Protecting Group Comprising a Purification Tag

Abstract

The present invention relates to compounds comprising a protecting group moiety-tag moiety conjugate, a method of purification and monoconjugates obtained from such method of purification.

1 . A compound having a moiety of formula (I): 
       
         
           
           
               
               
           
         
         wherein:
 the dashed line indicates attachment to the rest of the compound; 
 T is a tag moiety; and 
 PG is a protecting group moiety. 
 
       
     
     
         2 . The compound of  claim 1 ; 
       wherein PG is utilized for the reversible protection of an amine functional group and 
       wherein PG comprises a moiety of formula (Aa), (Ab), or (Ac): 
       
         
           
           
               
               
           
         
         wherein:
 the dashed line marked with the asterisk indicates attachment to T and the unmarked dashed line indicates attachment to the nitrogen of an amine functional group; 
 n is 0, 1, 2, 3, or 4; 
 —X— is a chemical bond or a spacer; 
 ═Y 1  is selected from the group consisting of ═O and ═S; 
 —Y 2 — is selected from the group consisting of —O— and —S—; 
 —Y 3 — and —Y 5 — are independently of each other selected from the group consisting of —O— and —S—; 
 —Y 4 — is selected from the group consisting of —O—, —NR 5 —, and —C(R 6 R 6a )—; 
 —R 2 , —R 3 , —R 4 , —R 5 , —R 6 , and R 6a  are independently of each other selected from the group consisting of —H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl; 
 —W— is selected from the group consisting of C 1-20 , alkyl optionally interrupted by one or more groups selected from the group consisting of —O—, —S— and —N(R 7 )—; 
 -Nu is a nucleophile selected from the group consisting of —N(R 7 R 7a ), —N(R 7 OH), —N(R 7 )—N(R 7a R 7b ), —S(R 7 ), —COOH, 
 
       
       
         
           
           
               
               
           
         
         
           -Nu′ is a nucleophile selected from the group consisting of —N(R 7 )—, —N(OH)—, —N(R 7 )—N(R 7a )—, —S—, —C(O)O—, 
         
       
       
         
           
           
               
               
           
         
         
           
             wherein:
 the unmarked dashed line indicates attachment to the rest of the moiety of formula (Ac); and 
 the dashed line marked with the asterisk indicates attachment to T; 
 
           
           —Ar— is selected from the group consisting of: 
         
       
       
         
           
           
               
               
           
         
         
           
             wherein:
 dashed lines indicate attachment to the rest of the prodrug, 
 —Z 1 — is selected from the group consisting of —O—, —S—, and —N(R 7 )—; and 
 —Z 2 — is —N(R 7 )—; and 
 
           
           —R 7 , R 7a , and —R 7b  are independently of each other selected from the group consisting of —H, C 1-6  alkyl, C 2-6  alkenyl, and C 2-6  alkynyl; 
         
         wherein the moiety of formula (Aa), (Ab), or (Ac) is optionally further substituted. 
       
     
     
         3 . The compound of  claim 1 ; 
       wherein PG is utilized for the reversible protection of an amine functional group; and 
       wherein PG of formula (I) preferably comprises a moiety of formula (C): 
       
         
           
           
               
               
           
         
         wherein:
 the dashed line marked with the asterisk indicates attachment to T of formula (I) and the unmarked dashed line indicates attachment to the nitrogen of an amine functional group; 
 X is C(R 4 R 4a ), N(R 4 ), O, C(R 4 R 4a )—C(R 5 R 5a ), C(R 5 R 5a )—C(R 4 R 4a ), C(R 4 R 4a )—N(R 6 ), N(R 6 )—C(R 4 R 4a ), C(R 4 R 4a )—O, or O—C(R 4 R 4a ); 
 X 1  is C or S(O); 
 X 2  is C(R 7 , R 7a ) or C(R 7 , R 7a )—C(R 8 , R 8a ); 
 R 1 , R 1a , R 2 , R 2a , R 3 , R 3a , R 4 , R 4a , R 5 , R 5a , R 6 , R 7 , R 7a , R 8 , and R 8a  are independently selected from the group consisting of H and C 1-6  alkyl; 
 optionally, one or more of the pair(s) R 1a /R 4 , R 1a /R 5a , R 4a /R 5a , R 4a /R 5a , and R 7a /R 8a  form a chemical bond; 
 optionally, one or more of the pair(s) R 1 /R 1a , R 2 /R 2a , R 4 /R 4a , R 5 /R 5a , R 7 /R 7a , and R 8 /R 8a  are joined together with the atom to which they are attached to form a C 3-8  cycloalkyl or a 4-membered to 7-membered heterocyclyl; 
 optionally, one or more of the pair(s) R 1 /R 4 , R 1 /R 5 , R 1 /R 6 , R 4 /R 5 , R 7 /R 8 , and R 2 /R 3  are joined together with the atoms to which they are attached to form a ring A; 
 optionally, R 3 /R 3a  are joined together with the nitrogen atom to which they are attached to form a 4-membered to 7-membered heterocycle; 
 A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10  cycloalkyl, 4-membered to 7-membered heterocyclyl, and 8-membered to 11-membered heterobicyclyl; 
 provided that one hydrogen of R 1 , R 1a , R 2 , R 2a , R 3 , R 3a , R 4 , R 4a , R 5 , R 5a , R 6 , R 7 , R 7a , R 8 , or R 8a  is replaced by -T of formula (I); and 
 
         wherein the moiety of formula (C) is optionally further substituted. 
       
     
     
         4 . The compound of  claim 1 ; 
       wherein PG is utilized for the reversible protection of a thiol functional group; and 
       PG is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         wherein:
 the dashed line marked with an asterisk indicates attachment to T and the unmarked dashed line indicates attachment to the rest of the compound; 
 Ar is an aromatic moiety which is optionally further substituted; 
 R 01 , R 03 , and R 04  are independently of each other a chemical bond or is C 1-50  alkyl, C 2-50  alkenyl, or C 2-50  alkynyl, wherein:
 C 1-50  alkyl, C 2-50  alkenyl, and C 2-50  alkynyl are optionally substituted with one or more R 3 , which are the same or different; and 
 C 1-50  alkyl, C 2-50  alkenyl, and C 2-50  alkynyl are optionally interrupted by one or more groups selected from the group consisting of -Q-, —C(O)O—, —O—, —C(O)—, —C(O)N(R 4 )—, —S(O) 2 N(R 4 )—, —S(O)N(R 4 )—, —S(O) 2 —, —S(O)—, —N(R 4 )S(O) 2 N(R 4 ) —, —S—, —N(R 4 )—, —OC(O)R 4 , —N(R 4 )C(O)—, —N(R 4 )S(O) 2 —, —N(R 4 )S(O)—, —N(R 4 )C(O)O—, —N(R 4 )C(O)N(R 4 )—, and —OC(O)N(R 4 R 4a ); 
 
 R 02  is —H, C 1-50  alkyl, C 2-50  alkenyl, or C 2-50  alkynyl, wherein:
 C 1-50  alkyl, C 2-50  alkenyl, and C 2-50  alkynyl are optionally substituted with one or more R 3 , which are the same or different; and 
 C 1-50  alkyl, C 2-50  alkenyl, and C 2-50  alkynyl are optionally interrupted by one or more groups selected from the group consisting of -Q-, —C(O)O—, —O—, —C(O)—, —C(O)N(R 4 )—, —S(O) 2 N(R 4 )—, —S(O)N(R 4 )—, —S(O)—, —S(O)—, —N(R 4 )S(O) 2 N 4a )—, —S—, —N(R 4 )—, —OC(O)R 4 , —N(R 4 )C(O)—, —N(R 4 )S(O) 2 —, —N(R 4 )S(O)—, —N(R 4 )C(O)O—, —N(R 4 )C(O)N(R 4 )—, and —OC(O)N(R 4 R 4a ); 
 
 Q is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10  cycloalkyl, 4-membered to 7-membered heterocyclyl, and 8 membered to 11-membered heterobicyclyl, wherein T is optionally substituted with one or more R 3 , which are the same or different;
 R 3  is halogen, —CN, oxo (═O), —COOR 5 , —OR 5 , —C(O)R 5 , —C(O)N(R 5 R 5a ), —S(O) 2 N(R 5 R 5a ), —S(O)N(R 5 R 5a ), —S(O) 2 R 5 , S(O)R 5 , —N(R 5 ) S(O) 2 N(R 5a R 5b ), —SR 5 , —N(R 5 R 5a ), —NO 2 , —OC(O)R 5 , —N(R 5 )C(O)R 5a , —N(R 5 )S(O) 2 R 5a , —N(R 5 )S(O)R 5a , —N(R 5 )C(O)OR 5a , —N(R 5 )C(O)N(R 5a R 5b ), —OC(O)N(R 5 R 5a ), or C 1-6  alkyl, wherein: 
 C 1-6  alkyl is optionally substituted with one or more halogen, which are the same or different; and 
 
 R 4 , R 4a , R 5 , R 5a , and R 5b  are independently selected from the group consisting of —H or C 1-6  alkyl, wherein:
 C 1-6  alkyl is optionally substituted with one or more halogen, which are the same or different. 
 
 
       
     
     
         5 . The compound of  claim 4 ; 
       wherein Ar is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein:
 dashed lines marked with an asterisk indicate attachment to T of formula (I) and the unmarked dashed lines indicate attachment to the rest of PG; 
 W is independently of each other O, S, or N; and 
 W′ is N; and 
 
         wherein Ar is optionally substituted with one or more substituent(s) independently selected from the group consisting of NO 2 , Cl, and F. 
       
     
     
         6 . The compound of  claim 4 ; 
       wherein PG is: 
       
         
           
           
               
               
           
         
         wherein:
 the dashed line marked with an asterisk indicates attachment to T; and 
 the unmarked dashed line indicates attachment to the rest of the compound. 
 
       
     
     
         7 . The compound of  claim 1 ;
 wherein T comprises at least one positive or negative charge.   
     
     
         8 . The compound of  claim 1 ;
 wherein T comprises at least one positive charge under aqueous conditions at pH 3 to 9.   
     
     
         9 . The compound of  claim 8 ;
 wherein the at least one positive charge is provided by an ammonium, phosphonium, or tertiary amine.   
     
     
         10 . The compound of  claim 1 ; 
       wherein T comprises:
 a polyamide comprising
 at least one component selected from the group consisting of tertiary amine, quaternary ammonium residue, and protonated ammonium residue; and 
 optionally further functional groups. 
 
 
     
     
         11 . The compound of  claim 1 ; 
       wherein T comprises a moiety of formula (a): 
       
         
           
           
               
               
           
         
         wherein:
 the dashed line indicates attachment to the rest of the compound; 
 R 1 , R 1a , R 1b , R 2 , R 2a , R 2b , R 3 , R 3a , R 3b , R 4 , R 4a , and R 4b  are independently of each other H or methyl; 
 
         each m is independently of each other 1, 2, 3, 4, 5, 6, 7, or 8; 
         each n is independently of each other 1, 2, 3, 4, 5, 6, 7, or 8; 
         each x is independently of each other 1, 2, 3, 4, 5, 6, 7, or 8; 
         each y is independently of each other 0, 1, 2, 3, 4, 5, 6, 7, or 8; and 
         SP is a spacer moiety. 
       
     
     
         12 . The compound of  claim 1 ; 
       wherein T comprises a moiety of formula (b): 
       
         
           
           
               
               
           
         
         wherein:
 the dashed line indicates attachment to PG of formula (I); 
 q is 0 or 1; and 
 SP is a spacer moiety. 
 
       
     
     
         13 . The compound of  claim 1 , wherein the compound comprises further comprising: 
       a moiety of formula compound comprising a moiety of formula (IIa): 
       
         
           
           
               
               
           
         
         wherein:
 the dashed line indicates attachment to the rest of the compound; 
 T and PG are as defined in  claim 1 ; 
 L 2  is a chemical bond or a spacer moiety; 
 L 1  is a reversible prodrug linker moiety. 
 
       
     
     
         14 . The compound of  claim 13 ; 
       wherein L 2  is a spacer moiety. 
     
     
         15 . The compound of  claim 13 ; 
       wherein L 2  of formula (IIa) is selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R y1 )—, —S(O) 2 N(R y1 )—, —S(O)N(R y1 )—, —S(O) 2 —, —S(O)—, —N(R y1 )S(O) 2 N(R y1a )—, —S—, —N(R y1 )—, —OC(OR y1 )(R y1a )—, —N(R y1 )C(O)N(R y1a )—, —OC(O)N(R y1 )—, C 1-50 alkyl, C 2-50  alkenyl, and C 2-50  alkynyl, wherein:
 -T-, C 1-20  alkyl, C 2-20  alkenyl, and C 2-20  alkynyl are optionally substituted with one or more R y2 , which are the same or different; and 
 C 1-20  alkyl, C 2-20  alkenyl, and C 2-20  alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R y3 )—, —S(O) 2 N(R y3 )—, —S(O)N(R y3 )—, —S(O) 2 —, —S(O)—, —N(R y3 )S(O) 2 N(R y3a )—, —S—, —N(R y3 )—, —OC(OR y3 ) (R y3a ), —N(R y3 )C(O)N(R y3a )—, and —OC(O)N(R y3a )—; 
 
       wherein:
 R y1  and R y1a  are independently of each other selected from the group consisting of —H, -T, C 1-10  alkyl, C 2-10  alkenyl, and C 2-10  alkynyl, wherein
 -T, C 1-10  alkyl, C 2-10  alkenyl, and C 2-10  alkynyl are optionally substituted with one or more R y2 , which are the same or different; and 
 C 1-10  alkyl, C 2-10  alkenyl, and C 2-10  alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R y4 )—, —S(O) 2 N(R y4 )—, —S(O)N(R y4 )—, —S(O) 2 —, —S(O)—, —N(R y4 )S(O) 2 N(R y4a )—, —S—, —N(R y4 )—, —OC(OR y4 )(R 4a )—, —N(R y4 )C(O)N(R y4a )—, and —OC(O)N(R y4 )—; 
 
 each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10  cycloalkyl, 3-membered to 10-membered heterocyclyl, 8-membered to 11-membered heterobicyclyl, 8-membered to 30-membered carbopolycyclyl, and 8-membered to 30-membered heteropolycyclyl, wherein:
 each T is independently optionally substituted with one or more R y2 , which are the same or different; 
 
 R y2  is selected from the group consisting of halogen, —CN, oxo (═O), —COOR y5 , —OR y5 , —C(O)R y5 , —C(O)N(R y5 R y5a ), —S(O) N(R y5 R y5 ), S(O)N(R y5 R y5a ), —S(O) 2 R y5 , —S(O)R y5 , —N(R y5 ) S(O) 2 N(R y5a R y5b ), —SR y5 , —N(R y5 R y5a ), —NO 2 , —OC(O)R y5 , —N(R y5 )C(O)OR y5a , —N(R y5 )S(O) 2 R y5a , —N(R y5 )S(O)R y5a , —N(R y5 )C(O)OR y5 , —N(R y5 )C(O)N(R y5a R y5b ), —OC(O)N(R y5 R y5a ), and C 1-6  alkyl, wherein:
 C 1 , alkyl is optionally substituted with one or more halogen, which are the same or different; and 
 
 each R y3 , R y3a , R y4 , R y4a , R y5 , R y5a , and R y5b  is, independently of each other, selected from the group consisting of —H, and C 1 , alkyl; wherein:
 C 1-6  alkyl is optionally substituted with one or more halogen, which are the same or different. 
 
 
     
     
         16 . The compound of  claim 13 ; 
       wherein L 1  comprises a moiety of formula (b-iii): 
       
         
           
           
               
               
           
         
         wherein:
 the dashed line with marked with the asterisk indicates attachment to L 2  of formula (IIa); 
 the unmarked dashed line indicates attachment to the rest of the compound comprising the moiety of formula (IIa); and 
 the moiety of formula (b-iii) is attached to the rest of the compound through an amine functional group provided by said rest of the compound by forming an amide linkage between L 1  and the rest of the compound comprising the moiety of formula (IIa); and 
 
         wherein:
 X of formula (b-iii) is C(R 4 R 4a ), N(R 4 ), O, C(R 4 R 4a )—C(R 5 R 5a ), C(R 5 R 5a )—C(R 4 R 4a ), C(R 4 R 4a )—N(R 6 ), N(R 6 )—C(R 4 R 4a ), C(R 4 R 4a )—O, or O—C(R 4 R 4a ); 
 X 1  of formula (b-iii) is C or S(O); 
 X 2  of formula (b-iii) is C(R 7 , R 7a ) or C(R 7 , R 7a )—C(R 8 , R 8a ); 
 X 3  of formula (b-iii) is O, S, or N—CN; 
 R 1 , R 1a , R 2 , R 2a , R 3 , R 3a , R 4 , R 4a , R 5 , R 5a , R 6 , R 7 , R 7a , R 8 , and R 8a  of formula (b-iii) are independent of each other selected from the group consisting of H and C 1-4  alkyl; 
 optionally, one or more of the pairs R 1a /R 4a , R 1a /R 5a , R 4a /R 5a , and R 7a /R 8a  of formula (b-iii) form a chemical bond; 
 optionally, one or more of the pairs R 1 /R 1a , R 2 /R 2a , R 4 /R 4a , R 5 /R 5a , R 7 /R 7a , and R 8 /R 8a  of formula (b-iii) are joined together with the atom to which they are attached to form a C 3-10  cycloalkyl or a 4-membered to 7-membered heterocyclyl; 
 optionally, one or more of the pairs R 1 /R 4 , R 1 /R 5 , R 1 /R 6 , R 4 /R 5 , R 4 /R 6 , R 7 /R 8 , and R 2 /R 3  of formula (b-iii) are joined together with the atoms to which they are attached to form a ring A; 
 optionally, R 3 /R 3a  of formula (b-iii) are joined together with the nitrogen atom to which they are attached to form a 4-membered to 7-membered heterocycle; 
 A of formula (b-iii) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10  cycloalkyl, 4-membered to 7-membered heterocyclyl, and 8-membered to 11-membered heterobicyclyl; and 
 
       
       wherein the moiety of formula (b-iii) is:
 substituted with L 2  of formula (IIa); and 
 optionally further substituted; 
 provided that:
 the hydrogen marked with the asterisk in formula (b-iii) is not replaced; and 
 R 3  and R 3a  of formula (b-iii) are, independently of each other, H or are connected to N through an SP 3 -hybridized carbon atom. 
 
 
     
     
         17 . The compound of  claim 1 ; 
       wherein the compound is of formula (IIIa):
   (T-PG-L 2 -L 1 ) x -PM  (IIIa);
 
 wherein:
 T and PG are as defined in  claim 1 ; 
 L 2  is a chemical bond or a spacer moiety; 
 L 1  reversible prodrug linker moiety. 
 x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and 
 PM is a moiety having a molecular weight of at least 1 kDa. 
 
 
     
     
         18 . The compound of  claim 17 ;
 wherein PM is a protein.   
     
     
         19 . The compound of  claim 17 ;
 wherein PM comprises and antibody or antibody fragment.   
     
     
         20 . A method of purification, comprising the steps of:
 (i) providing a mixture comprising a multitude of conjugates selected from the group consisting of formula (III) and formula (IIIa) which differ by their value for x, where the formulas (III) and (IIIa) are:
   (T-PG-X 1 -L-X 2 ) x -PM  (III); and
 
   (T-PG-L 2 -L 1 ) x -PM  (IIIa);
 
   wherein:
 each T is, independently of each other, a tag moiety; and 
 each PG is, independently of each other, a protecting group moiety; 
 X 1  and X 2  are each, independently of each other, a linkage; 
 L and L 2  are each, independently of each other, a chemical bond or a spacer moiety; 
 L 1  is a reversible prodrug linker moiety; 
 each x is, independently of each other, an integer from 1-20; and 
 each PM is, independently from each other, a moiety having a molecular weight of at least 1 kDa; 
   (ii) subjecting the mixture of step (i) to a purification method suitable for the tag moiety T; and   (iii) isolating a fraction that comprises compounds of formula (III) or (IIIa) in which at least 80% of all compounds of formula (III) or (IIIa) have the same value for x.   
     
     
         21 . The method of  claim 20 ;
 wherein T comprises a charged moiety and the purification method of step (ii) is ion exchange chromatography.   
     
     
         22 . A monoconjugate obtained from the method of  claim 20 , in which x in step (iii) is 1.