Synthesis and evaluation of 8-hydroxyquinolin derivatives substituted with (benzo[d][1,2]selenazol-3(2H)-one) as effective inhibitor of metal-induced Aβ aggregation and antioxidant

Article abstract of DOI:10.1016/j.bmc.2016.08.017

A series of 8-hydroxyquinolin derivatives substituted with (benzo[d][1,2]selenazol-3(2H)-one) at the 2-position were synthesized and evaluated for treatment of Alzheimer's disease. In vitro assays demonstrated that most of the target compounds exhibit significant inhibition of Cu(II)-induced Aβ_1–42aggregation, rapid H₂O₂scavenging and glutathione peroxidise (GPx)-like catalytic activity. Among these molecules, compound 9a is the most potent peroxide scavenger that possesses the ability to scavenge most H₂O₂within 200–220 min and possesses GPx-like activity (v₀= 106.0 μM·min^?1), enabling modulation of metal-induced Aβ aggregation.

Full text of DOI:10.1016/j.bmc.2016.08.017

Accepted Manuscript
Synthesis and evaluation of 8-hydroxyquinolin derivatives substituted with
(benzo[d][1,2]selenazol-3(2H)-one) as effective inhibitor of metal-induced
Aβ aggregation and antioxidant
Bo Wang, Zhiren Wang, Hong Chen, Chuan-Jun Lu, Xingshu Li
PII:
S0968-0896(16)30624-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.08.017
BMC 13200
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 June 2016
10 August 2016
11 August 2016
Please cite this article as: Wang, B., Wang, Z., Chen, H., Lu, C-J., Li, X., Synthesis and evaluation of 8-
hydroxyquinolin derivatives substituted with (benzo[d][1,2]selenazol-3(2H)-one) as effective inhibitor of metal-
induced Aβ aggregation and antioxidant, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/
j.bmc.2016.08.017
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Synthesis and evaluation of 8-hydroxyquinolin derivatives
substituted with (benzo[d][1,2]selenazol-3(2H)-one) as effective
inhibitor of metal-induced Aβ aggregation and antioxidant
Bo Wanga, Zhiren Wanga, Hong Chenb, Chuan-Jun Lub,∗ and Xingshu Lia,∗
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
bCollege of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China
Abstract: A series of 8-hydroxyquinolin derivatives substituted with (benzo[d][1,2]selenazol-3(2H)-one) at
the 2-position were synthesized and evaluated for treatment of Alzheimer’s disease. In vitro assays
demonstrated that most of the target compounds exhibit significant inhibition of Cu(II)-induced Aβ1−42
aggregation, rapid H2O2 scavenging and glutathione peroxidise (GPx)-like catalytic activity. Among these
molecules, compound 9a is the most potent peroxide scavenger that possesses the ability to scavenge most
H2O2 within 200-220 minutes and possesses GPx-like activity (ν0 = 106.0 µM•min−1), enabling modulation
of metal-induced Aβ aggregation.
1. Introduction
Alzheimer's disease (AD) is the most common form of dementia. AD affects approximately 44 million
people worldwide,1 and there will be more than 115 million people with AD in 2050.2 Many different
treatment approaches to AD have been developed over the past several decades based on the disease’s
hallmarks, such as β-amyloid (Aβ) deposits, τ-protein aggregation, low levels of acetylcholine, inflammation,
and dyshomeostasis of biometals and oxidative stress.3-5 However, there is still no drug that can definitively
cure Alzheimer’s disease. The limited therapeutic options approved by the U.S. Food and Drug
Administration include cholinesterase inhibitors (tacrine, donepezil, rivastigmine, galantamine) and the
NMDA receptor antagonist (memantine).6-7 Because of the complex interconnected network of multiple
factors and etiological hallmarks, the strategy of multitarget-directed ligands for the treatment of this fatal
neurodegenerative disorder should be appropriate. 8
Selenium is an essential micronutrient required for cellular antioxidant systems, and there are
twenty-five selenoproteins in the human body that exert specific biological functions9 (e.g., glutathione
peroxidises are important antioxidants in neuronal defence against oxidative stress). Selenium is vital for the
∗Corresponding author. Tel./fax: +086-0571-88320412; e-mail: luchuanjun@zjut.edu.cn
∗Corresponding author. Tel./fax: +086-020-39943050; e-mail: lixsh@mail.sysu.edu.cn

brain but its levels decrease with age.10-11 Much evidence has shown that selenium participates in the
neuropathology of disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis
and epilepsy.12 Recently, diphenyldiselenide [(PhSe)2] and p,p’-methoxyl-diphenyldiselenide [(MeOPhSe)2]
were used to treat Alzheimer’s-type dementia in a rat model.13-14 Compared with selenium, organoselenium
has been shown to have a higher biodisposability and biological activity, in regards to their inorganic
status.15 Therefore, considering both the role of selenium in nutrition and pathology of a neurodegenerative
disorder, it is desirable to develop a new organoselenium for the treating AD.
Studies showed that excessive biometals, such as iron, zinc, and copper, exist in the brains of AD
patients, which can readily bind to Aβ via three histidine residues, H6, H13, and H14, and facilitate Aβ
−Aβ interactions
aggregation. Thus, the development of metal chelator to modulate the metal
and the metal
distribution in the brain has become a promising approach in recent years. Clioquinol (CQ), of which
8-hydroxyquinoline moiety represents a key feature, exhibit moderate affinity for Cu and Zn, and
demonstrated a significant improvement in cognition and memory in clinical trials.16-17 We recently reported
a series of hybrids based on the fusion of donepezil and ebselen, and the “selenoxy-chinolin” with a high
GPx antioxidant ability. 18 In this study, we report on the synthesis and evaluation of 8-hydroxyquinolin
derivatives substituted with (benzo[d][1,2]selenazol-3(2H)-one) in the 2-position as an antioxidant and an
inhibitor of metal-induced Aβ aggregation (Fig. 1).
Figure 1. Design strategy for compounds 9a-9m
2. Chemistry
The synthesis routes of the hybrids of quinolin-8-ol and ebselen are summarized in the following Scheme
1. First, oxidized by m-CPBA, quinolin-8-ol 1 can be easily converted to oxide 2. After refluxing with
dimethylsulfate and then treating with NH4OH, compound 2 was converted into 2-aminoquinolin-8-ol 3,

which was further protected with Tert-butyldimethylsilyl chloride (TBSCl). On the other hand, diazotization
of substituted 2-aminobenzoic acids in the presence of sodium nitrite hydrogen chloride followed by Na2Se2
gave diselenide intermediates 6. 6 can be easily converted to chloride 7 with SOCl2, which was connected
with the above hydroxyl protected 2-aminoquinolin-8-ol 4 to provide 8. Finally, the deprotection of
compounds 8 provide the target quinolin-8-ol and ebselen hybrids.
Scheme 1. Synthesis of 9a-9m. Reagents and conditions: (a) m-CPBA, DCM, rt. (b) 1) Dimethylsulfatereflux; 2) NH4OH, -15 oC.
(c) TBSCl, DIPEA, THF, rt. (d). 1) H2O, NaNO2, HCl, 0-5 °C, 30 min; 2) Na2Se2, 50°C. (e) SOCl2, reflux. (f) anhydrous CH2Cl2,
triethylamine, rt. (g) K2CO3, MeOH. (h) TBAF, THF, rt.
3. Results and Discussion
3.1. Glutathione Peroxidase-Like Activity.

Oxidative stress is a prominent feature of AD.19 GPx, a selenoenzyme, which modulates the cellular
peroxide concentration in aerobic organisms by catalysing the reduction of hydroperoxides. Many
organoselenium compounds are reported to mimic the function of GPx.20 To evaluate the GPx-like
activity of the synthesized hybrids of quinolin-8-ol and ebselen, we applied the coupled reductase
method, which uses glutathione (GSH) as the thiol cofactor and three different peroxides hydrogen
peroxide (H2O2), cumene hydroperoxide (Cum-OOH), or tert-butyl hydroperoxide (t-BuOOH) as
substrates, and ebselen as the reference compound. The results in Table 1 indicate that some of the
hybrids provide potent activity in all three peroxide systems when compared with the reference
compounds ebselen (entry 14). Among them, compound 9j, bearing a CF3 in the R4 position, exhibited
the best GPx-like activity (entry 10, H2O2, v0 = 161.4 µM•min−1; t-BuOOH, v0 = 31.7 µM•min−1;
Cum-OOH, v0 = 62.0 µM•min−1). The preliminary analysis of the relationship between the structure
and the activity showed that the GPx-like activity was associated with both the substitutes and the
position on the ebselen moiety. The chlorine substituted in the R4 position (9g) exhibited the highest
activity in this series (entry 7, 8 and 9). Furthermore, it was found that when F and CF3 as the
substituent groups, the R1 or R4 position substitution of the benzoselenazol-3(2H)-one moiety were
indispensable for high GPx activity. Compounds 9f, which bears an F atom in the R1 position, gave the
best activity (entry 6) among the four F-substituted hybrids. Compound 9m, the CF3 substituted in the
R1 position also yielded very good activity (entry 13, H2O2, v0 = 132.0 µM•min−1; t-BuOOH, v0 = 30.0
µM•min−1; Cum-OOH, v0 = 31.7 µM•min−1). In addition, compound 9a, bearing a hydroxyl in the R3
position, provided much better activity (entry 1) compared with 9b (entry 2).
Table 1. Initial rates for the reduction of peroxide by GSH in the presence of ebselen and the synthetic
compounds.
v0 (µM·min-1) SDa
Entry Compound
R1
R2
R3
R4
H2O2
BuOOH
24.3 2.7
17.1 3.3
31.4 6.5
15.9 2.1
11.1 2.6
Cum-OOH
1
2
3
4
5
9a
9b
9c
9d
9e
H
H
H
H
H
H
H
H
H
F
OH
H
H
H
F
106.0 5.8
49.9 5.6
102.4 14.8
64.3 2.5
77.3 1.8
63.4 2.5
14.0 3.5
47.9 2.4
20.4 3.8
6.4 3.1
H
F
H
H
H

6
7
F
H
H
H
H
H
H
Cl
H
151.7 5.9
155.0 3.2
45.0 5.1
45.0 11.1
161.4 6.4
69.9 7.4
64.2 12.0
132.0 7.1
112.6 4.0
23.7 2.6
24.1 2.4
35.1 1.2
23.9 3.6
23.3 4.1
31.7 2.4
5.6 2.3
55.5 7.4
52.1 0.84
6.9 1.1
9f
9g
8
H
H
Cl
H
9h
9
H
Cl
H
H
7.1 1.8
9i
10
11
12
13
14
15
H
H
CF3
H
62.0 5.8
8.0 1.3
9j
H
H
CF3
H
9k
H
CF3
H
H
16.1 1.9
30.0 2.4
22.6 1.23
4.4 0.75
10.6 1.7
31.7 4.8
47.6 4.0
5.7 2.0
9l
CF3
H
H
9m
Ebselen
Control
a Reactions were carried out in phosphate buffer (100 mM, pH 7.5) at 25 °C, with 1 mM ethylenediaminetetraacetate (EDTA), 2
b
mM GSH, 0.4 mM NADPH, 1.3 unit/mL 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 deviation obtained.
3.2. Scavenging Peroxide in the Presence of GSH Catalysed by the Hybrids.
On the base of the GPx-like activity assay of the synthesized hybrids, to further evaluate the catalysed
antioxidant activity of the hybrids, kinetic reactions were conducted with ebselen (control) in the presence of
peroxide and GSH with the synthesized hybrids.21 Fig. 2 shows the scavenging rates of the tested
compounds to H2O2, Cum-OOH and t-BuOOH. The full scavenging of H2O2 was within 200-220 minutes in
the presence of catalyst 9a, and 9c, 9f, 9g, 9j and ebselen gave 60%, 71%, 62%, 65%, and 68% of the
scavenging rates, respectively. Under the same conditions, the full scavenging of Cum-OOH and t-BuOOH
were slightly slower than that of H2O2, which need 300 minutes in the presence of catalyst 9a (9c, 9f, 9g, 9j
and ebselen gave 30%, 46%, 31%, 38% and 67% scavenging for t-BuOOH, and 47%, 74%, 66%, 68% and
77% scavenging for Cum-OOH, respectively). As indicated in Fig. 2, compound 9a exhibited significantly
better scavenging activity for peroxides than reference compound ebselen.
A
B

Figure 2. Scavenging of peroxides (t-BuOOH, H2O2 and Cum-OOH) by GSH over time in the presence and absence of a
catalyst. (A) Scavenging of peroxide. (B) 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). Values are reported as the means for three independent experiments.
3.3. Radical Scavenging Activity Determined in the ORAC assay
In order to further evaluate the antioxidant activity of the synthesized compounds, of which certain of the
hybrids exhibited potent GPx-like activity, we performed an oxygen radical absorbance capacity assay
method using fluorescein (ORAC-FL) with the vitamin E analogue Trolox as our standard. The results in
Table 2 show that the hybrids 9a, 9c and 9f exhibited good ORAC-FL values of 2.3-3.6. Among the
compounds, 9f, which features a F atom at the R1 position, displayed the highest activity (3.6). Compounds
9g, 9j and 9m, featuring a Cl atom at the R4 position, a CF3 group in the R4 position (9j) or the R1 position
(9m), gave weaker activities than the vitamin E analogue Trolox. These results indicate that the substituent
groups play the more important role in ORAC-FL values than the position of the substituent groups.
Table 2. Oxygen Radical Absorbance Capacity (ORAC, Trolox Equivalents) for the optimal
compounds.
Compound ORACa
CQ
9a
9c
0.5 0.1
2.6 0.3
2.3 0.3
3.6 0.6
0.9 0.2
0.2 0.1
0.3 0.1
9f
9g
9j
9m
aData are expressed as µmol of trolox equivalent/µmol of tested compound.. The means standard deviation (SD) of three
independent experiments.

3.4. Modulation of Metal-Induced Aβ Aggregation by the Synthesized Compounds.
The remarkably high levels of biometal (Fe, Cu, and Zn) should have the neurotoxic potential in
AD-affected brains.22-23 To evaluate the modulation ability of metal-induced Aβ aggregation of the
synthesized compounds, we performed the thioflavin T (ThT) fluorescence assay. As Fig. 3 shows Cu2+
induces Aβ aggregation, the fluorescence unites increased from 100% (Aβ alone) to 160% (Aβ + Cu2+).
When CQ or the synthesized compounds were incubated with Aβ and Cu2+, the fluorescence unites in some
samples were significantly decreased (Aβ+Cu2++CQ, 87%; Aβ+Cu2++9a, 60%; Aβ+Cu2++9c, 82%;
Aβ+Cu2++9j, 79%, respectively), indicating these compounds had effects on the modulation of Cu2+-induced
Aβ aggregation. Especially, compound 9a possesses a greater effect on the control of Cu( II )-mediated Aβ
aggregation than CQ.
Figure 3. Inhibition experiments of Cu2+-induced Aβ1-42 aggregation in the presence of 9a, 9c, 9f, 9g, 9j and 9m. Experimental
conditions: Aβ1–42 (25 µM); CuSO4 (25 µM); CQ, or tested compounds (50 µM); HEPES (20 µM) and NaCl (150 µM); pH 6.6;
10 h, 37 °C.
3.5. Metal-Chelating Properties of 9a.
As compound 9a exhibited the potential to be an efficient multifunctional agent, we selected it for further
studies. Its ability to chelate biometals such as Cu(II), Fe(II), Fe(III), and Zn(II) was determined by UV-vis
spectroscopy (Fig. 4). Pure 9a in buffer exhibits a maximum absorption wavelength at 301 nm. It shifted to
316 nm after CuSO4 was added to a solution, indicating the formation of a 9a-Cu(II) complex. Similarly, the
maximum absorption at 304 nm for 9a+Zn2+, 297 nm for 9a+Fe2+ and 300 nm for 9a+Fe3+ also show the
coordination of compound 9a with these metals.

Figure 4. UV absorbance spectrum of compound 9a (50 µM) in the presence of CuSO4, ZnCl2, FeSO4 or FeCl3.The metal salts
(50 µM) in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4).
3.6. In Vitro Blood−Brain Barrier Permeation Assay.
It is essential for a successful central nervous system (CNS) drug to have good blood-brain barrier (BBB)
permeability. To determine the potential in vivo BBB permeability of these hybrids of quinolin-8-ol and
ebselen, we used the parallel artificial membrane permeation assay of the BBB (PAMPA-BBB) for the
evaluation. First, we compared the permeability of thirteen commercial drugs with known values in order to
validate the assay (see Supporting Information, Table S1). We obtained the linear correlation of Pe (exp) =
−1.0773 (R2
1.4574Pe (lit.)
= 0.9427) (see the Supporting Information), which plots the experimental data
versus the reported values. From this equation, and considering the limit previously stablished by Di et al.
for BBB permeability, compounds with a permeability greater than 4.7 × 10−6 cm•s-1 were determined to be
capable of crossing the BBB. As shown in the Pe values in Table 3, all the tested compounds could cross the
BBB.
Table 3. Permeability (Pe×10-6•cm s-1) as determined by the PAMPA-BBB assay shows the predicted
penetration of the CNSa, b for target compounds.
Prediction
Compound
Pe(10-6cm•s-1)
16.6 1.2
5.9 0.1
dispramine
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
9a
9c
9f
7.4 0.1
11.1 0.4
7.8 0.7
9g
9j
10.1 0.9
13.3 1.2
9m
b
aCompounds were dissolved in DMSO at 5 mg mL-1 and diluted with PBS/EtOH (70:30). Values are expressed as the means

SD of three independent experiments, compounds with a permeability, Pe> 4.7 × 10-6 cm•s-1 being considered capable of crossing
the BBB by passive diffusion.
4. Conclusions
Employing a multitarget-directed ligand MTDL strategy, we developed a series of 8-hydroxyquinolin
derivatives substituted with (benzo[d][1,2]selenazol-3(2H)-one) at the 2-position for the treatment of AD.
These novel compounds possess the main pharmacophores of clioquinol and ebselen, and provided a
significant inhibition ability of the Cu(II)-induced Aβ aggregation of clioquinol, and the scavenging peroxide
ability of ebselen. Among the synthesized compounds, 9a exhibited excellent capacity for scavenging of
H2O2, good GPx-like, and antioxidant activity. Furthermore, it could effectively modulate the metal-induced
Aβ aggregation and cross the BBB in vitro. All of these properties highlight 9a as the multifunctional,
leading compound for the treatment of AD.
5. Experimental
5.1. General
The NMR spectra were recorded using TMS as the internal standard on a Bruker Avance III spectrometer
at 400.132 (1H NMR) and 100.614 (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. All the reactions were followed by thin-layer
chromatography (TLC) on glass-packed precoated silica gel plates 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 spectrometers 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 HPLC, conducted on a Shimadzu LC-20AT series system, TC-C18
column (4.6×250 mm, 5 µm), eluted with CH3CN/H2O=80/20, at a flow rate of 0.5 mL/min.
8-hydroxyquinoline 1-oxide (2) and 2-Amino-8-hydroxyquinoline (3) were synthesized according to a
method described previously.24
5.2. Synthesis
5.2.1.
8-((tert-butyldimethylsilyl)oxy)quinolin-2-amine(4)

To a solution of 2-amino-8-hydroxyquinoline (3) (1.61 g, 10 mmol), ethyldiisopropylamine (2.54 g, 20
mmol) and 4-dimethylaminopyridine (120 mg, mmol) in 30 mL of anhydrous THF,
1
t-butyldimethylchlorosilane (1.8 g, 12 mmol) was added dropwise, at room temperature. After the
completion of the reaction (monitored by TLC), the mixture was diluted with dichloromethane, washed with
water followed by brine, and dried over anhydrous sodium sulphate. Evaporation of the solvent, at reduced
pressure, yielded the crude product, which was then purified by silica-column chromatography. White solid,
92% yield. 1H NMR (400 MHz, Chloroform-d) δ 7.84 (d, J = 8.7 Hz, 1H, Ar-H), 7.33 – 7.19 (m, 1H, Ar-H),
7.11 (t, J = 7.7 Hz, 1H, Ar-H), 7.06 (dd, J = 7.6, 1.6 Hz, 1H, Ar-H), 6.69 (d, J = 8.7 Hz, 1H, Ar-H), 4.64 (s,
2H, -NH2), 1.07 (s, 9H, C-CH3), 0.25 (s, 6H Si-CH3).
5.2.2.
Synthesis of 6a-6m and 7a-7m
Compound 6a-6m and 7a-7m were synthesized according to literature procedures.25
5.2.3.
General Procedures for the Synthesis of 8a-8m
To a solution of 1.0 equiv of compound 4, 10.0 equiv of anhydrous triethylamine in anhydrous
dichloromethane, was added 1.5 equiv of the compound 7 in anhydrous DCM dropwise. After stirring the
reaction at room temperature for 4 h, saturated NaHCO3 was added. The mixture was then extracted with
CH2Cl2, washed with brine, dried over anhydrous Na2SO4, and concentrated reduced under pressure to
provide the crude product, which was purified by silica-column chromatography with an CH2Cl2/CH3OH
gradient (starting from 2.5% up to 5% CH3OH).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-3-oxo-2,3-dihydrobenzo[d][1,2]selenazol-5-yl
acetate (8a). Yellow solid, 73% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.86 (d, J = 9.0 Hz, 1H, Ar-H),
8.17 (d, J = 9.0 Hz, 1H, Ar-H), 7.81 (d, J = 2.4 Hz, 1H, Ar-H), 7.68 (d, J = 8.5 Hz, 1H, Ar-H), 7.42 (m, 2H,

Ar-H), 7.34 (t, J = 7.9 Hz, 1H, Ar-H), 7.14 (dd, J = 7.5, 1.3 Hz, 1H, Ar-H), 2.36 (s, 3H,O-Ac), 1.18 (s, 9H,
C-CH3), 0.35 (s, 6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one (8b). Yellow solid,
75% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.90 (d, J = 9.0 Hz, 1H, Ar-H), 8.17 (d, J = 9.0 Hz, 1H,
Ar-H), 8.09 (d, J = 7.8 Hz, 1H, Ar-H), 7.73 – 7.59 (m, 2H, Ar-H), 7.48 – 7.39 (m, 2H, Ar-H), 7.33 (t, J = 7.8
Hz, 1H, Ar-H), 7.14 (d, J = 7.6 Hz, 1H, Ar-H), 1.18 (s, 9H, C-CH3), 0.36 (s, 6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-4-fluorobenzo[d][1,2]selenazol-3(2H)-one
(8c).
1
yellow solid, 70% yield. H NMR (400 MHz, Chloroform-d) δ 8.85 (d, J = 8.9 Hz, 1H, Ar-H), 8.47 (d, J =
8.8 Hz, 1H, Ar-H), 8.16 (d, J = 9.0 Hz, 1H, Ar-H), 7.60 – 7.52 (m, 2H, Ar-H), 7.33 (t, J = 7.7 Hz, 1H, Ar-H),
7.05 (m, 2H, Ar-H), 1.17 (s, 9H, C-CH3), 0.35 (s, 6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-5-fluorobenzo[d][1,2]selenazol-3(2H)-one
(8d).
1
yellow solid, 72% yield. H NMR (400 MHz, Chloroform-d) δ 8.78 (d, J = 8.8 Hz, 1H, Ar-H), 8.10 (d, J =
8.9 Hz, 1H, Ar-H), 7.71 (d, J = 8.3 Hz, 1H, Ar-H), 7.56 (dd, J = 7.6, 4.1 Hz, 1H, Ar-H), 7.35 (m, 2H, 1-H,
Ar-H), 7.32 – 7.23 (m, 1H, Ar-H), 7.07 (d, J = 7.5 Hz, 1H, Ar-H), 1.11 (s, 9H, C-CH3), 0.28 (s, 6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-6-fluorobenzo[d][1,2]selenazol-3(2H)-one
(8e).
Yellow solid, 75% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.87 (d, J = 8.9 Hz, 1H, Ar-H), 8.19 (d, J =
9.0 Hz, 1H, Ar-H), 8.08 (dd, J = 8.7, 5.2 Hz, 1H, Ar-H), 7.45 (dd, J = 8.2, 1.3 Hz, 1H, Ar-H), 7.41 (dd, J =
7.9, 2.2 Hz, 1H, Ar-H), 7.36 (t, J = 7.9 Hz, 1H, Ar-H), 7.21 – 7.10 (m, 2H, 2-H, Ar-H), 1.20 (s, 9H, C-CH3),
0.38 (s, 6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-7-fluorobenzo[d][1,2]selenazol-3(2H)-one
(8f).
1
Yellow solid, 73% yield. H NMR (400 MHz, Chloroform-d) δ 8.86 (d, J = 9.0 Hz, 1H, Ar-H), 8.21 (d, J =
9.0 Hz, 1H, Ar-H), 7.92 (dd, J = 7.7, 1.1 Hz, 1H, Ar-H), 7.52 – 7.42 (m, 2H, 17-H, Ar-H), 7.41 – 7.32 (m, 2H,
Ar-H), 7.17 (dd, J = 7.6, 1.4 Hz, 1H, Ar-H), 1.21 (s, 9H, C-CH3), 0.38 (s, 6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-4-chlorobenzo[d][1,2]selenazol-3(2H)-one
(8g).
Yellow solid, 76% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.89 (d, J = 9.0 Hz, 1H, Ar-H), 8.17 (d, J =
9.0 Hz, 1H, Ar-H), 7.58 (d, J = 7.4 Hz, 1H, Ar-H), 7.50 (t, J = 7.8 Hz, 1H, Ar-H), 7.45 – 7.38 (m, 2H, Ar-H),
7.34 (t, J = 7.9 Hz, 1H, Ar-H), 7.14 (d, J = 8.6 Hz, 1H, Ar-H), 1.18 (s, 9H, C-CH3), 0.35 (s, 6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-5-chlorobenzo[d][1,2]selenazol-3(2H)-one
(8h).
1
Yellow solid, 78% yield. H NMR (400 MHz, Chloroform-d) δ 8.87 (d, J = 9.0 Hz, 1H, Ar-H), 8.19 (d, J =
9.0 Hz, 1H, Ar-H), 8.07 (dd, J = 1.8, 0.8 Hz, 1H, Ar-H), 7.63 (m, 2H, Ar-H), 7.45 (dd, J = 8.2, 1.4 Hz, 1H,

Ar-H), 7.36 (t, J = 7.8 Hz, 1H, Ar-H), 7.16 (dd, J = 7.5, 1.4 Hz, 1H, Ar-H), 1.20 (s, 9H, C-CH3), 0.38 (s, 6H,
Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-6-chlorobenzo[d][1,2]selenazol-3(2H)-one
(8i).
1
Yellow solid, 73% yield. H NMR (400 MHz, Chloroform-d) δ 8.84 (d, J = 8.9 Hz, 1H, Ar-H), 8.17 (d, J =
9.0 Hz, 1H, Ar-H), 7.99 (d, J = 8.3 Hz, 1H, Ar-H), 7.70 (d, J = 1.7 Hz, 1H, Ar-H), 7.47 – 7.37 (m, 2H, Ar-H),
7.34 (t, J = 7.8 Hz, 1H, Ar-H), 7.14 (dd, J = 7.6, 1.3 Hz, 1H, Ar-H), 1.18 (s, 9H, C-CH3), 0.35 (s, 6H,
Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-4-(trifluoromethyl)benzo[d][1,2]selenazol-3(2H)-on
e (8j). Yellow solid, 75% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.91 (d, J = 9.0 Hz, 1H, Ar-H), 8.17 (d,
J = 9.0 Hz, 1H, Ar-H), 7.88 (d, J = 7.9 Hz, 1H, Ar-H), 7.79 (d, J = 7.6 Hz, 1H, Ar-H), 7.69 (t, J = 7.8 Hz, 1H,
Ar-H), 7.43 (dd, J = 8.1, 1.3 Hz, 1H, Ar-H), 7.34 (t, J = 7.8 Hz, 1H, Ar-H), 7.14 (dd, J = 7.6, 1.4 Hz, 1H,
Ar-H), 1.18 (s, 9H, C-CH3), 0.35 (s, 6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-5-(trifluoromethyl)benzo[d][1,2]selenazol-3(2H)-on
e (8k). yellow solid, 70% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.85 (d, J = 8.8 Hz, 1H, Ar-H), 8.34 (s,
1H, Ar-H), 8.19 (d, J = 8.9 Hz, 1H, Ar-H), 7.83 (d, J = 9.2 Hz, 2H, Ar-H), 7.44 (d, J = 8.0 Hz, 1H, Ar-H),
7.35 (t, J = 7.7 Hz, 1H, Ar-H), 7.15 (d, J = 7.5 Hz, 1H, Ar-H), 1.18 (s, 9H, C-CH3), 0.36 (s, 6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-6-(trifluoromethyl)benzo[d][1,2]selenazol-3(2H)-on
e (8l). Yellow solid, 70% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.86 (d, J = 9.0 Hz, 1H, Ar-H), 8.19
(dd, J = 8.6, 2.5 Hz, 2H, Ar-H), 7.99 (s, 1H, Ar-H), 7.67 (d, J = 8.2 Hz, 1H, Ar-H), 7.44 (dd, J = 8.1, 1.2 Hz,
1H, Ar-H), 7.36 (t, J = 7.9 Hz, 1H, Ar-H), 7.15 (dd, J = 7.6, 1.3 Hz, 1H, Ar-H), 1.18 (s, 9H, C-CH3), 0.36 (s,
6H, Si-CH3).
2-(8-((tert-butyldimethylsilyl)oxy)quinolin-2-yl)-7-(trifluoromethyl)benzo[d][1,2]selenazol-3(2H)-on
e (8m). Yellow solid, 75% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.81 (d, J = 8.9 Hz, 1H, Ar-H), 8.25
(d, J = 7.7 Hz, 1H, Ar-H), 8.20 (d, J = 9.0 Hz, 1H, Ar-H), 7.89 (d, J = 7.6 Hz, 1H, Ar-H), 7.57 (t, J = 7.4 Hz,
1H, Ar-H), 7.44 (dd, J = 8.1, 1.1 Hz, 1H, Ar-H), 7.35 (t, J = 7.9 Hz, 1H, Ar-H), 7.14 (dd, J = 7.6, 1.2 Hz, 1H,
Ar-H), 1.20 (s, 9H, C-CH3), 0.35 (s, 6H, Si-CH3).
5.2.4.
5-hydroxy-2-(8-hydroxyquinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one (9a)

To solution 8a in MeOH, 3.0 eq K2CO3 was added at room temperature and the reaction was monitored by
TLC. After the reaction completed, the solution was diluted with water, and then filtrated. The precipitation
was washed with water to give the crude product, which was purified by recrystallization (THF/MeOH =
1/9). Yellow solid, 55% yield. Mp 265.9-266.5 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.81 (s, 1H, -OH),δ
8.74 (d, J = 9.0 Hz, 1H, Ar-H), 8.34 (d, J = 9.1 Hz, 1H, Ar-H), 7.86 (d, J = 8.6 Hz, 1H, Ar-H), 7.44 – 7.26 (m,
3H, Ar-H), 7.16 (m, 2H, Ar-H ). 13C NMR (101 MHz, DMSO-d6) δ 165.0, 156.1, 151.7, 150.1, 138.5, 136.8,
131.4, 128.7, 127.3, 126.7, 125.7, 121.9, 117.9, 113.7, 112.8, 112.6. [M+H]+ for C16H10N2O3Se pred.
358.9930, meas.358.9923.
5.2.5.
General Procedures for the Synthesis of 9b-9m
9b:
R1=R2=R3=R4=H
9c:
9d:
9e:
9f:
9g:
9h:
9i:
9j:
9k:
9l:
R1=R2=R3=H, R4=F
R1=R2=R4=H, R3=F
R1=R3=R4=H, R2=F
R2=R3=R4=H, R1=F
R1=R2=R3=H, R4=Cl
R1=R2=R4=H, R3=Cl
R1=R3=R4=H, R2=Cl
R1=R2=R3=H, R4=CF3
R1=R2=R4=H, R3=CF3
R1=R3=R4=H, R2=CF3
R1
R3
Se
R4
N
N
OH
R2
O
9b-9m
9m:
R2=R3=R4=H, R1=CF3
To solution 8 (1.0 equiv) in anhydrous THF, 1.2 equiv tetrabutylammoniumfluoride (TBAF) was added
dropwise at room temperature and the reaction monitored by TLC. After completion of the reaction, the
solution was diluted with water, and then filtrated to yield the precipitation, which was washed again with
water, and dried under reduced pressure to provide 9.
2-(8-hydroxyquinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one (9b). Yellow solid, 83% yield. Mp
265.8-268.4 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H, -OH), 8.65 (d, J = 9.0 Hz, 1H, Ar-H), 8.26 (d,
J = 9.0 Hz, 1H, Ar-H), 7.99 (d, J = 8.0 Hz, 1H, Ar-H), 7.83 (d, J = 7.7 Hz, 1H, Ar-H), 7.58 (t, J = 7.5 Hz, 1H,
Ar-H), 7.36 (t, J = 7.5 Hz, 1H, Ar-H), 7.29 (dd, J = 8.2, 1.6 Hz, 1H, Ar-H), 7.24 (t, J = 7.7 Hz, 1H, Ar-H),
7.06 (dd, J = 7.4, 1.6 Hz, 1H, Ar-H). 13C NMR (101 MHz, DMSO-d6) δ 165.1, 151.8, 150.1, 140.1, 138.7,
136.9, 132.9, 130.6, 127.7, 127.4, 126.1, 125.9, 118.0, 113.8, 112.9. [M+H]+ for C16H10N2O2Se pred.
342.9981, meas. 342.9975.
4-fluoro-2-(8-hydroxyquinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one (9c). Yellow solid, 87% yield.
1
Mp 270.8-273.2 °C. H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H, -OH), 8.67 (d, J = 8.9 Hz, 1H, Ar-H),
8.36 (d, J = 9.0 Hz, 1H, Ar-H), 7.91 (d, J = 7.9 Hz, 1H, Ar-H), 7.65 (td, J = 8.1, 4.8 Hz, 1H, Ar-H), 7.39 (dd,
J = 8.2, 1.6 Hz, 1H, Ar-H), 7.35 (t, J = 7.7 Hz, 1H, Ar-H), 7.25 – 7.12 (m, 2H, Ar-H). 13C NMR (101 MHz,

DMSO-d6) δ 162.6 (d, J = 3.7 Hz), 162.4 (d, J = 262.6 Hz), 152.2, 150.3, 143.0, 139.2, 137.3, 134.6 (d, J =
8.6 Hz), 127.9, 126.4, 122.5 (d, J = 4.2 Hz), 118.7 (d, J = 10.2 Hz), 118.4, 114.2, 113.4 (d, J = 7.2 Hz),
113.2. [M+H]+ for C16 H9N2O2FSe pred. 360.9887, meas. 360.9860.
5-fluoro-2-(8-hydroxyquinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one(9d). Yellow solid, 83% yield.
Mp 292.9-296.2 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H, -OH), 8.71 (d, J = 9.0 Hz, 1H, Ar-H),
8.35 (d, J = 9.0 Hz, 1H, Ar-H), 8.10 (dd, J = 8.8, 5.0 Hz, 1H, Ar-H), 7.67 (dd, J = 8.6, 2.8 Hz, 1H, Ar-H),
7.57 (td, J = 8.9, 2.8 Hz, 1H, Ar-H), 7.41 (d, J = 8.0 Hz, 1H, Ar-H), 7.36 (t, J = 7.7 Hz, 1H, Ar-H), 7.16 (dd,
J = 7.3, 1.7 Hz, 1H, Ar-H). 13C NMR (101 MHz, DMSO-d6) δ 163.9, 161.1 (d, J = 242.9 Hz), 151.8, 149.9,
138.7, 136.8, 135.2, 132.1 (d, J = 7.4 Hz), 127.9 (d, J = 7.7 Hz), 127.4, 125.9, 120.8 (d, J = 23.3 Hz), 117.9,
113.6, 113.3 (d, J = 22.9 Hz), 112.9. [M+H]+ for C16 H9N2O2FSe pred. 360.9887, meas. 360.9872.
6-fluoro-2-(8-hydroxyquinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one (9e). Yellow solid, 82% yield.
Mp 262.6-263.5 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H, -OH), 8.69 (d, J = 9.0 Hz, 1H, Ar-H),
8.35 (d, J = 9.0 Hz, 1H, Ar-H), 7.95 (dd, J = 8.6, 5.4 Hz, 1H, Ar-H), 7.86 (dd, J = 9.1, 2.5 Hz, 1H, Ar-H),
7.40 (dd, J = 8.2, 1.5 Hz, 1H, Ar-H), 7.36 (t, J = 7.5 Hz, 1H, Ar-H), 7.31 (td, J = 8.7, 2.4 Hz, 1H, Ar-H),
7.16 (dd, J = 7.3, 1.5 Hz, 1H, Ar-H). 13C NMR (101 MHz, DMSO-d6) δ 165.5 (d, J = 251.5 Hz), 164.5,
152.3, 150.4, 143.0 (d, J = 10.5 Hz), 139.2, 137.3, 130.6 (d, J = 10.1 Hz), 127.9, 127.8, 126.3, 118.5, 114.7
(d, J = 23.9 Hz), 114.1, 113.4, 112.9 (d, J = 26.4 Hz). [M+H]+ for C16 H9N2O2FSe pred. 360.9887, meas.
360.9869.
7-fluoro-2-(8-hydroxyquinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one (9f). Yellow solid, 82% yield.
Mp 214.6-216.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H, -OH), δ 8.68 (d, J = 9.0 Hz, 1H, Ar-H),
8.40 (d, J = 9.0 Hz, 1H, Ar-H), 7.80 (dd, J = 7.6, 1.0 Hz, 1H, Ar-H), 7.61 (t, J = 8.7 Hz, 1H, Ar-H), 7.54 (td,
J = 7.8, 5.1 Hz, 1H, Ar-H), 7.45 – 7.31 (m, 2H, Ar-H), 7.18 (dd, J = 6.9, 2.0 Hz, 1H, Ar-H). 13C NMR (101
MHz, DMSO-d6) δ 164.0, 157.7 (d, J = 244.1 Hz), 152.1, 149.2, 139.8, 136.7, 133.1 (d, J = 5.9 Hz), 129.4 (d,
J = 6.5 Hz), 128.2, 126.8, 125.5 (d, J = 25.1 Hz), 124.7 (d, J = 3.0 Hz), 119.7 (d, J = 19.6 Hz), 118.5, 113.8,
113.7. [M-H]- for C16H9N2O2FSe pred. 358.9741, meas. 358.9738.
4-chloro-2-(8-hydroxyquinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one (9g). Yellow solid, 78% yield.
Mp 240.3-242.6 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H, -OH), 8.70 (d, J = 9.0 Hz, 1H, Ar-H),
8.36 (d, J = 9.0 Hz, 1H, Ar-H), 8.09 (d, J = 7.9 Hz, 1H, Ar-H), 7.59 (t, J = 7.8 Hz, 1H, Ar-H), 7.46 (d, J =
7.7 Hz, 1H, Ar-H), 7.39 (d, J = 7.9 Hz, 1H, Ar-H), 7.35 (t, J = 7.7 Hz, 1H, Ar-H), 7.16 (d, J = 7.3 Hz, 1H,

Ar-H). 13C NMR (101 MHz, DMSO-d6) δ 163.1, 152.2, 150.2, 143.9, 139.2, 137.2, 134.9, 133.4, 128.6,
128.0, 126.4, 125.7, 125.3, 118.4, 114.4, 113.4. [M+H]+ for C16H9N2O2ClSe pred.376.9589, meas. 376.9572.
5-chloro-2-(8-hydroxyquinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one (9h). Yellow solid, 81% yield.
Mp 288.3-291.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H, -OH), 8.71 (d, J = 8.9 Hz, 1H, Ar-H),
8.37 (d, J = 9.0 Hz, 1H, Ar-H), 8.11 (d, J = 8.5 Hz, 1H, Ar-H), 7.88 (d, J = 2.3 Hz, 1H, Ar-H), 7.74 (dd, J =
8.5, 2.3 Hz, 1H, Ar-H), 7.40 (d, J = 7.9 Hz, 1H, Ar-H), 7.36 (t, J = 7.7 Hz, 1H, Ar-H), 7.17 (dd, J = 7.3, 1.8
Hz, 1H, Ar-H). 13C NMR (101 MHz, DMSO) δ 163.6, 151.9, 151.7, 149.7, 138.7, 136.7, 132.58, 132.2,
131.1, 127.8, 127.4, 126.7, 125.9, 117.9, 113.6, 112.9. [M+H]+ for C16H9N2O2ClSe pred.376.9589, meas.
376.9566.
6-chloro-2-(8-hydroxyquinolin-2-yl)benzo[d][1,2]selenazol-3(2H)-one (9i). Yellow solid, 76% yield.
Mp 289.8-293.6 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H, -OH), 8.70 (d, J = 8.9 Hz, 1H, Ar-H),
8.36 (d, J = 9.0 Hz, 1H, Ar-H), 8.32 (d, J = 8.4 Hz, 1H, Ar-H), 8.11 (d, J = 1.9 Hz, 1H, Ar-H), 7.99 (dd, J =
8.5, 2.0 Hz, 1H, Ar-H), 7.45 – 7.31 (m, 2H, 1-H, Ar-H), 7.16 (dd, J = 7.2, 1.7 Hz, 1H, Ar-H). 13C NMR (101
MHz, DMSO) δ 164.0, 151.9, 149.9, 141.9, 138.7, 138.0, 136.8, 129.6, 129.2, 127.5, 126.4, 125.9, 125.3,
117.9, 113.6, 113.0. [M+H]+ for C16H9 N2O2ClSe pred.376.9589, meas. 376.9569.
2-(8-hydroxyquinolin-2-yl)-4-(trifluoromethyl)benzo[d][1,2]selenazol-3(2H)-one (9j). Yellow solid,
86% yield. Mp 252.7-255.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H, -OH), 8.74 (d, J = 9.0 Hz, 1H,
Ar-H), 8.46 (dd, J = 7.4, 1.7 Hz, 1H, Ar-H), 8.39 (d, J = 9.1 Hz, 1H, Ar-H), 7.93 – 7.74 (m, 2H, Ar-H), 7.43
(dd, J = 8.2, 1.6 Hz, 1H, Ar-H), 7.38 (t, J = 7.8 Hz, 1H, Ar-H), 7.19 (dd, J = 7.4, 1.5 Hz, 1H, Ar-H). 13C
NMR (101 MHz, DMSO-d6) δ 162.6, 152.3, 150.1, 144.2, 139.3, 137.3, 132.4, 130.9, 129.4 (q, J = 32.9 Hz),
128.1, 127.1, 126.5, 125.2 (d, J = 6.6 Hz), 123.6 (q, J = 273.6 Hz), 118.5, 114.1, 113.5. [M+H]+ for
C17H9N2O2F3Se pred. 410.9855 meas. 410.9835.
2-(8-hydroxyquinolin-2-yl)-5-(trifluoromethyl)benzo[d][1,2]selenazol-3(2H)-one (9k). Yellow solid,
89% yield. Mp 288.7-289.5 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H, -OH), 8.70 (d, J = 8.9 Hz, 1H,
Ar-H), 8.36 (d, J = 9.0 Hz, 1H, Ar-H), 8.32 (d, J = 8.4 Hz, 1H, Ar-H), 8.11 (d, J = 1.9 Hz, 1H, Ar-H), 7.99
(dd, J = 8.5, 2.0 Hz, 1H, Ar-H), 7.45 – 7.31 (m, 2H, Ar-H), 7.16 (dd, J = 7.2, 1.7 Hz, 1H, Ar-H). 13C NMR
(101 MHz, DMSO-d6) δ 164.3, 152.3, 150.2, 145.8, 139.3, 137.3, 131.7, 129.1 (d, J = 3.8 Hz), 128.0, 127.8,
127.4 (q, J = 32.8 Hz), 126.5, 124.5 (q, J = 271.94 Hz), 124.4 (d, J = 3.8 Hz), 118.5, 114.1, 113.5. [M+H]+
for C17H9N2O2F3Se pred. 410.9855, meas. 410.9852.

2-(8-hydroxyquinolin-2-yl)-6-(trifluoromethyl)benzo[d][1,2]selenazol-3(2H)-one (9l). Yellow solid,
81%. Mp 291.3-295.2 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H, -OH), 8.70 (d, J = 9.0 Hz, 1H,
Ar-H), 8.45 (d, J = 1.7 Hz, 1H, Ar-H), 8.36 (d, J = 9.0 Hz, 1H, Ar-H), 8.09 (d, J = 8.1 Hz, 1H, Ar-H), 7.75
(dd, J = 8.2, 1.7 Hz, 1H, Ar-H), 7.44 – 7.30 (m, 2H, Ar-H), 7.16 (dd, J = 7.1, 1.8 Hz, 1H, Ar-H). 13C NMR
(101 MHz, DMSO-d6) δ 163.7, 151.8, 149.7, 141.1, 138.8, 136.7, 134.0, 132.3 (q, J = 31.6 Hz), 128.6, 127.5,
126.0, 123.8 (d, J = 273.0 Hz), 123.0 (d, J = 4.2 Hz), 122.6 (d, J = 3.3 Hz), 117.9, 113.6, 113.0. [M+H]+ for
C17H9N2O2F3Se pred. 410.9855, meas. 410.9848.
2-(8-hydroxyquinolin-2-yl)-7-(trifluoromethyl)benzo[d][1,2]selenazol-3(2H)-one (9m). Yellow solid,
81% yield. Mp 263.0-265.9 °C.1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H, -OH), 8.71 (d, J = 9.0 Hz, 1H,
Ar-H), 8.43 (d, J = 9.0 Hz, 1H, Ar-H), 8.22 (d, J = 7.7 Hz, 1H, Ar-H), 8.16 (d, J = 7.7 Hz, 1H, Ar-H), 7.74 (t,
J = 7.7 Hz, 1H, Ar-H), 7.44 – 7.37 (m, 2H, Ar-H), 7.18 (dd, J = 6.9, 2.1 Hz, 1H, Ar-H). 13C NMR (101 MHz,
DMSO-d6) δ 163.2, 151.6, 148.7, 139.5, 136.1, 136.0, 132.2, 131.5, 131.2 (q, J = 3.7 Hz), 127.7, 127.6,
126.4, 124.5 (q, J = 33.1 Hz), 124.2 (q, J = 273.4 Hz), 118.1, 113.4, 113.2. [M+H]+ for C17H9N2O2F3Se pred.
410.9855, meas. 410.9842.
5.3. Biological Assays
5.3.1.
The tested compounds and fluorescein (FL) stock solution were diluted with 75 mM phosphate buffer (pH
7.4) to 10 µM (or 20 µM) and 0.117 µM, respectively. The solution of
Oxygen Radical Absorbance Capacity (ORAC-FL) Assay26,27
( )-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was diluted with the same buffer to
the concentration of 100, 80, 60, 50, 40, 20, and 10 µM. The solution of
2,2'-azobis-(amidinopropane)dihydrochloride (AAPH) was prepared by dissolving AAPH (108.4 mg) in 10
mL of 75 mM phosphate buffer (pH 7.4) to a final concentration of 40 mM. The mixture of the tested
compound (20 µL) and FL (120 µL; 70 nM, final concentration) was pre-incubated for 10 min at 37 oC, and
then 60 µL of the AAPH solution was added, and a blank using phosphate buffer instead of the tested
compound was used as the reference. The fluorescence was recorded every minute for 120 min (excitation,
485 nm; emission, 520 nm). All samples were prepared in triplicate, and at least three independent runs were
performed. The antioxidant curves (fluorescence versus time) were normalized to the curve of the blank. The
area under the fluorescence decay curve (AUC) was calculated as following equation:

. Where f0 is the initial fluorescence reading at 0 min and fi is the fluorescence
reading at time i. The net AUC was calculated by the expression: AUCsample – AUCblank. Regression equations
between net AUC and Trolox concentrations were calculated. ORAC-FL value for each sample were
calculated using the standard curve with the ORAC-FL value of the tested compound expressed as Trolox
equivalents.
5.3.2.
Coupled Reductase Assay
The GPx-like activity of the organoselenium compounds was determined using a spectrophotometric
method at 340 nm, as described previously by Wilson et al.28 The test sample contained GSH (2 mM), EDTA
(1 mM), glutathione reductase (1.3 unite/mL) and NADPH (0.4 mM) in 100 mM potassium phosphate buffer,
pH 7.5. To test the mixture at 25 °C, GPx samples (80 µM) were added and the reaction initiated uponthe
addition of peroxide (1.6 mM). The initial reduction rates were calculated from the oxidation rate of NADPH
at 340 nm, and the initial reduction rate was determined at least three times and calculated from the first
5%-10% of the reaction using 6.22 mM–1·cm–1 as the extinction coefficient for NADPH.
5.3.3.
In Vitro Blood-Brain Barrier (BBB) Permeation Assay
The BBB penetration of compounds were evaluated using the parallel artificial membrane permeation
assay (PAMPA) as described previouslyby Di et al.29 Porcine brain lipid (PBL) was obtained from Avanti
Polar Lipids. Thirteen commercial drugs with known values were purchased from Sigma and Alfa Aesar and
used to validate the assay. The donor microplate (PVDF membrane, pore size 0.45 mm) and acceptor
microplate were both from Millipore. The 96-well UV plate (COSTAR®) was from Corning Incorporated.
The acceptor 96-well microplate was filled with 300 µL PBS/EtOH (7:3), and the filter membrane was
impregnated with 4 µL PBL in dodecane (20 mg/mL). After the compounds were dissolved in DMSO at 5
mg/mL and diluted 50-fold in PBS/EtOH (7:3) to a final concentration of 100 µg/mL, 200 µL of the solution
was added to the donor wells. The acceptor filter plate was carefully placed on the donor plate to form a
sandwich, and left without disturbing for 10 h at 25°C. The donor plate was carefully removed, and the
concentration of compounds in the acceptor wells was determined using the UV plate reader (Flexstation®
3). Every sample was analysed at five wavelengths in four wells and at least three independent runs. Pe was
calculated by the following expression: Pe = -Vd×Va/[(Vd+Va)A×t] × ln(1-drugacceptor/drugequilibrium) where Vd
is the volume of donor well; Va, volume in acceptor well; A, filter area; t, permeation time; drugacceptor, the
absorbance obtained in the acceptor well; drugequilibrium, the theoretical equilibrium absorbance. The results

are given as the mean standard deviation.
5.3.4. Metal-Chelating Study
The chelating studies were performed with a UV-Vis spectrophotometer. All compounds are dissolved in
the buffer that was used for the experiment. The absorption spectra of each compound (50 µM, final
concentration) with or without CuSO4, FeSO4, or ZnCl2 (50 µM, final concentration) for 30 min in buffer (20
mM HEPES, 150 mM NaCl, pH 7.4) and were recorded at room temperature.
5.3.5.
ThT Assay
Aβ1–42 (Millipore, counter ion: NaOH) was dissolved in ammonium hydroxide (1% v/v) to yield a stock
solution (2000 µM), which was aliquoted into small samples and stored at -80 oC.
For the inhibition of copper-mediated Aβ1–42 aggregation experiment, the Aβ stock solution was diluted
in 20 µM HEPES (pH 6.6) with 150 µM NaCl. The mixture of the peptide (10 µL, 25 µM, final
concentration) with or without copper (10 µL, 25 µM, final concentration) and the tested compound (10 µL,
50 µM, final concentration) was incubated at 37 oC for 24 h. Next, 20 µL of the sample was diluted to a final
volume of 200 µL with 50 mM glycine-NaOH buffer (pH 8.0) containing thioflavin T (5 µM). Next, the
fluorescence intensities were recorded five minutes later (excitation, 450 nm; emission, 485 nm).30
Acknowledgments
We thank the National Natural Science Foundation of China (21402175) for financial support of this study.
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