Send Orders for Reprints to reprints@benthamscience.net 1573-4064/14 $58.00+.00 © 2014 Bentham Science Publishers 5-Aryl-1,3,4-thiadiazole-based Hydroxamic Acids as Histone Deacetylase
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1573-4064/14 $58.00+.00 © 2014 Bentham Science Publishers
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; b School of Medicine and Pharmacy, Hanoi
Na-tional University, Hanoi, Vietnam; c Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul
Na-tional University, Seoul 151-742, Korea; d Korea Research Institute of Bioscience and Biotechnology, Cheongwon, Chungbuk 363-883, Korea; e College 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
hy-droxamic 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
ana-logues of SAHA and evaluated their biological activities Most of the compounds in this series, e.g compounds with
5-aryl moiety being 2-furfuryl (5a), 5-bromofuran-2-yl (5b), 5-methylfuran-2-yl (5c), thiophen-2-yl (5d),
5-methylthiophen-2-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 4 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
affini-ties towards HDAC2 and 8
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
deacety-lation and acetydeacety-lation 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
en-zymes have been identified in human Based on their
ho-mologies 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, 9 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
demon-strated that these enzymes are not only involved in the
regu-lation 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
Pharmaceu-tical 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
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 (Vori-nostat), trichostatin A, LBH-589 (Panobi(Vori-nostat), PXD-101, MGCD0103 (Mocetinostat), MS-27-275 (Entinostat), and oxamflatin, among others [7, 8] Of these, SAHA (Vori-nostat, 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 cur-rently 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 multi-ple 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
Trang 2domain 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
phar-macophore In our previous report we have also
demon-strated that benzothiazole,
5-substitutedphenyl-1,3,4-thiadiazole and isatin-based systems could be excellent
sub-stitutions 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-thiadiazole-based hydroxamic acids In this paper, the results from this
study are reported
MATERIALS AND METHODS Chemistry
All products were homogenous, as examined by thin-layer 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
N OH
O O
CH3
CH3 N
H3C
CH 3
H
N OH O
O
Trichostatin A SAHA
N
O
O
N H
O
H
NH2
Oxamflatin
O
NHOH
H S O
O
NHOH H
HN
LBH-589
CH3
O
N
N N
O
H
NH 2
N
MGCD0103
Romidepsine
O
NHOH
NHSO 2 Ph
O HN
NH NH NH
O
O
O
O
CH3
CH 3
H 3 C
H3C
CH 3
S O S
Fig (1) Structures of some HDAC inhibitors
A
R
S
N N
6
B
C
S
N N
6 R
SRG
ZBG
Zn 2+
Aminoacid Chain
N
NHOH 4
R
D
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)
Trang 35-Aryl-1,3,4-thiadiazole-based Hydroxamic Acids as Histone Deacetylase Inhibitors Medicinal Chemistry, 2014, Vol 10, No ?? 3
follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
mul-tiplet 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.,
Ocean-side, CA), respectively Reagents and solvents were
pur-chased 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-thiadiazol-2-yl)-N 1-hydroxyoctandiamides (5a-i) was carried out as
illustrated in (Scheme 1) Details are described below
Synthesis of N 8 -[5-(furan-2-yl)-1,3,4-thiadiazol-2-yl]-N 1
-hydroxyoctanediamide (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, 2
mmoL) and thiosemicarbazide (0.218 g, 2.4 mmoL) in
etha-nol (15 mL) were added two drops of acetic acid were
added The resulting solution was refluxed for about 4 h
Upon completion, the reaction mixture was cooled to room
temperature and water (15 mL) was added to induce
precipi-tation 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,
di-luted by water (15 mL), alkalized by a NaOH 10% solution
(tested by litmus paper) and extracted with methylene
ride (40 mL) The extracts were pooled and methylene
chlo-ride was evaporated under reduced pressure The residue was
rescrystallized from ethanol to give compound 3a as a
yel-lowish 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)-8-oxooctanoate (4a)
Compound 4a was obtained from 3a by the following
procedure: 1,1’-carbonyldiimidazole (CDI) (162 mg, 1
mmoL) was dissolved in dichloromethane (DCM) and
suberic monomethyl ester acid (1.9 mL, 1 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+]
N 8 -[5-(Furan-2-yl)-1,3,4-thiadiazol-2-yl]-N 1 -hydroxyoctan-ediamide (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, 7 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 1 h and 30 min at -5°C and poured slowly into 30 ml of cold water, acidified with HCl 5% to
pH 5 The precipitate appeared was filtered and washed Re-crystallization 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 = 3 Hz, H-3), 6.72 (1H, s, H-4), 2.47-2.50 (2H, m, CH2), 1.93 (2H, t,
J = 7 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,
DMSO-d6, ppm): 171.66 (C-5’), 169.09 (C-8), 157.66 (C-1), 152.38 2’), 145.31 5’’), 145.17 2’’), 112.52 (C-4’’), 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
N 1 -[5-(5-Bromofuran-2-yl)-1,3,4-thiadiazol-2-yl]-N 8 -hydro-xyoctanediamide (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), 1.26-1.28 (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 7), 32.21 2), 28.25 6), 28.21 3), 24.94 (C-5), 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
N 8 -Hydroxy-N 1 -[5-(5-methylfuran-2-yl)-1,3,4-thiadiazol-2-yl]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]-.1
H-NMR (500 MHz, DMSO-d6, ppm): 12.64 (1H,s,NH), 10.38 (2H, d, OH, NH), 7.04 (1H, d, J = 3 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, CH ), 1.94(1H, t, J = 7 Hz, CH ),
Trang 41.58-1.60 (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 5’’), 143.57 2’’), 112.03 4’’), 108.85
(C-3’’), 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
N 8 -Hydroxy-N 1 -[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,
DM-SO-d6, ppm): 171.61 (C-5’), 169.10 (C-8), 157.79 (C-1),
156.10 2’), 132.32 5’’), 129.11 2’’), 128.99
(C-4’’), 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
N 1 -[5-(5-Bromothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-N 8
-hy-droxyoctanediamide (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,
DM-SO-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 5’), 169.08 8), 158.08
1), 155.252’), 134.04 4’’), 131.71 2’’), 129.61
(C-3’’), 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
N 8 -Hydroxy-N 1
-[5-(5-methylthiophen-2-yl)-1,3,4-thiadiazol-2-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),
2.45-2.50 (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 2’’), 129.91 5’’), 129.10 4’’), 126.83
(C-3’’), 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 (CH3) 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
N 1 -Hydroxy-N 8 -[5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl]octa-nediamide (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,
DM-SO-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
N 1 -Hydroxy-N 8 -[5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl]octa-nediamide (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,
DMSO-d6, 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 = 1 Hz,
6), 8.32 (1H, d, J = 8.00 Hz, 4), 7.55 - 7.57 (1H, m,
H-5); 2.50-2.52 (2H, m, CH2), 1.93 (2H, t, J = 7 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 (8), 159.39 (1), 151.64 (2’), 147.88 (2”, C-6”), 134.85 4”), 127.00 3”), 124.00 5”),35.31 (C-7), 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
N 1 -Hydroxy-N 8 -[5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl]octa-nediamide (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]- 1
H-NMR (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, Manas-sas, VA, USA) Cells were plated at 9 103 cells/well in
Trang 596-5-Aryl-1,3,4-thiadiazole-based Hydroxamic Acids as Histone Deacetylase Inhibitors Medicinal Chemistry, 2014, Vol 10, No ?? 5
well plates, incubated overnight, and treated with samples
for 48 h Test compounds were dissolved in dimethyl
sulfox-ide (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], 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1%
SDS, and 1 mM phenylmethylsulfonyl fluoride) Protein
concentrations in the lysates were determined using a
Bio-Rad protein assay kit (Bio-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
histone-H3, -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
(Amer-sham 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
stud-ies 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
ac-ids (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
pro-ceeded smoothly using ferric chloride in ethanol to give
2-amino-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 straight-forwardly 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 com-pounds were further evaluated at 5 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 prolifera-tion inhibiprolifera-tion) 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 5 of the 1,3,4-thiadiazole 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 gener-ally 2- to 6-fold lower than that of compound 6 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 intro-duced 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 6 (bearing a phenyl substituent), demonstrating
H
N NH 2
S
Ar S
N N
NH 2
Ar S
N N
N OCH3
O O
N N
N
H NHOH
O O
6
4a-i
Suberic acid monomethyl ester
5a-i
H2NCSNHNH2
H + , EtOH
FeCl3.6H2O
CDI, DMF
EtOH
NH 2 OH.HCl NaOH, MeOH
Scheme 1 Synthetic pathway for N 1 -hydroxy-N8-(5-aryl-1,3,4-thiadiazol-2-yl)octandiamides (5a-i)
Trang 6that 2-pyridyl, 3-pyridyl and phenyl moieties could be
ex-changeable at position 5 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
ex-periment, we evaluated the HDAC inhibition by the
com-pounds at 1 μM in a whole cell system with primary
antibod-ies against acetyl histone-H3, -H4, and GAPDH
(Glyceral-dehyde 3-phosphate dehydrogenase) The results showed
that in the presence of compounds 5a, 5b, and 5d at 1 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,
indicat-ing that HDAC activity was not inhibited, leadindicat-ing to a com-plete 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 4 cancer cell lines higher than 10 μM,
compound 5i showed IC50 values only in the range of
3.00-4.31 μM In contrast, compounds 5a, 5b and 5d showed
much stronger cytotoxicity with IC50 values observed in be-low micromolar range in all 4 cancer cell lines tested (Table
1). Amongthe compounds above, only in case of compound
Table 1 HDAC inhibition and cytotoxicity of the compounds synthesized
Ar S
N N
N NHOH
O O
6
5a-i
Cytotoxicity (IC 50 ,1μM)/Cell Lines2
Weight
SW620 MCF-7 PC3 AsPC-1
LogP3
5a
O
338.38 0.29 0.60 0.42 0.44 0.72
5b
O
5c
O
5d
S
354.45 0.28 0.35 0.45 0.28 1.17
5e
S
5f
S
5g
N
5h
N
349.41 0.59 0.37 0.90 1.04 0.16
5i
N
1 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%.; 2
Cell lines: SW620, colon cancer; MCF-7, breast cancer; PC3, prostate cancer; AsPC-1, pancreatic cancer; 3
Estimated by Software KOWWIN v1.67; 4
Data reported in reference 13; 5 SAHA, suberoylanilide acid, a positive control
Trang 75-Aryl-1,3,4-thiadiazole-based Hydroxamic Acids as Histone Deacetylase Inhibitors Medicinal Chemistry, 2014, Vol 10, No ?? 7
Fig (3) Effect of the compounds synthesized on histone
acetyla-tion 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
acetyla-tion 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
5g the HDAC inhibition at 1 μM was not finely correlated
with the cytotoxicity However, when assayed at higher
con-centrations of 3 μM, compound 5g showed clear and strong inhibition of HDAC, as seen in (Fig 4) At 3 μM, compound 5i also caused moderate inhibitory effects towards the en-zymes HDAC (Fig 4) Compound 5e, however, did not show any inhibition of histone-H3 deacetylation (Fig 4)
even at 3 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 per-formed with purified types of HDAC, the present results do 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 bioac-tivity 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 ob-servation suggests that high logP values may not be favor-able 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
com-pounds 5a and 5d are colored in blue and red Interaction parts of the HDAC2 were shown as a stick model with carbon, nitrogen, and
oxy-gen colored as green, blue and red, respectively Predicted hydrooxy-gen bonds are represented as black line
Trang 8Docking Study
To gain some insights into the interaction between these
compounds and HDAC, we implemented docking
experi-ments using the active site of HDAC Initially we selected
the structure of HDAC8 in complex with SAHA as a
dock-ing 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
com-plex with SAHA (PDB ID: 4LXZ) has been published by
Lauffer and co-workers [21] Since H3 and
histone-H4 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
ex-periments with SAHA to the crystal structures of HDAC2
and HDAC8 using AutoDock Vina program [19] after
SAHA was removed from the complex structures, as
de-scribed previously [12-14] It was found from docking
ex-periments that the compounds, as represented by 5a and 5d,
were located in the active site (Fig 5) with stabilization
en-ergy 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]
De-tailed 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
bond-ing 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
com-pound 5a
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-1,3,4-thiadiazole 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
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 toler-able, but on the thiophenyl moiety, only a 5-methyl group was acceptable Docking study performed with two
repre-sentative 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 inves-tigation
CONFLICT OF INTEREST
The authors confirm that this article content has no con-flict 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 Re-search 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 Re-search Center (GCRC) funded by the Korea government (MSIP, Grant number 2011-0030001)
REFERENCES
[1] De Ruijter, A.J.M.; Gennip, A.H.V.; Caron, H.N.; Kemp, S.; Kuilenburg, A.B.P.V Histone deacetylases (HDACs):
characteriza-tion of the classical HDAC family Biochem J., 2003, 370,
737-739
[2] Johnstone, R.W., Histone-deacetylase inhibitors: Novel drugs for
the treatment of cancer Nature Rev Drug Disc., 2002, 1, 287-299
[3] Marks, P.A.; Rifkind, R.A.; Richon, V.M.; Breslow, R.; Miller, T.; Kelly, W.K Histone deacetylases and cancer: Causes and
Thera-pies Nature Rev Cancer., 2001, 1, 194-202
[4] Witt, O.; Deubzer, H.E.; Milde, T.; Oehme, I HDAC family: What
are the cancer relevant targets? Cancer Lett., 2009, 277, 8-21
[5] Dokmanovic, M.; Marks, P.A Histone deacetylase inhibitors:
discovery and development as anticancer agents Expert Opin
In-vesti Drug., 2005, 14, 1497-1511
[6] Glaser, K.B HDAC inhibitors: clinical update and
mechanism-based potential Biochem Pharmacol., 2007, 74, 659-671
[7] Dallavalle, S.; Cincinelli, R.; Nannei, R.; Merlini, L.; Morini, G.; Penco, S.; Pisano, C.; Vesci, L.; Barbarino, M.; Zuco, V.; De Ce-sare, M.; Zunino, F Design, synthesis, and evaluation of biphenyl-4-yl-acrylohydroxamic acid derivatives as histone deacetylase
(HDAC) inhibitors Eur J Med Chem., 2009, 44, 1900-1912
[8] 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
On-col., 2009, 35, 909-920
[9] 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 his-tone deacetylase homologue bound to the TSA and SAHA
inhibi-tors Nature 1999, 401, 188-193
[10] Valente, S.; Mai, A Small-molecule inhibitors of histone deacety-lase for the treatment of cancer and non-cancer diseases: a patent
review (2011-2013) Expert Opin Ther Pat., 2014, 24, 401-415
[11] West, A.C.; Johnstone, R.W New and emerging HDAC inhibitors
for cancer treatment J Clin Invest., 2014, 124, 30-39
[12] 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 Ben-zothiazole-containing hydroxamic acids as histone deacetylase
Trang 9in-5-Aryl-1,3,4-thiadiazole-based Hydroxamic Acids as Histone Deacetylase Inhibitors Medicinal Chemistry, 2014, Vol 10, No ?? 9
hibitors and antitumor agents Bioorg Med Chem Lett., 2001, 21,
7509-7512
[13] 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-substitutedphenyl-1,3,4-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)
[14] 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
deacety-lase inhibitors and antitumor agents Eur J Med Chem., 2013, 70,
477-486
[15] 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
[16] You, Y.J.; Kim, Y.; Nam, N.H.; Bang, S.C.; Ahn, B.Z Alkyl and
carboxylalkyl esters of 4-demethyl-4-deoxypodophyllotoxin:
syn-thesis, cytotoxic, and antitumor activity Eur J Med Chem., 2004,
39, 189-193
[17] Kim, Y.; You, Y.J.; Nam, N.H.;, Ahn, B.Z Prodrugs of
4-demethyl-4-deoxypodophyllotoxin: synthesis and evaluation of the
antitumor activity Bioorg Med Chem Lett., 2002, 12, 3435-3438
[18] 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
anti-cancer drug screening Cancer Res., 1992, 52, 3029-3034
[19] Trott, O.; Olson, A.J AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient
optimi-zation, and multithreading J Comput Chem., 2010, 31, 455-461
[20] Somoza, J.R.; Skene, R.J.; Katz, B.A.; Mol, C.; Ho, J.D.; Jennings, A.J., Luong, C., Arvai, A., Buggy, J.J.; Chi, E.; Tang, J.; Sang, B.C.; Verner, E.; Wynands, R.; Leahy, E.M.; Dougan, D.R.; Snell, S.; Navre, M.; Knuth, M.W.; Swanson, R.V.; McRee, D.E.; Tari, L.W Structural snapshots of human HDAC8 provide insights into
the class I histone deacetylases Structure 2004, 12, 1325-1334
[21] 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
[22] Schuttelkopf, A.W.; van Aalten, D.M PRODRG: a tool for
high-throughput crystallography of protein-ligand complexes Acta
crystallographica Section D, Biological crystallography 2004, 60,
1355-1363
[23] Holm, L.; Rosenstrom, P Dali server: conservation mapping in 3D
Nucleic Acids Res., 2010, 38, W545-549