5 aryl 1 3 4 thiadiazole based hydroxamic acids as histone deacetylase inhibitors and antitumor agents synthesis bioevaluation and docking study

Send Orders for Reprints to reprints@benthamscience.net Medicinal Chemistry, 2014, 10, 000-000 5-Aryl-1,3,4-thiadiazole-based Hydroxamic Acids as Histone Deacetylase Inhibitors and Antitumor Agents: Synthesis, Bioevaluation and Docking Study Tran Lan Huonga, Do Thi Mai Dunga, Dao Thi Kim Oanha, Tran Thi Bich Lana, Phan Thi Phuong Dunga,*, Vu Duc Loib, Kyung Rok Kimc, Byung Woo Hanc, Jieun Yund, Jong Soon Kangd, Youngsoo Kime, Sang-Bae Hane,* and Nguyen-Hai Nama,* 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; dKorea Research Institute of Bioscience and Biotechnology, Cheongwon, Chungbuk 363-883, Korea; eCollege of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea Abstract: The search for newer histone deacetylase (HDAC) inhibitors has attracted a great deal of interest of medicinal chemists worldwide, especially after the first HDAC inhibitor (Zolinza®, widely known as SAHA or Suberoylanilide hydroxamic acid) was approved by the FDA for the treatment of T-cell lymphoma in 2006 As a continuity of our ongoing research in this area, we designed and synthesized a series of 5-aryl-1,3,4-thiadiazole-based hydroxamic acids as analogues of SAHA and evaluated their biological activities Most of the compounds in this series, e.g compounds with 5aryl moiety being 2-furfuryl (5a), 5-bromofuran-2-yl (5b), 5-methylfuran-2-yl (5c), thiophen-2-yl (5d), 5-methylthiophen2-yl (5f) and pyridyl (5g-i), were found to have potent anticancer cytotoxicity with IC50 values of generally 5- to 10-fold lower than that of SAHA in human cancer cell lines assayed Those compounds with potent cytotoxicity were also found to have strong HDAC inhibition effects Docking studies revealed that compounds 5a and 5d displayed high affinities towards HDAC2 and Keywords: Histone deacetylase (HDAC) inhibitors, 5-aryl-1,3,4-thiadiazole, cytotoxicity, heterocycle INTRODUCTION Histon deacetylases (HDACs) are enzymes that catalyze a deacetylation process of acetylated histones Since deacetylation and acetylation are associated with an open chromatin configuration and a permissive gene transcription state, these enzymes, together with histone acetylases, play important roles in gene transcriptions [1, 2] Currently, 18 HDAC enzymes have been identified in human Based on their homologies to yeast HDACs, human HDACs are divided into four classes Class I has four members, namely HDAC 1, 2, 3, and 8; Class II includes HDACs 4, 5, 6, 7, and 10 Class III HDACs comprising Sirt1-7, known as Sirtuins, are NAD+-dependent enzymes; and Class IV which has only one member, HDAC11, exhibits both class I and class II HDACs’ properties [1,2] In the past decade, a number of studies have demonstrated that these enzymes are not only involved in the regulation of chromatin structure and gene expression, but they can also regulate cell-cycle progression and carcinogenic *Address correspondence to these authors at the Department of Pharmaceutical Chemistry, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam; Tel/Fax: +84-4-3832332; E-mail: namnh@hup.edu.vn; and College of Pharmacy, Chungbuk National University, 12 Gaesin, Heungduk, Cheongju, Chungbuk 361-763, Korea; Tel/Fax: +82-43-261-2815; E-mail: shan@chungbuk.ac.kr 1573-4064/14 $58.00+.00 process which in turn involves in the formation of malignant tumors [1-3] HDACs have therefore become an attractive target for anticancer drug discovery [3-6] Intensive efforts of medicinal chemists worldwide have resulted in the finding of a variety of HDAC inhibitors, such as SAHA (Vorinostat), trichostatin A, LBH-589 (Panobinostat), PXD-101, MGCD0103 (Mocetinostat), MS-27-275 (Entinostat), and oxamflatin, among others [7, 8] Of these, SAHA (Vorinostat, trade name Zolinza®) was approved by the FDA in October 2006 to treat several types of lymphoma, including cutaneous T-cell lymphoma [9] The second HDAC inhibitor approved for use in clinical setting is romidepsin (trade name Istodax®) Romidepsin was approved by FDA in November 2009 for the treatment of cutaneous T-cell lymphoma To date, more than a dozen of other HDAC inhibitors are currently in some phase of clinical trials, either as monotherapy or in combination with other chemotherapeutic agents or radiation, in patients with hematologic and solid tumors, including breast, lung, pancreas, bladder, renal cancers, glioblastoma, melanoma, lymphomas, leukemias, and multiple myeloma [10, 11] It has been shown that a majority of HDAC inhibitors share a common pharmacophore motif which consists of three distinct domains The first domain is a metal binding head group (ZBG), which interacts with the Zn2+ ion at the bottom of the active binding site of the enzyme The second © 2014 Bentham Science Publishers Medicinal Chemistry, 2014, Vol 10, No ?? Huong et al O H N O N H O O H3C O CH3 NH Oxamflatin N H N O CH3 O OH S O NH NHSO2Ph CH3 CH3 S NH O H3C Romidepsine O Trichostatin A CH3 CH3 HN NHOH SAHA O H3C O OH NHOH N H N N N N H H N NH2 HN MGCD0103 CH3 LBH-589 O O O O N H H N N NH2 H N O S NHOH O O MS-27-275 PXD-101 Fig (1) Structures of some HDAC inhibitors A O B Aminoacid Chain O N R S SRG N H NHOH O N C O N S N H NHOH R D HON O O NHOH Zn 2+ N ZBG R Fig (2) A pharmacophore motif of HDACIs (A) and benzothiazole-, 5-substitutedphenyl-1,3,4-thiadiazole-, and isatin-based hydroxamic acids as HDACIs (B, C, D) domain is a long aliphatic linker, which occupies the narrow hydrophobic tubular channel The third domain is known as a surface recognition group (SRG) (a cap group) (Fig 2) [9] The surface recognition domain is essential for recognizing and binding to aminoacid chains at the entrance of the active pocket of enzymes To date, diverse surface recognition groups have been investigated based on this common pharmacophore In our previous report we have also demonstrated that benzothiazole, 5-substitutedphenyl-1,3,4thiadiazole and isatin-based systems could be excellent substitutions for a phenyl ring in SAHA (Fig 2) [12-14] As a continuity of our search for new HDAC inhibitors we have designed and synthesized a series of 5-aryl-1,3,4-thiadiazolebased hydroxamic acids In this paper, the results from this study are reported MATERIALS AND METHODS Chemistry All products were homogenous, as examined by thinlayer chromatography (TLC), performed on Whatman® 250
m Silica Gel GF Uniplates and visualized under UV light at
254 nm Melting points were determined by a Gallenkamp Melting Point apparatus (LabMerchant, London, United Kingdom) and are uncorrected Nuclear magnetic resonance spectra (1H NMR) were recorded on a Bruker DPX 500 MHz FT NMR spectrometer using tetramethylsilane as an internal standard and dimethyl sulfoxide-d6 (DMSO-d6) as solvent unless otherwise indicated Chemical shifts were reported in parts per million (ppm) downfield from tetramethylsilane as an internal standard Splitting patterns were designated as 5-Aryl-1,3,4-thiadiazole-based Hydroxamic Acids as Histone Deacetylase Inhibitors follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet Electron ionization (EI), Electrospray ionization (ESI) and high-resolution mass spectra were obtained using PE Biosystems API 2000 (Perkin Elmer, Palo Alto, CA) and Mariner® mass spectrometers (Azco Biotech, Inc., Oceanside, CA), respectively Reagents and solvents were purchased from Aldrich, Fluka Chemical Corp (Milwaukee, WI, USA) or other certified chemical companies and were used directly without additional steps of purification Media, sera and other reagents used for cell culture were purchased from GIBCO Co Ltd (Grand Island, New York, NY) The synthesis of the series of N8-(5-aryl-1,3,4-thiadiazol2-yl)-N1-hydroxyoctandiamides (5a-i) was carried out as illustrated in (Scheme 1) Details are described below Synthesis of N8-[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N1hydroxyoctanediamide (5a) 2-(Furan-2-yl-methylene)hydrazinecarbothioamide (2a) Compound 5a was synthesized using furfuraldehyde (1a) as the starting material To the mixture of 1a (0.17 mL, mmoL) and thiosemicarbazide (0.218 g, 2.4 mmoL) in ethanol (15 mL) were added two drops of acetic acid were added The resulting solution was refluxed for about h Upon completion, the reaction mixture was cooled to room temperature and water (15 mL) was added to induce precipitation The precipitate was filtered and washed with water (2 times), dried at 50-55°C to give a white-yellowish solid (0.32g, 95%) of 2a mp: 143-146°C; Rf = 0.74 (DCM/MeOH = 9/1) 5-(Furan-2-yl)-1,3,4-thiadiazol-2-amine (3a) Compound 3a was prepared from 2a using FeCl3 reagent The synthesis was carried out as following: a suspension of FeCl3.12H2O in ethanol was slowly added to the solution of 2a (2 mmoL) in ethanol The mixture was refluxed for 15 min, then, it was allowed to cool to room temperature, diluted by water (15 mL), alkalized by a NaOH 10% solution (tested by litmus paper) and extracted with methylene chloride (40 mL) The extracts were pooled and methylene chloride was evaporated under reduced pressure The residue was rescrystallized from ethanol to give compound 3a as a yellowish solid Yield: 84%; mp: 195-197°C; Rf = 0.71 (DCM/MeOH = 9/1) IR (KBr, cm-1): 3253 (NH2), 1615, 1525, 1475 (C=C) Methyl 8-(5-(furan-2-yl)-1,3,4-thiadiazol-2-ylamino)-8oxooctanoate (4a) Compound 4a was obtained from 3a by the following procedure: 1,1’-carbonyldiimidazole (CDI) (162 mg, mmoL) was dissolved in dichloromethane (DCM) and suberic monomethyl ester acid (1.9 mL, mmoL) was added The mixture was stirred for 10 min, a solution of 3a (180 mg) in DMF (2 mL) was added The reaction mixture was stirred at 60°C for 24 h DCM was evaporated under reduced pressure, then the mixture was poured into 20 mL of cold water The precipitate appeared was filtered and washed, dried at 70°C to give a white-pink solid Yield: 62%; mp: 155-158°C; Rf = 0.69 (DCM/MeOH = 9/1) IR (KBr, cm-1): 3250 (NH), 2914 (CH2), 1725 (C=O), 1600, 1575, 1480 (C=C); CI-MS (m/z): 377.55 [M+] Medicinal Chemistry, 2014, Vol 10, No ?? N8-[5-(Furan-2-yl)-1,3,4-thiadiazol-2-yl]-N1-hydroxyoctanediamide (5a) Compound 5a was obtained from 4a Synthesis 5a was carried out by the following procedure: To a solution of 4a (169 mg, 0.5 mmoL) in a mixture of MeOH (5 mL) and DMF (3 mL) was added NH2OH.HCl (490 mg, mmoL) Ultrasound was used to dissolve the mixture The mixture was cooled in a mixture of salt and crushed ice and NaOH (400 mg, 10 mmoL) in H2O (1 mL) was added The reaction mixture was stirred for h and 30 at -5°C and poured slowly into 30 ml of cold water, acidified with HCl 5% to pH The precipitate appeared was filtered and washed Recrystallization from EtOH gave white crystals The product was dried at 40°C for 24 h in a vacuum oven Yield: 55.0%; mp: 200.0-201.0°C; Rf = 0.52 (DCM/MeOH= 9/1) IR (KBr, cm-1): 3350 (OH, acid), 3152 (NH), 2923, 2865, 2850 (CH, CH2), 1673, 1637 (C=O), 1567 (C=C) CI-MS (m/z): 337.0 [M-H]-, 322 [M-OH]-.1H-NMR (500 MHz, DMSO-d6, ppm):
10.35 (1H, s, NH), 7.93 (1H, s, H-5), 7.18 (1H, d, J = Hz, H-3), 6.72 (1H, s, H-4), 2.47-2.50 (2H, m, CH2), 1.93 (2H, t, J = Hz, CH2), 1.58-1.61 (2H, m, CH2), 1.47-1.49 (2H, m, CH2), 1.26-1.28 (4H, m, CH2) 13C NMR (125 MHz, DMSOd6, ppm):
171.66 (C-5’), 169.09 (C-8), 157.66 (C-1), 152.38 (C-2’), 145.31 (C-5’’), 145.17 (C-2’’), 112.52 (C4’’), 110.75 (C-3’’), 34.80 (C-7), 32.19 (C-2), 28.24 (C-3), 28.21 (C-6), 24.93 (C-5), 24.40 (C-4) Anal Calcd For C14H18N4O4S (338.38): C, 49.69; H, 5.36; N, 16.56 Found: C, 49.61; H, 5.41; N, 16.62 Compounds 5b-i were synthesized via intermediates 2b-i, 3b-i and 4b-i by similar procedures as described above for 5a All final compounds were crystallized from ethanol Here only data for the final compound series are described N1-[5-(5-Bromofuran-2-yl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctanediamide (5b) White crystals; Yield: 58.0%; mp: 178.0-179.0°C; Rf = 0.58 (DCM/MeOH = 9/1) IR (KBr, cm-1): 3400 (OH), 3183, 3071 (NH), 2938, 2856 (CH2), 1693, 1611 (C=O), 1580, 1563, 1519 (C=C) ESI-MS (m/z): 416.0 [M-H]- 1H-NMR (500 MHz, DMSO-d6, ppm):
12.70 (1H, s, NH), 10.33 (1H, s, NH), 7.23 (1H, d, J = 3.5 Hz, H-3), 6.83 (1H, d, J = 3.5 Hz, H-4), 2.47-2.50 (2H, m, CH2), 1.93 (2H, t, J = 7.0 Hz, CH2), 1.57-1.61 (2H, m, CH2), 1.45-1.51 (2H, m, CH2), 1.261.28 (4H, m, CH2) 13C NMR (125 MHz, DMSO-d6, ppm):
171.71 (C-5’), 169.13 (C-8), 157.82 (C-1), 151.33 (C-2’), 147.01 (C-2”), 124.45 (C-5”), 114.59 (C-3”), 113.57 (C-4”), 34.81 (C-7), 32.21 (C-2), 28.25 (C-6), 28.21 (C-3), 24.94 (C5), 24.41 (C-4) Anal Calcd For C14H17BrN4O4S (417.28): C, 40.30; H, 4.11; N, 13.43 Found: C, 40.44; H, 4.23; N, 13.37 N8-Hydroxy-N1-[5-(5-methylfuran-2-yl)-1,3,4-thiadiazol-2yl]octanediamide (5c) White crystals; Yield: 57.0%; mp: 200.5-201.0°C; Rf = 0.54 (DCM/MeOH= 9/1) IR (KBr, cm-1): 3416 (OH, acid), 3158, 3032 (NH), 2925, 2856 (CH, CH2), 1718, 1694 (C=O), 1651, 1567, 1552 (C=C) ESI-MS (m/z): 351.4 [M-H]-.1HNMR (500 MHz, DMSO-d6, ppm):
12.64 (1H,s,NH), 10.38 (2H, d, OH, NH), 7.04 (1H, d, J = Hz, H-3), 6.32 (1H, d, J=2.5Hz, H-4), 2.47-2.50 (2H, m, CH2), 2.36 (3H,s,CH3), 2.18 (1H, t, J = 7.5 Hz, CH2a), 1.94(1H, t, J = Hz, CH2b), Medicinal Chemistry, 2014, Vol 10, No ?? 1.58-1.60 (2H, m, CH2), 1.46-1.48 (2H, m, CH2), 1.26-1.27 (4H, m, CH2) 13C NMR (125 MHz, DMSO-d6, ppm):  174.47 (C-5’), 171.64 (C-8), 157.15 (C-1), 154.60 (C-2’), 152.54 (C-5’’), 143.57 (C-2’’), 112.03 (C-4’’), 108.85 (C3’’), 34.81 (C-7), 33.63 (C-2), 32.21 (C-3), 28.19 (C-6), 24.95 (C-5), 24.42 (C-4), 13.38 (C-CH3) Anal Calcd For C15H20N4O4S (352.14): C, 51.12; H, 5.72; N, 15.90 Found: C, 51.23; H, 5.77; N, 16.01 N8-Hydroxy-N1-[5-(5-thiophen-2-yl)-1,3,4-thiadiazol-2-yl] octanediamide (5d) White crystals; Yield: 60.1%; mp: 165.5-167.0°C; Rf = 0.50 (DCM/MeOH = 9/1) IR (KBr, cm-1): 3361 (OH, acid), 3154 (NH), 2931, 2854 (CH, CH2), 1674, 1637 (C=O), 1563 (C=C) CI-MS (m/z): 353 [M-H]- 1H-NMR (500 MHz, DMSO-d6, ppm):  12.65 (1H, s, NH), 10.34 (1H, s, NH), 8.67 (1H, s, OH), 7.75 (1H, d, J = 4.5 Hz, H-5), 7.69 (1H, d, J = 2.5 Hz, H-3), 7.19 (1H, s, H-4), 2.48 (2H, m, CH2), 1.93 (2H, t, J = 7Hz, CH2), 1.58-1.60 (2H, m, CH2), 1.47-1.49 (2H, m, CH2),1.26 (4H, m, CH2) 13C NMR (125 MHz, DMSO-d6, ppm):  171.61 (C-5’), 169.10 (C-8), 157.79 (C-1), 156.10 (C-2’), 132.32 (C-5’’), 129.11 (C-2’’), 128.99 (C4’’), 128.39 (C-3’’), 34.80 (C-7), 32.21 (C-2), 28.28 (C-6), 28.22 (C-3), 24.96 (C-5), 24.45 (C-4) Anal Calcd For C14H18N4O3S2 (354.45): C, 47.44; H, 5.12; N, 15.81 Found: C, 47.39; H, 5.23; N, 15.77 N1-[5-(5-Bromothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-N8-hydroxyoctanediamide (5e) White crystals; Yield: 65.1%; mp: 167.5-169.0°C; Rf = 0.62 (DCM/MeOH = 9/1) IR (KBr, cm-1): 3411 (OH, acid), 3143 (NH), 2933 (CH, CH2), 1692, 1631 (C=O), 1565 (C=C) CI-MS (m/z): 432 [M-H]- 1H-NMR (500 MHz, DMSO-d6, ppm):  12.68 (1H, s, OH), 10.33 (1H, s, NH), 7.53 (1H, s, H-3), 7.30 (1H, d, J = 2.5 Hz), 2.46 - 2.48 (2H, m, CH2), 1.91 (2H, t, J = 6,5 Hz, CH2), 1.57 (2H, m, CH2), 1.46 (2H, m, CH2), 1.24 (4H, m, CH2) 13C NMR (125 MHz, DMSO-d6, ppm):  171.68 (C-5’), 169.08 (C-8), 158.08 (C1), 155.25(C-2’), 134.04 (C-4’’), 131.71 (C-2’’), 129.61 (C3’’), 114.73 (C-5”), 34.79 (C-7), 32.20 (C-2), 28.67 (C-6), 28.21 (C-3), 24.95 (C-5), 24.43 (C-4) Anal Calcd For C14H17BrN4O3S2 (333.34): C, 38.80; H, 3.95; N, 12.93 Found: C, 38.91; H, 3.88; N, 12.85 N8-Hydroxy-N1-[5-(5-methylthiophen-2-yl)-1,3,4-thiadiazol2-yl]octanediamide (5f) White crystals; Yield: 65.0%; mp: 201.5-202.0°C; Rf = 0.50 (DCM/MeOH= 9/1) IR (KBr, cm-1): 3163, 3035 (NH), 2914, 2853 (CH, CH2), 1692 (C=O), 1642, 1612, 1573 (C=C) ESI-MS (m/z): 367.4 [M-H]-.1H-NMR (500 MHz, DMSO-d6, ppm):  12.57 (1H, s, NH), 10.34 (1H, s, NH), 7.45 (1H, d, J = 3.5 Hz, H-3), 6.88 (1H, d,J=3.5, H-4), 2.452.50 (5H, m, C-CH2 ,C-CH3), 1.93 (2H, t, J = 7.5 Hz, CH2), 1.57-1.60 (2H, m, CH2), 1.46-1.49 (2H, m, CH2), 1.26-1.27 (4H, m, CH2) 13C NMR (125 MHz, DMSO-d6, ppm):  171.58 (C-5’), 169.18 (C-8), 157.40 (C-1), 156.24 (C-2’), 142.97 (C-2’’), 129.91 (C-5’’), 129.10 (C-4’’), 126.83 (C3’’), 34.81 (C-7), 32.23 (C-2), 28.28 (C-3), 28.23 (C-6), 24.97 (C-5), 24.47 (C-4), 15.09 (CH 3) Anal Calcd For C15H20N4O3S2 (368.07): C, 48.89; H, 5.47; N, 15.21 Found: C, 48.95; H, 5.51; N, 15.33 Huong et al N1-Hydroxy-N8-[5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl]octanediamide (5g) White crystals; Yield: 63.0%; mp: 212.0-213.0°C; Rf = 0.55 (DCM/MeOH = 9/1) IR (KBr, cm-1): 3231(OH, acid), 3161 (NH), 3022 (Car-H), 2930, 2855 (CH, CH2), 1693, 1667 (C=O), 1557 (C=C) CI-MS (m/z): 347.45 [M-2H]-, 332.82 [M-OH]- 1H-NMR (500 MHz, DMSO-d6, ppm):  12.62 (1H, s, NH), 10.35 (1H, s, NH), 8.67 (1H, s, OH), 8.66 (1H, s, H-6), 8.19 (1H, d, J = 7.5 Hz, H-3), 7.98 (1H, t, J = 7.0 Hz, H-4), 7.51 (1H, s, H-5), 2.48 -2.50 (2H, m, CH2), 1.94 (2H, t, J = 7.0 Hz, CH2), 1.59-1.60 (2H, m, CH2), 1.47-1.50 (2H, m, CH2),1.27 (4H, m, CH2) 13C NMR (125 MHz, DMSO-d6, ppm):  171.71 (C-5’), 169.12 (C-8), 163.48 (C-1), 160.03 (C-2”), 149.92 (C-2’), 149.17 (C-6”), 137.72 (C-4”), 125.26 (C-5”), 119.81 (C-3”), 34.89 (C-7), 32.21 (C-2), 28.27 (C-6), 28.24 (C-3), 24.95 (C-5), 24.41 (C-4) Anal Calcd For C15H19N5O5S2 (349.41): C, 51.56; H, 5.48; N, 20.04 Found: C, 51.67; H, 5.53; N, 19.85 N1-Hydroxy-N8-[5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl]octanediamide (5h) White powder; Yield: 62.0%; mp: 201.5-203.0°C; Rf = 0.57 (DCM/MeOH = 9/1).IR (KBr, cm-1): 3251(OH, acid), 3155 (NH), 3008 (Caren-H), 2911, 2853 (CH, CH2), 1695, 1638 (C=O), 1576, 1554 (C=C) ESI-MS (m/z): 471.4253 [M+Na]+, 348.3974 [M+H]+ 1H-NMR (500 MHz, DMSOd6, ppm):  12.73 (1H, s, NH), 10.34 (1H, s, NH), 9.11 (1H, d, J = 1.5 Hz, H-2), 8.70 (1H, s, OH), 8.69 (1H, d, J = Hz, H-6), 8.32 (1H, d, J = 8.00 Hz, H-4), 7.55 - 7.57 (1H, m, H5); 2.50-2.52 (2H, m, CH2), 1.93 (2H, t, J = Hz, CH2), 1.61 (2H, m, CH2), 1.48 (2H, m, CH2),1.24 - 1.27 (4H, m, CH2) 13 C NMR (125 MHz, DMSO-d6, ppm):  172.19 (C-5’), 169.56 (C-8), 159.39 (C-1), 151.64 (C-2’), 147.88 (C-2”, C6”), 134.85 (C-4”), 127.00 (C-3”), 124.00 (C-5”),35.31 (C7), 32.68 (C-2), 28.76 (C-6), 28.70 (C-3), 25.44 (C-5), 24.93 (C-4) Anal Calcd For C15H19N5O5S2 (349.41): C, 51.56; H, 5.48; N, 20.04 Found: C, 51.50; H, 5.58; N, 20.24 N1-Hydroxy-N8-[5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl]octanediamide (5i) White solids; Yield: 67.0%; mp: 170.0-172.0°C; Rf = 0.52 (DCM/MeOH = 9/1).IR (KBr, cm-1): 3265(OH, acid), 3139 (NH), 3009 (Caren-H), 2852 (CH, CH2), 1701, 1646 (C=O), 1620, 1547 (C=C) CI-MS (m/z): 348.4 [M-H]- 1HNMR (500 MHz, DMSO-d6, ppm):  12.85 (1H, s, NH), 10.42 (1H, s, NH), 8.74 (3H, s, H-2, H-6, OH), 7.90 (2H, s, H-3, H-5); 2.50 (2H, m, CH2), 1.94 (2H, m, CH2), 1.60 (2H, m, CH2), 1.48 (2H, m, CH2),1.26 (4H, m, CH2) 13C NMR (125 MHz, DMSO-d6, ppm):  171.86 (C-5’), 169.10 (C-8), 159.52 (C-1), 150.70 (C-2’, C-2”, C-6”), 137.20 (C-4”), 120.79 (C-3”, C5”), 34.83 (C-7), 32.19 (C-2), 28.27(C-6), 28.21 (C-3), 24.95 (C-5), 24.42 (C-4) Anal Calcd For C15H19N5O5S2 (349.41): C, 51.56; H, 5.48; N, 20.04 Found: C, 51.63; H, 5.56; N, 20.37 Cytotoxicity Assays Four human cancer cell lines, PC3 (prostate cancer), SW620 (colon cancer), MCF-7 (breast adenocarcinoma), and AsPC-1 (pancreatic cancer) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) Cells were plated at
103 cells/well in 96- 5-Aryl-1,3,4-thiadiazole-based Hydroxamic Acids as Histone Deacetylase Inhibitors Ar CHO H2NCSNHNH2 Ar H+, EtOH 1a-i O N Ar NH2 S N S Ar 3a-i O O N OCH3 NH2 EtOH N N H N FeCl3.6H2O S 2a-i Suberic acid monomethyl ester CDI, DMF N H N Medicinal Chemistry, 2014, Vol 10, No ?? NH2OH.HCl NaOH, MeOH Ar O N S 4a-i N H NHOH 5a-i Scheme Synthetic pathway for N1-hydroxy-N8-(5-aryl-1,3,4-thiadiazol-2-yl)octandiamides (5a-i) well plates, incubated overnight, and treated with samples for 48 h Test compounds were dissolved in dimethyl sulfoxide (DMSO) Cytotoxicity was measured by the method as described in literature [15] with slight modifications [16,17] The IC50 values were calculated according to the Probits method [18] The values reported for these compounds are averages of three separate determinations Western Blot Assay For Western blot assay, the total protein extracts were first prepared by lysing cells in RIPA buffer (50 mM Tris-Cl [pH 8.0], mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, and mM phenylmethylsulfonyl fluoride) Protein concentrations in the lysates were determined using a BioRad protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer's instructions Samples were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes The membranes were incubated with blocking buffer (Tris-buffered saline containing 0.2% Tween-20 and 3% nonfat dried milk) and probed with the primary antibodies against acetyl histoneH3, -H4, and GAPDH) After washing, membranes were probed with horseradish peroxidase-conjugated secondary antibodies Detection was performed using an enhanced chemiluminescent protein (ECL) detection system (Amersham Biosciences, Little Chalfont, UK) Docking Studies AutoDock Vina program [19] (The Scripps Research Institute, CA, USA) was used in the docking studies Initial structures of HDAC8 [20] and HDAC2 [21] (complexed with SAHA) were obtained from the Protein Data Bank (PDB) (PDB ID: 1T69 and PDB ID: 4LXZ, respectively) and coordinates for the compounds were generated using the GlycoBioChem PRODRG2 Server (http://davapc1.bioch dundee.ac.uk/prodrg/) [22] The grid maps for docking studies 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 [12-14] AutoDock Vina program was run with eight-way multithreading and the other parameters were default settings in AutoDock Vina program RESULTS AND DISCUSSION Chemistry A series of 5-aryl-1,3,4-thiadiazole-based hydroxamic acids (5a-i) were synthesized via a 4-step pathway (Scheme 1) In the first step, the thiosemicarbazones 2a-i were obtained by condensation of simple benzaldehydes 1a-i with thiosemicarbazide Intramolecular cyclization of 2a-i proceeded smoothly using ferric chloride in ethanol to give 2amino-5-aryl-1,3,4-thiadiazole derivatives 3a-i Coupling of 3a-i with suberic acid monomethyl ester using 1,1’carbodiimidazole (CDI) as a carboxylic activating reagent generated the ester intermediates 4a-i Reaction of the esters 4a-i with hydroxylamine in alkaline conditions gave the final products 5a-i in generally good yields The structures of the obtained compounds were straightforwardly and unambiguously confirmed by spectral studies, including IR, MS, 1H NMR and 13C NMR, and elemental analysis Bioactivity Initially we examined to see whether the synthesized compounds show cytotoxicity in human cancer cell lines The SRB (sulforhodamine B) cell proliferation assay was employed to evaluate the antiproliferative activity of the compounds The compounds 5a-i were first screened at the concentration of 30
M for cell growth inhibition against SW620 (human colon cancer) cell line It was found that all compounds 5a-i inhibited the growth of SW620 cells by more than 50% at this concentration Therefore, the compounds were further evaluated at concentrations (30, 10, 3, 1, 0.3
M) in the same SW620 and three more human cancer cell lines, including MCF-7 (breast adenocarcinoma), PC-3 (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 1) From (Table 1), it could be seen that the 2-furfuryl and thiophen-2-yl groups attached to position of the 1,3,4thiadiazole scaffold seemed to be more favorable for the cytotoxicity, compared to the phenyl moiety, as evidenced by the IC50 values of the compounds 5a and 5d were generally 2- to 6-fold lower than that of compound in four cell lines tested 5-Bromo or 5-methyl substitutions (compounds 5b, 5c) were tolerable for the cytotoxicity in case of furfuryl ring However, for compound 5d with a thiophen-2-yl group, only 5-methyl substitution (compound 5f) on the thiophene moiety was acceptable, while 5-bromo substituent introduced on the thiophene ring led to the loss of cytotoxicity against all four cell lines assayed Three compounds 5g, 5h and 5i bearing 2-, 3- and 4-pyridyl moieties showed potent cytoxicity, with compound 5g (bearing 2-pyridyl) and 5h (bearing 3-pyridyl) exhibited comparable cytotoxicity to compound (bearing a phenyl substituent), demonstrating Medicinal Chemistry, 2014, Vol 10, No ?? Huong et al Table HDAC inhibition and cytotoxicity of the compounds synthesized O N N Ar S N H O NHOH 5a-i Cpd Code Ar 5a Molecular Weight Cytotoxicity (IC50,1 μ M)/Cell Lines2 LogP3 SW620 MCF-7 PC3 AsPC-1 338.38 0.29 0.60 0.42 0.44 0.72 427.28 0.25 0.31 0.45 0.42 1.61 352.38 0.65 0.39 0.59 0.88 1.27 354.45 0.28 0.35 0.45 0.28 1.17 433.34 >10 >10 >10 >10 2.06 368.45 0.46 0.39 0.43 0.86 1.72 349.41 0.57 0.52 0.45 0.31 0.16 349.41 0.59 0.37 0.90 1.04 0.16 349.41 3.56 3.00 6.02 4.31 0.16 384.42 0.70 1.80 0.88 2.71 1.35 264.32 3.70 6.42 4.31 3.66 1.44 O 5b 5c Br O H3C O 5d S 5e 5f Br S H3C S N 5g 5h N 5i N 64 SAHA5 The concentrations (μM) of compounds that produces a 50% reduction in cell growth, the numbers represent the averaged results from triplicate experiments with deviation of less than 10%.; 2Cell lines: SW620, colon cancer; MCF-7, breast cancer; PC3, prostate cancer; AsPC-1, pancreatic cancer; 3Estimated by Software KOWWIN v1.67; 4Data reported in reference 13; 5SAHA, suberoylanilide acid, a positive control that 2-pyridyl, 3-pyridyl and phenyl moieties could be exchangeable at position of the 1,3,4-thiadiazole scaffold Compound 5i with 5-(4-pyridyl)-1,3,4-thiadiazole system still displayed comparable cytotoxicity to SAHA in four cell lines evaluated Next, we set to examine the effects of the representative compounds, including 5a, 5b, 5d, 5e, 5g and 5i, on the HDAC activity using the Western blot assay In the first experiment, we evaluated the HDAC inhibition by the compounds at μM in a whole cell system with primary antibodies against acetyl histone-H3, -H4, and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) The results showed that in the presence of compounds 5a, 5b, and 5d at μM, the acetylation of histone-H3 and histone-H4 clearly in- creased by a level similar to that caused by the presence of SAHA (Fig 3), indicating that the HDAC activity had been inhibited Meanwhile, in the presence of compounds 5e and 5i, the acetyl-H3 and acetyl-H4 were not observed, indicating that HDAC activity was not inhibited, leading to a complete deacetylation of histones H3 and H4 These results were found to be very well correlated with the cytotoxicity, e.g compound 5e was the least cytotoxic ones with the IC50 values against all cancer cell lines higher than 10 μM, compound 5i showed IC50 values only in the range of 3.004.31 μM In contrast, compounds 5a, 5b and 5d showed much stronger cytotoxicity with IC50 values observed in below micromolar range in all cancer cell lines tested (Table 1) Among the compounds above, only in case of compound 5-Aryl-1,3,4-thiadiazole-based Hydroxamic Acids as Histone Deacetylase Inhibitors Fig (3) Effect of the compounds synthesized on histone acetylation in SW620 cells Cells were treated with compounds (1 μM) for 24 hrs Levels of acetylated histone-H3 and -H4 in total cell lysates were determined by Western immunoblot analysis GAPDH is glyceraldehyde 3-phosphate dehydrogenase Fig (4) Effect of the compounds synthesized on histone acetylation in SW620 cells Cells were treated with compounds (3 μM) for 24 hrs Levels of acetylated histone-H3 in total cell lysates were determined by Western immunoblot analysis Medicinal Chemistry, 2014, Vol 10, No ?? 5g the HDAC inhibition at μM was not finely correlated with the cytotoxicity However, when assayed at higher concentrations of μM, compound 5g showed clear and strong inhibition of HDAC, as seen in (Fig 4) At μM, compound 5i also caused moderate inhibitory effects towards the enzymes HDAC (Fig 4) Compound 5e, however, did not show any inhibition of histone-H3 deacetylation (Fig 4) even at μM Thus, it is likely that the incorporation of the bulky bromine group on the thiophene ring was deleterious for the binding of compound 5e to HDAC These results were found correlated well with cytotoxicity profile of 5e Though a more comprehensive experiment needs to be performed with purified types of HDAC, the present results suggest that inhibition of HDAC could be a prominent mechanism of these compounds’ cytotoxicity Regarding drug-like properties of the compounds, it could be seen that all compounds have a molecular weight of less than 500 KDa and logP values from 0.16 to 2.06 (Table 1) The numbers of hydrogen bond donors (3) and hydrogen bond acceptors (8) fall within Lipinsky’s rule of five Though no clear correlation between logP values and bioactivity of the compounds was observed, it could be noted that compound 5e with the highest logP value (2.06) in the series was not active up to 10 μM in all cell lines tested This observation suggests that high logP values may not be favorable for cytotoxicity of the compounds in this series Fig (5) Stereo-view presentations of the actual binding poses of SAHA and simulated docking poses of compound 5a (A) and 5d (B) to HDAC2 SAHA is represented as a stick model with carbon, nitrogen, and oxygen atoms in yellow, blue and red, respectively Compounds 5a, and 5d are shown as a stick model with carbon atoms colored in cyan and magenta, respectively Nitrogen and oxygen atoms of compounds 5a and 5d are colored in blue and red Interaction parts of the HDAC2 were shown as a stick model with carbon, nitrogen, and oxygen colored as green, blue and red, respectively Predicted hydrogen bonds are represented as black line 8 Medicinal Chemistry, 2014, Vol 10, No ?? Docking Study To gain some insights into the interaction between these compounds and HDAC, we implemented docking experiments using the active site of HDAC Initially we selected the structure of HDAC8 in complex with SAHA as a docking template because its crystal structure is available (PDB ID: 1T69) [20] HDAC8 shares high structural similarity (DALI Z score = 40.4 and r.m.s.d = 2.1 A) [23] and amino acid sequence similarity (46%) to HDAC4 During the course of our study, the crystal structure of HDAC2 in complex with SAHA (PDB ID: 4LXZ) has been published by Lauffer and co-workers [21] Since histone-H3 and histoneH4 deacetylation is regulated by HDAC1 and HDAC2, we decided to focus our efforts on additional docking of these compounds to HDAC2 We executed control docking experiments with SAHA to the crystal structures of HDAC2 and HDAC8 using AutoDock Vina program [19] after SAHA was removed from the complex structures, as described previously [12-14] It was found from docking experiments that the compounds, as represented by 5a and 5d, were located in the active site (Fig 5) with stabilization energy lower than that of SAHA For example, stabilization energies of predicted binding modes on HDAC2/HDAC8 were calculated to be -7.6/-6.9 and -7.5/-7.0 kcal/mol for compounds 5a and 5d, respectively, while the values for SAHA were -6.3/-4.4 kcal/mol (r.m.s.d distance from the original SAHA in the crystal structure : 0.609/2.056 Å) A more careful look revealed that the long aliphatic parts of all compounds were docked in SAHA binding site of HDAC2 and HDAC8 Although the line-ups were not matched perfectly, the ends of branch chains were found to direct straight to zinc and zinc binding site of the enzymes’ active site, similar to the binding mode of SAHA [20] Detailed analysis of the binding interactions of compounds with HDAC2 showed that in general, the thiadiazole moiety seemed to form a stacking interaction and hydrogen bonding with amine group of
nitrogen of His33 (predicted bond distance is 3.5 Å) In the case of compound 5a, it was found that the furane ring could form an additional hydrogen bonding with a carboxyl group on side chain of Glu103 (predicted bond distance is 3.6 Å), leading to higher binding affinity of this compound to the enzyme However, for compound 5d, a thiophene could not form a hydrogen bond with Glu103, so thiophene and thiadiazole seemed to make a balance between stacking interaction, electrostatic charge, and gauche of two sulfurs As a result, the predicted positions of thiophene and thiadiazole in compound 5d are different from those in compound 5a Huong et al phenyl, while 2-pyridyl and 3-pyridyl were found equally good as a surrogate for the phenyl group Substituents like 5-bromo and 5-methyl on the furfuryl moiety were tolerable, but on the thiophenyl moiety, only a 5-methyl group was acceptable Docking study performed with two representative compounds 5a (bearing a 2-furfuryl) and 5d (bearing thiophen-2-yl) revealed that these compounds had higher binding affinities to HDAC2 and HDAC8 compared to SAHA All of the compounds designed and synthesized possessed good drug-like properties From this study, several compounds (such as 5a-d, 5f-h) turn out to be promising candidates that warrant further investigation CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest ACKNOWLEDGEMENTS We acknowledge the principal financial supports from a National Foundation for Science and Technology of Vietnam (NAFOSTED), Grant number 104.01-2013.16, and the Medical Research Center program through the National Research Foundation of Korea, Grant number 2010-0029480 The docking study was supported by the National Research Foundation of Korea (NRF) grant for the Global Core Research Center (GCRC) funded by the Korea government (MSIP, Grant number 2011-0030001) REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] CONCLUSION In this study, a series of 5-aryl-1,3,4-thiadiazole-based hydroxamic acids analogous to SAHA have been designed, synthesized and evaluated for antitumor cytotoxicity and HDAC inhibitory activity It was found that the 5-aryl-1,3,4thiadiazole scaffold was replaceable for a phenyl ring in SAHA In many instances, this scaffold proved to be more favorable for cytotoxicity of the compounds Among the 5-aryl substituents, 2-furfuryl (compound 5a) and thiophen-2-yl (compound 5d) were found to have positive influence on the bioactivity compared to the [9] [10] [11] [12] 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 Johnstone, R.W., Histone-deacetylase inhibitors: Novel drugs for the treatment of cancer Nature Rev Drug Disc., 2002, 1, 287-299 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? 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