Use of organometallic ruthenium compounds for the prevention and / or treatment of cancer-related diseases and / or conditions in individuals - Patents.com

Abstract

The present invention relates to a compound of the general formula (I): JPEG2025508999000018.jpg54170 (wherein A is selected from the group consisting of CF3SO3, PF6, Cl, BPh4, NO3 or BF4) in all possible racemic and stereoisomers.

Description

[Technical field]

[0001] The present invention relates to organometallic ruthenium compounds for use in the prevention and / or treatment of diseases and / or conditions associated with cancer in an individual, more particularly colorectal cancer, pancreatic ductal adenocarcinoma, non-small cell lung cancer, ovarian and endometrial cancer. [Background technology]

[0002] Colorectal cancer (CRC) is a significant cause of morbidity and mortality worldwide and is the second leading cause of cancer deaths, with an estimated 1.1 million cases and over 500,000 deaths worldwide in 2020 (Sung et al., 2021).

[0003] Several oncogenes are involved in CRC oncogenesis, with the most common mutations found in KRAS (40%), PIK3CA (20%) and BRAF (15%) (Laszlo et al., 2021; Laurent-Puig et al., 2009). These mutations activate key signaling pathways involved in CRC progression, namely the MAPK and PI3K signaling pathways, which are responsible for fundamental cellular processes such as proliferation, apoptosis, autophagy, and invasion / motility (Berg & Soreide, 2012).

[0004] KRAS is the most frequently mutated oncogene in cancer, and KRAS mutations are commonly associated with poor prognosis and resistance to treatment. More specifically, KRAS mutations are prominent in cancers such as CRC, pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC) (Haigis, 2017).

[0005] Additionally, KRAS can be mutated in a low percentage of almost every cancer type, but mutation rates higher than 10% characterize only ovarian and endometrial cancers (Timar & Kashofer, 2020).

[0006] The choice of treatment method for cancer is very important since each cancer responds differentially to different methods. Currently, four approaches are used: surgery, chemotherapy, radiation therapy and targeted therapy.

[0007] For CRC, for example, surgery is the mainstay treatment, followed by chemotherapy with 5-fluorouracil (5-FU). 5-FU is the most widely used drug to treat CRC and can be used alone or in combination with other drugs. However, it has a low success rate of 10%-15% due to severe side effects and resistance (Cazzanelli et al., 2016; Pardini et al., 2011; Van Cutsem et al., 2016).

[0008] Platinum anticancer drugs are the group of anticancer drugs with the most success stories in the field of medicinal inorganic chemistry. Cisplatin was the first to be discovered and is the most used drug of the group. Despite the clinical success of cisplatin, it has many associated drawbacks. Chemotherapy with cisplatin is frequently accompanied by severe side effects and its activity is limited to a narrow spectrum of tumors due to acquired and intrinsic resistance to the treatment (Bergamo et al., 2012).

[0009] CRC was one of the first cancer types where targeted therapy (anti-EGFR and anti-VEGFR) was introduced, and testing for RAS mutations became a routine molecular pathology activity, leading to several studies on KRAS clonality and primary / metastatic comparisons. For example, the compounds bevacizumab, cetuximab, and panitumumab are typically used to target EGFR and VEGFR, respectively (Lee & Sun, 2016; Zaniboni, 2015). These receptors activate the MAPK and PI3K signaling pathways, which are the most important signaling pathways in CRC carcinogenesis. However, recent studies have demonstrated that tumors with mutations in key downstream effectors of the EGFR signaling pathway, such as KRAS, BRAF, and PIK3CA, do not respond to EGFR antibodies, raising a clinically relevant problem that needs to be overcome (Hong et al., 2016; Temraz et al., 2015).

[0010] Due to the severe side effects of available treatments and the acquisition of resistance in some tumors, research has been directed towards the development of compounds based on other metals. Among the several metal complexes investigated so far, ruthenium (hereafter also referred to by its chemical symbol Ru) compounds appear as some of the most promising metallodrugs. Among other features, Ru-based drugs have shown good anticancer activity, various modes of action, chemical stability, structural diversity, diverse ligand binding modes and redox properties are achievable, and some complexes are quite selective for cancer cells (Moreira et al., 2019; Thota et al., 2018). Currently, there are several ruthenium complexes in clinical trials for use in cancer treatment (Alsaab et al., 2020; Thota et al., 2018).

[0011] A primary goal of researchers investigating therapeutic approaches to treat cancer is to discover new anticancer drugs with higher efficacy, reduced toxicity, no cross-resistance, and improved pharmacological characteristics.

[0012] Despite recent advances in cancer therapy, much remains to be done regarding available drugs and their side effects. In the case of CRC, treatment remains limited, and patients with CRC harboring mutations in KRAS do not respond to available treatments (Hong et al., 2016).

[0013] The present invention therefore proposes new and original organometallic ruthenium compounds for use in the prevention and / or treatment of diseases and / or conditions associated with cancer in an individual, more particularly colorectal cancer, pancreatic ductal adenocarcinoma, non-small cell lung cancer, ovarian and endometrial cancer. Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention relates to a compound of the general formula (I): [ka] (In the formula, A is selected from the group consisting of CF3SO3, PF6, Cl, BPh4, NO3 or BF4. An organometallic ruthenium compound having the formula: The organometallic ruthenium compounds relate to organometallic ruthenium compounds in all possible racemic and stereoisomeric forms.

[0015] The lack of specific anticancer drugs to target KRAS in cancers harboring this mutation highlights the need to develop new targeted drugs, which represents a major challenge and relevant clinical concern that needs to be resolved in cancer treatment.

[0016] Over the years, several strategies have attempted to indirectly target KRAS in three distinct ways. The first one investigated was targeting KRAS membrane localization, inhibiting farnesyltransferase by blocking KRAS post-translational modifications. However, these approaches do not distinguish between mutant and wild-type KRAS proteins and may affect numerous other proteins that undergo prenylation and post-translational processing by the same enzyme (Ryan & Corcoran, 2018a).

[0017] Another approach for the treatment of KRAS-driven tumors has been targeting the RAS GTP / GDP cycle using inhibitors of guanine nucleotide exchange factors such as Sos1, but the binding activity of these inhibitors to KRAS was weak, and these inhibitors did not translate into clinical use ( Nagasaka et al., 2020 ).

[0018] The final approach has been to target KRAS downstream effectors using inhibitors against BRAF, MEK, ERK, AKT or mTOR proteins, however, considerable crosstalk between MAPK and PI3K signaling pathways and associated drug resistance has resulted in limited clinical benefit (Liu et al., 2021).

[0019] KRAS mutations are found in 40% of CRCs, with codon 12 and 13 changes being the most frequent. The most common amino acid substitutions in CRCs with KRAS mutations are G12D (13%), G12V (9%), and G13D (7%) (Laszlo et al., 2021; Li et al., 2020). KRAS mutations are also present in 80% of PDAC, 35% of NSCLC, and 10% of ovarian and endometrial cancers, with NRAS, another important member of the RAS family, being the second most frequently mutated (Christensen et al., 2020; Haigis, 2017; Timar & Kashofer, 2020).

[0020] Selective targeting of specific mutations in KRAS may provide a more direct approach to inhibit oncogenic KRAS mutant function while sparing wild-type protein function.

[0021] In recent years, two KRAS G12C Inhibitors, sotrasib and adagrasib, have been approved for clinical use in non-small cell lung cancer (NSCLC; Liu et al., 2021). However, G12C Mutations are present in only 3% of CRCs with KRAS mutations, and these inhibitors are not effective in CRCs with mutated KRAS ( Laszlo et al., 2021 ).

[0022] Recent studies have demonstrated that potent and selective KRAS G12D Inhibitors were identified and the antitumor benefits were demonstrated in mouse animal models (Xiaolun Wang et al., 2022; Kemp SB, 2023). Such results support the potential for the development of effective therapeutic tools, as specific, potent, and durable effects were achieved in promoting tumor regression in transplanted and spontaneous pancreatic cancer models.

[0023] In recent years, more and more KRAS inhibitors have entered clinical trials, but they all target KRAS G12C To the best of our knowledge, no KRAS inhibitors are available to specifically target KRAS hotspot mutations in CRC. [Means for solving the problem]

[0024] Surprisingly, the present invention solves the problems of the prior art by identifying organometallic ruthenium compounds having formula (I) for use in the prevention and / or treatment of diseases and / or conditions associated with cancer in an individual, more particularly colorectal cancer, pancreatic ductal adenocarcinoma, non-small cell lung cancer, ovarian and endometrial cancer.

[0025] Over the last few years, we have been devoted to a family of “piano stool” compounds (Ru(II)-cyclopentadienyl complexes) as potential anticancer drugs for chemotherapy ( Valente et al., 2021 ).

[0026] So far, the anticancer efficacy of a particular organometallic ruthenium compound having formula (I), namely PMC79, has only been evaluated in the ovarian cancer cell line A2780, and in the breast cancer-derived cell lines MCF7 and MDA-MB-231 (Moreira et al., 2019). The anticancer efficacy of this compound was found to be promising, with the median inhibitory concentration (IC) values ​​for both models being 1.0 and 1.2 times higher than those for the control. 50 ) were lower (3.9 μM, 5.9 μM, and 2.1 μM for A2780, MCF7, and MDA-MB-231, respectively), and for breast cancer cells, IC 50 The values ​​were lower than those of cisplatin (36 μM and 110 μM for MCF7 and MDA-MB-231, respectively). 2 N,N')(η 5 -cyclopentadienyl)(triphenylphosphine)ruthenium(II) triflate.

[0027] Results also revealed that in breast cancer cell lines, PMC79 inhibited proliferation by reducing colony-forming ability and induced apoptosis demonstrated by increased Annexin V stained cells. PMC79 also induced changes in mitochondrial morphology with edematous and disorganized mitochondria, as well as alterations in the actin cytoskeleton with cytoskeletal disorganization, cell scattering, and reduced cell-to-cell cytoskeletal extension. Furthermore, results revealed that PMC79 preferentially distributed to the membrane fraction (Moreira et al., 2019).

[0028] Some Ru(II)-cyclopentadienyl complexes from our group have already been tested in CRC cell lines, but not PMC79 (Morais et al., 2016; Valente et al., 2021). The results show that even very small structural differences result in dramatic biological responses (Morais et al., 2016; Valente et al., 2021). Also, the same ruthenium compounds can be highly cytotoxic or not cytotoxic at all, depending on the genetic background of the cells (Teixeira et al., 2021). Therefore, considering that PMC79 has only been tested in ovarian and breast cancer models, and because of the genetic differences and distinct therapeutic approaches for these cancers, this same compound would not be expected to have application in CRC.

[0029] As mentioned above, KRAS mutations are the most frequent oncogenic alteration present in CRC (40%) as well as PDAC (80%), NSCLC (35%) and ovarian and endometrial cancer (10%) (Christensen et al., 2020; Haigis, 2017; Timar & Kashofer, 2020). Therefore, in addition to CRC, it is expected that this compound may also have application in PDAC, NSCLC, and ovarian and endometrial cancer models due to the high incidence of KRAS mutations in these cancers.

[0030] For the purpose of providing an understanding of the principles according to embodiments of the invention, reference will be made to the embodiments shown in the drawings and the terminology used to describe them. It should be understood that in no case is it intended to limit the scope of the invention to the contents of the drawings. Any subsequent modifications or alterations of the features of the invention shown herein, and any additional applications of the principles and embodiments of the invention shown, that would normally occur to a person skilled in the art having this specification in hand, are deemed to be within the scope of the invention as claimed. [Brief description of the drawings]

[0031] [Figure 1]FIG. 1 shows the results of a colony formation assay in SW480 colorectal cancer cell line after incubation with compounds according to the invention. [Diagram 2] Figure 2 is a graph showing cell cycle results in SW480 colorectal cancer cell line after incubation with compounds according to the invention. [Diagram 3] 1 is a graph showing the results of a CFSE assay in SW480 cells after incubation with compounds according to the invention. [Figure 4] FIG. 1 is a graph showing the results of Annexin V / propidium iodide in SW480 colorectal cancer cell line after incubation with compounds according to the invention. [Diagram 5] FIG. 1 shows the results of a terminal transferase dUTP nick end labeling (TUNEL) assay in SW480 cells after incubation with compounds according to the invention. [Figure 6] 1 is a graph showing the results of wound healing in SW480 cells after incubation with compounds according to the invention. [Figure 7] 1 is a graph showing the analysis of cellular distribution in SW480 cells after incubation with compounds according to the invention. [Figure 8] FIG. 2 shows the analysis of F-actin staining to assess how the cytoskeleton of SW480 cancer cells is affected by compounds according to the invention. [Figure 9] FIG. 1 shows a Western blot analysis of SW480 cell line examining the evaluation of expression of KRAS, pAKT, total-AKT, pERK and total-ERK proteins induced by compounds according to the invention. [Figure 10] FIG. 1 shows a Western blot analysis of RKO cell line examining the evaluation of expression of KRAS, pAKT, total-AKT, pERK and total-ERK proteins induced by compounds according to the invention. [Figure 11]FIG. 1 shows a Western blot analysis of the LS174T cell line examining the evaluation of expression of KRAS, pAKT, total-AKT, pERK and total-ERK proteins induced by compounds according to the invention. [Figure 12] FIG. 1 shows a Western blot analysis of SW480 cell line examining the evaluation of expression of KRAS protein induced by siRNA targeting KRAS and compounds according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] In a first aspect the present invention relates to a compound of general formula (I): [ka] (In the formula, A is selected from the group consisting of CF3SO3, PF6, Cl, BPh4, NO3 or BF4. wherein said organometallic ruthenium compounds are in all possible racemic and stereoisomeric forms.

[0033] In a preferred embodiment according to the first aspect of the present invention, the organometallic complex of formula (I) ({2,2'-bipyridine}-4,4'-diyldimethanol-k 2 N,N')(η 5 -cyclopentadienyl)(triphenylphosphine)ruthenium(II) triflate (compound PMC79).

[0034] In a preferred embodiment according to the invention, the individual is a mammal, in particular a human.

[0035] In another embodiment, the organometallic ruthenium compounds for use are in a composition comprising a pharma- ceutically acceptable excipient. The term "pharmaceutically acceptable" means that which is generally safe, non-toxic, and not biologically or otherwise undesirable and is useful in preparing pharmaceutical compositions, including those that are acceptable for veterinary and human pharmaceutical use.

[0036] In another embodiment, the organometallic ruthenium compound for use is in a composition to be administered by oral, rectal or parenteral injection route, preferably by parenteral route. The term "parenteral injection" refers to administration by injection under or through one or more layers of the skin or mucosa of an individual. More preferably, said parenteral administration is selected from the group consisting of intravenous administration, intramuscular administration, intradermal administration, or subcutaneous administration.

[0037] In other embodiments, the organometallic ruthenium compounds for use are in compositions in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, sprays, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions or sterile packaged powders.

[0038] The organometallic ruthenium compounds for use according to the invention inhibit KRAS and the downstream signalling pathways MAPK and PI3K. Inhibition of KRAS and the downstream signalling pathways MAPK and PI3K preferably results in inhibition of cell proliferation and induction of apoptosis.

[0039] In a preferred embodiment of the invention, the KRAS inhibitor is G12V and KRAS G12D Targeting mutations.

[0040] Examples and Experimental Methods Synthesis of PMC79 The compounds were synthesized using a protocol developed by the inventors (Corte-Real, Karas, Girio et al., 2019). Alternatively, the synthesis can be carried out in methanol instead of dichloromethane.

[0041] Cell lines and culture conditions The non-cancerous NCM460 cell line, derived from normal colonic epithelial mucosa, was obtained from INCELL (Moyer et al., 1996), and the colorectal cancer-derived cell lines, SW480, LS174T, and RKO, were obtained from the American Type Culture Collection (ATCC). All cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2. NCM460 and SW480 cells were grown in RPMI medium, LS174T and RKO cells were grown in DMEM, all supplemented with 10% FBS and 1% penicillin / streptomycin. SW480 cells were routinely cultured at 1 × 10 5 Cells were seeded at a concentration of 1000 cells / ml.

[0042] Sulforhodamine B assay CRC cells were seeded in 24-well test plates and 24 hours after seeding, the cells were incubated with various concentrations of PMC79 for 48 hours. Two negative controls were performed: in control (1), cells were incubated with growth medium only, and in DMSO control (2), cells were exposed to the highest concentration of DMSO used (up to 0.1% DMSO (v / v) per well) to remove any influence of this solvent on the results. After 48 hours of treatment, sulforhodamine B was performed as previously described. Results were expressed relative to the negative control (1), which was considered as 100% cell growth.

[0043] Colony formation assay SW480 cell line was seeded in 6-well plates at a concentration of 500 cells / ml. 24 hours after seeding, the cells were diluted with 1 / 4 IC 50 and 1 / 2IC 50Cells were incubated with PMC79 at 0.05 mg / mL for 48 h. Negative control cells were treated with DMSO 0.1%. After 48 h of incubation, the medium was replaced with fresh medium without PMC79. Medium was refreshed every 3 days. Nine days after treatment was stopped, cells were stained as previously described (Corte-Real, Karas, Bras et al. 2019).

[0044] Carboxyfluorescein succinimidyl ester (CFSE) assay SW480 cells were incubated with the dye and seeded according to the procedure recommended by the CFSE package insert (BD Horizon™). After 24 hours, the cells were incubated with IC 50 and 2 × I.C. 50 The cells were incubated with PMC79 at 0 h and collected at time 0 and acquired on a flow cytometer. After 48 and 72 h, both floating and attached cells were collected, washed with PBS, centrifuged at 500 rpm for 10 min, and the pellet was resuspended in PBS before acquisition on the flow cytometer.

[0045] Cell cycle analysis SW480 cell line was seeded in 6-well plates. After 24 hours, the cells were incubated with IC 50 PMC79. Negative control cells were treated with DMSO 0.1%. After 24 and 48 h, cells were harvested and processed according to the method described in (Teixeira et al., 2018). Data analysis and quantification of cell cycle phases were performed using FlowJo 10.7.2 software.

[0046] Annexin V / propidium iodide assay SW480 were seeded in 6-well plates, and 24 h after seeding, the cells were cultured using IC 50 Cells were exposed to PMC79 compound at 0.05 mg / mL for 1 h. Negative control cells were treated with DMSO 0.1%. After 48 h, cells were harvested and stained as previously described (Corte-Real, Karas, Bras et al., 2019). Acquisition was performed using a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA).

[0047] TUNEL assay The SW480 cell line was seeded in 6-well plates, and 24 h after seeding, the cells were cultured using IC 50 Cells were exposed to 100% PMC79. Negative control cells were treated with DMSO 0.1%. After 48 h, cells were treated according to (Teixeira et al., 2018). Slides were kept at -20 °C until visualization under a fluorescent microscope (Leica DM 5000B, Leica Microsystems, Wetzlar, Germany).

[0048] Wound healing assay SW480 cell line at 7 × 10 5 After 24 hours, a wound was made at the tip and the cells were incubated with IC 50 and 2 × I.C. 50 The cells were incubated with PMC79 for 12 hours. Negative control cells were treated with DMSO 0.1%. Each condition was photographed at 0, 4, 8, and 12 hours. ImageJ 1.53a software was used to analyze the data and measure the wound size at each time point.

[0049] Cellular distribution measured by ICP-MS analysis SW480 cells were seeded in 100 mm Petri dishes, and after 24 h, the cells were incubated with PMC79 and their IC 50 The cells were incubated with 101Ru at a concentration equal to the 101Ru value. After 48 h of incubation, the cells were washed with ice-cold PBS and processed to obtain a cell pellet. Cytoplasmic, membrane / particulate, cytoskeleton, and nuclear fractions were extracted using the Fraction-PREP (BioVision, USA) cell fractionation kit according to the manufacturer's protocol. After digestion of the samples, the Ru(101Ru) content in each fraction was measured by Thermo X-Series Quadrupole ICPMS (Thermo Scientific) and using the same procedure as previously described (Corte-Real et al., 2013).

[0050] F-actin staining with phalloidin 568 SW480 cell line was seeded in 12-well plates with one coverslip per well. After 24 h, the cells were incubated with IC 50 Cells were exposed to 100 μg / ml PMC79 and negative control cells were treated with DMSO 0.1%. After 48 h of incubation, cells were fixed and stained as previously described (Pilon et al., 2020). Representative images were obtained at 600× magnification on a fluorescence microscope (Olympus Motorized BX63F Upright Microscope) (Olympus (C) Co., Tokyo, Japan).

[0051] Immunoblot analysis Preparation of total protein extracts of colorectal cancer cell lines (RKO, LS174T, and SW480), SDS-PAGE, and Western blot were performed as previously described ( Alves et al., 2015 ).

[0052] antibody The antibodies used were anti-KRAS and β-actin (Sigma); anti-phospho-p44 / 42 MAPK (Thr202 / Tyr204) (pERK), anti-p44 / 42 total (ERK), anti-phospho-Akt (Ser473) (pAKT) and anti-Akt total (Cell Signaling) (AKT); GAPDH (Gene Tex).

[0053] Proteomics Research SW480 cell line was seeded in 100 mm Petri dishes and 24 h after seeding, cells were treated with PMC79 compound for protein extraction. Samples were processed for LC-MS / MS analysis (Chiva et al., 2018; Olivella et al., 2021). Protein identification and validation, as well as gene ontology, localization and network analysis were performed as previously described (Perkins et al., 1999; Beer et al., 2017; Perez-Riverol et al., 2022).

[0054] statistical analysis Results were obtained from at least three independent experiments and expressed as the mean ± SD. One-way ANOVA with Dunnett's post-hoc test or Tukey's post-hoc test and two-way ANOVA with Dunnett's post-hoc test were used to analyze the results. All different statistical analyses were performed using GraphPad Prism version 8 for macOS, GraphPad Software, La Jolla California USA (http: / / www.graphpad.com).

[0055] Examples and Results PMC79 has selective anti-cancer effects in colorectal cancer cells Evaluate the anticancer potential and IC of PMC79 compounds 50 To determine the .alpha.-amino acid sequence, a sulforhodamine B assay was used.

[0056] PMC79 has an IC of 40 μM 50 (Table 1) and was found to inhibit cell growth in SW480 CRC cell line, whereas the non-cancerous cell line NCM460 showed a higher IC 50 The value is shown (44 μM).

[0057] Table 1 shows the IC values ​​determined by sulforhodamine B after 48 hours of incubation. 50 Values ​​and selectivity indices are shown. The non-cancerous NCM460 cell line, derived from normal colonic epithelial mucosa, was used to determine the selectivity index against the colorectal cancer cell line SW480. Values ​​represent the mean ± SD of at least three independent experiments.

[0058] [Table 1]

[0059] The selectivity index was also determined, demonstrating that PMC79 is selective for CRC cells (SI=1.1). Some authors support that a selectivity index higher than 1 indicates cytotoxicity against cancer cells over healthy non-tumor cells. This result suggests that PMC79 is a good candidate to be investigated as a potential anti-cancer drug for CRC treatment.

[0060] PMC79 reduces proliferation of colorectal cancer cells Several techniques were used to study the effect of PMC79 on proliferation. Colony formation assays were performed to determine the 1 / 4IC 50 and 1 / 2IC 50 After exposure to PMC79 for a period of time, the cells' ability to recover and form colonies was assessed. The results showed that PMC79 significantly reduced the clonogenic potential of SW480 cells in a dose-dependent manner (Figure 1).

[0061] [Figure 1] shows 1 / 4IC in SW480 cell line. 50 and 1 / 2IC 50 Analysis of colony formation ability after 48 h of incubation with. A) Images of colony formation assay in SW480 cell line. B) Graphical representation of colony formation ability in SW480 cells. Values ​​represent the mean ± SD of at least three independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test. ****P ≤ 0.0001 compared to negative control.

[0062] To assess cell cycle changes induced by PMC79, SW480 cells were incubated with IC 50 The cells were incubated with PMC79 for 24 and 48 hours. At 24 hours, it was observed that PMC79 induced cell cycle arrest at the G0 / G1 phase, but after 24 hours, an increase in the sub-G1 population was observed, indicating that PMC79 induced cell death after 48 hours (Figure 2).

[0063] [Figure 2] shows the IC 50Cell cycle analysis using propidium iodide after 24 and 48 h of incubation with PMC79 of 1000 cells / mL. Values ​​represent the mean ± SD of at least three independent experiments. Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparison test. *P ≤ 0.05; ****P ≤ 0.0001 compared to negative control.

[0064] The effect on proliferation was also studied using the carboxyfluorescein succinimidyl ester assay (CFSE). CFSE is a dye that allows cell division to be monitored by flow cytometry; the dye binds covalently to protein amine groups within the cell, resulting in a long-lasting fluorescent adduct. With each cell division, the fluorescence of CFSE-stained cells decreases to half of the initial fluorescence. Thus, if cell proliferation is inhibited, we observe an increase in intracellular fluorescence. SW480 cells were treated with IC 50 and 2 × I.C. 50 The cells were incubated with the PMC79 compound for 48 and 72 hours. The results showed a decrease in proliferation under all conditions, supporting the anti-proliferative effect of PMC79 in CRC cells (Figure 3).

[0065] [Figure 3] shows the IC 50 and 2 × I.C. 50 Quantification of median CFSE fluorescence intensity normalized to T0 after 48 and 72 h of incubation with CFSE-positive cells is shown. Values ​​represent the mean ± SD of at least three independent experiments. Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparison test. Compared with negative control **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

[0066] PMC79 induces apoptosis in colorectal cancer cells The effect of PMC79 compounds on apoptosis was evaluated by flow cytometry using an Annexin V / propidium iodide (AV / PI) assay. CRC cells were treated with IC 50The results of incubation for 48 hours with PMC79 showed that treatment with PMC79 caused a significant increase in the percentage of apoptotic cells compared to the negative control (AV+ / PI-; AV+ / PI+) (74%) (Figure 4). For necrotic cells (AV- / PI+), an increase in this type of cell death (10%) was also observed, although to a lesser extent compared to the percentage of apoptotic cells.

[0067] [Figure 4] shows the apoptotic cell death analysis. A) IC 50 A) Graphical representation of Annexin V fluorescein isothiocyanate (AV-FITC) and propidium iodide (PI) assays in SW480 cells after 48 h of incubation with IgG. Values ​​represent the mean ± SD of at least three independent experiments. Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparison test. **P ≤ 0.01; ****P ≤ 0.0001 compared to negative control. B) Graphical representation of the total percentage of apoptotic (early apoptotic (AV+PI-) and late apoptotic (AV+PI+)) and necrotic cells (AV-PI+). Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparison test. **P ≤ 0.01; ****P ≤ 0.0001 compared to negative control.

[0068] SW480 and IC 50 The level of late apoptosis was also studied using the TUNEL assay after 48 hours of incubation with PMC79. We observed an increase in the number of apoptotic cells (TUNEL positive) in PMC79-treated cells (Figure 5). The presence of apoptotic bodies, a phenotypic change typical of apoptosis, was observed (Velma et al., 2016) (Figure 5a). This phenotypic change is one of the hallmarks of apoptosis, confirming that cell death induced by PMC79 is apoptotic.

[0069] [Figure 5] shows the level of TUNEL-positive cells. SW480 cells were treated with PMC79 IC 50After 48 h of incubation with , the cells were analyzed by TUNEL assay. a) Representative images (×600) of the TUNEL assay are shown. DAPI (40,6-diamidino-2-phenylindole), FITC (fluorescein isothiocyanate) and overlay were obtained by fluorescence microscopy. b) Analysis of the TUNEL assay in SW480 cells. Values ​​represent the mean ± SD of at least three independent experiments. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test. ****P≦0.0001 compared to negative control.

[0070] These results are also consistent with the data obtained with cell cycle analysis, showing an increased percentage of cells in the sub-G1 population after 48 h of incubation with PMC79. Overall, the results showed that cells exposed to PMC79 were primarily undergoing apoptosis.

[0071] PMC79 reduces colorectal cancer cell motility To determine the effect of PMC79 on cell migration, a wound-healing assay was used. SW480 cells were treated with IC 50 and 2 × I.C. 50 The wounds were incubated with 100 μg of PMC79 and the wound size was measured every 4 hours until the completion of the 12-hour incubation. PMC79 dose-dependently reduced the percentage of wound closure, implying a reduction in cell motility (Figure 6). This result indicates that PMC79 reduces the migration of SW480 cells.

[0072] [Figure 6] shows the PMC79 IC 50 and 2 × I.C. 50 Figure 1 shows a study of motility in SW480 cells using a wound-healing assay after 12 h of incubation with IL-1. Values ​​represent the mean ± SD of at least three independent experiments. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test. *P ≤ 0.05; ***P ≤ 0.001 compared to negative control.

[0073] PMC79 preferentially localizes to membrane fractions Intracellular distribution of PMC79 was performed using a commercial kit as described in the experimental section. SW480 cells were treated with IC 50 The cells were incubated at 100°C for 48 hours. Cytoplasmic, membrane, nuclear, and cytoskeletal fractions were extracted, and the results showed that PMC79 was primarily retained in the membrane fraction of colorectal cancer cells (Figure 7).

[0074] [Figure 7] shows the results of ICP-MS. 50 Figure 1 shows the analysis of PMC79 cell distribution after 48 h of incubation with IgG1 and IgG2a. Values ​​represent the mean ± SD of at least three independent experiments.

[0075] PMC79 induces changes in the actin cytoskeleton in colorectal cancer cells To assess the effect of PMC79 compound on the actin cytoskeleton structure of SW480 cells, F-actin organization and morphology were analyzed using phalloidin. 50 The cells were treated with PMC79 at 100 nm for 48 hours and then stained with phalloidin-AlexaFluor® 568.

[0076] PMC79 appeared to affect cell-cell adhesion and the establishment of cell-cell contacts, with changes in cell phenotype, i.e., circularity and cell dispersion ([Figure 8]). In addition, PMC79 appeared to affect cytoskeletal organization, inducing filopodia-like protrusions. A decrease in the number of cells present in the PMC79 condition was also observed compared to the negative control. This may be due to the decreased proliferation and increased apoptosis induced by PMC79.

[0077] [Figure 8] shows an IC 50 Analysis of F-actin staining performed for 48 h using PMC79 with values ​​of 0.01 and 0.01. Representative images (×600) of DAPI (4',6-diamidino-2-phenylindole), phalloidin-AlexaFluor 568 and overlay were obtained with a fluorescent microscope. Results are from at least three independent experiments.

[0078] PMC79 inhibits KRAS, AKT and ERK protein expression in colorectal cancer cells harboring KRAS mutations KRAS downstream signaling pathways (MAPK-ERK and PI3K-AKT) are involved in regulating survival, proliferation and motility of CRC cells. Therefore, it is of utmost importance to understand whether PMC79 compounds can affect KRAS signaling pathway by analyzing the expression of KRAS and downstream molecules AKT and ERK protein involved in PI3K and MAPK pathways, respectively.

[0079] Cisplatin and three other related ruthenium-derived compounds were used as a comparison. Cisplatin is known to be a platinum drug that is widely used in cancer treatment. PMC78, LCR134 and LCR220 are ruthenium-cyclopentadienyl compounds that are distinct from PMC79 and have different functionalization to target cancer cells.

[0080] [ka]

[0081] Prior to protein extraction and Western blot analysis, SW480 cells were incubated with the IC 50 The subjects were exposed to the same concentration for 48 hours.

[0082] The results demonstrated that PMC79 could inhibit the expression of KRAS, pERK, total-ERK, pAKT and total-AKT to a very high extent in SW480 cells carrying KRAS mutation (Figure 9). This was not observed when the cells were exposed to cisplatin or other ruthenium-derived compounds (PMC78, LCR134, LCR220), revealing that PMC79 could inhibit mutant KRAS and block MAPK-ERK and PI3K-AKT, which are the most important signaling pathways in CRC carcinogenesis.

[0083] [Figure 9] shows an IC 50Western blot analysis of KRAS, pAKT, total-AKT, pERK and total-ERK proteins in SW480 cells after 48 h incubation with cisplatin, PMC78, PMC79, LCR134 and LCR220 at 100 μg / ml. GAPDH immunoblot was used as a loading control. Results are from at least three independent experiments.

[0084] We also investigated the effect of PMC79 in RKO cell lines harboring wild-type KRAS, and no effect was observed on the expression of KRAS, pERK, total-ERK, pAKT, or total-AKT proteins. The same was observed for cisplatin and the compounds PMC78, LCR134, and LCR220 (Figure 10).

[0085] [Figure 10] shows an IC 50 Western blot analysis of KRAS, pAKT, total-AKT, pERK and total-ERK proteins in RKO cells after 48 h incubation with cisplatin, PMC78, PMC79, LCR134 and LCR220 at 100 mM concentrations. GAPDH immunoblot was used as a loading control. Results are from at least three independent experiments.

[0086] We also evaluated the effect of PMC79 in a different cell line harboring a KRAS mutation using LS174T cells. The results demonstrated that PMC79 could also inhibit the expression of KRAS, pERK, total-ERK, pAKT and total-AKT in this cell line (Figure 11).

[0087] [Figure 11] shows Western blot analysis of KRAS, pAKT, total-AKT, pERK and total-ERK proteins in LS174T cells after 48 hours of incubation with PMC79. GAPDH immunoblot was used as a loading control. Results are from at least three independent experiments.

[0088] To compare KRAS inhibition by PMC79 with molecules known to specifically target and inhibit KRAS, we used siRNA for KRAS to compare the degree of inhibition induced by both molecules (Alves et al., 2015). The results demonstrated that PMC79 exhibited inhibition of KRAS expression levels similar to KRAS-specific siRNA ([Figure 12]).

[0089] [Figure 12] shows a Western blot analysis of KRAS protein in SW480 cells. SW480 cells were left untransfected (control blank) or transfected with control siRNA or siRNA targeted against KRAS. Six hours after transfection, the cells were treated with IC 50 The cells were incubated with PMC79 for 48 h at 4 °C for 1 h. GAPDH immunoblots were used as loading controls. Results are from at least three independent experiments.

[0090] Overall, these results suggest that PMC79 is a novel marker for the expression of mutant KRAS, particularly KRAS G12V and KRAS G12D It was demonstrated to be a potent inhibitor of mutations.

[0091] PMC79 reduces the levels of all proteins in the KRAS signaling pathway Considering the results obtained for the inhibition of the expression of some proteins involved in the KRAS signaling pathway, IC 50 Proteomic analysis was performed using SW480 extracts treated with PMC79 for 48 h.

[0092] Consistent with previous results, proteomic studies revealed that all proteins involved in the KRAS signaling pathway, i.e., EGFR, KRAS, MEK1, MEK2, ERK1, PIK3, AKT1, AKT2, and MTOR, were less abundant in PMC79 than in control conditions (Table 2). Only two proteins, i.e., MEK1 (**P≦0.01), AKT1 (*P≦0.05), showed a significant decrease.

[0093] Table 2 shows the IC 50 Figure 1 shows proteomic analysis of proteins involved in MAPK and PI3K signaling pathways in SW480 cells after 48 h incubation with PMC79. Values ​​represent the average of at least three independent experiments.

[0094] [Table 2]

[0095] Proteomic studies supported previous results confirming that PMC79 inhibits the expression levels of KRAS downstream signaling proteins.

[0096] Consideration In the present invention, the inventors have discovered that an organometallic ruthenium compound is capable of inhibiting KRAS G12V and KRAS G12D demonstrated promising anti-cancer properties for the treatment of CRC harboring mutations. PMC79 was found to be selective in inhibiting CRC cell survival, exhibiting a selectivity index of 1.1, indicating that the anti-cancer effect on cancer cells is greater than that on normal cells that are not damaged by PMC79 (Ferreira et al., 2017).

[0097] The anticancer efficacy of compounds is primarily based on the ability of the drugs to inhibit proliferation, metastasis and induce apoptotic cell death in cancer cells, which generally correlate with favorable results in most preclinical and clinical studies (Kim, 2005).

[0098] Overall, the results showed that PMC79 inhibits proliferation in KRAS-mutated CRCs by reducing colony-forming ability, inducing G0 / G1 cell cycle arrest, and increasing CFSE fluorescence. Furthermore, PMC79 induces apoptotic cell death, as demonstrated by an increase in Annexin V- and TUNEL-positive cells, along with the presence of apoptotic bodies.

[0099] In addition, PMC79 induces changes in the actin cytoskeleton of CRC cells. The cytoskeleton is known to be responsible for several cellular processes such as cell division, cell motility, and cell death (Fletcher & Mullins, 2010). PMC79 was found to affect the cytoskeletal organization of cells, with cell scattering and obvious filopodia-like protrusions (Bornschl, 2013). These motility-like structures are associated with cell contraction among others. Results of wound healing assays also showed a decrease in motility in cells exposed to PMC79, supporting the role of filopodia-like protrusions in inhibiting cell motility. Inhibition of motility is also important for inhibiting the metastatic potential of CRC cells.

[0100] Here, the inventors have shown that a preferred compound according to the invention (PMC79) exhibits anticancer properties achieved by increasing apoptosis while decreasing proliferation rate and cell migration, three key features of cancer (Hanahan, 2022). These phenotypic properties associated with PMC79 are crucial for its efficacy as a potent therapeutic agent against CRCs with KRAS mutations, reducing the oncogenic potential of these CRCs and thus inhibiting tumor progression.

[0101] KRAS downstream signaling pathways (MAPK-ERK and PI3K-AKT) play important roles in CRC carcinogenesis and are involved in survival and proliferation processes that affect the progression of the disease. The main aim of any treatment is to inactivate the appropriate proteins involved in the signaling pathways, which may simultaneously result in inhibition of cell proliferation, metastasis and induction of apoptosis.

[0102] Nothing was known about the interaction of ruthenium-derived compounds with the KRAS signaling pathway (Mahmud et al., 2021). Here, the inventors studied the expression levels of KRAS and downstream signaling proteins and showed for the first time that the organometallic ruthenium compound according to the invention, namely PMC79, inhibits the expression of KRAS signaling.

[0103] The subcellular distribution of PMC79 showed that the compound preferentially distributed to the membrane fraction, which may explain its KRAS inhibitory effect, as KRAS needs to be attached to the plasma membrane in order to be localized and activated at the membrane level ( Cazzanelli et al., 2018 ; Laszlo et al., 2021 ).

[0104] KRAS activating mutations are the most frequent oncogenic alteration in CRC, yet therapeutic options in these types of cancer are scarce (Laurent-Puig et al., 2009; Pereira et al., 2022). A series of strategies have attempted to indirectly target KRAS, but these efforts have been unsuccessful in clinical trials over the past three decades (Ryan & Corcoran, 2018). Recently, two direct KRAS G12C Inhibitors, namely sotrasib and adagrasib, have been developed that target KRAS G12C It acts by selectively forming a covalent bond with cysteine ​​12 within the switch-II pocket of the protein, thereby locking KRAS in an inactive state; Liu et al., 2021). Preclinical studies have demonstrated that sotrasib and adagrasib inhibit KRAS in NSCLC and PDAC. G12C They showed that they selectively impair the viability of mutant cell lines, reduce proliferation, induce apoptosis, and inhibit p-ERK protein levels (Canon et al., 2019; Hallin et al., 2020). However, neither sotrasib nor adagrasib affected PI3K signaling, which may explain why these KRAS G12C This explains the acquisition of resistance to the inhibitors (Hallin et al., 2020). Furthermore, sotorasib and adagrasib have no effect on cell lines harboring other KRAS mutations in vitro and in vivo (Liu et al., 2021).

[0105] In addition to sotorasib and adagrasib, KRAS inhibitors such as ARS-1620, GDC-6036, D-1553, 1_AM, and ARS-853 G12C Development of other covalent inhibitors of KRAS G12CThese findings may provide new opportunities for selective targeting of various advanced solid tumors harboring KRAS mutations (Liu et al., 2021; Lito et al., 2016; Zeng et al., 2017). Despite the discovery of all these inhibitors, G12C The mutations are not hotspot mutations in CRC and are present in only 3% of CRCs with KRAS mutations, which explains why they are only used in the clinic for the treatment of NSCLC ( Laszlo et al., 2021 ; Liu et al., 2021 ).

[0106] Recently, KRAS G12D Inhibitors have been discovered, but their application has only been studied in vitro and in vivo on PDAC models (Xiaolun Wang et al., 2022; Kemp SB, 2023).

[0107] In the present invention, it was demonstrated that PMC79 inhibits KRAS and both KRAS signaling pathways (MAPK and PI3K) in cells carrying KRAS mutations, but not in cells with wild-type KRAS (Figure 10). This simultaneous inhibition of the MAPK and PI3K pathways is an advantage over known inhibitors and may prevent potential resistance issues in the future.

[0108] In addition, it was demonstrated that PMC79 inhibits KRAS similarly to siRNA designed to specifically target KRAS.

[0109] Inhibition of KRAS using siRNA has been investigated as an alternative approach to target KRAS mutations in CRC, but has been challenged by the difficulty of finding an efficient delivery system that would increase cellular uptake and reduce off-target effects of RNAi ( Jebelli et al., 2021 ).

[0110] Overall, the results compiled here support the finding that KRAS mutations, in particular KRAS G12V and KRAS G12D We support the use of PMC79 as a novel, specific and potent KRAS signaling inhibitor in mutation-harboring CRC.

[0111] As mentioned above, KRAS mutations are particularly common in CRC (40%), but are also frequent in PDAC (80%), NSCLC (35%), and ovarian and endometrial cancers (10%). Given the difficulty in finding treatments that specifically target KRAS, sotrasib and adagrasib have been shown to inhibit KRAS in NSCLC and PDAC. G12C By inhibiting KRAS, PMC79 has provided a new solution for the treatment of cancers with KRAS mutations; Liu et al., 2021). Given that these inhibitors have shown good results in cell lines from various cancer models, our results support the hypothesis that PMC79 may also inhibit KRAS in other types of cancers with KRAS mutations, such as PDAC, NSCLC, and ovarian and endometrial cancers.

[0112] As used in this specification, the expressions "approximately" and "approximately" refer to an interval of values ​​around 10% of a specified figure.

[0113] As used in this specification, the expression "substantially" means that the actual value is within an interval of approximately 10% of the desired value, variable or relevant limit, particularly within approximately 5% of the desired value, variable or relevant limit, or particularly within 1% of the desired value, variable or relevant limit.

[0114] The above subject matter is provided as an illustration of the present invention and therefore cannot be construed as limiting it. The terminology used herein for the purpose of describing the preferred embodiments of the present invention should not be construed as limiting the present invention. As used herein, the singular definite and indefinite articles are intended to be construed as including the plural, unless the context of the present specification clearly indicates otherwise. It will be understood that the expressions "comprise" and "include" as used in this specification specify the presence of features, elements, components, steps, and related operations, but do not exclude the possibility that other features, elements, components, steps, and operations are also contemplated.

[0115] All modifications must be considered within the scope of protection of the present invention, provided that they do not alter the essential characteristics of the following claims.

[0116] References Below is a list of references. (References) TIFF2025508999000007.tif216160TIFF2025508999000008.tif230157TIFF20255089990 00009.tif234157TIFF2025508999000010.tif241158TIFF2025508999000011.tif239158 TIFF2025508999000012.tif235158TIFF2025508999000013.tif235158TIFF20255089990 00014.tif232158TIFF2025508999000015.tif236158TIFF2025508999000016.tif174158

Claims

1. 2. A compound of the general formula (I): for use in the prevention and / or treatment of diseases and / or conditions associated with colorectal cancer, pancreatic ductal adenocarcinoma, non-small cell lung cancer, and ovarian and endometrial cancer in an individual: 【Chemistry 1】 (In the formula, A is CF 3 SO 3 , P.F. 6 , Cl, BPh 4 , NO 3 or BF 4 (selected from the group consisting of wherein said organometallic ruthenium compound is in all possible racemic and stereoisomeric forms.

2. ({2,2'-bipyridine}-4,4'-diyldimethanol-k 2 N,N')(η 5 2. The organometallic ruthenium compound for use according to claim 1, which is -cyclopentadienyl)(triphenylphosphane)ruthenium(II) triflate.

3. 3. The organometallic ruthenium compound for use according to claim 1 or 2, wherein the individual is a mammal, in particular a human.

4. 4. The organometallic ruthenium compound for use according to any one of claims 1 to 3, in a composition comprising a pharma- ceutically acceptable excipient.

5. 5. The organometallic ruthenium compound for use according to claim 4, wherein the composition is administered by oral, rectal or parenteral injection route, preferably by parenteral route.

6. 6. The organometallic ruthenium compound for use according to claim 5, wherein said parenteral administration is selected from the group consisting of intravenous administration, intramuscular administration, intradermal administration, or subcutaneous administration.

7. 7. The organometallic ruthenium compound for use according to any one of claims 4 to 6, wherein the composition is in a form selected from the group consisting of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, sprays, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, or sterile packaged powders.

8. 8. An organometallic ruthenium compound for use according to any one of claims 1 to 7 by inhibiting KRAS and the downstream signalling pathways MAPK and PI3K.

9. 9. The organometallic ruthenium compound for use according to claim 8, wherein said KRAS and downstream signalling pathways MAPK and PI3K result in inhibition of cell proliferation and induction of apoptosis.

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