Antiprotozoal saponins

Abstract

Saponins of formula a stereoisomeric form thereof or a pharmaceutically acceptable addition salt thereof, wherein R 1 to R 12 have the meaning given in the description, can be isolated from plants of the family Myrsinaceae and used to decrease the infectiousness of and reduce the mortality associated with protozoan parasites of the genus Leishmania which are responsible for a group of conditions known as leishmaniases.

1 . A process for the isolation of triterpene saponins from plants belonging to the family Myrsinaceae, characterized in that said process comprises the steps of 
 (a) exracting the dried plant parts with an alcohol and concentrating the extract,    (b) removing the apolar fraction from the extract by liquid-liquid extraction with an apolar solvent, and    (c) further purifying the saponins in the alcohol extract by liquid-liquid extraction, filtration and chromatography.    
     
     
         2 . A process according to  claim 1  wherein the alcohol is methanol, ethanol, isopropanol, butanol, each optionally admixed with water.  
     
     
         3 . A process according to  claim 1  wherein the saponins of the alcohol extract are further purified by 
 (c6) extracting the aqueous fraction with butanol satured with water,  
 (c7) evaporating the organic layer to dryness,  
 (c8) washing the residue in a ketone, and  
 (c9) filtering off the crude saponin mixture.  
 
     
     
         4 . A process according to  claim 1  wherein the saponins are isolated from the plant species  Maesa balansae , and the chromatography comprises straight phase chromatography liquid chromatography on silicagel or reversed-phase liquid chromatography with gradient eluent system using 
 A: 0.5% ammonium acetate in water  
 B: methanol  
 C: acetonitrile  
 wherein at t=0, (A:B:C)=(60:20:20) and t=end, (A:B:C)=(0:50:50).  
 
     
     
         5 . A triterpene saponin obtainable by a process according to anyone of  claims 1  to  4 .  
     
     
         6 . A triterpene saponin according to  claim 5  wherein said saponin is isolated from the plant species  Maesa balansae , and the chromatography comprises reversed-phase liquid chromatography with gradient eluent system using 
 A: 0.5% ammonium acetate in water  
 B: methanol  
 C: acetonitrile  
 wherein at t=0, (A:B:C)=(60:20:20) and t=end, (A:B:C)=(0:50:50), and wherein said saponin has the following characteristics:  
 Compound 1: MW=1532, λ max =228.6 nm, λ max2 =273.3 nm;  
 Compound 2: MW=1510, λ max =223.9 nm, λ max2 =274.5 nm;  
 Compound 3: MW=1532, λ max =279.2 nm, λ max2 =223.9 nm;  
 Compound 4: MW=1510, λ max =280.4 nm, λ max2 =222.7 nm;  
 Compound 5: MW=1574, λ max =276.8 nm, λ max2 =225.0 nm; or  
 Compound 6: MW=1552, λ max =279.2 nm, λ max2 =223.9 nm.  
 
     
     
         7 . A triterpene saponin having the formula  
       
         
           
           
               
               
           
         
       
       wherein R 2  is —(C═O)C 6 H 5  or —O(C═O)C(CH 3 )═CHCH 3 , 
 R 3  is (E) or (Z) —O(C═O)CH═CH—C 6 H 5 , and  
 R 4  is hydrogen or —C═O)CH 3 .  
 
     
     
         8 . A compound according to  claim 7  wherein 
 in compound 1, 
 R 2  is —O(C═O)C 6 H 5 ,  
 R 3  is (Z) —O(C═O)CH═CH—C 6 H 5 ,  
 R 4  is hydrogen;  
 
 in compound 2, 
 R 2  is —O(C═O)C(CH 3 )═CHCH 3 ,  
 R 3  is (Z) —O(C═O)CH═CH—C 6 H 5 ,  
 R 4  is hydrogen;  
 
 in compound 3, 
 R 2  is —O(C═O)C 6 H 5 ,  
 R 3  is (E) —O(C—O)CH═CH—C 6 H 5 ,  
 R 4  is hydrogen;  
 
 in compound 4, 
 R 2  is —O(C═O)C(CH 3 )═CHCH 3 ,  
 R 3  is (E) —O(C═O)CH═CH—C 6 H 5 ,  
 R 4  is hydrogen;  
 
 in compound 5, 
 R 2  is —O(C═O)C 6 H 5 ,  
 R 3  is (E) —O(C═O)CH═CH—C 6 H 5 ,  
 R 4  is —(C═O)CH 3 ;  
 
 in compound 6, 
 R 2  is —O(C═O)C(CH 3 )═CHCH 3 ,  
 R 3  is (E) —O(C═O)CH═CH—C 6 H 5 ,  
 R 4  is —(C═O)CH 3 .  
 
 
     
     
         9 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and as an active ingredient a triterpene saponin as defined in  claim 5 ,  6 ,  7  or  8 .  
     
     
         10 . A composition according to  claim 7  adapted for parenteral administration.  
     
     
         11 . Use of one or more triterpene saponins for the preparation of a pharmaceutical composition for treating leishmaniases in hosts infected by Leishmania species, characterized in that the saponin has the formula  
       
         
           
           
               
               
           
         
       
       a stereoisomeric form thereof or a pharmaceutically acceptable addition salt thereof, wherein 
 R 1  is hydrogen, —(C═O)C 1-5 alkyl, —(C═O)C 2-5 alkenyl, —(C═O)C 2-5 alkenyl substituted with phenyl, a monosaccharide group or an oligosaccharide group;  
 R 2  is hydrogen, hydroxy, —O(C═O)C 1-5 alkyl, —O(C═O)C 2-5 alkenyl, —O(C═O)C 6 H 5 , or —O(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 3  is hydrogen, hydroxy, —O(C═O)C 1-5 alkyl, —O(C═O)C 2-5 alkenyl, —O(C═O)C 6 H 5 , or —O(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 4  is hydrogen, C 1-6 alkyl, —(C═O)C 1-5 alkyl, —(C═O)C 2-5 alkenyl, —(C═O)C 6 H 5 , or —(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 5  is CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)CH 3 , CHO, COOH; or  
 R 5  and R 2  form a divalent radical of formula —C(═O)—O—;  
 R 6  and R 7  are hydrogen; or taken together they form a bond; or  
 R 5  and R 6  form a divalent radical of formula  
 —CH 2 —O—  (a), —CH(OR 13 )—O—  (b), —C(═O)—O—  (c),  wherein R 13  is hydrogen, C 1-6 alkyl or —(C═O)C 1-5 alkyl;    
 R 8α  and R 8β  each independently represent CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)C 1-5 alkyl, CHO, CH(OCH 3 ) 2 , CH═NOH, COOH; 
 R 8β  and R 3  form a divalent radical of formula —C(═O)—O—;  
 
 R 8β  and R 5  form a divalent radical of formula —CH 2 O—CHOH—;  
 R 9  is CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)C 1-5 alkyl, CHO, COOH;  
 R 10  is CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)C 1-5 alkyl, CHO, COOH;  
 R 11 , is hydrogen, hydroxy or O—C(═O)C 1-5 alkyl; or R 10  and R 11 , form a divalent radical of formula —CH 2 O—; and  
 R 12  is CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)CH 3 , CHO, CH═NOH, or COOH.  
 
     
     
         12 . Use according to  claim 11  wherein 
 R 1  is hydrogen, —(C═O)C 1-5 alkyl, or an oligosaccharide group;  
 R 3  is hydrogen, hydroxy, —O(C═O)C 1-5 alkyl, —O(C═O)C 2-5 alklenyl, —O(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 4  is hydrogen, C 1-6 alkyl, —(C═O)C 1-5 alkyl, —(C═O)C 2-5 alkenyl;  
 R 5  is CH 2 OH, CH 2 O—C(═O)CH 3 , CHO; and  
 R 6  and R 7  taken together form a bond; or  
 R 5  and R 6  form a divalent radical of formula  
 —CH 2 —O—  (a), —CH(OR 13 )—O—  (b), —C(═O)—O—  (c),  wherein R) 13  is hydrogen, C 1-6 alkyl or —(C═O)C 1-5 alkyl; and    
 R 7  is hydrogen;  
 R 8β  represents CH 3 , CH 2 OH, CHO, CH(OCH 3 ) 2 , CH═NOH, COOH;  
 R 8α  represents CH 3 ;  
 R 8β  and R 3  form a divalent radical of formula —C(═O)—O—; or  
 R 8β  and R 5  form a divalent radical of formula —CH 2 O—CHOH—;  
 R 10  is CH 3 , CH 2 OH;  
 R 11  is hydrogen, hydroxy or O—C(═O)C 1-5 alkyl; or  
 R 10  and R 11  form a divalent radical of formula —CH 2 O—; and  
 R 12  is CH 3 , CH 2 OH, CH 2 O—C(═O)CH 3 , CHO, or CH═NOH.  
 
     
     
         13 . Use according to  claim 12  wherein 
 R 1  is hydrogen or an oligosaccharide group;  
 R 2  is hydrogen, hydroxy, —O(C═O)C 1-5 alkyl, —O(C═O)C 2-5 alkenyl, —O(C═O)C 6 H 5 , or —O(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 3  is hydrogen, hydroxy, —O(C═O)C 1-5 alkyl, —O(C═O)C 2-5 alkenyl, —O(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 4  is hydrogen, C 1-6 alkyl, —(C═O)C 1-5 alkyl, —(C═O)C 2-5 alkenyl, —(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 5  is CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)CH 3 , CHO, COOH; and  
 R 6  and R 7  taken together form a bond; or  
 R 5  and R 6  form a divalent radical of formula  
 —CH 2 —O—  (a), —CH(OR 13 )—O—  (b), —C(═O)—O—  (c),  wherein R 13  is hydrogen; and    
 R 7  is hydrogen;  
 R 8α  and R 8β  both represent CH 3 ;  
 R 9  is CH 3 ;  
 R 10  is CH 3 ;  
 R 11  is hydrogen; and  
 R 12  is CH 3 .  
 
     
     
         14 . A method of alleviating clinical manifestations of, and curing disorders known as leishmaniases attributable to infection by protozoan parasites of the genus Leishmania in both men and animals, comprising administering to an infected host a therapeutically effective amount of a compound of formula:  
       
         
           
           
               
               
           
         
       
       a stereoisomeric form thereof or a pharmaceutically acceptable addition salt thereof, wherein 
 R 1  is hydrogen, —(C═O)C 1-5 alkyl, —(C═O)C 2-5 alkenyl, —(C═O)C 2-5 alkenyl substituted with phenyl, a monosaccharide group or an oligosaccharide group;  
 R 2  is hydrogen, hydroxy, —O(C═O)C 1-5 alkyl, —O(C═O)C 2-5 alkenyl, —O(C═O)C 6 H 5 , or —O(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 3  is hydrogen, hydroxy, —O(C═O)C 1-5 alkyl, —O(C═O)C 2-5 alkenyl, —O(C═O)C 6 H 5 , or —O(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 4  is hydrogen, C 1-6 alkyl, —(C═O)C 1-5 alkyl, —(C═O)C 2-5 alkenyl, —(C═O)C 6 H 5 , or —(C═O)C 2-5 alkenyl substituted with phenyl;  
 R 5  is CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)CH 3 , CHO, COOH; or  
 R 5  and R 2  form a divalent radical of formula —C(═O)—O—;  
 R 6  and R 7  are hydrogen; or taken together they form a bond; or  
 R 5  and R 6  form a divalent radical of formula  
 —CH 2 —O—  (a), —CH(OR 13 )—O—  (b), —C(═O)—O—  (c),  wherein R 13  is hydrogen, C 1-6 alkyl or —(C═O)C 1-5 alkyl;    
 R 8α  and R 8β  each independently represent CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)C 1-5 alkyl, CHO, CH(OCH 3 ) 2 , CH═NOH, COOH; 
 R 8β  and R 3  form a divalent radical of formula —C(═O)—O—;  
 
 R 8β  and R 5  form a divalent radical of formula —CH 2 O—CHOH—;  
 R 9  is CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)C 1-5 alkyl, CHO, COOH;  
 R 10  is CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)C 1-5 alkyl, CHO, COOH;  
 R 11  is hydrogen, hydroxy or O—C(═O)C 1-5 alkyl; or R 10  and R 11  form a divalent radical of formula —CH 2 O—; and  
 R 12  is CH 3 , CH 2 OH, CH 2 OCH 3 , CH 2 O—C(═O)CH 3 , CHO, CH═NOH, or COOH.