4-(Aminoalkylamino)-3-benzimidazole-quinolinones as potent CHK-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.03.059

CHK-1 is one of the key enzymes regulating checkpoints in cellular growth cycles. Novel 4-(amino-alkylamino)-3-benzimidazole-quinolinones were prepared and assayed for their ability to inhibit CHK-1. These compounds are potent cell permeable CHK-1 inhibit

Full text of DOI:10.1016/j.bmcl.2006.03.059

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3121–3124
4-(Aminoalkylamino)-3-benzimidazole-quinolinones
as potent CHK-1 inhibitors
Zhi-Jie Ni,* Paul Barsanti, Nathan Brammeier, Anthony Diebes, Daniel J. Poon,
Simon Ng, Sabina Pecchi, Keith Pfister, Paul A. Renhowe, Savithri Ramurthy,
Allan S. Wagman, Dirksen E. Bussiere, Vincent Le, Yasheen Zhou,
Johanna M. Jansen, Sylvia Ma and Thomas G. Gesner
Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608, USA
Received 25 January 2006; revised 9 March 2006; accepted 20 March 2006
Available online 5 April 2006
Abstract—CHK-1 is one of the key enzymes regulating checkpoints in cellular growth cycles. Novel 4-(aminoalkylamino)-3-benz-
imidazole-quinolinones were prepared and assayed for their ability to inhibit CHK-1. These compounds are potent cell permeable
CHK-1 inhibitors and showed synergistic effect with a DNA-damaging agent, camptothecin.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Upon DNA damage by ultraviolet light, radiation or
cytotoxic drugs, the cellular response is to arrest the cell
cycle at one of three checkpoints (G1/S, intra-S or G2/
M) to either permit DNA repair or to initiate apoptosis.
The serine/threonine checkpoint kinase, (CHK-1), regu-
lates both the G2/M and intra-S checkpoints, and plays
an important role in cell-cycle progression,1–4 especially
for p53-defective cancer cells. Since cell-cycle arrest is a
mechanism by which tumor cells can overcome the dam-
age induced by cytotoxic agents, abrogation of the G2/
M checkpoint with novel small molecule compounds
may increase the sensitivity of p53-deficient tumors to
chemotherapy.5,6 Importantly, in contrast to many cur-
rent therapies for cancer, this mechanism potentially
carries with it only a low risk of toxicity against non-
malignant cells, as CHK-1 inhibition is most effective
in p53-defective cancer cells. Thus, a major advantage
of CHK-1 inhibitors as a treatment for cancer is their
selective activity in conjunction with cytotoxics, such
as DNA-damaging reagents.
rently in clinical trials.10 Other CHK-1 inhibitors report-
ed in the literature include indazole–imidazole
analogs,11 and more selective UCN-01 analogs, such
as ICP-1.12 Recently a series of novel CHK-1 inhibitors
were reported, including N-aryl-N0-pyrazinylurea,13 1,3-
dihydro-indolone,14 and furanopyrimidine.15 Here, we
report 4-(aminoalkylamino)-3-benzimidazole-quinolin-
one derivatives as potent CHK-1 inhibitors with syner-
gistic effects with DNA-damaging agents in tumor cells.
The synthesis of 4-(aminoalkylamino)-3-benzimidazole-
quinolinone is shown in Scheme 1. Reaction of ethyl
2-benzimidazole-acetate with 1-N-p-methoxy-benzyl
(PMB) protected isatoic anhydride16 in the presence of
a base in THF yielded 4-hydroxy-3-benzimidazole-quin-
olinone A in good yields. Conversion of the 4-hydroxy
compound to the corresponding bis-triflate B was
accomplished with triflic anhydride in the presence of
pyridine at ꢀ5 ꢁC. Reaction of the bis-triflate with alkyl-
amines via a SNAr reaction was carried out in acetoni-
trile with Hunig’s base at 80 ꢁC. Deprotection of the
¨
Several CHK-1 inhibitors have been reported in the lit-
erature. Of these, UCN-01 (7-hydroxystaurosporine) is
a potent inhibitor of CHK-1 (Ki = 5.6 nM)7–9 and has
modest selectivity among other kinases.7 UCN-01 is cur-
PMB group and removal of the triflate group on the
benzimidazole of the intermediate C occurred in one
pot under acidic conditions (trifluoroacetic acid and
concentrated HCl; v/v, 7:1) at 90 ꢁC to yield 4-alkylami-
no-3-benzimidazole-quinolinone (4-alkyl ABIQ) com-
pounds in good yields.17
Keywords: CHK-1 kinase inhibitor; Kinase; Synergy.
*
Unsubstituted 4-aminobenzimidazolequinolone 1 has
moderate activity (IC50 = 0.73 lM) against CHK-1. A
Corresponding author. Tel.: +1 510 923 8387; fax: +1 510 923
3360; e-mail: zhi-jie_ni@chiron.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.059

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Z.-J. Ni et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3121–3124
OEt
OH
N
O
N
1) LHMDS
N
R1
H
O
N
N
H
N
O
O
R1
2)
PMB
A
O
PMB
OTf
N
R2NH2
N
Tf2O
Py
R1
Tf
Hünig's Base
N
O
PMB
B
NHR2
N
NHR2
N
TFA:HCl
N
N
R1
R1
H
Tf
N
O
N
H
O
PMB
C
LHMDS: Lithium bis(trimethylsilyl)amide
Hünig's base: Diisopropylethylamine
Scheme 1. Preparation of CHK-1 inhibitors.
NH group at C-4 is favorable for the potency. Presum-
ably, a hydrogen-bond donor at C-4 or simply a small
size hydrogen is important for keeping the two bicycles
in a co-planar configuration. Replacement of the C-4
amine group with a hydroxy group reduced the potency
by 4-fold, while the 4-diethylamino analog 3 was
inactive.
alkyl group, especially its stereochemistry. For example,
compound 5 was 60-fold less potent than its enantiomer
4 although the effect was less striking for the enantio-
meric pair 9 and 10.
To better understand the interactions of these inhibitors
with the CHK-1 protein, a co-crystal structure of com-
pound 11 with CHK-1 was obtained.18 The co-crystal
structure showed that the inhibitor formed hydrogen
bonds with Glu85 and Cys87 in the hinge region,
explaining the general kinase affinity of this ABIQ scaf-
fold. Significantly improved potency for the amino
groups at 4-position was derived from the strong inter-
actions of the tertiary-amine of 11 with an acidic patch
formed by Glu91 and Glu134, as shown in Figure 1.19
This charge–charge interaction also greatly improved
selectivity of this series of inhibitors against many other
kinases (cdk and cdc data are shown below).20 Several
inhibitors in the literature occupied the same ATP-bind-
ing pocket. However, none of the known inhibitors has
fully utilized the acidic patch.14,15
R
B
N
O
5
4
3
6
7
N
H
A
N
H
8
1
1 R = NH2, IC50 = 0.731 μM
2 R = OH, IC50 = 2.95 μM
3 R = NEt2, IC50 > 30 μM
The affinity of ABIQ analogs was sensitive to substitu-
tions. The hydrogen bond donor–acceptor–donor
(DAD) motif of the quinolinone and benzimidazole
interacts with the lower hinge of the ATP-binding pock-
et. Methylation at N-1 eliminated the hydrogen-bond
interaction with the protein and abolished activity. Sim-
ple 4-alkylamino groups were tolerated with moderate
success (data not shown). Furthermore, an aminoalkyl-
amine, e.g., (S)-3-aminoquinuclidine, introduced at the
4-position of product 4, led to a dramatic increase in
potency. To further explore the SAR of the C-4 alkyl-
amine group and the effect of substituents on the A-ring,
a set of amino-containing ABIQs was prepared. Table 1
highlights the inhibitory properties of representative
amine-containing alkyl ABIQs. The inhibition of
CHK-1 activity was dependent on the nature of amino-
The ability of the aminoalkyl ABIQ to inhibit CHK-1
activity was also sensitive to the substitution on the
A-ring of the quinolinone. Thus, modification of the
substitutions on the A-ring provided a series of potent
ABIQ-containing CHK-1 inhibitors (IC50 < 1 nM).
X-ray crystal structure shows a hydrophobic space near
C-6. Thus, substitutions at position C-6 are well tolerat-
ed. Medium size groups, such as methyl and chloro,
enhanced the activity more than 10-fold (compounds
6–9 and 11–13). Multiple substitutions on the A-ring
diminished the affinity (compound 14), while any group
at position C-8 (methyl in compound 15) was detrimen-
tal to the activity.

Z.-J. Ni et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3121–3124
3123
Table 1. IC50 of CHK-1 inhibitors
R2
NH
N
O
N
R1
H
N
H
Compound
R1
H
R2
IC50 (nM)
7.6
N
N
N
4
5
6
7
8
9
H
482
0.35
1.4
6-Me
6-Me
6-Me
6-Cl
H
N
HN
3.3
Figure 1. X-ray co-crystal structure of ABIQ 11 with CHK-1.
H
N
0.50
120
100
80
60
40
20
0
H
N
10
11
12
13
14
15
6-Cl
7.2
N
6-Cl
0.32
1.04
3.3
H
N
6-Cl
HN
6-Cl
N
N
1
2
)
3
4
5,6-di-Cl
8-Me
21.7
138
)
)
O
M
S
M
M
n
n
n
M)
M
3
.
n
5
3
.
D
2
(
1
3
.
1
3
3
(
1
(
T
P
3
(
T
1
P
1
C
1
1
C
+
Figure 2. Synergestic effect of compound 11 with CPT.
An ideal CHK-1 inhibitor would be minimally cytotox-
ic, while enhancing the anti-tumor effect of a real cyto-
toxic agent that would be used in combination with
the inhibitor. Our CHK-1 inhibitors have shown excel-
lent selectivity over a panel of kinases including those
regulating the cell cycle, but these compounds have also
shown cytotoxicity to tumor cell lines. Compound 11
had an IC50 of 0.32 nM for CHK-1 with an EC50 of
80 nM (breast cell line MDA-MB-435), while its selectiv-
ity ratio for cdk2/cyclin A, cdk4/cyclin D, and cdc2/
cyclin B was over 500-, 5000-, and 1500-fold, respective-
ly.21 More importantly, this class of compounds was
synergistic with known cytotoxic agents. When
MDA-MB-435 cells were treated with compound 11
and camptothecin (CPT), an inhibitor of topoisomerase
1, compound 11 significantly accelerated cell death
(Fig. 2).22,23 This represents a new example of small
molecule CHK-1 inhibitors having synergistic activity
with traditional cytotoxic agents.
In summary, we have identified a novel class of small
molecule CHK-1 inhibitors. The current synthetic
approach provided us with easy access to structurally
diverse
4-(aminoalkylamino)-3-benzimidazole-quino-
linones. These inhibitors were potent, selective, and able
to enhance anti-tumor effect in conjunction with cyto-
toxic agents. Their PK properties in rodents and in vivo
efficacy data will be reported in due course.
References and notes
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2002, 37, 115.

3124
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2. (a) Zhou, B.-B. S.; Sausville, E. A. Prog. Cell Cycle Res.
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for 4 h, saturated aqueous sodium bicarbonate was
added. The aqueous layer was extracted with DCM
twice. The combined organic layers were washed with
1 M citric acid, 1 M sodium bicarbonate, water, and
brine, dried over sodium sulfate, and concentrated to
give 1-[(4-methoxyphenyl)methyl]-2-oxo-3-{1-[(trifluoro-
methyl)-sulfonyl]benzimidazol-2-yl}-6-chloro-4-hydroquin-
olyl(trifluoromethyl)sulfonate, which was used for the
next reaction without further purification. A solution of
the bis-triflate prepared above, (S)-3-aminoquinuclidine
6. Koniaras, K.; Cuddihy, A. R.; Christopoulos, H.; Hogg,
A.; O’Connell, M. J. Oncogene 2001, 20, 7453.
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H. J. Antibiot. 1987, 40, 1782; (b) Graves, P. R.; Yu, L.;
Swartz, J. K.; Gales, J.; Sausville, E. A.; O’Connor, P. M.;
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9. (a) Bunch, R. T.; Eastman, A. Clin. Cancer Res. 1996, 2,
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11. Agouron: Reich, S.H.; Belcman, T. M; Kephart, S. E.;
Romines, W. H.; Wallaces, M. B. WO0153268, 2001.
12. For CHK-1 inhibitor, ICP-1, see: Eastman, A.; Kohn, E.
A.; Brown, M. K.; Rathman, J.; Livingstone, M.; Blank,
D. H.; Gribble, G. W. Mol. Cancer Therap. 2002, 1, 1067.
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3118.
(1.2 equiv), and Hunig’s base (4 equiv) in acetonitrile
¨
was heated at 80 ꢁC for 20 h. After cooling down to
room temperature, the reaction mixture was diluted with
ethyl acetate, washed with saturated aqueous sodium
bicarbonate, water, and brine, dried over sodium sulfate,
and concentrated to give
a
a solid. The solid was
dissolved in mixture of trifluoroacetic acid and
concentrated HCl (7:1), and heated at 90 ꢁC for 20 h.
The reaction was diluted with water and ethyl acetate,
and slowly basified to pH 9 with saturated aqueous
sodium bicarbonate. The aqueous layer was extracted
with ethyl acetate. The combined organic layers were
washed with water and brine, and dried over sodium
sulfate, and concentrated. The product was purified with
preparative HPLC to give the desired product 11.
18. The CHK1 complex with compound 11 has been assigned
the PDB ID code: 2GDO.
19. Le, V. et al. manuscript in preparation.
20. Chiron: Barsanti, P.; Bussiere, D.; Harrison, S.; Heise, C.;
Jazan, E.; Machajewski, T.; McBride, C.; McCrea, W.;
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Ramurthy, S.; Renhowe, P.; Shafer, C.; Wagman, A.;
Wiesmann, M. WO0418419, 2004.
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Aardalen, K.;Embry, M.; Ma, S.; Moler, E.; Ni, Z.-J.;
Menezes, D.; Hibner, B.; Gesner, T. G.; Schwartz, G. K.
Cancer Res. manuscript submitted.
22. Cell Synergy Assay: MDA-MB-435 cells (5· 103)
were plated onto
a 96-well plate in 100 lL cell
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Capiris, T.; Kesten, S. J.; Herzig, D. J. J. Med. Chem.
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17. General procedure for the preparation of 4-[((3S)-
quinuclidin-3-yl)amino]-3-benzimidazol-2-yl-6-chloro-hydro-
quinolin-2- one (11): 3-(1H-benzimidazole-2yl)-4-hydrox-
yl-1-(4-methoxybenzyl)-6-chloro-1H- quinolinone (1 equiv)
was suspended in dichloromethane (DCM) in the
presence of pyridine (20 equiv). The mixture was
warmed to dissolve the solid. The mixture was cooled
to ꢀ5 ꢁC and triflic anhydride (8 equiv) in DCM was
added dropwise. After the reaction was stirred at ꢀ5 ꢁC
culture medium and incubated for 4 h at 37 ꢁC
under 5% CO2. DMSO (vehicle control), CPT alone,
compound 11 alone, and compound 11 + CPT, all in
100 lL cell culture medium, were added, resulting in
the final concentrations indicated. The cells were
incubated further for 48 h. MTS was added to the
cultures, permitted to develop for 4 h at 37 ꢁC under
5% CO2, then read on
a microplate reader at
490 nm. The ODs were corrected for background
absorbance by subtracting the values obtained for
wells containing tissue culture medium alone. Percent
viable cells were calculated by: {[A490 (DMSO-treated
cells) ꢀ A490
(test
sample)]/A490
(DMSO-treated
cells)} · 100%. Synergy was defined as the effect
observed by the combination of the two compounds
being greater than the sum of the effect of each
compound alone.
23. Detailed isobologram analysis of the synergistic effect will
be published elsewhere (see Ref. 21).