N. Zhang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2825±2828
Table 2. Inhibitory activity of compounds 6, 10, and 11
2827
fold between the 50% ecacious concentration and the
toxic concentration. The data do indicate a dierence in
IC50 values for enzyme activity and eects on cell pro-
liferation on the order of 50±100-fold. Higher IC50
values in cells relative to enzyme may suggest a problem
with availability and uptake in cells, or alternatively,
re¯ect that net inhibition of the MEK/ERK pathway is
in the context of cell speci®c biochemistry (e.g. com-
pensatory alternative growth signals). Separate studies
to be reported elsewhere, show that these compounds
do inhibit ERK phosphorylation in tumor cells at con-
centrations less than 1 mM.
Compd
OPh
IC50 (nM)
Coupled MEK9
IC50 (nM)
Direct MEK10
6
10
11
para
meta
ortho
9
95
>10,000
30
100
>10,000
Values are means of two experiments.
Table 3. Cell growth inhibitory activity of compounds 6, 13, and 14
in three human colon tumor lines13
Raf inhibition or greater sensitivity of response in cou-
pled systems.11 However, more direct evidence for
selective MEK inhibition by some of these compounds
comes from data showing more potent inhibition of
MEK phosphorylation of ERK than Raf phosphoryla-
tion of MEK as measured by phosphoimaging of
[33P]ATP bands on SDS-PAGE gels (data not shown).
In addition, in vitro selectivity for other enzymes was
studied. The most active compounds, 6 and 13, were
tested in an ERK2 kinase assay,12 and found to have
greatly reduced activity (IC50>1 0 mM). They were also
found to have little eect on certain tyrosine kinases
{EGFR (epidermal growth factor receptor), ECK (epi-
thelial cell kinase), and KDR (VEGF related kinase
insert domain-containing receptor)} or serine/threonine
kinases such as cyclin dependent kinases (cdk2, cdk4)
and AKT (target for PI3K) (data not shown). Studies
on inhibition of other related MAPK kinase family
members is ongoing.
Compd
IC50 (mM)
Colo205
IC50 (mM)
Lovo
IC50 (mM)
SW620
6
13
14
1.43
0.63
0.36
0.63
0.25
0.19
1.50
0.70
0.38
In conclusion, we have developed a series of 3-cyano-4-
(phenoxyanilino)cyanoquinolines as MEK (MAP
kinase kinase) inhibitors. The most active compounds
have alkoxy groups at both the 6- and 7- positions.
Compounds with a methoxy group at the 6-position and
a morpholinoalkoxy group at the 7- position maintain
potent enzymatic activity compared with the methoxy
analogues. These compounds represent novel structures
with MEK inhibitory activity. The lead compounds
show low nanomolar IC50's against MAP kinase kinase,
and have potent activity on several tumor cell lines.
The cyanoquinoline core is necessary for inhibition of
MEK activity. Compound 15, the quinazoline analogue
of compound 6, was found to be weakly active
(IC50>100 mM).
References and Notes
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Compounds 6, 13, and 14 were tested for their ability to
inhibit cell growth in Colo205, Lovo, and SW620
human colon tumor lines in culture.13 The activity of
these compounds is shown in Table 3. Compounds 13
and 14, with a morpholinoalkoxy group at the 7-posi-
tion, showed better cell inhibitory activity than the cor-
responding dimethoxy compound, 6. Cells are checked
microscopically for morphological and cytotoxic chan-
ges at each concentration. Viability is checked using
trypan blue dye staining and counting on a hemacyt-
ometer. Compounds are not cytotoxic at concentrations
near the IC50 value; there is no evidence of cytotoxicity
at concentrations below 5 mM. Compounds 13 and 14
showed evidence of cytotoxicity only at concentrations
greater than 5 mM. This represents a window of 10±15