDMQ20 induced a dose-dependent increase in ROS in treated HeLa cells.

[0039] Induction of mitochondrial inner membrane potential (ΔΨm) by TDMQ20 The orientation of the mitochondrial membrane (electronegativity within the organelle) enables the internal transport of cations and the external transport of anions, thereby promoting the accumulation of cations within mitochondria. This electrochemical gradient promotes ATP synthesis. However, during the process of cell apoptosis, the mitochondrial membrane potential (ΔΨm) decreases. This process is related to the opening of mitochondrial permeable pores and the loss of the electrochemical gradient. Therefore, ΔΨm is a fundamental parameter of mitochondrial function and can be used as an indicator of cell health. This is because mitochondria are involved in the process of cell apoptosis (DR. Green et al., Science 1998, 281, 1309-1312).

[0040] The JC-1 dye, a fluorescent membrane-permeable agent, accumulates in mitochondria in a voltage-dependent manner to form JC-1 aggregates, which then diffuse into the mitochondria during depolarization to form monomers. Both JC-1 monomers and JC-1 aggregates exhibit green fluorescence (peak emission at 527 nm) in healthy (non-apoptotic) cells and apoptotic cells, respectively. JC-1 aggregates further exhibit red fluorescence (peak emission at 590 nm), which is characteristic of apoptotic cells. A higher ΔΨm corresponds to a greater redshift of the dye (more aggregates formed). Conversely, a lower mitochondrial ΔΨm corresponds to a lower red / green ratio of the fluorescent label (fewer aggregates formed). Therefore, the red / green fluorescence intensity ratio has significant importance for mitochondrial integrity and function, and mitochondrial depolarization is indicated by a decrease in the red / green fluorescence intensity ratio (LDZorova et al., Anal. Biochem. 2018, 552, 50-59; F. Sivandzade et al., Bio-Protocol. 2019, 9, e3128).

[0041] This method is used to evaluate mitochondrial membrane potential in HeLa cells incubated with various concentrations of TDMQ20 for 48 hours. Quantification was performed by flow cytometry processed using FlowJov.X software after JC-1 staining.

[0042] The results are shown in Figure 5. As the TDMQ20 concentration increases, the red / green fluorescence ratio decreases sharply (Figure 5B), indicating a breakdown of the mitochondrial membrane potential and suggesting that a rapid change has occurred in the mitochondria.

[0043] Regarding the results TDMQ20 showed varying degrees of cytotoxicity against five test cancer cell lines. It was particularly effective against non-small cell lung cancer A549, cervical cancer HeLa cells, and liver cancer HepG2. 50 The values ​​were in the range of 14-16 μM, significantly lower than those of the control drug 5-FU, and this effect is dose-dependent. Furthermore, TDMQ20 showed significantly higher selectivity for non-cancerous HaCaT cells and cancer cells than 5-FU.

[0044] Further studies have shown that TDMQ20 inhibits in vitro migration of HeLa cells at 3–15 μM. Cell cloning experiments and cell scratch assays have shown that this drug inhibits cancer cell proliferation and metastasis in vivo. This effect of TDMQ20 is dose-dependent in vitro.

[0045] As studies on the mechanism of action of TDMQ20 in vitro on HeLa cells have shown, TDMQ20 (i) enhances ROS production, (ii) significantly reduces mitochondrial membrane potential ΔΨm, and (iii) induces cellular apoptosis. In fact, these three phenomena are interrelated and therefore predictable. Permeability of the mitochondrial outer membrane and release of cytochrome c promote the activation of intracellular caspases and the execution of cellular apoptosis. The first target of activated caspases is the permeable mitochondria themselves, leading to disruption of electron transport, loss of ΔΨm, decrease in ATP levels, ROS production (due to loss of electron directionality in the respiratory chain), and loss of mitochondrial structural integrity. Therefore, a decrease in ΔΨm is important in the process of cellular apoptosis (Q. Chen et al., Blood 1998, 92, 4545-4553; J.-E. Ricci et al., Cell 2004, 117, 773-786).

[0046] The data obtained strongly demonstrate that TDMQ20 is a promising drug candidate for several human cancers.

[0047] Clearly, the specific embodiments described above are merely further details regarding the objectives, technical means, and beneficial effects of the present invention. Furthermore, the above description is merely a specific example of the present invention and does not limit it. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are all within the scope of protection of the present invention.

Claims

1. Use of a compound of formula (I) in the manufacture of a therapeutic agent for the treatment and / or prevention of metastasis of cancer, which is lung cancer, cervical cancer, liver cancer, or malignant melanoma. Equation (I).

2. The use according to claim 1, characterized in that the therapeutic agent comprises a pharmaceutically acceptable salt.

3. The use according to claim 2, characterized in that the salt is in the form of a hydrochloride salt.

4. The use of the therapeutic agent according to claim 1 or 2, characterized in that it inhibits the proliferation or migration of cancer cells.

5. The use of the therapeutic agent according to claim 4, characterized in that it inhibits the proliferation or migration of cancer cells by inducing ROS production, mitochondrial damage, and apoptosis.

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