Discovery of checkpoint kinase inhibitor (S)-5-(3-fluorophenyl)-N- (piperidin-3-yl)-3-ureidothiophene-2-carboxamide (AZD7762) by structure-based design and optimization of thiophenecarboxamide ureas

Article abstract of DOI:10.1021/jm300025r

Checkpoint kinases CHK1 and CHK2 are activated in response to DNA damage that results in cell cycle arrest, allowing sufficient time for DNA repair. Agents that lead to abrogation of such checkpoints have potential to increase the efficacy of such compounds as chemo- and radiotherapies. Thiophenecarboxamide ureas (TCUs) were identified as inhibitors of CHK1 by high throughput screening. A structure-based approach is described using crystal structures of JNK1 and CHK1 in complex with 1 and 2 and of the CHK1-3b complex. The ribose binding pocket of CHK1 was targeted to generate inhibitors with excellent cellular potency and selectivity over CDK1and IKKβ, key features lacking from the initial compounds. Optimization of 3b resulted in the identification of a regioisomeric 3-TCU lead 12a. Optimization of 12a led to the discovery of the clinical candidate 4 (AZD7762), which strongly potentiates the efficacy of a variety of DNA-damaging agents in preclinical models.

Full text of DOI:10.1021/jm300025r

Article
pubs.acs.org/jmc
Discovery of Checkpoint Kinase Inhibitor (S)-5-(3-Fluorophenyl)-N-
(piperidin-3-yl)-3-ureidothiophene-2-carboxamide (AZD7762) by
Structure-Based Design and Optimization of Thiophenecarboxamide
Ureas
Vibha Oza,*,† Susan Ashwell,† Lynsie Almeida,† Patrick Brassil,† Jason Breed,‡ Chun Deng,†,×
Thomas Gero,† Michael Grondine,† Candice Horn,†,# Stephanos Ioannidis,† Dongfang Liu,†,○ Paul Lyne,†
Nicholas Newcombe,‡ Martin Pass,‡ Jon Read,‡ Shannon Ready,†,∞ Sian Rowsell,§ Mei Su,†
̂
Dorin Toader,† Melissa Vasbinder,† Dingwei Yu,† Yan Yu,† Yafeng Xue,∥ Sonya Zabludoff,†,●
and James Janetka†,⊥
†AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
‡AstraZeneca R&D Alderley Park, Cheshire, SK10 4TG, United Kingdom
§AstraZeneca R&D, Macclesfield SK10 2NA, United Kingdom
∥AstraZeneca R&D Molndal, SE-43183 Molndal, Sweden
̈
̈
ABSTRACT: Checkpoint kinases CHK1 and CHK2 are activated in
response to DNA damage that results in cell cycle arrest, allowing
sufficient time for DNA repair. Agents that lead to abrogation of such
checkpoints have potential to increase the efficacy of such compounds as
chemo- and radiotherapies. Thiophenecarboxamide ureas (TCUs) were
identified as inhibitors of CHK1 by high throughput screening. A
structure-based approach is described using crystal structures of JNK1
and CHK1 in complex with 1 and 2 and of the CHK1−3b complex. The
ribose binding pocket of CHK1 was targeted to generate inhibitors with
excellent cellular potency and selectivity over CDK1and IKKβ, key
features lacking from the initial compounds. Optimization of 3b resulted in the identification of a regioisomeric 3-TCU lead 12a.
Optimization of 12a led to the discovery of the clinical candidate 4 (AZD7762), which strongly potentiates the efficacy of a
variety of DNA-damaging agents in preclinical models.
tumor cells over normal cells in the context of DNA damage,
and CHK1 inhibitors are highly sought after for use as chemo
or radiopotentiating agents. UCN-01 represents the first
example of CHK1 inhibitor.3a However, in addition to
CHK1, it hits a number of other kinases including PKC,
CDK1, and CDK2. Because of the toxicity of UCN-01 ascribed
to its polypharmacology, as well as unfavorable pharmacoki-
netic properties, the design and synthesis of many small
molecule CHK1 selective inhibitors have been undertaken by a
large number of research groups including ours.3 Culmination
of these efforts has been identification of several clinical
candidates in the area, namely, PF-477736, SCH-90077, XL-
844, and LY2606368 to name a few.3n In the publication, we
describe structure guided efforts toward optimization of a TCU
class of CHK1 inhibitors to identify AZD7762.
INTRODUCTION
■
The checkpoint kinases CHK1 and CHK2 play a key role in the
modulation of cell cycle checkpoints to ensure the integrity of
genetic information transfer and function during cell-cycle
progression and DNA repair following DNA damage. CHK1
and CHK2 are structurally related Ser/Thr protein kinases that
are phosphorylated by ATM and/or ATR kinases following
DNA damage. Subsequent downstream phosphorylation events
ultimately result in cell-cycle arrest. Inhibition of the checkpoint
kinases, in particular CHK1, has been shown to abrogate DNA
damage induced cell cycle arrest, allowing cells to advance
through the cell cycle without repairing DNA damage leading
to mitotic catastrophe and cell death or apoptosis.1 Tumor cells
are more sensitive to combination treatment with a DNA
damaging agent (chemo- or radiotherapy) and a CHK1
inhibitor compared to normal cells because of the propensity
of tumor cells to have genetic alterations in DNA damage
signaling and repair (DDR), such as p53 mutations, which
make the tumor cells more dependent on the remaining DDR
machinery.2 Thus, abrogation of these checkpoints is
hypothesized to lead to an increased and selective killing of
Received: January 6, 2012
Published: May 2, 2012
© 2012 American Chemical Society
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Figure 1. Evolution of the TCU class of checkpoint kinase inhibitors.
suggested that while substitution of the amide would not be
tolerated in JNK1, CDK1, or IKKβ because of the structural
differences, it would be in CHK1, thus opening a key avenue
RESULTS AND DISCUSSION
■
In a prior communication, we reported preliminary results on
the discovery and SAR of the thiophenecarboxamide urea
(TCU) class of checkpoint kinase inhibitors.3b In the present
report, we explain in detail our X-ray crystal structure guided
approach, which resulted in the rational design of lead
compounds 3a and 3b. We further describe the lead
optimization of 2-TCU 3b, resulting in the discovery of a
new class of isomeric thiophene checkpoint kinase inhibitors
and the final selection of the optimized clinical candidate 3-
TCU 4 (Figure 1).
X-ray Crystal Structure-Guided Identification of Lead
Compound 3. TCUs have been previously described as potent
ATP-competitive IKKβ inhibitors.4 We solved an X-ray crystal
structure of TCU 1 bound to JNK1, which has been used as a
surrogate for IKKβ which shows three atoms of the inhibitor,
namely, the amide carbonyl, amide NH, and distal urea NH,
making hydrogen bonds to the hinge region (Figure 2a, Table
1). We subsequently found that the TCU class of kinase
inhibitors adopts a unique binding mode in CHK1.
Approximately 70 compounds binding to CHK1 kinase are
deposited in the Protein Data Bank. Most bind in the ATP
binding site and form interactions with hinge, and some extend
into the sugar pocket. Other compounds recently described
bind in an allosteric pocket.3n Our X-ray crystal structure of
TCU 2 bound to CHK1 shows that compound 2 is rotated by
approximately 135° relative to compound 1 around an axis
perpendicular to the thiophene ring (Figure 2b and Figure 2c).
In this alternative binding mode, only two atoms of the
inhibitor, the external NH of the urea and the carbonyl of the
urea, form hydrogen bonds to the hinge region, while the aryl
group now points toward the solvent channel. The protein−
inhibitor contacts for deposited structures are summarized in
Table 1. The TCU binding mode exemplified in the JNK1−1
complex structure is not compatible with the DFG-in
conformation observed in the CHK1−2 complex. Asp169
from the DFG motif adopts a different conformation in the
JNK1 structure compared with the corresponding Asp148 in
CHK1. The conformation of the CHK1 aspartate (D148)
would result in a steric clash with the aryl group were 2 to
adopt the same binding mode as seen for 1 in JNK1 (Figure
2c). The structures of 1 and 2 bound to JNK1 and CHK1
Figure 2. X-ray crystal structures of JNK1 and CHK1 in complex with
selected compounds. The structures are oriented with the N-terminal
lobe toward the top left, the C-terminal lobe toward the bottom right,
and the hinge region on the bottom left. (a) X-ray crystal structure of
1 bound to JNK1 (PDB accession code 3PVE). Protein backbone
cartoon and carbon atoms of the inhibitor are colored pink. Hydrogen
bonds are indicated in magenta. (b) X-ray crystal structure of 2 bound
to CHK1 (PDB accession code 2YDI). Protein backbone and carbon
atoms of the inhibitor are colored pale blue. Hydrogen bonds are
colored blue. Final electron density (2Fo − Fc) contoured at 1.0σ is
shown as a wire mesh. (c) Superposition of X-ray crystal structures of
1 with JNK1 and 2 with CHK1. The TCU binding mode differs
between the two structures with the hinge binding donor−acceptor
changing from the amide in the JNK1 structure to the urea in the
CHK1 structure.
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Table 1. Polar contacts between inhibitor and protein for deposited structures
compd
protein residue/atom
inhibitor functional group
inhibitor atom
distance (Å)
location
hinge
protein
1
E109/O
M111/N
M111/O
E85/O
amide
N1
O2
N18
N3
O2
N2
O1
N4
N4
N4
N4
O2
N2
N2
N2
3.13
2.68
3.13
2.9
JNK1
1
amide
hinge
JNK1
1
urea
hinge
JNK1
2
urea
hinge
CHK1
CHK1
CHK1
CHK1
CHK1
CHK1
CHK1
CHK1
CHK1
CHK1
CHK1
CHK1
2
C87/N
urea
2.84
2.77
2.84
2.73
2.83
2.94
2.95
2.78
2.78
2.82
2.97
hinge
3b
3b
3b
3b
3b
4
E85/O
urea
hinge
C87/N
urea
hinge
D148/OD2
E134/O
N135/OD1
E85/O
piperidine
piperidine
piperidine
urea
ribose pocket
ribose pocket
ribose pocket
hinge
4
C87/N
urea
hinge
4
D148/OD2
E134/O
E135/OD1
piperidine
piperidine
piperidine
ribose pocket
ribose pocket
ribose pocket
4
4
for inhibitor design with the aim of optimizing interactions with
CHK1 as previously described.3b,5
CHK1 for cyclic amines and the importance of correct chirality
of the 3S-aminopiperidine moiety. In addition to the two
hydrogen bonding interactions with the hinge region of the
adenine binding site, the protonated piperidine nitrogen of 3b
makes three polar interactions at the edge of the ribose binding
pocket (Figure 3). These include a charge−charge or salt-
bridge interaction with the carboxylate moiety of Asp-148, and
charge−dipole interactions with the backbone carbonyl of Glu-
134 and amide side chain of Asn-135 (see Table 1 for distance
information). The backbone carbonyl interaction offers
tetrahedral hydrogen bonding geometry (hydrogen atoms are
not revealed by X-ray crystallography at this resolution). These
three interactions, the charge−charge interaction and two
charge−dipole interactions, explain why basic groups are
preferred at this position. The preference for the S-enantiomer
(3b) is presumably due to better positioning of the charged
piperidine nitrogen to make these polar interactions. The R-
enantiomer of 3b (not shown) showed a 24-fold drop in
potency (IC50 = 0.17 μM compared to 0.008 μM, Table 2).
Targeting the ribose binding pocket not only allowed us to
generate analogues with improved potency at the enzyme and
cellular level against CHK1 but also afforded selectivity against
CDK1 as predicted from the initial X-ray crystal structure of 2
in complex with CHK13b and based on the difference in the
binding modes of the TCU scaffold seen in CHK1 and JNK1.
Thus, utilizing this structure-based design approach, we were
able to generate high quality initial leads such as 3a and 3b
bearing substituted amides from starting hit 2 and were
subsequently poised to undergo an optimization campaign for
delivering a preclinical candidate CHK1 inhibitor for cancer
treatment in combination with DNA-damaging agents.
Generation of the X-ray structure of 3b bound to CHK1
validated our hypothesis for amide substitution resulting in key
interactions surrounding the ribose binding pocket.
We recently reported on the structure-guided hit to lead
identification, synthesis, and initial SAR of TCUs as CHK1
inhibitors,3b work that led to the discovery of lead inhibitor 3b.
A library of compounds bearing both derivatized amides and 5-
aryl substitutions was designed and synthesized to explore
potential interactions with residues in the CHK1 P-loop or in
the vicinity of the ribose binding pocket.3b,10 From this initial
SAR campaign and testing of a diverse set of compounds, it was
evident that cyclic amine substitutions, such as homopiperidine
or piperidine amide, yielded the most potent compounds as
exemplified by 3a and 3b (Figure 1). Additionally, we
determined that there was a strong stereochemical preference
for the S-isomer, suggesting that 3a and 3b make specific
interaction(s) in the CHK1 ATP-binding site in or in the
vicinity of the ribose sugar pocket as predicted.
X-ray Crystal Structure of Lead 3b Bound to CHK1. We
obtained an X-ray crystal structure of 3b bound to CHK1
(Figure 3) which provided an explanation for the preference of
Optimization of the TCU Series of CHK1 Inhibitors.
We have previously reported the synthesis of 3a and 3b.3b,7
Several approaches to the lead optimization of 3b were
implemented. These included the generation of substituted
ureas in an attempt to derive additional interactions with the
Leu-84 gatekeeper residue to gain further selectivity for CHK1.
Analogues of 3b containing a variety of alkyl and aryl
substitution on the urea were synthesized, allowing us to
quickly determine this to be an unproductive avenue, as
compounds were much less potent and solubility was not
Figure 3. X-ray crystal structure of lead TCU Inhibitor 3b in complex
with CHK1 kinase (PDB accession code 2YDJ). The protein is
oriented with the N-terminal lobe toward the top left, the C-terminal
lobe toward the bottom right, and the hinge region on the bottom left.
Protein backbone cartoon and carbon atoms of the protein are colored
wheat, while carbon atoms of the inhibitor are colored green.
Hydrogen bonds and salt bridges are shown as dotted blue lines. Final
electron density (2Fo − Fc) contoured at 1σ is shown as a wire mesh.
The polar contacts are summarized in Table 1.
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a
Table 2. SAR of 2-TCU and 3-TCU CHK1 Inhibitor Libraries
a
Depicted is a comparison of selected 3-TCUs with their 2-TCU matched pairs. 2-TCUs were generated as previously described.3b 3-TCUs were
synthesized using the method described for 4 in Scheme 1. EC50-CPT represents cellular activity in cells pretreated with a DNA damaging agent,
camptothecin, prior to compound treatment. EC50-Alone represents cellular activity without any pretreatment. IC50 represents activity of the
compound in a CHK1 biochemical assay.
consistent with iv administration (data not shown). In another
strategy, we pursued modification of the thiophene scaffold
including ring replacement with a phenyl and a variety of
heterocyclic rings. A matched pair analysis comparing analogues
of 3b containing a thiophene ring with those containing
heterocycles or a phenyl ring indicated that the thiophene core
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a
Scheme 1. Synthesis of 4
a
Reagents and conditions: (a) TCA−NCO, THF, 90%; (b) bromine, acetic acid, reflux, 97%; (c) NH3−MeOH (2 M), 99%; (d) tert-butyl
(3S)-3-aminopiperidine-1-carboxylate, AlMe3, THF, 40%; (e) 4-fluorophenylboronic acid, Pd(Ph3P)4, Cs2CO3, 1,4-dioxane/H2O, 88%; (f) HCl/1,4-
dioxane (4 M), rt, 4 h 84%.
was optimal for maintaining enzyme and cellular potency
against CHK1 (data not shown). As a result of this campaign,
however, we did identify a thiophene isomer with equivalent
potency against CHK1.
Discovery of 3-Thiophenecarboxamide Urea (3-TCU)
Inhibitors. Compound 3b contains a carboxamide at the 3-
position and a urea at the 2-position of the thiophene, and thus,
we refer to this scaffold as a 2-TCU. We envisioned that the
regioisomer analogous to JNK1 inhibitor 1 having the urea in
the 3-position and the amide in the 2-position could potentially
bind to CHK1 in a similar fashion as the 2-TCUs, offering an
additional opportunity for optimization.
In order to test this hypothesis, the matched pair 3-TCU 12a
was synthesized using the method depicted in Scheme 1 for our
Figure 4. Comparison of lead 2-TCU and 3-TCU matched pairs.
clinical candidate 4. Reaction of commercially available methyl
3-aminothiophene-2-carboxylate (5) with trichloroacetyl iso-
cyanate resulted in 6, which was brominated in acetic acid to
give 7. Interestingly, while these bromination conditions
proceeded at room temperature in the case of the positional
isomer used to generate 3b,3b bromination of 6 required
heating to effect its transformation to 7. Deprotection of the
urea with ammonia in methanol led to the formation of the
corresponding thiophene ester 8. Weinreb amidation con-
ditions similar to those previously reported for the 2-TCU
series3b were applied to 8 using tert-butyl (3S)-3-amino-
piperidine-1-carboxylate to generate thiophene amide 9.
Reaction of 9 with 3-fluorophenylboronic acid under Suzuki
cross-coupling conditions resulted in arylthiophene 10. Final
deprotection of the Boc group yielded the desired 3-TCU 12a
as the hydrochloride salt. Using the same synthetic sequence,
we generated a select number of analogues to derive SAR
relative to the 2-TCU series. As shown in Figure 4, a head to
head comparison of 2-TCU 3b (IC50 = 7 nM) and matched
pair 3-TCU 12a (IC50 = 12 nM) reveals the compounds to be
equivalent in their in vitro potency in both the biochemical
CHK1 kinase assay and the HT29 cell abrogation assay
described previously,3b,6 as expected by our prediction of
similar binding modes. However, the 3-TCU series exemplified
by 12a offer the added benefit of a 4-fold higher free compound
fraction in the human plasma protein binding assay, a general
trend observed across a broad range of the 3-TCUs.
Identification of Clinical Candidate 4 from Optimiza-
tion of 3-TCU 12a. The CHK1−3b crystal structure shows
that the phenyl ring on the 5-thienyl position projects into the
CHK1 solvent channel. This position is therefore predicted to
be tolerant of many substituents and functionality, allowing
modulation of physicochemical and pharmacokinetic proper-
ties. As a final lead optimization strategy for the 3-TCU lead
12a, we thus carried out a multifactorial compound library
campaign mainly focused on this position. This library was
designed with the intent of increasing selectivity for CHK1 over
other kinases while at the same time increasing oral
bioavailability and improving pharmacokinetics and efficacy in
the hollow fiber PD model and mouse animal models. Guided
by X-ray structural information, we installed a diverse array of
desired substituted aryl rings on the thiophene 5-position which
were tolerable to CHK1 binding.
To that end, the key bromothiophene intermediate 9 shown
in Scheme 1 was generated by Weinreb amidation8,9 and
allowed us to quickly generate a large library of 5-position
analogues using the Suzuki cross-coupling reaction as the
means to introduce diversity. A subset of commercially available
boronic acid and boronate esters were chosen to ensure
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diversity in the final compound set and consisted of
representative heterocyclic and aryl derivatives. Approximately
100 compounds were prioritized for synthesis and biological
evaluation. The data for selected matched pairs are summarized
in Table 2.
The X-ray crystal structure of 4 bound to CHK1, shown in
Figure 5, confirmed the predicted binding mode. The urea at
compounds show activity in the absence of DNA damage, in
most cases there is a >100-fold increase in potency in the
pretreated group for both the 2-TCU and 3-TCU series. A
number of potent compounds were identified in our in vitro
assays, paying special attention to physical properties like
solubility in addition to protein binding consistent with the iv
route of administration. Thermodynamic solubility data were
generated at pH 7.4 (0.1 M phosphate buffer). These
compounds were further differentiated by means of our in
vivo cascade that includes pharmacodynamic (PD) activity and
efficacy. Nude mouse pharmacokinetic (PK) profiles for
selected matched pairs are summarized in Table 3.
Table 3. Mouse PK (Nude) of Selected Matched Pairs
R = 4-F
Figure 5. X-ray crystal structure of 4 in complex with CHK1 kinase
(PDB accession code 2YDK) reveals that the binding mode of 3b was
preserved as predicted. The orientation is as in Figure 3. Protein
backbone cartoon and carbon atoms are colored wheat, while carbon
atoms of the inhibitor are colored green. Hydrogen bonds and salt
bridges are shown as dotted blue lines. Final electron density (2Fo −
Fc) contoured at 1σ is shown as a wire mesh. The polar contacts are as
summarized in Table 1. The figure was generated using PYMOL.22
11c,
R = 3-F R = 2,3-di-F
TCU
nude mouse
12c
11b, 4
11f, 12f
2-TCU: 11c, 11b, AUC (μM·h)
1.5
3.8
1.7
11f
Cl
81
33
71
(mL min−1 kg−1
)
)
T1/2 (h)
1.2
6.1
1.46
86
1
1.2
4.5
1.4
85
Vss (l/kg)
3-TCU: 12c, 4, 12f AUC (μM·h)
Cl
2.8
2.5
50
the thiophene 3-position makes identical interactions to the
hinge region as in the 2-TCU 3b, albeit with the thiophene
sulfur atom directed away from the hinge, and the 3-
fluorophenyl substituent at the 5-position is directed toward
the solvent channel.
(mL min−1 kg−1
T1/2 (h)
1.2
8.5
1.1
3.9
1.4
7.7
Vss (l/kg)
Analogues 12a−j and 4 were found to be very potent against
CHK1 in vitro. The potencies of the 3-TCU series (12a−j and
4) and their corresponding 2-TCU matched pairs10a (11a−j
and 3b) were generally almost identical. Determination of the
3-TCU series SAR was derived from a Topliss-like10b
examination of size, functionality, and electronic variation
through comprehensive substitutions on the phenyl ring of 12a.
For example, when the ortho, meta, and para fluorine
substitutions were compared (Table 2), it was evident that
substitution at the ortho position (12b) was less favorable with
regard to kinase activity but meta (4) and para (12c) analogues
had improved CHK1 activity and similar potency. Size had
minimal effect on activity, as demonstrated by the finding that
chloro, cyano, and other electron withdrawing functional
groups did not result in any significant change in activity, but
we did find much variation on cellular potency. Electron
donating groups such as dimethylamino and methoxy tended to
be slightly less potent and followed similar trends as reported
for the 2-TCU series.3b Examination of heterocyclic replace-
ments of the aryl group resulted in compounds that maintained
potency in both the enzyme and cellular assays, similar to that
reported earlier in the 2-TCU series3b (data not shown).
All compounds were tested both alone and in combination
with campothecin in the HT29 cellular checkpoint abrogation
assay3a to determine the extent of abrogation directly related to
CHK1 inhibition versus off-target cellular activities. While some
A comparison of PK parameters for 2-TCUs (11c, 11b, and
11f) and their 3-TCU matched pairs (12c, 4, 12f)
demonstrates that while they are equivalent in their PK
properties, 3-F substituted positional isomers 11b and 4
consistently show lower clearance. 3-TCU 4 could be further
differentiated from 11b by its higher solubility (>1 mM
compared to 779 μM) and lower plasma protein binding
(hPPB, see Figure 7B), an important consideration given the
planned iv route of administration. Several 2- and 3-TCUs were
screened in a high throughput pharmacodynamic (PD) assay
which aided the prioritization of compounds for efficacy studies
(see below). These data, along with the overall safety and
selectivity profile, enabled the selection of 4 as the clinical
candidate.6
Evaluation of 4 in the Hollow Fiber PD Assay. The
hollow fiber PD assay, described in detail in an earlier
publication,6a was used to determine the in vivo PD activity
of selected CHK1 inhibitors to assist prioritization of
compounds for testing in xenograft efficacy models. Briefly,
cells were treated with the DNA-damaging agent topotecan to
induce cell cycle arrest in the G2 phase. Following cell cycle
arrest, the hollow fibers are filled with cells and implanted
under the skin of the animal. This arrest is stable for up to 30 h
after implantation in the absence of treatment with a CHK1
inhibitor. Compounds are then administered at 10 mg/kg using
iv dosing. Finally, fibers are retrieved after 30 h, and the cell
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cycle profile is determined with propidium iodide staining using
flow cytometry. In animals dosed with CHK1 inhibitors, the G2
checkpoint is overcome causing a reduction in the number of
cells in G2 and a concomitant increase in the number of cells
re-entering the G1 phase. The “odds value” is then calculated as
the ratio of the G2/G1 populations for topotecan alone and the
CHK1 inhibitor combination. From the latter, we calculated an
odds ratio (OR) that allowed quantitative assessment of the
degree of checkpoint abrogation. The greater the OR, the
higher is the level of checkpoint abrogation. Shown in Table 4
efficacy of DNA damage inducing chemotherapeutics by 4 was
observed.
Kinase Selectivity Profile of Preclinical Candidate 4.
The kinase selectivity of 4 was checked by profiling against a
large panel of diverse Ser/Thr and Tyr protein kinases from
Upstate/Millipore in which examples of each kinase class are
represented. Key data for the screen are summarized in Figure
7C. In general, kinases showing less than 10-fold selectivity
were from the same kinase family (CAMK) as CHK1 or were
Src-family tyrosine kinases (Yes, Fyn, Lyn, Hck, and Lck but
not Src itself). Following preliminary profiling, expanded
screening was biased toward CAMK and tyrosine kinase family
members. Data generated from in-house and external screening
in the Upstate/Millipore kinase panel indicated good selectivity
(>10-fold) for CHK1 kinase versus 164 kinases. Importantly,
selectivity was shown in the cyclin-dependent kinase 1
(CDK1)/cyclin B1 scintillation proximity assay (>1000-fold)
and against other CDKs tested. A greater than 100-fold
selectivity against multiple protein kinase C isoforms, CDKs,
p38 (all isoforms), and MAPKAPK2 clearly differentiated the
activity profile of 4 from UCN-01, a known inhibitor of CHK1.
Compound 4 is an equally potent inhibitor of CHK1 and
CHK2 in vitro; hence, the phenotypic response cannot be
presumed to be arising from CHK1 inhibition alone based on
data from the experiments presented herein. Literature
precedent, however, has indicated a major role for CHK1
versus CHK2 when both kinases were assessed simulta-
neously.11,12
Table 4. PD Activity of Selected 2-TCUs and 3-TCUs
a
a
OR at 10
OR at 10
b
b
2-TCU
mg/kg (iv)
control 3-TCU
mg/kg (iv)
control
<0.05
c
3b
4.56
2.14
2.89
1.91
1.59
0.00
12a
12b
4
3.12
2.04
5.01
4.61
2.96
3.79
11a
0.013
0.001
0.37
0.024
0.000
0.000
0.001
<0.05
d
11b
11d
11g
11i
12d
12g
12i
0.138
0.002
2.62
a
b
OR = odds ratio. Control = compound alone run in the absence of a
c
d
DNA damaging agent. Average of eight runs. Average of two runs.
are the OR values for selected 2- and 3-TCUs demonstrating
the superiority of 4 over others in the series based on its high
OR value and lack of activity in the control arm of the
experiment (which reflects the extent of CHK1 selectivity
shown by the inhibitor).
Treatment with compound 4 resulted in the abrogation of
the G2 checkpoint in a dose dependent manner, as shown in
Figure 6.6a In general, our data show a good correlation
On the basis of the comparison of the key data presented in
Figure 7 against other compounds in the 2-TCU and 3-TCU
series combined with its efficacy and tolerability profile,
compound 4 was selected as a clinical candidate and entered
phase I in combination with gemcitabine or irinotecan.
CONCLUSION
■
TCUs were identified as potent ATP-competitive CHK1
inhibitors in an HTS campaign of the AstraZeneca compound
collection followed by expedient hit to lead identification and
optimization employing protein−ligand crystal structures to
rationally guide compound design and optimization. TCUs are
known inhibitors of IKK2 and JNK1 kinases. The X-ray crystal
structure of the original HTS hit 2 in complex with CHK1
highlighted substitution at the amide moiety as a means to
achieve productive interactions with key residues in the ribose
binding pocket, thereby increasing the potency. Substitution on
the amide was additionally advantageous in affording selectivity
over CDKs, IKKβ, and JNK1, where by virtue of an alternative
binding mode, substitution at the amide is not tolerated.
Optimization of the 2-TCU by pursuing amide libraries led to
the identification of the 3-(S)-piperidinylamide analogue 3b.
The crystal structure of CHK1 in complex with 3b revealed key
polar interactions in the ribose pocket, which account for the
increase in potency and enantiomeric preference at this
position. Exploration of alternative scaffolds led us to focus
on a positional isomer of 3b, which resulted in the rational
design of lead 3-TCU 12a and corresponding 3-TCU subseries,
which we found to be of equivalent potency compared to the 2-
TCUs with the added benefit of an improved DMPK profile
and in vivo efficacy. A Suzuki library of 5-substituted 3-TCUs,
designed to target the CHK1 solvent channel and thus
modulate PK and physicochemical properties, resulted in the
discovery of a large number of tolerated substitutions and
identified a 3-fluorophenyl moiety as an optimal analogue of
Figure 6. Dose dependent increase in the odds ratio (OR) observed
with AZD7762 (iv administration).
between the relative potency of inhibitors as measured in the
cellular checkpoint abrogation assay and the observed PD
effect. An exposure of 8 h above EC50 was required in order to
obtain maximal PD activity.6a There were also many examples
of compounds showing potency in vitro and yet lacking in vivo
activity in the PD model. We were able to explain this
discrepancy in the majority of cases through differences in PK
profiles among analogues.
Antitumor Efficacy Potentiation of DNA Damaging
Chemotherapeutics by 4.6a The ability of 4 to potentiate
the efficacy of both gemcitabine and irinotecan was evaluated in
mouse and rat xenograft models and is described in detail in an
earlier publication.6a In each case, strong potentiation of the
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Figure 7. (A) Structure of AZD7762. (B) In vitro and in vivo profile. (C) Kinase family selectivity profile.
spectrometer (compounds were detected by mass spectrometry in
APCI+ ionization) using a Luna 3 μm C8 (2) column (100A, 30−2.0
mm), eluting with mixtures of water−acetonitrile (ranging from 5% to
98% acetonitrile/water with 0.1% formic acid), using a UV detector at
220, 240, and 254 nm. The purity of all final compounds was at least
95%. Reverse phase chromatography was performed with Gilson
systems using a YMC-AQC18 reverse phase HPLC column with
dimensions 20 mm/100 and 50 mm/250 in water/MeCN with 0.1%
TFA as mobile phase. Silica gel chromatography was performed using
either glass column packed with silica gel (200−400 mesh, Aldrich
Chemical) or prepacked silica gel cartridges (Biotage and ISCO
CombiFlash systems)
Methyl 3-({[(Trichloroacetyl)amino]carbonyl}amino)-
thiophene-2-carboxylate (6). To a stirred solution of methyl 3-
aminothiophene-2-carboxylate (17.0 g, 108 mmol) in anhydrous THF
(216 mL) was added trichloroacetyl isocyanate (12.9 mL, 108 mmol)
slowly over a period of 20 min. After the addition was complete, a
precipitate formed and the mixture stirred for an additional 1 h at
room temperature. The desired product was obtained by filtration to
give 6 (36.8 g, 99%) as an off-white solid and was used without further
purification for synthesis of 7. LC/MS (APCI, ES): (M + H)+ 345.
Methyl 5-Bromo-3-({[(trichloroacetyl)amino]carbonyl}-
amino)thiophene-2-carboxylate (7). To a solution of 6 (10.11 g,
29.38 mmol) in glacial acetic acid (146 mL) at 0 °C was added slowly
a solution of bromine (46.4 mL, 90 mmol) in glacial acetic acid (38
mL). The resulting cloudy solution was heated to 70 °C for 3 h when
the mixture was cooled to room temperature. The desired product was
collected by filtration and the filtrate was washed with ether and dried
under vacuum to obtain the desired product as a white solid (12 g,
97%), which was used for preparation of 8 without further purification.
LC/MS (APCI, ES): (M + H)+ 424.
the 5-phenyl group. Hybridization of these key structural
features led to the identification of the development candidate
4 with suitable in vitro, in vivo, and safety profiles. An
additional key feature of 4 is >1 mM solubility, an important
consideration given the clinical route of administration is
intravenous. The development candidate 4 and the related
TCU class of CHK1 inhibitors strongly potentiate the effects of
DNA damage caused by cytotoxic agents such as gemcitabine
or irinotecan as well as when combined with radiation therapy
in a variety of preclinical models.6d,13 Clinical trials of 4 were
terminated after a full review of program data. Termination of
the study was made after a full review of program data and
assessment of the current risk−benefit profile. Both CHK1 and
CHK2 checkpoint kinase inhibitors remain an active area of
research and clinical development.
EXPERIMENTAL SECTION
■
General. All solvents used are anhydrous and commercially
available. All commercially available materials were used without
further purification unless otherwise stated. H NMR spectra were
1
recorded on Bruker 300 or 400 MHz NMR spectrometers. Chemical
shifts are expressed in parts per million (ppm, δ) relative to
tetramethylsilane (TMS) as an internal standard using deuterated
chloroform as a solvent unless otherwise indicated. All NMR chemical
shifts are reported as δ values in parts per million (ppm). The splitting
pattern abbreviations are as follows: s, singlet; d, doublet; t, triplet; q,
quartet; br, broad; br s, broad singlet; m, unresolved multiplet due to
the field strength of the instrument; dd, doublet of doublet; dt, doublet
of triplet; ddd, doublet of doublet of doublet. Analytical mass spectra
were run with an electron energy of 70 eV in the chemical ionization
(CI) mode using a direct exposure probe, where the indicated
ionization was effected by electron impact (EI), electrospray (ESP), or
atmospheric pressure chemical ionization (APCI). Values for M + H
are given; generally, only ions that indicate the parent mass are
reported. The purity of all final compounds was confirmed by analysis
with an Agilent 1100 liquid chromatography unit (LC) with an Agilent
1100 mass spectrometry detector (MSD) or a Waters ZQ mass
Methyl 3-[(Aminocarbonyl)amino]-5-bromothiophene-2-
carboxylate (8). A solution of 7 (12 g, 28.3 mmol) in dry MeOH
(60 mL) was purged with gaseous NH3 at 0 °C until the solution
became clear. The mixture was stirred for 20 min at room temperature,
and evaporation gave the desired product (7.8 g, 99%) as white solid.
1H NMR (DMSO-d6): δ 9.35 (s, 1H), 8.10 (s, 1H), 6.90 br s, 2H),
3.82 (s, 3 H). LC/MS (APCI, ES): (M + H)+ 280.
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tert-Butyl (3S)-3-[({3-[(Aminocarbonyl)amino]-5-bromo-2-
thienyl}carbonyl)-amino]piperidine-1-carboxylate (9). To a
solution of 8 (1.6 g, 5.7 mmol) in dry THF (30 mL) was added a
solution of Me3Al/tert-butyl (3S)-3-aminopiperidine-1-carboxylate in
THF (18 mL) (preformed by the addition of Me3Al (17.2 mL, 34.4
mmol) to a solution of tert-butyl (3S)-3-aminopiperidine-1-carboxylate
(3.7 g, 17.2 mmol) in THF at −78 °C, and the resulting yellow
solution was stirred at this temperature for 15 min), and the resulting
deep yellow solution was warmed to room temperature and stirred
overnight. The reaction mixture was cooled with ice, and a saturated
solution of sodium potassium tartrate was added to quench the
reaction. The mixture was partitioned between EtOAc and H2O (3:1
ratio). The aqueous layer was extracted with EtOAc (3×), and the
combined organic extracts were washed with H2O, brine and dried
over MgSO4. Evaporation gave a pale orange solid. Purification by
reverse phase HPLC using a gradient of 5−95% MeCN in 0.1% TFA
containing water gave the desired product as an off-white solid (1.0 g,
2 H), 1.92 (d, 2 H), 2.92 (m, 2 H), 3.17 (d, 1 H), 3.30 (d, 1 H), 4.21
(s, 1 H), 7.03 (s, 2 H) 7.25 (t, 2 H) 7.59 (t, 2 H), 7.92 (s, 1 H), 8.31
(d, 1 H), 9.07 (d, 2 H), 10.90 (s, 1 H). LC/MS (APCI, ES): (M + H)+
363.
5-(3-Chlorophenyl)-2-ureidothiophene-3-carboxylic Acid
1
(S)-Piperidin-3-ylamide (11d). H NMR (DMSO-d6): δ 10.94 (s,
1H), 9.10 (s, 1H), 8.30 (d, 1H), 8.06 (s, 1H), 7.61 (s, 1H), 7.49 (d,
1H), 7.42 (t, 1H), 7.30 (d, 1H), 7.09 (s, 2H), 4.21 (s, 1H), 3.32 (d,
1H), 3.17 (d, 1H), 2.92 (t, 2H), 1.93 (d, 2H), 1.68 (m, 2H). LC/MS
(APCI, ES): (M + H)+ 379.
5-(3,5-Difluorophenyl)-2-ureidothiophene-3-carboxylic Acid
1
(S)-Piperidin-3-ylamide (11e). H NMR (DMSO-d6): δ 10.95 (s,
1H), 9.10 (s, 1H), 8.98 (s, 1H), 8.32 (d, 1H), 8.16 (s, 1H), 7.26 (d,
2H), 7.12 (t, 2H), 4.22 (s, 1H), 3.28 (d, 1H), 3.17 (d, 1H), 2.95 (m,
2H), 1.91 (d, 2H), 1.65 (m, 2H). LC/MS (APCI, ES): (M + H)+ 381.
5-(2,3-Difluorophenyl)-2-ureidothiophene-3-carboxylic Acid
1
(S)-Piperidin-3-ylamide (11f). H NMR (DMSO-d6): δ 11.01 (s,
1
1H), 9.22 (s, 1H), 9.14 (s, 1H), 8.46 (d, 1H), 8.10 (s, 1H), 7.81 (t,
1H), 7.29 (m, 2H), 7.11 (br, 2H), 4.20 (s, 1H), 3.30 (d, 1H), 3.14 (d,
1H), 2.92 (m, 2H), 1.90 (d, 2H), 1.69 (m, 2H). LC/MS (APCI, ES):
(M + H)+ 381.
40%). H NMR (DMSO-d6): δ 10.06 (s, 1H), 8.02 (s, 1H), 7.89 (d,
1H), 6.71 (br s, 2H), 4.04 (m, 1H), 3.72 (m, 2H), 2.74 (m, 2H), 1.81
(m, 1H), 1.68 (m, 1H), 1.52 (m, 1H), 1.37 (s, 9H), 1.34 (m, 1H). LC/
MS (APCI, ES): (M + H)+ 448.
5-(2,4-Difluorophenyl)-2-ureidothiophene-3-carboxylic Acid
tert-Butyl (3S)-3-({[3-[(Aminocarbonyl)amino]-5-(3-fluoro-
phenyl)-2-thienyl]carbonyl}amino)piperidine-1-carboxylate
(10). In a round bottomed flask, 9 (1 g, 2.24 mmol), 3-
fluorobenzeneboronic acid (0.5 g, 3.36 mmol), and cesium carbonate
(2.9 g, 8.96 mmol) were dissolved in a mixture of water (3 mL) and
1,4-dioxane (20 mL). The solution was degassed under low vacuum
and nitrogen. Palladium(0) tetrakistriphenylphosphine was added
(0.26 g, 0.22 mmol) to the solution, and the colorless reaction mixture
was heated at 75 °C with stirring for 1 h. The aqueous layer was
separated. Ethyl acetate was added (20 mL), and the resulting solution
was dried over MgSO4 (anhydrous). After solvent evaporation the
residual solid was subjected to flash chromatography on silica gel (40
g) with 2−5% methanol in DCM over a 30 min gradient. The main
fraction contained traces of triphenylphosphine oxide and was
resubjected to flash chromatography under the same conditions to
afford a light yellow solid (0.92 mg, 88%). 1H NMR (CDCl3): δ 10.35
(s, 1H), 8.30 (s, 1H), 7.43−7.28 (m, 3H), 7.05 (m, 1H), 5.91 (br s,
1H), 4.79 (br s, 2H), 4.05 (m, 1H), 3.53−3.55 (m, 2H), 3.36−3.29(m,
2H), 1.89−1.49 (m, 13H). LC/MS (APCI, ES): (M + H)+ 463.
5-(2-Fluorophenyl)-2-ureidothiophene-3-carboxylic Acid
(S)-Piperidin-3-ylamide Hydrochloride (4). Intermediate 10
(0.92 g, 1.98 mmol) was dissolved in methanol (7 mL), and 4 M
HCl in dioxane (10 mL, 40 mmol) was added while cooling at 0 °C.
The solution was stirred for 3 h. The solvent was evaporated under
reduced pressure, and the residue was taken in methanol (20 mL) and
evaporated to dryness. This procedure was repeated 5 times to yield a
white powder. The solid was dissolved in water (10 mL) and filtered,
and solvent was evaporated by lyophilization over 48 h to obtain a
1
(S)-Piperidin-3-ylamide (11g). H NMR (DMSO-d6): δ 10.97 (s,
1H), 9.30 (s, 1H), 9.18 (s, 1H), 8.45 (d, 1H), 8.02 (s, 1H), 7.78 (q,
1H), 7.38 (t, 1H), 7.18 (t, 1H), 7.06 (br, 2H), 4.23 (s, 1H), 3.29 (d,
1H), 3.14 (d, 1H), 2.92 (m, 2H), 1.90 (d, 2H), 1.69 (m, 2H). LC/MS
(APCI, ES): (M + H)+ 381.
5-(3,4-Dichlorophenyl)-2-ureidothiophene-3-carboxylic
Acid (S)-Piperidin-3-ylamide (11h). 1H NMR (DMSO-d6): δ 10.96
(s, 1H), 9.30 (s, 1H), 9.19 (s, 1H), 8.43 (d, 1H), 8.18 (s, 1H), 7.79 (s,
1H), 7.63 (d, 1H), 7.51 (d, 1H), 7.10 (br, 2H), 4.24 (s, 1H), 3.29 (d,
1H), 3.13 (d, 1H), 2.94 (m, 2H), 1.92 (d, 2H), 1.70 (m, 2H). LC/MS
(APCI, ES): (M + H)+ 413.
5-(4-Cyanophenyl)-2-ureidothiophene-3-carboxylic Acid (S)-
1
Piperidin-3-ylamide (11i). H NMR (DMSO-d6): δ 10.98 (s, 1 H),
9.03 (s, 2 H), 8.37 (d, 1 H), 8.22 (s, 1 H), 7.84 (d, 2 H), 7.72 (d, 2 H),
7.14 (s, 2 H), 4.20 (s, 1 H), 3.31 (d, 1 H), 3.17 (d, 1 H), 2.93 (t, 2 H),
1.93 (d, 2 H), 1.67 (m, 2 H). LC/MS (APCI, ES): (M + H)+ 370.
5-(3-Cyanophenyl)-2-ureidothiophene-3-carboxylic Acid (S)-
Piperidin-3-ylamide (11j). 1H NMR (DMSO-d6): δ 10.94 (s, 1 H),
9.01 (s, 2 H), 8.28 (d, 1 H), 8.11 (s, 1 H), 7.98 (s, 1 H), 7.83 (d, 2 H),
7.71 (d, 1 H), 7.61 (t, 1 H), 7.12 (s, 2 H), 4.18 (s, 1 H), 3.18 (d, 2 H),
2.91 (m, 2 H), 1.93 (d, 2 H), 1.68 (m, 2 H). LC/MS (APCI, ES): (M
+ H)+ 370.
5-Phenyl-3-ureidothiophene-2-carboxylic Acid (S)-Piperi-
1
din-3-ylamide (12a). H NMR (DMSO-d6): δ 10.02 (s, 1H), 9.39
(brs, 2H), 8.25 (s, 1H), 8.18(d, 1H), 7.6 (d, 2H), 7.58−7.31 (m, 3H),
4.19 (m, 1H), 3.34−3.12 (m, 2H), 2.83 (m, 2H), 1.90−1.51 (m, 4H).
LC/MS (APCI, ES): (M + H)+ 345.
1
5-(2-Fluorophenyl)-3-ureidothiophene-2-carboxylic Acid
light yellow solid (0.67 g, 84%). H NMR (DMSO-d6): δ 10.98 (s,
1
(S)-Piperidin-3-ylamide (12b). H NMR (DMSO-d6): δ 9.95 (s, 1
1H), 9.29 (s, 1H), 9.20 (s, 1H), 8.43 (d, 1H), 8.06 (s, 1H), 7.77 (m,
2H), 7.29 (m, 3H), 7.08 (br, 2H), 4.25 (s, 1H), 3.29 (d, 1H), 3.14 (d,
1H), 2.92 (m, 2H), 1.93 (d, 2H), 1.67 (m, 2H). LC/MS (APCI, ES):
(M + H)+ 363.
H), 8.66 (s, 2 H), 7.84−8.02 (m, 1 H) 8.15 (d, 1 H), 7.70−7.86 (m, 1
H), 7.22−7.57 (m, 3 H), 7.10 (s, 1 H), 6.69 (s, 1 H), 3.79−4.58 (m, 2
H), 3.11−3.36 (m, 2 H), 2.77−3.05 (m, 1 H), 1.74−2.21 (m, 3 H),
1.47−1.75 (m, 1 H). LC/MS (APCI, ES): (M + H)+ 363.
Application of this two-step procedure described above for
synthesizing 4 from 10 enabled generation of compounds 11a−j
and 12a−j as hydrochloride salts.
5-(4-Fluorophenyl)-3-ureidothiophene-2-carboxylic Acid
1
(S)-Piperidin-3-ylamide (12c). H NMR (DMSO-d6): δ 9.94 (s, 1
H), 8.73 (s, 2 H), 8.22 (s, 1 H), 8.08 (d, J = 7.54 Hz, 1 H), 7.56−7.76
(m, 2 H), 7.19−7.44 (m, 2 H), 6.68 (s, 2 H), 4.05−4.28 (m, 1 H),
3.07−3.48 (m, 2 H), 2.67−3.00 (m, 2 H), 1.88 (s, 2 H), 1.53−1.68
(m,, 2 H). LC/MS (APCI, ES): (M + H)+ 363.
5-(2-Fluorophenyl)-2-ureidothiophene-3-carboxylic Acid
1
(S)-Piperidin-3-ylamide (11a). H NMR (DMSO-d6): δ 10.98 (s,
1H), 9.29 (s, 1H), 9.20 (s, 1H), 8.43 (d, 1H), 8.06 (s, 1H), 7.77 (m,
2H), 7.29 (m, 3H), 7.08 (br, 2H), 4.25 (s, 1H), 3.29 (d, 1H), 3.14 (d,
1H), 2.92 (m, 2H), 1.93 (d, 2H), 1.67 (m, 2H). LC/MS (APCI, ES):
(M + H)+ 363
5-(3-Chlorophenyl)-3-ureidothiophene-2-carboxylic Acid
1
(S)-Piperidin-3-ylamide (12d). H NMR (DMSO-d6): δ 9.85 (s, 1
H), 8.58 (s, 2 H), 8.30 (s, 1 H), 8.07−8.14 (m, 1 H), 8.05 (s, 1 H),
7.88 (d, 1 H), 7.81 (d, 1 H), 7.62 (t,1 H), 6.65 (s, 1 H), 3.92−4.29 (m,
1 H), 3.08−3.28 (m, 2 H), 2.70−2.87 (m, 2 H), 1.72−1.91 (m, 2 H),
1.44−1.71 (m, 2 H). LC/MS (APCI, ES): (M + H)+ 379.
5-(3,5-Difluorophenyl)-3-ureidothiophene-2-carboxylic Acid
(S)-Piperidin-3-ylamide (12e). 1H NMR (DMSO-d6): δ 9.89 (s,
1H), 9.19 (br s, 2H), 8.32 (s, 1H), 8.30 (d, 1H), 7.28 (m, 3H), 6.62
5-(3-Fluorophenyl)-2-ureidothiophene-3-carboxylic Acid
1
(S)-Piperidin-3-ylamide (11b). H NMR (DMSO-d6): δ 10.94 (s,
1 H), 8.98 (d, 2 H), 8.27 (d, 2 H), 8.03 (s, 1 H), 7.40 (m, 3 H), 7.08
(t, 2 H), 4.19 (s, 1H), 3.30 (m, 1H), 3.18 (d, 1H), 2.93 (m, 2H), 1.93
(d, 2H), 1.67 (m, 2H). LC/MS (APCI, ES): (M + H)+ 363.
5-(4-Fluorophenyl)-2-ureidothiophene-3-carboxylic Acid
1
(S)-Piperidin-3-ylamide (11c). H NMR (DMSO-d6): δ 1.68 (m,
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(br s, 1H), 4.18 (m, 1H), 3.18 (m, 2H), 2.78 (m, 2H), 1.89−1.42 (m,
4H). LC/MS (APCI, ES): (M + H)+ 381.
XLfit (model 205) with the curve bottom constrained to 0 and the top
constrained to 100 by nocodazole alone treatment.
In Vivo Studies. In vivo studies are described in detail in ref 6a
5-(2,3-Difluorophenyl)-3-ureidothiophene-2-carboxylic Acid
(S)-Piperidin-3-ylamide (12f). 1H NMR (DMSO-d6): δ 9.89 (s,
1H), 9.02 (br s, 2H), 8.35 (s, 1H), 8.20 (d, 1H), 7.68−7.20 (m, 3H),
6.68 (br s, 1H), 4.15 (m, 1H), 3.18 (m,2H), 2.80 (m, 2H), 1.89−1.50
cited in this report.
Overexpression and Purification of JNK1 Kinase. An N-
terminally truncated form of JNK1 (9-364) was expressed in TOP10
E.coli cells, making use of the expression plasmid pBADHisA resulting
in an expression construct bearing an N-terminal His-tag for
purification ease that can be removed via thrombin cleavage.
Expression was started at 28 °C at an OD600 of around 0.8 by
addition of 0.2% (w/v) arabinose. The cells were harvested by
centrifugation 6−18 h after induction, and the pellets were stored at
−80 °C. A pellet corresponding to 1 L of culture was suspended in 100
mL of ice-cold lysis buffer (50 mM Tris/HCl, pH 7.5, 500 mM NaCl,
10 mM imidazole, 10% glycerol, 1 mM DTT). After cell breakage
through ultrasonication, cell debris was removed by centrifugation and
the supernatant was collected. Then 10 mL of Ni2+-NTA Superflow
resin (Qiagen) equilibrated in lysis buffer was added to the cleared
lysate and incubated for 1 h at 4 °C on a shaking platform. The matrix
was washed with 75−150 mL of lysis buffer, and JNK1 protein was
eluted with 25 mL of elution buffer (50 mM Tris/HCl, pH 7.5, 500
mM NaCl, 300 mM imidazole, 10% glycerol, and 1 mM DTT). After
overnight dialysis at 4 °C against 2 L of 50 mM Tris/HCl, pH 7.5, 500
mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM DTT, the His-tag
was released by thrombin treatment (Pharmacia, 10 U/mg target
protein) for 2 h and passed over a Ni2+-NTA column (Qiagen, 10 mL
column volume) in the same buffer. The flow-through was collected,
concentrated to about 8−10 mL, and applied directly onto a
Superdex75 XK26/60 column (2 mL/min flow rate) equilibrated
with 25 mM Tris/HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM
DTT at 12 °C. Fractions of pure JNK1 eluting at around 160 mL
retention volume were pooled and concentrated to 9.5 mg/mL while
exchanging into a storage buffer consisting of 25 mM HEPES, pH 7.0,
plus 5 mM DTT. The final yield was typically in the range of 80−120
mg/L.
+
(m, 4H). LC/MS (M + H) 381.
5-(2,4-Difluorophenyl)-3-ureidothiophene-2-carboxylic Acid
1
(S)-Piperidin-3-ylamide (12g). H NMR (DMSO-d6): δ 9.89 (s,
1H), 8.90 (br s, 2H), 8.32 (s, 1H), 8.17 (d, 1H), 7.78 (q, 1H), 7.48 (t,
1H), 7.22 (t, 1H), 6.68 (br s, 1H), 4.18 (m, 1H), 3.18 (m, 2H), 2.78
(m, 2H), 1.89−1.42 (m, 4H). LC/MS (APCI, ES): (M + H)+ 381.
5-(3,4-Dichlorophenyl)-3-ureidothiophene-2-carboxylic
1
Acid (S)-Piperidin-3-ylamide (12h). H NMR (DMSO-d6): δ 9.82
(s, 1H), 9.12 (s, 1H), 8.98 (br s, 1H), 8.22 (s, 1H), 8.17 (d, 1H), 7.78
(s, 1H), 7.70 (d, 1H), 7.50 (s, 1H), 6.62 (br s, 1H), 4.18 (m, 1H), 3.18
(m, 2H), 2.78 (m, 2H), 1.89−1.42 (m, 4H). LC/MS (APCI, ES): (M
+ H)+ 414.
5-(4-Cyanophenyl)-3-ureidothiophene-2-carboxylic Acid (S)-
1
Piperidin-3-ylamide (12i). H NMR (DMSO-d6): δ 9.82 (s, 1H),
8.98 (br s, 1H), 8.32 (s, 1H), 8.19 (d, 1H), 7.82 (d, 2H), 7.76 (d, 2H),
6.62 (br s, 1H), 4.18 (m, 1H), 3.18 (m, 2H), 2.78 (m, 2H), 1.89−1.42
(m, 4H). LC/MS (APCI, ES): (M + H)+ 370.
5-(3-Cyanophenyl)-3-ureidothiophene-2-carboxylic Acid (S)-
1
Piperidin-3-ylamide (12j). H NMR (DMSO-d6): δ 9.86 (s, 1 H),
8.43−8.76 (m, 2 H), 8.06 (d, 2 H), 7.56−7.67 (m, 1 H), 7.40−7.47
(m, 2 H) 7.54 (s, 1 H), 6.64 (s, 1 H), 3.91−4.25 (m, 1 H), 3.20 (dd, 4
H), 2.62−2.88 (m, 2 H), 1.76−1.87 (m, 2 H), 1.43−1.66 (m, 2 H).
LC/MS (APCI, ES): (M + H)+ 370.
CHK1 Kinase Assay.6a Recombinant human CHK1 was expressed
as a glutathione S-transferase fusion in insect cells using a baculovirus
vector and purified by glutathione affinity chromatography. A synthetic
peptide substrate for CHK1 was synthesized by Bachem (N-
biotinylaminohexanoyl-KKVSRSGLYRSPMPENLNRPR). Com-
pounds were screened at a single concentration (10 μmol/L) for the
primary assay using a standard scintillation proximity assay protocol.
For follow-up, a dose response was determined (IC50). Final assay
concentrations of peptide and ATP (cold + 40 nCi [33P]ATP) were
0.8 and 1 μmol/L, respectively. In brief, compound and buffer
containing peptide and kinase and ATP were added sequentially to a
384-well assay plate. The plate was incubated for 2 h. Reaction was
stopped by the addition of buffer containing EDTA and scintillation
proximity assay beads (Amersham), and plates were read using a
TopCount reader (Packard). Data analysis was carried out using
proprietary software.
Crystallization, Ligand Soaking, and Structure Determina-
tion of 1 in Complex with JNK1. Crystals of JNK1 kinase were
grown using the hanging-drop vapor diffusion method with a reservoir
containing 15% PEG2000 MME, 100 mM HEPES, pH 7, and 10 mM
DTT and 1 mM AMP-PNP. Crystals were soaked for 24 h in mother
liquor containing 5 mM 1 and 10% DMSO. Data were collected at
beamline 711, MAX Lab, Lund, Sweden, at 100 K. The data were
processed with MOSFLM15 and programs in the CCP4 suite.16
Refinement and model rebuilding were carried out using CNX,17
Autobuster,18 and COOT.19
For kinetic analysis, a filter binding assay was used. The assay
reaction contained the reagents listed above but with the following
concentrations of ATP [0−600 μmol/L (cold + Ci [33P]ATP),
determined Km], Chk1 (0.5 nmol/L), and 4 (0, 1.5, 5, 10 nmol/L).
The mixture was incubated at room temperature for 20 min. The
reaction was stopped by the addition of EDTA. The mixture was
transferred to streptavidin FlashPlate (Perkin-Elmer), incubated at
room temperature for 1 h, and aspirated out, and wells were washed
with PBS. Plates were counted using a TopCount reader, and data
were analyzed using proprietary software.
Overexpression and Purification of CHK1 Kinase. Checkpoint
kinase (residues 1−276 and 1−289) protein production was as
previously described.14
Cocrystallization CHK1 in Complex with 2, 3, and 4.
Cocrystals of 2 in complex with CHK1 kinase (1−289) and 3b and
4 in complex with CHK1 kinase (1−276) were prepared as previously
described.14
Data Collection and Structure Solution of CHK1 Kinase
Complexes. Diffraction data for the CHK1−2 complex were
collected at Daresbury on beamline PX9.6 equipped with an ADSC
Quantum 4 detector, using a Si111 monochromatic wavelength of 0.86
Å at 100 K. Diffraction data for the CHK1−3b complex were collected
at the ESRF on beamline ID14-4 equipped with an ADSC CCD
detector, using a Si111 monochromatic wavelength of 1.0052 Å.
Diffraction data for the CHK1−4 complex were collected on a Rigaku
MM007 rotating anode X-ray generator equipped with a Saturn 92
CCD detector, using Osmic Varimax HF mirrors and Cu Kα radiation.
Data for the CHK1−2, and CHK1−3b complexes were processed
using MOSFLM15 and were scaled, merged, and reduced using CCP4
software.16 Data for CHK1−4 complex were processed using d*trek.20
The structures were solved by molecular replacement using
coordinates of the CHK1 kinase domain as a trial model using
CCP4 software.16 Protein and inhibitor were modeled into the
electron density using Quanta,21 COOT,19 and Flynn.24 The models
were refined using CNX17 and the CCP4 program Refmac5.23
Checkpoint Abrogation Assay.6a HT29 cells (3 × 105) were
seeded in 96-well plates and incubated overnight. Cells were treated
for 2 h with camptothecin (topoisomerase I inhibitor, 0.07 μg/mL) to
induce the G2 checkpoint. Cells were then treated for 20 h with vehicle
(0.5% DMSO) or caffeine (4 mmol/L, positive control) plus
nocodazole (1 μg/mL) or a 12-point titration of 4 (12.5 μmol/L to
6 nmol/L) plus nocodazole. Nocodazole alone-treated cells with no
camptothecin pretreatment were used to determine the maximum
mitotic index. Cells were fixed with 3.7% formaldehyde for 1 h,
permeabilized with PBS containing 0.05% Triton X, and incubated
with anti-phH3 antibody for 2 h followed by Alexa Fluor 488 anti-
rabbit (Molecular Probes) and Hoechst stain for 1 h. Mitotic index
was determined on the ArrayScan and expressed as the percentage of
cells undergoing mitosis. The EC50 was calculated by concentration−
response curve fitting using three-variable logistical equations within
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Table 5. Crystallographic Data Collection and Refinement Statistics for Complexes of 1 with c-Jun NH2-Terminal Kinase 1 and
for 2, 3b, and 4 with Checkpoint Kinase 1
parameter
1
2
3b
4
PDB code
protein
3PZE
2YDI
CHK1
P21
2YDJ
2YDK
CHK1
P21
JNK1
CHK1
P212121
space group
cell a, b, c (Å)
cell β (deg)
P212121
49.5, 71.3, 106.7
45.1, 66.0 58.2,
93.8
41.6, 70.8, 104.1
42.0, 64.9, 120,
98.8
resolution limit (Å)
completeness (%)
reflections, unique
multiplicity
32.3−2.0 (2.11−2.0)
96.7 (94.7)
25419
37.2−1.6 (1.69−1.6)
87.6 (39.7)
39316
23.9−1.9 (2.0−1.9)
94.92 (83.8)
23512
43.8−1.85 (1.92−1.85)
84.4 (78.7)
46087
3.5 (3.5)
2.2 (1.2)
2.39 (2.1)
2.44 (2.35)
0.089 (0.39)
5.8 (2.2)
Rmerge (%)
0.096 (0.83)
8.0 (1.5)
0.052 (0.20)
6.2 (4.0)
0.076 (0.38)
5.9 (2)
I/σ(I)
final R for all reflections (work/free) (%)
no. of atoms: protein/ligand/solvent
rmsd values
20.4/24.0
16.7/20.1
18.1/22.6
22.8/27.4
2817/18/197
2216/24/332
2118/24/292
4114/50/414
bond length (Å)
bond angle (deg)
temp factors
0.010
1.12
0.010
1.27
0.012
1.37
0.01
1.24
protein (Å2)
36.5
45.7
42.7
21.9
30.0
35.4
28.3
24.9
37.7
30.5
27.3
39.1
ligand (Å2)
solvent (Å2)
a
Ramachandran plot
most favored (%)
96.8
2.9
0
91.4
8.6
0
89.1
9.6
90
9
additional allowed (%)
generously allowed (%)
disallowed (%)
0.9
0.8
0.2
0.3
0
0.4
a
Percentage of residues in regions of the Ramachandran plot, according to PROCHECK.25 Numbers in parentheses refer to the highest resolution
shell.
Atomic coordinates and structure factors for the JNK1 complex
Julie Tucker and Richard Pauptit for their critical evaluation of
this manuscript.
with compound 1 and the CHK1 complexes with compounds 2, 3b,
and 4 have been deposited in the Protein Data Bank (PDB codes
3PZE, 2YDI, 2YDJ, and 2YDK, respectively) together with structure
factors and detailed experimental conditions. Crystallographic statistics
for the data processing and refinement are shown in Table 5.
ABBREVIATIONS USED
■
APCI, atmospheric pressure chemical ionization; ATP,
adenosine 5-triphosphate; CDCl3, deuterated chloroform; CI,
chemical ionization; CYP450, cytochrome P450; EI, electron
impact; ESP, electrospray; GST, glutathione S-transferase; IHC,
immunohistochemistry; iv, intravenous; LC, liquid chromatog-
raphy; PHH3, phosphohistone H3; TMS, tetramethylsilane;
HTS, high throughput screen; TCU, thiophenecarboxamide
urea; 2-TCU, 2-uriedothiophenecarboxamide urea; 3-TCU, 3-
uriedothiophenecarboxamide urea; DMSO, dimethylsulfoxide;
EtOAc, ethyl acetate; DCM, dichloromethane; DTT, dithio-
thrietol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesul-
fonic acid; THF, tetrahydrofuran; Rochelle’s salt, sodium
potassium tartrate; MgSO4, magnesium sulfate
AUTHOR INFORMATION
Corresponding Author
*Phone: +1 978-394-3088. E-mail: vibha.oza@gmail.com.
■
Present Addresses
⊥Department of Biochemistry and Molecular Biophysics,
Washington University School of Medicine, 660 S. Euclid
Avenue, Box 8231, St. Louis, MO 63110, United States.
#756 Central Park Drive West, 235, Plainfield, IN 46168,
United States.
∞AstraZeneca-MedImmune, One MedImmune Way, Gaithers-
burg, MD 20878, United States.
×AstraZeneca-Innovation Center China (ICC), 898 Halei
Road, Zhangjiang Hi-Tech Park, Pudong New Area 201203,
China.
REFERENCES
■
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○Eleven Cobble Court, Unionville, Connecticut, CT 06085,
United States.
●4310 Trias Street, San Diego, CA 92103, United States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Stefan Geschwindner for the data on JNK1,
Anne White and Anna Valentine for protein production, and
Tina Howard for crystallization support. The authors also thank
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