Design, synthesis, and biological evaluation of histone deacetylase inhibitors possessing glutathione peroxidase-like and antioxidant activities against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2018.10.022

A series of hybrids containing the pharmacophores of the histone deacetylase (HDAC) inhibitor, SAHA, and the antioxidant ebselen were designed and synthesized as multi-target-directed ligands against Alzheimer's disease. An in vitro assay indicated that some of these molecules exhibit potent HDAC inhibitory activity and ebselen-related pharmacological effects. Specifically, the optimal compound 7f was found to be a potent HDAC inhibitor (IC₅₀ = 0.037 μM), possessing rapid hydrogen peroxide scavenging activity and glutathione peroxidase-like activity (ν₀ = 150.0 μM min^?1) and good free oxygen radical absorbance capacity (value of ORAC: 2.2). Furthermore, compound 7f showed significant protective effects against damage induced by H₂O₂ and the ability to prevent ROS accumulation in PC12 cells.

Full text of DOI:10.1016/j.bmc.2018.10.022

Accepted Manuscript
Design, synthesis, and biological evaluation of histone deacetylase inhibitors
possessing glutathione peroxidase-like and antioxidant activities against Alz-
heimer’s disease
Jinhui Hu, Baijiao An, Tingting Pan, Zhengcunxiao Li, Ling Huang, Xingshu
Li
PII:
S0968-0896(18)31179-9
https://doi.org/10.1016/j.bmc.2018.10.022
BMC 14581
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 June 2018
22 October 2018
23 October 2018
Please cite this article as: Hu, J., An, B., Pan, T., Li, Z., Huang, L., Li, X., Design, synthesis, and biological evaluation
of histone deacetylase inhibitors possessing glutathione peroxidase-like and antioxidant activities against
Alzheimer’s disease, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.022
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Design, synthesis, and biological evaluation of histone deacetylase inhibitors
possessing glutathione peroxidase-like and antioxidant activities against
Alzheimer’s disease
Jinhui Hu, Baijiao An, Tingting Pan, Zhengcunxiao Li, Ling Huang,* and Xingshu Li
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006,
China
Abstract
A series of hybrids containing the pharmacophores of the histone deacetylase (HDAC)
inhibitor, SAHA, and the antioxidant ebselen were designed and synthesized as multi-
target-directed ligands against Alzheimer’s disease. An in vitro assay indicated that
some of these molecules exhibit potent HDAC inhibitory activity and ebselen-related
pharmacological effects. Specifically, the optimal compound 7f was found to be a
potent HDAC inhibitor (IC50 = 0.037 μM), possessing rapid hydrogen peroxide
scavenging activity and glutathione peroxidase-like activity (ν0 = 150.0 μM min−1)
and good free oxygen radical absorbance capacity (value of ORAC: 2.2). Furthermore,
compound 7f showed significant protective effects against damage induced by H2O2
and the ability to prevent ROS accumulation in PC12 cells.
Highlights
Compounds 7f exhibits potent HDAC6 inhibitory activity and ebselen-related
pharmacological effects.
Compound 7f has significant protective effects against damage induced by H2O2.
Compound 7f prevents ROS accumulation in PC12 cells.

Keywords
Alzheimer’s disease, histone deacetylase inhibitors, glutathione peroxidase-like
activity, antioxidant activities, multi-target-directed ligands
Introduction
Due to its complex pathogenesis and multifactorial progression, Alzheimer’s disease
(AD) is still one of the most difficult diseases to treat in the world today.[1] Currently,
the primary therapeutic options for the treatment of AD are acetylcholinesterase
inhibitors (AChEIs), namely tacrine, donepezil, rivastigmine, and galantamine.
However, these drugs can only slow down the progression of the disease to a certain
extent but fail to achieve the goal of healing.[2] Because of the aging population,
which is more prone to AD, it is very important to search for and to develop new
drugs for the treatment of this disease.[3]
HDACs are epigenetic modulators that deacetylate lysine residues in histone and
non-histone substrates; they can be divided into four groups: class I (HDACs 1, 2, 3,
and 8), class IIa (HDACs 4, 5, 7, and 9), class IIb (HDACs 6 and 10), and class IV
(HDAC11).[4] In normal cells, there is a balance between histone-acetylation and
deacetylation achieved by the activities of histone acetyltransferases (HATs) and
HDACs, respectively.[5] Breaking that balance may lead to some diseases.[6] For
example, class I HDACs play a critical role in tumorigenesis through the formation of
complexes with other proteins. Additionally, class I HDACs and HDAC6 are
implicated in AD memory-related dysfunction.[7, 8] Therefore, the inhibition of
histone deacetylases (HDACs) has attracted much attention for use as a treatment for

neurodegenerative disorders.[9] Studies have indicated that HDAC2 and HDAC6 are
overexpressed in the cortex and hippocampus of AD patients.[10, 11] Aberrant
accumulation of insoluble hyperphosphorylated tau (p-tau) is a hallmark of
neurodegenerative diseases including AD.[12] Acetylation of tau at Lys280 can
promote p-tau aggregation and prevent p-tau from being degraded, leading to
impaired cognitive function.[13] Inhibition of the activity of or depletion of HDAC6
can reduce p-tau levels, alleviating the features of neurological disorders caused by
tau.[14, 15] A recent study demonstrated that tubastatin A, a selective HDAC6
inhibitor, can alleviate behavioral deficits in a mouse with AD without obvious
adverse effects.[16] Vorinostat (SAHA), an FDA-approved HDAC inhibitor, can
enhance synaptic function and plasticity, exert an anti-inflammatory effect and
reinstate epigenetic balance in aging and AD models.[17, 18]
Oxidative stress is recognized as a contributing factor in aging and in the
progression of multiple neurodegenerative diseases including AD.[19] One of the
possible mechanisms of AD that has emerged in recent years is based on observations
linking mitochondrial dysfunction, which has been solidly confirmed in AD,[20, 21]
and the increased production of reactive oxygen species (ROS), leading to the
development of the disease.[22] HDAC6, a deacetylase specific for redox regulatory
proteins, plays a significant role in redox regulation and in the cellular stress
response.[23] Inhibition of HDAC6 can alleviate Aβ-induced ROS elevation caused
by the acetylation of Prx1.[24] In addition, recent evidence indicates that
overexpression of HDAC6 induces ROS generation through upregulation of NADPH

oxidase expression and activity, and through significantly enhanced expression of
several pro-inflammatory cytokines with the simultaneous deacetylation of α-
tubulin.[25] Antioxidant protection is beneficial to AD patients because the
endogenous antioxidant protection system declines rapidly with age.
Selenium (Se) is an essential trace mineral nutrient for humans. Selenium levels
decrease with age, and that decrease may play various roles in the progression of
AD.[26, 27] In our previous work, a serious of selenium-containing compounds were
synthesized using the multitarget-directed ligands (MTDLs) design strategy,[28-30]
including “ selenpezil” compounds,[31] clioquinol - ebselen hybrids,[32, 33]. All
those molecules are proved to possess a remarkable ability to mimic the enzymatic
properties of GPx and scavenge hydrogen peroxide. Based on advances in HDAC6
inhibitors used for the treatment of AD, and the role of selenium in providing
protection from free radical-induced cell damage, we herein report the synthesis and
evaluation of hybrids for the treatment of AD based on the fusion of the
pharmacophores of marketed HDAC inhibitors and the antioxidant ebselen.
N
O
H
N
OH
NH
OH
HN
N
O
HN
Tubastatin A
Panobinostat
O
N
H
H
N
N
N
H
N
OH
OH
N
N
H
O
O
N
SAHA
Quisinostat
O
N
O
O
N
Se
Linker
Se
HN OH
Ebselen
Figure 1. Known HDAC inhibitors (Tubastatin A, Panobinostatat, Quisinostat, SAHA),

antioxidant ebselen and the design strategy for hybrid compounds 7a-7k, 12a-12b and 18a-18c.
RESULTS AND DISCUSSION
Rational Design
HDAC6 is a zinc-dependent enzyme; the classical pharmacophore for HDAC6
consists of three parts: a hydrophobic recognition cap, a linker and a hydroxamic
moiety for the zinc binding domain. The literature contains reports indicating that
ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) has antioxidant and anti-
inflammatory activity and some inhibitory activity for both class I and IIa HDACs.[34]
To fuse the key structural features of ebselen and HDAC6 pharmacophores into one
molecule, we propose that the ebselen core could serve as a cap group, which is
attached by various linkers to the hydroxamic moiety. These linkers come from well-
known HDAC inhibitors, such as the flexible alkyl chain from Vorinostat, or the
arylalkyl chains from Tubastatin A[35] and Panobinostat[36] and the pyrimidyl group
from Quisinostat[37], resulting in a series of novel ebselen derivatives, identified as
7a-k, 12a-b and 18a-c (Figure 1).
Chemistry
Routes for the synthesis of the target compounds 7a-k, 12a-b and 18a-c are
summarized in Schemes 1-3. Commercially available 2-aminobenzoic acid was
reacted with NaNO2 in the presence of HCl at 0-5 °C, followed by reaction with
Na2Se2 to produce 2,2'-diselanediyldibenzoic acid, intermediate 2. The reaction of
intermediate 2 with thionyl chloride provided 3, which reacted with the amine 4e and
4g-j, obtained by the method reported in the literature (see Supporting Information),

to produce intermediates 5a-k. Target compounds 7a-k were obtained by the
hydrolysis of intermediates 5a-k, followed by treatment with NH2OTHP / EDC /
HOBt to mediate amide formation and by HCl to mediate deprotection (Scheme 1).
O
O
CO2H
c
a
b
Cl
Cl
OH
NH2
A
(4a-k)
H2N
COOR
Se
Se
2
1
3
2
O
O
O
e, f
d
N
Se
A
N
Se
A
N
Se
A
NH
OH
COOR
COOH
O
5a-k R = CH3 or C2H5
(
)
6a-k
7a-k
A:
n
O
n
n = 4,5,6
n = 3,5,6
a - c
d
e
f
g - i
j
k
Scheme 1 Synthesis of 7a-k. Reagents and conditions: (a) H2O, NaNO2, HCl, 0-5 °C, 30 min,
Na2Se2, 50 °C, 3 h, 71 %; (b) SOCl2, reflux, 2 h; (c) amine 4a-k, anhydrous CH2Cl2, triethylamine,
rt, 4h, 58 ~ 80 %, two steps; (d) LiOH, CH3OH / H2O (3:1), overnight, 84 ~ 92 %; (e) EDC·HCl,
HOBt, THPONH2, NMM, DMF, rt, overnight; (f) HCl (1.0 M), CH3OH / CH3CN, room
temperature for 3 h, 51 ~ 66 %, two steps.
Synthesis routes for the target compounds 12a-b using a two step procedure are listed
in Scheme 2. Intermediate 3 reacted with Boc-protected piperidin-4-ylmethanamine to
produce intermediate 8. Compounds 9a-b were obtained by deprotection of the Boc
group. Compounds 9a-b were reacted with ethyl 2-bromopyrimidine-5-carboxylate to
produce 10a-b. Intermediates 10a-b were converted to the target products 12a-b using
the procedure employed in the synthesis of products 7a-k.

O
O
O
a
b
c
n
N
Se
Cl
Cl
n
N
Se
Se
NH
N
3
Boc
9a, n = 0
9b, n = 1
8a, n = 0
8b, n = 1
O
O
O
e, f
d
n
N
Se
N
Se
N
Se
n
n
N
N
N
N
N
N
N
O
N
N
O
NH
OH
10a, n = 0
10b, n = 1
11a, n = 0
11b, n = 1
12a, n = 0
12b, n = 1
OH
O
O
Scheme 2 Synthesis of 12a-b. Reagents and conditions: (a) anhydrous CH2Cl2, triethylamine,
room temperature, 4 h, 78 %; (b) con. HCl, CH3OH, 8 h, 74 ~ 81%; (c) ethyl 2-bromopyrimidine-
5-carboxylate, K2CO3, acetone, 5 h, 64 ~71 %; (d) LiOH, CH3OH / H2O (3:1), overnight, 85 ~
89%; (e) EDC·HCl, HOBt, THPONH2, NMM, DMF, room temperature, overnight; (f) HCl (1.0
M), CH3OH / CH3CN, room temperature for 3 h, 51 ~ 65%, two steps.
The synthesis of the target compounds 18a-c is summarized in Scheme 3. Using F-,
OH- or CF3-substituted 2-aminobenzoic acid derivatives as the starting materials, acid
chlorides 15a-c were obtained, and these were reacted with methyl 4-(aminomethyl)
benzoate hydrochloride and the hydrolyzed to produce acids 17a-c. Finally, 18a-c
were obtained using the two step procedure described above.
O
O
R
R
R
CO2H
Se
a, b
c
d
Cl
Cl
OH
Se
NH2
13a, R = F
2
14a, R = F
15a, R = F
13b, R = OH
13c, R = CF3
14b, R = OAc
14c, R = CF3
15b, R = OAc
15c, R = CF3
O
O
O
OH
NH
O
OH
O
O
e
f, g
O
R
R
R
N
Se
N
Se
N
Se
17a, R = F
16a, R = F
18a, R = F
17b, R = OH
17c, R = CF3
16b, R = OH
16c, R = CF3
18b, R = OH
18c, R = CF3
Scheme 3 Synthesis of 18a-c. Reagents and conditions : (a) for compound 14a and 14c, H2O,
NaNO2, HCl, 0-5 °C, 30 min, Na2Se2, 50 °C, 3 h, 55 ~ 72%; for compound 14b, (a) H2O, NaNO2,

HCl, 0-5 °C, 30 min, Na2Se2, 50 °C, 3 h, 61 %; (b) acetic anhydride, pyridine, 110 °C, 3 h, 81 %;
(c) SOCl2, reflux; (d) methyl 4-(aminomethyl)benzoate hydrochloride, anhydrous CH2Cl2,
triethylamine, room temperature, 4 h, 66 ~ 73 %; (e) LiOH, CH3OH / H2O (3:1), overnight, 81 ~
91 %; (f) EDC·HCl, HOBt, THPONH2, NMM, DMF, room temperature, overnight; (g) HCl (1.0
M), CH3OH / CH3CN, room temperature for 3 h, 51 ~ 65 %, two steps.
HDAC Inhibitory Activity
The HDAC6 enzyme, which is related to the incidence of AD, was used to screen for
HDAC inhibitory activities in the synthesized compounds.[38, 39] The results, shown
in Table 1, indicate that all of the target compounds exhibited more potent activity
(IC50 values ranged from 0.019 to 1.97 μM) than ebselen (> 10 μM). Further structural
and activity studies demonstrated that increasing the flexible chain length between the
hydroxamic acid and the benzo[d][1,2]selenazol-3(2H)-one moiety tends to be
beneficial for HDAC6 inhibition potency (IC50: 7a, four carbon chain, 0.356 μM; 7b,
five carbon chain, 0.181 μM). Compound 7c, which possesses a six-carbon chain,
gave the best result (0.019 μM). Compound 7d exhibited the weakest activity (IC50:
1.97 μM) within the series, which suggests that a phenyl group instead of a flexible
chain seems unfavorable for activity. It is interesting that 7f, which contains a benzyl
group instead of a phenyl group, exhibited very good activity with an IC50 value of
0.037 μM. Other compounds, such as 7e and 7g-j, with a styryl, an alkoxyphenyl or
an alkylphenyl group between the hydroxamic acid and the benzo[d][1,2]selenazol-
3(2H)-one moiety, also provided good inhibitory activity (IC50: 0.508 -1.145 μM).
Strikingly, the ethyl-linked phenyl derivative 7k, (IC50 values of 0.863 μM), exhibited
far less potent activity against HDAC6 than the corresponding methylene-linked

compound 7f (0.037 μM). Compounds 12a and 12b, with a 2-(piperidin-1-yl)
pyrimidinyl or a 2-(methylene piperidin-1-yl)pyrimidinyl group, respectively,
between the hydroxamic acid and the benzo[d][1,2]selenazol-3(2H)-one moiety,
exhibited IC50 values of 0.968 μM and 0.444 μM, respectively, further indicating that
larger groups are disadvantageous for inhibitory activity. Finally, compounds 18a,
18b and 18c with F, OH or CF3 substitutes on the benzo[d][1,2]selenazol-3(2H)-one
moiety, had IC50 values of 0.178, 0.189 and 0.584 μM, respectively, which is much
weaker than that of the lead compound 7f, suggesting that the hydrophilicity and
electron-withdrawing strength of the HDACi cap group were not beneficial to HDAC
inhibitory activity. Out of concern for HDAC isoform selectivity, we also performed
enzyme inhibition assays against HDAC3, which is representative of class I HDACs
and associated with the onset of tumors. Compound 7c with linear alkyl chains
showed moderate inhibitory activity against HDAC3 (IC50 of 0.164 μM). While
compound 7f with benzyl group was far less potent against HDAC3 (IC50 of 1.30 μM).
Most of the other synthesized compounds exhibited less than 50% inhibition at a
concentration of 1.0 μM.
Table 1. HDAC inhibitory activities, free oxygen radical scavenging abilities and
glutathione peroxidase-like activities of target compounds 7a-k, 12a-b and 18a-c
O
O
n1
N
O
X
O
Se
N
Se
O
O
OH
NH
n1
O
O
n1
N
N
Se
N
N
H
N
Se
N
OH
NH
N
O
OH
NH
n2
Se
H
N
O
X
12a b
-
7k
OH
7d j
18a c
-
-
7a c
-
O

%
GPx-like ν0 (μM·min-1) c ± SD
Inhibition of
HDAC6a
IC50 (μM) ±
SD
inhibition
compd
X
n1 n2
of
ORACb
t-
Cum-
OOH
H2O2
HDAC3 at
1μM
BuOOH
7a
7b
-
-
4
5
-
-
0.356 ± 0.17
0.181 ± 0.03
63.16
76.35
0.164 ±
0.05e
0.9 ± 0.1
1.1 ± 0.2
79.8 ± 3.5
75.1 ± 1.1
24.1 ± 2.6 29.7 ± 2.8
30.6 ± 3.1 43.9 ± 3.4
7c
-
6
-
0.019 ± 0.0080
1.1 ± 0.1
83.0 ± 0.7
17.0 ± 1.3 50.7 ± 2.8
7d
7e
-
0
0
0
0
1.97 ± 0.20
40.90
48.63
1.30 ±
0.70e
1.1 ± 0.1
1.3 ± 0.1
121.5 ± 4.6 27.9 ± 0.3 76.9 ± 1.7
57.2 ± 1.5. 38.8 ± 1.5 106.0 ± 1.
CH=CH
0.679 ± 0.20
7f
-
1
0
0.037 ± 0.010
2.2 ± 0.2
150.2 ± 3.6 33.9 ± 2.8 98.0 ± 4.3
7g
O
O
O
-
0
0
0
0
-
3
5
6
2
-
0.903 ± 0.021
0.508 ± 0.035
0.181 ± 0.022
1.145 ± 0.69
0.863 ± 0.034
0.968 ± 0.076
0.444 ± 0.054
0.178 ± 0.071
0.188 ± 0.089
25.13
75.61
100.63
39.76
40.34
44.06
44.61
45.66
39.63
1.6 ± 0.3
1.0 ± 0.1
0.8 ± 0.1
2.1 ± 0.1
1.9 ± 0.3
1.3 ± 1.3
0.9 ± 0.1
1.3 ± 0.2
2.6 ± 0.2
74.1 ± 2.4
45.3 ± 0.4
44.6 ± 1.0
82.5 ± 2.6
12.6 ± 0.9 30.1 ± 3.0
7h
7.4 ± 1.8
9.4 ± 3.0
21.4 ± 3.0
26.9 ± 3.5
7i
7j
19.3 ± 3.4 20.3 ± 1.7
(±)7k
12a
12b
18a
18b
-
160.0 ± 0.8 26.4 ± 1.5 83.9 ± 1.8
140.4 ± 2.6 20.3 ± 0.8 71.0 ± 2.3
147.3 ± 3.2 35.3 ± 3.3 44.2 ± 0.3
-
0
1
-
-
-
-
F
OH
-
80.8 ± 1.9
30.1 ± 2.7 42.6 ± 2.0
-
-
175.5 ± 2.6 32.3 ± 3.3 102.7 ± 5.

18c
CF3
-
-
-
-
0.584 ± 0.046
0.025 ± 0.012
48.63
0.517 ±
0.12
NT
0.6 ± 0.1
77.9 ± 2.4
32.4 ± 1.8 27.5 ± 0.8
SAHA
-
1.0 ± 0.04 34.9 ± 0.4
5.2 ± 0.8
NT
5.3 ± 0.6
NT
Tubastatin A
Ebselen
0.020 ± 0.010
NT
NT
-
-
-
-
-
-
11.5 ± 1.1
-
NT
0.7 ± 0.15 134.0 ± 2.7 27.1 ± 0.6 62.4 ± 0.5
Control d
-
-
34.3 ± 1.0
4.6 ± 1.1
5.1 ± 0.5
b
aValues are expressed as mean ± SD of at least two independent duplicate experiments. Values
c
are expressed as mean ± standard deviation (SD) of at least three independent experiments.
Assay conditions: reactions were carried out in phosphate buffer (100 mM, pH 7.5) at 25 °C with
1 mM ethylene diamine tetraacetate (EDTA), 2 mM GSH, 0.4 mM NADPH, 1.3 unit·mL-1
glutathione reductase (GR), 80 μM selenium compounds, and 1.6 mM peroxide. All values were
determined in triplicate for the initial 10 s, and the average values with standard deviations were
obtained. d Control values were obtained from the reduction of peroxide by GSH in the absence of
the compound. NT: not tested. eIC50 of HDAC3 (μM)
Free Oxygen Radical Scavenging Ability.
To evaluate the antioxidant activity of the synthesized compounds, a free oxygen
radical absorbance capacity (ORAC) assay was performed using the vitamin E
analogue Trolox as the standard.[40, 41] As shown in Table 1, most of the target
compounds demonstrated an antioxidant activity similar to the vitamin E analogue
Trolox, and compounds 7f, 7k and 18b, which feature a phenylmethylene chain
between the hydroxamic acid and the benzo[d][1,2]selenazol-3(2H)-one moiety or an
OH group in the benzene ring of the benzo[d][1,2]selenazol-3(2H)-one moiety,
exhibited better results, with an ORAC-FL value of 1.9 ~ 2.6. ORAC-FL value of
compound 7j with 4-ethylphenyl linker was better than that of analogue 7e with 4-

vinylphenyl linker, which was unclear and need further investigation.
Glutathione peroxidase-like activity
To evaluate the GPx-like activity of the compounds, the coupled reductase method
was applied, using glutathione as the thiol cofactor and three different peroxides,
hydrogen peroxide, cumene hydroperoxide, or tert-butyl hydroperoxide (see list of
abbreviation), as substrates and ebselen as the reference compound (Table 1).[42, 43]
Within the series of compounds, 7f and 7k, with their associated phenyl methylene
group or phenyl methylmethylene group, respectively, demonstrated good catalytic
peroxidase-like activity, with values that were slightly better than ebselen’s value (7f,
H2O2, ν0 = 150.2 μM·min-1; t-BuOOH, ν0 = 33.9 μM·min-1; Cum-OOH, ν0 = 98.0
μM·min-1. (±)-7k, H2O2, ν0 = 160.0 μM·min-1; t-BuOOH, ν0 = 26.4 μM·min-1; Cum-
OOH, ν0 = 83.9 μM·min-1. Ebselen, H2O2, ν0 = 134.0 μM·min-1; t-BuOOH, ν0 = 27.1
μM·min-1; Cum-OOH, ν0 = 62.4 μM·min-1). Pyrimidylhydroxamic derivatives 12a
and 12b showed catalytic activities similar to ebselen (12a, H2O2, ν0 = 140.4 μM·min-
1; t-BuOOH, ν0 = 20.3 μM·min-1; Cum-OOH, ν0 = 71.0 μM·min-1; 12b, H2O2, ν0 =
147.3 μM·min-1; t-BuOOH, ν0 = 35.3 μM·min-1; Cum-OOH, ν0 = 44.2 μM·min-1). In
the free oxygen radical absorbance capacity (ORAC) assay, 18b, which has a OH
group in the benzene ring of the benzo[d][1,2]selenazol-3(2H)-one moiety, exhibited
the highest activity among the compounds (H2O2, ν0 = 175.5 μM·min-1; t-BuOOH, ν0
= 32.3 μM·min-1; Cum-OOH, ν0 = 102.7 μM·min-1). Moreover, when F and CF3 are
used as the substituent groups instead of OH, the catalytic activity was much weaker
than the catalytic activity of ebselen (18a, H2O2, ν0 = 80.8 μM·min-1; t-BuOOH, ν0 =

30.1 μM·min-1; Cum-OOH, ν0 = 42.6 μM·min-1; 18c, H2O2, ν0 = 77.9 μM·min-1; t-
BuOOH, ν0 = 32.4 μM·min-1; Cum-OOH, ν0 = 27.5 μM·min-1).
Catalytic activity of scavenging peroxide in the presence of GSH
To further investigate the catalytic antioxidant activity of ebselen derivatives 7c, 7f
and 18b, kinetic reactions utilizing H2O2, t-BuOOH and Cum-OOH were conducted
using ebselen and SAHA as the reference compounds. Figure 2 shows the scavenging
rates of these compounds for the peroxides. From this figure, it could be seen that
H2O2, t-BuOOH and Cum-OOH were fully scavenged in the presence of catalyst 18b
within 180 min (Figure 2A, B and C). Compound 7c, 7f and ebselen had scavenging
rates of 41 %, 62 % and 51 % for H2O2 and rates of 41 %, 78 % and 71 % for t-
BuOOH, respectively (Figure 2D). Under the same conditions, the scavenging rates of
7c, 7f and ebselen for Cum-OOH were lower than for H2O2 and t-BuOOH; the rates
for Cum-OOH were 25 %, 37 % and 29 %, respectively. Taken together, these data
indicate that compound 18b exhibited strong scavenging activity for peroxides.
Compound 7f provided a good scavenging activity, with a value that was slightly
better than the value of the reference compound ebselen.

Figure 2. Scavenging of peroxides (H2O2, t-BuOOH and Cum-OOH) by GSH over time in the
presence and absence of synthesized compounds. (A) Scavenging of H2O2. (B) Scavenging of t-
BuOOH. (C) Scavenging of Cum-OOH. (D) The maximal scavenging peroxide ability. Assay
conditions: reactions were carried out in phosphate buffer (100 mM, pH 7.5) at 25 °C with EDTA
(1 mM), GSH (0.25 mM), NADPH (0.50 mM), GR (1.3 unit·mL-1), peroxide (0.20 mM) and
catalyst (10 μM). All data represent the mean ± SE of three independent experiments. Key: ***, P
< 0.001 vs the vehicle group.
Protecting PC12 cells from H2O2- and 6-OHDA-induced damage
To study the protective effects of synthesized compounds against oxidative
damage to cells, cytotoxicity assays assessing the viability of the PC12 cell line using
the MTT assay were performed for compounds 7f, 18b, tubastatin A, ebselen or co-
administration of tubastatin A and ebselen at 10 and 20 μM concentrations. The
results are summarized in Figure 3A. Cells treated with compounds 7f, 18b, tubastatin
A, ebselen or co-administration of tubastatin A and ebselen at a concentration of 20
μM did not show any significant cytotoxicity. Hydrogen peroxide is an endogenous

cellular signaling molecule that can generate free radicals. With Hydrogen peroxide
treatment at a concentration of 400 μM, an approximately 50 % reduction in cell
viability was observed, compared to the control (Figure 3B). As shown in Figure 3C
Cell viability was dramatically increased to approximately 90% compared to the
control by the addition of compound 7f or co-incubation with tubastatin A and
ebselen at 5 μM concentrations (p < 0.001). Compound 18b, tubastatin A and ebselen
showed similar protective effects against H2O2 induced damage at 10 μM (p < 0.001,
p < 0.01, p < 0.01). Interestingly, the compound 7f exhibited a more favorable
protective effect on PC12 cells, comparing to tubastatin A or ebselen alone. Cell
viability of co-administration with tubastatin A and ebselen was significantly higher
than that of tubastatin A or ebselen alone. It is well known that 6-OHDA is a potent
neurotoxin that induces neuronal death via uncoupling of mitochondrial oxidative
phosphorylation, resulting in energy deprivation. 6-OHDA at a concentration of 200
μM for 12 h on PC12 cells caused approximately 50 % cell death when compared to
the control group (Figure 3B). Compounds 7f and co-incubation with tubastatin A and
ebselen showed significant protective effects against damage induced by 6-OHDA at
5 μM (p < 0.001) (Figure 3D). Compound 18b, tubastatin A or ebselen alone also
showed protective effects on PC12 cells at 10 μM (p < 0.01, p < 0.05, p < 0.001).
Moreover, compound 7f and co-administration with tubastatin A and ebselen
exhibited stronger protective effects against H2O2-induced cellular damage on PC12
cells, comparing to tubastatin A alone at 5 μM (p < 0.05, p < 0.01). Taken together,
these data indicate that 7f and co-administration tubastatin A and ebselen can
remarkedly protect PC12 cells from H2O2 and 6-OHDA induced cell damage.
Comparing to the treatment of tubastatin A or ebselen alone, pharmacological
HDAC6 inhibition and antioxidant activity show synergistical effects on PC12 cells

protection against oxidative damage.
Figure 3. Protective effect of 7f and 18b against H2O2-induced PC12 cell damage. (A)
Cytotoxicity evaluation of tested compounds in PC12 cells. The cells were treated with the
indicated concentrations of compounds for 24 h, and the cytotoxicity of the compounds was
determined by the MTT assay. (B) The cells were treated with various concentrations of H2O2 and
6-OHDA for 12 h, and the cell viability was determined by the MTT assay. Concentrations of 400
μM H2O2 and 200 μM 6-OHDA were used to induce cell damage in PC12 cells. (C) (D)
Protection against H2O2- and 6-OHDA-induced PC12 cell damage. PC12 cells (1 × 104 cells/well)
were plated in a 96-well plate for 1 d and subsequently treated with 7f, 18b, ebselen, tubastantin A
or ebselen and tubastantin A for another 24 h. After replacement with fresh medium containing
H2O2 and 6-OHDA, followed by continued incubation for the indicated times, cell viability was
measured by the MTT assay. All data represent the mean ± SD of three independent experiments.
Key: ***, p < 0.001 vs the vehicle group; ##, p < 0.01 vs the H2O2- or 6-OHDA-treated group; ##,
p < 0.01 vs the 6-OHDA or H2O2-treated group. $, p < 0.05, $$, p < 0.01, $$$, p < 0.001 vs the
each of columns. Tub A = tubastantin A.
Preventing ROS accumulation in PC12 cells
2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) is a cell-membrane-

permeable dye. After diffusing into cells, DCFH-DA is deacetylated by cellular
esterases, becoming non-fluorescent 2’,7’-dichlorodihydrofluorescin, which is rapidly
converted to highly fluorescent 2’,7’-dichlorofluorescein upon reaction with ROS.
Stimulation of the cells with H2O2 leads to an intracellular burst of ROS, when
compared with the negligible ROS level of the control, as shown in Figure 4A. The
ROS levels were significantly decreased when 7f was incubated with cells in the
presence of H2O2 at 5 μM. Co-incubation with ebselen and tubastatin A could
effectively reduce ROS levels at 5 μM. The quantifying assays in Figure 4B show the
concentration-dependent protective effect of compounds against H2O2-induced
intracellular oxidative stress. The ROS accumulation induced by H2O2 was
remarkably reduced by pretreatment of 7f or co-administration of ebselen and
tubastatin A at 5 μM (p < 0.001). Moreover, compound 7f and co-incubation with
ebselen and tubastatin A exhibited better capability preventing ROS accumulation in
PC12 cells compared to ebselen and tubastatin A alone at 5 μM (p < 0.001).

Figure 4. Prevention of ROS accumulation in PC12 cells by 7f, tubastatin A, ebselen and co-
administration of tubastatin A and ebselen. Pretreatment of the cells with compounds (5 or 10 µM)
significantly alleviates ROS accumulation induced by H2O2 (A, B) as determined by DCFH-DA
staining. The fluorescence images (A) were acquired by inverted fluorescence microscopy. The
ROS level, shown in image (B), was quantified by a fluorescence microplate reader. Key: ***, p <
0.001 vs the vehicle group; ##, p < 0.01, ###, p < 0.001 vs the model group; $$$, p < 0.001 vs the
each of columns.
Western blot.
To investigate the HDAC6-inhibitory activity of compound 7f in cell, Western
Blot (WB) was performed by assessing the changes in the acetylated α-tubulin/total α-
tubulin ratio in PC12 cells. The influence of compounds 7f on the acetylation of α-
tubulin in PC12 cells is shown in Figure 5. Treatment with compound 7f led to
upregulation of acetyl-α-tubulin expression in a dose-dependent manner, which
corresponds to the functionality of HDAC6 inhibitor.
Figure 5. Incubation of PC12 cells with 0.01, 0.1, 1, 10 μM of compound 7f results in an
increased acetyl-α-tubulin expression.
Conclusion
Based on the multifaceted conditions that exist in the AD state, we developed a
series of novel molecules possessing HDAC inhibitory activity, free oxygen radical
scavenging activity and glutathione peroxidase-like activity and studied the in vitro

activities of the molecules. The results indicate that most of the synthesized molecules
exhibit potent HDAC inhibitory activity at the sub-micromolar level as shown by the
IC50 values. Some of the compounds (such as 7c, 7f, 7k, 12a-b and 18b) provided
comparable or better glutathione peroxidase-like activity than that of the reference
compound ebselen. The results also demonstrated that some of the compounds (7f, 7k
and 18b) exhibited better free oxygen radical scavenging ability, with ORAC values
of 1.9 - 2.6. Furthermore, 7f showed significant protective effects against damage
induced by H2O2 and significant preventive effects against ROS accumulation in
PC12 cells. Based on the above findings, we believe that further research into the use
of compound 7a as an anti-AD lead compound is warranted.
Experimental
Chemistry. General methods. The NMR spectra were recorder using TMS as the
internal standard on a Bruker Avance III spectrometer at 400 (1H NMR) and 100 (13C
NMR) MHz or 500 (1H NMR) and 125 (13C NMR) MHz. MS spectra were generated
on an Agilent LC-MS 6120 instrument with an ESI and APCI mass selective detector.
The melting points were determined using an SRS-Opti Melt automated melting point
instrument. The reactions were followed by thin-layer chromatography (TLC) and
visualized in an iodine chamber or with a UV lamp. Flash column chromatography
was performed using silica gel (200-300 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd. The high-resolution mass spectra were obtained using a Shimadzu
LCMS-IT-TOF or LTQ Orbitrap XL (Thermo Scientific) mass spectrometer. The
purity (≥ 95 %) of the samples was determined by an Agilent 1100 series HPLC

system using C18 column (Agilent Eclipse XDB-C18 5 µm. 4.6 mm × 150 mm
column), eluted with CH3CN / H2O (0.1% formic acid + 10 mM NH4OAc), at a low
rate of 0.2 mL/min. A gradient of 90 % acetonitrile was run and then decreased to 10 %
acetonitrile over 45 min. The gradient was returned to 90 % acetonitrile over 5 min
and run isocratically for 10 min.
Synthesis of 2,2′-diselenobisbenzoic acid (2) and derivatives (14a-c).
Compounds 2 and 14a–c were synthesized according to literature procedures.[44]
General Procedure for the Synthesis of 2-(chloroseleno)benzoyl chloride (3)
and derivatives (15a-c). 2,2′-Diselenobisbenzoic acid or its derivatives 14a-c (0.01
mol) was added to a solution of thionyl chloride (20 mL) containing 0.5 mL of DMF
as a catalyst. After the mixture was stirred at 85 °C for 3 h, the solvent was
evaporated under a vacuum, and the crude product was purified by recrystallization
from hexane.
General Procedure for the Synthesis of 1,2-benzisoselenazol-3(2H)-one
derivatives 5a-k, 8a-b and 16a-c. To a stirred solution of the corresponding amine (2
mmol) in anhydrous CH2Cl2 containing dry triethylamine (5 mmol) a solution of 3 or
15a-c (2.5 mmol) in anhydrous CH2Cl2 (5 mL) was added dropwise over 15 min at
0 °C. After stirred for another 5 h at room temperature, water was added, and the
mixture was extracted with CH2Cl2. The combined organic phase was dried (Na2SO4)
and filtered to obtain the crude products, which were purified by column
chromatography using silica gel and a CH2Cl2 / MeOH mixture. (CH2Cl2 : MeOH =
100:1)

methyl 5-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)pentanoate (5a) Yellow oil, yield
1
79 %. H NMR (400 MHz, CDCl3) δ 8.04 (dd, J = 7.9, 1.4 Hz, 1H), 7.66 (d, J = 7.9
Hz, 1H), 7.61 – 7.52 (m, 1H), 7.43 (d, J = 7.4 Hz, 1H), 3.86 (dd, J = 8.1, 6.2 Hz, 2H),
3.65 (s, 3H), 2.43 – 2.26 (m, 2H), 1.82 – 1.60 (m, 2H), 1.43 (ddt, J = 8.8, 6.4, 3.3 Hz,
2H).
methyl 6-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)hexanoate (5b) Yellow oil, yield
1
80 %. H NMR (400 MHz, CDCl3) δ 8.04 (dd, J = 7.9, 1.4 Hz, 1H), 7.66 (d, J = 7.9
Hz, 1H), 7.61 – 7.52 (m, 1H), 7.43 (d, J = 7.4 Hz, 1H), 3.86 (dd, J = 8.1, 6.2 Hz, 2H),
3.65 (s, 3H), 2.43 – 2.26 (m, 2H), 1.82 – 1.60 (m, 4H), 1.43 (ddt, J = 8.8, 6.4, 3.3 Hz,
2H).
methyl 7-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)heptanoate (5c) Yellow oil, yield
1
77 %. H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 7.7 Hz, 1H), 7.63 (d, J = 7.8 Hz,
1H), 7.61 – 7.51 (m, 1H), 7.46 – 7.38 (m, 1H), 3.86 (t, J = 7.2 Hz, 2H), 2.31 (t, J = 7.5
Hz, 2H), 1.79 – 1.68 (m, 2H), 1.65 – 1.58 (m, 2H), 1.45 – 1.33 (m, 4H).
methyl 4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)benzoate (5d) Yellow oil, yield
1
67 %. H NMR (400 MHz, CDCl3) δ 8.15 – 8.10 (m, 2H), 8.09 (s, 1H), 7.79 (d, J =
8.7 Hz, 2H), 7.72 – 7.64 (m, 2H), 7.49 (ddd, J = 8.0, 5.5, 2.7 Hz, 1H), 3.93 (s, 3H).
ethyl (E)-3-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenyl)acrylate (5e) Yellow
oil, yield 58 %.1H NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 8.0 Hz, 1H), 7.93 (d, J
= 7.6 Hz, 1H), 7.84 – 7.60 (m, 6H), 7.50 (t, J = 7.4 Hz, 1H), 6.63 (d, J = 16.0 Hz, 1H),
4.21 (q, J = 7.0 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H).
methyl 4-((3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)benzoate (5f) Yellow oil,

1
yield 68 %. H NMR (400 MHz, DMSO-d6) δ 8.03 (d, J = 7.9 Hz, 1H), 7.92 (d, J =
8.3 Hz, 2H), 7.86 (dd, J = 7.7, 1.3 Hz, 1H), 7.62 (ddd, J = 8.3, 7.2, 1.5 Hz, 1H), 7.48 –
7.38 (m, 3H), 4.99 (s, 2H), 3.93 (s, 3H).
ethyl 4-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenoxy)butanoate (5g) Yellow
1
oil, yield 69 %. H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 7.8 Hz, 1H), 7.76 – 7.59
(m, 2H), 7.47 (dd, J = 12.8, 8.0 Hz, 3H), 6.94 (d, J = 8.9 Hz, 2H), 4.17 (q, J = 7.1 Hz,
2H), 4.04 (t, J = 6.1 Hz, 2H), 2.54 (t, J = 7.3 Hz, 2H), 2.22 – 2.05 (m, 2H), 1.28 (t, J =
7.1 Hz, 3H).
ethyl 6-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenoxy)hexanoate (5h) Yellow
1
oil, yield 61 %. H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 7.8 Hz, 1H), 7.72 – 7.58
(m, 2H), 7.46 (t, J = 8.4 Hz, 3H), 6.93 (d, J = 8.7 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H),
3.98 (t, J = 6.4 Hz, 2H), 2.34 (t, J = 7.4 Hz, 2H), 1.87 – 1.77 (m, 2H), 1.71 (dt, J =
14.7, 7.2 Hz, 4H), 1.52 (dd, J = 15.3, 8.2 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).
ethyl 7-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenoxy)heptanoate (5i) Yellow
oil, yield 65 %.1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 7.8 Hz, 1H), 7.64 (d, J =
5.8 Hz, 2H), 7.46 (t, J = 7.7 Hz, 3H), 6.93 (d, J = 8.8 Hz, 2H), 4.22 – 4.05 (m, 2H),
4.05 – 3.91 (m, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.86 – 1.76 (m, 2H), 1.67 (dt, J = 14.9,
7.3 Hz, 3H), 1.49 – 1.37 (m, 3H), 1.26 (t, J = 7.1 Hz, 3H).
ethyl 3-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenyl)propanoate (5j) Yellow
1
oil, yield 71 %. H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 7.9 Hz, 1H), 7.65 (d, J =
5.7 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.46 (ddd, J = 7.9, 6.1, 2.2 Hz, 1H), 7.27 (d, J =
7.2 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 2.97 (t, J = 7.7 Hz, 2H), 2.64 (t, J = 7.7 Hz, 2H),

1.25 (t, J = 7.1 Hz, 3H).
(±) methyl 4-(1-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)ethyl)benzoate ((±)-5k)
Yellow oil, yield 61 %. 1H NMR (500 MHz, CDCl3) δ 8.07 (d, J = 1.0 Hz, 1H), 8.03
(d, J = 8.4 Hz, 2H), 7.64 – 7.55 (m, 2H), 7.49 (d, J = 8.3 Hz, 2H), 7.43 (ddd, J = 8.1,
6.0, 2.3 Hz, 1H), 5.98 (q, J = 6.9 Hz, 1H), 3.91 (s, 3H), 1.76 (d, J = 6.9 Hz, 3H).
(S) methyl 4-(1-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)ethyl)benzoate ((S)-5k)
1
Yellow oil, yield 64 %. H NMR (400 MHz, CD3OD) δ 8.08 – 7.99 (m, 2H), 7.99 –
7.86 (m, 2H), 7.61 (s-, 1H), 7.52 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 7.1 Hz, 1H), 5.85 (q,
J = 6.9 Hz, 1H), 1.79 (d, J = 7.0 Hz, 3H).
tert-butyl 4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)piperidine-1-carboxylate (8a)
white solid, yield 73 %. 1H NMR (400 MHz, CDCl3) δ 8.05 (dt, J = 7.8, 1.0 Hz, 1H),
7.72 – 7.52 (m, 2H), 7.43 (ddd, J = 8.0, 7.1, 1.2 Hz, 1H), 4.65 (tt, J = 11.6, 4.1 Hz,
1H), 4.38 – 3.96 (m, 2H), 2.89 (t, J = 13.1 Hz, 2H), 2.31 – 1.92 (m, 2H), 1.49 (s, 9H).
tert-butyl
4-((3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)piperidine-1-
1
carboxylate (8b) white solid, yield 73 %. H NMR (500 MHz, CDCl3) δ 8.05 (d, J =
7.8 Hz, 1H), 7.65 – 7.57 (m, 2H), 7.44 (t, J = 7.4 Hz, 1H), 2.70 (t, J = 10.7 Hz, 2H),
1.94 (tdt, J = 11.0, 7.2, 3.5 Hz, 1H), 1.75 – 1.66 (m, 4H), 1.45 (s, 2H), 1.45 (s, 9H),
1.28 – 1.20 (m, 2H).
methyl 4-((5-fluoro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)benzoate (16a)
Yellow oil, yield 76 %. 1H NMR (400 MHz, DMSO-d6) 7.88 (dd, J = 8.6, 5.4 Hz, 1H),
7.81 (dd, J = 9.2, 2.4 Hz, 1H), 7.73 (d, J = 7.8 Hz, 2H), 7.37 (d, J = 7.9 Hz, 2H), 7.30
(td, J = 8.8, 2.5 Hz, 1H), 4.95 (s, 2H), 3.85 (s, 3H).

methyl 4-((5-hydroxy-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)benzoate (16b)
Yellow oil, yield 69 %. 1H NMR (400 MHz, CDCl3) δ 8.09 (dt, J = 7.8, 1.2 Hz, 1H),
8.06 – 8.00 (m, 2H), 7.67 – 7.56 (m, 2H), 7.41 (d, J = 8.2 Hz, 2H), 5.07 (s, 2H), 3.91
(s, 3H).
methyl
4-((3-oxo-5-(trifluoromethyl)benzo[d][1,2]selenazol-2(3H)-yl)methyl)
1
benzoate (16c) Yellow oil, yield 60 %. H NMR (500 MHz, DMSO-d6) 8.29 (d, J =
8.4 Hz, 1H), 8.07 (s, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 7.7 Hz, 2H), 7.42 (d, J
= 7.8 Hz, 2H), 5.04 (s, 2H), 3.90 (s, 3H).
General Method for the Preparation of Compounds 9a-b. To a solution of 8a or
8b (2.0 mmol) in MeOH, was slowly added 2 M hydrochloric acid (1.0 mL). After
stirred at room temperature for 5 hours, the solution was concentrated, diluted with
water and adjusted (pH = 8) with saturated sodium bicarbonate solution. The crude
product was obtained by filtration and used for next step without any further
purification.
2-(piperidin-4-yl)benzo[d][1,2]selenazol-3(2H)-one (9a) Yellow oil, yield 71 %.
1H NMR (400 MHz, CDCl3) δ 8.05 (dd, J = 7.8, 1.3 Hz, 1H), 7.64 (d, J = 7.7 Hz, 1H),
7.57 (td, J = 8.1, 7.6, 1.4 Hz, 1H), 7.46 – 7.39 (m, 1H), 4.61 (tt, J = 11.5, 4.3 Hz, 1H),
3.28 – 3.15 (m, 2H), 2.94 – 2.74 (m, 2H), 2.08 (ddd, J = 12.0, 4.6, 2.2 Hz, 2H), 1.65
(qd, J = 12.1, 4.4 Hz, 2H).
2-(piperidin-4-ylmethyl)benzo[d][1,2]selenazol-3(2H)-one (9b) Yellow oil, yield
1
84 %. H NMR (400 MHz, CD3OD) δ 7.94 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 7.7 Hz,
1H), 7.65 – 7.51 (m, 1H), 7.46 – 7.32 (m, 1H), 3.72 (d, J = 7.2 Hz, 2H), 3.26 (s, 1H),

3.17 (t, J = 16.8 Hz, 2H), 2.81 – 2.64 (m, 2H), 2.01 (ddd, J = 14.7, 7.4, 3.7 Hz, 1H),
1.79 (d, J = 13.1 Hz, 2H), 1.48 – 1.29 (m, 2H).
General Method for the Preparation of Compounds 10a-10b To a solution of 9a
or 9b (2.0 mmol) and ethyl 2-chloropyrimidine-5-carboxylate (2.2 mmol) in acetone,
K2CO3 (3.0 mmol) was added. After stirred at room temperature overnight, the
reaction mixture was quenched with water, followed by extraction with CH2Cl2. The
combined organic layer was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel chromatography to afford
yellow oil (CH2Cl2 / CH3OH = 100 : 1) .
ethyl
2-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)piperidin-1-yl)pyrimidine-5-
1
carboxylate (10a) Yellow oil, yield 64 %. H NMR (500 MHz, DMSO-d6) δ 8.81 (s,
2H), 8.05 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 7.5 Hz, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.43
(t, J = 7.1 Hz, 1H), 4.93 (d, J = 13.0 Hz, 2H), 4.62 (t, J = 10.7 Hz, 1H), 4.28 (d, J =
6.7 Hz, 2H), 3.16 (t, J = 12.9 Hz, 2H), 2.05 (d, J = 11.8 Hz, 2H), 1.67 (q, J = 11.6 Hz,
2H), 1.30 (t, J = 6.8 Hz, 3H).
ethyl
2-(4-((3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)piperidin-1-
1
yl)pyrimidine-5- carboxylate (10b) Yellow oil, yield 71 %. H NMR (400 MHz,
CDCl3) δ 8.81 (s, 2H), 8.05 (d, J = 7.7 Hz, 1H), 7.62 (q, J = 8.0 Hz, 2H), 7.44 (t, J =
7.2 Hz, 1H), 4.90 (d, J = 13.5 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.77 (d, J = 7.1 Hz,
2H), 3.08 – 2.82 (m, 2H), 2.22 – 2.08 (m, 1H), 1.87 (d, J = 11.3 Hz, 2H), 1.36 (t, J =
7.1 Hz, 3H), 1.34 – 1.23 (m, 2H).
General Method for the Preparation of Compounds 6a-k, 11a-b and 17a-d To

a solution of 5a-i, 10a-b or 16a-d (1.0 mmol) in MeOH/H2O (3 : 1) at 0 °C, LiOH
(5.0 mmol) was slowly added. The solvent was concentrated in vacuo, diluted with
H2O, and the pH then adjusted to 1-2 with 1 N HCl. The suspension was filtered to
afford the crude product which was used for the next step without further purification.
5-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)pentanoic acid (6a) Yellow oil, yield
84 %. 1H NMR (500 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.81
(d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 3.73 (t, J = 6.9
Hz, 2H), 2.26 (t, J = 7.3 Hz, 2H), 1.69 – 1.60 (m, 2H), 1.51 (dd, J = 14.8, 7.4 Hz, 2H).
6-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)hexanoic acid (6b) Yellow oil, yield
1
85 %. H NMR (400 MHz, CDCl3) δ 8.04 (dd, J = 7.9, 1.4 Hz, 1H), 7.66 (d, J = 7.9
Hz, 1H), 7.61 – 7.52 (m, 1H), 7.43 (d, J = 7.4 Hz, 1H), 3.86 (dd, J = 8.1, 6.2 Hz, 2H),
2.43 – 2.26 (m, 2H), 1.82 – 1.60 (m, 5H), 1.43 (ddt, J = 8.8, 6.4, 3.3 Hz, 2H).
7-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)heptanoic acid (6c) Yellow oil, yield
84 %. 1H NMR (400 MHz, DMSO-d6) δ 12.00 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.80
(d, J = 7.6 Hz, 1H), 7.60 (t, J = 7.3 Hz, 1H), 7.42 (t, J = 7.3 Hz, 1H), 3.70 (t, J = 6.7
Hz, 2H), 2.19 (t, J = 7.1 Hz, 2H), 1.62 (m, 2H), 1.49 (m, 2H), 1.30 (m, 4H).
4-((3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)benzoic acid (6d) Yellow solid.
yield 90 %. 1H NMR (500 MHz, DMSO-d6) δ 13.17 – 12.66 (m, 1H), 8.11 (d, J = 8.1
Hz, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H),
7.71 (t, J = 7.7 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H).
(E)-3-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenyl)acrylic acid (6e) White
solid, yield 90 %. 1H NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 8.1 Hz, 1H), 7.92 (d,

J = 7.7 Hz, 1H), 7.81 – 7.67 (m, 5H), 7.61 (d, J = 16.0 Hz, 1H), 7.49 (t, J = 7.4 Hz,
1H), 6.53 (d, J = 16.0 Hz, 1H).
4-((3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)benzoic acid (6f) White solid,
yield 92 %. 1H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H), 8.03 (d, J = 7.9 Hz, 1H),
7.92 (d, J = 8.3 Hz, 2H), 7.86 (dd, J = 7.7, 1.3 Hz, 1H), 7.62 (ddd, J = 8.3, 7.2, 1.5 Hz,
1H), 7.48 – 7.38 (m, 3H), 4.99 (s, 2H).
4-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenoxy)butanoic acid (6g) White
solid, yield 90 %. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 8.07 (d, J = 8.0 Hz,
1H), 7.88 (dd, J = 7.8, 0.9 Hz, 1H), 7.75 – 7.59 (m, 1H), 7.47 (dd, J = 11.3, 4.9 Hz,
3H), 7.08 – 6.96 (m, 2H), 4.02 (t, J = 6.4 Hz, 2H), 2.40 (t, J = 7.3 Hz, 2H), 1.96 (t, J =
6.9 Hz, 2H).
6-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenoxy)hexanoic acid (6h) White
solid, yield 89 %. 1H NMR (400 MHz, DMSO-d6) δ 12.01 (s, 1H), 8.08 (d, J = 8.1 Hz,
1H), 7.89 (d, J = 7.6 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.48 (dd, J = 7.8, 5.8 Hz, 3H),
7.00 (d, J = 8.9 Hz, 2H), 3.99 (t, J = 6.4 Hz, 2H), 2.25 (t, J = 7.3 Hz, 2H), 1.83 – 1.67
(m, 2H), 1.58 (dt, J = 15.0, 7.3 Hz, 2H), 1.44 (dt, J = 9.2, 7.3 Hz, 2H).
7-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenoxy)heptanoic acid (6i) White
solid, yield 90 %. 1H NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 8.07 (d, J = 8.0 Hz,
1H), 7.88 (d, J = 7.6 Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H), 7.47 (dd, J = 7.7, 5.5 Hz, 3H),
7.00 (d, J = 8.9 Hz, 2H), 3.99 (t, J = 6.4 Hz, 2H), 2.22 (t, J = 7.3 Hz, 2H), 1.83 – 1.65
(m, 2H), 1.62 – 1.49 (m, 2H), 1.44 – 1.33 (m, 4H).
3-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenyl)propanoic acid (6j) White

solid, yield 92 %. 1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 8.09 (d, J = 8.0 Hz,
1H), 7.95 – 7.87 (m, 1H), 7.73 – 7.65 (m, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.50 – 7.46
(m, 1H), 7.28 (d, J = 8.4 Hz, 2H), 2.85 (t, J = 7.5 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H).
4-(1-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)ethyl)benzoic acid (6k) White solid,
yield 88 %. 1H NMR (400 MHz, CD3OD) δ 8.08 – 7.99 (m, 2H), 7.99 – 7.86 (m, 2H),
7.61 (s, 1H), 7.52 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 7.1 Hz, 1H), 5.85 (q, J = 6.9 Hz,
1H), 1.79 (d, J = 7.0 Hz, 3H).
2-(4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)piperidin-1-yl)pyrimidine-5-
1
carboxylic acid (11a) White solid, yield 88 %. H NMR (400 MHz, DMSO-d6) δ
12.86 (s, 1H), 8.79 (s, 2H), 8.05 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 7.4 Hz, 1H), 7.65 –
7.58 (m, 1H), 7.43 (t, J = 7.5 Hz, 1H), 4.93 (d, J = 13.4 Hz, 2H), 4.66 – 4.54 (m, 1H),
3.15 (t, J = 12.0 Hz, 2H), 2.05 (d, J = 10.0 Hz, 2H), 1.66 (qd, J = 12.3, 4.1 Hz, 2H).
2-(4-((3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)piperidin-1-yl)pyrimidine-5-
carboxylic acid (11b) White solid, yield 91 %. 1H NMR (500 MHz, CD3OD) δ 8.78 (s,
2H), 7.94 (dd, J = 8.0, 4.6 Hz, 2H), 7.63 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H),
3.77 (d, J = 7.2 Hz, 2H), 3.00 (t, J = 12.6 Hz, 2H), 2.26 – 2.11 (m, 1H), 1.82 (d, J =
13.8 Hz, 2H), 1.46 – 1.01 (m, 4H).
4-((5-fluoro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)benzoic acid (17a)
White solid, yield 88 %. 1H NMR (400 MHz, DMSO-d6) δ 12.90 (s, 1H), 7.88 (dd, J
= 8.6, 5.4 Hz, 1H), 7.81 (dd, J = 9.2, 2.4 Hz, 1H), 7.73 (d, J = 7.8 Hz, 2H), 7.37 (d, J
= 7.9 Hz, 2H), 7.30 (td, J = 8.8, 2.5 Hz, 1H), 4.95 (s, 2H).
4-((5-hydroxy-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)methyl)benzoic acid (17b)

White solid, yield 85%. 1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 9.76 (s, 1H), 7.91
(d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.6 Hz, 1H), 7.39 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 2.6 Hz, 1H),
7.10 (dd, J = 8.6, 2.6 Hz, 1H), 4.95 (s, 2H).
4-((3-oxo-5-(trifluoromethyl)benzo[d][1,2]selenazol-2(3H)-yl)methyl)benzoic acid
(17c) White solid, yield 87%. 1H NMR (500 MHz, DMSO-d6) δ 12.96 (s, 1H), 8.29 (d,
J = 8.4 Hz, 1H), 8.07 (s, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 7.7 Hz, 2H), 7.42
(d, J = 7.8 Hz, 2H), 5.04 (s, 2H).
General Method for the Preparation of Compounds 7a-k, 12a-b and 18a-c. To
a solution of acid 6a-k, 11a-b or 17a-c (0.5 mmol) in CH2Cl2 at 0 °C, 1-
hydroxybenzotriazole (0.51 mmol), 1-(3-Dimethylaminopropyl)-3- ethylcarbodiimide
hydrochloride (EDCI) (0.51 mmol) and N-methylmorpholine (1.5 mmol) were added.
After the reaction mixture was stirred at room temperature for 1 hour, NH2OTHP
(0.55 mmol) was added. After stirred for another 4 hours, the reaction was quenched
with water, followed by extraction with CH2Cl2. The combined organic layer was
dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue
was purified by silica gel chromatography (CH2Cl2 / CH3OH = 50 : 1) to afford
yellow oil which was treated with 1 M hydrochloride in the presence of CH3OH /
CH3CN (1 : 3). The mixture was stirred at room temperature for 16 h, then
concentrated under reduced pressure to give a beige residue, which was purified by
recrystallization in CH3OH to afford the N-hydroxyamide derivatives.
N-hydroxy-5-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)pentanamide
(7a).
Pale
yellow oil, yield: 66 %. 1H NMR (500 MHz, CD3OD) δ 7.92 (d, J = 8.1 Hz, 2H), 7.61