4-Anilino-3-cyanobenzo[g]quinolines as kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00776-4

A series of 4-anilino-3-cyanobenzo[g]quinolines was prepared as potent kinase inhibitors. Compared with their bicyclic 4-anilino-3-cyanoquinoline analogues, the tricyclic 4-anilino-3-cyanobenzo[g]quinolines are less active against EGF-R kinase, equally active against MAPK kinase (MEK), and more active against Src kinase. For Src kinase inhibition, the best activity is obtained when both the 7- and 8-positions are substituted with alkoxy groups. Several of these kinase inhibitors show potent growth inhibitory activity in tumor cells.

Full text of DOI:10.1016/S0960-894X(01)00776-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 423–425
4-Anilino-3-cyanobenzo[g]quinolines as Kinase Inhibitors
Nan Zhang,a,* Biqi Wu,a Allan Wissner,a Dennis W. Powell,a Sridhar K. Rabindran,b
Constance Kohlerb and Frank Boschellib
aChemical Sciences, Wyeth-Ayerst Research, Pearl River, NY 10965, USA
bDiscovery Oncology, Wyeth-Ayerst Research, Pearl River, NY 10965, USA
Received 17 September 2001; accepted 13 November 2001
Abstract—A series of 4-anilino-3-cyanobenzo[g]quinolines was prepared as potent kinase inhibitors. Compared with their bicyclic
4-anilino-3-cyanoquinoline analogues, the tricyclic 4-anilino-3-cyanobenzo[g]quinolines are less active against EGF-R kinase,
equally active against MAPK kinase (MEK), and more active against Src kinase. For Src kinase inhibition, the best activity is
obtained when both the 7- and 8-positions are substituted with alkoxy groups. Several of these kinase inhibitors show potent
growth inhibitory activity in tumor cells. # 2002 Elsevier Science Ltd. All rights reserved.
Protein kinases, which transfer phosphoryl groups from
ATP to tyrosine, serine, and threonine residues on pro-
teins, play a key role in signal transduction pathways in
the regulation of cell growth and cell cycling in response
to external stimuli. However, under certain conditions,
as a result of over-expression, mutation, or, in the case
of receptor kinases, co-expression of the ligand and the
receptor, these enzymes can become hyper-activated,
resulting in uncontrolled cell proliferation. Inhibition of
kinase activity presents a unique opportunity to block
uncontrolled cell growth and, therefore, has potential
therapeutic utility in developing cancer treatments. We
recently reported 4-anilino-3-cyanoquinolines as potent
inhibitors of Epidermal Growth Factor Receptor (EGF-
R) kinase,1 Mitogen-Activated Protein Kinase (MAPK)
kinase,2 and Src kinase.3 The differential selectivity for
these kinases depends on the nature of the anilino group
at the 4-position. Dialkoxy substituents at the 6- and 7-
positions increase activity, while alkoxy substituents at
the 5- and 8-positions lead to decreased activity. These
results suggest there is room to expand the bicyclic core
from the 6- and 7-positions. We thus introduced a novel
tricyclic 3-cyanobenzo[g]quinoline core by fusing an
additional phenyl ring to the 6- and 7- positions of the
3-cyanoquinoline core. We now report the synthesis and
inhibitory activity of this series of 4-anilino-3-cyano-
benzo[g]quinolines in EGF-R kinase, MAPK kinase, and
Src kinase. The activity of the corresponding bicyclic 4-
anilino-3-cyanoquinolines is also shown for comparison.
As shown in Scheme 1, the synthesis of these tricyclic 4-
anilino-3-cyanobenzo[g]quinolines started from the cor-
responding 6,7-dimethoxynaphthalene-2,3-dicarboxylic
anhydride4 or 6-methoxynaphthalene-2,3-dicarboxylic
anhydride5 1. Treatment with sodium methoxide in
methanol opened the anhydrides, yielding mono-acid
Scheme 1. (a) NaOCH3, CH3OH; (b) (PhO)2PON3, then ꢀacetone/
.
H2O; (c) DMF DMA, reflux; (d) LiCH2CN, THF, À78 C, then
*Corresponding author. Tel.: +1-845-732-3425; fax: +1-845-732-
5561; e-mail: zhangn@war.wyeth.com
HOAc, rt; (e) POCl3, reflux; (f) ArNH2, EtOCH2CH2OH, reflux; (g)
pyridinium chloride, 215 ꢀC.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00776-4

424
N. Zhang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 423–425
mono-esters 2 (a 1:1 mixture of regio-isomers in the case
of asymmetric mono-methoxy substitution). The car-
boxylic acid group was then converted to an amino group
via Curtius rearrangement. We found that quenching the
isocyanate intermediate with a large quantity of wet ace-
tone led to the desired amino naphthoate directly. In the
case of mono-methoxy substitution, the regio-isomers
were separated at this amino naphthoate stage. The
amino group was then converted to the corresponding
core, the corresponding bicyclic analogues, 4-anilino-3-
cyanoquinolines, were also tested at the same time for
comparison.
We first introduced a 3-chloro-4-fluoroanilino group,
one of the best anilino groups for EGF-R kinase inhi-
bition,1 at the 4-position of the tricyclic core. Compared
with its bicyclic analogue 10, compound 9 is less active
as an EGF-R kinase inhibitor (Table 1). The compounds
were evaluated for their ability to inhibit the growth of
certain cell lines. Two human carcinoma cell lines were
used: A431 (epidermoid), which highly over-expresses
EGF-R, and SW620 (colon), which serves as a control
line expressing low levels of EGF-R. Compound 10
showed a 10-fold increase in activity in A431 cells versus
the control SW620 cells, while compound 9 showed a 3-
fold increase. These results suggest that both com-
pounds inhibit A431 cell growth, at least in part, by
interfering with the function of the EGF-R receptor.
.
formamidine 4 in refluxing DMF DMA. Addition of 2
equiv of lithium anion of acetonitrile followed by acid-
catalyzed cyclization with acetic acid gave the 3-cyano-
benzo[g]quinolones 5. Chlorination with POCl3 fol-
lowed by replacement of the chloride with anilines
provided the desired 4-anilino-3-cyanobenzo[g]quino-
lines 7. Demethylation of selected compounds was
accomplished in pyridinium chloride at 215 ꢀC, provid-
ing the corresponding hydroxy analogues 8.
The tricyclic 4-anilino-3-cyanobenzo[g]quinolines (7 and
8), with appropriate anilino groups at the 4-position,
were tested against three different kinases: EGF-R
kinase, MAPK kinase (MEK), and Src kinase. They
were also tested in tumor cell lines relevant to these
kinases. To evaluate the effect of fusing an additional
ring to form the tricyclic 3-cyanobenzo[g]quinoline
We next studied the effect of the tricyclic core on
MAPK kinase (MEK) inhibition. We introduced a 4-
phenoxyanilino group, one of the best anilino groups
for MAPK kinase inhibition,2 at the 4-position. As
shown in Table 2, the tricyclic compound 11 showed
activity comparable to that of the the bicyclic com-
pound 12 in both the enzymatic assay and the inhibition
of LOVO human colon tumor cell growth. This result
indicates that ring expansion of the bicyclic 3-cyano-
quinoline does not decrease potent MAPK kinase inhi-
bitory activity.
Table 1. EGF-R kinase and cell growth inhibitory activity of 9 and 10
As shown in Table 3, in contrast to the effects on EGF-R
kinase and MAPK kinase (MEK), the tricyclic 3-
cyanobenzo[g]quinoline core provides better potency
against Src kinase activity compared to the bicyclic
analogue. Compound 13, with a 4-chloro-5-methoxy-2-
methylanilino group, one of the most active anilino
groups for Src kinase inhibitory activity, at the 4-posi-
tion,3b was prepared. Compared with its bicyclic 3-
cyanoquinoline analogue 14, the tricyclic 3-cyano-
benzo[g]quinoline 13 was found to be more potent in
the enzymatic assay (3Â) as well as in the Src-trans-
formed fibroblast cell proliferation assay (10Â).
Compd
IC50 (mM)
EGF-R kinase
IC50 (mM)
A431 cell
IC50 (mM)
SW620 cell
9
10
18.0
5.17
0.68
1.03
1.99
9.89
IC50 values for EGF-R inhibition represent means of three separate
determinations with variations less than 30% of the means.
Table 2. MAPK kinase (MEK) and cell growth inhibitory activity of
11 and 12
Table 3. Src kinase and cell growth inhibitory activity of 13 and 14
Compd
IC50 (mM)
IC50 (mM)
LOVO cell
Compd
IC50 (mM)
Src kinase
IC50 (mM)
Src-transformed fibroblast cell
coupled MEK4
11
12
0.010
0.009
0.67
0.63
13
14
0.0014
0.0040
0.21
2.4
IC50 values for MEK inhibition represent means of three separate
determinations with variations less than 10% of the means.
IC50 values for Src inhibition represent means of three separate deter-
minations with variations less than 20% of the means.

N. Zhang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 423–425
Table 4. Src kinase inhibitory activity of 15–19
425
fusing an additional ring onto the 6- and 7-positions of
the quinoline core to form the tricyclic 4-anilino-3-cy-
anobenzo[g]quinolines. Presumably, these tricyclic 4-an-
ilino-3-cyanobenzo[g]quinolines are competitive inhibitors
with ATP as are the bicyclic analogues.1a,6 The tricyclic
core is inferior to the corresponding bicyclic 3-cyano-
quinoline core with respect to EGF-R kinase inhibition.
For MAPK kinase inhibition, the tricyclic 3-cyano-
benzo[g]quinoline with a 4-phenoxyanilino group at the
4-position of the quinoline provides activity equivalent
to that of the corresponding bicyclic 3-cyanoquinoline.
However, the tricyclic 3-cyanobenzo[g]quinoline with
one of the Src optimal anilino groups at the 4-position is
a more potent inhibitor of Src kinase than the corre-
sponding bicyclic 3-cyanoquinoline. SAR studies of the
substitutions at the 7- and 8-positions of the tricyclic
core on Src kinase inhibition showed patterns similar to
the SAR of 6- and 7-substituted bicyclic cyanoquinoline
analogues, thus suggesting a similar mode of binding to
the enzyme for both the bicyclic and tricyclic cores.
Compd
R1
R2
IC50 (mM)
15
16
17
18
19
OCH3
H
OCH3
OH
OCH3
OCH3
OCH3
H
OH
OCH3
0.018
30% inh. @ 0.1
30% inh. @ 0.1
10% inh. @ 0.01
0.038
IC50 values for Src inhibition represent means of at least two separate
determinations with variations less than 20% of the means.
The structure–activity relationship of 7- and 8-substit-
ution patterns of this series of tricyclic 3-cyano-
benzo[g]quinolines against Src kinase activity was
explored by comparing compound 15 with its mono-
methoxy analogues 16 and 17, and its demethylated ana-
logue 18. Instead of a 4-chloro-5-methoxy-2-methylan-
ilino, a 2,4-dichloroanilino group was introduced at the
4-position to avoid complications in the last demethyl-
ation step in the synthesis of 18. The activity of these
compounds against Src enzyme is shown in Table 4.
References and Notes
1. (a) Wissner, A.; Berger, D. M.; Boschelli, D. H.; Floyd,
M. B., Jr; Greenberger, L. M.; Gruber, B. C.; Johnson, B. D.;
Mamuya, N.; Nilakantan, R.; Reich, M. F.; Shen, R.; Tsou,
H.-R.; Upeslacis, E.; Wang, Y. F.; Wu, B.; Ye, F.; Zhang, N.
J. Med. Chem. 2000, 43, 3244. (b) Torrance, C. J.; Jackson,
P. E.; Montgomery, E.; Kinzler, K. W.; Vogelstein, B.; Wiss-
ner, A.; Nunes, M.; Frost, P.; Discafani, C. M. Nat. Med.
2000, 6, 1024.
2. (a) Zhang, N.; Wu, B.; Powell, D.; Wissner, A.; Floyd,
M. B.; Kovacs, E. D.; Toral-Barza, L.; Kohler, C. Bioorg.
Med. Chem. Lett. 2000, 10, 2825. (b) Zhang, N.; Wu, B.;
Powell, D.; Wissner, A.; Kovacs, E. D.; Toral-Barza, L.;
Kohler, C. In Frontiers of Biotechnology & Pharmaceuticals;
Science: New York, 2000; Vol. 1, p 305. (c) Zhang, N.; Wu,
B.; Eudy, N.; Wang, Y.; Ye, F.; Powell, D.; Wissner, A.;
Feldberg, L. R.; Kim, S. C.; Mallon, R.; Kovacs, E. D.; Toral-
Barza, L.; Kohler, C. Bioorg. Med. Chem. Lett. 2001, 11, 1407.
3. (a) Boschelli, D. H.; Wang, Y. D.; Ye, F.; Wu, B.; Zhang,
N.; Dutia, M.; Powell, D.; Wissner, A.; Arndt, K.; Weber,
J. M.; Boschelli, F. J. Med. Chem. 2001, 44, 822. (b) Boschelli,
D. H.; Ye, F.; Wang, D. Y.; Dutia, M.; Johnson, S.; Wu, B.;
Miller, K.; Powell, D. W.; Arndt, K.; Discafani, C.; Etienne,
C.; Gibbons, J.; Grod, J.; Lucas, J.; Weber, J. M.; Boschelli, F.
J. Med. Chem. 2001, 44, 3965. (c) Wang, Y. D.; Miller, K.;
Boschelli, D. H.; Ye, F.; Wu, B.; Floyd, M. B.; Powell, D. W.;
Wissner, A.; Weber, J. M.; Boschelli, F. Bioorg. Med. Chem.
Lett. 2000, 10, 2477.
Several conclusions can be reached from the Src kinase
inhibition results. First, the tricyclic 3-cyano-
benzo[g]quinoline 15 was again found to be more potent
in the enzymatic assay (2Â) than its bicyclic 3-cyano-
quinoline analogue 19. As for the anilino group at the 4-
position on the tricyclic core, we found the same trend
as in the case of the bicyclic 3-cyanoquinoline core,3b
that is, the 4-chloro-5-methoxy-2-methylanilino sub-
stituted compound is more potent than the 2,4-dichloro-
anilino substituted compound (13 vs 15). Compound 15,
with 7,8-dimethoxy substitutions, is more potent than
the corresponding mono-methoxy analogues (16 and
17) or the 7,8-dihydroxy analogue (18). Similar conclu-
sions were reached with the bicyclic 3-cyanoquinoline
series,3a where 6,7-dimethoxy substitutions were found
to be much more active than the corresponding mono-
methoxy analogues or 6,7-dihydroxy analogue. These
similarities in structure–activity relationships strongly
suggest that the tricyclic 3-cyanobenzo[g]quinolines
bind to the Src enzyme in a fashion similar to the
bicyclic 3-cyanoquinolines.
4. McOmie, J. F. W.; Perry, D. H. Synthesis 1973, 7, 416.
5. Brown, F. K.; Brown, P. J.; Bickett, D. M.; Chambers,
C. L.; Davies, H. G.; Deaton, D. N.; Drewry, D.; Foley, M.;
McElroy, A. B.; Gregson, M.; McGeehan, G. M.; Myers,
P. L.; Norton, D.; Salovich, J. M.; Schoenen, F. J.; Ward, P. J.
Med. Chem. 1994, 37, 674.
In summary, as part of a continuing effort to develop
potent and selective kinase inhibitors, we have extended
our work on the bicyclic 4-anilino-3-cyanoquinolines by
6. Unpublished results.