Abstract:
This invention is directed to a composition comprising 4-chloro-3,6-dinitrobenzotrifluoride and related compounds and method for using it to treat infections caused by organisms that have microtubules.

Description:
This application takes priority from Provisional Application No. 60/042,267, filed Mar. 31, 1997. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to chloralin (4-chloro-3,5 dinitrobenzotrifluoride) and related compounds for use against parasites having microtubules, including Gardia, Plasmodium, Trypanosome and Leishmania parasites. 
     BACKGROUND OF THE INVENTION 
     Leishmaniasis is a disease that presents a major public health problem worldwide, with approximately 12 million to 40 million persons estimated to be infected. Previously the treatment of choice has been pentavalent antimony in the form of sodium stribogluconate or megluminic antimonate. Both agents are administered intravenously and produce severe adverse side effects. Hospitalization of the patient during treatment is required. Clinical failures are not uncommon. Drugs that are more easily administered and are less toxic are required. 
     Microtubule inhibitors have been exploited previously as antihelminthic drugs, in cancer therapy and as herbicides. Trifluralin, a microtubule inhibiting herbicide, has been shown to inhibit Leishmania species. The mechanism of action of these microtubule inhibitors in plants has been studied. Dinitroaniline herbicides such as oryzalin and trifluralin interact directly with the major microtubule protein, tubulin, leading to disruption of mitosis. 
     Although antimicrobial dinitroaniline herbicides show great potential as antiprotozoal compounds, disputed indications of potential carcinogenicity will probably keep trifluralin from being developed for human use. 
     DESCRIPTION OF THE INVENTION 
     It is the purpose of this invention to provide improved means for treating infections caused by organisms that have microtubules by administration of 4-chloro-3, 6-dinitrobenzotrifluoride and related compounds which may be administered at a dosage sufficient to attain a blood concentration of 0.5 μM to 500 μM. 
     Materials and Methods 
     3-chloro-3,5-dinitrobenzotrifluoride (compound 1), 4-chloro-3-nitrobenzotrifluoride (compound 4), 3-amino-4-chlorobenzotrifluoride (compound 5), 2-bromo-3,5(bis(trifluoromethyl)aniline (compound 6), 4-methoxy-3-nitrobenzotrifluoride (compound 7), and 2-nitro-4-(trifluoromethyl)thiophenol (compound 8) were obtained from Aldrich Chemical Company and were used to without further purification. Trifluralin was obtained from Reidel de Haen and was used without further purification. 4-chloro-3-nitro-5-sulfonylbenzotrifluoride (compound 2) and 4-chloro-3-nitro-5-carboxybenzotrifluoride (compound 3) were obtained from the Walter Reed Army Institute of Research inventory and were used without further purification. All of the agents were initially dissolved in dimethyl sulfoxide and were then diluted at 100 fold in parasitic culture medium before being tested against Leishmania species. 
     Of the seven compounds tested, the chloralin and compounds 2, 4, and 8 were found to be much more effective than trifluralin. 
     A comparison of the antiparasitic activities of analogues with electron withdrawing groups of different strengths but the same leaving group Cl, showed a correlation between the strength of the electron-withdrawing group and the activity of the compound against Leishmania promastigotes. 
     Compounds of the invention are of the formula:                           
     wherein R 4  is a leaving group such as chloro, alkoxy, bromo, an amino which forms a primary or secondary amine, hydroxy or thiol; R 3  and R 5  are H, nitro, amino, CF 3 , carboxy or sulfonyl group, wherein at least one of R 3  and R 5  is nitro. The compounds, when tested, showed the following concentrations to be effective at the IC 50  level: 
     
       
         
               
               
               
               
             
           
               
                   
                 TABLE I 
               
               
                   
                   
               
             
             
               
                   
                 Chloralin (4-chloro-3,5 dinitrobenzotrifluoride) 
                 .89 
                 μM 
               
               
                   
                 4-chloro-3-nitro-5-sulfonylbenzotrifluoride 
                 6.1 
                 μM 
               
               
                   
                 4-chloro-3-nitro-5-carboxybenzotrifluoride 
                 120 
                 μM 
               
               
                   
                 4-chloro-3-nitrobenzotrifluoride 
                 35 
                 μM 
               
               
                   
                 3-amino-4-chlorobenzotrifluoride 
                 77 
                 μM 
               
               
                   
                 2-bromo-3,5-bis(trifluoromethyl)aniline 
                 26 
                 μM 
               
               
                   
                 4-methoxy-3-nitrobenzotrifluoride 
                 68 
                 μM 
               
               
                   
                 2-nitro-4-(trifluoromethyl)thiophenol 
                 15 
                 μM 
               
               
                   
                   
               
             
          
         
       
     
     Of particular value are the compounds wherein R 4  is chloro and R 5  and R 3  are nitro or a sulfur containing substituent such as sulfonyl. 
     The active agents may be administered systemically to attain the effective blood concentration of 0.5 μM to 500 μM concentration in the blood. For example, the active agents may be administered orally, intramuscularly or, in the case of a severely ill patient, intravenously, in appropriate pharmaceutical carrier. Additionally, for cutaneous infections, the active agents may, for example, be administered as salves, ointments, gels, or lotions to the affected areas. Furthermore, the compounds, when given with transdermal carriers, may be administered dermally for systemic effect. 
     Compositions for administration are exemplified. However, such examples should not be viewed as limiting the invention. 
    
    
     EXAMPLE 1 
     Composition for parenteral use or use as spray: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 4-chloro-3,5 dinitro benzotrifluoride 
                 10 mg. 
               
               
                   
                 phosphate buffered saline 
                  5 ml. 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE 2 
     Composition for parenteral use: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 4,chloro-3-nitro-5-sulfonylbenzotrifluoride 
                 10 mg. 
               
               
                   
                 10% glucose in 1/2 normal saline 
                 10 ml 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE 3 
     Composition for topical use 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 2-nitro-4-(trifluoromethyl)thiophenol 
                 50 mg 
               
               
                   
                 propylene glycol 
                  5 ml 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE 4 
     For transdermal application, a patch composed of trilaminate of an adhesive matrix sandwiched between a non-permeable backing and a protective covering layer is prepared in the following manner: 
     To a pressure-sensitive silicone adhesive composition BIOPSA™ Q7-2920 (Dow Corning Corp., Midland, Mich., U.S.A.) in cyclohexane (50% w/v) is added sufficient amounts of Compound 2 to provide a composition containing 10% compound 2. The adhesive is applied to a polyester film to provide in successive layers to provide about 2 mg of active agent per cm 2 . The film containing the adhesive is then made into patches of 10 cm 2 . The patches would be covered with a protective layer to be removed before application of the patch. Patches may be prepared containing permeation enhancers such as cyclodextrin, butylated hydroxyanisole, or butylated hydroxytoluene. 
     EXAMPLE 5 
     The following composition is useful for oral administration: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 2-nitro-4-(trifluoromethyl)thiophenol 
                  10 mg 
               
               
                   
                 starch 
                 495 mg 
               
               
                   
                   
               
             
          
         
       
     
     The composition is placed in a capsule for oral administration