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  • This structure guided the variation


    This structure guided the variation of the compounds aiming either at enhanced interaction with CK1 or to exclude interaction with CK1 (R=Me, 15). We chose the 5-cloro substituted oxoindole as backbone as it is supposed to show enhanced metabolic stability, which is commonly used in medicinal chemistry, for example, Pfizer’s Carprofen (). All compounds were tested in the cellular Aβ generation assay. The four most potent GSIs were subsequently investigated on their CK1δ activity to investigate the influence of CK1δ on the γ-secretase. A further aim of this investigation was the identification of selective GSIs or even GSMs, void off cross-activity on CK1δ or related kinases. The Knoevenagel condensation of these IC261-derivatives utilizes an oxoindole-derivative, a respective aldehyde and piperidine as a base. The reaction is carried out under microwave irradiation at 100°C for 30min to provide the products in moderate to good yields. The -isomer was enriched in the subsequent re-crystallization. The proportion of the / isomers was analyzed by HPLC–MS signal CU CPT 4a synthesis and the HPLC signals were definitely assigned to the molecular mass. (, see Supplementary data) The assignment of the two isomers to the two HPLC signals was established by H NMR-spectroscopy. The isomerization of the pure -isomer to the equilibrium of - and -isomers was monitored by HPLC–MS for (), H NMR-spectroscopy and 2-dimensional-NMR-spectroscopy for (, see Supplementary data) to be complete within 2days in methanol solution, which compares to the assay conditions: buffered HO, 24h. Thus the cellular data are obtained for / mixtures regardless of the purity of the initial isomer. IC261 () is a competitive ATP-binding-site inhibitor. The interaction with this binding-site was reported by Mashhoon et al. based on the co-crystallization (PDB: ) of CK1 with IC261. features both a hydrogen-bond-donor in form of the indole-amin and three methoxy-substituents as hydrogen-bond-acceptors, which can be divided in two - and one -substitution. The structure analysis suggests two hydrogen-bonds of the indole-amine with Asp and Leu. Notable interactions were assigned to the -methoxy-group and Lys and an intermolecular interaction with a benzene hydrogen and interactions of the p-methoxy-group with Ser and Asp (). The symmetric substitution pattern with two -methoxy-groups enables interactions of both rotamers. Both the -isomer purity and the atrop-isomerism found in the co-crystal were captured from the equilibrium conformations in solution. The influence of the o-substitution was determined by a derivative bearing just one -methoxy-group () and several derivatives where the methoxy-groups were replaced by fluorine (), nitro () or sulfonic acid (). Just one out of these derivatives ( Aβ=62%, Aβ=76%, Aβ=65%) displayed a significant decrease in Aβ-levels comparable to the activity of IC261 (Aβ=55%, Aβ=77%, Aβ=77%, ). The single o-substituent and the absence of a p-substituent may cause these minor changes in potency of . A complete loss of activity was observed for those derivatives which lacked -methoxy-groups. This observation demonstrates the essential role of the -methoxy-group which fits exactly into the ATP-binding-site. This assumption was confirmed by introduction of an benzo[δ][1,3]dioxole () which did not CU CPT 4a synthesis display any significant activity on Aβ-levels. This group bears oxygen in the -position, but has an additional methylene bridge to another oxygen in the -position. The activity loss can be explained by sterical hindrance of the dioxole group, which may displace Asp thus disrupting essential hydrogen-bonds. Secondary, m-substitution is detrimental to activity, this was additionally tested by two fluorines () in the m-positions, which displayed surprisingly increased Aβ generation of up to 112% at a concentration of 5μM. This may be due to inverse modulation; such a switch from straight to inverse γ-secretase modulation on a common scaffold was observed previously.,