OF DNA METHYLTRANSFERASES

The present invention concerns new inhibitors of DNA methyltransferases as well as preparation process thereof.

Epigenetic modifications are known to control gene expression. In mammals, methylation of deoxycytidines was shown to be a major factor of the epigenetic regulation and occurs at CpG sites, which are enriched in so-called CpG islands often located in genes promoters (R. S. Illingworth, A. P. Bird, FEBS Lett 2009, 583, 1713; S. L. Berger, T. Kouzarides, R. Shiekhattar, A. Shilatifard, Genes Dev 2009, 23, 781 ; M. Weber, I. Hellmann, M. B. Stadler, L. Ramos, S. Paabo, M. Rebhan, D. Schubeler, Nat Genet 2007, 39, 457). Noteworthy, hypermethylation of the promoters induces gene silencing. The addition of a methyl group on the carbon-5 position of the cytosine is catalysed by the C5 DNA methyltransferases (DNMT)( X. Cheng, R. M. Blumenthal, Structure 2008, 16, 341 ; .M. G. Goll, T. H. Bestor, Annu Rev Biochem 2005, 74, 481 ; A. Jeltsch, Curr Top Microbiol Immunol 2006, 301, 203). Mammalian DNMTs are divided in two major families: DNMT1 , DNMT3A, 3B and 3L. At the exception of DNMT3L, which is catalytically inactive, all share a common catalytic mechanism using S-adenosyl-L-methionine (SAM) as methyl donor. The impairment of epigenetic information can compromise the normal development of an organism and is involved in various diseases, such as cancer (M. Esteller, N Engl J Med 2008, 358, 1 148; S. Sharma, T. K. Kelly, P. A. Jones, Carcinogenesis 2010, 31, 27). Cancerous cells often present two types of aberrant DNA methylation: a global hypomethylation leading to genomic instability, and a specific hypermethylation of the promoters of certain genes, such as tumor suppressor genes, which silences them. Interestingly, epigenetic modifications are reversible and it has been shown that inhibitors of DNMTs are able to demethylate the promoters and reactivate tumor suppressor genes (N. Yu, M. Wang, Curr Med Chem 2008, 15, 1350; A. Mai, L. Altucci, Int J Biochem Cell Biol 2009, 41, 199; M. Szyf, Annu Rev Pharmacol Toxicol 2009, 49, 243).

Today, two families of DNMT inhibitors are known (C. B. Yoo, P. A. Jones, Nat Rev Drug Discov 2006, 5, 37): nucleoside and non-nucleoside analogues. Among the latter, procainamide, used in last thirty years as antiarrhytmic, was shown to induce significant demethylation of genomic DNA in human cancer cells together with its analogue procaine - an anaesthetic (B. H. Lee, S. Yegnasubramanian, X. Lin, W. G. Nelson, J Biol Chem 2005, 280, 40749; E. Cornacchia, J. Golbus, J. Maybaum, J. Strahler, S. Hanash, B. Richardson, J Immunol 1988, 140, 2197). These compounds are described to bind to CpG sequences and thereby disturb the DNA methylation (A. Villar-Garea, M. F. Fraga, J. Espada, M. Esteller, Cancer Res 2003, 63, 4984; S. Castellano, D. Kuck, M. Sala, E. Novellino, F. Lyko, G. Sbardella, J Med Chem 2008, 51, 2321 ).

An aim of the present invention is to provide new DNA methyltransferases inhibitors, said inhibitors having an improved activity of inhibition.

The present invention relates to a com ound having the following formula (I):

wherein:

- Ri and R ₂ are independently from each other chosen from alkyl groups having from 1 to 12 carbon atoms, or may form together with the nitrogen atom bearing them a N-cycloalkyl group having from 3 to 12 carbon atoms,

- R ₃ , R ₄ , R5 and R ₆ are independently from each other chosen from the group consisting of: H, halogen, nitro, R _a , OR _a , SR _a , NR _a R _b , COR _a , CONR _a R _b and COOR _a , R _a and R _b being independently from each other H or an alkyl group having from 1 to 12 carbon atoms,

- X ₂ is -CH ₂ - or -CH2-CH2-X3-, X ₃ being O or NH,

- n is an integer comprised from 2 to 20, and

- R is an inhibitor of DNA methyltransferases,

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

The compounds of the present invention are procainamide / procaine derivatives having an activity of inhibition of DNA methyltransferases.

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.

In particular, in the present invention, procainamide was used as DNA binder to guide an inhibitor of DNMT to CpG rich regions, in order to increase its local concentration at CpG sites. This strategy has already been successfully used to direct the action of DNA topoisomerases I and II inhibitors to specific DNA sites (P.

B. Arimondo, C. Bailly, A. S. Boutorine, V. A. Ryabinin, A. N. Syniakov, J. S. Sun, T. Garestier, C. Helene, Angew Chem Int Ed Engl 2001 , 40, 3045; P. B. Arimondo, A. Boutorine, B. Baldeyrou, C. Bailly, M. Kuwahara, S. M. Hecht, J. S. Sun, T. Garestier, C. Helene, J Biol Chem 2002, 277, 3132; M. Duca, D. Guianvarc'h, K. Oussedik, L. Halby, A. Garbesi, D. Dauzonne, C. Monneret, N. Osheroff, C. Giovannangeli, P. B. Arimondo, Nucleic Acids Res 2006, 34, 1900; K. Oussedik, J.

C. Francois, L. Halby, C. Senamaud-Beaufort, G. Toutirais, S. Dallavalle, Y. Pommier, C. Pisano, P. B. Arimondo, FASEB J 2010; V. Stierle, M. Duca, L. Halby, C. Senamaud-Beaufort, M. L. Capobianco, A. Laigle, B. Jolles, P. B. Arimondo, Mol Pharmacol 2008, 73, 1568).

The term "alkyl" means a saturated or unsaturated aliphatic hydrocarbon group which may be straight or branched having 1 to 12, and preferably 1 to 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 1 to 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, cycloalkyi, hydroxy (OH), alkoxy, amino (NH ₂ ), acylamino (NHCOAlk), aroylamino (NHCOAr), carboxy (COOH).

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

The term "cycloalkyi" as employed herein includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom capable of substitution may be substituted by a substituent. Examples of cycloalkyi moieties include, but are not limited to, cyclohexyl and adamantyl.

The term "halo" refers to the atoms of the group 17 of the periodic table (halogens) and includes in particular fluorine, chlorine, bromine, and iodine atom.

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.

Preferably, in formula (I), Ri and R ₂ are identical, and most preferably they represent an ethyl group.

According to a particular embodiment of the present invention, in formula (I), Ri and R ₂ may form a ring together with the nitrogen atom bearing them. In particular, N, R and R ₂ may form a piperidine ring.

Preferably, in formula (I), R ₃ to R ₆ are H.

According to another preferred embodiment, in formula (I), at least one of R ₃ to R ₆ is other than H. Most preferably, one of R ₃ to R ₆ is N0 ₂ or CI.

Preferably, in formula (I), one of R ₃ to R ₆ is N0 ₂ or CI and the three other groups are H.

Preferably, in formula (I), X ₂ is -CH ₂ -. A preferred group of compounds of the present invention have the following formula (1-1 ):

n, Ri to R ₆ and R being as defined in formula (I).

Preferably, in formula (1-1 ), R ₃ to R ₆ are H.

Compounds of formula (1-1 ) are derivatives of procainamide and correspond to compounds of formula (I) wherein X is -NH- and X ₂ is -CH ₂ -.

Another preferred group of compounds of the present invention have the following formula

Preferably, in formula (I-2), R ₃ to R ₆ are H.

Compounds of formula (I-2) are derivatives of procaine and correspond to compounds of formula (I) wherein is -O- and X ₂ is -CH ₂ -.

The present invention also relates to a com ound having the following formula

wherein:

n and R are as defined above in formula (I).

Compounds of formula (I I) correspond to compounds of formula (I) wherein R and R ₂ are ethyl, R ₃ to R ₆ are H, is -NH- and X ₂ is -CH ₂ -.

In formula (I), (1-1 ), (I-2) or (I I), R is chosen from the known DNA methyltransferases inhibitors.

According to a particular embodiment, R is chosen from the group consisting of: S-adenosine methionine or analogues thereof, indole derivatives such as N- phthalyl-L-tryptophan (RG1 08), phthalimide derivatives, cytosine or derivatives thereof such as 5-azacytidine, polyphenol derivatives such as epigallocatechin 3- gallate (EGCG), and phthalazine derivatives such as hydralazine.

R may also be chosen among flavones and flavanones derivatives such as those described in the European patent application 09 305 840.2. According to a preferred embodiment, in formula (I), R is a phthalimide derivative (for example containing nitro, dimethylamino, methoxy, dimethoxy, methyl).

Preferably, R may be represented by one of the following formulae:

wherein R to R ₆ are aryl substituents, preferably nitro, amine (such as dimethylamino), alcoxy (such as methoxy, dimethoxy), and alkyl (such as methyl).

According to a preferred embodiment, the compounds of the present invention have the formula (I), (1-1 ), (I-2) or (II) wherein R comprises a phthalimide group.

A particularly preferred group of compounds of the invention consists of

wherein X is a bond or a group - NHCOCH(R')-, R' having the below formula:

Compounds of formula (III) correspond to compounds having formula (I) wherein R and R ₂ are ethyl, R ₃ to R ₆ are H, X is -NH-, X ₂ is -CH ₂ -, and R is a group of formula:

Another preferred group of compounds of the invention consists of compounds having the following formula (IV):

Compounds of formula (IV) correspond to compounds having formula (I) wherein and R ₂ are ethyl, R ₃ to R ₆ are H, is -NH-, X ₂ is -CH ₂ -, and R is a group of formula:

Most preferably, in formula (IV), n is comprised from 4 to 14, and is preferably 10 or 12.

Preferably, the present invention also relates to a compound having the following

Compounds of formula (V) correspond to compounds having formula (I) wherein and R ₂ are ethyl, R ₃ to R ₆ are H, is -NH-, X ₂ is -CH ₂ -, and R is a group of formula:

Most preferably, in formula (V), n is 10 or 12.

The present invention also relates to a drug comprising a compound as defined above having formula (I). It also relates to a drug comprising a compound as defined above having formula (II), (III), (IV) or (V).

The present invention also relates to a pharmaceutical composition comprising a compound having formula (I), in association with at least one pharmaceutically acceptable excipient. It also relates to a pharmaceutical composition comprising a compound having formula (1-1 ), (I-2), (II), (III), (IV) or (V), in association with at least one pharmaceutically acceptable excipient. The present invention also relates to the compound of formula (I) for its use for the prevention and/or the treatment of cancer, parasitic diseases or neurological disorders.

The present invention also relates to the compound of formula (1-1 ), (I-2), (II), (III), (IV) or (V) for its use for the prevention and/or the treatment of cancer, parasitic diseases or neurological disorders.

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.

As parasitic diseases, one may in particular cite Trypanomiasis diseases, and more particularly sleeping sickness (or Human African Trypanosomiasis) and Chagas disease (or American Trypanosomiasis). In particular, the compounds of the invention are potent antiparasite agent against Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense, as well as against Trypanosoma cruzi. Neglected tropical diseases continue to cause significant morbidity and mortality in the developing world. Of the 1 ,556 new drugs approved between 1975 and 2004, 21 (1 .3%) were specifically developed for tropical diseases and tuberculosis, even though these diseases account for 1 1 .4% of the global disease burden. Among them, sleeping sickness or Human African Trypanosomiasis (HAT) is endemic in 36 African countries and around 60 million people are at risk of being infected. The disease is caused by two trypanosome subspecies, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, a parasitic disease transmitted by the tsetse fly in sub-Saharan Africa. Existing treatments of the disease are toxic (5% inducing death) or difficult to administer. Pentamidine and, in a lesser extent, suramine are used at the first stage of the sickness (haemolymphatic phase). Pentamidine injections protect against gambiense, but not against rhodesiense. At the second stage (neurological phase), melarsoprol, a highly toxic and dangerous agent, is administrated, and more recently eflornithine, for gambiense only. Today it is urgent to find a new treatment less toxic and more efficient, and to bypass the resistance that is currently appearing. Therefore there is an urgent need to discover new chemical entities against this neglected disease, in addition to an efficient prevention. Based on the inhibition of DNA methylation, the inventors have shown that the compounds of the invention have interesting antiproliferative properties on T brucei gambiense.

American trypanosomiasis or Chagas disease is transmitted by the parasite Trypanosoma cruzi transmitted to humans by blood-sucking triatomine bugs (http:/7wwv¾ ^f .cdc,gov/chagas/gen Jnfo/index.htmi). It affects central and south America, with 40 % of the disease present in Brazil. Antiparasitic treatment is most effective early in the course of infection. Drugs of choice include azole or nitro derivatives such as benznidazole (RADANIL ^® ) or nifurtimox (LAMPIT ^® ). Both agents have severe side-effects and are not able to induce a complete elimination of T cruzi from the body, especially in chronically infected patients, and resistance to these drugs has been reported.

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 present invention also relates to a method for preparing the compound having formula (I), comprising the reaction of a nosyl derivative having the following formula (VI):

P , Pi2, Xi , P13, P14, P15, and R ₆ being as defined above in formula (I),

with the bromo compound having the following formula (VII):

R, n and X ₂ being as defined above in formula (I).

The synthesis of the compounds of the invention is carried out as a one pot reaction.

Preferably, the reaction of (VI) with (VII) is carried out in presence of a weak base, such as any weak inorganic or organic base, and in particular CsC0 ₃ , K ₂ C0 ₃ , Na ₂ C0 ₃ , triethylamine, imidazole, and pyridine. Preferably, this reaction is carried out in K ₂ CO ₃ .

The method of the invention also comprises a treatment step with thiophenol and water.

The compound of formula (VI) is obtained from the reaction of 2- nitrobenzylsulfonyl chloride with a compound having formula (VIII) as follows:

PM , PI ₂ , R ₃ , R ₄ , R ₅ , R ₆ and X being as defined above in formula (I).

The preparation of (VI) is preferably carried out in dichloromethane at room temperature. This preparation step may also be carried out in DMF or acetonitrile. EXAMPLES

CHEMICAL SYNTHESIS OF COMPOUNDS OF THE INVENTION

Materials

The solvents are obtained from Carlo-Erba-SDS; 1 ,14-dibromotetradecane is obtained from Epsilon Chimie (Brest) and the other reagents are obtained from Sigma.

Example 1 : Synthesis of compounds (8)-(13)

The phtalimido derivatives of procainamide 8 to 13 are compounds of formula (IV) wherein n is 2, 4, 6, 8, 10 or 12, respectively.

Scheme 2. a) K ₂ C0 ₃ , Kl, CH ₃ CN 18h reflux then thiophenol, K ₂ C0 ₃ , CH ₃ CN 3h reflux, 79% for n=2, 82% for n=4, 75% for n=6, 83% for n=8, 89% for n=10, 85% for n=12.

Synthesis of intermediate compound 2

N-(2-(diethylamino)ethyl)-4-(2-nitrophenylsulfonamido)benzam ide 2

To a solution of procainamide 1 (hydrochloride salt) (5g; 18.5mmol)(Sigma) in dichloromethane (50ml_) and TEA (5ml_) was added 2-nitrophenylsufoxide chloride (5g; 23mmol) and DMAP (0.25g; 2mmol). The mixture was stirred overnight at room temperature. The solvent was removed by vacuum and the residue was purified by flash chromatography silica gel with dichloromethane/methanol(ammoniac 1 .75%) 9:1 to give 2 as a pale yellow amorphous solid (hygroscopic) (4.3g; yield 55%).

¹ H NMR (400 MHz, DMSO) δ 8.36 (m, 1 H _p11 ), 7.94 (m, 1 H _n3 ), 7.82 (m, 1 H _n6 ), 7.71 (m, 2H _n4+n5 ), 7.64 (d, J = 8.7 Hz, 2H _p8 ), 7.01 (d, J = 8.7 Hz, 2H _p9 ), 3.44 (m, 2H _p4 ), 2.94 (m, 6H _p2+p3 ), 1 .10 (t, J = 7.2 Hz, 6H _p1 ).

¹³ C (100 MHz, DMSO) δ 171 .5 (C _p6 ), 153.3 (C _p7 ), 151 .3 (C _n2 ), 139.4 (Cm), 138.4 (C _n5 ), 137.1 (C _n4 ), 134.9 (C _n3 ), 133.4 (C _n6 ), 131 .8 (C _p10 ), 129.2 (C _p8 ), 124.4 (C _p7 ), 55.8 (C _p4 ), 52.0 (C _p3 ), 40.6 (C _p2 ), 14.9 (C _p1 )

HRMS-ESI(m/z) calculated for Ci ₉ H ₂₄ N ₄ 0 ₅ S [M+H] ⁺ : 421 .15402; Found: 421 .15374.

Synthesis of compounds 8-13

To a solution of the nosylate 2 (160 mg; 0.38 mmol) in dry acetonitrile (3 mL) was added K ₂ C0 ₃ (160 mg; 1 .16 mmol), a catalytic amount of Kl and the desired bromoalkylpthtalimide (0.19 mmol). The mixture was refluxed overnight then thiophenol (50 μΙ_; 0.48 mmol) was added and the solution was heated at 80 °C for 2h. Solvent was removed and the residue was purified by flash chromatography silica gel with dichloromethane/methanol (ammonia 1 .75%) 98:2 as eluent.

Compounds 8 to 13 are compounds of above formula (A) wherein n is 0, 2, 4, 6, 8 or 10, respectively.

N-(2-(diethylamino)ethyl)-4-(3-(1 ,3-dioxoisoindolin-2-yl)butylamino) benzamide 8

(n= 0) from 54 mg of N-(4-bromobutyl)phthalimide, compound 8 was obtained as a yellow oil with a yield of 79%.

¹ H NMR (400 MHz, CDCI ₃ ) δ 7.77 (m, 2H _ph3 ), 7.64 (m, 2H _ph4 ), 7.54 (dm, J = 8.7Hz, 2H _p8 ), 6.71 (m, 1 H _p5 ), 6.48 (dm, J = 8.7Hz, 2H _p9 ), 4.02 (m, 1 H _p11 ), 3.66 (t, J =7.2Hz, 2H _I5 ), 3.38 (dd, J = 5.4, 1 1 .4 Hz, 2H _p4 ), 3.13 (m, 2H _n ), 2.56 (t, J = 6.1 Hz, 2H _p3 ), 2.49 (q, J = 7.2Hz, 4H _p2 ), 1 .74 (m, 2H _I4 ), 1 .60 (m, 2H _I2 ), 0.97 (t, J = 7.3Hz, 6H _p1 ).

¹³ C (100 MHz, CDCI ₃ ) 168.5 (C _ph1 ), 167.2 (C _p6 ), 150.7 (C _p7 ), 134.0 (C _ph4 ), 132.0 (C _ph2 ), 128.6 (Cpe), 123.3 (C _ph3 ), 122.9 (C _p10 ), 1 1 1 .7 (C _p9 ), 51 .5 (C _p3 ); 46.8 (C _p2 ), 43.0 (C _n ), 37.6 (C _l5 ), 37.1 (C _p4 ), 26.5; 26.2 (C _l2+I4 ), 12.0 (C _p1 ).

HRMS-ESI (m/z) calculated for C ₂₅ H ₃₃ 0 ₃ N ₄ [M+H] ⁺ : 437.25472; Found: 437.25487.

N-(2-(diethylamino)ethyl)-4-(3-(1 ,3-dioxoisoindolin-2-yl)hexylamino) benzamide 9

(n= 2) from 59 mg of N-(6-bromohexyl)phthalimide, compound 9 was obtained as a pale yellow oil with a yield of 82%. ¹ H NMR (400 MHz, CDCI ₃ ) δ 7.75 (m, 2H _ph3 ), 7.62 (m, 2H _ph4 ), 7.52 (dm, J= 8.7Hz, 2H _p8 ), 6.68 (m, 1H _p5 ), 6.44 (dm, J= 8.7Hz, 2H _p9 ), 3.98 (m, 1H _p11 ), 3.61 (t, J=7.2Hz, 2H _I5 ), 3.37 (dd, J= 5.4, 11.4 Hz, 2H _p4 ), 3.04 (m, 2H _n ), 2.55 (t, J= 6.1Hz, 2H _p3 ), 2.47 (q, J= 7.2Hz, 4H _p2 ), 1.55 (m, 4H _I2+I4 ), 1.31 (m, 4H _I3 ), 0.95 (t, J= 7.3Hz, 6H _p1 ).

¹³ C (100 MHz, CDCI ₃ ) 168.5 (C _ph i), 167.3 (C _p6 ), 150.9 (C _p7 ), 133.9 (C _ph4 ), 132.1 (C _ph2 ), 128.6 (C _p8 ), 123.2 (C _ph3 ), 122.7 (C _p10 ), 111.6 (C _p9 ), 51.5 (C _p3 ); 46.8 (C _p2 ), 43.3 (C _n ), 37.8 (C _l5 ), 37.1 (C _p4 ), 29.1; 28.5; 26.5 (C _l2+I3+I4 ), 12.0 (C _p1 ).

HRMS-ESI (m/z) calculated for C ₂₇ H ₃₇ 0 ₃ N ₄ [M+H] ⁺ : 465.28602; Found: 465.28588.

N-(2-(diethylamino)ethyl)-4-(3-(1,3-dioxoisoindolin-2-yl) octylamino) benzamide 10

(n= 4) from 64 mg of N-(8-bromooctyl)phthalimide, compound 10 was obtained as a yellow foam with a yield of 75%.

¹ H NMR (400 MHz, CDCI ₃ ) δ 7.75 (m, 2H _ph3 ), 7.62 (m, 2H _ph4 ), 7.54 (dm, J= 8.7Hz, 2H _p8 ), 6.68 (m, 1H _p5 ), 6.47 (dm, J= 8.7Hz, 2H _p9 ), 3.95 (m, 1H _p11 ), 3.60 (t, J=7.2Hz, 2H _I5 ), 3.38 (dd, J= 5.5, 11.5 Hz, 2H _p4 ), 3.04 (m, 2H _n ), 2.56 (t, J= 6.1Hz, 2H _p3 ), 2.48 (q, J= 7.3Hz, 4H _p2 ), 1.55 (m, 4H _I2+I4 ), 1.31 (m, 8H _I3 ), 0.95 (t, J= 7.3Hz, 6H _p1 ).

¹³ C (100 MHz, CDCI ₃ ) 168.5 (C _ph1 ), 167.3 (C _p6 ), 151.0 (C _p7 ), 133.9 (C _ph4 ), 132.2 (C _ph2 ), 128.6 (C _p8 ), 123.1 (C _ph3 ), 122.7 (C _p10 ), 111.6 (C _p9 ),51.6 (C _p3 ); 46.8 (C _p2 ), 43.5 (Cn), 38.0 (C _l5 ), 37.1 (C _p4 ), 29.3-26.7 (C _l2+I3+I4 ), 12.0 (C _p1 ).

HRMS-ESI(m/z) calculated for C ₂₉ H ₄₁ 0 ₃ N ₄ [M+H] ⁺ : 493.31732; Found: 493.31695.

N-(2-(diethylamino)ethyl)-4-(3-(1,3-dioxoisoindolin-2-yl) decylamino) benzamide 11

(n= 6) from 70 mg of N-(10-bromodecyl)phthalimide, compound 11 was obtained as a pale yellow solid with a yield of 83%.

¹ H NMR (400 MHz, CDCI ₃ ) δ 7.76 (m, 2H _ph3 ), 7.63 (m, 2H _ph4 ), 7.55 (d, J= 8.7Hz, 2H _p8 ), 6.70 (m, 1H _p5 ), 6.49 (d, J= 8.8Hz, 2H _p9 ), 3.95 (m, 1H _p11 ), 3.60 (t, J =7.2Hz, 2H _I5 ), 3.38 (dd, J= 5.6, 11.3 Hz, 2H _p4 ), 3.05 (dd, J= 6.8, 12.3Hz, 2H _n ), 2.55 (t, J = 5.9Hz, 2H _p3 ), 2.47 (q, J= 7.1Hz, 4H _p2 ), 1.55 (m, 4H _I2+I4 ), 1.22 (m, 12H _I3 ), 0.95 (t, J = 7.1Hz, 6H _p1 ).

¹³ C (100 MHz, CDCI ₃ ) 168.5 (C _ph1 ), 167.3 (C _p6 ), 151.0 (C _p7 ), 133.9 (C _ph4 ), 132.2 (C _ph2 ), 128.6 (C _p e), 123.1 (C _ph3 ), 122.6 (C _p10 ), 111.6 (C _p9 ),51.5 (C _p3 ); 46.8 (C _p2 ), 43.5 (Cn), 38.0 (C _l5 ), 37.1 (C _p4 ), 29.4-26.8 (C _l2+I3+I4 ), 12.0 (C _p1 ).

HRMS-ESI (m/z) calculated for C ₃ iH ₄₅ 0 ₃ N ₄ [M+H] ⁺ : 521.34862; Found: 521.34819.

N-(2-(diethylamino)ethyl)-4-(3-(1,3-dioxoisoindolin-2-yl) dodecylamino) benzamide 12

(n= 8) from 75 mg of N-(12-bromododecyl)phthalimide, compound 12 was obtained as a pale yellow solid with a yield of 89%.

¹ H NMR (400 MHz, CDCI ₃ ) δ 7.74 (m, 2H _ph3 ), 7.62 (m, 2H _ph4 ), 7.54 (d, J= 8.7Hz, 2H _p8 ), 6.66 (m, 1H _p5 ), 6.48 (d, J= 8.7Hz, 2H _p9 ), 3.97 (m, 1H _p11 ), 3.59 (t, J =7.2Hz, 2H| ₅ ), 3.38 (dd, J= 5.6, 11.5 Hz, 2H _p4 ), 3.05 (m, 2H _n ), 2.56 (t, J= 6.0 Hz, 2H _p3 ), 2.47 (q, J= 7.1Hz, 4H _p2 ), 1.55 (m, 4H _I2+I4 ), 1.23 (m, 16H _I3 ), 0.96 (t, J= 7.1 , 6H _p1 ).

¹³ C (100 MHz, CDCI ₃ ) 168.4 (C _ph1 ), 167.2 (C _p6 ), 151.0 (C _p7 ), 133.8 (C _ph4 ), 132.2 (C _ph2 ), 128.5 (C _p8 ), 123.1 (C _ph3 ), 122.6 (C _p10 ), 111.6 (C _p9 ),51.5 (C _p3 ); 46.8 (C _p2 ), 43.5 (Cn), 38.0 (C _l5 ), 37.2 (C _p4 ), 29.5-27.0 (C _l2+l3+ i ₄ ), 12.0 (C _p1 ).

HRMS-ESI (m/z) calculated for C ₃₃ H ₄₉ 0 ₃ N ₄ [M+H] ⁺ : 549.37992; Found: 549.37938.

N-(2-(diethylamino)ethyl)-4-(3-(1,3-dioxoisoindolin-2-yl) tetradecylamino) benzamide 13

(n= 10) from 80 mg of N-(14-bromotetradecyl)phthalimide, compound 13 was obtained as a pale yellow solid with a yield of 85%.

¹ H NMR (400 MHz, CDCI ₃ ) δ 7.73 (m, 2H _ph3 ), 7.61 (m, 4H _ph4+p8 ), 7.12 (m, 1H _p5 ), 6.48 (d, J= 8.8Hz, 2H _p9 ), 4.15 (m, 1H _p11 ), 3.58 (t, J =7.1 Hz, 2H _I5 ), 3.46 (dd, J= 5.6, 11.3 Hz, 2H _p4 ), 3.03 (dd, J= 6.6, 12.5Hz, 2H _h ), 2.74 (t, J= 5.8Hz, 2H _p3 ), 2.65 (q, J = 7.2Hz, 4H _p2 ), 1.53 (m, 4H _I2+I4 ), 1.20 (m, 20H _I3 ), 1.04 (t, J= 7.1Hz, 6H _p1 ).

¹³ C (100 MHz, CDCI ₃ ) 168.4 (C _ph i), 167.9 (C _p6 ), 151.3 (C _p7 ), 133.8 (C _ph4 ), 132.1 (C _ph2 ), 128.5 (Cpe), 123.1 (C _ph3 ), 121.6 (C _p10 ), 111.5 (C _p9 ), 52.2 (C _p3 ); 47.3 (C _p2 ), 43.5 (Cn), 38.0 (C _l5 ), 36.8 (C _p4 ), 29.5-26.8 (C _l2+I3+I4 ), 11.3 (C _p1 ).

HRMS-ESI (m/z) calculated for C ₃₅ H ₅₃ 0 ₃ N ₄ [M+H] ⁺ : 577.41122; Found: 577.41085.

Example 2: Synthesis of compounds (17)-(19)

17; n=4

Lor D18; n=10

19; n=12

Scheme 3: a) NH ₂ -NHCH ₃ , MeOH, 18h, RT, 69% for n= 10 and 68% for n=12. b) RG108 (D or L configuration), DCC, NHS, DMF, 4h, RT, 32% for n=10, 46% for n=12. Synthesis of intermediate compounds 14-16

4-(3-aminohexylamino)-N-(2-(diethylamino)ethyl)benzamide 14

To a solution of compound 9 (65 mg; 0.14 mmol) in ethanol (1 mL) was added 100 μΙ_ of methylhydrazine. The mixture was stirred at room temperature overnight. The solvent was removed and the residue was taking off with toluene and coevaporated until methylhydrazine was completely removed. The residue was finally purified by flash chromatography silica gel with dichloromethane/methanol (ammonia 1 .75%) 97:3 to give 14 as a pale yellow foam (31 mg; 65%).

¹ H NMR(400 MHz, DMSO) δ 8.45(t, J = 5.5Hz, 1 H _p5 ), 7.63(d, J = 8.7Hz, 2H _p8 ), 6.55(d, J = 8.7Hz, 2H _p9 ), 3.58(m, 3H _p11+p4 ), 3.17(m, 6H _p2+l1 ), 3.03(t, J = 7.04Hz, H _p3 ), 2.77(m, 2H _NH2 ), 1 .53(m, 4H _I2+I4 ), 1 .22(m, 4H _I3+I5 ).

¹³ C(100 MHz, DMSO) 172.3(C _p6 ), 157.1 (C _p7 ), 133.9(C _p8 ), 125.2(C _p10 ), 1 15.9(C _p9 ), 55.1 (C _p3 ) ; 52.2(C _p2 ), 47.7(d), 44.0(C _I5 ), 39.3(C _p4 ), 34.3-31 .0(C _l2+l3+ , ₄ ), 13.6(C _p1 ).

MS (m/z) calculated for C ₂₅ H ₄₇ 0N ₄ [M+H] ⁺ : 334.3; Found: 334.3.

4-(3-aminododecylamino)-N-(2-(diethylamino)ethyl)benzamid e 15

To a solution of compound 12 (72 mg; 0.14 mmol) in ethanol (1 mL) was added 100 μΙ_ of methylhydrazine. The mixture was stirred at room temperature overnight. The solvent was removed and the residue was taking off with toluene and coevaporated until methylhydrazine was completely removed. The residue was finally purified by flash chromatography silica gel with dichloromethane/methanol (ammonia 1 .75%) 97:3 to give 15 as a pale yellow foam (41 mg; 69%).

¹ H NMR(400 MHz, DMSO) δ 8.43(t, J = 5.5Hz, 1 H _p5 ), 7.63(d, J = 8.7Hz, 2H _p8 ), 6.55(d, J = 8.7Hz, 2H _p9 ), 3.57(m, 3H _p11+p4 ), 3.20(m, 6H _p2+l1 ), 3.03(t, J = 7.04Hz, H _p3 ), 2.77(m, 2H _NH2 ), 1 .53(m, 4H _I2+I4 ), 1 .26(m, 16H _I3+I5 ).

¹³ C(100 MHz, DMSO) 172.2(C _p6 ), 157.0(C _p7 ), 134.0(C _p8 ), 125.0(C _p10 ), 1 15.8(C _p9 ), 55.2(C _p3 ) ; 52.0(C _p2 ), 47.6(d), 44.1 (C _l5 ), 39.4(C _p4 ), 34.3-31 .0(C _I2+I3+I4 ), 13.6(C _p1 ).

HRMS-ESI(m/z) calculated for d ₅ H ₄₇ ON ₄ [M+H] ⁺ : 419.37444; Found: 419.37429. 4-(3-aminotetradecylamino)-N-(2-(diethylamino)ethyl)benzamid e 16

To a solution of compound 13 (81 mg; 0.14 mmol) in ethanol (1 mL) was added 100 μΙ_ of methylhydrazine. The mixture was stirred at room temperature overnight. The solvent was removed and the residue was taking off with toluene and coevaporated until methylhydrazine was completely removed. The residue was finally purified by flash chromatography silica gel with dichloromethane/methanol (ammonia 1 .75%) 97:3 to give 16 as a pale yellow foam (43mg; 68%).

¹ H NMR(400 MHz, DMSO) δ 8.43(t, J = 5.5Hz, 1 H _p5 ), 7.65(d, J = 8.7Hz, 2H _p8 ), 6.53(d, J = 8.7Hz, 2H _p9 ), 3.56(m, 3H _p11+p4 ), 3.21 (m, 6H _p2+l1 ), 3.1 (t, J = 7.04Hz, H _p3 ), 2.77(m, 2H _NH2 ), 1 .54(m, 4H _I2+I4 ), 1 .24(m, 20H _I3+I5 ).

¹³ C(100 MHz, DMSO) 172.2(C _p6 ), 157.1 (C _p7 ), 134.0(C _p8 ), 125.0(C _p10 ), 1 15.8(C _p9 ), 54.9(C _p3 ) ; 52.1 (C _p2 ), 47.7(d), 44.1 (C _l5 ), 39.4(C _p4 ), 35.0 -31 .0(C _I2+I3+I4 ), 13.6(C _p1 ).

MS-ESI(m/z) calculated for C ₂₅ H ₄₇ 0N ₄ [M+H] ⁺ : 446.4; Found: 446.4. Synthesis of compounds 17-19

To a solution of D or L RG108 (80 mg; 0.24 mmol) in DMF (500 μΙ) was added NHS (41 mg; 0.36 mmol), DCC (50 mg; 0.24 μηιοΙ) and Dipea (40 μΙ_; 0.40 μηιοΙ). The mixture was stirred at room temperature for 2h then filtered to removed DCU and then 14 (46 mg; 0.05 mmol), 15 (41 mg; 0.1 mmol) or 16 (43 mg; 0.1 mmol) was added and the mixture was stirred at room for 2h. The solvent was removed and the residue was purified by flash chromatography on silica gel with dichloromethane/methanol (ammonia 0.5%) 99:1 as eluent to give 17 as a pale yellow solid (52 mg; yield 58%), 18D or 18L as a pale yellow solid (24 mg; yield 32%) or 19 as a pale yellow solid (35 mg; yield 48%). N-(2-(diethylamino)ethyl)-4-(3-(2-(1,3-dioxoisoindolin-2-yl) -3-(1H-indol-3^ yl)propanamido)hexylamino)benzamide 17

¹ H NMR (400 MHz, CDCI ₃ ) δ 8.69 (s _b , 1H _t1 ), 7.69 (m, 4Hp _h4+ , ₆₊ ,7 ₊ p8 ₊ ph3), 7.21 (m, 2H _t1+t4 ), 7.01 (m, 4H _t2+t5+t6+p5 ), 6.47 (dm, J= 8.8Hz, 2H _p9 ), 6.30 (m, 1H _t13 ), 5.16(t, J = 7.1Hz, 1H _t11 ), 4.18 (m, 1H _p11 ), 3.65 (m, 4Η, _10+ρ4 ), 3.13 (m, 2H _I5 ), 3.00 (m, 2H _n ), 2.54 (m, 2H _p3 ), 2.85 (m, 4H _p2 ), 1.45 (m, 2H _I4 ), 1.33 (m, 2H _I2 ), 0.95 (m, 8H _p1+l3 ).

¹³ C (100 MHz, CDCI ₃ ) 168.7 (C,i ₂ ), 168.2 (C _ph i), 167.4 (C _p6 ), 151.0 (C _p7 ), 136.3 (C _ph2 ), 134.1 (C _ph4 ), 131.6 (C _t8 ), 128.6 (C _p8 ), 126.9 (C _t3 ), 123.4 (C _ph3 ), 122.9 (C _t2 ), 122.5 (C _p io), 122.2 (C _t5ort6 ), 119.6 (C _t5ort6 ), 118.6 (C _t7 ), 111.6 (C _p9 ), 111.3 (C _t9 ), 111.0 (C _t4 ), 55.0 (C,ii), 51.5 (C _p3 ); 46.8 (C _p2 ), 43.5 (d), 39.9 (C _l5 ), 37.1 (C _p4 ), 29.5-25.5 (C| ₂₊ i ₃₊ i ₄₊ tio), 12.0 (C _p ).

HRMS-ESI(m/z) calculated for C ₃₈ H ₄₇ 0 ₄ N ₆ [M+H] ⁺ : 651.3653; Found: 651.36598.

N-(2-(diethylamino)ethyl)-4-(3-(2-(1.3-dioxoisoindolin-2- yl)-3-(1H-indol-3- yl)propanamido)dodecylamino)benzamide 18

¹ H NMR (400 MHz, CDCI ₃ ) δ 8.49 (s _b , 1H _t1 ), 7.66 (m, 2H _ph3 ), 7.54 (m, 4H _ph4+t6+t7 ), 7.52 (dm, J= 8.8Hz, 2H _p8 ), 7.21 (s, 1H _t1 ), 7.19 (s, 1H _t4 ), 7.01 (m, 2H _t5+t6 ), 6.93 (d, J = 2.4Hz, 1H _t2 ), 6.70 (m, 1H _p5 ), 6.46 (dm, J= 8.8Hz, 2H _p9 ), 6.25 (m, 1H _t13 ), 5.16 (dd, J= 7.0Hz, 9.1Hz, 1H _t11 ), 3.95 (m, 1H _p11 ), 3.65 (m, 2H _t10 ), 3.36 (dd, J= 5.6, 11.6 Hz, 2H _p4 ), 3.13 (dd, J= 7.1Hz, 12.9Hz, 2H _I5 ), 3.04 (dd, J= 7.0, 12.6Hz, 2H _n ), 2.54 (t, J =

6.1Hz, 2H _p3 ), 2.48 (q, J= 7.1Hz, 4H _p2 ), 1.53 (m, 2H _I4 ), 1.24 (m, 18H _I3+I2 ), 0.95 (t, J = 7.1Hz, 6H _p1 ).

¹³ C (100 MHz, CDCI ₃ ) 168.7 (C _t12 ), 168.2 (C _ph1 ), 167.4 (C _p6 ), 151.0 (C _p7 ), 136.3 (C _ph2 ), 134.1 (C _ph4 ), 131.6 (C _t8 ), 128.6 (C _p8 ), 126.9 (C _t3 ), 123.4 (C _ph3 ), 122.9 (C _t2 ), 122.5 (C _p io), 122.2 (C _t5ort6 ), 119.6 (C _t5ort6 ), 118.6 (C _t7 ), 111.6 (C _p9 ), 111.3 (C _t9 ), 111.0

(C _t4 ), 55.0 (C _t11 ), 51.5 (C _p3 ); 46.8 (C _p2 ), 43.5 (C _h ), 39.9 (C _l5 ), 37.1 (C _p4 ), 29.5-25.5

(C| ₂₊ | ₃₊ | ₄₊ tio), 12.0 (C _p i).

HRMS-ESI(m/z) calculated for C ₄₄ H ₅₉ 0 ₄ N ₆ [M+H] ⁺ : 735.45923; Found: 735.45973. N-(2-(diethylamino)ethyl)-4-(3-(2-(1 ,3-dioxoisoindolin-2-yl)-3-(1 H-indol-3- yl)propanamido)tetradecylamino)benzamide 19

¹ H NMR(400 MHz, CDCI ₃ ) δ 8.49(s _b , 1H _t1 ), 7.66(m, 2H _ph3 ), 7.54(m, 4H _ph4+t6+t7 ), 7.52(dm, J= 8.8Hz, 2H _p8 ), 7.21 (s, 1H _t1 ), 7.19(s, 1H _t4 ), 7.01 (m, 2H _t5+t6 ), 6.93(d, J = 2.4Hz, 1H _t2 ), 6.70(m, 1H _p5 ), 6.46(dm, J= 8.8Hz, 2H _p9 ), 6.25(m, 1H _t13 ), 5.16(dd, J = 7.0Hz, 9.1Hz, 1H _t11 ), 3.95(m, 1H _p11 ), 3.65(m, 2H _t10 ), 3.36(dd, J= 5.6, 11.6 Hz, 2H _p4 ),

3.13(dd, J= 7.1Hz, 12.9Hz, 2H _I5 ), 3.04(dd, J= 7.0, 12.6Hz, 2H _h ), 2.54(t, J= 6.1Hz, 2H _p3 ), 2.48(q, J= 7.1Hz, 4H _p2 ), 1.53(m, 2H _I4 ), 1.24(m, 22H _I3+I2 ), 0.95(t, J= 7.1Hz, 6H _p1 ).

¹³ C(100 MHz, CDCI ₃ ) 168.7(C _t12 ), 168.2(C _ph1 ), 167.4(C _p6 ), 151.0(C _p7 ), 136.3(C _ph2 ), 134.1(C _ph4 ), 131.6(C _t8 ), 128.6(C _p8 ), 126.9(C _t3 ), 123.4(C _ph3 ), 122.9(C _t2 ), 122.5(C _p10 ),

122.2(C _t5ort6 ),119.6(C _t5ort6 ), 118.6(C _t7 ), 111.6(C _p9 ), 111.3(C _t9 ), 111.0(C _t4 ), 55.0(C _t11 ), 51.5(C _p3 ) ; 46.8(C _p2 ), 43.5(d), 39.9(C _I5 ), 37.1 (C _p4 ), 29.5-25.5(C _l2+l3+l4+t10 ), 12.0(C _p1 ). HRMS-ESI(m/z) calculated for C4 ₆ H ₆ 30 ₄ N ₆ [M+H] ⁺ : 763.49053; Found: 763.49146. calculated for [M+Na] ⁺ : 795.51675 ; Found: 795.51693.

Example 3: Synthesis of compound (20)

Compound (20) is also named FM23 and is a compound obtained from the conjugation of a flavone and a procainamide derivative.

The flavone 23 is obtained as described in B. Bauvois et al., J Med Chem 46, 3900 (Aug 28, 2003).

To a solution of flavone 23 (5 mg; 12.4 μηιοΙ) in DMF (100 μΙ) was added HOBT (7 mg), DCC (10 mg; 48 μηιοΙ), Dipea (20 μΙ_; 20 μηιοΙ) and compound 11 (15 mg, 38 μηιοΙ). The mixture was stirred at room temperature overnight. The solvent was removed and the residue was purified by preparative HPLC C-18 column in a linear gradient 0-90% acetonitrile with 0.1 % TFA to afford FM23 (0.8 mg; yield 4%) as dark yellow foams.

Mass spectroscopy (m/z) calculated for C ₄ i H ₅₃ N ₆ 09 [M+H] ⁺ : 773.4 found: 773.4.

BIOLOGICAL RESULTS Materials and Methods

Cell lines: DU145, a human prostate cancer cell line, and HCT1 16, a human breast cancer cell line, were obtained from ATCC (USA). MTS assay: Cytotoxicity was assessed after 72h using CellTiter96 Aqueous Non-Radioactive Cell Proliferation Assay following the manufacturer's instructions (Promega). C5 DNA methyltransferase activity assay: incorporation of ³ H-methyl groups in duplex DNA using methyl- ³ H SAM was measured with the C-terminal catalytic domain of the murine DNMT3A (623-908) and the C-terminal domain of DNMT3L (208-421 ) as described in (D. Jia, R. Z. Jurkowska, X. Zhang, A. Jeltsch, X. Cheng, Nature 2007, 449, 248) or in hemimethylated duplex DNA with human DNMT1 produced in bacculovirus system as described in B. H. Lee, S. Yegnasubramanian,

X. Lin, W. G. Nelson, J Biol Chem 2005, 280, 40749.

Methyltransferase assays: A comparison was carried out on the murine catalytic DNMT3A/3L complex, on the bacterial EcoDam N-6 DNA methyltransferase and on the catalytic domain of the human G9a histone H3K9 methyltransferase according to the protocols described in (J. R. Horton, K. Liebert, M. Bekes, A. Jeltsch, X. Cheng, J Mol Biol 2006, 358, 559; P. Rathert, Dhayalan A, Murakami M, Zhang X, Tamas R, Jurkowska R, Komatsu Y, Shinkai Y, Cheng X, Jeltsch A., Nat Chem Biol. 2008, 4, 344; M. Roth, A. Jeltsch, Biol Chem 2000, 381, 269).

NMR saturation transfer difference (STD) experiments: NMR experiments were recorded on a Bruker Avance III NMR spectrometer operating at a ¹ H frequency of 500 MHz and equipped with a TCI cryoprobe. Ligands were tested as detailed in the Supplementary Information.

Docking: Ligand structures were prepared for the docking process with Marvin (Chemaxon). Dock 6.4 was used for the molecular docking of the ligand in the the X- ray crystal structure of Dnmt3A (PDB: 2QRV, chain A). Default parameters have been used. Chimera has been used for the graphical visualisation.

DNMT1 and DNMT3A/3L activity

The phtalimides (8 to 13) and RG108 amide derivatives (17, 18 and 19) were tested on the DNMT1 and DNMT3A/3L activity at 10 μΜ. The FM23 (20) was also tested on the DNMT3A/3L activity at 10 μΜ. Some compounds showed a greatly increased activity compared to the parent compound 1 (procainamide). The inhibition profile on the two enzymes was comparable. Interestingly, in the phtalimide family, as the length of the linker increased, the inhibition activity increased up to compound 12 with 12 carbon atoms linker. Compounds 12 and 13 resulted among the most potent inhibitors together with the RG108 conjugates 18L/D and 19 bearing longer linker arms

Table 1. Biochemical and biological activity of the procainamide conjugates. Inhibition of human DNMT1 and murine catalytic complex DNMT3a/3L are reported as IC50 (μΜ) and the cytotoxicity on DU145 and HCT116 cell lines is reported as TC50 (μΜ).

DNMT3A/3L DNMT1 ^[al

Compounds HCT116 ^[bl DU145 ^[bl

IC50 (μΜΓ IC50 (μΜ) TC50 (μΜ) TC50 (μΜ)

1 >500 >300 430 ± 44 700 ± 68

L-RG108 >500 >250 424 ± 63 657 ± 51 phtalimide >500 > 1000 N.D. N.D.

8 450 ± 50 > 100 180 ± 10 313 ±53

9 185 ±20 > 100 33 ± 10 72 ± 13

10 26 ±2 > 100 4.3 ±0.8 24 ± 10

11 26 ±5 77 ±9 10 ± 5 25 ± 14

12 8.2 ± 1.9 4.9 ±1.3 2.1 ±0.6 8.5 ±2.1

13 9.7 ±2.0 8.5 ±2.1 4.8 ±2.1 5.8 ± 1.1

15 N.D. N.D. 27 ±7 43 ± 0.7

17 89 ±5 55 ±4 N.D. N.D.

18L 4.2 ± 0.8 21 ±9 8.5 ±3.1 7.3 ±0.5

18D 10.5 ± 1.7 21 ±2 2.9 ±1.3 5.9 ±2.6

19 9.0 ± 2.6 18 ± 3 3.7 ± 1.8 2.1 ±0.6

20 15 N.D. N.D. N.D.

[a] mean value (± SE) of the concentration at which 50% of inhibition of the enzyme activity is observed [b] mean value (± SE) of the concentration at which 50% of inhibition of cellular proliferation is observed. N.D.= not determined Interestingly, on DNMT3A/3L, the L configuration was slightly more potent. On DNMT1 , compounds 18 and 19 are 3-4 folds less active than compound 12.

This suggests that the presence of a hydrophobic group may be necessary for the activity. Next the inventors investigated by saturation transfer difference (STD) NMR spectroscopy experiments which moiety of the conjugate interacts with the enzyme. In this context, it has been recently proposed by modelling (N. Singh, A. Duenas-Gonzalez, F. Lyko, J. L. Medina-Franco, ChemMedChem 2009, 4, 792) that the procainamide could interact also with DNMT1 and not only the DNA substrate. The STD experiments showed that procainamide does not interact with DNMT1 , DNMT3A or DNMT3A/3L whereas phtalimide or RG108 interact with enzymes. Clearly, these results suggest that the procainamide moiety of the conjugate acts as DNA binder and directs the action of the phtalimide or RG108 on the enzyme. Finally, docking simulations in the X-ray crystal structure of the catalytic DNMT3A of compound 12 and 18L positioned the phtalimide or L-RG108 moiety deep in the catalytic pocket while the procainamide moiety protrudes outwards.

In addition, the selectivity of the conjugates for the C5 DNA methyltransferases was addressed. As shown in Table 2, compounds 12 and 13 are also very specific for the C5 DNA methyltransferases. Finally, the cytotoxicity of the most potent compounds was determined on two tumor cell lines, colon cancer cells HCT1 16 and prostate cancer cells DU145. The cytotoxicity profile obtained on the two cancer cell lines was similar to the one of the inhibition of the methylation activity (see above Table 1 ). Again in the phtalamide family, as the length of the linker increased, the cytotoxicity increased up to compound 12. Once more, the compounds that showed the highest cytotoxicity were phtalamide conjugates 12 and 13, and the long amido conjugates of RG108 (18 and 19).

Table 2. Methyltransferase selectivity. Inhibition of murine catalytic complex

DNMT3a/3L, EcoDam and G9A are reported as IC50 ^[a] (μΜ).

DNMT3A/3L EcoDam G9a ^[bl

Compounds

IC50 (μΜ) IC50 (μΜ) IC50 (μΜ)

8 410 1 240 > 1000 > 1000

11 210 ± 20 N.D. N.D.

12 8.5 ± 4.2 > 1000 70 ± 10

13 9.2± 3.6 200 ± 40 160 ± 50 [a] mean value (± SE) of the concentration at which 50% of inhibition of the enzyme activity is observed

In conclusion, the above results show that the compounds of the invention are potent inhibitors of DNMT1 and DNMT3a/3L, based on the conjugation of procainamide to RG108 or phtalimide. Certain conjugates are at least 50 times more active than the parent compounds. For the RG108 conjugates, the attachment site on the RG108 was extremely important: conjugation on the carboxyl group gave among the most active compounds. It was also shown that the length of the linker arm plays an important role and the 12 carbon atom linker (compounds 12 and 18) is the most promising. STD experiments and docking simulations suggested that the procainamide moiety does not interact with the enzyme in agreement with its binding as DNA ligand to position the phtalimide or L-RG108 to interact with the enzyme. Interestingly, 12 and 13 showed a very good selectivity for C5 DNA methyltransferases when compared to bacterial DNA (EcoDam) and mammalian histone G9a methyltransferases. In addition, conjugates 18 and 19 presented a slight selectivity between DNMT3A/3L and DNMT1 Interestingly, the most potent inhibitors showed also a μΜ cytotoxicity on the cancerous cell lines DU145 and HCT1 16.

Antiparasite activity

The compounds of the invention have been tester for their growth inhibition activity on the blood forms of Trypanosoma brucei brucei.

Assay for in vitro inhibition of T. brucei growth

Bloodstream forms of the Trypanosoma brucei brucei strain 93 and T b. gambiense "Feo" (ITMAP 1893) were cultured in HMI9 medium supplemented with 10% FCS at 37 ^< C under an atmosphere of 5% C0 ₂ (Hirumi H and Hirumi K. (1994) Parasitol. Today 10, 80-4). In all experiments, log-phage cell cultures were harvested by centrifugation at 3,000 x g and immediately used. Drug assays were based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable cells (Raz, B., Iten, M., Grether-Buhler, Y., Kaminsky, R., Brun, R. (1997) Acta Trop. 68, 139) and were performed according to the manufacturer recommendations (AlamarBlue ^R Assay, Invitrogen Corporation). Drug stock solutions were prepared in pure DMSO. T. brucei bloodstream forms (10 ⁵ cells/ml) were cultured as described above in 96-well plates (200 μΙ_ per well) either in the absence or in the presence of different concentrations of inhibitors. After 48-h incubation, AlamarBlue ^R solution (20 μΙ_) was added in each well and fluorescence was measured at 530 nm excitation and 590 nm emission wavelengths after 4-h incubation. Each inhibitor concentration was tested in triplicate and the experiment repeated at least twice. The percentage of inhibition of parasite growth rate was calculated by comparing the fluorescence of parasites maintained in the presence of drug to that of in the absence of drug. Inhibition of parasite growth measured using AlamarBlue ^R was systematically comforted by direct microscopic observations of cultures. Table 3 below shows the obtained results:

These results show that the compound 13 is very efficient at blocking the in vitro proliferation of the bloodstream forms of non human pathogenic Trypanosoma brucei brucei.

The compounds of the invention have also been tested on Trypanosoma brucei gambiense.

Compound 13 show an acceptable selectivity compared to rat L6 and human MRC5 cells (Table 4).

Table 4 - Mean concentration (IC50 in μΜ) and SD at which 50% of the proliferation of Trypanosoma brucei gambiense, is inhibited. Pentamidine is the drug of reference.

Cytotox Cytotox Index of Index of

Compounds IC50 SD (IC50) on (IC50) on Selectivity Selectivity

L6 MRC5 L6 MRC5

Procainamide >50 >50 >50

9 >50 >50 >50

10 7.9 0.8 36.3 36.6 4.6 4.7

1 1 3.3 0.5 8.3 21 .4 2.5 6.6

12 2.1 0.1 16.3 32.7 7.6 15.3 13 0.519 0.048 8.8 18.7 17.0 36.1

Pentamidine 0.005 0.001 13.4 58.7 2851 .1 12489.4