Title of Invention	A NOVEL ANTIMICROBIAL COMPOUND USEFUL FOR TREATMENT OF DISEASES LIKE TUBEERCULOSIS AND LEPROSY
Abstract	The present invention relates to new antimicrobial compounds, their synthesis and their use for treatment of mammalian infections. The present invention aims at the generation of new compounds with activity against mycobacteria as potential new tuberculosis drugs to overcome problems concerning resistance and drug intolerance. This aim has been solved by providing compounds of the formula 1 wherein R1 and R2 are, independently of each other, NO2, NR7R8, NHOR9, COOR9, CN, CONR10R11, CHO, F, Cl, Br, SO2NR12R13, lower alkoxy, OCF3, mono-, di or trifluoromethyl; R3 and R4 are, independently of each other, H, a saturated or unsaturated, linear or branched aliphatic radical having 1 -3 chain members, F, Cl, Br, lower alkoxy; R5 is H, a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-7 chain members; R6 is a radical: wherein X is saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-5 chain members, or R5 and R6 together represent bivalent radicals wherein n is 1-4: R7- R13 are, independently of each other H or a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1 -5 chain members, phenyl, benzyl or R7 and R8 together, R

Title of Invention

A NOVEL ANTIMICROBIAL COMPOUND USEFUL FOR TREATMENT OF DISEASES LIKE TUBEERCULOSIS AND LEPROSY

Abstract

The present invention relates to new antimicrobial compounds, their synthesis and their use for treatment of mammalian infections. The present invention aims at the generation of new compounds with activity against mycobacteria as potential new tuberculosis drugs to overcome problems concerning resistance and drug intolerance. This aim has been solved by providing compounds of the formula 1 wherein R1 and R2 are, independently of each other, NO2, NR7R8, NHOR9, COOR9, CN, CONR10R11, CHO, F, Cl, Br, SO2NR12R13, lower alkoxy, OCF3, mono-, di or trifluoromethyl; R3 and R4 are, independently of each other, H, a saturated or unsaturated, linear or branched aliphatic radical having 1 -3 chain members, F, Cl, Br, lower alkoxy; R5 is H, a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-7 chain members; R6 is a radical: wherein X is saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-5 chain members, or R5 and R6 together represent bivalent radicals wherein n is 1-4: R7- R13 are, independently of each other H or a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1 -5 chain members, phenyl, benzyl or R7 and R8 together, R

Full Text	New antimicrobial compounds, their synthesis and their use for treatment of mammalian infections Description The present invention relates to novel benzothiazin derivatives and their use as antibacterial agents in infectious diseases of mammals (humans and animals) caused by bacteria, especially diseases like tuberculosis (TB) and leprosy caused by mycobacteria. Thiazinone, their derivatives and their use as antibacterial agents, especially against mycobacteria (TB), laid open for public in AR 24 25 67 Al, AU 37 04 400 Al, CA 13 22 551 Cl or EP 0 245 901 B1 for instance. As known, there is a threatening worldwide increase in tuberculosis infections with mycobacteria which developed resistance against the available therapeutics (B.R.Bloom, J.L.Murray, Tuberculosis: commentary on a reemergent killer. Science 1992, 257, 1055-1064). Extremely dangerous is the development of multidrug resistant (MDR) mycobacteria. These are mycobacteria, resistant at least against two of the most active tuberculosis drugs, isoniazid and rifampicin, but also against streptomycin, pyranzinamid and ethambutol. The proportion of MDR-TB in some countries is already more than 20%. Even more threatening became the situation since the first cases of XDR-TB (extremly resistant TB) were diagnosed last year in South Africa. Now XDR-TB is already spread over all continents. Mycobacteria causing XDR-TB are resistant against the first line TB drugs Rifampicin, Isoniazid, Pyrazinamid, Ethambutol and additionally against the second line chinolones and aminoglycosides. (Nature Med. 2007,13, 295-298) Together with the increased number of TB diseases generally, worldwide it causes about 2.000.000 deaths annually. For the treatment of such diseases, like (TB) or leprosy, there is an urgent need for new drugs with new mechanisms of actions, especially to overcome drug resistance and to overcome the known dramatic side effects of the available drugs. Object of the invention The present invention aims at the generation of new compounds with activity against mycobacteria as potential new tuberculosis drugs to overcome problems concerning resistance and drug intolerance. Solution of the technical problem This aim has been solved by providing compounds of the formula I wherein R1 and R2 are, independently of each other, NO2, NR7R8, NHOR9, COOR9, CN, CONR10R11, CHO, F, Cl, Br, SO2NR12R13, lower alkoxy, OCF3, mono-, di or trifluoromethyl; R3 and R4 are, independently of each other, H, a saturated or unsaturated, linear or branched aliphatic radical having 1-3 chain members, F, Cl, Br, lower alkoxy; R5 is H, a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-7 chain members; R6 is a radical: wherein X is saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-5 chain members, or R5 and R6 together represent bivalent radicals wherein n is 1-4: R7- R13 are, independently of each other H or a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-5 chain members, phenyl, benzyl or R7 and R8 together, R10 and R11 together, R12 and R13 together represent a linear or branched aliphatic bivalent radical having 1-7 chain members; R14 and R15 are, independently of each other, H, linear or branched aliphatic radical having 1-5 chain members, F, Cl, Br, NO2, NH2, CF3. In a preferred embodiment the invention concerns compounds of the formula (I) selected from the group consisting of 2-[4-(2-Rl4,5-R15-phenyl)piperazin-l-yl]-8-nitro-6-(trifluoromethyl)-4H- -1,3-benzothiazin-4-one, 2-[4-(2-R14,6-R15-phenyl)piperazin-l-yl]-8-nitro-6-(trifluoromethyl)-4H- -1,3 -benzothiazin-4-one, 2-[4-(3-R14,5-R15-phenyl)piperazin-l-yl]-8-nitro-6-(trifluoromethyl)-4H- -1,3 -benzothiazin-4-one, 2-[benzyl(methyl)amino]-8-nitro-6-R2-4H-1,3-benzothiazin-4-one, 2-[benzyl(R5)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4- one, 2-[benzyl(R5)amino]-8-nitro-6-R2-4H-1,3-benzothiazin-4-one, 2-[benzyl(R5)amino]-8-nitro-6-chloro-4H-1,3-benzothiazin-4-one, 2-[benzyl(R5)amino]-8-nitro-6-fluoro-4H-1,3-benzothiazin-4-one, 2-[benzyl(R5)amino]-8-R1-6-(trifluoromethyl)-4H-1,3-benzothiazin-4- one, wherein R1, R5, R14 and R15 have the above meanings.The present invention is even more particularly concerned with at least one compound selected from the group consisting of 2-[4-(4-Chlorophenyl)piperazin-1-yl]-6,8-dinitro-4H-1,3-benzothiazin-4- one, 2-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-8-nitro-6-(trifluorome- thyl)-4H-1,3-benzothiazin-4-one, 8-Nitro-6-(trifluoromethyl)-2- {4-[3-(trifluoromethyl)phenyl]piperazin-1- yl}-4H-1,3-benzothiazin-4-one, 2-[Benzyl(ethyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothia- zin-4-one, 2-[Benzyl(methyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothia- zin-4-one, 2-[4-(2-Fluorophenyl)piperazin-1-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3- benzothiazin-4-one, 2-(4-Benzylpiperazin-1 -yl)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzo- thiazin-4-one, 2-(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one, 2-{Methyl[(1R)-1 -phenylethyl]amino} -8-nitro-6-(trifluoromethyl)-4H- 1,3-benzothiazin-4-one, 2-[Benzyl(methyl)amino]-6-chloro-8-nitro-4H-1,3-benzothiazin-4-one. We used 4 different methods for the synthesis of new and novel 1,3- benzothiazin-4-one derivatives. Methods A, B and C propose to use as starting material well known polysubstituted 2-chloro(bromo)- benzcarboxamides, many of them described in the literature or can be easily prepared by analogues methods (Isaew S. G. Farm. Zh. (Kiev), 2000, 52; Makosza M., Nizamov S. Org. Prep, and Proced. Int., 1997, 29, 707; Nerin C, Tornes A. R., Domento C, Cacho J. J. Agr. and Food Chem., 1996, 44, 4009; Thiel W., Mayer R., Jauer E.-A., Modrow H., Dost H. J. Prakt. Chem., 1986, 328, 497; Yokoyama M., Yoshida S., Imamoto T. Synthesis, 1982, 591; Romanowski J., Eckstein Z. Pol. J. Chem., 1984, 58, 263; Nisato D., Sacilotto R., Frigerio M., Boveri S., Palmisano G., Lesma G. Org. Prep. Proced. Int., 1985, 17, 75; Oikawa N., Nakagawa Y., Nishimura K., Ueno T., Fujita T., Pestic. Sci., 1994, 41, 139; Welch D.E., Baron R.R., J. Med. Chem., 1969, 12, 299; Fuller R.W., Molloy B.B., Day W.A., Roush B.W., March M.M., J. Med. Chem., 1973, 16, 101 and many others). Method A The starting 2-chlorobenzcarboxamides were treated by 1,0-1,2 equimolar quantity of the metal salts of dithiocarbamates in alcohols, aceton or in their mixture under 0-50°C for a period of about one quater of an hour to about 24 hours. Preferably, this reaction is conducted in alcohol at room temperature. The reaction mixture was diluted by water and solid 2-dithiocarbamoylbenzcarboxamide was filtered off. For the next step it is possible to use crude product or to recrystallize it from a suitable organic solvent. 2-Dithiocarbamoylbenzcarboxamide was treated by light alkaly (e.g. Na2HPO4, NaHCO3, Na2CO3, etc) in water, alcohols or in a mixture of water/alcohol at a temperature of 50-100°C for 2-36 hours. Preferably, this reaction is conducted in a mixture of water/alcohol at 50-75°C for about 24 hours. When the reaction is complete, the 2-substituted-4H-1,3-benzothiazin-4-one is obtained by conventional recovery procedures, e.g. trituration with ethylacetate or dilution with water, filtration and recrystallization from a suitable organic solvent. Method B This method proposes to use excess of metal dithiocarbamate as light alkaly in the benzothiazinone cyclization and not to isolate 2- dithiocarbamoylbenzcarboxamide. So, the starting 2- chlorobenzcarboxamides can be treated with a 1,2-2,5 equimolar quantity of the metal salts of dithiocarbamates in alcohols, aceton or in their mixture at 20-80°C for a period of about 3-36 hours. Preferably, this reaction is conducted in alcohol or in a mixture of water/alcohol at 50-75°C for about 24 hours. The aimed 2-substituted-4H-1,3- benzothiazin-4-one is obtained by recovery procedures from method A. Method C Method C This method uses as starting material 2-chlorobenzencarboxamides. These compounds were treated with 1,1-2,0 fold excess of metal salts of alkylxantogenate, for example commercial available potassium ethylxantogenate, at a temperature of 20-100°C in different alcohols, acetone, acetonitrile or other suitable organic solvents for a period of about one half hour to about 24 hours. Preferably, this reaction is conducted in alcohol at room temperature for about 24 hours. The isolated 2-alkoxy-4H-1,3-benzothiazin-4-one was treated with the corresponding amine HNR5R6 in acetic acid, alcohols, ethylacetate, DMF, aceton or acetonitril for a period of up to 48 hours for full exchange of the alkoxy group to the corresponding amine. After the process is completed the reaction mixture can be evaporated and diluted by water or it can be diluted by water directly. The aimed 2- NR5R6-4H- 1,3-benzothiazin-4-one is recovered by customary isolation procedures, e.g. filtration and recrystallization from a suitable organic solvent. Method D The classical method of l,3-benzothiazin-4-one synthesis by use of the reaction of thiocyanate salts with 2-chloroarylchloroanhydride and a subsequent treatment of the reaction mass with the corresponding amine is usable too. This method is well described in the scientific literature, for example: J. Imrich, P. Kristian, Coll. Czech. Chem. Commun., 1982, 47, 3268-3282; D. Koscik, P. Kristian, J. Gonda, E.Dandarova, Coll. Czech. Chem. Commun., 1983, 48, 3315-3328; D. Koscik, P. Kristian, O. Forgac, Coll. Czech. Chem. Commun., 1983, 48, 3427- 3432; T. H. Cronin, H. - J .E. Hess, Pat. US 3522247. Surprisingly the compounds of the invention exhibit strong antibacterial activity, especially against mycobacteria with minimal inhibitory concentrations (MIC) in the range of growing mycobacteria, of including multiresistant strains, determined by the classical method and of 2.0 - 50.0 ng/ml for M. tuberculosis H37Rv determined by the Alamar Blue method. Surprisingly the compounds of the invention demonstrate a high level of selectivity for mycobacteria only which reduces the potential for adverse side effects dramatically. The compounds of the invention are non-mutagenic at 5 mg/ml in the SOS chromotest (M.Isidori, M. Lavorgna, A.Nardelli, L.Pascarella, A. Parella, Sci. Total Environ., 2005, 346, 87-98; M. Bombardier, N. Bermingham, R. Legault, A. Fouquet, Chemosphere, 2001, 42, 931-44; D.A. Widdick, D.I. Edwards, Mutat. Res., 1991, 259, 89-93). Thus, the compounds of the invention are useful for the treatment of tuberculosis infections and other mycobacterial infections, in humans and in animals. Accordingly, the invention concerns pharmaceutical compositions comprising a compound of the formula I. The invention relates furthermore to a compound of the formula I for use in a method for the treatment of bacterial infections in mammals. Preferred compounds of the formula I for use in such method are those specifically listed above. The compounds of the invention are formulated for use by preparing a diluted solution or suspension in pharmaceutically acceptable aqueous, organic or aqueous-organic medium for topical or parenteral administration by intravenous, subcutaneous or intramuscular injection, or for intranasal application; or are prepared in tablet, capsule or aqueous suspension form with conventional excipients for oral administration or as suppositorium. The compounds can be used in dosages from 0.001 - 1000 mg/kg body weight. The examples which follow in the subsequent experimental part serve to illustrate the invention but should not be construed as a limitation thereof. The structures of the compounds of the invention were established by modes of synthesis and elementary analysis, and by nuclear magnetic resonance and mass spectra. Embodiments Starting materials Chemicals and solvents were purchased from Alfa-Aesar (GB) or from Aldrich Co. (Sigma-Aldrich Company, St-Louis, US) and were used in the synthesis without additional purification. Melting points were determined according to the BP procedure and are uncorrected (Electrothermal 9001, GB). If analyses are indicated only by the symbols of the elements, analytical results are within ±0.3% of the theoretical values (Carlo-Erba 5500, Italy). NMR spectra were determined with a Varian Unity Plus 400 (USA). Shifts for 1H NMR are reported in ppm downfield from TMS (5). Mass spectra were obtained using a Finnigan SSQ-700 (USA) instrument with direct injection. Reactions and purity of compounds were controlled by TLC using Silicagel 60 F254 aluminium sheets (Merck Co, Germany). Example 1 2-[4-(4-Chlorophenyl)piperazin-1 -yl]-6,8-dinitro-4H-1,3 -benzothiazin-4- one, (compound 1) 0.5 g of 2-chloro-3,5-dinitrobenzcarboxamide was dissolved in 25 ml ethanol. The reaction mixture was treated with of 0.39 g of 4-(4- chlorophenyl)-piperazine dithiocarbamate sodium salt dihydrate and stored for 6 h at room temperature. The reaction mixture was poured into 50 ml of cooled water and the resulting yellow precipitate was filtered off. Pure final product was obtained after recrystallization from ethanol. 2-Aminocarbonyl-4,6-dinitrophenyl-4-(4-chlorophenyl)piperazine-1 - carbodithioate is a light yellow crystalline solid. Yield 68 %. mp 178- 180°C. MS m/z 481 (M+). Anal. Calcd. for C18H16ClN5O5S2: C, 44.86; H, 3.35; N, 14.53; S, 13.31 Found: C, 44.71; H, 3.36; N, 14.62; S, 13.35 0.5 g of 2-aminocarbonyl-4,6-dinitrophenyl-4-(4-chlorophenyl)pipe- razine-1-carbodithioate was dissolved in 25 ml ethanol. The reaction mixture was treated with of 0.2 g of Na2HPO4 x 12H2O and refluxed for 6 h. Reaction mixture was cooled in the refrigerator and the ligth yellow precipitate was filtered off and washed with 50 ml water and 30 ml methanol. Pure final product was obtained after recrystallization twice from ethanol. 2-( 1,4-2-[4-(4-Chlorophenyl)piperazin-1 -yl]-6,8-dinitro- 4H-1,3-benzothiazin-4-one is a light yellow crystalline solid. Yield 38 %.mp 279-281°C (EtOH) MS m/z 447 (M+). 1H NMR (DMSO-d6/CDCl3) d 9.08 and 8.95 (two 1H, two s, 2CH), 6.88 and 6.71 (two 2H, d, C6H4Cl), 3,68 and 3.30 (two 4H, m, N(CH2CH2)2N) ppm. Anal. Calcd. for C18H14ClN5O5S: C, 48.27; H, 3.15; N, 15.04; S, 7.16 Found: C, 48.34; H, 3.22; N, 14.97; S, 7.23 Example 2 2-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-8-nitro-6-(trifluorome- thyl)-4H-1,3-benzothiazin-4-one, (compound 2) Following the procedure of Example 1 using of 2-chloro-3-nitro-5- trifluoromethylbenzcarboxamide as starting material. Light yellow crystalline solid. Yield 44%. mp 158-161°C (DMF/water) MS m/z 484 (M+). 1H NMR (DMSO-d6/CDCl3) d 8.80 and 8.77 (two 1H, two s, 2CH), 7.32 (1H, s, CH), 9.95 and 6.73 (two 1H, d, two CH), 3,65 and 3.29 (two 4H, m, N(CH2CH2)2N), 2.29 (3H, s, CH3) ppm. Anal. Calcd. for C20H16CIF3N4O3S: C, 49.54; H, 3.33; N, 11.55; S, 6.61 Found: C, 49.45; H, 3.40; N, 11.47; S, 6.83 Example 3 8-Nitro-6-(trifluoromethyl)-2- {4-[3-(trifluoromethyl)phenyl]piperazin-1 - yl}-4H -1,3-benzothiazin-4-one, (compound 3) Following the procedure of Example 1 using of 2-chloro-3-nitro-5- trifluoromethylbenzcarboxamide as starting material. Light yellow crystaline solid. Yield 33%. mp 201-203°C (EtOH). MS m/z 504 (M+). 1H NMR (DMSO-d6/CDCl3) d 8.80 and 8.77 (two 1H, two s, 2CH), 7.61 (1H, s, CH), 7.39 and 7.03 (two 1H, d, two CH), 3,66 and 3.31 (two 4H, m, N(CH2CH2)2N) ppm. Anal. Calcd. for C20H14F6N4O3S: C, 47.62; H, 2.80; N, 11.11; S, 6.36 Found: C, 47.74; H, 2.91; N, 11.29; S, 6.53 Example 4 2-[Benzyl(ethyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothia- zin-4-one, (compound 4) A suspension of 1.2 g 2-chloro-3-amino-5-trifluoromethyl- benzcarboxamide in 45 ml ethanol was treated with 2.0 g of benzyl(ethyl)dithiocarbamate sodium salt dihydrate and refluxed for 14 h. The dark red reaction mixture was diluted with 70 ml of water, cooled in the refrigerator for 6 hours, the ligth yellow precipitate was filtered off and washed with 50 ml ester. Pure final product was obtained after column chromatography (hexane/aceton 3:1). 2-[Benzyl(ethyl)amino]-8- nitro-6-(trifluoromethyl)-4H-1,3-benzothia-zin-4-one is a light yellow crystalline solid. Yield 40 %. mp 94-97°C. MS m/z 409 (M+). lH NMR (DMSO-d6/CDCl3) d 8.80 and 8.77 (two 1H, two s, 2CH), 7.41-7.25 (5H, m, C6H5), 4.62 (2H, s, CH2), 3.43 (2H, q, CH2), 1.01 (3H,t,CH3)ppm. Anal. Calcd. for C18H14F3N3O3S: C, 52.81; H, 3.45; N, 10.26; S, 7.83 Found: C, 52.73; H, 3.38; N, 10.44; S, 7.89 Example 5 2-[Benzyl(methyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothia- zin-4-one, (compound 5) Following the procedure of Example 4. Light yellow crystalline solid. Yield 47%. mp 120-124°C (EtOH/water). MS m/z 395 (M+). 1H NMR (DMSO-d6/CDCl3) d 8.81 and 8.77 (two 1H, two s, 2CH), 7.40-7.25 (5H, m, C6H5), 4.64 (2H, s, CH2), 2.87 (3H, s, CH3) ppm. Anal. Calcd. for C17H12F3N3O3S: C, 51.64; H, 3.06; N, 10.63; S, 8.11 Found: C, 51.76; H, 3.13; N, 10.41; S, 8.34 Example 6 2-[4-(2-Fluorophenyl)piperazin-1 -yl]-8-nitro-6-(trifluoromethyl)-4H-1,3- benzothiazin-4-one, (compound 6) Following the procedure of Example 1. Light yellow crystalline solid. Yield 37%. mp 164-168°C (i-PrOH). MS m/z 454 (M+). 1H NMR (DMSO-d6/CDCl3) d 8.81 and 8.77 (two 1H, two s, 2CH), 6.76 (3H, m, 3CH), 6.11 (1H, m, CH), 3,68 and 3.30 (two 4H, m, N(CH2CH2)2N) ppm. Anal. Calcd. for C19H14F4N4O3S: C, 50.22; H, 3.11; N, 12.33; S, 7.06 Found: C, 50.08; H, 3.21; N, 12.46; S, 7.09 Example 7 2-(4-Benzylpiperazin-1 -yl)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzo- thiazin-4-one, (compound 7) Following the procedure of Example 4. Yellow crystalline solid. Yield 51%. mp 161-163°C (EtOH/DMF). MS m/z 450 (M+). 1H NMR (DMSO-d6/CDCl3) d 8.80 and 8.76 (two 1H, two s, 2CH), 7.19-7.28 (5H, m, Ph), 3.48 (2H, s, CH2), 3,38 and 3.09 (two 4H, m, N(CH2CH2)2N) ppm. Anal. Calcd. for C17H18N4O7S: C, 53.33; H, 3.80; N, 12.44; S, 7.12 Found: C, 53.29; H, 4.01; N, 12.48; S, 7.06 Example 8 2-(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one, (compound 8) A suspension of 2.5 g 2-chloro-3-nitro-5-trifluoromethylbenzcar- boxamide in 25 ml ethanol was treated with of 1.75 g of sodium ethylxantogenate and stored for 24 h at room temperature. The reaction mixture was poured into 50 ml of cooled water and the resulting yellow precipitate was filtered off. Pure 2-ethoxy-8-nitro-6-(trifluoromethyl)- 4H-1,3-benzothiazin-4-one was obtained after recrystallization from ethanol/water as white crystalline solid. Yield 58 %. mp 146-148°C. MS m/z 320 (M+). Anal. Calcd. for C11H7F3N2O4S: C, 41.26; H, 2.20; N, 8.75; S, 10.01 Found: C, 41.34; H, 2.22; N, 8.87; S, 10.27 A solution of 0.7 g of 2-ethoxy-8-nitro-6-(trifluoromethyl)-4H-1,3- benzothiazin-4-one in 15 ml acetic acid was treated with 0.4 ml of benzylamine and refluxed for 14 h. The reaction mixture was evaporated and the residue was treated by 10 ml water, the yellow precipitate was filtered off and washed with 50 ml water. Pure final product was obtained after recrystallization twice from ethanol/DMF. 2- (Benzylamino)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one is a light yellow crystalline solid. Yield 73 %. mp 192-194°C. MS m/z 381(M+). 1H NMR (DMSO-d6/CDCl3) d 9.27 (1H, broad s, NH), 8.80 and 8.75 (two 1H, two s, 2CH), 7.74-7.49 (5H, m, Ph), 4.49 (2H, s, CH2) ppm. Anal. Calcd. for C16H10F3N3O3S: C, 50.39; H, 2.64; N, 11.02; S, 8.41 Found: C, 50.42; H, 2.61; N, 10.89; S, 8.64 Example 9 2-{Methyl[(1R)-1-phenylethyl]amino} -8-nitro-6-(trifluoromethyl)-4H- 1,3-benzothiazin-4-one, (compound 9) Following the procedure of Example 1. Light yellow crystalline solid. Yield 54%. mp 110-113°C (purification by column chromatography aceton/hexane 5:1). MS m/z 409 (M+). 1H NMR (DMSO-d6/CDCl3) d 8.82 and 8.76 (two 1H, two s, 2CH), 7.84, 7.43, 7.15 (5H, 3 m, Ph), 4.84 (H, m, CH), 3.07 (3H, s, NCH3), 1.39(3H,d,CH3)ppm. Anal. Calcd. for C18H14F3N3O3S: C, 52.81; H, 3.45; N, 10.26; S, 7.83 Found: C, 52.73; H, 3.46; N, 10.19; S, 7.92 Example 10 2-[Benzyl(methyl)amino]-6-chloro-8-nitro-4H-1,3-benzothiazin-4-one, (compound 10) Following the procedure of Example 8. Light yellow crystalline solid. Yield 64%. mp 138-141°C (purification by column chromatography aceton/hexane 4:1). MS m/z 361 (M+). 1H NMR (DMSO-d6/CDCl3) d 8.37 and 8.23 (two 1H, two s, 2CH), 7.45-7.35 (5H, m, Ph), 4.62 (2H, 2, CH2), 2.87 (3H, s, CH3) ppm. Anal. Calcd. for C16H12ClN3O3S: C, 53.11; H, 3.34; N, 11.61; S, 8.86 Found: C, 53.19; H, 3.30; N, 11.52; S, 8.89 Example 11 Determination of the in vitro inhibitory activity of the compounds of the invention against mycobacteria. The antibacterial activities of the compounds against Mycobacterium smegmatis SG 987, M. aurum SB66, M. vaccae MET 10670 and M. fortuitum B were tested by determination of minimal inhibitory concentrations (MIC) by the broth micro dilution method in Mueller- Hinton broth (Difco) according to the NCCLS guidelines [National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; 5th Ed.; Villanova, Ed.; Approved standard Document M7-A5. NCCLS, (2000)]. The results are presented in Table 1. Example 12 Activity against M. tuberculosis H37Rv was tested by the following method for determination of minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC): Strains were inoculated onto solid Lowenstein-Jensen medium. After 21 days, the cultures grown were used to prepare an inoculum suspension corresponding to 5 x 108 microbial cells/ml. With 0,2 ml of that suspension tubes with 2 ml liquid Shkolnikova medium, containing corresponding concentrations of compounds under study - from 100,0 to 0,195 µg/ml, were inoculated. After 14 days of incubation at 37 °C the tubes with liquid medium were centrifuged for 15min. at 3000 RPM. After discarding the supernatant, the sediment was resuspended in 0,8 ml of sterile 0,9% NaCl. 0,1 ml of the suspension was used to prepare smears subsequently stained by the Ziehl-Neelsen method. The remaining sediment was inoculated in 0,2 ml volumes into three tubes with solid drug free Lowenstein-Jensen medium to determine minimal bactericidal concentrations (MBC). The results were read after 21-28 days of cultivation at 37 °C. Controls were tubes cultured with test- strains not treated with the studied agents. Minimal bactericidal concentration of drugs (MBC) was considered as the drug concentration completely inhibiting the growth of mycobacteria on the solid medium. The bacteriostatic effect (MIC) was characterized by the presence of only individual mycobacteria in the smear and a strong decrease in the number of colonies grown on solid media compared to the controls. The results are presented in Table 2. Table 2: Antimicrobial activity of compounds of the formula I against Mycobacterium tuberculosis H37Rv and clinical isolates HSRE resistant strain and XTB strain as determined by minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) Example 13 Activity against M. tuberculosis H37Rv was determined by the resazurin reduction assay (MIC96) too. The method was described in detail in: P. Quillardet, O. Huisman, R. D'Ari, M. Hofhung, Proc. Natl. Acad. Sci. USA, 1982, 79, 5971-5; J.C. Palomino, A. Martin, M. Camacho, H. Guerra, J. Swings, F. Portaels, Antimicrob. Agents Chemother., 2002, 46, 2720-2. The results are presented in Table 3. We claim 1. A compound of formula (I) or a salt thereof, wherein R1 and R2 are independently from each other NO2, NR7R8, NHOR9, COOR9, CN, CONR10R11, CHO, F, Cl, Br, SO2NR12R13, lower alkoxy, OCF3, mono-, di or trifluoromethyl; R3 and R4 are independently from each other H, a saturated or unsaturated, linear or branched aliphatic radical having 1-3 chain members, F, Cl, Br, or lower alkoxy; R5 is H, a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-7 chain members; R6 is a radical: wherein X is a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-5 chain members, or R5 and R6 together represent bivalent radicals: wherein n is 1-4; R7-R13 are independently from each other H or a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-5 chain members, phenyl, benzyl or R7 and R8 together, R10 and R11 together, R12 and R13 together represent a linear or branched aliphatic bivalent radical having 1-7 chain members; R14 and R15 are independently from each other H, a linear or branched aliphatic radical having 1-5 chain members, F, Cl, Br, NO2, NH2, or CF3. 2. A compound according to formula (I) of claim 1, selected from the group consisting of: A) 2-[4-(2-R14, 5-R15-Phenyl)piperazin-1-yl]-8-nitro-6- (trifluoromethyl)-4H-1,3-benzothiazin-4-one, wherein R14 represents H and R15 represents F, Cl, or Br, B) 2-[4-(3-R14, 5-R15-Phenyl)piperazin-1-yl]-8-nitro-6- (trifluoromethyl)-4H-1,3-benzothiazin-4-one, wherein R14 represents H and R15 represents F or Cl, C) 2-[Benzyl(methyl)amino]-8-nitro-6-R2-4H-1,3- benzothiazin-4-one, wherein R2 represents CF3, Cl or F. D) 2- [Benzyl (R5) amino]-8-R1-6-(trifluoromethyl)-4H-1,3- benzothiazin-4-one, wherein R1 represents CF3, NO2, NHOH or NR7R8 and R5, R7 and R8 have the same meaning as defined in claim 1, E) 6-R2-2-[Methyl(2-phenylethyl)amino]-8-nitro-4H-1,3- benzothiazin-4-one, wherein R2 has the same meaning as defined in claim 1, F) 6-Trifluoromethyl-2-[methyl(2-phenylethyl)amino]-8- R1-4H-1,3-benzothiazin-4-one, wherein R1 has the same meaning as defined in claim 1, G) 2-[4-(2-R14, 6-R15-Phenyl)piperazin-1-yl]-8-nitro-6-- (trifluoromethyl)-4H-1,3-benzothiazin-4-one, wherein R14 and R15 have the same meaning as defined in claim 1, H) 2-[Benzyl (R5)amino]-8-nitro-6-(trifluoromethyl)-4H- 1,3-benzothiazin-4-one, wherein R5 has the same meaning as defined in claim 1, I) 2-[Benzyl (R5)amino]-8-nitro-6-R2-4H-1, 3-benzothiazin- 4-one, wherein R2 and R5 have the same meaning as defined in claim 1, J) 2-[Benzyl(R5)amino]-8-nitro-6-chloro-4H-1,3- benzothiazin-4-one, wherein R5 has the same meaning as defined in claim 1, K) 2-[Benzyl (R5) amino]-8-nitro-6-fluoro-4H-1,3- benzothiazin-4-one according to formula (I) of claim 1, wherein R5 has the same meaning as defined in claim 1. 3. A compound according to formula (I) of claim 1, selected from the group consisting of: 2-{Methyl[(1R)-1-phenylethyl)amino}-8-nitro-6- (trifluoromethyl)-4H-1,3-benzothiazin-4-one, 2- [4- (4-Chlorophenyl)piperazin-1-yl]-6,8-dinitro-4H-1,3- benzothiazin-4-one, 2-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-8-nitro-6- (trifluoromethyl)-4H-1,3-benzothiazin-4-one, 8-Nitro-6-(trifluoromethyl)-2-{4-[3- (trifluoromethyl)phenyl]piperazin-1-yl}-4H-1,3-benzothiazin-4- one, 2-[Benzyl(ethyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1, 3- benzothiazin-4-one, 2-[Benzyl(methyl)amino]-8-nitro-6-(trifluoromethyl)-4H- 1,3-benzothiazin-4-one, 2-[4-(2-Fluorophenyl)piperazin-1-yl]-8-nitro-6- (trifluoromethyl)-4H-1,3-benzothiazin-4-one, 2-(4-Benzylpiperazin-1-yl)-8-nitro-6-(trifluoromethyl)-4H- 1,3-benzothiazin-4-one, 2-(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H-1, 3- benzothiazin-4-one, and 2-[Benzyl(methyl)amino]-6-chloro-8-nitro-4H-1, 3- benzothiazin-4-one, or a salt thereof. 4. Use of a compound of formula (I) or a salt thereof according to any of the preceding claims for the preparation of a pharmaceutical composition. 5. Use of a compound of formula (I) or a salt thereof according to any of the preceding claims for the preparation of a medicament for the therapeutic or prophylactic treatment of microbial infection in mammals. 6. Use according to claim 5, wherein the microbial infection in mammals is a tuberculosis or leprosy infection. 7. Pharmaceutical composition comprising a compound according to any of the preceding claims. 8. A compound according to any of the preceding claims or a salt thereof for use in a method for the therapeutic or prophylactic treatment of microbial infection in mammals. 9. A compound according to any of the preceding claims or a salt thereof for use in a method for the therapeutic or prophylactic treatment of tuberculosis or leprosy infection in mammals. 10. Method for the preparation of 2-NR5R6-4H-1, 3-benzothiazin- 4-ones according to formula (I) of claim 1, comprising reacting 2-chlorobenzcarboxamides with 1.1 to 2.5 times in excess of metal salts of alkylxanthogenate at a temperature of 20-100°C in a solvent and subsequently treating the resulting 2-alkoxy-4ff-1,3-benzothiazin-4-one with an amine HNR5R6 in a solvent at a temperature of 20-100°C. The present invention relates to new antimicrobial compounds, their synthesis and their use for treatment of mammalian infections. The present invention aims at the generation of new compounds with activity against mycobacteria as potential new tuberculosis drugs to overcome problems concerning resistance and drug intolerance. This aim has been solved by providing compounds of the formula 1 wherein R1 and R2 are, independently of each other, NO2, NR7R8, NHOR9, COOR9, CN, CONR10R11, CHO, F, Cl, Br, SO2NR12R13, lower alkoxy, OCF3, mono-, di or trifluoromethyl; R3 and R4 are, independently of each other, H, a saturated or unsaturated, linear or branched aliphatic radical having 1 -3 chain members, F, Cl, Br, lower alkoxy; R5 is H, a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-7 chain members; R6 is a radical: wherein X is saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-5 chain members, or R5 and R6 together represent bivalent radicals wherein n is 1-4: R7- R13 are, independently of each other H or a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1 -5 chain members, phenyl, benzyl or R7 and R8 together, R

Full Text

New antimicrobial compounds, their synthesis and their use for
treatment of mammalian infections
Description
The present invention relates to novel benzothiazin derivatives and their
use as antibacterial agents in infectious diseases of mammals (humans
and animals) caused by bacteria, especially diseases like tuberculosis
(TB) and leprosy caused by mycobacteria.
Thiazinone, their derivatives and their use as antibacterial agents,
especially against mycobacteria (TB), laid open for public in AR 24 25
67 Al, AU 37 04 400 Al, CA 13 22 551 Cl or EP 0 245 901 B1 for
instance.
As known, there is a threatening worldwide increase in tuberculosis
infections with mycobacteria which developed resistance against the
available therapeutics (B.R.Bloom, J.L.Murray, Tuberculosis:
commentary on a reemergent killer. Science 1992, 257, 1055-1064).
Extremely dangerous is the development of multidrug resistant (MDR)
mycobacteria. These are mycobacteria, resistant at least against two of
the most active tuberculosis drugs, isoniazid and rifampicin, but also
against streptomycin, pyranzinamid and ethambutol. The proportion of
MDR-TB in some countries is already more than 20%. Even more
threatening became the situation since the first cases of XDR-TB
(extremly resistant TB) were diagnosed last year in South Africa. Now
XDR-TB is already spread over all continents. Mycobacteria causing
XDR-TB are resistant against the first line TB drugs Rifampicin,
Isoniazid, Pyrazinamid, Ethambutol and additionally against the second
line chinolones and aminoglycosides. (Nature Med. 2007,13, 295-298)
Together with the increased number of TB diseases generally, worldwide
it causes about 2.000.000 deaths annually.
For the treatment of such diseases, like (TB) or leprosy, there is an
urgent need for new drugs with new mechanisms of actions, especially to
overcome drug resistance and to overcome the known dramatic side
effects of the available drugs.
Object of the invention
The present invention aims at the generation of new compounds with
activity against mycobacteria as potential new tuberculosis drugs to
overcome problems concerning resistance and drug intolerance.
Solution of the technical problem
This aim has been solved by providing compounds of the formula I

wherein R1 and R2 are, independently of each other, NO2, NR7R8,
NHOR9, COOR9, CN, CONR10R11, CHO, F, Cl, Br, SO2NR12R13, lower
alkoxy, OCF3, mono-, di or trifluoromethyl;
R3 and R4 are, independently of each other, H, a saturated or unsaturated,
linear or branched aliphatic radical having 1-3 chain members, F, Cl, Br,
lower alkoxy;
R5 is H, a saturated or unsaturated, halogenated or unhalogenated, linear
or branched aliphatic radical having 1-7 chain members;
R6 is a radical:

wherein X is saturated or unsaturated, halogenated or unhalogenated,
linear or branched aliphatic radical having 1-5 chain members, or
R5 and R6 together represent bivalent radicals wherein n is 1-4:

R7- R13 are, independently of each other H or a saturated or unsaturated,
halogenated or unhalogenated, linear or branched aliphatic radical
having 1-5 chain members, phenyl, benzyl or R7 and R8 together, R10 and
R11 together, R12 and R13 together represent a linear or branched aliphatic
bivalent radical having 1-7 chain members;
R14 and R15 are, independently of each other, H, linear or branched
aliphatic radical having 1-5 chain members, F, Cl, Br, NO2, NH2, CF3.
In a preferred embodiment the invention concerns compounds of the
formula (I) selected from the group consisting of
2-[4-(2-Rl4,5-R15-phenyl)piperazin-l-yl]-8-nitro-6-(trifluoromethyl)-4H-
-1,3-benzothiazin-4-one,
2-[4-(2-R14,6-R15-phenyl)piperazin-l-yl]-8-nitro-6-(trifluoromethyl)-4H-
-1,3 -benzothiazin-4-one,
2-[4-(3-R14,5-R15-phenyl)piperazin-l-yl]-8-nitro-6-(trifluoromethyl)-4H-
-1,3 -benzothiazin-4-one,
2-[benzyl(methyl)amino]-8-nitro-6-R2-4H-1,3-benzothiazin-4-one,
2-[benzyl(R5)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-
one,
2-[benzyl(R5)amino]-8-nitro-6-R2-4H-1,3-benzothiazin-4-one,
2-[benzyl(R5)amino]-8-nitro-6-chloro-4H-1,3-benzothiazin-4-one,
2-[benzyl(R5)amino]-8-nitro-6-fluoro-4H-1,3-benzothiazin-4-one,
2-[benzyl(R5)amino]-8-R1-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-
one,
wherein R1, R5, R14 and R15 have the above meanings.The present
invention is even more particularly concerned with at least one
compound selected from the group consisting of
2-[4-(4-Chlorophenyl)piperazin-1-yl]-6,8-dinitro-4H-1,3-benzothiazin-4-
one,
2-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-8-nitro-6-(trifluorome-
thyl)-4H-1,3-benzothiazin-4-one,
8-Nitro-6-(trifluoromethyl)-2- {4-[3-(trifluoromethyl)phenyl]piperazin-1-
yl}-4H-1,3-benzothiazin-4-one,
2-[Benzyl(ethyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothia-
zin-4-one,
2-[Benzyl(methyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothia-
zin-4-one,
2-[4-(2-Fluorophenyl)piperazin-1-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-
benzothiazin-4-one,
2-(4-Benzylpiperazin-1 -yl)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzo-
thiazin-4-one,
2-(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one,
2-{Methyl[(1R)-1 -phenylethyl]amino} -8-nitro-6-(trifluoromethyl)-4H-
1,3-benzothiazin-4-one,
2-[Benzyl(methyl)amino]-6-chloro-8-nitro-4H-1,3-benzothiazin-4-one.
We used 4 different methods for the synthesis of new and novel 1,3-
benzothiazin-4-one derivatives. Methods A, B and C propose to use as
starting material well known polysubstituted 2-chloro(bromo)-
benzcarboxamides, many of them described in the literature or can be
easily prepared by analogues methods (Isaew S. G. Farm. Zh. (Kiev),
2000, 52; Makosza M., Nizamov S. Org. Prep, and Proced. Int., 1997,
29, 707; Nerin C, Tornes A. R., Domento C, Cacho J. J. Agr. and Food
Chem., 1996, 44, 4009; Thiel W., Mayer R., Jauer E.-A., Modrow H.,
Dost H. J. Prakt. Chem., 1986, 328, 497; Yokoyama M., Yoshida S.,
Imamoto T. Synthesis, 1982, 591; Romanowski J., Eckstein Z. Pol. J.
Chem., 1984, 58, 263; Nisato D., Sacilotto R., Frigerio M., Boveri S.,
Palmisano G., Lesma G. Org. Prep. Proced. Int., 1985, 17, 75; Oikawa
N., Nakagawa Y., Nishimura K., Ueno T., Fujita T., Pestic. Sci., 1994,
41, 139; Welch D.E., Baron R.R., J. Med. Chem., 1969, 12, 299; Fuller
R.W., Molloy B.B., Day W.A., Roush B.W., March M.M., J. Med.
Chem., 1973, 16, 101 and many others).

Method A
The starting 2-chlorobenzcarboxamides were treated by 1,0-1,2
equimolar quantity of the metal salts of dithiocarbamates in alcohols,
aceton or in their mixture under 0-50°C for a period of about one quater
of an hour to about 24 hours. Preferably, this reaction is conducted in
alcohol at room temperature. The reaction mixture was diluted by water
and solid 2-dithiocarbamoylbenzcarboxamide was filtered off. For the
next step it is possible to use crude product or to recrystallize it from a
suitable organic solvent. 2-Dithiocarbamoylbenzcarboxamide was
treated by light alkaly (e.g. Na2HPO4, NaHCO3, Na2CO3, etc) in water,
alcohols or in a mixture of water/alcohol at a temperature of 50-100°C
for 2-36 hours. Preferably, this reaction is conducted in a mixture of
water/alcohol at 50-75°C for about 24 hours. When the reaction is
complete, the 2-substituted-4H-1,3-benzothiazin-4-one is obtained by
conventional recovery procedures, e.g. trituration with ethylacetate or
dilution with water, filtration and recrystallization from a suitable
organic solvent.
Method B
This method proposes to use excess of metal dithiocarbamate as light
alkaly in the benzothiazinone cyclization and not to isolate 2-
dithiocarbamoylbenzcarboxamide. So, the starting 2-
chlorobenzcarboxamides can be treated with a 1,2-2,5 equimolar
quantity of the metal salts of dithiocarbamates in alcohols, aceton or in
their mixture at 20-80°C for a period of about 3-36 hours. Preferably,
this reaction is conducted in alcohol or in a mixture of water/alcohol at
50-75°C for about 24 hours. The aimed 2-substituted-4H-1,3-
benzothiazin-4-one is obtained by recovery procedures from method A.
Method C

Method C
This method uses as starting material 2-chlorobenzencarboxamides.
These compounds were treated with 1,1-2,0 fold excess of metal salts of
alkylxantogenate, for example commercial available potassium
ethylxantogenate, at a temperature of 20-100°C in different alcohols,
acetone, acetonitrile or other suitable organic solvents for a period of
about one half hour to about 24 hours. Preferably, this reaction is
conducted in alcohol at room temperature for about 24 hours. The
isolated 2-alkoxy-4H-1,3-benzothiazin-4-one was treated with the
corresponding amine HNR5R6 in acetic acid, alcohols, ethylacetate,
DMF, aceton or acetonitril for a period of up to 48 hours for full
exchange of the alkoxy group to the corresponding amine. After the
process is completed the reaction mixture can be evaporated and diluted
by water or it can be diluted by water directly. The aimed 2- NR5R6-4H-
1,3-benzothiazin-4-one is recovered by customary isolation procedures,
e.g. filtration and recrystallization from a suitable organic solvent.
Method D
The classical method of l,3-benzothiazin-4-one synthesis by use of the
reaction of thiocyanate salts with 2-chloroarylchloroanhydride and a
subsequent treatment of the reaction mass with the corresponding amine
is usable too. This method is well described in the scientific literature,
for example: J. Imrich, P. Kristian, Coll. Czech. Chem. Commun., 1982,
47, 3268-3282; D. Koscik, P. Kristian, J. Gonda, E.Dandarova,
Coll. Czech. Chem. Commun., 1983, 48, 3315-3328; D. Koscik,
P. Kristian, O. Forgac, Coll. Czech. Chem. Commun., 1983, 48, 3427-
3432; T. H. Cronin, H. - J .E. Hess, Pat. US 3522247.
Surprisingly the compounds of the invention exhibit strong antibacterial
activity, especially against mycobacteria with minimal inhibitory
concentrations (MIC) in the range of growing mycobacteria, of including multiresistant strains, determined by the classical method and
of 2.0 - 50.0 ng/ml for M. tuberculosis H37Rv determined by the
Alamar Blue method. Surprisingly the compounds of the invention
demonstrate a high level of selectivity for mycobacteria only which
reduces the potential for adverse side effects dramatically.
The compounds of the invention are non-mutagenic at 5 mg/ml in the
SOS chromotest (M.Isidori, M. Lavorgna, A.Nardelli, L.Pascarella, A.
Parella, Sci. Total Environ., 2005, 346, 87-98; M. Bombardier, N.
Bermingham, R. Legault, A. Fouquet, Chemosphere, 2001, 42, 931-44;
D.A. Widdick, D.I. Edwards, Mutat. Res., 1991, 259, 89-93).
Thus, the compounds of the invention are useful for the treatment of
tuberculosis infections and other mycobacterial infections, in humans
and in animals.
Accordingly, the invention concerns pharmaceutical compositions
comprising a compound of the formula I.
The invention relates furthermore to a compound of the formula I for use
in a method for the treatment of bacterial infections in mammals.
Preferred compounds of the formula I for use in such method are those
specifically listed above.
The compounds of the invention are formulated for use by preparing a
diluted solution or suspension in pharmaceutically acceptable aqueous,
organic or aqueous-organic medium for topical or parenteral
administration by intravenous, subcutaneous or intramuscular injection,
or for intranasal application; or are prepared in tablet, capsule or aqueous
suspension form with conventional excipients for oral administration or
as suppositorium.
The compounds can be used in dosages from 0.001 - 1000 mg/kg body
weight.
The examples which follow in the subsequent experimental part serve to
illustrate the invention but should not be construed as a limitation
thereof.
The structures of the compounds of the invention were established by
modes of synthesis and elementary analysis, and by nuclear magnetic
resonance and mass spectra.
Embodiments
Starting materials
Chemicals and solvents were purchased from Alfa-Aesar (GB) or from
Aldrich Co. (Sigma-Aldrich Company, St-Louis, US) and were used in
the synthesis without additional purification. Melting points were
determined according to the BP procedure and are uncorrected
(Electrothermal 9001, GB). If analyses are indicated only by the symbols
of the elements, analytical results are within ±0.3% of the theoretical
values (Carlo-Erba 5500, Italy). NMR spectra were determined with a
Varian Unity Plus 400 (USA). Shifts for 1H NMR are reported in ppm
downfield from TMS (5). Mass spectra were obtained using a Finnigan
SSQ-700 (USA) instrument with direct injection. Reactions and purity of
compounds were controlled by TLC using Silicagel 60 F254 aluminium
sheets (Merck Co, Germany).
Example 1
2-[4-(4-Chlorophenyl)piperazin-1 -yl]-6,8-dinitro-4H-1,3 -benzothiazin-4-
one, (compound 1)
0.5 g of 2-chloro-3,5-dinitrobenzcarboxamide was dissolved in 25 ml
ethanol. The reaction mixture was treated with of 0.39 g of 4-(4-
chlorophenyl)-piperazine dithiocarbamate sodium salt dihydrate and
stored for 6 h at room temperature. The reaction mixture was poured into
50 ml of cooled water and the resulting yellow precipitate was filtered
off. Pure final product was obtained after recrystallization from ethanol.
2-Aminocarbonyl-4,6-dinitrophenyl-4-(4-chlorophenyl)piperazine-1 -
carbodithioate is a light yellow crystalline solid. Yield 68 %. mp 178-
180°C. MS m/z 481 (M+).
Anal. Calcd. for C18H16ClN5O5S2: C, 44.86; H, 3.35; N, 14.53; S, 13.31
Found: C, 44.71; H, 3.36; N, 14.62; S, 13.35
0.5 g of 2-aminocarbonyl-4,6-dinitrophenyl-4-(4-chlorophenyl)pipe-
razine-1-carbodithioate was dissolved in 25 ml ethanol. The reaction
mixture was treated with of 0.2 g of Na2HPO4 x 12H2O and refluxed for
6 h. Reaction mixture was cooled in the refrigerator and the ligth yellow
precipitate was filtered off and washed with 50 ml water and 30 ml
methanol. Pure final product was obtained after recrystallization twice
from ethanol. 2-( 1,4-2-[4-(4-Chlorophenyl)piperazin-1 -yl]-6,8-dinitro-
4H-1,3-benzothiazin-4-one is a light yellow crystalline solid. Yield 38
%.mp 279-281°C (EtOH)
MS m/z 447 (M+).
1H NMR (DMSO-d6/CDCl3) d 9.08 and 8.95 (two 1H, two s, 2CH), 6.88
and 6.71 (two 2H, d, C6H4Cl), 3,68 and 3.30 (two 4H, m,
N(CH2CH2)2N) ppm.
Anal. Calcd. for C18H14ClN5O5S: C, 48.27; H, 3.15; N, 15.04; S, 7.16
Found: C, 48.34; H, 3.22; N, 14.97; S, 7.23
Example 2
2-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-8-nitro-6-(trifluorome-
thyl)-4H-1,3-benzothiazin-4-one, (compound 2)
Following the procedure of Example 1 using of 2-chloro-3-nitro-5-
trifluoromethylbenzcarboxamide as starting material. Light yellow
crystalline solid. Yield 44%. mp 158-161°C (DMF/water)
MS m/z 484 (M+).
1H NMR (DMSO-d6/CDCl3) d 8.80 and 8.77 (two 1H, two s, 2CH), 7.32
(1H, s, CH), 9.95 and 6.73 (two 1H, d, two CH), 3,65 and 3.29 (two 4H,
m, N(CH2CH2)2N), 2.29 (3H, s, CH3) ppm.
Anal. Calcd. for C20H16CIF3N4O3S: C, 49.54; H, 3.33; N, 11.55; S, 6.61
Found: C, 49.45; H, 3.40; N, 11.47; S, 6.83
Example 3
8-Nitro-6-(trifluoromethyl)-2- {4-[3-(trifluoromethyl)phenyl]piperazin-1 -
yl}-4H -1,3-benzothiazin-4-one, (compound 3)
Following the procedure of Example 1 using of 2-chloro-3-nitro-5-
trifluoromethylbenzcarboxamide as starting material. Light yellow
crystaline solid. Yield 33%. mp 201-203°C (EtOH).
MS m/z 504 (M+).
1H NMR (DMSO-d6/CDCl3) d 8.80 and 8.77 (two 1H, two s, 2CH), 7.61
(1H, s, CH), 7.39 and 7.03 (two 1H, d, two CH), 3,66 and 3.31 (two 4H,
m, N(CH2CH2)2N) ppm.
Anal. Calcd. for C20H14F6N4O3S: C, 47.62; H, 2.80; N, 11.11; S, 6.36
Found: C, 47.74; H, 2.91; N, 11.29; S, 6.53
Example 4
2-[Benzyl(ethyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothia-
zin-4-one, (compound 4)
A suspension of 1.2 g 2-chloro-3-amino-5-trifluoromethyl-
benzcarboxamide in 45 ml ethanol was treated with 2.0 g of
benzyl(ethyl)dithiocarbamate sodium salt dihydrate and refluxed for 14
h. The dark red reaction mixture was diluted with 70 ml of water, cooled
in the refrigerator for 6 hours, the ligth yellow precipitate was filtered off
and washed with 50 ml ester. Pure final product was obtained after
column chromatography (hexane/aceton 3:1). 2-[Benzyl(ethyl)amino]-8-
nitro-6-(trifluoromethyl)-4H-1,3-benzothia-zin-4-one is a light yellow
crystalline solid. Yield 40 %. mp 94-97°C.
MS m/z 409 (M+).
lH NMR (DMSO-d6/CDCl3) d 8.80 and 8.77 (two 1H, two s, 2CH),
7.41-7.25 (5H, m, C6H5), 4.62 (2H, s, CH2), 3.43 (2H, q, CH2), 1.01
(3H,t,CH3)ppm.
Anal. Calcd. for C18H14F3N3O3S: C, 52.81; H, 3.45; N, 10.26; S, 7.83
Found: C, 52.73; H, 3.38; N, 10.44; S, 7.89
Example 5
2-[Benzyl(methyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothia-
zin-4-one, (compound 5)
Following the procedure of Example 4. Light yellow crystalline solid.
Yield 47%. mp 120-124°C (EtOH/water).
MS m/z 395 (M+).
1H NMR (DMSO-d6/CDCl3) d 8.81 and 8.77 (two 1H, two s, 2CH),
7.40-7.25 (5H, m, C6H5), 4.64 (2H, s, CH2), 2.87 (3H, s, CH3) ppm.
Anal. Calcd. for C17H12F3N3O3S: C, 51.64; H, 3.06; N, 10.63; S, 8.11
Found: C, 51.76; H, 3.13; N, 10.41; S, 8.34
Example 6
2-[4-(2-Fluorophenyl)piperazin-1 -yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-
benzothiazin-4-one, (compound 6)
Following the procedure of Example 1. Light yellow crystalline solid.
Yield 37%. mp 164-168°C (i-PrOH).
MS m/z 454 (M+).
1H NMR (DMSO-d6/CDCl3) d 8.81 and 8.77 (two 1H, two s, 2CH), 6.76
(3H, m, 3CH), 6.11 (1H, m, CH), 3,68 and 3.30 (two 4H, m,
N(CH2CH2)2N) ppm.
Anal. Calcd. for C19H14F4N4O3S: C, 50.22; H, 3.11; N, 12.33; S, 7.06
Found: C, 50.08; H, 3.21; N, 12.46; S, 7.09
Example 7
2-(4-Benzylpiperazin-1 -yl)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzo-
thiazin-4-one, (compound 7)
Following the procedure of Example 4. Yellow crystalline solid. Yield
51%. mp 161-163°C (EtOH/DMF).
MS m/z 450 (M+).
1H NMR (DMSO-d6/CDCl3) d 8.80 and 8.76 (two 1H, two s, 2CH),
7.19-7.28 (5H, m, Ph), 3.48 (2H, s, CH2), 3,38 and 3.09 (two 4H, m,
N(CH2CH2)2N) ppm.
Anal. Calcd. for C17H18N4O7S: C, 53.33; H, 3.80; N, 12.44; S, 7.12
Found: C, 53.29; H, 4.01; N, 12.48; S, 7.06
Example 8
2-(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one,
(compound 8)
A suspension of 2.5 g 2-chloro-3-nitro-5-trifluoromethylbenzcar-
boxamide in 25 ml ethanol was treated with of 1.75 g of sodium
ethylxantogenate and stored for 24 h at room temperature. The reaction
mixture was poured into 50 ml of cooled water and the resulting yellow
precipitate was filtered off. Pure 2-ethoxy-8-nitro-6-(trifluoromethyl)-
4H-1,3-benzothiazin-4-one was obtained after recrystallization from
ethanol/water as white crystalline solid. Yield 58 %. mp 146-148°C.
MS m/z 320 (M+).
Anal. Calcd. for C11H7F3N2O4S: C, 41.26; H, 2.20; N, 8.75; S, 10.01
Found: C, 41.34; H, 2.22; N, 8.87; S, 10.27
A solution of 0.7 g of 2-ethoxy-8-nitro-6-(trifluoromethyl)-4H-1,3-
benzothiazin-4-one in 15 ml acetic acid was treated with 0.4 ml of
benzylamine and refluxed for 14 h. The reaction mixture was evaporated
and the residue was treated by 10 ml water, the yellow precipitate was
filtered off and washed with 50 ml water. Pure final product was
obtained after recrystallization twice from ethanol/DMF. 2-
(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one is
a light yellow crystalline solid. Yield 73 %. mp 192-194°C.
MS m/z 381(M+).
1H NMR (DMSO-d6/CDCl3) d 9.27 (1H, broad s, NH), 8.80 and 8.75
(two 1H, two s, 2CH), 7.74-7.49 (5H, m, Ph), 4.49 (2H, s, CH2) ppm.
Anal. Calcd. for C16H10F3N3O3S: C, 50.39; H, 2.64; N, 11.02; S, 8.41
Found: C, 50.42; H, 2.61; N, 10.89; S, 8.64
Example 9
2-{Methyl[(1R)-1-phenylethyl]amino} -8-nitro-6-(trifluoromethyl)-4H-
1,3-benzothiazin-4-one, (compound 9)
Following the procedure of Example 1. Light yellow crystalline solid.
Yield 54%. mp 110-113°C (purification by column chromatography
aceton/hexane 5:1).
MS m/z 409 (M+).
1H NMR (DMSO-d6/CDCl3) d 8.82 and 8.76 (two 1H, two s, 2CH),
7.84, 7.43, 7.15 (5H, 3 m, Ph), 4.84 (H, m, CH), 3.07 (3H, s, NCH3),
1.39(3H,d,CH3)ppm.
Anal. Calcd. for C18H14F3N3O3S: C, 52.81; H, 3.45; N, 10.26; S, 7.83
Found: C, 52.73; H, 3.46; N, 10.19; S, 7.92
Example 10
2-[Benzyl(methyl)amino]-6-chloro-8-nitro-4H-1,3-benzothiazin-4-one,
(compound 10)
Following the procedure of Example 8. Light yellow crystalline solid.
Yield 64%. mp 138-141°C (purification by column chromatography
aceton/hexane 4:1).
MS m/z 361 (M+).
1H NMR (DMSO-d6/CDCl3) d 8.37 and 8.23 (two 1H, two s, 2CH),
7.45-7.35 (5H, m, Ph), 4.62 (2H, 2, CH2), 2.87 (3H, s, CH3) ppm.
Anal. Calcd. for C16H12ClN3O3S: C, 53.11; H, 3.34; N, 11.61; S, 8.86
Found: C, 53.19; H, 3.30; N, 11.52; S, 8.89
Example 11
Determination of the in vitro inhibitory activity of the compounds of the
invention against mycobacteria.
The antibacterial activities of the compounds against Mycobacterium
smegmatis SG 987, M. aurum SB66, M. vaccae MET 10670 and M.
fortuitum B were tested by determination of minimal inhibitory
concentrations (MIC) by the broth micro dilution method in Mueller-
Hinton broth (Difco) according to the NCCLS guidelines [National
Committee for Clinical Laboratory Standards: Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically; 5th
Ed.; Villanova, Ed.; Approved standard Document M7-A5. NCCLS,
(2000)]. The results are presented in Table 1.

Example 12
Activity against M. tuberculosis H37Rv was tested by the following
method for determination of minimal inhibitory concentrations (MIC)
and minimal bactericidal concentrations (MBC):
Strains were inoculated onto solid Lowenstein-Jensen medium. After 21
days, the cultures grown were used to prepare an inoculum suspension
corresponding to 5 x 108 microbial cells/ml. With 0,2 ml of that
suspension tubes with 2 ml liquid Shkolnikova medium, containing
corresponding concentrations of compounds under study - from 100,0 to
0,195 µg/ml, were inoculated. After 14 days of incubation at 37 °C the
tubes with liquid medium were centrifuged for 15min. at 3000 RPM.
After discarding the supernatant, the sediment was resuspended in 0,8 ml
of sterile 0,9% NaCl. 0,1 ml of the suspension was used to prepare
smears subsequently stained by the Ziehl-Neelsen method. The
remaining sediment was inoculated in 0,2 ml volumes into three tubes
with solid drug free Lowenstein-Jensen medium to determine minimal
bactericidal concentrations (MBC). The results were read after 21-28
days of cultivation at 37 °C. Controls were tubes cultured with test-
strains not treated with the studied agents.
Minimal bactericidal concentration of drugs (MBC) was considered as
the drug concentration completely inhibiting the growth of mycobacteria
on the solid medium. The bacteriostatic effect (MIC) was characterized
by the presence of only individual mycobacteria in the smear and a
strong decrease in the number of colonies grown on solid media
compared to the controls. The results are presented in Table 2.
Table 2: Antimicrobial activity of compounds of the formula I against
Mycobacterium tuberculosis H37Rv and clinical isolates HSRE resistant
strain and XTB strain as determined by minimal inhibitory concentrations
(MIC) and minimal bactericidal concentrations (MBC)

Example 13
Activity against M. tuberculosis H37Rv was determined by the resazurin
reduction assay (MIC96) too. The method was described in detail in: P.
Quillardet, O. Huisman, R. D'Ari, M. Hofhung, Proc. Natl. Acad. Sci.
USA, 1982, 79, 5971-5; J.C. Palomino, A. Martin, M. Camacho, H.
Guerra, J. Swings, F. Portaels, Antimicrob. Agents Chemother., 2002,
46, 2720-2. The results are presented in Table 3.
We claim
1. A compound of formula (I)

or a salt thereof,
wherein R1 and R2 are independently from each other NO2, NR7R8,
NHOR9, COOR9, CN, CONR10R11, CHO, F, Cl, Br, SO2NR12R13, lower
alkoxy, OCF3, mono-, di or trifluoromethyl;
R3 and R4 are independently from each other H, a saturated or
unsaturated, linear or branched aliphatic radical having 1-3
chain members, F, Cl, Br, or lower alkoxy;
R5 is H, a saturated or unsaturated, halogenated or
unhalogenated, linear or branched aliphatic radical having 1-7
chain members;
R6 is a radical:

wherein X is a saturated or unsaturated, halogenated or
unhalogenated, linear or branched aliphatic radical having 1-5
chain members, or
R5 and R6 together represent bivalent radicals:

wherein n is 1-4;
R7-R13 are independently from each other H or a saturated or
unsaturated, halogenated or unhalogenated, linear or branched
aliphatic radical having 1-5 chain members, phenyl, benzyl or
R7 and R8 together, R10 and R11 together, R12 and R13 together
represent a linear or branched aliphatic bivalent radical
having 1-7 chain members;
R14 and R15 are independently from each other H, a linear or
branched aliphatic radical having 1-5 chain members, F, Cl,
Br, NO2, NH2, or CF3.
2. A compound according to formula (I) of claim 1, selected
from the group consisting of:
A) 2-[4-(2-R14, 5-R15-Phenyl)piperazin-1-yl]-8-nitro-6-
(trifluoromethyl)-4H-1,3-benzothiazin-4-one, wherein R14
represents H and R15 represents F, Cl, or Br,
B) 2-[4-(3-R14, 5-R15-Phenyl)piperazin-1-yl]-8-nitro-6-
(trifluoromethyl)-4H-1,3-benzothiazin-4-one, wherein R14
represents H and R15 represents F or Cl,
C) 2-[Benzyl(methyl)amino]-8-nitro-6-R2-4H-1,3-
benzothiazin-4-one, wherein R2 represents CF3, Cl or F.
D) 2- [Benzyl (R5) amino]-8-R1-6-(trifluoromethyl)-4H-1,3-
benzothiazin-4-one, wherein R1 represents CF3, NO2, NHOH or
NR7R8 and R5, R7 and R8 have the same meaning as defined in
claim 1,
E) 6-R2-2-[Methyl(2-phenylethyl)amino]-8-nitro-4H-1,3-
benzothiazin-4-one, wherein R2 has the same meaning as defined
in claim 1,
F) 6-Trifluoromethyl-2-[methyl(2-phenylethyl)amino]-8-
R1-4H-1,3-benzothiazin-4-one, wherein R1 has the same meaning
as defined in claim 1,
G) 2-[4-(2-R14, 6-R15-Phenyl)piperazin-1-yl]-8-nitro-6--
(trifluoromethyl)-4H-1,3-benzothiazin-4-one, wherein R14 and R15
have the same meaning as defined in claim 1,
H) 2-[Benzyl (R5)amino]-8-nitro-6-(trifluoromethyl)-4H-
1,3-benzothiazin-4-one, wherein R5 has the same meaning as
defined in claim 1,
I) 2-[Benzyl (R5)amino]-8-nitro-6-R2-4H-1, 3-benzothiazin-
4-one, wherein R2 and R5 have the same meaning as defined in
claim 1,
J) 2-[Benzyl(R5)amino]-8-nitro-6-chloro-4H-1,3-
benzothiazin-4-one, wherein R5 has the same meaning as defined
in claim 1,
K) 2-[Benzyl (R5) amino]-8-nitro-6-fluoro-4H-1,3-
benzothiazin-4-one according to formula (I) of claim 1,
wherein R5 has the same meaning as defined in claim 1.
3. A compound according to formula (I) of claim 1, selected
from the group consisting of:
2-{Methyl[(1R)-1-phenylethyl)amino}-8-nitro-6-
(trifluoromethyl)-4H-1,3-benzothiazin-4-one,
2- [4- (4-Chlorophenyl)piperazin-1-yl]-6,8-dinitro-4H-1,3-
benzothiazin-4-one,
2-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-8-nitro-6-
(trifluoromethyl)-4H-1,3-benzothiazin-4-one,
8-Nitro-6-(trifluoromethyl)-2-{4-[3-
(trifluoromethyl)phenyl]piperazin-1-yl}-4H-1,3-benzothiazin-4-
one,
2-[Benzyl(ethyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1, 3-
benzothiazin-4-one,
2-[Benzyl(methyl)amino]-8-nitro-6-(trifluoromethyl)-4H-
1,3-benzothiazin-4-one,
2-[4-(2-Fluorophenyl)piperazin-1-yl]-8-nitro-6-
(trifluoromethyl)-4H-1,3-benzothiazin-4-one,
2-(4-Benzylpiperazin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-
1,3-benzothiazin-4-one,
2-(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H-1, 3-
benzothiazin-4-one, and
2-[Benzyl(methyl)amino]-6-chloro-8-nitro-4H-1, 3-
benzothiazin-4-one,
or a salt thereof.
4. Use of a compound of formula (I) or a salt thereof
according to any of the preceding claims for the preparation
of a pharmaceutical composition.
5. Use of a compound of formula (I) or a salt thereof
according to any of the preceding claims for the preparation
of a medicament for the therapeutic or prophylactic treatment
of microbial infection in mammals.
6. Use according to claim 5, wherein the microbial infection
in mammals is a tuberculosis or leprosy infection.
7. Pharmaceutical composition comprising a compound according
to any of the preceding claims.
8. A compound according to any of the preceding claims or a
salt thereof for use in a method for the therapeutic or
prophylactic treatment of microbial infection in mammals.
9. A compound according to any of the preceding claims or a
salt thereof for use in a method for the therapeutic or
prophylactic treatment of tuberculosis or leprosy infection in
mammals.
10. Method for the preparation of 2-NR5R6-4H-1, 3-benzothiazin-
4-ones according to formula (I) of claim 1, comprising
reacting 2-chlorobenzcarboxamides with 1.1 to 2.5 times in
excess of metal salts of alkylxanthogenate at a temperature of
20-100°C in a solvent and subsequently treating the resulting
2-alkoxy-4ff-1,3-benzothiazin-4-one with an amine HNR5R6 in a
solvent at a temperature of 20-100°C.

The present invention relates to new antimicrobial compounds, their
synthesis and their use for treatment of mammalian infections. The
present invention aims at the generation of new compounds with activity
against mycobacteria as potential new tuberculosis drugs to overcome
problems concerning resistance and drug intolerance. This aim has been
solved by providing compounds of the formula 1 wherein R1 and R2 are,
independently of each other, NO2, NR7R8, NHOR9, COOR9, CN, CONR10R11,
CHO, F, Cl, Br, SO2NR12R13, lower alkoxy, OCF3, mono-, di or
trifluoromethyl; R3 and R4 are, independently of each other, H, a saturated
or unsaturated, linear or branched aliphatic radical having 1 -3 chain
members, F, Cl, Br, lower alkoxy; R5 is H, a saturated or unsaturated,
halogenated or unhalogenated, linear or branched aliphatic radical having
1-7 chain members; R6 is a radical: wherein X is saturated or unsaturated,
halogenated or unhalogenated, linear or branched aliphatic radical having
1-5 chain members, or R5 and R6 together represent bivalent radicals
wherein n is 1-4: R7- R13 are, independently of each other H or a
saturated or unsaturated, halogenated or unhalogenated, linear or
branched aliphatic radical having 1 -5 chain members, phenyl, benzyl or
R7 and R8 together, R

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Patent Number

278976

Indian Patent Application Number

261/KOLNP/2010

PG Journal Number

02/2017

Publication Date

13-Jan-2017

Grant Date

05-Jan-2017

Date of Filing

21-Jan-2010

Name of Patentee

LEIBNIZ-INSTITUT FÜR NATURSTOFF-FORSCHUNG UND INFEKTIONSBIOLOGIE E.V. HANS-KNÖLL-INSTITUT

Applicant Address

BEUTENBERGSTRASSE 11-A, 07745 JENA, GERMANY

Inventors:

#	Inventor's Name	Inventor's Address
1	MÖLLMANN, UTE	SCHLENDORFER OBERWEG 17 07749 JENA GERMANY
2	MAKAROV, VADIM	OLIMPISKI PROSPECT 10/1/17 MOSCOW, 129090 RUSSIA
3	STEWART, T. COLE	CHEMIN DE LA COCARDE 17BIS 1024 ECUBLENS SWITZERLAND

PCT International Classification Number

A61K31/5415; A61P31/06; A61P31/08

PCT International Application Number

PCT/EP2008/005142

PCT International Filing date

2008-06-25

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	07013899.5	2007-07-16	EUROPEAN UNION