Abstract:
Method of treating a tumor in a patient in need thereof by administering to the patient an effective amount of 4-[6-(diethylaminomethyl)naphth-2-ylmethyloxycarbamoyl]benzohydrozamic acid.

Description:
This application is the U.S. National Phase of International Application PCT/EP2003/014171, filed 10 Dec. 2003, which designated the U.S. PCT/EP2003/014171 claims priority to Italian Application No. MI2003A000064 filed 17 Jan. 2003. The entire content of these applications are incorporated herein by reference. 
     DESCRIPTION 
     The present invention relates to the use of hydroxamic acid derivatives containing an amidobenzoic moiety for the preparation of anti-tumour medicaments. 
     BACKGROUND OF THE INVENTION 
     Hydroxamic acid derivatives containing an amidobenzoic moiety are disclosed in EP 901465 as potential medicaments with anti-inflammatory and immuno-suppressive activity, ascribable to the inhibition of the production of pro-inflammatory cytokines, in particular Tumour Necrosis Factor and interleukin-1-beta. 
     Said derivatives are represented by the following general formula (I) 
                                
wherein
     R′ is hydrogen or (C 1-4 )alkyl;   A is adamantyl or a mono-, bi- or tricyclic residue, which can optionally be partially or completely unsaturated, contain one or more heteroatoms selected from the group consisting of N, S or O, and optionally substituted with hydroxy, alkanoyloxy, primary, secondary or tertiary amino, amino(C 14 )alkyl, mono- or di-(C 1-4 )alkyl-amino(C 1-4 )alkyl, halogen, (C 14 )alkyl, tri(C 1-4 )alkylammonium-(C 14 )alkyl;              is a 1 to 5 carbon atoms chain optionally containing a double bond or a NR′ group wherein R′ is as defined above;   R is hydrogen or phenyl;   X is an oxygen atom or a NR′ group wherein R′ is either as defined above or absent;   r and m are independently 0, 1 or 2;   B is a phenylene or a cyclohexylene ring;   Y is hydroxy or an amino(C 1-5 )alkyl chain optionally interrupted by an oxygen atom;
 
with the proviso that a tricyclic group as defined for A is fluorenyl only when, at the same time, X is different from Ol and Y is different from hydroxy, unless said fluorenyl is substituted with a tri(C 14 )alkylammonium(C 14 )alkyl group.
   

     Preferred compounds of formula (I) are those in which R′ is hydrogen, A is optionally substituted phenyl or 1- or 2-naphthyl, cyclohexyl, 1- or 2-1,2,3,4-tetrahydronaphthyl, adamantyl, quinolinyl, isoquinolinyl, 1- or 2-indenyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl. 
     Most preferred are the compounds (I) in which A is phenyl or 1- or 2-naphthyl, R is phenyl when A is phenyl or is hydrogen when A is 1- or 2-naphthyl. 
     Said compounds can be prepared according to methods disclosed in EP 901465, herein incorporated by reference. 
     SUMMARY OF THE INVENTION 
     It has now been found that the compounds of formula (I) are endowed with anti-tumour activity both in vitro and in vivo. 
     The invention relates therefore to the use of compounds of formula (I) 
                                
wherein
     R′ is hydrogen or (C 1-4 )alkyl;   A is adamantyl or a mono-, bi- or tricyclic residue, which can optionally be partially or completely unsaturated, optionally containing one or more heteroatoms selected from the group consisting of N, S or O, and optionally substituted substituted with hydroxy, alkanoyloxy, primary, secondary or tertiary amino, amino(C 14 )alkyl, mono- or di-(C 1-4 )alkyl-amino(C 1-4 )alkyl, halogen, (C 14 )alkyl, tri(C 1-4 )alkylammonium-(C 14 )alkyl;              is a 1 to 5 carbon atoms chain optionally containing a double bond or a NR′ group wherein R′ is as defined above;   R is hydrogen or phenyl;   X is an oxygen atom or a NR′ group wherein R′ is either as defined above or absent;   r and m are independently 0, 1 or 2;   B is a phenylene or a cyclohexylene ring;   Y is hydroxy or an amino(C 1-5 )alkyl chain optionally interrupted by an oxygen atom;
 
with the proviso that a tricyclic group as defined for A is fluorenyl only when, at the same time, X is different from O and Y is different from hydroxy, unless said fluorenyl is substituted with a tri(C 14 )alkylammonium(C 14 )alkyl group for the preparation of anti-tumor medicaments.
   

     Compounds (I) can be used for the treatment of neoplasias of different origin, in particular of melanomas, colon, lung and breast carcinomas, neuroblastomas, sarcomas, various forms of leukaemia (erythroleukaemia, acute promyelocytic leukaemia) and the like, at daily single or multiple doses ranging from 1 to 500 mg, depending on the disease and pharmaceutical and toxicological characteristics of the considered compound, which can be administered as suitable formulations through the oral, parenteral or topical route, for example through direct perfusion at the site of the tumour lesion. Moreover, the compounds of formula (I) can be administered in combination with other known antineoplastic agents, according to polychemotherapy protocols. 
     The activity of the compounds of formula (I) was evidenced in vitro, on cultured tumour cell lines, and in vivo, on the experimental model of the murine melanoma B16-BL6. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention will be further described with reference to the accompanying FIGURE which shows plots of tumor weights versus Days. 
     
    
    
     EXAMPLES 
     The following examples illustrate the invention in greater detail. 
     Example 1 
     In Vitro Activity 
     The anti-tumour effect of the compound of example 12 of EP 901465, 4-[6-(diethylaminomethyl)naphth-2-ylmethyloxycarbamoyl)] benzohydroxamic acid hydrochloride (ITF 2357), was tested in vitro by measuring the capacity of the compound to inhibit the growth of tumour cell lines of different histotype deriving from both solid tumours and haematological tumours. The cell lines used were: A549 (pulmonary carcinoma), MDA-MB435 (breast carcinoma) and KG-1 (myeloid leukaemia). 
     A549 and MDA-MB435 cells (respectively grown in E-MEM and D-MEM culture medium, supplemented with 10% of fetal calf serum) were seeded on 96 wells-flat bottom-plates (5000 cells/well) and allowed to adhere for 4 hours at 37° C. under 5% CO 2  atmosphere. Thereafter ITF 2357 at various doses was added to each well (4 replicates). After further 36 hours, tritiated thymidine (1 μCi/well) was added to each well and left therein for the following 12 hours. After this time, the cells were washed 3 times with culture medium and then solubilized with 1N NaOH for 30 minutes. 
     A liquid scintillation beta counter was used for measurement of the radioactivity contained in each sample, which is directly proportional to DNA synthesis and therefore to cell proliferation. KG-1 cells, grown in RPMI 1640 culture medium, supplemented with 5% fetal calf serum, were seeded in 96 wells-flat bottom-plates (250000 cells/well). ITF 2357 at various doses was immediately added and after 36 hours tritiated thymidine was added to each well (1 μCi/well) and left therein for the following 12 hours. At the end the cells were collected by a cell harvester and the radioactivity contained in each sample which is directly proportionated to DNA synthesis and therefore to cell proliferation was measured by a liquid scintillation beta counter. 
     The effect of different doses of ITF 2357 was measured as percent of inhibition of radioactivity incorporation compared with untreated control cells. The concentration capable of inducing a 50% cell growth inhibition (IC 50 ) was then determined by means of linear regression. 
     The results obtained are summarized in the following table: 
     
       
         
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Cell line 
               
             
          
           
               
                   
                 A549 
                 MDA-MB435 
                 KG-1 
               
               
                   
                   
               
             
          
           
               
                   
                 IC 50 (nM) 
                 495 
                 73 
                 552 
               
               
                   
                   
               
             
          
         
       
     
     The results obtained show that ITF 2357 inhibits in vitro, at very low dosages (range 10-9M), the growth of the cell lines employed. ITF 2357 in particular inhibits cell growth both of cells from solid tumours (A549 and MDA-MB435) and from leukaemias (KG-1), therefore suggesting its use on tumours of different histotype. 
     Example 2 
     In Vitro Activity 
     Cell lines derived from human solid tumours of different histotype and stabilised in vitro were used in the following experiment. In particular, three cell lines from head and neck tumours (KB, Ca127 and Hep2), two cell lines derived from colon carcinomas (HT-29 and LoVo) and four cell lines derived from melanomas (Colo38, Pes41, Pes43 and Anad) were studied. 
     The cells were grown according to conventional methods in flasks containing synthetic culture medium added with fetal serum, at 37° C. under 5% CO 2  atmosphere, then seeded in 96 wells plates and allowed to adhere for some hours. ITF 2357 was added in triplicate at increasing doses to each well and the cells were incubated for further 72 hours. Viable cells were labelled by dyeing with sulforhodamine B and their amount was determined by spectrophotometric evaluation of the dye content in each well. The effect of ITF 2357 was calculated as percentage of inhibition, at each concentration, of the dye incorporation in wells containing ITF 2357 compared with control wells (cells without drug). IC 50  values of ITF 2357 were calculated by Software Calcusyn (Biosoft) according to dose-response curves. 
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                   
                 ITF 2357 
               
               
                   
                 Cell line 
                 Tumour origin 
                 IC 50  (μM) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 KB 
                 Head-neck 
                 0.64 
               
               
                   
                 Cal27 
                 Head-neck 
                 3.4 
               
               
                   
                 Hep-2 
                 Head-neck 
                 1 
               
               
                   
                 HT-29 
                 Colon carcinoma 
                 0.7 
               
               
                   
                 LoVo 
                 Colon carcinoma 
                 2.5 
               
               
                   
                 Colo38 
                 Melanoma 
                 2.38 
               
               
                   
                 Pes41 
                 Melanoma 
                 1.6 
               
               
                   
                 Pes43 
                 Melanoma 
                 1.4 
               
               
                   
                 Anad 
                 Melanoma 
                 5.7 
               
               
                   
                   
               
             
          
         
       
     
     Example 3 
     In Vivo Activity 
     The anti-tumour effect of ITF 2357 was studied measuring the capacity of the compound to reduce the growth of the murine melanoma B16-BL6. 
     B16-BL6 tumour is a highly metastatic variant (Sciumbata T. et al. Invasion and Metastasis 1996; 16: 132-143) of the native tumour B16 and it grows subcutaneously in the syngenic mouse C57BL/6 (Gutman M et al. Cancer Biother. 1994; 9(2): 163-170). 
     B16-BL6 tumour cells were inoculated subcutaneously in female C57BL/6 mice (10 animals/group, weight 20-22 grams) at the dose of 2×10 5  cells/mouse. ITF 2357 dissolved in water was administered orally, at the indicated doses, 10 minutes before the inoculum of the tumour cells and then daily for 6 days a week. The tumour growth was expressed as tumour weight, measuring twice a week, by means of a calibre, the two perpendicular diameters of the nodules. The weight was calculated according to the formula: (diameter 1×diameter 2) 2 /2 as described in Giavazzi R. et al. Cancer Res. 1986; 46: 1928-1933. 
     The results obtained are reported in the enclosed FIGURE. 
     It can be observed that ITF 2357 exerts a dose-dependent inhibitory effect on the tumour growth, since it reduced of about 50% the volume of the tumoural nodule after 15 days of treatment.