Design, synthesis and biological evaluation of saccharin-based N-hydroxybenzamides as histone deacetylases (HDACs) inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.07.008

We report the development of a novel series of saccharin-based N-hydroxybenzamides as histone deacetylases inhibitors. Among them, 6j exhibited potent HDACs inhibitory activity against Hela nuclear extract. Further biological evaluation found 6i showed similar antiproliferative activities in vitro compared with the approved SAHA.

Full text of DOI:10.1016/j.bmc.2015.07.008

Accepted Manuscript
Design, synthesis and biological evaluation of saccharin-based N-hydroxyben-
zamides as histone deacetylases (HDACs) inhibitors
Huansheng Fu, Leiqiang Han, Xuben Hou, Yanyan Dun, Lei Wang, Xiaowei
Gong, Hao Fang
PII:
S0968-0896(15)00575-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.008
BMC 12437
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 February 2015
22 June 2015
7 July 2015
Please cite this article as: Fu, H., Han, L., Hou, X., Dun, Y., Wang, L., Gong, X., Fang, H., Design, synthesis and
biological evaluation of saccharin-based N-hydroxybenzamides as histone deacetylases (HDACs) inhibitors,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis and biological evaluation of saccharin-based
N-hydroxybenzamides as histone deacetylases (HDACs) inhibitors
Huansheng Fua, Leiqiang Hana, Xuben Houa, Yanyan Duna, Lei Wanga, Xiaowei
Gongb, Hao Fanga*
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmacy, Shandong University, 44 West Wenhua Rd, Ji’nan
250012, PR China
bShandong Provincial Key Laboratory of Neuroprotective Drug, Shandong Qidu
Pharmaceutical Co., Ltd, 250012, China
ABSTRACT
We report the development of
a
novel series of saccharin-based
N-hydroxybenzamides as histone deacetylases inhibitors. Among them, 6j exhibited
potent HDACs inhibitory activity against Hela nuclear extract. Further biological
evaluation found 6i showed similar antiproliferative activities in vitro compared with
the approved SAHA.
Keywords：Saccharin; N-Hydroxybenzamide; Histone deacetylases inhibitor
1. Introduction
As the basic composition unit of nucleosome, histones can be covalently
modified at N-terminal, the so called post-translational modifications, which can
adjust gene expression without changing the primary DNA sequence. Acetylation of
* Corresponding author. Phone/Fax: +86-531-88382731. E-mail : haofangcn@sdu.edu.cn.

lysines is the important one of them. It is regulated by the function of histone acetyl
transferases (HATs) and histone deacetylases (HDACs). HDACs have a significant
effect on cell cycle, cell growth, differentiation, senescence, apoptosis,
immunogenicity, immune suppression and angiogenesis1 and their dysregulated
expression has been observed in malignancies2, neurological disorders3, viral
infection4, inflammation5 and so on. Therefore, HDACs have been identified as
potential drug targets for a broad range of human disorders especially malignancies.
So far, three HDACs inhibitors have been approved (Figure 1): Vorinostat (SAHA)
and Romidepsin (FK228) for the treatment of cutaneous T-cell lymphoma (CTCL) in
2006 and 2009, respectively6, and Belinostat (PXD101) for the treatment of peripheral
T-cell lymphoma (PTCL) in 20147. Generally, the pharmacophore of HDACs
inhibitors is constituted of three motifs: a zinc binding group (ZBG), a linker, and a
surface recognition motif.
Figure 1. Approved HDACs inhibitors
In
our
previous
work,
we
reported
the
discovery
of
as
1,2-dihydrobenzo[d]isothiazol-3-one-1,1-dioxide
(saccharin)
derivatives

submicromolar HDACs inhibitors.8 Some of them showed higher inhibitory potency
and comparative or better antiproliferative activities compared with SAHA. The
results indicated that saccharin derivatives could be used as lead compounds to
develop potent HDACs inhibitors as anticancer agents. Also, N-hydroxybenzamide
has been widely studied as efficient ZBG of HDACs inhibitors.9-11 And our previous
studies showed N-hydroxybenzamide derivatives had good inhibitory activities
against HDACs and antiproliferative activities in the xenograft models.12-14 In our
on-going studies, saccharin-based N-hydroxybenzamide derivatives were developed
as HDACs inhibitors. Although one compound (R = H, linker = CH2, ZBG =
4-substituted N-hydroxybenzamide, Figure 2) was mentioned in a patent15, variation
of R group or linker and related SAR were not reported. In this article, we reported
the synthesis and preliminary biological evaluation of these saccharin-based
N-hydroxybenzamide derivatives.
Figure 2. HDACs inhibitor pharmacophore model and our saccharin-based
N-hydroxybenzamide derivatives.
2. Chemistry
The synthesis of our newly designed HDACs inhibitors is outlined in Schemes
1-3. Coupling reaction of bromoalkylcarboxylic acids (1a-1d) and substituted amines
(2a-2c) afforded the key intermediates 3a-3j. Then, the target compounds 6a-6r were
obtained through alkylation with saccharins (4a-4d) and a mixed anhydride approach

according to literature procedures8.
As shown in Scheme 2, final N-hydroxybenzamides 8a-8d were prepared
following the similar procedures as Scheme 1. More 6-substituted saccharin
derivatives were synthesized from 7b, followed by reduction, acylation and finally
converted to target compounds 11a–11g in Scheme 3.
Scheme 1. Synthesis of compounds 6a-6r. Reagents: (a) oxalyl chloride, NaHCO3, THF; (b)
saccharin or 6-substituted saccharin (4a-4d), NaHCO3, DMF; (c) ClCOO-iBu, Et3N, NH2OH,
THF.
Scheme 2. Synthesis of compounds 8a-8d. Reagents: (a) NaHCO3, DMF; (b) ClCOO-iBu, Et3N,
NH2OH, THF.
Scheme 3. Synthesis of compounds 11a-11g. Reagents: (a) H2, Pd/C, THF; (b) (i) R3CO2H, SOCl2;
(ii) NaHCO3, THF; (c) ClCOO-iBu, Et3N, NH2OH, THF.

3. Results and discussion
HDACs inhibitory activities of the compounds were evaluated by the Color de
Lys assay used HeLa nuclear extract as the enzyme source. The results were
summarized as IC50 values in Table 1.
As shown in Table 1, when the aromatic ring A was meta substituted, the
derivatives 6a-6e did not show inhibitory activities against HeLa nuclear extract.
However, most of the compounds with para substituted N-hydroxybenzamide
exhibited good HDACs inhibitory activities. In addition, the linker had a significant
impact on the inhibitory activities against HDACs. For example, all the target
compounds with the linker of different N-methylene alkylamides (6p-6r) displayed
poor inhibition on HDACs (IC50 > 2 µM). But some target compounds with
propanamide (6f) or butylamide group (6j) possessed similar inhibitory activities
compared with positive control SAHA. Besides, most of the target compounds with
methylene as the linker (8a-8d, 11a-11g) also exhibited good inhibitory activities.
It needs to be noted that substitution at 6-position of saccharin (R) could also
influence the target compounds’ potency. For example, IC50 value of 6j (R = H) or 6l
(R = I) was lower than 0.5 µM. While the nitro and phenyl were introduced to
6-position, compounds 6k (R = NO2) and 6m (R = Ph) displayed poor inhibition
against HDACs. According to the data of target compounds 8a-8d and 11a-11g,
phenyl (8d, IC50 = 0.386 µM) or benzamido (11a, IC50 = 0.380 µM) in 6-position
substitution gave much better inhibitory activities compared with 8a (IC50 = 1.398 µM)
without substitution.

Table 1. In vitro HDACs activity
Position of
substituents on A
meta
IC50 of
HDACsa (µM)
>5
Compd
R
Linker
H
-(CH2)2CONH-
-(CH2)3CONH-
-(CH2)4CONH-
-(CH2)5CONH-
-(CH2)5CONH-
-(CH2)2CONH-
-(CH2)2CONH-
-(CH2)2CONH-
-(CH2)2CONH-
-(CH2)3CONH-
-(CH2)3CONH-
-(CH2)3CONH-
-(CH2)3CONH-
-(CH2)4CONH-
-(CH2)4CONH-
-(CH2)2CONHCH2-
-(CH2)4CONHCH2-
-(CH2)5CONHCH2-
-CH2-
6a
6b
H
>5
meta
H
>5
6c
meta
H
>5
6d
meta
NO2
>5
6e
meta
H
0.300 0.021
2.02 0.77
0.523 0.161
1.70 0.651
0.233 0.136
1.66 1.45
0.419 0.243
1.51 1.23
0.305 0.028
>5
6f
para
NO2
6g
para
I
6h
para
Ph
6i
para
H
6j
para
NO2
6k
para
I
6l
para
Ph
6m
6n
para
I
para
Ph
6o
para
H
2.79 1.00
3.40 0.84
2.65 0.38
1.398 0.554
>5
6p
para
H
6q
para
H
6r
para
H
NO2
8a
para
-CH2-
8b
para
I
-CH2-
1.335 0.330
0.386 0.062
0.380 0.069
>5
8c
para
Ph
-CH2-
8d
para
Ph-CONH-
-CH2-
11a
11b
11c
11d
11e
11f
11g
SAHA
para
4-diPh-CONH-
3,4,5-triOCH3-Ph-CONH-
4-CF3-Ph-CONH-
4-F-Ph-CONH-
Bn-CONH-
-CH2-
para
-CH2-
1.710 0.039
1.413 0.085
0.646 0.209
0.530 0.202
1.297 0.213
0.193 0.082
para
-CH2-
para
-CH2-
para
-CH2-
para
4-CH3-Ph-CH=CH-CONH-
-CH2-
para
a Inhibitory data are means of no fewer than two independent determinations and expressed with
standard deviations.

To study the interaction between the inhibitors and HDACs, we docked
compound 6j and SAHA in the active site of HDAC 2 (PDB entry: 4LXZ) using
Surflex Dock. The results suggested that 6j possessed a similar binding mode to
SAHA in the active site of HDAC2. Both The hydroxamic acid moiety of SAHA and
the N-hydroxybenzamide moiety of 6j could chelate to the Zn2+ ion as well as form
hydrogen bond with Tyr308 in the HDAC2 active site. (Figure 3a) In addition, the
π-π interactions of aromatic ring in 6j with Phe155 and His183 could further increase
the interaction with HDAC 2. (Figure 3b) The above interactions might contribute to
the potency of 6j against HDACs.
a
b
Figure 3. (a) Proposed binding mode of 6j.
(b) The interplays between compound 6j and HDAC2 in the active site by software:
Molecular Operating Environment.
To further test the saccharin N-hydroxybenzamide HDACs inhibitors at the
cellular level, we screened the potent compounds (IC50 of HDACs < 2.0 µM) with
MDA-MB-231 (Figure 4). And three compounds 6i, 6j and 6m were selected to
evaluate their antiproliferative activities in vitro using cancer cell lines of
MDA-MB-231, KG1 and PC-3 by MTT assay (Table 2). According to the inhibition
data, all tested compounds showed potent antiproliferative activities. Among them,

target compound 6i displayed similar activities compared with SAHA.
Figure 4. Inhibitions of selected compounds to MDA-MB-231 at 10 µM.
Table 2. Antiproliferative activities of representative compounds
IC50 (µM) a
Compd
MDA-MB-231
3.45 0.59
8.71 0.28
9.90 0.90
4.61 0.80
KG1
PC-3
8.79 0.63
>25
5.23 0.64
NDb
6i
6j
11.6 2.7
2.75 0.40
>25
6m
SAHA
10.4 0.9
a Inhibitory data are means of three independent determinations and expressed with standard
deviations. b ND = no data.
As some reports showed that inhibition of HDACs could induce apoptosis in
carcinoma cells16, the ability of compounds 6i and 6j inducing human breast cancer
cell (MDA-MB-231) apoptosis was studied and compared with that of SAHA (Figure
5). The flow cytometry analysis results showed that 6i, 6j and SAHA could induce
apoptosis in a dose-dependent manner after treated for 24 h, and the apoptosis rates at
1µM were 16.76% (6i), 7.50% (6j) and 11.32% (SAHA), respectively. These data
were consistent with that in MTT assay.

Figure 5. Inducing apoptosis of MDA-MB-231 by 6i, 6j and SAHA at 1 µM and 4 µM after 24 h.
4. Conclusions
We have designed and synthesized a novel series of saccharin-based
N-hydroxybenzamides as HDACs inhibitors. The preliminary enzyme assay showed
that the structure of the linker and substituents at 6-position of saccharin ring had
significant effects on the HDACs inhibitory activities. Target compound 6j exhibited
potent HDACs inhibitory activity against Hela nuclear extract. And 6i had similar in
vitro antiproliferative activities compared with SAHA. These results suggest
saccharin-based N-hydroxybenzamide derivatives could be used as lead compounds to
develop novel antitumor agents in the future.
5. Experimental section
5.1. Chemistry
All materials and reagents from commercial suppliers were analytical and used
without further purification. All reactions were monitored by thin-layer
chromatography (TLC) on 0.25 mm silica gel plates (60GF-254) and visualized with

UV light, ninhydrin, chloride ferric or iodine vapor. Melting points were recorded by
1
the RY-1G electrothermal melting point apparatus without correction. H NMR and
13CNMR spectra were obtained on Bruker spectrometer (600 MHz, 400MHz and 300
MHz). The chemical shifts were expressed as δ values (parts per million) relative to
TMS 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 spectrums
afforded on an Agilent 6510 Quadrupole Time-of-Flight LC/MS deliver.
General Procedure for the Preparation of 6a-6r, 8a-8d and 11a-11g
Under ice bath, the substituted benzoic acid (1.0 mmol) was dissolved in
anhydrous THF (20 ml), and Et3N (0.17 ml, 1.2 mmol) and isobutyl chloroformate
(0.15 ml, 1.2 mmol) was added in sequence. The resulting solution was stirred for 10
min at 0 °C. Then a solution of NH2OH.HCl (0.28 g, 4.0 mmol) and Et3N (0.55 ml,
4.0 mmol) in anhydrous methanol (1 ml) was added. The resulting mixture was stirred
for 3–8 h at RT. After removing the solvents, the resulting residue was dissolved in
ethyl acetate (20 ml), and washed with HCl (1 M, 10×3 ml), H2O (10×3 ml) and
saturated sodium chloride (10×3 ml), respectively. The crude product was purified by
column chromatography (DCM/MeOH=50:1).
5.1.1. 3-(3-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)propanamido)-N-hydro
1
xybenzamide (6a) Yield 20%, mp 199-201 °C. H NMR (300 MHz, DMSO-d6) δ
11.16 (s, 1H), 10.22 (s, 1H), 9.00 (d, J = 1.8 Hz, 1H), 8.31 (dd, J = 6.9 Hz, 0.9 Hz,
1H), 8.14-8.12 (m, 1H), 8.09-7.98 (m, 2H), 7.96 (s, 1H), 7.76-7.72 (m, 1H), 7.40-7.32
(m, 2H), 4.05 (t, J = 7.2 Hz, 2H), 2.86 (t, J = 7.2 Hz, 2H), 2.11-2.01 (m, 2H). 13C

NMR (75 MHz, DMSO-d6) δ 168.12, 164.28, 158.40, 139.14, 136.81, 135.82, 135.28,
133.52, 128.69, 126.34, 125.09, 121.77, 121.54, 121.28, 118.29, 34.94, 34.86. HRMS
(AP-ESI) m/z calcd for C17H15N3O6S [M + H]+ 390.0754, found 390.0750.
5.1.2. 3-(4-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)butanamido)-N-hydro
1
xybenzamide (6b) Yield 12%, mp 180 °C (dec.). H NMR (300 MHz, DMSO-d6) δ
11.15 (s, 1H), 10.06 (s, 1H), 9.00 (s, 1H), 8.32 (dd, J = 6.9 Hz, 0.9 Hz, 1H), 8.14-8.11
(m, 1H), 8.09-8.00 (m, 2H), 7.97 (s, 1H), 7.75-7.71 (m, 1H), 7.38-7.31 (m, 2H), 3.81
(t, J = 7.2 Hz, 2H), 2.47 (t, J = 7.2 Hz, 2H), 2.11-2.01 (m, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 170.46, 164.42, 158.71, 139.44, 136.87, 135.86, 135.35, 133.60, 128.75,
126.49, 125.18, 121.66, 121.60, 121.15, 118.22, 38.30, 33.19, 23.82. HRMS (AP-ESI)
m/z calcd for C18H17N3O6S [M + H]+ 404.0911, found 404.0907.
5.1.3 3-(5-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)pentanamido)-N-hydro
1
xybenzamide (6c) Yield 12%, mp 180 °C (dec.). H NMR (300 MHz, DMSO-d6) δ
11.16 (s, 1H), 10.05 (s, 1H), 9.01 (s, 1H), 8.31 (d, J = 7.2 Hz, 1H), 8.13-7.98 (m, 4H),
7.77-7.73 (m, 1H), 7.39-7.32 (m, 2H), 3.76 (t, J = 6.9 Hz, 2H), 2.39 (t, J = 6.9 Hz,
2H), 1.88-1.65 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ 170.99, 164.31, 158.59,
139.38, 136.77, 135.74, 135.23, 133.49, 128.64, 126.32, 125.05, 121.52, 121.48,
121.02, 118.07, 38.47, 35.68, 27.48, 22.24. HRMS (AP-ESI) m/z calcd for
C19H19N3O6S [M + H]+ 418.1067, found 418.1062.
5.1.4.
3-(6-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)hexanamido)-N-
1
hydroxybenzamide (6d) Yield 30%, mp 156-158 °C. H NMR (300 MHz, DMSO-d6)
δ 11.14 (s, 1H), 10.00 (s, 1H), 8.99 (s, 1H),8.30 (dd, J = 7.2 Hz, 0.6 Hz, 1H),

8.12-8.09 (m, 1H), 8.08-7.99 (m, 2H), 7.97 (s, 1H), 7.76-7.72 (m, 1H), 7.37-7.30 (m,
2H), 3.73 (t, J = 7.2 Hz, 2H), 2.32 (t, J = 7.2 Hz, 2H), 1.82-1.72 (m, 2H), 1.69-1.60
(m, 2H), 1.45-1.35 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 171.25, 164.34, 158.58,
139.44, 136.78, 135.74, 135.23, 133.48, 128.63, 126.32, 125.05, 121.55, 121.47,
120.99, 118.10, 38.59, 36.14, 27.69, 25.76, 24.50. HRMS (AP-ESI) m/z calcd for
C20H21N3O6S [M + H]+ 432.1224, found 432.1219.
5.1.5.
N-hydroxy-3-(6-(6-nitro-1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
1
hexanamido)benzamide (6e) Yield 27%, mp 169-171 °C. H NMR (300 MHz,
DMSO-d6) δ 11.15 (s, 1H), 10.00 (s, 1H), 9.25 (d, J = 1.8 Hz, 1H), 9.00 (s, 1H), 8.71
(dd, J = 8.4 Hz, 2.1 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.97 (s, 1H), 7.75-7.72 (m, 1H),
7.37-7.30 (m, 2H), 3.78 (t, J = 7.2 Hz, 2H), 2.33 (t, J = 7.2 Hz, 2H), 1.84-1.74 (m,
2H), 1.70-1.60 (m, 2H), 1.47-1.37 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 171.22,
164.31, 157.05, 151.81, 139.43, 137.67, 133.48, 130.98, 130.25, 128.62, 126.78,
121.54, 120.97, 118.11, 117.93, 36.11, 27.57, 25.72, 24.47. HRMS (AP-ESI) m/z
calcd for C20H20N4O8S [M + H]+ 477.1075, found 477.1061.
5.1.6.
4-(3-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)propanamido)-N-
1
hydroxybenzamide (6f) Yield 26%, mp 208 °C (dec.). H NMR (300 MHz,
DMSO-d6) δ 11.09 (s, 1H), 10.29 (s, 1H), 8.31 (dd, J = 6.9 Hz, 0.9 Hz, 1H), 8.13 (dd,
J = 6.9 Hz, 0.9 Hz, 1H), 8.09-7.98 (m, 2H), 7.89 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 8.7
Hz, 2H), 4.06 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 168.33, 163.94, 158.41, 141.49, 136.80, 135.82, 135.29, 127.67, 127.30,
126.33, 125.09, 121.54, 118.51, 35.00, 34.81. HRMS (AP-ESI) m/z calcd for

C17H15N3O6S [M + H]+ 390.0754, found 390.0753.
5.1.7.
N-hydroxy-4-(3-(6-nitro-1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
1
propanamido)benzamide (6g) Yield 30%, mp >250 °C. H NMR (300 MHz,
DMSO-d6) δ 11.09 (s, 1H), 10.30 (s, 1H), 9.27 (d, J = 1.8 Hz, 1H), 8.93 (s, 1H), 8.72
(dd, J = 8.4 Hz, 1.8 Hz, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.62
(d, J = 8.7 Hz, 2H), 4.09 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H). 13C NMR (75
MHz, DMSO-d6) δ 168.21, 164.02, 156.87, 151.83, 141.44, 137.66, 130.96, 130.28,
127.63, 127.34, 126.78, 118.50, 118.00, 35.33, 34.80. HRMS (AP-ESI) m/z calcd for
C17H14N4O8S [M + H]+ 435.0605, found 435.0616.
5.1.8.
N-hydroxy-4-(3-(6-iodo-1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
1
propanamido)benzamide (6h) Yield 33%, mp 250 °C (dec.). H NMR (300 MHz,
DMSO-d6) δ 11.08 (s, 1H), 10.30 (s, 1H), 8.98 (s, 1H), 8.82 (s, 1H), 8.37 (d, J = 8.1
Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.62 (d, J = 8.7 Hz, 2H),
4.02 (t, J = 7.2Hz, 2H), 2.85 (t, J = 7.2 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ
168.25, 163.87, 158.07, 144.04, 141.42, 137.81, 129.88, 127.59, 126.29, 125.72,
118.49, 103.90, 34.86. HRMS (AP-ESI) m/z calcd for C22H17N3O6S [M + H]+
515.9721, found 515.9722.
5.1.9. 4-(3-(1,1-dioxido-3-oxo-6-phenylbenzo[d]isothiazol-2(3H)-yl)propanamido)
1
-N-hydroxybenzamide (6i) Yield 25%, mp 245 °C (dec.). H NMR (300 MHz,
DMSO-d6) δ 11.10 (s, 1H), 10.31 (s, 1H), 8.94 (s, 1H), 8.67 (d, J = 1.2 Hz, 1H), 8.30
(dd, J = 8.1 Hz, 1.2 Hz, 1H), 8.17 (d, J = 8.1 Hz, 1H), 7.89 (dd, J = 8.1 Hz, 1.5 Hz,
2H), 7.71 (d, J = 9.0 Hz, 2H), 7.64 (d, J = 9.0 Hz, 2H), 7.59-7.51 (m, 3H), 4.07 (t, J =

7.2Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 168.30, 163.90,
158.19, 147.38, 141.46, 137.94, 136.99, 133.16, 129.47, 129.25, 127.63, 127.49,
127.27, 125.58, 124.91, 119.41, 118.49, 35.02, 34.86. HRMS (AP-ESI) m/z calcd for
C23H19N3O6S [M + H]+ 466.1067, found 466.1062.
5.1.10. 4-(4-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)butanamido)-N-hydro
1
xybenzamide (6j) Yield 27%, mp 229-230 °C. H NMR (300 MHz, DMSO-d6) δ
11.09 (s, 1H), 10.15 (s, 1H), 8.93 (d, J = 1.2 Hz, 1H), 8.32 (d, J = 7.5 Hz, 1H),
8.13-7.98 (m, 3H), 7.70 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 8.7 Hz, 2H), 3.81 (t, J = 7.2
Hz, 2H), 2.50 (t, J = 7.2 Hz, 2H), 2.10-2.01 (m, 2H). 13C NMR (75 MHz, DMSO-d6)
δ 170.52, 163.92, 158.58, 141.70, 136.74, 135.73, 135.21, 127.62, 127.01, 126.36,
125.04, 121.47, 118.28, 38.15, 33.11, 23.59. HRMS (AP-ESI) m/z calcd for
C18H17N3O6S [M + H]+ 404.0911, found 404.0910.
5.1.11.
N-hydroxy-4-(4-(6-nitro-1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
1
butanamido)benzamide (6k) Yield 35%, mp 172-174°C. H NMR (300 MHz,
DMSO-d6) δ 11.09 (s, 1H), 10.16 (s, 1H), 9.28 (d, J = 1.8 Hz, 1H), 8.93 (s, 1H), 8.72
(dd, J = 8.4 Hz, 1.8 Hz, 1H), 8.33 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.63
(d, J = 8.7 Hz, 2H), 3.86 (t, J = 7.2 Hz, 2H), 2.50 (t, J = 7.2 Hz, 2H), 2.12-2.02 (m,
2H). 13C NMR (75 MHz, DMSO-d6) δ 170.54, 163.94, 157.07, 151.79, 141.71,
137.64, 131.09, 130.24, 127.64, 127.04, 126.77, 118.31, 117.93, 38.75, 33.08, 23.50.
HRMS (AP-ESI) m/z calcd for C18H16N4O8S [M + H]+ 449.0762, found 449.0764.
5.1.12.
N-hydroxy-4-(4-(6-iodo-1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
1
butanamido)benzamide (6l) Yield 40%, mp 220 °C (dec.). H NMR (300 MHz,

DMSO-d6) δ 11.11 (s, 1H), 10.14 (s, 1H), 8.95 (s, 1H), 8.83 (s, 1H), 8.37 (d, J = 8.1
Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 8.7 Hz, 2H),
3.79 (t, J = 6.9 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H), 2.09-1.99 (m, 2H). 13C NMR (75
MHz, DMSO-d6) δ 170.53, 163.88, 158.29, 144.01, 141.70, 137.80, 129.86, 127.62,
127.02, 126.29, 125.82, 118.31, 103.78, 38.26, 33.10, 23.56. HRMS (AP-ESI) m/z
calcd for C18H16IN3O6S [M + H]+ 529.9877, found 529.9875.
5.1.13. 4-(4-(1,1-dioxido-3-oxo-6-phenylbenzo[d]isothiazol-2(3H)-yl)butanamido)-
1
N-hydroxybenzamide (6m) Yield 22%, mp 240 °C (dec.). H NMR (300 MHz,
DMSO-d6) δ 11.09 (s, 1H), 10.15 (s, 1H), 8.93 (s, 1H), 8.69 (d, J = 1.5 Hz, 1H), 8.30
(dd, J = 8.1 Hz, 1.5 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.92-7.86 (m, 2H), 7.70 (d, J =
8.7 Hz, 2H), 7.64 (d, J = 8.7 Hz, 2H), 7.59-7.49 (m, 3H), 3.83 (t, J = 6.9 Hz, 2H),
2.50 (t, J = 6.9 Hz, 2H), 2.12-2.03 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 170.75,
164.13, 158.62, 147.55, 141.92, 138.12, 137.22, 133.35, 130.52, 129.68, 129.47,
127.83, 127.72, 127.23, 125.79, 125.21, 119.60, 118.49, 38.42, 33.33, 23.85. HRMS
(AP-ESI) m/z calcd for C24H21N3O6S [M + H]+ 480.1224, found 480.1219.
5.1.14.
N-hydroxy-4-(5-(6-iodo-1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
1
pentanamido)benzamide (6n) Yield 24%, mp 230-231 °C. H NMR (300 MHz,
DMSO-d6) δ 11.08 (s, 1H), 10.11 (s, 1H), 8.92 (s, 1H), 8.82 (d, J = 1.2 Hz, 1H), 8.36
(dd, J = 8.1 Hz, 1.2 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.63
(d, J = 8.7 Hz, 2H), 3.73 (t, J = 6.9 Hz, 2H), 2.39 (t, J = 6.9 Hz, 2H), 1.82-1.67 (m,
4H). 13C NMR (75 MHz, DMSO-d6) δ 171.15, 163.87, 158.24, 143.99, 141.72,
137.77, 129.82, 127.60, 126.96, 126.27, 125.73, 118.24, 103.76, 38.52, 35.68, 27.38,

22.11. HRMS (AP-ESI) m/z calcd for C19H18IN3O6S [M + H]+ 544.0034, found
544.0037.
5.1.15. 4-(5-(1,1-dioxido-3-oxo-6-phenylbenzo[d]isothiazol-2(3H)-yl)pentanamido)
1
-N-hydroxybenzamide (6o) Yield 28%, mp 220-221 °C. H NMR (300 MHz,
DMSO-d6) δ 11.08 (s, 1H), 10.12 (s, 1H), 8.92 (s, 1H), 8.68 (d, J = 1.5 Hz, 1H), 8.30
(dd, J = 8.1 Hz, 1.5 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.91-7.86 (m, 2H), 7.70 (d, J =
8.7 Hz, 2H), 7.64 (d, J = 8.7 Hz, 2H), 7.59-7.48 (m, 3H), 3.77 (t, J = 6.9 Hz, 2H),
2.41 (t, J = 6.9 Hz, 2H), 1.84-1.67 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ 171.26,
163.99, 158.46, 147.40, 141.83, 137.98, 137.09, 133.21, 129.53, 129.33, 127.70,
127.57, 127.06, 125.63, 125.00, 119.45, 118.34, 38.59, 35.80, 27.57, 22.24. HRMS
(AP-ESI) m/z calcd for C25H23N3O6S [M + H]+ 494.1380, found 494.1378.
5.1.16. 4-((3-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)propanamido)methyl)
1
-N-hydroxybenzamide (6p) Yield 27%, mp 208-210 °C. H NMR (300 MHz,
DMSO-d6) δ 11.15 (s, 1H), 8.97 (d, J = 1.8 Hz, 1H), 8.57 (t, J = 6.0 Hz, 1H), 8.31 (d,
J = 6.9 Hz, 1H), 8.10 (dd, J = 7.5 Hz, 1.5 Hz, 1H), 8.05 (td, J = 7.5 Hz, 1.5 Hz, 1H),
8.00 (td, J = 7.5 Hz, 1.5 Hz, 1H), 7.67 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H),
4.31 (d, J = 6.0 Hz, 2H), 3.97 (t, J = 7.5 Hz, 2H), 2.69 (t, J = 7.5 Hz, 2H). 13C NMR
(75 MHz, DMSO-d6) δ 169.00, 164.12, 158.37, 142.57, 136.82, 135.79, 135.26,
131.31, 127.13, 126.91, 126.36, 125.07, 121.52, 41.93, 35.12, 33.89. HRMS (AP-ESI)
m/z calcd for C18H17N3O6S [M + H]+ 404.0911, found 404.0913.
5.1.17. 4-((5-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)pentanamido)methyl)-
1
N-hydroxybenzamide (6q) Yield 22%, mp 162-164 °C. H NMR (300 MHz,

DMSO-d6) δ 11.16 (s, 1H), 8.98 (s, 1H), 8.38 (t, J = 6.0 Hz, 1H), 8.31 (dd, J = 7.5 Hz,
0.6 Hz, 1H), 8.10 (td, J = 7.5 Hz, 1.2 Hz, 1H), 8.06-7.89 (m, 2H), 7.69 (d, J = 8.4 Hz,
2H), 7.29 (d, J = 8.4 Hz, 2H), 4.30 (d, J = 6.0 Hz, 2H), 3.73 (t, J = 7.2 Hz, 2H), 2.21
(t, J = 7.2 Hz, 2H), 1.80-1.70 (m, 2H), 1.67-1.58 (m, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 171.87, 164.16, 158.59, 142.96, 136.80, 135.76, 135.24, 131.26, 126.99,
126.93, 126.33, 125.06, 121.50, 41.78, 38.52, 34.68, 27.59, 22.54. HRMS (AP-ESI)
m/z calcd for C20H21N3O6S [M + H]+ 432.1224, found 432.1217.
5.1.18. 4-((6-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)hexanamido)methyl)-
N-hydroxybenzamide (6r) Yield 20%, mp 86-88 °C. 1H NMR (300 MHz, DMSO-d6)
δ 11.07 (s, 1H), 9.01 (s, 1H), 8.35 (t, J = 6.0 Hz, 1H), 8.31 (d, J = 7.2 Hz, 1H),
8.13-7.97 (m, 3H), 7.70 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 4.29 (d, J = 6.0
Hz, 2H), 3.72 (t, J = 7.2 Hz, 2H), 2.17 (t, J = 7.2 Hz, 2H), 1.80-1.70 (m, 2H),
1.64-1.54 (m, 2H), 1.41-1.31 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 172.04,
164.05, 158.57, 142.97, 136.78, 135.75, 135.23, 131.22, 126.94, 126.88, 126.30,
125.04, 121.47, 41.73, 38.61, 35.08, 27.67, 25.80, 24.71. HRMS (AP-ESI) m/z calcd
for C21H23N3O6S [M + H]+ 446.1380, found 446.1384.
5.1.19.
4-((1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)methyl)-N-hydroxyl
benzamide (8a) Yield 35%, mp 164-166 °C. 1H NMR (600 MHz, DMSO-d6) δ 11.22
(s, 1H), 9.05 (s, 1H), 8.35 (d, J = 7.2 Hz, 1H), 8.13 (d, J = 7.2 Hz, 1H), 8.07 (t, J = 7.2
Hz, 1H), 8.01 (t, J = 7.2 Hz, 1H), 7.72 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 7.8 Hz, 2H),
13
4.98 (s, 2H). C NMR (100 MHz, DMSO-d6) δ 163.86, 158.62, 138.20, 136.81,
135.92, 135.32, 132.28, 127.66, 126.19, 127.08, 125.22, 121.64, 41.24. HRMS

(AP-ESI) m/z calcd for C15H12N2O5S [M + H]+ 333.0540, found 333.0540.
5.1.20.
N-hydroxy-4-((6-nitro-1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
1
methyl)benzamide (8b) Yield 25%, mp 210-212 °C. H NMR (600 MHz, DMSO-d6)
δ 11.23 (s, 1H), 9.32 (s, 1H), 9.06 (s, 1H), 8.72 (d, J = 8.4 Hz, 1H), 8.34 (d, J = 8.4 Hz,
1H), 7.73 (d, J = 7.8 Hz, 2H), 7.51 (d, J = 7.8 Hz, 2H), 5.02 (s, 2H). 13C NMR (100
MHz, DMSO) δ 163.82, 157.14, 151.86, 137.81, 137.72, 132.36, 130.97, 130.27,
127.77, 127.09, 126.97, 118.09, 41.76. HRMS (AP-ESI) m/z calcd for C15H11N3O7S
[M + H]+ 378.0390, found 378.0386.
5.1.21.
N-hydroxy-4-((6-iodo-1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
methyl) benzamide (8c) Yield 46%, mp 190-192 °C. 1H NMR (600 MHz, DMSO-d6)
δ 11.21 (s, 1H), 9.04 (s, 1H), 8.87 (s, 1H), 8.37 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 7.8 Hz,
1H), 7.72 (d, J = 7.8 Hz, 2H), 7.47 (d, J = 7.8 Hz, 2H), 4.95 (s, 2H). 13C NMR (100
MHz, DMSO) δ 163.82, 158.31, 144.08, 138.04, 137.83, 132.28, 129.99, 127.66,
127.06, 126.44, 125.64, 103.96, 41.29. HRMS (AP-ESI) m/z calcd for C15H11IN2O5S
[M + H]+ 458.9506, found 458.9509.
5.1.22.
4-((1,1-dioxido-3-oxo-6-phenylbenzo[d]isothiazol-2(3H)-yl)methyl)-N-
1
hydroxybenzamide (8d) Yield 55%, mp 198-200 °C. H NMR (300 MHz, DMSO-d6)
δ 11.22 (s, 1H), 9.05 (d, J = 1.5 Hz, 1H), 8.73 (d, J = 1.5 Hz, 1H), 8.31 (dd, J = 8.1 Hz,
1.5 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.92-7.89 (m, 2H), 7.73 (d, J = 8.4 Hz, 2H),
7.60-7.49 (m, 5H), 5.00 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.86, 158.44,
147.52, 138.28, 137.99, 136.99, 133.23, 132.27, 129.51, 129.28, 127.64, 127.53,
127.09, 125.76, 124.82, 119.57, 41.30. HRMS (AP-ESI) m/z calcd for C21H16N2O5S

[M + H]+ 409.0853, found 409.0853.
5.1.23. 4-((6-benzamido-1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)methyl)-N-
hydroxybenzamide (11a) Yield 46%, mp 128-130 °C. 1H NMR (600 MHz,
DMSO-d6) δ 11.30 (s, 1H), 11.06 (s, 1H), 9.06 (s, 1H), 8.69 (s, 1H), 8.25 (d, J = 8.4
Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H ), 8.00 (d, J = 7.2 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H),
7.65 (t, J = 7.2 Hz, 1H), 7.58 (t, J = 7.2 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 4.97 (s, 2H).
13C NMR (100 MHz, DMSO-d6) δ 166.53, 163.83, 158.34, 145.87, 138.33, 138.05,
133.82, 132.42, 132.26, 128.60, 127.96, 127.73, 127.10, 126.37, 125.36, 120.24,
110.98, 41.31. HRMS (AP-ESI) m/z calcd for C22H17N3O6S [M + H]+ 452.0911,
found 452.0918.
5.1.24. N-(2-(4-(hydroxycarbamoyl)benzyl)-1,1-dioxido-3-oxo-2,3-dihydrobenzo
[d]isothiazol-6-yl)-[1,1'-biphenyl]-4-carboxamide (11b) Yield 44%, mp 238-240 °C.
1H NMR (600 MHz, DMSO-d6) δ 11.23 (s, 1H), 11.10 (s, 1H), 9.06 (s, 1H), 8.72 (s,
1H), 8.28 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 8.4 Hz, 2H), 7.90
(d, J = 8.4 Hz, 2H), 7.78 (d, J = 7.8 Hz, 2H), 7.74 (d, J = 7.8 Hz, 2H), 7.54-7.49 (m,
4H), 7.43 (t, J = 7.2 Hz, 1H), 4.98 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 166.09,
163.88, 158.33, 145.88, 143.91, 138.86, 138.33, 138.05, 132.47, 132.30, 129.06,
128.67, 128.31, 127.71, 127.11, 126.95, 126.74, 126.36, 125.33, 120.20, 110.97,
41.30. HRMS (AP-ESI) m/z calcd for C28H21N3O6S [M + H]+ 528.1224, found
528.1224.
5.1.25. N-(2-(4-(hydroxycarbamoyl)benzyl)-1,1-dioxido-3-oxo-2,3-dihydrobenzo
1
[d]isothiazol-6-yl)-3,4,5-trimethoxybenzamide (11c) Yield 45%, mp 228-230 °C. H

NMR (600 MHz, DMSO-d6) δ 11.23 (s, 1H), 10.90 (s, 1H), 9.05 (s, 1H), 8.63 (s, 1H),
8.25 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.49 (d, J
= 8.4 Hz, 2H), 7.33 (s, 2H), 4.97 (s, 2H), 3.89 (s, 6H), 3.75 (s, 3H). 13C NMR (100
MHz, DMSO-d6) δ 166.97, 165.85, 158.35, 152.73, 145.86, 141.07, 138.00, 129.53,
128.81, 127.85, 127.73, 127.12, 126.33, 125.47, 120.18, 111.22, 105.79, 60.18, 56.23,
41.31. HRMS (AP-ESI) m/z calcd for C25H23N3O9S [M + H]+ 542.1228, found
542.1226.
5.1.26. 4-((1,1-dioxido-3-oxo-6-(4-(trifluoromethyl)benzamido)benzo[d]isothiazol-
1
2(3H)-yl)methyl)-N-hydroxybenzamide (11d) Yield 35%, mp 245-247 °C. H NMR
(600 MHz, DMSO-d6) δ 11.25 (s, 1H), 11.23 (s, 1H), 9.06 (s, 1H), 8.68 (s, 1H), 8.24
(d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 2H), 8.16 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 8.4
Hz, 2H), 7.74 (d, J = 7.8 Hz, 2H), 7.49 (d, J = 7.8 Hz, 2H), 4.98 (s, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 165.37, 163.89, 158.28, 145.46, 138.30, 138.03, 137.61,
132.31, 132.02 (q, J = 31.8 Hz), 128.89, 127.72, 127.11, 126.42, 125.57 (q, J = 3.3
Hz), 125.55, 123.79 (q, J = 271.0 Hz), 120.61, 111.21, 41.32. HRMS (AP-ESI) m/z
calcd for C23H16F3N3O6S [M + H]+ 520.0785, found 520.0785.
5.1.27. 4-fluoro-N-(2-(4-(hydroxycarbamoyl)benzyl)-1,1-dioxido-3-oxo-2,3-dihy
1
drobenzo[d]isothiazol-6-yl)benzamide (11e) Yield 47%, mp 244-246 °C. H NMR
(600 MHz, DMSO-d6) δ 11.23 (s, 1H), 11.06 (s, 1H), 9.06 (s, 1H), 8.67 (s, 1H), 8.24
(d, J = 7.8 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.11-8.09 (m, 2H), 7.74 (d, J = 7.8 Hz,
2H), 7.49 (d, J = 7.8 Hz, 2H), 7.42 (t, J = 8.4 Hz, 2H), 4.97 (s, 2H). 13C NMR (100
MHz, DMSO-d6) δ 165.36, 164.54 (d, J = 249.0 Hz), 163.89, 158.31, 145.77, 138.32,

138.03, 130.82 (d, J = 9.2 Hz), 132.30, 130.25 (d, J = 2.7 Hz), 127.71, 127.11, 126.35,
125.37, 120.28, 115.60 (d, J = 21.9 Hz), 111.01, 41.30. HRMS (AP-ESI) m/z calcd
for C22H16FN3O6S [M + H]+ 470.0817, found 470.0818.
5.1.28. 4-((1,1-dioxido-3-oxo-6-(2-phenylacetamido)benzo[d]isothiazol-2(3H)-yl)
1
methyl)-N-hydroxybenzamide (11f) Yield 46%, mp 198-200 °C. H NMR (300
MHz, DMSO-d6) δ 11.23 (s, 1H), 9.06 (s, 1H), 8.54 (d, J = 1.5 Hz, 1H), 8.07 (d, J =
8.4 Hz, 1H), 7.97 (dd, J = 8.4 Hz, 1.5 Hz, 1H), 7.71 (d, J = 8.1 Hz, 2H), 7.45 (d, J =
8.1 Hz, 2H), 7.38-7.25 (m, 5H), 4.94 (s, 2H), 3.78 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 170.60, 163.91, 158.28, 145.67, 138.33, 138.24, 134.99, 132.29, 129.23,
128.38, 127.70, 127.11, 126.78, 126.53, 124.34, 119.94, 109.80, 43.28, 41.28. HRMS
(AP-ESI) m/z calcd for C23H19N3O6S [M + H]+ 466.1067, found 466.1056.
5.1.29.
(E)-4-((1,1-dioxido-3-oxo-6-(3-(p-tolyl)acrylamido)benzo[d]isothiazol-2
1
(3H)-yl)methyl)-N-hydroxybenzamide (11g) Yield 53%, mp >250 °C. H NMR
(300 MHz, DMSO-d6) δ 11.23 (s, 1H), 11.07 (s, 1H), 9.06 (s, 1H), 8.66 (d, J = 1.8 Hz,
1H), 8.09 (d, J = 8.4 Hz, 1H), 7.97 (dd, J = 8.4 Hz, 1.8 Hz, 1H), 7.73 (d, J = 8.1 Hz,
2H), 7.65(d, J = 15.6 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.27
(d, J = 8.1 Hz, 2H), 6.77 (d, J = 15.6 Hz, 1H), 4.96 (s, 2H), 2.35 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 164.76, 163.88, 158.29, 145.86, 142.25, 140.28, 138.34,
138.27, 132.28, 131.51, 129.66, 128.01, 127.67, 127.10, 126.50, 124.39, 119.90,
119.83, 109.83, 41.26, 20.99. HRMS (AP-ESI) m/z calcd for C25H21N3O6S [M + H]+
492.1224, found 492.1221.
5.2. In vitro HDACs assay

HDACs assays were performed according to the Color de Lys assay kits
instruction. HDACs came from HeLa nuclear extracts (mainly including HDAC 1 and
HDAC 2), and the substrate was an acetylated histone peptide. On the 96-well plate,
HDACs (5 µL/well), different concentrations of compound (10 µL/well) and substrate
(25 µL/well) were added successively. After being incubated at 37 °C for 30 min, the
mixture (50 µL/well) of Color de Lys Developer and TSA was put in and then the
system reacted another 30 min. Enzyme activity was determined 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
according to reference17. The molecular structures of 6j was optimized using Concord
method and then assigned with AM1-BCC charges.18 The top-scored conformation
was selected as the best docking result.
5.4. MTT assay
Human breast cancer cell (MDA-MB-231), myelogenous leukemia cell (KG1)
and prostatic cancer cell (PC-3) were cultured in RPMI1640 medium (10% FBS) at
37 °C in 5% CO2 humidified incubator, respectively. Cancer cells were plated in
96-well plate (5000/ well), cultured for 8 h in complete growth medium, then treated
with different concentrations of compounds for 48 h. After addition of MTT
(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide, 5 mg/mL) and
further incubation for 4 h, formazan was extracted by adding 150 µL DMSO and
antiproliferative activity was measured in a microtiter-plate reader at 570 nm.

5.5 Apoptosis assay
MDA-MB-231 cells (1.2 × 105) were treated with different concentrations of 6i,
6j or SAHA for 24 h. Cells were harvested and washed with PBS. After resuspended
in 195 µL Annexin V-FITC binding buffer, cells were added 5 µL Annexin V-FITC
and 10 µL propidium iodide (PI). Then the cells were incubated for 10 min at room
temperature, kept on ice and analyzed by flow cytometry.
Acknowledgments
Thanks for the help in docking studies by Prof. Renxiao Wang from Shanghai
Institute of Organic Chemistry in China. This work was supported by National
Natural Science Foundation China (No. 81373281), Shandong Natural Science Fund
for Distinguished Young Scholars (No. JQ201319), and the Program for New Century
Excellent Talents in University (No. NCET-12-0337).
Supplementary data
Supplementary data associated with this article can be found, in the online
version, at
References
1
2
3
4
West, A. C.; Johnstone, R. W. J. Clin. Invest. 2014, 124, 30.
Weichert, W. Cancer Lett. 2009, 280, 168.
Kazantsev, A. G.; Thompson, L. M. Nat. Rev. Drug Discov. 2008, 7, 854.
Archin, N. M.; Liberty, A. L.; Kashuba, A. D.; Choudhary, S. K.; Kuruc, J. D.; Crooks, A. M.; Parker, D.
C.; Anderson, E. M.; Kearney, M. F.; Strain, M. C.; Richman, D. D.; Hudgens, M. G.; Bosch, R. J.; Coffin, J.
M.; Eron, J. J.; Hazuda, D. J.; Margolis, D. M. Nature 2012, 487, 482.
5
6
Royce, S. G.; Karagiannis, T. C. Curr. Opin. Allergy Clin. Immunol. 2014, 14, 44.
Wagner, F. F.; Wesmall yi, U. M.; Lewis, M. C.; Holson, E. B. Neurotherapeutics : the journal of the
American Society for Experimental NeuroTherapeutics 2013, 10, 589.
7
8
9
Poole, R. M. Drugs 2014, 74, 1543.
Han, L.; Wang, L.; Hou, X.; Fu, H.; Song, W.; Tang, W.; Fang, H. Bioorg. Med. Chem. 2014, 22, 1529.
Zhang, L.; Zhang, Y.; Chou, C. J.; Inks, E. S.; Wang, X.; Li, X.; Hou, J.; Xu, W. ChemMedChem 2014, 9,

638.
10 Su, H.; Yu, L.; Nebbioso, A.; Carafa, V.; Chen, Y.; Altucci, L.; You, Q. Bioorg. Med. Chem. Lett. 2009,
19, 6284.
11 Blackburn, C.; Barrett, C.; Chin, J.; Garcia, K.; Gigstad, K.; Gould, A.; Gutierrez, J.; Harrison, S.;
Hoar, K.; Lynch, C.; Rowland, R. S.; Tsu, C.; Ringeling, J.; Xu, H. J. Med. Chem. 2013, 56, 7201.
12 Jiao, J.; Fang, H.; Wang, X.; Guan, P.; Yuan, Y.; Xu, W. Eur. J. Med. Chem. 2009, 44, 4470.
13 Feng, J.; Fang, H.; Wang, X.; Jia, Y.; Zhang, L.; Jiao, J.; Zhang, J.; Gu, L.; Xu, W. Cancer Biol. Ther.
2011, 11, 477.
14 Jiao, J.; Fang, H.; Xu, W. F. Drug Discov. Ther. 2008, 2, 211.
15 Gomez, V., J. A.; Dominguez, S., J. F. ; Tabraue, C., M. PCT Int. Appl. WO 2008003800 A1 2008.
16 Yang, L.; Liang, Q.; Shen, K.; Ma, L.; An, N.; Deng, W.; Fei, Z.; Liu, J. Biomed. Pharmacother. 2015,
71, 70.
17 Hou, X.; Li, R.; Li, K.; Yu, X.; Sun, J. P.; Fang, H. J. Med. Chem. 2014, 57, 9309.
18 Hou, X.; Du, J.; Zhang, J.; Du, L.; Fang, H.; Li, M. J. Chem. Inf. Model. 2013, 53, 188.

Graphical abstract