1
858
R. Zhu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1853–1859
has
nitrosoureas.
a
higher AGT dependency than the chloroethyl
idea of toxicity enhancement as a consequence of a dual function
1
4,16,20
agent whose primary action sensitizes cells to its secondary action.
Although AGT protects tumor cells against chemotherapeutic
agents that alkylate the O-6 position of DNA guanine, it is also a po-
tential source of vulnerability if it can be selectively depleted in can-
cers while being spared in normal tissue. Current efforts to ablate
The nature and yield of the chloroethylating and methylating
species generated from 1 and 2 depend upon the location of the
chloroethyl and methyl groups and upon the mechanism of activa-
tion of each of these analogs. In 1, the chloroethylating species is
generated by base-catalyzed activation of 90CE; whereas, in 2,
the chloroethylating species results from the fragmentation of
the N-nitrosourea like moiety (Fig. 1). In contrast, the methylating
species results from the N-alkyl-N-nitroso component of 1, and
from the sulfonylhydrazine component of 2. These features inevi-
tably result in significant differences in the cytotoxic properties
of these agents. Of the analogs examined, 1 was the most cytotoxic,
displaying substantially greater toxicity in AGT expressing and
non-expressing cells than either laromustine or 2.
6
AGT have employed global depletion agents such as O -BG, which
also deplete AGT in normal tissue, with the result being that little
net therapeutic benefit is realized in patients because normal tissue
3
1–33
is also sensitized to the alkylating agent.
These findings under-
score the importance of therapeutically generating, or at least main-
taining, any pre-existing tumor AGT deficit with respect to normal
tissue. The selective sensitization/destruction of tumors through
3
4,35
AGT ablation and AGT deficit exploitation strategies
would be
substantially enhanced by the availability of clinical agents that
selectively alkylate at the O-6 position of guanine, thereby reducing
non-specific and non-therapeutic toxicities. This preferential
destruction would be particularly efficacious if the O-6 position of
the guanine alkylating agent also exhibited tumor selectivity. Hence,
one of our long term goals is the development of highly cytotoxic
The cytotoxic effects of methylation and 2-chloroethylation at
guanine O-6 differ in important respects. O -Methylguanine is rap-
idly titrated by AGT and O -methylguanine lesions only persist and
6
6
result in cytotoxic actions if the number of methylations exceed
the number of AGT molecules.14 If unrepaired, methylation lethal-
ity appears to be due to the ‘mismatched repair machinery’, initiat-
ing apoptosis due to failed repair.2 In the absence of functional
mismatch repair the guanine O-6 methylations lead to point muta-
tions which require a substantial number to be lethal. In contrast,
the O -chloroethylguanine lesion has a limited AGT window of re-
pair because it undergoes a spontaneous rearrangement to produce
the highly lethal cross-link which then cannot be repaired by
AGT. It is therefore likely that the efficient delivery of two differ-
ent DNA guanine O-6 alkyl lesions by 1 accounts for its superior
cytotoxicity compared to laromustine in AGT deficient cell lines.
If similar secondary non-AGT dependent repair mechanism(s) are
involved in the repair of both O-6 lesion types, competition for
these repair mechanism(s) may allow more time for O-6 chloroe-
thylations to progress to highly lethal cross-links. In cell lines with
high AGT activity, which are normally very resistant to agents such
as laromustine, the profoundly greater cytotoxicity seen with 1
compared to laromustine (Fig. 3) appears to be largely a conse-
6
guanine O specific dual function agents, with short half lives such
9
as 1, that are incorporated into a targeted platform such as that em-
9
7,8
ployed by KS900 and KS119. More optimal segregation/pre-re-
lease of the AGT ablating methylating activity can be engineered
into molecules by making the primary methylation event occur with
a significantly shorter half-life than the secondary chloroethylation
event. Highly cytotoxic dual function (methylating and chloroethy-
lating) agents, or combinations of single agents that emulate these
two functions, especially if sequentially delivered, may be highly
useful candidates as targeted prodrugs. The approximate 10-fold in-
crease in overall potency is likely to be a major advantage when
using tumor activated prodrugs since the quantity of agent selec-
tively delivered to the tumor is limited.
6
3
0
Acknowledgments
This work was supported in part by US Public Health Service
Grants CA122112, and CA129186 from the National Cancer Insti-
tute, and a Grant from the National Foundation for Cancer
Research.
6
quence of AGT ablation, as most if not all of the O -methylguanine
lesions generated will rapidly eliminate protective AGT. Thus, the
addition of an efficient AGT titrating methylating component to
laromustine, while eliminating its weakly AGT attenuating carba-
moylating activity, markedly enhanced its cytotoxicity. Interest-
ingly, compound, 2, which also generated both a methylating and
chloroethylating species, while being more cytotoxic than laro-
mustine, was not comparably cytotoxic to 1, emphasizing the
importance of the relative locations of the alkylating groups. Ear-
lier work in our laboratory has clearly demonstrated that 90CE
gives a much higher yield of cross-links than the nitrosoureas
BCNU or CCNU. Therefore, we expected that the chloroethylating
species generated by 1 would give a higher yield of cross-links than
those from 2, in keeping with the relative preferences of these clas-
ses of chloroethylating agents for the guanine O-6 position. In addi-
tion, the greater absolute yields of methylating species in the case
of 1, as measured by methanol yield, should result in superior sen-
sitization by AGT ablation. AGT ablation studies support these con-
tentions since 1 showed greater depletion of AGT activity at 2 h
than 2. Furthermore, it is possible that the faster and early predom-
inant release of the methylating species in the case of 1 may more
efficiently sensitize cells to the more cytotoxic chloroethylating
species by depleting AGT without initially competing with the
chloroethyl lesions to the same extent, as would be in the case of
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