P Zhang et aL/ European Journal of medicinal Chemistry 61(2013)95-103 Table 2 GSK-3B inhibitory activities of compounds 6a-6y. ry activity ( inhibition) of compound 6v(100 HM)against severa R rosine loh Cdk-1/cyclin B 1.5 EPH-A2 -24.3 GFR-Ba-81.8 PKC 12.0 Erbb Su11248 as reference inhibitor for Flt-1(87.1% inhibition), KDR(89.7% inhibi for EGFR(86.9% inhibition). ErbB2(79.4% 2-Thienyl hibition)and ErbB4(82.8% for EPH-A2(83. 1% inhibition), Abl (90.0% d pD173074 as reference inhibitor for RON(93.4% inhibition). 3-Pyridyl 2.2.5. Molecular docking In order to gain an in-depth understanding on the interaction 2-F-Bn 2-CI-Bn mechanism for BTZs within the non-AtP binding pocket of GSK-3B, docking study of compound 6v was performed utilizing the gold 5.0 [21] software. The GSK-3B crystal structure as PDB ID of 1PYX 4-CH,0- was chosen as the model of receptor because it was lately proven to 3-COOMe-Bn be the best one for the non- atP binding in a docking study with three GSK-3B crystal structures(PDB ID: 1PYX, 1Q41, and 1Q4L [22]. The ligand was prepared by minimizing the energy of compound 6v using Sybyl 6.9 31 with the MMFF94 force, and the CI-Ph 2-NOz-B binding site was defined as a sphere of 10 A radius around the 4-Br-Ph 2-NOz-B 78 TDZD-8 residue arg 209, which is suggested to be a key residue for GSK-3B binding process [22]. The suggested binding mode was shown in value of at least two separate determinations, each determina- Fig. 4 and several key interactions were observed. Compound 6v TDZD-8, the first reported non-ATP competitive GSK-3B inhibitor, was used as was located between residues Arg 209 and Ser 236, and its BTZ ring reference compound in this study. ound with Arg 209 by cation-T interaction In addition, there were two hydrogen bonds formed between 6v and the binding pocket. compounds with methyl moiety or without substituent at C2 did One hydrogen bond was attributed to the oxygen of carbonyl on not show any activities(6h, 6i). However, the inhibition potency BIZ ring with Arg 209, and the other one interacted between nitro as enhanced considerably when aromatic groups, such as thienyl, group of the compound and Ser 236. Finally,, C2-subsitituted group furyl, phenyl and benzyl(6f, 6g 6k and 6v) were introduced to O2, of the inhibitor was extended to the hydrophobic region consisting suggesting favorable hydrophobic interactions with the enzyme. of three amino residues (Leu 169, Pro 331 and Thr 330). This In a summary the presence of both a bulky hydrophobic docking mode can well explain the inhibitory activity of compound substituent at N5 position and an aromatic group at C2 position in 6v to GSK-3B. It is worth to note that this result is also consistent the BTZ scaffold(61, 6t, 6u and 6v)seems favorable to increase the with the recently proposed hypothesis by Martinez et al. 221. GSK-3B inhibitory activity. which assumed that three key residues(Arg 209, Thr 235, Ser 236) A2.5 25μM 1.5 25μM control 1[Gs-2] pre-incubation time E+l (min) Fig 3. Kinetic data determined for the compound 6v. (A)ATP concentrations varied from 0.5 uM to 8 uM in the reaction mixture: GS-2 concentration was kept constant at 6.25 HM epicted in the plot. (B)GS-2 concentrations varied from 0.78 uM to 12.5 M in the reaction mixture: ATP concentration was kept constant at 2 HM; ompound concentrations were depicted in the plot. (C)Time dependent GSK-3B inhibition of 6v. Each point was the mean of two separate experiments and each experimentcompounds with methyl moiety or without substituent at C2 did not show any activities (6h, 6i). However, the inhibition potency was enhanced considerably when aromatic groups, such as thienyl, furyl, phenyl and benzyl (6f, 6g, 6k and 6v) were introduced to C2, suggesting favorable hydrophobic interactions with the enzyme. In a summary, the presence of both a bulky hydrophobic substituent at N5 position and an aromatic group at C2 position in the BTZ scaffold (6l, 6t, 6u and 6v) seems favorable to increase the GSK-3b inhibitory activity. 2.2.5. Molecular docking In order to gain an in-depth understanding on the interaction mechanism for BTZs within the non-ATP binding pocket of GSK-3b, a docking study of compound 6v was performed utilizing the GOLD 5.0 [21] software. The GSK-3b crystal structure as PDB ID of 1PYX was chosen as the model of receptor because it was lately proven to be the best one for the non-ATP binding in a docking study with three GSK-3b crystal structures (PDB ID: 1PYX, 1Q41, and 1Q4L) [22]. The ligand was prepared by minimizing the energy of compound 6v using Sybyl 6.9 [31] with the MMFF94 force, and the binding site was defined as a sphere of 10 A radius around the residue Arg 209, which is suggested to be a key residue for GSK-3b binding process [22]. The suggested binding mode was shown in Fig. 4 and several key interactions were observed. Compound 6v was located between residues Arg 209 and Ser 236, and its BTZ ring bound with Arg 209 by cation-p interaction. In addition, there were two hydrogen bonds formed between 6v and the binding pocket. One hydrogen bond was attributed to the oxygen of carbonyl on BTZ ring with Arg 209, and the other one interacted between nitro group of the compound and Ser 236. Finally, C2-subsitituted group of the inhibitor was extended to the hydrophobic region consisting of three amino residues (Leu 169, Pro 331 and Thr 330). This docking mode can well explain the inhibitory activity of compound 6v to GSK-3b. It is worth to note that this result is also consistent with the recently proposed hypothesis by Martinez et al. [22], which assumed that three key residues (Arg 209, Thr 235, Ser 236) Table 1 GSK-3b inhibitory activities of compounds 6ae6y. Compound R1 R2 IC50 (mM)a 6a 2-Thienyl Et >100 6b 2-Thienyl i Pr >100 6c 2-Thienyl n Bu >100 6d 2-Thienyl Cyclohexthylmethyl >100 6e 2-Thienyl Benzoyl >100 6f 2-Thienyl Bn 47.5 6g 2-Furyl Bn 77.2 6h H Bn >100 6i Me Bn >100 6j 3-Pyridyl Bn >100 6k Ph Bn 42.7 6l Ph 2-NO2eBn 25.0 6m Ph 2-CNeBn >100 6n Ph 2-F-Bn >100 6o Ph 2-Cl-Bn >100 6p Ph 2-Br-Bn 73.9 6q Ph 2-CH3eBn 76.1 6r Ph 4-CH3OeBn 73.7 6s Ph 3-COOHeBn >100 6t Ph 3-COOMeeBn 27.8 6u Ph 3-ClePhCOCH2 37.8 6v PhCH2 2-NO2eBn 23.0 6w 4-FePh 2-NO2eBn 81.5 6x 4-ClePh 2-NO2eBn 71.3 6y 4-BrePh 2-NO2eBn 67.8 TDZD-8b e e 1.4 a IC50, the mean value of at least two separate determinations, each determina￾tion was mean of triplicate experiments. b TDZD-8, the first reported non-ATP competitive GSK-3b inhibitor, was used as reference compound in this study. Fig. 3. Kinetic data determined for the compound 6v. (A) ATP concentrations varied from 0.5 mM to 8 mM in the reaction mixture; GS-2 concentration was kept constant at 6.25 mM; compound concentrations were depicted in the plot. (B) GS-2 concentrations varied from 0.78 mM to 12.5 mM in the reaction mixture; ATP concentration was kept constant at 2 mM; compound concentrations were depicted in the plot. (C) Time dependent GSK-3b inhibition of 6v. Each point was the mean of two separate experiments and each experiment performed in triplicate. Table 2 Inhibitory activity (% inhibition) of compound 6v (100 mM) against several protein kinases. Serine/ threonine kinases % Inhibition Tyrosine kinases % Inhibition Tyrosine kinases % Inhibition Cdk-1/cyclin B 1.5 Flt-1a 0.6 ErbB4b 0 CK-II 0 KDRa 5.3 EPH-A2c
24.3 PKA 0 PDGFR-ba
81.8 Ablc 18.1 PKCa 12.0 EGFRb 0 RONd 0 e e ErbB2b 2.7 e e a Su11248 as reference inhibitor for Flt-1 (87.1% inhibition), KDR (89.7% inhibi￾tion) and PDGFR-b (82.1% inhibition). b BIBW2992 as reference inhibitor for EGFR (86.9% inhibition), ErbB2 (79.4% inhibition) and ErbB4 (82.8% inhibition). c Dasatinib as reference inhibitor for EPH-A2 (83.1% inhibition), Abl (90.0% inhibition). d PD173074 as reference inhibitor for RON (93.4% inhibition). 98 P. Zhang et al. / European Journal of Medicinal Chemistry 61 (2013) 95e103