Improved antiproliferative activity of 1,3,4-thiadiazole-containing histone deacetylase (HDAC) inhibitors by introduction of the heteroaromatic surface recognition motif

Article abstract of DOI:10.1016/j.bmc.2014.09.039

A series of 1,3,4-thiadiazole-containing hydroxamic acids, in accord with the common pharmacophore of histone deacetylase (HDAC) inhibitors (a Zn²⁺ binding moiety-a linker-a surface recognition motif), was identified as submicromolar HDAC inhibitors by our group. In this study, we continued our efforts to develop 1,3,4-thiadiazole bearing hydroxamate analogues by modifying the surface recognition motif. We found that 1,3,4-thiadiazoles having a heteroaromatic substituent showed better HDAC inhibitory activity in enzymatic assay and higher antiproliferative potency in cellular assay compared to SAHA.

Full text of DOI:10.1016/j.bmc.2014.09.039

Accepted Manuscript
Improved antiproliferative activity of 1,3,4-thiadiazole-containing histone de-
acetylase (HDAC) inhibitors by introduction of the heteroaromatic surface
recognition motif
Peng Guan, Lei Wang, Xuben Hou, Yichao Wan, Wenfang Xu, Weiping Tang,
Hao Fang
PII:
S0968-0896(14)00685-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.09.039
BMC 11826
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 July 2014
17 September 2014
18 September 2014
Please cite this article as: Guan, P., Wang, L., Hou, X., Wan, Y., Xu, W., Tang, W., Fang, H., Improved
antiproliferative activity of 1,3,4-thiadiazole-containing histone deacetylase (HDAC) inhibitors by introduction of
the heteroaromatic surface recognition motif, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/
10.1016/j.bmc.2014.09.039
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Improved antiproliferative activity of 1,3,4-thiadiazole-containing histone
deacetylase (HDAC) inhibitors by introduction of the heteroaromatic surface
recognition motif
Peng Guana, Lei Wanga, Xuben Houa, Yichao Wana, Wenfang Xua, Weiping Tangb,
Hao Fanga∗
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmacy, Shandong University, Jinan, Shandong, 250012,
P.R. China
bDivision of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin,
Madison 53705, USA.
Abstract
A series of 1,3,4-thiadiazole-containing hydroxamic acids, in accord with the
common pharmacophore of histone deacetylase (HDAC) inhibitors (a Zn2+ binding
moiety-a linker-a surface recognition motif), was identified as submicromolar HDAC
inhibitors by our group. In this study, we continued our efforts to develop
1,3,4-thiadiazole bearing hydroxamate analogues by modifying the surface
recognition motif. We found that 1,3,4-thiadiazoles having a heteroaromatic
substituent showed better HDAC inhibitory activity in enzymatic assay and higher
antiproliferative potency in cellular assay compared to SAHA.
Keywords: 1,3,4-thiadiazole; hydroxamic acid; histone deacetylase inhibitor; surface
recognition motif; antiproliferation
1. Introduction
Histone deactylases are a family of epigenetic enzymes which catalyze the removal
of acetyl moieties from N-acetyllysine residues of histones and non-histone proteins,
many of which are responsible for regulation of gene expression.1 Histone
deacetylases have been found mechanistically linked to tumor onset and progression，
the inhibition of which induces cell cycle or growth arrest, differentiation and
apoptosis of malignant cells.2 Histone deactylase (HDAC) inhibitors demonstrated
prominent antitumor efficacy on broad spectrum neoplasms in preclinical and clinical
studies.2,3 Strikingly, vorinostat (suberoylanilide hydroxamic acid, SAHA)4 and
romidepsin (depsipeptide)5 were approved for the treatment of cutaneous T-cell
lymphoma by FDA in 2006 and 2009, respectively. (Figure 1)
∗ Corresponding author. Phone: +86-531-88382731. Fax: +86-531-88382548. E-mail: haofangcn@sdu.edu.cn.
1

Figure 1. Chemical structures of SAHA and depsipeptide.
In general, the pharmacophore for HDAC inhibitors is composed of a Zn2+ binding
moiety, a linker, and a surface recognition motif. (Figure 2) In the prior work, we
reported the discovery of 1,3,4-thiadiazole based hydroxamates as submicromolar
HDAC inhibitors, which are comparable to SAHA.6 (Figure 2) The linker length (n)
and the surface recognition motif (R) were systematically varied to find the optimum
geometric parameters. The structure-activity relationship (SAR) indicated that the
presence of 5 or 6 carbon units between the hydroxamate group and the
1,3,4-thiadiazole ring (n = 5 or 6) is optimal for enzymatic efficacy. Compound 1,
containing a phenyl in the surface recognition domain (R = phenyl), showed
approximately 2-fold greater potency against HDAC enzymes than SAHA. However,
none of these active HDAC inhibitors outperformed SAHA as an antiproliferative
agent of cancer cells. In an effort to increase the proliferative inhibition of
1,3,4-thiadiazole bearing hydroxamates, while maintaining n as 5 or 6, we
investigated the embellishment and replacement of the surface recognition motif
phenyl in 1.
Figure 2. Pharmacophoric characteristics of HDAC inhibitors instantiated by the
chemical structure of 1,3,4-thiadiazole-containing hydroxamates.
2. Chemistry
The synthesis of the 1,3,4-thiadiazole bearing hydroxamic acids is shown in
Scheme 1. The 5-substituted-1,3,4-thiadiazol-2-amines 59-81 were prepared by a
facile one-pot reaction of the corresponding carboxylic acids, N-aminothiourea, and
phosphoryl chloride. Saponification of dimethylesters 82-83 furnished the
2

monomethyl esters 84-85 in good yields. Sequential treatment of 84-85 with refluxing
thionyl chloride and then 59-81 gave 86-119, which were converted to hydroxamic
acids 2-35 according to reported procedures by treating with freshly prepared
potassium hydroxylamine.7
Scheme 1. Reagents and conditions: (a) NH2NHCSNH2, POCl3, 75 °C, 0.5 h then
H2O, reflux, 4 h; (b) KOH, CH3OH, 0 °C to rt, 4 h; (c) i) SOCl2, reflux, 2 h; ii) Et3N,
THF, 0 °C to rt, overnight; (d) NH2OH·HCl, KOH, CH3OH, rt,1 h.
3. Results and Discussion
First, we probed the effect of positional and structural modifications of the phenyl
on the potency in enzymatic assays. The inhibitory data of total HDAC activity in
HeLa nuclear extract in comparison with SAHA as a positive control were compiled
in Tables 1. Unfortunately, this class of derivatives was all less active towards HDAC
enzymes than SAHA, suggesting substitution of the phenyl gave no advantage in
terms of enzymatic inhibition. However, some interesting trends can be observed: 1)
addition of a bulky group on the phenyl led to a significant loss in HDAC inhibitory
activity; 2) compounds with an electron-withdrawing group exhibited generally
poorer HDAC inhibition than those with an electron-donating group. Additionally, a
tendency in enzymatic potency of para-substitution < meta-substitution <
ortho-substitution was noted.
Given that the phenyl-substituted derivatives did not display higher enzymatic
potency than SAHA, we turned our attention to a set of alternative aromatic rings.
Replacement of the phenyl with a naphthalenyl group resulted in a considerable loss
in HDAC inhibition, while heteroaromatic analogues possessed better or similar
activity compared to SAHA. Compounds 33 and 35 showed the best activity, with
IC50 values of approximately 300 nM, suggesting a preference for a heteroaromatic
ring adjacent to the 1,3,4-thiadiazole ring as a surface recognition motif. Pyridine- and
thiophene- containing compounds had improved HDAC inhibitory efficacy in
comparison to SAHA.
Table 1. HDAC inhibitory activity of 1,3,4-thiadiazole hydroxamic acid derivatives.
3

IC50 of HDACs
(nM)a
compd
R
n
4-morpholino-Ph
4-N(CH3)2-Ph
4-OCH3-Ph
3,4-OCH3-Ph
4-CH3-Ph
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
5
5
5
5
5
5
6
6
6
6
6
6
>5000
>5000
2
3
437 55
487 56
422 53
>5000
4
5
6
(1,1'-biphenyl)-4-yl
4-F-Ph
7
741 52
988 81
847 43
507 19
1285 127
875 45
721 55
709 40
1154 92
>5000
8
4-Cl-Ph
9
3-Cl-Ph
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
SAHA
2-Cl-Ph
4-Br-Ph
4-I-Ph
4-SO2CH3-Ph
4-NO2-Ph
4-CF3-Ph
4-morpholino-Ph
(1,1'-biphenyl)-4-yl
3-Br-Ph
>5000
1215 297
526 42
>5000
2-Br-Ph
4-I-Ph
4-SO2CH3-Ph
4-CF3-Ph
598 51
>5000
naphthalen-1-yl
naphthalen-2-yl
pyridin-3-yl
pyridin-4-yl
furan-2-yl
3186 475
>5000
325 33
411 39
434 33
411 43
>5000
thiophen-2-yl
naphthalen-1-yl
naphthalen-2-yl
pyridin-3-yl
pyridin-4-yl
furan-2-yl
>5000
386 53
286 8
436 53
310 42
416 20
thiophen-2-yl
4

a Inhibitory data are means of three independent determinations and expressed with
standard deviations.
Recently, genetic studies suggested that HDAC1 and HDAC2 should be most
closely related to tumor cell survival.8 Therefore inhibition of HDAC1 and HDAC2
might be helpful to block cancer cell proliferation. In our studies, the enzyme for
HDAC inhibitory assay came from HeLa nuclear extracts which mainly include
HDAC1 and HDAC2. Considering that the crystal structure of HDAC2 and SAHA
complex has been reported (PDB code 4LXZ), further molecular docking was
performed using most active compound 33 and the program of Surflex-Dock (Figure
3). The result showed that the hydroxamic acid of compound 33 could chelate Zn2+
ion in the active site of HDAC2, which indicated that compound 33 could have a
similar binding mode to SAHA. On the other hand, 1,3,4-thiadiazole of 33 could form
additional π-π interaction with Tyr209 and hydrogen bond with Phe210, which
suggested that interactions between the surface recognition motif and HDAC may
lead to the better potency of compound 33 compared with SAHA.
Figure 3. (A) Docked compound 33 (depicted in atom type) into the crystal structure
of HDAC2 complexed with SAHA (depicted in red). (B) Binding mode of compound
33 in the active site of HDAC2.
To further ascertain the utility of heteroaromatic HDAC inhibitors at the cellular
level, the efficacy of pyridine and thiophene analogues on the viability of cancer cells
was measured by MTT assay. Table 2 showed their IC50 values and SAHA was used
as a reference. The 6 carbon unit linker (compound 32, 33 and 35) afforded
appreciably potent cell growth inhibition in the same order of magnitude with SAHA.
It was noteworthy that the thiophene variant 35 exerted higher antiproliferative
activity toward all three cell lines than SAHA. In addition, the pyridine derivative 32
was more active towards the MDA-MB-231 cell than SAHA.
5

Table 2. Antiproliferative activities of heteroaromatic compound towards
MDA-MB-231, K562 and PC3 cell lines.
IC50 (µM)a
IC50 of
compd
R
n
HDACs
(nM)
MDA-MB-231
K562
PC3
pyridin-4-yl
thiophen-2-yl
pyridin-3-yl
pyridin-3-yl
pyridin-4-yl
thiophen-2-yl
6
6
5
6
5
5
286 8
310 42
325 33
386 53
411 39
411 43
416 20
4.69 0.61
1.21 0.18
11.7 2.80
1.87 0.07
40.1 10.6
15.2 0.82
2.29 0.19
4.15 1.19
1.56 0.06
>60
7.75 1.04
3.60 0.83
54.4 7.08
7.98 1.19
>60
33
35
26
4.36 0.85
>60
32
27
>60
>60
29
1.61 0.08
5.79 1.23
SAHA
a Inhibitory data are means of three independent determinations and expressed with
standard deviations.
4. Conclusions
Through an optimization process of the surface recognition motif of
1,3,4-thiadiazole-containing hydroxamate HDAC inhibitors, we discovered that
heteroaromatic derivatives had higher or similar inhibitory activity in both enzymatic
and cellular assays compared to SAHA. Thiophene-containing compound 35 was
identified as a more potent inhibitor of HDAC enzymes and tumor cell growth than
SAHA, representing a highly promising antitumor agent with the potential for the
treatment of human cancers.
5. Experimental section
5.1. Chemistry
All starting materials, reagents, and solvents from commercial suppliers were used
without further purification unless otherwise noted. All reactions were monitored by
thin layer chromatography (TLC) on 0.25 mm silica gel (60GF-254) plates and
visualized with iodine vapor, UV light, or ferric chloride. Melting points were
determined by the RY-1 electrothermal melting point apparatus without correction.
ESI-MS spectras were recorded on an Aglient-1100 series LC/MSD trap spectrometer.
13
1H NMR and C NMR spectra were recorded on a Brucker DRX spectrometer (300
MHz). The chemical shifts were expressed by δ values (parts per million) with
tetramethylsilane (TMS) as internal standard and significant data were reported in an
order of multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet) and number of
protons. HRMS spectra were performed on an Agilent 6510 Quadrupole
Time-of-Flight LC/MS deliver.
6

5.1.1. 5-(4-Morpholinophenyl)-1,3,4-thiadiazol-2-amine (59)
A mixture of 4-morpholinobenzoic acid (5.18 g, 25 mmol) and N-aminothiourea
(2.28 g, 25 mmol) in POCl3 (7 ml) was stirred vigrously at 75 °C for 0.5 h. After
addition of H2O (30 ml), the reaction mixture was heated under reflux for 4 h and
basified to pH 8 by 50% NaOH solution. The mixture was filtered and the filter cake
was recystallized from ethanol to yield 3.90 g of compound 59 as a yellow crystal.
1
Yield: 59%, mp: 293-295 °C (EtOH). ESI-MS m/z: 263.2 [M+H]+; H NMR
(DMSO-d6) δ 3.19 (t, J = 5.4 Hz, 4H), 3.74 (t, J = 5.4 Hz, 4H), 7.00 (m, 2H), 7.23 (s,
2H) , 7.59 (m, 2H).
The synthetic procedures of compounds 60-81 were the same as that described
above.
5.1.1.1. 5-(4-(Dimethylamino)phenyl)-1,3,4-thiadiazol-2-amine (60)
Yield: 97%, mp: 249-250 °C (EtOH). ESI-MS m/z: 221.3 [M+H]+; 1H NMR (CDCl3)
δ 2.96 (s, 6H), 6.74-6.76 (m, 2H), 7.15 (s, 2H), 7.54-7.55 (m, 2H).
5.1.1.2. 5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-amine (61)
1
Yield: 82%, mp: 219-220 °C (EtOH). ESI-MS m/z: 208.2 [M+H]+; H NMR
(DMSO-d6) δ 3.80 (s, 3H), 7.01-7.03 (m, 2H), 7.23 (s, 2H), 7.67-7.69 (m, 2H).
5.1.1.3. 5-(3,4-Dimethoxyphenyl)-1,3,4-thiadiazol-2-amine (62)
1
Yield: 77%, mp: 195-197 °C (EtOH). ESI-MS m/z: 238.2 [M+H]+; H NMR
(DMSO-d6) δ 3.80 (s, 3H), 3.82 (s, 3H), 7.02 (d, J = 8.4 Hz, 1H), 7.19-7.21 (dd, J1 =
8.4 Hz, J2 = 1.8 Hz, 1H), 7.31 (s, 2H), 7.36 (d, J = 1.8 Hz, 1H).
5.1.1.4. 5-(p-Tolyl)-1,3,4-thiadiazol-2-amine (63)
1
Yield: 98%, mp: 194-195 °C (EtOH). ESI-MS m/z: 192.3 [M+H]+; H NMR
(DMSO-d6) δ 2.34 (s, 3H), 7.27 (d, J = 8.4 Hz, 2H), 7.35 (s, 2H), 7.64 (d, J = 8.4 Hz,
2H).
5.1.1.5. 5-([1,1'-Biphenyl]-4-yl)-1,3,4-thiadiazol-2-amine (64)
1
Yield: 43%, mp: 245-247 °C (EtOH). ESI-MS m/z: 254.2 [M+H]+; H NMR
(DMSO-d6) δ 7.40 (t, J = 7.8 Hz, 1H), 7.45 (s, 2H), 7.48-7.51 (m, 2H), 7.72-7.73 (m,
2H), 7.77-7.78 (m, 2H), 7.83-7.85 (m, 2H).
5.1.1.6. 5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-amine (65)
1
Yield: 84%, mp: 238-240 °C (EtOH). ESI-MS m/z: 196.3 [M+H]+; H NMR
(DMSO-d6) δ 7.30-7.33 (m, 2H), 7.43 (s, 2H), 7.80-7.82 (m, 2H).
5.1.1.7. 5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-amine (66)
1
Yield: 98%, mp: 226-228 °C (EtOH). ESI-MS m/z: 212.2 [M+H]+; H NMR
(DMSO-d6) δ 7.48 (s, 2H), 7.52-7.54 (m, 2H), 7.77-7.78 (m, 2H).
7

5.1.1.8. 5-(3-Chlorophenyl)-1,3,4-thiadiazol-2-amine (67)
Yield: 75%, mp: 212-214 °C (EtOH). ESI-MS m/z: 212.1 [M+H]+; 1H NMR (CDCl3)
δ 7.49-7.50 (m, 2H), 7.54 (s, 2H), 7.70-7.71 (m, 1H), 7.80 (s, 1H).
5.1.1.9. 5-(2-Chlorophenyl)-1,3,4-thiadiazol-2-amine (68)
Yield: 88%, mp: 190-192 °C (EtOH). ESI-MS m/z: 212.1 [M+H]+; 1H NMR (CDCl3)
δ 7.45-7.50 (m, 4H), 7.60-7.62 (m, 1H), 7.99-8.01 (m, 1H).
5.1.1.10. 5-(4-Bromophenyl)-1,3,4-thiadiazol-2-amine (69)
1
Yield: 98%, mp: 216-218 °C (EtOH). ESI-MS m/z: 256.1 [M+H]+; H NMR
(DMSO-d6) δ 7.49 (s, 2H), 7.66-7.67 (m, 2H), 7.70-7.71 (m, 2H).
5.1.1.11. 5-(4-Iodophenyl)-1,3,4-thiadiazol-2-amine (70)
1
Yield: 77%, mp: 240-241 °C (EtOH). ESI-MS m/z: 304.2 [M+H]+; H NMR
(DMSO-d6) δ 7.48 (s, 2H), 7.54-7.55 (m, 2H), 7.83-7.84 (m, 2H).
5.1.1.12. 5-(4-(Methylsulfonyl)phenyl)-1,3,4-thiadiazol-2-amine (71)
1
Yield: 41%, mp: 241-242 °C (EtOH). ESI-MS m/z: 256.1 [M+H]+; H NMR
(DMSO-d6) δ 3.26 (s, 3H), 7.65 (s, 2H), 7.99-8.02 (m, 4H).
5.1.1.13. 5-(4-Nitrophenyl)-1,3,4-thiadiazol-2-amine (72)
1
Yield: 53%, mp: 256-258 °C (EtOH). ESI-MS m/z: 223.4 [M+H]+; H NMR
(DMSO-d6) δ 7.74 (s, 2H), 8.02 (d, J = 9.0 Hz, 2H), 8.30 (d, J = 9.0 Hz, 2H).
5.1.1.14. 5-(4-(Trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-amine (73)
1
Yield: 75%, mp: 242-243 °C (EtOH). ESI-MS m/z: 246.3 [M+H]+; H NMR
(DMSO-d6) δ 7.61 (s, 2H), 7.82-7.83 (m, 2H), 7.97-7.98 (m, 2H).
5.1.1.15. 5-(3-Bromophenyl)-1,3,4-thiadiazol-2-amine (74)
1
Yield: 89%, mp: 222-224 °C (EtOH). ESI-MS m/z: 256.1 [M+H]+; H NMR
(DMSO-d6) δ 7.43 (t, J = 7.8 Hz, 1H), 7.54 (s, 2H), 7.62-7.64 (m, 1H), 7.74 (d, J =
7.8 Hz, 1H), 7.93 (t, J = 1.8 Hz, 1H).
5.1.1.16. 5-(2-Bromophenyl)-1,3,4-thiadiazol-2-amine (75)
Yield: 85%, mp: 202-203 °C (EtOH). ESI-MS m/z: 256.1 [M+H]+; 1H NMR (CDCl3)
δ 7.39-7.42 (td, J1 = 7.8 Hz, J2 = 1.8 Hz, 1H), 7.45 (s, 2H), 7.49-7.51 (td, J1 = 7.8 Hz,
J2 = 1.2 Hz, 1H), 7.77-7.79 (dd, J1 = 7.8 Hz, J2 = 1.2 Hz, 1H), 7.86-7.88 (dd, J1 =
7.8 Hz, J2 = 1.8 Hz, 1H).
5.1.1.17. 5-(Naphthalen-1-yl)-1,3,4-thiadiazol-2-amine (76)
1
Yield: 62%, mp: 208-210 °C (EtOH). ESI-MS m/z: 228.2 [M+H]+; H NMR
(DMSO-d6) δ 7.49 (s, 2H), 7.58-7.66 (m, 3H), 7.74-7.76 (m, 1H), 8.04 (t, J = 8.4 Hz,
2H), 8.80 (d, J = 9.0 Hz, 1H).
8

5.1.1.18. 5-(Naphthalen-2-yl)-1,3,4-thiadiazol-2-amine (77)
Yield: 95%, mp: 230-231 °C (EtOH). ESI-MS m/z: 228.3 [M+H]+; H NMR
(DMSO-d6) δ 7.49 (s, 2H), 7.55-7.59 (m, 2H), 7.95-7.96 (m, 1H), 7.99-8.00 (m, 2H),
8.03-8.05 (m, 1H), 8.24 (s, 1H).
1
5.1.1.19. 5-(Pyridin-3-yl)-1,3,4-thiadiazol-2-amine (78)
1
Yield: 70%, mp: 234-236 °C (EtOH). ESI-MS m/z: 179.2 [M+H]+; H NMR
(DMSO-d6) δ 7.50-7.52 (m, 1H), 7.56 (s, 2H), 8.13-8.15 (m, 1H), 8.61-8.62 (dd, J1 =
4.8 Hz, J2 = 1.8Hz, 1H), 8.95 (d, J = 2.4 Hz, 1H).
5.1.1.20. 5-(Pyridin-4-yl)-1,3,4-thiadiazol-2-amine (79)
1
Yield: 62%, mp: 238-241 °C (EtOH). ESI-MS m/z: 179.1 [M+H]+; H NMR
(DMSO-d6) δ 7.70 (s, 2H), 7.71-7.72 (m, 2H), 8.64-8.65 (m, 2H).
5.1.1.21. 5-(Furan-2-yl)-1,3,4-thiadiazol-2-amine (80)
1
Yield: 97%, mp: 247-249 °C (EtOH). ESI-MS m/z: 168.3 [M+H]+; H NMR
(DMSO-d6) δ 6.65-6.66 (m, 1H), 6.95 (d, J = 3.6 Hz, 1H), 7.45 (s, 2H), 7.83-7.84 (m,
1H).
5.1.1.22. 5-(Thiophen-2-yl)-1,3,4-thiadiazol-2-amine (81)
1
Yield: 61%, mp: 204-206 °C (EtOH). ESI-MS m/z: 184.2 [M+H]+; H NMR
(DMSO-d6) δ 7.12-7.14 (m, 1H), 7.41-7.42 (dd, J1 = 3.6 Hz, J2 = 0.6 Hz, 1H), 7.43 (s,
2H), 7.63-7.64 (dd, J1 = 4.8 Hz, J2 = 0.6 Hz, 1H).
5.1.2. 7-Methoxy-7-oxoheptanoic acid (84)
A solution of KOH (5.87 g, 104.65 mmol) in CH3OH (150 ml) was added dropwise
to dimethyl heptanedioate 82 (16.94 g, 90 mmol) at 0 °C. The reaction mixture was
allowed to be stirred for 4 h at room temperature. After removal of the solvent under
reduced pressure, Et2O (100 ml) and H2O (200 ml) were added and the organic phase
was concentrated to give 82 as yellow oil (5.08 g, 30%). The aqueous phase was
acidified to pH 3 by concentrated HCl and extracted with Et2O (100 ml×3). The
combined organic layer was washed with brine (100 ml×3) and dried over MgSO4.
The solvent was concentrated in vacuum. Filtration and purification with silica gel
column chromatography gave 5.96 g (38%) of compound 84 as colorless oil. ESI-MS
1
m/z: 173.3 [M-H]-; H NMR (DMSO-d6) δ 1.23-1.31 (m, 4H), 1.44-1.57 (m, 4H),
2.19 (t, J = 7.2 Hz, 2H), 2.29 (t, J = 7.2 Hz, 2H), 3.58 (s, 3H), 11.97 (s, 1H).
The synthetic procedures of compounds 85 were the same as that described above.
5.1.2.1. 8-Methoxy-8-oxooctanoic acid (85)
1
Yield: 44%. ESI-MS m/z: 187.4 [M-H]-; H NMR (DMSO-d6) δ 1.21-1.32 (m, 4H),
1.43-1.53 (m, 4H), 2.18 (t, J = 7.2 Hz, 2H), 2.29 (t, J = 7.2 Hz, 2H), 3.58 (s, 3H),
11.97 (s, 1H).
5.1.3.
Methyl
9

7-((5-(4-morpholinophenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (86)
A soln. of 84 (1.74 g, 10 mmol) in SOCl2 (4 ml) was refluxed for 2 h. The removal
of SOCl2 under reduced pressure yielded orange oil, which was dissolved in
dichloromethane. The resulting solution was added dropwise to a solution of 59 (2.10
g, 8 mmol) and Et3N (3.5 mL, 25 mmol) in dichloromethane (65 mL) at 0 °C. The
resulting reaction mixture was stirred overnight at room temperature, washed by 1M
H3PO4 (3×80 mL) and brine (3×80 mL), and dried (MgSO4). Concentration and
recrystallization from EtOAc gave 1.22 g of compound 86 as a colorless crystal. Yield:
1
36%, mp: 214-216 °C (EtOAc). ESI-MS m/z: 419.4 [M+H]+; H NMR (CDCl3) δ
1.47-1.57 (m, 2H), 1.68-1.78 (m, 2H), 1.82-1.92 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H),
2.83 (t, J = 7.5 Hz, 2H), 3.28 (t, J = 4.8 Hz, 4H), 3.63 (s, 3H), 3.89 (t, J = 4.8 Hz, 4H),
6.97 (d, J = 8.7 Hz, 2H), 7.83 (d, J = 8.7 Hz, 2H), 13.27 (s, 1H).
The synthetic procedures of compounds 87-119 were the same as that described
above.
5.1.3.1.
Methyl
7-((5-(4-(dimethylamino)phenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate
(87)
Yield: 62%, mp: 192-194 °C (EtOAc). ESI-MS m/z: 377.4 [M+H]+; 1H NMR (CDCl3)
δ 1.48-1.57 (m, 2H), 1.68-1.78 (m, 2H), 1.81-1.92 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H),
2.84 (t, J = 7.5 Hz, 2H), 3.05 (s, 6H), 3.63 (s, 3H), 6.75 (d, J = 9.0 Hz, 2H), 7.79 (d, J
= 9.0 Hz, 2H), 13.32 (s, 1H).
5.1.3.2.
Methyl
7-((5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (88)
Yield: 59%, mp: 177-179 °C (EtOAc). ESI-MS m/z: 364.4 [M+H]+; 1H NMR (CDCl3)
δ 1.47-1.57 (m, 2H), 1.68-1.78 (m, 2H), 1.82-1.92 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H),
2.84 (t, J = 7.5 Hz, 2H), 3.63 (s, 3H), 3.88 (s, 3H), 6.98-7.03 (m, 2H), 7.84-7.89 (m,
2H), 13.31 (s, 1H).
5.1.3.3.
Methyl
7-((5-(3,4-dimethoxyphenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (89)
Yield: 55%, mp: 179-181 °C (EtOAc). ESI-MS m/z: 394.3 [M+H]+; 1H NMR (CDCl3)
δ 1.47-1.57 (m, 2H), 1.67-1.77 (m, 2H), 1.82-1.92 (m, 2H), 2.32 (t, J = 7.5 Hz, 2H),
2.84 (t, J = 7.5 Hz, 2H), 3.63 (s, 3H), 3.96 (s, 3H), 3.97 (s, 3H), 6.96 (d, J = 8.4 Hz,
1H), 7.45-7.49 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 7.51 (d, J = 1.8 Hz, 1H), 13.15 (s,
1H).
5.1.3.4. Methyl 7-oxo-7-((5-(p-tolyl)-1,3,4-thiadiazol-2-yl)amino)heptanoate (90)
Yield: 53%, mp: 160-162 °C (EtOAc). ESI-MS m/z: 348.3 [M+H]+; 1H NMR (CDCl3)
δ 1.47-1.57 (m, 2H), 1.68-1.78 (m, 2H), 1.82-1.92 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H),
2.42 (s, 3H), 2.85 (t, J = 7.5 Hz, 2H), 3.63 (s, 3H), 7.29 (d, J = 8.1 Hz, 2H), 7.82 (d, J
= 8.1 Hz, 2H), 13.28 (s, 1H).
10

5.1.3.5.
Methyl
7-((5-([1,1'-biphenyl]-4-yl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (91)
Yield: 60%, mp: 181-183 °C (EtOAc). ESI-MS m/z: 410.4 [M+H]+; 1H NMR (CDCl3)
δ 1.50-1.60 (m, 2H), 1.70-1.80 (m, 2H), 1.85-1.95 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H),
2.88 (t, J = 7.5 Hz, 2H), 3.63 (s, 3H), 7.37-7.43 (m, 1H), 7.46-7.51 (m, 2H), 7.64-7.66
(m, 2H), 7.72 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 13.37 (s, 1H).
5.1.3.6.
Methyl
7-((5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (92)
Yield: 80%, mp: 200-201 °C (EtOAc). ESI-MS m/z: 352.4 [M+H]+; 1H NMR (CDCl3)
δ 1.46-1.56 (m, 2H), 1.68-1.78 (m, 2H), 1.82-1.92 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H),
2.82 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.17-7.23 (m, 2H), 7.91-7.96 (m, 2H), 12.98 (s,
1H).
5.1.3.7.
Methyl
7-((5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (93)
Yield: 75%, mp: 201-204 °C (EtOAc). ESI-MS m/z: 368.2 [M+H]+; 1H NMR (CDCl3)
δ 1.47-1.57 (m, 2H), 1.68-1.78 (m, 2H), 1.83-1.93 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H),
2.84 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.45-7.50 (m, 2H), 7.85-7.89 (m, 2H), 13.32 (s,
1H).
5.1.3.8.
Methyl
7-((5-(3-chlorophenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (94)
Yield: 72%, mp: 172-173 °C (EtOAc). ESI-MS m/z: 368.3 [M+H]+; 1H NMR (CDCl3)
δ 1.47-1.58 (m, 2H), 1.68-1.79 (m, 2H), 1.83-1.93 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H),
2.83 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.41-7.48 (m, 2H), 7.77-7.80 (dt, J1 = 6.9 Hz,
J2 = 1.8 Hz, 1H), 7.97 (s, 1H), 13.02 (s, 1H).
5.1.3.9.
Methyl
7-((5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (95)
Yield: 67%, mp: 155-157 °C (EtOAc). ESI-MS m/z: 368.2 [M+H]+; 1H NMR (CDCl3)
δ 1.45-1.55 (m, 2H), 1.66-1.76 (m, 2H), 1.82-1.92 (m, 2H), 2.32 (t, J = 7.2 Hz, 2H),
2.84 (t, J = 7.2 Hz, 2H), 3.63 (s, 3H), 7.40-7.46 (m, 2H), 7.51-7.57 (m, 1H), 8.14-8.19
(m, 1H), 13.17 (s, 1H).
5.1.3.10.
Methyl
7-((5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (96)
Yield: 57%, mp: 201-202 °C (EtOAc). ESI-MS m/z: 412.4 [M+H]+; 1H NMR (CDCl3)
δ 1.47-1.57 (m, 2H), 1.68-1.78 (m, 2H), 1.82-1.93 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H),
2.84 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.61-7.66 (m, 2H), 7.78-7.83 (m, 2H), 13.24 (s,
1H).
5.1.3.11.
Methyl
7-((5-(4-iodophenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (97)
11

Yield: 67%, mp: 207-209 °C (EtOAc). ESI-MS m/z: 460.3 [M+H]+; 1H NMR (CDCl3)
δ 1.46-1.56 (m, 2H), 1.67-1.77 (m, 2H), 1.82-1.92 (m, 2H), 2.33 (t, J = 7.5 Hz, 2H),
2.83 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.64-7.68 (m, 2H), 7.82-7.86 (m, 2H), 13.17 (s,
1H).
5.1.3.12.
Methyl
7-((5-(4-(methylsulfonyl)phenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate
(98)
Yield: 39%, mp: 219-221 °C (EtOAc). ESI-MS m/z: 412.4 [M+H]+; 1H NMR (CDCl3)
δ 1.48-1.58 (m, 2H), 1.69-1.79 (m, 2H), 1.84-1.94 (m, 2H), 2.35 (t, J = 7.2 Hz, 2H),
2.84 (t, J = 7.2 Hz, 2H), 3.12 (s, 3H), 3.66 (s, 3H), 8.08-8.11 (m, 2H), 8.15-8.17 (m,
2H), 12.94 (s, 1H).
5.1.3.13.
Methyl
7-((5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (99)
Yield: 46%, mp: 229-239 °C (EtOAc). ESI-MS m/z: 379.4 [M+H]+; 1H NMR (CDCl3)
δ 1.48-1.58 (m, 2H), 1.69-1.79 (m, 2H), 1.85-1.95 (m, 2H), 2.36 (t, J = 7.5 Hz, 2H),
2.84 (t, J = 7.5 Hz, 2H), 3.66 (s, 3H), 8.11-8.16 (m, 2H), 8.36-8.40 (m, 2H), 12.86 (s,
1H).
5.1.3.14.
Methyl
7-oxo-7-((5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)amino)heptanoate
(100)
Yield: 61%, mp: 195-196 °C (EtOAc). ESI-MS m/z: 402.4 [M+H]+; 1H NMR (CDCl3)
δ 1.47-1.57 (m, 2H), 1.68-1.79 (m, 2H), 1.84-1.94 (m, 2H), 2.35 (t, J = 7.2 Hz, 2H),
2.84 (t, J = 7.2 Hz, 2H), 3.64 (s, 3H), 7.77 (d, J = 8.1 Hz, 2H), 8.07 (d, J = 8.1 Hz,
2H), 12.98 (s, 1H).
5.1.3.15.
Methyl
8-((5-(4-morpholinophenyl)-1,3,4-thiadiazol-2-yl)amino)-8-oxooctanoate (101)
Yield: 33%, mp: 235-237 °C (EtOAc). ESI-MS m/z: 433.5 [M+H]+; 1H NMR (CDCl3)
δ 1.31-1.48 (m, 4H), 1.54-1.64 (m, 2H), 1.73-1.83 (m, 2H), 2.22 (t, J = 7.5 Hz, 2H),
2.75 (t, J = 7.5 Hz, 2H), 3.21 (t, J = 4.8 Hz, 4H), 3.57 (s, 3H), 3.82 (t, J = 4.8 Hz, 4H),
6.91 (d, J = 9.0 Hz, 2H), 7.76 (d, J = 9.0 Hz, 2H), 13.18 (s, 1H).
5.1.3.16.
Methyl
8-((5-([1,1'-biphenyl]-4-yl)-1,3,4-thiadiazol-2-yl)amino)-8-oxooctanoate (102)
Yield: 51%, mp: 201-204 °C (EtOAc). ESI-MS m/z: 424.5 [M+H]+; 1H NMR (CDCl3)
δ 1.43-1.58 (m, 4H), 1.62-1.72 (m, 2H), 1.83-1.93 (m, 2H), 2.30 (t, J = 7.5 Hz, 2H),
2.85 (t, J = 7.5 Hz, 2H), 3.62 (s, 3H), 7.38-7.43 (m, 1H), 7.46-7.51 (m, 2H), 7.65-7.67
(m, 2H), 7.74 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 13.08 (s, 1H).
5.1.3.17.
Methyl
8-((5-(3-bromophenyl)-1,3,4-thiadiazol-2-yl)amino)-8-oxooctanoate (103)
12

Yield: 38%, mp: 200-202 °C (EtOAc). ESI-MS m/z: 426.2 [M+H]+; 1H NMR (CDCl3)
δ 1.39-1.56 (m, 4H), 1.62-1.71 (m, 2H), 1.81-1.91 (m, 2H), 2.30 (t, J = 7.5 Hz, 2H),
2.81 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.37 (t, J = 7.8 Hz, 1H), 7.60-7.64 (m, 1H),
7.81-7.85 (m, 1H), 8.13 (t, J = 1.8 Hz, 1H), 12.94 (s, 1H).
5.1.3.18.
Methyl
8-((5-(2-bromophenyl)-1,3,4-thiadiazol-2-yl)amino)-8-oxooctanoate (104)
Yield: 68%, mp: 133-135 °C (EtOAc). ESI-MS m/z: 426.2 [M+H]+; 1H NMR (CDCl3)
δ 1.35-1.52 (m, 4H), 1.58-1.67 (m, 2H), 1.79-1.89 (m, 2H), 2.27 (t, J = 7.5 Hz, 2H),
2.81 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 7.32-7.38 (td, J1 = 7.8 Hz, J2 = 1.8 Hz, 1H),
7.44-7.49 (td, J1 = 7.8 Hz, J2 = 1.2 Hz, 1H), 7.73-7.76 (dd, J1 = 7.8 Hz, J2 = 1.2 Hz,
1H), 7.97-8.01 (dd, J1 = 7.8 Hz, J2 = 1.8 Hz, 1H), 12.93 (s, 1H).
5.1.3.19. Methyl 8-((5-(4-iodophenyl)-1,3,4-thiadiazol-2-yl)amino)-8-oxooctanoate
(105)
Yield: 77%, mp: 198-200 °C (EtOAc). ESI-MS m/z: 474.2 [M+H]+; 1H NMR (CDCl3)
δ 1.40-1.54 (m, 4H), 1.60-1.70 (m, 2H), 1.80-1.90 (m, 2H), 2.29 (t, J = 7.5 Hz, 2H),
2.79 (t, J = 7.5 Hz, 2H), 3.66 (s, 3H), 7.65-7.68 (m, 2H), 7.83-7.86 (m, 2H), 12.81 (s,
1H).
5.1.3.20.
Methyl
8-((5-(4-(methylsulfonyl)phenyl)-1,3,4-thiadiazol-2-yl)amino)-8-oxooctanoate
(106)
Yield: 40%, mp: 234-236 °C (EtOAc). ESI-MS m/z: 426.4 [M+H]+; 1H NMR (CDCl3)
δ 1.41-1.52 (m, 4H), 1.64-1.71 (m, 2H), 1.80-1.90 (m, 2H), 2.31 (t, J = 7.2 Hz, 2H),
2.75 (t, J = 7.2 Hz, 2H), 3.12 (s, 3H), 3.66 (s, 3H), 8.07-8.10 (m, 2H), 8.14-8.17 (m,
2H), 11.96 (s, 1H).
5.1.3.21.
Methyl
8-oxo-8-((5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)amino)octanoate
(107)
Yield: 49%, mp: 202-203 °C (EtOAc). ESI-MS m/z: 416.4 [M+H]+; 1H NMR (CDCl3)
δ 1.38-1.56 (m, 4H), 1.61-1.71 (m, 2H), 1.82-1.92 (m, 2H), 2.30 (t, J = 7.5 Hz, 2H),
2.82 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 7.76-7.79 (m, 2H), 8.05-8.08 (m, 2H), 12.91 (s,
1H).
5.1.3.22.
Methyl
7-((5-(naphthalen-1-yl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (108)
Yield: 75%, mp: 169-170 °C (EtOAc). ESI-MS m/z: 384.3 [M+H]+; 1H NMR (CDCl3)
δ 1.43-1.54 (m, 2H), 1.63-1.73 (m, 2H), 1.84-1.94 (m, 2H), 2.25 (t, J = 7.5 Hz, 2H),
2.88 (t, J = 7.5 Hz, 2H), 3.61 (s, 3H), 7.55-7.61 (m, 3H), 7.83-7.86 (dd, J1 = 7.2 Hz,
J2 = 0.9 Hz, 1H), 7.92-7.97 (m, 1H), 8.01 (d, J = 8.1 Hz, 1H), 8.75-8.78 (m, 1H),
13.21 (s, 1H).
13

5.1.3.23.
Methyl
7-((5-(naphthalen-2-yl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate (109)
Yield: 33%, mp: 200-201 °C (EtOAc). ESI-MS m/z: 384.4 [M+H]+; 1H NMR (CDCl3)
δ 1.51-1.61 (m, 2H), 1.71-1.81 (m, 2H), 1.86-1.96 (m, 2H), 2.36 (t, J = 7.5 Hz, 2H),
2.88 (t, J = 7.5 Hz, 2H), 3.62 (s, 3H), 7.55-7.59 (m, 2H), 7.87-7.97 (m, 3H), 8.09-8.13
(m, 1H), 8.35 (s, 1H), 12.93 (s, 1H).
5.1.3.24.
Methyl
7-oxo-7-((5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl)amino)heptanoate (110)
Yield: 60%, mp: 173-174 °C (EtOAc). ESI-MS m/z: 335.5 [M+H]+; 1H NMR (CDCl3)
δ 1.47-1.57 (m, 2H), 1.68-1.78 (m, 2H), 1.83-1.93 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H),
2.83 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.47-7.51 (m, 1H), 8.27 (d, J = 8.1 Hz, 1H),
8.74 (d, J = 4.2 Hz, 1H), 9.22 (s, 1H), 12.89 (s, 1H).
5.1.3.25.
Methyl
7-oxo-7-((5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)amino)heptanoate (111)
Yield: 51%, mp: 170-172 °C (EtOAc). ESI-MS m/z: 335.5 [M+H]+; 1H NMR (CDCl3)
δ 1.48-1.58 (m, 2H), 1.69-1.79 (m, 2H), 1.84-1.94 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H),
2.84 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 7.83-7.85 (m, 2H), 8.79-8.81 (m, 2H), 13.01 (s,
1H).
5.1.3.26. Methyl 7-((5-(furan-2-yl)-1,3,4-thiadiazol-2-yl)amino)-7-oxoheptanoate
(112)
Yield: 60%, mp: 179-181 °C (EtOAc). ESI-MS m/z: 324.4 [M+H]+; 1H NMR (CDCl3)
δ 1.45-1.55 (m, 2H), 1.67-1.77 (m, 2H), 1.80-1.90 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H),
2.78 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 6.57-6.59 (m, 1H), 7.07-7.08 (dd, J1 = 3.6 Hz,
J2 = 0.6 Hz, 1H), 7.59-7.60 (m, 1H), 12.77 (s, 1H).
5.1.3.27.
Methyl
7-oxo-7-((5-(thiophen-2-yl)-1,3,4-thiadiazol-2-yl)amino)heptanoate (113)
Yield: 49%, mp: 175-177 °C (EtOAc). ESI-MS m/z: 340.4 [M+H]+; 1H NMR (CDCl3)
δ 1.48-1.58 (m, 2H), 1.69-1.79 (m, 2H), 1.81-1.91 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H),
2.80 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.12-7.15 (m, 1H), 7.46-7.48 (dd, J1 = 5.1 Hz,
J2 = 1.2 Hz, 1H), 7.50-7.51 (dd, J1 = 3.6 Hz, J2 = 0.9 Hz, 1H), 12.93 (s, 1H).
5.1.3.28.
Methyl
8-((5-(naphthalen-1-yl)-1,3,4-thiadiazol-2-yl)amino)-8-oxooctanoate (114)
Yield: 77%, mp: 191-193 °C (EtOAc). ESI-MS m/z: 398.4 [M+H]+; 1H NMR (CDCl3)
δ 1.36-1.50 (m, 4H), 1.52-1.62 (m, 2H), 1.82-1.92 (m, 2H), 2.19 (t, J = 7.5 Hz, 2H),
2.87 (t, J = 7.5 Hz, 2H), 3.62 (s, 3H), 7.56-7.62 (m, 3H), 7.83-7.86 (dd, J1 = 7.2 Hz,
J2 = 1.2 Hz, 1H), 7.92-7.97 (m, 1H), 8.01 (d, J = 8.4 Hz, 1H), 8.76-8.08 (m, 1H),
13.19 (s, 1H).
5.1.3.29.
Methyl
14

8-((5-(naphthalen-2-yl)-1,3,4-thiadiazol-2-yl)amino)-8-oxooctanoate (115)
Yield: 60%, mp: 199-200 °C (EtOAc). ESI-MS m/z: 398.4 [M+H]+; 1H NMR (CDCl3)
δ 1.41-1.57 (m, 4H), 1.63-1.73 (m, 2H), 1.84-1.94 (m, 2H), 2.30 (t, J = 7.5 Hz, 2H),
2.85 (t, J = 7.5 Hz, 2H), 3.63 (s, 3H), 7.54-7.61 (m, 2H), 7.88-7.98 (m, 3H), 8.10-8.14
(m, 1H), 8.35 (s, 1H), 12.81 (s, 1H).
5.1.3.30. Methyl 8-oxo-8-((5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl)amino)octanoate
(116)
Yield: 42%, mp: 165-166 °C (EtOAc). ESI-MS m/z: 349.4 [M+H]+; 1H NMR (CDCl3)
δ 1.38-1.56 (m, 4H), 1.60-1.70 (m, 2H), 1.82-1.92 (m, 2H), 2.29 (t, J = 7.5 Hz, 2H),
2.85 (t, J = 7.5 Hz, 2H), 3.64 (s, 3H), 7.45-7.50 (m, 1H), 8.23-8.27 (m, 1H), 8.73-8.75
(m, 1H), 9.19 (d, J = 1.8 Hz, 1H), 13.32 (s, 1H).
5.1.3.31. Methyl 8-oxo-8-((5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)amino)octanoate
(117)
Yield: 50%, mp: 186-187 °C (EtOAc). ESI-MS m/z: 349.4 [M+H]+; 1H NMR (CDCl3)
δ 1.39-1.56 (m, 4H), 1.61-1.71 (m, 2H), 1.82-1.92 (m, 2H), 2.30 (t, J = 7.5 Hz, 2H),
2.82 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 7.81-7.83 (m, 2H), 8.78-8.80 (m, 2H), 12.89 (s,
1H).
5.1.3.32. Methyl 8-((5-(furan-2-yl)-1,3,4-thiadiazol-2-yl)amino)-8-oxooctanoate
(118)
Yield: 67%, mp: 160-171 °C (EtOAc). ESI-MS m/z: 338.5 [M+H]+; 1H NMR (CDCl3)
δ 1.38-1.53 (m, 4H), 1.60-1.70 (m, 2H), 1.78-1.88 (m, 2H), 2.30 (t, J = 7.5 Hz, 2H),
2.78 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 6.58-6.59 (m, 1H), 7.06-7.07 (dd, J1 = 3.6 Hz,
J2 = 0.6 Hz, 1H), 7.60-7.61 (m, 1H), 12.82 (s, 1H).
5.1.3.33.
Methyl
8-oxo-8-((5-(thiophen-2-yl)-1,3,4-thiadiazol-2-yl)amino)octanoate (119)
Yield: 62%, mp: 163-164 °C (EtOAc). ESI-MS m/z: 354.3 [M+H]+; 1H NMR (CDCl3)
δ 1.40-1.56 (m, 4H), 1.61-1.71 (m, 2H), 1.79-1.89 (m, 2H), 2.31 (t, J = 7.5 Hz, 2H),
2.78 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 7.13-7.15 (m, 1H), 7.48 (d, J = 5.1 Hz, 1H),
7.51 (d, J = 3.6 Hz, 1H), 12.90 (s, 1H).
5.1.4.
N1-hydroxy-N7-(5-(4-morpholinophenyl)-1,3,4-thiadiazol-2-yl)heptanediamide
(2)
Solution A was prepared by dissolution of hydroxylamine hydrochloride (4.67 g,
67 mmol) in methanol (24 mL). Potassium hydroxide (6.60 g, 100 mmol) was diluted
in methanol (14 mL) to develop solution B, which was added dropwise to the solution
A at 0 °C . The reaction mixture was stirred for 0.5 h at 0 °C and filtered to give a
solution of hydroxylamine in methanol. 86 (0.42 g, 1 mmol) was dissolved in the
solution of hydroxylamine in methanol (10 mL). The resulting mixture was stirred for
1 h at room temperature and then adjusted to pH 7 by concentrated HCl. After
15

concentration under reducd pressure, the residue was washed with water to afford
1
0.34 g of compound 2 as a white solid. Yield: 81%, mp: 239-240 °C. H NMR
(DMSO-d6, 300 MHz, δ, ppm) 1.22-1.32 (m, 2H), 1.46-1.66 (m, 4H), 1.95 (t, J = 7.2
Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H), 3.23 (t, J = 4.5Hz, 4H), 3.75 (t, J = 4.5Hz, 4H),
13
7.03-7.06 (m, 2H), 7.76-7.79 (m, 2H), 8.65 (s, 1H), 10.32 (s, 1H), 12.46 (s, 1H); C
NMR (DMSO-d6, 75 MHz, δ, ppm) 24.32, 24.80, 28.05, 32.07, 34.70, 47.35, 65.87,
114.58, 120.28, 127.84, 152.32, 157.12, 161.83, 168.99, 171.26; HRMS (AP-ESI) m/z
calcd for C19H25N5O4S [M+H]+ 420.1700, found: 420.1707.
The synthetic procedures of compounds 3-35 were the same as that described
above.
5.1.4.1.
N1-(5-(4-(dimethylamino)phenyl)-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptanediami
de (3)
1
Yield: 98%, mp: 214-215 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.22-1.32 (m,
2H), 1.46-1.66 (m, 4H), 1.95 (t, J = 7.2 Hz, 2H), 2.44-2.52 (m, 2H), 2.99 (s, 6H), 6.79
(d, J = 9.0 Hz, 2H), 7.72 (d, J = 9.0 Hz, 2H), 8.64 (s, 1H), 10.32 (s, 1H), 12.40 (s, 1H);
13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.33, 24.79, 28.05, 32.07, 34.70, 39.70,
111.98, 117.40, 127.89, 151.58, 156.64, 162.26, 168.97, 171.17; HRMS (AP-ESI) m/z
calcd for C17H23N5O3S [M+H]+ 378.1594, found: 378.1594.
5.1.4.2.
N1-hydroxy-N7-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)heptanediamide (4)
1
Yield: 85%, mp: 212-213 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.22-1.32 (m,
2H), 1.46-1.67 (m, 4H), 1.95 (t, J = 7.2 Hz, 2H), 2.46-2.49 (m, 2H), 3.83 (s, 3H),
13
7.05-7.10 (m, 2H), 7.85-7.89 (m, 2H), 8.64 (s, 1H), 10.32 (s, 1H), 12.51 (s, 1H); C
NMR (DMSO-d6, 75 MHz, δ, ppm) 24.30, 24.79, 28.04, 32.07, 34.69, 55.35, 114.68,
122.75, 128.38, 157.63, 160.98, 161.43, 168.99, 171.35; HRMS (AP-ESI) m/z calcd
for C16H20N4O4S [M+H]+ 365.1278, found: 365.1282.
5.1.4.3.
N1-(5-(3,4-dimethoxyphenyl)-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptanediamide
(5)
1
Yield: 89%, mp: 213-215 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.22-1.32 (m,
2H), 1.46-1.66 (m, 4H), 1.95 (t, J = 7.5 Hz, 2H), 2.46-2.49 (m, 2H), 3.83 (s, 3H), 3.86
(s, 3H), 7.08 (d, J = 8.4 Hz, 1H), 7.43-7.46 (dd, J1 = 8.4 Hz, J2 = 2.1 Hz, 1H), 7.50 (d,
J = 2.1 Hz, 1H), 8.65 (s, 1H), 10.32 (s, 1H), 12.54 (s, 1H); 13C NMR (DMSO-d6, 75
MHz, δ, ppm) 24.30, 24.79, 28.04, 32.07, 34.69, 55.58, 55.64, 109.31, 112.02, 120.29,
122.81, 149.10, 150.79, 157.70, 161.62, 168.97, 171.37; HRMS (AP-ESI) m/z calcd
for C17H22N4O5S [M+H]+ 395.1384, found: 395.1388.
5.1.4.4. N1-hydroxy-N7-(5-(p-tolyl)-1,3,4-thiadiazol-2-yl)heptanediamide (6)
1
Yield: 72%, mp: 201-202 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.22-1.32 (m,
2H), 1.46-1.67 (m, 4H), 1.95 (t, J = 7.2 Hz, 2H), 2.37 (s, 3H), 2.49 (t, J = 7.2 Hz, 2H),
16

7.34 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H), 8.64 (s, 1H), 10.32 (s, 1H), 12.55 (s,
1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 20.90, 24.29, 24.79, 28.04, 32.07, 34.70,
126.75, 127.51, 129.82, 140.36, 157.97, 161.67, 168.99, 171.41; HRMS (AP-ESI)
m/z calcd for C16H20N4O3S [M+H]+ 349.1329, found: 349.1334.
5.1.4.5.
N1-(5-([1,1'-biphenyl]-4-yl)-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptanediamide (7)
1
Yield: 78%, mp: 254-256 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.24-1.34 (m,
2H), 1.48-1.58 (m, 2H), 1.58-1.68 (m, 2H), 1.96 (t, J = 7.5 Hz, 2H), 2.49-2.54 (m,
2H), 7.39-7.44 (m, 1H), 7.51 (t, J = 7.2 Hz, 2H), 7.74-7.77 (m, 2H), 7.84 (d, J = 8.4
Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 8.66 (s, 1H), 10.33 (s, 1H), 12.58 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.82, 25.31, 28.57, 32.59, 35.29, 127.18, 127.91,
127.97, 128.54, 129.55, 129.77, 139.51, 142.47, 158.94, 161.73, 169.49, 172.09;
HRMS (AP-ESI) m/z calcd for C21H22N4O3S [M+H]+ 411.1485, found: 411.1492.
5.1.4.6. N1-(5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptanediamide
(8)
1
Yield: 77%, mp: 299-302 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.23-1.33 (m,
2H), 1.47-1.67 (m, 4H), 1.95 (t, J = 7.5 Hz, 2H), 2.48-2.52 (m, 2H), 7.33-7.41 (m,
2H), 7.96-8.03 (m, 2H), 8.65 (s, 1H), 10.32 (s, 1H), 12.60 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.27, 24.78, 28.03, 32.06, 34.70, 116.20, 116.50,
126.82, 126.86, 129.09, 129.21, 158.38, 160.53, 161.60, 164.89, 168.98, 171.51;
HRMS (AP-ESI) m/z calcd for C15H17FN4O3S [M+H]+ 353.1078, found: 353.1072.
5.1.4.7. N1-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptanediamide
(9)
1
Yield: 81%, mp: 313-316 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.22-1.33 (m,
2H), 1.47-1.67 (m, 4H), 1.95 (t, J = 7.2 Hz, 2H), 2.48-2.53 (m, 2H), 7.57-7.62 (m,
2H), 7.94-7.98 (m, 2H), 8.65 (s, 1H), 10.32 (s, 1H), 12.63 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.26, 24.78, 28.03, 32.06, 34.72, 128.52, 129.10,
129.36, 135.05, 158.61, 160.46, 168.96, 171.57; HRMS (AP-ESI) m/z calcd for
C15H17ClN4O3S [M+H]+ 369.0783, found: 369.0779.
5.1.4.8. N1-(5-(3-chlorophenyl)-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptanediamide
(10)
1
Yield: 92%, mp: 285-288 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.23-1.33 (m,
2H), 1.47-1.57 (m, 2H), 1.57-1.67 (m, 2H), 1.95 (t, J = 7.2 Hz, 2H), 2.48-2.53 (m,
2H), 7.53-7.61 (m, 2H), 7.87-7.91 (dt, J1 = 6.9 Hz, J2 = 1.8 Hz, 1H), 7.99 (d, J = 1.8
13
Hz, 1H), 8.65 (s, 1H), 10.32 (s, 1H), 12.66 (s, 1H); C NMR (DMSO-d6, 75 MHz, δ,
ppm) 24.26, 24.78, 28.03, 32.06, 34.73, 125.71, 126.07, 130.21, 131.24, 132.17,
133.97, 158.84, 160.14, 168.96, 171.62; HRMS (AP-ESI) m/z calcd for
C15H17ClN4O3S [M+H]+ 369.0783, found: 369.0785.
5.1.4.9. N1-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptanediamide
17

(11)
1
Yield: 98%, mp: 210-212 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.23-1.33 (m,
2H), 1.47-1.57 (m, 2H), 1.57-1.67 (m, 2H), 1.96 (t, J = 7.5 Hz, 2H), 2.49-2.54 (m,
2H), 7.50-7.59 (m, 2H), 7.67-7.70 (m, 1H), 8.09-8.12 (m, 1H), 8.65 (d, J = 1.2 Hz,
1H), 10.32 (s, 1H), 12.67 (s, 1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.25,
24.79, 28.03, 32.07, 34.64, 127.83, 128.99, 130.55, 130.83, 131.02, 131.74, 157.59,
159.92, 168.97, 171.64; HRMS (AP-ESI) m/z calcd for C15H17ClN4O3S [M+H]+
369.0783, found: 369.0783.
5.1.4.10.
N1-(5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptanediamide
(12)
1
Yield: 87%, mp: 318-322 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.23-1.32 (m,
2H), 1.47-1.67 (m, 4H), 1.95 (t, J = 7.5 Hz, 2H), 2.48-2.52 (m, 2H), 7.71-7.75 (m,
2H), 7.87-7.91 (m, 2H), 8.66 (s, 1H), 10.32 (s, 1H), 12.68 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.28, 24.79, 28.04, 32.07, 34.76, 123.75, 128.68,
129.46, 132.26, 158.71, 160.50, 168.97, 171.62; HRMS (AP-ESI) m/z calcd for
C15H17BrN4O3S [M+H]+ 413.0278, found: 413.0270.
5.1.4.11. N1-hydroxy-N7-(5-(4-iodophenyl)-1,3,4-thiadiazol-2-yl)heptanediamide
(13)
1
Yield: 80%, mp: 309-312 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.22-1.32 (m,
2H), 1.46-1.67 (m, 4H), 1.95 (t, J = 7.5 Hz, 2H), 2.47-2.52 (m, 2H), 7.70-7.75 (m,
2H), 7.88-7.92 (m, 2H), 8.64 (s, 1H), 10.32 (s, 1H), 12.61 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.27, 24.79, 28.03, 32.07, 34.74, 97.26, 128.57, 129.71,
138.08, 158.58, 160.79, 168.98, 171.58; HRMS (AP-ESI) m/z calcd for
C15H17IN4O3S [M+H]+ 461.0139, found: 461.0140.
5.1.4.12.
N1-hydroxy-N7-(5-(4-(methylsulfonyl)phenyl)-1,3,4-thiadiazol-2-yl)heptanediami
de (14)
1
Yield: 69%, mp: 270-272 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.23-1.33 (m,
2H), 1.47-1.57 (m, 2H), 1.58-1.68 (m, 2H), 1.96 (t, J = 7.5 Hz, 2H), 2.50-2.54 (m,
2H), 3.29 (s, 3H), 8.05-8.08 (m, 2H), 8.20-8.23 (m, 2H), 8.65 (s, 1H), 10.32 (s, 1H),
12.73 (s, 1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.27, 24.78, 28.03, 32.06,
34.77, 43.35, 127.62, 127.99, 134.72, 141.96, 159.37, 160.01, 168.97, 171.77; HRMS
(AP-ESI) m/z calcd for C16H20N4O5S2 [M+H]+ 413.0948, found: 413.0952.
5.1.4.13. N1-hydroxy-N7-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)heptanediamide
(15)
1
Yield: 95%, mp: 296-299 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.23-1.33 (m,
2H), 1.47-1.57 (m, 2H), 1.58-1.68 (m, 2H), 1.96 (t, J = 7.5 Hz, 2H), 2.53 (t, J = 7.5
Hz, 2H), 8.20-8.24 (m, 2H), 8.33-8.37 (m, 2H), 8.65 (s, 1H), 10.32 (s, 1H), 12.77 (s,
1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.23, 24.77, 28.03, 32.06, 34.73,
18

124.47, 127.94, 136.02, 148.17, 159.58, 168.97, 171.76; HRMS (AP-ESI) m/z calcd
for C15H17N5O5S [M+H]+ 380.1023, found: 380.1027.
5.1.4.14.
N1-hydroxy-N7-(5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)heptanediami
de (16)
1
Yield: 82%, mp: 283-286 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.23-1.33 (m,
2H), 1.47-1.57 (m, 2H), 1.58-1.68 (m, 2H), 1.95 (t, J = 7.2 Hz, 2H), 2.49-2.54 (m,
2H), 7.88-7.90 (m, 2H), 8.15-8.18 (m, 2H), 8.64 (s, 1H), 10.32 (s, 1H), 12.70 (s, 1H);
13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.24, 24.78, 28.03, 32.06, 34.72, 122.06,
125.67, 126.15, 126.20, 127.54, 129.56, 129.98, 130.41, 130.84, 133.97, 159.10,
160.12, 168.98, 171.65; HRMS (AP-ESI) m/z calcd for C16H17F3N4O3S [M+H]+
403.1046, found: 403.1054.
5.1.4.15.
N1-hydroxy-N8-(5-(4-morpholinophenyl)-1,3,4-thiadiazol-2-yl)octanediamide (17)
1
Yield: 76%, mp: 224-226 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.27-1.28 (m,
4H), 1.44-1.54 (m, 2H), 1.56-1.63 (m, 2H), 1.94 (t, J = 7.2 Hz, 2H), 2.47 (t, J = 7.2
Hz, 2H), 3.23 (d, J = 4.8 Hz, 4H), 3.75 (d, J = 4.8 Hz, 4H), 7.04 (d, J = 8.7 Hz, 2H),
7.77 (d, J = 8.7 Hz, 2H), 8.64 (s, 1H), 10.31 (s, 1H), 12.44 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.47, 24.92, 28.20, 28.25, 32.18, 34.81, 47.37, 65.88,
114.59, 120.30, 127.84, 152.33, 157.16, 161.80, 169.04, 171.33; HRMS (AP-ESI) m/z
calcd for C20H27N5O4S [M+H]+ 434.1857, found: 434.1864.
5.1.4.16.
N1-(5-([1,1'-biphenyl]-4-yl)-1,3,4-thiadiazol-2-yl)-N8-hydroxyoctanediamide (18)
1
Yield: 88%, mp: 265-266 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.28-1.29 (m,
4H), 1.45-1.54 (m, 2H), 1.58-1.65 (m, 2H), 1.95 (t, J = 7.5 Hz, 2H), 2.49-2.53 (m,
2H), 7.39-7.44 (m, 1H), 7.48-7.53 (m, 2H), 7.74-7.77 (m, 2H), 7.84 (d, J = 8.4 Hz,
13
2H), 8.03 (d, J = 8.4 Hz, 2H), 8.64 (s, 1H), 10.32 (s, 1H), 12.62 (s, 1H); C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.44, 24.92, 28.21, 28.25, 32.18, 34.83, 126.67, 127.41,
127.46, 128.04, 129.04, 129.23, 139.00, 141.98, 158.33, 161.27, 169.04, 171.57;
HRMS (AP-ESI) m/z calcd for C22H24N4O3S [M+H]+ 425.1642, found: 425.1645.
5.1.4.17. N1-(5-(3-bromophenyl)-1,3,4-thiadiazol-2-yl)-N8-hydroxyoctanediamide
(19)
1
Yield: 70%, mp: 245-247 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.28-1.29 (m,
4H), 1.45-1.54 (m, 2H), 1.57-1.64 (m, 2H), 1.94 (t, J = 7.5 Hz, 2H), 2.47-2.52 (m,
2H), 7.49 (t, J = 7.8 Hz, 1H), 7.70-7.73 (m, 1H), 7.92 (d, J = 7.8 Hz, 1H), 8.12 (t, J =
1.8 Hz, 1H), 8.63 (s, 1H), 10.32 (s, 1H), 12.63 (s, 1H); 13C NMR (DMSO-d6, 75 MHz,
δ, ppm) 24.45, 24.92, 28.20, 28.24, 32.18, 34.91, 122.40, 126.07, 128.86, 131.46,
132.44, 133.06, 159.07, 159.92, 169.04, 171.79; HRMS (AP-ESI) m/z calcd for
C16H19BrN4O3S [M+H]+ 427.0434, found: 427.0439.
19

5.1.4.18. N1-(5-(2-bromophenyl)-1,3,4-thiadiazol-2-yl)-N8-hydroxyoctanediamide
(20)
1
Yield: 80%, mp: 165-167 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.28-1.29 (m,
4H), 1.45-1.54 (m, 2H), 1.57-1.64 (m, 2H), 1.94 (t, J = 7.2 Hz, 2H), 2.49-2.54 (m,
2H), 7.45-7.50 (td, J1 = 7.8 Hz, J2 = 1.8 Hz, 1H), 7.53-7.58 (td, J1 = 7.8 Hz, J2 = 1.2
Hz, 1H), 7.83-7.86 (dd, J1 = 7.8 Hz, J2 = 1.2 Hz, 1H), 7.93-7.96 (dd, J1 = 7.8 Hz, J2
= 1.8 Hz, 1H), 8.63 (d, J = 1.2 Hz, 1H), 10.31 (s, 1H), 12.65 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.41, 24.92, 28.20, 28.25, 32.18, 34.75, 121.31, 128.19,
131.06, 131.63, 131.86, 133.76, 159.05, 159.72, 169.05, 171.67; HRMS (AP-ESI)
m/z calcd for C16H19BrN4O3S [M+H]+ 427.0434, found: 427.0430.
5.1.4.19. N1-hydroxy-N8-(5-(4-iodophenyl)-1,3,4-thiadiazol-2-yl)octanediamide
(21)
1
Yield: 89%, mp: 284-286 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.27-1.28 (m,
4H), 1.44-1.53 (m, 2H), 1.56-1.66 (m, 2H), 1.93 (t, J = 7.2 Hz, 2H), 2.47-2.51 (m,
2H), 7.70-7.75 (m, 2H), 7.88-7.92 (m, 2H), 8.63 (d, J = 1.5 Hz, 1H), 10.31 (s, 1H),
12.64 (s, 1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.93, 25.42, 28.70, 28.75,
32.69, 35.32, 97.80, 129.10, 130.21, 138,61, 159.02, 161.34, 169.55, 172.10; HRMS
(AP-ESI) m/z calcd for C16H19IN4O3S [M+H]+ 475.0295, found: 475.0284.
5.1.4.20.
N1-hydroxy-N8-(5-(4-(methylsulfonyl)phenyl)-1,3,4-thiadiazol-2-yl)octanediamide
(22)
1
Yield: 66%, mp: 253-255 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.28-1.29 (m,
4H), 1.47-1.54 (m, 2H), 1.60-1.65 (m, 2H), 1.94 (t, J = 7.5 Hz, 2H), 2.50-2.54 (m,
2H), 3.29 (s, 3H), 8.05-8.08 (m, 2H), 8.20-8.23 (m, 2H), 8.64 (s, 1H), 10.31 (s, 1H),
12.75 (s, 1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.41, 24.91, 28.18, 28.23,
32.18, 34.83, 43.35, 127.63, 127.98, 134.70, 141.97, 159.30, 160.05, 169.04, 171.76;
HRMS (AP-ESI) m/z calcd for C17H22N4O5S2 [M+H]+ 427.1104, found: 427.1098.
5.1.4.21.
N1-hydroxy-N8-(5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)octanediamid
e (23)
1
Yield: 86%, mp: 281-284 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.28-1.29 (m,
4H), 1.45-1.54 (m, 2H), 1.58-1.65 (m, 2H), 1.95 (t, J = 7.2 Hz, 2H), 2.50-2.54 (m,
2H), 7.89 (d, J = 8.1 Hz, 2H), 8.17 (d, J = 8.1 Hz, 2H), 8.64 (s, 1H), 10.32 (s, 1H),
12.71 (s, 1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.40, 24.91, 28.19, 28.24,
32.17, 34.84, 122.08, 125.69, 126.13, 126.18, 126.23, 126.28, 127.56, 129.56, 129.99,
130.41, 130.84, 134.00, 159.16, 160.12, 169.03, 171.74; HRMS (AP-ESI) m/z calcd
for C17H19F3N4O3S [M+H]+ 417.1203, found: 417.1203.
5.1.4.22.
N1-hydroxy-N7-(5-(naphthalen-1-yl)-1,3,4-thiadiazol-2-yl)heptanediamide (24)
1
Yield: 99%, mp: 200-201 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.25-1.35 (m,
20

2H), 1.48-1.58 (m, 2H), 1.59-1.69 (m, 2H), 1.97 (t, J = 7.5 Hz, 2H), 2.53 (t, J = 7.5
Hz, 2H), 7.61-7.69 (m, 3H), 7.85 (d, J = 6.6 Hz, 1H), 8.04-8.09 (m, 1H), 8.12 (d, J =
8.1 Hz, 1H), 8.65-8.70 (m, 2H), 10.34 (s, 1H), 12.70 (s, 1H); 13C NMR (DMSO-d6, 75
MHz, δ, ppm) 24.33, 24.80, 28.05, 32.07, 34.74, 125.30, 125.48, 126.62, 126.78,
127.58, 128.55, 129.22, 129.96, 130.69, 133.54, 158.69, 160.94, 168.96, 171.66;
HRMS (AP-ESI) m/z calcd for C19H20N4O3S [M+H]+ 385.1329, found: 385.1323.
5.1.4.23.
N1-hydroxy-N7-(5-(naphthalen-2-yl)-1,3,4-thiadiazol-2-yl)heptanediamide (25)
1
Yield: 83%, mp: 209-210 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.25-1.34 (m,
2H), 1.48-1.58 (m, 2H), 1.59-1.69 (m, 2H), 1.97 (t, J = 7.2 Hz, 2H), 2.50-2.55 (m,
2H), 7.58-7.64 (m, 2H), 7.96-8.02 (m, 1H), 8.04-8.14 (m, 3H), 8.52 (s, 1H), 8.66 (s,
1H), 10.33 (s, 1H), 12.65 (s, 1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.32,
24.79, 28.05, 32.08, 34.76, 123.54, 126.96, 127.03, 127.40, 127.69, 127.73, 128.54,
128.89, 132.86, 133.65, 158.38, 161.78, 168.98, 171.54; HRMS (AP-ESI) m/z calcd
for C19H20N4O3S [M+H]+ 385.1329, found: 385.1334.
5.1.4.24. N1-hydroxy-N7-(5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl)heptanediamide
(26)
1
Yield: 86%, mp: 233-234 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.23-1.33 (m,
2H), 1.47-1.57 (m, 2H), 1.58-1.68 (m, 2H), 1.96 (t, J = 7.2 Hz, 2H), 2.49-2.54 (m,
2H), 7.55-7.59 (m, 1H), 8.31-8.35 (m, 1H), 8.65 (s, 1H), 8.69-8.71 (dd, J1 = 4.8 Hz,
13
J2 = 1.5 Hz, 1H), 9.12 (d, J = 2.1 Hz, 1H), 10.32 (s, 1H), 12.69 (s, 1H); C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.26, 24.78, 28.02, 32.06, 34.71, 124.26, 126.48,
134.30, 147.37, 151.12, 158.89, 168.99, 171.64; HRMS (AP-ESI) m/z calcd for
C14H17N5O3S [M+H]+ 336.1125, found: 336.1128.
5.1.4.25.
(27)
N1-hydroxy-N7-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)heptanediamide
1
Yield: 98%, mp: 230-231 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.23-1.33 (m,
2H), 1.47-1.57 (m, 2H), 1.58-1.68 (m, 2H), 1.96 (t, J = 7.2 Hz, 2H), 2.50-2.55 (m,
2H), 7.90-7.92 (m, 2H), 8.65 (s, 1H), 8.72-8.74 (m, 2H), 10.33 (s, 1H), 12.79 (s, 1H);
13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.22, 24.77, 28.02, 32.06, 34.72, 120.75,
137.14, 150.69, 159.50, 159.53, 168.97, 171.75; HRMS (AP-ESI) m/z calcd for
C14H17N5O3S [M+H]+ 336.1125, found: 336.1125.
5.1.4.26. N1-(5-(furan-2-yl)-1,3,4-thiadiazol-2-yl)-N7-hydroxyheptanediamide (28)
1
Yield: 65%, mp: 198-200 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.22-1.32 (m,
2H), 1.46-1.67 (m, 4H), 1.95 (t, J = 7.5 Hz, 2H), 2.47-2.52 (m, 2H), 6.72-6.74 (m,
1H), 7.19 (d, J = 3.0 Hz, 1H), 7.94 (d, J = 1.2 Hz, 1H), 8.64 (s, 1H), 10.32 (s, 1H),
12.67 (s, 1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.21, 24.77, 28.02, 32.05,
34.66, 110.75, 112.50, 145.12, 145.31, 152.38, 157.56, 168.98, 171.54; HRMS
(AP-ESI) m/z calcd for C13H16N4O4S [M+H]+ 325.0965, found: 325.0965.
21

5.1.4.27. N1-hydroxy-N7-(5-(thiophen-2-yl)-1,3,4-thiadiazol-2-yl)heptanediamide
(29)
1
Yield: 88%, mp: 206-208 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.22-1.32 (m,
2H), 1.46-1.66 (m, 4H), 1.95 (t, J = 7.5 Hz, 2H), 2.49 (t, J = 7.5 Hz, 2H), 7.19-7.22
(m, 1H), 7.67-7.70 (dd, J1 = 3.6 Hz, J2 = 0.9 Hz, 1H), 7.75-7.77 (dd, J1 = 5.1 Hz, J2
13
= 0.9 Hz, 1H), 8.64 (s, 1H), 10.32 (s, 1H), 12.63 (s, 1H); C NMR (DMSO-d6, 75
MHz, δ, ppm) 24.26, 24.78, 28.03, 32.06, 34.68, 128.35, 128.94, 129.07, 132.29,
156.05, 157.75, 168.97, 171.53; HRMS (AP-ESI) m/z calcd for C13H16N4O3S2
[M+H]+ 341.0737, found: 341.0748.
5.1.4.28. N1-hydroxy-N8-(5-(naphthalen-1-yl)-1,3,4-thiadiazol-2-yl)octanediamide
(30)
1
Yield: 90%, mp: 201-203 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.30-1.31 (m,
4H), 1.46-1.55 (m, 2H), 1.59-1.66 (m, 2H), 1.95 (t, J = 7.5 Hz, 2H), 2.53 (t, J = 7.5
Hz, 2H), 7.61-7.69 (m, 3H), 7.87-7.90 (dd, J1 = 6.9 Hz, J2 = 0.9 Hz, 1H), 8.04-8.08
(m, 1H), 8.12 (d, J = 8.4 Hz, 1H), 8.65-8.69 (m, 2H), 10.32 (s, 1H), 12.66 (s, 1H); 13C
NMR (DMSO-d6, 75 MHz, δ, ppm) 24.48, 24.92, 28.21, 28.26, 32.19, 34.82, 125.30,
125.48, 126.62, 126.78, 127.58, 128.55, 129.22, 129.96, 130.70, 133.54, 158.67,
160.96, 169.05, 171.68; HRMS (AP-ESI) m/z calcd for C20H22N4O3S [M+H]+
399.1485, found: 399.1490.
5.1.4.29. N1-hydroxy-N8-(5-(naphthalen-2-yl)-1,3,4-thiadiazol-2-yl)octanediamide
(31)
1
Yield: 98%, mp: 237-239 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.29-1.30 (m,
4H), 1.46-1.55 (m, 2H), 1.59-1.66 (m, 2H), 1.95 (t, J = 7.2 Hz, 2H), 2.52 (t, J = 7.2
Hz, 2H), 7.58-7.64 (m, 2H), 7.97-8.02 (m, 1H), 8.04-8.14 (m, 3H), 8.51 (s, 1H), 8.67
13
(s, 1H), 10.33 (s, 1H), 12.63 (s, 1H); C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.99,
25.44, 28.73, 28.77, 32.70, 35.41, 124.04, 127.44, 127.52, 127.89, 128.23, 129.04,
129.39, 133.37, 134.15, 158.99, 162.23, 169.56, 172.14; HRMS (AP-ESI) m/z calcd
for C20H22N4O3S [M+H]+ 399.1485, found: 399.1490.
5.1.4.30.
(32)
N1-hydroxy-N8-(5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl)octanediamide
1
Yield: 86%, mp: 197-198 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.28-1.29 (m,
4H), 1.45-1.54 (m, 2H), 1.58-1.65 (m, 2H), 1.95 (t, J = 7.5 Hz, 2H), 2.51 (t, J = 7.5
Hz, 2H), 7.55-7.59 (m, 1H), 8.31-8.35 (m, 1H), 8.64 (s, 1H), 8.69-8.71 (dd, J = 4.8 Hz,
J = 1.2 Hz, 1H), 9.12 (d, J = 1.8 Hz, 1H), 10.32 (s, 1H), 12.69 (s, 1H); 13C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.41, 24.91, 28.18, 28.24, 32.18, 34.81, 124.26, 126.49,
134.30, 147.38, 151.13, 158.89, 169.04, 171.68; HRMS (AP-ESI) m/z calcd for
C15H19N5O3S [M+H]+ 350.1281, found: 350.1275.
5.1.4.31. N1-hydroxy-N8-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)octanediamide
(33)
1
Yield: 97%, mp: 248-250 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.28-1.29 (m,
22

4H), 1.45-1.54 (m, 2H), 1.58-1.65 (m, 2H), 1.95 (t, J = 7.5 Hz, 2H), 2.52 (t, J = 7.5
Hz, 2H), 7.90-7.92 (m, 2H), 8.64 (s, 1H), 8.72-8.74 (m, 2H), 10.32 (s, 1H), 12.65 (s,
1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.38, 24.91, 28.18, 28.23, 32.17, 34.82,
120.75, 137.15, 150.69, 159.52, 169.05, 171.79; HRMS (AP-ESI) m/z calcd for
C15H19N5O3S [M+H]+ 350.1281, found: 350.1289.
5.1.4.32. N1-(5-(furan-2-yl)-1,3,4-thiadiazol-2-yl)-N8-hydroxyoctanediamide (34)
1
Yield: 98%, mp: 203-204 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.27-1.28 (m,
4H), 1.44-1.54 (m, 2H), 1.56-1.63 (m, 2H), 1.94 (t, J = 7.2 Hz, 2H), 2.47-2.52 (m,
2H), 6.72-6.73 (m, 1H), 7.18 (d, J = 3.3 Hz, 1H), 7.94 (d, J = 1.5 Hz, 1H), 8.63 (s,
1H), 10.31 (s, 1H), 12.63 (s, 1H); 13C NMR (DMSO-d6, 75 MHz, δ, ppm) 24.41,
24.91, 28.19, 28.23, 32.17, 34.80, 128.32, 128.89, 129.02, 132.32, 156.02, 157.82,
169.06, 171.59; HRMS (AP-ESI) m/z calcd for C14H18N4O4S [M+H]+ 339.1122,
found: 339.1130.
5.1.4.33. N1-hydroxy-N8-(5-(thiophen-2-yl)-1,3,4-thiadiazol-2-yl)octanediamide
(35)
1
Yield: 87%, mp: 205-206 °C. H NMR (DMSO-d6, 300 MHz, δ, ppm) 1.27-1.29 (m,
4H), 1.44-1.54 (m, 2H), 1.56-1.63 (m, 2H), 1.94 (t, J = 7.5 Hz, 2H), 2.47-2.51 (m,
2H), 7.19-7.22 (m, 1H), 7.68-7.70 (dd, J1 = 3.6 Hz, J2 = 0.9 Hz, 1H), 7.74-7.76 (dd,
13
J1 = 5.1 Hz, J2 = 0.9 Hz, 1H), 8.63 (s, 1H), 10.31 (s, 1H), 12.51 (s, 1H); C NMR
(DMSO-d6, 75 MHz, δ, ppm) 24.38, 24.90, 28.19, 28.23, 32.17, 34.80, 110.70, 112.49,
145.16, 145.27, 152.33, 157.69, 169.06, 171.65; HRMS (AP-ESI) m/z calcd for
C14H18N4O3S2 [M+H]+ 355.0893, found: 355.0897.
5.2. In vitro HDAC assay
According to the instruction of Color de Lys assay kits, HeLa cell nuclear extracts
(5 µL/well) and different concentrations of compounds (10 µL/well) were incubated at
37 °C for 5 minutes in the 96-well plate. After addition of Color de Lys substrate (25
µL), the resulting mixture reacted at 37 °C for 30 minutes. At the end Color de Lys
Developer (50 µL/well) was added. After incubation for 15 minutes, enzymatic
activity was measured in a microtiter-plate reader at 405 nm.
5.3. Molecular Docking
Surflex-Dock program in Sybyl 7.3 was used with default values except specially
referred to. The structure of compound 33 was optimized using Powell’s method and
then assigned with AM1-BCC atomic charges. The active site was generated based on
the ligand in the co-crystal structure of HDAC2-SAHA (PDB code: 4LXZ). The
top-scored conformation was selected as the best docking result.
5.4. MTT Assay
MDA-MB-231 breast cancer cell, K562 chronic myelogenous leukaemia cell and
PC3 prostate cancer cell were cultured in RPMI1640 medium (10% FBS) in 5% CO2
humidified incubator at 37 °C. Cancer cells were plated at 5,000 cells per well in
23

96-well plates and treated with 60, 20, 6.67, 2.22, 0.74, 0.25 µM of compounds for 48
h. After addition of 5 mg/mL MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl
-2H-tetrazolium bromide) solution (20 µL) and further incubation for 4 h, DMSO (150
µL) was added for extraction of utiformazan. Antiproliferative activity was
determined in a microtiter-plate reader at 570 nm.
Acknowledgments
This work was supported by National Natural Science Foundation China (No.
81373281), Shandong Natural Science Fund for Distinguished Young Scholars (No.
JQ201319), the Program for New Century Excellent Talents in University (No.
NCET-12-0337), Independent Innovation Foundation of Shandong University,
IIFSDU (No. 2012JC003), and Program for Changjiang Scholars and Innovative
Research Team in University, PCSIRT (No. IRT13028).
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