Design, synthesis and docking study of 4-arylpiperazine carboxamides as monoamine neurotransmitters reuptake inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.06.043

Rational drug design method has been used to generate 4-arylpiperazine carboxamides in an effort to develop safer, more potent and effective monoamine neurotransmitters reuptake inhibitors. Out of twenty-seven synthesized compounds, compound 9 displayed potent monoamine neurotransmitter reuptake inhibitory activity against HEK cells transfected with hSERT or hNET. A Surflex-Dock docking model of 9 was also studied.

Full text of DOI:10.1016/j.bmc.2018.06.043

Accepted Manuscript
Design, synthesis and docking study of 4-arylpiperazine carboxamides as mono-
amine neurotransmitters reuptake inhibitors
Suresh Paudel, Ningning Sun, Daulat Bikram Khadka, Goon Yoon, Kyeong-
Man Kim, Seung Hoon Cheon
PII:
S0968-0896(18)30679-5
https://doi.org/10.1016/j.bmc.2018.06.043
BMC 14435
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 April 2018
29 June 2018
30 June 2018
Please cite this article as: Paudel, S., Sun, N., Khadka, D.B., Yoon, G., Kim, K-M., Cheon, S.H., Design, synthesis
and docking study of 4-arylpiperazine carboxamides as monoamine neurotransmitters reuptake inhibitors,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.06.043
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Design, synthesis and docking study of 4-arylpiperazine carboxamides as
monoamine neurotransmitters reuptake inhibitors
Suresh Paudela, Ningning Suna, Daulat Bikram Khadkaa, Goon Yoonb, Kyeong-Man Kima*,
Seung Hoon Cheona*
aCollege of Pharmacy and Research Institute of Drug Development, Chonnam National
University, Gwangju 61186, Republic of Korea
bCollege of Pharmacy and Natural Medicine Research Institute, Mokpo National University,
Jeonnam 58554, Republic of Korea
 Corresponding author. Tel.: +82625302929; fax: +82625302911; e-mail: shcheon@jnu.ac.kr
(S.H. Cheon); Tel.: +82625302936; fax: +82625302949; e-mail: kmkim@jnu.ac.kr (K. M. Kim).
Abstract:
Rational drug design method has been used to generate 4-arylpiperazine carboxamides in an
effort to develop safer, more potent and effective monoamine neurotransmitters reuptake
inhibitors. Out of twenty-seven synthesized compounds, compound 9 displayed potent
monoamine neurotransmitter reuptake inhibitory activity against HEK cells transfected with
hSERT or hNET. A Surflex-Dock docking model of 9 was also studied.
Keywords: 4-arylpiperazine carboxamide, serotonin reuptake inhibitor, norepinephrine reuptake
inhibitor, docking.

1. Introduction:
Monoamines neurotransmitters are involved in broad range of brain functions, including
1
regulation of mood, cognition, attention, appetite, sleep and rewarded processing. In depressed
patients, a deficiency of monoamine neurotransmitters, serotonin (5-HT), norepinephrine (NE)
and dopamine (DA) in brain suggests that regulation of monoamine neurotransmitters level is
essential for the treatment of depression. 2-5 By inhibiting monoamine transporters, monoamine
neurotransmitter reuptake inhibitors exhibit their antidepressant activity.6 Therefore, these
monoamine neurotransmitters represent a promising target for the development of novel
antidepressants.7
5-HT or dual 5-HT and NE reuptake inhibitors are clinically prescribed antidepressants. 8-9
However, these drugs have several limitations and cause side effects, such as delayed onset of
action, insomnia, sexual dysfunction.10-11. Trazodone, 5-HT reuptake inhibitor is comparatively
safer than conventional antidepressant. However, it possesses side effects like irregular heart rate,
and priapism.12-13 Nefazodone, atypical antidepressant can cause severe liver damage. 14-15
Tandospirone can result in insomnia, dysphoria, tachycardia.16 Another antidepressant drug,
buspirone can cause metabolic acidosis (in diabetes patients), could not be used with monoamine
oxidase (MAO) inhibitors and in patients with severely compromised liver and/or renal function
(Fig. 1A).17-18
New therapeutic agents with improved efficacy, safety and time to onset without side effects are
still desirable.19-21 Considering these facts, our research group has studied and developed 4-
benzylpiperidine carboxamides and their analogues, as potent monoamine reuptake inhibitors
(Fig. 1B). The basic essential features of the neurotransmitter reuptake inhibitors consist of
variable aromatic region (Ar1), piperazine/piperidine-aromatic region (Ar2), a linker and
functional group (FG) 22-26. Thus, we designed 4-arylpiperazine carboxamides as possible
monoamine neurotransmitter reuptake inhibitors based on these fundamentals components and
with aim to overcome limitations and side effects of traditional antidepressants (Fig. 1C). 4-
arylpiperazine derivative is a hybrid structure of 4-benzylpiperidine arylamide and
arylpiperazine-tetrazoles. Three carbon units in the linker was considered for the design of new
dual reuptake inhibitors since previously reported our compounds with three carbon units

showed better inhibitory activities than the compounds with two carbon units. 4-benzylpiperidine
carboxamides which differ in substitution at Ar1 and Ar2 were considered to explore their
impact on inhibition of neurotransmitter reuptake. The present study provides a detailed
description of design, synthesis, biological evaluation and docking study of arylpiperazine
carboxamides as monoamine reuptake inhibitors.
Fig. 1. (A) Known antidepressants: Trazodone, tandospirone, nefazodone and buspirone. (B) Arylpiperazine-
tetrazole and 4-benzylpiperidine carboxamide (C) Design of newer arylpiperazine carboxamides 1–27.

2. Chemistry
Various aromatic carboxylic acids 28a–28f were refluxed overnight with excess thionyl chloride in
benzene or toluene and the residue obtained by evaporating the solvent was further reacted with 3-
bromopropylamine hydrobromide in the presence of triethylamine (TEA) to give different amides 29a–
29f (Scheme 1). Alternatively, amidation reaction between aromatic carboxylic acid 28g and 3-
bromopropylamine hydrobromide using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDCI.HCl) as a coupling reagent gave corresponding amides 29g. Various aromatic amines 30h–30r
were reacted with bis(2-chloroethyl)amine hydrochloride to obtain 4-arylpiperazines hydrochloride salts
31h–31r (31h–31k were commercially available). The substitution reaction of 29a–29g with 4-
substituted arylpiperazine (31h–31r) gave carboxamide derivatives 1–27.

Scheme 1: Synthesis of 4-arylpiperazine carboxamides 1-27. Reagents and conditions: (i) SOCl2,
benzene/toluene, reflux (ii) CH2Cl2, TEA, 0 °C–r.t. (iii) EDCI.HCl, DMAP, TEA, r.t. (iv) diethylene
glycol monomethyl ether, 150 °C (v) DMSO, TEA, 100 °C.
3. Results and discussion

In vitro neurotransmitter reuptake inhibition experiments of the synthesized compounds were conducted
in human embryonic kidney 293 (HEK-293) cells transfected with human serotonin transporter (hSERT)
or human norepinephrine transporter (hNET) or human dopamine transporter (hDAT) using a
neurotransmitter uptake assay. The IC50 values for neurotransmitter reuptake inhibitory activity of the
compounds are presented in the Table 1 and 2.
Table 1. Serotonin (5-HT), Norepinephrine (NE) and dopamine (DA) reuptake inhibition (IC50) by
substituted arylpiperazines 1–12.
Compound
R1
X
N
N
C
N
N
C
N
N
C
N
N
C
-
Y
C
N
C
C
N
C
C
N
C
C
N
C
-
R2
5-HT (µM)
> 10
> 10
> 10
> 10
> 10
4.27
NE (µM) DA (µM)
3,5-dichlorophenyl
-
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
0.26
> 10
> 10
2.55
2.55
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
-
1
,,
,,
-
2
3
2,3-(Cl)2
-
4
1-naphthyl
,,
-
5
,,
2,3-(Cl)2
6
-
9.64
7
2-naphthyl
,,
-
> 10
0.74
8
,,
9
2,3-(Cl)2
-
6.94
10
2-quinolyl
,,
,,
-
-
> 10
1.17
11
12
2,3-(Cl)2
-
0.20
Venlafaxine·HCl

-
-
-
-
-
3.24
1.20
Bupropion
Initially, 12 arylpiperazine carboxamides (1–12) were prepared and analyzed for their neurotransmitter
(5-HT, NE and DA) reuptake inhibitory effects. About half of compounds were active for 5-HT and two
compounds (9 and 12) were active for NE. Unfortunately, the dopamine reuptake inhibitory activity of
arylpiperazine carboxamides was not satisfactory at 10 µM. Thus, additional carboxamide derivatives
were analyzed only for 5-HT and NE reuptake inhibitory activity.
Based on biological data of compounds reported by our group, our initial efforts focused on synthesis of
compounds with three carbon linker, aromatic rings R1 (3,5-dichlorophenyl, 2-quinolyl, 1-naphthyl and 2-
naphthyl) and substituted arylpiperazines R2 (1-(2-pyridyl)piperazine, 1-(2-pyrimidyl)piperazine and 1-
(2,3-dichlorophenyl)piperazine) (1-12). As expected, arylpiperazine carboxamides containing 2-naphtyl
and 1-(2,3-dichlorophenyl) piperazine (9, IC50 = 0.74 µM for 5-HT, 0.26 µM for NE) showed a greater
neurotransmitter reuptake inhibitory activity than other analogues. Furthermore, arylpiperazine
carboxamides bearing 2-quinolyl and 1-(2,3-dichlorophenyl) piperazine (12, IC50 = 1.17 µM for 5-HT,
2.55 µM for NE) displayed desirable 5-HT and NE reuptake inhibitory activity. Therefore, we focused on
the effects of substitution of keeping 2-naphthyl and 2-quinolyl as aromatic rings (R1) and various
substituted arylpiperazines R2 (1-(4-methylphenyl)piperazine, 1-(4-(trifluoromethyl)phenyl)piperazine, 1-
(2-methoxyphenyl)piperazine, 1-(4-methoxyphenyl)piperazine, 1-(4-chlorophenyl)piperazine, 1-(2,4-
dichlorophenyl)piperazine, 1-(3,4-dichlorophenyl)piperazine) (20-34). The monoamine neurotransmitter
inhibitory activity of (1-(4-methylphenyl)piperazine containing 2-naphthyl (13, IC50 = 0.46 µM for 5-HT,
2.69 µM for NE) was less than its analogues 14 (IC50 = 0.28 µM for 5-HT, >10 µM for NE) for 5-HT,
although it was greater with NE.
Table 2. Serotonin (NE) and norepinephrine (NE) reuptake inhibition (IC50) by arylpiperazine
carboxamides 13–27.

Compound
R1
R2
5-HT (µM)
0.46
NE (µM)
2.69
> 10
> 10
> 10
> 10
6.07
> 10
> 10
> 10
> 10
> 10
1.34
3.66
4.96
3.15
2.55
2-naphthyl
4-Me
4-Cl
13
,,
0.28
14
> 10
15
2-quinolyl
-
,,
4-Me
2-OMe
4-OMe
4-Cl
0.23
16
,,
> 10
17
,,
0.23
18
,,
1.32
19
,,
6.85
20
2,4-(Cl)2
3,4-(Cl)2
3-CF3
2,3-(Cl)2
4-Cl
,,
0.273
0.7414
> 10
21
22
benzo(b)furanyl
,,
23
0.55
24
benzo(b)thiophenyl
,,
0.40
25
2,3-(Cl)2
2-OMe
2,3-(Cl)2
-
4.46
26
4-biphenyl
,,
-
1.01
27
0.20
Venlafaxine·HCl

In general, unsubstituted phenyl piperazines containing 2-quinolyl (15, IC50 > 10 µM for 5-HT and NE)
was less potent than substituted arylpiperazine with 2-quinolyl (16-21, IC50 - -
>10 µM for NE). Interestingly, the activity of 2-methoxycompounds (17, IC50 = >10 µM for 5-HT
and NE) was significantly less than 4-methoxycompounds (18, IC50 = 0.23 µM for 5-HT, 6.07 µM for
NE). Furthermore, among di-halogen substituted compounds, 2,3-(Cl)2 substituted derivative (12, IC50 =
1.17 µM for 5-HT, 2.55 µM for NE) were greater than its analogues (2,4-(Cl)2 substituted derivative 20,
IC50 = 6.85 µM for 5-HT, >10 µM for NE) and (3,4-(Cl)2 substituted derivative 21, IC50 = 0.27 µM for 5-
HT, >10 µM for NE).
To further elucidate the possible effects on monoamine neurotransmitter reuptake inhibition, we
substituted aromatic rings R1 with Benzo[b]furanyl, Benzo[b]thiophenyl and 4-biphenyl and substituted
arylpiperazine R2 with 1-(4-(trifluoromethyl)phenyl)piperazine, 1-(2-methoxyphenyl)piperazine, 1-(4-
chlorophenyl)piperazine and 1-(2,3-dichlorophenyl)piperazine (22-27). The 5-HT and NE reuptake
inhibitory activities of compounds containing fused heterocyclic ring were in the following order: 2-
benzo[b]furanyl (22, IC50 = 0.74 µM for 5-HT, >10 µM for NE, 23, IC50 = >10 µM for 5-HT and NE) <
2-benzo[b]thiophenyl (24 IC50 = 0.55 µM for 5-HT, 1.34 µM for NE, 25, IC50 = 0.40 µM for 5-HT, 3.66
µM for NE). Furthermore, the activity of derivatives containing 4-biphenyl and 2-
methoxyphenyl)piperazine (26, IC50 = 4.46 µM for 5-HT, 4.96 µM for NE) was less than its analogues
(27, IC50 = 1.01 µM for 5-HT, 3.15 µM for NE).
3.2. Docking study
Compound 9 was selected for docking study as it is active against both 5-HT and NE
because not only compound 16 but 18 is also potentially active against 5-HT. A hypothetical
binding mode of SERT and compound 9 as shown in figure 2B. The docking study shows that
the compound 9 interacted with SERT in the same unique manner as paroxetine. The
naphthalene moiety of the compound occupied the central binding site to have hydrophobic

interaction with Ile172 and Tyr176. The CO of the amide had H-bond with Tyr95. The 3 Carbon
unit linker was particularly important as it bends the compound for optimal placement in the L-
shaped pocket. The linker was optimally placed in the region of the pocket walled by Phe335,
Gly338 and Val501.The terminal phenyl group faced towards allosteric site and was surrounded
by hydrophobic amino acids like Tyr175 and Leu99
Figure 2. (A) Binding mode of hSERT-paroxetine (PDB ID: 5I6X). (B) Hypothetical binding
mode of hSERT-9. H-bonds are displayed as brown discontinuous lines and ionic bond as solid
red lines.
4. Conclusion
In summary, a series of arylpiperazine carboxamides were designed, synthesized and evaluated
for neurotransmitter reuptake inhibitory activity. The synthesis of arylpiperazine carboxamides
involved two main steps: amidation and substitution. Most of 4-arylpiperazine carboxamides
showed micromolar neurotransmitter reuptake inhibitory action against HEK cell lines as
possible neurotransmitter reuptake inhibitors transfected with human serotonin transporter
(hSERT). Docking study was performed with Surflex-Dock program available in Sybyl-X 2.1.1

(wint_os5x) using three dimensional structural of human SERT and antidepressant paroxetine
(PDB ID: 5I6X). Furthermore, studies on 4-arylpiperazine carboxamides and its related diverse
structures could be carried via receptor-based drug design for the development of
neurotransmitter reuptake inhibitors with more specificity and less toxicity.
5. Experimental Section
5.1. Chemistry
A Thomas Hoover melting point apparatus was used for the determination of the melting
point (mp) by the capillary method, and the values were uncorrected. Varian Unity Plus 300
MHz, Varian Unity Inova 500 MHz and Bruker BioSpin GmbH spectrometers were used to
obtain 1H and 13C NMR data, which are reported in ppm downfield from the peak of the internal
standard, tetramethylsilane. The data are reported as chemical shift, multiplicity (s: singlet, d:
doublet, t: triplet, q: quartet, quin: quintet, m: multiplet, dd: doublet of doublets), coupling
constant in Hz and number of protons. Mass spectra were obtained using an Advion Expression
CMS, and recorded in a positive ion mode with an electrospray (ESI) source. Merck silica gel 60
(70–230 mesh) was used for column chromatography. Plates coated with silica gel 60 F254
(Merck) were used for thin-layer chromatography. Reagents were purchased from Sigma–
Aldrich and Alfa Aesar and used without further purification.
5.1.1. N-(3-Bromopropyl)-3,5-dichlorobenzamide (29a). Compound 28a (191 mg, 1 mmol)
was refluxed in excess of thionyl chloride (3 mL) overnight. Excess of thionyl chloride was
evaporated and the residue was dissolved in CH2Cl2, 3-bromopropylamine hydrobromide (328
mg, 1.5 mmol was added followed by triethylamine (TEA; 0.42 mL, 3 mmol). The reaction
mixture was stirred at room temperature. After the reaction was completed, the reaction mixture
was diluted with CH2Cl2 and sequentially washed with water, 1N HCl and saturated NaHCO3.
The organic layer was dried over MgSO4, filtered and concentrated. The obtained product was
purified by column chromatography with n-hexane: Ethyl acetate (EtOAc) = 4:1 to obtain 29a,
(236 mg, 76%) as white solid. Retention factor Rf = 0.85 (n-hexane: EtOAc = 1:1). Mp = 98 °C.

1H NMR (300 MHz, CDCl3): δ (d J = 1.8 Hz, 2H), 7.49 (s, J = 1.8 Hz, 1H), 6.38 (s, 1H),
3.62 (q, J = 6.4 Hz, 2H), 3.49 (t, J = 6.3 Hz, 2H), 2.21 (quin, J = 6.5 Hz, 2H).
5.1.2 N-(3-Bromopropyl)-1-naphthamide (29b). The procedure described for the preparation
of 29a was used with compound 28b (500 mg, 2.90 mmol), thionyl chloride (10 mL) in presence
of toluene (10 mL), 3-bromopropylamine hydrobromide (952mg, 4.35) and TEA (1.21 mL, 8.7
mmol) to obtain 29b (626 mg 74%) as white solid. Rf = 0.74 (n-hexane: EtOAc = 1:1). Mp =
104 °C. 1H NMR (300 MHz, CDCl3): δ 8 4–8.21 (m, 1H), 7.88–7.82 (m, 2H), 7.52–7.49 (m,
3H),7.40–7.35 (m, 1H), 6.41 (s, 1H), 3.59 (q, J = 6.4 Hz, 2H), 3.46 (t, J = 6.4 Hz, 2H), 2.18
(quin, J = 6.4 Hz, 2H).
5.1.3 N-(3-Bromopropyl)quinoline-2-carboxamide (29c). The procedure described for the
preparation of 29a was used with compound 28c (550 mg, 3.17 mmol), thionyl chloride (8 mL),
3-bromopropylamine hydrobromide (1040 mg, 4.75mmol and TEA (1.32 mL, 9.51 mmol) to
obtain 29c (697 mg, 75%) as white solid Rf = 0.87 (n-hexane: EtOAc = 1:1). Mp = 94 °C. 1H
NMR (300 MHz, CDCl3): δ 8 4 (s 1H), 8.30 (s, 2H), 7.88 (ddd, J = 8.1, 1.5, 0.6 Hz, 1H), 7.77
(ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.62 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.59 (d, J = 6.9 Hz, 1H),
3.74–3.66 (m, 2H), 3.53 (t, J = 6.6 Hz, 2H), 2.27 (quin, J = 6.6, 2H).
5.1.4 N-(3-Bromopropyl)benzo(b)furan-2-carboxamide (29d). The procedure described for
the preparation of 29a was used with compound 28d (300 mg, 1.85 mmol), thionyl chloride (5
mL) in presence of benzene (10 mL), 3-bromopropylamine hydrobromide (607mg, 2.77 mmol),
TEA (0.77 mL, 5.55 mmol) to obtain 29d (480 mg, 92%) as white solid. Rf = 0.72 (n-hexane:
EtOAc = 1:1). Mp = 88 °C. 1H NMR (300 MHz, CDCl3): δ (ddd J = 7.8, 1.2, 0.6 Hz, 1H),
7.48–7.45 (m, 2H), 7.39 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H), 7.29–7.24 (m, 1H), 7.04 (s, 1H), 3.64 (q,
J = 6.5 Hz, 2H), 3.49 (t, J = 6.6 Hz, 2H), 2.21 (quin, J = 6.5 Hz, 2H).
5.1.5 N-(3-Bromopropyl)benzo[b]thiophene-2-carboxamide (29e). The procedure described
for the preparation of 29a was used with compound 28e (220 mg, 1.23 mmol), thionyl chloride
(3 mL) in presence of toluene (5 mL), 3-bromopropylamine hydrobromide (402mg, 1.84 mmol)

and TEA (0.51 mL, 3.69 mmol to obtain 29e (325 mg, 89%) as white solid. Rf = 0.74 (n-hexane:
1
EtOAc = 1:1). Mp = 122 °C. H NMR (300 MHz, CDCl3): δ 8 –7.79 (m, 3H), 7.43–7.38 (m,
2H), 6.47 (s, 1H), 3.64 (q, J = 6.4 Hz, 2H), 3.51 (t, J = 6.3 Hz, 2H), 2.23 (quin, J = 6.4 Hz, 2H).
5.1.6 N-(3-Bromopropyl)-1,2-dihydro-[1,1′-biphenyl]-4-carboxamide (29f). The procedure
described for the preparation of 29a was used with compound 28f (1000mg, 4.99 mmol), thionyl
chloride (20 mL) in presence of benzene (20 mL), 3-bromopropylamine hydrobromide (1637 mg,
7.48 mmol and TEA (2 mL, 14.97 mmol) to obtain 29f (952 mg, 60%) as white solid. Rf = 0.62
(n-hexane: EtOAc = 1:1). Mp = 135 °C. 1H NMR (300 MHz, CDCl3): δ 8 –7.82 (m, 2H),
7.67–7.58 (m, 4H), 7.50–7.36 (m, 3H), 6.42 (s, 1H), 3.69–3.61 (m, 2H), 3.51 (t, J = 6.4 Hz, 2H),
2.20 (quin, J = 6.5, 2H).
5.1.7 N-(3-Bromopropyl)-2-naphthamide (29g). The 28g (250 mg, 1.45 mmol) was dissolved
in CH2Cl2 (5 mL). EDCI.HCl (333 mg, 1.74 mmol) was added to reaction mixture followed by
DMAP (8.9 mg, 0.07mmol). To the reaction solution 3-bromopropylamine hydrobromide (317
mg, 1.45 mmol) was added and stirred for 30 minutes. TEA (0.66 mL, 4.78 mmol) was poured
slowly drop wise and stirred reaction at room temperature. After the reaction was completed, the
reaction mixture was diluted with CH2Cl2 and washed with water. The organic layer was dried
over MgSO4, filtered and concentrated. The obtained product was purified by column
chromatography with Hexane: EtOAc (4:1) to obtain 29g (266 mg, 63%) as white solid. Rf =
0.79 (n-hexane: EtOAc = 1:1). Mp = 124 °C. 1H NMR (300 MHz, CDCl3): δ 8.28 (s, 1H, 1-H),
7.92–7.80 (m, 4H), 7.60–7.51 (m, 2H), 6.54 (s, 1H), 3.68 (q, J = 6.4 Hz, 2 Hz), 3.53 (t, J = 6.3,
2H), 2.25 (quin, J = 6.6 Hz, 2H)
5.1.8. 1-Phenylpiperazine hydrochloride (31l)
A mixture of aniline 30l (279.4 mg, 3 mmol), bis(2-chloroethyl)amine hydrochloride (535.5 mg, 3 mmol)
and diethylene glycol monomethyl ether (0.75 mL) was heated at 150 °C for about 12 h. The reaction

mixture was cooled to room temperature and dissolved in methanol (4 mL), followed by the addition of
diethyl ether (150 mL). The precipitate formed was recovered by filtration and washed with diethyl ether
to obtain 31l as an HCl salt (530 mg, 89%). The HCl salt was used for the next reaction without further
purification.
5.1.9. 1-(4-Methylphenyl)piperazine hydrochloride (31m)
The procedure described for the preparation of 31l was used with compound 30m (321.2 mg, 3 mmol),
bis(2-chloroethyl)amine hydrochloride (535.5 mg, 3 mmol), and diethylene glycol monomethyl ether
(0.75 mL) to obtain 31m as an HCl salt (562 mg, 88%). The HCl salt was used for the next reaction
without further purification.
5.1.10. 1-(2-Methoxyphenyl)piperazine hydrochloride (31n)
The procedure described for the preparation of 31l was used with compound 30n (369.4 mg, 3 mmol),
bis(2-chloroethyl)amine hydrochloride (535.5 mg, 3 mmol) and diethylene glycol monomethyl ether (0.75
mL to obtain 31n as an HCl salt ((510 mg, 74%). The HCl salt was used for the next reaction without
further purification.
5.1.11. 1-(4-Methoxyphenyl)piperazine hydrochloride (31o)
The procedure described for the preparation of 31l was used with compound 30o (369.4 mg, 3 mmol),
bis(2-chloroethyl)amine hydrochloride (535.5 mg, 3 mmol) and diethylene glycol monomethyl ether (0.75
mL) to obtain 31o as an HCl salt (549 mg, 80%). The HCl salt was used for the next reaction without
further purification.

5.1.12. 1-(4-Chlorophenyl)piperazine hydrochloride (31p)
The procedure described for the preparation of 31l was used with compound 30p (321.4 mg, 3 mmol),
bis(2chloroethyl)amine hydrochloride (535.5 mg, 3 mmol) and diethylene glycol monomethyl ether (0.75
mL) to obtain 31p as an HCl salt (630 mg, 90%). The HCl salt was used for the next reaction without
purification.
5.1.13. 1-(2,4-Dichlorophenyl)piperazine hydrochloride (31q)
The procedure described for the preparation of 31l was used with compound 30q (486.1 mg, 3 mmol),
bis(2-chloroethyl)amine hydrochloride (535.5 mg, 3 mmol) and diethylene glycol monomethyl ether (0.75
mL) to obtain 31q as an HCl salt (640 mg, 80%). The HCl salt was used for the next reaction without
purification.
5.1.14. 1-(3,4-Dichlorophenyl)piperazine hydrochloride (31r)
The procedure described for the preparation of 31l was used with compound 30r (486.1 mg, 3 mmol),
bis(2-chloroethyl)amine hydrochloride (535.5 mg, 3 mmol) and diethylene glycol monomethyl ether (0.75
mL) to obtain 31r as an HCl salt (627 mg, 78%). The HCl salt was used for the next reaction without
purification.
5.1.15. 3,5-Dichloro-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)benzamide (1)
The mixture of 29a (110 mg, 0.35 mmol), 31h (0.1 mL, 0.70 mmol), TEA (0.19 mL, 1.4 mmol) and
DMSO (1 mL) was stirred at 100 oC. After the reaction was completed, the water was added to the

reaction mixture and extracted with ethyl acetate. The organic layer was dried over MgSO4, filtered and
concentrated. The obtained product was purified by column chromatography on silica gel with n-hexane:
EtOAc: MeOH (10:1.5:0.5) to obtain to obtain 1 (68 mg, 50%) as white solid. Rf = 0.29 (n-hexane:
EtOAc: MeOH = 2.5:1.5:1). Mp = 107–110 °C. 1H NMR (300 MHz, CDCl3): δ 8 (s ) 8 (dd J =
5.4, 1.2 Hz, 1H), 7.70 (d, J = 1.8 Hz, 2H), 7.52–7.46 (m, 1H), 7.38 (t, J = 1.8 Hz, 1H), 6.67–6.63 (m, 2H),
3.58 (qi, J = 4.0 Hz, 6H), 2.66–2.62 (m, 6H), 1.83 (qi, J = 5.7 Hz, 2H). ; 13C NMR (125 MHz, CDCl3): δ
164.4, 159.4, 147.9, 137.5, 135.3, 131.0, 125.6, 113.7, 107.1, 58.8, 53.3, 45.2, 41.4, 41.0, 23.6. MS
(ESI): m/z 415.1 (M+Na) +.
5.1.16. 3,5-Dichloro-N-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)propyl)benzamide (2)
The procedure described for the preparation of 1 was used with compound 29a (200 mg, 0.64 mmol), 31i
(0.18 mL, 1.28 mmol), TEA (0.35 mL, 2.56 mmol) and DMSO (1 mL) to obtain 2 (151 mg, 60%) as
white solid. Rf = 0.29 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 110–112 °C. 1H NMR (300 MHz,
CDCl3): δ 8 (s ) 8 (d J = 4.8 Hz, 2H), 7.72 (d, J = 1,8 Hz, 2H), 7.41 (t, J = 1.95 Hz, 1H), 6.51
(t, J = 4.6 Hz, 1H), 3.87 (t, J = 5.1 Hz, 4H), 3.59 (qi, J = 5.6 Hz, 2H), 2.65–2.57 (m, 6H), 1.83 (qi, J =
5.85 Hz, 2H) ; 13C NMR (125 MHz, CDCl3): δ 4.5, 161.5, 157.7, 137.5, 135.3, 131.0, 125.6, 110.1,
58.6, 53.3, 43.6, 41.2, 40.9, 23.7. MS (ESI): m/z 394.6 (M+H) +.
5.1.17. 3,5-Dichloro-N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)benzamide (3)
The procedure described for the preparation of 1 was used with compound 29a (70 mg, 0.23 mmol), 31j
(123 mg, 0.46 mmol), TEA (0.12 mL, 0.92 mmol) and DMSO (1 mL) to obtain 3 (53 mg, 50%) as white
solid. Rf = 0.61 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 117–121 °C. 1H NMR (300 MHz, CDCl3):
δ 4 (d J = 1.8 Hz, 2H), 7.48 (t, J = 1.8 Hz, 1H), 7.18–7.12 (m, 2H), 7.00–6.93 (m, 1H), 6.76 (s, 1H),
3.57 (q, J = 5.6 Hz, 2H), 3.09 (s, 4H), 2.71–2.67 (m, 6H), 126–1.25 (m, 2H); 13C NMR (125 MHz,

CDCl3): δ 4 8 4 8 9 9 5.5, 131.2, 127.5, 125.7, 124.8, 118.6, 56.3, 53.0,
51.3, 41.0, 36.5, 29.7. MS (ESI): m/z 482.2 (M+Na) +.
5.1.18. N-(3-(4-(Pyridin-2-yl)piperazin-1-yl)propyl)-1-naphthamide (4).
The procedure described for the preparation of 1 was used with compound 29b (80 mg, 0.27 mmol), 31h
(0.08 mL, 0.54 mmol), TEA (0.15 mL, 1.08 mmol) and DMSO (2 mL) to obtain 4 (45 mg, 44%) as light
brown liquid. Rf = 0.67 (CH2Cl2: MeOH = 10:1). 1H NMR (300 MHz, CDCl3): δ 8 (d J = 7.8 Hz, 1H),
8.15–8.12 (m, 1H), 7.84–7.78 (m, 3H), 7.57–7.29 (m, 5H), 6.61–6.57 (m, 1H), 6.48 (d, J = 8.7 Hz, 1H),
3.60 (q, J = 5.9 Hz, 2H), 3.25 (t, J = 4.9 Hz, 4H), 2.55–2.47 (m, 6H), 1.82 (quin, J = 6.2 Hz, 2H) ; 13C
NMR (125 MHz, CDCl3): δ 9 4 9 4 8 4 34.8, 133.6, 130.3, 130.1, 128.2, 126.8, 126.2,
125.4, 124.7, 124.6, 113.3, 106.9, 57.6, 52.9, 44.9, 40.8, 40.0, 24.9. MS (ESI): m/z 397.9 (M+Na) +.
5.1.19. N-(3-(4-(Pyrimidin-2-yl)piperazin-1-yl)propyl)-1-naphthamide (5).
The procedure described for the preparation of 1 was used with compound 29b (80 mg, 0.27 mmol), 31i
(0.04 mL, 0.32 mmol), TEA (0.11 mL, 0.81 mmol) and DMSO (1 mL) to obtain 5 (50 mg, 49%) as light
brown liquid. Rf = 0.66 (CH2Cl2: MeOH = 10:1). 1H NMR (300 MHz, CDCl3): δ 8 (d J = 7.8 Hz, 1H),
8.26 (d, J =4.5 Hz, 2H), 7.88–7.82 (m, 2H), 7.73 (s, 1H), 7.62–7.40 (m, 4H), 6.46 (t, J = 4.8 Hz, 1H), 3.68
(q, J = 5.8 Hz, 2H), 3.57 (t, J = 4.95 Hz, 4H), 2.58 (t, J = 6.15 Hz, 2H), 2.48 (t, J = 4.95, 4H), 1.87 (quin,
J = 6.1 Hz, 2H). ; 13C NMR (125 MHz, CDCl3): δ 8 4
53.0, 41.0, 31.9, 29.7, 24.9. MS (ESI): m/z 376.5 (M+H) +.
5.1.20. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)-1-naphthamide (6).
The procedure described for the preparation of 1 was used with compound 29b (90 mg, 0.31 mmol), 31j
(99 mg, 0.37 mmol), TEA (0.13 mL, 0.93 mmol) and DMSO (2 mL) to obtain 6 (60 mg, 44%) as white
solid. Rf = 0.53 (CH2Cl2: MeOH = 10:1). Mp = 124–126 °C. 1H NMR (300 MHz, CDCl3): δ 8.34 (d, J =

7.8 Hz, 1H), 8.11 (s, 1H), 7.88–7.82 (m, 2H), 7.61–7.38 (m, 4H),7.14–7.05 (m, 2H), 6.53 (dd, J = 7.5, 1.8
Hz, 1H), 3.64 (q, J = 5.7 Hz, 2H), 2.59 (t, J = 6 Hz, 10H), 1.82 (quin, J = 6 Hz, 2H). ; 13C NMR (125
MHz, CDCl3): δ 9 4 134.8, 133.6, 130.4, 130.1, 128.2, 126.9, 126.3, 125.4, 124.7, 124.6,
109.8, 57.7, 53.0, 43.4, 40.9, 40.1, 24.9. MS (ESI): m/z 464.2 (M+Na) +.
5.1.21 N-(3-(4-(Pyridin-2-yl)piperazin-1-yl)propyl)-2-naphthamide (7).
The procedure described for the preparation of 1 was used with compound 29g (130 mg, 0.44 mmol), 31h
(0.08 mL, 0.52 mmol), TEA (0.18 mL, 1.32 mmol) and DMSO (1 mL) to obtain 7 (75 mg, 45%) as white
solid. Rf = 0.61 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 116–120 °C. 1H NMR (300 MHz, CDCl3):
δ 8 (s ) 8 (s ) 8 (dddd J = 4.95, 2.1, 1.8, 0.9 Hz, 1H), 7.91–7.87 (m, 1H), 7.83–7.75 (m,
3H), 7.51–7.44 (m, 2H), 7.37 (ddd, J = 8.1, 6.9 , 1.2 Hz, 1H), 6.68–6.63 (m, 1H), 6.59 (d, J = 8.4 Hz, 1H),
3.65 (q, J = 8.4 Hz, 2H), 3.57 (t, J = 5.1 Hz, 4H), 2.64 (t, J = 5.25 Hz, 6H), 1.87 (quin, J = 5.9 Hz, 2H) ;
13C NMR (125 MHz, CDCl3): δ 9 9 4 9 4 9 8 8
127.4, 127.2, 126.6, 123.7, 113.6, 107.1, 58.3, 53.2, 45.3, 40.9, 40.8, 24.4. MS (ESI): m/z 375.2.2
(M+H) +.
5.1.22. N-(3-(4-(Pyrimidin-2-yl)piperazin-1-yl)propyl)-2-naphthamide (8).
The procedure described for the preparation of 1 was used with compound 29g (130 mg, 0.44 mmol), 31i
(0.07 mL, 0.52 mmol), TEA (0.18 mL, 1.32 mmol) and DMSO (2 mL) to obtain 8 (70 mg, 42%) as light
brown liquid. Rf = 0.58 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). 1H NMR (300 MHz, CDCl3): δ 8 4–8.27
(m, 4H), 7.90–7.82 (m, 4H), 7.54–7.42 (m, 2H), 6.5 (t, J = 4.8 Hz, 1H), 3.86 (t, J = 4.95 Hz, 4H), 3.66 (q,
J = 5.7 Hz, 2H), 2.64–2.57 (m, 6H), 1.87 (q, J = 6 Hz, 2H) ; 13C NMR (125 MHz, CDCl3): δ 4
157.7, 134.6, 132.6, 132.0, 128.7, 128.3, 127.7, 127.4, 127.3, 126.6, 123.6, 110.0, 58.4, 53.3, 43.7, 40.9,
40.8, 24.5. MS (ESI): m/z 398.4 (M+Na) +.
5.1.23. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)-2-naphthamide (9).

The procedure described for the preparation of 1 was used with compound 29g (130 mg, 0.44 mmol), 31j
(141 mg, 0.52 mmol), TEA (0.18 mL, 1.32 mmol) and DMSO (2 mL) to obtain 9 (80 mg, 41%) as light
brown liquid. Rf = 0.61 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). 1H NMR (300 MHz, CDCl3): δ 8 4
(s,1H), 8.34 (s, 1H), 7.95–7.86 (m , 4H), 7.58–7.45 (m, 2H), 7.18–7.14 (m, 1H), 7.05 (t, J = 8.1 Hz, 1H),
6.68 ( dd, J = 8.1, 1.5 Hz, 1H), 3.67 (q, J = 5.6 Hz, 2H), 3.03 (s, 4H), 2.71–2.67 (m, 6H), 1.86 ( quin, J =
5.85, 2H) ; 13C NMR (125 MHz, CDCl3): δ 4 9 8 4 4 0 132.6, 132.3, 128.8, 128.3,
127.8, 127.5, 127.4, 127.2, 126.6, 124.8, 124.1, 118.5, 58.3, 53.4, 51.2, 40.9, 29.7, 24.3. MS (ESI): m/z
442.2 (M+H) +.
5.1.24. N-(3-(4-(Pyridin-2-yl)piperazin-1-yl)propyl)quinoline-2-carboxamide (10).
The procedure described for the preparation of 1 was used with compound 29c (150 mg, 0.51 mmol), 31h
(0.09 mL, 0.61 mmol), TEA (0.21 mL, 1.53 mmol) and DMSO (1.5 mL) to obtain 10 (95mg, 49%) as
white solid. Rf = 0.61 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 134–136 °C. 1H NMR (300 MHz,
CDCl3): δ 9 (s ) 8 –8.26 (m, 3H), 7.93–7.82 (m, 2H), 7.56–7.45 (m, 3H), 6.67–6.63 (m, 2H),
3.72–3.65 (m, 6H), 2.64 (q, J = 5.9 Hz, 6H), 1.91(quin, J = 6.3 Hz, 2H). ; 13C NMR (125 MHz, CDCl3): δ
164.6, 159.7, 150.1, 148.0, 146.5, 137.4, 137.3, 129.9, 129.5, 129.2, 127.7, 119.0, 113.3, 110.0, 107.2,
57.7, 53.2, 45.3, 41.0, 39.5, 25.8. MS (ESI): m/z 376.8 (M+H) +.
5.1.25. N-(3-(4-(Pyrimidin-2-yl)piperazin-1-yl)propyl)quinoline-2-carboxamide (11).
The procedure described for the preparation of 1 was used with compound 29c (150 mg, 0.51 mmol), 31i
(0.09 mL, 0.61 mmol), TEA (0.21 mL, 1.53 mmol) and DMSO (1.5 mL) to obtain 11 (85mg, 44%) as
light brown liquid. Rf = 0.22 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). 1H NMR (300 MHz, CDCl3): δ 9
(s, 1H), 8.34–8.27 (m, 4H), 7.87–7.86 (m, 1H), 7.85–7.84 (m, 1H), 7.63–7.53 (m, 2H), 6.50 (t, J = 5.8 Hz,
1H), 3.96 (t, J = 5.1 Hz, 4H),3.68 (q, J = 6 Hz, 2H), 2.63–2.57 (m, 6H), 1.91 (quin, J = 6.15 Hz, 2H). ; 13C
NMR (125 MHz, CDCl3): δ 4 1.8, 157.7, 150.1, 146.5, 137.4, 130.0, 129.4, 129.3, 127.8, 127.7,
119.0, 109.8, 57.5, 53.2, 43.8, 41.0, 39.2, 29.7, 26.0. MS (ESI): m/z 377.4 (M+H) +.

5.1.26. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)quinoline-2-carboxamide (12).
The procedure described for the preparation of 1 was used with compound 29c (150 mg, 0.51 mmol), 31j
(160 mg, 0.61 mmol), TEA (0.21 mL, 1.53 mmol) and DMSO (1.5 mL) to obtain 12 (102 mg, 45%) as
white solid. Rf = 0.55 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 62–65 °C. 1H NMR (300 MHz,
CDCl3): δ 9 (s ) 8 –8.28 (m, 2H), 8.10 (d, J = 8.1 Hz, 1H), 7.89–7.85 (m, 1H), 7.68–7.56 (m,
2H), 7.19–7.08 (m, 2H), 6.92–6.88 (m, 1H), 3.68 (q, J = 6 Hz, 2H), 3.19–3.16 (m, 4H), 2.69–2.61 (m, 6H),
1.90 (quin, J = 6.3 Hz, 2H) ; 13C NMR (125 MHz, CDCl3): δ 4 4 4 4 9
129.8, 129.6, 129.1, 127.8, 127.7, 127.3, 127.2, 124.3, 119, 118.6, 57.5, 53.4, 51.1, 39.4, 25.8. MS (ESI):
m/z 443.5 (M+H) +.
5.1.27. N-(3-(4-(p-Tolyl)piperazin-1-yl)propyl)-2-naphthamide (13).
The procedure described for the preparation of 1 was used with compound 29g (300 mg, 1.02 mmol),
31m (285 mg, 1.22 mmol), TEA (0.42 mL, 3.06 mmol) and DMSO (2 mL) to obtain 13 (175 mg, 42%) as
1
white solid. Rf = 0.63 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 130–134 °C. H NMR (400 MHz,
CDCl3): δ 8 4 (s ) 8 4 (s ) 9 –7.78 (m, 4H), 7.53–7.49 (m, 1H), 7.41–7.37 (m, 1H), 7.07 (d, J
= 6 Hz, 2H), 6.81–6.78 (m, 2H), 3.67–3.63 (m, 2H), 3.19–3.17 (m, 4H), 2.70–2.64 (m, 6H), 2.29 (s,
3H),1.87–1.83 (m, 2H) ; 13C NMR (100 MHz, CDCl3): δ 149.8, 134.7, 132.6, 132.0, 129.0, 128.8,
128.4, 127.7, 127.5, 127.1, 126.6, 124.9, 123.9, 117.4, 58.5, 53.6, 49.4, 41.0, 24.3. MS (ESI): m/z 388.6
(M+H) +.
5.1.28. N-(3-(4-(4-Chlorophenyl)piperazin-1-yl)propyl)-2-naphthamide (14).
The procedure described for the preparation of 1 was used with compound 29g (300 mg, 1.02 mmol), 31p
(260 mg, 1.22 mmol), TEA (0.42 mL, 3.06 mmol) and DMSO (2 mL) to obtain 14 (182 mg, 44%) as

white solid. Rf = 0.66 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 147–149 °C. 1H NMR (400 MHz,
CDCl3): δ 8 9 (s ) 8 (s ) 9 –7.74 (m, 4H), 7.54–7.37 (m, 2H), 7.22–7.18 (m, 2H), 6.80–6.76
(m, 2H), 3.68–3.64 (m, 2H), 3.18 ( t, J = 3.6 Hz, 4H), 2.70–2.65 (m, 6H), 1.90–1.84 (m, 2H) ; 13C NMR
(100 MHz, CDCl3): δ 49 4 132.6, 131.9, 129.7, 129.6, 128.9, 128.3, 127.7, 127.4, 127.3,
126.6, 123.9, 116.7, 58.6, 53.6, 50, 41.2, 24.3, 20.5. MS (ESI): m/z 430.2 (M+Na) +.
5.1.29. N-(3-(4-Phenylpiperazin-1-yl)propyl)quinoline-2-carboxamide (15).
The procedure described for the preparation of 1 was used with compound 29c (200 mg, 0.68 mmol), 31l
(240 mg, 0.81 mmol), TEA (0.28 mL, 2.04 mmol) and DMSO (2 mL) to obtain 15 (150 mg, 98%) as
white solid. Rf = 0.61 (CH2Cl2: MeOH = 10:1). Mp = 164–167 °C. 1H NMR (500 MHz, CDCl3): δ 9 (s
1H), 8.34–8.28 (m, 2H), 7.85 (d, J = 0.6 Hz, 1H), 7.84 (d, J = 0.9 Hz, 1H), 7.57–7.46 (m, 2H), 7.31–7.26
(m, 2H), 6.97–6.87 (m, 3H), 3.71–3.67 (m, 2H), 3.38 ( t, J = 3 Hz, 4H), 2.70 (t, J = 3 Hz, 4H), 2.65–2.63
(m, 2H), 1.94–1.89 (m, 2H). ; 13C NMR (125 MHz, CDCl3): δ 4 4 4 9 9
129.7, 129.2, 129.1, 127.7, 127.6, 119.6, 119.0, 116.0, 57.7, 53.4, 49.0, 40.9, 39.4, 29.7, 25.8. MS (ESI):
m/z 397.0 (M+Na) +.
5.1.30. N-(3-(4-(p-Tolyl)piperazin-1-yl)propyl)quinoline-2-carboxamide (16).
The procedure described for the preparation of 1 was used with compound 29c (300 mg, 1.02 mmol),
31m (260 mg, 1.22 mmol), TEA (0.42 mL, 3.06 mmol) and DMSO (2 mL) to obtain 16 (158 mg, 40%) as
white solid. Rf = 0.60 (n-hexane: EtOAc: MeOH = 2.5:1.5:1).Mp = 134–136 °C. 1H NMR (400 MHz,
CDCl3): δ 9 4 (s ) 8 –8.27 (m, 2H), 7.99 (d, J = 6 Hz, 1H), 7.86 –7.84 (m, 1H), 7.58–7.50 (m, 2H),
7.08 (d, J = 6 Hz, 2H), 6.85 (d, J = 6.3 Hz, 2H), 3.69–3.65 (m, 2H), 3.31 ( t, J = 3.6 Hz, 4H), 2.70–2.61
(m, 6H), 2.29 (s, 3H), 1.94–1.88 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 4 49 4

137.3, 129.9, 129.8, 129.6, 129.2, 129.1, 127.73, 127.7, 119.0, 116.5, 57.6, 53.5, 49.6, 39.4, 29.7, 25.9,
20.5. MS (ESI): m/z 389.3 (M+H) +.
5.1.31. N-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propyl)quinoline-2-carboxamide (17).
The procedure described for the preparation of 1 was used with compound 29c (150 mg, 0.51 mmol), 31n
(117 mg, 0.61 mmol), TEA (0.21 mL, 1.53 mmol) and DMSO (1.5 mL) to obtain 17 (100mg, 48%) as
white solid. Rf = 0.55 (CH2Cl: MeOH = 10:1). Mp = 147–150 °C. 1H NMR (500 MHz, CDCl3): δ 9 (s
1H), 8.34–8.28 (m, 2H), 7.86 (d, J = 0.9 Hz, 1H), 7.85 (d, J = 0.9 Hz, 1H), 7.64–7.56 (m, 2H), 7.03–6.86
(m, 4H), 3.86 (s, 3H), 3.70–3.67 (m, 2H), 3.23 (s, 4H), 2.73 (s, 4H), 2.65–2.62 (m, 2H), 1.93–1.88 (m,
2H). ; 13C NMR (125 MHz, CDCl3): δ 64.6, 152.2, 150.2, 146.5, 141.2, 137.3, 129.93, 129.9, 129.2,
127.7, 122.9, 120.9, 119.1, 118.3, 111.2, 57.7, 55.3, 53.7, 50.5, 41.0, 39.5, 25.9. MS (ESI): m/z 405.4
(M+H) +.
5.1.32. N-(3-(4-(4-Methoxyphenyl)piperazin-1-yl)propyl)quinoline-2-carboxamide (18).
The procedure described for the preparation of 1 was used with compound 29c (300 mg, 1.02 mmol), 31o
(280 mg, 1.22 mmol), TEA (0.42 mL, 3.06 mmol) and DMSO (2 mL) to obtain 18 (198 mg, 48%) as
white solid. Rf = 0.61 (n-hexane: EtOAc: MeOH = 2.5:1.5:1).Mp = 145–150 °C. 1H NMR (400 MHz,
CDCl3): δ 9 (s ) 8 –8.28 (m, 2H), 8.02–7.84 (m, 2H), 7.59–7.52 (m, 2H), 6.93–6.84 (m, 4H),
3.80 (s, 3H), 3.70–3.65 (m, 2H), 3.25 (t, J = 3.6 Hz, 4H), 2.69–2.61 (m, 6H), 1.93–1.87 (m, 2H); 13C
NMR (100 MHz, CDCl3): δ 4 4 9 49 8 4 9 9 9 8 9 9.0,
118.1, 114.4, 57.7, 55.6, 53.6, 50.4, 41.3, 39.1, 25.9, 15.95. MS (ESI): m/z 405.0 (M+H) +.
5.1.33. N-(3-(4-(4-Chlorophenyl)piperazin-1-yl)propyl)quinoline-2-carboxamide (19).

The procedure described for the preparation of 1 was used with compound 29c (300 mg, 1.02 mmol), 31p
(285 mg, 1.22 mmol), TEA (0.42 mL, 3.06 mmol) and DMSO (2 mL) to obtain 19 (192 mg, 46%) as
white solid. Rf = 0.50 (CH2Cl: MeOH = 10:1). Mp = 150–155 °C. 1H NMR (500 MHz, CDCl3): δ 9 (s
1H), 8.34–8.29 (m, 2H), 7.94–7.92 (m, 1H), 7.86 (dd, J = 4.8, 0.6 Hz, 1H), 7.59–7.55 (m, 1H), 7.51–7.47
(m, 1H), 7.23–7.21 (m, 2H), 6.87–6.84 (m, 2H), 3.71–3.65 (m, 2H), 3.33 (t, J = 3 Hz, 4H), 2.69 (t, J = 3
Hz, 4H), 2.64 (t, J = 3.9 Hz, 2H), 1.93–1.88 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 4 49 9
146.4, 137.3, 129.9, 129.6, 129.2, 128.9, 128.9, 127.75, 127.7, 124.3, 119, 117.2, 117.1, 57.7, 54.9, 53.3,
49.0, 48.9, 46, 39.5, 25.9. MS (ESI): m/z 409.9 (M+H) +.
5.1.34. N-(3-(4-(2,4-Dichlorophenyl)piperazin-1-yl)propyl)quinoline-2-carboxamide (20).
The procedure described for the preparation of 1 was used with compound 29c (300 mg, 1.02 mmol), 31q
(327 mg, 1.22 mmol), TEA (0.42 mL, 3.06 mmol) and DMSO (2 mL) to obtain 20 (190 mg, 42%) as
white solid. Rf = 0.50 (CH2Cl: MeOH = 10:1). Mp = 125–129 °C. 1H NMR (500 MHz, CDCl3): δ 8 99 (s
1H), 8.34–8.29 (m, 2H), 8.09 (dd, J = 5.1, 0.3 Hz, 1H), 7.88 (dd, J = 4.8, 0.6 Hz, 1H), 7.68–7.59 (m, 2H),
7.36 (d, J = 1.5 Hz, 1H), 7.15 (dd, J = 5.4, 1.5 Hz, 1H), 6.90 (d, J = 5.1 Hz, 1H), 3.70–3.66 (m, 2H),
3.16–3.15 (m, 4H), 2.69 (s, 4H), 2.63 (t, J = 3.9 Hz, 2H), 1.92–1.87 (m, 2H). ; 13C NMR (125 MHz,
CDCl3): δ 4 48 4 4 9 9 9 9 4 9 8 8 8
127.4, 121.2, 119.1, 57.5, 53.4, 51.1, 41.0, 39.3, 29.7, 26.0. MS (ESI): m/z 443.3 (M+H) +.
5.1.35. N-(3-(4-(3,4-Dichlorophenyl)piperazin-1-yl)propyl)quinoline-2-carboxamide (21).
The procedure described for the preparation of 1 was used with compound 29c (300 mg, 1.02 mmol), 31r
(327 mg, 1.22 mmol), TEA (0.42 mL, 3.06 mmol) and DMSO (2 mL) to obtain 21 (203 mg, 45%) as
white solid. Rf = 0.61 (n-hexane: EtOAc: MeOH = 2.5:1.5:1).Mp = 145–150 °C. 1H NMR (400 MHz,
CDCl3): δ 9 (s ) 8 –8.20 (m, 2H), 7.84–7.77 (m, 2H), 7.51–7.41 (m, 2H), 7.21–7.19 (m, 1H),

6.87 (d, J = 2.1 Hz, 1H), 6.68–6.65 (m, 1H), 3.63–3.58 (m, 2H), 3.26–3.23 (m, 4H), 2.60–2.53 (m, 6H),
1.85–1.78 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 49 149.1, 145.4, 136.4, 131.7, 129.4, 128.9,
128.5, 128.2, 126.8, 126.7, 121.0, 118.0, 116.1, 114.3, 56.7, 52.1, 47.5, 38.5, 24.8. MS (ESI): m/z 443.8
(M+H) +.
5.1.36. N-(3-(4-(4-(Trifluoromethyl)phenyl)piperazin-1-yl)propyl)benzofuran-2-carboxamide (22).
The procedure described for the preparation of 1 was used with compound 29d (200 mg, 0.70 mmol), 31k
(244 mg, 1.06 mmol), TEA (0.39 mL, 2.8 mmol) and DMSO (2 mL) to obtain 22 (150 mg, 49%) as white
solid. Rf = 0.29 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 171–174 °C. 1H NMR (300 MHz, CDCl3):
δ 8 8 (s ) –7.43 (m, 4H), 7.24–7.10 (m, 2H), 6.99–6.94 (m, 3H), 3.64 (q, J = 4.5 Hz, 2H), 3.46 (t,
J = 4.8 Hz, 4H), 2.73–2.65 (m, 6H), 1.18 (quin, J = 5.6 Hz, 2H). ; 13C NMR (125 MHz, CDCl3): δ 8 8
154.5, 153.3, 149.3, 127.6, 126.52, 126.5, 126.4, 123.6, 122.6, 114.6, 111.3, 110.1, 58.3, 53.0, 47.9, 41.0,
40.2, 29.7, 24.4. MS (ESI): m/z 432.8 (M+H) +.
5.1.37. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)benzofuran-2-carboxamide (23)
The procedure described for the preparation of 1 was used with compound 29d (300 mg, 1.07 mmol), 31j
(343 mg, 1.28 mmol), TEA (0.44 mL, 3.2 mmol) and DMSO (2 mL) to obtain 23 (150 mg, 32%) as liquid.
Rf = 0.61 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). 1H NMR (400 MHz, CDCl3): δ (d J = 5.7 Hz, 1H),
7.47–7.32 (m, 3H), 7.24–7.20 (m, 2H), 7.13–7.04 (m, 2H), 6.95 (s, 1H), 6.80–6.77 (m, 1H), 4.24–4.21 (m,
2H), 3.60–3.51 (m, 6H), 2.90–2.87 (m, 4H), 1.99–191 (m, 2H); 13C NMR (100 MHz, CDCl3): δ
157.9, 154.5, 153.7, 149.8, 147.7, 133.1, 131.2, 129.9, 127.8, 126.5, 125.9, 124.1, 122.7, 121.7, 117.7,
110.7, 109.3, 65.0, 62.2, 50.2, 35.4, 28.2, 22.1. MS (ESI): m/z 432.4 (M+H) +.
5.1.38. N-(3-(4-(4-Chlorophenyl)piperazin-1-yl)propyl)benzo[b]thiophene-2-carboxamide (24).

The procedure described for the preparation of 1 was used with compound 29e (300 mg, 1.0 mmol), 31p
(280 mg, 1.2 mmol), TEA (0.41 mL, 3 mmol) and DMSO (2 mL) to obtain 24 (182 mg, 44%) as white
solid. Rf = 0.66 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 139–142 °C. 1H NMR (400 MHz, CDCl3):
δ 8 9 (s ) 8–7.62 (m,2H), 7.64–7.62 (m, 1H), 7.40–7.31 (m, 2H), 7.22–7.20 (m, 2H), 6.83–6.81
(m, 2H), 3.61 (q, J = 4.2 Hz, 2H), 3.25–3.22 (m, 4H), 2.69–2.61 (m, 6H), 1.86–1.80 (m, 2H); 13C NMR
(100 MHz, CDCl3): δ 49 8 4 8 9 8 129, 126.2, 125.2, 124.9, 124.8, 122.7, 117.3,
58.3, 53.3, 49.3, 41, 24.3. MS (ESI): m/z 414.1 (M+H) +.
5.1.39. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)benzo[b]thiophene-2-carboxamide (25).
The procedure described for the preparation of 1 was used with compound 29e (300 mg, 1.0 mmol), 31j
(280 mg, 1.2 mmol), TEA (0.41 mL, 3 mmol) and DMSO (2 mL) to obtain 25 (224 mg, 50%) as light
brown liquid. Rf = 0.66 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). 1H NMR (400 MHz, CDCl3): δ 8.30 (s,
1H), 7.84–7.77 (m, 3H), 7.44–7.36 (m,2H), 7.19 (dd, J = 6, 1.2 Hz, 1H), 7.13 (t, J = 6 Hz, 1H), 6.87 (dd,
J = 6, 1.2 Hz, 1H), 3.65–3.61 (m, 2H), 3.13 (s, 4H),2.73–2.66 (m, 6H), 1.87–1.81 (m, 2H); 13C NMR (100
MHz, CDCl3): δ 162.2, 151.1, 140.5, 139.0, 138.9, 134.2, 127.7, 127.5, 126.2, 125.2, 124.9, 124.88,
124.84, 122.7, 118.5, 58.5, 53.5, 51.3, 41.1, 30.9, 24.2. MS (ESI): m/z 448.3 (M+H) +.
5.1.40. N-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propyl)-[1,1'-biphenyl]-4-carboxamide (26).
The procedure described for the preparation of 1 was used with compound 29f (300 mg, 0.94 mmol), 31n
(274 mg, 1.12 mmol), TEA (0.39 mL, 2.82 mmol) and DMSO (2 mL) to obtain 26 (234 mg, 58%) as
white solid. Rf = 0.66 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 102–105 °C. 1H NMR (300 MHz,
CDCl3): δ 8 49 (t J = 4.2 Hz, 1H), 7.94–7.90 (m, 2H), 7.64–7.54 (m, 4H), 7.47–7.34 (m, 3H), 7.04–6.97
(m, 1H), 6.88–6.85 (m, 3H), 3.86 (s, 3H), 3.65–3.60 (q, J = 5.5 Hz, 2H), 3.09 (s, 4H), 2.72–2.64 9m, 6H),
1.83 (quin, J = 5.7 Hz, 2H). ; 13C NMR (125 MHz, CDCl3): δ 4 9 4 4

128.9, 128.8, 127.9, 127.6, 127.5, 127.2, 127.1, 123.2, 121.0, 118.1, 111.2, 58.5, 55.4, 53.6, 50.6, 41.0,
24.2. MS (ESI): m/z 452.0 (M+Na) +.
5.1.41. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)-[1,1'-biphenyl]-4-carboxamide (27).
The procedure described for the preparation of 1 was used with compound 29f (300 mg, 0.94 mmol), 31j
(301 mg, 1.12 mmol), TEA (0.39 mL, 2.82 mmol) and DMSO (2 mL) to obtain 27 (242 mg, 55%) as
white solid. Rf = 0.61 (n-hexane: EtOAc: MeOH = 2.5:1.5:1). Mp = 129–131 °C. 1H NMR (400 MHz,
CDCl3): δ 8 (s ) 9 –7.88 (m, 2H), 7.65–7.57 (m, 4H), 7.47–7.36 (m, 3H), 7.15 (dd, J = 6.1, 1.2
Hz, 1H), 7.04 (t, J = 6 Hz, 1H), 6.83 (dd, J = 6, 1.2 Hz, 1H), 3.65–3.61 (m, 2H), 3.03 (s, 4H), 2.70–2.65
(m, 6H), 1.87–1.81 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 4 9 4 .0, 134.1, 134.0,
128.9, 128.0, 127.64, 127.6, 127.5, 127.1, 127.1, 124.8, 118.4, 58.3, 53.4, 51.3, 40.8, 24.5. MS (ESI):
m/z 490.1 (M+Na) +.
5.2. Neurotransmitter uptake assay
The assay was performed following the method described in the literature with slight
modification.22 HEK-293 cells were cultured in a medium supplemented with fetal bovine serum
and transfected with hSERT or hNET or hDAT. Radiolabeled [3H]-5-HT (PerkinElmer,
Waltham, MA, USA), [3H]-NE (PerkinElmer, Waltham, MA, USA) and [3H]-DA (PerkinElmer)
were used at a concentration of 20 nM in the assay. Radioactivity was measured using a Wallac
1450 MicroBeta® TriLux liquid scintillation counter (PerkinElmer). Venlafaxine hydrochloride,
bupropion, and GBR12909 were used as reference neurotransmitter reuptake inhibitors.
5.3. Docking
The docking study was performed in Sybyl-X 2.1.1 (winnt_os5x) using the Surflex-Dock
program. The structure of hSERT-paroxetine was downloaded from Protein Data Bank (PDB ID:

5I6X).27 Maltose, cholesterol, N-acetylglucosamine and chloride ion were deleted. The ligand
(paroxetine) was extracted. Hydrogens were added and minimized using the MMFF94s force
field with MMFF94 charges, by using a conjugate gradient method, distance-dependent
dielectric constant and converging to 0.01 kcal/mol·Å. Protomol, an idealized representation of a
ligand that makes every potential interaction with the binding site, was generated on the basis of
ligand mode. Compounds intended for docking study including 9 and paroxetine were
constructed in Sybyl; energy was minimized with MMFF94s force field and MMFF94 charged
and stored in Sybyl database. The compounds in the Sybyl database were docked into the
binding site by Surflex-Dock on the basis of protomol constructed earlier. The extracted
paroxetine was considered as a reference molecule. The docking protocol was able to reproduce
the position of paroxetine (manually constructed and stored in the Sybyl database) in the binding
site with 0.43 Å root-mean-square deviations (RMSD) of the heavy atoms of the extracted
paroxetine.
Acknowledgment
This research was supported by Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education (grant No.
2012R1A1A2006613) and the Bio & Medical Technology Development Program of the National
Research Foundation (NRF) & funded by the Korean government (MSIT) (NRF-
2017M3A9G2077568). The authors would like to thank the Korea basic science institute
Gwangju center for performing the 1H NMR and 13C NMR.
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