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O
O
HO
1) S-S
Reduction
HO
NH
O
NH
S
O
R
OCH
3
S
S
2) Intramolecular
Michael
Addition
OCH
3
O
H
H
O
O
OH
CH
3
O
NH
Bergman
Cyclization
Hydrogen
Abstraction
S
O
Double-strand
DNA breaks
t
1/2
=4.5s
O
H
Figure 5.2
Mechanism of DNA damage. The bridgehead alkene prevents Bergman
cyclization until reduction of the S-S bond at an undetermined intracel-
lular location allows the resultant thiol to eliminate it by Michael addition.
The half-life of 4.5 s allows the Michael adduct to sample the DNA and,
perhaps, transit from the cytoplasm to the nucleus.
Me
Me
H
O
Me
Me
N
Antibody
S-S-NAc-Gamma
Antibody
N
H
S-S-NAc-Gamma
O
n
n
Amide Conjugate
Carbohydrate Conjugate
Figure 5.3
Initial conjugate types. These conjugates differ primarily in the hydrolytic
release afforded by the hydrazone in the carbohydrate conjugate. Disulfide
reduction of the highly stabilized disulfide could play a role in the release
of calicheamicin, but the primary route of release of a calicheamicin
derivative from the amide conjugate is likely to be lysosomal proteolysis of
the antibody to release a zwitterionic derivative of lysine.
13
usually in the lysosomes, in a way that releases an active cytotoxic agent. Two
different types of linkers were effective with the CTM01 antibody (Figure 5.3),
one that attached to oxidized carbohydrates on the antibody through hydrazone
formation, and a second attached to lysines on the antibody.
12
These are loosely
referred to as the carbohydrate conjugates and the amide conjugates. Initial
work with the anti-CD33 antibody P67.6 showed that the N-acetylcalicheamicin
was a preferred derivative and that stabilizing the disulfide by the introduction
of two flanking methyl groups was again beneficial, but interesting differences in
activity that depended on the nature of the linker were seen.
5.4 The Original Anti-CD33 Conjugate
Oxidation of the P67.6 antibody with periodate follow by reaction with N-acetyl-
g-calicheamicin dimethylhydrazide (DMH) gave a carbohydrate conjugate that
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