Difluoro-dioxolo-benzoimidazol-benzamides as potent inhibitors of CK1δ and ε with nanomolar inhibitory activity on cancer cell proliferation

Article abstract of DOI:10.1021/jm500600b

Deregulation of CK1 (casein kinase 1) activity can be involved in the development of several pathological disorders and diseases such as cancer. Therefore, research interest in identifying potent CK1-specific inhibitors is still increasing. A previously p

Full text of DOI:10.1021/jm500600b

Article
pubs.acs.org/jmc
Difluoro-dioxolo-benzoimidazol-benzamides As Potent Inhibitors of
CK1δ and ε with Nanomolar Inhibitory Activity on Cancer Cell
Proliferation
Julia Richter,† Joachim Bischof,† Mirko Zaja,*,‡ Hella Kohlhof,‡ Olaf Othersen,‡ Daniel Vitt,‡
Vanessa Alscher,† Irmgard Pospiech,† Balbina García-Reyes,† Sebastian Berg,† Johann Leban,‡,§
and Uwe Knippschild*,†
†Department of General and Visceral Surgery, Ulm University Hospital, Albert-Einstein-Allee 23, D-89081 Ulm, Germany
‡4SC Discovery GmbH, Am Klopferspitz 19a, D-82152 Planegg-Martinsried, Germany
S
* Supporting Information
ABSTRACT: Deregulation of CK1 (casein kinase 1) activity can be involved in
the development of several pathological disorders and diseases such as cancer.
Therefore, research interest in identifying potent CK1-specific inhibitors is still
increasing. A previously published potent and selective benzimidazole-derived
CK1δ/ε-specific inhibitor compound with significant effects on several tumor
cell lines was further modified to difluoro-dioxolo-benzoimidazole derivatives
displaying remarkable inhibitory effects and increased intracellular availability. In
the present study, we identified two heterocyclic molecules as new CK1-specific
inhibitor compounds with favorable physicochemical properties and notable
selectivity in a kinome-wide screen. Being compared to other CK1 isoforms,
these compounds exhibited advanced isoform selectivity toward CK1δ.
Moreover, newly designed compounds showed increased growth inhibitory
activity in a panel of different tumor cell lines as determined by analyses of cell
viability and cell cycle distribution. In summary, presented lead optimization
resulted in new highly selective CK1δ-specific small molecule inhibitors with increased biological activity.
use of CK1-specific inhibitors is considered to prove therapeutic
potential in the cure of these diseases.8,12,13
INTRODUCTION
■
Among the first protein kinases having been described in
literature are members of the ubiquitously expressed serine/
threonine-specific CK1 family.1−4 The human CK1 family
consists of at least six different isoforms (α, γ1, γ2, γ3, δ, and
ε) and related variants which can originate from alternative
splicing.5−8 Within the kinase domain, all CK1 isoforms are
highly conserved with a homology reaching between 52% and
98%, whereas the highly related isoforms CK1δ and ε
demonstrate the highest homology. However, all isoforms and
variants differ significantly in the length and composition of their
N- and C-terminal sequences.
Members of the CK1 family are able to phosphorylate multiple
substrates. This enables CK1 protein kinases themselves to take
important functions in various cellular processes such as DNA
repair, cell cycle progression, cytokinesis, differentiation, and
apoptosis.8−11 The activity of several CK1 isoforms (especially
those of CK1α, δ, and ε) is furthermore linked to central signal
integration molecules like p53 or β-catenin.8,9 Because of the
extraordinary important position of CK1 in these signal
transduction pathways, a tight regulation of CK1 expression
and activity is required. Deregulation of CK1 isoforms can
contribute to the pathogenesis of certain diseases like cancer,
neurodegenerative diseases, and inflammatory disorders, and the
Ideally, these inhibitors are targeting CK1 in a highly isoform-
dependent fashion in order to specifically affect only a single or
few of the important cellular functions of CK1 family members.
So far, numerous CK1-specific inhibitors have been developed,
with IC26114 and D447615 being best characterized for in vitro
and in vivo application. IC261 already demonstrated therapeutic
potential in a subcutaneous mouse xenotransplantation model
for pancreatic cancer.16 However, the cellular effects of this
inhibitor have at least in part been shown to be similar to the
spindle poison colchicine.17 In general, inhibitor compounds
which exhibit increased efficacy and selectivity in vitro might only
have limited potential in vivo due to poor solubility and limited
permeability through the cell membrane. Therefore, the design
of highly isoform-selective inhibitors with increased solubility in
a physiological environment remains a demanding challenge.
We previously reported potent and selective benzimidazole-
derived CK1 inhibitor compounds showing remarkable effects
on several tumor cell lines.18 We now further modified the
benzimidazole moiety in these analogues to a difluoro-dioxolo-
benzoimidazole to obtain analogues that are more potent. These
Received: April 17, 2014
© XXXX American Chemical Society
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Table 1. Structures of CK1-Specific Inhibitors and IC50 Values for CK1δkd, CK1δ, and CK1ε Using Substrate GST-p531‑64
a
Structure and IC50 data adopted from Bischof et al., 2012 for the purpose of comparison.18
analogues allowed us to reduce the size of the molecule to obtain
better biophysical properties. Structure−activity relationship, X-
ray structure of CK1δ and the lead analogue, biochemical
characterization, and inhibitory effects on the proliferation on
various established tumor cell lines are presented for a
representative set of these new analogues.
B
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Table 2. Isoform Specific Inhibition of CK1 Isoforms α, γ3, δkd, δ, and ε by Compounds 1 and 2 Using Substrate α-Casein
CK1α [μM]
CK1γ3 [μM]
CK1δkd [μM]
CK1δ [μM]
CK1ε [μM]
1
2
0.16 0.02
1.36 0.22
0.81 0.25
0.80 0.13
0.02 0.01
0.17 0.04
0.07 0.02
0.13 0.03
0.19 0.04
0.16 0.06
Figure 1. Characterization of the biological activity of compound 1 on CK1δ transcription variants and a M82F gatekeeper mutant. (A) Inhibitor
potency directly depends on the kinase’s phosphorylation state. Expression of recombinant kinase in Escherichia coli at 15 °C for 14 h results in a lower
phosphorylation state of the kinase compared to expression at 37 °C for 2 h. This difference in phosphorylation state directly influences the potency of
compound 1 (indicated by the IC50 value) which was tested on mouse GST-CK1δ TV1 and mouse GST-CK1δ TV2 which both were expressed at 15 °C
for 14 h or at 37 °C for 2 h. (B) The potential of compound 1 to inhibit the kinase activity of CK1δkd was assayed in the presence of increasing ATP
concentrations in order to prove the ATP-competitive properties of this compound class. Compound 1 was used at its determined IC50 concentration
for CK1δkd of approximately 0.02 μM. (C) Compound 1 was used at the determined IC50 concentration to inhibit GST-wt CK1δ (wt) or a GST-
CK1δM82F (M82F) gatekeeper mutant. Phosphate incorporation into the substrate GST-p531−64 (FP267) was quantified by Cherenkov counting.
Obtained data were normalized toward the respective DMSO control reactions. Error bars in A−C represent the standard error of the mean.
compared to that against CK1δ (0.02 μM). Furthermore, to
confirm CK1δ isoform-selectivity of compound 1, its inhibitory
activity on GST-CK1α and GST-CK1γ3 was determined using
α-casein as substrate. In comparison with CK1δ and ε (also
assayed using α-casein), isoforms GST-CK1α and GST-CK1γ3
showed lower sensitivity toward compound 1 (Table 2 and
Supporting Information Figures 1−6).
RESULTS
■
We previously reported 2-benzamido-N-(1H-benzo[d]imidazol-
2-yl)thiazole-4-carboxamide derivatives as potent and specific
CK1δ inhibitors. The most active inhibitor was a compound
bearing a 2-(2-(trifluoromethoxy)benzamido)thiazole-4-carbox-
amide moiety on one side and a substituted benzimidazole
moiety on the other, with a trifluoromethyl group or halogen
being of benefit for activity. This ATP-competitive inhibitor
showed specific hydrophobic interactions between the trifluor-
omethoxy-group and the CK1δ enzyme (compound Bischof-5,
Table 1).18
To maintain the inhibitory effects of this class of inhibitors
toward CK1δ while improving solubility and intracellular
availability, we kept the 2-(2-(trifluoromethoxy)benzamido)-
thiazole-4-carboxamide moiety constant and screened different
readily available benzimidazoles (data not shown). We found a
difluoromethyldioxolo group on the benzimidazole being the
most favorite group (compound 1, Table 1).
Biological Activity of Compound 1. IC50 values against
CK1δkd, GST-CK1δ, GST-CK1δ TV1 (transcription variant 1),
GST-CK1δ TV2, and CK1ε were determined in in vitro kinase
assays using the GST-p531−64 fusion protein (FP267) as
substrate. Indeed, compound 1 showed much lower IC50 values
against CK1δkd (0.01 μM) and GST-CK1δ (0.02 μM) than all
previously published benzimidazole-derived compounds (Table
1).18 Indicating strong selectivity for CK1δ, the IC50 value for
compound 1 and CK1ε (0.21 μM) was nearly 10-fold increased
Because expression of recombinant CK1δ transcription
variants (TV) in bacteria at different temperatures influences
the kinase’s phosphorylation status,18 kinase activity, and
sensitivity of GST-CK1δ TV1 and GST-CK1δ TV2 toward
compound 1 was analyzed. GST-CK1δ TV1 and TV2 induced at
15 °C for 14 h are 1.5- to 4-fold more active and incorporate
more radioactive phosphate into the substrate than CK1δ
transcription variants induced at 37 °C for 2 h. Furthermore,
GST-CK1δ TV1 and GST-CK1δ TV2 expressed at 15 °C for 14
h are more sensitive toward inhibition by compound 1 (Figure
1A). Analysis of the ability of compound 1 to inhibit CK1δkd in
the presence of different amounts of ATP revealed that
compound 1 acts as an ATP-competitive inhibitor (Figure 1B).
Because methionine 82 plays an important role as gatekeeper
residue in the docking mode of isoxazoles to the ATP binding
pocket,19 we also compared the ability of compound 1 to inhibit
GST-wt CK1δ and the GST-CK1δM82F gatekeeper mutant.
Whereas GST-wt CK1δ activity was decreased by 40% in the
presence of 1 in in vitro kinase assays, activity of GST-CK1δM82F
was less affected (20% reduction) (Figure 1C).
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Figure 2. Determination of selectivity of compound 1. To determine target selectivity, a panel of 442 protein kinases was screened for inhibition by
compound 1 at a concentration of 10 μM. (A) Targets showing less than 35% of control activity in the presence of 1. (B) Illustration of phylogenetic
relations of targets listed in (A). Image generated using TREEspot Software Tool and reprinted with permission from KINOMEscan, a division of
DiscoveRx Corporation. Copyright 2010 DiscoveRx Corporation.
Figure 3. X-ray structure of compound 1 in the ATP binding pocket of CK1δ (PDB ID code 4TW9; only chain A depicted). For better visibility, only
kinase-typical structural elements of the backbone (hinge-region in yellow, glycine-rich loop in green, DFG-motif in cyan, and catalytic region in red),
selected residues (nonpolar hydrogen atoms omitted), and heavy atoms of solvent (within 4.5 Å of polar ligand atoms) are pictured. Dotted lines in black
and gray illustrate classical and π−hydrogen bonds. Solid black lines represent distance measurements in Å. The protein surface within 4.5 Å of
compound 1, clipped to allow unobstructed view, portrays hydrophobic, polar, and hydrogen bond interaction possibilities in green, blue, and magenta,
respectively.
Furthermore, characterization of the target selectivity of
Diego, USA) with a panel of 442 protein kinases showed that
compound 1 (10 μM) in a KINOMEscan (KINOMEscan, San
CK1 isoforms were potently targeted by compound 1 (CSNK1E
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0.1%, CSNK1A1L 0.6%, CSNK1A1 3.4%, and CSNK1D 5.8%
relative to controls), while most other kinases were not
significantly bound by 1 (Figure 2). However, compound 1
additionally targeted wild-type and mutant forms of FLT3 kinase
(FLT3 5.4%, FLT3(D835Y) 3.8%, and FLT3(N841I) 3.1%) to a
similar extent as CK1 isoforms.
Binding Mode of Compound 1 to CK1δ. The binding
mode of compound 1 was determined by cocrystallization of
compound 1 and CK1δ protein as described in Experimental
Procedures. The tertiary structure of the protein is well
conserved in comparison to CK1δ as described by Longenecker
and colleagues (1CKI).20 The DFG-motif exhibits the “in”-
conformation. For the analysis of ligand interactions, hydrogen
atoms were added to the X-ray structure using the structure
preparation function with standard parameters of the MOE
software.21
Hinge residues Glu83 and Leu85 are hydrogen bonded via the
backbone to the benzimidazole (aromatic hydrogen and
nitrogen), the nitrogen of the amide bond, and aromatic
hydrogen of the thiazole ring of compound 1, respectively. π−
Hydrogen bonds are observed for the three aromatic regions of
compound 1 with Ile23, Leu84, Leu85, Pro87, and the CH2-
group of Asp91. The slight torque of the ligand ends observed
between the two chains present in the unit cell of the X-ray is
responsible for some minor changes of the π−hydrogen bonds
(e.g., thiazole vs benzene). As further consequence of this torque
the CH2-group of Asp91 is forming a direct hydrogen bond to
compound 1 in chain A, whereas in chain B the contact is
mediated by a water molecule. Further remarkable are the close
contacts of one fluoride of the difluoro-benzodioxolane to the
oxygen atoms of Tyr56 and water with distances between 2.5 and
2.7 Å and the link between Asp149 and compound 1 via a sodium
ion (Na+ to oxygen distance 3.97 Å). The trifluoromethyl group
is pointed into the dent with hydrophobic interaction surface
consisting of Pro87, Asp91, Leu92, Phe95, Leu293, and Phe295.
Solvent hydrogen bonds are observed in the X-ray structure for
most of the capable functional groups (Figure 3).
Figure 4. Comparison of X-ray structures of compound 1 (PDB ID code
4TW9, dark gray) and compound Bischof-5 (PDB ID code 4TWC, light
gray) in the ATP binding pocket of CK1δ (only chain B depicted). For
better visibility, only kinase-typical structural elements of the backbone
(hinge-region in yellow, glycine-rich loop in green, Hyd1 in orange,
DFG-motif in cyan, α-C helix in purple, and catalytic region in red),
Tyr56 and the compounds are pictured. Structures were prepared using
the structure preparation function with standard parameters of the MOE
software.21 Compound 1 exhibits a 0.43 Å (chain B; 0.21 Å in chain A)
shorter fluorine−tyrosine oxygen distance of 2.51 Å (chain B; 2.72 Å in
chain A) than compound Bischof-518 and highly improved hydrogen
bond geometry, e.g., C−F−OTyr angle of 155.3° versus 125.7° (chain
B; 174.6° versus 116.1° in chain A), as no additional close hydrogen
donors for germinal coordination are available.
Table 3. EC50 Values of Most Effective Compounds for
Selected Cell Lines
EC50 value [μM] for compound
cell line
1
2
8
AC1M88
A2780
1.41 0.17
1.66 0.21
0.83 0.05
1.62 0.21
1.57 0.17
1.27 0.09
1.87 0.16
1.93 0.11
3.97 0.17
3.50 0.38
0.18 0.01
0.11 0.01
0.11 0.01
0.07 0.01
0.13 0.02
0.15 0.01
0.36 0.07
0.26 0.02
0.70 0.08
0.35 0.08
0.40 0.10
0.28 0.05
BxPC3
Calu6
0.36 0.07
nd
Compound 1 binds to the ATP binding site as depicted in
Figure 3 in an identical pose observed for a related inhibitor
molecule reported by Bischof et al. in 2012 (see Table 1,
compound Bischof-5).18 A superposition of new compound 1
and compound Bischof-5 is presented in Figure 4. In case of the
gatekeeper mutant GST-CK1δM82F and compound Bischof-5,
previously an additional π−hydrogen could be observed, formed
with the mutated gatekeeper Phe82.18 For the difluoro-dioxolo-
benzoimidazol based compounds presented in the present
manuscript, this interaction could not be detected due to reasons
of steric hindrance.
Colo357
HT29
nd
0.46 0.03
MCF7
nd
nd
nd
nd
MiaPaCa
PancTu-1
Panc1
dose and cell line dependent differences. Whereas the amount of
dead cells only slightly increased up to 16.8% (BxPC3), the
amount of G2-arrested cells increased 2−4-fold upon inhibitor
treatment (BxPC3 35%, MCF7 40%, and HT29 62%).
Efficacy of Compound 1 in Cell Culture. MTT assays were
performed to determine the EC50 values of compound 1 for
selected established tumor cell lines. Whereas BxPC3 and HT29
cells were most sensitive to the treatment with compound 1
(EC50 = 0.8 μM and 1.2 μM, respectively), A2780 and MCF7
cells showed higher EC50 values (1.6 and 1.87 μM, respectively).
The pancreatic tumor cell lines MiaPaCa, Panc1, and PancTu
were less sensitive to the treatment, as indicated by EC50 values of
1.9, 3.5, and 3.9 μM, respectively (Table 3, Figure 5A).
Additionally, FACS analyses were performed to compare the
effects of compound 1 with those of vehicle only (DMSO) with
respect to cell cycle distribution of A2780, BxPc3, HT29, and
MCF7 cells. All cell lines were treated with compound 1 in two
different concentrations (EC50 concentration and 3-fold of EC50
concentration) for 48 h. The results shown in Figure 5B indicate
Reduction of Molecular Size and Improvement of
Physicochemical Properties. Having established a robust
binding interaction with difluoro-dioxolo-benzoimidazole bear-
ing compounds, we aimed to optimize the lead structure by
keeping this feature and the difluoro-dioxolo-benzoimidazole
scaffold in place. We significantly reduced the molecular size by
acylating the 2-amino group of the benzimidazole with simple
and mostly commercially available phenyl acyl residues bearing
different substituents in the meta position, however, still keeping
the CK1δ and ε inhibitory activity (Scheme 1A). Synthesis of 2,2-
difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazole-6-
amine, which served as a building block, is shown in Scheme 1B.
Apart from meta-substituted compounds also para-substituted
counterparts were synthesized. However, in a preliminary
radiometric protein kinase filter binding assay (performed by
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Figure 5. Activity of compound 1 in cell culture. (A) Various cancer cell lines were treated with compound 1 at increasing concentrations and cell
viability was measured in MTT viability assays. Raw data were analyzed and EC50 values calculated using GraphPad Prism 6. Error bars represent the
standard error of the mean. (B) Cell cycle FACS analyses of A2780, BxPC3, HT29, and MCF7 cells were performed in the presence of different
concentrations of compound 1 (EC50 concentration and 3-fold of EC50 concentration) for 48 h, stained with propidium iodide, and analyzed in a flow
cytometer. Control and DMSO-treated cells showed a normal cell cycle distribution of asynchronously proliferating cells. Treatment with compound 1
led to an increase in the amount of dead cells as well as to an increased percentage of cells being arrested in G2 phase of the cell cycle.
Reaction Biology Corporation, Malvern, USA), best results could
be obtained for meta-substituted compounds, thereby providing
a rationale for focusing on meta-substituted derivatives (for
example compound 2, CK1δ IC50 = 0.034 μM vs para-substituted
counterpart CK1δ IC50 = 0.201 μM; and compound 5, CK1δ
decided to replace the ester group with a heterocyclic substituent
that might be isosteric (compound 9, Table 1).
Biological Activity of Compounds 2−9. Initially,
advanced compounds were in vitro screened for their capability
to inhibit CK1δkd, GST-CK1δ, or CK1ε in 10 μM concentration
using GST-p531−64 as substrate (Figure 6). In this first screening,
preservation of the CK1 inhibitory effect of the further developed
compounds could be confirmed. Changes in inhibitory activity
and CK1 isoform selectivity could also be foreseeable by the
results of this screen. Subsequently, also IC50 values were
determined for CK1δkd, GST-CK1δ, and CK1ε in order to
further characterize compounds 2−9 (Supporting Information
Figures 4−6). Remarkable selective inhibitory activity on CK1δ
with IC50 values below 100 nM could be observed for compound
2 (0.07 μM for CK1δkd vs 0.52 μM for CK1ε, see Table 1 and
Supporting Information Figures 4, 5, and 6). To further
characterize isoform selectivity of compounds 2−9, all
compounds were additionally tested for their inhibitory
IC50 = 0.018 μM vs para-substituted counterpart CK1δ IC50
=
0.106 μM, respectively). Synthesis of ortho-substituted com-
pounds remains extremely challenging due to the lack of
appropriate available building blocks and only provided
insufficient results (data not shown).
Meta substitution of the phenyl ring led to active compounds
like the 3-methoxybenzamide derivative 2 and the 3-
(dimethylamino)benzamide derivative 5. Next we introduced a
nitrogen atom into the phenyl ring. This led to nicotinamide 3
and isonicotinamides 4 and 6 with improved solubility and still
moderate activity on CK1. As even the 3-(methylcarbamoyl)-
benzamide derivative 8 showed moderate activity on CK1, we
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Scheme 1. Syntheses of Final Products and Building Block: (A) Synthesis of Final Products (1-9), (B) Synthesis of Building Block
a
(14)
a
(A) (a) HBTU, DIPEA, DMAP, DMF, room temperature, 18 h; (b) pyridine, room temperature, 18 h. The 2,2-difluoro-5H-
[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-amine (14) served as building block and could be easily synthesized from commercially available
5-amino-2,2-difluoro-1,3-benzodioxole as shown in (B). (B) (a) Acetic anhydride, toluene, 100 °C, 2 h; (b) glacial acetic acid, fuming nitric acid,
room temperature, 22 h, then 60 °C, 18 h; (c) sodium methylate, methanol, 0 °C, 20 min, then 5 °C, 25 min; (d) Raney Nickel, methanol,
hydrogen, room temperature, 1 h; (e) cyanogen bromide, methanol, room temperature, 18 h.
acts as ATP-competitive inhibitor (Figure 7A). For compound 2
also inhibition of the CK1δM82F gatekeeper mutant was analyzed.
Figure 7. Characterization of the biological properties of compound 2.
Figure 6. Effect of tested inhibitor compounds on CK1 kinase activity.
(A) The potential of compound 2 to inhibit the kinase activity of
Advanced inhibitor compounds were first tested in screening assays on
CK1δ kinase domain (CK1δkd), GST-CK1δ, and CK1ε, showing
CK1δkd was assayed in the presence of increasing ATP concentrations
in order to prove the ATP-competitive properties of this compound
class. Compound 2 was used at its determined IC50 concentration for
CK1δkd of approximately 0.07 μM. (B) Compound 2 was used at the
distinct effects on kinase activity and isoform specific differences. The
fusion protein GST-p531−64 was used as substrate, compounds were
used at a concentration of 10 μM. Results are shown as normalized bar
graph, error bars indicate the standard error of the mean.
determined IC50 concentration to inhibit GST-wt CK1δ (wt) or a GST-
CK1δM82F (M82F) gatekeeper mutant. Phosphate incorporation into
the substrate GST-p531−64 (FP267) was quantified by Cherenkov
counting. Obtained data were normalized toward the respective DMSO
control reactions. Error bars in (A) and (B) represent the standard error
of the mean.
capability against GST-CK1α and GST-CK1γ3 using α-casein as
substrate. Initial screening at 10 μM concentration detected weak
inhibitory activity on GST-CK1γ3, whereas GST-CK1α shows
higher sensitivity toward the tested compounds (Table 2,
Supporting Information Figure 1).
Here, compound 2 only shows marginal differences in the
inhibition of GST-wt CK1δ and GST-CK1δM82F (Figure 7B).
Characterization of the specificity of compound 2 using the
same panel of 442 kinases, as already shown for 1, demonstrated,
that 2 is still potently targeting CK1 isoforms (CSNK1A1L
0.45%, CSNK1D 0.35%, CSNK1E 0.15%, and CSNK1G2 8.2%
of control) but also members of the DYRK (DYRK1B 3.4% and
DYRK2 8.3%) and CLK families of protein kinases (CLK1 5%,
CLK2 5.7%, and CLK4 3.3%), as well as FLT3 and FTL3
mutants (FLT3 8.6%, FLT3(D835H) 6.5%, FLT3(D835Y)
7.4%, FLT3(K663Q) 7.9%, and FLT3(N841I) 0%) (Figure 8).
Efficacy of Compounds 2−9 in Cell Culture. An initial
screen of compounds 2−9 at 100 nM concentration identified
Determination of IC50 values of compound 2 for GST-CK1α,
GST-CK1γ3, GST-CK1δ, and CK1ε using α-casein as substrate
confirmed isoform selectivity toward CK1δ and CK1ε, as
indicated by 3−6-fold higher IC50 values for CK1α and CK1γ3
than for CK1δ or ε, respectively (CK1α, 1.3 μM; CK1γ3, 0.8 μM,
Table 2). Additionally to the initially performed 10 μM
screening, IC50 values of the most potent compounds for
CK1α and CK1γ3 were determined (Supporting Information
Figures 2 and 3).
To prove the ATP-competitive properties of compound 2,
kinase activity of CK1δkd was assayed in the presence of
increasing ATP concentrations, revealing that compound 2 also
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Figure 8. Determination of selectivity of compound 2. To determine target selectivity, a panel of 442 protein kinases was screened for inhibition by
compound 2 at a concentration of 10 μM. (A) Targets showing less than 35% of control activity in the presence of 2. (B) Illustration of phylogenetic
relations of targets listed in (A). Image generated using TREEspot Software Tool and reprinted with permission from KINOMEscan, a division of
DiscoveRx Corporation. Copyright 2010 DiscoveRx Corporation.
Figure 9. In vitro proliferation screening of compound 2. In an in vitro proliferation screen, the ability of compound 2 to affect the growth of 82
established tumor cell lines was performed by Oncolead (Karlsfeld, Germany). Presented data refer to selected cell lines of colon, pancreas, breast, and
ovary origin. The Z-score indicates the distance of the determined values to the mean calculated for all cell lines.
compounds 2 and 8 as the most potent compounds able to
inhibit growth of BxPc3 cells (Supporting Information Figure 7).
Compound 2 was subsequently used in a proliferation screen to
test its growth inhibitory ability in 82 established tumor cell lines.
Results indicate inhibitory effects on tumor cell growth in
selected cell lines of colon, pancreas, breast, and ovary origin
H
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Figure 10. Activity of compound 2 in cell culture. (A) Several established cancer cell lines were treated with compound 2 at increasing concentrations
and cell viability was measured in MTT viability assays. Raw data were analyzed and EC50 values calculated using GraphPad Prism 6. Error bars represent
the standard error of the mean. (B) Cell cycle FACS analyses of A2780, BxPC3, HT29, and MCF7 cells were performed in the presence of different
concentrations of compound 2 (EC50 concentration and 3-fold of EC50 concentration) for 48 h, stained with propidium iodide, and analyzed in a flow
cytometer. Control and DMSO-treated cells showed a normal cell cycle distribution of asynchronously proliferating cells. Treatment with compound 2
resulted in an increased amount of dead cells as well as in an increase in the percentage of cells being arrested in G2 phase of the cell cycle. Compared to
compound 1, these effects could already be observed at rather low concentrations of compound 2.
(Figure 9). To improve the significance of the proliferation
screen data, MTT assays were performed to determine EC50
values for compounds 2 and 8 on several cell lines (Table 3 and
Supporting Information Figure 8). Being compared to
compound 1, compounds 2 and 8 show increased potential to
affect the viability of all tested tumor cell lines (Table 3).
Compound 2 shows EC50 values between 0.11 μM (BxPC3) and
0.36 μM (MCF7) (Figure 10A). In comparison to compound 1,
which shows an EC50 of 0.826 μM for BxPC3 and 1.874 μM for
MCF7, the potential to be active in cell culture dramatically
increased for compound 2. Additionally carried out FACS
analyses using two different concentrations of compound 2
(EC50 concentration and 3-fold of EC50 concentration) led to
similar results as already shown for compound 1. However,
effects of compound 2 were significantly stronger than previously
observed for 1 and could be obtained from much lower
concentrations used to treat the cultured cells. Treatment with 2
at the determined EC50 concentration (0.1−0.4 μM) led to 21%
(A2780), 25% (HT29), or 31% (MCF7) of dead cells, while the
higher tested concentrations (0.3−1.2 μM) resulted in 33%
(A2780), 49% (BxPC3), 51% (MCF7), and 91% (HT29) of cells
being arrested in G2 phase of the cell cycle (Figure 10B).
DISCUSSION AND CONCLUSIONS
■
We previously reported 2-benzamido-N-(1H-benzo[d]imidazol-
2-yl)thiazole-4-carboxamide derivatives as potent and specific
CK1 inhibitors. We furthermore demonstrated that substitutions
on the phenyl ring of the benzimidazole, such as a trifluoromethyl
group or halogen, are of benefit for inhibitory activity.18
We now found a difluoromethyldioxolo group on the
benzimidazole adding even more to activity and specificity
toward CK1 (compound 1, Table 1) with significantly improved
binding geometry being compared to previously published
compounds (Bischof-5,18 see Figure 4). For compound 1,
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reduced distance to interacting Tyr56 (2.51 vs 2.94 Å for
compound Bischof-5) as well as an additional interaction with
Asp149 could be achieved. Compound 1 showed increased
effects on CK1δ compared to CK1ε and only displayed similar
strong binding to FLT3 wild-type and mutant kinases in a
kinome-wide scan.
Some of the performed structural optimization carried out
using compound 1 indeed kept the improved binding properties
of the difluoro-dioxolo-benzoimidazole scaffold and the specific
inhibitory effects on CK1δ, with compound 2 showing best
effects on CK1δ activity and retained selectivity over CK1ε
(IC50CK1δ = 0.14 μM vs IC50CK1ε = 0.52 μM). We also performed
a KINOMEscan of compound 2 in order to learn more about its
kinase inhibition profile. In addition to its activity on CK1, this
scan also indicated increased targeting of CLK and DYRK
kinases as well as FLT3 and FLT3 mutants. However,
considering the significant reduction in compound complexity
(related to improved physicochemical properties), compound 2
still displays remarkable target selectivity being compared to
complex-structured compound 1.
For both compounds 1 and 2, the ATP-competitive properties
could be confirmed. However, depending on the compound’s
binding mode, its access to the ATP binding site of the kinase
might be influenced by compound- or kinase-specific inter-
actions, respectively. As expected, IC50 values not only depended
on the specific kinase isoform but also on the substrate used for in
vitro assays. This can be clearly seen by comparing the different
IC50 values determined for compounds 1 and 2 using either GST-
p531−64 or α-casein as substrates for CK1δkd, CK1δ, and CK1ε.
For some earlier described isoxazole-derived CK1-specific
inhibitor compounds mutation of the CK1δ gatekeeper residue
methionine 82 to the more bulky phenylalanine effectively
abolished inhibitor binding to CK1δ.19 Thereby, the more
spacious phenylalanine prevents access of the inhibitor molecules
to the kinase’s hydrophobic selectivity pocket.19,22 As illustrated
in Figure 3, compound 1 is not accessing this hydrophobic
pocket and does not establish any remarkable interaction with
the gatekeeper residue Met82. The conclusions drawn from this
X-ray-based models could successfully be confirmed in kinase
reactions using both wild-type and gatekeeper mutant CK1δ
kinase. These results only detected minor differences for the
inhibition of GST-CK1δ and GST-CK1δM82F by compounds 1 or
2. In the case of compound Bischof-5, an additional π−hydrogen
bond could be formed with the mutated gatekeeper Phe82.18
However, due to steric hindrance, this interaction could not be
observed with the difluoro-dioxolo-benzoimidazol based com-
pounds presented in this article.
Moving to cell culture based characterization approaches, an
anticancer screen on a battery of 82 tumor cell lines using
compound 2 revealed an antiproliferative effect on a selected
number of tumor cell lines. More detailed analyses of compounds
1 or 2 using cell lines A2780, BxPC3, HT29, and MCF7 detected
cell line- and concentration-specific effects in MTT and cell cycle
analyses. The observed differences might originate from cell line-
specific differences in p53 expression and mutational status as
well as from WNT-related differences because CK1 isoforms in
general are essentially involved in the regulation of p53- and
WNT-related pathways.8,9 However, comparing the effects of
compounds 1 and 2 on the presented set of cell lines, compound
2 turned out to be much more effective in MTT assays and cell
cycle analyses. This massive increase in cell culture efficacy can be
explained by successfully optimized physicochemical properties
of compound 2, which in contrast to compound 1 exhibits
improved intracellular availability.
In summary, with compound 1, we characterized a potent CK1
isoform-specific small molecule inhibitor which could be
successfully optimized for being inhibitory active and available
within the cell. Following extensive biological and pharmaco-
logical characterization and further development, new derivatives
of compound 2 might represent interesting substances for
therapeutic application in personalized medicine.
EXPERIMENTAL PROCEDURES
General Procedures. Analytical TLC: Merck aluminum sheets,
silica gel 60 F254. Preparative TLC: Merck PLC plates, silica gel 60 F254
■
,
0.5, 1.0, or 2.0 mm. Flash chromatography: Acros silica gel 60A, 0.035−
0.070 mm. Flash Master Personal or Flash Master II, Jones
1
Chromatography, UK. H NMR spectra were recorded with Bruker
Avance at 300 MHz; concentration, 1−5 mg/mL; temperature, 305 K.
The 13C NMR spectra at 75.5 MHz; concentration, 5−20 mg/mL;
temperature, 305 K. The residual solvent peaks were used as internal
standards (DMSO-d6, δH 2.49, δC 39.5; CDCl3, δH 7.24, δC 77.0;
CD3OD, δH 3.30, δC 49.0). Alternatively, TMS was used as a standard
(indicated with TMS). For all tested and final compounds where no
analytical purity is mentioned, compounds were confirmed >95% pure
via HPLC methods. Analytical LC/ESI-MS: Waters Waters 2700
Autosampler, 1 × Waters 1525 multisolvent delivery system, 5 μL
sample loop. Column: Phenomenex Onyx Monolythic C18 50 mm × 2
mm, with stainless steel 2 μm prefilter. Eluent A, H2O + 0.1% HCOOH;
eluent B, MeCN. Gradient, 5% B to 100% B within 3.80 min, then
isocratic for 0.20 min, then back to 5% B within 0.07 min, then isocratic
for 0.23 min; flow, 0.6 mL/min and 1.2 mL/min. Waters Micromass ZQ
4000 single quadrupole mass spectrometer with electrospray source. MS
method, MS4_15 minPM-80−800−35 V; positive/negative ion mode
scanning, m/z 80−800 in 0.5 s; capillary voltage, 3.50 kV; cone voltage,
50 V; multiplier, 650; source block and desolvation gas temperature, 120
and 300 °C, respectively. Waters 2487 dual λ absorbance detector, set to
254 nm. Software, Waters Masslynx V 4.0. Sensitive reactions were
performed under nitrogen. Commercial solvents were used without any
pretreatment. In general, final compounds obtained as solids were
triturated with diisopropylether (DIPE).
Synthesis of Final Compounds. Synthesis of N-(2,2-Difluoro-5H-
[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-2-(2-
(trifluoromethoxy)benzamido)thiazole-4-carboxamide (1). 2,2-Di-
fluoro-5H-[1,3]dioxolo [4′,5′:4,5]benzo[1,2-d]imidazol-6-amine hy-
drobromide (14) (965 mg, 3.28 mmol) and 2-(2-(trifluoromethoxy)-
benzamido)thiazole-4-carboxylic acid (16) (1.09 g, 3.28 mmol) were
dissolved in 10 mL of dry DMF, 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluroniumhexafluorophosphate (HBTU) (1.24 g, 3.28 mmol)
and N,N-diisopropylethylamine (DIPEA) (1.14 mL, 6.55 mmol) were
added, and the reaction mixture was stirred at room temperature for 18
h. The reaction mixture was then concentrated in vacuo. The residue
was dissolved in EtOAc and was washed twice with an aqueous 5%
sodium hydrogen carbonate solution, aqueous 5% citric acid solution,
and water. The organic layer was dried over magnesium sulfate and was
concentrated in vacuo. The product was purified by silica gel flash
chromatography with petrol ether/ethyl acetate. The product was
obtained as a light-yellow solid (104 mg, 0.20 mmol, 6% yield). 1H NMR
(DMSO-d6 + CCl4) δ ppm 7.48 (s, 2H, CH-arom), 7.54−7.59 (m, 2H,
CH-arom), 7.70−7.76 (m, 1H, CH-arom), 7.80−7.83 (dd, 1H, CH-
arom), 8.35 (s, 1H, CH-thiazol), 11.14 (s, 1H, NH), 12.49 (s, 1H, NH),
13.12 (s, 1H, NH).
Synthesis of N-(2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-yl)-3-methoxybenzamide (2). 3-Methoxy-benzoyl chlor-
ide (0.35 mL, 2.47 mmol) was added to the suspension of 2,2-difluoro-
5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-amine hydro bro-
mide (14) (485 mg, 1.65 mmol) in 5 mL of dry pyridine. The resulting
mixture was stirred at room temperature for 18 h. The reaction mixture
was poured into ice water. The precipitate was refluxed in a mixture of
EtOH/H2O/KOH (10 mL/15 mL/0.45 g) during 20 min. The reaction
mixture was cooled down, diluted with 30 mL water, and neutralized
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with HOAc. The precipitate was filtered off and washed with water. It
was recrystallized from water/MeOH/acetone. The crude product was
again recrystallized from acetonitrile. The product was obtained as a
light-beige solid (241 mg, 0.69 mmol, 42% yield). 1H NMR (400 MHz,
DMSO-d6) δ ppm 3.89 (s, 3H, CH3), 7.15−7.24 (m, 2H, CH-arom),
7.41−7.51 (m, 3H, CH-arom), 7.64−7.72 (m, 2H, CH-arom), 11.62−
12.94 (bs, 2H, NH+NH).
amine hydrobromide (14) (100 mg, 0.34 mmol) was added. Then 2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate (HBTU) (129 mg, 0.34 mmol), 4-dimethylaminopyridine (4
mg, 0.03 mmol), and N,N-diisopropylethylamine (0.15 mL, 0.85 mmol)
were added. The reaction mixture was stirred at room temperature for
18 h. The reaction mixture was poured into ice−water, and the resulting
precipitate was filtered off and dried. The crude solid was purified by a
preparative TLC (PLC silica gel 60 F254, 2 mm, eluent: DCM:MeOH
95:5). The product was obtained as a light-yellow solid (21 mg, 0.05
mmol, 16% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.31 (t, J =
73.76 Hz, 1H), 7.44 (dd, J = 2.13 Hz, J = 8.13 Hz, 1H), 7.47 (s, 2H), 7.61
(t, J = 7.98 Hz, 1H), 7.89 (bs, 1H), 7.98 (dt, J = 1.21 Hz, J = 7.84 Hz,
1H), 12.38 (bs, 2H).
Synthesis of Methyl 3-((2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]-
benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate (8). To a solution of
mono-methyl isophthalate (118 mg, 0.65 mmol) in 2 mL of N,N-
dimethylformamide, 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-amine hydrobromide (14) (211 mg, 0.72 mmol), 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) (248 mg, 0.65 mmol), 4-dimethylaminopyridine (8 mg, 0.07
mmol), and N,N-diisopropylethylamine (0.28 mL, 1.63 mmol) were
added. The reaction mixture was stirred at room temperature for 18 h. It
was poured into ice water. The formed precipitate was filtered off and
purified by preparative TLC (DCM/MeOH 95:5). The product was
obtained as a light-yellow solid (28 mg, 0.08 mmol, 11% yield). 1H NMR
(400 MHz, DMSO-d6) δ ppm 3.91 (s, 3H), 7.48 (s, 2H), 7.71 (t, J = 7.77
Hz, 1H), 8.19 (d, J = 7.74 Hz, 1H), 8.36 (d, J = 7.80 Hz, 1H), 8.68 (s,
1H), 12.45 (bs, 2H).
Synthesis of N-(2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-yl)-3-(1H-tetrazol-5-yl)benzamide (9). To a solution of
3-(1H-tetrazol-5-yl)benzoic acid (92.7 mg, 0.487 mmol) in dry DMF (5
mL), 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-
amine hydrobromide (14) (143.35 mg, 0.487 mmol) was added.
Then 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluor-
ophosphate (HBTU) (184.8 mg, 0.487 mmol), 4-dimethylaminopyr-
idine (5.956 mg, 0.049 mmol), and N,N-diisopropylethylamine (0.212
mL, 1.219 mmol) were added. The reaction mixture was stirred at room
temperature for 18 h. The reaction mixture was poured into ice−water,
and the resulting slimy solid was filtered off. The crude product was
dissolved with ethyl acetate and water. The organic layer was washed
twice with an aqueous 5% citric acid solution. The organic layer was
dried over magnesium sulfate and concentrated in vacuo. The crude
product was washed with methanol, ethyl acetate, and a little bit of water.
The solid was filtered off. The product was obtained as a beige solid (21
mg, 0.05 mmol, 11% yield). NMR (400 MHz, DMSO-d6) δ ppm 7.48 (s,
2H), 7.56 (t, J = 7.76 Hz, 1H), 8.00 (d, J = 8.22 Hz, 1H), 8.21 (dt, J =
1.32 Hz, J = 7.73 Hz 1H), 8.68 (t, J = 1.53 Hz, 1H), 12.32 (bs, 2H).
Intermediate Synthesis. Synthesis of N-(2,2-Difluorobenzo[d]-
[1,3]dioxol-5-yl)acetamide (10). A solution of 5-amino-2,2-difluoro-
1,3-benzodioxole (26.0 g, 150.186 mmol) in dry toluene (410 mL) and
acetic anhydride (16.2 mL, 1.15 equiv) was stirred at 100 °C for 2 h.
Subsequently, the solvent was removed under reduced pressure. The
crude product was dissolved in 100 mL of methanol to remove traces of
acetic anhydride. The solvent was subsequently evaporated. The
obtained crude product was recrystallized from toluene. The obtained
product was filtered off and dried under high vacuum to obtain greyish-
beige crystals (30.5 g, 92.5% yield, 98% purity). 1H NMR (DMSO-d6 +
CCl4): 2.04 (3H, s, CH3), 7.20−7.23 (1H, dd, CH-arom), 7.30−7.33
(1H, s, CH-arom), 7.74−7.75 (1H, d, CH-arom), 10.12 (1H, s, NH).
Synthesis of N-(2,2-Difluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)-
acetamide (11). N-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)acetamide
(10) (11.39 g, 52.938 mmol) was dissolved in glacial acetic acid (50.4
mL). To the resulting mixture, a mixture of fuming nitric acid (6.1 mL,
3.35 equiv) in glacial acetic acid (20.2 mL, 0.28 equiv) was added
dropwise. After addition, the reaction mixture was stirred at room
temperature for 22 h. The reaction mixture was then stirred at 60 °C for
18 h. Subsequently, the reaction mixture was poured into a mixture of ice
and water. The resulting precipitate was filtered off by suction filtration.
The crude product was then purified by column chromatography on a
silica gel flash column (eluent DCM:MeOH 95:5). The product was
Synthesis of N-(2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-yl)-5-methoxynicotinamide (3). To a solution of 5-
methoxynicotinic acid (100 mg, 0.65 mmol) in 2 mL of N,N-
dimethylformamide, 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-amine hydrobromide (14) (211 mg, 0.72 mmol), 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate
(HBTU) (248 mg, 0.65 mmol), 4-dimethylaminopyridine (8 mg, 0.07
mmol), and N,N-diisopropylethylamine (0.28 mL, 1.63 mmol) were
added. The reaction mixture was stirred at room temperature for 18 h. It
was poured into ice water. The formed precipitate was washed with
water, DCM, acetone, and MeOH. The product was obtained as a light-
1
yellow solid (165 mg, 0.47 mmol, 73% yield). H NMR (400 MHz,
DMSO-d6) δ ppm 3.92 (s, 3H, CH3), 7.48 (s, 2H), 8.02 (dd, J = 1.83 Hz,
J = 2.79 Hz, 1 H), 8.50 (d, J = 2.82 Hz, 1H), 8.82 (d, J = 1.74 Hz, 1H),
12.43 (bs, 2H).
Synthesis of N-(2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-yl)-2-methoxyisonicotinamide (4). To a solution of 2-
methoxy-4-pyridinecarboxylic acid (100 mg, 0.65 mmol) in 2 mL of
N,N-dimethylformamide, 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]-
benzo[1,2-d]imidazol-6-amine hydrobromide (14) (211 mg, 0.72
mmol), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (HBTU) (248 mg, 0.65 mmol), 4-dimethylaminopyr-
idine (8 mg, 0.07 mmol), and N,N-diisopropylethylamine (0.28 mL,
1.63 mmol) were added. The reaction mixture was stirred at room
temperature for 18 h. It was poured into ice water. The formed
precipitate was washed with water, DCM, acetone, and MeOH. The
product was obtained as a light-yellow solid (74 mg, 0.21 mmol, 33%
yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 3.92 (s, 3H, CH3), 7.42
(bs, 1H), 7.48 (bs, 2H), 7.56 (d, J = 4.80 Hz, 1H), 8.35 (d, J = 5.28 Hz,
1H), 12.44 (bs, 2H).
Synthesis of N-(2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-yl)-3-(dimethylamino)benzamide (5). To a solution of
3-(dimethylamino)benzoic acid (108 mg, 0.65 mmol) in 2 mL of N,N-
dimethylformamide, 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-amine hydrobromide (14) (211 mg, 0.72 mmol), 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) (248 mg, 0.65 mmol), 4-dimethylaminopyridine (8 mg, 0.07
mmol), and N,N-diisopropylethylamine (0.28 mL, 1.63 mmol) were
added. The reaction mixture was stirred at room temperature for 18 h. It
was poured into ice water. The formed precipitate was filtered off and
purified by preparative TLC (DCM/MeOH 95:5). The product was
obtained as a light-yellow solid (37 mg, 0.1 mmol, 16% yield). 1H NMR
(400 MHz, DMSO-d6) δ ppm 2.99 (s, 6H), 6.97 (dt, J = 7.14 Hz, J = 2.30
Hz, 1H), 7.30−7.39 (m, 2H), 7.47 (s, 2H), 11.93 (bs, 1H), 12.50 (bs,
1H).
Synthesis of N-(2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-yl)-2-(dimethylamino)isonicotinamide (6). To a solu-
tion of 2-dimethylamino-isonicotinic acid (109 mg, 0.65 mmol) in 2 mL
of N,N-dimethylformamide, 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]-
benzo[1,2-d]imidazol-6-amine hydrobromide (14) (211 mg, 0.72
mmol), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (HBTU) (248 mg, 0.65 mmol), 4-dimethylaminopyr-
idine (8 mg, 0.07 mmol), and N,N-diisopropylethylamine (0.28 mL,
1.63 mmol) were added. The reaction mixture was stirred at room
temperature for 18 h. It was poured into ice water. The formed
precipitate was filtered off. The product was obtained as a yellow solid
1
(100 mg, 0.28 mmol, 43% yield). H NMR (400 MHz, DMSO-d6) δ
ppm 3.10 (s, 6H), 7.10 (dd, J = 1.32 Hz, J = 5.16 Hz, 1H), 7.27 (bs, 1H),
7.47 (s, 2H), 8.24 (dd, J = 0.50 Hz, J = 5.16 Hz, 1H), 12.35 (bs, 2H).
Synthesis of N-(2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-
d]imidazol-6-yl)-3-(difluoromethoxy)benzamide (7). To a solution of
3-(difluoromethoxy)benzoic acid (64 mg, 0.34 mmol) in 5 mL of dry
DMF, 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-
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isolated and concentrated in vacuo to obtain a yellow solid (6.7 g, 49%
yield, 100% purity). 1H NMR (DMSO-d6 + CCl4): 2.09 (3H, s, CH3),
7.76 (1H, s, CH-arom), 8.15 (1H, s, CH-arom), 10.33 (1H, s, NH).
Synthesis of 2,2-Difluoro-6-nitrobenzo[d][1,3]dioxol-5-amine
(12). N-(2,2-Difluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)acetamide (11)
(6.76 g, 25.985 mmol) was dissolved in methanol (676 mL). The
reaction mixture was cooled to 0 °C. Then sodium methylate (ca. 25% in
methanol) (30.3 mL, 5 equiv) was added and the resulting mixture was
stirred at 0 °C for 20 min and subsequently at 5 °C for 25 min. The
reaction was then interrupted by adding glacial acetic acid (37.2 mL, 25
equiv). The solvent was removed under reduced pressure, whereupon a
liquid−oily residue formed. The last traces of solvent, together with the
remaining glacial acetic acid, were removed by a 2-fold coevaporation
with toluene. Upon addition of toluene, a white solid precipitated, which
was filtered off by suction filtration. The filtrate was concentrated in
vacuo and dried under reduced pressure. The crude product was purified
by column chromatography on a silica gel flash column (eluent
DCM:MeOH 95:5). The first spot as observed by thin layer
chromatography under the same eluent conditions was isolated and
the fractions containing the product were collected, concentrated in
4-carboxylate (15) (10 g, 28 mmol, 1 equiv) was dissolved in THF (20
mL), and an aqueous 2 M NaOH solution (110 mL) was added at RT.
The reaction mixture was stirred at room temperature for 24 h. THF was
then removed under reduced pressure. The residual aqueous phase was
acidified to pH = 1−2 using a 15% aqueous HCl solution. The
precipitate was collected by filtration, washed with water, and dried. The
product was obtained as a white solid (10.4 g, 31 mmol, yield >90%). 1H
NMR (DMSO-d6): 7.51−7.57 (2H, m, CH-arom), 7.68−7.74 (1H, m,
CH-arom), 7.78−7.81 (1H, dd, CH-arom), 8.06 (1H, s, CH-thiazole),
13.01 (1H, s, NH).
X-ray Analysis. Crelux (CRELUX GmbH, Martinsried, Germany)
produced cocrystals of human CK1δ variant R13N with compound 1
that diffract to 2.4 Å resolution on a Bruker AXS Microstar generator
equipped with Montel mirrors. Crystals were obtained using sitting drop
vapor diffusion set-ups. The diluted protein solution (1 mg/mL) was
incubated at 20 °C for 2 h with 15 mM of compound 1 and then
concentrated to 13.5 mg/mL. Then 0.4 μL of protein solution (13.5 mg/
mL in 50 mM HEPES, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 5 mM
β-octyl glucoside, pH 7.5) was mixed with 0.4 μL of reservoir solution
(0.1 M NaCl, 1.4 M (NH4)2SO4, 0.1 M Bis-Tris, pH 5.8) and
equilibrated over 60 μL of reservoir solution. Crystals appeared after 1−
3 days.
1
vacuo, and dried to obtain an orange powder. H NMR (DMSO-d6;
CCl4): 6.95 (1H, s, CH-arom), 7.79 (2H, s, NH2), 7.95 (1H, s, CH-
arom).
The structure was determined using the published CK1δ structure
(PDB accession code 1CKI) as starting model. Several rounds of
alternating manual rebuilding and refinement with REFMAC523,24
resulted in the final model with two receptors in the unit cell.
A Na+ ion modeled in the vicinity of the compound was placed to best
explain observed strong difference electron density features. However, it
has to be kept in mind that with X-ray crystallographic methods, and in
particular at the current resolution, an unambiguous assignment of the
exact nature of the bound ion cannot be made. We opted for Na+ based
on its vicinity to a likely negatively charged aspartate residue (Asp149).
Plasmids. Vectors for bacterial expression of mouse CK1δ
transcription variants 1 (TV1; FP1170) and 2 (TV2; FP1171) were
generated as described elsewhere.18 For the expression of rat CK1δ and
rat CK1δM82F as glutathione S-transferase fusion proteins, the plasmids
pGEX-2T-CK1δ (FP449)25 and pGEX-2T-CK1δM82F (FP1153)19 were
used. Expression of GST-fused bovine CK1α and human CK1γ3 TVX6
was carried out in E. coli expressing plasmids pGEX-2T-CK1α
(FP296)16 or pGEX-2T-CK1γ3, respectively (FP1054; subcloned
from pGBKT7-CK1γ3).26
Synthesis of 2,2-Difluorobenzo[d][1,3]dioxole-5,6-diamine (13).
2,2-Difluoro-6-nitrobenzo[d][1,3]dioxol-5-amine (12) (770 mg, 3.53
mmol) was dissolved in dry methanol (100 mL) under an argon
atmosphere and hydrogenated with an excess of Raney Nickel at room
temperature for 1 h. The reaction mixture was filtered over Celite 545
and washed with methanol. The solvent was evaporated and
concentrated in vacuo. A purple solid precipitated and was dried
under reduced pressure. The crude greyish product (530 mg, 53% yield)
was purified by column chromatography on a silica gel flash column
(eluent DCM: MeOH 95:5). The selected fractions were combined and
concentrated in vacuo. The crude product was precipitated as a
hydrochloride with 1.25 M hydrogen chloride solution in ethanol and an
excess of ethyl acetate. A solid precipitated, and the suspension was
stirred overnight. Subsequently, the solid was filtered off. Afterward, the
obtained 2,2-difluorobenzo[d][1,3]dioxole-5,6-diamine hydrochloride
was dissolved in water and extracted with ethyl acetate. The aqueous
layer was alkalized to pH 8−10 and extracted with ethyl acetate. The
organic layer was then dried over magnesium sulfate, concentrated in
Plasmid pGEX-2T-p531−64 was used to express mouse GST-p531−64
(FP267) for being used as substrate in in vitro kinase reactions.27
Overexpression and Purification of Glutathione S-Trans-
ferase Fusion Proteins. Expression and purification of the GST-
fusion proteins mouse GST-p531−64 (FP267), bovine GST-CK1α
(FP296), human GST-CK1γ3 TVX6 (FP1054), rat GST-CK1δ
(FP449), rat GST-CK1δM82F (FP1153), mouse GST-CK1δ TV1
(FP1170), and mouse GST-CK1δ TV2 (FP1171) were carried out as
described elsewhere.28 Expression of fusion proteins FP1170 and
FP1171 was either performed at 37 °C for 2 h or at 15 °C for 14 h.
FP296, FP1054, FP449, and FP1153 were always expressed at 15 °C for
14 h.
1
vacuo, and dried to obtain a brown solid (98% purity). H NMR
(DMSO-d6 + CCl4): 4.52 (4H, s, 2 × NH2), 6.52 (2H, s, CH-arom).
Synthesis of 2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]-
imidazol-6-amine hydrobromide (14). To a solution of 2,2-
difluorobenzo[d][1,3]dioxole-5,6-diamine (13) (4.91 g, 0.0261 mol)
in dry methanol (98 mL), cyanogen bromide (3.23 g, 1.3 equiv) was
added. The reaction mixture was stirred at room temperature for 18 h.
The reaction mixture was subsequently concentrated in vacuo. The
residue was then washed with dichloromethane and the precipitate was
filtered off and dried to obtain a brown solid (6.72 g, 88% yield, 98%
purity). 1H NMR (DMSO-d6 + CCl4): 7.47 (2H, s, 2 × CH-arom), 8.46
(2H, s, NH2), 12.58 (1H, s, NH).
In Vitro Kinase Assays. In vitro kinase assays were carried out in the
presence of various potential inhibitors of CK1δ at an ATP
concentration of 0.01 mM and DMSO solvent control as described
previously.29 Where indicated, higher ATP concentrations (0.05, 0.1,
and 0.25 mM) were used. Fusion protein mouse GST-p531−64 (FP267)
or α-casein (C6780; Sigma-Aldrich, St. Louis, USA) were used as
substrates. Bovine GST-CK1α (FP296), human GST-CK1γ3 TVX6
(FP1054), recombinant CK1δ kinase domain (CK1δkd, NEB, Frankfurt
am Main, Germany), rat GST-CK1δ (FP449), rat GST-CK1δM82F
(FP1153), mouse GST-CK1δ TV1 (FP1170), mouse GST-CK1δ
TV2 (FP1171), and recombinant human CK1ε (Invitrogen, Karlsruhe,
Germany) were used as sources of enzyme. Phosphorylated proteins
were separated by SDS-PAGE, and the protein bands were visualized on
dried gels by autoradiography. The phosphorylated protein bands were
excised, and phosphorylation was quantified by Cherenkov counting.
Dose−response analyses were carried out using GraphPad Prism 6
(GraphPad Software, La Jolla, USA) statistical software.
Synthesis of Ethyl 2-(2-(Trifluoromethoxy)benzamido)thiazole-4-
carboxylate (15). To a solution of ethyl 2-aminothiazole-4-carboxylate
(12 g, 70 mmol, 1 equiv) in dry THF (300 mL), DIPEA (250 mL, 209
mmol, 3 equiv) was added. A solution of 2-(trifluoromethoxy)-benzoyl
chloride (19 g, 84 mmol, 1.2 equiv) in THF (50 mL) was then added
dropwise at 0 °C. The reaction mixture was stirred at room temperature
for 24 h. Water (50 mL) was then added, and THF was removed under
reduced pressure. The obtained residue was extracted with DCM. The
organic layer was dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash column
chromatography on silica gel (PE/EtOAc 80:20). The product was
obtained as a white solid (12 g, 33 mmol, 48% yield). 1H NMR (DMSO-
d6): 1.28−1.33 (3H, t, CH3), 4.26−4.33 (2H, q, CH2), 7.51−7.57 (2H,
m, CH-arom), 7.68−7.74 (1H, m, CH-arom), 7.78−7.81 (1H, dd, CH-
arom), 8.14 (1H, s, CH-thiazole), 13.11 (1H, s, NH).
Synthesis of 2-(2-(Trifluoromethoxy)benzamido)thiazole-4-car-
boxylic Acid (16). Ethyl 2-(2-(trifluoromethoxy)benzamido)thiazole-
L
dx.doi.org/10.1021/jm500600b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
KINOMEscan: High-Throughput Kinase Selectivity Profiling.
KINOMEscan analyses were performed by Ambit Biosciences
Corporation, San Diego, USA (now part of DiscoveRx Corporation,
Fremont, USA) to determine binding constants of compounds 1 and 2
to 442 eukaryotic kinases.
Present Address
§For J.L.: Medical University of Vienna, Department of
Pediatrics, Waehringer Guertel 18, AT-1090 Vienna, Austria.
Author Contributions
Cell Lines. The human anaplastic adenocarcinoma cell line Calu6,30
the human breast cancer cell line MCF7,31 as well as the pancreatic
cancer cell lines Colo357,32 MiaPaCa,33 PancTu-1,34 and Panc135 were
grown in Dulbecco’s Modified Eagle’s Medium (DMEM). The human
colon adenocarcinoma cell line HT2930 was grown in McCoy’s 5A
medium while the human ovary adenocarcinoma cell line A278036 was
cultivated in RPMI-1640 medium. The human pancreatic adenocarci-
noma cell line BxPC337 was grown in DMEM:RPMI (1:1), whereas the
human extravillous trophoblast/choriocarcinoma hybrid cell line
AC1M88,38,39 generated by fusion of extravillous trophoblasts with
AC1-1, a mutant of the choriocarcinoma cell line Jeg-3, was cultivated in
DMEM/F-12 (1:1). All media were supplemented with 10% fetal calf
serum (FCS; Biochrom, Berlin, Germany), 100 units/mL penicillin, 100
μg/mL streptomycin (Gibco, Karlsruhe, Germany), and 2 mM
glutamine. All cells were grown at 37 °C in a humidified 5% carbon
dioxide atmosphere.
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript. J.R. and J.B. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Jacqueline Kruger and Mario Muller for excellent
■
̈
̈
technical support. We also thank Dr. Ulrike Hars from CRELUX
GmbH for the X-ray crystallographic analysis. The plasmid used
to express GST-CK1α fusion protein was kindly provided by Dr.
David Meek, University of Dundee. This work was supported by
a grant to Uwe Knippschild from the Deutsche Forschungsge-
meinschaft (DFG) (KN356/6-1).
MTT. Cells were seeded at a concentration of 5 × 104 cells/mL in 96-
well cell culture plates and allowed to attach for 24 h at 37 °C and 5%
CO2. To investigate the cytotoxic effects of compounds 1 and 2, cells
were treated with various concentrations (0.01−10 μM) of inhibitor,
with untreated and DMSO-treated cells serving as control. After an
incubation period of 48 h at 37 °C, 10 μL of MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 12 mM MTT
solved in PBS) were added, followed by further incubation for 4 h at 37
°C. Media containing MTT was then removed carefully, and 100 μL of
0.04 N HCl in 2-propanol were added to dissolve formazan crystals by
shaking plates for 30 min on an orbital shaker. Finally, the resulting
purple solution was spectrophotometrically measured at 570 nm.
Experiments were repeated three times with four replicates per assay.
Flow Cytometry and Cell Cycle Analysis. Subconfluent
AC1M88, A2780, BxPC3, Calu6, Colo357, HT29, MCF7, MiaPaCa,
PancTu-1, and Panc1 cells were treated with two different
concentrations (EC50 concentration and 3-fold of EC50 concentration)
of compounds 1 or 2 for 48 h. Untreated and DMSO-treated cells served
as controls. Cells were harvested, washed once with PBS, and prepared
for cell cycle analysis using the “Cycle Test Plus Kit” (BD, San Jose,
USA). Cell cycle profiles were obtained using a FACScan flow cytometer
and CellQuest software (BD Biosciences, San Jose, USA).
ABBREVIATIONS USED
■
CK1α, casein kinase 1 alpha; CK1γ3, casein kinase 1 gamma 3;
CK1δkd, CK1δ kinase domain; CK1δ, casein kinase 1 delta;
CK1ε, casein kinase 1 epsilon; nd, not determined
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* Supporting Information
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Corresponding Authors
*For U.K.: phone, 0049 731 500 53580; fax, 0049 731 500
53582; e-mail, uwe.knippschild@uniklinik-ulm.de.
*For M.Z.: phone, 0049 89 700763 92; fax, 0049 89 700763 29;
e-mail, mirko.zaja@4sc.com.
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