P Zhang er al/ European Joumal of Medicinal Chemistry 61(2013)95-103 ADME properties are difficult to predict accurately just by in silico and so the predicted properties should be used with caution. For our study, more experiments should be done to explore the real 3. Conclusion ARG 209 SER 26 n summary, we have disclosed a novel series of BTZ compounds as non-ATP competitive GSK-3B inhibitors Based on the structure modification of the hits found from a structure-based docking screening, twenty five btz derivatives were successfully synthe- sized and the in vitro luminescent bioassay showed that about half of them can inhibit GSK-3B with ICso values in micromolar level Among them, three compounds 61, 6t and 6v have ICso values of 25.0 uM, 27.8 HM and 23.0 HM respectively. As the representative compound 6v is proved to reversibly inhibit GSK-3B as non-ATP competitive mechanism through kinetic analysis. Moreover, it almost has not shown any inhibitory activity to a whole panel of other 13 protein kinases, which to some extent suggests good Fig 4. Suggested binding mode for compound 6v in GSK-3B(PDB ID: IPYX) specificity of BIZ compounds with respect to GSK-3B. The results of our primary SAR study also remind us that the nature of BTZ moiety In potentially related to the activ and certain substituents would be important for retaining GSK-3B ore, it is hoped that the prese inhibition. The presence of both a bulky hydrophobic substituent at study of more potent Gsk-3B seem crucial to increase the inhibitory activity. Finally, the binding mode of compound 6v to a non-atP binding pocket of GSK-3B was established by molecular docking and several key interactions were Finally, the adme descriptors module available in Discovery bserved. In this binding mode the affinity seems mainly attrib- Studio(DS)3.0[23 was used to predict a range of drug-like ited to the hydrogen-bonds interaction by the carbonyl and nitro properties for the compound 6v. This protocol uses the qsar groups of inhibitor 6v with Arg 209 and Ser 236 respectively, which models and is suitable to estimate the adme related properties for let the compound more closely bind to the pocket of enzyme and small molecules. The following properties, and classes of proper- hoped that the inhibitory potency and specificity of BTZs toward ties, can be computed such as human intestinal absorption( HIA), GSK-3B shou queous solubility, blood-brain barrier penetration(BBB), cyto low them worth to further study as potenti therapeutic candidates for severe unmet human diseases where chrome P450(CYP450)2D6 inhibition and plasma protein binding. GSK-3B is up-regulated. The resulted data were listed in Table 3. It showed compound 6v might have good intestinal absorption after oral administration and nedium ability to cross the blood-brain barrier (BBB), but its ity predicted was not so good. In addition, 4. Experi compound 6v was likely to inhibit CYP2D6 enzyme, which belongs 4.1. Chemistry to CYP450 enzyme family. The results also showed that the binding between inhibitor and plasma protein is less than 90%, which Reagents were purchased from commercial sources and then proteins in the blood. However, it is a common experience that chromatography was carried out at medium pressure using silica Co. Ltd. All the reactions were monitored by thin layer chroma- 3 tography(TLC)on silica gel. H NMR andC NMR spectra were ADME properties of compound 6v predicted. obtained on Mercury Plus 400 spectrometers working at 400 MHz nd 100 MHz, respectively. Chemical shifts(O)are reported in parts per million(ppm) relative to internal tetramethylsilane(TMs)andJ values are reported in Hertz. MS spectra were recorded on al 00 Agilent LC-MS 1100 instrument with an ESI mass selective detector. Melting points were determined by an SGW X-4 thermometer and AlogP98 were uncorrected(slide method). Level 0 means inhibitor has good human intestinal absorption(HlA)after o 4.1.1. General procedure for the synthesis of 3-substituted acrylic eous solubility of inhibitor is not very good and its drug- A mixture of substituted carbaldehyde(200 mmol), propen dioic acid (20.8 g. 200 mmol) in a solution of pyridin e(10 ml d Level 2 means inhibitor has medium ability to cross the blood-brain barrier 120 mmol )and piperidine(1 ml)was warmed at reflux for 2 h. The e Level 1 means inhibitor likely to inhibit CYP2D6 enzyme. resultant solution was poured into 2 M HCl ag. and then cooled to f Levelo means the binding between inhibitor and plasma protein is less than 90% room temperature. The present solid was collected by filtration, (No markers flagged and Alog P98 4.0). washed with water and recrystallized from ethanol/ water.in an allosteric binding site are potentially related to the active conformation of GSK-3b. Therefore, it is hoped that the present work will be helpful for further study of more potent GSK-3b inhibitors. 2.2.6. Predicting ADME properties Finally, the ADME descriptors module available in Discovery Studio (DS) 3.0 [23] was used to predict a range of drug-like properties for the compound 6v. This protocol uses the QSAR models and is suitable to estimate the ADME related properties for small molecules. The following properties, and classes of proper￾ties, can be computed such as human intestinal absorption (HIA), aqueous solubility, bloodebrain barrier penetration (BBB), cyto￾chrome P450 (CYP450) 2D6 inhibition and plasma protein binding. The resulted data were listed in Table 3. It showed compound 6v might have good intestinal absorption after oral administration and medium ability to cross the bloodebrain barrier (BBB), but its aqueous solubility predicted was not so good. In addition, compound 6v was likely to inhibit CYP2D6 enzyme, which belongs to CYP450 enzyme family. The results also showed that the binding between inhibitor and plasma protein is less than 90%, which means the compound is unlikely to be highly bound to carrier proteins in the blood. However, it is a common experience that ADME properties are difficult to predict accurately just by in silico and so the predicted properties should be used with caution. For our study, more experiments should be done to explore the real properties of these compounds. 3. Conclusion In summary, we have disclosed a novel series of BTZ compounds as non-ATP competitive GSK-3b inhibitors. Based on the structure modification of the hits found from a structure-based docking screening, twenty five BTZ derivatives were successfully synthe￾sized and the in vitro luminescent bioassay showed that about half of them can inhibit GSK-3b with IC50 values in micromolar level. Among them, three compounds 6l, 6t and 6v have IC50 values of 25.0 mM, 27.8 mM and 23.0 mM respectively. As the representative, compound 6v is proved to reversibly inhibit GSK-3b as non-ATP competitive mechanism through kinetic analysis. Moreover, it almost has not shown any inhibitory activity to a whole panel of other 13 protein kinases, which to some extent suggests good specificity of BTZ compounds with respect to GSK-3b. The results of our primary SAR study also remind us that the nature of BTZ moiety and certain substituents would be important for retaining GSK-3b inhibition. The presence of both a bulky hydrophobic substituent at N5 position and an aromatic group at C2 position in BTZ scaffold seem crucial to increase the inhibitory activity. Finally, the binding mode of compound 6v to a non-ATP binding pocket of GSK-3b was established by molecular docking and several key interactions were observed. In this binding mode, the affinity seems mainly attrib￾uted to the hydrogen-bonds interaction by the carbonyl and nitro groups of inhibitor 6v with Arg 209 and Ser 236 respectively, which let the compound more closely bind to the pocket of enzyme and contributed to the inhibitory activity. According to our work, it is hoped that the inhibitory potency and specificity of BTZs toward GSK-3b should allow them worth to further study as potential therapeutic candidates for severe unmet human diseases where GSK-3b is up-regulated. 4. Experimental 4.1. Chemistry Reagents were purchased from commercial sources and then used without further purification except special case. Flash column chromatography was carried out at medium pressure using silica gel (200e300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. All the reactions were monitored by thin layer chroma￾tography (TLC) on silica gel. 1 H NMR and 13C NMR spectra were obtained on Mercury Plus 400 spectrometers working at 400 MHz and 100 MHz, respectively. Chemical shifts (d) are reported in parts per million (ppm) relative to internal tetramethylsilane (TMS) and J values are reported in Hertz. MS spectra were recorded on an Agilent LC-MS 1100 instrument with an ESI mass selective detector. Melting points were determined by an SGW X-4 thermometer and were uncorrected (slide method). 4.1.1. General procedure for the synthesis of 3-substituted acrylic acid A mixture of substituted carbaldehyde (200 mmol), propene￾dioic acid (20.8 g, 200 mmol) in a solution of pyridine (10 ml, 120 mmol) and piperidine (1 ml) was warmed at reflux for 2 h. The resultant solution was poured into 2 M HCl aq. and then cooled to room temperature. The present solid was collected by filtration, washed with water and recrystallized from ethanol/water. Fig. 4. Suggested binding mode for compound 6v in GSK-3b (PDB ID: 1PYX). Table 3 ADME properties of compound 6v predicted. Value Level Absorptiona NVb 0 Solubilityc
4.619 2 BBBd
0.06 2 CYP2D6e 0.722 1 PPBf NV 0 AlogP98f 3.733 0 a Level 0 means inhibitor has good human intestinal absorption (HIA) after oral administration. b NV means no value was given. c Level 2 means the aqueous solubility of inhibitor is not very good and its drug￾likeness properties are low. d Level 2 means inhibitor has medium ability to cross the blood
brain barrier (BBB). e Level 1 means inhibitor likely to inhibit CYP2D6 enzyme. f Level 0 means the binding between inhibitor and plasma protein is less than 90% (No markers flagged and AlogP98 < 4.0). P. Zhang et al. / European Journal of Medicinal Chemistry 61 (2013) 95e103 99