Methods of preparation of conjugates

Patent metadata

Field	Value
Publication number	US-9353127-B2
Application number	US-201213397205-A
Country	US
Kind code	B2
Filing date	Feb 15, 2012
Priority date	Feb 15, 2011
Publication date	May 31, 2016
Grant date	May 31, 2016

Abstract

The present invention is directed to methods of preparing a conjugate of a cell-binding agent and a drug (such as a cytotoxic compound). The methods comprise the use of an imine reactive compound to enable efficient conjugations of cytotoxic compounds with cell binding agents.

First claim

The invention claimed is:

1. 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 modified cytotoxic compound with a modified CBA at a pH of about 4 to about 9, wherein:
a) the modified CBA comprises a residue of a bifunctional crosslinking agent bonded to the CBA, and the residue comprises the linking group and a thiol-reactive group; and
b) the modified cytotoxic compound is produced by reacting an imine-containing compound represented by the following formula:

or a pharmaceutically acceptable salt thereof, with an imine reactive reagent, wherein:
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 —(CH 2 CH 2 O) n—R c , 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;
R c is —H or a substituted or unsubstituted linear or branched alkyl having 1 to 4 carbon atoms;
R 1 , R 2 , R 3 , R 4 , R 1 ′, R 2 ′, R 3 ′ and R 4 ′ 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 —(OCH 2 CH 2 ) n—R c , halogen, guanidinium [—NH(C═NH)NH 2 ], —OR, —NR′R″, —NO 2 , —NCO, —NR′COR″, —SR, a sulfoxide represented by —SOR′, a sulfone represented by —SO 2 R′, a sulfonate —SO 3 − M + , a sulfate —OSO 3 − M 30 , a sulfonamide represented by —SO 2 NR′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 —(CH 2 CH 2 O) n —R c , 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, —R, —NHR, —NR 2 , —COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(CH 2 CH 2 O) n —R c , 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, CH 2 , BH, SO and SO 2;
R 6 is —H, —R, —OR, —SR, —NR′R″, —NO 2 , or, halogen;
Z and Z′ are independently selected from —(CH 2 ) n′ —, —(CH 2 ) n ′ —CR 7 R 8 —(CH 2 ) na′ —, —(CH 2 ) n′ —NR 9 —(CH 2 ) na′ —, —(CH 2 ) n′ —O—(CH 2 ) na′ — and —(CH 2 ) n′ —S—(CH 2 ) na′ —;
n′ and na′ are the same or different, and are selected from 0, 1, 2 and 3;
R 7 and R 8 are the same or different, and are each independently selected from —H, —OH, —SH, —COOH, —NHR′, a polyethylene glycol unit —(OCH 2 CH 2 ) 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;
R 9 is independently selected from —H, an optionally substituted linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit —(OCH 2 CH 2 ) n —;
A and A′ are the same or different, and are independently selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR′S—, —N(R 5 )— and —CRR′N(R 5 )—,
R 5 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 (—OCH 2 CH 2 ) n —;
L is absent, or when present, comprises a thiol group, or is a polyethylene glycol unit (—OCH 2 CH 2 ) 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, provided that the modified cytotoxic compound comprises a thiol group; and
the imine reactive reagent is selected from the group consisting of sulfites (H 2 SO 3 , H 2 SO 2 or a salt of HSO 3 − , SO 3 2− or HSO 2 − formed with a cation), metabisulfite (H 2 S 2 O 5 or a salt of S 2 O 5 2− formed with a cation), mono, di, tri, and tetra-thiophosphates (PO 3 SH 3 , PO 2 S 2 H 3 , POS 3 H 3 , PS 4 H 3 or a salt of PO 3 S 3− , PO 2 S 2 3− , POS 3 3− or PS 4 3− formed with a cation), thio phosphate esters ((R i O) 2 PS(OR i ), R i SH, R i SOH, R i SO 2 H, R i SO 3 H), hydroxyl amine, hydrazine, NH 2 O—R i , R i ′NH—R i , NH 2 —R i , NH 2 —CO—NH 2 , NH 2 —C(═S)—NH 2 ′ thiosulfate (H 2 S 2 O 3 or a salt of S 2 O 3 2− formed with a cation), dithionite (H 2 S 2 O 4 or a salt of S 2 O 4 2− formed with a cation), phosphorodithioate (P(═S)(OR k )(SH)(OH) or a salt thereof formed with a cation), hydroxamic acid (R k C(═O)NHOH or a salt formed with a cation), hydrazide (R k CONHNH 2 ), formaldehyde sulfoxylate (HOCH 2 SO 2 H or a salt of HOCH 2 SO 2 − formed with a cation), glycated nucleotide, fludarabine or a mixture thereof, wherein R i and R i′ are each independently a linear or branched alkyl having 1 to 10 carbon atoms and are substituted with at least one substituent selected from —N(R j ) 2 , —CO 2 H, —SO 3 H, and —PO 3 H; R j is a linear or branched alkyl having 1 to 6 carbon atoms; R k is a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl.

2. 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 the CBA with an imine-containing cytotoxic compound, an imine reactive reagent, and a bifunctional crosslinking agent comprising the linking group to form the conjugate, wherein the imine-containing cytotoxic compound is represented by the following formula:

wherein:
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 —(CH 2 CH 2 O) n —R c , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 1

Assignees

• Fishkin Nathan
• Miller Michael
• Li Wei
• Singh Rajeeva
• Immunogen Inc

Inventors

• Fishkin Nathan
• Miller Michael
• Li Wei
• Singh Rajeeva

Classifications

• A61K47/6849

  the antibody targeting a receptor, a cell surface antigen or a cell surface determinant · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07D487/04 Primary

  Ortho-condensed systems · CPC title

• A61K2039/505

  comprising antibodies · CPC title