Design, Synthesis and Biological Investigation of Flavone Derivatives as Potential Multi-Receptor Atypical Antipsychotics

Article abstract of DOI:10.3390/molecules25184107

The design of a series of novel flavone derivatives was synthesized as potential broad-spectrum antipsychotics by using multi-receptor affinity strategy between dopamine receptors and serotonin receptors. Among them, 7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl) piperidin- 1-yl) butoxy)-2,2-dimethylchroman-4-one (6j) exhibited a promising preclinical profile. Compound 6j not only showed high affinity for dopamine D₂, D₃, and serotonin 5-HT_1A, 5-HT_2A receptors, but was also endowed with low to moderate activities on 5-HT_2C, α₁, and H₁ receptors, indicating a low liability to induce side effects such as weight gain, orthostatic hypotension and QT prolongation. In vivo behavioral studies suggested that 6j has favorable effects in alleviating the schizophrenia-like symptoms without causing catalepsy. Taken together, compound 6j has the potential to be further developed as a novel atypical antipsychotic.

Full text of DOI:10.3390/molecules25184107

molecules
Article
Design, Synthesis and Biological Investigation of
Flavone Derivatives as Potential Multi-Receptor
Atypical Antipsychotics
Lanchang Gao 1,†, Zhengge Yang 1,†, Jiaying Xiong 1, Chao Hao 1, Ru Ma 2, Xin Liu 1,
Bi-Feng Liu 1, Jian Jin 2, Guisen Zhang 1,2,* and Yin Chen 2,
*
1
Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of
Science and Technology, Wuhan 430074, China; D201577449@hust.edu.cn (L.G.); charles@long-ou.com (Z.Y.);
bear@hust.edu.cn (J.X.); d201880507@hust.edu.cn (C.H.); xliu@mail.hust.edu.cn (X.L.);
bfliu@mail.hust.edu.cn (B.-F.L.)
Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine
Pharmaceutical Compound Screening, School of Pharmacy, Jiangsu Ocean University, Lianyungang 222005,
China; 12111030011@fudan.edu.cn (R.M.); 2019000016@jou.edu.cn (J.J.)
2
*
Correspondence: gszhang@mail.hust.edu.cn (G.Z.); 2019000015@jou.edu.cn (Y.C.);
Tel.: +86-27-8779-2235 (G.Z.); +86-0518-8589-5791 (Y.C.); Fax: +86-27-8779-2170 (G.Z.)
These authors contributed equally to this work.
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 August 2020; Accepted: 28 August 2020; Published: 8 September 2020
Abstract: The design of a series of novel flavone derivatives was synthesized as potential
broad-spectrum antipsychotics by using multi-receptor affinity strategy between dopamine receptors
and serotonin receptors. Among them, 7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl) piperidin- 1-yl)
butoxy)-2,2-dimethylchroman-4-one (6j) exhibited a promising preclinical profile. Compound
6j not only showed high affinity for dopamine D2, D3, and serotonin 5-HT1A, 5-HT2A receptors, but
was also endowed with low to moderate activities on 5-HT2C, α1, and H1 receptors, indicating a low
liability to induce side effects such as weight gain, orthostatic hypotension and QT prolongation.
In vivo behavioral studies suggested that 6j has favorable effects in alleviating the schizophrenia-like
symptoms without causing catalepsy. Taken together, compound 6j has the potential to be further
developed as a novel atypical antipsychotic.
Keywords: dopamine; serotonin; multi-target; atypical antipsychotics
1. Introduction
Schizophrenia is a severe neuropsychiatric disorder that affects over 1% of the world’s
population [
1]. The clinical symptoms of schizophrenia are categorized into three general types,
including positive, negative, and cognitive symptoms [2]. Currently, antipsychotics are the mainstay
in schizophrenia treatment. For example, the first-generation antipsychotics (FGAs), namely typical
antipsychotics including chlorpromazine and haloperidol, are all dopamine D2 receptor antagonists
and effective for the treatment of positive symptoms, but invalid for the negative symptoms and
cognitive impairment [
effects such as extrapyramidal symptoms (EPS), hyperprolactinemia, and cognitive impairments [
The second-generation antipsychotics (SGAs), also known as atypical antipsychotics, including
clozapine [ ], and risperidone [ ], have antagonistic action on both dopamine and serotonin receptors,
especially with high affinity for 5-HT1A and 5-HT2A receptors, which contributes to various kinds
of therapeutic advantages, such as fewer EPSs than FGAs [ ]. However, the SGAs also produce
3
,4]. However, the strong antagonism of D2 receptor results in serious side
5].
6
7
8
many adverse metabolic and cardiovascular effects [
9], such as weight gain, hyperprolactinemia,
Molecules 2020, 25, 4107; doi:10.3390/molecules25184107
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4107
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constipation, QT prolongation, and also show limited effects on negative symptoms and cognitive
impairments [10 11]. Fortunately, these defects could be avoided by developing a multi-receptor ligand
,
with a “selective” multi-receptor profile which can precisely modulates several specific targets to get a
better effect and safety in the therapy of schizophrenia [12,13].
To verify this multi-receptor affinity strategy, we have previously developed several novel potential
antipsychotics, such as compounds
1 and 2 shown in Figure 1, both of which could significantly
alleviate the positive symptoms of schizophrenia, are associated with lower weight gain and lower
prolactin levels, and have obvious therapeutic effects. In order to obtain versatile molecules with better
therapeutic effects and less side effects, in this paper, a series of new compounds was designed and
synthesized by means of a polypharmacological strategy and molecular hybridization method on the
basis of our previous research [14,15]. The design concept of new compounds as shown in Figure 1,
where the general structure contains a flavone-like moiety, a heterocyclic or arylpiperazine (piperidine)
moiety and a flexible linker. By optimizing the combination of the benzocaprolactam fragment of
brexpiprazole and the coumarin derivative scaffolds of compounds 1 and 2, they were transformed into
a flavone-like fragment. The choice of heterocyclic or arylpiperazine (piperidine) originates from the
commercial atypical antipsychotics [16], such as risperidone, brexpiprazole, aripiprazole. The linker
flexibility of the new compounds also be evaluated as our previous SAR studies have proved it to play
an important role in modulating the receptor function profiles [14,15,17].
Ar:
N
N
N
N
N
N
N
N
R
N
N
N
O
N
N
N
N
N
O
S
N
S
N
N
N
O
N
O
CH3
F
N
O
O
O
F
F
N
N
N
O
N
O
O
O
Ar
Hybridization
X
Risperidone
1
2
N
O
S
Cl
Base moiety
Fragment of possible modifications
O
O
O
F
N
N
N
O
N
O
H
N
O
Brexpiprazole
Q:
O
O
O
O
R3
R5
R2
R1
R4
O
N
Q
F
Linker
O
N
O
Q2
Q1
Flavones-like moiety
Figure 1.
Design of new flavone-linker-based heterocyclic arylpiperazine(piperidine) and
arylpiperazine(piperidine) derivatives.
Among these derivatives, compound 6j [7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl) iperidin-1-yl)
butoxy)-2,2-dimethylchroman-4-one] exhibited a favorable polypharmacological antipsychotic profile.
In vitro, it showed much higher potency for the desired targets (D2, 5-HT1A, 5-HT2A, and D3) than
other off-target receptors (5-HT2C, α1, and H1). Further in vivo behavioral studies suggested that it
has favorable effects in alleviating schizophrenia-like symptoms without causing catalepsy and a low
liability to induce side-effects. Thus, compound 6j has the potential to be developed as a novel atypical
antipsychotic candidate to treat schizophrenia.

Molecules 2020, 25, 4107
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2. Results and Discussion
2.1. Chemistry
New compounds were synthesized as shown in Schemes 1–3.
In Scheme 1, 2,4-
dihydroxyacetophenone (
presence of pyrrolidine and acetonitrile, and the intermediates 7a and 7b were prepared using a
similar process. Compounds 5a 5b and 8a 8d were obtained through alkylation of and 7a 7b with
1-bromo-3-chloropropane or 1-bromo-4-chlorobutane. Compounds 6a 6j and 9a 9k were obtained by
coupling 5a 5b and 8a 8d with different arylpiperazines or heterocyclic arylpiperidines in the presence
of K2CO3 in acetonitrile. As shown in Scheme 2, the new compounds 11a 11e and 14a 14b were
synthesized following similar approaches to those described in Scheme 1, that is treatment of resorcinol
and its derivatives 10a 10e with trifluoromethanesulfonic acid in the presence of 3-chloropropionic
acid, and then treatment with sodium hydroxide solution to afford 11a 11e. Compounds 14a and
14b were synthesized starting from resorcinol and its derivatives by treatment with crotonic acid
in the presence of anhydrous zinc chloride. The preparation of 12a 12e and 15a 15b used the
same procedure as the synthesis of 5a 5b. Subsequently the intermediates 12a 12e and 14a 14b
were coupled with 6-fluoro-3-(piperidin-4-yl) benzo[d]isoxazole to yield compounds 13a 13d and
16a 16b. Compound 21 was synthesized in five steps as described in Scheme 3. The intermediate
3) was converted into intermediate 4 by reaction with acetone in the
–
-
4
–
–
–
–
-
–
–
-
-
–
–
-
–
–
-
–
17 was synthesized from 2,4-dihydroxypropiophenone. Hydrolysis of 17 under basic conditions
(NaHCO3/MeOH) produced 18. Intermediate 19 was prepared by reduction with Pd/C, followed by
reaction with 1-bromo-4-chlorobutane to afford 20. The target compound 21 was obtained by coupling
20 with 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole in the presence of K2CO3 in acetonitrile.
O
O
O
ⅱ
ⅲ
O
Cl
OH
n
N
O
n
X
5a: n=1
5b: n=2
4
6a-6k: n=2
6l,6m: n=1
Ar
6a-6i,6k: X as N
6j,6l: X as C
ⅰ
O
O
O
O
ⅰ
ⅱ
H3C
HO
O
OH
O
Cl
m
OH
n
n
3
8a: n=1, m=1
8b: n=2, m=1
8c: n=1, m=2
8d: n=2, m=2
7a: n=1
7b: n=2
ⅲ
O
O
O
N
m
X
Ar
n
9a, 9b: n=1, m=1
9c, 9d: n=1, m=2
9e, 9f, 9g: n=2, m=1
9h,9i,9j, 9k: n=2, m=2
9a,9b,9e,9f,9i,9l:X as N
9c,9d,9g,9g,9k:X as C
Scheme 1.
Reagents and conditions: (i) (a) acetone, cyclopentanone or cyclohexanone,
pyrrolidine, CH3CN, 50 ◦C, 12 h; (b) hydrochloric acid solution; (ii) 1-bromo-3-chloropropane
or 1-bromo-4-chlorobutane, K2CO3, DMF, rt, 24 h; (iii) 4-aromatic ring substituted piperazine
(
6a-6i,6k,9a,9b,9e,9f,9i,9l) or 4-aromatic ring substituted piperidine (6j,6l,9c,9d,9g,9h,9k) CH3CN,
K2CO3, KI, 70 ◦C.

Molecules 2020, 25, 4107
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Scheme 2. Reagents and conditions: (i) (a) trifluoromethanesulfonic acid, 3-chloropropionic acid,
80 ◦C, 1 h; (b) sodium hydroxide solution(2 N); (ii) 1-bromo-4-chlorobutane, K2CO3, DMF, rt, 24 h;
(iii) CH3CN, K2CO3, KI, 70 ◦C, 10 h; (iv) crotonic acid, anhydrous zinc chloride, 180 ◦C, 30 min.
Scheme 3. Reagents and conditions: (i) acetic anhydride, sodium acetate anhydrous, reflux, 14 h;
(ii) sodium bicarbonate solution, MeOH, rt, 3 h; (iii) Pd/C, H2, rt; (iv) 1-bromo-4-chlorobutane, K2CO3,
DMF, rt, 24 h; (v) 6-fluoro-3-(piperidin-4-yl)-3a,7a-dihydrobenzo[d]isoxazole, CH3CN, K2CO3, KI,
70 ◦C.
2.2. In Vitro Evaluation and Structure-Activity Relationship (SAR)
At beginning of this study, the 7-hydroxy-2,2-dimethylchroman-4-one group was selected as a
privileged structure derived from coumarin and flavone. The influence of various heterocyclic and
aryl piperazines(piperidine) on the activities of D2, 5-HT1A and 5-HT2A receptors were primarily
evaluated and the results are summarized in Table 1. Among those compounds, 6a
–6d bearing
phenylpiperazine and substituted phenylpiperazines (-F, -CF3, -OCH3) showed moderate affinity for

Molecules 2020, 25, 4107
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5-HT1A and 5-HT2A receptors and lower affinity for D2 receptors. When the phenylpiperazine was
replaced with a heterocyclic piperazine, compound 6e bearing a pyripiperazine devoid of affinity for
D2 receptor resulted. Compound 6f with pyrimidine almost lost the affinity for all the three receptors.
Compound 6g bearing a 1-(benzo[d][
receptor. Interestingly, replacing the phenylpiperazine with a benzoheterocycle-piperazines(piperidine),
such as benzothiazole-piperazine and benzisothiazole- piperazine, gave compounds 6h and 6i
1,3] dioxol-5-ylmethyl) piperazine showed lower affinity for the D2
,
which showed significantly increased for all three receptors. Especially, compound 6j bearing a
6-fluorobenzo[d]isoxazole-piperidine moiety displayed excellent D2 (Ki = 8.1 nM), 5-HT1A (Ki = 9.7
nM) and 5-HT2A (Ki = 3.2 nM) receptor potency.
Table 1. Binding Affinities for D2, 5-HT1A, and 5-HT2A Receptors of Compounds.
Receptor Affinity Ki ± SEM (nM) a
Compound
NR1R2
D2
5-HT1A
5-HT2A
6a
553.5 ± 68.7
118.9 ± 17.4
89.8 ± 11.3
6b
776.8 ± 84.7
155.6 ± 19.5
269.1 ± 30.3
72.5 ± 8.4
6c
1128.6 ± 108.4
147.4 ± 16.9
6d
2351.5 ± 317.4
275.2 ± 25.2
86.8 ± 5.6
6e
6f
4587.3 ± 510.3
>10,000 b
627.4 ± 75.3
735.6 ± 82.7
216.5 ± 20.8
722.1 ± 64.9
1135.1 ± 151.7
379.5 ± 39.1
6g
1135.2 ± 140.4
6h
6i
43.1 ± 5.7
26.2 ± 2.7
25.3 ± 2.8
81.7 ± 9.5
55.2 ± 4.9
19.5 ± 2.2
6j
12.1 ± 1.5
9.7 ± 1.3
3.2 ± 0.4
a
Ki values are taken from three experiments, expressed as means SEM. b The Ki values were not calculated because
the inhibition percentages at 10 mM were too low.

Molecules 2020, 25, 4107
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According to the above results, the 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole moiety was
selected as a privileged fragment, as our previous studies have proved that compounds bearing this
pharmacophore show favorable activities for D2, 5-HT1A and 5-HT2A receptors [14,15,17]. Next the
effect of substituents on the 7-hydroxy-2,2-dimethylchroman-4-one group (Table 2) was investigated,
such as five-membered rings, six-membered rings, methyl and chlorine groups. First, when dimethyl
was replaced with a cyclopentyl ring, the obtained compound 9d had reduce activities on the D2,
5-HT1A and 5-HT2A receptors (9d vs. 6j). Similarly, enlargement of the five-membered ring to a
cyclohexyl ring (compound 9h) slightly lowered the affinity for the three receptors compared with
6j. Removing the dimethyl or replacing the dimethyl with a methyl (13a and 16a) showed a negative
impact on all the three receptors compared with compound 6j. Meanwhile, when the two methyl
substituents at the 5 and 6 positions (compounds 21) of 7-hydroxy-2,2-dimethylchroman-4-one group
were changed simultaneously, it resulted in increased activities for the 5-HT2A receptor but decreased
activity for the D2 and 5-HT1A receptors. We further investigated the influence of a methyl group
substituted at different positions on the phenyl ring. The activities of the substitution of methyl at
the 2- (13c), 3- (13b) and 8- (13d) positions of the phenyl ring were retained, but were slightly lower
than without a substituent (13a). According the results, when the methyl was substituted at the 6
position of 7-hydroxy-2,2-dimethylchroman-4-one group (16a) and the 8-chloro substituted derivative
16b displayed decreased activity for the 5-HT1A receptor (16a vs. 16b).
Table 2.
Binding Affinities for D2, 5-HT1A, and 5-HT2A Receptors of Compounds and
Reference Antipsychotics.
Receptor Affinity Ki ± SEM (nM) a
Compound
Structure
D2
5-HT1A
5-HT2A
O
O
6k
3157.4 ± 466.4
18.6 ± 2.3
115.8 ± 15.8
33.1 ± 4.5
6.7 ± 0.7
N
O
O
N
6l
11.5 ± 1.2
9a
3853.8 ± 327.9
3793.4 ± 408.7
476.1 ± 57.5
384.9 ± 49.4
72.0 ± 6.8
61.7 ± 8.6
9b
9c
18.9 ± 2.1
15.7 ± 1.9
10.4 ± 1.4
8.2 ± 0.8
8.3 ± 0.9
8.5 ± 0.7
9d
9e
9f
2054.9 ± 229.5
914.6 ± 90.3
312.1 ± 41.4
48.3 ± 5.2
93.5 ± 7.9
76.7 ± 9.4

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Table 2. Cont.
Receptor Affinity Ki ± SEM (nM) a
Compound
Structure
D2
5-HT1A
5-HT2A
9g
9h
18.4 ± 1.8
7.7 ± 0.9
9.1 ± 1.1
7.3 ± 0.4
17.0 ± 2.3
154.2 ± 19.6
81.7 ± 9.7
5.6 ± 0.3
92.1 ± 8.8
76.3 ± 7.1
9i
9j
67.5 ± 7.4
35.8 ± 5.2
9k
13a
13b
13c
35.1 ± 4.7
22.5 ± 2.6
28.9 ± 3.4
25.8 ± 3.9
213.3 ± 39.5
13.1 ± 1.7
24.6 ± 2.9
18.1 ± 1.3
75.6 ± 8.6
9.6 ± 0.8
10.5 ± 1.3
15.9 ± 2.3
13d
22.9 ± 3.1
17.0 ± 2.2
13.7 ± 1.9
16a
16b
21
19.7 ± 2.7
21.4 ± 2.4
28.4 ± 3.2
10.1 ± 1.3
78.8 ± 8.9
16.2 ± 1.5
7.9 ± 0.5
8.1 ± 1.1
2.4 ± 1.9
Clozapine
Risperidone
132.5 ± 13.7
3.5 ± 0.4
188.5 ± 18.3
191.5 ± 16.9
12.7 ± 1.8
0.17 ± 0.03
a
Ki values obtained from three experiments, recorded as means ± SEM.
As the compounds bearing a cyclopentyl ring (9c and 9d) and a cyclohexyl ring (9g
and 9h) moiety showed higher potency for the three receptors (Tables 1 and 2), we therefore
attempted to expand the structural transformation of 6-fluoro-3-(piperidin-4-yl) benzo[d]isoxazole
fragment. When the privileged structure 6-fluoro-3-(piperidin-4-yl) benzo[d]isoxazole was replaced
with phenylpiperazine derivatives and heterocyclic piperazines, such as 1-(2-methoxyphenyl)
piperazine (9a
,9b,9e),1-(2-(trifluoromethyl)phenyl)piperazine (9f), 1-(2,3-dimethylphenyl)piperazine
(9i), 1-(2,3-dichlorophenyl)piperazine (9j) and 3-(piperidin-4-yl)benzo[d]isothiazole (9k), all of the
resulting compounds fail to improve the affinities for the D2, 5-HT1A and 5-HT2A receptors,
indicating that the 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole was the optimum structure for this
type of compound.

Molecules 2020, 25, 4107
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Our previous SAR studies reveal that the linker length and flexibility play an important role in the
regulation of the receptor potency, and the lengths of the linker generally considered appropriate were
three carbons and four carbons 1415. Therefore, investigation of chain length was focused on three
carbons and four carbons. As shown in Tables 1 and 2, most of the compounds with chain lengths of
four carbons exhibited higher affinities for the three receptors than that of three carbon derivatives
(6j vs. 6l; 9a vs. 9b; 9g vs. 9h), for example, elongation of the linker of 9c from three carbons to four
carbons resulted in improved activities for D2 and 5-HT1A but had a negative effect on the 5-HT2A
receptor (9c vs. 9d). This indicated that the appropriate length of the linker was four carbons.
Overall, compounds 6j, 9d, 9g and 9h demonstrated high affinity for the D2, 5-HT1A and 5-HT2A
receptors (D2, Ki < 20 nM; 5-HT1A, Ki <10 nM; 5-HT2A, Ki < 10 nM) and the potency ratio between D2
and 5-HT1A, 5-HT2A was less than 5, which means the compounds have balanced receptor activity
profiles. Therefore, compounds 6j, 9d, 9g and 9h were selected for further evaluation.
A vast number of clinical studies has demonstrated that atypical antipsychotics can induce a
series of adverse effects in the treatment of schizophrenia, such as weight gain, hyperglycaemia and
QTc prolongation [18
off-target receptors, mainly focused on histamine H1, 5-HT2C, and adrenergic α1
–
20]. These adverse side effects have been proved to be associated with several
21 22]. For instance,
[
,
the synergistic effects of H1 and 5-HT2C antagonism may cause weight gain and hyperglycaemia
while antagonism on the adrenergic α1 receptor may trigger orthostatic hypotension [23]. Therefore,
the selected compounds were further evaluated for these receptors in the present study. As shown
in Table 3, compounds 6j
and lower activities for 5-HT2c receptors compared with risperidone (α1, Ki = 2.8
2.7 nM), suggesting that 6j 9d 9g and 9h had low liability to
,
9d
,
9g and 9h exhibited moderate activities for the α1 and H1 receptors
±
0.4 nM; H1,
Ki = 26.1 ± 2.8 nM; 5-HT2c, Ki = 19.7
±
,
,
elicit treatment-associated adverse effects. In addition, the selected compounds were subjected to
additional studies of binding to the D3 receptor, as the effect of atypical antipsychotic drugs on the D3
receptor is related to cognitive and motivational behaviors [24], and several recent pharmacological
studies strongly support the idea that antagonism on the D3 receptor might reduce catalepsy [25].
As shown in Table 3, compounds 6j, 9d, 9g and 9h had high affinities for the D3 receptor, which may
be beneficial for the treatment of schizophrenia.
Table 3. Activities on D3, 5-HT2C, α1 and H1 Receptors of compounds and reference antipsychotics.
Receptor Affinity Ki ± SEM (nM) a
hERG
IC50 (nM)
Compound
D3
5-HT2C
α
H1
1
6j
9d
9g
25.4 ± 3.1
42.6 ± 5.3
35.2 ± 2.8
38.6 ± 3.7
10.9 ± 1.9
265.2 ± 18.5
565.8 ± 75.4
425.6 ± 47.8
401.1 ± 55.6
528.2 ± 68.5
19.7 ± 2.7
237.2 ± 86.4
161.7 ± 57.1
138.2 ± 39.8
101.5 ± 50.6
2.8 ± 0.4.
938.5 ± 101.3
784.2 ± 98.3
909.3 ± 78.5
819.5 ± 91.8
26.1 ± 2.8
3769.8 ± 297.5
3380.3 ± 352.3
3015.5 ± 290.8
3982.6 ± 411.5
168.5 ± 23.1
9h
Risperidone
Clozapine
16.1 ± 3.1
33.6 ± 3.9
4.3 ± 0.5
2750.4 ± 317.2
a
Ki values obtained from three experiments, recorded as means ± SEM.
Cardiotoxicity is an important drug evaluation indicator, which is often caused by the blockade of
the human ether-a-go-go-related gene (hERG) potassium channel [26 27]. To assess the cardiotoxicity
of compounds 6j 9d 9g and 9h, their inhibitory actions on hERG were assessed in vitro. As shown
,
,
,
in Table 3, all of the tested compounds exhibited lower levels of inhibition on hERG compared to
clozapine and risperidone, indicating that they have low risks for QT interval prolongation.
To sum up, all the in vitro evaluations results have shown that compound 6j exhibited higher
affinity for D2, D3, 5-HT1A and 5-HT2A receptors and weaker affinity for 5-HT2c, α1 and H1 receptors
than other candidates, therefore, compound 6j was then subjected to further investigation.

Molecules 2020, 25, 4107
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2.3. Acute Toxicity
To assess the safety of compound 6j, its acute toxicity was assayed in vivo in terms of LD50 values.
As shown in Table 4, compound 6j showed a high safety threshold, even exceeds the maximum dose
(LD50 > 2000 mg/kg). The results indicated that compound 6j possesses high safety performance and
low acute toxicity.
Table 4. In Vivo Pharmacological Profile of Compound.
MK-80 b
Compound
APO a
CAT c
CAT/APO
CAT/MK-801
LD50
6j
0.19
17.92
0.046
0.16
2.28
0.011
56.84
50.0
0.92
b
299.16
21.93
20
355.25
5.58
83.63
>2000
150.0
82.1
Clozapine
Risperidone
a
APO: apomorphine-induced climbing (ED50, mg/kg, po). MK-801: MK-801-induced hyperactivity (ED50, mg/kg,
po). CAT: catalepsy (ED50, mg/kg, po).
c
2.4. Intrinsic Activity of Compound 6j
As shown in Table 5, compound 6j stimulated the D2L, D3, 5-HT1A, 5-HT2A receptors in the
agonist assay and showed weak agonist activity, the efficacy of the reference compounds less than
10%, respectively. In the antagonist assay, the efficacy of compound 6j blocked the four receptors more
than 90%. Thus, 6j functioned as an antagonist at the D2L (IC50 = 8.9 nM), D3 (IC50 = 31.5 nM), 5-HT1A
(IC50 = 201.4 nM), 5-HT2A (IC50 = 195.5 nM) receptors.
Table 5. Activities of Compound 6j and Reference Compounds to D2L, D3, 5-HT1A, 5-HT2A Receptors.
Activation (10
µM, %) (n = 3)
Inhibition (10
µM, %) (n = 3)
IC50
(nM)
Receptor
Compd
EC50 (nM)
D2L
dopamine
SCH23390
6j
dopamine
spiperone
6j
5-HT
WAY-100635
6j
101.3 ± 5.3
19.1
99.1 ± 3.2
97.4 ± 4.1
31.6
8.9
4.5 ± 0.6
D3
98.4 ± 4.4
2.5
2.3
98.8 ± 4.8
97.5 ± 2.7
93.9
31.5
5.9 ± 0.7
5-HT1A
5-HT2A
99.7 ± 3.8
98.4 ± 3.7
99.1 ± 4.5
11.2
201.4
0.8 ± 0.1
5-HT
ketanserin
6j
100.2 ± 2.6
20.4
98.2 ± 2.6
99.8 ± 2.2
87.6
195.5
1.4 ± 0.2
2.5. Behavioral Studies
As schizophrenic patients show many psychiatric and somatic symptoms in clinical manifestations,
more intuitive and visual models to evaluate the antipsychotic-like activities of the new compounds
are required. Nowadays, there are several models commonly used to assess the antipsychotic-like
efficacy of potential compounds, such as apomorphine (APO)-induced hyperlocomotion [28,29] and
MK-801-induced hyperactivity models [30]. In the APO-induced hyperlocomotion model, the D2
receptor agonist apomorphine is commonly used to induce behavioral agitation in mice to simulate the
positive symptoms of schizophrenia, but this can be reversed by compounds with antipsychotic-like
efficacies. In the MK-801-induced hyperactivity model, the non-competitive NMDA receptor antagonist
MK-801 can induce the schizophrenia-like symptoms in healthy animals, but compounds with
antipsychotic-like efficacies can significantly reverse these symptoms. In addition, the catalepsy
test model is used to predict the propensity of antipsychotics to induce EPS in humans [31]. This
model has become a common and important animal model for assessing the potential therapeutic

Molecules 2020, 25, 4107
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effect in schizophrenia [31]. In this study, 6j was subjected to these models to verify its potential
antipsychotic activity.
2.5.1. Apomorphine-Induced Hyperlocomotion
As shown in Figure 2, compound 6j induced a dose-dependent response in APO-induced
hyperlocomotion model, with an ED50 value of 0.19 mg/kg (Table 4). Mice in the control groups
received clozapine and risperidone inhibit the APO-induced climbing with ED50 values of 17.92 and
0.046 mg/kg, respectively. The comparison of results indicates that compound 6j has certain efficacy
against the positive symptoms of psychosis.
7
Apomorphine-induced climbing
#
6
Vehicle
5
Vehicle+APO
*
APO+6j
4
3
*
2
**
1
0
APO+0.3
Vehicle
APO+0.03 APO+0.1
Vehicle+APO APO+0.01
Dose (mg/kg)
Figure 2. Effect of compound 6j on APO-induced climbing in mice (10/group). The score was shown as
means
±
SEM. Statistical significances were performed by the nonparametric two-tailed Mann-Whitney
#
U-test: p < 0.05 vs. vehicle treatment; * p < 0.05 and ** p < 0.01 vs. apomorphine treatment.
2.5.2. MK-801-Induced Hyperactivity
In the MK-801-induced hyperactivity model, compound 6j significantly and dose dependently
attenuated the increased locomotor activity (Figure 3) with an ED50 value of 0.16 mg/kg (Table 4). In the
control group, mice treated with clozapine and risperidone with ED50 values of 2.28 and 0.011 mg/kg,
respectively. The comparison of the results shows that compound 6j was more potent than clozapine.
MK-801-induced hyperactivity
30000
#
Vehicle
25000
Vehicle+MK-801
*
20000
15000
10000
5000
0
MK-801+6j
**
**
**
Dose(mg/kg)
Figure 3. Effect of compound 6j on MK-801-induced hyperactivity in mice (10/group). The distance
expressed as the means
±
SEM. Statistical evaluation was conducted by two-way ANOVA followed by
#
Tukey’s test for multiple comparisons. p < 0.05 vs. vehicle treatment; ** p < 0.01 and * p < 0.05 vs.
MK-801 treatment.

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2.5.3. Catalepsy
In the catalepsy test model, compound 6j displayed a high threshold for catalepsy with an ED50
value of 56.84 mg/kg, but the ED50 values of clozapine and risperidone were 50.0 mg/kg and 0.92 mg/kg,
the comparison of results indicates that 6j has a lower incidence of EPS (Table 4).
In addition, compound 6j displayed a wide therapeutic index (TI) range (299.16–352.5) based on
its efficacy (APO and MK-801 models) and its side effects (catalepsy), while the TI of clozapine range
from 5.58 to 21.93, and 20.0 to 83.63 of risperidone (Table 4). The result demonstrated that 6j has a
higher safety margin than clozapine and risperidone.
3. Materials and Methods
3.1. General Information
All solvents were from commercial sources and used without further purification. The purity of
all the reagents and the test compounds was more than 95%. Melting points were determined in open
1
capillary tubes and are uncorrected. H-NMR spectra were recorded on an Avance III 400 spectrometer
(Bruker, Karlsruhe, Germany) at 400 MHz (1H) using CDCl3 and DMSO-d6 as solvents. Chemical
shifts recorded as δ values (ppm), using tetramethylsilane (TMS) as the internal reference; Coupling
constants (J) are given in Hz. Proton multiplicities are labeled as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), and br (broad signal). Thin layer chromatography (TLC) was performed
using silica gel GF254. Flash column chromatography was carried out using silica gel.
3.2. Instrumentation
High performance liquid chromatography (HPLC) methods: LC-20AD spectrometer (Shimadzu,
Kyoto, Japan); column, Shimadzu VP-ODS (4.6 mm
acetonitrile 0.01 mol/L NH4H2PO4 (0.2% Et3N, pH = 3.0) (10:90); B: acetonitrile 0.01 mol/L NH4H2PO4
(0.1% Et3N, pH = 3.0)(70:30); flow rate, 1.0 mL/min; sample size, 10
L; column temperature, 40 ◦C;
×
250 mm, 5 µm) C18-253; mobile phase, A:
µ
UV detection condition, 210 nm; High resolution mass spectra (HRMS) spectrometer, Agilent 6530
Q-TOF LC/MS and Agilent 1290 Infinity (G4212A) spectrometer (Agilent, Santa Carla, CA, USA). The
HPLC conditions as following: column X BridgeR Shield RP18 (4.6
×
150 mm, 3.5 µm, Waters, Milford
MA USA), C18-330; mobile phase: A, 5mmol/L NH4OAc (pH = 6.0)(10:90); B, MeOH; eluent, 30%
A and 70% B (V:V); flow rate, 1.0 mL/min; column temperature, 35 ◦C; UV detection, 254 nm. Mass
spectrometry conditions: Dual AJS ESI, positive; Gas temperature, 350 ◦C; Fragmentor, 80 KV; Drying
gas, 7 mL/min; Nebulizer, 45 psi; Sheath gas temperature, 350 ◦C; Capillary Voltage, 4000 V; Sheath gas
flow, 11 L/min; Nozzle Voltage, 500 V. Low resolution mass spectrometry (LRMS) and high performance
liquid chromatography-mass spectrometry (HPLC-MS) were obtained using an Agilent MS/1200 HPLC
liquid chromatograph/mass spectrometer (JXZX-FXS-064).Agilent ZORBAX SB-C18 (4.6
m); ESI source: scan mode, positive/negative; Rage, 50–1000; Nitrogen gas flow, 11 L/min; Column
pressure: 3000–3000 eV. Chromatographic parameter: Mobile phase, 50% acetonitrile, 50% water; UV
×
150 mm,
5
µ
−
detection, 210 nm, 245 nm; injection volume, 1.0 µ
L; flow rate, 0.3 mL/min; column temperature, 30 ◦C.
3.3. Synthesis
3.3.1. General Procedures for the Preparation of Intermediates 4 and 7
Compound 7a and 7b were synthesized by the following reaction: 2,4-dihydroxyacetophenone
4,
and pyrrolidine were added to a suitable a◦mount of acetonitrile and then corresponding ketone was
added and the mixture was stirred at 50 C for 12 h. Then, it was cooled down and poured into
an ice-cold 2 M solution of hydrochloric acid and water, stirred for another 30 min, filtered and the
obtained solid was recrystallized from EtOH (95%) to give 4 and 7a–7b.

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3.3.2. General Procedures for the Preparation of Intermediates 5 and 8
To a suspension of intermediate
4 and 7 in DMF, 1.5 equivalents of K2CO3 and
1-bromo-3-chloropropane or 1-bromo-4-chlorobutane were added and the suspension was stirred for
24 h at room temperature and then filtered. The filtrate was evaporated in vacuo followed by addition
of dichloromethane (DCM), washed with water and dried over anhydrous Na2SO4, the solvent was
evaporated in vacuo and the residue was separated by silica gel column chromatography to afford 5a
,
5b and 8a–8d.
3.3.3. General Procedures for the Preparation of Target Compounds 6 and 9
A mixture of intermediates 5 or 8, anhydrous potassium carbonate, CH3CN and a catalytic amount
of potassium iodide (KI) was stirred at 70 ◦C for 10 h. The mixture was filtered and the filtrate was
concentrated under reduced pressure, the residue was dissolved in DCM, washed with water and
dried with anhydrous MgSO4, the solvent was removed under reduced pressure and the residue was
separated by silica gel column chromatography to afford 6a-6l and 9a-9k.
3.3.4. General Procedures for the Preparation of Intermediates 11, 12, 14, and 15
A mixture of resorcinol or a derivative, 3-chloropropionic acid and trifluoromethanesulfonic acid
was stirred at 80 ◦C for 1 h. The reaction mixtures were cooled to room temperature and poured into
water, extracted with ethyl acetate (EA), The combined organic phase was washed with water and
dried over anhydrous MgSO4, the solvent was removed under reduced pressure and the residue was
added to a certain amount of sodium hydroxide solution (2N) at 0–5 ◦C, then stirred for 2 h. The pH
was adjusted to above 2 with hydrochloric acid solution and stand for 30 min, the formed intermediates
11a–11d were collected by filtration, washed with water and dried in vacuo.
A mixture of resorcinol or 2-chlororesorcinol, crotonic acid and anhydrous zinc chloride was
stirred at 180 ◦C for 30 min. The reaction mixtures were cooled to room temperature and poured into
water, extracted with EA, the combined organic layer was washed with water, brine, and dried over
MgSO4, the solvents were removed under reduced pressure and the crude products was purified via
column chromatography to give pure products 14a–14b.
Intermediates 12 and 15 were prepared as described for compound
5 using 11 or 14,
1-bromo-4-chlorobutane and anhydrous K2CO3 in DMF; the crude products were purified via
column chromatography.
3.3.5. General Procedures for the Preparation of Target Intermediates 13 and 16
The target compounds 13a
–13d and 16a–16b were prepared as described for compound 6 using
12a
–
12e 15a 15b and K2CO3, KI, 6-fluoro-3-(piperidin-4-yl) benzo[d]isoxazole in acetonitrile, the
,
–
crude products were purified via column chromatography.
3.3.6. General Procedures for the Preparation of Intermediates 17, 18 and 19
1-(2,4-Dihydroxyphenyl)propan-1-one (16.6 g) and sodium acetate anhydrous (8.3 g) were added
to acetic anhydride (70 mL), the reaction mixture was stirred at reflux for 14 h and cooled to room
temperature, diluted with water and extracted with DCM, the organic layer was separated and
washed with water, dried over MgSO4, concentrated in vacuo to give intermediate 17 as a yellow
solid. The intermediate 17 was dissolved in a solution (50 mL) of saturated sodium bicarbonate and
MeOH (V:V = 1:1), the suspension was stirred at rt for 3 h, the product intermediate 18 obtained by
filtration. Intermediate 18 was dissolved in MeOH, and then Pd/C added and stirred at rt for 10 h
under hydrogen atmosphere, filtered and the filtrate concentrated in vacuo to give intermediate 19.

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3.3.7. The Preparation of Intermediate 20 and Target Compound 21
The intermediate 20 and target compound 21 were prepared as described for intermediate 15 and
target compound 16.
3.3.8. The Characteristics and Spectroscopic Data of the Target Compounds
2,2-Dimethyl-7-(4-(4-phenylpiperazin-1-yl)butoxy) chroman-4-one (6a).Pale-white solid; m. p. 131–133 ◦C;
yield 80.3%; 1H-NMR (CDCl3)
δ 7.84 (d, J = 8.8 Hz, 1H), 7.32 (dd, J = 8.6, 7.4 Hz, 2H), 6.98 (dd, J = 8.8,
0.9 Hz, 2H), 6.91 (t, J = 7.3 Hz, 1H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H), 6.42 (d, J = 2.3 Hz, 1H), 4.07 (t, J = 6.3
Hz, 2H), 3.31–3.21 (m, 4H), 2.71 (s, 2H), 2.70–2.65 (m, 4H), 2.56–2.43 (m, 2H), 1.89 (dd, J = 14.4, 6.3
Hz, 2H), 1.83–1.69 (m, 2H), 1.50 (d, J = 2.0 Hz, 6H). HRMS (ESI) calculated for C25H33N2O3 [M + H]+
,
409.2486; found, 409.2479.
7-(4-(4-(2-Fluorophenyl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one (6b). Pale-white solid; m. p.
135–137 ◦C; yield 78.8%; 1H-NMR (CDCl3)
δ 7.84 (d, J = 8.8 Hz, 1H), 7.19–6.87 (m, 4H), 6.57 (dd, J = 8.8,
2.3 Hz, 1H), 6.41 (d, J = 2.3 Hz, 1H), 4.06 (t, J = 6.3 Hz, 2H), 3.27–3.06 (m, 4H), 2.71–2.97 (m, 6H),
2.58–2.40 (m, 2H), 1.93–1.71 (m, 4H), 1.49 (s, 6H). HRMS (ESI) calculated for C25H32FN2O3 [M + H]+,
427.2391; found, 427.2390.
2,2-Dimethyl-7-(4-(4-(3-(trifluoromethyl) phenyl) piperazin-1-yl) butoxy) chroman-4-one (6c). Pale-white
solid; m. p. 139–140 ◦C; yield 76.2%; 1H-NMR (CDCl3)
δ 7.84 (d, J = 8.8 Hz, 1H), 7.40 (t, J = 8.0 Hz,
1H), 7.31 (s, 1H), 7.20 - 7.02 (m, 2H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H), 6.41 (dd, J = 5.0, 2.7 Hz, 1H),
4.07 (dd, J = 8.0, 4.4 Hz, 2H), 3.33 (s, 4H), 2.68 (d, J = 24.9 Hz, 6H), 2.56 (s, 2H), 1.96–1.85 (m, 2H),
1.83–1.71 (m, 2H), 1.50 (d, J = 2.7 Hz, 6H). HRMS (ESI) calculated for C26H32F3N2O3 [M + H]+, 477.2360;
found, 477.2358.
7-(4-(4-(2-Methoxyphenyl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one (6d).Off-white solid; m. p.
128–130 ◦C; yield 75.5%; 1H-NMR (CDCl3)
δ 7.80 (t, J = 4.0 Hz, 1H), 7.02–6.87 (m, 4H), 6.57 (m, 1H),
6.41 (d, J = 4.0 Hz, 1H), 4.09 (d, J = 4.0 Hz, 2H), 3.87 (s, 2H), 2.70-2.60 (m, 4H), 2.05 (d, J = 8.0 Hz, 2H),
2.68 (d, J = 22.9 Hz, 2H), 2.67–2.58 (m, 4H), 2.57–2.42 (m, 2H), 1.89 (dd, J = 14.4, 6.3 Hz, 2H), 1.45 (s,
6H). HRMS (ESI) calculated for C26H34N2O4 [M + H]+, 438.2519; found, 438.2511.
2,2-Dimethyl-7-(4-(4-(pyridin-2-yl) piperazin-1-yl) butoxy) chroman-4-one (6e).Pale-white solid; m. p.
1
127–129 ◦C; yield 73.2%; H-NMR (CDCl3)
δ 8.33 - 8.17 (m, 1H), 7.83 (t, J = 8.1 Hz, 1H), 7.53 (ddd,
J = 8.9, 7.1, 2.0 Hz, 1H), 6.79–6.62 (m, 2H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H), 6.42 (d, J = 2.3 Hz, 1H), 4.07
(t, J = 6.3 Hz, 2H), 3.67–3.53 (m, 4H), 2.68 (d, J = 22.9 Hz, 2H), 2.67–2.58 (m, 4H), 2.57–2.42 (m, 2H),
1.89 (dd, J = 14.4, 6.3 Hz, 2H), 1.78 (dd, J = 14.9, 8.1 Hz, 2H), 1.49 (s, 6H). HRMS (ESI) calculated for
C24H32N3O3 [M + H]+, 410.2438; found, 410.2438.
2,2-Dimethyl-7-(4-(4-(pyrimidin-2-yl) piperazin-1-yl) butoxy) chroman-4-one (6f).Pale-white solid; m. p.
128–131 ◦C; yield 69.7%; 1H-NMR (CDCl3)
δ 8.35 (d, J = 4.7 Hz, 2H), 7.83 (d, J = 8.8 Hz, 1H), 6.64–6.52
(m, 2H), 6.42 (d, J = 2.3 Hz, 1H), 4.06 (t, J = 6.3 Hz, 2H), 3.90 (s, 4H), 2.71 (s, 2H), 2.56 (d, J = 20.1 Hz,
4H), 2.51 (d, J = 7.4 Hz, 2H), 1.94 - 1.82 (m, 2H), 1.82 - 1.70 (m, 2H), 1.49 (s, 6H). HRMS (ESI) calculated
for C23H31N4O3 [M + H] +, 411.2391; found, 411.2390.
7-(4-(4-(Benzo[d] [
1
,
3
] dioxol-5-ylmethyl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one(6g).White solid;
m. p. 112–114 ◦C; yield 69.4%; 1H-NMR (CDCl3)
δ
7.82 (d, J = 8.8 Hz, 1H), 6.89 (s, 1H), 6.78 (d, J = 0.8
Hz, 2H), 6.55 (dd, J = 8.8, 2.3 Hz, 1H), 6.39 (d, J = 2.3 Hz, 1H), 5.97 (s, 2H), 4.16 (q, J = 7.1 Hz, 2H), 4.03
(t, J = 6.4 Hz, 2H), 3.45 (s, 2H), 2.70 (s, 2H), 2.46 (m, 6H), 1.94–1.60 (m, 4H), 1.49 (s, 6H), 1.30 (t, J = 7.1
Hz, 2H). HRMS (ESI) calculated for C27H35N2O5 [M + H]+, 467.2540; found, 467.2529.
7-(4-(4-(Benzo[b]thiophen-4-yl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one(6h).Pale white solid; m.
p. 117119 ◦C; yield 67.7%; 1H-NMR (CDCl3)
δ 7.85 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.45
(q, J = 5.6 Hz, 2H), 7.32 (dd, J = 9.6, 6.1 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.59 (dd, J = 8.8, 2.3 Hz,
1H), 6.43 (d, J = 2.3 Hz, 1H), 4.27 - 3.97 (m, 2H), 3.26 (s, 4H), 2.75 (d, J = 30.9 Hz, 4H), 2.64–2.41 (m,

Molecules 2020, 25, 4107
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2H), 2.02–1.70 (m, 4H), 1.50 (s, 6H), 1.31 (t, J = 7.1 Hz, 2H). HRMS (ESI) calculated for C27H33N2O3S
[M + H]+, 465.2206; found, 465.2201.
7-(4-(4-(Benzo[d]isothiazol-3-yl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one (6i). Pale white solid;
1
m. p. 119–120 ◦C; yield 65.8%; H-NMR (CDCl3)
δ 7.96 (d, J = 8.2 Hz, 1H), 7.85 (t, J = 8.1 Hz, 2H),
7.52 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H), 7.41 (ddd, J = 8.0, 7.0, 0.9 Hz, 1H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H),
6.43 (d, J = 2.3 Hz, 1H), 4.08 (t, J = 6.3 Hz, 2H), 3.71–3.50 (m, 4H), 2.86–2.66 (m, 4H), 2.64–2.44 (m,
2H), 1.98–1.68 (m, 4H), 1.50 (s, 6H), 1.31 (t, J = 7.1 Hz, 2H). HRMS (ESI) calculated for C26H32N3O3S
[M + H]+, 466.2195; found, 466.2189.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl) piperidin-1-yl) butoxy)-2,2-dimethylchroman- 4-one (6j). Pale white
solid; m. p. 116–117 ◦C; yield 70.2%; 1H-NMR (CDCl3)
δ 7.80 (d, J = 8.8 Hz, 1H), 7.70 (dd, J = 8.7, 5.0
Hz, 1H), 7.25 (dd, J = 8.5, 1.9 Hz, 1H), 7.06 (td, J = 8.9, 2.1 Hz, 1H), 6.57 (dd, J = 8.8, 2.4 Hz, 1H), 6.40
(d, J = 2.4 Hz, 1H), 4.23–4.13 (m, 2H), 3.86 (dd, J = 5.4, 4.2 Hz, 2H), 3.74 (d, J = 5.8 Hz, 2H), 3.15 – 3.01
(m, 3H), 2.69 (dd, J = 11.2, 5.4 Hz, 4H), 2.26 (dd, J = 11.3, 3.0 Hz, 2H), 2.15 – 2.02 (m, 4H), 1.45 (s, 6H).
HRMS (ESI) calculated for C27H32FN2O4 [M + H]+, 467.2341; found, 467.2335.
7-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propoxy)-2,2-dimethylchroman-4-one (6k). Pale-white solid; m.
p. 103–105 ◦C; yield 71.5%; 1H-NMR (CDCl3)
δ 7.80 (d, J = 8.0 Hz, 1H), 7.02–6.87(m, 4 H), 6.54–6.57
(m, 1H), 6.40 (d, J =4 Hz, 1H), 3.88 (s, 3H), 3.13 (s, br, 4H), 2.70–2.67 (m, 8H), 2.60 (t, J = 8.0 Hz, 2H),
2.05-2.03 (m, 2H), 1.46 (s, 6H). HRMS (ESI) calculated for C25H33FN2O4 [M + H]+, 425.2435; found,
425.2433.
7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)-2,2-dimethylchroman-4-one (6l). Pale-white
solid; m. p. 116–118 ◦C; yield 73.4%; 1H-NMR (CDCl3)
δ 7.80 (d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H),
7.27–7.25 (m, 1H), 7.09-7.05 (m, 1H), 6.57-6.54 (m, 1H), 6.40 (d, J = 4.0 Hz, 1H), 4.10 (t, J = 8.0 Hz,
2H), 3.13-3.09 (m, 3H), 2.68 (s, 2H), 2.59 (t, J = 8.0 Hz, 2H), 2.21-2.00 (m, 8H), 1.46 (s, 6H).HRMS (ESI)
calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2182.
7-(3-(4-(2-Methoxyphenyl) piperazin-1-yl) propoxy) spiro[chromane-2,10-cyclopentan]-4-one (9a). Off-white
solid; m. p. 128–130 ◦C; yield 68.5%; 1H-NMR (CDCl3)
δ 7.81 (d, J = 8.0 Hz, 1H), 7.04–6.88(m, 4H),
6.58–6.55 (m, 1H), 6.40 (d, J = 4.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H), 3.89 (s, 3H), 3.13 (s, br, 4H), 2.79 (s,
2H), 2.71 (s, br, 4H), 2.61 (t, J = 8.0 Hz, 2H), 2.03–2.12 (m, 4H), 1.87-1.91 (m, 2H), 1.74-1.65 (m, 4H).
HRMS (ESI) calculated for C27H35FN2O4 [M + H]+, 451.2519; found, 451.2511.
7-(4-(4-(2-Methoxyphenyl) piperazin-1-yl) butoxy) spiro[chromane-2,10-cyclopentan]- 4-one (9b). Off-white
1
solid; m. p. 96–98 ◦C; yield 76.4%; H-NMR (CDCl3)
δ 7.80 (d, J = 8.0 Hz, 1H), 7.03–6.86 (m, 4H),
6.56–6.53 (m, 1H), 6.37 (d, J = 4.0 Hz, 1H), 4.03 (t, J = 8.0 Hz, 2H), 3.88 (s, 3H), 3.12 (s, br, 4H), 2.78 (s,
2H), 2.68 (s, br, 4H), 2.49 (t, J = 8.0 Hz, 2H), 2.10–2.05 (m, 2H), 1.87–1.84 (m, 4H), 1.75–1.69 (m, 6H).
HRMS (ESI) calculated for C27H35FN2O4 [M + H]+, 451.2519; found, 451.2511. HRMS (ESI) calculated
for C28H37N2O4[M+H]+, 465.2748; found, 465.2745.
7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)spiro[chromane-2,10-cyclopentan]-4-one (9c).
White solid; m. p. 91–93 ◦C; yield 70.2%; 1H-NMR (CDCl3)
δ 7.80 (d, J = 8.0 Hz, 1H), 7.72-7.69(m, 1H),
7.26–7.24 (m, 1H), 7.09-7.04 (m, 1H), 6.57–6.54(m, 1H), 6.41 (d, J = 4.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H),
3.11-3.08 (m, 3H), 2.78 (s, 2H), 2.58 (t, J = 8.0 Hz, 2H), 2.23-1.86 (m, 12H), 1.74-1.64 (m, 4H). HRMS (ESI)
calculated for C28H32FN2O4 [M + H]+, 479.2341; found, 479.2338.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)spiro[chromane-2,10-cyclopentan]-4-one (9d).
◦
Pale-white solid; m. p. 103–105 C; yield 68.6%; 1H-NMR (CDCl3)
δ 7.80 (d, J = 8.0 Hz, 1H),
7.69–7.73(m, 1H), 7.26–7.24 (m, 1H), 7.09–7.04 (m, 1H), 6.56–6.54 (m, 1H), 6.38 (d, J = 4.0 Hz, 1H), 4.04
(t, J = 8.0 Hz, 2H), 3.12-3.08 (m, 3H), 2.78 (s, 2H), 2.48 (t, J = 8.0 Hz, 2H), 2.19-2.07 (m, 8H), 1.90-1.63 (s,
10H). HRMS (ESI) calculated for C29H34FN2O4 [M + H]+, 493.2497; found, 493.2496.

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7-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propoxy)spiro[chromane-2,10-cyclohexan]-4-one (9e). Pale-white
solid; m. p. 105–107 ◦C; yield 71.4%; 1H-NMR (CDCl3)
7.79 (d, J = 8.0 Hz, 1H), 6.85–7.00 (m, 4H),
δ
6.56–6.53 (m, 1H), 6.44 (d, J = 4.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H), 3.87 (s, 3H), 3.12 (m, 4H), 2.70 (s, br,
4H), 2.64 (s, 2H), 2.60 (t, J = 8.0 Hz, 2H), 2.07-1.97 (m, 4H), 1.72-1.66 (m, 2H), 1.52-1.45 (m, 4H), 1.34–1.30
(s, 2H). HRMS (ESI) calculated for C28H37N2O4 [M + H]+, 465.2748; found, 465.2740.
7-(3-(4-(2-(Trifluoromethyl)phenyl)piperazin-1-yl)propoxy)spiro[chromane-2,10-cyclohexan]-4-one
Off-white solid; m. p. 138–140 ◦C; yield 74.7%; 1H-NMR (CDCl3)
7.80–7.73 (m, 1H), 7.42–7.34 (m,
(9f).
δ
1H), 7.19–7.06 (m, 3H), 6.56–6.52 (m, 1H), 6.41 (d, J = 4.0 Hz, 1H), 4.05 (t, J = 8.0 Hz, 2H), 3.31-3.28 (m,
2H), 2.70–2.52 (m, 6H), 2.30–2.05 (m, 4H), 1.99–1.48 (m, 10H), 1.36–1.32 (m, 2H). HRMS (ESI) calculated
for C28H34F3N2O3 [M + H]+, 503.2516; found, 503.2511.
7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)spiro[chromane-2,10-cyclohexan]-4-one (9g).
Pale-white solid; m. p. 96–98 ◦C; yield 67.1%; 1H-NMR (CDCl3)
δ 7.79(d, J = 8.0 Hz, 1H), 7.74–7.70
(m, 1H), 7.26–7.24 (m, 1H), 7.10–7.05 (m, 1H), 6.58-6.55 (m, 1H), 6.45 (d, J = 4.0 Hz, 1H), 4.11 (t, J = 8.0
Hz, 2H), 3.12–3.10 (m, 3H), 2.66 (s, 2H), 2.61 (t, J = 8.0 Hz, 2H), 2.2–1.98 (m, 12H), 1.74–1.48 (m, 4H),
1.36-1.33 (m, 2H). HRMS (ESI) calculated for C29H34FN2O4 [M + H]+, 493.2497; found, 493.2495.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)spiro[chromane-2,10-cyclohexan]-4-one
White solid; m. p. 126–128 ◦C; yield 72.9%; 1H-NMR (CDCl3)
7.78(d, J = 8.0 Hz, 1H), 7.70–7.73 (m,
(9h).
δ
1H), 7.26–7.24 (m, 1H), 7.09–7.04 (m, 1H), 6.55–6.52 (m, 1H), 6.42 (d, J = 4.0 Hz, 1H), 4.05 (t, J = 8.0
Hz, 2H), 3.13-3.06 (m, 3H), 2.65 (s, 2H), 2.49(t, J = 8.0 Hz, 2H), 2.18-1.98 (m, 8H), 1.86-1.84 (m, 2H),
1.75-1.69 (m, 4H), 1.53-1.50 (m, 4H), 1.37-1.31 (m, 2H). HRMS (ESI) calculated for C30H36FN2O4 [M +
H]+, 507.2654; found, 507.6259.
7-(4-(4-(2,3-Dimethylphenyl)piperazin-1-yl)butoxy)spiro[chromane-2,10-cyclohexan]-4-one (9i). White solid;
◦
1
m. p. 138–140 C; yield 74.3%; H-NMR (CDCl3)
δ 7.81–7.76 (m, 1H), 7.10–6.92(m, 3H), 6.56–6.52
(m, 1H), 6.42 (d, J = 4.0 Hz, 1H), 4.06(t, J = 8.0 Hz, 2H), 3.06 (s, br, 4H), 2.66 (s, 2H), 2.60 (t, J = 8.0
Hz, 2H), 2.29 (s, 3H), 2.23 (s, 3H), 2.07–1.48 (m, 16H), 1.37–1.31 (m, 2H).HRMS (ESI) calculated for
C30H41N2O3 [M + H]+,477.3112; found, 477.3110.
7-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butoxy)spiro[chromane-2,10-cyclohexan]-4-one(9j). White solid; m.
p. 119–121 ◦C; yield 70.7%; 1H-NMR (CDCl3)
δ 7.81–7.76(m, 1H), 7.10–6.92 (m, 3H), 6.56–6.52 (m, 1H),
6.42 (d, J = 4.0 Hz, 1H), 4.06 (t, J = 8.0 Hz, 2H), 3.00 (s, br, 4H), 2.66 (s, 2H), 2.54(t, J = 8.0 Hz, 3H), 2.29
(s, 3H), 2.01-1.48 (m, 12H), 1.37–1.33 (m, 2H). HRMS (ESI) calculated for C28H35Cl2N2O3 [M + H]+,
517.2019; found, 517.2013.
7-(4-(4-(Benzo[d]isothiazol-3-yl)piperidin-1-yl)butoxy)spiro[chromane-2,10-cyclohexan]-4-one (9k). Pale-white
solid; m. p. 129–130 ◦C; yield 62.8%; 1H-NMR (CDCl3)
δ 7.79 (d, J = 8.0 Hz, 2H), 7.19–7.16 (m, 2H),
7.49–7.45 (m, 1H), 6.99–6.96 (m, 1H), 6.56–6.53 (m, 1H), 6.42 (d, J = 4.0 Hz, 1H), 4.05 (t, J = 8.0 Hz, 2H),
3.11(s, br, 4H), 2.70 (s, br, 4H), 2.66(s, 2H), 2.52 (t, J = 8.0 Hz, 2H), 2.00-1.48 (m, 12H), 1.34-1.31 (m, 2H).
HRMS (ESI) calculated for C30H37N2O3S[M + H]+, 505.2519; found, 505.2514.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)chroman-4-one (13a). Colorless oily liquid;
yield 58.1%; 1H-NMR (CDCl3)
δ 7.80(d, J = 8.0 Hz, 1H), 7.75–7.71 (m, 1H), 7.26–7.24 (m, 1H), 7.11–7.06
(m, 1H), 6.56–6.53 (m, 1H), 6.38 (d, J = 4.0 Hz, 1H), 4.42 (t, J = 8.0 Hz, 2H), 4.06 (t, J = 8.0 Hz, 2H),3.06–2.95
(m, 4H), 2.81–2.78 (m, 1H), 2.52–2.43 (m, 4H), 1.92–1.69 (m, 6H), 1.41-1.35 (m, 2H). HRMS (ESI) calculated
for C25H28FN2O4 [M + H]+, 439.2018; found, 439.2017.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-5-methylchroman-4-one (13b). Pale-white solid;
m. p. 85–87 ◦C; yield 63.6%; 1H-NMR (CDCl3)
δ 7.75–7.71 (m, 1H), 7.25–7.23 (m, 1H), 7.06–7.11 (m,
1H), 6.54–6.57 (m, 1H), 6.39 (d, J = 4.0 Hz, 1H), 4.45 (t, J = 8.0 Hz, 2H), 4.07 (t, J = 8.0 Hz, 2H), 3.07–2.96
(m, 4H), 2.80–2.77 (m, 1H), 2.52–2.43 (m, 4H), 2.39 (s, 3H), 1.91–1.67 (m, 6H), 1.40–1.33 (m, 2H). HRMS
(ESI) calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2182.

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7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-6-methylchroman-4-one (13c). Pale-white solid;
m. p. 91–93 ◦C; yield 60.3%; 1H-NMR (CDCl3)
7.80 (d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.26-7.24 (m,
δ
1H), 7.12–7.07 (m, 1H), 6.38 (d, J = 4.0 Hz, 1H), 4.46 (t, J = 8.0 Hz, 2H), 4.06 (t, J = 8.0 Hz, 2H), 3.08–2.99
(m, 4H), 2.80–2.77 (m, 1H), 2.52–2.43 (m, 4H), 2.38 (s, 3H), 1.92–1.68 (m, 6H), 1.41–1.35 (m, 2H). HRMS
(ESI) calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2182.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-8-methylchroman-4-one (13d). Pale-white solid;
m.p. 88–90 ◦C; yield 67.0%; 1H-NMR (CDCl3)
δ 7.80 (d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.26–7.24 (m,
1H), 7.12–7.07 (m, 1H), 6.57–6.54 (m, 1H), 4.45 (t, J = 8.0 Hz, 2H), 4.06 (t, J = 8.0 Hz, 2H), 3.08–3.01 (m,
4H), 2.82–2.79 (m, 1H), 2.55–2.44 (m, 4H), 2.40 (s, 3H), 1.91–1.69 (m, 6H), 1.41–1.36 (m, 2H). HRMS (ESI)
calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2179.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-2-methylchroman-4–one (16a). Pale-white solid;
m. p. 95–97 ◦C; yield 63.4%; 1H-NMR (CDCl3)
δ 7.80(d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.27–7.25
(m, 1H), 7.10–7.05 (m, 1H), 6.57–6.54 (m,1H), 6.39 (d, J = 4.0 Hz, 1H), 4.41–4.38 (m, 1H), 4.05 (t, J = 8.0
Hz, 2H), 3.06–3.01 (m, 3H), 2.81–2.78 (m, 2H), 2.51–2.41 (m, 4H), 1.93–1.67 (m, 6H), 1.50–1.47 (m, 5H).
HRMS (ESI) calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2180.
8-Chloro-7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-2-methylchroman-4-one (16b). Pale-
white solid; m. p. 99–101 ◦C; yield 64.7%; 1H-NMR (CDCl3)
δ 7.81(d, J = 8.0 Hz, 1H), 7.74–7.71 (m,
1H), 7.26–7.23 (m, 1H), 7.10–7.05 (m, 1H), 6.56–6.54 (m,1H), 4.40–4.38 (m, 1H), 4.05 (t, J = 8.0 Hz, 2H),
3.05–3.01 (m, 3H), 2.81–2.78 (m, 2H), 2.51–2.41 (m, 4H), 1.92–1.67 (m, 6H), 1.50–1.46 (m, 5H). HRMS
(ESI) calculated for C26H29ClFN2O4 [M + H]+, 487.1794; found, 487.1785.
7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-2,3-dimethylchroman-4-one (21). Pale-white
1
solid; m. p. 82–84 ◦C; yield 86.1%; H-NMR (CDCl3)
δ 7.80 (d, J = 8.0 Hz, 1H), 7.75–7.71 (m, 1H),
7.26–7.24 (m, 1H), 7.13–7.08 (m, 1H), 6.58–6.54 (m, 1H), 6.38 (d, J = 4.0 Hz, 1H), 4.45 (m, 1H), 4.05 (t,
J = 8.0 Hz, 2H), 3.69 (m, 1H), 3.03-3.01(m, 2H), 2.82-2.78 (m, 1H), 2.55–2.44 (m, 4H), 1.92–1.69 (m, 6H),
1.41–1.28 (m, 8H). HRMS (ESI) calculated for C27H32FN2O4 [M + H]+, 467.2341; found, 467.2335.
3.4. Biological Studies
Ethics Statement
Chinese Kun Ming (KM) Mice (20
±
2.0 g) and Sprague-Dawley (SD) rats (250
±
5.0 g) were used
as experimental animals in this study. Animals were housed under standardized light and temperature
conditions and received standard rat chow and tap water at libitum. Animals were randomly assigned
to different experimental groups and each group was kept in a separate cage. All the research involving
animals in this study follows the guidelines of the bylaws on experiments on animals, and has been
approved by the Ethics and Experimental Animal Committee of Jiangsu Ocean University (Project
identification code:2020002, date of approval: 8 January 2020). For the procedural details of the
biological studies see the Supplementary Materials.
4. Conclusions
In summary, a series of new flavone derivatives were synthesized based on heterocyclic
and aromatic piperazines (piperidine), and their antipsychotic activities were evaluated. Of these
compounds, 6j was favorable for binding to multiple acceptors with the KI value of D2 (12.1 nM),
D3 (25.4 nM), 5-HT1A (9.7 nM) and 5-HT2A (3.2 nM), respectively. On the other hand, 6j showed
low potency for 5-HT2C, α1, H1 receptors and human ether-a-go-go-related gene (hERG) channel
(IC50 3769.8 nM), which are closely related to the adverse effects of existing antipsychotics.
In addition, compound 6j exhibited significant inhibition of schizophrenia-like symptoms (APO
and MK-801-induced motor behavior) without causing catalepsy. Furthermore, compound 6j with
its high value of ED50 (58.4 mg/kg) in catalepsy and LD50 (>2000 mg/kg) acts at a low dose in

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apomorphine-induced climbing test (ED50 0.19 mg/kg), which results in a wider therapeutic index
range compared with the typical antipsychotics clozapine and risperidone. Thus, compound 6j has
great potential in the treatment of schizophrenia as a novel multi-target antipsychotic drug.
Supplementary Materials: Receptor Binding Studies; Intrinsic activity assessment; hERG Affinity; Acute toxicity;
Behavioral studies; 1H-NMR, 13C-NMR and HR-MS of 6j.
Author Contributions: Data curation, L.G., Z.Y., and J.X.; Investigation, L.G., J.X., C.H., J.J. and Z.Y.; Methodology,
L.G., Z.Y., Y.C., R.M., and G.Z.; writing—original draft preparation, L.G. and Y.C.; writing—review and editing,
Y.C., and G.Z.; supervision, G.Z., Y.C., B.-F.L. and X.L. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was undertaken as part of the Six Talent Peak project in Jiangsu Province (grant
2019-SWYY-128) and the Key Research and Development Program of Lianyungang High-Tech District (ZD201916).
Acknowledgments: We are grateful for support Jiangsu Nhwa Pharmaceutical Co., Ltd. and the Priority Academic
Program Development of Jiangsu Higher Education Institutions of China.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).