Design, synthesis and biological activities of piperidine-spirooxadiazole derivatives as α7 nicotinic receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2020.112774

α: 7 nicotinic acetylcholine receptors (nAChRs) expressed in the nervous and immune systems have been suggested to play important roles in the control of inflammation. However, the lack of antagonist tools specifically inhibiting α7 nAChR impedes the validation of the channel as therapeutic target. To discover a selective α7 antagonist, we started a pharmacophore-based virtual screening and identified a piperidine-spirooxadiazole derivative T761–0184 that acts as a α7 antagonist. A series of novel piperidine-spirooxadiazole derivatives were subsequently synthesized and evaluated using two-electrode voltage clamp (TEVC) assay in Xenopus oocytes. Lead compounds from two series inhibited α7 with their IC₅₀ values ranging from 3.3 μM to 13.7 μM. Compound B10 exhibited α7 selectivity over other α4β2 and α3β4 nAChR subtypes. The analysis of structure-activity relationship (SAR) provides valuable insights for further development of selective α7 nAChR antagonists.

Full text of DOI:10.1016/j.ejmech.2020.112774

European Journal of Medicinal Chemistry 207 (2020) 112774
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological activities of piperidine-
spirooxadiazole derivatives as
a
7 nicotinic receptor antagonists
,
,
,
Han Zhang a 1, Xiaomeng He a 1, Xintong Wang b 1, Bo Yu a, Siqi Zhao a, Peili Jiao a,
Hongwei Jin a, Zhenming Liu a *, KeWei Wang c *, Liangren Zhang a *, Lihe Zhang a
,
,
,
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, PR China
b Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, PR China
c Department of Pharmacology, Qingdao University School of Phamacy, Qingdao, 266021, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
a
: 7 nicotinic acetylcholine receptors (nAChRs) expressed in the nervous and immune systems have
been suggested to play important roles in the control of inflammation. However, the lack of antagonist
tools specifically inhibiting 7 nAChR impedes the validation of the channel as therapeutic target. To
discover a selective 7 antagonist, we started a pharmacophore-based virtual screening and identified a
piperidine-spirooxadiazole derivative T761e0184 that acts as a 7 antagonist. A series of novel
Received 16 July 2020
Received in revised form
18 August 2020
a
a
Accepted 18 August 2020
a
piperidine-spirooxadiazole derivatives were subsequently synthesized and evaluated using two-
electrode voltage clamp (TEVC) assay in Xenopus oocytes. Lead compounds from two series inhibited
Keywords:
a7 nAChR
a
7 with their IC50 values ranging from 3.3
mM to 13.7 mM. Compound B10 exhibited a7 selectivity over
Piperidine-spirooxadiazole derivatives
Antagonists
SAR
other 2 and 4 nAChR subtypes. The analysis of structure-activity relationship (SAR) provides
a
4
b
a3b
valuable insights for further development of selective
a
7 nAChR antagonists.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
perspectives [5e7].
The a
7 nAChR is a member of ligand-gated Ca2þ channel that is
Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-
loop receptor superfamily, which primarily mediate signal trans-
duction of the neurotransmitter acetylcholine (ACh) at the ganglion
and neuromuscular junctions [1]. These receptors are composed of
highly expressed in hippocampus, cortex and several subcortical
limbic regions, also expressed in immune cells and endothelial
cells, contributing to cognition, attention, working memory, reward
pathways and inflammatory modulation [7,8]. A plentiful of evi-
muscle-type (
a
1,
b
1,
d
,
g
, and ε) and neuronal (
a
2ꢀ
a
10 and
b
2ꢀ
b
4)
dences have supported the enhancement of a7 nAChR as potential
therapeutic strategies for many diseases such as schizophrenia,
Alzheimer’s disease and inflammatory disorders [9e11]. However,
subunits, each subunit contains a large N-terminal extracellular
domain, followed by four transmembrane spans with intervening
intracellular domains [2]. Functional pentameric nAChR can be
subdivided in homopentamers, which are formed by five identical
subunits such as
combination of different subunits, such as
nAChR [3,4]. Among them, two neuronal receptors
activation of
nAChR expression promote cancer cell proliferation and
metastasis in lung, colon, and bladder tissues [12]. Thus, selective
inhibitors of 7 nAChR can inhibit cancer cell growth through
reversing proangiogenic effects of nicotine [13,14]. Moreover, the
inhibition of 7 nAChR has explored as potential treatment for
a7 nAChR may not be always beneficial. High levels of
a7
a7 nAChR, or heteropentamers that result from the
a3b
4 nAChR and
7 and
a4
4
b
2
2
a
a
a
b
have been identified as primary interests from the drug discovery
a
organophosphorus nerve agent intoxication and smoking cessation
[15,16].
Activation of the
a7 nAChR has been widely studied, which
* Corresponding authors.
provides us information for the design of its antagonists. The pri-
mary orthosteric ligand binding pocket for agonists and competi-
tive antagonists is localized in the extracellular domain at the
interface of two adjacent
ACh is an endogenous ligand that binds to the orthosteric ligand
E-mail addresses: zhanghan930506@163.com (H. Zhang), xhe@bjmu.edu.cn
(X. He), wangxintong@bjmu.edu.cn (X. Wang), boyu@bjmu.edu.cn (B. Yu),
1811210124@bjmu.edu.cn (S. Zhao), pelly@bjmu.edu.cn (P. Jiao), jinhw@bjmu.edu.
cn (H. Jin), zmliu@bjmu.edu.cn (Z. Liu), wangkw@bjmu.edu.cn (K. Wang),
liangren@bjmu.edu.cn (L. Zhang), zdszlh@bjmu.edu.cn (L. Zhang).
a7 subunits (loops AeC, loops DeE) [17].
1
These authors contributed equally.
https://doi.org/10.1016/j.ejmech.2020.112774
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
binding site to activate calcium influx, yielded a EC50 value of
~100e200 M [18]. Over the past three decades, a variety of
orthosteric agonists have been developed for the 7 nAChR as po-
tential therapeutics, such as GTS-21, JN403 and so on [19e21].
Thereinto, several 7 nAChR selective agonists (e.g., AQW051, SSR-
important part of many drug discovery strategies [33]. Considering
that the 7 nAChR orthosteric modulators share general pharma-
cophoric features and many agonists already have typical phar-
macophoric elements and drug-like properties. Besides, the
m
a
a
a
discovery of
ported. Therefore, we developed a pharmacophore model based on
the structures of 11 reported 7 nAChR agonists and conducted
a7 nAChR antagonists by its agonists has been re-
180711 and RG3487) have advanced to clinical trials for schizo-
phrenia and Alzheimer’s disease (Fig. 1) [22e24]. The general fea-
a
tures of these reported
basic fragment, which can be protonated at physiological pH and
form cation- interaction with the receptor; a hydrophobic aro-
matic moiety providing interaction and a linker [25e27]. In
addition, several allosteric modulators, e.g. AVL3288, showed
different structural features compared with orthosteric modulators
(Fig. 1) [28]. Therefore, development of a7 nAChR modulators has
attracted increasing attention for both academic functional studies
and clinical applications.
a
7 nAChR orthosteric agonists include: a
virtual screening with the aid of pharmacophore-based model. As a
result, piperidine-spirooxadiazole derivative T761e0184 was
discovered to be a novel a7 nAChR antagonist. To elucidate the
structure-activity relationship (SAR) and discover more active
compounds, a variety of T761e0184 derivatives were further
designed by modification of substituted groups at 3- and 8-position
of piperidine-spirooxadiazole scaffold, especially introduction of
aromatic moiety (R1) at 3-position of the scaffold to form potential
p
p
-p
p-p stacking effect. The a7 nAChR antagonistic activities of all
Although extensive studies have been conducted to discover
synthesized compounds were assessed by two-electrode voltage
clamp (TEVC) assay in Xenopus oocytes. In addition, the antago-
nistic activities of the representative compound over other related
nAChR subtypes were also investigated. Herein, we report the
synthesis and biological investigation of these compounds, which
selective
tagonists is relatively limited (Fig. 1).
methyllycaconitine (MLA) are two frequently used
potent antagonists, which obtained from natural sources, also
inhibit some other nAChR subtypes [20]. Besides, several 7 nAChR
a
7 nAChR agonists, the development of
a
7 selective an-
-BTX) and
7 nAChR
a-Bungarotoxin (
a
a
a
will definitely promote the discovery of novel selective a7 nAChR
antagonists discovered by derivatization or structural modification
antagonists [34].
of agonists, have been reported with antagonistic activities at
micromolar levels (IC50 ¼ 3.7e300
mM), including triazolylamide
2. Results and discussions
derivatives, quinoline derivatives and nicotine analogues
[27,29,30]. Moreover, phenothiazine-class antipsychotics such as
phenothiazine derivative and amphetamine-type stimulants
2.1. Computationally guided discovery of
antagonists
a7 nAChR orthosteric
amphetamine have also been reported as
with IC50 values of 0.4 M and 12 M, respectively [31,32]. Overall,
the reported 7 nAChR antagonists are still limited and lack of
enough antagonistic activity and/or specificity. Therefore, there is
an urgent need for the discovery of novel 7 nAChR antagonists,
a7 nAChR antagonists,
m
m
In an effort to discover novel selective ligands of
pharmacophore-based virtual screening approach was employed
(Fig. 2). We first selected 11 reported 7 nAChR agonists according
to structural diversity and rigidity as training sets (Fig. S1), and
constructed a pharmacophore model based on the common fea-
tures of selected compounds using HipHop program (Discovery
Studio, Accelrys, Inc.). The selected model contained 1 hydrogen-
bond receptor, 1 hydrophobic center and 1 positively ionized
a7 nAChR,
a
a
a
which will not only provide direct molecular tools for the phar-
macological researches related to this channel, but also lead to the
potential clinical therapeutic agents for related diseases.
Pharmacophore-based virtual screening approach has been an
Fig. 1. Chemical structures of representative a7 nAChR modulators.

H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
3
Fig. 2. Flow chart illustrating the computationally guided discovery process of a7 nAChR orthosteric antagonists.
group. Since the HipHop model produced a large number of false
positive results, we established a recursive partitioning (RP) model.
Then we used the afore mentioned two models to perform virtual
screening of the commercial small-molecule database ChemDiv
(http://www.chemdiv.org/). In order to remove unwanted struc-
tures and accelerate the process of virtual screening, the “rule of
five” filter was applied before the pharmacophore-based database
searching. In the end, we obtained 13 compounds for biological
evaluation.
adjacent subunits, in which the piperidine N atom formed three
cation- interactions with residues TYR191, TRP145 and TYR184 in
the form of nitrogen cations, as a novel positive charge center; The
8-position p-fluorobenzyl formed a interaction with TRP145.
Through comparing with the structural characteristics and binding
mode of known 7 nAChR modulators, we found that the 3-
position cyclopropyl was replaced by aromatic moiety might in-
crease the hydrophobic interaction and the potential stacking
p
p-p
a
p-p
effect, suggesting us a feasible modified strategy to enhance its
TEVC was used to evaluate the activities of these 13 compounds
potency.
in Xenopus oocytes expressing human
showed inhibitory effects on ACh induced
(Fig. S2). For the purpose of calculating inhibition ratio, 100
compound was co-applied with 100 M ACh, and the resulting
currents were compared to those induced by ACh alone. Among
these novel ligands for the nAChR, the piperidine-
a
7 nAChR, 10 of them
a
7 nAChR currents
2.2. Chemistry
mM
m
Further structural optimizations were performed on T761e0184
to discover more active antagonists of a7 nAChR. Considering the
a
7
structural similarity with known modulators, we retained the
piperidine-spirooxadiazole skeleton of T761e0184 and modified
the structure mainly at its 3-position (R1) and 8-position (R2),
which are called Class A and B, respectively (Fig. 2). The general
synthetic routes for preparation of Class A and B are described in
Scheme 1 and Scheme 2.
Different aryl substituted nitriles (1a-1o) were reacted with
hydroxylamine hydrochloride via addition reaction in ethanol to
afford N-hydroxylformamidines (2a-2o), which were then cyclized
by reacting with N-benzylpiperidone using p-toluene sulfonic acid
as catalyst in toluene to form target compounds A1-A15 [36]. The
biaryl derivatives A16-A36 was prepared from A7 or A13 through
Suzuki coupling reaction with substituted boric acids under the
catalytic system of Pd(PPh3)4 [37].
spirooxadiazole derivative T761e0184 was selected for further
investigation due to its potency (Fig. 3).
To investigate the binding mode of T761e0184 targeting
orthosteric site of
ried out by using Induced Fit Docking module of Schrodinger Suite
a
7 nAChR, molecular docking studies were car-
€
€
(Schrodinger, LLC, New York, NY, USA). Although the three-
dimensional (3D) structure of human
a7 nAChR has not been
solved, several structures of the extracellular region of the ho-
mologous acetylcholine binding protein (AChBP) or chimera are
available, which shares 64% sequence identity and 71% similarity
with native
a7 nAChR [35]. So, T761e0184 was docked to the crystal
structure of human
a7-AChBP chimera (PDB ID code: 3SQ6). The
docking result (Fig. S3) indicated that T761e0184 bound into the
classic aromatic pocket and the piperidine-spirooxadiazole moiety
could be matched well to the orthosteric binding site between two
Scheme 2 outlines the synthesis of compounds B1eB15. Treat-
ment of piperidone with organic halides (3a, 3b) in the presence of
Fig. 3. Inhibition of
a
7 currents by T761e0184 in two-electrode voltage clamp recordings of Xenopus oocytes expressing human
a7 nAChRs. Representative traces of currents
recorded in response to 100
m
M ACh (left), 100 M T761e0184 (middle), and co-application of 100 M T761e0184 and ACh (right) using two-electrode voltage clamp.
m
m

4
H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
Scheme 1. Synthesis of compounds A1-A36. Reagents and conditions: (a). NH2OHꢁ HCl, pyridine, EtOH, reflux, 2e6 h; (b) 1-Benzylpiperidin-4-one, TsOHꢁH2O, toluene, reflux,
overnight; (c) Aryl boric acids, Na2CO3, Pd(PPh3)4, DMF, H2O, 80e120 ꢂC, 6e20 h.
Scheme 2. Synthesis of compounds B1eB15. Reagents and conditions: (a). Piperidin-4-one, Na2CO3, CH3CN, r.t., 45 min, 50 ꢂC, 45 min, 70 ꢂC, 2 h-overnight; (b) 2a, 2f, 2i, 2l or 2m,
TsOHꢁH2O, toluene, reflux, overnight; (c) Aryl boric acids, Na2CO3, Pd(PPh3)4, DMF, H2O, 80e120 ꢂC, 6e20 h.
sodium carbonate produced substituted piperidones (4a, 4b). As
aforementioned, cyclization reactions were accomplished from
synthesized or commercially available substituted piperidones (4a-
4e) and N-hydroxyformamidines (2a, 2f, 2i, 2l or 2m) to give
compounds B1eB12. And the biaryl derivatives B13eB15 were
obtained via Suzuki coupling reaction starting from B10 or B12.
tested compounds (Tables 1e3). Of them, 24 compounds reduced
more than 80% ACh-induced 7 nAChR currents increase compared
with the vehicle-treated cells. Their inhibitory activities at 30
were further tested, 9 compounds inhibited more than 70% ACh-
induced currents. The concentration-dependent response curves
of several compounds that exhibited most potent inhibitory activ-
a
m
M
ities at the concentration of 30
values. Five compounds, i.e. A2, A7, B9, B10 and B15, gave IC50
values of 3.3e13.7 M (Fig. 4, Fig. 5).
mM were plotted to calculate IC50
2.3. Inhibitory activity and SAR
m
As shown in Table 1 and Fig. 4, when the p-fluorobenzyl group at
R2-position of piperidine-spirooxadiazole was replaced with
benzyl, compound A1 maintained the inhibitory activity. Therefore,
we retained benzyl at R2-position and investigated the inhibitory
activities of various aromatic substitution groups at R1-position of
piperidine-spirooxadiazole to discover more active compounds.
Totally, 51 compounds were synthesized and their biological
activities were investigated by TEVC. The 100
each compound was individually tested by co-application with
100 M Ach similar with the case of T761e0184, a varied degree of
mM inhibition rate of
m
antagonistic response of ACh-induced currents was observed,
whereas no significant potentiation was exerted by any of the

H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
5
Table 1
Inhibition of
a
7 nAChR currents by piperidine-spirooxadiazole derivatives through modifications in R1 on
a7 nAChR currents investigated by TEVC.
Compds
R1 100 30
m
M Inhibition (%)a M Inhibition (%)a
m
IC50
(
m
M)
Agonism
A1
83.9 5.0
98.2 0.3
52.2 8.7
70.4 2.0
e
A2
A3
A4
A5
A6
A7
A8
A9
13.7
e
e
e
e
e
þ
e
e
75.4 4.0
83.1 2.0
78.3 5.5
90.7 2.4
91.5 2.0
79.6 6.5
79.1 2.2
e
65.9 2.2
e
77.4 3.8
90.8 2.0
6.0
e
e
A10
A11
A12
94.5 4.0
74.9 1.7
68.7 3.3
65.1 2.7
e
e
e
e
e
A13
A14
A15
90.3 2.7
91.1 4.6
69.8 8.1
63.7 5.2
76.6 1.8
e
e
e
e
T761e0184
83.9 5.1
52.8 1.6
e
“-“: no activity.
“–“: not measured.
a
Percentage inhibition at test concentration, and data are the mean SEM from 3 to 4 determinations (n ¼ 3e4); currents were recorded in TEVC.
Replacing cyclopropyl group at R1-position of piperidine-
spirooxadiazole with phenyl (A2), thiazol-2-yl (A10), 6-
bromopyridin-3-yl (A13) and isoquinolin-3-yl (A14) resulted in
higher potency than A1, while the other substitution groups, i.e.
thiophen-2-yl (A11), pyridin-2-yl (A12) and 1H-indazol-3-yl (A15)
exhibited lower inhibitory activity than A1. This difference might be
related to the electronic density or spatial size of the aromatic
moiety. Among them, phenyl substituted compound A2 was the
activation current. “-“: no agonistic activity; “þ“: weak agonistic
effect, less than 20% of 100 M ACh.
m
A1-A15: R2 ¼ benzyl; T761e0184: R2 ¼ p-fluorobenzyl
Since the biaryl group was a common motif among a7 orthos-
teric modulators, such as agonist AQW051 (Fig. 1) [24], we also
replaced cyclopropyl with various biaryl groups at the R1-position
of piperidine-spirooxadiazole. As shown in Table 2, replacing the
bromine atom of A7 or A13 with phenyl, substituted phenyl, thio-
phen and pyridin, decreased inhibitory activity was observed for a
large proportion of resulting compounds (A16-A19, A21, A24-A31,
A33, A35). But biaryl bearing some 30 substitution displayed
maintained or higher potency than A1, such as 30-methoxy (A20,
A34), 30-hydroxy (A22), 30-amino (A23, A36) and pyridin-3-yl
(A32). These results implied that electron-donating group at 30-
position of biaryl was beneficial to increase the inhibitory activities.
most potent antagonist with an IC50 value of 13.7 mM (Fig. 4), which
lead us to further investigate the SAR of substituted phenyl de-
rivatives. The introduction of m-methylphenyl (A3), m-fluo-
rophenyl (A4), p-fluorophenyl (A5), p-methoxyphenyl (A8) or 2,4-
dimethoxyphenyl (A9) substituent into the R1-position of
piperidine-spirooxadiazole decreased the inhibitory activity
compared with A2. However, strong inhibitory activities were
observed for p-chlorophenyl (A6) and p-bromophenyl (A7)
substituted piperidine-spirooxadiazole derivatives, particularly A7
The ratio was tested as a parameter with a ratio of 100
activation current. “-“: no agonistic activity; “þ“: weak agonistic
effect, less than 20% of 100 M ACh.
To further study the SAR of R2-position substituted piperidine-
spirooxadiazole derivatives, we retained the substituent at R1-
mM ACh-
showed an IC50 value of 6.0 mM (Fig. 4). These results demonstrated
that the replacement of cyclopropyl with aromatic moieties (p-
chlorophenyl or p-bromophenyl) were favorable for improving
potency as we expected.
m
The ratio was tested as a parameter with a ratio of 100 mM ACh-

6
H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
Table 2
Inhibition of
a
7 nAChR currents by 3-biaryl piperidine-spirooxadiazole derivatives.
R1 100
M Inhibition (%)a
Compds
m
30
m
M Inhibition (%)a
IC50
(
mM)
Agonism
A16
71.4 8.4
72.0 2.6
80.1 2.7
26.2 11.3
95.6 0.8
73.0 9.5
e
e
e
A17
A18
A19
A20
A21
e
e
e
e
e
e
e
e
e
e
e
30.0 10.4
e
76.9 5.1
e
A22
A23
A24
A25
A26
A27
A28
A29
A30
80.4 2.3
93.5 1.9
81.1 14.5
67.0 0.8
34.9 5.5
42.4 11.6
52.8 12.2
40.5 9.8
36.5 5.0
59.0 1.7
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
60.1 3.3
39.5 2.8
e
e
e
e
e
e
A31
A32
71.1 2.1
e
e
e
e
e
81.1 14.5
65.0 4.4
A33
A34
75.2 2.7
89.9 2.2
e
e
e
e
e
54.7 2.5
A35
58.2 5.1
e
e
e

H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
7
Table 2 (continued )
Compds
R1
100
m
M Inhibition (%)a
30
m
M Inhibition (%)a
IC50
(
mM)
Agonism
A36
87.2 3.6
61.8 1.1
e
e
“-“: no activity.
“–“: not measured.
a
Percentage inhibition at test concentration, and data are the mean SEM from 3 to 4 determinations (n ¼ 3e4); currents were recorded in TEVC.
Table 3
Inhibition of a7 nAChR currents by piperidine-spirooxadiazole derivatives through modifications in R2.
Compds
R1
R2
100
m
M Inhibition (%)a
30
m
M Inhibition (%)a
IC50
(m
M)
Agonism
B1
73.5 7.4
75.1 1.4
58.1 6.3
e
e
e
B2
B3
e
e
e
e
e
e
e
B4
B5
B6
B7
B8
B9
B10
70.2 8.4
80.0 5.3
80.0 5.2
18.8 3.5
58.2 9.4
96.7 1.3
97.4 0.3
e
e
e
e
e
e
e
e
39.3 10.2
37.9 6.9
e
e
e
e
e
e
ꢀ CH3
ꢀ CH3
90.4 0.2
91.8 2.1
4.4
5.4
B11
B12
ꢀ CH3
ꢀ CH3
87.4 2.6
83.4 3.8
69.7 3.6
44.1 2.1
e
e
e
e
B13
B14
B15
ꢀ CH3
ꢀ CH3
ꢀ CH3
93.2 0.5
78.2 4.4
93.4 0.8
81.3 4.6
e
e
e
e
–
e
86.1 3.0
3.3
“-“: no activity.
“–“: not measured.
a
Percentage inhibition at test concentration, and data are the mean SEM from 3 to 4 determinations (n ¼ 3e4); currents were recorded in TEVC.
position which was favorable to the inhibitory activity and inves-
tigated the biological activities of different substituted derivatives
at R2-position (Table 3 and Fig. 5). By replacing the benzyl group at
R2-position of piperidine-spirooxadiazole with tert-butox-
ycarbonyl, ethoxycarbonyl and 2-/4-pyridylmethyl groups, com-
pounds B1eB8 were formed but decreased inhibitory activities
were observed. Meanwhile, the replacement of benzyl group at R2-
position with methyl (B9, B10) resulted in improved inhibitory
activities compared with corresponding R1-substituted derivatives
withdrawing groups at R2-position of piperidine-spirooxadiazole,
it was not conducive to the protonation of the piperidine N atom
connected to it under physiological conditions, suggesting us to
synthesize methyl substituted compounds (B11eB15), and com-
pound B15 expectedly exhibited strong inhibitory activity with an
IC50 ¼ 3.3
mM (Fig. 5). These results indicated that methyl substit-
uent at R2-position of piperidine-spirooxadiazole derivatives were
favorable to the inhibitory activity.
Subsequently, we investigated the binding mode of represen-
tative compound B10 into the orthosteric binding pocket of human
(A2, A7), showed IC50 values of 4.4
mM and 5.4 mM, respectively
(Fig. 5). These probably might be the presence of electron-
a7-AChBP chimera (PDB ID code: 3SQ6) by molecular docking as

8
H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
Fig. 4. Concentration-dependent inhibition of
a7 AChR currents by A2 and A7 in TEVC assay. Data were presented as the mean SEM from four independent experiments (n ¼ 4).
Fig. 5. Inhibition of
a7 currents by B9, B10 and B15. (a) Inhibition activity of 10 mM and 100 mM B10 against the a7 nAChR-mediated currents. Concentration-dependent inhibition of
a
7 AChR currents by B9 (b), B10 (c) and B15 (d). Data were presented as the mean SEM from four independent experiments (n ¼ 4).
previously described in 2.1. As shown in Fig. 6, B10 matched better
to the orthosteric binding site between two adjacent subunits
compared with T761e0184. Because B10 not only maintained three
2.4. Inhibitory activities of compound B10 on other related ligand-
gated ion channels
cation-
through the piperidine N atom as positive charge center, but also
formed a new interaction with TRP53 by aromatic moiety at R1-
position of the piperidine-spirooxadiazole. This binding mode was
more similar to that of known 7 AChR selective modulators,
p
interactions with residues TYR191, TRP145 and TYR184
To evaluate the selectivity of representative compound B10, we
examined the effects of B10 on
subtypes with distributions similar to that of
[38]. As shown in Fig. 7, the expression of 4 and
Xenopus oocytes produced a robust current in response to 100
Ach. The application of B10 (10 M) hardly induced 4 and
nAChR currents, and the lack of effects on 4 and 2 nAChR
were further confirmed by the co-application of B10 with Ach,
which resulted in a mild reduction of 4 and 2 currents by
20% and 24%, respectively. The concentration-dependent response
curves of B10 on 4 and 2 nAChR were plotted to calculate
IC50 values. Compound B10 showed only weak non-concentration-
dependent inhibition on the 4 currents, and gave an IC50 value
of 32.0 M on the 2 nAChR, which displayed about 6-fold over
7 nAChR IC50 (5.4 M). As a conclusion of channel selectivity
a
4b
2 and
a
3
b
4 nAChR by TEVC,
7 nAChR in brain
2 nAChR in
p-
p
a
a
3
b
a4b
a
mM
verifying the introduction of aromatic moiety increased the
antagonistic activity.
m
a3
b
a4b2
a3
b
a4b
Taken together, we found that aromatic moieties (p-bromo-
phenyl, 30 electron-donating groups substituted biaryl) at R1-posi-
tion and methyl at R2-position of the piperidine-spirooxadiazole
were favorable for the inhibitory activity. Our investigation illus-
trated that piperidine-spirooxadiazole derivatives were a series of
a3
b
a4b
a3
b
a4b
novel potent
tivity of the substituents on the piperidine-spirooxadiazole scaffold
played key roles in the inhibitory activity against 7 AchR.
a7 AChR antagonists, and the size and electronega-
a3b
m
a4b
a
a
m

H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
9
Fig. 6. 2D (a) and 3D (b) protein-ligand contacts of B10 predicted through molecular docking. It was docked well with the orthosteric site between subunit D (including key amino
acid residues W145, Y184 and Y191, etc.) and subunit E (including amino acid residues W53, Q55 and L116, etc.).
Fig. 7. Selective inhibition of
a7 over
a3
b4 and
a
4b
2 nAChRs by B10 in TEVC assay. (a) Inhibition of
a3
b4 currents by 10
mM B10 against. (b) Inhibition of a4b2 currents by 10 mM
B10. (c) Summary for concentration-dependent inhibition of
iments (n ¼ 4), and ***P < 0.005, *P < 0.05.
a7, a3b4 and a4b2 nAChR currents by B10. Data were presented as the mean SEM from four independent exper-

10
H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
experiments, B10 exhibited selectivity over
3. Conclusion
a
3
b
4 and
a
4
b
2 nAChR.
room temperature and extracted with EtOAc three times. The
combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated under reduced pres-
sure. The crude product was purified by silica gel column chro-
matography eluting with PE/EtOAc/TEA (20/10/1) to afford A16-
A36.
In this study, we discovered a potent
a
7 nAChR antagonist
piperidine-spirooxadiazole
derivative
T761e0184 by
pharmacophore-based virtual screening. For exploring the effect of
3- and 8-position moiety on the antagonistic activity and finding
more potent agents, 51 T761e0184 derivatives were designed and
synthesized. All of them exhibited moderate to excellent antago-
4.2.1.1. 8-Benzyl-3-cyclopropyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-
ene (A1). White powder. Yield: 32.0%. Mp: 141e142 ꢂC. 1H NMR
(400 MHz, CDCl3)
d 7.31 (s, 5H), 4.08 (s, 1H), 3.53 (s, 2H), 2.69e2.36
nism against the
(3-methyl-8-aryl) with IC50 values of 4.4
respectively. Docking studies explained the importance of aromatic
moieties at the 8-position. More importantly, B10 showed
selectivity over other 2 and 4 nAChR subtypes. The findings
of this study provided valuable insights for the further develop-
ment of selectivity 7 nAChR antagonists.
a
7 nAChR, especially compounds B9, B10 and B15
(m, 4H), 2.13e1.86 (m, 2H), 1.85e1.68 (m, 2H), 1.59e1.50 (m, 1H),
m
M, 5.4 M and 3.3 M,
m
m
1.01e0.83 (m, 4H). 13C NMR (101 MHz, CDCl3)
d 158.5, 138.1, 129.1,
128.2, 127.1, 94.8, 62.6, 50.5, 36.2, 5.9, 5.8. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C16H22N3O: 272.1763, found: 272.1766.
a
7
a
4b
a3b
4.2.1.2. 8-Benzyl-3-phenyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-ene
a
(A2). Yellow powder. Yield: 46.2%. Mp: 179e180 ꢂC. 1H NMR
(400 MHz, CDCl3)
(s,1H), 3.56 (s, 2H), 2.76e2.48 (m, 4H), 2.16e2.02 (m, 2H),1.97e1.84
(m, 2H). 13C NMR (101 MHz, CDCl3)
155.3, 138.3, 130.7, 129.0,
d
7.68 (d, J ¼ 7.4 Hz, 2H), 7.55e7.26 (m, 8H), 4.50
4. Experimental section
d
4.1. Materials and methods
128.6, 128.2, 127.1, 126.3, 125.9, 96.0, 77.2, 62.7, 50.5, 36.6. HRMS
(ESI-TOFþ) m/z [MþH]þ calcd for C19H22N3O: 308.1763, found:
308.1768.
All of chemicals and solvents used were obtained from com-
mercial sources. The solvents were dried by standard procedures.
Thin layer chromatography (Silica gel GF254, Qingdao Haiyang
Chemical Co., Ltd, Qingdao, China) was employed to monitor re-
action process, and silica column chromatography was carried out
to purify crude product (Silica gel 200e300 mesh, Shanghai San-
pont Co., Ltd, Shanghai, China). 1H NMR spectra and 13C NMR
spectra were recorded at 400 MHz using Bruker Avance III spec-
trometer (Bruker CO., Switzerland) in CDCl3 or DMSO‑d6 solution
with tetramethylsilane as internal standard and chemical shift
values were given in ppm. The NMR data was processed by software
MestReNova (Ver.6.1.0, mestrelab research S.L.). The splitting peak
was designed as s, singlet; d, doublet; dd, double doublet; t, triplet;
m, multiplet; br, broad. The high resolution mass (HRMS) was
measured on FT-MS-Bruker APEX IV mass spectrometer.
4.2.1.3. 8-Benzyl-3-(m-tolyl)-1-oxa-2,4,8-triazaspiro[4.5]dec-2-ene
(A3). Yellow powder. Yield: 30.0%. Mp: 180e181 ꢂC. 1H NMR
(400 MHz, CDCl3)
1H), 3.56 (s, 2H), 2.79e2.44 (m, 4H), 2.37 (s, 3H), 2.13e1.98 (m, 2H),
1.92e1.84 (m, 2H). 13C NMR (101 MHz, CDCl3)
155.4, 138.4, 138.3,
d 7.63e7.43 (m, 2H), 7.38e7.26 (m, 7H), 4.49 (s,
d
131.5, 129.0, 128.5, 128.2, 127.1, 126.9, 125.8, 123.4, 95.9, 62.7, 50.5,
36.6, 21.3. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C20H24N3O:
322.1919, found: 322.1916.
4.2.1.4. 8-Benzyl-3-(3-fluorophenyl)-1-oxa-2,4,8-triazaspiro[4.5]dec-
2-ene (A4). White powder. Yield: 24.1%. Mp: 144e145 ꢂC. 1H NMR
(400 MHz, CDCl3)
(m, 4H), 7.31e7.26 (m, 1H), 7.18e7.11 (m, 1H), 4.49 (s, 1H), 3.56 (s,
2H), 2.68e2.54 (m, 4H), 2.11e2.03 (m, 2H), 1.93e1.86 (m, 2H). 13
NMR (101 MHz, CDCl3)
d 7.50e7.43 (m, 1H), 7.42e7.36 (m, 2H), 7.35e7.32
C
4.2. Chemical synthesis
d
164.0, 162.7 (d, J ¼ 246.9 Hz), 154.6, 138.4,
130.5, 130.4, 129.2, 128.4, 127.2, 122.1 (d, J ¼ 3.2 Hz), 117.8 (d,
J ¼ 21.2 Hz), 113.5 (d, J ¼ 23.6 Hz), 96.6, 62.8, 50.6, 36.7. HRMS (ESI-
TOFþ) m/z [MþH]þ calcd for C19H21FN3O: 326.1669, found:
326.1671.
4.2.1. General procedure for the synthesis of compounds A1ꢀA36
The syntheses of compounds A1-A36 were mainly referred to
literature methods [36,37].
A mixture of hydroxylamine hydrochloride (103 mg, 1.5 mmol)
and pyridine (158 mg, 2 mmol) in ethanol (5 mL) was stirred at
room temperature for 15 min, followed by the addition of different
aryl substituted nitriles 1a-1o (1 mmol). The reaction mixture was
refluxed for 2e6 h and the solvent was evaporated under reduced
pressure. The residue was dissolved in EtOAc and washed with
water three times, dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The crude product was
purfied by silica gel column chromatography eluting with DCM/
MeOH/NH3ꢁH2O (200/4/1) to afford 2a-2o.
4.2.1.5. 8-Benzyl-3-(4-fluorophenyl)-1-oxa-2,4,8-triazaspiro[4.5]dec-
2-ene (A5). White powder. Yield: 24.8%. Mp:169e170 ꢂC. 1H NMR
(400 MHz, DMSO‑d6)
d
7.72 (dd, J ¼ 8.9, 5.5 Hz, 2H), 7.47 (s, 1H),
7.34e7.30 (m, 5H), 7.28e7.22 (m, 1H), 3.51 (s, 2H), 2.81e2.52 (m,
2H), 2.47e2.27 (m, 2H), 1.90e1.64 (m, 4H). 13C NMR (101 MHz,
CDCl3)
d
164.12 (d, J ¼ 251.4 Hz), 162.8, 154.5, 138.2, 129.1, 128.5,
128.4, 128.3, 127.1, 122.2, 122.2 (d, J ¼ 3.2 Hz), 122.1, 115.9, 115.7,
96.2, 62.7, 50.5, 36.5. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for
C19H21FN3O: 326.1669, found: 326.1663.
TsOHꢁH2O (37.5 mg, 0.2 mmol) was added to a solution of 2a-2o
(1 mmol) and N-benzylpiperidone (285 mg, 1.5 mmol) in toluene
(5 mL). The mixture was refluxed overnight. After completion, the
mixture was extracted with EtOAc three times. The combined
organic layers were washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography
eluting with DCM/MeOH/NH3ꢁH2O (100/3/1) to afford A1-A15.
Pd(PPh3)4 (113 mg, 0.1 mmol) was added to a degassed mixture
of A7 or A13 (1 mmol), substituted boric acids (2 mmol), K2CO3
(483 mg, 3.5 mmol) in dioxane (4 mL) and water (2 mL) under
argon. The mixture was heated at 80e120 ꢂC for 6e20 h, cooled to
4.2.1.6. 8-Benzyl-3-(4-chlorophenyl)-1-oxa-2,4,8-triazaspiro[4.5]
dec-2-ene (A6). Yellow powder. Yield: 40.3%. Mp: 148e149 ꢂC. 1H
NMR (400 MHz, Methanol-d4)
d 7.70e7.62 (m, 2H), 7.49e7.42 (m,
2H), 7.39e7.30 (m, 4H), 7.30e7.23 (m, 1H), 3.58 (s, 2H), 2.83e2.69
(m, 2H), 2.56e2.43 (m, 2H), 2.01e1.84 (m, 4H). 13C NMR (101 MHz,
CDCl3)
d 154.4, 138.3, 136.7, 129.0, 128.9, 128.2, 127.6, 127.1, 124.5,
96.4, 62.6, 50.5, 36.6. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for
C19H21N3OCl: 342.1373, found: 342.1371.
4.2.1.7. 8-Benzyl-3-(4-bromophenyl)-1-oxa-2,4,8-triazaspiro[4.5]
dec-2-ene (A7). Yellow powder. Yield: 47.3%. Mp: 176e177 ꢂC. 1H

H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
11
NMR (400 MHz, CDCl3)
d
7.55 (s, 4H), 7.37e7.27 (m, 5H), 4.37 (s,1H),
2.19e2.07 (m, 2H), 2.00e1.91 (m, 2H). 13C NMR (101 MHz, CDCl3)
3.56 (s, 2H), 2.66e2.51 (m, 4H), 2.14e2.00 (m, 2H), 1.93e1.84 (m,
d
155.8, 151.9, 138.5, 135.6, 138.4, 131.1, 129.0, 128.5, 128.2, 127.6,
2H). 13C NMR (101 MHz, CDCl3)
d
154.5, 138.3, 131.9, 129.0, 128.2,
127.4, 127.0, 119.1, 106.5, 96.6, 62.7, 50.6, 36.5. HRMS (ESI-TOFþ) m/z
127.8, 127.1,125.0,124.9, 96.4, 62.6, 50.5, 36.6. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C19H21BrN3O: 386.0868, found: 386.0864.
[MþH]þ calcd for C22H23N4O: 359.1872, found: 359.1867.
4.2.1.15. 8-Benzyl-3-(1H-indazol-3-yl)-1-oxa-2,4,8-triazaspiro[4.5]
dec-2-ene (A15). Yellow powder. Yield: 45.0%. Mp: 150e151 ꢂC. 1H
4.2.1.8. 8-Benzyl-3-(4-methoxyphenyl)-1-oxa-2,4,8-triazaspiro[4.5]
dec-2-ene (A8). Yellow powder. Yield: 27.7%. Mp: 150e151 ꢂC. 1H
NMR (400 MHz, CDCl3)
d
11.00 (brs, 1H), 8.19 (d, J ¼ 8.2 Hz, 1H), 7.45
NMR (400 MHz, CDCl3)
d
7.62 (d, J ¼ 8.8 Hz, 2H), 7.38e7.26 (m, 5H),
(t, J ¼ 8.9 Hz, 2H), 7.37e7.27 (m, 6H), 5.36 (s, 1H), 3.58 (s, 2H),
2.74e2.57 (m, 4H), 2.17e2.07 (m, 2H), 2.01e1.93 (m, 2H). 13C NMR
6.91 (d, J ¼ 8.8 Hz, 2H), 4.41 (s, 1H), 3.83 (s, 3H), 3.55 (s, 2H),
2.77e2.40 (m, 4H), 2.14e1.97 (m, 2H), 1.95e1.78 (m, 2H). 13C NMR
(101 MHz, CDCl3)
d 151.5, 141.0, 138.0, 133.5, 129.1, 128.3, 127.7,
(101 MHz, CDCl3)
d 161.5, 155.0, 148.6, 138.4, 129.0, 128.2, 127.9,
127.2, 122.6, 122.3, 120.7, 109.8, 95.5, 62.7, 50.6, 36.3. HRMS (ESI-
127.0, 118.3, 114.0, 95.7, 62.7, 55.3, 50.6, 36.5. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C20H24N3O2: 338.1869, found: 338.1866.
TOFþ) m/z [MþH]þ calcd for C20H22N5O: 348.1811, found: 348.1815.
4.2.1.16. 3-([1,10-Biphenyl]-4-yl)-8-benzyl-1-oxa-2,4,8-triazaspiro
4.2.1.9. 8-Benzyl-3-(2,4-dimethoxyphenyl)-1-oxa-2,4,8-triazaspiro
[4.5]dec-2-ene (A16). Yellow powder. Yield: 66.7%. Mp: 195e196 ꢂC.
[4.5]dec-2-ene (A9). Yellow powder. Yield: 29.9%. Mp: 157e158 ꢂC.
1H NMR (400 MHz, CDCl3)
d 7.75 (m, 2H), 7.70e7.42 (m, 8H),
1H NMR (400 MHz, CDCl3)
d
7.80 (d, J ¼ 8.4 Hz, 1H), 7.37e7.29 (m,
7.37e7.31 (m, 4H), 4.61 (s, 1H), 3.60 (s, 2H), 2.83e2.53 (m, 4H),
4H), 6.52 (d, J ¼ 8.4 Hz, 1H), 6.49e6.43 (m, 1H), 5.78 (s, 1H), 3.86 (s,
3H), 3.82 (s, 3H), 3.55 (s, 2H), 2.67e2.46 (m, 4H), 2.10e2.02 (m, 2H),
2.18e2.03 (m, 2H), 2.01e1.89 (m, 2H). 13C NMR (101 MHz, CDCl3)
d
155.1, 143.5, 140.0, 132.1, 129.2, 128.9, 128.3, 127.9, 127.3, 127.2,
1.92e1.82 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 162.7, 158.0, 154.4,
127.0, 126.8, 124.7, 95.9, 62.5, 50.4, 36.3. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C25H26N3O: 384.2076, found: 384.2072.
138.5, 130.6, 129.0, 128.2, 127.0, 107.1, 105.6, 98.8, 95.7, 62.7, 55.8,
55.5, 50.7, 36.2. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for
C
21H26N3O3: 368.1974, found: 368.1968.
4.2.1.17. 8-Benzyl-3-(20-methyl-[1,10-biphenyl]-4-yl)-1-oxa-2,4,8-
4.2.1.10. 8-Benzyl-3-(thiazol-2-yl)-1-oxa-2,4,8-triazaspiro[4.5]dec-
triazaspiro[4.5]dec-2-ene (A17). Yellow powder. Yield: 32.3%%. Mp:
2-ene (A10). Yellow powder. Yield: 23.3%. 1H NMR (400 MHz,
199e200 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.72 (d, J ¼ 8.4 Hz, 2H),
CDCl3)
5H), 5.38 (s, 1H), 3.56 (s, 2H), 2.71e2.43 (m, 4H), 2.17e2.03 (m, 2H),
2.00e1.88 (m, 2H). 13C NMR (101 MHz, CDCl3)
153.5, 151.6, 146.2,
d
7.87 (d, J ¼ 4.0 Hz,1H), 7.45 (d, J ¼ 4.0 Hz,1H), 7.41e7.27 (m,
7.40e7.27 (m, 7H), 7.26e7.12 (m, 4H), 4.64 (s, 1H), 3.55 (s, 2H),
2.79e2.50 (m, 4H), 2.24 (s, 3H), 2.10e2.03 (m, 2H), 1.97e1.86 (m,
d
2H). 13C NMR (101 MHz, CDCl3)
d 155.2, 144.5, 140.8, 138.3, 135.2,
143.2, 129.0, 128.3, 127.1, 121.7, 97.7, 62.6, 50.4, 36.4. HRMS (ESI-
TOFþ) m/z [MþH]þ calcd for C16H19N4OS: 315.1280, found:
315.1283.
130.4, 129.5, 129.5, 129.0, 128.3, 127.7, 127.1, 126.1, 125.9, 124.4, 96.1,
62.7, 50.6, 36.6, 20.4. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for
C26H28N3O: 398.2232, found: 398.2230.
4.2.1.11. 8-Benzyl-3-(thiophen-2-yl)-1-oxa-2,4,8-triazaspiro[4.5]dec-
4.2.1.18. 8-Benzyl-3-(30-methyl-[1,10-biphenyl]-4-yl)-1-oxa-2,4,8-
2-ene (A11). White solid. Yield: 10.4%. Mp: 193e194 ꢂC. 1H NMR
triazaspiro[4.5]dec-2-ene (A18). White powder. Yield: 38.7%. Mp:
186e187 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.74 (d, J ¼ 8.1 Hz, 2H), 7.62
(400 MHz, CDCl3)
d 7.57e7.24 (m, 7H), 7.06 (s, 1H), 4.65 (s, 1H), 3.56
(s, 2H), 2.75e2.45 (m, 4H), 2.19e1.98 (m, 2H), 1.97e1.81 (m, 2H). 13
C
(d, J ¼ 8.1 Hz, 2H), 7.45e7.27 (m, 8H), 7.20 (d, J ¼ 7.2 Hz, 1H), 4.58 (s,
NMR (101 MHz, CDCl3)
d 151.3, 138.2, 129.0, 128.3, 128.3, 127.8,
1H), 3.58 (s, 2H), 2.86e2.53 (m, 4H), 2.43 (s, 3H), 2.13e2.05 (m, 2H),
127.5, 127.4, 127.1, 96.4, 62.6, 50.5, 36.4. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C17H20N3OS: 314.1327, found: 314.1334.
1.97e1.86 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 155.1, 143.6, 140.0,
138.5, 138.3, 129.0, 128.8, 128.6, 128.3, 127.8, 127.3, 127.1, 126.7,
124.6, 124.2, 96.1, 62.7, 50.5, 36.6, 21.5. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C26H28N3O: 398.2232, found: 398.2232.
4.2.1.12. 8-Benzyl-3-(pyridin-2-yl)-1-oxa-2,4,8-triazaspiro[4.5]dec-
2-ene (A12). Yellow powder. Yield: 22.2%. Mp: 142e143 ꢂC. 1H NMR
(400 MHz, CDCl3)
d
8.56 (d, J ¼ 5.0 Hz, 1H), 7.95 (d, J ¼ 8.0 Hz, 1H),
4.2.1.19. 8-Benzyl-3-(30-isopropyl-[1,10-biphenyl]-4-yl)-1-oxa-2,4,8-
7.73 (t, J ¼ 7.8 Hz, 1H), 7.34e7.25 (m, 6H), 5.67 (s, 1H), 3.55 (s, 2H),
2.71e2.53 (m, 4H), 2.12e2.04 (m, 2H), 1.95e1.87 (m, 2H). 13C NMR
triazaspiro[4.5]dec-2-ene (A19). White powder. Yield: 74.8%. Mp:
148e149 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.75 (d, J ¼ 8.4 Hz, 2H),
(101 MHz, CDCl3)
d 155.4, 148.9, 145.2, 138.3, 136.6, 129.0, 128.2,
127.1, 124.9, 121.9, 96.8, 62.7, 50.5, 36.5. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C18H21N4O: 309.1715, found: 309.1714.
7.63 (d, J ¼ 8.4 Hz, 2H), 7.45 (s, 1H), 7.44e7.37 (m, 2H), 7.37e7.26 (m,
6H), 4.57 (s, 1H), 3.58 (s, 2H), 2.99 (m, J ¼ 6.9 Hz, 1H), 2.75e2.55 (m,
4H), 2.11e2.04 (m, 2H), 1.98e1.88 (m, 2H), 1.32 (s, 3H), 1.30 (s, 3H).
13C NMR (101 MHz, CDCl3)
d 155.1, 149.6, 143.8, 140.0, 138.2, 132.0,
4.2.1.13. 8-Benzyl-3-(6-bromopyridin-3-yl)-1-oxa-2,4,8-triazaspiro
129.0, 128.9, 128.3, 127.3, 127.1, 126.7, 126.0, 125.3, 124.6, 96.1, 62.6,
[4.5]dec-2-ene (A13). Yellow powder. Yield: 38.8%. Mp: 169e170 ꢂC.
50.5, 36.5, 34.2, 24.0. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for
1H NMR (400 MHz, CDCl3)
d
8.60 (s, 1H), 7.88 (d, J ¼ 8.0 Hz, 1H), 7.53
C28H32N3O: 426.2545, found: 426.2543.
(d, J ¼ 8.4 Hz, 1H), 7.41e7.27 (m, 5H), 4.55 (s, 1H), 3.56 (s, 2H),
2.70e2.52 (m, 4H), 2.09e2.02 (m, 2H), 1.94e1.88 (m, 2H). 13C NMR
(101 MHz, CDCl3)
d
152.4, 147.4, 144.1, 138.1, 136.0, 129.0, 128.3,
4.2.1.20. 8-Benzyl-3-(30-methoxy-[1,10-biphenyl]-4-yl)-1-oxa-2,4,8-
128.2, 127.1, 121.7, 97.1, 62.6, 50.3, 36.5. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C18H20BrN4O: 387.0820, found: 387.0817.
triazaspiro[4.5]dec-2-ene (A20). Yellow powder. Yield: 15.6%. 1H
NMR (400 MHz, CDCl3)
d
7.77e7.69 (m, 2H), 7.57 (dd, J ¼ 18.8,
8.4 Hz, 4H), 7.42e7.32 (m, 4H), 7.31e7.27 (m, 1H), 6.99 (d, J ¼ 8.4 Hz,
2H), 4.54 (s, 1H), 3.86 (s, 3H), 3.62 (s, 2H), 2.81e2.57 (m, 4H),
2.14e2.06 (m, 2H), 2.00e1.94 (m, 2H). 13C NMR (101 MHz, CDCl3)
4.2.1.14. 8-Benzyl-3-(isoquinolin-3-yl)-1-oxa-2,4,8-triazaspiro[4.5]
dec-2-ene (A14). Yellow powder. Yield: 26.2%. Mp: 140e141 ꢂC. 1H
NMR (400 MHz, CDCl3)
d
9.19 (s, 1H), 8.37 (s, 1H), 7.99 (d, J ¼ 8.0 Hz,
d 159.6, 155.2, 143.1, 132.4, 129.2, 128.3, 128.1, 127.4, 126.8, 126.7,
1H), 7.88 (d, J ¼ 8.0 Hz, 1H), 7.74 (t, J ¼ 7.6 Hz, 1H), 7.66 (t, J ¼ 8.0 Hz,
123.9, 119.5, 114.3, 95.7, 62.5, 55.3, 50.5, 36.3. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C26H28N3O2: 414.2182, found: 414.2180.
1H), 7.37e7.26 (m, 5H), 5.78 (s, 1H), 3.57 (s, 2H), 2.77e2.46 (m, 4H),

12
H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
4.2.1.21. 8-Benzyl-3-(30,50-dimethoxy-[1,10-biphenyl]-4-yl)-1-oxa-
7.59e7.53 (m, 4H), 7.42e7.28 (m, 5H), 7.14 (t, J ¼ 8.2 Hz, 2H), 4.62 (s,
1H), 3.58 (s, 2H), 2.78e2.49 (m, 4H), 2.12e2.03 (m, 2H), 1.96e1.88
2,4,8-triazaspiro[4.5]dec-2-ene (A21). Yellow powder. Yield: 20%.
Mp: 158e159 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.84e7.69 (m, 2H),
(m, 2H). 13C NMR (101 MHz, CDCl3)
d
162.8 (d, J ¼ 247.8 Hz),155.0,
7.66e7.56 (m, 2H), 7.44e7.27 (m, 5H), 6.72 (s, 2H), 6.49 (s, 1H), 4.52
(s, 1H), 3.85 (s, 6H), 3.57 (s, 2H), 2.79e2.47 (m, 4H), 2.16e2.01 (m,
142.4, 138.0, 132.0 (d, J ¼ 10.1 Hz), 129.1, 128.7, 128.6, 128.3, 127.1,
126.8, 124.7, 115.8 (d, J ¼ 21.5 Hz), 96.1, 62.6, 50.5, 36.5. HRMS (ESI-
TOFþ) m/z [MþH]þ calcd for C25H25FN3O: 402.1982, found:
402.1978.
2H), 1.98e1.83 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 161.1, 155.1,
143.4, 143.4, 142.2, 138.3, 129.0, 128.2, 127.3, 127.1, 126.7, 125.0,
105.4, 99.7, 96.2, 62.7, 55.4, 50.5, 36.6. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C27H30N3O3: 444.2287, found: 444.2290.
4.2.1.28. 8-Benzyl-3-(40-chloro-[1,10-biphenyl]-4-yl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-ene (A28). Yellow powder. Yield: 24.6%. Mp:
4.2.1.22. 4’-(8-Benzyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-en-3-yl)-
[1,10-biphenyl]-3-ol (A22). White powder. Yield: 96.2%. Mp:
228e229 ꢂC. 1H NMR (400 MHz, CDCl3)
7.59 (d, J ¼ 8.4 Hz, 2H), 7.53 (d, J ¼ 8.4 Hz, 2H), 7.42 (d, J ¼ 8.4 Hz,
2H), 7.34 (d, J ¼ 4.4 Hz, 5H), 4.50 (s, 1H), 3.58 (s, 2H), 2.74e2.54 (m,
4H), 2.13e2.04 (m, 2H), 1.98e1.88 (m, 2H). 13C NMR (101 MHz,
d
7.75 (d, J ¼ 8.4 Hz, 2H),
194e195 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.65 (d, J ¼ 7.8 Hz, 2H), 7.49
(d, J ¼ 7.8 Hz, 2H), 7.43e7.27 (m, 6H), 7.14e6.97 (m, 2H), 6.89 (d,
J ¼ 7.4 Hz, 1H), 4.88 (s, 1H), 3.68 (s, 2H), 3.09e2.39 (m, 4H),
CDCl3)
d 155.0, 142.2, 138.4, 138.1, 134.1, 129.1, 129.1, 128.3, 127.2,
2.22e1.84 (m, 4H). 13C NMR (101 MHz, Methanol-d4)
d
157.3, 156.1,
127.1, 126.8, 125.0, 96.1, 62.6, 50.5, 36.5. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C25H25ClN3O: 418.1686, found: 418.1685.
143.6, 141.3, 129.7, 129.7, 128.3, 127.7, 127.0, 126.6, 124.1, 118.2, 114.7,
113.6, 95.1, 62.2, 50.0, 35.1. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for
C
25H26N3O2: 400.2025, found: 400.2025.
4.2.1.29. 8-Benzyl-3-(40-methoxy-[1,10-biphenyl]-4-yl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-ene (A29). Yellow powder. Yield: 70.7%. Mp:
4.2.1.23. 4’-(8-Benzyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-en-3-yl)-
[1,10-biphenyl]-3-amine (A23). White powder. Yield: 30.1%. Mp:
230e231 ꢂC. 1H NMR (400 MHz, CDCl3)
7.57 (dd, J ¼ 17.6, 8.4 Hz, 4H), 7.39e7.26 (m, 5H), 6.99 (d, J ¼ 8.4 Hz,
2H), 4.54 (s, 1H), 3.86 (s, 3H), 3.57 (s, 2H), 2.78e2.48 (m, 4H),
2.14e2.02 (m, 2H), 1.97e1.86 (m, 2H). 13C NMR (101 MHz, CDCl3)
d
7.72 (d, J ¼ 8.4 Hz, 2H),
184e185 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.72 (d, J ¼ 8.0 Hz, 2H), 7.59
(d, J ¼ 8.0 Hz, 2H), 7.41e7.27 (m, 5H), 7.24e7.16 (m, 1H), 6.98 (d,
J ¼ 7.6 Hz, 1H), 6.90 (s, 1H), 6.70 (d, J ¼ 7.6 Hz, 1H), 4.57 (s, 1H), 3.77
(brs, 2H), 3.58 (s, 2H), 2.72e2.54 (m, 4H), 1.97e1.87 (m, 2H),
d
159.6, 155.2, 143.0, 138.2, 132.4, 129.1, 128.3, 128.1, 127.1, 126.7,
126.7, 124.0, 114.3, 96.0, 62.7, 55.3, 50.5, 36.5. HRMS (ESI-TOFþ) m/z
1.97e1.87 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 155.2, 146.8, 143.7,
[MþH]þ calcd for C26H28N3O2: 414.2182, found: 414.2177.
141.2, 138.1, 129.8, 129.1, 128.3, 127.2, 127.1, 126.6, 124.6, 117.5, 114.6,
113.6, 96.0, 62.6, 50.5, 36.5, 29.7. HRMS (ESI-TOFþ) m/z [MþH]þ
calcd for C25H27N4O: 399.2185, found: 399.2183.
4.2.1.30. 8-Benzyl-3-(4’-(trifluoromethyl)-1,2-dihydro-[1,10-
biphenyl]-4-yl)-1-oxa-2,4,8-triazaspiro
Yellow powder. Yield: 34.0%. Mp: 226e227 ꢂC. 1H NMR (400 MHz,
CDCl3)
[4.5]dec-2-ene
(A30).
4.2.1.24. 4’-(8-Benzyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-en-3-yl)-
N,N-dimethyl-[1,10-biphenyl]-3-amine (A24). Yellow powder. Yield:
d
7.79 (d, J ¼ 8.4 Hz, 2H), 7.70 (s, 4H), 7.64 (d, J ¼ 8.4 Hz, 2H),
7.40e7.27 (m, 5H), 4.51 (s, 1H), 3.57 (s, 2H), 2.73e2.48 (m, 4H),
45.5%. Mp: 174e175 ꢂC. 1H NMR (400 MHz, CDCl3)
d 7.74 (d,
2.13e2.03 (m, J ¼ 16.4 Hz, 2H), 1.98e1.87 (m, 2H). 13C NMR
J ¼ 8.3 Hz, 2H), 7.63 (d, J ¼ 8.3 Hz, 2H), 7.46e7.26 (m, 6H), 7.00e6.87
(101 MHz, CDCl3) d 154.9, 143.5, 141.9, 138.3, 129.0, 128.3, 127.5,
(m, 2H), 6.77 (dd, J ¼ 8.3, 2.0 Hz, 1H), 4.58 (s, 1H), 3.57 (s, 2H), 3.01
127.4, 127.1, 126.9, 125.8, 125.8, 125.7, 96.3, 62.7, 50.5, 36.6. HRMS
(ESI-TOFþ) m/z [MþH]þ calcd for C26H25F3N3O: 452.1950, found:
452.1947.
(s, 6H), 2.74e2.46 (m, 4H), 2.11e2.05 (m, 2H), 1.96e1.86 (m, 2H). 13
C
NMR (101 MHz, CDCl3)
d 155.2, 150.9, 144.5, 141.0, 138.3, 129.5,
129.1, 128.3, 127.5, 127.1, 126.6124.5, 115.5, 112.1, 111.2, 96.1, 62.7,
50.5, 40.6, 36.6. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C27H31N4O:
427.2484, found: 427.2491.
4.2.1.31. 8-Benzyl-3-(4-(thiophen-2-yl)phenyl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-ene (A31). Yellow powder. Yield: 33.3%. Mp:
215e216 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.71 (d, J ¼ 8.3 Hz, 2H), 7.62
4.2.1.25. 8-Benzyl-3-(3’-(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1-
oxa-2,4,8-triazaspiro[4.5]dec-2-ene (A25). Yellow powder. Yield:
(d, J ¼ 8.3 Hz, 2H), 7.51 (s, 1H), 7.40 (s, 2H), 7.44e7.24 (m, 5H), 4.56
(s,1H), 3.57 (s, 2H), 2.78e2.51 (m, 4H), 2.10e2.03 (m, 2H), 1.94e1.86
38.5%. Mp: 193e194 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.89e7.67 (m,
4H), 7.67e7.51 (m, 4H), 7.33 (d, J ¼ 23.2 Hz, 5H), 4.56 (s, 1H), 3.62 (s,
2H), 2.78e2.53 (m, 4H), 2.15e2.05 (m, 2H), 2.03e1.94 (m, 2H). 13
NMR (101 MHz, CDCl3)
(m, 2H). 13C NMR (101 MHz, CDCl3)
d 155.1, 141.2, 138.3,138.0, 129.0,
128.2, 127.1, 126.8, 126.6, 126.5, 126.0, 124.5, 121.2, 109.9, 96.1, 62.7,
50.5, 36.6. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C23H24N3OS:
390.1640, found: 390.1631.
C
d
154.9, 141.9, 140.8, 131.3 (q, J ¼ 32.3 Hz),
130.3, 129.4, 129.2, 128.4, 127.4, 126.9, 124.1 (q, J ¼ 284.8 Hz), 124.6,
124.6, 124.5, 123.8, 123.8, 96.0, 62.5, 50.4, 36.2. HRMS (ESI-TOFþ) m/
z [MþH]þ calcd for C26H25F3N3O: 452.1950, found: 452.1941.
4.2.1.32. 8-Benzyl-3-(4-(pyridin-3-yl)phenyl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-ene (A32). Yellow powder. Yield: 99%. Mp:
173e174 ꢂC. 1H NMR (400 MHz, CDCl3)
d 8.79 (s, 1H), 8.59 (d,
4.2.1.26. 8-Benzyl-3-(40-methyl-[1,10-biphenyl]-4-yl)-1-oxa-2,4,8-
J ¼ 4.4 Hz, 1H), 7.85 (d, J ¼ 8.0 Hz, 1H), 7.79 (d, J ¼ 8.2 Hz, 2H), 7.57
(d, J ¼ 8.1 Hz, 2H), 7.43e7.26 (m, 6H), 5.13 (s, 1H), 3.55 (s, 2H),
2.67e2.53 (m, 4H), 2.11e2.01 (m, 2H), 1.97e1.85 (m, 2H). 13C NMR
triazaspiro[4.5]dec-2-ene (A26). Yellow powder. Yield: 19.2%. Mp:
213e214 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.73 (d, J ¼ 8.2 Hz, 2H), 7.62
(d, J ¼ 8.0 Hz, 2H), 7.51 (d, J ¼ 7.8 Hz, 2H), 7.39e7.26 (m, 7H), 4.48 (s,
1H), 3.57 (s, 2H), 2.73e2.53 (m, 4H), 2.40 (s, 3H), 2.15e2.05 (m, 2H),
(101 MHz, CDCl3) d 155.0, 148.9, 148.1, 139.8, 138.3, 135.6, 134.3,
129.0, 128.2, 127.2, 127.1, 127.1, 125.8, 123.7, 96.3, 62.7, 50.5, 36.6.
HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C24H25N4O: 385.2028,
found: 385.2027.
1.96e1.87 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 155.1, 143.4, 138.2,
137.8, 137.1, 129.6, 129.1, 128.3, 127.1, 127.0, 126.9, 126.7, 96.0, 62.7,
50.5, 36.5, 21.1. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C26H28N3O:
398.2232, found: 398.2225.
4.2.1.33. 8-Benzyl-3-(4-(pyridin-4-yl)phenyl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-ene (A33). Yellow powder. Yield: 86%. Mp:
4.2.1.27. 8-Benzyl-3-(40-fluoro-[1,10-biphenyl]-4-yl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-ene (A27). Yellow powder. Yield: 33.3%. Mp:
211e212 ꢂC. 1H NMR (400 MHz, CDCl3)
d
8.65 (d, J ¼ 5.8 Hz, 2H), 7.80
(d, J ¼ 8.3 Hz, 2H), 7.64 (d, J ¼ 8.3 Hz, 2H), 7.48 (d, J ¼ 6.0 Hz, 2H),
203e204 ꢂC. 1H NMR (400 MHz, CDCl3)
d 7.81e7.71 (m, 2H),
7.32 (d, J ¼ 4.5 Hz, 5H), 5.05 (s, 1H), 3.55 (s, 2H), 2.73e2.43 (m, 4H),

H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
13
2.14e1.99 (m, 2H), 1.97e1.83 (m, 2H). 13C NMR (101 MHz, CDCl3)
chromatography eluting with PE/EtOAc/TEA (20/10/1) to afford
d
154.9, 150.3, 147.2, 140.1, 138.3, 129.0, 128.2, 127.2, 127.1126.7,
B13eB15.
121.6, 121.5, 96.5, 62.7, 50.5, 36.6. HRMS (ESI-TOFþ) m/z [MþH]þ
calcd for C24H25N4O: 385.2028, found: 385.2022.
4.2.2.1. tert-Butyl 3-phenyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-ene-8-
carboxylate (B1). White powder. Yield: 87.8%. 1H NMR (400 MHz,
4.2.1.34. 8-Benzyl-3-(4-(2-methoxypyridin-4-yl)phenyl)-1-oxa-
CDCl3)
d
7.65 (d, J ¼ 7.6 Hz, 2H), 7.49e7.39 (m, 1H), 7.38e7.28 (m,
2,4,8-triazaspiro[4.5]dec-2-ene (A34). White powder. Yield: 37.5%.
2H), 5.11 (s, 1H), 3.97e3.68 (m, 2H), 3.41e3.16 (m, 2H), 2.07e1.86
Mp: 198e199 ꢂC. 1H NMR (400 MHz, CDCl3)
d
8.23 (d, J ¼ 5.2 Hz,
(m, 2H), 1.85e1.68 (m, 2H), 1.44 (s, 9H). 13C NMR (101 MHz, CDCl3)
1H), 7.78 (d, J ¼ 8.4 Hz, 2H), 7.65 (d, J ¼ 8.4 Hz, 2H), 7.39e7.27 (m,
5H), 7.10 (d, J ¼ 5.6 Hz, 1H), 6.95 (s, 1H), 4.45 (s, 1H), 3.99 (s, 3H),
3.58 (s, 2H), 2.72e2.55 (m, 4H), 2.12e2.05 (m, 2H), 1.95e1.85 (m,
d 155.5, 154.6, 130.8, 128.6, 126.4, 125.6, 95.8, 79.8, 77.2, 41.2, 36.3,
28.4. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C17H24N3O3: 318.1818,
found: 318.1817.
2H). 13C NMR (101 MHz, CDCl3)
d 164.9, 154.8, 149.9, 147.4, 140.4,
138.0, 129.0, 128.3, 127.2, 127.1, 126.9, 115.1, 108.5, 96.3, 62.6, 53.6,
50.5, 36.6,14.1. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C25H27N4O2:
415.2134, found: 415.2130.
4.2.2.2. Ethyl 3-phenyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-ene-8-
carboxylate (B2). White powder. Yield: 44.1%. mp: 184e185 ꢂC. 1H
NMR (400 MHz, CDCl3)
d
7.68 (d, J ¼ 7.8 Hz, 2H), 7.53e7.34 (m, 3H),
4.55 (brs, 1H), 4.15 (q, J ¼ 7.2 Hz, 2H), 4.01e3.82 (m, 2H), 3.39 (t,
4.2.1.35. 8-Benzyl-3-(6-(3-methoxyphenyl)pyridin-3-yl)-1-oxa-
J ¼ 11.6 Hz, 2H), 2.17e1.97 (m, 2H), 1.87e1.68 (m, 2H), 1.27 (t,
2,4,8-triazaspiro[4.5]dec-2-ene (A35). White powder. Yield: 97.5%.
J ¼ 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 155.4, 130.9, 128.7,
Mp: 148e149 ꢂC. 1H NMR (400 MHz, CDCl3)
d
8.92 (s, 1H), 8.05 (s,
126.3, 125.6, 95.6, 61.5, 41.0, 36.3, 14.6. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C15H20N3O3: 290.1505, found: 290.1507.
1H), 7.76 (s, 1H), 7.70e7.29 (m, 8H), 6.99 (s, 1H), 4.69 (s, 1H), 3.89 (s,
3H), 3.57 (s, 2H), 2.91e2.41 (m, 4H), 2.15e2.02 (m, 2H), 1.97e1.89
(m, 2H). 13C NMR (101 MHz, CDCl3)
d
160.1, 158.9, 153.2, 147.1, 139.7,
4.2.2.3. Ethyl 3-(4-bromophenyl)-1-oxa-2,4,8-triazaspiro[4.5]dec-2-
138.2, 134.5, 129.9, 129.1, 128.3, 127.1, 120.6, 120.3, 119.4, 115.7, 112.1,
ene-8-carboxylate (B3). White powder. Yield: 87.8% yield. Mp:
96.6, 62.6, 55.4, 50.4, 36.5. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for
214e215 ꢂC. 1H NMR (400 MHz, CDCl3)
d 7.55 (s, 4H), 4.44 (s, 1H),
C
25H27N4O2: 415.2134, found: 415.2129.
4.15 (q, J ¼ 7.0 Hz, 2H), 3.99e3.86 (m, 2H), 3.39 (t, J ¼ 12.8 Hz, 2H),
2.09e1.98 (d, J ¼ 12.8 Hz, 2H), 1.83e1.72 (m, 2H), 1.27 (t, J ¼ 7.2 Hz,
4.2.1.36. 3-(5-(8-Benzyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-en-3-yl)
pyridin-2-yl)aniline (A36). White powder. Yield: 32.3%.
Mp:173e174 ꢂC. 1H NMR (400 MHz, CDCl3)
8.91 (s, 1H), 8.03 (d,
H). 13C NMR (101 MHz, CDCl3)
d 155.3, 154.7, 132.0, 127.8, 125.2,
124.6, 96.0, 61.5, 41.0, 36.3, 14.6. HRMS (ESI-TOFþ) m/z [MþH]þ
d
calcd for C15H19BrN3O3: 368.0611, found: 368.0611.
J ¼ 10.6 Hz, 1H), 7.73 (d, J ¼ 8.3 Hz, 1H), 7.40 (s, 1H), 7.36e7.26 (m,
7H), 6.77 (d, J ¼ 7.1 Hz, 1H), 4.55 (s, 1H), 3.80 (s, 2H), 3.57 (s, 2H),
2.82e2.43 (m, 4H), 2.14e2.00 (m, 2H), 1.98e1.86 (m, 2H). 13C NMR
4.2.2.4. 3-Phenyl-8-(pyridin-2-ylmethyl)-1-oxa-2,4,8-triazaspiro
[4.5]dec-2-ene (B4). Yellow powder. Yield: 49.3%. 1H NMR
(101 MHz, CDCl3)
d
159.3, 153.2, 147.0, 147.0,139.4, 138.3, 134.4,
(400 MHz, CDCl3)
d
8.67 (d, J ¼ 4.8 Hz, 1H), 8.03 (t, J ¼ 7.8 Hz, 1H),
129.8, 129.0, 128.3, 127.1, 120.4, 120.2, 117.3, 116.4, 113.5, 96.6, 62.6,
50.5, 36.6. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C24H26N5O:
400.2137, found: 400.2132.
7.70e7.62 (m, 2H), 7.62e7.57 (m, 1H), 7.50 (d, J ¼ 7.8 Hz, 1H),
7.47e7.36 (m, 3H), 5.17 (s, 1H), 4.05 (s, 2H), 3.36e3.23 (m, 2H), 2.99
e 2-82 (m, 2H), 2.66e2.49 (m, 2H), 2.23e2.17 (m, 2H). 13C NMR
(101 MHz, CDCl3)
d 158.5, 155.4, 149.3, 136.4, 130.6, 128.6, 126.3,
4.2.2. General procedure for the synthesis of compounds B1eB15
A mixture of piperidin-4-one (104 mg, 1.05 mmol), 3a, 3b
(1 mmol) and Na2CO3 (318 mg, 3 mmol) in CH3CN (3 mL) was
stirred at room temperature for 45 min, followed by stirred at 50 ꢂC
for 45 min, then heated to 70 ꢂC for an additional 2e10 h. After
completion, the solvent was evaporated under reduced pressure.
The residue was dissolved in EtOAc and washed with water three
times, dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure. The crude product was purfied by silica gel
column chromatography eluting with DCM/MeOH (100/1) to afford
4a, 4b.
TsOHꢁH2O (37.5 mg, 0.2 mmol) was added to a solution of 2a, 2f,
2i, 2l or 2m (1 mmol) and 4a, 4b or commercially available piper-
idones 4c-4e (1.5 mmol) in toluene (5 mL). The mixture was
refluxed overnight. After completion, the mixture was extracted
with EtOAc three times. The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure. The crude product was purified by silica
gel column chromatography eluting with DCM/MeOH/NH3ꢁH2O
(100/3/1) to afford B1eB12.
125.9, 123.1, 122.0, 95.9, 64.2, 50.8, 36.5. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C18H21N4O: 309.1715, found: 309.1717.
4.2.2.5. 8-(Pyridin-2-ylmethyl)-3-(thiazol-2-yl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-ene (B5). Yellow powder. Yield: 30.1%. Mp:
148e149 ꢂC. 1H NMR (400 MHz, CDCl3)
7.84 (d, J ¼ 2.8 Hz, 1H), 7.64 (t, J ¼ 7.2 Hz, 1H), 7.51e7.31 (m, 2H),
7.23e7.01 (m, 1H), 5.53 (s, 1H), 3.68 (s, 2H), 2.91e2.53 (m, 4H),
2.21e2.00 (m, 2H), 2.00e1.85 (m, 2H). 13C NMR (101 MHz, CDCl3)
d
8.55 (d, J ¼ 3.6 Hz, 1H),
d
158.4, 153.5, 151.6, 149.3, 143.1, 136.4, 123.1, 122.0, 121.7, 97.6, 64.1,
50.6, 36.4. HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C15H18N5OS:
316.1232, found: 316.1234.
4.2.2.6. 3-(Isoquinolin-3-yl)-8-(pyridin-2-ylmethyl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-ene (B6). Yellow powder. Yield: 42.1%. Mp:
184e185 ꢂC. 1H NMR (400 MHz, CDCl3)
d 9.17 (s, 1H), 8.57 (d,
J ¼ 4.2 Hz,1H), 8.35 (s,1H), 7.97 (d, J ¼ 8.2 Hz,1H), 7.86 (d, J ¼ 8.2 Hz,
1H), 7.72 (t, J ¼ 7.2 Hz, 1H), 7.64 (t, J ¼ 7.8 Hz, 2H), 7.39 (d, J ¼ 7.8 Hz,
1H), 7.18e7.10 (m, 1H), 5.80 (s, 1H), 3.71 (s, 2H), 2.75e2.64 (m, 4H),
2.17e2.08 (m, 2H), 2.03e1.97 (m, 2H). 13C NMR (101 MHz, CDCl3)
Pd(PPh3)4 (113 mg, 0.1 mmol) was added to a degassed mixture
of B10 or B12 (1 mmol), substituted boric acids (2 mmol), K2CO3
(483 mg, 3.5 mmol) in dioxane (4 mL) and water (2 mL) under
argon. The mixture was heated at 80e120 ꢂC for 6e20 h, cooled to
room temperature and extracted with EtOAc three times. The
combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated under reduced pres-
sure. The crude product was purified by silica gel column
d 158.6, 155.8, 151.9, 149.3, 138.5, 136.4, 135.6, 131.1, 129.0, 128.5,
127.6, 127.3, 123.1, 122.0, 119.1, 96.4, 64.2, 50.8, 36.5. HRMS (ESI-
TOFþ) m/z [MþH]þ calcd for C21H22N5O: 360.1824, found: 360.1821.
4.2.2.7. 8-(Pyridin-4-ylmethyl)-3-(thiazol-2-yl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-ene (B7). Yellow powder. Yield: 19.7%. Mp:
231e232 ꢂC. 1H NMR (400 MHz, CDCl3)
7.87 (d, J ¼ 3.2 Hz, 1H), 7.46 (d, J ¼ 3.2 Hz, 1H), 7.28 (d, J ¼ 2.8 Hz,
d
8.55 (d, J ¼ 5.6 Hz, 2H),

14
H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
2H), 5.40 (s, 1H), 3.55 (s, 2H), 2.71e2.53 (m, 4H), 2.15e2.04 (m, 2H),
2.70e2.56 (m, 4H), 2.37 (s, 3H), 2.13e2.03 (m, 2H), 1.99e1.92 (m,
2H). 13C NMR (101 MHz, CDCl3)
155.3, 146.9, 143.7, 141.1, 129.8,
127.2, 126.7, 124.5, 117.4, 114.7, 113.6, 95.3, 52.7, 45.8, 36.3. HRMS
(ESI-TOFþ) m/z [MþH]þ calcd for C19H23N4O: 323.1872, found:
323.1869.
2.01e1.88 (m, 2H). 13C NMR (101 MHz, CDCl3)
d
153.4, 151.6, 149.8,
d
147.6, 143.2, 123.6, 121.8, 97.4, 61.3, 50.5, 36.4. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C15H18N5OS: 316.1232, found: 316.1231.
4.2.2.8. 3-(Isoquinolin-3-yl)-8-(pyridin-4-ylmethyl)-1-oxa-2,4,8-
triazaspiro[4.5]dec-2-en (B8). Yellow powder. Yield: 50.1%. Mp:
4.2.2.15. 3-(4-(2-Methoxypyridin-4-yl)phenyl)-8-methyl-1-oxa-
157e158 ꢂC. 1H NMR (400 MHz, CDCl3)
d
9.20 (s, 1H), 8.55 (d,
2,4,8-triazaspiro[4.5]dec-2-ene (B15). Yellow powder. Yield: 50.0%.
J ¼ 5.6 Hz, 2H), 8.37 (s, 1H), 8.00 (d, J ¼ 8.0 Hz, 1H), 7.90 (d,
J ¼ 8.2 Hz, 1H), 7.75 (t, J ¼ 8.0 Hz, 1H), 7.67 (t, J ¼ 7.6 Hz, 1H), 7.30 (d,
J ¼ 5.8 Hz, 2H), 5.76 (s, 1H), 3.57 (s, 2H), 2.69e2.57 (m, 4H),
2.17e2.12 (m, 2H), 2.00e1.88 (m, 2H). 13C NMR (101 MHz, CDCl3)
Mp: 225e226 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
1H), 7.89 (d, J ¼ 8.4 Hz, 2H), 7.80 (d, J ¼ 8.4 Hz, 2H), 7.72 (s, 1H), 7.35
(d, J ¼ 6.6 Hz, 1H), 7.16 (s, 1H), 3.90 (s, 3H), 2.97e2.79 (m, 2H),
2.75e2.55 (m, 2H), 2.41 (s, 3H), 2.04e1.83 (m, 4H). 13C NMR
d
8.25 (d, J ¼ 5.4 Hz,
d
155.9, 151.9, 149.8, 147.8, 138.4, 135.6, 131.1, 129.1, 128.5, 127.6,
(101 MHz, DMSO‑d6) d 164.9, 154.9, 149.6, 148.0, 139.2, 127.5, 127.2,
127.4, 123.7119.1, 96.2, 61.4, 50.7, 36.5. HRMS (ESI-TOFþ) m/z
126.9, 115.4, 108.0, 95.0, 55.3, 53.7, 52.0, 35.3. HRMS (ESI-TOFþ) m/z
[MþH]þ calcd for C19H23N4O2: 339.1820, found: 339.1821.
[MþH]þ calcd for C21H22N5O: 360.1824, found: 360.1826.
4.2.2.9. 8-Methyl-3-phenyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-ene
4.3. TEVC recording in xenopus oocytes
(B9). Yellow powder. Yield: 39.0%. Mp: 177e178 ꢂC. 1H NMR
(400 MHz, CDCl3)
d
7.68 (d, J ¼ 6.8 Hz, 2H), 7.52e7.34 (m, 3H), 4.55
Similar to our previously reported protocol [39,40], oocytes
were harvested from Xenopus laevis female clawed frogs after
anesthesia, and washed twice in the Ca2þ-free OR2 solution
(82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.4)
before transferred to ~25-mL tubes. Oocytes were then treated with
2 mg/mL collagenase in OR2 solution (Sigma type II, Sigma-Aldrich
Inc, St Louis, MO, USA) for 20 min at room temperature under
gentle rotation. The stage V and VI oocytes were selected for mi-
croinjections. For TEVC recordings in oocytes, capped cRNAs were
transcribed in vitro using the T3 mMESSAGEmMACHINE Kit
(Ambion, Austin, TX, USA) following the linearization of plasmids in
pBluescript KSM vectors. The oocytes were injected with 46 nL of
(s, 1H), 2.79e2.56 (m, 4H), 2.37 (s, 3H), 2.14e2.08 (m, 2H),
2.02e1.93 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 155.3, 130.8, 128.7,
126.3, 125.8, 95.2, 52.7, 45.7, 36.3. HRMS (ESI-TOFþ) m/z [MþH]þ
calcd for C13H18N3O: 232.1450, found: 232.1447.
4.2.2.10. 3-(4-Bromophenyl)-8-methyl-1-oxa-2,4,8-triazaspiro[4.5]
dec-2-ene (B10). Yellow powder. Yield: 53.3%. mp: 230e231 ꢂC. 1H
NMR (400 MHz, CDCl3)
d 7.65e7.35 (m, 4H), 5.13 (s, 1H), 2.92e2.57
(m, 4H), 2.41 (s, 3H), 2.14e1.96 (m, 4H). 13C NMR (101 MHz, CDCl3)
d
154.8, 131.8, 127.9, 125.0, 124.7, 95.2, 52.6, 45.5, 35.8. HRMS (ESI-
TOFþ) m/z [MþH]þ calcd for C13H17BrN3O: 310.0555, found:
310.0554.
cRNA solution containing approximately 20 ng human
cRNA using a micro-injector (Drummond Scientific, Broomall, PA,
USA). For the expression of heteromeric rat 4 and rat a4b2
a7 nAChR
4.2.2.11. 8-Methyl-3-(thiazol-2-yl)-1-oxa-2,4,8-triazaspiro[4.5]dec-
a3b
2-ene (B11). Yellow powder. Yield: 37.1%. Mp: 182e183 ꢂC. 1H NMR
nAChRs, approximately total of 2 ng cRNAs were injected in a 1:1
combination of each subunit into oocytes that were incubated at
16 ꢂC in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM
MgCl2, 5 mM 4-(2-hydroxyethyl)1-piperazineethanesulphonic acid
(HEPES), pH 7.4 adjusted with NaOH). Recordings were made
24e72 h post-injections. Oocytes were impaled with two micro-
(400 MHz, CDCl3)
d 7.86 (s, 1H), 7.46 (s, 1H), 5.52 (s, 1H), 3.03e2.48
(m, 4H), 2.35 (s, 3H), 2.16e2.04 (m, 2H), 2.04e1.92 (m, 2H). 13C NMR
(101 MHz, CDCl3)
d 153.3, 151.6, 143.2, 121.8, 96.9, 52.6, 45.6, 36.1.
HRMS (ESI-TOFþ) m/z [MþH]þ calcd for C10H15N4OS: 239.0967,
found: 239.0967.
electrodes (0.5e1.0 MU) filled with 3 M KCl in a 40-mL recording
4.2.2.12. 3-(6-Bromopyridin-3-yl)-8-methyl-1-oxa-2,4,8-triazaspiro
chamber. The membrane potential was held at ꢀ90 mV using
standard voltage clamp procedures. Currents were recorded in
Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1.8 mM
CaCl2, 1 mM MgCl2, 0.0005 mM atropine) at room temperature
(22 1 ꢂC) using a GeneClamp 500B amplifier (Axon Instruments,
Union City, CA, USA).
[4.5]dec-2-ene (B12). White powder. Yield: 17.2%. mp: 176e177 ꢂC.
1H NMR (400 MHz, DMSO‑d6)
d
8.66 (s, 1H), 7.98 (d, J ¼ 8.4 Hz, 1H),
7.78 (d, J ¼ 8.4 Hz, 2H), 2.73e2.57 (m, 2H), 2.48e2.36 (m, 2H), 2.27
(s, 3H), 1.89e1.76 (m, 4H). 13C NMR (101 MHz, DMSO‑d6)
152.6,
d
148.0, 143.3, 137.0, 128.7, 122.3, 96.2, 52.3, 45.6, 36.0. HRMS (ESI-
TOFþ) m/z [MþH]þ calcd for C12H16N4OBr: 311.0507, found:
311.0508.
4.4. Data analysis
4.2.2.13. 3-(30-methoxy-[1,10-biphenyl]-4-yl)-8-methyl-1-oxa-2,4,8-
All data were expressed as the means SEM. Statistical signif-
icance was assessed by Student’s t-test and one-way ANOVA using
the Prism version 5.0 software. P < 0.05 was considered statistically
significant. In the TEVC recordings, currents were quantified by
measuring peak current amplitudes and analyzed using Patch-
Master and Origin 9.0 software. The concentrationꢀresponse
curves were fitted to the Hill equation as follows:
Inormalized ¼ 1e1/(1 þ (IC50/C)nH).
triazaspiro[4.5]dec-2-ene (B13). Yellow powder. Yield: 66.7% yield.
Mp: 225e226 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 7.83e7.70 (m, 4H),
7.53 (s, 1H), 7.43e7.36 (m, 1H), 7.32e7.26 (m, 1H), 7.26e7.22 (m,
1H), 6.97 (dd, J ¼ 8.2, 2.4 Hz, 1H), 3.83 (s, 3H), 2.78e2.59 (m, 2H),
2.47e2.32 (m, 2H), 2.29 (s, 3H), 1.92e1.76 (m, 4H). 13C NMR
(101 MHz, DMSO‑d6)
d 160.2, 155.0, 142.2, 141.0, 130.5, 127.3, 127.1,
125.4, 119.4, 114.0, 112.6, 95.3, 57.7, 55.6, 52.4, 36.0. HRMS (ESI-
TOFþ) m/z [MþH]þ calcd for C20H24N3O2: 338.1869, found:
338.1868.
4.5. Virtual screening
4.2.2.14. 4’-(8-methyl-1-oxa-2,4,8-triazaspiro[4.5]dec-2-en-3-yl)-
[1,10-biphenyl]-3-amine (B14). Brown solid. Yield: 77.8%. Mp:
4.5.1. Preparation of compound library
In brief, ChemDiv database was prepared by a homemade pro-
tocol in Pipeline Pilot v7.5 (PP 7.5, Accelrys, Inc.), in which the
molecules formed 3D coordinates. The prepared database was
filtered by a basic standard in FILTER (OpenEye Scientific Software,
225e226 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.71 (d, J ¼ 8.2 Hz, 2H),
7.58 (d, J ¼ 8.2 Hz, 2H), 7.22 (t, J ¼ 7.8 Hz,1H), 6.97 (d, J ¼ 7.6 Hz,1H),
6.89 (s, 1H), 6.70 (d, J ¼ 7.8 Hz, 1H), 4.76 (s, 1H), 3.74 (brs, 2H),

H. Zhang et al. / European Journal of Medicinal Chemistry 207 (2020) 112774
15
Inc) to eliminate molecules with unsatisfied properties according to
“rule of five”. Subsequently, the database was prepared with
OMEGA (OpenEye Scientific Software, Inc) to generate up to 500
conformations for each molecule.
Foundation of China (81373272, 21772005, 81872730) and the
Beijing Natural Science Foundation (7202088, 7172118). We are
very appreciative of Dr. Wei Huang (Chinese Academy of Medical
Sciences, Beijing) for their help in this work.
4.5.2. Pharmacophore-based search
For the pharmacophore generation, 3D structures of training set
(11 selected compounds) were generated and energy minimized by
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112774.
€
€
using LigPrep module of Schrodinger Suite (Schrodinger, LLC, New
York, NY, USA). Optimized conformations of training set were
submitted to the common feature pharmacophore generation
procedure in Discovery Studio 2.5 (DS 2.5, Accelrys, Inc.). The
HipHop module in Catalyst was used to identify and overlay com-
mon features shared by the training set. The MaxOmitFeat of all
compounds in training set were set to 1, and the Principal of
compound nicotine and Epibatidine were set to 0, compound TC-
1698 and 7aa were set to 1, other compounds were set to 2.
Hydrogen bonding acceptor, Hydrogen bonding doner, POS Charge,
PosIonizable and Hydrophobic, were selected as pharmocophoric
features based on the chemical features present in the training set.
The Minimum features were set to 3, and the Minimum inter-
feature distance was set to 1. According to the matching of the
training set compounds, we selected the Hip06 model from the
total 10 pharmacophores developed, which contained 1 hydrogen-
bond receptor, 1 hydrophobic center and 1 positively ionized group.
The HipHop model was used to search prepared ChemDiv database.
The results were ranked by ‘Fit Value’ score and the top 10% com-
pounds were retained.
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