J. B. Smaill et al. / Bioorg. Med. Chem. Lett. 18 (2008) 929–933
933
Table 5. Modulation of Cdc2 Y15 phosphorylation by compound 14
in HT-29 tumours in nude mice
pTyr15 antibody. The results are given in Table 5, and
show that the DNA damage caused by the topo I poison
CPT-11 results, as expected, in a substantial increase in
phosphorylation on Cdc2 Y15. This can be lowered by
14, only marginally at 20 mg/kg but substantially (50%
reduction) at 40 mg/kg, although the latter dose gener-
ated significant oedema at the injection site, and is likely
too high for multi-dose regimens.
Doseb (mg/kg)
Cdc2 PY15a
Control CPT-11 only 14 only CPT-11 + 14
40
20
10
100
100
100
250
298
266
57
113
73
124
258
263
a Intensity of PY15 signal in Western blots in relation to normalised
controls set at 100%.
b Drug given in two doses 3 h apart.
In summary, the data show that a range of solubilising
bases can be tolerated at the 8-position of the carbazole
chromophore, providing potent Wee1/Chk1 inhibitors
of sufficient solubility to evaluate in cellular and
in vivo assays. These compounds were able to inhibit
phosphorylation of the target Cdc2 kinase on tyrosine-
15, and to substantially potentiate the cytotoxicity of
cisplatin in p53-negative cells in culture through abroga-
tion of the G2/M checkpoint.
There was little differentiation in these assays between
the compounds, including the (CH2)4- and O(CH2)3-
linked ones, and compound 2. Compound 14 was con-
sidered of particular interest, showing the greatest po-
tency in both the PY15 and MI assays (Table 2) and
possessing acceptable aqueous solubility as the HCl salt
in water (2.2 mg/mL). This compares3 with a solubility
of <3 lg/mL for compound 2.
Acknowledgments
Compounds 14 and 15 were also evaluated for their ability
to sensitise tumour cells to the cytotoxic effects of cis-
platin, which was used as a typical DNA-damaging agent
(Table 3). In HT-29 human colon cancer cells, concentra-
tions of those well below their IC50s for growth inhibition
resulted in significant (ꢀ4- to 7-fold) increases in the tox-
icity of cisplatin. The table reports a single experiment
that was typical of a number using different cell lines
and with different DNA-damaging agents.
We thank the Cancer Society Auckland and the Maurice
Wilkins Centre for Molecular Biodiscovery for contin-
ued support.
References and notes
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Selected compounds were screened against a panel of 28
kinases at a concentration of 10 lM,7 as shown in Table
4. As a class, the C-8 solubilised 9-hydroxypyrrolo[3,4-
c]carbazole-1,3(2H,6H)-diones appear also to be inhibi-
tors of MKK1, AMPK, Chk1 (as described above),
PHOS kinase and Lck. Significant inhibition of MAP-
KAP-K1a, MSK1, PKCa, PKBa, SGK and S6K1 was
also observed for a number of examples. This lack of
selectivity may in part be responsible for the relatively
potent single agent cytotoxicity described in Table 3
and the low MTDs achieved in vivo (see below).
The pharmacodynamic effect of compound 14 was also
studied in vivo in female nu/nu mice bearing HT-29 hu-
man tumour xenografts. Animals were treated with
CPT-11 (75 mg/kg iv; a high dose, but within the ac-
cepted range8,9), followed at 24 and 27 h with 14 (iv)
at a range of doses. Tumours were excised at 30 h, then
disaggregated, and the levels of Cdc2 Y15 phosphoryla-
tion were measured by Western blotting, using anti-
7. Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P. Biochem.
J. 2000, 351, 95.
8. El-Galley, R.; Keane, T. E.; Sun, C. Urol. Oncol. 2003, 21,
49.
9. Hardman, W. E.; Moyer, M. P.; Cameron, I. L. Br. J.
Cancer 1999, 81, 440.