Abstract:
The present invention relates to linear and cyclic guanidine derivatives, method of preparation and uses thereof, pharmaceutical compositions to be used as antifungal agents, in particular against  Candida  species.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a 371 of PCT/IB2009/051032 filed Mar. 12, 2009, which claims the benefit of U.S. Application No. 61/036,165 filed Mar. 13, 2008, the contents of each of which are incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     The present invention relates to linear and cyclic guanidine derivatives, method of preparation and uses thereof, pharmaceutical compositions to be used as antifungal agents, in particular against  Candida  species. 
     BACKGROUND OF THE INVENTION 
     The opportunistic human pathogen  Candida albicans  and other non- albicans  species have acquired considerable clinical significance as infectious agents in immunocompromised patients, being important causes of morbidity and mortality. The recommended therapy relies on fluconazole, voriconazole and caspofungin. In fact, also many of the new possible antifungal agents that can be found in the literature possess an azole core. 
     The pathogenic species of  Candida  derive their relevance not only from the severity of the infections but also from their ability to develop resistance against a variety of antifungal agents. In fact, widespread and prolonged use of azoles has led to the rapid development of multidrug resistance, which poses a major hurdle in antifungal therapy. Many of the currently available drugs have become ineffective against new or re-emerging fungi because of the rapid development of resistance. These problems have give rise to the need to develop new effective antifungal agents. Accordingly, in the last years, new structural classes of antifungal agents were reported, among which guanidine derivatives proved to have very interesting inhibitory activity. As an example, guazatine (a mixture of guanidines and polyamines used in agriculture as fungicide) was classified as a moderately hazardous antifungal agent, while results from in vivo animal studies demonstrated a high potential for guazatine and related compounds as antifungal agents. Authors have recently reported that components of guazatine are able to act toward albicans and non-albicans  Candida  species. 
     SUMMARY OF THE INVENTION 
     The present invention concerns novel cyclic guanilated derivatives of different polyamines. On the basis of the results obtained with components of guazatine, new cyclic guanidine derivatives of different polyamines have been synthesized, and their biological evaluation against 8 clinical isolates and 3 reference species of  Candida  ( C. albicans  ATCC 60193,  C. krusei  ATCC 14243,  C. parapsilosis  ATCC 34136) has been carried out. The new compounds object of the invention possess an excellent antifungal activity, and they
         1. are very active against different species of  Candida;      2. have a low toxicity   3. are active also against drug resistant strains of  Candida.          

     It is further object of the invention to use any of the compounds as a medicament. 
     It is further object of the invention to use any of the compounds as an anti-infectious agent, preferably as antifungal agent, more preferably as antifungal agent against  Candida  species, even more preferably wherein the  Candida  species belong to the group of  C. albicans, C. krusei, C. parapsilosis.    
     It is further object of the invention to provide a pharmaceutical composition comprising any of the compounds claimed herein or a mixture of any of them, and appropriate excipients and diluents. The expert in the field shall select appropriate excipients and diluents according to the way of administration (topic, oral, parenteral, etc.). In a preferred embodiment the composition further comprises at least one other compound with antifungal activity. 
     It is further object of the invention to provide a process for the preparation of a compound comprising the following steps: 
     a) reaction of a suitable amine R 1 —NH 2  with a suitable S-methylisothiourea in a suitable solvent for obtaining compound 2 
                                
wherein
 
n 1  and n 2  are 4 or 6, n 2  can be n 1  or n 2  can be different from n 1 ;
 
R 1 ═H, propargyl, cyclopropylmethyl, γ-methylallyl, β-methylallyl, γ,γ-dimethylallyl, methyl or ethyl;
 
R 2 ═H, propargyl, cyclopropylmethyl, γ-methylallyl, β-methylallyl, γ,γ-dimethylallyl, methyl or ethyl;
 
b) extract and/or purify compound 2 as obtained under a);
 
c) allow compound 2 to react with an appropriate N,N′-bis(tert-butoxycarbonyl)-N-(alkyl)-S-methylisothiourea under conditions suitable for obtaining the compound 3, or 5-10 shown below:
 
                                                          
wherein
 
R 2 ═H, propargyl, cyclopropylmethyl, γ-methylallyl, β-methylallyl, γ,γ-dimethylallyl, methyl or ethyl;
 
R 3 ═H, methyl, ethyl or benzyl;
 
R 4 -R 23 ═H, methyl or ethyl; R 4 -R 23  can be same or different;
 
d) purify compounds 3, 5-10 as under c);
 
e) allow compounds 3, 5-10 to react under suitable conditions for obtaining compounds claimed herein;
 
f) extract and/or purify the compounds as obtained under e).
 
     It is further object of the invention an intermediate of any of formula 3, 5-10: 
     
       
                 
         
             
             
         
      
       
                 
         
             
             
         
       
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Chemistry 
     The compounds described in this invention can be synthesised as described below: 
     
       
                 
         
             
             
         
      
     
     Examples of the synthetic procedures: 
     
       
                 
         
             
             
         
      
     
     Example 1 
     Preparation of 1-amino-17-[N 2 ,N 3 -bis(tert-butoxycarbonyl)guanidino]-9-azaheptadecane (12) 
     To a stirred solution of 1,17-diamino-9-azaheptadecane 3 (4.9 g, 15.06 mmol) in THF/CH 3 OH 5/3 (80 mL) at 50° C., a solution of N,N′-bis(tert-butoxycarbonyl)-S-methylisothiourea (1.456 g, 5.02 mmol) in THF (25 mL) was added dropwise over 1 h. After 16 h, the reaction mixture was concentrated under reduced pressure and the residue was purified by flash chromatography (6% methanol, 4% triethylamine, 90% ethyl acetate), affording 12 as a pale yellow oil, 3.51 g (70%). 
     Example 2 
     General Procedure for the Preparation of 14 
     To a stirred solution of 12 (1.5 mmol) in THF (15 mL) at 60° C., a solution of the appropriate N,N′-bis(tert-butoxycarbonyl)-N-(alkyl)-S-methylisothiourea (1 mmol) in THF (5 mL) was added dropwise. The reaction mixtures were stirred at 60° C. for 16 h, cooled at rt and concentrated under reduced pressure. The crude mixtures were purified by flash chromatography, affording 14a-b as yellow oils. 
     NMR data for compounds 14a-b 
     Tert-butyl N-[(2Z)-5-(8-{[(1Z)-{[benzyl][(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}octyl)-4-oxo-1,3,5-triazacyclotridecane-2-ylidene]carbamate (14a) 
       1 H NMR (CDCl 3 ) δ 12.06 (NH, br s), 8.73 (NH, br s), 8.06 (NH, br s), 7.26-7.24 (5H, m), 4.82 (2H, s), 3.47-3.32 (2H, m), 3.25-3.18 (2H, m), 3.00-2.93 (4H, m), 2.01-1.82 (4H, m), 1.48 (9H, s), 1.43 (9H, s), 1.41 (9H, s), 1.29-1.11 (20H, m). MS (ESI): m/z=772.1 [M+H] + . 
     Tert-butyl N-[(2Z)-5-(8-{[(1Z)-{[propargyl][(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}octyl)-4-oxo-1,3,5-triazacyclotridecan-2-ylidene]carbamate (14b) 
       1 H NMR (CDCl 3 ) δ 8.09 (NH, br s), 4.45 (2H, s), 3.50 (1H, s), 3.45-3.20 (4H, m), 3.25-3.15 (4H, m), 2.01-1.82 (4H, m), 1.48 (9H, s), 1.43 (9H, s), 1.41 (9H, s), 1.32-1.11 (20H, m). MS (ESI): m/z=720.2 [M+H] + . 
     Example 3 
     General Procedure for Synthesis of Compounds 15a-15e 
     Compounds 14a-14e were treated with a 10% solution of freshly distilled TFA in dry DCM (30 mL for 1 mmol) and the reaction mixtures was stirred at rt under argon. After 24 h, the reaction mixtures were concentrated under reduced pressure giving the desired compounds as tri trifluoroacetate salts (brown oils), in quantitative yield. The mixtures were purified by semipreparative HPLC affording the final compounds as tri triformiate salts. NMR data for compounds 15a-15e 
     1-[benzyl]-3-[8-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)octyl]guanidine tri trifluoroacetate (15a) 
       1 H NMR (CD 3 ) 2 CO δ 8.25 (NH, br s), 7.75 (NH, br s), 7.44 (NH, br s), 7.35-7.32 (5H, m), 4.55-4.52 (2H, d, J=5 Hz), 3.75-3.44 (4H, m), 3.32-3.29 (4H, m), 1.64-1.57 (8H, m), 1.30 (16 H, br s). MS (ESI): m/z=472.1 [M+H] + . 
     1-[propargyl]-3-[8-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)octyl]guanidine tri trifluoroacetate (15b) 
       1 H NMR (CD 3 ) 2 CO δ8.26 (NH, br s), 7.60 (NH, br s), 7.44 (NH, br s), 4.16-4.14 (2H, m), 3.48-3.45 (4H, m), 3.33-3.27 (4H, m), 2.89 (1H, s), 1.68-1.59 (8H, m), 1.35-1.29 (16H, m). MS (ESI): m/z=420.1 [M+H] + . 
     1-[cyclopropylmethyl]-3-[8-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)octyl]guanidine tri trifluoroacetate (15c) 
       1 H NMR (CD 3 ) 2 CO δ 8.24 (NH, br s), 7.77 (NH, br s), 7.20 (NH, br s), 5.77-5.45 (2H, m), 3.74-3.71 (2H, m), 3.30-3.25 (4H, m), 3.19-3.12 (4H, m), 1.70-1.46 (12H, m), 1.34-1.26 (16H, m). MS (ESI): m/z=436.2 [M+H] + . 
     1[β-methylallyl]-3-[8-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)octyl]guanidine tri trifluoroacetate (15d) 
       1 H NMR (CD 3 ) 2 CO δ 8.30 (NH, br s), 7.58 (NH, br s), 4.93-4.88 (2H, d, J=10 Hz), 3.84-3.82 (2H, m), 3.46-3.40 (4H, m), 3.37-3.20 (4H, m), 1.72 (3H, s), 1.62 (8H, br s), 1.32-1.27 (16H, m). MS (ESI): m/z 436.3 [M+H] + . 
     1-[γ,γ-dimethylallyl]-3-[8-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)octyl]guanidine tri trifluoroacetate (15e) 
       1 H NMR (CD 3 ) 2 CO δ 8.22 (NH, br s), 7.90 (NH, br s), 5.28-5.25 (1H, m), 3.74-3.71 (2H, m), 3.40-3.30 (4H, m), 3.20-3.10 (4H, m), 1.65-1.44 (14H, m), 1.30-1.28 (16H, m). MS (ESI): m/z=450.3 [M+H] + . 
     Biological Tests 
     Determination of MICs by AFST-EUCAST Standard Methodology 
     Assay medium. The assay medium was RPMI 1604 without NaHCO 3  and with L-glutamine (Sigma Aldrich, Italy), buffered at pH 7.0 with 0.165 M morpholinepropanesulphonic acid (Sigma Aldrich, Italy) and supplemented with 2% (w/v) glucose. The medium, prepared as double-strength solution, was sterilized by filtration and diluted 1:2 (v/v) with the fungal inoculum prepared in sterile distilled water. 
     Preparation of Inoculate 
     The yeast isolates were grown on Sabouraud dextrose agar (Oxoid, Madrid, Spain) for 48 h at 37° C. before testing. Suspensions were prepared by combining five distinct colonies of each culture of &gt;1 mm diameter. A spectrophotometric procedure for inoculum preparation was used. The final inoculum suspension, prepared in sterile distilled water, contained between 0.5·10 5  and 2.5·10 5  cfu/mL. 
     Antifungal Agents 
     Stock solutions of test compounds were prepared in 100% dimethyl sulfoxide. Stock solutions were prepared as 100× concentration relative to the highest concentration in the antifungal activity test, and frozen at −70° C. until used. 
     Susceptibility Testing 
     Sterile plastic micro titration plates containing flat-bottomed wells were used. The plates contained serial dilution of the antifungal agents with a volume of assay medium of 100 μL/well. Two drug-free medium wells were used as sterility and growth controls. The trays were inoculated with 100 μL/well of the final inoculum, with the exception of sterility control wells. The range of concentrations tested for each drug was 1.25-80 μM. The microtitration plates were incubated at 37° C. for 24 h. The minimal inhibitory concentrations (MICs) were determined at 24 h both visually and spectrophotometrically. 
     Visual Endpoint Determination 
     By visual endpoint determination, MICs were determined according to a S-1-R scale, with S (susceptible) indicating an optical clear culture, I (intermediate susceptibility) indicating a slightly hazy culture, and R (resistant) indicating no reduction in turbidity. The MIC 50  was defined as the lowest concentration of a drug which correspond to a S culture. 
     Spectrophotometric Endpoint Determination 
     Microtitration plates were stirred using a microtitration plate shaker before reading to ensure uniform turbidity. MICs were obtained by measuring the absorbance at 450 nm with a microtitration plate reader. The value of the blank was subtracted from reading of the rest of the wells. Two endpoints were defined for each antifungal agent tested, with MIC 80  indicating the lowest drug concentration resulting in a reduction of growth of 80% or more (determined spectrophotometrically) compared with the growth of the control, and MIC 50  indicating the lowest drug concentration resulting in a reduction in growth of 50% (determined spectrophotometrically) compared with the growth of the control. The MIC 50  was also defined as the spectrophotometric endpoint. 
     Results 
     The results of the biological tests are shown in Table 1 and Table 2. Compound 15e, bearing the most bulky side chain (a prenyl group), showed interesting activity toward  C. albicans  (20-40 μM),  C. krusei  and  C. tropicalis , while  C. parapsilosis  and  C. glabrata  were low sensitive to such a compound (40-80 μM). Reducing the size of the unsaturated chain to a butenyl group (15c), activity underwent a significant increase showing very good values toward  C. albicans  (2.5 μM against all strains) and  C. tropicalis  (1.25 μM).  C. krusei  strains were also sensitive, but at a lower concentration (10 μM). Changing the butenyl chain into a methylpropenyl moiety (15d) caused a dramatic loss of activity against all fungal strains. The best activity for this compound was found toward  C. albicans  (MIC=20 μM toward the standard and 15T strains). A further reduction of the side chain size to a propargyl moiety as in 15b restored a good activity toward  C. albicans  (with the exception of  C. albicans  4T that was resistant to such a compound) and  C. tropicalis  (5 μM). Finally, aromatisation of the side chain to a benzyl group (15a) led to activity data comparable to those found for the butenyl derivative 15c. In summary, both the butenyl and benzyl derivatives showed the best values for antifungal activity, followed by the propargyl compound that retained interesting activity toward a wide number of fungal strains. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Antifungal activity of guazatine components 
               
               
                 and linear and cyclic guanidino derivatives. 
               
             
          
           
               
                   
                 Antifungal activity, expressed as MIC 50  (μM) a   
               
             
          
           
               
                   Candida  species 
                 15a 
                 15b 
                 15c 
                 15d 
                 15e 
                 F 
               
               
                   
               
             
          
           
               
                   C. albicans  ATCC 60193 
                 2.5 
                 2.5 
                 2.5 
                 20 
                 40 
                 0.8 
               
               
                   C. albicans  4T 
                 2.5 
                 80 
                 2.5 
                 40 
                 20 
                 209 
               
               
                   C. albicans  53T 
                 2.5 
                 5 
                 2.5 
                 40 
                 20 
                 418 
               
               
                   C. albicans  15T 
                 5 
                 2.5 
                 2.5 
                 20 
                 20 
                 209 
               
               
                   C. krusei  ATCC 14243 
                 20 
                 80 
                 10 
                 40 
                 10 
                 209 
               
               
                   C. krusei  193T 
                 10 
                 40 
                 10 
                 80 
                 20 
                 418 
               
               
                 
                   C. parapsilosis 
                 
                 80 
                 40 
                 &gt;80 
                 &gt;80 
                 &gt;80 
                 6.5 
               
               
                 ATCC 34136 
                   
                   
                   
                   
                   
                   
               
               
                   C. parapsilosis  64E 
                 20 
                 40 
                 20 
                 &gt;80 
                 &gt;80 
                 32 
               
               
                   C. parapsilosis  81E 
                 20 
                 80 
                 40 
                 &gt;80 
                 40 
                 13 
               
               
                   C. glabrata  70E 
                 40 
                 80 
                 40 
                 80 
                 80 
                 209 
               
               
                   C. tropicalis  86E 
                 2.5 
                 5 
                 1.25 
                 40 
                 20 
                 52 
               
               
                   
               
               
                   a MIC values were determined at 24 h both visually and spectrophotometrically. 
               
               
                 F is fluconazole. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
             
             
               
                 Antifungal activity of guazatine components and linear and cyclic guanidino derivatives. 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
           
               
                   
                 Antifungal activity, expressed as MIC 50  (μM) a   
               
             
          
           
               
                   Candida  species 
                 12c 
                 12f 
                 13a 
                 13b 
                 13d 
                 13e 
                 13f 
               
               
                   
               
               
                   C. albicans  ATCC 60193 
                 20 
                 80 
                 2.5 
                 2.5 
                 2.5 
                 20 
                 40 
               
               
                   C. albicans  4T 
                 10 
                 80 
                 2.5 
                 80 
                 1.25 
                 40 
                 20 
               
               
                   C. albicans  53T 
                 10 
                 80 
                 2.5 
                 5 
                 2.5 
                 40 
                 20 
               
               
                   C. albicans  15T 
                 20 
                 40 
                 5 
                 2.5 
                 1.25 
                 20 
                 20 
               
               
                   C. krusei  ATCC 14243 
                 5 
                 40 
                 20 
                 80 
                 5 
                 40 
                 10 
               
               
                   C. krusei  193T 
                 10 
                 20 
                 10 
                 40 
                 5 
                 80 
                 20 
               
               
                   C. parapsilosis  ATCC 34136 
                 80  
                 &gt;80 
                 80 
                 40 
                 5 
                 &gt;80 
                 &gt;80 
               
               
                   C. parapsilosis  64E 
                 5 
                 &gt;80 
                 20 
                 40 
                 5 
                 &gt;80 
                 &gt;80 
               
               
                   C. parapsilosis  81E 
                 20 
                 40 
                 20 
                 80 
                 5 
                 &gt;80 
                 40 
               
               
                   C. glabrata  70E 
                 20 
                 80 
                 40 
                 80 
                 20 
                 80 
                 80 
               
               
                   C. tropicalis  86E 
                 5 
                 20 
                 2.5 
                 5 
                 1.25 
                 40 
                 20 
               
               
                   
               
               
                   a MIC values were determined at 24 h both visually and spectrophotometrically. 
               
             
          
         
       
     
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