Selective sulfonation of benzodiazepine derivatives

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:
• —NR5—P1—C(≡O)—(CRaRb)s—C(≡O)— (B1′)
• wherein:
• Ra and Rb, for each occurrence, are each independently —H, (C1-C3)alkyl or a charged substituent or an ionizable group Q;
• R5 is —H or an optionally substituted linear or branched alkyl having 1 to 10 carbon atoms;
• P1 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;
• R1, R2, R3, R4, R1′, R2′, R3′, and R4′ are all —H;
• R6 is OMe;
• X1′ and Y1′ 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;
• R23 and R24, for each occurrence, are independently H or an optionally substituted alkyl;
• m′ is an integer between 0 and 10;
• and
• Rh′ is H or an optionally substituted alkyl.