Design and synthesis of N -benzimidazol-2-yl- N' -sulfonyl acetamidines

N -Sulfonyl- N' -benzimidazol-2-yl acetamidines have been designed as CK1 inhibitors. Binding modes in the ATP pocket of CK1  were determined by molecular modeling. The synthetic approach involves sequential acylation of 2-aminobenzimidazoles followed by reaction of amides with Lawesson’s reagent and iminosulfonylation of thioamides with sulfonyl azides. The iminosulfonylation was carried out in boiling ethanol with an equivalent ratio of azides and thioamides. The synthesized compounds were tested for their ability to inhibit CK1 isoforms in vitro and to inhibit the growth of tumor cell lines. Among the synthesized compounds, two products showed inhibitory abilities towards CK1δ and CK1ε.


Introduction
The benzimidazole nucleus is a constituent of numerous therapeutic agents, and its derivatives exhibit various types of bioactivity including anticancer, antimicrobial, antiviral, anti-inflammatory, antiparasitic, antioxidant, proton pump inhibiting, antihypertensive, etc. 1 Modification by an amino group, nitrogen atoms of imidazole ring, and CH bonds of benzene ring also results in formation of biologically active compounds. 1Richter et al. have found that N-aminoacylated benzimidazoles inhibit сasein kinase 1 (CK1) and exhibit nanomolar anticancer activity. 2On the other hand, N-sulfonyl amidines have been introduced as osteoclast differentiation inhibitors, anti-resorptive agents 3 , and dopamine transport inhibitors. 4 It is therefore a worthwhile challenge to develop previously unknown hybrid molecules containing both a benzimidazole and an N-sulfonylamidine group, with the aim of finding new biologically active compounds.

Results and Discussion
Herein, we report a novel synthetic approach to new benzimidazole derivatives bearing N-sulfonyl acetamidine groups, and preliminary data on their inhibition of casein kinases.The design of the structures planned for the CK1 inhibition activity testing, along with the plan to synthesize the desired compounds, are shown in Scheme 1.A series of 2-aminobenzimidazoles bearing various substituents including a dioxole ring, the use of acetic anhydride, acetyl chloride and trifluoroacetic anhydride as acylating reagents, and the use of mesyl azide and various other sulfonyl azides demonstrated the variety of the structures of the planned compounds.Acylation (A), thionation (T) and iminosulfonylation (IS) were steps applied for the retrosynthesis of target molecules.

Scheme 1. Retrosynthetic design of N-benzimidazol-2-yl-N'-sulfonyl acetamidines.
6][7][8] To introduce R 4 of a small size acetic anhydride and trifluoroacetic anhydride were selected.Reactions corresponding to step T involve Lawesson's reagent.They have been applied for the synthesis of 2-thiocarbamoyl benzimidazoles in which R 4 is only aryl. 9The general method for the synthesis of N-sulfonylamidines involves alkylation followed by treatment of alkyl thioimidates with amines 10 and then with sulfonyl chlorides (step IS).However, the low yield of the target compounds forced us to rule out this method from consideration.Recently a N-sulfonylamidines synthesis based on a Cu catalyzed three component reaction 11 was developed and applied to the synthesis of benzimidazol-2-yl-N-sulfonylamidines bearing R 4 aryl and long alkyl groups 12 but cannot be used for the synthesis of amidines of acetic acid (R 4 = Me) designed for testing of biological activity.4][15][16][17][18] This approach has successfully been applied to the synthesis of N-sulfonyl amidines from cyclic thioamides 13 and active methylene thioamides, 4,14 and therefore it was selected as the method of choice for the synthesis of the target compounds.
The starting amides 1a-h were prepared in high yields in the reaction of commercially available 2aminobenzimidazoles with acetyl chloride (or acetic anhydride) and trifluoroacetic anhydride for the synthesis of acetamido (1a-f) and trifluoroacetamido (1g,h) benzimidazoles, respectively.][21] To prepare the targeted sulfonyl amidines 4 we have studied reactions of thioamides 2a-h with a variety of sulfonyl azides 3a-g bearing methyl, aryl, or heteroaryl moieties in various reaction conditions.First we tried to avoid any solvent.The corresponding protocol was successfully used earlier to prepare sulfonylamidines from methylene active thioamides. 4,14However we were not able to isolate any new compounds apart from the initial reagents and some sort of tar.The darkening of the reaction mass took place while no nitrogen evolution being a characteristic feature of azidation of thioamide 4,14 was fixed.The adding a few drops of DMF did not improve the situation.Thioamides 2c,d did not react with sulfonyl azides 1a,b in water, though this solvent was successfully used to prepare other types of amidine. 14,16The reason may involve the very low solubility of thioamides 2 in water.Luckily, the reaction of (benzimidazol-2-yl)ethanethioamides 2a-h with azides 3a-g occurred when the mixture of thioamides 2 and sulfonyl azides 3 in equivalent ratio (for the reaction of thioamide 2f with azide 3c 5.0 equiv. of the azide was used) was heated in boiling ethanol for 2-10 h.We have also found that thioamides of trifluoroacetic acid 2g,h did not react with sulfonyl azides under any conditions.(Scheme 2; Figure 1) The structures of all new compounds were reliably confirmed by the combination of 1 H and 13 C NMR spectroscopy including 2D HMBC and HSQC experiments, as well as mass spectrometry.Thus, 1 H NMR spectra of amides 1d,f, thioamides 2a-h, and amidines 4a-o display methyl group singlets and benzene ring protons at 2.16-2.71and 7.04-8.10ppm, respectively.The characteristic signals in the 13 C NMR spectra at 201.3-203.7 ppm of the thioamides 2a-h, corresponding to carbons of the thioamide group, were observed.Signals of carbons of amidine groups of compounds 4a-o appear at 163.3-167.5 ppm, i.e. close to signals for methylene active N-sulfonyl amidines. 4,14heme 2. Synthesis of (benzimidazol-2-yl)ethanethioamides 2a-h and (benzimidazol-2-yl)acetamidines 4a-o.Additional proof for the proposed structures of compounds 4a-o was achieved by X-ray analysis of a single crystal of 4o which was successfully obtained by crystallization from ethyl acetate (Figure 2).The compound crystallized in the centrosymmetric space group of the triclinic system.The molecule has a Г-like configuration.The bonds lengths and interatomic angles in the molecules do not show any significant deviations from standard.The amidine moiety is fixed in the plane of the heterocycle in the limits 0.1 Å by intramolecular Hbonds, the C-atom of CF 2 -group deviated from the least-squares plane of the heterocycle by 0.2 Å.The fluorophenyl substituent deviated from the plane of the heterocycle, the dihedral angle between the planes of the rings is 72 о and the interatomic angle N(2)-S(1)-C( 8) is 102.55(15)о .In the crystal the molecules form dimeric intermolecular H-bonds NH ... N with participation of the amidine NH-group and N-atom of the imidazole, and NH ... O H-bonds between imidazole and O-atom of the SO 2 -group.

Molecular Modeling
Energetically minimized ligand conformations were docked (see Experimental part) into the active site of CK1 and CK1, respectively.Binding poses were determined and subsequently ranked based on their calculated binding affinities.While we did not obtain a plausible binding mode for CK1, the top ranked binding poses and the corresponding 2D ligand interaction diagrams for CK1 are shown in Figure 3. Consistent with the binding mode of benzimidazole inhibitors in CK1 determined by X-ray crystallography in our study a highly similar situation could be obtained for 2c.Herein, the 2-amino-benzimidazole core is involved in key H-bonds towards the hinge motif Leu85.The CF 3 motif occupies the hydrophic pocket surrounded by Ile148/23, Met80, Tyr56, and Pro66.On the other site, the thioacetate moiety is oriented towards the hydrophobic region which opens to the solvent exposed area.Thus, based on the binding mode of hit fragment 2c, a structure based optimization approach towards more potent CK1 inhibitors will be developed.

Inhibitory effects of selected compounds on the kinase activity of CK1 isoforms
Selected compounds were initially screened for their activity against different CK1 isoforms against a concentration of 10 µM ATP.Under these conditions, the synthesized compounds did not show activities on CK1α and CK1γ3.Modest activities were found for 2c and 4l which slightly inhibit CK1δ and CK1ε respectively.Hit compound 2c showed an IC 50 value of approximately 7 µM for CK1δ and 4l an IC 50 of 4.86 µM for CK1ε (Figure 4).Although these values are significantly weaker than IC 50 values of known highly potent CK1-specific inhibitors, 22 our results suggest that our scaffolds might be improved for further development of potent CK1 isoform kinase inhibitors.X-Ray analysis.The single-crystal X-ray diffraction data for 4o were collected with a "Xcalibur 3" diffractometer (Oxford Diffraction) with CCD detector applying the standard procedure (CuK α -irradiation (λ = 1.54184Å), graphite monochromator, ω-scans with step 1 о at T = 295(2) K).Empirical absorption correction was applied.The structure was solved by direct method with SHELX97 and was refined by full-matrix least squares on F 2 using SHELX97. 23ynthesis.]24 Ethyl 2-acetamido-1H-benzimidazole-5-carboxylate (1d).A solution of ethyl 2-amino-1H-benzimidazole-5carboxylate (1.20 g, 5.85 mmol) in excess of acetic anhydride (5 mL) was refluxed for 1 h.Water (30 mL) was added to the cooled reaction mixture and the resulting suspension was stirred for 30 min.The formed precipitate was filtered off, washed with water, dried and crystallized from ethyl acetate.Colorless powder, yield 1.02 g (70%), mp 293-295 o С. 1

Molecular modeling
Molecular modeling was performed on a DELL Precision T5500 eight core workstation.For visualization Maestro, version 10.4, 2014 (Schrödinger LLC, New York, NY, USA) was used. 25Protein structures were prepared prior to docking by Schrödinger Protein Preparation Wizard synchronizing the following modules: Epik, 26 Impact, and Prime. 27Water molecules beyond 5 Å from hetero atoms have been deleted.H-bond optimization was performed in a standard sampling, the Root-mean-square deviation for atomic positions cutoff of heavy atoms in subsequent protein minimization was set to 0.3 Å.By using this workflow, for CK1δ, we generated a model based on the high-resolution structure PDB 4TWC, 28 and for CK1 based on PDB 4HNI. 29igands were prepared by MacroModel 30 to generate energetically minimized structures.Ionization and tautomeric states were processed by LigPrep utilizing Hammet and Taft methodology-based Epik.Additionally, Epik state penalties were implemented.Receptor grid were generated by Glide SP settings. 29Energetically minimized ligand conformations were docked into the active site of the protein, respectively.Binding poses were determined and subsequently ranked based on their calculated binding affinities.

Biological testing. Materials and methods
In vitro kinase assays.In vitro kinase assays were performed with different CK1 isoforms and selected compounds at an ATP concentration of 10 µM and using DMSO controls as described previously. 32,33Bovine GST-CK1α (FP296) 34 human GST-CK1γ (FP1054) 35 human GST-CK1δ TV1 (FP1417), 36 and recombinant human GST-CK1ε (FP455) 37 were used as sources of enzyme.Phosphorylated proteins were separated by SDS-PAGE and stained with Coomassie.α-casein served as a substrate for all kinase assay reactions.Phosphate incorporation was detected by autoradiography of dried gels.The phosphorylated protein bands were cut out and quantified by Cherenkov counting.Dose-response analyses were carried out using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla CA, USA) statistical software.In vitro kinase assays were performed in the presence or absence of the selected compounds.CK1α, CK1γ3, CK1δ, and CK1ε were used as sources of enzyme and α-casein as substrate.All compounds were used at a concentration of 10 µM.Kinase reactions were separated by SDS-PAGE and quantification of phosphate incorporation was performed by Cherenkov counting.Results are shown as normalized bar graphs using DMSO as a control for 100% kinase activity (dotted line).Error bars represent the standard deviation (SD) (DMSO: dimethyl sulfoxide).Cell culture.The glioblastoma cell line DK-MG 38 was obtained from the Leibniz Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen).Cells were grown in RPMI medium.The medium was 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.The cultures were kept at 37 o C in a humidified 5% carbon dioxide atmosphere.Cell viability assay.Cells were seeded at a concentration of 5 x 10 4 cells/ml in 96-well cell culture plates and allowed to attach overnight at 37 o C and 5% CO 2 .To investigate the effects of selected compounds on cancer cell proliferation, cells were treated with 10 µM of each compound, with untreated and DMSO-treated cells serving as a control.All media were exchanged every two days for fresh treated or control media.After an incubation period of 7 days at 37 o C, 10 µl of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 12 mM solution in PBS] were added, followed by further incubation for 4 h at 37 o C. Media containing MTT was then removed carefully and 100 µl of 0.04 N HCl in isopropanol were added.To dissolve the formazan crystals, the plates were placed for 30 min on an orbital shaker.The resulting purple solution was spectrophotometrically measured at 570 nm with TECAN Spectra II Plate Reader using Magellan3 (TECAN) as software.IC 50 values for 2c and 4l were evaluated for their inhibition of CK1δ and CK1ε, respectively (Figure 4).Determination of the 50% inhibitory concentration (IC 50 ) curves of these compounds on the kinase activities was performed using serial dilutions with CK1δ and CK1ε as enzymes.Kinase reactions were separated by SDS-PAGE and the phosphate incorporation into α-casein was measured by Cherenkov counting.Obtained data were normalized towards their respective DMSO control reactions.Dose-response analyses were performed using GraphPad Prism 6, curves are shown as mean.Error bars represent the standard deviation (SD) (DMSO: dimethyl sulfoxide).

Figure 2 .Scheme 3 .
Figure 2. Compound 4o according to XRD data in the thermal ellipsoids of the 50% probability level.

Figure 3 .
Figure 3. Modeled binding modes of 2c in the ATP-binding pocket of CK1δ (pdb 4TWC).Key amino acid residues and ligand-active site interactions are shown (left).Right: corresponding 2D ligand interaction diagram.