Design, synthesis and preliminary bioactivity studies of 1,3,4-thiadiazole hydroxamic acid derivatives as novel histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.04.032

Histone deacetylase (HDAC) inhibitors have emerged as a new class of anticancer agents, targeting the biological processes including cell cycle, apoptosis and differentiation. In the present study, a series of 1,3,4-thiadiazole based hydroxamic acids were developed as potent HDAC inhibitors. Some of them showed good inhibitory activity in HDAC enzyme assay and potent growth inhibition in some tumor cell lines. Among them, compound 6i (IC₅₀ = 0.089 μM), exhibited better inhibitory effect compared with SAHA (IC₅₀ = 0.15 μM).

Full text of DOI:10.1016/j.bmc.2012.04.032

Bioorganic & Medicinal Chemistry 20 (2012) 3865–3872
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and preliminary bioactivity studies of 1,3,4-thiadiazole
hydroxamic acid derivatives as novel histone deacetylase inhibitors
Peng Guan a, Feng’e Sun a, Xuben Hou a, Feng Wang a, Fan Yi b, Wenfang Xu a, Hao Fang a,
⇑
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, PR China
b Department of Pharmacology, Shandong University, School of Medicine, Ji’nan, Shandong 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylase (HDAC) inhibitors have emerged as a new class of anticancer agents, targeting the
biological processes including cell cycle, apoptosis and differentiation. In the present study, a series of
1,3,4-thiadiazole based hydroxamic acids were developed as potent HDAC inhibitors. Some of them
showed good inhibitory activity in HDAC enzyme assay and potent growth inhibition in some tumor cell
Received 25 February 2012
Revised 13 April 2012
Accepted 16 April 2012
Available online 21 April 2012
lines. Among them, compound 6i (IC50 = 0.089
SAHA (IC50 = 0.15 M).
l
M), exhibited better inhibitory effect compared with
l
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
1,3,4-Thiadiazole
Histone deacetylase inhibitors
Anticancer
1. Introduction
tain cellular localization owing to their ability to shuttle between
nucleus and cytoplasm.
Histone deacetylases are highlighted as one of the pivotal mem-
bers in tumor epigenome in response to their corroborated compe-
tence to deacetylate histone as well as non-histone proteins. In this
way histone deacetylases involve in chromatin remodeling and
assembly, DNA repair and recombination and the consequent reg-
ulation of gene expression.1 Indeed, extensive studies have re-
cently revealed that histone deacetylases can be tethered
mechanistically to the oncogenesis, maintenance and progression
of cancer.2
Eighteen histone deacetylases have been identified in the mam-
malian genome and grouped to four classes based on their homol-
ogy to the respective yeast transcriptional control factor sequence.
Class III HDAC (Surtuin1 to -7) share domains with yeast SIR2 pro-
tein and their dependence on NAD+ for deacetylase activity atten-
uates concerns here. Classical HDAC comprising Classes I, II, and IV
HDAC family members are Zn2+-dependent: Class I HDAC (HDAC1,
-2, -3, and -8) are closely related to yeast RPD3; Class II HDAC,
including Class IIa (HDAC4, -5, -7, and -9) and Class IIb (HDAC6
and -10), possess sequence similarity to yeast Hda1; HDAC11 is
homologs of both RPD3 and Hda1, consequently defining Class IV
HDAC. Classes I and IV HDAC are pervasively expressed in diverse
tissues and generally localized to the nucleus. Nevertheless, Class II
HDAC, which are restricted to certain cell types, display an uncer-
Histone deacetylase (HDAC) inhibitors induce, to a variable ex-
tent, cell cycle and growth arrest, differentiation or apoptosis of
malignant cells of in vitro models and in vivo xenografts. Strikingly,
their anticancer efficacy has been clinically substantiated in broad
spectrum of neoplasm types from both haematological and solid
origins.2,3 Suberoylanilide hydroxamic acid (SAHA, Vorinostat)4
(Fig. 1) and depsipeptide (FK228, Romidepsin)5 have been ap-
proved by FDA for the treatment of cutaneous T-cell lymphoma
in 2006 and 2009, respectively.
Most of the HDAC inhibitors include three parts: a Zn2+ binding
group, a linker domain and a surface recognition motif (Fig. 1). Pre-
viously, we developed N-phenylpropionamide6 and tetrahydroiso-
quinoline-based hydroxamic acids7–9 as novel HDAC inhibitor
scaffolds and the latter exhibited higher in vivo antitumor activity
than SAHA. In our ongoing study, a 1,3,4-thiadiazole ring has been
O
H
N
OH
N
H
O
Zinc
binding
group
Surface
recognition
motif
Linker
⇑
Corresponding author. Tel.: +86 531 88382731; fax: +86 531 88382548.
E-mail address: haofangcn@sdu.edu.cn (H. Fang).
Figure 1. The chemical structure of SAHA and pharmacophoric characteristics of
HDAC inhibitors exemplified by SAHA.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.032

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P. Guan et al. / Bioorg. Med. Chem. 20 (2012) 3865–3872
Figure 2. (a) The docking modes of compound 6i (depicted in atom type) and SAHA (depicted in green) in the active site of HDAC1 (left) and their top views with surface
representations (right). (b) Hydrogen bond interactions between 6i (a) and SAHA (b) and HDAC1.
introduced as a substitution of the rigid tetrahydroisoquinoline
fragment. This paper will describe the synthesis, enzyme inhibition
and antiproliferative activities against some tumor cell lines of the
1,3,4-thiadiazole hydroxamic acid derivatives.
Compounds 10a–10d were prepared (Scheme 2) from commer-
cially available diethyl oxalate 7, following the synthetic approach
described in Scheme 1. However, compounds 13a and 13b could
not be obtained using the same method. Thus key intermediates
12a–12b were prepared (Scheme 3) from succinic anhydride 11. Fi-
nal hydroxamic acids were generated by the mixed anhydride ap-
proach according to literature procedures.6
2. Chemistry
Synthesis of the 2-amino-1,3,4-thiadiazole-based hydroxa-
mates is described in Schemes 1–3. The 5-substituted-1,3,4-thia-
3. Results and discussion
diazol-2-amines 2a–2d were prepared via
a facile one-pot
reaction of N-aminothiourea with phosphoryl chloride. Saponifica-
tion of the dimethyl esters 3a–3d furnished the monomethyl esters
4a–4d in good yields. Sequential treatment of 4a–4d with refluxing
thionyl chloride and then 2a–2d gave 5a–5p, which were con-
verted to hydroxamic acids 6a–6p by a freshly prepared potassium
hydroxylamine according to reported procedures.10
HDAC inhibitory activity of the 1,3,4-thiadiazole based
hydroxamates were assessed by the Color de Lys assay and the re-
sults are tabulated as IC50 values in Table 1. According to the data
in Table 1, both the length of the linker chain (n) and the substitu-
tion in 1,3,4-thiadiazole (R) play a role in potency. For example,
compounds with the linker comprised of five or six methylene
N
N
a
R
COOH
R
NH2
S
1a-1d
2a-2d
O
O
N
O
O
N
N
N
c
d
OH
R
N
H
O
R
( )n
N
H
( )n
N
H
S
S
O
O
O
O
b
( )n
O
O
( )n
O
OH
5a-5p
6a-6p
3a-3d
4a-4d
Scheme 1. Reagents: (a) NH2NHCSNH2, POCl3, then H2O; (b) KOH, CH3OH; (c) (i) SOCl2; (ii) Et3N, THF; (d) NH2OK, CH3OH.

P. Guan et al. / Bioorg. Med. Chem. 20 (2012) 3865–3872
3867
O
O
O
O
H
H
N
a
b
S
c
S
N
R
OH
O
Cl
R
O
N
H
O
O
N
N
N
O
N
O
O
O
7
8
9a-9d
10a-10d
Scheme 2. Reagents: (a) (i) CH3CO2K, H2O; (ii) SOCl2, Et2O; (b) 2a–2d, Et3N, THF; (c) NH2OK, CH3OH.
O
O
H
N
H
N
S
a
S
O
b
OH
R
R
O
O
OH
N
H
N
N
N
N
O
O
11
12a-12b
13a-13b
Scheme 3. Reagents: (a) 2a, or 2d, CH3CN; (b) i-BuCOOCl, N-methylmorpholine, DMF then NH2OH, CH3OH.
Table 1
HDAC inhibitory activity of 1,3,4-thiadiazole hydroxamate derivatives
H
N
H
N
S
( )n
OH
R
N
O
O
N
Compd
R
n
IC50 of HDACa
>5
(l
M)
Compd
R
n
IC50 of HDACa
0.27 0.004
(lM)
6a
3
6m
6
6b
6c
6d
6e
6f
3
3
3
4
4
4
4
5
5
5
5
1.87 0.41
2.71 0.25
0.16 0.03
1.03 0.04
1.12 0.01
3.49 0.04
1.70 0.40
0.089 0.005
0.22 0.04
0.33 0.05
>5
6n
6
6
6
0
0
0
0
2
2
0.26 0.05
6o
0.32 0.05
6p
3.21 0.10
10a
10b
10c
10d
13a
13b
>5
>5
>5
>5
>5
>5
6g
6h
6i
6j
O
H
N
6k
6l
OH
N
H
SAHA
0.15 0.02
O
a
All of the compounds were assayed three times, and their inhibition results are means of the three independent assays and expressed with standard deviations.
units, such as 6i–6k and 6m–6o, inhibited HDAC in nanomolar
range, while the rest showed only micromolar activity. In addition,
the phenyl and benzyl substitution in 1,3,4-thiadiazole were more
potent than the substitution of phenethyl and (E)-styryl except for
compound 6d, which exhibited as good enzyme inhibitory activity
as SAHA. In general, the substitutions in 1,3,4-thiadiazole ring have
a small effect on the inhibitory activities against HDAC compared
with the linker between zinc binding group and 1,3,4-thiadiazole
ring.
To further validate the utility of this set of structures at the cel-
lular level, the effect of the exposure of selected compounds was
tested on the viability of MDA-MB-231 breast cancer cell and
K562 chronic myelogenous leukemia cell. We evaluated com-
pounds 6i, 6d, 6j and 6n using MTT assay. The IC50 values were

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P. Guan et al. / Bioorg. Med. Chem. 20 (2012) 3865–3872
Table 2
5.1.1. 5-Phenyl-1,3,4-thiadiazol-2-amino (2a)
Antiproliferative activities of representative compounds against MDA-MB-231 and
K562 cell lines
A stirring mixture of benzoic acid (6.10 g, 50 mmol), N-amin-
othiourea (4.55 g, 50 mmol) and POCl3 (13 ml) was heated at
75 °C for 0.5 h. After cooling down to room temperature, water
(55 ml) was added. The reaction mixture was refluxed for 4 h. After
cooling, the mixture was basified to pH 8 by the dropwise addition
of 50% NaOH solution under stirring. The precipitate was filtered
and recrystallized from ethanol to yield 8.14 g of the target com-
pound 2a as a colorless crystal. Yield: 92%, mp: 224–226 °C. ESI-
MS m/z: 178.2 [M+H]+; 1H NMR (DMSO-d6) d 7.42–7.45 (m, 3H),
7.47 (m, 2H), 7.75 (m, 2H).
a
Compd
IC50 of HDAC (
l
M)
IC50
MDA-MB-231
(lM)
K562
6i
6d
6j
6n
0.089 0.005
0.16 0.03
0.22 0.04
0.26 0.05
0.15 0.02
5.52 2.62
5.90 2.75
6.14 1.94
2.98 0.47
1.32 0.46
12.9 3.18
6.75 2.37
12.2 5.04
9.12 1.10
1.69 0.04
SAHA
a
All of the compounds were assayed three times, and their inhibition results are
means of the three independent assays and expressed with standard deviations.
Compounds 2b–2d were synthesized following the procedure
described above.
summarized in Table 2. Among these compounds, all of them could
inhibit the cell proliferation effectively and compound 6n showed
similar antiproliferative activity against the MDA-MB-231 cell
compared with SAHA. It seems that these compounds possessed
better growth inhibition towards the MDA-MB-231 cell than the
K562 cell.
In order to understand the interaction between these inhibitors
and HDAC, we modeled the three dimensional structure of HDAC1
(HDAC1 and HDAC2 exhibit a sequence identity of approx. 91% in
N-terminal region including the deacetylase catalytic domain) in
the MODELLER program11 following the method in published pa-
per12 and then docked compound 6i and SAHA in the active site
of homology HDAC1 model using AutoDock4.213 (Fig. 2). The result
suggested 6i had a similar binding mode to SAHA in the active site
of HDAC1. Both of them did not only chelate Zn2+ ion with the
hydroxamic acid moiety but also had three hydrogen bonds with
His132, Gln252 and Gly293, respectively. It was worth noting that
the nitrogen atom of 1,3,4-thiadiazole in 6i formed two hydrogen
bonds with Phe197.
5.1.1.1. 5-Benzyl-1,3,4-thiadiazol-2-amine (2b).
Yield: 90%,
mp: 193–195 °C. ESI-MS m/z: 192.4 [M+H]+; 1H NMR (DMSO-d6)
d 4.15 (s, 2H), 7.06 (s, 2H), 7.24–7.28 (m, 3H), 7.34 (m, 2H).
5.1.1.2. 5-Phenethyl-1,3,4-thiadiazol-2-amine (2c).
Yield:
89%, mp: 78–80 °C. ESI-MS m/z: 206.1 [M+H]+; 1H NMR (DMSO-
d6) d 2.95 (t, J = 7.8 Hz, 2H), 3.12 (t, J = 7.8 Hz, 2H), 7.02 (s, 2H),
7.20 (m, 1H), 7.24 (m, 2H), 7.29 (m, 2H).
5.1.1.3. (E)-5-Styryl-1,3,4-thiadiazol-2-amine (2d).
Yield:
81%, mp: 236–238 °C. ESI-MS m/z: 204.2 [M+H]+; 1H NMR
(DMSO-d6) d 7.06 (d, J = 16.2 Hz, 1H), 7.30–7.36 (m, 2H), 7.39 (m,
2H), 7.45 (s, 2H), 7.64 (m, 2H).
5.1.2. 5-Methoxy-5-oxopentanoic acid (4a)
A solution of KOH (5.87 g, 104.65 mmol) in MeOH (150 ml) was
added to dimethyl glutarate (13.15 g, 90 mmol), and the mixture
was stirred for 4 h at rt. The solvent was then removed, and Et2O
(100 ml) and H2O (200 ml) were added. The organic layer was sep-
arated, washed with brine, dried (MgSO4), and concentrated under
reduced pressure to afford 3a as a yellow oil (4.61 g, 32%). The
aqueous layer was acidified with concentrated HCl to pH 3, and ex-
tracted with Et2O (3 Â 100 ml). The combined organic phase was
washed with brine (3 Â 100 ml) and dried (MgSO4). The solvent
was removed to give a mixture of a white solid and an oil. Filtration
and concentration in vacuum and purification with silica gel col-
umn chromatography gave 5.79 g (44%) of 4a as a colorless oil.
4. Conclusions
In summary, we have designed and synthesized a novel series of
1,3,4-thiadiazole based hydroxamic acid HDAC inhibitors which
have different linkers and substitutions in 1,3,4-thiadiazole ring
as the surface recognition motif. The strong enzymatic inhibition
of compound 6d, 6i–6k and 6m–6o suggested that the presence
of 5–6 carbon units between the Zn2+ binding group and the
1,3,4-thiadiazole ring is optimal for potency. These results suggest
that 1,3,4-thiadiazole hydroxic acid derivatives could be used as
lead compounds to develop new potent HDAC inhibitors.
5.1.2.1. 6-Methoxy-6-oxohexanoic acid (4b).
was synthesized following the procedure described above. Yield:
47%.
Compound 4b
5. Experimental section
5.1.2.2. 7-Methoxy-7-oxoheptanoic acid (4c).
Compound 4c
was synthesized following the procedure described in Section
5.1.2. Yield: 40%.
5.1. Chemistry: general procedures
Unless otherwise noted, all starting materials, reagents and sol-
vents were obtained from commercial suppliers and used without
further purification. All reactions except those in aqueous media
were carried out by standard techniques for the exclusion of mois-
ture. All reactions were monitored by thin-layer chromatography
on 0.25 mm silica gel plates (60GF-254) and visualized with UV
light, chloride ferric or iodine vapor. Melting points were deter-
mined on an electrothermal melting point apparatus without cor-
rection. ESI-MS was determined on an Aglient-1100 series LC/MSD
trap spectrometer. 1H NMR spectrums were obtained on a Brucker
DRX spectrometer (600 MHz). The chemical shifts are expressed in
d values (parts per million) relative to tetramethylsilane (TMS) as
internal standard. Significant 1H NMR data are reported in the fol-
lowing order: multiplicity (s, singlet; d, doublet; t, triplet; m, mul-
tiplet) number of protons. HRMS spectrums were conducted on an
Agilent 6510 Quadrupole Time-of-Flight LC/MS deliver.
5.1.2.3. 8-Methoxy-8-oxooctanoic acid (4d).
was synthesized following the procedure described in Section
5.1.2. Yield: 35%.
Compound 4d
5.1.3. Methyl 5-oxo-5-((5-phenyl-1,3,4-thiadiazol-2-
yl)amino)pentanoate (5a)
A soln of 4a (1.46 g, 10 mmol) in SOCl2 (4 ml) was refluxed for
1 h. The SOCl2 was removed under reduced pressure to afford an
orange oil, which was added dropwise to a stirred solution of 2a
(1.42 g, 8 mmol) and Et3N (3.5 ml, 25 mmol) in anhydrous tetrahy-
drofuran (65 ml) at 0 °C. The reaction mixture was allowed to be
stirred overnight at room temperature. The solvent was removed
in vacuum and the residue was diluted with dichloromethane
(150 ml), washed with 1 M H3PO4 (3 Â 80 ml) and brine
(3 Â 80 ml) and dried over with MgSO4. Filtration and concentra-
tion in vacuum and recrystallization from AcOEt gave 1.29 g of

P. Guan et al. / Bioorg. Med. Chem. 20 (2012) 3865–3872
3869
5a as a white crystal. Yield: 53%, mp: 182–185 °C. ESI-MS m/z:
306.4 [M+H]+; 1H NMR (CDCl3) d 2.18–2.23 (m, 2H), 2.56 (t,
J = 7.2 Hz, 2H), 2.93 (t, J = 7.2 Hz, 2H), 3.65 (s, 3H), 7.49–7.50 (m,
3H), 7.94–7.95 (m, 2H), 13.63 (s, 1H).
Compounds 5b–5p were synthesized following the procedure
described above.
5.1.3.10.
yl)amino)heptanoate (5k).
Methyl
7-oxo-7-((5-phenethyl-1,3,4-thiadiazol-2-
Yield: 58%, mp: 112–115 °C. ESI-
MS m/z: 362.5 [M+H]+; 1H NMR (CDCl3) d 1.43–1.48 (m, 2H),
1.67–1.72 (m, 2H), 1.78–1.83 (m, 2H), 2.32 (t, J = 7.5 Hz, 2H), 2.74
(t, J = 7.5 Hz, 2H), 3.13 (t, J = 7.7 Hz, 2H), 3.35 (t, J = 7.7 Hz, 2H),
3.63 (s, 3H), 7.21–7.24 (m, 3H), 7.29–7.32 (m, 2H), 13.32 (s, 1H).
5.1.3.11.
yl)amino)heptanoate (5l).
(E)-Methyl 7-oxo-7-((5-styryl-1,3,4-thiadiazol-2-
5.1.3.1. Methyl 5-((5-benzyl-1,3,4-thiadiazol-2-yl)amino)-5-oxo-
Yield: 51%, mp: 176–178 °C. ESI-
pentanoate (5b).
Yield: 68%, mp: 159–161 °C. ESI-MS m/z:
MS m/z: 360.3 [M+H]+; 1H NMR (CDCl3) d 1.50–1.54 (m, 2H),
1.71–1.76 (m, 2H), 1.83–1.88 (m, 2H), 2.36 (t, J = 7.5 Hz, 2H), 2.81
(t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 7.28 (d, J = 16.2 Hz, 1H), 7.35–7.37
(m, 1H), 7.40–7.43 (m, 3H), 7.55 (m, 2H), 13.27 (s, 1H).
320.3 [M+H]+; 1H NMR (CDCl3) d 2.08–2.13 (m, 2H), 2.47 (t,
J = 7.5 Hz, 2H), 2.81 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 4.33 (s, 2H),
7.28–7.35 (m, 5H), 13.41 (s, 1H).
5.1.3.2.
yl)amino)pentanoate (5c).
Methyl
5-oxo-5-((5-phenethyl-1,3,4-thiadiazol-2-
5.1.3.12.
yl)amino)octanoate (5m).
Methyl
8-oxo-8-((5-phenyl-1,3,4-thiadiazol-2-
Yield: 71%, mp: 131–133 °C. ESI-
Yield: 49%, mp: 157–159 °C. ESI-
MS m/z: 334.4 [M+H]+; 1H NMR (CDCl3) d 2.09–2.14 (m, 2H), 2.48
(t, J = 7.2 Hz, 2H), 2.80 (t, J = 7.2 Hz, 2H), 3.12 (t, J = 8.0 Hz, 2H),
3.34 (t, J = 8.0 Hz, 2H), 3.65 (s, 3H), 7.22–7.24 (m, 3H), 7.30–7.32
(m, 2H), 13.39 (s, 1H).
MS m/z: 348.3 [M+H]+; 1H NMR (CDCl3) d 1.40–1.45 (m, 2H),
1.49–1.54 (m, 2H), 1.63–1.68 (m, 2H), 1.84–1.89 (m, 2H), 2.29 (t,
J = 7.5 Hz, 2H), 2.86 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.49–7.52 (m,
3H), 7.93–7.94 (m, 2H), 13.56 (s, 1H).
5.1.3.3.
yl)amino)pentanoate (5d).
(E)-Methyl
5-oxo-5-((5-styryl-1,3,4-thiadiazol-2-
5.1.3.13. Methyl 8-((5-benzyl-1,3,4-thiadiazol-2-yl)amino)-8-
Yield: 55%, mp: 217–219 °C. ESI-
oxooctanoate (5n).
Yield: 70%, mp: 112–114 °C. ESI-MS m/
MS m/z: 332.3 [M+H]+; 1H NMR (DMSO-d6) d 1.87–1.89 (m, 2H),
2.39 (t, J = 7.5 Hz, 2H), 2.55 (t, J = 7.5 Hz, 2H), 3.60 (s, 3H), 7.37
(m, 1H), 7.41–7.47 (m, 3H), 7.52 (d, J = 16.2 Hz, 1H), 7.72 (m, 2H),
12.59 (s, 1H).
z: 362.4 [M+H]+; 1H NMR (CDCl3) d 1.37–1.45 (m, 4H), 1.60–1.65
(m, 2H), 1.74–1.79 (m, 2H), 2.29 (t, J = 7.2 Hz, 2H), 2.74 (t,
J = 7.2 Hz, 2H), 3.64 (s, 3H), 4.34 (s, 2H), 7.28–7.35 (m, 5H), 13.32
(s, 1H).
5.1.3.4.
yl)amino)hexanoate (5e).
Methyl
6-oxo-6-((5-phenyl-1,3,4-thiadiazol-2-
5.1.3.14.
yl)amino)octanoate (5o).
Methyl
8-oxo-8-((5-phenethyl-1,3,4-thiadiazol-2-
Yield: 65%, mp: 203–205 °C. ESI-
Yield: 56%, mp: 129–130 °C. ESI-
MS m/z: 320.4 [M+H]+; 1H NMR (CDCl3) d 1.82–1.94 (m, 4H), 2.44
(t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 3.65 (s, 3H), 7.48–7.50
(m, 3H), 7.93–7.95 (m, 2H), 13.62 (s, 1H).
MS m/z: 376.5 [M+H]+; 1H NMR (CDCl3) d 1.37–1.46 (m, 4H),
1.61–1.66 (m, 2H), 1.76–1.81 (m, 2H), 2.29 (t, J = 7.5 Hz, 2H), 2.74
(t, J = 7.5 Hz, 2H), 3.13 (t, J = 7.8 Hz, 2H), 3.35 (t, J = 7.8 Hz, 2H),
3.64 (s, 3H), 7.21–7.24 (m, 3H), 7.29–7.32 (m, 2H), 13.34 (s, 1H).
5.1.3.5. Methyl 6-((5-benzyl-1,3,4-thiadiazol-2-yl)amino)-6-oxo-
hexanoate (5f).
Yield: 60%, mp: 138–139 °C. ESI-MS m/z:
5.1.3.15.
yl)amino)octanoate (5p).
(E)-Methyl 8-oxo-8-((5-styryl-1,3,4-thiadiazol-2-
334.4 [M+H]+; 1H NMR (CDCl3) d 1.73–1.84 (m, 4H), 2.39 (t,
J = 7.4 Hz, 2H), 2.77 (t, J = 7.4 Hz, 2H), 3.64 (s, 3H), 4.34 (s, 2H),
7.27–7.35 (m, 5H), 13.40 (s, 1H).
Yield: 69%, mp: 168–170 °C. ESI-
MS m/z: 374.4 [M+H]+; 1H NMR (CDCl3) d 1.39–1.44 (m, 2H),
1.47–1.51 (m, 2H), 1.64–1.69 (m, 2H), 1.81–1.86 (m, 2H), 2.32 (t,
J = 7.2 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 3.64 (s, 3H), 7.27 (t, 1H,
J = 16.8 Hz), 7.41–7.47 (m, 4H), 7.55 (m, 2H), 13.40 (s, 1H).
5.1.3.6.
Methyl
6-oxo-6-((5-phenethyl-1,3,4-thiadiazol-2-
yl)amino)hexanoate (5g).
Yield: 67%, mp: 160–162 °C. ESI-
MS m/z: 348.4 [M+H]+; 1H NMR (CDCl3) d 1.74–1.86 (m, 4H), 2.40
(t, J = 7.2 Hz, 2H), 2.76 (t, J = 7.2 Hz, 2H), 3.13 (t, J = 8.0 Hz, 2H),
3.35 (t, J = 8.0 Hz, 2H), 3.63 (s, 3H), 7.21–7.24 (m, 3H), 7.29–7.32
(m, 2H), 13.39 (s, 1H).
5.1.4. N1-Hydroxy-N5-(5-phenyl-1,3,4-thiadiazol-2-
yl)glutaramide (6a)
Preparation of hydroxylamine in methanol solution: hydroxyl-
amine hydrochloride (4.67 g, 67 mmol) was dissolved in methanol
(24 ml) to form solution A. Potassium hydroxide (5.61 g,
100 mmol) was dissolved in methanol (14 ml) to form solution B.
To the solution A at 0 °C was added solution B dropwise. The mix-
ture was stirred for 30 min at 0 °C, and the solid was filtered out to
afford a solution of hydroxylamine in methanol. To a flask contain-
ing 5a (0.61 g, 2 mmol) was added the solution of hydroxylamine
in methanol (10 ml). The mixture was stirred at room temperature
for 1 h. Then it was adjusted to pH 7 with concentrated HCl. The
mixture was concentrated to give a residue washed with water
and dichloromethane to afford 0.54 g of 6a as a white solid. Yield:
88%, mp: 189–191 °C. 1H NMR (DMSO-d6) d 1.81–1.88 (m, 2H), 2.02
(t, J = 7.3 Hz, 2H), 2.54 (t, J = 7.3 Hz, 2H), 7.52–7.54 (m, 3H), 7.93–
7.95 (m, 2H), 8.72 (s, 1H), 10.39 (s, 1H), 13.64 (s, 1H); HRMS (AP-
ESI) m/z Calcd for C13H14N4O3S [M+H]+ 307.0859. Found: 307.0854.
Compounds 6a–6p were synthesized following the procedure
described above.
5.1.3.7.
(E)-Methyl
6-oxo-6-((5-styryl-1,3,4-thiadiazol-2-
yl)amino)hexanoate (5h).
Yield: 70%, mp: 200–203 °C. ESI-
MS m/z: 346.2 [M+H]+; 1H NMR (CDCl3) d 1.80–1.91 (m, 4H), 2.45
(t, J = 7.4 Hz, 2H), 2.84 (t, J = 7.4 Hz, 2H), 3.66 (s, 3H), 7.25 (d,
J = 16.8 Hz, 1H), 7.34–7.41 (m, 4H), 7.55 (m, 2H), 13.51 (s, 1H).
5.1.3.8.
Methyl
7-oxo-7-((5-phenyl-1,3,4-thiadiazol-2-
yl)amino)heptanoate (5i).
Yield: 69%, mp: 160–162 °C. ESI-
MS m/z: 334.4 [M+H]+; 1H NMR (CDCl3) d 1.51–1.56 (m, 2H),
1.71–1.76 (m, 2H), 1.86–1.91 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H), 2.87
(t, J = 7.5 Hz, 2H), 3.63 (s, 3H), 7.49–7.51 (m, 3H), 7.93–7.94 (m,
2H), 13.58 (s, 1H).
5.1.3.9. Methyl 7-((5-benzyl-1,3,4-thiadiazol-2-yl)amino)-7-
oxoheptanoate (5j).
Yield: 52%, mp: 122–123 °C. ESI-MS m/
z: 348.4 [M+H]+; 1H NMR (CDCl3) d 1.42–1.47 (m, 2H), 1.66–1.71
(m, 2H), 1.76–1.81 (m, 2H), 2.31 (t, J = 7.5 Hz, 2H), 2.74 (t,
J = 7.5 Hz, 2H), 3.64 (s, 3H), 4.34 (s, 2H), 7.28–7.35 (m, 5H), 13.27
(s, 1H).
5.1.4.1. N1-(5-Benzyl-1,3,4-thiadiazol-2-yl)-N5-hydroxyglutara-
mide (6b).
d 1.76–1.81 (m, 2H), 1.96 (t, J = 7.3 Hz, 2H), 2.44 (t, J = 7.3 Hz,
Yield: 91%, mp: 168–170 °C. 1H NMR (DMSO-d6)

3870
P. Guan et al. / Bioorg. Med. Chem. 20 (2012) 3865–3872
2H), 4.33 (s, 2H), 7.26–7.37 (m, 5H), 8.69 (s, 1H), 10.35 (s, 1H),
12.40 (s, 1H); HRMS (AP-ESI) m/z Calcd for C14H16N4O3S [M+H]+
321.1016. Found: 321.1025.
1H), 12.29 (br s, 1H); HRMS (AP-ESI) m/z Calcd for C17H22N4O3S
[M+H]+ 363.1485. Found: 363.1489.
5.1.4.11. (E)-N1-Hydroxy-N7-(5-styryl-1,3,4-thiadiazol-2-yl)hep-
5.1.4.2. N1-Hydroxy-N5-(5-phenethyl-1,3,4-thiadiazol-2-yl)glut-
tanediamide (6l).
Yield: 90%, mp: 198–200 °C. 1H NMR
aramide (6c).
Yield: 76%, mp: 166–167 °C. 1H NMR (DMSO-
(DMSO-d6) d 1.23–1.30 (m, 2H), 1.47–1.55 (m, 2H), 1.57–1.65 (m,
2H), 1.95 (t, J = 7.3 Hz, 2H), 2.49 (t, J = 7.3 Hz, 2H), 7.36 (m, 1H),
7.40–7.47 (m, 3H), 7.71 (d, J = 16.5 Hz, 2H), 7.71 (m, 2H), 8.67 (s,
1H), 10.33 (s, 1H), 12.55 (s, 1H); HRMS (AP-ESI) m/z Calcd for
d6) d 1.78–1.82 (m, 2H), 1.99 (t, J = 7.3 Hz, 2H), 2.45 (t, J = 7.3 Hz,
2H), 3.03 (t, J = 7.7 Hz, 2H), 3.29 (t, J = 7.7 Hz, 2H), 7.18–7.31 (m,
5H), 8.68 (s, 1H), 10.36 (s, 1H), 12.33 (s, 1H); HRMS (AP-ESI) m/z
Calcd for C15H18N4O3S [M+H]+ 335.1172. Found: 335.1172.
C
17H20N4O3S [M+H]+ 361.1329. Found: 361.1338.
5.1.4.3. (E)-N1-Hydroxy-N5-(5-styryl-1,3,4-thiadiazol-2-yl)glut-
aramide (6d).
5.1.4.12. N1-Hydroxy-N8-(5-phenyl-1,3,4-thiadiazol-2-yl)octane-
diamide (6m).
Yield: 91%, mp: 204–205 °C. 1H NMR (DMSO-
Yield: 89%, mp: 238–242 °C. 1H NMR (DMSO-
d6) d 1.80–1.87 (m, 2H), 2.02 (t, J = 7.3 Hz, 2H), 2.53 (t, J = 7.3 Hz,
2H), 7.36 (m, 1H), 7.40–7.47 (m, 3H), 7.52 (d, J = 16.1 Hz, 1H),
7.71 (m, 2H), 8.72 (s, 1H), 10.39 (s, 1H), 12.56 (s, 1H); HRMS (AP-
ESI) m/z Calcd for C15H16N4O3S [M+H]+ 333.1016. Found: 333.1016.
d6) d 1.26–1.28 (m, 4H), 1.45–1.52 (m, 2H), 1.58–1.63 (m, 2H),
1.94 (t, J = 7.4 Hz, 2H), 2.48 (t, J = 7.4 Hz, 2H), 7.51–7.56 (m, 3H),
7.92–7.95 (m, 2H), 8.67 (s, 1H), 10.34 (s, 1H), 12.63 (s, 1H); HRMS
(AP-ESI) m/z Calcd for C16H20N4O3S [M+H]+ 349.1329. Found:
349.1336.
5.1.4.4. N1-Hydroxy-N6-(5-phenyl-1,3,4-thiadiazol-2-yl)adipa-
mide (6e).
Yield: 90%, mp: 192–196 °C. 1H NMR (DMSO-d6)
5.1.4.13. N1-(5-Benzyl-1,3,4-thiadiazol-2-yl)-N8-hydroxyoctane-
d 1.52–1.64 (m, 4H), 1.98 (t, J = 7.0 Hz, 2H), 2.52 (t, J = 7.0 Hz,
2H), 7.51–7.54 (m, 3H), 7.91–7.94 (m, 2H), 8.57 (s, 1H), 10.28 (s,
1H), 12.52 (s, 1H); HRMS (AP-ESI) m/z Calcd for C14H16N4O3S
[M+H]+ 321.1016. Found: 321.1024.
diamide (6n).
Yield: 70%, mp: 161–163 °C. 1H NMR (DMSO-
d6) d 1.22–1.23 (m, 4H), 1.44–1.47 (m, 2H), 1.53–1.56 (m, 2H),
1.92 (t, J = 7.4 Hz, 2H), 2.41 (t, J = 7.4 Hz, 2H), 4.33 (s, 2H), 7.25–
7.37 (m, 5H), 8.64 (s, 1H), 10.31 (s, 1H), 12.37 (br s, 1H); HRMS
(AP-ESI) m/z Calcd for C17H22N4O3S [M+H]+ 363.1485. Found:
363.1495.
5.1.4.5.
mide (6f).
N1-(5-Benzyl-1,3,4-thiadiazol-2-yl)-N6-hydroxyadipa-
Yield: 87%, mp: 184–185 °C. 1H NMR (DMSO-d6)
d 1.44–1.57 (m, 4H), 1.94 (t, J = 7.0 Hz, 2H), 2.42 (t, J = 7.0 Hz,
2H), 7.26–7.36 (m, 5H), 8.65 (s, 1H), 10.33 (s, 1H), 12.36 (s, 1H);
HRMS (AP-ESI) m/z Calcd for C15H18N4O3S [M+H]+ 335.1172.
Found: 335.1179.
5.1.4.14.
yl)octanediamide (6o).
N1-Hydroxy-N8-(5-phenethyl-1,3,4-thiadiazol-2-
Yield: 74%, mp: 165–166 °C. 1H
NMR (DMSO-d6) d 1.24–1.25 (m, 4H), 1.44–1.51 (m, 2H), 1.53–
1.58 (m, 2H), 1.93 (t, J = 7.4 Hz, 2H), 2.42 (t, J = 7.4 Hz, 2H), 3.03
(t, J = 7.7 Hz, 2H), 3.28 (t, J = 7.7 Hz, 2H), 7.17–7.31 (m, 5H), 8.63
(br s, 1H), 10.31 (br s, 1H), 12.30 (br s, 1H); HRMS (AP-ESI) m/z
Calcd for C18H24N4O3S [M+H]+ 377.1642. Found: 377.1647.
5.1.4.6. N1-Hydroxy-N6-(5-phenethyl-1,3,4-thiadiazol-2-yl)adi-
pamide (6g).
Yield: 94%, mp: 186–188 °C. 1H NMR (DMSO-
d6) d 1.47–1.59 (m, 4H), 1.95 (t, J = 7.0 Hz, 2H), 2.43 (t, J = 7.0 Hz,
2H), 3.03 (t, J = 7.7 Hz, 2H), 3.29 (t, J = 7.7 Hz, 2H), 7.18–7.31 (m,
5H), 8.65 (s, 1H), 10.33 (s, 1H), 12.31 (s, 1H); HRMS (AP-ESI) m/z
Calcd for C16H20N4O3S [M+H]+ 349.1329. Found: 349.1321.
5.1.4.15.
yl)octanediamide (6p).
(E)-N1-Hydroxy-N8-(5-styryl-1,3,4-thiadiazol-2-
Yield: 95%, mp: 206–207 °C. 1H
NMR (DMSO-d6) d 1.28–1.29 (m, 4H), 1.46–1.53 (m, 2H), 1.58–
1.65 (m, 2H), 1.94 (t, J = 7.3 Hz, 2H), 2.47 (t, J = 7.3 Hz, 2H), 7.35
(m, 1H), 7.40–7.45 (m, 3H), 7.49 (d, J = 16.5 Hz, 1H), 7.69 (m, 2H),
8.54 (s, 1H), 10.25 (s, 1H), 12.44 (s, 1H); HRMS (AP-ESI) m/z Calcd
for C18H22N4O3S [M+H]+ 375.1485. Found: 375.1487.
5.1.4.7. (E)-N1-Hydroxy-N6-(5-styryl-1,3,4-thiadiazol-2-yl)adipa-
mide (6h).
Yield: 84%, mp: 210–213 °C. 1H NMR (DMSO-d6) d
1.50–1.64 (m, 4H), 1.98 (t, J = 7.1 Hz, 2H), 2.47 (t, J = 7.1 Hz, 2H),
7.36 (m, 1H), 7.40–7.46 (m, 3H), 7.51 (d, J = 16.1 Hz, 1H), 7.71 (m,
2H), 8.66 (s, 1H), 10.35 (s, 1H), 12.49 (s, 1H); HRMS (AP-ESI) m/z
Calcd for C16H18N4O3S [M+H]+ 347.1172. Found: 347.1179.
5.1.5. Ethyl 2-chloro-2-oxoacetate (8)
A mixture of potassium acetate (20 g), water (30 ml) and diethyl
oxalate (29.2 g, 0.2 mol) was stirred at 70–80 °C for 2 h. The reac-
tion mixture was cooled and condensed to 30 ml. After ethanol
(50 ml) and diethyl ether (150 ml) were added, the solution was
filtrated to give 23.61 g of potassium monoethyl oxalate (76%).
SOCl2 (30 g, 0.25 mol) was added slowly to this dry potassium salt
(20.6 g, 0.13 mol) previously mixed with diethyl ether (20 ml) in an
ice bath. The mixture was then heated under reflux for 15 h. The
white solid of produced potassium chloride was filtrated, the fil-
trate was fractionated and a fraction of 125–130 °C was collected
to give 12.22 g of 8 (69%).
5.1.4.8.
tanediamide (6i).
N1-Hydroxy-N7-(5-phenyl-1,3,4-thiadiazol-2-yl)hep-
Yield: 80%, mp: 172–174 °C. 1H NMR
(DMSO-d6) d 1.23–1.31 (m, 2H), 1.47–1.53 (m, 2H), 1.58–1.65 (m,
2H), 1.95 (t, J = 7.3 Hz, 2H), 2.47 (t, J = 7.3 Hz, 2H), 7.52–7.55 (m,
3H), 7.92–7.95 (m, 2H), 8.68 (s, 1H), 10.34 (s, 1H), 12.60 (s, 1H);
HRMS (AP-ESI) m/z Calcd for C15H18N4O3S [M+H]+ 335.1172.
Found: 335.1178.
5.1.4.9. N1-(5-Benzyl-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptane-
diamide (6j).
Yield: 79%, mp: 247–249 °C. 1H NMR (DMSO-
d6) d 1.20–1.25 (m, 2H), 1.45–1.57 (m, 4H), 1.92 (t, J = 7.3 Hz,
2H), 2.41 (t, J = 7.3 Hz, 2H), 4.33 (s, 2H), 7.25–7.39 (m, 5H), 8.65
(s, 1H), 10.31 (s, 1H), 12.37 (br s, 1H); HRMS (AP-ESI) m/z Calcd
for C16H20N4O3S [M+H]+ 349.1329. Found: 349.1325.
5.1.6. Ethyl 2-oxo-2-((5-phenyl-1,3,4-thiadiazol-2-
yl)amino)acetate (9a)
Eight (1.36 g, 10 mmol) was added dropwise to a stirred solu-
tion of 2a (1.77 g, 10 mmol) and Et3N (3.5 ml, 25 mmol) in anhy-
drous tetrahydrofuran (65 ml) at 0 °C. The reaction mixture was
allowed to be stirred overnight at room temperature. The solvent
was removed in vacuum and the residue was diluted with dichlo-
romethane (150 ml), washed with 1M H3PO4 (3 Â 80 ml) and brine
(3 Â 80 ml) and dried over with MgSO4. Filtration and concentra-
tion in vacuum and recrystallization from methanol gave 1.41 g
5.1.4.10.
yl)heptanediamide (6k).
N1-Hydroxy-N7-(5-phenethyl-1,3,4-thiadiazol-2-
Yield: 84%, mp: 176–177 °C. 1H
NMR (DMSO-d6) d 1.20–1.28 (m, 2H), 1.45–1.61 (m, 4H), 1.93 (t,
J = 7.3 Hz, 2H), 2.42 (t, J = 7.3 Hz, 2H), 3.03 (t, J = 7.8 Hz, 2H), 3.28
(t, J = 7.8 Hz, 2H), 7.17–7.31 (m, 5H), 8.65 (br s, 1H), 10.31 (br s,

P. Guan et al. / Bioorg. Med. Chem. 20 (2012) 3865–3872
3871
of 5a as a white crystal. Yield: 51%, mp: 194–196 °C. ESI-MS m/z:
304.3 [M+H]+; 1H NMR (DMSO-d6) d 1.35 (t, J = 7.2 Hz, 3H), 4.34–
4.37 (m, 2H), 7.44–7.58 (m, 3H), 7.98–8.00 (m, 2H), 13.68 (br s,
1H).
MS m/z: 304.3 [M+H]+; 1H NMR (DMSO-d6) d 2.61 (t, J = 6.6 Hz,
2H), 2.75 (t, J = 6.6 Hz, 2H), 7.37 (m, 1H), 7.41–7.46 (m, 3H), 7.52
(d, J = 16.8 Hz, 1H), 7.72 (m, 2H), 12.30 (br s, 1H), 12.63 (s, 1H).
5.1.9. N1-Hydroxy-N4-(5-phenyl-1,3,4-thiadiazol-2-
5.1.6.1.
Ethyl
2-((5-benzyl-1,3,4-thiadiazol-2-yl)amino)-2-
yl)succinamide (13a)
oxoacetate (9b).
Yield: 58%, mp: 131–133 °C. ESI-MS m/z:
To a À20 °C cooled solution of 12a (2.12 g, 7 mmol) and N-
methylmorpholine (1.6 ml, 14 mmol) in anhydrous N,N-dimethyl-
formamide (15 ml) was added ClCOOBu-i (1.1 ml, 8.4 mmol), and
the mixture was stirred for 0.5 h. The solid was filtered out and fil-
trate was added to freshly prepared NH2OK in methanol (5 ml,
1.54 mol/L). The resulting mixture was stirred at room tempera-
ture overnight, then was filtered and the residue was washed with
water to give 1.29 g of 13a as a white solid. Yield: 63%, mp: 197–
198 °C. 1H NMR (DMSO-d6) d 2.35 (t, J = 6.8 Hz, 2H), 2.75 (t,
J = 6.8 Hz, 2H), 7.53–7.55 (m, 3H), 7.93–7.94 (m, 2H), 8.76 (s, 1H),
10.47 (s, 1H), 12.68 (s, 1H); HRMS (AP-ESI) m/z Calcd for
292.4 [M+H]+; 1H NMR (DMSO-d6) d 1.30 (t, J = 7.2 Hz, 3H), 4.29–
4.32 (m, 2H), 4.42 (s, 2H), 7.27–7.39 (m, 5H), 13.48 (br s, 1H).
5.1.6.2.
Ethyl
2-oxo-2-((5-phenethyl-1,3,4-thiadiazol-2-
yl)amino)acetate (9c).
Yield: 64%, mp: 135–138 °C. ESI-MS
m/z: 306.4 [M+H]+; 1H NMR (DMSO-d6) d 1.31 (t, J = 7.2 Hz, 3H),
3.06 (t, J = 7.8 Hz, 2H), 3.35 (t, J = 7.8 Hz, 2H), 4.29–4.33 (m, 2H),
4.42, 7.19–7.31 (m, 5H), 13.40 (br s, 1H).
5.1.6.3.
(E)-Ethyl
2-oxo-2-((5-styryl-1,3,4-thiadiazol-2-
Yield: 48%, mp: 191–193 °C. ESI-MS
yl)amino)acetate (9d).
C
12H12N4O3S [M+H]+ 293.0703. Found: 293.0698.
m/z: 304.3 [M+H]+; 1H NMR (DMSO-d6) d 1.34 (t, J = 7.2 Hz, 3H),
7.94–7.95 (m, 2H), 7.38–7.39 (m, 1H), 7.42–7.45 (m, 2H), 7.56
(m, 2H), 7.74–7.75 (m, 2H), 13.60 (s, 1H).
5.1.9.1. (E)-N1-Hydroxy-N4-(5-styryl-1,3,4-thiadiazol-2-yl)succ
inamide (13b). Compound 12b was synthesized following
the procedure described above. Yield: 49%, mp: 196–197 °C. 1H
NMR (DMSO-d6) d 2.35 (t, J = 7.0 Hz, 2H), 2.74 (t, J = 7.0 Hz, 2H),
7.36 (m, 1H), 7.40–7.45 (m, J = 16.3 Hz, 3H), 7.51 (d, J = 16.3 Hz,
1H), 7.71 (m, 2H), 8.73 (s, 1H), 10.45 (s, 1H), 12.57 (br s, 1H); HRMS
(AP-ESI) m/z Calcd for C14H14N4O3S [M+H]+ 319.0859. Found:
319.0853.
5.1.7. N1-Hydroxy-N2-(5-phenyl-1,3,4-thiadiazol-2-yl)oxalamide
(10a)
To a flask containing 9a (0.55 g, 2 mmol) was added the solution
of hydroxylamine in methanol (10 ml). The mixture was stirred at
room temperature for 0.5 h. The precipitate was filtrated out and
washed with water and dichloromethane to afford 0.82 g of 10a
as a white solid. Yield: 88%, mp: 192–193 °C. 1H NMR (DMSO-d6)
d 7.53–7.56 (m, 3H), 7.96–7.99 (m, 2H), 9.57 (s, 1H), 11.93 (s,
1H), 13.23 (br s, 1H); HRMS (AP-ESI) m/z Calcd for C10H8N4O3S
[M+H]+ 265.0390. Found: 265.0395.
5.2. In vitro HDAC assay
We performed assays according to the kit instruction. HDAC
came from HeLa cell nucleus extracts, mainly including HDAC1
and HDAC2, and the substrate was a type of [3H] acetylated histone
peptide. The tested compounds and the control drug SAHA were
diluted to various concentrations. On the 96-well plate, HDAC
5.1.7.1.
mide (10b).
N1-(5-Benzyl-1,3,4-thiadiazol-2-yl)-N2-hydroxyoxala-
Yield: 91%, mp: 174–175 °C. 1H NMR (DMSO-
d6) d 4.39 (s, 2H), 7.26–7.37 (m, 5H), 9.52 (s, 1H), 11.84 (s, 1H),
12.95 (br s, 1H); HRMS (AP-ESI) m/z Calcd for C11H10N4O3S
[M+H]+ 279.0546. Found: 279.0551.
(5
trations of samples and 25
30 min, Color de Lys Developer (50
l
L/well) were incubated at 37 °C with 10
L of substrate. After reacting for
L/well) was added. Then, after
lL of various concen-
l
l
15 min the ultraviolet absorption of the wells was measured on a
microtiter-plate reader at 405 nm. The inhibition rates were calcu-
lated from the ultraviolet absorption readings of inhibited wells re-
lated to those of control wells. Finally, the IC50 values were
determined using a regression analysis of the concentration/inhibi-
tion data.
5.1.7.2.
yl)oxalamide (10c).
(DMSO-d6) d 3.05 (t, J = 7.6 Hz, 2H), 3.35 (t, J = 7.6 Hz, 2H), 7.18–
7.31 (m, 5H), 9.52 (br s, 1H), 11.85 (br s, 1H), 12.90 (br s, 1H);
HRMS (AP-ESI) m/z Calcd for C12H12N4O3S [M+H]+ 293.0703.
Found: 293.0707.
N1-Hydroxy-N2-(5-phenethyl-1,3,4-thiadiazol-2-
Yield: 89%, mp: 164–166 °C. 1H NMR
5.3. MTT Assay
5.1.7.3. (E)-N1-Hydroxy-N2-(5-styryl-1,3,4-thiadiazol-2-yl)oxala-
mide (10d).
Yield: 93%, mp: 195–196 °C. 1H NMR (DMSO-d6)
MDA-MB-231 and K562 cells were respectively cultured in
RPMI1640 medium containing 10% FBS at 37 °C in 5% CO2 humid-
ified incubator. Cell proliferation was determined by the MTT (3-
[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide)
assay. Briefly, cells were plated in a 96-well plate at 10,000 cells
per well, cultured for 4 h in complete growth medium, then treated
with 2000, 400, 80, 16, 3.2, 0.64 mg/mL of compounds for 48 h.
0.5% MTT solution was added to each well. After further incubation
for 4 h, formazan formed from MTT was extracted by adding
d 7.37 (m, 1H), 7.43 (m, 2H), 7.51 (d, J = 16.5 Hz, 1H), 7.57 (d,
J = 16.5 Hz, 1H), 7.73 (m, 2H), 9.56 (br s, 1H), 11.91 (s, 1H), 13.14
(br s, 1H); HRMS (AP-ESI) m/z Calcd for C12H10N4O3S [M+H]+
291.0546. Found: 291.0552.
5.1.8. 4-Oxo-4-((5-phenyl-1,3,4-thiadiazol-2-yl)amino)butanoic
acid (12a)
Succinic anhydride (2 g, 0.02 mol) was added to a solution of 2a
(3.54 g, 0.02 mol) in hot acetonitrile (150 ml). The solution was
heated for 1 h under reflux. After cooling, the precipitated product
was collected to give 2.88 g of 12a as a white solid. Yield: 52%, mp:
267–270 °C. ESI-MS m/z: 278.3 [M+H]+; 1H NMR (DMSO-d6) d 2.60
(t, J = 6.6 Hz, 2H), 2.74 (t, J = 6.6 Hz, 2H), 7.53–7.55 (m, 3H), 7.93–
7.94 (m, 2H), 12.25 (s, 1H), 12.70 (s, 1H).
200 lL DMSO and mixing for 15 min. Optical density was read with
an microtiter-plate reader at 570 nm.
5.4. Docking study
AutoDock 4.2 was used for all docking calculations. One hun-
dred runs were performed using Lamarckian Genetic Algorithm
(LGA) with default parameters. The molecular structures were gen-
erated with Sybyl/Sketch module and optimized using semi-
empirical MOPAC/AM1 method and then assigned with AM1-BCC
5.1.8.1. 4-Oxo-4-((5-phenyl-1,3,4-thiadiazol-2-yl)amino)buta-
noic acid (12b).
Compound 11b was synthesized following
the procedure described above. Yield: 59%, mp: 228–232 °C. ESI-

3872
P. Guan et al. / Bioorg. Med. Chem. 20 (2012) 3865–3872
charges. Results differing less than 0.5 Å in positional root-mean-
square deviation (RMSD) were clustered together. Conformations
in first cluster with the most favorable free binding energy were
selected as the best docking result.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
1. Minucci, S.; Pelicci, P. G. Nat. Rev. Cancer 2006, 6, 38.
2. Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug Disc. 2006, 5, 769.
3. Venugopal, B.; Evans, T. R. Curr. Med. Chem. 2011, 18, 1658.
4. Marks, P. A.; Breslow, R. Nat. Biotechnol. 2007, 25, 84.
5. VanderMolen, K. M.; McCulloch, W.; Pearce, C. J.; Oberlies, N. H. J. Antibiot.
2011, 64, 525.
6. Jiao, J.; Fang, H.; Wang, X.; Guan, P.; Yuan, Y.; Xu, W. Eur. J. Med. Chem. 2009, 44,
4470.
7. Zhang, Y.; Feng, J.; Liu, C.; Zhang, L.; Jiao, J.; Fang, H.; Su, L.; Zhang, X.; Zhang, J.;
Li, M.; Wang, B.; Xu, W. Bioorg. Med. Chem. 2010, 18, 1761.
8. Zhang, Y.; Feng, J.; Jia, Y.; Wang, X.; Zhang, L.; Liu, C.; Fang, H.; Xu, W. J. Med.
Chem. 2011, 54, 2823.
Acknowledgments
This work was supported by this work was supported by Na-
tional Natural Foundation Research Grant (Grant No. 21172133),
Shandong Provincial Natural Science Foundation (Grant No.
ZR2010HM028), Shandong Natural Science Fund for Distinguished
Young Scholars (Grant No. JQ201121) and Independent Innovation
Foundation of Shandong University (IIFSDU, Grant No. 2010JC17)
in China.
9. Zhang, Y.; Fang, H.; Feng, J.; Jia, Y.; Wang, X.; Xu, W. J. Med. Chem. 2011, 54,
5532.
10. Qian, C. C.; Xiong; Zhai, Haixiao. WO/2009/036016, 2009.
11. Fiser, A.; Sali, A. Methods Enzymol. 2003, 374, 461.
12. Wang, D. F.; Helquist, P.; Wiech, N. L.; Wiest, O. J. Med. Chem. 2005, 48, 6936.
13. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D.
S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.032.