WO2011029956A1 - Flavones and flavanones derivates as dna methyltransferases inhibitors - Google Patents

Abstract

The present invention relates to a compound of formula (I) or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers, for its use in the prevention and/or treatment of cancer, developmental diseases, neurodegenerative diseases or Trypanomiasis diseases by inhibition of DNA-methyltransferases.

Description

Flavones and flavanones derivates as DNA methyltransferases inhibitors

The present invention concerns flavones and flavanones derivates as DNA methyltransferases inhibitors. The invention also concerns a method of high- throughput screening of DNA methyltransferases inhibitors.

During the last decades, flavones and flavanones derivatives have been extensively studied for their medicinal applications. In this regard, the article of Dauzonne et al. (Eur J Med Chem, 1997, 32, 71 -82) discloses a series of 3- aminoflavones for their antiproliferative activity; and the article of Bauvois et al. (J. Med. Chem., 2003, 46, 3900-3913) relates to the synthesis and biological evaluation of flavone derivatives as reversible inhibitors of aminopeptidase N (APN/CD13).

The search for the best strategy to fight against the tumor is slowly evolving into a tailor-maid treatment, optimized for each patient thanks to pharmacogenomics. By the way, cancer is still a fatal disease in half of the detected cases. There is an urgent need to develop alternative solutions, by designing innovative concepts that do not only propose new technical solutions to the same questions, but indeed call for a simultaneous expansion of cancer treatment understanding.

Cancer cells are out of control. Throughout tumorigenesis they acquire (or are naturally selected thanks to) some key modifications that hack their natural protections against abnormal cell proliferation and boost their malignant characteristics: independence to growth signals, tissue invasion, unlimited replicative potential, etc. Proto-oncogenes and oncogenes are able to transform a normal cell into an over-clocked system: much more active but also more fragile. Indeed, cancer cells need an important nutrients and oxygen supply, triggered by a leaky and imperfect neovascular system. They are also addicted to specific metabolisms; they poorly repair their DNA damages, etc. These are various strategic options to impact the disease that chemotherapy already explores. An innovative choice would be to awake the cancer cell's sleeping defences against tumor progression, for instance by reactivating the silenced tumor-suppressor genes. The idea is there to revert the pathological phenotype of malignant cells, not necessarily to kill them, but to force them to adopt a more normal behavior: senescence, differentiation, cell death, etc. Epigenetics is of great interest to transform this concept into reality. Epigenetics describes the cellular regulations of genes expression that are heritable without being coded within the DNA sequence: DNA methylation, histones modifications (e.g. methylation and acetylation) and nucleosomes positioning. Epigenetic modifications such as DNA methylation are involved in the control of gene expression and play an important role in pathologies, as for example in cancer.

A general disruption in the epigenetic landscape is virtually associated with every human cancer (M. Esteller, N Engl J Med 358, 1 148; Mar 13, 2008). For instance, the genome of malignant cells is hypomethylated, which is responsible for aberrant expression of repeated sequences such as transposons and indeed global genomic instability. At the same time, most of the tumors harbor very early a specific hypermethylation in the promoter region of tumors-suppressor genes that leads to their silencing. This aberrant inactivation is reversible by blocking the DNA methylation process that is managed by a super-family of enzymes called DNA methyl-transferases (DNMTs) (X. Cheng, R. M. Blumenthal, Structure 16, 341 ; Mar, 2008). In mammals, DNMTs catalyse the transfer of a methyl group from the cofactor S-Adenosyl Methionine (SAM) to the position 5 of cytosines belonging to CpG dinucleotides. Indeed, DNMTs inhibitors have already proven their ability to bring back malignant cells to a more "normal epigenetic state" and thus, to reactivate genes that are essential to fight against tumorigenesis ( C. B. Yoo, P. A. Jones, Nat Rev Drug Discov 5, 37; Jan, 2006). Epigenetic drugs form a very attractive new class of chemotherapy agents.

One distinguishes two types of DNMT inhibitors. Cytosine analogs have been known for twenty years. FDA has approved in 2004 and 2006 5-azacytidine (Vidaza™) and 5-aza-2'-deoxycytidine (Dacogen™), respectively, for the treatment of myelodysplasic syndromes. Cytosine analogues are currently tested against many types of solid tumors. They share a structure analogy and have a unique mode of action, by acting as suicide substrates. Active DNMTs have a catalytic cysteine that forms a transient covalent complex with DNA during its methylation: cytosine analogues block definitively the enzyme at this step of the reaction. Indeed, these molecules need to be integrated into the genome during replication to be active. Even if they show a very promising activity, cytosines analogues are still highly toxic. Furthermore, some of them are chemically instable. More recently a second class of molecules, non-nucleotidic DNMT inhibitors, have been discovered (C. B. Yoo, P. A. Jones, Nat Rev Drug Discov 5, 37; Jan, 2006). Indeed, they show a much broader chemical diversity. Hydralazine is developed as a treatment against brain and ovarian tumors. Other agents like the green tea's EGCG or the RG108 (a molecule discovered through a virtual screening) are studied. All these drugs do not need to be integrated into DNA to be active but their mechanisms remains mainly unknown. Their specificity for the DNMTs is also to be demonstrated.

Since the arsenal of DNMTs inhibitors is still very incomplete, there is an urgent need to design more efficient and less toxic inhibitors.

An aim of the present invention is thus to provide new DNMTs inhibitors.

Another aim of the present invention is to provide a new high throughput assay to screen for DNMTs inhibitors.

The present invention relates to a com ound of formula (I):

wherein:

^■ Ri is chosen from the group consisting of:

- H,

halogen,

(CrC₆)alkoxy,

- -ArCHO, wherein Ai is an alkylene radical comprising from 1 to 12 carbon atoms,

- -A₁-CO₂H, wherein Ai is as defined above, and

- alkenyl comprising from 2 to 12 carbon atoms, and preferably having 3 carbon atoms;

^■ R₂ is chosen from the group consisting of:

- H,

halogen,

- OH,

- N0₂;

^■ R₃ is chosen from the group consisting of:

H, and

- (C C₆)alkoxy,

^■ X is C=O or CHOH;

^■ — is either a single bond or a double bond; ^■ — is either none or a single bond, provided that when— is a double bond, then— is none;

^■ R₄ is OH, N0₂ or a halogen atom;

^■ R₅ ^a is a halogen atom and R₅ ^b is H, or none when— is a double bond;

^■ R₆ is chosen from the group consisting of:

- H,

halogen,

C0₂R, wherein R is an alkyl group comprising from 1 to 12 carbon atoms, and

- C0₂CH₂Ph;

^■ R₇ is chosen from the group consisting of:

- H,

halogen,

- OH,

N0₂ and

- (Ci-C₆)alkoxy;

^■ R₈ is chosen from the group consisting of:

- H,

halogen,

- N0₂,

- OH,

(CrC₆)alkoxy, and

- OCH₂Ph;

^■ R₉ is chosen from the group consisting of:

- H,

(CrC₆)alkoxy, and

^■ R₁₀ is chosen from the group consisting of:

- H,

halogen,

- OH,

(CrC₆)alkoxy, and

- N0₂;

or its pharmaceutically acceptable salts, hydrates, or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers, for its use in the prevention and/or treatment of cancer, developmental diseases, neurodegenerative diseases or Trypanomiasis diseases by inhibition of DNA-methyltransferases.

The term "alkenyl" as employed herein includes partially unsaturated, nonaromatic, hydrocarbon groups having 2 to 12 carbons, preferably 2 to 6 carbons.

The term "alkylene" as employed herein refers to a divalent radical comprising from 1 to 12 carbon atoms, and preferably from 1 to 6 carbon atoms. Said radical may be represented by the formula (CH₂)_n wherein n is an integer varying from 1 to 12, and preferably from 1 to 6.

The term "halogen" refers to the atoms of the group VII of the periodic table and includes in particular fluorine, chlorine, bromine, and iodine atom.

The term "alkoxy" refers to an -O-alkyl radical.

The term "alkyl" means a saturated or unsaturated aliphatic hydrocarbon group which may be straight or branched having about 1 to about 12 carbon atoms in the chain. Preferred alkyl groups have 1 to about 6 carbon atoms in the chain. "Branched" means that one or lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. «Lower alkyl» means about 1 to about 4 carbon atoms in the chain which may be straight or branched. The alkyl may be substituted with one or more «alkyl group substituants», which may be the same or different, and include for instance halo, cycloalkyl, hydroxy, alkoxy, amino, acylamino, aroylamino, carboxy.

The compounds herein described may have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well-known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a compound are intended, unless the stereochemistry or the isomeric form is specifically indicated.

"Pharmaceutically acceptable" means it is, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to salts which retain the biological effectiveness and properties of the compounds of the invention and which are not biologically or otherwise undesirable. In many cases, the compounds of the invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. For a review of pharmaceutically acceptable salts see Berge, et al. ((1977) J. Pharm. Sd, vol. 66, 1 ). The expression "non-toxic pharmaceutically acceptable salts" refers to non-toxic salts formed with nontoxic, pharmaceutically acceptable inorganic or organic acids or inorganic or organic bases. For example, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, fumaric, methanesulfonic, and toluenesulfonic acid and the like.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

While it is possible for the compounds of the invention having formula (I) to be administered alone it is preferred to present them as pharmaceutical compositions. The pharmaceutical compositions, both for veterinary and for human use, useful according to the present invention comprise at least one compound having formula (I) as above defined, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients.

In certain preferred embodiments, active ingredients necessary in combination therapy may be combined in a single pharmaceutical composition for simultaneous administration.

As used herein, the term "pharmaceutically acceptable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified. In particular, the pharmaceutical compositions may be formulated in solid dosage form, for example capsules, tablets, pills, powders, dragees or granules.

The choice of vehicle and the content of active substance in the vehicle are generally determined in accordance with the solubility and chemical properties of the active compound, the particular mode of administration and the provisions to be observed in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets. To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.

The pharmaceutical compositions can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, rectal, nasal, buccal, ocular, sublingual, transdermal, rectal, topical, vaginal, parenteral (including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intraperitoneal. It will be appreciated that the preferred route may vary with for example the condition of the recipient.

The formulations can be prepared in unit dosage form by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Total daily dose of the compounds of the invention administered to a subject in single or divided doses may be in amounts, for example, of from about 0.001 to about 100 mg/kg body weight daily and preferably 0.01 to 10 mg/kg/day. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated. The compounds of the invention are drugs that inhibit DNA methylation and may therefore be useful for the treatment of tumors and proliferative diseases, such as coronary restenosis and neoplastic diseases, particularly colon carcinoma, familiary adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer, prostate carcinoma, melanoma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acute myeolid leukemia (AML), chronic myeloid leukemia (CML), hepatocellular carcinoma, neuroblastoma, intestine carcinoma, rectum carcinoma, colon carcinoma, oesophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tong carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroidea carcinoma, papillary thyroidea carcinoma, renal carcinoma, kidney parenchyma carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, pancreatic carcinoma, testis carcinoma, breast carcinoma, urinary carcinoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, hepatocellular carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, or plasmocytoma.

They may be also used for the treatment of developmental disorders such as Prader-Willi-Syndrome, Angelman-Syndrome (Happy Puppet Syndrome), Beckwith- Wiedemann-Syndrome, and neurodegenerative diseases.

The term "neurodegenerative disease" is used throughout the specification to identify a disease which is caused by damage to the central nervous system and can be identified by neuronal death. Exemplary neurodegenerative diseases include HIV-associated Dementia, multiple sclerosis, Alzheimer's Disease, Parkinson's Disease, amyotrophic lateral sclerosis, schizophrenia and Pick's Disease.

As used herein, the term "neurodegenerative disease" shall be taken to mean a disease that is characterized by neuronal cell death. The neuronal cell death observed in a neurodegenerative disease is often preceded by neuronal dysfunction, sometimes by several years. Accordingly, the term "neurodegenerative disease" includes a disease or disorder that is characterized by neuronal dysfunction and eventually neuronal cell death. Often neurodegenerative diseases are also characterized by increased gliosis (e.g., astrocytosis or microgliosis) in the region/s of neuronal death. Cellular events observed in a neurodegenerative disease often manifest as a behavioral change (e.g., deterioration of thinking and/or memory) and/or a movement change (e.g., tremor, ataxia, postural change and/or rigidity). Examples of neurodegenerative disease include, for example, FTLD, amyotrophic lateral sclerosis, ataxia (e.g., spinocerebellar ataxia or Friedreich's Ataxia), Creutzfeldt-Jakob Disease, a polyglutamine disease (e.g., Huntington's disease or spinal bulbar muscular atrophy), Hallervorden-Spatz disease, idiopathic torsion disease, Lewy body disease, multiple system atrophy, neuroanthocytosis syndrome, olivopontocerebellar atrophy, Pelizaeus-Merzbacher disease, progressive supranuclear palsy, syringomyelia, torticollis, spinal muscular atrophy or a trinucleotide repeat disease (e.g., Fragile X Syndrome).

The family of enzymes 'DNA-methyltransferases' is described in X. Cheng, R. M. Blumenthal, Structure 16, 341 (Mar, 2008). DNA methyltransferases are the enzymes responsible for DNA methylation that in mammals catalyze the transfer of a methyl group from the AdoMet co-factor to the postion 5 of cytidine in a CpG context.

The inhibition of said DNA-methyltransferases may be measured by the test as described below and alternatively as in M. Roth, A. Jeltsch, Biol Chem 381 , 269, 2000.

According to an advantageous embodiment, in the above-mentioned formula (I), R₄ is N0₂ or a halogen atom.

Preferably, in the above-mentioned formula (I), Ri is chosen from the group consisting of: H, halogen such as Br, OCH₃, CH₂CHO, CH₂COOH, and CH₂CH=CH₂.

Preferably, in the above-mentioned formula (I), R₂ is chosen from the group consisting of: H, CI, Br, OH, and N0₂, and more preferably from the group consisting of: H, CI, Br, and N0₂.

Preferably, in the above-mentioned formula (I), R₃ is H or OCH₃.

Preferably, in the above-mentioned formula (I), R₄ is OH, N0₂ or CI, more preferably N0₂ or CI, and most preferably is N0₂.

Preferably, in the above-mentioned formula (I), R₆ is chosen from the group consisting of: H, F, C0₂CH₃, and C0₂CH₂Ph.

Preferably, in the above-mentioned formula (I), R₇ is chosen from the group consisting of: H, CI, OH, N0₂, and OCH₃, and more preferably from the group consisting of: H, CI, N0₂, and OCH₃. Preferably, in the above-mentioned formula (I), R₈ is chosen from the group consisting of: H, F, CI, N0₂, OH, OCH₃, and OCH₂Ph, and more preferably from the group consisting of: H, F, CI, N0₂ and OCH₃.

Preferably, in the above-mentioned formula (I), R₉ is chosen from the group consisting of: H, OCH₃, and C₆H₄-C0₂H.

Preferably, in the above-mentioned formula (I), Ri₀ is chosen from the group consisting of: H, F, CI, OH, OCH₃, and N0₂.

A preferred group of compounds according to the invention are compounds having formula (I) as defined above, wherein at most two, and preferably at most one, of the groups Ri , R₂, and R₃ is other than H.

A preferred group of compounds according to the invention are compounds having formula (I) as defined above, wherein at most three, preferably two, and more preferably at most one, of the groups R₆, R₇, R₈, Rg, and R ₀ is other than H.

The present invention also relates to a compound having formula (1-1 ):

wherein Ri , R₂, R₃, R₄, R₆, R₇, Rs, Rg and Ri₀ are as defined above for formula (l),for its use as mentioned above.

The compounds of formula (1-1 ) are compounds of formula (I) wherein X is C=0, — is a double bond, — is none, and R₅ ^a and R₅ ^b are none.

Preferably, in the above-mentioned formula (1-1 ), R₄ is N0₂ or CI.

The present invention also relates to a compound having formula (I-2):

wherein R_{1 ;} R₂, R₃,

formula (I), for its use as mentioned above.

The compounds of formula (I-2) are compounds of formula (I) wherein X is C=0, — is a single bond,— is a single bond, and R₅ ^b is H. In formula (I-2) above, R₄ is preferably N0₂.

In formula (I-2) above, R₅ ^a is preferably a halogen atom, and in particular is CI. The compounds of formula (I-2) may also be represented by the following formula:

Thus, the present

wherein Ri , R₂, R

for its use as mentioned above.

The compounds of formula (1-2-1 ) may also be represented by the following formula:

A preferred group of

invention are compounds having formula (1-2-1 ) as defined above, wherein at most two, and preferably at most one, of the groups Ri , R₂, and R₃ is other than H.

A preferred group of compounds according to the invention are compounds having formula (1-2-1 ) as defined above, wherein at most three, preferably two, and more preferably at most one, of the groups R₆, R₇, R₈, Rg, and R ₀ is other than H.

Preferably, in formula (1-2-1 ), R₂ and R₃ are H.

Preferably, in formula (1-2-1 ), R_{1 ;} R₂ and R₃ are H.

A group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein Ri is an alkenyl group as defined above, and in particular CH₂CH=CH₂. A subgroup of preferred compounds of the invention consists of those as defined above wherein R₂ and R₃ are H.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein Ri and R₃ are H, and R₂ is halogen, such as CI, N0₂ or alkoxy such as OCH₃.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein Ri and R₂ are halogen, and in particular Br.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein R^ and R₂ are H. A subgroup of this group of preferred compounds consists of compounds wherein R₃ is alkoxy, and preferably OCH₃.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein R^ is alkoxy, and preferably OCH₃. A subgroup of this group of preferred compounds consists of compounds wherein R₂ and R₃ are H.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein R₆ to Ri₀ are H.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein R₆ and R₈ to Ri₀ are H. A subgroup of this group of preferred compounds consists of compounds wherein R₇ is halogen such as CI, N0₂, or alkoxy such as OCH₃.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein R₈ is halogen, and in particular CI, and R₆,

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein three of the groups R₆ to R ₀ are alkoxy such as OCH₃, the two other groups being H.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein at most three of the groups R₆ to R ₀ are alkoxy such as OCH₃, the other groups being H.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein two of the groups R₆ to R ₀ are alkoxy such as OCH₃, the three other groups being H.

Another group of compounds of the invention consists of compounds having formula (1-2-1 ) as defined above wherein one of the groups R₆ to Ri₀ are alkoxy such as OCH₃, the other groups being H.

 The present invention also relates to a compound having formula (I-3):

wherein R_{1 ;} R₂, R₃, R₄, R₅ ^a, R₅ ^b, R₆, R₇, Rs, Rg and R ₀ are as defined above for formula (I), for its use as mentioned above.

The compounds of formula (I-3) are compounds of formula (I) wherein X is CHOH, — is a single bond, and — is a single bond.

A preferred group of compounds of formula (I-3) consists of compounds of formula (I-3) wherein R₅ ^b is H.

Preferably, in the above-mentioned formula (I-3), R₄ is N0₂ or CI.

The compounds of formula (I-3) may also be represented by the following formula:

According to an advantageous embodiment, the present invention also relates to the compound having formula (I-4):

wherein R_{1 ;} R₂, R₃, R₆, R₇, R₈, R₉ and R ₀ are as defined above for formula (I), for its use as defined above.

The compounds of formula (I-4) are compounds of formula (1-1 ) wherein R₄ is

N0₂. A preferred group of compounds according to the invention are compounds having formula (I-4) as defined above, wherein Ri is chosen from the group selected from: CH₂CHO, CH₂C0₂H, and CH₂CH=CH₂.

Another preferred group of compounds according to the invention are compounds having formula (I-4) as defined above, wherein at least one of the groups R₇, R₈, Rg and Ri₀ is a (CrC₆)alkoxy group.

One of the most advantageous groups of compounds according to the invention consists of compounds having formula (I-4) as defined above, wherein:

- Ri is chosen from the group selected from: CH₂CHO, CH₂C0₂H, and CH₂CH=CH₂, and

- at least one of the groups R₇, R₈, R₉ and R ₀ is a (CrC₆)alkoxy group, and preferably OCH₃.

Preferred compounds having formula (I-4) as defined above are compounds having formula (1-4-1 ) as follows:

wherein Ri , R₆, R₇, Rs, Rg and Ri₀ are as defined above.

A preferred group of compounds according to the invention are compounds having formula (1-4-1 ) as defined above, wherein at most two, and preferably at most one, of the groups R₆, R₇, Rs, Rg, and Ri₀ is other than H.

A preferred group of compounds of formula (1-4-1 ) consists of compounds wherein R_7i R₉ or R ₀ is N0₂.

Another preferred group of compounds of formula (1-4-1 ) consists of compounds wherein R₇ is N0₂.

Preferably, in formula (1-4-1 ), is H, CH₂CHO or CH₂C0₂H.

Preferred compounds having formula (I-4) are as follows:

(1 1 ) (19) (23)

(106) (107) Among the compounds useful for the use according to the invention, the compounds having formula (I-5) may also be mentioned:

wherein Ri , R₂, R₃, Re, R₇, Rs, Rg and Ri₀ are as defined above.

The compounds of formula (I-5) are compounds of formula (1-1 ) wherein R₄ is

CI.

A preferred group of compounds according to the invention consists of compounds having formula (I-5), wherein Ri , R₂ and R₃ are H.

Among said compounds having formula (I-5), the following preferred compounds may be mentioned:

R₈ and R ₀ being as defined above.

Preferably, in formula (1-5-1 ), R₈ is N0₂.

Preferably, in formula (I-5-2), R ₀ is halogen, and in particular Preferred compounds having formula (I-5) are as follows:

The present invention also relates to the compound of formula (I-6):

wherein R_{1 ;} R₂, R₃, R₆, R₇, R₈, R₉ and R ₀ are as defined above for formula (I), for its use as mentioned above.

The compounds of formula (I-6) are compounds of formula (I-3) wherein R₄ is N0₂, R₅ ^a is CI and R₅ ^b is H.

A preferred group of compounds according to the invention consists of compounds of formula (I-6) as defined above, wherein Ri , R₂ and R₃ are H.

Another preferred group of compounds according to the invention consists of compounds having formula (I-6) as defined above, wherein R₆, R₈, R₉ and Ri₀ are H.

Among those compounds, the following compounds may be mentioned:

wherein R₇ is as defined above for formula (I).

One of the most advantageous groups of compounds according to the invention consists of compounds of formula (I-6) as defined above, wherein:

- R₆, R₈, R₉ and R ₀ are H.

Another preferred group of compounds according to the invention consists of compounds of formula (I-6) as defined above, wherein R₇ is N0₂.

Advantageous compounds according to the invention are compounds having formula (I-6) as defined above, wherein:

- R₇ is N0₂.

Preferred compounds having formula (I-6) are as follows:

The present invention also relates to the compound having the following formu

for its use as mentioned above.

The present invention relates to the below preferred compounds for their use in the prevention and/or treatment of cancer, developmental disorders, neurodegenerative diseases or Trypanomiasis diseases by inhibition of DNA- methyltransferases:

(23)

(68)

The present invention also relates to a compound of formula (I) as defined above for its use in the prevention and/or the treatment of developmental diseases or Trypanomiasis diseases.

The present invention also relates to a compound of formula (1-4) as defined above for its use in the prevention and/or the treatment of neurodegenerative diseases.

The present invention also relates to a compound of formula (1-2) as defined above for its use in the prevention and/or the treatment of neurodegenerative diseases.

The present invention also relates to a com ound having formula (1-5):

wherein R_{1 ;} R₂, R_3, R₆, R₇, s, Rg, and R ₀ are as defined above for formula (I), or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers.

According to an advantageous embodiment, in formula (I-5), Ri , R₂ and R₃ are

H. According to an advantageous embodiment, in formula (I-5), only one of groups R₆, R₇, R₈, Rg, and Ri₀ are other than H, the others being H. Among those preferred compounds, one may mention compounds having formula (1-5-1 ) or (I-5-2) as mentioned above.

The present invention also relates to compounds having formula (I-4-2) as follows:

wherein R₆, R₇, R₈, R₉, and R ₀ are as defined above for formula (I), or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers.

The compounds of formula (I-4-2) are compounds of formula (1-4-1 ) as defined above wherein is CH₂CHO.

A preferred group of compounds according to the invention are compounds having formula (I-4-2) as defined above, wherein at most two, and preferably at most one, of the groups R₆, R₇, R₈, Rg, and Ri₀ are other than H.

A preferred group of compounds of formula (I-4-2) consists of compounds wherein R₇ is N0₂.

Another preferred group of compounds of formula (I-4-2) consists of compounds wherein R₈ is OCH₂Ph and/or R₉ is C₆H₄COOH.

The present invention also relates to a compound having formula (I-2-3):

wherein i

- H,

halogen,

- (C C₆)alkoxy,

- -ArCHO, wherein Ai is an alkylene radical comprising from 1 to 12 carbon atoms, and - -A1 -CO2H , wherein Ai is as defined above,

and R₂, R₃, Re, R7, Rs, Rg and Ri ₀ are as defined above.

Another group of compounds of the invention consists of compounds having formula (I-2-3) as defined above wherein Ri and R₃ are H, and R₂ is halogen, such as CI, N0₂ or alkoxy such as OCH₃.

Another group of compounds of the invention consists of compounds having formula (I-2-3) as defined above wherein Ri and R₂ are halogen, and in particular Br.

Another group of compounds of the invention consists of compounds having formula (I-2-3) as defined above wherein and R₂ are H. A subgroup of this group of preferred compounds consists of compounds wherein R₃ is alkoxy, and preferably OCH₃.

Another group of compounds of the invention consists of compounds having formula (I-2-3) as defined above wherein is alkoxy, and preferably OCH₃. A subgroup of this group of preferred compounds consists of compounds wherein R₂ and R₃ are H.

The present invention also relates to a compound having formula (I-2-2):

wherein Ri , R₂, R₃, R₆, R₇, Rg and Ri ₀ are as defined above,

or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers.

Preferably, in formula (I-2-2), R₆, R₉ and R ₀ are H.

More preferably, in formula (I-2-2), R₆, R₉ and R ₀ are H and R₇ is halogen such as I.

Preferably, in formula (I-2-2), at most one of the groups Ri , R₂, and R₃ is other than H.

The present invention also relates to the following compounds as such: (47), (48), (50), (52), (58), (59), (60), (61 ), (62), (63), (64), (65), (66), (67), (68), (69), (70), (71 ), (72), (73), (74), (75), (76), (77), (79), (80), (81 ), (82), (83), (1 13), and (1 14).

The present invention also relates to a pharmaceutical composition comprising a compound having formula (I-5), (I-4-2), (I-2-3) or (I-2-2) as mentioned above, in association with at least one pharmaceutically acceptable excipient.

It also relates to a pharmaceutical composition comprising a compound having formula (1 1 ), (38), (59), (60), (82), (99) or (102) as mentioned above, in association with at least one pharmaceutically acceptable excipient.

The present invention also relates to a method for screening a DNA methyltransferase inhibitor comprising the following steps:

- a step of coating a solid support with avidin, streptavidin or neutravidin, - a step of incubating the coated solid support with a double-modified DNA duplex containing a fluorophore at the 5' or 3' end of one strand of the duplex and biotin at the 5' or 3' end of the other strand,

- a step of incubating the support coated with avidin, streptavidin or neutravidin and the double-modified DNA duplex with a DNA methyltransferase and a compound to be tested for its property to inhibit said DNA methyltransferase in the presence of the enzyme cofactor SAM, the compound to be tested and the DNA methyltransferase being added simultaneously or successively, said incubation step being followed by a washing step,

- a step of incubating the support with a restriction enzyme sensitive to the methylation status of the DNA, said incubation being carried out in such conditions that the restriction enzyme is active, said incubation step being followed by a step of washing the support, and

- a step of measuring the fluorescence signal, being understood that said compound is a DNA methyltransferase inhibitor when the measured fluorescence signal is lower than the fluorescence signal measured in the absence of the compound to be tested.

This screening method is based on methylation-sensitive DNA cleaving restriction enzyme and fluorescence measurement (see Figure 1 ).

Each step of the above-mentioned screening method is followed by washing steps, in particular with PBS, eventually in association with Tween 20.

The step of incubating the support with a restriction enzyme is carried out in the presence of a restriction buffer having the appropriate pH and salt concentration.

The method of the invention is based on the detection of the cleavage of a fluorescence labelled DNA duplex by restriction enzymes that are active only if the substrate is not methylated in their specific recognition site containing a CpG. Therefore, a preferred embodiment is as follows: 96-well plates coated through biotin/avidin interactions with a double-modified DNA duplex containing the fluorophore, FAM, at one end and the biotin at the other end. The duplex can be methylated by the DNMT on a single CpG site and then cleaved after a washing step by the appropriate restriction enzyme. When a hit molecule has inhibited the DNMT during the methylation step of the assay, restriction will be active, the cleaved FAM-containing duplex is washed away and one observes a loss of fluorescence signal. On the contrary, if the tested compound is inactive, the DNMT methylates the DNA substrate and protects it against further cleavage. In this case, the fluorescence signal remains intact in the corresponding well of the plate at the end of the assay.

In comparison to previous tests in the literature, the method of the invention presents several technical improvements. Roth and Jeltsch's test (M. Roth, A. Jeltsch, Biol Chem 381 , 269, 2000) is based on the use of radioactivity and it necessitates the release of the DNA target from the surface for reading. In the method of the invention, after a washing step, the fluorescence signal is read immediately without any further step. The high sensitivity and selectivity is given by the restriction enzyme and does not involve the measurement of the radioactive methyl groups incorporated on the DNA substrate. Woo et al. (Y. H. Woo, P. T. Rajagopalan, S. J. Benkovic, Anal Biochem 340, 336, 2005) have developed an ELISA assay for Hhc I DNA MTase that is less efficient, more expensive and more laborious. Li et al. (J. Li, H. Yan, K. Wang, W. Tan, X. Zhou, Anal Chem 79, 1050, 2007) have developed a fluorescence screening assay based on the use of an hairpin duplex containing a fluorophore (TAMRA) at one end and the extinction molecule DABCYL at the other end, and a single CpG site with a restriction enzyme methylation sensitive. In this case, since there are no washing steps between the methylation and restriction reaction fluorescent compounds cannot be screened and false negative may be molecules that inhibit the restriction step or that just bind DNA. Therefore in the present screening, the set-up is such that there are washing steps after each reaction that are fundamental to eliminate all the limitations and drawbacks above and be applicable to all compounds without any restriction.

The assay of the invention can be understood as a high throughput method to find inhibitors of whether methyltransferase activity or a well defined DNMT. The same method can also be applied to other DNA modifying enzymes (such as DNA topoisomerases).

The present invention also relates to a kit for screening a DNA methyltransferase inhibitor, comprising: (1 ) a solid support coated with avidin, (2) a double-modified DNA duplex containing a fluorophore at the 5' end of one strand of the duplex and biotin at the 5' end of the other strand, (3) a DNA methyltransferase and its cofactor SAM, and (4) a restriction enzyme.

The kit of the present invention may also contain instructions for use to carry out the method for screening DNA methyltransferase inhibitors.

It may also comprise PBS, eventually in association with Tween 20, for washing steps, such as PBST (PBS ^"I X - 0.5% Tween 20). It may also comprise a restriction buffer having the appropriate pH and salt concentration, such as a buffer comprising 10 mM Bis-Tris-Propane-HCI, 10 mM MgCI₂, and 1 mM DTT (pH 7.0). It may also comprise a reaction buffer such as a buffer comprising 20 mM HEPES pH 7.2, 50 mM KCI, and 1 mM EDTA).

The invention will be further illustrated in view of the following examples.

SYNTHESIS OF COMPOUNDS

Melting points were measured on a Kofler hot stage apparatus and are uncorrected. Infrared spectra were recorded on a Perkin-Elmer RX 1 FT spectrophotometer as deuteriochloroform solutions or KBr discs. The H-NMR (300 MHz) were recorded on a Varian AC 300 spectrometer. Chemical shifts are expressed as parts per million downfield from tetramethylsilane. Splitting patterns have been designated as follows: s (singlet), d (doublet), dd (doublet of doublet), ddd (doublet of doublet of doublet), t (triplet), dt (doublet of triplet), m (multiplet), br. (broad signal). Coupling constants (J values) are listed in hertz (Hz). Mass spectra were obtained with a Nermag-Ribermag R10-10C spectrometer applying a desorption chemical ionization technique using ammonia as the reagent gas

Example 1 : Synthesis of compound (11 )

The 3-nitro-2-(3-nitrophenyl)-4-oxo-4H-1 -benzopyran-8-acetaldehyde (1 1 ) has been synthesized by ozonolysis starting from 8-allyl-3-nitro-2-(3-nitrophenyl)-4/- -1 - benzopyran-4-one formerly obtained as described in Bauvois, B.; Puiffe M. L; Bongui J. B.; Paillat S.; Monneret C, Dauzonne D. J. Med. Chem. 2003;46(18):3900-3913.

3-Nitro-2-(3-nitrophenyl)-4-oxo-4H-1-benzopyran-8-acetaldehyde (11 )

Yield 65%; mp 172-173^ recrystallized from a benzene/cyclohexane mixture.

IR (CDCI₃) v_max (cm-¹): 3090, 2926, 2833, 1766, 1731 ,1672, 1604, 1541 , 1483, 1445, 1376, 1350, 1 178, 1 139, 1020.

¹H NMR (CDCI₃) δ: 4.10 (s, 2H), 7.57 (t, J = 7.7 Hz, 1 H), 7.70 (d, J = 7.4 Hz, 1 H), 7.77 (t, J = 8.1 Hz, 1 H), 7.97 d, J = 7.9 Hz, 1 H), 8.31 (d, J = 8.0 Hz, 1 H), 8.48 (dd, J_{1 =} 2.0 Hz, J₂= 8.2 Hz, 1 H), 8.57 (br s, 1 H), 9.90 (s, 1 H).

MS (m/z): 355 (M + H)⁺.

Example 2: Synthesis of compound (59)

The chloronitro derivative 59 has been prepared from 3-allyl-2- hydroxybenzaldehyde and (Z)-4-benzyloxy-1 -(2-chloro-2-nitroethenyl)-3-iodo- benzene via the corresponding 3-chloro-3,4-dihydro-4-hydroxy-3-nitro-2H-1 - benzopyrane (Dauzonne, D.; Demerseman P. A convenient synthesis of 3-chloro- 3,4-dihydro-4-hydroxy-3-nitro-2-phenyl-2h-1 -benzopyrans Synthesis 1990, 66-70) according to a previously published procedure (Dauzonne, D.; Grandjean, C. Synthesis of 2-aryl-3-nitro-4H-1 -benzopyran-4-ones. Synthesis 1992, 677-680).

8-Allyl-2-(4-benzyloxy-3-iodophenyl)-3-chloro-2,3-dihydro-3-nitro-4H-1- benzopyran-4-one (59)

Yield 87%; mp 106-107°C recrystallized from a benzene/heptane mixture.

IR (CDCI₃) Vmax (cm ¹): 3070, 2917, 1712, 1599, 1579, 1488, 1478, 1451 , 1344, 1288, 1260, 1213, 1081 , 1044.

¹H NMR (CDCI₃) δ: 3.35-3.53 (m, 2H), 5.02.-5.21 (m, 2H), 5.18 (s, 2H), 5.82- 6.02 (m, 2H), 6.19 (s, 1 H), 6.86 (d, J = 8.6 Hz, 1 H), 7.18 (t, J = 7.7 Hz, 1 H), 7.31 - 7.52 (m, 6H), 7.55 (dd, J_{1 =} 1 .6 Hz, J₂= 7.4 Hz, 1 H), 7.93 (dd, J_{1 =} 1 .7 Hz, J₂= 8.0 Hz, 1 H), 7.99 (d, J = 2.2 Hz, 1 H).

MS (m/z): 576 - 578 (M + H)⁺.

Example 3: Synthesis of compound (60)

The chloronitro derivative 60 has been prepared according to the same procedure as described in example 2 starting from salicylaldehyde and the same (Z)- -chloro- -nitrostyrene.

2-(4-Benzyloxy-3-iodophenyl)-3-chloro-2,3-dihydro-3-nitro-4H-1-benzo pyran-4-one (60)

Yield 94%; mp 178-179°C recrystallized from a benzene/heptane mixture.

IR (KBr) Vmax (cm ¹): 3038, 1712, 1605, 1567, 1497, 1474, 1464, 1407, 1393, 1341 , 1291 , 1248, 1221 , 1 152

¹H NMR (CDCI₃) δ: 5.18 (s, 2H), 6.19 (s, 1 H), 6.85 (d, J = 8.6 Hz, 1 H), 7.17- 7.50 (m, 8H), 7.69 (ddd, J₁ = 1 .7 Hz, J₂ = 7.3 Hz, J₃ = 8.5 Hz, 1 H), 8.03 (d, J = 1 .8 Hz, 1 H), 8.06 (dd, J₁ = 1 .4 Hz, J₂ = 8.0 Hz, 1 H).

MS (m/z): 536 - 538 (M + H)⁺.

Example 4: Synthesis of compound (82)

The chloronitro derivative 82 has been prepared according to the same procedure as described in example 2 starting from salicylaldehyde and (Z)-1 -(2- chloro-2-nitroethenyl)-2,4-dimethoxybenzene. 3- Chloro-2,3-dihydro-2-(2,4-dimethoxyphenyl)-3-nitro-4H-1-benzopyran-4-one (82)

Yield 85%; mp 1 13-1 1 °C recrystallized from a benzene/heptane mixture. IR (CDCI₃) Vmax (cm ¹): 3010, 2967, 2941 , 2841 , 1710, 1608, 1581 , 1510, 1466, 1296, 1226, 1212, 1 162, 1 153, 1 127, 1 1 16, 1068, 1037.

¹H NMR (CDCI₃) δ: 3.72 (s, 3H), 3.84 (s, 3H), 6.42 (d, J = 2.4 Hz, 1 H), 6.65 (s, 1 H), 7.13 (dd, J₁ = 0.6 Hz, J₂ = 8.4 Hz, 1 H), 7.22 (ddd, J₁ = 1 .0 Hz, J₂ = 7.4 Hz, J₃ = 8.1 Hz, 1 H), 7.65 (ddd, J₁ = 1 .7 Hz, J₂ = 7.2 Hz, J₃ = 8.7 Hz, 1 H), 7.74 (d, J = 8.7 Hz, 1 H), 8.07 (dd, J₁ = 1 .7 Hz, J₂ = 7.9 Hz, 1 H).

MS (m/z): 364 - 366 (M + H)⁺.

Example 5: Synthesis of compound (38)

The synthesis of the 2'-benzyloxy-5'-(3-nitro-4-oxo-4H-chromen-2-yl)biphenyl-

4- carboxylic acid (38) has been carried out using a three-step procedure starting from 60 (example 3). Formation, under basic conditions, of the 2-(4-benzyloxy-3- iodophenyl)-3-nitro-4H-1 -benzopyran-4-one in 98% yield followed by Pallado- catalyzed condensation of this iodoflavone with 4-formylphenylboronic acid in the presence of potassium carbonate provided the 2'-benzyloxy-5'-(3-nitro-4-oxo-4H- chromen-2-yl)biphenyl-4-carbox-aldehyde in 75% yield. Subsequent oxidation of this latter formyl derivative with oxone in acidic medium gave the wanted acid 38 in 93% yield.

2'-Benzyloxy-5'-(3-nitro-4-oxo-4H-chromen-2-yl)biphenyl-4-carboxylic acid (38)

MP: 252-255 ^°C.

IR(KBr) Vmax (cm ¹): 3200, 2889, 1695, 1660, 1618, 1601 , 1569, 1524, 1499,

1466, 1374, 1361 , 1273, 1 1 19.

¹H NMR (DMSO-d₆) δ: 5.30 (s, 2H), 7.30-7.42 (m, 5H), 7.49 (brd, J = 9.4 Hz,

1 H), 7.59-7.70 (m, 3H), 7.77-7.80 (m, 2H), 7.86-8.00 (m, 4H), 8.16 (brd, J = 7.8 Hz,

1 H), 13.01 (s, 1 H)

MS (m/z): 494 (M + H)⁺.

Example 6: Synthesis of compounds (99) and (102)

The 3-chloroflavones 99 and 102 have been isolated as minor by-products (4% and 5%, respectively) in large scale synthesis of the corresponding 3- nitroflavones (Dauzonne, D.; Folleas, B.; Martinez, L; Chabot, G. G. Synthesis and in vitro cytotoxicity of a series of 3-aminoflavones. Eur. J. Med. Chem. 1997, 32, 71 - 82.)

3-Chloro-2-(4-nitrophenyl)- 4H-1-benzopyran-4-one (99)

Mp 196-197^<Ό recrystallized from a benzene/heptane mixture.

IR (CDCI₃) Vmax (cm ¹): 1652, 1618, 1608, 1595, 1526, 1492, 1468, 1348, 1230, 1217, 1 1 10, 1086.

¹H NMR (CDCI₃) δ: 7.47-7.59 (m, 2H), 7.77 (ddd, J₁ = 1 .6 Hz, J₂ = 7.1 Hz, J₃ =

8.7 Hz, 1 H), 8.09-8.15 (m, 2H), 8.31 (dd, J₁ = 1 .6 Hz, J₂ = 8.0 Hz, 1 H), 8.37-8.44 (m, 2H).

MS (m/z): 302 - 304 (M + H)⁺.

3-Chloro-2-(2-fluorophenyl)- 4H-1-benzopyran-4-one (102)

mp 189-190°C recrystallized from a benzene/heptane mixture

IR (CDC ) Vmax (cm ¹): 1766, 1740, 1668, 1605, 1488, 1478, 1458, 1326, 1301 , 1286, 1244, 1227, 1 178, 1 158, 1 144, 1 108, 1097, 1053, 1020.

¹H NMR (CDCI₃) δ: 7.18-7.39 (m, 4H), 7.58-7.67 (m, 1 H), 7.75-7.86 (m, 2H),

8.08 (dt, J₁ = 1 .8 Hz, J₂ = 7.6 Hz, 1 H).

MS (m/z): 275 - 277 (M + H)⁺.

Example 7: Synthesis of compounds (113) and (114)

Compounds 113 and 114 were synthesized starting from the appropriate reagents according to the methodology depicted in B. Bauvois et al., J Med Chem 46, 3900 (Aug 28, 2003).

3-Chloro-2,3-dihydro-2-(3-fluorophenyl)-3-nitro-4H-1 -benzopyran-4-one (113): Yield 95%; mp 123.5 -124.5^<€ recrystallized from heptane, IR (CDCI₃) v_max (cm ¹): 1713, 1609, 1580, 1475, 1466, 1451 , 1293, 1223, 1212, 1 153, 1 1 17, 1070. H NMR (CDCI₃) δ: 6.30 (s, 1 H), 7.15-7.47 (m, 6H), 7.71 (ddd, J₁ = 1 .7 Hz, J₂ = 7.2 Hz, J₃ = 8.4 Hz, 1 H), 8.07 (dd, J₁ = 1 .5 Hz, J₂ = 7.8 Hz, 1 H). MS (m/z): 322 - 324 (M + H)+.

[3-Chloro-2-(2-fluorophenyl)-3-nitro-4-oxo-chroman-8-yl]acetic acid (114):

Yield 72%; mp 210-212°C recrystallized from toluene. IR (KBr) v_max (cm ¹): 1714, 1580, 1351 . ¹H NMR (DMSO-d₆) δ: 3.68 (s, 2H,), 6.97 (s, 1 H), 7.27-7.41 (m, 3H), 7.54 -7.62 (m, 1 H), 7.44 -7.81 (m, 2H), 7.90-7.94 (dd, 1 H, J, = 1 .7Hz, J₂ = 8.0Hz); 12.50 (s,1 H), exchangeable with D₂0). MS (m/z): 380 -382 (M + H)⁺.

Example 8: Other compounds

Compounds 46 and 51 are described in D. Dauzonne, Demerseman. P., Synthesis, 66 (1990). Compounds 41 , 47, 48, 52, 64, 65, 66, 67, 68, 69, 70, 74, 75, 76, 77, 78, 79, 80 and 83 are described in D. Dauzonne, C. Grandjean, Synthesis, 677 (1992).

Compounds 45, 53, 61 , 62, 63, 71 , 72, 73 and 81 are described in D. Dauzonne, B. Folleas, L. Martinez, G. G. Chabot, Eur. J. Med. Chem. 32, 71 (1997)

Compounds 19, 23, 44, 50, 54, 55, 56, 57, 106 and 107 in B. Bauvois et al., J Med Chem 46, 3900 (Aug 28, 2003).

Compound 58 is described in M. Pham et al., Drug Metab. Dispos. 35, 2023 (2007).

5-azacytidine was bought from Calbiochem and conserved in water at -20 °C. All the drugs are dissolved in 100% DMSO and conserved at -20 °C.

SCREENING METHOD

Substrate design:

Short DNA duplexes have been made upon hybridization of complementary oligonucleotides (Eurogentec, Belgium) bearing a FAM and a biotin, respectively at the 5' end of each strand. The substrates have a single CpG site included in a flanking sequence that is simultaneously adequate for the DNA methyltransferase and the methylation-sensible restriction enzyme. The substrates for the assay with DNTM3A/3L, M.Sss I and DNMT1 have respectively the following sequences (the CpG site is in bold):

DNTM3A/3L and M.Sss I:

FAM- 5' GCTATATATACGTACTGTGAACCCTACCAGACATGCACTG 3' (SEQ ID NO: 1 ) 3' CGATATATATGCATGACACTTGGGATGGTGTGTACGTGAC 5'-biotin (SEQ ID NO: 2)

FAM - 5' AGCCCGGGTAGGGTTCA 3' (SEQ ID NO: 3)

3' TCGGGCCCATCCCAAGT 5' - biotin (SEQ ID NO: 4) DNTM1 :

FAM - 5' GCATATATATGAmCGATCCTGTAGGTCACTACCAGACATGCACTG 3' (SEQ ID NO: 10) 3' CGTATATATACTGCTAGGACATCCAGTGATGGTCTGTACGTGAC 5 -biotin (SEQ ID NO: 1 1 ) wherein mC is 5-methyl-deoxycytidine

Plate coating:

Costar™ 96-Well high binding EIA/RIA plates (ref. 9018) have been coated with 1 μρ/ννθΙΙ of avidin (Sigma) in 100 μΙ_ of 100 mM NaHC0₃, pH 9.60 at 4°C overnight. The plate has been then washed five times with PBST (PBS 1 X - 0.5% Tween 20), 500 mM NaCI. The plate can be stored 2 weeks at 4°C. To further coat the substrate on the plate, 100pmol/well of DNA has been incubated in 100 μΙ_ of PBST at room temperature for at least 30 min. The plate is finally ready-to-use after being washed three times with PBST, 500 mM NaCI and three times with PBST. DNMT3a/3L methylation reactions:

The C-terminal catalytic domain of the murine DNMT3a (623-908) and the C- terminal domain of DNMT3L (208-421 ), obtained as described in (Jia, D., Jurkowska, R.Z., Zhang, X., Jeltsch, A. and Cheng, X. (2007). Nature 449, 248-51 .) have been preincubated together at 200 nM during 20 minutes at room temperature in reaction buffer (20 mM HEPES pH 7.2, 50 mM KCI, 1 mM EDTA) in presence of the tested compound in total volume of 55 μΙ-Λ/vell of a Greiner™ 96 well V-form transfer microplate. Tested compounds can be added whether after the preincubation of the enzymatic mix or during this complex formation step in each well to reach optimal inhibition.

50 μΙ_ out of total 57 μΙ_ of each well in the transfer plate has been transferred into the corresponding well of the testing plate, coated with the DNA substrate. Freshly dissolved AdoMet (Sigma) has been added to start the reaction at the final concentration of 20 μΜ. Methylation has been achieved during 1 hour at 37 ^<C. Each well has been then washed three times with PBST (PBS 1 X - 0.5% Tween 20), 500 mM NaCI and three times with PBST.

Methylation reaction by DNMT1

The human GST-tagged DNMT1 was bought from BPS Bioscience. The enzyme was incubated at 350 nM in reaction buffer (20 mM HEPES pH 7.2, 50 mM KCI, 1 mM EDTA) and 20 μΜ SAM in the presence of the tested compound in a total volume of 50 μΙ_/ννβΙΙ of a testing plate. The methylation reaction was achieved at 37°C during 120 min. Each well was washed as described.

Methylation conditions with M.Sss I :

DNA methylation by the bacterial CpG methyltransferase M.Sss I (New England Biolabs) in the presence of the tested compound in a total volume of 50 μΙ_/ννβΙΙ during 1 hour at 37°C was realized in the following conditions: 50 nM M.Sssl, 20 μΜ SAM, 10 mM Tris-HCI pH 7.9, 50 mM NaCI, 10 mM MgCI₂, 1 mM DTT. Each well was washed as described.

Restriction conditions:

DNMT3a/3L assay and M.Sssl assay: 2 units/well of the methylation-sensible restriction enzyme HpyCH4 IV (New Englands Biolabs) have been incubated in total 50 μΙ_ volume of restriction buffer (10 mM Bis-Tris-Propane-HCI, 10 mM MgCI₂, 1 mM DTT, pH 7.0) during 1 hour at 37 ^<C. The plate has been then washed three times with PBST, 500 mM NaCI and three times with PBST.

M.Sssl assay: 2 units/well of the methylation-sensible restriction enzyme Hpa II (New Englands Biolabs) have been incubated in 50 μΙ_ total of the same restriction buffer. Washing conditions are the same as above.

DNMT1 assay: 2 units/well of the methylation-sensible restriction enzyme BfuCI (New Englands Biolabs) have been incubated in 50 μΙ_ total of the same restriction buffer. Washing conditions are the same as above.

Fluorescence detection and quantification, quality control :

The fluorescence signal of the DNA substrate has been measured on a Typhoon™ scanner (Amersham™). Fluorescence has been measured after the microplate coating with DNA to check if the amount of substrate is adequate, after methylation and after completion of the reaction. Up to ten plates can be scanned in a single scan. Quantification has been done automatically by measuring the sum of pixels in each well.

Two different quality indicators have been used to calibrate each experiment. The first one is the global percentage of methylation defined as 100^*((average signal of methylation controls - average signal of restriction controls)/( average signal of DNA controls)).

The second one is the Z-factor described in (J. H. Zhang, T. D. Chung, K. R. Oldenburg, J Biomol Screen 4, 67, 1999). IC₅o values have been evaluated graphically through the Kaleidagraph™ software.

For the determination of the IC₅₀ (concentration of drug needed to obtain 50% of inhibition), each experimental set was performed in triplicate and the results were plotted as relative methylation activity against the log of inhibitor concentration. IC₅₀ values were evaluated after fitting the dose-response plots by non-linear regression through the GraphPad Prism™ software with constrained variable slope equations.

Mean IC₅o are given ± the standard error defined as— , where SD_sampie is the

(^H -1)

standard deviation of the observed IC₅₀ values and n the number of experiments Complementary enzymatic tests:

The inhibitory effect of the hit molecules on the purified C-terminal domains of Dnmt3a and Dnmt3L has been tested also by an in vitro radioactive methylation assay. DNA methylation activity of the complex is measured by the incorporation of tritiated methyl groups from labeled S-[methyl-³H] AdoMet (specific activity 370 GBq / mmol, Perkin Elmer) into a biotinylated, hemi-methylated oligonucleotide substrate (biotin - GAAGCT GGACAG TAMeCGTC AAGAGA GTGCAA / TTGCAC TCTCTT GACGTA CTGTCC AGCTTC)(SEQ ID NO: 5 / SEQ ID NO: 6) using the avidin-biotin methylation kinetic assay as described (M. Roth, A. Jeltsch, Biol Chem 381 , 269, 2000). The methylation reactions were carried out in the methylation buffer (20 mM HEPES, pH 7.5, 50 mM KCI, 1 mM EDTA, 25 μg/mL bovine serum albumin (BSA)), using 2 μΜ DNA, 5.5 μΜ of labeled AdoMet and 0.5 μΜ of both proteins. Decreasing amounts of inhibitor in DMSO (500 μΜ, 250 μΜ, 100 μΜ, 50 μΜ, 20 μΜ, 1 μΜ, no inhibitor) were pre-incubated with Dnmt3a/C and Dnmt3L/C at room temperature for 30 min. The methylation reactions were started by the addition of substrate DNA and allowed to proceed for 5 min. Similar experiments were carried out with the purified bacterial EcoDam N-6 DNA methyltransferase (J. R. Horton, K. Liebert, M. Bekes, A. Jeltsch, X. Cheng, J Mol Biol 358, 559, 2006) and the catalytic domain of the human G9a histone H3K9 methyltransferase (P. Rathert, A. Dhayalan, H. Ma, A. Jeltsch, Mol Biosyst 4, 1 186, 2008). For EcoDam, 0.5 μΜ enzyme and 2 μΜ DNA (biotin - GACAGG CCGMeATC ACTGTCGC / GCGACA GTGATC GGCCTGTC)(SEQ ID NO: 7 / SEQ ID NO: 8) were used and the reaction was carried out in the EcoDam methylation buffer (100 mM HEPES pH 8.0, 1 mM EDTA, 0.5 mM DTT, 0.2 mg/mL BSA); whereas for G9a, 0.1 μΜ of enzyme, 20 μΜ of the histone H3 peptide tail (biotin - MARTKQTARKSTGGKAPRKQ)(SEQ ID NO: 9) were used and the methylation buffer contained 50 mM Tris/HCI pH 9.0, 5 mM MgCI₂, 4 mM DTT. Each experimental set was performed in triplicate and the results were plotted as relative methylation activity (expressed in %) against the inhibitor concentration. To obtain the apparent inhibition constant (Ki) data were fitted to the following equation: signal (Ci) = BL+1 OOxKi / (Ki+Ci) where BL is baseline, Ci is the concentration of inhibitor and Ki is the apparent inhibition constant.

Cellular studies:

Human cancer cells (DU145, LNCaP, PC3, HCT1 16 and MCF7) were purchased from ATCC. These cell lines were grown in the RPMI1640, DMEM or MEM medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg mL streptomycin at 37^<C with 5% C0₂.

Cells were seeded in 6-wells plate and treated between 3 and 10 days with the compounds that were removed 12h before harvesting. 5azacytidine was used at 1 μΜ and added every day, compounds 1 1 and 23 were used at 100 μΜ and compounds 47 and 62 at 50 μΜ. They were added only once.

Radioactive enzymatic tests:

DNA methylation activity of the DNMT3a/3L complex was measured by the incorporation of tritiated methyl groups from labeled S-[methyl-³H] SAM (specific activity 2.9 TBq/mmol, Perkin Elmer) into a DNA duplex substrate containing 8 CpG sites, with the following sequence 5 'AGGGGACGAAGGAGGGAAGGAAGGGC- AAGGCGGGGGGGGCTCTGCGAGAGCGCGCCCAGCCCCGCCTTCGGGCCCCA CAG (SEQ ID NO: 12). The methylation reactions were carried out in the methylation buffer (20 mM HEPES pH 7.2, 50 mM KCI, 1 mM EDTA), using 200 nM DNA, 280 nM of radiolabeled SAM and 0.5 μΜ of both proteins.

Similar experiments were carried out using the purified bacterial EcoDam N-6 DNA methyltransferase and the catalytic domain of the human G9a histone H3K9 methyltransferase, as described in (Horton, J.R., Liebert, K., Bekes, M., Jeltsch, A. & Cheng, X. Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase. J Mol Biol 358, 559-70 (2006)) and (Rathert, P., Cheng, X. & Jeltsch, A. Continuous enzymatic assay for histone lysine methyltransferases. Biotechniques 43, 602, 604, 606 passim (2007)), respectively. Each experimental set was performed in triplicate and the results were plotted as relative methylation activity (expressed in %) against the inhibitor concentration. To obtain the apparent inhibition constant (IC₅₀) data were fitted to the following equation : signal (C,) = BL+I OOx IC₅₀/( IC₅₀+Ci) where BL is baseline, C, is the concentration of inhibitor and IC₅₀ is the concentration to obtain 50% of apparent inhibition.

Zebrafish experiments

Fertilized eggs were obtained from natural mating of adult zebrafish maintained under standard conditions (Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 3rd Edition, (Eugene, OR, University of Oregon Press, 385 (Book), 1 995)). Embryos were incubated in an aqueous solution of the various substrates (from stage 1 6 cells to analysis). Embryos were manually dechorionated before malformation scored or 5mC detected with a specific antibody (anti-5-methylcytosine antibody, Calbiochem). Errors bars are statistical errors estimated as (Vp(1 -pJ/n) where p is the percentage of embryos exhibiting a phenotype and n the total number of embryos investigated (or Vl/n when p=0 or 1 ). For immunohistochemistry, embryos were treated as previously described (MacKay, A.B., Mhanni, A.A., McGowan, R.A. & Krone, P.H. Immunological detection of changes in genomic DNA methylation during early zebrafish development. Genome 50, 778-85 (2007)) with minor modifications (after incubation with the anti-5-methylcytosine antibody, the embryos were washed again and incubated first with biotinylated anti-mouse secondary antibody (GE Healthcare) and then with streptavidine fluorescein (GE Healthcare).

Results of the screening method

The C-terminal domains of the murine DNMT3a and DNMT3L have been preincubated together with each tested compound in a transfer 96 well microplate for 25 minutes. Next the wells have been transferred into the testing plate that has been previously coated with the DNA fluorescent substrate. SAM has finally been added to start the methylation reaction. After one hour of incubation at 37 °C, plates have been washed to avoid drugs interference with next steps of the assay. Then a restriction mix has been added in each well. After 30 min of incubation at 37°C, plates have been washed again and final signal has been measured and quantified by a Typhoon™ scanner.

The screening of 1 14 molecules gave insight about the advantages of such a new assay. These molecules had a potential interest because some show chemical proximity with genistein, a soybean isoflavone that has an antiangiogenic activity. Genistein is an inhibitor of several enzymatic activities: tyrosine kinases, topoisomease II and DNMTs.

1 14 flavones and flavanones derivates have been tested for their ability to inhibit the DNMT3a/3L complex.

The screen of the first 52 molecules at 500 μΜ detected several active compounds against the DNMT3a/3L complex. The IC₅₀ of ten hit molecules have been evaluated (in triplicate at least) through the same assay but using drugs concentration ranges in each line of the 96 well plate. Compounds n °46 and 47 were the most active inhibitors of this first screening with promising submicromolar IC₅₀ (concentration at which 50% of inhibition is observed) of 417 nM and 169 nM, respectively. The chemical structures of these two compounds and other hit molecules share the same chloro-nitro motif at the C3 position in the ring B of the flavanone skeleton. Then 55 parent compounds harboring the same or similar chemical characteristic have been screened, leading to 27 new hit molecules, the majority of them showing submicromolar activity. In table 1 below are reported the structure and IC₅₀ of the most potent compounds. At this step, the best molecule displayed a very promising in vitro activity with IC₅₀ = 85 nM ± 8% (compound 62).

The most interesting compounds were tested in a classical methylation assay based on the incorporation of radioactively labeled ³H-SAM in a DNA duplex. The results are globally comparable since the best molecules in the screening assay (bearing the 3-chloro-3-nitro motif) are also the best in the radioactive assay.

Table 1 - Comparison of IC₅₀ of some interesting compounds measured on

Genistein >500 >100

n.d. = not determined

The selectivity and mechanism of action of these new potential DNMT3a/3L inhibitors by complementary experimental approaches have also been studied.

First, the HTS was applied to the human DNMT1 and the bacterial methyltransferase M. Sssl. The chemical library was screened at 5 μΜ. Globally, a similar profile was observed on DNMT1 and M.Sssl as on the catalytic DNMT3a/3L complex with little differences.

Then it was investigated whether they could affect the activity of other methyltransferase activities in order to get insights in the mechanism of inhibition. The most representing compounds were tested against the histone methyltransferase (HMTK) G9a that methylates histone tails on their lysine residues (Rathert et al., 2007) and the bacterial DNA (adenine N-6)-methyltransferase EcoDam that methylates DNA at GATC sites (Urig, S. et al. The Escherichia coli dam DNA methyltransferase modifies DNA in a highly processive reaction. J Mol β/Ό/ 319, 1085-96 (2002)). A biotin/avidin microplate kinetic assay based on ³H-SAM incorporation was used for the quantitative analysis of methyltransferases activities in vitro (Roth, M. & Jeltsch, A. Biotin-avidin microplate assay for the quantitative analysis of enzymatic methylation of DNA by DNA methyltransferases. Biol Chem 381 , 269-72 (2000)). Interestingly, the flavone 11 showed a very good specificity for the DNMT3a/3L complex compared to the other two methyltransferases, with an apparent IC₅₀ of 15 μΜ against DNMT3a/3L and of 352 μΜ against G9a HMTK and 290 μΜ against EcoDam. The nitro-chloro flavanone 62 showed specificity for the DNA methyltransferases (IC₅₀ = 14.8, 131 .4 and 6.5 μΜ for DNMT3a/3L, G9a HMTK and EcoDam, respectively). On the other hand, the nitro analogs 70 and 47 were less selective with an apparent IC₅o = 9-5 and 4.5 μΜ against DNMT3a/3L, more efficient against EcoDam with IC₅₀ = 2.2 and 0.9 μΜ and finally showed an excellent inhibition of the HMTK G9a, IC₅₀ = 0.5 and 1 μΜ, respectively.

Molecular docking studies were carried out on the three enzymes. Compounds 11 , 47 62, 69, 70 and 71 were docked with Dock 6.4 on the crystal structure of the murine catalytic DNMT3a/3L complex (PDB: 2QRV)( Jia, D., Jurkowska, R.Z., Zhang, X., Jeltsch, A. & Cheng, X. Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature 449, 248-51 (2007)). Since the flavanones exist in the racemic form, both enantiomers (R,S) and (S,R) were docked. Clearly, all compounds were found in the catalytic pocket, either in the SAM pocket or closer to the DNA pocket. Interestingly, flavone derivative 11 made a H-bond with an amino acid close to the catalytic cysteine. Differences were observed between the enantiomers of the flavanones: the (S,R) molecules stretched in the DNA pocket whereas the (R,S) molecules tended to be docked in the SAM pocket, except for 47 and 71 for which both enantiomers were found closer to the DNA pocket. Strikingly, the N0₂ group at the C3 position shared by all the compounds was always located in the same pocket formed by Pro705, Phe636, Gly 638, Glu660 and Arg 887. This was also found for all enantiomers. Interestingly, the potent inhibitors 69 and 71 made a H-bond with the catalytic cysteine in position 706. Analog docking studies were done in the crystal structure of EcoDam (PDB: 2G1 P) and G9a (PDB: 3K5K). In this case, the minimal conformation for all compounds corresponded to the one in the SAM pocket of the enzymes.

Since the docking studies suggested that the compounds should have a mixed inhibition mechanism, partially competing with the SAM cofactor as sinefungin, the apparent K_m and V_max of the DNMT3a/3L complex were determined in the presence of the inhibitors as a function of the SAM concentration. Indeed, compounds 11 , 47 and 62 gave typical plots of a mixed inhibition model: with increasing concentration of the inhibitor the apparent K_m increased and the V_max decreased, proving that the molecule had a different affinity for the DNMT3a/3L complex depending whether or not a SAM molecule was bound. This result has two major implications. First, the DNMTs inhibitors of the invention are not pure SAM competitors. Since SAM is the common cofactor that provides the methyl group of all methyltransferase activities in the cell, SAM competition would be unfavorable for selectivity. Second, the mixed inhibition profile is characteristic for a binding inside the active site of the enzyme.

Finally, it is known that inhibition of DNA methylation at the blastula period induces the loss of tail and abnormal patterning of somites in zebrafish (Martin, C.C., Laforest, L, Akimenko, M.A. & Ekker, M. A role for DNA methylation in gastrulation and somite patterning. Dev Biol 206, 189-205 (1999)). In this context, it has been chosen to investigate the biological effect of the molecules 11 , 23, 47, 62 and 69 on the development of zebrafish. Embryos were treated at 16-cells stage with different concentration of the drugs directly added in embryo medium in 24 wells plates. The phenotype was analyzed 6, 24 and 48 h after treatment. 5- azactidine (5-azaC) was used as a positive control and embryos incubated with vehicle (DMSO) as a negative control. It has been found that 100 μΜ 5-azaC (n = 240) induced the expected aberrant morphology: short or absent tail (59%) as well as abnormal somites in comparison to DMSO treated embryos that induced no remarkable phenotype in 90% of the embryos (n =185). At 25 μΜ, compounds 47 and 62 killed all the embryos (n= 40 and 58). Strikingly at 2.5 μΜ these molecules (n=223 and 237) induced a phenotypic profile comparable to the one observed upon 5-azaC treatment: a third of the embryos had no or short tails. Interestingly, genistein induced only high cytotoxicity (13.5%) and a growth delay (36.5%). Compound 69, the most active on the purified catalytic DNMT3a/3L complex, did not give any phenotype at 2.5 μΜ. Global DNA methylation was assessed by fluorescent immunolabelling with an antibody directed against 5-Me-dC. A decrease in signal was observed upon treatment with 5-aza-C and compounds 62 and 47. Compounds 81 and 69 did not affect global methylation.

In conclusion, most of the compounds of the invention showed submicromolar activity in vitro and were well more potent than the references compounds RG108 and genistein (IC₅o of 315 μΜ and > 500 μΜ, respectively). Compound 69, a nitro- chloro flavanone derivate, was the most active molecule with an apparent IC₅₀ = 370 nM. Docking studies located it in the catalytic pocket of DNMT3a, as it is the case for most of the tested molecules in this study.

SAR studies showed the importance of the nitro on position 3 of the flavones and the nitro-chloro motif on the position 3 of the flavanones. In agreement, docking studies suggested that this N0₂ is always positioned in a pocket close to (< 5 A) amino acids Pro705, Phe636, Gly 638, Glu660 and Arg 887. SAR investigations also revealed the positive influence of electron-withdrawing substituents on the ring B, the highest effect was observed with a N0₂ in ortho (compound 69). A potential H bond with the catalytic cytosine 706 was found in the minimal conformation by docking. A synergy was also observed when electron-withdrawing groups are on ring B and A (compound 71 ).

Docking studies and SAM competition experiments strongly indicated that the DNMT inhibitors of the invention have a mix inhibition profile, since they interact both with the DNA and SAM pocket. Furthermore, kinetics experiments with the methyltransferase G9a HMTK and the bacterial DNMT EcoDam showed a good specificity of compound 11 for the DNMT3a/3L complex, a specificity of compound 62 for the DNA methyltransferases and a poorer selectivity of compounds 47 and 70. Interesting, nitro-flavone 11 is the most specific compound for DNMT3a/3L and its minimal energy conformation is found mainly in the DNA pocket, differently from what is observed with all the other compounds. However it is also the less potent inhibitor with an IC50 of 9.5 μΜ.

For the biological activity of the compounds, the most active inhibitors were tested for their effect on zebrafish development. In fact, zebrafish is getting more and more used as a cancer model (Feitsma, H. & Cuppen, E. Zebrafish as a cancer model. Mol Cancer Res 6, 685-94 (2008)). In particular, great epigenetic changes occur in the first steps of development of the zebrafish embryo (Mhanni, A. A. & McGowan, R.A. Global changes in genomic methylation levels during early development of the zebrafish embryo. Dev Genes Evol 214, 412-7 (2004)). For example, it has been recently demonstrated that the no tail gene, which is necessary for notochord and tail formation undergoes a dynamic regulation of its expression by specific de novo methylation of its CpG island (Yamakoshi, K. & Shimoda, N. De novo DNA methylation at the CpG island of the zebrafish no tail gene. Genesis 37, 195-202 (2003)). Interestingly, compounds 47 and 62 gave the same short axis phenotype as 5-azaC on zebrafish embryo development. Furthermore, fluorescent immunolabeling detecting 5-methyl-deoxycytidine (5- MedC) confirmed these results: treatment with 5-azaC or compounds 47 and 62 induced a decrease in the fluorescence signal associated with 5-MedC.

Finally, the screening method of the invention is robust, versatile and can be applied to all types of methyltransferases. This method has several technical improvements compared to the HTS of the literature. It does not use radioactivity and allows direct readout of the fluorescent signal, after washing and restriction digest. Woo et al. (Woo, Y.H., Rajagopalan, P.T. & Benkovic, S.J. A nonradioactive DNA methyltransferase assay adaptable to high-throughput screening. Anal Biochem 340, 336-40 (2005)) have developed a non-radioactive ELISA assay for Hhcl DNA MTase activity that is however less efficient, more expensive and more laborious to perform. Li et al. (Li, J., Yan, H., Wang, K., Tan, W. & Zhou, X. Hairpin fluorescence DNA probe for real-time monitoring of DNA methylation. Anal Chem 79, 1050-6 (2007)) developed a fluorescence screening assay based on the use of an hairpin duplex containing a fluorophore (TAMRA) at one end and the extinction molecule DABCYL at the other end, and a single CpG site within the recognition site for a methylation sensitive restriction enzyme. Recently a HTS based on a comparable system not using DNA hairpins was published (Ye, Y. & Stivers, J.T. Fluorescence-based high-throughput assay for human DNA (cytosine-5)- methyltransferase 1 . Anal Biochem 401 , 168-72 (2010)). In these tests, the main limitation is that the washing steps cannot be used (the hairpin or double modified DNA duplex would be washed away). Thus, fluorescent drugs can interfere with the final quantification of fluorescence signal and yield false positives and negatives, since the tested compounds remain in each well throughout the assay. In addition, certain compounds might also inhibit the restriction step, increasing further the occurrence of false negative results. This hairpin-based test has another technical difficulty: the DNMT(s) of interest and the appropriate methylation-sensitive restriction enzyme must be compatible in terms of reaction buffer, which is not always possible and affects the versatility of the method. Surface immobilization of the DNA substrate in the screening method of the invention enables to do extensive washing at every step, preventing all these drawbacks.

Claims

1. A compound of formula I):

wherein:

^■ Ri is chosen from the group consisting of:

- H,

halogen,

- (C C₆)alkoxy,

- -ArCHO, wherein Ai is an alkylene radical comprising from 1 to 12 carbon atoms,

- -A₁-CO₂H, wherein Ai is as defined above, and

- alkenyl comprising from 2 to 12 carbon atoms, and preferably having 3 carbon atoms;

^■ R₂ is chosen from the group consisting of:

- H,

halogen,

- OH, and

- N0₂;

^■ R₃ is chosen from the group consisting of:

H, and

- (C C₆)alkoxy,

^■ X is C=O or CHOH;

^■ — is either a single bond or a double bond;

^■ — is either none or a single bond, provided that when— is a double bond, then— is none;

^■ R₄ is OH, N0₂ or a halogen atom;

^■ R₅ ^a is a halogen atom and R₅ ^b is H, or none when— is a double bond;

^■ R₆ is chosen from the group consisting of: - H,

halogen,

- C0₂R, wherein R is an alkyl group comprising from 1 to 12 carbon atoms, and

- C0₂CH₂Ph;

^■ R₇ is chosen from the group consisting of:

- H,

halogen,

- OH,

N0₂ and

- (Ci-C₆)alkoxy;

^■ R₈ is chosen from the group consisting of:

- H,

halogen,

- N0₂,

- OH,

(CrC₆)alkoxy, and

- OCH₂Ph;

^■ R₉ is chosen from the group consisting of:

- H,

(CrC₆)alkoxy, and

CeH₄-C0₂H;

^■ Rio is chosen from the group consisting of:

- H,

halogen,

- OH,

(CrC₆)alkoxy, and

- N0₂;

or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers,

for its use in the prevention and/or treatment of cancer, developmental diseases, neurodegenerative diseases or Trypanomiasis diseases by inhibition of DNA-methyltransferases.

2. The compound having formula (1-1 ):

wherein R_{1 ;} R₂, R₃, R₄, R₆, R₇, Rs, Rg and R ₀ are as defined in claim 1 , for the use according to claim 1 .

3. The compound having formula (I-2):

wherein Ri , R₂, R

for the use according to claim 1 .

4. The compound having formula (I-2) for its use according to claim 3, wherein R₄ is N0₂ and R₅ ^a is CI.

5. The compound having formula (I-3):

wherein R_{1 ;} R₂, R₃,

for the use according to claim 1 .

6. The compound having formula (1-4):

wherein R_{1 ;} R₂, R₃

for the use according to claim 1 .

7. The compound having formula (I-4) for its use according to claim 6, wherein is chosen from the group selected from: CH₂CHO, CH₂C0₂H, and CH₂CH=CH₂.

8. The compound having formula (I-4) for its use according to claim 6 or 7, wherein at least one of the groups R₇, R₈, R₉ and R ₀ is a (CrC₆)alkoxy group.

9. The compound having formula (I) or (1-1 ) for its use according to claim 1 or 2, having formula (I-5):

wherein Ri , R₂, R₃, R₆, R₇, R₈, R₉ and Ri₀ are as defined in claim 1 .

10. The compound having formula (I-5) for its use according to claim 9, wherein R _; R₂ and R₃ are H.

wherein Ri , R₂, R₃, Re, R₇, Rs, Rg and Ri ₀ are as defined in claim 1 , for its use according to claim 1 .

A compound having formula (I-4-2)

wherein:

^■ R₆ is chosen from the group consisting of:

- H ,

halogen,

- C0₂R, wherein R is an alkyl group comprising from 1 to 12 carbon atoms, and

- C0₂CH₂Ph;

^■ R₇ is chosen from the group consisting of:

- H ,

halogen,

- OH ,

N0₂ and

(CrC₆)alkoxy;

^■ R₈ is chosen from the group consisting of:

- H ,

halogen,

- N0₂,

- OH ,

(CrC₆)alkoxy, and

- OCH₂Ph;

^■ R₉ is chosen from the group consisting of:

- H ,

(CrC₆)alkoxy, and

^■ Ri o is chosen from the group consisting of:

- H , halogen,

- OH,

(CrC₆)alkoxy, and

- N0₂;

wherein at least one of R₆, R₇, R₈, Rg and Ri₀ is other than H,

or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers.

A compound having formula (I-2-3)

^■ Ri is chosen from the group consisting of:

- H,

halogen,

(CrC₆)alkoxy,

- -ArCHO, wherein Ai is an alkylene radical comprising from 1 to 12 carbon atoms, and

- -A₁-CO₂H, wherein Ai is as defined above;

^■ R₂ is chosen from the group consisting of:

- H,

halogen,

- OH, and

- N0₂;

^■ R₃ is chosen from the group consisting of:

H, and

- (C C₆)alkoxy,

^■ R₆ is chosen from the group consisting of:

- H,

halogen,

- C0₂R, wherein R is an alkyl group comprising from 1 to 12 carbon atoms, and - C0₂CH₂Ph;

^■ R₇ is chosen from the group consisting of:

- H,

halogen,

- OH,

N0₂ and

(CrC₆)alkoxy;

^■ R₈ is chosen from the group consisting of:

- H,

halogen,

- N0₂,

- OH,

(CrC₆)alkoxy, and

- OCH₂Ph;

^■ R₉ is chosen from the group consisting of:

- H,

(CrC₆)alkoxy, and

- C₆H₄-C0₂H; and

^■ R₁₀ is chosen from the group consisting of:

- H,

halogen,

- OH,

(CrC₆)alkoxy, and

- N0₂;

or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers.

A compound having formula (I-2-2)

wherein:

Ri is chosen from the group consisting of: - H ,

halogen,

(CrC₆)alkoxy,

- -A1 -CHO, wherein Ai is an alkylene radical comprising from 1 to 12 carbon atoms,

- -A1 -CO2H , wherein Ai is as defined above, and

- alkenyl comprising from 2 to 12 carbon atoms, and preferably having 3 carbon atoms;

■ R₂ is chosen from the group consisting of:

- H ,

halogen,

- OH , and

- N0₂;

■ R₃ is chosen from the group consisting of:

- H , and

- (d-C₆)alkoxy,

■ R₆ is chosen from the group consisting of:

- H ,

halogen,

- C0₂R, wherein R is an alkyl group comprising from 1 to 12 carbon atoms, and

- C0₂CH₂Ph;

■ R₇ is chosen from the group consisting of:

- H ,

- halogen,

- OH ,

N0₂ and

- (Ci -C₆)alkoxy;

■ R₉ is chosen from the group consisting of:

- H,

(CrC₆)alkoxy, and

■ R ₀ is chosen from the group consisting of:

- H ,

- halogen,

- OH , (CrC₆)alkoxy, and

- N0₂;

or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers.

15. A pharmaceutical composition comprising a compound according to any one of claims 12 to 14, in association with at least one pharmaceutically acceptable excipient.

16. A method for screening a DNA methyltransferase inhibitor comprising the following steps:

- a step of coating a solid support with avidin, streptavidin or neutravidin,

- a step of incubating the coated solid support with a double-modified DNA duplex containing a fluorophore at the 5' or 3' end of one strand of the duplex and biotin at the 5' or 3' end of the other strand,

- a step of incubating the support coated with avidin, streptavidin or neutravidin and the double-modified DNA duplex with a DNA methyltransferase and a compound to be tested for its property to inhibit said DNA methyltransferase in the presence of the enzyme cofactor SAM, said incubation step being followed by a washing step,

- a step of incubating the support with a restriction enzyme, said incubation being carried out in such conditions that the restriction enzyme is active, said incubation step being followed by a step of washing the support, and

- a step of measuring the fluorescence signal, being understood that said compound is a DNA methyltransferase inhibitor when the measured fluorescence signal is lower than the fluorescence signal measured in the absence of the compound to be tested.

17. A kit for screening a DNA methyltransferase inhibitor, comprising: (1 ) a solid support coated with avidin, streptavidin or neutravidin, (2) a double-modified DNA duplex containing a fluorophore at the 5' or 3' end of one strand of the duplex and biotin at the 5' or 3' end of the other strand, (3) a DNA methyltransferase and its cofactor SAM, and (4) a restriction enzyme.