P Zhang er al/ European Joumal of Medicinal Chemistry 61(2013)95-10 3a-3j 5a与 6a-6r,6u-6y Scheme 1.(a)CH2(COOH), pyridine, piperidine, 2 h, reflux:( b)2-aminobenzenethiol, 4 A molecular sieve, 6h, 180C: (c)RX, X=CI or Br or l,NaH,DMF,0.5h,-10C. 2.2. Biological evaluation and SAR study preincubation time were measured and the results were shown in Fig 3C. Normally, the inhibition effect of reversible inhibitors does 2.2.1. GSK-3B inhibitio not increase at different incubation time. while an irreversible All the newly prepared BTZ derivatives were evaluated for their inhibitor increases the inhibition percentage as it increases the time GSK-3B inhibitory activity by a recently well described Kinase-GloTm of incubation with the enzyme As could be seen in Fig. 3C, the luminescent technique, which is regarded as a safer nonradioactive activities of 6v were almost kept unchanged with the increasing of assay 20. GS-2 is the most utilized substrate of GSK-3B as a small the pre-incubation time, which indicates compound 6v acts as peptide similar to skeletal muscle glycogen synthase. It is a reversible GSK-3B inhibitor. composed of 26-amino acids and contains a prephosphorylated serine residue. In our assay, we used the prephosphorylated 12-2 23. Kinase selectivity studies amino acids polypeptide substrate 65 HSSPHQ(pS)EDEEE,which High selectivity of protein kinase inhibitors is critical to avoid is as same as the one used by Andrea Baki in a high throughput despread effects in a potential therapy. Thus for evaluating the GSK-3B enzyme was incubated with ATP and GS-2 in the presence sentative compound 6v was assayed against a panel of other 13 or absence of the tested compound, and then the amount of ATP kinases. These kinases include four serine/threonine kinases as emaining in solution, which inversely correlates to kinase activity, Cdk-1/cyclin B, CK-ll, PKA and PKCa which are highly close to GSK was quantified following the kinase reaction. The ICso values are 3B, and nine tyrosine kinases as Flt-1, KDR, PDGFR-B, EPH-A2, EGFR, listed in Table 1 and it can be seen that some of compounds really ErbB2, ErbB4, RON and Abl, which play important role in cancer inhibit GSK-3B in micromolar scale. Among them the best three signaling pathways. All of the inhibitory assays were carried out at compounds 61, 6t and 6v have the ICso values of 25.0 uM, 27. 8 uM a 100 uM of 6v concentration. The serine/ threonine kinases inhi- and 23.0 uM, respectively bition were performed at Millipore Corporation(Dundee, UK) by KinaseProfilerTM service, and the tyrosine kinases screen was 2. 2. 2. Mode of inhibition assayed utilizing an ELISA approach as described in Experimental In order to explore the biological mechanism of BTZ derivatives, section. The results listed in Table 2 showed that compound 6v compound 6v was used to study the kinetic features. We firstly tested almost displayed no inhibitory activity against the whole set of whether a competition effect exists between 6v and ATP. That is, kinases, which to some extent demonstrates that BTZs might have eping the concentration ofGS-2 unchanged the enzyme inhibitor good specificity with respect to GSK-3B activities of compound 6v were measured at its two differer concentrations separately while AtP concentrations varied. The 2. 2. 4. Structure-activity relationships (SAR)analysis results are showed in Fig 3A and it can be seen from the double In order to explore the features between the chemical reciprocal plotting of the data, in which compound 6v acts as a non structures and their GSK-3B inhibition, a preliminary structure ATP competitive inhibitor. Then we explored that whether there is activity relationship of BTzs was analyzed based on the preceding a competitive relationship between 6v and GS-2. In these experi- in vitro data, which were listed in Table 1. The substituents ments, GS-2 concentrations varied and atP concentration was kept attached to the n5 of the btz ring seemed to be important for unchanged as the activities of 6v were measured at concentrations of keeping the activities, of which the benzyl group was the best one 25 HM and 50 uM separately. The results were showed in Fig. 3B and(6f vs 6a-e)in the test set. Furthermore, ortho-nitro group and the double reciprocal plotting of the data in this figure indicates that meta-carbomethoxy group could dramatically contribute to the compound 6v acts as a noncompetitive inhibitor of GS-2 inhibitory potency as attached to the benzyl moiety( 61, 6t). To further investigate the interaction feature of Blzs to the On the other hand. the size and nature of the substituents r enzyme, the inhibitory activities of compound 6v at different attached to the c2 should also be crucial for inhibition. The b COOMe Scheme 2. Reagents and conditions: (a)conc. Ha1, 100oC,7 h; (b)SoCl, MeOH, reflux.2.2. Biological evaluation and SAR study 2.2.1. GSK-3b inhibition All the newly prepared BTZ derivatives were evaluated for their GSK-3b inhibitory activity by a recently well described Kinase-Glo luminescent technique, which is regarded as a safer nonradioactive assay [20]. GS-2 is the most utilized substrate of GSK-3b as a small peptide similar to skeletal muscle glycogen synthase. It is composed of 26-amino acids and contains a prephosphorylated serine residue. In our assay, we used the prephosphorylated 12- amino acids polypeptide substrate 650HSSPHQ (pS)EDEEE, which is as same as the one used by Andrea Baki in a high throughput luminescent assay for looking for GSK-3b inhibitors [20]. Briefly, GSK-3b enzyme was incubated with ATP and GS-2 in the presence or absence of the tested compound, and then the amount of ATP remaining in solution, which inversely correlates to kinase activity, was quantified following the kinase reaction. The IC50 values are listed in Table 1 and it can be seen that some of compounds really inhibit GSK-3b in micromolar scale. Among them the best three compounds 6l, 6t and 6v have the IC50 values of 25.0 mM, 27.8 mM and 23.0 mM, respectively. 2.2.2. Mode of inhibition In order to explore the biological mechanism of BTZ derivatives, compound 6v was used to study the kinetic features.We firstly tested whether a competition effect exists between 6v and ATP. That is, keeping the concentration of GS-2 unchanged, the enzyme inhibitory activities of compound 6v were measured at its two different concentrations separately while ATP concentrations varied. The results are showed in Fig. 3A and it can be seen from the double reciprocal plotting of the data, in which compound 6v acts as a non￾ATP competitive inhibitor. Then we explored that whether there is a competitive relationship between 6v and GS-2. In these experi￾ments, GS-2 concentrations varied and ATP concentration was kept unchanged as the activities of 6v were measured at concentrations of 25 mM and 50 mM separately. The results were showed in Fig. 3B and the double reciprocal plotting of the data in this figure indicates that compound 6v acts as a noncompetitive inhibitor of GS-2. To further investigate the interaction feature of BTZs to the enzyme, the inhibitory activities of compound 6v at different preincubation time were measured and the results were shown in Fig. 3C. Normally, the inhibition effect of reversible inhibitors does not increase at different incubation time, while an irreversible inhibitor increases the inhibition percentage as it increases the time of incubation with the enzyme. As could be seen in Fig. 3C, the activities of 6v were almost kept unchanged with the increasing of the pre-incubation time, which indicates compound 6v acts as a reversible GSK-3b inhibitor. 2.2.3. Kinase selectivity studies High selectivity of protein kinase inhibitors is critical to avoid widespread effects in a potential therapy. Thus for evaluating the selectivity of BTZ compounds as potential inhibitors, the repre￾sentative compound 6v was assayed against a panel of other 13 kinases. These kinases include four serine/threonine kinases as Cdk-1/cyclin B, CK-II, PKA and PKCa which are highly close to GSK- 3b, and nine tyrosine kinases as Flt-1, KDR, PDGFR-b, EPH-A2, EGFR, ErbB2, ErbB4, RON and Abl, which play important role in cancer signaling pathways. All of the inhibitory assays were carried out at a 100 mM of 6v concentration. The serine/threonine kinases inhi￾bition were performed at Millipore Corporation (Dundee, UK) by KinaseProfiler service, and the tyrosine kinases screen was assayed utilizing an ELISA approach as described in Experimental section. The results listed in Table 2 showed that compound 6v almost displayed no inhibitory activity against the whole set of kinases, which to some extent demonstrates that BTZs might have good specificity with respect to GSK-3b. 2.2.4. Structureeactivity relationships (SAR) analysis In order to explore the main features between the chemical structures and their GSK-3b inhibition, a preliminary structuree activity relationship of BTZs was analyzed based on the preceding in vitro data, which were listed in Table 1. The substituents R2 attached to the N5 of the BTZ ring seemed to be important for keeping the activities, of which the benzyl group was the best one (6f vs 6aee) in the test set. Furthermore, ortho-nitro group and meta-carbomethoxy group could dramatically contribute to the inhibitory potency as attached to the benzyl moiety (6l, 6t). On the other hand, the size and nature of the substituents R1 attached to the C2 should also be crucial for inhibition. The a bc Scheme 1. (a) CH2(COOH)2, pyridine, piperidine, 2 h, reflux; (b) 2-aminobenzenethiol, 4 A molecular sieve, 6 h, 180 C; (c) R2 X, X ¼ Cl or Br or I, NaH, DMF, 0.5 h,
10 C. a b Scheme 2. Reagents and conditions: (a) conc. HCl, 100 C, 7 h; (b) SOCl2, MeOH, reflux. P. Zhang et al. / European Journal of Medicinal Chemistry 61 (2013) 95e103 97