Discovery of aryl-piperidine derivatives as potential antipsychotic agents using molecular hybridization strategy

Article abstract of DOI:10.1016/j.ejmech.2020.112214

Schizophrenia is a chronic, disabling mental disorder that affects about one percent of world's population. Drugs acting on multiple targets have been demonstrated to provide superior efficacy in schizophrenia than agents acting on single target. In this study, based on FW01, a selective potent 5-HT_1A receptor agonist discovered via dynamic pharmacophore-based virtual screening, molecular hybridization strategy was employed to optimize its in vitro activity over D₂ and 5-HT_2A receptors. The optimized compound 9f was found to show dual potent D₂ and 5-HT_2A receptors antagonistic activity. In addition, compound 9f showed good in vivo metabolic stability with t_1/2 of 2 h in ICR mice and good capability to penetrate the blood-brain barrier with K_p value of 4.03. These results demonstrated that the dual D₂ and 5-HT_1A receptor antagonist 9f could serve as a promising lead compound to discover potent antipsychotic agents.

Full text of DOI:10.1016/j.ejmech.2020.112214

Journal Pre-proof
Discovery of aryl-piperidine derivatives as potential antipsychotic agents using
molecular hybridization strategy
Chen Zhu, Xinwei Li, Bangyi Zhao, Weiqing Peng, Wei Li, Wei Fu
PII:
S0223-5234(20)30181-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112214
EJMECH 112214
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 December 2019
Revised Date: 5 March 2020
Accepted Date: 5 March 2020
Please cite this article as: C. Zhu, X. Li, B. Zhao, W. Peng, W. Li, W. Fu, Discovery of aryl-piperidine
derivatives as potential antipsychotic agents using molecular hybridization strategy, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112214.
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2020 Published by Elsevier Masson SAS.

Discovery of Aryl-piperidine Derivatives as Potential Antipsychotic
Agents Using Molecular Hybridization Strategy
#
#
Chen Zhu , Xinwei Li , Bangyi Zhao, Weiqing Peng, Wei Li, Wei Fu*
School of Pharmacy & Minhang Hospital, Fudan University, Shanghai 201301, P.
R. China
#
These authors contributed equally in this work.
*Corresponding author: Tel & Fax: +86-21-51980096, E-mail address: wfu@fudan.edu.cn
(Wei Fu)
Abstract
Schizophrenia is a chronic, disabling mental disorder that affects about one percent
of world’s population. Drugs acting on multiple targets have been demonstrated to
provide superior efficacy in schizophrenia than agents acting on single target. In this
study, based on FW01, a selective potent 5-HT1A receptor agonist discovered via
dynamic pharmacophore-based virtual screening, molecular hybridization strategy
was employed to optimize its in vitro activity over D and 5-HT2A receptors. The
2
optimized compound 9f was found to show dual potent D and 5-HT2A receptors
2
antagonistic activity. In addition, compound 9f showed good in vivo metabolic
stability with t1/2 of 2 h in ICR mice and good capability to penetrate the blood-brain
barrier with K value of 4.03. These results demonstrated that the dual D and
p
2
5
-HT receptor antagonist 9f could serve as a promising lead compound to discover
1A
potent antipsychotic agents.
Keywords
D receptor antagonist, 5-HT2A receptor antagonist, molecular hybridization,
2
multi-target strategy, antipsychotic
1
. Introduction
Schizophrenia is a chronic, disabling mental disorder with high risk of suicide,
1

1
affecting more than one percent of world’s population. The symptoms of
schizophrenia can be divided into three categories: positive symptoms, including
hallucination and delusion; negative symptoms, such as motivation and diminished
2
, 3
expression; and cognitive deficits like deficit in learning, memory and attention.
The dopaminergic hypothesis of schizophrenia postulates a hyperactivity of
dopamine in the mesolimbic pathway, and a hypofunctionality of dopamine in other
4
brain areas like the prefrontal cortex. Typical antipsychotics, such as haloperidol
(
Figure 1), producing therapeutic effects by the blockage of D receptor, are
2
effective for controlling positive symptoms, but exert severe extrapyramidal side
5
effects (EPS) and hyperprolactinemia. The D binding property is the key
2
therapeutic mechanism of typical antipsychotics in ameliorating at least the positive
6
symptoms of schizophrenia. Accumulating reports showed that multiple receptors,
including dopamine receptors and serotonin receptors in the central nervous system
(CNS), contribute to the complicated pathophysiological behaviors and various
7
, 8
symptoms of schizophrenia.
Atypical antipsychotics, such as clozapine and risperidone (Figure 1), have
equivalent or superior efficacy than typical antipsychotics but with lower EPS and
hyperprolactinemia. The general idea is that the blockage of 5-HT2A receptor
enhances dopamine efflux, compensates for the effect of blocking dopamine
receptors, thus dampening the deleterious effect associated with the blockade of D2
9
, 10
receptor.
Previous studies showed that 5-HT2A receptor antagonist could enhance
the antipsychotic effect of typical and atypical antipsychotics, and was effective in
11, 12
attenuating negative and cognitive symptoms of schizophrenia.
As for the role of
5
5
-HT , accumulated data supported the assertion that there is key involvement of
1
A
-HT1A receptors in the pathophysiology and treatment-related facets of
schizophrenia, particularly negative symptomatology. The combined D receptor
2
antagonists and 5-HT1A receptor agonists SLV313 (Figure 1) augmented
extracellular microdialysate dopamine and acetylcholine levels in medial prefrontal
1
3
cortex. The enhanced dopaminergic and cholinergic tone induced by the stimulation
of 5-HT1A receptor would translate into efficacy in the cognitive and negative
2

1
4
symptom domains. The allosteric methods were expected to expedite the discovery
1
5,16,17
of dopamine and serotonin regulators.
Though atypical antipsychotics are widely used in clinical practice, they still remain
1
8, 19
some severe side effects such as weight gain and cardiovascular risk.
Figure 1. Structures and biological activity of antipsychotics
In this study, based on FW01, a selective potent 5-HT receptor agonist discovered
1
A
by dynamic pharmacophore-based virtual screening, molecular hybridization strategy
was employed with the eventual aim to discover promising multi-target antipsychotic
agents with dual D and 5-HT antagonistic action.
2
2A
2
2
. Result and discussion
.1 Compound design
Our previous study discovered a selective 5-HT1A receptor agonist FW01 (Figure 1)
2
0
through the dynamic pharmacophore-based virtual screening. FW01 displayed high
binding affinity for 5-HT1A receptor with K of 51.9±16.4 nM, moderate affinity for
i
5
-HT2A receptor with K of 206.71±7.46 nM and weak affinity for dopamine D3
i
receptor with K of 2161.35±25.55 nM. FW01 bound to 5-HT receptor via multiple
i
1A
interaction forces. Nitrogen of indole and piperazine formed hydrogen bonding with
S5.42, Y7.43 and N7.39, respectively. Protonated nitrogen of piperazine participated
in the salt bridge with D3.32 (Figure 2). We employed hybridization strategy with an
aim to discover multi-target antipsychotic agents towards both serotonin receptors
and dopamine receptors, especially D receptor.
2
3

Figure 2. Binding mode of FW01 (green, sticks) with 5-HT1A receptor (cartoon).
The binding pocket of the dopamine receptor was divided into two parts, the
orthosteric binding site (OBS), in which dopamine was also thought to bind,
enclosed by transmembrane segments (TMs) 3, 5, 6, and 7, and a second binding site
2
1
(
SBS) enclosed by TMs 1, 2, 3 and 7.
,3-dichloro-4-phenylpiperazine terminus ( ) of compound
receptor agonist, maintained high binding affinities for both D and D receptors,
In Newman’s study, the
2
Ⅱ
Ⅰ, a highly selective D3
2
3
while the isolated 2-butyl-indole amide terminus (Ⅲ) showed weak binding affinities
2
2
for D and D receptors. The predicted binding pose of compound showed that
Ⅰ
2
3
the 2,3-dichloro-4-phenylpiperazine can penetrate into the OBS, with the 2-indole
terminus extended into SBP, suggesting that the binding in the OBS is critical to
potency and efficacy for dopamine receptors (Figure 3B). The docking results of
FW01 with D receptor represented that FW01 was inactive for the indole moiety of
2
FW01 collides with amino acid residues in the OBS (Figure 3D). Based on this
finding, we considered replacing the indolepiperazine moiety of the FW01 with
phenylpiperazine moiety of D antagonist
Ⅰto introduce the efficacy for D receptor
2
2
(Figure 3A). Considering the substitution on the benzene moiety may affect activity
for D receptor by altering the binding pose within the OBS, the o-methoxy
2
substitution on the benzene can constrain the bioactive conformation of benzene,
compounds 5a-5h were designed. Next, we studied the binding mode of atypical
antipsychotic iloperidone with D2 receptor. It showed that the
6
-fluoro-1,2-benzisoxazolyl piperidine moiety bound in the OBS (Figure 3C).
4

Therefore, we replaced the indolepiperazine moiety of the FW01 with the
-fluoro-1,2-benzisoxazolyl piperidine moiety of iloperidone in order to introduce
the dual D and 5-HT receptors antagonist activity. Meanwhile, we reversed the
6
2
2A
atom order of the amide bond of FW01 to avoid the possible toxic aniline of the
newly compounds. Besides, we changed the cyclohexyl ring to alkyl substituents, in
order to reduce the ClogP value of new compounds (Figure 3E). Finally, compounds
9
a-9f were designed, which is more structurally related to iloperidone and the dual
D /5-HT receptors antagonistic activity were successfully introduced.
2
2A
Figure 3. (A) Structures of lead compounds. (B) Electrostatic potential surface of compound

(pink) with D receptor. (C) Electrostatic potential surface of iloperidone (blue) with D receptor.
2 2
(D) Electrostatic potential surface of FW01 (green) with D2 receptor. (E) Design of
aryl-piperazine/piperidine derivatives.
2
.2 Chemical synthesis
Synthesis of 5a-l was shown in scheme 1. Commercially available arylpiperazines
were first acylated with 3-chloropropionyl chloride to obtain compound 2, which was
5

subjected to reduction by borane-methyl sulfide complex to give compound 3.
Compound 4, which was obtained via nucleophilic substitution of compound 3 with
corresponding amines, underwent condensation with benzoic acid to give 5a-l.
O
a
N
Cl
b
N
Cl
Aryl
N
NH
N
N
Aryl
Aryl
1
2
3
O
c
R
1
d
N
N
N
N
H
N
N
R
1
Aryl
Aryl
4
5 (a - l)
O
Aryl
N
N
=
R
1
= propyl
isopropyl
cyclopropylmethyl
oxetanyl
N
N
N
N
N
N
N
Cl
Cl
Scheme 1. Reagents and conditions: (a) 3-chloropropionyl chloride, TEA, DCM, r.t., 10 min; (b)
borane-methyl sulfide complex, THF, 80� , 2 h; (c) R -NH , K CO , CH CN, 75� , 8 h; (d)
1
2
2
3
3
benzoic acid, HBTU, HOBT, DIEA, DCM, r.t., 6 h.
Synthesis of 9a-i were shown in scheme 2. Commercially available
-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole underwent alkylation with
-bromo-3-chloropropane to give 7, followed by amination with corresponding
6
1
amines to afford compound 8. 9a-d were obtained by condensation of 8 with benzoic
acid and 9e-i were obtained by reaction with corresponding arylsulfonyl chloride
1
with 8. Structure of all compounds were confirmed by ESI-MS, HRMS, H NMR
1
3
and C NMR.
6

F
e
f
R1
NH
N
Cl
N
N
F
F
H
O
N
N
N
O
O
6
7
8
O
R = propyl
N
N
R
1
F
g
isopropyl
1
cyclopropylmethyl
oxetanyl
N
N
O
9
(a-d)
(e-i)
R
2
h
O
R = H, F, Cl, CF , CN
S
2
3
N
N
F
O
R
1
O
9
Scheme 2. Reagents and conditions: (e) 1-bromo-3-chloropropane, K CO , DMF, r.t., 17 h; (f)
2
3
R -NH , K CO , CH CN, 75� , 8 h; (g) benzoic acid, HBTU, HOBT, DIEA, DCM, r.t., 6 h; (h)
1
2
2
3
3
benzenesulfonyl chloride derivatives, DIEA, DCM, r.t., 10 h.
2
.3 Biological evaluation
After these new derivatives were synthesized, they were subjected to evaluation for
the functional activity toward 5-HT , 5-HT and D receptors. To our delight, as
1
A
2A
2
shown in Table 1, in addition to 5-HT1A receptor agonistic activity, 5a-5d displayed
moderate D receptor and weak 5-HT2A receptor antagonistic activity, which
2
confirmed the reliability of the design idea. However, in sharp contrast to FW01,
which served as a selective 5-HT1A receptor agonist, 5e~5h only showed moderate
antagonistic activity for D receptor and no activity for 5-HT and 5-HT receptors.
2
1A
2A
Compound 5g showed moderate D receptor antagonistic activity, indicating that
2
cyclopropylmethyl is superior to n-propyl, iso-propyl and oxetanyl. Compounds
5
i~5l bearing pyridine-2-yl displayed as selective 5-HT1A receptor agonists, with
similar activity profile to FW01. 5j and 5k bearing isopropyl and cyclopropylmethyl,
respectively, had higher potency than 5i and 5l. Different activity profile of
o-methoxylphenyl and pyridine-2-yl against 5-HT , 5-HT and D receptors
1
A
2A
2
suggested that this part plays a pivotal role in potency and efficacy.
As for 9a~9d that bear 6-fluorobenzo[d]isoxazo-2-lyl, they showed greatly reduced
activity for 5-HT1A receptor, and greatly improved antagonistic activity for D2
receptor and 5-HT2A receptor comparing with 5i~5l. Among 9a~9d, while 9c
displayed highest binding affinity for D receptor, 9b displayed highest binding
2
7

affinity for 5-HT receptor. Due to high activity for 5-HT receptor and D receptor,
2
A
2A
2
we next evaluated in vitro metabolic stability of 9a~9d in mice hepatic microsome
assay. As shown in Table 1, compound 9d was most stable among tested compounds
with t1/2 of 173.2 min, indicating that oxetanyl was metabolically stable superior to
isopropyl, cyclopropylmethy and propyl.
Table 1 Biological activity of compounds 5a~5l, and 9a~9d for D , 5-HT and 5-HT2A
2
1A
receptors.
D2
5-HT1A
5-HT2A
in vitro
Compounds
FW01
Aryl
X
R1
t1/2
IC50 (nM)
EC50 (nM)
IC50 (nM)
a
a
--
--
N
--
2161.35±25.55
7
206.71±7.46
3249
--
--
5
5
a
484.7
528
451.6
b
N
N
N
N
N
N
N
N
N
N
N
46.1
2008
--
5
c
768.1
2177
--
--
4
59.2
175.1
5
d
1878
466.9
823.5
288.7
3765
6935
NA
583.2
5
e
NA
NA
NA
NA
--
--
--
--
--
--
--
--
5
f
5
g
NA
NA
5
h
NA
NA
5
i
54.5
22.8
24.9
373.8
7549
NA
5
j
5
k
NA
7719
NA
5
l
NA
8

9
9
a
C
C
C
C
18.5
59.3
NA
NA
NA
NA
73.1
11.6
40.8
18.8
6.2
31.5
6.9
b
9
c
10.8
9
d
156.0
173.2
a: Ki value; NA: no activity.
However, compounds 9a~9d displayed moderate antagonistic activity for D receptor.
2
We next made an attempt to improve D and 5-HT receptor activity by substitution
2
2A
of benzoyl group with arylsulfonyl group while keeping oxetanyl constant. As shown
in Table 2, all compounds showed improved antagonistic activity for D receptor but
2
varying activity for 5-HT2A receptor. Compound 9e bearing phenylsulfonyl was
found to have highest activity for D receptor as well as 5-HT2A receptor.
2
Substitutions of H with F, Cl, CF or CN were also tolerated with minor reduced
3
activity for D receptor and moderate reduced activity for 5-HT receptor.
2
2A
Table 2 Biological activity of compounds 9e~9i for D , 5-HT and 5-HT2A receptors.
2
1A
D2
5-HT1A
5-HT2A
Compounds
R2
IC50 (nM)
EC50 (nM)
IC50 (nM)
9
e
1.9
3.0
2.0
2.3
3.6
NA
9.3
9
f
NA
NA
NA
NA
15.1
34.6
92.7
11.3
9
9
g
h
9
i
NA: no activity
Given potential metabolically labile of para-phenyl of 9e, reduced activity of 9g and
9
h for 5-HT2A receptor, potential hydrolysis of 9i in vivo, 9f was finally chosen to
9

evaluate its in vivo metabolic stability in ICR mice. As showed in Table 3, 9f was
rapidly eliminated with t1/2 of 0.54 h after i.v. injection. However, 9f displayed
improved t1/2 with 2.0 h after p.o. administration. Despite its relatively higher t , its
1/2
oral availability was low with F of 6.3%.
Table 3. Pharmacokinetics data of compound 9f measured in ICR mice (n = 6/group).
Dose
AUC0-24h
(ng×h/ml)
829.1
t1/2
(h)
Cmax
(ng/ml)
-
Tmax
(h)
-
F
(%)
-
(
mg/kg)
5
(i.v.)
0.54
2.00
2
5 (p.o.)
259.4
141.7
0.25
6.3
We also tested its blood brain barrier (BBB) penetration ability in ICR mice. As
showed in Table 4, 9f displayed K value of 1.72 after 1 hour and 4.03 after 4 hours
p
later, demonstrating that 9f was easy to pass BBB. As showed in Figure S1,
compound 9f displayed sedative effect in the forced swimming test, the mice keep
immobile within 2 hours after administration of 9f (i.g.).
New second-generation antipsychotic medications, iloperidone, asenapine and
lurasidone, approved by The Food and Drug Administration (FDA), are potent dual
2
3,24,25,26
5
-HT2A and D receptors antagonists (Table S1).
Among them, lurasidone
2
2
6
shows weak 5-HT1A agonistic activity. Ileridone and lurasidone is indicated for the
2
7,28
treatment of schizophrenia in adults,
asenapine has regulatory approval for the
29
indications of schizophrenia and bipolar mania/mixed episodes. In this work, 9f
shows potent dual D and 5-HT2A receptors antagonistic activity in vitro, good
2
capability to penetrate the blood-brain barrier with K value of 4.03 and potent
p
sedative activity in vivo, we speculate 9f has potential to treat schizophrenia,
especially to treat the positive symptom of schizophrenia. In the course of the next
research, further pharmacological studies would be carried on 9f to test its
antipsychotic effect.
2
.4 SAR discussion
Among all the above compounds，compounds 5a~5d bearing 2,3-dichlorophenyl
1
0

showed moderate D and weak 5-HT2A receptor antagonistic activity, moderate
2
5
-HT1A agonistic activity. Compounds 5e~5h bearing 2-methoxyphenyl served as
moderate selective D receptor antagonists. Compounds 5i~5l bearing pyridine-2-yl
2
displayed as selective 5-HT1A receptor agonists. Compounds 9a~9i bearing
6
-fluorobenzo[d]isoxazo-2-lyl displayed potent antagonistic activity towards D and
2
5
-HT2A receptors. To understand the structure and activity relationship (SAR)
between compounds 5a~5d, 5e~5h, 5i~5l and 9a~9i, the binding modes of
representative compound from every group 5c, 5g, 5h and 9f with dopamine and
serotonin subtypes were studied by molecular docking. Docking of compounds 5c,
5
g, 5h and 9f was performed using the GOLD 5.0.1 suit to the binding sites of the
X-ray structure of D receptor (PDB code: 6CM4), 5-HT1A receptor that was
2
obtained by homology modeling from the X-ray structure of 5-HT1B receptor (PDB
3
0,31,32
code: 4IAR) and 5-HT2A receptor (PDB code: 6A93).
The results were shown
in Figure 4.
For compound 5c, the protonated nitrogen atom interacts with residue D3.32 and the
benzene in the tail part formed π-π interaction with W7.40 of D receptor (Figure 4A).
2
In the active pocket of the 5-HT1A receptor, the protonated nitrogen atom of 5c had
interaction with residue D3.32 of D receptor, the benzene in the head part formed π-π
2
interaction with F6.52 and the benzene in the tail part of 5c formed π-π interaction
with W7.40 (Figure 4B). In the case of binding mode of compound 5c with 5-HT2A
receptor (Figure 4C), the protonated nitrogen atom formed a hydrogen bond with
residue D3.32 and the benzene in the head part formed π-π interaction with F6.52.
Thus compound 5c was found to display more potent activity toward 5-HT receptor
1
A
than that of D and 5-HT2A receptor. In terms of a selective and moderate D receptor
2
2
antagonist 5g, the protonated nitrogen atom formed strong salt bridge with D3.32, the
benzene in the head formed π-π interaction with F5.47 and F6.52, the benzene in the
tail formed π-π interaction with W100 (Figure 4D). While in terms of the binding
mode of 5g with 5-HT1A and 5-HT2A receptors, the phenylpiperazine moiety of 5g is
not able to reach into the deep pocket of receptors due to the existence of
2
-methoxyphenyl, leading to the cyclopropylmethyl in 5g collapsed with C187 and
11

C227 in the active pocket of 5-HT and 5-HT receptors, respectively (Figure 4E
1
A
2A
and 4F). Thus, 5g lost activity toward 5-HT and 5-HT receptors. For selective
1
A
2A
5
-HT receptor agonist 5k, the cyclopropylmethyl collapsed with V3.29 in the pocket
1A
of D receptor (Figure 4G), which is caused by the lack of conformational constraint
2
since there is no substituent in the head part. While in terms of 5k with 5-HT1A
receptor (Figure 4H), the protonated nitrogen atom of 5k formed strong salt bridge
with D3.32 of 5-HT1A receptor, the benzene in the head formed π-π interaction with
W6.48 and F6.52. However, in terms of binding of 5k with 5-HT , the only
2
A
interaction comes from the interaction of its protonated nitrogen atom with D3.32 in
-HT2A receptor. In contract, 9f showed potent activity toward D and 5-HT
5
2
2A
receptors, while it missed the activity to 5-HT . In terms of the binding of 9f with D
2
1
A
receptor (Figure 4J), the protonated nitrogen atom of 9f formed strong salt bridge
with residue D3.32. The benzisoxazole moiety of 9f inserted deeply into the OBS. The
oxygen atom of the benzisoxazole moiety of 9f formed a hydrogen bond with T3.37
and the oxygen atom of the oxetanyl moiety also formed a hydrogen bond with T7.39.
As for the docking of 9f with 5-HT2A receptor (Figure 4L), the protonated nitrogen
atom formed a hydrogen bond with the carboxylic acid side chain of D3.32 of 5-HT2A
receptor. The oxygen atom of the benzisoxazole moiety formed strong hydrogen bonds
with T3.37 and S5.46, and the oxygen atom of the sulfonamide moiety also formed a
hydrogen bond with the NH group of L229. While in the case of the interaction of 9f
with 5-HT1A receptor (Figure 4K), the benzoisoxazole moiety of 9f collapsed with
F6.44 and C3.36 in the active pocket of 5-HT , that is why 9f missed the binding
1
A
affinity for 5-HT receptor.
1
A
1
2

Figure 4. (A) The binding mode of compound 5c with D receptor. (B) The binding mode of
2
compound 5c with 5-HT1A receptor. (C) The binding mode of compound 5c with 5-HT2A receptor.
(
D) The binding mode of compound 5g with D receptor. (E) Electrostatic potential surface of
2
compound 5g with 5-HT1A receptor. (F) Electrostatic potential surface of compound 5g with
-HT2A receptor. (G) Electrostatic potential surface of compound 5k with D receptor. (H) The
5
2
binding mode of compound 5k with 5-HT1A receptor. (I) The binding mode of compound 5k with
-HT2A receptor. (J) The binding mode of compound 9f with D receptor. (K) Electrostatic
5
2
potential surface of compound 9f with 5-HT1A receptor. (L) The binding mode of compound 9f
with 5-HT2A receptor. Compound 5c was shown as blue sticks. Compound 5g was shown as pink
sticks. Compound 5k was shown as green sticks. Compound 9f was shown as white sticks.
3
. Conclusion
In summary, a series of aryl-piperazine/piperidine compounds were designed,
1
3

synthesized and evaluated activity over D , 5-HT and 5-HT receptors. Among all
2
1A
2A
the compounds, compound 9f, displayed potent antagonist activity towards D and
2
5
-HT2A receptors, good in vivo metabolic stability with t1/2 of 2 h in ICR mice after
oral administration and nice capability to penetrate the blood-brain barrier with Kp
value of 4.03. Molecular docking studies elucidated the driving force for the strong
association of 9f with D and 5-HT receptors. Thus, 9f could be used as a lead
2
2A
compound for further pharmacological studies and to discover potent antipsychotic
agents acting on both D and 5-HT receptors.
2
. Experimental protocols
.1. Chemistry
2A
4
4
Reagents and solvents were purchased from Adamas-beta and used without further
purification. Analytical thin-layer chromatography was performed on HSGF 254
(0.15-0.2 mm thickness, Yantai Jiangyou Company, Yantai, Shandong, China).
1
Column chromatography was carried out on silica gel (200-300 mesh). H NMR
spectra was recorded on a Brucker AMX-400/600 instrument, using TMS as an
internal standard and the chemical shifts were reported in parts per million (ppm).
Proton-coupling patterns were described as singlet, doublet, triplet, quartet, multiplet,
and coupling constants (J) values are given in hertz (Hz). Mass spectra were given
with an electric ionization (ESI) produced by HP5973N analytical mass spectrometer.
All tested compounds had a purity of >95% determined by HPLC.
4
.1.1. General procedures for the preparation of compound 2.
To a solution of corresponding aryl-piperazine (2.6 mmol) and triethylamine (3.1
mmol) in anhydrous dichloromethane (5 ml), 3-chloropropionyl chloride (3.1 mmol)
was added at 0°C and then stirred at room temperature for 10 min. After completion
of the reaction, 20 ml water was added and the aqueous phase was washed with
dichloromethane (3×15 ml). The organic phases were combined, washed with
saturated brine, and dried over anhydrous Na SO , filtered and evaporated under
2
4
reduced pressure. The residue was purified by column chromatography (petroleum
ether: ethyl acetate = 5:1, V/V) to yield compound 2.
4
.1.2. General procedures for the preparation of compound 3.
1
4

To a solution of 2 (2.0 mmol) in 20 ml anhydrous tetrahydrofuran, borane-methyl
sulfide complex (8.0 mmol) was added at 0°C under N protection over 30 min and
2
then stirred at 70 � for 2 h. 40 ml methanol was then added and the reaction mixture
was stirred at 70 � for another 1 h. After completion of the reaction, the solvent was
evaporated and 40 ml dichloromethane was added and the organic phase was washed
with water (3×20 ml), saturated brine. The organic phases were combined and dried
over anhydrous Na SO , filtered and evaporated under reduced pressure. The residue
2
4
was purified by column chromatography (petroleum ether: ethyl acetate = 5:1, V/V)
to yield compound 3.
4
.1.3. General procedures for the preparation of compound 4.
To a solution of 3 (2.0 mmol) in acetonitrile (5 ml), potassium carbonate (3.0 mmol)
and corresponding amine (3.0 mmol) were added at room temperature. The reaction
0
mixture was refluxed at 85 C for 8 h. After reaction was completed, the solvent was
evaporated and 40 ml dichloromethane was added and the organic phase was washed
with water (3×20 ml), saturated brine, dried over anhydrous Na SO , filtered and
2
4
evaporated under reduced pressure. The residue was purified by silica gel column
chromatography (dichloromethane: methanol = 40:1, V/V) to yield compound 4.
4
.1.4. General procedures for the preparation of compounds 5a~5l.
To a solution of 4 (2.0 mmol) in dichloromethane (6 ml), benzoic acid (2.4 mmol),
HBTU (3.0 mmol), 1-hydroxybenzotriazole (3.0 mmol) and N,
N-diisopropylethylamine (4.0 mmol) were added. The reaction mixture was stirred at
room temperature for 6 h. After completion of reaction, 10 ml dichloromethane was
added and the organic phase was washed with water (3×10 ml), saturated brine, dried
over anhydrous Na SO , filtered and evaporated under reduced pressure. The residue
2
4
was purified by chromatography (dichloromethane: methanol = 40:1, V/V) to yield
compounds 5a~5l.
4
.1.4.1. N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)-N-propylbenzamide (5a).
Compound 5a was prepared from
-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-propylpropan-1-amine and benzoic acid,
3
1
and obtained as yellowish oil. Yield 80.9%; H NMR (400 MHz, DMSO-d ) δ 7.43
6
1
5

(d, J = 4.0 Hz, 3H), 7.30-7.34 (m, 4H), 7.12 (d, J = 32.0 Hz, 1H), 3.41 (d, J = 20.0
Hz, 3H), 3.17 (d, J = 32.0 Hz, 2H), 3.00 (s, 1H), 2.78 (s, 2H), 2.58 (s, 1H), 2.42 (s,
1
0
1
H), 2.22 (d, J = 48.0 Hz, 4H), 1.80 (s, 1H), 1.61 (s, 3H), 1.49 (s, 1H), 0.92 (s, 2H),
13
.67 (s, 1H). C NMR (151 MHz, DMSO-d ) δ 170.6, 151.3, 137.6, 132.8, 129.0,
6
28.6, 128.5, 126.4, 126.1, 124.5, 119.7, 54.7, 52.6, 51.0, 45.9, 40.2, 25.5, 20.6, 11.4.
+
+
HRMS (ESI ) m/z [M+H] calculated: 434.1760, found: 434.1761.
.1.4.2. N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)-N-isopropylbenzamide
5b). Compound 5b was prepared from
4
(
3
-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-isopropylpropan-1-amine and benzoic
1
acid, and obtained as yellowish oil. Yield 81.7%; H NMR (400 MHz, DMSO-d ) δ
6
7
3
.43 (d, J = 4.0 Hz, 3H), 7.30-7.34 (m, 4H), 7.13 (d, J = 28.0 Hz, 1H), 3.80 (s, 1H),
.30 (s, 1H), 3.16 (s, 1H), 3.01 (s, 3H), 2.76 (s, 1H), 2.57 (s, 2H), 2.42 (s, 2H), 2.13
1
3
(
d, J = 20.0 Hz, 2H), 1.81 (s, 1H), 1.53 (s,1H), 1.24 (s, 2H), 1.10 (m, 4H). C NMR
(
151 MHz, DMSO-d ) δ 170.4, 151.3, 137.9, 132.8, 129.0, 128.6, 126.1, 126.0, 124.5,
6
+
+
1
4
4
19.7, 55.6, 52.9, 51.1, 50.0, 38.4, 26.2, 20.8. HRMS (ESI ) m/z [M+H] calculated:
34.1760, found: 434.1764.
.1.4.3.
N-(cyclopropylmethyl)-N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)benzamid
(5c). Compound 5c was prepared from
e
N-(cyclopropylmethyl)-3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propan-1-amine and
1
benzoic acid, and obtained as yellowish oil. Yield 76.2%; H NMR (400 MHz,
DMSO-d ) δ 7.43-7.44 (m, 3H), 7.30-7.36 (m, 4H), 7.13 (d, J = 32.0 Hz, 1H), 3.56 (s,
6
1
H), 3.31 (s, 1H), 3.04 (d, J = 28.0 Hz, 3H), 2.79 (s, 2H), 2.59 (s, 2H), 2.43 (s, 2H),
.28 (s, 2H), 2.17 (s, 1H), 1.84 (s, 1H), 1.65 (s, 1H), 1.00 (d, J = 76.0 Hz, 1H), 0.48
2
13
(
d, J = 20.0 Hz, 2H), 0.31 (s, 1H), 0.05 (s, 1H). C NMR (151 MHz, DMSO-d ) δ
6
1
5
70.7, 151.3, 137.5, 132.8, 129.1, 128.6, 128.5, 126.5, 126.1, 124.5, 119.7, 55.1, 53.3,
+
+
2.6, 51.0, 48.2, 40.2, 10.4, 3.6. HRMS (ESI ) m/z [M+H] calculated: 446.1760,
found: 446.1766.
.1.4.4.
N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)-N-(oxetan-3-yl)benzamide (5d).
4
1
6

Compound
5d
was
prepared
from
N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)oxetan-3-amine and benzoic acid,
1
and obtained as yellowish oil. Yield 76.7%; H NMR (400 MHz, DMSO-d ) δ 7.46
6
(d, J = 4.0 Hz, 3H), 7.30-7.32 (m, 3H), 7.10 (s, 1H), 4.89-4.92 (m, 1H), 4.67 (d, J =
13
8
.0 Hz, 4H), 3.41 (s, 2H), 2.78 (s, 6H), 2.27 (s, 5H), 1.19 (s, 1H). C NMR (151
MHz, DMSO-d ) δ 151.3, 136.6, 132.8, 129.8, 129.8, 128.6, 128.6, 127.0, 126.1,
6
+
+
1
4
4
24.5, 119.6, 74.9, 55.1, 52.7, 51.0, 40.2, 25.6. HRMS (ESI ) m/z [M+H] calculated:
48.1553, found: 448.1554.
.1.4.5. N-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-N-propylbenzamide (5e).
Compound
5e
was
prepared
from
3
-(4-(2-methoxyphenyl)piperazin-1-yl)-N-propylpropan-1-amine and benzoic acid,
1
and obtained as yellowish oil. Yield 73.8%; H NMR (400 MHz, DMSO-d ) δ 7.42 (s,
6
3
2
2
=
H), 7.32-7.34 (m, 2H), 6.82-6.92 (m, 4H), 3.76 (d, J = 8.0 Hz, 3H), 3.38-3.43 (m,
H), 3.13-3.24 (m, 3H), 2.97 (s, 2H), 2.75 (s, 2H), 2.53 (s, 1H), 2.39 (s, 1H), 2.24 (s,
H), 2.13 (t, J = 8.0 Hz, 1H), 1.79 (s, 1H), 1.58-1.63 (m, 2H), 1.48 (s, 1H), 1.00 (t, J
1
3
8.0 Hz, 1H), 0.91 (t, J = 8.0 Hz, 1H), 0.67 (t, J = 8.0 Hz, 1H). C NMR (151 MHz,
DMSO-d ) δ 170.4, 152.1, 141.4, 137.9, 129.0, 128.6, 126.0, 122.5, 121.0, 118.0,
6
+
+
1
12.1, 67.4, 55.8, 53.1, 50.2, 40.2, 38.5, 26.2, 20.8. HRMS (ESI ) m/z [M+H]
calculated: 396.2646, found: 396.2648.
.1.4.6. N-isopropyl-N-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)benzamide
5f). Compound 5f was prepared from
4
(
N-isopropyl-3-(4-(2-methoxyphenyl)piperazin-1-yl)propan-1-amine and benzoic acid,
1
and obtained as a yellowish oil. Yield 79.5%; H NMR (400 MHz, DMSO-d ) δ 7.43
6
(
d, J = 4.0 Hz, 3H), 7.32-7.34 (m, 2H), 6.87-6.93 (m, 4H), 3.77 (s, 3H), 3.29 (s, 2H),
.16 (s, 1H), 2.97 (s, 3H), 2.73 (s, 1H), 2.55 (s, 2H), 2.40 (s, 2H), 2.11 (d, J = 16.0
Hz, 2H), 1.80 (s, 1H), 1.23-1.24 (m, 2H), 1.10 (d, J = 8.0 Hz, 4H), 0.99-1.02 (m, 1H).
3
1
3
C NMR (151 MHz, DMSO-d ) δ 170.6, 152.1, 141.4, 137.6, 129.0, 128.5, 126.4,
6
+
1
22.5, 121.0, 118.0, 112.1, 55.5, 54.8, 52.9, 50.1, 46.8, 25.5, 20.6, 11.4. HRMS (ESI )
+
m/z [M+H] calculated: 396.2646, found: 396.2647.
4
.1.4.7.
1
7

N-(cyclopropylmethyl)-N-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)benzamide
5g). Compound 5g was prepared from
(
N-(cyclopropylmethyl)-3-(4-(2-methoxyphenyl)piperazin-1-yl)propan-1-amine and
1
benzoic acid, and obtained as yellowish oil. Yield 68.8%; H NMR (400 MHz,
DMSO-d ) δ 7.43 (s, 3H), 7.34 (d, J = 4.0 Hz, 2H), 6.82-6.92 (m, 4H), 3.76 (s, 3H),
6
3
.55 (s, 1H), 3.30 (s, 2H), 3.07 (s, 1H), 2.97 (s, 2H), 2.76 (s, 2H), 2.54 (s, 1H), 2.39
(
(
s, 1H), 2.25 (s, 2H), 2.14 (s, 1H), 1.82 (s, 1H), 1.64 (s, 1H), 0.90-1.09 (m, 2H), 0.47
13
d, J = 24.0 Hz, 2H), 0.30 (s, 1H), 0.05 (s, 1H). C NMR (151 MHz, DMSO-d ) δ
6
1
5
70.7, 152.1, 141.4, 137.5, 129.1, 128.5, 126.5, 122.5, 121.0, 118.0, 112.1, 55.5, 55.1,
+
+
3.2, 50.2, 40.2, 24.6, 9.9, 3.6. HRMS (ESI ) m/z [M+H] calculated: 408.2646,
found: 408.2649.
.1.4.8. N-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-N-(oxetan-3-yl)benzamide
5h). Compound 5h was prepared from
4
(
N-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)oxetan-3-amine and benzoic acid,
1
and obtained as yellowish oil. Yield 78.4%; H NMR (400 MHz, DMSO-d ) δ 7.46 (s,
6
5
H), 6.85-6.92 (m, 4H), 4.90 (t, J = 8.0 Hz, 1H), 4.62-4.68 (m, 4H), 3.75 (s, 3H),
.57 (s, 1H), 3.42 (s, 1H), 2.74 (s, 5H), 2.17 (d, J = 44.0 Hz, 5H), 1.81 (s, 1H), 1.46
3
1
3
(
s, 1H). C NMR (151 MHz, DMSO-d ) δ 151.8, 141.1, 136.3, 129.4, 128.2, 126.7,
6
+
1
22.1, 120.7, 117.7, 111.8, 74.6, 55.1, 52.6, 49.8, 39.9, 28.9, 26.4, 25.3. HRMS (ESI )
+
m/z [M+H] calculated: 410.2438, found: 410.2442.
4
.1.4.9.
N-propyl-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)benzamide
5i was prepared
(5i).
Compound
from
N-propyl-3-(4-(pyridin-2-yl)piperazin-1-yl)propan-1-amine and benzoic acid, and
1
obtained as yellowish oil. Yield 65.5%; H NMR (400 MHz, DMSO-d ) δ 8.10 (s,
6
1
H), 7.52 (s, 1H), 7.42-7.44 (m, 3H), 7.34 (s, 2H), 6.75-6.83 (dd, J = 20.0 Hz, J =
1 2
8
.0 Hz, 1H), 6.63 (s, 1H), 3.47 (s, 3H), 3.26 (s, 4H), 3.13 (s, 1H), 2.43 (d, J = 36.0
Hz, 2H), 2.16 (d, J = 24.0 Hz, 4H), 1.80 (s, 1H), 1.62 (s, 2H), 1.48 (s, 1H), 0.92 (d, J
1
3
=
8.0 Hz, 2H), 0.67 (s, 1H). C NMR (151 MHz, DMSO-d ) δ 170.6, 159.2, 147.7,
6
1
37.6, 129.0, 128.5, 126.4, 113.1, 107.2, 54.8, 52.4, 45.9, 44.7, 40.2, 25.4, 20.6, 11.4.
+
+
HRMS (ESI ) m/z [M+H] calculated: 367.2492, found: 367.2496.
1
8

4
.1.4.10. N-isopropyl-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)benzamide (5j).
Compound 5j was prepared from
N-isopropyl-3-(4-(pyridin-2-yl)piperazin-1-yl)propan-1-amine and benzoic acid, and
1
obtained as yellowish oil. Yield 74.3%; H NMR (400 MHz, DMSO-d ) δ 8.11 (s,
6
1
H), 7.52 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 4.0 Hz, 3H), 7.34 (d, J = 4.0 Hz, 2H), 6.83
(
(
d, J = 8.0 Hz, 1H), 6.63 (s, 1H), 3.80 (s, 1H), 3.48 (s, 1H), 3.16-3.30 (m, 4H), 2.40
1
3
s, 2H), 2.08 (s, 2H), 1.54 (s, 1H), 1.24 (s, 1H), 1.09-1.11 (m, 5H). C NMR (151
MHz, DMSO-d ) δ 170.4, 159.2, 147.7, 137.9, 137.6, 129.0, 128.6, 126.0, 113.1,
6
+
+
1
3
4
07.2, 55.7, 52.6, 50.0, 44.8, 38.4, 26.2, 20.8. HRMS (ESI ) m/z [M+H] calculated:
67.2492, found: 367.2498.
.1.4.11.
N-(cyclopropylmethyl)-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)benzamide (5k).
Compound
5k
was
prepared
from
and
N-(cyclopropylmethyl)-3-(4-(pyridin-2-yl)piperazin-1-yl)propan-1-amine
1
benzoic acid, and obtained as yellowish oil. Yield 62.3%; H NMR (400 MHz,
DMSO-d ) δ 8.10 (s, 1H), 7.50-7.53 (m, 1H), 7.43-7.44 (m, 3H), 7.36 (s, 2H),
6
6
3
.76-6.84 (dd, J = 24.0 Hz, J = 2.0 Hz, 1H), 6.63 (s, 1H), 3.51 (d, J = 36.0 Hz, 4H),
1 2
.27 (s, 4H), 3.07 (s, 1H), 2.43 (d, J = 36.0 Hz, 2H), 2.16 (d, J = 24.0 Hz, 3H), 1.84
(s, 1H), 1.65 (s, 1H), 1.00 (d, J = 72.0 Hz, 1H), 0.47 (d, J = 24.0 Hz, 2H), 0.31 (s,
1
3
1
H), 0.05 (s, 1H). C NMR (151 MHz, DMSO-d ) δ 170.7, 159.2, 147.7, 137.6,
6
1
37.5, 129.1, 128.5, 126.5, 113.1, 107.2, 55.5, 52.7, 48.2, 44.9, 40.2, 24.6, 10.4, 3.6.
+
+
HRMS (ESI ) m/z [M+H] calculated: 379.2492, found: 379.2497.
.1.4.12. N-(oxetan-3-yl)-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)benzamide (5l).
Compound 5l was prepared from
4
N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)oxetan-3-amine and benzoic acid, and
1
obtained as yellowish oil. Yield 64.6%; H NMR (400 MHz, DMSO-d ) δ 8.08-8.10
6
(
m, 1H), 7.46-7.53 (m, 6H), 6.79 (s, 1H), 6.61-6.64 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H),
1 2
4
2
1
.89-4.92 (m, 1H), 4.66 (t, J = 8.0 Hz, 4H), 3.44 (s, 2H), 3.25 (s, 3H), 2.42 (s, 2H),
1
3
.16 (s, 5H), 1.47 (s, 2H). C NMR (151 MHz, DMSO-d ) δ 159.2, 147.7, 137.6,
6
36.6, 129.8, 128.5, 127.0, 113.1, 107.2, 74.9, 54.8, 52.5, 44.7, 40.2, 25.6. HRMS
1
9

+
+
(
ESI ) m/z [M+H] calculated: 381.2285, found: 381.2292.
4
.1.5. General procedures for the preparation of compound 7.
To a solution of 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole (45 mmol) and
potassium carbonate (91 mmol) in N,N-dimethylformamide (80 ml) in ice-water bath,
a solution of 1-bromo-3-chloropropane in N,N-dimethylformamide (20 ml) was
dropped into the stirred suspension for 30 min. Reaction continued for 17 h at room
temperature. After completion of the reaction, the reaction mixture was poured into
water and washed with ethyl acetate (5×40 ml). The organic phases were combined
and was dried over anhydrous Na SO , filtered and evaporated under reduced
2
4
pressure. The residue was purified by chromatography (petroleum ether: ethyl acetate
2:1, V/V) to yield compound 7..
.1.6. General procedures for the preparation of compound 8.
=
4
To a solution of 7 (2.0 mmol) in acetonitrile (6 ml), potassium carbonate (2.4 mmol)
and corresponding amine (2.4 mmol) were added. The reaction mixture was refluxed
for 8 h at 85℃. After completion of the reaction, acetonitrile was removed and 40 ml
dichloromethane was added and the organic phase was washed with water (3×20 ml),
saturated brine, dried over anhydrous Na SO , filtered and evaporated under reduced
2
4
pressure. The residue was purified by chromatography (dichloromethane: methanol =
4
4
0:1, V/V) to yield compound 8.
.1.7. General procedures for the preparation of compound 9a-d.
To a solution of 8 (1.0 mmol) in dichloromethane (5 ml), benzoic acid (1.2 mmol),
HBTU (1.5 mmol), 1-hydroxybenzotriazole (1.5 mmol) and
N,N-diisopropylethylamine (2.0 mmol) were added. The reaction mixture was stirred
at room temperature for 6 h. After completion of the reaction, 10 ml dichloromethane
was added and the organic phase was washed with water (3×10 ml), saturated brine,
dried over anhydrous Na SO , filtered and evaporated under reduced pressure. The
2
4
residue was purified by chromatography (dichloromethane: methanol = 40:1, V/V) to
yield compounds 9a~9d.
4
.1.7.1.
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)-N-propylbenzamide
2
0

(
9a).
Compound
9a
was
prepared
from
and
3
-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-N-propylpropan-1-amine
1
benzoic acid, and obtained as yellowish oil. Yield 60.3%; H NMR (400 MHz,
DMSO-d ) δ 7.97 (d, J = 36.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.33 (s, 3H), 7.42 (s,
6
3
4
H), 3.17 (d, J = 32.0 Hz, 3H), 3.03 (s, 2H), 2.67 (s, 1H), 2.39 (s, 1H), 2.08 (d, J =
0.0 Hz, 3H), 1.80-1.88 (m, 4H), 1.61 (s, 4H), 1.48 (s, 1H), 0.91 (s, 2H), 0.66 (s, 1H).
1
3
C NMR (151 MHz, DMSO-d ) δ 170.6, 164.6, 163.2, 161.5, 137.6, 129.0, 128.4,
6
1
26.3, 123.9, 117.4, 112.7, 97.6, 55.0, 52.8, 46.9, 40.2, 33.6, 30.3, 25.6, 20.5, 11.4.
+
+
HRMS (ESI ) m/z [M+H] calculated: 424.2395 found: 424.2401.
.1.7.2.
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)-N-isopropylbenzamid
4
e
(9b).
Compound
9b
was
prepared
from
3
-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-N-isopropylpropan-1-amine and
1
benzoic acid, and obtained as yellowish oil. Yield 64.4%; H NMR (400 MHz,
DMSO-d ) δ 7.99 (d, J = 28.0 Hz, 1H), 7.69-7.22 (dd, J = 12.0 Hz, J = 4.0 Hz, 1H),
6
1
2
7
2
.43 (s, 3H), 7.27-7.34 (m, 3H), 3.80 (s, 1H), 3.29 (s, 2H), 3.10 (d, J = 52.0 Hz, 3H),
1
3
.40 (s, 1H), 2.08 (s, 4H), 1.83 (s, 4H), 1.55 (s, 1H), 1.25 (s, 2H), 1.11 (s, 4H). C
NMR (151 MHz, DMSO-d ) δ 170.4, 164.6, 163.2, 161.5, 138.0, 128.9, 128.6, 126.0,
6
+
1
23.9, 117.4, 112.7, 97.5, 55.9, 53.1, 50.0, 38.5, 33.7, 30.3, 26.4, 20.8. HRMS (ESI )
+
m/z [M+H] calculated: 424.2395, found: 424.2303.
4
.1.7.3.
N-(cyclopropylmethyl)-N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl
benzamide (9c). Compound 9c was prepared from
)
N-(cyclopropylmethyl)-3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propan-1-
1
amine and benzoic acid, and obtained as yellowish oil. Yield 61.6%; H NMR (400
MHz, DMSO-d ) δ 7.98 (d, J = 32.0 Hz, 1H), 7.69-7.72 (dd, J = 8.0 Hz, J = 4.0 Hz,
6
1
2
1H), 7.43 (d, J = 8.0 Hz, 3H), 7.36 (d, J = 4.0 Hz, 2H), 7.30 (s, 1H), 3.56 (s, 1H),
3.04-3.16 (m, 4H), 2.68 (s, 1H), 2.40 (s, 1H), 2.11 (d, J = 28.0 Hz, 4H), 1.86 (s, 4H),
1.64 (s, 2H), 0.91-1.10 (m, 1H), 0.48 (d, J = 24.0 Hz, 2H), 0.31 (s, 1H), 0.06 (s, 1H).
1
3
C NMR (151 MHz, DMSO-d ) δ 170.7, 164.6, 163.2, 161.5, 137.5, 129.0, 128.5,
6
2
1

1
26.4, 123.9, 117.4, 112.7, 97.6, 55.0, 53.1, 48.2, 40.1, 33.6, 30.3, 25.6, 10.3, 3.5.
+
+
HRMS (ESI ) m/z [M+H] calculated: 436.2395, found: 436.2400.
.1.7.4.
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)-N-(oxetan-3-yl)benza
4
mide
(9d).
Compound
9d
was
prepared
from
and
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)oxetan-3-amine
1
benzoic acid, and obtained as yellowish solid. Yield 62.0%; H NMR (400 MHz,
DMSO-d ) δ 7.93 (s, 1H), 7.69-7.72 (dd, J = 12.0 Hz, J = 4.0 Hz, 1H), 7.45 (s, 5H),
6
1
2
7
.28-7.33 (m, 1H), 4.91 (p, J = 8.0 Hz, 1H), 4.50-4.69 (m, 4H), 3.43 (s, 1H), 3.04 (s,
1
3
2
H), 2.64 (s, 2H), 2.11 (s, 3H), 1.87 (s, 4H), 1.52 (d, J = 44.0 Hz, 3H). C NMR
(
151 MHz, DMSO-d ) δ 164.6, 163.2, 162.9, 161.4, 136.6, 129.7, 128.5, 127.0, 123.9,
6
+
1
17.3, 112.7, 97.6, 74.9, 55.0, 52.9, 40.2, 33.6, 30.2, 25.8. HRMS (ESI ) m/z
+
[
M+H] calculated: 438.2187, found: 438.2191.
.1.8. General procedure for the preparation of compounds 9e~9i.
4
To a solution of 8 (1.0 mmol) in anhydrous dichloromethane (5 ml) in ice-water bath,
corresponding benzenesulfonyl chloride derivatives (1.5 mmol) and
N,N-diisopropylethylamine (1.5 mmol) were added. The reaction mixture was stirred
at room temperature for 10 h. After completion of the reaction, 10 ml
dichloromethane was added and the organic phase was washed with water (3×10 ml),
saturated brine, dried over Na SO , filtered and evaporated under reduced pressure.
2
4
The residue was purified by chromatography (dichloromethane: methanol = 40:1,
V/V) to yield compounds 9e~9i.
4
.1.8.1
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)-N-(oxetan-3-yl)benze
nesulfonamide
(9e).
Compound
9e
was
prepared
from
and
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)oxetan-3-amine
1
benzenesulfonyl chloride, and obtained as yellowish solid. Yield 54.2%; H NMR
(
400 MHz, DMSO-d ) δ 7.99-8.02 (dd, J =8.0 Hz, J = 4.0 Hz, 1H), 7.80 (d, J = 8.0
6 1 2
Hz, 2H), 7.69-7.75 (m, 2H), 7.64 (t, J = 8.0 Hz, 2H), 7.26-7.31 (td, J = 8.0 Hz, J =
1
2
4
.0 Hz, 1H), 4.80 (p, J = 4.0 Hz, 1H), 4.59-4.62 (dd, J = 8.0 Hz, J = 4.0 Hz, 2H),
1 2
2
2

4
.49-4.53 (dd, J = 8.0 Hz, J = 4.0 Hz, 2H), 3.11-3.20 (m, 3H), 2.93 (d, J = 12.0 Hz,
1 2
1
3
2
H), 2.34 (t, J = 8.0 Hz, 2H), 2.02-2.09 (m, 4H), 1.71-1.84 (m, 4H). C NMR (151
MHz, DMSO-d ) δ 164.6, 163.2, 162.9, 161.5, 138.0, 133.4, 129.8, 127.1, 123.9,
6
+
1
17.4, 112.7, 97.6, 75.0, 54.9, 53.0, 51.9, 43.6, 40.2, 33.6, 30.3, 27.4. HRMS (ESI )
+
m/z [M+H] calculated: 474.1857, found: 474.1863.
4
4
.1.8.2.
-Fluoro-N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)-N-(oxetan-3
-
yl)benzenesulfonamide
(9f).
Compound
9f
was
prepared
from
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)oxetan-3-amine
and
1
4
-fluorobenzenesulfonyl chloride, and obtained as yellowish solid. Yield 63.0%; H
NMR (400 MHz, DMSO-d ) δ 7.99-8.03 (dd, J =8.0 Hz, J = 4.0 Hz, 1H), 7.87-7.90
6
1
2
(m, 2H), 7.71 (d, J = 8.0 Hz, 1H), 7.46-7.50 (m, 2H), 7.29 (t, J = 8.0 Hz, 1H), 4.79 (p,
J = 8.0 Hz, 1H), 4.61 (t, J = 8.0 Hz, 2H), 4.53 (t, J = 8.0 Hz, 2H), 3.12-3.21 (m, 3H),
2
4
1
.94 (d, J = 8.0 Hz, 2H), 2.36 (t, J = 8.0 Hz, 2H), 2.03-2.10 (m, 4H), 1.74-1.85 (m,
1
3
H). C NMR (151 MHz, DMSO-d ) δ 165.3, 164.3, 162.9, 162.6, 161.2, 134.1,
6
30.0, 123.6, 117.1, 116.7, 112.4, 97.2, 74.7, 54.6, 52.7, 51.5, 43.3, 33.3, 30.0, 27.1.
+
+
HRMS (ESI ) m/z [M+H] calculated: 492.1763, found: 492.1767.
4
4
.1.8.3.
-Chloro-N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)-N-(oxetan-3
-
yl)benzenesulfonamide
(9g).
Compound
9g
was
prepared
from
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)oxetan-3-amine
and
1
4
-chlorobenzenesulfonyl chloride, and obtained as yellowish solid. Yield 63.0%; H
NMR (400 MHz, DMSO-d ) δ 7.99-8.02 (dd, J =8.0 Hz, J = 4.0 Hz, 1H), 7.81-7.83
6
1
2
(
dd, J =8.0 Hz, J = 4.0 Hz, 2H), 7.71 (d, J = 8.0 Hz, 1H), 7.46-7.50 (m, 2H), 7.29 (t,
1 2
J = 8.0 Hz, 1H), 4.79 (p, J = 8.0 Hz, 1H), 4.61 (t, J = 8.0 Hz, 2H), 4.53 (t, J = 8.0 Hz,
H), 3.12-3.21 (m, 3H), 2.94 (d, J = 8.0 Hz, 2H), 2.36 (t, J = 8.0 Hz, 2H), 2.03-2.10
2
1
3
(
m, 4H), 1.74-1.85 (m, 4H). C NMR (151 MHz, DMSO-d ) δ 164.3, 162.9, 162.6,
6
1
4
5
61.2, 138.1, 136.6, 129.5, 128.8, 123.6, 117.1, 112.4, 97.2, 74.7, 54.6, 52.7, 51.5,
+
+
3.4, 33.3, 30.0, 27.1. HRMS (ESI ) m/z [M+H] calculated: 508.1468, found:
08.1471.
2
3

4
.1.8.4.
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)-N-(oxetan-3-yl)-4-(tri
fluoromethyl)benzenesulfonamide (9h). Compound 9h was prepared from
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)oxetan-3-amine
and
4
6
-(trifluoromethyl)benzenesulfonyl chloride, and obtained as yellowish solid. Yield
1
4.8%; H NMR (400 MHz, DMSO-d ) δ 7.99-8.05 (m, 5H), 7.69-7.72 (dd, J = 8.0
6
1
Hz, J = 1.6 Hz, 2H), 7.69-7.72 (dt, J = 8.0 Hz, J = 4.0 Hz, 3H), 7.26-7.31 (td, J =
2
1
2
1
8
.0 Hz, J = 4.0 Hz, 1H), 4.81 (p, J = 8.0 Hz, 1H), 4.61 (t, J = 8.0 Hz, 2H), 4.54 (t, J
2
=
8.0 Hz, 2H), 3.20 (t, J = 8.0 Hz, 2H), 3.11-3.14 (m, 1H), 2.93 (d, J = 12.0 Hz, 2H),
13
2
.35 (t, J = 8.0 Hz, 2H), 2.02-2.10 (m, 4H), 1.72-1.85 (m, 4H). C NMR (151 MHz,
DMSO-d ) δ 164.3, 162.9, 162.6, 161.2, 141.7, 132.8, 127.9, 126.6, 123.6, 117.1,
6
+
+
1
12.4, 97.2, 74.6, 54.6, 52.7, 51.5, 43.5, 33.3, 30.0, 27.1. HRMS (ESI ) m/z [M+H]
calculated: 542.1731, found: 542.1730.
4
4
.1.8.5.
-Cyano-N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)-N-(oxetan-3-
yl)benzenesulfonamide
(9i).
Compound
9i
was
prepared
from
N-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propyl)oxetan-3-amine
and
1
4
-cyanobenzenesulfonyl chloride, and obtained as yellowish solid. Yield 66.9%; H
NMR (400 MHz, DMSO-d ) δ 8.12 (d, J = 8.0 Hz, 2H), 7.98-8.02 (m, 3H), 7.69-7.72
6
(dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 7.26-7.31 (td, J = 8.0 Hz, J = 4.0 Hz, 1H), 4.87
1 2 1 2
(p, J = 8.0 Hz, 1H), 4.62 (t, J = 8.0 Hz, 2H), 4.54 (t, J = 8.0 Hz, 2H), 3.25 (t, J = 8.0
Hz, 2H), 3.14 (t, J = 12.0 Hz, 1H), 2.94 (d, J = 12.0 Hz, 2H), 2.35 (t, J = 8.0 Hz, 2H),
1
3
2
1
5
.02-2.10 (m, 4H), 1.73-1.85 (m, 4H). C NMR (151 MHz, DMSO-d ) δ 164.3,
6
62.9, 162.6, 161.2, 141.9, 133.6, 127.6, 123.6, 117.1, 115.5, 112.4, 97.3, 74.6, 54.5,
+
+
2.7, 51.5, 43.4, 33.3, 30.0, 27.2. HRMS (ESI ) m/z [M+H] calculated: 499.1810,
found: 499.1812.
4
4
.2. Biological Evaluation
.2.1. Ultra Lance cAMP assay
The Ultra Lance cAMP assay was performed to evaluate the function of synthesized
compounds to D receptor and 5-HT1A receptor, using HEK-293 cells expressing the
2
2
4

human D receptor and 5-HT1A receptor. Reference compounds for D receptor and
2
2
5
-HT1A receptor were risperidone, and 8-OH-DPAT, respectively. Procedures for
Ultra Lance cAMP assay are: (1) Transfer compound to assay plate by Echo
compound total volume 100 µl; (2) Collect cells with stimulation buffer; (3)
(
Transfer 10 µl of cell solution to assay plate; (4) Centrifuge at 600 rpm for 3 min and
incubate 60 min at room temperature; (5) Add 5 µl 4X Eu-cAMP tracer solution and
5
µl 4X ULight™-anti-cAMP solution to assay plate; (6) Centrifuge at 600 rpm for 3
min and incubate 60 min at room temperature; (7) Read plate on EnVision. The IC50
values were calculated by nonlinear regression using a sigmoidal function.
4
.2.2. Fluorometric Imaging Plate Reader (FLIPR) assay
Using CHO-K1 cells expressing human 5-HT2A receptor, The FLIPR assay was
performed to evaluate the function of synthesized compounds to 5-HT2A receptor.
Reference compounds for 5-HT2A receptor was risperidone. Procedures for FLIPR
assay are : (1) Add 50 µl of the cell suspension to each well on the assay plate and
place the assay plate in 37� , 5% CO incubate for 16-24 h; (2) Remove cell plates
2
from the incubator or centrifuge, remove the supernatant and add 30 µl 1* dye; (3)
Place the assay plates in the 37� , 5% CO incubator for 1 h; (4) add 30 µl assay
2
buffer into the assay plate and then shake for 20-40 min; (5) Place the plates on the
FLIPR and 15 µl /well of test compound is added and calcium flux signal is
measured. After 15 min add 22.5 µl /well of inducer and calcium flux signal is
measured. The IC values were calculated by nonlinear regression using a sigmoidal
5
0
function.
.2.3. In vitro metabolic stability assay
4
The in vitro metabolic stability assay was performed to evaluate metabolic stability
of these newly discovered compounds in incubation with mouse liver microsomes.
Compounds were first dissolved in methanol to obtain the 1 mM stock solution. The
working solution was prepared by diluting 10 µl from stock solution with 90 µl
methanol and 900 µl water. The incubation system was consisted of 40 µl working
solution, 10 µl 20 mg/ml mouse liver microsomes and 310 µl 0.1 M PBS (pH = 7.4)
and incubated at 37� . Add 40 µl 10 mM NADPH to start the reaction and timing. 50
2
5

µl incubation medium was collected at 0 min, 5 min, 10 min, 20 min, 40 min, 60 min
and then added to 100 µl cold methanol, centrifuge at 4000 rpm for 15 min under 4� .
The supernatant was analyzed by liquid chromatography tandem with spectrometry
(
LC-MS/MS).
4
4
.3. Molecular Modeling
.3.1. Homology Modeling
The amino acid sequence of 5-HT1A receptor were downloaded from the
UniProtKB database (Entry code: P08908), and sequence similarity search was
performed using NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
The structure (PDB code: 4IAR) of 5-HT receptor was selected as the template to
1
B
construct the agonistic conformation of 5-HT1A receptor. Sequence alignment of
-HT receptor and 5-HT receptor was carried out using Discovery Studio 2016
5
1
B
1A
(hereafter abbreviated to DS). Homology modeling was performed with DS. Ten
models were generated after loop refinement and the one with the lowest Discrete
Optimized Protein Energy (DOPE) score was submitted to energy minimization
(
100 steps steepest descent with backbone constrained). The PROCHECK program
http://servicesn.mbi.ucla.edu/PROCHECK) was used to evaluate the
(
stereochemical quality of 5-HT receptor.
1
A
4
.3.2. Molecular Docking Operations
Molecular docking was carried out using GOLD 5.0.1. The binding site was
defined to include all residues within a 15.0 Å radius of the conserved D3.32 Cγ
carbon atom of D 5-HT and 5-HT receptors. A hydrogen-bond constraint was
2
,
1A
2A
set between the protonated nitrogen atom (N1) of ligand and D3.32 of D 5-HT
1A
2
,
and 5-HT2A receptor. Ten conformations were produced for each ligand, and
Gold-Score was used as scoring function. Other parameters were set as standard
default. High-scoring complexes were inspected visually to identify the most
reasonable solution. FW01 was docked with 5-HT and D receptors, respectively.
1
A
2
Compound 1 and iloperidone was docked with D receptor, respectively.
2
Compounds 5c, 5g, 5k and 9f were docked with D , 5-HT2A and 5-HT1A receptors
2
respectively.
2
6

Acknowledgments
This work is supported by grants from National Natural Science Foundation of China
(
NO. 81773635) and Shanghai Science and Technology Development Funds
14431900500).
(
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