Synthesis and biological evaluation of 3-ethylidene-1,3-dihydro-indol-2- ones as novel checkpoint 1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.09.064

Chk1 inhibitors have emerged as a novel class of neoplastic agents for abrogating the G2 DNA damage checkpoint arrest. Analogs of the Chk1 inhibitor, 3-ethylidene-1,3-dihydro-indol-2-one, were synthesized and tested in vitro for their inhibitory activitie

Full text of DOI:10.1016/j.bmcl.2005.09.064

Bioorganic & Medicinal Chemistry Letters 16 (2006) 421–426
Synthesis and biological evaluation of
3-ethylidene-1,3-dihydro-indol-2-ones as novel
checkpoint 1 inhibitors
Nan-Horng Lin,* Ping Xia, Peter Kovar, Chang Park, Zehan Chen, Haiying Zhang,
Saul H. Rosenberg and Hing L. Sham
Cancer Research, R-47B, Global Pharmaceutical Products Division, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064-3500, USA
Received 22 August 2005; revised 21 September 2005; accepted 22 September 2005
Available online 18 October 2005
Abstract—Chk1 inhibitors have emerged as a novel class of neoplastic agents for abrogating the G2 DNA damage checkpoint
arrest. Analogs of the Chk1 inhibitor, 3-ethylidene-1,3-dihydro-indol-2-one, were synthesized and tested in vitro for their inhibitory
activities. The most promising compound identified from this series is analog 28, which possesses potent enzymatic and cellular
activities.
Ó 2005 Elsevier Ltd. All rights reserved.
In response to DNA damage, cells arrest at various cell
cycle checkpoints (G1, S, or G2) to initiate repair pro-
grams. Upon completion of these programs, cells pro-
ceed through the cell cycle. If the DNA damage is not
repairable, they undergo apoptosis.1–3 Most tumor cells,
through the loss of p53 or Rb function, are deficient in
the G1 checkpoint and must arrest at either S or G2
to repair DNA damage.4–8 If the S and G2 checkpoints
are abrogated, the cells will undergo premature mitotic
entry/premature chromosome condensation (PCC),
leading to apoptosis, mitotic catastrophe, and cell death.
In contrast, normal cells will arrest at G1 in response to
DNA damage and will be less affected by S and G2
checkpoint abrogation.9–15
the Cdc25C substrate Cdc2, and inactivation of cyclin
B/Cdc2 complex, which results in cell cycle arrest at G2.
Recently, we and others have demonstrated that Chk1
also plays a key role in the S checkpoint induced by var-
ious DNA damaging agents.20–24 Activation of Chk1
through phosphorylation by ATM or ATR kinase in re-
sponse to various types of DNA damage induces degra-
dation of Cdc25A. The resulting diminished cellular
level of Cdc25A has been shown to be directly responsi-
ble for the S-phase arrest. Correspondingly, elimination
of Chk1 with siRNA leads to abrogation of both
Cdc25A proteolysis and the S- or G2 checkpoints.20,24
Collectively, these results demonstrate that Chk1 plays
an important role in both the S- and G2 checkpoints,
largely mediated by Cdc25A.
Checkpoint kinase 1 (Chk1), a serine/threonine kinase,
was initially identified as a link between DNA damage
and cell cycle arrest at G2.16,17 In response to DNA dam-
age, Chk1 phosphorylates and inactivates the Cdc25 fam-
ily of phosphatases (Cdc25A, B, and C). It was discovered
that upon phosphorylation by Chk1, Cdc25C is rapidly
deactivated through its sequestration to the cytosol.18,19
The consequences of this are hyperphosphorylation of
Cancer cells often lack one or more genes for G1 check-
point control, either through loss of p53 or Rb function,
or by overexpression of proto-oncogenes (cyclins and
CDKs).25 Inhibition of the remaining checkpoints, at S
and G2, by Chk1 inhibitors should make tumor cells
selectively more sensitive to anticancer therapies, such
as c-radiation or DNA damaging drugs.12,26–28 In addi-
tion, tumor cells deficient in p53 are usually more resis-
tant to cancer therapeutics. Thus, Chk1 inhibitors can
potentially enhance the cytotoxicity of DNA damaging
agents and overcome this resistance. There is ample
Keywords: Kinase; Checkpoint 1; Inhibitor.
*
Corresponding author. Tel.: +1 847 938 8797; fax: +1 847 935
5466; e-mail: nanhorng.lin@abbott.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.064

422
N.-H. Lin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 421–426
experimental evidence to demonstrate that the abroga-
tion of the S- and G2 checkpoints, through inactivation
of Chk1, can selectively sensitize cells deficient in the G1
checkpoint to DNA damage.12–16,26,29,30
Indolinone analogs were prepared using the reaction
sequences shown in Schemes 1 and 2. The non-commer-
cially available starting keto acid 2 was prepared from
the corresponding toluene by reaction with dimethyl-
malonate in the presence of sodium (Scheme 1) Oxida-
tion of the keto acid with hydrogen peroxide followed
by reduction of the nitro group to an amine gave the
6-bromo-indolinone 4. The indolinone was deprotonat-
ed with piperidine, and addition of the resultant anion
to the pyrrole aldehyde 5 provided the desired ethylidene
indolinone 6. Coupling of the substituted indolinone
bromides 6 with various substituted phenylboronic acids
such as 7 gave the indolinone analogs 8 that are
described in Tables 1–3.
We and others have disclosed ureas as a new class of
small molecule Chk1 inhibitors.31–34 To continue our
search for a structurally different Chk1 inhibitor that
possesses potent activity against Chk1 enzyme, we initi-
ated an extensive medicinal chemistry effort based on
high throughput screen hits. In this paper, we report
the synthesis and preliminary structure–activity relation-
ship of a series of these compounds, 3-ethylidene-1,3-
dihydro-indol-2-one.
O
Me
OH
ii
i
CO2H
O
NO2
Br
NO2
O
Br
Br
R1
NO2
1
2
3
Me
R2
HN
R2
CHO
iv
iii
O
+
R1
O
N
Me
Br
N
H
H
N
4
5
6
Br
H
Me
R2
HN
MeO
HO
R1
v
O
O
+
B
N
O
H
7
8
HO
OMe
Scheme 1. Reagents: (i) Na, dimethylmalonate; (ii) H2O2, NaOH; (iii) Zn, H2SO4; (iv) piperidine, MeOH; (v) Pd(PPh3)4, CsF.
R1
R2
R1
R1
R2
R2
i
ii
CH3
CH3
CH3
CHO
CO2H
N
N
N
R1
R2
iii
CH3
CHO
N
R1
R1
R1
R2
R2
R2
v
iv
OH
CH3
CH3
CH3
CO2Et
CHO
N
N
N
Scheme 2. Reagents: (i) Heat, neat or DMF; (ii) POCl3, DMF; (iii) CF3CO2H, CH(OEt)3; (iv) LiAlH4; (v) MnO2.

N.-H. Lin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 421–426
Table 1. Inhibition data for indolinone analogsa Table 2. Inhibition data for pyrrole analogsa
423
Ar2
5
6
NH
O
HO
MeO
N
H
O
N
Ar
H
Compound
Cx
C5
Ar2
IC50 (nM)
2839
Compound
Ar
IC50 (nM)
9
5'
N
H
13
7
4'
HO
3'
OMe
10
11
C5
C5
>50,000
3465
MeO
MeO
N
H
17
>10,000
OMe
N
18
19
20
29
1033
10
H
HO
OMe
MeO
12
C5
>50,000
H2N
HO
OMe
13
14
C6
C6
7
N
H
21
22
23
>10,000
>10,000
>10,000
F
92
F
N
H
F3CO
Me
15
C6
1306
N
H
a See Ref. 34 for assay method.
OMe
MeO
16
C6
>50,000
corresponding ester followed by oxidation of the resul-
tant alcohol with manganese oxide.
a See Ref. 34 for assay method.
In this study, several phenyl and pyrrole analogs of 3-
ethylidene-1,3-dihydro-indol-2-one were tested for their
inhibitory activities against Chk1 enzyme. The effects
of varying the substituents at the C4-position of the
pyrrole moiety have been studied in detail. We have also
investigated the impact of positional isomers and
substituents on phenyl ring on the inhibitory potency.
Scheme 2 illustrates three routes that were used to pre-
pare the substituted pyrrole aldehydes. Thus, commer-
cially available substituted pyrrole acids were thermally
decarboxylated to provide pyrrole intermediates, which
could be formylated with phosphorous oxychloride in
dimethylformamide to obtain the corresponding alde-
hydes. Alternatively, the pyrrole aldehydes were formed
directly from the corresponding acids by treatment with
trifluoroacetic acid and triethyl orthoformate. Finally,
aldehydes could also be obtained by reduction of the
The effects of moving the 3-methoxy-4-hydroxyphenyl
moiety from the C5 position to the C6 position are
shown in Table 1. With the exception of dimethoxy
analogs (12 and 16), it reveals that C5-substituted
3-methoxy-4-hydroxylphenyl analogs are consistently
less potent than the corresponding C6-substituted

424
N.-H. Lin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 421–426
Table 3. Inhibition data for pyrrole analogsa
Table 3 (continued)
Compound
Ar
IC50 (nM)
Ar
O
33
25
N
N
H
H
HO
OMe
15
34
1306
445
Compound
Ar
IC50 (nM)
N
3
H
Me
2
13
7
5
1
N
H
CN
N
Me
N
H
a See Ref. 34 for assay method.
24
25
120
Me
N
H
compounds against the Chk1 enzyme in the indolinone
series. For instance, C5-substituted compound 9 shows
a 405-fold decrease in potency compared with the corre-
sponding C6-substituted analog 13. A similar SAR
trend is also observed when the pyrrole moiety is re-
placed with an indole group (10 vs. 14 and 11 vs. 15).
The data also indicate that compound 13, with a pyrrole
group at C3 ethylidene, is more potent than the corre-
sponding indole analogs 14 and 15. Replacement of in-
dole with dimethoxyphenyl results in a huge reduction
in inhibitory potency. Since compound 13 provides the
most potent inhibitor, we then turned our attention to
the modification of 3-methoxy-4-hydroxyphenyl ring
with various substituents. It was hoped that this change
would increase enzymatic potency.
3465
Me
Me
N
H
Ph
26
27
>10,000
3972
N
H
N
H
14
28
92
As indicated in Table 2, replacement of the hydroxyl
group at the C40- position of the phenyl ring (18) with
an electron-donating amino group (19) causes a 36-fold
decrease in potency. Further reduction is observed when
the hydroxyl group is replaced with a methyl group. The
introduction of electron-withdrawing groups such as flu-
oro or trifluoromethoxy groups at the C4 position
resulted in complete loss of inhibition (cf. 21 and 22).
Apparently, electronic effects do not play an important
role in determining the enzymatic potency. In contrast,
shifting the methoxy group from the C30 position to
the C20 position of phenyl ring has only minimal impact
on the enzymatic potency (13 vs. 20). However, intro-
duction of methoxy groups to the C30, C40, and C50
positions (17) caused a huge reduction in potency. These
SAR studies clearly indicate that the 3-methoxy-4-
hydroxyphenyl moiety is still the best group for the C6
position of indolinone ring.
N
H
4
N
N
H
N
29
30
18
75
N
H
CO2Me
Me
Me
N
H
NM2
31
32
1280
Since compound 13 provides the most potent enzymatic
activity, we then focused our attention on SAR studies
of the pyrrole ring in the indolinone series (Table 3).
We have introduced various functionalities to the C30-,
C40-, and C50 positions of pyrrole ring. The inhibitory
activity at the C30-position of the pyrrole appears to
be governed by steric effects. As Table 3 indicates,
the phenyl analog (26) that has a large group at the
Me
Me
N
H
OH
Me
47
N
H
(continued on next page)

N.-H. Lin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 421–426
425
C30-position is less potent than either the methyl (24) or
isopropyl (25) analogs. In addition, the dimethyl com-
pound 24 is 17-fold less potent than the parent pyrrole
analog 13.
structure of compound 13 indicates that the 30-OMe
group forms a hydrogen bond with Lys38 of the back-
bone protein of the Chk1 enzyme, and the 40-OH func-
tionality forms another H-bond with Glu55. In addition,
the hinge region of compound 13 forms two additional
hydrogen bonds with both Glu85 and Cys 87 as shown
in Figure 1.
Table 3 also reveals that replacement of the pyrrole ring
with an imidazole moiety is reasonably well tolerated
(13 vs. 29), but reduction of the pyrrole ring to a pyrrol-
idine moiety results in a drastic reduction of potency in
the enzymatic assay (13 vs. 27). It is interesting to note
that introduction of polar long chained aliphatic func-
tionalities to the C40-position provides more potent
Chk1 inhibitors compared with the C40-unsubstituted
analog (30 vs. 24 and 32 vs. 24). The same SAR trend
was observed with 20-isopropyl-50-methyl pyrrole analog
(31 vs. 25).
The most potent compounds identified were submitted
for enzymatic assays against other serine/threonine ki-
nases. As shown in Table 4, analog 13 is the most
selective Chk1 inhibitor identified in this study. It does
not have any inhibitory activity against the other en-
zymes tested. Compound 28 has 91-fold selectivity
against CDC2 enzyme, whereas compound 29 shows
only 14-fold selectivity. This demonstrates that the
group connected to the olefinic moiety in the indoli-
none series plays an important role in determining
the enzymatic selectivity against other kinases.
As described previously, replacement of the pyrrole
ring with an indole moiety results in a 13-fold de-
crease in potency in the inhibition of Chk1 enzyme
(13 vs. 14). In contrast to this result, replacement of
the pyrrole ring with an azaindole moiety has only
minimal impact on the enzymatic potency (13 vs.
28). Interestingly, the inhibitory activities of indole
analogs appear to be governed by steric effects. Hence,
as previously observed, the C70-methyl analog 15 is
less potent than compound 14. A similar SAR trend
was also observed with azaindole analogs. Thus, intro-
ducing a cyano group to azaindole moiety results in a
decrease in potency of greater than 100-fold (18 vs.
34). The most potent compound identified in our
SAR studies of this series is the azaindole analog 28
with an IC50 value of 4 nM.
The cellular activity of the most potent compound 28
was evaluated in the MTS assay. For routine screen-
ing, the antiproliferative EC50Õs of the compounds
are determined in HeLa cells (a p53-deficient human
cervical cancer cell line), either alone or in the pres-
ence of 150 nM of doxorubicin, a topoisomerase II
inhibitor in clinical use known to confer G2/M check-
point arrest at this concentration in HeLa cells. In the
latter assay, the EC50s are calculated from the percent-
ages of inhibition by the test compounds at varying
concentrations above the background inhibition by
150 nM doxorubicin. Thus, the ratio of the EC50s of
the compound alone and the compound with
150 nM doxorubicin represents the ability of the com-
pound to sensitize HeLa cells. Compound 28 exhibits
significant antiproliferative activity as a single agent
(EC50 = 0.01 lM), probably indicating that this com-
pound may inhibit other targets, consistent with its
inferior selectivity. Compound 28 also shows potent
antiproliferative activity (EC50 = 0.18 lM) in the pres-
ence of doxorubicin (150 nM). To confirm the target,
additional characterization of the compound using
other cellular assays is required.
To understand how these Chk1 inhibitors interact with
the enzyme, the potent Chk1 inhibitor 13 was submitted
for X-ray crystallographic study.35 The X-ray co-crystal
GLU85 CO
CYS87 N
GLU55
COO
In summary, we have shown that variation of the sub-
stituent pattern at the phenyl or pyrrole rings of a 6-
substituted indolinone compound of structure 8 alters
the inhibitory properties of these compounds. The as-
say data indicate that substituents are not well tolerat-
ed at the C5-position of the phenyl ring (Table 1). In
general, compounds having a hydroxyl group at the
C40-position as shown in Table 2 possess potent inhib-
itory activities against the Chk1 enzyme. Unsubstitut-
ed pyrrole or azaindole analogs possesses the most
potent activity against the Chk1 enzyme. Introduction
of substituents to either the pyrrole or the azaindole
Figure 1. X-ray co-crystal structure of 13 with Chk1 enzyme.
Table 4. Inhibition data of Chk1 inhibitors against various kinases
Compound Chk1 Chk2
CDC2
CHK2
CK2
EGFR
MAPKAP2 PKCc
PKCd
SGK
SRC
Tie2
13
28
29
7
4
21730 >50000 >50000 >50000 >50000 >50000
>50000 >50000 >50000 >50000 >50000
22679 454
14649 251
368
NT
41690
NT
NT >50000
>50000 NT
NT
NT
>50000 >50000 1500
>50000 2377 NT
>50000 23273
18

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N.-H. Lin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 421–426
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