Altes Target, Neue Hits - Entwicklung von Inhibitoren für die PIM1-Kinase

Within this work, new compounds for inhibiting the PIM1 kinase were synthesized and investigated in three projects based on different preliminary work. The elucidation of the binding modes of promising candidates using protein crystallography allowed for the directed further development of the corresponding molecular structures using methods of rational drug design. The aim was to identify novel binding motifs in order to make them accessible for hit-to-lead optimization and thus provide starting points for the development of clinically useful PIM1 inhibitors. In the first project, structural proposals from a virtual screening approach were to be synthesized for which, despite intensive studies, no synthesis route could yet be established. After a renewed retrosynthetic analysis, a modification of the key reaction enabled the synthesis of at least two of the target compounds. Of these, compound 2.26 showed moderate activity (IC50 = 228 µM) against the target enzyme, but at the same time exhibited a very well-defined binding mode in the crystal structure and was therefore selected for subsequent optimization. Accordingly, proposals for improving the structure were developed and implemented, taking into account the observed binding position. This included comparative analyses of already published inhibitors as well as considerations on the context of structure-based drug design. Unfortunately, no substantial increase in affinity could yet be achieved, but the gained insights into the binding properties of the structural motif will support the future development of high-affinity derivatives. The protein crystal structure of quinoxaline fragment 3.2 was chosen as the starting point for the second project. Using in silico methods, a number of derivatives were generated from this fragment and investigated via docking. After manual evaluation of the binding mode, promising specimen were synthesized and their biological activity tested. However, these compounds showed no appreciable activity against the PIM1 kinase, casting doubt on the validity of the binding mode from the initial fragment crystal structure. Re-examination of the fragment by X-ray crystallography revealed an alternative binding mode, which had not been considered for generating and evaluating the docking results. In order to circumvent the presumed impairment of the docking hypotheses by the inconsistent binding mode of the fragment, a systematic development via SAR was pursued instead. This process identified compound 3.37 with an IC50 of 89 µM as a promising candidate for further optimization. In the third project, fragment-based structure proposals from a virtual screening were examined for their synthetic feasibility and a series of target compounds and corresponding analogues were synthesized. Compound 4.70 with an IC50 of 26.4 µM was identified as a suitable starting point for further optimization. Based on the crystal structure of 4.70 in complex with PIM1, various structural classes were derived that were intended to combine the relevant structural features with easier synthetic accessibility. These included the group of stilbenes (4.77) and indolin-2-on 4.131. For the stilbenes, an IC50 between 1.60 µM and 4.32 µM was achieved depending on the substitution pattern. Furthermore, different binding modes could be determined for cis- and trans-isomers on the basis of the respective protein crystal structures, although the cis-isomers exhibited an unstable configuration. In order to simplify the synthesis, cyanostilbene 4.95 was derived from this substance class, which showed an IC50 of 1.72 µM. Due to the convenient synthetic access, this class of compounds represents an excellent starting point to strive for optimization of the already achieved activity through SAR or computer-assisted modification. Based on the effort to stabilize the cis-stilbenes in their configuration, accordingly substituted five-membered heterocycles were developed. Among those, triazole 4.114 with an IC50 of 4.49 µM also represents a suitable starting point for the further development of inhibitors. The indolin-2-one-compound 4.131 showed the highest affinity of all compounds investigated in this work with an IC50 of 0.60 µM. Utilizing a SAR, the significance of individual structural features for the affinity could be determined; in the course of this, positions for the directed further development were also identified. By using the crystal structure of a suitable analogue, it was also possible to determine the binding mode of this substance class, so that computer-assisted methods as well as structure-based drug design can be applied for further optimization. Overall, a number of hits with activity against the PIM1 kinase within the single-digit micromolar range were identified in the course of this work. The development of reliable synthesis routes combined with the systematic analysis of the relevant structural motifs also laid the foundation for subsequent efforts to optimize these compounds into high-affinity inhibitors.