Glycogen synthase kinase-3β (GSK-3β) represents a relevant drug target for the treatment of neurodegenerative pathologies including Alzheimer’s disease. We herein report on the optimization of a novel class of GSK-3β inhibitors based on the tofacitinib-derived screen hit 3-((3R,4R)-3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile (1). We synthesized a series of 19 novel 7-chloro-9H-pyrimido[4,5-b]indole-based derivatives and studied their structure–activity relationships with focus on the cyanoacetyl piperidine moiety. We unveiled the crucial role of the nitrile group and its importance for the activity of this compound series. A successful rigidization approach afforded 3-(3aRS,7aSR)-(1-(7-chloro-9H-pyrimido[4,5-b]indol-4-yl)octahydro-6H-pyrrolo[2,3-c]pyridin-6-yl)-propanenitrile (24), which displayed an IC50 value of 130 nM on GSK-3β and was further characterized by its metabolic stability. Finally, we disclosed the putative binding modes of the most potent inhibitors within the ATP binding site of GSK-3β by 1 µs molecular dynamics simulations.

In small molecule binding, water is not a passive bystander but rather takes an active role in the binding site, which may be decisive for the potency of the inhibitor. Here, by addressing a high-energy water, we improved the IC 50 value of our co-crystallized glycogen synthase kinase-3β (GSK-3β) inhibitor by nearly two orders of magnitude. Surprisingly, our results demonstrate that this high-energy water was not displaced by our potent inhibitor (S)-3-(3-((7-ethynyl-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile ((S)-15, IC 50 value of 6 nM). Instead, only a subtle shift in the location of this water molecule resulted in a dramatic decrease in the energy of this high-energy hydration site, as shown by the WaterMap analysis combined with microsecond timescale molecular dynamics simulations. (S)-15 demonstrated both a favorable kinome selectivity profile and target engagement in a cellular environment and reduced GSK-3 autophosphorylation in neuronal SH-SY5Y cells. Overall, our findings highlight that even a slight adjustment in the location of a high-energy water can be decisive for ligand binding.

NO2BF4‐Nitrierungen der Amide (I) in Ethylacetat (auch in aliphatischen Ethern oder Phosphorsäureestern) liefern die Nitroamide (II) in höherer Ausb. als Nitrierungen in Acetonitril oder Methylenchlorid.

Glycogen synthase kinase-3β (GSK-3β) is a potential target in the field of Alzheimer’s disease drug discovery. We recently reported a new class of 9H-pyrimido[4,5-b]indole-based GSK-3β inhibitors, of which 3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile (1) demonstrated promising inhibitory potency. However, this compound underwent rapid degradation by human liver microsomes. Starting from 1, we prepared a series of amide-based derivatives and studied their structure–activity relationships against GSK-3β supported by 1 µs molecular dynamics simulations. The biological potency of this series was substantially enhanced by identifying the eutomer configuration at the stereocenter. Moreover, the introduction of an amide bond proved to be an effective strategy to eliminate the metabolic hotspot. The most potent compounds, (R)-3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)-3-oxopropanenitrile ((R)-2) and (R)-1-(3-((7-bromo-9Hpyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propan-1-one ((R)-28), exhibited IC50 values of 480 nM and 360 nM, respectively, and displayed improved metabolic stability. Their favorable biological profile is complemented by minimal cytotoxicity and neuroprotective properties.

Here we describe the synthesis of N-(6-chloro-3-nitropyridin-2-yl)5-(1-methyl-1H-pyrazol-4-yl)isoquinolin-3-amine via a three-step procedure including a Buchwald–Hartwig arylamination with benzophenone imine and a highly regioselective nucleophilic aromatic substitution. The title compound was analyzed by nuclear magnetic resonance spectroscopy (1H, 13C, HSQC, HMBC, COSY, DEPT90 and NOESY), high resolution mass spectrometry (ESI-TOF-HRMS) and infrared spectroscopy (ATR-IR) and its structure was confirmed by single crystal X-ray diffraction. The inhibitory potency of the title compound was evaluated for selected kinases harboring a rare cysteine in the hinge region (MPS1, MAPKAPK2 and p70S6Kβ/S6K2).