Abstract:
Piperazinyl derivatives of formula (I) and their use as a drug, particularly for the treatment of cancer, are disclosed. Also disclosed are pharmaceutical compositions comprising the piperazinyl derivatives, and methods for synthesizing the piperazinyl derivatives.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation-in-part of U.S. application Ser. No. 14/369,407, filed Jun. 27, 2014, now allowed, which is a U.S. national stage application under 35 U.S.C. §371 of International Application PCT/EP2012/077059 (published as WO 2013/098393), filed Dec. 28, 2012, which claims priority to Application FR 1162586, filed Dec. 30, 2011. Benefit of the filing date of each of these prior applications is hereby claimed. Each of these prior applications is hereby incorporated by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention concerns piperazinyl compounds particularly useful in the treatment of cancer, compositions containing the same and their method of preparation. 
       BACKGROUND OF THE INVENTION 
       [0003]    With lengthening lifetimes cancer, one of the leading causes of mortality in the world, affects an increasingly greater number of persons and remains difficult to treat. 
         [0004]    The developing resistance to anticancer agents is a serious problem which considerably curbs the treatment of numerous types of cancer. Lowered tolerance to an agent is often accompanied by cross-resistance to a variety of other agents. This multiple resistance to anticancer agents known as Multidrug Resistance, MDR, is caused by numerous mechanisms of which only a very small number have been well characterized. These mechanisms include an increase in drug efflux, an increase in cell detoxifying capability, alteration of molecular targets affected by these anticancer agents, modification of the DNA repair system and modification of apoptotic routes (Baguley, Mol. Biotechnol., 2010, 46, 308-316; Gatti et al., Methods Mol. Med. 2005, 111, 127-148; Longley et al., J. Pathol. 2005, 205, 275-292; Kohno et al., Eur. J. Cancer 2005, 41, 2577-2586). 
         [0005]    The development of anticancer treatments able to avoid these resistance mechanisms is a major challenge and up until the present time the initiated trials have given few results. 
         [0006]    Anticancer agents more particularly intended for the treatment of chemotherapy-resistant cancer are described in WO 2009/150248. They meet the following general formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R1 and R2, together with the nitrogen atom which carries them, may form a heterocycle such as a piperazinyl group optionally substituted, the only exemplified compounds being optionally substituted on the nitrogen atom of the piperazine. 
       SUMMARY OF THE INVENTION 
       [0007]    The inventors of this patent application have surprisingly discovered that the insertion of a substituent X at alpha position of the second nitrogen atom of piperazine (see formula (I) below) allows an improvement in the physicochemical properties of the compounds, in particular their solubility, their pharmacokinetic properties and biological activities. 
         [0008]    The subject of the present patent application is therefore more particularly a substituted piperazinyl compound of following general formula (I): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and the pharmaceutically acceptable salts thereof, its stereoisomers or mixtures of stereoisomers in any proportion, in particular an enantiomer mixture and notably a racemic mixture, where:
       X is a (C 1 -C 6 ) alkyl, phenyl, benzyl, C(O)OR5 or C(O)NHR5 group;   R1 is a hydrogen atom or a C(O)H, C(O)R6 or C(O)OR6 group;   R2 is a hydrogen atom or a (C 1 -C 6 )alkyl group;   or R2 together with R1 or X forms a saturated hydrocarbon chain to form a 5 or 6-membered ring, in particular a 5-membered ring;
           R3 is a hydrogen or halogen atom or a (C 1 -C 6 )alkyl or (C 1 -C 6 )alkoxy group;   R4 is a hydrogen or halogen atom, CN, NO 2 , or a (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, aryloxy, benzyloxy or heteroaryloxy group, the said group optionally being substituted by one or more halogen atoms;   Ar is a thiophenyl group or a phenyl group optionally substituted by one or more halogen atoms; and   R5 and R6 independently of one another are a (C 1 -C 6 )alkyl, aryl-(C 1 -C 6 )alkyl or aryl group, the said group optionally being substituted by one or more halogen atoms.   
               
 
         [0017]    By &lt;&lt;halogen&gt;&gt; in the meaning of the present invention is meant a fluorine, bromine, chlorine or iodine atom. Advantageously it is a fluorine, bromine or chlorine atom. 
         [0018]    By &lt;&lt;alkyl&gt;&gt; group in the meaning of the present invention is meant any saturated, straight-chain or branched hydrocarbon group, advantageously having 1 to 6, preferably 1 to 4 carbon atoms. These may particularly be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, neopentyl or n-hexyl groups. Advantageously it is a methyl, ethyl, isopropyl, tert-butyl or isobutyl group. 
         [0019]    In some cases, the alkyl group may optionally be substituted by one or more halogen atoms, in particular bromine, chlorine and fluorine and advantageously fluorine. In this case the group will particularly be the —CF 3  group. 
         [0020]    By &lt;&lt;alkoxy&gt;&gt; group in the meaning of the present invention is meant an alkyl group such as defined above linked to the remainder of the molecule via an oxygen atom. Examples of alkoxy group are the methoxy, ethoxy, isopropoxy or tert-butoxy groups. Advantageously it is the methoxy or tert-butoxy group, and further advantageously the methoxy group. 
         [0021]    In some cases, the alkoxy group can be substituted by one or more fluorine atoms. In this case, it is advantageously the —OCHF 2  or —OCF 3  group, in particular —OCF 3 . 
         [0022]    By &lt;&lt;aryl&gt;&gt; group in the meaning of the present invention is meant an aromatic group preferably having 5 to 10 carbon atoms and comprising one or more fused rings. Advantageously it is the phenyl group. 
         [0023]    By &lt;heteroaryl&gt;&gt; group in the meaning of the present invention is meant any aryl group such as defined above in which one or more carbon atoms have been replaced by one or more heteroatoms, advantageously 1 to 4 and more advantageously 1 to 2, such as sulfur, nitrogen or oxygen atoms for example. Advantageously it is a furyl, thiophenyl, pyridinyl, pyrimidyl, quinolinyl, 1,2,3-thiadiazolyl benzoimidazolyl, indazolyl or 1,2,3-benzotriazolyl group. 
         [0024]    By &lt;aryloxy group in the meaning of the present invention is meant an aryl group such as defined above linked to the remainder of the molecule via an oxygen atom. It is advantageously a phenyloxy group. 
         [0025]    By &lt;&lt;heteroaryloxy&gt;&gt; group in the meaning of the present invention is meant a heteroaryl group such as defined above linked to the remainder of the molecule via an oxygen atom. It is advantageously a pyridinyloxy group. 
         [0026]    By &lt;&lt;aryl-(C 1 -C 6 )alkyl group in the meaning of the present invention is meant an aryl group such as defined above linked to the remainder of the molecule via an alkyl group such as defined above comprising 1 to 6 carbon atoms. Advantageously it is a benzyl or 1-phenethyl group, and more advantageously benzyl. 
         [0027]    In the present invention by &lt;&lt;pharmaceutically acceptable&gt;&gt; is meant that which is useful for the preparation of a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and is acceptable for veterinary use and human pharmacopeia use. 
         [0028]    By &lt;&lt;pharmaceutically acceptable salts of a compound in the present invention is meant salts which are pharmaceutically acceptable as defined herein and which have the desired pharmacological activity of the parent compound. Such salts comprise: 
         [0000]    (1) hydrates and solvates;
 
(2) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and similar; or formed with organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethane-sulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphtalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and similar, advantageously it is hydrochloric acid; and
 
(3) the salts formed when an acid proton present in the parent compound is either replaced by a metal ion e.g. an alkaline metal ion (Na + , K +  or Li +  for example), an alkaline-earth metal ion (such as Ca 2+  or Mg 2+ ) or an aluminium ion; or it is coordinated with an organic or inorganic base. Acceptable organic bases comprise diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and similar. Acceptable inorganic bases comprise aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.
 
         [0029]    In the present invention by &lt;&lt;stereoisomers&gt;&gt; it is meant to designate diastereoisomers or enantiomers. They are therefore optical isomers. The stereoisomers which are not images of one another in a mirror are therefore designated as &lt;&lt;diastereoisomers&gt;&gt;, and the stereoisomers which are non-superimposable images in a mirror are designated as &lt;&lt;enantiomers&gt;&gt;. 
         [0030]    A carbon atom linked to four non-identical substituents is called a chiral centre. 
         [0031]    An equimolar mixture of two enantiomers is called a racemic mixture. 
         [0032]    The compounds of the present invention can in particular meet the following formula (I-bis): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    the nitrogen atom carrying the X group then being of (S) configuration. 
         [0033]    Advantageously X is a (C 1 -C 6 )alkyl, in particular (C 1 -C 4 )alkyl, phenyl or benzyl group. 
         [0034]    Advantageously R1 is a hydrogen atom or a C(O)R6 or C(O)OR6 group, in particular a hydrogen atom. 
         [0035]    Advantageously R2 is a hydrogen atom or a (C 1 -C 6 )alkyl group e.g. methyl. 
         [0036]    Advantageously R3 is a hydrogen atom or a (C 1 -C 6 )alkyl group e.g. methyl. 
         [0037]    Advantageously R4 is a hydrogen or halogen atom, or a (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy or aryloxy group, the said group optionally being substituted by one or more halogen atoms, fluorine in particular. 
         [0038]    Advantageously Ar is a thiophenyl group or a phenyl group substituted by one or more fluorine atoms such as 4-fluoro-phenyl. 
         [0039]    According to one particular embodiment of the invention, X is a (C 1 -C 6 )alkyl, phenyl, benzyl, C(O)OR5, C(O)NHR5 group; R1 is a hydrogen atom; R2 is a hydrogen atom or a (C 1 -C 6 )alkyl group, advantageously (C 1 -C 4 )alkyl or together with R1 or X forms a saturated hydrocarbon chain to form a 5-membered ring; R3 is a hydrogen or halogen atom or a (C 1 -C 6 )alkyl group, in particular (C 1 -C 3 )alkyl, or a (C 1 -C 6 )alkoxy e.g. methoxy; R4 is a halogen atom, CN, NO 2  or a (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, aryloxy, benzyloxy or heteroaryloxy group, the said group optionally being substituted by one or more halogen atoms; Ar is a thiophenyl group or a phenyl group optionally substituted by a halogen; and R5 and R6 independently of one another are a (C 1 -C 6 )alkyl, aryl-(C 1 -C 6 )alkyl or aryl group, the said group optionally being substituted by one or more halogen atoms. 
         [0040]    More advantageously, X is a (C 1 -C 6 )alkyl, phenyl, benzyl, C(O)OR5, C(O)NHR5 group; R1 is a hydrogen atom; R2 is a hydrogen atom or a C 1 -C 6 )alkyl group, advantageously (C 1 -C 4 )alkyl; R3 is a hydrogen or halogen atom or a (C 1 -C 6 )alkyl group, in particular (C 1 -C 3 )alkyl, or a (C 1 -C 6 )alkoxy, e.g. methoxy; R4 is a halogen atom or a (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, aryloxy, benzyloxy or heteroaryloxy group, the said group optionally being substituted by one or more halogen atoms; Ar is a thiophenyl group or phenyl group optionally substituted by a halogen; and R5 and R6 independently of one another are a (C 1 -C 6 )alkyl, aryl-(C 1 -C 6 )alkyl or aryl group, the said group optionally being substituted by one or more halogen atoms. 
         [0041]    Further advantageously, X is a (C 1 -C 6 )alkyl, phenyl or benzyl group; R1 and R2 are a hydrogen atom; R3 is a hydrogen or halogen atom or a (C 1 -C 6 )alkyl group, in particular (C 1 -C 3 )alkyl; R4 is a halogen atom or a (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, aryloxy or benzyloxy group, the said group optionally being substituted by one or more halogen atoms; Ar is a thiophenyl group or a phenyl group optionally substituted by a halogen; and R5 and R6 independently of one another are a (C 1 -C 6 )alkyl, aryl-(C 1 -C 6 )alkyl or aryl group, the said group optionally being substituted by one or halogen atoms. 
         [0042]    Preferably X is a (C 1 -C 6 )alkyl, phenyl or benzyl group; R1 and R2 are a hydrogen atom; R3 is a hydrogen atom or a (C 1 -C 6 )alkyl group, in particular (C 1 -C 3 )alkyl; R4 is a halogen atom or a (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, aryloxy or benzyloxy group, the said group optionally being substituted by one or more halogen atoms; Ar represents a thiophenyl group or a phenyl group optionally substituted by a fluorine atom such as 4-fluoro-phenyl; and R5 and R6 independently of one another are a (C 1 -C 6 )alkyl, aryl-(C 1 -C 6 )alkyl or aryl group, the said group optionally being substituted by one or more fluorine atoms. 
         [0043]    In particular it is one of the compounds in Examples I-1a to I-63 described in the experimental part below, or one of the pharmaceutically acceptable salts thereof, their stereoisomers or mixtures of stereoisomers in any proportion, in particular an enantiomer mixture and especially a racemic mixture. 
         [0044]    The present invention also concerns a compound of formula (I) such as defined above for use thereof as drug intended in particular for the treatment or prevention of cancer, and particularly to treat chemotherapy-resistant cancer. 
         [0045]    The present invention also concerns the use of a compound of formula (I) such as defined above to produce a drug particularly intended to treat or prevent cancer, in particular to treat chemotherapy-resistant cancer. 
         [0046]    The present invention also concerns a method for treating or preventing cancer, in particular chemotherapy-resistant cancer, comprising the administration of a sufficient amount of formula (I) compound such as defined above to a patient in need thereof. 
         [0047]    The cancer may be more particularly selected from breast cancer, leukaemia (such as acute promyelocytic leukaemia), colon cancer (such as colon adenocarcinoma), pancreatic cancer, ovarian cancer, melanoma, lung cancer, central nervous system (CNS) cancer, prostate cancer, renal cancer, head and neck cancer and hepatocarcinoma, and more particularly a chemotherapy-resistant cancer. 
         [0048]    A further subject of the invention is a pharmaceutical composition comprising at least one formula (I) compound such as defined above in association with one or more pharmaceutically acceptable excipients. 
         [0049]    In one particular embodiment, this composition may comprise at least one other active ingredient. 
         [0050]    In particular this or these active ingredient(s) may be anticancer agents conventionally used to treat cancer. These anticancer agents can be selected in particular from among cisplatin and the derivatives thereof such as carboplatin and oxalyplatin; taxanes such as taxol, taxotere, paclitaxel and docetaxel; vinca alkaloids such as vinblastine, vincristine and vinorelbine; purine analogues such as mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine; topoisomerase I inhibitors such as camptothecin compounds e.g. irinotecan and topotecan; topoisomerase II inhibitors such as epipodophyllotoxin, podophyllotoxin and the derivatives thereof e.g. etoposide and teniposide; anti-tumour nucleoside derivatives such as 5-fluorouracil, leucovorin, gemcitabine or capecitabine; alkylating agents such as nitrogen mustards e.g. cyclophosphamide, mechlorethamine, chlorambucil and melphalan, nitroso-ureas such as carmustin, lomustin and streptozocin, alkylsulfonates such as busulfan, ethylenimines and methylmelamines such as thiotepa and hexamethylmelamine, and tetrazines such as dacarbazine; derivatives of anti-tumour anthracyclines such as daunorubicin, adriamycin, doxil, idarubicin and mitoxantrone; molecules targeting the IGF-I receptor such as picropodophyllin; derivatives of tetracarcin such as tetrocarcin A; corticosteroids such as prednisone; antibodies such as trastuzumab (anti-HER2 antibody), rituximab (anti-CD20 antibody), gemtuzamab, cetuximab, pertuzumab and bevacizumab; antagonists or selective modulators of oestrogen receptors such as tamoxifen, fulvestrant, toremifene, droloxifene, faslodex and raloxifene; aromatase inhibitors such as exemestane, anastrozole, letrozole and vorozole; differentiating agents such as retinoids e.g. retinoic acid and vitamin D and agents blocking the metabolism of retinoic acid such as accutane; DNA methyl-transferase inhibitors such as azacytidine and decitabine; antifolates such as permetrexed disodium; antibiotics such as antinomycin D, bleomycin, mitomycin C, actinomycin D, carminomycin, daunomycin and plicamycin; antimetabolites such as chlofarabine, aminopterin, cytosine arabinoside, floxuridine and methotrexate; apoptosis-inducing agents and anti-angiogenic Bcl-2 inhibitors such as YC 137, BH 312, ABT 737, gossypol, HA 14-1, TW 37 and decanoic acid; agents binding to tubulin such as combrestatin, derivatives of colchicine and nocodazole; kinase inhibitors such as flavoperidol, imatinib mesylate, erlotinib and gefitinib; farnesyl transferase inhibitors such as tipifarnib; inhibitors of histone-deacetylases such as sodium butyrate, suberoylanilide hydroxamic acid, depsipeptide, NVP-LAQ824, R306465, JNJ-26481585 and trichostatin A; inhibitors of the ubiquitin-proteasome system such as MLN.41, bortezomib and yondelis; and telomerase inhibitors such as telomestatin. 
         [0051]    The compounds of the invention can be given via oral, sublingual, parenteral, sub-cutaneous, intramuscular, intravenous, transdermal, local or rectal route. 
         [0052]    In the pharmaceutical compositions of the present invention for oral, sublingual, parenteral, sub-cutaneous, intramuscular, intravenous, transdermal, local or rectal route, the active ingredient can be administered in unit administration forms, in a mixture with conventional pharmaceutical carriers, to animals or to human beings. Suitable unit administration forms include forms via oral route such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual and buccal administration forms, parenteral, sub-cutaneous, intramuscular, intravenous, intranasal or intraocular administration forms, and rectal administration forms. 
         [0053]    When a solid composition is prepared in tablet form, the main active ingredient is mixed with a pharmaceutical carrier such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or analogues. It is possible to coat the tablets with sucrose or other suitable materials, or they can be treated so that they have sustained or delayed release and continuously release a predetermined amount of active ingredient. 
         [0054]    A capsule preparation is obtained by mixing the active ingredient with a diluent and pouring the mixture obtained into soft or hard capsules. 
         [0055]    A preparation in syrup or elixir form can contain the active ingredient together with a sweetener, an antiseptic and taste enhancer and suitable colouring agent. 
         [0056]    Water-dispersible powders or granules can contain the active ingredient in a mixture with dispersing agents or wetting agents, or suspending agents, and also with taste enhancers or sweeteners. 
         [0057]    For rectal administration, recourse is made to suppositories prepared with binders which melt at rectal temperature e.g. cocoa butter or polyethylene glycols. 
         [0058]    For parenteral, intranasal or intraocular administration use is made of aqueous suspensions, of saline isotonic solutions or sterile, injectable solutions which contain pharmacologically compatible dispersing agents and/or wetting agents. 
         [0059]    The active ingredient can also be formulated in microcapsule form optionally with one or more additive carriers. 
         [0060]    The compounds of the invention can be used at doses of between 0.01 mg and 1000 mg per day, given in a single daily dose or in several doses throughout the day e.g. twice daily in equal doses. The daily administered dose is advantageously between 5 mg and 500 mg, more advantageously between 10 mg and 200 mg. It may be necessary to use doses outside these ranges which persons skilled in the art will know how to determine. 
         [0061]    A further subject of the invention is a pharmaceutical composition comprising: 
         [0062]    (i) at least one formula (I) compound such as defined above; and 
         [0063]    (ii) at least one other active ingredient 
         [0000]    as combination products for simultaneous, separate or time-staggered use. 
         [0064]    It is effectively frequent for cancer to be treated with bi- or tri-therapy. It may be useful in particular to associate the molecules of the invention with one or more anticancer compounds first allowing treatment of the cancer and secondly preventing the onset of resistant cancer cells. 
         [0065]    In particular, this or these active ingredient(s) may be anticancer agents usually used to treat cancer. These anticancer agents can be selected in particular from among cisplatin and its derivatives such as carboplatin and oxalyplatin; taxanes such as taxol, taxotere, paclitaxel and docetaxel; vinca alkaloids such as vinblastine, vincristine and vinorelbine; purine analogues such as mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine; topoisomerase I inhibitors such as camptothecin compounds e.g. irinotecan and topotecan; topoisomerase II inhibitors such as epipodophyllotoxin, podophyllotoxin and the derivatives thereof such as etoposide and teniposide; anti-tumour nucleoside derivatives such as 5-fluorouracil, leucovorin, gemcitabine or capecitabine; alkylating agents such as nitrogen mustards e.g. cyclophosphamide, mechlorethamine, chlorambucil and melphalan, nitroso-ureas such as carmustin, lomustin and streptozocin, alkylsulfonates such as busulfan, ethylenimines and methylmelamines such as thiotepa and hexamethylmelamine, and tetrazines such as dacarbazine; anti-tumour anthracycline derivatives such as daunorubicin, adriamycin, doxil, idarubicin and mitoxantrone; molecules targeting the IGF-I receptor such as picropodophyllin; tetracarcin derivatives such as tetrocarcin A; corticosteroids such as prednisone; antibodies such as trastuzumab (anti-HER2 antibody), rituximab (anti-CD20 antibody), gemtuzamab, cetuximab, pertuzumab and bevacizumab; antagonists or selective modulators of oestrogen receptors such as tamoxifen, fulvestrant, toremifene, droloxifene, faslodex and raloxifene; aromatase inhibitors such as exemestane, anastrozole, letrozole and vorozole; differentiating agents such as retinoids e.g. retinoic acid and vitamin D and agents blocking the metabolism of retinoic acid such as accutane; DNA methyl-transferase inhibitors such as azacytidine and decitabine; antifolates such as permetrexed disodium; antibiotics such as antinomycin D, bleomycin, mitomycin C, actinomycin D, carminomycin, daunomycin and plicamycin; antimetabolites such as chlofarabine, aminopterin, cytosine arabinoside, floxuridine and methotrexate; apoptosis-inducing agents and anti-angiogenic agents of Bcl-2 inhibitors such as YC 137, BH 312, ABT 737, gossypol, HA 14-1, TW 37 and decanoic acid; agents binding to tubulin such as combrestatin, derivatives of colchicine an nocodazole; kinase inhibitors such as flavoperidol, imatinib mesylate, erlotinib an gefitinib; farnesyl transferase inhibitors such as tipifarnib; inhibitors of histone-deacetylases such as sodium butyrate, suberoylanilide hydroxamic acid, depsipeptide, NVP-LAQ824, R306465, JNJ-26481585 and trichostatin A; inhibitors of the ubiquitin-proteasome system such as MLN.41, bortezomib and yondelis; and telomerase inhibitors such as telomestatin. 
         [0066]    A further subject of the invention is a pharmaceutical composition such as defined above, for use thereof as drug to treat or prevent cancer in particular, and particularly chemotherapy-resistant cancer. 
         [0067]    The cancer may be more particularly selected from breast cancer, leukaemia (such as acute promyelocytic leukaemia), colon cancer (such as colon adenocarcinoma), pancreatic cancer, ovarian cancer, melanoma, lung cancer, central nervous system (CNS) cancer, prostate cancer, renal cancer, head and neck cancer and hepatocarcinoma, and more particularly a chemotherapy-resistant cancer. 
         [0068]    The present invention also concerns a method for preparing a formula (I) compound such as defined above comprising the following successive steps:
       a) reacting an amine of following formula (II):       
 
         [0000]    
       
                 
         
             
             
         
       
       
         
           
             
               
                 where X, R1, R2, R3, R4 and Ar are such as previously defined, R1 not representing a hydrogen atom; 
                 with chloroacetyl chloride in the presence of a base to give a formula (I) compound where R1≠H; and 
               
             
             b) optionally deprotecting the nitrogen atom carrying the R1≠H group to give a formula (I) compound where R1=H. 
           
         
       
     
         [0073]    Step a): 
         [0074]    The base used for this step is preferably a weak base such as NaHCO 3 . 
         [0075]    The amine of formula (II) can be obtained by reaction of a piperazine of following formula (III): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where X, R1 and R2 are as previously defined, R1 not representing a hydrogen atom, with an acid of following formula (IV): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R3, R4 and Ar are as previously defined. 
         [0076]    This reaction can be conducted under peptide coupling conditions well known to skilled persons. 
         [0077]    Coupling is therefore preferably performed in the presence of a coupling agent such as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), carbonyldiimidazole (CDI), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), optionally associated with a coupling auxiliary such as N-hydroxy succinimide (NHS), N-hydroxy benzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), l-hydroxy-7-azabenzotriazole (HAt) or N-hydroxysulfosuccinimide (sulfo NHS). Preferably it is HBTU. 
         [0078]    A base such as diisopropyl-ethylamine (DIPEA) may also be present. 
         [0079]    The piperazine of formula (III) is either obtained commercially or prepared following methods well known to persons skilled in the art. 
         [0080]    The acid of formula (IV) can be prepared using the following successive steps:
       i) reacting a ketoester of following formula (V):       
 
         [0000]    
       
                 
         
             
             
         
       
       
         
           
             
               
                 where Ar is as previously defined and R represents a (C 1 -C 6 )alkyl group e.g. ethyl, with an aniline of following formula (VI): 
               
             
           
         
       
     
         [0000]    
       
                 
         
             
             
         
       
       
         
           
             
               
                 where R3 and R4 are as previously defined, 
                 to give an imine of following formula (VII): 
               
             
           
         
       
     
         [0000]    
       
                 
         
             
             
         
       
       
         
           
             
               
                 where R, R3, R4 and Ar are as previously defined; 
               
             
             ii) reducing the imine of formula (VII) obtained at the preceding step to give an amine of following formula (VIII): 
           
         
       
     
         [0000]    
       
                 
         
             
             
         
       
       
         
           
             
               
                 where R, R3, R4 and Ar are as previously defined; and 
               
             
             iii) saponifying the ester function of the formula (VIII) compound obtained at the preceding step to give the acid of formula (IV). 
           
         
       
     
         [0089]    Step i) can be conducted in the presence of an acid such as paratoluene sulfonic acid (PTSA). The reaction can be performed in a polar solvent such as toluene. Preferably the reaction medium is heated under reflux using Dean-Stark apparatus to remove the water as and when it is formed during the reaction. 
         [0090]    The ketoester (V) used for this reaction is either obtained commercially or prepared via Friedel-Crafts reaction using ethyl oxalyl chloride and the corresponding aromatic in the presence of a Lewis acid such as aluminium chloride AlCl 3 . 
         [0091]    The aniline (VI) used for this reaction is either obtained commercially or prepared using methods well known to skilled persons. 
         [0092]    Reducing step ii) can be performed in the presence of a reducing agent well known to skilled persons such as sodium cyanoborohydride. 
         [0093]    Saponification step iii) can be performed under conditions well known to skilled persons, in particular in the presence of a base such as NaOH, KOH or LiOH. 
         [0094]    Step b): 
         [0095]    This step is preferably conducted with a formula (I) compound in which R1=CO 2 R6, such as CO 2 tBu, via treatment with an acid such as HCl. 
         [0096]    The compound thus obtained can be separated from the reaction medium using methods well known to skilled persons, e.g. by extraction, evaporation of the solvent or by precipitation and filtration. 
         [0097]    The compound may also be purified if necessary using techniques well known to skilled persons, e.g. by recrystallization if the compound is crystalline, by distillation, by silica gel chromatography or high performance liquid chromatography (HPLC). 
         [0098]    The method of the present invention to prepare compounds of the present invention where R1≠H is shown in the following reaction scheme: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0099]    The following examples illustrate the invention but are not limiting thereof. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0100]      FIG. 1  gives the time-plasma concentration curves for a mouse given compound I-43 dia2 administered via intravenous route (IV) at a dose of 10 mg/kg or via oral route (PO) at a dose of 30 mg/kg. 
           [0101]      FIG. 2  presents the results of cell proliferation inhibition of 3 human cancer cell lines established from head &amp; neck (HNSCC) and 4 human cancer cell lines established from hepatocarcinoma (HCC) by compound I-43b. 
           [0102]      FIG. 3  presents the mean growth percent of the NCI 60 cell lines in the presence of compound I-43b at a concentration of 10 M. 
           [0103]      FIGS. 4A, 4B and 4C  represent the percentage growth of the NCI 60 cell lines in the presence of compound I-43b at five different concentrations. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0104]    The following examples are set forth as representative of certain aspects and advantages relating to the present disclosure. These examples are not to be construed as limiting the scope of the invention, as other equivalent embodiments will be apparent in view of the present disclosure and appended claims. 
       EXAMPLES 
     I—Synthesis of Compounds of the Invention 
       [0105]    In the following section two different nomenclatures were adopted when the two diastereoisomers of a compound of the invention were separated:
       a/b each designating the particular structure of a single diastereoisomer;   dia1/dia2 respectively designating the least polar and most polar diastereoisomer in the chromatographic system used.       
 
         [0108]    The particular stereochemistry of each of the diastereoisomers was not determined. Therefore, it was impossible to allocate the particular structure a and b to each isolated diastereoisomer dia1 and dia2. This is why a double nomenclature is used. 
         [0109]    The following abbreviations are used in this section: 
       TLC Thin Layer Chromatography 
     DCM Dichloromethane 
     DIEA Diisopropylethylamine 
       [0110]    HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
 
LCMS Liquid Chromatography coupled with Mass Spectrometer
 
       NMR Nuclear Magnetic Resonance 
       [0111]    RT Room temperature 
       Examples I-1a and I-1b 
     Diastereoisomers of the tert-butyl ester of 4-[2-[(2-chloro-acetyl)-(4-phenoxy-phenyl)-amino]-2-(4-fluoro-phenyl)-acetyl]-(S)-2-isopropyl-piperazine-1-carboxylic acid 
       [0112]    
       
                 
         
             
             
         
       
     
       Stage 1: Ethyl ester of (4-fluoro-phenyl)-oxo-acetic acid (1) 
       [0113]    To a solution of aluminium chloride (21.13 g; 160 mmol) in DCM (200 mL) at 0° C. under argon, ethyl oxalyl chloride (17.9 mL; 160 mmol) was added dropwise for 10 min. The medium was left under agitation for 10 minutes. Fluorobenzene (14.7 mL; 160 mmol) diluted in 30 mL of DCM, was added dropwise at 0° C. The medium was left under agitation at room temperature for 12 hours. The medium was washed with water and the organic phase dried over MgSO 4 . After evaporation, the recovered oil was purified by flash chromatography on silica gel eluting with cyclohexane-ethyl acetate 90:10. 
         [0114]    A yellow oil was recovered (17.08 g; 54%). 
         [0115]      1 H NMR (300 MHz, CDCl 3 ): δ 8.04-8.14 (m; 1.8H); 7.15-7.24 (m; 1.9H); 4.46 (q; J=7.2 Hz; 2.0H); 1.44 (t; J=7.2 Hz; 3.0H). 
       Stage 2: Ethyl ester of (4-fluoro-phenyl)-[(Z)-4-phenoxy-phenylimino]-acetic acid (2) 
       [0116]    To a solution of 1 (3.92 g; 20 mmol) in toluene (25 mL) were successively added paratoluene sulfonic acid (200 mg; 1 mmol) and 4-phenoxyphenyl-aniline (3.70 g; 20 mmol) in the presence of a molecular sieve. The medium was placed under reflux in DeanStark apparatus for 20 hours. The medium was washed in water and the organic phase dried over MgSO 4 . After evaporation, the recovered oil was purified by flash silica gel chromatography eluting with cyclohexane-ethyl acetate 90:10. 
         [0117]    Recovery of a yellow oil (6.27 g; 86%). 
         [0118]    LCMS [M+H]=364 (C 22 H 18 FNO 3 ) 
       Stage 3: Ethyl ester of (4-fluoro-phenyl)-(4-phenoxy-phenylamino)-acetic acid (3) 
       [0119]    To a solution of 2 (6.27 g; 17.26 mmol) in methanol (75 mL) and acetic acid (7.5 mL), sodium cyanoborohydride (1.63 g; 26 mmol) was added. The medium was left under agitation for 1 hour at RT. The methanol was partly evaporated, the solution neutralized with Na 2 CO 3  with the addition of water if necessary. The medium was extracted with DCM and the organic phase dried over MgSO 4 . After evaporation, the recovered oil was purified by flash chromatography on silica gel eluting with cyclohexane-ethyl acetate 95:5. 
         [0120]    Recovery of a yellow oil (5.91 g; 93%). 
         [0121]    LCMS [M+H]=366 (C 22 H 20 FNO 3 ) 
         [0122]      1 H NMR (300 MHz, CDCl 3 ): δ 7.45-7.54 (m; 1.9H); 7.23-7.31 (m; 1.9H); 6.97-7.11 (m; 2.9H); 6.81-6.94 (m; 3.9H); 6.54 (d; J=9.0 Hz; 2.0H); 5.01 (br; 1.0H); 4.90 (br; 0.9H); 4.10-4.32 (m; 2.0H); 1.23 (t; J=7.0 Hz; 3.0H). 
       Stage 4: (4-fluoro-phenyl)-(4-phenoxy-phenylamino)-acetic acid (4) 
       [0123]    To a solution of 3 (8.04 g; 22 mmol) in 130 mL of acetonitrile was added 66 mL of a 1 M solution of LiOH (3 eq). The reaction medium was left under agitation for 2 to 3 hours, completion of the reaction being controlled by TLC (cyclohexane-ethyl acetate 60:40). The acetonitrile was partly evaporated, the medium acidified with a 1 M solution of HCl with the addition of 200 mL of water. The medium was filtered and the recovered solid washed three times in water and dried in vacuo in a drying oven in the presence of P 2 O 5 . 
         [0124]    Recovery of a white powder (7.17 g; 97%). 
         [0125]    LCMS [M+H]=338 (C 20 H 16 FNO 3 ) 
         [0126]      1 H NMR (300 MHz, DMSO): δ 7.55 (dd; J=8.5 Hz; J=5.6 Hz; 2.1H); 7.28 (t; J=7.9 Hz; 2.1H); 7.20 (t; J=8.5 Hz; 2.1H); 6.99 (t; J=7.0 Hz; 1.1H); 6.74-6.90 (m; 4.0H; 6.62-6.70 (m; 2.0H); 5.10 (s 1.0H). 
       Stage 5: Tert-butyl ester of 4-[2-(4-fluoro-phenyl)-2-(4-phenoxy-phenylamino)-acetyl]-(S)-2-isopropyl-piperazine-1-carboxylic acid (5) 
       [0127]    To a solution of 4 (7.17 g; 21.2 mmol) in DCM (150 mL) in the presence of one equivalent of DIEA (3.7 mL) was added a solution of Boc-alpha-(S)-isopropyl-piperazine hydrochloride (5.63 g; 21.26 mmol) in the presence of 1 eq of DIEA (3.7 mL) in 50 mL of DCM, followed by HBTU (8.06 g; 21.2 mmol). The medium was left under agitation for 12 hours. The medium was washed with water and the organic phase dried over MgSO 4 . After evaporation the recovered oil was purified by flash chromatography on silica gel eluting with cyclohexane-ethyl acetate 80:20. 
         [0128]    Recovery of a white foam (11.90 g; 100%). 
         [0129]    LCMS [M+H]=548 (C 32 H 38 FN 3 O 4 ) 
       Stage 6 Tert-butyl ester of 4-[2-[(2-chloro-acetyl)-(4-phenoxy-phenyl)-amino]-2-(4-fluoro-phenyl)-acetyl]-(S)-2-isopropyl-piperazine-1-carboxylic acid 
       [0130]    To a solution of 5 (11.86 g; 22.66 mmol) in 250 mL of DCM in the presence of NaHCO 3  (7.30 g; 87.0 mmol) the chloroacetyl chloride (3.45 mL; 43.3 mmol) was added. The medium was left under agitation for 12 hours. The medium was washed with water and the organic phase dried over MgSO 4 . After evaporation the recovered oil was purified by flash chromatography on silica gel with cyclohexane-ethyl acetate gradient of 95-5′ to 50-50 to obtain two diastereoisomers separately in the form of colourless foam: 
       Least Polar Diastereoisomer (I-1 Dia1) 
       [0131]    (3.80 g; 28%) 
         [0132]    LCMS [M+H]=625 (C 34 H 39 ClFN 3 O 5 ) 
         [0133]      1 H NMR (300 MHz, CDCl 3 ): δ 7.92-8.01 (m; 1.0H); 7.30-7.40 (m; 2.0H); 7.10-7.18 (m; 1.1H); 7.01-7.09 (m; 1.1H); 6.84-7.00 (m; 6.1H); 6.55-6.65 (m; 1.1H); 6.32-6.48 (m; 2.1H); 4.72 (d; J=13.5 Hz; 0.5H) 4.63 (d; J=13.5 Hz; 0.4H); 3.52-3.96 (m; 4.0H); 3.10-3.27 (m; 0.5H); 2.85-3.07 (m; 0.4H); 2.23-2.85 (m; 0.5H+0.7H+0.4H); 1.87-2.14 (m; 0.6H); 1.42 (s; 8.7H); 1.17 (d; J=6.6 Hz; 1.0H); 1.03 (d; J=6.6 Hz; 1.3H); 0.88 (d; J=6.6 Hz; 1.1H); 0.69 (d; J=6.6 Hz; 1.3H). 
       Most Polar Diastereoisomer (I-1 Dia2) 
       [0134]    (3.29 g; 24%) 
         [0135]    LCMS [M+H]=625 (C 34 H 39 ClFN 3 O 5 ) 
         [0136]      1 H NMR (300 MHz, CD 2 Cl 2 ): δ 7.85-8.00 (m; 1.0H); 7.36 (t; J=7.6 Hz; 2.1H); 6.99-7.21 (m; 3.2H); 6.81-6.98 (m; 5.2H); 6.63 (br; 1.1H); 6.35-65.5 (m; 2.1H); 4.65 (d; J=13.1 Hz; 0.6H) 4.42 (d; J=13.1 Hz; 0.3H); 3.50-4.16 (m; 4.9H); 3.00-3.43 (m; 0.9H); 2.57-2.90 (m; 1.9H); 1.98-2.18 (m; 0.7H); 1.36-1.49 (m; 10.0H); 1.73 (d; J=6.5 Hz; 2.1H); 0.90 (d; J=6.5 Hz; 2.1H); 0.63 (d; J=6.5 Hz; 1.0H); 0.20 (d; J=6.5 Hz; 0.9H). 
       Examples I-2a and I-2b 
     Diastereoisomers of the tert-butyl ester of 4-[-2-[(2-chloro-acetyl)-(4-phenoxy-phenyl)-amino]-2-(4-fluoro-phenyl)-acetyl]-(R)-2-isopropyl-piperazine-1-carboxylic acid 
       [0137]    
       
                 
         
             
             
         
       
     
       Stage 1: Tert-butyl ester of 4-[2-(4-fluoro-phenyl)-2-(4-phenoxy-phenylamino)-acetyl]-(R)-2-isopropyl-piperazine-1-carboxylic acid (6) 
       [0138]    To a solution of (4-fluoro-phenyl)-(4-phenoxy-phenylamino)-acetic acid 4 (253 mg; 0.75 mmol) in DCM (10 mL) in the presence of one equivalent of DIEA (131 μL) was added a solution of Boc-alpha-(R)-isopropyl-piperazine (171 mg; 0.75 mmol) in the presence of 1 eq of DIEA (131 μL) in 5 mL of DCM, followed by HBTU (285 mg; 0.75 mmol). The medium was left under agitation for 12 hours. The medium was washed with water and the organic phase dried over MgSO 4 . After evaporation the recovered oil was purified by flash chromatography on silica gel eluting with cyclohexane-ethyl acetate 80:20. 
         [0139]    Recovery of a white foam (369 mg; 90%). 
         [0140]    LCMS [M+H]=548 (C 32 H 38 FN 3 O 4 ) 
       Stage 2: Ter-butyl ester of 4-[-2-[(2-chloro-acetyl)-(4-phenoxy-phenyl)-amino]-2-(4-fluoro-phenyl)-acetyl]-(R)-2-isopropyl-piperazine-1-carboxylic acid 
       [0141]    Both diastereoisomers were prepared from 6 following the same operating mode as for the preparation in Example 1 (stage 6). 
         [0142]    Separate recovery of the two diastereoisomers in the form of a colourless foam. 
       Least Polar Diastereoisomer (I-2 Dia1) (195 mg; 42%) 
       [0143]    LCMS [M+H]=625 (C 34 H 39 ClFN 3 O 5 ) 
         [0144]      1 H NMR (300 MHz, CD 2 Cl 2 ): δ 7.85-8.00 (m; 1.0H); 7.36 (t; J=7.6 Hz; 2.0H); 6.99-7.21 (m; 3.1H); 6.81-6.98 (m; 4.9H); 6.63 (br; 1.0H); 6.35-6.55 (m; 2.1H); 4.65 (d; J=13.0 Hz; 0.7H) 4.42 (d; J=13.0 Hz; 0.2H); 3.50-4.16 (m; 4.9H); 3.00-3.43 (m.; 0.8H); 2.57-3.90 (m; 2.0H); 1.98-2.18 (m; 0.8H); 1.36-1.49 (m; 10.5H); 1.73 (d; J=6.5 Hz; 2.0H); 0.90 (d; J=6.5 Hz; 2.0H); 0.63 (d; J=6.5 Hz; 0.8H); 0.20 (d; J=6.5 Hz; 0.8H). 
       Most Polar Diastereoisomer (I-2 Dia2) (122 mg; 26%) 
       [0145]    LCMS [M+H]=625 (C 34 H 39 ClFN 3 O 5 ) 
         [0146]      1 H NMR (300 MHz, CDCl 3 ): δ 7.92-8.01 (m; 1.0H); 7.30-7.40 (m; 2.0H); 7.10-7.18 (m; 1.0H); 7.01-7.09 (m; 1.1H); 6.84-7.00 (m; 6.0H); 6.55-6.65 (m; 1.1H); 6.32-6.48 (m; 2.1H); 4.72 (d; J=13.5 Hz; 0.4H) 4.63 (d; J=13.5 Hz; 0.3H); 3.52-3.96 (m; 4.7H); 3.10-3.27 (m; 0.7H); 2.85-3.07 (m; 0.5H); 2.23-2.85 (m; 0.5H+0.6H+0.8H); 1.87-2.14 (m; 0.9H); 1.42 (s; 8.6H); 1.17 (d; J=6.6 Hz; 1.4H); 1.03 (d; J=6.6 Hz; 2.1H); 0.88 (d; J=6.6 Hz; 2.1H); 0.69 (d; J=6.6 Hz; 1.6H). 
       Examples I-3a and I-3b 
     Hydrochloride of the diastereoisomers of 2-chloro-N-[1-(4-fluoro-phenyl)-2-((S)-3-isopropyl-piperazin-1-yl)-2-oxo-ethyl]-N-(4-phenoxy-phenyl)-acetamide 
       [0147]    
       
                 
         
             
             
         
       
     
         [0148]    To a solution of the I-1 dia2 diastereoisomer (3.24 g; 5.2 mmol) in 50 mL of DCM the HCl gas was added by bubbling. The reaction medium was left under agitation for 12 hours at RT. The DCM was evaporated and the residual oil precipitated in ether. 
         [0149]    The example I-3 dia2 was obtained in the form of a white power after filtration: (2.53 g; 87%). 
         [0150]    LCMS [M+H]=524 (C 29 H 32 Cl 2 FN 3 O 3 ) 
         [0151]      1 H NMR (300 MHz, DMSO): δ 8.60-9.35 (m; 1.6H); 7.77 (br; 0.8H); 7.30-7.40 (m; 2.0H); 7.00-7.23 (m; 5.1H); 6.80-7.00 (m; 3.1H); 6.54-6.76 (m; 3.0H); 4.56 (d; J=13.3 Hz; 1.0H); 3.88-4.16 (m; 3.0H); 3.00-3.30 (m; 3.1H); 2.65-2.96 (m; 1.7H); 1.52-2.00 (m; 1.6H); 1.00 (t; J=7.4 Hz; 2.4H); 0.59 (dd; J=15.6 Hz; J=6.7 Hz; 3.5H) 
         [0152]    Applying the same procedure starting from example I-1 dia1, example I-3 dia1 was obtained in the form of a white powder after filtration: (63 mg). 
         [0153]    LCMS [M+H]=524 (C 29 H 32 Cl 2 FN 3 O 3 ) 
         [0154]      1 H NMR (300 MHz, DMSO): δ 8.65-9.6 (br; 1.2H); 7.77 (br.; 0.8H); 7.30-7.40 (m; 2.0H); 6.36-7.25 (m; 11.7H); 4.40-4.60 (m; 0.8H); 4.00-4.12 (m; 2.0H); 3.76-3.98 (m; 0.9H); 3.37-3.63 (m; 0.9H); 2.65-3.30 (m; 4.0H); 1.77-2.06 (m; 1.6H); 0.89-1.06 (m; 6.1H). 
       Examples I-4a and I-4b 
     Hydrochloride of the diastereoismers of 2-Chloro-N-[1-(4-fluoro-phenyl)-2-((R)-3-isopropyl-piperazin-1-yl)-2-oxo-ethyl]-N-(4-phenoxy-phenyl)-acetamide 
       [0155]    
       
                 
         
             
             
         
       
     
         [0156]    The same protocol was followed as for Examples I-3a and I-3b starting from each of the diastereoisomers I-2a and I-2b. 
         [0000]    Starting from the First Diastereoisomer of Example I-2 (I-4 Dia1): 
         [0157]    Recovery of a white powder after filtration: (95 mg) 
         [0158]    LCMS [M+H]=524 (C 29 H 32 Cl 2 FN 3 O 3 ) 
         [0159]      1 H NMR (300 MHz, DMSO): δ 8.65-9.6 (br; 1.3H).; 7.77 (br; 0.4H); 7.30-7.40 (m; 2.0H); 6.36-7.25 (m; 11.8H); 4.40-4.60 (m; 0.9H); 4.00-4.12 (m; 2.0H); 3.76-3.98 (m; 1.0H); 3.37-3.63 (m; 1.0H); 2.65-3.30 (m; 3.8H); 1.77-2.06 (m; 1.8H); 0.89-1.06 (m; 6.1H). 
         [0000]    Starting from the Second Diastereoisomer of Example I-2 (I-4 Dia2): 
         [0160]    Recovery of a white powder after filtration: (95 mg) 
         [0161]    LCMS [M+H]=524 (C 29 H 32 Cl 2 FN 3 O 3 ) 
         [0162]      1 H NMR (300 MHz, DMSO): δ 8.60-9.35 (m; 1.7H); 7.77 (br; 0.9H); 7.30-7.40 (m; 2.0H); 7.00-7.23 (m; 5.0H); 6.80-7.00 (m; 3.0H); 6.54-6.76 (m; 2.9H); 4.56 (d; J=13.3 Hz; 1.0H); 3.88-4.16 (m; 3.0H); 3.00-3.30 (m; 3.1H); 2.65-2.96 (m; 1.7H); 1.52-2.06 (m; 1.9H); 1.00 (t; J=7.2 Hz; 2.4H); 0.59 (dd; J=15.4 Hz; J=6.7 Hz; 3.3H). 
       Examples I-5a and I-5b 
     Diastereoisomers of the tert-butyl ester of 4-[-2-[(2-chloro-acetyl)-(2-methyl-4-phenoxy-phenyl)-amino]-2-(4-fluoro-phenyl)-acetyl]-(S)-2-isopropyl-piperazine-1-carboxylic acid 
       [0163]    
       
                 
         
             
             
         
       
     
       Stage 1: Tert-butyl ester of 4-[2-(4-fluoro-phenyl)-2-(2-methyl-4-phenoxy-phenylamino)-acetyl]-(S)-2-isopropyl-piperazine-1-carboxylic acid (8) 
       [0164]    To a solution of (4-fluoro-phenyl)-(2-methyl-4-phenoxy-phenylamino)-acetic acid (9.29 g; 26.4 mmol) in DCM (150 mL) in the presence of one equivalent of DIEA (4.6 mL) was added a solution of Boc-alpha-(S)-isopropyl-piperazine hydrochloride (7.00 g; 26.4 mmol) in the presence of 1 eq of DIEA (4.6 mL) in 50 mL of DCM, followed by HBTU (10.00 g; 26.4 mmol). The medium was left under agitation for 12 hours. The medium was washed with water and the organic phase dried over MgSO 4 . After evaporation the recovered oil was purified by flash chromatography on silica gel eluting with cyclohexane-ethyl acetate 80:20. 
         [0165]    Recovery of a white foam (14.13 g; 95%). 
         [0166]    LCMS [M+H]=562 (C 33 H 40 FN 3 O 4 ) 
       Stage 2: Tert-butyl ester of 4-[-2-[(2-chloro-acetyl)-(2-methyl-4-phenoxy-phenyl)-amino]-2-(4-fluoro-phenyl)-acetyl]-(S)-2-isopropyl-piperazine-1-carboxylic acid 
       [0167]    To a solution of 8 (14.13 g; 25.1 mmol) in 250 mL of DCM in the presence of NaHCO 3  (8.40 g; 100.0 mmol) was added chloroacetyl chloride (4.00 mL; 50.0 mmol). The medium was left under agitation for 12 hours. The medium was washed with water and the organic phase dried over MgSO 4 . After evaporation the recovered oil was purified by flash chromatography on silica gel eluting with cyclohexane-ethyl acetate 90:10 gradually up to 50:50. 
         [0168]    Recovery of both diastereoisomers in the form of a colourless foam: 
       Least Polar Diastereoisomer (I-5 Dia1) (3.83 g; 24%) 
       [0169]    LCMS [M+H]=639 (C 35 H 41 ClFN 3 O 5 ) 
         [0170]      1 H NMR (300 MHz, CD 2 Cl 2 ): δ 7.94-8.57 (m; 1.0H); 7.35 (t; J=7.9 Hz; 2.0H); 7.07-7.27 (m; 3.0H); 6.74-6.95 (m; 5.0H); 6.58 (br d; J=2.6 Hz; 1.1H); 6.51 (br s; 0.2H); 6.41 (s; 0.8H); 6.31 (br s; 0.3H); 4.62 (d; J=13.5 Hz; 0.7H) 4.39 (d; J=13.5 Hz; 0.3H); 3.53-4.05 (m; 4.8H); 3.04-3.46 (m; 0.8H); 2.41-2.96 (m; 2.1H); 2.04-2.23 (m; 0.8H); 1.82-1.95 (m; 2.2H); 1.43 (br s; 10.1H); 1.07 (d; J=6.5 Hz; 2.1H); 0.90 (d; J=6.5 Hz; 2.3H); 0.63 (d; J=6.5 Hz; 1.0H); 0.29 (d; J=6.5 Hz; 0.8H). 
       Most Polar Diastereoisomer (I-5 Dia2) (4.40 g; 27%) 
       [0171]    LCMS [M+H]=639 (C 35 H 41 ClFN 3 O 5 ) 
         [0172]      1 H NMR (300 MHz, CDCl 3 ): δ 8.00-8.10 (m; 1.0H); 7.30-7.40 (m; 2.1H); 6.98-7.18 (m; 3.2H); 6.73-6.90 (m; 5.3H); 6.52-6.58 (m; 1.0H); 6.34-6.39 (m; 1.0H); 4.71 (d; J=13.5 Hz; 0.7H); 4.49 (d; J=13.5 Hz; 0.4H); 3.50-4.00 (m; 4.7H); 3.10-3.30 (m; 0.7H); 2.86-3.07 (m; 0.4H); 2.54-2.85 (m; 1.5H); 2.30-2.47 (m; 0.4H); 1.80-1.87 (m; 2.8H); 1.54-1.60 (m; 2.5H); 1.42 (br s; 8.8H); 1.19 (d; J=6.6 Hz; 1.1H); 1.00 (d; J=6.6 Hz; 1.5H); 0.88 (d; J=6.6 Hz; 1.2H); 0.64 (d; J=6.6 Hz; 1.5H). 
       Examples I-6a and I-6b 
     Diastereoisomers of the tert-butyl ester of 4-[-2-[(2-chloro-acetyl)-(4-phenoxy-phenyl)-amino]-2-(4-fluoro-phenyl)-acetyl]-(R)-2-isopropyl-piperazine-1-carboxylic acid 
       [0173]    
       
                 
         
             
             
         
       
     
         [0174]    These two diastereoisomers were prepared in the same manner as in the preceding example in the form of colourless foam: 
       Least Polar Diastereoisomer (I-6 Dia1) (97 m; 30%) 
       [0175]    LCMS [M+H]=639 (C 35 H 41 ClFN 3 O 5 ) 
         [0176]      1 H NMR (300 MHz, CD 2 Cl 2 ): δ 7.94-8.57 (m; 0.9H); 7.35 (t; J=7.9 Hz; 1.9H); 7.05-7.25 (m; 3.1H); 6.72-6.93 (m; 5.0H); 6.58 (br d; J=2.6 Hz; 1.1H); 6.41 (s; 0.8H); 6.31 (br s; 0.3H); 4.63 (d; J=13.5 Hz; 0.8H) 4.40 (d; J=13.5 Hz; 0.3H); 3.51-4.06 (m; 4.8H); 3.03-3.45 (m; 1.0H); 2.41-2.96 (m; 1.6H); 2.02-2.21 (m; 0.8H); 1.82-1.95 (m; 2.1H); 1.43 (br s; 10.1H); 1.07 (d; J=6.5 Hz; 2.1H); 0.90 (d; J=6.5 Hz; 2.3H); 0.63 (d; J=6.5 Hz; 1.0H); 0.29 (d; J=6.5 Hz; 0.8H). 
       Most Polar Diastereoisomer (I-6 Dia2) (90 mg; 28%) 
       [0177]    LCMS [M+H]=639 (C 35 H 41 ClFN 3 O 5 ) 
         [0178]      1 H NMR (300 MHz, CDCl 3 ): δ 8.00-8.10 (m; 1.0H); 7.30-7.40 (m; 2.1H); 6.98-7.18 (m; 3.1H); 6.73-6.90 (m; 5.1H); 6.52-6.58 (m; 1.0H); 6.34-6.39 (m; 1.0H); 4.70 (d; J=13.5 Hz; 0.7H); 4.49 (d; J=13.5 Hz; 0.4H); 3.50-4.00 (m; 4.8H); 3.10-3.30 (m; 0.7H); 2.86-3.07 (m; 0.4H); 2.54-2.85 (m; 1.5H); 2.30-2.47 (m; 0.4H); 1.80-1.87 (m; 2.8H); 1.54-1.60 (m; 2.6H); 1.42 (br s; 8.6H); 1.20 (d; J=6.6 Hz; 1.1H); 1.01 (d; J=6.6 Hz; 1.5H); 0.89 (d; J=6.6 Hz; 1.2H); 0.64 (d; J=6.6 Hz; 1.5H). 
       Examples I-7a and I-7b 
     Hydrochloride of the diastereoisomers of 2-chloro-N-[-1-(4-fluoro-phenyl)-2-((S)-3-isopropyl-piperazin-1-yl)-2-oxo-ethyl]-N-(2-methyl-4-phenoxy-phenyl)-acetamide 
       [0179]    
       
                 
         
             
             
         
       
     
         [0180]    To a solution of one of the diastereoisomers I-5a and I-5b in 50 mL of DCM the HCl gas was added by bubbling. The reaction medium was left under agitation for 12 hours at RT. The DCM was evaporated and the residual oil precipitated in ethyl ether. 
         [0000]    Starting from the First Diastereoisomer of Example I-5 (I-7 Dia1): 
         [0181]    Recovery of a white powder after filtration: (26 mg) 
         [0182]    LCMS [M+H]=538 (C 30 H 34 Cl 2 FN 3 O 3 ) 
         [0183]      1 H NMR (300 MHz, DMSO): δ 8.79-9.33 (m; 1.3H); 7.83 (t; J=9.0 Hz; 1.0H); 7.24-7.40 (m; 4.0H); 6.97-7.15 (m; 3.1H); 6.73-6.89 (m; 3.2H); 6.64 (d; J=2.7 Hz; 0.9H); 6.51-6.59 (m; 1.0H); 4.40-4.55 (br m; 1.1H); 3.86-4.09 (m; 3.6H); 3.45-3.60 (m; 0.7H); 2.78-3.05 (m; 2.8H); 1.79-2.00 (m; 4.5H); 1.61-1.77 (m; 0.7H); 0.97 (d; J=6.7 Hz; 6.0H). 
         [0000]    Starting from the Second Diastereoisomer of Example I-5 (I-7 Dia2): 
         [0184]    Recovery of a white powder after filtration: (2.62 g) 
         [0185]    LCMS [M+H]=538 (C 30 H 34 Cl 2 FN 3 O 3 ) 
         [0186]      1 H NMR (300 MHz, DMSO): δ 8.78-9.51 (m; 1.9H); 7.82 (t; J=8.9 Hz; 0.9H); 7.20-7.41 (m; 4.0H); 6.97-7.16 (m; 3.1H); 6.71-6.90 (m; 3.1H); 6.61-6.70 (m; 1.9H); 4.46-4.60 (br m; 1.0H); 3.85-4.15 (m; 3.1H); 3.00-3.30 (m; 3.0H); 2.57-2.96 (m; 1.8H); 1.43-1.98 (m; 4.3H); 1.00 (dd; J=8.8 Hz; J=7.0 Hz; 2.7H); 0.71 (d; J=6.8 Hz; 1.6H); 0.65 (d; J=6.8 Hz; 1.5H). 
       Example I-8 
     Hydrochloride of 2-chloro-N-[-1-(4-fluoro-phenyl)-2-((R)-3-isopropyl-piperazin-1-yl)-2-oxo-ethyl]-N-(2-methyl-4-phenoxy-phenyl)-acetamide 
       [0187]    
       
                 
         
             
             
         
       
     
         [0188]    The same protocol as in the preceding example was followed starting from each of the diastereoisomers of Example I-6. 
         [0000]    Starting from the First Diastereoisomer of Example I-6 (I-8 Dia1): 
         [0189]    Recovery of a white powder after filtration: (34 mg; 56%) 
         [0190]    LCMS [M+H]=538 (C 30 H 34 Cl 2 FN 3 O 3 ) 
         [0191]      1 H NMR (300 MHz, DMSO): δ 8.79-9.33 (m; 1.3H); 7.83 (t; J=9.0 Hz; 0.9H); 7.24-7.40 (m; 4.0H); 6.97-7.15 (m; 3.1H); 6.73-6.89 (m; 3.1H); 6.64 (d; J=2.7 Hz; 1.0H); 6.51-6.59 (m; 1.0H); 4.41-4.56 (br m; 1.1H); 3.86-4.09 (m; 3.4H); 3.45-3.60 (m; 0.7H); 2.78-3.05 (m; 2.8H); 1.79-2.00 (m; 4.4H); 1.61-1.77 (m; 0.8H); 0.97 (d; J=6.7 Hz; 6.0H). 
         [0000]    Starting from the Second Diastereoisomer of Example 16 (I-8 Dia2): 
         [0192]    Recovery of a white powder after filtration: (30 mg; 54%) 
         [0193]    LCMS [M+H]=538 (C 30 H 34 Cl 2 FN 3 O 3 ) 
         [0194]      1 H NMR (300 MHz, DMSO): δ 8.78-9.51 (m; 1.5H); 7.82 (t; J=8.9 Hz; 1.0H); 7.20-7.41 (m; 4.0H); 6.97-7.16 (m; 3.1H); 6.71-6.90 (m; 3.2H); 6.61-6.70 (m; 1.9H); 4.46-4.60 (br m; 1.0H); 3.85-4.15 (m; 3.1H); 3.00-3.30 (m; 2.9H); 2.57-2.96 (m; 1.8H); 1.43-1.98 (m; 4.3H); 1.00 (dd; J=8.8 Hz; J=7.0 Hz; 2.7H); 0.71 (d; J=6.8 Hz; 1.6H); 0.65 (d; J=6.8 Hz; 1.5H). 
         [0195]    Using the same operating modes and the same separation modes by silica chromatography as above, the following examples were prepared from diversely substituted anilines and piperazines. They were isolated either in the form of a mixture of two or four diastereoisomers (one example number for the same chemical structure) or in the form of separate diastereoisomers. In this latter case the nomenclature a/b was used to designate each of the diastereoisomers. 
         [0000]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
         [0196]    The following examples were obtained by replacing the ethyl ester of (4-fluoro-phenyl)-oxo-acetic acid by ethyl thiophene-2-glyoxylate and following the same operating modes as previously. 
         [0000]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
       II—Pharmacological Study of the Compounds of the Present Invention 
       [0197]    1) Cytotoxicity Tests 
         [0000]    Assay with Various Compounds According to the Invention on MCF 7, MCF-7/Adr, HL-60, HL-60/R10, HT29, Mia Paca2, Panc-1 and SK-OV-3 cell lines: 
         [0198]    The effects of the compounds of the invention on the proliferation of cancer cells were studied on different human cancer cell lines of different tissue origin (MCF-7: breast cancer, MCF-7/adr adriamycin-resistant breast cancer, HL-60: acute promyelocytic leukaemia, HL-60/R10: doxorubicin-resistant acute promyelocytic leukaemia, HT29: colon adenocarcinoma, Mia Paca2: pancreatic tumour, Panc-1: pancreatic exocrine tumour, SK-OV-3: cisplatin- and adriamycin resistant ovarian cancer). The cancer cells used for this study were incubated at 37° C. in the presence of one of the compounds of the invention added to the culture medium at different concentrations. 
         [0199]    The cancer cell lines were obtained from ATCC (American Type Culture Collection) for MCF-7, from Hôpital de la Pitié Salpetrière for MCF-7/adr and from Oncodesign (Dijon, France) for HL-60, HL-60/R10, HT29, MiaPaCa2, Panc-1 and SK-OV-3. They were cultured in RPMI 1640 medium containing 2 mM L-Glutamine supplemented with 10% foetal calf serum (Lonza; Verviers, Belgium). All the cell lines were held in culture at 37° C. in a humid atmosphere containing 5% CO 2 . Cell proliferation was assessed using the ViaLight® Plus Assay Kit (Lonza; Verviers, Belgium) following the manufacturer&#39;s instructions. The cells were seeded in 96-well culture plates compatible with luminescence read-off (white plates with transparent bottom) in a proportion of 5 000 to 10 000 cells per well in 100 μl of culture medium. After a pre-incubation time of 24 hours at 37° C., the compounds of the invention dissolved in dimethylsulfoxide (DMSO) were individually added to each well in a proportion of 0.5 μl per well. After 72 hours&#39; incubation at 37° C. in a humid atmosphere containing 5% CO 2 , 50 μl of lysis buffer were added to each well and 15 minutes later 100 μl of ATP measuring agent were added. The plates were read under a luminometer to evaluate cell viability. The data obtained was processed by computer to obtain the EC 50  value i.e. the concentration value of each of the compounds which induces 50% cell viability compared with a control value (0.5% DMSO alone). 
         [0200]    The results obtained are given in following Tables 1 and 2. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Results obtained with cell lines HL-60, HL60/R10, 
               
               
                 MCF-7 and MCF-7/adr (EC 50  expressed in nM) 
               
             
          
           
               
                   
                 EC 50  (nM) 
                   
               
             
          
           
               
                 Example No 
                 HL-60 
                 HL60/R10 
                 MCF-7 
                 MCF-7/adr 
               
               
                   
               
             
          
           
               
                 I-1 dia1 
                 1799 
                 −219 
                 −2500 
                 338 
               
               
                 I-1 dia2 
                 824 
                 23 
                 1304 
                 39 
               
               
                 I-2 dia1 
                 728 
                 40 
                 2091 
                 49 
               
               
                 I-2 dia2 
                 2061 
                 472 
                 2500 
                 645 
               
               
                 I-3 dia1 
                 1311 
                 15 
                 927 
                 55 
               
               
                 I-3 dia2 
                 504 
                 2 
                 301 
                 9 
               
               
                 I-4 dia1 
                 648 
                 4 
                 315 
                 13 
               
               
                 I-4 dia2 
                 1321 
                 62 
                 863 
                 166 
               
               
                 I-7 dia1 
                 2500 
                 604 
                 2500 
                 618 
               
               
                 I-7 dia2 
                 1938 
                 26 
                 1954 
                 98 
               
               
                 I-8 dia1 
                 2029 
                 54 
                 1740 
                 148 
               
               
                 I-8 dia2 
                 1737 
                 312 
                 2410 
                 477 
               
               
                 I-9 
                 771 
                 99 
                 2500 
                 108 
               
               
                 I-10 
                 641 
                 131 
                 2500 
                 151 
               
               
                 I-11 
                 939 
                 68 
                 2500 
                 102 
               
               
                 I-12 
                 2321 
                 82 
                 1787 
                 421 
               
               
                 I-13 
                 1989 
                 88 
                 2500 
                 532 
               
               
                 I-14 
                 1848 
                 95 
                 2500 
                 232 
               
               
                 I-16 dia1 
                 1478 
                 450 
                 2500 
                 527 
               
               
                 I-16 dia2 
                 1913 
                 317 
                 2500 
                 443 
               
               
                 I-17 dia1 
                 2313 
                 353 
                 2500 
                 898 
               
               
                 I-17 dia2 
                 1604 
                 49 
                 1994 
                 152 
               
               
                 I-18 dia1 
                 1596 
                 140 
                 2500 
                 485 
               
               
                 I-18 dia2 
                 352 
                 44 
                 591 
                 152 
               
               
                 I-19 dia1 
                 516 
                 97 
                 2500 
                 128 
               
               
                 I-19 dia2 
                 453 
                 24 
                 2500 
                 35 
               
               
                 I-20 dia1 
                 196 
                 80 
                 1571 
                 73 
               
               
                 I-20 dia2 
                 1478 
                 139 
                 2500 
                 165 
               
               
                 I-21 dia1 
                 2447 
                 13 
                 1950 
                 93 
               
               
                 I-21 dia2 
                 630 
                 8 
                 869 
                 10 
               
               
                 I-22 dia1 
                 1995 
                 17 
                 569 
                 131 
               
               
                 I-22 dia2 
                 1149 
                 18 
                 430 
                 34 
               
               
                 I-23 
                 559 
                 12 
                 nd 
                 nd 
               
               
                 I-24 
                 626 
                 15 
                 nd 
                 nd 
               
               
                 I-25 dia1 
                 1122 
                 43 
                 nd 
                 nd 
               
               
                 I-25 dia2 
                 819 
                 103 
                 nd 
                 nd 
               
               
                 I-26 dia1 
                 2447 
                 13 
                 1950 
                 93 
               
               
                 I-26 dia2 
                 630 
                 8 
                 869 
                 10 
               
               
                 I-27 dia1 
                 1995 
                 17 
                 569 
                 131 
               
               
                 I-27 dia2 
                 1149 
                 18 
                 430 
                 34 
               
               
                 I-28 
                 623 
                 206 
                 nd 
                 nd 
               
               
                 I-29 
                 939 
                 38 
                 1032 
                 54 
               
               
                 I-30 
                 202 
                 10 
                 1006 
                 44 
               
               
                 I-31 
                 766 
                 64 
                 2500 
                 89 
               
               
                 I-32 
                 175 
                 78 
                 2500 
                 58 
               
               
                 I-33 
                 2500 
                 157 
                 2500 
                 394 
               
               
                 I-34 
                 1123 
                 30 
                 1775 
                 100 
               
               
                 I-35 
                 300 
                 249 
                 632 
                 863 
               
               
                 I-36 
                 372 
                 28 
                 2500 
                 24 
               
               
                 I-37 
                 967 
                 154 
                 2500 
                 156 
               
               
                 I-38 
                 2500 
                 189 
                 2500 
                 851 
               
               
                 I-39 
                 1814 
                 9 
                 776 
                 52 
               
               
                 I-40 dia1 
                 2129 
                 248 
                 2500 
                 371 
               
               
                 I-40 dia2 
                 2500 
                 487 
                 2500 
                 827 
               
               
                 I-43 dia2 
                 1886 
                 20 
                 1424 
                 126 
               
               
                 I-44 
                 215 
                 7 
                 nd 
                 nd 
               
               
                 I-45 
                 2136 
                 17 
                 nd 
                 nd 
               
               
                 I-46 dia1 
                 2500 
                 318 
                 2500 
                 544 
               
               
                 I-46 dia2 
                 449 
                 10 
                 1184 
                 58 
               
               
                 I-47 dia1 
                 936 
                 54 
                 nd 
                 nd 
               
               
                 I-47 dia2 
                 849 
                 8 
                 nd 
                 nd 
               
               
                 I-48 
                 1991 
                 10 
                 nd 
                 nd 
               
               
                 I-49 
                 1166 
                 321 
                 nd 
                 nd 
               
               
                 I-50 dia1 
                 2102 
                 259 
                 nd 
                 Nd 
               
               
                 I-50 dia2 
                 841 
                 8 
                 nd 
                 nd 
               
               
                 I-51 dia1 
                 145 
                 96 
                 nd 
                 nd 
               
               
                 I-51 dia2 
                 3 
                 1 
                 nd 
                 nd 
               
               
                 I-52 dia1 
                 2500 
                 194 
                 nd 
                 nd 
               
               
                 I-52 dia2 
                 223 
                 24 
                 nd 
                 nd 
               
               
                 I-53 dia1 
                 1981 
                 645 
                 2064 
                 462 
               
               
                 I-53 dia2 
                 547 
                 305 
                 nd 
                 nd 
               
               
                 I-58 
                 1058 
                 11 
                 870 
                 88 
               
               
                 I-59 
                 956 
                 16 
                 745 
                 106 
               
               
                   
               
               
                 nd = non-determined 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Results obtained with other cell lines (EC 50  expressed in nM) 
               
             
          
           
               
                   
                 EC 50  (nM) 
                   
               
             
          
           
               
                   
                 Example No 
                 HT29 
                 Mia PaCa2 
                 Panc-1 
                 SK-OV-3 
               
               
                   
                   
               
             
          
           
               
                   
                 I-3 dia2 
                 1 
                 2 
                 1 
                 1 
               
               
                   
                 I-7 dia2 
                 8 
                 13 
                 5 
                 7 
               
               
                   
                 I-43 dia2 
                 10 
                 18 
                 7 
                 9 
               
               
                   
                 I-46 dia2 
                 6 
                 10 
                 4 
                 6 
               
               
                   
                 I-50 dia2 
                 9 
                 2200 
                 9 
                 11 
               
               
                   
                   
               
             
          
         
       
     
         [0201]    Following Tables 3 and 4 illustrate the gain in cytotoxic activity on the resistant HL60/R10 line, obtained with the compounds having a piperazine substituted at alpha position of nitrogen 4 of the piperazine compared with a non-substituted piperazine and/or substituted at another position of the piperazine. The best cytotoxic activity is obtained with the absolute configuration (S) of this substitution. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Results obtained with different substitutions of piperazine 
               
             
          
           
               
                   
                 EC 50  (nM) 
               
             
          
           
               
                 Example 
                 HL-60 
                 HL-60/R10 
               
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 1627 
                 226 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 2321 
                 82 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 1989 
                 88 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 1848 
                 95 
               
               
                   
               
             
          
         
       
     
         [0000]                                                          TABLE 4                   Results obtained with different substitutions of piperazine                EC 50  (nM)            Example   HL-60   HL-60/R10                                                  579   21                                             1058   11                                             662   65                                             966   16                                             311   38                    
Assay with Compound I-43b on the NCI 60 Tumor Cell Lines:
 
         [0202]    Compound I-43b has been tested in the NCI 60 cell line panel. This screen utilizes 60 different human tumor cell lines, representing leukemia, melanoma, lung cancer, colon cancer, CNS cancer, ovarian cancer, breast cancer, prostate cancer and renal cancer. 
         [0203]    The 60 human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37° C., 5% CO 2 , 95% air and 100% relative humidity for 24 h prior to addition of the tested drug. 
         [0204]    After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Compound I-43b is solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/ml gentamicin. Aliquots of 100 μl of these different drug dilutions are added to the appropriate microtiter wells already containing 100 μl of medium, resulting in the required final drug concentrations. 
         [0205]    Following drug addition, the plates are incubated for an additional 48 h at 37° C., 5% CO 2 , 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4° C. The supernatant is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentrations levels. Percentage growth inhibition is calculated as: 
         [0000]      [( Ti−Tz )/( C−Tz )]×100 for concentrations for which  Ti&gt;/=Tz  
 
         [0000]      [( Ti−Tz )/ Tz]× 100 for concentrations for which  Ti&lt;Tz.    
         [0206]    Three dose response parameters are calculated for each experimental agent. Growth inhibition of 50% (GI50) is calculated from [(Ti−Tz)/(C−Tz)]×100=50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) is calculated from Ti=Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment is calculated from [(Ti−Tz)/Tz]×100=−50. Values are calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested. 
         [0207]    The output from the single dose screen is reported as a mean graph on  FIG. 3 . Compound I-43b shows selective growth inhibition on all these tumor cell lines at the concentration of 10 μM. This screen is unique in that the complexity of a 60 cell line dose response produced by compound I-43b results in a biological response pattern which can be utilized in pattern recognition algorithms (COMPARE program. See: http://dtp.cancer.gov/docs/compare/compare.html). Using these algorithms, it has been possible to determine that the response pattern of compound I-43b is unique and not similar to that of any of the standard prototype compounds included in the NCI database. 
         [0208]    In addition, Compound I-43b has been evaluated against the 60 cell panel at five concentration levels to determine the concentration inhibiting 50% of cell proliferation of each cell line. The results are presented on  FIGS. 4A, 4B and 4C  (with X axis: Log 10  sample concentration (Molar)−Y axis: Percentage Growth). 
         [0000]    Assay with Compound I-43b on HNSCC and HCC: 
         [0209]    Compound I-43b has been tested in 3 human cancer cell lines established from Head &amp; Neck (HNSCC) and in 4 human cancer cell lines established hepatocarcinoma (HCC). In all cases, compound I-43b inhibited cell proliferation in a concentration dependent manner (see results on  FIG. 2 ). Cell proliferation has been evaluated using the MTT assay after 72 hours of treatment. 
         [0210]    2) Determination of Aqueous Solubility 
         [0211]    Aqueous solubility is a major physicochemical parameter for improving the ADME properties (Absorption, Distribution, Metabolism and Excretion) in a molecule (Drug-like properties: concepts, structure design and methods, Edward Harvel Kerns, Li Di; Academic Press, 2008). 
         [0212]    The aqueous solubility of each compound was measured at pH 7.4. It was measured using HPLC on the supernatants obtained by centrifugation after saturation of the media with excess compound after an agitation time of 24 h and at a temperature of 20° C. The preparation and treatment of the samples was robotized. 
         [0213]    Table 5 shows the gain in aqueous solubility obtained for a compound of the invention I-58 compared with a non-substituted piperazine or substituted at another position. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Aqueous solubility obtained with different piperazine substitutions. 
               
             
          
           
               
                   
                   
                 Solubility 
               
               
                   
                 Example 
                 (μg/mL) 
               
               
                   
                   
               
             
          
           
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 68 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 203 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 105 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 152 
               
               
                   
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 50 
               
               
                   
                   
               
             
          
         
       
     
         [0214]    3) Pharmacokinetic Parameters in Mice 
         [0215]    The pharmacokinetic behaviour of compounds is a pre-requisite for reasonable use thereof in in vivo experimentation. The compounds were given in DMSO solution via intravenous route (IV) or oral route (PO) to balb/c mice. Blood samples were taken at times ranging from 5 minutes to 6 hours, the plasmas were collected and the concentration of the compounds in each sample was assayed by LC/MS/MS. The data obtained allowed the plotting of time-concentration curves and determination of fundamental parameters such as plasma half-life of the compound (T½), area under curve at a given time (AUCt) and the maximum concentration obtained (Cmax). Table 6 shows the gain contributed by piperazine substitution on the pharmacokinetic parameters of the compounds administered via intravenous route at a dose of 10 mg/kg. 
         [0216]      FIG. 1  gives the time-plasma concentration curves in a mouse after administration of I-43 dia2 via IV and PO route. Compound I-43 dia2 therefore shows good bioavailability in a mouse, in particular via oral route. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Pharmacokinetic parameters obtained with various piperazine substitutions. 
               
             
          
           
               
                   
                 Cmax 
                 AUCt 
                 AUCinf 
                   
               
               
                 Example 
                 (ng/mL) 
                 (ng/mL*h) 
                 (ng/mL*h) 
                 t½ (h) 
               
               
                   
               
             
          
           
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 1469.50 
                 1278.25 
                 1321.68 
                 1.38 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 3797.6 
                 3287.08 
                 3837.61 
                 2.64 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 1424.37 
                 1677.94 
                 2057.38 
                 2.47