Synthesis, docking, 3-D-qsar, and biological assays of novel indole derivatives targeting serotonin transporter, dopamine D2 receptor, and mao-a enzyme: In the pursuit for potential multitarget directed ligands

Article abstract of DOI:10.3390/molecules25204614

A series of 27 compounds of general structure 2,3-dihydro-benzo[1,4]oxazin-4-yl)-2-{4-[3-(1H-3indolyl)-propyl]-1-piperazinyl}-ethanamides, Series I: 7(a-o) and (2-{4-[3-(1H-3-indolyl) -propyl]-1-piperazinyl}-acetylamine)-N-(2-morfolin-4-yl-ethyl)-fluorinated benzamides Series II: 13(a-l) were synthesized and evaluated as novel multitarget ligands towards dopamine D₂ receptor, serotonin transporter (SERT), and monoamine oxidase-A (MAO-A) directed to the management of major depressive disorder (MDD). All the assayed compounds showed affinity for SERT in the nanomolar range, with five of them displaying Ki values from 5 to 10 nM. Compounds 7k, Ki = 5.63 ± 0.82 nM, and 13c, Ki = 6.85 ± 0.19 nM, showed the highest potencies. The affinities for D₂ ranged from micro to nanomolar, while MAO-A inhibition was more discrete. Nevertheless, compounds 7m and 7n showed affinities for the D₂ receptor in the nanomolar range (7n: Ki = 307 ± 6 nM and 7m: Ki = 593 ± 62 nM). Compound 7n was the only derivative displaying comparable affinities for SERT and D₂ receptor (D₂/SERT ratio = 3.6) and could be considered as a multitarget lead for further optimization. In addition, docking studies aimed to rationalize the molecular interactions and binding modes of the designed compounds in the most relevant protein targets were carried out. Furthermore, in order to obtain information on the structure-activity relationship of the synthesized series, a 3-D-QSAR CoMFA and CoMSIA study was conducted and validated internally and externally (q² = 0.625, 0.523 for CoMFA and CoMSIA and r²_ncv = 0.967, 0.959 for CoMFA and CoMSIA, respectively).

Full text of DOI:10.3390/molecules25204614

molecules
Article
Synthesis, Docking, 3-D-Qsar, and Biological Assays
of Novel Indole Derivatives Targeting Serotonin
Transporter, Dopamine D2 Receptor, and Mao-A
Enzyme: In the Pursuit for Potential Multitarget
Directed Ligands
1
2
3
Christopher Cerda-Cavieres , Gabriel Quiroz , Patricio Iturriaga-Vásquez
,
1
1
1
4
Julio Rodríguez-Lavado , Jazmín Alarcón-Espósito , Claudio Saitz , Carlos D. Pessoa-Mahana ,
Hery Chung 4 , Ramiro Araya-Maturana
Claudia Ojeda-Gómez , Miguel Reyes-Parada
5
6,7
6
, Jaime Mella-Raipán
, David Cabezas ,
and Hernán Pessoa-Mahana 1,*
8
9,10,
*
1
Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias Químicas y Farmacéuticas,
Universidad de Chile, Casilla 233, Santiago 8380494, Chile; cdc.cavieres@gmail.com (C.C.-C.);
julio.rodriguez@ciq.uchile.cl (J.R.-L.); jazmin.alarc@gmail.com (J.A.-E.); clsaitz@ciq.uchile.cl (C.S.)
Programa de Doctorado en Farmacología, Universidad de Chile, Santiago 8380453, Chile;
gquirozn@ciq.uchile.cl
2
3
4
Departamento de Ciencias Qu
de la Frontera, Temuco 4811230, Chile; patricio.iturriaga@ufrontera.cl
Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile, Santiago
820244, Chile; cpessoa@puc.cl (C.D.P.-M.); chung.hery@gmail.com (H.C.)
Instituto de Química y Recursos Naturales, Universidad de Talca, Talca 3460000, Chile; raraya@utalca.cl
Instituto de Qu mica y Bioqu mica, Facultad de Ciencias, Universidad de Valpara so, Av. Gran Bretaña 1111,
Valparaíso 2360102, Chile; jaime.mella@uv.cl (J.M.-R.); jaime.mella@uc.cl (D.C.)
ímicas y Recursos Naturales, Facultad de Ingeniería y Ciencias, Universidad
7
5
6
í
í
í
7
Centro de Investigación Farmacopea Chilena (CIFAR), Universidad de Valparaíso, Av. Gran Bretaña 1111,
Valparaíso 2360102, Chile
Colegio Instituto San Martín, Hermanos Maristas, Curicó 3341965, Chile; claudia-ojeda@gmail.com
8
9
Centro de Investigación Biomédica y Aplicada (CIBAP), Escuela de Medicina, Facultad de Ciencias Médicas,
Universidad de Santiago de Chile, Santiago 9170002, Chile
Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Talca 3467987, Chile
Correspondence: miguel.reyes@usach.cl (M.R.-P.); hpessoa@ciq.uchile.cl (H.P.-M.)
1
0
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Tomasz Plech and Marta Andres-Mach
Received: 27 August 2020; Accepted: 3 October 2020; Published: 10 October 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: A series of 27 compounds of general structure 2,3-dihydro-benzo[1,4]oxazin-4-yl)-2-
{
-
4-[3-(1H-3indolyl)-propyl]-1-piperazinyl}-ethanamides, Series I:
propyl]-1-piperazinyl}-acetylamine)-N-(2-morfolin-4-yl-ethyl)-fluorinated benzamides Series II:
) were synthesized and evaluated as novel multitarget ligands towards dopamine D2
7(a–o) and (2-{4-[3-(1H-3-indolyl)
13(a–l
receptor, serotonin transporter (SERT), and monoamine oxidase-A (MAO-A) directed to the
management of major depressive disorder (MDD). All the assayed compounds showed affinity
for SERT in the nanomolar range, with five of them displaying Ki values from 5 to 10 nM.
Compounds 7k
,
Ki = 5.63 ± 0.82 nM, and 13c, Ki = 6.85
±
0.19 nM, showed the highest potencies.
The affinities for D ranged from micro to nanomolar, while MAO-A inhibition was more discrete.
2
Nevertheless, compounds 7m and 7n showed affinities for the D receptor in the nanomolar range
2
(
7n
:
Ki = 307 ± 6 nM and 7m: Ki = 593
±
62 nM). Compound 7n was the only derivative displaying
comparable affinities for SERT and D receptor (D /SERT ratio = 3.6) and could be considered as
2
2
a multitarget lead for further optimization. In addition, docking studies aimed to rationalize the
molecular interactions and binding modes of the designed compounds in the most relevant protein
targets were carried out. Furthermore, in order to obtain information on the structure–activity
relationship of the synthesized series, a 3-D-QSAR CoMFA and CoMSIA study was conducted and
Molecules 2020, 25, 4614; doi:10.3390/molecules25204614
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4614
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2
2
validated internally and externally (q = 0.625, 0.523 for CoMFA and CoMSIA and r ncv = 0.967,
.959 for CoMFA and CoMSIA, respectively).
0
Keywords: polypharmacology; SERT; dopamine D receptor; 3-indolylpropylpiperazines; multitarget;
2
docking; QSAR
1
. Introduction
Major depressive disorder (MDD) is a common, chronic, recurring, heterogeneous, and potentially
life-threatening disease affecting up to 20% of the world population, according to World Health
Organization [ ]. MDD is triggered by a complex pattern of genetic, epigenetic, and environmental
factors and, despite its prevalence, it is not being properly treated [ ]. As a result, such a devastating
1
2–4
disorder is enormously costing, economically, socially, and individually [5,6].
Since the monoamine hypothesis of depression was developed in the 1950s [
7], antidepressant
drug discovery has been a very active research field [
8], although MDD’s underlying mechanism and
neurological basis are not yet fully understood [ 11]. The global antidepressants market is expected
9
–
to grow from $14.3 billion in 2019 to about $28.6 billion in 2020 as mental health issues are expected to
surge because of the Covid-19 pandemic making an impact on the global economy.
Thus far, the vast majority of marketed antidepressants act through the serotoninergic and
norepinephrinergic systems, elevating synaptic levels of the corresponding neurotransmitters by
blocking the monoamine transporters [12–15]. Many adverse effects associated with first-generation
antidepressants (mainly tricyclic and monoamine oxidase inhibitors, –MAOi–) were partially overcome
by the arrival of second-generation antidepressants, including selective serotonin reuptake inhibitors
(SSRIs), norepinephrine reuptake inhibitors (NRIs), and dual antidepressants (SNRIs), with the latter
being advantageous as they can treat a wide range of symptoms [16 19]. However, much less approved
–
antidepressants do target the dopamine system, notwithstanding dopamine has also been implicated
in the pathophysiology of depression [20].
Inside the central nervous system (CNS), dopamine plays pivotal roles in executive functions,
motor control, emotion, cognition, reinforcement, and reward, among others [21
related to the pathophysiology of several CNS disorders, such as Parkinson’s disease, multiple sclerosis,
schizophrenia, drug addiction, and MDD [24 26]. Even though depression has been historically
–23]. Its dysfunction is
–
associated with misregulation within the serotonin/norepinephrine system, in the last years, an
increasing number of voices have been raised supporting that dopamine system dysfunction strings
along with it [24,27,28].
Due to the aforementioned causes, MDD is nowadays recognized as a complex dynamic system [29
]
endowed with a complex pathophysiology. For this reason, depression symptoms and its underlying
causes are more likely to be treated by a multitarget approach, by using promiscuous drugs also
defined as multi-target directed ligands (MTDLs): A single drug molecule that selectively targets
multiple receptors, so that different pathways conducting to the disease can be modified by using a
single molecule [20,30–32].
Indole derivatives have always been recognized as serotoninergic modulators, given their related
structural connection. Furthermore, in our research group, we have extensive experience in the synthesis
of indolylpropyl-piperazine derivatives, which have been successfully employed by us [33
other groups [38 39] as very potent serotonin transporter (SERT) ligands. On the other hand, molecular
structures containing morpholine [40] or benzoxazinone [41 45] cores have been reported as MAOi
–37] and
,
–
or dopamine D receptor modulators, respectively. In the light of such a background, we decided
2
to conduct an exploratory study by merging the aforementioned indolylpropyl-piperazines and
morpholine/benzoxazinone units. Herein, we describe the synthesis, docking, QSAR, and biological

Molecules 2020, 25, 4614
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evaluation of two series of molecular hybrids targeting SERT, MAO-A, and D receptor in the pursuit
2
of promising multi-target leads for potential MDD treatment.
2
2
2
. Results and Discussion
.1. Chemistry Series I
,3-Dihydro-benzo[b][1,4] oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide
derivatives 7a–o
The synthetic approach for the preparation of the target compounds 7a
–
o
is outlined below.
) obtained in a six-step
(R = H, F, Br) obtained by
) in a 42–89%
The reaction took place between piperazine benzoxazine derivatives
sequence and 82–95% yields (Scheme 1), with 3-indolyl tosylates 1a
6
c
(a–c
–
reported literature procedures [37
,
46] to give nine final compounds
7(a
,
b,
c,
g,
h
,
i
,
m
,
n,
o
yield (Scheme 2).
Scheme 1. Synthesis of piperazinylbenzoxazine derivatives
6 (a–c). Reagents and experimental conditions.
◦ ◦ ◦
a) Fe /(H O/EtOH/CH COOH) 1:1:1/60 C; (b) 2-chloroacetyl chloride, CH CN, K CO , 80 C;
2 3 3 2 3
(
(
◦ ◦
c) THF/LiAlH /N /0 C; (d) 2-chloroacetyl chloride-THF, 0 C; (e) N-Boc-piperazine, CH CN, K CO ,
4 2 3 2 3
◦ ◦
0 C; (f) TFA- CH Cl , 0 C.
2 2
8
◦
Scheme 2. Synthesis of Series I. Reagents and conditions: K CO , CH CN, 80 C, Yield (42–89%).
2
3
3
The synthesis of the derivatives 7d–f and 7j–l was accomplished by a different synthetic strategy,
involving indolylpiperazines 9a
and 4j to give the aforementioned six final compounds (7d
Scheme 3).
–
b
[
35], which were reacted with N-chloroacetyl benzoxazines 4d
–f
–
l
–f
and 7j ) with good to excellent yields
–l
(

Molecules 2020, 25, 4614
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◦
Scheme 3. Synthesis of Series I. Route 2. Reagents and conditions: K CO , CH CN, 80 C, Yield (63–91%).
2
3
3
In summary, 15 compounds were synthesized for Series I in yields ranging from 42% to 91%.
2
.2. Chemistry Series II
(
2-{4-[3-(1H-Indol-3-yl)-propyl]-1-piperazinyl}-acetylamine)-N-(2-morpholin-4-yl-ethyl)-fluorinated
benzamides
The synthetic pathway of this series involved the fluorinated benzamide derivatives 12a–d, which
were obtained from commercially available isomeric fluoro nitrobenzoic acids in a three-step sequence
with good to excellent yields as shown in Scheme 4.
◦
Scheme 4. Synthesis of derivatives 12a–d. Reagents and experimental conditions: (a) SOCl , 60 C;
2
◦ ◦
b) 2-aminoethyl morpholine, anhydrous THF; (c) Fe /(H O/EtOH/CH COOH)/60 C; (81–98%) (d)
2 3
(
◦
2
-chloroacetylchloride, anhydrous THF, 0 C, Yield (93–97%).
The fluorinated benzamides derivatives 12a
–d were finally connected to indolylpropylpiperazines
9
a–c to achieve the expected compounds 13a–l, with yields ranging from 47% to 85% (Scheme 5).

Molecules 2020, 25, 4614
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◦
Scheme 5. Synthesis of Series II derivatives 13a–l. Reagents and conditions: K CO , CH CN, 80 C,
2 3 3
Yield (47–85%).
2
.3. Pharmacology Series I
Table 1 summarizes the affinity of compounds 7a–o for SERT, D receptor, and MAO-A.
2
Most compounds were potent and clearly selective as SERT ligands, showing in all cases affinities in
the nanomolar range, whereas the affinities for D and MAO-A ranged from micromolar to much
2
higher values, respectively. A detailed analysis of SERT activities indicates that a C-5 substitution
of the indole ring with halogens (fluorine or bromine; compounds 7g or 7m) leads to more potent
compounds than the unsubstituted derivative (7a). On the other hand, the presence of a halogen
atom at C-6 of the benzoxazine ring increased the affinity (e.g., 7c
the most potent compounds were those exhibiting a dual halogen substitution pattern (7i
,
7d, and 7e vs. 7a). Accordingly,
7j, and 7k),
,
with Ki values below 10 nM. The C-7 halogen substitution on the benzoxazine ring gave no consistent
effects, slight increases (7b and 7h) or decreases (7n) of affinity were observed, as compared with
the corresponding C-7 unsubstituted compounds (7a, 7g, and 7m, respectively). The D receptor
2
affinity for this series indicates that no conclusive structure–activity relationships can be extracted for
these compounds. Nevertheless, it is apparent that dihalogenated derivatives, bearing one halogen
atom at the C-5 of the indole ring and the other at either the C-6 or C-7 of the benzoxazine moiety
(7h–7k
,
7m–7o), resulted in more potent compounds than their corresponding monohalogenated or
unsubstituted counterparts (7a
–
7e, 7g). Moreover, the presence of a methoxyl group at the C-6 of the
benzoxazine ring has almost no effect on the affinity of the compounds for D receptor. It is worth
2
mentioning that the dihalogenated compound 7n was the only derivative displaying comparable
affinities for SERT and D receptor (D /SERT ratio = 3.6) and could be considered as a potential leader
2
2
in the search of more potent multitarget compounds.

Molecules 2020, 25, 4614
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Table 1. Affinities, measured as Ki values at the serotonin transporter (SERT), D receptor, and percent
2
of monoamine oxidase-A (MAO-A) inhibition (at 100
Series I).
µM) of indolepiperazinyl benzoxazine derivatives
(
%
Inhibition
Compound
R
R1
R2
SERT (nM)
D (nM)
2
MAO-A at 100 µM
Fluoxetine
Haloperidol
-
-
-
-
-H
-H
-F
-Cl
-Br
-OCH3
-H
-H
-F
-Cl
-Br
-OCH3
-H
-H
-F
-
-
-H
-F
3.15 ± 0.32
N.D.
N.D.
N.D.
N.D.
27%
43%
40%
18%
38%
58%
41%
0%
3.88 ± 0.44
4840 ± 68
4740 ± 131
2387 ± 163
1265 ± 182
910 ± 263
4407 ± 647
2315 ± 64
2317 ± 163
1681 ± 79
902 ± 4
7
a
-H
-H
-H
-H
-H
-H
-F
-F
-F
-F
-F
307.10 ± 30.46
180.80 ± 19.97
68.78 ± 1.02
81.39 ± 4.97
36.57 ± 0.55
412.20 ± 57.86
9.63 ± 0.31
7.61 ± 0.02
7.51 ± 0.12
12.30 ± 2.31
5.63 ± 0.82
12.60 ± 1.52
50.18 ± 2.47
84.44 ± 2.03
34.89 ± 0,32
7
b
7
c
-H
-H
-H
-H
-H
-F
-H
-H
-H
-H
-H
-F
7
d
7
e
7
f
7
g
7
h
7
7
i
j
0%
20%
20%
52%
30%
23%
26%
7
k
1041 ± 232
3322 ± 256
593 ± 62
7
l
-F
7
m
-Br
-Br
-Br
7
n
307 ± 6
7
o
-H
1152 ± 227
Data represent the mean
not determined.
±
SEM of three experiments, each with triplicate samples. All Ki values are in nM. N.D.
2
.4. Docking Simulation Series I
Considering the pharmacological results, docking studies aimed to rationalize the molecular
interactions and binding modes of the designed compounds were carried out only in the human SERT
hSERT) and in selected cases at the D receptor.
(
2
2
.4.1. hSERT
The most potent compounds 7g
which favors the following stabilizing interactions: A
–donor aromatic residue Tyr176, a coulombic interaction between the protonated piperazine N-1 with
the Asp98 residue, and a –cation interaction for the protonated piperazine with Tyr95. Furthermore,
,
7h
,
7i, and 7
k
showed a common docking pose (Figure 1),
π–π interaction between the indole ring and the
π
π
aromatic interactions were also observed for the benzoxazine ring with the residues Phe341 and Phe335.
These drug–target interactions are in agreement with those described in the crystal structure of the
hSERT in complex with the inhibitor (S)-citalopram [47,48].

Molecules 2020, 25, 4614
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A
B
C
D
Figure 1. Docking poses in SERT obtained for compounds 7g
in orange, and 7k
(
A
) in yellow, 7h
(
B
) in magenta, 7i
(
C
)
+
(
D
) in blue. Nearby residues <5 Å (grey sticks) and Na atoms (pink spheres) are
shown. Dotted lines represent ionic interactions and aromatic interactions are shown with green lines.
The relevance of the fluorinated substitution on the indole ring is clearly evidenced by comparison
of compounds 7f and 7l. Both derivatives share the same substitution pattern in the benzoxazine ring,
differing only by the presence of a fluorine atom at the indole moiety, which induces a different docking
pose for 7f. Thus, the least potent compound of the series (7f) adopted a binding mode in which both
indole and piperazine ring interactions are clearly less favored than the C-5 fluorinated counterpart
(Figure 2). Compounds showing intermediate affinities (7a
–7e and 7m–7o) exhibited docking poses
between the most and least favorable binding modes (not shown).
A
B
ASP98
SER439
TYR95
TYR176
LEU443
5
,69Å
3
,84Å
SER336
ALA173
PHE341
ILE172
ALA169
PHE335
VAL501
Figure 2. Comparison of the docking poses in SERT obtained for compounds 7f
) in dark green. Nearby residues <5 Å (grey sticks) and Na+ atoms (pink spheres) are shown.
Dotted lines represent ionic interactions and aromatic interactions are shown with green lines.
(A) in light green and
7l (B

Molecules 2020, 25, 4614
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2
.4.2. D Receptor
2
Docking simulations showed that compounds of this series adopt, at the D receptor, a binding
2
mode similar to that experimentally determined for the atypical antipsychotic risperidone [49]. Thus,
the indole moiety appears located into the deep hydrophobic sub-pocket of the orthosteric site, lined
by Cys118, Thr119, Ser197, Phe198, and Trp386, while the protonated piperazine N-1 locates in a
favorable position to establish a coulombic interaction with Asp114 (Figure 3). Furthermore, the
benzoxazine portion extends to the additional hydrophobic sub-pocket lined by Val91, Trp100, Phe110,
and Tyr408, in a similar fashion to that observed in the crystal structure for the pyrimidinone moiety
of risperidone. Interestingly, this general binding mode was observed for both the most and the
least potent compounds of this series (7a, 7b, and 7m, 7n), respectively (Figure 3A,B). Therefore, it is
tempting to speculate that the higher affinity showed by brominated derivatives (7m and 7n) is due to
the formation of a halogen bond between the bromine and a hydroxyl group of an adjacent residue
(e.g., Ser197). As observed (Figure 3), this could also change the position of the benzoxazine moiety,
favoring its interactions at the more external hydrophobic sub-pocket.
TRP100
TRP100
VAL91
VAL91
PHE110
TYR408
TYR408
PHE110
THR412
THR412
PHE389
PHE389
ASP114
ASP114
2
.70Å
2.70Å
TYR416
TYR416
VAL115
TRP386
VAL115
TRP386
PHE198
CYS118
CYS118
PHE198
THR119
THR119
SER197
SER197
A
B
Figure 3. Docking poses in D obtained for compounds 7a in yellow and 7b in blue (
A
), and compounds
2
7
m in magenta and 7n in green (B). Nearby residues < 5 Å (grey sticks) are shown. Dotted lines
represent ionic interactions. Yellow segmented line represents the deep hydrophobic sub-pocket.
Residues lining the more external hydrophobic sub-pocket are shown in orange.
2
.5. Pharmacology Series II
Table 2 summarizes the affinity of Series II compounds 13a
As in the case of indole benzoxazine derivatives (Series I), most indole morpholine ethylbenzamides
Series II) were potent SERT ligands, showing much lower affinities for D receptor and virtually no
–13l for SERT, D receptor, and MAO-A.
2
(
2
effect upon MAO-A activity. Regarding SERT activity, and in agreement with our previous studies,
halogen substitution at C-5 of the indole ring with fluorine or bromine (compounds 13a ) conducted
an increase in affinity as compared with the unsubstituted analogues 13i , with the fluoro derivatives
3a being the most potent of the series. On the other hand, when the acetanilide portion, connected
–g
–
l
1
–d
to the indolylpropylpiperazinyl fragment, was functionalized with a fluorine atom (at C-2) and a
morpholino ethylcarboxamide, the best affinities were obtained when the bulkier substituent was
located at meta position (compounds 13c, 13g, and 13k).

Molecules 2020, 25, 4614
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Table 2. Affinities, measured as Ki values at SERT, D receptor, and percent of MAO-A inhibition (at
2
1
00 µM) of indolepiperazinylmorpholinoethyl benzamide derivatives (Series II).
Position
Position
% Inhibition
MAO-A at 100 µM
Compound
R
(Indolylpropyl
SERT (nM)
D2 (nM)
F
Piperazineacetamide)
Fluoxetine
Haloperidol
-
-
F
F
F
F
Br
Br
Br
Br
H
H
H
H
-
-
-
-
3.15 ± 0.32
N.D.
N.D.
3.88 ± 0.44
5455 ± 25
3100 ± 42
592.60 ± 30.22
9060 ± 29
5619 ± 31
1832 ± 33
765 ± 16
N.D.
N.D.
19%
42%
28%
30%
39%
39%
16%
9.4%
23%
44%
34%
34%
1
3a
3b
3c
3d
3e
3f
3g
3h
2
3
5
2
2
3
5
2
2
3
5
2
4
4
2
5
4
4
2
5
4
4
2
5
16.04 ± 0.21
23.78 ± 0.46
6.83 ± 0.19
98.14 ± 1.24
222.60 ± 16.44
58.85 ± 1.14
12.62 ± 0.42
199.60 ± 14.53
9945 ± 383
109.90 ± 3.26
26.81 ± 0.91
52.16 ± 3.24
1
1
1
1
1
1
1
4700 ± 168
48910 ± 1279
2339 ± 45
4169 ± 335
2000 ± 34
13i
13j
13k
13l
Data represent the mean
N.D. no determined.
±
SEM of three experiments, each with triplicate samples. All Ki values are in nM range.
2
.6. Docking Simulation Series II
Similar to the analysis of Series I and considering the pharmacological results, docking studies
were carried out only in hSERT. In this series, seven compounds exhibited Ki values between 7 and
0 nM (13a 13b 13c 13f 13g 13k, and 13l). Docking simulations showed that compounds with the
6
,
,
,
,
,
lowest Ki values (13c and 13g) share a common binding mode into the S1 site of the SERT, which is
similar to that described for compounds of Series I (Figure 4A). Thus, the piperazine N-1 can establish
ionic and π–cation interactions with Asp98 and Tyr176, respectively, while the indole moiety can
participate in aromatic interactions with Tyr176 and Phe341. Interestingly, the ethylmorpholinic chain
extends towards the extracellular vestibule (also known as the S2 site). On the other hand, for the
compounds with the lowest affinities (13e and 13i), docking simulations showed that the piperazine
N-1 was located farther away from Asp98 and Tyr95, making the possible ionic interactions with
these residues unlikely or much weaker (Figure 4B). The analysis of the docking poses indicates
that the most potent compounds, i.e., those having a 5,2-substitution pattern (13c
exhibited an extended conformation at the binding site, while the least potent compounds (13e and
3i, showing a 2,4-substitution pattern) adopted a more constrained binding mode, impairing the most
relevant interactions.
, 13g, and 13k)
1

Molecules 2020, 25, 4614
10 of 30
A
B
Figure 4. Docking poses in SERT obtained for compounds 13c in cyan and 13g in purple (
A
), and 13e
+
in light blue, and 13i in orange (
B
). Nearby residues < 5 Å (grey sticks) and Na atoms (pink spheres)
are shown. Dotted lines represent ionic interactions, orange lines represent
and aromatic interactions are shown with green lines.
π–cation interactions,
2
.7. 3-D-QSAR Study
To systematize the structure–activity relationship of the synthesized molecules, we carried out a
-D-QSAR study of the CoMFA and CoMSIA type. The complete series of 27 molecules was divided
3
into training (19 compounds) and test sets (8 compounds) in a ratio of 70:30, selecting the test set
2
compounds at random to avoid bias. The q values for the best models were 0.625 and 0.523 for
CoMFA and CoMSIA, respectively while the r2ncv values were 0.967 and 0.959 for CoMFA and CoMSIA,
respectively. The statistical summary, as well as the tables of affinities for both models and their
respective graphs, are incorporated in the Supplementary Material.
The steric contour map of CoMFA (Figure 5A) shows a green polyhedron on the bromine atom of
compound 7k, the most active of the series. This means that the insertion of bulky atoms or groups
in this position is favorable for biological activity. This is consistent with docking studies showing
that compounds of series I place halogen into the void space close to lipophilic residues like Trp100
and Tyr408. In the case of compounds of series II, the meta-substituted benzamides placed the chain
towards the green region, not the ortho-substituted ones, so it is preferable that the chains are in the
meta-position. This is confirmed in the docking of these compounds, in which better accommodation
is observed in the SERT binding site. On the other hand, the electrostatic contour map (Figure 5B)
shows three blue polyhedra of significant size. This means that the presence of positively charged
atoms in these positions would be favorable for affinity. Such polyhedra are located on the carbon
atom bonded to the halogen in the case of series I, suggesting that the presence of electronegative
atoms bonded to the aforementioned carbon is favorable. The second blue polyhedron is localized on
the oxygen atom of the carbonyl group belonging to the ortho-substituted series II amide-compounds.
Therefore, oxygen atom remotion would be favorable for affinity. Finally, the third polyhedron is
observed on the oxygen atom of the morpholine ring in the ortho-substituted compounds for series
II, indicating that changing the morpholine by a piperazine or piperidine ring should lead to better
affinities. Furthermore, alkyl chains substitutions at the ortho-position in the benzamide ring resulted
in less favorable affinities compared to meta substitutions as was experimentally corroborated.

Molecules 2020, 25, 4614
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Figure 5. CoMFA (A,B) and CoMSIA (C,D) contour maps for the entire series of compounds evaluated
in SERT. The compound with the best SERT affinity (7k) is depicted. A. Steric contour map. Green means
that large groups are favorable for activity. B and D. Electrostatic contour maps. Red means electron-rich
atoms are favorable and blue means positively charged atoms are favorable. C. Hydrophobic map.
Gray means that hydrophilic groups are favorable for activity and yellow means that hydrophobic
groups are favorable.
The hydrophobic contour map of CoMSIA (Figure 5C) showed a gray polyhedron at position C5
of the indole ring, meaning that the presence of hydrophilic groups is favorable for affinity. In fact,
the C5 fluorine-substituted indoles displayed the best affinities of the series. Other polar groups like
OH, NH , or NR would also be interesting to evaluate at this position. Similarly, a yellow polyhedron
2
2
located on the bromine atom of the benzoxazine framework (compound 7k) means that the presence of
lipophilic groups is favorable for activity. In concordance, halogens like Cl, Br, and I would be the most
appropriated substituents and groups, such as aromatic rings, alkyl, and/or alkoxy chains, could also
be explored. In the case of compounds of series II, a yellow polyhedron is located on the amide group
of the meta-substituted compounds; therefore, the replacement of the amide by a less-polar function,
such as a ketone or ester, would be an interesting option to explore. On the other hand, the electrostatic
contour map of CoMSIA (Figure 5D) showed two polyhedra around compound 7k. A red polyhedron
on the halogen atom at position 5 of the indole ring means that the presence of electron-rich atoms is
favorable for affinity. It is interesting to note that the blue polyhedron intersecting the carbon atom of
indole at position 5 is complementary to the red polyhedron. In consequence, the presence of a positive
charge on the indole ring is favorable for activity. Other potential electron-withdrawing groups to
be explored are CN, NO , and COR. Docking studies showed π–stacking interaction between the
2
π-deficient indole ring with Tyr176, Phe341, and Trp386 residues.

Molecules 2020, 25, 4614
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3
. Materials and Methods
3
.1. General Methods
1
Melting points were determined on a hot-stage apparatus and were uncorrected. The H and
C-NMR spectra were obtained on a Bruker DRX-300 spectrometer (300 and 75 MHz, respectively) in
13
CDCl , DMSO-d , and CD COCD -d . Chemical shifts were recorded in ppm (δ) relative to TMS as
3
6
3
3
6
an internal standard. J values are given in Hz. Micro-analyses were carried out on a Fisons EA 1108
analyzer. High-resolution mass spectra were recorded on a DSA–TOFAxION 2 TOF MS (Perkin Elmer,
Shelton, CT, USA), positive mode. Silica gel Merck 60 (70–230 mesh) and aluminum sheets coated with
silica gel 60 F254 were used for column and TLC chromatography, respectively.
3
.1.1. General Procedure for the Synthesis of [4-[2-(3,4-Dihydro-2H-benzo[b][1,4]oxazin-4-yl)-2-oxo-
ethyl]-piperazine-1-yl] tert-butylcarbamate Derivatives 5a–c
[
4-[2-(3,4-Dihydro-2H-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl] tert-butylcarbamate (5a) as
a Model
To a solution containing 2-Chloro-1-(2,3-dihydrobenzo[b][1,4]oxazin-4-yl) ethanamide 4a (1.5 g;
.09 mmol) in dry CH CN (60 mL), N-Boc-piperazine (1321 mg; 7.09 mmol) and anhydrous K CO
7
(
3
2
3
◦
980 mg; 7.09 mmol) were added. The mixture was stirred at 80 C for 24 h. After this time, the mixture
was diluted with water (100 mL) and the solution extracted with EtOAc (100 mL 3), dried over
anhydrous Na SO , and concentrated under reduced pressure. The organic crude was purified by
×
2
4
silica gel column chromatography with EtOAc as eluent, to provide 5a (2152 mg; 84% yield) as a white
◦
1
0
0
solid. m.p.: 133.0–134.0 C; H-NMR (CDCl ):
2
2
δ
1.45 (s, 9H, H-4 ), 2.52 (t, 4H, H-3 ,J = 4.8 Hz), 3.39 (s,
H, H-1 ), 3.45 (t, 4H, H-2 , J = 4.5 Hz), 3.99 (t, 2H, H-3, J = 4.8 Hz), 4.31 (t, 2H, H-2, J = 4.4 Hz), 6.91 (m,
H, H-6 and H-8), 7.08 (t, 1H, H-7, J = 7.3 Hz), and 7.99 (br. s, 1H, H-5) ppm. 13C-NMR (CDCl3):
27.9
3
0
0
δ
(
3X), 42.6, 43.2 (2X), 52.4 (2X), 55.4, 66.4, 79.3, 116.8, 119.7, 123.5, 125.6, 146.3, 154.2, 167.3, and 167.5
+
ppm. HRMS: (EI) Calculated for C H N O (M ) = 362.20799. Found: 362.2113.
19
27
3
4
[
4-[2-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl]
tert-butylcarbamate (5b)
2-Chloro-1-(7-fluoro-2,3-dihidro-benzo [1,4]oxazin-4-yl)-ethanamide 4b (1.5 g; 6.53 mmol),
N-Boc-piperazine (1216 mg; 6.53 mmol), and anhydrous K CO (902 mg; 6.53 mmol), to afford
2
3
◦
1
0
5
2
b
(1983 mg; 80% yield) as a white solid. m.p.: 126.6–128.5 C; H-NMR (CDCl ):
.51 (t, 4H, H-2 , J = 4.9 Hz), 3.35 (s, 2H, H-1 ), 3.45 (t, 4H, H-3 , J = 4.8 Hz), 3.97 (t, 2H, H-3, J = 4.8
δ
1.46 (s, 9H, H-4 ),
3
0
0
0
Hz), 4.30 (t, 2H, H-2, J = 4.1 Hz), 6.58–6.66 (m, 2H, H-6, and H-8), and 8.03 (br. s, 1H, H-5) ppm.
1
3
2
C-NMR (CDCl3):
δ
27.9 (3X), 42.5 43.2 (2X), 52.4 (2X), 62.1, 66.4, 79.3, 103.8 (d, J
= 27 Hz), 106.7 (d,
C-F
= 6 Hz), 154.2, 155.6
0
0
2
= 22.5 Hz), 121.8 (d, 3JC-F = 9.3 Hz), 124.6 (d, J
4
= 8.2 Hz), 147.3 (d, 3
J
J
C-F
1
C-F
C-F
+
(
d, J
= 222 Hz), and 167 ppm. HRMS: (EI) Calculated for C H FN O (M ) = 380.19856. Found:
C-F
19 26
3
4
3
80.2047.
[
4-[2-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl]
tert-butylcarbamate (5c)
2-Chloro-1-(6-fluoro-2,3-dihidro-benzo [1,4]oxazin-4-yl)-ethanamide 4c (1.5 g; 6.53 mmol),
N-Boc-piperazine (1216 mg; 6.53 mmol), and anhydrous K CO (902 mg; 6.53 mmol), to afford
2
3
◦
1
0
5
2
4
c
(2033 mg; 82% yield) as a white solid. m.p.: 113.4–114.5 C; H-NMR (CDCl ):
.53 (t, 4H, H-2 , J = 4.8 Hz), 3.35 (s, 2H, H-1 ), 3.46 (t, 4H, H-3 , J = 4.8 Hz), 3.98 (t, 2H, H-3, J = 4.9 Hz),
.27 (t, 2H, H-2, J = 4.4 Hz), 6.77–6.87 (m, 2H, H-6 and H-8), and 7.82 (br. s, 1H, H-5) ppm. C-NMR
δ
1.46 (s, 9H, H-4 ),
3
0
0
0
13
2
0
(
CDCl ):
δ
27.9 (3X), 42.5 43.4 (2X), 52.3 (2X), 62.1, 65.6, 79.3, 110.1 (d, J
= 28.5 Hz), 112.2 (d,
C-F
3
0
2
3
4
= 4.9 Hz), 142.2 (d, 3 JC-F = 9.3 Hz),154.2, 155.5
J
= 23.6 Hz), 117.1 (d, J
= 9.3 Hz), 125.7 (d, J
C-F C-F
C-F
1
+
(
d, J
= 238 Hz), and 167.1 ppm. HRMS: (EI) Calculated for C H FN O (M ) = 380.19856. Found:
C-F
19 26
3
4
3
80.2043.

Molecules 2020, 25, 4614
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3
.1.2. General Procedure for the Synthesis of 1-(2,3-dihydro-benzo[1,4]oxazin-4-yl)-2-piperazin-1-yl-
ethanamide Derivatives 6a–c
Synthesis of 1-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide (6a) as a Model
mixture of [4-[2-(3,4-Dihydro-2H-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl]
tert-butylcarbamate 5a (2 g; 5.53 mmol) in dry CH Cl (20 mL) and trifluoroacetic acid (12 mL)
A
2
2
◦
was stirred at 0 C, for 4 h. After this time, dry CH Cl (200 mL) was added and neutralized
2
2
with solid NaHCO (10 g) to later filter on celite. The mixture was finally diluted with a saturated
3
solution of NaHCO (200 mL), extracted with EtOAc (8
×
50 mL), dried over anhydrous Na SO ,
2 4
3
and concentrated under vacuum to obtain pure 6a (867 mg; 82% yield) as an unstable yellow light
1
0 0
solid, highly hygroscopic; H-NMR (DMSO-d ):
δ 2.37 (m, 4H, H-2 ), 2.69 (m, 4H, H-3 ), 3.29 (s, 2H,
6
0
H-1 ), 3.89 (t, 2H, H-3, J = 4.4 Hz), 4.25 (t, 2H, H-2, J = 4.1 Hz), 6.85 (m, 2H, H-6 and H-8), 7.0 (t, 1H,
H-7, J = 7.6 Hz) and 7.85 (br. s, 1H, H-5) ppm. 13C-NMR (DMSO-d6):
δ40.5, 45.2 (2X), 53.2 (2X), 62.0,
6
6.1, 73.5, 116.7, 119.6, 123.8, 125.1, 146.2, and 167.8 ppm.
1
-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide (6b)
4-[2-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl]tertbutyl
carbamate 5b (2 g; 5.27 mmol) and trifluoroacetic acid (12 mL), in dry CH Cl (20 mL) reacted to
[
2
2
1
provide 6b (1398 mg; 95% yield) as a brown light hygroscopic compound; H-NMR (CDCl ):
δ
2.5 7
3
0
0
0
0
(m, 4H, H-2 ), 2.94 (t, 4H, H-3 , J = 4.4 Hz), 3.18 (br. s, 1H, H-4 ), 3.33 (s, 2H, H-1 ), 3.97 (t, 2H, H-3,
J = 4.8 Hz), 4.31 (t, 2H, H-2, J = 4.2 Hz), 6.59–6.65 (m, 2H, H-6, and H-8), and 8.0 (br. s, 1H, H-5)
1
3
2
ppm. C-NMR (CDCl ):
δ
45.6 (2X), 53.8 (2X), 60.4, 66.9, 77.3, 104.2 (d, J
= 24 Hz), 107.2 (d,
3
4
C-F
0
0
2
3
= 9.4 Hz), 143.4 (d, 3 JC-F = 15 Hz), 155.7 (d, J
1
JC-F = 23 Hz), 114.5 (d, J
= 7 Hz), 125.1 (d, J
C-F
C-F
C-F
=
250 Hz), and 171.2 ppm.
1
-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide (6c)
4-[2-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl]tertbutyl
carbamate 5c (2 g; 5.27 mmol) and trifluoroacetic acid (12 mL), in dry CH Cl (20 mL) reacted to
[
2
2
1
provide 6c (1369 mg; 93% yield) as a brown light hygroscopic compound; H-NMR (CDCl ):
4
J = 4.5 Hz), 6.76–6.87 (m, 2H, H-7 and H-8) and 7.71 (br. s, 1H, H-5) ppm; C-NMR (CDCl ):
δ
2.62 (m,
3
0
0
0
0
H, H-2 ), 2.99 (t, 4H, H-3 , J = 4.6 Hz), 3.35 (s, 2H, H-1 ), 3.97 (m, 3H, H-3 and H-4 ), 4.28 (t, 2H, H-2,
13
δ 45.6
3
0
2
= 28 Hz), 112.8 (d, JC-F = 32 Hz), 117.6 (d, 3JC-F = 9.4 Hz),
= 6.6 Hz), 157.6(d, J
2
(
1
2X), 53.8 (2X), 60.4, 66.9, 77.2, 110.6 (d, J
C-F
0
26.1 (d, 3 JC-F = 11 Hz), 142.8 (d, J
4
1
= 237 Hz), and 171.2 ppm.
C-F
C-F
3
.1.3. General Procedure for the Synthesis of 2,3-dihydro-benzo[b][1,4] oxazin-4-yl)-2-{4-[3-(1H-3-
indolyl)-propyl]-1-piperazinyl} ethanamide Derivatives 7a–c, 7g–i, and 7m–o. Method 1
1
-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl} ethanamide (7a
)
as a Model
To a stirred solution of 3-(1H-3-indolyl)-propyl-4-methylbencensulfonate 1a (254 mg. 0.77 mmol),
in dry CH CN (50 mL), benzoxazine 6a (200 mg, 0.77 mmol) and anhydrous K CO (106 mg; 0.77 mmol)
3
2
3
◦
were added. The mixture was heated at 80 C for 24 h. After this time, the resulting mixture was poured
into water (100 mL), extracted with EtOAc (4 50 mL), dried over anhydrous Na SO , and concentrated
under reduced pressure. The organic crude was purified by column chromatography EtOAc/MeOH
6:1) to give compound 7a (217 mg; 70% yield) as a yellow light solid; m.p.: 124.3–125.8 C; H-NMR
DMSO-d6):
J = 7.4 Hz), 3.33 (s, 2H, H-7 ), 3.90 (t, 2H, H-3 , J = 3.8 Hz), 4.26 (t, 2H, H-2 , J = 3.7 Hz), 6.85 (m, 2H,
H-6 and H-8 ), 6.95 (t, 1H, H-7 , J = 7.7 Hz), 7.03 (m, 2H, H-5 and H-6), 7.09 (d, 1H, H-2, J = 1.4 Hz),
.33 (d, 1H, H-4, J = 7.8 Hz), 7.49 (d, 1H, H-7, J = 7.7 Hz), 7.90 (br. s, 1H, H-5 ), and 10.79 (br. s, 1H,
H-1) ppm. C-NMR (DMSO-d6):
×
2
4
◦
1
(
(
0 0 0 0 0
δ 1.77 (q, 2H, H-2 , J = 7.0 Hz), 2.22–2.50 (m, 10H, H-3 , H-4 and H-5 ), 2.67 (t, 2H, H-1 ,
0
00
00
00
00
00
00
7
13
δ 22.9, 27.6, 39.2, 40.8, 53.0 (2X), 53.2 (2X), 58.0, 66.6, 111.8, 114.8,

Molecules 2020, 25, 4614
14 of 30
1
17.2, 118.5, 118.7, 120.1, 121.2, 122.6, 124.3, 126.0, 127.7, 128.5, 136.7, 146.7, and 168.3 ppm. HRMS: (EI)
+
Calculated for C H N O (M ) = 419.5313. Found: 419.4426.
25
30
4
2
1
-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7b)
To a solution of 3-(1H-3-indolyl)-propyl-4-methylbencensulfonate 1a (237 mg; 0.72 mmol), in dry
CH CN (50 mL), 7-fluoro-benzoxazine 6b (200 mg; 0.72 mmol) and anhydrous K CO (100 mg;
3
2
3
0
.72 mmol) were added. The chromatographic purification provided 7b (242 mg; 80% yield) as a
◦
1
0
δ 1.76 (q, 2H, H-2 , J = 7.3 Hz), 2.22–2.48
yellow light solid. m.p.: 124.0–125.3 C; H-NMR (DMSO-d ):
6
0 0 0 0 0 00
m, 10H, H-3 , H-4 , and H-5 ), 2.67 (t, 2H, H-1 , J = 7.4 Hz), 3.31 (s, 2H, H-7 ), 3.90 (m, 2H, H-3 ), 4.29
m, 2H, H-2 ), 6.68–6.78 (m, 2H, H-6 and H-8 ), 6.95 (t, 1H, H-5 or H-6, J = 7.1 Hz), 7.04 (t, 1H, H-6 or
(
(
0
0
00
00
H-5, J = 7.0 Hz), 7.09 (d, 1H, H-2, J = 1.8 Hz), 7.32 (d, 1H, H-4, J = 8.0 Hz), 7.49 (d, 1H, H-7, J = 7.7 Hz),
0
0
13
7
(
1
.92 (br. s, 1H, H-5 ), and 10.83 (br. s, 1H, H-1) ppm. C-NMR (DMSO-d6):
δ
22.9, 27.6, 53.0 (2X), 53.3
0
2
= 26 Hz), 106.9 (d, 2 JC-F = 23 Hz), 111.8, 114.8, 118.6 (d, J
3
2X), 58.1, 67, 104.0 (d, J
21.2, 122.6, 123.2, 125.6 (d, J
= 18 Hz),
C-F
C-F
0
3
4
= 9 Hz), 127.7, 136.7, 143.2, 144, 147.6, 147.8 (d, JC-F = 13 Hz), 159.5
C-F
1
+
(
d, J
= 240 Hz), and 168.2 ppm. HRMS: (EI) Calculated for C H FN O (M ) = 437.5318. Found:
C-F
25 29
4
2
4
37.5301.
1
-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7c)
To a solution of 3-(1H-3-indolyl)-propyl-4-methylbencensulfonate 1a (237 mg; 0.72 mmol), in
dry CH CN (50 mL), 6-fluoro-benzoxazine 6c (200 mg; 0.72 mmol) and anhydrous K CO (100 mg;
3
2
3
0
.72 mmol) were added. The crude mixture was column chromatographed to provide pure 7c (269
◦
1
0
mg; 89% yield) as a yellow light solid. m.p.: 123.8–125.1 C; H-NMR (DMSO-d ):
J = 7.1 Hz), 2.30–2.54 (m, 10H, H-3 , H-4 , and H-5 ), 2.73 (t, 2H, H-1 , J = 7.4 Hz), 3.40 (s, 2H, H-7 ),
δ
1.83 (q, 2H, H-2 ,
6
0
0
0
0
0
0
0
00
00
00
3
1
.98 (t, 2H, H-3 , J = 4.0 Hz), 4.30 (t, 2H, H-2 , J = 3.7 Hz), 6.91–6.97 (m, 2H, H-7 and H-8 ), 7.01 (td,
H, H-5 or H-6, J = 7.3 Hz, J = 1.0 Hz), 7.11 (td, 1H, H-6 or H-5, J = 7.0 Hz, J = 1.1 Hz), 7.15 (d, 1H,
H-2, J = 2.1 Hz), 7.38 (d, 1H, H-4, J = 8.0 Hz), 7.55 (d, 1H, H-7, J = 7.7 Hz), 7.95 (br. d, 1H, H-5 , J = 11.7
o
m
o
m
0
0
13
Hz), and 10.81 (br. s, 1H, H-1) ppm. C-NMR (DMSO-d6):
δ
22.9, 27.6, 53.0 (2X), 53.3 (2X), 58.1, 66.1,
0
2
= 29 Hz), 111.9 (d, 2 JC-F = 25 Hz), 114.8, 117.9 (d, J
3
1
1
10.3 (d, J
= 9 Hz), 118.5, 118.7, 121.2, 122.6,
= 2 Hz), 155.5 (d, J
C-F
C-F
0
26.9 (d, 3 JC-F = 12 Hz), 127.7, 128.7, 129.9, 136.7, 138.3, 143 (d, J
4
1
= 239 Hz),
C-F
C-F
+
and 168.7 ppm. HRMS: (EI) Calculated for C H FN O (M ) = 437.5318. Found: 437.5084.
25
29
4
2
1
-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7g)
To a solution of 3-(5-Fluoro-1H-3-indolyl)-propyl-4-methylbencensulfonate 1b (268 mg; 0.77 mmol)
in CH CN (50 mL), 1-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide 6a (200 mg;
3
0
.77 mmol) and anhydrous K CO (106 mg; 0.77 mmol) were added. The crude mixture was column
2 3
◦
chromatographed to provide pure 7g (252 mg; 78% yield) as a yellow light solid. m.p.: 168.4–171.3 C;
1
0 0 0 0
H-NMR (DMSO-d ):
δ 1.76 (q, 2H, H-2 , J = 6.9 Hz), 2.30–2.51 (m, 10H, H-3 , H-4 and H-5 ), 2.63
6
0
0
00
00
(
t, 2H, H-1 , J = 7.3 Hz), 3.34 (s, 2H, H-7 ), 3.89 (m, 2H, H-3 ), 4.26 (t, 2H, H-2 , J = 3.6 Hz), 6.75 (td, 1H,
0
0
00
00
H-6, J
o
= 10.4 Hz, J
m
= 2.7 Hz), 6.80–6.91 (m, 2H, H-6 , and H-8 ), 7.01 (t, 1H, H-7 , J = 7.4 Hz), 7.18
(
d, 1H, H-2, J = 1.8 Hz), 7.23 (dd, 1H, H-7, J
o
= 10.2 Hz, J
m
= 2.3 Hz), 7.30 (dd, 1H, H-4, J
o
= 8.9 Hz,
0
0
13
Jm = 4.5 Hz), 7.88 (br. s, 1H, H-5 ), and 10.89 (s, 1H, H-1) ppm. C-NMR (DMSO-d ):
δ
22.6, 27.2, 52.7
6
0
2
2
3
(2X), 53.1 (2X), 57.7, 66.1, 103.4 (d, J
= 23 Hz), 104.2, 109.3 (d, J
= 26 Hz),112.6 (d, J
= 9.9
C-F
C-F
C-F
0
4
3
Hz), 115.1 (d, J
1
= 4.9 Hz), 117.2, 120.1, 124.3, 124.9, 125.7, 126.6, 127.9 (d, JC–F = 9.9 Hz), 133.4,
C-F
1
+
46.7, 147.9, 157.3 (d, J
= 234 Hz), and 168.2 ppm. HRMS: (EI) Calculated for C H FN O (M ) =
C-F 25 29 4 2
4
37.5318. Found: 437.5312.

Molecules 2020, 25, 4614
15 of 30
1
-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7h)
To a solution of 3-(5-Fluoro-1H-3-indolyl)-propyl-4-methylbencensulfonate 1b (268 mg; 0.72 mmol)
in CH CN (50 mL), 1-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide
3
6b (200 mg; 0.72 mmol) and anhydrous K CO (100 mg; 0.72 mmol) were added. The crude mixture
2 3
was column chromatographed to provide pure 7h (205 mg; 65% yield) as a yellow light solid. m.p.:
◦ 0 0 0
68.1–169.9 C; H-NMR (DMSO-d ): δ 1.75 (q, 2H, H-2 , J = 7.0 Hz), 2.25–2.52 (m, 10H, H-3 , H-4 and
6
H-5 ), 2.64 (t, 2H, H-1 , J = 7.4 Hz), 3.35 (s, 2H, H-7 ), 3.92 (t, 2H, H-3 , J = 3.8 Hz), 4.25 (t, 2H, H-2 ,
1
1
0
0
0
00
00
0
0
00
J = 3.7 Hz), 6.85–6.93 (m, 3H, H-6, H-6 , and H-8 ), 7.19 (d, 1H, H-2, J = 2.1 Hz), 7.24 (dd, 1H, H-7,
00
Jo = 10.2 Hz, J = 2.3 Hz), 7.31 (dd, 1H, H-4, J = 8.8 Hz, J = 4.5 Hz), 7.92 (br. d, 1H, H-5 , J = 9.6 Hz),
m o m
1
3
and 10.88 (s, 1H, H-1) ppm. C-NMR (DMSO-d6):
δ
22.7, 27.5, 52.9 (2X), 53.2 (2X), 57.8, 66.1, 103.4 (d,
0
00
2
000
2
2
= 26 Hz), 110.3 (d, JC-F = 28 Hz), 112.0 (d, 2 JC-F = 25 Hz), 112.6 (d,
JC-F = 23 Hz), 109.3 (d, J
C-F
0
00
3
3J
= 9.9 Hz), 115.1 (d, J
4
= 4.9 Hz), 117.9 (d, JC-F = 9 Hz), 124.9, 126, 126.9 (d, JC–F = 9 Hz), 127.9
C-F
3
C-F
0
00
0
0
1
4
1
(
d, 3 JC–F = 9.9 Hz), 128.5, 133.3, 143.0 (d, J
= 2.3 Hz), 155.5 (d, J
= 234 Hz), 157.0 (d, J
=
C–F
C-F
C-F
+
2
31 Hz), and 168.7 ppm. HRMS: (EI) Calculated for C H F N O (M ) = 455.5222. Found: 455.5198.
25
28
2
4
2
1
-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7i)
To a solution of 3-(5-Fluoro-1H-3-indolyl)-propyl-4-methylbencensulfonate 1b (268 mg; 0.72 mmol)
in CH CN (50 mL), 1-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide 6c
3
(
200 mg; 0.72 mmol) and anhydrous K CO (100 mg; 0.72 mmol) were added to afford pure 7i (196
2
3
◦
1
0
mg; 62% yield) as a yellow light solid. m.p.: 166.8–168.1 C; H-NMR (DMSO-d ):
J = 7.0 Hz), 2.22–2.55 (m, 10H, H-3 , H-4 and H-5 ), 2.64 (t, 2H, H-1 , J = 7.4 Hz), 3.35 (s, 2H, H-7 ), 3.92
δ
1.75 (q, 2H, H-2 ,
6
0
0
0
0
0
00
00
00
00
(
(
t, 2H, H-3 , J = 3.8 Hz), 4.25 (t, 2H, H-2 , J = 3.7 Hz), 6.83–6.94 (m, 3H, H-6, H-7 and H-8 ), 7.19
d, 1H, H-2, J = 2.1 Hz), 7.24 (dd, 1H, H-7, J
o
= 10.2 Hz, J
m
= 2.3 Hz), 7.31 (dd, 1H, H-4, J
o
= 8.8 Hz,
0
0
13
Jm = 4.5 Hz), 7.92 (br. d, 1H, H-5 , J = 9.6 Hz), and 10.88 (s, 1H, H-1) ppm. C-NMR (DMSO-d6):
δ
2
0
2
2
22.7, 27.5, 52.9 (2X), 53.2 (2X), 57.8, 66.1, 103.4 (d, J
= 23 Hz), 109.3 (d, J
= 26 Hz), 110.3 (d,
= 4.9 Hz), 117.9
C-F
3
C-F
4
0
0
000
J
= 28 Hz), 112.0 (d, 2 JC-F = 24.7 Hz), 112.6 (d, J
= 9.9 Hz), 115.1 (d, J
C-F
C-F
C-F
0
0
0
000
3
3
3
(
(
d, JC-F = 8.8 Hz), 124.9, 126.9 (d, JC–F = 13 Hz), 126, 127.9 (d, JC–F = 9.9 Hz), 129, 133.3, 143.0
d, JC-F = 2.2 Hz), 155.5 (d, J
0
0
4
1
= 234 Hz), 157.0 (d, 1 JC–F = 231 Hz), and 168.6 ppm. HRMS: (EI)
C-F
+
Calculated for C H F N O (M ) = 455.5222. Found: 455.5205.
25
28
2
4
2
1
-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7m)
To a solution of 3-(5-Bromo-1H-3-indolyl)-propyl-4-methylbencensulfonate 1c (314 mg; 0.77 mmol)
in CH CN (50 mL), 1-(2,3-Dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide 6a (200 mg;
3
0
.77 mmol) and anhydrous K CO (106 mg; 0.77 mmol) were added. The crude mixture was column
2 3
◦
chromatographed to provide pure 7m (157 mg; 42% yield) as a yellow light solid. m.p.: 90.5–92.4 C;
1
0 0 0 0
H-NMR (DMSO-d ):
δ 1.74 (q, 2H, H-2 , J = 7.1 Hz), 2.25–2.51 (m, 10H, H-3 , H-4 , and H-5 ), 2.65 (t,
6
0
0
00
00
2
6
H, H-1 , J = 7.1 Hz), 3.34 (s, 2H, H-7 ), 3.90 (t, 2H, H-3 , J = 7.1 Hz), 4.27 (t, 2H, H-2 , J = 7.1 Hz),
0
0
00
00
.81–6.90 (m, 2H, H-6 , and H-8 ), 7.02 (t, 1H, H-7 , J = 7.6 Hz), 7.10–7.20 (m, 2H, H-2, and H-6), 7.30
00
(
d, 1H, H-7, J = 8.5 Hz), 7.68 (d, 1H, H-4, J = 1.6 Hz), 7.85 (br. s, 1H, H-5 ), and 11.04 (s, 1H, H-1) ppm.
1
3
C-NMR (DMSO-d6):
δ 22.4, 27.6, 52.9 (2X), 53.2 (2X), 57.6, 66.6, 111.2, 113.9, 114.7, 117.2, 120.1, 121.1,
1
23.6, 124.3, 124.5, 125.7, 126, 126.6, 128.5, 129.6, 135.3, 146.7, and 168.2 ppm. HRMS: (EI) Calculated for
+
C H BrN O (M ) = 498.4374. Found: 498.4362.
25
29
4
2
1
-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7n)
To a solution of 3-(5-Bromo-1H-3-indolyl)-propyl-4-methylbencensulfonate 1c (294 mg; 0.72 mmol)
in CH CN (50 mL), 1-(7-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide
3

Molecules 2020, 25, 4614
16 of 30
6b (200 mg; 0.72 mmol) and anhydrous K CO (100 mg; 0.72 mmol) were added. The crude mixture
2 3
was column chromatographed to provide pure 7n (180 mg; 50% yield) as a yellow light solid. m.p.:
◦ 0 0 0
6.1–98.1 C; H-NMR (DMSO-d ): δ 1.73 (q, 2H, H-2 , J = 6.6 Hz), 2.18–2.51 (m, 10H, H-3 , H-4 and
6
H-5 ), 2.64 (t, 2H, H-1 , J = 7.0 Hz), 3.32 (s, 2H, H-7 ), 3.90 (m, 2H, H-3 ), 4.29 (m, 2H, H-2 ), 6.65–6.81
1
9
0
0
0
00
00
00 00
(
m, 2H, H-6 and H-8 ), 7.10–7.20 (m, 2H, H-2, and H-6), 7.30 (d, 1H, H-7, J = 8.5 Hz), 7.68 (d, 1H, H-4,
0
0
13
J = 1.0 Hz), 7.95 (br. s, 1H, H-5 ), and 11.05 (s, 1H, H-1) ppm. C-NMR (DMSO-d ):
2X), 53.2 (2X), 57.6, 66.6, 110.3 (d, J
δ 22.4, 27.6, 52.9
6
0
2
2
(
(
1
= 28.5 Hz), 111.2, 112.0 (d, J
= 25 Hz),113.8, 114.7, 117.9
C-F
C-F
0
3
3
4
d, J
= 8.8 Hz), 121.1, 123.6, 124.4, 126.9 (d, J
= 12 Hz), 129.6, 135.3, 143.0 (d, J
= 2.3 Hz),
C-F
C-F
C-F
+
1
55.5 (d, J
= 233 Hz), and 168.7 ppm. HRMS: (EI) Calculated for C H BrFN O (M ) = 516.4278.
C-F
25 28
4
2
Found: 516.4267.
1
-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7o)
To a solution of 3-(5-Bromo-1H-3-indolyl)-propyl-4-methylbencensulfonate 1c (294 mg; 0.72 mmol)
in CH CN (50 mL), 1-(6-Fluoro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-(1-piperazinyl) ethanamide
3
6c (200 mg; 0.72 mmol) and anhydrous K CO (100 mg; 0.72 mmol) were added. The crude mixture
2 3
was column chromatographed to provide pure 7o (251 mg; 70% yield) as a yellow light solid. m.p.:
◦ 0 0 0
7.2–98.8 C; H-NMR (DMSO-d ): δ 1.74 (q, 2H, H-2 , J = 6.7 Hz), 2.21–2.52 (m, 10H, H-3 , H-4 and
6
H-5 ), 2.65 (t, 2H, H-1 , J = 7.0 Hz), 3.35 (s, 2H, H-7 ), 3.92 (m, 2H, H-3 ), 4.24 (m, 2H, H-2 ), 6.84–6.93
1
9
0
0
0
00
00
00 00
(
m, 2H, H-7 and H8 ), 7.10–7.21 (m, 2H, H-2 and H-6), 7.30 (d, 1H, H-7, J = 8.6 Hz), 7.68 (s, 1H, H-4),
0
0
13
7
5
.85 (br. d, 1H, H-5 , J = 7.8 Hz), and 11.04 (s, 1H, H-1) ppm. C-NMR (DMSO-d ):
2.9 (2X), 53.2 (2X), 57.7, 66.1, 110.3 (d, J
δ
22.4, 27.6,
6
0
2
2
= 28.5 Hz), 111.2, 112.0 (d, J
= 22.5 Hz), 113.8, 114.7,
C-F
C-F
0
3
3
1
1
17.9 (d, J
= 8.8 Hz), 121.1, 123.6, 124.4, 126, 126.9 (d, JC-F = 12.1 Hz), 128.5, 129.6, 132.2, 135.3,
C-F
4
1
38.2, 143.0 (d, J
= 2.2 Hz), 155.5 (d, J
= 233 Hz), and 168.7 ppm. HRMS: (EI) Calculated for
C-F
C-F
+
C H BrFN O (M ) = 516.4278. Found: 516.4272.
25
28
4
2
3
.1.4. General procedure for the synthesis of 2,3-Dihydro-benzo[1,4]oxazin-4-yl)-2-{4-[3-(1H-3-
indolyl)-propyl]-1-piperazinyl} ethanamides 7d–f and 7j–l. Method 2
Synthesis of 1-(6-Chloro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-
piperazinyl} ethanamide. Compound (7d) as a Model
To a stirred solution of 3-(3-Piperazin-1-yl-propyl)-1H-indol 9a (224 mg; 0.92 mmol), in dry
CH CN (50 mL), 2-Chloro-1-(6-chloro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4e (227 mg;
3
0
8
.92 mmol), and anhydrous K CO (127 mg; 0.92 mmol) were added. The mixture was heated at
2 3
◦
0 C for 24 h. After this time, the resulting mixture was poured into water (100 mL) and extracted
50 mL), dried over anhydrous Na SO , and concentrated under reduced pressure.
with EtOAc (4
×
2
4
The organic crude was purified by column chromatography EtOAc/MeOH (6:1) to give pure 7d (290 mg;
7
J = 7.0 Hz), 2.20–2.52 (m, 10H, H-3 , H-4 and H-5 ), 2.68 (t, 2H, H-1 , J = 7.3 Hz), 3.32 (s, 2H, H-7 ), 3.91
◦
1
0
1.6% yield) as a yellow light solid. m.p.: 135.3–136.1 C; H-NMR (DMSO-d ):
δ
1.78 (q, 2H, H-2 ,
6
0
0
0
0
0
00
00
00
(t, 2H, H-3 , J = 3.7 Hz), 4.27 (t, 2H, H-2 , J = 3.8 Hz), 6.90 (d, 1H, H-8 , J = 8.8 Hz), 6.96 (t, 1H, H-5
0
0
or H-6, J = 7.3 Hz), 7.02–7.12 (m, 3H, 2-H, H-6 or H-5 and H-7 ), 7.34 (d, 1H, H-4, J = 8.0 Hz), 7.50
00
13
(
d, 1H, H-7, J = 7.8 Hz), 8.09 (br. s, 1H, H-5 ), and 10.75 (br. s, 1H, H-1) ppm. C-NMR (DMSO-d6):
δ
22.9, 27.6, 39.3, 40.5, 52.9 (2X), 53.2 (2X), 58.0, 66.4, 111.8, 114.9, 118.5, 118.6, 118.7, 121.2, 122.6, 123.5,
+
1
23.6, 125.2, 127.6, 127.7, 136.8, 145.5, and 168.6 ppm. HRMS: (EI) Calculated for C H ClN O (M )
25
29
4
2
=
453.9864. Found: 453.9856.
1
-(6-Bromo-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7e)
To a solution of 3-(3-Piperazin-1-yl-propyl)-1H-indol 9a (241 mg; 0.99 mmol) in CH CN (50 mL),
3
2
-Chloro-1-(6-Bromo-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4d (287 mg; 0.99 mmol) and
anhydrous K CO (137 mg; 0.99 mmol) were added to afford 7e (298 mg; 62.6% yield) as a yellow light
2
3

Molecules 2020, 25, 4614
17 of 30
◦
1
0
δ 1.77 (q, 2H, H-2 , J = 6.7 Hz), 2.27–2.51 (m, 10H,
solid. m.p.: 132.7–134.1 C; H-NMR (DMSO-d ):
6
0
0
0
0
0
00
H-3 , H-4 and H-5 ), 2.68 (t, 2H, H-1 , J = 7.3 Hz), 3.32 (s, 2H, H-7 ), 3.91 (m, 2H, H-3 ), 4.27 (t, 2H,
H-2 , J = 3.8 Hz), 6.86 (d, 1H, H-8 , J = 8.8 Hz), 6.96 (td, 1H, H-5 or H-6, J
.05 (td, 1H, H-6 or H-5, J = 7.3 Hz, J = 1.0 Hz), 7.10 (d, 1H, H-2, J = 1.9 Hz), 7.19 (dd, 1H, H-7 ,
00
00
o m
= 7.3 Hz, J = 1.0 Hz),
0
0
7
o
m
Jo = 8.7 Hz, J
m
= 2.1 Hz), 7.33 (d, 1H, H-4, J = 8.0 Hz), 7.50 (d, 1H, H-7, J = 7.8 Hz), 8.20 (br. s, 1H,
00
13
H-5 ), and 10.77 (br.s, 1H, H-1) ppm. C-NMR (DMSO-d6):
δ 22.9, 27.6, 39.3, 40.5, 52.9 (2X), 53.2 (2X),
5
8.0, 66.5, 111.1, 111.8, 114.8, 118.5, 118.7, 119.1, 121.2, 122.6, 125.4, 126.3, 127.6, 128.1, 136.7, 146.0, and
+
1
68.6 ppm. HRMS: (EI) Calculated for C H BrN O (M ) = 498.4373. Found: 498.4364.
25 29 4 2
1
-(6-Methoxy-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7f)
To a solution of 3-(3-Piperazin-1-yl-propyl)-1H-indol 9a (282 mg; 1.16 mmol) in CH CN (50 mL),
3
2
-Chloro-1-(6-Methoxy-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4f (280 mg; 1.16 mmol) and
anhydrous K CO (160 mg; 1.16 mmol) were added to provide pure 7f (400 mg; 79% yield) as a yellow
2
3
◦
1
0
δ 1.75 (q, 2H, H-2 , J = 7.0 Hz), 2.23–2.49 (m,
light solid. m.p.: 123.6–125.1 C; H-NMR (DMSO-d ):
6
0 0 0 0 0 00
1
0H, H-3 , H-4 and H-5 ), 2.65 (t, 2H, H-1 , J = 7.4 Hz), 3.31 (s, 2H, H-7 ), 3.65 (s, 3H, H-6 ), 3.86 (t, 2H,
00
00
00
H-3 , J = 3.8 Hz), 4.18 (t, 2H, H-2 , J = 3.8 Hz), 6.63 (dd, 1H, H-7 , J
H, H8 , J = 8.9 Hz), 6.94 (t, 1H, H-5 or H-6, J = 7.0 Hz), 7.03 (t, 1H, H-6 or H-5, J = 6.9 Hz), 7.08 (d,
o
= 8.9 Hz, J
m
= 2.3 Hz), 6.79 (d,
00
1
1
0
0
H, H-2, J = 1.9 Hz), 7.31 (d, 1H, H-4, J = 8.0 Hz), 7.48 (d, 1H, H-7, J = 7.8 Hz), 7.61 (br. s, 1H, H-5 ),
22.8, 27.6, 39.3, 40.5, 53.0 (2X), 53.2 (2X), 55.8,
8.0, 66.1, 109.3, 111.7, 114.8, 117.3, 118.4, 118.7, 121.1, 122.5, 126.8, 127.4, 127.6, 136.7, 140.7, 152.6, and
and 10.74 (br. s, 1H, H-1) ppm. 13C-NMR (DMSO-d6):
δ
5
1
+
68.3 ppm. HRMS: (EI) Calculated for C H N O (M ) = 449.5673. Found: 449.5669.
26 32 4 3
1
-(6-Chloro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7j)
To a solution of 5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indol 9b (340 mg; 1.3 mmol) in CH CN
3
(
50 mL), 2-Chloro-1-(6-chloro-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4e (320 mg; 1.3 mmol)
and anhydrous K CO (180 mg 1.3 mmol) were added to afford pure compound 7j (540 mg; 91% yield)
2
3
◦
1
0
0
as a yellow light solid. m.p.: 120.5–122.8 C; H-NMR (DMSO-d ):
.21–2.50 (m, 10H, H-3 , H-4 and H-5 ), 2.64 (t, 2H, H-1 , J = 7.4 Hz), 3.33 (s, 2H, H-7 ), 3.91 (t, 2H,
H-3 ,J = 3.7 Hz), 4.27 (t, 2H, H-2 , J = 3.7 Hz), 6.85–6.92 (m, 2H, H-6 and H-8 ), 7.07 (dd, 1H, H-7 ,
δ
1.75 (q, 2H, H-2 , J = 7.1 Hz),
6
0
0
0
0
2
00
00
00
00
Jo = 8.7 Hz, J
m
= 2.3 Hz), 7.19 (d, 1H, H-2, J = 1.7 Hz), 7.24 (dd, 1H, H-7, J
o
= 10.2 Hz, J
m
= 2.3 Hz), 7.31
0
0
13
(
(
dd, 1H, H-4, J
DMSO-d6):
26.3 Hz), 112.6 (d, J
o
= 8.9 Hz, J
m
= 4.7 Hz), 8.07 (br. s, 1H, H-5 ), and 10.88 (br. s, 1H, H-1) ppm. C-NMR
0
2
= 23 Hz), 109.3 (d, 2 JC-F
δ
22.7, 27.5, 39.3, 40.5, 52.9 (2X), 53.2 (2X), 57.8, 66.4, 103.4 (d, J
C-F
3
4
=
= 9.9 Hz), 114.4, 115.1 (d, J
= 5.4 Hz), 118.7, 123.5, 124.8, 125.2, 127.5, 127.9
C-F
C-F
0
3
1
(
d, J
= 9.3 Hz), 133.3, 145.5, 157.0 (d, J
= 231 Hz), and 168.6 ppm. HRMS: (EI) Calculated for
C–F
C–F
+
C H ClFN O (M ) = 471.9769. Found: 471.9767.
25
28
4
2
1
-(6-Bromo-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl}
ethanamide (7k)
To a solution of 5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indol 9b (784 mg; 3 mmol) in CH CN (50
3
mL), 2-Chloro-1-(6-bromo-2,3-dihydro-benzo[b][1,4]oxazin-4-yl) ethanamide 4d (870 mg; 3 mmol) and
anhydrous K CO (180 mg 1.3 mmol) were added to afford pure compound 7k (1130 mg; 75.3% yield)
2
3
◦
1
0
0
as a yellow light solid. m.p.: 102.8–103.9 C; H-NMR (DMSO-d ):
.26–2.56 (m, 10H, H-3 , H-4 and H-5 ), 2.69 (t, 2H, H-1 , J = 7.3 Hz), 3.38 (s, 2H, H-7 ), 3.97 (t, 2H,
H-3 ,J = 3.4 Hz), 4.33 (t, 2H, H-2 , J = 3.6 Hz), 6.89–6.99 (m, 2H, H-6, and H-8 ), 7.13 (dd, 1H, H-7 ,
δ
1.80 (q, 2H, H-2 , J = 7.0 Hz),
6
0
0
0
0
2
00
00
00
00
Jo = 8.7 Hz, J
m
= 2.3 Hz), 7.24 (d, 1H, H-2, J = 1.9 Hz), 7.30 (dd, 1H, H-7, J
o
= 10.2 Hz, J
m
= 2.3 Hz), 7.37
0
0
13
(
dd, 1H, H-4, J
o
= 8.8 Hz, J
m
= 4.5 Hz), 8.13 (br. s, 1H, H-5 ), and 10.93 (br. s, 1H, H-1) ppm. C-NMR
2
(
DMSO-d ):
JC-F = 26 Hz), 112.6 (d, J
δ
22.7, 27.5, 39.3, 40.5, 52.9 (2X), 53.2 (2X), 57.9, 66.4, 103.4 (d, J
= 23.1 Hz), 109.3 (d,
C-F
6
0
2
3
4
= 9.9 Hz), 115.1 (d, J
= 4.9 Hz), 118.7, 123.4, 123.5, 124.8, 125.3, 127.6,
C-F
C-F

Molecules 2020, 25, 4614
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0
1
1
27.9 (d, 3 JC–F = 9.3 Hz), 133.3, 145.5, 157.0 (d, J
= 231 Hz), and 168.6 ppm. HRMS: (EI) Calculated
C–F
+
for C H BrFN O (M ) = 516.4278. Found: 516.4270.
25
28
4
2
1
-(6-Methoxy-2,3-dihydro-benzo[b][1,4]oxazin-4-yl)-2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-
piperazinyl} ethanamide (7l)
To a solution of 5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indol 9b (379 mg; 1.45 mmol) in
CH CN (50 mL), 2-chloro-1-(6-methoxy-2,3-dihydro-benzo[b][1,4] oxazin-4-yl) ethanamide 4f (350
3
mg; 1.45 mmol) and anhydrous K CO (200 mg; 1.45 mmol) were added to afford pure compound
2
3
◦
1
7
2
l
(535 mg; 81.6% yield) as a yellow light solid. m.p.: 94.5–97.3 C; H-NMR (DMSO-d ):
H, H-2 , J = 7.0 Hz), 2.2–2.50 (m, 10H, H-3 , H-4 and H-5 ), 2.64 (t, 2H, H-1 , J = 7.4 Hz), 3.34 (s, 2H,
H-7 ), 3.68 (s, 3H, H-6 ), 3.89 (t, 2H, H-3 ,J = 4.5 Hz), 4.21 (t, 2H, H-2 , J = 3.9 Hz), 6.65 (dd, 1H, H-7 ,
= 2.9 Hz), 6.81 (d, 1H, H-8 , J = 8.9 Hz), 6.89 (td, 1H, H-6, J
d,1H, H-2, J = 2.2 Hz), 7.25 (dd, 1H, H-7, J
4.6 Hz), 7.62 (br. s, 1H, H-5 ), and 10.88 (br. s, 1H, H-1) ppm. C-NMR (DMSO-d ):
δ
1.75 (q,
6
0
0
0
0
0
0
00
00
00
00
00
Jo = 8.9 Hz, J
(
m
o
= 9.2 Hz, J
m
= 2.5 Hz), 7.19
= 8.8 Hz, J
22.7, 27.6,
o
= 10.1 Hz, J
m
= 2.5 Hz), 7.32 (dd, 1H, H-4, J
o
m
0
0
13
=
δ
6
0
2
2
0
3
9.3, 40.5, 53.1 (2X), 53.3 (2X), 55.9, 57.9, 66.2, 103.4 (d, J
= 23.2 Hz), 109.3 (d, JC-F = 26 Hz), 109.3,
C-F
12.6 (d, 3JC-F = 10 Hz), 115.2 (d, J
4
= 4.4 Hz), 117.4, 124.9, 126.9, 127.9 (d, JC–F = 10 Hz), 128.6,
3
1
1
C-F
1
33.4, 140.7, 152.7, 157.0 (d, J
= 231 Hz), and 168.4 ppm. HRMS: (EI) Calculated for C H FN O
26 31 4 3
C–F
+
(M ) = 467.5577. Found: 467.5567.
3
.1.5. General Procedure for the Synthesis of N-(2-morpholin-4-yl-ethyl)-benzamides nitro-fluorinated
Derivatives 10a–d
4
-Fluoro-N-(2-morpholin-4-yl-ethyl)-2-nitro-benzamide (10a) as a Model
To a solution of 4-Fluoro-2-nitro-benzoyl chloride (828 mg; 4.07 mmol) in dry THF (50 mL),
◦
2
-morpholin-4-yl-ethylamine (0.53 mL; 4.07 mmol) was added. The mixture was stirred at 0 C under
nitrogen atmosphere for 2 h. After this time, saturated solution of NaHCO was added (100 mL) and
3
the mixture was extracted with EtOAc (3
×
60 mL). The organic layers were dried over anhydrous
Na SO filtered, and concentrated under vacuum conditions to give a crude form, which was purified
2
4
by column chromatography with EtOAc/MeOH (6:1) as eluent to give 10a (963 mg; 80% yield) as a
◦
1
0
yellow light solid. m.p.: 149.8–151.5 C; H-NMR (acetone-d ):
(
δ
2.32 (t, 4H, H-4 , J = 4.6 Hz), 2.42
6
0
0
0
t, 2H, H-3 , J = 6.3 Hz), 3.35 (q, 2H, H-2 , J = 6.3 Hz), 3.46 (t, 4H, H-5 , J = 4.5 Hz), 7.45 (td, 1H, H-5,
0
Jo = 8.2 Hz, J
m
= 2.5 Hz), 7.59 (dd, 1H, H-6, J
o
3
= 8.5 Hz, J
m
= 5.5 Hz), 7.65 (br. s, 1H, H-1 ), and 7.70
1
(dd, 1H, H-3, J
o
= 8.5 Hz, J
m
= 2.6 Hz) ppm. C-NMR (acetone-d6):
= 22 Hz), 129.1 (d, 4JC-F = 3.3 Hz), 130.5 (d, 3JC-F = 9 Hz), 148.1 (d,
C-F
= 8.2 Hz), 161.7 (d, 1JC-F = 251 Hz), and 164.2 ppm. HRMS: (EI) Calculated for C H FN O
4
δ
36.1, 53 (2X), 56.6, 66 (2X), 111.3
0
(
3
d, 2J
= 27 Hz), 119.6 (d, 2 J
C-F
0
J
C-F
+
13 16
3
(M ) = 298.1203. Found: 298.1198.
4
-Fluoro-N-(2-morpholin-4-yl-ethyl)-3-nitro-benzamide (10b)
-Fluoro-3-nitro-benzoyl chloride (553 mg; 2.72 mmol), and 2-morpholin-4-yl-ethylamine (0.35 mL;
4
◦
1
2
.72 mmol), to afford 10b (712 mg; 88% yield) as a yellow light solid. m.p.: 132.5–133.5 C; H-NMR
0
0
0
(acetone-d ):
δ
2.32 (t, 4H, H-4 , J = 4.4 Hz), 2.43 (t, 2H, H-3 , J = 6.6 Hz), 3.37–3.50 (m, 6H, H-2 and
6
0
0
H-5 ), 7.48 (m, 1H, H-5), 7.92 (br. s, 1H, H-1 ), 8.16 (m, 1H, H-2), and 8.45 (dd, 1H, H-6, J
Jm = 2.3 Hz) ppm
o
= 7.2 Hz,
= 21.4 Hz),
= 3.9 Hz),
13
2
0
3
.
C-NMR (acetone-d ):
= 1.6 Hz), 131.6 (d, J
= 266 Hz), and 163 ppm. HRMS: (EI) Calculated for C H FN O (M ) = 298.1203,
13 16 3 4
δ
36.4, 53.1 (2X), 56.9, 66.1 (2X), 118.1 (d, J
6
C-F
0
4
3
2
1
1
24.6 (d, J
56.2 (d, J
= 3.8 Hz), 134.2 (d, J
= 9.9 Hz), 150.4 (d, J
C-F C-F
C-F
C-F
1
+
C-F
Found: 298.1201.
5
-Fluoro-N-(2-morpholin-4-yl-ethyl)-2-nitro-benzamide (10c)
-Fluoro-2-nitro-benzoyl chloride (803 mg; 3.94 mmol), and 2-morpholin-4-yl-ethylamine (0.51 mL;
5
◦
1
3
.94 mmol), to afford 10c (951 mg; 81% yield) as a yellow light solid. m.p.: 103.4–104.5 C; H-NMR

Molecules 2020, 25, 4614
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0
0
0
(
acetone-d ):
δ
2.33 (t, 4H, H-4 , J = 4.6 Hz), 2.44 (t, 2H, H-3 , J = 6.3 Hz), 3.36 (m, 2H, H-2 ), 3.46 (t, 4H,
6
0
0
H-5 , J = 4.5 Hz), 7.23–7.37 (m, 2H, H-3, and H-4), 7.67 (br. s, 1H, H-1 ), and 8.02 (dd, 1H, H-6, J
= 4.8 Hz) ppm. C-NMR (acetone-d ):
16.5 (d, 2 JC-F = 23.6 Hz), 126.8 (d, J
o
= 9.1
13
2
Hz, J
1
1
m
δ
36.1, 53 (2X), 56.5, 66 (2X), 115.6 (d, J
= 9.9 Hz), 136.0 (d, JC-F = 8.8 Hz), 143 (d, J
= 25.3 Hz),
= 3.3 Hz),
C-F
6
C-F
0
0
3
3
4
C-F
1
+
63.9, and 163.9 (d, J
= 256 Hz) ppm. HRMS: (EI) Calculated for C H FN O (M ) = 298.1203.
13 16 3 4
C-F
Found: 298.1201.
2
-Fluor-N-(2-morpholin-4-yl-ethyl)-5-nitro-benzamide (10d)
-Fluoro-5-nitro-benzoyl chloride (825 mg; 4.05 mmol), and 2-morpholin-4-yl-ethylamine (0.53 mL;
2
◦
1
4
.05 mmol), to afford 10d (902 mg; 75% yield) as a yellow light solid. m.p.: 126.4–127.2 C; H-NMR
0 0 0
(
acetone-d6): δ 2.35 (t, 4H, H-4 , J = 4.5 Hz), 2.46 (t, 2H, H-3 , J = 6.3 Hz), 3.43 (q, 2H, H-2 , J = 6.5 Hz),
0 0
.49 (t, 4H, H-5 , J = 4.5 Hz), 7.43 (m, 1H, H-3), 7.66 (br. s, 1H, H-1 ), 8.30 (m, 1H, H-4), and 8.56 (dd,
3
1
3
1H, H-6, J
o
= 6.3 Hz, J
m
= 3 Hz) ppm. C-NMR (acetone-d ):
δ
36.1, 52.9 (2X), 56.2, 66.1 (2X), 117.4
6
0
2
2
4
3
(
d, J
= 26.9 Hz), 123.6 (d, JC-F = 16.5 Hz), 126.3 (d, J
= 4.9 Hz), 127.5 (d, J
= 11 Hz), 144(d,
C-F
C-F
C-F
0
3
1
J
= 9 Hz), 160.4, and 162.8 (d, J
= 259 Hz) ppm. HRMS: (EI) Calculated for C H FN O
C-F 13 16 3 4
C-F
+
(M ) = 298.1203. Found: 298.1201.
3
.1.6. General Procedure for the Synthesis of N-(2-morpholin-4-yl-ethyl)-benzamides
amino-fluorinated Derivatives 11a–d
2
-Amino-N-(2-morpholin-4-yl-ethyl)-4-fluoro-benzamide (11a) as a Model
To a mixture containing water-acetic acid-ethanol (1:1:1), 4-Fluoro-N-(2-morpholin-4-yl-ethyl)-2-
nitro-benzamide 10a (1 g; 3.36 mmol) and iron powder (734 mg; 13.1 mmol) were added. The resulting
◦
mixture was heated and stirred for 3 h at 70 C. After this time, the mixture was filtered to remove excess
metallic iron, transferred to a flask containing a mixture of EtOAc/H O (400 mL, 1:1), and neutralized
2
with NaHCO (10 gr). The aqueous phase was extracted with EtOAc (50 mL
×
3). The organic layer was
3
dried over anhydrous Na SO and concentrated under vacuum to give a crude, which was purified
2
4
by column chromatography with EtOAc/MeOH (6:1) to give 11a (891 mg; 98% yield) as a yellow
◦
1
0
0
light solid. m.p.: 120.3–121.7 C; H-NMR (CDCl ):
δ
2.46 (t, 4H, H-4 , J = 4.4 Hz), 2.55 (t, 2H, H-3 ,
3
0
0
J = 6.1 Hz), 3.46 (q, 2H, H-2 , J = 5.6 Hz), 3.69 (t, 4H, H-5 , J = 4.7 Hz), 5.72 (br. s, 2H, H-2), 6.28–6.35
m, 2H, H-3 and H-5), 6.57 (br. s, 1H, H-1 ), and 7.25 (dd, 1H, H-6, J
C-NMR (CDCl ): δ 36.7, 53.3 (2X), 56.7, 66.9 (2X), 102.3 (d, J
0
(
o
= 9.3 Hz, J
m
= 3.2 Hz) ppm.
0
1
3
2
2
= 24.2 Hz), 103.9 (d, JC-F = 23 Hz),
= 11.7 Hz), 165.3 (d, J
3
C-F
0
4
3
3
1
1
12.4 (d, J
= 2.2 Hz), 129.3 (d, J
= 11 Hz), 151.1 (d, J
= 249 Hz),
C-F
C-F
C-F
C-F
+
and 168.6 ppm. HRMS: (EI) Calculated for C H FN O (M ) = 268.1461. Found: 268.1454.
13
18
3
2
3
-Amino-N-(2-morpholin-4-yl-ethyl)-4-fluoro-benzamide (11b)
4
-Fluoro-N-(2-morpholin-4-yl-ethyl)-3-nitro-benzamide 10b (1 g; 3.36 mmol) and iron powder
◦
(
734 mg; 13.1 mmol) to afford 11b (726 mg; 81% yield) as a yellow light solid. m.p.: 147.4–149.1 C;
H-NMR (DMSO-d ):
.63 (t, 4H, H-5 , J = 4.3 Hz), 5.39 (br. s, 2H, H-3), 6.99–7.15 (m, 2H, H-2 and H-4), 7.31 (dd, 1H, H-6,
Jo = 8.9 Hz, J
1
0
0
0
δ
2.40–2.51 (m, 4H, H-4 ), 2.57 (br. s, 2H, H-3 ), 3.40 (q, 2H, H-2 , J = 6.6 Hz),
6
0
3
0
13
m
= 1.6 Hz), and 6.57 (br. t, 1H, H-1 , J = 5.2 Hz), ppm. C-NMR (DMSO-d ):
6
δ
36.9,
0
2
3
= 3.3 Hz), 116 (d, 3 JC-F = 6 Hz), 131.7
= 242 Hz), and 168.3 ppm. HRMS: (EI)
C-F
5
(
3.7 (2X), 57.8, 66.6 (2X), 115.0 (d, J
= 30 Hz), 115.1 (d, J
= 13.2 Hz), 152.5 (d, J
C-F
C-F
0
4
= 2.8 Hz), 136.7 (d, 2
1
d, J
J
C-F
C-F
+
Calculated for C H FN O (M ) = 268.1461. Found: 268.1458.
13
18
3
2
2
-Amino-N-(2-morpholin-4-yl-ethyl)-5-fluoro-benzamide (11c)
5
-Fluoro-N-(2-morpholin-4-yl-ethyl)-2-nitro-benzamide 10c (1 g; 3.36 mmol) and iron powder
◦
(
734 mg; 13.1 mmol) to afford 11c (697 mg; 78% yield) as a yellow light solid. m.p.: 104.8–105.6 C;
H-NMR (CDCl3):
J = 5.6 Hz), 3.69 (t, 4H, H-5 , J = 4.6 Hz), 5.28 (br. s, 2H, H-2), 6.55–6.63 (br. m, 2H, H-3, and H-1 ),
1
0
0
0
δ
2.47 (t, 4H, H-4 , J = 4.4 Hz), 2.55 (t, 2H, H-3 , J = 6.1 Hz), 3.47 (q, 2H, H-2 ,
0
0

Molecules 2020, 25, 4614
20 of 30
6
.93 (td, 1H, H-6, J
o
= 8.6 Hz, J
m
= 2.9 Hz), and 7.0 (dd, 1H, H-4, J
o
= 9.1 Hz, J
m
= 2.7 Hz) ppm.
13
2
3
C-NMR (CDCl ):
δ
35.9, 53.3 (2X), 56.8, 67 (2X), 113.1 (d, J
= 23 Hz), 116.6 (d, J
= 1.5 Hz), and 155.5 (d, J
= 5.1 Hz), 118.4
3
C-F
C-F
0
0
d, JC-F = 7.3 Hz), 119.5 (d, 2 JC-F = 23 Hz), 144.8 (d, J
3
4
1
= 236 Hz),
C-F
(
C-F
+
1
68.3 ppm. HRMS: (EI) Calculated for C H FN O (M ) = 268.1461. Found: 268.1459.
13 18 3 2
5
-Amino-N-(2-morpholin-4-yl-ethyl)-2-fluoro-benzamide (11d)
2
-Fluoro-N-(2-morpholin-4-yl-ethyl)-5-nitro-benzamide 10d (1 g; 3.36 mmol) and iron powder
◦
(
734 mg; 13.1 mmol) to afford 11d (798 mg; 89% yield) as a yellow light solid. m.p.: 106.8–107.9 C;
H-NMR (CDCl ):
H-2 ), 3.59 (t, 4H, H-5 , J = 4.7 Hz), 6.59 (m, 1H, H-6), 6.78 (m, 1H, H-3), 7.23 (dd, 1H, H-4, J
1
0
0
δ
2.37 (t, 4H, H-4 , J = 4.4 Hz), 2.45 (t, 2H, H-3 , J = 6.1 Hz), 3.24–3.50 (br. m, 4H, H-5
3
0
0
o
= 6.4 Hz,
36.3, 53.2 (2X), 56.5, 67 (2X), 116.6
0
13
J
m
= 3.2 Hz), and 7.28 (br. s, 1H, H-1 ) ppm. C-NMR (CDCl ):
δ
3
0
2
4
= 1.5 Hz), 119.1 (d, 3JC-F = 8.8 Hz), 121.3 (d, 2 JC-F = 13.2 Hz), 143.1
(
(
(
d, J
= 26.4 Hz), 117.0 (d, J
C-F
C-F
0
3
1
d, JC-F = 2.2 Hz), 154.2 (d, J
M ) = 268.1461. Found: 268.1457.
= 238 Hz), and 163.4 ppm. HRMS: (EI) Calculated for C H FN O
13 18 3 2
C-F
+
3
.1.7. General Procedure for the Synthesis of (2-Chloro-acetylamino)-N-(2-morpholin-4-yl-ethyl)
fluorinated Benzamides Derivatives 12a–d
2
-(2-Chloro-acetylamino)-4-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide (12a) as a Model
To a solution of 2-amino-N-(2-morpholin-4-yl-ethyl)-4-fluoro-benzamide 11a (400 mg; 1.5 mmol)
in dry THF (60 mL), 2-chloro-acetylchloride (0.12 mL; 1.5 mmol) was added. The mixture was stirred
◦
at 0 C under nitrogen atmosphere for 2 h. After this time, a saturated solution of NaHCO (100
3
mL) was added. The mixture was extracted with EtOAc (3 X 100 mL). The organic layers were dried
over anhydrous Na SO , filtered, and concentrated under vacuum conditions to give a crude, which
2
4
was purified by column chromatography EtOAc/MeOH (6:1), to give 12a (479 mg; 93% yield) as a
◦
1
0 0
δ 2.48 (t, 4H, H-4 , J = 4.4 Hz), 2.58 (t, 2H, H-3 ,
white solid. m.p.: 124.8–126.3 C; H-NMR (CDCl ):
3
0
0
00
J = 5.9 Hz), 3.51 (q, 2H, H-2 , J = 5.4 Hz), 3.69 (t, 4H, H-5 , J = 4.6 Hz), 4.13 (s, 2H, H-1 ), 6.81 (td, 1H,
= 2.7 Hz), 6.88 (br. s, 1H, H-1 ), 7.46 (dd, 1H, H-5, J
H, H-3, Jo = 11.6 Hz, Jm = 2.7 Hz), and 12.24 (br. s, 1H, H-2) ppm. C-NMR (CDCl ): δ 36, 43.2, 53.3
3
0
H-5, J
o
= 7.7 Hz, J
m
o
= 8.8 Hz, J
m
= 6.1 Hz), 8.42 (dd,
13
1
(
3J
0
2
2
= 28 Hz), 110.6 (d, JC-F = 22 Hz), 116.8 (d, 4JC-F = 3.7 Hz), 128.4 (d,
2X), 56.5, 66.9 (2X), 108.7 (d, J
C-F
0
= 10.3 Hz), 141 (d, 3 JC-F = 12.5 Hz), 164.7 (d, J
1
= 252 Hz), 165.5, and 167.8 ppm. HRMS: (EI)
C-F
C-F
+
Calculated for C H ClFN O (M ) = 344.1177. Found: 344.1173.
15
19
3
3
3
-(2-Chloro-acetylamino)-4-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide (12b)
-Amino-N-(2-morpholin-4-yl-ethyl)-4-fluoro-benzamide 11b (400 mg; 1.5 mmol) and
-chloro-acetylchloride (0.12 mL; 1.5 mmol), to afford 12b (482 mg; 94% yield) as a white solid.
3
2
◦
1
0 0
δ 2.48 (t, 4H, H-4 , J = 4.4 Hz), 2.57 (t, 2H, H-3 , J = 6.1 Hz),
m.p.: 129.1–130.8 C; H-NMR (CDCl ):
3
0
0
00
0
3
7
.50 (q, 2H, H-2 , J = 5.6 Hz), 3.71 (t, 4H, H-5 , J = 4.7 Hz), 4.20 (s, 2H, H-1 ), 6.89 (br. s, 1H, H-1 ),
.14–7.19 (m, 1H, H-5), 7.64–7.68 (m, 1H, H-2), 8.57 (br. s, 1H, H-3), and 8.67 (dd, 1H, H-6, Jo = 7.3 Hz
,
1
3
2
J
m
= 2 Hz) ppm. C-NMR (CDCl ):
δ
36.2, 42.9, 53.3 (2X), 56.6, 66.9 (2X), 115.4 (d, J
= 19.8 Hz),
C-F
3
3
0
0
4
= 8.8 Hz), 125.4 (d, 2 JC-F = 11 Hz), 131.4 (d, 3 JC-F = 2.9 Hz),
1
1
19.5 (d, J
54.3 (d, J
= 1.5 Hz), 125.2 (d, J
C-F
C-F
1
+
= 250 Hz), 164.1, and 166 ppm. HRMS: (EI) Calculated for C H ClFN O (M ) =
C-F
15 19
3
3
3
44.1177. Found: 344.1176.
2
-(2-Chloro-acetylamino)-5-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide (12c)
-Amino-N-(2-morpholin-4-yl-ethyl)-5-fluoro-benzamide 11c (400 mg; 1.5 mmol) and
-chloro-acetylchloride (0.12 mL; 1.5 mmol), to afford 12c (496 mg, 97% yield) as a white solid.
2
2
◦
1
0 0
δ 2.55 (t, 4H, H-4 , J = 4.4 Hz), 2.65 (t, 2H, H-3 , J = 6.1 Hz), 3.57
m.p.: 98.5–99.8 C; H-NMR (CDCl ):
3
0
0
00
0
(
c, 2H, H-2 , J = 5.6 Hz), 3.77 (t, 4H, H-5 , J = 4.7 Hz), 4.20 (s, 2H, H-1 ), 6.93 (br. s, 1H, H-1 ), 7.19–7.27
o m
(m, 2H, H-4 and H-6), 8.61 (dd, 1H, H-3, J = 8.9 Hz, J = 5.1 Hz), and 11.8 (br. s, 1H, H-2) ppm.

Molecules 2020, 25, 4614
21 of 30
1
3
2
4
C-NMR (CDCl ):
δ
36.1, 43.1, 53.3 (2X), 56.5, 66.9 (2X), 113.3 (d, J
= 24 Hz), 119.2 (d, J
= 22 Hz),
3
C-F
C-F
0
0
3
2
3
1
1
1
22.7 (d, J
= 5.9 Hz), 123.6 (d, J
= 7.3 Hz), 134.8 (d, J
= 2.9 Hz), 158.2 (d, J
= 245 Hz),
C-F
C-F
C-F
C-F
+
65.1, and 167.3 ppm. HRMS: (EI) Calculated for C H ClFN O (M ) = 344.1177. Found: 344.1164.
15
19
3
3
5
-(2-Chloro-acetylamino)-2-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide (12d)
-Amino-N-(2-morpholin-4-yl-ethyl)-2-fluoro-benzamide 11d (400 mg; 1.5 mmol) and
-chloro-acetylchloride (0.12 mL; 1.5 mmol), to afford 12d (483 mg; 94% yield) as a white solid.
5
2
◦
1
0 0
δ 2.48 (t, 4H, H-4 , J = 4.2 Hz), 2.57 (t, 2H, H-3 , J = 5.9 Hz),
m.p.: 144.1–145.1 C; H-NMR (CDCl ):
3
0
0
00
3
7
.54 (q, 2H, H-2 , J = 5.4 Hz), 3.69 (t, 4H, H-5 , J = 4.4 Hz), 4.15 (s, 2H, H-1 ), 7.05–7.13 (m, 1H, H-3),
0
.47 (br. s, 1H, H-1 ), 7.9 (dd, 1H, H-4, J
o
= 6.5 Hz, J
m
= 2.7 Hz), 8.03–8.09 (m, 1H, H-6), and 8.70 (br. s,
13
2
1H, H-5) ppm. C-NMR (CDCl ):
δ
36.4, 42.9, 53.2 (2X), 56.3, 67 (2X), 116.9 (d, J
= 2.2 Hz), 125.2 (d, J
= 26.4 Hz), 121.5
= 2.9 Hz), 157.4
3
C-F
0
0
2
= 13.2 Hz), 123.1 (d, 4J
3
3
(
(
d, J
d, J
= 8.8 Hz), 133.9 (d, J
C-F
C-F
C-F
C-F
1
+
= 246 Hz), 162.6, and 164.4 ppm. HRMS: (EI) Calculated for C H ClFN O (M ) = 344.1177.
15 19 3 3
C-F
Found: 344.1173.
3
.1.8. General Procedure for the Synthesis of (2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-
acetylamino)-N-(2-morpholin-4-yl-ethyl)-fluorinated Benzamides Derivatives 13a–l
4
-Fluoro-2-(2-{4-[3-(5-fluor-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-
ethyl) Benzamide (13a) as a Model
To a stirred solution of 5-fluoro-3-(3-piperazin-1-yl-propyl)-1H-indole 9b (152 mg; 0.58 mmol),
in dry CH CN (50 mL), 2-(2-chloro-acetylamino)-4-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12a
3
(
199 mg; 0.58 mmol) and anhydrous K CO (80 mg; 0.58 mmol) were added. The mixture was heated
2 3
◦
at 80 C for 24 h. After this time, the resulting mixture was poured into water (100 mL), extracted
with EtOAc (4 50 mL), dried over anhydrous Na SO , and concentrated under reduced pressure.
The organic crude was purified by column chromatography EtOAc/MeOH (6:1) to give 13a (298
mg; 62.6%) as a yellow light solid. m.p.: 91.3–93.1 C; H-NMR (DMSO-d ):
J = 7.4 Hz), 2.26–2.56 (m, 16H, H-3 , H-4 , H-5 , H-9 and H-1 ), 2.66 (t, 2H, H-1 , J = 7.3 Hz), 3.13
×
2
4
◦
1
0
δ 1.78 (q, 2H, H-2 ,
6
0
0
0
000
00
000
0
0
0
(
(
s, 2H, H-6 ), 3.45–3.62 (m, 6H, H-8 , and H-2 ), 6.90 (td, 1H, H-6, J
o
= 9.2 Hz, J
m
= 2.5 Hz), 7.01
0
0
td, 1H, H-5 , Jo = 8.4 Hz, J
m
= 2.7 Hz), 7.20 (d, 1H, H-2, J = 2.1 Hz), 7.25 (dd, 1H, H-7, J
= 4.7 Hz), 7.68–7.76 (m, 1H, H-3 ), 8.40 (dd, 1H, H-6 ,
Jm = 2.6 Hz), 8.62 (t, 1H, H-7 , J = 5.5 Hz), 10.87 (s, 1H, H-1), and 12.24 (br. s, 1H, H-7 )
o
= 10.2 Hz,
0
0
00
Jm = 2.5 Hz), 7.32 (dd, 1H, H-4, J
o
= 8.8 Hz, J
m
0
0
0
Jo = 12.3 Hz
,
ppm. 1 C-NMR (DMSO-d6):
3
δ
22.2, 27.0, 36.4, 52.4 (2X), 53.0 (2X), 53.2 (2X), 57.1, 57.5, 61.8, 66.1 (2X),
0
00
000
02.7 (d, 2JC-F = 23.1 Hz), 106.5 (d, J
2
= 28.2 Hz), 108.8 (d, 2 JC-F = 26 Hz), 109.1 (d, 2 JC-F = 22.3
1
C-F
4
0
0
3
3
4
Hz), 112.1 (d, J
9
(
= 9.5 Hz) 114.7 (d, J
= 4.8 Hz), 118.2 (d, JC-F = 2.9 Hz), 124.3, 127.4 (d, JC-F
=
C-F
3
C-F
0
0
000
.5 Hz), 130.2 (d, JC–F = 9.9 Hz), 132.9, 140.2 (d, 3 JC-F = 12.1 Hz), 156.5 (d, J
1
= 231 Hz), 163.3
C–F
0
1
+
d, JC-F = 246 Hz), 166.8, and 169.8 ppm. HRMS: (EI) Calculated for C H F N O (M ) = 569.3051.
30 38 2 6 3
Found: 569.3050.
4
-Fluoro-3-(2-{4-[3-(5-fluor-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-
yl-ethyl) Benzamide (13b)
5-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indole 9b (152 mg; 0.58 mmol), 3-(2-chloro-acetylamino)-
4
0
-fluor-N-(2-morpholin-4-yl-ethyl)-benzamide 12b (199 mg; 0.58 mmol), and anhydrous K CO (80 mg;
2
◦
3
1
.58 mmol), to afford 13b (150 mg; 47% yield) as a yellow light solid. m.p.: 92.2–93.9 C; H-NMR
0 0 0 0 00 000
DMSO-d ): δ 1.78 (q, 2H, H-2 , J = 7.4 Hz), 2.27–2.56 (m, 16H, H-3 , H-4 , H-5 , H-9 and H-1 ), 2.66
6
(
(
(
0
0
0
000
t, 2H, H-1 , J = 7.3 Hz), 3.19 (s, 2H, H-6 ), 3.33–3.36 (m, 2H, H-8 ), 3.57 (t, 4H, H-2 , J = 4.4 Hz), 6.89
td, 1H, H-6, J
o
= 9.2 Hz, J
m
= 2.5 Hz), 7.20 (d, 1H, H-2, J = 1.9 Hz), 7.26 (dd, 1H, H-7, J
o
= 10.1 Hz,
0
0
00
00
J
m
= 2.4 Hz), 7.29–7.42 (m, 2H, H-4 and H-5 ), 7.59–7.67 (m, 1H, H-2 ), 8.40–8.50 (m, 2H, H-6 , and
00
0
13
H-7 ), 9.68 (br. s, 1H, H-7 ), and 10.87 (s, 1H, H-1) ppm. C-NMR (DMSO-d6):
δ 22.7, 27.5, 37.1, 53.3
00
2
= 23.5 Hz), 109.3 (d, 2
(2X), 53.4 (2X), 53.7 (2X), 57.7, 57.9, 61.6, 66.7 (2X), 103.4 (d, J
J
= 26.4 Hz),
= 6.6 Hz),
C-F
C-F
0
0
0
3
4
2
4
112.6 (d, J
= 9.5 Hz), 115.2 (d, J
= 4.4 Hz), 115.6 (d, J
= 19.8 Hz), 123 (d, J
C-F
C-F
C-F
C-F

Molecules 2020, 25, 4614
22 of 30
0
00
0
3
1
2
24.5 (d, 3JC-F = 8.1 Hz), 124.9, 126.2 (d, 2 JC-F = 11.7 Hz), 127.9 (d, J
= 9.5 Hz), 131.6 (d, 3JC-F
=
C–F
0
1
.9 Hz), 133.4, 155.3 (d, 1JC-F = 248 Hz), 157.0 (d, JC–F = 230.4 Hz). 165.6, and 169.1 ppm. HRMS: (EI)
Calculated for C H F N O (M ) = 569.3051. Found: 569.3046.
+
30
38
2
6
3
2
-Fluoro-5-(2-{4-[3-(5-fluor-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-
ethyl) Benzamide (13c)
5
-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indole 9b (152 mg; 0.58 mmol), 5-(2-Chloro-acetylamino)-
2
-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12d (199 mg; 0.58 mmol), and anhydrous K CO
2
3
◦
(
80 mg; 0.58 mmol), to afford 13c (178 mg; 55% yield) as a yellow light solid. m.p.: 91.8–93.2 C;
H-NMR (DMSO-d ):
1
0
0
δ
1.76 (q, 2H, H-2 , J = 7.4 Hz), 2.32 (t, 2H, H-3 , J = 6.9 Hz), 2.36–2.56 (m, 14H,
6
0
0
00
000
0
0
00
H-4 , H-5 , H-9 and H-1 ), 2.65 (t, 2H, H-1 , J = 7.3 Hz), 3.11 (s, 2H, H-6 ), 3.36–3.42 (m, 2H, H-8 ),
0
00
3.57 (t, 4H, H-2 , J = 4.5 Hz), 6.89 (td, 1H, H-6, J
o
= 8.2 Hz, J
m
= 2.6 Hz), 7.17–7.28 (m, 3H, H-2, H-7
0
0
00
00
and H-3 ), 7.31 (dd, 1H, H-4, J
Jo = 6.5 Hz, J = 2.8 Hz), 8.13–8.24 (m, 1H, H-7 ), 9.90 (br. s, 1H, H-7 ), and 10.89 (s, 1H, H-1) ppm.
C-NMR (DMSO-d6): 22.7, 27.6, 37, 53.1 (2X), 53.4 (2X), 53.7 (2X), 57.4, 57.9, 62.3, 66.7 (2X), 103.4 (d,
= 22.7 Hz), 109.3 (d, 2 JC-F = 26.5 Hz), 112.6 (d, JC-F = 9.9 Hz), 115.2 (d, J
o m
= 8.8 Hz, J = 4.6 Hz), 7.73–7.81 (m, 1H, H-6 ), 7.92 (dd, 1H, H-4 ,
0
0
0
m
13
δ
0
00
2J
3
4
= 4.4 Hz), 116.7
C-F
C-F
0
0
0
000
2
d, 2 JC-F = 23.8 Hz), 121.33 (d, JC-F = 2.2 Hz), 123.6 (d, JC-F = 8.3 Hz), 124.2 (d, J
4
3
= 9.5 Hz),
C-F
(
0
00
0
24.9, 127.9 (d, 3 JC–F = 9.4 Hz), 133.6, 135.5 (d, 3 JC-F = 3.3 Hz), 155.4 (d, J
1
= 245 Hz), 157 (d,
C–F
1
0
1
+
JC-F = 231 Hz), 163.7, and 168.9 ppm. HRMS: (EI) Calculated for C H F N O (M ) = 569.3051.
30
38
2
6
3
Found: 569.3047.
5
-Fluoro-2-(2-{4-[3-(5-fluoro-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-
ethyl) Benzamide (13d)
5
-Fluoro-3-(3-piperazin-1-yl-propyl)-1H-indole 9b (152 mg; 0.58 mmol), 2-(2-chloro-
acetylamino)-5-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12c (199 mg; 0.58 mmol), and anhydrous
K CO (80 mg; 0.58 mmol), to afford 13d (188 mg; 59% yield) as a yellow light solid. m.p.: 94.2–95.4
2
3
◦
1
0 0 0 0 00
δ 1.79 (q, 2H, H-2 , J = 7.2 Hz), 2.32–2.51 (m, 16H, H-3 , H-4 , H-5 , H-9 , and
C; H-NMR (DMSO-d ):
6
0
000
0
00
000
H-1 ), 2.66 (t, 2H, H-1 , J = 7.3 Hz), 3.10 (s, 2H, H-6 ), 3.50–3.60 (m, 6H, H-8 , and H-2 ), 6.89 (td, 1H,
H-6, J
o
= 9.2 Hz, J
m
= 2.5 Hz), 7.20 (d, 1H, H-2, J = 2.0 Hz), 7.25 (dd, 1H, H-7, J
o
= 10.1 Hz, J
m
= 2.5
0
0
00
00
Hz), 7.29–7.40 (m, 2H, H-4 and H-4 ), 7.49 (dd, 1H, H-3 , J
o
= 9.5 Hz, J
m
= 3.0 Hz), 8.53 (dd, 1H, H-6 ,
0
0
0
Jo = 9.2 Hz, J
m
= 5.4 Hz), 8.69 (t, 1H, H-7 , J = 5.4 Hz), 10.89 (s, 1H, H-1), and 11.62 (br. s, 1H, H-7 )
ppm. 13C-NMR (DMSO-d ):
δ 22.2, 26.9, 36.4, 52.4 (2X), 52.9 (2X), 53.2 (2X), 57, 57.4, 61.7, 66.1 (2X),
6
0
00
2
2
2
3
1
1
1
02.9 (d, J
14.6 (d, J
= 22.3 Hz), 108.8 (d, JC-F = 26.3 Hz), 112.1 (d, J
= 9.9 Hz), 114.5 (d, JC-F = 23.8 Hz),
C-F
C-F
0
00
0
00
4
= 5 Hz), 118 (d, 2 JC-F = 22 Hz), 122.1 (d, JC-F = 7 Hz), 123.8 (d, JC-F = 6.2 Hz), 124.3,
3
3
C-F
0
00
0
0
1
27.4 (d, 3 JC–F = 9.5 Hz), 132.9, 134.4 (d, 4 JC-F = 2.2 Hz), 152.4 (d, J
1
= 231 Hz), 156.5 (d, JC-F
C–F
+
=
223 Hz), 166.3, and 169 ppm. HRMS: (EI) Calculated for C H F N O (M ) = 569.3051. Found:
30 38 2 6 3
5
69.3048.
4
-Fluoro-2-(2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-
ethyl) Benzamide (13e)
5
-Bromo-3-(3-piperazin-1-yl-propyl)-1H-indole 9c (187 mg; 0.58 mmol), 2-(2-chloro-acetylamino)-4-
fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12a (199 mg; 0.58 mmol), and anhydrous K CO (80 mg;
2
◦
3
1
0
(
.58 mmol), to afford 13e (236 mg; 66% yield) as a yellow light solid. m.p.: 92.1 -93.8 C; H-NMR
0 0 0 0
DMSO-d ): δ 1.78 (q, 2H, H-2 , J = 7.4 Hz), 2.35 (t, 2H, H-3 , J = 6.9 Hz), 2.38–2.52 (m, 14H, H-4 , H-5 ,
6
0
0
000
0
0
00
H-9 and H-1 ), 2.67 (t, 2H, H-1 , J = 7.3 Hz), 3.13 (s, 2H, H-6 ), 3.40–3.42 (m, 2H, H-8 ), 3.54 (t, 4H,
H-2 , J = 4.5 Hz), 7.0 (td, 1H, H-5 , J
0
00
00
o m
= 8.3 Hz, J = 2.8 Hz), 7.12–7.20 (m, 2H, H-2, and H-6), 7.31 (d,
0
0
1H, H-7, J = 8.6 Hz), 7.62 (d, 1H, H-4, J = 1.7 Hz), 7.77 (dd, 1H, H-3 , J
o
= 8.8 Hz, J
m
= 6.6 Hz), 8.39
0
0
00
(dd, 1H, H-6 , J
o
= 12.3 Hz, J
m
= 2.7 Hz), 8.70 (t, 1H, H-7 , J = 5.4 Hz), 11.1 (s, 1H, H-1), and 12.1 (br.
0
13
s, 1H, H-7 ) ppm. C-NMR (DMSO-d6):
δ
22.8, 27.4, 36.9, 52.6 (2X), 52.7 (2X), 53.7 (2X), 56.2, 57.6,
0
2.2, 66.6 (2X), 107.1 (d, 2JC-F = 26.5 Hz), 109.7 (d, J
2
= 22.7 Hz), 111.2, 113.3, 118.6 (d, J
4
= 4.9
C-F
6
C-F

Molecules 2020, 25, 4614
23 of 30
0
Hz), 118.7, 121.6, 123.7, 124.4, 129.6, 130.8 (d, 3JC-F = 9.9 Hz), 135.4, 140.7 (d, 3 JC-F = 12.2 Hz), 163.8 (d,
1
+
JC-F = 246 Hz), 167.3, and 170.4 ppm. HRMS: (EI) Calculated for C H BrFN O (M ) = 629.2251.
30
38
6
3
Found: 629.2246.
4
-Fluoro-3-(2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-
ethyl) Benzamide (13f)
5
-Bromo-3-(3-piperazin-1-yl-propyl)-1H-indole 9c (187 mg; 0.58 mmol), 3-(2-chloro-acetylamino)-
4
-fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12b (199 mg; 0.58 mmol), and anhydrous K CO
2
3
◦
(
80 mg; 0.58 mmol), to afford 13f (193 mg; 54% yield) as a yellow light solid. m.p.: 85.2–86.8 C;
H-NMR (DMSO-d ):
1
0
0
0
0
00
δ
0
1.78 (q, 2H, H-2 , J = 6.5 Hz), 2.30–2.55 (m, 16H, H-3 , H-4 , H-5 , H-9 , and
6
0
00
0
00
000
H-1 ), 2.68 (t, 2H, H-1 , J = 7.3 Hz), 3.20 (s, 2H, H-6 ), 3.40–3.44 (m, 2H, H-8 ), 3.57 (t, 4H, H-2 ,
J = 4.4 Hz), 7.13–7.21 (m, 2H, H-2, and H-6), 7.31 (d, 1H, H-7, J = 10.1 Hz), 7.34–7.42 (m, 1H, H-5 ),
0
0
00 00 00
7
.59–7.67 (m, 1H, H-2 ), 7.62 (d, 1H, H-4, J = 1.6 Hz), 8.37–8.52 (m, 2H, H-6 and H-7 ), 9.69 (br. s, 1H,
0
13
H-7 ), and 11.0 (s, 1H, H-1) ppm. C-NMR (DMSO-d ):
δ
22.2, 26.9, 36.4, 52.4 (2X), 52.9 (2X), 53.2 (2X),
= 21 Hz), 121.1, 123.1, 123.28 (d, J
6
2
4
5
7, 57.4, 61.7, 66.1 (2X), 111.3, 113.8, 114.7, 115.6 (d, J
= 2.9 Hz),
C-F
C-F
0
0
3
2
3
123.7, 124.5 (d, J
= 9.5 Hz), 126.2 (d, J
= 11 Hz), 129.7, 131.63 (d, J
= 3.7 Hz), 135.7, 155.3
C-F
C-F
C-F
1
+
(d, J
= 248 Hz), 165.6, and 169 ppm. HRMS: (EI) Calculated for C H BrFN O (M ) = 629.2251.
C-F 30 38 6 3
Found: 629.2250.
2
-Fluoro-5-(2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-
ethyl) Benzamide (13g)
5
-Bromo-3-(3-piperazin-1-yl-propyl)-1H-indole 9c (187 mg; 0.58 mmol), 5-(2-chloro-acetylamino)-2-
fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12d (199 mg; 0.58 mmol), and anhydrous K CO (80
2
1
3
◦
mg; 0.58 mmol), to afford 13g (249 mg; 70% yield) as a yellow light solid. m.p.: 73.2–74.5 C; H-NMR
0
0
0
0
00
000
(
(
DMSO-d ):
δ
1.79 (q, 2H, H-2 , J = 7.2 Hz), 2.32–2.51 (m, 16 H, H-3 , H-4 , H-5 , H-9 , and H-1 ), 2.66
t, 2H, H-1 , J = 7.3 Hz), 3.10 (s, 2H, H-6 ), 3.50–3.60 (m, 6H, H-8 , and H-2 ), 6.89 (td, 1H, H-6, J
6
0
0
00
000
o
= 9.2
Hz, J
m
= 2.5 Hz), 7.20 (d, 1H, H-2, J = 2.0 Hz), 7.25 (dd, 1H, H-7, J
o
= 10.1 Hz, J
m
= 2.5 Hz), 7.29–7.40
0
0
00
00
(m, 2H, H-4, and H6 ), 7.49 (dd, 1H, H-4 , J
o
= 9.5 Hz, J
m
= 3.0 Hz), 8.53 (dd, 1H, H-3 , J
o
= 9.2 Hz,
0
0
0
13
Jm = 5.4 Hz), 8.69 (t, 1H, H-7 , J = 5.4 Hz), 10.89 (s, 1H, H-1), and 11.62 (br. s, 1H, H-7 ) ppm. C-NMR
(
1
DMSO-d ):
δ
22.4, 27.5, 36.9, 52.9 (2X), 53 (2X), 53.6 (2X), 574, 57.6, 62.1, 66.7 (2X), 111.3, 113.8, 114.7,
6
2
0
3
0
3
= 3.7 Hz), 123.6 (d, JC-F = 7.3 Hz), 123.7, 124.2 (d, 2 JC-F
16.7 (d, J
= 23.5 Hz), 121.1, 121.4 (d, J
C-F
C-F
4
1
=
15.4 Hz), 124.5, 129.6, 135.3, 135.4 (d, J
= 2.2 Hz), 155.4 (d, J
= 245 Hz), 163.7, and 168.9 ppm.
C-F
C-F
+
HRMS: (EI) Calculated for C H BrFN O (M ) = 629.2251. Found: 629.2248.
30
38
6
3
5
-Fluoro-2-(2-{4-[3-(5-bromo-1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-
ethyl) Benzamide (13h)
5
-Bromo-3-(3-piperazin-1-yl-propyl)-1H-indole 9c (187 mg; 0.58 mmol), 2-(2-chloro-acetylamino)-5-
fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12c (199 mg; 0.58 mmol), and anhydrous K CO (80 mg;
2
◦
3
1
0
(
.58 mmol), to afford 13h (303 mg; 85% yield) as a yellow light solid. m.p.: 73.8–74.7 C; H-NMR
0 0 0 0 00 000
DMSO-d ): δ 1.71–1.84 (m, 2H, H-2 ), 2.25–2.56 (m, 16H, H-3 , H-4 , H-5 , H-9 , and H-1 ), 2.67 (t, 2H,
6
0
0
00
000
H-1 , J = 7.2 Hz), 3.14 (s, 2H, H-6 ), 3.40–3.43 (m, 2H, H-8 ), 3.57 (t, 4H, H-2 , J = 4.5 Hz), 7.13–7.27 (m,
00
3H, H-2, and H-6 and H-3 ), 7.31 (d, 1H, H-7, J = 8.5 Hz), 7.69 (d, 1H, H-4, J = 1.7 Hz), 7.73–7.81 (m,
00 00 00 0
1
H, H-6 ), 7.92 (dd, 1H, H-4 , J = 6.4 Hz, J = 2.7 Hz), 8.15–8.22 (m, 1H, H-7 ), 9.9 (br. s, 1H, H-7 ),
o m
and 11 (s, 1H, H-1) ppm. 13C-NMR (DMSO-d ):
δ
22.4, 27.5, 36.9, 52.8 (2X), 53.3 (2X), 53.7 (2X), 57.5,
6
0
2
2
57.7, 62.2, 66.6 (2X), 111.3, 113.8, 114.7, 115.1 (d, J
= 24.2 Hz), 118.5 (d, JC-F = 21.3 Hz), 121.1, 122.6
C-F
0
3
= 7.3 Hz), 123.7, 124.3 (d, 3 JC-F = 5.9 Hz), 124.4, 129.6, 134.9 (d, J
4
(
d, J
= 2.2 Hz), 135.4, 157.3 (d,
C-F
C-F
1
+
JC-F = 241 Hz), 166.9, and 169.5 ppm. HRMS: (EI) Calculated for C H BrFN O (M ) = 629.2251.
30
38
6
3
Found: 629.2249.

Molecules 2020, 25, 4614
24 of 30
4
-Fluoro-2-(2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl)
Benzamide (13i)
3-(3-Piperazin-1-yl-propyl)-1H-indole 9a (150 mg; 0.62 mmol), 2-(2-chloro-acetylamino)-4-fluoro-
N-(2-morpholin-4-yl-ethyl)-benzamide 12a (213 mg; 0.62 mmol), and anhydrous K CO (86 mg;
2
◦
3
1
0
.62 mmol), to afford 13i (249 mg; 75% yield) as a yellow light solid. m.p.: 80.5–81.8 C; H-NMR
0 0 0 00 000 0
(
2
6
DMSO-d6): δ 1.86 (m, 2H, H-2 ), 2.25–2.49 (m, 12H, H-3 , H-4 , H-9 and H-1 ), 2.58 (m, 4H, H-5 ),
0 0 00 000
.7 (t, 2H, H-1 , J = 7.4 Hz), 3.15 (s, 2H, H-6 ), 3.35–3.40 (m, 2H, H-8 ), 3.54 (t, 4H, H-2 , J = 4.3 Hz),
0
0
.93–7.09 (m, 3H, H-5 and H-6 and H-5 ), 7.12 (d, 1H, H-2, J = 1 Hz), 7.33 (d, 1H, H-7, J = 8 Hz), 7.51 (d,
0
0
00
1H, H-4, J = 7.8 Hz), 7.76 (dd, 1H, H-3 , J
o
= 8.6 Hz, J
m
= 6.7 Hz), 8.39 (dd, 1H, H-6 , J
o
= 12.3 Hz,
0
0
0
13
J
m
= 2.6 Hz), 8.68 (t, 1H, H-7 , J = 5 Hz), 10.8 (s, 1H, H-1), and 12.1 (br. s, 1H, H-7 ) ppm. C-NMR
2
(
DMSO-d6):
δ
22.5, 27.7, 36.9, 52.9 (2X), 53.5 (2X), 53.7 (2X), 55.4, 57.6, 62.3, 66.6 (2X), 107 (d, J
= 28
C-F
3
0
Hz), 109.7 (d, 2 JC-F = 22 Hz), 111.8, 114.5, 118.6 (d, J
4
= 2.9 Hz), 121.3, 122.6, 127.6, 130.8 (d, J
=
C-F
C-F
0
3
1
10.9 Hz), 134, 136.8, 140.8 (d, J
= 12.5 Hz), 141.4, 163.8 (d, J
= 245 Hz), 167.3, and 170.3 ppm.
C-F
C-F
+
HRMS: (EI) Calculated for C H FN O (M ) = 551.3145. Found: 551.3139.
30
39
6
3
4
-Fluoro-3-(2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl)
Benzamide (13j)
3-(3-Piperazin-1-yl-propyl)-1H-indole 9a (150 mg; 0.62 mmol), 3-(2-chloro-acetylamino)-4-
fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12b (213 mg; 0.62 mmol), and anhydrous K CO (86 mg;
2
◦
3
1
0
.62 mmol), to afford 13j (268 mg; 81% yield) as a yellow light solid. m.p.: 70.2–71.9 C; H-NMR
0 0 0 0 00 000
DMSO-d ): δ 1.81 (q, 2H, H-2 , J = 7.5 Hz), 2.21–2.53 (m, 16H, H-3 , H-4 , H-5 , H-9 , and H-1 ), 2.7
6
(
(
(
0
0
00
000
t, 2H, H-1 , J = 7.4 Hz), 3.19 (s, 2H, H-6 ), 3.38–3.40 (m, 2H, H-8 ), 3.57 (t, 4H, H-2 , J = 4.5 Hz), 6.96
td, 1H, H-5 or H-6, J = 7.5 Hz, J = 1 Hz), 7.06 (td, 1H, H-6 or H-5, J = 7.4 Hz, J = 1 Hz), 7.11 (d,
H, H-2, J = 2.1 Hz), 7.32–7.40 (m, 2H, H-7 and H-5 ), 7.51 (d, 1H, H-4, J = 7.7 Hz), 7.61–7.66 (m, 1H,
o
m
o
m
0
0
1
00
00
00
0
13
H-2 ), 8.40–8.52 (m, 2H, H-6 and H-7 ), 9.69 (br. s, 1H, H-7 ), and 10.8 (s, 1H, H-1) ppm. C-NMR
(
(
DMSO-d ):
δ
22.4, 27.1, 36.6, 52.8 (2X), 52.9 (2X), 53.2 (2X), 57.2, 57.5, 61.1, 66.2 (2X), 111.3, 114.3, 115.1
6
0
2
3
3
d, J
= 20.1 Hz), 118.1 (d, J
= 10.6 Hz), 120.7, 122.1, 122.4, 123.0, 124.0 (d, JC-F = 8.1 Hz), 125.7 (d,
C-F
C-F
0
2
4
1
JC-F = 11.8 Hz), 127.2, 131.1 (d, J
= 3.3 Hz), 136.3, 154.7 (d, J
= 249 Hz), 165.1, and 168.5 ppm.
C-F
C-F
+
HRMS: (EI) Calculated for C H FN O (M ) = 551.3145. Found: 551.3140.
3
0
39
6
3
2
-Fluoro-5-(2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl)
Benzamide (13k)
3-(3-Piperazin-1-yl-propyl)-1H-indole 9a (150 mg; 0.62 mmol), 5-(2-chloro-acetylamino)-2-
fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12d (213 mg; 0.62 mmol), and anhydrous K CO (86 mg;
2
◦
3
1
0
.62 mmol), to afford 13k (200 mg; 60.7% yield) as a yellow light solid. m.p.: 69.7–71.5 C; H-NMR
0
0
0
0
00
000
(
(
6
DMSO-d ):
δ
1.81 (q, 2H, H-2 , J = 6.8 Hz), 2.25–2.54 (m, 16H, H-3 , H-4 , H-5 , H-9 , and H-1 ), 2.7
6
0
0
00
000
t, 2H, H-1 , J = 7.2 Hz), 3.13 (s, 2H, H-6 ), 3.39 (q, 2H, H-8 , J = 6.4 Hz), 3.58 (t, 4H, H-2 , J = 4.2 Hz),
.96 (t, 1H, H-5 or H-6, J = 7.2 Hz), 7.06 (t, 1H, H-6 or H-5, J = 6.9 Hz), 7.11 (d, 1H, H-2, J = 1.2 Hz), 7.24
00
t, 1H, H-3 , J = 9.8 Hz), 7.34 (d, 1H, H-4, J = 8.0 Hz), 7.51 (d, 1H, H-7, J = 7.7 Hz), 7.76–7.81 (m, 1H,
(
00
00
00 0
H-6 ), 7.94 (dd, 1H, H-4 , J = 6.3 Hz, J = 2.6 Hz), 8.15–8.22 (m, 1H, H-7 ), 9.90 (br. s, 1H, H-7 ), and
o m
1
6
0.8 (s, 1H, H-1) ppm. 13C-NMR (DMSO-d ):
2.2, 66.7 (2X), 111.8, 114.8, 116.7 (d, J
δ
22.9, 27.5, 37.0, 53.0 (2X), 53.2 (2X), 53.7 (2X), 57.4, 57.9,
6
2
4
= 24.2 Hz), 118.5, 118.7, 121.2, 121.4 (d, J
= 2.2 Hz), 112.6,
C-F
C-F
0
3
= 8.1 Hz), 124.1 (d, 2 JC-F = 16.1 Hz), 127.7, 135.4 (d, J
3
1
1
23.6 (d, J
= 2.9 Hz), 136.8, 155.4 (d, J
C-F
C-F
C-F
+
=
245 Hz), 163.7, and 168.9 ppm. HRMS: (EI) Calculated for C H FN O (M ) = 551.3145. Found:
30
39
6
3
5
51.3142.
5
-Fluoro-2-(2-{4-[3-(1H-3-indolyl)-propyl]-1-piperazinyl}-acetylamino)-N-(2-morpholin-4-yl-ethyl)
Benzamide (13l)
3-(3-Piperazin-1-yl-propyl)-1H-indole 9a (200 mg; 0.82 mmol), 2-(2-chloro-acetylamino)-5-
fluoro-N-(2-morpholin-4-yl-ethyl)-benzamide 12c (281 mg; 0.82 mmol), and anhydrous K CO (113 mg;
2
3

Molecules 2020, 25, 4614
25 of 30
◦
1
0
.82 mmol), to afford 13l (263 mg; 60% yield) as a yellow light solid. m.p.: 68.1–69.6 C; H-NMR
0 0 0 0 00 000
DMSO-d ): δ 1.80 (q, 2H, H-2 , J = 7.6 Hz), 2.30–2.52 (m, 16H, H-3 , H-4 , H-5 , H-9 and H-1 ), 2.69
6
(
(
(
0
0
00
000
t, 2H, H-1 , J = 7.4 Hz), 3.18 (s, 2H, H-6 ), 3.70–3.41 (m, 2H, H-8 ), 3.56 (t, 4H, H-2 , J = 4.5 Hz), 6.96
td, 1H, H-5 or H-6, J = 7.8 Hz, J = 1.0 Hz), 7.05 (td, 1H, H-6 or H-5, J = 7 Hz, J = 1.1 Hz), 7.11 (d, 1H,
H-2, J = 2.1 Hz), 7.33 (t, 1H, H-4 , J = 8.4 Hz), 7.39 (d, 1H, H-4, J = 8.7 Hz), 7.50 (d, 1H, H-7, J = 7.8 Hz),
o
m
o
m
0
0
00 00 00 0
7
.59–7.66 (m, 1H, H-3 ), 8.40–8.48 (m, 2H, H-6 and H-7 ), 9.68 (br. s, 1H, H-7 ), and 10.75 (s, 1H, H-1)
1
3
ppm. C-NMR (DMSO-d ):
δ
22.9, 27.7, 37.1, 53.3 (2X), 53.4 (2X), 53.8 (2X), 57.8, 58.1, 61.6, 66.7 (2X),
6
2
0
= 19.4 Hz), 118.6 (d, 2
J
= 17.7 Hz), 121.2, 122.6, 123.0, 124.5 (d, J
3
1
11.7, 113.4, 114.9, 115.6 (d, J
C-F
C-F
C-F
0
3
4
1
=
8.3 Hz),126.2 (d, J
= 11.6 Hz), 127.7, 131.63 (d, J
= 2.8 Hz), 136.7, 155.3 (d, J
= 249 Hz),
C-F
C-F
C-F
+
1
3
3
65.5, and 169.1 ppm. HRMS: (EI) Calculated for C H FN O (M ) = 551.3145. Found: 551.3141.
30 39 6 3
.2. Biological Assay
.2.1. Reagents
[3
Ci/mmol; NET856), membrane from clonal cell line HEK-293 that overexpresses SERT (Code:
RBHSTM400UA), and membrane from CHO-K1 clonal cell line that overexpresses D receptor
H]Paroxetine (20.8 Ci/mmol; Code NET869), [ H]-Methylspiperone (specific activity 64.1
3
2
(Code: RBHD2CM400UA) were purchased from Perkin-Elmer (Boston, MA, USA). Fluoxetine and
haloperidol were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used were
of analytical grade.
3
.2.2. SERT Binding
To determine the binding of all compounds at SERT, competitive binding assays were performed
according to previously reported procedures with some modifications [36]. Briefly, assays were carried
out in a total volume of 0.5 mL containing 9
µ
g protein of membrane from a clonal cell line HEK-293 that
3
overexpresses SERT, 50 mM Tris buffer, pH 7.4, 120 mM NaCl, 5 mM KCl, 2 nM [ H]-paroxetine (specific
activity 20.8 Ci/mmol, PerkinElmer), and the compounds to be tested at different concentrations
− −4 ◦
9
(
10 –10 M). After 1 h at 27 C, incubations were stopped by rapid filtration through Whatman GF/C
filters presoaked in 0.5% polyethyleneimine, which were washed five times with 3 mL of ice-cold
buffer, dried, and put in Eppendorf tubes with scintillation liquid. Radioactivity was counted by a
liquid scintillation counter (MicroBeta 2450 microplate counter, PerkinElmer). Control curve was
performed with fluoxetine in the same experimental conditions. Non-specific binding was determined
with 10 µM fluoxetine.
3
.2.3. D Receptor Binding
2
To determine the binding of all compounds at D receptor, competitive binding assays were
2
performed according to provider indications with some modifications. Briefly, assays were carried out
in a total volume of 0.5 mL containing 3 µg protein of membrane from a CHO-K1 clonal cell line that
overexpresses D receptor, 50 mM Tris buffer, pH 7.4, 120 mM NaCl, 5 mM KCl, 5 mM MgCl , 1 mM
2
2
3
EDTA, 0.5 nM [ H]-methylspiperone (specific activity 64.1 Ci/mmol, PerkinElmer), and the compounds
−
9
−4
◦
to be tested at different concentrations (10 –10 M). After 2 h at 27 C, incubations were stopped
by rapid filtration through Whatman GF/C filters presoaked in 0.5% polyethyleneimine, which were
washed five times with 3 mL of ice-cold wash buffer (50 mM Tris buffer, pH 7.4, 154 mM NaCl), dried,
and put in Eppendorf tubes with scintillation liquid. Radioactivity was counted as described before.
Control curve was performed with haloperidol in the same conditions. Non-specific binding was
determined with 10 µM haloperidol.
Analysis of data: All curves were fitted using the sigmoidal dose–response inhibition curve
(
variable slope) equation built into GraphPad PRISM 5.01 (GraphPad Software Inc., San Diego, CA,
USA). The analysis gives the IC50 value (i.e., the drug concentration inhibiting specific binding by 50%)
to calculate Ki (affinity constant) by the Cheng–Prussof equation (Ki = IC /(1 + ([radioligand]/Kd
50

Molecules 2020, 25, 4614
26 of 30
3
(
radioligand))). The Kd values used correspond to 0.13 nM to [ H] paroxetine on SERT [50], and 0.1 nM
3
to [ H]-methylspiperone on D (provided by the manufacturer). The IC and Ki values correspond to
2
50
the results of three independent experiments, each in triplicate. All data are expressed as the mean
SEM.
±
3
.2.4. MAO-A Inhibition
All experimental procedures were approved by the Ethics Committee of the University of Santiago
de Chile and the Science Council (FONDECYT) of Chile and followed internationally accepted
guidelines (NIH Guide for the Care and Use of Laboratory Animals).
The effects of the compounds on rat MAO-A activity were studied following a previously reported
methodology [50
100 M) was used as the selective substrate for MAO-A. This compound and its metabolite were
detected by HPLC with electrochemical detection. As an exploratory evaluation, the percentage of
MAO-A inhibition in the presence of 100 M of the different compounds was determined, with the
idea of evaluating in detail those compounds showing an inhibitory activity in the range of 70–100%.
,51], using a crude rat brain mitochondrial suspension as a source of enzyme. Serotonin
(
µ
µ
3
.2.5. Molecular Docking
Molecular docking studies for the two families of compounds were performed on two different
protein targets (SERT and D receptor). All dockings were carried out at pH 7.4 in the crystal
2
structures of human SERT (hSERT PDB: 5I73) [47] and human D receptor [hD PDB: 6CM4) [49].
2
2
All compounds were modelled using the Spartan’14 Software (Wavefunction, Inc. Irvine, CA) and
geometry optimization calculations were carried out using the software package at the Hartree-Fock
level using the 6-31G* basis set. Docking studies were performed using AutoDockv4.2 [52] software
suite with Autodock Tools ADT 1.5.6 [52
proteins. Grid maps were calculated using the autogrid option with a grid volume of 70
points with a grid spacing of 0.375 Å and centered on the coordinates x, y, z: 33.3 184.5; 0.565
,
53] following the standard docking procedure for rigid
70 70
9.397;
×
×
−
and 37.019 28.136 for the SERT and D receptor respectively. Docking simulations were performed with
2
a Lamarckian genetic algorithm (LGA) and binding energies were estimated according to the internal
scoring function implemented by the program; 250 independent runs per ligand were carried out with
an initial population of 300 individuals. Default settings were used for all other parameters. The lowest
free-energy resulting complexes were selected and further analyzed using the Visual Molecular
Dynamic (VMD) visualization program [54]. Validation of the docking protocol was performed using
the co-crystallized ligands (S)-citalopram and risperidone for SERT and D , respectively.
2
3
.2.6. QSAR Methods
CoMFA and CoMSIA studies were performed with Sybyl X-1.2 software [55] installed in a Windows
10 environment on a PC with an Intel Core i7 CPU. The geometric optimization, field calculation,
and charges calculation were performed as previously reported [56] (Figure S1 and Table S3 in
Supplementary Material) [57]. The internal validation of the models was done by calculating the
2
2
cross-validation coefficient q [58]. The models with the highest value of q were selected and then
subjected to external validation [59 61] (Table S2). In all cases, the best models passed the validation
–
limits [59] (Table S2). The regression graphs of each model and the tables of experimental versus
calculated values are in the Supplementary Material (Table S3, Figure S2).
4
. Conclusions
According to these results, the design of hybrid or bifunctional compounds, i.e., molecules that
incorporate two pharmacophores known to act at different receptors into a single chemical entity,
is an attractive approach for the development of agents having a targeted polypharmacological
profile [19
,62,63]. In the present work, we attempted to combine SERT effects previously demonstrated
for indolylalkylpiperazine derivatives, functionalizing the parent scaffold with structural fragments of

Molecules 2020, 25, 4614
27 of 30
drugs with known activity upon D receptor or MAO-A. Unexpectedly, the synthesized compounds did
2
not show, in most cases, a multitarget profile, since they exhibited a high affinity for SERT while showing
almost no effect at D receptor or MAO-A. This indicates that this strategy, although plausible, requires
2
a very fine design of the fragments to be connected and how these are going to be linked. Beyond
these considerations, our results highlight the remarkable stability of the indolylpropylpiperazine
skeleton as SERT ligand, which exhibits a high affinity by this target, apparently regardless of the type
of the associated moiety [34–36]. We think that this represents an important feature for the design
of polypharmacological molecules, in which an effect upon SERT is pursued. Docking and QSAR
results allowed us to rationalize the high SERT affinity observed for compounds in both studied
families. Thus, the presence of a halogen at the C-5 position of the indole ring and fluorine atoms at
the benzoxazine (Series I) or acetanilide (Series II) moieties probably induces electronic deprotection of
the corresponding aromatic rings, favoring stronger
aromatic residues at the binding site.
π–π interactions of these frameworks with donor
Interestingly, one of the compounds (7n) showed a promissory multitarget profile, being the only
derivative showing a relatively high and comparable affinity for SERT and D receptor (Ki = 84.4
2
and 307 nM, respectively). Even though at this time it is difficult to determine the molecular aspects
underlying this pharmacological promiscuity, it is clear that for polypharmacological drugs, a similar
affinity for different receptors is the most relevant characteristic, and therefore 7n stands as a very
attractive lead for further optimization.
Supplementary Materials: Supplementary materials are available online. Table S1. Statistical parameters and
Field combinations for CoMFA and CoMSIA. Table S2. Summary of external validation parameters for CoMFA
and CoMSIA. Table S3. Experimental and predicted pKi and residual values for analyzed compounds according
to CoMFA and CoMSIA. Figure S1. The superimposed structures of all compounds used in the CoMFA/CoMSIA
models. Figure S2. Plots of experimental versus predicted pKi values for the training and test set molecules for
CoMFA (A, B) and CoMSIA (C, D) models. Figure S3. hSERT affinity curves for compounds of Series I (7a
7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o, and fluoxetine), displaying IC50 values. Each determination was
made in triplicate and the data were expressed as the mean SD. Figure S4. D2 affinity curves for compounds
of Series I (7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o, and haloperidol), displaying IC50 values.
Each determination was made in triplicate and the data were expressed as the mean SD. Figure S5. hSERT
affinity curves for compounds of Series II (13a 13b 13c 13d 13e 13f 13g 13h 13i 13j 13k 13l, and fluoxetine),
displaying IC50 values. Each determination was made in triplicate and the data were expressed as the mean SD.
13l
, 7b,
7
c
,
,
,
,
,
,
,
,
,
,
,
,
±
,
,
,
,
,
,
,
,
,
,
,
,
,
,
±
,
,
,
,
,
,
,
,
,
,
,
±
Figure S6. D2 affinity curves for compounds of Series II (13a
,
13b
,
13c
,
13d
,
13e
,
13f
,
13g
,
13h
,
13i
,
13j
,
13k
,
,
and haloperidol), displaying IC50 values. Each determination was made in triplicate and the data expressed as the
mean ± SD.
Author Contributions: Conceptualization, H.P.-M. and M.R.-P.; methodology, C.C.-C., J.R.-L., J.A.-E., C.S., C.O.-G.,
G.Q. and P.I.-V.; software, J.M.-R., D.C., C.D.P.-M. and H.C.; validation, H.P.-M., R.A.-M. and M.R.-P.; formal
analysis, H.P.-M., C.C.-C., R.A.-M., C.S., P.I.-V., J.M.-R., C.D.P.-M., H.C. and M.R.-P.; resources, H.P.-M., J.M.-R.,
P.I.-V. and M.R.-P.; data curation, H.P.-M., C.C.-C., J.M.-R., H.C. and M.R.-P.; writing—original draft preparation,
H.P.-M., C.C.-C. and M.R.-P.; writing—review and editing, H.P.-M., J.R.-L., J.A.-E., J.M.-R., H.C. and M.R.-P.;
supervision, H.P.-M. and M.R.-P.; project administration, H.P.-M., J.M.-R., P.I.-V. and M.R.-P.; funding acquisition,
H.P.-M., P.I.-V. and M.R.-P. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by FONDECYT (Chile) Grant numbers: 1170269 (H. Pessoa-Mahana), 1170662
(
M. Reyes-Parada) and FONDECYT Postdoctoral Grant 3180602 (J. Alarcón-Espósito). G. Quiroz was fellow of the
CONICYT National Ph.D. Scholar Fellowship.
Acknowledgments: The authors are grateful to Claudia Sanhueza for her valuable technical assistance. Funding:
This work was supported by FONDECYT Grant numbers: 1170269 (H. Pessoa-Mahana), 1170662 (M. Reyes-Parada);
FONDECYT Postdoctoral Grant 3180602 (J. Alarc
ón-Espósito). G. Quiroz was fellow of the CONICYT National
Ph.D. Scholar Fellowship.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Depression Fact Sheet; World Health Organization (WHO): Geneva, Switzerland, 2020; Available online:
https://www.who.int/news-room/fact-sheets/detail/depression (accessed on 10 September 2020).
Miller, N.; Perich, T.; Meade, T. Depression, mania and self-reported creativity in bipolar disorder.
Psychiatry Res. 2019, 276, 129–133. [CrossRef] [PubMed]
2
.

Molecules 2020, 25, 4614
28 of 30
3
.
Watters, A.J.; Carpenter, J.S.; Harris, A.W.F.; Korgaonkar, M.S.; Williams, L.M. Characterizing neurocognitive
markers of familial risk for depression using multi-modal imaging, behavioral and self-report measures.
J. Affect. Disord. 2019, 253, 336–342. [CrossRef] [PubMed]
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
.
.
.
.
.
.
McIntosh, A.M.; Sullivan, P.F.; Lewis, C.M. Uncovering the Genetic Architecture of Major Depression. Neuron
2
019, 102, 91–103. [CrossRef]
Coretti, S.; Rumi, F.; Cicchetti, A. The Social Cost of Major Depression. A Systematic Review. Rev. Eur. Stud.
019, 11, 73. [CrossRef]
2
Collins, P.Y.; Patel, V.; Joestl, S.S.; March, D.; Insel, T.R.; Daar, A.S.; Bordin, I.A.; Costello, E.J.; Durkin, M.;
Fairburn, C.; et al. Grand challenges in global mental health. Nature 2011, 475, 27–30. [CrossRef]
Hirschfeld, R.M. History and evolution of the monoamine hypothesis of depression. J. Clin. Psychiatry 2000
,
6
1 (Suppl. 6), 4–6.
Berton, O.; Nestler, E.J. New approaches to antidepressant drug discovery: Beyond monoamines. Nat. Rev.
Neurosci. 2006, 7, 137–151. [CrossRef]
Hasler, G. Pathophysiology of depression: Do we have any solid evidence of interest to clinicians? World
Psychiatry 2010, 9, 155–161. [CrossRef]
0. Lazar, M.A.; McIntyre, R.S. Chapter 34 Novel Therapeutic Targets for Major Depressive Disorder.
In Neurobiology of Depression; Academic Press: London, UK, 2019; pp. 383–400.
1. Hendrie, C.; Pickles, A. The failure of the antidepressant drug discovery process is systemic. J. Psychopharmacol.
2
013, 27, 407–416. [CrossRef]
2. Brummelte, S.; Mc Glanaghy, E.; Bonnin, A.; Oberlander, T.F. Developmental changes in serotonin signaling:
Implications for early brain function, behavior and adaptation. Neuroscience 2017, 342, 212–231. [CrossRef]
3. David, D.J.; Gardier, A.M. The pharmacological basis of the serotonin system: Application to antidepressant
response. L’Encephale 2016, 42, 255–263. [CrossRef] [PubMed]
4. Javelot, H. Psychopharmacology of anxiety and depression: Historical aspects, current treatments and
perspectives. Ann. Pharm. Fr. 2016, 74, 93–118. [CrossRef] [PubMed]
5. Daut, R.A.; Fonken, L.K. Circadian regulation of depression: A role for serotonin. Front. Neuroendocrinol.
2
019, 54, 100746. [CrossRef] [PubMed]
6. Hillhouse, T.M.; Porter, J.H. A brief history of the development of antidepressant drugs: From monoamines
to glutamate. Exp. Clin. Psychopharmacol. 2015, 23, 1–21. [CrossRef]
7. Milelli, A.; Turrini, E.; Catanzaro, E.; Maffei, F.; Fimognari, C. Perspectives in Designing Multifunctional
Molecules in Antipsychotic Drug Discovery. Drug Dev. Res. 2016, 77, 437–443. [CrossRef]
8. Mendonça J
thienobenzo- diazepines: Multi-target drugs for CNS disorders. Mini Rev. Med. Chem. 2015, 15, 630–647.
CrossRef]
únior, F.J.B.; Scotti, L.; Ishiki, H.; Botelho, S.P.S.; Da Silva, M.S.; Scotti, M.T. Benzo- and
[
1
9. Reyes-Parada, M.; Iturriaga-Vasquez, P. The development of novel polypharmacological agents targeting
the multiple binding sites of nicotinic acetylcholine receptors. Expert Opin. Drug Discov. 2016, 11, 969–981.
[CrossRef]
2
0. Subbaiah, M.A.M. Triple Reuptake Inhibitors as Potential Therapeutics for Depression and Other Disorders:
Design Paradigm and Developmental Challenges. J. Med. Chem. 2018, 61, 2133–2165. [CrossRef]
2
1. Ju
and Its Dysfunction as a Consequence of Oxidative Stress. Oxid. Med. Cell. Longev. 2016, 2016, 9730467.
CrossRef]
2. Berridge, K.C. The debate over dopamine’s role in reward: The case for incentive salience. Psychopharmacology
007, 191, 391–431. [CrossRef]
árez Olguín, H.; Calderón Guzmán, D.; Hernández García, E.; Barragán Mejía, G. The Role of Dopamine
[
2
2
2
2
3. Wise, R.A. Dopamine, learning and motivation. Nat. Rev. Neurosci. 2004, 5, 483–494. [CrossRef] [PubMed]
4. Belujon, P.; Grace, A.A. Dopamine System Dysregulation in Major Depressive Disorders. Int. J.
Neuropsychopharmacol. 2017, 20, 1036–1046. [CrossRef] [PubMed]
2
5. Brisch, R.; Saniotis, A.; Wolf, R.; Bielau, H.; Bernstein, H.-G.; Steiner, J.; Bogerts, B.; Braun, K.; Jankowski, Z.;
Kumaratilake, J.; et al. The Role of Dopamine in Schizophrenia from a Neurobiological and Evolutionary
Perspective: Old Fashioned, but Still in Vogue. Front. Psychiatry 2014, 5, 47. [CrossRef] [PubMed]
2
6. Meder, D.; Herz, D.M.; Rowe, J.B.; Lehéricy, S.; Siebner, H.R. The role of dopamine in the brain—Lessons
learned from Parkinson’s disease. Neuroimage 2019, 190, 79–93. [CrossRef] [PubMed]

Molecules 2020, 25, 4614
29 of 30
2
2
7. Dunlop, B.W.; Nemeroff, C.B. The Role of Dopamine in the Pathophysiology of Depression. Arch. Gen.
Psychiatry 2007, 64, 327–337. [CrossRef]
8. Gold, M.S.; Blum, K.; Febo, M.; Baron, D.; Modestino, E.J.; Elman, I.; Badgaiyan, R.D. Molecular role of
dopamine in anhedonia linked to reward deficiency syndrome (RDS) and anti- reward systems. Front. Biosci.
(Schol. Ed.) 2018, 10, 309–325. [CrossRef]
2
9. Cramer, A.O.J.; van Borkulo, C.D.; Giltay, E.J.; van der Maas, H.L.J.; Kendler, K.S.; Scheffer, M.; Borsboom, D.
Major Depression as a Complex Dynamic System. PLoS ONE 2016, 11, e0167490. [CrossRef]
3
0. Liu, W.; Wang, H.; Li, X.; Xu, Y.; Zhang, J.; Wang, W.; Gong, Q.; Qiu, X.; Zhu, J.; Mao, F.; et al. Design,
synthesis and evaluation of vilazodone-tacrine hybrids as multitarget-directed ligands against depression
with cognitive impairment. Bioorg. Med. Chem. 2018, 26, 3117–3125. [CrossRef]
3
1. Dessalew, N.; Mikre, W.; Hymete, A. Multitarget Selective Antidepressants Design: Latest Developments,
Opportunities and Challenges. Cent. Nerv. Syst. Agents Med. Chem. 2008, 8, 131–142. [CrossRef]
2. Millan, M.J. On “polypharmacy” and multi-target agents, complementary strategies for improving the
treatment of depression: A comparative appraisal. Int. J. Neuropsychopharmacol. 2014, 17, 1009–1037.
3
[CrossRef]
3
3
3
3. Rodr guez-Lavado, J.; Gallardo-Garrido, C.; Mallea, M.; Bustos, V.; Osorio, R.; Hödar-Salazar, M.; Chung, H.;
í
Araya-Maturana, R.; Lorca, M.; Pessoa-Mahana, C.D.; et al. Synthesis, in vitro evaluation and molecular
docking of a new class of indolylpropyl benzamidopiperazines as dual AChE and SERT ligands for
Alzheimer’s disease. Eur. J. Med. Chem. 2020, 198, 112368. [CrossRef] [PubMed]
4. Pessoa-Mahana, H.; Silva-Matus, P.; Pessoa-Mahana, C.D.; Chung, H.; Iturriaga-Vásquez, P.; Quiroz, G.;
Möller-Acuña, P.; Zapata-Torres, G.; Saitz-Barr a, C.; Araya-Maturana, R.; et al. Synthesis and Docking of
í
Novel 3-Indolylpropyl Derivatives as New Polypharmacological Agents Displaying Affinity for 5-HT(1A)
R/SERT. Arch. Pharm. (Weinh.) 2017, 350. [CrossRef]
5. Pessoa-Mahana, H.; González-Lira, C.; Fierro, A.; Zapata-Torres, G.; Pessoa-Mahana, C.D.; Ortiz-Severin, J.;
Iturriaga-V squez, P.; Reyes-Parada, M.; Silva-Matus, P.; Saitz-Barr
á
ía, C.; et al. Synthesis, docking and
pharmacological evaluation of novel homo- and hetero-bis 3-piperazinylpropylindole derivatives at SERT
and 5-HT1A receptor. Bioorg. Med. Chem. 2013, 21, 7604–7611. [CrossRef] [PubMed]
3
3
6. Ojeda-G
ndez-Rojas, C. Synthesis and biological screening of novel indolalkyl arenes targeting the serotonine
transporter. Arch. Pharm. (Weinh.) 2014, 347, 174–184. [CrossRef]
7. Pessoa-Mahana, H.; N ñez, C.U.; Araya-Maturana, R.; Barr a, C.S.; Zapata-Torres, G.; Pessoa-Mahana, C.D.;
Iturriaga-Vasquez, P.; Mella-Raip n, J.; Reyes-Parada, M.; Celis-Barros, C. Synthesis, 5-hydroxytryptamine1A
ómez, C.; Pessoa-Mahana, H.; Iturriaga-Vásquez, P.; Pessoa-Mahana, C.D.; Recabarren-Gajardo, G.;
M
é
ú
í
á
receptor affinity and docking studies of 3-[3-(4-aryl-1-piperazinyl)-propyl]-1H-indole derivatives.
Chem. Pharm. Bull. (Tokyo) 2012, 60, 632–638. [CrossRef]
3
3
8. Prins, L.H.A.; Petzer, J.P.; Malan, S.F. Inhibition of monoamine oxidase by indole and benzofuran derivatives.
Eur. J. Med. Chem. 2010, 45, 4458–4466. [CrossRef]
9. Heinrich, T.; Böttcher, H.; Gericke, R.; Bartoszyk, G.D.; Anzali, S.; Seyfried, C.A.; Greiner, H.E.;
Van Amsterdam, C. Synthesis and structure–activity relationship in a class of indolebutylpiperazines
as dual 5-HT(1A) receptor agonists and serotonin reuptake inhibitors. J. Med. Chem. 2004, 47, 4684–4692.
[CrossRef]
4
0. Mathew, B.; Baek, S.C.; Thomas Parambi, D.G.; Lee, J.P.; Mathew, G.E.; Jayanthi, S.; Vinod, D.; Rapheal, C.;
Devikrishna, V.; Kondarath, S.S.; et al. Potent and highly selective dual-targeting monoamine oxidase-B
inhibitors: Fluorinated chalcones of morpholine versus imidazole. Arch. Pharm. (Weinh.) 2019, 352, 1800309.
[CrossRef]
4
4
4
1. Weichert, D.; Stanek, M.; Hübner, H.; Gmeiner, P. Structure-guided development of dual β2
adrenergic/dopamine D2 receptor agonists. Bioorg. Med. Chem. 2016, 24, 2641–2653. [CrossRef]
2. Männel, B.; Hübner, H.; Möller, D.; Gmeiner, P.
β-Arrestin biased dopamine D2 receptor partial agonists:
Synthesis and pharmacological evaluation. Bioorg. Med. Chem. 2017, 25, 5613–5628. [CrossRef]
3. Szabo, M.; Lim, H.D.; Herenbrink, C.K.; Christopoulos, A.; Lane, J.R.; Capuano, B. Proof of concept study for
designed multiple ligands targeting the dopamine D2, serotonin 5-HT2A, and muscarinic M1 acetylcholine
receptors. J. Med. Chem. 2015, 58, 1550–1555. [CrossRef] [PubMed]
4
4. Ghanbarpour, A.; Hadizadeh, F.; Piri, F.; Rashidi-Ranjbar, P. Synthesis, conformational analysis and
antidepressant activity of moclobemide new analogues. Pharm. Acta Helv. 1997, 72, 119–122. [CrossRef]

Molecules 2020, 25, 4614
30 of 30
4
4
5. Bonnet, U. Moclobemide: Therapeutic use and clinical studies. Cns Drug Rev. 2003, 9, 97–140. [CrossRef]
PubMed]
ndez-Rojas, C.; Quiroz, G.; Fa
Gallardo-Toledo, E.; Saitz-Barr
evaluation of potential acetylcholinesterase inhibitors based on a benzoxazine core. Arch. Pharm. (Weinh.)
018, 351, 1800024. [CrossRef] [PubMed]
[
M
6.
é
ú
ndez, M.; Gallardo-Garrido, C.; Pessoa-Mahana, C.D.; Chung, H.;
í
a, C.; Araya-Maturana, R.; Kogan, M.J.; et al. Synthesis and biological
2
4
4
4
5
7. Coleman, J.A.; Green, E.M.; Gouaux, E. X-ray structures and mechanism of the human serotonin transporter.
Nature 2016, 532, 334–339. [CrossRef]
8. Wang, H.; Goehring, A.; Wang, K.H.; Penmatsa, A.; Ressler, R.; Gouaux, E. Structural basis for action by
diverse antidepressants on biogenic amine transporters. Nature 2013, 503, 141–145. [CrossRef]
9. Wang, S.; Che, T.; Levit, A.; Shoichet, B.K.; Wacker, D.; Roth, B.L. Structure of the D2 dopamine receptor
bound to the atypical antipsychotic drug risperidone. Nature 2018, 555, 269–273. [CrossRef]
0. Scorza, M.C.; Carrau, C.; Silveira, R.; Zapata-Torres, G.; Cassels, B.K.; Reyes-Parada, M. Monoamine Oxidase
Inhibitory Properties of Some Methoxylated and Alkylthio Amphetamine Derivatives: Structure–Activity
Relationships. Biochem. Pharmacol. 1997, 54, 1361–1369. [CrossRef]
5
1. Tatsumi, M.; Groshan, K.; Blakely, R.D.; Richelson, E. Pharmacological profile of antidepressants and related
compounds at human monoamine transporters. Eur. J. Pharmacol. 1997, 340, 249–258. [CrossRef]
2. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4
and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30,
5
2
785–2791. [CrossRef]
5
3. Sanner, M.F. Python: A programming language for software integration and development. J. Mol. Graph.
Model. 1999, 17, 57–61. [PubMed]
5
4. Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular dynamics. J. Mol. Graph. 1996, 14, 33–38.
[CrossRef]
5
5
5. SYBYL-X 1.2; Tripos International: St. Louis, MO, USA, 2010.
6. Lorca, M.; Valdes, Y.; Chung, H.; Romero-Parra, J.; Pessoa-Mahana, C.D.; Mella, J. Three-Dimensional
Quantitative Structure-Activity Relationships (3D-QSAR) on a Series of Piperazine-Carboxamides Fatty Acid
Amide Hydrolase (FAAH) Inhibitors as a Useful Tool for the Design of New Cannabinoid Ligands. Int. J.
Mol. Sci. 2019, 20, 2510. [CrossRef] [PubMed]
5
5
7. Klebe, G.; Abraham, U.; Mietzner, T. Molecular similarity indices in a comparative analysis (CoMSIA) of drug
molecules to correlate and predict their biological activity. J. Med. Chem. 1994, 37, 4130–4146. [CrossRef]
[PubMed]
8. Clark, M.; Cramer III, R.D.; Van Opdenbosch, N. Validation of the general purpose tripos 5.2 force field.
J. Comput. Chem. 1989, 10, 982–1012. [CrossRef]
5
9. Golbraikh, A.; Tropsha, A. Beware of q2! J. Mol. Graph. Model. 2002, 20, 269–276. [CrossRef]
6
0. Tropsha, A. Best Practices for QSAR Model Development, Validation, and Exploitation. Mol. Inform. 2010
,
2
9, 476–488. [CrossRef]
6
6
6
1. Roy, K.; Chakraborty, P.; Mitra, I.; Ojha, P.K.; Kar, S.; Das, R.N. Some case studies on application of “r(m)2”
metrics for judging quality of quantitative structure-activity relationship predictions: Emphasis on scaling of
response data. J. Comput. Chem. 2013, 34, 1071–1082. [CrossRef]
2. Butini, S.; Nikolic, K.; Kassel, S.; Brückmann, H.; Filipic, S.; Agbaba, D.; Gemma, S.; Brogi, S.; Brindisi, M.;
Campiani, G.; et al. Polypharmacology of dopamine receptor ligands. Prog. Neurobiol. 2016, 142, 68–103.
[
CrossRef]
3. Turnaturi, R.; Aric
New players in an old game. Eur. J. Med. Chem. 2016, 108, 211–228. [CrossRef]
ò
, G.; Ronsisvalle, G.; Parenti, C.; Pasquinucci, L. Multitarget opioid ligands in pain relief:
Sample Availability: Samples of the compounds are available from the authors.
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