Abstract:
The use of N 6 -(ferrocenmethyl)quinazoline-2,4,6-triamine (H2), its derivatives, and prodrugs that present antimicrobial (antibiotic, microbicide), antiparasitic (parasiticide), antiprotozoal (protozoacide), and antileishmanial (leishmanicide) activities, as well as its use as a drug in vertebrates (humans and animals).

Description:
RELATED APPLICATIONS 
     The present application is filed under 35 U.S.C. §371 as the U.S. national phase of International Application PCT/MX2011/000025, filed Feb. 21, 2011, which designated the U.S. and claims priority to the Mexican Application Serial No. MX/a/2010/002868, filed on Apr. 26, 2010, by Norma del Carmen Galindo Sevilla et al., the entire disclosure of both of which is incorporated herein by reference. 
     BACKGROUND 
     The use of N 6 -(ferrocenmethyl)quinazoline-2,4,6-triamine as an antimicrobial, antibiotic, microbicide, bacteriological, bacteriostatic, antiparasitic, antiprotozoal, or antileishmanial agent has not been previously reported. 
     Since the emergence of leishmaniasis in 1885, few agents have been described and used in the treatment of this disease, and these agents have variable efficiency and effectiveness. Therapeutic options are rare and include expensive drugs that are difficult to obtain, lack a coordinated registry, and may be toxic or ineffective. Antimonials, for example (including the meglumine antimoniate), were introduced in 1940 and continue to be the treatment of choice for cutaneous leishmaniasis, although the treatment regimens are longer than 20 days and can induce pancreatitis (the most frequent reason that treatment is discontinued) as well as serious electrocardiographic changes. Amphotericin B, which is nephrotoxic and hypercalcemic, is also used (Alvar J, et al., 1997.  Clin. Microbiol. Rev.  10: 298-319; Alvar J, et al., 2008.  Clin. Microbiol. Rev.  21: 334-359). 
     Other compounds used as antiparasites, such as metronidazole, present variable results, which in general reflects a lack of evidence regarding these drugs. Recently, the in vitro leishmanicidal activity of hydroxyurea was described (Martinez-Rojano H, et al., 2008.  Antimicrob. Agents Chemother.  52: 3642-3647), although in vivo evidence has not been reported. 
     THE SUBJECT MATTER OF THE INVENTION 
     The present invention refers to the human or veterinary use of a compound that contains N 6 -(ferrocenmethyl)quinazoline-2,4,6-triamine, as well as its derivatives and prodrugs, as an antimicrobial (antibiotic, microbicide), antiparasitic (parasiticide), antiprotozoal (protozoacide), or antileishmanial (leishmanicide) agent. 
     The N 6 -(ferrocenmethyl)quinazoline-2,4,6-triamine compound, which we refer to as H2, presents antimicrobial, antiparasitic, and leishmanicide activity from 0.1 μg/ml to greater than 100 μg/ml. H2 can be used in the treatment of infections caused by microorganisms, parasites, and protozoa, including members of the  Leishmania  genus in particular. 
    
    
     
       DESCRIPTION OF FIGURES 
         FIG. 1 : The biological activity of H2 toward the in vitro growth of  Leishmania mexicana  strain MHOM/MX/01/Tab3. A growth curve collected after 72 hours of parasite cultivation in a Neubauer chamber in the presence of the H2 is shown. The H2 concentration is shown on the horizontal axis, whereas the number of parasites/ml is shown on the vertical axis. The experiment was performed at room temperature using high-glucose Dulbecco&#39;s Modified Eagle&#39;s Medium with 10% fetal bovine serum. 
         FIG. 2 : Photograph obtained using an inverted microscope showing the inhibition of  Leishmania  growth by H2. The parasite culture shown in the left image was grown under the same conditions described in  FIG. 1  but with the absence of H2. The right image shows a culture grown in the presence of 1 μg/ml of the H2 compound, indicating a clear lack of growth and destruction of the parasite in the presence of H2. 
         FIG. 3 : A plot showing the H2 prodrug activity toward the in vitro growth of  Leishmania Mexicana . To obtain a higher sensitivity in this experiment, the MNYC/BZ/62/M379 reference strain, which has a higher sensitivity to H2 than does MHOM/MX/01/Tab3, was used. The number of parasites/ml was determined at 72 hours of incubation using 100 mM of each compound in four replicates. The culture conditions were the same as described in  FIG. 1 . 
         FIG. 4 : An example of biological activity of H2 parenterally administered at a dose of 0.1 mg/mL in 100 μl of physiological saline solution; a) and c) correspond to the activity prior to treatment, b) is a control using only a saline solution, and d) is the condition with H2. Images b) and d) were collected at 14 days after treatment. 
         FIG. 5 : An example of the biological activity of the HA2 prodrug dissolved in drinking water and orally administrated at a dose of 1 mg/mL ad libitum for 3 days; a) before treatment, b) three months after treatment, c) six months after treatment, and d) seven months after treatment. 
     
    
    
     DESCRIPTION OF THE INVENTION 
     Description of the Compound 
     The compound N 6 -(ferrocenmethyl)quinazoline-2,4,6-triamine (H2) is a solid substance at room temperature and atmospheric pressure. Its contains carbon, hydrogen, nitrogen and iron (II) and has a molecular weight of 374 a.m.u., a condensed molecular formula C 19 H 19 N 5 Fe, and the following chemical structure: 
     
       
                 
         
             
             
         
      
     
     H2 presents the following physicochemical properties: 
     Melting point: 210.6-211° C. 
     R f : 0.53 (2-butanol/acetic acid/water 80:20:5) 
     Infrared spectrum (KBr): 3369 and 3244 (N—H), 1693 and 1668 (C═O), 823 (C—H ferrocene). 
     Proton nuclear magnetic resonance spectrum (DMSO-d 6 ): 3.98 ppm (t, J=6, 2H, CH 2 ), 4.10 ppm (t, J=3, 2H, ferrocene), 4.20 ppm (s, 5H, ferrocene), 4.32 ppm (t, J=3, 2H, ferrocene), 5.3 ppm (t, J=6, 2H, CH 2 ), 5.51 ppm (s, 2H, NH 2 ), 6.96 ppm (d, J=2.4, 1H, quinazoline), 7.02 ppm (s, 1H, NH 2 ), 7.04 ppm (br., s, 1H, quinazoline), 7.049 ppm (br, s, 1H, quinazoline). 
     Elemental analysis for C 19 H 20 FeN 5 : Calculated: C, 61.14; H, 5.13; N, 18.76. Measured: C, 61.14; H, 4.92; N, 18.03. 
     H2 synthesis is initiated by the condensation of N,N′-(6-aminoquinazoline-2,4-diyl)diacetamide with ferrocencarboxaldehyde in dimethylformamide (DMF). Subsequent reduction with sodium borohydride (NaBH 4 ) gives HA2, which produces H2 in a 62% yield when hydrolyzed in a methanolic sodium hydroxide solution. 
     
       
                 
         
             
             
         
      
     
     It is also possible to prepare H2 prodrugs, i.e., compounds with the same base structure that form H2 when metabolized in a living organism. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 
               
             
             
               
                   
               
               
                 H2 Prodrugs 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
           
               
                 No. 
                 R 1   
                 R 2   
                 R 3   
               
               
                   
               
               
                 HA2 
                 NHC(O)CH 3   
                 NHC(O)CH 3   
                 H 
               
               
                 2 
                 NHCOCH 2 CH 2 COOH 
                 NH 2   
                 H 
               
               
                 3 
                 NH 2   
                 NHCOCH 2 CH 2 COOH 
                 H 
               
               
                 4 
                 NHCOCH 2 CH 2 COOH 
                 NHCOCH 2 CH 2 COOH 
                 H 
               
               
                 5 
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 NH 2   
                 H 
               
               
                 6 
                 NH 2   
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 H 
               
               
                 7 
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 H 
               
               
                   
               
             
          
         
       
     
     These compounds can be obtained using the following process: 
     The synthesis of prodrugs 2 and 3 is initiated by reacting one equivalent of succinic anhydride with H2 in DMF. After stirring at room temperature until the reactants are consumed, the resulting suspension is separated by filtration. The mixture of the obtained compounds (2 and 3) is separated by open column chromatography using silica gel as the stationary phase and chloroform as the mobile phase. The synthesis of prodrugs 4 and 7 is initiated by reacting two equivalents of succinic anhydride with H2 in DMF. After stirring at room temperature until the reactants are consumed, the suspended solution is separated by filtration to obtain prodrug 4, which is reacted with dicyclohexylcarbodiimide and hydroxyurea in DMF at room temperature for 72 hours. The reaction mixture is separated by open column chromatography using silica gel as the stationary phase and a chloroform/methanol gradient as the mobile phase to obtain prodrug 7. 
                                
Pharmaceutical Composition:
 
     As part of the invention, the pharmaceutical compositions of H2, derivatives and prodrugs are also presented along with the pharmaceutically acceptable excipients. The following excipients can be employed for the compound synthesis: low-molecular-weight carboxymethylcellulose, high-molecular-weight carboxymethylcellulose, ethanol, Tween 20, Tween 80, Cremophor, polyethylene glycol, propylene glycol, glycerol, triethanolamine, lactose, alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, heptakis, methyl-beta-cyclodextrin, and gamma-cyclodextrin. 
     Administration Routes: 
     The administration of H2, derivatives or prodrugs to biological organisms is performed by any pharmaceutical route that is used and accepted for that purpose. 
     Biological Activity: 
     At greater than 5 μg/ml, H2 is lethal to  Leishmania  in less than 5 hours. The in vitro effect is apparent at 30 minutes after application. The parasite structure is modified such that it loses its characteristic form, loses refringence, becomes spherical, and is incapable of multiplying. H2 has a CL 50  of 2.6 μm/ml for the  Leishmania mexicana  MHOM/MX/01/Tab3 strain. The mechanism of cellular damage could not be identified by means of annexin for concentrations greater than 10 μg/ml; it is thought that a necrosis process rather than apoptosis is likely involved. 
     In comparison to other compounds with leishmanicidal activity, such as meglumine antimoniate, metronidazole, or hydroxyurea, H2 kills the total amount of parasites more quickly at up to 10-fold faster than any previously described compounds at doses that are ten-fold lower or less. H2 also presents activity against other protozoa, including  Trypanosoma, Plasmodium, Entamoeba , and  Giardia , as well as metazoan parasites and microorganisms in general. 
     Cytotoxicity against murine cells was not found in in vitro studies or in vivo studies using oral, parenteral, or dermal administration in mice. The compound was designed to specifically inhibit the activity of vital protozoan enzymes without activity in the human versions. 
     EXAMPLES 
     1) Synthesis 
     Using a 50 ml Florence flask equipped with magnetic stirring, a Vigreux column, and a nitrogen atmosphere, 0.31 g of ferrocencarboxaldehyde (0.00143), 0.3 g of N,N′-(6-aminoquinazoline-2,4-diyl)diacetamide (1 eq.), 1 ml of DMF, and a drop of acetic acid were combined. The mixture was stirred at 85° C. for 45 minutes. The mixture was cooled to 0° C. using an ice-water bath, and 0.0671 g (2 eq.) of NaBH 4  was slowly added. The ice bath was removed, and stirring was continued for 12 hours at room temperature. The DMF was evaporated in a rotatory evaporator, and a saturated solution of Na 2 CO 3  was added to the residue. The yellow precipitate that formed was separated by filtration and rinsed several times with water. After drying at room temperature, the solid was rinsed several times with diisopropyl ether to obtain 0.3239 g of HA2 with a 48% yield, R f =0.76 (CHCl 3 /MeOH 80:20) and p.f.=218-220° C. HA2 was hydrolyzed with one equivalent of a methanolic sodium hydroxide solution to obtain a precipitate that was separated by filtration. The solid was cleaned in methanol with activated carbon. From this procedure, 0.32 g of a yellow compound (H2) was obtained at a 62% yield, R f =0.53 (2-butanol/acetic acid/water 80:20:5) and p.f.=210.6-211° C. 
     2) Biological Activity 
     H2 (3 μg/ml) eliminates more than 90% of the parasites in  Leishmania mexicana  cultures (Tab3 or M379 strain) with 10 6  parasites/ml in Dulbecco&#39;s medium modified with 4.5 mg/mL glucose and 10% fetal bovine serum. 
     3) Pharmaceutical Preparation 
     To prepare a suspension of H2, 10 mg of the substance was dissolved in 1 ml of DMF. Subsequently, 100 μl of the solution was diluted with water (1:10) to obtain a suspension for oral administration to rodents. 
     4) H2 Derivatives 
     
       
         
               
             
               
               
               
               
             
           
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
           
               
                 No. 
                 R 1   
                 R 2   
                 R 3   
               
               
                   
               
               
                  8 
                 NHC(O)CH 3   
                 NHC(O)CH 3   
                 CH 3   
               
               
                  9 
                 NHCOCH 2 CH 2 COOH 
                 NH 2   
                 CH 3   
               
               
                 10 
                 NH 2   
                 NHCOCH 2 CH 2 COOH 
                 CH 3   
               
               
                 11 
                 NHCOCH 2 CH 2 COOH 
                 NHCOCH 2 CH 2 COOH 
                 CH 3   
               
               
                 12 
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 NH 2   
                 CH 3   
               
               
                 13 
                 NH 2   
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 CH 3   
               
               
                 14 
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 CH 3   
               
               
                 15 
                 NHC(O)CH 3   
                 NHC(O)CH 3   
                 CH 3 CH 2   
               
               
                 16 
                 NHCOCH 2 CH 2 COOH 
                 NH 2   
                 CH 3 CH 2   
               
               
                 17 
                 NH 2   
                 NHCOCH 2 CH 2 COOH 
                 CH 3 CH 2   
               
               
                 18 
                 NHCOCH 2 CH 2 COOH 
                 NHCOCH 2 CH 2 COOH 
                 CH 3 CH 2   
               
               
                 19 
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 NH 2   
                 CH 3 CH 2   
               
               
                 20 
                 NH 2   
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 CH 3 CH 2   
               
               
                 21 
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 NHCOCH 2 CH 2 COONHC(NH)NH 2   
                 CH 3 CH 2   
               
               
                 HO2 
                 NH 2   
                 OH 
                 H 
               
               
                 HO4 
                 OH 
                 NH 2   
                 H 
               
               
                 HO24 
                 OH 
                 OH 
                 H 
               
               
                   
               
             
          
         
       
     
     5) Prodrug Synthesis 
     In a 50 ml Florence flask equipped with magnetic stiffing, a Vigreux column, and a nitrogen atmosphere, 0.31 g of ferrocencarboxaldehyde (0.00143), 0.3 g of N,N′-(6-aminoquinazoline-2,4-diyl)diacetamide (1 eq.), 1 ml of DMF, and a drop of acetic acid were combined. The mixture was stirred at 85° C. for 45 minutes. The mixture was cooled to 0° C. in an ice-water bath, and 0.0671 g (2 eq.) of NaBH 4  was slowly added. The ice bath was removed, and stirring was continued for 12 hours at room temperature. Subsequently, the DMF was evaporated in a rotary evaporator. A saturated solution of Na 2 CO 3  was added to the residue. The yellow precipitate that formed was separated by filtration and rinsed several times with water. After drying at room temperature, the solid was rinsed several times with diisopropyl ether to obtain 0.3239 g of HA2 at 48% yield, R f =0.76 (CHCl 3 /MeOH 80:20) and p.f.=218-220° C. 
     6) Biological Activity of the HA2 Prodrug and the Derivatives HO2 and HO4 
     The following is a list of  Leishmania mexicana  growth inhibition activity by the prodrugs compared to H2 and a control. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Compound 
                 % of  Leishmania  Growth Inhibition 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 0 
               
               
                   
                 HO2 
                 20 
               
               
                   
                 HO4 
                 25 
               
               
                   
                 FBC 
                 32 
               
               
                   
                 HA2 
                 24 
               
               
                   
                 H2 
                 100 
               
               
                   
                   
               
               
                   
                 FBC: N-(ferrocenmethyl)aniline