EP3880165A1

EP3880165A1 - Use of an organometallic compound as a dna-demethylating agent

Info

Publication number: EP3880165A1
Application number: EP19835442.5A
Authority: EP
Inventors: Stéphanie GRANDEMANGE; Antonio MONARI; Philippe Gros; Pierre François CARTRON
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Lorraine
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Lorraine
Priority date: 2018-11-12
Filing date: 2019-11-12
Publication date: 2021-09-22
Also published as: FR3088200B1; US20220031652A1; CA3118628A1; WO2020099777A1; FR3088200A1

Abstract

The invention relates to the non-therapeutic use, as a DNA-demethylating agent, of at least one organometallic compound comprising an iron atom bound to two terpyridine groups. The invention also relates to an organometallic compound comprising an iron atom bound to two terpyridine groups, to be used as a drug.

Description

USE OF AN ORGANOMETALLIC COMPOUND AS A DEMETHYLATION AGENT

DNA

TECHNICAL FIELD TO WHICH THE INVENTION RELATES

The present invention relates generally to the non-therapeutic use and the therapeutic use of compounds having a demethylating effect on DNA.

It therefore relates, first of all, to the non-therapeutic use as an agent for demethylation of DNA of organometallic iron-based complexes to form, for example, biotechnology tools, such as sequencing reagents.

Then, 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.

TECHNOLOGICAL BACKGROUND

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.

The term 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.

In particular, 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. Among DNA methyltransferases, 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). On the other hand, DNMT2 does not show DNA methyltransferase activity. DNA methylation leads to repression of gene expression (Bird, 2002). There is also an enzyme (peptidyl arginine deiminase 4) which, by decreasing the arginine residues of histone H3, can interfere with methylation (Cuthbert et al., 2004).

To date, the development of DNA demethylating agents (also known by the English term “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.

For example, for many cancers, certain tumor suppressor genes, such as, for example, p16, have been shown to be hypermethylated (Herman et al., 1995). The consequence of this hypermethylation is the inactivation of this gene regulating the cell cycle. Inhibitors blocking DNA methyltransferase activity cause the hypomethylation of these tumor suppressor genes and thus have anticancer properties (Santini et al., 2001).

At present, 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 ).

1) 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. However, 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. However, zebularin exhibits better stability (Zhou et al., 2002; Cheng et al., 2003) and lower toxicity (Cheng et al., 2004).

2) The inhibitors of the enzymatic activity of DNMTs (such as RG108, procaine, nanaomycin and procainamide) 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.

3) Inhibitors of S-adenosylhomocysteine hydrolase lead to a drop in S-adenosylmethionine which is the methyl donor allowing methyltransferases to catalyze the methylation of DNA cytosines (Cladribine, Fludarabine and Clofarabine) (Bonate et al ., 2006; Jeha et al., 2004).

4) The inhibitors of 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.

5) 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. As the protein complexes formed with DNMTs can vary depending on the target genes, 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. For example, 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).

To date, the search for molecules capable of modulating the level of DNA methylation has therefore focused solely on inhibitors of the enzymes responsible for this process, namely DNMTs.

Also known from the state of the art, the publication by Zohreh Naseri et al. 201 1, Pergamon Press, which discloses organometallic complexes comprising the ligand 4 '- (2-thienyl) -2,2', 6 ', 2 ”-terpyridine. A solvate of a corresponding iron complex (Fe (thioterpy) ₂ (NO ₃ ) ₂ .MeOH) is described as compound 3 in the publication. However, according to the results presented in this publication, this iron 3 complex has negligible activity towards the microorganisms tested, does not demonstrate a cytotoxic effect on the leukemia and fibrosarcoma cell lines tested (Table 7), and its activity of trapping superoxide ions is not remarkable.

The publication by Uttara et al. 2012, Journal of Inorganic Biochemistry, describes photocytotoxic terpyridine iron complexes (complexes 1, 1 a, 2, 2a, 3 and 3a). According to the biological results provided, 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.

In this context, there is a need in the prior art to develop new DNA demethylating agents for therapeutic and non-therapeutic purposes.

There is in particular a need in the prior art to develop new DNA demethylating agents which are effective, which preferably have reduced or lesser adverse effects compared to the demethylating agents of the prior art (preferably , low cell cytotoxicity), while being easy to implement and advantageously having a moderate cost price.

An object of the present invention is thus to propose new compounds which avoid, all or in part, the abovementioned drawbacks. OBJECT OF THE INVENTION

The Applicant has thus focused on the development of new demethylating compounds capable of interacting with DNA.

In particular, it has developed a new class of demethylating agents capable of causing direct demethylation of DNA by simple chemical reactions in cell-free and cellular systems.

The Applicant has indeed demonstrated that certain organometallic compounds comprising an iron atom linked to two terpyridine groups allow direct demethylation of DNA.

These compounds could in particular be used either for non-therapeutic applications, for example, as reaction reagents when DNA demethylation is required, or for therapeutic applications.

In this case, the Applicant has demonstrated that the compounds according to the invention had an antiproliferative action, in particular on cancer cells. In addition, as shown by the experimental tests below (in particular examples 9 and 10), the compounds according to the invention are non-cytotoxic and thus exhibit a cytostatic effect.

To this end, 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 :

R ¹ to R ⁶ , identical or different, are chosen from: a hydrogen atom; a linear or branched alkyl chain of formula C _n Hh _{n + i} with n = 1 to 12; a COOH group; an SO3H group; a PO3H group; an aromatic cycle of formula (II) to (VI) below:

(II) (III) (IV) (V) (VI) where

Y are each independently of the others a CH group or a nitrogen atom (the two Y groups can be identical or different);

R ⁷ is chosen from a group OC _Y H2 _{Y + I} with y = 1 to 10; SC _W H2 _{W + I with} w = 1 to 10;

N (C _m H2m + i) 2 with m = 1 to 6, COOH; SO3H or PO2H3;

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.

Finally, the present invention could be a pharmaceutical composition which could comprise, in a pharmaceutically acceptable carrier, at least one organometallic compound as defined above. 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.

DETAILED DESCRIPTION OF AN EXAMPLE OF IMPLEMENTATION

The description which follows, given by way of nonlimiting examples, will make it clear what the invention consists of and how it can be carried out.

I. Non-therapeutic use of the compound or compounds of general formula (I)

As mentioned above, 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:

in which :

R ¹ to R ⁶ , identical or different, are independently chosen from: a hydrogen atom; a linear or branched alkyl chain of formula C _n Hh _{n + i} with n = 1 to 12; a COOH group; an SO ₃ H group; a PO ₃ H group; an aromatic cycle of formula (II) to (VI) below:

(II) (III) (IV) (V) (VI) where Y are each independently of the others a CH group or a nitrogen atom, that is to say the two groups Y can be identical or different;

R ⁷ is chosen from a group OC _Y H2 _{Y + I} with y = 1 to 10; SC _W H2 _{W + I with} w = 1 to 10; N (C _m H2m + i) 2 with m = 1 to 6, COOH; SO3H; PO2H3;

Z denotes an oxygen atom, a sulfur atom or an NH group;

X is a counterion.

According to the invention, 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. In addition, 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.

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.

According to the invention, the term “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.

According to a first preferred embodiment, R ¹ to R ⁶ may correspond to a hydrogen atom.

According to this mode, the organometallic compound of formula (I) can correspond to the following formula (IX):

This compound of formula (IX) will be named hereinafter AIM1.

According to a second preferred embodiment, R ¹ , R ³ , R ⁴ , 6 ⁶ may be identical and correspond to a hydrogen atom, and R ² and R ⁵ may be identical and denote an aromatic ring of formula (II) as described above with Y = CH and thus correspond to a benzene of the following formula (Vil):

(VII)

According to this mode, the organometallic compound of formula (I) can correspond to the following formula (X):

This compound of formula (IX) will be named hereinafter AIM2.

According to a third preferred embodiment, R ¹ , R ³ , R ⁴ , R ⁶ are identical and correspond to a hydrogen atom, and R ² and R ⁵ are identical and denote an aromatic ring of formula (II) where a Y = CH and the other Y = N and correspond to a pyridine radical of formula (VIII) below: where the dotted line represents a CC bond between the carbon carried by the pyridine radical and the carbon carried by the terpyridine group of the organometallic compound of formula (I).

According to this mode, the organometallic compound of formula (I) can correspond to the following formula (XI):

This compound of formula (IX) will be named hereinafter AIM3.

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.

According to one embodiment of the invention, the organometallic compound of formula (I) excludes the compound or compounds where R ¹ = R ³ = R ⁴ = R ⁶ = H and where R ² = R ⁵ is chosen from an aromatic ring of formula (VI) in which Z is a sulfur atom or an aromatic ring of formula (II) or (III) with Y = CH.

Preferably, the counterion X is chosen from: Cl, Br, I, the hexafluorophosphate of formula PF ₆ , preferably the counterion is PF ₆ .

Advantageously, the organometallic compound (s) are capable of causing direct demethylation of DNA.

Without being bound by any theory, it would seem that the demethylation of DNA is due either to a nucleophilic attack of 5-methylcytosines (5mC) thus causing their deamination, and / or to an oxidation mediated by iron which would allow a conversion of 5mC into 5-hydroxymethylcytosine (5hmC).

Thus, the 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.

By way of example, 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.

The synthesis of the 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.

In particular, the synthesis of the compound (s) according to the invention can be carried out according to the method described below.

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. For example, 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).

Diagram 1

In particular, a solution comprising FeCh (106 mg, 0.84 mmol) and methanol (20 ml) is added to a solution comprising the appropriate terpyridine ligand (1.68 mmol) and methanol (20 ml). The mixture is then stirred for 10 min. Ethyl ether (100 mL) is then added leading to a purple solid collected by filtration, washed with ether and dried under vacuum.

AIM1. Yield: 90%. ¹ H NMR (200 MHz, CD ₃ OD): d = 7.85 (d, J = 8.1 Hz, 4H), 7.49 (t, J = 8.2 Hz, 2H), 7.41 (d, J = 8.2 Hz, 4H), 6.68 (m, 4H), 5.90 (d, J = 3.8 Hz,

8H) ppm. HRMS (ESI) calcd for C ₃ oH ₂ 2FeN ₆ Cl2 m / z = 261 .0627 [M - 2 Cl] ²⁺ . Found: 261 .0683.

AIM2. Yield: 69%. ¹ H NMR (200 MHz, DMSO-d6) 9.70 (s, 4H), 9.09 (d, J = 8.5 Hz, 4H), 8.57 (d, J = 7.4 Hz, 2H), 8.05 (t, J = 7.4 Hz , 4H), 7.85 (t, J = 7.2 Hz, 4H), 7.78 (t, J = 7.0 Hz, 2H,), 7.3 (d, J = 5.1 Hz, 4H), 7.2 (t, J = 6.3 Hz, 4H). HRMS (ESI) calcd for

C42H ₃ oFeN ₆ Cl2 m / z = 337.0936 [M - 2 Cl] ²⁺ . Found: 337.0902.

AIM3. Yield: 83%. ¹ H NMR (200 MHz, CD ₃ CN): d = 9.27 (s, 2H), 9.08 (d, J = 5.8 Hz, 2H), 8.65 (d, J = 8.1 Hz, 2H), 8.38 (d, J = 5.8 Hz, 2H), 7.97 (dt, J = 6.7 Hz, 1 .2 Hz, 2H), 7.22-7.10 (m, 4H) ppm. HRMS (ESI) calcd for C ^ HzsFeNsCh m / z = 338.0888 [M - 2 Cl] ²⁺ . Found: 338.0906.

I I. Therapeutic use of the compound (s) of general formula (I)

As already mentioned previously, 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.

Of course, the characteristics described above for the organometallic compound (s) of formula (I) for non-therapeutic use are included here for therapeutic use (namely for their use as a medicament).

In particular, according to one embodiment of the invention, the organometallic compound of formula (I) excludes the compound or compounds where R ¹ = R ³ = R ⁴ = R ⁶ = H and where R ² = R ⁵ is chosen from an aromatic ring of formula (V) or (VI) in which Z is a sulfur atom or an aromatic ring of formula (II) or (III) with Y = CH.

Preferably, the organometallic compound (s) according to the invention can be used to treat diseases linked to cellular hyperproliferation, in particular cancers.

In particular, 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.

In addition, the organometallic compound (s) can be used to treat tumors resistant to other anticancer agents.

Due to the demethylating capacity of these compounds, the Applicant has also discovered that the 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.

Also, the present invention may be intended to form a pharmaceutical composition comprising, in a pharmaceutically acceptable carrier, at least one organometallic compound of formula (I) as defined above.

Of course, the characteristics described above for the 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.

In the context of the invention, 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. The choice of such supports essentially depends on the route of administration envisaged.

In general, 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.

Techniques for preparing pharmaceutical compositions can be easily found by those skilled in the art, for example in the work Remmington's Pharmaceutical Sciences, Mid. Publish. Co, Easton, PA, USA.

Physiologically acceptable adjuvants, vehicles and excipients are also described in the book "Handbook of Pharmaceutical Excipients, Second Edition, American Pharmaceutical Association, 1994".

To formulate a pharmaceutical composition, a person skilled in the art can advantageously refer to the latest edition of the European Pharmacopoeia or the Pharmacopoeia of the United States of America (USP).

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).

Thus, 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.

For oral administration, a solid pharmaceutical composition is preferred, for example in the form of tablets, capsules or capsules.

In the liquid form, 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.

According to a first aspect, the oral forms are particularly preferred. In a second aspect, the intravenous, intramuscular, subcutaneous, and inhalation routes are preferred.

For example, for injections, the compositions are generally in the form of liquid suspensions, which can be injected by means of syringes or infusions, for example. In this regard, 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.

It is understood that the 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.

Typically, 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. In addition, repeated injections can be made. On the other hand, for chronic treatments, prolonged and / or delayed release systems may be advantageous.

By way of example, for oral administration, 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).

Such compounds are, for example, magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, gelatin, cellulose materials, etc. The 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.

Generally, in order to treat a patient with a pathology linked to a deregulation of epigenetic modifications (such as cancer), 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.

In the context of the invention, the term “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.

Thus, the 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.

Also, the 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. For example, it would be possible to provide a pretreatment with a compound according to the invention, followed by treatment with an anticancer agent.

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 Applicant has in fact surprisingly demonstrated that the compounds according to the invention made it possible to sensitize cancer cells to anticancer agents, or even to act in synergy with them (examples 1 1 and 12).

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.

The invention is further illustrated, without being limited, by the appended figures and the following examples where:

- Figure 1: shows the result of an molecular modeling experiment of the demethylating capacity of the compound AIM3 according to the invention;

- Figure 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;

- Figure 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;

- Figure 4: shows the evaluation of the overall demethylating power of the AIM3 molecule on T98G cells and shows the absence of a dose effect of the demethylating power of the AIM3 molecule following its incubation for 1 h (p = 0.2578);

- Figure 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);

- Figure 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);

- Figure 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);

- Figure 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;

- Figure 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. For cells 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;

- Figure 10: corresponds to the experimental plate design of Example 11; - Figure 11: is a graphic representation of the cell viability of

MDA-MB 231 treated with different concentrations of the AIM2 compound according to the invention and / or tamoxifen;

- Figure 12: shows a set of graphs showing the sensitivity of different breast tumor lines to AIM2, AIM3 compounds, conventional chemotherapy agents and their combinations;

- Figure 13: shows the experiment plan of Example 13;

- Figure 14: shows the inhibition of proliferation by the molecules AIM2 and AIM3 which is reversible in certain cells; from left to right are presented the results for MDA-MB-231 (triple negative breast cancer cells), MCF10A (healthy breast epithelial cells) and T47R + Doxo (doxorubicin resistant breast cancer cells) treated with AIM2 (panel of top) or AIM3 (bottom panel) for 72 hours of pre-treatment then reseeded for an additional 72 hours; the different conditions correspond to: T = without prior treatment or during these 72 hours; AIM2 / 3 + NT cells pretreated with AIM2 or AIM3 and then without treatment; AIM2 / 3 + AIM2 / 3 cells pretreated with AIM2 or AIM3 and then returned in the presence of AIM2 or AIM3 for 72 hours;

- Figure 15: represents the experience conditions of Example 14;

- Figure 16: represents the tested molecules of Example 14;

- Figure 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;

- Figure 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;

- Figure 19: shows the inhibition of the proliferation of T47D cells by the different molecules AIM1, AIM2, AIM3 and Ru after 48 hours of treatment; and

- Figure 20: shows the inhibition of proliferation of MDA-MB-231 cells by the different molecules AIM1, AIM2, AIM3 and Ru after 48 hours of treatment. III. EXAMPLES

In order to biologically confirm these data, a study of the influence of the molecules AIM3 and AIM2 on DNA methylation was carried out in acellular and cellular systems.

EXAMPLE 1 Solubilization of the AIM2 and AIM3 compounds according to the invention for their use on cells

For the various tests described below, the compounds AIM2 and AIM3 according to the invention were previously dissolved in the following manner (Table 1):

Table 1

For the AIM2 compound, daughter solutions can precipitate at 4 ° C. It is thus necessary to incubate them at 37 ° C for 10 min before their use.

EXAMPLE 2 Molecular modeling of the demethylating capacity of the compound AIM3 according to the invention

In order to demonstrate the demethylating action of the organometallic compounds according to the invention, the Applicant has carried out a molecular modeling of the demethylating capacity of the AIM3 compound according to the invention.

For this purpose, the energy profile linked to the demethylation reaction was carried out using quantum chemistry calculations. In particular, 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.

Subsequently, the transition state, that is to say the maximum energy conformation, was also optimized, which made it possible to estimate the energy barrier which must be overcome in order to be able to complete the reaction in this case of demethylation. 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”. In addition, 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.

As shown in FIG.1, the results obtained revealed that the compound AIM3 and the other organometallic compounds of formula (I) could cause direct demethylation of DNA via favorable thermodynamic reactions.

EXAMPLE 3 Measurement of the level of 5-methylacytosine (5 mC) by ELISA (Fiq. 2 (a))

Acellular test:

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).

In particular, methylated DNA (500ng, ref # N4007 NEB, genomic DNA of HeLa cell hypermethylated in vitro by the enzyme M.Sssi) is incubated for 1 h at 37 ° C either in the presence of water (Ctrl), either in the presence of AIM2 (2 mM) or AIM3 (2 mM), or in the presence of 10 μg of his-tagged-TET2 protein.

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).

Cell test:

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 .

5mC-ELISA (Zymo Research)

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 μL of washing buffer (supplied by the kit). Then, 200 μL 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%).

As shown in Fig. 2 (a), the measurement of the methylcytosine level (test

ELISA) present at the DNA probe revealed a significant drop in methylcytosines after treatment with the molecules AIM3 and AIM2.

EXAMPLE 4 Determination of the radiolabelled methyl groups (Fig. 2 (b))

In addition, the demethylating activity of AIM2 (2mM) and the his-tagged protein-

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.

Protocol

This DMB experiment is carried out as described by Yokochi and Robertson

(2004).

A 96-well plate is pretreated beforehand with 10 mg / ml of BSA (bovine serum albumin) for 30 min.

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).

After an incubation of 30 min at 37 ° C, 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 μg of nuclear extract isolated using the kit, nuclear extract kit (Active Motif, # 40410). After the incubations, 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.

Results (Fig. 2 (b))

As shown in Fig. 2 (b), the compound AIM2 exhibits a demethylating action of DNA.

EXAMPLE 5 Experiment on U251 Glioblastoma Cells (Fiq.3)

In parallel, in order to verify the demethylating power of these organometallic molecules according to the invention, experiments on U251 glioblastoma cells were carried out.

Protocol

The experiment carried out on U251 cells consisted in treating the latter for 1 or 4 hours with 2mM of AIM2, AIM3 or 5aza-2-deoxycytidine which is a known and commonly used demethylating agent.

Following the treatments, the DNA was extracted and subjected to an ELISA test in order to determine the level of methylated cytosines (protocol, see EXAMPLE 3).

Results:

As indicated in FIG. 3, 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.

EXAMPLE 6 Experiment on another cell line of glioblastoma T98G (Fiq.4)

Furthermore, the demethylating action of AIM3 has also been observed on another glioblastoma cell line, called T98G (Figure 4).

Protocol

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.

Results:

As indicated in FIG. 4, in these cells the demethylating power of the AIM3 molecule is visible after 1 h of treatment at a concentration of 2 mM. However, an increase in the concentration of this molecule (of the order of 5 to 10 mM) does not increase its demethylating power. Indeed, a 2mM dose of AIM3 has a demethylating effect which is similar to that of a 10 mM dose of AIM3.

EXAMPLE 7 Experiment on MDA-MB231 cells (Fiq.5)

An enzymatic digestion experiment was also carried out in order to determine the percentage of overall demethylation induced by the molecules AIM2 and AIM3 according to the invention.

Protocol

1. Cell culture

1.1. Culture conditions and treatments

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 seeding is carried out in 6 cm Petri dishes (4 ml medium / dish) according to the following conditions (Table 2):

Table 2

1.2. Conditions for taking up molecules

5aza2deoxycytidine (Sigma A3656)

The powder is taken up at 100 mM in DMSO (Sigma). This stock solution is aliquoted (2 mL) and stored at -80 ° C.

Intermediate dilutions are made in the culture medium.

AIM 2

The powder (717.07 g / mol) is taken up at 1 mM in PBS (Fisher) containing

20% ethanol. This stock solution is stored at 4 ° C.

Intermediate dilutions are made in PBS. AIM 3

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.

Intermediate dilutions are made in PBS

NB: Stock solutions of AIM 2 and AIM3 precipitate at 4 ° C. It is enough to incubate them for 5 minutes at 37 ° C. in order to re-solubilize them.

1.3. Cell harvesting procedure

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.

1.4. Planning

Table 3

2. Lysis and extraction of DNA

2.1. Cell lysis

Each pellet is taken up in 100 μl of PBS, to which are added in order:

100 pL water

- 150 pL SDS 0.1%

50 pL Proteinase K 10mg / mL (Sigma)

Lysis takes place at 37 ° C overnight. 2.2. DNA extraction

Add 2 volumes of phenol / chloroform 5: 1 to the lysate

Vortex 30 seconds

Centrifuge at 16,000 G for 5 minutes at room temperature

Recover the aqueous phase (supernatant)

Add 1 volume of 23: 1 chloroform / isoamylalcohol

Centrifuge at 16,000 G for 5 minutes at room temperature

Recover the aqueous phase (supernatant)

Add 1/10 of the volume of 3M sodium acetate

Then 1 volume of cold absolute ethanol

Vortex 10 seconds

Leave to precipitate overnight at -20 ° C

Centrifuge at 12,000 G for 15 minutes at 4 ° C

Wash the pellet with 500 μL of 70% ethanol

Centrifuge at 12,000 G for 5 minutes at 4 ° C

Let the base dry (open tube cover) until it becomes translucent

Dissolve the pellet in sterile water (approximately 100 pL, volume to be adjusted according to the size of the pellet obtained)

Dosing the DNA at 260 nm

Store DNA at -20 ° C

3. Enzymatic digestion with MSPI and Hpall (Fisher, ER0541 and ER051 1)

1 pg of DNA from each condition is digested with MSPI and Hpall according to the following protocol:

2 pL Buffer TANGO 10X

1 pg DNA

Water qs 16 pL

2 pL Hpall or Mspl (= 20 U)

Incubate at 37 ° C overnight, then 20 minutes at 80 ° C (enzyme inactivation)

4. Migration of digested DNA on agarose gel

Add 4pL of 6X deposition blue to each digestion volume (20pL)

(6X blue deposit: 10mM TrisHCI pH 7.6, 60mM EDTA, 60% glycerol, 0.03% blue bromophenol, 0.03% xylene cyanol FF)

Migrate all of each digestion on a 1% agarose gel + 2pL BET 10mg / mL at 90V for 40 minutes.

The intensity of each strip is measured using the Quantity One software.

Calculation% of DNA demethylation: (Hpall - Mspl) Treated x 100 / (Hpall - Mspl) N _OP Treated Results:

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

Crystal violet protocol

Table 4

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 μl 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. Then they are colored for 30 min using 100 μl of a 0.1% crystal violet solution (dilution 1/5 in H2O of the stock solution: 0.5% crystal violet 2% ethanol). The cells are then washed 3 times with 100 μL of FhO.

The next step is to dissolve the purple crystal in order to obtain a colored solution which can be dosed. Thus, 100 μl 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).

Results:

At the biological level, it was determined that the molecules AIM2 and AIM3 had an antiproliferative action on normal mammary epithelial cells (MCF-10A) and on mammary tumor cells (MDA-MB231 and Hs578T) (FIGS. 6 and 7). These results make it possible to define the median inhibitory concentration (IC50) of the molecules AIM2 (Figure 6) and AIM3 (Figure 7). The IC50 of the AIM2 molecule in MCF-10A cells is between 0.5 and 1 mM and in MDA-MB231 cells between 1 and 1.5 mM; the IC50 of the AIM3 molecule in MCF-10A cells is between 1 and 1.5 mM and in MDA-MB231 cells between 1.5 and 2 mM. EXAMPLE 9 Influence of the compounds AIM2 and AIM3 according to the invention on the proliferation of breast epithelial cells MCF-10A by flow cytometry (Fiq.8): absence of cellular toxicity

In order to define this antiproliferative action, a study of the cell cycle of MCF-10A mammary epithelial cells by flow cytometry was also carried out (Figure 8).

Protocol: cell cycle

1. Inoculate the cells in 60mm boxes.

The concentration of the cells is presented in the table below.

Table 5

2. Treat cells according to treatment time.

3. Collect the medium from each box in a 15ml tube.

4. Wash cells with 2mL PBS.

5. Collect the PBS in the tube.

6. Add 1 mL of 1 ^c trypsin to each dish and incubate them in the incubator for

5 minutes for MDA-MB231 and Hs578T, and 15 minutes for MCF-10A.

7. Inhibit trypsin with the medium and recover the cells in the tube.

8. Resuspend the cells well and count using a TC10 ™ Automated Cell Counter (Bio-Rad).

9. Take 1x10 ⁶ cells in a new 15ml tube, centrifuge at 500 G for 5 minutes.

10. Aspirate the supernatant and wash the cells with 2ml of PBS.

1 1. Centrifuge at 500 G for 5 minutes.

12. Aspirate the supernatant, take up the pellet in 300 ml of PBS and separate the cells well with a 200 ml pipette.

13. Add 700mI of cold absolute ethanol dropwise while vortexing in the tube.

14. Put the cells in the fridge at -20 ° C for at least 30 minutes to ensure permeabilization and correct fixation.

15. Take 500 µL of cells (5x10 ⁵ cells) from each suspension and centrifuge at

800G for 10 minutes.

16. Gently aspirate the supernatant. 17. Resume each pellet in 500 mI of DNA stainning solution containing 10mI of RNase A and 2mI of propidium iodide (PI, 50 pg / ml). The unmarked pellets are taken up in 500 μl of PBS.

18. 10 minute incubation in the dark.

19. Reading at FACS.

Results:

As shown in Fig. 8, 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.

EXAMPLE 10 Influence on Cell Proliferation (PARP Test Cleaved by Western The absence of cell death has been confirmed by experiments in which the absence of the PARP protein cleaved by western blot has been verified.

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.

Protocol:

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.

Table 6

6 hours after their seeding, 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. At the end of the chosen treatment time, 24, 48, 72, 96, 120, 144 hours, the treated and untreated cells are detached by the action of trypsin. 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.

b). For treatments longer than 72 hours, the media are renewed every 72 hours with or without Al Ms.

vs). In the proliferation reversibility test (red curves in the figure), the treatment is stopped after 72 hours and the media are renewed without adding AIMs molecules.

Results:

Thus, the molecules AIM2 and AIM3 have a cytostatic action in the different cell lines used (MCF-10A, MDA-MB231 and Hs578).

As indicated in FIG. 9, 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). On the other hand, in order to determine the reversibility of these treatments, a 72h treatment was also carried out with 2mM of molecule, followed by a change of medium without adding any molecule. In this experiment, it appears that the change in environment allows the cells to proliferate again and this in a much more marked manner concerning healthy mammary epithelial cells (MCF10A). This result confirms the previous observations demonstrating the non-cytotoxicity of these molecules and therefore their cytostatic effect.

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:

- their insensitivity to chemotherapy agents commonly used in the treatment of breast cancer

- the lack of targeted treatment for these tumors.

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.

From a mechanistic point of view, 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. The resistance of MDA-MB231 cells to tamoxifen has been shown to be due to a lack of expression of ERa. In 2000, 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.

In addition, treatment with a demethylating agent (5-azadesoxycytidine) made it possible to re-induce the expression of ERa in MDA-MB231 cells (link: the synergie inhibitory effect of 5-aza- 2-deoxycytidine and tamoxifen on estrogen receptor alpha negative breast cancer eye (in vitro).

As the results of the use on 5aza cells are very promising, clinical trials have been carried out for the treatment of certain leukemias associated with hypermethylation of specific genes. Although the overall survival of the patients is markedly improved, the fact remains that the use of 5-azadesoxycytidine is associated with serious side effects (neutropenia, thrombocytopenia, anemia, pyrexia), due to the absence specificity of the action of this molecule. Part of the work carried out in oncology is thus to generate and study new demethylating agents with improved specificity of action.

It is in this context that the compounds according to the invention have been developed, including the compound AIM2 which, as shown in the tests above, has a demethylating action, both in vitro and in cellulo.

Faced with this mode of action, 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.

First, the synergy of action between our AIM2 molecule and tamoxifen was studied.

Chou-Talalay tests were carried out by determining the number of cells attached after treatments using the AIM2 compound and / or using tamoxifen.

Protocol: AIM2 / Tamoxifen Chou Talalav synergism experiment

For this, MDA-MB231 cells were seeded in a 96-well plate at a cell density of 8,000 cells / well in a volume of 100 μL / 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. At the end of the treatments, a marking with violet crystal is carried out in order to count the living cells.

The protocol is as follows:

- vacuum wells

- wash the wells with 100pL of PBS

- add 50 μL of 0.1% violet crystal and incubate for 30 minutes at room temperature

- rinse 3 times with distilled water

- add 100 μL of 10% acetic acid per well and incubate with shaking

10 minutes

- measure the absorbance at 595 nm in a VICTOR Multilabel Plate Reader (PerkinElmer).

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.

Results:

The results show that 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. In order to know whether this decrease in cell viability under the AIM2 + Tam condition is explained by a synergy between the 2 molecules, 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.

The equation on which the software is based is as follows: fa / fu = l (D / Dm) J ^m where:

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

(IC50)

m corresponds to the shape of the dose-response curve.

You just need to enter the 3 datasets software:

- the values corresponding to the effect of molecule A at the given dose - the values corresponding to the effect of molecule B at the given dose

- the values corresponding to the effect of molecule A and of molecule B at the combination of given doses.

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

- if the Cl at a given dose combination is greater than 1, then there is an antagonism between molecule A and molecule B.

The results (expressed in the form of an index or Cl combination) are presented in Table 7 below:

Table 7

As the results show, all the combinations of doses (except the combination of 10,000 nM of AIM2 and 750 nM of TAM) make it possible to observe a synergy (Cl <1) between tamoxifen and AIM2.

These results therefore make it possible to conclude that a pre-treatment using AIM2 makes it possible to sensitize the MDA-MB231 cells to tamoxifen, which are basic insensitive. EXAMPLE 12: MTT test (viability) to demonstrate the effect of the compounds according to the invention on the sensitivity to anticancer agents (Fiq.12)

Subsequently, on the basis of the resensitization of MDA-MB231 cells to tamoxifen in the presence of AIM2, the Applicant studied whether a treatment with the compounds AIM2 and AIM3 made it possible to increase the sensitivity of different lines of mammary tumors in the face of different chemotherapy agents conventionally used: 5-fluorouracil and doxorubicin.

Protocol:

For this, 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. After 3 hours of incubation at 37 ° C, 100 µL of 25% SDS is added to each well and the plates are incubated at 37 ° C overnight. The absorbance is measured at 570nm. The results presented correspond to the mean ± the error standard to average from three independent experiments. The differences between the cells treated at different times are significant for a p <0.05.

Results:

The results show that a 24h treatment with AIM2 / 3 has no significant effect on viability, regardless of the breast cancer line taken into account. However, when treatment with AIM2 / 3 is followed by treatment with 5Fu or doxorubicin, cell viability decreases significantly, in all lines. This effect appears after 24 hours of treatment, but is accentuated even more during 48-hour treatments.

These results therefore suggest that the compounds according to the invention sensitize breast tumor cells to chemotherapy agents, indicating a possible therapeutic use of these molecules.

EXAMPLE 13 Proliferation test following a pre-treatment with AIM2 or AIM3 2 uM 72h (Fig. 13 and Fig. 141

Culture condition:

The different cell lines are cultivated in an incubator at 37 ° C, 5% CC> 2 and 95% humidity according to the following nutrient media:

Table 8

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.

Pre-treatment of cell lines

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:

Table 9

The cells are then treated 24 hours after seeding with the molecules

AIM2 or AIM3 at a concentration of 2 mM for 72 hours.

Cell seeding following pre-treatment with AIM

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).

The action of 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:

Table 10

Cell count

Each day for 72 hours, 3 boxes of a cell type and a given treatment are counted.

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.

As shown in Fig. 14, the results obtained indicate that the proliferation inhibition observed following treatment with AIM2 and AIM3 is reversible in MCF10A cells and not in MDA-MB-231 cells. Reversibility is also observed in T47D R + doxo cells treated with AIM2 but not with AIM3. EXAMPLE 14 Study of the effect of the processing of the molecules AIM1, AIM2, AIM3 and Ru (Fiq.16) at 2 μM on different cell lines for 48 h or 72 h

Culture condition

Table 11

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. Experiments plan

Each cell line is seeded in 96-well plates (100 ml_ medium / well) according to the following conditions:

Table 12

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.

After 48h or 72h of treatment, the medium from each well is removed and then washing is carried out with 100 μl of PBS.

The cells are then fixed with 100 μL of 4% paraformaldehyde for 20 minutes, followed by two washes with 500 μL of PBS.

Purple Crystal Essay

The percentage of adhered cells is demonstrated by staining with violet crystal.

100 μL of 0.1% violet crystal (dilution to 1/5 in H2O of the stock solution: 0.5% violet crystal 2% 0.22 μm filtered ethanol) is added to each well.

After 30 minutes of incubation at room temperature, the wells are rinsed 3 times with distilled water and then properly dried by tapping on a sheet of absorbent paper.

100 µL of 10% acetic acid is then added to each well.

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).

Results

All of these results shown in Figs. 17 to 20, indicates that the molecules AIM2 and AIM3 induce a significant inhibition of the proliferation of the different cell types tested. The AIM1 molecule is also capable of causing a decrease in cell proliferation even if it is less marked than for the AIM2 and AIM3 molecules. On the other hand, the molecule (comparative example) called Ru (which corresponds to the molecule AIM2 whose iron has been replaced by ruthenium) does not cause inhibition of proliferation under the conditions tested.

Claims

1. 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) below:

in which :

(II) (III) (IV) (V) (VI) where

Y are each independently of the others a CH group or a nitrogen atom; R ⁷ is chosen from a group OC _Y H2 _{Y + I} with y = 1 to 10; SC _W H2 _{W + I with} w = 1 to 10;

N (C _m H2m + i) 2 with m = 1 to 6, COOH; SO3H or PO2H3;

Z denotes an oxygen atom, a sulfur atom or an NH group;

X is a counterion.

2. Non-therapeutic use according to claim 1, in which R ¹ to R ⁶ correspond to a hydrogen atom.

3. Non-therapeutic use according to claim 1, in which R ¹ , R ³ , R ⁴ , 6 ⁶ are identical and correspond to a hydrogen atom, and R ² and R ⁵ are identical and denote a benzene of formula ( Vil) following:

4. Non-therapeutic use according to claim 1, in which R ¹ , R ³ , R ⁴ , 6 ⁶ are identical and correspond to a hydrogen atom, and R ² and R ⁵ are identical and denote a pyridine radical of formula (VIII) following:

(VIII)

where the dotted line represents a CC bond between the carbon carried by the pyridine radical and the carbon carried by the terpyridine group of the organometallic compound of formula (I).

5. Non-therapeutic use according to any one of the preceding claims, in which the counterion X is chosen from: Cl, Br or I

6. Non-therapeutic use according to any one of the preceding claims, in which said organometallic compound is capable of causing direct demethylation of DNA.

7. Organometallic compound comprising an iron atom linked to two terpyridine groups, having a general formula (I) defined according to any one of claims 1 to 6 for its use as a medicament.

8. Organometallic compound according to claim 7, for its use in the treatment of diseases linked to cellular hyperproliferation.

9. Organometallic compound according to claim 7 or 8, for its use in the treatment of cancers.

10. Organometallic compound one of claims 7 to 9, for its use in the treatment of glioblastomas, promyelocytic leukemias, cancers of the prostate, ovaries, lungs, breasts, digestive tracts, in particular the liver, pancreas, head and neck, colon, non-Hodgkin lymphoma or melanoma.

1 1. Organometallic compound according to one of claims 7 to 10, for its use in the treatment of tumors resistant to cisplatin, 5-fluorouracil, tomoxifene or even doxorubicin or other anticancer compounds.

12. Organometallic compound according to claim 7 or 8, for its use in the treatment of inflammation, obesity, neurodegenerative diseases or cardiovascular diseases.