The discovery of potent cRaf1 kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(99)00668-X

A series of benzylidene-1H-indol-2-one (oxindole) derivatives was synthesized and evaluated as cRaf-1 kinase inhibitors. The key features of the molecules were the donor/acceptor motif common to kinase inhibitors and a critical acidic phenol flanked by two substitutions. Diverse 5-position substitutions provided compounds with low nanomolar kinase enzyme inhibition and inhibited the intracellular MAPK pathway. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(99)00668-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 223±226
The Discovery of Potent cRaf1 Kinase Inhibitors
Karen Lackey,* Michael Cory, Ronda Davis, Stephen V. Frye, Philip A. Harris,
Robert N. Hunter, David K. Jung, O. Bradley McDonald, Robert W. McNutt,
Michael R. Peel, Randy D. Rutkowske, James M. Veal and Edgar R. Wood
Glaxo Wellcome, Inc., 5 Moore Drive, Research Triangle Park, NC 27709, USA
Received 1 October 1999; accepted 19 November 1999
AbstractÐA series of benzylidene-1H-indol-2-one (oxindole) derivatives was synthesized and evaluated as cRaf-1 kinase inhibitors.
The key features of the molecules were the donor/acceptor motif common to kinase inhibitors and a critical acidic phenol ¯anked
by two substitutions. Diverse 5-position substitutions provided compounds with low nanomolar kinase enzyme inhibition and
inhibited the intracellular MAPK pathway. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Workers at Kanegafuchi Chem. Ind. Co. reported the
signal inhibitory properties of cinnamamides (ST 638)
and their ``cyclised'' derivatives, substituted oxindoles
(ST 280 and ST 458).9 Further studies provided data
that suggested that seemingly small substituent changes
around a core template could provide remarkably dif-
ferent selectivity and potency pro®les in kinase inhibi-
tion.10 The facile synthetic routes and diversity of
substituents that could be introduced made this an
attractive starting point to discover a cRaf1 kinase
inhibitor. In this report, we will describe the cRaf1 SAR
of this series of compounds and demonstrate the intra-
cellular inhibition of cRaf1 kinase for representative
compounds.
Mutation, overexpression, and receptor-mediated
hyperactivation of ras genes play major roles in the
formation of human tumors.1±5 The protein kinase
cRaf1 is required for Ras signal transduction and is the
®rst enzyme in a MAP kinase cascade consisting of the
three protein kinases cRaf1, MEK and ERK. Disrup-
tion of this cascade at various points inhibits ras signal
transduction and transformation in many experimental
systems.1±5 cRaf1 also contributes to tumorigenicity by
negatively regulating apoptosis through direct inter-
action with bcl-2 family members.6±8 Thus, selective
inhibitors of the protein kinase cRaf1 may prove to be
e€ective anticancer agents.
Chemistry
SAR for the benzylidine oxindoles was rapidly eluci-
dated through both solution phase parallel synthetic
methods and discrete compound synthesis. The chemi-
cal libraries were designed to take advantage of the
facile mixed aldol condensation reaction between an
aryl aldehyde and a substituted oxindole.11 Initially we
were interested in elucidating the SAR requirements of
the benzylidene portion of the molecule. To this end we
built focused compound sets utilizing commercially
available oxindole and 5-chlorooxindole. Large sets of
diversely substituted aryl-aldehydes were reacted in
solution phase, parallel synthetic fashion using one
equivalent of each monomer in toluene in the presence
of catalytic p-toluenesulfonic acid at 105 ꢀC for 24 h.
Abbreviations: SAR, structure±activity relationships; EGF(R), endo-
thelial growth factor (receptor); MAPK, mitogen activated protein
kinase; ERK, extracellular-signal-regulated kinase; MEK, MAPK/
ERK kinase; FGF, ®broblast growth factor.
*Corresponding author. Tel.: +1-919-483-6240; fax: +1-919-483-
6053; e-mail: kel8725@glaxowellcome.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00668-X

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K. Lackey et al. / Bioorg. Med. Chem. Lett. 10 (2000) 223±226
The products were collected by parallel ®ltration meth-
ods to provide more than 450 compounds. The structure
and purity of the desired products were evaluated using
HPLC and MS analyses.
Once the preferred benzylidine substitution was deter-
mined, the emphasis of our synthetic e€orts was shifted
to elucidating the SAR of the aryl ring of the oxindole
and to improving the potency and cellular ecacy of the
molecules. Since the synthetic methods have been well
described,12 this discussion will be limited to high-
lighting the key transformations used to create chemical
diversity in the 5-position of the oxindole. Scheme 1
depicts the three principal methods that we used.
Selective acylation utilizing a variety of acyl chlorides
was achieved by using aluminum chloride with DMF.
Palladium-catalyzed couplings were carried out using
5-bromooxindole and aryl stannane reagents. Palla-
dium-catalyzed carbonylations a€orded esters that were
subsequently hydrolyzed and coupled with diverse
amines under standard amide bond forming reactions.
Acylation of oxindole with chloroacetylchloride readily
a€orded the 5-chloromethyl ketone derivative that was
subjected to aldol condensation with the desired aro-
matic aldehyde or treated with thioureas and thioa-
mides to provide functionalized 5-thiazole oxindoles.
Figure 1. Summary of SAR.
kinase.15 For optimal potency and selectivity, the ben-
zylidene ring needed to be substituted with a 4-phenol
¯anked by two substituents. Substitution in the 2-posi-
tion of the benzylidine aryl ringÐfor example, methyl,
bromo, methoxy, or nitroÐrendered the compounds
signi®cantly less potent in the enzyme assay.
A signi®cant correlation was found between the acidity
of the phenol and the potency of cRaf1 inhibition
(Fig. 2). The measured pKa where X and Y were varied
in the 3-(4-hydroxybenzylidine)-5-chloro-oxindole series
ranged over greater than 4 units (X=Y=OMe, pKa
9.8; X=Y=Cl, pKa 5.5).16 Compounds with greater
acidity were proportionally more potent cRaf1 kinase
inhibitors.
Biological Activity
The screening strategy for evaluating cRaf1 inhibitors
involved a two step process: (1) use of a cascade assay of
three nonhomologous kinases, Raf/MEK/ERK2 and
(2) target identi®cation for the potent cascade inhibi-
tors.13 This strategy provided a facile screen with
potency and selectivity information. Over 2000 com-
pounds in the benzylidene oxindole series were eval-
uated in this assay system and the SAR is summarized
in Figure 1. The discussion that follows will move
counter-clockwise around the oxindole template of
Figure 1 beginning with the NH/carbonyl which is a
prototypical donor/acceptor motif found in many
protein kinase enzyme inhibitors.14 Accordingly, N-
methylation of the amide NH abolished raf kinase inhi-
bition consistent with the reported H-bond pattern of
substituted oxindoles bound to FGF receptor tyrosine
The double bond in this series can exist in an E or Z
con®guration, and both isomers were typically observed
in the NMR spectra.17 However, in the presence of
water, the compounds with a pKa below 7 (correspond-
ing to the more potent inhibitors) rapidly equilibrated
to a 1:1 ratio of E- and Z-isomers. We found that for
compounds where X=alkoxy and Y=halo (pKa ꢁ7.0),
the equilibrium E:Z ratio was pH dependent. The rela-
tive isomer ratio was easily evaluated by HPLC and
could be varied by adjusting the pH of the mobile
Scheme 1. Key synthetic transformations for diverse oxindole
monomers.
18
Figure 2.
.

K. Lackey et al. / Bioorg. Med. Chem. Lett. 10 (2000) 223±226
225
phase. If the double bond was saturated forming an sp3
carbon at position 3, the raf kinase inhibition was lost.
steric hindrance into this area of the binding pocket.
Substitution of the 4,5-position with fused ring hetero-
cycles, for example the benzthiazole (6) or quinoline
analogues, produced compounds which were 8-to 20-
fold more potent than the unsubstituted parent (12) (see
Table 1). The 6- and 7-position could be substituted
with halogens and other small groups with moderate
improvements in enzyme potency.
Compounds substituted with 4-iodo and 4-methyl
groups were synthesized to enforce the Z-con®guration
in an attempt to determine the active geometric isomer.
While these compounds were signi®cantly less active,
this may also be explained as due to the introduction of
The 5-position of the oxindole served as an ideal posi-
tion to substitute for enhanced potency of the com-
pounds. In addition, the 5-position proved to be
remarkably tolerant of a wide variety of functionality
including halogens, esters, amides, aryl groups, hetero-
aryl groups, and acyl groups. A representative set of
raf inhibitors is shown in Table 1. Compounds 1±11
showed 5100-fold selectivity for raf kinase versus
CDK1, CDK2, c-src, ERK2, MEK, p38, Tie2,
VEGFR2, and c-fms.
Table 1. Representative data
Compds
Structure
Cascade assay
IC50, mMa
0.009
EMAPKb
%I [cmpd]
R
I
X
The inhibition of cRaf1 kinase activity should result in
the down-regulation of MAP kinase activity. A cell-
based mechanistic assay, EMAPK, measures the inhibi-
tion of EGF stimulated MAP kinase activation.19 The
assay was used to demonstrate that the compounds
penetrated the cellular membrane and inhibited the
desired target. In general, the 5-aryl, heteroaryl, halo,
acyl and ester substitutions were e€ective intracellular
raf kinase inhibitors. Amides and aminosulfonyl groups
(e.g. 11) in the 5-position were signi®cantly less e€ective.
It is important to note that these compounds did not
inhibit the upstream target EGFR tyrosine kinase as
measured by the lack of inhibition of auto-phosphor-
ylation in the same cellular system.20
1
2
Br
90%
5 uM
84%
Br
0.018
15 uM
3
4
5
6
Br
Br
Br
Br
0.046
0.011
0.025
0.016
94%
15 uM
52%
10 uM
55%
10 uM
Conclusion
66%
15 uM
cRaf1 inhibitors are predicted to be broad-spectrum
antitumor agents and have the potential to enhance
existing cancer chemotherapies. We have discovered
potent cRaf1 kinase inhibitors that contain a pharma-
cophore composed of an acidic phenol and a donor/
acceptor binding group (NH-CO). These inhibitors are
e€ective at blocking the intracellular MAP kinase path-
way. The details of the cellular and in vivo ecacy
properties of these compounds will be reported in the
future.21
7
8
Br
Br
0.065
0.013
81%
10 uM
53%
10 uM
9
Cl
Cl
0.032
0.040
81%
15 uM
Acknowledgement
10
40%
15 uM
We would like to thank Douglas J. Minick for measur-
ing the pKa values of our compounds.
11
12
Br
Br
0.029
1.30
0%
15 uM
References and Notes
H
±
aValues are means of greater than three experiments for enzyme assay
of Raf/MEK/ERK and the target of enzyme inhibition was validated
to be cRaf1 kinase.
bCellular evaluation of compounds.19 Percent inhibition (%I) of
MAPK activation at the concentration listed.
1. Daum, G.; Eisenmann-Tappe, I.; Fries, H.-W.; Troppmair,
J.; Rapp, U. R. TIBS 1994, 19, 474.
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279.

226
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1H NMR trends
4. Kolch, W.; Heidecker, G.; Lloyd, P.; Rapp, U. R. Nature
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unpublished results.
13C NMR trends
11. A similar approach was undertaken by workers at Sugen in
their identi®cation of VEGFR TK inhibitor: Sun, L.; Tran,
N.; Tang, F.; Harald, A.; Hirth, P.; McMahon, G.; Tang, C. J.
Med. Chem. 1998, 41, 2588.
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A.; Hunter, R. N., III; Veal, J. M.; Dickerson, S.; Peel, M. R.
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16. PKa values were determined using a spectrophotometric
method developed in our laboratory. Similar methods are
discussed in Albert, A.; Serjeant, E. P. The determination of
Ionization Constants, 2nd ed., Edinburgh, T.&A. Constable
Ltd., 1971.
17. E/Z isomers were assigned using 1H NMR chemical shifts,
NOE experiments, and 3 bond carbon19-proton coupling con-
stants.
18. pIC50=11.63 0.74*pKa; r2=0.92, F=206.81, n=20,
P>0.01.
19. EMAPK: The EMAPK assay measures the EGF stimulated
MAP kinase activation in a cellular assay format. Cells were
serum starved for 24 h. cRaf1 inhibitors were pre-incubated
with the cells for 60 min. The cells were stimulated with EGF
(100 nM) for 5 min. The cells were lysed in SDS polyacrylamide
gel sample bu€er and the protein samples were resolved by
SDS-PAGE. ERK activation was evaluated by Western Blot-
ting using Anti-Phospho ERK (New England BioLabs).
20. EGF receptor autophosphorylation was evaluated in a
manner similar to that cited in note 13 using Anti-PY20.
21. Results of a variety of cellular assay systems with a subset
of these compounds: Wood, E. R.; Crosby, R. M.; Davenport,
E. A.; Gilmer, T. M.; Hunter, B. N.; Keith, B. R.; Lee, A. V.;
McDonald, O. B.; Mullin, R. J.; Rusnak, D. W.; Lackey, K.
E. Am. Assoc. Cancer Res. 1999, 40, 724.