Compounds, compositions, and methods for controlling biofilms

Abstract

The present invention provides nitrogen heterocyclic compounds, compositions, and methods for controlling biofilms, i.e., disrupting biofilms, preventing biofilm formation, enhancing biofilms, or modifying biofilms. Methods for screening test compounds for control of biofilms and devices for use therein are also provided.

1 . A method of enhancing the formation or existence of a biofilm on a substrate, comprising: 
 contacting the substrate with a compound having structure A:                        wherein n is 1-4; and    when n is 1, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H, C 6 -C 20  alkyl or alkenyl;  
 wherein when R is H, one of R 1  and R 2  is other than H.;  
   when n is 2, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H; C 4 -C 20  alkyl or alkenyl;  
 wherein when R is H, one of R 1  and R 2  is other than H;  
   when n is 3, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H; C 5 -C 20  alkyl or alkenyl;  
 wherein when R is H, one of R 1  and R 2  is other than H; or  
   when n is 4, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H; C 1 -C 20  alkyl or C 2 -C 20  alkenyl;  
     or a salt thereof, or a stereoisomer thereof;    or contacting the biofilm with a compound having structure B:                        wherein 
 n and m are independently 1, 2, 3, or 4, 
 X is C 1 -C 20  alkyl, or C 2 -C 20  alkenyl;  
 R 1  and R 4  are independently H, alkyl, acyl or alkoxycarbonyl, and  
 R 2  and R 3  are independently H, or alkyl;  
 
 with the proviso that when n and m are 2, X is C 8 -C 20  alkyl, or C 8 -C 20  alkenyl;  
     or a salt thereof, or a stereoisomer thereof;    for a time sufficient to control the biofilm on the substrate.    
   
   
       2 . The method of  claim 1  wherein the compound has structure A and n is 1.  
   
   
       3 . The method of  claim 2  where controlling is enhancement of biofilm formation or enhancement of existing biofilm, and wherein R is alkoxycarbonyl, R 1  is H, and R 2  is C 10 -C 12  alkyl.  
   
   
       4 . The method of  claim 2  where controlling is prevention of biofilm formation or dispersion of existing biofilm, and wherein R and R 1  are H, and R 2  is C 6 -C 12  alkyl.  
   
   
       5 . The method of  claim 1  wherein the compound has structure A and n is 2.  
   
   
       6 . The method of  claim 5  where controlling is enhancement of biofilm formation or enhancement of existing biofilm, and wherein R is alkoxycarbonyl or acyl, R 1  is H, and R 2  is C 6 -C 12  alkyl.  
   
   
       7 . The method of  claim 5  where controlling is prevention of biofilm formation or dispersion of existing biofilm, and wherein R and R 1  are H, and R 2  is C 10 -C 12  alkyl.  
   
   
       8 . The method of  claim 5  wherein R is H, R 1  is H, and R 2  is C 10  alkyl.  
   
   
       9 . The method of  claim 1  wherein the compound has structure A and n is 3.  
   
   
       10 . The method of  claim 9  wherein R is H or alkoxycarbonyl, R 1  is H, and R 2  is C 8  alkyl.  
   
   
       11 . The method of  claim 9  wherein controlling is enhancement of formation of biofilm, and wherein R is alkoxycarbonyl, acyl, or H, R 1  is H, and R 2  is C 8  alkyl.  
   
   
       12 . The method of  claim 1  wherein the compound has structure A and n is 4.  
   
   
       13 . The method of  claim 1  wherein the compound has structure B and n and m are 1.  
   
   
       14 . The method of  claim 1  wherein the compound has structure B and n and m are 2.  
   
   
       15 . The method of  claim 14  wherein X is C 12  alkyl, and R 1 , R 2 , R 3 , and R 4  are H.  
   
   
       16 . The method of  claim 14  where controlling is enhancement of biofilm formation or enhancement of existing biofilm, and wherein X is C 12  alkyl, R 2  and R 3  are H, and R 1  and R 4  are alkoxycarbonyl.  
   
   
       17 . The method of  claim 14  where controlling is prevention of biofilm formation or dispersion of existing biofilm, and wherein X is C 12  alkyl, and R 1 -R 4  are H.  
   
   
       18 . The method of  claim 1  wherein the compound has structure B and n and m are 3.  
   
   
       19 . The method of  claim 1  wherein the compound has structure B and n and m are 4.  
   
   
       20 . A compound for enhancing the formation or existence of a biofilm on a substrate having structure A:  
     
       
         
         
             
             
         
       
       wherein n is 1, 3, or 4; and  
       when n is 1, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H, C 6 -C 20  alkyl or alkenyl;  
 wherein when R is H, one of R 1  and R 2  is other than H;  
 
       when n is 3, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H; C 5 -C 20  alkyl or alkenyl;  
 wherein when R is H, one of R 1  and R 2  is other than H; or  
 
       when n is 4, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H; C 5 -C 20  alkyl or alkenyl;  
 
       or a salt thereof, or a stereoisomer thereof.  
     
   
   
       21 . The compound of  claim 20  wherein n is 1.  
   
   
       22 . The compound of  claim 20  wherein n is 3.  
   
   
       23 . The compound of  claim 20  wherein n is 4.  
   
   
       24 . A compound having structure B:  
     
       
         
         
             
             
         
       
       wherein 
 n and m are independently 1, 2, 3, or 4, 
 X is C 1 -C 20  alkyl, or C 2 -C 20  alkenyl;  
 R 1  and R 4  are independently H, alkyl, acyl or alkoxycarbonyl, and  
 R 2  and R 3  are independently H, or alkyl;  
 
 with the proviso that when n and m are 2, X is C 8 -C 20  alkyl, or C 8 -C 20  alkenyl;  
 
       or a salt thereof, or a stereoisomer thereof.  
     
   
   
       25 . The compound of  claim 24  wherein n and m are 1.  
   
   
       26 . The compound of  claim 24  wherein n and m are 2.  
   
   
       27 . The compound of  claim 26  wherein X is C 12  alkyl, and R 1 , R 2 , R 3 , and R 4  are H.  
   
   
       28 . The compound of  claim 24  wherein n and m are 3.  
   
   
       29 . The compound of  claim 24  wherein n and m are 4.  
   
   
       30 . A composition comprising the compound of  claim 20  and a carrier.  
   
   
       31 . A composition comprising the compound of  claim 24  and a carrier.  
   
   
       32 . An apparatus for testing biofilm control on a substrate surface, the apparatus comprising: 
 a first body having a platform holding a removable substrate surface lacking projections, the substrate surface for growing biofilm thereon; and    a second body adapted to receive the first body in alignment, the second body having a plurality of individual wells;    wherein the wells are in fluid tight communication with the substrate surface of the first body when the first and second bodies are assembled and in use.    
   
   
       33 . An apparatus for testing biofilm control on a substrate surface, the apparatus comprising: 
 first and third bodies, each body having a platform holding a removable substrate surface lacking projections, the substrate surface for growing biofilm thereon; and    a second body having a first side and a second side, the second body adapted to receive the first and third bodies in sandwich alignment to the first side and second side, the second body having a plurality of individual openings extending from the first side to the second side providing a well open at each end and in fluid tight communication with the substrate surface of the first and third bodies when the apparatus is assembled and in use.    
   
   
       34 . A method of screening a test compound for control of biofilm on a substrate surface, comprising: 
 obtaining the apparatus of  claim 33;     incubating a biofilm forming microorganism with the test compound in a well of the apparatus under conditions that allow biofilm formation on the substrate surface absent the test compound;    comparing biofilm formation on the substrate surface in the presence of the test compound with biofilm formation on the substrate surface in the absence of the test compound,    wherein when biofilm formation in the presence of the compound is less than in the absence of the test compound, then the test compound has inhibitory activity for biofilm formation on the substrate surface, and    wherein when biofilm formation in the presence of the compound is greater than in the absence of the test compound, then the test compound has stimulatory activity for biofilm formation on the substrate surface.    
   
   
       35 . A method of screening a test compound for dispersion of an existing biofilm on a substrate surface, comprising: 
 obtaining the apparatus of  claim 33;     incubating a biofilm forming microorganism in a well of the apparatus under conditions to form biofilm on the substrate surface;    contacting the biofilm on the substrate surface with a test compound;    comparing biofilm amount on the substrate surface in the presence of the test compound with biofilm amount on the substrate surface in the absence of the test compound,    wherein when biofilm amount in the presence of the compound is less than in the absence of the test compound, then the test compound has activity for biofilm dispersion on the substrate surface.    
   
   
       36 . A method for amplifying an effect of a compound having biofilm dispersion activity on amount of dispersed biofilm, comprising: 
 incubating a biofilm with a compound having biofilm dispersion activity for a time sufficient to allow the compound to act to disperse biofilm;    adding a base to the biofilm;    incubating for a time sufficient to amplify the effect of the compound; and    determining the amount of biofilm present;    wherein the effect of the compound is amplified in the presence of the base as compared to the effect in the absence of a base.    
   
   
       37 . A method of preventing biofilm formation or dispersing an existing biofilm, comprising: 
 contacting the substrate with a compound having structure A:                        wherein n is 1; and    R and R 1  are H;    R 2  is C 6 -C 12  alkyl.      
   
   
       38 . The method of  claim 38  wherein R is H, R 1  is H, and R 2  is C 10  alkyl.  
   
   
       39 . A compound for preventing biofilm formation or dispersing an existing biofilm having structure A:  
     
       
         
         
             
             
         
       
       wherein n is 1, 3, or 4; and  
       when n is 1, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H, C 6 -C 20  alkyl or alkenyl;  
 wherein when R is H, one of R 1  and R 2  is other than H;  
 
       when n is 3, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H; C 5 -C 20  alkyl or alkenyl;  
 wherein when R is H, one of R 1  and R 2  is other than H; or  
 
       when n is 4, 
 R is H, alkyl, acyl, or alkoxycarbonyl; and  
 R 1  and R 2  are independently H; C 5 -C 20  alkyl or alkenyl;  
 
       or a salt thereof, or a stereoisomer thereof.