Smart Pro-Drugs of Serine Protease Inhibitors

Abstract

The present invention relates to prodrugs of protease inhibitors, such as inhibitors of the proteosome, DPP IV, FAPα and the like. These“pro-inhibitors” are activated, i.e., cleaved, by an “activated protease” to release an active inhibitor moiety in proximity to a “target protease”. The identity of activating protease and target protease can be the same (such as pro-inhibitors being referred to as “Target-Activated Smart Protease Inhibitors” or “TASPI”) or different (e.g., “Target-Directed Smart Protease Inhibitors” or “TDSPI”). After activation of the pro-inhibitor, the active inhibitor moiety can self-inactivate by, e.g., intramolecular-cyclization or cis-trans isomerization.

1 . A pro-inhibitor represented by the general formula (I) or a solvate, pharmaceutically functional derivative or pharmaceutically acceptable salt thereof:
   A-G  (I)
   wherein
 A represents an “address moiety”, e.g., a peptidyl moiety which is a substrate for an activating protease; 
 A and G are covalently linked by a bond that is cleaved by the activating protease; and 
 G represents an “inhibitor moiety”, e.g., which inhibits the proteolytic activity of a target protease, 
 wherein, the inhibitor moiety G, when cleaved from A by the activating serine protease, undergoes reversible conformation-dependent inactivation and/or inhibits the target protease with a Ki of 100 nM or less. 
   
     
     
         2 . The pro-inhibitor of  claim 1 , wherein A represents a C-terminally linked peptide or peptide analog which is a substrate for the activating enzyme. 
     
     
         3 . The pro-inhibitor of  claim 2 , wherein A is a dipeptidyl or tripepidyl moiety. 
     
     
         4 . The pro-inhibitor of  claim 1 , wherein at least one residue of A is a non-naturally occurring amino acid analog. 
     
     
         5 . The pro-inhibitor of  claim 1 , wherein A is a substrate of DPP IV. 
     
     
         6 . The pro-inhibitor of  claim 1 , wherein G is a dipeptidyl moiety having an electrophilic functional group that can form a covalent adduct with a residue in the active site of a protease replacing the carboxyl terminus of the dipeptidyl moiety. 
     
     
         7 . The pro-inhibitor of  claim 6 , wherein G is represented in the general formula (II):
   Xaa 1 -Xaa 2 -W  (II)
   wherein
 Xaa1 and Xaa2 each independently represent an amino acid residue; 
 W represents —CN, —CH═NR 5 , 
   
       
         
           
           
               
               
           
         
         
           R 5  represents H, an alkyl, an alkenyl, an alkynyl, —C(X 1 )(X 2 )X 3 , —(CH 2 )m-R 6 , —(CH 2 )n-OH, —(CH 2 )n-O-alkyl, —(CH 2 )n-O-alkenyl, —(CH 2 )n-O-alkynyl, —(CH 2 )n-O—(CH 2 )m-R 6 , —(CH 2 )n-SH, —(CH 2 )n-S-alkyl, —(CH 2 )n-S-alkenyl, —(CH 2 )n-S-alkynyl, —(CH 2 )n-S—(CH 2 )m-R 6 , —C(O)C(O)NH 2 , —C(O)C(O)OR 7 ; 
           R 6  represents, independently for each occurrence, a substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; 
           R 7  represents, independently for each occurrence, hydrogen, or a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; Y 1  and Y 2  can independently or together be OH, or a group capable of being hydrolyzed to a hydroxyl group, including cyclic derivatives where Y 1  and Y 2  are connected via a ring having from 5 to 8 atoms in the ring structure (such as pinacol or the like); 
           R 50  represents O or S; 
           R 51  represents N 3 , SH 2 , NH 2 , NO 2  or —OR 7 ; 
           R 52  represents hydrogen, a lower alkyl, an amine, —OR 7 , or a pharmaceutically acceptable salt, or R 51  and R 52  taken together with the phosphorous atom to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; 
           X 1  represents a halogen; 
           X 2  and X 3  each represent a hydrogen or a halogen; 
           m is zero or an integer in the range of 1 to 8; and n is an integer in the range of 1 to 8. 
         
       
     
     
         8 . The pro-inhibitor of  claim 1 , wherein the target protease is a serine protease. 
     
     
         9 . The pro-inhibitor of  claim 1 , wherein the activating protease is a serine protease. 
     
     
         10 . The pro-inhibitor of  claim 1 , wherein the activating protease is a metalloprotease. 
     
     
         11 . The pro-inhibitor of  claim 1 , wherein the activating protease is a cysteine protease. 
     
     
         12 . The pro-inhibitor of  claim 1 , wherein G is an inhibitor of a target protease which, when cleaved from the pro-inhibitor by the activating protease, inhibits the target protease with a K i  of 100 nM (10 −7 M) or less. 
     
     
         13 . The pro-inhibitor of  claim 1 , wherein the K i  for the inactive conformer is at least 5 times greater than the K i  for the active conformer of the inhibitor moiety G. 
     
     
         14 . The pro-inhibitor of  claim 1 , wherein the therapeutic index for the pro-inhibitor is at least 2 times greater than the therapeutic index for the inhibitor moiety G administered alone. 
     
     
         15 . The pro-inhibitor of  claim 1 , provided in a pharmaceutical preparation that is substantially pyrogen-free. 
     
     
         16 . The pro-inhibitor of  claim 15 , wherein a single administration of the pharmaceutical preparation can produce a therapeutically effective amount of the inhibitor moiety G for a period of at least 4 hours. 
     
     
         17 . The pro-inhibitor of  claim 1 , wherein G undergoes reversible pH-dependent inactivation. 
     
     
         18 . The pro-inhibitor of  claim 2 , wherein the amino terminus of A is blocked with an amino-terminal protecting group. 
     
     
         19 . The pro-inhibitor of  claim 1 , wherein the target protease is a post-prolyl cleaving protease. 
     
     
         20 . The pro-inhibitor of  claim 19 , wherein the target protease is selected from the group consisting of DPP IV, DPP II, Prolyl oligopeptidase (PO), Fibroblast Activating Protein (FAP), and prolyl carboxypeptidase. 
     
     
         21 . The protease inhibitor of  claim 1 , represented in the general formula: 
       
         
           
           
               
               
           
         
         wherein 
         A represents a 4-8 membered heterocycle including the N and the Cα carbon; 
         W represents a functional group which reacts with an active site residue of the targeted protease to form a covalent adduct, as for example, —CN, —CH═NR 5 , 
       
       
         
           
           
               
               
           
         
         
           R 1  represents a C-terminally linked peptide or peptide analog which is a substrate for an activating enzyme; 
           R 2  is absent or represents one or more substitutions to the ring A, each of which can independently be a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, —(CH 2 ) m —R 6 , —(CH 2 ) m —OH, —(CH 2 ) m —O-lower alkyl, —(CH 2 ) m —O-lower alkenyl, —(CH 2 ) n —O—(CH 2 ) m —R 6 , —(CH 2 ) m —SH, —(CH 2 ) m —S-lower alkyl, —(CH 2 ) m —S-lower alkenyl, —(CH 2 ) n —S—(CH 2 ) m —R 6 ; 
           R 3  represents, independently for each occurrence, a hydrogen or a substituent which does not conjugate the electron pair of the nitrogen from which it pends, such as a lower alkyl; 
           R 4  represents hydrogen or a small hydrophobic group such as a halogen, a lower alkyl, a lower alkenyl, or a lower alkynyl; 
           R 5  represents H, an alkyl, an alkenyl, an alkynyl, —C(X 1 )(X 2 )X 3 , —(CH 2 )m-R 6 , —(CH 2 )n-OH, —(CH 2 )n-O-alkyl, —(CH 2 )n-O-alkenyl, —(CH 2 )n-O-alkynyl, —(CH 2 )n-O—(CH 2 )m-R 6 , —(CH 2 )n-SH, —(CH 2 )n-S-alkyl, —(CH 2 )n-S-alkenyl, —(CH 2 )n-S-alkynyl, —(CH 2 )n-S—(CH 2 )m-R 6 , —C(O)C(O)NH 2 , —C(O)C(O)OR 7 ; 
           R 6  represents, for each occurrence, a substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; 
           R 7  represents, for each occurrence, hydrogen, or a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; and 
           Y 1  and Y 2  can independently or together be OH, or a group capable of being hydrolyzed to a hydroxyl group, including cyclic derivatives where Y 1  and Y 2  are connected via a ring having from 5 to 8 atoms in the ring structure (such as pinacol or the like), 
           R 50  represents O or S; 
           R 51  represents N 3 , SH 2 , NH 2 , NO 2  or —OR 7 ; 
           R 52  represents hydrogen, a lower alkyl, an amine, —OR 7 , or a pharmaceutically acceptable salt, or R 51  and R 52  taken together with the phosphorous atom to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure 
           X 1  represents a halogen; 
           X 2  and X 3  each represent a hydrogen or a halogen; 
           m is zero or an integer in the range of 1 to 8; and n is an integer in the range of 1 to 8. 
         
       
     
     
         22 . The pro-inhibitor of  claim 21 , wherein R 2  is absent, or represents a small hydrophobic group. 
     
     
         23 . The pro-inhibitor of  claim 7 , wherein W represents: 
       
         
           
           
               
               
           
         
       
     
     
         24 . The pro-inhibitor of  claim 23 , wherein R 5  is a hydrogen or —C(X 1 )(X 2 )X 3 , wherein X 1  is a fluorine, and X 2  and X 3 , if halogens, are also fluorine. 
     
     
         25 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pro-inhibitor of  claim 1 , or a pharmaceutically acceptable salt thereof. 
     
     
         26 . A method for treating a patient suffering from a disorder for which inhibition of an active protease provides therapeutic benefit, comprising the administration of a therapeutically effective amount of a pro-inhibitor of  claim 1  having an inhibitor moiety G that inhibits the active protease. 
     
     
         27 . (canceled) 
     
     
         28 . A packaged pharmaceutical comprising one or more pro-inhibitors of  claim 1  formulated in a pharmaceutically acceptable excipient, in association with instructions (written and/or pictorial) describing the recommended dosage and/or administration of the formulation to a patient.