Synthesis, Biological Evaluation and Molecular Modeling of Substituted Indeno[1,2-b]indoles as Inhibitors of Human Protein Kinase CK2

Due to their system of annulated 6-5-5-6-membered rings, indenoindoles have sparked great interest for the design of ATP-competitive inhibitors of human CK2. In the present study, we prepared twenty-one indeno[1,2-b]indole derivatives, all of which were tested in vitro on human CK2. The indenoindolones 5a and 5b inhibited human CK2 with an IC50 of 0.17 and 0.61 µM, respectively. The indeno[1,2-b]indoloquinone 7a also showed inhibitory activity on CK2 at a submicromolar range (IC50 = 0.43 µM). Additionally, a large number of indenoindole derivatives was evaluated for their cytotoxic activities against the cell lines 3T3, WI-38, HEK293T and MEF.

The key bond lengths and angles of the 2-hydroxyindane-1,3-dione moiety of 4d' are very similar to those given in the literature for similarly substituted 2-hydroxyindane-1,3-dione derivatives [20]. The hydroxyindane-1,3-dione system (C13-C14-C15-C16-C17-C18-C19-C20-C21) of compound 4d' is nearly planar with a mean out-of-plane deviation of 0.076 Å with the largest deviation of 0.173(2) Å for atom C13. In this compound, the cyclohexane moiety (C7-C8-C9-C10-C11-C12) is almost planar with a maximum deviation from planarity of 0.278(2) Å found for C7. 4d' contains a cation and an anion in the solid state. The cation is formed by the ammonium function on the isopropylamine molecule. The anion is formed after hydrolysis of the intermediate imine; according to the C-O and C-C bond lengths (ranging from 1.252(3) to 1.255(3) Å, and from 1.399(3) to 1.408(3) Å, respectively) the negative charge is delocalized over C10 and the two adjacent carbonyl functions. In the crystal, the molecules are linked together by intermolecular N-H···O hydrogen bonding between the carbonyl groups of the cyclohexane-dione system and the ammonium group of the isopropylamine. An intramolecular O-H···O hydrogen bond between the hydroxyl group and the carbonyl oxygen of the cyclohexane-dione was also noticed in the solid-state conformation.
To optimize the reaction conditions, 4d was only isolated from the mother liquor and used without further purification for the next step.  . Crystal structure of 4d' with our numbering scheme; displacement ellipsoids are drawn at the 30% probability level. Drawing was performed using the OLEX2 graphical interface [21].

Scheme 2. Synthesis of indeno
After taking into account the results in our pharmacomodulation works, the introduction of a CH3 group at position 7 (D-ring) allowed us to identify compounds 5a, 6a, and 7a as potent CK2 inhibitors. Furthermore, a second substituent, the i-C3H7 group at position 7, showed a favorable effect on CK2 inhibition (e.g., compounds 5b and 6b).
The assumption that indeno [1,2-b]indoles as small and planar molecules exhibit their inhibitory effect towards CK2 by interacting with the ATP binding pocket had already been verified in a previous investigation [13]. In order to validate the ATP competitive mode of inhibition for the CK2 inhibitors described here, the IC50 values of the two most potent inhibitors, 5a and 7a ( Figure 5), were determined at six different ATP concentrations ranging from 6 to 600 µM. IC50 values recorded for the two inhibitors at different ATP concentrations (0.044, 0.097, 0.17, 0.44, 0.76 and 1.17 µM for 5a and 0.13, 0.34, 0.43, 1.27, 3.32 and 5.16 µM for 7a) were observed to linearly increase with the concentration of ATP, thus indicating the ATP competitive mode of CK2 inhibition. The obtained IC50 value at the highest ATP concentration of 600 µM was 27-fold higher for 5a and 40-fold higher for 7a when compared to the IC50 values obtained at the lowest ATP concentration of 6 µM. The IC50 values obtained at the different ATP concentrations were further used to determine the Ki values of 5a and 7a. For this purpose, reaction rates at different inhibitor concentrations were plotted in a Lineweaver-Burk diagram against the varying ATP concentration, resulting in 1 ⁄ as given by the intercepts with the abscissa in both graphs. For determining the Ki values, were plotted against the different inhibitor concentrations as shown for 7a in Figure 6. The Ki value with negative sign was obtained by the intercept with the abscissa and was found to be 144 ± 22 nM with a regression coefficient of R 2 = 0.9941. For 5a, IC50 values obtained with the highest ATP concentrations at 300 and 600 µM appeared to be not in the linear range in the plot, what precluded the determination of the corresponding Ki value. These IC50 values were therefore excluded for this compound. Although the resulting plot exhibited a poor regression coefficient of R 2 = 0.8253, the Ki value for 5a could be obtained by this plot and was found to be 27 ± 12 nM.  were plotted against the corresponding inhibitor concentrations.
Four cell lines of various origins were used to evaluate the cytotoxicity of our target compounds 5-7. The selected cell lines were (i) NIH-3T3, a cell line originally established from the primary mouse embryonic fibroblast cells, (ii) MEF, primary mouse embryonic fibroblasts (used at their early passages) prepared as previously described [24], (iii) WI-38, a human diploid cell line derived from normal embryonic lung tissue, and (iv) HEK293T, a transformed variant of the human embryonic kidney cell line HEK293. After 96 h, cytotoxicity was evaluated using the WST-1 assay. For each compound tested, the EC50 (effective concentration of drug needed to inhibit cell growth/viability by 50%) was generated from the dose-response curves for each cell line. Seventeen compounds were evaluated on the cell lines and then compared to TBB used as a standard. Among the three best CK2 inhibitors (5a, 5b, 7a), compounds 5a and 5b displayed no marked cytotoxic activity, except for compound 5b on the MEF cell line (EC50 = 4.4 µM). After 96 h their EC50 values were superior to 10 µM, similar to TBB. No data were obtained with compound 7a due to the lack of solubility. Four compounds, belonging to the sub-series of D-ring paraquinone derivatives (7c, 7d, 7f, 7g), displayed cytotoxic activity (0.4 < EC50 µM < 8.8), especially on HEK293T.  In order to understand observed differences in the inhibitory activity of compounds 5a, 7a, 5h (R1 = R2 = H) [13] and 7h (R1 = R2 = H) [14] (Table S1 in the supplementary data), the compounds were structurally aligned with the program vROCS [24] onto an inhibitor with pyridocarbazolone scaffold cocrystallized with CK2 (PDB code: 3OWJ [12]). Subsequently, the compounds were energy minimized in the presence of the enzyme with the program Moloc [26], keeping all protein atoms fixed. For this the co-crystallized water molecule 429 was removed, as it clashed with the ligands (distance ≤ 2.2 Å); in contrast, water molecule 382 was kept as its distance to the ligands was  4 Å. In addition, the minimization was performed with all binding site residues within 5 Å of the ligands (L45-R47, S51, V53, V66, K68, I95, F113-V116, H160-N161, M163, and I174-W176) allowed to move, in order to investigate if the ligand binding would cause conformational changes within the binding site. The resulting protein structures showed only minor differences ( Figure S1 in the supplementary data) that are within the experimental uncertainty of the structure determination (root mean-square deviation of the heavy atoms of the binding pocket < 0.4 Å); these differences are mainly caused by optimized intramolecular hydrogen-bonding of the CK2 residues. Thus, we focused on the binding modes of 5a, 7a, 5h and 7h obtained with fixed protein atoms for a better comparison to the crystal structure. The obtained binding modes of 5a, 7a, 5h and 7h agree very well with the one of the co-crystallized inhibitor ( Figure S2 in the supplementary data). These binding modes provide explanations as to the observed differences in the inhibitory activity ( Figure 7): (I) A carbonyl oxygen at position 6 points towards the negatively charged side chain of Asp175 resulting in the vicinity of two hydrogen bond acceptor groups. This unfavorable interaction increases the IC50 value of 7h [14] by a factor of 15 compared to 5h [13]. (II) Methyl substitution in position 7, first, decreases the polarity of the carbonyl group and, second, partially shields the group from solvation in the unbound state, making desolvation effects upon binding less costly. This results in an increase in inhibitory activity for 7-methyl derivatives of the paraquinone series as observed for 7h compared to its methylated analogue 7a (IC50 values 5.55 and 0.43 µM, respectively). (III) The methyl group also makes contacts with the Cα atom of Gly46 (Figure 7). A complete burial of a methyl group can increase a binding constant by up to 10-fold [27]. Here, the methyl group becomes only partially buried, which can explain the only two-fold increase of the inhibitory activity of 5a compared to 5h.

General
Melting points were measured in capillary tubes using a BUCHI 510 apparatus and were uncorrected. IR spectra were recorded on a Perkin Elmer 1310 spectrometer and a Spectrum One spectrometer using KBr pellets ( cm −1 ). 1 H-NMR and 13 C-NMR spectra (broadband decoupling and DEPT-135) were recorded on a Brucker Avance 400 (400 MHz for 1 H and 100 MHz for 13 C) or a Brucker Avance 500 spectrometer (500 MHz for 1 H and 125 MHz for 13 C) using CDCl3 or DMSO-d6 as solvents. NMR analysis of compounds 4-7 was performed with the same experiments described in [22]. Chemical shifts (δ) are referred to that of the solvent. Low resolution mass spectra were recorded on an Agilent 1290 Infinity system equipped with an Agilent 1260 DAD detector and an Agilent 6120 Quadrupole mass detector with an ESI source in positive mode. HRMS spectra were performed on a Q-Tof Micro Water with an ESI source in positive mode. Flash chromatography was performed on 230-400-mesh silica.

Preparation of Recombinant Human CK2 Holoenzyme
The preparation of the human recombinant CK2 holoenzyme was performed according to a protocol described previously [29]. For the expression of the α-subunit (CSNK2A1) and β-subunit (CSNK2B) of the human protein kinase CK2 a pT7-7 expression system in Escherichia coli BL21(DE3) was used. Newly transformed starter cultures were grown overnight at 37 °C in LB-medium to the stationary phase. New medium was inoculated with the separate starter cultures for both subunits and cultivated until an OD500 of 0.6 was reached. Expression was induced by addition of IPTG (1 mM final concentration) and carried out at 30 °C for 5-6 h for CSNK2A1 and at 37 °C for 3 h for CSNK2B. Cells were harvested by centrifugation (6000× g for 10 minutes at 4 °C) and disrupted by sonication (three times 30 seconds on ice). Preparations were then centrifuged to remove the cells debris and the bacterial extracts for both subunits were combined and purified by a three-column procedure. Fractions exhibiting CK2 activity were combined and analyzed by SDS-PAGE and Western Blot.

Capillary Electrophoresis Based Assay for the Testing of Inhibitors of the Human CK2
Testing of the inhibitors of the human CK2 was performed by the recently established capillary electrophoresis based CK2 activity assay of Gratz et al. [23]. Therefore, 2 µL of the dissolved inhibitors (stock solution in DMSO) were mixed with 78 µL of CK2 supplemented kinase buffer which was composed of 1 µg CK2 holoenzyme, 50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2 and 1 mM DTT. The reaction was initiated by the addition of 120 µL assay buffer, which was composed of 25 mM Tris/HCl (pH 8.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µM ATP and 0.19 mM of the substrate peptide RRRDDDSDDD. The reaction was carried out for 15 minutes at 37 °C and stopped by the addition of 4 µL EDTA (0.5 M). Subsequently the reaction mixture was analyzed by a PA800 capillary electrophoresis from Beckman Coulter (Krefeld, Germany). Acetic acid (2 M, adjusted with conc. HCl to a pH of 2.0) was used as the electrolyte for electrophoretic separation. The separated substrate and product peptide were detected at 214 nm using a DAD-detector. Pure solvent was used as negative control (0% inhibition), assays devoid of CK2 were used as positive control (100% inhibition). For primary testing an inhibitor concentration of 10 µM was used. Compounds that revealed at least 50% inhibition at 10 µM were used for IC50 determinations. For the determination of IC50, inhibition was determined using nine inhibitor concentrations ranging from 0.001 µM to 100 µM. IC50 were calculated from the resulting dose-response curves.

Mode of Inhibition and Determination of Ki
The CE based CK2 activity assay as described was used to investigate whether 5a and 7a inhibit CK2 by an ATP competitive mode. For this purpose, enzymatic reactions and IC50 determinations were performed as described in Section 3.3.2., but varying compositions of the assay buffer with respect to the ATP concentration were used. IC50 values were measured at six different final ATP concentrations ranging from 6 to 600 µM. The obtained IC50 values were subsequently used to determine the Ki values of 5a and 7a. For this purpose, reaction velocities at different inhibitor concentrations were plotted in a Lineweaver-Burk diagram against the varied ATP concentrations and the corresponding 1 ⁄ were obtained by the intercepts with the abscissa. Finally the values as obtained were plotted against the different inhibitor concentrations. By this method, the Ki values with a negative sign were given by the intercepts of the linear slope with the abscissa.