Selective sulfonation of benzodiazepine derivatives

Abstract

The invention relates to novel methods of preparing cell-binding agent-cytotoxic agent conjugates, wherein the cytotoxic agent is an imine-containing cytotoxic agent bearing a maleimide group. In some embodiments, the cell-binding agent (CBA) is covalently linked to the cytotoxic agent through an engineered Cys, such as an engineered Cys in the heavy chain CH3 domain, at a position corresponds to the EU/OU numbering position 442 (or C442) on an antibody CBA. The invention also provides conjugates prepared by the methods of the present invention, compositions and methods useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal using the conjugates of the invention.

1 . A method of preparing a cell-binding agent-cytotoxic agent conjugate comprising the steps of:
 (a) reacting an imine-moiety in an imine-containing cytotoxic agent represented by the following formula:   
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt thereof, with sulfur dioxide, a bisulfite salt or a metabisulfite salt in an aqueous solution at a pH of 1.9 to 5.0 to form a modified cytotoxic agent comprising a modified imine moiety represented by the following formula: 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt thereof; and
 (b) reacting the modified cytotoxic agent with a cell-binding agent to form the cell-binding agent-cytotoxic agent conjugate, 
 wherein: 
 
       
         
           
           
               
               
           
         
         
           wherein: 
           L″ and L′″ are both —H; 
           L′ is represented by the following formula:
   —NR 5 —P 1 —C(≡O)—(CR a R b ) s —C(≡O)—  (B1′)
 
 wherein: 
 R a  and R b , for each occurrence, are each independently —H, (C 1 -C 3 )alkyl or a charged substituent or an ionizable group Q; 
 R 5  is —H or an optionally substituted linear or branched alkyl having 1 to 10 carbon atoms; 
 P 1  is an amino acid residue or a peptide containing between 2 to 20 amino acid residues; and 
 s is an integer from 1 to 6; 
 
           R 1 , R 2 , R 3 , R 4 , R 1 ′, R 2 ′, R 3 ′, and R 4 ′ are all —H; 
           R 6  is OMe; 
           X 1′  and Y 1′  are both —H; and 
           A and A′ are —O—; and 
         
         L is represented by the following structural formula: 
       
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein:
 s1 is the site covalently linked to D; 
 s2 is the site covalently linked to the maleimide group; 
 R 23  and R 24 , for each occurrence, are independently H or an optionally substituted alkyl; 
 m′ is an integer between 0 and 10; and 
 
         R h′  is H or an optionally substituted alkyl. 
       
     
     
         2 - 12 . (canceled) 
     
     
         13 . The method of  claim 1 , wherein:
 0.5 to 5 equivalents of the bisulfite salt or 0.25 to 2.5 equivalents of the metabisulfite salt is reacted with 1 equivalent of the imine-containing cytotoxic agent;   0.8 to 2.0 equivalents of the bisulfite salt or 0.4 to 1.0 equivalents of the metabisulfite salt is reacted with 1 equivalent of the imine-containing cytotoxic agent;   1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to 0.8 equivalents of the metabisulfite salt is reacted with 1 equivalent of the imine-containing cytotoxic agent; or   1.4 equivalents of the bisulfite salt or 0.7 equivalent of the metabisulfite salt is reacted with 1 equivalent of the imine-containing cytotoxic agent.   
     
     
         14 - 19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the reaction of step (a) is carried out in a mixture of an organic solvent and water. 
     
     
         21 . The method of  claim 20 , wherein the reaction of step (a) is carried out in a mixture of dimethylacetamide (DMA) and water. 
     
     
         22 . The method of  claim 20 , wherein the reaction of step (a) is carried out in a mixture of DMA and water, wherein the volume ratio of DMA and water is 1:1. 
     
     
         23 - 25 . (canceled) 
     
     
         26 . The method of  claim 1 , wherein in step (a), the imine-containing cytotoxic agent is reacted with sodium bisulfite. 
     
     
         27 . The method of  claim 1 , wherein in step (a), the imine-containing cytotoxic agent is reacted with sodium metabisulfite. 
     
     
         28 . The method of  claim 1 , wherein the modified cytotoxic agent is not purified before reacting with the cell-binding agent in step (b). 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 1 , wherein the reaction of step (b) is carried out at a pH of 4 to 9, a pH of 5 to 8.5, or a pH of 5.5 to 6.5. 
     
     
         31 - 47 . (canceled) 
     
     
         48 . The method of  claim 1 , wherein R a  and R b  are both H and R 5  is H or Me. 
     
     
         49 . (canceled) 
     
     
         50 . The method of  claim 48 , wherein the charged substituent or an ionizable group Q is i) —SO 3 H, —Z′—SO 3 H, —OPO 3 H 2 , —Z′—OPO 3 H 2 , —PO 3 H 2 , —Z′—PO 3 H 2 , —CO 2 H, —Z′—CO 2 H, —NR 11 R 12 , or —Z′—NR 11 R 12 , or a pharmaceutically acceptable salt thereof, or, ii) —N + R 14 R 15 R 16 X −  or —Z′—N + R 14 R 15 R 16 X − ; Z′ is an optionally substituted alkylene, an optionally substituted cycloalkylene or an optionally substituted phenylene; R 11  and R 12 , for each occurrence, are independently H or an optionally substituted alkyl; R 14  to R 16  are each independently an optionally substituted alkyl; and X −  is a pharmaceutically acceptable anion. 
     
     
         51 . The method of  claim 50 , wherein Q is —SO 3 H or a pharmaceutically acceptable salt thereof. 
     
     
         52 . The method of  claim 1 , wherein P 1  is a peptide containing 2 to 10 amino acid residues. 
     
     
         53 . The method of  claim 52 , wherein P 1  is a peptide containing 2 to 5 amino acid residues. 
     
     
         54 . The method of  claim 53 , wherein P 1  is Gly-Gly-Gly, Ala-Val, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -tosyl-Arg, Phe-N 9 -nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe and Gln-Ala. 
     
     
         55 . The method of  claim 54 , wherein P 1  is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala. 
     
     
         56 - 66 . (canceled) 
     
     
         67 . The method of  claim 1 , wherein D is represented by the following structural formula: 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt thereof. 
     
     
         68 . (canceled) 
     
     
         69 . The method of  claim 1 , wherein R 23  and R 24  are both H; and m′ is an integer between 1 and 6. 
     
     
         70 . The method of  claim 69 , wherein R h′  is H. 
     
     
         71 . The method of  claim 1 , wherein L is represented by the following structural formula: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof. 
       
     
     
         72 - 167 . (canceled)