Exploration of certain 1,3-oxazole- and 1,3-thiazole-based hydroxamic acids as histone deacetylase inhibitors and antitumor agents

Article abstract of DOI:10.1016/j.bioorg.2020.103988

Several novel series of hydroxamic acids bearing 2-benzamidooxazole/thiazole (5a-g, 6a-g) or 2-phenylsulfonamidothiazole (8a-c) were designed and synthesized. The compounds were obtained straightforwards via a two step pathway, starting from commercially

Full text of DOI:10.1016/j.bioorg.2020.103988

Journal Pre-proofs
Exploration of Certain 1,3-Oxazole- and 1,3-Thiazole-Based Hydroxamic
Acids as Histone Deacetylase Inhibitors and Antitumor Agents
Duong Tien Anh, Pham-The Hai, Le-Thi-Thu Huong, Eun Jae Park, Hye Won
Jun, Jong Soon Kang, Joo-Hee Kwon, Do Thi Mai Dung, Vu Tran Anh, Van
Thi My Hue, Sang-Bae Han, Nguyen-Hai Nam
PII:
S0045-2068(20)31285-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103988
YBIOO 103988
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
8 March 2020
14 April 2020
29 May 2020
Please cite this article as: D. Tien Anh, P-T. Hai, L-T. Huong, E. Jae Park, H. Won Jun, J. Soon Kang, J-H.
Kwon, D. Thi Mai Dung, V. Tran Anh, V. Thi My Hue, S-B. Han, N-H. Nam, Exploration of Certain 1,3-
Oxazole- and 1,3-Thiazole-Based Hydroxamic Acids as Histone Deacetylase Inhibitors and Antitumor Agents,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103988
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Exploration of Certain 1,3-Oxazole- and 1,3-Thiazole-Based Hydroxamic Acids
as Histone Deacetylase Inhibitors and Antitumor Agents
Duong Tien Anh,a Pham-The Hai,a Le-Thi-Thu Huong,a Eun Jae Park, bHye Won
Jun,b Jong Soon Kang,c Joo-Hee Kwon,c Do Thi Mai Dung,a Vu Tran Anh,a Van Thi
My Hue,*,a Sang-Bae Han,*,b Nguyen-Hai Nam,*,a
aHanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam
bCollege of Pharmacy, Chungbuk National University, 194-31, Osongsaengmyung-1,
Heungdeok, Cheongju, Chungbuk, 28160, Republic of Korea
cLaboratory Animal Resource Center, Korea Research Institute of Bioscience and
Biotechnology, Cheongju, Chungbuk, Republic of Korea
Abstract
Several novel series of hydroxamic acids bearing 2-benzamidooxazole/thiazole (5a-g,
6a-g) or 2-phenylsulfonamidothiazole (8a-c) were designed and synthesized. The
compounds were obtained straightforwards via a two step pathway, starting from
commercially available ethyl 2-aminooxazole-4-carboxylate or ethyl 2-
aminothiazole-4-carboxylate. Biological evaluation showed that these hydroxamic
acids generally exhibited good cytotoxicity against three human cancer cell lines
(SW620, colon; PC-3, prostate; NCI-H23, lung cancer), with IC50 values in low
micromolar range and comparable to that of SAHA. These compounds also
comparably inhibited HDACs with IC50 values in sub-micromolar range (0.010-0.131
µM) and some compounds (e.g 5f, IC50, 0.010 µM) were even more potent than
SAHA (IC50, 0.025 µM) in HDAC inhibition. Representative compounds 6a and 8a
appeared to arrest the SW620 cell cycle at G2 phase and significantly induced both
early and late apoptosis of SW620 colon cancer cells. Docking experiments on
HDAC2 and HDAC6 isozymes revealed favorable interactions at the tunnel of the
HDAC active site which positively contributed to the inhibitory activity of
synthesized compound. The binding affinity predicted by docking program showed
good correlation with the experimental IC50 values. This study demonstrates that
simple 1,3-oxazole- and 1,3-thiazole-based hydroxamic acids are also promising as
antitumor agents and HDAC inhibitors and these results should provide valuable
information for further design of more potent HDAC inhibitors and antitumor agents.
Keywords: Histone deacetylase (HDAC) inhibitors, hydroxamic acids, docking, 1,3-
oxazole, 1,3-thiazole.
*Corresponding authors: Tel.: +84-4-39330531; Fax: +84-4-39332332; Emails:
namnh@hup.edu.vn (N.H. Nam; V.T.M. Hue); shan@chungbuk.ac.kr (S.B. Han).
-1-

1. Introduction
Acetylation and deacetylation of the acetyl groups on the lysine amino tails of histone
proteins are two opposite processes controlling the epigenetic balance [1].
Acetylation is catalyzed by histone acetyltransferases (HATs), while histone
deacetylases (HDACs) are responsible for the catalytic removal of the acetyl groups
on the histone lysine amino tails.[1] In mammalials, eighteen isoforms of HDACs
have been described. These isoforms are divided into four classes (I-IV).[2, 3] It has
been well documented that isoforms in class I (HDACs 1-3, 8) and class II (HDACs
4-6, 7, 9, 10) actively promote cell proliferation and block apoptosis, while
preventing cellular differentiation, thus causing the cells growing continuously [2, 3].
HDAC isoforms 4, 6, 7 and 10 of class II have also been demonstrated to promote
angiogenesis and cellular migration, two processes important for tumor metastatis [2,
3]. Due to their clear implication in the initiation and development of verious
maligancies, HDACs have become a validated target for current anticancer drug
design and development.[1-3] As a result, a number of structurally diverse HDAC
inhibitors have been identified through intensive efforts of medicinal chemists
worldwide. These include hydroxamic acids (e.g. SAHA or suberoylanilide
hydroxamic acid), cyclic peptide (e.g. depsipeptide), short-chain fatty acids (e.g.
valproic acid, phenylbutyric acid), and benzamides [4-9].
The first HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA, Zoinza®), was
approved by the U.S. FDA in October 2006 to treat cutaneous T cell lymphoma.
Since then, the U.S. FDA has approved three more HDAC inhibitors, including
romidepsin (Istodax®), belinostat (PXD101), and panobinostat (LBH-589,
Farydak®), for use in clinical settings [7-10]. Most recently, in 2015,
chidamide (HBI-8000, tucidinostat, Epidaza®), was approved by the Chinese FDA
for relapsed or refractory peripheral T cell lymphoma [11]. A dozen of other HDAC
inhibitors, such as mocetinostat (MGCD0103), entinostat (MS-27-527), and
givinostat (ITF2357) (Figure 1), are currently undergoing clinical trials for several
types of cancer at different phases [12-14]. Except romidepsin, which is naturally
occuring, all other compounds are synthetic.
-2-

O
H3C
CH3
H
CH3
O
O
O
O
N
NHOH
O
S
N
N
NHOH
O
H
S
H
O
O
HN
O
SAHA (vorinostat)
S
PXD-101 (belinostat)
H
N
N
H
O
CH3
H3C
O
O
Romidepsin
NH2
N
H
NHOH
H
H
N
N
N
Chidamide
HN
O
LBH-589 (panobinostat)
CH3
F
O
N
O
N
NH2
N
N
NH2
H
H
H
H
N
N
N
N
MS-275 (entinostat)
O
O
MGCD0103 (mocetinostat)
C2H5
N
H
C2H5
O
N
O
NHOH
ITF2357 (givinostat)
Figure 1. Structures of some HDAC inhibitors approved or under clinical trials
During the last decade, we have explored numerous approaches to discover novel
HDAC inhibitors as antitumor agents, including experimental and theoretical
methods [15-23]. Several series of heterocycle-based benzothiazole-, 5-aryl-3,4,5-
thiadiazole-, or 2-oxoindoline-based hydroxamic acids as analogues of SAHA have
been investigated [15-21]. Additionally, some series of N-hydroxybenzamides and N-
hydroxypropenamides as analogues of PXD-101 (belinostat) or ITF2357 (givinostat)
have also been synthesized and evaluated for HDAC inhibition and human cancer
cell cytotoxicity [20, 21, 24]. All those compound series were found to exhibit very
potent HDAC inhibitory activity as well as cytotoxicity [15-23]. Some compounds
also exerted in vivo antitumor effects in nude mice inoculated inoculated with PC-3
human prostate cancer cells [16]. Continuing our investigation, we expanded our
interest into small molecule hydroxamic acids bearing diverse heterocycles such as
1,2-oxazole, 1,3-oxazole indole, pyrazole (Figure 2A). It was expected that the
-3-

heterocycles could creat more interaction with the aminoacid chains located in the
enzyme active binding site. The results showed that compounds II, VI, and VII
displayed significant cytotoxicity and HDAC inhibition in Hela nuclear extract assay
(Figure 2A). Of these, compound II appeared to be the most promising one. Inspired
by these results, we have designed and synthesized several analogues of compound II
(Figure 2B). This paper describes the results obtained from the synthesis, biological
evaluation and docking study of these novel hydroxamic acids bearing 2-benzamido-
1,3-oxazole/1,3-thiazole (5a-g, 6a-g) or 2-phenylsulfonamido-1,3-thiazole (8a-c).
(A)
O
O
O
O
O
O
N
NHOH
NHOH
O
NHOH
N
H
N
N
N
H
O
O
H
N
III
I
II
O
O
O
NHOH
NH
NHOH
O
N
NHOH
N
N
N
N
N
H3C
H
IV
O
H
O
VI
V
O
O
O
NHOH
O
NHOH
N
N
N
H
N
NH
N
H
H3C
VII
VIII
O
(B)
O
N
NHOH
N
H
X
R
O
5
6
(X = O)
(X = S)
O
N
NHOH
O
N
O
H
O
O
N
NHOH
II
S
N
S
R
H
8
Figure 2. (A) Some small molecule hydroxamic acids bearing 1,2-oxazole, 1,3-
oxazole, indole, pyrazole; (B) Rational design of 1,3-oxazole hydroxamic acids as
histone deacetylase inhibitors
-4-

2. Materials and Methods
2.1. Chemistry
Thin layer chromatography performed using Whatman® 250 m Silica Gel GF
Uniplates and visualized under UV light at 254 and 365 nm, was used to check the
progress of reactions and preliminary evaluation of compounds’ homogeneity.
Melting points were measured using a Gallenkamp Melting Point Apparatus
(LabMerchant, London, United Kingdom) and were uncorrected. Purification of
compounds was carried out using crystallization methods and/or open silica gel
column flash chromatography employing Merck silica gel 60 (240 to 400 mesh) as
stationary phase. Nuclear magnetic resonance spectra (1H NMR) were recorded on a
Bruker 500 MHz spectrometer with DMSO-d6 as solvent unless otherwise indicated.
Tetramethylsilane was used as an internal standard. Chemical shifts are reported in
parts per million (ppm), downfield from tetramethylsilane. Mass spectra with
different ionization modes including electron ionization (EI), electrospray ionization
(ESI), were recorded using PE Biosystems API2000 (Perkin Elmer, Palo Alto, CA,
USA) and Mariner® (Azco Biotech, Inc. Oceanside, CA, USA) mass spectrometers,
respectively. The elemental (C, H, N) analyses were performed on a Perkin Elmer
model 2400 elemental analyzer. All reagents and solvents were purchased from
Aldrich or Fluka Chemical Corp. (Milwaukee, WI, USA) or Merck unless noted
otherwise. Solvents were used directly as purchased unless otherwise indicated.
The synthesis of novel hydroxamic acids bearing 2-benzamidooxazole/thiazole (5a-g,
6a-g) or 2-phenylsulfonamidothiazole (8a-c) was carried out as illustrated in Scheme
1. Details are described below.
General procedures for the synthesis of compounds 5a-g
To a solution of commercially available ethyl 2-aminooxazole-4-carboxylate (2
mmol) in 10 ml DCM, DMAP (244 mg, 2 mmol) was added. After stirring for 10
minutes, a respective benzoyl chloride (2.2 mmol) was added. The reaction mixture
was again stirred at 50℃ for 12 hours. After the reaction was finished (monitored by
TLC), the solvent was removed under reduced pressure. A solution of NaHCO3 5%
was gradually added to adjust pH to 7, which led to the formation of white solids.
The solids were filtered, washed with cold water and dried at 60℃. The crude product
was further purified by column chromatography (DCM/methanol = 95:5) to give the
corresponding derivative 3, yields 67-82%.
Each of the intermediate esters 3 (1 mmol) was dissolved in methanol (10 mL). Then,
-5-

hydroxylamine.HCl (685 mg, 10 mmol) was added, followed by dropwise addition of
a solution of NaOH (400 mg in 1 mL of water). The mixture was stirred at -5℃ until
the reaction completed (1-2 h, checked by TLC). At the end of this reaction, the
resulting reaction mixture was poured into ice-cold water, neutralised to pH~7 and
acidified by dropwise addition of a solution of HCl 5% to induce the maximum
precipitation. The precipitates were filtered, dried and re-crystalised in methanol to
give the corresponding target compounds 5a-g.
2-Benzamido-N-hydroxyoxazole-4-carboxamide (5a)
White solid; Yield: 65%. mp: 175-176 oC. Rf = 0.47 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3362 (NH), 3181, 3119 (OH); 3030, 3009 (CH, aren); 1686,
1655 (C=O); 1624, 1599, 1553 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.67
(1H, s, NHOH), 11.04 (1H, s, CO-NH), 9.11 (1H, s, NHOH), 8.44 (1H, s, H-5), 7.97
(2H, dd, J = 8.00 Hz, J’ = 1.00 Hz, H-2’, H-6’), 7.66 (1H, t, J = 7.50 Hz, H-4’), 7.56
13
(2H, t, J = 7.75 Hz, H-3’, H-5’). C NMR (125 MHz, DMSO-d6, ppm): δ 165.92,
158.23, 153.43, 139.09, 134.58, 133.23, 132.93, 129.12, 128.57. HR-MS (ESI) m/z:
248.0670 [M+H]+.
2-(2-Chlorobenzamido)-N-hydroxyoxazole-4-carboxamide (5b)
White solid; Yield: 72%. mp: 191-192 oC. Rf = 0.49 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3460 (NH); 3254, 3123 (OH); 2982 (CH, aren); 1651 (C=O);
1
1595, 1557, 1530 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 12.12 (1H, s,
NHOH), 11.00 (1H, s, CO-NH), 9.30 (1H, s, NHOH), 8.67 (1H, s, H-5), 7.79 (4H, m
13
, H-3’, H-4’, H-5’, H-6’). C NMR (125 MHz, DMSO-d6, ppm): δ 163.60, 161.04,
153.44, 142.38, 135.26, 133.06, 132.44, 132.02, 131.08, 129.60, 127.71. HR-MS
(ESI) m/z: 282.0279 [M+H]+.
2-(3-Chlorobenzamido)-N-hydroxyoxazole-4-carboxamide (5c)
White solid; Yield: 76%. mp: 193-194 oC. Rf = 0.49 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3356 (NH); 3156 (OH); 2988, 2872, 2805 (CH, aren); (1686,
1
1659 (C=O); 1616, 1564 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.00 (1H,
s, CO-NH), 9.11 (1H, s, NHOH), 8.44 (1H, s, H-5), 8.02 (1H, s, H-2’), 7.93 (1H, d , J
= 7.50 Hz, H-4’), 7.73 (1H, d, J = 7.50 Hz, H-6’), 7.59 (1H, t, J = 7.75 Hz, H-5’). 13C
NMR (125 MHz, DMSO-d6, ppm): δ 164.58, 158.20, 153.48, 138.96, 135.16, 134.54,
133.86, 132.91, 131.09, 128.38, 127.36. HR-MS (ESI) m/z: 282.0281 [M+H]+.
2-(4-Chlorobenzamido)-N-hydroxyoxazole-4-carboxamide (5d)
White solid; Yield: 75%. mp: 198-199 oC. Rf = 0.49 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3354 (NH); 3156 (OH); 3134, 2988, 2843 (CH, aren); 1690,
1
1661 (C=O); 1614, 1568 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.74 (1H,
s, NHOH), 11.02 (1H, s, CO-NH), 9.11 (1H, s, NHOH), 8.44 (1H, s, H-5), 7.99 (2H,
-6-

13
d, J = 8.50 Hz, H-2’. H-6’), 7.63 (2H, d, J = 8.50 Hz, H-3’, H-5’). C NMR (125
MHz, DMSO-d6, ppm): δ 164.96, 158.21, 153.45, 139.03, 138.06, 134.55, 131.84,
130.56, 129.21. HR-MS (ESI) m/z: 282.0281 [M+H]+.
2-(4-Fluorobenzamido)-N-hydroxyoxazole-4-carboxamide (5e)
White solid; Yield: 77%. mp: 179-180 oC. Rf = 0.45 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3358 (NH); 3154 (OH); 2988, 2884 (CH, aren); 1686, 1661
1
(C=O); 1603, 1570, 1504 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.61 (1H,
s, NHOH), 10.92 (1H, s, CO-NH), 9.01 (1H, s, NHOH), 8.35 (1H, s, H-5), 7.97 (2H,
dd, J = 8.50 Hz, J’ = 5.50 Hz, H-2’, H-6’), 7.31 (2H, t, J = 8.75 Hz, H-3’, H-5’). 13C
NMR (125 MHz, DMSO-d6, ppm): δ 166.19, 164.81, 164.20, 158.23, 153.37, 139.11,
134.58, 131.54, 131.46, 129.44, 116.24, 116.07. HR-MS (ESI) m/z: 264.0433
[M+H]+.
N-Hydroxy-2-(4-methylbenzamido)oxazole-4-carboxamide (5f)
White solid; Yield: 74%. mp: 185-186 oC. Rf = 0.44 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3374 (NH); 3161, 3125 (OH); 3042, 2988, 2864 (CH, aren);
1680, 1647 (C=O); 1624, 1551, 1506 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ
11.46 (1H, s, NHOH), 10.92 (1H, s, CO-NH), 9.01 (1H, s, NHOH), 8.635 (1H, s, H-
5), 7.80 (2H, d, J = 7.50 Hz, H-2’, H-6’), 7.27 (2H, d, J = 7.50 Hz, H-3’, H-5’), 2.31
13
(3H, s, CH3). C NMR (125 MHz, DMSO-d6, ppm): δ 165.78, 158.26, 153.54,
143.51, 139.11, 134.58, 130.10, 129.64, 128.64, 21.55. HR-MS (ESI) m/z: 260.0680
[M+H]+.
N-Hydroxy-2-(4-methoxybenzamido)oxazole-4-carboxamide (5g)
White solid; Yield: 65%. mp: 181-182 oC. Rf = 0.43 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3460, 3368 (NH); 3163, 3121 (OH); 3009, 2847 (CH, aren);
1676, 1651 (C=O); 1597, 1551, 1506 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ
11.47 (1H, s, NHOH), 11.01 (1H, s, CO-NH), 9.10 (1H, s, NHOH), 8.43 (1H, s, H-5),
7.98 (2H, d, J = 8.50 Hz, H-2’, H-6’), 7.08 (2H, d, J = 9.00 Hz, H-3’, H-5’), 3.85
13
(3H, s, OCH3). C NMR (125 MHz, DMSO-d6, ppm): δ 165.25, 163.27, 158.28,
153.67, 139.07, 134.55, 130.71, 124.93, 114.38, 56.02. HR-MS (ESI) m/z: 278.0776
[M+H]+.
General procedures for the synthesis of compounds 6a-g
Compounds 6a-g were synthesized via a two-step pathway as illustrated in scheme 1.
The procedures were similar to that described for compound 5a-g with was used
commercially available ethyl 2-aminothiazole-4-carboxylate instead of ethyl 2-
aminoesters.
2-Benzamido-N-hydroxythiazole-4-carboxamide (6a)
-7-

White solid; Yield: 68%. mp: 189-188 oC. Rf = 0.52 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3339 (NH); 3262, 3111 (OH); 3057, 2994 (CH, aren); 1655
1
(C=O); 1601, 1545 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 12.64 (1H, s,
NHOH), 10.73 (1H, s, CO-NH), 9.05 (1H, s, NHOH), 8.01 (2H, d, J = 8.00 Hz, H-2’,
H-6’), 7.71 (1H, d, J = 2.50 Hz, H-5), 7.57 (1H, t, J = 7.50 Hz, H-4’), 7.47 (2H, t , J
13
= 7.75 Hz, H-3’, H-5’). C NMR (125 MHz, DMSO-d6, ppm): δ165.98, 159.80,
158.99, 143.72, 133.24, 132.29, 129.12, 128.64, 117.50. HR-MS (ESI) m/z: 262.0302
[M+H]+.
2-(2-Chlorobenzamido)-N-hydroxythiazole-4-carboxamide (6b)
White solid; Yield: 72%. mp: 198-199 oC. Rf = 0.54 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3337 (NH); 3175, 3117 (OH); 3057, 2967, 2872 (CH, aren);
1680, 1655 (C=O); 1589, 1545 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 12.80
(1H, s, NHOH), 10.75 (1H, s, CO-NH), 9.02 (1H, s, NHOH), 7.75 (1H, s, H-5), 7.58-
13
7.37 (4H, m, H-3’, H-4’, H-5’, H-6’). C NMR (125 MHz, DMSO-d6, ppm): δ
165.80, 159.56, 158.02, 143.84, 134.65, 132.48, 130.70, 130.24, 129.90, 127.72,
117.88. HR-MS (ESI) m/z: 295.9898 [M+H]+.
2-(3-Chlorobenzamido)-N-hydroxythiazole-4-carboxamide (6c)
White solid; Yield: 74%. mp: 201-202 oC. Rf = 0.54 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3393 (NH); 3125 (OH); 3065, 2967 (CH, aren); 1651 (C=O);
1599, 1549 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 8.07 (1H, d, J = 1.50 Hz,
H-2’), 7.96 (1H, d, J = 8.00 Hz, H-6’), 7.77 (1H, s, H-5), 7.61 (1H, dd, J = 8.00 Hz,
J’ = 1.50 Hz, H-4’), 7.49 (1H, t, J = 7.75 Hz, H-5’). 13C NMR (125 MHz, DMSO-d6,
ppm): δ 164.76, 159.71, 157.59, 143.62, 134.43, 133.90, 132.91, 131.05, 128.48,
127.40, 117.62. HR-MS (ESI) m/z: 295.9903 [M+H]+.
2-(4-Chlorobenzamido)-N-hydroxythiazole-4-carboxamide (6d)
White solid; Yield: 74%. mp: 207-208 oC. Rf = 0.54 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3395, 3332 (NH); 3142 (OH); 3061, 2968, 2803 (CH, aren);
1
1651 (C=O); 1593, 1547 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 12.75 (1H,
s, NHOH), 10.75 (1H, s, CO-NH), 9.07 (1H, s, NHOH), 8.02 (2H, d, J = 9.00 Hz, H-
13
2’, H-6’), 7.72 (1H, s, H-5), 7.54 (2H, d, J = 8.50 Hz, H-3’, H-5’). C NMR (125
MHz, DMSO-d6, ppm): δ 165.06, 159.74, 158.94, 143.73, 138.11, 131.18, 130.60,
129.22, 117.59. HR-MS (ESI) m/z: 295.9905 [M+H]+.
2-(4-Fluorobenzamido)-N-hydroxythiazole-4-carboxamide (6e)
White solid; Yield: 75%. mp: 188-189 oC. Rf = 0.45 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3399, 3321 (NH); 3142 (OH); 3075, 2984, 2820 (CH, aren);
1657 (C=O); 1601, 1547, 1510 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 12.46
(1H, s, NHOH), 10.73 (1H, s, CO-NH), 9.07 (1H, s, NHOH), 8.10 (2H, dd, J = 8.50
Hz, J’ = 5.50 Hz, H-2’, H-6’), 7.73 (1H, s, H-5), 7.31 (2H, t, J = 8.75 Hz, H-3’, H-
-8-

5’). 13C NMR (125 MHz, DMSO-d6, ppm): δ 166.22, 164.92, 164.22, 159.82, 158.95,
143.69, 131.64, 131.57, 128.82, 128.80, 117.47, 116.25, 116.08. HR-MS (ESI) m/z:
280.0204 [M+H]+.
N-Hydroxy-2-(4-methylbenzamido)thiazole-4-carboxamide (6f)
White solid; Yield: 72%. mp: 179-180 oC. Rf = 0.49 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3447 (NH); 3221 (OH); 3107, 2974, 2903 (CH, aren); 1649
1
(C=O); 1609, 1541 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 12.64 (1H, s,
NHOH), 10.70 (1H, s, CO-NH), 9.05 (1H, s, NHOH), 7.92 (2H, d, J = 8.50 Hz, H-2’,
H-6’), 7.70 (1H, s, H-5), 7.27 (2H, d, J = 8.00 Hz, H-3’, H-5’), 2.30 (3H, s, CH3). 13C
NMR (125 MHz, DMSO-d6, ppm): δ 165.80, 159.88, 159.20, 143.70, 143.55, 129.67,
129.43, 128.69, 117.41, 21.57. HR-MS (ESI) m/z: 276.0454 [M+H]+.
N-Hydroxy-2-(4-methoxybenzamido)thiazole-4-carboxamide (6g)
White solid; Yield: 76%. mp: 197-198 oC. Rf = 0.48 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3337 (NH); 3215, 3179, 3117 (OH); 2992, 2837 (CH, aren);
1661 (C=O); 1605, 1547, 1501 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 12.50
(1H, s, NHOH), 10.67 (1H, s, CO-NH), 9.04 (1H, s, NHOH), 8.02 (2H, d, J = 8.50
Hz, H-2’, H-6’), 7.69 (1H, s, H-5), 7.00 (2H, d, J = 8.50 Hz, H-3’, H-5’), 3.77 (3H, s,
13
OCH3). C NMR (125 MHz, DMSO-d6, ppm): δ 165.25, 163.31, 159.92, 159.14,
143.66, 130.78, 124.25, 117.26, 114.42, 56.02. HR-MS (ESI) m/z: 294.0539 [M+H]+.
General procedures for the synthesis of compounds 8a-c
Compounds 9a-c were synthesized via a two-step pathway as illustrated in scheme 1.
The procedures were similar to that described for compound 6 with was used
respective benzenesulfonyl chloride instead of respective benzoyl chloride.
N-Hydroxy-2-(phenylsulfonamido)thiazole-4-carboxamide (8a)
White solid; Yield: 65%. mp: 193-194 oC. Rf = 0.38 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3248, 3117 (OH); 3061, 2982, 2905 (CH, aren); 1645 (C=O);
1
1549 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 13.07 (1H, s, NHOH), 11.22
(1H, s, SO2-NH), 10.86 (1H, s, NHOH), 9.24 (1H, s, H-5), 8.01 (2H, d, J = 8.00 Hz,
H-2’, H-6’), 7.57 (1H, t, J = 7.50 Hz, H-4’), 7.47 (2H, d, J = 7.75 Hz, H-3’, H-5’).
13C NMR (125 MHz, DMSO-d6, ppm): δ 159.13, 156.10, 141.50, 137.44, 129.63,
128.21, 126.36, 111.50. HR-MS (ESI) m/z: 297.9965 [M+H]+.
2-((4-Chlorophenyl)sulfonamido)-N-hydroxythiazole-4-carboxamide (8b)
White solid; Yield: 68%. mp: 221-222 oC. Rf = 0.39 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3179 (OH); 3098, 2990, 2903 (CH, aren); 1663 (C=O); 1580,
1
1520 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 13.07 (1H, s, NHOH), 11.22
(1H, s, SO2-NH), 10.86 (1H, s, NHOH), 9.24 (1H, s, H-5), 7.72 (2H, d, J = 8.50 Hz,
13
H-2’, H-6’), 7.53 (2H, d, J = 8.50 Hz, H-3’, H-5’). C NMR (125 MHz, DMSO-d6,
-9-

ppm): δ 159.13, 156.09, 141.50, 137.44, 129.63, 128.21, 126.35, 111.50. HR-MS
(ESI) m/z: 331.9579 [M+H]+.
N-Hydroxy-2-((4-methylphenyl)sulfonamido)thiazole-4-carboxamide (8c)
White solid; Yield: 66%. mp: 214-215 oC. Rf = 0.42 (DCM : MeOH : AcOH = 90 : 5
: 1). IR (KBr, cm-1): 3181, 3127 (OH); 3075, 2974, 2920 (CH, aren); 1649 (C=O);
1
1595, 1539, 1516 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.18 (1H, s, SO2-
NH), 10.86 (1H, s, NHOH), 9.24 (1H, s, H-5), 7.60 (2H, s, J = 8.00 Hz, H-2’, H-6’),
7.26 (2H, d, J = 8.50 Hz, H-3’, H-5’), 2.27 (3H, s, CH3). 13C NMR (125 MHz, DMSO-
d6, ppm): δ 159.12, 156.19, 142.99, 139.61, 129.93, 129.91, 126.36, 117.65, 21.42.
HR-MS (ESI) m/z: 312.0122 [M+H]+.
2.2. Biology
2.2.1. Cytotoxicity assay
The cytotoxicity of the synthesized compounds was evaluated against three human
cancer cell lines, including SW620 (colon cancer), PC3 (prostate cancer), and NCI-
H23 (lung cancer). The cell lines were purchased from a Cancer Cell Bank at the
Korea Research Institute of Bioscience and Biotechnology (KRIBB). The media, sera
and other reagents that were used for cell culture in this assay were obtained from
GIBCO Co. Ltd. (Grand Island, New York, USA). The cells were culture in DMEM
(Dulbecco’s Modified Eagle Medium) until confluence. The cells were then
trypsinized and suspended at 3 × 104 cells/mL of cell culture medium. On day 0, each
well of the 96-well plates was seeded with 180 μL of cell suspension. The plates were
o
then incubated in a 5% CO2 incubator at 37 C for 24 h. Compounds were initially
dissolved in dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations by
culture medium. Then 20 μL of each compounds’ samples, which were prepared as
described above, were added to each well of the 96-well plates, which had been
seeded with cell suspension and incubated for 24-h, at various concentrations. The
plates were further incubated for 48 h. Cytotoxicity of the compounds was measured
by the colorimetric method, as described previously [25] with slight modifications
[25-28]. The IC50 values were calculated using a Probits software and were averages
of three independent determinations (SD ≤ 10%) [29].
2.2.2. HDAC enzymes assay
The HDAC enzymatic assay was performed using a Fluorogenic HDAC Assay Kit
(Abcam. MA, USA) according to the manufacturer’s instructions. Briefly, HDAC
enzymes were incubated with vehicle or various concentrations of the assayed
samples or SAHA for 30 min at 37oC in the presence of an HDAC fluorimetric
substrate. The HDAC assay developer (which produces a fluorophore in reaction
-10-

mixture) was added, and the fluorescence was measured using VICTOR
(PerkinElmer, Waltham, MA, USA) with excitation at 360 nm and emission at 460
nm. The measured activities were subtracted by the vehicle-treated control enzyme
activities and IC50 values were calculated using GraphPad Prism (GraphPad Software,
San Diego, CA, USA).
2.2.3. Cell cycle analysis
U937 human lymphoma cells (5 × 105/ml per well) were plated in 6-well culture
plates and allowed to grow for 24 h. The cells were treated with compounds at
different concentrations for 24 h or 48 h, and then harvested. The harvested cells
were washed twice with ice-cold PBS, fixed in 75% ice-cold ethanol, and stained
with propidium iodide (PI) in the presence of RNase at room temperature for 30 min.
The stained cells were analyzed for DNA content using a FACScalibur flow
cytometer (BD Biosciences, San Jose, CA, USA) and the data were processed using
Cell Quest Pro software (BD Biosciences).
2.2.4. Apoptosis assay
The Annexin V-FITC/PI dual staining assay was used to determine the percentage of
apoptotic cells. U937 cells (5 × 105/ml per well) were plated in 6-well culture plates
and allowed to grow for 24 h. The cells were treated with compounds different
concentrations for 24 h or 48 h, and then harvested. The harvested cells were washed
twice with ice-cold PBS and incubated in the dark at room temperature in 100 ml of
1× binding buffer containing 1 µl Annexin V-FITC and 12.5 mL PI. After 15 min
incubation, cells were analyzed for percentage undergoing apoptosis using a
FACScalibur flow cytometer (BD Biosciences). The data were processed using Cell
Quest Pro software (BD Biosciences).
2.3. Molecular docking studies
The chemical structures of docking compounds were built and energy-optimized
within an rms gradient of 0.1 kcal.mol-1.Å-1 using MOE 2009.10 package, applying
the 94s variant of the Merck Molecular force field (MMFF94s) [30]. The hydroxamic
acids were deprotonated taking into account the charge transfer mechanism of HDAC
inhibitor [31]. The crystal structure of HDAC enzymes were retrieved from the
Protein Data Bank (PDB ID: 4LXZ and 5EEN) [32, 33]. For protein preparation we
employed the same procedures reported previously [20, 24]. The flexible-ligand
rigid-protein settings were used for docking experiments using MOE Triangle
Matcher placement method, keeping 30 poses for conformational analysis. Only
conformers that can form bidentate chelation with zinc ion are selected. To
-11-

determining binding affinity, London dG scoring (E_score1) and affinity scoring
function (E_score2) implemented into MOE 2009.10 were used [30]. The energy
minimization using the molecular mechanics force field method was performed for
refining the final conformation. Regarding other parameters, default configurations
were used.
3. Results and Discussions
3.1. Chemistry
The target hydroxamic acids bearing 2-benzamidooxazole (5a-g) or 2-
benzamidothiazole (6a-g) were obtained straightforwards via a two step pathway,
starting from commercially available ethyl 2-aminooxazole-4-carboxylate (1) or ethyl
2-aminothiazole-4-carboxylate (2). The first step was a simple amidification between
compounds 1 and 2 with benzoyl chloride and its different derivatives. The reactions
proceeded smoothly in dichloromethane with gentle heating to afford the benzamides
3a-g and 4a-g in quantitative yields. The second step involved an acyl nucleophilic
substitution between the ester intermediates 3a-g, 4a-g and hydroxylamine. The
reaction was optimized in methanol. Sodium hydroxide was used as a base to liberate
hydroxylamine from its corresponding hydrochloride salt. The second step afforded
the target hydroxamic acids 5a-g and 6a-g in moderate to good yields.
Three hydroxamic acids bearing 2-phenylsulfonamidothiazole structure (8a-c) were
reached by a similar two-step pathway, in which benzenesulfonyl chloride or its
derivatives were used instead of benzoyl chlorides. All reactions occurred in the same
manner.
The structures of the synthesized compounds were determined straightforwardly
based on analysis of spectroscopic data, including IR, MS, 1H and 13C NMR.
-12-

O
O
O
O
N
O
NHOH
N
OC2H5
c
N
a
N
OC2H5
N
X
R
H
H2N
R
X
H
5
)
(X = O
X
3
4
)
(X = O
(X = S)
6
(X = S)
1
2
)
(X = O
(X = S)
a
b
c
d)
) R = H
e
) R = 4-F
) R = 2-Cl
) R = 3-Cl
R = 4-Cl
f
) R = 4-CH3
g
) R = 4-OCH3
O
O
O
O
O
N
NHOH
O
O
N
OC2H5
S
c
S
b
N
N
OC2H5
N
R
S
H
R
S
H
H2N
S
8
7
2
a
) R = H
b
) R = 4-Cl
c
) R = 4-CH3
Scheme
1.
Synthesis
of
novel
hydroxamic
acids
bearing
2-
benzamidooxazole/thiazole (5a-g, 6a-g) or 2-phenylsulfonamidothiazole (8a-c).
Reagents and conditions: a) Benzoyl chloride or its derivatives, DCM, DMAP, 50 ℃;
b) ArSO2Cl, DCM, DMAP, 50 ℃; c) H2N-OH.HCl, NaOH, MeOH.
3.2. Bioactivity
The synthesized compounds were evaluated for their HDAC inhibitory ability using
Hela cell nuclear extract assay and cytotoxicity against three human cancer cell lines,
including SW620 (colon cancer), PC3 (prostate cancer), and NCI-H23 (lung cancer).
SAHA was used as a positive control. The results are presented in Table 1. As shown
in Table 1, in general, the synthesized compounds exhibited good cytotoxicity against
all three cancer cell lines tested with IC50 values of less than 10 µM in all cases. In
series 5a-g, addition of the chloro substitutuent at either positions 2, 3 or 4 of the
phenyl ring seemed to slightly enhanced cytoxicity in PC3 cells of the resulting
compounds 5b-d (IC50 values, 2.33-3.48 µM), as compared to 5a (IC50 value, 4.35
µM). In almost all other cases, the addition of substituents, either electron releasing
(Cl, F) or electron withdrawing groups (CH3, OCH3) slightly decreased the
cytotoxicity. Generally, however, compounds bearing electron releasing groups (Cl,
F) (compounds 5b-e) were still found to be more potent than compounds bearing
electron withdrawing groups (CH3, OCH3) (compounds 5f, g). Compound 5d could
be considered as the most potent in term of cytotoxicity within series 5a-g. It could
be postulated that the presence of halogens would be favorable for the compounds to
be more cellular permeable, as manifested by higher logP values. However, other
complicate factors, e.g. their electronic influences on the interactions of compounds
with HDAC binding site, might also come into play.
-13-

Table 1. Inhibition of HDAC activity and cytotoxicity of the synthesized compounds
against several cancer cell lines
O
O
3
3
N
4
4
O
O
O
N
2'
NHOH
NHOH
2'
5'
2
2
1'
1'
S
3'
3'
N
N
5
5
R
4'
X
S
R
4'
H
H
1
1
6' 5
6
)
6'
(X = O
5'
8
(X = S)
Cytotoxicity (IC50,2 M)/Cell
Cpd.
code
X
HDAC
(Hela
lines3
Ar
LogP1
extract)
Inhibition
SW620
PC3
NCI-H23
(IC50,2 M)
0.068±0.0 1.98±0. 4.35±0.
12 13 58
0.056±0.0 2.31±0. 2.33±0.
10 14 05
0.029±0.0 3.22±0. 3.35±0.
03 39 10
0.069±0.0 2.40±0. 3.48±0.
11 05 01
0.038±0.0 5.36±0. 5.44±0.
01 39 86
0.010±0.0 6.68±0. 5.11±0.
01 91 10
0.069±0.0 3.43±0. 7.50±0.
03 38 91
0.037±0.0 4.22±0. 8.48±1.
02 17 11
0.051±0.0 5.99±0. 3.78±0.
01 48 53
0.038±0.0 3.02±0. 5.02±0.
07 20 82
0.033±0.0 4.42±0. 6.57±0.
01 56 50
0.035±0.0 3.47±0. 4.64±0.
02 21 43
0.131±0.0 2.21±0. 4.34±0.
52 01 64
0.073±0.0 6.54±0. 6.05±0.
09 01 08
0.059±0.0 2.28±0. 2.11±0.
06 07 09
O
O
O
O
O
O
O
S
H
0.16
0.80
0.80
0.80
0.36
0.71
0.24
1.07
1.72
1.72
1.72
1.27
1.62
1.15
0.25
0.90
5a
5b
3.29±0.20
4.92±0.30
3.79±0.43
2.70±0.23
5.80±0.77
3.87±0.46
5.65±0.53
6.14±0.27
7.69±0.83
5.02±0.37
6.78±0.23
6.24±0.02
3.29±0.51
6.41±0.08
1.57±0.05
2-Cl
3-Cl
4-Cl
4-F
5c
5d
5e
5f
4-CH3
4-OCH3
H
5g
6a
6b
S
2-Cl
6c
6d
6e
6f
S
3-Cl
S
4-Cl
S
4-F
S
4-CH3
4-OCH3
H
S
6g
S
8a
8b
S
4-Cl
0.036±0.0 2.17±0. 1.96±0. 1.45±0.10
-14-

04
15
01
8c
S
4-CH3
264.32
0.33
1.44
0.044±0.0 1.91±0. 2.24±0.
1.73±0.08
1.44±0.17
00
18
1.12±0. 1.82±0.
10 09
34
0.025±0.00
SAHA4
2
2
1Calculated by KOWWIN v1.67 software; The concentration (M) of compounds
that produces a 50% reduction in enzyme activity or cell growth, the numbers
represent the averaged results from triplicate experiments; 3Cell lines: SW620, colon
4
cancer; PC3, prostate cancer; NCI-H23, lung cancer; SAHA, suberoylanilide acid,
a positive control.
Thiazole compounds (series 6a-g) seemed to be slightly less potent than the oxazole
ones (series 5a-g) in term of cytotoxicity. It was hard to delineate the structure-
cytotoxicity relationships within series 6a-g. In this series, compound 6g bearing 4-
methoxy substituent on the phenyl ring appeared to be the most cytotoxic one, but
still less potent than SAHA. Noteworthy, three compounds of sulfonamide type (8a-
c) exhibited stronger cytotoxicity compared to benzamide compounds (series 5a-g,
6a-g). The cytotoxicity of compounds 8a-c was comparable to that of SAHA. These
results suggest the sulfonamide functionality appears to be more favorable for the
bioactivity of these compound types and further exploration of the hydroxamic acids
incorporating sulfonamide functionality similar to compounds 8a-c should warranted.
Regarding the HDAC inhibition, compounds 5a, 6a and 8a dislayed potent activity
with IC50 values being 0.068, 0.037 and 0.059 µM, respectively. Generally, in series
5a-g introduction of the substituents on the phenyl ring of the benzamide moeity
retained or in some cases even enhanced the HDAC inhibitory activity. Compound 5f
bearing 4-methyl substituent was the most potent HDAC inhibitor with IC50 value as
low as 0.010 µM. In series 6a-g, a clearer structure-HDAC inhibition relationship
was observed. Electron-withdrawing substituents seemed to retain or enhance the
HDAC inhibitory activity (such as -Cl, compounds 6c, 6d, IC50 values 0.038, 0.033
µM), respectively, meanwhile, electron-releasing substituents (such as -CH3, -OCH3,
compounds 6f, 6g, IC50 values 0.252, 0.628 µM).
Although generally less potent than SAHA, based on the IC50 values, these
compounds were still very potent in term of HDAC inhibition. And given their
structral simplicity, further investigation of this type of compounds should be
warranted.
The results from SRB assays demonstrate that the compounds in three series 5a-g,
6a-g and 8a-c displayed relatively potent cytotoxicity against three human cancer cell
lines. We next decided to investigate whether these compounds affect the cell cycle
-15-

and apoptosis. Three representative compounds, including 5a, 6a and 8a, were
selected and the flow cytometry was used to analyze the effects of compounds on the
cell cyle. SW620 human colon cancer cells were treated with each compound at
different concentrations for 24 h and then DNA contents were analyzed. It was found
that, at 10 µM, all compounds did not affect the cell cycle (Figure 3A). At 50 µM,
compounds 5a, 6a and 8a mainly increased the cell death population (7.08, 15.55,
and 15.83%, respectively, vs. 6.69% of the VH, Figure 3B), while their effects on
other phases of the cell cycle were still not clearly observable. We decided to
increase the compounds concentration to 100 µM and found that, at this
concentration, compounds 6a and 8a significantly arrested cells at G2 phase of the
cell cycle (20,53 and 26.99%, respectively, vs. 6.21% of the VH, Figure 3C).
Next, to investigate whether the compounds induce apoptosis we used an Annexin V-
FITC/propidium iodide (PI) dual staining assay. Three representative compounds,
including 5a, 6a and 8a were chosen. Phosphatidylserine (PS) is one component of
the cell membrane which plays a key role in cell cycle signaling, especially in
relation to apoptosis. During early apoptosis, PS, which locates on the cytosolic
(inner) side of the cell membrane, translocates to the extracellular (outer) surface of
the cell membrane. Propidium iodide (PI) is a fluorescent intercalating agent.
Normally, the membrane of live cells is not permeable to PI. However, the membrane
of dead cells or cells in the latter stages is permeable to PI and their nuclei stain red.
Annexin V intrinsically has a high affinity for PS, therefore, annexin V fluorescently
labelled with fluorescein isothiocyanate (FITC) has been used to identify early
apoptotic cells. We treated SW620 cells with different concentrations of compounds
5a, 6a and 8a for 24 h, then stained the cells with Annexin V-FITC and PI. The
results illustrated in Figure 4 show that, at 10 µM, all three compounds did not affect
the cell cycle (Figure 4A). At 50 µM, compounds 6a and 8a slightly increased the
early apoptotic cell population (Figure 4B). However, at 100 µM concentration, the
induction of both early and late apoptosis in SW620 cells was clearly observed with
compounds 6a and 8a (Figure 4C).
Consistent with the effects on the cell cycle and apoptosis, compounds 6a and 8a at
50 µM concentration also significantly caused morphological changes of SW620
cells (Figure 5A). These effects were much more pronounced at higher concentration
of 100 µM (Figure 5B).
-16-

SAHA
VH
UN
A)
Dead: 8.88
G0/G1: 56.27
S: 32.61
Dead: 32.49
G0/G1: 50.61
S: 14.00
Dead: 13.45
G0/G1: 52.21
S: 31.41
G2: 1.76
G2: 2.24
G2: 2.34
t
6a
5a
8a
C o u n
Dead: 13.47
G0/G1: 53.03
S: 30.90
Dead: 14.55
G0/G1: 49.89
S: 32.14
Dead: 11.44
G0/G1: 50.96
S: 34.30
G2: 2.15
G2: 2.60
G2: 2.72
DNA content
B)
SAHA
VH
UN
Dead: 3.06
G0/G1: 41.10
S: 36.70
Dead: 47.82
G0/G1: 33.43
S: 14.41
Dead: 2.06
G0/G1: 43.42
S: 37.24
G2: 7.40
G2: 2.41
G2: 7.77
t
6a
5a
8a
C o u n
Dead: 25.79
G0/G1: 30,01
S: 34.24
Dead: 1.71
G0/G1: 48.50
S: 38.29
Dead: 1.82
G0/G1: 51.48
S: 41.51
G2: 5.77
G2: 5.00
G2: 2.95
DNA content
C)
SAHA
VH
UN
Dead: 2.38
G0/G1: 48.73
S: 34.27
Dead: 64.10
G0/G1: 18.56
S: 12.69
Dead: 2.05
G0/G1: 47.74
S: 36.75
G2: 6.55
G2: 2.95
G2: 6.21
t
5a
6a
8a
C o u n
Dead: 11.03
G0/G1: 3.11
S: 33.12
Dead: 1.52
G0/G1: 49.07
S: 38.74
Dead: 19.03
G0/G1: 5.05
S: 36.66
G2: 26.99
G2: 5.11
G2: 20.53
DNA content
Figure 3. Cell cycle analysis of representative compounds 5a, 6a and 8a. SW620
(human colon cancer) cells (5 x 105 cells/well in 6-well plate, pre-incubated for 2 h)
were treated with compounds (10 M) and SAHA (10 M) for 24 h (A). SW620
(human colon cancer) cells (2 x 105 cells/well in 6-well plate, pre-incubated for 2 h)
were treated with compounds (50 M) and SAHA (1 M) for 24 h (B). SW620
(human colon cancer) cells (2 x 105 cells/well in 6-well plate, pre-incubated for 24 h)
were treated with compounds (100 M) and SAHA (10 M) for 48 h (C). The
harvested cells were stained with propidium iodide (PI) in the presence of RNase and
-17-

then were analyzed for DNA content. UN: untreated, VH: vehicle (DMSO. 0.05%).
VH
SAHA
UN
A)
0.13%
0.31% 6.92%
43.11%
1.93%
3.62%
2.04%
3.36%
0.13%
5a
6a
8a
2.76%
4.26%
0.20%
1.91%
3.06%
0.22% 2.10%
3.74%
0.18%
Annexin V
B)
VH
SAHA
UN
1.71%
4.32% 10.23%
10.42%
2.12%
4.15%
3.28%
2.34%
2.47%
8a
5a
6a
3.02%
5.22%
3.10% 3.42%
1.75%
2.39%
2.96%
3.33%
4.16%
Annexin V
C)
SAHA
VH
UN
1.39%
3.14% 79.28%
8.63%
0.46%
0.82%
1.68%
0.37%
0.78%
8a
6a
10.23%
12.73%
5a
3.88%
2.90%
1.42%
3.76%
0.56%
1.14%
12.80%
Annexin V
Figure 4. Apoptosis (Annexin V/PI) analysis of representative compounds 5a, 6a
and 8a. SW620 (human colon cancer) cells (5 x 105 cells/well in 6-well plate, pre-
incubated for 2 h) were treated with compounds (10 M) and SAHA (10 M) for 24
h (A). SW620 (human colon cancer) cells (2 x 105 cells/well in 6-well plate, pre-
incubated for 2 h) were treated with compounds (50 M) and SAHA (1 M) for 24 h
(B).SW620 (human colon cancer) cells (2 x 105 cells/well in 6-well plate, pre-
incubated for 24 h) were treated with compounds (100 M) and SAHA (10 M) for
-18-

48 h (C). The harvested cells were incubated with Annexin V-FITC and PI. UN:
untreated, VH: vehicle (DMSO. 0.05%). Area 1 = PI positive population, Area 2:
Annexin V-postivie population.
SAHA
A)
VH
UN
8a
6a
5a
B)
UN
VH
SAHA
5a
6a
8a
Figure 5. Morphology changes of cells treated with representative compounds 5a, 6a
and 8a or SAHA. SW620 (human colon cancer) cells (2 x 105 cells/well in 6-well
plate, pre-incubated for 2 h) were treated with compounds (50 M) and SAHA (1
M) for 24 h (A). SW620 (human colon cancer) cells (2 x 105 cells/well in 6-well
plate, pre-incubated for 24 h) were treated with compounds (100 M) and SAHA (10
M) for 48 h (B). Then, the cells were photographed using an Imaging Device:
Biostation with 40X lens. Scale bar: 20 µm.
3.3. Molecular docking studies
-19-

In order to further explore the structure-activity relationship of HDAC inhibition
activity of synthesized compounds, docking simulations were carried out using MOE
software. To do so, two crystal structures of HDAC enzymes were selected: (i)
HDAC2 in complex with SAHA (PDB ID: 4LXZ) [32], and (ii) HDAC6 in complex
with Belinostat (PDB ID: 5EEN) [33] which shares similar structure features with our
designed compounds. By using two isozymes of different classes (HDAC2 belongs to
class I meanwhile HDAC6 class IIB) we could reveal some evidences related to the
selectivity of synthesized compounds.
Firstly, the docking procedures were validated by redocking the co-crystal ligand the
active site of the enzymes. For HDAC2, the redocked and co-crystal SAHA
conformations were highly superimposed with RMSD = 0.3770Å. As can be
observed in Figure 6A, the key H-bonding interactions with the residues Asp104,
His145, His146 and Tyr308 were well conserved. In addition, the hydroxamic group
of the redocked SAHA formed typical bidentate chelation with the cofactor zinc ion
in the binding site which is the key element of the experimental binding model.
According HDAC6, we also obtained good docking results. The deviation between
redocked and co-crystal binding poses was quite low (RMSD = 0.8103Å). All the key
interactions with His573, His574, Phe643 and Tyr745 were conserved (see Figure
6B). The hydroxamate group of Belinostat also formed stable chelate complex with
zinc ion of HDAC6. The docking scores calculated for redocked SAHA and
Belinostat were shown in Table 2. From the results obtained, we can conclude that
the currently applied docking protocols are suitable for using in the subsequent
docking studies.
Figure 6. Superposition between: (A) redocked (blue) and co-crystal (yellow) SAHA
in HDAC2 (PDB ID: 4LXZ), and (B) redocked (pink) and co-crystal (green)
-20-

Belinostat in HDAC6 (PDB ID: 5EEN). The binding pockets are colored by
hydrogen bond type.
In the next steps, all the 17 synthesized compounds (5a-g, 6a-g, 8a-c) were docked
into the binding site of HDAC2 and HDAC6 enzymes. Most of the HDAC inhibitors
conform to a Cap-Linker-Chelator pharmacophoric model [31, 34]. The cap group
can interact with the external surface of the active site, connecting with the zinc
binding group (chelator) through a linker that accommodate the tubular access of the
active site [6]. Our compounds were mainly designed based the modifications made
on the 1,3-oxazole and 1,3-thiazole rings of the linker groups. This substitution might
affect the interactions with the residues in the long tunnel in the HDAC pocket. For
HDAC2, all the compounds formed multiple pi-stacking interactions with Phe155,
His183 and Phe210 which is different from that of SAHA, meanwhile the key H-
bonding interactions are similar to SAHA. As can be seen in the Figure 6B, all the
compounds were well accomodated in the binding site of HDAC2 and formed
bidentate chelation with zinc ion in the same fashion of redocked SAHA. Table 2
showed the similarly close distances (1.95-2.73Å) between hydroxamate groups and
zinc ion of all the compounds.
Likewise, all compounds were found to be lying deep into the binding pocket of
HDAC6. They could form chelation with zinc ion in the same manner of Belinostat.
With exception of 5c, 6b and 6f, compounds 5a-g and 6a-g formed three H-bonds
with His573, His574, and Gly582. Meanwhile compounds 8a-b have additional H-
bond between sulfonyl group of linker with His614. As can be observed in the Table
2, the docking scores calculated for lingands in complexes with HDAC6 were
slightly lower than HDAC2, suggesting the higher affinity of synthesized compounds
towards HDAC6 compared to HDAC2 isozyme.
-21-

Figure 7. Topological presentation of the binding modes and interactions of
compounds SAHA (redocked), 5f, 6d, and 8b in HDAC2 active site.
Figure 8. Topological presentation of the binding modes and interactions of
compounds Belinostat (redocked), 5f, 6d, and 8b in HDAC6 active site.
-22-

Figure 7 and 8 illustrates the comparison between the binding modes of some
representative compounds (5f, 6d, 8b) and reference compounds (SAHA and
Belinostat). These compounds are the most potential compounds according to HDAC
inhibition from each series. For HDAC2, all the compounds could form 3 to 4 H-
bonding interactions with His145, His146, His183 and Tyr308 (Figure 7). The van
der Waals interactions involved in holding ligands within the active site include pi-
interactions between the 1,3-oxazole and 1,3-thiazole linker moieties and aromatic
rings of Phe155, His183 and Phe210. In addition, the capping group showed
numerous interactions with residues at the rim of the pocket, such as Asp104, Tyr209
and Leu276. Recently, we have investigated the N-hydroxybenzamide scaffold as
linker and observed very similar results to those obtained in this study [20, 24, 35].
On the other hand, the interactions between three synthesized compounds were quite
similar to Belinostat. As can be reveal in Figure 8, the pi-stacking interactions with
hydrophobic residues Phe583 and Phe643 appeared to be important for stabilizing the
ligand-protein complexes. In addition compound 8b showed higher H-bonding
interactions with HDAC6 compared to Belinostat, 5f and 6d. Considering the
importance of isoform selectivity, especially to avoid cardiac side effects in screening
novel HDAC inhibitors, it would be of high interest to experimentally investigate the
inhibitory selectivity of our synthesized compounds towards HDAC enzymes in the
future.
We continued analyzing the correlations between the docking scores and the IC50
values. Our scoring calculations were based on two common function applied in the
MOE package, including the London and affinity scoring functions. As can be seen
in Figure 8, for both systems (HDAC2 and HDAC6), the binding affinities calculated
via London scoring function showed higher correlation with experimental values (R2
= 0.76 and 0.71) compared to that calculated using other method (R2 = 0.65 and
0.61). These findings agreed with previous results and once again confirmed the
ability of London scoring function to quantitatively predict the experimental HDAC
inhibitory activity [24].
Table 2. Docking scores of all compounds with HDAC2 and HDAC6 enzymes.
Cpd. code
HDAC2
HDAC6
E_Score1* E_score2* Distance E_Score E_score Distance
*
*
to Zn2+*
1
2
to Zn2+
-
=O
-
=O
OH
OH
5a
-14.535
-7.005
1.97 2.34 -15.737
-9.516 1.95 2.4
-23-

5b
-16.471
-17.872
-14.772
-15.463
-19.695
-15.442
-16.404
-18.134
-16.261
-17.190
-16.958
-12.592
-13.644
-15.085
-17.921
-15.479
-19.102
-7.435
-8.851
-6.966
-7.126
-9.430
-7.149
-7.521
-7.783
-7.483
-8.122
-8.058
-6.834
-7.111
-7.791
-8.958
-7.493
-7.757
2.01 2.3 -15.901
1.96 2.32 -18.671
1.95 2.39 -15.945
1.96 2.33 -17.974
1.97 2.34 -20.014
1.96 2.32 -17.308
1.96 2.43 -17.269
1.97 2.47 -17.266
2.04 2.47 -17.391
2.03 2.43 -17.768
1.97 2.47 -16.518
1.98 2.48 -13.674
1.97 2.48 -14.814
1.99 2.45 -18.328
1.96 2.73 -16.960
1.98 2.60 -16.165
1.96 2.54
-9.425 1.94 2.46
-10.438 1.95 2.48
-9.607 1.95 2.41
-9.513 1.95 2.41
-10.974 2.07 2.21
-9.869 1.95 2.38
-9.732 1.95 2.38
-9.887 1.95 2.37
-9.846 1.95 2.37
-9.971 1.96 2.47
-9.901 1.96 2.48
-9.482 2.02 2.31
-9.772 1.95 2.36
-10.071 1.95 2.52
-10.259 1.95 2.48
-10.189 1.99 2.23
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
6g
8a
8b
8c
SAHA
Belinostat
-19.030
-10.885 1.96 2.30
*
The docking score (kcal/mol) calculated from the London (with refinement) and
affinity scoring function from MOE software
** Distances (Å) from oxygen atoms (-O and =OH) of hydroxamate group to zinc ion.
-24-

Figure 9. Correlation analysis of IC50 values and docking scores determined for all
the ligands bound to HDAC2 (A-B) and HDAC6 (C-D).
4. Conclusions
In conclusion, we have reported here seventeen simple hydroxamic acids bearing 1,3-
oxazole- and 1,3-thiazole scallfods and evaluated their cytotoxicity as well as their
HDAC inhibition. Overall, the compounds were potent HDAC inhibitors. Some
compounds (with IC50 values as low as 0.010 µM) were even more potent than
SAHA. These compounds also exhibited good cytotoxicity with IC50 values in a
range from 1.45 to 7.69 µM. Representative compounds 6a and 8a appeared to arrest
the SW620 cell cycle at G2 phase and significantly induced both early and late
apoptosis of SW620 colon cancer cells. Docking experiments on HDAC2 and
HDAC6 isozymes revealed some pi-stacking interactions formed between the 1,3-
oxazole and 1,3-thiazole linker moieties toward hydrophobic residues at the tunnel of
the HDAC active site which positively contributed to the inhibitory activity of
synthesized compounds. The binding affinity predicted by docking program showed
good correlation with the experimental IC50 values. This study demonstrates that
simple 1,3-oxazole- and 1,3-thiazole-based hydroxamic acids are also promising as
-25-

antitumor agents and HDAC inhibitors. Also, these results should provide valuable
information for further design of more potent HDAC inhibitors and antitumor agents.
Acknowledgements
We acknowledge the principal financial supports from the National Foundation for
Science and Technology of Vietnam (NAFOSTED, Grant number 104.01-2017.301).
The work was also partly supported by a grant funded by the Korean Government
(NRF-2017R1A5A2015541) for more indepth-biological evaluation and mechanism
study.
Declaration of interest
The authors declarate no conflict of interest.
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