P Zhang et aL/ European Journal of Medicinal Chemistry 61(2013)95-103 (m, 1H). 2.86-267(m, 2H). C NMR(100 MHz, DMSO-d6)6 170.3, (Dundee, UK). The prephosphorylated polypeptide substrate GS-2 167.6, 145.7, 144.3, 138.2, 136.6, 132.6, 1312, 129.1, 129. 1. 128.9, 128.5, was synthesized by GL Biochem Ltd (Shanghai, China). Kinase-Glo 128.0, 127.6, 126.7, 126.5, 1248, 52.3, 50.3, 41.7: ESI-MS(positive ): 390. 1 Luminescent Kinase Assay(catalog number V6713)was obtained (M+1)+ from Promega Corporation(Madison, WI). ATP 2Na was purchased from Roche. TDZD-8(catalog number 098K4602V)was supplied by 4.1.3.20. 2, 3-Dihydro-2-phenyl-5-(3-carbomethoxybenzyl)-1. 5- Sigma-Aldrich(St. Louis, MO) Assay buffer contained 50 mM benzothiazepin- 4(5H)-one(6t). To a solution of 6s(0.04 g, 0. 1 mmol) HEPES (pH 7.5). 1 mM EDTA, 1 mM EGTA, and 15 mM magnesium in methanol (2 ml) was added SoCl2(0.1 ml) dropwise at0Cunder acetate. Glow-type luminescence was recorded by Fluoroskan N2 and the reaction mixture was stirred under reflux for 2 h. Then the Ascent Fl (Thermo Electron, US). mixture was cooled to room temperature. After the organic solvent was evaporated, the residue was purified by chromatography 4.2. 1. Inhibition of GsK-3B (petroleum ether/ethyl acetate/methanol 15: 5.5: 0.6) to give pale- The measurement of GSK-3B inhibition was performed in assay yellow solid, Yield: 89%6; mp 110.9-1122C: H NMR (400 MHz, buffer using black 96-well plates according to the Kinase-Glo assay CDCI)8798(s, 1H). 7.92(d, J=7.6 Hz, 1H), 7.60(d, J=6.4 Hz, 2H), method of Baki [20. In a typical assay, 4 Lof interest compound with 7.46-7.16(m, 9H), 5.41(d, J=15.2 Hz, 1H), 5.00(d, J= 15.2 Hz, 1H), different concentration(dissolved in DMSO)was diluted by 14 uL of 4.89(dd,=5.6Hz, 12.4 Hz, 1H),3.91(s, 3H).2.98-2.86(m, 2H);c assay buffer, and 2 uL(20 ng)of enzyme solution were added to each NMR(100 MHZ, CDCI3)8 170.8, 167.0, 145.8, 143.9, 137.4, 136.7, 132.6, well followed by 20 uL of assay buffer containing 12.5 HM substrate 60.3, 130.2, 129.0, 128.8, 128.6, 128.6, 127.7, 127.5, 1273, 126. 1. 124.0, and 4 HM ATP. After 30 min of incubation at 30oC, the enzymatic 529,521,514420:ESMS( positive):4042(M+1)+ reaction was stopped with 40 uL of Kinase-Glo reagent. Glow-type luminescence was recorded after 10 min. The activity is propor- 4.1.3.21. 2,3-Dihydro-2-phenyl-5-(2-(3-chlorophenyl)-2-oxoethyl)- tional to the difference of the total and consumed ATP. The inhibitory 1, 5-benzothiazepin-4(5H)-one(6u). Yield: 65%: mp 60.2-63.9 C: activities were calculated on the basis of maximal activities measured H NMR(400 MHZ, CDCl3)68.01(t,J=1.6 Hz, 1H). 7.91(d, in the absence of inhibitor. The ICso value was defined as the J=7.8 Hz, 1H). 7.65-716(m, 11H), 5.76(d, 17.6 Hz, 1H). 4.61(d, concentration of each compound that reduces 50% the enzymatic Iz, 1H). 4.82(dd, J=5.1 Hz, 12.9 Hz, 1H), 3.05-283(m, 2H): activity with respect to that without inhibitors 13cNMR(100 MHz, CDCl3)d1928,1705,1467,1438,1365,1363 1352,1338,130.6,130.2.1288,1284,127.8,127.5,127.0,126.3,422. Kinetic analysis on GSK-38 1261.1237,559,528,416;ESMS( positive):4081(M+1)+ The protocol of the whole kinetic experiments was much to the one of GSK-3B inhibition tests. The activities of compound 6v 4. 22. 2, 3-Dihydro-2-benzyl-5-(2-nitrobenzyl)-1, 5-benzothiazepin- were measured separately at its two different concentrations as 4 (5H-one( 6v) Yield: 95%: mp 135.0-1369C: H NMR(400 MHz, 25 HM and 50 HM. In the experiments for testing the relationship CDCl)88.37-6.55(m, 12H), 5.86-4.86(m, 2H). 4. 26-3.62(m, 1H). between 6v and ATP, the concentration of substrate GS-2 was kept 2.69(dddd, J=111.3, 95.2, 19.1, 9.8 Hz, 4H): CNMR (100 MHz, CDCl3) unchanged at 6. 25 HM, while the concentration of ATP was set at 61717,1480.1461,1382,1378,1371,1359,1336,133.0,1328,1305,0.5μM,1pM,2pM,4 HM and8 uM separately.Then, in the 129.6, 129.3, 128.5. 1279, 127. 1, 126.8, 126.4, 124.9, 123. 2, 51.2, 49.4, following experiments for testing the relationship between 6v and 441,403,373: ESI-MS( positive)}4052(M+1) GS-2, the concentration of ATP was kept unchanged at 2 uM while GS-2 concentration was set at 0.78 HM, 1.56 HM, 3. 13 HM, 6.25 AM 4.1.3.23. 2, 3-Dihydro-2-(4-fluorophenyl)-5-(2-nitrobenzyl-1, 5- and 12.5 uM separately. Double-reciprocal plotting of the data was enzothiazepin-4(5H)-one (6w). Yield: 91%: mp 166.5-1688C:H depicted in Fig3 NMR(400MHz,CDCl3)68.37-655(m,12H).5.53(q,J=173H Reversibility of compound 6v was determined by evaluating its 2H). 4.88(dd, J= 12.7, 5.4 Hz, 1H), 3. 13-2.46(m, 2H): C NMr activity to the enzyme at different incubation time. The incubation (100 MHz, CDCl3)8 170.7, 148. 1. 145.8 139.5, 136.8, 133.6, 132.9, time was set at 0 min, 5 min and 10 min while concentration of 130.8,130.0.129,.1288.128.1,127.7,126.8,125.0.1246,123.5, compound6 v was kept unchanged at12.5 HM and25 uM sepa- rately. The inhibition effects of compound 6v in these conditions were shown in Fig 3C. 4. 24. 2, 3-Dihydro-2-(chlorophenyl)-5-(2-nitrobenzyl)-1.5- benzothiazepin-4(5H)-one(6x). Yield: 88%: mp 164.8-1674C:H 4.2.3. Selectivity studies of tyrosine kinases inhibition NMR(400MHz,CDCl3)b8.29-6.77(m,12H.5.53(q,J=172Hz The experimental procedures for the inhibition of nine tyrosine 2H). 4.85(dd, J= 12.7, 5.4 Hz, 1H). 3.15-2.69(m, 2H): C NMR kinases were conducted using ELISA method reported by Geng et al. (100MHz,CDCl)6170.6,148.1,145.8.1421,136.8,1336,1326,[30]20μg/ ml of Poly(Gu,Tyr)4:1( Sigma) was pre- coated as 130.9.129.6,129.0.128.1,1275.1274.1267,125.0.123.5.52.2,49.4, a substrate in96- well plates.50μLof10 M ATP solution diluted 41.9: ESI-MS(positive ) 4250(M+1). with kinase reaction buffer (50 mM HEPES PH 7.4, 50 mM MgCl2. 0.5 mM MnCl, 0. 2 mM Na3VO4 and 1 mM DTT) was added to each 4.1.3.25. 2,3-Dihydro-2-(bromophenyl)-5-(2-nitrobenzyl)-1, 5- well. Various concentrations of compounds diluted in 10 uL of 1% benzothiazepin-4(5H)-one(6y). Yield: 93%: mp 178.0-1815C:H DMsO(v/v)were added to each reaction well, with 1% DMso (v/v) NMR(400 MHZ, CDCI3)88.38-6.78(m, 12H), 5.53(q,= 17.2 Hz, used as the negative control. The kinase reaction was initiated by 2H). 4.84(dd, J=12.7, 5.3 Hz, 1H), 3. 29-2.60(m, 2H): C NMR the addition of purified tyrosine kinase proteins diluted with 40 HL (100 MHz, CDCI3)8 170.6, 148. 1, 145.8, 142. 1, 136.8, 133.6, 132.6, of kinase reaction buffer solution. After incubation for 60 min at 130.9, 129.6, 129.0, 128. 1, 127.5, 127.4, 126.7, 125.0, 123.5, 52.2, 49.4, 37oC, the plate was washed three times with Phosphate Buffered 49:ES-Ms( positive):4690.4710(M+1) Saline(pbs)containing 0. 1% Tween 20(T-PBS). Next, 100 uL of anti phospho tyrosine(PY99 )antibody(1: 500 diluted in 5 mg/mL BSA 4. 2. Biological evaluation T-PBS)was added After 30 min incubation at 37 .C, the plate was washed three times. A solution of 100 uL of horseradish peroxidase- Human recombinant glycogen synthase kinase-3B(catalog conjugated goat anti-mouse IgG(1: 2000 diluted in 5 mg/mL BSAT- number 14-306)was purchased from Millipore Corporation PBS)was added. The plate was reincubated at 37C for 30 min, and(m, 1H), 2.86e2.67 (m, 2H). 13C NMR (100 MHz, DMSO-d6) d 170.3, 167.6, 145.7, 144.3, 138.2, 136.6, 132.6, 131.2, 129.1, 129.1, 128.9, 128.5, 128.0,127.6,126.7,126.5,124.8, 52.3, 50.3, 41.7; ESI-MS (positive): 390.1 (M þ 1)þ. 4.1.3.20. 2,3-Dihydro-2-phenyl-5-(3-carbomethoxybenzyl)-1,5- benzothiazepin-4(5H)-one (6t). To a solution of 6s (0.04 g, 0.1 mmol) in methanol (2 ml) was added SOCl2 (0.1 ml) dropwise at 0 C under N2 and the reaction mixture was stirred under reflux for 2 h. Then the mixture was cooled to room temperature. After the organic solvent was evaporated, the residue was purified by chromatography (petroleum ether/ethyl acetate/methanol 15:5.5:0.6) to give pale￾yellow solid, Yield: 89%; mp 110.9e112.2 C; 1 H NMR (400 MHz, CDCl3) d 7.98 (s, 1H), 7.92 (d, J ¼ 7.6 Hz, 1H), 7.60 (d, J ¼ 6.4 Hz, 2H), 7.46e7.16 (m, 9H), 5.41 (d, J ¼ 15.2 Hz, 1H), 5.00 (d, J ¼ 15.2 Hz, 1H), 4.89 (dd, J ¼ 5.6 Hz, 12.4 Hz, 1H), 3.91 (s, 3H), 2.98e2.86 (m, 2H); 13C NMR (100 MHz, CDCl3) d 170.8, 167.0, 145.8, 143.9, 137.4, 136.7, 132.6, 130.3, 130.2, 129.0, 128.8, 128.6, 128.6, 127.7, 127.5, 127.3, 126.1, 124.0, 52.9, 52.1, 51.4, 42.0; ESI-MS (positive): 404.2 (M þ 1)þ. 4.1.3.21. 2,3-Dihydro-2-phenyl-5-(2-(3-chlorophenyl)-2-oxoethyl)- 1,5-benzothiazepin-4(5H)-one (6u). Yield: 65%; mp 60.2e63.9 C; 1 H NMR (400 MHz, CDCl3) d 8.01 (t, J ¼ 1.6 Hz, 1H), 7.91 (d, J ¼ 7.8 Hz, 1H), 7.65e7.16 (m, 11H), 5.76 (d, J ¼ 17.6 Hz, 1H), 4.61 (d, J ¼ 17.6 Hz, 1H), 4.82 (dd, J ¼ 5.1 Hz, 12.9 Hz, 1H), 3.05e2.83 (m, 2H); 13C NMR (100 MHz, CDCl3) d 192.8, 170.5, 146.7, 143.8, 136.5, 136.3, 135.2, 133.8, 130.6, 130.2, 128.8, 128.4, 127.8, 127.5, 127.0, 126.3, 126.1, 123.7, 55.9, 52.8, 41.6; ESI-MS (positive): 408.1 (M þ 1)þ. 4.1.3.22. 2,3-Dihydro-2-benzyl-5-(2-nitrobenzyl)-1,5-benzothiazepin- 4(5H)-one (6v). Yield: 95%; mp 135.0e136.9 C; 1 H NMR (400 MHz, CDCl3) d 8.37e6.55 (m, 12H), 5.86e4.86 (m, 2H), 4.26e3.62 (m, 1H), 2.69 (dddd, J ¼ 111.3, 95.2, 19.1, 9.8 Hz, 4H); 13C NMR (100 MHz, CDCl3) d 171.7, 148.0, 146.1, 138.2, 137.8, 137.1, 135.9, 133.6, 133.0, 132.8, 130.5, 129.6, 129.3, 128.5, 127.9, 127.1, 126.8, 126.4, 124.9, 123.2, 51.2, 49.4, 44.1, 40.3, 37.3; ESI-MS (positive): 405.2 (M þ 1)þ. 4.1.3.23. 2,3-Dihydro-2-(4-fluorophenyl)-5-(2-nitrobenzyl)-1,5- benzothiazepin-4(5H)-one (6w). Yield: 91%; mp 166.5e168.8 C; 1 H NMR (400 MHz, CDCl3) d 8.37e6.55 (m, 12H), 5.53 (q, J ¼ 17.3 Hz, 2H), 4.88 (dd, J ¼ 12.7, 5.4 Hz, 1H), 3.13e2.46 (m, 2H); 13C NMR (100 MHz, CDCl3) d 170.7, 148.1, 145.8, 139.5, 136.8, 133.6, 132.9, 130.8, 130.0, 129.1, 128.8, 128.1, 127.7, 126.8, 125.0, 124.6, 123.5, 115.6, 52.2, 49.4, 42.2; ESI-MS (positive): 409.1 (M þ 1)þ. 4.1.3.24. 2,3-Dihydro-2-(chlorophenyl)-5-(2-nitrobenzyl)-1,5- benzothiazepin-4(5H)-one (6x). Yield: 88%; mp 164.8e167.4 C; 1 H NMR (400 MHz, CDCl3) d 8.29e6.77 (m, 12H), 5.53 (q, J ¼ 17.2 Hz, 2H), 4.85 (dd, J ¼ 12.7, 5.4 Hz, 1H), 3.15e2.69 (m, 2H); 13C NMR (100 MHz, CDCl3) d 170.6, 148.1, 145.8, 142.1, 136.8, 133.6, 132.6, 130.9, 129.6, 129.0, 128.1, 127.5, 127.4, 126.7, 125.0, 123.5, 52.2, 49.4, 41.9; ESI-MS (positive): 425.0 (M þ 1)þ. 4.1.3.25. 2,3-Dihydro-2-(bromophenyl)-5-(2-nitrobenzyl)-1,5- benzothiazepin-4(5H)-one (6y). Yield: 93%; mp 178.0e181.5 C; 1 H NMR (400 MHz, CDCl3) d 8.38e6.78 (m, 12H), 5.53 (q, J ¼ 17.2 Hz, 2H), 4.84 (dd, J ¼ 12.7, 5.3 Hz, 1H), 3.29e2.60 (m, 2H); 13C NMR (100 MHz, CDCl3) d 170.6, 148.1, 145.8, 142.1, 136.8, 133.6, 132.6, 130.9, 129.6, 129.0, 128.1, 127.5, 127.4, 126.7, 125.0, 123.5, 52.2, 49.4, 41.9; ESI-MS (positive): 469.0, 471.0 (M þ 1)þ. 4.2. Biological evaluation Human recombinant glycogen synthase kinase-3b (catalog number 14e306) was purchased from Millipore Corporation (Dundee, UK). The prephosphorylated polypeptide substrate GS-2 was synthesized by GL Biochem Ltd (Shanghai, China). Kinase-Glo Luminescent Kinase Assay (catalog number V6713) was obtained from Promega Corporation (Madison, WI). ATP$2Na was purchased from Roche. TDZD-8 (catalog number 098K4602V) was supplied by SigmaeAldrich (St. Louis, MO). Assay buffer contained 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 15 mM magnesium acetate. Glow-type luminescence was recorded by Fluoroskan Ascent Fl (Thermo Electron, US). 4.2.1. Inhibition of GSK-3b The measurement of GSK-3b inhibition was performed in assay buffer using black 96-well plates according to the Kinase-Glo assay method of Baki[20]. In a typical assay, 4 mL of interest compound with different concentration (dissolved in DMSO) was diluted by 14 mL of assay buffer, and 2 mL (20 ng) of enzyme solution were added to each well followed by 20 mL of assay buffer containing 12.5 mM substrate and 4 mM ATP. After 30 min of incubation at 30 C, the enzymatic reaction was stopped with 40 mL of Kinase-Glo reagent. Glow-type luminescence was recorded after 10 min. The activity is propor￾tional to the difference of the total and consumed ATP. The inhibitory activities were calculated on the basis of maximal activities measured in the absence of inhibitor. The IC50 value was defined as the concentration of each compound that reduces 50% the enzymatic activity with respect to that without inhibitors. 4.2.2. Kinetic analysis on GSK-3b The protocol of the whole kinetic experiments was much similar to the one of GSK-3b inhibition tests. The activities of compound 6v were measured separately at its two different concentrations as 25 mM and 50 mM. In the experiments for testing the relationship between 6v and ATP, the concentration of substrate GS-2 was kept unchanged at 6.25 mM, while the concentration of ATP was set at 0.5 mM, 1 mM, 2 mM, 4 mM and 8 mM separately. Then, in the following experiments for testing the relationship between 6v and GS-2, the concentration of ATP was kept unchanged at 2 mM while GS-2 concentration was set at 0.78 mM, 1.56 mM, 3.13 mM, 6.25 mM and 12.5 mM separately. Double-reciprocal plotting of the data was depicted in Fig. 3. Reversibility of compound 6v was determined by evaluating its activity to the enzyme at different incubation time. The incubation time was set at 0 min, 5 min and 10 min while concentration of compound 6v was kept unchanged at 12.5 mM and 25 mM sepa￾rately. The inhibition effects of compound 6v in these conditions were shown in Fig. 3C. 4.2.3. Selectivity studies of tyrosine kinases inhibition The experimental procedures for the inhibition of nine tyrosine kinases were conducted using ELISA method reported by Geng et al. [30]. 20 mg/ml of Poly (Glu, Tyr) 4:1 (Sigma) was pre-coated as a substrate in 96-well plates. 50 mL of 10 mM ATP solution diluted with kinase reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4 and 1 mM DTT) was added to each well. Various concentrations of compounds diluted in 10 mL of 1% DMSO (v/v) were added to each reaction well, with 1% DMSO (v/v) used as the negative control. The kinase reaction was initiated by the addition of purified tyrosine kinase proteins diluted with 40 mL of kinase reaction buffer solution. After incubation for 60 min at 37 C, the plate was washed three times with Phosphate Buffered Saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100 mL of anti￾phospho tyrosine (PY99) antibody (1:500 diluted in 5 mg/mL BSA T-PBS) was added. After 30 min incubation at 37 C, the plate was washed three times. A solution of 100 mL of horseradish peroxidase￾conjugated goat anti-mouse IgG (1:2000 diluted in 5 mg/mL BSA T￾PBS) was added. The plate was reincubated at 37 C for 30 min, and 102 P. Zhang et al. / European Journal of Medicinal Chemistry 61 (2013) 95e103