Synthesis and biological evaluation of a new class of multi-target heterocycle piperazine derivatives as potential antipsychotics

Article abstract of DOI:10.1039/d1ra02426d

In this study, we designed and synthesized a novel series of multi-receptor ligands as polypharmacological antipsychotic agents by using a multi-receptor affinity strategy. Among them,3wcombines a multi-receptor mechanism with high mixed affinities for D<

Full text of DOI:10.1039/d1ra02426d

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Synthesis and biological evaluation of a new class
of multi-target heterocycle piperazine derivatives
as potential antipsychotics†
Cite this: RSC Adv., 2021, 11, 16931
Lanchang Gao,‡ab Chao Hao,‡b Ru Ma,a Jiali Chen,b Guisen Zhangab
a
*
and Yin Chen
In this study, we designed and synthesized a novel series of multi-receptor ligands as polypharmacological
antipsychotic agents by using a multi-receptor affinity strategy. Among them, 3w combines a multi-
receptor mechanism with high mixed affinities for D2, 5-HT1A, 5-HT2A and H3 receptors, and low efficacy
at the off-target receptors (5-HT2C, H1 and a1 receptor) and human ether-a-go-go-related gene (hERG)
`
channel. In addition, compound 3w exhibits favorable antipsychotic drug-like activities in in vivo
assessment. An animal behavioral study revealed that compound 3w significantly reverses apomorphine-
induced climbing and MK-801-induced hyperactivity, and avoidance behavior in the CAR test, with
a high threshold for catalepsy. Moreover, compound 3w demonstrates memory enhancement in a novel
object recognition task and low liabilities for weight gain and hyperprolactinemia in a long-term
metabolic adverse effects model. Thus, 3w was selected as an antipsychotic candidate for further
development.
Received 26th March 2021
Accepted 26th April 2021
DOI: 10.1039/d1ra02426d
rsc.li/rsc-advances
improvement of negative cognitive symptoms and in promoting
relapse prevention and cognitive impairment. However,
1. Introduction
patients who take antipsychotic drugs still suffer from long-
term side effects, such as QT (Q wave and the end of the T
wave on electrocardiograms) interval prolongation, hyper-
prolactinemia and weight gain.4 Therefore, there is still a great
clinical need for the development of safer, more effective novel
antipsychotics.
Schizophrenia is a chronic and complex psychotic mental
disorder that affects around 1% of people. It is characterized by
a combination of positive, negative and cognitive impairment.1
Although current antipsychotics on the market have brought
about great progress in the treatment of schizophrenia, such as
typical antipsychotics (chlorpromazine and haloperidol, Fig. 1)
that have been proven to be effective treatments in controlling
positive symptoms, their strong and nonselective blockade of
dopaminergic transmission causes numerous side effects, such
as tardive dyskinesia (TD), extrapyramidal symptoms (EPS) and
hyperprolactinemia, and even exacerbates negative and cogni-
tive symptoms.2,3 Atypical antipsychotics, such as clozapine and
risperidone, are less tightly bound to the dopamine D2 receptor.
Besides this, their affinities for various 5-hydroxytryptamine (5-
HT) receptors means that they display more clinical advantages
over typical antipsychotics in the treatment of positive symp-
toms, as well as having small effect size advantages in the
From the perspective of targets, activation of the 5-HT1A
receptor in the frontal cortex will enhance the functions of the
mesocortical dopamine pathway, which may improve the
negative symptoms and cognitive decits in patients who have
schizophrenia.5,6 An inverse agonist of 5-HT2A could counteract
excessive D2 receptor blockade, which is not only conducive to
alleviating extrapyramidal effects but also enhances the efficacy
against negative symptoms.7–9 Also, there is plenty of evidence
that strongly supports that combined effects on D2 and 5-HT2A
receptors are benecial to the improvement of both negative
symptoms and symptoms positive of schizophrenia.10,11
Currently, a large number of literature studies have detailed the
design of multi-target ligands that can simultaneously modu-
late and balance their activities at several specic targets to
overcome the shortcomings of conventional antischizophrenic
drugs, such as the novel antipsychotics aripiprazole,12 brexpi-
prazole and cariprazine,13,14 which differ from previous anti-
psychotics in that they are more inclined to achieve balance and
coordinate multiple biological targets (Fig. 1). For example,
both brexpiprazole and cariprazine modulate and stabilize DA
(dopamine) neurotransmission via synergistic effects rather
aJiangsu 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. E-mail: 2019000015@jou.
edu.cn; Fax: +86-518-85586628; Tel: +86-518-85586628
bDepartment of Biomedical Engineering, College of Life Science and Technology,
Huazhong University of Science and Technology, Wuhan 430074, China
† Electronic supplementary information (ESI) available: Receptor binding studies;
hERG affinity; intrinsic activity; acute toxicity; behavioral studies; 1H NMR, 13C
NMR and HR-MS of 3w. See DOI: 10.1039/d1ra02426d
‡ These authors contributed equally to this work.
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Fig. 1 The structure of representative antipsychotic drugs and representative compounds from our previous study.
than by complete antagonism of D2 alone.15,16 In addition, these giving them an advantage over most of the conventional anti-
drugs are partial agonists of D2, 5-HT1A, 5-HT2A, and 5-HT2C schizophrenic drugs.23,24
(particularly of 5-HT1A and 5-HT2A) and exhibit higher levels of
Inspired by these ndings, here we describe a series of new
17,18
intrinsic activity at 5-HT1A
.
These combined actions effec- compounds prepared using a molecular hybridization method,
tively relieve the behavioral and psychological symptoms of the design concept of which is shown in Fig. 2. Their general
schizophrenia, signicantly reduce severe side effects, are structure consists of benzothiophenylpiperazine, which is
associated with a favorable safety prole, and improve cognitive a crucial DA and 5-HT pharmacophore of the novel antipsy-
symptoms to some extent.19
chotic brexpiprazole, and
a
special pharmacophore
As schizophrenia has varied and numerous different symp-
toms, developing multi-target ligands with
a
poly-
pharmacological prole has become a widely used therapeutic
approach.20 Also, exploring novel multi-target ligands that can
accurately modulate and balance activities at dopaminergic and
serotoninergic receptors, especially the D2, 5-HT1A and 5-HT2A
receptors, and simultaneously decrease the affinity for other off-
target receptors associated with side effects has been our long-
standing research interest.21,22 In our previous study, we
explored a series of multi-target ligands, as shown in Fig. 1,
where compounds A and B exhibited a high affinity for dopa-
mine D2 and serotonin 5-HT1A, 5-HT2A, 5-HT6 receptors, and low
affinity for 5-HT2C, histamine H1, and adrenergic a1, which can
be regarded as related to side effects. Both compound A and B
`
showed negligible effects on the human ether-a-go-go-related
gene channel (hERG; associated with QT interval prolonga-
tion). In the study of animal models, compounds A and B show
favorable anti-schizophrenic activity, such as markedly inhibi-
ted APO (apomorphine)-induced hyperlocomotion, and MK-
801-induced hyperactivity. In addition, compounds A and B
display a high threshold for acute toxicity, a low tendency to
induce catalepsy, as well as negligible side effects (hyper-
prolactinemia and weight gain) when compared to risperidone.
To our satisfaction, compounds A and B display precognition
properties in the novel object recognition task (NOR) in rats,
Fig. 2 The design of new benzothiophenylpiperazine derivatives. The
privileged structure benzothiophenylpiperazine acts as a base moiety,
and aralkylpyrrolidine and piperidine or piperazine fragments that
connect the benzene rings via a methylene group act as variable
modifiers. The two parts are connected via a flexible chain to form new
derivatives.
16932 | RSC Adv., 2021, 11, 16931–16941
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Scheme 1 Reagents and conditions: (i) K2CO3, 1-bromo-3-chloropropane, acetone, rt, 24 h; (ii) acetone, K2CO3, 2-bromoethanol, reflux, 10 h;
(iii) SOCl2, DCM, reflux, 3 h; (iv) pyrrolidine/piperidine/morpholine/N-methylpiperazine/4-piperazinyl quinoline, (CH3COO)3BHNa, DCM, 0 ꢁC–rt,
24 h or MeOH, 0 ꢁC–rt, 24 h; (v) K2CO3, KI, CH3CN, 80 ꢁC, 12 h.
(pyrrolidine, piperidine or morpholine connected to a benzene derivatives in the presence of sodium triacetoxyborohydride.
ring via a methylene group), derived from reported H3 and 5- The desired compounds 3a–3y were synthesized via the reaction
HT6 receptor antagonists (pitolisant, compounds C and E SB- of 1a or 1b and 2a–2y using anhydrous potassium carbonate as
7424) that showed potential to improve cognitive decits in a base and a trace of potassium iodide as a catalyst.
clinical trials,25–27 expect that a mixed DA/5-HT/H3 receptor
affinity prole is benecial for the cognitive function of
schizophrenia. Finally, the two central pharmacophores are 2.2. Biological studies
connected via an appropriate linker, according to the literature,
2.2.1. Ethics statement. Chinese Kun Ming (KM) mice (20
as well as our own experience. Besides this, the effects that
ꢀ 2.0 g) and Sprague-Dawley (SD) rats (250 ꢀ 5.0 g) were used as
different substituents on the benzene ring of the new
experimental animals in this study. The animals were housed
under standardized light and temperature conditions and
received standard rat chow and tap water ad libitum. Animals
compounds have on the receptor affinity were investigated.
Subsequently, these new derivatives were subject to in vitro
evaluation and in vivo behavioral studies. Among these new
were randomly assigned to different experimental groups and
derivatives, compound 3w demonstrates a high level of multi-
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
target activities at D2, 5-HT1A, 5-HT2A, H3 and lacks the recep-
tors associated with side effects (5-HT2C, a1, and H1).28–30 In
behavioral studies, compound 3w was found to signicantly
attenuate MK-801-induced hyperlocomotion as well as
Ocean University (Project identication code: 2020002, date of
apomorphine-induced climbing, exhibiting negligible liability
approval: 8 January 2020). For the procedural details of the
in terms of inducing weight gain, and resulted in no signicant
biological studies see the ESI.†
hyperprolactinemia compared to risperidone. In addition, NOR
testing demonstrated that compound 3w shows pro-cognitive
properties in rats, which conform to the original expectations.
Thus, the present study identies compound 3w as a potential
antipsychotic candidate for the treatment of schizophrenia.
2.3. In vitro evaluation of new compounds
2.3.1. Structure–affinity
relationships
studies.
As
mentioned above, the novel compounds in the present study
were rationally designed based on available multi-target anti-
psychotic drugs and the privileged structures of the reported H3
and 5-HT6 receptor antagonists, and the SARs of the new
derivatives were preliminarily evaluated in vitro (the affinities
2. Results and discussion
2.1. Chemistry
The general procedures for the synthesis of the intermediates for D2, 5-HT1A, 5-HT2A, 5-HT6, H3 receptors). The compounds
(1a, 1b, 2a–2y) and novel compounds 3a–3y are illustrated in with the best activities were selected and subjected to safety and
Scheme 1. The substitution of alkyl groups into the aryl- behavioral studies.
piperazines was achieved through the reaction of benzothio-
The effect of modications on the functional activities of the
phenylpiperazine with 1-bromo-3-chloropropane in the pres- ve target receptors was preliminarily evaluated at the begin-
ence of anhydrous potassium carbonate to afford 1a. The ning of this study. As the results in Table 1 show, all of the
benzothiophenylpiperazine reacts with 2-bromoethanol compounds in the present study (3a–3y) exhibit affinities for D2,
following reux in a mixture of thionyl chloride and dichloro- 5-HT1A and 5-HT2A to some extent, especially compounds 3t and
methane to given intermediate 1b. The intermediates 2a–2y 3w, which exhibit high affinity for the D2, 5-HT1A and 5-HT2A
were obtained using p-hydroxybenzaldehyde and its derivatives receptors, attributed to the privileged structure of
to react with pyrrolidine, piperidine, morpholine and their benzothiophenylpiperazine.
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Table 1 Binding affinities for the D2, 5-HT1A, 5-HT2A, 5-HT6 and H3 receptors of the compounds 3a–3y
Receptor affinity Ki ꢀ SEMa (nM)
Cmpd
3a
R1
n
1
1
R2
NR3R4
D2
5-HT1A
5-HT2A
5-HT6
H3
CH3
H
H
94.1 ꢀ 5.6
154.7 ꢀ 10.5
163.8 ꢀ 13.0
205.6 ꢀ 23.0
276.4 ꢀ 24.2
463.5 ꢀ 43.0
481.9 ꢀ 52.2
61.3 ꢀ 3.9
66.9 ꢀ 3.0
3b
CH3
95.6 ꢀ 11.2
100.1 ꢀ 7.0
98.7 ꢀ 6.8
3c
3d
3e
3f
F
1
1
1
1
1
0
H
F
154.6 ꢀ 15.2
144.1 ꢀ 10.4
157.3 ꢀ 9.8
184.6 ꢀ 16.3
149.0 ꢀ 11.7
354.6 ꢀ 19.3
258.6 ꢀ 14.0
262.4 ꢀ 17.5
248.6 ꢀ 22.9
268.5 ꢀ 20.2
128.7 ꢀ 15.1
156.9 ꢀ 12.6
382.8 ꢀ 34.6
354.2 ꢀ 65.8
418.5 ꢀ 85.4
375.6 ꢀ 58.3
462.4 ꢀ 55.7
764.3 ꢀ 94.6
92.9 ꢀ 3.6
54.4 ꢀ 2.0
58.9 ꢀ 5.5
75.30 ꢀ 8.5
62.5 ꢀ 5.0
164.3 ꢀ 14.5
H
Cl
H
Cl
H
H
101.2 ꢀ 10.1
120.1 ꢀ 15.0
99.5 ꢀ 8.4
H
3g
3h
OCH3
OCH3
82.5 ꢀ 5.8
3i
H
1
1
1
1
1
1
H
91.1 ꢀ 4.5
138.2 ꢀ 30.1
249.6 ꢀ 37.5
197.4 ꢀ 15.5
110.2 ꢀ 8.9
125.2 ꢀ 10.2
134.7 ꢀ 10.5
305.4 ꢀ 28.2
394.5 ꢀ 38.2
285.4 ꢀ 15.7
213.2 ꢀ 24.3
253.1 ꢀ 23.1
185.6 ꢀ 20.1
324.4 ꢀ 27.5
286.1 ꢀ 19.3
175.4 ꢀ 25.3
145.9 ꢀ 11.5
152.71 ꢀ 15.6
384.0 ꢀ 51.3
385.4 ꢀ 51.0
403.7 ꢀ 87.8
389.5 ꢀ 63.2
364.5 ꢀ 55.6
375.4 ꢀ 42.2
53.0 ꢀ 6.3
85.0 ꢀ 9.8
103.2 ꢀ 24.6
88.4 ꢀ 10.1
81.5 ꢀ 6.5
89.4 ꢀ 10.6
3j
CH3
H
H
3k
3l
CH3
H
F
3m
3n
H
F
Cl
H
3o
3p
3q
3r
H
1
1
1
1
1
1
1
Cl
H
H
H
H
F
155.3 ꢀ 13.0
144.2 ꢀ 15.0
138.2 ꢀ 20.7
90.2 ꢀ 12.0
78.5 ꢀ 16.5
24.6 ꢀ 8.7
200.4 ꢀ 22.1
228.5 ꢀ 25.8
210.3 ꢀ 31.6
14.6 ꢀ 2.0
176.5 ꢀ 14.5
197.6 ꢀ 16.9
205.5 ꢀ 24.7
12.4 ꢀ 1.3
395.7 ꢀ 48.8
415.5 ꢀ 81.3
438.3 ꢀ 58.1
295.9 ꢀ 42.6
304.0 ꢀ 38.6
322.2 ꢀ 53.9
356.3 ꢀ 67.2
95.7 ꢀ 8.2
49.3 ꢀ 36.5
47.2 ꢀ 6.4
56.2 ꢀ 5.1
61.0 ꢀ 5.8
54.2 ꢀ 7.3
43.1 ꢀ 3.5
OCH3
H
CH3
F
3s
3t
14.8 ꢀ 28.6
11.7 ꢀ 1.9
11.5 ꢀ 1.5
H
12.0 ꢀ 2.4
3u
H
Cl
85.6 ꢀ 9.6
145.9 ꢀ 15.3
156.7 ꢀ 19.1
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Table 1 (Contd.)
Receptor affinity Ki ꢀ SEMa (nM)
Cmpd
R1
n
R2
H
NR3R4
D2
5-HT1A
5-HT2A
5-HT6
H3
3v
OCH3
1
73.0 ꢀ 6.4
128.4 ꢀ 12.6
118.4 ꢀ 10.4
341.7 ꢀ 64.5
48.2 ꢀ 4.2
3w
3x
H
H
1
1
H
H
17.5 ꢀ 1.9
16.7 ꢀ 1.8
5.6 ꢀ 6.5
336.4 ꢀ 65.3
313.6 ꢀ 61.0
12.1 ꢀ 1.5
86.2 ꢀ 15.0
109.2 ꢀ 12.4
119.5 ꢀ 15.4
39.4 ꢀ 26.0
3y
H
1
H
30.8 ꢀ 5.6
58.5 ꢀ 4.2
83.4 ꢀ 9.7
33.1 ꢀ 5.8
753.1 ꢀ 98.5
a
Ki values obtained from three experiments, recorded as means ꢀ SEM.
The effects of N-heterocycles (aralkyl pyrrolidine, piperidine, and electron-withdrawing (–F, –Cl, –OCH3) groups were intro-
morpholine and piperazine fragments) and 8-(piperazin-1-yl) duced at different positions of the benzene ring, of the
quinoline connected to a benzene ring were assessed, and the compounds 3a–3i that bear N-methyl pyrrolidinyl substituents,
results suggest that the N-heterocycles play a signicant role in the substituted compounds 3a–3f display decreased affinities
the SARs. According to the results, compounds (3a–3h) with for three receptors (D2, 5-HT1A and 5-HT2A) compared with 3i,
pyrrolidineyl disubstitution show higher affinity for the D2 with only the –OCH3-substituted derivative 3g showing
receptor than compounds with piperidyl substitution (3j–3q), increased affinity for the 5-HT1A receptor. However, the pres-
but show lower affinity for the D2 receptor than compounds ence of different substituents on the benzene ring does not have
with morpholinyl, piperazinyl and 8-(piperazin-1-yl)quinolineyl any obvious impact on the affinity for the 5-HT6 and H3 recep-
substitution. Similar results were also observed for the 5-HT1A tors. In terms of the compounds bearing N-methyl piperidyl
receptor. Except for the compounds with piperazinyl (3x) and 8- substituents (3j–3q), meta and ortho positioned –CH3
(piperazin-1-yl)quinolineyl substitution (3y), most of the substituted 3j and 3k exhibit diminished affinities for the 5-
compounds show moderate to low affinity for the 5-HT2A HT1A and 5-HT2A receptors. However, when –F, –Cl, –CH3, and
receptor. Compounds 3a–3x show moderate to high affinity for –OCH3 groups are introduced to the N-methyl morpholinyl
the H3 receptor, but only 3y exhibits high affinity for the 5-HT6 substituted derivatives, the formed compounds 3r–3v show
receptor, which may be due to the introduction of the privileged a decline in their affinities for the D2 receptor. Compared to
structures in the H3 and 5-HT6 receptor antagonists.
compound 3w, –F and –CH3-substituted 3r–3t show improved
In the present study, the effects that different electron- affinities for 5-HT1A, but a negative effect for 5-HT2A receptors.
withdrawing group of the substituents of the benzene ring, such Disappointingly, –Cl and –OCH3 group substituted 3u and 3v
as –F, –Cl, –CH3, and –OCH3 groups, have on the various exhibit a dramatic decline in affinities for the D2, 5-HT1A and 5-
receptors were also evaluated. When electron-donating (–CH3) HT2A receptors, and compounds with –F, –Cl, –CH3, and –OCH3
Table 2 Activities on the 5-HT2C, H1 and a1 receptors (Ki nM) of the candidate and reference antipsychotics
Receptor affinity Ki ꢀ SEMa (nM)
Cmpd
5-HT2C
a1
H1
hERG IC50 (nM)
3t
3w
430.4 ꢀ 52.3
572.2 ꢀ 91.5
30.1 ꢀ 2.8
398.2 ꢀ 49.5
405.5 ꢀ 60.8
51.4 ꢀ 3.6
668.3 ꢀ 32.5
819.6 ꢀ 48.7
23.8 ꢀ 1.5
1158
1765
1480
Risperidone
a
Ki values obtained from three experiments, recorded as means ꢀ SEM.
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substituents (3s–3v) show slight differences in affinity for the 5- potassium channel mediates the delayed rectied potassium
HT6 receptor and less H3 affinity. current and drugs that inhibit the hERG channel may cause
Based on the literature and our previous experience, the prolonged QT intervals34 and increase the occurrence of
space between benzothiophenylpiperazine and the phenoxy potentially lethal Torsades de Pointes arrhythmia.35 Thus, the
structure features three carbons. On replacement of the chain inhibition of hERG is an effective and widely used indicator for
lengths of 3g from three carbons to two carbons, the generated the prediction of the cardiotoxicity of candidate drugs. To
compound 3h displays diminished affinities for the 5-HT1A, 5- predict the cardiotoxicity of compounds 3t and 3w, their
HT2A, 5-HT6 and H3 receptors, but slightly enhanced affinity for inhibitory actions on the hERG channel were evaluated in
D2 receptor.
a patch-clamp assay in vitro. Compound 3w (IC50 ¼ 1765 nM)
In order to obtain compounds that show balance and high displays a lower hERG channel inhibition than compound 3t
affinity, as well as high selectivity for further biological evalua- (IC50 ¼ 1158 nM) (Table 2), which suggests that compound 3w
tion, herein, we set up three primary selection lter conditions: has a low propensity to elicit treatment-induced QT interval
(a) high potency for the D2, 5-HT1A and 5-HT2A receptors (D2, Ki prolongation than 3t and risperidone (IC50 ¼ 1480 nM).
# 30 nM; 5-HT1A, Ki # 20 nM; and 5-HT2A, Ki # 20 nM); (b) an
To sum up, compound 3w displays excellent in vitro proles
affinity for H3 # 15 nM and 5-HT6 # 350 nM (c) with balanced with favorable affinity for the desired target receptors (D2, 5-
activities at the D2, 5-HT1A and 5-HT2A receptors, with a potency HT1A, 5-HT2A, H3 and 5-HT6) and lower affinity for the off-target
ratio for any two receptors of #2. As a result, compounds 3t and receptors (5-HT2C, H1 and a1) and hERG channel. Therefore,
3w were selected for further biological evaluation, including compound 3w was subjected to intrinsic activity, safety assess-
safety and behavioral studies.
ment and animal behavioral studies to verify its effect on
2.3.2. The receptor selectivity of the selected compounds. schizophrenia.
Compared to conventional antipsychotics, atypical antipsy-
chotics signicantly reduce the possibilities of unwanted effects
2.5. Intrinsic activity
such as EPS and tardive dyskinesia (TD; 47). However, these
drugs still have pharmacological effects on off-target receptors,
such as 5-HT2C, H1, and a1, which are related to many
Compound 3w was chosen for further functional characteriza-
tion because of its excellent in vitro activity and favorable safety
prole. The results (ESI† Table 1) show that compound 3w
displays feeble agonist activity against the D2, 5-HT1A, 5-HT2A, 5-
HT6 and H3 receptors. In the antagonist assays, compound 3w
shows potent antagonism of the ve receptors, higher than 85%
efficiency. Compound 3w shows potent D2 (IC50 ¼ 11.6 nM)
antagonism, and moderate 5-HT1A (IC50 ¼ 218.5 nM), 5-HT2A
(IC50 ¼ 141.8 nM), 5-HT6 (IC50 ¼ 413.5 nM) and H3 (IC50 ¼ 232.3
nM) antagonism.
medication-related
adverse
events.31
For
example,
antipsychotic-induced weight gain is probably caused by
a synergistic effect on the 5-HT2C and H1 receptors32 and the
inhibition of the a1 receptor is presumed to be associated with
orthostatic hypotension and rhythm problems.33 Thus, assays of
the 5-HT2C, H1 and a1 receptors were also conducted in this
study. As the results in Table 2 show, the affinities of
compounds 3t and 3w for the 5-HT2c, H1, and a1 receptors are
far lower than those of risperidone and haloperidol, and 3w (5-
HT2c, Ki ¼ 572.2 nM; a1, Ki ¼ 450.5 nM; H1, Ki ¼ 819.6 nM)
exhibits lower affinities for the three receptors than 3t (5-HT2c,
Ki ¼ 430.4 nM; a1, Ki ¼ 398.2 nM; H1, Ki ¼ 668.3 nM), which
suggests that 3w is less likely to be associated with side effects
such as weight gain, as well as with a lower incidence of
orthostatic hypotension.
2.6. Acute toxicity
The acute toxicities of 3w were assessed based on lethal dose,
50% (LD50) analyses (Table 3). The LD50 value of 3w is over
1000 mg kgꢂ1, whereas those of haloperidol and risperidone are
21.0 and 82.0 mg kgꢂ1, respectively. Thus, 3w has a better safety
prole than haloperidol and risperidone.
2.4. hERG channel blockade
2.7. Evaluations of antipsychotic drug-like activities in
animal models
Cardiotoxicity is a serious side effect that usually occurs due to
the interactions between drugs and various voltage-gated ion Based on in vivo and acute oral toxicity evaluation, compound
channels in the heart, particularly the hERG channel. The hERG 3w was chosen as a candidate for in vivo behavioral study and
Table 3 In vivo pharmacological profile of the compounds
Cmpd
LD50
APOa
MK-801b
CATc
CARd
CAT/APO
CAT/MK-801
3w
>1000
21.0
82.0
0.28
0.09
0.05
0.17
0.11
0.02
27.5
0.12
0.51
2.1
—
0.65
91.07
5.67
10.4
161.76
4.63
32.5
Haloperidol
Risperidone
a
APO: apomorphine-induced climbing (ED50, mg kgꢂ1, po (per os)). b MK-801: MK-801-induced hyperactivity (ED50, mg kgꢂ1, po). c CAT: catalepsy
(ED50, mg kgꢂ1, po). CAR: conditioned avoidance response (ED50, mg kgꢂ1, po).
d
16936 | RSC Adv., 2021, 11, 16931–16941
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Fig. 3 (A) Effect of compound 3w on APO-induced climbing in mice (10 per group). The scores are shown as means ꢀ SEM. Statistical
significances were performed using a nonparametric two-tailed Mann–Whitney U-test: *p < 0.05 and **p < 0.01 versus apomorphine treatment;
#p < 0.05 versus vehicle treatment. (B) Effect of compound 3w on MK-801-induced hyperactivity in mice (10 per group). The distance expressed
as the means ꢀ SEM. Statistical evaluation was performed via two-way ANOVA followed by Tukey's test for multiple comparisons. *p < 0.05 and
**p < 0.01 vs. MK-801 treatment; #p < 0.05 vs. vehicle treatment.
long-term side effects evaluation to verify its antipsychotic-like 3w has the antipsychotic-like efficacy of attenuating the nega-
activities.
2.7.1. Apomorphine-induced climbing. The climbing
tive and cognitive symptoms of schizophrenia.
2.7.3. Potential for catalepsy. A signicant risk factor
behavior induced by D2 receptor agonists, such as apomorphine associated with schizophrenia treatment is that antipsychotics
is commonly used as a rodent model of psychoses.36 Therefore, have the tendency to trigger EPS side effects in humans. To
compounds normalizing climbing behavior demonstrate predict the likelihood that a novel agent will produce EPS-like
antipsychotic-like properties. According to the results of this side effects, the rodent catalepsy test has become a commonly
model (Table 3 and Fig. 3A), compound 3w reverses APO- used model in antipsychotic drug discovery studies.38 Thus,
induced climbing, with an ED50 (50% effective dose) value of compound 3w was subject to the horizontal bar test in mice to
0.28 mg kgꢂ1. As positive control drugs, haloperidol and ris- predict its tendency for striatal-mediated side effects and
peridone display signicant inhibitory effect on the climbing
behavior, with ED50 values of 0.09 and 0.05 mg kgꢂ1, respec-
tively. This indicates that compound 3w is a potent antagonist
of the D2 receptor in vitro, which would likely alleviate or
eliminate the positive symptoms of schizophrenia.
2.7.2. MK-801-induced hyperlocomotion.
N-Methyl-D-
aspartate (NMDA) receptors are involved in many important
physiological functions in the central nervous system (CNS) and
are also important targets in the therapy of several neuropsy-
chiatric diseases. Moreover, abnormalities in this receptor
system may cause CNS dysfunction and many non-competitive
NMDA receptor antagonists (such as PCP, MK-801 and ket-
amine) produce schizophrenia-like symptoms (negative and
cognitive symptoms) in healthy animals. However, compounds
with antipsychotic-like efficacies signicantly reverse these
symptoms.37 Therefore, the NMDA receptor antagonist MK-801-
induced hyperactivity model is oen used to assess the poten-
tial antipsychotic-like efficacy of new compounds. In this assay,
compound 3w attenuates the MK-801-induced hyperactivity
signicantly in mice, with an ED50 value of 0.17 mg kgꢂ1. In the
control group, mice that receive oral administrations of halo-
peridol and risperidone show decreased locomotor activities,
with ED50 values of 0.11 and 0.02 mg kgꢂ1, respectively (Table 3
and Fig. 3B). To sum up, these results indicate that compound
Fig.
4 Effect of compound 3w on the conditioned avoidance
response in rats (10 per group). The percentage of avoidance is shown
as the mean ꢀ SEM, and the level of significance is *p < 0.05 and **p <
0.01 vs. the vehicle group. Statistical analysis was performed using the
SPSS software.
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cataleptogenic potential. In this test, haloperidol (ED50: 0.51 mg ED50 of 2.1 and 0.65 mg kgꢂ1. This indicates that compound 3w
kgꢂ1) and risperidone (ED50: 1.15 mg kgꢂ1) induce signicant shows high efficacy towards DA receptors in vitro, which may
cataleptic effects (Table 3), which is in line with their strong bring about therapeutic effects in the treatment of
antagonistic effect on the D2 receptor in vitro. In comparison, schizophrenia.
compound 3w exhibits a low potential cataleptogenic effect
2.7.5. Memory study. The NOR task is based on an animal's
(ED50: 27.5 mg kgꢂ1), which infers that 3w has a higher innate preference for novelty, and this model is oen used to
threshold for catalepsy and lower incidence of EPS. In addition, predict the potential of a novel drug for cognitive enhance-
compound 3w exhibits a broad therapeutic index that ranges ment.41,42 In this study, oral administration of compound 3w
from 91.07 to 161.76, based on its efficacy (apomorphine and (1.0–10.0 mg kgꢂ1) has no obvious effect on the total exploration
MK-801 models) and its adverse effects (catalepsy). In the time during the acquisition trial (Fig. 5A) compared with the
positive control groups, the therapeutic index of haloperidol is blank and control groups (risperidone and rivastigmine). In the
between 4.63 and 5.67, while the therapeutic index of risper- retention trial (Fig. 5B), the rats that received rivastigmine
idone is in the range of 10.4–32.5.
exhibit a longer exploration (0.1 to 3.0 mg kgꢂ1) time for novel
2.7.4. Conditioned avoidance response (CAR) tests. The things compared with the vehicle conditions, suggesting that
conditioned avoidance response (CAR) test is a sensitive the memory acquired from the acquisition trial is preserved.
method for evaluating antipsychotics that have a high affinity However, there is no signicant difference in the exploration
for DA receptors and it can also be used to screen for antipsy- times for the novel and familiar objects when the rats are given
chotics that primarily affect neurotransmitter receptors.39,40 In risperidone (0.02 to 0.2 mg kgꢂ1) and a low dose of compound
this model, the results show that risperidone and compound 3w 3w (0.1 mg kgꢂ1), indicating that risperidone and a low dose of
effectively suppress the CAR in rats (Table 3 and Fig. 4), with an compound 3w have little or no effect on memory enhancement.
Fig. 5 Effects of compound 3w on the NOR testing in rats (10 per group). Experimental results acquired 60 min after oral administration of
vehicle risperidone (0.02, 0.06, 0.2 mg kgꢂ1), rivastigmine (0.1, 0.3, 1.0 mg kgꢂ1), and 3w (0.1, 0.3, 1.0 mg kgꢂ1). (A) Exploration times in the
acquisition trial; (B) exploration times for the familiar and a novel object during acquisition trials; and (C) novelty discrimination index (NDI) in the
retention trial. Data are presented as the mean ꢀ SEM (n ¼ 10). **p < 0.01 and *p < 0.05 compared with a familiar object via a paired test; #p < 0.05
and ##p < 0.01 compared with the vehicle group.
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Fig. 6 Effects of 3w and risperidone on body weight (A) and serum prolactin (PRL) (B) in mice after 28 days of administration (10 per group). The
weight gain and prolactin are presented as the means ꢀ SEM. Statistical evaluation was performed via a student's t test. *p < 0.05 and **p < 0.01
versus the vehicle group.
Additionally, the rats receiving 0.3 and 1.0 mg kgꢂ1 oral doses of inhibitory activity. The multi-receptor antagonist prole of 3w
compound 3w exhibit an obvious increase in the novelty means that it has a signicant effect in the inhibition of
discrimination index (NDI) as well as those that received 0.1 and schizophrenia-like symptoms, including reversing APO-induced
0.3 mg kgꢂ1 of rivastigmine, whereas under the vehicle condi- hyperlocomotion and MK-801-induced hyperactivity in mice
tions or dosed with 0.02 to 0.2 mg kgꢂ1 of risperidone the rats and restraining the CAR in rats, and has a high threshold in
do not show increased NDI (Fig. 5C). To sum up, these results terms of acute toxicity and a lower tendency to induce catalepsy.
demonstrate that compound 3w improves recognition memory Moreover, compared to the representative conventional anti-
in this model.
psychotic risperidone and the memory-enhancing rivastigmine,
Taken together, compound 3w exhibits good anti- compound 3w demonstrates superior effectiveness in cognitive
schizophrenic activity in terms of the above-mentioned behav- enhancement in NOR tests. Taken together, compound 3w is
ioral testing, especially in terms of memory enhancement in the similar to the compounds A and B that were reported previously
NOR testing, which is probably due to the addition of H3 by our research group, and is in alignment with our original
receptor antagonism to its mixed dopamine and 5-hydroxy- expectations. Thus, compound 3w was selected for the treat-
tryptamine receptor antagonist prole.
ment of schizophrenia and deserves further development.
Funding
2.8. Weight gain and serum prolactin levels
To determine whether compound 3w has adverse long-term
metabolic adverse, it was assessed in terms of its ability to
induce weight gain and hyperprolactinemia in an animal model
of chronic administration. Aer 28 days, the mice given
compound 3w exhibit negligible weight gain, while the mice
given risperidone show signicant weight gain (Fig. 6A and B).
Besides this, risperidone induces a signicant increase in
serum prolactin levels, but compound 3w does not, which
means that 3w shows a lower incidence of treatment-related
side effects than the representative atypical antischizophrenic
drug risperidone, which might result from its desirable selec-
tivity prole against the 5-HT2C and H1 receptors that are related
to the adverse effects of marketed antipsychotics.
We are grateful for support from the National Natural Science
Foundation of China (No. 81602971).
Conflicts of interest
The authors declare no conict of interest.
Acknowledgements
We are grateful for support from Jiangsu Nhwa Pharmaceutical
Co., Ltd. and the Priority Academic Program Development of
Jiangsu Higher Education Institutions of China.
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