Optical molecular sensors for cytochrome P450 activity

Abstract

The invention provides a compound, useful as an optical probe or sensor of the activity of at least one cytochrome P450 enzyme, and methods of using the compound to screen candidate drugs, and candidate drugs identified by these methods. The optical probe of the invention is a compound having the generic structure Y-L-Q, wherein Y is selected from the group consisting of Q as herein defined, saturated C 1 -C 20 alkyl, unsaturated C 1 -C 20 alkenyl, unsaturated C 1 -C 20 alkynyl, substituted saturated C 1 -C 20 alkyl, substituted unsaturated C 1 -C 20 alkenyl, substituted unsaturated C 1 -C 20 alkynyl, C 1 -C 20 cycloalkyl, C 1 -C 20 cycloalkenyl, substituted saturated C 1 -C 20 cycloalkyl, substituted unsaturated C 1 -C 20 cycloalkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; L is selected from the group of (—OCR 2 H) p —, wherein for each p, all R 2 are separately selected from the group consisting of a hydrogen atom, saturated C 1 -C 20 alkyl, unsaturated C 1 -C 20 alkenyl, unsaturated C 1 -C 20 alkynyl, substituted saturated C 1 -C 20 alkyl, substituted unsaturated C 1 -C 20 alkenyl, substituted unsaturated C 1 -C 20 alkynyl, C 1 -C 20 cycloalkyl, C 1 -C 20 cycloalkenyl, substituted saturated C 1 -C 20 cycloalkyl, substituted unsaturated C 1 -C 20 cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and p is a positive integer no greater than twelve; and Q is a chemical moiety that gives rise to optical properties in its hydroxy or hydroxylate, phenol or phenoxide form that are different from the optical properties that arise from its ether form. Most preferably, p is one, R 2 is hydrogen, and Q is the ether form of a phenoxide fluorophore.

1 - 20 . (canceled) 
     
     
         21 . A process for preparing a compound useful as a sensor for cytochrome P450 activity having the structure
   R 3 -L-Q   
       said process comprising the step of reacting a compound of the formula
   H-Q 
 
       in the presence of DMF/K 2 CO 3  or diisopropylethylamine/DMF at temperatures at or slightly above the freezing point of water, with a compound of the formula
   R 3 -L-X 
 
       wherein:
 X is a suitable leaving group selected from the group consisting of a halogen atom, a tosyl group, a mesyl group, and a triflate group; 
 R 3  is selected from the group consisting of Q as herein defined, R 1  of the known fluorogenic cytochrome P450 substrates, saturated C 1 -C 20  alkyl, unsaturated C 1 -C 20  alkenyl, unsaturated C 1 -C 20  alkynyl, substituted saturated C 1 -C 20  alkyl, substituted unsaturated C 1 -C 20  alkenyl, substituted unsaturated C 1 -C 20  alkynyl, C 1 -C 20  cycloalkyl, C 1 -C 20  cycloalkenyl, substituted saturated C 1 -C 20  cycloalkyl, substituted unsaturated C 1 -C 20  cycloalkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; wherein if R 3  is selected from Q as herein defined, L is L′, wherein L′ is selected from the group consisting of —CR 4 H)(—OCR 2 H) p —, —(CR 4 H)(—O(substituted ortho-phenyl)CR 2 H) p —, —(CR 4 H)(—O(substituted meta-phenyl)CR 2 H) p —, and —CR 4 H)(—O(substituted para-phenyl)CR 2 H) p —, wherein each R 2  and each R 4  is separately selected from the group consisting of a hydrogen atom, saturated C 1 -C 20  alkyl, unsaturated C 1 -C 20  alkenyl, unsaturated C 1 -C 20  alkynyl, substituted saturated C 1 -C 20  alkyl, substituted unsaturated C 1 -C 20  alkenyl, substituted unsaturated C 1 -C 20  alkynyl, C 1 -C 20  cycloalkyl, C 1 -C 20  cycloalkenyl, substituted saturated C 1 -C 20  cycloalkyl, substituted unsaturated C 1 -C 20  cycloalkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl, and p is a positive integer no greater than twelve; 
 L has the chemical structure L′, as herein defined, or (—OCR 2 H) p —, wherein each R 2  is separately selected from the group consisting of a hydrogen atom, saturated C 1 -C 20  alkyl, unsaturated C 1 -C 20  alkenyl, unsaturated C 1 -C 20  alkynyl, substituted saturated C 1 -C 20  alkyl, substituted unsaturated C 1 -C 20  alkenyl, substituted unsaturated C 1 -C 20  alkynyl, C 1 -C 20  cycloalkyl, C 1 -C 20  cycloalkenyl, substituted saturated C 1 -C 20  cycloalkyl, substituted unsaturated C 1 -C 20  cycloalkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl, and p is a positive integer no greater than twelve; 
 Q is attached to L through an ether linkage via the oxygen atom indicated by the following arrow, and has a structure selected from the group consisting of the following structures: 
 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein: 
         m is a positive integer no greater than five; 
         R a , R b , R c , R d , R e , R f , R g , R h , R i , R j , R k , and R l , are each separately selected from the group consisting of a hydrogen atom, a halogen atom, C 1 -C 20  alkyl, substituted C 1 -C 20  alkyl, perhalogenated alkyl, cyloalkyl, substituted cycloalkyl, aryl, substituted aryl, benzyl, heteroatyl, substituted heteroaryl, cyano, nitro, azido, —SR s , —OR o , —NR n1 R n2 R n2 , —N + R n1 R n2 R n3 , —P + R n1 R n2 R n3 , —COR c , —C(═NOR o )R C , —CSR C , —OCOR C , —OCONR n1 R n2 , —OCO 2 R c , —CONR n1 R n2 , —C(═N)NR n1 R n2 , —CO 2 R o , —SO 2 NR n1 R n2 , —SO 3 R o , —SO 2 R o , —PO(OR o ) 2 , —NR n1 CSNR n2 R n3 , —NR n1 C(═N)NR n2 R n3 , —NR n1 CONR n2 R n3 , —NR n1 COR c  and —NR n1 S(═O) 2 R S ; 
         R n1 , R n2 , R n3 R o  and R s , are each separately selected from the group consisting of a hydrogen atom, C 1 -C 20 , alkyl, substituted C 1 -C 20  alkyl, cyloalkyl, substituted cycloalkyl, aryl, substituted aryl, benzyl, heteroaryl, substituted heteroaryl and may constitute parts of an aliphatic or aromatic heterocycle; 
         R c  is selected from the group consisting of a hydrogen atom, C 1 -C 20  alkyl, substituted C 1 -C 20  alkyl, perhalogenated alkyl, cyloalkyl, substituted cycloalkyl, aryl, substituted aryl, benzyl, heteroaryl, substituted heteroaryl, cyano and may constitute parts of an aliphatic or aromatic homo- or heterocycle; 
         A is selected from the group consisting of an oxygen atom, a sulfur atom, SO, SO 2 , C(CH 3 ) 2  and C(CF 3 ) 2 ; 
         E and E′ are separately selected from the group consisting of an oxygen atom, a sulfur atom and NR n1 ; 
         G is selected from the group consisting of an oxygen atom, a sulfur atom, and NR n1 R n2 , wherein if G is selected from NR n1 R n2 , G and R c , as well as G and R d , may constitute parts of a heterocycle; and 
         T is selected from the group consisting of an oxygen atom and NR n1 . 
       
     
     
         22 . The process of  claim 21 , wherein R 3  is selected from the group consisting of saturated C 1 -C 20  alkyl, unsaturated C 1 -C 20  alkenyl, unsaturated C 1 -C 20  alkynyl, substituted saturated C 1 -C 20  alkyl, substituted unsaturated C 1 -C 20  alkenyl, substituted unsaturated C 1 -C 20  alkynyl, C 1 -C 20  cycloalkyl, C 1 -C 20  cycloalkenyl, substituted saturated C 1 -C 20  cycloalkyl, substituted unsaturated C 1 -C 20  cycloalkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl. 
     
     
         23 . The process of  claim 22 , wherein R 3  is selected from the group consisting of C 1 -C 8  alkyl, C 1 -C 8  alkenyl, substituted C 2 -C 8  alkyl, substituted C 2 -C 8  alkenyl, alkoxyalkyl, aryl, substituted aryl, tertiary and quaternary aminoalkyl, and guanidiniumalkyl groups. 
     
     
         24 . The process of  claim 23 , wherein R 3  is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, octyl, and benzyl. 
     
     
         25 . The process of  claim 21 , wherein T is an oxygen atom and G is an oxygen atom. 
     
     
         26 . The process of  claim 21 , wherein m is a positive integer no greater than two. 
     
     
         27 . The process of  claim 21 , wherein m equals one. 
     
     
         28 . The process of  claim 21 , wherein Q is a phenoxide fluorophore. 
     
     
         29 . The process of  claim 28 , wherein Q is a phenoxide fluorophore selected from the group consisting of a 7-hydroxycoumarin derivative, a resorufin, and a fluorescein. 
     
     
         30 . The process of  claim 21 , wherein R 2  and R 4  are hydrogen atoms for all values of p. 
     
     
         31 . The process of  claim 21 , wherein p is equal to one.