Methods of preparation of conjugates

A method for preparing a conjugate comprising a cell-binding agent (CBA) conjugated to a cytotoxic compound with a linking group, the method comprising:

• a) reacting a modified cytotoxic compound with a bifunctional crosslinking agent comprising the linking group, a group reactive with the CBA (such as a thiol group, a maleimide group, a haloacetamide group, or an amine group), and a group reactive with the modified cytotoxic compound, to form a second modified cytotoxic compound covalently bonded to a residue of the bifunctional crosslinking agent, wherein the residue comprises the linking group and the group reactive with the CBA;
• wherein the modified cytotoxic compound is represented by one of the following formulas, or a pharmaceutically acceptable salt thereof:
• wherein:
• Y is a leaving group, and is a sulfite (HSO3, HSO2 or a salt of HSO3−, SO32− or HSO2− formed with a cation), metabisulfite (H2S2O5 or a salt of S2O52− formed with a cation), mono-, di-, tri-, and tetra-thiophosphate (PO3SH3, PO2S2H2, POS3H2, PS4H2 or a salt of PO3S3−, PO2S23−, POS33− or PS43− formed with a cation), thio phosphate ester (RiO)2PS(ORi), RiS—, RiSO, RiSO2, RiSO3, thiosulfate (HS2O3 or a salt of S2O32− formed with a cation), dithionite (HS2O4 or a salt of S2O42− formed with a cation), phosphorodithioate (P(═S)(ORk′)(S)(OH) or a salt thereof formed with a cation), hydroxamic acid (Rk′C(═O)NOH or a salt formed with a cation), formaldehyde sulfoxylate (HOCH2SO2− or a salt of HOCH2SO2− formed with a cation, such as HOCH2SO2−Na+) or a mixture thereof, wherein Ri is a linear or branched alkyl having 1 to 10 carbon atoms and is substituted with at least one substituent selected from —N(Rj)2, —CO2H, —SO3H, and —PO3H;
• Ri can be further optionally substituted with a substituent for an alkyl described herein;
• Rj is a linear or branched alkyl having 1 to 6 carbon atoms;
• Rk′ is a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl;
• X′ is selected from —H, an amine-protecting group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH2CH2O)n—Rc, an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, and an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P;
• Y′ is selected from —H, an oxo group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionally substituted 6- to 18-membered aryl, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, an optionally substituted 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms;
• Rc is —H or a substituted or unsubstituted linear or branched alkyl having 1 to 4 carbon atoms;
• R1, R2, R3, R4, R1′, R2′, R3′ and R4′ are each independently selected from the group consisting of —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(OCH2CH2)n—Rc, halogen, guanidinium [—NH(C═NH)NH2], —OR, —NR′R″, —NO2, —NCO, —NR′COR″, —SR, a sulfoxide represented by —SOR′, a sulfone represented by —SO2R′, a sulfonate —SO3−M+, a sulfate —OSO3−M+, a sulfonamide represented by —SO2NR′R″, cyano, an azido, —COR′, —OCOR′, —OCONR′R″;
• R, for each occurrence, is independently selected from the group consisting of —H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH2CH2O)n—Rc, an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P;
• R′ and R″ are each independently selected from —H, —OH, —OR, —NHR, —NR2, —COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH2CH2O)n—Rc, and an optionally substituted 3-18-membered heterocyclic ring having 1 to 6 heteroatoms independently selected from O, S, N and P;
• n is an integer from 1 to 24;
• W is selected from C═O, C═S, CH2, BH, SO and SO2;
• R6 is —H, —R, —OR, —SR, —NR′R″, —NO2, or, halogen;
• Z and Z′ are independently selected from —(CH2)n′—, —(CH2)n′—CR7R8—(CH2)na′—, —(CH2)n′—NR9—(CH2)na′—, —(CH2)na′—O—(CH2)na′— and —(CH2)n′—S—(CH2)na′—;
• n′ and na′ are the same or different, and are selected from 0, 1, 2 and 3;
• R7 and R8 are the same or different, and are each independently selected from —H, —OH, —SH, —COOH, —NHR′, a polyethylene glycol unit —(OCH2CH2)n—, an amino acid, a peptide unit bearing 2 to 6 amino acids, an optionally substituted linear, branched or cyclic alkyl having from 1 to 10 carbon atoms;
• R9 is independently selected from —H, an optionally substituted linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(OCH2CH2)n—;
• A and A′ are the same or different, and are independently selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRRiS—, —N(R5)— and —CRR′N(R5)—,
• R5 for each occurrence is independently —H or an optionally substituted linear or branched alkyl having 1 to 10 carbon atoms;
• D and D′ are the same or different, and are independently absent or selected from the group consisting of an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, an amino acid, a peptide bearing 2 to 6 amino acids, and a polyethylene glycol unit (—OCH2CH2)n—;
• L is absent, or when present, comprises the thiol group, or is a polyethylene glycol unit (—OCH2CH2)n—, a linear, branched or cyclic alkyl or alkenyl having 1 to 10 carbon atoms, a phenyl group, a 3- to 18-membered heterocyclic ring or a 5- to 18-membered heteroaryl ring having 1 to 6 heteroatoms independently selected from O, S, N and P, wherein the alkyl, alkenyl, phenyl, or heterocyclic or heteroaryl ring is optionally substituted;
• and,
• b) reacting the second modified cytotoxic compound with the CBA through the group reactive with the CBA, at a pH of about 4 to about 9, to form the conjugate.

A method for preparing a conjugate comprising a cell-binding agent (CBA) conjugated to a cytotoxic compound with a linking group, the method comprising reacting a second modified cytotoxic compound with the CBA at a pH of about 4 to about 9, wherein the second modified cytotoxic compound comprises:

• a) a residue of a bifunctional crosslinking agent bonded to the cytotoxic compound, and the residue comprises the linking group and a reactive group selected from a reactive ester and a thiol-reactive group, and,
• b) a group represented by:
• wherein:
• Y is a leaving group, and is a sulfite (HSO3, HSO2 or a salt of HSO3−, SO32− or HSO2− formed with a cation), metabisulfite (H2S2O5 or a salt of S2O52− formed with a cation), mono-, di-, tri-, and tetra-thiophosphate (PO3SH3, PO2S2H2, POS3H2, PS4H2 or a salt of PO3S3−, PO2S23−, POS33− or PS43− formed with a cation), thio phosphate ester (RiO)2PS(ORi), RiS—, RiSO, RiSO2, RiSO3, thiosulfate (HS2O3 or a salt of S2O32− formed with a cation), dithionite (HS2O4 or a salt of S2O42− formed with a cation), phosphorodithioate (P(═S)(ORk′)(S)(OH) or a salt thereof formed with a cation), hydroxamic acid (Rk′C(═O)NOH or a salt formed with a cation), formaldehyde sulfoxylate (HOCH2SO2− or a salt of HOCH2SO2− formed with a cation, such as HOCH2SO2−Na+) or a mixture thereof, wherein Ri is a linear or branched alkyl having 1 to 10 carbon atoms and is substituted with at least one substituent selected from —N(Rj)2, —CO2H, —SO3H, and —PO3H;
• Ri can be further optionally substituted with a substituent for an alkyl described herein;
• Rj is a linear or branched alkyl having 1 to 6 carbon atoms;
• Rk′ is a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl.