Identification of inhibitors of checkpoint kinase 1 through template screening

Article abstract of DOI:10.1021/jm900314j

Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment

Full text of DOI:10.1021/jm900314j

4810 J. Med. Chem. 2009, 52, 4810–4819
DOI: 10.1021/jm900314j
Identification of Inhibitors of Checkpoint Kinase 1 through Template Screening
Thomas P. Matthews,† Suki Klair,§ Samantha Burns,† Kathy Boxall,† Michael Cherry,§ Martin Fisher,§ Isaac M. Westwood,‡
Michael I. Walton,† Tatiana McHardy,† Kwai-Ming J. Cheung,† Rob Van Montfort,‡ David Williams,§ G. Wynne Aherne,†
Michelle D. Garrett,† John Reader,§ and Ian Collins*,†
†Cancer Research UK Centre for Cancer Therapeutics and ‡Sections of Cancer Therapeutics and Structural Biology, The Institute of Cancer
Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K., and §Sareum Ltd, 2 Iconix Park, Pampisford, Cambridge CB22 3EG, U.K.
Received March 12, 2009
Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1
abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to
genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit
generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization.
The sequential combination of in silico low molecular weight template selection, a high concentration
biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13
template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate
counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum
performance of the high concentration bioassay. One low molecular weight, weakly active purine
template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyr-
idines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon
cancer cells.
Introduction
culminating in cell cycle arrest in the S and G2/M phase.8,9
Activated CHK1 signals to several proteins to bring about
cell cycle arrest, in particular the CDC25 family of phospha-
tases. CDC25 phosphatases are essential to dephosphorylate
and activate the cyclin dependent kinase (CDK)/cyclin com-
plexes that drive cell cycle progression. CHK1-mediated-
phosphorylation of the CDC25 proteins has been shown to
block their ability to dephosphorylate CDKs, through inhi-
biting the direct interaction of CDC25 proteins with CDKs,
sequestration in the cytoplasm, or ubiquitin-mediated
degradation, thus leading to cell cycle arrest.10 Because cells
lacking intact p53-dependent G1 checkpoints are particularly
reliant on S and G2/M checkpoints, they are more sensitive to
chemotherapeutic treatment in the presence of a CHK1
inhibitor.7 Inhibition of CHK1 by siRNA5,11,12 or small
molecules7,13-20 has been shown to abrogate cell cycle arrest,
leading to premature mitotic entry and enhanced tumor cell
death following DNA damage by a range of chemotherapeu-
tics. In particular, a number of ATP-competitive inhibitors
of CHK1 have been shown to sensitize tumors to DNA-
damaging agents in preclinical animal models18-21 and
ATP-competitive CHK1 inhibitors are now in phase I clinical
trials.19-22
Several distinct chemotypes have been identified as CHK1
inhibitors, and these have been extensively reviewed14,21-23
(Chart 1). A collection of scaffolds have been developed based
on the indolocarbazole natural product kinase inhibitor
staurosporine and its analogue UCN-01.24 Most other pub-
lished CHK1 inhibitors originate from hits discovered by high
throughput screening (HTS) of large conventional compound
libraries.15-17 In some cases, HTS was combined with pre-
liminary in silico screening to reduce the size of the biochem-
ical screen and enhance the hit rate.25,26 The development of
The genomic integrity of replicating cells is maintained by a
network of sensors that monitor DNA and control ordered
progression through the cell cycle. In response to DNA
damage, this network serves to activate checkpoints at various
stages of the cell cycle that delay progression, allowing cells to
repair the damage or directing them to programmed cell death
if the damage is too severe.1,2 Many well established cancer
chemotherapies and radiotherapy exert their therapeutic ef-
fect through damage to DNA. The activation of cell cycle
checkpoints provides an opportunity for cancer cells, as well
as normal cells, to repair DNA damage and therefore reduce
the effect of, or become resistant to, DNA-damaging treat-
ments.3 The G1 checkpoint mediated by p53 is a major barrier
to genomic instability, but over half of all cancers are func-
tionally defective for the p53 pathway.4 These tumor cells are
more reliant on the S and G2/M checkpoints to repair
damage. Therefore drugs that abrogate the DNA damage-
induced S and G2/M checkpoints should selectively sensitize
p53 deficient cancer cells to genotoxic cancer therapies.3,5-7
Checkpoint kinase 1 (CHK1a) is a serine/threonine kinase
that is phosphorylated and activated by the upstream kinase
ataxia-telangiectasia mutated and rad3-related (ATR) in re-
sponse to DNA damage. ATR itself is activated by DNA
damage or replication stress that causes double- or
single-strand breaks in DNA, initiating a signaling cascade
*To whom correspondence should be addressed. Phone þ44 208
7224317. Fax: þ44 208 7224126. E-mail: ian.collins@icr.ac.uk.
a Abbreviations: AlphaScreen, amplified luminescent proximity
homogeneous assay screen; ATR, ataxia-telangiectasia mutated and
rad3-related; CDK, cyclin dependent kinase; CHK1, checkpoint kinase
1; HTS, high throughput screening; LE, ligand efficiency.
r
pubs.acs.org/jmc
Published on Web 07/02/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4811
Table 1. Properties Defining the Templates
Chart 1. Structures of Selected Published Clinical Candidate
CHK1 Inhibitors
property
template values
no. of heavy atoms
CLogP
8-24
-2 to 4
1-6
0-6
1-3
total H-bond donors and acceptors
rotatable bonds
no. of rings
small molecule CHK1 inhibitors from these sources has been
aided by the availability of the X-ray crystal structure of the
enzyme27 and protein-ligand complexes.15-17
Figure 1. CHK1 ATP-binding site pharmacophore used for the
virtual screen of commercially available templates.
Fragment-based screening offers an alternative approach
for small molecule hit generation.28 A potential benefit of
fragment screening is that chemical diversity space can be
probed effectively using a smaller number of molecules than is
possible with conventional HTS libraries. Low molecular
weight fragments may have weak binding affinities, outside
the range of conventional biochemical assays, necessitating
the use of biophysical screening techniques (NMR, X-ray
crystallography, or surface plasmon resonance) to detect
interactions with the target protein. Despite the low affinity
of the fragment hits, they are often bound with high ligand
efficiency (LE) and thus serve as good platforms for elabora-
tion to more potent ligands.29 Structural data on the binding
mode of the fragments is essential, however, if the weak initial
hits are to be progressed rapidly to tractable lead compounds.
We have used a template-based approach to identify new
inhibitors of CHK1, which balances the efficiency of a
biochemical assay for screening compound activity with the
desirabilityofstartingwithsmall, ligand-efficient structures to
maximize the probability of developing a “druglike” inhibitor
during optimization. The templates are therefore low molec-
ular weight structures, which are large enough to have detect-
able binding at high micromolar levels in a high concentration
biochemical assay yet small enough to benefit from the higher
coverage of diversity space offered by fragments.30 Hits from
the high concentration CHK1 kinase assay were validated by
crystallography of the bound complex to provide the neces-
sary structural information, enabling progression to more
potent inhibitors.
overlap between the property space proposed for fragment
libraries employed in biophysical screening and that proposed
for “leadlike” compounds in HTS.31 The upper limits to size,
lipophilicity, and functional group count allow for addition of
atoms during lead optimization while still remaining in “drug-
like” chemical space. Approximately 15000 templates were
identified from commercially available compound collections
using these library filters. To select appropriate templates for
biochemical screening, a pharmacophore search was per-
formed using the pharmacophore tools within MOE (Chemi-
cal Computing Group)32 to identify compounds with the
features of potential kinase inhibitors.
Template structures were matched to five conserved fea-
tures within the kinase ATP site in its active conformation, as
illustrated in Figure 1. The query was built from an overlay of
the structure of multiple published ligands in CHK1 using a
combination of the protein and ligand structures to define the
position of the pharmacophore points. A match to at least
three of the five points defined was required to warrant a hit.
Volume constraints were also applied to limit the scope of the
search, with the intent of reducing the number of false
positives identified in the pharmacophore search. To allow
for some leeway in movement of individual protein residues,
˚
exclusion volume spheres defined by radii of 2.0 A were placed
at each of the protein heavy atom positions surrounding the
ATP-binding site in the structure of CHK1 (PDB 1IA8).
These volumes were amalgamated into a single volume defini-
tion, and molecules were rejected if an atom from the phar-
macophore-matching pose fell within the excluded volume.
Hits from the search were clustered by structural type, focus-
ing on the core rings of each compound, and representatives
from each cluster were chosen by visual inspection. In total,
361 templates were selected for biochemical screening.
Results and Discussion
The structural features and calculated physicochemical
parameters used to select the template screening set in this
work are outlined in Table 1. In general, these cover the

4812 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Matthews et al.
The focused set of 361 compounds was evaluated in a 384-
well format Amplified Luminescent Proximity Homogeneous
Assay Screen (AlphaScreen)33 for inhibition of CHK1 kinase
activity, measured by the reduction in phosphorylation of a
CHK1 substrate peptide. The assay was adapted for screening
athigh ligand concentration (250μM), with each test plate run
under three different conditions. First, compounds were
tested six times in the standard AlphaScreen format to achieve
a high level of confidence. The screen was then run in triplicate
withthe inclusion ofa detergent(0.01% Triton X)toeliminate
potential false positives due to aggregation of poorly soluble
compounds at the high concentration.34 Finally, the plates
were counterscreened three times in the presence of phos-
phorylated substrate peptide to eliminate compounds inter-
fering with the AlphaScreen signal. False positives in
AlphaScreen assays have been observed due to compound
fluorescence or reaction with the singlet oxygen generated in
the screen.35
Chart 2. Chemical Structures of the Nine Template Hits
Validated by CHK1 Crystallographya
Two levels of hits were classified from this screening data.
The first set of compounds selected inhibited CHK1 activity
by more than 25% at 250 μM in at least 5/6 of the replicate
AlphaScreen assays and retained this activity in at least 2/3 of
the assays in the presence of detergent. Compounds were not
classified as hits if apparent inhibition was observed in
the presence of phosphorylated peptide. This identified nine
high-confidence hits showing inhibition of CHK1 between 35
and 79% at 250 μM. To maximize the potential output from
the screen, 13 further compounds were selected that inhibited
CHK1 by more than 25% at 250 μM in at least 3/6 of the
multiple assay runs while not giving inhibition in the assay
interference counter screen, but showed some reduction in
activity when assayed in the presence of detergent.
a Compounds 1 and 2 were lower confidence hits from the bioassay,
which showed some nonspecific inhibition, while compounds 3-9 were
high confidence hits that showed similar activity in the presence and
absence of Triton X-100 detergent in the bioassay.
The 22 template hits from the bioassay were validated using
X-ray crystallography by soaking into crystals of the apo-
CHK1 kinase domain. Hits were grouped by structural
classes, and where a group contained several compounds,
representative members were selected for crystallography.
Soaking experiments were therefore set up with eight of the
high confidence hits and seven of the lower confidence hits.
From these 15 chemically diverse structures, nine (1-9) were
confirmed to bind in the ATP-binding site of CHK1 by X-ray
crystallography (Chart 2, Table 2). The final hit-rate of the
screen was approximately 0.06% from the virtual template set
(2.5% from the 361 assayed compounds). The overall screen-
ing cascade is summarized in Figure 2. Importantly, the
validation rate by crystallography was much greater for the
high confidence hits than for the lower confidence compounds
that showed a reduction in activity when assayed in the
presence of detergent. Of the nine high confidence hits,
soaking experiments were carried out with eight distinct
chemotypes, of which seven were confirmed by crystallo-
graphic analysis (compounds 3-9). In contrast, of the 13
lower confidence hits, seven distinct chemotypes were selected
for X-ray crystallography but only two compounds, 1 and 2,
were confirmed as ATP-competitive binders to CHK1. This
supports the application of counter screens for potential
compound aggregation to increase the useful output of
low affinity, high concentration screens of this type. Although
the hit 2 was found to bind in the ATP-binding site of CHK1,
this compound failed to show CHK1 inhibition at high
concentration in a DELFIA-based kinase assay developed
to support medicinal chemistry progression of the compounds
(Table 2). Thus the significantly lower activity of 2 recorded
in the AlphaScreen in the presence of detergent is more
Table 2. CHK1 Inhibition by Crystallographically Validated Template
Hits 1-9 in High Concentration AlphaScreen and DELFIA Assays
AlphaScreen
þ0.01%
AlphaScreen %
inhibition
at 250
((SD) μMa
Triton-X100 DELFIA
(% inhibition at (% inhibition
crystallographically
validated hits
250 μM ( SD)b at 100 μM)c
1
2
3
4
5
6
26.4 ((6.9)
40.9 ((8.4)
41.9 ((2.5)
50.1 ((5.0)
51.4 ((1.2)
38.5 ((3.2)
6.5 ((3.2)
14.2 ((3.3)
41.5 ((1.7)
52.8 ((1.1)
43.1 ((3.0)
45.3 ((1.6)
nd
0%
ndd
nd
27%
IC50 = 52 μM
(46, 57)e
33%e
7
8
9
36.9 ((1.7)
35.7 ((1.3)
75.7 ((1.0)
39.9 ((4.5)
33.5 ((12.8)
63.0 ((0.7)
32%
IC50 = 42 μM
(28, 58)e
a Mean ((SD) for n=6 determinations. Positive control staurospor-
ine gave 73 ((9.9) % inhibition @ 25 nM. b Mean ((SD) for n=3
determinations. Positive control staurosporine gave 73 ((15) % inhibi-
tion @ 25 nM. c Single determination. Standard inhibitor staurosporine
gave IC50=2.1 ((1.8) nM. d nd=not determined. e Mean of 2 indepen-
dent determinations, individual values in parentheses.
representative of the targeted biological potency of the com-
pound. In contrast, the biological activities of the high con-
fidence hits 5-9 in the initial screens were all confirmed in the
second assay format.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4813
Figure 2. Summary of the template screening cascade and hit
validation.
All nine validated hits bound in the ATP pocket. However,
the core structures, binding modes and degree of hydrogen
bonding to the kinase hinge region, varied. Templates 1-4
showed only weak, single hydrogenbondcontacts to the hinge
region despite the presence of multiple hydrogen-bond donors
and acceptors in the molecules. In contrast, templates 5-9
were more firmly anchored in the binding site with bi- or
tridentate hydrogen bonding to the hinge region. These latter
hits were considered more promising as the presence of
stronger polar interactions to the starting template should
reduce the chance of encountering major changes to the
binding mode during the elaboration of the inhibitors.36 The
CHK1 crystal structures of five of these validated hits, 5-9,
are shown in Figure 3. A structural overlay of the hits shows
that they occupied similar space relative to the hinge region of
the kinase (Figure 3F). The thiazolo[5,4-c]azepin-4-one 5
adopted a bidentate hinge-binding pose, with the lactam
forming hydrogen bonds to the backbone amides of Glu85
and Cys87 in CHK1 (Figure 3A). The core of 5 occupied the
cleft of the ATP-binding site with the pendant acetamide
substitutent located close to Glu91 on the solvent-exposed
specificity surface. The tricycle 6 also adopted a bidentate
hinge-binding pose, with two hydrogen bonds from the
pyrimidinone functionality to Glu85 and Cys87 (Figure 3B).
The pyrimidinone was also hydrogen-bonded through a
neighboring water molecule to the side chain of Ser147. The
water molecule was situated at the mouth of a highly con-
served interior pocket of CHK1 defined principally by resi-
dues Val68, Asn59, and Glu55 in which a trio of water
molecules is typically observed.37 The remainder of the tri-
cycle was directed toward the solvent with the cyclohexane
located near the specificity surface formed by residues Gly89,
Gly90, and Glu91. Unique among this set of hits, the ami-
nothiazole 7 bound to the hinge region in a tridendate manner
with the primary amine hydrogen-bonding to Glu85, and the
thiazole nitrogen and tert-butyl substituted amine to the
amino and carbonyl functionality of Cys87, respectively
(Figure 3C). The phenyl ketone of 7 was pointed into the
ribose-binding pocket, while the tert-butylamino group
Figure 3. Crystal structures of hits 5 (A), 6 (B), 7 (C), 8 (D), and 9
(E) in the ATP binding pocket of CHK1. Panel F: Overlay of hits 5-
9 relative to the hinge-region of CHK1, orange = 5, gray = 6, red =
7, cyan = 8, green = 9.
emerged from the ATP-site near the specificity surface. The
cocrystal structure of 8 bound to CHK1 showed bidentate
hydrogen bonding between the imidazolone motif of the
ligand and the backbone amides of Glu85 and Cys87
(Figure 3D). The pendant thiophene was directed over the
specificity surface toward solvent, while the thiomethylether
pointed toward the water-filled pocket within CHK1. The
initial elaboration of the purine 9, which binds in the ATP site
of CHK1 (Figure 3E) and has an IC50 of 42 μM and a
calculated LE = 0.42 kcal mol-1 heavy atom-1, is discussed
below as an example of several weakly active but ligand
efficient template hits from this screen that were progressed
to more potent CHK1 inhibitors using structure-based drug
design.
The crystal structure of 4-(9H-purin-6-yl)morpholine 9
bound to CHK1 confirmed the expected hydrogen bonds
between N-3 and N-9 of the purine with the backbone amides
of Cys87 and Glu85, respectively (Figure 4A). This binding
mode has been reported for other purine-derived kinase
inhibitors.38,39 Importantly, this is not equivalent to the
binding of the purine moiety of ATP or ADP, where the
heterocycle is in a very different orientation, with hydrogen
bonding to the hinge region from N-1 of the purine and a 6-
NH2 substituent. The morpholine substituent of 9 did not
appear to make specific contacts with the protein backbone
but extended toward the ribose pocket. A limited number of
analogues containing small polar side chains at C-2 or C-3
directed toward the ribose pocket were synthesized, including
carboxylic acid, N-(tert-butyloxycarbonyloxy)amine, and
amine groups. From these, the racemic morpholine analogue

4814 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Matthews et al.
Table 3. Biological Activities and Ligand Efficiencies of Analogues of
the Template Hit 9
Figure 4. Details of the binding of 9 (A), 10 (B), 12 (C), and 14 (D)
to CHK1 kinase domain. Residues and side chains not involved in
binding are omitted for clarity. Water molecules detected in the
interior pocket of CHK1 or hydrogen bonded to the inhibitors are
shown as red spheres. Hydrogen bonds to the inhibitors are shown
as dashed green lines.
10 bearing a methylamino group at C-2 gave increased
potency and maintained LE relative to the hit (10, LE =
0.42 kcal mol-1 heavy atom-1) (Table 3). A crystal structure
confirmed the purine remained as the hinge binding compo-
nent, while the pendant primary amine formed a network of
polar interactions in the ribose pocket of CHK1 (Figure 4B).
The aminebounddirectly tothe backbone carbonyl of Glu134
and interacted with the side chains of Asn135 and Ser147
mediated by a water molecule. The amine was also close
˚
(ca. 3.4 A) to the terminal carboxylate of Glu91.
We sought to alter the purine heterocyclic core at an early
stage to explore novel inhibitors that retained the key nitro-
gens atoms to interact with the hinge region while providing
new substitution vectors to explore additional regions of the
binding site. Comparison of the crystal structures of the
purines 9 and 10 with the structures of the other template hits
5-8 indicated that the purines lacked a substitution vector
toward the kinase specificity surface (Figure 3F). This was
introduced through switching to the pyrazolopyridine scaf-
fold, resulting initially in the more potent ester 11. Although
pyrazolopyridines are well-known as kinase inhibitor scaf-
folds,46 the introduction of the 4-(2-aminomethylmorpholin-
4-yl) substituentled tonovel compounds.47 The reducedLE of
11 was recovered when the ester was replaced by a 5-bromo
substituent to give 12. The crystal structure of 12 showed a
changein theorientation and conformation ofthe morpholine
ring to deliver the pendant amine for direct interaction with
the carboxylate of Glu91 (Figure 4C). The morpholine
adopted a twisted conformation with respect to the plane of
the bicycle as a result of a steric clash with the 5-bromo
substituent. A possibleweakinteraction witha watermolecule
a Mean of 2 independent determinations in DELFIA format, indivi-
dual values in parentheses. Standard inhibitor staurosporine gave
IC50=2.1 ((1.8) nM. b Single determination.
The single enantiomers of 14 were synthesized and tested but
showed no appreciable enantiodiscrimination for binding to
CHK1, emphasizing the conformational flexibility of the
2-aminomethylmorpholine.
The pyrazolopyridines were profiled for selectivity and
cellular activity (Table 4). Some selectivity was seen for
inhibition of CHK1 over the unrelated checkpoint kinase
CHK2 (8-60-fold). The possible difference in therapeutic
outcome of dual CHK1 and CHK2 inhibition, as opposed
to selective CHK1 inhibition, remains to be determined.
siRNA studies suggest that the CHK1 inhibitor phenotype
dominates and is not necessarily enhanced by the simultaneous
˚
at the edge of the interior pocket was also detected (ca. 3.5 A).
Substitution at C-5 of the pyrazolopyridine by thiophene
(13) or phenyl (14) retained the submicromolar activity and
acceptable LE. The crystal structure of 14 showed the mor-
pholine ring adopting a near-identical conformation to that
observed in 12 but with the pendant phenyl adopting a torsion
ofca. 56°. Inrefining the structuresofracemic10, 12, and 14, it
was generally possible to obtain a reasonable fit for either
enantiomer of the morpholine side chains to the observed
electron density by changes to the conformation of the ring.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4815
Scheme 1a
Table 4. Selectivity and Cellular Data for Selected Compounds
abrogation
CDK1c % of G2 check
CHK1a
CHK2b inhibition
pointd
96 h SRBe
no. IC50 (μM) IC50 (μM) at 100 μM IC50 (μM)
GI50 (μM)
11
12
1.2
0.39
74
7.4
3
37
19 (17, 20) 64 (60, 68)
5 (3.9, 6)
16 (14, 18)
(0.33, 0.45)f
0.29
13
4.6
5.9
48
43
6 (7, 5)
>50
10.5 (10, 11)
40g
R-14 0.70
(0.52, 0.88)f
a Single determination in DELFIA format. Standard inhibitor staur-
osporine gave IC50=2.1 ((1.8) nM. b Single determination in DELFIA
format. Standard inhibitor staurosporine gave IC50 = 27 ((8) nM
c Single determination in DELFIA format. Standard inhibitor stauros-
porine gave IC50=18 ((4) nM d Mean of 2 independent determinations
in HT29 cells treated with etoposide to induce G2/M arrest, individual
values in parentheses. Standard inhibitor staurosporine gave IC50
=
0.0017 ((0.0004) μM. e Mean of 2 independent determinations in HT29
cells, individual values in parentheses. Assay standard etoposide gave
GI50 = 0.73 ((0.13) μM. f Mean of 2 independent determinations,
individual values in parentheses. g single determination.
a Reagents and conditions: (i) POCl3, 110°C; (ii) tert-butyl morpho-
lin-2-ylmethylcarbamate, Et3N, n-BuOH, 100°C (53% over 2 steps); (iii)
4M HCl-dioxane, rt (8%); (iv) POCl3, 60°C (79%); (v) tert-butyl
morpholin-2-ylmethylcarbamate, Et3N, n-BuOH, 100°C (49%); (vi)
ArB(OH)2, Pd(dppf)Cl2, Na2CO3, H2O, MeCN, 140°C, microwave;
(vii) CF3CO2H, 40°C (16-41% over 2 steps).
inhibition of CHK2.11 Compounds 11-14 showed little in-
hibition of the cell cycle kinase CDK1. This is important for
the interpretation of cellular checkpoint abrogation assays as
inhibition of CDK1 will lead to interruptions in the cell cycle,
independent of CHK1-mediated effects.40 The compounds
were assessed in p53-/- HT29 colon cancer cells for their
ability to abrogate the G2-M checkpoint arrest induced by
treatment with etoposide. HT29 and certain other p53-/-
colon cancer cell lines have been shown to have increased
sensitivity to CHK1 inhibitors in vitro and in vivo.41 Com-
pounds 11-13 abrogated the G2-M checkpoint in HT29 cells
at concentrations 10-20-fold above the CHK1 inhibitory
IC50 values. These compounds were also active in the growth
inhibition assay, with some 2-3-fold selectivity for the
CHK1-mediated cellular effect over general cytotoxicity.
The phenyl substituted analogue (R)-14 did not show similar
cellular activity, with no abrogation of the G2-M checkpoint
detectable. We speculate that this might reflect undetermined
off-target activities of the compound and underlines the
importance of measuring cellular activity as well as inhibition
of target biochemical activity in the progression of hits.
quickly generate submicromolar pyrazolopyridine inhibitors
with good ligand efficiency. The pyrazolopyridine core pos-
sesses substitution patterns that should allow access to the key
regions in CHK1 that are associated with highly specific
inhibition. Although the pyrazolopyridines represent early
stage leads, the compounds have appropriate CHK1-
mediated cellular activity and successfully abrogate the G2-
M checkpoint HT29 colon cancer cells. This demonstrates the
ability of the template screen to efficiently identify progres-
sible hit matter suitable for further optimisation.
Experimental Section
Synthetic Chemistry. (4-(9H-Purin-6-yl)morpholin-2-yl)-
methanamine (10) was prepared by direct nucleophilic aromatic
substitution of 6-chloropurine with commercially available
racemic tert-butyl morpholin-2-ylmethylcarbamate, followed
by acid deprotection. Compound 11 was prepared from ethyl
4-chloro-1H-pyrazolo[3,4-b]pyridine-5-carboxylate42 in a simi-
lar manner. The pyrazalopyridine bicyclic core was prepared
from paramethoxybenzyl protected pyrazolopyridone 15 as
previously described by Misra43 (Scheme 1). Chlorination of
15 using phosphorus oxychloride at 110 °C also removed the
paramethoxybenzyl group, and introduction of the morpholin-
2-ylmethylamine by SNAr displacement followed by deprotec-
tion yielded 12. An advanced intermediate 16 with the para-
methoxybenzyl protecting group still intact was formed by
lowering the temperature of the chlorination step to 60 °C,
followed by nucleophilic substitution as before. Suzuki cou-
plings to the protected 5-bromopyrazolopyridine 16, followed
by dual deprotection by stirring overnight at 40 °C in trifluoro-
acetic acid, gave the thienyl and phenyl substituted compounds
13 and 14.
tert-Butyl morpholin-2-ylmethylcarbamate was prepared as
either enantiomer starting from the appropriate 3-amino-1,
2-propanediol 17 as described by Brenner et al.44 (Scheme 2). For
example, the 2-hydroxymethylmorpholine 1844 was O-tosylated.
Displacement of the tosylate by azide, reduction, and protection
of the primary amine gave the bis-carbamate 19. Selective
deprotection of the secondary amine was achieved by exposure
to acid at low temperature (5 °C) to give 20, from which the
Conclusions
We have shown that the template screening strategy de-
scribed above is an effective means to generate new inhibitors
of the checkpoint kinase CHK1. The combination of in silico
template design, a high concentration AlphaScreen biochem-
ical assay for primary hit detection, followed by confirmation
using protein-ligand X-ray crystallography provided multi-
ple validated template hits. This proved an attractive hit
generation approach for CHK1 and is likely to be more
widely applicable for other targets for which a robust high
concentration bioassay is available. Our data reinforces the
value of appropriate counter-screening to rule out nonspecific
aggregation by test compounds in the high concentration
bioassay. Although some poorly active templates can indeed
bevalid hits, andweak activityisexpected for moleculesin this
molecular weight range, a much better validated hit rate was
observed from those primary hits where no nonspecific in-
hibitory effect was detected. We have shown the rapid pro-
gression of one purine template hit using the initial structural
information to give a more potent inhibitor and the subse-
quent application of iterative structure-based design to

4816 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Matthews et al.
Scheme 2a
microwave reactor at 140 °C for 2 ꢀ 5 min. The solvent was
removed in vacuo, and the residue was purified by preparative
HPLC to afford tert-butyl(4-(9H-purin-6-yl)morpholin-2-yl)
methylcarbamate (0.035 g, 0.10 mmol, 46%). LC-MS m/z
1
357 (M þ Naþ), Rt =2.05 min. H NMR (MeOH-d4) δ 1.48
(9H, s), 3.02 (1H, t, J=12.0 Hz), 3.26 (2H, dt, J=6.0, 1.0 Hz),
3.30 (1H, m), 3.62 (1H, m), 3.70 (1H, dd, J=11.5, 2.5 Hz), 4.05
(1H, ddd, J=11.5, 3.5, 1.5 Hz), 5.28 (2H, m), 6.80 (1H, t, J = 5.5
Hz), 8.04 (1H, s), 8.25 (1H, s). 4 M HCl in dioxane (0.1 mL, 0.40
mmol) was added to a suspension of a portion of the carbamate
(0.020 g, 0.06 mmol) in CH2Cl2 (1 mL), and the mixture was
stirred at rt for 4.5 h. The suspension was then dissolved in
MeOH and loaded onto an MP-TsOH resin cartridge (500 mg).
The cartridge was washed with MeOH, and the product was
eluted with 2 M ammonia in MeOH. The solvent was removed in
vacuo to afford (4-(9H-purin-6-yl)morpholin-2-yl)methana-
mine (10) (0.013 g, 0.06 mmol, 93%). LC-MS m/z 235 (Mþ
Hþ), Rt = 0.68 min. Hi-Res LC-MS calcd for C10H15N6O,
235.13019, found 235.13017 (M þ Hþ). 1H NMR (MeOH-d4) δ
2.77-2.88 (2H, m), 3.05 (1H, dd, J=13.0, 10.5 Hz), 3.30 (1H,
m), 3.62 (1H, m), 3.73 (1H, ddd, J=11.5, 11.5, 2.5 Hz), 4.08 (1H,
ddd,=11.5, 3.5, 1.5 Hz), 5.25 (2H, m), 8.05 (1H, s), 8.25 (1H, s).
Ethyl 4-(2-(Aminomethyl)morpholino)-1H-pyrazolo[3,4-b]-
pyridine-5-carboxylate (11). A solution of tert-butyl morpho-
lin-2-ylmethylcarbamate (0.134 g, 0.62 mmol), ethyl 4-chloro-
1H-pyrazolo[3,4-b]pyridine-5-carboxylate,42 (0.142 g, 0.63
mmol) and Et3N (0.26 mL, 1.87 mmol) in n-BuOH (2.5 mL)
was heated at 120 °C in a microwave reactor for 60 min. The
mixture was cooled, and the yellow solid was collected by
filtration, redissolved in EtOAc (15 mL), and washed with brine
(10 mL) and water (10 mL). The organic layer was concentrated
to give ethyl 4-(2-((tert-butoxycarbonylamino)methyl)morpho-
lino)-1H-pyrazolo[3,4-b]pyridine-5-carboxylate as a yellow so-
lid (0.11 g, 0.27 mmol, 44%). LC-MS m/z 406 (M þ Hþ), Rt=
4.12 min. 1H NMR (500 MHz, DMSO-d6) δ 1.30 (3H, t, J=7.5
Hz), 1.40 (9H, s), 2.95-3.20 (4H, m), 3.54 (1H, d, J=13 Hz),
3.60-3.75 (3H, m), 3.94 (1H, d, J=11.5 Hz), 4.25 (2H, q, J=7.5
Hz), 7.00 (1H, s, broad), 8.24 (1H, s), 8.52 (1H, s). A portion of
the carbamate (0.028 g, 0.07 mmol) was dissolved in methanol (3
mL), and CF3CO2H (2 mL) was added. The solution was
refluxed for 16 h. The mixture was concentrated and the residue
was purified by ion exchange chromatography on SCX-II acidic
resin (2 g) eluting with MeOH then 2 M NH3-MeOH. The basic
fractions were combined and concentrated to give 11 as a yellow
oil (0.020 g, 0.065 mmol, 93%). LC-MS m/z 306 (M þ Hþ), Rt=
1.22 min. Hi-Res LC-MS calcd for C19H28N5O5 406.2085,
a Reagents and conditions: (i) TsCl, Et3N, DMAP, CH2Cl2, 0°C f rt
(85%); (ii) NaN3, DMF, 80°C; (iii) NH4þHCO2-, Pd/C, MeOH, rt; (iv)
Cbz-Cl, Et3N, CH2Cl2, rt (42% over 3 steps); (v) 4M HCl-dioxane,
0°C f 5°C (83%); (vi) 5-bromo-4-chloro-1-(4-methoxybenzyl)-1H-
pyrazolo[3,4-b]pyridine, Et3N, n-BuOH, 160°C, microwave (67%);
(vii) PhB(OH)2, Pd(dppf)Cl2, Na2CO3, H2O, MeCN, 140°C, microwave
(48%); (viii) CF3CO2H, 40°C (55%).
synthesis followed a similar path to that for the racemate. The
intermediate 20 was reacted with 5-bromo-4-chloro-1-(4-methoxy-
benzyl)-1H-pyrazolo[3,4-b]pyridine to give the coupled product
21. Suzuki coupling to 21 and double N-deprotection with
trifluoroacetic acid gave R-14. The S-enantiomer of 14 was
prepared in similar fashion.
General Synthetic Chemistry. Reactions were carried out
under N2. Organic solutions were dried over MgSO4 or Na2SO4.
Starting materials and solvents were purchased from commer-
cial suppliers and were used without further purification.
Microwave reactions were carried out using a Biotage Initiator
60 microwave reactor. Flash silica chromatography was per-
formed using Merck silica gel 60 (0.025-0.04 mm). Ion ex-
change chromatography was performed using Isolute Flash
SCX-II (acidic) or Flash NH2 (basic) resin cartridges. 1H
NMR spectra were recorded on a Bruker AMX500 instrument
using internal deuterium locks. Chemical shifts (δ) are reported
relative to TMS (δ = 0) and/or referenced to the solvent in
which they were measured. Combined HPLC-MS analyses
were recorded using a Waters Alliance 2795 separations module
and Waters/Micromass LCT mass detector with electrospray
ionization (þve or -ve ion mode as indicated) and with HPLC
performed using Supelco DISCOVERY C18, 50 mm ꢀ 4.6 mm
or 30 mm ꢀ 4.6 mm i.d. columns, at a temperature of 22 °C with
gradient elution of 10-90% MeOH/0.1% aqueous formic acid
at a flow rate of 1 mL/min and a run time of 6 min. Compounds
were detected at 254 nm using a Waters 2487 dual λ absorbance
detector. All tested compounds gave g95% purity as deter-
mined by this method. All purified synthetic intermediates gave
g95% purity as determined by this method except where
indicated in the text. High-resolution mass spectra were mea-
sured on an Agilent 6210 ToF HPLC-MS with a Phenomenex
Gemini 3 μm C18 (3 cm ꢀ 4.6 mm i.d.) column.
1
found 406.2097 (M þ Hþ). H NMR (500 MHz, CD3OD) δ
1.40 (3H, t, J=7.5 Hz), 2.75-2.90 (2H, m), 3.25 (1H, dd, J=10,
12.5 Hz), 3.45-3.55 (1H, m), 3.65 (1H, d, J=12.5 Hz), 3.75 (1H,
d, J=11.5 Hz), 3.84-3.90 (2H, m), 4.05 (1H, d, J=11 Hz), 4.30
(2H, q, J=7.5 Hz), 8.28 (1H, s), 8.65 (1H, s).
(4-(5-Bromo-1H-pyrazolo[3,4-b]pyridin-4-yl)morpholin-2-
yl)methanamine (12). A suspension of 5-bromo-1-(4-methoxy-
benzyl)-1H-pyrazolo[3,4-b]pyridin-4(7H)-one (15)43 (0.10 g,
0.30 mmol) in POCl3 (2.5 mL, 27 mmol) was heated at 110 °C
for 3 h. The solution was cooled, and volatile components were
removed by evaporation. The remaining syrup was poured into
ice-water and extracted with EtOAc (3ꢀ20 mL). The com-
bined organic layers were washed with brine, dried, and con-
centrated to give a crude mixture containing 5-bromo-
4-chloro-1H-pyrazolo[3,4-b]pyridine as the major component.
LC-MS m/z 232, 234 (M þ Hþ), Rt=4.43 min, purity=52%.
The crude material was added to a solution of tert-butyl
morpholin-2-ylmethylcarbamate (0.098 g, 0.45 mmol) and
Et3N (0.14 mL, 0.99 mmol) in n-BuOH (1.87 mL) and was
heated at 100 °C for 18 h. The mixture was cooled, concen-
trated, and purified by preparative TLC, eluting with 5%
MeOH-CH2Cl2 to give tert-butyl (4-(5-bromo-1H-pyrazolo
[3,4-b]pyridin-4-yl)morpholin-2-yl)methylcarbamate as a yel-
low powder (0.066 g, 53% over two steps). LC-MS m/z 412,
(4-(9H-Purin-6-yl)morpholin-2-yl)methanamine (10). A solu-
tion of tert-butyl morpholin-2-ylmethylcarbamate (0.115 g,
0.53 mmol), 6-chloro-9H-purine (0.035 g, 0.23 mmol), and
Et3N (0.058 mL, 0.42 mmol) in NMP (1.1 mL) was heated in a

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4817
414 (M þ Hþ), Rt=4.60 min. 1H NMR (500 MHz, CDCl3) δ
1.47 (9H, s), 3.20 (1H, t, J=11 Hz), 3.26-3.32 (1H, m), 3.37-
3.56 (1H, m), 3.88-3.42 (2H, m), 3.82-3.86 (3H, m), 4.07 (1H,
d, J = 11.5 Hz), 8.18 (1H, s), 8.48 (1H, s). A portion of the
carbamate (0.033 g, 0.08 mmol) was dissolved in dioxane (0.5
mL), and 4 M HCl in dioxane (0.5 mL, 2.0 mmol) was added.
The solution was stirred at rt for 18 h. The solution was
concentrated, and the residue was dissolved in MeOH and
purified by ion exchange chromatography on Isolute SCX II
acidic resin (2 g), eluting with MeOH and then 1 M NH3-
MeOH. Preparative TLC, eluting with 1:20:79 Et3N:MeOH:
CH2Cl2, gave 12 as an off-white amorphous powder (0.002 g,
0.0064 mmol, 8%). LC-MS m/z 312, 314 (M þ Hþ), Rt=1.63
min. Hi-Res LC-MS calcd for C11H15BrN5O 312.04545,
m/z 310 (M þ Hþ), Rt =0.67 min. Hi-Res LC-MS calcd for
1
C17H19N5O 310.16624, found 310.16629 (M þ Hþ). H NMR
(500 MHz, MeOH-d4) δ 2.89-2.95 (2H, m), 3.05 (1H, dd, J=
10.0, 12.0 Hz), 3.20-3.26 (1H, m), 3.47-3.49 (1H, m), 3.60-
3.65 (1H, m), 3.74-3.79 (1H, m), 3.86-3.89 (1H, m), 7.37-7.41
(1H, m), 7.51 (2H, t, J=7.5 Hz), 7.56 (2H, dd, J=1.5, 7.5 Hz),
8.14 (1H, s), 8.32 (1H, s).
Acknowledgment. We thank Dr. N. Proisy for initial syn-
thetic work in the preparation of 18 and Dr. A. Mirza and M.
Richards for assistance in the spectroscopic characterisation
of test compounds. This work was supported by Cancer
Research UK [CUK] grant numbers C309/A2187, C309/
A8274 and C309/A8365. We acknowledge NHS funding to
the NIHR Biomedical Research Centre.
1
found 312.04543 (M þ Hþ). H NMR (500 MHz, MeOH-d4)
δ 2.85-2.93 (2H, m), 3.20 (1H, dt, J=2.0, 10.0 Hz), 3.41-3.46
(1H, m), 3.81-3.86 (2H, m), 3.89-3.94 (2H, m), 4.10-4.13
(1H, m), 8.29 (1H, s), 8.40 (1H, s).
Supporting Information Available: Experimental procedures
for the preparation of compounds 19, 20, 21, (R)-14, and (S)-14.
Experimental procedures and CHK1 inhibitory data for the
preparation of additional analogues related to compounds 9 and
10. Methods for high concentration CHK1 AlphaScreen, DEL-
FIA enzyme inhibition assays for CHK1, CHK2, and CDK1,
G2 checkpoint abrogation assay, SRB cytotoxicity assay. Meth-
ods for crystal structure determination of CHK1-inhibitor
complexes, crystallographic information on the structures of
5-10, 12, 14. This material is available free of charge via the
Internet at http://pubs.acs.org.
(4-(5-(Thiophen-3-yl)-1H-pyrazolo[3,4-b]pyridin-4-yl)morpholin-
2-yl)methanamine (13). A suspension of 1543 (0.20 mg, 0.60 mmol)
in POCl3 (5 mL, 54 mmol) was heated at 60 °C for 18 h. The
mixture was cooled, and volatile components were removed by
evaporation. The remaining syrup was poured into ice-water and
extracted with EtOAc (3 ꢀ 20 mL). The combined extracts were
washed with brine, dried, and concentrated to give 5-bromo-4-
chloro-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-b]pyridine as an off-
white amorphous powder (0.166 g, 0.47 mmol, 79%). LC-MS m/z
352, 354 (M þ Hþ), Rt=5.70 min, purity=91%. 1H NMR (500
MHz, MeOH-d4) δ 3.70 (3H, s), 5.61 (2H, s), 6.86 (2H, d, J=8.5
Hz), 7.22 (2H, d, J=8.5 Hz), 8.29 (1H, s), 8.79 (1H, s). A portion of
the material (0.10 g, 0.28 mmol) was dissolved in warmed n-BuOH
(1.2 mL), and tert-butyl morpholin-2-ylmethylcarbamate (0.135 g,
0.62 mmol) and Et3N (0.18 mL, 1.31 mmol) were added. The
reaction mixture was heated in a microwave reactor at 160 °C for 3
h. The mixture was cooled and concentrated. Flash column
chromatography on silica, eluting with a gradient of EtOAc-
hexanes, gave 16 as a white amorphous powder (0.074 g, 49%).
LC-MS m/z 532, 534 (M þ Hþ), Rt=5.49 min. 1H NMR (500
MHz, MeOH-d4) δ 1.45 (9H, s), 3.11-3.15 (1H, m), 3.19-3.27
(2H, m), 3.36-3.41 (1H, m), 3.52-3.59 (1H, m), 3.73 (3H, s),
3.77-3.94 (3H, m), 4.03-4.05 (1H, m), 5.54 (2H, s), 6.82 (2H, d,
J=8.5 Hz), 7.22 (2H, d, J=8.5 Hz), 8.24 (1H, s), 8.42 (1H, s). A
portion of 16 (16 mg, 0.030 mmol) was dissolved in warmed MeCN
(0.56 mL) before 3-thienylboronic acid (8 mg, 0.060 mmol),
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