(S)-3-(3-((7-Ethynyl-9H-pyrimido[4,5-b]indol-4-yl)amino)piperidin-1-yl)propanenitrile

: The title compound ( S )-3-(3-((7-ethynyl-9 H -pyrimido[4,5- b ]indol-4-yl)amino)piperidin-1-yl)propanenitrile ( 2 ) was synthesized in ﬁve steps, starting from 4-chloro-7-iodo-9 H -pyrimido[4,5-b ]indole ( 3 ), and was characterized by 1 H-NMR, 13 C-NMR, MS and HPLC. Moreover, its structure was conﬁrmed by single crystal X-ray diffraction. Pyrimido[4,5- b ]indole 2 demonstrated an IC 50 value of 2.24 µ M in a NanoBRET TM TE intracellular glycogen synthase kinase-3 β assay.


Introduction
Glycogen synthase kinase-3β (GSK-3β) is a highly multi-tasking serine/threonine kinase, which has been associated with various pathologic conditions including type II diabetes, cancer, cardiac hypertrophy and neurodegeneration [1]. Consequently, this disease-relevant enzyme is under ongoing investigation as a potential target for the development of novel drug candidates [2].
We recently reported on the optimization of pyrimido [4,5-b]indole-based inhibitors of GSK-3β, leading to the discovery of lead compound 1, which displayed a micromolar IC 50 value in a NanoBRET TM target engagement (TE) intracellular GSK-3β assay (Promega) ( Figure 1) [3,4]. To assess the contribution of the methyl group to the cellular target engagement potency of 1, this substituent was removed, resulting in the title compound 2.

X-ray Structure
The three-dimensional network within the crystal packing of compound 2 features diverse inter-and intramolecular polar interactions ( Figure 3). These include bidentate hydrogen bonds between the tricyclic scaffolds of adjacent molecules, as well as intramolecular interactions between the secondary aryl amine N-H group and the piperidine nitrogen.

Biological Evaluation
Pyrimido[4,5-b]indole 2 was evaluated in a NanoBRET TM TE intracellular GSK-3β assay [4], where it displayed a two-fold higher IC 50 value compared to parent compound 1 ( Table 1). These data indicate that the removal of the methyl group at the piperidinyl amino function resulted in reduced cellular target engagement potency.

General
All reagents and solvents were of commercial quality and utilized without further purification. The chromatographic retention times of all the reported compounds and the purity of the title compound 2 were determined on an Agilent 1100 Series HPLC system, equipped with an ultraviolet diode array detector (detection at 254 and 230 nm) from Agilent Technologies (Santa Clara, CA, USA). The chromatographic separation was carried out on a XBridgeTM C18 column (150 × 4.6 mm, 5 µm) from Waters (Milford, MA, USA). The injection volume was 5 µL and the flow was 1.5 mL/min, using the following gradient: 0.01 M KH 2 PO 4 , pH 2.3 (mobile phase A), MeOH (mobile phase B), 40% B to 85% B in 8 min; 85% B for 5 min; 85% B to 40% B in 2 min; stop time 16 min. Column chromatography was performed on Geduran Si 60 40-63 µm silica from Merck (Darmstadt, Germany) or commercial 50 µm silica columns from Interchim (Montluçon, France), using an Interchim PuriFlash XS520Plus automated flash chromatography system. NMR spectra were measured on an Avance 300 MHz or an Avance 400 MHz NMR spectrometer from Bruker (Billerica, BA, USA). Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane. All spectra were calibrated against the (residual proton) peak of the deuterated solvent. Mass spectrometry was carried out on an Agilent 6540 UHD Accurate-Mass Q-TOF liquid chromatography coupled electrospray ionization mass spectrometer (LC-ESI-MS) from Agilent Technologies (Santa Clara, CA, USA), at the analytical department of the University of Regensburg. X-ray diffraction data were collected on a STOE IPDS 2T diffractometer (STOE & Cie, Darmstadt, Germany) using monochromated Mo Kα radiation (0.71073 Å). (4) 4-Chloro-7-iodo-9H-pyrimido [4,5-b]indole (3) (170.0 mg, 0.52 mmol), tert-butyl (S)-3aminopiperidine-1-carboxylate (206.6 mg, 1.03 mmol) and DIPEA (200.0 mg, 1.55 mmol) were stirred in n-BuOH at 120 • C for 24 h. After cooling down to room temperature (rt), the mixture was diluted with EtOAc (40 mL) and washed with brine (3 × 20 mL). The organic layer was dried over MgSO 4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO 2 , DCM-MeOH gradient elution from 97.5-2.5 to 93.5-6.5) to obtain 193 mg of the title compound as a brown solid (76% yield).

Biological Evaluation
The NanoBRET TM TE intracellular GSK-3β assay was performed as previously described [4], with minor modifications in the preparation of the HEK293T cells. One day prior to the experiment, cells were treated with trypsin and centrifuged (210 g, 5 min). Afterwards, cells were resuspended in Leibovitz' L-15 medium supplemented with 5% FCS and 10 mM HEPES and were adjusted to a density of 300,000 cells per mL. Meanwhile, the transfection reagent was prepared. For a 96-well plate, 4 µg of transfection carrier DNA (Promega, Fitchburg, WI, USA) and 0.45 µg of NanoLuc ® -GSK3B Fusion vector (Promega) were diluted in 450 µL of L-15 and 13.5 µL of X-tremeGENE TM HP (Roche Diagnostics, Mannheim, Germany) were added. The DNA complex was allowed to form by incubating it for 20 min at rt. Subsequently, the lipid DNA complex was added to 9 mL of the cell suspension. Then, 80 µL of this cell suspension were added to each well of a white 96-well plate (Brand, Wertheim, Germany). The plate was incubated at 37 • C for 24 h (no additional CO 2 ).