Synthesis, bioevaluation and docking study of 5-substitutedphenyl-1,3,4-thiadiazole-based hydroxamic acids as histone deacetylase inhibitors and antitumor agents

Article abstract of DOI:10.3109/14756366.2013.832238

Since the first histone deacetylase (HDAC) inhibitor (Zolinza, widely known as suberoylanilide hydroxamic acid; SAHA) was approved by the Food and Drug Administration for the treatment of T-cell lymphoma in 2006, the search for newer HDAC inhibitors has attracted a great deal of interest of medicinal chemists worldwide. As a continuity of our ongoing research in this area, we designed and synthesized a series of 5-substitutedphenyl-1,3,4-thiadiazole-based hydroxamic acids as analogues of SAHA and evaluated their biological activities. A number of compounds in this series, for example, N¹-hydroxy-N⁸-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)octandiamide (5b), N¹-hydroxy-N⁸-(5-(3-chlorophenyl-1,3,4-thiadiazol-2-yl)octandiamide (5c) and N¹-hydroxy-N⁸-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)octandiamide (5d), were found to possess potent anticancer cytotoxicity and HDAC inhibition effects. Compounds 5b-d were generally two- to five-fold more potent in terms of cytotoxicity compared to SAHA against five cancer cell lines tested. Docking studies revealed that these hydroxamic acid displayed higher affinities than SAHA toward HDAC8.

Full text of DOI:10.3109/14756366.2013.832238

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2013 Informa UK Ltd. DOI: 10.3109/14756366.2013.832238
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ORIGINAL ARTICLE
Synthesis, bioevaluation and docking study of 5-substitutedphenyl-
1,3,4-thiadiazole-based hydroxamic acids as histone deacetylase
inhibitors and antitumor agents
Nguyen-Hai Nam1, Tran Lan Huong1, Do Thi Mai Dung1, Phan Thi Phuong Dung1, Dao Thi Kim Oanh1,
Sang Ho Park2, Kyungrok Kim2, Byung Woo Han2, Jieun Yun3, Jong Soon Kang3, Youngsoo Kim4, and
Sang-Bae Han4
2
1Department of Pharmaceutical Chemistry, Hanoi University of Pharmacy, Hanoi, Vietnam, Research Institute of Pharmaceutical Sciences,
Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul 151-742, Korea, 3Bioevaluation Center, Korea Research Institute of
4
Bioscience and Biotechnology, Cheongwon, Chungbuk 363-883, Korea, and Department of Pharmacy, College of Pharmacy, Chungbuk National
University, Cheongju, Chungbuk 361-763, Korea
Abstract
Keywords
Since the first histone deacetylase (HDAC) inhibitor (ZolinzaÕ, widely known as suberoylanilide
hydroxamic acid; SAHA) was approved by the Food and Drug Administration for the treatment
of T-cell lymphoma in 2006, the search for newer HDAC inhibitors has attracted a great deal of
interest of medicinal chemists worldwide. As a continuity of our ongoing research in this area,
we designed and synthesized a series of 5-substitutedphenyl-1,3,4-thiadiazole-based hydro-
xamic acids as analogues of SAHA and evaluated their biological activities. A number of
compounds in this series, for example, N1-hydroxy-N8-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-
yl)octandiamide (5b), N1-hydroxy-N8-(5-(3-chlorophenyl-1,3,4-thiadiazol-2-yl)octandiamide (5c)
and N1-hydroxy-N8-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)octandiamide (5d), were found to
possess potent anticancer cytotoxicity and HDAC inhibition effects. Compounds 5b–d were
generally two- to five-fold more potent in terms of cytotoxicity compared to SAHA against five
cancer cell lines tested. Docking studies revealed that these hydroxamic acid displayed higher
affinities than SAHA toward HDAC8.
5-phenyl-1,3,4-thiadiazole, cytotoxicity,
heterocycle, histone deacetylase (HDAC)
inhibitors
History
Received 9 July 2013
Revised 31 July 2013
Accepted 1 August 2013
Published online 10 September 2013
Introduction
Intensive efforts of medicinal chemists have resulted in the
finding of a variety of HDAC inhibitors, such as trichostatin A,
suberoylanilide hydroxamic acid (SAHA; vorinostat), MS-27-275
(entinostat), LBH-589 (panobinostat), PXD-101 and oxamflatin,
among others (Figure 1)7–9. Of these, SAHA has been approved
by the Food and Drug Administration in 2006 to treat several
types of lymphoma, including cutaneous T-cell lymphoma. To
date, at least 12 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 lung, breast, pancreas,
renal, bladder cancers, melanoma, glioblastoma, leukemias,
lymphomas and multiple myeloma.
The common pharmacophore motif of a majority of HDAC
inhibitors consists of three distinct domains: a metal binding head
group, which interacts with the Zn2þ ion at the bottom of the
active binding site of the enzyme; a long aliphatic linker, which
occupies the narrow hydrophobic tubular channel; and a surface
recognition group (SRG) (a cap group) (Figure 2)9. The surface
recognition domain is essential for recognizing and binding to
aminoacid chains at the entrance of the active pocket of enzymes.
Based on this pharmacophore, a number of SRGs have been
studied. In our previous report, we have also demonstrated that a
thiazole and benzothiazole system could be excellent substitution
for a phenyl ring in SAHA10. As a continuity of our search for
Histone deacetylases (HDACs) constitute a group of enzymes that
have important roles in gene transcription because acetylation or
deacetylation is associated with an open chromatin configuration
and a permissive gene transcription state1. To date, 18 HDAC
enzymes have been identified in human and they are subdivided
into four classes based on their homologies to yeast HDACs. Class
I includes HDAC 1–3 and 8; class II includes HDACs 4–7, 9 and
10. Class III HDACs, known as sirtuins, include Sirt1–7, which
are NADþ-dependent enzymes; class IV has only one member,
HDAC11, exhibits properties of both classes I and II HDACs1.
A number of evidences have demonstrated that these enzymes
are not only involved in the regulation of chromatin structure and
gene expression but also they can also regulate cell-cycle
progression and carcinogenic process, which in turn involves in
the formation of malignant tumors2,3. HDACs have, therefore,
become an attractive target for anticancer drug discovery4–6
.
Address for correspondence: Nguyen Hai Nam, Department of Pharma-
ceutical Chemistry, Hanoi University of Pharmacy, 13-15 Le Thanh Tong,
Hanoi, Vietnam. E-mail: namnh@hup.edu.vn
Sang-Bae Han, Department of Pharmacy, College of Pharmacy,
Chungbuk National University, 12 Gaesin, Heungduk, Cheongju,
Chungbuk 361-763, Korea. E-mail: shan@chungbuk.ac.kr

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N.-H. Nam et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
O
O
O
H
N
OH
OH
N
H
N
H
CH
CH
3
H C
3
3
O
N
SAHA
Trichostatin A
CH
O
3
O
NHOH
O
N
H
NH
2
H
N
N
Oxamflatin
O
O
MS-27-275
NHSO Ph
2
O
H
NHOH
O
N
H
N
S
NHOH
O
HN
PXD-101
LBH-589
CH
3
Figure 1. Structures of some HDAC inhibitors.
Figure 2. A pharmacophore motif of HDACIs
(A) and benzothiazole/5-substitutedphenyl-
1,3,4-thiadiazole-based hydroxamic acids as
HDACIs (B and C).
SRG
(A)
(B)
O
O
N
R
N
NHOH
NHOH
6
H
S
(C)
R
O
O
N
N
Aminoacid Chain
N
6
H
S
new HDAC inhibitors, we have designed and synthesized a
The synthesis of the series of N1-hydroxy-N8-(5-substituted-
series of 5-substitutedphenyl-1,3,4-thiadiazole-based hydroxamic phenyl-1,3,4-thiadiazol-2-yl)octandiamides (5a–o) was carried
acids (Figure 2). In this article, the results from this study are out as illustrated in Scheme 1. Details are described as follows.
reported.
Synthesis of N1-hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)
Materials and methods
octanediamide (5a)
Chemistry
2-Phenylidenehydrazinecarbothioamide (2a)
All products were homogenous, as examined by thin-layer
chromatography, performed on WhatmanÕ 250 mm 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 tetra-
methylsilane 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 follows: s, singlet; d, doublet; t, triplet; q, quartet;
and m, multiplet. Electron ionization, electrospray ionization
(ESI) and high-resolution mass spectra were obtained using PE
Biosystems API 2000 (Perkin Elmer, Palo Alto, CA) and
MarinerÕ mass spectrometer (Azco Biotech, Inc., Oceanside,
CA), respectively. Reagents and solvents were purchased from
Aldrich or Fluka Chemical Corp. (Milwaukee, WI) 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 compound 5a used benzaldehyde (1a) as the
starting material. About two drops of acetic acid was added to
the mixture of 1a (0.2 mL; 2 mmoL) and thiosemicarbazide
(0.218 g; 2.4 mmoL) in ethanol (15 mL). The mixture was
refluxed for about 4 h. Upon completion, the reaction mixture
was cooled to room temperature. Water (15 mL) was added to
induce precipitation. The precipitate was filtered and washed
with water (two times), dried at 60 ꢀC to yield a white–yellow
solid (0.33 g, 91.2%) of 2a. mp: 165–163 ꢀC; Rf ¼ 0.54
(dichloromethane/methanol (DCM/MeOH) ¼ 9/1).
5-Phenyl-1,3,4-thiadiazol-2-amine (3a)
Compound 3a was obtained from 2a, using Fe3þ reagent. The
synthesis was carried out as follows: a suspension of FeCl3.12H2O
in ethanol was slowly added to the solution of 2a 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.

DOI: 10.3109/14756366.2013.832238
5-substitutedphenyl-1,3,4-thiadiazole-based hydroxamic acids as histone deacetylase inhibitors
3
N
N
H
CHO
NH
N
NH
2
2
FeCl .6H O
H NCSNHNH
3
2
N
S
2
2
R
R
N
EtOH
R
+
H , EtOH
S
1a-o
2a-o
3a-o
O
O
O
O
N
N
N
N
H
OCH
3
Suberic acid
monomethyl ester
6
N
NHOH
6
H
S
NH OH.HCl
S
2
R
R
CDI, DMF
NaOH, MeOH
4a-o
5a-o
Scheme 1. Synthetic pathway for N1-hydroxy-N8-(5-substitutedphenyl-1,3,4-thiadiazol-2-yl)octandiamides (5a–o).
The residue was recrystallized from ethanol to give compound 3a N1-Hydroxy-N8-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)octane-
as a yellow solid. Yield: 72.5%; mp: 225–227 ꢀC; Rf ¼ 0.66 diamide (5b)
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3277 (NH2), 1514, 1468
White crystals; yield: 62.1%; mp: 163.5–165.0 ꢀC; Rf ¼ 0.52
(C¼C).
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3288 (OH, acid), 3030
(NH), 2927 (CH, CH2), 2857 (CH, CH2), 1650, 1630 (C¼O) and
Methyl 8-oxo-8-[(5-phenyl-1,3,4-thiadiazol-2-yl)amino]octanoate
1584 (C¼C). CI-MS (m/z): 381.5 [M ꢁ H]ꢁ. 1H-NMR (500 MHz,
(4a)
DMSO-d6, ppm): ꢁ 12.67 (1 H, s, NH), 10.34 (1 H, s, NH), 8.68
Compound 4a was obtained as follows: 1,10-carbodiimidazole (1 H, brs, OH), 8.09 (1 H, d, J ¼ 7.50 Hz), 7.68 (1 H, d,
(CDI) (162 mg; 1 mmoL) was dissolved in DCM and suberic J ¼ 7.75 Hz), 7.50–7.58 (2 H, m), 2.18–2.21 (2 H, m, CH2),
monomethyl ester acid (1.9 mL; 1 mmoL) was added. The mixture 1.92–1.95 (2 H, m, CH2), 1.58–1.62 (2 H, m, CH2), 1.46–1.51
was stirred for 10 min, and a solution of 3a (180 mg) in DMF (2 H, m, CH2) and 1.25–1.30 (4 H, m, CH2). 13C NMR (125 MHz,
(2 mL) was added. The reaction mixture was stirred at 60 ꢀC for DMSO-d6, ppm): ꢁ 171.7 (C-500), 169.1 (C-8), 159.9 (C-1), 157.6
24 h. DCM was evaporated under reduced pressure, then the (C-20), 131.7 (C-100), 131,0 (C-200), 130.8 (C-400), 130.5 (C-600),
mixture was poured into 20 mL of cold water. The precipitate 128.9 (C-300), 127.8 (C-500), 34.7 (C-7), 33.6 (C-2), 28.2 (C-3, C-
appeared was filtered and washed, dried at 70 ꢀC to give a white– 6), 24.9 and 24.3 (C-4, C-5). Anal. Calcd. for C16H19ClN4O3S
pink solid. Yield: 58.5%; mp: 162.0–164.0 ꢀC; Rf ¼ 0.80 (DCM/ (382.87): C, 50.19; H, 5.00; and N, 14.63. Found: C, 50.15; H,
MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3162 (NH), 2853 (CH2), 1737 5.11; and N, 14.45.
(C¼O), 1562, 1436 (C¼C); CI-MS (m/z): 347.0 [Mþ].
N1-hydroxy-N8-(5-(3-chlorophenyl)-1,3,4-thiadiazol-2-yl)octane-
diamide (5c)
N1-Hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octanediamide
(5a)
White crystals; yield: 62.1%; mp: 163.5–165.0 ꢀC; Rf ¼ 0.52
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3430 (OH), 3040 (NH),
Compound 5a was obtained from 4a. Synthesis of 5a was carried
out as follows: To a solution of 4a (173 mg, 0.5 mmoL) in a
mixture of MeOH (5 mL) and DMF (3 mL) was added
2927, 2862 (CH, CH2), 1645, 1635 (C¼O) and 1567 (C¼C).
CI-MS (m/z): 381.6 [M ꢁ H]ꢁ, 340.5 [M ꢁ C3H5]ꢁ. 1H-NMR
NH2OH.HCl (490 mg, 7 mmoL). Ultrasound was used to dissolve
the mixture. The mixture was cooled in a mixture of salt and
(500 MHz, DMSO-d6, ppm): ꢁ 12.69 (1 H, s, NH), 10.36 (1 H, s,
NH), 8.68 (1 H, brs, OH), 7.97 (1 H, s), 7.88 (1 H, d, J ¼ 6.0 Hz),
crushed ice, and NaOH (17 mmoL) in H2O (1 mL) was added.
7.55–7.57 (2 H, m), 1.92–1.95 (2 H, m, CH2), 1.57–1.60 (2 H, m,
The reaction mixture was stirred for 1 h and 30 min at 0 ꢀC and
CH2), 1.48–1.51 (2 H, m, CH2) and 1.23–1.26 (6 H, m, CH2). 13
C
poured slowly into 30 mL of cold water, acidified with HCl 5%
to pH 5. 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: 52.0%; mp:
202.5–204.0 ꢀC; Rf ¼ 0.62 (DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1):
3251 (OH, acid), 3050 (NH), 2927, 2865 (CH, CH2), 1650, 1629
(C¼O) and 1558 (C¼C). CI-MS (m/z): 347.1 [M ꢁ H]ꢁ. 1H-NMR
(500 MHz, DMSO-d6, ppm): ꢁ 12.61 (1 H, s, NH), 10.36 (1 H, s,
NH), 8.65 (1 H, brs, OH), 7.92–7.93 (2 H, m), 7.51–7.52 (3 H, m),
2.39–2.42 (2 H, m, CH2), 1.91–1.94 (2 H, m, CH2), 1.56–1.59
(2 H, m, CH2), 1.46–1.49 (2 H, m, CH2) and 1.25–1.26 (4 H, m,
NMR (125 MHz, DMSO-d6, ppm): ꢁ 171.7 (C-50), 169.2 (C-8),
160.2 (C-1), 158.8 (C-20), 134.0 (C-100), 132.2 (C-300), 131.3
(C-200), 130.3 (C-600), 126.1 (C-500), 125.8 (C-400), 34.9 (C-7), 32.2
(C-2), 28.2 (C-3, C-6), 25.0 and 24.5 (C-4, C-5). Anal. Calcd. for
C16H19ClN4O3S (382.87): C, 50.19; H, 5.00; and N, 14.63.
Found: C, 50.21; H, 5.04; and N, 14.59.
N1-hydroxy-N8-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)octane-
diamide (5d)
White powder; yield: 58.4%; mp: 231.5–233.0 ꢀC; and Rf ¼ 0.53
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3340 (OH acid), 3171
(NH), 2936, 2853 (CH, CH2), 1630, 1620 (C¼O) and 1581
(C¼C). CI-MS (m/z): 381.2 [M ꢁ H]ꢁ and 340.1 [M ꢁ C3H5]ꢁ.
1H-NMR (500 MHz, DMSO-d6, ppm): ꢁ 12.66 (1 H, s, NH), 10.36
(1 H, s, NH), 8.69 (1 H, brs, OH), 7.94 (2 H, d, J ¼ 8.0 Hz); 7.58
(2 H, d, J ¼ 8.0 Hz), 1.92–1.95 (2 H, m, CH2), 1.57–1.61 (2 H, m,
13
CH2). C NMR (125 MHz, DMSO-d6, ppm): ꢁ 171.6 (C-500),
169.2 (C-8), 161.7 (C-1), 158.4 (C-20), 130.5 (C-100), 130.3
(C-200,C-600), 129.4 (C-300, C-500), 126.9 (C-400), 32.3 (C-7), 34.8
(C-2), 28.3 (C-3, C-6), 25.0, 24.5 (C-4, C-5). Anal. Calcd. for
C16H20N4O3S (348.13): C, 55.16; H, 5.79; N, 16.08. Found: C,
55.21; H, 5.75; N, 16.14.
Compounds 5b–o were synthesized via intermediates 2b–o, 3b–
o and 4b–o by similar procedures as described above for 5a. Only
data for the final compound series are described in this study.
CH2), 1.45–1.50 (2 H, m, CH2) and 1.26–1.27 (6 H, m, CH2). 13
C
NMR (125 MHz, DMSO-d6, ppm): ꢁ 171.7 (C-50), 169.2 (C-8),
160.6 (C-1), 158.6 (C-20), 135.1 (C-400), 129.4 (C-100), 129.1

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J Enzyme Inhib Med Chem, Early Online: 1–8
(C-300, C-500), 128.6 (C-200, C-600), 34.9 (C-7), 33.2 (C-2), 28.3 [M þ H]þ. 1H-NMR (500 MHz, DMSO-d6 þ CDOD3, ppm): ꢁ
(C-3, C-6), 25.0 and 24.4 (C-4, C-5). Anal. Calcd. for 7.85 (2H, d, J ¼ 8.00 Hz), 7.05 (2 H, d, J ¼ 8.00 Hz), 3.81 (3 H,
C16H19ClN4O3S (382.87): C, 50.19; H, 5.00; and N, 14.63. s, OCH3); 2.46–2.50 (2 H, m, CH2), 1.95 (2 H, t, CH2), 1.56–
Found: C, 50.25; H, 5.10; and N, 14.48.
1.60 (2 H, m, CH2), 1.46–1.50 (2 H, m, CH2) and 1.26–1.30
(4 H, m, CH2). 13C NMR (125 MHz, DMSO-d6 þ CDOD3, ppm):
ꢁ 171.7 (C-500), 169.0 (C-8), 161.3 (C-1), 160.9 (C-400), 158.0
(C-20), 128.4 (C-200, C-600), 122.9 (C-100), 114.7 (C-300, C-500),
55.4 (OCH3), 35.0 (C-7), 32.2 (C-2), 28.2 (C-3, C-6), 25.0 and
24.3 (C-4, C-5). Anal. Calcd. for C17H22N4O4S (378.45): C,
53.95; H, 5.86; and N, 14.80. Found: C, 53.90; H, 5.84; and N,
14.83.
N1-hydroxy-N8-(5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl)octane-
diamide (5e)
White crystals; yield: 60.0%; mp: 212.0–213.0 ꢀC; Rf ¼ 0.55
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3400 (OH), 3239, 3054
(NH), 2939, 2853 (CH2), 1687, 1628 (C¼O), 1597, 1557 and
1515 (C¼C). CI-MS (m/z): 756.0 [2M þ Na]þ, 363.8 [M ꢁ 2H]ꢁ.
1H-NMR (500 MHz, DMSO-d6, ppm): ꢁ 12.63 (1 H, s, NH), 10.42
(1 H, s, NH), 7.97 (2 H, d, J ¼ 8.0 Hz), 7.34 (2 H, d, J ¼ 8.0 Hz),
2.47–2.50 (2 H, m, CH2), 1.93–1.96 (2 H, m, CH2), 1.56–1.60
N1-hydroxy-N8-(5-(4-(dimethylamino)phenyl)-1,3,4-thiadiazol-2-
yl)octanediamide (5i)
(2 H, m, CH2), 1.46–1.50 (2 H, m, CH2) and 1.25–1.26 (4 H, m, White powder; yield: 61.0%; mp: 180.0–181.0 ꢀC; and Rf ¼ 0.45
CH2). C NMR (125 MHz, DMSO-d6, ppm): ꢁ 171.6 (C-50), (DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3350 (OH), 3227 (NH),
13
169.2 (C-8), 162.3 (C-1), 160.6 (C-400), 158.3 (C-20), 129.2 (C-300, 2926, 2853 (CH2), 1652 (C¼O), 1609, 1570 and 1539 (C¼C).
C-500), 126.2 (C-100), 116.4 (C-200, C-600), 34.8 (C-7), 33.6 (C-2), CI-MS (m/z): 389.0 [M ꢁ 2H]ꢁ. H-NMR (500 MHz, DMSO-d6,
1
28.3 (C-3, C-6) and 24.3 (C-4, C-5, overlap). Anal. Calcd. for ppm): ꢁ 12.41 (1 H, s, NH), 10.37 (1 H, brs, NH), 7.71 (2 H, d,
C16H19FN4O3S (366.41): C, 52.45; H, 5.23; and N, 15.29. Found: J ¼ 7.80 Hz), 6.79 (2 H, d, J ¼ 7.80 Hz), 2.98 (6 H, s, 2CH3), 2.46–
C, 52.50; H, 5.27; and N, 15.32.
2.50 (2 H, m, CH2), 1.95 (2 H, t, CH2), 1.58–1.61 (2 H, m, CH2),
1.47–1.50 (2 H, m, CH2), 1.27–1.31 (4 H, m, CH2). 13C NMR
(125 MHz, DMSO-d6, ppm): ꢁ 171.3 (C-50), 169.1 (C-8), 162.2
(C-1), 156.6 (C-20), 151.5 (C-400), 127.8 (C-200, C-600), 114.4 (C-
300, C-500), 40.2 (2CH3), 34.8 (C-7), 32.2 (C-2), 28.2 (C-3, C-6)
and 24.3 (C-4, C-5, overlap). Anal. Calcd. for C18H25N5O3S
(391.17): C, 55.22; H, 6.44; and N, 17.89. Found: C, 55.25; H,
6.41; and N, 17.85.
N1-hydroxy-N8-(5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl)octane-
diamide (5f)
White powder; yield: 60.5%; mp: 202.5–204.0 ꢀC; Rf ¼ 0.57
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3400 (OH), 3163, 3049
(NH), 2923, 2850 (CH2), 1691, 1616 (C¼O), 1571, 1557 and
1469 (C¼C). CI-MS (m/z): 425.9 [M ꢁ H]ꢁ. 1H-NMR
(500 MHz, DMSO-d6, ppm): ꢁ 12.68 (1 H, s, NH), 10.37 (1 H,
s, NH), 7.86–7.88 (2 H, d, J ¼ 8.00 Hz), 7.71 (2 H, d,
J ¼ 8.00 Hz), 2.48–2.50 (2 H, m, CH2), 1.94–2.16 (2 H, m,
CH2), 1.59–1.61 (2 H, m, CH2), 1.45–1.49 (2 H, m, CH2) and
1.27–1.29 (4 H, m, CH2). 13C NMR (125 MHz, DMSO-d6, ppm):
ꢁ 174.3 (C-50), 171.6 (C-8), 160.6 (C-1), 158.6 (C-20), 132.3 (C-
300, C-500), 129.4 (C-100), 128.7 (C-200, C-600), 128.6 (C-300, C-500),
123.8 (C-400), 34.7 (C-7), 33.6 (C-2), 32.1, 28.2 (C-3, C-6), 25.0
and 24.3 (C-4, C-5). Anal. Calcd. for C16H19BrN4O3S (427.32):
C, 44.97; H, 4.48; and N, 13.11. Found: C, 45.03; H, 4.51; and
N, 13.07.
N1-hydroxy-N8-(5-(2-nitrophenyl)-1,3,4-thiadiazol-2-yl)octane-
diamide (5j)
Yellowish solid; yield: 52.0%; mp: 175.0–176.5 ꢀC; and Rf ¼ 0.50
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3300 (OH), 3097, 3012
(NH), 2935, 2866 (CH2), 1709, 1698 (C¼O), 1537 and 1470
(C¼C). CI-MS (m/z): 391.5 [M ꢁ H]ꢁ; 1H-NMR (500 MHz,
DMSO-d6, ppm): ꢁ 12.76 (1 H, s, NH), 10.34 (1 H, s, NH), 8.06
(1 H, d, J ¼ 7.50 Hz), 7.88 (1 H, d, J ¼ 7.5 Hz), 7.84 (1 H, t,
J ¼ 7.50 Hz), 7.78 (1 H, t, J ¼ 7.50 Hz), 2.50–2.53 (2 H, m, CH2),
1.94–1.91 (2 H, m, CH2), 1.58–1.63 (2 H, m, CH2), 1.46–1.52
(2 H, m, CH2) and 1.25–1.29 (4 H, m, CH2). 13C NMR (125 MHz,
DMSO-d6, ppm): ꢁ 171.9 (C-50), 169.1 (C-8), 159.7 (C-1), 156.8
(C-20), 148.5 (C-200), 133.1 (C-500), 131.9 (C-100), 131.5 (C-400),
124.5 (C-600), 123.4 (C-300), 34.8 (C-7), 32.2 (C-2), 28.2 (C-3,
C-6), 25.0 and 24.3 (C-4, C-5). Anal. Calcd. for C16H19N5O5S
(393.11): C, 48.85; H, 4.87; and N, 17.80. Found: C, 48.88; H,
4.91; and N, 17.77.
N1-hydroxy-N8-(5-(4-methylphenyl)-1,3,4-thiadiazol-2-yl)octane-
diamide (5g)
White solid; yield: 65.0%; mp: 170.0–172.0 ꢀC; Rf ¼ 0.52 (DCM/
MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3500 (OH), 3162, 3023 (NH),
2929, 2862 (CH2), 1702, 1650, 1615 (C¼O), 1560 and 1463
(C¼C). CI-MS (m/z): 359.8 [M ꢁ 2H]ꢁ and 344.9 [M ꢁ OH]ꢁ.
1H-NMR (500 MHz, DMSO-d6, ppm): ꢁ 12.59 (1 H, s, NH), 10.36
(1 H, s, NH), 7.80 (2 H, d, J ¼ 8.50 Hz), 7.32 (2 H, d, J ¼ 8.50 Hz),
2.47–2.50 (2 H, m, CH2), 2.19–2.16 (1 H, t, CH2b), 2.35 (3 H, s,
-CH3), 1.90–1.94 (1 H, t, CH2a), 1.58–1.61 (2 H, m, CH2), 1.47–
1.50 (2 H, m, CH2) and 1.27–1.29 (4 H, m, CH2). 13C NMR
(125 MHz, DMSO-d6, ppm): ꢁ 174.4 (C-50), 169.1 (C-8), 161.7
(C-1), 158.0 (C-20), 140.1 (C-100), 129.9 (C-300, C500), 127.5
(C-400), 126.8 (C-200, C-600), 34.8 (C-7), 33.6 (C-2), 32.2 (CH3),
28.3, 28.2 (C-3, C-6), 24.3 and 24.3 (C-4, C-5). Anal. Calcd. for
C17H22N4O3S (362.14): C, 56.33; H, 6.12; and N, 15.46. Found:
C, 56.36; H, 6.13; and N, 15.43.
N1-hydroxy-N8-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)octane-
diamide (5k)
Yellowish powder; yield: 53.5%; mp: 170.0–172.0 ꢀC; and
Rf ¼ 0.52 (DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3410 (OH),
2925, 2853 (CH2), 1691, 1624 (C¼O), 1599, 1547 and 1519
(C¼C). CI-MS (m/z): 391.5 [M ꢁ H]ꢁ; 1H-NMR (500 MHz,
DMSO-d6, ppm): ꢁ 10.42 (1 H, s, NH), 9.70 (1 H, brs, OH), 8.32
(2 H, d, J ¼ 8.30 Hz), 8.16 (2 H, d, J ¼ 8.30 Hz), 2.45 (2 H, t,
CH2), 2.06 (1 H, t, CH2b), 1.95 (1 H, t, CH2a), 1.58–1.59 (2 H, m,
CH2), 1.47 (2 H, m, CH2) and 1.21–1.26 (4 H, m, CH2). 13C NMR
(125 MHz, DMSO-d6, ppm): ꢁ 173.9 (C-50), 169.7 (C-8), 162.9
(C-1), 152.2 (C-20), 147.7 (C-400), 137.6 (C-100), 127.1 (C-200,
C-600), 124.2 (C-300, C-500), 40.0 (C-7), 34.9 (C-2), 28.2 (C-3, C-6)
and 24.3 (C-4, C-5, overlap). Anal. Calcd. for C16H19N5O5S
(393.11): C, 48.85; H, 4.87; and N, 17.80. Found: C, 48.91; H,
4.86; and N, 17.82. HRMS (ESI) calcd for [M–H] C16H19N5O5S,
m/z: 392.1029, observed: 392.1021.
N1-hydroxy-N8-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)octa-
nediamide (5h)
White crystals; yield: 62.5%; mp: 178.0–179.0 ꢀC; Rf ¼ 0.58
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3400 (OH), 3183, 3071
(NH), 2938, 2856 (CH2), 1693, 1611 (C¼O), 1580, 1563 and
1519 (C¼C). ESI-MS (m/z): 401.3369 [M þ Na]þ and 377.3847

DOI: 10.3109/14756366.2013.832238
5-substitutedphenyl-1,3,4-thiadiazole-based hydroxamic acids as histone deacetylase inhibitors
5
13
N1-hydroxy-N8-(5-(2,6-dichlorophenyl)-1,3,4-thiadiazol-2-yl)oc-
tanediamide (5l)
CH2). C NMR (125 MHz, DMSO-d6, ppm): ꢁ 171.5 (C-50),
169.1 (C-8), 161.6 (C-1), 158.3 (C-200), 153.4 (C-300, C-500), 139.4
(C-400), 125.7 (C-100), 104.2 (C-200, C-600), 116.8 (C-100), 108.9
(C-500), 60.1 (-OCH3), 56.1 (-2OCH3), 34.8 (C-7), 32.2 (C-2), 28.3
(C-3, C-6), 24.5 and 24.3 (C-4, C-5). Anal. Calcd. for
C16H20N4O3S (438.16): C, 52.04; H, 5.98; and N, 12.78. Found:
C, 52.05; H, 5.97; and N, 12.76.
White solid; yield: 50.0%; mp: 175.0–176.0 ꢀC; and Rf ¼ 0.56
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3350 (OH), 3155 (NH),
2927, 2856 (CH2), 1701, 1629 (C¼O) and 1534 (C¼C). CI-MS
1
(m/z): 416.3 [M ꢁ H]ꢁ, 499.3 [M ꢁ OH]ꢁ; H-NMR (500 MHz,
DMSO-d6, ppm): ꢁ 10.46 (1 H, s, NH), 9.71 (1 H, s, br, NH), 8.75
(1 H, brs, OH), 7.67 (2 H, d, J ¼ 7.30 Hz), 7.61 (1 H, t,
J ¼ 7.30 Hz), 2.50–2.54 (2 H, m, CH2), 1.93–1.95 (2 H, m,
Cytotoxicity assays
CH2), 1.59–1.61 (2 H, m, CH2), 1.47–1.49 (2 H, m, CH2) and Human cancer cell lines, NCI-H460 (lung cancer), PC3 (prostate
1.22–1.29 (4 H, m, CH2). 13C NMR (125 MHz, DMSO-d6, ppm): cancer), SW620 (colon cancer), MCF-7 (breast adenocarcinoma)
ꢁ 172.0 (C-50), 169.0 (C-8), 160.5 (C-1), 155.3 (C-20), 135.1 and AsPC-1 (pancreatic cancer) cell lines were obtained from the
(C-200, C-600), 132.9 (C-400), 128.7 (C-300, C-500), 34.9 (C-7), 32.2 American Type Culture Collection (Manassas, VA). Cells were
(C-2), 28.2 (C-3, C-6), 25.0 and 24.3 (C-4, C-5). Anal. Calcd. for plated at 9 ꢂ 103 cells/well in 96-well plates, incubated overnight
C16H18Cl2N4O3S (416.05): C, 46.05; H, 4.35; and N, 13.43. and treated with samples for 48 h. Compounds were dissolved in
Found: C, 46.21; H, 4.39; and N, 13.40.
DMSO. Cytotoxicity was measured by the method as described in
literature11 with slight modifications12. The final concentration of
DMSO in the wells containing cells was 0.01%, and this
concentration did not affect the cell growth11,12. The IC50
values were calculated according to the Probit’s method13. The
values reported for these compounds are averages of three
separate determinations.
N1-hydroxy-N8-(5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-thiadiazol-2-
yl)octanediamide (5m)
White solid; yield: 55.0%; mp: 190.5–192.0 ꢀC; Rf ¼ 0.54 (DCM/
MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3340–3300 (OH), 3161, 3032
(NH), 2923, 2854 (CH2), 1692, 1637 (C¼O), 1574, 1505 and 1458
(C¼C). CI-MS (m/z): 393.12 [M]þ, 391.12 [M ꢁ H]ꢁ and 376.13
1
[M ꢁ OH]þ; H-NMR (500 MHz, DMSO-d6 þ CDOD3, ppm): ꢁ
Western blot assay
7.46 (1 H, s), 7.39 (1 H, d, J ¼ 7.50 Hz), 7.01 (1 H, d, J ¼ 7.50 Hz),
6.10 (2 H, s, CH2), 2.43–2.47 (2 H, m, CH2), 2.15–2.18 (1 H, m,
CH2), 1.90–1.94 (1 H, m, CH2), 1.59–1.61 (2 H, m, CH2), 1.48–
1.49 (2 H, m, CH2) and 1.18–1.27 (4 H, m, CH2). 13C NMR
(125 MHz, DMSO-d6 þ CDOD3, ppm): ꢁ 171.4 (C-50), 169.1 (C-
8), 161.7 (C-1), 157.8 (C-20), 149.2 (C-300), 148.0 (C-400), 124.3 (C-
100), 121.8 (C-600), 108.9 (C-500), 106.4 (C-200), 101.8 (-OCH2O-),
34.8 (C-7), 32.2 (C-2), 28.3, 28.2 (C-3, C-6), 25.0 and 24.4 (C-4, C-
5). Anal. Calcd. for C17H20N4O5S (392.12): C, 52.03; H, 5.14; and
N, 14.28. Found: C, 52.09; H, 5.11; and N, 14.25.
Total protein extracts were prepared by lysing cells in radio-
immunoprecipitation buffer (50 mM Tris-Cl [pH 8.0], 5 mM
EDTA, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate
(SDS) and 1 mM phenylmethylsulfonyl fluoride). Protein con-
centrations in the lysates were determined using a Bio-Rad
protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA)
according to the manufacturer’s instructions. Samples were
separated on SDS polyacrylamide gels and transferred to nitro-
cellulose 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 glyceraldehyde 3-phosphate
dehydrogenase (GAPDH). After washing, membranes were
probed with horseradish peroxidase-conjugated secondary anti-
bodies. Detection was performed using an enhanced chemilumin-
escent protein detection system (Amersham Biosciences, Little
Chalfont, UK).
N1-hydroxy-N8-(5-(2,3,4-trimethoxyphenyl)-1,3,4-thiadiazol-2-
yl)octanediamide (5n)
White solid; yield: 50.0%; mp: 175.0–176.0 ꢀC; and Rf ¼ 0.51
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3424 (OH), 3171, 3044
(NH), 2940, 2860 (CH2), 1688, 1659 (C¼O), 1594, 1576 and
1552 (C¼C). CI-MS (m/z): 437.3 [M ꢁ H]ꢁ; 1H-NMR (500 MHz,
DMSO-d6, ppm): ꢁ 12.30 (1 H, s, NH), 10.32 (1 H, s, NH), 8.60
(1 H, brs, OH), 7.94 (1 H, d, J ¼ 7.50 Hz), 6.99 (1 H, d, Docking studies
J ¼ 7.50 Hz), 3.93 (3 H, s, CH3), 3.87 (3 H, s, CH3), 3.82 (3 H,
s, CH3), 2.48–2.50 (2 H, m, CH2), 1.95–1.98 (2 H, m, CH2), 1.58–
1.61 (2 H, m, CH2), 1.49–1.51 (2 H, m, CH2) and 1.25–1.28 (4 H,
AutoDock Vina program14 was used in the docking studies. Initial
structure of HDAC8 was obtained from the Protein Data Bank
(PDB) (PDB ID: 1T69)15, and coordinates for the compounds
were generated using the GlycoBioChem PRODRG2 Server
(http://davapc1.bioch.dundee.ac.uk/prodrg/)16. The grid maps for
docking studies were centered on the SAHA binding site and
13
m, CH2). C NMR (125 MHz, DMSO-d6, ppm): ꢁ 174.5 (C-50),
169.1 (C-8), 159.6 (C-1), 156.2 (C-200), 155.1 (C-400), 150.1
(C-200), 141.6 (C-300), 121.9 (C-600), 116.8 (C-100), 108.9 (C-500),
60.8 (-OCH3), 60.5 (-OCH3), 56.1 (-OCH3), 34.8 (C-7), 32.2
(C-2), 28.3 (C-3, C-6), 24.5 and 24.3 (C-4, C-5). Anal. Calcd. for
C16H20N4O3S (438.16): C, 52.04; H, 5.98; and N, 12.78. Found:
C, 52.09; H, 5.95; and N, 12.74.
˚
comprised 26 ꢂ 26 ꢂ 22 points with 1.0 A spacing after SAHA
was removed from the complex structure, as described previ-
ously10. AutoDock Vina program was run with four-way multi-
threading, and the other parameters were default settings in
Autodock Vina Program (The Scripps Research Institute, La Jolla,
CA).
N1-hydroxy-N8-(5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-
yl)octanediamide (5o)
White solid; yield: 53.5%; mp: 177.0–178.0 ꢀC; and Rf ¼ 0.54
(DCM/MeOH ¼ 9/1). IR (KBr, cmꢁ1): 3480 (OH), 3210 (NH),
2939, 2860 (CH2), 1708, 1651 (C¼O), 1572, 1515 and 1467
(C¼C). CI-MS (m/z): 437.41 [M ꢁ H]ꢁ; 1H-NMR (500 MHz,
DMSO-d6, ppm): ꢁ 12.59 (1 H, s, NH), 10.34 (1 H, s, NH), 8.67
(1 H, brs, OH), 7.18 (2 H, s), 3.87 (6 H, s, OCH3), 3.72 (3 H, s,
CH3), 2.47–2.50 (2 H, m, CH2), 1.91–1.94 (2 H, m, CH2), 1.59–
1.60 (2 H, m, CH2), 1.47–1.49 (2 H, m, CH2), 1.27–1.28 (4 H, m,
Results and discussion
Chemistry
A series of 5-substitutedphenyl-1,3,4-thiadiazole-based hydroxa-
mic acids (5a–o) were synthesized via a four-step pathway
(Scheme 1). In the first step, the thiosemicarbazones 2a–o were
obtained by condensation of simple benzaldehydes 1a–o with
thiosemicarbazide. Intramolecular cyclization of 2a–o proceeded

6
N.-H. Nam et al.
Table 1. HDAC inhibition and cytotoxicity of the compounds synthesized.
J Enzyme Inhib Med Chem, Early Online: 1–8
O
O
N
N
N
H
NHOH
6
S
R
5a-o
Cytotoxicity (IC50,* mM)/Cell linesy
Compound
code
Molecular
weight
R
SW620 MCF-7
PC3
AsPC-1 NCI-H460 Log pz
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
H
384.42
382.86
382.86
382.86
366.41
427.32
362.45
378.45
391.49
393.42
393.42
417.31
390.46
438.50
438.50
264.32
0.70
0.34
0.45
1.62
3.58
0.72
11.52
1.46
6.61
19.23
26.71
16.21
1.89
2.29
4.31
3.70
1.80
1.23
1.23
0.73
8.05
1.27
20.78
2.46
6.67
31.18
76.25
16.29
2.15
4.15
3.50
6.42
0.88
0.42
1.76
0.84
2.92
0.83
14.90
1.63
7.35
14.69
25.57
15.18
2.54
1.77
1.89
4.31
2.71
0.63
1.10
1.34
1.88
0.08
6.38
0.80
7.16
33.47
76.25
14.95
3.04
2.11
6.02
3.66
1.07
0.11
1.94
1.50
4.79
1.42
13.16
1.97
8.18
16.69
76.25
14.38
3.34
3.21
10.20
2.77
1.35
1.99
1.99
1.99
1.55
2.24
1.90
1.43
1.53
1.17
1.17
2.64
1.41
0.86
0.86
1.44
2-Cl
3-Cl
4-Cl
4-F
4-Br
4-CH3
4-OCH3
4-N(CH3)2
2-NO2
4-NO2
2,6-Cl2
3,4-CH2OCH2-
2,3,4-(OCH3)3
3,4,5-(OCH3)3
SAHAô
*The concentrations (mM) of compounds that produce a 50% reduction in cell growth, the numbers represent the
averaged results from triplicate experiments with deviation of less than 10%.
yCell lines: SW620, colon cancer; MCF-7, breast cancer; PC3, prostate cancer; AsPC-1, pancreatic cancer; and
NCI-H460, lung cancer.
zEstimated by Software Kowwin v1.67 (US EPA, EPI suite v 4.0).
ôSAHA, suberoylanilide hydroxamic acid, a positive control.
smoothly using ferric chloride in ethanol to give 2-amino-5- moiety led to a better analogue than SAHA in term of
phenyl-1,3,4-thiadiazole derivatives 3a–o. Coupling of 3a–o with cytotoxicity. It should be mentioned that in the course of our
suberic acid monomethyl ester using CDI as a carboxylic study, we noticed an article by Guan et al.17 in which compound
activating reagent generated the ester intermediates 4a–o. 5a was reported. In this article, compound 5a was reported to
Reaction of the esters 4a–o with hydroxylamine in alkaline inhibit HDAC (came from HeLa cell nucleus extracts, containing
conditions gave the final products 5a–o in generally good yields. mainly HDAC1 and HDAC2) with IC50 value of 0.27 mM).
The presence of a nitro substituent on the phenyl ring was found However, no cytotoxic data for this compound were revealed.
to somewhat lower the yield of overall process.
The presence of one halogen atom on the phenyl ring, either at
The structures of the obtained compounds were straightfor- position 2, 3 or 4, retained the cytotoxicity or even slightly
wardly and unambiguously confirmed by spectral studies, enhanced it (compounds 5b–d, 5f). It seemed that substitution at
including IR, MS, 1H NMR and 13C NMR and elemental analysis. position 2 was the most favorable for cytotoxicity. For example,
compound 5b and 5c with a chlorine substituent at position 2 and
3, respectively, were more cytotoxic than compound 5a in SW620
cell line (IC50 values of 0.34 and 0.45 mM versus 0.70 mM).
Bioactivity
The sulforhodamine B cell proliferation assay was used to
evaluate the antiproliferative activity of the synthesized com-
pounds. The compounds 5a–o were first screened at 30 mM for
cell growth inhibition against SW620 (human colon cancer) cell
line. It was found that all compounds inhibited the growth of
SW620 cells by more than 50% at this concentration. Therefore,
the compounds were subjected to further evaluation at five
concentrations (30, 10, 3, 1, 0.3 mM) in SW620 and four human
cancer cell lines more, including MCF-7 (breast adenocarcin-
oma), PC-3 (prostate cancer), AsPC-1 (pancreas cancer) and NCI-
H460 (lung 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.
Compound 5b was even 10-fold more active than 5a in NCI-460
cell line (IC50 value of 0.11 mM versus 1.07 mM). Especially,
compound 5f was about 30-times more potent than compound 5a
in AsPC-1 cells (IC50 value of 0.08 mM versus 2.71 mM). Of
exception was compound 5e, bearing a 4-fluorine substituent,
which showed decreased cytotoxicity compared to compound 5a.
Nevertheless, the cytotoxicity of these compounds were still
comparable to that of SAHA in five cell lines tested.
The introduction of an additional chlorine at position 6
(compound 5l), however, led to a dramatic decrease in cytotox-
icity. Within the series, two compounds bearing nitro substituents
(compounds 5j and 5 k) were the least cytotoxic ones. Compound
5 g, with a 4-methyl substituent, and compound 5i, with a bulkier
4-dimethylamino group on the phenyl ring, showed almost 10-fold
decrease of cytotoxicity compared to compound 5a. However, it
was observed that the 3,4-methylenedioxy, 4-methoxy or poly-
methoxy substitutions were relatively tolerable for cytotoxic
activity of this compound series, as evidenced that compounds 5h
Data in Table 1 demonstrated that compound 5a showed strong
cytotoxicity against all five cell lines tested. Especially on SW620
and PC3 cell lines, this compound was approximately five-fold
more potent than SAHA. Thus, the introduction of the 1,3,4-
thiadiazole heterocycle between the phenyl ring and the amide

DOI: 10.3109/14756366.2013.832238
5-substitutedphenyl-1,3,4-thiadiazole-based hydroxamic acids as histone deacetylase inhibitors
7
and 5m–o were equally active or even slightly more cytotoxic
than SAHA against all five cancer cell lines assayed.
Next, we examined the effects of the compounds on the HDAC
activity using the Western blot assay. Evaluation of HDAC
inhibition in a whole cell system with primary antibodies against
acetyl histone-H3, -H4, and GAPDH revealed that the acetylation
of histone-H3 and histone-H4 increased in the presence of
compounds 5a–f, 5h, 5i and 5m–o (Figure 3), indicating that the
HDAC activity had been inhibited. Meanwhile, in the presence of
compounds 5g, 5j–l, the acetyl-H3 and acetyl-H3 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 well correlated with the cytotoxicity, e.g. four
compounds 5g, 5j–l were the least cytotoxic ones with the IC50
values against all five cancer cell lines higher than 11.52 mM. In
contrast, other compounds like 5a–f, 5h, 5i and 5m–o showed much
Figure 3. Effect of the compounds synthesized on histone acetylation in
SW620 cells. Cells were treated with compounds (10 mM) for 24 h. Levels
of acetylated histone-H3 and -H4 in total cell lysates were determined by
Western immunoblot analysis. VH denotes vehicle treatment (blank
control). SAHA (suberoylanilide hydroxamic acid) is a positive control.
Figure 4. Stereoview of the actual binding pose of SAHA and simulated docking pose of compound 5b (A) and 5c (B) to HDAC8. SAHA, compounds
5b, 5c, and the interaction parts of the HDAC8 were shown as a stick model.

8
N.-H. Nam et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Grant number 104.01-2012.17 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). The authors report no conflict
of interest.
stronger cytotoxicity with IC50 values observed in low to sub-
micromolar range in all five cancer cell lines tested (Table 1). The
results demonstrate 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 0.86 to 2.64. The numbers of
hydrogen bond donors (3) and hydrogen bond acceptors (8) fall
within Lipinsky’s rule of five (Table 1). However, no clear
correlation between logP values and bioactivity of the compounds
was observed.
References
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To speculate the interaction between these compounds and HDAC,
we implemented docking experiments using the active site of
HDAC. We selected the structure of HDAC8 in complex with
SAHA as a docking template because its crystal structure is
available (PDB ID: 1T69)15, and it shares high structural similarity
(DALI Z score ¼ 40.4 and r.m.s.d. ¼ 2.1 A)18 and amino acid
sequence similarity (46%) to HDAC4. We performed control
docking experiments with SAHA to the crystal structure of HDAC8
using AutoDock Vina program14 after SAHA was removed from
the complex structure, as described previously10. It was found from
docking experiments that two compounds, 5b and 5c, were located
in the active site (Figure 4) with more stable energy than that of
SAHA and their aliphatic linkers seemed to interact with the
residues in the active site tunnel of HDAC8 as the inhibition mode
of SAHA15. Stabilization energies of predicted binding modes were
ꢁ6.4 and ꢁ7.1 kcal/mol for compounds 5b and 5c, respectively,
while the value for SAHA was ꢁ4.4 kcal/mol. Thus, based on the
docking results, compounds 5b and 5c showed higher affinities
than SAHA toward HDAC8.
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Conclusion
In this study, fifteen 5-substitutedphenyl-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 presence of a 1,3,4-
thiadiazole heterocycle between the phenyl ring and the amide
moiety could be favorable for cytotoxicity. In general, halogen,
methoxy or polymethoxy substituents on the phenyl ring were
tolerated and, in some cases, led to increase in cytotoxicity. It was
also found that the cytotoxicity of the compounds was well
correlated with the inhibition of HDAC. Docking study performed
with two representative compounds 5b (bearing 2-chlorophenyl)
and 5c (bearing 3-chlorophenyl) revealed that these compounds
had higher binding affinities to HDAC8 compared to SAHA. All
of the compounds designed and synthesized possessed good drug-
like properties. From this study, several compounds, such as 5b
and 5c, were promising that warrant further investigation.
accuracy of docking with
a new scoring function, efficient
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17. Guan P, Sun F, Hou X, et al. Design, synthesis and preliminary
bioactivity studies of 1,3,4-thiadiazole hydroxamic acid derivatives
as novel histone deacetylase inhibitors. Bioorg Med Chem 2012;20:
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Declaration of interest
18. Holm L, Rosenstrom P. Dali server: conservation mapping in 3D.
Nucleic Acids Res 2010;38:W545–9.
We acknowledge the principal financial supports from a National
Foundation for Science and Technology of Vietnam (NAFOSTED),