Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/28—Compounds containing heavy metals
  • A61K31/295—Iron group metal compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
  • A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
  • A61K8/4926—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having six membered rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/06—Preparations for care of the skin for countering cellulitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/58—Metal complex; Coordination compounds

Definitions

• the present invention relates generally to the non-therapeutic use and the therapeutic use of compounds having a demethylating effect on DNA.
• the invention also relates to the therapeutic use of these iron-based organometallic complexes as a medicament, for example for the treatment of proliferative pathologies, in particular of cancers.
• Cancers are the second leading cause of death after cardiovascular disease in industrialized countries. Aside from direct DNA mutations of essential genes, the expansion of a cancer cell is frequently associated with epigenetic changes, that is, not directly encoded by the DNA sequence.
• epigenetics indeed includes a whole set of modifications responsible for the regulation of gene expression which can be transmitted during cell division and which do not involve any change in the DNA sequence (Flintoft, 2017). This term thus groups together various processes, such as DNA methylation, post-translational modifications of histones and expression of non-coding RNA.
• the regulation of gene expression is a phenomenon that influences most aspects of cell biology (embryogenesis, development, cell differentiation, as well as cell death) in multicellular organisms.
• Deregulation of epigenetic changes, both in their maintenance and in their coordination, is frequently observed for various pathologies, such as inflammation, obesity, neurodegenerative and cardiovascular diseases, as well as cancers. Consequently, the search for molecules capable of regulating these processes is of major interest.
• DNA methylation is an epigenetic process. It is the result of a methylation of cytosines to 5-methylcytosines in the CG dimers of DNA, in particular in the CpG islets. About 80% of these regions are methylated in the human genome.
• the DNA methyltransferase (DNMTs) enzymes catalyze the addition of a methyl group originating from the donor Sadenosylmethionine on the carbon 5 of a cytosine.
• DNMT1 which is an enzyme involved in the maintenance of epigenetic information (Leonhardt et al., 1992; Liu et al., 1998), takes as its model the half-methylated DNA strand during the replication while the enzymes DNMT3a and 3b can act on an unmethylated strand and therefore modify the epigenetic information (Okano et al., 1999).
• DNMT2 does not show DNA methyltransferase activity. DNA methylation leads to repression of gene expression (Bird, 2002).
• DNA demethylating agents also known by the English term “DNA hypomethylating agent”
• DNA hypomethylating agent is based on strategies leading to indirect demethylation of DNA, in particular via inhibition enzymes responsible for the methylation thereof, namely the DNMTs described above.
• tumor suppressor genes such as, for example, p16
• p16 certain tumor suppressor genes
• Inhibitors blocking DNA methyltransferase activity cause the hypomethylation of these tumor suppressor genes and thus have anticancer properties (Santini et al., 2001).
• the different demethylating agents of DNA can be classified according to their mode of action into five different categories: 1) non-methylatable nucleoside analogs (decitabine, azacytidine), 2) inhibitors of catalytic activity (procainamide), 3) inhibitors of S-adenosylmethionine hydrolase (caldribine), 4) inhibitors of expression of DNMTs (MG98) and 5) inhibitors of the interaction of DNMTSs with their partners (UP peptide ).
• Non-methylatable nucleoside analogs such as 5-azacytidine, Decitabine (DAC or 5-aza-2'-deoxycytidine), Zebularine (dZTP), 5-fluoro-2'- deoxycytidine (FdCyd), SGI-1 10 (modified decitabine) and CP-4200 (a derivative of 5-azacytidine) allow the capture of DNMTs on DNA having incorporated these analogs thus leading to their degradation.
• these non-methylatable nucleoside analogs such as 5-azacytidine, or 5-aza-2’-deoxycytidine, in addition to having significant side effects, generally show high toxicity and low stability.
• zebularin exhibits better stability (Zhou et al., 2002; Cheng et al., 2003) and lower toxicity (Cheng et al., 2004).
• the inhibitors of the enzymatic activity of DNMTs are synthetic compounds which have an affinity for regions of DNA rich in CpG thus blocking the activity of DNMT1. These molecules have several advantages over nucleotide analogs because their use is not limited to highly proliferative cells since they directly inhibit DNMTs without requiring their incorporation into DNA.
• DNMT synthesis such as antisense oligonucleotides and microRNAs (MG98) (Amato, 2007; Winquist et al., 2006) are based on the fact that hypermethylation is very often associated with an increase in l expression of DNMTs. Despite a strong enthusiasm for this strategy, at present, phase I or II clinical trials have revealed a lack of efficacy of these oligonucleotides.
• the inhibitors of the interaction of DNMTs with their partners relate to peptide molecules which are capable of disturbing the association of DNMTs with some of their protein partners necessary for their functioning.
• this approach therefore makes it possible to avoid (compared to the other modes of action mentioned above) the induction of a global hypomethylation which can be at the origin significant side effects.
• specific inhibition of the DNMT3A / ISGF3y complex has been shown to restore sensitivity to temozolomide and thus reduce tumor growth of glioblastomas (Cheray et al., 2013, 2014; Hervouet et al., 2010).
• complex 1 has the lowest DNA binding affinity among the three complexes tested, has no cleavage activity. DNA under UV-A or under green light.
• Document FR 1 463 870 describes the use of organometallic complexes for the dyeing of keratin materials.
• An object of the present invention is thus to propose new compounds which avoid, all or in part, the abovementioned drawbacks.
• the Applicant has thus focused on the development of new demethylating compounds capable of interacting with DNA.
• the Applicant has demonstrated that the compounds according to the invention had an antiproliferative action, in particular on cancer cells.
• the compounds according to the invention are non-cytotoxic and thus exhibit a cytostatic effect.
• the subject of the present invention is the non-therapeutic use, as DNA demethylation agent, of at least one organometallic compound comprising an iron atom linked to two terpyridine groups of general formula ( I) following: in which :
• Y are each independently of the others a CH group or a nitrogen atom (the two Y groups can be identical or different);
• Z denotes an oxygen atom, a sulfur atom or an NH group
• the dotted line represents a C-C bond at the point of attachment of the aromatic cycle of formula (II) to (VI) to the terpyridine groups of the organometallic compound of formula (I);
• X is a counterion
• the present invention also relates to an organometallic compound comprising an iron atom linked to two terpyridine groups, having a general formula (I) defined above for its use as a medicament.
• the present invention could be a pharmaceutical composition which could comprise, in a pharmaceutically acceptable carrier, at least one organometallic compound as defined above.
• a pharmaceutically acceptable carrier for the rest of the description, unless otherwise specified, the indication of a range of values “from X to Y” or between “X to Y”, in the present invention, is understood to mean as including the X and Y values.
• the present invention relates to the non-therapeutic use, as DNA demethylating agent, of at least one organometallic compound comprising an iron atom linked to two terpyridine groups of formula general (I) below:
• Z denotes an oxygen atom, a sulfur atom or an NH group
• the dotted line represents a C-C bond at the point of attachment of the aromatic cycle of formula (II) to (VI) to the terpyridine groups of the organometallic compound of formula (I);
• X is a counterion
• the terpyridine groups are ligands which are particularly suitable for the iron atom which make it possible to form a relatively stable organometallic compound and which would therefore be resistant even in a biological environment.
• the particular configuration of the terpyridine ligands gives remarkable rigidity to the organometallic compound according to the invention.
• the compounds of the invention may be in the form of salts, solvates and / or pharmaceutically acceptable prodrugs.
• the pro-drugs are variants of the compounds of the invention which can be transformed in vivo into compounds of general formula (I) according to the invention.
• the term “pharmaceutically acceptable salts or solvates” refers to salts or solvates which retain the desired biological activity as described above of the organometallic compounds of the invention and which expose to very little, even no unwanted toxicological effects.
• alkyl designates a linear or branched, saturated or unsaturated hydrocarbon radical, advantageously having from 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyle, neopentyle, n-hexyle, etc. Groups having from 1 to 6 atoms and in particular from 1 to 4 carbon atoms are preferred.
• R 1 to R 6 may correspond to a hydrogen atom.
• organometallic compound of formula (I) can correspond to the following formula (IX):
• R 1 , R 3 , R 4 , 6 6 may be identical and correspond to a hydrogen atom
• organometallic compound of formula (I) can correspond to the following formula (X):
• R 1 , R 3 , R 4 , R 6 are identical and correspond to a hydrogen atom
• organometallic compound of formula (I) can correspond to the following formula (XI):
• the two terpyridine groups of this compound AIM3 thus each carry a pyridine nucleus, the nitrogen atom making it possible to induce a nucleophilic effect.
• the counterion X is chosen from: Cl, Br, I, the hexafluorophosphate of formula PF 6 , preferably the counterion is PF 6 .
• the organometallic compound (s) are capable of causing direct demethylation of DNA.
• organometallic compounds of formula (I) according to the invention can in particular be used in order to form biotechnology tools, for example as sequencing reagents where demethylation of DNA is required.
• the compound or compounds of the invention can be used as a biotechnology tool, such as for example as a reagent for carrying out demethylation experiments in vitro on cells, generally tumor cells, originating from a sample taken from an animal. These experiments would make it possible, for example, to confirm the presence or not of DNA methylation phenomenon on certain genes, to block the cell proliferation of these cells and therefore to sensitize them to chemotherapy agents.
• "By animal” according to the invention means mammals, birds, fish, insects, etc., as well as humans.
• organometallic compound (s) of formula (I) according to the invention and in particular of the compounds AIM1 to AIM3 has previously been described in publications, such as the publication of EC Constable, AMW Cargill Thompson, Dalton Trans, 1992, 2947 -2950 for the compounds AIM1 and AIM3 and the publication by H. Krass, EA Plummer, JM Haider, PR Barker, NW Alcock, Z. Pikramenou, MJ, Hannon, DG Kurth, Angew. Chem. Int. Ed. 40 (2001) 3862-3865 for the compound AIM2.
• the terpyridine ligands required for the synthesis of the organometallic compounds according to the invention were prepared using the Krohnke reaction between 2-acetylpyridine and an appropriate aryl aldehyde compound in the presence of ammonia.
• the homoleptic complexes AIM1, AIM2 and AIM3 are obtained with a respective yield of 90, 69 and 83% by reacting the appropriate terpyridine ligand (2 equivalents) with FeCh in acetonitrile (scheme 1).
• the organometallic compound (s) of formula (I) according to the invention having a demethylating action of DNA can also represent an active substance for therapeutic purposes, as will be described below.
• the present invention thus relates to an organometallic compound comprising an iron atom linked to two terpyridine groups, having a general formula (I) as defined above for its use as a medicament.
• organometallic compound (s) of formula (I) for non-therapeutic use are included here for therapeutic use (namely for their use as a medicament).
• the organometallic compound (s) according to the invention can be used to treat diseases linked to cellular hyperproliferation, in particular cancers.
• the organometallic compound (s) according to the invention can be used to treat glioblastomas, promyelocytic leukemias, cancers of the prostate, ovaries, lungs, breasts, digestive tracts, in particular the liver, pancreas , head and neck, colon, non-Hodgkin's lymphoma or melanoma, etc.
• the compounds of the invention exhibit an antiproliferative effect with respect to tumor and healthy cells. These compounds cause an accumulation of cells in the G2 / M or G0 / G1 phase. Cell death can be observed but only after long treatments of minimum 96 hours. Thus, these compounds can be used to reduce tumor proliferation.
• organometallic compound (s) can be used to treat tumors resistant to other anticancer agents.
• organometallic compound (s) according to the invention could be used to treat other diseases linked to deregulation of epigenetic modifications, such as to treat inflammation, obesity, neurodegenerative diseases or cardiovascular diseases.
• the present invention may be intended to form a pharmaceutical composition
• a pharmaceutical composition comprising, in a pharmaceutically acceptable carrier, at least one organometallic compound of formula (I) as defined above.
• organometallic compound (s) of formula (I) for non-therapeutic use or for use as a medicament are included here for therapeutic use as a medicament.
• the term “pharmaceutically acceptable carrier” designates substances such as excipients, vehicles, adjuvants, buffers which are conventionally used, in combination with one or more active principles (here, the organometallic compound or compounds according to the invention), for the preparation of a medicament.
• active principles here, the organometallic compound or compounds according to the invention.
• the choice of such supports essentially depends on the route of administration envisaged.
• the pharmaceutically acceptable carrier is an excipient, a vehicle and / or an adjuvant chosen from the group consisting of pharmaceutically acceptable excipients, vehicles and / or adjuvants.
• a person skilled in the art may in particular advantageously refer to the fourth edition 2002 of the European Pharmacopoeia, or even to the edition USP 25-NF 20 of the American Pharmacopoeia (U. S. Pharmacopeia).
• the pharmaceutical composition can comprise one or more agents or vehicles chosen from dispersants, solubilizers, stabilizers, preservatives, etc.
• the pharmaceutical composition can be a human or veterinary pharmaceutical composition.
• the pharmaceutical composition may be in a liquid form, or, preferably, in a solid form.
• a solid pharmaceutical composition is preferred, for example in the form of tablets, capsules or capsules.
• a pharmaceutical composition is preferred in the form of an aqueous suspension.
• a pharmaceutical composition can thus be presented in a form for oral administration, by inhalation, parenteral or injection such as for example by intravenous, intramuscular, subcutaneous, trans-dermal, intra-arterial, etc.
• the oral forms are particularly preferred.
• the intravenous, intramuscular, subcutaneous, and inhalation routes are preferred.
• the compositions are generally in the form of liquid suspensions, which can be injected by means of syringes or infusions, for example.
• the compounds are generally dissolved in an acceptable support which comprises saline, physiological, isotonic, buffered solutions, etc., compatible with pharmaceutical use and known to those skilled in the art.
• Agents or vehicles which can be used in liquid and / or injectable formulations are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc.
• injection rate and / or the dose injected can be adapted by a person skilled in the art depending on the patient, the pathology concerned, the mode of administration, etc.
• the organometallic compound (s) according to the invention are administered in doses which can vary between 0.1 pg and 100 mg / kg of body weight, more generally between 0.01 and 10 mg / kg, typically between 0.1 and 10 mg / kg.
• doses which can vary between 0.1 pg and 100 mg / kg of body weight, more generally between 0.01 and 10 mg / kg, typically between 0.1 and 10 mg / kg.
• repeated injections can be made.
• prolonged and / or delayed release systems may be advantageous.
• solid pharmaceutical forms as pharmaceutical support can comprise, as vehicles, adjuvants or excipients, at least one diluting agent, a flavoring, a solubilizing agent, a lubricating agent. , a suspending agent, a disintegrating agent and an encapsulating agent, the identity and the function of these different classics being fully documented in the European Pharmacopoeia or in the Pharmacopoeia of the United States of America (USP).
• USP European Pharmacopoeia or in the Pharmacopoeia of the United States of America
• compositions in liquid form can also comprise water, optionally in admixture with propylene glycol or polyethylene glycol, and optionally also coloring agents, flavorings, stabilizers and thickening agents.
• the method comprises a step during which said patient is administered a therapeutically effective amount of a purified dry extract of the or organometallic compounds according to the invention, or alternatively of the pharmaceutical composition described above.
• treatment designates preventive, curative, palliative treatment, as well as the management of patients (reduction of suffering, improvement of lifespan, slowing down the progression of the disease , reduction of tumor growth, etc.).
• the treatment can also be carried out in combination with other chemical or physical agents or treatments (chemotherapy, radiotherapy, gene therapy, etc.) ⁇
• the treatments and drugs of the invention are particularly intended for humans.
• organometallic compound (s) according to the invention and / or the pharmaceutical composition may, for example, be used in the treatment of cancers in combination with an anticancer treatment using radiation, such as radiotherapy.
• organometallic compound (s) according to the invention and / or the pharmaceutical composition can for example be used in the treatment of cancers in combination with at least one other anticancer chemical agent, conditioned and administered in a combined, separate or sequential manner.
• the other anticancer chemical agent can for example be cisplatin, carboplatin, taxoter, taxol, tomoxifene, doxorubicin or 5-fluorouracil and is advantageously taxol, 5-fluorouracil or else tomoxifene or doxorubicin .
• the present invention thus makes it possible to inhibit in vivo, in vitro or ex vivo the proliferation of tumor cells, comprising bringing said tumor cells into contact with the pharmaceutical composition.
• the tumor cells can in particular be those of the pathologies specified above.
• FIG. 2 shows the evaluation of the demethylating power of the compounds AIM2 and AIM3 according to the invention in an in vitro system, (a) ELISA tests were carried out in order to measure the level of methylated cytosines present on a fragment of DNA after action of TET2 demethylase or AIM2 and AIM3 molecules; (b) the demethylating activity of AIM2 (2mM) and of the his-tagged-TET2 protein (10pg) was measured by an assay of the incorporation of radiolabelled methyl groups at the level of a biotynilized double stranded DNA previously methylated by the enzyme MSsI;
• FIG. 3 represents the evaluation of the overall demethylating power of the molecules AIM2 and AIM3 in U251 cells: an ELISA test was carried out in order to measure the level of methylated cytosines present in the DNA of untreated U251 cells versus treated for 1 or 4h with 2mM of 5aza-2-deoxycytidine, AIM2 or AIM3;
• FIG. 5 shows the evaluation of the overall demethylating power of the molecules AIM2 and AIM3 according to the invention on MDA-MB231 cells: a quantification by densitometry made it possible to obtain a quantitative value of the intensity of the bands present in the framed part of the gel (the calculation of the percentage of demethylation was calculated using the formula indicated);
• FIG. 6 shows the influence of the AIM2 molecule according to the invention on the proliferation of MCF-10A breast epithelial cells and MDA-MB231 and Hs578T mammary tumor cells: the cells were seeded, after 24 hours of culture the cells were treated for 48 hours with varying concentrations of the AIIM2 molecule; the determination of the number of adherent cells was carried out by staining with violet crystal and quantified by a measurement of the absorbance at 595 nm; the results correspond to the average of 4 independent experiments, the standard errors to the average (SEM) are indicated and the significance of the data was calculated by comparing the treated vs untreated cells (Student test);
• FIG. 7 shows the influence of the AIM3 molecule according to the invention on the proliferation of breast epithelial cells MCF-10A and mammary tumors
• MDA-MB231 and Hs578T the cells were seeded, after 24 h of culture the cells were treated for 48 h with variable concentrations of the molecule AIIM3; the determination of the number of adherent cells was carried out by staining with violet crystal and quantified by a measurement of the absorbance at 595 nm; the results correspond to the average of 4 independent experiments, the standard errors to the average (SEM) are indicated - the significance of the data was calculated by comparing the treated vs untreated cells (Student test);
• FIG. 8 is an analysis of the cell cycle of MCF-10A cells treated or not with 0.5 mM AIM2 or AIM3; the cells were seeded and treated with 0.5 mM AIM2 or AIM3 for 0, 16, 24, 48 and 72 h; after detachment the cells are fixed and permeabilized in a 70% cold ethanol solution; labeling with propidium iodide is then carried out in order to label the DNA and the fluorescence is quantified by flow cytometry;
• FIG. 9 shows an analysis of the number of cells treated or not with 2mM AIM2 or AIM3.
• the cells were seeded and treated with 2mM AIM2 or AIM3 for 0, 24, 48, 72, 96, 120, 144, 168 and 192h. After detachment, the cells are counted using a Malassez type counting cell.
• the medium of the NT or AIM2 or AIM3 cells is renewed every 72 hours.
• AIM2 (72) + NT or AIM3 (72) + NT the culture medium contains the indicated molecule only for 72 hours and after that it is replaced by a medium without molecules in order to test the reversibility of the effect;
• MDA-MB 231 treated with different concentrations of the AIM2 compound according to the invention and / or tamoxifen;
• FIG. 12 shows a set of graphs showing the sensitivity of different breast tumor lines to AIM2, AIM3 compounds, conventional chemotherapy agents and their combinations;
• FIG. 17 shows the inhibition of the proliferation of T47D R + doxo cells by the different molecules AIM1, AIM2, AIM3 and Ru after 48 and 72 hours of treatment;
• FIG. 18 shows the inhibition of the proliferation of glioblastoma cells U251 and U87 by the different molecules AIM1, AIM2, AIM3 and Ru after 72 hours of treatment;
• FIG. 19 shows the inhibition of the proliferation of T47D cells by the different molecules AIM1, AIM2, AIM3 and Ru after 48 hours of treatment;
• daughter solutions can precipitate at 4 ° C. It is thus necessary to incubate them at 37 ° C for 10 min before their use.
• the Applicant has carried out a molecular modeling of the demethylating capacity of the AIM3 compound according to the invention.
• the energy profile linked to the demethylation reaction was carried out using quantum chemistry calculations.
• a model system comprising a methylated cytosine and AIM3 was used.
• the geometries of the reagents and products have been optimized using the density functional theory "DFT" using wB97XD as the exchange-correlation functional and LANL2DZ as the orbital base.
• the transition state that is to say the maximum energy conformation
• the energy profile of the reaction in space connecting the reactants and the products via the transition state was also estimated by analyzing the energy variation according to a reaction coordinate, i.e. the distance between methyl and the peripheral nitrogen of AIM3.
• the effects of the solvent, water were taken into account using a model of the polarizable continuum "PCM”.
• All molecular modeling calculations were carried out using Gaussian 09 software version D01 and using standard molecular modeling techniques, in particular detailed in numerous works such as “Chris J. Cramer Essential of Computational Chemistry Wiley eds”.
• those skilled in the art can refer to the Gaussian 09 manual for a detailed illustration of the geometry optimization techniques that have been used.
• the cell-free experiments consist in incubating methylated DNA (Qiagen, France) at 37 ° C. for 1 h with either 2 mM of molecules AIM2 and AIM3, or with the recombinant TET2 protein which is a protein known for its demethylating role and serving as positive control or either with water (Ctrl).
• methylated DNA 500ng, ref # N4007 NEB, genomic DNA of HeLa cell hypermethylated in vitro by the enzyme M.Sssi
• methylated DNA 500ng, ref # N4007 NEB, genomic DNA of HeLa cell hypermethylated in vitro by the enzyme M.Sssi
• the DNA is then used to carry out a 5mC-ELISA test (Zymo Research) i.e. making it possible to measure the level of 5-methylcytosines (see below).
• the cells of U251 gliomas or T98G glioblastomas were seeded at 2.10 6 respectively. 24 h after seeding, the cells were treated for 1 or 4 h with the molecules AIM2 or AIM3 at a rate of 0, 2, 5, 10 mM. After the treatment, the culture medium is replaced by a medium without molecules and the cells are kept in culture for 48 hours. The cells are then mechanically detached with a scraper in order to carry out a total DNA extraction via the use of the QiaCube automaton (Qiagen) and the QIAamp DNA Mini QIAcube Kit (Qiagen), without modification with the instructions provided by Qiagen .
• QiaCube automaton Qiagen
• Qiagen QIAamp DNA Mini QIAcube Kit
• the DNA (100 ng) collected as indicated above is first denatured at 98 ° C for 5 min (use of the MyCycler BioRad thermocycler), before being deposited in the wells of the plate: 96 wells provided for this purpose (Kit 5mC-ELISA, Zymo) for 1 h of incubation at 37 ° C. The supernatant is then removed and the wells are washed 3 times with 200 ⁇ L of washing buffer (supplied by the kit). Then, 200 ⁇ L of washing buffer are incubated in each well for 30 min at 37 ° C.
• the revelation / quantification step of 5- methylcytosines is carried out with the incubation (1 h, 37 ° C) of the anti-5mC antibody provided by the kit (ref / A3001-30), diluted to 1/2000. After 3 washes (200 ml of washing buffer provided by the kit), the primary antibody is revealed following the incubation (1 h, 37 ° C) of the coupled secondary antibody HRP (D5325-3-30). Finally, after 3 washes (200 ml of washing buffer supplied by the kit), the ELISA is revealed by adding 100 ml of "HRP developer" (supplied by the kit). The ELISA plate is read at 405-450 nm (Victor plate reader, Perkin Elmer). In parallel, a standard range is carried out with methylated DNA whose percentage of 5 mC is known (range: 0, 5, 10, 25, 50, 75, 100%).
• TET2 (10pg) (positive control) was measured by an assay for the incorporation of methyl groups radiolabelled in a double-stranded DNA biotynilized and previously methylated by the enzyme M.SSsl.
• a 96-well plate is pretreated beforehand with 10 mg / ml of BSA (bovine serum albumin) for 30 min.
• BSA bovine serum albumin
• the magnetic beads (10 mg / ml, Dynal) are washed three times with the TENT2M buffer (20 mM Tris, pH 8.0, 2 mM EDTA, 0.01% Triton X-100, 2 M NaCl). The concentration of these beads is adjusted to 2.5 mg / ml with the TENT2M buffer and 1/10 of the volume with the non-radioactive form of AdoMet (100 mg / ml; Sigma).
• the methylation reaction (40 mI) contains 200 U of the enzyme M.Sssi (NEB # M0226) 125 nM DNA oligonucleotides, 200ng DNA, and 900 nM tritium- labeled AdoMet (Amersham Bioscience, 1 mCi / ml) in the reaction medium (50 mM Tris, pH 8.0, 5 mM EDTA, 10% glycerol, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride).
• the reactions are stopped by adding an equivalent volume of magnetic beads in suspension and gentle shaking for 15 min at room temperature.
• the magnetic beads are separated using a magnet and washed successively with 300, 200, 150, and 100 mI TENT1 M (10 mM Tris, pH 8.0, 1 mM EDTA, 0.005% Triton X-100, 1 M NaCI ).
• the magnetic beads are resuspended in 25 mI of TENT1 M in the presence of “putative demethylating agents” or with 100 ⁇ g of nuclear extract isolated using the kit, nuclear extract kit (Active Motif, # 40410).
• the magnetic beads are separated using a magnet and washed successively with 300, 200, 150, and 100 mI of TENT1 M (10 mM Tris, pH 8.0, 1 mM EDTA, 0.005% Triton X- 100, 1 M NaCl). Then the level of incorporated tritium is measured in a scintillation counter.
• the compound AIM2 exhibits a demethylating action of DNA.
• the molecules AIM2 and AIM3 exhibit demethylating power after 1 h and 4 h of incubation clearly greater than that of 5aza-2-deoxycytidine which is the known and commonly used demethylating agent, but which requires a incorporation into DNA.
• T98G glioblastoma cell line
• T98G cells The experiment carried out on T98G cells consisted in treating the latter for 1 h with different concentrations of the compound AIM3 according to the invention, namely 0; 2; 5 and 10 mM.
• the cells of the breast cancer line MDA-MB 231 are cultured in RPMI 1640 medium (Gibco) containing 2 mM L-Glutamine (Sigma), 0.1 mg / ml of gentamicin (Sigma) and 10% of fetal calf serum ( Sigma) decomplemented 30 minutes at 56 ° C. They are incubated at 37 ° C and 5% CO2.
• the powder is taken up at 100 mM in DMSO (Sigma). This stock solution is aliquoted (2 mL) and stored at -80 ° C.
• the powder (717.07 g / mol) is taken up at 1 mM in PBS (Fisher) containing
• the powder (719.06 g / mol) is taken up at 5 mM in PBS containing 20% ethanol. This stock solution is stored at 4 ° C.
• the cell layer is washed with 2mL of PBS, then trypsinized with 0.5mL of EDX 1X trypsin (Sigma) for 5 minutes at 37 ° C. The action of trypsin is then inhibited by the addition of 4 mL of culture medium.
• the cell suspension is finally centrifuged at 200 G for 5 minutes and the pellet stored at -20 ° C.
• the intensity of each strip is measured using the Quantity One software.
• the data presented in FIG. 5 confirm that, like 5-azacytidine, the treatment of MDA-MB231 cells with the molecules AIM2 and AIM3 makes it possible to reduce the overall percentage of methylation of the genome.
• EXAMPLE 8 Influence of the compounds AIM2 and AIM3 according to the invention on the proliferation of breast epithelial cells MCF-10A and mammary tumor MDA-MB231 and Hs578T (Fig. 6 and Fig. 71
• the cells are seeded according to the densities indicated above (Table 4). After 24 hours of culture, the culture medium is replaced by medium containing or not containing the molecules AIM2 or AIM3 at concentrations of 0.5; 1; 1.5; 2 and 2.5 mM in four copies for 48 hours. After these 48 hours of treatment, the culture medium is removed and the cells are washed with a 1 ⁇ PBS solution. They are then fixed in 100 ⁇ l of culture medium containing 4% of paraformaldehyde and incubated for 20 min at room temperature. The cells are then rinsed with a 1X PBS solution.
• the next step is to dissolve the purple crystal in order to obtain a colored solution which can be dosed.
• 100 ⁇ l of a 10% acetic acid solution is added, then the plates are incubated with shaking until the total solubilization of the Violet Crystal.
• An absorbance is then read at 595 nm using a plate reader (VICTOR).
• the concentration of the cells is presented in the table below.
• the results obtained indicate that treatment with 0.5 mM AIM2 and AIM3 induces a blockage of the cell cycle in G1 phase.
• the absence of cells in the subG1 phase indicates that these molecules do not exhibit cellular toxicity under these treatment conditions.
• the effect of these molecules on cell proliferation was also evaluated as a function of the treatment time and no longer of the dose of molecule used.
• the 2mM dose which is the one capable of causing DNA demethylation, was chosen.
• the cells are seeded in 60mm boxes at a rate of 4 ml / box on the 1st day at the concentration indicated in the table below.
• the organometallic complexes AIM2 and AIM3 at 2 mM or the solvent (0.04% ethanol) for the untreated cells are added to the culture medium.
• the treated and untreated cells are detached by the action of trypsin.
• the cells After inactivation of trypsin with culture medium containing 10% fetal calf serum, the cells are washed in PBS and counted after staining with Trypan Blue so as to count only the living cells (a fraction of 10 ml of the suspension is mixed with 10 ml of 0.4% Trypan blue (Thermo Scientific) The cells are counted using a Malassez counting cell (Marienfeld-Superior). at). Prior to treatment, the Al Ms molecules are preheated in a heating block (Thermo Scientific) at 37 ° C. with stirring for 10 minutes in order to guarantee total solubilization.
• a heating block Thermo Scientific
• the media are renewed every 72 hours with or without Al Ms.
• the molecules AIM2 and AIM3 have a cytostatic action in the different cell lines used (MCF-10A, MDA-MB231 and Hs578).
• a treatment with a dose of 2 mM inhibits the proliferation of healthy mammary epithelial cells (MCF10A), but also of breast cancer cells (MDA-MB231 and HS578T) (cf. curves square points compared to the untreated controls round).
• MDF10A healthy mammary epithelial cells
• MDA-MB231 and HS578T breast cancer cells
• a 72h treatment was also carried out with 2mM of molecule, followed by a change of medium without adding any molecule.
• MCF10A healthy mammary epithelial cells
• EXAMPLE 1 1 Sensitizing effect of compounds according to the invention vis-à-vis chemotherapy in mammary tumor cells (Fiq.10 and Fig.1 1)
• Breast cancer actually groups different subcategories based on the expression of 3 molecular markers: the estrogen receptor (ERa), the progesterone receptor (PR) and the transmembrane receptor belonging to the family of receptors for EGF (HER2). Some tumors are thus called triple negative, because they do not express any of the 3 molecular markers. This type of tumor poses many problems in their treatment because:
• One of the major challenges in the treatment of breast cancer consists in trying to re-sensitize triple negative breast tumors to chemotherapy agents.
• One of the aims is thus to restore the sensitivity of cells triple negative breast cancer, MDA-MB231, to a chemotherapy agent, tamoxifen.
• tamoxifen is a selective estrogen receptor (ERa) modulator, that is to say that it binds specifically to estrogen receptors, but without activating a signaling pathway.
• ERa selective estrogen receptor
• the resistance of MDA-MB231 cells to tamoxifen has been shown to be due to a lack of expression of ERa.
• Yoshida and his collaborators showed that the hypermethylation of 2 promoter regions (promoter A and B) upstream of the gene coding ERa is inversely correlated to the expression of ERa in MDA-MB231 cells.
• the Applicant has studied whether the compounds according to the invention make it possible to demethylate the ERa promoter in MDA-MB231 cells, in order to re-induce its expression, and to resensitize cells to tamoxifen.
• Chou-Talalay tests were carried out by determining the number of cells attached after treatments using the AIM2 compound and / or using tamoxifen.
• MDA-MB231 cells were seeded in a 96-well plate at a cell density of 8,000 cells / well in a volume of 100 ⁇ L / well.
• the experimental plate design is shown in Figure 10.
• Treatment with AIM2 is done for 24 hours, followed by 48 hours of treatment with tamoxifen.
• a marking with violet crystal is carried out in order to count the living cells.
• the protocol is as follows:
• the plate triplicates are averaged, and the results are expressed as a function of the untreated control which is set to 1 for each condition.
• MDA-MB231 cells which are ER negative, are insensitive to tamoxifen.
• a treatment with AIM2 induces a reduction in cell viability of the order of 50% from the lowest dose (2000 nM).
• Pretreatment with AIM2 followed by treatment with tamoxifen also induces a decrease in cell viability, which seems greater than in the presence of the molecule AIM2 only.
• the Applicant used the CompuSync software which is based on the Chou-Talalay method via the principle of effect median of the law of mass action.
• fa and fu correspond respectively to the fractions of cells affected and not affected by the treatments
• D and Dm correspond respectively to the dose used and the median dose
• m corresponds to the shape of the dose-response curve.
• the software then generates combination indices (Cl): - if the Cl at a given dose combination is less than 1, then there is a synergy of action between molecule A and molecule B
• the breast cancer cells, MCF-7, MDA-MB231 and Hs578T, as well as a line of normal breast cells, MCF10A were seeded at a density of 1 ⁇ 10 4 cells / well in a 96-well plate. well. After 24 hours of inoculation, the cells are brought into contact with 2mM of AIM2 / 3 for 1 hour followed by 50mM of 5-fluorouracil or 50ng / ml of doxorubicin for 24 or 48 hours. Following these treatments, the media are removed and 0.5 mg / mL of MTT is added to each well.
• the different cell lines are cultivated in an incubator at 37 ° C, 5% CC> 2 and 95% humidity according to the following nutrient media:
• the different culture media are alternately supplemented with 0.1 mg / mL of Gentamicin (Sigma G1272) and 1% of Penicillin-Steptomycin (Sigma P4333).
• the fetal calf serum (SVF) and the horse serum are decomplemented beforehand for 30 minutes at 56 ° C.
• the doxorubicin-resistant line T47D is cultured in the presence of 12.5 ng doxorubicin / ml in order to maintain the resistance. However, doxorubicin is not added to the culture medium during the pre-treatment phases and the proliferation test.
• the cells are seeded in 100 mm boxes (10 ml medium / box) for the Untreated conditions and in 150 mm boxes (20 ml medium / box) for the conditions treated according to the following quantities:
• the cells are then treated 24 hours after seeding with the molecules
• AIM2 or AIM3 at a concentration of 2 mM for 72 hours.
• the cells are detached with 1 mL (10 mm dishes) or 2 ml (150 mm dishes) of trypsin - EDTA 1X for 5 minutes at 37 ° C (15 minutes of incubation are necessary to detach the MCF10A cells).
• trypsin is then inhibited by 10 ml of culture medium and then the cell suspension is centrifuged at 200 G for 5 minutes.
• Each pellet is taken up in 10 ml of medium and then the cells are counted using a Mallasez cell.
• the cells are then seeded in 60 mm dishes (4 ml medium / dish) as follows and according to the experimental plan of Fig. 13:
• the cells After washing with 2 ml of PBS, the cells are detached with 1 ml of 1X trysin-EDTA at 37 ° C for 10 minutes (20 minutes for the MCF10A line).
• the tryspine is then inhibited by the addition of 4 ml of medium. Then the cells are pelletized at 200G for 5 minutes. The pellets are taken up in a precise volume of PBS depending on the size of the pellet.
• the cells are correctly separated by several round trips to the P200 and counted using a Mallasez cell.
• the different cell lines are cultivated in an incubator at 37 ° C, 5% CC> 2 and 95% humidity according to the following nutrient media:
• the different culture media are alternately supplemented with 0.1 mg / mL of Gentamicin (Sigma G1272) and 1% of Penicillin-Steptomycin (Sigma P4333).
• Fetal calf serum (SVF) and horse serum are decomplemented beforehand for 30 minutes at 56 ° C.
• the doxorubicin resistant line T47D is cultured in the presence of 12.5 ng doxorubicin / ml in order to maintain the resistance, however doxorubicin is not added to the culture medium during the proliferation test.
• Each cell line is seeded in 96-well plates (100 ml_ medium / well) according to the following conditions:
• Each treatment condition is performed in a tripicat as shown in Figure 15.
• the cells are treated with the molecules represented in FIG. 16 (AIM1, AIM2, AIM3 according to the invention and Ru as a comparative example) at 2 mM 24 h after seeding.
• the medium from each well is removed and then washing is carried out with 100 ⁇ l of PBS.
• the cells are then fixed with 100 ⁇ L of 4% paraformaldehyde for 20 minutes, followed by two washes with 500 ⁇ L of PBS.
• the percentage of adhered cells is demonstrated by staining with violet crystal.
• the wells are rinsed 3 times with distilled water and then properly dried by tapping on a sheet of absorbent paper.
• the plate is placed under stirring until the total solubilization of the violet crystal crystals (approximately 10 minutes).
• the absorbance is read using a plate reader at 595 nm (see Fig. 17 to 20).

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