Patent Publication Number: US-2011059977-A1

Title: USE OF CANTHIN-6-ONE AND ITS ANALOGS IN THE TREATMENT OF MYCOBACTERIA-LINKED PATHOLOGIES ( amended

Description:
This invention relates to the use of canthin-6-one and its analogs in the preparation of a drug for the treatment or prevention of pathologies linked to, or caused by mycobacteria, particularly tuberculosis. 
     Canthin-6-one is a known compound which has been isolated from plants such as:  Ailanthus altissima  (Simaroubaceae) by Ohmoto et al., Chem. Pharm. Bull., 1976, 24, 1532-1536;  Brucea antidysenterica  (Simaroubaceae) by Fukamiya et al., Planta Med., 1987, 53, 140-143;  Eurycoma harmandiana  (Simaroubaceae) by Kachanapoom et al., Phytochemistry, 2001, 56, 383-386;  Peganum nigellastrum  (Zygophyllaceae) by Ma et al., Phytochemistry, 2000, 53, 1075-1078. 
     Canthin-6-one and some of its derivatives show interesting pharmacological activities, and notably an antifungal activity (Thouvenel et al., 2003; Soriano-Agatón et al., 2005), and also a trypanocidal activity in Chagas&#39; disease (WO 2004/050092). 
     Mycobacteria, among other bacteria, have been thoroughly studied because they are responsible for two serious diseases: tuberculosis and leprosy. 
     Tuberculosis is an infectious, contagious and endemic disease, with a highly marked respiratory tropism, due to  Mycobacterium tuberculosis  (or Koch&#39;s bacillus).  Mycobacterium tuberculosis  belongs to the genus mycobacteria, and so does the leprosy bacillus ( Mycobacterium leprae ). 
     It is thought that tuberculosis kills some two million people each year (WHO, 2006). Moreover tuberculosis is more and more often associated with infections by the HIV virus (responsible for ca. 13% of deaths by AIDS worldwide); hence the importance of an anti-tuberculosis treatment for immunosuppressed patients, and above all AIDS patients. 
     Pulmonary tuberculosis is the most frequent variant, but other parts of the body may be infected (kidney, joints, genital organs, pericardium, brain . . . ). 
     Tuberculosis contamination is essentially due to the release of bacilli into the atmosphere, via droplets which are suspended in the air. Inhalation of a small number of droplets is enough for the infection of one individual. Transmission by food (ingestion of meat or milk which have been contaminated by  Mycobacterium bovis ) has all but disappeared. 
     The treatment of any form of tuberculosis (respiratory tract tuberculosis, bone and joints tuberculosis) rests upon the regular uptake of antibiotics. With any patient, a manifest primary infection with radiological or systemic signs must be considered as a tuberculosis and treated as such. 
     The standard treatment rests on the daily uptake, during 6 months, of isoniazide and rifampicine, together with the administration of pyrazinamide and ethambutol during the first two months (Working Group of the French High Council of Public Hygiene). The first aim of this polytherapy is to exert an influence upon the various bacilli populations, and to obtain a cure in six months&#39; time. The second aim is to prevent the selection of mutants which are resistant, and which could cause a resistant bacilli relapse. 
     However to this day the standard treatment is not always enough to prevent the appearance of resistant strains. 
     One of the aims of the invention is to provide a new class of compounds which are active for the treatment of pathologies which are associated with or caused by mycobacteriae. 
     One of the aims of the invention is to provide new drugs against tuberculosis with pharmacological properties whose efficiency is comparable to that of already used drugs, but which make it possible to remedy the emergence of strains which are resistant to the drugs in use. 
     One of the aims of the invention is to provide new drugs against tuberculosis which are active with immunosuppressed patients. 
     In this invention it has been found surprisingly that canthin-6-one and its derivatives have antimycobacterial properties. 
     This invention relates to the use, in the preparation of a drug for the treatment or prevention of pathologies which are linked to or caused by mycobacteria, of at least one compound having the following Formula (I): 
     
       
         
         
             
             
         
       
     
     in which:
         B represents:
           a nitrogen atom, or   a N-oxide N + —O − , group, or   a group having the formula N + —R ou   
               

     
       
         
         
             
             
         
       
     
     R representing an alkyl group, which is linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms, which may be substituted, for example by at least one halogen atom, and X −  represents an anion which may be chosen among mineral or organic anions,
         R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7  and R 8  represent independently:
           a hydrogen atom,   an alkyl group which is linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   a halogen atom chosen among chlorine, fluorine, bromine and iodine,   a halogenoalkyl group, in which the alkyl chain is linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms, and in which the halogen atom(s) are chosen among fluorine, chlorine, bromine or iodine,   a hydroxyl group,   a nitro —NO group,   a cyano —CN group,   a —SH group,   a carboxylic acid —COOH group,   an amide —CONH 2  group,   an amine —NH 2  group,   an alcoxy —OR a , group, wherein R a  represents an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   an alkylester —COOR b  group, wherein R b  represents an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   a secondary (—NHCOR c ) or tertiary   (—N(COR d )COR c ) alkylamide group, wherein R c  and R d  independently represent an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   a secondary (—NHR e ) or tertiary (—NR e R f ) alkylamine group, wherein R e  and R f  independently represent an alkyl group which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   an alkylthio (—SR g ) group, wherein R g  represents an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   a C 2 -C 6  heterocyclic group, comprising from 1 to 4 heteroatoms chosen among sulphur, nitrogen and oxygen,   a —SO 2 —NR h R i , group or a —NR h —SO 2 —R i  group, wherein R h -R i , independently represent an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   
           the groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7  and R 8  may also form the following intramolecular cyclisations:
           1/ cyclisation between R 1  and R 2  and/or   2/ cyclisation between R 3  and R 4  and/or   3/ cyclisation between R 5  and R 6  and/or   4/ cyclisation between R 6  and R 7  and/or   5/ cyclisation between R 7  and R 8 ,   6/ cyclisation between R 3  and B, notably when B represents N + —R,   7/ cyclisation between R 2  and B, notably when B represents N + —R,   
               

     said thus formed cycles being notably aromatic cycles comprising from 5 to 30 carbon atoms, and which may contain at least one heteroatom chosen among: O, N, S, the aromatic cycles being for example chosen among benzene, naphthalene, pyridine, pyrrole, thiophene, furan, pyrazine, 
     wherein said cycles may be substituted by
         a hydrogen atom,   a linear, branched or cyclic, saturated or unsaturated alkyl group, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   a halogen atom chosen among chlorine, fluorine, bromine and iodine,   a halogenoalkyl group, wherein the alkyl chain is linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms, and wherein the halogen atom(s) are chosen among fluorine, chlorine, bromine and iodine,   a hydroxyl group,   a nitro —NO group,   a cyano —CN group,   a —SH group, —   a carboxylic acid —COOH group,   a —CONH 2  group,   an amine —NH 2  group,   a —OR a  group, wherein R a  represents an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   an alkylester —COOR b  group, wherein R b  represents an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   a secondary (—NHCOR c ) or tertiary   (—N(COR d )COR c ) alkylamide group, wherein R c  and R d  independently represent an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   a secondary (—NHR e ) or tertiary (—NR e R f ) alkylamine group, wherein R e  and R f  independently represent an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   an alkylthio (—SR g ) group, wherein R g  represents an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms,   a C 2 -C 6  heterocyclic group, comprising from 1 to 4 heteroatoms chosen among sulphur, nitrogen and oxygen,   a —SO 2 —NR h R i  group or a —NR h —SO 2 —R i  group, wherein R h  and R i  independently represent an alkyl group, which may be linear, branched or cyclic, saturated or unsaturated, comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms.       

     Canthin-6-one corresponds to Formula (I) wherein the substituents R 1 -R 8  represent a hydrogen atom and B represent nitrogen. 
     By mycobacteria is meant all species of the genus  Mycobacterium , classified in the order of Actinomycetales, whose common characteristic is to be detected by Ziehl-Neelsen staining, and to be acid-alcohol-resistant (for example Timo Ulrichs et al. J. Pathol. 2005, 205:633-640 &lt;&lt;Modified immunohistological staining allows detection of Ziehl-Neelsen-negative  Mycobacterium tuberculosis  organisms and their precise localization in human tissue&gt;&gt;). 
     According to an advantageous embodiment the mycobacteria as included in the invention belong to the group comprising  Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium smegmatis, Mycobacterium africanum, Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium avium intracellulaire, Mycobacterium scrofulaceum, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei, Mycobacterium ulcerans  and  Mycobacterium abcessus.    
     According to an advantageous embodiment the invention relates to the use of the hereabove Formula (I) compounds, in the preparation of a drug for the treatment or prevention of pathologies which are linked to or caused by  Mycobacterium tuberculosis  or  Mycobacterium bovis.    
     Advantageously the invention relates to the use of a hereabove defined Formula (I) compound, in the preparation of a drug for the treatment or the prevention of the following pathologies: 
     Tuberculosis, and particularly
         Tuberculosis of the respiratory tract (pulmonary tuberculosis, tuberculous lymphadenitis, larynx, trachea and bronchi tuberculosis, tuberculous pleurisy and tuberculous primary infection of the respiratory tract),   Tuberculosis of the nervous system (tuberculous meningitis, tuberculous polyneuritis, tuberculous myelitis),   Tuberculosis of the bone and joints,   Tuberculosis of the urogenital tract,   Peripheral tuberculous adenopathy,   Tuberculosis of the intestine, the peritoneum and the mesenteric glands   Tuberculosis of the skin and the subcutaneous cellular tissue   Tuberculosis of the eye and ear   Tuberculosis of the adrenal glands   Acute miliary tuberculosis       

     Buruli&#39;s ulcer, 
     Nosocomial infections, 
     Leprosy, 
     Cutaneous infections, 
     Soft tissue infections, 
     Osteomyelites, 
     Localized abscesses, 
     Lipoid pneumoniae. 
     According to another advantageous embodiment, in the Formula (I) compounds wherein B represents 
     
       
         
         
             
             
         
       
     
     X −  is chosen among Cl − , Br − , I − , S − , PO 3   − , NO 3   − , 
     acetate, oxalate, tartrate, succinate, maleate, fumarate, gluconate, citrate, malate, ascorbate, benzoate. 
     According to an advantageous embodiment the invention relates to the use, such as hereabove defined, of Formula (I) compounds wherein B represents a nitrogen atom and the compounds correspond to the following Formula (II): 
     
       
         
         
             
             
         
       
     
     According to an advantageous embodiment, the invention relates to the use, as hereabove defined, of Formula (I) compounds wherein B represents a N-oxide NO group, and the compounds correspond to the following Formula (III): 
     
       
         
         
             
             
         
       
     
     According to an advantageous embodiment, the invention relates to the use, as hereabove defined, of Formula (I) compounds, wherein B represents a group having the Formula, 
     
       
         
         
             
             
         
       
     
     X −  and R being such as hereabove defined, and the compounds correspond to the following Formula (IV): 
     
       
         
         
             
             
         
       
     
     R notably represents a methyl or ethyl group, for instance substituted with at least one halogen atom such as F, Cl, Br, or I. 
     According to an advantageous embodiment, the invention relates to the use, as hereabove defined, of Formula (I) compounds, wherein R 3  and R 4  form a benzene cycle between them, and the compounds correspond to the following Formula (I-1): 
     
       
         
         
             
             
         
       
     
     According to an advantageous embodiment of the invention, the Formula (I) compounds are chosen among: 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     wherein R 1 , R 8 , B and X −  have the hereabove given meanings. 
     According to another advantageous embodiment, the Formula (I) compounds as used are such that at least one of radicals R 5 -R 8 , and notably R 6 , represents a halogen atom, notably a fluorine atom. 
     According to another advantageous embodiment of the invention, the Formula (I) compound which is used is canthin-6-one, which corresponds to the following formula; 
     
       
         
         
             
             
         
       
     
     According to an advantageous embodiment of the invention the Formula (I) compounds which are used are chosen among one of the following compounds: 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     The above-defined Formula (I) compounds are advantageously delivered at a dose from ca, 0.01 mg/kg/day to ca. 100 mg/kg/day, preferably of ca. 0.1-50 mg/kg/day, and advantageously from ca. 1 to ca. 20 mg/kg/day. 
     No toxicity has been observed in the various experiments for determining the lethal dose (LD50). Assays have been made with mice by intraperitoneally delivering a dose of 200 mg/kg/day of a Formula (I) compound. All tested mice survived. According to an advantageous embodiment of the invention the Formula (I) compounds as used in the invention are delivered orally. 
     The invention also relates to the compound N-oxide-benzo[e]canthin-6-one, corresponding to the following formula: 
     
       
         
         
             
             
         
       
     
     This compound is a new compound. 
     The invention also relates to a pharmaceutical composition comprising as an active substance the compound N-oxide-benzo[e]canthin-6-one, corresponding to the following formula: 
     
       
         
         
             
             
         
       
     
     in association with a pharmaceutically acceptable excipient. 
     The compounds as used in the invention may be prepared according to a process as described in Patent Application WO 2004/050092. In this application canthin-6-one has been isolated from plant extracts which contain it, and notably from the bark of the trunk of a Rutacea identified as  Zanthoxylum chiloperone  var.  angustifolium.    
     Canthin-6-one and its derivatives may be prepared by total chemical synthesis or hemisynthesis, as described in &lt;&lt;Soriano-Agatón et al., 2005; Journal of Natural Products, Volume 68, Number 11, November, 2005&gt;&gt;. 
     The following examples are given for illustrating in greater detail the various aspects of the invention, but they must not be construed as being forms which limit the invention. 
    
    
     EXAMPLE 1 
     Synthesis of canthin-6-one and its Analogs 
     Canthin-6-one and its analogs are obtained by total synthesis. A rapid access pathway to the general skeleton, allowing pharmacomodulations, has been developed. 
     The following Scheme follows this strategy for obtaining canthin-6-one 
     
       
         
         
             
             
         
       
     
     1-Synthesis of canthin-6-one from Tryptamine 
     4-[2-(1-H-3-indolyl)ethylcarbamoyl]-butanoic acid 
     In CH 2 Cl 2  (50 tryptamine (0.01 mole) is dissolved and succinic anhydride (1.1 eq.) is gradually added. This solution is agitated during 18 hours at room temperature, then concentrated under reduced pressure. The obtained solid is triturated with a minimum amount of CH 2 Cl 2 , filtrated and dried (&gt;97%). 
     Methyl 4-[2-(1H-3-indolyl)ethylcarbamoyl]-butanoate 
     4-[2-(1-H-3-indolyl)ethylcarbamoyl]-butanoic acid (0.008 mole) is dissolved in methanol (50 mL). Amberlyst H-15® resin (20% m/m) is added and the mixture is refluxed during 18 hours. After elimination of the resin by filtration and concentration under reduced pressure the obtained solid is triturated in ethly ether, then filtrated and dried (&gt;97%). 
     Methyl 4-(3-4-dihydro-9H-β-carboline-1-yl)-butanoate 
     Methyl 4-[2-(1H-3-indolyl)ethylcarbamoyl]-butanoate (0.90 mmole) is dissolved in benzene (10 mL). POCl 3  (3 eq.) is added dropwise at 5° C. The mixture is refluxed during 1 hour, then concentrated under reduced pressure and used as such in the next step. 
     Canthin-6-one 
     In CH 2 Cl 2  (25 mL) methyl 4-(3-4-dihydro-9H-β-carboline-1-yl)-butanoate (0.50 mmole) is dissolved and DBU (diazabicycloundecene, 3 eq.) is added. This solution is agitated during 18 hours at room temperature, then washed with a saturated aqueous NaHCO 3  solution. The organic phase is dried (Na 2 SO 4 ), then concentrated under reduced pressure. Canthin-6-one is purified by a silica gel column chromatography (elution:CH 2 Cl 2 /MeOH 99:1, 60% two steps). 
     2-Synthesis of canthin-6-one Derivatives 
     Canthin-6-one analogs may be synthesized by using techniques such as described in &lt;&lt;Soriano-Agatón et al., 2005; Journal of Natural Products, Volume 68, Number 11, November, 2005&gt;&gt;. 
     The compound N-oxide-benzo[e]canthin-6-one is prepared according to the following protocol: 
     Benzo[e]canthin-6-one (50 mg-0.2 mmole) is dissolved in CH 2 Cl 2  (25 mL), then m-chloroperbenzoic acid (3 eq.) is added. This solution is agitated during 18 hours at room temperature. Water (25 mL) is added and the bi-phase medium is agitated during 1 hr, The organic phase is then washed with a saturated aqueous NaHCO 3  solution (3×20 mL), then dried (Na 2 SO 4 ) and concentrated under reduced pressure. The obtained product is purified by silica gel column chromatography (elution:CH 2 Cl 2 /MeOH 9:1) to give the N-oxide-benzo[e]canthin-6-one in the form of a powder (40 mg-70%). 
     
       
         
         
             
             
         
       
     
     3-Analytic Data for canthin-6-one and its Analogs 
       
     
       
         
           
               
             
               
                   
               
             
            
               
                 Canthin-6-one: 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 microcrystalline yellow powder 
               
               
                 melting point: 161-163° C. 
               
               
                 IR υ max  cm −1 : 1,673 (- 
               
               
                 MS (IE): m/z [M + Na] +  243 
               
               
                 MSHR (IC): for C 14 H 8 N 2 ONa 
               
               
                 [M + Na] +  243.0534, found: m/z 243.0532 
               
               
                 R f : 0.6 (CH 2 Cl 2 /MeOH 9:1) 
               
               
                   
               
               
                 MNR  1 H (400 MHz, CDCl 3 ): 
               
            
           
           
               
               
               
               
               
            
               
                 δ (ppm) 
                 multiplicity 
                 coupling  n J (Hz) 
                 integration 
                 attribution 
               
               
                   
               
               
                 6.87 
                 d 
                   3 J 9.8 Hz 
                 1H 
                 H-5 
               
               
                 7.41 
                 t 
                   3 J 8.0 Hz 
                 1H 
                  H-10 
               
               
                 7.58 
                 t 
                   3 J 8.0 Hz 
                 1H 
                 H-9 
               
               
                 7.80 
                 d 
                   3 J 5.0 Hz 
                 1H 
                 H-1 
               
               
                 7.90 
                 d 
                   3 J 9.8 Hz 
                 1H 
                 H-4 
               
               
                 7.94 
                 d 
                   3 J 8.0 Hz 
                 1H 
                  H-11 
               
               
                 8.50 
                 d 
                   3 J 8.0 Hz 
                 1H 
                 H-8 
               
               
                 8.70 
                 d 
                   3 J 5.0 Hz 
                 1H 
                 H-2 
               
               
                   
               
            
           
           
               
            
               
                 MNR  13 C (100 MHz, CDCl 3 ): 116.1 (C-1); 117.0 (C-8); 122.4 
               
               
                 (C-11); 124.1 (C-11a); 125.4 (C-10); 128.7 (C-5); 129.9 (C-11b); 
               
               
                 130.6 (C-9); 131.7 (C-3b); 135.9 (C-3a); 139.1 (C-7a); 139.3 (C-4); 
               
               
                 145.6 (C-2); 159.2 (C-6). 
               
               
                   
               
               
                 10-methoxy-canthin-6-one: 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 microcrystalline yellow powder 
               
               
                 Melting point: 203-205° C. 
               
               
                 IR υ max  cm −1 : 1,673 (—C═O) 
               
               
                 MS (IC): m/z [M + H] +  251 
               
               
                 MSHR (IC): for C 15 H 11 N 2 O 2   
               
               
                 [M + H] +  251.0821, found: m/z 251.0825 
               
               
                 R f : 0.6 (CH 2 Cl 2 /MeOH 9:1) 
               
               
                   
               
               
                 RMN  1 H (400 MHz, CDCl 3 ): 
               
            
           
           
               
               
               
               
               
            
               
                 δ (ppm) 
                 multiplicity 
                 coupling  n J (Hz) 
                 integration 
                 attribution 
               
               
                   
               
               
                 3.96 
                 s 
                   
                 3H 
                 —OMe 
               
               
                 6.98 
                 d 
                   3 J 9.8 Hz 
                 1H 
                 H-5 
               
               
                 7.27 
                 dd 
                   3 J 9.0 Hz.  4 J 2.1 Hz 
                 1H 
                 H-9 
               
               
                 7.57 
                 d 
                   4 J 2.1 Hz 
                 1H 
                  H-11 
               
               
                 7.96 
                 d 
                   3 J 5.0 Hz 
                 1H 
                 H-1 
               
               
                 8.05 
                 d 
                   3 J 9.8 Hz 
                 1H 
                 H-4 
               
               
                 8.55 
                 d 
                   3 J 9.0 Hz 
                 1H 
                 H-8 
               
               
                 8.81 
                 d 
                   3 J 5.0 Hz 
                 1H 
                 H-2 
               
               
                   
               
            
           
           
               
            
               
                 MNR  13 C (100 MHz, CDCl 3 ): 56.0 (—OMe); 106.7 (C-11); 116.4 
               
               
                 (C-1); 118.1 (C-8); 118.3 (C-9); 125.8 (C-11a); 129.4 (C-5); 131.0 
               
               
                 (C-11b); 132.5 (C-3b); 133.8 (C-7a); 136.0 (C-3a); 138.7 (C-4); 
               
               
                 145.0 (C-2); 158.1 (C-10); 159.2 (C-6). 
               
               
                   
               
               
                 Benzo[e]canthin-6-one: 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 microcrystalline white powder 
               
               
                 Melting point: 228-230° C. 
               
               
                 IR υ max  cm −1 : 1,681 (—C═O) 
               
               
                 MS (IE): m/z [M + H] +  271 
               
               
                 MSHR (IC): for C 18 H 11 N 2 O 2   
               
               
                 [M + H] +  271.0871, found: m/z 271.2073 
               
               
                 R f : 0.6 (CH 2 Cl 2 /MeOH 9:1) 
               
               
                   
               
               
                 MNR  1 H (400 MHz, CDCl 3 ): 
               
            
           
           
               
               
               
               
               
            
               
                 δ (ppm) 
                 multiplicity 
                 coupling  n J (Hz) 
                 integration 
                 attribution 
               
               
                   
               
               
                 7.51 
                 t 
                   3 J 8.0 Hz 
                 1H 
                  H-10 
               
               
                 7-68-7.76 
                 m 
                   
                 2H 
                 H-14, H-9, 
               
               
                 7.87 
                 t 
                   3 J 8.0 Hz 
                 1H 
                  H-13 
               
               
                 7.91 
                 d 
                   3 J 5.0 Hz 
                 1H 
                 H-1 
               
               
                 8.10 
                 d 
                   3 J 8.0 Hz 
                 1H 
                  H-11 
               
               
                 8.61 
                 d 
                   3 J 8.0 Hz 
                 1H 
                  H-15 
               
               
                 8.75 
                 m 
                   
                 2H 
                 H-12. H-8  
               
               
                 8.81 
                 d 
                   3 J 5.0 Hz 
                 1H 
                 H-2 
               
               
                   
               
            
           
           
               
            
               
                 MNR  13 C (100 MHz, CDCl 3 ): 115.1 (C-1); 117.5 (C-8); 122.4 
               
               
                 (C-11); 123.5 (C-12); 124.9 (C-11a); 125.3 (C-10); 129.2 (C-15); 
               
               
                 129.5 (C-3a); 130.0 (C-14); 130.4 (C-3b); 130.5 (C-11b); 130.6 
               
               
                 (C-9); 133.5 (C-13); 134.7 (C-4); 136.0 (C-5); 139.4 (C-7a); 144.9 
               
               
                 (C-2); 159.4 (C-6). 
               
               
                   
               
               
                 Pyrazine[e]canthin-6-one: 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 microcrystalline brown 
               
               
                 Melting point: 250-253° C. 
               
               
                 IR υ max  cm −1 : 1,699 (—C═O) 
               
               
                 MS (IE): m/z [M + Na] +  295 
               
               
                 MSHR (IC): for C 16 H 8 N 4 ONa 
               
               
                 [M + Na] +  295.0596, found: m/z 295.0593 
               
               
                 R f : 0.4 (CH 2 Cl 2 /MeOH 9:1) 
               
               
                   
               
               
                 MNR  1 H (400 MHz, CDCl 3 ): 
               
            
           
           
               
               
               
               
               
            
               
                 δ (ppm) 
                 multiplicity 
                 coupling  n J (Hz) 
                 integration 
                 attribution 
               
               
                   
               
               
                 7.58 
                 t 
                   3 J 8.0 Hz 
                 1H 
                  H-10 
               
               
                 7.78 
                 t 
                   3 J 8.0 Hz 
                 1H 
                 H-9 
               
               
                 8.09 
                 d 
                   3 J 5.2 Hz 
                 1H 
                 H-1 
               
               
                 8.15 
                 d 
                   3 J 8.0 Hz 
                 1H 
                  H-11 
               
               
                 8.82 
                 d 
                   3 J 8.0 Hz 
                 1H 
                 H-8 
               
               
                 9.02 
                 d 
                   3 J 5.2 Hz 
                 1H 
                 H-2 
               
               
                 9.07 
                 d 
                   3 J 1.6 Hz 
                 1H 
                  H-13 
               
               
                 9.15 
                 d 
                   3 J 1.6 Hz 
                 1H 
                  H-14 
               
               
                   
               
            
           
           
               
            
               
                 MNR  13 C (100 MHz, CDCl 3 ): 117.3 (C-1); 117.8 (C-8); 122.7 
               
               
                 (C-11); 124.7 (C-11a); 126.2 (C-10); 131.4 (C-9); 132.1 (C-11b); 
               
               
                 134.1 (C-3b); 139.0 (C-7a); 141.6 (C-3a); 146.0 (C-2); 146.7 (C-13); 
               
               
                 147.4 (C-4); 147.4 (C-5); 148.7 (C-14); 157.1 (C-6). 
               
               
                   
               
               
                 N-oxide-canthin-6-one: 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 microcrystalline yellow 
               
               
                 Melting point: 243-245° C. 
               
               
                 IR υ max  cm −1 : 16S77 (—C═O) 
               
               
                 MS (IC): m/z [M + Na] +  237 
               
               
                 MSHR (IC): for C 14 H 9 N 2 O 
               
               
                 [M + H] +  237.0586, found: m/z 237.0584 
               
               
                 R f : 0.6 (CH 2 Cl 2 /MeOH 9:1) 
               
               
                   
               
               
                 MNR  1 H (400 MHz, CDCl 3 ): 
               
            
           
           
               
               
               
               
               
            
               
                 δ (ppm) 
                 multiplicity 
                 coupling  n J (Hz) 
                 integration 
                 attribution 
               
               
                   
               
               
                 6.92 
                 d 
                   3 J 10.0 Hz  
                 1H 
                 H-5 
               
               
                 7.53 
                 t 
                   3 J 8.0 Hz 
                 1H 
                  H-10 
               
               
                 7.65 
                 t 
                   3 J 8.0 Hz 
                 1H 
                 H-9 
               
               
                 7.82 
                 d 
                   3 J 6.6 Hz 
                 1H 
                 H-1 
               
               
                 7.99 
                 d 
                   3 J 8.0 Hz 
                 1H 
                  H-11 
               
               
                 8.34 
                 d 
                   3 J 6.6 Hz 
                 1H 
                 H-2 
               
               
                 8.38 
                 d 
                   3 J 10.0 Hz  
                 1H 
                 H-4 
               
               
                 8.65 
                 d 
                   3 J 8.0 Hz 
                 1H 
                 H-8 
               
               
                   
               
            
           
           
               
            
               
                 MNR  13 C (100 MHz, CDCl 3 ): 117.4 (C-1); 117.7 (C-8); 121.8 
               
               
                 (C-11); 123.7 (C-11a); 126.3 (C-5); 128.4 (C-2); 129.1 (C-3b); 129.6 
               
               
                 (C-10); 130.0 (C-9); 132.0 (C-11b); 133.0 (C-4); 134.0 (C-3b); 140.0 
               
               
                 (C-7-a); 160.1 (C-6). 
               
               
                   
               
               
                 N-methyl-canthin-6-one iodide: 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 microcrystalline orange powder 
               
               
                 Melting point: 238-241° C. 
               
               
                 IR υ max  cm −1 : 1,684 (—C═O) 
               
               
                 MS (IC): m/z [M] +  235 
               
               
                 MSHR (IC): for C 15 H 11 N 2 O 
               
               
                 [M] +  235.0871, found: m/z 235.0873 
               
               
                   
               
               
                 MNR  1 H (400 MHz, δ 6  DMSO): 
               
            
           
           
               
               
               
               
               
            
               
                 δ (ppm) 
                 multiplicity 
                 coupling  n J (Hz) 
                 integration 
                 attribution 
               
               
                   
               
               
                 4.64 
                 s 
                   
                 3H 
                 —Me 
               
               
                 7.41 
                 d 
                   3 J 10.0 Hz  
                 1H 
                 H-5 
               
               
                 7.73 
                 t 
                   3 J 7.8 Hz 
                 1H 
                  H-10 
               
               
                 7.96 
                 t 
                   3 J 7.8 Hz 
                 1H 
                 H-9 
               
               
                 8.57 
                 m 
                   
                 3H 
                 H-8. H-11. 
               
               
                   
                   
                   
                   
                 H-4 
               
               
                 8.90 
                 d 
                   3 J 6.3 Hz 
                 1H 
                 H-1 
               
               
                 9.18 
                 d 
                   3 J 6.3 Hz 
                 1H 
                 H-2 
               
               
                   
               
            
           
           
               
            
               
                 MNR  13 C (50 MHz, δ 6  DMSO): 44.3 (C—Me); 116.8 (C-8); 119.1 
               
               
                 (C-1); 122.5 (C-11a); 125.7 (C-11); 127.4 (C-10); 130.2 (C-4); 130.2 
               
               
                 (C-3a); 133.3 (C-3b); 133.7 (C-5); 134.7 (C-9); 136.1 (C-11b); 141.4 
               
               
                 (C-7a); 142.7 (C-2); 158.0 (C-6). 
               
               
                   
               
               
                 N-bromoéthyl-canthin-6-one bromide: 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 pale green microcrystalline powder 
               
               
                 Melting point: &gt;260° C. 
               
               
                 IR υ max  cm −1 : 1,682 (—C═O) 
               
               
                 MS (IC): m/z [M] +  248 
               
               
                 MSHR (IC): for C 16 H 12 N 2 O 
               
               
                 [M] +  248.0950, found: m/z 248.0953 
               
               
                   
               
               
                 MNR  1 H (200 MHz, D 2 O): 
               
            
           
           
               
               
               
               
               
            
               
                 δ (ppm) 
                 multiplicity 
                 coupling  n J (Hz) 
                 integration 
                 attribution 
               
               
                   
               
               
                 4.19 
                 t 
                   3 J 5.8 Hz 
                 2H 
                     H-2′ 
               
               
                 5.51 
                 t 
                   3 J 5..8 Hz  
                 2H 
                     H-1′ 
               
               
                 7.12 
                 d 
                   3 J 10.2 Hz  
                 1H 
                 H-5 
               
               
                 7.41 
                 t 
                   3 J 8.0 Hz 
                 1H 
                  H-10 
               
               
                 7.62 
                 t 
                   3 J 8.0 Hz 
                 1H 
                 H-9 
               
               
                 8.06-8.16 
                 m 
                   
                 2H 
                 H-8, H-11 
               
               
                 8.27 
                 d 
                   3 J 10.2 Hz  
                 1H 
                 H-4 
               
               
                 8.41 
                 d 
                   3 J 6.4 Hz 
                 1H 
                 H-1 
               
               
                 8.81 
                 d 
                   3 J 6.4 Hz 
                 1H 
                 H-2 
               
               
                   
               
            
           
           
               
            
               
                 N-oxide-benzo[e]canthin-6-one 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 yellow powder 
               
               
                 Melting point: &gt;250° C. 
               
               
                 IR ν max  cm −1 : 1,676 (—C═O) 
               
               
                 MS (ES): m/z [M] +  286 
               
               
                 MSHR (ES): for C 18 H 11 N 2 O 2   
               
               
                 [M] +  286.0742, found: m/z 286.0741 
               
               
                 R f : 0.6 (CH 2 Cl 2 /MeOH 9:1) 
               
               
                   
               
               
                 RMN  1 H (200 MHz, D 2 O): 
               
            
           
           
               
               
               
               
               
            
               
                 δ (ppm) 
                 multiplicity 
                 coupling  n J (Hz) 
                 integration 
                 attribution 
               
               
                   
               
               
                 7.50 
                 t 
                   3 J 8.0 Hz 
                 1H 
                  H-10 
               
               
                 7.72 
                 t 
                   3 J 8.0 Hz 
                 1H 
                 H-9 
               
               
                 7.82-7.93 
                 m 
                   
                 2H 
                 H-14, H-13 
               
               
                 8.08 
                 d 
                   3 J 8.0 Hz 
                 1H 
                  H-11 
               
               
                 8.19 
                 d 
                   3 J 6.7 Hz 
                 1H 
                 H-1 
               
               
                 8.47-8.57 
                 m 
                   
                 2H 
                  H-8, H-15 
               
               
                 8.67 
                 d 
                   3 J 6.7 Hz 
                 1H 
                 H-2 
               
               
                 9.23 
                 d 
                   3 J 8.0 Hz 
                 1H 
                  H-12 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 2 
     Methodology of Biological Assays 
     The antimycobacterial activity of canthin-6-one and some twenty canthin-6-one analog molecules was assessed on  Mycobacterium bovis  BCG,  Mycobacterium smegmatis, Mycobacterium tuberculosis , and  Mycobacterium avium.  
 
 M. bovis  is very close to  M. tuberculosis  (Institut Pasteur, 2002); above 99% identity exists between the two genomes.
 
       M. smegmatis  is a bacterium with a rapid growth (Group IV in the Runyon classification of bacteria), with a length of 3-5 μm, sometimes curved, with a tendency to lose its acid-fast character in cultures aged over 5 days.  M. smegmatis  may be cultured in Middlebrook 7H10 gelose and on a Löwenstein-Jensen medium, and may grow on a MacConkey gelose without hexamethyl pararosaniline chloride. It assimilates M-inositol, D-mannitol, L-rhamnose and D-sorbitol.  M. smegmatis  reacts to ethambutol (5 μg/mL), and is rifampicine-(25 μg/mL) and isoniazide-resistant (10 μg/mL). 
       M. avium  is a slow-growth bacterium, strictly aerobic and without pigmentation.  M. avium  may be cultured on a Löwenstein-Jensen medium or a Coletsos medium. 
     All molecules under test were prepared by chemical synthesis.
 
The antimycobacterial properties of canthin-6-one and its analogs were shown by assessing the inhibitory activity on  Mycobacterium bovis  BCG,  Mycobacterium smegmatis, Mycobacterium tuberculosis , and  Mycobacterium avium , as expressed with the MIC (Minimum Inhibitory Concentration).
 
     In Vitro Assay on  Mycobacterium bovis  BCG 
     The liquid culture medium which is used is a 7H9 medium (Middlebrook 7H9 Difco) to which is added Tween 80 and OADC (Oleic acid, Albumin Fraction V, Dextrose and Catalase) (complement for Middlebrook OADC culture medium). 
     Assays are made in a sterile atmosphere with MICROTEST™ flat bottom 96 well plates. The attenuated  Mycobacterium bovis  strain which is used is Strain 040812 from Institut Pasteur, 180 μL of a BCG suspension with 1.10 5  cfu (colony forming units)/mL are poured into each cup (except for the outer cups, which are filled with sterile water). 
     The molecules to be assayed are diluted in the culture medium (for the hardly soluble molecules dilutions are made with a maximum concentration of 1% DMSO (dimethyl sulfoxide)). 20 μL of each dilution are poured into the corresponding cups. All test are carried out three times. 
     The various controls as left on each plate are the following:
         Positive control BCG alone in the culture medium (200 μL);   Negative control culture medium alone (200 μL);   Control with solvent: BCG (198 μL) with DMSO (10 μL).   Reference antibiotics which are used are isoniazide and ethambutol. The plates are then wrapped in Parafilm® and left in an incubator during 6 days at 37° C.
 
A first reading of the results is made with a reverse microscope, at the smallest magnification. The MIC corresponds to the last concentration for which there is no bacterial veil.
       

     Results are confirmed by MTT coloration (methylthiazoyltetrazolium bromide). The MTT is prepared extemporaneously in a 5 mg/mL buffered phosphate solution, and 50 μL of this solution are added in each well. The plates are then incubated during 4 hours at 37° C. and sheltered from light. After this period 50 μL DMSO/ethanol 50/50 (v/v) are added and the plates are brought back to 37° C. during 24 hrs. Finally the last step is a reading of the optical density (OD) at 570 nm. The MIC may be determined by observing a notable increase in the OD, corresponding to the presence of living bacterial colonies. 
     The MIC of isoniazide is determined by this method 
       MIC=0.10 μg/mL
 
       and for ethambutol: 
       MIC=5 μg/mL
 
                     TABLE                  In vitro activity of canthin-6-one and       analogs on  Mycobacterium   bovis  BCG                         Minimal Inhibitory           Concentration                         MOLECULES   MIC (μg/mL)   MIC (μmol/L)                                      7.5   34.1                                       2.5   &lt;   CMI   &lt;   5 *     10.6   &lt;   CMI   &lt;   21.2 *                                      7.5   31.9                                     10     42.5                                      7.5   30.0                                       2.5   &lt;   CMI   &lt;   5 *     9.2   &lt;   CMI   &lt;   18.4 *                                     10     34.8                                        1   &lt;   CMI   &lt;   5 *     3.0   &lt;   CMI   &lt;   15.3 *                                     10     36.8                                     5    24.5                                      0.1    0.73               *In bold type, equivalent activity to that of ethambutol.            
As may be deduced from the Table of results, several molecules which were tested have shown Minimum Inhibitory Concentrations (MIC in μg) which were equivalent to that of antituberculous reference molecules, viz. ethambutol or pyrazinamide, with MICs between 1 to 5 μg/mL.
 
     Canthin-5-one and its analogs have shown in the above-described activity assays a surprising efficiency against  Mycobacterium bovis  BCG. 
     The other compounds have shown appreciable anti-mycobacterial properties. 
     In Vitro Assays on  Mycobacterium smegmatis    
     The mycobacteria inoculum, 2-3.4×10 4  cfu (Colony Forming Units)/mL is prepared from the mc 2 155 strain of  M. smegmatis  and cultured in Dubos medium (Difco, USA) to which is added 10% OADC (Oleic acid, Albumin Fraction V, Dextrose and Catalase), 100 μl of the inoculum are poured into each wells. 
     In a first step the molecules which were the most active on  M. bovis (canthin-6-one, N-oxide-canthin-6-one, benzo[e]canthin-6-one and N-bromoethyl-canthin-6-one bromide) were tested, and in a second step all canthin-6-one analogs were tested. 
     All molecules to be tested are prepared in dimethylsulfoxide (DMSO) at a concentration of 50 mg/ml and frozen until use. 
     20 μl of each dilution for each molecule are prepared in 100 μl of the Dubos culture medium (Difco, USA), and then distributed into 96 well plates, at a concentration between 100-0.75 μg/ml. 
     Control wells containing DMSO alone and ethambutol (Etibi®, Myambutol®) or ofloxacine (synthesis antibiotic belonging to the fluoroquinolone family) are included at a concentration between 1 μg/l-1 ng/ml. 
     The plates are covered, sealed and incubated at 37° C. during 48 hours. 
     Minimal Inhibitory Concentrations (MIC) are determined using the resazurin coloration microdilution method. The powdery resazurin salt (Sigma) is prepared at a concentration of 0.01% (weight/vol) in distilled water, and the solution is then sterilized by 0.22 μm membrane filtration, and kept at 4° C. during one week. 
     Into the plates as incubated during 48 hours, 30 μl of a resazurin solution are added in each well. The plates are then incubated at 37° C. during 24 hours. The colour variation from blue to pink is indicative of a decrease in resazurin which corresponds to the development of the bacterial colony. The MIC is determined as being the lowest concentration to prevent a colour change. The optical density of each well is measured at 530-630 nm using a micro-plate reader, The MIC is determined by drawing the dose-optical density response curve. 
     The ofloxacin MIC is determined with this method 
       CMI=1 μg/ml,
 
       and for ethambutol 
       CMI=0.5 μg/ml.
 
     Several molecules have shown an efficiency which is equivalent to that of antituberculous reference molecules. 
     Canthin-6-one and its analogs have shown themselves to be efficient, in the hereabove-described activity tests, against  Mycobacterium smegmatis.    
     In Vitro Tests on  Mycobacterium tuberculosis    
     Slow growing mycobacteria such as  M. tuberculosis  H37Rv are cultured in Löwenstein-Jensen medium (Difco Laboratories, USA). 
     In a first step the molecules which were the most active on  M. bovis  and which are hereabove mentioned have been tested, and in a second step all canthin-6-one analogs have been analyzed. 
     The molecules to be tested are diluted in a culture medium—for scarcely soluble molecules dilutions are made with a maximum concentration of 1% DMSO (dimethylsulfoxide). All tests are repeated three times. 
     The Minimum Inhibitory Concentrations are determined in a 7H11 agar medium to which is added 10% OADC (Oleic acid, Albumin Fraction V, Dextrose and Catalase) containing 10 3 -10 5  cfu (colony forming unit)/ml. 
     The mycobacterial colonies are enumerated after 21-30 days incubation at 37° C. The Minimum Inhibitory Concentration is determined as being the smallest concentration of canthin-6-one derivatives reducing their growth by at least 1% as compared with an untreated control colony. 
     The MIC of rifampicin (antituberculous antibiotic belonging to the rifamycin family) is: 
       MIC=0.1 μg/ml
 
       and for ofloxacin: 
       MIC=1 μg/ml.
 
     Several molecules have shown an efficiency which is equivalent to that of antituberculous reference molecules. 
     Canthin-6-one and its analogs have shown, in the hereabove-described activity tests, to be efficient against  Mycobacterium tuberculosis.    
     In Vitro Tests on  Mycobacterium avium    
     Slow growing mycobacteria such as  M. avium  101 are cultured in Löwenstein-Jensen medium (Difco Laboratories, USA). 
     In a first step the molecules which are the most active on  M. bovis  and which are hereabove cited have been tested, and in a second step all canthin-6-one analogs have been analyzed. 
     The molecules to be tested are diluted in a culture medium—for the scarsely soluble molecules dilutions are made with a maximum concentration of 1% DMSO (dimethylsulfoxide). All tests are repeated three times. 
     The Minimum Inhibitory Concentrations are determined in a 7H11 agar medium to which is added 10% OADC (Oleic acid, Albumin Fraction V, Dextrose and Catalase) containing 10 3 -10 5  cfu (colony forming units)/ml. 
     The mycobacterial colonies are enumerated after 21-30 days incubation at 37° C. The Minimal Inhibitory Concentration is determined as being the smallest canthin-6-one derivatives concentration which reduced growth by at least 1%, as compared with an untreated control colony. 
     The MIC of isoniazide (antituberculous derivative of isonicotinic acid) is: 
       MIC=0.1 μg/ml
 
       and for ofloxacin 
       MIC=6.2 μg/ml.
 
     Several molecules have shown an efficiency which is equivalent to that of antituberculous reference molecules. 
     Canthin-6-one and its analogs have shown, in activity tests such as hereabove described, to be efficient against  Mycobacterium avium.    
     EXAMPLE 3 
     In Vivo Tests on Mycobacterium bovis BCG 
     Test Animals: 
     Animals used in these tests are C57B1/6 female mice. 
     Infection: 
     200 μL of a BCG suspension at a concentration of 5×10 5  VU/mL (VU: viable units) in a physiological saline solution are inoculated to the various mice after 7 days housing. 
     Treatment: 
     Treatment begins 15 days after BCG inoculation and for a duration of 5 days (once a day). All tested molecules (canthin-6-one and its analogs) are suspended in a physiological saline solution (with 1% DMSO), and 200 μL are given to the mice intraperitoneally. 
     The reference product which is used is isoniazide at a dose of 25 mg/kg/day, and the molecules are tested at a dose of 20 mg/kg/day. 
     Parameters Under Study: 
     The antituberculous activity is assessed by counting the number of bacteria at the spleen, liver and lungs levels. 
     These organs are removed at the end of treatment after killing the mice. The organs are later comminuted in a Sauton buffer and laid onto 7H11 geloses to which OADC is added (Oleic acid, Albumin Fraction V, Dextrose and catalase). 
     Counting on each box is carried out 2-3 weeks after incubation at 37° C. 
     Several molecules have shown an efficiency which is equivalent to that of isoniazide. 
     Canthin-6-one and its analogs have shown, in the hereabove described in vivo activity tests, to be efficient against  Mycobacterium bovis  BOG.