G. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2293–2298
Table 3. Results of the MTS and FACS cellular assays (lM)
2297
Compound
IC50 (nM)
MTSa (lM)
FACSb (lM)
Cpd alone
Cpd/Doxc
Ratio
EC50
Cpd/Doxd
Ratio
7
5
>58.9
8.65
15.0
0.89
24.0
>59.3
0.44
12.2
0.41
0.76
0.22
4.66
6.55
>3.9
9.7
8a
8b
8f
3
22
23
8
>59.3
>59.3
>59.3
>58.9
7.86
>2.5
>10
>10
>10
1.71
8i
>136
>4.8
19.1
2.0
>5.9
8j
9
8u
15
19a
29a
29d
7
12
10
10
13
1.54
0.83
>10
3.66
>10
0.14
0.58
1.45
>71
6.3
3.8
3.1
14.5
16.4
>6.9
2.5
a Tested using HeLa cells.
b Tested using H1299 cells.
c The Dox concentration was 100 nM.
d The Dox concentration was 500 nM.
for cytotoxicity. FACS analysis measures abrogation of
the G2 checkpoint as an indicator of Chk1-based cellu-
lar mechanism for the compounds. These data, along
with the enzymatic assay data (IC50’s), are presented
in Table 3 as IC50 values at which the compound reduces
cell growth or decreases G2 cells by half. We have
defined an ideal result as a compound possessing little
or no antiproliferative activity when dosed alone
(>59.3 lM in the MTS assay and >10 lM in the FACS
assay), but possessing high antiproliferative activity in
the presence of doxorubicin (Dox) (ideally 61 lM), thus
providing the highest ratio.
the compounds showed activity in both assays. Of the
compounds tested, 8i showed the best overall results
among all three assays possessing an IC50 of 8 nM,
MTS single agent activity for compound alone of
>59.3 lM and combination activity with doxorubicin
of 0.44 lM, and a ratio of single agent to combination
activity of >10.0 lM/1.7 lM in the FACS assay. In
addition, compounds 8a and 8u also showed moderate
to good activity having 9.7- and 19.1-fold MTS single/
combination ratios, respectively.
In summary, we have modified R4 of the urea phenyl
ring with a variety of chemical structures, and R2 and
R5 to some degree. Several compounds showed very
promising results as potent and selective Chk1 inhibi-
tors. In particular, compound 8i showed very good re-
sults throughout the enzymatic and cellular assays.
Although we have progressed toward our goal of finding
a potent and selective Chk1 inhibitor, additional work is
needed to obtain greater insight into the SAR of these
compounds.
From the data in Table 3, it is seen that good activity in
the enzymatic assay does not always translate into good
cellular activity. Factors such as cell permeability can
contribute to these differences. Compounds 7, 8b, 8f,
and 8j all have potent IC50’s ranging from 5 to 23 nM,
however, compounds 7, 8b, and 8j have only very weak
combination cellular activity: 15, 24.0, and 12.2 lM,
respectively. Compound 8f did not show any combina-
tion or single agent cellular activity in either the MTS
or FACS assay. Overall, the two cellular assays corrob-
orate well with each other. Compound 8i had no single
compound activity and showed strong combination
activity in the MTS assay. FACS analysis also showed
little single compound activity and had some combina-
tion activity. An apparent discrepancy between the
MTS and FACS assays was observed for compounds
15, 19a, and 29a. However, the different cell lines and
compound concentrations used in the two assays could
explain the limited variations. The MTS assay uses
HeLa cells that are more sensitive than the H1299 cells
used in the FACS assay. For example, a single com-
pound EC50 value of 0.83 lM for compound 19a in
the MTS assay became 3.66 lM in the FACS assay.
Moreover, the MTS assay measures proliferation, while
FACS analysis evaluates Chk1-based cellular mecha-
nism. The two parameters may vary in the cell lines.
The single compound activities for compounds 15 and
29a in the MTS assay were not detected in the FACS as-
say because the maximum concentration for FACS was
only 10 lM. For combination treatments, all three of
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