Synthesis and evaluation of novel 2,3-dihydrobenzo[b][1,4]dioxin- and indolealkylamine derivatives as potential antidepressants

Article abstract of DOI:10.1002/ardp.201300238

A series of 2,3-dihydrobenzo[b][1,4]dioxin- and indolealkylamine derivatives were synthesized and the target compounds were evaluated for their binding affinities at the 5-HT_1A receptor and serotonin transporter. Antidepressant-like activities of the compounds were screened using the tail suspension and forced swim tests in mice. Preliminary results indicated that the target compounds exhibited high binding affinities at the 5-HT_1A receptor and serotonin transporter, and produced marked antidepressant-like effects. The best example from this study, compound 5, exhibited high binding affinities for the 5-HT_1A receptor (K_i = 96 nM) and serotonin transporter (K_i = 9.8 nM). The intrinsic activity of compound 5 showed agonistic property to the 5-HT_1A receptor and inhibition of the 5-HT transporter. Furthermore, compound 5 exhibited greater antidepressant efficacy than fluoxetine and showed acceptable pharmacokinetic properties. A series of 3-(2-(4-((2,3-dihydrobenzo[b][1,4]dioxin-3-yl)methyl) piperazin-1-yl)ethyl-1H-indole derivatives were synthesized and the target compounds were evaluated for their antidepressant activities in vitro and in vivo. The compounds described were also evaluated for dual 5-HTT and 5-HT _1A receptor activities.

Full text of DOI:10.1002/ardp.201300238

32
Arch. Pharm. Chem. Life Sci. 2014, 347, 32–41
Full Paper
Synthesis and Evaluation of Novel 2,3-Dihydrobenzo[b][1,4]-
dioxin- and Indolealkylamine Derivatives as Potential
Antidepressants
Songlin Wang1, Yin Chen2, Xinghua Liu2, Xiangqing Xu2, Xin Liu1, Bi-Feng Liu1, and
Guisen Zhang1, 2
1
Department of Biomedical Engineering, Systems Biology Theme, College of Life Science and Technology,
Huazhong University of Science and Technology, Wuhan, China
Jiangsu Nhwa Pharmaceutical Co., Ltd., Xuzhou, Jiangsu, China
2
A series of 2,3-dihydrobenzo[b][1,4]dioxin- and indolealkylamine derivatives were synthesized and the
target compounds were evaluated for their binding affinities at the 5-HT1A receptor and serotonin
transporter. Antidepressant-like activities of the compounds were screened using the tail suspension
and forced swim tests in mice. Preliminary results indicated that the target compounds exhibited
high binding affinities at the 5-HT1A receptor and serotonin transporter, and produced marked
antidepressant-like effects. The best example from this study, compound 5, exhibited high binding
affinities for the 5-HT1A receptor (Ki ¼ 96 nM) and serotonin transporter (Ki ¼ 9.8 nM). The intrinsic
activity of compound 5 showed agonistic property to the 5-HT1A receptor and inhibition of the 5-HT
transporter. Furthermore, compound 5 exhibited greater antidepressant efficacy than fluoxetine and
showed acceptable pharmacokinetic properties.
Keywords: Antidepressants / 2,3-Dihydrobenzo[b][1,4]dioxin / Dual 5-HT1A/5-HT transporter activity /
Indolealkylamine
Received: June 22, 2013; Revised: September 1, 2013; Accepted: September 5, 2013
DOI 10.1002/ardp.201300238
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
Recent research showed that the effects of the treatment
with SSRIs can be augmented by concomitant use of a 5-HT1A
antagonist. Several groups have already reported their
strategies for creation of a single molecular entity combining
both 5-HT1A receptor antagonist and 5-HT transporter (5-HTT)
inhibitor [5–10]. Another possibility is that the effects of
an SSRI can be augmented by the coadministration of a 5-HT1A
agonist. Buspirone, a 5-HT1A receptor partial agonist, can
be an effective augmentation agent alongside treatment
with SSRIs [11]. The agents with the 5-HT1A receptor agonist
and the 5-HTT inhibitor properties could produce the
same therapeutic benefits, which is evidenced not only by
the number of scientific studies but also by the fact that some
of them are currently used for treatment of depression [12,
13]. For example, vilazodone acting as a 5-HT reuptake
inhibitor and 5-HT1A receptor partial agonist could produce
more efficacious antidepressant therapeutic benefits [14, 15].
Selective serotonin reuptake inhibitors (SSRIs) such as
fluoxetine, citalopram, paroxetine, and sertraline have
achieved great success in the treatment of depression. These
antidepressants have fewer side effects than either tricyclic
antidepressants or non-selective monoamine oxidase inhib-
itors. However, there are some limitations associated with the
use of SSRIs including a long onset of action and moderate
patient response [1–4], which led to the idea to develop a new
generation of more efficacious antidepressant drugs.
Correspondence: Dr. Guisen Zhang, Department of Biomedical Engi-
neering, Systems Biology Theme, College of Life Science and Technology,
Huazhong University of Science and Technology, 1037 Luoyu Road,
Wuhan 430074, China.
E-mail: gszhang@mail.hust.edu.cn
Fax: þ86 27 87792170
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2014, 347, 32–41
Dihydrobenzodioxin and Indolealkylamine Derivatives as Antidepressants
33
Several researches have revealed the agents containing an
aryloxyethylamine and a 3-indolealkyamine linked through a
common basic nitrogen as having potent binding affinities
for both the 5-HT1A receptor and 5-HTT [17–21]. As a similar
strategy, we designed a series of derivatives containing
a 1-((2,3-dihydrobenzo[b][1,4]dioxin-3-yl)methyl)piperazine
moiety and a straight chain aminoalkyl indole moiety
linked through a nitrogen (Fig. 1). Aminoalkyl indole
moieties have been used successfully to produce 5-HTT
pharmacophore moieties [22], while benzodioxanmethyl-
piperazine moieties have served as 5-HT1A pharmacophore
moieties. We report here the synthesis and behavioral
investigation of new compounds as novel and potential
antidepressants, characterized by potent binding affinities
for the 5-HT1A receptor and 5-HTT. Compounds were also
investigated in vivo in mice using the tail suspension test
(TST) and the forced swimming test (FST). Among them,
compound 5 was chosen for further biological investiga-
tion. The results revealed that compound 5 validated our
approach to potential antidepressants.
Figure 1. Design strategy and structure of 3-(2-(4-((2,3-
dihydrobenzo[b][1,4]dioxin-3-yl)methyl)piperazin-1-yl)ethyl)-1H-in-
dole derivatives.
Results and discussion
Therefore, the concept of developing a dual-acting agent
modulating both the 5-HT1A receptor and 5-HTT in one
molecule has been proven practicable for the discovery of
novel antidepressants [16].
Chemistry
The general methods used for synthesis of compounds 1–24
are summarized in Scheme 1. 4-Substituted phenylhydrazine
hydrochloride derivatives were reacted with 2,3-dihydrofuran
Scheme 1. Reagents and conditions: (a) 2,3-dihydrofuran or 3,4-dihydro-2H-pyran, 4% H2SO4, DMAc, 100°C; (b) CBr4, triphenylphosphine,
CH3CN, r.t.; (c) NaBH4, TiCl4, 1,2-dimethoxyethane, 0°C; (d) 4-toluene sulfonyl chloride, pyridine, r.t.; (e) piperazine, ethanol, refluxed;
(f) 2a–h, K2CO3, acetone, refluxed.
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34
S. Wang et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 32–41
or 3,4-dihydro-2H-pyran in dilute sulfuric acid and
N,N-dimethylacetamide at 100°C to give 3-substituted indoles
(1a–h) [23]. The 3-substituted indoles were brominated with
carbon tetrabromide to obtain 3-bromoalkylindole (2a–h) [16].
The combined reagent TiCl4/NaBH4 was used to reduce methyl
3-substituted 1,4-benzodioxan-2-carboxylate to alcohols
(3a–c). Reaction of the alcohols (3a–c) with 4-toluene sulfonyl
chloride generated the 2-tosyloxymethyl-1,4-benzodioxan
derivatives (4a–c). Reaction of the 2-tosyloxymethyl-1,4-
benzodioxan derivatives with excess piperazine afforded
the piperazine derivatives (5a–c) [24]. Treatment of equimolar
quantities of 3-bromoalkylindoles (2a–h) with the piperazine
derivatives (5a–c) resulted in the formation of the title
compounds (1–24).
Ki ¼ 8.8 nM; compound 5, Ki ¼ 9.8 nM) and the 5-HT1A receptor
(compound 5, Ki ¼ 96 nM). The 5-substituent derivatives
exhibited excellent binding affinity at the 5-HTT (compound
2, Ki ¼ 5.1 nM; compound 6, Ki ¼ 4.8 nM) and moderate to
weak affinity for the 5-HT1A receptor. The 5-unsubstituted
derivative had slightly higher affinity for the 5-HT1A receptor
when tethered by a two-carbon chain (Ki of 96 nM for
compound 5 vs. 155 nM for compound 1). However, the
5-substituted derivatives had little effect on the binding
affinities at the 5-HT1A receptor and the 5-HTT, when tethered
by a two-carbon side chain (compounds 1–4 vs. compounds
5–8). These results revealed that compounds 1–8 exhibited
high affinity for the 5-HTT and the 5-fluoro derivatives had the
highest affinity for the 5-HTT. Unfortunately, introduction of a
substitution on the indole moiety led to a significant decrease
in the binding affinity at the 5-HT1A receptor (compounds 2–4
vs. compound 1; compounds 6–8 vs. compound 5). As
depicted in Table 1, compounds 1, 2, and 5 exhibited
excellent binding affinity at the 5-HTT and moderate binding
affinity at the 5-HT1A receptor.
In vitro studies
Compounds were evaluated in vitro to determine their binding
affinities for the 5-HTT and the 5-HT1A receptors. According to
Table 1, the 5-unsubstituent derivatives (among compounds
1–8) had potent affinities at the 5-HTT (compound 1,
Table 1. Binding affinities for the 5-HTT, 5-HT1A, 5-HT2A, 5-HT2C, a1, a2, and D2 for compounds 1–24.
Binding data
Kia) (mean ꢀ SEM), nM
Percentage of inhibitionb), 10 mM
Compounds
n
R1
R2
5-HTT
5-HT1A
5-HT2A (%)
5-HT2C (%)
a1
a2
D2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2
2
2
2
1
1
1
1
2
2
2
2
1
1
1
1
2
2
2
2
1
1
1
1
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
F
Cl
CH3
H
8.8 ꢀ 0.6
5.1 ꢀ 0.5
6.6 ꢀ 0.4
9.2 ꢀ 0.9
9.8 ꢀ 1.6
4.8 ꢀ 0.2
8.9 ꢀ 0.2
22 ꢀ 1.8
155 ꢀ 13
208 ꢀ 11
597 ꢀ 43
1691 ꢀ 324
96 ꢀ 15
72
79
85
77
77
65
85
73
80
81
71
65
86
76
76
70
57
72
79
85
80
77
65
85
43
52
37
47
39
16
65
44
61
35
53
42
66
36
34
11
39
26
53
31
64
38
28
49
1442 ꢀ 217a)
1056 ꢀ 58a)
3102 ꢀ 154a)
34%
66%
64%
74%
74%
64%
54%
73%
60%
34%
36%
n.a.
5%
21%
33%
17%
26%
27%
n.a.
11%
9%
n.a.
17%
34%
8%
22%
27%
31%
15%
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
1477 ꢀ 214a)
F
568 ꢀ 65
577 ꢀ 58
1732 ꢀ 332
885 ꢀ 64
602 ꢀ 45
2263 ꢀ 311
1815 ꢀ 203
454 ꢀ 85
40 ꢀ 9
27%
29%
36%
Cl
CH3
H
71 ꢀ 6.2
32%
F
23 ꢀ 1.3
1116 ꢀ 203a)
Cl
CH3
H
147 ꢀ 1.8
495 ꢀ 12
5.7 ꢀ 0.2
1.7 ꢀ 0.1
450 ꢀ 65
634 ꢀ 107
15.6 ꢀ 2.3
35.7 ꢀ 4.4
57.6 ꢀ 6.5
74.0 ꢀ 8.3
125 ꢀ 13
217 ꢀ 29
481 ꢀ 31
709 ꢀ 45
9.2 ꢀ 0.1
9%
24%
2349 ꢀ 109a)
16%
6%
F
34%
Cl
CH3
H
1033 ꢀ 156
1269 ꢀ 182
115 ꢀ 12
533 ꢀ 31
58%b)
23%
19%
28%
31%
n.a.
35%
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
F
Cl
CH3
H
F
Cl
CH3
21%
1216 ꢀ 241
39%
82 ꢀ 3
850 ꢀ 86a)
201 ꢀ 18
65%b)
36%
20%
22%
794 ꢀ 24
Fluoxetine
8-OH-DPAT
7 ꢀ 0.1
n.d: no activity.
a)
Ki ¼ IC50/(1 þ C/Kd).
b)
Percentage of inhibition (%) ¼ (total binding ꢁ specific binding) ꢂ 100%/(total binding ꢁ non-specific binding).
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Arch. Pharm. Chem. Life Sci. 2014, 347, 32–41
Dihydrobenzodioxin and Indolealkylamine Derivatives as Antidepressants
35
Next, we investigated the effects of introduction of a methyl
group on the 1,4-dioxane ring (compounds 9–16). The 5-fluoro
derivatives showed high binding affinities at both the 5-HTT
and 5-HT1A receptors. The 5-chloro (compounds 11 and 15) and
5-methyl derivatives (compounds 12 and 16) displayed weak
binding affinity at the 5-HT1A receptor and moderate binding
affinity for the 5-HTT. The 5-unsubstituent derivatives showed
potent affinity for the 5-HTT. In terms of the carbon chain
length of the aminoalkyl indole, the derivatives tethered by a
two-carbon chain exhibited increased binding affinity at the
5-HT1A receptor (compounds 9–12 vs. compounds 13–16).
When tethered by a two-carbon chain, the 5-unsubstituent
and 5-fluoro derivatives showed increased binding affinities at
both the 5-HTT and the 5-HT1A receptor (compound 9 vs.
compound 13; compound 10 vs. compound 14). Finally,
compound 14 had excellent binding affinities for both the
5-HTT (Ki ¼ 1.7 nM) and 5-HT1A receptor (Ki ¼ 40 nM).
pharmacological models are commonly used for evaluation of
antidepressant activities.
The potential antidepressant activities of these compounds
were assayed at a dose of 80 mg/kg in the TST in mice. As
shown in Table 2, compounds 5, 14, and 17 showed notable
antidepressant activities. Compounds 5, 14, 17, and fluoxetine
significantly decreased the immobility time by 63.4%, 54.4%,
41.7%, and 54.8%, respectively. Compound 5 was more
effective than compounds 14, 17, and fluoxetine.
Furthermore, studies of the active compounds at doses of
10, 20, and 40 mg/kg revealed that compounds 5 and 14
produced a distinct antidepressant-like effect in the TST
(Table 3). Compounds 5, 14, and fluoxetine had their ED50 at
doses of 18.6, 41.4, and 49.1 mg/kg, respectively.
The ED50 dose of compound 5 was significantly lower than
that of compound 14. Compound 5 was selected for further
Introduction of a phenyl on the 1,4-dioxane ring (com-
pounds 17–24) decreased the binding affinity for the 5-HTT.
Substitution on the indole moiety led to significant decrease
in binding affinities at both the 5-HTT and 5-HT1A receptors
(compounds 18–20 vs. compounds 17, 22–24 vs. compound
21). In terms of carbon chain length, the derivatives tethered
by a two-carbon chain were found to have the decreased
binding affinity at the 5-HTT and the increased binding
affinity at the 5-HT1A receptor (compounds 17–20 vs.
compounds 21–24). Finally, compound 17 showed potent
binding affinity at the 5-HTT (Ki ¼ 15.6 nM) and moderate
affinity at the 5-HT1A receptor (Ki ¼ 115 nM).
Table 2. The effects of compounds in the tail suspension test in
mice.
Immobility time (s)
Compounds
Dose (mg/kg, po)
Mean ꢀ SEM
Vehicle
Fluoxetine
1
2
5
14
17
–
108
80
80
80
80
80
80
48.8 ꢀ 4.2a)
93.2 ꢀ 3.5
95.5 ꢀ 4.4
39.5 ꢀ 4.2b)
49.2 ꢀ 5.3a)
63.0 ꢀ 4.1a)
Results are represented as mean ꢀ SEM with n ¼ 10 in each group.
Values are significant at a)p < 0.05 and b)p < 0.01, when compared
with vehicle group.
Several researches have revealed that the effects of the
treatment with SSRIs can be augmented by concomitant
use of other monoaminergic receptor ligands, such as the
5-HT2A [25, 26], 5-HT2C [27], a2-adrenergic receptors [28, 29],
and the D2 receptor [30]. Compounds were evaluated to
determine their binding affinities for these monoaminergic
receptors (see in Supporting Information). As shown in
Table 1, compounds 1–24 exhibited weak affinity or no
activity at these monoaminergic receptors. Additionally, the
receptor binding pharmacology for the 5-HT1A receptor
exhibits significant overlap with the a1-adrenergic recep-
tor [31]. The binding affinity for the a1-adrenergic receptor
was also determined. All the compounds demonstrated good
selectivity over the a1-adrenergic receptor. Thus, it seems
evident that the antidepressant effects should be mediated
by the 5-HT1A receptor and the 5-HTT.
Table 3. The effect of compounds 5 and 14 given orally at graded
doses in the tail suspension test in mice.
Immobility time (s)
Dose
Compounds
(mg/kg, po)
Mean ꢀ SEM
Vehicle
Fluoxetine
–
92.2
10
20
40
–
63.3 ꢀ 4.1a)
61.3 ꢀ 3.8a)
57.5 ꢀ 4.2a)
95.6
Vehicle
5
10
20
40
10
20
40
50.5 ꢀ 5.2a)
48.5 ꢀ 3.3b)
28.9 ꢀ 4.6b)
69.3 ꢀ 3.9a)
57.4 ꢀ 4.5a)
54.4 ꢀ 6.3a)
In vivo studies
14
Compounds 1, 2, 5, 14, and 17 exhibited high binding affinity
at the 5-HT1A receptor with Ki values of 40–208 nM, and
excellent binding affinity at the 5-HTT with Ki values of 1.7–
15.6 nM. These compounds were evaluated further in vivo
using the TST, the FST, and the locomotor activity. These
Results are represented as mean ꢀ SEM with n ¼ 10 in each group.
Values are significant at a)p < 0.05 and b)p < 0.001, when
compared with vehicle group.
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S. Wang et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 32–41
investigation in vivo in the FST. As shown in Table 4,
compound 5 at doses of 20, 40, and 80 mg/kg produced a
distinct antidepressant-like effect. These results also confirm
that compound 5 exhibited potential antidepressant-like
activity.
Locomotor activity test was conducted to distinguish the
false positive effects in the animal models. To check possible
occurrence of drug-induced changes in the locomotor activity,
which may have contributed to behavior in the TST and the
FST, the influence of compound 5 on locomotor activity was
examined. Compound 5 showed a sedative effect in mice
without the production of hyperactivity, when the locomotor
activity was recorded for 10 min (Table 5).
Table 6. The intrinsic activities the 5-HT1A receptor and the 5-HT
transporter.
5-HT1A receptor
5-HT transporter
Activation
EC50 value
Inhibition
IC50 value
(nM)
Inhibition
IC50, value
(nM)
Compounds
(nM)
5
5-HT
WAY-100635
Fluoxetine
127.0 ꢀ 12.5
–
40.9 ꢀ 3.4
1.98 ꢀ 0.3
–
1.64 ꢀ 0.2
–
–
–
–
–
35.5 ꢀ 3.1
Result are represented as mean ꢀ SEM with n ¼ 3 in each group.
The TST and the FST showed that compounds had
significant antidepressant-like activities, as we hypothesized.
Compound 5, as a result, may have potential in pharmaco-
therapy for the treatment of depressive disorders.
significant activation threshold in an agonist assay on the
5-HT1A receptor (EC50 ¼ 127 nM) and inhibited the 5-HT
transporter with a high activity (IC50 ¼ 40.9 nM). The results
indicate that compound 5 acts as 5-HT1A receptor agonist and
5-HTT inhibitor.
Intrinsic activity of compound 5 at 5-HT1A receptor and
serotonin transporter
The intrinsic activity of compound 5 was selected for further
investigation. The assays were carried out according to the
previously published procedure [32, 33] (see in Supporting
Information). As shown in Table 6, compound 5 showed
Ether-a-gogo-related gene (hERG) KC channels
Investigation of hERG channel blockade is an important step
in the drug discovery trajectory in the pharmaceutical
industry. Patch-clamp technique method was used for in vitro
assay for hERG inhibition (see in Supporting Information).
Compound 5 showed relatively moderate inhibition against
hERG and the IC50 value of 1.4 mM, which is only slightly lower
than the reference; IC50 value for fluoxetine was reported to
be 3.1 mM [34]. The results indicate compound 5 was identified
to possess moderate hERG affinity.
Table 4. The effects of compound 5 and fluoxetine in the forced
swim test in mice.
Immobility time (s)
Dose
Compounds
(mg/kg, po)
Mean ꢀ SEM
Vehicle
Fluoxetine
5
–
109.9
80
20
40
80
53.7 ꢀ 5.6a)
52.6 ꢀ 3.8a)
45.6 ꢀ 4.5a)
33.5 ꢀ 3.2b)
Pharmacokinetic properties of compound 5
Compound 5 was selected based on its in vitro profile for in vivo
characterization. Supporting Information Table S1 highlights
the pharmacokinetic parameters of compound 5 in rat.
Intravenous administration to rats (10 mg/kg) resulted in
detectable plasma levels (t1/2 ¼ 4.1 h), and oral administration
to rats (30 mg/kg) resulted in a t1/2 of 5.0 h. The area under
the curve (AUC) value was 5200 mg h/L after intravenous
administration versus 6090 mg h/L after oral administration.
The Cmax value after oral dosing was 591 mg/L, and after
intravenous administration was 1660 mg/L. The Tmax value
after oral dosing was 4.7 h. These results confirm that
compound 5 is a promising candidate for antidepressant
activity with good oral bioavailability (F ¼ 39%).
Results are represented as mean ꢀ SEM with n ¼ 10 in each group.
Values are significant at a)p < 0.05 and b)p < 0.001, when
compared with vehicle group.
Table 5. Locomotor activities of the mice treated with compounds
(20 mg/kg) or vehicle were counted for 10 min by activity analyzer.
Dose
(mg/kg, po)
Compounds
Locomotor activity
Vehicle
Fluoxetine
5
–
955.2
20
20
40
843.5 ꢀ 325.9a)
684.5 ꢀ 337.5a)
394.6 ꢀ 243.3b)
Conclusion
In summary, 3-(2-(4-((2,3-dihydrobenzo[b][1,4]dioxin-3-yl)methyl)-
piperazin-1-yl)ethyl)-H-indole derivatives were synthesized (com-
pounds 1–24). The derivatives were considered to possess dual
Results are represented as mean ꢀ SEM with n ¼ 10 in each group.
Values are significant at a)p < 0.05 and b)p < 0.01, when compared
with control group.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 32–41
Dihydrobenzodioxin and Indolealkylamine Derivatives as Antidepressants
37
biological activities. In our work, compound 5 exhibited high
binding affinities at both the 5-HT1A receptor and 5-HTT. The
tests of intrinsic activity showed that compound 5 was a mixed
5-HT1A receptor agonist and 5-HTT inhibitor. The TST and FST
revealed that compound 5 possessed potential antidepressant
activity. Moreover, compound 5 exhibited acceptable pharma-
cokinetic properties and relatively moderate hERG affinity.
Our preliminary results are sufficiently promising to warrant
that compound 5 is being used as a tool to identify a
development candidate for promising antidepressant.
carboxylate in 20 mL anhydrous 1,2-dimethoxyethane was
added dropwise at 0°C. The reaction mixture was stirred at
room temperature for 6–14 h. Then the reaction was quenched by
the addition of water with ice cooling. The mixture was extracted
with ether (100 mL ꢂ 3). The extracts were washed with water and
dried. After evaporating to dryness under reduced pressure, the
residue was purified by column chromatography, eluting with
ethyl acetate/petroleum ether 50:50 v/v.
General procedure for the preparation of 2-tosyloxymethyl-
1,4-benzodioxan derivatives (4a–c)
A solution of 0.05 mol (2-substituted 2,3-dihydro-benzo[b][1,4]-
dioxin-3-yl)methanol (3a–c) and 0.05 mol p-TsCl in 60 mL anhy-
drous pyridine was added in a 150-mL flask at 0°C in an ice-water
bath. The reaction mixture was stirred 18 h at room temperature.
The solution was diluted with ether and washed with dilute
hydrochloric acid and water, and dried. After evaporating to
dryness under reduced pressure, the residue was purified by
column chromatography, eluting with ethyl acetate/petroleum
ether 1:99 v/v.
Experimental
General
1H NMR spectra are recorded at 400 MHz on a Varian Inova
Unity 200 spectrometer in CDCl3 or DMSO-d6 solution.
Chemical shifts are given in d values (ppm), using tetrame-
thylsilane (TMS) as the internal standard; coupling constants (J)
were given in Hz. Reagents were all of analytical grade or of
chemical purity. Electron impact mass spectra were obtained
on Agilent 1100 LC–MS. Column chromatographic purification
was carried out using silica gel. Melting points were determined
in open capillary tubes and are uncorrected. The purity of
the tested compounds was assessed by HPLC analysis and was
above 98%.
General procedure for the preparation of piperazine
derivatives (5a–c)
A solution of 0.05 mol 2-tosyloxymethyl-1,4-benzodioxan deriva-
tives (4a–c) and 0.1 mol piperazine in 100 mL ethanol was added
in a 250-mL flask. The reaction mixture was refluxed in an oil bath
for 36–48 h. The solvent was evaporated under reduced pressure.
The residue was suspended in 5% NaOH and extracted with CHCl3
(100 mL ꢂ 3). The extracts were washed with water and dried.
After evaporating to dryness under reduced pressure, the residue
was purified by column chromatography, eluting with ethyl
acetate/petroleum ether 10:90 v/v.
General procedure for the synthesis of 3-substituted
indoles (1a–h)
A solution of 0.05 mol 4-substituted phenylhydrazine hydro-
chloride in 75 mL 4% H2SO4 and 75 mL dimethylacetamide was
added in a 250-mL flask. The solution was heated to 100°C in an
oil bath. Then, 0.05 mol dihydrofuran or dihydropyran was added
dropwise. The reaction mixture was stirred at 100°C for 2–3 h.
Then the mixture was cooled to room temperature, extracted
with ethyl acetate (80 mL ꢂ 3) and washed with water. After
evaporating to dryness under reduced pressure, the residue was
purified by column chromatography, eluting with ethyl acetate/
petroleum ether 20:80 v/v.
General procedure for the preparation of compounds 1–24
A mixture of 10 mmol piperazine derivatives (5a–c), 25 mmol
K2CO3, 10 mmol 3-bromoalkylindole (2a–h), and a catalytic
amount of KI in 45 mL anhydrous acetonitrile was added in a
100-mL flask. The mixture was refluxed for 8–12 h in an oil bath.
After filtering, the resulting filtrate was evaporated under
reduced pressure. The residue was suspended in water (30 mL)
and extracted with dichloromethane (30 mL ꢂ 3). Evaporation
and purification on silica-gel column (methanol/methylene
dichloride 2:98 v/v) yielded compounds 1–24.
General procedure for the preparation of
3-bromoalkylindoles (2a–h)
A
solution of 0.025 mol triphenylphosphine in 80 mL dry
3-(3-(4-((2,3-Dihydrobenzo[b][1,4]dioxin-3-yl)methyl)-
acetonitrile was added in
a 250-mL flask. A solution of
0.02 mol 3-substituted indoles (1a–h) in 75 mL dry acetonitrile
was added. Then a solution of 0.027 mol carbontetrabromide in
25 mL dry acetonitrile was added dropwise. The reaction mixture
was stirred at room temperature for 2–3 h. After evaporating to
dryness under reduced pressure, the residue was purified by
column chromatography, eluting with ethyl acetate/petroleum
ether 10:90 v/v.
piperazin-1-yl)propyl)-1H-indole (1)
Yield: 72%; conversion to the HCl salt generated an off-white
1
solid; m.p.: 244°C (dec.); H NMR (DMSO-d6) d: 10.87 (s, 1H), 7.54
(d, 1H, J ¼ 7.8 Hz), 7.35 (d, 1H, J ¼ 8.0 Hz), 7.19 (d, 1H, J ¼ 1.8 Hz),
7.10 (t, 1H, J ¼ 14.8 Hz), 7.02 (t, 1H, J ¼ 14.8 Hz), 6.94–6.82 (m, 4H),
4.72–4.29 (m, 2H), 4.07–3.98 (m, 1H), 3.74–3.29 (m, 10H), 3.27–
3.10 (m, 2H), 2.75 (t, 2H, J ¼ 14.8 Hz), 2.16–2.04 (m, 2H); ESI-MS m/z
392.1 (MþH)þ.
General procedure for the preparation of (2-substituted 2,3-
dihydro-benzo[b][1,4]dioxin-3-yl)methanol (3a–c)
A mixture of 0.055 mol titanium tetrachloride and 0.165 mol
sodium borohydride in 80 mL anhydrous 1,2-dimethoxyethane
was added in a 250-mL flask at 0°C in an ice-water bath. Then a
solution of 0.05 mol methyl 3-substituted 1,4-benzodioxan-2-
5-Fluoro-3-(3-(4-((2,3-dihydrobenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)propyl)-1H-indole (2)
Yield: 69%; conversion to the HCl salt generated an off-white
solid; m.p.: 252°C (dec.); 1H NMR (DMSO-d6) d: 10.98 (s, 1H), 7.36–7.30
(m, 2H), 7.29–7.26 (m, 1H), 6.99–6.83 (m, 5H), 4.68–4.29 (m, 2H),
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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38
S. Wang et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 32–41
4.08–4.00 (m, 1H), 3.74–3.22 (m, 10H), 3.26–3.03 (m, 2H), 2.72 (t, 2H,
J ¼ 14.8 Hz), 2.13–2.01 (m, 2H); ESI-MS m/z 410.1 (MþH)þ.
(d, 1H, J ¼ 7.8 Hz), 7.34 (d, 1H, J ¼ 8 Hz), 7.19 (d, 1H, J ¼ 1.8 Hz), 7.07
(t, 1H, J ¼ 14.8 Hz), 6.98–6.82 (m, 5H), 4.48–4.39 (m, 1H), 4.21–4.12
(m, 1H), 3.88–3.28 (m, 10H), 3.23–3.09 (m, 2H), 2.75 (t, 2H,
J ¼ 14.8 Hz), 2.15–2.01 (m, 2H), 1.38–1.18 (m, 3H); ESI-MS m/z 406.1
(MþH)þ.
5-Chloro-3-(3-(4-((2,3-dihydrobenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)propyl)-1H-indole (3)
Yield: 65%; conversion to the HCl salt generated an off-white
1
5-Fluoro-3-(3-(4-((2,3-dihydro-2-methylbenzo[b][1,4]-
solid; m.p.: 247°C (dec.); H NMR (DMSO-d6) d: 11.10 (s, 1H), 7.60
dioxin-3-yl)methyl)piperazin-1-yl)propyl)-1H-indole (10)
Yield: 69%; conversion to the HCl salt generated an off-white
solid; m.p.: 249°C (dec.); 1H NMR (DMSO-d6) d: 11.00 (s, 1H), 7.37–
7.30 (m, 2H), 7.30–7.26 (m, 1H), 6.99–6.83 (m, 5H), 4.49–4.41
(m, 1H), 4.21–4.15 (m, 1H), 3.88–3.28 (m, 10H), 3.26–3.05 (m, 2H),
2.72 (t, 2H, J ¼ 14.8 Hz), 2.14–2.02 (m, 2H), 1.36–1.20 (m, 3H);
ESI-MS m/z 424.1 (MþH)þ.
(d, 1H, J ¼ 1.8 Hz), 7.36 (d, 1H, J ¼ 8.6 Hz), 7.28 (d, 1H, J ¼ 2.0 Hz),
7.10–7.05 (m, 1H), 6.95–6.83 (m, 4H), 4.66–4.29 (m, 2H), 4.06–3.99
(m, 1H), 3.88–3.28 (m, 10H), 3.24–3.06 (m, 2H), 2.72 (t, 2H,
J ¼ 14.8 Hz), 2.13–2.01 (m, 2H); ESI-MS m/z 426.0 (MþH)þ.
3-(3-(4-((2,3-Dihydrobenzo[b][1,4]dioxin-3-yl)methyl)-
piperazin-1-yl)propyl)-5-methyl-1H-indole (4)
Yield: 63%; conversion to the HCl salt generated an off-white
1
5-Chloro-3-(3-(4-((2,3-dihydro-2-methylbenzo[b][1,4]-
solid; m.p.: 244°C (dec.); H NMR (DMSO-d6) d: 10.72 (s, 1H), 7.32
(s, 1H), 7.23 (d, 1H, J ¼ 8.0 Hz), 7.13 (d, 1H, J ¼ 2.0 Hz), 6.96–6.85
(m, 5H), 4.71–4.30 (m, 2H), 4.07–3.99 (m, 1H), 3.84–3.28 (m, 10H),
3.24–3.12 (m, 2H), 2.73 (t, 2H, J ¼ 14.8 Hz), 2.13–2.01 (m, 2H), 2.39
(s, 3H); ESI-MS m/z 406.1 (MþH)þ.
dioxin-3-yl)methyl)piperazin-1-yl)propyl)-1H-indole (11)
Yield: 58%; conversion to the HCl salt generated an off-white
1
solid; m.p.: 245°C (dec.); H NMR (DMSO-d6) d: 11.10 (s, 1H), 7.60
(d, 1H, J ¼ 1.8 Hz), 7.36 (d, 1H, J ¼ 8.6 Hz), 7.28 (d, 1H, J ¼ 1.8 Hz),
7.10–7.05 (m, 1H), 6.99–6.83 (m, 4H), 4.47–4.41 (m, 1H), 4.21–4.15
(m, 1H), 3.88–3.28 (m, 10H), 3.23–3.05 (m, 2H), 2.73 (t, 2H,
J ¼ 14.8 Hz), 2.14–2.02 (m, 2H), 1.38–1.21 (m, 3H); ESI-MS m/z 440.1
(MþH)þ.
3-(2-(4-((2,3-Dihydrobenzo[b][1,4]dioxin-3-yl)methyl)-
piperazin-1-yl)ethyl)-1H-indole (5)
Yield: 45%; conversion to the HCl salt generated an off-white
1
solid; m.p. 238°C (dec.); H NMR (DMSO-d6) d: 10.97 (s, 1H), 7.63
3-(3-(4-((2,3-Dihydro-2-methylbenzo[b][1,4]dioxin-3-yl)-
(d, 1H, J ¼ 7.9 Hz), 7.37 (d, 1H, J ¼ 8.0 Hz), 7.24 (d, 1H, J ¼ 2.1 Hz),
7.10 (t, 1H, J ¼ 14.8 Hz), 7.02 (t, 1H, J ¼ 14.8 Hz), 6.94–6.82 (m, 4H),
4.55–4.32 (m, 2H), 4.07–3.98 (m, 1H), 3.74–3.29 (m, 12H), 3.20–3.09
(m, 2H); ESI-MS m/z 378.1 (MþH)þ.
methyl)piperazin-1-yl)propyl)-5-methyl-1H-indole (12)
Yield: 55%; conversion to the HCl salt generated an off-white
solid; m.p.: 239°C (dec.); 1H NMR (DMSO-d6) d: 10.73 (s, 1H), 7.43–
7.39 (m, 1H), 7.27–7.23 (m, 1H), 7.20–7.16 (m, 1H), 7.05–6.83
(m, 5H), 4.49–4.41 (m, 1H), 4.21–4.15 (m, 1H), 3.88–3.28 (m, 10H),
3.29–3.03 (m 2H), 2.73 (t, 2H, J ¼ 14.8 Hz), 2.39 (s, 3H), 2.14–2.02
(m, 2H), 1.38–1.21 (m, 3H); ESI-MS m/z 420.1 (MþH)þ.
5-Fluoro-3-(2-(4-((2,3-dihydrobenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)ethyl)-1H-indole (6)
Yield: 58%; conversion to the HCl salt generated an off-white
solid; m.p.: 223°C (dec.); 1H NMR (DMSO-d6) d: 11.10 (s, 1H), 7.48–
7.43 (m, 1H), 7.40–7.34 (m, 1H), 7.34–7.30 (m, 1H), 6.99–6.83
(m, 5H), 4.72–4.31 (m, 2H), 4.08–4.00 (m, 1H), 3.88–3.28 (m, 12H),
3.22–3.08 (m, 2H); ESI-MS m/z 396.1 (MþH)þ.
3-(2-(4-((2,3-Dihydro-2-methylbenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)ethyl)-1H-indole (13)
Yield: 58%; conversion to the HCl salt generated an off-white
1
solid; m.p.: 234°C (dec.); H NMR (DMSO-d6) d: 10.97 (s, 1H), 7.63
(d, 1H, J ¼ 7.8 Hz), 7.37 (d, 1H, J ¼ 8.0 Hz), 7.23 (d, 1H, J ¼ 2.1 Hz),
7.13–6.98 (m, 2H), 6.96–6.85 (m, 4H), 4.49–4.41 (m, 1H), 4.21–4.15
(m, 1H), 3.88–3.28 (m, 12H), 3.22–3.10 (m, 2H), 1.37–1.20 (m, 3H);
ESI-MS m/z 392.1 (MþH)þ.
5-Chloro-3-(2-(4-((2,3-dihydrobenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)ethyl)-1H-indole (7)
Yield: 52%; conversion to the HCl salt generated an off-white
solid; m.p.: 225°C (dec.); 1H NMR (DMSO-d6) d: 11.20 (s, 1H), 7.72
(d, 1H, J¼ 1.8 Hz), 7.39 (d, 1H, J¼ 8.6 Hz), 7.33 (d, 1H, J ¼ 2.1 Hz), 7.12–
7.08 (m, 1H), 6.96–6.85 (m, 4H), 4.67–4.31 (m, 2H), 4.08–4.00 (m, 1H),
3.88–3.28 (m, 12H), 3.20–3.12 (m, 2H); ESI-MS m/z 412.0 (MþH)þ.
5-Fluoro-3-(2-(4-((2,3-dihydro-2-methylbenzo[b][1,4]-
dioxin-3-yl)methyl)piperazin-1-yl)ethyl)-1H-indole (14)
Yield: 54%; conversion to the HCl salt generated an off-white
solid; m.p.: 232°C (dec.); 1H NMR (DMSO-d6) d: 11.10 (s, 1H), 7.48–
7.43 (m, 1H), 7.40–7.34 (m, 1H), 7.34–7.30 (m, 1H), 6.99–
6.83 (m, 5H), 4.49–4.41 (m, 1H), 4.21–4.15 (m, 1H), 3.88–3.28
(m, 12H), 3.22–3.09 (m, 2H), 1.38–1.21 (m, 3H); ESI-MS m/z 410.1
(MþH)þ.
3-(2-(4-((2,3-Dihydrobenzo[b][1,4]dioxin-3-yl)methyl)-
piperazin-1-yl)ethyl)-5-methyl-1H-indole (8)
Yield: 64%; conversion to the HCl salt generated an off-white
1
solid; m.p.: 226°C (dec.); H NMR (DMSO-d6) d: 10.85 (s, 1H), 7.41
(s, 1H), 7.25 (d, 1H, J ¼ 8.0 Hz), 7.18 (d, 1H, J ¼ 1.8 Hz), 6.96–6.85
(m, 5H), 4.72–4.31 (m, 2H), 4.08–4.00 (m, 1H), 3.88–3.28 (m, 12H),
3.26–3.03 (m, 2H), 2.39 (s, 3H); ESI-MS m/z 392.1 (MþH)þ.
5-Chloro-3-(2-(4-((2,3-dihydro-2-methylbenzo[b][1,4]-
dioxin-3-yl)methyl)piperazin-1-yl)ethyl)-1H-indole (15)
3-(3-(4-((2,3-Dihydro-2-methylbenzo[b][1,4]dioxin-3-yl)-
Yield: 51%; conversion to the HCl salt generated an off-white
1
methyl)piperazin-1-yl)propyl)-1H-indole (9)
solid; m.p.: 234°C (dec.); H NMR (DMSO-d6) d: 11.19 (s, 1H), 7.70
Yield: 66%; conversion to the HCl salt generated an off-white
(d, 1H, J ¼ 1.8 Hz), 7.39 (d, 1H, J ¼ 8.6 Hz), 7.32 (d, 1H, J ¼ 2.0 Hz),
1
solid; m.p.: 240°C (dec.); H NMR (DMSO-d6) d: 10.86 (s, 1H), 7.54
7.12–7.07 (m, 1H), 6.93–6.81 (m, 4H), 4.47–4.40 (m, 1H), 4.20–4.12
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 32–41
Dihydrobenzodioxin and Indolealkylamine Derivatives as Antidepressants
39
(m, 1H), 3.88–3.28 (m, 12H), 3.16–3.02 (m, 2H), 1.37–1.20 (m, 3H);
ESI-MS m/z 426.1 (MþH)þ.
5-Fluoro-3-(2-(4-((2,3-dihydro-2-phenylbenzo[b][1,4]-
dioxin-3-yl)methyl)piperazin-1-yl)ethyl)-1H-indole (22)
Yield: 44%; conversion to the HCl salt generated an off-white
solid; m.p.: 205°C (dec.); 1H NMR (DMSO-d6) d: 11.10 (s, 1H), 7.64–
7.54 (m, 1H), 7.53–7.42 (m, 5H), 7.39–7.33 (m, 1H), 7.32–7.29
(m, 1H), 7.10–6.83 (m, 5H), 5.17–5.09 (m, 1H), 4.83–4.63 (m, 1H),
3.88–3.28 (m, 12H), 3.25–3.10 (m, 2H); ESI-MS m/z 472.2 (MþH)þ.
3-(2-(4-((2,3-Dihydro-2-methylbenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)ethyl)-5-methyl-1H-indole (16)
Yield: 45%; conversion to the HCl salt generated an off-white
solid; m.p.: 221°C (dec.); 1H NMR (DMSO-d6) d: 10.83 (s, 1H), 7.4
(s, 1H), 7.25 (d, 1H, J ¼ 8.0 Hz), 7.18 (d, 1H, J ¼ 2.0 Hz), 6.99–6.83
(m, 5H), 4.49–4.41 (m, 1H), 4.21–4.15 (m, 1H), 3.88–3.28 (m, 12H),
3.19–3.08 (m, 2H), 2.39 (s, 3H), 1.38–1.21 (m, 3H); ESI-MS m/z 406.1
(MþH)þ.
5-Chloro-3-(2-(4-((2,3-dihydro-2-phenylbenzo[b][1,4]-
dioxin-3-yl)methyl)piperazin-1-yl)ethyl)-1H-indole (23)
Yield: 61%; conversion to the HCl salt generated an off-white
solid; m.p.: 219°C (dec.); 1H NMR (DMSO-d6) d: 11.08 (s, 1H), 7.61–
7.56 (m, 2H), 7.51–7.40 (m, 4H), 7.35–7.28 (m, 1H), 7.28–7.24
(m, 1H), 7.11–6.82 (m, 5H), 5.17–5.09 (m, 1H), 4.83–4.63 (m, 1H),
3.88–3.28 (m, 12H), 3.26–3.11 (m, 2H); ESI-MS m/z 488.2 (MþH)þ.
3-(3-(4-((2,3-Dihydro-2-phenylbenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)propyl)-1H-indole (17)
Yield: 75%; conversion to the HCl salt generated an off-white
solid; m.p.: 214°C (dec.); 1H NMR (DMSO-d6) d: 10.98 (s, 1H),
7.63–7.51 (m, 2H), 7.51–7.40 (m, 4H), 7.37–7.31 (m, 1H), 7.22–
7.15 (m, 1H), 7.11–6.82 (m, 6H), 5.17–5.09 (m, 1H), 4.83–4.63
(m, 1H), 3.88–3.28 (m, 10H), 3.23–3.08 (m, 2H), 2.74 (t, 2H,
J ¼ 14.8 Hz), 2.14–2.02 (m, 2H); ESI-MS m/z 468.3 (MþH)þ.
3-(2-(4-((2,3-Dihydro-2-phenylbenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)ethyl)-5-methyl-1H-indole (24)
Yield: 42%; conversion to the HCl salt generated an off-white
solid; m.p.: 203°C (dec.); 1H NMR (DMSO-d6) d: 11.10 (s, 1H), 7.61–
7.56 (m, 1H), 7.51–7.40 (m, 4H), 7.35–7.28 (m, 1H), 7.25–7.20
(m, 1H), 7.14–7.10 (m, 1H), 7.11–6.82 (m, 5H), 5.17–5.09 (m, 1H),
4.83–4.63 (m, 1H), 3.88–3.28 (m, 12H), 3.18–3.10 (m, 2H), 2.38
(s, 3H); ESI-MS m/z 468.3 (MþH)þ.
5-Fluoro-3-(3-(4-((2,3-dihydro-2-phenylbenzo[b][1,4]-
dioxin-3-yl)methyl)piperazin-1-yl)propyl)-1H-indole (18)
Yield: 53%; conversion to the HCl salt generated an off-white
solid; m.p.: 234°C (dec.); 1H NMR (DMSO-d6) d: 10.97 (s, 1H), 7.61–
7.56 (m, 1H), 7.51–7.40 (m, 4H), 7.36–7.24 (m, 3H), 7.11–6.82
(m, 5H), 5.17–5.09 (m, 1H), 4.83–4.63 (m, 1H), 3.88–3.28 (m, 10H),
3.14–3.04 (m, 2H), 2.70 (t, 2H, J ¼ 14.8 Hz), 2.10–1.96 (m, 2H);
ESI-MS m/z 486.3 (MþH)þ.
In vitro binding assays
The following specific radioligands were used: 5-HTT, [3H]-
paroxetine; serotonin 5-HT1A receptor, [3H]8-OH-DPAT; 5-HT2A
,
[3H]ketanserine; 5-HT2C receptor, [3H]mesulergine; a1-adrenergic
receptor, [3H]prazosin; a2-adrenergic receptor, [3H]rauwolscine;
and D2 receptor, [3H]spiperone. Tissue sources are from the adult
Sprague-Dawley rat. All the new compounds were dissolved in 5%
DMSO. Compounds were tested at least three times over a 6 log
concentration range (10ꢁ5 to 10ꢁ10 M). IC50 values were deter-
mined by nonlinear regression analysis using Hill equation curve
fitting. Ki values were calculated based on the Cheng and Prussoff
equation: Ki ¼ IC50/(1 þ C/Kd), where C represents the concentra-
tion of the hot ligand used and Kd its receptor dissociation
constant, and were calculated for each labeled ligand. Mean Ki
values and SEM are reported for at least three independent
experiments.
5-Chloro-3-(3-(4-((2,3-dihydro-2-phenylbenzo[b][1,4]-
dioxin-3-yl)methyl)piperazin-1-yl)propyl)-1H-indole (19)
Yield: 67%; conversion to the HCl salt generated an off-white
solid; m.p.: 228°C (dec.); 1H NMR (DMSO-d6) d: 11.08 (s, 1H),
7.61–7.56 (m, 2H), 7.51–7.40 (m, 4H,), 7.35–7.28 (m, 1H),
7.28–7.24 (m, 1H), 7.11–6.82 (m, 5H), 5.17–5.09 (m, 1H),
4.83–4.63 (m, 1H), 3.88–3.28 (m, 10H), 3.22–3.03 (m, 2H),
2.72 (t, 2H, J ¼ 14.8 Hz), 2.10–1.96 (m, 2H); ESI-MS m/z 502.2
(MþH)þ.
3-(3-(4-((2,3-Dihydro-2-phenylbenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)propyl)-5-methyl-1H-indole (20)
Yield: 54%; conversion to the HCl salt generated an off-white
solid; m.p.: 221°C (dec.); 1H NMR (DMSO-d6) d: 10.71 (s, 1H), 7.61–
7.56 (m, 1H), 7.51–7.40 (m, 4H), 7.35–7.28 (m, 1H), 7.25–7.20
(m, 1H), 7.14–7.10 (m, 1H), 7.11–6.82 (m, 5H), 5.17–5.09 (m, 1H),
4.83–4.63 (m, 1H), 3.88–3.28 (m, 10H), 3.22–3.10 (m, 2H), 2.70
(t, 2H, J ¼ 14.8 Hz), 2.11–1.92 (m, 2H), 2.38 (s, 3H); ESI-MS m/z 482.3
(MþH)þ.
5-HTT binding assay
The assay was carried out according to the published procedure
with slight modifications [35]. [3H]Paroxetine (22.9 Ci/mmol,
Perkin Elmer Life Sciences, Boston, MA, USA) was used for
labeling 5-HTT. Rat cerebral cortex was homogenized in 20
volumes of ice-cold Tris–HCl buffer (50 mM, pH 7.4) using an
ULTRA TURRAX homogenizer and centrifuged at 20,000g for
10 min. The pellet was incubated in Tris–HCl buffer for 10 min at
37°C. The suspension was then washed twice by centrifugation
and resuspended in same conditions. The final pellet was
resuspended in Tris–HCl buffer containing 150 mM NaCl and
5 mM KCl. The final incubation mixture consisted of 900 mL of the
tissue suspension, 50 mL of 1 nM [3H]paroxetine, 50 mL of 10 mM
paroxetine for non-specific binding, 50 mL of new compounds or
reference drug for specific binding. The tubes were incubated at
22°C for 60 min. The incubation was followed by a rapid vacuum
filtration through Whatman GF/B glass filters, and the filters were
3-(2-(4-((2,3-Dihydro-2-phenylbenzo[b][1,4]dioxin-3-yl)-
methyl)piperazin-1-yl)ethyl)-1H-indole (21)
Yield: 52%; conversion to the HCl salt generated an off-white
solid; m.p.: 213°C (dec.); 1H NMR (DMSO-d6) d: 10.98 (s, 1H),
7.66–7.54 (m, 2H), 7.52–7.40 (m, 4H), 7.39–7.34 (m, 1H), 7.27–
7.18 (m, 1H), 7.14–6.83 (m, 6H), 5.17–5.09 (m, 1H), 4.83–4.63
(m, 1H), 3.88–3.28 (m, 12H), 3.26–3.11 (m, 2H); ESI-MS m/z 454.3
(MþH)þ.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

40
S. Wang et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 32–41
washed twice with 5 mL cold buffer and transferred to
scintillation vials. Scintillation fluid (3.0 mL) was added and
the radioactivity bound was measured using a Beckman LS 6500
liquid scintillation counter.
keep its head above the surface. The total duration of immobility
was recorded during the last 4 min of a 6-min test session.
Locomotor activity [39]
Locomotor activity was assessed in automated activity frames
equipped with an automated video tracking system. Fluoxetine
and the test compounds were orally administered by intragastric
administration 1 h before test start. Mice were acclimated to
the test chamber for 30 min before starting the experiment.
Locomotor activity was then measured for 10 min. The horizontal
distance traveled in centimeters by the animals was analyzed.
5-HT1A binding assay
[3H]8-OH-DPAT (187.4 Ci/mmol, Perkin Elmer Life Sciences) was
used for labeling the 5-HT1A receptor. The assay was carried out
according to the previously published procedure [36]. Rat cerebral
cortex was homogenized in 20 volumes of ice-cold Tris–HCl buffer
(50 mM, pH 7.7) using an ULTRA TURRAX homogenizer, and was
then centrifuged at 32,000g for 10 min at 4°C. The pellet was
incubated in Tris–HCl buffer at 37°C for 10 min and then
centrifuged at 32,000g for 10 min at 4°C. The final pellet was
resuspended in Tris–HCl buffer containing 10 mM pargyline, 4 mM
CaCl2, and 0.1% ascorbic acid. The final incubation mixture
consisted of 900 mL of the tissue suspension, 50 mL of 0.5 nM [3H]8-
OH-DPAT, 50 mL of 10 mM serotonin for non-specific binding, 50 mL
of new compounds or the reference drug for specific binding. The
tubes were incubated at 37°C for 30 min. The incubation was
followed by a rapid vacuum filtration through Whatman GF/B
glass filters, and the filters were washed twice with 5 mL cold
buffer and transferred to scintillation vials. Scintillation fluid
(3.0 mL) was added and the radioactivity bound was measured
using a Beckman LS 6500 liquid scintillation counter.
Statistics
Data obtained from pharmacological experiments were analyzed
by the program Statistical Package for the Social Science (SPSS).
Results are expressed as mean ꢀ SEM; n represents the number of
animals. A p-value of <0.05 was considered statistically significant.
The authors gratefully acknowledge the financial support from the
Jiangsu Nhwa Pharmaceutical and the Huazhong University of Science
and Technology Analytical and Testing Center.
The authors have declared no conflict of interest.
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