Inhibitors of Mycobacterium Tuberculosis Malate Synthase, Methods of Making and Uses Thereof

Abstract

The present invention provides aryl- or heteroaryl-diketo acid compounds effective to inhibit an activity of a Mycobacterial malate synthase enzyme or to inhibit a malate synthase activity in other bacteria having the enzyme. The compounds may be phenyl- naphthyl-, or thienyl-substituted diketo acids and carboxylate derivatives thereof. Also provided are methods of treating tuberculosis or other pathophysiological conditions associated with a malate synthase enzyme with the inhibitory compounds and methods of in silico design of the inhibitory compounds. In addition, the present invention provides the inhibitory compounds designed by this method. Furthermore, three-dimensional X-ray crystal structures of the Mycobacterial malate synthase complexed with the inhibitory compounds are provided. Further still a method for stabilizing an aromatic or heteroaromatic diketo acid or its prodrug or close analog in solution by derivatizing at least the ortho position on the aromatic ring is provided.

What is claimed is: 
     
         1 . A substituted diketo acid compound having the chemical structure comprising: 
       
         
           
           
               
               
           
         
         wherein R 1  is a thienyl, bithiophenyl, imidazolyl, benzothienyl, furanyl, benzofuranyl, pyrimidinyl, pyrrolyl, or pyridyl or substituted derivatives thereof; and 
         R 2  is H, OH, OC 1-6  alkyl, NH 2 , NHC 1-6 alkyl, or N(C 1-6 alkyl) 2 ; or 
       
       a pharmacologically acceptable salt thereof. 
     
     
         2 . The substituted diketo acid compound of  claim 1 , wherein R 1  is a 2- or 3-thienyl or (3-methyl)-2-thienyl substituted with R 8  at C5, wherein R 8  is one or more of R 3  at C2, R 4  at C3, R 5  at C4, R 6  at C5, or R 7  at C6, phenoxyphenyl, or 2-thienyl. 
     
     
         3 . The substituted diketo acid compound of  claim 2 , wherein R 3 , R 4 , R 5 , R 6 , and R 7  independently are H, OH, NO 2 , C 1-6 alkyl, C 1-6 alkoxy, O(C 1-6 alkyl)O(C 1-6 alkyl), Br, F, Cl, I, Ph, PhBr, PhF, PhCH 2 , PhOCH 3 , Ph(CH 2 ) 2 , CF 3 , CH 3 SO 2 , imidazolyl, or wherein R 1  and R 8  together are benzothienyl, 
     
     
         4 . The substituted diketo acid compound of  claim 3 , wherein one or more of R 3 , R 4 , R 5 , R 6  or R 7  is independently methoxy, Br, F, Cl, I, or methyl and R 2  is OH or OC 1-6 alkyl. 
     
     
         5 . The substituted diketo acid compound of  claim 1 , wherein R 1  is a 2-furanyl substituted with methyl at C5. 
     
     
         6 . A pharmaceutical composition comprising the substituted diketo acid compound of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         7 . A method for inhibiting an activity of a malate synthase enzyme in a bacterium, comprising:
 contacting the bacterium with an amount of the compound of  claim 1  effective to inhibit the activity of the malate synthase enzyme.   
     
     
         8 . The method of  claim 7 , wherein the bacterium is a  Mycobacterium.    
     
     
         9 . The method of  claim 8 , wherein the  Mycobacterium  is  Mycobacterium tuberculosis.    
     
     
         10 . A method for treating tuberculosis in a subject, comprising:
 administering one or more times a pharmacologically effective amount of one or more of the compounds of  claim 1  to the subject.   
     
     
         11 . The method of  claim 9 , further comprising:
 administering one or more times a pharmacologically effective amount of one or more other tuberculosis drugs.   
     
     
         12 . The method of  claim 11 , wherein the other tuberculosis drugs are isoniazid, rifampicin, pyrazinamide, or ethambutol. 
     
     
         13 . The method of  claim 11 , wherein the other tuberculosis drugs are administered concurrently or consecutively. 
     
     
         14 . A method for designing a potential inhibitory compound of a  Mycobacterium  malate synthase enzyme, comprising:
 identifying a compound in silico that interacts with the malate synthase active site, said identification based at least in part on a computer model of a crystalline structure of the malate synthase enzyme; and   screening the potential compound for inhibition of CoA production by Mycobacterial malate synthase.   
     
     
         15 . The method of  claim 14 , wherein said screening comprises:
 contacting a Mycobacterial malate synthase in the presence of an acetate carbon source with the potential compound; and   measuring production of CoA in the presence and absence of the potential compound; wherein a decrease in a level of CoA production in the presence of the compound compared to a level in the absence of the compound indicates that the potential compound is an inhibitor of the malate synthase.   
     
     
         16 . The method of  claim 15 , further comprising measuring growth inhibition of a  Mycobacteria  culture in the presence of the screened inhibitory compound compared to growth of a control in the absence thereof. 
     
     
         17 . The method of  claim 14 , wherein the potential compound comprises a carboxylate moiety positioned to contact a Mg2+ ion comprising the malate synthase active site. 
     
     
         18 . The method of  claim 14 , wherein the potential compound comprises a ketoacid positioned to bind a Mg2+ ion and a 1,3-diketo moiety positioned to form one or more hydrogen bonds with an amino acid residue that is or is functionally equivalent to an Arg-239 residue of native  Mycobacterium tuberculosis  malate synthase, both of the a Mg2+ ion and the functionally equivalent residue comprising the malate synthase active site. 
     
     
         19 . The method of  claim 14 , wherein the potential compound comprises a substituted phenyl moiety positioned to overlap an acetyl CoA binding site within the malate synthase. 
     
     
         20 . The method of  claim 14 , wherein the  Mycobacterium  is  Mycobacterium tuberculosis.    
     
     
         21 . An inhibitory compound designed by the method of  claim 14 . 
     
     
         22 . A three-dimensional X-ray crystal structure comprising a Mycobacterial malate synthase complexed to an inhibitory compound designed by the method of  claim 14 . 
     
     
         23 . A method for increasing the stability of an aromatic or heteroaromatic diketo acid compound in solution, comprising:
 derivatizing the aromatic ring or the heteroaromatic ring comprising the diketo acid or a prodrug thereof in at least an ortho position with a substituent effective to disrupt coplanarity of the aromatic ring with the diketo acid moiety thereby stabilizing the aromatic diketo acid compound.   
     
     
         24 . The method of  claim 23 , wherein the aromatic ring is a phenyl or naphthyl moiety and the heteroaromatic ring is a thienyl moiety. 
     
     
         25 . The method of  claim 23 , wherein the stabilizing substituent is OH, NO 2 , C 1-6 alkyl, C 1-6 alkoxy, O(C 1-6 alkyl)O(C 1-6 alkyl), Br, F, Cl, I, Ph, PhCH 2 , PhOCH 3 , Ph(CH 2 ) 2 , CF 3 , CH 3 SO 2 , or imidazolyl. 
     
     
         26 . The method of  claim 23 , wherein the stabilized aromatic or heteroaromatic diketo acid compound inhibits an activity of a bacterial malate synthase upon contact therewith in vitro or in vivo. 
     
     
         27 . The method of  claim 26 , wherein the bacterial malate synthase comprises a  Mycobacterium.    
     
     
         28 . A substituted diketo acid compound that is:
 (Z)-methyl 4-([2,2′-bithiophen]-5-yl)-2-hydroxy-4-oxobut-2-enoate;   (Z)-2-hydroxy-4-(3-methyl-5-phenylthiophen-2-yl)-4-oxobut-2-enoic acid;   (Z)-methyl 2-hydroxy-4-(3-methyl-5-phenylthiophen-2-yl)-4-oxobut-2-enoate   (Z)-methyl 2-hydroxy-4-(5-(4-methoxyphenyl)thiophen-2-yl)-4-oxobut-2-enoate;   (Z)-2-hydroxy-4-(5-(4-methoxyphenyl)thiophen-2-yl)-4-oxobut-2-enoic acid;   (Z)-methyl 4-(5-(4-fluorophenyl)thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoate;   (Z)-4-(5-(4-fluorophenyl)thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoic acid;   (Z)-methyl 2-hydroxy-4-oxo-4-(5-(4-(trifluoromethyl)phenyl)thiophen-2-yl)but-2-enoate   (Z)-2-hydroxy-4-oxo-4-(5-(4-(trifluoromethyl)phenyl)thiophen-2-yl)but-2-enoic acid   (Z)-methyl 2-hydroxy-4-(5-(4-hydroxyphenyl)thiophen-2-yl)-4-oxobut-2-enoate   (Z)-2-hydroxy-4-(5-(4-hydroxyphenyl)thiophen-2-yl)-4-oxobut-2-enoic acid;   (Z)-methyl 4-(benzo[b]thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoate;   (Z)-4-(benzo[b]thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoic acid;   (Z)-methyl 4-(5-(4-chlorophenyl)thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoate;   (Z)-4-(5-(4-chlorophenyl)thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoic acid   (Z)-methyl 2-hydroxy-4-oxo-4-(5-(4-phenoxyphenyl)thiophen-2-yl)but-2-enoate;   (Z)-2-hydroxy-4-oxo-4-(5-(4-phenoxyphenyl)thiophen-2-yl)but-2-enoic acid;   (Z)-methyl 2-hydroxy-4-(5-methylfuran-2-yl)-4-oxobut-2-enoate;   (Z)-2-hydroxy-4-(5-methylfuran-2-yl)-4-oxobut-2-enoic acid;   (Z)-methyl 4-(furan-2-yl)-2-hydroxy-4-oxobut-2-enoate;   (Z)-4-(furan-2-yl)-2-hydroxy-4-oxobut-2-enoic acid;   (Z)-4-(benzofuran-2-yl)-2-hydroxy-4-oxobut-2-enoic acid;   (Z)-methyl 4-(5-(4-bromophenyl)thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoate;   (Z)-4-(5-(4-bromophenyl)thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoic acid;   (Z)-methyl 2-hydroxy-4-oxo-4-(pyridin-2-yl)but-2-enoate;   (Z)-2-hydroxy-4-oxo-4-(pyridin-2-yl)but-2-enoic acid;   (Z)-methyl 2-hydroxy-4-oxo-4-(pyridin-4-yl)but-2-enoate;   (Z)-2-hydroxy-4-oxo-4-(pyridin-4-yl)but-2-enoic acid;   (Z)-methyl 2-hydroxy-4-oxo-4-(pyrimidin-2-yl)but-2-enoate;   (Z)-2-hydroxy-4-oxo-4-(pyrimidin-2-yl)but-2-enoic acid;   (Z)-methyl 2-hydroxy-4-oxo-4-(thiophen-3-yl)but-2-enoate; or   (Z)-2-hydroxy-4-oxo-4-(thiophen-3-yl)but-2-enoic acid.