Anti-infective compounds

Abstract

The present invention relates to 4H-pyrido[1,2-a]pyrimidin-4-one compounds and their use in the treatment of bacterial infections, in particular Tuberculosis.

We claim: 
     
         1 . A screening method comprising the steps of:
 (a) batch infection of host cells with fluorescently labeled  M. tuberculosis  mycobacteria;   (b) removing free unbound mycobacteria;   (c) adding compounds that are to be tested to a multi-well plate;   (d) seeding said host cells infected with fluorescently labeled  M. tuberculosis  mycobacteria into said multi-well plate containing said compounds;   (e) incubating said multi-well plate containing host cells infected with fluorescently labeled  M. tuberculosis  mycobacteria and said compounds;   (f) fluorescently labeling said host cells; and   (g) analyzing said multi-well plate using automated confocal microscopy.   
     
     
         2 . The method of  claim 1 , wherein the screening method searches for compounds that interfere with the multiplication of  M. tuberculosis  within said host cells. 
     
     
         3 . The method of  claim 1 , wherein the host cells are macrophages. 
     
     
         4 . The method of  claim 3 , wherein the macrophages are live macrophages. 
     
     
         5 . The method of  claim 1 , wherein the automated confocal fluorescence microscopy measures intracellular mycobacterial growth. 
     
     
         6 . The method of  claim 1 , wherein step (g) comprises determining for each compound
 a total host cell number,   a percentage of infected host cells; and/or   a percent inhibition of infection.   
     
     
         7 . The method of  claim 6 , wherein the host cells are macrophages. 
     
     
         8 . The method of  claim 6 , wherein step (g) further comprises analyzing the total host cell number to determine if it is high or low; and wherein
 (i) a low total cell number is indicative for the compound toxicity and/or of the unrestricted growth of  M. tuberculosis  inside macrophages;   and/or   (ii) a high (total) cell number is indicative that the compound is not toxic and prevents mycobacterial growth.   
     
     
         9 . The method of  claim 1 , further comprising the step of using control(s) selected from at least one of the following
 adding a compound with known anti-tuberculosis activity instead of the compounds in step (c) as a positive control; and   adding DMSO or antibiotic control(s) instead of the compounds in step (c) as negative control.   
     
     
         10 . The method of  claim 9 , wherein the compound with known anti-tuberculosis activity is selected from isoniazid, rifampin, ethionamide, and moxifloxacin. 
     
     
         11 . The method of  claim 1 , wherein the  M. tuberculosis  mycobacteria are labeled with GFP. 
     
     
         12 . The method of  claim 1 , wherein the host cells are labeled with SYTO 60. 
     
     
         13 . The method of  claim 1 , wherein after step (b) an incubation with an antibiotic is carried out. 
     
     
         14 . The method of  claim 13 , wherein the antibiotic is amykacin. 
     
     
         15 . The method of  claim 1 , wherein the screening method is used for the high throughput screening (HTS) of large scale chemical libraries. 
     
     
         16 . A compound that interferes with the multiplication of  M. tuberculosis  within host cells identified in the method of  claim 1 . 
     
     
         17 . A compound that interferes with the multiplication of  M. tuberculosis  within host cells identified in the method of  claim 3 . 
     
     
         18 . The compound of  claim 17 , which is represented by formula 
       
         
           
           
               
               
           
         
       
     
     
         19 . A compound that interferes with the multiplication of  M. tuberculosis  within host cells identified in the method of  claim 9 . 
     
     
         20 . A compound that interferes with the multiplication of  M. tuberculosis  within host cells identified in the method of  claim 13 .