Design, synthesis and evaluation of clioquinol-ebselen hybrids as multi-target-directed ligands against Alzheimer's disease

Article abstract of DOI:10.1039/c5ra26797h

A novel series of compounds obtained by fusing the metal-chelating agent clioquinol and the antioxidant ebselen were designed, synthesized and evaluated as multi-target-directed ligands against Alzheimer's disease (AD). Specifically, compared with their parent compounds clioquinol and ebselen, these hybrids demonstrated significant potency in inhibiting self- and Cu(ii)-induced amyloid-β (Aβ) aggregation and acted as remarkable antioxidants and biometal chelators. In addition, the hybrids showed considerable improvements in ebselen-related pharmacological properties, including the ability to mimic glutathione peroxidase and scavenge H₂O₂. Of these molecules, compound 10h was identified as a potential lead compound for AD therapy. Importantly, this compound was found to possess rapid H₂O₂ scavenging activity and glutathione peroxidase-like (GPx-like) activity. Moreover, compound 10h was able to efficiently disassemble preformed self- and Cu(ii)-induced Aβ aggregates. Furthermore, 10h was able to penetrate the central nervous system (CNS) and did not exhibit any acute toxicity in mice at doses up to 2000 mg kg^-1.

Full text of DOI:10.1039/c5ra26797h

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Design, synthesis and evaluation of clioquinol–
ebselen hybrids as multi-target-directed ligands
against Alzheimer's disease†
Cite this: RSC Adv., 2016, 6, 7139
*
*
Zhiren Wang, Wenrui Li, Yali Wang, Xiruo Li, Ling Huang and Xingshu Li
A novel series of compounds obtained by fusing the metal-chelating agent clioquinol and the antioxidant
ebselen were designed, synthesized and evaluated as multi-target-directed ligands against Alzheimer's
disease (AD). Specifically, compared with their parent compounds clioquinol and ebselen, these hybrids
demonstrated significant potency in inhibiting self- and Cu(II)-induced amyloid-b (Ab) aggregation and
acted as remarkable antioxidants and biometal chelators. In addition, the hybrids showed considerable
improvements in ebselen-related pharmacological properties, including the ability to mimic glutathione
peroxidase and scavenge H2O2. Of these molecules, compound 10h was identified as a potential lead
compound for AD therapy. Importantly, this compound was found to possess rapid H2O2 scavenging
activity and glutathione peroxidase-like (GPx-like) activity. Moreover, compound 10h was able to
efficiently disassemble preformed self- and Cu(II)-induced Ab aggregates. Furthermore, 10h was able to
penetrate the central nervous system (CNS) and did not exhibit any acute toxicity in mice at doses up
Received 15th December 2015
Accepted 4th January 2016
DOI: 10.1039/c5ra26797h
www.rsc.org/advances
to 2000 mg kgꢀ1
.
generate reactive oxygen species (ROS) in the presence of some
transition metals and form stable dityrosine cross-linked
dimers, which are generated by free radical attack under
oxidative conditions. The interaction of Ab peptides with dys-
regulated redox-active metal ions, such as remarkably high
levels of iron (1 mM), zinc (1 mM), and copper (0.4 mM),
promote Ab aggregation and overproduction of ROS.5 Addi-
tionally, AD patients exhibit a signicant disease-dependent
increase in oxidative markers due to decreased protection of
neurons from mitochondrial oxidative stress, resulting in
higher levels of oxidative stress.6–8 Therefore, successful inhi-
bition of Ab aggregation, modulation of the biometals in the
brain and protection of neuronal cells from oxidative damage
could potentially prevent AD.
Because of the multifactorial pathogenesis of AD, in recent
years, many multitarget-directed ligands (MTDLs) acting on
diverse targets have been developed for the treatment of AD.9–15
Recent studies have indicated that the strategies using MTDLs
have provided a great deal of potential advantages such as
improving safety (reduced risk of drug–drug interactions) and
therapeutic effects (simultaneously inhibiting multiple targets)
and facilitating clinical studies (reduced uncertainty in phar-
macokinetics).12 The successful multitarget-directed lead
compound ladostigil, which combines the carbamate moiety of
rivastigmine with rasagiline, has undergone phase II clinical
studies as an anti-AD agent (Fig. 1).16
Introduction
Alzheimer's disease (AD), a multifaceted and devastating neuro-
degenerative disorder, is one of the public health challenges
facing our generation. Currently, more than 44 million people
suffer from AD worldwide, and the global cost is estimated at US
$604 billion.1 The etiology of AD has not been fully detailed at
present, but multiple factors are known to play important roles
in its pathogenesis, including low levels of acetylcholine,
b-amyloid (Ab) deposits, s-protein aggregation, oxidative stress,
inammation and dyshomeostasis of biometals.2 However, the
only therapeutic options approved by the US Food and Drug
Administration (FDA) are cholinesterase inhibitors (donepezil,
rivastigmine, and galantamine) and an N-methyl-D-aspartate
(NMDA) receptor antagonist (memantine), which result in
modest improvement in AD symptoms but have little efficacy in
preventing progression of the neurodegenerative disorder.3
The production of oligomeric aggregates of Ab in the brain
and the accumulation of intracellular neurobrillary tangles are
among the clearest indicators of the pathogenesis of AD, and
these two events are thought to initiate the disease, ultimately
leading to neuronal loss and dementia.3,4 Ab can also efficiently
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006,
China. E-mail: Huangl72@mail.sysu.edu.cn; lixsh@mail.sysu.edu.cn; Fax: +86-20-
3994-3051; +86-20-3994-3050; Tel: +86-20-3994-3051; +86-20-3994-3050
Clioquinol (CQ, Fig. 1) and its second-generation analogue
PBT2 (Fig. 1), 8-hydroxyquinoline derivatives with moderate
affinity for Cu and Zn that inhibit metal-induced Ab aggregation
† Electronic supplementary information (ESI) available: NMR spectrums,
LC-HRMS spectrums, computation studies, BBB-assay and GPx-like of target
compounds. See DOI: 10.1039/c5ra26797h
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Fig. 1 Structures of clioquinol, PBT2, ebselen, SE-518, SE-5i, riva-
stigmine, rasagiline and ladostigil.
Scheme 1 Synthesis of 2a–2c, 3b,3c, 4b,4c. Reagents and conditions:
(a) H2O, NaNO2, H2SO4, 15–18 ꢁC; (b) H2O, HNO3; (c) H2O, KOH,
NaClO, rt; (d) CH2Cl2 : hexane ¼ 1 : 1, (Boc)2O, DIPEA, DMAP, 50 ꢁC,
72 h; (e) EtOH, Na2S$9H2O; (f) H2O, KOH, KI, I2, rt.
and ROS generation, have been used for the treatment of AD
and have shown evidence of slowing cognitive deterioration and
signicantly decreasing plasma Ab1–42 levels.17–19
HNO3 to generate 2a. Compounds 2b (X ¼ Cl) and 2c (X ¼ I)
were prepared from 2a using previously published methods,25–27
and the phenolic hydroxy group of 2b and 2c were protected by
the reacting them with di-tert-butyl dicarbonate ester to give
compounds 3b (X ¼ Cl) and 3c (X ¼ I). Reduction of 3b and 3c
with Na2S$9H2O in EtOH provided 4b (X ¼ Cl) and 4c (X ¼ I),
respectively (Scheme 1).
The target compounds were obtained through the route
described in Schemes 2 and 3. First, Na2Se2 was reacted with the
corresponding benzenediazonium salts, which were prepared
from commercially available substituted 2-amino benzoic acids
The fundamental small molecule utilized in cellular antiox-
idant defence mechanisms is glutathione (GSH), which protects
cells by reducing harmful peroxides under the catalysis of
glutathione peroxidase (GPx). The small, organoselenium
compound ebselen (Fig. 1), which possesses the catalytic ability
to detoxify ROS, has been demonstrated to be an outstanding
GPx mimic and shown signicant benecial effects in a primate
model of neuro-degenerative diseases.20,21
Previously, we designed and synthesized a series of “selen-
pezil” compounds (SE-518 and SE-5i, Fig. 1) by fusing the
pharmacophores of donepezil and ebselen, which inhibit
acetylcholinesterase (AChE) and mimic GPx function.22,23 In
addition, a novel series of selenium-containing clioquinol
compounds, which exhibit signicant antioxidation and inhi-
bition of metal-induced Ab aggregation effects, were also
investigated in our previous study.24 Considering that both
redistributing biometal and enhancing antioxidation in the
brain are signicant approaches for the treatment of AD, in this
work (Fig. 2), we fused the pharmacophores of clioquinol and
ebselen to one molecule, which does not require the major
structural modications necessary for other MTDLs but results
in strong Ab aggregation inhibitory activity and a remarkable
ability to mimic the enzymatic properties of GPx.
Results and discussion
Scheme 2 Synthesis of 6a–6p, 7a–7p, 8a–8p (X ¼ Cl), 9a–9o (X ¼ I),
10a–10p (X ¼ Cl) and 11a–11o (X ¼ I). Reagents and conditions: (a)
H2O, NaNO2, HCl, 0–5 ꢁC, 30 min, Na2Se2, 50 ꢁC; (b) SOCl2, reflux; (c)
anhydrous CH2Cl2, triethylamine, rt; (d) CH2Cl2, HCl (g), 0–5 ꢁC.
Chemistry
The synthetic route of the CQ–ebselen hybrids is described in
Schemes 1–3. Commercially available quinolin-8-ol (1) rst
reacted with NaNO2/H2SO4 and was subsequently oxidized by
Scheme 3 Synthesis of 6q, 7q, 8q (X ¼ Cl), 9q (X ¼ I), 10q (X ¼ Cl) and
11q (X ¼ I). Reagents and conditions: (a) H2O, NaNO2, HCl, 0–5 ꢁC,
Fig. 2 Drug design strategy for multi-target-directed ligands (CQ–ebselen 30 min, Na2Se2, 50 ꢁC; (b) SOCl2, reflux; (c) anhydrous CH2Cl2,
hybrids).
triethylamine, 16 h; (d) CH2Cl2, HCl (g), 0–5 ꢁC.
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(5a–5q) and HNO2 to give 2,2-diselenobisbenzoic acids 6a–6q. much better catalytic activity compared with its analogue 11a
Subsequent reaction with SOCl2 under reux provided 2-chlor- (entry 2, X ¼ I and R ¼ H, H2O2: 59.8, t-BuOOH: 6.8, Cum-OOH:
oselenobenzoyl chlorides 7a–7q, which were reacted with the 28.1 mM minꢀ1), which bears an iodine on the oxine moiety. The
corresponding amine in anhydrous CH2Cl2 to provide key same tendency was also observed between 10e (entry 8, X ¼ Cl
intermediates 8a–8q (X ¼ Cl) and 9a–9q (X ¼ I). The hydro- and R1 ¼ F, H2O2: 151.8, t-BuOOH: 24.3, Cum-OOH: 113.3 mM
chloride compounds 10a–10q (X ¼ Cl) and 11a–11q (X ¼ I) were minꢀ1) and 11e (entry 9, X ¼ I and R1 ¼ F, H2O2: 141.7,
obtained by removal of the Boc group under HCl gas.
t BuOOH: 22.8 and Cum-OOH: 107.3 mM minꢀ1), as well as
between 10f vs. 11f, 10g vs. 11g, and 10h vs. 11h (Table 1).
The investigations of classical GPx-like mechanism and its
rate-determining step (Scheme 4) demonstrated that the weak
intramolecular nonbonding Se/O interaction in the selenenyl
Glutathione peroxidase-like activity and computational
studies
GPx, a mammalian antioxidant enzyme, is able to decompose sulde was favorable for the thiol to attack the sulfur atom
both metabolic and exogenous sources of ROS to H2O in the cell instead of selenium atom in the selenenyl sulde to obtain the
by using GSH or other related thiols as cofactors. Small selenol intermediate and facilitated the GPx-like cycle.28,30–33
molecular mimics of GPx have been identied as potential
therapeutics in studies of anti-inammation and neurodegen- of the CQ–ebselen hybrids, density functional theory (DFT)
erative disease.20,21
calculations and natural bond orbital (NBO) analyses were
To obtain better insight into the different GPx-like activities
The GPx-like activity of the clioquinol–ebselen hybrids was carried out for the key intermediate selenenyl suldes. The
evaluated by the classical method of GSH–GSSG coupled geometries of the selenenyl suldes were fully optimized using
reductase (see ESI†).28,29 The initial reduction rates (n0) for calculations at the B3LYP/6-311+g(d) level. As indicated by the
hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BuOOH) results in Table 2, weak intramolecular nonbonding Se/O
and cumene hydroperoxide (Cum-OOH) by reduced glutathione interactions were present in the selenenyl suldes 10d-S (R4 ¼
(GSH) in the presence and absence of the test compounds were Cl, 3.48 kcal molꢀ1), 10h-S (R4 ¼ F, 2.27 kcal molꢀ1), and 10l-S
calculated from a linear t spanning the rst 5–10% of the (R4 ¼ CF3, 2.79 kcal molꢀ1), but there was a strong Se/O
reaction by following the oxidation of reduced nicotinamide interaction in 10p-S (R1 ¼ OMe, 17.69 kcal molꢀ1). These results
adenine dinucleotide (NADPH) in phosphate buffer at 340 nm. led to the increased GPx-like activity of 10d, 10h and 10l, but
Most of the compounds provided much better or comparable weaker activity of 10p. Weak noncovalent Se/O interactions
catalytic activity compared with ebselen (Table 1, entry 29. were also observed in 10e-S (R1 ¼ F, 1.15 kcal molꢀ1), 10i-S (R1 ¼
H2O2: 124.3, t BuOOH: 27.4 and Cum-OOH: 61.3 mM minꢀ1), SE- CF3, 0.67 kcal molꢀ1) and 10m-S (R1 ¼ OMe, 0.57 kcal molꢀ1).
518 (entry 32. H2O2: 124.1, t BuOOH: 16.3 and Cum-OOH: 74.4 However, substituent groups in the R1 position, which were next
mM minꢀ1) and SE-5i (entry 33. H2O2: 122.7, t BuOOH: 23.1 and to the selenium–sulfur bond, may have caused steric hindrance
Cum-OOH: 56.6 mM minꢀ1). Specically, the results in Table 1 and prevented GSH from attacking the selenenyl suldes,
show that the substituent groups, such as Cl, F and CF3, in the resulting in moderate GPx-like activity by 10e, 10i and 10m.
R4 position of the benzoselenazol-3(2H)-one moiety were Replacement of the chlorine group on the oxine moiety with an
indispensable for high GPx activity. For example, compounds iodine group increased the Se/O interaction from 2.27 kcal
10d (entry 7. R4 ¼ Cl, H2O2: 219.7, t BuOOH: 31.0 and Cum- molꢀ1 (10h-S, R4 ¼ F, X ¼ Cl) to 2.83 kcal molꢀ1 (11h-S, R4 ¼ F,
OOH: 181.6 mM minꢀ1), 10h (entry 14. R4 ¼ F, H2O2: 228.0, X ¼ I), which gave rise to a decrease in GPx-like activity.
t BuOOH: 55.0 and Cum-OOH: 172.1 mM minꢀ1) and 10l (entry
20. R4 ¼ CF3, H2O2: 212.8, t BuOOH: 39.5 and Cum-OOH: 163.0
Determination of the catalytic parameters
mM minꢀ1), with Cl, F and CF3 in the R4 position of the ben-
zoselenazol-3(2H)-one moiety, exhibited much better results Kinetic experiments were conducted to further understand the
than other analogs with substituent groups removed or shied catalytic behavior of the target compound, with ebselen as the
to the R1, R2 or R3 position (such as 10a, entry 1; 10b,10c, entry control compound. Lineweaver–Burk (double reciprocal) plots
3, 5; 10e–10g, entry 8, 10 and 12; 10i–10k, 16, 17 and 19). for 10h were obtained by plotting the reciprocal of the initial
However, replacement of the substituent group in the R4 posi- rate (1/n0) against the reciprocal of the substrate concentration
tion with methoxy, i.e., 10p (entry 26, R4 ¼ OMe, H2O2: 128.4, (1/[substrate]) and were used to determine the catalytic
t-BuOOH: 20.2 and Cum-OOH: 73.6 mM minꢀ1), resulted in parameters.28,29 These catalytic parameters, including
a
substantial decrease in GPx-like activity. In addition, maximum velocity (Vmax), Michaelis constant (KM), catalytic
compounds with substituent groups in the R1 position, such as constant (kcat) and catalytic efficiency (h), were obtained for the
10e (F, entry 8), 10i (CF3, entry 16), and 10m (OMe, entry 21), reduction of H2O2, t-BuOOH and Cum-OOH in the presence of
exhibited better catalytic activity than compounds 10f,10g (R2 or the hybrid 10h and ebselen. It is evident from Table 3 that the
R3 ¼ F), 10j,10k (R2 or R3 ¼ CF3) and 10n,10o (R2 or R3 ¼ OMe), KM value of 10h (0.64, 1.16 and 0.75 mM at various peroxide
respectively. The presence of a chlorine group on the oxine concentrations; 0.47, 0.45 and 0.40 mM at various GSH
moiety was shown to be favorable for GPx-like activity compared concentrations) was much lower compared with the KM value
with the presence of an iodine group. For instance, 10a (entry 1, obtained for ebselen (1.32, 3.33 and 1.11 mM at various
X ¼ Cl and R ¼ H, H2O2: 97.8, t-BuOOH: 16.1, Cum-OOH: 47.5 peroxide concentrations; 1.05, 0.83 and 1.18 mM at various GSH
mM minꢀ1), with a chlorine group in the oxine moiety, exhibited concentrations). Furthermore, the
h
values that were
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Table 1 Initial rates, n0 (mM minꢀ1) for the reduction of peroxide by GSH in the presence of clioquinol and ebselen hybrid derivatives
Paper
GPx-like v0 (mM minꢀ1) ꢂ SDa
Entry
Compound
R1
R2
R3
R4
X
H2O2
t-BuOOH
Cum-OOH
1
2
3
4
5
6
7
8
10a$HCl
11a$HCl
10b$HCl
11b$HCl
10c$HCl
11c$HCl
10d$HCl
10e$HCl
11e$HCl
10f$HCl
11f$HCl
10g$HCl
11g$HCl
10h$HCl
11h$HCl
10i$HCl
10j$HCl
11j$HCl
10k$HCl
10l$HCl
10m$HCl
10n$HCl
11n$HCl
10o$HCl
11o$HCl
10p$HCl
10q$HCl
11q$HCl
Ebselen
H
H
H
H
H
H
H
F
F
H
H
H
H
H
H
CF3
H
H
H
H
OMe
H
H
H
H
H
—
—
—
—
H
H
Cl
Cl
H
H
H
H
H
F
F
H
H
H
H
H
CF3
CF3
H
H
H
OMe
OMe
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
F
F
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
F
F
H
H
H
H
CF3
H
H
H
H
H
OMe
—
—
—
—
—
—
—
—
Cl
I
Cl
I
Cl
I
Cl
Cl
I
Cl
I
Cl
I
Cl
I
Cl
Cl
I
Cl
Cl
Cl
Cl
I
Cl
I
Cl
Cl
I
—
—
—
—
—
—
97.8 ꢂ 2.1
59.8 ꢂ 3.7
78.4 ꢂ 7.4
63.3 ꢂ 17
16.1 ꢂ 2.5
6.8 ꢂ 1.2
20.2 ꢂ 2.9
4.3 ꢂ 1.1
9.7 ꢂ 2.1
5.6 ꢂ 1.7
31.0 ꢂ 1.5
24.3 ꢂ 3.2
22.8 ꢂ 5.2
14.0 ꢂ 1.9
2.0 ꢂ 0.7
31.6 ꢂ 1.7
12.7 ꢂ 1.4
55.0 ꢂ 4.5
24.7 ꢂ 0.9
15.3 ꢂ 1.5
3.6 ꢂ 1.0
17.0 ꢂ 6.9
13.7 ꢂ 2.4
39.5 ꢂ 2.4
38.3 ꢂ 0.3
10.2 ꢂ 1.2
13.9 ꢂ 2.0
10.7 ꢂ 1.1
2.9 ꢂ 1.4
20.2 ꢂ 2.9
12.2 ꢂ 3.3
5.4 ꢂ 0.4
27.4 ꢂ 0.1
4.7 ꢂ 0.8
28.1 ꢂ 0.5
16.3 ꢂ 1.2
23.1 ꢂ 2.8
5.0 ꢂ 0.8
47.5 ꢂ 2.7
28.1 ꢂ 1.8
73.2 ꢂ 3.3
28.3 ꢂ 1.6
45.9 ꢂ 2.9
22.3 ꢂ 3.4
181.6 ꢂ 11.6
113.3 ꢂ 5.6
107.3 ꢂ 5.9
79.1 ꢂ 2.0
48.0 ꢂ 0.5
123.3 ꢂ 0.9
32.2 ꢂ 3.3
172.1 ꢂ 3.4
98.1 ꢂ 5.1
113.2 ꢂ 7.8
46.0 ꢂ 4.7
23.4 ꢂ 1.8
86.0 ꢂ 7.2
163.0 ꢂ 3.3
135.5 ꢂ 0.3
40.3 ꢂ 3.0
18.2 ꢂ 1.0
42.9 ꢂ 4.4
25.4 ꢂ 6.3
73.6 ꢂ 4.3
11.6 ꢂ 0.7
15.7 ꢂ 2.6
61.3 ꢂ 0.1
5.9 ꢂ 1.5
90.9 ꢂ 10.6
40.3 ꢂ 5.8
219.7 ꢂ 3.9
151.8 ꢂ 10.1
141.7 ꢂ 6.7
102.7 ꢂ 3.1
86.5 ꢂ 1.1
147.0 ꢂ 0.88
59.9 ꢂ 7.5
228.0 ꢂ 9.8
193.6 ꢂ 4.2
103.5 ꢂ 10.8
83.6 ꢂ 7.4
65.4 ꢂ 4.2
108.5 ꢂ 4.1
212.8 ꢂ 5.3
155.4 ꢂ 5.4
75.5 ꢂ 5.3
56.2 ꢂ 1.8
76.6 ꢂ 10.6
30.0 ꢂ 2.5
128.4 ꢂ 4.3
43.1 ꢂ 5.1
44.1 ꢂ 2.8
124.3 ꢂ 3.4
37.6 ꢂ 0.3
126.1 ꢂ 2.9
124.1 ꢂ 6.8
122.7 ꢂ 3.6
37.2 ꢂ 0.7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
H
CF3
H
H
H
H
OMe
OMe
H
—
—
—
—
—
—
—
—
—
—
—
—
Clioquinol
Ebselen + clioquinol
SE-518
59.9 ꢂ 0.6
74.4 ꢂ 5.2
56.6 ꢂ 4.6
5.3 ꢂ 1.1
—
—
—
—
—
—
SE-5i
Controlb
a
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 per mL glutathione reductase (GR), 80 mM selenium compounds, and 1.6 mM peroxide. All values were
b
determined in triplicate for the initial 10 s, and the average values with standard deviation were obtained. Control values were obtained from
the reduction of peroxide by GSH in the absence of compound.
determined for 10h at various thiol concentrations for each
peroxide concentration were found to be approximately three to
ve times higher than those of ebselen. Overall, these obser-
vations revealed that the hybrid 10h results in signicant GPx-
like activity compared with ebselen.
Hydrogen peroxide scavenging activity
Cellular damage in the nervous system by free radical species
has been implicated in AD pathology.20,34 Exogenous free radi-
cals, generated by H2O2, are highly reactive species capable of
causing damage to lipids, protein and DNA.35 Experimental
Scheme 4 The classical GPx-like cycle and the rate-determining step
in the classical GPx-like cycle.
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Table 2 Summary of density functional theory (DFT) calculations and natural bond orbital (NBO) analysis on the selenenyl sulfides at B3LYP/6-
311+G(d,p) level on the B3LYP/6-311+G(d) level-optimized geometries
qO/Se–S [ꢁ]
qSe
qS(ꢀ)
ESe/O [kcal molꢀ1
]
˚
Selenenyl suldes
R
X
rSe/O [A]
10a-S
10b-S
10c-S
10d-S
10e-S
10f-S
10g-S
10h-S
11h-S
10i-S
10j-S
10k-S
10l-S
10m-S
10n-S
10o-S
10p-S
R ¼ H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Ia
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
2.609
2.611
2.618
2.677
2.973
2.601
2.620
2.681
2.679
3.036
2.625
2.608
2.795
3.124
2.616
2.628
2.479
175.8
175.8
175.4
175.4
152.9
175.8
175.5
175.8
176.0
152.0
175.4
175.8
174.2
144.0
175.9
175.2
176.4
0.366
0.374
0.371
0.367
0.315
0.377
0.365
0.388
0.389
0.309
0.375
0.383
0.335
0.299
0.364
0.355
0.390
0.030
0.026
0.025
0.027
0.010
0.028
0.025
0.035
0.038
0.029
0.020
0.024
0.005
0.013
0.028
0.030
0.047
10.38
13.51
14.74
3.48
R2 ¼ Cl
R3 ¼ Cl
R4 ¼ Cl
R1 ¼ F
1.15
R2 ¼ F
14.21
15.81
2.27
2.83
0.67
13.56
11.21
2.79
R3 ¼ F
R4 ¼ F
R4 ¼ F
R1 ¼ CF3
R2 ¼ CF3
R3 ¼ CF3
R4 ¼ CF3
R1 ¼ OMe
R2 ¼ OMe
R3 ¼ OMe
R4 ¼ OMe
0.57
13.97
10.41
17.69
a
Using Lanl2dz as the basis set for the I atom and 6-311+G(d,p) for C, H, O, N, F, S, Se atoms.
Table 3 Effect of peroxide and GSH concentrations on Vmax (maximum velocity), KM (Michaelis constant), kcat (catalytic constant), and h (catalytic
efficiency) of ebselen and 11h
Compound
Peroxide
Vmax (mM minꢀ1
)
KM (mM)
kcat (minꢀ1
)
h (mMꢀ1 minꢀ1
)
Effect of peroxide concentrationa
a
Ebselen
H2O2
222.71 ꢂ 3.46
58.55 ꢂ 3.00
141.44 ꢂ 8.10
351.52 ꢂ 5.42
102.21 ꢂ 6.24
246.54 ꢂ 6.17
1.32 ꢂ 0.04
3.33 ꢂ 0.17
1.11 ꢂ 0.06
0.64 ꢂ 0.11
1.16 ꢂ 0.04
0.75 ꢂ 0.03
2.78 ꢂ 0.20
0.73 ꢂ 0.03
1.77 ꢂ 0.10
4.39 ꢂ 0.18
1.28 ꢂ 0.06
3.08 ꢂ 0.07
2.10 ꢂ 0.1
0.22 ꢂ 0.01
1.59 ꢂ 0.09
6.86 ꢂ 0.41
1.10 ꢂ 0.08
4.10 ꢂ 0.09
t-BuOOHa
Cum-OOHa
a
10h$HCl
H2O2
t-BuOOHa
Cum-OOHa
Effect of thiol concentrationb
b
Ebselen
H2O2
187.96 ꢂ 2.77
43.01 ꢂ 0.60
75.64 ꢂ 4.21
290.09 ꢂ 9.21
68.83 ꢂ 3.43
201.21 ꢂ 4.87
1.05 ꢂ 0.01
0.83 ꢂ 0.01
1.18 ꢂ 0.06
0.47 ꢂ 0.08
0.45 ꢂ 0.03
0.40 ꢂ 0.05
2.35 ꢂ 0.03
0.54 ꢂ 0.07
0.95 ꢂ 0.05
3.62 ꢂ 0.11
0.86 ꢂ 0.08
2.51 ꢂ 0.06
2.23 ꢂ 0.02
0.65 ꢂ 0.08
0.80 ꢂ 0.04
7.70 ꢂ 0.23
1.91 ꢂ 0.03
6.27 ꢂ 0.15
t-BuOOHb
Cum-OOHb
b
10h$HCl
H2O2
t-BuOOHb
Cum-OOHb
a
Assay conditions: reactions were carried out at 25 ꢁC in phosphate buffer (100 mM, pH 7.5) containing 2 mM GSH, 0.4 mM NADPH, 1 mM EDTA,
1.3 U mLꢀ1 GR, variable peroxide, and 80 mM test compound. b Assay conditions: reactions were carried out at 25 ꢁC in phosphate buffer (100 mM,
pH 7.5) containing variable GSH, 0.4 mM NADPH, 1 mM EDTA, 1.3 U mLꢀ1 GR, 1.6 mM peroxide, and 80 mM test compound.
evidence has indicated that oxidative stress is a key event in AD 10g, 10h, 10l and 11h were selected for a horseradish peroxidase
onset and progression, and ROS- and reactive nitrogen species assay (HRPA); clioquinol and ebselen were used as reference
(RNS)-mediated injury have been observed in AD brain.36 compounds.24 As the results in Fig. 3 indicate, all compounds
Therefore, the antioxidant effects of H2O2 detoxication by exhibited signicantly better scavenging activity for hydrogen
antioxidants are signicant for AD patients. To further evaluate peroxide than CQ, ebselen and ebselen + CQ. Compounds 10d,
the antioxidant activity of the hybrids, compounds 10d, 10f, 10f, 10g, 10h, 10l and 11h demonstrated complete scavenging
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hybrids 10h and 11h, containing a uorine group in the R4
position of the benzoselenazol-3(2H)-one moiety, showed better
inhibition than the other hybrids (10h, IC50 ¼ 9.6 mM; 11h, 8.1
mM). Shiing the uorine group of the benzoselenazol-3(2H)-
one moiety to R1, R2 and R3 to generate 10e–10g and 11e–11g
resulted in less potent activity than the parent compounds (10e–
10g gave 9.9–15.6 of the IC50 values, and 11e–11g showed
9.5–10.5 mM of the IC50 values), showing that the uorine group
in the R4 position of the benzoselenazol-3(2H)-one moiety was
important. Replacement of the F in 10h and 11h with an H, Cl,
CF3 or OCH3 group resulted in compounds 10a–10d, 10i–10p,
11a–11c and 11j–11o (exhibited IC50 values of 10.1–12.7, 11.1–
16.6, 9.9–10.6 and 8.6–9.3 mM), which provided less potent
activity than 10h and 11h. Compounds 10q and 11q, which have
a naphthaselenazol-3(2H)-one structure, also exhibited weaker
Fig. 3 Scavenging effects on hydrogen peroxide in the HRPA assay
(H2O2 ¼ 100 mM). Values reported are means ꢂ SD of three inde-
pendent experiments. ***p < 0.001 in comparison to ebselen.
inhibitory activity than 10h and 11h, respectively (10q, IC50
¼
13.8 mM; 11q, 8.5 mM). The presence of a halogen on the oxine
moiety, especially iodine, was also shown to be important for
inhibitory activity. Compounds 10h and 11h, with a chlorine
and iodine group on the oxine moiety, exhibited IC50 values of
9.6 mM and 8.1 mM, respectively.
activity for hydrogen peroxide at concentrations of 80–200 mM.
These results were signicantly better than our previous
compounds SE-518 and SE-5i, which were unable to completely
scavenge H2O2 at concentrations lower than 200 mM.22,23
Furthermore, 10h and 11h maintained the effects at concen-
trations as low as 50 mM. In contrast, CQ, ebselen and ebselen +
CQ showed low activity even at high concentrations.
Oxygen radical absorbance capacity
The antioxidant activity of the target compounds was also deter-
mined using an oxygen radical absorbance capacity assay with
uorescein (ORAC-FL).42,43 Trolox, a vitamin E analogue respon-
Inhibition of self-mediated Ab1–42 aggregation
The inhibition activity of the target compounds against Ab1–42 sible for capturing radicals, was used as a standard. As shown in
aggregation was determined via thioavin T (ThT) uorescence Table 4, most of the target compounds demonstrated higher
assay,37–41 using curcumin and resveratrol as reference antioxidant activity (ORAC-FL values of 0.6–3.5 Trolox equivalents)
compounds (Table 4). All the hybrid compounds exhibited more than ebselen, CQ and ebselen + CQ. Notably, compound 10q with
signicant inhibition of Ab aggregation than ebselen, CQ and the naphthaselenazol-3(2H)-one moiety exhibited the best antiox-
ebselen + CQ. Specically, the results demonstrated that the idant activity (10q, ORAC-FL value ¼ 3.5). The structure–activity
Table 4 Inhibition of Ab1–42 aggregation, Oxygen Radical Absorbance Capacity (ORAC, Trolox equivalents)
Inhibition of Ab1–42
Inhibition of Ab1–42
Compound
aggregation IC50a (mM)
ORACb
Compound
aggregation IC50 (mM) SDa
ORACb
10a$HCl
10b$HCl
10c$HCl
10d$HCl
10e$HCl
10f$HCl
10g$HCl
10h$HCl
10i$HCl
10j$HCl
10k$HCl
10l$HCl
10m$HCl
10n$HCl
10o$HCl
10p$HCl
10q$HCl
11.7 ꢂ 0.3
11.2 ꢂ 0.1
10.2 ꢂ 1.0
10.1 ꢂ 0.9
12.3 ꢂ 0.5
9.9 ꢂ 1.1
2.6 ꢂ 0.1
2.6 ꢂ 0.3
2.3 ꢂ 0.2
2.6 ꢂ 0.1
2.5 ꢂ 0.1
2.4 ꢂ 0.3
2.7 ꢂ 0.3
2.6 ꢂ 0.1
2.5 ꢂ 0.2
2.3 ꢂ 0.4
2.4 ꢂ 0.1
2.6 ꢂ 0.3
2.5 ꢂ 0.1
2.5 ꢂ 0.3
2.9 ꢂ 0.1
2.6 ꢂ 0.2
3.5 ꢂ 0.1
11a$HCl
11b$HCl
11c$HCl
11e$HCl
11f$HCl
11g$HCl
11h$HCl
11j$HCl
11n$HCl
11o$HCl
11q$HCl
CQ
9.9 ꢂ 0.2
10.6 ꢂ 0.4
10.5 ꢂ 0.3
10.3 ꢂ 0.7
10.5 ꢂ 0.2
9.5 ꢂ 0.2
8.1 ꢂ 0.3
9.3 ꢂ 0.2
8.9 ꢂ 0.5
8.6 ꢂ 0.4
8.5 ꢂ 0.5
>50
1.0 ꢂ 0.1
1.0 ꢂ 0.1
0.8 ꢂ 0.1
0.9 ꢂ 0.1
1.5 ꢂ 0.3
0.9 ꢂ 0.1
0.6 ꢂ 0.2
0.9 ꢂ 0.2
1.3 ꢂ 0.2
0.9 ꢂ 0.1
1.3 ꢂ 0.1
0.6 ꢂ 0.1
0.2 ꢂ 0.1
0.7 ꢂ 0.1
n.t.c
15.6 ꢂ 0.1
9.6 ꢂ 0.4
15.3 ꢂ 1.6
14.2 ꢂ 0.3
16.6 ꢂ 1.3
12.1 ꢂ 0.7
14.7 ꢂ 0.8
13.8 ꢂ 1.6
11.1 ꢂ 0.1
11.3 ꢂ 1.7
13.8 ꢂ 0.5
Ebselen
>100
>50
Ebselen + CQ
Curcumin
Resveratrol
18.3 ꢂ 1.3
15.1 ꢂ 1.6
n.t.c
a
The thioavin-T uorescence method was used for the inhibition of Ab1–42 (concentration 25 mM) aggregation. The concentration (mM) required
for 50% inhibition was determined from dose–response curves. Experiments were performed in triplicate. b The means ꢂ SD of three independent
experiments. Data are expressed as mmol of Trolox equivalent/mmol of tested compound. c n.t. means ‘not tested’.
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relationship of the halogen on the oxine moiety observed with the compared with CQ (67%; 40%) and ebselen + CQ (68%; 39%). In
GPx-like activity was also observed with the antioxidant activity. accordance with the ThT binding assay results, TEM images
The presence of a halogen on the oxine moiety, especially iodine, indicated that more well-structured Cu(II)-induced Ab brils
was unfavorable for antioxidant activity. Compounds 10h and 11h, (Fig. 5Bc and d, sequence 3) were generated compared with the
with a chlorine or iodine group on the oxine moiety, showed samples of Ab alone, CQ, 10h and ebselen + CQ (Fig. 5Bc and d,
ORAC-FL values of 2.6 and 0.6, respectively.
sequence 2, 4, 5 and 6, respectively). Overall, the results of both
the ThT uorescence and TEM assays revealed that 10h was able
to control self- and Cu(II)-mediated Ab aggregation and disas-
semble preformed self- and Cu(II)-induced aggregates more
effectively than CQ, ebselen and ebselen + CQ.
Metal-chelating properties
The biometal-chelating (such as Cu2+, Zn2+, Fe2+ and Fe3+)
ability of 10h was studied by UV-visible spectrometry.24 New
absorption features were detected in the UV-vis spectra for
compound 10h upon the addition of CuSO4 (426 nm) or ZnCl2
(263 nm) FeSO4 (260 nm) and FeCl3 (264 nm), indicating that
10h was able to interact with Cu2+/Zn2+/Fe2+/Fe3+ to form
complexes (Fig. 4). The binding stoichiometry of the Cu2+–10h
complex was determined by following the absorption changes at
426 nm. The presence of an isosbestic point indicated the
formation of a unique Cu2+–10h complex (Fig. 4B), and titration
analysis was consistent with a 1 : 2 Cu2+/ligand molar ratio
(Fig. 4B inset).
Neuronal antioxidative effects against the production of ROS
induced by t-BuOOH
To evaluate the intracellular antioxidant efficacy of the optimal
compound, we carried out a cellular anti-oxidative activity assay
in human SH-SY5Y cells with Trolox as the reference
compound, which was able to directly determine intracellular
ROS production based on the uorescence probe of dichloro-
uorescein diacetate (DCFH-DA). First, as a colorimetric
MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide] assay showed that compound 10h (1–10 mM, 100% cell
viability; 25 mM, 75%; 50 mM, 37%) did not exhibit any toxicity at
1–10 mM (Fig. 6), we used 10 mM as the maximum concentration
of 10h in the intracellular antioxidant assay. As shown in Fig. 7,
aer the cells were treated with t-BuOOH, intracellular ROS
production increased from 32% (PBS) to 100% (t-BuOOH).
However, signicantly less ROS production was found in the
10h-treated cells (10 mM, 61%; 5 mM, 69%; 1 mM, 82%), which
also exhibited more potent intracellular antioxidant efficacy
than CQ (10 mM, 90%; 5 mM, 93%; 1 mM, 97%), ebselen (10 mM,
76%; 5 mM, 82%; 1 mM, 94%) and ebselen + CQ (10 mM, 70%; 5
mM, 79%; 1 mM, 92%). These results indicate that compound
10h has the potential to be an efficient multifunctional agent,
showing antioxidant activity, for the treatment of AD.
Modulation of self- and metal-induced Ab aggregation and
disaggregation of Ab aggregates by 10h
To evaluate the capacity of compound 10h to inhibit Ab1–42
aggregation and disassemble preformed Ab1–42 aggregates, ThT
uorescence (to measure the formation of Ab aggregates) and
transmission electron microscopy (TEM) assays (to determine
the morphological changes in the Ab species) were performed.
The results in Fig. 5A show that compound 10h inhibited 65%
(Fig. 5Aa) of Ab1–42 aggregation and disaggregated 67%
(Fig. 5Ab) of preformed Ab1–42 aggregates. In contrast, CQ (30%;
19%), ebselen (less than 10%) and ebselen + CQ (32%; 20%)
exhibited relatively weak effects compared with 10h. These
observations were further demonstrated by TEM, and smaller
and shorter Ab brils were observed in the presence of 10h
(Fig. 5Ac and d, sequence 5) compared with the controls,
ebselen, CQ and ebselen + CQ (Fig. 5Ad and e, sequence 2, 3, 4
and 6, respectively).
Blood–brain barrier permeation assay
To evaluate the brain penetration of the hybrids, a parallel
articial membrane permeability (PAMPA) assay, as previously
described, was used for a blood–brain barrier (PAMPA-BBB)
assay.24 The in vitro permeability (Pe) of target compounds and
13 commercial drugs through a porcine polar brain lipid were
detected using a mixture of PBS and ethanol at a ratio of 70 : 30
(Table S13, see ESI†). A plot of experimental permeability versus
reported values was determined for the assay validation, which
produced a good linear correlation, Pe(exp) ¼ 1.4574Pe(bibil) ꢀ
1.0773 (R2 ¼ 0.9427) (see ESI, Fig. S1†). From this equation, and
considering the limit established by Di et al. for blood–brain
barrier permeation, we were convinced that compounds with
permeabilities > 4.7 ꢃ 10ꢀ6 cm sꢀ1 could cross the blood–brain
barrier (see ESI, Table S14†). On the basis of the measured
permeability, which is presented in Table 5, most of the
compounds would be expected to be able to cross the blood–
brain barrier. The structure–activity relationship study showed
that compounds 10b–10l and 10q, which have hydrophobic
groups, such as uorine, chlorine, triuoromethyl and naph-
thaline, on the benzoselenazol-3(2H)-one moiety, had much
Additionally, as indicated by the results presented in Fig. 5Ba
and b, less Cu(II)-triggered Ab aggregation was found in the
presence of 10h (inhibition: 78%; disaggregation: 58%)
Fig. 4 (A) UV absorbance spectrum of compound 10h (50 mM) alone
or in the presence of CuSO4, ZnCl2, FeSO4 or FeCl3 (50 mM) in buffer
(20 mM HEPES, 150 mM NaCl, pH 7.4). (B) UV-vis titration of
compound 10h (80 mM) with Cu2+ in buffer (20 mM HEPES, 150 mM
NaCl, pH 7.4) at room temperature.
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Fig. 5 Inhibition and disaggregation experiments in the presence of 10h. (A) self-induced Ab1–42 aggregation experiments including (a and b)
top: scheme for the inhibition or disaggregation experiments. Bottom: The fluorescence intensity of the ThT binding assay. Statistical significance
was analyzed by ANOVA: (**) p < 0.01, (***) p < 0.001. (c and d) TEM image analysis of the inhibition of Ab1–42 aggregation and disaggregation of
Ab1–42 fibrils. Serial number: (1) fresh Ab1–42, (2) Ab1–42 alone, (3) Ab1–42 + ebselen, (4) Ab1–42 + CQ, (5) Ab1–42 + 10h, (6) Ab1–42 + ebselen + CQ.
Experimental conditions: Ab1–42 (25 mM); CQ, ebselen or 10h (25 mM); PBS (50 mM); pH 7.4; 37 ꢁC. (B) Cu2+-induced Ab1–42 aggregation
experiments including (a and b) top: scheme for the inhibition or disaggregation experiments. Bottom: The fluorescence intensity of the ThT
binding assay. Statistical significance was analyzed by ANOVA: (*) p < 0.05, (***) p < 0.001. (c and d) TEM image analysis of the inhibition of Cu2+
-
,
induced Ab1–42 aggregation and disaggregation of Cu2+-induced Ab1–42 fibrils. Serial number: (1) fresh Ab1–42, (2) Ab1–42 alone, (3) Ab1–42 + Cu2+
(4) Ab1–42 + Cu2+ + CQ, (5) Ab1–42 + Cu2+ + 10h, (6) Ab1–42 + ebselen + CQ. Experimental conditions: Ab1–42 (25 mM); CuSO4 (25 mM); CQ, or 10h
(50 mM); HEPES (20 mM) and NaCl (150 mM); pH 6.6; 37 ꢁC.
higher Pe values. However, for compounds with weak hydro- that compound 10h, which contains a chlorine group on the
phobic groups, such as 10a, and 10m–10p, the Pe values were oxine, would be able to penetrate the BBB and reach its bio-
slightly lower. The effect of halogens, especially chlorine, on the logical targets in the CNS, whereas the ability of 11h to pene-
oxine moiety was favorable for brain penetration. We expected trate the BBB was questionable. Therefore, given that brain
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was perhaps the most promising multifunctional anti-AD agent,
was evaluated using this assay. Twenty KM mice (common
closed-colony mice that are most widely used in biomedical
research in China) were randomly divided into four groups, and
the test compound was given in single oral doses of 0, 677, 1333,
or 2000 mg kgꢀ1. Aer administration of the compound, mice
were observed continuously for the rst 4 h for any abnormal
behavior and mortality changes, intermittently for the next 24 h,
and occasionally thereaer for 14 days for the onset of any
delayed effects. All mice were sacriced on the 14th day of drug
administration and examined macroscopically for possible
damage to the heart, liver, and kidneys. Animals treated with
compound 10h did not show any acute toxicity or mortality
either immediately or during the post-treatment period.
Furthermore, no signicant abnormal changes were observed
during the experimental period in terms of water or food
consumption or body weight. Therefore, compound 10h was
Fig. 6 Effect of 10h on cell viability in SH-SY5Y cells.
non-toxic and well tolerated at doses of up to 2000 mg kgꢀ1
.
Conclusions
In conclusion, our study involved the synthesis of a new series
of multi-target-directed ligands for the treatment of AD that
were designed as fusions of clioquinol and ebselen. These
compounds were evaluated as potential inhibitors of Ab1–42
aggregation, GPx mimics, superfast antioxidant catalysts
against H2O2, and metal-chelating ligands. Of the synthesized
compounds, 10h exhibited the most potent inhibition of Ab1–42
aggregation, signicant GPx-like activity, excellent anti-
oxidation, and disaggregation of Ab brils generated by self-
and Cu(II)-induced Ab aggregation. According to the results of
an in vitro blood–brain barrier model assay, this compound was
able to penetrate the CNS, and toxicity tests in mice showed that
10h had no acute toxicity at doses of up to 2000 mg kgꢀ1. These
Fig. 7 Inhibition of t-BuOOH-induced ROS production by Trolox, CQ,
ebselen, ebselen + CQ, and 10h determined by DCFH-DA. Data are the
mean ꢂ SD of at least three independent experiments performed in
sextuplicate and expressed as percent of control (cells only treated
with t-BuOOH). Statistical comparisons with control (only t-BuOOH)
were performed by one-way ANOVA (levels of significance *p < 0.05;
**p < 0.01; ***p < 0.001, ns p > 0.05).
penetration is the primary requirement for successful anti-AD
drugs, 10h was chosen for further evaluation.
Acute toxicity of compound 10h
The acute toxicity prole is considered a very important crite- properties highlight the potential of compound 10h to be a lead
rion in the development of new drugs. Compound 10h, which compound for the treatment of AD.
Table 5 Permeability (Pe ꢃ 10ꢀ6 cm sꢀ1) determined by the PAMPA-BBB assay for target compounds and predicted penetration of the CNS
Compounda
Pe (ꢃ10ꢀ6 cm sꢀ1
)
Prediction
Compounda
Pe (ꢃ10ꢀ6 cm sꢀ1
)
Prediction
b
b
10a$HCl
10b$HCl
10c$HCl
10d$HCl
10e$HCl
10f$HCl
10g$HCl
10h$HCl
10i$HCl
10j$HCl
10k$HCl
10l$HCl
10m$HCl
10n$HCl
10o$HCl
8.6 ꢂ 0.1
14.3 ꢂ 0.5
14.6 ꢂ 0.1
7.1 ꢂ 0.4
12.1 ꢂ 0.2
12.9 ꢂ 0.1
13.2 ꢂ 0.1
6.4 ꢂ 0.1
12.8 ꢂ 0.7
14.2 ꢂ 0.1
13.6 ꢂ 0.2
8.7 ꢂ 0.6
4.9 ꢂ 0.3
10.7 ꢂ 0.4
10.2 ꢂ 0.2
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
10p$HCl
10q$HCl
11a$HCl
11b$HCl
11c$HCl
11e$HCl
11f$HCl
11g$HCl
11h$HCl
11j$HCl
11n$HCl
11o$HCl
11q$HCl
Hydrocortisone
Chlorpromazine
3.1 ꢂ 0.6
20.1 ꢂ 1.0
8.1 ꢂ 0.1
10.3 ꢂ 0.1
10.2 ꢂ 0.2
11.3 ꢂ 0.2
7.8 ꢂ 0.2
10.3 ꢂ 0.2
4.5 ꢂ 0.2
13.6 ꢂ 0.2
10.2 ꢂ 0.6
7.3 ꢂ 0.1
9.8 ꢂ 0.2
1.9 ꢂ 0.1
5.8 ꢂ 0.3
CNSꢂ
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNSꢂ
CNS+
CNS+
CNS+
CNS+
CNSꢀ
CNS+
Compounds were dissolved in DMSO at 5 mg mLꢀ1 and diluted with PBS/EtOH (70 : 30). Values are expressed as the means ꢂ SD of three
a
b
independent experiments, compounds with permeabilities Pe > 4.7 ꢃ 10ꢀ6 cm sꢀ1 could cross the blood–brain barrier by passive diffusion.
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MHz, DMSO-d6) d 162.58, 147.88, 137.91, 135.70, 134.30, 133.17,
125.95, 123.42, 79.40. LC/MS (ESI): 314.9 [M ꢀ H]ꢀ.
Experimental
The NMR spectra were recorder using TMS as the internal stan-
dard on a Bruker AvanceIII 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. The reac-
tions 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
spectra were obtained using a Shimadzu LCMS-IT-TOF or LTQ
Orbitrap XL (Thermo Scientic) 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 ꢃ 250
mm, 5 mm), eluted with CH3OH/PBS (25 mM NaH2PO4 pH 3.0),
General procedures for the synthesis of 3b,3c
DMAP (2.10 g, 17.20 mmol) and DIPEA (12 mL, 68.90 mmol)
were added at room temperature to a stirred suspension of
nitroxoline (10.0 g, 34.45 mmol) and di-tert-butyl dicarbonate
(15.03 g, 68.90 mmol) in a 1 : 2 mixture of hexanes–DCM
(500 mL). The mixture was stirred for 72 h at 50 ꢁC, ltered, and
concentrated under reduced pressure to provide a crude
product which was puried by ash column chromatography on
silica gel (eluent : petroleum ether/DCM ¼ 8 : 2).
tert-Butyl(7-chloro-5-nitroquinolin-8-yl)carbonate (3b).
Yellow solid, 62% yield. Rf ¼ 0.58 (petroleum/EtOAc ¼ 10/1). 1H
NMR (400 MHz, CDCl3) d 9.05 (d, J ¼ 8.8 Hz, 2H), 8.50 (s, 1H),
7.62 (dd, J ¼ 8.7, 4.1 Hz, 1H), 1.59 (s, 9H). 13C NMR (101 MHz,
CDCl3) d 156.60, 150.93, 145.35, 139.91, 132.26, 128.07, 127.26,
123.75, 121.40, 88.22, 29.86.
30/70, at a ow rate of 0.5 mL minꢀ1
.
tert-Butyl(7-iodo-5-nitroquinolin-8-yl)carbonate (3c). Yellow
1
solid, 76% yield. Rf ¼ 0.67 (petroleum/EtOAc ¼ 10/1). H NMR
5-Nitroquinolin-8-ol (2a)
(400 MHz, CDCl3) d 9.04 (dd, J ¼ 8.7, 1.3 Hz, 1H), 8.98 (d, J ¼ 2.5
Hz, 1H), 8.82 (s, 1H), 7.64 (dd, J ¼ 8.8, 4.1 Hz, 1H), 1.66 (s, 9H).
13C NMR (101 MHz, CDCl3) d 161.82, 150.24, 143.59, 140.13,
135.31, 132.45, 123.98, 122.51, 92.06, 89.15, 30.69.
Compound 2a was synthesised according to the literature.25,27
Yellow solid, 80% yield. Rf ¼ 0.30 (CH2Cl2/CH3OH ¼ 20/1).
1H NMR (400 MHz, DMSO-d6) d 9.14 (d, J ¼ 8.8 Hz, 1H), 9.02
(d, J ¼ 3.5 Hz, 1H), 8.54 (d, J ¼ 8.7 Hz, 1H), 7.88 (dd, J ¼ 8.7, 3.9
Hz, 1H), 7.21 (d, J ¼ 8.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
d 160.91, 149.08, 137.35, 134.80, 132.35, 129.06, 125.17, 122.57,
110.02. LC/MS (ESI): 189.0 [M ꢀ H]ꢀ.
General procedures for the synthesis of 4b,4c
To a solution of tert-butyl(7-chloro-5-nitroquinolin-8-yl)carbonate
(3b) or tert-butyl(7-iodo-5-nitroquinolin-8-yl)carbonate (3c) (1.0
equiv.) in 20 mL of ethanol was added 5.0 equiv. of Na2S$9H2O in
one portion. The mixture was stirred at room temperature for 3 h.
5-Amino-7-chloroquinolin-8-yl tert-butyl carbonate (4b).
7-Chloro-5-nitroquinolin-8-ol (2b)
Compound 2b was synthesised according to the literature.26 2a
(12.0 g, 63.1 mmol) and KOH (3.5 g, 63.1 mmol) was dissolved in
1000 mL distilled water, 100 mL of NaClO (7.5%) solution was
added dropwise at room temperature. The reaction was moni-
tored by TLC, aer the completion of the reaction, the solution
was adjusted to pH ¼ 6.0 with acetic acid, ltrated, the
precipitation was washed with 400 mL distilled water, dried in
the air to get the product as a yellow solid. 71% yield. Rf ¼ 0.32
1
Yellow solid, 86%Yield. Rf ¼ 0.54 (petroleum/EtOAc ¼ 5/1). H
NMR (400 MHz, CDCl3) d 8.89 (dd, J ¼ 4.1, 1.4 Hz, 1H), 8.08 (dd,
J ¼ 8.5, 1.5 Hz, 1H), 7.30 (dd, J ¼ 8.5, 4.1 Hz, 2H), 6.81 (s, 1H),
1.50 (s, 9H). 13C NMR (101 MHz, CDCl3) d 149.89, 146.16,
142.36, 137.93, 129.77, 129.62, 119.60, 118.72, 111.46, 83.60,
29.65. LC/MS (ESI): 251.1 [M ꢀ CO2 + H]+.
5-Amino-7-iodoquinolin-8-yl tert-butyl carbonate (4c).
1
Yellow solid, 84% yield. Rf ¼ 0.67 (petroleum/EtOAc ¼ 5/1). H
1
(CH2Cl2/CH3OH ¼ 20/1). H NMR (400 MHz, DMSO-d6) d 9.34
NMR (400 MHz, CDCl3) d 8.86 (dd, J ¼ 4.0, 1.5 Hz, 1H), 8.08 (dd,
J ¼ 8.5, 1.5 Hz, 1H), 7.34 (dd, J ¼ 8.5, 4.1 Hz, 1H), 7.23 (s, 1H),
3.96 (s, 2H), 1.57 (s, 9H). 13C NMR (101 MHz, CDCl3) d 149.25,
147.65, 144.54, 138.43, 130.78, 129.91, 119.92, 119.51, 96.58,
84.30, 30.45. LC/MS (ESI): 343.0 [M ꢀ CO2 + H]+.
(d, J ¼ 8.5 Hz, 1H), 8.99 (d, J ¼ 3.7 Hz, 1H), 8.64 (s, 1H), 7.97 (dd,
J ¼ 8.8, 4.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 159.14,
147.69, 136.05, 135.77, 131.41, 130.06, 125.79, 122.71, 115.71.
LC/MS (ESI): 223.0 [M ꢀ H]ꢀ.
7-Iodo-5-nitroquinolin-8-ol (2c)
General procedures for the synthesis of 6a–6q and 7a–7q
Compound 2c was synthesised according to the literature.25 2a
(12.0 g, 63.1 mmol) and KOH (10.6 g, 189.5 mmol) was dissolved
in 1000 mL distilled water. A mixture of I2 (24.0 g, 94.6 mmol) in
KI (15.7 g, 94.6 mmol) was added in portion at room tempera-
ture. Aer the completion of the reaction, the product was
precipitated out by adjusted pH ¼ 6.0 with acetic acid, ltrated,
Compound 6a–6q and 7a–7q were synthesized according to
literature procedures.44
General procedures for the synthesis of 8a–8q (X ¼ Cl) and
9a–9q (X ¼ I)
washed with 500 mL distilled water, dried in the air. Yellow To a solution of 20 mL of anhydrous DCM, 10.0 equiv. of
1
solid, 74% yield. Rf ¼ 0.34 (CH2Cl2/CH3OH ¼ 20/1). H NMR anhydrous triethylamine and 1.0 equiv. of 4b or 4c, a solution of
(400 MHz, DMSO-d6) d 9.28 (d, J ¼ 8.6 Hz, 1H), 8.92 (d, J ¼ 3.6 1.5 equiv. of the corresponding chloride in 2 mL of anhydrous
Hz, 1H), 8.83 (s, 1H), 7.92 (dd, J ¼ 8.8, 4.3 Hz, 1H). 13C NMR (101 DCM was added dropwise. The reaction was stirred at room
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temperature for 16 h. Saturated NaHCO3 was added to the CDCl3) d 8.95 (dd, J ¼ 4.0, 1.5 Hz, 1H), 8.13 (dd, J ¼ 8.6, 5.2 Hz,
mixture, which was then extracted with DCM, washed with 1H), 8.00 (dd, J ¼ 8.5, 1.5 Hz, 1H), 7.67 (s, 1H), 7.44 (dd, J ¼ 7.7,
brine, dried over anhydrous Na2SO4, and concentrated under 2.0 Hz, 1H), 7.39 (dd, J ¼ 8.5, 4.1 Hz, 1H), 7.23 (dd, J ¼ 8.6, 2.1
reduced pressure. The residue was puried by silica-column Hz, 1H), 1.59 (s, 9H). 13C NMR (101 MHz, CDCl3) d 166.57 (d, 1JC–F
chromatography.
¼ 80.9 Hz), 164.42 (s), 151.51 (s), 150.60 (s), 146.07 (s), 140.81 (d,
tert-Butyl(7-chloro-5-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl) 2JC–F ¼ 10.2 Hz), 131.91 (s), 131.49 (d, 3JC–F ¼ 9.8 Hz), 129.71 (s),
quinolin-8-yl)carbonate (8a). Pale yellow solid, 69% yield. Rf ¼ 129.13 (d, 4JC–F ¼ 44.2 Hz), 126.04 (s), 122.03 (s), 121.74 (s), 115.42
0.53 (petroleum/EtOAc ¼ 3/1). 1H NMR (400 MHz, CDCl3) d 8.92 (d, 5JC–F ¼ 23.5 Hz), 111.22 (d, 6JC–F ¼ 26.2 Hz), 86.12 (s), 29.86 (s).
(d, J ¼ 2.6 Hz, 1H), 8.13 (d, J ¼ 7.7 Hz, 1H), 7.99 (d, J ¼ 8.5 Hz, LC/MS (ESI): 451.0 [M ꢀ CO2 + H]+.
1H), 7.72 (d, J ¼ 7.9 Hz, 1H), 7.69–7.63 (m, 2H), 7.50 (t, J ¼ 7.4
tert-Butyl(7-chloro-5-(5-uoro-3-oxobenzo[d][1,2]selenazol-
Hz, 1H), 7.35 (dd, J ¼ 8.5, 4.0 Hz, 1H), 1.57 (s, 9H). 13C NMR (101 2(3H)-yl)quinolin-8-yl)carbonate (8g). Pale yellow solid, 81%
1
MHz, CDCl3) d 167.16, 151.30, 150.50, 146.05, 139.24, 132.89, yield. Rf ¼ 0.31 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz,
132.05, 129.69, 129.61, 129.51, 128.86, 126.77, 126.06, 125.68, CDCl3) d 8.97 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.00 (dd, J ¼ 8.5, 1.7 Hz,
124.25, 121.64, 85.96, 29.85. LC/MS (ESI): 433.0 [M ꢀ CO2 + H]+. 1H), 7.84 (dd, J ¼ 8.1, 2.6 Hz, 1H), 7.70 (dd, J ¼ 8.8, 4.4 Hz, 1H),
tert-Butyl(7-chloro-5-(6-chloro-3-oxobenzo[d][1,2]selenazol- 7.67 (s, 1H), 7.45 (td, J ¼ 8.6, 2.7 Hz, 1H), 7.40 (dd, J ¼ 8.5, 4.2
2(3H)-yl)quinolin-8-yl)carbonate (8b). Pale yellow solid, 63% Hz, 1H), 1.57 (s, 9H). 13C NMR (101 MHz, CDCl3) d 166.29 (d,
yield. Rf ¼ 0.34 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, 1JC–F ¼ 3.3 Hz), 163.35 (s), 160.89 (s), 151.48 (s), 150.59 (s),
1
2
CDCl3) d 8.95 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.06 (d, J ¼ 8.4 Hz, 1H), 146.02 (s), 133.87 (d, JC–F ¼ 2.0 Hz), 131.90 (s), 129.57 (s),
7.98 (dd, J ¼ 8.5, 1.7 Hz, 1H), 7.74 (d, J ¼ 1.4 Hz, 1H), 7.67 (s, 129.39 (s), 128.86 (s), 127.40 (d, 3JC–F ¼ 8.0 Hz), 125.93 (d, 4JC–F
5
1H), 7.50 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.39 (dd, J ¼ 8.5, 4.1 Hz, 1H), ¼ 2.0 Hz), 125.86 (s), 121.74 (s), 121.26 (d, JC–F ¼ 24.1 Hz),
1.59 (s, 9H). 13C NMR (101 MHz, CDCl3) d 166.25, 151.54, 115.52 (d, JC–F ¼ 23.4 Hz), 86.11 (s), 29.84 (s). LC/MS (ESI):
6
150.61, 146.06, 140.35, 139.70, 131.86, 130.41, 129.67, 129.22, 451.0 [M ꢀ CO2 + H]+.
128.91, 127.66, 125.97, 124.21, 124.11, 121.76, 86.14, 29.85. LC/
tert-Butyl(7-chloro-5-(4-uoro-3-oxobenzo[d][1,2]selenazol-
MS (ESI): 467.0 [M ꢀ CO2 + H]+.
2(3H)-yl)quinolin-8-yl)carbonate (8h). Pale yellow solid, 78%
1
tert-Butyl(7-chloro-5-(5-chloro-3-oxobenzo[d][1,2]selenazol- yield. Rf ¼ 0.30 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz,
2(3H)-yl)quinolin-8-yl)carbonate (8c). Pale yellow solid, 74% CDCl3) d 8.95 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.04 (dd, J ¼ 8.5, 1.6 Hz,
1
yield. Rf ¼ 0.33 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, 1H), 7.66 (s, 1H), 7.64 (dd, J ¼ 8.1, 4.7 Hz, 1H), 7.49 (d, J ¼
CDCl3) d 8.94 (d, J ¼ 2.9 Hz, 1H), 8.12 (s, 1H), 7.97 (d, J ¼ 8.0 Hz, 7.6 Hz, 1H), 7.40 (dd, J ¼ 8.5, 4.1 Hz, 1H), 7.16 (dd, J ¼ 9.5,
1H), 7.66 (d, J ¼ 4.7 Hz, 3H), 7.38 (dd, J ¼ 8.5, 4.1 Hz, 1H), 1.58 8.4 Hz, 1H), 1.59 (s, 9H). 13C NMR (101 MHz, DMSO-d6)
(s, 9H). 13C NMR (101 MHz, CDCl3) d 165.83, 151.95, 150.53, d 164.25 (d, JC–F ¼ 3.4 Hz), 163.78 (s), 161.18 (s), 151.30 (s),
1
2
144.68, 142.46, 136.79, 133.68, 133.41, 132.33, 132.10, 130.93, 150.28 (s), 145.71 (s), 143.43 (s), 133.74 (d, JC–F ¼ 8.3 Hz),
129.46, 129.21, 126.96, 126.90, 125.91, 125.25, 122.48, 84.79, 132.96 (s), 131.12 (s), 129.30 (s), 128.27 (s), 126.42 (s), 122.67
27.64. LC/MS (ESI): 467.0 [M ꢀ CO2 + H]+.
(d, 3JC–F ¼ 3.8 Hz), 122.59 (s), 115.18 (d, 5JC–F ¼ 11.5 Hz), 113.41
tert-Butyl(7-chloro-5-(4-chloro-3-oxobenzo[d][1,2]selenazol- (d, 6JC–F ¼ 19.5 Hz), 85.28 (s), 30.10 (s). LC/MS (ESI): 451.0 [M ꢀ
2(3H)-yl)quinolin-8-yl)carbonate (8d). Pale yellow solid, 58% CO2 + H]+.
1
yield. Rf ¼ 0.37 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz,
tert-Butyl(7-chloro-5-(3-oxo-7-(triuoromethyl)benzo[d][1,2]
CDCl3) d 8.96 (d, J ¼ 3.9 Hz, 1H), 8.03 (dd, J ¼ 8.5, 2.5 Hz, 1H), selenazol-2(3H)-yl)quinolin-8-yl)carbonate (8i). White solid,
7.66 (t, J ¼ 4.1 Hz, 1H), 7.63 (dd, J ¼ 10.6, 3.7 Hz, 1H), 7.58 (dd, J 75% yield. Rf ¼ 0.40 (petroleum/EtOAc ¼ 5/1). 1H NMR (400
¼ 7.8, 4.2 Hz, 1H), 7.52 (dd, J ¼ 7.7, 3.1 Hz, 1H), 7.41 (dd, J ¼ 8.5, MHz, CDCl3) d 8.98 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.35 (d, J ¼ 7.6 Hz,
4.2 Hz, 1H), 1.58 (s, 9H). 13C NMR (101 MHz, CDCl3) d 164.99, 1H), 8.06–7.96 (m, 2H), 7.74–7.64 (m, 2H), 7.42 (dd, J ¼ 8.5, 4.1
151.46, 150.54, 146.04, 141.87, 137.48, 132.57, 131.95, 129.84, Hz, 1H), 1.60 (s, 9H). 13C NMR (101 MHz, CDCl3) d 165.78 (s),
129.32, 129.09, 128.86, 126.16, 122.91, 121.78, 120.77, 86.07, 151.68 (s), 150.68 (s), 146.09 (s), 135.96 (d, 1JC–F ¼ 2.0 Hz), 133.04
29.83. LC/MS (ESI): 467.0 [M ꢀ CO2 + H]+.
(s), 131.81 (s), 130.63 (dd, 2JC–F ¼ 6.9, 3.4 Hz), 129.68 (s), 128.93
tert-Butyl(7-chloro-5-(7-uoro-3-oxobenzo[d][1,2]selenazol- (s), 128.67 (s), 127.53 (s), 127.30 (s), 125.90 (s), 125.89 (dd, 3JC–F
2(3H)-yl)quinolin-8-yl)carbonate (8e). Pale yellow solid, 66% ¼ 68.7, 34.9 Hz), 121.84 (s), 86.17 (s), 29.87 (s). LC/MS (ESI):
yield. Rf ¼ 0.27 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, 501.0 [M ꢀ CO2 + H]+.
1
CDCl3) d 8.97 (dd, J ¼ 4.1, 1.5 Hz, 1H), 8.06–7.94 (m, 2H), 7.70 (s,
tert-Butyl(7-chloro-5-(3-oxo-6-(triuoromethyl)benzo[d][1,2]
1H), 7.55 (tt, J ¼ 10.0, 5.0 Hz, 1H), 7.46 (t, J ¼ 8.3 Hz, 1H), 7.41 selenazol-2(3H)-yl)quinolin-8-yl)carbonate (8j). White solid,
(dd, J ¼ 8.5, 4.1 Hz, 1H), 1.60 (s, 9H). 13C NMR (101 MHz, CDCl3) 85% yield. Rf ¼ 0.43 (petroleum/EtOAc ¼ 5/1). 1H NMR (400
1
d 166.17 (d, JC–F ¼ 2.4 Hz), 158.76 (s), 156.31 (s), 151.63 (s), MHz, CDCl3) d 8.96 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.28 (d, J ¼ 8.1 Hz,
150.65 (s), 146.06 (s), 131.85 (s), 129.74 (s), 129.20 (s), 128.99 (s), 1H), 8.05 (s, 1H), 7.98 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.78 (d, J ¼ 8.1
128.91 (d, 2JC–F ¼ 3.1 Hz), 128.18 (d, 3JC–F ¼ 4.4 Hz), 125.97 (s), Hz, 1H), 7.69 (s, 1H), 7.40 (dd, J ¼ 8.5, 4.1 Hz, 1H), 1.60 (s, 9H).
125.21 (d, 4JC–F ¼ 3.1 Hz), 121.82 (s), 118.58 (d, 5JC–F ¼ 19.0 Hz), 13C NMR (101 MHz, CDCl3) d 166.21 (s), 151.62 (s), 150.63 (s),
86.12 (s), 29.89 (s). LC/MS (ESI): 451.0 [M ꢀ CO2 + H]+.
146.00 (s), 140.13 (s), 134.56 (q, JC–F ¼ 32.7 Hz), 131.69 (s),
1
tert-Butyl(7-chloro-5-(6-uoro-3-oxobenzo[d][1,2]selenazol- 129.84 (s), 129.57 (s), 128.93 (d, 2JC–F ¼ 9.5 Hz), 128.61 (s), 125.84
2(3H)-yl)quinolin-8-yl)carbonate (8f). Pale yellow solid, 82% (s), 123.61 (d, 3JC–F ¼ 3.1 Hz), 123.46 (q, 4JC–F ¼ 273.6 Hz), 122.16
1
yield. Rf ¼ 0.32 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz,
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(d, 5JC–F J ¼ 3.4 Hz), 121.82 (s), 86.19 (s), 29.78 (s). LC/MS (ESI): CDCl3) d 8.93 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.06 (dd, J ¼ 8.5, 1.6 Hz,
501.0 [M ꢀ CO2 + H]+.
1H), 7.64 (s, 1H), 7.60 (t, J ¼ 8.1 Hz, 1H), 7.37 (dd, J ¼ 8.5, 4.1 Hz,
tert-Butyl(7-chloro-5-(3-oxo-5-(triuoromethyl)benzo[d][1,2] 1H), 7.25 (d, J ¼ 7.9 Hz, 1H), 6.93 (d, J ¼ 8.3 Hz, 1H), 4.00 (s, 3H),
selenazol-2(3H)-yl)quinolin-8-yl)carbonate (8k). White solid, 1.59 (s, 9H). 13C NMR (101 MHz, CDCl3) d 166.00, 161.80,
81% yield. Rf ¼ 0.41 (petroleum/EtOAc ¼ 5/1). 1H NMR (400 151.09, 150.41, 146.03, 141.61, 133.71, 132.32, 130.03, 129.78,
MHz, CDCl3) d 8.98 (d, J ¼ 4.0 Hz, 1H), 8.44 (s, 1H), 8.06–7.92 128.82, 126.34, 121.60, 116.12, 113.35, 108.49, 85.84, 56.01,
(m, 2H), 7.89 (d, J ¼ 8.4 Hz, 1H), 7.70 (s, 1H), 7.42 (ddd, J ¼ 8.5, 29.85. LC/MS (ESI): 463.0 [M ꢀ CO2 + H]+.
4.1, 0.5 Hz, 1H), 1.60 (s, 9H). 13C NMR (101 MHz, CDCl3)
tert-Butyl(7-chloro-5-(3-oxonaphtho[2,3-d][1,2]selenazol-
d 166.14 (s), 151.68 (s), 150.64 (s), 146.05 (s), 143.39 (s), 131.74 2(3H)-yl)quinolin-8-yl)carbonate (8q). Pale yellow solid, 67%
1
(s), 129.67 (q, 1JC–F ¼ 33.4 Hz), 129.64 (s), 129.03 (dd, 2JC–F ¼ 6.5, yield. Rf ¼ 0.41 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz,
3
4
3.0 Hz), 128.93 (d, JC–F ¼ 6.3 Hz), 126.57 (q, JC–F ¼ 4.0 Hz), CDCl3) d 8.96 (d, J ¼ 3.9 Hz, 1H), 8.76 (s, 1H), 8.13 (s, 1H), 8.12–
5
126.19 (s), 125.87 (s), 125.19 (s), 123.61 (q, JC–F ¼ 272.4 Hz), 8.06 (m, 2H), 7.92 (d, J ¼ 8.2 Hz, 1H), 7.74 (s, 1H), 7.72–7.65 (m,
121.80 (s), 86.23 (s), 29.83 (s). LC/MS (ESI): 501.0 [M ꢀ CO2 + H]+. 1H), 7.64–7.56 (m, 1H), 7.40 (dd, J ¼ 8.5, 4.1 Hz, 1H), 1.60 (s,
tert-Butyl(7-chloro-5-(3-oxo-4-(triuoromethyl)benzo[d][1,2] 9H). 13C NMR (101 MHz, CDCl3) d 167.15, 151.85, 150.57,
selenazol-2(3H)-yl)quinolin-8-yl)carbonate (8l). White solid, 144.40, 142.46, 135.95, 133.02, 132.35, 131.62, 130.95, 130.44,
73% yield. Rf ¼ 0.37 (petroleum/EtOAc ¼ 5/1). 1H NMR (400 129.71, 129.12, 128.97, 127.21, 126.98, 126.63, 126.06, 124.08,
MHz, CDCl3) d 8.96 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.01 (dd, J ¼ 8.5, 1.6 122.87, 122.34, 84.69, 27.65. LC/MS (ESI): 483.0 [M ꢀ CO2 + H]+.
Hz, 1H), 7.95 (d, J ¼ 8.1 Hz, 1H), 7.91 (d, J ¼ 7.6 Hz, 1H), 7.80 (t, J
tert-Butyl(7-iodo-5-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)
¼ 7.8 Hz, 1H), 7.69 (s, 1H), 7.42 (dd, J ¼ 8.5, 4.1 Hz, 1H), 1.58 (s, quinolin-8-yl)carbonate (9a). Pale yellow solid, 73% yield. Rf ¼
9H). 13C NMR (101 MHz, CDCl3) d 164.42 (s), 151.42 (s), 150.57 0.37 (petroleum/EtOAc ¼ 5/1). 1H NMR (400 MHz, CDCl3) d 8.92
(s), 145.95 (s), 142.28 (s), 131.84 (s), 131.65 (s), 131.40 (dd, 1JC–F
=
(dd, J ¼ 4.1, 1.7 Hz, 1H), 8.22–8.13 (m, 1H), 8.04 (s, 1H), 8.02 (dd,
67.3, 33.5 Hz), 129.80 (s), 129.31 (s), 128.83 (s), 128.62 (s), 126.07 J ¼ 8.5, 1.7 Hz, 1H), 7.74 (dd, J ¼ 1.5, 1.0 Hz, 1H), 7.73 (d, J ¼ 1.0
(s), 125.45 (q, 2JC–F = 6.5 Hz), 122.72 (q, 3JC–F = 274.1 Hz), 121.96 Hz, 1H), 7.59–7.51 (m, 1H), 7.42 (dd, J ¼ 8.5, 4.1 Hz, 1H), 1.67 (s,
(s), 121.89 (s), 86.16 (s), 29.79 (s). LC/MS (ESI): 501.0 [M ꢀ CO2 + 9H). 13C NMR (101 MHz, CDCl3) d 164.70, 149.88, 137.50,
H]+.
135.05, 132.95, 132.45, 132.20, 131.15, 129.63, 129.12, 126.83,
tert-Butyl(7-chloro-5-(7-methoxy-3-oxobenzo[d][1,2]selena- 125.77, 124.74, 124.13, 121.97, 94.23, 86.81, 30.67. LC/MS (ESI):
zol-2(3H)-yl)quinolin-8-yl)carbonate (8m). Pale yellow solid, 524.9 [M ꢀ CO2 + H]+.
71% yield. Rf ¼ 0.38 (petroleum/EtOAc ¼ 5/1). 1H NMR (400
tert-Butyl(5-(6-chloro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)-
MHz, CDCl3) d 8.95 (dd, J ¼ 4.0, 1.6 Hz, 1H), 8.01 (dd, J ¼ 8.5, 1.6 7-iodoquinolin-8-yl)carbonate (9b). Pale yellow solid, 65% yield.
1
Hz, 1H), 7.77 (d, J ¼ 7.7 Hz, 1H), 7.68 (s, 1H), 7.52 (t, J ¼ 7.9 Hz, Rf ¼ 0.41 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, CDCl3)
1H), 7.39 (dd, J ¼ 8.5, 4.1 Hz, 1H), 7.16 (d, J ¼ 8.0 Hz, 1H), 4.02 d 8.89 (d, J ¼ 2.4 Hz, 1H), 8.03 (d, J ¼ 8.3 Hz, 1H), 7.99 (s, 1H),
(s, 3H), 1.60 (s, 9H). 13C NMR (101 MHz, CDCl3) d 167.36, 154.21, 7.95 (d, J ¼ 7.3 Hz, 1H), 7.73 (s, 1H), 7.46 (d, J ¼ 8.3 Hz, 1H), 7.39
151.24, 150.54, 146.04, 132.09, 130.00, 129.56, 128.84, 128.69, (dd, J ¼ 8.4, 4.0 Hz, 1H), 1.62 (s, 9H). 13C NMR (101 MHz, CDCl3)
126.84, 126.04, 121.68, 121.23, 114.96, 112.71, 111.68, 85.94, d 165.90, 151.71, 150.97, 148.13, 141.66, 137.07, 133.41, 133.08,
56.11, 29.88. LC/MS (ESI): 463.0 [M ꢀ CO2 + H]+.
131.94, 129.24, 127.64, 127.29, 127.14, 125.37, 122.49, 120.59,
tert-Butyl(7-chloro-5-(6-methoxy-3-oxobenzo[d][1,2]selena- 95.59, 84.15, 30.82. LC/MS (ESI): 558.9 [M ꢀ CO2 + H]+.
zol-2(3H)-yl)quinolin-8-yl)carbonate (8n). White solid, 74%
tert-Butyl(5-(5-chloro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)-
1
yield. Rf ¼ 0.37 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, 7-iodoquinolin-8-yl)carbonate (9c). Pale yellow solid, 67% yield.
1
CDCl3) d 8.95 (d, J ¼ 3.9 Hz, 1H), 8.11–7.98 (m, 2H), 7.67 (s, 1H), Rf ¼ 0.42 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, CDCl3)
7.39 (dd, J ¼ 8.4, 4.2 Hz, 1H), 7.17 (d, J ¼ 2.1 Hz, 1H), 7.07 (dd, J d 8.91 (d, J ¼ 4.0 Hz, 1H), 8.14 (s, 1H), 8.04–7.93 (m, 2H), 7.67
¼ 8.6, 2.1 Hz, 1H), 3.95 (s, 3H), 1.59 (s, 9H). 13C NMR (101 MHz, (dd, J ¼ 15.6, 8.5 Hz, 2H), 7.41 (dd, J ¼ 8.5, 4.0 Hz, 1H), 1.66 (s,
CDCl3) d 166.85, 163.79, 151.26, 150.46, 146.10, 140.95, 132.13, 9H). 13C NMR (101 MHz, CDCl3) d 165.94, 151.61, 150.51,
130.70, 129.88, 129.71, 128.87, 126.22, 121.59, 118.61, 114.96, 149.71, 141.49, 137.01, 136.54, 133.57, 133.30, 132.62, 132.17,
107.64, 85.93, 55.88, 29.86. LC/MS (ESI): 463.0 [M ꢀ CO2 + H]+. 129.30, 127.00, 126.95, 125.39, 122.75, 89.98, 84.70, 27.73. LC/
tert-Butyl(7-chloro-5-(5-methoxy-3-oxobenzo[d][1,2]selena- MS (ESI): 558.9 [M ꢀ CO2 + H]+.
zol-2(3H)-yl)quinolin-8-yl)carbonate (8o). White solid, 71%
tert-Butyl(5-(7-uoro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)-
1
yield. Rf ¼ 0.35 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, 7-iodoquinolin-8-yl)carbonate (9e). Pale yellow solid, 68% yield.
1
CDCl3) d 8.95 (dd, J ¼ 4.0, 1.6 Hz, 1H), 8.01 (dd, J ¼ 8.5, 1.6 Hz, Rf ¼ 0.31 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, CDCl3)
1H), 7.68 (s, 1H), 7.63 (d, J ¼ 2.6 Hz, 1H), 7.59 (d, J ¼ 8.7 Hz, 1H), d 8.95–8.89 (m, 1H), 8.04 (d, J ¼ 1.4 Hz, 1H), 7.98 (t, J ¼ 7.9 Hz,
7.39 (dd, J ¼ 8.6, 4.2 Hz, 1H), 7.35 (dd, J ¼ 8.8, 2.7 Hz, 1H), 3.93 2H), 7.54 (ddd, J ¼ 12.9, 7.8, 1.5 Hz, 1H), 7.45 (d, J ¼ 8.2 Hz, 1H),
(s, 3H), 1.60 (s, 9H). 13C NMR (101 MHz, CDCl3) d 167.14, 159.27, 7.43–7.39 (m, 1H), 1.66 (s, 9H). 13C NMR (101 MHz, CDCl3)
1
151.24, 150.51, 146.02, 132.09, 130.32, 129.88, 129.50, 128.83, d 166.16 (d, JC–F ¼ 2.4 Hz), 158.77 (s), 156.92 (s), 156.32 (s),
126.68, 126.01, 125.13, 122.67, 121.63, 111.15, 85.95, 55.78, 149.98 (s), 144.47 (s), 137.58 (s), 131.98 (s), 129.50 (s), 128.93 (d,
29.85. LC/MS (ESI): 463.0 [M ꢀ CO2 + H]+.
2JC–F ¼ 6.1 Hz), 128.22 (d, 3JC–F ¼ 4.5 Hz), 127.07 (s), 125.22 (d,
tert-Butyl(7-chloro-5-(4-methoxy-3-oxobenzo[d][1,2]selena- 4JC–F ¼ 3.0 Hz), 122.09 (s), 118.56 (d, 5JC–F ¼ 19.0 Hz), 94.19 (s),
zol-2(3H)-yl)quinolin-8-yl)carbonate (8p). White solid, 82% 86.92 (s), 30.69 (s). LC/MS (ESI): 543.0 [M ꢀ CO2 + H]+.
1
yield. Rf ¼ 0.24 (petroleum/EtOAc ¼ 3/1). H NMR (400 MHz,
7150 | RSC Adv., 2016, 6, 7139–7158
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tert-Butyl(5-(6-uoro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)-
RSC Advances
tert-Butyl(7-iodo-5-(5-methoxy-3-oxobenzo[d][1,2]selenazol-
7-iodoquinolin-8-yl)carbonate (9f). Pale yellow solid, 69%yield. 2(3H)-yl)quinolin-8-yl)carbonate (9o). White solid, 72% yield. Rf
Rf ¼ 0.37 (petroleum/EtOAc ¼ 5/1). 1H NMR (400 MHz, ¼ 0.38 (petroleum/EtOAc ¼ 5/1). 1H NMR (400 MHz, CDCl3)
CDCl3) d 8.91 (dd, J ¼ 4.0, 1.6 Hz, 1H), 8.12 (dd, J ¼ 8.6, 5.2 d 8.91 (dd, J ¼ 4.0, 1.5 Hz, 1H), 8.02 (s, 1H), 8.00 (dd, J ¼ 8.5, 1.6
Hz, 1H), 8.01 (s, 1H), 7.98 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.47–7.36 Hz, 1H), 7.63 (d, J ¼ 2.5 Hz, 1H), 7.58 (d, J ¼ 8.7 Hz, 1H), 7.41
(m, 2H), 7.23 (dd, J ¼ 8.6, 2.2 Hz, 1H), 1.65 (s, 9H). 13C NMR (dd, J ¼ 8.5, 4.1 Hz, 1H), 7.36 (dd, J ¼ 8.7, 2.6 Hz, 1H), 3.93 (s,
1
(101 MHz, CDCl3) d 166.53 (d, JC–F ¼ 87.4 Hz), 164.42 (s), 3H), 1.66 (s, 9H). 13C NMR (101 MHz, CDCl3) d 167.10, 159.26,
2
156.80 (s), 149.88 (s), 144.47 (s), 140.76 (d, JC–F ¼ 10.2 Hz), 156.53, 149.85, 144.43, 137.34, 132.23, 130.29, 130.18, 127.12,
3
137.55 (s), 132.02 (s), 131.49 (d, JC–F ¼ 9.8 Hz), 129.66 (s), 126.69, 125.10, 122.70, 121.92, 111.16, 94.27, 86.72, 55.82,
4
127.15 (s), 121.98 (s), 115.38 (d, JC–F ¼ 23.6 Hz), 111.16 (d, 30.66. LC/MS (ESI): 592.9 [M ꢀ CO2 + H]+.
5JC–F ¼ 26.2 Hz), 94.13 (s), 86.87 (s), 30.64 (s). LC/MS (ESI):
tert-Butyl(7-iodo-5-(3-oxonaphtho[2,3-d][1,2]selenazol-
542.9 [M ꢀ CO2 + H]+.
2(3H)-yl)quinolin-8-yl)carbonate (9q). Pale yellow solid, 68%
1
tert-Butyl(5-(5-uoro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)- yield. Rf ¼ 0.40 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz,
7-iodoquinolin-8-yl)carbonate (9g). Pale yellow solid, 75% yield. CDCl3) d 8.93 (dd, J ¼ 4.1, 1.7 Hz, 1H), 8.76 (s, 1H), 8.15–8.06 (m,
1
Rf ¼ 0.38 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, CDCl3) 4H), 7.94 (d, J ¼ 8.3 Hz, 1H), 7.73–7.68 (m, 1H), 7.64–7.59 (m,
d 8.92 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.02 (s, 1H), 7.99 (dd, J ¼ 8.5, 1.6 1H), 7.43 (dd, J ¼ 8.5, 4.1 Hz, 1H), 1.69 (s, 9H). 13C NMR (101
Hz, 1H), 7.86 (dd, J ¼ 8.1, 2.6 Hz, 1H), 7.69 (dd, J ¼ 8.7, 4.4 Hz, MHz, CDCl3) d 167.03, 156.60, 149.90, 144.52, 137.47, 135.93,
1H), 7.49 (td, J ¼ 8.5, 2.7 Hz, 1H), 7.42 (dd, J ¼ 8.5, 4.1 Hz, 1H), 132.43, 132.23, 131.64, 130.39, 130.16, 129.75, 128.90, 127.18,
1.66 (s, 9H). 13C NMR (101 MHz, CDCl3) d 166.10 (d, 1JC–F ¼ 2.8 127.15, 126.57, 124.34, 122.74, 121.96, 94.34, 86.81, 30.66. LC/
Hz), 163.39 (s), 160.93 (s), 156.82 (s), 149.94 (s), 144.45 (s), MS (ESI): 575.0 [M ꢀ CO2 + H]+.
137.45 (s), 133.58 (s), 132.03 (s), 129.67 (s), 127.40 (d, 2JC–F ¼ 7.9
3
Hz), 127.01 (s), 125.64 (d, JC–F ¼ 7.5 Hz), 122.03 (s), 121.35 (d,
4JC–F ¼ 24.2 Hz), 115.73 (d, 5JC–F ¼ 23.4 Hz), 94.17 (s), 86.92 (s),
General procedure for the synthesis of 10a–10q and 11a–11q
30.66 (s). LC/MS (ESI): 542.9 [M ꢀ CO2 + H]+.
To a solution of 300 mg of 8 (X ¼ Cl), or 9 (X ¼ I) in 12 mL
tert-Butyl(5-(4-uoro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)-
CH2Cl2, HCl gas was bubbled at room temperature for 4 h. The
reaction was stirred under an HCl atmosphere overnight at 0 ꢁC.
The mixture was ltered and dried under reduced pressure to
yield the product.
7-iodoquinolin-8-yl)carbonate (9h). Pale yellow solid, 65% yield.
1
Rf ¼ 0.35 (petroleum/EtOAc ¼ 5/1). H NMR (400 MHz, CDCl3)
d 8.91 (d, J ¼ 3.6 Hz, 1H), 8.09–7.96 (m, 2H), 7.65 (td, J ¼ 7.9, 4.7
Hz, 1H), 7.48 (d, J ¼ 7.9 Hz, 1H), 7.42 (dd, J ¼ 8.4, 4.1 Hz, 1H),
7.21–7.12 (m, 1H), 1.66 (s, 9H). 13C NMR (101 MHz, DMSO-d6)
2-(7-Chloro-8-hydroxyquinolin-5-yl)benzo[d][1,2]selenazol-
3(2H)-one hydrochloride (10a). Yellow solid, 89% yield. Mp
246.7–247.6 ꢁC. Rf ¼ 0.25 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR (400
MHz, DMSO-d6) d 9.03 (d, J ¼ 3.3 Hz, 1H), 8.50 (d, J ¼ 8.0 Hz, 1H),
8.31 (d, J ¼ 8.0 Hz, 1H), 7.89 (d, J ¼ 7.2 Hz, 1H), 7.80 (dd, J ¼ 8.5,
4.6 Hz, 1H), 7.72 (s, 1H), 7.65 (t, J ¼ 7.1 Hz, 1H), 7.47 (t, J ¼ 7.2
Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 166.52, 147.79, 146.76,
141.60, 137.36, 135.13, 131.54, 129.27, 128.01, 127.84, 127.59,
127.07, 125.84, 125.58, 122.47, 117.96. [M + H]+ for C16H9ClN2-
O2Se pred. 376.9591, meas. 376.9591; HPLC purity: 98.31%.
6-Chloro-2-(7-chloro-8-hydroxyquinolin-5-yl)benzo[d][1,2]
selenazol-3(2H)-one hydrochloride (10b). Yellow solid, 91%
yield. Mp 258.2–259.1 ꢁC. Rf ¼ 0.35 (CH2Cl2/CH3OH ¼ 10/1). 1H
NMR (400 MHz, DMSO-d6) d 9.02 (d, J ¼ 3.4 Hz, 1H), 8.64 (d, J ¼
1.4 Hz, 1H), 8.27 (d, J ¼ 8.0 Hz, 1H), 7.86 (d, J ¼ 8.2 Hz, 1H), 7.77
(dd, J ¼ 8.5, 4.6 Hz, 1H), 7.69 (s, 1H), 7.50 (dd, J ¼ 8.2, 1.5 Hz,
1H). 13C NMR (101 MHz, DMSO-d6) d 165.57, 146.84, 145.30,
143.70, 139.87, 136.45, 133.21, 129.56, 128.91, 128.49, 127.65,
126.79, 126.02, 125.77, 122.48, 119.66. [M + H]+ for C16H8Cl2-
N2O2Se pred. 410.9201, meas. 410.9201; HPLC purity: 99.80%.
5-Chloro-2-(7-chloro-8-hydroxyquinolin-5-yl)benzo[d][1,2]
selenazol-3(2H)-one hydrochloride (10c). Yellow solid, 96%
yield. Mp 219.7–221.6 ꢁC. Rf ¼ 0.36 (CH2Cl2/CH3OH ¼ 10/1). 1H
NMR (400 MHz, DMSO-d6) d 8.99 (dd, J ¼ 4.4, 1.3 Hz, 1H), 8.51
(d, J ¼ 8.7 Hz, 1H), 8.16 (dd, J ¼ 8.5, 1.1 Hz, 1H), 7.83 (d, J ¼ 2.2
1
d 164.26 (d, JC–F ¼ 3.6 Hz), 163.77 (s), 161.18 (s), 155.60 (s),
150.53 (s), 144.27 (s), 143.34 (s), 137.12 (s), 133.62 (d, 2JC–F ¼ 8.3
Hz), 132.98 (s), 131.19 (s), 127.32 (s), 122.63 (s), 115.17 (d, 3JC–F
¼ 11.4 Hz), 113.36 (d, 4JC–F ¼ 19.4 Hz), 95.41 (s), 85.75 (s), 30.86
(s). LC/MS (ESI): 543.0 [M ꢀ CO2 + H]+.
tert-Butyl(7-iodo-5-(3-oxo-6-(triuoromethyl)benzo[d][1,2]
selenazol-2(3H)-yl)quinolin-8-yl)carbonate (9j). Pale yellow
1
solid, 78% yield. Rf ¼ 0.48 (petroleum/EtOAc ¼ 5/1). H NMR
(400 MHz, CDCl3) d 8.92 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.26 (d, J ¼ 8.2
Hz, 1H), 8.05 (s, 1H), 8.02 (s, 1H), 7.96 (dd, J ¼ 8.5, 1.7 Hz, 1H),
7.77 (d, J ¼ 8.1 Hz, 1H), 7.42 (dd, J ¼ 8.5, 4.1 Hz, 1H), 1.65 (s,
9H). 13C NMR (101 MHz, CDCl3) d 165.94 (s), 157.01 (s), 149.97
(s), 144.46 (s), 139.71 (s), 137.50 (s), 134.73 (q, 1JC–F ¼ 32.9 Hz),
131.86 (s), 130.06 (s), 129.30 (s), 128.55 (s), 126.97 (s), 126.14
(dd, 2JC–F ¼ 192.1, 81.2 Hz), 123.72 (d, 3JC–F ¼ 3.3 Hz), 122.09 (s),
121.81 (dd, 4JC–F ¼ 7.4, 3.5 Hz), 94.12 (s), 87.00 (s), 30.64 (s). LC/
MS (ESI): 592.9 [M ꢀ CO2 + H]+.
tert-Butyl(7-iodo-5-(6-methoxy-3-oxobenzo[d][1,2]selenazol-
2(3H)-yl)quinolin-8-yl)carbonate (9n). White solid, 81% yield. Rf
¼ 0.37 (petroleum/EtOAc ¼ 5/1). 1H NMR (400 MHz, CDCl3)
d 8.90 (dd, J ¼ 4.0, 1.5 Hz, 1H), 8.10–7.98 (m, 3H), 7.41 (dd, J ¼
8.5, 4.1 Hz, 1H), 7.16 (d, J ¼ 1.7 Hz, 1H), 7.07 (dd, J ¼ 8.3, 1.7 Hz,
1H), 3.95 (s, 3H), 1.66 (s, 9H). 13C NMR (101 MHz, CDCl3)
d 166.93, 163.69, 156.49, 149.83, 144.47, 141.14, 137.53, 132.29,
130.60, 130.20, 127.32, 121.91, 118.62, 114.96, 107.68, 94.27,
86.73, 55.89, 30.66. LC/MS (ESI): 592.9 [M ꢀ CO2 + H]+.
Hz, 1H), 7.72–7.71 (m, 1H), 7.70–7.68 (m, 1H), 7.66 (s, 1H). 13
C
NMR (101 MHz, DMSO-d6) d 165.14, 148.54, 147.88, 140.21,
136.67, 135.34, 131.26, 130.96, 129.89, 128.98, 128.77, 127.11,
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126.66, 125.22, 122.43, 116.57. [M + H]+ for C16H8Cl2N2O2Se 5.2 Hz, 1H), 7.76 (d, J ¼ 8.3 Hz, 1H), 7.63 (td, J ¼ 8.1, 5.0 Hz, 1H),
pred. 410.9201, meas. 410.9204; HPLC purity: 96.60%.
7.56 (d, J ¼ 8.3 Hz, 1H), 7.24 (dd, J ¼ 10.6, 8.2 Hz, 1H). 13C NMR
4-Chloro-2-(7-chloro-8-hydroxyquinolin-5-yl)benzo[d][1,2] (101 MHz, DMSO-d6) d 163.72 (d, 1JC–F ¼ 3.1 Hz), 161.84 (d, 2JC–F
selenazol-3(2H)-one hydrochloride (10d). Yellow solid, 92% ¼ 259.8 Hz), 147.58 (s), 144.53 (s), 144.21 (s), 143.13 (s), 131.88
yield. Mp 244.5–245.7 ꢁC. Rf ¼ 0.35 (CH2Cl2/CH3OH ¼ 10/1). 1H (d, JC–F ¼ 8.3 Hz), 130.58 (s), 128.94 (s), 127.21 (d, JC–F ¼ 8.7
3
4
NMR (400 MHz, DMSO-d6) d 9.05 (dd, J ¼ 4.5, 1.5 Hz, 1H), 8.56 Hz), 123.41 (d, 5JC–F ¼ 3.3 Hz), 122.58 (s), 116.13 (d, 6JC–F ¼ 10.8
(dd, J ¼ 8.1, 0.7 Hz, 1H), 8.28 (dd, J ¼ 8.6, 1.4 Hz, 1H), 7.80 (dd, J Hz), 115.59 (s), 112.47 (d, JC–F ¼ 19.8 Hz). [M + H]+ for C16H8-
7
¼ 8.6, 4.6 Hz, 1H), 7.75 (s, 1H), 7.60 (t, J ¼ 7.9 Hz, 1H), 7.51 (dd, J ClFN2O2Se pred. 394.9496, meas. 394.9498; HPLC purity:
¼ 7.8, 1.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 164.92, 97.39%.
148.67, 147.96, 145.27, 136.90, 136.33, 134.82, 131.85, 130.06,
2-(7-Chloro-8-hydroxyquinolin-5-yl)-7-(triuoromethyl)
128.57, 127.71, 126.55, 126.15, 123.08, 117.82. [M + H]+ for benzo[d][1,2]selenazol-3(2H)-one hydrochloride (10i). Yellow
C
16H8Cl2N2O2Se pred. 410.9201, meas. 410.9204; HPLC purity: solid, 76% yield. Mp 298.5–299.0 ꢁC. Rf ¼ 0.38 (CH2Cl2/CH3OH
98.34%.
¼ 10/1). 1H NMR (400 MHz, DMSO-d6) d 9.00 (d, J ¼ 3.8 Hz, 1H),
2-(7-Chloro-8-hydroxyquinolin-5-yl)-7-uorobenzo[d][1,2] 8.29 (d, J ¼ 7.6 Hz, 2H), 8.16 (d, J ¼ 8.4 Hz, 1H), 7.90 (s, 1H), 7.85
selenazol-3(2H)-one hydrochloride (10e). Yellow solid, 91% (t, J ¼ 7.7 Hz, 1H), 7.67 (dd, J ¼ 8.4, 4.2 Hz, 1H). 13C NMR (101
yield. Mp 281.3–281.9 ꢁC. Rf ¼ 0.31 (CH2Cl2/CH3OH ¼ 10/1). 1H MHz, DMSO-d6) d 165.81 (s), 164.87 (s), 146.55 (s), 144.15 (s),
1
NMR (400 MHz, DMSO-d6) d 9.06 (d, J ¼ 4.3 Hz, 1H), 8.27 (dd, J 136.02 (s), 133.44 (d, JC–F ¼ 32.8 Hz), 131.69 (s), 131.19 (s),
2
¼ 8.5, 1.1 Hz, 1H), 7.91 (s, 1H), 7.86 (d, J ¼ 7.4 Hz, 1H), 7.78 (dd, 130.84 (s), 128.35 (s), 128.18 (s), 127.15 (s), 124.81 (dd, JC–F
¼
3
J ¼ 8.5, 4.4 Hz, 1H), 7.72 (t, J ¼ 8.5 Hz, 1H), 7.65 (td, J ¼ 7.8, 5.1 41.1, 22.7 Hz), 124.50 (dd, JC–F ¼ 271.8, 36.9 Hz), 121.77 (s),
Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 166.36 (d, 1JC–F ¼ 1.9 115.09 (s), 110.59 (s). [M + H]+ for C17H8ClF3N2O2Se pred.
Hz), 157.94 (d, 2JC–F ¼ 246.3 Hz), 149.55 (s), 149.21 (s), 136.90 (s), 444.9464, meas. 444.9475; HPLC purity: 98.02%.
135.50 (s), 131.00 (s), 129.48 (d, 3JC–F ¼ 6.5 Hz), 129.07 (d, 4JC–F
¼
2-(7-Chloro-8-hydroxyquinolin-5-yl)-6-(triuoromethyl)
4.6 Hz), 126.16 (s), 126.12 (d, 5JC–F ¼ 24.6 Hz), 125.72 (s), 125.13 benzo[d][1,2]selenazol-3(2H)-one hydrochloride (10j). Yellow
(d, 6JC–F ¼ 2.6 Hz), 123.47 (s), 119.35 (d, 7JC–F ¼ 19.7 Hz), 117.14 solid, 88% yield. Mp 211.4–212.3 ꢁC. Rf ¼ 0.40 (CH2Cl2/CH3OH
(s). [M + H]+ for C16H8ClFN2O2Se pred. 394.9496, meas. ¼ 10/1). 1H NMR (400 MHz, DMSO-d6) d 9.02 (s, 2H), 8.28 (d, J ¼
394.9504; HPLC purity: 98.81%.
7.8 Hz, 1H), 8.06 (d, J ¼ 8.0 Hz, 1H), 7.82–7.73 (m, 2H), 7.71 (s,
2-(7-Chloro-8-hydroxyquinolin-5-yl)-6-uorobenzo[d][1,2] 1H). 13C NMR (101 MHz, DMSO-d6) d 165.36 (s), 146.70 (s),
selenazol-3(2H)-one hydrochloride (10f). Yellow solid, 92% 145.20 (s), 142.93 (s), 140.26 (s), 132.90 (s), 132.16 (s), 131.13 (q,
yield. Mp 217.6–218.6 ꢁC. Rf ¼ 0.32 (CH2Cl2/CH3OH ¼ 10/1). 1H 1JC–F ¼ 31.5 Hz), 129.64 (s), 128.42 (s), 128.36 (s), 125.75 (s),
NMR (400 MHz, DMSO-d6) d 8.98 (d, J ¼ 3.1 Hz, 1H), 8.26 (dd, J 124.68 (d, 2JC–F ¼ 4.0 Hz), 124.08 (q, 3JC–F ¼ 273.0 Hz), 122.52 (s),
¼ 9.6, 2.2 Hz, 1H), 8.13 (d, J ¼ 7.8 Hz, 1H), 7.91 (dd, J ¼ 8.4, 5.6 122.23 (d, JC–F ¼ 3.0 Hz), 119.91 (s). [M + H]+ for C17H8ClF3-
4
Hz, 1H), 7.69 (dd, J ¼ 8.6, 4.4 Hz, 1H), 7.65 (s, 1H), 7.32 (td, J ¼ N2O2Se pred. 444.9464, meas. 444.9463; HPLC purity: 99.12%.
8.6, 2.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 165.56 (s),
2-(7-Chloro-8-hydroxyquinolin-5-yl)-5-(triuoromethyl)
164.02 (d, 1JC–F ¼ 248.8 Hz), 147.10 (s), 145.67 (s), 144.30 (d, 2JC–F benzo[d][1,2]selenazol-3(2H)-one hydrochloride (10k). Yellow
¼ 10.7 Hz), 139.16 (s), 133.77 (s), 129.64 (s), 129.51 (d, 3JC–F ¼ 6.9 solid, 90% yield. Mp 228.1–228.9 ꢁC. Rf ¼ 0.36 (CH2Cl2/CH3OH
Hz), 128.40 (s), 125.75 (s), 125.27 (s), 122.47 (s), 119.16 (s), ¼ 10/1). 1H NMR (400 MHz, DMSO-d6) d 9.07 (dd, J ¼ 4.5, 1.1 Hz,
113.92 (d, 4JC–F ¼ 7.2 Hz), 113.67 (d, 5JC–F ¼ 3.1 Hz). [M + H]+ for 1H), 8.86 (d, J ¼ 8.5 Hz, 1H), 8.38 (d, J ¼ 8.5 Hz, 1H), 8.12 (s, 1H),
C
16H8ClFN2O2Se pred. 394.9496, meas. 394.9498; HPLC purity: 8.01 (dd, J ¼ 8.6, 1.7 Hz, 1H), 7.82 (dd, J ¼ 8.6, 4.6 Hz, 1H), 7.77
98.43%.
(s, 1H). 13C NMR (101 MHz, DMSO-d6) d 165.89 (s), 147.73 (s),
2-(7-Chloro-8-hydroxyquinolin-5-yl)-5-uorobenzo[d][1,2] 147.12 (s), 145.57 (s), 141.13 (s), 133.16 (s), 130.18 (s), 129.89 (s),
selenazol-3(2H)-one hydrochloride (10g). Yellow solid, 90% 129.31 (s), 128.94 (s), 127.63 (d, 1JC–F ¼ 2.8 Hz), 127.11 (d, 2JC–F
¼
yield. Mp 250.9–252.0 ꢁC. Rf ¼ 0.31 (CH2Cl2/CH3OH ¼ 10/1). 1H 32.3 Hz), 126.28 (s), 124.76 (dd, 3JC–F ¼ 272.6, 27.8 Hz), 124.36 (d,
NMR (400 MHz, DMSO-d6) d 9.01 (d, J ¼ 3.5 Hz, 1H), 8.52 (dd, J 4JC–F ¼ 3.6 Hz), 123.07 (s), 120.62 (s). [M + H]+ for C17H8ClF3-
¼ 8.7, 5.0 Hz, 1H), 8.21 (d, J ¼ 8.4 Hz, 1H), 7.74 (dd, J ¼ 8.4, 4.4 N2O2Se pred. 444.9464, meas. 444.9471; HPLC purity: 97.98%.
Hz, 1H), 7.68 (s, 1H), 7.62 (dd, J ¼ 8.4, 2.4 Hz, 1H), 7.56 (td, J ¼
2-(7-Chloro-8-hydroxyquinolin-5-yl)-4-(triuoromethyl)benzo
8.8, 2.5 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 165.44 (d, 1JC–F [d][1,2]selenazol-3(2H)-one hydrochloride (10l). Yellow solid,
¼ 2.6 Hz), 162.27 (d, 2JC–F ¼ 36.3 Hz), 159.86 (d, 3JC–F ¼ 36.9 Hz), 75% yield. Mp 229.1–230.5 ꢁC. Rf ¼ 0.50 (CH2Cl2/CH3OH ¼
147.26 (s), 145.99 (s), 138.75 (s), 136.74 (s), 134.05 (s), 129.94 (d, 10/1). 1H NMR (400 MHz, DMSO-d6) d 8.97 (dd, J ¼ 4.2, 1.3 Hz,
4JC–F ¼ 7.4 Hz), 129.33 (s), 128.21 (s), 125.58 (s), 122.47 (s), 1H), 8.50 (d, J ¼ 8.6 Hz, 1H), 8.27 (d, J ¼ 7.9 Hz, 1H), 7.82 (d, J ¼
119.21 (d, 5JC–F ¼ 23.0 Hz), 118.88 (s), 113.05 (d, 6JC–F ¼ 22.7 Hz). 8.2 Hz, 1H), 7.70–7.65 (m, 3H). 13C NMR (101 MHz, DMSO-d6)
[M + H]+ for C16H8ClFN2O2Se pred. 394.9496, meas. 394.9495; d 164.38 (s), 149.75 (d, JC–F ¼ 2.4 Hz), 144.31 (s), 132.89 (s),
1
2
HPLC purity: 98.21%.
131.70 (s), 131.10 (s), 129.96 (s), 129.50 (dd, JC–F ¼ 72.1, 39.4
2-(7-Chloro-8-hydroxyquinolin-5-yl)-4-uorobenzo[d][1,2] Hz), 125.93 (s), 125.37 (d, 3JC–F ¼ 6.1 Hz), 123.65 (q, 4JC–F ¼ 273.7
selenazol-3(2H)-one hydrochloride (10h). Yellow solid, 87% Hz), 123.59 (s), 122.93 (s), 115.17 (s). [M + H]+ for C17H8ClF3-
yield. Mp 256.9–257.4 ꢁC. Rf ¼ 0.34 (CH2Cl2/CH3OH ¼ 10/1). 1H N2O2Se pred. 444.9464, meas. 444.9480; HPLC purity: 98.01%.
NMR (400 MHz, DMSO-d6) d 9.13 (dd, J ¼ 5.2, 1.2 Hz, 1H), 8.75
2-(7-Chloro-8-hydroxyquinolin-5-yl)-7-methoxybenzo[d][1,2]
(dd, J ¼ 8.6, 1.0 Hz, 1H), 8.41 (d, J ¼ 8.1 Hz, 1H), 8.04 (dd, J ¼ 8.6, selenazol-3(2H)-one hydrochloride (10m). Yellow solid, 77%
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yield. Mp 253.7–254.9 ꢁC. Rf ¼ 0.36 (CH2Cl2/CH3OH ¼ 10/1). 1H 7.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 166.45, 152.29,
NMR (400 MHz, DMSO-d6) d 8.86 (d, J ¼ 2.6 Hz, 1H), 7.88 (d, J ¼ 148.31, 141.39, 136.35, 135.40, 135.22, 135.20, 131.58, 127.85,
8.4 Hz, 1H), 7.70 (s, 1H), 7.59 (d, J ¼ 4.3 Hz, 2H), 7.55 (dd, J ¼ 127.62, 126.90, 126.17, 125.87, 122.60, 80.92. [M + H]+ for C16-
8.6, 4.1 Hz, 1H), 7.45–7.39 (m, 1H), 4.00 (s, 3H). 13C NMR (101 H9IN2O2Se pred. 468.8947, meas. 468.8950; HPLC purity:
MHz, DMSO-d6) d 167.26, 154.58, 149.10, 140.37, 132.39, 130.48, 96.73%.
129.30, 128.33, 127.07, 126.19, 122.88, 120.89, 115.20, 113.95,
6-Chloro-2-(8-hydroxy-7-iodoquinolin-5-yl)benzo[d][1,2]sele-
56.80. [M + H]+ for C17H11ClN2O3Se pred. 406.9696, meas. nazol-3(2H)-one hydrochloride (11b). Red solid, 92% yield. Mp
406.9699; HPLC purity: 98.32%.
218.2–219.1 ꢁC. Rf ¼ 0.41 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR (400
2-(7-Chloro-8-hydroxyquinolin-5-yl)-6-methoxybenzo[d][1,2] MHz, DMSO-d6) d 8.92 (d, J ¼ 2.9 Hz, 1H), 8.52 (s, 1H), 8.03 (d, J
selenazol-3(2H)-one hydrochloride (10n). Yellow solid, 91% ¼ 8.4 Hz, 1H), 7.86 (d, J ¼ 8.2 Hz, 1H), 7.82 (s, 1H), 7.66 (dd, J ¼
yield. Mp 254.2–245.5 ꢁC. Rf ¼ 0.34 (CH2Cl2/CH3OH ¼ 10/1). 1H 8.4, 4.3 Hz, 1H), 7.52 (dd, J ¼ 8.2, 1.6 Hz, 1H). 13C NMR (101
NMR (400 MHz, DMSO-d6) d 9.01 (dd, J ¼ 4.4, 1.5 Hz, 1H), 8.15 MHz, DMSO-d6) d 165.47, 151.49, 147.80, 143.45, 136.46, 134.47,
(dd, J ¼ 8.5, 1.3 Hz, 1H), 7.96 (d, J ¼ 2.3 Hz, 1H), 7.81 (d, J ¼ 8.6 128.94, 128.43, 128.19, 128.04, 127.46, 126.62, 126.22, 126.07,
Hz, 1H), 7.73 (dd, J ¼ 8.6, 4.4 Hz, 1H), 7.70 (s, 1H), 7.08 (dd, J ¼ 122.56, 82.41. [M + H]+ for C16H8ClIN2O2Se pred. 502.8557,
8.6, 2.4 Hz, 1H), 3.88 (s, 3H). 13C NMR (101 MHz, DMSO-d6) meas. 502.8556; HPLC purity: 97.68%.
d 166.24, 162.08, 147.49, 146.18, 143.73, 138.12, 134.56, 129.38,
5-Chloro-2-(8-hydroxy-7-iodoquinolin-5-yl)benzo[d][1,2]sele-
128.71, 128.29, 125.74, 122.42, 121.10, 118.44, 113.72, 110.82, nazol-3(2H)-one hydrochloride (11c). Red solid, 84% yield. Mp
55.55. [M + H]+ for C17H11ClN2O3Se pred. 406.9696, meas. 228.4–229.6 ꢁC. Rf ¼ 0.43 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR (400
406.9696; HPLC purity: 96.52%.
MHz, DMSO-d6) d 8.96 (s, 1H), 8.58 (d, J ¼ 7.3 Hz, 1H), 8.20 (d, J
2-(7-Chloro-8-hydroxyquinolin-5-yl)-5-methoxybenzo[d][1,2] ¼ 7.9 Hz, 1H), 7.87 (s, 1H), 7.81 (s, 1H), 7.76–7.71 (m, 1H), 7.68
selenazol-3(2H)-one hydrochloride (10o). Yellow solid, 87% (d, J ¼ 8.5 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 165.06,
yield. Mp 221.3–222.5 ꢁC. Rf ¼ 0.36 (CH2Cl2/CH3OH ¼ 10/1). 1H 151.66, 147.92, 140.39, 136.30, 134.67, 131.09, 130.87, 130.13,
NMR (400 MHz, DMSO-d6) d 9.00 (d, J ¼ 3.2 Hz, 1H), 8.30 (d, J ¼ 129.19, 128.16, 126.56, 126.11, 122.56, 82.03. [M + H]+ for C16-
8.8 Hz, 1H), 8.16 (d, J ¼ 7.8 Hz, 1H), 7.73 (dd, J ¼ 8.5, 4.4 Hz, H8ClIN2O2Se pred. 502.8557, meas. 502.8556; HPLC purity:
1H), 7.67 (s, 1H), 7.39 (d, J ¼ 2.5 Hz, 1H), 7.30 (dd, J ¼ 8.8, 2.7 98.12%.
Hz, 1H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO-d6) d 166.21,
7-Fluoro-2-(8-hydroxy-7-iodoquinolin-5-yl)benzo[d][1,2]sele-
158.26, 146.91, 145.34, 139.70, 133.31, 132.71, 129.46, 129.07, nazol-3(2H)-one hydrochloride (11e). Yellow solid, 81% yield.
128.80, 128.31, 125.71, 122.45, 120.19, 119.55, 109.96, 55.45. [M Mp 205.2–206.3 ꢁC. Rf ¼ 0.32 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR
+ H]+ for C17H11ClN2O3Se pred. 406.9696, meas. 406.9694; (400 MHz, DMSO-d6) d 8.99 (dd, J ¼ 4.2, 1.3 Hz, 1H), 8.13 (dd, J ¼
HPLC purity: 98.45%.
8.5, 1.3 Hz, 1H), 8.05 (s, 1H), 7.86 (d, J ¼ 7.5 Hz, 1H), 7.77–7.69
2-(7-Chloro-8-hydroxyquinolin-5-yl)-4-methoxybenzo[d][1,2] (m, 2H), 7.65 (td, J ¼ 7.8, 5.0 Hz, 1H). 13C NMR (101 MHz,
1
2
selenazol-3(2H)-one hydrochloride (10p). Yellow solid, 76% DMSO-d6) d 166.30 (d, JC–F ¼ 2.0 Hz), 157.92 (d, JC–F ¼ 246.0
1
ꢁ
yield. Mp 263.4–264.2 C. Rf ¼ 0.31 (CH2Cl2/CH3OH ¼ 5/1). H Hz), 154.53 (s), 149.45 (s), 138.00 (s), 136.52 (s), 134.14 (s),
NMR (400 MHz, DMSO-d6) d 8.95 (s, 1H), 7.94 (d, J ¼ 8.2 Hz, 1H), 129.48 (d, 3JC–F ¼ 6.5 Hz), 129.09 (d, 4JC–F ¼ 4.7 Hz), 126.76 (s),
5
7.71 (d, J ¼ 7.7 Hz, 1H), 7.61–7.57 (m, 3H), 7.04 (d, J ¼ 7.8 Hz, 126.11 (s), 126.02 (d, JC–F ¼ 24.4 Hz), 125.14 (s), 123.60 (s),
1H), 3.85 (s, 3H). 13C NMR (101 MHz, DMSO-d6) d 165.76, 119.29 (d, 6JC–F ¼ 19.8 Hz), 80.21 (s). [M + H]+ for C16H8FIN2O2Se
161.26, 150.25, 149.98, 143.34, 139.20, 133.28, 133.15, 129.69, pred. 486.8852, meas. 486.8857; HPLC purity: 99.21%.
126.48, 125.88, 122.97, 118.16, 115.36, 114.52, 108.99, 56.06. [M
6-Fluoro-2-(8-hydroxy-7-iodoquinolin-5-yl)benzo[d][1,2]sele-
+ H]+ for C17H11ClN2O3Se pred. 406.9696, meas. 406.9706; nazol-3(2H)-one hydrochloride (11f). Yellow solid, 89% yield.
HPLC purity: 99.01%.
Mp 209.5–210.3 ꢁC. Rf ¼ 0.32 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR
2-(7-Chloro-8-hydroxyquinolin-5-yl)naphtho[2,3-d][1,2]sele- (400 MHz, DMSO-d6) d 8.93 (d, J ¼ 3.0 Hz, 1H), 8.30 (dd, J ¼ 9.7,
nazol-3(2H)-one hydrochloride (10q). Yellow solid, 81% yield. 2.1 Hz, 1H), 8.08 (d, J ¼ 7.7 Hz, 1H), 7.90 (dd, J ¼ 8.4, 5.6 Hz,
Mp 229.9–231.1 ꢁC. Rf ¼ 0.42 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR 1H), 7.83 (s, 1H), 7.68 (dd, J ¼ 8.4, 4.3 Hz, 1H), 7.31 (td, J ¼ 8.6,
(400 MHz, DMSO-d6) d 9.04 (d, J ¼ 3.3 Hz, 1H), 8.93 (s, 1H), 8.56 2.3 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 165.47 (s), 164.06
(s, 1H), 8.35 (d, J ¼ 8.2 Hz, 1H), 8.16 (d, J ¼ 8.1 Hz, 1H), 7.98 (d, J (d, 1JC–F ¼ 249.0 Hz), 152.08 (s), 148.20 (s), 143.90 (d, 2JC–F ¼ 10.9
¼ 8.2 Hz, 1H), 7.79 (dd, J ¼ 8.6, 4.6 Hz, 1H), 7.76 (s, 1H), 7.67– Hz), 136.32 (s), 135.35 (d, 3JC–F ¼ 22.8 Hz), 129.65 (d, 4JC–F ¼ 9.7
7.62 (m, 1H), 7.59–7.53 (m, 1H). 13C NMR (101 MHz, DMSO-d6) Hz), 127.95 (s), 126.18 (s), 124.98 (s), 122.57 (s), 113.99 (s),
5
d 166.25, 147.35, 145.92, 138.61, 135.92, 134.85, 134.27, 130.94, 113.72 (d, JC–F ¼ 6.6 Hz), 113.41 (s), 81.15 (s). [M + H]+ for
129.28, 129.05, 128.62, 127.98, 127.82, 127.19, 125.70, 125.62,
C16H8FIN2O2Se pred. 486.8852, meas. 486.8854; HPLC purity:
125.45, 122.41, 118.84. [M + H]+ for C20H11ClN2O2Se pred. 98.41%.
426.9747, meas. 426.9753; HPLC purity: 99.81%.
5-Fluoro-2-(8-hydroxy-7-iodoquinolin-5-yl)benzo[d][1,2]sele-
2-(8-Hydroxy-7-iodoquinolin-5-yl)benzo[d][1,2]selenazol- nazol-3(2H)-one hydrochloride (11g). Yellow solid, 92% yield.
3(2H)-one hydrochloride (11a). Yellow solid, 85% yield. Mp Mp 236.7–237.9 ꢁC. Rf ¼ 0.38 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR
218.5–219.9 ꢁC. Rf ¼ 0.33 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR (400 (400 MHz, DMSO-d6) d 8.93 (dd, J ¼ 4.2, 1.4 Hz, 1H), 8.46 (dd, J ¼
MHz, DMSO-d6) d 8.94 (dd, J ¼ 4.3, 1.4 Hz, 1H), 8.43 (d, J ¼ 8.0 8.7, 5.0 Hz, 1H), 8.05 (d, J ¼ 8.5 Hz, 1H), 7.84 (s, 1H), 7.67 (dd, J
Hz, 1H), 8.09 (d, J ¼ 8.5 Hz, 1H), 7.88 (d, J ¼ 7.7 Hz, 1H), 7.85 (s, ¼ 8.5, 4.3 Hz, 1H), 7.62 (dd, J ¼ 8.5, 2.5 Hz, 1H), 7.57 (td, J ¼ 8.9,
1H), 7.69 (dd, J ¼ 8.6, 4.4 Hz, 1H), 7.67–7.63 (m, 1H), 7.47 (t, J ¼ 2.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 165.33 (d, 1JC–F
¼
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3.2 Hz), 162.44 (s), 160.03 (s), 150.83 (s), 147.35 (s), 137.73 (s), 1H), 7.66 (d, J ¼ 7.4 Hz, 1H), 7.57 (t, J ¼ 7.3 Hz, 1H). 13C NMR
136.68 (s), 133.56 (s), 129.96 (d, 2JC–F ¼ 7.4 Hz), 129.34 (d, 3JC–F
¼
(101 MHz, DMSO-d6) d 166.18, 151.69, 147.98, 136.39, 136.17,
7.3 Hz), 128.65 (s), 126.27 (s), 122.56 (s), 119.17 (d, 4JC–F ¼ 22.7 135.66, 134.87, 130.95, 129.09, 128.48, 128.00, 127.86, 127.56,
5
Hz), 113.02 (d, JC–F ¼ 22.8 Hz), 83.82 (s). [M + H]+ for C16H8- 127.20, 126.18, 125.74, 125.25, 122.54, 82.01. [M + H]+ for C20-
FIN2O2Se pred. 486.8852, meas. 486.8851; HPLC purity: 98.24%. H11IN2O2Se pred. 518.9103, meas. 518.9100; HPLC purity:
4-Fluoro-2-(8-hydroxy-7-iodoquinolin-5-yl)benzo[d][1,2]sele- 98.76%.
nazol-3(2H)-one hydrochloride (11h). Yellow solid, 94% yield.
Mp 218.0–219.1 ꢁC. Rf ¼ 0.40 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR
Biological assays
(400 MHz, DMSO-d6) d 8.94 (dd, J ¼ 4.1, 1.3 Hz, 1H), 8.20 (d, J ¼
ThT assay.40 Ab1–42 (Millipore, counter ion: NaOH) was dis-
8.1 Hz, 1H), 8.07 (dd, J ¼ 8.5, 1.3 Hz, 1H), 7.86 (s, 1H), 7.68 (dd, J solved in ammonium hydroxide (1% v/v) to give a stock solution
¼ 8.6, 4.3 Hz, 1H), 7.66–7.61 (m, 1H), 7.24 (dd, J ¼ 10.4, 8.1 Hz, (2000 mꢁM), which was aliquoted into small samples and stored
1H). 13C NMR (101 MHz, DMSO-d6) d 163.70 (d, 1JC–F ¼ 3.1 Hz), at ꢀ80 C.
2
161.83 (d, JC–F ¼ 259.8 Hz), 151.27 (s), 147.60 (s), 144.06 (s),
For the inhibition of self-mediated Ab1–42 aggregation
experiment, the Ab stock solution was diluted with 50 mM
137.07 (s), 136.97 (s), 134.06 (d, 3JC–F ¼ 2.7 Hz), 131.96 (d, 4JC–F
¼
8.2 Hz), 128.08 (s), 126.44 (s), 123.27 (d, 5JC–F ¼ 3.1 Hz), 122.62 phosphate buffer (pH 7.4) to 50 mM before use. A mixture of the
(s), 115.97 (d, 6JC–F ¼ 11.4 Hz), 112.47 (d, 7JC–F ¼ 19.6 Hz), 83.02 peptide (10 mL, 25 mM, nal concentration) with or without the
(s). [M + H]+ for C16H8FIN2O2Se pred. 486.8852, meas. 486.8849; tested compound (10 mL) was incubated at 37 ꢁC for 48 h. Blanks
HPLC purity: 98.39%.
using 50 mM phosphate buffer (pH 7.4) instead of Ab with or
2-(8-Hydroxy-7-iodoquinolin-5-yl)-6-(triuoromethyl)benzo without inhibitors were also carried out. The sample was
[d][1,2]selenazol-3(2H)-one hydrochloride (11j). Yellow solid, diluted to a nal volume of 200 mL with 50 mM glycine–NaOH
91% yield. Mp 201.2–202.3 ꢁC. Rf ¼ 0.44 (CH2Cl2/CH3OH ¼ 10/ buffer (pH 8.0) containing thioavin T (5 mM). Then the uo-
1
1). H NMR (400 MHz, DMSO-d6) d 8.94 (s, 2H), 8.08 (t, J ¼ 8.4 rescence intensities were recorded ve minutes later (excitation,
Hz, 2H), 7.86 (s, 1H), 7.79 (d, J ¼ 7.7 Hz, 1H), 7.68 (dd, J ¼ 8.2, 450 nm; emission, 485 nm). None of the tested compounds
4.2 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 165.29 (s), 151.03 displayed uorescence signals under the experimental condi-
(s), 147.41 (s), 142.72 (s), 137.57 (s), 136.73 (s), 133.70 (s), 131.97 tions. The percent inhibition of aggregation was calculated by
(s), 131.17 (q, 1JC–F ¼ 31.3 Hz), 128.41 (s), 128.30 (s), 126.27 (s), the expression (1 ꢀ IFi/IFc) ꢃ 100%, in which IFi and IFc are the
124.54 (d, 2JC–F ¼ 4.0 Hz), 124.07 (q, 3JC–F ¼ 273.0 Hz), 122.60 (s), uorescence intensities obtained for Ab in the presence and
122.27 (d, 4JC–F ¼ 3.1 Hz), 83.59 (s). [M + H]+ for C17H8F3IN2O2Se absence of inhibitors aer subtracting the background,
pred. 536.8821, meas. 536.8819; HPLC purity: 97.49%.
respectively.
2-(8-Hydroxy-7-iodoquinolin-5-yl)-6-methoxybenzo[d][1,2]
For the inhibition of copper-mediated Ab1–42 aggregation
selenazol-3(2H)-one hydrochloride (11n). Yellow solid, 95% experiment, the Ab stock solution was diluted in 20 mM HEPES
yield. Mp 195.4–196.7 ꢁC. Rf ¼ 0.31 (CH2Cl2/CH3OH ¼ 10/1). 1H (pH 6.6) with 150 mM NaCl. The mixture of the peptide (10 mL, 25
NMR (400 MHz, DMSO-d6) d 8.92 (d, J ¼ 2.9 Hz, 1H), 7.98 (d, J ¼ mM, nal concentration) with or without copper (10 mL, 25 mM,
8.4 Hz, 1H), 7.86 (d, J ¼ 2.0 Hz, 1H), 7.82 (s, 1H), 7.79 (d, J ¼ 8.5 nal concentration) and the tested compound (10 mL, 50 mM,
Hz, 1H), 7.65 (dd, J ¼ 8.4, 4.3 Hz, 1H), 7.06 (dd, J ¼ 8.6, 2.2 Hz, nal concentration) was incubated at 37 ꢁC for 24 h. Then 20 mL
1H), 3.86 (s, 3H). 13C NMR (101 MHz, DMSO-d6) d 166.20, of the sample was diluted to a nal volume of 200 mL with
162.12, 151.63, 147.89, 143.52, 136.62, 136.20, 134.66, 128.75, 50 mM glycine–NaOH buffer (pH 8.0) containing thioavin T
128.29, 126.36, 122.56, 120.88, 113.82, 110.59, 82.09, 55.57. [M + (5 mM). The detection method was the same as that of self-
H]+ for C17H11IN2O3Se pred. 498.9052, meas. 498.9053; HPLC mediated Ab1–42 aggregation experiment.
purity: 98.58%.
For the disaggregation of self-induced Ab brils experiment,
2-(8-Hydroxy-7-iodoquinolin-5-yl)-5-methoxybenzo[d][1,2] the Ab stock solution was diluted with 10 mM phosphate buffer
selenazol-3(2H)-one hydrochloride (11o). Yellow solid, 83% (pH 7.4). The peptide (15 mL, 50 mM) was incubated at 37 ꢁC for 24
yield. Mp 222.9–223.8 ꢁC. Rf ¼ 0.36 (CH2Cl2/CH3OH ¼ 10/1). 1H h. The tested compound (15 mL, 50 mM) was then added and
NMR (400 MHz, DMSO-d6) d 8.92 (dd, J ¼ 4.1, 1.3 Hz, 1H), 8.20 incubated at 37 ꢁC for another 24 h. Then 20 mL of the sample was
(d, J ¼ 7.2 Hz, 1H), 7.99 (d, J ¼ 7.8 Hz, 1H), 7.82 (s, 1H), 7.66 (dd, diluted to a nal volume of 200 mL with 50 mM glycine–NaOH
J ¼ 8.5, 4.2 Hz, 1H), 7.39 (d, J ¼ 2.5 Hz, 1H), 7.32 (dd, J ¼ 8.6, 2.4 buffer (pH 8.0) containing thioavin T (5 mM). The detection
Hz, 1H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO-d6) d 166.25, method was the same as above.
158.36, 152.29, 152.27, 148.26, 136.37, 135.26, 132.10, 128.49,
For the disaggregation of copper-induced Ab brils experi-
127.91, 127.73, 126.11, 122.62, 120.59, 110.10, 81.02, 55.48. [M + ment, the Ab stock solution was diluted in 20 mM HEPES (pH
H]+ for C17H11IN2O3Se pred. 498.9054, meas. 498.9053; HPLC 6.6) with 150 mM NaCl. The mixture of the peptide (10 mL, 25
purity: 96.38%.
mM, nal concentration) with copper (10 mL, 25 mM, nal
2-(8-Hydroxy-7-iodoquinolin-5-yl)naphtho[2,3-d][1,2]selena- concentration) was incubated at 37 ꢁC for 24 h. The tested
zol-3(2H)-one hydrochloride (11q). Yellow solid, 86% yield. Mp compound (10 mL, 50ꢁmM, nal concentration) was then added
232.2–233.1 ꢁC. Rf ¼ 0.38 (CH2Cl2/CH3OH ¼ 10/1). 1H NMR (400 and incubated at 37 C for another 24 h.50 Then 20 mL of the
MHz, DMSO-d6) d 8.95 (dd, J ¼ 4.3, 1.4 Hz, 1H), 8.84 (s, 1H), 8.58 sample was diluted to a nal volume of 200 mL with 50 mM
(s, 1H), 8.18 (d, J ¼ 8.2 Hz, 1H), 8.14 (dd, J ¼ 8.5, 1.0 Hz, 1H), glycine–NaOH buffer (pH 8.0) containing thioavin T (5 mM).
8.00 (d, J ¼ 8.4 Hz, 1H), 7.91 (s, 1H), 7.69 (dd, J ¼ 8.6, 4.3 Hz, The detection method was the same as above.
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TEM assay.45 For the metal-free experiment, Ab stock solu- Computational methods
RSC Advances
tion was diluted with a 10 mM phosphate buffer (pH ¼ 7.4); for
the copper-induced experiment, Ab stock solution was diluted
with 20 mM HEPES (pH ¼ 6.6) and 150 mM NaCl. The sample
preparation was same as that for the ThT assay.
Aliquots (10 mL) of the samples were placed on a carbon-
coated copper/rhodium grid for 2 min. Each grid was stained
with uranyl acetate (1%, 5 mL) for 2 min. Aer draining off the
excess staining solution, the specimen was transferred for
imaging in a transmission electron microscope (JEOL JEM-
1400). All compounds are solubilized in the buffer which was
used for the experiment.
All calculations were performed using the Gaussian 09 suite of
quantum chemical programs. The hybrid Becke 3-Lee–Yang–
Parr (B3LYP) exchange correlation–functional was applied for
DFT calculations. The computational models were fully opti-
mized using DFT calculations at B3LYP/6-311+g(d) level of
theory and characterized by frequency calculation. As gluta-
thione was very exible, the G group replaced with a Me group
according to the reported method in literature.28,30–33 Orbital
interactions were analyzed using the natural bond orbital (NBO)
method at the B3LYP/6-311+g(d,p) level.
Oxygen radical absorbance capacity (ORAC-FL) assay46,47 The
tested compound and uorescein (FL) stock solution were
diluted with 75 mM phosphate buffer (pH 7.4) to 10 mM(or 20
mM) and 0.117 mM, respectively. The solution of (ꢂ)-6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was
diluted with the same buffer to 100, 80, 60, 50, 40, 20, and 10 mM.
The solution of 2,20-azobis-(amidinopropane)dihydrochloride
(AAPH) was prepared before the experiment by dissolving 108.4
mg AAPH in 10 mL 75 mM phosphate buffer (pH 7.4) to a nal
concentration of 40 mM. The mixture of the tested compound
(20 mL) and FL (120 mL; 70 nM, nal concentration) was pre-
incubated for 10 min at 37 ꢁC, and then 60 mL of the AAPH
solution was added. The uorescence was recorded every minute
for 120 min (excitation, 485 nm; emission, 520 nm). A blank
using phosphate buffer instead of the tested compound was also
carried out. All reaction mixtures were prepared triple and at
least three independent runs were performed for each sample.
The antioxidant curves (uorescence versus time) were normal-
ized to the curve of the blank. The area under the uorescence
decay curve (AUC) was calculated as following equation:
Horseradish peroxidase assay
The hydrogen peroxide scavenging property was measured
using the horseradish peroxidase assay.49 Test compounds of
200 mL of different concentrations were incubated with 100 mL
of 1 mM H2O2. Then, 0.1 M phosphate buffer (pH 7.4, 100 mL)
and 0.1 M NaCl (50 mL) were added. The solution was incubated
for 10 h at 37 ꢁC. Then, a solution containing red phenol (0.2 mg
mLꢀ1) and horseradish peroxidase (0.5 mg mLꢀ1) in 0.1 M
phosphate buffer (500 mL) was added. Aer 15 min, 1 M NaOH
solution (50 mL) was added to quench the reaction, and the
absorbance was recorded at 610 nm. The result is expressed as
the percentage of reduction of H2O2. The percentage of H2O2
scavenging activity was calculated according to the following
formula: 1 ꢀ (Asample ꢀ Ablank)/(Acontrol ꢀ Ablank) ꢃ 100, where
Asample is the absorbance of the samples, Ablank is the absor-
bance of the blank, and Acontrol is the absorbance of the control.
Neuronal antioxidative effects against the production of ROS
induced by t-BuOOH
AUC ¼ 1 þ i¼20ð fi=f0Þ
X
The evaluation of neuronal antioxidative effects against the
production of ROS induced by t-BuOOH was carried out
according to our previous reported method.24 The cytotoxicity
was evaluated with the colorimetric MTT [3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Human
neuron-like cells, SH-SY5Y, were routinely grown at 37 ꢁC in
a humidied incubator with 5% CO2 in Dulbecco's modied
Eagle's medium (DMEM, GIBCO) supplemented with 10%
foetal calf serum (FCS, GIBCO), 1 mM glutamine, 50 mg mLꢀ1
penicillin and 50 mg mLꢀ1 streptomycin. SH-SY5Y cells were
seeded at 5 ꢃ 104 cells per well in 96-well plates. Aer 24 h, the
medium was removed and replaced with the tested compounds
i¼1
where f0 is the initial uorescence reading at 0 min and fi is the
uorescence 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 by using the
standard curve which means the ORAC-FL value of tested
compound expressed as Trolox equivalents.
Coupled reductase assay
The GPx-like activity of the organoselenium compounds was at different concentrations for 24 h at 37 ꢁC. Then, the cells were
determined using a spectrophotometric method at 340 nm as incubated with MTT (2.5 mg mLꢀ1) in PBS for 4 h. Aer the
described by Wilson et al.48 The test mixture contained GSH (2 removal of MTT, the formazan crystals were dissolved in DMSO.
mM), EDTA (1 mM), glutathione reductase (1.3 unite per mL) The amount of formazan was measured (570 nm). Cell viability
and NADPH (0.4 mM) in 100 mM potassium phosphate buffer, was expressed as percentage of control cells and calculated by
pH 7.5. GPx samples (80 mM) were added to the test mixture at the formula Ft/Fnt ꢃ 100, where Ft ¼ absorbance of treated
25 ꢁC, and the reaction was initiated by the addition of peroxide neurones and Fnt ¼ absorbance of untreated neurones. SH-SY5Y
(1.6 mM). The initial reduction rates were calculated from the cells were seeded at 1 ꢃ 104 cells per well in 96-well plates; aer
oxidation rate of NADPH at 340 nm. The initial reduction rate 24 h, the medium was removed and replaced with tested
ꢁ
was determined at least 3 times and calculated from the rst 5– compounds at 37 C and kept for another 24 h. The cells were
10% of the reaction using 6.22 mMꢀ1 cmꢀ1 as the extinction washed with PBS and incubated with 5 mM of DCFH-DA
coefficient for NADPH.
(a uorescent probe) in PBS at 37 ꢁC in 5% CO2 for 30 min.
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Aer removal of DCFH-DA and further washing, the cells were to 19 : 00) at 20–22 ꢁC with a relative humidity of 60–70%.
exposed to 0.1 mM t-BuOOH (a compound used to induce Sterile food and water were provided according to institutional
oxidative stress) in PBS for 30 min. At the end of the incubation, guidelines. Prior to each experiment, mice were fasted over-
the uorescence of the cells from each well was measured night and allowed free access to water. Compound 10h was
(lexcitation ¼ 485 nm, lemission ¼ 535 nm) with a multifunctional suspension in 0.5% carboxymethyl cellulose sodium (CMC-Na)
microplate reader (Molecular Devices, Flex Station 3). The salt solution (677, 1333, and 2000 mg kgꢀ1) and given via oral
antioxidant activity was expressed as percentage of control cells administration according to the divided experimental groups.
and calculated by the formula (Ft ꢀ Fnt)/(F0t ꢀ Fnt) ꢃ 100, where Aer the administration of the compounds, the mice were
Ft ¼ absorbance of treated neurons with tested compound, F0t ¼ observed continuously for the rst 4 h for any abnormal
absorbance of treated neurons without tested compound and behavior and mortality changes, intermittently for the next 24 h,
Fnt ¼ absorbance of neurons not treated with t-BuOOH.
and occasionally thereaer for 14 days for the onset of any
delayed effects. All animals were sacriced on the 14th day aer
drug administration and were macroscopically examined for
possible damage to the heart, liver, and kidneys.
In vitro blood–brain barrier permeation assay
The blood–brain barrier penetration of compounds was evalu-
ated using the parallel articial membrane permeation assay
(PAMPA) described by Di et al.50–52 Commercial drugs were
purchased from Sigma and Alfa Aesar. Porcine brain lipid (PBL)
was obtained from Avanti Polar Lipids. 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 lled with 300 mL PBS/EtOH (7 : 3), and the lter membrane
was impregnated with 4 mL PBL in dodecane (20 mg mLꢀ1).
Compounds were dissolved in DMSO at 5 mg mLꢀ1 and diluted
50-fold in PBS/EtOH (7 : 3) to a nal concentration of 100 mg
mLꢀ1. Then, 200 mL of the solution was added to the donor
wells. The acceptor lter plate was carefully placed on the donor
plate to form a sandwich, which was le undisturbed for 10 h at
25 ꢁC. Aer incubation, 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 analyzed at ve wavelengths in four wells and in at
least three independent runs. Pe was calculated by the following
Metal-chelating study
The chelating studies were performed with a UV-Vis spectro-
photometer. All compounds are solubilized in the buffer which
was used for the experiment. The absorption spectra of each
compound (50 mM, nal concentration) alone or in the presence
of CuSO4, FeSO4, or ZnCl2 (50 mM, nal concentration) for 30
min in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were
recorded at room temperature.
For the stoichiometry of the compound-Cu2+ complex, a xed
amount of 10h (80 mM) was mixed with growing amounts of
copper ion (0–128 mM), and the difference UV-vis spectra were
examined to investigate the ratio of ligand/metal in the complex.
Statistical analysis
The results are expressed as the mean ꢂ SD of at least three
independent experiments. Data were subjected to Student's t test
or one-way analysis of variance (ANOVA) followed by Dunnett's test.
p values less than 0.05 were accepted to indicate the signicance.
expression: Pe ¼ ꢀVd ꢃ Va/[(Vd + Va)A ꢃ t] ꢃ ln(1 ꢀ drugacceptor
/
drugequilibrium) where Vd is the volume of donor well; Va, volume
Acknowledgements
,
in acceptor well; A, lter area; t, permeation time; drugacceptor
the absorbance obtained in the acceptor well; drugequilibrium, the
theoretical equilibrium absorbance. The results are given as the
mean ꢂ standard deviation. In the experiment, 13 quality
control standards (Table S13, see ESI†) of known BBB perme-
ability were included to validate the analysis set. A plot of the
experimental data versus literature values gave a strong linear
correlation, Pe(exp) ¼ 1.4574Pe(lit) ꢀ 1.0773 (R2 ¼ 0.9427)
(Fig. S1†). From this equation and the limit established by Di
et al. (Pe(lit) ¼ 4.0 ꢃ 10ꢀ6 cm sꢀ1) for blood–brain barrier
permeation, we concluded that compounds with a permeability
greater than 4.7 ꢃ 10ꢀ6 cm sꢀ1 could cross the blood–brain
barrier (Table S14†).
We thank the National Natural Science Foundation of China
(No. 21302235, 20972198), Guangdong Natural Science Foun-
dation (2014A030313124) and the Fundamental Research
Funds for the Central Universities (15ykpy04) for nancial
support of this study. We also thank the National Super-
computing Center in Guangzhou for providing the computa-
tional resources. This research is also supported in part by the
Guangdong Province Key Laboratory of Computational Science
and the Guangdong Province Computational Science Innovative
Research Team.
Notes and references
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