Selective sulfonation of benzodiazepine derivatives

Patent metadata

Field	Value
Publication number	US-10287256-B2
Application number	US-201715820648-A
Country	US
Kind code	B2
Filing date	Nov 22, 2017
Priority date	Nov 23, 2016
Publication date	May 14, 2019
Grant date	May 14, 2019

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.

First claim

We claim:

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 D is an imine-containing benzodiazepine compound; and L is a linker.

2. The method of claim 1 , wherein D is an imine-containing indolinobenzodiazepine or an imine-containing pyrrolobenzodiazepine (PBD) compound.

3. The method of claim 1 , wherein the reaction of step (a) is carried out at a pH of 2.9 to 4.0, a pH of 2.9 to 3.7, a pH of 3.1 to 3.5, a pH of 3.2 to 3.4, or a pH of 3.3.

4. The method of claim 1 , wherein the reaction of step (a) is carried out in the presence of a buffer solution.

5. 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
wherein 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.

6. The method of claim 1 , wherein the reaction of step (a) is carried out at:
(i) a pH of 2.9 to 3.7 and 1.0 to 1.8 equivalents of the bisulfite salt or 0.5 to 0.9 equivalent of the metabisulfite salt is reacted with 1 equivalent of the imine-containing cytotoxic agent;
(ii) a pH of 3.1 to 3.5 and 1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to 0.8 equivalent of the metabisulfite salt is reacted with 1 equivalent of the imine-containing cytotoxic agent; or
(iii) a pH of 3.3 and 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.

7. The method of claim 1 , wherein the reaction of step (a) is carried out in a mixture of an organic solvent and water.

8. The method of claim 7 , wherein the reaction of step (a) is carried out in a mixture of dimethylacetamide (DMA) and water.

9. The method of claim 1 , wherein in step (a), the imine-containing cytotoxic agent is reacted with sodium bisulfite or sodium metabisulfite.

10. The method of claim 1 , wherein the modified cytotoxic agent is not purified before reacting with the cell-binding agent in step (b).

11. 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.

12. The method of claim 1 , wherein D is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein:
one of L′, L″, and L′″ is represented by the following formula:
—Z 1 —P 1 —Z 2 —R x1 —C(═O)—  (A′), or

—N(R e )—R x1 —C(═O)—  (D′);

and the other two are each independently selected from —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 , halogen, guanidinium [—NH(C═NH)NH 2 ], —OR, —NR′R″, —NO 2 , —NR′COR″, —SR, —SOR′, —SO 2 R′, —SO 3 H, —OSO 3 H, —SO 2 NR′R″, cyano, an azido, —COR′, —OCOR′, and —OCONR′R″;
one of the Z 1 and Z 2 is —C(═O)—, and the other is —NR 5 —;
P 1 is an amino acid residue or a peptide containing between 2 to 20 amino acid residues;
R x1 is an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;
R e is —H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms or —(CH 2 —CH 2 —O) n —R k , wherein R k is a —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (e.g., —NHR 101 ) or tertiary amino (—NR 101 R 102 ) group or a 5- or 6-membered nitrogen containing heterocycle, wherein R 101 and R 102 are each independently a linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms;
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, —OR, —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- to 18-membered heterocyclic ring having 1 to 6 heteroatoms independently selected from O, S, N and P;
R c is —H or an optionally substituted linear or branched alkyl having 1 to 4 carbon atoms;
n is an integer from 1 to 24;
X 1 ′ 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 1 ′ 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 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 —(CH 2 CH 2 O) n —R c , halogen, guanidinium [—NH(C═NH)NH 2 ], —OR, —NR′R″, —NO 2 , —NCO, —NR′COR″, —SR, —SOR′, —SO 2 R′, —SO 3 − H, —OSO 3 H, —SO 2 NR′R″, cyano, an azido, —COR′, —OCOR′, and —OCONR′R″;
R 6 is —H, —R, —OR, —SR, —NR′R″, —NO 2 , or halogen;
G is —CH— or —N—;
A and A′ are the same or different, and are independently selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —

Assignees

• Immunogen Inc

Inventors

• Hilderbrand Scott A
• Hutchins Benjamin M

Classifications

• A61K47/6849

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

• C07K16/2866

  against receptors for cytokines, lymphokines, interferons · CPC title

• C07D243/26 Primary

  Preparation from compounds already containing the benzodiazepine skeleton · CPC title

• C07K16/30

  from tumour cells · CPC title

• C07K2317/526

  CH3 domain · CPC title