Synthesis and antidepressant-like activity of novel aralkyl piperazine derivatives targeting SSRI/5-HT1A/5-HT7

Article abstract of DOI:10.1016/j.ejmech.2017.12.063

A series of novel aralkyl piperazine derivatives were synthesized, and evaluated for their serotonin reuptake inhibitory and 5-HT_1A/5-HT₇ receptors affinities activity. Antidepressant activities in vivo of the compounds were screened using the forced swimming test (FST) and tail suspension test (TST). The results indicated that compounds 21k (RUI, IC₅₀ = 31 nM; 5-HT_1A, 5-HT₇, k_i = 62, 12 nM) and 21n (RUI, IC₅₀ = 25 nM; 5-HT_1A, 5-HT₇, k_i = 28, 3.3 nM) exhibited high affinities for the 5-HT_1A/5-HT₇ receptors coupled with potent serotonin reuptake inhibition. Specifically, the most promising compound 21n possessed a good oral pharmacokinetic properties and an acceptable hERG profile, and showed potent antidepressant-like effect in the FST and TST models.

Full text of DOI:10.1016/j.ejmech.2017.12.063

Accepted Manuscript
Synthesis and antidepressant-like activity of novel aralkyl piperazine derivatives
targeting SSRI/5-HT /5-HT
7
1A
Zheng-Song Gu, Ai-nan Zhou, Ying Xiao, Qing-Wei Zhang, Jian-Qi Li
PII:
S0223-5234(17)31093-0
10.1016/j.ejmech.2017.12.063
EJMECH 10041
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 November 2017
Revised Date: 16 December 2017
Accepted Date: 16 December 2017
Please cite this article as: Z.-S. Gu, A.-n. Zhou, Y. Xiao, Q.-W. Zhang, J.-Q. Li, Synthesis and
antidepressant-like activity of novel aralkyl piperazine derivatives targeting SSRI/5-HT /5-HT ,
1A
7
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.063.
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ACCEPTED MANUSCRIPT
Synthesis and antidepressant-like activity of novel
aralkyl piperazine derivatives targeting
SSRI/5-HT /5-HT
1
A
7
Zheng-Song Gu, Ai-nan Zhou, Ying Xiao, Qing-Wei Zhang*, Jian-Qi Li*
Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical
Industry, China State Institute of Pharmaceutical Industry, 285 Gebaini Road, Shanghai 201203,
PR China
*
Corresponding author: Tel.: +86-021-20572128. E-mail: lijianqb@126.com; sipiqingwei@163.com
Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis and antidepressant-like activity of
novel aralkyl piperazine derivatives targeting
SSRI/5-HT /5-HT
1
A
7
Zheng-Song Gu, Ai-Nan Zhou, Ying Xiao, Qing-Wei Zhang*, Jian-Qi Li*
Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical
Industry, China State Institute of Pharmaceutical Industry, 285 Gebaini Road, Shanghai 201203,
PR China
*
Corresponding author: Tel.: +86-021-20572128. E-mail: lijianqb@126.com; sipiqingwei@163.com
Abstract
A series of novel aralkyl piperazine derivatives were synthesized, and evaluated
for their serotonin reuptake inhibitory and 5-HT1A /5-HT receptors affinities activity.
7
Antidepressant activities in vivo of the compounds were screened using the forced
swimming test (FST) and tail suspension test (TST). The results indicated that
compounds 21k (RUI, IC = 31 nM; 5-HT , 5-HT , k = 62, 12 nM) and 21n (RUI,
50
1A
7
i
IC50 = 25 nM; 5-HT , 5-HT , k = 28, 3.3 nM) exhibited high affinities for the
1A
7
i
5
-HT /5-HT receptors coupled with potent serotonin reuptake inhibition.
1A 7
Specifically, the most promising compound 21n possessed a good oral
pharmacokinetic properties and an acceptable hERG profile, and showed potent
antidepressant-like effect in the FST and TST models.
Key words
Antidepressant, serotonin reuptake inhibitory, 5-HT receptor, 5-HT receptor
1
A
7

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1
. Introduction
Depression is a debilitating disease that is characterized by low mood, slow
[1]
thought and mental disorder . According to the World Health Organization (WHO),
depression, especially major depression disorder, would be the second leading cause
[2]
of disability worldwide by the year 2020 . Over the past six decades, about six major
[3]
classes of monoamine based drugs have been developed in the therapy of depression .
Despite the extensive use of antidepressant drugs, there are still significant unmet
needs in the treatment of depression, including a long onset of action, moderate
patient response, and numerous adverse effects such as nausea/emesis, weight gain,
[4-5]
insomnia and sexual dysfunction . Recently, there has been important achievement
of antidepressant development, which interacts with dual or multiple targets. The
combination of serotonin transporter (SERT) inhibition with various 5-HT receptor
[6]
subtypes seems to be one of the most valuable approach to antidepressive therapy .
Recent clinical research has demonstrated that pindolol could accelerate
antidepressants onset time and enhance SSRIs beneficial effects by affecting 5-HT1A
[7]
receptor . Similarly, the approved antidepressant vilazodone with high affinity and
selectivity for 5-HT transporter and 5-HT1A receptor displayed fast antidepressant
[8]
efficacy with minimal adverse effects . Moreover, the combined administration of
low doses of an SSRI with selective 5-HT receptor antagonist (SB-269970) was also
7
[
9]
found active in behavioral models of depression . Additionally, recent research has
indicated that the vortioxetine showed obvious cognitive enhancement effects through
[10]
blockade of 5-HT receptor
. Hence, it stands to reason that agents with dual or
7
multiple binding affinities to SERT/5-HT /5-HT may be beneficial as treatment
1
A
7
options for depression and other cognitive impairment disorders.
Earlier study by Mewshaw et al. showed that aryloxylethylindolealkylamine
[11]
derivative I displayed high affinities for the SERT and 5-HT receptor . Venkatesan
1
A
et al. reported that the benzofuran derivative II exhibited potent binding affinity for
[
12]
the 5-HT1A receptor and SERT . Moreover, our previous research reported that the
aryl piperazine benzo[b][1,4]oxazine derivative III exhibited high affinities for
[
13]
5
-HT1A receptor coupled with moderate serotonin reuptake inhibitory (Fig. 2).

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Meanwhile, considerable research efforts have also been directed toward the
identification of novel compounds targeting 5-HT1A and 5-HT receptor. Leopoldo et
7
al. disclosed that 2-biphenyl piperazine derivatives IV-VI showed high affinity for
[14-16]
5
-HT1A and 5-HT receptors
(Fig. 2). Brasili et al. focused on the development
7
[17]
of novel potent 5-HT1A ligands , and reported that 1,4-dithiaspiro[4.5]decane
derivative VII and 1-(2-methoxyphenyl)piperazine derivatives VIII-X exhibited high
[18-21]
affinity and moderate to good selectivity for 5-HT R (Fig. 3)
.
1
A
Our lab has been engaged in multiple receptor-targeting in order to extend the
scope and utility of CNS agents. Herein we designed and synthesized a series of novel
aralkyl piperazine derivatives by modifying the scaffold of compound II. As shown in
Fig. 4, we first explored the effect of different aromatic ring substituents at the N1
position on the affinity for 5-HT1A and 5-HT receptors. Additionally, we examined
7
whether serotonin reuptake inhibition was improved by replacing the benzofuran
moiety with indole moiety. From these studies, we have discovered a new series of
compounds that exhibited high binding affinities at 5-HT /5-HT receptors coupled
1
A
7
with potent 5-HT reuptake inhibitory activity, and also demonstrated excellent oral
bioavailability and marked antidepressant-like activity in animal behavioral models.
2
. Results and discussion
2
.1 Chemistry
The synthetic routes of target compounds were outlined in Schemes 1-5. In
scheme 1, a series of benzofuran-3-yl piperazine derivatives (6a-d) were prepared.
The starting material benzofuran-3(2H)-one 1 was converted into ethyl ester 2 via the
Wittig reaction. Then intermediate 2 was reduced with lithium tetrahydroaluminate
into the corresponding alcohol 3. Activation of alcohol 3 with 4-toluenesulfonyl
chloride in the presence of triethylamine at room temperature provided intermediate 4.
The resulting compound 4 was then treated with benzyl piperazine derivatives 5 via
SN2 mechanism to yield 6a-d. The benzofuran-2-yl piperazine derivative 11 was
constructed by coupling of 2-iodophenol 7 with but-3-yn-1-ol 8, and subsequent
activation, substitution with 1-([1,1'-biphenyl]-2-yl)piperazine 5c (Scheme 2).

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According to scheme 3, the 1H-indole-5-carbonitrile piperazine analog 15 was
prepared. The 2-(5-bromo-1H-indol-3-yl)ethanol 12 was treated with CuCN to yield
alcohol 13. The target compound 15 was afforded by bromination of 13 with
tetrabromomethane and triphenylphosphine, and substitution with 1-([1,1'-biphenyl]-
2
-yl)-piperazine 5c.
The preparation of the desired 5-fluoro-1H-indole piperazine derivatives 21 was
depicted in scheme 4. The alcohols 18a and 18b were obtained by the Fischer indole
synthesis reaction of 4-Fluorophenylhydrazine hydrochloride 17 with
2
,3-dihydrofuran 16a and 3,4-dihydro-2H-pyran 16b, respectively. Bromination of
compound 18a with tetrabromomethane and triphenylphosphine provided compound
9a. Activation of alcohol 18b with 4-toluenesulfonyl chloride in the presence of
triethylamine at room temperature provided compound 19b. The desired compounds
1a-n were offered by nucleophilic substitution reaction of intermediates 19a-b with
1
2
aryl piperazine derivatives 20. Finally, the four carbon linker compound 26 was
prepared (scheme 5). The acylation of the aromatic ring 22 with 4-chlorobutanoyl
chloride provided compound 23. Then intermediate 23 was reduced with triethylsilane
and trifluoroacetic acid to yield compound 24. The compound 25 was obtained by
substitution of intermediate 24 with aryl piperazine derivative 20f. The deprotection
of intermediate 25 with 4N aqueous NaOH solution gave the desired compound 26.
The synthesis of commercially unavailable biphenyl piperazine intermediates
2
0a-i was shown in scheme 6. The biphenyl amine compounds 27 were treated with
bis(2-chloroethyl)amine hydrochloride 28 via SN2 mechanism to obtain 20a-i.
2
.2 Biology
All synthesized compounds were tested for their inhibition of 5-HT reuptake and
binding affinities at 5-HT /5-HT receptors in vitro assays. The following specific
1
A
7
3
radioligands and tissue sources were used: (1) 5-HT1A receptors, [ H] 8-OH-DPAT,
3
human recombinant (HEK-293 cells); (2) 5-HT receptors, [ H] LSD, human
7
3
recombinant (CHO cells); (3) Rat serotonin transporter, [ H] serotonin, rat brain
synaptosomes. All compounds were initially screened at 10 µM concentration, and

ACCEPTED MANUSCRIPT
selective compounds (inhibition >90%) were then assayed to obtain their IC and Ki
5
0
values. Vortioxetine was used as a reference. The detailed results were summarized in
Table 1.
The role of aromatic ring substitution at the N1 position was first explored. The
methoxyl 6a and methylthio 6b analogs were selectively inhibitory for 5-HT1A
receptor and 5-HT reuptake (inhibition ratio > 90%). Interestingly, the 2-phenyl
analog 6c displayed high potency for 5-HT reuptake inhibition [RUI, IC = 210 nM]
5
0
and kept more than 90% inhibition for 5-HT1A and 5-HT receptors, whereas the
7
3
-phenyl analog 6d showed less than 80% inhibition for 5-HT receptor at the
7
concentration of 10 µM. These results showed the importance of phenyl group at the
R1 position in interacting with 5-HT receptor. When the 2-phenyl feature remained,
7
the replacement of the benzofuran-3-yl moiety with the benzofuran-2-yl moiety
(
compound 11) dramatically decreased inhibition of 5-HT reuptake [RUI, IC = 2900
50
nM]. Meanwhile, the replacement of benzofuran moiety with the 5-carbonitrile-indole
moiety (compound 15) [RUI, IC = 190 nM] or 5-fluoro-indole moiety (compound
5
0
2
1a) [RUI, IC50 = 110 nM] increased inhibition of 5-HT reuptake. In addition,
compound 21a displayed high affinities for both the 5-HT1A and 5-HT receptors
7
[
5-HT , k = 0.53 nM; 5-HT , k = 0.26 nM]. The results showed that the
1A i 7 i
5
-fluoro-indole-3-yl could be a preferred substituent (Table 1).
In the next research, we focused on the modification of biphenyl moiety of
compound 21a. We first introduced different substituents into the pair position of the
phenyl group (compounds 21b-e). As shown in Table 1, none of these pair substituted
compounds exhibited potency for 5-HT reuptake inhibition except 4-fluoro derivative
2
1b [RUI, IC = 590 nM]. Meanwhile, the activity of ortho and meta substituted
50
phenyl compounds was also explored (compounds 21f-h). Compared with 21a, the
-fluorophenyl analog 21f showed a small decrease in affinity for 5-HT /5-HT
7
3
1
A
receptors [5-HT , k = 5.6 nM; 5-HT , k = 0.79 nM] and reduction in inhibition of
1
A
i
7
i
5
-HT reuptake [RUI, IC = 150 nM]. Similarly, the 3-methylphenyl analog 21g also
50
exhibited high potency for 5-HT reuptake inhibition [RUI, IC50 = 160 nM].
Furthermore, the 2-fluorophenyl analog 21h displayed a small increase in inhibition

ACCEPTED MANUSCRIPT
of 5-HT reuptake [RUI, IC = 96 nM] but a considerable reduction in affinity for
5
0
5
-HT /5-HT receptors [5-HT , k = 25 nM; 5-HT , k = 2.3 nM] (21h v 21a).
1A 7 1A i 7 i
The influence of the chain length on 5-HT reuptake inhibition and 5-HT1A
5-HT receptors affinities was also explored. Surprisingly, the three carbon chain
/
7
analog 21i displayed outstanding affinity for 5-HT /5-HT receptors [5-HT , ki =
1
A
7
1A
1
7
3 nM; 5-HT , ki = 1.1 nM] and excellent 5-HT reuptake inhibitory activity [IC =
7 50
.6 nM]. Similarly, 3-fluorophenyl analog 21j exhibited higher potency for 5-HT
reuptake inhibition [RUI, IC50 = 42 nM] (21j v 21f). However, the compound 26
showed dramatically decreased inhibition of 5-HT reuptake when the linker was
elongated to four carbons [RUI, IC = 120 nM] (26 v 21k). Those results suggested
5
0
that the distance of linker chain had a great influence on the 5-HT reuptake activity.
Finally, the effect of R substituent in the central benzene ring of compound 21j
2
was explored. The 4-fluoro substituted analog 21k showed a small increase in
inhibition of 5-HT reuptake [RUI, IC = 31 nM] but a big reduction in affinities for
5
0
the 5-HT1A and 5-HT receptors [5-HT , ki = 62 nM; 5-HT , ki = 12 nM] (21k vs
7
1A
7
2
1j). In contrast, the 3-fluoro 21l and 5-fluoro 21m analogs displayed higher potency
for the 5-HT /5-HT receptors and the 5-HT reuptake inhibition compared to 21k. As
1
A
7
the above mentioned (21f vs 21a), the replacement of the 3-fluorophenyl moiety with
the phenyl moiety (compound 21n) at R1 position displayed increased inhibition of
5
-HT reuptake [RUI, IC = 25 nM] and binding affinities for 5-HT /5-HT receptors
50 1A 7
[
5-HT , ki = 28 nM; 5-HT , ki = 3.3 nM] (21n vs 21k).
1A 7
With the above information in hand, the potent compounds 21a-b, 21f, 21h and
1j-n were selected for metabolic stability evaluation in vitro. The results are shown
2
in Table 2. The stability values of compounds 21a-b and 21f suggested that the
presence of an fluorine atom at the meta and pair position of phenyl group could
improve metabolic stability. However, the introduction of fluorine atom at the ortho
position of phenyl group reduced metabolic stability (21h vs 21a). Interestingly,
compound 21j showed increased metabolic stability when the length of the linker
extended from two to three carbons (21j vs 21f). Moreover, the metabolic stability
influence of the R substituent in the central benzene ring was also explored (21k-n).
2

ACCEPTED MANUSCRIPT
The 3-fluoro 21l and 5-fluoro 21m analogs displayed stabilities equal to 21j.
Interestingly, the 4-fluoro substituted compound 21k showed significant improvement
in metabolic stability (21k vs 21j). Similarly, compound 21n displayed metabolic
stability higher than 21j. Those results suggested that the introduction of a fluoro
group into the 4-position of the central benzene ring could improve metabolic stability
significantly.
On the basis of in vitro studies, the compounds 21k and 21n were selected for
further evaluation in the mouse forced swim test (FST). The compounds were
administered orally once daily for 7 days at doses of 10, 20 and 40 mg/kg/day (PO),
respectively. Vortioxetine was acted as a positive control (40 mg/kg/day, PO). The
results are shown in Fig. 5. Compared with vehicle, the selected compounds 21k and
2
1n reduced immobility times in the FST in a dose-dependent manner that was
statistically significant at 20 and 40 mg/kg doses (PO). The positive control,
vortioxetine, also produced a statistically significant reduction of immobility time at
4
0 mg/kg dose.
The most promising compound 21n was further characterized in the tail
suspension test (TST). The results are shown in Fig. 6. Venlafaxine was used as a
reference compound (40 mg/kg/day, PO). The compound 21n was administered orally
once daily for 7 days at doses of 10, 20 and 40 mg/kg/day (PO). Compared to the
control group, compound 21n was dose dependently reduced the immobility time in
the TST, which was statistically significant at 20 and 40 mg/kg doses.
Compound 21n was also tested in the hERG channel binding assay to ascertain if
the compound can cause cardiac toxicity. The data showed that compound 21n
displayed acceptable hERG safety profile (IC =16.66µM).
5
0
It was suggested that many 5-HT R ligands showed high affinity for
1
A
[22]
α1-adrenoceptors due to their high degree of homology (approximately 45%)
.
The receptor selectivity of 5-HT1A subtype and a1-adrenoceptor was measured. The
data showed that compound 21n exhibited equal affinity for a1-adrenoceptor (Ki = 28
nM) (Table 3).
Finally, the pharmacokinetic properties of 21n were measured in male SD rats

ACCEPTED MANUSCRIPT
(Table 4). After administration of 2 mg/kg (PO) 21n to SD rats, a Cmax of 86.7 ng/mL
was obtained at 2 h. The elimination half-life of 21n after oral administration was 6.1
h. Compound 21n also had a potent orally bioavailable (F = 68.5 %) in rats.
3
. Conclusion
In summary, a new series of novel aralkyl-piperazine derivatives were
investigated, and as our continuing effort to identify novel compounds targeting
SSRI/5-HT /5-HT for the treatment of depressive disorders. Our SAR studies
1
A
7
revealed that the position of aromatic heterocyclic (6c vs 11) and the length of the
linker (21a vs 21i) were clearly important for 5-HT inhibitory activity. The data
showed that the metabolic stability of the compounds was related to the presence of
fluorine atoms in well-defined regions of molecule (21k and 21n). To confirm the
potential in vivo antidepressant effect of this series of compounds, compounds 21k
and 21n were selected for FST profiling in mice. The two selected compounds
showed a dose-dependent reduction of the immobility time in the FST. The most
promising compound 21n displayed dose dependently reduced the immobility time in
TST model. In addition, compound 21n also had good oral pharmacokinetic
properties in rats, and had an acceptable hERG profile. Our data lay a foundation for
successful future development of novel aralkyl-piperazine derivatives as next
generation of antidepressants.
4
. Experimental protocols
4
.1 Chemistry
Unless specified otherwise, all starting materials, reagents and solvents were
commercially available. Nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker NMR AVANCE 600 (600 MHz) or a Varian INOVA-400 (400 MHz)
spectrometer with TMS as an internal standard. Chemical shift (δ values) and
coupling constants (J values) are given in ppm and Hz, respectively. Signal
multiplicities are characterized as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), and br (broad). ESI mass spectra and HRMS were performed on an

ACCEPTED MANUSCRIPT
Agilent 6210 TOF spectrometer. The progress of all reactions was monitored using
TLC on precoated silica gel plates GF254 (Qingdao Haiyang Chemical, China). Silica
gel column chromatography was performed with Silica gel 60G (Qingdao Haiyang
Chemical, China). The chromatograms were viewed under UV light at 254 and/or 265
nm. Uncorrected melting points were determined on an electrothermal melting point
apparatus. Solvents and reagents were used without any pretreatment.
4
.1.1 Procedure A. ethyl 2-(benzofuran-3-yl)acetate (2)
A mixture of benzofuran-3(2H)-one (4g, 29.82 mmol) and (Carbethoxymethylene)
triphenylphosphorane (15.58g, 44.73 mmol) in toluene (200 mL) was refluxed for
8h under N atmosphere. After cooling to room temperature, the solvent was
-
4
2
removed under reduced pressure. The residue was purified by column
chromatography on silica gel (PE/EtOAc = 50:1, V/V) to give compound 2 as
[23]
+
colourless oil (4.56 g, 75%) . MS (ESI) m/z: 227.06 [M+Na] .
4
.1.2 Procedure B. 2-(benzofuran-3-yl)ethanol (3)
To a stirred suspension of LiAlH (1.49 g, 39.17 mmol) in dry THF (50 mL) was
4
added slowly a solution of ethyl 2-(benzofuran-3-yl)acetate (2) (4 g, 19.58 mmol ) in
o
dry THF (20 mL) at 0 C. After the addition, the mixture was stirred at room
temperature for 4h and quenched with saturated NH Cl solution. The mixture was
4
extracted with DCM and washed with water and brine. The organic layer was dried
over anhydrous Na SO , filtered and concentrated. The residue was purified by
2
4
column chromatography on silica gel (PE/EtOAc = 5:1, V/V) to give compound 3 as
[23]
+
yellow oil (2.86 g, 90%) . MS (ESI) m/z: 185.11 [M+Na] .
4
.1.3 Procedure C. 2-(benzofuran-3-yl)ethyl 4-methylbenzenesulfonate (4)
To a stirred solution of p-toluenesulfonyl chloride (4.41 g, 23.13 mmol), triethylamine
3.12 g, 30.83 mmol) and 4-dimethylaminopyridine (188 mg, 1.54 mmol) in dry
CH Cl (60 mL) was added slowly a solution of benzofuran-ethanol (3) (2.5 g, 15.41
(
2
2
o
o
mmol) in dry CH Cl (25 mL) at 0 C. The reaction mixture was stirred for 1 h at 0 C
2
2

ACCEPTED MANUSCRIPT
and left for overnight at room temperature. The reaction mixture was washed with
water and brine, dried over anhydrous Na SO , filtered and concentrated. The residue
2
4
was purified by column chromatography on silica gel (PE/EtOAc = 5:1, V/V) to give
[24]
+
compound 4 as a white solid (3.41 g, 70%) . MS (ESI) m/z: 339.07 [M+Na] .
4
.1.4 Procedure D. General procedure for the preparation of compounds 6a-d
A mixture of 2-(benzofuran-3-yl)ethyl 4-methylbenzenesulfonate (4) (1.58 mmol),
phenylpiperazine derivatives (5) (1.89 mmol) and K CO (3.16 mmol) in CH CN (50
2
3
3
o
mL) was stirred at 80 C for 12 h. The solvent was removed under reduced pressure,
then CH Cl (50 mL) was added. The mixture was washed with water and brine, dried
2
2
over anhydrous Na SO , filtered and concentrated. The residue was purified by
2
4
column chromatography on silica gel (DCM/MeOH = 100/1, V/V) to give compound
. The compound 6 was dissolved in EA (15 mL), then hydrogen chloride ethyl
acetate solution (2N, 1 mL) was added dropwise. The mixture was stirred at r.t. for
2h, then filtered. The residue was washed with EtOAc and EtOH separately，dried in
vacuo to give compound 6 hydrochloride.
6
1
4
.1.4.1
1-(2-(benzofuran-3-yl)ethyl)-4-(2-methoxyphenyl)piperazine
(6a)
hydrochloride.
o
+ 1
Yield 70 % as white solid. Mp: 228-229 C. MS (ESI) m/z: 337.31 [M+H] . H NMR
(
400 MHz, DMSO) δ 7.93 (s, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H),
.40 – 7.28 (m, 2H), 7.08 – 6.90 (m, 4H), 3.81 (s, 3H), 3.68 (d, J = 11.4 Hz, 2H), 3.53
d, J = 12.3 Hz, 2H), 3.48 – 3.39 (m, 2H), 3.31 – 3.23 (m, 4H), 3.14 (t, J = 11.7 Hz,
7
(
1
3
2
1
4
H). C NMR (100 MHz, DMSO) δ 154.59, 151.83, 142.70, 138.62, 127.14, 124.63,
24.00, 122.63, 120.85, 120.01, 118.54, 115.63, 112.09, 111.37, 55.42, 54.16, 50.71,
+
6.97, 17.85. HRMS calcd for C H N O [M+H] , 337.1916; found, 337.1918.
2
1
24
2
2
4
.1.4.2
1-(2-(benzofuran-3-yl)ethyl)-4-(2-(methylthio)phenyl)piperazine
(6b)
hydrochloride.
Yield 68 % as a white solid. Mp: 253-254 C. MS (ESI) m/z: 353.29 [M+H] . H NMR
o
+ 1

ACCEPTED MANUSCRIPT
(400 MHz, DMSO) δ 7.93 (s, 1H), 7.84 (d, J = 7.1 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H),
7
.38 – 7.29 (m, 2H), 7.22 – 7.12 (m, 4H), 3.71 (d, J = 8.9 Hz, 2H), 3.52 – 3.45 (m,
13
2
H), 3.34 (d, J = 9.6 Hz, 2H), 3.27 – 3.19 (m, 6H), 2.41 (s, 3H). C NMR (100 MHz,
DMSO) δ 154.61, 147.42, 142.72, 134.30, 127.14, 125.12, 125.00, 124.65, 124.56,
1
22.64, 120.03, 119.60, 115.61, 111.39, 54.20, 51.30, 47.91, 17.94, 13.52. HRMS
+
calcd for C H N OS [M+H] , 353.1688; found, 353.1687.
2
1
24
2
4
.1.4.3
hydrochloride.
Yield 65 % as a white solid. Mp: 237-239 C. MS (ESI) m/z: 383.21 [M+H] . H NMR
400 MHz, DMSO) δ 7.89 (s, 1H), 7.77 (d, J = 7.2 Hz, 1H), 7.66 (d, J = 7.1 Hz, 2H),
.58 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.38 – 7.24 (m, 5H), 7.16 (t, J = 8.3
Hz, 2H), 3.51 (d, J = 10.9 Hz, 2H), 3.44 – 3.36 (m, 2H), 3.16 – 3.13 (m, 4H), 3.06 –
1-([1,1'-biphenyl]-2-yl)-4-(2-(benzofuran-3-yl)ethyl)piperazine
(6c)
o
+ 1
(
7
1
3
2
1
.92 (m, 4H). C NMR (100 MHz, DMSO) δ 154.57, 148.25, 142.68, 140.13, 134.03,
31.28, 128.56, 128.26, 127.08, 124.63, 123.51, 122.61, 119.95, 118.57, 115.58,
+
1
11.38, 54.11, 50.74, 47.35, 17.83. HRMS calcd for C H N O [M+H] , 383.2123;
2
6
26
2
found, 383.2124.
4
.1.4.3
1-([1,1'-biphenyl]-3-yl)-4-(2-(benzofuran-3-yl)ethyl)piperazine
(6d)
hydrochloride.
o
+
1
Yield 65 % as a white solid. Mp: 211-212 C. MS (ESI) m/z: 383.22 [M+H] . H
NMR (400 MHz, DMSO) δ 7.93 (s, 1H), 7.81 (d, J = 6.9 Hz, 1H), 7.70 – 7.65 (m,
2
1
3
H), 7.59 (d, J = 7.8 Hz, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.40 – 7.28 (m, 4H), 7.26 (s,
H), 7.16 (d, J = 7.6 Hz, 1H), 7.04 (dd, J = 8.1, 2.2 Hz, 1H), 4.00 (d, J = 8.5 Hz, 2H),
1
3
.71 (d, J = 6.7 Hz, 2H), 3.52 – 3.43 (m, 2H), 3.31 – 3.18 (m, 6H). C NMR (100
MHz, DMSO) δ 154.59, 149.83, 142.69, 141.27, 140.47, 129.70, 128.76, 127.42,
1
5
27.14, 126.85, 124.64, 122.63, 120.02, 118.68, 115.67, 115.10, 114.49, 111.38, 54.04,
+
0.32, 45.49,17.87. HRMS calcd for C H N O [M+H] , 383.2123; found, 383.2125.
2
6
26
2
4
.1.5 Procedure E. 2-(benzofuran-2-yl)ethanol (9)

ACCEPTED MANUSCRIPT
To a stirred mixture of 2-iodophenol (5.7 g, 25.94 mmol), palladium acetate (292 mg,
1
.29 mmol), CuI (247 mg, 1.29 mmol) and triphenylphosphine (340 mg, 1.29 mmol)
in dry triethylamine (60 ml) was added 3-butyn-1-ol (2 g, 28.5 mmol) under
nitrogen atmosphere. The reaction mixture was then stirred for overnight at room
temperature. The mixture was concentrated, diluted with EtOAc (100 mL). The
EtOAc layer was washed with water and brine, dried over anhydrous Na SO , filtered
2
4
and concentrated. The residue was purified by column chromatography on silica gel
[24]
(
PE/EA = 3/1, V/V) to give compound 9 as yellow oil (3.7 g, 88%) . MS (ESI) m/z:
+
1
85.09 [M+Na] .
4
.1.6 Procedure F. 2-(benzofuran-2-yl)ethyl 4-methylbenzenesulfonate (10)
To a stirred solution of p-toluenesulfonyl chloride (3.53 g, 18.51 mmol), triethylamine
2.5 g, 24.70 mmol) and 4-dimethylaminopyridine (150 mg, 1.23 mmol) in dry
CH Cl (40 mL) was added slowly a solution of benzofuran-ethanol (9) (2 g, 12.33
(
2
2
o
o
mmol) in dry CH Cl (20 mL) at 0 C. The reaction mixture was stirred for 1 h at 0 C
2
2
and left for overnight at room temperature. The reaction mixture was washed with
water and brine, dried over anhydrous Na SO , filtered and concentrated. The residue
2
4
was purified by column chromatography on silica gel (PE/EA= 5:1, V/V) to give
[24]
+
compound 10 as a white solid ( 2.73 g, 70%) . MS (ESI) m/z: 339.07 [M+Na] .
4
.1.7 Procedure G. 1-([1,1'-biphenyl]-2-yl)-4-(2-(benzofuran-2-yl)ethyl)piperazine
(11)
A mixture of 2-(benzofuran-2-yl)ethyl 4-methylbenzenesulfonate (10) (420 mg, 1.33
mmol), phenylpiperazine derivatives (5c) (400 mg, 1.46 mmol) and K CO (367 mg,
2
3
o
2
.66 mmol) in CH CN (30 mL) was Stirred at 80 C for 12 h. The solvent was
3
removed under reduced pressure, then CH Cl (50 mL) was added. The mixture was
2
2
washed with water and brine, dried over anhydrous Na SO , filtered and concentrated.
2
4
The residue was purified by column chromatography on silica gel (DCM/MeOH =
00/1, V/V) to give compound 11. The compound 11 was dissolved in EtOAc (15
mL), then hydrogen chloride ethyl acetate solution (2N, 1 mL) was added dropwise.
1

ACCEPTED MANUSCRIPT
The mixture was stirred at r.t. for 12h, then filtered. The residue was washed with EA
and EtOH separately，dried in vacuo to give compound 11 hydrochloride as white
o
+ 1
solid (378 mg, 68 %). Mp: 204-205 C. MS (ESI) m/z: 383.24 [M+H] . H NMR (600
MHz, DMSO) δ 7.65 (d, J = 7.2 Hz, 2H), 7.59 (d, J = 7.4 Hz, 1H), 7.52 (d, J = 8.0 Hz,
1
2
3
H), 7.45 (t, J = 7.7 Hz, 2H), 7.36 – 7.32 (m, 2H), 7.28 – 7.22 (m, 3H), 7.16 – 7.12 (m,
H), 6.75 (s, 1H), 3.51 – 3.46 (m, 4H), 3.33 – 3.31 (m, 2H), 3.13 (d, J = 12.3 Hz, 2H),
1
3
.04 (t, J = 12.0 Hz, 2H), 2.96 (dd, J = 19.4, 9.2 Hz, 2H). C NMR (100 MHz,
DMSO) δ 154.15, 154.06, 148.22, 140.10, 133.98, 131.26, 128.57, 128.53, 128.26,
1
4
28.13, 127.05, 123.87, 123.49, 122.85, 120.72, 118.54, 110.75, 103.65, 52.73, 50.84,
+
7.26, 22.65. HRMS calcd for C H N O [M+H] , 383.2123; found, 383.2127.
2
6
26
2
4
.1.8 Procedure H. 3-(2-hydroxyethyl)-1H-indole-5-carbonitrile (13)
To a solution of 2-(5-bromo-1H-indol-3-yl)ethanol (12) (6 g, 24.99 mmol) in
anhydrous DMF (200 mL) was added CuCN (22.38 g, 249.9 mmol). The reaction
o
mixture was charged with N , then stirred at 130 C for 48h. After cooling to room
2
temperature, the mixture was concentrated under reduced pressure, then EtOAc (100
mL) was added. The resulting mixture was filtered, the filtrate was washed with water
and brine, dried over Na SO , filtered and concentrated. The residue was purified by
2
4
column chromatography on silica gel (PE/EA= 5:1, V/V) to give compound 13 as
o
- 1
white solid (1.86 g, 40%). Mp: 125-126 C. MS (ESI) m/z: 185.06 [M-H] . H NMR
(
400 MHz, CDCl ) δ 7.99 (s, 1H), 7.43 – 7.42 (m, 2H), 7.23 (d, J = 2.2 Hz, 1H), 3.93
3
(dd, J = 11.4, 5.9 Hz, 2H), 3.03 (t, J = 6.3 Hz, 2H).
4
.1.9 Procedure I. 3-(2-bromoethyl)-1H-indole-5-carbonitrile (14)
To a solution of 3-(2-hydroxyethyl)-1H-indole-5-carbonitrile (13) (1.5 g, 8.05 mmol)
in CH Cl (60 mL) was added successively CBr (4 g, 12.08 mmol) and PPh (3.11 g,
2
2
4
3
11.84 mmol). The mixture was stirred at room temperature for 5h. After the
completion of reaction, the solvent was concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (PE/EA= 5:1, V/V) to
o
give compound 14 as an white solid (1.2 g, 60%). Mp: 129-130 C. MS (ESI) m/z:

ACCEPTED MANUSCRIPT
+
1
2
70.99 [M+Na] . H NMR (400 MHz, CDCl ) δ 7.95 (s, 1H), 7.44 (s, 2H), 7.25 (d, J
3
=
2.2 Hz, 1H), 3.64 (t, J = 7.2 Hz, 2H), 3.34 (t, J = 7.2 Hz, 2H).
4
.1.10 Procedure J. 3-(2-(4-([1,1'-biphenyl]-2-yl)piperazin-1-yl)ethyl)-1H-indole-5-
carbonitrile hydrochloride (15)
A mixture of 3-(2-bromoethyl)-1H-indole-5-carbonitrile (14) (500 mg, 2.0 mmol),
phenylpiperazine derivatives (5c) (526 mg, 2.2 mmol) , K CO (555 mg, 4.0 mmol)
2
3
o
and KI (34 mg, 0.2 mmol) in DMF (40 mL) was Stirred at 100 C for 12 h. The
solvent was removed under reduced pressure, then DCM (50 mL) was added. The
mixture was washed with water and brine, dried over anhydrous Na SO , filtered and
2
4
concentrated. The residue was purified by column chromatography on silica gel
DCM/MeOH = 100/1, V/V) to give compound 15. The compound 15 was dissolved
(
in EA (15 mL), then hydrogen chloride ethyl acetate solution (2N, 1 mL) was added
dropwise. The mixture was stirred at r.t. for 12h, then filtered. The residue was
washed with EtOAc and ethanol separately，dried in vacuo to give compound 15
o
hydrochloride as white solid (533 mg, 60 %). Mp: 238 - 239 C. MS (ESI) m/z: 407.23
+
1
[
M+H] . H NMR (600 MHz, DMSO) δ 11.60 (s, 1H), 8.23 (s, 1H), 7.66 (d, J = 7.4
Hz, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.48 – 7.44 (m, 4H), 7.36 (dd, J = 13.0, 6.8 Hz, 2H),
7
3
.26 (d, J = 6.5 Hz, 1H), 7.16 (t, J = 8.5 Hz, 2H), 3.51 (d, J = 11.0 Hz, 2H), 3.33 –
.31 (m, 2H), 3.22 – 3.12 (m, 4H), 3.05 (t, J = 11.9 Hz, 2H), 2.95 (dd, J = 20.4, 10.2
1
3
Hz, 2H). C NMR (100 MHz, DMSO) δ 148.27, 140.14, 137.95, 134.03, 131.28,
28.56, 128.25, 127.09, 126.53, 126.05, 124.41, 123.89, 123.49, 120.82, 118.57,
12.80, 110.56, 100.55, 55.25, 50.70, 47.37, 19.19. HRMS calcd for C H N
4
1
1
2
7
26
+
[
M+H] , 407.2236; found, 407.2242.
4
.1.11 Procedure K. General procedure for the preparation of compounds 18a-b
(4-fluorophenyl)hydrazine hydrochloride (17) (16.26 g, 100 mmol) was dissolved in a
mixture of N,N-dimethylacetamide (140 mL) and 4% m/m aqueous H SO (140 mL)
2
4
o
and heated to 100 C. 2,3-dihydrofuran (16a) or 3,4-dihydro-2H-pyran (16b) (100
mmol) was then added dropwise over 5 min, and the solution was stirred for 3h at 100

ACCEPTED MANUSCRIPT
o
C. After cooling to room temperature, the mixture was extracted with EtOAc (3×100
mL), the combined organic layer was washed with H O (3×100 mL), dried over
2
anhydrous Na SO , filtered and concentrated. The residue was purified by column
2
4
chromatography on silica gel (PE/EtOAc = 5:1, V/V) to give compound 18.
4
.1.11.1 2-(5-fluoro-1H-indol-3-yl)ethanol (18a)
Starting with 2,3-dihydrofuran (16a), Procedure K was followed to afford compound
[25]
-
1
8a as an orange oil (11.64 g, 65 %) . MS (ESI) m/z: 178.06 [M-H] .
4
.1.11.2 3-(5-fluoro-1H-indol-3-yl)propan-1-ol (18b)
Starting with 3,4-dihydro-2H-pyran (16b), Procedure K was followed to afford
[26]
-
compound 18b as an orange oil (11.59 g, 60 %) . MS (ESI) m/z: 192.08 [M-H] .
4
.1.12 Procedure L. 3-(2-bromoethyl)-5-fluoro-1H-indole (19a)
To a solution of 2-(5-fluoro-1H-indol-3-yl)ethanol (18a) (10 g, 55.8 mmol) in CH Cl
2
2
(
200 mL) was added successively CBr (27.76 g, 83.71 mmol) and PPh (21.52 g,
4
3
o
8
2.03 mmol) at 0 C. The mixture was stirred at room temperature for 5h. After the
completion of reaction, the solvent was concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (PE/EA= 5:1, V/V) to
[27]
+
give compound 19a as a yellow oil (9.46 g, 70%) . MS (ESI) m/z: 242.06 [M+H] .
4
.1.13 Procedure M. 3-(5-fluoro-1H-indol-3-yl)propyl 4-methylbenzenesulfonate
(19b)
To a stirred solution of p-toluenesulfonyl chloride (11.84 g, 62.11 mmol),
triethylamine (7.86 g, 77.63 mmol) and 4-dimethylaminopyridine (630 mg, 5.17
mmol) in dry CH Cl (150 mL) was added slowly a solution of 3-(5-fluoro-1H-
2
2
o
indol-3-yl)propan-1-ol (18b) (10 g, 51.75 mmol) in dry CH Cl (50 mL) at 0 C. The
2
2
o
reaction mixture was stirred for 1 h at 0 C and left for overnight at room temperature.
The reaction mixture was washed with water and brine, dried over anhydrous Na SO ,
2
4
filtered and concentrated. The residue was purified by column chromatography on

ACCEPTED MANUSCRIPT
silica gel (PE/EA= 5:1, V/V) to give compound 19b as a pale-yellow solid (11.69 g,
[26]
+
6
5%) . MS (ESI) m/z: 370.09 [M+Na] .
4
.1.14 Procedure N. General procedure for the preparation of compounds 21a-h
A
mixture of 3-(2-bromoethyl)-5-fluoro-1H-indole (19a) (4.13 mmol),
phenylpiperazine derivatives (20) (4.54 mmol) , K CO (8.26 mmol) and KI (0.41
2
3
o
mmol) in DMF (40 mL) was stirred at 100 C for 12 h. The solvent was removed
under reduced pressure, then DCM (50 mL) was added. The mixture was washed with
water and brine, dried over anhydrous Na SO , filtered and concentrated. The residue
2
4
was purified by column chromatography on silica gel (DCM/MeOH = 100/1, V/V) to
give compound 21. The compound 21 was dissolved in EA (15 mL), then hydrogen
chloride ethyl acetate solution (2N, 2 mL) was added dropwise. The mixture was
stirred at r.t. for 12h, then filtered. The residue was washed with EtOAc and EtOH
separately，dried in vacuo to give compound 21 hydrochloride.
4
.1.14.1
3-(2-(4-([1,1'-biphenyl]-2-yl)piperazin-1-yl)ethyl)-5-fluoro-1H-indole
hydrochloride (21a)
o
+ 1
Yield 61 % as a white solid. Mp: 259 - 260 C. MS (ESI) m/z: 400.25 [M+H] . H
NMR (400 MHz, DMSO) δ 11.09 (s, 1H), 7.66 (d, J = 7.3 Hz, 2H), 7.50 – 7.40 (m,
3
H), 7.39 – 7.23 (m, 5H), 7.16 (t, J = 8.0 Hz, 2H), 6.94 (td, J = 9.2, 2.4 Hz, 1H), 3.50
1
3
(
d, J = 10.6 Hz, 2H), 3.32 – 3.28 (m, 2H), 3.18 – 2.90 (m, 8H). C NMR (150 MHz,
DMSO) δ 158.22 (d, J = 229.7 Hz), 148.79 (s), 140.64 (s), 134.53 (s), 133.38 (s),
1
1
1
31.78 (s), 129.07 (s), 129.03 (s), 128.76 (s), 127.58 (s), 127.43 (d, J = 9.9 Hz),
25.81 (s), 123.99 (s), 119.07 (s), 112.97 (d, J = 9.5 Hz), 109.98 (d, J = 3.9 Hz),
09.82 (d, J = 26.0 Hz), 103.71 (d, J = 23.0 Hz), 55.81 (s), 51.16 (s), 47.86 (s), 19.92
+
(
s). HRMS calcd for C H N F [M+H] , 400.2189; found, 400.2195.
2
6
26
3
4
.1.14.2
5-fluoro-3-(2-(4-(4'-fluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)ethyl)-1H-
indole hydrochloride (21b)
Yield 56 % as a white solid. Mp: 258-259 C. MS (ESI) m/z: 418.23[M+H] . H NMR
o
+ 1

ACCEPTED MANUSCRIPT
(400 MHz, DMSO) δ 11.10 (s, 1H), 7.71 (dd, J = 8.6, 5.7 Hz, 2H), 7.44 (dd, J = 10.0,
2
.3 Hz, 1H), 7.39 – 7.33 (m, 2H), 7.31 – 7.23 (m, 4H), 7.16 (t, J = 7.4 Hz, 2H), 6.94
(
(
td, J = 9.2, 2.5 Hz, 1H), 3.51 (d, J = 11.1 Hz, 2H), 3.33 – 3.26 (m, 2H), 3.17 – 3.02
13
m, 6H), 2.96 (dd, J = 20.2, 10.5 Hz, 2H). C NMR (150 MHz, DMSO) δ 161.70 (d,
J = 242.6 Hz), 157.22 (d, J = 230.0 Hz), 148.84 (s), 136.81 (s), 133.71 (s), 133.38 (s),
1
1
4
31.66 (s), 130.87 (d, J = 7.7 Hz), 129.16 (s), 127.43 (d, J = 9.8 Hz), 125.80 (s),
24.16 (s), 119.31 (s), 115.87 (d, J = 21.0 Hz), 112.97 (d, J = 9.7 Hz), 109.98 (d, J =
.2 Hz), 109.82 (d, J = 26.0 Hz), 103.70 (d, J = 23.0 Hz), 55.87 (s), 51.19 (s), 47.94
+
(
s), 19.91 (s). HRMS calcd for C H N F [M+H] , 418.2095; found, 418.2094.
2
6
25
3 2
4
.1.14.3
5-fluoro-3-(2-(4-(4'-methyl-[1,1'-biphenyl]-2-yl)piperazin-1-yl)ethyl)-1H-
indole hydrochloride (21c)
Yield 52 % as a white solid. Mp: 246-247 C. MS (ESI) m/z: 414.25[M+H] . H NMR
400 MHz, DMSO) δ 11.10 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.44 (dd, J = 10.0, 2.2
Hz, 1H), 7.39 – 7.29 (m, 3H), 7.27 – 7.22 (m, 3H), 7.14 (t, J = 7.6 Hz, 2H), 6.94 (td, J
9.2, 2.4 Hz, 1H), 3.51 (d, J = 10.5 Hz, 2H), 3.33 – 3.27 (m, 2H), 3.16– 3.09 (m, 4H),
o
+ 1
(
=
1
3
3
2
.05 – 2.92 (m, 4H), 2.35 (s, 3H). C NMR (150 MHz, DMSO) δ 157.22 (d, J =
30.0 Hz), 148.78 (s), 137.72 (s), 136.63 (s), 134.44 (s), 133.39 (s), 131.75 (s), 129.67
(s), 128.77 (s), 128.59 (s), 127.44 (d, J = 9.8 Hz), 125.80 (s), 123.97 (s), 119.02 (s),
112.97 (d, J = 9.6 Hz), 110.01 (d, J = 4.2 Hz), 109.81 (d, J = 26.0 Hz), 103.71 (d, J =
2
3.0 Hz), 55.81 (s), 51.20 (s), 47.83 (s), 21.32 (s), 19.93 (s). HRMS calcd for
+
C H N F [M+H] , 414.2346; found, 414.2343.
27
28
3
4
.1.14.4 5-fluoro-3-(2-(4-(4'-methoxy-[1,1'-biphenyl]-2-yl)piperazin-1-yl)ethyl)-1H-
indole hydrochloride (21d)
o
+ 1
Yield 53 % as a white solid. Mp: 224 - 225 C. MS (ESI) m/z: 430.23[M+H] . H
NMR (400 MHz, DMSO) δ 11.09 (s, 1H), 7.61 (d, J = 8.7 Hz, 2H), 7.43 (dd, J = 10.0,
2
.3 Hz, 1H), 7.36 (dd, J = 8.8, 4.6 Hz, 1H), 7.34 – 7.28 (m, 2H), 7.23 (dd, J = 7.4, 1.4
Hz, 1H), 7.12 (dd, J = 12.4, 4.9 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 6.94 (td, J = 9.2,
.4 Hz, 1H), 3.80 (s, 3H), 3.53 (d, J = 5.3 Hz, 2H), 3.33 – 3.27 (m, 2H), 3.18 – 3.07
2

ACCEPTED MANUSCRIPT
1
3
(
m, 4H), 3.03 – 2.94 (m, 4H). C NMR (150 MHz, DMSO) δ 158.73 (s), 157.22 (d, J
230.0 Hz), 148.72 (s), 134.24 (s), 133.38 (s), 132.76 (s), 131.60 (s), 129.89 (s),
28.54 (s), 127.44 (d, J = 9.9 Hz), 125.81 (s), 123.99 (s), 119.03 (s), 114.46 (s),
12.97 (d, J = 9.6 Hz), 109.98 (d, J = 3.0 Hz), 109.82 (d, J = 26.0 Hz), 103.70 (d, J =
=
1
1
2
3.0 Hz), 55.88 (s), 55.50 (s), 51.26 (s), 47.80 (s), 19.92 (s). HRMS calcd for
+
C H N FO [M+H] , 430.2295; found, 430.2301.
27
28
3
4
.1.14.5 2'-(4-(2-(5-fluoro-1H-indol-3-yl)ethyl)piperazin-1-yl)-[1,1'-biphenyl]-4-
carbonitrile hydrochloride (21e)
o
+ 1
Yield 49 % as a white solid. Mp: 274-275 C. MS (ESI) m/z: 425.23 [M+H] . H
NMR (600 MHz, DMSO) δ 11.10 (s, 1H), 7.89 (q, J = 8.3 Hz, 4H), 7.43 (dd, J = 14.4,
8
2
3
2
.1 Hz, 2H), 7.36 (dd, J = 8.7, 4.5 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.23 – 7.19 (m,
H), 6.94 (t, J = 9.0 Hz, 1H), 3.51 (d, J = 11.3 Hz, 2H), 3.32 – 3.28 (m, 2H), 3.15 –
13
.03 (m, 6H), 2.99 – 2.92 (m, 2H). C NMR (150 MHz, DMSO) δ 157.22 (d, J =
30.0 Hz), 148.97 (s), 145.50 (s), 133.38 (s), 133.02 (s), 132.99 (s), 131.68 (s), 130.18
(s), 129.90 (s), 127.43 (d, J = 9.8 Hz), 125.79 (s), 124.37 (s), 119.54 (d, J = 20.3 Hz),
112.97 (d, J = 9.6 Hz), 110.11(s), 109.98 (d, J = 3.6 Hz), 109.83 (d, J = 26.0 Hz),
1
03.70 (d, J = 23.1 Hz), 55.87 (s), 51.07 (s), 48.20 (s), 19.91 (s). HRMS calcd for
+
C H N F [M+H] , 425.2142; found, 425.2143.
27
25
4
4
.1.14.6
5-fluoro-3-(2-(4-(3'-fluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)ethyl)-1H-
indole hydrochloride (21f)
o
+
1
Yield 54 % as a white solid. Mp: 264-265 C. MS (ESI) m/z: 418.25 [M+H] . H
NMR (400 MHz, DMSO) δ 11.09 (s, 1H), 7.56 – 7.48 (m, 2H), 7.47 – 7.42 (m, 2H),
7
.40 – 7.34 (m, 2H), 7.31 – 7.27 (m, 2H), 7.22 – 7.15 (m, 3H), 6.94 (td, J = 9.2, 2.5
Hz, 1H), 3.52 (d, J = 11.2 Hz, 2H), 3.32 – 3.28 (m, 2H), 3.16 – 3.02 (m, 6H), 2.99 –
1
3
2
.90 (m, 2H). C NMR (150 MHz, DMSO) δ 162.74 (d, J = 241.8 Hz), 157.22 (d, J =
2
30.0 Hz), 148.85 (s), 143.03 (d, J = 7.8 Hz), 133.38 (s), 131.82 (s), 130.94 (d, J = 8.4
Hz), 129.56 (s), 127.44 (d, J = 9.8 Hz), 125.82 (s), 125.02 (s), 124.11 (s), 119.29 (s),
15.50 (d, J = 21.6 Hz), 114.36 (d, J = 20.6 Hz), 112.97 (d, J = 9.6 Hz), 109.97 (d, J =
1

ACCEPTED MANUSCRIPT
3
.8 Hz), 109.81 (d, J = 26.1 Hz), 103.70 (d, J = 23.0 Hz), 55.82 (s), 51.18 (s), 48.02
+
(
s), 19.92 (s). HRMS calcd for C H N F [M+H] , 418.2095; found, 418.2096.
2
6
25
3 2
4
.1.14.7
5-fluoro-3-(2-(4-(3'-methyl-[1,1'-biphenyl]-2-yl)piperazin-1-yl)ethyl)-1H-
indole hydrochloride (21g)
o
+
1
Yield 48 % as a white solid. Mp: 262-263 C. MS (ESI) m/z: 414.22 [M+H] . H
NMR (400 MHz, DMSO) δ 11.08 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.44 – 7.40 (m,
2
H), 7.38 – 7.29 (m, 4H), 7.24 (dd, J = 7.5, 1.4 Hz, 1H), 7.17 – 7.12 (m, 3H), 6.94 (td,
J = 9.2, 2.5 Hz, 1H), 3.52 (d, J = 11.0 Hz, 2H), 3.33 – 3.29 (m, 2H), 3.18 – 3.06 (m,
1
3
4
H), 3.03 – 2.88 (m, 4H), 2.38 (s, 3H). C NMR (100 MHz, DMSO) δ 156.71 (d, J =
2
29.9 Hz), 148.24 (s), 140.10 (s), 137.67 (s), 134.02 (s), 132.86 (s), 131.26 (s), 128.81
(s), 128.39 (d, J = 5.2 Hz), 127.71 (s), 126.93 (d, J = 9.9 Hz), 125.31 (s), 123.38 (s),
118.47 (s), 112.46 (d, J = 9.7 Hz), 109.46 (d, J = 4.7 Hz), 109.30 (d, J = 26.0 Hz),
1
03.20 (d, J = 23.0 Hz), 55.33 (s), 50.74 (s), 47.28 (s), 21.10 (s), 19.38 (s). HRMS
+
calcd for C H N F [M+H] , 414.2346; found, 414.2351.
2
7
28
3
4
.1.14.8
5-fluoro-3-(2-(4-(2'-fluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)ethyl)-1H-
indole hydrochloride (21h)
o
+
1
Yield 57 % as a white solid. Mp: 184-185 C. MS (ESI) m/z: 418.24 [M+H] . H
NMR (400 MHz, DMSO) δ 11.09 (s, 1H), 7.57 – 7.50 (m, 1H), 7.48 – 7.39 (m, 3H),
7
.38 – 7.15 (m, 7H), 6.93 (td, J = 9.2, 2.4 Hz, 1H), 3.49 (d, J = 11.6 Hz, 2H), 3.30 –
13
3
.24 (m, 2H), 3.15 – 2.99 (m, 6H), 2.77 (dd, J = 18.5, 8.4 Hz, 2H). C NMR (150
MHz, DMSO) δ 159.30 (d, J = 244.0 Hz), 157.21 (d, J = 229.8 Hz), 149.76 (s),
1
33.35 (s), 132.03 (s), 131.85 (s), 130.10 (s), 129.93 (d, J = 7.7 Hz), 129.77 (s),
27.77 (d, J = 15.0 Hz), 127.41 (d, J = 9.6 Hz), 125.82 (s), 124.99 (s), 124.05 (s),
1
119.88 (s), 116.35 (d, J = 22.2 Hz), 112.95 (d, J = 9.8 Hz), 109.92 (d, J = 6.6 Hz),
1
09.81 (d, J = 26.6 Hz), 103.70 (d, J = 23.0 Hz), 55.77 (s), 51.41 (s), 48.23 (s), 19.91
+
(
s). HRMS calcd for C H N F [M+H] , 418.2095; found, 418.2091.
2
6
25
3 2
4
.1.15 Procedure O. General procedure for the preparation of compounds 21i-n

ACCEPTED MANUSCRIPT
A mixture of 3-(5-fluoro-1H-indol-3-yl)propyl 4-methylbenzenesulfonate (19b) (2.87
mmol), phenylpiperazine derivatives (20) (3.16 mmol) and K CO (5.75 mmol) in
2
3
o
CH CN (40 mL) was stirred at 80 C for 12 h. The solvent was removed under
3
reduced pressure, then CH Cl (50 mL) was added. The mixture was washed with
2
2
water and brine, dried over anhydrous Na SO , filtered and concentrated. The residue
2
4
was purified by column chromatography on silica gel (DCM/MeOH = 100/1, V/V) to
give compound 21. The compound 21 was dissolved in EA (15 mL), then hydrogen
chloride ethyl acetate solution (2N, 2 mL) was added dropwise. The mixture was
stirred at r.t. for 12h, then filtered. The residue was washed with EA and EtOH
separately，dried in vacuo to give compound 21 hydrochloride.
4
.1.15.1
3-(3-(4-([1,1'-biphenyl]-2-yl)piperazin-1-yl)propyl)-5-fluoro-1H-indole
hydrochloride (21i)
o
+ 1
Yield 69 % as a white solid. Mp: 174-175 C. MS (ESI) m/z: 414.25 [M+H] . H
NMR (600 MHz, DMSO) δ 10.99 (s, 1H), 7.64 (d, J = 7.3 Hz, 2H), 7.44 (t, J = 7.6 Hz,
2
8
3
2
H), 7.36 – 7.28 (m, 4H), 7.27 – 7.22 (m, 2H), 7.14 (t, J = 7.4 Hz, 1H), 7.10 (d, J =
.0 Hz, 1H), 6.91 (td, J = 9.2, 2.4 Hz, 1H), 3.48 – 3.34 (m, 4H), 3.10 – 3.05 (m, 4H),
.00 (t, J = 11.7 Hz, 2H), 2.84 (dd, J = 20.7, 9.4 Hz, 2H), 2.69 (t, J = 7.3 Hz, 2H),
1
3
.06 – 2.00 (m, 2H). C NMR (150 MHz, DMSO) δ 157.12 (d, J = 229.4 Hz), 148.75
(s), 140.62 (s), 134.49 (s), 133.45 (s), 131.75 (s), 129.04 (s), 129.01 (s), 128.75 (s),
1
27.64 (d, J = 9.9 Hz), 127.56 (s), 125.17 (s), 123.95 (s), 119.01 (s), 113.60 (d, J = 4.4
Hz), 112.78 (d, J = 9.8 Hz), 109.51 (d, J = 26.0 Hz), 103.51 (d, J = 22.8 Hz), 55.88 (s),
+
5
1.31 (s), 47.76 (s), 24.11 (s), 22.29 (s). HRMS calcd for C H N F [M+H] ,
2
7
28
3
4
14.2346; found, 414.2350.
4
.1.15.2 5-fluoro-3-(3-(4-(3'-fluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)propyl)-1H-
indole hydrochloride (21j)
o
+
1
Yield 67 % as a white solid. Mp: 232-233 C. MS (ESI) m/z: 432.25 [M+H] . H
NMR (600 MHz, DMSO) δ 10.99 (s, 1H), 7.52 – 7.45 (m, 2H), 7.42 (d, J = 10.3 Hz,
1
H), 7.39 – 7.24 (m, 5H), 7.20 – 7.12 (m, 3H), 6.91 (td, J = 9.2, 2.3 Hz, 1H), 3.41 (d,

ACCEPTED MANUSCRIPT
J = 11.5 Hz, 2H), 3.13 – 3.06 (m, 4H), 3.02 (t, J = 11.9 Hz, 2H), 2.85 (dd, J = 20.4,
1
3
9
.5 Hz, 2H), 2.69 (t, J = 7.4 Hz, 2H), 2.03 (dt, J = 15.4, 7.6 Hz, 2H). C NMR (150
MHz, DMSO) δ 162.72 (d, J = 241.8 Hz), 157.12 (d, J = 229.5 Hz), 148.81 (s),
1
43.00 (d, J = 7.7 Hz), 133.46 (s), 133.29 (s), 131.79 (s), 130.92 (d, J = 8.4 Hz),
29.54 (s), 127.64 (d, J = 9.6 Hz), 125.16 (s), 125.00 (s), 124.06 (s), 119.22 (s),
1
115.47 (d, J = 21.6 Hz), 114.34 (d, J = 20.7 Hz), 113.62 (d, J = 4.4 Hz), 112.78 (d, J =
9
.6 Hz), 109.50 (d, J = 26.0 Hz), 103.51 (d, J = 22.8 Hz), 55.87 (s), 51.32 (s), 47.89
+
(
s), 24.12 (s), 22.28 (s). HRMS calcd for C H N F [M+H] , 432.2251; found,
2
7
27
3
2
4
32.2248.
4
1
.1.15.3 3-(3-(4-(3',5-difluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)propyl)-5-fluoro-
H-indole hydrochloride (21k)
o
Yield 67 % as a white solid, the purity was 98.1% by HPLC analysis. Mp: 243-244 C.
+
1
MS (ESI) m/z: 450.40 [M+H] . H NMR (600 MHz, DMSO) δ 10.98 (s, 1H), 7.54 (d,
J = 7.7 Hz, 1H), 7.51 – 7.43 (m, 2H), 7.33 (dd, J = 8.7, 4.6 Hz, 1H), 7.30 (dd, J = 10.0,
2
.2 Hz, 1H), 7.26 (s, 1H), 7.23 – 7.13 (m, 4H), 6.91 (td, J = 9.2, 2.4 Hz, 1H), 3.40 (d,
J = 11.2 Hz, 2H), 3.13 – 3.07 (m, 2H), 3.04 – 2.97 (m, 4H), 2.83 (q, J = 14.0 Hz, 2H),
1
3
2
.69 (t, J = 7.3 Hz, 2H), 2.03 (dt, J = 14.9, 7.4 Hz, 2H). C NMR (150 MHz, DMSO)
δ 162.66 (d, J = 242.0 Hz), 158.99 (d, J = 239.0 Hz), 157.11 (d, J = 229.5 Hz), 145.33
s), 141.51 (d, J = 7.8 Hz), 135.44 (d, J = 6.6 Hz), 133.45 (s), 130.95 (d, J = 8.3 Hz),
27.64 (d, J = 9.6 Hz), 125.15 (s), 121.23 (d, J = 8.1 Hz), 118.21 (d, J = 22.8 Hz),
15.71 (d, J = 21.5 Hz), 115.57 (d, J = 20.7 Hz), 114.87 (d, J = 20.7 Hz), 113.61 (d, J
4.4 Hz), 112.78 (d, J = 9.6 Hz), 109.50 (d, J = 26.0 Hz), 103.50 (d, J = 22.7 Hz),
5.84 (s), 51.34 (s), 48.19 (s), 24.13 (s), 22.27 (s). HRMS calcd for C H N F
(
1
1
=
5
27
26
3 3
+
[
M+H] , 450.2157; found, 450.2158.
4
1
.1.15.4 3-(3-(4-(3',6-difluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)propyl)-5-fluoro-
H-indole hydrochloride (21l)
o
+
Yield 61 % as a white solid. Mp: 197-199 C. MS (ESI) m/z: 450.24 [M+H] .
1
H NMR (400 MHz, DMSO) δ 10.97 (s, 1H), 7.56 – 7.47 (m, 1H), 7.43 – 7.19 (m,

ACCEPTED MANUSCRIPT
7
H), 7.01 (dd, J = 18.4, 8.8 Hz, 2H), 6.91 (td, J = 9.2, 2.5 Hz, 1H), 3.38 – 3.34 (m,
13
2
H), 3.11 – 2.97 (m, 6H), 2.76 – 2.65 (m, 4H), 2.06 – 1.95 (m, 2H). C NMR (150
MHz, DMSO) δ 162.51 (d, J = 242.0 Hz), 160.05 (d, J = 242.0 Hz), 157.11 (d, J =
2
29.5 Hz), 150.80 (d, J = 4.7 Hz), 135.47 (d, J = 8.0 Hz), 133.44 (s), 130.83 (d, J =
.3 Hz), 130.54 (d, J = 10.4 Hz), 127.63 (d, J = 9.6 Hz), 126.64 (s), 125.15 (s), 121.34
8
(
d, J = 15.2 Hz), 117.22 (d, J = 21.8 Hz), 115.43 (s), 115.01 (d, J = 20.8 Hz), 113.61
d, J = 4.4 Hz), 112.77 (d, J = 9.6 Hz), 110.89 (d, J = 23.1 Hz), 109.49 (d, J = 26.0
(
Hz), 103.50 (d, J = 22.7 Hz), 55.83 (s), 51.23 (s), 47.80 (s), 24.10 (s), 22.26 (s).
+
HRMS calcd for C H N F [M+H] , 450.2157; found, 450.2158.
2
7
26
3 3
4
1
.1.15.5 3-(3-(4-(3',4-difluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)propyl)-5-fluoro-
H-indole hydrochloride (21m)
o
+
1
Yield 64 % as a white solid. Mp: 233-234 C. MS (ESI) m/z: 450.20 [M+H] . H
NMR (400 MHz, DMSO) δ 10.97 (s, 1H), 7.52 – 7.44 (m, 2H), 7.42 – 7.23 (m, 5H),
7
.21– 7.15 (m, 1H), 7.01 – 6.87 (m, 3H), 3.40 (d, J = 12.4 Hz, 2H), 3.15 – 3.06 (m,
13
4
H), 3.04 – 2.81 (m, 4H), 2.69 (t, J = 7.3 Hz, 2H), 2.08 – 1.94 (m, 2H). C NMR
(
150 MHz, DMSO) δ 162.84 (d, J = 243.9 Hz), 162.78 (d, J = 242.0 Hz), 157.11 (d, J
229.5 Hz), 150.64 (d, J = 8.6 Hz), 142.22 (d, J = 7.8 Hz), 133.45 (s), 133.37 (s),
31.08 (d, J = 8.1 Hz), 129.20 (s), 127.63 (d, J = 9.8 Hz), 125.16 (s), 124.90 (s),
15.41 (d, J = 21.8 Hz), 114.45 (d, J = 20.7 Hz), 113.60 (d, J = 4.2 Hz), 112.77 (d, J =
=
1
1
9
.5 Hz), 110.03 (d, J = 20.7 Hz), 109.50 (d, J = 26.1 Hz), 106.63 (d, J = 23.4 Hz),
1
03.50 (d, J = 22.7 Hz), 55.84 (s), 51.04 (s), 47.60 (s), 24.12 (s), 22.27 (s). HRMS
+
calcd for C H N F [M+H] , 450.2157; found, 450.2157.
2
7
26
3 3
4
.1.15.6
indole hydrochloride (21n)
Yield 68 % as a white solid, the purity was 98.3% by HPLC analysis. Mp: 215-216 C.
5-fluoro-3-(3-(4-(5-fluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)propyl)-1H-
o
+
1
MS (ESI) m/z: 432.24 [M+H] . H NMR (400 MHz, DMSO) δ 10.98 (s, 1H), 7.68 –
.64 (m, 2H), 7.45 (t, J = 7.5 Hz, 2H), 7.40 – 7.25 (m, 4H), 7.20 – 7.08 (m, 3H), 6.91
td, J = 9.2, 2.5 Hz, 1H), 3.37 (d, J = 12.1 Hz, 2H), 3.12 – 3.05 (m, 2H), 3.02 – 2.97
7
(

ACCEPTED MANUSCRIPT
1
3
(
m, 4H), 2.89 – 2.77 (m, 2H), 2.69 (t, J = 7.4 Hz, 2H), 2.08 – 1.98 (m, 2H). C NMR
(100 MHz, DMSO) δ 158.48 (d, J = 238.7 Hz), 156.60 (d, J = 229.4 Hz), 144.75 (s),
1
38.70 (s), 136.16 (d, J = 7.5 Hz), 132.94 (s), 128.57 (s), 128.34 (s), 127.57 (s),
27.12 (d, J = 9.7 Hz), 124.66 (s), 120.44 (d, J = 8.5 Hz), 117.57 (d, J = 22.7 Hz),
1
114.48 (d, J = 21.5 Hz), 113.08 (d, J = 4.6 Hz), 112.27 (d, J = 9.7 Hz), 109.00 (d, J =
2
5.9 Hz), 103.00 (d, J = 22.7 Hz), 55.34 (s), 50.79 (s), 47.54 (s), 23.60 (s), 21.77 (s).
+
HRMS calcd for C H N F [M+H] , 432.2251; found, 432.2249.
2
7
27
3 2
4
.1.16 Procedure P. 4-chloro-1-(5-fluoro-1-tosyl-1H-indol-3-yl)butan-1-one 23
To an ice-cold solution of 5-fluoro-1-tosyl-1H-indole 22 (3 g, 10.36 mmol) in
CH Cl (45 mL) was added AlCl (1.52 g, 11.40 mmol), followed by the addition of
2
2
3
4
-chlorobutanoyl chloride (1.6 g, 11.40 mmol) in CH Cl (15 mL). After the addition,
2 2
the mixture was stirred at r.t. for 3h. Then reaction mixture was poured slowly into
ice-water, then stirred for 15 min. The organic layer was separated, washed with brine,
dried over Na SO , filtered and concentrated. The residue was purified by column
2
4
chromatography on silica gel (PE/EtOAc = 5/1, V/V) to give compound 23. Yield
o
+ 1
(
2.65 g, 65 %) as a white solid. Mp: 127-129 C. MS (ESI) m/z: 394.07 [M+H] . H
NMR (400 MHz, CDCl ) δ 8.29 (s, 1H), 8.00 (dd, J = 9.1, 2.6 Hz, 1H), 7.88 (dd, J =
3
9.1, 4.3 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.11 (td, J = 9.0,
2.6 Hz, 1H), 3.68 (t, J = 6.2 Hz, 2H), 3.10 (t, J = 7.0 Hz, 2H), 2.39 (s, 3H), 2.31 –
2.18 (m, 2H).
4
.1.17 Procedure Q. 3-(4-chlorobutyl)-5-fluoro-1-tosyl-1H-indole 24
To a stirred solution of 4-chloro-1-(5-fluoro-1-tosyl-1H-indol-3-yl)butan-1-one 23
(
2.5g, 6.34 mmol) in trifluoroacetic acid (45 mL) was added dropwise HSiEt (2.2 g,
3
o
o
1
9.04 mmol) at 0 C. After the addition, the mixture was stirred at 0 C for 30 min,
o
then heated to 50 C for 3 h. The reaction mixture was concentrated under reduced
pressure, then water (40 mL) was added. The resulting mixture was stirred at r.t. for
3
0 min, much yellow solid precipitated. The mixture was filtered, the residue was
washed with water, dried in vacuo to give an pale-yellow solid. The solid was

ACCEPTED MANUSCRIPT
recrystallized from EtOAc to give the desired product. Yield (1.39g, 58 %) as a white
o
+ 1
solid. Mp: 104 - 105 C. MS (ESI) m/z: 402.05 [M+Na] . H NMR (400 MHz, CDCl )
3
δ 7.92 (dd, J = 9.0, 4.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.35 (s, 1H), 7.21 (d, J = 8.1
Hz, 2H), 7.10 (dd, J = 8.8, 2.5 Hz, 1H), 7.03 (td, J = 9.0, 2.5 Hz, 1H), 3.59 – 3.50 (m,
2
H), 2.64 (dd, J = 6.9, 6.2 Hz, 2H), 2.34 (s, 3H), 1.83 – 1.79 (m, 4H).
4
5
.1.18 Procedure R. 3-(4-(4-(3',5-difluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)butyl)-
-fluoro-1-tosyl-1H-indole 25
A mixture of 3-(4-chlorobutyl)-5-fluoro-1-tosyl-1H-indole 24 (1g, 2.63 mmol),
phenylpiperazine derivatives 20f (0.79 g, 2.89 mmol) , K CO (0.73 g, 5.26 mmol)
2
3
o
and KI (44 mg, 0.26 mmol) in DMF (30 mL) was stirred at 100 C for 12 h. The
solvent was removed under reduced pressure, then DCM (50 mL) was added. The
mixture was washed with water and brine, dried over anhydrous Na SO , filtered and
2
4
concentrated. The residue was purified by column chromatography on silica gel
(DCM/MeOH = 100/1, V/V) to give compound 25 as a yellow solid. Yield (1.21 g,
o
+ 1
7
5 %). Mp: 64 - 65 C. MS (ESI) m/z: 618.23 [M+H] . H NMR (400 MHz, CDCl ) δ
3
7
.90 (dd, J = 9.0, 4.4 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.43 – 7.31 (m, 4H), 7.19 (d, J
=
8.1 Hz, 2H), 7.10 (dd, J = 8.8, 2.5 Hz, 1H), 7.04 – 6.96 (m, 5H), 2.80 (t, J = 4.7 Hz,
H), 2.59 (t, J = 7.4 Hz, 2H), 2.37 – 2.29 (m, 7 H), 1.69 – 1.62 (m, 4H), 1.54 – 1.47
m, 2H).
4
(
4
5
.1.19 Procedure S. 3-(4-(4-(3',5-difluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)butyl)-
-fluoro-1H-indole hydrochloride 26
A
mixture of 3-(4-(4-(3',5-difluoro-[1,1'-biphenyl]-2-yl)piperazin-1-yl)butyl)-
5
-fluoro-1-tosyl-1H-indole 25 (1.2 g, 1.94 mmol), 4N NaOH solution (4.8 mL) in the
o
co-solvent of EtOH (30 mL) and THF (5 mL) was stirred at 80 C for 12h. After
cooling to room temperature, the solvent was evaporated, then the residue was diluted
with CH Cl (60 mL). The mixture was washed with water and brine, dried over
2
2
anhydrous Na SO , filtered and concentrated. The residue was purified by column
2
4
chromatography on silica gel (DCM/MeOH = 100/1, V/V) to give compound 26. The

ACCEPTED MANUSCRIPT
compound 26 was dissolved in EA (30 mL), then hydrogen chloride ethyl acetate
solution (2N, 2 mL) was added dropwise. The mixture was stirred at r.t. for 12h, then
filtered. The residue was washed with EtOAc and EtOH separately，dried in vacuo to
give compound 26 hydrochloride as white solid. Yield (0.56 g, 58 %). Mp: 249-250
o
+ 1
C. MS (ESI) m/z: 464.23 [M+H] . H NMR (400 MHz, DMSO) δ 7.57 – 7.43 (m,
3
H), 7.32 (dd, J = 8.8, 4.6 Hz, 1H), 7.28 – 7.14 (m, 6H), 6.89 (td, J = 9.2, 2.5 Hz, 1H),
.37 (d, J = 12.0 Hz, 2H), 3.15 – 2.94 (m, 6H), 2.89 – 2.74 (m, 2H), 2.67 (t, J = 7.1
3
1
3
Hz, 2H), 1.76 – 1.56 (m, 4H). C NMR (150 MHz, DMSO) δ 162.66 (d, J = 242.0
Hz), 158.99 (d, J = 239.0 Hz), 157.06 (d, J = 229.4 Hz), 145.34 (s), 141.51 (d, J = 7.8
Hz), 135.49 (d, J = 7.2 Hz), 133.43 (s), 130.94 (d, J = 8.3 Hz), 127.71 (d, J = 9.6 Hz),
1
25.13 (s), 125.02 (s), 121.27 (d, J = 8.1 Hz), 118.20 (d, J = 23.0 Hz), 115.72 (d, J =
2.2 Hz), 115.58 (d, J = 21.6 Hz), 114.86 (d, J = 20.7 Hz), 114.65 (d, J = 4.4 Hz),
2
112.72 (d, J = 9.6 Hz), 109.35 (d, J = 25.8 Hz), 103.40 (d, J = 22.7 Hz), 55.71 (s),
5
1.28 (s), 48.16 (s), 27.36 (s), 24.52 (s), 23.29 (s). HRMS calcd for C H N F
28
28
3 3
+
[
M+H] , 464.2314; found, 464.2318.
4
.1.20 Procedure T. General procedure for the preparation of compounds 20a-h
Ａmixture of biphenyl amine 27 (90 mmol), Bis(2-chloroethyl)amine hydrochloride
2
8 (90 mmol), K CO (90 mmol) and KI (90 mmol) in xylene (450 mL) was stirred at
2 3
o
1
40 C for 48 h. After cooling to room temperature, the solvent was evaporated, then
DCM (150 mL) was added. The mixture was washed with water and brine, dried over
anhydrous Na SO , filtered and concentrated. The residue was purified by column
2
4
chromatography on silica gel (DCM/MeOH = 20/1, V/V) to give compound 20.
4
.1.20.1 1-(2'-fluoro-[1,1'-biphenyl]-2-yl)piperazine (20a)
o
+
Yield (22 %) as a yellow solid. Mp: 63-64 C. MS (ESI) m/z: 257.23 [M+H] .
1
H NMR (400 MHz, CDCl ) δ 7.47 (td, J = 7.5, 1.8 Hz, 1H), 7.36 – 7.31 (m, 1H),
3
7
2
.31 – 7.23 (m, 2H), 7.19 – 7.13 (m, 1H), 7.13 – 7.06 (m, 3H), 2.80 – 2.77 (m, 4H),
.71 – 2.68 (m, 4H).

ACCEPTED MANUSCRIPT
4
.1.20.2 1-(3'-fluoro-[1,1'-biphenyl]-2-yl)piperazine (20b)
o
+ 1
Yield (38%) as a white solid. Mp: 223-224 C. MS (ESI) m/z: 257.18 [M+H] . H
NMR (400 MHz, CDCl ) δ 7.41 – 7.30 (m, 3H), 7.28 – 7.26 (m, 1H), 7.24 – 7.23 (m,
3
1
H), 7.15 (t, J = 7.1 Hz, 1H), 7.07 – 6.98 (m, 2H), 3.13 – 3.06 (m, 8H).
4
.1.20.3 1-(3'-methyl-[1,1'-biphenyl]-2-yl)piperazine (20c)
o
+
Yield (41 %) as a pale-yellow solid. Mp: 172-174 C. MS (ESI) m/z: 253.21 [M+H] .
1
H NMR (400 MHz, CDCl ) δ 7.36 (d, J = 7.7 Hz, 1H), 7.31 – 7.28 (m, 2H), 7.28 –
3
7
–
.27 (m, 1H), 7.25 – 7.23 (m, 1H), 7.16 – 7.09 (m, 2H), 7.01 (d, J = 7.4 Hz, 1H), 3.15
3.10 (m, 4H), 3.07 – 3.04 (m, 4H), 2.39 (s, 3H).
4
.1.20.4 1-(4'-methyl-[1,1'-biphenyl]-2-yl)piperazine (20d)
o
+ 1
Yield (39%) as a white solid. Mp: 253 - 254 C. MS (ESI) m/z: 253.19 [M+H] . H
NMR (400 MHz, CDCl ) δ 7.42 (d, J = 8.0 Hz, 2H), 7.29 (dd, J = 7.7, 1.5 Hz, 1H),
3
7
7
.24 (dd, J = 7.6, 1.6 Hz, 1H), 7.21 (d, J = 7.9 Hz, 2H), 7.13 (td, J = 7.5, 0.8 Hz, 1H),
.01 (d, J = 7.9 Hz, 1H), 3.13 – 3.10 (m, 4H), 3.07 – 3.05 (m, 4H), 2.38 (s, 3H).
4
.1.20.5 2'-(piperazin-1-yl)-[1,1'-biphenyl]-4-carbonitrile (20e)
o
+ 1
Yield (33%) as a white solid. Mp: 259-260 C. MS (ESI) m/z: 264.14 [M+H] . H
NMR (400 MHz, CDCl ) δ 7.74 – 7.66 (m, 4H), 7.38 (td, J = 8.1, 2.0 Hz, 1H), 7.26 –
3
7
.17 (m, 2H), 7.10 (d, J = 7.9 Hz, 1H), 3.16 – 3.04 (m, 8H).
4
.1.20.6 1-(3',5-difluoro-[1,1'-biphenyl]-2-yl)piperazine (20f)
o
+ 1
Yield (36%) as a white solid. Mp: 253 - 254 C. MS (ESI) m/z: 275.14 [M+H] . H
NMR (400 MHz, CDCl ) δ 7.43 – 7.36 (m, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.22 (d, J =
3
9
.8 Hz, 1H), 7.08 – 6.96 (m, 4H), 3.09 (s, 8H).
4
.1.20.7 1-(3',6-difluoro-[1,1'-biphenyl]-2-yl)piperazine (20g)
o
+
Yield (27%) as a pale-yellow solid. Mp: 181 - 182 C. MS (ESI) m/z: 275.22 [M+H] .
1
H NMR (400 MHz, CDCl ) δ 7.42 (dd, J = 14.0, 7.9 Hz, 1H), 7.33 – 7.27 (m, 1H),
3

ACCEPTED MANUSCRIPT
7
.22 (d, J = 7.6 Hz, 1H), 7.15 (d, J = 9.7 Hz, 1H), 7.06 (td, J = 8.4, 2.3 Hz, 1H), 6.93
(t, J = 8.7 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 3.18 – 3.15 (m, 4H), 3.07 – 3.05 (m, 4H).
4
.1.20.8 1-(3',4-difluoro-[1,1'-biphenyl]-2-yl)piperazine (20h)
o
+ 1
Yield (23%) as a yellow solid. Mp: 104-105 C. MS (ESI) m/z: 275.17 [M+H] . H
NMR (400 MHz, CDCl ) δ 7.42 – 7.35 (m, 1H), 7.29 (s, 1H), 7.22 – 7.18 (m, 2H),
3
7
1
.03 (td, J = 8.4, 1.9 Hz, 1H), 6.86 (td, J = 8.2, 2.4 Hz, 1H), 6.76 (dd, J = 10.5, 2.4 Hz,
H), 3.17 – 3.10 (m, 8H).
4
.1.20.91-(5-fluoro-[1,1'-biphenyl]-2-yl)piperazine (20i)
o
+ 1
Yield (38%) as a white solid. Mp: 254 - 255 C. MS (ESI) m/z: 257.14 [M+H] . H
NMR (400 MHz, CDCl ) δ 7.51-7.48 (m, 2H), 7.44 – 7.38 (m, 2H), 7.36 – 7.30 (m,
3
1
H), 6.99 (d, J = 7.5 Hz, 3H), 3.06 (s, 8H).
4
4
4
.2 Biology evaluation
.2.1 In vitro binding assays
.2.1.1 5-HT reuptake
The synaptosomes (150 µg) are incubated for 15 min at 37°C with 0.1 µCi
H]serotonin in the absence (control) or presence of the test compound or the
3
[
reference compound in a buffer containing 106.2 mM NaCl, 4.5 mM KCl, 2.25 mM
MgSO , 1.08 mM NaH PO , 22.5 mM NaHCO , 9.9 mM glucose, 9 µM EGTA and
4
2
4
3
4
5 µM ascorbic acid (pH 7.4). Basal control activity is determined by incubating the
same mixture for 15 min at 4°C in the presence of 10 µM imipramine to block the
uptake. Following incubation, the samples are filtered rapidly under vacuum through
glass fiber filters (GF/B, Packard) and rinsed twice with ice-cold incubation buffer
3
using a 96-sample cell harvester (Unifilter, Packard) to eliminate free [ H]serotonin.
The filters are dried and the retained radioactivity is measured in a scintillation
counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).The
3
results are expressed as a percent inhibition of the control uptake of [ H]serotonin.
The standard inhibitory reference compound is imipramine, which is tested in each

ACCEPTED MANUSCRIPT
experiment at several concentrations to obtain an inhibition curve from which its IC50
[28]
value is calculated
.
4
.2.1.2 5-HT receptor
1A
Cell membrane homogenates (36 µg protein) are incubated for 60 min at 22°C
3
with 0.3 nM [ H]8-OH-DPAT in the absence or presence of the test compound in a
buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO , 0.5 mM EDTA and 2
4
µg/ml aprotinine. Nonspecific binding is determined in the presence of 10 µM
8
-OH-DPAT. Following incubation, the samples are filtered rapidly under vacuum
through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed
several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester
(Unifilter, Packard). The filters are dried then counted for radioactivity in a
scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0,
Packard). The results are expressed as a percent inhibition of the control radioligand
specific binding. The standard reference compound is 8-OH-DPAT, which is tested in
each experiment at several concentrations to obtain a competition curve from which
[29]
its IC is calculated
.
5
0
4
.2.1.3 5-HT receptor
7
Cell membrane homogenates (24 µg protein) are incubated for 120 min at 22°C
3
with 4 nM [ H] LSD in the absence or presence of the test compound in a buffer
containing 50 mM Tris-HCl (pH 7.4), 10 mM MgSO and 0.5 mM EDTA.
4
Nonspecific binding is determined in the presence of 10 µM 5-HT. Following
incubation, the samples are filtered rapidly under vacuum through glass fiber filters
(GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50
mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are
dried then counted for radioactivity in a scintillation counter (Topcount, Packard)
using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a
percent inhibition of the control radioligand specific binding. The standard reference
compound is 5-HT, which is tested in each experiment at several concentrations to