Guanine riboswitch binding compounds and their use as antibiotics

Abstract

The present invention includes novel compounds and pharmaceutically acceptable formulations of said compounds which exhibit antibiotic activity against microorganisms bearing a guanine riboswitch that controls the expression of the guaA gene, including organisms which are resistant to certain antibiotic families, and which are useful as antibacterial agents for treatment or prophylaxis of bacterial infections in animals or in humans, in particular but not limited to infections of the mammary gland, or their use as antiseptics, agents for sterilization or disinfection.

1 . A compound of the general formula 1.0, 
       
         
           
           
               
               
           
         
         wherein, when said compound is bound to a guanine riboswitch, R3 may serve as a hydrogen bond acceptor but cannot be ribosylated; 
         wherein  represents a single or double bond; 
         wherein R3 is —N═, —S—, —CH— or —O—; 
         wherein R6 is a carbon atom; 
         wherein R1, R2, R4 or R5 are identical or different and are independently —NR8-, —CHR8-, ═CR8-, —O(═O)— or —C(═NR8)-; 
         wherein R8 is —H, —NH 2 , —OH, —SH, a halide, fluorine, chlorine, bromine, iodine, —CO 2 H, —CO 2 -alkyl, —CO 2 -aryl, —C(O)NH 2 , —CH 3 , —POOH, —SO 2 alkyl, —SO 2 aryl, —SH, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted benzyloxy, substituted or unsubstituted —NHalkyl, substituted or unsubstituted —NHalkoxy, -substituted or unsubstituted NHC(O)alkyl, substituted or unsubstituted —NHCO 2 alkyl, substituted or unsubstituted —NH—NHalkyl, substituted or unsubstituted NH—NHalkoxy, substituted or unsubstituted —NH—SO 2 alkyl, —NHC(O)NH 2 , —NH—NH 2 , —NH—SO 2 —R9, —NHCO 2 CH 2 —R9, —NH—OR9, —NH 2 —R9, —NH—NH—R9, —NHR9, —NH—CH 2 —R9, or —NH—NH—CH 2 —R9, wherein R9 is: 
       
       
         
           
           
               
               
           
         
         wherein n is an integer from 1 to 5; 
         wherein R10 is —H, —NH 2 , —OH, alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine or iodine; 
         wherein, R7 is ═O, —OH, —SH, —NH 2 , —CO 2 H, —CO 2 -alkyl, —CO 2 -aryl, —C(O)NH 2 , -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine, chlorine, bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl, —NHCO 2 alkyl, ═NR8, ═NR9, —NHCO 2 CH 2 —R9, —NHC(O)NH 2 , —NH—NH 2 , —NH—NHalkyl, —NH—NHalkoxy, —SO 2 alkyl, —SO 2 aryl, —NH—SO 2 alkyl, —NH—SO 2 —R9, —NH—OR9, —NH—R9, —NH—NH—R9, —NH—NH—CH 2 —R9, or —NH—CH 2 —R9, wherein R8 and R9 are as defined above, 
         with the proviso that the compound is not: 
         4-hydroxy-2,5,6-triaminopyrimidine (1.01); 
         2,4-diamino-6-hydroxypyrimidine (1.13); or 
         4,5-diamino-6-hydroxy-2-mercaptopyrimidine (1.16). 
       
     
     
         2 . A compound of the general formula 2.0, 
       
         
           
           
               
               
           
         
         wherein, when said compound is bound to a guanine riboswitch, R9 may serve as a hydrogen bond donor but cannot be ribosylated; 
         wherein represents a single or double bond; 
         wherein R3 is —N═, —S—, or —O—; 
         wherein R1 or R2 are identical or different and are independently —NR11-, —CHR11-, ═CR11-, —C(═O)— or —C(═NR11)-; 
         wherein R11 is —H, —NH 2 , —OH, —SH, —CO 2 H, —CO 2 -alkyl, —CO 2 -aryl, —C(O)NH 2 , —CH 3 , —POOH, —SO 2 alkyl, —SO 2 aryl, —SH, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted benzyloxy, substituted or unsubstituted —NHalkyl, substituted or unsubstituted —NHalkoxy, substituted or unsubstituted —NHC(O)alkyl, substituted or unsubstituted —NHCO 2 alkyl, substituted or unsubstituted —NH—NHalkyl, substituted or unsubstituted —NH—NHalkoxy, substituted or unsubstituted —NH—SO 2 alkyl, —NHC(O)NH 2 , —NH—N H 2 , —NH—SO 2 —R12, —NHCO 2 CH 2 —R12, —NH—OR12, —NH 2 —R12, —NH—NH—R12, —NHR12, —NH—CH 2 —R12, or —NH—NH—CH 2 —R12; 
         wherein R12 is: 
       
       
         
           
           
               
               
           
         
         wherein n is in integer from 1 to 5; 
         wherein R13 is at least one of —H, —NH 2 , —OH, alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine, iodine; 
         wherein R4, R5 and R6 are carbon atoms; 
         wherein R7 is —═N—, —NH—, —CH 2 —; —O— or —S—; 
         wherein R8 is —CH 2 , —O—, —S—, —CHR13- or —CR13=, wherein R13 is as defined above; 
         wherein R9 is —CH 2 —; —O—, —S—, or —P(OOH)—, —N═; 
         wherein R10 is ═O, —OH, —SH, —NH 2 , —CO 2 H, —CO 2 -alkyl, —CO 2 -aryl, —C(O)NH 2 , -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine, chlorine, bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl, —NHCO 2 alkyl, ═NR11, ═NR12, —NHCO 2 CH 2 —R12, —NHC(O)NH 2 , —NH—NH 2 , —NH—NHalkyl, —NH—NHalkoxy, —SO 2 alkyl, —SO 2 aryl, —NH—SO 2 alkyl, —NH—SO 2 —R12, —NH—OR12, —NH—R12, —NH—NH—R12, —NH—NH—CH 2 —R12, or —NH—CH 2 —R12; 
         wherein R11 and R12 are as defined above, 
         with the proviso that the compound is not: guanine, hypoxanthine, xanthine or 5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17). 
       
     
     
         3 . A compound of the general formula 3.0, 
       
         
           
           
               
               
           
         
         wherein, when the compound is bound to a guanine riboswitch, R10 may serve as a hydrogen bond donor but cannot be ribosylated; 
         wherein represents a single or double bond; 
         wherein R3 is —N═, —S—, or —O—; 
         wherein R10 is —N═, —CH 2 —; —O—, —S—, or —P(OOH)—; 
         wherein R1, R2 and R9are identical or different and are independently —NR12-, —CHR12-, ═CR12-, —O(═O)— or —C(═NR12)-; 
         wherein R12 is —H, —NH 2 , —OH, —SH, —CO 2 H, —CO 2 -alkyl, —CO 2 -aryl, —C(O)NH 2 , —CH 3 , —POOH, —SO 2 alkyl, —SO 2 aryl, —SH, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted benzyloxy, -substituted or unsubstituted NHalkyl, substituted or unsubstituted —NHalkoxy, substituted or unsubstituted —NHC(O)alkyl, substituted or unsubstituted —NHCO 2 alkyl, substituted or unsubstituted —NH—NHalkyl, substituted or unsubstituted —NH—NHalkoxy, substituted or unsubstituted —NH—SO 2 alkyl, —NHC(O)NH 2 , —NH—NH 2 , —NH—SO 2 —R13,—NHCO 2 CH 2 —R13, —NH—OR13, —N+H2-R13, —NH—NH—R13, —NHR13, —NH—CH 2 —R13, or —NH—NH—CH 2 —R13; 
         wherein R13 is: 
       
       
         
           
           
               
               
           
         
         wherein n is an integer from 1 to 5; 
         wherein R14 is —H, —NH 2 , —OH, alkoxy, —N-morpholino, a halide, fluorine, chlorine, bromine, or iodine; 
         wherein R4, R5 and R6 are carbon atoms; 
         wherein R7 and R8 are identical or different and are independently —CH═N—, —CH 2 —O—, CH 2 —S—, or CH 2 SO 2 —; 
         wherein R11 is ═O, —OH, —SH, —NH 2 , —CO 2 H, —CO 2 -alkyl, —CO 2 -aryl, —C(O)NH 2 , -alkoxy, -aryloxy, -benzyloxy, a halide, fluorine, chlorine, bromine, iodine, —NHalkyl, —NHalkoxy, —NHC(O)alkyl, —NHCO 2 alkyl, ═NR12, ═NR13, —NHCO 2 CH 2 —R13, —NHC(O)NH 2 , —NH—NH 2 , —NH—NHalkyl, —NH—NHalkoxy, —SO 2 alkyl, —SO 2 aryl, —NH—SO 2 alkyl, —NH—SO 2 —R13, —NH—OR13, —NH—R13, —NH—NH—R13, —NH—NH—CH 2 —R13, or —NH—CH 2 —R13; and 
         wherein R12 and R13 are as defined above. 
       
     
     
         4 . The compound of  claim 2 , wherein:
 (i) R1 is —NH—;   (ii) R2 is ═CNH 2 —;   (iii) R3 is —N═;   (iv) R7 is —N═ or —S—;   (v) R8 is —CR13=;   (vi) R9 is —O— or —N═;   (vii) R10 is ═O or ═NH; or   (viii) any combination of (i) to (vii).   
     
     
         5 . The compound of  claim 2 , wherein said compound is of the general formula 2.0a, 
       
         
           
           
               
               
           
         
         wherein R2, R7, R8, R9 and R10 are as defined in  claim 2 . 
       
     
     
         6 . The compound of  claim 5 , wherein R2 is ═CNH 2 —. 
     
     
         7 . The compound of  claim 5 , wherein R7 is —N═ or —S—. 
     
     
         8 . The compound of  claim 5 , wherein R8 is —CR13=. 
     
     
         9 . The compound of  claim 5 , wherein R9 is —O— or —N═. 
     
     
         10 . The compound of  claim 5 , wherein R10 is ═O or ═NH. 
     
     
         11 . A compound of the general formula 3.0a, 
       
         
           
           
               
               
           
         
         wherein R2, R7, R10 and R11 are as defined in  claim 3 . 
       
     
     
         12 . The compound of  claim 1 , wherein
 (i) R1 is —NH—;   (ii) R2 is —C(NH 2 )═ or ═C(SH)—;   (iii) R3 is —N═;   (iv) R4 is ═CNH 2 —;   (v) R5 is —CH═ or —C(NH 2 )═;   (vi) R6 is —C═;   (vii) R7 is ═NH or ═O; or   (viii) any combination of (i) to (vii).   
     
     
         13 . The compound of  claim 1 , wherein said compound is of the general formula 1.0a, 
       
         
           
           
               
               
           
         
         wherein R2 and R7 are as defined in  claim 1 , 
         wherein R5 is —CH═ or —C(NH 2 )═, and wherein R4 is —C(NH 2 )═. 
       
     
     
         14 . The compound of  claim 13 , wherein R7 is ═O or ═NH. 
     
     
         15 . The compound of  claim 13 , wherein R2 is —C(NH 2 )═ or —C(SH)═. 
     
     
         16 . A composition comprising the compound defined in  claim 1 , and
 (a) an antibiotic;   (b) an antiseptic;   (c) a disinfectant;   (d) a diluent;   (e) an excipient;   (f) a pharmaceutically acceptable carrier; or   (g) any combination of (a)-(f).   
     
     
         17 . A composition comprising
 (i) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01);   (ii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16)   (iii) 2,4-diamino-6-hydroxypyrimidine (formula 1.13); or   (iv) 5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17); and   (a) an antibiotic;   (b) an antiseptic;   (c) a disinfectant;   (d) a pharmaceutically acceptable carrier; or   (e) any combination of (a)-(d).   
     
     
         18 . The composition of  claim 16 , wherein said composition is a pharmaceutical composition. 
     
     
         19 . A method of preventing or treating a microbial infection in a subject, said method comprising administering to said subject a therapeutically effective amount of a non ribosylable ligand of a guanine riboswitch, wherein said microbial infection is caused by a pathogen bearing the guanine riboswitch, and wherein the guanine riboswitch controls the expression of guaA. 
     
     
         20 . The method of  claim 19 , wherein said non ribosylable ligand of a guanine riboswitch is
 (i) the compound defined in  claim 1 ;   (ii) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01);   (iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16);   (iv) 2,4-diamino-6-hydroxypyrimidine (formula 1.13); or   (v) 5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).   
     
     
         21 . The method of  claim 19 , wherein said subject is an animal. 
     
     
         22 . The method of  claim 21 , wherein said subject is a cow or a human. 
     
     
         23 . The method of  claim 19 , wherein said pathogen is a bacteria belonging to the genus  Staphylococcus  or  Clostridium.    
     
     
         24 . The method of  claim 23 , wherein said bacteria is  Staphylococcus aureus,  methicillin-resistant  Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Clostridium botulinum  or  Clostridium difficile.    
     
     
         25 . The method of  claim 19 , wherein said infection is a mammary gland infection. 
     
     
         26 .- 33 . (canceled) 
     
     
         34 . A method of disinfecting and/or sterilizing an object of a pathogen, said method comprising applying an effective amount of a non ribosylable ligand of a guanine riboswitch to said object, wherein said pathogen bears a guanine riboswitch that controls the expression of guaA. 
     
     
         35 . The method of  claim 34 , wherein said non ribosylable ligand of a guanine riboswitch is
 (i) the compound defined in  claim 1 ;   (ii) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01);   (iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16);   (iv) 2,4-diamino-6-hydroxypyrimidine (formula 1.13); or   (v) 5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).   
     
     
         36 . The method of  claim 34 , wherein said object is an animal or milk. 
     
     
         37 - 39 . (canceled) 
     
     
         40 . A method of selecting a pathogen treatable by
 (i) the compound defined in  claim 1 ;   (ii) 4-hydroxy-2,5,6-triaminopyrimidine(formula 1.01);   (iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16);   (iv) 2,4-diamino-6-hydroxypyrimidine (formula 1.13);   (v) 5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17); or   (vi) the composition defined in  claim 16 ,   said method comprising determining whether said pathogen bears a guanine riboswitch that controls the expression of guaA.   
     
     
         41 . A method of identifying a compound for preventing or treating a microbial infection caused by a pathogen bearing a guanine riboswitch that controls the expression of guaA, said method comprising contacting a guanine riboswitch with said compound; determining whether said compound binds to said guanine riboswitch; wherein the binding of said compound to said guanine riboswitch is an indication that said compound is suitable for preventing or treating said microbial infection. 
     
     
         42 . The method of  claim 41 , wherein said guanine riboswitch is the guanine xpt riboswitch from  Streptococcus pyogenes  (STPY-xpt). 
     
     
         43 . The method of  claim 41 , further comprising contacting said guanine riboswitch with guanine or a guanine-like ligand. 
     
     
         44 . The method of  claim 43 , further comprising determining whether said compound may be ribosylated. 
     
     
         45 . A method for preventing the development of multi-drug resistance of a bacteria in a subject, or treating a multidrug resistance of a bacteria in the subject said method comprising administering a non ribosylable ligand of a guanine riboswitch to the subject, wherein the bacteria bears a guanine riboswitch that controls the expression of guaA. 
     
     
         46 . The method of  claim 45 , wherein said non ribosylable ligand of a guanine riboswitch is:
 (i) the compound defined in  claim 1 ;   (ii) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01);   (iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16);   (iv) 2,4-diamino-6-hydroxypyrimidine (formula 1.13); or   (v) 5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17).   
     
     
         47 .- 49 . (canceled) 
     
     
         50 . A kit comprising:
 (i) the compound defined in  claim 1 ;   (ii) 4-hydroxy-2,5,6-triaminopyrimidine (formula 1.01);   (iii) 4,5-diamino-6-hydroxy-2-mercaptopyrimidine (formula 1.16);   (iv) 2,4-diamino-6-hydroxypyrimidine (formula 1.13);   (v) 5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one (2.17); or   (vi) the composition defined in  claim 16 ,   and instructions to use same in the prevention or treatment of a microbial infection.   
     
     
         51 . A method for preparing the compound of formula 2.02 
       
         
           
           
               
               
           
         
         said method comprising 
         (a) reacting diethylacetamido malonate with guanidine hydrochloride in the presence of sodium methoxide and methanol to obtain a solid; 
         (b) dissolving the solid of (a) in an aqueous solution; 
         (c) subjecting the solution of (b) to an acidic precipitation to obtain a 5-formamino-2-amino-4,6-dihydroxypyrimidine precipitate; 
         (d) dissolving the 5-formamino-2-amino-4,6-dihydroxypyrimidine precipitate in an acid; and 
         (e) precipitating the solution of (d) to obtain the compound of formula 2.02. 
       
     
     
         52 . The method of  claim 51 , wherein said reacting is performed under reflux. 
     
     
         53 . The method of  claim 51 , wherein said acidic precipitation of (b) is performed using a hydrochloric acid (HCl) solution. 
     
     
         54 . The method of  claim 53 , wherein said HCl solution is a 50% HCl solution. 
     
     
         55 . The method of  claim 51 , wherein dissolving of (d) is performed using sulfuric acid. 
     
     
         56 . The method of  claim 55 , wherein said sulfuric acid is 12 M sulfuric acid. 
     
     
         57 . The method of  claim 51 , further comprising washing said solid of (a) with methanol and chloroform. 
     
     
         58 . The method of  claim 51 , wherein said aqueous solution is water. 
     
     
         59 . The method of  claim 51 , wherein said precipitating of (e) is performed using tetrahydrofuran (THF). 
     
     
         60 . - 70 .l (canceled)