Synthesis and biological evaluation of a series of multi-target N-substituted cyclic imide derivatives with potential antipsychotic effect

Article abstract of DOI:10.1016/j.ejmech.2017.12.099

In the present study, a series of multi-target N-substituted cyclic imide derivatives which possessed potent dopamine D₂, serotonin 5-HT_1A and 5-HT_2A receptors properties were synthesized and evaluated as potential antipsychotics. Among these compounds, (3aR,4R,7S,7aS)-2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione hydrochloride (3d) held a promising pharmacological profile. 3d not only showed potent and balanced in vitro activities on D₂/5-HT_1A/5-HT_2A receptors, but also endowed with low to moderate activities on 5-HT_2C, H₁, α_1A, M₃ receptors and hERG channel, suggesting a low liability to induce side effects such as weight gain, orthostatic hypotension and QT prolongation. In animal behavioral studies, 3d reduced phencyclidine-induced hyperlocomotion with a high threshold for catalepsy induction. Compound 3d was selected as a potential antipsychotic candidate for further development.

Full text of DOI:10.1016/j.ejmech.2017.12.099

Accepted Manuscript
Synthesis and biological evaluation of a series of multi-target N-substituted cyclic
imide derivatives with potential antipsychotic effect
Mingshuo Xu, Yu Wang, Feipu Yang, Chunhui Wu, Zhen Wang, Bin Ye, Xiangrui
Jiang, Qingjie Zhao, Jianfeng Li, Yongjian Liu, Junchi Zhang, Guanghui Tian, Yang
He, Jingshan Shen, Hualiang Jiang
PII:
S0223-5234(17)31130-3
10.1016/j.ejmech.2017.12.099
EJMECH 10077
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 October 2017
Revised Date: 29 December 2017
Accepted Date: 30 December 2017
Please cite this article as: M. Xu, Y. Wang, F. Yang, C. Wu, Z. Wang, B. Ye, X. Jiang, Q. Zhao, J. Li,
Y. Liu, J. Zhang, G. Tian, Y. He, J. Shen, H. Jiang, Synthesis and biological evaluation of a series of
multi-target N-substituted cyclic imide derivatives with potential antipsychotic effect, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.099.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of a series of multi-target N-substituted cyclic
imide derivatives with potential antipsychotic effect
a
a
c
a
c
a
Mingshuo Xu a, b, Yu Wang , Feipu Yang , Chunhui Wu , Zhen Wang , Bin Ye , Xiangrui Jiang ,
a
a
c
a
c
Qingjie Zhao , Jianfeng Li , Yongjian Liu , Junchi Zhang , Guanghui Tian , Yang He a, *, Jingshan
Shen a, *, Hualiang Jiang a
a CAS Key Laboratory for Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
b University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
c Topharman Shanghai Co., Ltd, 1088 Chuansha Road, Shanghai 201209, China
Abstract
In the present study, a series of multi-target N-substituted cyclic imide derivatives which
possessed potent dopamine D2, serotonin 5-HT1A and 5-HT2A receptors properties were synthesized and
evaluated
as
potential
antipsychotics.
Among
these
compounds,
(3aR,4R,7S,7aS)-2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahydro-1H-4,7-me
thanoisoindole-1,3(2H)-dione hydrochloride (3d) held a promising pharmacological profile. 3d not
only showed potent and balanced in vitro activities on D2/5-HT1A/5-HT2A receptors, but also endowed
with low to moderate activities on 5-HT2C, H1, α1A, M3 receptors and hERG channel, suggesting a low
liability to induce side effects such as weight gain, orthostatic hypotension and QT prolongation. In
animal behavioral studies, 3d reduced phencyclidine-induced hyperlocomotion with a high threshold
for catalepsy induction. Compound 3d was selected as a potential antipsychotic candidate for further
development.
Keywords: antipsychotic, cyclic imide, multi-target, 5-HT1A receptor
1 Introduction
Schizophrenia is a severe neuropsychiatric disorder affecting about 1% of the world’s
population[1]. It is characterized by three broad categories of core symptoms, e.g. positive symptoms
(delusions, hallucinations and thought disorder), negative symptoms (alogia, affective flattening,
anhedonia, avolition and apathy) and cognitive impairment[2]. Typical antipsychotics (dopamine D2
receptor antagonists) were proved to be effective merely in the treatment of positive symptoms[3].
Besides, nonselective inhibition of dopaminergic transmission can cause severe side effects, such as
extrapyramidal symptoms (EPS), tardive dyskinesia (TD), hyperprolactinemia[4] and deterioration of

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the negative and cognitive symptoms[5, 6]. In an effort to improve the efficacy and safety profile, a
class of multi-target drugs called atypical antipsychotics was discovered (Fig. 1). Atypical
antipsychotics bind not only to D2 receptor, but also various 5-hydroxytryptamine (5-HT) receptors.
These polypharmacological antipsychotic agents have demonstrated some clinical advantage over
typical D2 receptor antagonists in the treatment of negative symptoms and cognitive impairment[7, 8].
Even though atypical antipsychotics cause less EPS and are better tolerated in patients, they tend to
result in other unwanted side effects like weight gain, diabetes mellitus, hyperlipidemia and QT interval
prolongation after chronic medication[9]. As a result, development of novel antipsychotics with
broader therapeutic spectrum as well as minimal side effects is of great significance.
Fig. 1. structure of representative atypical antipsychotic drugs.
5-HT receptors are important therapeutic targets for many central nervous system diseases[10].
D2R/5-HT2AR dual antagonism is shared by all atypical antipsychotics[11]. It is asssumed that this
characteristic contributes to the substantial reduction in EPS for atypical antipsychotics[3, 12]. In the
last few years, considerable attention has been given to 5-HT1A receptor as the new generation of
atypical antipsychotics such as aripiprazole, brexpiprazole and cariprazine (Fig. 1) all exhibit affinity
for this receptor. 5-HT1A receptor is largely distributed throughout the brain, and it plays an important
role in the treatment of various neuropsychiatric disorders, such as schizophrenia[13, 14], anxiety[15,
16] and major depressive disorder[17, 18]. Additional 5-HT1A receptor agonistic activity was proved to
improve the safety profile and therapeutic effect of atypical antipsychotics. First, 5-HT1AR agonism
could alleviate the EPS side effect of antipsychotics by activating 5-HT1AR located in primary motor
cortex and dorsolateral striatum regions[19]. Second, 5-HT1A partial agonist as adjunctive therapy
could improve positive symptoms[20], which is the core symptoms of schizophrenzia. Furthermore,
5-HT1AR agonists induced dopamine release in the prefrontal cortex, thus potentiated the function of
mesocortical dopamine pathway[14]. As a consequence, negative symptoms and cognitive impairment

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might be ameliorated[21, 22]. In summary, 5-HT1AR agonism is of great benefit for the improvement of
current antipsychotic therapy.
As schizophrenia is a complex psychiatric disease with numerous different symptoms, introducing
multi-target drugs with polypharmacological profile has become a widely used therapeutic
approach[23]. Thus, developing novel muti-target antipsychotics that act on dopaminergic and
serotoninergic receptors with potent and balanced activities as well as with low activities for receptors
associated with side effects has been our long-standing research interest. Especiallly, the emphasis was
put on effect on 5-HT1A receptor due to its potential therapeutic benefit. 5-HT1A receptor agonists and
partial agonists with cyclic imide moieties (e.g. buspirone and tandospirone) have demonstrated
clinical efficacy in the treatment of psychiatric disorders. The unique feature of these compounds is a
cyclic imide fragment connected to a nitrogen-containing base moiety through a proper linker. On the
other side, marketed antipsychotic drugs, such as risperidone and brexpiprazole, are D2R/5-HT2AR dual
ligands. The arylpiperazine or arylpiperdine fragments of these antipsychotics were also shared by
many other compounds which targeted to serotonin receptors[24, 25]. By applying molecular
hybridization method for multi-target drug design, a series of new N-substituted cyclic imide
derivatives (Fig. 2) was synthesized and their activites on D2R, 5-HT1AR and 5-HT2AR were evaluated.
Among these compounds, compound 3d exhibited best in vitro activities, and demonstrated potent
antipsychotic effect in in vivo behavioral studies. As a result, compound 3d had the potential to be
developed as an antipsychotic drug.
Fig. 2. Design of new N-substituted cyclic imide derivatives.
2. Results and discussion
2.1. Chemistry

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The synthesis of compounds 3a–3v was depicted in Scheme 1. As illustrated in Scheme 1,
commercially available cyclic imide 1a–1k was first alkylated with corresponding α,ω-dihaloalkanes
under the condition of K2CO3 in N,N-dimethyl formamide (DMF) to afford intermediate 2a–2n.
Synthesis of compounds 3a–3v was accomplished by coupling 2a–2n with corresponding
arylpiperazines or arylpiperidines in the presence of K2CO3 and KI in DMF/H2O system.
Compounds 4a and 4b were obtained by dihydroxylation of olefin compounds 3r and 3d
respectively in the presence of potassium permanganate (Scheme 2)[26]. Subsequent acetylation of
compound 4b with Ac2O afforded ester derivative 5a.
The synthesis of phthalimide derivates 8a–8e was shown in Scheme 3. Commercially available
6a–6e were alkylated with 1,4-dibromobutane to provide intermediates 7a–7e, which were then
coupled with 1-(benzo[b]thiophen-4-yl)piperazine hydrochloride under mild base condition to give 8a–
8e.
1
The structure of intermediates 2a–2n, 7a–7e was confirmed by analysis of H-NMR as described
1
in supporting information. The structure of final compounds was confirmed by analysis of H-NMR
and ESI-MS spectra as described in Section 4.

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Scheme 1. Reagents and conditions: (i) 1-bromo-4-chlorobutane for 2a–2k, 1,2-dibromoethane for 2l,
1-bromo-3-chloropropane for 2m, 1-bromo-5-chloropentane for 2n, K2CO3, DMF, rt, overnight; (ii)
arylpiperazine hydrochloride, K2CO3, KI, DMF/H2O, 95 °C, 12 h.
Scheme 2. Reagents and conditions: (i) KMnO4, acetone, 0 °C then rt, 30 min; (ii) Ac2O, pyridine, rt,
24 h.
Scheme 3. Reagents and conditions: (i) 1,4-dibromobutane, K2CO3, acetone, reflux, 2 h; (ii)
1-(benzo[b]thiophen-4-yl)piperazine hydrochloride, K2CO3, KI, MeCN, reflux, 5 h.
Table 1
Functional acivities on D2, 5-HT1A and 5-HT2A receptors of compound 3a–3j.
D2R
5-HT1AR
5-HT2AR
IC50 (nM)
Cmpd
Base
Cyclic imide
IC50 (nM)
EC50 (nM)
4.8
> 100000
91.3
5.2
3a
3b
80.2
130

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6.1
6.9
3.6
3.3
10.6
2.4
3.6
6.1
14
27.5
24.6
956
3c
3d
3e
3f
6.4
9.6
2.5
5.3
9.8
2.0
4.2
82.3
32.0
854
3g
3h
3i
2840
35.8
3j
2.2. In vitro receptor functional activities and structure-activity relationship (SAR)
The influence of base moiety on functional activities of three target receptors was first evaluated
at the beginning of our study. The results were summarized in Table 1. As anticipated on the basis of
our previous study, compound 3a bearing (6-fluorobenzo-[d]isoxazol-3-yl)piperidine as base moiety
was devoid of 5-HT1AR agonistic activity. Compound 3b with 1-(benzo[b]thiophen-6-yl)piperazine as
base moiety possessed moderate but balanced activity on three receptors (IC50 or EC50 value ≈ 100 nM).
When changing piperazine substituent on benzothiophene ring from 6–position to 4–position, the
activities on three receptors showed a remarkable elevation for D2R and 5-HT1AR (3c vs 3b).
Compound 3d, the olefin analogue of 3c, displayed slightly enhanced agonistic activity on 5-HT1AR,
while retained high D2R and 5-HT2AR potency. Replacement of benzothiophene with its bioisostere,
benzo[d]thiazole, led to a sharp decline in 5-HT2AR antagonistic activity with little change on D2R and
5-HT1AR activities.(3e vs 3d). Introduction of a fluorine atom at 2-position of benzothiophene ring
resulted in a decrease in 5-HT1AR and 5-HT2AR potency by 2-fold, while D2R potency increased
slightly (3f vs 3d). Interestingly, shifting the relative position of piperazine ring on benzothiophene ring

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from 4-position to 7-position made nearly no change on receptor profile (3g vs 3d). Transforming
benzothiophene moiety to oxygen-containing benzoheterocycles like 2,3-dihydrobenzo[b][1,4]dioxine
and 2,2-difluorobenzo[d][1,3]dioxole was proved to be detrimental for 5-HT2AR antagonistic activities
as IC50 values of compounds 3h and 3i dropped to micromolar level. Finally, Compound 3j bearing
4-(1,2-benzisothiazol-3-yl)piperazine moiety showed excellent activities for all the three receptors.
Encouraged by the above results, we proceeded with our investigation by introducing different
linkers or cyclic imide moieties. 1-(benzo[b]thiophen-4-yl)piperazine was chosen as potential
pharmacophoric moiety as previous studies demonstrated that compounds bearing this pharmacophoric
core exhibited high activities for D2, 5-HT1AR and 5-HT2AR (3c and 3d in Table 1). Previous SAR
studies on psychotropic drugs have suggested that the linker length and flexibility play an important
role in determining receptor function and the tetramethylene space was generally considered as the
optimal length of spacer[27-29]. Therefore, investigation of linker moiety was focused on chain length
around four carbons. As shown in Table 2, the functional activities for D2R gradually increased as
chain length increasing from two to four carbons. Further elongation of the spacer from four carbons to
five carbons caused decrease in the D2R antagonistic activity (3m vs 3d). However, this was not the
case for 5-HT1AR and 5-HT2AR, as the potency trend following the order: (CH2)4 > (CH2)2 > (CH2)3 >
(CH2)5. Notably, compound 3m was completely devoid of 5-HT1AR agonism activity. Taken together,
four carbon chain was proved to be optimal as expected and maintained unchanged in the next
modification.
Table 2
Functional acivities on D2, 5-HT1A and 5-HT2A receptors of compounds 3d and 3k–3m.
D2R
IC50 (nM)
119
5-HT1AR
EC50 (nM)
13.8
5-HT2AR
IC50 (nM)
82.4
Cmpd
linker
3k
3l
(CH2)2
(CH2)3
(CH2)4
32.4
107
113
6.4
3.6
24.6
3d

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28
> 100000
314
3m
(CH2)5
Imide moiety is the common fragment of the azapirone psychotropic drugs, which is thought to
modulate the activity and selectivity for 5-HT1A receptor[30]. We therefore decided to explore
structural transformation of this part extensively. These compounds could be roughly divided into two
types: succinimide derivatives (compounds 3n–3r, 4a–4b, 5a, 8a–8e) and glutarimide derivatives
(compound 3s–3v). The data on functional activities were summarized in Table 3.
Table 3
Functional acivities on D2, 5-HT1A and 5-HT2A receptors of compound 3n–3v, 4a–4b, 5a and 8a–8e.
D2R
5-HT1AR
5-HT2AR
IC50 (nM)
Cmpd
IC50 (nM)
EC50 (nM)
8.4
> 100000
11.6
10.4
3n
80.6
1660
3o
O
N
13
4.3
8.5
9.4
14.1
8.8
1.7
1.1
34
3p
3q
3r
4a
4b
O
0.9
0.6
10.5
17.4
84.9
81.5

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44.6
131
38.1
213
30.1
543
7.2
3.1
21.3
1.1
546
683
318
254
282
7250
134
25.9
29.1
103
5a
8a
8b
8c
8d
8e
3s
13.3
5.8
47.9
1.6
10.9
24.4
11
1.24
7.7
3t
3u
3v
1.2
The SAR study of succinimide derivatives was emphasized on the influence of substituent size
and electric properities of the fused rings. As shown in Table 3, compound 3n with gem-dimethyl
substitution displayed good potency for D2R and 5-HT2AR, but it was completely devoid of 5-HT1AR
agonistic effect with EC50 > 100 µM. In contrast, compound 3o with a p-tolyl substituent exhibited
outstanding 5-HT1AR potency, but bulky substituent appeared to be detrimental for D2R and 5-HT2AR
activities. Replacement of the gem-dimethyl substituted cyclopropyl ring with cyclopentyl ring resulted
in higher potency for all the three receptors (3p). Further enlargement of the cyclopentyl ring to a
six-membered cyclohexyl ring had an obviously negative impact on 5-HT2AR activity (compound 3q
vs 3p). Compared with compound 3q, its olefin analogue 3r exhibited stronger activities on 5-HT1AR
and 5-HT2AR while retaining antagonistic activity on D2R, which was consistent with the result of

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compounds 3c and 3d (Table 1). This indicated that introduce of C-C double bond had some advantage,
and we presumed that the decrease in molecular flexibility of olefine compound might account for
higher potency for 5-HT1AR. However, replacement of the cyclohexyl ring with more rigid phenyl ring
led to reduced activities on all three receptors (compound 8a vs compound 3r). We speculated that the
potency decline after aromatization might be due to electronic factors as well as steric factors.
We further investigated the effect of introducing halogen atoms to the phthalimide moiety
(compounds 8b–8e, Table 3). According to the results, the 4-flouro substituted derivative 8b and
4-chloro substituted derivative 8d displayed improved activities for all the three receptors. By contrast,
5-flouro substituted derivative 8c showed increased activities for 5-HT1AR and 5-HT2AR receptors
while decreased activity for D2R, and 5-chloro substituted derivative 8e exhibited a dramatic decline in
activities for all three receptors. On the whole, the potency trend for three receptors followed the order:
ortho-substitution (8b, 8d) > no substitution (8a) > meta-substitution (8c, 8e); F-substitution >
Cl-substitution (8b vs 8d, 8c vs 8e). Disappointingly, none of these phthalimide derivatives showed
comparable activity as compounds 3q and 3r, which suggested that aromatic rings were inferior to
aliphatic rings for succinimide derivatives. Further oxidation of C-C double bond of compounds 3d and
3r gave corresponding dihydroxyl compounds 4b and 4a with activities on three receptors decreasing
to some extent. Biacetylation of hydroxyl groups had a positive effect on 5-HT1AR potency, albeit
activities on D2R and 5-HT2AR reduced (5 vs 4a).
Glutarimide derivatives 3s–3v modified in the spiro moiety gave interesting results as well. As
shown in Table 3, compound 3t with ethyl methyl disubstitution showed higher potency for 5-HT2AR
than compound 3s with gem-dimethyl substitution, although their activities on D2R and 5-HT1AR were
comparable. After formal ring closure, compound 3u showed weaker activities than 3t for D2R and
5-HT1AR while potency for 5-HT2AR retained. Enlargement of the cyclopentyl spiro moiety of 3u gave
compound 3v, which was less active on 5-HT2AR while slightly more active on D2R and 5-HT1AR.
In order to juggle the potency and selectivity of synthesized compounds for further biological
evaluation, two primary selection creteria were set: ( a ) high potency for D2R, 5-HT1AR and 5-HT2AR
(IC50 or EC50 value < 30 nM); ( b ) to obtain a balanced receptor activity profiles, the potency ratio
between any two of the three receptors should be no greater than 10. As a result, compounds 3c, 3d, 3t
and 3u were selected to assess their liability to cause antipsychotic-induced side effects.
2.3. In vitro receptor functional activities on safety profile and hERG affnities

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Although atypical antipsychotics have low liability to cause serious adverse effects (EPS and TD),
they are associated with a wide range of other adverse effects, such as weight gain, hyperglycaemia,
dyslipidaemia and cardiovascular disease[9]. These side effects were associated with unintentional
engagement of multiple pharmacological targets, which led to undesired consequences and poor
medication adherence. Therefore, further characterization of pharmacological profiles of candidate
compounds is essential.
Various receptors were involved in the emergence of these side effects, including 5-HT2C receptor,
M3 cholinoceptor, α1A adrenergic receptor and H1 histaminergic receptor[31-33]. 5-HT2C and H1
receptors were closely related to the contol of feeding and body weight, and might be responsible for
weight gain side effect of antipsychotic drugs. According to Table 4, all selected compounds showed
moderate activities for H1 receptor, and they displayed much weaker potency for 5-HT2C receptor than
risperidone. On the basis of the above results, compounds 3c, 3d, 3t and 3u had low potential to elicit
antipsychotic-induced weight gain. Inhibition of peripheral α1A adrenergic receptor was presumed to be
associated with antipsychotic-induced orthostatic hypotension. Compounds 3c, 3d, 3t and 3u showed
moderate α1A receptor antagonistic activities, comparable to that of aripiprazole and weaker than that of
risperidone, suggesting that they had low liability to cause orthostatic hypotension. Pancreatic M3
cholinoceptor appeared to control cholinergic-dependent insulin release, blocking which might lead to
hyperglycaemia and type II diabetes mellitus. For example, olanzapine and clozapine exhibited the
highest clinical incidence of diabetes side effect, both of which are potent M3 receptor antagonists[34].
All selected compounds as well as risperidone and aripiprazole were devoid of M3 receptor
antagonistic activity, indicating a low propensity to elicit hyperglycaemia.
Morever, blocking human ether-a-go-go-related gene (hERG) potassium channel causes QT
interval prolongation, which is associated with potentially fatal arrhythmia called Torsades de
Pointes[35]. As a result, inhibitory potency for hERG has become a major safety concern for drug
discovery. As shown in Table 4, all tested compounds exhibited quite weak hERG blocking potency,
indicating that they held low propensity to elicit treatment-induced QT interval prolongation.
On the basis of what was discussed above, we drew the conclusion that compounds 3c, 3d, 3t and
3u had low potential to cause serious adverse effects associated with antipsychotic drugs.
Table 4
Activities on 5-HT2C, H1, α1A, M3 receptors and hERG of seleceted compounds and reference

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antipsychotics.
Cmpd
5-HT2C
IC50 (nM)
188
H1
IC50 (nM)
174
α1A
IC50 (nM)
124
M3
hERG
IC50 (nM)
7520
IC50 (nM)
> 100000
> 100000
> 100000
> 100000
> 100000
> 100000
3c
3d
341
195
178
7850
606
253
376
6210
3t
400
305
375
2630
3u
RISa
ARIb
1.81
454
10.9
1330
1380
420
170
3860
a RIS = Risperidone.
b ARI = Aripiprazole.
2.4. Behavioral studies
Based on the in vitro pharmacological profile, compounds 3c, 3d, 3t and 3u were then subjected
to in vivo behavioral study to verify their effects on schizophrenia.
2.4.1. PCP-induced hyperlocomotion
Phencyclidine (PCP), a N-methyl-D-aspartic acid receptor (NMDAR) antagonist, has been found
to simulate schizophrenia[36], and this effect can be reversed by antipsychotics[37]. Currently,
PCP-induced hyperlocomotion is a widely used model to assess antipsychotic-like efficacy of potential
antipsychotic agents. In this assay, compounds 3c, 3d, 3t and 3u all attenuated PCP-induced
hyperlocomotion significantly in ICR mice at the dose of 3 mg/kg, among which 3d exhibited most
potent inhibitory effect. Moreover, compound 3d produced significant dose-dependent responses with
an ED50 value of 1.80 mg/kg (Fig. 3), which was consistent with its potent in vitro D2R antagonistic
activity. On the basis of the data presented, compound 3d clearly differentiated itself from 3c, 3t and
3u, justifying further in vivo evaluation.

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7000
6000
5000
4000
3000
2000
1000
0
###
**
**
**
**
**
**
***
Fig. 3. Effects of compounds 3c, 3d, 3t, 3u and aripiprazole on PCP-induced hyperlocomotion in mice
(7 mg/kg, ip, 6/group). Animals were habituated for 10 min before the 75 min measurement period.
Results were expressed as the means ± SEM of distance traveled. Statistical evaluation was performed
by two-way ANOVA followed by student’s t test for multiple comparisons. *p < 0.05 versus PCP
treatment; **p < 0.01 versus PCP treatment; ***p < 0.001 versus PCP treatment; ###p < 0.001 versus
vehicle treatment.
2.4.2. Catalepsy
Catalepsy is a frequently used model to predict liability of antipsychotics to induce EPS side effect
in humans[38]. As shown in Table 5, compound 3d exhibited low potential for catalepsy with an ED50
value of 14.10 mg/kg. The therapeutic index of compound 3d based on its efficacy and side effect
(catalepsy) was 7.83, while the therapeutic index of aripiprazole was 2.43. Compound 3d had a higher
threshold for catalepsy compared with aripiprazole, which might translate into lower clinical EPS
liability.
Table 5
In vivo pharmacological profile of compound 3d.

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Cmpd
3d
PCPa
1.80
2.93
CATb
14.10
7.13
CAT/PCP
7.83
2.43
ARI
a PCP: PCP-induced hyperlocomotion (ED50, mg/kg, ig).
b CAT: Catalepsy (ED50, mg/kg, ig).
c ARI = Aripiprazole.
2.4.3. Quipazine-induced head twitch response in mice
Quipazine-induced head twitch response (HTR) in mice was 5-HT2AR-dependent, so it was an
important model to verify the in vivo efficacy of 5-HT2AR antagonists. As shown in Table 6, compound
3d exhibited dose-dependent inhibitory effect on HTR (Table 6). Particularly, HTR in mice was
completely inhibited by compound 3d at the dose of 3 mg/kg, which was superior to aripiprazole
(reduction ratio = 70.8%).
Table 6
Effect of compound 3d on Quipazine-induced head twitches response in mice.
Head twitchesa
Treatment
Dose (mg/kg)
% of HTR reduction
(number of episodes)
13.0 ± 2.1
8.4 ± 2.2*
0***
Vehicle
3d
0
1
3
3
35.4%
100%
70.8%
3d
ARIb
3.8 ± 1.2
a
Values represent the number of head twitches (mean ± SEM) during the 15 min test.
b ARI = Aripiprazole
* p < 0.05 versus Quipazine-treated group.
** p < 0.01 versus Quipazine-treated group.
*** p < 0.001 versus (±)-Quipazine-treated group.
2.5. Evaluation of pharmacokinetics
The pharmacokinetic properties of 3d were measured in rats (Table 7). The area under the curve
(AUC) value of compound 3d was 3144.58 ng × h/mL after intravenous administration vs 1002.26 ng ×
h/mL after oral administration. Then, the oral bioavailability of compound 3d was caclulated as ratio of

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AUC (po) to AUC (iv). Oral bioavailability of 3d was 32%. The elimination half-lifves of 3d were 2.66
h and 3.26 h after intravenous and oral administration, respectively. The Cmax value after oral dosing of
3d was 85.95 ng/mL, and Tmax value was 5.33 h. These encouraging preclinical data suggested that 3d
possessed acceptable drug-like human pharmacokinetic properties.
Further metabolite profiling studies of compound 3d in rats and subsequent structural analysis of
metabolites revealed an important metabolite 4b which formed in significant amounts (~21% based on
AUCs) relative to the parent compound 3d. As compound 4b was found to be slightly less potent for
D2, 5-HT1A and 5-HT2A receptors than parent compound 3d in the previous discussion, it might
contribute significantly to the in vivo pharmacology of 3d.
Table 7
plasma pharmacokinetic data following the administration of Compound 3d in rats (n = 3/group).
dose
Cmax
(ng/mL)
2658.99
85.95
Tmax
(h)
t1/2
(h)
AUC0-24h
(ng × h/mL)
3144.58
F (%)
(mg/kg)
10 (iv)
10 (po)
10 (po)
0.083
5.33
3.67 a
2.66
3.26
11.15 a
-
32
-
1002.26
16.63a
210.47 a
a plasma pharmacokinetic data of metabolite 4b following the administration of compound 3d in rats.
In summary, a series of new N-substituted cyclic imide compounds were synthesized and
evaluated for their antipsychotic activities. The efforts to explore SAR provided some beneficial clues
to the development of multi-target antipsychotic drugs. Among the synthesized compounds, 3d
exhibited not only potent and balanced in vitro activities on D2R/5-HT1AR/5-HT2AR, but also favorable
in vivo profiles on behavioral assays. Besides, with low to moderate potency for M3, 5-HT2C, H1 and
α1A receptor and hERG channel, compound 3d had low risk of inducing side effects associated with
these targets. Moreover, compound 3d exhibited acceptable pharmacokinetic properties. As a result,
compound 3d had the potential to be developed into novel multi-target antipsychotic drug.
4. Experimental
4.1. Chemistry experimental
All chemicals and solvents were purchased from commercial suppliers (e.g. Sinopharm Chemical
Reagent Co., Ltd.) and were used without further purification. Nuclear magnetic resonance (NMR)

ACCEPTED MANUSCRIPT
1
spectra were recorded on Varian Mercury Plus-300 (300 MHz for H NMR), Bruker AVANCE III 400
1
(400 MHz for H NMR, 101 MHz for 13C NMR) or Bruker AVANCE III 500 (126 MHz for 13C
NMR)spectrometer with TMS in DMSO-d6 or CDCl3 solution as an internal standard. Chemical shifts
were given in δ values (ppm) and coupling constants (J) were given in Hz. Signal multiplicities were
characterized as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad). High
resolution mass spectra (HRMS) were obtained using Agilent G6520 Q-TOF mass spectrometer; Low
resolution mass spectra (LRMS) were obtained using Finnigan MAT95 mass spectrometer. TLC on
silica gel GF254 was used to monitor the progress of all reactions. Column chromatographic
purification was carried out using silica gel (200–300 mesh). The HPLC conditions were as follows:
column, Agilent ZORBAX SB-C18 (4.6×150mm, 5µm); mobile phase, 0.02 mol/L sodium
1-octanesulfonate (PH = 2.0 adjusted by 85% H3PO4)/acetonitrile 65/35–30/70; UV detection, 220 nm;
injection volume, 10 µL; flow rate, 1.0 mL/min; column temperature, 30 °C.
4.1.1 General procedure for the preparation of compounds of 2a–2n.
To a suspension of corresponding cyclic imide 1a–1k (4 mmol), potassium carbonate (4.4 mmol)
in DMF (5 mL), 1-bromo-4-chlorobutane (1,2-dibromoethane (5 eq) for 2l, 1-bromo-3-chloropropane
for 2m, 1-bromo-5-chloropentane for 2n) (4.4 mmol) was added dropewise at 0 °C. The mixture was
stirred at room temperature overnight and then partitioned between ethyl acetate (EA, 20 mL) and
water (30 mL). The organic layer was washed successively with water (3 × 30 mL), saturated brine,
and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum and the residue was
purified by column chromatography using petroleum ether(PE) : EA (8:1) as eluent to give 2a–2n as
colorless oil.
4.1.2 General procedure for the preparation of compounds of 3a–3v.
Arylpiperazine or arylpiperidine (0.2 mmol), 2a–2n (0.3 mmol), potassium iodide (0.2 mmol),
potassium carbonate (0.7 mmol) were added to DMF (2 mL)/H2O (0.5 mL), and then the reaction
mixture was heated at 95 °C for 12 h. After cooling to room temperature, the reaction mixture was
partitioned between ethyl acetate (EA, 2 mL) and water (30 mL). The organic layer was washed
successively with water (3 × 20 mL), saturated brine, and dried over anhydrous Na2SO4. The solvent
was evaporated under vacuum and the residue was purified by column chromatography using DCM :
MeOH (80:1) as eluent to afford colorless oil. The oil was dissolved in ethyl acetate and acidified with
1 M hydrochloride in ethyl acetate solution, keeping stirring for 30 min. Then the resulting solid was

ACCEPTED MANUSCRIPT
collected by filtration to give (3a–3v) as a white solid in the hydrochloride salt form.
4.1.2.1.
(3aR,4S,7R,7aS)-2-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butyl)hexahydro-1H-4,7-metha
noisoindole-1,3(2H)-dione
(3a).
The
title
compound
was
prepared
from
6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole hydrochloride and 2a as a white solid (free base). Yield
1
69%; H NMR (300 MHz, DMSO-d6) δ 8.00 (dd, J = 8.7, 5.3 Hz, 1H), 7.67 (dd, J = 9.1, 2.2 Hz, 1H),
7.26 (td, J = 9.1, 2.2 Hz, 1H), 2.96 – 2.86 (m, 2H), 2.64 (s, 2H), 2.53 – 2.44 (m, 5H), 2.30 (t, J = 6.8 Hz,
2H), 2.13 – 1.92 (m, 4H), 1.89 – 1.72 (m, 2H), 1.62 – 1.24 (m, 8H), 1.15 (d, J = 10.7 Hz, 1H), 0.97 (d,
J = 10.7 Hz, 1H). MS (ESI) m/z = 440.4 ([M+H]+).
4.1.2.2.
(3aR,4S,7R,7aS)-2-(4-(4-(benzo[b]thiophen-6-yl)piperazin-1-yl)butyl)hexahydro-1H-4,7-methanoisoin
dole-1,3(2H)-dione hydrochloride (3b). The title compound
was prepared from
1
1-(benzo[b]thiophen-6-yl)piperazine and 2a as a white solid. Yield 62%; H NMR (400 MHz,
DMSO-d6) δ 11.01 (s, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.50 (d, J = 5.4 Hz, 1H),
7.31 (d, J = 5.4 Hz, 1H), 7.17 (dd, J = 8.8, 2.2 Hz, 1H), 3.85 (d, J = 12.8 Hz, 2H), 3.52 (d, J = 11.6 Hz,
2H), 3.37 (t, J = 7.2 Hz, 2H), 3.29 – 3.04 (m, 5H), 2.67 (s, 2H), 1.78 – 1.64 (m, 2H), 1.62 – 1.41 (m,
5H), 1.38 – 1.25 (m, 2H), 1.16 (d, J = 10.7 Hz, 2H), 0.99 (d, J = 10.7 Hz, 2H). MS (ESI) m/z = 438.5
([M+H]+).
4.1.2.3.
(3aR,4S,7R,7aS)-2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)hexahydro-1H-4,7-methanoisoin
dole-1,3(2H)-dione
hydrochloride
(3c).
The
title
compound
was
prepared
from
1
1-(benzo[b]thiophen-4-yl)piperazine hydrochloride and 2a as a white solid. Yield 75%; H NMR (300
MHz, DMSO-d6) δ 11.20 (s, 1H), 7.76 (d, J = 5.0 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 5.0 Hz,
1H), 7.31 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H), 3.64 – 3.45 (m, 4H), 3.38 (t, J = 7.0 Hz, 2H),
3.33 – 3.21 (m, 4H), 3.20 – 3.08 (m, 2H), 2.67 (s, 2H), 1.83 – 1.66 (m, 2H), 1.65 – 1.43 (m, 4H), 1.37 –
1.21 (m, 4H), 1.16 (d, J = 10.3 Hz, 1H), 0.99 (d, J = 10.3 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ
178.57, 146.49, 140.59, 133.30, 126.52, 125.04, 121.72, 117.69, 112.54, 54.82, 51.05, 48.28, 47.97,
39.06, 37.27, 32.85, 27.38, 24.58, 20.50. MS (ESI) m/z = 438.4 ([M+H]+). HRMS (ESI) m/z calcd for
C25H32N3O2S ([M+H]+), 438.2210; found 438.2214.
4.1.2.4.

ACCEPTED MANUSCRIPT
(3aR,4R,7S,7aS)-2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahydro-1H-4,7-me
thanoisoindole-1,3(2H)-dione hydrochloride (3d). The title compound was prepared from
1-(benzo[b]thiophen-4-yl)piperazine hydrochloride and 2b as a white solid. Yield 52%; 1H NMR (400
MHz, DMSO-d6) δ 11.12 (s, 1H), 7.78 (d, J = 5.5 Hz, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 5.5 Hz,
1H), 7.33 (t, J = 7.9 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 6.33 – 6.32 (m, 2H), 3.57 – 3.52 (m, 4H), 3.41 (t,
J = 7.2 Hz, 2H), 3.33 – 3.22 (m, 4H), 3.20 – 3.09 (m, 4H), 2.73 (s, 2H), 1.82 – 1.70 (m, 2H), 1.58 –
1.51 (m, 2H), 1.40 (d, J = 9.7 Hz, 1H), 1.16 (d, J = 9.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ
177.85, 146.68, 140.77, 137.81, 133.48, 126.69, 125.22, 121.90, 117.86, 112.72, 55.02, 51.25, 48.47,
47.48, 44.64, 42.68, 37.44, 24.72, 20.70. MS (ESI) m/z = 436.4 ([M+H]+). HRMS (ESI) m/z calcd for
C25H30N3O2S ([M+H]+), 436.2053; found 436.2053.
4.1.2.5.
(3aR,4R,7S,7aS)-2-(4-(4-(benzo[d]thiazol-4-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahydro-1H-4,7-meth
anoisoindole-1,3(2H)-dione hydrochloride (3e). The title compound was prepared from
1
4-(piperazin-1-yl)benzo[d]thiazole hydrochloride and 2b as a white solid. Yield 45%; H NMR (300
MHz, DMSO-d6) δ 10.72 (s, 1H), 9.32 (s, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 6.98
(d, J = 7.9 Hz, 1H), 6.33 – 6.28 (m, 2H), 4.19 (d, J = 10.4 Hz, 2H), 3.57 (d, J = 9.6 Hz, 2H), 3.39 (t, J =
7.1 Hz, 2H), 3.33 – 3.07 (m, 8H), 2.70 (s, 2H), 1.80 – 1.64 (m, 2H), 1.59 – 1.45 (m, 2H), 1.37 (d, J =
9.8 Hz, 1H), 1.13 (d, J = 9.8 Hz, 1H). MS (ESI) m/z = 437.3 ([M+H]+).
4.1.2.6.
(3aR,4R,7S,7aS)-2-(4-(4-(2-fluorobenzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahydro-1H
-4,7-methanoisoindole-1,3(2H)-dione hydrochloride (3f). The title compound was prepared from
1-(2-fluorobenzo[b]thiophen-4-yl)piperazine 2,2,2-trifluoroacetate and 2b as a white solid. Yield 61%;
1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.17
(d, J = 3.0 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.36 – 6.31 (m, 2H), 3.54 (d, J = 11.8 Hz, 2H), 3.50 –
3.38 (m, 4H), 3.31 – 3.22 (m, 2H), 3.21 – 3.09 (m, 6H), 2.73 (s, 2H), 1.79 – 1.66 (m, 2H), 1.55 (p, J =
7.5 Hz, 2H), 1.40 (d, J = 9.7 Hz, 1H), 1.16 (d, J = 9.7 Hz, 1H). MS (ESI) m/z = 454.4 ([M+H]+).
4.1.2.7.
(3aR,4R,7S,7aS)-2-(4-(4-(benzo[b]thiophen-7-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahydro-1H-4,7-me
thanoisoindole-1,3(2H)-dione hydrochloride (3g). The title compound was prepared from
1-(benzo[b]thiophen-7-yl)piperazine hydrochloride and 2b as a white solid. Yield 75%; 1H NMR (400

ACCEPTED MANUSCRIPT
MHz, DMSO-d6) δ 10.37 (s, 1H), 7.77 (d, J = 5.4 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 5.4 Hz,
1H), 7.38 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 7.8 Hz, 1H), 6.35 – 6.30 (m, 2H), 3.72 – 3.53 (m, 4H), 3.41 (t,
J = 7.2 Hz, 2H), 3.34 – 3.15 (m, 6H), 3.12 (s, 2H), 2.72 (s, 2H), 1.73 (s, 2H), 1.55 (q, J = 7.6 Hz, 2H),
1.40 (d, J = 9.6 Hz, 1H), 1.15 (d, J = 9.6 Hz, 1H). MS (ESI) m/z = 436.4 ([M+H]+).
4.1.2.8.
(3aR,4R,7S,7aS)-2-(4-(4-(2,3-dihydrobenzo[b][1,4]dioxin-5-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahy
dro-1H-4,7-methanoisoindole-1,3(2H)-dione hydrochloride (3h). The title compound was prepared
from 1-(2,3-dihydrobenzo[b][1,4]dioxin-5-yl)piperazine hydrochloride and 2b as a white solid. Yield
1
63%; H NMR (400 MHz, DMSO-d6) δ 11.15 – 10.69 (m, 1H), 6.75 (t, J = 8.1 Hz, 1H), 6.57 (dd, J =
8.1, 1.4 Hz, 1H), 6.51 (dd, J = 8.1, 1.4 Hz, 1H), 6.35 – 6.29 (m, 2H), 4.29 – 4.19 (m, 4H), 3.47 (d, J =
8.7 Hz, 4H), 3.39 (t, J = 7.2 Hz, 2H), 3.17 – 2.99 (m, 8H), 2.71 (s, 2H), 1.70 (d, J = 10.3 Hz, 2H), 1.57
– 1.45 (m, 2H), 1.38 (d, J = 9.7 Hz, 1H), 1.14 (d, J = 9.7 Hz, 1H). MS (ESI) m/z = 438.4 ([M+H]+).
4.1.2.9.
(3aR,4R,7S,7aS)-2-(4-(4-(2,2-difluorobenzo[d][1,3]dioxol-4-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahy
dro-1H-4,7-methanoisoindole-1,3(2H)-dione hydrochloride (3i). The title compound was prepared
from 1-(2,2-difluorobenzo[d][1,3]dioxol-4-yl)piperazine hydrochloride and 2b as a white solid. Yield
65%; 1H NMR (400 MHz, DMSO-d6) δ 11.07 – 10.58 (m, 1H), 7.14 (t, J = 8.3 Hz, 1H), 6.98 (d, J = 8.3
Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 6.32 (s, 2H), 3.72 (d, J = 13.0 Hz, 2H), 3.53 (d, J = 12.0 Hz, 2H),
3.45 – 3.22 (m, 4H), 3.21 – 3.04 (m, 6H), 2.71 (s, 2H), 1.79 – 1.63 (m, 2H), 1.51 (p, J = 7.5 Hz, 2H),
1.39 (d, J = 9.7 Hz, 1H), 1.14 (d, J = 9.7 Hz, 1H). MS (ESI) m/z = 460.4 ([M+H]+).
4.1.2.10.
(3aR,4R,7S,7aS)-2-(4-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahydro-1H-4,7-m
ethanoisoindole-1,3(2H)-dione hydrochloride (3j). The title compound was prepared from
3-(piperazin-1-yl)benzo[d]isothiazole hydrochloride and 2b as a white solid. Yield 37%; 1H NMR (400
MHz, DMSO-d6) δ 11.01 (s, 1H), 8.17 – 8.07 (m, 2H), 7.60 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H),
6.32 (s, 2H), 4.06 (d, J = 13.4 Hz, 2H), 3.62 – 3.36 (m, 6H), 3.31 – 3.07 (m, 6H), 2.71 (s, 2H), 1.80 –
1.65 (m, 2H), 1.53 (p, J = 7.5 Hz, 2H), 1.39 (d, J = 9.6 Hz, 1H), 1.14 (d, J = 9.6 Hz, 1H). MS (ESI) m/z
= 437.3 ([M+H]+).
4.1.2.11
(3aR,4R,7S,7aS)-2-(2-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)-3a,4,7,7a-tetrahydro-1H-4,7-me

ACCEPTED MANUSCRIPT
thanoisoindole-1,3(2H)-dione hydrochloride (3k). The title compound was prepared from
1
1-(benzo[b]thiophen-4-yl)piperazine hydrochloride and 2l as a white solid. Yield 64%; H NMR (300
MHz, DMSO-d6) δ 11.42 (s, 1H), 7.76 (d, J = 5.5 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 5.5 Hz,
1H), 7.31 (t, J = 7.8 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.32 (s, 2H), 3.84 (t, J = 6.3 Hz, 2H), 3.68 (d, J
= 10.3 Hz, 2H), 3.53 (d, J = 10.9 Hz, 2H), 3.44 – 3.20 (m, 6H), 3.10 (s, 2H), 2.78 (s, 2H), 1.38 (d, J =
9.6 Hz, 1H), 1.22 (d, J = 9.6 Hz, 1H). MS (ESI) m/z = 408.0 ([M+H]+).
4.1.2.12.
(3aR,4R,7S,7aS)-2-(3-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)propyl)-3a,4,7,7a-tetrahydro-1H-4,7-m
ethanoisoindole-1,3(2H)-dione hydrochloride (3l). The title compound was prepared from
1-(benzo[b]thiophen-4-yl)piperazine hydrochloride and 2m as a white solid. Yield 85%; 1H NMR (300
MHz, DMSO-d6) δ 11.36 (s, 1H), 7.76 (d, J = 5.5 Hz, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 5.5 Hz,
1H), 7.31 (t, J = 7.9 Hz, 1H), 6.95 (d, J = 7.9 Hz, 1H), 6.32 (s, 2H), 3.64 – 3.40 (m, 6H), 3.37 – 3.07 (m,
8H), 2.72 (s, 2H), 2.07 – 1.92 (m, 2H), 1.39 (d, J = 9.8 Hz, 1H), 1.18 (d, J = 9.8 Hz, 1H). MS (ESI) m/z
= 421.9 ([M+H]+).
4.1.2.13.
(3aR,4R,7S,7aS)-2-(5-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)pentyl)-3a,4,7,7a-tetrahydro-1H-4,7-m
ethanoisoindole-1,3(2H)-dione hydrochloride (3m). The title compound was prepared from
1-(benzo[b]thiophen-4-yl)piperazine hydrochloride and 2n as a white solid. Yield 56%; 1H NMR (400
MHz, DMSO-d6) δ 10.80 (s, 1H), 7.76 (d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 5.5 Hz,
1H), 7.32 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H), 6.35 – 6.28 (m, 2H), 3.62 – 3.48 (m, 4H), 3.38 (t,
J = 7.2 Hz, 2H), 3.35 – 3.17 (m, 4H), 3.17 – 3.06 (m, 4H), 2.71 (s, 2H), 1.84 – 1.71 (m, 2H), 1.52 (p, J
= 7.4 Hz, 2H), 1.39 (d, J = 9.7 Hz, 1H), 1.35 – 1.25 (m, 2H), 1.13 (d, J = 9.6 Hz, 1H). MS (ESI) m/z =
450.4 ([M+H]+).
4.1.2.14.
3-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2,4-dion
e hydrochloride (3n). The title compound was prepared from 1-(benzo[b]thiophen-4-yl)piperazine
hydrochloride and 2c as a white solid. Yield 66%; 1H NMR (300 MHz, DMSO-d6) δ 10.71 (s, 1H), 7.77
(d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 5.5 Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H), 6.96 (d,
J = 7.9 Hz, 1H), 3.64 – 3.46 (m, 4H), 3.38 – 3.10 (m, 8H), 2.54 (s, 2H), 1.80 – 1.65 (m, 2H), 1.50 (p, J
= 7.5 Hz, 2H), 1.20 (s, 3H), 1.12 (s, 3H). MS (ESI) m/z = 412.4 ([M+H]+).

ACCEPTED MANUSCRIPT
4.1.2.15.
3-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-1-(p-tolyl)-3-azabicyclo[3.1.0]hexane-2,4-dione
hydrochloride (3o). The title compound was prepared from 1-(benzo[b]thiophen-4-yl)piperazine
1
hydrochloride and 2d as a white solid. Yield 53%; H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H),
7.76 (d, J = 5.4 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.48 (d, J = 5.4 Hz, 1H), 7.36 (d, J = 7.7 Hz, 2H),
7.31 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 7.7 Hz, 2H), 6.96 (d, J = 7.7 Hz, 1H), 3.62 – 3.46 (m, 4H), 3.40 –
3.19 (m, 6H), 3.20 – 3.08 (m, 2H), 2.93 (dd, J = 8.3, 3.5 Hz, 1H), 2.29 (s, 3H), 2.04 (t, J = 4.1 Hz, 1H),
1.92 (dd, J = 8.3, 4.5 Hz, 1H), 1.79 – 1.67 (m, 2H), 1.59 – 1.47 (m, 2H). MS (ESI) m/z = 474.3
([M+H]+).
4.1.2.16.
2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)tetrahydrocyclopenta[c]pyrrole-1,3(2H,3aH)-dion
e hydrochloride (3p). The title compound was prepared from 1-(benzo[b]thiophen-4-yl)piperazine
hydrochloride and 2e as a white solid. Yield 70%; 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 7.77
(d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 5.5 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 6.97 (d,
J = 7.8 Hz, 1H), 3.62 – 3.48 (m, 4H), 3.40 (t, J = 7.0 Hz, 2H), 3.34 – 3.08 (m, 8H), 1.97 – 1.61 (m, 7H),
1.53 (p, J = 7.3 Hz, 2H), 1.29 – 1.12 (m, 1H). MS (ESI) m/z = 412.3 ([M+H]+).
4.1.2.17. 2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)hexahydro-1H-isoindole-1,3(2H)-dione
hydrochloride (3q). The title compound was prepared from 1-(benzo[b]thiophen-4-yl)piperazine
hydrochloride and 2f as a white solid. Yield 55%; 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 7.77
(d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 5.5 Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H), 6.97 (d,
J = 7.9 Hz, 1H), 3.67 – 3.48 (m, 4H), 3.42 (t, J = 6.9 Hz, 2H), 3.24 – 3.08 (m, 4H), 3.01 – 2.90 (m, 2H),
1.83 – 1.48 (m, 8H), 1.48 – 1.19 (m, 4H). MS (ESI) m/z = 426.4 ([M+H]+).
4.1.2.17.
2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione
hydrochloride (3r). The title compound was prepared from 1-(benzo[b]thiophen-4-yl)piperazine
1
hydrochloride and 2g as a white solid. Yield 45%; H NMR (300 MHz, DMSO-d6) δ 11.32 (s, 1H),
7.76 (d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 5.5 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H),
6.96 (d, J = 7.8 Hz, 1H), 5.96 – 5.84 (m, 2H), 3.58 – 3.47 (m, 4H), 3.39 (t, J = 6.8 Hz, 2H), 3.35 – 3.06
(m, 8H), 2.47 – 2.35 (m, 2H), 2.18 (dt, J = 14.6, 4.4 Hz, 2H), 1.78 – 1.59 (m, 2H), 1.51 (q, J = 7.0 Hz,
2H). MS (ESI) m/z = 424.4 ([M+H]+).

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4.1.2.18.
1-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-4,4-dimethylpiperidine-2,6-dione
hydrochloride (3s). The title compound was prepared from 1-(benzo[b]thiophen-4-yl)piperazine
1
hydrochloride and 2h as a white solid. Yield 62%; H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H),
7.77 (d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 5.5 Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H),
6.97 (d, J = 7.9 Hz, 1H), 3.69 (t, J = 7.3 Hz, 2H), 3.62 – 3.47 (m, 4H), 3.36 – 3.10 (m, 6H), 2.55 (s, 4H),
1.80 – 1.66 (m, 2H), 1.49 (p, J = 7.6 Hz, 2H), 0.99 (s, 6H). MS (ESI) m/z = 414.3 ([M+H]+).
4.1.2.19.
1-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-4-ethyl-4-methylpiperidine-2,6-dione
hydrochloride (3t). The title compound was prepared from 1-(benzo[b]thiophen-4-yl)piperazine
hydrochloride and 2i as a white solid. Yield 47%; 1H NMR (300 MHz, DMSO-d6) δ 11.20 (s, 1H), 7.77
(d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 5.6 Hz, 1H), 7.31 (t, J = 7.9 Hz, 1H), 6.96 (d,
J = 7.9 Hz, 1H), 3.68 (t, J = 7.3 Hz, 2H), 3.59 – 3.45 (m, 4H), 3.26 (d, J = 9.0 Hz, 4H), 3.20 – 3.06 (m,
2H), 2.66 – 2.42 (m, 4H), 1.83 – 1.65 (m, 2H), 1.48 (p, J = 7.4 Hz, 2H), 1.31 (q, J = 7.4 Hz, 2H), 0.92
(s, 3H), 0.81 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 172.05, 146.50, 140.59, 133.30,
126.51, 125.04, 121.71, 117.68, 112.53, 54.99, 51.03, 48.31, 43.51, 37.91, 32.44, 31.40, 24.76, 23.46,
20.58, 7.80. MS (ESI) m/z = 428.4 ([M+H]+). HRMS (ESI) m/z calcd for C24H34N3O2S ([M+H]+),
428.2366; found 428.2372.
4.1.2.20.
8-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-8-azaspiro[4.5]decane-7,9-dione
hydrochloride (3u). The title compound was prepared from 1-(benzo[b]thiophen-4-yl)piperazine
hydrochloride and 2j as a white solid. Yield 59%; 1H NMR (300 MHz, DMSO-d6) δ 11.27 (s, 1H), 7.76
(d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 5.5 Hz, 1H), 7.31 (t, J = 7.9 Hz, 1H), 6.96 (d,
J = 7.9 Hz, 1H), 3.67 (t, J = 7.2 Hz, 2H), 3.61 – 3.42 (m, 4H), 3.38 – 3.20 (m, 4H), 3.19 – 3.08 (m, 2H),
2.63 (s, 4H), 1.83 – 1.67 (m, 2H), 1.67 – 1.57 (m, 4H), 1.54 – 1.36 (m, 6H). 13C NMR (126 MHz,
DMSO-d6) δ 172.22, 146.51, 140.60, 133.31, 126.52, 125.05, 121.72, 117.69, 112.54, 55.03, 51.08,
48.34, 43.83, 37.92, 36.85, 24.82, 23.68, 20.58. MS (ESI) m/z = 440.4 ([M+H]+). HRMS (ESI) m/z
calcd for C25H34N3O2S ([M+H]+), 440.2366; found 440.2366.
4.1.2.21.
3-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-3-azaspiro[5.5]undecane-2,4-dione
hydrochloride (3v). The title compound was prepared from 1-(benzo[b]thiophen-4-yl)piperazine
1
hydrochloride and 2k as a white solid. Yield 61%; H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H),
7.77 (d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 5.5 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H),
6.97 (d, J = 7.8 Hz, 1H), 3.67 (t, J = 7.4 Hz, 2H), 3.61 – 3.47 (m, 4H), 3.32 – 3.12 (m, 6H), 2.61 (s, 4H),

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1.79 – 1.64 (m, 2H), 1.56 – 1.24 (m, 12H). MS (ESI) m/z = 454.4 ([M+H]+).
4.1.3. preparation of compounds 4a, 4b and 5a.
4.1.3.1.
2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-5,6-dihydroxyhexahydro-1H-isoindole-1,3(2H)-di
one (4a)
Free base of 3r (424 mg, 1 mmol) was dissolved in acetone (10 mL), cooling to 0 °C, and
potassium permanganate aqueous solution (25 mL, contain potassium permanganate 158 mg) was
added dropwise. Then the reaction mixture was stirred at room temperature for 30 min, extracted with
ethyl acetate (40 mL). The aqueous layer was filtered, saturated with NaCl, exacted with ethyl acetate
(2 × 30 mL). The organic phase was combined, dried over anhydrous Na2SO4, purified by column
chromatography using DCM : MeOH (100:1 – 10:1) as eluent to give 4a (200 mg, 44%) as a white
foamed solid. 1H NMR (300 MHz, DMSO-d6) δ 7.69 (d, J = 5.4 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.39
(d, J = 5.4 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 6.89 (d, J = 7.8 Hz, 1H), 4.70 – 4.64 (m, 2H), 3.51 – 3.41
(m, 2H), 3.37 (d, J = 6.3 Hz, 2H), 3.13 – 2.94 (m, 6H), 2.58 (s, 4H), 2.36 (t, J = 6.7 Hz, 2H), 2.00 –
1.83 (m, 2H), 1.71 – 1.32 (m, 6H). MS (ESI) m/z = 458.4 ([M+H]+).
4.1.3.2.
(3aR,4S,5S,6R,7R,7aS)-2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-5,6-dihydroxyhexahydro-
1H-4,7-methanoisoindole-1,3(2H)-dione (4b)
Following the same procedure as 4a, compound 4b was prepared from the free base of 3d as a
white solid. Yield 35%; 1H NMR (400 MHz, Chloroform-d) δ 7.57 (d, J = 7.8 Hz, 1H), 7.41 (d, J = 5.5
Hz, 1H), 7.36 (d, J = 5.5 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 3.87 – 3.82 (m,
2H), 3.58 – 3.49 (m, 2H), 3.29 (s, 4H), 2.88 (s, 4H), 2.68 – 2.53 (m, 6H), 1.84 (d, J = 11.8 Hz, 1H),
1.73 – 1.58 (m, 4H), 0.99 (d, J = 11.8 Hz, 1H). MS (ESI) m/z = 470.4 ([M+H]+).
4.1.3.3.
(3aR,4S,5S,6R,7R,7aS)-2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-1,3-dioxooctahydro-1H-4
,7-methanoisoindole-5,6-diyl diacetate (5a)
4b (127 mg, 0.27 mmol) was dissolved in pyridine (3 mL), and then Ac2O (127 µL, 1.35 mmol)
was added dropwise, stirred at room temperature for 24 h. The solvent was evaporated under vacuum,
and the residue was partitioned between ethyl acetate (30 mL) and water (20 mL). The organic layer
was washed successively with water and saturated brine, dried over anhydrous Na2SO4, purified by

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column chromatography using DCM : MeOH (100:1 – 20:1) as eluent to give 5a (100 mg, 67%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.70 (d, J = 5.5 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.41
(d, J = 5.5 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 4.87 (s, 2H), 3.40 (t, J = 6.8 Hz,
2H), 3.08 (s, 4H), 2.89 (s, 2H), 2.75 – 2.28 (m, 6H), 2.01 (s, 6H), 1.73 (d, J = 11.7 Hz, 1H), 1.59 – 1.33
(m, 4H), 1.06 (d, J = 11.7 Hz, 1H). MS (ESI) m/z = 554.3 ([M+H]+).
4.1.4. Preparation of compounds 8a–e.
4.1.4.1. 2-(4-bromobutyl)isoindoline-1,3-dione (7a)
Phthalimide 6a (148 mg, 1 mmol), 1,4-dibromobutane (1.080 g, 5 mmol), potassium carbonate
(276 mg, 2 mmol) was added to acetone (3 mL), and the reaction mixture was refluxed for 2 h. After
cooling to room temperature, the reaction mixture was purified by column chromatography using PE :
acetone (40:1) as eluent to give 7a (185 mg, 66%) as a white solid.
4.1.4.2. 2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)isoindoline-1,3-dione (8a)
7a (89 mg, 0.32 mmol), 1-(benzo[b]thiophen-4-yl)piperazine hydrochloride (107 mg, 0.42 mmol),
potassium carbonate (88 mg, 0.64 mmol) and potassium iodide (27 mg, 0.16 mmol) was added into
MeCN (5 mL). The reaction mixture was refluxed for 5 h, cooled to room temperature, and then
purified by column chromatography using DCM : MeOH (70:1) as eluent to give 8a (117 mg, 87%) as
light yellow oil. 1H NMR (300 MHz, Chloroform-d) δ 7.90 – 7.79 (m, 2H), 7.76 – 7.66 (m, 2H), 7.54
(d, J = 8.1 Hz, 1H), 7.43 – 7.35 (m, 2H), 7.30 – 7.22 (m, 1H), 6.88 (dd, J = 7.7, 0.9 Hz, 1H), 3.74 (t, J
= 7.0 Hz, 2H), 3.23 – 3.11 (m, 4H), 2.77 – 2.61 (m, 4H), 2.55 – 2.43 (m, 2H), 1.85 – 1.52 (m, 4H). MS
(ESI) m/z = 420.4 ([M+H]+).
4.1.4.3.
2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-4-fluoroisoindoline-1,3-dione
hydrochloride (8b)
The title compound was prepared from 6b following the procedure described for the synthesis of
compound 8a as a white solid in hydrochloride salt form. Yield 97%; 1H NMR (300 MHz, DMSO-d6) δ
10.61 (s, 1H), 7.95 – 7.84 (m, 1H), 7.80 – 7.63 (m, 4H), 7.48 (d, J = 5.5 Hz, 1H), 7.31 (t, J = 7.8 Hz,
1H), 6.96 (d, J = 7.8 Hz, 1H), 3.72 – 3.47 (m, 6H), 3.40 – 3.10 (m, 6H), 1.89 – 1.60 (m, 4H). MS (ESI)
m/z = 438.3 ([M+H]+).
4.1.4.4.
2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-5-fluoroisoindoline-1,3-dione
hydrochloride (8c)
The title compound was prepared from 6c following the procedure described for the synthesis of

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compound 8a as a white solid in hydrochloride salt form. Yield 60%; 1H NMR (300 MHz, DMSO-d6) δ
10.61 (s, 1H), 7.95 – 7.84 (m, 1H), 7.80 – 7.63 (m, 4H), 7.48 (d, J = 5.5 Hz, 1H), 7.31 (t, J = 7.8 Hz,
1H), 6.96 (d, J = 7.8 Hz, 1H), 3.72 – 3.47 (m, 6H), 3.40 – 3.10 (m, 6H), 1.89 – 1.60 (m, 4H). MS (ESI)
m/z = 438.3 ([M+H]+).
4.1.4.5. 2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-4-chloroisoindoline-1,3-dione (8d)
The title compound was prepared from 6d following the procedure described for the synthesis of
1
compound 8a as a light yellow solid in hydrochloride salt form. Yield 55%; H NMR (300 MHz,
DMSO-d6) δ 7.89 – 7.78 (m, 3H), 7.68 (d, J = 5.5 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 5.5 Hz,
1H), 7.27 (t, J = 7.8 Hz, 1H), 6.87 (d, J = 7.8 Hz, 1H), 3.60 (t, J = 6.9 Hz, 2H), 3.04 (s, 4H), 2.58 (s,
4H), 2.38 (t, J = 7.1 Hz, 2H), 1.65 (p, J = 7.1 Hz, 2H), 1.49 (p, J = 7.1 Hz, 2H). MS (ESI) m/z = 454.3
([M+H]+).
4.1.4.6.
2-(4-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)butyl)-5-chloroisoindoline-1,3-dione
hydrochloride (8e)
The title compound was prepared from 6e following the procedure described for the synthesis of
compound 8a as a white solid in hydrochloride salt form. Yield 40%; 1H NMR (300 MHz, DMSO-d6) δ
10.28 (s, 1H), 7.98 (s, 1H), 7.91 (s, 2H), 7.77 (d, J = 5.5 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.48 (d, J =
5.6 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H), 3.73 – 3.44 (m, 6H), 3.14 (d, J = 20.1
Hz, 6H), 1.87 – 1.59 (m, 4H). MS (ESI) m/z = 454.2 ([M+H]+).
4.2. Biological evaluation
4.2.1. Functional activity Assays
All the compounds were screened on 5-HT1A agonist & D2 antagonist mode assays using
LANCE® Ultra. Some compounds were screened on H1, M3, α1A, 5-HT2A, 5-HT2C antagonist mode
assays using FLIPR. Refenrence compounds for D2, 5-HT1A, 5-HT2A, 5-HT2C, H1, M3, α1A receptor
functional assays were risperidone, 8-OH-DPAT, risperidone, methysergide, pyrilamine, atropine and
WB4101 respectively.
Lance Ultra cAMP assay:
1) Transfer compound to assay plate by Echo;
2) Collect cells with stimulation buffer;
3) Reaction: a) Transfer 10 µl of cell solution to assay plate, b) Centrifuge at 600 rpm for 3 minutes and
incubate 60 minutes at room temperature, c) Add 5 µl 4X Eu-cAMP trace solution and 5 µL 4X

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ULightTM-anti-cAMP solution to assay plate, d) Centrifuge at 600 rpm for 3 minutes and incubate 60
minutes at room temperature.
4) Reading plate on EnVision.
FLIPR assay:
1) Seed cells at the density of 10K cell/well, 37°C, 5% CO2 incubates for 16-24 hours;
2) Loading cells with 30 uL of Calcium 5 and 37°C, 5% CO2 incubates for 1 hours;
3) Tranfer compound to compound plate with 30 µL Assay Buffer by Echo;
4) Add 15 µL/well of compound and incubate for 10min at room temperature;
5) Add 22.5 µL/well of inducer and measure calcium flux signal with FLIPR.
4.2.2. hERG assay
Selected compounds was tested for effect on hERG potassium channels by automated patch clamp
method (QPatchHTX, Sophion, Stockholm, Sweden) at WuXi AppTec (Shanghai, China).
4.2.3. Behavioral studies
4.2.3.1. PCP-induced hyperlocomotion.
Male ICR mice (18~22 g, 6 mice in each group) were used. Animals were individually placed into
a Plexiglas open field arena (40 × 40 × 45 cm) for 45 min after intragastric administration of the test
compounds (1, 3 and 10 mg/kg) or aripiprazole (3 mg/kg), animals were treated with PCP (7 mg/kg,
i.p.), and placed back into the experimental apparatus. Animals were habituated for 10 min before the
75 min measurement period. Results are expressed as the means ± SEM of distance traveled. Statistical
evaluation was performed by two-way ANOVA followed by student’s t test for multiple comparisons.
*p < 0.05 versus PCP treatment; **p < 0.01 versus PCP treatment; ***p < 0.001 versus PCP treatment;
###p < 0.001 versus vehicle treatment.
4.2.3.2. Catalepsy test
Mice were orally dosed with vehicle or test compounds. Catalepsy was evaluated on a metal bar
0.3 cm in diameter positioned 4.5 cm above the tabletop. The test consisted in positioning the animal
with its forepaws on the bar and recording how long it remained hanging onto the bar. A mean
immobility score of 20 s was used as the criterion for the presence of catalepsy.
4.2.3.3. Quipazine-induced head twitches
ICR mice (18~30 g, 7 mice/dose/test compound) were fasted for 12 h and then placed in
individual cells of a clear Lucite box. Varing dose of test compounds or normal saline were given i.g.

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30 min before s.c. administration of quipazine (5 mg/kg). The behavioral observation was initiated 30
min after quipazine treatment, The number of stereotyped head twitch movements was counted for
each mouse by a trained observer over a 15 min period. The results were shown as means ± SEM and
compared with one-way analysis of variance, with intergroup comparisons for individual compounds
being calculated with Dunnett’s test.
4.2.3.4. Pharmacokinetic study
Pharmacokinetic studies were performed in male SD rats (3 in each group) weighing 200g ~ 250 g.
Pharmacokinetic parameters were obtained by single intravenous (i.v.) and p.o. administration of the
test compound 10 mg/kg. The test compound was dissolved in mixed solution of DMSO (5%), PEG400
(40%) and normal saline (55%). Heparinized samples of blood were collected at 5 min, 15min, 30 min,
1 h, 2 h, 4 h, 8 h and 24 h after oral or intravenous administration. Plasma was obtained after
centrifugation (8000 rpm, 6min, 2~8 °C) and stored frozen at −80°C for analysis. Plasma samples were
analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
Acknowledgements
This work was supported by Special Foundation of Chinese Academy of Sciences for strategic
pilot technology (Grant No. XDA12040208) and R&D Program of Shanghai Municipal Science and
Technology Commission (Grant No. 14431905500). The authors gratefully acknowledge Topharman
Shanghai Co., Ltd for collaboration.
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