Synthesis and biological evaluation of new multi-target 3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives with potential antidepressant effect

Article abstract of DOI:10.1016/j.ejmech.2019.111736

A series of novel 3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives were synthesised and evaluated for their 5-HT_1A/D₂/5-HT_2A/5-HT₆/5-HT₇ receptor affinity and serotonin reuptake inhibition. Most of the evaluated compounds displayed high affinities for 5-HT_1A receptors (e.g., 4c K_i = 2.3 nM, 4l K_i = 3.2 nM). The antidepressant activity of the selected compounds was screened in vivo using the forced swim test (FST). The results indicate that compound MW005 (agonist of the pre- and postsynaptic 5-HT_1A receptor) exhibited promising affinities for the 5-HT_1A/SERT/D₂/5-HT₆/5-HT₇ receptors and showed an antidepressant-like activity in the FST model.

Full text of DOI:10.1016/j.ejmech.2019.111736

European Journal of Medicinal Chemistry 183 (2019) 111736
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of new multi-target 3-(1H-indol-
-yl)pyrrolidine-2,5-dione derivatives with potential antidepressant
effect
3
Martyna Z. Wr oꢀ bel a , Andrzej Chodkowski , Franciszek Herold , Monika Marciniak ,
,
*
a
a
a
a
b
b
c
Maciej Dawidowski , Agata Siwek , Gabriela Starowicz , Katarzyna Stachowicz ,
c
b
,
c
d
d
Bernadeta Szewczyk , Gabriel Nowak , Mariusz Belka , Tomasz B a˛ czek ,
Grzegorz Satała , Andrzej J. Bojarski , Jadwiga Turło
Department of Drug Technology and Pharmaceutical Biotechnology, Faculty of Pharmacy, Medical University of Warsaw, 1 Banacha Street, 02-097,
Warszawa, Poland
Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688, Krak oꢀ w, Poland
Department of Neurobiology, Maj Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343, Krak oꢀ w, Poland
Department of Pharmaceutical Chemistry, Medical University of Gda nꢀ sk, 80-416, Gda nꢀ sk, Poland
Department of Medicinal Chemistry, Maj Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343, Krak oꢀ w, Poland
e
e
a
a
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 August 2019
Received in revised form
A series of novel 3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives were synthesised and evaluated for
their 5-HT1A/D
evaluated compounds displayed high affinities for 5-HT1A receptors (e.g., 4c K
The antidepressant activity of the selected compounds was screened in vivo using the forced swim test
FST). The results indicate that compound MW005 (agonist of the pre- and postsynaptic 5-HT1A receptor)
2
/5-HT2A/5-HT
6
/5-HT
7
receptor affinity and serotonin reuptake inhibition. Most of the
i
¼ 2.3 nM, 4l K ¼ 3.2 nM).
i
23 September 2019
Accepted 24 September 2019
Available online 26 September 2019
(
exhibited promising affinities for the 5-HT1A/SERT/D
2
/5-HT
6
/5-HT
7
receptors and showed an
antidepressant-like activity in the FST model.
Keywords:
©
2019 Elsevier Masson SAS. All rights reserved.
Multitarget directed ligand
5
5
-HT1A agonists
-HT1A/SERT dual activity
Serotonin reuptake inhibitors
receptor ligands
Antidepressants
D
2
1
. Introduction
receptor ligands seem to be particularly promising [2]. Nowadays,
the following partial agonists of the 5-HT1A receptor are used as
medications e buspirone (anxiolytic), vilazodone and vortioxetine
(antidepressants), methysergide (antimigraine drug), cariprazine,
aripiprazole, lurasidone and brexpiprazole (neuroleptics) (Fig. 1) e
whereas many others are at various stages of clinical trials (Fig. 2)
[3,4]. This shows how useful 5-HT1A receptor ligands are for phar-
macotherapies, especially for psychiatric illness. Currently, the
studies are mostly focused on the use of 5-HT1A agonists in the
treatment of the most frequently diagnosed mental disease -
depression, and also as atypical neuroleptics [5,6].
Serotonergic transmission disorders in the central nervous
system (CNS) play a very important role in the pathomechanisms of
affective diseases, anxiety diseases, addictions, suicidal tendencies,
migraine, bulimia and obesity, as well as sleep and memory dis-
orders [1]. Up to now, many chemical substances with high affinity
for serotonergic receptors have been described, and 5-HT1A
Abbreviations: SAR, structure-activity relationship; 8-OH-DPAT, (±)-8-hydroxy-
2
-(di-N-propylamino)tetralin; K
i
, inhibitor constant; SSRIs, selective serotonin re-
Depression is the leading cause of disability worldwide,
affecting over 300 million people [11]. The most severe disadvan-
tages of the most frequently used SSRI medications are their long
latency period and low effectiveness (around one-third of patients
do not respond to pharmacotherapy) [2]. Thus, there is an
uptake inhibitors; hERG, Human Ether-a-go-go-related Gene; HEK293, human
embryonic kidney cell line 293; FST, forced swim test; PBS, phosphate-buffered
saline; P-gp, P-glycoprotein 1.
*
Corresponding author.
E-mail address: martyna.wrobel@wum.edu.pl (M.Z. Wr oꢀ bel).
https://doi.org/10.1016/j.ejmech.2019.111736
223-5234/© 2019 Elsevier Masson SAS. All rights reserved.
0

2
M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
Fig. 1. Approved drugs, serotonin 1A (5-HT1A) receptor partial agonist [7e10].
Fig. 2. The chemical structures of 5-HT1A receptor ligands as clinical candidates for atypical antipsychotics and antidepressants [3,4].
important need to search for new strategies for treatment of this
disease. A hypothesis of shortening of the latency period of SSRI
medications, involving the co-administration of substances acting
as SERT inhibitors and 5-HT1A antagonists, has appeared and has
been confirmed in pre-clinical studies. A decreased amount of
endogenous serotonin released as a consequence of a single
administration of SSRI medications was mostly abolished by a
simultaneous administration of a 5-HT1A autoreceptor antagonist e
either pindolol or the more selective WAY-100635 [12e15]. As has
been demonstrated in further studies, a return to activity of sero-
tonergic neurons is tightly associated with desensitisation of
somatodendritic 5-HT1A autoreceptors [16]. Studies on the devel-
opment of drugs with shorter latency periods were started on a
group of 5-HT1AR agonists. Simultaneous administration of SSRI
and a 5-HT1A receptor agonist or a partial agonist causes an im-
mediate increase in neurotransmission in the serotonergic system
by the stimulation of postsynaptic receptors. At the same time,
release of endogenous serotonin is inhibited by a negative feedback
activated by the stimulation of somatodendritic autoreceptors [17].
Wilcox described the clinical action of buspirone and gepirone as
partial agonists of the 5-HT1A receptor [18]. Simultaneous admin-
istration of buspirone and an SSRI caused a significant increase in
the effectiveness of therapy, when compared to administration of
the SSRI alone in patients suffering from depression, who had not
previously responded to other treatments [19].
Additionally, many research results suggest, that it is implau-
sible that a single 5-HT receptor can enhance actions of SSRI on
depressive symptoms, and therefore, ligands of a single receptor
are unlikely to overcome the limitations of current treatments [2].
On this basis, a novel strategy for treating depression requires
searching for drugs with a polypharmacological receptor profile
[20,21]. Research into new antidepressants targeting SERT/5-HT1A
5-HT is particularly promising, as simultaneous administration of
SSRI medications and the selective 5-HT antagonist (SB-269970)
/
7
7
increased the SSRIs’ antidepressant activity in animal models [22].
Moreover, contemporaneous research have indicated that vorti-
oxetine has cognitive enhancement effects as only one antide-
pressant; this activity has been linked to the blocking of the 5-HT
receptor [21,22]. Quite recently, another serotonin receptor, 5-HT R,
has begun to attract considerable interest as a valuable target
7
6

M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
3
affecting learning and memory processes. In fact, 5-HT
agents have been indicated to improve cognitive function in a
statistically significant manner [23,24].
6
receptor
of compounds with high binding affinities for 5-HT1AR and with
multi-target profiles because such compounds are regarded as
promising for the pharmacotherapy of depression. Therefore, the
synthesised derivatives were evaluated for their 5-HT1A receptor
affinity and serotonin reuptake inhibition. Selected compounds
2
The role of the 5-HT2A and D receptors in the therapy of both
schizophrenia and depression is well established [25e27]. An
example of an antidepressant with a pharmacological profile
featuring high-affinity partial agonism at 5-HT1A and antagonism at
2 6 7
were then tested for their affinity for D , 5-HT2A, 5-HT , and 5-HT
receptors, as well as in a panel of tests comprising metabolic sta-
bility, bioavailability prediction and hERG channel modulation as-
says. Finally, the most promising compounds, possessing desirable
receptor profiles, were subjected to in vivo testing in an animal
behavioural model of depression.
2 7
D , 5-HT2A, and 5-HT receptors is lurasidone (Fig. 1), approved by
the US Food and Drug Administration in June 2013 for the acute
treatment of bipolar depression [28]. Therefore, ligands with pol-
ypharmacological receptor profiles and with high affinity for the 5-
HT1A receptor as new antidepressants deserves particular attention.
In our previous paper, we described the synthesis and biological
evaluation of pyrrolidine-2,5-dione derivatives with very good
binding affinities for SERT and 5-HT1A receptors [29]. The SAR
analysis revealed that the presence of a 1,2,3,6-tetrahydropyridine
or piperidine moiety in the pharmacophore part of the molecule
has a significant impact. One of the main observations was that
compounds with a fully hydrogenated pyridine ring exhibited
better binding to the 5-HT1A receptor. Out of this series, 1-{4-[4-(5-
fluoro-1H-indol-3-yl)piperidin-1-yl]butyl}-3-(1H-indol-3-yl)pyr-
rolidine-2,5-dione (MW005) [29] was identified as a potent ligand
2. Results and discussion
2.1. Chemistry
Final compounds 4aer and 6aed were obtained via multi-step
synthesis, according to Scheme 1 and Scheme 2, respectively. In
order to carry out the synthesis, series of new synthons were
necessary: N-bromobutyl, N-bromopropyl and N-bromoethyl de-
rivatives of 3-(5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione
(2aec) or 3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (2def).
with multi-receptor profile (K
i
[nM] 5-HT1A ¼ 7.5, SERT ¼ 505,
Other intermediates, 1aeb and 1-(4-bromobutyl)-3-(1H-indol-3-
yl)pyrrolidine-2,5-dione (2g), as well as 3-piperidin-4-yl-1H-
indole (3aec) and 3-piperidin-3-yl-1H-indole derivatives (5aec)
were synthesised according to previously described procedures (for
more information see Supplementary data) [29,30,33e35].
Compounds 1a and 1b were obtained by reaction of maleimide
with 5-methoxyindole or 5-fluoroindole, respectively, in glacial
acetic acid (Scheme 1). This is a modification of the Michael addi-
tion, described by Macor et al. and Bergman et al. [33e35]. Macor
et al. described the differences in the time and yield of this reaction
as depending on the substituent in the C5 position of the 1H-indole
D
2
¼ 14, 5-HT2A ¼ 71, 5-HT
6
¼ 63 and 5-HT ¼ 196); therefore, it
7
was chosen as a starting point for design of further derivatives.
Hence, we designed and synthesised a series of 3-(1H-indol-3-yl)
pyrrolidine-2,5-dione derivatives (4aer) having a two-, three- or
four-methylene spacer, methoxy or fluoro substituent in the C5
position at the 1H-indole ring (R
uent at 3-piperidin-4-yl-1H-indole in the pharmacophore part of
the molecule (R ).
1
) and a different type of substit-
2
¼ eH, eF or eOCH
3
Keeping in mind the concept of developing ligands with high
affinity for 5-HT1A, we conducted a new exploratory study focused
on the replacement of the 3-piperidin-4-yl-1H-indole moiety with
1
(R ): 5-methoxyindole (electron donating group) was less active
3
-piperidin-3-yl-1H-indole in compounds 6aed (Fig. 3). This res-
and 5-fluoroindole (electron withdrawing group) was more active
in the reaction [35]. Our results confirm this observation. The
resulting derivatives 1a and 1b were subjected to N-alkylation with
idue is more similar in its structure to serotonin, and its confor-
mational constraints aimed to increase the activity of compounds
by increasing the affinity for the 5-HT1A receptor [30e32].
In the current project, we focused our attention on a new series
1,4-dibromobutane, 1,3-dibromopropane or 1,2-dibromoethane at
the N1 position, in the presence of potassium carbonate in acetone.
Fig. 3. Design strategy for a novel series of pyrrolidine-2,5-dione derivatives resulting from the optimisation of compound MW005.

4
M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
Scheme 1. The synthetic pathways to the investigated 3-piperidin-4-yl-1H-indole derivatives 4aer (series of 3-piperidin-4-yl-1H-indole derivatives). Reagents and conditions: (i)
CH COOH (ii) BrCH (CH CH CO , acetone (iii) K CO , KI(cat.), CH CN.
Br (n ¼ 0, 1 or 2), K
3
2
2
)
n
2
2
3
2
3
3
2 3 3
Scheme 2. The synthesis of the investigated compounds 6aed (series of 3-piperidin-3-yl-1H-indole derivatives). Reagents and conditions: (i) K CO , CH CN.
Intermediates 2aef were obtained in high yields (Scheme 1). Final
ligands 4aer and 6aed were synthesised by N-alkylation of com-
pounds 2aef or 2g with synthons 3aec or 5aec (Scheme 1 and
Scheme 2), according to previously described procedures [29]. To
obtain analytical samples, all final compounds 4aer and 6aed were
purified by column chromatography or via crystallisation.
four methylene spacer was crucial for affinity to the 5-HT1A re-
ceptor in this group of derivatives. Analogues with a shorter
aliphatic chain had, on average, 30 times higher K
i
values (e.g., 4r: K
¼ 3.2 nM). This is in contrast
i
˃ 5000 nM vs 4o: K
i
¼ 157.0 nM vs 4l: K
i
with our previous results based on a similar class of compounds,
which indicated good 5-HT1A binding affinity of ligands with a two-
carbon linker [29].
Next, we focused on the modification of the 3-(1H-indol-3-yl)
pyrrolidine-2,5-dione moiety. We decided to leave the pyrrolidine-
2,5-dione ring, because, as a cyclic imide and as a lipophilic portion
of the molecule, it has an important effect on the stabilisation of the
2
.2. In vitro receptor assay and structureeactivity relationship
(
SAR)
Our approach focused on the development of poly-
ligandereceptor complex via dipoleedipole and pep interactions
pharmacological agents with high affinity for 5-HT1A/SERT re-
ceptors, which would possess additional receptor activity.
Therefore, the synthesised compounds were initially tested for
their in vitro affinity for the 5-HT1A receptor and inhibition of SERT,
using a radioligand binding assay. Next, the selected structural
analogues of compound MW005, compounds 4aer, were tested for
[5,36,37]. We synthesised compounds 4aei that possessed the
methoxy group (R ), whereas compounds 4jer had a fluorine atom
1
as a substituent (R
1
), at the C-5 position of the 1H-indole ring. We
noticed that when comparing the compounds 4aei (R ¼ eOCH )
1
3
to their 5-fluoro analogues 4jer (R
had a higher affinity for the 5-HT1A receptor.
1
¼ eF), all but one (4d vs 4m),
their D
presented in Table 1 and Table 2.
In general, all the newly synthesised compounds, excluding 4q
and 4r, showed very high to moderate affinity for 5-HT1A
¼ 2.3e336 nM).
Keeping in mind the results of our previous study [29], we
2
, 5-HT2A, 5-HT
6
and 5-HT
7
receptor affinities. The results are
Finally, we examined the type of substituent in the C-5 position
of the 3-piperidin-4-yl-1H-indole (R
2
) pharmacophore part of the
molecule conferring optimal selectivity and affinity for 5-HT1A
.
R
Following our previous studies of 3-(1H-indol-3-yl)pyrrolidine-2,5-
dione derivatives docking to the binding site of the 5-HT1A receptor,
the affinity and selectivity of the investigated group of compounds
(K
i
determined the length of the alkyl linker that is optimal for 5-
HT1AR binding of the synthesised compounds. For this purpose,
we obtained derivatives of 3-(1H-indol-3-yl)pyrrolidine-2,5-dione
were predominantly determined by the electronegativity of the R
substituent [29,38]. Therefore, we assumed that compounds with
the eF or eOCH substituent (R ), as a result of their electronega-
2
3
2
4
aer having a two-, three- or four-methylene spacer between the
imide and 3-piperidin-4-yl-1H-indole moiety. We observed the
best K values for 4c (2.3 nM) and 4l (3.2 nM) (Table 1). Generally,
tivity and local inductive effects (in the case of fluorine), should
make protonated piperidine a much more effective proton donor,
i

M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
5
Table 1
5-HT1A receptor and SERT binding affinities of 3-piperidin-4-yl-1H-indole derivatives 4aer and 5-HT1A receptor binding affinities of 3-piperidin-3-yl-1H-indole derivatives
6aed.
compound
R
1
R
2
n
K
i
± SEM [nM]
5-HT1A
SERT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
6
6
6
6
a
b
c
d
e
f
g
h
i
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
F
F
F
F
F
F
F
F
F
3
3
3
3
3
3
3
3
3
H
F
OCH
H
F
OCH
H
F
OCH
H
F
OCH
H
F
OCH
H
F
OCH
H
F
OCH
OCH
2
2
2
1
1
1
0
0
0
2
2
2
1
1
1
0
0
0
2
2
2
2
6.3 ± 0.1
4.9 ± 0.1
2.3 ± 0.8
705.0 ± 62.7
17.5 ± 1.2
308.0 ± 29.0
523.0 ± 33.7
30.0 ± 2.9
111.0 ± 10.3
141.8 ± 14.4
221.0 ± 14.6
63.0 ± 7.0
51.0 ± 2.1
32.4 ± 1.9
201.0 ± 7.6
150.5 ± 13.0
140.0 ± 14.0
156.0 ± 15.4
102.0 ± 7.4
314.0 ± 15.2
1313.0 ± 150.0
NT
3
3
3
3
3
3
204.1 ± 13.7
246.0 ± 8.3
68.7 ± 7.8
48.7 ± 4.2
222.0 ± 23.0
240.0 ± 22.8
13.0 ± 1.0
7.0 ± 0.3
j
k
l
3.2 ± 0.2
m
n
o
p
q
r
a
b
c
143.2 ± 12.1
336.0 ± 23.5
157.0 ± 14.7
320.0 ± 33.0
700.0 ± 71.0
>5000
122.0 ± 11.9
183.0 ± 10.9
78.0 ± 8.0
52.0 ± 2.6
5.5 ± 0.5
H
H
H
OCH
NT
NT
NT
NT
3
3
d
3
serotonin
imipramine
NT
2.5 ± 0.3
NT - not tested.
Table 2
2 6 7
5-HT1A, D , 5-HT2A, 5-HT and 5-HT receptor binding affinities of selected 3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives.
compound
R
1
R
2
n
K
i
± SEM [nM]
5-HT1A
D
2
5-HT2A
5-HT
6
5-HT
7
MW005
MW011
H
H
OCH
OCH
OCH
F
F
2
2
2
2
2
2
2
2
7.5 ± 0.5
2.2 ± 0.7
6.3 ± 0.1
4.9 ± 0.1
2.3 ± 0.8
13.0 ± 1.0
7.0 ± 0.3
3.2 ± 0.2
14 ± 2
71 ± 9
498 ± 23
31 ± 4
63 ± 5
159 ± 9
81 ± 6
196 ± 14
345 ± 24
25 ± 3
OCH
H
F
OCH
H
F
3
3
3
161 ± 14
16 ± 2
4
4
4
4
4
4
a
b
c
j
k
l
3
3
3
a
a
a
641 ± 93a
320 ± 23
50±6a
62±7
118±9
85 ± 11
217 ± 13
190 ± 16
499 ± 65
427 ± 32
a
a
a
50±7
29±2
a
a
a
a
F
F
19±2
20±3
1944 ± 231
209 ± 26
301 ± 24
1158 ± 175
a
a
a
458 ± 32a
OCH
71±8
a
SD.
and, as a result, able to form a strong hydrogen bond with the 5-
HT1A receptor binding pocket. The current results reveal that the
influence of the type of R substituent in the 3-(1H-indol-3-yl)
2
pyrrolidine-2,5-dione derivatives on 5-HT1A binding can be ar-
ranged, for the four-methylene spacer series, in descending order:
Next, we replaced the 3-piperidin-4-yl-1H-indole moiety with
its 3-piperidin-3-yl-1H-indole regioisomer, which resulted in
compounds 6aed. As was mentioned in the Introduction, at the
initial stage of the study we checked whether series 6 shows any
affinity for the 5-HT1A receptor. The results for compounds (4aer)
show that a four-methylene spacer coupled with the 5-fluoro-4-
OCH
fluence falls in the opposite order for the ethyl-linker series. For the
three-methylene linker series, no clear influence of the R sub-
3
(4c, 4l) > F (4b, 4k) > H (4a, 4j). Surprisingly, the binding in-
piperidin-4-yl-1H-indole (R
2
¼ eF) or 5-methoxy-4-piperidin-4-
2
yl-1H-indole (R ) residue is optimal for high affinity for
2
¼ eOCH
3
stituent was observed. Nevertheless, it should be noted that the
results of the binding studies did not indicate significant differ-
ences in affinity for 5-HT1A arising from the type of substituent in
the pharmacophore part of the molecule, particularly within the
most potent four-methylene spacer series (4aec and 4jel).
5-HT1A; therefore, we decided to unchanged this length of the
linker. As shown in Table 1, compounds 6aed displayed good af-
finities for the 5-HT1A receptor, with the corresponding K ranging
i
from 52 nM to 183.0 nM. On the basis of these results, it can be
summarised that derivatives 6 are potent 5-HT1A ligands and

6
M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
should be explored in the future.
Compounds selected for the metabolic stability test differ in
substituents in R (-H, eOCH , eF) and R (-H, eF, eOCH ) posi-
We synthesised 3-(1H-indol-3-yl)pyrrolidine-2,5-dione de-
rivatives in order to improve both SERT inhibition and affinity for 5-
HT1A. Although most of the compounds from the 4aer series
display moderate SERT inhibitory activities, some of them show 5-
1
3
2
3
tions. Surprisingly, these structural differences have only a minor
impact on their metabolic stability. It was reported that substitu-
tion of the aromatic ring by fluorine can improve stability by
blocking oxidation reactions, whereas the methoxy substituent
usually easily undergoes the O-dealkylation reaction decreasing
stability significantly [39]. In this case, another structural element is
probably responsible for its susceptibility to undergo first phase
metabolism. We applied two chemoinformatic tools, namely Xen-
osite and SOMP - Site Of Metabolism Prediction to explore possible
origins of experimental observations [39,40]. The results for
MW005 are summarised in Fig. 5. Xenosite with its human liver
microsome model for CYP-mediated oxidations identified two
carbon atoms in the alkyl chain as the most probable site vulner-
able for biotransformation. Similarly, SOMP, equipped in several
models for different CYP isoenzymes pointed out the same posi-
tions. With the SOMP algorithm atoms are ranked by deltaP value
that is calculated as difference between probabilities that the atom
is or is not a site of metabolism (deltaP > 0.5 are shown). The
biotransformation of the above-mentioned sites of metabolism
leads to N-dealkylation and, as a consequence, the degradation of a
molecule into two smaller parts, which was previously reported
several times for similar compounds [41,42].
HT1A/SERT dual activities: 4b (5-HT1A
K
i
¼ 4.9 nM, SERT
K
i
¼ 17.5 nM) and 4k (5-HT1A ¼ 7.0 nM, SERT K
K
i
i
¼ 32.4 nM). These
results confirmed our previous observation that in order to achieve
dual affinity for 5-HT1A and SERT in this group of compounds, the
introduction of a four-methylene spacer along with the 5-fluoro-3-
piperidin-4-yl-1H-indole residue is highly recommended. This can
be explained by differences in the ligandereceptor interaction
2
depending on the R substituents, that is, electronegativity and
spatial size (i.e., van der Waals radius), according to the results
described for reference compound MW005 (Fig. 3) in the previous
paper [29]. Moreover, the relatively small volume of the SERT
binding pocket favours compounds with a eF substituent rather
3
than those bearing the larger eOCH group.
In order to verify the potential of the investigated group of de-
rivatives as multi-target agents, we tested selected MW005 ana-
logues bearing four-methylene spacers for their binding affinities
2 6 7
for D , 5-HT2A, 5-HT and 5-HT receptors using in vitro assays. The
results are summarised in Table 2. The affinities of the previously
reported derivatives MW005 and MW011 are also included for
comparison [29]. Compounds 4a and 4j displayed high potency for
5
-HT1A, 5-HT2A, 5-HT
a high affinity for D R. Compound 4j seemed particularly promising
because of its good affinity for all the tested receptors (K [nM]: 5-
HT1A ¼ 13, SERT ¼ 51, D ¼ 190, 5-HT2A ¼ 50, 5-HT ¼ 29 and 5-
HT
¼ 50). The compounds reported by us in a previous study
exhibited average to low affinities for 5-HT receptors [29]. In
contrast, the current results reveal that the presence of the fluoro or
methoxy group (R ) substituents increases affinity for 5-HT (4a
¼ 25 nM, 4j K ¼ 50 nM). On the other hand, the derivatives
MW011 and 4c, bearing the 5-methoxy-4-piperidin-4-yl-1H-indole
¼ eOCH ) moiety, are clearly most selective for 5HT1AR.
6
and 5-HT
7
receptors. In addition, 4a showed
2.4. In vivo assay
2
i
Next, the in vivo tests, i.e. the induced hypothermia and forced
swim tests (FST), in mice, for the same set of compounds (MW005,
4a, 4b, 4c, 4j, 4k and 4l) were carried out. This allowed us to specify
their agonist-antagonist properties in reference to pre- and post-
synaptic 5-HT1A receptors.
Compound 8-OH-DPAT, a 5-HT1A receptor agonist, induces hy-
pothermia in mice, an effect mediated through the somatodendritic
5-HT1A receptor [43,44]. This hypothermia is abolished by
WAY100635, an antagonist of 5-HT1A receptors [45]. Hence the
hypothermia produced by the compounds tested in mice (and
reduced by WAY100635) was regarded as a measure of presynaptic
2
6
7
7
1
7
K
i
i
(
R
2
3
2.3. Metabolic stability
5-HT1A receptor agonistic activity (Table 3 and Table 4). All tested
Because of the multireceptor profile of MW005 (5-HT1A, SERT,
compounds, like 8-OH-DPAT, induced hypothermia in mice, and
this hypothermia (Table 3) is abolished by WAY100635 (Table 4),
therefore all tested compounds were classified as presynaptic
agonists.
D
2
and 5-HT
tested. Additionally, compounds with the highest 5-HT1A affinity (K
nM] < 15: 4a, 4b, 4c, 4j, 4k and 4l) were selected for a metabolic
7
), its metabolic stability and in vivo activity was also
i
[
stability assay. The compounds were tested in an in vitro metabolic
stability assay involving human liver microsomes and NADPH as a
cofactor of first phase metabolism. All compounds showed rapid
biotransformation with only minor differences between tested
derivatives (Fig. 4).
To determine the postsynaptic 5-HT1A receptor agonistic effect
of the tested 5-HT1A ligands, their ability to reduce immobility time
in the FST was measured (Fig. 6 and Fig. 7). Only compound MW005
shortened immobility time in the FST and decreased activity in the
spontaneous locomotor activity test at 10 mg/kg dose (Fig. 7),
Fig. 4. Metabolic stability of selected derivatives expressed as metabolic half-life in an in vitro test. The box shows the average value and the whiskers represent standard deviation
n ¼ 3).
(

M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
7
Fig. 5. Results for in silico site of metabolism prediction for MW005. A: Xenosite output as graphical representation with colour scale bar showing the probability of undergoing
CYP-mediated oxidation (HLM Model). B: SOMP output with selected deltaP values for CYP isoforms and associated positions in a molecule. Stereochemistry of chiral centres in the
molecule is forced by internal algorithm of Xenosite and cannot be changed by end-user. (For interpretation of the references to colour in this figure legend, the reader is referred to
the Web version of this article.)
therefore it can be classified as an agonist of the pre- and post-
synaptic 5-HT1A receptor with antidepressant activity. The in vivo
tests results were summarised in Table 5.
adrenoceptors. It was suggested that 5-HT1A ligands exhibit low
selectivity due to a high degree of homology (approximately 45%)
between 5-HT1A and
1
a -adrenoceptors, and it might cause unde-
Effects of MW005 on the immobility time in the FST in CD-1
mice. MW005 was administered i.p. 30 min before the FST. Values
expressed as the means ± S.E.M. were evaluated by one-way
ANOVA. **P < 0.01, vs vehicle group, n ¼ 10; MW005 (10 mg/kg
i.p.) was administered 30 min before the locomotor activity test.
Values are expressed as mean ± S.E.M. (n ¼ 3e6), followed by t-test.
sirable side effects such as hypotensive effects and worsened out-
comes of the clinical state [49]. Compound MW005 displayed high
affinities for both the 5-HT1A and
a
1
receptors (K
i
¼ 7.5 ± 0.5 and
K
i
¼ 30 ± 3 nM, respectively). In summary, MW005 presented
bioavailability and cardiac toxicity results that merit further opti-
misation of the compound to improve its permeability, oral phar-
macokinetic properties and cardiac safety.
*
p < 0.05 vs vehicle group.
2.5. The bioavailability prediction and cardiac toxicity in vitro
assays
3. Conclusions
Concerning the results described above, early in vitro bioavail-
Administering of antidepressant and neuroleptic drugs with
polypharmacological profiles have become a widely used thera-
peutic approach. In the present work, we described synthesis, re-
ceptor affinity, functional profile, metabolic stability and
behavioural evaluation of a new series of 3-(1H-indol-3-yl)pyrro-
lidine-2,5-dione derivatives. Among them, compound 4c exhibited
ability assays (the Bioavailability Panel) were performed at Eurofins
Pharma Discovery Services for the most promising compound
MW005. This panel consists of the aqueous solubility, human
plasma protein binding and Caco-2 permeability assays, which are
used for the prediction of ADME properties, that have to be
considered during decision making in the drug development pro-
cess. The results are summarised in Table 6 Compound MW005
displayed good aqueous solubility (simulated intestinal fluid (SIF) e
the highest binding affinities at 5-HT1AR (K
pound 4b displayed the best double binding 5-HT1A/SERT activity
(5-HT1A ¼ 17.5 nM). On the other hand, MW005
¼ 4.9 nM, SERT K
and 4j showed polypharmacological profiles potentially beneficial
for the treatment of depression (MW005 e K
[nM]: 5-HT1A ¼ 7.5,
SERT ¼ 505, D ¼ 14, 5-HT2A ¼ 71, 5-HT ¼ 63, 5-HT ¼ 196; 4j e K
[nM]: 5-HT1A ¼ 13, SERT ¼ 51, D ¼ 190, 5-HT2A ¼ 50, 5-HT ¼ 29, 5-
HT
¼ 50). Our SAR studies revealed that A) the four-methylene
spacer was crucial for affinity for the 5-HT1A receptor, B) the
presence of the methoxy group (R ) substituent increased affinity
i
¼ 2.3 nM), and com-
K
i
i
1
90.6
mM; phosphate-buffered saline, pH 7.4 (PBS) e 136.5
mM;
simulated gastric fluid (SGF) e 151.0
mM), high plasma protein
i
binding (95%) and low Caco-2 permeability (Papp ꢀ 2.5 e for more
2
6
7
i
details see Supplementary data). Drugs that are completely absor-
2
6
bed in humans are characterised by permeability coefficients of
7
ꢂ6
>
0
<
1 ꢁ 10 cm/s; >1% but <100% have permeability coefficients of
ꢂ6
.1e1.0 ꢁ 10 cm/s, while drugs and peptides that are absorbed at
1
ꢂ7
1% have permeability coefficients of ꢀ1 ꢁ 10 cm/s [46]. The final
for the 5-HT1A receptor and C) considering the four-carbon linker
series, the binding results did not indicate significant differences in
affinity for 5-HT1A upon varying the type of substituent in the
efflux ratio (ER ¼ basolateral to apical [BA]/apical to basolateral
[
AB] permeability) was ER ꢀ 2.5, which means that compound
MW005 is not considered to be a P-gp substrate [47].
2
pharmacophore part of the molecule (R ). However, in order to
Compound MW005 was further characterised in the hERG
channel automated patch-clamp assay to verify if it has potential to
trigger cardiac toxicity. Blocking the human ether-a-go-go-related
gene (hERG) potassium channel causes QT interval prolongation,
which is associated with potentially fatal arrhythmia, called Tor-
sades de Pointes [48]. This study was performed at Eurofins Pharma
Discovery Services. Compound MW005 was tested at three con-
centrations (10.0, 1.0 and 0.1 mM) showing 96.4, 84.7 and 45.1%
inhibition of tail current, respectively. This indicates that MW005
has a high propensity for hERG channel blocking. Additionally, we
obtain dual affinity for 5-HT1A/SERT in this group of compounds,
the introduction of a four-methylene spacer and a 5-fluoro-3-
piperidin-4-yl-1H-indole residue was crucial. The presence of the
fluoro or methoxy group (R
1
) substituents increased the affinity of
the compounds for 5-HT . These efforts led to the selection of
7
MW005, which combined a multi-receptor mechanism with
prominent 5-HT1AR pre- and postsynaptic agonism, good metabolic
stability and dose-dependent reduction of the immobility time in
the FST. Nevertheless, the presented bioavailability study and the
cardiac toxicity results suggested a need for further optimisation to
improve oral pharmacokinetic properties and cardiac safety.
determined the affinity of compound MW005 for
1
a -

8
M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
Table 3
The effect of the tested compounds: MW005, 4a, 4b, 4c, 4j, 4k and 4l on the body temperature in mice.
0
Treatment
Dose (mg/kg)
D
t ± SEM ( C)
30 min
60 min
90 min
120 min
Vehicle
MW005
e
0.4 ± 0.1
0.3 ± 0.1
0.5 ± 0.1
0.4 ± 0.3
0.3 ± 0.1
0.5 ± 0.3
0.1 ± 0.2
0.0 ± 0.3
ꢂ0.4 ± 0.2
0.1 ± 0.1
0.2 ± 0.3
0.2 ± 0.2
0.3 ± 0.2
ꢂ0.3 ± 0.2
ns
5
c
c
c
10
15
20
ꢂ1.4 ± 0.1
ꢂ2.0 ± 0.2
ꢂ2.2 ± 0.1
ꢂ0.5 ± 0.3
ꢂ0.8 ± 0.4
ꢂ1.0 ± 0.3
b
c
a
p < 0.0001
p ¼ 0.0002
p ¼ 0.0496
Vehicle
e
ꢂ0.2 ± 0.0
ꢂ0.2 ± 0.2
ꢂ0.2 ± 0.1
ꢂ0.0 ± 0.3
ꢂ0.4 ± 0.2
ꢂ0.3 ± 0.1
0.0 ± 0.2
ꢂ0.1 ± 0.2
ꢂ0.1 ± 0.1
ꢂ0.4 ± 0.2
ꢂ0.9 ± 0.2
4a
5
c
1
2
0
0
ꢂ1.3 ± 0.4
ꢂ2.9 ± 0.1
ꢂ0.4 ± 0.1
c
c
b
b
ꢂ1.5 ± 0.2
p < 0.0001
ꢂ1.1 ± 0.2
p ¼ 0.0006
p < 0.0001
p ¼ 0.0084
Vehicle
e
ꢂ0.1 ± 0.1
ꢂ0.1 ± 0.2
ꢂ0.1 ± 0.1
0.4 ± 0.2
ꢂ0.1 ± 0.5
0.5 ± 0.2
ns
ꢂ0.2 ± 0.2
0.1 ± 0.2
0.3 ± 0.3
0.6 ± 0.1
ns
ꢂ0.1 ± 0.1
ꢂ0.4 ± 0.2
0.4 ± 0.2
0.6 ± 0.1
p ¼ 0.002
4b
5
b
10
ꢂ1.3 ± 0.6
ꢂ1.8 ± 0.4
c
20
p < 0.0001
Vehicle
e
0.1 ± 0.2
0.2 ± 0.2
0.1 ± 0.2
0.3 ± 0.2
0.6 ± 0.1
0.2 ± 0.2
ꢂ0.1 ± 0.1
0.2 ± 0.2
0.6 ± 0.2
0.0 ± 0.2
0.4 ± 0.2
0.4 ± 0.2
0.3 ± 0.2
ꢂ0.5 ± 0.4
p ¼ 0.0249
4c
5
a
a
1
1
2
0
5
0
ꢂ0.5 ± 0.2
ꢂ0.9 ± 0.2
ꢂ3.1 ± 0.2
c
c
0.5 ± 0.2
c
ꢂ1.2 ± 0.4
ꢂ0.6 ± 0.3
p < 0.0001
p < 0.0001
p ¼ 0.0008
Vehicle
e
ꢂ0.1 ± 0.1
ꢂ0.4 ± 0.1
ꢂ0.6 ± 0.3
ꢂ0.2 ± 0.1
ꢂ0.2 ± 0.2
0.1 ± 0.2
0.1 ± 0.2
ꢂ0.2 ± 0.2
0.0 ± 0.2
ꢂ0.4 ± 0.2
ꢂ0.2 ± 0.4
ns
0.1 ± 0.1
4j
5
ꢂ0.3 ± 0.2
ꢂ0.2 ± 0.2
ꢂ0.4 ± 0.2
a
1
1
2
0
5
0
c
b
ꢂ1.8 ± 0.3
ꢂ2.3 ± 0.3
ꢂ0.7 ± 0.3
c
b
ꢂ0.7 ± 0.3
ꢂ0.6 ± 0.3
p ¼ 0.0214
p < 0.0001
p ¼ 0.0011
Vehicle
e
ꢂ0.1 ± 0.1
0.0 ± 0.2
0.2 ± 0.2
ꢂ0.1 ± 0.2
ꢂ0.4 ± 0.3
ns
0.0 ± 0.2
0.4 ± 0.2
0.3 ± 0.2
0.1 ± 0.2
ns
0.1 ± 0.1
0.3 ± 0.2
0.4 ± 0.2
0.5 ± 0.2
ns
a
4k
5
0.4 ± 0.2
c
10
ꢂ1.0 ± 0.2
c
20
ꢂ2.0 ± 0.2
p < 0.0001
Vehicle
e
0.1 ± 0.4
0.4 ± 0.1
0.1 ± 0.1
0.6 ± 0.1
0.2 ± 0.2
ꢂ0.0 ± 0.1
0.6 ± 0.1b
0.2 ± 0.1
0.3 ± 0.0
a
0.8 ± 0.1a
0.3 ± 0.2
4l
5
c
1
2
0
0
ꢂ1.1 ± 0.2
c
c
ꢂ3.2 ± 0.2
ꢂ1.4 ± 0.3
ꢂ0.6 ± 0.3
ꢂ0.2 ± 0.3
p < 0.0001
p < 0.0001
p ¼ 0.0002
p ¼ 0.0224
Vehicle
WAY100635
e
0.2 ± 0.1
0.1 ± 0.2
0.1 ± 0.2
0.1 ± 0.2
0.1 ± 0.2
ꢂ0.1 ± 0.2
ꢂ0.1 ± 0.1
ns
0.1 ± 0.2
ꢂ0.2 ± 0.1
0.3 ± 0.3
ns
0.1
5
c
c
8-OH-DPAT
ꢂ1.7 ± 0.2
ꢂ1.1 ± 0.2
p < 0.0001
p < 0.005
The investigated compounds were administered (ip) 30 min before test. The absolute mean body temperatures were within a range 37 ± 0.5 ꢃC; n ¼ 7e8 mice per group, a
p < 0.05, b p < 0.01, cp < 0.001 vs. respective vehicle.
4
4
4
. Experimental protocols
(500 MHz for 1H NMR, 125 MHz for 13C NMR) spectrometer in
acetoneeD , CDCl or CD OD. Chemical shifts ( ) were expressed in
6
3
3
d
.1. Chemistry
parts per million (ppm) relative to tetramethylsilane used as the
internal reference. The following abbreviations are used to describe
peak patterns where appropriate: s (singlet), bs (broad singlet),
d (doublet), dd (doublet of doublets), t (triplet), td (triplet of dou-
blets), pt (pseudo triplet), 4d (quartet of doublets), m (multiplet), q
(quartet), qu (quintet), * - coupling with fluorine nucleus. Coupling
constants (J) are in hertz (Hz).
.1.1. General remarks
All solvents and reagents were purchased from commercial
sources and were used without any purification. Melting points
were determined on an Electrothermal IA9200 apparatus (Cole-
Parmer Ltd., Stone, Staffordshire, UK) with open capillary tubes and
were uncorrected. The purity (>95%) and homogeneity of the
compounds were routinely confirmed. NMR spectra were recorded
on a Varian INOVA 500 MHz (Varian, Palo Alto, CA, USA) (500 MHz
The High-Resolution Mass Spectrometry (HRMS) was per-
formed using Micromass LCT TOF (Waters Corporation, Milford,
MA, USA) mass spectrometer equipped with ESI ionization source,
TOF analyser and MCP detector. Samples as 1 mg/L concentration of
tested compounds were prepared in methanol. MS detection set-
1
13
for H NMR, 125 MHz for C NMR), Varian Unity Plus 200 MHz
13
(
Varian, Palo Alto, CA, USA) (50 MHz for C NMR) or Bruker
ꢃ
AVANCE III HD 500 MHz (Bruker BioSpin, Rheinstetten, Germany)
tings were as follows: source temperature 80 C, desolvation

M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
9
Table 4
Table 5
The effect of WAY100635 (0.1 mg/kg s.c.) on the hypothermia induced by com-
pounds MW005, 4a, 4b, 4c, 4j, 4k and 4l.
Functional in vivo 5-HT1A receptor activity of the investigated compounds.
Compound
5-HT1A receptor functional activity
0
Treatment and dose (mg/kg)
D
t ± SEM ( C)
Presynaptic
Postsynaptic
3
0 min
60 min
MW005
Agonist
Agonist
Agonist
Agonist
Agonist
Agonist
Agonist
Agonist
Vehicle þ vehicle
ꢂ0.0 ± 0.1
ꢂ1.4 ± 0.1
ꢂ0.1 ± 0.3
0.3 ± 0.1
ꢂ0.5 ± 0.3
0.7 ± 0.3
ns
4a
4b
4c
4j
c
f
Vehicle þ MW005 (10)
WAY100635 (0.1) þ MW005 (10)
No agonists activity
p < 0.0001
No agonists activity
No agonists activity
4k
Vehicle
ꢂ0.0 ± 0.1
ꢂ0.1 ± 0.2
ꢂ0.4 ± 0.2
ꢂ0.2 ± 0.2
ns
4l
b
Vehicle þ 4a (10)
ꢂ1.3 ± 0.4
ꢂ0.4 ± 0.3
d
WAY100635 þ 4a (10)
p ¼ 0.0036
ꢃ
temperature 150 C, desolvation gas flow rate 200 L/h, cone gas
flow 100 L/h, capillary potential 3.50e5.00 kV, cone potential
26e50 V, extraction cone potential 4e20 V, RF Lens 260e350 V.
Nitrogen was used for both nebulizing and drying gas. The data
were obtained in a scan mode ranging from 20 to 2000 m/z in time
Vehicle
0.1 ± 0.1
0.4 ± 0.0
ꢂ0.1 ± 0.5
0.4 ± 0.2
ns
b
Vehicle þ 4b (10)
ꢂ1.3 ± 0.6
ꢂ0.1 ± 0.1
p ¼ 0.0064
WAY100635 þ 4b (10)
1
.0 s intervals. Data acquisition software was MassLynx V 3.5
(Waters).
Flash column chromatography was carried out on Merck silica
Vehicle
0.1 ± 0.3
0.1 ± 0.3
0.2 ± 0.2
0.3 ± 0.3
ns
c
Vehicle þ 4c (15)
ꢂ0.9 ± 0.2
e
WAY100635 þ 4c (15)
ꢂ0.1 ± 0.2
p ¼ 0.0006
ꢂ0.1 ± 0.2
gel 60 (230e400 mesh ASTM) using dichloromethane/methanol as
the solvent (99:1, 98:2, 97:3, 95:5 [v/v]). Thin layer chromatography
was run on Merck silica gel (Kieselgel 60 F254) plates (Merck,
Darmstadt, Germany), with mobile phases of dioxane, toluene,
ethanol and 25% NH
methanol, diethyl ether and NH
Vehicle
ꢂ0.0 ± 0.1
ꢂ0.7 ± 0.3
0.0 ± 0.2
p ¼ 0.0308
c
Vehicle þ 4j (15)
ꢂ1.8 ± 0.3
ꢂ0.8 ± 0.3
e
WAY100635 þ 4j (15)
p < 0.0001
4
OH (6.0:3.2:0.5:0.2 [v/v]) or chloroform,
OH (18.0:4.0:3.6:0.4). Compounds
4
Vehicle
0.1 ± 0.1
0.4 ± 0.0
were visualised by UV light (254 nm). Room temperature refers to
2
c
Vehicle þ 4k (10)
ꢂ1.0 ± 0.2
0.4 ± 0.2f
p < 0.0001
ꢂ0.1 ± 0.2
ꢃ
0e25 C. Intermediate 1-(4-bromobutyl)-3-(1H-indol-3-yl)pyrro-
b,e
WAY100635 þ 4k (10)
0.8 ± 0.2
lidine-2,5-dione (2g), derivatives of 3-piperidin-4-yl-1H-indole
3aec) and 3-piperidin-3-yl-1H-indole derivatives (5aec), as well
p ¼ 0.0012
(
Vehicle
0.1 ± 0.4
0.0 ± 0.2
0.2 ± 0.2
0.5 ± 0.2
ns
as compounds MW005 and MW011 (Scheme 1, Scheme 2 and
Table 2) were obtained following the protocol described in our
previous paper (for more information see Supplementary data)
c
Vehicle þ 4l (10)
ꢂ1.1 ± 0.2
d
WAY100635 þ 4l (10)
ꢂ0.4 ± 0.2
p < 0.0001
1
13
[29,30]. Atom numbering, H NMR and C NMR spectra of all
WAY100635 was administered 15 min before the tested compounds.
synthesised compounds is available in Supplementary data, for
atom numbering see Fig. 8.
a
p < 0.05 vs vehicle, bp < 0.01 vs vehicle, p < 0.001 vs vehicle.
c
d
e
f
p < 0.05 vs compound group, p < 0.01 vs compound group, p < 0.001 vs compound
group.
4
1
.1.2. General procedure for the preparation of 3-(5-substituted-
H-indol-3-yl)pyrrolidine-2,5-dione (1aeb)
A
mixture of 5-fluoro-1H-indole or 5-methoxy-1H-indole
0.10 mol) and 10% molar excess of maleimide (12.87 g, 0.11 mol) in
acetic acid (125 mL) was refluxed while stirring for about 48 h and
8 h, respectively for 1a and 1b. The completion of the reaction was
(
6
assigned chromatographically (TLC). The reaction mixture was
cooled, the solvent was removed under vacuum and the crude
product was crystallized as described below to give compound
(1aeb) [37e39].
4
.1.2.1. 3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione
(1a).
Fig. 6. Effect of compounds: 4b, 4j and 4k on the FST in CD-1 mice.
The crude product (brown oil) was dissolved in a few mL of cold
Fig. 7. Effect of MW005 on the FST in CD-1 mice and effect of MW005 on locomotor activity in mice.

10
M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
Table 6
In vitro bioavailability data for MW005.
Assay
MW005 concentration
200
Property
Reference
195.4 M (metoprolol)
a
(
PBS)
m
M
136.5
190.6
151.0
95%
m
m
m
M
M
M
m
b
Aqueous solubility
(SIF)
(
SGF)c
Protein bindingd
Permeability
10
10
m
m
M
M
85% (warfarin)
e
e
ꢂ6
ꢂ6
ꢂ6
A-B
B-A
0.6 [10 cm/s]
0.3 [10 cm/s] (ranitidine)
ꢂ6
1.5 [10 cm/s]
2.9 [10 cm/s] (ranitidine)
Solubility study was performed in a 200
m
M; concentration.
a
aqueous solubility at buffer pH7.4.
b
aqueous solubility at simulated intestinal fluid.
aqueous solubility in simulated gastric fluid.
plasma human.
c
d
e
Caco-2, pH 6.5/7.4.
0
3
0
x
x
x
1
12.4 (C7 , d*, J ¼ 9.8),133.1 (C7 a), 32.7 (C2 ), 29.9 (C2 ), 38.0 (C3 ).
4
2
.1.3.2. 1-(3-bromopropyl)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-
,5-dione (2b). The title compound was isolated as a dark yellow
oil. Yield: 77% (2.18 g); ESI-HRMS m/z calcd for C15
H
14FBrN
2 2
O Na
þ
1
(
M
þ
Na) 375.0120, found 375.0120;
H NMR (500 MHz,
3
¼9.5, 3J
4
acetoneeD
(
7.15 (C2 H, d,
(
6
):
d
4.26 (C3H, 4d,
J
1
2
¼ 5.0, J ¼ 0.5), 2.90
2
3
2 3
C4H(1), dd, J ¼ 18.5, J ¼ 5.0), 3.27 (C4H(2), dd, J ¼ 18.5, J ¼ 9.5),
0
3 3 4
0
Fig. 8. General atom numbering for 1H NMR and 13C NMR spectra.
J ¼ 3.0), 7.09 (C4 H, dd,
J
H-F ¼ 9.0, J ¼ 2.5), 6.97
0
3
4
0
3 4
C6 H, td, J ¼ 9.5, J ¼ 2.5), 7.28 (C7 H, dd, J ¼ 9.0, JH-F ¼ 4.5), 3.77
x
3
x
3
x
3
(
C1 H
2
, t, J ¼ 7.0), 2.23 (C2 H
2
, q, J ¼ 7.0), 3.40 (C3 H
2
, q, J ¼ 6.5),
0
13
methanol, then dichloromethane was added to clear opacity. The
8.25 (N1 H, bs); C NMR (125 MHz, acetoneeD ):
d
178.0 (C2), 38.0
6
0
ꢃ
0
4
mixture was left for one week at 4 C. The precipitated solid was
(C3), 36.1 (C4), 176.0 (C5), 123.7 (C2 ), 111.6 (C3 , d*, J ¼ 4.9), 126.1
3 2
0
0
0
filtered, washed with dichloromethane and dried. The title com-
(C3 a, d*, J ¼ 9.8), 103.6 (C4 , d*, J ¼ 23.9), 158.0 (C5 , d*,
1
0
2
0
3
pound was isolated as orange solid. Yield: 61% 5.28 g, m.p.
J ¼ 235.9), 111.3 (C6 , d*, J ¼ 26.4), 112.4 (C7 , d*, J ¼ 9.3), 133.1
x x x
ꢃ
ꢃ
0
1
96e199 C (lit. 190e195 C [34]).
(C7 a), 30.6 (C1 ), 29.8 (C2 ), 38.0 (C3 ).
4
.1.2.2. 3-(5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione
(1b).
4.1.3.3. 1-(2-bromoethyl)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-
dione (2c). The title compound was isolated as a yellow oil. Yield:
Crystallisation from ethyl acetate. The title compound was isolated
as a light brown powder. Yield: 77% 6.92 g, m.p. 180e190 C (lit.
ꢃ
þ
50% (3.1 g); ESI-HRMS m/z calcd for C14H12FBrN O Na (M þ Na)
2
2
ꢃ
1
180 C, melts with decomposition [33]).
6
360.9964, found 360.9977; H NMR (500 MHz, acetoneeD ): d 4.46
3
3
2
3
(
C3H, dd,
J
1
¼9.5,
J
2
¼ 5.0), 2.90 (C4H(1), dd, J ¼ 18.5, J ¼ 5.5),
3 3
2
0
4
.1.3. General procedure for the preparation of 1-(4-bromoalkyl)-3-
3.36 (C4H(2), dd, J ¼ 18.5, J ¼ 5.5), 7.42 (C2 H, d, J ¼ 2.0), 7.31
0
3 4 5 3
0
(
5-substituted-1H-indol-3-yl)pyrrolidine-2,5-dione (2aef)
(C4 H, 4d, J ¼ 8.5,
J
H-F ¼ 4.5, J ¼ 0.5), 6.94 (C6 H, td,
J
H-F ¼ 9.0,
4
3
0
x
3
4
5
x
The mixture of appropriate 3-(5-substituted-1H-indol-3-yl)
J ¼ 2.5), 7.42 (C7 H, 4d, J ¼ 8.5, J ¼ 4.5, J ¼ 0.5), 3.97 (C1
2
H , t,
6
):
3
13
pyrrolidine-2,5-dione (1aeb) (0.01 mol) and 1,4-dibromobutane
J ¼ 7.0), 3.69 (C2
H
2
, t, J ¼ 6.5); C NMR (125 MHz, acetoneeD
1
(
(
0.05 mol), 1,3-dibromopropane (0.05 mol) or 1,2-dibromoethane
0.10 mol) and K CO (0.02 mol) and 100 mL of acetone was stir-
d
178.4 (C2), 38.6 (C3), 36.8 (C4), 158.5 (C5, d*, J ¼ 230.8), 125.8
4
0
2
(C2'), 112.6 (C3', d*, J ¼ 4.9), 127.6 (C3 a), 104.5 (C4', d*, J ¼ 23.4),
2
3
3
0
x
x
red and refluxed for 4e6 h (14 h for 1-(2-bromoethyl)-3-(5-fluoro-
H-indol-3-yl)pyrrolidine-2,5-dione (2c)). Reaction time was
113.5 (C7', d*, J ¼ 9.75), 134.5 (C7 a), 40.9 (C1 ), 28.9 (C2 ).
1
monitored using TLC. After cooling, the mixture was filtered, and
the filtrate was evaporated to dryness. The crude residue was pu-
4.1.3.4. 1-(4-bromobutyl)-3-(5-methoxy-1H-indol-3-yl)pyrrolidine-
2,5-dione (2d). The title compound was isolated as a dark yellow
ꢃ
crystals. Yield: 64% (1.86 g); m.p. 103e107 C; ESI-HRMS m/z calcd
rified by flash chromatography using CH
CH Cl as an eluent. Proper fractions were identified by TLC and
evaporated to dryness.
2
Cl
2
/MeOH (98:2 v/v) or
þ
1
2
2
for C17H19BrN O Na (M þ Na) 401.0477, found 401.0473; H NMR
2
3
3 3
(500 MHz, CDCl ):
3
d
4.26 (C3H, dd, J ¼9.5, J ¼ 5.0), 2.92 (C4H(1),
2 3
1
2
2
3
dd, J ¼ 18.0, J ¼ 5.0), 3.25 (C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.06
0
3
0
4
0
3
4.1.3.1. 1-(4-bromobutyl)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-
(C2 H, d, J ¼ 2.5), 6.87 (C4 H, d, J ¼ 2.0), 7.25 (C7 H, dd, J ¼ 8.5,
5
0
3
4
x
3
dione (2a). The title compound was isolated as a yellow crystals.
J ¼ 0.5), 6.88 (C6 H, dd, J ¼ 8.5, J ¼ 2.5), 3.64 (C1 H
2
, t, J ¼ 7.0),
x
x
x
3
ꢃ
Yield: 92% (1.69 g); m.p. 108e130 C (melts with decomposition);
1.88 (C2 H
2
, m), 1.82 (C3 H
2
, m), 3.42 (C4 H
2
, t, J ¼ 6.5), 3.83
þ
13
ESI-HRMS m/z calcd for C16
H
16FBrN
2
O
2
Na (M þ Na) 389.0277,
(OCH
3
, s), 8.14 (NH, bs); C NMR (125 MHz, CDCl
3
):
d
178.1 (C2),
1
0
0
0
found 389.0275; H NMR (500 MHz, acetoneeD
6
):
d
4.24 (C3H, 4d,
38.0 (C3), 36.2 (C4), 176.4 (C5), 122.7 (C2 ), 111.2 (C3 ), 126.2 (C3 a),
3
3
2
3
0 0 0 0 0
J
1
¼9.5,
J
2
¼ 5.0), 2.88 (C4H(1), dd, J ¼ 18.0, J ¼ 5.0), 3.25
100.6 (C4 ), 154.4 (C5 ), 112.4 (C6 ), 112.8 (C7 ), 131.8 (C7 a), 38.1
2
3
0
3
0
0
x
x
x
x
(
C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.12 (C2 H, d, J ¼ 2.5), 7.08 (C4 H,
3
(C1 ), 29.9 (C2 ), 26.5 (C3 ), 32.7 (C4 ), 55.9 (OCH ).
3
4
0
3
4
dd, JH-F ¼ 9.5, J ¼ 2.5), 6.96 (C6 H, td, J ¼ 9.0, J ¼ 2.5), 7.26 (C7 H,
3
4
x
3
x
dd, J ¼ 8.5, JH-F ¼ 4.5), 3.65 (C1 H
2
, t, J ¼ 7.0), 1.88 (C2 H
2
, m), 1.82
4.1.3.5. 1-(3-bromopropyl)-3-(5-methoxy-1H-indol-3-yl)pyrrolidine-
x
x
3
0
13
(
(
C3 H
2
, m), 3.42 (C4 H
):
2
, t, J ¼ 6.5), 8.32 (N1 H, bs); C NMR
2,5-dione (2e). The title compound was isolated as a dark yellow
ꢃ
125 MHz, acetoneeD
6
d
178.0 (C2), 38.1 (C3), 36.1 (C4), 176.1 (C5),
crystals. Yield: 82% (2.42 g); m.p. 141e143 C; ESI-HRMS m/z calcd
0
0
4
0
3
þ
1
1
23.8 (C2 ), 111.6 (C3 , d*, J ¼ 4.9), 126.0 (C3 a, d*, J ¼ 9.7), 103.6
for C16
(500 MHz, CDCl
H
17BrN
2
O
3
):
Na (M þ Na) 387.0320, found 387.0316; H NMR
0
2
0
1
0
2
3
3
(
C4 , d*, J ¼ 23.9),158.0 (C5 , d*, J ¼ 235.8),111.3 (C6 , d*, J ¼ 26.3),
3
d
4.26 (C3H, dd, J
1
¼9.5, J
2
¼ 5.0), 2.92 (C4H(1),

M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
11
2
3
2
3
dd, J ¼ 18.5, J ¼ 5.0), 3.26 (C4H(2), dd, J ¼ 18.5, J ¼ 9.5), 7.05
compound was isolated as a yellow solid. Yield: 46.5% (0.12 g); m.p.
0
3
0
4
0
3
ꢃ
þ
(
C2 H, d, J ¼ 2.5), 6.86 (C4 H, d, J ¼ 2.5), 7.25 (C7 H, dd, J ¼ 8.5,
111e119 C; ESI-HRMS m/z calcd for C30
517.2612, found: 517.2615; H NMR (500 MHz, acetoneeD
H
32FN
4
O
3
H (M þ H)
5
0
3
4
x
3
1
J ¼ 0.5), 6.88 (C6 H, dd, J ¼ 8.5, J ¼ 2.5), 3.75 (C1 H
2
, t, J ¼ 7.0),
6
): 4.51
d
x
3
x
3
3
3
2
3
2
.22 (C2 H
2
, q, J ¼ 7.0), 3.39 (C3 H
2
, t, J ¼ 7.0), 3.83 (OCH
3
, s), 8.18
(C3H, dd,
J
1
¼9.5,
J
2
¼ 5.0), 2.88 (C4H(1), dd, J ¼ 18.0, J ¼ 5.0),
NH, bs); 13C NMR (125 MHz, CDCl
):
d
0
178.1 (C2), 38.0 (C3), 36.2
3.37 (C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.35 (C2 H, d, J ¼ 2.5), 7.04
2
3
0
3
(
(
1
3
0
0
0
0
4
0
3
4
0
C4), 176.3 (C5), 122.7 (C2 ), 111.0 (C3 ), 126.2 (C3 a), 100.5 (C4 ),
(C4 H, d, J ¼ 2.5), 6.80 (C6 H, dd, J ¼ 8.5, J ¼ 2.5), 7.33 (C7 H, dt,
3 5 x 3 x
0
0
0
0
x
54.4 (C5 ), 112.4 (C6 ), 112.8 (C7 ), 131.7 (C7 a), 38.1 (C1 ), 29.7
J ¼ 8.5, J ¼ 0.5), 3.61 (C1 H
2
, t, J ¼ 7.0), 1.88 (C2 H
2
, m), 1.73
, t), 3.48 (CaH(E), CeH(E), pt), 2.93
CaH(A), CeH(A), m), 2.34 (CbH(E), CdH(E), pk), 2.14 (CbH(A),
x
x
x
3
x
(
C2 ), 30.7 (C3 ), 55.9 (OCH
3
).
(C3 H
2
, q, J ¼ 7.5), 3.03 (C4 H
2
(
3
4
2
.1.3.6. 1-(2-bromoethyl)-3-(5-methoxy-1H-indol-3-yl)pyrrolidine-
,5-dione (2f). The title compound was isolated as a dark yellow
crystals. Yield: 55% (1.54 g); m.p. 145e148 C; ESI-HRMS m/z calcd
for C15
CdH(A), pd), 3.13 (CcH, m), 7.22 (C2”H, d, J ¼ 2.0), 7.42 (C4”H, dd,
3
4
3
4
J
H-F ¼ 10.5, J ¼ 2.5), 6.89 (C6”H, td, J ¼ 9.0, J ¼ 2.5), 7.39 (C7”H,
ꢃ
3
4
5
0
3
, s), 10.11 (N1 H, bs),
4d, J ¼ 8.5,
J
H-F ¼ 4.0, J ¼ 0.5), 3.80 (OCH
þ
1
H
15BrN
2
O
3
Na (M þ Na) 373.0164, found 373.0165; H NMR
10.17 (N1”H, bs).
3
3
5
(
(
500 MHz, CDCl
3
):
d
4.30 (C3H, 4d, J
1
¼9.5, J
2
¼ 5.0, J ¼ 0.5), 2.96
2
3
2
3
C4H(1), dd, J ¼ 18.5, J ¼ 5.0), 3.28 (C4H(2), dd, J ¼ 18.5, J ¼ 9.5),
4.1.5.3. 1-{4-[4-(5-methoxy-1H-indol-3-yl)piperidin-1-yl]butyl}-3-
(5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione (4c). The title
compound was isolated as a yellow solid. Yield: 60.8% (0.16 g); m.p.
0
3
0
4
0
7
.09 (C2 H, d, J ¼ 2.5), 6.91 (C4 H, d, J ¼ 2.5), 7.25 (C7 H, dd,
5 3 4 x
3
0
J ¼ 8.5, J ¼ 0.5), 6.89 (C6 H, dd, J ¼ 9.0, J ¼ 2.5), 4.04 (C1 H
2
, t,
C
3
x
3
13
ꢃ
þ
J ¼ 6.5), 3.62 (C2 H
2
, t, J ¼ 6.5), 3.84 (OCH
3
, s), 8.14 (NH, bs);
105e114 C; ESI-HRMS m/z calcd for C31
H
36
N
4
O
4
H (M þ H)
1
NMR (125 MHz, CDCl
3
0
):
d
177.7 (C2), 38.1 (C3), 36.2 (C4), 175.9 (C5),
529.2815, found: 529.2829; H NMR (500 MHz, CDCl
3
): 4.21 (C3H,
d
0
0
0
0
3
3
2
3
1
22.9 (C2 ), 110.9 (C3 ), 126.2 (C3 a), 100.6 (C4 ), 154.4 (C5 ), 112.4
dd,
J
1
¼9.5,
J
2
¼ 5.0), 2.87 (C4H(1), dd, J ¼ 18.0, J ¼ 5.0), 3.20
0 0 0
x
x
2
3
0
3
(
C6 ), 112.9 (C7 ), 131.7 (C7 a), 40.2 (C1 ), 27.6 (C2 ), 56.0 (OCH ).
3
(C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.04 (C2 H, d, J ¼ 2.0), 6.85e6.89
0
0
0
3
x
(
C4 H, C6 H, C”4H, m), 7.23 (C7 H, d, J ¼ 8.5), 3.64 (C1 H
2
, t,
3
x
3
x
3
4
(
.1.4. Procedure for the synthesis of 3-piperidin-4-yl-1H-indoles
3aec)
The starting compounds 3aec were obtained according to pre-
viously described procedures [29,50,51]. For more details see
Supplementary data.
J ¼ 7.5), 1.69 (C2 H
2
, q, J ¼ 7.5), 1.58 (C3 H
2
, q, J ¼ 7.5), 2.43
x
3
(C4 H
2
, t, J ¼ 7.5), 3.02 (CaH(E), Ce(E), m), 2.12 (CaH(A), Ce(A), m),
2.0 (CbH(E), CdH(E), m), 1.81 (CbH(A), CdH(A), m), 2.76 (CcH, tt,
3
4
3
3
3
J
1
¼12.0, J
2
¼ 3.5), 6.97 (C2”H, d, J ¼ 2.5), 6.83 (C6”H, dd, J ¼ 9.0,
3
0
J ¼ 2.5), 7.21 (C7”H, d, J ¼ 8.5), 3.82 (C5 -OCH
3 3
, s), 3.84 (C5”-OCH ,
0
13
s), 8.55 (N1 H, bs), 8.11 (N1”H, bs); C NMR (125 MHz, CDCl
3
0
):
0
4
.1.5. General procedure for the synthesis of derivatives 3-(1H-
indol-3-yl)pyrrolidine-2,5-dione (4aer)
d
178.4 (C2), 38.1 (C3), 36.2 (C4), 176.5 (C5), 122.8 (C2 ), 111.1 (C3 ),
126.2 (C3 a), 100.6 (C4 ), 154.3 (C5 ), 112.4 (C6 ), 112.7 (C7 ), 131.8
(C7 a), 38.8 (C1 ), 25.9 (C2 ), 24.0 (C3 ), 58.3 (C4 ), 54.3 (Ca, Ce),
0 0 0 0 0
0
x
x
x
x
A mixture of appropriate derivatives of 1-(4-bromoalkyl)-3-(5-
substituted-1H-indol-3-yl)pyrrolidine-2,5-dione
0.5 mmol), the appropriate derivatives of 5-substitued-3-(piper-
idin-4-yl)-1H-indoles (3aec) (0.5 mmol), K CO (1.0 mmol), a cat-
(2aef)
32.6 (Cb, Cd), 33.3 (Cc), 120.7 (C2”), 120.8 (C3”), 126.9 (C3”a), 101.1
(C4”), 133.7 (C5”), 111.9 (C6”), 111.9 (C7”), 131.6 (C7”a), 55.9 (5 -
OCH ), 56.1 (5”-OCH ).
3 3
0
(
2
3
alytic amount of KI and 35 mL acetonitrile was stirred and refluxed
for 4e5 h. Reaction time was monitored using TLC. After cooling,
the mixture was filtered, and the filtrate was evaporated to dryness.
The crude residue was purified by flash chromatography using
4.1.5.4. 1-{3-[4-(1H-indol-3-yl)piperidin-1-yl]propyl}-3-(5-
methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione (4d). The title com-
pound was isolated as a yellow solid. Yield: 67.0% (0.16 g); m.p.
ꢃ
þ
CH
2
Cl
2
/MeOH (98:2, 97:3, 95:5, 90:10 v/v) as an eluent. Proper
111e116 C; ESI-HRMS m/z calcd for C29
485.2553, found: 485.2569; H NMR (500 MHz, CDCl
H
32
N
4
O
3
H (M þ H)
1
fractions were identified by TLC and evaporated to dryness giving
analytically pure compounds 4aer.
3
): 4.25 (C3H,
d
3
3
5
2
3
4d, J
3
1
¼9.0, J
2
¼ 5.0, J ¼ 0.5), 2.91 (C4H(1), dd, J ¼ 18.5, J ¼ 5.0),
2
3
0
0
0
.25 (C4H(2), dd, J ¼ 18.5, J ¼ 9.5), 6.88 (C2 H, C4 H, C6 H, m), 7.26
0
3 x 3 x
4
1
.1.5.1. 1-{4-[4-(1H-indol-3-yl)piperidin-1-yl]butyl}-3-(5-methoxy-
H-indol-3-yl)pyrrolidine-2,5-dione (4a). The title compound was
(C7 H, d, J ¼ 9.5), 3.69 (C1 H
2
, t, J ¼ 7.0), 1.92 (C2 H
2
, m), 2.48
x
3
(C3 H
2
, t, J ¼ 7.0), 3.04 (CaH(E), CeH(E), m), 2.13 (CaH(A), CeH(A),
ꢃ
isolated as a yellow solid. Yield: 76.6% (0.19 g); m.p. 111e117 C; ESI-
HRMS m/z calcd for C30
m), 2.03 (CbH(E), CdH(E), m), 1.81 (CbH(A), CdH(A), m), 2.82 (CcH,
þ
3
3
3
H
33
N
4
O
3
H (M þ H) 499.2709, found:
tt, J
1
¼11.5, J
2
¼ 3.5), 7.08 (C2”H, C5”H, m), 7.61 (C4”H, d, J ¼ 8.0),
99.2703; 1H NMR (500 MHz, CDCl
):
d
4.21 (C3H, dd,
3
J
1
¼9.0,
7.17 (C6”H, m,
3
J
1
¼8.0,
3
J
2
¼ 7.0, J ¼ 1.5), 7.34 (C7”H, dt, J ¼ 8.0,
13
4
3
4
3
3
2
3
4
5
0
J
2
¼ 4.5), 2.87 (C4H(1), dd, J ¼ 18.0, J ¼ 4.5), 3.20 (C4H(2), dd,
J ¼ J ¼ 1.0), 3.83 (OCH
3
, s), 8.25 (N1 H, bs), 8.06 (N1”H, bs);
C
2
3
0
x
0
0
4
J ¼ 18.0, J ¼ 9.5), 6.86 (C2 H, C6 H, m), 6.89 (C4 H, d, J ¼ 2.0), 7.23
NMR (125 MHz, CDCl
3
0
):
d
178.2 (C2), 37.5 (C3), 36.3 (C4), 176.5 (C5),
0
3
3
x
3
0
0
0
0
(
C7 H, d, J ¼ 9.0), 3.63 (C1 H
2
, t, J ¼ 7.0), 1.68 (C2 H
2
, q, J ¼ 7.0),
122.8 (C2 ), 111.3 (C3 ), 126.3 (C3 a), 100.7 (C4 ), 154.3 (C5 ), 112.4
x
3
x
3
0
0
0
x
x
x
1.59 (C3 H
2
, q, J ¼ 7.0), 2.45 (C4 H
2
, t, J ¼ 7.5), 3.02 (CaH(E), Ce(E),
(C6 ), 112.6 (C7 ), 131.8 (C7 a), 38.1 (C1 ), 25.1 (C2 ), 56.2 (C3 ), 54.2
(Ca, Ce), 32.7 and 32.8 (Cb, Cd), 33.3 (Cc), 119.7 (C2”), 121.1 (C3”),
126.6 (C3”a), 119.0 (C4”), 119.1 (C5”), 121.9 (C6”), 111.2 (C7”), 136.4
m), 2.13 (CaH(A), Ce(A), m), 2.0 (CbH(E), CdH(E), m), 1.83 (CbH(A),
3
3
CdH(A), m), 2.80 (CcH, tt,
J
1
¼12.0,
J
2
¼ 3.5), 6.97 (C2”H, d,
3 3
3
4
3
3
J ¼ 2.5), 7.60 (C4”H, d, J ¼ 8.0), 7.08 (C5”H, m,
J
1
¼8.0,
J
2
¼ 7.0,
3
(C7”a), 55.9 (OCH ).
3
3
4
J ¼ 1.0), 7.16 (C6”H, m,
J ¼ 8.5), 3.82 (OCH , s), 8.57 (N1 H, bs), 8.25 (N1”H, bs); C NMR
125 MHz, CDCl ): 178.4 (C2), 38.1 (C3), 36.2 (C4), 176.6 (C5), 122.8
J
1
¼8.0,
J
2
¼ 7.0, J ¼ 1.0), 7.33 (C7”H, d,
0
13
3
4.1.5.5. 1-{3-[4-(5-fluoro-1H-indol-3-yl)piperidin-1-yl]propyl}-3-(5-
methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione (4e). The title com-
pound was isolated as a yellow solid. Yield: 59.7% (0.15 g); m.p.
(
(
3
d
0
0
0
0
0
0
x
C2 ), 111.1 (C3 ), 126.2 (C3 a), 100.6 (C4 ), 154.3 (C5 ), 112.4 (C6 ),
0
0
x
x
x
ꢃ
þ
112.7 (C7 ), 131.8 (C7 a), 38.6 (C1 ), 25.8 (C2 ), 23.8 (C3 ), 58.2 (C4 ),
106e117 C; ESI-HRMS m/z calcd for C29
H
31FN
4
O
3
H (M þ H)
1
5
1
(
4.1 and 54.2 (Ca, Ce), 32.4 and 32.4 (Cb, Cd), 33.1 (Cc), 119.8 (C2”),
20.8 (C3”), 126.6 (C3”a), 119.0 (C4”), 119.0 (C5”), 121.9 (C6”), 111.3
C7”), 136.4 (C7”a), 55.9 (OCH ).
503.2458, found: 503.2476; H NMR (500 MHz, acetoneeD
(C3H, 4d,
6
): 4.38
d
3
3
4
2
J
1
¼9.5,
J
2
¼ 4.5, J ¼ 0.5), 2.85 (C4H(1), dd, J ¼ 18.0,
3
2 3 3
0
3
J ¼ 5.0), 3.32 (C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.31 (C2 H, d, J ¼ 2.5),
0
4
0
3
4
0
6
.99 (C4 H, d, J ¼ 2.0), 6.80 (C6 H, dd, J ¼ 9.0, J ¼ 2.5), 7.32 (C7 H,
3 x 3 x x
4
.1.5.2. 1-{4-[4-(5-fluoro-1H-indol-3-yl)piperidin-1-yl]butyl}-3-(5-
d, J ¼ 8.5), 3.64 (C1 H
2
, t, J ¼ 7.0), 1.85 (C2 H
2 2
, m), 2.45 (C3 H , t,
3
methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione (4b). The title
J ¼ 7.0), 3.03 (CaH(E), CeH(E), pd), 2.12 (CaH(A), CeH(A), m), 1.99

12
M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
3
ꢃ
þ
(
CbH(E), CdH(E), m), 1.78 (CbH(A), CdH(A), m), 2.78 (CcH, tt,
J
A-
m.p. 178e182 C; ESI-HRMS m/z calcd for C29
H
32
N
4
O
4
Na (M þ Na)
): 4.38
3
A-E ¼ 3.5), 7.15 (C2”H, d, J ¼ 2.5), 7.29 (C4”H, dd, 3JH-
3
1
A
¼ 12.0,
J
523.2310, found: 523.2321; H NMR (500 MHz, acetoneeD
6
d
4
3
4
3
3
2
3
F
¼ 12.0, J ¼ 3.0), 6.87 (C6”H, td, J ¼ 9.0, J ¼ 2.5), 7.35 (C7”H, 4d,
(C3H, dd,
J
1
¼9.0,
J
2
¼ 4.0), 2.79 (C4H(1), dd, J ¼ 18.0, J ¼ 4.5),
3
4
5
0
3
, s), 10.05 (N1 H, bs), 10.09
2
4
0
3
J ¼ 9.0, JH-F ¼ 4.5, J ¼ 0.5), 3.79 (OCH
N1”H, bs).
3.32 (C4H(2), dd, J ¼ 18.0, J ¼ 9.0), 7.33 (C2 H, d, J ¼ 2.5), 6,81
0
4
0
x
3
4
0
(
(C4 H, d, J ¼ 2.5), 6.80 (C6 H, dd, J ¼ 8.5, J ¼ 2.5), 7.31 (C7 H, dd,
3
5
3
x
3
J ¼ 8.5, J ¼ 0.5), 3.71 (C1 H
2
, t, J ¼ 7.0), 2.60 (C2 H
2
, t, J ¼ 7.0),
4
.1.5.6. 1-{3-[4-(5-methoxy-1H-indol-3-yl)piperidin-1-yl]propyl}-3-
(4f). The title
compound was isolated as a yellow solid. Yield: 93.8% (0.24 g); m.p.
3.10 (CaH(E), CeH(E), pd), 2.17 (CaH(A), CeH(A), m), 1.98 (CbH(E),
3
3
(
5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione
CdH(E), m), 1.71 (CbH(A), CdH(A), pk), 2.77 (CcH, tt, JA-A ¼ 12.0, JA-
3 3
E
¼ 4.0), 7.02 (C2''H, d, J ¼ 2.0), 7.04 (C4''H, d, J ¼ 2.5), 6.74 (C6''H,
3 4 3 5
ꢃ
þ
1
5
dd,
(
00e109 C; ESI-HRMS m/z calcd for C30
H
34
N
4
O
4
H (M þ H)
): 4.24 (C3H,
¼ 5.0), 2.90 (C4H(1), dd, J ¼ 18.5, J ¼ 5.0), 3.22
dd, J ¼ 8.5, J ¼ 2.5), 7.25 (C7''H, dd, J ¼ 8.5, J ¼ 0.5), 3.78 (OCH
3
',
1
0
15.2658, found: 515.2678; H NMR (500 MHz, CDCl
3
d
s), 3.79 (OCH
3
'', s), 9.77 (N1 H, bs), 10.08 (N1''H, bs).
3
3
2
3
J
1
¼9.5,
J
2
2
3
0
3
C4H(2), dd, J ¼ 18.5, J ¼ 9.5), 7.05 (C2 H, d, J ¼ 2.5), 6.85e6.89
4.1.5.10. 1-{4-[4-(1H-indol-3-yl)piperidin-1-yl]butyl}-3-(5-fluoro-
1H-indol-3-yl)pyrrolidine-2,5-dione (4j). The title compound was
0
0
0
3
5
x
(
C4 H, C6 H, m), 7.23 (C7 H, dd, J ¼ 8.5, J ¼ 0.5), 3.68 (C1 H
2
, t,
3
x
x
3
J ¼ 7.0), 1.92 (C2 H
2
, m), 2.49 (C3 H
2
, t, J ¼ 7.5), 3.04 (CaH(E),
isolated as
a
light yellow solid. Yield: 42.0% (0.1 g); m.p.
ꢃ
þ
Ce(E), m), 2.14 (CaH(A), Ce(A), m), 2.0 (CbH(E), CdH(E), m), 1.79
122e130 C; ESI-HRMS m/z calcd for C29
487.2509, found: 487.2499; H NMR (500 MHz, acetoneeD
H
31FN
4
O
2
H (M þ H)
3
3
1
(
CbH(A), CdH(A), m), 2.75 (CcH, tt, J
1
¼11.5, J
2
¼ 3.5), 7.03 (C2”H,
6
):
d
4.55
3
3
3
3
2
3
d, J ¼ 2.5), 6.81e6.84 (C4”H, C6”H, m), 7.21 (C7”H, d, J ¼ 8.5), 3.81
(C3H, dd,
J
1
¼9.5,
J
2
¼ 5.0), 2.90 (C4H(1), dd, J ¼ 18.0, J ¼ 5.0),
0
0
3
2
3
0
3
(
(
(
C5 -OCH
3
, s), 3.84 (C5”-OCH
3
, s), 8.44 (N1 H, d, J ¼ 1.0), 8.08
3.38 (C4H(2), dd, J ¼ 18.00, J ¼ 9.5), 7.50 (C2 H, d, J ¼ 2.5), 7.30
13
0
3
4
0
3
4
N1”H, bs); C NMR (125 MHz, CDCl
3
):
d
178.3 (C2), 37.4 (C3), 36.3
(C4 H, dd, JH-F ¼ 10.0, J ¼ 2.5), 6.93 (C6 H, td, J ¼ 9.0, J ¼ 2.5), 7.41
3 4 x 3 x
0
0
0
0
0
C4), 176.5 (C5), 122.9 (C2 ), 111.2 (C3 ), 126.3 (C3 a), 100.7 (C4 ),
54.3 (C5 ), 112.4 (C6 ), 112.5 (C7 ), 131.8 (C7 a), 38.1 (C1 ), 24.9
C2 ), 56.1 (C3 , 5”-OCH ), 54.2(Ca, Ce) 32.4 and 32.5 (Cb, Cd), 33.2
3
Cc), 120.7 (C2”), 120.6 (C3”), 126.9 (C3”a), 101.1 (C4”), 153.7 (C5”),
(C7 H, dd, J ¼ 9.0, J H-F ¼ 4.5), 3.60 (C1 H
2
, t, J ¼ 7.0), 1.95 (C2 H
2
,
0
0
0
0
x
x
3
x
3
1
(
(
1
m), 1.71 (C3 H
2
, q, J ¼ 7.0), 3.12 (C4 H , t, J ¼ 8.0), 3.55 (CaH(E),
2
x
x
CeH(E), m), 3.07 (CaH (A), Ce(A), m), 2.47 (CbH(E), CdH(E), m) 2.16
3
3
(CbH(A), Cd(A), pd) 3.22 (CcH, tt,
J
A-A ¼ 12.0,
J
A-E ¼ 3.5), 7.14
0
3
3
3
11.9 (C6”, C7”), 131.6 (C7”a), 55.9 (5 -OCH
3
).
(C2”H, d, J ¼ 2.0), 7.74 (C4”H, d, J ¼ 8.0), 7.00 (C5”H, m,
J
1
¼8.0,
3
4
3
3
4
J
2
¼ 7.0, J ¼ 1.0), 7.10 (C6”H, m,
J
1
¼8.0,
J
2
¼ 7.0, J ¼ 1.5), 7.41
3
4
5
0
4
1
.1.5.7. 1-{2-[4-(1H-indol-3-yl)piperidin-1-yl]ethyl}-3-(5-methoxy-
H-indol-3-yl)pyrrolidine-2,5-dione (4g). The title compound was
(C7”H, dt, J ¼ 8.0, J ¼ J ¼ 1.0), 10.08 (N1 H, bs), 10.41 (N1”H, bs);
13
C NMR (50 MHz, acetoneeD
6
):
d 179.1 (C2), 38.2 (C3), 36.9 (C4),
0
0
0
0
0
isolated as
1
a
light brown solid. Yield: 41.7% (0.10 g); m.p.
177.1 (C5), 125.8 (C2 ), 112.7 (C3 , d*), 127.9 (C3 a, d*), 104.5 (C4 , d*,
ꢃ
þ
2
1
0
2
04e108 C; ESI-HRMS m/z calcd for C28
71.2389, found: 471.2396; H NMR (500 MHz, acetoneeD
H
30
N
4
O
3
H (M þ H)
J ¼ 23.8), 158.4 (C5 , d*, J ¼ 232.4), 110.7 (C6 , d*, J ¼ 26.5), 113.5
1
0
x
3 x x x
0
4
6
):
d
4.36
(C7 , d*, J ¼ 9.9), 134.4 (C7 a), 38.8 (C1 ), 25.6 (C2 ), 21.8 (C3 ), 57.0
C3H, 4d, 3
J
¼9,5,
3
J
2
¼ 4.5, J ¼ 0.5), 2.81 (C4H(1), dd, J ¼ 18.0,
4
2
(C4 ), 53.5 (Ca, Ce), 30.4 (Cb, Cd), 32.3 (Cc), 119.8 (C2”), 119.1 (C3”),
(
1
3
2
3
0
0
J ¼ 5.0), 3.30 (C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.04 (C2 H, C4 H, m),
127.3 (C3”a), 119.5 (C4”), 121.6 (C5”), 122.2 (C6”), 112.4 (C7”), 137.8
(C7”a).
0
x
3
4
0
3
5
6
3
.81 (C6 H, dd, J ¼ 8.5, J ¼ 2.5), 7.31 (C7 H, dd, J ¼ 9.0, J ¼ 0.5),
3
x
3
.72 (C1 H
2
, t, J ¼ 7.0) 2.61 (C2 H
2
, t, J ¼ 6.5), 3.10 (CaH(E), CeH(E),
m), 2.18 (CaH(A), CeH(A), m),1.98 (CbH(E), CdH(E), m),1.73 (CbH(A),
4.1.5.11. 1-{4-[4-(5-fluoro-1H-indol-3-yl)piperidin-1-yl]butyl}-3-(5-
fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (4k). The title com-
pound was isolated as a light yellow solid. Yield: 68.0% (0.17 g); m.p.
3
CdH(A), m), 2.80 (CcH, m*), 7.32 (C2"H, d, J ¼ 2.5), 7.58 (C4"H, dd,
3
4
3
3
4
J ¼ 7.5, J ¼ 1.0), 6.98 (C5"H, m,
J
1
¼7.5,
J
2
¼ 7.0, J ¼ 1.0), 7.07
3
3
4
3
ꢃ
(
C6"H, m,
J
1
¼8.0,
J
2
¼ 7.0, J ¼ 1.0), 7.36 (C7"H, dt, J ¼ 8.0,
120e134 C (melts with decomposition); ESI-HRMS m/z calcd for
4
5
0
þ
1
J ¼ J ¼ 1.0), 3.79 (OCH
3
, s), 9.92 (N1 H, bs), 10.09 (N1"H, bs).
C
29
30
H N
4
O
2
F
2
H (M þ H) 505.2415, found: 505.2392; H NMR
3 3
(
500 MHz, acetoneeD
6
):
d
4.44 (C3H, 4d,
J
1
¼10.0,
J
2
¼ 4.5,
4
2
2
3
4
.1.5.8. 1-{2-[4-(5-fluoro-1H-indol-3-yl)piperidin-1-yl]ethyl}-3-(5-
methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione (4h). The title com-
pound was isolated as a light brown solid. Yield: 57.2% (0.14 g); m.p.
J ¼ 0.5), 2.87 (C4H(1), dd, J ¼ 18.0, J ¼ 5.0), 3.33 (C4H(2), dd,
3
0
3
0
3
J ¼ 18.00, J ¼ 9.5), 7.44 (C2 H, d, J ¼ 2.5), 7.26 (C4 H, dd,
J
H-
4
0
3
4
0
F
¼ 10.0, J ¼ 2.5), 6.93 (C6 H, td, J ¼ 9.5, J ¼ 2.5), 7.42 (C7 H, 4d,
ꢃ
þ
3
J ¼ 9.0, 4J H-F ¼ 4.5, J ¼ 0.5), 3.58 (C1 H
5 x 3 x
2
03e204 C; ESI-HRMS m/z calcd for C28
H
29FN
4
O
3
Na (M þ Na)
2
, t, J ¼ 7.0), 1.68 (C2 H
2
,
1
x
x
3
5
(
11.2132, found: 511.2121; H NMR (500 MHz, acetoneeD
6
): 4.38
d
m), 1.61 (C3 H
2
, m) 2.54 (C4 H
2
, t, J ¼ 7.0), 3.10 (CaH(E), CeH(E),
C3H, dd, 3
J
1
¼9.0,
3
J
2
¼ 4.5), 2.81 (C4H(1), dd, J ¼ 18.0, J ¼ 4.5),
2
3
pd), 2.29 (CaH (A), Ce(A), pt), 2.00 (CbH(E), CdH(E), pd), 1.88
2
3
0
3
3
3
3
.32 (C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.33 (C2 H, d, J ¼ 2.5), 7.04
(CbH(A), Cd(A), m), 2.83 (CcH, tt,
JA-A ¼ 12.0,
J
A-E ¼ 3.5), 7.18
0
4
0
3
3
0
3
3
4
(
C4 H, d, J ¼ 2.5), 6.87 (C6 H, td, JH-F ¼ 2.5, J ¼ 9.0), 7.28 (C7 H, dd,
(C2”H, d, J ¼ 2.5), 7.32 (C4”H, dd, JH-F ¼ 10.5, J ¼ 2.5), 6.87 (C6”H,
3
4
x
3
x
3
4
3
4
5
J ¼ 10.0, J ¼ 2.0), 3.72 (C1 H
CaH(E), CeH(E), m), 2.19 (CaH(A), CeH(A), m), 1.97 (CbH(E), CdH(E),
m), 1.70 (CbH(A), CdH(A), m), 2.77 (CcH, m*), 7.13 (C2''H, pd,
2
, t, J ¼ 6.5), 2.61 (C2 H
2
, ps), 3.09
td,
J
1
¼9.0, J ¼ 2.5), 7.36 (C7”H, 4d, J ¼ 9.0,
J
H-F ¼ 5.0, J ¼ 0.5),
0
13
(
10.14 (N1 H, bs), 10.43 (N1”H, bs); C NMR (125 MHz, acetoneeD
6
):
0
0
d
178.8 (C2), 38.7 (C3), 36.8 (C4), 177.0 (C5), 125.7 (C2 ), 112.9 (C3 ,
3
3
3
4
4
0
3
0
2
J ¼ 2.0), 7.26 (C4''H, d, JH-F ¼ 9.0), 6.80 (C6''H, dd, J ¼ 9.0, J ¼ 2.5),
d*, J ¼ 4.4), 127.7 (C3 a, d*, J ¼ 9.8), 104.4 (C4 , d*, J ¼ 23.4), 158.4
3
4
0
0
1 2 3
0
0
7
1
3
1
.35 (C7''H, dd, J ¼ 9.0, J H-F ¼ 4.5), 3.79 (OCH
3
', s), 10.03 (N1 H, bs),
(C5 , d*, J ¼ 232.5), 110.7 (C6 , d*, J ¼ 26.3), 113.5 (C7 , d*, J ¼ 9.7),
0.08 (N1''H, bs); 13C NMR (125 MHz, acetoneeD
):
d
178.8 (C2),
134.5 (C7 a), 39.0 (C1 ), 26.3 (C2 ), 24.3 (C3 ), 58.5 (C4 ), 54.7 (Ca,
0
x x x x
6
0
4
8.8 (C3), 37.3 (C4), 177.0 (C5), 123.2 (C2'), 112.8 (C3'), 127.7 (C3 a),
Ce), 33.1, 33.1 (Cb, Cd), 34.0 (Cc), 123.3 (C2”), 121.2 (C3”, d*, J ¼ 4.9),
0
3
2
01.6 (C4'), 155.0 (C5'), 112.6 (C6') 113.1 (C7'), 133.0 (C7 a), 36.9
C1 ), 56.2 (C2 ), 55.0 (Ca), 33.9 (Cb), 34.3 (Cc), 33.9 (Cd), 55.0 (Ce),
127.9 (C3”a, d*, J ¼ 9.8), 104.2 (C4” d*, J ¼ 23.4), 158.1 (C5”, d*,
1 2 3
x
x
(
J ¼ 231.4), 110.0 (C6”, d*, J ¼ 26.3), 113.0 (C7”, d*, J ¼ 9.8), 134.4
4
1
24.2 (C2''), 113.0 (C3'', d*, J ¼ 9.7), 127.9 (C3''a, d*), 104.0 (C4'', d*,
(C7”a).
2
1
2
J ¼ 23.4), 158.0 (C5'', d*, J ¼ 230.2), 110.0 (C6'', d*, J ¼ 26.1), 113.1
3
4
0
(
C7'', d*, J ¼ 6.2), 134.3 (C7''a, d*, J ¼ 18.5), 56.0 (C5 -OCH
3
).
4.1.5.12. 1-{4-[4-(5-methoxy-1H-indol-3-yl)piperidin-1-yl]butyl}-3-
5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (4l). The title com-
(
4
.1.5.9. 1-{2-[4-(5-methoxy-1H-indol-3-yl)piperidin-1-yl]ethyl}-3-
pound was isolated as a light yellow solid. Yield: 64.0% (0. 16 g);
ꢃ
þ
(
5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione (4i). The title
m.p. 80e85 C; ESI-HRMS m/z calcd for C30
H N
33 4
O
3
FH (M þ H)
1
compound was isolated as a light brown solid. Yield: 84.0% (0.21 g);
517.2615, found: 517.2630; H NMR (500 MHz, acetoneeD
6
): d 4.46

M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
13
2
3
(
3
C3H, dd, 3
J
1
¼9.0, 3J
2
¼ 5.0), 2.88 (C4H(1), dd, J ¼ 17.5, J ¼ 5.0),
compound was isolated as a yellow solid. Yield: 55.7% (0.13 g); m.p.
2
3
0
3
ꢃ
þ
.34 (C4H(2), dd, J ¼ 17.5, J ¼ 9.0), 7.46 (C2 H, d, J ¼ 2.5), 7.27
123e126 C; ESI-HRMS m/z calcd for C29
H
31
N
4
O
3
FH (M þ H)
0
0
3
4
0
3
4
1
(
(
(
(
(
C4 H, dd, JH-F ¼ 10.0, J ¼ 3.0), 6.93 (C6 H, td, J ¼ 9.0, J ¼ 2.5), 7.42
503.2458, found: 503.2440; H NMR (500 MHz, acetoneeD
6
):
3
4
5
x
3
3
3
2
C7 H, dd, J ¼ 9.0, J H-F ¼ 4.0, J ¼ 0.5), 3.58 (C1 H
2
, t, J ¼ 6.0), 1.69
d
4.42 (C3H, dd,
J
1
¼9.5,
J
2
¼ 5.0), 2.86 (C4H(1), dd, J ¼ 18.0,
x
x
x
3
2
3
0
3
C2 H
2
, C3 H
2
, m), 2.65 (C4 H
2
, bs) 3.19 (CaH(E), CeH(E), pd), 2.44
J ¼ 5.0), 3.33 (C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.45 (C2 H, d, J ¼ 2.0),
0
3 4 3 4
0
CaH(A), Ce(A), bs), ~2.0** (CbH(E), CdH(E), CbH(A), CdH(A)), 2.98
7.26 (C4 H, dd, JH-F ¼ 10.0, J ¼ 2.5), 6.94 (C6 H, td, J ¼ 9.5, J ¼ 2.5),
3
4
0
3
4
x
3
CcH, m), 7.07 (C2”H, d, J ¼ 2.0), 7.16 (C4”H, d, J ¼ 2.5), 6.75 (C6”H,
7.42 (C7 H, dd, J ¼ 9.0,
J
H-F ¼ 4.5), 3.64 (C1 H
2
, t, J ¼ 7.0), 1.88
3
4
3
x
x
dd, J ¼ 8.5, J ¼ 2.5), 7.27 (C7”H, d, J ¼ 8.0), 3.81 (OCH
3
, s), 9.84
(C2 H
2
, CbH(A), CdH(A), m), 2.52 (C3 H
2
, bs), 3.09 (CaH(E), CeH(E),
0
13
(
d
N1 H, bs), 10.38 (N1”H, bs); C NMR (125 MHz, acetoneeD
6
):
pd), 2.21 (CaH(A), Ce(A), bs), 2.01 (CbH(E), CdH(E), pd), 2.82 (CcH,
0
0
3
4
178.8 (C2), 38.7 (C3), 36.8 (C4), 177.0 (C5), 125.7 (C2 ), 112.9 (C3 ,
m), 7.04 (C2”H, d, J ¼ 2.0), 7.12 (C4”H, d, J ¼ 2.0), 6.74 (C6”H, dd,
4
0
3
0
2
3 4 3 5
d*, J ¼ 4.8), 127.8 (C3 a, d*, J ¼ 9.7), 104.4 (C4 , d*, J ¼ 24.0), 158.4
J ¼ 9.0, J ¼ 2.0), 7.25 (C7”H, dd, J ¼ 8.5, J ¼ 0.5), 3.80 (OCH
3
, s),
0
1
0
2
0
3
0
13
(
1
C5 , d*, J ¼ 232.4), 110.7 (C6 , d*, J ¼ 26.4), 113.5 (C7 , d*, J ¼ 9.7),
9.79 (N1 H, bs), 10.35 (N1”H, bs); C NMR (125 MHz, acetoneeD
6
):
0
x
x
x
x
0
0
34.5 (C7 a), 38.8 (C1 ), 26.2 (C2 ), 23.8 (C3 ),58.25 (C4 ), 54.6 (Ca,
d
178.7 (C2), 38.7 (C3), 36.8 (C4), 176.9 (C5), 125.7 (C2 ), 112.9 (C3 ,
4
0
3
0
2
Ce), 23.6 (Cb, Cd), 33.9 (Cc), 127.9 (C2”), 120.4 (C3”), 122.0 (C3”a),
01.6 (C4”),154.6 (C5”), 112.8 (C6”), 112.4 (C7”), 133.0 (C7”a), 56.2
d*, J ¼ 4.4), 127.7 (C3 a, d*, J ¼ 9.7), 104.4 (C4 , d*, J ¼ 23.4), 158.4
0
1 2 3
0
0
1
(
(C5 , d*, J ¼ 232.5), 110.7 (C6 , d*, J ¼ 26.4), 113.5 (C7 , d*, J ¼ 9.7),
0
x x x
OCH ).
3
134.5 (C7 a), 37.8 (C1 ), 25.6 (C2 ), 56.7 (C3 ), 54.9 (Ca, Ce), 33.5 (Cb,
Cd), 34.3 (Cc), 128.0 (C2”), 120.9 (C3”), 121.8 (C3”a), 101.6 (C4”),
4
1
.1.5.13. 1-{3-[4-(1H-indol-3-yl)piperidin-1-yl]propyl}-3-(5-fluoro-
H-indol-3-yl)pyrrolidine-2,5-dione (4m). The title compound was
3
154.5 (C5”), 112.8 (C6”), 112.3 (C7”), 133.0 (C7”a), 56.0 (OCH ).
isolated as
a
light yellow solid. Yield: 51.9% (0.13 g); m.p.
4.1.5.16. 1-{2-[4-(1H-indol-3-yl)piperidin-1-yl]ethyl}-3-(5-fluoro-
1H-indol-3-yl)pyrrolidine-2,5-dione (4p). The title compound was
isolated as a light yellow solid. Yield: 38.2% (0.13 g); m.p.
ꢃ
þ
124e132 C; ESI-HRMS m/z calcd for C28
H
29
N
4
O
2
FH (M þ H)
1
4
(
73.2353, found: 473.2358; H NMR (500 MHz, acetoneeD
6
):
d
4.42
C3H, dd, 3
J
¼9.5,
3
J
¼ 5.0), 2.87 (C4H(1), dd, J ¼ 18.0, J ¼ 5.0),
2
3
123e124 C; ESI-HRMS m/z calcd for C27
481.2029, found: 481.2016; H NMR (500 MHz, acetoneeD
ꢃ
H
N
O
FNa (M þ Na)
þ
1
2
27
4
2
2
3
0
3
1
3
.33 (C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.44 (C2 H, d, J ¼ 2.5), 7.26
6
):
2
d
4.39
0
3
4
0
x
3
4
3
3
(
(
C4 H, dd, JH-F ¼ 10.0, J ¼ 2.5), 6.94 (C6 H, td, J ¼ 9.0, J ¼ 2.5),7.42
(C3H, dd,
J
1
¼9.5,
J
2
¼ 5.0), 2.85-2.78 (C4H(1), CcH, m, J ¼ 18.0,
0
3
4
3
x
3
2
4
0
3
C7 H, dd, J ¼ 9.0, J H-F ¼ 5.0), 3.64 (C1 H
2
, t, J ¼ 7.0), 1.87 (C2 H
, bs), 3.08 (CaH(E), CeH(E), pd), 2.19
CaH(A), Ce(A), bs), 2.02 (CbH(E), CdH(E), pd), 2.84 (CcH, m), 7.07
2
,
J ¼ 5.5), 3.30 (C4H(2), dd, J ¼ 17.5, J ¼ 9.5), 7.46 (C2 H, d, J ¼ 2.0),
x
0
0
3 4 3 4
0
CbH(A), CdH(A), m), 2.50 (C3 H
2
7.29 (C4 H, dd, JH-F ¼ 9.5, J ¼ 2.5), 6.93 (C6 H, td, J ¼ 9.0, J ¼ 2.5),
3 4 x x
(
(
7.41 (C7 H, dd, J ¼ 9.0, JH-F ¼ 4.5), 4.39 (C1 H
2 2
, m), 2.18 (C2 H , m),
3
3
3
C2”H, d, J ¼ 1.5), 7.61 (C4”H, d, J ¼ 8.0), 6.98 (C5”H, m,
J
1
¼8.0,
3.12 (CaH(E), CeH(E), m), 2.18 (CaH(A), CeH(A), m), 1.98 (CbH(E),
3
4
3
3
4
2
J
2
¼ 6.0, J ¼ 1.0), 7.07 (C6”H, m,
J
1
¼8.5,
J
2
¼ 6.0, J ¼ 1.0), 7.37
CdH(E), m), 1.73 (CbH(A), CdH(A), m), 7.04 (C2''H, d, J ¼ 2.0), 7.60
3
0
13
3
3
(
(
(
C7”H, d, J ¼ 8.0), 9.95 (N1 H, bs), 10.36 (N1”H, bs); C NMR
(C4"H, d, J ¼ 8.0), 6.97 (C5"H, m), 7.07 (C6''H, m), 7.36 (C7''H, d, JH-
4 13
0
125 MHz, acetoneeD
6
):
d
178.7 (C2), 38.7 (C3), 36.8 (C4), 176.9
F
¼ 9.5, J ¼ 2.5), 9.91 (N1 H, bs), 10.34 (N1''H, bs); C NMR
(125 MHz, acetoneeD ): 178.7 (C2), 37.1 (C3), 36.9 (C4), 176.8 (C5),
125.9 (C2'),113.3 (C3', d, J ¼ 4.8),127.5 (C3 a, d, J ¼ 9.6),104.4 (C4'),
0
2
0
4
0
3
C5), 125.7 (C2 ), 112.9 (C3 , d*, J ¼ 4.9), 127.7 (C3 a, d*, J ¼ 10.3),
6
d
0
0
1
0
3
0
3
1
04.4 (C4 , d*, J ¼ 23.4), 158.4 (C5 , d*, J ¼ 232.4), 110.7 (C6 , sd*,
2
0
3 x x
0
3
3
0
J ¼ 26.4), 113.5 (C7 , d*, J ¼ 9.7), 134.5 (C7 a), 37.8 (C1 ), 25.6 (C2 ),
158.5 (C5'), 110.7 (C6', d, J ¼ 26.5) 113.5 (C7', J ¼ 9.6), 134.5 (C7 a),
x
x x
5
6.8 (C3 ), 54.9 (Ca, Ce), 33.7 (Cb, Cd), 34.4 (Cc), 119.6 (C2”, C5”),
38.7 (C1 ), 56.2 (C2 ), 55.1 (Ca, Ce), 34.0 (Cb Cd), 34.5 (Cc), 121.0
3
121.0 (C3”), 127.7 (C3”a), 119.2 (C4”), 122.0 (C6”), 112.2 (C7”), 137.9
(C2''), 121.4 (C3''), 127.8 (C3''a), 104.4 (C4'', d, J ¼ 23.4), 119.6 (C5''),
(
C7”a).
3
122.0 (C6''), 112.7 (C7''), 137.9 (C7''a), 56.2 (C5''-OCH ).
4
.1.5.14. 1-{3-[4-(5-fluoro-1H-indol-3-yl)piperidin-1-yl]propyl}-3-
4.1.5.17. 1-{2-[4-(5-fluoro-1H-indol-3-yl)piperidin-1-yl]ethyl}-3-(5-
fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (4q). The title com-
pound was isolated as a light yellow solid. Yield: 56.5% (0.20 g).
Obtained from double amount of starting compounds.; m.p.
(
5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (4n). The title com-
pound was isolated as a yellow solid. Yield: 47.3% (0.12 g); m.p.
ꢃ
112e140 C (melts with decomposition); ESI-HRMS m/z calcd for
þ
1
ꢃ
þ
C
H
28 28
N
4
O
F
2 2
H (M þ H) 491.2259, found: 491.2260; H NMR
108e115 C; ESI-HRMS m/z calcd for C27
477.2102, found: 477.2091; H NMR (500 MHz, acetoneeD
H
26
N
4
O
2
F
2
H (M þ H)
3
3
1
(
500 MHz, acetoneeD
6
):
d
4.41 (C3H, 4d,
J
1
¼9.5,
J
2
¼ 5.0,
6
): d 4.39
4
2
2
3
3
3
2
3
J ¼ 0.5), 2.86 (C4H(1), dd, J ¼ 18.0, J ¼ 5.0), 3.33 (C4H(2), dd,
(C3H, dd,
J
1
¼9.5,
J
2
¼ 5.5), 2.83 (C4H(1), dd, J ¼ 18.0, J ¼ 5.5),
3
0
3
0
3
2 4 3
0
J ¼ 18.00, J ¼ 9.5), 7.43 (C2 H, d, J ¼ 2.0), 7.25 (C4 H, dd,
J
H-
3.30 (C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.45 (C2 H, d, J ¼ 2.0),
4
0
3
4
0
0
0
3
4
F
¼ 10.0, J ¼ 2.5), 6.94 (C6 H, td, J ¼ 9.5, J ¼ 2.5), 7.42 (C7 H, dd,
7.29e7.26 (C4 H, C4", m), 6.92 (C6 H, td, J ¼ 9.0, J ¼ 2.5), 7.41
3 4 x x
3
J ¼ 9.0, 4J H-F ¼ 4.5), 3.63 (C1 H
x
3
x
0
2
, t, J ¼ 7.0), 1.84 (C2 H
2
, m), 2.45
(C7 H, dd, J ¼ 8.5, JH-F ¼ 4.0), 3.72 (C1 H
2 2
, m), 2.62 (C2 H , m), 3.13
x
3
(
C3 H
2
, t, J ¼ 7.0), 3.04 (CaH(E), CeH(E), m), 2.12 (CaH(A), Ce(A), m),
(CaH(E), CeH(E), m), 2.18 (CaH(A), CeH(A), m), 1.97 (CbH(E), CdH(E),
3
3
3
1.79 (CbH(E), CdH(E), m), 1.76 (CbH(A), Cd(A), m), 2.78 (CcH, tt, JA-
m), 1.70 (CbH(A), CdH(A), pk), 2.77 (CcH, tt, JA-A ¼ 12.0, JA-E ¼ 3.5),
3 3 4
3
3
3
A
¼ 12.0,
J
A-E ¼ 4.0), 7.15 (C2”H, d, J ¼ 2.0), 7.29 (C4”H, dd,
J
H-
7.12 (C2''H, d, J ¼ 2.0), 6.87 (C6''H, td, J ¼ 9.0, J ¼ 2.5), 7.35 (C7''H,
3 4
4
3
4
0
3
', s), 8.74 (N1 H, bs), 10.34 (N1''H,
F
¼ 10.0, J ¼ 2.5), 6.87 (C6”H, td, J
1
¼9.5, J ¼ 2.5), 7.35 (C7”H, dd,
dd, JH-F ¼ 8.5, J ¼ 4.5), 3.78 (OCH
3
4
0
13
13
J ¼ 9.0,
J
H-F ¼ 4.5), 10.08 (N1 H, bs), 10.39 (N1”H, bs); C NMR
): 178.8 (C2), 37.9 (C3), 36.8 (C4),177.0 (C5),
bs); C NMR (125 MHz, acetoneeD
6
):
d
178.7 (C2), 37.0 (C3), 36.9
0
(
125 MHz, acetoneeD
6
0
d
(C4), 176.8 (C5), 125.9 (C2'), 113.2 (C3'), 127.4 (C3 a), 104.4 (C4'),
0
4
0
3
0
x
1
25.7 (C2 ), 112.9 (C3 , d*, J ¼ 4.9), 127.7 (C3 a, d*, J ¼ 9.7), 104.4
158.4 (C5'), 110.7 (C6') 113.5 (C7'), 134.5 (C7 a), 38.7 (C1 ), 56.2
0
2
0
1
0
2
x
(
1
(
C4 , d*, J ¼ 27.3), 158.4 (C5 , d*, J ¼ 232.5),110.7 (C6 , d*, J ¼ 26.3),
(C2 ), 54.9 (Ca, Ce), 34.0 (Cb), 33.8 (Cc, Cd), 123.2 (C2''), 121.6 (C3''),
0
3
0
x
x
13.5 (C7 , d*, J ¼ 9.7), 134.5 (C7 a), 38.7 (C1 ), 25.8 (C2 ), 56.9
128.1 (C3''a), 104.2 (C4''), 158.1 (C5''), 110.0 (C6''), 112.9 (C7''), 134.4
(C7''a).
x
C3 ), 55.0 (Ca, Ce), 33.8, 33.7 (Cb, Cd), 34.3 (Cc), 123.2 (C2”), 121.5
C3”, d*, 4J ¼ 4.9), 128.0 (C3”a, d*, J ¼ 9.8), 104.2 (C4”, d*,
3
(
2
1
2
J ¼ 26.8),158.1 (C5”, d*, J ¼ 231.5), 110.0 (C6”, d*, J ¼ 26.4), 113.0
4.1.5.18. 1-{2-[4-(5-methoxy-1H-indol-3-yl)piperidin-1-yl]ethyl}-3-
(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (4r). The title com-
pound was isolated as a yellow solid. Yield: 63.4% (0.23 g). Obtained
3
(
C7”, d*, J ¼ 9.2), 134.4 (C7”a).
ꢃ
4
3
.1.5.15. 1-{3-[4-(5-methoxy-1H-indol-3-yl)piperidin-1-yl]propyl}-
-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (4o). The title
from double amount of starting compounds.; m.p. 132e140 C; ESI-
HRMS m/z calcd for C28
29
H N
4
O
3
FH (M þ H)þ 489.2302, found:

14
M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
#
#
#
4
J
89.2318; 1H NMR (500 MHz, acetoneeD
6
):
d
4.40 (C3H, dd,
CaH(E), m ), 3.04e3.13 (CbH, CeH(E), m ), 2.83 (C4H(1), m ),
3
3
2
3
x
#
2
3
1
¼9.5,
J
2
¼ 5.0), 2.82 (C4H(1), dd, J ¼ 18.0, J ¼ 4.5), 3.30
2.55e2.65 (C4 H
2
, m ), 2.29 (CaH(A), t, J ¼ JA-A ¼ 11.5), 2.22
2
4
0
3
0
2
3 3
(
C4H(2), dd, J ¼ 18.0, J ¼ 9.5), 7.46 (C2 H, d, J ¼ 2.0), 6,93 (C6 H, td,
(CeH(A), td, J ¼ JA-A ¼ 11.5,
J
A-E ¼ 4.0), 2.02 (CcH(E), pd),
3
4
0
3
4
x
x x
, m); 13C NMR
2
J ¼ 9.0, J ¼ 2.5), 7.41 (C7 H, dd, J ¼ 9.0,
J
H-F ¼ 4.5), 3.71 (C1 H
2
,
1.72e1.85 (CdH
(125 MHz, CD
2
, m), 1.47e1.66 (CeH(A), C2 H
2
, C3 H
x
m), 2.61 (C2 H
2
, m), 3.11 (CaH(E), CeH(E), m), 2.17 (CaH(A), CeH(A),
3
OD):
d
0
180.9 (C2, s), 178.8 (C5, s), 158.8 (C5”, d,
1
3
m), 2.00 - 1.96 (CbH(E), CdH(E), m), 1.71 (CbH(A), CdH(A), m), 2.77
J ¼ 232.3), 138.4 (C7 , s), 134.6 (C7”a, s), 127.9 (C3”a, d, J ¼ 9.7),
3
3
3
0
#
5
(
CcH, tt, JA-A ¼ 12.0, JA-E ¼ 3.5), 7.01 (C2''H, d, J ¼ 2.5), 7.08 (C4''H,
127.0 and 127.0 (C3 a, 2s ), 124.1 and 124.0 (C2”, 2d, J ¼ 1.8 and
5 # #
4
3
4
3
0
0
0
d, J ¼ 2.0), 6.74 (C6''H, dd, J ¼ 8.5, J ¼ 2.0), 7.25 (C7''H, d, J ¼ 9.0),
J ¼ 1.9 ), 122.9 and 122.9 (C2 , 2s ), 120.3 (C6 , s), 119.2 (C4 , s),
# 4
0
13
0
0
9
.77 (OCH
3
, s), 9.76 (N1 H, bs), 10.34 (N1''H, bs); C NMR (125 MHz,
119.2 (C5 , s), 118.0 (C3”, 2d ), 113.2 (C7”, d, J ¼ 9.7), 112.8 (C7 , s),
# 2
0
acetoneeD
6
):
d
178.7 (C2), 37.1 (C3), 36.9 (C4), 158.5 (C5), 125.9
112.0 and 112.0 (C3 , 2s ), 110.6 (C6”, d, J ¼ 26.4), 104.0 (C4”, d,
2 # x
0
(
1
3
C2'), 113.4 (C3'), 127.5 (C3 a), 104.4 (C4'), 158.5 (C5'), 110.7 (C6')
J ¼ 23.5), 60.7 and 60.7 (Ca, 2s ), 58.8 (C4 , s), 54.3 (Ce, s), 39.5 and
x # x #
13.5 (C7', 3J ¼ 9.7), 134.5 (C7 a), 38.7 (C1 ), 55.9 (C2 ), 55.3 (Ca),
0
x
x
39.5 (C1 , 2s ), 39.3 and 39.2 (C3 , 2s ), 37.3 (C4, s), 34.3 (Cb, s), 31.4
#
#
x
4.0 (Cb), 34.4 (Cc), 33.9 (Cd), 55.0 (Ce), 121.7 (C2''), 121.2 (C3''),
and 31.4 (Cc, 2s ), 26.5 and 26.5 (Cd, 2s ), 25.6 (C2 , s), 23.8 and
23.7 (C3 , 2s ).
1
x
#
1
28.1 (C3''a), 101.6 (C4''), 154.5 (C5'', d*, J ¼ 230.2), 112.2 (C6''),
112.7 (C7''), 133.0 (C7''a), 56.2 (C5''-OCH ).
3
#
-
NMR spectra indicate that the sample contains two different
4
.1.6. General procedure for the synthesis of derivatives 3-(1H-
stereoisomers, hence the doubling of signals and the complex
appearance of multiplets.
indol-3-yl)pyrrolidine-2,5-dione (6aed)
A mixture of appropriate derivatives of 1-(4-bromobutyl)-3-(5-
methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione (2a) or 1-(4-
bromobutyl)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione
0.002 mol), the appropriate derivatives of 5-substitued-3-(piper-
idin-3-yl)-1H-indoles hydrochloride (5aec) (0.002 mol) and K CO
0.010 mol) and 50 mL acetonitrile were stirred and refluxed for
(2g)
4.1.6.3. 1-{4-[3-(5-methoxy-1H-indol-3-yl)piperidin-1-yl]butyl}-3-
(1H-indol-3-yl)pyrrolidine-2,5-dione (6c). The title compound was
isolated as a yellow solid. Yield: 51.8% (0.52 g). Obtained from
(
2
3
ꢃ
(
double amount of substrates; m.p. 88e114 C (melts with decom-
þ
5
e7 h. Reaction was monitored using TLC. After cooling, the
position); ESI-HRMS m/z calcd for
C
30
H
34
N
4
O
3
H
(M
þ
H)
mixture was filtered, and the filtrate was evaporated to dryness.
The crude residue was purified by flash chromatography using
499.2709, found: 499.2713; 1H NMR (500 MHz, CD
3
OD):
0 0
#
#
d
3
7.30e7.36 (C4 H, C7 H, m ), 7.23 (C7”H, m ), 7.13 (C2”H, d,
0
#
#
0
CH
2
Cl
2
/MeOH (98:2, 97:3 v/v) as an eluent. Proper fractions were
J ¼ 2.5), 7.04e7.09 (C6 H, m ), 7.03 (C4”H, m ), 6.93e6.98 (C2 H,
0
# # #
identified by TLC and evaporated to dryness giving analytically pure
compounds 6aed.
C5 H, m ), 6.76 (C6”H, m ), 4.20e4.26 (C3H, m ), 3.77 (OCH , s),
3
x
#
#
#
3.55 (C1 H
2
, m ), 3.19e3.25 (C4H(2), m ), 3.12e3.18 (CaH(E), m ),
.05e3.12 (CbH, m ), 3.01 (CeH(E), pd), 2.81 (C4H(1), m ), 2.50
#
#
3
x
#
#
4
.1.6.1. 1-{4-[3-(1H-indol-3-yl)piperidin-1-yl]butyl}-3-(1H-indol-3-
(C4 H
2
, m ), 2.07e2.17 (CaH(A), CeH(A), m ), 2.02 (CcH(E), pd),
#
x
x
#
13
yl)pyrrolidine-2,5-dione (6a). The title compound was isolated as a
1.70e1.82 (CdH
2
, m ), 1.45e1.65 (CcH(A), C2 H
2
, C3 H
2
, m );
C
ꢃ
yellow solid. Yield: 44.9% (0.42 g); m.p. 98e105 C; ESI-HRMS m/z
NMR (125 MHz, CD
3
OD):
d
180.8 (C2, s), 178.8 (C5, s), 154.9 (C5”, s),
þ
1
0
#
calcd for C29
H
32
N
4
O
2
H (M þ H) 469.2604, found: 469.2593;
H
138.4 (C7 a, s), 133.3 and 133.3 (C7”a, 2s ), 128.1 (C3”a, s), 127.0
#
0
0
0
#
0
#
NMR (500 MHz, CD
3
OD):
d
7.55 (C7”H, m ), 7.31e7.36 (C4 H, C7 H,
(C3 a, 2s ), 124.0 (C2”, s), 122.9 and 122.9 (C2 , 2s ), 122.6 and 122.6
#
3
0
#
#
0 0 0
#
C4”H, m ), 7.13 (C2”H, d, J ¼ 4.5), 7.05e7.10 (C6 H, C6”H, m ),
(C4”, 2s ), 120.3 (C6 , s), 119.2 (C4 , C5 , s), 117.9 and 117.9 (C3”, 2s ),
0
0
#
3
3
0
0
#
6
3
.93e7.01 (C2 H, C5 H, C5”H, m ), 4.23 (C3H, dd, J
1
¼9.5, J
2
¼ 4.5),
113.1 (C6”, s), 112.8 (C7”, s), 112.6 (C7 , s), 112.0 and 112.0 (C3 , 2s ),
101.4 (C4”, s), 61.1 and 61.1 (Ca, 2s ), 58.9 and 58.9 (C4 , 2s ), 56.3
x
#
#
x
#
.55 (C1 H
2
, m), 3.07e3.22 (CaH(E), CbH, C4H(2), m ), 2.96 (CeH(E),
2
3
x
#
x
#
pd), 2.80 (C4H(1), 4d, J ¼ 13.5, J ¼ 4.5), 2.40e2.46 (C4 H
2
, m ),
3
(OCH , s), 54.6 (Ce, s), 39.4 (C1 , s), 39.3 and 39.3 (C3, 2s ), 37.3 (C4,
#
#
#
1
2
.99e2.12 (CaH(A), CeH(A), CcH(E), m), 1.68e1.80 (CdH , m ), 1.60
s), 34.4 (Cb, s), 31.6 and 31.5 (Cc, 2s ), 26.6 and 26.6 (Cd, 2s ), 25.9
x
#
x
13
x
x
#
(
C2 H
2
, m ), 1.45e1.56 (C3 H
2
, CcH(A), m); C NMR (125 MHz,
(C2 , s), 24.0 and 24.0 (C3 , 2s ).
0
#
CD OD):
3
d
180.9 (C2, s), 178.8 (C5, s), 138.4 (C7 a, s), 138.1 (C7”a,
0
#
2
(
s#), 127.8 (C3”a, s), 127.0 (C3 a, s), 124.0 (C2”, 2s ), 123.0 and 123.0
- NMR spectra indicate that the sample contains two different
0
#
#
0
C2 , 2s ), 122.4 (C6”, s), 121.7 and 121.7 (C3”, 2s ), 120.3 (C6 , s),
stereoisomers, hence the doubling of signals and the appearance
of complex multiplets.
#
0
1
1
6
19.6 and 119.6 (C5”, 2s ), 119.4 (C4”, s), 119.2 (C5 , s), 118.6 and
0
#
0
0
#
18.6 (C4 , 2s ),112.8 (C7”, s),112.4 (C7 , s),112.1 and 112.0 (C3 , 2s ),
#
x
x
1.5 and 61.4 (Ca, 2s ), 59.2 (C4 , s), 54.7 (Ce, s), 39.4 (C1 , s), 39.4
C3, s), 37.3 (C4, s), 34.7 (Cb, s), 32.0 and 32.0 (Cc, 2s ), 26.7 (Cd,
#
(
2
4.1.6.4. 1-{4-[3-(5-methoxy-1H-indol-3-yl)piperidin-1-yl]butyl}-3-
(5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione (6d). The title
compound was isolated as a yellow solid. Yield: 48.3% (0.55 g); m.p.
#
x
x
#
s ), 26.1 (C2 , s), 24.3 and 24.2 (C3 , 2s ).
#
ꢃ
þ
-
NMR spectra indicate that the sample contains two different
102e110 C; ESI-HRMS m/z calcd for C31
36
H N
4
O
4
H (M þ H)
1
stereoisomers, hence the doubling of signals and appearance of
complex multiplets.
529.2815, found: 529.2811; H NMR (500 MHz, CD
3
OD): 7.19e7.24
d
0
0
#
3
#
(C7 H, C7”H, m ), 7.10 (C2”H, d, J ¼ 3.5), 7.02 (C4”H, m ), 6.97
4
0
3
0
(
C4 H, d, J ¼ 4.5), 6.85 (C2 H, d, J ¼ 2.0), 6.74e6.78 (C6 H, C6”H,
#
#
#
m ), 4.18e4.23 (C3H, m ), 3.77 and 3.77 (C5”-OCH
3
, 2s ), 3.74 and
0
#
x
#
#
4.1.6.2. 1-{4-[3-(5-fluoro-1H-indol-3-yl)piperidin-1-yl]butyl}-3-(1H-
3.74 (C5 -OCH
3
, 2s ), 3.54 (C1 H
2
, m ), 3.24 (C4H(2), m ), 3.16
(CaH(E), m ), 3.10 (CbH, m ), 3.05 (CeH(E), pd), 2.80 (C4H(1), m ),
#
#
#
indol-3-yl)pyrrolidine-2,5-dione (6b). The title compound was iso-
ꢃ
x
#
#
lated as a yellow solid. Yield: 49.2% (0.48 g); m.p. 94e120 C (melts
2.50e2.61 (C4 H
(CcH(E), CdH(E), m ), 1.44e1.63 (CcH(A), CdH(A), C2 H
m ); C NMR (125 MHz, CD
2
, m ), 2.14e2.25 (CaH(A), CeH(A), m ), 1.71e1.83
#
x
x
with decomposition); ESI-HRMS m/z calcd for C29
H
31
N
4
O
2
FH
OD):
H-F ¼ 2.0),
H-F ¼ 10.0, J ¼ 3.5, J ¼ 2.0), 7.17 (C2”H, d,
2
, C3 H
2
,
þ
1
#
13
(
d
M þ H) 487.2484, found: 487.2498; H NMR (500 MHz, CD
3
3
OD):
d
180.9 (C2, s), 178.8 (C5, s), 155.2
(C5 , s), 154.9 (C5”, s), 133.6 (C7 a, s), 133.2 (C7”a, s), 128.0 (C3”a, s),
127.5 and 127.5 (C3 a, 2s ), 124.5 (C2”, s), 122.6 and 122.6 (C2 , 2s ),
117.5 and 117.5 (C3”, 2s ), 113.5 (C6”, s), 113.1 (C7”, s), 112.9 and
0
0
3
4
0
0
7.32e7.37 (C4 H, C7 H, m), 7.29 (C7”H, dd, J ¼ 9.0,
J
3
4
5
0
#
0
#
7
.21e7.26 (C4”H, m,
J
3
0
0
#
0
#
#
#
J ¼ 2.5), 7.07 (C2 H, C6 H, m ), 6.96 (C5 H, m ), 6.86 (C6”H, m ),
#
x
#
0
#
0
#
0
4
.25e4.31 (C3H, m ), 3.55e3.60 (C1 H
2
, m ), 3.14e3.27 (C4H(2),
112.9 (C7 , 2s ), 112.7 and 112.6 (C3 , 2s ), 111.8 (C6 , s), 101.6 and

M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
15
#
0
#
#
1
5
(
01.6 (C4”, 2s ), 101.4 and 101.4 (C4 , 2s ), 60.8 and 60.8 (Ca, 2s ),
carried out according to a previously described, standard technique
x
0
3
8.7 (C4 , s), 56.3 (C5”-OCH
Ce, 2s ), 39.4 (C1 , s), 39.2 and 39.1 (C3, 2s ), 37.1 (C4, s), 34.2 (Cb,
s), 31.3 (Cc, s), 26.6 and 26.6 (Cd, 2s ), 25.6 (C2 , s), 23.7 and 23.7
3
, s), 56.3 (C5 -OCH
3
, s), 54.4 and 54.3
using 0.5 nM of [ H]-ketanserin as the radioligand and 10
mM of
#
x
#
methysergide to determine non-specific binding [71]. Following
20 min of incubation, the samples containing receptor homoge-
nates and the investigated compound (7e9 concentrations run in
triplicate) were rapidly filtered under vacuum through GF/B glass
fiber filters; the filters were washed extensively with an ice-cold
#
x
x
#
(
C3 , 2s ).
#
-
NMR spectra indicate that the sample contains two different
stereoisomers, hence the doubling of signals and appearance of
complex multiplets.
buffer using
measured by scintillation counting using a liquid scintillation
cocktail. The inhibition constants (K ) were calculated using the
a Brandel harvester. Bound radioactivity was
4
4
.2. Biology evaluation
i
Cheng-Prusoff equation. Results are expressed as the means of at
least three separate experiments.
.2.1. In vitro experiment
Preparation of solutions of test and reference compounds:
1
mM stock solutions of tested compounds were prepared in
4.2.1.4. Receptor binding experiments with HEK293 cells expressing
DMSO. Serial dilutions of compounds were prepared in 96-well
microplate in assay buffers using automated pipetting system
epMotion 5070 (Eppendorf). Each compound was tested in 6 con-
centrations from 10 to 5 to 10e10 M (final concentration).
6 7
human D2L, 5-HT and 5-HT receptors
4.2.1.4.1. Cell culture and preparation of cell membranes.
HEK293 cells with stable expression of human dopamine D2L or
serotonin 5-HT
6
and 5-HT7b receptors (prepared with the use of
ꢃ
Lipofectamine 2000) were maintained at 37 C in a humidified at-
4
.2.1.1. 5-HT1A binding assay. Radioligand binding was performed
mosphere with 5% CO
Medium containing 10% dialyzed fetal bovine serum and 500
G418 sulfate. For membrane preparation, cells were subcultured in
2
and grown in Dulbecco's Modifier Eagle
g/mL
using membranes from CHO-K1 cells stably transfected with the
human 5-HT1A receptor (PerkinElmer). All assays were carried out
m
in duplicates. 50
[
membranes (10
m
l working solution of the tested compounds, 50
H]-8-OH-DPAT (final concentration 1 nM) and 150 l diluted
g protein per well) prepared in assay buffer
, 0,5 mM EDTA, 0.1% ascorbic
acid) were transferred to polypropylene 96-well microplate using
6-wells pipetting station Rainin Liquidator (MettlerToledo). Se-
rotonin (10 M) was used to define nonspecific binding. Microplate
m
l
10 cm diameter dishes, grown to 90% confluence, washed twice
with prewarmed to 37 C phosphate buffered saline (PBS) and
3
ꢃ
m
m
pelleted by centrifugation (200 g) in PBS containing 0.1 mM EDTA
and 1 mM dithiothreitol. Prior to membrane preparation, pellets
(
50 mM Tris, pH 7.4, 10 mM MgSO
4
ꢃ
were stored at ꢂ80 C.
9
4.2.1.4.2. Radioligand binding assays. Cell pellets were thawed
and homogenized in 20 vol of assay buffer using an Ultra Turrax
tissue homogenizer and centrifuged twice at 35.00 g for 20 min at
m
was covered with a sealing tape, mixed and incubated for 60 min at
ꢃ
ꢃ
ꢃ
2
7 C. The reaction was terminated by rapid filtration through GF/C
4 C, with incubation for 15 min at 37 C in between. The compo-
sition of the assay buffers was as follows: for dopamine D2LR,
filter mate presoaked with 0.3% polyethyleneimine for 30 min. Ten
rapid washes with 200
performed using automated harvester system Harvester-96 MACH
III FM (Tomtec). The filter mates were dried at 37 C in forced air fan
ꢃ
m
l 50 mM Tris buffer (4 C, pH 7.4) were
50 mM Tris-HCl, 1 mM EDTA, 4 mM MgCl
1.5 mM CaCl and 0.1% ascorbate; for 5-HT
0.5 mM EDTA and 4 mM MgCl ; and for 5-HT7bR, 50 mM Tris-HCl,
4 mM MgCl , 10 M pargyline and 0.1% ascorbate. All assays were
2
, 120 mM NaCl, 5 mM KCl,
2
6
R, 50 mM Tris-HCl,
ꢃ
2
incubator and then solid scintillator MeltiLex was melted on filter
2
m
ꢃ
mates at 90 C for 4 min. Radioactivity was counted in MicroBeta2
incubated in a total volume of 200
m
L in 96-well microtitre plates
ꢃ
scintillation counter (PerkinElmer). Data were fitted to a one-site
curve-fitting equation with Prism 6 (GraphPad Software) and K
values were estimated from the ChengꢂPrusoff equation.
for 1 h at 37 C. The process of equilibration was terminated by
rapid filtration through Unifilter plates with a 96-well cell harvester
and radioactivity retained on the filters was quantified on a
Microbeta plate reader. For displacement studies, the assay samples
i
3
4.2.1.2. SERT binding assay. Radioligand binding was performed
contained radioligands such as [ H]-raclopride (74.4 Ci/mmol) for
3
using membranes from HEK-293 cells stably transfected with the
human serotonin transporter (PerkinElmer). All assays were carried
dopamine D2LR and 2 nM [ H]-LSD (85.2 Ci/mmol for 5-HT
6
R) or
3
0.6 nM [ H]-5-CT (39.2 Ci/mmol) for 5-HT
Non-specific binding was defined with 1
the D2L assay, whereas 10 M of 5-HT or 10
used in the 5-HT R or 5-HT R binding experiments, respectively.
Each compound was tested in triplicate at 7e8 concentrations
7
R.
out in duplicates. 50
m
l working solution of the tested compounds,
l [ H]-imipramine (final concentration 2 nM) and 150 l diluted
membranes (9 g protein per well) prepared in assay buffer (50 mM
m
M of (þ)butaclamol in
3
5
0
m
m
m
mM methiothepine were
m
6
7
Tris, pH 7.4, 5 mM KCl, 120 mM NaCl) were transferred to poly-
propylene 96-well microplate using 96-wells pipetting station
ꢂ11
ꢂ4
i
(10 e10 M). The inhibition constants (K ) were calculated from
Rainin Liquidator (MettlerToledo). Imipramine (10
m
M) was used to
the Cheng-Prusoff equation [52]. Membrane preparation and gen-
eral assay procedures for cloned receptors were adjusted to a 96-
microwell format based on protocols previously described by us
[53,54].
define nonspecific binding. Microplate was covered with a sealing
ꢃ
tape, mixed and incubated for 30 min at 27 C. The reaction was
terminated by rapid filtration through GF/C filter mate presoaked
with 0.5% polyethyleneimine for 30 min. Ten rapid washes with
ꢃ
2
00
m
l 50 mM Tris buffer, 154 mM NaCl (4 C, pH 7.4) were per-
4.2.1.5. ADRA1 receptor binding assay. Preparation of solutions of
test and reference compounds: 10 mM stock solutions of tested
compounds were prepared in DMSO. Serial dilutions of compounds
were prepared in 96-well microplate in assay buffers using auto-
mated pipetting system epMotion 5070 (Eppendorf). Each com-
pound was tested in 6 concentrations from 10 to 5 to 10e10 M (final
concentration).
formed using automated harvester system Harvester-96 MACH III
FM (Tomtec). The filter mates were dried at 37 C in forced air fan
incubator and then solid scintillator MeltiLex was melted on filter
mates at 90 C for 4 min. Radioactivity was counted in MicroBeta2
scintillation counter (PerkinElmer). Data were fitted to a one-site
curve-fitting equation with Prism 6 (GraphPad Software) and K
values were estimated from the ChengꢂPrusoff equation.
ꢃ
ꢃ
i
Radioligand binding was performed using tissue rat cortex. All
assays were carried out in duplicates. 50
m
l working solution of the
l [ H]-prazosin (final concentration 0.2 nM)
l tissue suspension prepared in assay buffer (50 mM Tris-
3
4
.2.1.3. 5-HT2A binding assay. Tissue preparations (rat cortex) and
competition binding experiments for 5-HT2A receptors were
tested compounds, 50
and 150
m
m

16
M.Z. Wr oꢀ bel et al. / European Journal of Medicinal Chemistry 183 (2019) 111736
HCl, pH 7.6) were transferred to polypropylene 96-well microplate
using 96-wells pipetting station Rainin Liquidator (MettlerToledo).
Phentolamine (10 mM) was used to define nonspecific binding.
data were analysed by Dunnett's test (when one drug was admin-
istrated) or by the Newman-Keuls test (when two drugs were
administrated).
Microplate was covered with a sealing tape, mixed and incubated
ꢃ
for 30 min at 30 C. The reaction was terminated by rapid filtration
4.2.3.1. Body temperature in mice. The effects of the tested com-
pounds: 4a, 4b, 4c, 4j, 4k and 4l given alone on the rectal body
temperature in mice (measured with an Ellab thermometer) were
recorded 30, 60, 90 and 120 min after their administration. In the
another experiment the effect of WAY100635 (0.1 mg/kg s.c. as
aqueous solution) on the hypothermia induced by tested com-
pounds and/or 8-OH-DPAT was measured. WAY100635 was
administered 15 min before the tested compounds and the rectal
body temperature was recorded 30 min and 60 min after injection.
through GF/B filter mate. Ten rapid washes with 200
buffer (4 C, pH 7.6) were performed using 96-well FilterMate
harvester (PerkinElmer, USA). The filter mates were dried at 37 C
in forced air fan incubator and then solid scintillator MeltiLex was
melted on filter mates at 90 C for 4 min. The radioactivity on the
filter was measured in MicroBeta TriLux 1450 scintillation counter
PerkinElmer, USA). Data were fitted to a one-site curve-fitting
equation with Prism 6 (GraphPad Software) and K
estimated from the ChengꢂPrusoff equation.
m
l 50 mM Tris
ꢃ
ꢃ
ꢃ
(
i
values were
4.2.3.2. Forced swim test in mice. The experiments were performed
4
.2.2. Metabolic stability
Stock solutions of studied compounds were prepared at con-
centration of 10 mM in DMSO. Working solutions were prepared
daily by dilution of stock with reaction buffer or acetonitrile, final
concentration of organic solvent did not exceed 1%. Incubation
mixture contained 1
Sigma-Aldrich) and 0.5 mg/mL of pooled human liver microsomes
HLM, Sigma-Aldrich) in potassium phosphate buffer (0.1 M, pH
.4). Incubation was carried out in thermostat at 37 C and started
according to the method of Porsolt et al. (1977). Mice were placed
individually into glass cylinders (height 25 cm, diameter 10 cm)
ꢃ
containing about 20 cm of water (23 C). The animals were left in
the cylinder for 6min. First 2min of test was count as adaptation
period, the total duration of immobility was measured during a 4-
min test. The mouse was estimated to be immobile when it
remained floating passively, performing slow motion to keep head
above the water. Tested compounds were administered 30 min
before the test.
mM of a studied derivative, 1 mM of NADPH
(
(
7
ꢃ
by addition of studied compound. 50 mL samples were taken at
starting point and after 5,10,15, 20, 30 min. Enzymatic reaction was
terminated by the addition of the equal volume of ice-cold aceto-
4
.2.3.3. Locomotor activity of mice (LA). The locomotor activity of
mice was measured in a 20-station photo beam activity system
Opto-M3 Activity Meter; Columbus Instruments) composed of
nitrile, containing 1
mM of buspirone hydrochloride as internal
(
standard. After collection, samples were immediately centrifuged
Plexiglas locomotor activity chambers (40 x 20 ꢁ 15 cm). Mice were
placed individually in the chamber, and the total number of
ambulation was recorded for the time adequate to the forced
swimming test.
(
10 min, 10000 rpm) and resulted supernatant was directly ana-
ꢃ
lysed or kept in ꢂ80 C until LC-MS analysis. Natural logarithm of a
compound over IS peak area ratio was plotted versus incubation
time. Metabolic half-time (t1/2) was calculated from the slope of the
linear regression.
4.2.4. The bioavailability assays
LC-MS analysis was performed on an Agilent 1260 system
coupled to SingleQuad 6120 mass spectrometer (Agilent Technol-
ogies, Santa Clara, CA, USA). Poroshell EC-C18 (2.1 mm ꢁ 150 mm,
The in vitro bioavailability prediction assays and the hERG
automated patch-clamp experiments were performed by Eurofins
Pharma Discovery Services according to the well-known methods.
Further methodological details are available on the company
website (www.eurofinsdiscoveryservices.com) and the appropriate
publications [ [55e58]]. For more details see also Supplementary
data.
2
.7 mm, Agilent Technologies, Santa Clara, CA, USA) was used in
reversed-phase mode with gradient elution starting with 90% of
phase A (0.1% formic acid in water) and 10% of phase B (0.1% dormic
acid in acetonitrile). The amount of phase B was linearly increased
to 100% in 15 min and equilibrated. Total analysis time was
ꢃ
2
1 min at 25 C, flow rate was 0.5 mL/min and the injection volume
Acknowledgements
was 20 mL. The mass spectrometer was equipped with electrospray
ion source and operated in positive ionization. Mass analyser was
This study was supported by the Polish National Science Centre
Grants No. 2013/09/B/NZ7/00748 and 2016/23/B/NZ7/02916. The
authors acknowledge the contribution of Joanna Polska for tech-
nical assistance.
þ
set individually to each compound to detect [Mþ H] protonated
molecule. MSD parameters of the ESI source were as follows:
nebulizer pressure 40 psig (N
gas temperature 300 C, capillary voltage 3.0 kV, fragmentor
voltage 150 V.
2
), drying gas 10 mL/min (N
2
), drying
ꢃ
Appendix A. Supplementary data
4
.2.3. In vivo experiments
The experiments were performed on male CD-1 mice (29e42 g).
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111736.
ꢃ
The animals were kept at a room temperature (21 ± 2 C) on a
natural day-night cycle (MayeNovember) and housed under
standard laboratory conditions. They had free access to food and
tap water before the experiment. Each experimental group con-
sisted of 6e8 animals/dose. All the animals were used only once. 8-
Hydroxy-2-(di-n-propylamino)tetralin hydro-bromide (8-OH-
DPAT, Research Biochemical Inc.) was used as aqueous solutions.
Compounds: 4a, 4b, 4c, 4j, 4k and 4l were suspended in a 10%
aqueous solution of dimethyl sulfoxide (DMSO). Vehicle group was
administered as 10% aqueous solution of DMSO. 8-OH-DPAT was
injected subcutaneously (sc), 4a, 4b, 4c, 4j, 4k and 4l were given
intraperitoneally (ip) in a volume of 10 mL/kg/mice. The obtained
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