Structure-based design of potent and selective 2-(quinazolin-2-yl)phenol inhibitors of checkpoint kinase 2

Article abstract of DOI:10.1021/jm101150b

Structure-based design was applied to the optimization of a series of 2-(quinazolin-2-yl)phenols to generate potent and selective ATP-competitive inhibitors of the DNA damage response signaling enzyme checkpoint kinase 2 (CHK2). Structure-activity relationships for multiple substituent positions were optimized separately and in combination leading to the 2-(quinazolin-2-yl) phenol 46 (IC₅₀ 3 nM) with good selectivity for CHK2 against CHK1 and a wider panel of kinases and with promising in vitro ADMET properties. Off-target activity at hERG ion channels shown by the core scaffold was successfully reduced by the addition of peripheral polar substitution. In addition to showing mechanistic inhibition of CHK2 in HT29 human colon cancer cells, a concentration dependent radioprotective effect in mouse thymocytes was demonstrated for the potent inhibitor 46 (CCT241533).

Full text of DOI:10.1021/jm101150b

580 J. Med. Chem. 2011, 54, 580–590
DOI: 10.1021/jm101150b
Structure-Based Design of Potent and Selective 2-(Quinazolin-2-yl)phenol Inhibitors
of Checkpoint Kinase 2
John J. Caldwell,*,† Emma J. Welsh,† Cornelis Matijssen,† Victoria E. Anderson,† Laurent Antoni,† Kathy Boxall,†
Frederique Urban,† Angela Hayes,† Florence I. Raynaud,† Laurent J. M. Rigoreau,§ Tony Raynham,§ G. Wynne Aherne,†
Laurence H. Pearl,‡, Antony W. Oliver,‡, Michelle D. Garrett,† and Ian Collins†
†Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K.,
‡Section of Structural Biology, Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, U.K., and
§Cancer Research Technology Discovery Laboratories London, Wolfson Institute for Biomedical Research, The Cruciform Building,
Gower Street, London WC1E 6BT, U.K. Present address: Genome Damage and Stability Centre, Science Park Road,
University of Sussex, Falmer, Brighton, East Sussex, BN1 9RQ, U.K.
Received September 6, 2010
Structure-based design was applied to the optimization of a series of 2-(quinazolin-2-yl)phenols to
generate potent and selective ATP-competitive inhibitors of the DNA damage response signaling
enzyme checkpoint kinase 2 (CHK2). Structure-activity relationships for multiple substituent posi-
tions were optimized separately and in combination leading to the 2-(quinazolin-2-yl)phenol 46
(IC50 3 nM) with good selectivity for CHK2 against CHK1 and a wider panel of kinases and with
promising in vitro ADMET properties. Off-target activity at hERG ion channels shown by the core
scaffold was successfully reduced by the addition of peripheral polar substitution. In addition to
showing mechanistic inhibition of CHK2 in HT29 human colon cancer cells, a concentration dependent
radioprotective effect in mouse thymocytes was demonstrated for the potent inhibitor 46 (CCT241533).
Introduction
cancer treatment modalities has prompted exploration of
CHK2 inhibition within this context.18-23 Many tumors
bypass normal cell cycle responses to DNA damage as a result
of defective p53 tumor suppressor function.24 In normal cells,
p53 is activated by CHK2 and other kinases in response to
double-strand DNA damage resulting in G1 cell cycle arrest
and apoptosis.25 Therefore, it could be expected that subse-
quent to a DNA damaging event, survival of normal cells
could be enhanced through CHK2 inhibition. The validity of
this approach has been tested by exposure of chk2-/- trans-
genic mice to ionizing radiation, which showed an increased
resistance to apoptosis.9,26 A radioprotective effect of CHK2
inhibition has also been demonstrated in isolated mouse
thymocytes as well as human lymphocytes.18,20 A further
potential use of a CHK2 inhibitor within a clinical context
hasbeen suggestedby the observationthat depletion of CHK2
by siRNA increases cancer cell sensitivity to poly(ADP
ribose)polymerase (PARP) inhibition.45
Single-agent inhibition of CHK2 may also provide an anti-
tumor effect in cancer cells, and therefore a therapeutic
approach, where CHK2 is highly activated, suggesting an
essential role for survival.6,16,17 Pommier et al. recently de-
monstrated antiproliferative activity of the small molecule
CHK2 inhibitor PV1019(2, Figure 1) as well as CHK2 siRNA
in cancer cell lines with high intrinsic CHK2 expression.27
Compound 2 also demonstrated in vitro potentiation of
the DNA-damaging chemotherapeutics topotecan and camp-
tothecin in OVCAR-4 and OVCAR-5 human tumor cells.
However, the generality of potentiation of DNA-damaging
agents by CHK2 inhibitors remains controversial6,16,17 be-
cause other pharmacological inhibitors of CHK2, such as
VRX0466617 (1, Figure 1), have not shown such effects.20,22
DNA damage in cells by external agents such as ionizing
radiation or genotoxic chemotherapy leads to an accumula-
tion of defective genetic material, which in turn can lead to
tumorigenesis or apoptosis.1,2 The typical cell cycle response
to DNA damage involves activation of signal transduction
pathways resulting in cell cycle arrest, proceeding to apoptosis
or activation of repair mechanisms depending on the severity
of the damage.3,4 One key transducer of this DNA damage
response is checkpoint kinase 2 (CHK2a), a serine/threonine
kinase, which is activated in response to double-strand breaks
in DNA.5-9 Activation of the DNA damage sensor ataxia
telangiectasia mutated (ATM) kinase in the presence of double-
strand breaks results in phosphorylation of inactive CHK2 at
Thr68.10,11 Consequent homodimerization of CHK2 induces
trans-autophosphorylation of Thr383 and Thr387, giving the
fully activated enzyme, followed by cis-autophosphorylation
on Ser516.12-15 Activated CHK2 kinase has been found to be
involved in a number of cell cycle events, including cell cycle
arrest, apoptosis, DNA repair, and mitosis.3,5,16,17
The involvement of CHK2 in multiple cell cycle and DNA
repair processes has made evaluation of the kinase as a poten-
tial anticancer drug target difficult,6 however, several possible
therapeutic contexts for CHK2 inhibition have been sug-
gested. In particular, the continuing use of DNA-damaging
*To whom correspondence should be addressed. Phone: þ44 207 352
8133 ext 4705. Fax: þ44 208 722 4126. E-mail: john.caldwell@icr.ac.uk.
a Abbreviations: ATM, ataxia and telangiectasia mutated; CHK1,
checkpoint kinase 1; CHK2, checkpoint kinase 2; DELFIA, dissocia-
tion-enhanced lanthanide fluorescent immunoassay; hERG, human
ether-a-go-go related gene; PAMPA, parallel artificial membrane per-
meability assay.
r
pubs.acs.org/jmc
Published on Web 12/27/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 581
Table 1. Investigation of Cyclic 4-Amino Substitution and Phenolic
Substitution
Figure 1. Reported ATP competitive, selective CHK2 inhibitors
1-4.
A small group of ATP-competitive, selective CHK2 inhib-
itors have been reported in the literature thus far (Figure 1)
based around isothiazoles 1,20,21 guanylhydrazones 2,22,27 and
2-arylbenzimidazoles 3.18,19 We have recently reported the
identification of 2-aminopyridines, such as 4, as CHK2
inhibitors.23 The discovery of further CHK2 selective inhibi-
tors and characterization of their pharmacology is important
to elucidate the consequences and therapeutic utility of CHK2
inhibition in cancer. In particular, the separation of CHK2
activity from inhibition of the distinct DNA damage response
signaling enzyme CHK1 is desirable to understand the
phenotype of CHK2 inhibition with small molecules.6 During
kinome profiling of early hits from an unrelated kinase screening
program, compounds containing the 2-(quinazolin-2-yl)phenol
moiety were identified as potential CHK2 inhibitors.28-30 Here,
we report a structure-based approach to the optimization of
novel 2-(quinazolin-2-yl)phenols to potent, selective CHK2
inhibitors and demonstrate their activity in cancer and non-
tumorigenic cell lines.
a Single determination in DELFIA assay format unless otherwise
noted. Standard inhibitor staurosporine gave mean ((SEM) IC50 = 27
((2.6) nM, n = 10. b Mean of n = 2 determinations, individual values in
parentheses. c Mean ((SEM), n = 3.
Results and Discussion
Screening of a small library of 2-(quinazolin-2-yl)phenols
identified 5 (Table 1) as a starting point for the development
of potent, selective quinazoline CHK2 inhibitors. As initial
attempts to crystallize 5 bound to the CHK2 enzyme proved
fruitless, synthetic efforts were first directed toward varying
the amine substituent at the 4-position to gain potency and aid
crystallization attempts. To this end, a range of acyclic aliphatic
diamines were attached, however, no increase in CHK2
potency was found. The use of cyclic bis-amines connected
through an exocyclic nitrogen to the quinazoline core led
to greater success (Table 1). Specifically, (S)-pyrrolidine 10
gave a 10-fold improvement in potency relative to 5 and was
also found to be 30-fold more active than the (R)-enantiomer
11. Comparable potency to 10 was observed with the
(R)-piperidine 13, although for the six-membered ring, the
(R)-stereochemistry was favored over the (S)-enantiomer 12.
The racemic 3-aminoazepine 16 was similarly active, although
a slight drop in activity was noted when the alternative
4-aminoazepine was introduced in 17, indicating a preference
for a constrained two-atom spacer between the two nitrogens.
The secondary nature of both the ring and exocyclic nitrogens
was shown to be important by the fall in activity of the
N-methylated compounds 14 and 15 compared to the unsub-
stituted analogue 10.
Further development of structures based around 10 ap-
peared promising, given both the availability and synthetic
tractability of 3-aminopyrrolidine intermediates. Importantly,

582 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Caldwell et al.
unfavorable and the two-carbon distance between the two
nitrogens was optimal.
Concurrent with the studies on the amine group, substitu-
tion of the phenol group of 5 was investigated through the
synthesis of a sequence of molecules containing fluorine at all
positions around the ring (6-9, Table 1). Whereas fluoro-
substitution at the 30- and 60-positions resulted in lower CHK2
inhibition, the 40-fluoro substituent 7 was tolerated and the 50-
fluoro substituent 8 increased the activity by 2-fold. This
structure activity was also consistent with the binding mode
of the quinazoline shown for 10, where the 50-position on the
phenolic ring appeared to offer most opportunity for further
substitution. A fall in activity from substitution at the
30-position would be expected due to close proximity to the
wall of the binding pocket. Substitution at the 60-position may
be unfavorable due to repulsion between the fluorine and
quinazoline N-3, disrupting the planar conformation of the
core scaffold.
The (S)-3-aminopyrrolidine template of 10 was used as the
core group for optimization of the 50-substituent of the
phenol. Replacement of the 50-proton with fluorine gave a
3-fold improvement in potency with18, also observed with the
chloride19(Table 2). A counterscreen of 18 againstthe CHK1
enzyme showed 17-fold selectivity for CHK2 relative to
CHK1. As some 2-(quinazolin-2-yl)phenols have been re-
ported as ligands for voltage-gated ion channels,32 an early
indication of potential hERG activity within the series was
sought.31 Compound 18 inhibited the hERG ion channel tail
current by 90% at a concentration of 10 μM, which when
titrated equated to an IC50 of 3 μM. It therefore became
necessary to evaluate and to reduce this undesirable activity
during further optimization of the series.33
Introduction of a small alkyl group at the 50-position was
tolerated, as in the methyl and trifluoromethyl analogues 20
and 22, however, activity decreased with larger lipophilic
groups such as isopropyl (21). From a range of five-membered
heterocyclesadded to replacethe 50-alkylsubstituent, themost
significant improvementin activityrelativeto10wasseenwith
the addition of a 4- or 3-pyrazole substituent, 23 and 24,
reaching low nanomolar activity against the CHK2 enzyme in
the instance of 24. We have previously shown the potential for
pyrazole substituents to interact productively with the con-
served catalytic residues within other protein kinases.47 Inter-
estingly, improved relative selectivity against CHK1 was
observed for the pyrazol-3-yl isomer 24. A further benefit of
the pyrazole substituent was a reduction in hERG inhibition
to levels comparable to those of DMSO control for 23, while
24 showed a hERG IC50 of 16 μM (Table 2).
The crystal structure of 24 bound to CHK2 is shown in
Figure 2B (PDB 2XM9). An additional interaction between
the pyrazole and Lys249 was observed and is likely to account
for the increase in CHK2 potency when compared to 10,
although a similar improvement in potency was surprisingly
not seen for 4-pyrazole 23.
In relation to the selectivity observed for 24, the similarities
between CHK2 and CHK1 were demonstrated by a sequence
alignment (see Supporting Information) showing the highly
conserved nature of the ATP-binding pockets when compar-
ing the two enzymes. Surrounding the pocket, 10 out of 12
residues are identical, with the remaining two differing only by
single methyl substituents. In an attempt to rationalize the
improved selectivity of 24 for CHK2 over CHK1, the crystal
structure of 24 bound to CHK2 was overlaid with a reported
CHK1 structure (PDB 2C3I)34 using Pymol35 and assuming a
Figure 2. Molecular details for the binding of (A) 10 (PDB 2XM8),
(B) 24 (PDB 2XM9), (C) 46 (PDB 2XBJ) to CHK2 (gray, C-R
backbone “tube”). Nitrogen, oxygen, and fluorine atoms are col-
ored blue, red, and gray, respectively. Blue spheres represent water
molecules, and magenta spheres a magnesium ion. Residues of the
glycine-rich loop, when ordered, are indicated by the transparent
blue section of backbone “tube”. Ligands, in each case, are shown in
“stick” representation, with carbon atoms colored orange. Repre-
sentative electron density from omit maps are shown in each case by
the “chicken-wire” mesh, contoured at 1.4 (A) or 2.5 (B,C) σ. (D)
Pymol overlay of the crystal structure of 24 in CHK2 (gray) with
that of CHK1 (green; PDB 2C3I). Dashed lines represent hydrogen
bonds formed by the CHK2 side chains Lys240 and Glu273 to the
pyrazole. Parts (A-C) were produced using CCP4MG.44
we were able to obtain the crystal structure of 10 complexed
with CHK2 (Figure 2, PDB 2XM8). This showed that 10
bound within the ATP pocket of the enzyme, with an intramo-
lecular hydrogen bond present between the phenolic hydrogen
and the quinazoline N-1, forming a planar, pseudotetracyclic
core scaffold. The quinazoline was sandwiched between the
lipophilic side chains of Val234 and Leu354, with the side
chains of Ala247, Leu301, and Leu303 also contributing to a
hydrophobic surface surrounding the core. An intermolecular
hydrogen bond was formed between the phenol oxygen and the
amide NH of Met304 in the hinge region of the kinase, while the
6- and 7-positions of the quinazoline were directed toward the
solvent exposed part of the enzyme. The pendant 3-aminopyr-
rolidine group occupied the ribose pocket, with the protonated
pyrrolidine nitrogen forming a charge-assisted hydrogen bond
with the side chain carbonyl of Asn352. The exocyclic nitrogen
of 10 was close to the side chain of Glu308. In subsequent
structures solved, the equivalent exocyclic nitrogen in other
analogues was observed to form a charge-assisted hydrogen
bond to this residue (Figure 2B,C). These interactions are
consistent with the observed structure-activity (Table 1),
where N-methylation of either nitrogen was found to be

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 583
Table 2. Optimization of the Phenol Substituent
CHK2
IC50 ( μM)a
CHK1
IC50 ( μM)b
CHK2/1
selectivity
hERG
(inh@10 μM) (%)c
no.
R
10
18
19
20
21
22
23
24
H
0.16 (0.14, 0.18)
0.060 (0.07, 0.05)
0.070 (0.11, 0.03)
0.092 ((0.023)e
1.7f
0.201 ((0.078)e
0.065 (0.079, 0.050)
0.009 (0.006, 0.012)
4.5 (4.2, 4.7)
0.99 (1.2, 0.78)
nd
28
17
nd
nd
nd
7
ndd
90
nd
nd
nd
96
9
-F
-Cl
-Me
-iPr
-CF3
pyrazol-4-yl
pyrazol-3-yl
nd
nd
1.32 (1.30, 1.35)
1.91 (2.56, 1.25)
1.07 (1.1, 1.05)
29
119
23
a Two independent determinations in DELFIA assay format, individual values in parentheses. Standard inhibitor staurosporine gave mean ((SEM)
IC50 = 27 ((2.6) nM, n = 10. b Two independent determinations in DELFIA assay format, individual values in parentheses. Standard inhibitor
staurosporine gave mean ((SEM) IC50 = 2.1 ((0.41) nM, n = 26. c Mean of multiple determinations (n = 2-8) in a high-throughput cell-based
electrophysiology assay for inhibition of hERG tail current.31 DMSO 0.3% aqueous vehicle negative control gave 9-20% inhibition. One μM cisapride
positive control gave 89-99% inhibition. d nd = not determined e Mean ((SEM) for n = 3 determinations. f n = 1 determination.
comparable hinge-binding mode for the inhibitor (Figure 2D).
Closeoverlappingof the sequenceswithonlyminor changesin
the positioning of the Glu-Lys salt bridge was seen. However,
without the corresponding crystal structure of 24 in CHK1,
analysis of suchsubtle structuraldifferences, and whetherthey
are significant, is difficult. A comparison of the crystal
structures of the inhibitor debromohymenialdesine bound to
both CHK2 and CHK1 has been included in the Supporting
Information. Again, this shows similar differences to those
addition of peripheral polar groups has been identified as a
general strategy for reducing hERG activity,33 the ethyl ester
group in 31 was converted to both the methyl and (1,1-
dimethyl)methyl alcohols 32 and 33. In this instance, the more
substituted analogue 33 gave a significant improvement in
CHK2 activity, as well as retaining high CHK1 selectivity (ca.
120-fold) and maintaining low hERG inhibition.
From the binding of the quinazoline core in the CHK2
enzyme shown by X-ray crystallography (Figure 2), the 6- and
7- positions of the quinazoline presented further opportunity
for the addition of groups to enhance both potency and
physicochemical properties within the series. A number of
mono- and disubstituted quinazolines were therefore synthe-
sized (Table 4). Generally, it was found that a single substitu-
tion at the 6-position, such as methoxy 34, methoxyethoxy 36,
or alcohol 38 analogues, did not improve CHK2 potency. The
addition of the peripheral alcohol in 38 was, however, found
to reduce hERG activity. Mono substitution at the 7-position
was found to give increased CHK2 activity, giving a 2- to
4-fold improvement relative to nonsubstituted compounds.
However, substantial hERG inhibition remained, even with
the alcohol group present in 39. Combining substitution at
both the 6- and 7-positions was found to be beneficial to both
increase CHK2 activity and reduce hERG inhibition, as seen
for compounds 40 and 41. The additive effect on potency of
appropriate phenol substitution was demonstrated again by
the synthesis of the corresponding fluorophenol analogues 42,
43, and 44, giving compounds active against CHK2 in the
10-30 nM activity range with reduced hERG activity for 44
(hERG IC50 = 44 μM). It was also observed that substitution
of the quinazoline core at C-6 and/or C-7 had minimal effect
on the selectivity for CHK2 over CHK1, as might be antici-
pated from the orientation of these groups out into solvent.
The various regions of the scaffold had been independently
optimized for CHK2 potency, selectivity over CHK1, and
reduced hERG inhibition, and it now remained to synthesize
the key combinations (Table 5). Combining the 3-pyrazolyl
group of 24 and the substituted pyrrolidine of 33 gave the
nanomolar CHK2 inhibitor 49. Selectivity over CHK1 was
˚
shown in Figure 2D, with a 2.0 A elongation of the Lys-Glu salt
bridge observed. It remains to be seen if these small differences
do indeed account for the difference in selectivity of 24 for CHK2
over CHK1. However, the improvement in selectivity observed
for 24 does suggest that differences in this region of the CHK2
enzyme in comparison to CHK1 can be exploited for selectivity.
Having increased potency against the CHK2 enzyme
through optimization of the phenol substitution pattern, it
was anticipated that this could be combined with additional
modification of the pyrrolidine functionality of 10. From the
X-ray of 10, the 4- or 5-positions of the 3-aminopyrrolidine
appeared to offer possible vectors for further substitution.
Retaining the 3-(S)-aminopyrrolidine chirality optimal in 10,
the diasteroisomeric esters 25 and 26 were made (Table 3).
Both showed a reduction in activity, although 26 had im-
proved selectivity against CHK1. Using the methyl ester as a
synthetic handle, a range of simple amides were prepared,
such as dimethylamide 27, but no improvement in activity
relative to 10 was found. Replacement of the 5-ester by the
(1,1-dimethyl)methyl alcohol 28 was also unproductive
despite the presence of the potency-enhancing 5-fluorophenol
substitution. The simpler methyl alcohol 29 gave only a
marginal improvement in activity in comparison to 18. With
no improvements found in selectivity against CHK1 or sig-
nificant reduction of hERG inhibition, substitution at the
pyrrolidine 4-position was investigated instead. The ethyl
ester group in 31 was found to be tolerated and was preferred
to the enantiomer 30. Addition of this functionality at the
4-position reduced hERG activity and in the case of 31 gave
an improvement in selectivity against CHK1. Given that

584 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Table 3. Modifications to the Pyrrolidine Substituent
Caldwell et al.
a Mean of n = 2 determinations in DELFIA assay format, individual values in parentheses. Standard inhibitor staurosporine gave mean ((SEM)
IC50 = 27 ((2.6) nM, n = 10. b Mean of n = 2 determinations in DELFIA assay format, individual values in parentheses. Standard inhibitor
staurosporine gave mean ((SEM) IC50 = 2.1 ((0.41) nM, n = 26. c Mean of multiple determinations (n = 2-8) in a high-throughput cell-based
electrophysiology assay for inhibition of hERG tail current.31 DMSO 0.3% aqueous vehicle negative control gave 9-20% inhibition. One μM cisapride
positive control gave 89-99% inhibition. d Single determination. e nd = not determined. f Mean ((SEM) for n = 3 determinations.
also found to be additive, with near 600-fold selectivity and
low hERG activity (49, hERG IC50 = 31 μM). The isomeric
pyrazol-4-yl substituted analogue 48 also gave a potent
compound with minimal hERG activity, although with some-
what reduced selectivity for CHK2 over CHK1, mirroring the
structure-activityseen for23and 24. Potent CHK2 inhibitors
were also obtained when combining the (1,1-dimethyl)methyl
alcohol substituted pyrrolidines with the 6,7-disubstituted
quinazoline core. The 6,7-dimethoxyquinazoline 46 was the
most potent CHK2 inhibitor identified in the series, with
selectivity (63-fold) over CHK1 and low hERG inhibition
(46, hERG IC50 = 22 μM). The 6-methoxy, 7-methoxyethoxy
variant 45 gave similar CHK2 and hERG activities, however,
it also had lower selectivity over CHK1 (21-fold). The methy-
lene alcohol 47 was found to be a less potent CHK2 inhibitor
than 46, with lower selectivity.
The crystal structure of 46 bound to CHK2 was solved and
compared to earlier compounds in the series (Figure 2C).

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 585
Table 4. Addition of Solubilizing Groups at C-6 and C-7 of the Quinazoline
no.
R1
R6
-OMe
R7
CHK2 IC50 ( μM)a
CHK1 IC50 ( μM)b
ndd
CHK2/1 selectivity
hERG (inh@ 10 μM) (%)c
34
35
36
37
38
39
40
41
42
43
44
H
H
H
H
H
H
H
H
F
-H
0.20 (0.21, 0.19)
0.029 (0.039, 0.019)
0.28 (0.21, 0.35)
0.070 ((0.005)e
0.60 (0.23, 0.97)
0.078 ((0.026)e
0.028 (0.033, 0.024)
0.12 (0.042, 0.19)
0.012 (0.015, 0.010)
0.011 (0.005, 0.017)
0.030 (0.018, 0.043)
nd
30
18
12
43
14
31
3
nd
86
100
61
28
77
46
22
68
65
25
-H
-OMe
0.87 (0.65, 1.08)
5.05 (2.0, 8.1)
0.85 (0.90, 0.79)
26 ((11)e
1.1 (1.4, 0.87)
0.87 ((0.30)e
0.33 (0.32, 0.34)
0.20 ((0.03)e
0.14 (0.14, 0.13)
0.26 ((0.09)e
-O(CH2)2OMe
-H
-(CH2)3OH
-H
-OMe
-OMe
-OMe
-H
-H
-O(CH2)2OMe
-H
-(CH2)3OH
-OMe
-O(CH2)2OMe
-OMe
-O(CH2)2OMe
-O(CH2)2OMe
17
13
9
F
F
-OMe
a Mean of n = 2 determinations in DELFIA assay format, individual values in parentheses. Standard inhibitor staurosporine gave mean ((SEM)
IC50 = 27 ((2.6) nM, n = 10. b Mean of n = 2 determinations in DELFIA assay format, individual values in parentheses. Standard inhibitor
staurosporine gave mean ((SEM) IC50 = 2.1 ((0.41) nM, n = 26. c Mean of multiple determinations (n = 2-8) in a high-throughput cell-based
electrophysiology assay for inhibition of hERG tail current.31 DMSO 0.3% aqueous vehicle negative control gave 9-20% inhibition. One μM cisapride
positive control gave 89-99% inhibition. d nd = not determined. e Mean ((SEM) for n = 3 determinations.
Overall, the binding mode was found to be very highly
conserved relative to previous compounds, with all of the
key hydrogen bond interactions maintained. The potency
gained with 46 therefore appears to be due to the presence
of the two methoxy substituents occupying the solvent ex-
posed region of the enzyme, and contributions from the
isopropyl alcohol substituent, which may participate in a
second intramolecular hydrogen bond to the quinazoline
exocyclic NH in 46. Although no specific interactions between
the alcohol group and enzyme were observed, an increase in
order within the glycine-rich P-loop was inferred from the
more clearly defined electron density in the crystal structure
compared to the complexes of 10 and 24.
Evaluation of the wider kinase inhibitory profile of the two
most potent compounds, 46 and 49, was undertaken (see
Supporting Information).36 A screen against a total of 85
kinases representative of various branches of the human
kinome showed 46 to be the more selective, strongly inhibiting
(>90% inhibition @ 1 μM) only 3 of the 85 kinases tested,
highlighting PHK and MARK3 along with CHK2. In con-
trast, 49 was found tobeless selective, strongly inhibiting 19of
the 85 enzymes in the panel (>90% inhibition @ 1 μM). This
perceived difference in selectivity profile was further sup-
ported through calculation of Gini coefficients,37 taking into
account the total inhibitoryactivities of the compounds across
the panel, where 46 gave a value of 0.53, compared to 0.29 for
49. Interestingly, the selectivity for CHK2 over CHK1 is not
representative of a general level of kinase selectivity for this
series of compounds.
was observed from ca. 1 μM concentration of the inhibitor 46
(Figure 3). Activation of CHK2 in response to DNA damage
caused by etoposide was detected by phosphorylation of
CHK2 Thr68, regardless of the presence of the CHK2 in-
hibitor. However, inhibition of CHK2 itself is clearly con-
firmed by the reduction of the Ser516 autophosphorylation at
increasing concentrations of 46, such that the signal is com-
pletely abolished at 1 μM concentration of the compound.
Selected compounds were evaluated for effects in human
cancer cells and in vitro pharmacokinetic properties (Table 6).
In general, there was a correlation of the concentration at
which rescue of etoposide-induced CHK2 bandshift was
observed with the potency of the inhibitors in the CHK2
biochemical assay. An exception was 49, which was appar-
ently less effective in the CHK2 mechanistic assay than
anticipated from the in vitro potency. The passive diffusion38
of these 2-(quinazolin-2-yl)phenols across a membrane of 2%
phosphatidylcholine in dodecane was generally assessed as
high. Cytotoxicityin HT29cellswassimilarforallcompounds
and did not appear to correlate strongly with inhibition of the
etoposide-induced CHK2 bandshift. It is therefore likely that
a component of the cytotoxicity is related to off-target effects
other than CHK2 inhibition. Some support for this hypoth-
esis is given by the higher cytotoxicity of 49, despite the
apparently weaker inhibitory effects on CHK2 in cells. Inter-
estingly, 49 was significantly less selective than the analogue
46 in the wider kinase selectivity profiling. All the compounds
showed good intrinsic metabolic stability when incubated in
mouse liver microsomes, which included UDP-glucuronosyl-
transferase and therefore informed on aspects of both primary
and secondary metabolism.39 The combination of moderate-
to-high passive permeability and low intrinsic metabolism
indicated promising in vitro pharmacokinetic properties for
the compounds.
A qualitative assay for inhibition of CHK2 in HT29 human
colon cancer cells was developed based on the ability of
the compounds to prevent an electrophoresis gel bandshift
of the CHK2 protein following phosphorylation induced by
the DNA damaging topoisomerase II inhibitor etoposide.
HT29 cells were treated with the CHK2 inhibitor for one
hour before addition of etoposide. Inhibition of the bandshift
On the basis of the potency and selectivity of 46 in in
vitro and cellular assays, this compound was chosen for

586 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Caldwell et al.
Table 5. Optimal Combinations of Quinazoline, Pyrrolidine, and Phenol Substitution
a Mean of n = 2 determinations in DELFIA assay format, individual values in parentheses. Standard inhibitor staurosporine gave mean ((SEM)
IC50 = 27 ((2.6) nM, n = 10. b Mean of n = 2 determinations in DELFIA assay format, individual values in parentheses. Standard inhibitor
staurosporine gave mean ((SEM) IC50 = 2.1 ((0.41) nM, n = 26. c Mean of multiple determinations (n = 2-8) in a high-throughput cell-based
electrophysiology assay for inhibition of hERG tail current.31 0.3% DMSO aqueous vehicle negative control gave 9-20% inhibition. One μM cisapride
positive control gave 89-99% inhibition. d nd = not determined. e Mean ((SEM) of n = 3 determinations.
further studies. Radioprotectant effects of CHK2 inhibitors
toward nontumorigenic murine and human cells have been
reported.9,18,20,26 Here, isolated mouse thymocytes were in-
cubated with varying concentrations of 46 for an hour before
being exposed to ionizing radiation. A protective effect of 46
was indeed observed, in a concentration dependent manner
(Figure 4), with complete reduction of radiation-induced
apoptosis observed in the presence of 20 μM concentration
of 46.
Figure 3. Effect of 46 on CHK2 bandshift and biomarkers for
CHK2 activation (CHK2-pT68) and autophosphorylation (CHK-
pS516) in HT29 cells. Cells were treated with compound for 1 h
followed by 50 μM etoposide for 5 h.
Conclusions
The development of a series of 2-(quinazolin-2-yl)phenols
as ATP-competitive CHK2 inhibitors was achieved by stepwise

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 587
Table 6. Cellular Activities and in Vitro ADME Properties of Selected Inhibitors
10
18
0.060
10
43
0.011
24
0.009
49
46
0.003
CHK2 IC50 ( μM)a
0.16
n.d.c
1.5
0.008
10
0.3
CHK2 bandshift MEC ( μM)b
5
1.3
1
0.5
1
1.4
cytotoxicity in HT29 cells GI50 ( μM)d
maximum passive permeability (Pe) (ꢀ 10-6 cm/s)e
mouse microsomal turnover (% metabolized at 30 min) f
1.8
56 (pH 5)
25
n.d.
27
29 (pH 7.4)
25
37 (pH 6.5)
27
8 (pH 5)
22
11 (pH 5)
23
a See Tables 1-5. b Minimum effective concentration (MEC) for complete inhibition of the etoposide-induced CHK2 bandshift, see footnote to
Figure 3. c nd = not determined. d Single determination using SRB colorimetric assay.40 e Measured at three pH (5, 6.5 and 7.4) in PAMPA.38 The pH
corresponding to maximum permeability is in parentheses. f Percent decrease in parent compound as measured by liquid chromatography-mass
spectrometry.39
confirms the radioprotectant effects of pharmacological in-
hibition of CHK2 in normal tissue.18,20,22 These 2-(quina-
zolin-2-yl)phenol CHK2 inhibitors are potent and selective
chemical tool compounds suitable for use in probing the
consequences of CHK2 inhibition in cancer cells.41 In partic-
ular, the antiproliferative effect of a combination of the
selective small molecule CHK2 inhibitor 46 (CCT241533)
with PARP inhibitors will be reported elsewhere.46
Experimental Section
Synthetic Chemistry. Synthesis of the requisite quinazolines
was achieved by the two general methods outlined in Schemes 1
and 2. The first method used was the reaction of anthraniloni-
Figure 4. Mouse thymocytes were preincubated for 1 h with in-
dicated doses of 46 before exposure to 5Gy IR. At 16 h post-IR,
apoptosis was assessed by Annexin V-FITC FACS. Results shown
are mean ((SD) for n = 3 determinations. Statistics: * P < 0.05 and
** P < 0.01 significantly different from no drug control.
triles with 2-methoxybenzoyl chlorides to give the correspond-
ing amides, which were cyclized under basic conditions to give
quinazolines of general structure 5028 (Scheme 1). Deprotection
of the anisole and displacement of the 4-chloro substituent with
an appropriate amine provided the desired targets 51. This route
is exemplified in Scheme 1 by the synthesis of 49, where sub-
sequent modification of the phenol group to install a pyrazole by
a Suzuki-Miyaura reaction was also carried out.
optimization of multiple substitutions around the core struc-
ture, strongly guided by close analysis of crystal structures of
representative compounds bound within the ATP pocket of
the CHK2 enzyme. Pleasingly, by combining the separately
optimized components around the core quinazoline structure,
a generally additive effect with respect to CHK2 potency was
observed, resulting in low nanomolar enzyme inhibitors. An
early awareness of possible hERG liabilities associated with
the 2-(quinazolin-2-yl)phenol scaffold allowed only structural
features tending toward reducing hERG activity to be pro-
gressed. The addition of polar functionality around the
periphery of the core was effective in reducing this off-target
activity without detriment to the kinase inhibitory potency.
Interestingly, hERG activity was only sensitive to polar
substitution at specific positions rather than a reduction being
associated with generally increasing polar surface area. Great-
er than 500-fold selectivity against the related DNA damage
response signaling enzyme CHK1 was achieved with pyrazo-
lyl substituents that appear to target differences between the
two kinases in the region of the conserved lysine and the
C-helix. However, high selectivity for CHK2 inhibition over
CHK1 did not predict for high specificity for CHK2 versus a
wider selection of protein kinases. Potent CHK2 inhibitors
from this series were shown to have favorable in vitro ADME
properties, in particular good passive membrane permeabil-
ity, and the expected inhibition of CHK2 was demonstrated in
a mechanistic cell-based assay. Although cytotoxicity toward
a human colon cancer cell line in vitro was observed, compar-
ison of the activity of compounds with varying CHK2 target
inhibition potencies in cells suggested an off-target compo-
nent to the antiproliferative effects. Finally, a concentration-
dependent radioprotective effect toward mouse thymocytes
by the optimized compound 46 was observed. This further
An alternative general method is shown in Scheme 2. Quina-
zoline-2,4-diones 55, either commercially available or synthe-
sized from the relevant 2-nitroesters,43 were activated by
reaction with phosphorus oxychloride. Selective displacement
by amines at the 4-position followed by Suzuki-Miyaura
coupling provided the desired compounds 57. The synthesis of
46 from 6,7-dimethoxyquinazolin-2,4-dione is shown as a spe-
cific example. The particular method used to synthesize the
quinazoline cores for all compounds and details of further struc-
tural elaborations are described in the Supporting Information.
General Experimental. Starting materials and solvents were
purchased from commercial suppliers and were used without
further purification. Microwave reactions were carried out in a
Biotage Initiator 60 microwave reactor. Organic solutions were
dried over MgSO4 or Na2SO4. Flash silica chromatography was
performed using Merck silica gel 60 (0.025-0.04 mm). Ion
exchange chromatography was performed using Isolute Flash
SCX-II (acidic) or Flash NH2 (basic) resin cartridges. Auto-
mated MPLC was performed on a Biotage SP1 Instrument using
prepacked silica cartridges and UV-triggered fraction collection
(254 nm). 1H NMR spectra were recorded on a Bruker AMX500
instrument using an internal deuterium lock. 13C NMR spectra
were recorded on a Bruker AMX500 instrument at 126 MHz
using an internal deuterium lock. Chemical shifts (δ) are refer-
enced to the solvent in which they were measured. GC-MS
analyses were performed on a Thermo Trace/Finnigan Polaris Q
system with a Zebron 15 m ꢀ 0.25 mm i.d. column with 2.5 μm
film thickness using helium as the carrier gas at a flow rate of
1.8 mL/min and measuring the sample mass following ioniza-
tion by EI or CI. Combined HPLC-MS analyses were recorded
using a Waters Alliance 2795 separations module and Waters/
Micromass LCT mass detector with electrospray ioniza-
tion (þve or -ve ion mode as indicated) and with HPLC
performed using Phenomenex Gemini C18, 50 mm ꢀ 4.6 mm

588 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Caldwell et al.
Scheme 1a
a Reagents and conditions: (i) S(O)Cl2; (ii) 54, Hunigs base, CHCl3; (iii) 20% w/v aq NaOH, H2O2; (iv) P(O)Cl3; (v) BBr3, CH2Cl2; (vi) 52,42 Et3N,
CH2Cl2; (vii) MeMgBr, THF; (viii) 53, Pd(PPh3)4, K3PO4, DMA, H2O; (ix) 4 M HCl in dioxane, MeOH.
Scheme 2a
a Reagents and conditions: (i) P(O)Cl3, reflux; (ii) 52,42 Et3N, CH2Cl2, reflux; (iii) 58, Pd(PPh3)4, Na2CO3, toluene, H2O; (iv) MeMgBr, THF; (v) 4 M
HCl in dioxane, MeOH.
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 3.5, 6, or 10 min as
indicated. Compounds were detected at 254 nm using a Waters
2487 dual absorbance detector. All tested compounds gave
>95% purity as determined by this method.
4-Fluoro-2-(4-((3S,4R)-4-(2-hydroxypropan-2-yl)pyrrolidin-
3-ylamino)-6,7-dimethoxyquinazolin-2-yl)phenol (46). A solution
of 6,7-dimethoxyquinazoline-2,4-diol (1.000 g, 4.500 mmol) in
P(O)Cl3 (20 mL) was refluxed for 14 h. The reaction mixture
was concentrated and dissolved in CH2Cl2 (200 mL). The
organic phase was washed with iced H2O (100 mL) and brine
(100 mL) and then concentrated to give crude 2,4-dichloro-6,
7-dimethoxyquinazoline. The crude material was dissolved in
CH2Cl2 (14 mL), (3R,4S)-1-tert-butyl 3-ethyl 4-aminopyrroli-
dine-1,3-dicarboxylate hydrochloride (0.900 g, 3.053 mmol),42
and Et3N (1.7 mL, 12.273 mmol) added and the resulting
solution refluxed for 4 days. The reaction was cooled, H2O
(100 mL) added, and the aqueous extracted with CH2Cl2 (2 ꢀ
100 mL). Organic layers were combined, dried over MgSO4, and
the crude product purified by silica column chromatography
(67% EtOAc in hexanes) to give (3R,4S)-1-tert-butyl 3-ethyl
4-(2-chloro-6,7-dimethoxyquinazolin-4-ylamino)pyrrolidine-1,3-di-
carboxylate (1.189 g, 81%). LC-MS (3.5 min) m/z 481, 483 [M þ
Hþ], Rt 2.69 min. 1H NMR (500 MHz, CDCl3) δ 7.17 (s, 1H), 6.91
(s, 1H), 5.90 (brd, J= 6.5 Hz, 1H), 5.06 (pent, J= 6.5 Hz, 1H),4.22

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 589
(q, J = 7.0 Hz, 1H), 4.13-4.06 (m, 1H), 4.01 (s, 3H), 3.99 (s, 3H),
3.87-3.69 (m, 2H), 3.51-3.31 (m, 1H), 3.25-3.18 (m, 1H), 1.50
(s, 9H), 1.25 (t, J = 7.0 Hz, 1H). (3R,4S)-1-tert-Butyl 3-ethyl
4-(2-chloro-6,7-dimethoxyquinazolin-4-ylamino)pyrrolidine-1,3-di-
carboxylate (0.962 g, 2.000 mmol), 5-fluoro-2-hydroxyphenylboro-
nic acid (0.344 g, 2.200 mmol), Na2CO3 (0.636 g, 6.000 mmol), and
Pd(PPh3)4 (0.114 g, 0.100 mmol) were dissolved in toluene (5 mL)
and H2O (2 mL) and N2 bubbled through the resulting biphasic
mixture for 30 min. The mixture was then heated at 100 °C for 24 h.
The mixture was cooled and H2O (100 mL) added. The aqueous
layer was extracted with EtOAc (2 ꢀ 100 mL). Organic extracts were
combined, dried over MgSO4, and concentrated. Purification by
silica column chromatography (67% EtOAc in hexanes) gave
(3R,4S)-1-tert-butyl 3-ethyl 4-(2-(5-fluoro-2-hydroxyphenyl)-
6,7-dimethoxyquinazolin-4-ylamino)pyrrolidine-1,3-dicarboxylate
(0.969 g, 87%). LC-MS (3.5 min) m/z 557 [M þ Hþ], Rt 2.97 min.
1H NMR (500 MHz, CDCl3) δ 8.20 (dd, J = 10.0, 3.0 Hz, 1H),
7.20-7.15 (m, 1H), 7.10-7.02 (m, 2H), 6.95 (dd, J = 9.0, 4.5 Hz,
1H), 6.34 (br d, J = 5.0 Hz, 1H), 5.17-5.12 (m, 1H), 4.32-4.26
(m, 2H), 4.03 (s, 3H), 4.02 (s, 3H), 3.94-3.32 (m, 5H), 1.52 (s, 9H),
1.28 (t, J = 7.0 Hz, 1H). MeMgBr (5.7 mL of a 3 M solution in
Et2O) was added to a solution of (3R,4S)-1-tert-butyl 3-ethyl
4-(2-(5-fluoro-2-hydroxyphenyl)-6,7-dimethoxyquinazolin-
4-ylamino)pyrrolidine-1,3-dicarboxylate (0.969 g, 1.723 mmol) in
THF (17 mL) at 0 °C. After 1 h at rt, satd aq NH4Cl (100 mL) was
carefully added and the aqueous layer extracted with EtOAc (2 ꢀ
75 mL). The organic layer was dried, concentrated, and the resulting
crude material resubjected to the above reaction conditions due to
presence of unreacted SM and intermediate methyl ketone. The
resulting crude was purified by silica column chromatography
(EtOAc) and the isolated Boc protected amine stirred in a solution
of MeOH (20 mL) and HCl (10 mL of a 4 M solution in dioxane) for
24 h. The reaction was concentrated, and the resulting crude product
purified by SCX-2 Isolute column, washing first with MeOH and
finally with 1 M NH3 in MeOH. Purification by Biotage column
chromatography (silica KP-NH, 5% MeOH in CH2Cl2) gave
4-fluoro-2-(4-((3S,4R)-4-(2-hydroxypropan-2-yl)pyrrolidin-3-
ylamino)-6,7-dimethoxyquinazolin-2-yl)phenol 46 (0.212 g, 28%).
LC-MS (3.5 min) m/z 355 [M þ Hþ], Rt 1.92 min. 1H NMR (500
MHz, MeOD) δ 8.13 (dd, J = 10.0, 3.0 Hz, 1H), 7.51 (s, 1H), 7.09
(s, 1H), 7.03 (ddd, J = 9.0, 8.0, 3.0 Hz, 1H), 6.86 (dd, J = 9.0, 4.5
Hz, 1H), 5.06-5.02 (m, 1H), 3.98 (s, 3H), 3.97 (s, 3H), 3.40-3.25
(m, 2H), 3.05-2.94 (m, 2H), 2.46 (dd, J = 14.0, 8.0 Hz, 1H), 1.32
(s, 3H), 1.30 (s, 3H). 13C NMR (126 MHz, DMSO) δ 158.1, 157.2,
156.7, 154.6 (d, J= 233 Hz), 154.5, 148.8, 143.4, 120.0 (d, J=8Hz),
118.6 (d, J= 24 Hz), 118.3 (d, J=8Hz), 113.6(d, J= 8 Hz), 107.0,
106.2, 102.7, 69.5, 56.3, 55.9, 55.6, 54.5, 53.6, 48.1, 28.6, 28.1 HRMS
calcd for C22H27N4O4F (M þ Hþ) 443.2089, found 443.2096.
CHK2 binding sites. This material is available free of charge via
the Internet at http://pubs.acs.org.
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