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.

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.

The present invention provides methods of preparing compound of Formula (I), wherein the compounds are represented by the structure of Formula (I):

##STR00001##

wherein: R.sup.1 each is independently F, Cl, Br, I, OCH.sub.3, CN or NO.sub.2; R.sup.2 each is independently identical or different C.sub.1-C.sub.5 alkyl; n.sup.1 is an integer between 1 and 5; and n.sup.2 is an integer between 1 and 4. In addition, the present invention provides a compound represented by the following structures:

##STR00002##

wherein X comprises: chloride, acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydroiodide, maleate, 2-hydroxyethanesulfonate, lactate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, or undecanoate salt, or any combination thereof.

The present invention provides methods of preparing compound of Formula (I), wherein the compounds are represented by the structure of Formula (I):

##STR00001##

wherein: R.sup.1 each is independently F, Cl, Br, I, OCH.sub.3, CN or NO.sub.2; R.sup.2 each is independently identical or different C.sub.1-C.sub.5 alkyl; n.sup.1 is an integer between 1 and 5; and n.sup.2 is an integer between 1 and 4. In addition, the present invention provides a compound represented by the following structures:

##STR00002##

wherein X comprises: chloride, acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydroiodide, maleate, 2-hydroxyethanesulfonate, lactate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, or undecanoate salt, or any combination thereof.

The present invention provides methods of preparing compound of Formula (I), wherein the compounds are represented by the structure of Formula (I) wherein: R.sup.1 each is independently F, Cl, Br, I, OCH.sub.3, CN or NO.sub.2; R.sup.2 each is independently identical or different C.sub.1-C.sub.5 alkyl; n.sup.1 is an integer between 1 and 5; and n.sup.2 is an integer between 1 and 4. In addition, the present invention provides a compound represented by the following structures: Compound (1a) and Compound (2), wherein X comprises: chloride, acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydroiodide, maleate, 2-hydroxyethanesulfonate, lactate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, or undecanoate salt, or any combination thereof. ##STR00001##

The present invention provides methods of preparing compound of Formula (I), wherein the compounds are represented by the structure of Formula (I) wherein: R.sup.1 each is independently F, Cl, Br, I, OCH.sub.3, CN or NO.sub.2; R.sup.2 each is independently identical or different C.sub.1-C.sub.5 alkyl; n.sup.1 is an integer between 1 and 5; and n.sup.2 is an integer between 1 and 4. In addition, the present invention provides a compound represented by the following structures: Compound (1a) and Compound (2), wherein X comprises: chloride, acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydroiodide, maleate, 2-hydroxyethanesulfonate, lactate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, or undecanoate salt, or any combination thereof. ##STR00001##

The present invention provides salts represented by the structure of Formula (I): wherein Q is an ammonium cation; and processes comprising the use of the salts in the preparation of benzodiazepinone compounds, especially bis(fluoroalkyl) derivatives thereof.

##STR00001##

The present invention provides salts represented by the structure of Formula (I): wherein Q is an ammonium cation; and processes comprising the use of the salts in the preparation of benzodiazepinone compounds, especially bis(fluoroalkyl) derivatives thereof.

##STR00001##