Development of 6-substituted indolylquinolinones as potent Chek1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.08.053

Through a comparison of X-ray co-crystallographic data for 1 and 2 in the Chek1 active site, it was hypothesized that the affinity of the indolylquinolinone series (2) for Chek1 kinase would be improved via C6 substitution into the hydrophobic region I (HI) pocket. An efficient route to 6-bromo-3-indolyl-quinolinone (9) was developed, and this series was rapidly optimized for potency by modification at C6. A general trend was observed among these low nanomolar Chek1 inhibitors that compounds with multiple basic amines, or elevated polar surface area (PSA) exhibited poor cell potency. Minimization of these parameters (basic amines, PSA) resulted in Chek1 inhibitors with improved cell potency, and preliminary pharmacokinetic data are presented for several of these compounds.

Full text of DOI:10.1016/j.bmcl.2006.08.053

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5907–5912
Development of 6-substituted indolylquinolinones
as potent Chek1 kinase inhibitors
Shaei Huang,a,* Robert M. Garbaccio,a Mark E. Fraley,a Justin Steen,a
Constantine Kreatsoulas,a George Hartman,a Steve Stirdivant,b Bob Drakas,b
Keith Rickert,b Eileen Walsh,b Kelly Hamilton,b Carolyn A. Buser,b James Hardwick,b
Xianzhi Mao,b Marc Abrams,b Steve Beck,b Weikang Tao,b Rob Lobell,b
Laura Sepp-Lorenzino,b Youwei Yan,c Mari Ikuta,c Joan Zugay Murphy,c
Vinod Sardana,c Sanjeev Munshi,c Lawrence Kuo,c Michael Reillyd and Elizabeth Mahand
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Structural Biology, Merck Research Laboratories, West Point, PA 19486, USA
dDepartment of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
Received 5 July 2006; revised 9 August 2006; accepted 9 August 2006
Available online 20 September 2006
Abstract—Through a comparison of X-ray co-crystallographic data for 1 and 2 in the Chek1 active site, it was hypothesized that the
affinity of the indolylquinolinone series (2) for Chek1 kinase would be improved via C6 substitution into the hydrophobic region I
(HI) pocket. An efficient route to 6-bromo-3-indolyl-quinolinone (9) was developed, and this series was rapidly optimized for poten-
cy by modification at C6. A general trend was observed among these low nanomolar Chek1 inhibitors that compounds with multiple
basic amines, or elevated polar surface area (PSA) exhibited poor cell potency. Minimization of these parameters (basic amines,
PSA) resulted in Chek1 inhibitors with improved cell potency, and preliminary pharmacokinetic data are presented for several
of these compounds.
Ó 2006 Elsevier Ltd. All rights reserved.
Despite their inherent toxicity, DNA damaging agents
continue to remain central in clinical cancer chemother-
apy. Therefore, strategies directed at improving their
therapeutic index are warranted. Following DNA dam-
age, normal cells arrest and attempt repair at the cell
cycle checkpoint G1, via the tumor suppressor protein
p53, and at G2 and S via the checkpoint kinase
Chek1.1–3 Tumor cells, however often are deficient in
the G1 checkpoint due to loss of p53 function (estimated
50–70% of all cancers) and thus, must rely on the check-
point kinase Chek1 to induce arrest at the S and G2
phases for survival. These p53-deficient cancers should
be more vulnerable to Chek1 inhibition which results
in abrogation of DNA-damage-induced arrest and
premature progression into mitosis resulting in mitotic
catastrophe and apoptosis. To summarize, abrogation
of the S and G2 checkpoints should sensitize cancer cells
to DNA damaging agents in p53-deficient backgrounds
without enhancing toxicity toward non-malignant cells.
As such, Chek1 inhibitors4 have the potential to widen
the therapeutic window for clinically utilized DNA dam-
aging agents in p53-deficient tumors.
Through comparison of the inhibitor-bound crystal
structures (Figs. 1 and 25) of ATP-competitive HTS leads
Keywords: Chek1; Kinase; Quinolinone; PSA; Anticancer; Chek1 in-
hibitor; p53; Chekpoint escape; Pharmacokinetics; Indolylquinolinone.
*
Corresponding author. Tel.: +1 2156526909; fax: +1 2156526345;
E-mail: shaei_huang@merck.com
Figure 1. Chek1 inhibitor leads 1 and 2.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.053

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S. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5907–5912
afford (E)-methyl 2-amino-5-bromocinnamate 4 in 95%
yield. Quinolinone formation was effected by intramo-
lecular cyclization of 4 in ethylene glycol at 200 °C to
yield 6-bromo-quinolinone 5.12 Treatment of 5 with
POCl3 gave chloroquinoline 6. Regioselective deproto-
nation13 was accomplished by LDA and the resulting
anion reacted with 1-chloro-2-iodoethane to provide
the 3-iodo-quinoline 7. Hydrolysis of 7 gave 6-bromo-
3-iodo-quinolinone 8. Suzuki coupling of 8 with
Boc-protected-indolyl boronic acid afforded indolyl-
quinolinone 9.14 Removal of the TBS group, followed
by oxidation with Dess-Martin Periodinane yielded
aldehyde 10. Reductive amination with various amines
in the presence of NaBH(OAc)3 afforded 11. Deprotec-
tion of the Boc group, followed by Suzuki coupling with
various boronic acids gave indolylquinolinones 13.
Unsubstituted indolylquinolinone
2 has moderate
Chek1 inhibitory activity15 (IC50 = 144 nM). Through
positional scanning on the quinolinone with a CN
group, compound 17 (Table 1) confirmed that C6 was
the optimal locus for potency enhancement (>30-fold
potency increase over 2). Furthermore, conversion of
the nitrile 17 to the amide 18 resulted in an additional
6-fold enhancement of potency. Despite the intrinsic
potency of 17 and 18, these Chek1 inhibitors exhibited
only weak activity in our cellular checkpoint escape
assay16 (CEA) which measures a compound’s ability to
release H1299 tumor cells from Camptothecin-induced
cell cycle arrest. It was suspected that the presence of
two basic amines at the C50 position of the indole com-
promised cell permeability.17 Evaluation of the piperi-
dine derivative 19 revealed not only that cell potency
could be dramatically improved18 by removal of one
of these basic amines, but that the primary amine in
17 was not contributing to intrinsic potency. With the
discovery of the cell potency-enhancing C50 piperidine,
a more detailed exploration of the quinolinone C6
SAR was conducted.
Figure 2. X-ray crystallographic structures of 1 (salmon) and 2 (gray)
bound to Chek1 (light blue) and their hydrogen bonds are shown in
dashed green and blue lines, respectively. Three conserved water
molecules found in H1 are shown as red spheres with their hydrogen
bonding network (dashed red lines).
16 and 2 with Chek1, it was hypothesized that placement
of substituents at C6 of the indolylquinolinone (2) would
best mimic the potency-enhancing C6-CN in 1. This
substitution was envisioned to fill a hydrophobic pocket
(HI),7 which is adjacent to the Chek1 ATP-binding site
that has been utilized by others.8,9 In this paper, we report
the synthesis, preliminary structure–activity relationship
and pharmacokinetic properties of 6-substituted indolyl-
quinolinones. Another report has appeared that shows an
alternative path for optimization of this series for Chek1
via C4 substitution.10
Indolylquinolinone analogs were prepared using the
general reaction sequence shown in Scheme 1.11 4-Bro-
mo-2-iodoaniline reacted regioselectively with methyl
acrylate in a microwave promoted Heck-coupling to
A set of 6-substituted-50-piperidinyl-indolyquinolinones
was prepared (Table 2). With a bromide handle, Suzuki
Scheme 1. Synthesis of 6-substituted indolylquinolinones.

S. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5907–5912
Table 1. Inhibition data for indolylquinolinone analogs
5909
Compound
Structure
Chek1a IC50 (nM)
Cell EC50 (nM)
14
1000
2
15
16
140
580
190
17
18
4.0
3700
3900
0.70
19
4.6
170
a Tested at 0.1 mM ATP.
coupling and copper-mediated Ullmann coupling reac-
tions19 were used to survey potential groups. Amides
and 5-membered ring heterocycles such as 21–28 provid-
ed Chek1 inhibitors with excellent potency against the
enzyme, although some compounds (21, 27) again
appeared to have compromised cell potency. Of particu-
lar interest was the pyrazole-containing inhibitor 22
which best balanced intrinsic and cell-based potency.
A few phenols were also incorporated8,9 (30 and 31),
yet these resulted in compounds with diminished
potency. The general observation was made that potent
inhibitors with high PSA (>100 A ) yielded poor
cell potency, as in 21 and 27, possibly due to impaired
cell permeability. PSA has been previously correlated
to cell permeability21 and other drug transport proper-
ties.22 Experimental logP values for this series do not
correlate well with the loss in cell potency and were
within a ‘drug-like’ range (i.e., logP for 22 = 2.81).
H-bond acceptors (e.g., 21, 22, 25, 26, and 28). Remov-
ing the outer H-bond acceptor, as in 23, 24 or 29, leads
to a decrease in potency.
Following the selection of pyrazole as a suitable C6 sub-
stituent, efforts returned to the C50 indole position in or-
der to further optimize potency and to begin exploring
pharmacokinetics. Earlier efforts in this series23 indicated
that this group was a primary site of oxidative metabo-
lism and that improved PK profiles could be obtained
through reducing its oxidative potential. Cell potency/
permeability was easily influenced by the character of
this substitution, and again the trend was noted that
high molecular PSA resulted in poor cell potency (i.e.,
34, 35). The strategy that emerged was to combine
potency enhancing groups at C6 and C50 that minimized
PSA.
2
20
˚
Finally, the pharmacokinetic profile of these com-
pounds was studied in dog (Table 3). Plasma clearance
for compounds 22, 32–35 was moderate with half-lives
ranging from 1.5 to 4.4 h. Clearance was improved
with 36 by capping the pyrazole with a methyl group
albeit with reduced potency. Considering cellular
potency as well as PK profile, compounds 22 and 32
emerged out as lead compounds in this indolylquinoli-
none series.
An analysis of the binding mode of 22 allowed us to
hypothesize as to the unexpected potency of polar
substituents oriented toward the hydrophobic region I.
Given that X-ray crystallographic structures cannot re-
solve hydrogen atoms, we postulate that the pyrazole
in 22 hydrogen bonds to water in hydrophobic region
I and helps to stabilize these moieties in the binding
pocket. As can be seen in Figure 3,5 the pyrazole group
of 22 can hydrogen bond to either of the front two water
molecules, one of the acidic oxygens of Asp148, and the
basic nitrogen of Lys38 (not shown) in addition to the
backbone interactions made by this class of inhibitors
(see Figure 2). Furthermore, an analysis of the limited
SAR presented in Table 2 suggests the importance of
correctly coordinating these water molecules with
In summary, a series of potent Chek1 inhibitors were
discovered based on comparison of the X-ray structure
of ATP-competitive HTS leads 1 and 2 bound to
Chek1. An efficient synthetic route has been developed
to facilitate the SAR studies. A general trend was
observed that compounds with multiple basic amines,

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S. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5907–5912
Table 2. Inhibition data for C6-substituted indolylquinolinone analogs
R1
Cell EC50 (nM)
460
a
Chek1 IC50 (nM)
2
PSA (A )
˚
Compound
20
2.9
54
21
22
0.34
0.65
520
97
102
89
23
5.6
4.3
250
320
83
75
2419
25
1.1
130
190
74
69
26
0.64
6.4
2719
1300
103
2819
2919
0.74
14
230
880
89
88
30
29
20
6300
79
89
31
>10,000
a Tested at 0.1 mM ATP.
Figure 3. X-ray crystallographic structure of 22 (yellow) bound to Chek1 (tan). Hydrogen bonds between the inhibitor and Chek1 are shown in
dashed lines (maroon). Three conserved water molecules found in H1 are shown as red spheres with their hydrogen bonding network (dashed red
lines). Image on left is a detail of the pyrazole hydrogen-bonding environment.

S. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5907–5912
Table 3. Pharmacokinetic profiles of indolylquinolinones
5911
Compoundc
R2
Cell EC50 (nM)
dog PK
CL (mL/min/kg)
a
Chek1 IC50 (nM)
2
PSA (A )
˚
t1/2 (h)
22
32
33
34
35
36b
0.65
1.0
97
120
89
89
30
22
34
27
23
4.4
2.5
2.0
1.5
2.0
4.1
0.83
2.5
170
102
111
123
98
870
2.1
1142
420
3.0
6.5
a Tested at 0.1 mM ATP.
b Pyrazole is N-methylated in 36.
c Compounds dosed at 0.25 mpk iv (DMSO) in cassette format.
1
11. All compounds were characterized by H NMR and high
resolution mass spectrometry. Experimental procedures
will be published in a submitted patent by these authors.
12. Kondo, Y.; Inamoto, K.; Sakamoto, T. J. Comb. Chem.
2000, 2, 232.
or high PSA, exhibited diminished cell potency.
Minimization of these parameters, resulted in Chek1
inhibitors with improved cell potency. Among these
potent Chek1 inhibitors, compounds 22 and 32 were
found to be leading compounds with moderate clear-
ance in dogs following intravenous dosing.
13. Marsais, F.; Godard, A.; Queguiner, G. J. Heterocyclic
Chem. 1989, 26, 1589.
14. Fraley, M. E.; Arrington, K. L.; Buser, C. A.; Ciecko, P.
A.; Coll, K. E.; Fernandes, C.; Hartman, G. D.; Hoffman,
W. F.; Lynch, J. J.; McFall, R. C.; Rickert, K.; Singh, R.;
Smith, S.; Thomas, K. A.; Wong, B. K. Bioorg. Med.
Chem. Lett. 2004, 14, 351.
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15. Chek1 inhibitory activity was measured using a homoge-
neous time-resolved fluorescence assay which measures
phosphorylation of a biotinylated GSK-3 peptide as
described in Barnett et al., Biochem J. 2005, 385, 399.
For the construct, a naturally occurring exon 10 splice
variant of human Chek1 described in patent application
US20050266469(A1), containing primarily the kinase
domain, was expressed in baculovirus with a C-terminal
6-histidine tag. This protein was purified on a Ni affinity
column and used as it is for kinetic assays, or purified
further on Heparin and SEC columns for crystallography.
The Chek1 concentration was 0.5 nM and ATP was used
at 0.1 mM. IC50 values are reported as the averages of at
least two independent determinations; standard deviations
are within 25–50% of IC50 values.
16. NCI-H1299 lung carcinoma cells were arrested with 16-h
treatment of camptothecin, and then treated with Chek1
inhibitors for additional 8 h. Checkpoint escaped mitotic
index due to Chek1 inhibition was assessed by measuring
the mitotic-specific phosphorylation of nucleolin in Chek1
inhibitor-treated cells using an antibody-coated, bead-
based assay. In this assay, total nucleolin is captured on a
streptavidin-coated paramagnetic bead coupled with bio-
tinylated nucleolin monoclonal antibody 4E2 (Research
Diagnostics, Inc.). Phosphorylated nucleolin is detected by
an antibody complex consisting of a phospho-specific
nucleolin monoclonal antibody TG3 (Applied NeuroSo-
lutions, Inc.) and a ruthenylated goat anti-mouse IgM
antibody labeled with ruthenylation kit (BioVeris Corp).
5. Compounds 1, 2 or 22 were diffused into pre-formed apo
Chek1 crystals. The X-ray diffraction data were collected
from these Chek1 inhibitor complex crystals to 1.8, 2.0,
˚
and 1.7 A resolution with Rsym = 0.067, 0.093, 0.063 and
completeness = 96%, 94%, 98%, respectively. The complex
structures were refined to an R-factor of 0.24, 0.23, and
0.23, respectively. The detailed X-ray diffraction data and
refinement statistics are listed under PDB code 2HXL,
2HXQ, and 2HY0 at the protein data bank.
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S. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5907–5912
The electrochemiluminescent complex is quantified with
19. For preparation of triazole, 28, 29 and tetrazole, 27, see:
(a) Wu, Y-J.; He, H.; L’Heureuk, A. Tetrahedron Lett.
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BioVeris M-8 Analyzer. The EC50 of checkpoint escape
mediated by Chek1 inhibition was determined with 10-
point series diluted Chek1 inhibitor-treated tetraplicate
cell samples.
17. Chek1 inhibitors in another report also showed dimin-
ished cell potency and the authors also suspect cell
permeability: Li, G.; Hasvold, L. A.; Tao, Z.-F.; Wang,
G. T.; Gwaltney, S. L., II; Patel, J.; Kovar, P.; Credo, R.
B.; Chen, Z.; Zhang, H.; Park, C.; Sham, H. L.; Sowin, T.;
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20. PSA calculations are done using the method pub-
lished by Clark. Clark, D. E. J. Pharm. Sci. 1999,
88, 807.
21. Papageorgiou, C.; Gamenish, G.; Borer, X. Bioorg. Med.
Chem. Lett. 2001, 11, 1549.
22. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43,
3714.
18. Note that Chek1 inhibitory activity is measured at Km for
ATP (0.1 mM). In the cell assay, the ATP concentration is
2.0 mM resulting in an inherent 10-fold potency shift
between these assays.
23. Fraley, M. E.; Hoffman, W. F.; Arrington, K. L.;
Hungate, R. W.; Hartman, G. D.; McFall, R. C.; Coll,
K. E.; Rickert, K.; Thomas, K. A.; McGaughey, G. B.
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