Specific tetradentate copper chelators as anticancer agents

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 tetradentate monoquinoline (TDMQ) copper chelators as anti-cancer drugs. [Background technology]

[0002] Cancer is the second leading cause of death worldwide after cardiovascular disease. Over the years, various cancer treatments have been developed, including surgery, radiation therapy, chemotherapy, gene therapy, and immunotherapy. Due to the complexity of cancer and the ability of cancer cells to develop drug resistance, the development of new treatments in all these various therapeutic areas, 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 among the various chemotherapy-based approaches developed, this class of metal ligands has not been extensively studied. For example, copper ion concentrations in metastatic cancer and malignant glioma were 1.5-fold and 1.3-fold higher than in control groups, respectively (D. Yoshida et al., J. Neurooncol., 1993, 16, 109-115). Furthermore, angiogenesis has been demonstrated to be 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 demonstrated to inhibit angiogenesis in various cancer cells and xenograft systems (L. Finney et al., Clin. Exp. Pharmacol. Physiol. 2009, 36, 88-94). Copper chelators, such as tetrathiomolybdate, D-penicillamine, or trientine, have been used in xenograft mouse studies (Q. Pan et al., Cancer Res., 2002, 62, 4854-4859) and in several anticancer clinical trials, but 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, the present invention utilizes effective and selective copper chelators as anti-cancer agents. Summary of the Invention

[0005] The copper-specific tetradentate ligands, designated TDMQ, first developed in this study are potential therapeutic agents for treating Alzheimer's disease (AD), as copper homeostasis is disrupted in the brains of patients with AD (Y. Liu et al. Acc. Chem. Res., 2019, 52, 2026-2035). These ligands were then used to reduce cancer cell exposure to copper ions, as these metal ions are involved in tumor growth and tumor angiogenesis (Ishida et al. and Fnu et al., cited in their entirety). Our research group has obtained 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 has been reported in the above patent and is publicly available (W. Zhang et al., ChemMedChem, 2018, 13, 864-704). Among various TDMQ ligands (see structure above), TDMQ20 was selected as the best drug candidate, as it showed favorable pharmacological properties in a mouse AD model (J. Zhao et al., ACS Chem. Neurosci., 2021, 12, 140-149). JPEG2025141778000002.jpg38170 General formula of the reported TDMQ ligand

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

[0008] In the present invention, the cytotoxic and antiproliferative activities of TDMQ20 against several cancer cell lines are disclosed.

[0009] Further, in accordance with the present invention, compounds and methods for treating cancer or inhibiting cancer metastasis are disclosed.

[0010] In view of the above-mentioned medical need for more effective compounds applicable to the treatment of cancer, the present invention relates to a series of highly selective copper chelators, designated TDMQ, which have cytotoxic activity against several cancer cell lines in vitro.

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

[0012] Furthermore, the chelating agent disclosed in the present invention has significantly better selectivity for cancer cells and non-cancerous HaCaT cells than 5-FU.

[0013] The mechanism of action of TDMQ ligands in HeLa cancer cell lines was investigated, and it was found that TDMQ20 induces ROS (reactive oxygen species), mitochondrial damage, and cancer cell apoptosis. TDMQ20 can also inhibit cancer cell migration.

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

[0015] [Figure 1]This shows the effect of compound TDMQ20 on HeLa cells' colony-forming ability after 14 days of treatment. (A) Representative images of colonies formed after 14 days of treatment of HeLa cells with increasing concentrations of TDMQ20. (B) Quantitative analysis of the clonogenesis assay (**p<0.01, ***p<0.0001). "Control" indicates untreated cells. [Figure 2] Figure 1 shows the inhibitory effect of 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 images of scratches formed by HeLa cells after treatment with TDMQ20. (B) Quantitative analysis of scratch closure after treatment with TDMQ20 (****: p<0.0001 compared to control). "Control" indicates untreated cells. Scale factor: 250 μM. [Figure 3] 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) was performed using Annexin V / propidium iodide (PI) staining and flow cytometry. "Control" indicates untreated cells. (B) Histograms show the percentage of apoptotic cells (Q2 + Q3) in flow cytometry images. Compared with the control group, "ns" indicates p>0.05, and ***: p<0.0001. [Figure 4] Figure 1 shows TDMQ20-induced ROS production in HeLa cells, detected by DCF fluorescence after 48 h incubation (***p<0.001, ***p<0.0001). [Figure 5] (A) Red / green fluorescence of JC-1 stained HeLa cells treated with various concentrations of TDMQ20, an indirect measure of mitochondrial membrane potential (ΔΨm) (flow cytometry detection). (B) Histograms show data from A as mean ± SD of three independent experiments. **** indicates p<0.0001 compared to the control group. DETAILED DESCRIPTION OF THE INVENTION

[0016] The following examples of the present invention will clearly and completely illustrate the technical solutions of the present invention. Obviously, the following examples are only some of the examples of the present invention, and are not all of the examples. Based on the examples of the present invention, any other examples that can be obtained by those skilled in the art without creative efforts should fall within the scope of protection of the present invention.

[0017] Unless otherwise specified, the test methods used in the examples of the present invention are all conventional methods, 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 "tetradentate monoquinoline copper chelator" refers to TDMQ. Based on previous studies, TDMQ may be applicable as a "copper chelator" for the treatment of Alzheimer's disease.

[0020] 5-FU stands for 5-fluorouracil and is currently used clinically as an anticancer drug.

[0021] Cancer primarily refers to various malignant tumors, including, but not limited to, malignant melanoma, lung cancer, breast cancer, cervical cancer, colon cancer, melanoma, cutaneous squamous cell carcinoma, liver cancer, osteosarcoma, prostate cancer, and uveal melanoma. The structural formula of the compound TDMQ20 is as follows: JPEG2025141778000003.jpg40170

[0022] Example 1: Pharmacological activity of TDMQ ligands against various cancer cell lines Cytotoxicity against cancer cells, selectivity against non-cancer cells The cytotoxicity of several compounds from the TDMQ series, particularly TDMQ20, was evaluated using several human cancer cell lines, with the clinically used anticancer drug 5-fluorouracil (5-FU) as a control.

[0023] The human cancer cell lines targeted were 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 served as a non-cancer reference cell line.

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

[0025] The cell activity was dependent on the concentration of TDMC20, and the anti-cancer activity of TDMC20 was evaluated for each cell line (IC 50 The drug concentration was determined as the concentration at which the proliferation of the cells was inhibited by 50%.

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

[0027] Table 1: Viability of several cell lines treated with TDMQ20 for 48 hours measured by MTT assay. JPEG2025141778000004.jpg23170 Results are the mean ± SD (μM) of at least three independent experiments.

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

[0029] These results demonstrate that TDMQ20 has dose-dependent cytotoxicity against several human cancer cell lines. The cytotoxicity was higher than that of the control drug 5-FU (4-fold higher than that against lung adenocarcinoma A549 cells). Compared to non-cancerous HaCaT cells, TDMQ20 exhibits selective cytotoxicity against cancer cells (SI ≥ 2.5).

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

[0031] The colony-forming ability of HeLa cells treated with TDMQ20 is shown in FIG.

[0032] Cell scratch assay: The migration ability of HeLa cells in the presence of TDMQ20 was assessed using a wound healing assay monitored by optical microscopy. After 24 hours of drug treatment, the cell migration speed was measured by the width of a scratch formed in the cell monolayer. This method simulated cell migration during the in vitro scratch healing process. For 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 and sealed scratches generated in the cell monolayer, demonstrating the anti-cell migration effect of this drug.

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

[0035] Example 2: Inhibitory mechanism of TDMQ ligand on HeLa cancer cell line Promotion of TDMQ20 on cell apoptosis The pro-apoptotic activity of TDMQ20 in HeLa cells was assessed after 48 hours of incubation with the drug using the annexin V-FITC / PI method (FITC and PI represent fluorescein-5-isothiocyanate and propidium iodide, respectively). After staining, viable cells were either very scarce or non-fluorescent (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 apoptotic cells (quadrant Q2), late apoptotic cells (quadrant Q3), and necrotic cells (quadrant Q1), was 51%, 15%, and 9%, respectively. Thus, TDMQ20 induced apoptosis in HeLa cells in a dose-dependent manner.

[0036] Induction of TDMQ20 leads to excessive production of reactive oxygen species (ROS) Cells continuously produce reactive oxygen species during aerobic metabolic processes. ROS production plays an important protective and functional role in the immune system. Cells are equipped with a powerful antioxidant defense system to counteract the overproduction of ROS. Oxidative damage is thought to play an important role in many human diseases, including cancer, and oxidative damage occurs in cells when ROS production exceeds the cells' natural antioxidant defenses.

[0037] The most direct method for measuring intracellular ROS production is the use of 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. Stained cells were analyzed by flow cytometry using FlowJo vX software (E. Eruslanov et al., Methods Mol. Biol. 2010, 594, 57-52).

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

[0039] Induction of mitochondrial inner membrane potential collapse (ΔΨm) by TDMQ20 The orientation of the mitochondrial membrane (electronegativity within the organelle) allows the inward transport of cations and the outward transport of anions, thereby promoting the accumulation of cations within the mitochondria. This electrochemical gradient promotes ATP synthesis. However, during the process of cell apoptosis, the mitochondrial membrane potential (ΔΨm) decreases. This process is associated with the opening of the mitochondrial permeability pore 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, since mitochondria are involved in the apoptotic process of cells (DR Green et al., Science 1998, 281, 1309-1312).

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

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

[0042] The results are shown in Figure 5. As the TDMQ20 concentration increased, the red / green fluorescence ratio decreased rapidly (Figure 5B), indicating a collapse of the mitochondrial membrane potential and the occurrence of rapid changes in the mitochondria.

[0043] About the results TDMQ20 showed varying degrees of cytotoxicity against the five cancer cell lines tested. It was particularly effective against non-small cell lung cancer A549, cervical cancer HeLa cells, and liver cancer HepG2, with IC 50 The concentrations of TDMQ20 were in the range of 14-16 μM, significantly lower than those of the control drug 5-FU, and the effect was dose-dependent. Furthermore, TDMQ20 was significantly more selective for non-cancerous HaCaT cells and cancer cells than 5-FU.

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

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

[0046] The data obtained strongly validate TDMQ20 as a select drug candidate for several human cancers.

[0047] Obviously, the above specific embodiments are merely a further detailed description of the objectives, technical means and beneficial effects of the present invention. It should be noted that the above descriptions are merely specific examples of the present invention and do not limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention are all included within the protection scope of the present invention.

Claims

1. A method for treating cancer and preventing cancer metastasis, comprising administering an effective amount of a compound of formula (I). Formula (I) In the formula, Y is a group represented by the formula: (CH ２ ) ｎ -NH-(CH ２ ) ｍ -N(CH ３ ) ２ n and m represent 1, 2, or 3; R ５ , R ６ and R ７ may be the same or different and each independently represents a hydrogen atom, a chlorine atom, a fluorine atom or trifluoromethyl.

2. In the formula (I), n=m=2, R ５ =R ７ = Cl, R ６ The method of claim 1, wherein TDMQ20 =H is a preferred molecule for producing a cancer therapeutic agent, and the therapeutic agent includes an acceptable salt of the TDMQ series, with the hydrochloride salt form being the preferred form.

3. The method of claim 1 or 2, wherein the compound inhibits cancer cell proliferation or migration.

4. The method according to claim 3, characterized in that it inhibits the proliferation or migration of cancer cells by inducing ROS production, mitochondrial damage and apoptosis.

5. 4. The method of claim 3, wherein the cancer cells are in a mammal.

6. 6. The method of claim 5, wherein the mammal is a human.

7. 3. The method according to claim 1 or 2, wherein the compound of formula (I) which is a drug includes acceptable salts of the TDMQ series, the hydrochloride form of the compound being the preferred form.

8. The method of claim 1, wherein cancer refers to a malignant tumor, including, but not limited to, malignant melanoma, lung cancer, breast cancer, cervical cancer, colon cancer, melanoma, cutaneous squamous cell carcinoma, liver cancer, osteosarcoma, prostate cancer, and uveal melanoma.

9. 7. The method of claim 6, comprising the treatment or prevention of lung cancer, liver cancer, or malignant melanoma.

10. The method according to claim 6, characterized in that TDMQ20 exhibits high cytotoxicity in vitro against several human cancer lines, particularly non-small cell lung cancer A549, cancer HeLa cells, and liver cancer HepG2.

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