Specific tetradentate copper chelators as anticancer agents

Abstract

Copper chelators of the tetradentate monoquinoline (TDMQ) series are highly efficient against several cancer cell lines. One of these selective copper chelators, TDMQ20, is highly cytotoxic on the non-small cell lung carcinoma (A549), the cervix cancer HeLa and the hepatocarcinoma HepG2 cell cultures, with IC 50 values ranging from 14 to 16 mM in vitro, lower than those obtained with the reference drug 5-fluorouracil (5-FU). TDMQ20 also exhibits a significant antiproliferative activity on HeLa cells in vitro. The mechanism of its cytotoxicity and antiproliferative activity involved intracellular production of reactive oxygen species, drastic mitochondrial damages and induction of apoptosis. The selectivity of TDMQ20 for cancer cells with respect to non-cancer human cells is higher than that of 5-FU, the reference drug. These data strongly support the selection of TDMQ20 as drug-candidate to treat several human cancers.

Description

Specific tetradentate copper chelators as anticancer agentsTechnical Field

[0001] The present invention is related to the field of anticancer drugs. More specifically, the present invention supports the use of copper chelators of the tetradentate monoquinoline (TDMQ) series as anticancer agents.Background Art

[0002] Cancer is the second cause of deaths after cardiovascular diseases worldwide. Over the years, many different methods of cancer treatments have been developed, including surgery, radiation therapy, chemotherapy, gene therapy and immunotherapy. Because of the complexity of cancer and the capacity of cancer cells to create resistant strains, it is absolutely necessary to create new tools in all these different therapeutic areas, including chemotherapy (V. Schirrmacher,Intern. J. Oncology, 2019,54, 407-419; N. Vasanet al., Nature, 2019,575, 299-309; K. Bukowskiet al., Intern. J. Mol. Sci., 2020,21, 3233).

[0003] Among all the different approaches that have been developed in chemotherapy, the category of metal ligands has not been extensively explored, despite the fact that it has been known for a long time that the content of metal ions can be different in cancer cells compared to normal ones. For example, the concentration of copper ions in metastatic carcinoma and in malignant glioma is 1.5 and 1.3 times higher than that of controls, respectively (D. Yoshidaet al., J. Neurooncol., 1993,16, 109-115). In addition, it has been evidenced that angiogenesis is a copper-dependent process (E. Ursoet al.,J. Vasc. Res., 2015,52, 172–196; L. Chenet al.,Signal Transduct. Target. Ther., 2022,7, 378). Copper is involved in the tumor growth and dissemination (metastasis) of a large variety of cancer cells (S. Ishidaet al.,PNAS, 2013,110, 19507–19512; G. Fnuet al.,Front. Oncol. 2021,11, 765329), and depletion of copper has been shown to inhibit angiogenesis in a wide variety of cancer cell and xenograft systems (L. Finneyet al.,Clin. Exp. Pharmacol. Physiol.2009,36, 88-94). Assays in xenograft mice (Q. Panet al.,Cancer Res., 2002,62, 4854-4859), and some anticancer clinical trials have been performed with copper chelators, such as tetrathiomolybdate, D-penicillamine or trientine, but without real success, mainly due to the lack of metal specificity of these ligands and their potential toxicity (L. Finneyet al., Clin. Exp. Pharmacol. Physiol., 2009,36, 88-94; C. Santiniet al., Chem. Rev., 2014,114, 815-862; J. Yoshiiet al.,Int. J. Cancer, 2001,94, 768-773; S. Bremet al.,Neurooncol.July 2005, 246-253).

[0004] Consequently, our working hypothesis was to use highly effective and selective copper chelators as anticancer agents.Technical Problem

[0005] We initially developed a series of copper specific tetradentate ligands named TDMQ, as potential agents for the treatment of Alzheimer’s disease (AD), since it has been evidenced that the homeostasis of copper is disturbed in the brain of patients with AD (see Y. Liuet al. Acc. Chem. Res., 2019,52, 2026-2035). Then, we decided to explore the possibility of using these ligands to reduce the access of cancer cells to copper ions, since these metal ions are involved in tumor growth and angiogenesis of tumors (see Ishidaet al. and Fnuet al., cited in the section above). The TDMQ chelator series has been patented by our group (see Y. Liuet al.,China (GDUT-CNRS), 27 May 2016, n° 201610369550.X, Patent number US 10,807,957 B2. Approved on October 20, 2020. Canada, patent number 3 025 406, approved on June 1st, 2021. Japan, patent number 6889825, approved on May 26th, 2021. Europe, patent application pending).

[0006] The chemical syntheses of TDMQ ligands have been reported in the patents mentioned above, and have been published (W. Zhanget al., ChemMedChem, 2018, 13, 864-704). Among all various TDMQ ligands (see above structures), we have selected TDMQ20 as the best drug-candidate because of its suitable pharmacological profile evidenced in mouse AD-models (see J. Zhaoet al., ACS Chem. Neurosci., 2021,12, 140-149).

[0007]

[0008] Reported General formula of TDMQ ligands.

[0009] For TDMQ20: R = 5,7-dichloro-, n = m = 2.

[0010] The cytotoxic and antiproliferative activities of TDMQ20 against several cancer cell lines are reported in the present application.Technical Solution

[0011] The present disclosure compounds and methods to treat cancer or prevent metastasis.

[0012] In view of the above-mentioned medical need for more efficient compounds useful for the treatment of cancer, the present invention concerns a series of highly selective copper chelators named TDMQ having cytotoxic activitiesin vitroon several cancer cell lines.

[0013] In particular, the invention provides evidences that TDMQ20 displays various degrees of cytotoxic activity against the five tested cancer cell lines. This copper ligand was particularly efficient on the non-small cell lung carcinoma A549, the cervix cancer HeLa cells and the hepatocarcinoma HepG2, at lower concentrations than that required with the reference drug 5-FU. The cytotoxic effect of TDMQ20 against cancer cells is dose-dependent.

[0014] In addition, its selectivity for cancer cells compared to non-cancer HaCaT cells is significantly higher than that of 5-FU.Advantageous Effects

[0015] Investigations of the mechanism of action of TDMQ ligands on HeLa cancer cell lines show that TDMQ20 induces the production of ROS (reactive oxygen species), mitochondrial damage and cancer cell apoptosis. TDMQ20 also prevents cancer cell migration.

[0016] These results show that TDMQ20 has higher anti-cancer activities with a better safety window than 5-fluorouracil (5-FU), a drug which is currently used in the clinical treatment of several cancers, including breast cancer, colon or rectal cancer and stomach cancer. Description of Drawings

[0017] Figure 1. shows the effect of TDMQ20 on the colony-forming ability of HeLa cells at the 14thday after treatment. (A) Representative images of colonies formed by HeLa cells at the 14thday after treatment with increasing concentrations of TDMQ20. (B) Quantitative analysis of this clonogenicity assay. **p< 0.01, ****p< 0.0001 vs. control. “Control” stands for untreated cells.

[0018] FIG. 2 shows the inhibition of TDMQ20 (14.5 mM) on the migratory ability of HeLa cells after 24h-treatment, in the wound healing assay. (A) Representative images of wounds formed by HeLa cells after treatment with TDMQ20. (B) Quantitative analysis of the wound closure upon treatment by TDMQ20. ****p< 0.0001 vs. control. “Control” stands for untreated cells. Scale bar: 250 mm.

[0019] Figure 3. Pro-apoptotic activity of TDMQ20 on HeLa cells after 48 h of incubation. (A) Quantitative analysis of necrotic cells (quadrant Q1), early apoptotic cells (quadrant Q2), advanced apoptotic cells (quadrant Q3) and non-apoptotic cells (quadrant Q4), using annexin V / propidium iodide (PI) staining and detection by flow cytometry. “Control” stands for untreated cells. (B) Histograms represent the proportion of apoptotic cells (Q2 + Q3) within the flow cytometry charts; “ns” stands forp> 0.05, ****p< 0.0001vs. control.

[0020] Figure 4. ROS production induced by TDMQ20 in HeLa cells, detected by DCF fluorescence after 48 h of incubation. ***p< 0.001, ****p< 0.0001.

[0021] Figure 5. (A) Red / green fluorescence of HeLa cells treated with various concentrations of TDMQ20, and stained by JC-1, as an indirect measure of mitochondrial membrane potential (ΔΨm). Detection by flow cytometry. (B) Histograms presenting data of panel A as mean values of three independent experiments ± SD; ****p< 0.0001vs. control.Best Mode

[0022] TDMQ refers to a series compound of the formula (I).

[0023] The term “copper chelators of the tetradentate monoquinoline” refers to TDMQ. Based on prior research, TDMQ as a “copper chelator” can be used for the treatment of Alzheimer’s disease.

[0024] 5-FU stands for 5-fluorouracil which is currently clinically used as an anticancer agent.

[0025] Cancer mainly refers to various malignant tumors. The tumor may be, but is not limited to malignant melanoma, lung cancer, breast cancer, cervix cancer, colon cancer, cutaneous melanoma, cutaneous squamous carcinoma, hepatocellular carcinoma, osteosarcoma, prostate cancer, and uveal melanoma.

[0026] The structural formula of TDMQ20 is as follows :

[0027] EMBODIMENT 1: Pharmacological activity of TDMQ ligands on different cancer cell lines

[0028] Cytotoxicity to cancer cells, selectivity with respect to non-cancer cells

[0029] The cytotoxicity of several compounds from the TDMQ series, especially TDMQ20, was evaluated on several human cancer cell lines. The clinically used anticancer drug 5-fluorouracil (5-FU) was used as comparator.

[0030] The targeted human cancer lines were 1) the metastatic melanoma A375, 2) the non-small cell lung adenocarcinoma A549, 3) the melanoma COLO-829, 4) the cervical cancer HeLa, 5) the hepatocellular carcinoma HepG2. The human immortalized keratinocyte cell line HaCaT was used as non-cancer reference cell line.

[0031] The MTT test was used to measure the cell viability. In this test, the applied colorless tetrazolium salt is reduced by mitochondrial succinate dehydrogenase, thus producing the violet formazan derivative that is quantified by UV-visible spectrometry at 570 nm.

[0032] The cell viability was dependent on the concentration of TDMQ20, and the anti-cancer activity of TDMQ20 was quantified as the concentrations of drug inhibiting by 50 % the growth of each cell line (IC50) after a 48-h incubation period.

[0033] Pharmacological results are reported in Table 1.

[0034] Table 1. Viability of several cell lines treated with TDMQ20 for 48 h, determined by the MTT

[0035] assay. Results are mean values ± SD (mM) of at least three independent experiments.

[0036]

[0037] TDMQ20 exhibited higher cytotoxic activities (lower IC50values) than 5-FU against all the tested cancer cells. Its cytotoxic activity is especially high against the non-small cell lung carcinoma A549, the cervix cancer HeLa cells and the hepatocarcinoma HepG2, with IC50values in the range 14-16 mM, lower than that of the reference drug 5-FU. The cytotoxicity of TDMQ20 on the non-cancer cell line HaCaT (IC50= 41 mM) was lower than that of the reference drug 5-FU (IC50= 30 mM). The selectivity (SI) of TDMQ20 for A549 cells, HeLa cells and HepG2 cell lines with respect to non-cancer cells HaCaT [SI = IC50(HaCaT) / IC50(cancer cell)] was ranging from 2.5 to 2.8. This safety window is better than that of 5-FU.

[0038] These results indicate the dose-dependent cytotoxic activity of TDMQ20 against several human cancer cell lines. This activity is higher than that of the reference drug 5-FU (four times higher on lung adenocarcinoma A549 cells). The cytotoxicity of TDMQ20 is selective for cancer cells with respect to non-cancer HaCaT cells (SI ≥ 2.5).

[0039] Antiproliferative activity of TDMQ20 against HeLa cells

[0040] Colony-forming assay. The proliferation of HeLa cells exposed to various concentrations of TDMQ20 was evaluated at the 14thday after treatment. The number of HeLa cells in cultures treated with TDMQ20 drastically decreased with increasing drug concentration from 1.5 to 12 mM. At a concentration of TDMQ20 = 12 mM, HeLa cells were not detectable.

[0041] The colony-forming ability of HeLa cells treated by TDMQ20 is reported in Figure 1.

[0042] Wound healing assay (or “scratch assay”). The migratory ability of HeLa cells in the presence of TDMQ20 was evaluated using the wound healing assay monitored by optical microscopy. Measurement after 24 h of the width of the “wound” created in a cell monolayer quantify the migration rate of the cells to close the wound. This method mimics cell migration during wound healingin vivo. For cancer cells, cell migration is involved in several processes such as tumor invasion, neo-angiogenesis and metastasis (X. Wanget al.,BMC Pharmacol. Toxicol.2019,20, 4).

[0043] In vitro, TDMQ20 at 14.5 mM significantly inhibited HeLa cell migration to close a wound created in a cell monolayer, indicating that the drug exhibited a cell anti-migratory effect.

[0044] The antiproliferative activity of TDMQ20 in the colony-forming assay and in the “scratch assay” suggests that TDMQ20 should be able to inhibit tumor growth and metastasis.

[0045] EMBODIMENT 2: Mechanisms of activity of TDMQ ligands on HeLa cancer cell linesTDMQ20 promotes apoptosis

[0046] The pro-apoptotic activity of TDMQ20 in HeLa cells was evaluated after 48 h of incubation with the drug, using the annexin V-FITC / PI method (FITC and PI stand for fluoresceine-5-isothiocyanate and propidium iodide, respectively). After staining, live cells show little or no fluorescence (annexin V– / PI–), early apoptosis cells show green fluorescence (annexin V+ / PI–), late apoptosis cells and necrosis cells show red and green fluorescence (annexin V+ / PI+). Cells were detected by flow cytometry. IDEAS software was used for acquisition of data and FlowJo v. X software was used for analysis of data. Results are reported in Figure 3. The total proportion of apoptotic cells, including early apoptosis (quadrants Q2) or late apoptosis (quadrants Q3) or necrosis (quadrants Q1), accounted for 51%, 15% and 9% when HeLa cells were treated with TDMQ20 at the concentration of 14.5 mM, 7.3 mM and 3.7 mM, respectively. So, TDMQ20 induced the apoptosis of HeLa cells in a dose-dependent manner.

[0047] TDMQ20 induces overproduction of reactive oxygen species (ROS)

[0048] Cells constantly generate reactive oxygen species (ROS) during aerobic metabolism. The ROS generation plays an important protective and functional role in the immune system. Cells are equipped with a powerful antioxidant defense system to combat excessive production of ROS. Oxidative damage, thought to play an important role in many human diseases including cancer, occurs in cells when the generation of ROS overwhelms the cell natural antioxidant defenses.

[0049] The most straightforward technique for measuring the production of reduced oxygen species in cells is the use of the cell permeable fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA). In the presence of ROS, predominantly H2O2, H2DCF is rapidly oxidized to 2′,7′-dichlorofluorescein (DCF) which is highly fluorescent, with excitation and emission wavelengths of 498 and 522 nm, respectively. The stained cells were analyzed by flow cytometry and analyzed by the FlowJo v.Xsoftware (E. Eruslanovet al.,Methods Mol. Biol.2010,594, 57-52).

[0050] Results are reported in Figure 4. The fluorescence of DCF in HeLa cells incubated with TDMQ20 at 3.7 mM, 7.3 mM and 14.5 mM, was 125%, 138% and 150% of the control cells, after 48 h of incubation with the drug. So, TDMQ20 induced a dose-dependent increase of ROS production in treated HeLa cells.

[0051] TDMQ20 induces collapse of the mitochondrial inner transmembrane potential (DYm)

[0052] The direction of the mitochondrial membrane potential (with the interior of the organelle being electronegative) allows to produce inward transport of cations and outward transport of anions, thus promoting accumulation of cations in the mitochondria. This electrochemical gradient drives the synthesis of ATP. However, during apoptosis, the mitochondrial transmembrane potential (ΔΨm) decreases since the process is associated with the opening of the mitochondrial permeability pores and loss of the electrochemical gradient. Thus, ΔΨm is an essential parameter of the mitochondrial function that can be used as an indicator of cell health since mitochondria are inherently involved in the apoptotic process of cells (D. R. Greenet al.,Science1998,281, 1309-1312.

[0053] The fluorescent membrane-permeant JC-1 dye exhibits potential-dependent accumulation in mitochondria, which forms JC-1 aggregates, and it diffuses across mitochondria to form monomeric state upon depolarization. Both JC-1 monomers and JC-1 aggregates exhibit green fluorescence (peak emission at 527 nm) due to healthy (non-apoptotic) cells and apoptotic cells, respectively. The JC-1 aggregates also exhibit red fluorescence (peak emission at 590 nm), signature of apoptotic cells. The higher is the ΔΨm, the more elevated is the red shift of the dye (more aggregates formed); vice-versa, the lower is the ΔΨm of the mitochondria and the lower is the red to green ratio of the fluorescent marker (few aggregates formed). Therefore, the ratio of red / green fluorescence intensities is significant of mitochondrial integrity and function, and mitochondrial depolarization is indicated by a reduction in the red to green fluorescence intensity ratio. (L. D. Zorovaet al.,Anal. Biochem. 2018,552, 50-59 ; F. Sivandzadeet al., Bio-Protocol. 2019,9, e3128).

[0054] This method was used to evaluate the mitochondrial membrane potential in HeLa cells incubated with various concentrations of TDMQ20 for 48 h, using staining with JC-1, and quantification by flow cytometry treated with the FlowJo v. X software.

[0055] Results are reported in Figure 5. The drastic decrease of the red / green fluorescence ratio while the concentration of TDMQ20 increased (Figure 5B) indicates the collapse of the mitochondrial transmembrane potential, suggesting a drastic alteration of mitochondria.

[0056] Comments on the results

[0057] TDMQ20 displayed various degrees of cytotoxic activity against the five tested cancer cell lines. It was particularly efficient on the non-small cell lung carcinoma A549, the cervix cancer HeLa cells and the hepatocarcinoma HepG2, with IC50values in the range 14-16 mM, significantly lower than that of the reference drug 5-FU. This effect is dose-dependent. In addition, its selectivity for cancer cells compared to non-cancer HaCaT cells is significantly higher than that of 5-FU.

[0058] Further studies carried out indicate that TDMQ20 at 3-15 mM inhibits migration of HeLa cellsin vitro, in the colony-forming assay and in the wound-healing assay, suggesting that this drug could prevent cancer cells proliferation and metastasisin vivo. This effect of TDMQ20 is dose-dependentin vitro.

[0059] Preliminary investigations on the mechanism of action of TDMQ20 on HeLa cellsin vitroindicate that TDMQ20 (i) enhances ROS production, (ii) drastically decreases the mitochondrial transmembrane potential DYm, and (iii) induces apoptosis. In fact, the concomitance of these three phenomena is expected, because they are linked together. Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to the disruption of electron transport, the loss of ΔΨm, the decline in ATP levels, the production of ROS (due to electrons in the respiratory chain losing their way), and the loss of mitochondrial structural integrity. So, reduction of ΔΨm appears to be a key event during apoptosis (Q. Chenet al.,Blood1998,92, 4545-4553; J.-E. Ricciet al.,Cell2004,117, 773-786).

[0060] The obtained data strongly supports the selection of TDMQ20 as drug-candidate for several human cancers.

Claims

1.A method of treating cancer and preventing metastasis, comprising the administration of an effective amount of compound of the formula (I); ;Where Y represents a group with the following formula: (CH2)n–NH–(CH2)m–N(CH3)2, n and m represent 1 or 2 or 3, R5, R6 and R7 are the same or different, and independently represent a hydrogen atom, or a chlorine atom, or a fluorine atom, or a trifluoromethyl group.2.A method as claimed in claim 1, wherein TDMQ20 depicted by Formula (1) with n = m = 2, R5= R7 = Cl, R6= H, is the preferred TDMQ molecule to manufactdrugs for the treatment of cancer. The drug includes acceptable salts of the TDMQ series. The hydrochloride form is the preferred one.3.A method as claimed in claim 1 or 2, wherein the compound prevents cancer cell proliferation or migration.4.A method as claimed in claim 3, wherein prevents cancer cell proliferation or migration via the induction of the production of ROS (reactive oxygen species), mitochondrial damage and apoptosis. 5.A method as claimed in claim 3, wherein the cancer cell is in a mammal. 6.A method as claimed in claim 5, wherein the mammal is a human.7.A method as claimed in claim 1 or 2, wherein compound of the formula (I) as the drugs include acceptable salts of the TDMQ series. The hydrochloride form is the preferred one.8.A method as claimed in claim 1, wherein cancer refers to malignant tumors. The tumor may be, but is not limited to malignant melanoma, lung cancer, breast cancer, cervix cancer, colon cancer, cutaneous melanoma, cutaneous squamous carcinoma, hepatocellular carcinoma, osteosarcoma, prostate cancer, and uveal melanoma. 9.A method as claimed in claim 6, comprising treating or preventing lung cancer, cervix cancer and the hepatocarcinoma carcinoma, malignant melanoma.10.A method as claimed in claim 6, wherein TDMQ20 is highly cytotoxicin vitroon several human cancer lines, especially against the non-small cell lung carcinoma A549, the cervix cancer HeLa cells and the hepatocarcinoma HepG2.

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