DSpace at VNU: Novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides as Histone Deacetylase Inhibitors and Antitumor Agents

Send Orders for Reprints to reprints@benthamscience.ae Medicinal Chemistry, 2015, 11, 725-735 Novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides Histone Deacetylase Inhibitors and Antitumor Agents 725 as Do T M Dunga, Phan T P Dunga, Dao T K Oanh*,a, Pham T Haia, Le T T Huongb, Vu D Loib, Hyunggu Hahnc, Byung W Hanc, Jisung Kimd, Sang-Bae Han*,d and Nguyen-Hai Nam*,a a Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam; bSchool of Medicine and Pharmacy, Hanoi National University, Hanoi, Vietnam; cResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea; dCollege of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea Abstract: Histone deacetylases (HDAC) are currently a group of validated targets for anticancer drug discovery and development In our research program to find novel small molecules targeting these enzymes, we designed and synthesized two series of 3-hydroxyimino-2-oxoindoline- and 3methoxyimino-2-oxoindoline-based N-hydroxypropenamides (3a-g, 6a-g) The results show that these propenamides potently inhibited HDAC2 with IC50 values in sub-micromolar range, approximately 10-fold lower than that of SAHA (also known as suberoylanilohydroxamic acid) Evaluation of cytotoxicity of these compounds in three human cancer cell lines revealed that most of the synthesized compounds were up to 5-fold more cytotoxic than SAHA Docking studies showed that the compounds bound to HDAC2 at the binding site with higher binding affinities compared to SAHA Our present results demonstrate that these novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides are potential for further development as anticancer agents Keywords: Histone deacetylase (HDAC) inhibitors, hydroxamic acids, 3-substituted-2-oxoindoline, propenamide INTRODUCTION Histone deacetylases (HDAC) have become interesting molecular targets for anticancer drug design and development in the past decades [1-3] These enzymes are known to catalyze the removal of acetyl groups from lysine residues in the tails of histone proteins, which results in the condensation of chromatin and repression of transcription process [2,3] Hitherto, 18 different isoforms of HDACs have been identified in eukaryotes [4] These are categorized into four classes based on their relative sequence similarity Class I includes four members, namely HDAC1, 2, and HDAC8 Class II currently has six members including HDAC4, 5, 6, 7, and HDAC10 HDACs of class III are also known as Sirtuins The Sirtuins with seven members (Sirt1-7) are NAD+-dependent enzymes Finally, class IV HDAC has only one member (HDAC11) HDAC11 is known to have properties of both class II and class I HDAC2 [4] Among four classes, HDACs of class I are considered to be those of the most important isoforms and have been comprehensively investigated [4] Inhibition of different HDAC isoforms has been shown to result in a number of sequential events related to cell dif- *Address correspondence to this author at the Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam; Tel: +84-4-39330531; Fax: +84-4-39332332; E-mails: namnh@hup.edu.vn; oanhdtk@hup.edu.vn; shan@chungbuk.ac.kr 17-/15 $58.00+.00 ferentiation, apoptosis and cell cycle arrest in many types oftumor cells [5,6] The selective effects of HDAC inhibition on the growth of tumor cells have been clearly demonstrated not only in vitro but also in a number of in vivo preclinical models and clinical settings [7,8] Therefore, inhibition of HDACs has become an interesting approach in cancer treatment nowadays [9] As a result, hundreds of HDAC inhibitors have been reported by medicinal chemists recently These inhibitors range from short-chain fatty acids (like butyrate, phenylbutyrate or valproic acid) to diverse types of hydroxamic acids, or benzamides [10-16] Numerous HDAC inhibitors such as PXD-01 (Belinostat), LBH-589 (Panobinostat), MS-27-527 (Entinostat) (Fig 1) are currently under clinical trials at different phases and two HDAC inhibitors including SAHA (or vorinostat, tradename Zolinza®) (also known as suberoylanilohydroxamic acid) and romidepsin (tradename Istodax®) (Fig 1) have been approved for clinical applications In our research program to develop novel hydroxamic acids as potential inhibitors of HDACs and anticancer agents, we have focused our efforts on heterocyclic analogues of SAHA In our previous reports, we have described several series of hydroxamic acids incorporating a benzothiazole/thiazole system with very potent HDAC inhibitory activity as well as cytotoxicity (Fig 2) [17,18] One compound from this series, N1-(6-chlorobenzo[d]thiazol-2-yl)-N8 hydroxyoctanediamide (HUP00752), which possessed the most potent HDAC inhibition and cytotoxicity, has been © 2015 Bentham Science Publishers 726 Medicinal Chemistry, 2015, Vol 11, No Dung et al O O O H N N H O SAHA OH O O H3C NH O H N O S NHOH HN CH3 O O S PXD-101 NHOH H N CH3 LBH-589 O HN S O H3C Romidepsin O CH3 CH3 O N H H N N MS-27-275 NH2 O Fig (1) Structures of some HDAC inhibitors selected for further in vivo evaluation Preliminary evaluation in PC-3 prostate cancer cells xenografted mice model revealed that this compound exhibited in vivo antitumor efficacy equipotent as compared to SAHA [18] Currently, HUP00752 is selected for further development as a promising anticancer agent Inspired by this success, we expanded our research with a series of 5-substitutedphenyl/aryl-1,3,4thiadiazole-based hydroxamic acids (Fig 2) and these compounds were also very potent as HDAC inhibitors and possessed strong cytotoxicity [19,20] Continuing our research program, we have designed, synthesized and evaluated a novel series of 3-substituted-2-oxoindoline-based propenamides The current paper reports the results we obtained from the synthesis, biological evaluation and representative docking study from this study O O N R N NHOH n H (n = 1, 2, 4) S R = H, 6-CH3, 6-OCH3, 6-OC2H5, 6-SO2CH3, 6-NO2, 6-Cl, 6-CF3, 6-NO2 O N R O N N NHOH H S R = H, 2-Cl, 3-Cl, 4-Cl, 4-F, 4-Br, 2-NO2, 4-NO2, 2,6-Cl2, 4-CH3, 4-OCH3, 4-N(CH3)2, 3,4-CH2OCH22,3,4-(OCH3)3, 3,4,5-(OCH3)3 Fig (2) Structures of some benzothiazole-, 5-substitutedphenyl1,3,4-thiadiazole-based hydroxamic acids MATERIALS AND METHODS Chemistry Thin layer chromatography which was performed using Whatman® 250 m Silica Gel GF Uniplates and visualized under UV light at 254 and 365 nm, were used to check the progress of reactions and preliminary evaluation of compounds’ homogeneity In all cases, the compounds achieved purity of 97% or above, as estimated by HPLC method Melting points were measured using a Gallenkamp Melting Point Apparatus (LabMerchant, London, United Kingdom) and are uncorrected Purification of compounds were carried out using crystallization methods and/or open flash silica gel column chromatography employing Merck silica gel 60 (240 to 400 mesh) as stationary phase Nuclear magnetic resonance spectra (1H NMR) were recorded on a Bruker 500 MHz spectrometer with DMSO-d6 as solvent unless otherwise indicated Tetramethylsilane was used as an internal standard Chemical shifts are reported in parts per million (ppm), downfield from tetramethylsilane Mass spectra with different ionization modes including electron ionization (EI), Electrospray ionization (ESI), were recorded using PE Biosystems API2000 (Perkin Elmer, Palo Alto, CA, USA) and Mariner® (Azco Biotech, Inc Oceanside, CA, USA) mass spectrometers, respectively All reagents and solvents were purchased from Aldrich or Fluka Chemical Corp (Milwaukee, WI, USA) or Merck unless noted otherwise Solvents were used directly as purchased unless otherwise indicated The synthesis of two series of (E)-N-hydroxy-3-(4-(((Z)3-(hydroxyimino)-5/7-substituted-2-oxoindolin-1-yl)methyl) phenyl)propenamides (3a-g) and (E)-N-hydroxy-3-(4-(((Z)3-(methoxyimino)-5/7-substituted-2-oxoindolin-1-yl)methyl) phenyl)propenamides (6a-g) was carried out as illustrated in schemes 1-2 Details are described as follows: General Procedures for the Synthesis of (E)-N-hydroxy-3(4-(((Z)-3-(hydroxyimino)-5/7-substituted-2-oxoindolin-1yl)methyl)phenyl)propenamides (3a-g) A solution of 1a-g (1 mmol) in DMF (3 mL) was cooled to -5 oC and K2CO3 (165.5 mg, 1.2 mmol) was added The mixture was stirred for hour at -5 oC and 45 minutes at room temperature, then CH3OH (0.5 mL) and KI (8.3 mg, 0.05 mmol) were added After stirring for 15 minutes, a solution of (E)-methyl 4-bromomethylcinnamate (255 mg, mmol) in DMF (1 mL) was added and the resulting reaction mixture was stirred at 60 oC for 24 hours Upon completion, the reaction mixture was cooled, acidified with 10% HCl to pH ~4 and extracted with DCM (50 mL x 2) The extracts were pooled and DCM was evaporated under reduced pressure to give brown-yellowish oil residues (2a-g) which were used for the next step without further purification The brown-yellowish oils (2a-g) obtained above were each dissolved in a mixture of methanol/tetrahydrofuran (1/1, mL) and the resulting solution from each compound was cooled to -5 oC and hydroxylammonium chloride (195 mg, 10 mmol) was added NaOH (0.4 g, 10 mmol) was dissolved in mL of water, cooled to 0-5 oC and added to the mixture The mixture was stirred at -5 o C until compound 2ag reacted completely (30-60 minutes) The reaction mixture was neutralized to pH by dropwise addition of a solution of HCl 15% to induce precipitation The precipitate was fil- Novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides tered, washed with water, recrystallized from ethanol and dried at 60 oC to yield compounds 3a-g (E)-N-Hydroxy-3-(4-(((Z)-3-(hydroxyimino)-2-oxoindolin1-yl)methyl)phenyl)propenamide (3a) Yellow solid; Yield: 74% mp: 197-198oC Rf = 0.45 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3441 (NH), 3197 (OH), 3049 (C-H, aren), 2856 (CH, CH2), 1714, 1655 (C=O), 1608 (C=C), 1463 (C-N) ESI-MS (m/z): 339 [M+2H] - 1H-NMR (500 MHz, DMSO-d6, ppm): 8.01 (1H, d, J = 7.5 Hz, H-4”); 7.52 (2H, d, J = 8.5 Hz, H-2’, H-6’); 7.40 (1H, d, J = 15.5 Hz, H-3); 7.35 (2H, d, J = 8.5 Hz, H-3’, H-5’); 7.34 (1H, t, J = 7.5 Hz, H-5”); 7.07 (1H, t, J = 7.5 Hz, H-6”); 6.96 (1H, d, J = 8.0 Hz, H-7”); 6.45 (1H, d, J = 16.0 Hz, H-2); 4.95 (2H, s, -CH2-) 13C NMR (125 MHz, DMSO-d6, ppm): 163.36, 162.69, 143.41, 142.60, 137.79, 137.61, 134.15, 131.92, 127.88, 127.81, 126.91, 122.88, 119.21, 115.41, 109.55, 42.37 Anal Calcd For C18H15N3O4 (337.33): C, 64.09; H, 4.48; N, 12.46 Found: C, 64.11; H, 4.49; N, 12.41 (E)-3-(4-(((Z)-5-Fluoro-3-(hydroxyimino)-2-oxoindolin-1yl)methyl)phenyl)-N-hydroxypropenamide (3b) Yellowish solid; Yield: 72% mp: 198-199 oC Rf = 0.35 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3452 (NH), 3224 (OH), 3049 (C-H, aren) 2856 (CH, CH2), 1720, 1662 (C=O), 1615, 1513 (C=C), 1474 (C-N) ESI-MS (m/z): 357 [M+2H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 7.78 (1H, dd, J = 8.0 Hz, J’ = 2.5 Hz, H-4”); 7.52 (2H, d, J = 8.0 Hz, H-2’, H-6’); 7.41 (1H, d, J = 16.0 Hz, H-3); 7.35 (2H, d, J = 8.0 Hz, H-3’, H-5’); 7.21 (1H, td, J = 9.0 Hz, 3.0 Hz, H6”); 6.96 (1H, dd, J = 8.5 Hz, J’ = 4.0 Hz, H-7”); 6.44 (1H, d, J = 16.0 Hz, H-2); 4.95 (2H, s, -CH2-) 13C NMR (125 MHz, DMSO-d6, ppm): 163.35, 162.68, 159.05, 157.15, 143.14, 138.72, 137.81, 137.47, 134.17, 127.89, 127.81, 119.19, 117.91, 117.73, 116.06, 115.98, 113.80, 113.59, 110.49, 110.42, 42.46 Anal Calcd For C18H14FN3O4 (355.32): C, 58.81; H, 6.58; N, 13.72 Found: C, 58.78; H, 6.55; N, 13.83 (E)-3-(4-(((Z)-5-Chloro-3-(hydroxyimino)-2-oxoindolin-1yl)methyl)phenyl)-N-hydroxypropenamide (3c) Yellowish solid; Yield: 69% mp: 196-198 oC Rf = 0.38 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3279 (OH), 3069 (C-H, aren), 2924, 2847 (CH, CH2), 1716, 1656 (C=O), 1607, 1513 (C=C), 1463 (C-N) ESI-MS (m/z): 370.4 [M-H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 13.80 (1H, s, OH); 10.75 (1H, s, NH); 9.03 (1H, s, OH); 7.97 (1H, d, J = 2.0 Hz, H-4”); 7.52 (2H, d, J = 8.0 Hz, H-2’, H-6’); 7.43-7.40 (2H, m, H-6”, H-3); 7.35 (2H, d, J = 8.0 Hz, H-3’, H-5’); 6.99 (1H, d, J = 8.5 Hz, H-7”); 6.43 (1H, d, J = 15.5 Hz, H2); 4.96 (2H, s, H-7’a, H-7’b) 13C NMR (125 MHz, DMSOd6, ppm): 162.90, 162.67, 142.68, 141.39, 137.79, 137.21, 134.17, 131.40, 127.85, 127.75, 126.67, 126.24, 119.17, 116.51, 110.10, 42.46 Anal Calcd For C18H14ClN3O4 (371.78): C, 58.15; H, 3.80; N, 11.30 Found: C, 58.19; H, 3.76; N, 11.26 (E)-3-(4-(((Z)-5-Bromo-3-(hydroxyimino)-2-oxoindolin-1yl)methyl)phenyl)-N-hydroxypropenamide (3d) Yellow solid; Yield: 67% mp: 205-207 oC Rf = 0.47 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3431 Medicinal Chemistry, 2015, Vol 11, No 727 (NH), 3290 (OH ), 3089 (C-H, aren), 2925 (CH, CH2), 1715, 1656 (C=O), 1603, 1462 (C=C), 1433 (C-N) ESI-MS (m/z): 415.3 [M-H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 13.85 (1H, s, OH); 10.74 (1H, s, NH); 9.02 (1H, s, OH); 8.10 (1H, d, J = 2.0 Hz, H-4”); 7.56 (1H, dd, J = 8.5 Hz, J’ = 2.0 Hz, H-6”); 7.52 (2H, d, J = 8.0 Hz, H-2’, H-6’); 7.42 (1H, d, J = 15.5 Hz, H-3); 7.35 (2H, d, J = 8.0Hz, H-3’, H-5’); 6.95 (1H, d, J = 8.5 Hz, H-7”), 6.43 (1H, d, J = 15.5 Hz, H-2); 4.96 (2H, s, H-7’a, H-7’b) 13C NMR (125 MHz, DMSO-d6, ppm): 162.76, 162.64, 142.54, 141.77, 137.78, 137.16, 134.21, 134.14, 128.92, 127.82, 127.72, 119.16, 116.92, 114.30, 111.59, 42.41 Anal Calcd For C18H14BrN3O4 (416.23): C, 51.94; H, 3.39; N, 10.10 Found: C, 51.98; H, 3.42; N, 10.14 (E)-N-Hydroxy-3-(4-(((Z)-3-(hydroxyimino)-5-methyl-2oxoindolin-1-yl)methyl)phenyl)propenamide (3e) Pale yellow solid; Yield: 71% mp: 195-196 oC Rf = 0.46 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3226 (OH), 3049 (C-H, aren), 2921 (CH, CH2), 1711, 1660 (C=O), 1616, 1478 (C=C), 1438 (C-N) ESI-MS (m/z): 349.9 [M-H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 13.50 (1H, s, OH); 10.76 (1H, s, NH); 9.03 (1H, s, OH); 7.85 (1H, s, H-4”); 7.54 (2H, d, 8.0Hz, H-2’, H-6’); 7.41 (1H, d, J = 15.5 Hz, H3); 7.34 (2H, d, J = 8.0 Hz, H-3’, H-5’); 7.16 (1H, d, J = 7.5 Hz, H-7”); 6.85 (1H, d, J = 8.0 Hz, H-6”); 6.41 (1H, d, J = 16.0 Hz, H-2); 4.92 (2H, s, H-7’a, H-7’b); 2.25 (3H, s, 163.27, CH3) 13C NMR (125 MHz, DMSO-d6, ppm): 162.74, 143.58, 140.45, 137.89, 137.67, 134.08, 132.19, 131.94, 127.86, 127.76, 127.52, 119.11, 115.40, 109.35, 42.35, 20.52 Anal Calcd For C19H17N3O4 (351.36): C, 64.95; H, 4.88; N, 11.96 Found: C, 64.99; H, 4.51; N, 11.92 (E)-N-Hydroxy-3-(4-(((Z)-3-(hydroxyimino)-5-methoxy-2oxoindolin-1-yl)methyl)phenyl)propenamide (3f) Pale yellow solid; Yield: 75% mp: 201-202 oC Rf = 0.38 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3212 (OH), 3054 (C-H, aren), 2884 (CH, CH2), 1709, 1660 (C=O), 1625, 1514, 1480 (C=C), 1436 (C-N) ESI-MS (m/z): 368.3 [M+H]+ 1H-NMR (500 MHz, DMSO-d6, ppm): 13.61 (1H, s, OH); 7.69 (1H, d, J = 2.0Hz, H-4”); 7.52 (2H, d, J = 8.0 Hz, H-2’, H-6’); 7.41 (1H, d, J = 15.5 Hz, H-3); 7.34 (2H, d, J = 8.0 Hz, H-3’, H-5’); 6.94 (1H, dd, J = 8.5, J’ = 2.5Hz, H-6”); 6.87 (1H, d, J = 8.5 Hz, H-7”); 6.44 (1H, d, J = 15.5 Hz, H-2); 4.92 (2H, s, H-7’a, H-7’b), 3.74 (4H, s, 163.15, OCH3) 13C NMR (125 MHz, DMSO-d6, ppm): 162.70, 155.33, 143.70, 137.83, 137.67, 136.28, 134.12, 127.87, 127.80, 119.17, 116.90, 115.95, 113.28, 110.20, 55.68, 42.41 Anal Calcd For C19H17N3O5 (367.36): C, 62.12; H, 4.66; N, 11.44 Found: C, 62.17; H, 4.62; N, 11.47 (E)-3-(4-(((Z)-7-Chloro-3-(hydroxyimino)-2-oxoindolin-1yl)methyl)phenyl)-N-hydroxypropenamide (3g) Yellowish solid; Yield: 72% mp: 190-192 oC Rf = 0.44 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3214 (OH), 3054 (C-H, aren), 2855 (CH, CH2), 1717, 1659 (C=O), 1602, 1514, 1470 (C=C), 1442 (C-N) ESI-MS (m/z): 370.6 [M-2H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 13.90 (1H, s, OH); 10.74 (1H, s, NH); 9.01 (1H, s, OH); 8.10 (1H, dd, J = 8.5 Hz, J’ = 1.0 Hz, H-4”); 7.52 (2H, d, J = 8.0 Hz, H-2’, H-6’); 7.42 (1H, d, J = 15.5 Hz, H-3); 7.38 (1H, dd, J = 8.5 Hz, J’= 1.0 Hz, H-6”); 7.24 (2H, d, J = 8.0 Hz, H- 728 Medicinal Chemistry, 2015, Vol 11, No 3’, H-5’); 7.12 (1H, t, J = 8.0 Hz, H-5”), 6.42 (1H, d, J = 15.5 Hz, H-2), 5.30 (1H, s, H-7’a, H-7’b) 13C NMR (125 MHz, DMSO-d6, ppm): 163.90, 162.70, 142.11, 138.96, 138.43, 137.88, 133.80, 133.67, 127.76, 126.50, 125.95, 124.39, 118.91, 118.26, 114.58, 40.01 Anal Calcd For C18H14ClN3O4 (371.78): C, 58.15; H, 3.80; N, 11.30 Found: C, 58.19; H, 3.86; N, 11.25 General Procedures for the Synthesis of (E)-N-hydroxy-3(4-(((Z)-3-(methoxyimino)-5/7-substituted-2-oxoindolin-1yl)methyl)phenyl)propenamides (6a-g) A mixture of 1a-g (1 mmol) and methoxylamine hydrochloride (501 mg, mmol) in pyridine anhydrous (5 mL) was refluxed for hours The whole reaction mixture was then cooled to room temperature and poured into a cold solution of HCl 5M (30 mL) The precipitates (4a-g) were washed with water, dried at 40oC under vacuum for 24 hours and used directly for the next step without further purification Compounds 4a-g obtained above were dissolved in DMF (3 mL) The resulting solution was cooled to -5oC and K2CO3 (165.5 mg, 1.2 mmol) was added The mixture was stirred for hour at -5 oC and 45 minutes at room temperature, then CH3OH (0.5 mL) and KI (8.3 mg, 0.05 mmol) were added After stirring for 15 minutes, a solution of (E)methyl 4-bromomethylcinnamate (255 mg, mmol) in DMF (1 mL) was added and the resulting reaction mixture was stirred at 60 oC for 24 hours Upon completion, the reaction mixture was cooled, acidified with 10% HCl to pH ~ and extracted with DCM (50 mL x 2) The extracts were pooled and DCM was evaporated under reduced pressure Compounds 5a-g obtained as brown-yellow oils The brown-yellowish oils (5a-g) obtained above were each dissolved in a mixture of methanol/tetrahydrofuran (1/1, mL) and the resulting solution from each compound was cooled to -5 oC and hydroxylammonium chloride (195 mg, 10 mmoL) was added NaOH (0.4 g, 10 mmoL) was dissolved in mL of water, cooled to 0-5 oC and added to the mixture The mixture was stirred at -5 oC until compound 5a-g reacted completely (30-60 minutes) The reaction mixture was neutralized to pH by dropwise addition of a solution of HCl 15% to induce precipitation The precipitate was filtered, washed with water, recrystallized from ethanol, then dried at 60 oC to yield compounds 6a-g (E)-N-Hydroxy-3-(4-(((Z)-3-(methoxyimino)-2-oxoindolin1-yl)methyl)phenyl)propenamide (6a) Pale yellow solid; Yield: 76% mp: 207-208 oC Rf = 0.43 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3313 (NH), 3220 (OH), 3038 (C-H, aren), 2938 (CH, CH2), 1718, 1648 (C=O), 1622, 1607, 1516 (C=C), 1437 (C-N) ESI-MS (m/z): 349.4 [M-2H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 10.76 (1H, s, NH), 7.89 (1H, d, J = 7.5 Hz, H-4”), 7.52 (2H, d, J = 8.0 Hz, H-2’, H-6’), 7.44-7.35 (4H, m, H-3, H-3’, H-5’, H-5”), 7.07 (1H, t, J = 8.5 Hz, H-6”), 6.98 (1H, d, J = 8.0 Hz, H-7”), 6.44 (1H, d, J = 16.0 Hz, H-2), 4.95 (2H, s, H-7’a, H-7’b), 4.23 (3H, s, -NOCH3), 13C NMR (125 MHz, DMSO-d6, ppm): 162.63, 162.41, 143.25, 143.16, 137.75, 137.31, 134.13, 132.85, 127.81, 127.75, 127.37, 122.95, 119.16, 115.01, 109.79, 64.53, 42.40 Anal Calcd For C19H17N3O4 (351.36): C, 64.95; H, 4.88; N, 11.96 Found: C, 64.91; H, 4.92; N, 11.91 Dung et al (E)-3-(4-(((Z)-5-Fluoro-3-(methoxyimino)-2-oxoindolin-1yl)methyl)phenyl)-N-hydroxypropenamide (6b) Pale yellow solid; Yield: 67% mp: 194-195 oC Rf = 0.41 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3426 (NH), 3199 (OH), 2946 (CH, CH2), 1715 (C=O), 1614, 1601, 1513, 1476 (C=C), 1430 (C-N) ESI-MS (m/z): 368.0 [M-H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 10.76 (1H, s, NH), 9.03 (1H, s, OH), 7.69 (1H, d, J = 6.5 Hz, H-4”), 7.52 (2H, d, J = 7.0 Hz, H-2’, H-6’), 7.42 (1H, d, J = 15.5 Hz, H3), 7.36 (2H, d, J = 7.5 Hz, H-3’, H-5’), 7.27 (1H, t, J = 7.5Hz, H-6”), 6.99 (1H, s, H-7”), 6.44 (1H, d, J = 15.5 Hz, H-2), 4.95 (2H, s, H-7’a, H-7’b), 4.25 (3H, s, -NOCH3) 13C NMR (125 MHz, DMSO-d6, ppm): 162.66, 162.29, 158.99, 157.09, 142.87, 139.59, 137.75, 137.11, 134.17, 127.83, 127.76, 119.20, 118.99, 115.59, 115.51, 114.55, 114.34, 110.87, 110.81, 64.78, 42.53 Anal Calcd For C19H16FN3O4 (369.35): C, 61.79; H, 4.37; N, 11.38 Found: C, 61.83; H, 4.41; N, 11.34 (E)-3-(4-(((Z)-5-Chloro-3-(methoxyimino)-2-oxoindolin-1yl)methyl)phenyl)-N-hydroxypropenamide (6c) Yellow solid; Yield: 70% mp: 202-203 oC Rf = 0.37 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3426 (NH), 3224 (OH), 2942 (CH, CH2), 1722, 1661 (C=O), 1608, 1467 (C=C), 1444 (C-N) ESI-MS (m/z): 386.4 [M+H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 10.75 (1H, s, NH), 9.02 (1H, s, OH), 7.87 (1H, d, J = 2.0 Hz, H-4”), 7.52 (2H, d, J = 8.0 Hz, H-2’, H-6’), 7.46 (1H, dd, J = 8.5 Hz, J’ = 2.0 Hz, H-6”), 7.42 (1H, d, J = 16.0 Hz, H-3), 7.35 (2H, d, J = 8.0 Hz, H-3’, H-5’), 7.00 (1H, d, J = 8.0 Hz, H7”), 6.43 (1H, d, J = 15.5 Hz, H-2), 4.95 (2H, s, H-7’a, H7’b), 4.26 (3H, s, -NOCH3) 13C NMR (125 MHz, DMSO-d6, ppm): 162.62, 162.08, 142.41, 142.02, 137.72, 136.97, 134.17, 132.26, 127.81, 127.72, 126.79, 126.64, 119.20, 116.21, 111.34, 64.85, 42.52 Anal Calcd For C19H16ClN3O4 (385.80): C, 59.15; H, 4.18; N, 10.89 Found: C, 59.19; H, 4.21; N, 10.83 (E)-3-(4-(((Z)-5-Bromo-3-(methoxyimino)-2-oxoindolin-1yl)methyl)phenyl)-N-hydroxypropenamide (6d) Yellow solid; Yield: 65% mp: 190-191 oC Rf = 0.42 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3435 (NH), 2942 (CH, CH2), 1723, 1627 (C=O), 1604, 1516, 1466 (C=C), 1437 (C-N) ESI-MS (m/z): 429.9 [M-H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 8.00 (1H, s, H-4”), 7.65-7.51 (4H, m, H-6”, H-2’, H-6’, H-3), 7.43-7.34 (2H, m, H-3’, H5’), 6.96 (1H, d, J = 8.0 Hz, H-7”), 6.50-6.41 (1H, m, H-2), 4.98-4.95 (2H, m, H-7’a, H-7’b), 4.26 (3H, s, -NOCH3) 13C NMR (125 MHz, DMSO-d6, ppm): 167.43, 161.98, 143.27, 142.40, 142.38, 142.27, 137.78, 137.71, 135.08, 133.56, 129.32, 128.51, 127.79, 127.69, 127.64, 119.28, 119.19, 116.64, 114.43, 111.81, 64.86, 42.49 Anal Calcd For C19H16BrN3O4 (430.25): C, 53.04; H, 3.75; N, 9.77 Found: C, 53.11; H, 3.79; N, 9.72 (E)-N-Hydroxy-3-(4-(((Z)-3-(methoxyimino)-5-methyl-2oxoindolin-1-yl)methyl)phenyl)propenamide (6e) Pale yellow solid; Yield: 68% mp: 193-194 oC Rf = 0.44 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3428 (NH), 3296 (OH), 2938, 2856 (CH, CH2), 1709, 1669 Novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides (C=O), 1616, 1594, 1516, 1480 (C=C), 1441 (C-N) ESI-MS (m/z): 364.1 [M-H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 10.74 (1H, s, NH), 9.01 (1H, s, OH), 7.74 (1H, s, H-4”), 7.51 (2H, d, J = 8.0 Hz, H-2’, H-6’), 7.41 (1H, d, J = 16.0 Hz, H3), 7.43 (2H, d, J = 8.0 Hz, H-3’, H-5’), 7.20 (1H, d, J = 7.5 Hz, H-7”), 6.87 (1H, d, J = 8.0 Hz, H-6”), 6.42 (1H, d, J = 16.0 Hz, H-2), 4.93 (2H, s, H-7’a, H-7’b), 4.23 (3H, s, NOCH3), 2.26 (3H, s, -CH3) 13C NMR (125 MHz, DMSO-d6, ppm): 162.61, 162.39, 143.28, 141.03, 137.75, 137.38, 134.07, 133.05, 132.07, 127.83, 127.78, 127.70, 119.11, 115.05, 109.58, 64.48, 42.37, 20.40 Anal Calcd For C20H19N3O4 (365.38): C, 65.74; H, 5.24; N, 11.50 Found: C, 65.77; H, 5.21; N, 11.54 (E)-N-Hydroxy-3-(4-(((Z)-3-(methoxyimino)-5-methoxy-2oxoindolin-1-yl)methyl)phenyl)propenamide (6f) Pale yellow solid; Yield: 71% mp: 201-202 oC Rf = 0.42 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3413 (NH), 3214 (OH), 3018 (C-H, aren), 2942 (CH, CH2), 1699 (C=O), 1617, 1595, 1478 (C=C) ESI-MS (m/z): 379.4 [M2H]- 1H-NMR (500 MHz, DMSO-d6, ppm): 10.73 (1H, s, NH), 9.01 (1H, s, OH), 7.52 (2H, d, J = 8.0 Hz, H-2’, H-6’), 7.49 (1H, d, J = 2.5 Hz, H-4”), 7.41 (1H, d, J = 16.0 Hz, H3), 7.34 (2H, d, J = 8.0 Hz, H-3’, H-5’), 6.99 (1H, dd, J = 8.0 Hz, J’= 2.5Hz, H-6”), 6.90 (1H, d, J = 8.5 Hz, H-7”), 6.42 (1H, d, J = 16.0 Hz, H-2), 4.92 (2H, s, H-7’a, H-7’b), 4.24 (3H, s, =NOCH3), 3.72 (3H, s, -OCH3) 13C NMR (125 MHz, DMSO-d6, ppm): 162.63, 162.25, 155.32, 143.41, 137.76, 137.40, 136.82, 134.09, 127.79, 127.73, 119.12, 117.61, 115.57, 113.80, 110.40, 64.60, 55.69, 42.42 Anal Calcd For C20H19N3O5 (381.38): C, 62.99; H, 5.02; N, 11.02 Found: C, 63.06; H, 5.10; N, 10.91 (E)-3-(4-(((Z)-7-Chloro-3-(methoxyimino)-2-oxoindolin-1yl)methyl)phenyl)-N-hydroxypropenamide (6g) Pale yellow solid; Yield: 75% mp: 203-204 oC Rf = 0.39 (DCM : MeOH : AcOH = 90 : : 1) IR (KBr, cm-1): 3307 (NH), 3204 (OH), 3018 (C-H, aren), 2938 (CH, CH2), 1722, 1662 (C=O), 1624, 1601, 1514, 1470 (C=C), 1442 (C-N) ESI-MS (m/z): 383.3 [M-2H]- 1H-NMR (500 MHz, DMSOd6, ppm): 10.74 (1H, s, NH), 9.01 (1H, s, OH), 7.98 (1H, d, J = 7.5 Hz, H-4”), 7.51 (2H, d, J = 8.0 Hz, H-2’, H-6’), 7.447.41 (2H, m, H-3, H-6”), 7.25 (2H, d, J = 8.0 Hz, H-3’, H5’), 7.12 (1H, t, J = 8.0 Hz, H-5”), 6.42 (1H, d, J = 16.0 Hz, H-2), 5.28 (2H, s, H-7’a, H-7’b), 4.27 (3H, s, -NOCH3) 13C NMR (125 MHz, DMSO-d6, ppm): 163.12, 162.69, 141.91, 139.06, 138.74, 137.85, 134.68, 133.70, 127.76, 126.50, 126.44, 124.49, 118.94, 117.98, 114.79, 64.95, 44.14 Anal Calcd For C19H16ClN3O4 (385.80): C, 59.15; H, 4.18; N, 10.89 Found: C, 59.18; H, 4.21; N, 10.84 Cytotoxicity Assay The cytotoxicity of the synthesized compounds was evaluated against three human cancer cell lines, including SW620 (colon cancer), PC3 (prostate cancer), and AsPC-1 (pancreatic cancer) The cell lines were purchased from a Cancer Cell Bank at the Korea Research Institute of Bioscience and Biotechnology (KRIBB) The media, sera and other reagents that were used for cell culture in this assay were obtained from GIBCO Co Ltd (Grand Island, New York, USA) The cells were culture in DMEM (Dulbecco’s Medicinal Chemistry, 2015, Vol 11, No 729 Modified Eagle Medium) until confluence The cells were then trypsinized and suspended at 104 cells/mL of cell culture medium On day 0, each well of the 96-well plates was seeded with 180 L of cell suspension The plates were then incubated in a 5% CO2 incubator at 37 oC for 24 h Compounds were initially dissolved in dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations by culture medium Then 20 L of each compounds’ samples, which were prepared as described above, were added to each well of the 96-well plates, which had been seeded with cell suspension and incubated for 24-h, at various concentrations The plates were further incubated for 48 h Cytotoxicity of the compounds was measured by the colorimetric method, as described previously [21] with slight modifications [20,22,23] The IC50 values were calculated using a Probits method [24] and were averages of three independent determinations (SD 10%) Western Blot Analysis The Western blot analysis was performed as described previously [20] Briefly, the total protein extracts were firstly obtained by cell lysis using RIPA buffer (composed of 50 mM Tris-Cl [pH 8.0], mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, and mM phenylmethylsulfonyl fluoride) The protein concentrations present in the cell lysates as obtained above were measured by using a Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) following the manufacturer's procedures The assay samples were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes In the next step, the nitrocellulose membranes were further incubated in blocking buffer (which was made from Tris-buffered saline containing 0.2% Tween-20 and 3% nonfat dried milk) and probed with the primary antibodies against acetyl histone-H3, -H4, and GAPDH) After washing, the obtained membranes were further probed with horseradish peroxidase-conjugated secondary antibodies The detection was performed using an enhanced chemiluminescent protein (ECL) detection system (Amersham Biosciences, Little Chalfont, UK) HDAC2 Enzyme Assay The HDAC2 enzyme was purchased from BPS Bioscience (San Diego, CA, USA) and the enzymatic HDAC assay was performed using a Fluorogenic HDAC Assay Kit (BPS Bioscience) according to the manufacturer’s instructions Briefly, HDAC2 enzymes were incubated with vehicle or various concentrations of the assayed samples or SAHA for 30 at 37 o C in the presence of an HDAC fluorimetric substrate The HDAC assay developer (which produces a fluorophore in reaction mixture) was added, and the fluorescence was measured using VICTOR3 (PerkinElmer, Waltham, MA, USA) with excitation at 360 nm and emission at 460 nm The measured activities were subtracted by the vehicle-treated control enzyme activities and IC50 values were calculated using GraphPad Prism (GraphPad Software, San Diego, CA, USA) Docking Studies In the docking studies we used AutoDock Vina program [25] (The Scripps Research Institute, CA, USA) The structure of HDAC2 protein in complex with SAHA [26] was 730 Medicinal Chemistry, 2015, Vol 11, No Dung et al O O O O R O OCH3 a NH R N OH O N O b R N a, R = H b, R = 5-F NHOH c, R = 5-Cl d, R = 5-Br e, R = 5-CH3 f, R = 5-OCH3 g, R = 7-Cl Scheme Synthesis of 3-oxime-2-oxoindolin-based N-hydroxypropenamides Reagents and conditions: bromomethylcinnamate, K2CO3, KI, DMF, 60 oC, 24 hrs; b) NH2OH.HCl, NaOH, MeOH/THF, -5 oC, 30-60 O R H3CO O O R NH N b R O O c NHOH R 4- OCH3 a NH Methyl O H3CON H3CON O a) N N a, R = H b, R = 5-F c, R = 5-Cl d, R = 5-Br e, R = 5-CH3 f, R = 5-OCH3 g, R = 7-Cl Scheme Synthesis of 3-methoxime-2-oxoindolin-based N-hydroxypropenamides Reagents and conditions: a) NH2OCH3.HCl, pyridine, DMF; b) Methyl 4-bromomethylcinnamate, K2CO3, KI, DMF, 60 oC, 24 hrs; c) NH2OH.HCl, NaOH, MeOH/THF, -5 oC, 30-60 obtained from the Protein Data Bank (PDB) (PDB ID: 4LXZ) and the coordinates of the compounds were generated employing the GlycoBioChem PRODRG2 Server (http://davapc1.bioch.dundee.ac.uk/prodrg/) [27] For the docking studies, the grid maps were centered on the SAHA binding site and comprised 26 X 26 X 22 points with 1.0 Å spacing after SAHA was removed from the complex structure, as described previously [17-20] AutoDock Vina program was performed using eight-way multithreading and the other parameters were default settings in AutoDock Vina program RESULTS AND DISCUSSIONS Chemistry The synthesis of N-hydroxypropenamides 3a-g was illustrated in Scheme In the first step, isatin and its 5- or 7substituted derivatives were reacted in a molar equivalence of methyl 4-bromomethylcinnamate in dimethyl formamide under alkaline conditions (K2CO3) with a catalytic amount of KI to furnish the cinnamate intermediates 2a-h as brownyellow oils in good overall yields (85-96%) Next, nucleophilic acyl substitution of the cinnamates 2a-h by hydroxylamine under basic conditions gave the final propenamides 3a-h in good yields The 3-methoxyimino-2-oxoindoline N-hydroxypropenamides (6a-g) were synthesized using a similar pathway but in this case the isatins 1a-g were first converted to the 3methoxyimino-2-oxoindoline derivatives 4a-g, using methoxylamine hydrochloride under refluxing conditions in anhydrous pyridine (Scheme 2) The structures of the final products were unambiguously determined using spectroscopic methods, including IR, MS, H NMR and 13C NMR It was found that hydroxylamine reacted at both the ester and 3-oxo groups on the indoline moiety of the esters 2a-g, resulting in 3-hydroxyimino-2oxoindoline N-hydroxypropenamides (3a-g) This was evidenced from the mass spectral data and the downfield shifts of H-4’ ( 8.01-7.78 ppm) in the 1H NMR spectra of compounds 3a-g The downfield shift of H-4 indicated the presence of the hydroxyimino group at position on the indoline ring [20] Also, generally, in the 1H NMR spectra of compounds 3a-g measured in DMSO-d6, three broad singlets appeared around 9.02, 10.75, and 13.50-13.80 ppm, which were attributable to –OH, –NH (hydroxamic acid moiety), and -OH (of the 3-hydroxyimino moiety on the indoline ring) In the 13C NMR spectra of compounds 3a-g, the peaks attributable for 3-oxo carbon of the indoline ring, which should appear around 184.00-188.00 ppm [20], were not observed These evidences further confirmed the presence of the 3-hydroxyimino group on the indoline moiety The concurrent reaction of hydroxylamine at the 3-oxo groups on the indoline ring and the ester group has been observed under the same conditions previously [20] The reaction of hydroxylamine at the 3-oxo groups on the indoline ring occurred so readily even when the molar equivalence of hydroxylamine to compounds 2a-g was significantly It was found that when hydroxylamine.HCl was used at 1:1 molar Novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides Medicinal Chemistry, 2015, Vol 11, No 731 Fig (3) Effects of the compounds synthesized on histone acetylation in SW620 cells Cells were treated with compounds or SAHA at g/mL for 24 hrs Levels of acetyl-histone-H3 and -H4 in total cell lysates were determined by Western immunoblot analysis Table Inhibition of HDAC activity and cytotoxicity of the compounds synthesized against several cancer cell lines OR' O N O R NHOH N (R' = H) (R' = CH3) Cpd Code R 3a -H 337.32 3b 5-F 3c Cytotoxicity (IC50,2 M)/Cell lines3 HDAC2 inhibition (IC50,2 M) SW620 PC3 AsPC-1 2.11 0.116 2.26 1.33 0.95 355.32 2.27 0.152 0.91 1.29 1.46 5-Cl 371.78 2.67 0.086 0.90 0.47 0.81 3d 5-Br 416.23 2.94 0.120 1.41 0.91 0.63 3e 5-CH3 351.36 2.60 0.179 1.93 1.20 0.83 3f 5-OCH3 367.36 1.98 0.117 2.46 1.13 0.68 3g 7-Cl 371.78 2.67 0.447 2.24 1.94 1.68 6a -H 351.36 2.37 0.472 1.96 1.14 1.23 6b 5-F 369.35 2.53 0.267 0.71 0.93 0.56 6c 5-Cl 385.80 2.93 0.199 1.10 0.88 0.82 6d 5-Br 430.25 3.20 1.320 0.80 0.81 1.11 6e 5-CH3 365.38 2.86 0.175 1.23 1.37 1.79 6f 5-OCH3 381.38 2.25 0.254 5.25 4.31 4.55 7-Cl 385.80 2.93 1.399 3.15 4.54 3.58 264.32 1.44 1.840 3.26 1.75 3.19 6g SAHA Molecular Weight LogP1 Calculated by ChemDraw 9.0 software; The concentration ( M) of compounds that produces a 50% reduction in enzyme activity or cell growth, the numbers represent the averaged results from triplicate experiments with deviation of less than 10%.; 3Cell lines: SW620, colon cancer; PC3, prostate cancer; AsPC-1, pancreatic cancer; 4SAHA, suberoylanilide acid, a positive control equivalent to the corresponding ester intermediate, a mixture of many products together with the unreacted starting materials (the ester) were observed, which rendered difficult to isolate the target products 3a-g Bioactivity Initially, the compounds synthesized including 3a-g and 6a-g were assessed for their inhibition of histone-H3 and histone-H4 deacetylation using Western Blot analysis Because histone deacetylation is mainly governed by HDACs in general, the HDAC inhibitory effects of the compounds, therefore, could be indirectly extrapolated through their ability to inhibit the level of histone deacetylation As shown in (Fig 3), in the presence of SAHA, a known HDAC inhibitor, the levels of acetyl-histones H3 and acetyl-histones H4 appeared intense, indicating that HDACs had been strongly inhibited and were unable to remove the acetyl groups on 732 Medicinal Chemistry, 2015, Vol 11, No histones H3 and histones H4 Similarly, the levels of acetylhistone H3 and acetyl-histone H4 also appeared clearly in the presence of compounds 3a, 3b, 3d-f, 6a-c, 6e, and 6f, suggesting that HDAC activity had been greatly inhibited In the presence of compound 3c, acetyl-histones H3 and acetylhistones H4 were still observed, but at much less intense levels Thus, compound 3c was still active, although, it was weaker than the above compounds in this assay In contrast, the levels of acetyl-histones H3 and acetyl-histones H4 were almost not observed under the presence of compound 3g or compound 6g, indicating that HDACs were still active and had deacetylated histones H3 and histones H4 These results demonstrate that substitution of a chlorine (and possibly other substituents) at position was not favorable for bioactivity while position was tolerable for a diverse substituents When compounds 3a-g and 6a-g were evaluated in enzyme-based assay using HDAC2, it was found that all twelve compounds inhibited HDAC2 with IC50 values ranged from as low as 0.086 M to 1.399 M (Table 1), lower than that of SAHA (1.840 M) Thus, these compounds were up 21fold more potent than SAHA in term of HDAC2 inhibition Within these two compounds’ series, compounds 3g and 6g were the least active These results were in good agreement with Western blot assay Similar correlation was also observed with compound 6d In contrast, compound 3c was the most potent in the HDAC2 assay with IC50 value of 0.086 M, however, it was only weakly active in the Western Blot assay Since histones-H3 and histone-H4 are substrates of HDAC2 and HDAC3 [28], it was likely that HDAC3 was still not inhibited by compound 3c Similar explanation could also be applied for compounds 3g, 6d and 6g Next, the antiproliferative activity of the synthesized compounds was we measured using SRB (sulforhodamine B) cell proliferation assay Initially we screened the compounds for their effects on cell growth at 30 M using one cell line (SW620, a human colon cancer) From this screening, it was found that all compounds 3a-g, 6a-g completely inhibited the growth of SW620 cells at this concentration Therefore, all the compounds were subjected for further evaluation at concentrations (30, 10, 3, 1, 0.3 M) in SW620 and two more human cancer cell lines, including PC3 (prostate cancer) and AsPC-1 (pancreas cancer) cell lines The IC50 (the concentration that causes 50% of cell proliferation inhibition) values of each compounds were determined and summarized in Table SAHA was used as a positive control Data from Table clearly indicated that, with the exception of compounds 6f, all other compounds were generally equipotent or more potent than SAHA in term of cytotoxicity against all three cancer cell lines assayed Also in all three cell lines tested, compound 3a and compound 3b were equally cytotoxic, indicating that methylation of the oxime in 3a was tolerable for cytotoxicity Substitution of electron withdrawing groups (-F, -Cl, -Br) at position on the indoline moiety seemed to enhance cytotoxicity (compounds 3b-d, 6b-d), meanwhile, substitution at position seemed less favorable for bioactivity (compounds 3g, 6g) Among the electron donating groups (-CH3, -OCH3), it seemed that only the methyl group slightly enhanced cytotoxicity (compounds 3e, 6e vs compounds 3a, 6a), while the methoxy group did not (compound 3f) Concurrent presence Dung et al of the 5-methoxy substituent and methoxime on the indoline ring even decreased cytotoxicity (compound 6f) The results suggested that the bioactivity of these compounds might be very sensitive to the bulkiness of the substituents on the indoline moiety Regarding the relationships between the cytotoxicity and the inhibitory effects of the compounds on HDAC2 or histones-H3 and histones-H4 deacetylation, it could be seen that there was relatively good correlation within series 3a-h Compounds, which were active in the Western Blot analysis and showed good inhibition on HDAC2 activity, were also significantly cytotoxic against all three cancer cell lines (3be) or at least two of three cancer cell lines tested (3a,f) Meanwhile, compound 3g, which was inactive in the Western Blot analysis and showed the highest IC50 value against HDAC2, was the least cytotoxic compound in the series 3ag Similar correlation was also observed with compounds in series 6a-g, except compound 6d Compound 6d was not active in the Western Blot analysis and showed relatively high IC50 value against HDAC2 (1.320 M) However, against three cancer cell lines assay, this compound exhibited potent cytotoxicity It was possible that other HDAC types might have been affected by compound 6d, and subsequently still leading to cell growth inhibition in overall Of course, other mechanisms of cytotoxicity of compound 6d, besides HDAC inhibition, should not be excluded, and this is obviously an interesting topic for further study From these observations, it is clear that, in order to delineate a complete correlation between HDAC inhibition and cytotoxicity of these compounds, more experiments need to be performed with remaining HDAC subtypes Nevertheless, with the present results, it could be stated that these 3-substituted-2oxoindoline-based N-hydroxypropenamides are promising as potent HDAC inhibitors and cytotoxic agents Docking Studies To gain some insights into the interaction between these compounds and HDAC, we implemented docking experiments using the active site of HDAC Since histone-H3 and histone-H4 deacetylation is regulated principally by HDAC2 and HDAC3 [28], we decided to select the structure of HDAC2 in complex with SAHA as a docking template The crystal structure of HDAC2 in complex with SAHA (PDB ID: 4LXZ) has been reported by Lauffer and co-workers [26] We executed control docking experiments with SAHA to the crystal structures of HDAC2 using AutoDock Vina program [25] after SAHA was removed from the complex structure, as described previously [17-20] It was found from docking experiments that all the compounds synthesized by were located in the active site with binding affinities higher than that of SAHA, as manifested by stabilization energies ranging from -8.3 to -9.4 kcal/mol, significantly lower than that of SAHA For example, stabilization energies of predicted binding modes on HDAC2 were calculated to be -9.0 and -8.6 kcal/mol for compounds 3b and 6b, respectively, while the values for SAHA were -7.4 kcal/mol (r.m.s.d distance from the original SAHA in the crystal structure: 0.609/2.056 Å) It was found from the docking experiments that, a zinc ion (grey sphere) was coordinated by three residues of HDAC2, including Asp181, His183 and Asp269 All the synthesized compounds interacted with the zinc ion in a Novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides Medicinal Chemistry, 2015, Vol 11, No 733 Fig (4) Stereo-view presentations of the actual binding poses of SAHA and simulated docking poses of compounds 3b (A) and 3g (B) to HDAC2 SAHA is represented as a stick model with carbon, nitrogen, and oxygen atoms in pink, blue and red, respectively Compounds 3b and 3g are presented as a stick model with carbon colored in cyan and brown; nitrogen, and oxygen atoms colored in blue and red, respectively The most important parts for the enzyme for interaction of these compounds were shown as a stick model with carbon, nitrogen, and oxygen colored as grey, blue and red, respectively Zn2+ ion is shown as a bright gray sphere (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper) similar manner as SAHA did For all compounds, it was found that a benzene ring linking the indoline and Nhydroxypropenamide moieties was tightly stacked between Phe155 and Phe210 residues of the enzymes (Fig 4) and this stacking interaction could be the key factor attributing to the high binding affinities of the compounds with HDAC2 In these docking experiments, however, the indoline part was found to insignificantly interact with the enzymes Subsequently, very little variance in the binding affinities among the compounds with different substituted groups was observed For example, the stabilization energies of predicted binding modes on HDAC2 of compounds 3g and 6g were found to be -8.9 and -8.4 kcal/mol, respectively These values were almost similar to that of compounds 6b and 6g Thus, the different in the binding stabilization energies presently could not explain for the 3-fold difference in the IC50 values of compounds 3b and 3g or a 5-fold difference in the IC50 values of compounds 6b and 6g in the HDAC2 enzyme inhibition assay (Table 1) Nevertheless, the binding stabilization energy of compound 6g was still lower than that of SAHA (-8.4 kcal/mol vs -7/4 kcal/mol, respectively), which was likely explainable for the relatively higher inhibition of this compound (IC50, 1.399 M) compared to that of SAHA (IC50, 1.840 M) towards HDAC2 734 Medicinal Chemistry, 2015, Vol 11, No Dung et al CONCLUSION In conclusion, we have reported two series of 3substituted-2-oxoindoline-based N-hydroxypropenamides with strong HDAC2 inhibitory effects and potent cytotoxicity againsts several human cancer cell lines, including SW620 (human colon cancer), PC-3 (prostate cancer) and AsPC-1 (pancreas cancer) The results we obtained from this study clearly indicated that diverse substituents could be introduced at both positions and on the benzene ring of the 3-hydroxyimino-2-oxoindoline or 3-methoxyimino-2oxoindoline moiety while still retaining both HDAC inhibition and cytotoxicity of the resulting N-hydroxypropenamides Our present findings suggest that further manipulation on the indoline moiety is possible and promising to potentially produce more potent HDAC inhibitors with potential anticancer activity More extensive evaluation of several compounds in the series 3a-g and 6a-g against different HDAC isoforms and in-depth docking investigation are being performed to gain a quantitative correlation between the enzyme inhibitory effects and cytotoxicity of these compounds series CONFLICT OF INTEREST [11] [12] [13] [14] [15] [16] [17] [18] The authors confirm that this article content has no conflict of interest ACKNOWLEDGEMENTS We acknowledge the principal financial supports from the National Foundation for Science and Technology of Vietnam (NAFOSTED, Grant number 104.01-2014.55) The biological study was supported by the Medical Research Center program (MRC, Grant number 2008-0062275) and the docking study was supported by the Global Core Research Center (GCRC, Grant number 2012-0001193) from the National Research Foundation (NRF) of Korea [19] [20] [21] REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] Nam, N.H.; Parang, K Current targets for anticancer drugs discovery Curr Drugs Targets 2003, 4, 159-179 Marks, P.A.; Rifkind, R.A.; Richon, V.M.; Breslow, R.; Miller, T.; Kelly, W.K Histone deacetylases and cancer: Causes and Therapies Nature Rev Cancer., 2001, 1, 194-202 Witt, O.; Deubzer, H.E.; Milde, T.; Oehme, I HDAC family: What are the cancer relevant targets? Cancer Lett 2009, 277, 8-21 De Ruijter, A.J.M.; Gennip, A.H.V.; Caron, H.N.; Kemp, S.; Kuilenburg, A.B.P.V Histone deacetylases (HDACs): characterization of the classical HDAC family Biochem J., 2003, 370, 737739 Bolden, J.E.; Peart, M.J.; Johnstone, R.W Anticancer activities of histone deacetylase inhibitors Nat Rev Drug Discov 2006, 5, 769-784 Dokmanovic, M.; Marks, P.A Histone deacetylase inhibitors: discovery and development as anticancer agents Expert Opin Investi Drug., 2005, 14, 1497-1511 Johnstone, R.W., Histone-deacetylase inhibitors: Novel drugs for the treatment of cancer Nature Rev Drug Disc., 2002, 1, 287-299 Glaser, K.B HDAC inhibitors: clinical update and mechanismbased potential Biochem Pharmacol., 2007, 74, 659-671 West, A.C.; Johnstone, R.W New and emerging HDAC inhibitors for cancer treatment J Clin Invest., 2014, 124, 30-39 Dallavalle, S.; Cincinelli, R.; Nannei, R.; Merlini, L.; Morini, G.; Penco, S.; Pisano, C.; Vesci, L.; Barbarino, M.; Zuco, V.; De Cesare, M.; Zunino, F Design, synthesis, and evaluation of biphenyl- [22] [23] [24] [25] [26] [27] 4-yl-acrylohydroxamic acid derivatives as histone deacetylase (HDAC) inhibitors Eur J Med Chem., 2009, 44, 1900-1912 Bracker, T.U., Sommer, A., Fichtner, I., Faus, H., Haendler, B., Hess-Stumpp, H Efficacy of MS-275, a selective inhibitor of class I histone deacetylases, in human colon cancer models Int J Oncol., 2009, 35, 909-920 Finnin, M.S.; Donigian, J.R.; Cohen, A.; Richon, V.M.; Rifkind, R.A.; Marks, P.A.; Breslow, R.; Pavietich, N.P Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors Nature 1999, 401, 188-193 Valente, S.; Mai, A Small-molecule inhibitors of histone deacetylase for the treatment of cancer and non-cancer diseases: a patent review (2011-2013) Expert Opin Ther Pat., 2014, 24, 401-415 Li, J.; Li, G.; Xu, X Histone deacetylase inhibitors: an attractive strategy for cancer therapy Curr Med Chem 2013, 20, 18581886 Ververis, K.; Hiong, A.; Karagiannis, T.C.; Licciardi, P.V Histone deacetylase inhibitors (HDACIs): multitargeted anticancer agents Biologics 2013, 7, 47-60 Qiu T, Zhou L, Zhu W, Wang T, Wang J, Shu Y, Liu P Effects of treatment with histone deacetylase inhibitors in solid tumors: a review based on 30 clinical trials Future Oncol., 2013, 9, 255-269 Oanh, D.T.K.; Hai, H.V.; Hue, V.T.M.; Park, S.H.,; Kim, H.J.; Han, B.W.; Kim, H.S.; Hong, J.T.; Han, S.B.; Nam, N.H Benzothiazole-containing hydroxamic acids as histone deacetylase inhibitors and antitumor agents Bioorg Med Chem Lett., 2011, 21, 7509-7512 Tung, T.T.; Oanh, D.T.; Dung, P.T.; Hue, V.T.; Park, S.H.; Han, B.W.; Kim, Y.; Hong, J.T.; Han, S.B.; Nam, N.H New benzothiazole/thiazole-containing hydroxamic acids as potent histone deacetylase inhibitors and antitumor agents Med Chem 2013, 9, 10511057 Nam, N.H.; Huong, T.L.; Dung, D.T.M.; Oanh, D.T.K.; Dung, P.T.P.; Kim, K.R.; Han, B.W.; Kim, Y.S.; Hong, J.T.; Han, S.B Synthesis, bioevaluation and docking study of 5-substitutedphenyl1,3,4-thiadiazole-based hydroxamic acids as histone deacetylase inhibitors and antitumor agents J Enzyme Inhib Med Chem (2013) (published online, doi:10.3109/14756366.2013.832238) Nam, N.H.; Huong, T.L.; Dung, D.T.M.; Oanh, D.T.K.; Dung, P.T.P.; Quyen, D.; Kim, K.R.; Han, B.W.; Kim, Y.S.; Hong, J.T.; Han, S.B Novel isatin-based hydroxamic acids as histone deacetylase inhibitors and antitumor agents Eur J Med Chem., 2013, 70, 477-486 Skehan, P.; Storeng, R.; Scudiero, D.; Monk, A.; MacMahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenney, S.; Boyd M.R New colorimetric cytotoxicity assay for anticancer drug screening J Natl Cancer Inst., 1990, 82, 1107-1112 You, Y.J.; Kim, Y.; Nam, N.H.; Bang, S.C.; Ahn, B.Z Alkyl and carboxylalkyl esters of -demethyl-4-deoxypodophyllotoxin: synthesis, cytotoxic, and antitumor activity Eur J Med Chem., 2004, 39, 189-193 Kim, Y.; You, Y.J.; Nam, N.H.;, Ahn, B.Z Prodrugs of demethyl-4-deoxypodophyllotoxin: synthesis and evaluation of the antitumor activity Bioorg Med Chem Lett., 2002, 12, 3435-3438 Wu, L.; Smythe, A.M.; Stinson, S.F.; Mullendore, L.A.; Monks, A.; Scudiero, D.A.; Paull, K.D.; Koutsoukos, A.D.; Rubinstein, L.V.; Boyd, M.R.; Shoemaker, R.H Multidrug-resistant phenotype of disease-oriented panels of human tumor cell lines used for anticancer drug screening Cancer Res., 1992, 52, 3029-3034 Trott, O.; Olson, A.J AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading J Comput Chem., 2010, 31, 455-461 Lauffer, B.E.; Mintzer, R.; Fong, R.; Mukund, S.; Tam, C.; Zilberleyb, I.; Flicke, B.; Ritscher, A.; Fedorowicz, G.; Vallero, R.; Ortwine, D.F.; Gunzner, J.; Modrusan, Z.; Neumann, L.; Koth, C.M.; Lupardus, P.J.; Kaminker, J.S.; Heise, C.E.; Steiner, P Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability, J Biol Chem., 2013, 288, 26926-26943 Schuttelkopf, A.W.; van Aalten, D.M PRODRG: a tool for highthroughput crystallography of protein-ligand complexes Acta Novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides [28] crystallographica Section D, Biological Crystallogra., 2004, 60, 1355-1363 Pelzel, H.R.; Schlamp, C.L.; Nickells, R.W Histone H4 deacetylation plays a critical role in early gene silencing during Received: November 23, 2014 Revised: June 27, 2015 Medicinal Chemistry, 2015, Vol 11, No neuronal apoptosis BMC Neuroscience 2010, (http://www.biomedcentral.com/1471-2202/11/62) 11, 735 62 Accepted: June 27, 2015 ... Johnstone, R.W., Histone- deacetylase inhibitors: Novel drugs for the treatment of cancer Nature Rev Drug Disc., 2002, 1, 287-299 Glaser, K.B HDAC inhibitors: clinical update and mechanismbased potential... histone deacetylase homologue bound to the TSA and SAHA inhibitors Nature 1999, 401, 188-193 Valente, S.; Mai, A Small-molecule inhibitors of histone deacetylase for the treatment of cancer and. .. Han, S.B Synthesis, bioevaluation and docking study of 5-substitutedphenyl1,3,4-thiadiazole-based hydroxamic acids as histone deacetylase inhibitors and antitumor agents J Enzyme Inhib Med Chem