Difluoro-dioxolo-benzoimidazol-benzamides As Potent Inhibitors of

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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.

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INTRODUCTION

use of CK1-specific inhibitors is considered to prove therapeutic potential in the cure of these diseases.8,12,13 Ideally, these inhibitors are targeting CK1 in a highly isoformdependent 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 benzimidazolederived 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-dioxolobenzoimidazole to obtain analogues that are more potent. These

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 © XXXX American Chemical Society

Received: April 17, 2014

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dx.doi.org/10.1021/jm500600b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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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, Xray 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 1 2

CK1α [μM]

CK1γ3 [μM]

CK1δkd [μM]

CK1δ [μM]

CK1ε [μM]

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 GSTCK1δM82F (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.

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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). 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).

RESULTS We previously reported 2-benzamido-N-(1H-benzo[d]imidazol2-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-carboxamide 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 trifluoromethoxy-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 C



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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 compound 1 (10 μM) in a KINOMEscan (KINOMEscan, San

Diego, USA) with a panel of 442 protein kinases showed that CK1 isoforms were potently targeted by compound 1 (CSNK1E D



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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 CH2group 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). 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-dioxolobenzoimidazol based compounds presented in the present manuscript, this interaction could not be detected due to reasons of steric hindrance. 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

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 BxPC3 Calu6 Colo357 HT29 MCF7 MiaPaCa PancTu-1 Panc1

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 0.36 ± 0.07 nd nd 0.46 ± 0.03 nd nd nd nd

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%). Reduction of Molecular Size and Improvement of Physicochemical Properties. Having established a robust binding interaction with difluoro-dioxolo-benzoimidazole bearing 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 2,2difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazole-6amine, 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 E



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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 IC50 = 0.018 μM vs para-substituted counterpart CK1δ IC50 = 0.106 μM, respectively). Synthesis of ortho-substituted compounds 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

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 F



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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 (14)a

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. (A) The potential of compound 2 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 2 was used at its determined IC50 concentration for CK1δkd of approximately 0.07 μM. (B) Compound 2 was used at the determined IC50 concentration to inhibit GST-wt CK1δ (wt) or a GSTCK1δM82F (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.

Figure 6. Effect of tested inhibitor compounds on CK1 kinase activity. Advanced inhibitor compounds were first tested in screening assays on CK1δ kinase domain (CK1δkd), GST-CK1δ, and CK1ε, showing 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.

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). 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

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 G



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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.

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

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 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.

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).

(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

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DISCUSSION AND CONCLUSIONS We previously reported 2-benzamido-N-(1H-benzo[d]imidazol2-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, I



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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 pharmacological characterization and further development, new derivatives of compound 2 might represent interesting substances for therapeutic application in personalized medicine.

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 interactions, 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 GSTp531−64 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 compounds 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 linespecific 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

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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 Chromatography, UK. 1H 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-Difluoro-5H-[1,3]dioxolo [4′,5′:4,5]benzo[1,2-d]imidazol-6-amine hydrobromide (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)-1,1,3,3tetramethyluroniumhexafluorophosphate (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, CHarom), 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[1,2d]imidazol-6-yl)-3-methoxybenzamide (2). 3-Methoxy-benzoyl chloride (0.35 mL, 2.47 mmol) was added to the suspension of 2,2-difluoro5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-amine hydro bromide (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 J



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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). Synthesis of N-(2,2-Difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2d]imidazol-6-yl)-5-methoxynicotinamide (3). To a solution of 5methoxynicotinic acid (100 mg, 0.65 mmol) in 2 mL of N,Ndimethylformamide, 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2d]imidazol-6-amine hydrobromide (14) (211 mg, 0.72 mmol), 2-(1Hbenzotriazole-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 lightyellow solid (165 mg, 0.47 mmol, 73% yield). 1H 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[1,2d]imidazol-6-yl)-2-methoxyisonicotinamide (4). To a solution of 2methoxy-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 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 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[1,2d]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,Ndimethylformamide, 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2d]imidazol-6-amine hydrobromide (14) (211 mg, 0.72 mmol), 2-(1Hbenzotriazole-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[1,2d]imidazol-6-yl)-2-(dimethylamino)isonicotinamide (6). To a solution 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 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. The product was obtained as a yellow solid (100 mg, 0.28 mmol, 43% yield). 1H 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[1,2d]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-

amine hydrobromide (14) (100 mg, 0.34 mmol) was added. Then 2(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (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,Ndimethylformamide, 2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2d]imidazol-6-amine hydrobromide (14) (211 mg, 0.72 mmol), 2-(1Hbenzotriazole-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[1,2d]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-6amine hydrobromide (14) (143.35 mg, 0.487 mmol) was added. Then 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (184.8 mg, 0.487 mmol), 4-dimethylaminopyridine (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-difluoro1,3-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 greyishbeige 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 K



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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 vacuo, and dried to obtain an orange powder. 1H NMR (DMSO-d6; CCl4): 6.95 (1H, s, CH-arom), 7.79 (2H, s, NH2), 7.95 (1H, s, CHarom). 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 vacuo, and dried to obtain a brown solid (98% purity). 1H 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 2,2difluorobenzo[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). Synthesis of Ethyl 2-(2-(Trifluoromethoxy)benzamido)thiazole-4carboxylate (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 (DMSOd6): 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, CHarom), 8.14 (1H, s, CH-thiazole), 13.11 (1H, s, NH). Synthesis of 2-(2-(Trifluoromethoxy)benzamido)thiazole-4-carboxylic Acid (16). Ethyl 2-(2-(trifluoromethoxy)benzamido)thiazole-

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. 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 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-Transferase Fusion Proteins. Expression and purification of the GSTfusion 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. 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. L



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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. 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 adenocarcinoma 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. MTT. Cells were seeded at a concentration of 5 × 104 cells/mL in 96well 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-(4,5dimethylthiazol-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). In Vitro Proliferation Screening. In vitro screening of the effects of compound 2 on the proliferation of a panel of 82 cell lines was performed by Oncolead (Karlsfeld, Germany).

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Present Address §

For J.L.: Medical University of Vienna, Department of Pediatrics, Waehringer Guertel 18, AT-1090 Vienna, Austria. Author Contributions

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.

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ACKNOWLEDGMENTS We thank Jacqueline Krüger and Mario Müller 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 Forschungsgemeinschaft (DFG) (KN356/6-1).

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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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(1) Fish, K. J.; Cegielska, A.; Getman, M. E.; Landes, G. M.; Virshup, D. M. Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family. J. Biol. Chem. 1995, 270, 14875−14883. (2) Graves, P. R.; Haas, D. W.; Hagedorn, C. H.; DePaoli-Roach, A. A.; Roach, P. J. Molecular cloning, expression, and characterization of a 49kilodalton casein kinase I isoform from rat testis. J. Biol. Chem. 1993, 268, 6394−6401. (3) Ospina, B.; Fernandez-Renart, M. Characterization of three casein kinases type I from Dictyostelium discoideum. Biochim. Biophys. Acta 1990, 1052, 483−488. (4) Roof, D. M.; Meluh, P. B.; Rose, M. D. Kinesin-related proteins required for assembly of the mitotic spindle. J. Cell Biol. 1992, 118, 95− 108. (5) Burzio, V.; Antonelli, M.; Allende, C. C.; Allende, J. E. Biochemical and cellular characteristics of the four splice variants of protein kinase CK1alpha from zebrafish (Danio rerio). J. Cell. Biochem. 2002, 86, 805− 814. (6) Fu, Z.; Chakraborti, T.; Morse, S.; Bennett, G. S.; Shaw, G. Four casein kinase I isoforms are differentially partitioned between nucleus and cytoplasm. Exp. Cell Res. 2001, 269, 275−286. (7) Green, C. L.; Bennett, G. S. Identification of four alternatively spliced isoforms of chicken casein kinase I alpha that are all expressed in diverse cell types. Gene 1998, 216, 189−195. (8) Knippschild, U.; Kruger, M.; Richter, J.; Xu, P.; Garcia-Reyes, B.; Peifer, C.; Halekotte, J.; Bakulev, V.; Bischof, J. The CK1 Family: Contribution to Cellular Stress Response and Its Role in Carcinogenesis. Front. Oncol. 2014, 4, 96. (9) Cheong, J. K.; Virshup, D. M. Casein kinase 1: complexity in the family. Int. J. Biochem. Cell Biol. 2011, 43, 465−469. (10) Knippschild, U.; Wolff, S.; Giamas, G.; Brockschmidt, C.; Wittau, M.; Wurl, P. U.; Eismann, T.; Stoter, M. The role of the casein kinase 1 (CK1) family in different signaling pathways linked to cancer development. Onkologie 2005, 28, 508−514. (11) Price, M. A. CKI, there’s more than one: casein kinase I family members in WNT and hedgehog signaling. Genes Dev. 2006, 20, 399− 410. (12) Gill, C.; Walsh, S. E.; Morrissey, C.; Fitzpatrick, J. M.; Watson, R. W. Resveratrol sensitizes androgen independent prostate cancer cells to death-receptor mediated apoptosis through multiple mechanisms. Prostate 2007, 67, 1641−1653.

ASSOCIATED CONTENT

S Supporting Information *

More detailed IC50 and EC50 data as well as cell viability screening results for BxPC3 cells, molecular formula strings, and NMR spectra (PDF, CSV). This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

PDB ID codes used in the present manuscript are: 4TW9 compound 1 in complex with the human CK1δ variant R13N and 4TWC compound Bischof-5 in complex with the human CK1δ variant R13N.18

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*For U.K.: phone, 0049 731 500 53580; fax, 0049 731 500 53582; e-mail, [email protected]. *For M.Z.: phone, 0049 89 700763 92; fax, 0049 89 700763 29; e-mail, [email protected]. M



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