EP2794570A1

EP2794570A1 - Metal-chelating compounds having at least one polyamino chain

Info

Publication number: EP2794570A1
Application number: EP12810215.9A
Authority: EP
Inventors: François GABORIAU; David DENIAUD; Vincent CORCE; Karine JULIENNE APHECETCHE; Eric Renault
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite de Rennes 1; Universite de Nantes
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite de Rennes 1; Universite de Nantes
Priority date: 2011-12-20
Filing date: 2012-12-18
Publication date: 2014-10-29
Also published as: WO2013092556A1; US20140364454A1; FR2984317B1; CA2860520A1; FR2984317A1

Abstract

The invention relates to metal-chelating compounds comprising one or more 8-hydroxyquinoline units substituted in position 2 with a nitrogenous group comprising a polyamino chain, wherein the polyamino chain is N-substituted on said nitrogenous group, to the composition containing same, and to the uses thereof in the therapeutic field and in particular in the treatment of diseases associated with an abnormality in the regulation of metal metabolism resulting in a metal overload in human or animal cells.

Description

Metal chelating compounds having at least one polyamine chain.

The present invention relates to chimeric molecules associating one or more 8-hydroxyquinoline metal chelator groups with at least one polyamine chain. These compounds are hereinafter referred to by the acronym "Quilamine".

The invention also relates to the use in the therapeutic field of such Quilamines, in particular in the treatment or prevention of proliferative and / or metal-overload-related diseases such as cancers and neurodegenerative diseases in humans or in humans. animal.

Many metal ions from iron, copper, manganese, zinc, are involved in several biochemical and metabolic reactions of the body. The needs of the body generally vary according to the type of metal ions and the location of the biochemical and metabolic reactions involved. To ensure proper functioning of the body, these metal ions must be present at certain levels. to avoid overloads or deficiencies for example. In particular, overloads of metal ions such as iron and copper are harmful even toxic to the body because they catalyze the formation of reactive oxygen species. Overloads of metal ions can have various origins: genetic, metabolic, exogenous nutritional contributions or repeated blood transfusions. In particular, they can be linked to diseases such as cancers or tumors, neurodegenerative diseases, in which an imbalance in the homeostasis of metal ions occurs.

In the case of cancer, tumor cells proliferate uncontrollably and require abnormally high amounts of metal ions such as iron and copper, for their growth, compared to healthy cells.

The chelators initially proposed for the treatment of primary iron overloads such as Desferal®, and those more recently developed for the treatment of secondary overloads (Thalassemias), such as deferiprone® and deferasirox®, inhibit the proliferation of rat liver tumor cells. in culture (Richardson, DR, Potential of Iron Chelators as Effective Antiproliferative Agents, Can J Physiol Pharmacol, 1997, 75 (10-11): 1164-80). It has been shown that the antiproliferative activity of quilamine HQ1-44 is superior to that of deferasirox® in human tumor cells of hepatic and enterocyte origin. Desferal® inhibits the growth of melanoma cells in vitro and in vivo (Richardson, D., P. Ponka, and E. Baker, The ejfect of the iron (III) chelator, desferrioxamine, on iron and transferrin uptake by the human malignant melanoma cell. Cancer Res, 1994. 54 (3): p. 685-9) and hepatoma (Yamasaki, T., Terai S. and I. Sakaida, Deferoxamine for advanced hepatocellular carcinoma, The New England Journal of Medicine, 201 1, 365 (6): 576-77). In preliminary clinical trials, this chelator has been shown to be effective in the treatment of leukemias and neuroblastomas. In addition, the mode of action of cytotoxic chemotherapeutic agents, such as doxorubicin and Bleomycin, partially interferes with their chelating ability of iron and copper, as well as clioquinol, a derivative of 8-hydroxyquinoline (Ding, WQ, et al. Anticancer activity of the antibiotic clioquinol, Cancer Res, 2005. 65 (8): 3389-95).

Conversely, the addition of exogenous iron stimulates the proliferation of tumor cells. The need for iron during the cell cycle is illustrated by the hyperexpression on the plasma membrane of transferrin receptor proliferating cells that allows the entry of transferrin-bound iron into the cell. Ferroportin, an iron export protein that plays a key role in the systemic regulation of this metal, is a diagnostic marker for breast cancer. The increase in the expression of iron import proteins (DCYTB, DMT1 and T fR1) and the elimination of the expression of iron export proteins (HEPH and FPN), which lead to an increase in intracellular iron , have been described during the progression of colorectal cancer.

In addition, the involvement of polyamine metabolism in cellular replication and thus in proliferative processes makes this metabolism one of the preferred targets of antiproliferative drugs, along with the source of new circulating signals likely to reveal the existence of a neoplastic process within an organism.

The polyamines found not only inside the cells but also in the circulating state in the biological fluids of the body, such as blood, come from three main sources:

physiological cell proliferation (growth and / or renewal of the constituent cells of the organism) and tumoral,

- food,

- intestinal bacteria.

The biosynthesis and uptake of these ubiquitous molecules by the polyamine transport system (PTS) is strongly activated in tumor cells. The activity Antiproliferative treatment of iron chelators such as deferasirox® and O-trensox is associated with the intracellular iron depletion they cause and the inhibition of polyamine metabolism (Gaboriau, F., et al., Modulation of Cell Proliferation and polyamine metabolism in rat liver cell cultures by the iron chelator O-trensox, Biometals, 2006. 19 (6): 623-32).

The reactive oxygen species produced in iron and / or copper overload at the cerebral level cause alterations of the membrane lipids, proteins and DNA of the nerve cells involved in the etiology of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Elevated iron levels are observed in the central nervous system (CNS). Regardless of their involvement in the catalysis of reactive oxygen species, metals such as iron and copper also appear to play an important role in protein aggregation. They are therefore likely to provide a link between the two pathological processes of protein aggregation and oxidative damage characterizing neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD) and those with prions. Among the iron chelators for their effectiveness in inhibiting neurodegeneration, Clioquinol, which is currently in clinical trial, has the best protective efficacy in animal models of AD and PD.

The development of chelators of these metal ions, especially iron or copper, is an interesting research pathway in the treatment of proliferative diseases and / or related to metal overload in the body, such as cancers or certain neurodegenerative diseases. Chelators capable of inducing metal depletion in the cell large enough to create intracellular metal deficiency and to reduce cell proliferation and local production of reactive oxygen species are sought.

However, one of the problems of these chelators is that they are captured and complex metal ions indifferently in healthy and diseased cells. Even if the iron or copper needs of the tumor cells are higher than those of the normal cells, it is preferable to vectorise the chelator towards the tumor cells, in order to avoid too much toxicity of the treatments.

Studies have examined the role of polyamines as intracellular carriers of iron chelators. Natural polyamines, such as putrescine, spermine, spermidine, norspermine and norspermidine, as well as iron, copper, manganese and zinc are essential for cell growth. Polyamine deficiency caused by treatment with an inhibitor of polyamine synthesis, a Decontamination of the intestinal tract by an antibiotic and a depleted polyamine diet, slows tumor growth in animals (Seiler N. Thirty years of polyamine-related approaches to cancer therapy.) Retrospect and prospect. 2003, 4: 537-64).

Cancer cells have a particularly amplified metabolism (biosynthesis and uptake) of polyamines. The Polyamine Transport System (PTS), which transports polyamines, is particularly active in these cells. As the recognition and transport of polyamines is not specific for natural polyamines, it has made it possible to develop natural polyamine analogues that can be recognized and transported by PBS.

In the patent application EP 1 667 727, it is known to use benzylmaltol (deferiprone® or CP20) or poly (hydroxamic acid) chelators coupled to a polyamine chain in the treatment of cancer or a tumor, and in the treatment of a subject suffering from a metal overload condition.

However, these chelators have a low affinity for iron and are not entirely satisfactory for the development of a therapeutic treatment, for example in the treatment of cancers. In particular, the antiproliferative action of these chelators on cultured liver cells (HuH7 line) is not very effective.

The Applicant has now discovered a new family of compounds associating metal chelators and polyamine (s), called quilamines. These compounds effectively and selectively exert an antiproliferative action on the diseased cells and remedy the aforementioned drawbacks. The quilamines according to the invention are compounds comprising at least one 8-hydroxyquinoline unit substituted in the 2-position, that is to say in the alpha position of the constituent nitrogen atom of the 8-hydroxyquinoline ring, by at least one polyamine chain. Owing to their particular structure, the Quilamines according to the invention are capable of treating, in a targeted, less toxic and more efficient manner, proliferative diseases and / or related to a metal overload in human or animal cells, such as cancers. or neurodegenerative diseases compared to known chelators.

In particular, it has been observed that on the same type of cell line, a quilamine according to the invention has a strong antiproliferative activity that can even be greater than that of conventional iron chelators, for the same concentration of chelators. The quilamines according to the invention exhibit enhanced or at least unchanged antiproliferative activity in the presence of exogenous iron, compared to classical chelators of iron. They are therefore particularly effective for treating diseases related to sideroid overload in human or animal cells.

In addition, the Quilamines according to the invention have the advantage of being low in toxicity and suitable for various applications in the therapeutic field. They have a low cytotoxicity for healthy cells, particularly thanks to their high selectivity of recognition by the polyamine transport system, allowing them to selectively target the tumor cells.

Another object of the present application relates to the quilamines according to the invention as therapeutic treatment agents. The subject of the invention is also the Quilamines according to the invention as agents for treating proliferative diseases and / or neurodegenerative diseases linked to an overload of metal, for example iron and / or copper. Quilamines according to the invention are particularly useful as antiproliferative agents and / or cytotoxic agents of cancer or tumor cells.

The compounds according to the invention may be obtained according to preparation processes in which the polyamine chain (s) is (or are) coupled to the 8-hydroxyquinoleic motif (s) by steps of reductive amination, nucleophilic substitution or addition of Mickaël to give a quilamine according to the invention. These preparation methods have the advantage of being simple to perform. In particular, they make it possible to synthesize the quilamines according to the invention in less than fifteen steps, and preferably in less than twelve steps.

Other objects, aspects or features of the invention will become more clearly apparent from the description and examples.

The invention relates to compounds comprising at least one 8-hydroxyquinoline unit having in the 2-position, that is to say in the alpha position of the constituent nitrogen atom of the 8-hydroxyquinoline ring, at least one polyamine chain. .

More specifically, the quilamines according to the invention correspond to the following general formula (I), or to one of its conjugated forms, its salts or solvates:

(I) in which :

R ¹ , R ² , R ³ , R ⁴ and R ⁵ are chosen independently from one another from a hydrogen atom, a linear or branched, saturated, unsaturated, cyclic or aromatic hydrocarbon group comprising from 1 to 10 carbon atoms, carbon, a cyclic hydrocarbon group, aliphatic or aromatic, comprising from 4 to 12 carbon atoms, a halogen, a thiol, a hydroxyl, an amine preferably secondary or tertiary, an ether, a thioether;

R ⁶ is a hydroxyl;

a is equal to 0 or 1 with:

when a is equal to 0, M is a group corresponding to the following formula (Γ):

in which :

b being 0 or 1,

L represents an alkyl group having from one to four carbon atoms group C = O or C = S,

A ^{1 having} the general formula (X) or (Y):

in which :

R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are chosen independently from each other from hydrogen atoms, linear or branched, saturated, unsaturated, cyclic or aromatic hydrocarbon groups comprising from 1 to 12 carbon atoms, and protective groups of amino function,

B ¹ , B ² and B ³ are chosen independently of one another from linear or branched, saturated or unsaturated hydrocarbon groups comprising from 2 to 6 carbon atoms

i and j, identical or different, are equal to 0 or 1,

h is an integer from 0 to 4;

in which :

R ^'11, R ^12, R' ^13, R ^'14, R ^15, R ¹⁶ and R ¹⁷ are independently selected from each other from linear hydrocarbon groups or branched, saturated, unsaturated, cyclic or aromatic containing from 1 to 12 carbon atoms, and amino function protecting groups.

B and B are independently selected from linear or branched, saturated or unsaturated hydrocarbon groups having from 2 to 6 carbon atoms

B ' ³ is chosen from linear or branched, saturated or unsaturated hydrocarbon-based groups comprising from 2 to 6 carbon atoms, which can be interrupted by one or more secondary amino functions -NH- and / or one or more tertiary amino functions -NR ¹¹ -,

-NR ¹² -.

i and j, identical or different, are equal to 0 or 1,

h is an integer from 0 to 4;

and when a is equal to 1, M is a group corresponding to the following formula (I "): in which :

c and c ', which are identical or different, being equal to 0 or 1,

L and L ', identical or different, representing an alkyl group having 1 to 4 carbon atoms, a C = O or C = S group,

D ¹ and D ² , identical or different, representing a hydrocarbon group, linear or branched, comprising from 1 to 5 carbon atoms

A ² being the same as or different from A ¹ and having the formula (X) or (Y);

R ' ¹ , R' ² , R ' ³ , R' ⁴ , R ' ⁵ and R' ⁶ , are chosen independently from each other from a hydrogen atom, a saturated or unsaturated linear or branched hydrocarbon group, comprising: from 1 to 10 carbon atoms, a cyclic hydrocarbon group, aliphatic or optionally substituted aromatic, comprising from 4 to 12 carbon atoms, a halogen, a thiol, a hydroxyl, an amine preferably secondary or tertiary, an ether, a thioether.

The quilamine according to the invention is a metal chelator. It is capable of binding with a metal atom or metal ion by forming several (at least two) chelator-metal coordination bonds to form a metal complex, especially with a metal atom of the 3d series of transition metals, particularly the iron, copper, manganese and / or zinc. The quilamine according to the invention is especially capable of chelating an iron ion having a degree of oxidation equal to two (Fe (II) or Fe ²⁺ ) or three (Fe (III) or Fe ³⁺ ), an ion of copper having an oxidation state of two (Cu (II) or Cu ²⁺ ), a manganese ion having an oxidation state of two (Mn (II) or Mn ² ) or a zinc ion having a oxidation degree equal to two (Zn (II) or Zn ²⁺ ). Preferably, quilamine is an iron chelator.

The complexing affinity of quilamine with the metal ion in the body reflects the chelating or chelating ability of quilamine. This affinity can be evaluated by measuring the value of pW corresponding to -log of the concentration of free metal ion W, that is to say non-complexed by quilamine, for a ratio of the respective concentrations of Quilamine (10 micromoles per liter). ) and metal ion (1 micromole per liter) equal to 10 at 25 ° C and pH 7.4. The higher the pW value, the greater the affinity of the quilamine with the metal ion. The affinity of quilamine with iron in its ferric form (Fe (III)) and its ferrous form (Fe (II)) is measured respectively by pFe ³⁺ and pFe ²⁺ at physiological pH (pH = 7.4) at 25 ° C. Under these measurement conditions, the quilamine according to the invention has a pFe ²⁺ and / or pFe ³⁺ value at physiological pH which is strictly greater than that measured under identical conditions for the ligands of low molecular weight usually present in human cells. or animal complexing labile iron pool (Labil Iron Pool or LIP). The ligands of low molecular weight usually present in the cells may be for example albumin, citrate (pFe ³⁺ / citrate = 19.3 at pH = 7.4), ascorbate.

In particular, the pFe ²⁺ or pFe ³⁺ of the quilamine-iron complex is strictly greater than 20, and preferably ranging from 25 to 27 physiological pH.

At physiological pH, quilamine forms a quilamine-Fe (III) complex preferably having a ligand / metal stoichiometry of 2/1. The ligand / metal stoichiometry can for example be determined by the continuous variations method using the transition. characteristic in the absorption spectrum of Quilamine-Fe (III) complex at 580nm, for chelating and iron concentrations ranging from 0 to 500 micromoles per liter.

The polyamine chain (s) of the quilamine is or are recognized by the polyamine transport system of the cells and allows Quilamine to be transported to the cells.

Preferably, the polyamine chain or chains of the quilamine according to the invention is or are cationic or cationizable.

Cationic means that which comprises one or more quaternary amino functions, such that the global ionic charge carried by the amine chain is positive. In this sense, the polyamine chain may be mono or polycationic.

The term "cationizable" means that which comprises one or more primary and / or secondary amine functional groups, in particular in salt form, spontaneously ionizing into cation (s) in the medium for using quilamine, at physiological pH.

The polyamine chain or chains may comprise a putrescine, spermine, spermidine, or non-natural analog of these compounds such as homospermine, homospermidine, norspermine,

Advantageously, the polyamine chain or chains comprise a similar non-natural motif to putrescine, spermine or spermidine, such as homospermine, homospermidine, norspermine, allowing them to be recognized by the polyamine transport system, but without being degraded by the enzymes. of the oxidative retroconversion pathway.

In a first more preferred embodiment, a is zero and b is one.

In a second particularly preferred embodiment, a is zero, b is one, and L is CH ₂ ^'

In a third particularly preferred embodiment, a is zero, b is one, and L is CH ₂ -CH ₂ ^'.

In a fourth particularly preferred embodiment, a is zero, b is one, and L is a C = O ^'group.

In a fifth particularly preferred embodiment, a is zero, b is one, and L is a C = S ^'group.

In a sixth most preferred embodiment, when a is equal to one, c and c 'are identical, L and L' are identical, D ¹ and D ² are identical, and / or R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are identical to R ' ¹ , R' ² , R ' ³ , R' ⁴ , R ' ⁵ and R' ⁶ respectively. Preferably, R ' ⁶ is a hydroxyl.

In a seventh embodiment, the quilamine according to the invention meets the general formula (the exclusion of

following compound:

Preferably, B ¹ , B ² , B ³ are chosen from n-propyl and n-butyl groups and B ' ³ is chosen from n-propyl and n-butyl groups which may be interrupted by one or more secondary amino functions - NH- and / or one or more tertiary amino functions -NR ¹¹ -, -NR ¹² -.

In a particular embodiment, the group or groups A ¹ and A ² mentioned above can be chosen from:

a) those of formula (X) in which: B ¹ and B ³ denote n-butyl groups, i is equal to 1, j is equal to zero, h is an integer, even or odd, ranging from 1 to 4, R ¹³ and R ¹⁴ are hydrogen atoms, R ¹¹ being as previously defined; and

b) those of formula (X) in which: B ¹ and B ³ denote n-propyl groups, i is equal to 1, j is zero, h is an integer, even or odd, ranging from 1 to 4, R ¹³ and R ¹⁴ are hydrogen atoms, R ¹¹ being as previously defined;

In a preferred embodiment of the formulas (X) and (Y), B ³ and B ' ³ do not denote an n-propyl group and R ¹³ , R ¹⁴ , R' ¹³ , R ^'14 and R ¹⁷ do not denote simultaneously a hydrogen atom. In other words, preferably, the polyamine chain does not terminate with a primary n-propylamine group. Such polyamine chains are likely to be degraded by amine oxidase enzymes present in the serum (polyamine oxidase and semicarbazide sensitive amine oxidase) and generate a cytotoxic aldehyde and hydrogen peroxide.

In another preferred embodiment, the quilamine has the formula (I) wherein a is zero, b is one and L is CH ₂ and A ¹ or A ² is of the formula (X) wherein h = i = 1 and B ¹ is n-propyl. These Quilamines have a particularly important chelating power.

Among all the quilamines that can be used according to the invention, those corresponding to the formula (I) or to one of their conjugate forms, salts or solvates, in which:

R ¹ to R ⁵ are chosen independently from each other from a hydrogen atom, a saturated or unsaturated linear or branched hydrocarbon group comprising from 1 to 10 carbon atoms; R ⁶ is a hydroxyl; a is zero; b is equal to one; L is a C ₁ -C ₂ alkyl group; and the group A ¹ corresponds to the formula (X) in which R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are chosen independently from each other among hydrogen atoms, and amino protecting groups; B ¹ , B ² and B ^{3, which may be} identical or different, are n-propyl and / or n-butyl groups; i and j identical or different being equal to one or zero; h being equal to one.

In particular, those corresponding to the following formulas, or to one of their conjugate forms, salts, or solvates, are preferred:

(2- [4- (4-aminobutyl) aminobutyl] aminomethylquinoléin-8-ol)

(2- {4- [4- (4-aminobyl) aminobutylaminobutylaminomethyl) -inolin-8-ol)

(2- {4- [4- (3-aminopropyl) aminobutylaminobutylaminomethyl) -inolin-8-ol)

(2- [4- (3-aminopropyl) aminobutyl] aminomethylquinoléin-8-ol)

(2- {4- [3- (4-aminobutyl) aminopropyl] aminobutyl} aminomethylquinoléin-8-ol)

(2- {4- [3 - (3-aminopropyl) aminopropyl] aminobutyl} aminomethylquinolin-8-ol)

(2- [3- (4-aminobutyl) aminopropyl] aminomethylquinoléin-8-ol)

(2- {3- [4- (4-aminobutyl) aminobutyl] aminopropyl} aminomethylquinoléin-8-ol)

(2- {3- [4- (3-aminopropyl) aminobutyl] aminopropyl} aminomethylquinoléin-8-ol) OH (HQl-33)

(2- [3- (3-aminopropyl) aminopropyl] aminomethylquinoléin-8-ol)

(2- {3- [3- (4-aminobutyl) aminopropyl]} aminopropyl aminomethylquinoléin-8-ol)

(2- {3 - [3 - (3-aminopropyl) aminopropyl] aminopropyl} aminomethylquinolin-8-ol)

More preferably still, Quilamines HQ 1-44, HQ1-444, HQ1-443, HQ1-43, HQ1-434, HQ1-43, HQ1-33, HQ1-334, HQ1-333, or one of their conjugate forms, salts, or solvates.

The preferred quilamine is HQ 1-44 and its conjugated forms, salts or solvates. The acronym HQ1-44 describes the chelating unit HQ hydroxyquinoline, indexed 1 for the number of C¾ group linked to the polyamine chain. The number 44 (and respectively 444, 443, 43, 434, 433, 33, 334, 333, 34, 344, 343) specifies the number of carbon between each nitrogen atom of the polyamine chain.

The quinolines can be selected from any pharmaceutically acceptable salt. By "pharmaceutically acceptable salt" is meant in particular a salt with a pharmaceutically acceptable inorganic acid, such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic or hydrobromic acids; or a salt with a pharmaceutically acceptable organic acid, such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic or oxalic acids, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric. When the quilamine has a sufficiently acidic function at physiological pH to react with an organic or mineral base, quilamine can form a salt with said base.

The term "treatment" or "treating" refers to the ability of Quilamines to decrease the pool of excess metal ion (iron and / or copper) and / or to reduce polyamine levels in tumor cells and consequently to decrease and and / or inhibit the development of these cancerous or tumor cells and / or their symptoms.

The solvates of Quilamines consist of complexes or aggregates formed by one or more Quilamines or one of its salts as defined above, with one or more solvent molecules. The solvents may be, for example, water, methanol, ethanol, isopropanol or acetic acid. When the solvent is water, the solvate is a hydrate. Preferably, the solvates of Quilamines are hydrates such as hemihydrates, monohydrates, dihydrates, trihydrates, tetrahydrates.

The conjugated forms of Quilamines correspond to the forms of resonances due to the delocalization of one or more electronic doublets.

The quilamine according to the invention can be used as a therapeutic agent in humans and animals, in particular as an agent for treating proliferative diseases, neurodegenerative diseases, and / or linked to iron overload, copper , zinc and / or manganese, in human or animal cells. In particular, Quilamine can be used as an anti-tumor agent, for example in the treatment of hepatocarcinomas, lymphomas, melanomas, renal, ovarian carcinomas, breast, colon, and / or prostate carcinomas. , bladder, pancreas, and lungs in humans or animals. It can also be used to treat neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases.

The quilamine according to the invention can be used as an inhibitory agent for the endogenous synthesis of polyamines by the body. The polyamines result from the degradation of arginine, either directly by arginine decarboxylase which converts it to agmatine, or via the urea cycle which starts with arginine catalyzed transformation of arginine into ornithine. . The polyamines are then synthesized sequentially from ornithine which is decarboxylated initially by ornithine decarboxylase (ODC) to form putrescine. Spermidine is synthesized by spermidine synthase which makes the addition putrescine of an aminopropyl group provided by S-adenosyl methionine, decarboxylated by S-adenosyl methionine decarboxylase (SAMDC). Spermine is formed in the same way from spermidine by the addition of a second aminopropyl group. The main inhibitors of polyamine synthesis act at the ODC (α-difluoromethyl ornithine or DFMO), or SAMDC (CGP 48664).

Another subject of the invention relates to a composition comprising at least one quilamine as defined above.

The composition according to the invention may further comprise at least one excipient.

The excipient (s) that can be used are chemically inert and pharmacologically inactive auxiliary substances, in particular they do not influence the effects of quilamine and any additional active ingredients present in the composition. The excipient serves to formulate the composition of the invention in the form most suitable for the desired route of administration and optionally, where appropriate, to modulate the rate of release of the active substance (s) to the organism. Examples of excipients include: water and sucrose are the two excipients constituting the simple syrup - or, for dry forms, the modified starch (s) and the modified cellulose (s) are disintegrating agents used in dry forms (tablets, capsules, etc.) to accelerate the disintegration (or disintegration) of these once arrived in the stomach. For parenteral administration, the excipient may be an aqueous, sterile, isotonic to blood, pharmaceutically acceptable solvent or diluent, such as phosphate or saline acetate buffers, water, 5% dextrose solution. These formulations can be prepared as defined doses contained in a sterile glass vial and sealed according to the proven methods of the pharmacy. The excipient is preferably a phosphate buffered saline.

The composition according to the invention may be administered by any route of administration conventionally used in the therapeutic field, applied to humans or animals. In particular, it can be administered orally, sublingually, parenterally, subcutaneously, intramuscularly, intravenously, transdermally, locally, rectally or inhaled. Preferably, it is administered orally in a formulation in syrup, capsules or tablets.

The composition according to the invention preferably comprises a therapeutically effective quilamine content, that is to say such that the composition can be used as a therapeutic agent, in particular as a treatment agent for proliferative diseases and neurodegenerative and / or related to an overload of iron, copper, zinc and / or manganese, especially as antitumor agent, anti Parkinsonian or anti-Alzheimer, in humans or animals.

The composition according to the invention may further comprise at least one additional active ingredient different from the Quilamines and having in particular a marketing authorization.

As additional active ingredients, different from quilamines, there may be mentioned chemotherapeutic agents such as alkylating agents (Cis-Platinum), plant alkaloids (paclitaxel, epothilones), topoisomerase inhibitors (campthotecin, taxanes, alkaloids). Vinca family (Vinblastine, Vincristine, ...)), microtubule inhibitors (Bleomycin) and anti-metabolites (5-Fluoro uracil) and polyamine-like inhibitors such as Eflornithine (α-Difluoromethylornithine) and CGP 48664.

The composition according to the invention may also comprise at least one additive chosen from preservatives such as methyl methylhydroxybenzoate, chlorocresol, metacresol, phenol and benzalkonium chloride.

The composition may be a ready-to-use composition or a composition obtained by extemporaneous mixing of the quilamine (s) with one or more excipients and / or one or more additional active ingredients and / or one or more additives as mentioned above.

When the composition comprises at least one quilamine, and at least one additional active ingredient, the composition may be a combination product for simultaneous, separate or spread over time use of these various active principles and quilamines.

According to an advantageous variant, the additional active principle is chosen from an endogenous polyamine synthesis inhibiting agent and a chemotherapeutic agent.

According to a particularly advantageous variant, said endogenous polyamine synthesis inhibiting agent is an inhibitor of ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase or spermine synthase.

By "polyamine synthesis inhibiting agent" is meant a molecule capable of completely or partially blocking, directly or indirectly, at least one of the enzymes involved in the synthesis of polyamines in the human or animal body. Ornithine decarboxylase (EC 4.1.1.17) is a target enzyme for compounds that inhibit the biosynthesis of polyamines. The role of the inhibitor of polyamine biosynthesis is to stop or significantly reduce the endogenous production of polyamines in the organism treated with the product according to the present invention. Co-treatment with such inhibitors of the endogenous synthesis of polyamines makes it possible to enhance the antiproliferative activity of the quilamines according to the present invention by a conjugated deficiency of polyamines and of intracellular iron.

By "chemotherapeutic agent" have meant a chemical molecule to treat diseases such as cancer, neurodegenerative diseases or autoimmune diseases. The majority of chemotherapeutic substances work by stopping mitosis (cell division), effectively targeting dividing cells too rapidly or the synthesis and function of DNA. Some new agents do not act directly on DNA but directly target a molecular abnormality (leukemia, colon cancer).

The composition according to the invention as defined above can be used as a therapeutic agent in humans and animals, especially as an agent for treating proliferative diseases, neurodegenerative diseases and / or related to metal overload. in human or animal cells. In particular, it can be used as an agent for the treatment of hepatocarcinomas, lymphomas, melanomas, renal, ovarian carcinomas, breast, colon, and / or carcinomas of the prostate, bladder, prostate pancreas, and lungs in humans or animals. It can also be used to treat neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases.

The compounds according to the invention can be obtained by a process in which the polyamine chain (s) is (are) coupled to the 8-hydroxyquinoleic unit (s) by a reductive amination step, nucleophilic substitution. or addition of Mickael.

In particular, the method used comprises:

a reductive amination step between an aldehyde reagent and an amino reactant, one of the reagents being able to carry a precursor of the 8-hydroxyquinoline unit and the other being able to carry a precursor of the polyamine chain,

or a nucleophilic substitution step of an amino reactant carrying a precursor of the polyamine chain, on an electrophilic reagent carrying a precursor of the 8-hydroxyoxynoleic motif,

or a step of adding Mickaël between an alkene reagent bearing a precursor of the 8-hydroxyquinoline unit, and an amino reactant carrying a precursor of the polyamine chain.

In particular, said precursors of the 8-hydroxyquinoline unit comprise in position alpha of the constituent nitrogen atom of the 8-hydroxyquinoline ring a functional group suitable for reacting with the second reactant carrying the precursor of the polyamine chain.

For the preparation of a quilamine of formula (I) wherein a is equal to zero and b is equal to zero (denoted quilamine HQ'O), the preparation process comprises the following steps:

(i) reductive amination between a primary amine carrying the 8-hydroxyquinoline unit of formula (II)

(Π)

in which R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined in formula (I), and whose thiol, hydroxyl, primary and secondary amine functions, which may be present, are optionally protected by appropriate protective groups and a carboxaldehyde carrying a polyamine chain, of formula (ΙΓ)

(ΙΓ)

wherein A ' ¹ represents an amino hydrocarbon group corresponding to A ¹ as defined above, and whose primary and secondary amine functions are protected by appropriate protecting groups;

(ii) then deprotection of the protected functions in one or more steps.

In step (i), the reducing agent is chosen from hydrogen (H ₂ ) in the presence of a catalyst (palladium on activated carbon for example), a hydride (NaBH ₄ , NaBH ₃ CN or triacetoxyborohydride sodium NaBH (OAc) ₃ for example), and a hydrogen donor (formic acid, or a salt thereof for example). Preferably, sodium triacetoxyborohydride is used.

The thiol, hydroxyl, primary and secondary amine functions can be protected by means of well-known protective groups making them inactive with respect to the reductive amination reaction. The deprotection reactions corresponding to the various protected functions are also known per se by those skilled in the art.

As examples of protective groups of the hydroxyl functions, mention may be made of acetyl group (Ac), silyl derivatives or methyl. These functions can be deprotected respectively by an acid treatment, a fluoride anion or boron tribromide. By way of examples of protective groups of the primary and secondary amine functions of the polyamine chains, there may be mentioned, for example, tert-butyloxycarbonyl (Boc), benzyloxycarbonyl or benzyl group. These functions can be deprotected respectively by acid treatment with trifluoroacetic acid or hydrochloric acid, by hydrogenolysis in the presence of dihydrogen and palladium. When the 8-hydroxyquinole unit has primary or secondary amine functions, they can be protected and deprotected in the same way.

The carboxaldehyde bearing a polyamine chain, of formula (ΙΓ) can be obtained from a hydrocarbon monomer constituting the polyamine chain (for example B ¹ , B ² , B ³ in the case of formula (X)) comprising a terminal hydroxyl primary function and a terminal amine primary function, that is to say at the end of the chain. The polyamine chain may be obtained by an N-substitution on the primary amine of the first monomer, a second constituent monomer of the polyamine chain and bearing a nitrile function; reduction of nitrile function to primary amine; protection of the appropriate functions (secondary amine in particular); and then possibly repeating these steps until the desired polyamine chain is obtained. The carboxaldehyde is finally obtained by oxidizing the primary hydroxyl function of the resulting hydroxypolyamine intermediate.

The diagram below illustrates such a process for preparing a quilamine of HQ'O type (HQO-44, 8 "):

1 "

X is a leaving group such as

OTs, OMs, halogen

Z and 2 'identical or different

1) Reduction are groupings

protectors

2) Protection

For the preparation of a quilamine of formula (I) in which a is equal to zero, b is equal to one and L represents a CH ₂ group (denoted HQ'l), the preparation process comprises the following steps:

(i) reductive amination between a carboxaldehyde reagent bearing the 8-hydroxyquinoline unit, of formula

(III)

in which R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined in formula (I), and whose thiol, hydroxyl, primary and secondary amine functions, which may be present, are optionally protected by appropriate protective groups;

and a primary amine carrying a polyamine chain of formula (IIP)

(III ¹ )

wherein A ¹ is as defined in formula (ΙΓ);

FIRE I LLE OF REM PLACEM ENT (RULE 26) (ii) then deprotection of the protected functions in one or more steps.

The reducing agent may be chosen from those used in the preparation process described above. Preferably, sodium triacetoxyborohydride is used.

Thiol, hydroxyl, primary and secondary amine functions can be protected and deprotected as indicated in the previous preparation method.

The primary amine carrying a polyamine chain of formula (ΠΓ) can be obtained by a process of preparation similar to the carboxaldehyde of formula (ΙΓ) described above, from a hydrocarbon monomer constituting the polyamine chain (by example B ¹ , B ² , B ³ in the case of formula (X)) having two primary amine functions. One of the amine functions is protected by a protective group beforehand. Then, the polyamine chain can be obtained by an N-substitution on the unprotected primary amine of the first monomer, a second constituent monomer of the polyamine chain and carrying a nitrile function, reduction of the nitrile function to primary amine ; then protection of the appropriate functions (secondary amine in particular) before possible repetition of these steps until the desired polyamine chain is obtained.

The scheme below illustrates a process for the preparation of a HQ1 type quilamine (HQ1-44, 8) according to this embodiment from commercial hydroxyquinoline carbaldehyde 6:

Protection

H ₂ N, ZHN ..

NH ₂ NH ₂

, CN

X is a betting group such as

OTs, OMs, halogen

ZHN .. ^

^"' ^" XN

H

1) Protection

Z and Z 'identical or different

2) Reduction

are groupings

protectors

ZHN,. NH,

Z '

8

For the preparation of a quilamine of formula (I) in which a is equal to zero, b is equal to one and L represents a group CH ₂ -CH ₂ (denoted HQ '2) three methods of preparation can be envisaged.

According to a first variant, the process for preparing quilamine HQ'2 comprises the following steps:

(i) reductive amination between an acetaldehyde bearing the 8-hydroxyquinoline unit, of formula (IV)

(IV)

in which R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as in formula (I), and whose

SUBSTITUTE SHEET (RULE 26) thiol, hydroxyl, primary and secondary amine functions which may be present are optionally protected by appropriate protective groups;

and a primary amine carrying a polyamine chain, of formula (ΠΓ) as defined above;

(ii) then deprotection of the protected functions in one or more steps.

According to a second variant, the process for preparing quilamine HQ'2 comprises the following steps:

(i) addition of Mickaël between an alkene carrying the 8-hydroxyquinoline unit, of formula (V)

in which R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined in formula (I), and whose thiol, hydroxyl or amine functions that may be present are optionally protected by appropriate protective groups ;

(ii) then deprotection of the protected functions in one or more steps.

The primary and secondary thiol and amine functions can be protected and deprotected as indicated in the previous preparation method.

According to a third variant, the process for preparing quilamine HQ'2 comprises the following steps:

(i) nucleophilic substitution of an electrophilic reagent bearing the 8-hydroxyquinoline unit, of formula (VI) in which R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined in formula (I), and whose thiol, hydroxyl, primary and secondary amine functions which may be present are optionally protected by appropriate protective groups,

X represents a good leaving group, such as tosylate (OTs) or mesylate (OMs), or a halogen;

by a primary amine carrying a polyamine chain, of formula (ΠΓ) as defined above;

(ii) then deprotection of the protected functions in one or more steps.

The scheme below illustrates a process for preparing a HQ'2 quilamine according to each of the three variants of this embodiment from commercial hydroxyquinaldine 6:

ant

hydroxymethylation

The hydroxymethylation of compound 6 can be obtained by reacting it with butyllithium and then paraformaldehyde.

For the preparation of a quilamine of formula (I) in which a is equal to zero, b is equal to one and L represents a group C = O (denoted HQ '(CO)) this can be obtained by n' any conventional reaction to obtain an amide from a primary amine. By way of example, mention may be made of peptide coupling reactions such as the reaction of a carboxylic acid carrying the 8-hydroxyquinoline unit of formula (VII)

(VII)

in which R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined in formula (I), and whose thiol, hydroxyl, primary and secondary amine functions which may be present are optionally protected by appropriate protective groups,

with a primary amine carrying a polyamine chain, of formula (ΠΓ) as defined above, in the presence of a coupling agent after protection of other amines that may be present on the polyamine chain.

Useful cutting agents include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC or EDCI) and N, N'-dicyclohexylcarbodiimide (DCC). N-Hydroxybenzotriazole (HOBt) may be added to the reaction medium in order to activate the reaction.

The diagram below illustrates a process for preparing a quilamine HQ '(CO) according to this embodiment:

For the preparation of a quilamine of formula (I) wherein a is zero, b is one and L is C = S (denoted as HQ '(CS)), this may be obtained by i) thionation, using Lawesson's reagent or diphosphorus pentasulfide (P ₄ S ₁₀ ), of a quilamine HQ '(CO) whose thiol, hydroxyl, primary and secondary amine functions that may be present are optionally protected by appropriate protective groups, and then (ii) deprotection of the protected functions. The thiol, hydroxyl, primary and secondary amine functions are preferably protected and deprotected as indicated in the previous preparation method.

The diagram below illustrates a process for preparing a quilamine HQ '(CS) according to this embodiment: In a sixth embodiment, the process of the invention relates to the preparation of a quilamine of formula (I) in which a is equal to 1, c and c 'are identical, L and L' are identical, D ¹ and D ² are identical, and / or R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are identical to R ' ¹ ,

R ' ² , R ", R' ⁴ , R ° and R ', 6 respectively.

Said quilamine according to the invention can be obtained by (i) reductive amination of two equivalents of a carbaldehyde bearing an 8-hydroxyquinoleic unit with one equivalent of a primary diamine carrying a polyamine chain whose primary amine functions and secondary antibodies have been protected, and then (ii) deprotection of the protected functions, said carbaldehyde being in position 2 of the 8-hydroxyquinoline unit. The primary and secondary amino functions of the polyamine chain can be protected and deprotected as previously taught.

As useful carbaldehydes, mention may be made of the compounds corresponding to the formula

(VIII) following,

(VIII)

in which: L, c, D ¹ 'R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined in formula (I). The reagent contents used in each of the above embodiments can be selected and optimized to obtain the best yields.

The examples below serve to illustrate the various aspects of the invention.

EXAMPLES

EXAMPLE 1 Preparation of quilamine 8 '(2- [4- (4-aminobutyl) aminobutylaminomethyl) -inolin-8-ol

1) Preparation of the reagent 5 '(A ^^ - di-tert-butyloxycarbonylpermidine):

Synthesis of tert-butyl (4-aminobutyl) carbamate 4.4 grams of 1,4 diaminobutane (0.05 mol) are dissolved in 110 ml of a solution of triethylamine and methanol (10% by volume of TEA in methanol) under argon and at 0 ° C. A solution of di-terbutyl dicarbonate (3.63 g, 0.017 mol) and methanol (10 ml) is added dropwise into the mixture with vigorous stirring. The mixture is stirred at room temperature overnight. The solvents are evaporated under vacuum to obtain an oily residue which is dissolved in dichloromethane (100 ml) and washed with twice 100 ml of an aqueous solution of sodium hydroxide (10% by weight of NaOH in water)

The organic phase is dried and stripped of solvent in vacuo, and the product is purified by chromatography (1: 10.89NH ₄ OH: MeOH: CHCl ₃ ). The product 2 'obtained is a translucent oil (yield: 81%). R _f 0.4 (NH ₄ OH / MeOH / CHCl ₃ )

1H NMR (300MHz, CDCl ₃ ) δ 4.71 (s, 1H, NHCO), 3.10 (dt, J = 6Hz, 2H, CH ₂ ); 2.69 (t, Ji = 6Hz, 2H, CH ₂ ); 1.57-1.44 (m, 4H, 2CH ₂ ); 1.42 (s, 9H, CH ₃ ) ..

RM ¹³ C (75 MHz, CDCl ₃ ) δ 156.2; 79.2; 41.9; 40.8; 30.8, 28.5; 27.6. Synthesis of 4 'tert-butyl 4' (4- (3-cyanopropylamino) butyl] carbamate

To a solution of amine protected by tert-butoxycarbonyl V (2. 1 g, 0.01 mol) and anhydrous acetonitrile (75 ml) are added 5.14 grams of potassium carbonate. The suspension is mixed at room temperature for 10 minutes. The 4-bromobutyronitrile 3 '(1.65 g, 0.Olmol) is added to the mixture and the whole is stirred at 50 ° C for 24 hours. The mixture is filtered to remove most of the inorganic salts, and the acetonitrile is evaporated in vacuo. The oily residue obtained is purified by flash chromatography (1: 5: 94 NH ₄ OH: MeOH: CHCl ₃ ). The product 4 'obtained is a translucent oil (yield: 76%). R / 0.5 (1: 10:89 NH ₄ OH: MeOH: CHCl ₃ ).

1 H NMR (300 MHz, CDCl ₃ ) δ 4.77 (s, 1H, NHCO); 3.11 (q, Ji = 6Hz, 2H, CH ₂ ); 2.73 (t, J = 6.9 Hz, 2H, CH ₂ ); 2.61 (t, J = 6.6Hz, 2H, CH ₂ ); 2.44 (t, J = 7.2Hz, 2H, CH ₂ ); 1.80 (quintuplet, J = 6.9 Hz, 2H, CH ₂ ); 1.55-1.46 (m, 4H, 2CH ₂ ); 1.43 (s, 9H, 3CH ₃ ).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 156.4; 119.8; 78.9; 76.4; 49.3; 48.0; 40.4; 28.42 (3C); 27.8; 27.4; 25.8; 14.9. Synthesis of tert-butyl (4-aminobutyl- (4-tert-butoxycarbonylaminobutyl) carbamate a) Protection of amine 4 '

The aminonitrile 4 '(1.74 g, 6.8 mmol) is dissolved in 40 ml of a solution of methanol and triethylamine (10% by volume of TEA in methanol). Di-tertzo-butyldicarbonate (3.63 g, 0.017 mol) is added dropwise to the mixture with stirring.

The mixture is stirred at room temperature overnight. The solvents (MeOH and TEA) are evaporated under vacuum to obtain an oily residue which is dissolved in dichloromethane (100 ml) and washed twice with 30 ml of an aqueous solution of hydrogencarbonate (saturated solution in water) and twice with 30 ml of water.

The organic phase is dried with Na ₂ SO ₄ , filtered and purified by flash chromatography (80:20, dichloromethane: ethyl acetate). The intermediate product obtained is a translucent oil (95%)

1 H NMR (300 MHz, CDCl ₃ ) δ 4.57 (s, 0.8H, NHCO); 3.29 (t, J = 6.9 Hz, 2H, CH ₂ ); 3.03-3.23 (m, 4H, 2CH ₂ ); 2.34 (t, J = 7.2Hz, 2H, CH ₂ ); 1.87 (t, J = 6.9 Hz, 2H, CH ₂ ); 1.46-1.61 (m, 4H, 2xCH ₂ ); 1.45 (s, 9H, CH ₃ ); 1.43 (s, 9H, CH ₃ ).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 156.1; 157.7, 19.4; 80.1; 79.3, 47.2, 45.8, 40.2, 28.5, 27.8, 27.5, 25.6, 24.6, 14.8. b) Reduction of the nitrile function and obtaining of the product 5 '

Raney nickel (50% by weight in aqueous solution) is washed in ethanol (and permanently stored in the wet state in solvent because the compound is pyrophoric). The intermediate product obtained in the preceding step (2.00 g, 5.6 mmol) and NH ₄ OH are added. Argon is bubbled for 20 minutes. The suspension is hydrogenated at 20 bar for 24 hours, Raney nickel is filtered on celite (Raney nickel is permanently stored in the wet state with ethanol). Ethanol and NH ₄ OH are evaporated under vacuum and the oily residue is dissolved in CH ₂ Cl ₂ and washed with a 10% aqueous solution by weight of NaOH (3 * 50ml). The organic phase is dried with Na ₂ S0 ₄ , filtered and the solvent is evaporated under vacuum to give the 5 'product (98%).

1H NMR: (300MHz, MeOD) δ 3.21 (t, 4H, J = 7.2Hz,); 3.04 (t, 2H, J = 6.9Hz, 2CH ₂ ); 2.66 (t, 2H, J = 7.05Hz,); 1.62-1.51 (m, 8H); 1.46 (s, 9H), 1.43 (s, 9H).

¹³ C NMR (75MHz, MeOD) δ 158.5, 157.4, 80.8, 79.8, 42.2, 40.9, 30.8, 28.7, 28.3, 27.1, 26.6. Protection

Bo <¾0

HBOC

81%

2 '

4 '

1) Protection

98% Βθ <¾0

2) Ni Raney, H ₂

2) Preparation of reagent 7 (8-hydroxyquinoline-2-carbaldehyde):

Reagent 7 is a commercially available reagent. It can be obtained from product 6 according to the following reaction scheme:

3) Preparation of quilamine 8 '(2- [4- (4-aminobutyl) aminobutyllaminomethylquinolin-8-o!)

One equivalent of 8-hydroxyquinoline-2-carbaldehyde 7 is added to a solution of polyamines 5 '(1.2 eq) in 1,2-dichloroethane (10 ml) at room temperature under stirring under argon.

After 15 minutes, 3 equivalents of sodium triacetoxyborohydride are added and the

FIRE ILLE DE REM PLACEM ENT (RULE 26) The mixture is stirred for 12 hours until the aldehyde is consumed (followed by thin layer chromatography). The reaction is stopped by adding 10 ml of an aqueous solution of NaOH (1 mol / liter). The mixture is stirred for a further 20 minutes and the product is extracted with dichloromethane (3 times), dried over sodium sulfate and concentrated under reduced pressure. The residue is purified by chromatography on silica gel (chloroform / methanol / ammonia: (94: 5: 1)). A pale yellow oil is obtained (91%).

IR (KBr): ν = 3053, 2982, 2934, 1706, 1683, 1575, 1507, 1475, 1455, 1420, 1391, 1366, 1265, 1168, 1047 cm ^-1 .

1H NMR (400 MHz, [D ₄ ] MeOD): δ = 1.35-1.69 (m, 26H, 8C¾6C¾), 2.77 (bs, 2H, C¾), 3.03 (t, 2H, CH ₂ , J = 6.8 Hz) , 3.13-3.26 (m, 4H, 2C¾), 4.14 (s, 2H, C¾), 7.10 (dd, H, J = 7.5 Hz, J = 1.3 Hz), 7.34 (dd, HJ = 8.3 Hz, J = 1.3 Hz), 7.41 (dd, H, J = 8.1 Hz, J = 7.6 Hz), 7.45 (d, H, J = 8.5 Hz), 8.20 (d, H, J = 8.5 Hz).

¹³ C NMR (100 MHz, [D ₄ ] MeOD): δ = 26.5, 27.0, 27.2, 28.4, 28.7 (3C), 28.8 (3C), 40.9, 48.1, 49.9, 54.9, 79.8, 80.8, 1 12.2, 1 18.8, 122.1, 128.3, 129.4, 137.9, 139.2, 154.2, 157.4 (2C), 158.5.

High Resolution Mass Spectrometry (MALDI): calcd. for C 8 H ₄₄ N ₂ 0 ₄ ₅ [M + H] ⁺ 517.3384; found 517.3386

Protection :

The diprotected compound is dissolved in 5 ml of ethanol and then 6 ml of an aqueous hydrochloric acid solution (6 mol / l) is added at 0 ° C. The mixture is stirred for 24 hours and the solvent is evaporated. The solid is taken up in a minimum of ethanol and the precipitate is filtered under vacuum and dried for 6 hours in a pump (73%).

Melting point above 250 ° C.

IR (KBr): v = 3384, 2957, 2798, 1642, 1606, 1551, 1513, 1461, 1420, 1398, 1344, 1302, 1252-1055, 841 cm ^1.

¾ NMR (400 MHz, D ₂ 0): δ = 1.76-2.04 (m, 8H, 4C / J ₂ ), 3.06-3.22 (m, 6H, 3C / J ₂ ), 3.38 (dd, 2H, J = 7.75) Hz, J = 7.45 Hz, CH ₂ ), 4.78 (s, 2H, CH ₂ ), 7.36 (dd, 1H, J = 7.45 Hz, J = 1.49 Hz, H _7ar ), 7.60 (dd, 1H, J = 8.30 Hz, 7 = 1.49 Hz, H _5ar ), 7.65 (dd, 1H, J = 8.30 Hz, J = 7.45 Hz, H ₆ , 7.76 (d, 1H, J); = 8.60 Hz, H ₃ J, 8.64 (d, 1H, J = 8.60 Hz, 7J _4ar ) ppm.

¹³ C NMR (100 MHz, D ₂ 0): δ = 22.7, 22.8 (2C), 24.0, 38.9, 46.9, 47.0, 47.3, 49.7, 114.1, 119.4, 121.1, 128.9, 129.3, 134.5, 141.8, 148.5, 149.5 ppm.

High Resolution Mass Spectrometry (MALDI): C ₁₈ H ₂₉ N ₄ O [M + H-4HCl] ⁺ : Calculated: 317.2336; Found: 317.2348. Elemental analysis: C ₁₈ H ₃₂ Cl ₄ N ₄ O 1.2 H ₂ O: Calculated: C, 44.68; H, 7.17; N, 11.58. Found: C, 44.52; H, 6.97; N, 11.57

EXAMPLE 2 Preparation of Quilamine of the HQ '(CO) Type

Synthesis of N- (4 - ((4-aminobutyl) amino) butyl) -8-hydroxyquinoline-2-carboxamide

104 mg of 2-acid 8-hydroxyquinoline (0.552 mmol) are dissolved in anhydrous dichloromethane under argon. 80.8 mg of hydroxybenzotriazole (HOBT) (0.607 mmol) and 122 mg of dichlohexyl carbodiimide (DCC) (0.607 mmol) are added at 0 ° C. The solution is stirred one hour, and the amine 4 'is added (200mg, 0.552mmol). The mixture is stirred for 24 hours. The solvents are evaporated under vacuum and the residue is dissolved in dichloromethane, washed with NaHCO 3, dried over Na ₂ SO ₄ and filtered. The solvent is removed under vacuum and the product is purified by flash chromatography (dichloromethane: methanol, 97: 3). The protected product obtained is a beige solid (99% yield).

1H NMR (400MHz, MeOD) δ 8.39 (d, J = 8.5Hz, 1H); 8.18 (d, J = 8.5Hz, 1H); 7.53 (dd, J = 8.2Hz, J _d = 7.6Hz, 1H); 7.42 (dd, J = 8.2Hz, J = 1.0Hz, 1H); 7.16 (dd, J = 7.6 Hz, J = 1.0Hz H 1); 3.51 (t, J = 6.3Hz, 2H); 3.19 (t, J = 7.2Hz, 2H); 3.01 (t, 2H, J = 6.7Hz,); 1.74-1.31 (m, 26H). ¹³ C NMR (100MHz, MeOD) δ 166.6, 158.5, 157.4, 155.0, 148.8, 138.8, 138.4, 131.4, 130.5, 119.9, 118.9, 112.7, 80.8, 79.8, 48.1, 40.9, 40.3, 28.8, 28.7, 28.3, 27.9 , 27.0.

Protection:

The protected product (100 mg, 0.188 mmol) is dissolved in 1 ml of ethanol and then an excess of an aqueous solution of hydrochloric acid (6 mol / l) is added. The solution is mixed at room temperature for 24 hours, the solvents are evaporated under vacuum. The product obtained is a yellow solid (60 mg, 96% yield).

1H NMR (400MHz, D ₂ O) δ 8.16 (d, J = 8.7Hz, 1H); 7.74 (d, J = 8.7 Hz, 1H); 7.45 (dd, J = 8.4Hz, J = 7.7Hz, 1H); 7.29 (dd, J = 1Hz, J = 1Hz, 1H); 7.07 (dd, J = 1Hz, J = 7.7Hz, 1H); 3.48 (t, J = 6.6Hz, 2H); 3.23-3.03 (m, 6H); 1.91-1.73 (m, 8H). ¹³ C NMR (100MHz, MeOD) δ 165.5, 150.9, 145.8, 138.4, 135.4, 129.5, 129.4, 118.9, 18.3, 112.2, 47.9, 46.8, 38.9, 38.8, 25.7, 23.9, 23.2, 22.7.

The physico-chemical and biological properties of Quilamines according to the present invention have been studied and the results of these studies are presented below with reference to the figures according to which:

FIG. 1 shows the contribution of the polyamine chain in the iron chelation by the Quilamines according to the invention;

- Figure 2 shows the stoichiometry of the interaction of quilamine HQ1-44 with iron (L ₂ : Fei);

FIG. 3 shows the effects of quilamine HQ1-44 () and reference chelators, 8-hydroxyquinoline (HQ, □) and deferasirox ™ (ICL670, Δ) on the viability of hamster ovary cells, the line wild (CHO, solid curves) and the mutant strain deficient for the Polyamine Transport System (CHOMG, dashed lines);

FIG. 4 shows the influence of the exogenous micromolar iron (dashed lines) on the viability of hamster ovary cells (CHO), exposed to different concentrations of compounds, namely the quilamine HQ1-44 () and the chelators of reference, 8-hydroxyquinoline (HQ, □) and deferasirox ™ (ICL670, Δ);

- Figure 5 shows the effects of Quilamines and the reference chelator, 8-hydroxyquinoline (8-HQ) on the viability of tumor cells from human hepatocarcinoma (HepG2);

FIG. 6 shows the influence of STP on the viability of human hepatocarcinoma cells (HepG2) treated with quilamine HQ1-44;

- Figure 7 shows the effects of Quilamines and reference chelators, 8-hydroxyquinoline (8-HQ) and deferasirox ™ (ICL670) on the viability of tumor cells derived from human colon adenocarcinoma (Caco-2);

FIG. 8 shows the effect of quilamine HQ 1-44 and the reference chelator, deferasirox ™ (ICL670) on the expression of iron metabolism genes of human colon adenocarcinoma cells (Caco-2)

- Figure 9 shows the effect of Quilamine HQ 1-44 and the reference chelator, deferasirox ™ (ICL670) on iron metabolism of human colon adenocarcinoma cells (Caco-2):

FIG. 10 shows the effect of the quilamine HQ1-44 and the reference chelator, deferasirox ™ (ICL670) on the expression of genes of the polyamine metabolism of human colon adenocarcinoma cells (Caco-2);

- Figure 1 1 shows the effect of quilamine HQ1-44 and the reference chelator, deferasirox ™ (ICL670) on the polyamine metabolism of human colon adenocarcinoma cells (Caco-2).

I- Physicochemical properties of Quilamines 1. Interaction capacity with labile iron: Calcein test: (figurel)

Quilamines HQ 1-34, HQ1-344, HQ1-343, HQ1-33, HQ1-333, HQ1-44, HQ1-43, HQ 1-444, HQ 1-443 according to the invention were synthesized and tested. Each quilamine was measured for its ability to displace iron (III) complexed with calcein. Calcein is a fluorescent molecule which has a pFe3 + value of 20.3 at pH 7.4, close to the value measured for the ligands of low molecular weight present in human or animal cells and complexing the labile iron pool, such as the citrate which has a pFe ³⁺ of 19.3 at pH = 7.4 (LIP). The fluorescence of calcein is detected in a microplate, at a concentration of 0.1 micromol per liter, in solution in a HEPES (4- (2-hydroxyethyl) -1- piperazine ethanesulfonic acid) at pH 7.4, in a Fusion-type fluorescence fluorescence reader (Packard), under excitation at 450 nanometers and analysis of the fluorescence emission at 515 nanometers. The fluorescence of calcein is quenched in the presence of iron (III) in hydrochloride (chloride) form at a concentration of 1 micromole per liter. The addition of the quilamine at different concentrations of between 0.01 and 40 micromoles per liter causes the transfer of iron (III) complexed with calcein to quilamine, resulting in a restoration of the fluorescence of calcein. The dose-effect curves reflecting the percentage of restoration of the fluorescein of the calcein (relative to the fluorescence of the free molecule in the absence of iron), for each quilamine concentration, make it possible to deduce the Quilamine concentration causing the displacement. of 50% Fe (III) of the calcein and the restoration of its associated fluorescence (EC50). These EC50 concentration values are inversely proportional to the chelating capacity of the chelators and their ability to mobilize iron from the body's LIP.

Comparatively, it was measured under the same conditions the efficiency to move the iron (III) complexed with the calcein of two chelators of the prior art, 8-hydroxyquinoline and O-trensox, the latter being currently considered as the chelator higher affinity for iron.

With reference to FIG. 1, the tests showed that the Quilamines according to the invention have a better chelating capacity with respect to the two chelators tested for chelator concentrations of less than 0.2 micromole per liter. The comparison of the chelating efficiency of quilamines with that of the basic chelating unit, 8-hydroxyquinoline, shows that the polyamine chain enhances the interaction capacity of this chelator with Fe (III). In addition, it has been observed that HQ1-3 type quilamines (HQ1-34, HQ1-344, HQ1-343, HQ1-33, HQ1-333) have a better chelating capacity compared with HQ1-4 type quilamines ( HQ1-44, HQ1-43, HQ1-444, HQ1-443).

2. Study of the interaction of quilamine HQ1-44 with iron (Figure 2)

The interaction of quilamine HQ1-44 with iron (Fe3 +) was evaluated, thanks to the characteristic transition at 580nm observed in the absorption spectrum of the quilamine complex HQ1-44 with Fe (III). The continuous variation method consists of measuring the amount of complex formed between quilamine and Fe (III), deduced from the value of the absorption at 580 nm, by varying the ratio of quilamine / quilamine concen trations. + Fe (III) for a total Quilamine + Fe (III) concentration constant and equal to 100 micromoles per liter (Tris / HCl buffer 100 millimoles per liter, pH 7.4).

The continuous variations method showed that quilamine HQ1-44 forms a complex with Fe (III) with ligand / iron stoichiometry of 2/1 at physiological pH. Potentiometric measurements were made in thermostatically controlled titration cells at 25 ± 0.1 ° C, using a Metrohm 702 SM Titrino connected to a Metrohm 6.0233.100. The ligand solution is prepared at a concentration of ~ 1.5 x 10 ³ moles per liter, the Fe ³⁺ solution is made from FeCl ₃ and titrated by complexation with a standard solution of EDTA (ethylenediaminetetraacetic acid). The sample for the measurement contains approximately 0.03 millimoles per liter of ligand in a volume of 30 ml or the ionic strength is maintained at 0.1 mol per liter using KN0 ₃ as the supporting electrolyte. In titrations Fe ³⁺ is added to 0.45 or 0.9 equivalents of ligand. Each titration consists of 150-200 equilibrium points at a pH ranging from 2.0 to 1.5, and is repeated at least twice. The thermodynamic constants are calculated with the HYPERQUAD software and the species speciation diagram is made by the Hyss program.

Comparison of the pFe3 + of HQ1-44 (27 at pH 7.4) with that of o-Trensox, which forms a complex of very high affinity with Fe (III), of stoichiometry 1/1 (pFe3 + = 29.5 measured in the same pH conditions) confirms the strong chelating capacity of quilamine HQ1-44. It is also compared to that of the basic chelating unit, 8-hydroxyquinoline (pFe3 + = 20.6 measured under the same pH conditions), which confirms the strengthening of the chelating capacity of this chelator by the polyamine chain of quilamine HQ1-44.

II- Biological properties compared to healthy ovarian hamster cells 1. Involvement of STP in Quilamines uptake

The in vitro efficacy of different Quilamines was tested on a wild-type hamster ovary (CHO) cell line with PBS, and a CHO-MG mutant line lacking PBS. The two CHO and CHO-MG cell types were treated 24h after their seeding in 96-well microplates by concentrations of the different Quilamines of between 0.1 and 400 micromoles per liter. The cytostatic (antiproliferative) and cytotoxic effects of quilamine after 72 h of cell treatment were evaluated respectively by counting the cell nuclei after labeling the DNA with the Hoescht 33342 fluorescent intercalator and by analyzing the membrane damage, detected by the assay for lactate dehydrogenase (LDH) activity in the supernatants). ^Λ

The results obtained with quilamine HQ1-44 are shown in Figure 3, Figure 3A for the results on cell viability and Figure 3B for results on cellular toxicity.

The IC50 inhibitory concentration, corresponding to the concentration of Quilamines for 50% inhibition of cell proliferation, was deduced from the dose-response counting curves of living cell nuclei on the CHO and CHO-MG lines. These dose-effect curves are sometimes biphasic, as in the case of quilamine HQ1-44 which has an antiproliferative component (without concomitant release of LDH in the supernatant) in the range of concentrations between 0.4 and 3 micromoles per liter and second cytotoxic component accompanied by a membrane toxicity resulting in a release of LDH, for concentrations greater than 2 micromoles per liter. The lower the IC50 value, the more effective the anti-proliferative effect of the tested chelators.

In addition, the ratio of the IC50 inhibitory concentrations found for the CHO-MG line and the CHO line (IC50 ratio (CHO-MG / CHO)) was calculated for several Quilamines according to the invention. This report makes it possible to estimate the recognition selectivity of the different chelators by the STP: the higher this ratio, the better the chelator is recognized by the STP. When this ratio is close to 1, the tested chelator is not or little recognized by the STP.

The results were listed in Table 2 below and were compared with those obtained under the same conditions for two metal ion chelators of the prior art, 8-hydroxyquinoline and ICL670 (Deferasirox ™ sold by Novartis ).

Table 2: Influence of the Polyamine Transport System on the selectivity of the antiproliferative action of Quilamines compared to that of the reference chelators, 8-hydroxyquinoline (8-HQ) and deferasirox ™ (ICL670).

The compound HQ1-44 is characterized by an IC50 of 1.4 micromoles per liter on the CHO line and 344 micromoles per liter on the CHO-MG line. This compound has the most effective antiproliferative effect and is best recognized by STP.

In addition, poly (n-butylamine) chains (HQ1-44 and HQ 1-444) appear to be more selectively recognized by PBS than poly (n-propylamine) chains (HQ1-33).

Moreover, the quilamines of the invention tested all have excellent recognition by the STP compared to the known chelators 8-hydroxyquinoline and ICL670.

2. Effectiveness of Quilamines

The cytostatic or antiproliferative action and the 72-hour cytotoxic action of quilamine HQ1-44 compared with 8-hydroxyquinoline and ICL670 on the CHO lines, with or without exogenous iron, were compared. Fe (III) citrate form at a concentration of 20 micromoles per liter. The results obtained are shown in Figure 4, Figure 4A for cell viability results, and Figure 4B for cell toxicity results.

In the absence of exogenous iron, it is observed that 8-hydroxyquinoline causes a decrease in the number of viable cells associated with LDH release (cytotoxic effect). In particular, 8-hydroxyquinoline exerts a cytotoxic effect from a chelating concentration greater than or equal to 5 micromoles per liter. In the presence of iron Exogenous, there is no change in the cytotoxic action of 8-hydroxyquinoline.

The dose-response curves for ICL670 are biphasic, like that of quilamine HQ1-44. In the absence of exogenous iron, a cytostatic effect is observed without membrane alteration for chelating concentrations of between 3 and 10 micromoles per liter and a cytotoxic effect for chelating concentrations of greater than 10 micromoles per liter, resulting in the release of LDH. . After addition of the exogenous iron, an inhibition of the cytostatic and cytotoxic effects induced by this chelator is observed.

For quilamine HQ1-44, in the absence of exogenous iron, there is a cytostatic effect without membrane alteration for chelating concentrations of less than 3 micromoles per liter (ranging from 0.4 to 3 micromoles per liter) and a cytotoxic effect for chelating concentrations greater than 3 micromoles per liter resulting in the release of LDH. After addition of the exogenous iron, an improvement in the antiproliferative effect of quilamine (IC 50 = 0.6 micromol per liter instead of 1.4 previously measured) and a partial inhibition of the cytotoxicity of this compound at concentrations of chelator greater than 3 micromoles per liter.

Quilamine HQl-44 is therefore less toxic than 8-hydroxyquinoline and retains an antiproliferative action even in case of sideric overload unlike ICL670, which is of some interest for treating proliferative diseases and / or related to an overload. iron in the cells. The absence of inhibition of the antiproliferative effect of quilamine in the presence of exogenous iron suggests that the antiproliferative action of quilamine is not associated solely with its iron depletion capacity in cells. It could also be associated with the HQ1-44 inhibition capacity of polyamine metabolism, as shown below in Caco-2 cells (§4).

III- Biological properties compared to tumor cells

1. Efficacy of Quilamines in Hepatocyte Tumor Cells of the HepG2 Line

The efficacy of Quilamines was analyzed on hepatocyte cells of the HepG2 tumor line derived from a human hepatocarcinoma, and compared with that of ICL670, in the presence or absence of exogenous iron. The proliferating HepG2 cell cultures were treated 24h after their seeding in 96-well microplates by concentrations of the different Quilamines ranging from 0.1 to 400 micromoles per liter. The cytostatic (antiproliferative) and cytotoxic effects of quilamine after 72 hours of treatment cells were evaluated respectively by counting the cell nuclei after labeling the DNA with the Hoescht 33342 fluorescent intercalator or the mitochondrial succinate dehydrogenase (SDH) activity and by analyzing the membrane damage detected by the activity assay. lactate dehydrogenase (LDH) in the supernatants. The results obtained on the SDH activity are given in FIG.

The majority of Quilamines, such as ICL670, have, in the absence of exogenous iron, biphasic dose-effect curves with an antiproliferative component, for concentrations of between 1 and 10 micromoles per liter and a cytotoxic component associated with a release of LDH, for concentrations greater than 10 micromoles per liter. The percentage of HepG2 cells concerned by the antiproliferative effect and the IC50 values associated with this cytostatic effect, in the absence of exogenous iron, have been listed in Table 3 below.

Table 3: Antiproliferative effect of Quilamines compared to that of the reference chelator, deferasirox ™ (ICL670), in human hepatocarcinoma cells (HepG2).

The antiproliferative efficacy of HQ1-44 is highest after ICL670, followed by the compounds HQ1-443, HQ1-333 and HQ1-343.

In the presence of exogenous iron, inhibition of cytostatic and cytotoxic effects induced by ICL670 is observed, whereas for quilamine HQ1-44 only the cytotoxic effect is partially inhibited. As in the case of the cells of the CHO line, this absence of inhibition of the antiproliferative effect of Quilamines by the addition of exogenous iron seems to confirm that this effect is not only associated with the capacity of depletion of the intracellular iron of Quilamine. .

2. Involvement of PBS in the antiproliferative action of Quilamines in cells hepatocyte tumors of the HepG2 line (Figure 6)

The involvement of STP in the cytostatic and cytotoxic action of Quilamines has been analyzed in proliferating HepG2 hepatocyte tumor cells, after activation of STP or competitive inhibition of PBS by spermidine, a polyamine naturally present in the body and most effectively captured by the STP. The quilamine treatment conditions are identical to those described in paragraph III-1.

By inhibiting ornithine decarboxylase (ODC), the key enzyme in the biosynthesis of polyamines, depletion of intracellular polyamines and compensatory activation of PBS that fills the deficiency is caused. This activation of STP is done using the ODC inhibitor, alpha-difluoromethyl ornithine (DFMO).

The competitive inhibition of PBS by spermidine is caused by co-treatment of cells with Quilamines in the presence of spermidine at a concentration of 50 micromol per liter.

The results obtained with quilamine HQ1-44 are given in FIG.

It is observed that the activation of the STP by pretreatment with DFMO results in an amplification of its cytotoxicity at concentrations greater than 50 micromoles per liter (50-100 micromoles per liter).

It is observed that the competitive inhibition of the quilamine uptake by the PBS results in an inhibition of the cytostatic effects (Quilamine concentration range of less than 10 micromoles per liter) and cytotoxic effects (Quilamine concentration range of 50 to 100 micromoles per liter). This suggests the involvement of PBS in the effects of Quilamine HQ1-44 on hepatocyte HepG2 cells.

3. Efficacy of Quilamines in Entero-Cocytic Caco-2 Tumor Cells (Figure 7)

The effect of Quilamines on cell viability was analyzed on the enterocyte cells of the Caco-2 tumor line, derived from a carcinoma of the human colon and compared with the action of ICL670, in the presence or absence of exogenous iron. after activation (DFMO) or competitive inhibition by spermidine, of STP. The operating conditions used are identical to those used for the CHO / CHO-MG and HepG2 cells.

As in the case of the other cell lines in the proliferative phase, presented above, the dose-response curves of the action of Quilamines on the number of viable cells (number of nuclei) are biphasic with a cytostatic component for concentrations of less than 30 micromoles per liter and a cytotoxic component with release of LDH for concentrations greater than 30 micromoles per liter. Only this second cytotoxic component is observed when the cells of the Caco-2 line are treated after confluence (cessation of proliferation).

The results for the measurement of the antiproliferative effect of Quilamines, and of ICL670 on proliferating cells (cytostatic component), in the absence of exogenous iron, have been listed in Table 4 below. 8-hydroxyquinoline (8-HQ) has only one cytotoxic component with an IC50 of 5 micromoles per liter.

Table 4: Antiproliferative effect of Quilamines compared to those of the reference chelators, 1 to 8-hydroxyquinoline (8-HQ) and deferasirox ™ (ICL670), in human colon adenocarcinoma cells (Caco-2).

After addition of the exogenous iron, inhibition of cytostatic and cytotoxic effects induced by ICL670 is observed, whereas for quilamine HQ1-44 only the cytotoxic effect is partially inhibited. This lack of inhibition of the antiproliferative effect of quilamines by the addition of exogenous iron seems to confirm that this effect is not associated solely with the quilamine intracellular iron depletion capacity. 3. Antitumor action of Quilamines by intracellular iron depletion in enterocyte tumor cells of the Caco-2 line

After 72 hours' treatment of the Caco-2 cells, in the proliferative phase with the quilamine HQ1-44 and the ICL670 at the concentration of 10 micromoles per liter, the analysis is carried out using the Real Time-Polymerase Chain Reaction (RT) technique. -qPCR) the expression of the two main genes regulating intracellular iron metabolism, that of L-ferritin, iron storage protein, as well as that of the transferrin receptor, the iron-import protein of the membrane. The results are shown in Figure 8. The cDNAs (Deoxyribonucleic Acid Complementary) synthesized during reverse transcription are amplified by qPCR (amount of polymerase chain reaction) to quantify the expression of mRNA (messenger ribonucleic acid) genes involved in the metabolism of iron.

After 72 hours' treatment of Caco-2 cells, in the proliferative phase with quilamine HQ1-44 and ICL670, the biochemical analysis of intracellular iron (Fe2 + and Fe3 +), the two main proteins regulating iron metabolism, is performed. intracellular concentrations of L-ferritin, an iron storage protein, as well as transferrin-soluble receptor assay in cell supernatant, transferrin receptor concentration marker, iron of the membrane. The results are shown in Figure 9.

On Caco-2 cells in the proliferative phase, quilamine HQ1-44 and ICL670 at a concentration of 10 micromoles per liter do not significantly modify the expression of genes coding for the iron storage protein, L -ferritin (L-Fer), and for the main route of entry of iron into the cell, the transferrin receptor (RTrf). Exposure of cells to iron-saturated transferrin (holotransferrin) does not alter the expression of L-Ferritin and reduces transferrin receptor expression. The iron overload induced by exposure for 72 h to 20 micromoles per liter of iron-citrate is accompanied by a small increase in the expression of L-ferritin (not significant) and an inhibition of expressing of the transferrin receptor, in accordance with the IRL7IRP regulation of these two genes.

The biochemical analysis of L-ferritin and iron concentrations shows that Quilamine HQ1-44 and ICL670 at a concentration of 10 micromoles per liter cause intracellular iron depletion. Treatment with these chelators has no significant effect on the transferrin-soluble receptor. Conversely, exposure to Γ holotransferrin (iron-saturated Trf) and iron-citrate is accompanied by a marked increase in L-ferritin and intracellular iron.

5. Antitumor action of Quilamines by involvement of polyamine metabolism in enterocytic tumor cells of the Caco-2 line

After 72 hours treatment of Caco-2 cells, proliferative phase by Quilamine HQl-44 and ICL670 at a concentration of 10 micromoles per liter, RT-qPCR analysis of the expression of the main polyamine regulatory genes for metabolism, such as ornithine decarboxylase (ODC), is carried out. antizyme (OAZ1), S-adenosyl methionine decarboxylase (SAMDC) and polyamine oxidase (PAO). The results are shown in Figure 10.

After 72 hours' treatment of the Caco-2 cells, in the proliferative phase with the quilamine HQ1-44 and the ICL670 at the concentration of 10 micromoles per liter, the biochemical analysis is carried out by tandem mass spectrometry coupled with liquid chromatography. (LC / MSMS) intracellular polyamines such as putrescine, spermidine and spermine, after dansylation. The results are shown in Figure 11.

The treatment for 72 hours of Caco-2 proliferating cells, by the chelators HQ1-44 and ICL670 at the concentration of 10 micromoles per liter has no effect on the expression of the genes involved in the regulation of polyamine metabolism, such as that of the ODC of antizyme (OAZ1), S-adenosyl methionine decarboxylase (SAMDC) and that of polyamine oxidase (PAO). Citrate alone causes the reduction of the expression of genes involved in the polyamine biosynthesis pathway (ODC and SAMDC). On the other hand, the iron overload caused by holotransferrin or iron-citrate has no effect on the expression of the genes of the polyamine metabolism.

Iron overload does not alter the concentrations of natural polyamines (Putrescine, spermidine and spermine). On the other hand, the intracellular concentrations of putrescine (Put) and spermidine (Spd) are lowered after treatment with Quilamine HQ1-44 (-73% Put, -55% Spd) and to a lesser degree by ICL670 (-34 % Put, -15% Spd). This reduction in Put, associated with a smaller decrease in Spd and no effect on intracellular Spm, is comparable to the effect of DFMO, an inhibitor of ODC.

This reduction of intracellular polyamines, putrescine and spermidine, induced in Caco-2 proliferating cells by quilamine HQ1-44 and to a lesser extent with ICL670 at a concentration of 10 micromoles per liter, contributes to the antiproliferative effect of these cells. chelators, together with the iron depletion they cause. The influence of intracellular polyamine inhibition by HQ1-44 on cell proliferation explains the absence of inhibition of the antiproliferative effect of Quilamines by exogenous iron

Claims

A compound characterized in that it corresponds to the following general formula (I), or to one of its conjugated forms, its salts or solvates:

(I)

in which :

R ⁶ is a hydroxyl;

a is equal to 0 or 1 with:

when a is equal to 0, M is a group corresponding to the following formula (Γ):

in which :

b being 0 or 1,

L represents an alkyl group having from one to four carbon atoms, a group C = O or C = S,

A ^{1 having} the general formula (X) or (Y):

in which :

i and j, identical or different, are equal to 0 or 1,

h is an integer from 0 to 4;

in which :

B ¹ and B ² are chosen independently of one another from linear or branched hydrocarbon groups, saturated or unsaturated, comprising from 2 to 6 carbon atoms

-NR ¹² -.

i and j, identical or different, are equal to 0 or 1, h is an integer from 0 to 4;

c and c ', which are identical or different, being equal to 0 or 1,

R ' ¹ , R' ² , R ' ³ , R' ⁴ , R ' ⁵ and R' ⁶ , are chosen independently from each other from a hydrogen atom, a saturated or unsaturated linear or branched hydrocarbon group, comprising: from 1 to 10 carbon atoms, an optionally substituted cyclic, aliphatic or aromatic hydrocarbon group comprising from 4 to 12 carbon atoms, a halogen, a thiol, a hydroxyl, an amine, preferably a secondary or tertiary amine, an ether, a thioether .

2. Compound according to claim 1, characterized in that it is a metal chelator

3. Compound according to claim 2, characterized in that it is a chelator of iron, copper, manganese and / or zinc.

4. Compound according to any one of the preceding claims, characterized in that the polyamine chain or chains are cationic or cationizable.

5. A compound according to any one of the preceding claims, characterized in that a is equal to 1, c and c 'are identical, L and L' are identical, D ¹ and D ² are identical.

6. Compound according to any one of the preceding claims, characterized in that R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are identical to R ' ¹ , R' ² , R ' ³ , R' ⁴ , R ' ⁵ and R' ⁶ respectively.

7. Compound according to any one of claims 1 to 6, characterized in that R ⁶ is a hydroxyl.

8. Compound according to the preceding claim, characterized in that B ¹ , B ² , B ³ are chosen from n-propyl and n-butyl groups, and B ' ³ chosen from n-propyl and n-butyl groups which may be interrupted by one or more secondary amine functions -NH- and / or one or more tertiary amine functions -NR ¹¹ -, -NR ¹² -.

9.Compound according to any one of claims 1 to 8, characterized in that it corresponds to formula (I) in which: R ¹ to R ⁵ are chosen independently of one another from a hydrogen atom, a linear or branched hydrocarbon group, saturated or unsaturated, comprising from 1 to 10 carbon atoms; R ⁶ is a hydroxyl; a is zero; b is equal to one; L is a C ₁ -C ₂ alkyl group; and the group A ^{1 has} the formula (X) wherein R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently selected from hydrogen, and amine protecting groups; B ¹ , B ² and B ^{3, which may be} identical or different, are n-propyl and / or n-butyl groups; i and j identical or different being equal to one or zero; h being equal to one.

A compound according to any one of the preceding claims as a therapeutic agent in man and in animals.

11. Compound according to claim 10, as a treatment agent for proliferative diseases,

12. The compound of claim 10 as an agent for treating neurodegenerative diseases.

13. Compound according to claim 10 as an agent for the treatment of diseases related to an overload of iron, copper, zinc and / or manganese, in human or animal cells.

14. Compound according to claim 10, as inhibiting agent for the synthesis of polyamines in the body.

15. A composition comprising at least one compound as defined in any one of claims 1 to 14 and optionally at least one additional active ingredient, said compound (s) and any principle (s) ) active (s) of the composition can be used simultaneously, separately or spread over time.

16. Composition according to claim 15, characterized in that the additional active ingredient is chosen from inhibiting agents for the endogenous synthesis of polyamines or chemotherapeutic agents.