Optimization of a pyrazoloquinolinone class of Chk1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.07.051

The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduction of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from overlapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety in compound 52 (Chk1 IC₅₀ = 3.1 nM).

Full text of DOI:10.1016/j.bmcl.2007.07.051

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5989–5994
Optimization of a pyrazoloquinolinone class
of Chk1 kinase inhibitors
Edward J. Brnardic,a,* Robert M. Garbaccio,a Mark E. Fraley,a Edward S. Tasber,a
Justin T. Steen,a Kenneth L. Arrington,a Vadim Y. Dudkin,a George D. Hartman,a
Steven M. Stirdivant,b Bob A. Drakas,b Keith Rickert,b Eileen S. Walsh,b Kelly Hamilton,b
Carolyn A. Buser,b James Hardwick,b Weikang Tao,b Stephen C. Beck,b Xianzhi Mao,b
Robert B. Lobell,b Laura Sepp-Lorenzino,b Youwei Yan,c Mari Ikuta,c
Sanjeev K. Munshi,c Lawrence C. Kuoc and Constantine Kreatsoulasd
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 Molecular Systems, Merck Research Laboratories, West Point, PA 19486, USA
Received 17 April 2007; revised 3 July 2007; accepted 17 July 2007
Available online 19 August 2007
Abstract—The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduc-
tion of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from over-
lapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety
in compound 52 (Chk1 IC50 = 3.1 nM).
Ó 2007 Elsevier Ltd. All rights reserved.
Although effective, DNA damaging agents used in che-
motherapy are considered to be less than optimal in that
they show little selectivity for killing tumor cells over
normal proliferating cells. In response to DNA damage,
a normal cell activates checkpoints that delay cell cycle
transitions via the tumor suppressor protein p53 and
the checkpoint kinase Chk1, respectively. This delay
allows the cell to repair DNA before entering into mito-
sis.1–4 Many tumor cells, however, lack G1- and S-phase
checkpoints due to inactivation of the p53 tumor sup-
pressor gene (estimated 50–70% of all cancers), and rely
solely on Chk1 for arrest and repair. In these DNA
damaged tumor cells, Chk1 inhibition results in abroga-
tion of cell cycle arrest and allows premature mitotic
entry ultimately leading to cell death through mitotic
catastrophe and apoptotic pathways. Chk1 inhibitors
should therefore sensitize tumor cells deficient in p53
to DNA damaging agents.5,6 In this paper we report
efforts to maximize the potency of lead 1 (Fig. 1).
Our lead optimization efforts were initially guided by the
overlay of the X-ray crystal structures of Chk1 with
bound inhibitors 1 and 2 (Figs. 1 and 2).7 The X-ray
crystal structures of 1 and 2 indicated that both leads
Keywords: Chk1 kinase; Pyrazoloquinolinone; Oncology; Checkpoint
escape; DNA damage.
*
Figure 1. Leads 1 and 2.
Corresponding author. E-mail: edward_brnardic@merck.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.051

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E. J. Brnardic et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5989–5994
to provide 4-hydroxyquinolinones 6. Heating the 4-
hydroxyquinolinones 6 with hydrazine afforded the pyr-
azoloquinolinones 7 in good overall yield.
The initial set of compounds (Table 1) established the
optimal vector for filling this hydrophobic pocket.
Substitution at the 9-position was not tolerated as both
the 9-methyl (14) and 9-chloro (15) derivatives were
inactive.12,13 We rationalized that the substituent at
the 9-position collides with the hinge region of the
ATP pocket preventing the key hydrogen bonding
interactions with the peptide main chain. We were also
unable to improve activity with substitution at the
6- and 7-positions, as compounds 9 and 10 showed no
Table 1. Inhibitory activity of aryl modifications (8–17)
Figure 2. X-ray crystal structure with bound inhibitors 1 and 2.
were anchored to the adenine region8 of the Chk1 ATP
binding site through hydrogen bonds to the peptide
backbone at Glu85 and Cys87. Analysis and compari-
son of the bound structures for 1 and 2 indicated three
strategies for rapid optimization of potency in 1. The
first strategy aimed to fill an evident hydrophobic pocket
located between the aryl ring of 1 and the protein–sol-
vent interface. The second strategy focused on the intro-
duction of polar groups designed to drive potency
enhancement by interaction with the solvent front and
improve aqueous solubility. The final, and most provoc-
ative, strategy was suggested by the ionic interaction of
the tropane amine 2 with Glu91, an interaction not uti-
lized by 1. It was surmised that substitution on the 4-, 5-
or 6-positions of the pyrazoloquinolinone ring had the
potential to reach this key residue (Glu91).
Cmpd
R
Chk1
IC50 (nM)
Cell
EC50(nM)
8
H
1370
1490
1980
230
9
6-Me
7-CI
10
11
12
13
14
15
8-Me
8-CI
40
2980
8-NHCOCH3
9-Me
9-CI
7080
>100,000
28,860
16
17
29
710
To explore our first strategy9 we utilized anthranilic
acids10,11 to prepare our target molecules (Fig. 3).
Substituted anthranilic acids 3 were cyclized with ethyl
chloroacetate to provide benzoxazinones 4. These elec-
trophiles reacted with the sodium anion of ethyl aceto-
acetate to provide acrylates 5 which were cyclized and
decarboxylated upon treatment with sodium methoxide
130
1210
Figure 3. Synthesis of pyrazoloquinolinones from anthranilic acids.

E. J. Brnardic et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5989–5994
5991
significant change in potency. Substitution at the 8-posi-
tion was identified as the key position for improving po-
tency.14 The methyl derivative 11 offered a 6-fold
increase, while the chloro derivative 12 provided a great-
er than 30-fold increase in potency. As predicted, polar
groups such as the 8-amide (13) were less tolerated in the
hydrophobic pocket in that they displayed a significant
loss of potency. We were, however, pleased to find that
the 7,8-fused ring systems of 16 and 17 greatly enhanced
enzyme potency (up to 50-fold) and showed modest lev-
els of cell activity.15
ubility without detrimentally affecting enzyme affinity.
We prepared analogs with polar groups at the solvent
front utilizing the reaction scheme shown in Figure 4.
The nitrobenzene derivative 18 was Cbz-protected to
give 19 which was then reduced to aniline 20 with stan-
nous (II) chloride. Aniline 20 was iodinated with NIS to
provide a 1:2 mixture of iodo-isomers from which the
minor, desired ortho-iodoaniline 21 was isolated by
chromatography. Coupling reactions of iodoaniline 21
with protected pyrazole-4-carboxylic acids16 22 using
PyClu17 provided amide 23, which was alkylated with
ethyl bromide in the presence of cesium carbonate in
good yield. The tertiary amide 24 underwent Heck cycli-
zation18 at 90 °C to provide protected pyrazoloquinoli-
nones 25. Removal of the Cbz-protecting group under
hydrogenolysis conditions provided amine 26 which
could be elaborated with an electrophile to provide 27.
With the identification of potent cores via these modifi-
cations we turned our attention to our second strategy.
Molecular modeling suggested that substitution on the
fused cyclohexyl ring could project into solvent and sub-
stituents might be used to modify properties such as sol-
Figure 4. Synthesis of pyrazoloquinolinones from anilines.

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E. J. Brnardic et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5989–5994
Table 2. Inhibitory activity of compounds with polar groups at the
solvent front (29–31)
using cores 12, 16, and 17. The synthesis of our targets
employed a variety of reaction sequences. The alkylation
of pyrazoloquinolinones 3222 with alkylating agents and
cesium carbonate (Fig. 5) yielded a 3:1 mixture of N-
alkylated (33) versus O-alkylated (34) products which
were separated by silica gel chromatography. In order
to substitute the 4-position, the quinolinones were con-
verted to chloroquinolinones23 35 by heating with
PhPOCl2. Chlorides 35 were then reacted with amines
to afford aminoquinolinones 36. Compounds 38 were
derived from Suzuki coupling reactions with the tetrahy-
dronaphthyl aryl bromide24 37 and the appropriate
boronic acid (Fig. 6), followed by further derivitization
as required.
2
PSA(A )
˚
Cmpd
R
Chk1 Cell
IC50 (nM) EC50 (nM)
29
30
31
18
12
10
>10,000
>10,000
>10,000
103
115
123
Analysis of SAR (Table 3) indicated that substitution at
the 4- and 6-positions (39–44) did not provide improve-
ment in activity, indicating that the side chains were not
able to make productive interactions within the ATP
pocket. Gratifyingly, we found that substitution on the
5-position provided a significant increase in activity. A
three-carbon amine chain was found to be optimal
(46),25 as neither the two-carbon (45) nor the four-car-
bon (47) chain lengths provided equivalent potency
enhancement. Application of the propyl amine chain
to cores 16 and 17 gave compounds 48 and 49 which
showed excellent enzyme and cell-based potency.26 Since
49 displayed superior solubility properties to analog 48,
we focused on derivatives employing the tetrahydro-
naphthyl core. While 49 showed excellent potency and
The pyrazoloquinolinone was then deprotected using
trifluoroacetic acid to provide 28.
Our strategy to improve enzyme potency and aqueous
solubility through the introduction of polar groups
designed to reach the solvent front was successful.
Compounds 29–31 displayed enhanced aqueous
solubility and were found to be ꢀ10-fold more potent
than the comparison compound 17.19 However, these
compounds (29–31) were found to be inactive in our
cell-based assay, presumably due to poor cellular perme-
ability20 that may be a result of their high polarity
2
21
˚
(PSA > 100 A ) (Table 2).
Our third strategy focused on an attempt to reach the
key residue Glu91. Molecular modeling suggested the
possible interaction of Glu91 with substitution from
the 4-, 5-, or 6-positions. We explored this possibility
Figure 6. Synthesis of 6-substituted pyrazoloquinolinones.
Figure 5. Synthesis of 4- and 5-substituted pyrazoloquinolinones.

E. J. Brnardic et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5989–5994
Table 3. Inhibitory activity of compounds designed to access Glu91
5993
Cmpd
X
Structure
C
R
Chk1 IC50 (nM)
850
Cell EC50 (nM)
39
8-CI
40
41
42
43
44
45
46
47
48
49
50
51
52
8-CI
C
D
D
E
E
F
F
F
F
F
F
F
F
64
10,180
8-CI
60
8-CI
220
58
7, 8-Cy
7, 8-Cy
8-CI
290
36
980
160
1010
15
8-CI
1.2
20
8-CI
7, 8-Cy
7, 8-Cy
7, 8-Cy
7, 8-Cy
7, 8-Cy
0.66
1.1
14
94
1290
83
3.2
3.1
65
aqueous solubility, it demonstrated moderate binding to
the IKr potassium channel hERG (human Ether-a-go-go
Related Gene)27 with an IC50 = 5300 nM.28 We sought
to decrease the hERG activity by reducing the basicity
of the amine functionality29 of 49 (pKa = 10.2).30 Specif-
ically, we investigated modulating basicity through the
introduction of fluorine in the propyl chain (50–52).31
The difluoro derivative 50 (pKa = 7.6) suffered a reduc-
tion in potency perhaps as a result of a diminished ionic
interaction with Glu91. However, the mono fluoro
derivative 52 (pKa = 8.9) maintained good cell potency
(EC50 = 65 nM) and showed a modest improvement in
hERG off-target activity (IC50 = 13,200 nM).
In summary, we have described the use of X-ray crystal-
lography to rapidly optimize Chk1 potency (>1000-fold
improvement) from an HTS lead. Through the compar-
ison of two X-ray structures of HTS leads 1 and 2,

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B. D. et al. J. Med. Chem. 2006, 49, 4896; Prudhomme,
M. et al. Eur. J. Pharmacol. 2007, 554, 106.
potential hydrophobic and ionic interactions were iden-
tified. This effort culminated in the fusion of a cyclo-
hexyl ring and appendage of a propyl amine at N-5 to
produce inhibitor 52 with excellent Chk1 potency and
solubility properties.
15. Note that in the enzyme assay, the Chk1 inhibitory
activity is measured at Km for ATP (0.1 mM). However, in
the cell assay the ATP concentration is 2.0 mM resulting
in an inherent 10-fold shift in potency between these
assays.
16. Sridhar, R.; Sivaprasad, G.; Perumal, P. T. J. Heterocyclic
Chem. 2004, 41, 405.
Acknowledgment
17. N,N,N0,N0-Bis(tetramethylene)-chloroformamidinium
hexafluorophosphate.
We thank Carl Homnick for chiral separations of inter-
mediates to give 51 and 52.
18. We found it to be necessary to alkylate the amide in order
for the Heck cyclization to proceed.
19. The 1-ethyl group was found not to be potency enhancing
as shown in the comparison between compounds 1 and 8.
20. (a) Papageorgiou, C.; Camensich, G.; Borer, X. Bioorg.
Med. Chem. Lett. 2001, 11, 1549; (b) Huang, S. et al.
Bioorg. Med. Chem. Lett. 2006, 16, 5907.
21. PSA was calculated by the method of Clark, D. E. J.
Pharm. Sci. 1999, 88, 807.
22. Compounds 32 were prepared by treating 7 with (Boc)2O
and catalytic DMAP in DMF at 23 °C.
23. Daou, B.; Soufiaoui, M. Tetrahedron 1989, 45, 3351.
24. The tetrahydronaphthyl aryl bromide anthranilic acid
precursor was synthesized by the reduction of commer-
cially available naphthyl anthranilic acid with Raney
nickel, followed by bromination using Br2 in pyridine in
85% overall yield.
References and notes
1. Bartek, J.; Lukas, J. Cancer Cell 2003, 2, 421.
2. Zhou, B. S.; Bartek, J. Nat. Rev. Cancer 2004, 4, 1.
3. Tao, Z.; Lin, N. Anti-Cancer Agents Med. Chem. 2006, 6,
377.
4. Hapke, G.; Yin, M. B.; Rustum, Y. M. Cancer Metastasis
Rev. 2001, 20, 109.
5. Kawabe, T. Mol. Cancer Ther. 2004, 3, 513.
6. Prudhomme, M. Recent Patents Anti-Cancer Drug Discov.
2006, 1, 55.
7. PDB coordinates for 1: 2QHN, 2: 2QHM.
8. Traxler, P.; Furet, P. Pharmacol. Ther. 1999, 82, 195.
9. For full experimental detail on compounds described in
this paper, see WO2006074281.
25. Note that the Chk1 X-ray crystal structure with bound
˚
inhibitor 49 indicated a 2.80 A bond length between the
amine and side chain of Glu91.
10. Mitsos, C.; Zografos, A.; Igglessi-Markopoulou, O. Het-
erocycles 1999, 51, 1543.
11. Phadtare, S. K.; Kamat, S. K.; Panse, G. T. Indian J.
Chem., Sect. B 1983, 22, 496.
26. At this stage of our optimization efforts, we became aware
of independent efforts to optimize this series
(WO2005028474).
12. Chk1 inhibitory activity was measured using a homoge-
neous time-resolved fluorescence assay that detects phos-
phorylation of a biotinylated GSK-3 peptide as described
by Barnett, S. F. et al. Biochem. J. 2005, 385, 399, The
assay was run at 0.5 nM enzyme and 0.1 mM ATP. IC50
values are reported as averages of at least two independent
determinations; standard deviations are within +25–50%
of IC50 values..
27. Blockade of hERG has been implicated in the drug-
induced alteration of cardiac ventricular repolarization
that is typically manifested in prolongation of the heart
rate-corrected QT interval (QTc). Prolongation of QTc
interval may potentiate cardiac arrhythmia in patients and
lead to torsades de pointes or sudden death. See: DePonti,
F.; Poluzzi, E.; Cavalli, A.; Recanatini, M.; Montanaro,
N. Drug Safety 2002, 25, 263.
13. NCI-H1299 lung carcinoma cells were arrested with 16 h
treatment of camptothecin and then treated with Chk1
inhibitors for additional 8 h. Escape from cell cycle arrest
and progression into mitosis due to Chk1 inhibition were
measured by quantifying the mitosis-specific phosphory-
lation of nucleolin using an antibody-coated, bead-based
assay. For assay details, see: Fraley, M. E. et al. Bioorg.
Med. Chem. Lett. 2006, 16, 1775, Ref. 16. The values of
EC50 were measured with 10-point, half-log dilution series
and are reported as an average of tetraplicate
determinations.
28. The hERG IC50 values were determined by binding
competition experiments using membrane preparations
from human embryonic kidney cells that constitutively
express hERG.
29. Jamieson, C.; Moir, E. M.; Rankovic, Z.; Wishart, G. J.
Med. Chem. 2006, 49, 5029.
30. pKa was calculated using Marvin software, available from
ChemAxxon Ltd.
31. Bromides utilized in the synthesis of compounds 50–52
were generated from diethyl fluoromalonates via a multi-
step sequence; separation of enantiomers was carried out
using chiral normal phase chromatography and assign-
ment was based on an independent enantioselective
synthesis.
14. Potency enhancement via substitution at the 8-position of
the pyrazoloquinolinone was also observed at the corre-
sponding position of a series of indoles as noted in Palmer,