Fitness assay and associated methods

Abstract

The present invention provides an assay for determining the biochemical fitness of a biochemical species in a mutant replicating biological entity relative to its predecessor. The present invention further provides a continuous fluorogenic assay for measuring the anti-HIV protease activity of a protease inhibitor. The present invention also provides a method of administering a therapeutic compound that reduces the chances of the emergence of drug resistance in therapy. The present invention also provides a compound of the formula: or a pharmaceutically acceptable salt, a prodrug, a composition, or an ester thereof, wherein A is a group of the formula: R 1 , R 2 , R 3 , R 5 , or R 6 is H, or an optionally substituted and/or heteroatom-bearing alkyl, alkenyl, alkynyl, or cyclic group; Y and/or Z are CH 2 , O, S, SO, SO 2 , amino, amides, carbamates, ureas, or thiocarbonyl derivatives thereof, optionally substituted with an alkyl, alkenyl, or alkynyl group; n is from 1 to 5; X is a bond, an optionally substituted methylene or ethylene, an amino, 0 or S; Q is C(O), C(S), or SO 2 ; m is from 0 to 6; R 4 is OH, ═O (keto), NH 2 , or alkylamino, including esters, amides, and salts thereof; and W is C(O), C(S), S(O), or SO 2 . Optionally, R 5 and R 6 , together with the N—W bond of formula (I), comprise a macrocyclic ring.

1 - 62 . (canceled)  
     
     
         63 . A compound represented by a formula:  
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt, prodrug, or ester thereof, wherein: 
 A is a group having a formula:  
                     
 R 1  is H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroaralkyl, wherein at least one hydrogen atom is optionally substituted with a substituent selected from the group consisting of OR 7 , SR 7 , CN, NO 2 , N 3 , and a halogen, and wherein R 7  is H, unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl;  
 Y and Z, the same or different, are independently selected from the group consisting of CH 2 , O, S, SO, SO 2 , NR 8 , R 8 C(O)N, R 8 C(S)N, R 8 OC(O)N, R 8 OC—(S)N, R 8 SC(O)N, R 8 R 9 NC(O)N, and R 8 R 9 NC(S)N, wherein R 8  and R 9  each are selected from the group consisting of H, unsubstituted alkyl, unsubstituted alkenyl, and unsubstituted alkynyl;  
 n is an integer from 1 to 5;  
 X is a covalent bond, CHR 10 , CHR 10 CH 2 , CH 2 CHR 10 , O, NR 10 , or S, wherein R 10  is H, unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl;  
 Q is C(O), C(S), or SO 2 ;  
 R 2  is H, C 1 -C 6  alkyl, C 2 -C 6  alkenyl, or C 2 -C 6  alkynyl;  
 m is an integer from 0 to 6;  
 R 3  is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl wherein at least one hydrogen atom is optionally substituted with a substituent selected from the group consisting of alkyl, (CH 2 ) p R 11 , OR 12 , SR 12 , CN, N 3 , NO 2 , NR 12 R 13 , C(O)R 12 , C(S)R 12 , CO 2 R 12 , C(O)SR 12 , C(O)NR 12 R 13 , C(S)NR 12 R 13 , NR 12 C(O)R 13 , NR 12 C—(S)R 13 , NR 12 CO 2 R 13 , NR 12 C(O)SR 13 , and halogen, and wherein p is an integer from 0 to 5;  
 R 11  is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl wherein at least one hydrogen atom is optionally substituted with a substituent selected from the group consisting of a halogen, OH, OCH 3 , NH 2 , NO 2 , SH, and CN; and  
 R 12  and R 13  are independently selected from the group consisting of H, unsubstituted alkyl, unsubstituted alkenyl, and unsubstituted alkynyl;  
 R 4  is OH, ═O (keto), NH 2 , or NHCH 3 ;  
 R 5  is H, C 1 -C 6  alkyl radical, C 2 -C 6  alkenyl radical, or (CH 2 ) q R 14 , wherein q is an integer from 0 to 5, R 14  is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl radical wherein at least one hydrogen atom is optionally substituted with a substituent selected from the group consisting of a halogen, OH, OCH 3 , NH 2 , NO 2 , SH, and CN;  
 W is C(O), C(S), or SO 2 ; and  
 R 6  is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl radical wherein at least one hydrogen atom is optionally substituted with a substituent selected from the group consisting of halogen, OR 15 , SR 15 , S(O)R 15 , SO 2 NR 15 , SO 2 N 15 R 16 , SO 2 N(OH)R 15 , CN, CR 15 ═NR 16 , CR 15 ═N(OR 16 ), N 3 , NO 2 , NR 15 R 16 , N(OH)R 15 , C(O)R 15 , C(S)R 15 , CO 2 R 15 , C(O)SR 15 , C(O)NR 15 R 16 , C(S)—NR 15 R 16 , C(O)N(OH)R 15 , C(S)N(OH)R 15 , NR 15 C(O)R 16 , NR 15 C—(S)R 16 , N(OH)C(O)R 15 , N(OH)C(S)R 15 , NR 15 CO 2 R 16 , N(OH)—CO 2 R 15 , NR 15 C(O)SR 16 , NR 15 C(O)NR 16 R 17 , NR 15 C(S)NR 16 R 17 , N(OH)C(O)NR 15 R 16 , N(OH)C(S)NR 15 R 16 , NR 15 C(O)N(OH)R 16 , NR 15 C(S)N(OH)R 16 , NR 15 SO 2 R 16 , NHSO 2 NR 15 R 16 , NR 15 SO 2 NHR 16 , P(O) (OR 15 ) (OR 16 ), alkyl, alkoxy, alkylthio, alkylamino, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, aryloxy, arylamino, arylthio, aralkyl, aryloxyalkyl, arylaminoalkyl, aralkoxy, (aryloxy)alkoxy, (arylamino)alkoxy, (arylthio)alkoxy, aralkylamino, (aryloxy)alkylamino, (arylamino)alkylamino, (arylthio)alkylamino, aralkylthio, (aryloxy)alkylthio, (arylamino)alkylthio, (arylthio)alkylthio, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylthio, heteroaralkyl, heteroaralkoxy, heteroaralkylamino, and heteroaralkylthio, and wherein R 15 , R 16 , and R 17  are H, unsubstituted alkyl, or unsubstituted alkenyl,  
 wherein, when at least one hydrogen atom of R 6  is substituted with a substituent other than halogen, OR 15 , SR 15 , CN, N 3 , NO 2 , NR 15 R 16 , C(O)R 15 , C(S)R 15 , CO 2 R 15 , C(O)SR 15 , C(O)NR 15 R 16 , C(S)NR 15 R 16 , NR 15 C(O)R 16 , NR 15 C(S)R 16 , NR 15 CO 2 R 16 , NR 15 C(O)SR 16 , NR 15 C—(O)NR 16 R 17 , or NR 15 C(S)NR ≠ R 17 , at least one hydrogen atom on said substituent is optionally substituted with halogen, OR 15 , SR 15 , CN, N 3 , NO 2 , NR 15 R 16 , C(O)R 15 , C(S)R 15 , CO 2 R 15 , C(O)SR 15 , C(O, NR 15 R 16 , C(S)NR 15 R 16 , NR 15 C(O)R 15 , NR 15 C(S)R 16 , NR 15  CO 2 R 16 , NR 15 C(O)SR 16 , NR 15 C—(O)NR 16 R 17 , or NR 15 C(S)NR 16 R 17 ; or  
 R 5  and R 6  together comprise a 12- to 18-membered ring comprising at least one additional heteroatom in the ring skeleton which includes the N—W bond of formula (I); and  
 wherein said compound inhibits a multidrug-resistant retroviral protease.  
 
     
     
         64 . The compound of  claim 63  wherein A has the formula:  
       
         
           
           
               
               
           
         
       
     
     
         65 . The compound of  claim 63  or  64  wherein: 
 when R 1  is alkyl, it is a C 1 -C 6  alkyl;    when R 1  is alkenyl, it is a C 2 -C 6  alkenyl;    when R 1  is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, R 1  is a 4- to 7-membered ring;    when R 7 , R 8 , or R 9  is unsubstituted alkyl, it is a C 1 -C 6  unsubstituted alkyl;    when R 7 , R 8 , or R 9  is unsubstituted alkenyl, it is a C 1 -C 6  unsubstituted alkenyl;    R 3  is a 4- to 7-membered ring;    R 11  is a 4- to 7-membered ring;    when R 12  or R 13  is unsubstituted alkyl, it is a C 1 -C 6  unsubstituted alkyl;    when R 12  or R 13  is unsubstituted alkenyl, it is a C 2 -C 6  unsubstituted alkenyl;    when R 14  is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, R 14  is a 4- to 7-membered ring;    when R 6  is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, R 6  is a 4- to 7-membered ring;    when R 6  is substituted with a substituent that is alkyl, alkylthio, or alkylamino, the substituent comprises from one to six carbon atoms; and    when R 6  is substituted with a substituent that is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, the substituent is a 4- to 7-membered ring;    or a pharmaceutically acceptable salt, a prodrug, or an ester thereof.    
     
     
         66 . The compound of  claim 63  or  64  wherein Q is C(O), R 2  is H, and W is SO 2 , or a pharmaceutically acceptable salt, prodrug, or ester thereof.  
     
     
         67 . The compound of  claim 64  represented by a formula:  
       
         
           
           
               
               
           
         
       
     
     
         68 . The compound of  claim 67  represented by a formula:  
       
         
           
           
               
               
           
         
       
       wherein Ar is phenyl, optionally substituted with a substituent selected from the group consisting of methyl, amino, hydroxy, methoxy, methylthio, hydroxymethyl, aminomethyl, and methoxymethyl.  
     
     
         69 . The compound of  claim 68  represented by a formula:  
       
         
           
           
               
               
           
         
       
     
     
         70 . The compound of  claim 68  or  69  wherein X is oxygen.  
     
     
         71 . The compound of  claim 68  or  69  wherein R 5  is isobutyl.  
     
     
         72 . The compound of  claim 68  or  69  wherein Ar is phenyl substituted at the para position.  
     
     
         73 . The compound of  claim 68  or  69  wherein Ar is phenyl substituted at the meta position.  
     
     
         74 . The compound of  claim 68  or  69  wherein Ar is phenyl substituted at the ortho position.  
     
     
         75 . The compound of  claim 68  or  69  wherein Ar is selected from the group consisting of para-aminophenyl, para-toluyl, para-methoxyphenyl, meta-methoxyphenyl, and meta-hydroxymethylphenyl.  
     
     
         76 . The compound of  claim 69  represented by a formula  
       
         
           
           
               
               
           
         
       
     
     
         77 . A pharmaceutical composition comprising (a) compound of  claim 63 ,  64 ,  67 ,  68 , or  69  and (b) a pharmaceutically acceptable carrier.