Design and synthesis of arylamidine derivatives as serotonin/norepinephrine dual reuptake inhibitors

Article abstract of DOI:10.3390/molecules24030497

To improve the in vivo antidepressant activity of previously reported serotonin (5-HT) and norepinephrine (NE) dual reuptake inhibitors, three series of arylamidine derivatives were designed and synthesized. The in vitro 5-HT and NE reuptake inhibitory activities of these compounds were evaluated, and compound II-5 was identified as the most potent 5-HT (IC₅₀ = 620 nM) and NE (IC₅₀ = 10 nM) dual reuptake inhibitor. Compound II-5 exhibited potent antidepressant activity in the rat tail suspension test and showed an acceptable safety profile in a preliminary acute toxicity test in mice. Our results show that these arylamidine derivatives exhibit potent 5-HT/NE dual reuptake inhibition and should be explored further as antidepressant drug candidates.

Full text of DOI:10.3390/molecules24030497

molecules
Article
Design and Synthesis of Arylamidine Derivatives as
Serotonin/Norepinephrine Dual Reuptake Inhibitors
Hui Wen 1 , Wen Qin 2, Guangzhong Yang 1 and Yanshen Guo 1,
*
1
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College,
Beijing 100050, China; wenhui@imm.ac.cn (H.W.); chufm@imm.ac.cn (G.Y.)
National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention,
Beijing 100050, China; qinwen@ninh.chinacdc.cn
2
*
Correspondence: guoys@imm.ac.cn; Tel.: +86-10-63165249
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 15 January 2019; Accepted: 28 January 2019; Published: 30 January 2019
Abstract: To improve the in vivo antidepressant activity of previously reported serotonin (5-HT) and
norepinephrine (NE) dual reuptake inhibitors, three series of arylamidine derivatives were designed
and synthesized. The in vitro 5-HT and NE reuptake inhibitory activities of these compounds were
evaluated, and compound II-5 was identified as the most potent 5-HT (IC50 = 620 nM) and NE
(IC50 = 10 nM) dual reuptake inhibitor. Compound II-5 exhibited potent antidepressant activity in
the rat tail suspension test and showed an acceptable safety profile in a preliminary acute toxicity test
in mice. Our results show that these arylamidine derivatives exhibit potent 5-HT/NE dual reuptake
inhibition and should be explored further as antidepressant drug candidates.
Keywords: serotonin; norepinephrine; dual reuptake inhibitors; antidepressant
1. Introduction
Depression is a common mental illness that can be severe, chronic, and sometimes
life-threatening [
1
,2]. According to the World Health Organization, depression is one of the top
causes of disability worldwide and affects 350 million people [
3
]. Therefore, there is an urgent need to
develop antidepressants to improve the lives of people living with depression [4–7].
The biogenic amine transmitters, serotonin (5-HT), norepinephrine (NE), and dopamine (DA),
are closely related to symptoms of depression [8]. First-generation antidepressants were mainly
designed as single reuptake inhibitors, such as selective 5-HT reuptake inhibitors (SSRIs) and NE
reuptake inhibitors (NRIs). Several single reuptake inhibitors, such as fluoxetine (SSRI) and reboxetine
(NRI) (Figure 1), have poor safety and tolerability profiles. Second-generation antidepressants are dual
reuptake inhibitors, such as 5-HT/NE reuptake inhibitors (SNRIs) and NE/DA reuptake inhibitors
(NDRIs). Dual inhibitors currently on the market include bupropion (NDRI), venlafaxine (SNRI),
and duloxetine (SNRI) (Figure 1). Dual reuptake inhibitors have better antidepressant effects and
fewer side effects than single reuptake inhibitors [9]. SNRIs have a shorter working time and fewer
adverse reactions, and thus are regarded as better antidepressant drugs than SSRIs [10].
In our previous study, we used compounds with 5-HT or NE reuptake inhibition activities to
build a pharmacophore model of the characteristics of 5-HT and NE inhibitors (Figure 2) [11]. A series
of substituted arylamidine derivatives was discovered using the 5-HT and NE pharmacophore model
(Figure 3). One type of arylamidine derivative fitted the proposed 5-HT and NE pharmacophore
model. Subsequent pharmacological tests indicated that these compounds showed good 5-HT and NE
reuptake inhibition activity [12–14].
Molecules 2019, 24, 497; doi:10.3390/molecules24030497
www.mdpi.com/journal/molecules

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Figure 1. Structures of antidepressants with single or dual reuptake inhibition.
(a)
(b)
(c)
(d)
) and norepinephrine (NE)
Figure 2. Proposed pharmacophore model Hypo1 of serotonin (5-HT) (
a
(
b
) produced by the Hypo Gen module in Catalyst 4.10 software (Accelrys, San Diego, CA, USA).
The distance between the pharmacophore features is reported in angstroms. Mapping of an arylamidine
derivative onto the 5-HT ( ) and NE ( ) pharmacophore models. Green, blue, brown, and red contours
c
d
represent hydrogen-bond acceptor (HA), hydrophobic (HY), aromatic ring (RA), and positive ionizable
(PI) pharmacophore features, respectively.
Figure 3. Design of inhibitor series I–III based on arylamidine derivatives.
However, an in vivo study of antidepressant activity in rats showed that arylamidine derivatives
were less potent than the commercial SNRI antidepressants venlafaxine and duloxetine. We suspected
that the high rigidity of the planar structure in arylamidine derivatives, compared with the structures
of other SNRI inhibitors, such as venlafaxine and duloxetine, was a main cause of the poor
antidepressant activity in rats. To circumvent these problems with arylamidine derivatives, in this
work, we designed and synthesized 32 compounds in three series (I–III) of optimized arylamidine
derivatives. The pharmacological activity of the 32 compounds was evaluated in vitro and selected
compounds were evaluated in vivo.

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2. Results and Discussion
2.1. Chemical Design and Modeling Prediction
To increase the structural flexibility of the optimized compounds, we designed three series of
compounds (Figure 3). A methylene group (-CH2-) was inserted between the aromatic ring and
nitrogen atom to form the structure of series I. Then, based on series I, another two series of derivatives
were designed. In series II, the benzyl ring was replaced with a naphthalene ring and in series III,
the amidine ring was replaced with a naphthalene ring.
The compounds were docked into the 5-HT and NE pharmacophore models, and all three types
of molecules matched the model well. Compounds I-4 and II-4 are shown as examples of mapping on
the pharmacophore model in Figure 4. Generally, the binding, as in the 5-HT and NE pharmacophore
models, can be classified as aromatic ring (RA, brown), hydrophobic (HY, blue), positive ionizable
(PI, red), and hydrogen-bond acceptor (HA, green) features. Compound I-4 was docked in the 5-HT
model, and the two aromatic rings matched either the RA or HY features well. The nitrogen atoms
of the piperazine ring were recognized as an HA feature, and the amidine imino nitrogen atom was
recognized as a PI feature. Compound I-4 was also mapped on the NE model and the two aromatic
rings matched the HY feature, the nitrogen atoms of the piperazine ring were recognized as the PI
feature, and amidine imino nitrogen atom was recognized as the HA feature. We observed similar
mapping for compound II-4 in both models.
(a)
Figure 4. Mapping of compounds I-4 and II-4 on the serotonin (5-HT) pharmacophore model (
on the NE pharmacophore model ( ). Green, blue, brown, and red contours represent hydrogen-bond
(b)
(c)
(d)
a,c) and
b,d
acceptor (HA), hydrophobic (HY), aromatic ring (RA), and positive ionizable (PI) pharmacophore
features, respectively.
Our experience suggested that for the design of a new series of compounds (Figure 2), the major
pharmacodynamic domain in the arylamidine derivatives, including the (E) configuration, would have
to be retained. Only small changes to the structure of arylamidine derivatives are tolerated. Otherwise,
the new compounds would lose their inhibitory activities against both 5-HT and NE reuptake.
Thus, in the design, the key change was the addition of a methylene group, which broke the
original planar configuration and increased the flexibility of the molecules. This design strategy
balanced the pharmacological activity and the desired structural flexibility. Additionally, based on the
pharmacophore models, the space for the aromatic ring in the arylamidine derivatives can tolerate a
bigger aromatic fragment, such as an aphthalene ring. This might further favor the binding of the new
compounds to either 5-HT or NE.
In summary, the docking study showed that all of the designed compounds matched the 5-HT and
NE pharmacophore models well. The fragments in the compounds matched the corresponding binding
domains in both models. Similar binding modes were observed for the mapping of compounds I-4
and II-4 in both models. The docking results indicated that the designed compounds were likely to be
good dual reuptake inhibitors of 5-HT and NE.

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2.2. Chemistry Synthesis
The synthesis for series I–III is shown in Scheme 1. An aromatic acid (
1) was used as the starting
reagent to form an acyl chloride (
various aromatic methylamines to form a substituted amide (
was the synthesis of compound . All the final compounds had an (E) configuration, which was initially
formed in the production of acyl chloride . (E)-acyl chloride was obtained by the reaction between
amide and PCl5. During the synthesis of compound , piperazine could react with two molecules
of compound to produce disubstituted by-products. The amount of disubstituted by-products was
2) with yields of 75–95%. The intermediate (
2) was reacted with
3
). The most important step in the scheme
4
4
4
3
5
4
reduced by increasing the amount of piperazine in this reaction. Subsequently, all target compounds
were prepared as hydrochloride salts (E)-I, (E)-II, and (E)-III to obtain stable solids.
Scheme 1. Synthesis of (E)-I–(E)-III. (a) Oxalyl chloride, CH2Cl2; (b) Ar2-methylamine, triethylamine,
CH2Cl2; (c) PCl5, C2H4Cl2; (d) piperazine, CH2Cl2; (e) ethanol, HCl.
2.3. Structure Confirmation
1D-NOE NMR was used to confirm the configuration of the structure. The 1D-NOE spectrum
of compound I-11 (Figure 5) showed weak NOE enhancements between the Ha and Hb protons,
and Ha and Hc protons, indicating that the Ha proton was spatially close to the Hb and Hc protons.
Thus, the absolute configuration of the target compound was (E).
In the 1H-NMR spectra of several compounds, such as I-15, the Ar-CH2-N fragment exhibited
a singlet, where as in the spectra of other compounds, such as I-14, the same fragment produced a
double doublet. The only structural difference between I-14 and I-15 was the ortho- or para-chloro
substituent on the amidine ring (Figure 6). The difference between the spectra may arise from the weak
steric hindrance caused by the ortho-chloro substituent in I-14, but not by the para-chloro substituent
in I-15. The steric hindrance would make the two protons in the Ar-CH2-N fragment magnetically
inequivalent in I-14, producing the double doublet. This observation is also important because if
the configuration of the final compound was (Z), and there would be no steric hindrance, the two
Ar-CH2-N protons would not be magnetically inequivalent, and the double doublet would not appear.
Consequently, the steric hindrance indirectly indicated that the absolute configuration of the final
compound was (E).

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H
H
NO2
H
H
N
Cl
N
N
+HCl
H
Figure 5. 1D-NOE NMR of compound (E)-I-11.
Cl
H
H
N
Cl
N
N
H
+HCl
I-15 (D2O)
Cl
Cl
H'
H''
N
N
N
H
+HCl
I-14 (D2O)
1
Figure 6. H-NMR spectra of I-14 and I-15.
2.4. Biological Results and Discussion
2.4.1. In Vitro Test
All the target compounds were evaluated for their inhibitory activity against the reuptake of 5-HT
and NE at the cellular level. Rat brain tissue was used for the uptake test, which included 3H-5-HT and
3H-NE [15]. 1-[7,8-3H]-Noradrenaline and 5-hydroxy-[3H]-tryptamine trifluoroacetate were obtained
from Amersham Corporation. Duloxetine was used as the reference compound. All compounds were

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tested at a concentration of 10
IC50 values (Table 1).
µM, and potent compounds (inhibition >80%) were assayed to obtain
In series I, most compounds exhibited moderate dual reuptake inhibition activity. Compound
I-19 was the most potent dual inhibitor in this series (5-HT, IC50 = 830 nM; NE, IC50 = 560 nM),
and was comparable to the reference compound, duloxetine (5-HT, IC50 = 640 nM; NE, IC50 = 49 nM).
In series II, compounds II-4 (5-HT, IC50 = 130 nM; NE, IC50 = 880 nM) and II-5 (5-HT, IC50 = 620 nM;
NE, IC50 = 10 nM) were the most potent compounds. Most compounds in this series had improved
dual reuptake inhibition activity. In series III, compound III-1 also showed good potency for 5-HT and
NE transporters.
The series I compounds maintained the dual 5-HT and NE inhibitory activity after the insertion
of a methylene into the arylamidine derivative structure, although the general inhibitory activity
was moderate. Various substituents on the benzene ring did not dramatically improve the binding
of these compounds with the targets. In series II, a naphthalene ring replaced the benzyl ring.
Interestingly, some compounds in this series exhibited dramatically improved dual inhibitory activities.
Thus, the addition of a big hydrophobic fragment, that is, the naphthalene ring, improved binding to
the5-HT and NE transporters.
Table 1. Inhibition data for serotonin (5-HT) and norephinephrine (NE) reuptake for series I~III.
5-HT a
NE a
Compd.
R1
R2
b
b
(IC50
)
(IC50
)
86%
(640 nM)
97%
(49 nM)
Duloxetine
-
-
I-1
I-2
I-3
I-4
I-5
I-6
I-7
I-8
I-9
I-10
I-11
I-12
I-13
I-14
I-15
I-16
I-17
I-18
H
H
H
H
H
H
H
H
H
4-OCH3
4-NO2
2-Cl
3-Cl
4-Cl
65%
38%
48%
58%
47%
17%
38%
54%
65%
61%
50%
51%
67%
76%
48%
45%
43%
75%
82%
(830 nM)
72%
34%
67%
66%
65%
63%
57%
39%
35%
67%
51%
47%
49%
81%
84%
60%
68%
26%
27%
63%
78%
(560 nM)
35%
57%
4-F
2-F
4-CN
3-CN
4-CH3
2-Cl, 4-NO2
4-NO2
2-Cl
H
H
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3-Cl
4-Cl
4-F
2-F
4-CN
I-19
4-Cl
3-CN
I-20
I-21
4-Cl
4-OCH3
4-CH3
4-NO2

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Table 1. Cont.
5-HT a
NE a
(IC50)
Compd.
R1
R2
b
b
(IC50
)
R2
N
N
N
(E)-II
H
II-1
II-2
II-3
-
-
-
H
4-NO2
4-F
80%
57%
84%
90%
(130 nM)
81%
67%
52%
76%
78%
(880 nM)
92%
II-4
-
2-F
II-5
-
2-Cl
(620 nM)
(10 nM)
II-6
II-7
II-8
II-9
II-10
-
-
-
-
-
3-Cl
4-Cl
4-CN
3-CN
4-CH3
65%
62%
70%
88%
75%
59%
46%
54%
82%
63%
III-1
H
5-NO2
73%
66%
a
Percent inhibition measured at a concentration of 10 µM, b IC50 was tested for selected compounds.
2.4.2. In Vivo Test
Based on the in vitro results, compounds I-19
,
II-4, and II-5 were selected for the tail suspension
test (TST) in rats. The TST has been used widely as a preclinical model for screening antidepressant
activity, and it is sensitive to commercially available antidepressant drugs. In the assay, rats are
suspended by tail and the time that the animal is immobile is recorded. The compounds were
administered to male rats at a dose of 30 mg/kg (PO). Duloxetine was used as the positive control
(30 mg/kg, PO). The compounds all reduced the immobility times of rats compared with the negative
control vehicle group, indicating that all compounds exerted an antidepressant effect (Figure 7, Table 2).
However, the antidepressant effect of compound II-5 was the closest to that of duloxetine, although
it was still smaller. Both compound II-5 and duloxetine exhibited statistical significance for the
antidepressant effect. In addition, the safety profile of compound II-5 was also explored with a
preliminary acute toxicity test in mice at a single dose of 200 or 400 mg/kg (PO) (n = 6). Compound
II-5 did not result in the death of mice at doses of 200 and 400 mg/kg after 20 h, and thus the LD50
was >400 mg/kg (PO), indicating that the toxicity was low and that the safety profile was acceptable
(Table 3).
In summary, compound II-5 had the most potent in vitro 5-HT and NE dual inhibitory activity
of the three compounds used in the TST, and it also had the most potent in vivo pharmacological
antidepressant effect. Compound II-5 was reasonably safe for mice at a dose of 30 mg/kg. However,
the in vivo antidepressant effect of compound II-5 (5-HT, IC50 = 620 nM; NE, IC50 = 10 nM) was smaller
than that of leading antidepressant duloxetine (5-HT, IC50 = 640 nM; NE, IC50 = 49 nM), although they

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had similar in vitro activities. The less potent in vivo antidepressant effect of compound II-5 may arise
from its in vivo metabolism, which needs to be examined and optimized.
Table 2. The effect of four compounds in the rat test predictive of antidepressant activity.
Group
Dose (mg/kg)
Immobility Time (s)
Rate of Shorten Time (%)
Vehicle
Duloxetine
I-19
54 ± 18
11 ± 5
30
30
30
30
79.0
37.8
39.7
67.8
34 ± 17
33 ± 19
18 ± 7
II-4
II-5
Table 3. Acute toxicity of II-5 in mice.
Dose (mg/kg)
Mice (n)
Death (n)
Survival at 20 h
400
200
Vehicle
6
6
6
0
0
0
100%
100%
100%
***
***
Figure 7. Tail suspension test (TST) results of compounds I-19
,
II-4, and II-5 and duloxetine (single
dose of 30 mg/kg), (*** p < 0.001).
3. Experiment Section
3.1. Chemistry
Most reagents and solvents were obtained from commercial suppliers and used as received.
Some reactions were performed under an inert atmosphere (N2). 1H-NMR spectra were obtained
on an NMR spectrometer (Mercury, Varian, San Diego, CA, USA; 400MHz). Melting points were
measured with a melting point apparatus (Yanaco-50, YANACO company, Kyoto, Japan) and are
not corrected. Electro spray ionization (ESI) mass spectra and high-resolution mass spectroscopy
(HRMS) were performed with a liquid chromatograph/mass selective detector time-of-flight mass
spectrometer (LC/MSD TOF, Agilent Technologies, Santa Clara, CA, USA). The yields are of the
purified product and are not optimized. Silica gel column chromatography was performed with silica
gel 60G (Qingdao Haiyang Chemical, Qingdao, China). The procedure for preparing series I–III was
the same as for compound I-1. Purity was determined using HPLC, LC/MS and NMR spectroscopy.
All of the synthesized compounds have purity over 95%.

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General Procedure for Preparing (E)-N-((4-Methoxyphenyl)(piperazin-1-yl)methylene)-1-
phenylmethanamine hydrochloride (I-1)
Preparation of N-Benzyl-4-methoxybenzamide
One drop of dry N,N-dimethyl formamide and oxalyl chloride (0.62 g, 4.9 mmol) were added
dropwise to a solution of 4-methoxybenzoic acid (0.50 g, 3.3 mmol) in dry CH2Cl2 (20 mL). The reaction
mixture was stirred at room temperature for 2 h, and then distilled in vacuo to give the crude acyl
chloride, which was dissolved in dry CH2Cl2 (10 mL). Dry triethylamine (0.40 g, 3.9 mmol) was added
to a solution of benzylamine (0.35 g, 3.3 mmol) in dry CH2Cl2 (20 mL). The reaction mixture was
cooled to 0 ◦C, and the acyl chloride solution was added dropwise. After stirring at 20–25 ◦C for 4 h,
the reaction mixture was distilled in vacuo, and washed with a 10% solution of NaOH and water,
and dried. Silica gel column chromatography (hexane/ethyl acetate) afforded I-1 as a white powder
(0.75 g, 94.8% yield).
Preparation of (E)-N-((4-Methoxyphenyl)(piperazin-1-yl)methylene)-1-phenylmethanamine
hydrochloride (I-1)
N-Benzyl-4-methoxybenzam◦ide (0.50 g, 2.1 mmol) was dissolved in dry dichloroethane (10 mL),
and the solution was heated to 50 C for 30 min. PCl5 (0.44 g, 2.1 mmol) was added in one portion and
the reaction was stirred at 90 ◦C for 2 h. After most of the solvent was removed in vacuo, the residue
was dissolved in dry CH2Cl2 (10 mL) and was added dropwise to a solution of piperazine (0.54 g,
6 mmol) in dry CH2Cl2 (10 mL) at 0 ◦C. The reaction mixture was stirred at room temperature for 2 h,
and then distilled in vacuo. Et2O (50 mL) and saturated aqueous Na2CO3 (30 mL) were added to the
mixture. After the aqueous extract was separated, the organic extract was washed twice with saturated
aqueous Na2CO3 and 10% aqueous NaOH, and then extracted with 1 mol/L HCl. The aqueous extract
was washed twice with CH2Cl2 and Et2O, and was treated with 10% aqueous NaOH to increase the
pH to 10. The aqueous extract was re-extracted with Et2O (50 mL), and the Et2O extract was dried over
Na2SO4. After Et2O was removed under vacuum, the residue was dissolved in dry ethanol (5 mL),
and an ethanolic solution of HCl was added dropwise to adjust the pH to 2 to afford I-1 as a white
powder (0.18 g, 25% yield). Mp: 195–197 ◦C. 1H-NMR (400 MHz, D2O):
δ 3.37 (br, 2H, piperazinyl-H),
3.61 (br, 2H, piperazinyl-H), 3.66 (br, 2H,piperazinyl-H), 3.89 (s, 3H, -CH3), 4.13 (br, 2H, piperazinyl-H),
4.44 (s, 2H, -CH2-), 7.10–7.16 (m, 4H, ArH), 7.35–7.41 (m, 5H, ArH). HRMS calcd for C19H23N3O
(M + H)+, 310.1914; found, 310.1912.
(E)-N-((4-Nitrophenyl)(piperazin-1-yl)methylene)-1-phenylmethanamine hydrochloride (I-2): Yield 40%, Mp:
230–231 ◦C. 1H-NMR (400 MHz, D2O):
δ 3.37 (br, 2H, piperazinyl-H), 3.61 (br, 2H, piperazinyl-H),
3.66 (br, 2H, piperazinyl-H), 4.13 (br, 2H, piperazinyl-H), 4.44 (s, 2H, -CH2-), 7.10–7.16 (m, 4H, ArH),
7.35–7.41 (m, 5H, ArH). HRMS calcd for C18H20N4O2 (M + H)+, 325.1659; found, 325.1654.
(E)-N-((2-Chlorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanamine hydrochloride (I-3): Yield 18%, Mp:
◦
142–144 C.1H-NMR (400 MHz, D2O):
δ
3.40 (br, 2H,0 piperazinyl-H), 3.67 (br, 4H,0p0 iperazinyl-H),
4.24 (br, 2H, piperazinyl-H), 4.38 (d, 1H, J = 14.0 Hz, -CH -), 4.46 (d, 1H, J = 14.0 Hz, -CH -), 7.11–7.12 (m,
2H, ArH), 7.37–7.38 (m, 2H, ArH), 7.48 (d, 1H, J = 7.6 Hz, ArH), 7.55–7.58 (m, 2H, ArH), 7.70–7.73 (m,
2H, ArH). HRMS calcd for C18H20ClN3 (M + H)+, 314.1419; found, 314.1421.
(E)-N-((3-Chlorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanamine hydrochloride (I-4): Yield 66%, Mp:
◦
1
235–237 C. H-NMR (400 MHz, D2O):
δ 3.34 (t, 2H, J = 5.4 Hz, piperazinyl-H), 3.58–3.66 (m, 4H,
piperazinyl-H),040 .14 (t, 2H, J = 5.4 Hz, piperazinyl-H), 4.39 (d, 1H, J = 15.9 Hz, -CH0-), 4.45 (d, 1H,
J = 15.9 Hz, -CH -), 7.07–7.11 (m, 2H, ArH), 7.35–7.38 (m, 4H, ArH), 7.45 (s, 1H, ArH), 7.56–7.61 (m, 1H,
ArH), 7.73 (d, 1H, J = 7.2 Hz, ArH). HRMS calcd for C18H20ClN3 (M + H)+, 314.1419; found, 314.1411.

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(E)-N-((4-Chlorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanamine hydrochloride (I-5): Yield 65%, Mp:
182–184 ◦C. 1H- NMR (400 MHz, D2O):
3.35 (br, 2H, piperazinyl-H), 3.64 (br, 4H, piperazinyl-H),
δ
4.14 (br, 2H, piperazinyl-H), 4.42 (s, 2H, -CH2-), 7.11 (br, 2H, ArH), 7.35–7.42 (m, 5H, ArH), 7.63 (d, 2H,
J = 8.4 Hz, ArH). HRMS calcd for C18H20ClN3 (M + H)+, 314.1419; found, 314.1414.
(E)-N-((4-Fluorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanamine hydrochloride (I-6): Yield 33%, Mp:
220–222 ◦C. 1H-NMR (400 MHz, D2O):
δ 3.34 (br, 2H, piperazinyl-H), 3.58 (br, 2H, piperazinyl-H),
3.64 (br, 2H, piperazinyl-H), 4.12 (br, 2H, piperazinyl-H), 4.43 (s, 2H, -CH2-), 7.11 (br, 2H, ArH),
7.32–7.37 (m, 5H, ArH), 7.46–7.50 (m, 2H, ArH). HRMS calcd for C18H20FN3 (M + H)+, 298.1714; found,
298.1710.
(E)-N-((2-Fluorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanamine hydrochloride (I-7): Yield 22%, Mp:
182–184 ◦C. 1H-NMR (400 MHz, D2O):
δ 3.34 (br, 2H, piperazinyl-H), 3.59 (br, 2H, piperazinyl-H),
3.71 (br, 2H, piperazinyl-H), 4.17 (br, 2H, piperazinyl-H), 4.42 (d, 1H, J = 15.6 Hz, -CH00-), 4.49 (d,
1H, J = 15.6 Hz, -CH00-), 7.08–7.10 (m, 2H, ArH), 7.34–7.39 (m, 4H, ArH), 7.43–7.45 (m, 2H, ArH),
7.72–7.80 (m, 1H, ArH). HRMS calcd for C18H20FN3 (M + H)+, 298.1714; found, 298.1706.
(E)-4-((Benzylimino)(piperazin-1-yl)methyl)benzonitrile hydrochloride (I-8): Yield 38%, Mp: 205–207 ◦C.
1H-NMR (400 MHz, D2O):
δ 3.36 (br, 2H, piperazinyl-H), 3.61 (br, 4H, piperazinyl-H), 4.17 (br, 2H,
piperazinyl-H), 4.40 (s, 2H, -CH2-), 7.04–7.08 (m, 2H, ArH), 7.34–7.36 (m, 3H, ArH), 7.60 (d, 2H,
J = 8.4 Hz, ArH), 7.98 (d, 2H, J = 8.4 Hz, ArH). HRMS calcd for C19H20N4 (M + H)+, 305.1761; found,
305.1760.
(E)-3-((Benzylimino)(piperazin-1-yl)methyl)benzonitrile hydrochloride (I-9): Yield 60%, Mp: 190–192 ◦C.
1H-NMR (400 MHz, D2O):
δ 3.35 (br, 2H, piperazinyl-H), 3.61 (br, 4H, piperazinyl-H), 4.18 (br, 2H,
piperazinyl-H), 4.36 (d, 1H, J = 15.6 Hz, -CH0-), 4.44 (d, 1H, J = 15.6 Hz, -CH00-), 7.04–7.06 (m, 2H,
ArH), 7.31–7.41 (m, 3H, ArH), 7.72–7.81 (m, 3H, ArH), 8.07 (d, 1H, J = 6.9 Hz, ArH). HRMS calcd for
C19H20N4 (M + H)+, 305.1761; found, 305.1758.
(E)-1-Phe◦nyl-N-(piperazin-1-yl(p-tolyl)methylene)methanamine hydrochloride (I-10): Yield 50%, Mp:
1
172–174 C. H-NMR (400 MHz, D2O):
δ 2.42 (s, 3H, -CH3), 3.34 (t, 2H, J = 5.4 Hz, J = 5.1 Hz,
piperazinyl-H), 3.59 (t, 2H, J = 5.7 Hz, J = 5.1 Hz, piperazinyl-H), 3.64 (t, 2H, J = 5.7 Hz, piperazinyl-H),
4.12 (t, 2H, J = 6.0 Hz, piperazinyl-H), 4.42 (s, 2H, -CH2-), 7.09–7.12 (m, 2H, ArH), 7.30–7.37 (m, 5H,
ArH), 7.45 (d, 2H, J = 8.1 Hz, ArH). HRMS calcd for C19H23N3 (M + H)+, 294.1965; found, 294.1962.
(E)-N-((2-Chloro-4-nitrophenyl)(piperazin-1-yl)methylene)-1-phenylmethanamine hydrochloride
(I-11):
Yield 73%, Mp: 195–197 ◦C. 1H-NMR (400 MHz, D2O):
δ 3.39 (br, 2H, piperazinyl-H), 3.66 (br, 4H,
piperazinyl-H), 4.13 (br, 2H, piperazinyl-H), 4.35 (d, 1H, J = 15.6 Hz, -CH0-), 4.46 (d, 1H, J = 15.6 Hz,
00
-CH -), 7.05 (d, 2H, J = 7.8 Hz, ArH), 7.31–7.38 (m, 3H, ArH), 7.71 (d, 1H, J = 8.7 Hz, ArH), 8.35 (d, 1H,
J = 8.4 Hz, ArH), 8.57 (s, 1H, J = 8.7 Hz, ArH). HRMS calcd for C18H19ClN4O2 (M + H)+, 359.1269;
found, 359.1277.
(E)-1-(4-Chlorophenyl)-N-((4-nitrophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (I-12):
Yield 22%, Mp: 172–174 ◦C. 1H-NMR (400 MHz, D2O):
δ 3.35 (br, 2H, piperazinyl-H), 3.65 (br, 4H,
piperazinyl-H), 4.18 (br, 2H, piperazinyl-H), 4.39 (s, 2H, -CH2-), 7.01 (d, 2H, J = 8.4 Hz, ArH), 7.34 (d,
2H, J = 8.4 Hz, ArH), 7.67 (d, 2H, J = 8.7 Hz, ArH), 8.42 (d, 2H, J = 8.7 Hz, ArH). HRMS calcd for
C18H19ClN4O2 (M + H)+, 359.1269; found, 359.1271.
(E)-1-(4-Chlorophenyl)-N-((2-chlorophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (I-13):
Yield 56%, Mp: 181–183 ◦C. 1H-NMR (400 MHz, D2O):
δ
3.36 (br, 2H, piperazinyl-H), 3.61 (br, 4H,
piperazinyl-H), 4.14 (br, 2H, piperazinyl-H), 4.34 (d, 1H, J = 15.6 Hz, -CH0-), 4.42 (d, 1H, J = 15.6 Hz,

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00
-CH -), 7.01 (d, 2H, J = 8.4 Hz, ArH), 7.33 (d, 2H, J = 8.4 Hz, ArH), 7.42 (d, 1H, J = 7.8 Hz, ArH), 7.55 (t,
1H, J = 7.5 Hz, ArH), 7.64–7.72 (m, 2H, ArH). HRMS calcd for C18H19Cl2N3 (M + H)+, 348.1029; found,
348.1032.
(E)-1-(4-Chlorophenyl)-N-((3-chlorophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (I-14):
Yield 53%, Mp: 218–220 ◦C. 1H-NMR (400 MHz, D2O):
δ 3.34 (br, 2H, piperazinyl-H), 3.63 (br, 4H,
piperazinyl-H), 4.14 (br, 2H, piperazinyl-H), 4.35 (d, 1H, J = 15.9 Hz, -CH0-), 4.35 (d, 1H, J = 15.9 Hz,
00
-CH -), 7.03 (d, 2H, J = 8.7 Hz, ArH), 7.33–7.37 (m, 3H, ArH), 7.40 (s, 1H, ArH), 7.55–7.61 (m, 1H, ArH),
7.72 (d, 1H, J = 7.2 Hz, ArH). HRMS calcd for C18H19Cl2N3 (M + H)+, 348.1029; found, 348.1027.
(E)-1-(4-Chlorophenyl)-N-(◦(4-chlorophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (I-15):
1
Yield 36%, Mp: 157–159 C. H-NMR (400 MHz, D2O):
δ 3.33 (br, 2H, piperazinyl-H), 3.62 (br,4H,
piperazinyl-H), 4.13 (br, 2H, piperazinyl-H), 4.40 (s, 2H, -CH2-), 7.03 (d, 2H, J = 8.4 Hz, ArH), 7.35 (d,
2H, J = 8.1 Hz, ArH), 7.37 (d, 2H, J = 8.1 Hz, ArH), 7.63 (d, 2H, J = 8.4 Hz, ArH). HRMS calcd for
C18H19Cl2N3 (M + H)+, 348.1029; found, 348.1027.
(E)-1-(4-Chlorophenyl)-N-((4-fluorophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (I-16):
◦
1
Yield 20%, Mp: 165–167 C. H-NMR (400 MHz, D2O):
δ 3.34 (br, 2H, piperazinyl-H), 3.62 (br,
4H, piperazinyl-H), 4.13 (br, 2H, piperazinyl-H), 4.41 (s, 2H, -CH2-), 7.05 (d, 2H, J = 8.1 Hz, ArH),
7.31–7.37 (m, 4H, ArH), 7.42–7.47 (m, 2H, ArH). HRMS calcd for C18H19ClFN3 (M + H)+, 332.1324;
found, 332.1323.
(E)-1-(4-Chlorophenyl)-N-((2-fluorophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (I-17):
Yield 21%, Mp: 215–217 ◦C. 1H-NMR (400 MHz, D2O):
δ
3.26 (br, 2H, piperazinyl-H), 3.52 (br, 2H,
0
piperazinyl-H), 3.70 (br, 2H, piperazinyl-H), 4.18 (br, 2H, piperazinyl-H), 4.41 (d, 1H, J = 15.6 Hz, -CH -),
00
4.47 (d, 1H, J = 15.6 Hz, -CH -), 7.02 (d, 2H, J = 8.4 Hz, ArH), 7.32–7.38 (m, 3H, ArH), 7.41–7.43 (m, 2H,
ArH), 7.72–7.79 (m, 1H, ArH). HRMS calcd for C18H19ClFN3 (M + H)+, 332.1324; found, 332.1321.
(E)-1-(4-Chlorophenyl)-N-((4-cyanophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (I-18):
Yield 39%, Mp: 180–182 ◦C. 1H-NMR (400 MHz, D2O):
δ 3.35 (br, 2H, piperazinyl-H), 3.62 (br, 4H,
piperazinyl-H), 4.16 (br, 2H, piperazinyl-H), 4.37 (s, 2H, -CH2-), 7.01 (d, 2H, J = 8.4 Hz, ArH), 7.34 (d,
2H, J = 8.4 Hz, ArH), 7.58 (d, 2H, J = 8.7 Hz, ArH), 7.97 (d, 2H, J = 8.7 Hz, ArH). HRMS calcd for
C19H19ClN4 (M + H)+, 339.1371; found, 339.1368.
(E)-1-(4-Chlorophenyl)-N-(◦(3-cyanophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (I-19):
1
Yield 26%, Mp: 156–158 C. H-NMR (400 MHz, D2O):
δ 3.21 (br, 2H, piperazinyl-H), 3.49 (br,4H,
piperazinyl-H), 4.03 (br, 2H, piperazinyl-H), 4.21 (d, 1H, J = 15.9 Hz, -CH0-), 4.29 (d, 1H, J = 15.9 Hz,
00
-CH -), 6.85 (d, 2H, J = 8.7 Hz, ArH), 7.21 (d, 2H, J = 8.7 Hz, ArH), 7.56–7.67 (m, 3H, ArH), 7.93 (d, 1H,
J = 7.8 Hz, ArH). HRMS calcd for C19H19ClN4 (M + H)+, 339.1371; found, 339.1366.
(E)-1-(4-Chlorophenyl)-N-(piperazin-1-yl(p-tolyl)methylene)methanamine hydrochloride (I-20): Yield 57%,
Mp: 183–185 ◦C. 1H-NMR (400 MHz, D2O):
δ 2.41 (s, 3H, -CH3), 3.32 (br, 2H, piperazinyl-H), 3.58 (br,
2H, piperazinyl-H), 3.63 (br, 2H, piperazinyl-H), 4.11 (br, 2H, piperazinyl-H), 4.40 (s, 2H, -CH2-),
7.04 (d, 2H, J = 8.1 Hz, ArH), 7.27 (d, 2H, J = 8.4 Hz, ArH), 7.35 (d, 2H, J = 8.4 Hz, ArH), 7.43 (d, 2H,
J = 8.1 Hz, ArH). HRMS calcd for C19H22ClN3 (M + H)+, 328.1575; found, 328.1574.
(E)-1-(4-Methoxyphenyl)-N-((4-nitrophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (I-21):
Yield 15%, Mp: 210–212 ◦C. 1H-NMR (400 MHz, D2O):
δ 3.33 (br, 2H, piperazinyl-H), 3.59 (br, 4H,
piperazinyl-H), 3.81 (s, 3H, -OCH3), 4.15 (br, 2H, piperazinyl-H), 4.35 (s, 2H, -CH2-), 6.90 (d, 2H,
J = 8.7 Hz, ArH), 6.97 (d, 2H, J = 8.7 Hz, ArH), 7.66 (d, 2H, J = 9.3 Hz, ArH), 8.42 (d, 2H, J = 9.3 Hz,
ArH). HRMS calcd for C19H22N4O3 (M + H)+, 355.1765; found, 355.1766.

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(E)-1-(Naphthalen-1-yl)-N-(phenyl(piperazin-1-yl)methylene)methanamine hydrochloride (II-1): Yield 34%,
Mp: 187–189 ◦C. 1H-NMR (400 MHz, D2O):
3.33 (t, 2H, J = 5.4 Hz, piperazinyl-H), 3.54–3.68 (m, 4H,
δ
piperazinyl-H), 4.12 (t, 2H, J = 5.4 Hz, piperazinyl-H), 4.82 (s, 2H, -CH2-), 7.22 (d, 1H, J = 7.2 Hz, ArH),
7.40–7.44 (m, 3H, ArH), 7.51–7.57 (m, 4H, ArH), 7.60–7.68 (m, 2H, ArH), 7.87 (d, 1H, J = 8.1 Hz, ArH),
7.87 (d, 1H, J = 9.0 Hz, ArH). HRMS calcd for C22H23N3 (M + H)+, 330.1965; found, 330.1962.
(E)-1-(Naphthalen-1-yl)-N-((4-nitrophenyl)(piperazin-1-yl)methylene)methanamine hydrochloride (II-2):
◦
1
Yield 11%, Mp: 202–204 C. H-NMR (400 MHz, D2O):
δ 3.34 (br, 2H, piperazinyl-H), 3.60 (br,
4H, piperazinyl-H), 4.21 (br, 2H, piperazinyl-H), 4.84 (s, 2H, -CH2-), 7.20 (d, 1H, J = 6.9 Hz, ArH),
7.38–7.43 (m, 1H, ArH), 7.46–7.53 (m, 2H, ArH), 7.56–7.61 (m, 3H, ArH), 7.84–7.89 (m, 2H, ArH), 8.17 (d,
2H, J = 9.0 Hz, ArH). HRMS calcd for C22H22N4O2 (M + H)+, 375.1816; found, 375.1814.
(E)-N-((4-Fluorophenyl)(piperazin-1-yl)methylene)-1-(naphthalen-1-yl)methanamine hydrochloride (II-3):
◦
1
Yield 24%, Mp: 193–195 C. H-NMR (400 MHz, D2O):
δ 3.36 (br, 2H, piperazinyl-H), 3.58 (br,2H,
piperazinyl-H), 3.65 (t, 2H, J = 4.2 Hz, J = 5.7 Hz, piperazinyl-H), 4.12 (t, 2H, J = 5.1 Hz, J = 5.4 Hz,
piperazinyl-H), 4.87 (s, 2H, -CH2-), 7.22–7.28 (m, 3H, ArH), 7.41–7.48 (m, 3H, ArH), 7.52–7.60 (m, 2H,
ArH), 7.65–7.68 (m, 1H, ArH), 7.91 (d, 1H, J = 8.4 Hz, ArH), 7.95–7.98 (m, 1H, ArH). HRMS calcd for
C22H22FN3 (M + H)+, 348.1871; found, 348.1873.
(E)-N-((2-Fluorophenyl)(piperazin-1-yl)methylene)-1-(naphthalen-1-yl)methanamine hydrochloride (II-4):
◦
1
Yield 41%, Mp: 148–150 C. H-NMR (400 MHz, D2O):
δ 3.33 (br, 2H, piperazinyl-H), 3.56 (br,2H,
piperazinyl-H), 3.67 (br, 2H, piperazinyl-H), 4.12–4.21 (m, 2H, piperazinyl-H), 4.92 (s, 2H, -CH2-),
7.12 (br, 1H, ArH), 7.23–7.29 (m, 1H, ArH), 7.33–7.46 (m, 3H, ArH), 7.56 (br, 2H, ArH), 7.64–7.72 (m,
2H, ArH), 7.88–8.05 (m, 2H, ArH). HRMS calcd for C22H22FN3 (M + H)+, 348.1871; found, 348.1868.
(E)-N-((2-Chlorophenyl)(pip◦erazin-1-yl)methylene)-1-(naphthalen-1-yl)methanamine hydrochloride (II-5):
1
Yield 58%, Mp: 165–167 C. H-NMR (400 MHz, D2O):
δ
3.34 (br, 2H, piperazinyl-H), 3.63 (br,
4H, piperazinyl-H), 4.15 (br, 2H, piperazinyl-H), 4.89 (s, 2H, -CH2-), 7.07 (d, 1H, J = 6.6 Hz, ArH),
7.37–7.49 (m, 3H, ArH), 7.55–7.65 (m, 4H, ArH), 7.78–7.81 (m, 1H, ArH), 7.91 (d, 1H, J = 8.7 Hz, ArH),
7.98–8.04 (m, 1H, ArH). HRMS calcd for C22H22ClN3 (M + H)+, 364.1575; found, 364.1574.
(E)-N-((3-Chlorophenyl)(pi◦perazin-1-yl)methylene)-1-(naphthalen-1-yl)methanamine hydrochloride (II-6):
1
Yield 34%, Mp: 198–200 C. H-NMR (400 MHz, D2O):
δ 3.31 (br, 2H, piperazinyl-H), 3.65 (br,4H,
piperazinyl-H), 4.12 (br, 2H, piperazinyl-H), 4.78 (s, 2H, -CH2-),7.16 (d, 1H, J = 6.9 Hz, ArH),
7.29–7.32 (m, 2H, ArH), 7.39–7.62 (m, 6H, ArH), 7.87 (d, 1H, J = 8.4 Hz, ArH), 7.93 (d, 1H, J = 6.9 Hz,
ArH). HRMS calcd for C22H22ClN3 (M + H)+, 364.1575; found, 364.1576.
(E)-N-((4-Chlorophenyl)(piperazin-1-yl)methylene)-1-(naphthalen-1-yl)methanamine hydrochloride (II-7):
1
Yield 41%, Mp: 210–212 ◦C. H-NMR (400 MHz, D2O):
δ 3.19 (t, 2H, J = 5.1 Hz, J = 5.1 Hz,
piperazinyl-H), 3.43 (t, 2H, J = 6.9 Hz, J = 3.9 Hz, piperazinyl-H), 3.50 (t, 2H, J = 5.7 Hz, piperazinyl-H),
4.12 (t, 2H, J = 6.0 Hz, piperazinyl-H), 4.75 (s, 2H, -CH2-), 7.09 (d, 1H, J = 6.6 Hz, ArH), 7.20 (d,
2H, J = 8.4 Hz, ArH), 7.30–7.35 (m, 1H, ArH), 7.38 (d, 2H, J = 8.4 Hz, ArH), 7.41–7.54 (m, 3H, ArH),
7.76–7.85 (m, 2H, ArH). HRMS calcd for C22H22ClN3 (M + H)+, 364.1575; found, 364.1577.
(E)-4-(((Naphthalen-1-ylmethyl)imino)(piperazin-1-yl)methyl)benzonitrile hydrochloride (II-8): Yield 30%,
◦
Mp: 158–160 C. 1H-NMR (400 MHz, D2O):
δ 3.34 (br, 2H, piperazinyl-H), 3.63 (br,4H, piperazinyl-H),
4.18 (br, 2H, piperazinyl-H), 4.84 (s, 2H, -CH2-), 7.19 (d, 1H, J = 7.2 Hz, ArH), 7.40–7.63 (m, 6H, ArH),
7.77 (d, 2H, J = 8.4 Hz, ArH), 7.88 (d, 1H, J = 8.1 Hz, ArH), 7.94 (d, 1H, J = 9.0 Hz, ArH). HRMS calcd
for C23H22N4 (M + H)+, 355.1917; found, 355.1921.

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(E)-3-(((Naphthalen-1-ylmethyl)imino)(piperazin-1-yl)methyl)benzonitrile hydrochloride (II-9): Yield 11%,
◦
Mp: 168–170 C. 1H-NMR (400 MHz, D2O):
δ 3.16 (br, 2H, piperazinyl-H), 3.45 (br,4H, piperazinyl-H),
0
00
4.04 (br, 2H, piperazinyl-H), 4.72 (d, 1H, J = 13.5 Hz, -CH -), 4.78 (d, 1H, J = 13.5 Hz, -CH -), 6.85 (d, 1H,
J = 5.7 Hz, ArH), 7.01–7.05 (m, 2H, ArH), 7.24–7.29 (m, 2H, ArH), 7.35–7.52 (m, 3H, ArH), 7.73–7.90 (m,
3H, ArH). HRMS calcd for C23H22N4 (M + H)+, 355.1917; found, 355.1911.
(E)-1-(Naphthalen-1-yl)-N-(piperazin-1-yl(p-tolyl)methylene)methanamine hydrochloride (II-10): Yield 49%,
◦
Mp: 178–180 C. 1H-NMR (400 MHz, D2O):
δ 2.38 (s, 3H, -CH3), 3.19 (t, 2H, J = 5.4 Hz, piperazinyl-H),
3.42 (t, 2H, J = 5.7 Hz, piperazinyl-H), 3.51 (t, 2H, J = 5.4 Hz, piperazinyl-H), 3.96 (t, 2H, J = 5.4 Hz,
piperazinyl-H), 4.74 (s, 2H, -CH2-), 7.10–7.15 (m, 3H, ArH), 7.21 (d, 2H, J = 8.1 Hz, ArH), 7.30–7.35 (m,
1H, ArH), 7.37–7.44 (m, 2H, ArH), 7.46–7.52 (m, 1H, ArH), 7.77 (d, 1H, J = 8.1 Hz, ArH), 7.83 (d, 1H,
J = 9.3 Hz, ArH). HRMS calcd for C23H25N3 (M + H)+, 344.2121; found, 344.2108.
(E)-N-((5-Nitronaphthalen-1-yl)(piperazin-1-yl)methylene)-1-phenylmethanamine hydrochloride
Yield 14%, Mp: 173–175 ◦C. 1H-NMR (400 MHz, D2O):
3.14–3.26 (m, 2H, piperazinyl-H),
3.51—3.57 (m, 2H, piperazinyl-H), 3.71 (br, 2H, piperazinyl-H), 4.34 (br, 2H, piperazinyl-H), 4.23 (d,
(III-1):
δ
0
00
1H, J = 15.0 Hz, -CH -), 4.36 (d, 1H, J = 15.0 Hz, -CH -), 6.73 (d, 2H, J = 6.6 Hz, ArH), 7.06–7.16 (m, 3H,
ArH), 7.59 (t, 1H, J = 7.8 Hz, ArH), 7.80–7.86 (m, 2H, ArH), 7.94 (t, 1H, J=7.2Hz, ArH), 7.29 (d, 1H,
J = 7.8 Hz, ArH), 8.74 (d, 1H, J = 8.7 Hz, ArH). HRMS calcd for C22H22N4O2 (M + H)+, 375.1816; found,
375.1815.
3.2. Pharmacological Method
3.2.1. Inhibition of 5-HT or NE Reuptake Activity in Rat Brain Synaptosome
All animal experiments were conducted in compliance with the Care and Use of Laboratory
Animals, with the approval of Peking Union Medical College and Chinese Academy of Medical
Sciences’ Animal Studies Committee (Project code, 7102116; October 2010). Adult male Wistar rats
were used. Animals were killed by decapitation and the whole brain, with the exception of the
brainstem and cerebellum, was quickly removed. The procerebrum region was prepared, weighed,
and homogenized in 10 volumes of ice-cold 0.32 mol/L sucrose solution using a glass homogenizer.
The homogenate was centrifuged at 1000
used for the uptake experiments. In the assay, the tissue suspension (200
50 nmol/L 3H-NE or 3H-5-hydroxytryptamine (800
L) in Krebs-Henseleit bicarbonate buffer and a
L; 1
10−4, 1 10−5, 1 10−6, 1 10−7
×
g and 4 ◦C for 10 min. The supernatant was decanted and
L) was incubated with
µ
µ
solution of the drug at the appropriate conc◦entration (20
with the vehicle (20 µ
L) at 0 ◦C in an ice bath. After incubation, the tubes were immediately filtered
µ
×
×
×
×
,
−8
1
×
10 mol/L) or the vehicle (20
µ
L) at 37 C for 10 min. For each assay, three tubes were incubated
with a cell harvester. The fiberglass filter membrane was washed three times with ice-cold saline,
placed in a scintillation vial, and counted in liquid scintillation cocktail (4 mL). Active uptake was
◦
the difference between the counts per minute at 37 and 0 C. The percent inhibition at each drug
concentration was the mean of three measurements [15].
3.2.2. TST in Rats
Rats were administered the vehicle as a negative control, duloxetine (30 mg/kg) as a positive
control, or test compounds I-19, II-4, and II-5 (30 mg/kg). For two days before the formal experiment,
the rats in each group were given doses orally once a day in the morning and in the afternoon. The rats
were given doses orally once in each group in the formal experiment. After 60 min of administration,
the rats were suspended head-down more than 50 cm from the ground with a 12-cm-long piece of
insulating tape at a distance of 1 cm from the tail tip. The distance between adjacent rats was 15 cm.
The immobility time of the rats was recorded. The rate of change in the immobility time of the rats in
the test drug group and the positive control group was calculated and compared with the negative

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control group. Statistical analysis was performed using Prism 7.0 (GraphPad Software, San Diego, CA,
USA). The data of the immobility time of vehicle, duloxetine, I-19, II-4, and II-5 were analyzed by the
One-way ANOVA.
3.2.3. Acute Toxicity Test
Male ICR mice (20–25 g) were used from Institute of Materia Medica, Chinese Academy of Medical
Sciences, Beijing, China. Mice were randomly divided into two groups with six mice each. Mice were
orally given II-5 with a single dose 200 and 400 mg/kg or vehicle control, respectively. The mouse
death was monitored for 20 h after treatment.
4. Conclusions
In this study, we designed and synthesized three series of arylamidine derivatives to discover
potent reuptake inhibitors of 5-HT and NE transporters. Our results showed that series II compounds
with a large aromatic ring exhibited improved in vitro 5-HT and NE inhibitory activity, and compound
II-5 was the most potent dual inhibitor (5-HT, IC50 = 620 nM; NE, IC50 = 10 nM). Compounds
I-19, II-4, and II-5 were selected for TST profiling in rats to test the in vivo antidepressant effect.
These three compounds reduced the immobility time in the TST, indicating in vivo antidepressant
activity. Compound II-5 showed the most potent in vivo antidepressant activity and had an acceptable
safety profile. These arylamidine derivatives are interesting compounds to explore further as potential
antidepressant drug candidates.
Author Contributions: H.W. and Y.G. conceived and wrote the paper; H.W., W.Q., and G.Y. performed the
experiments and analyzed the data.
Funding: This work was financially supported by Beijing Natural Science Foundation of China (7102116).
Conflicts of Interest: The authors declare no conflict of interest.
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