Novel 3-(1,2,3,6-Tetrahydropyridin-4-yl)-1H-indole-Based Multifunctional Ligands with Antipsychotic-Like, Mood-Modulating, and Procognitive Activity

Article abstract of DOI:10.1021/acs.jmedchem.7b00839

The most troublesome aspects of behavioral and psychological symptoms of dementia (BPSD) are nowadays addressed by antidepressant, anxiolytic, and antipsychotic drugs, often administered off-label. Considering their modest effectiveness in dementia patients, the increased risk of adverse events and cognitive decline, there is an unmet need for well-tolerated and effective therapy of BPSD. We designed and synthesized multifunctional ligands characterized in vitro as high-affinity partial agonists of D₂R, antagonists of 5-HT₆R, and blockers of SERT. Moreover, the molecules activated 5-HT_1AR and blocked 5-HT₇R while having no relevant affinity for off-target M₁R and hERG channel. Compound 16 (N-{2-[4-(5-chloro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}-3-methylbenzene-1-sulfonamide) exhibited a broad antipsychotic-, antidepressant-, and anxiolytic-like activity, not eliciting motor impairments in mice. Most importantly, 16 showed memory-enhancing properties and it ameliorated memory deficits induced by scopolamine. The molecule outperformed most important comparators in selected tests, indicating its potential in the treatment of both cognitive and noncognitive (behavioral and psychological) symptoms of dementia.

Full text of DOI:10.1021/acs.jmedchem.7b00839

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Article
Novel 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole-
Based Multifunctional Ligands With Antipsychotic-
like, Mood-Modulating And Procognitive Activity
Adam Bucki, Monika Marcinkowska, Joanna #niecikowska, Krzysztof Wi#ckowski, Maciej H. Paw#owski,
Monika G#uch-Lutwin, Anna Grybo#, Agata Siwek, Karolina Pytka, Magdalena Jastrz#bska-
Wi#sek, Anna Partyka, Anna Weso#owska, Pawe# Mierzejewski, and Marcin Ko#aczkowski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00839 • Publication Date (Web): 01 Aug 2017
Downloaded from http://pubs.acs.org on August 1, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society.
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Novel 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole-Based Multifunctional
Ligands With Antipsychotic-like, Mood-Modulating And Procognitive Activity
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a*
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Adam Bucki , Monika Marcinkowska , Joanna Śniecikowska , Krzysztof Więckowski ,
a
a
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a
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Maciej Pawłowski , Monika Głuch-Lutwin , Anna Gryboś , Agata Siwek , Karolina Pytka ,
a
a
a
b
Magdalena Jastrzębska-Więsek , Anna Partyka , Anna Wesołowska , Paweł Mierzejewski
and Marcin Kołaczkowski a,c
a
Faculty of Pharmacy, Jagiellonian University Medical College,
9
Medyczna Street, 30-688 Kraków, Poland
b
Institute of Psychiatry and Neurology, 9 Sobieskiego Street, 02-957 Warsaw, Poland
c
Adamed Ltd., Pieńków 149, 05-152 Czosnów, Poland
*
Corresponding Author Information:
Phone: (48)126205460 Fax: (48)126205458.
E-mail: adam.bucki@uj.edu.pl
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ABSTRACT
The most troublesome aspects of behavioral and psychological symptoms of dementia
BPSD) are nowadays addressed by antidepressant, anxiolytic and antipsychotic drugs, often
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administered off-label. Considering their modest effectiveness in dementia patients,
the increased risk of adverse events and cognitive decline, there is an unmet need for well-
tolerated and effective therapy of BPSD.
We designed and synthesized multifunctional ligands characterized in vitro as high-affinity
partial agonists of D
2
R, antagonists of 5-HT
6
R and blockers of SERT. Moreover, the molecules
R and
activated 5-HT1AR and blocked 5-HT
7
R, while having no relevant affinity for off-target M
1
hERG channel. Compound 16 (N-{2-[4-(5-chloro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-
yl]ethyl}-3-methylbenzene-1-sulfonamide) exhibited a broad antipsychotic-, antidepressant- and
anxiolytic-like activity, not eliciting motor impairments in mice. Most importantly, 16 showed
memory-enhancing properties and it ameliorated memory deficits induced by scopolamine. The
molecule outperformed most important comparators in selected tests, indicating its potential in
the treatment of both cognitive and non-cognitive (behavioral and psychological) symptoms of
dementia.
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INTRODUCTION
Dementia refers to cognitive issues such as memory loss and thinking inability of variable
pathophysiology, the most common being Alzheimer’s disease (AD) and vascular dementia.1
Regardless of their origin, in 90% of patients cognitive decline is complicated by combinations
of behavioral and psychological symptoms of dementia (BPSD), such as agitation, restlessness,
aggression, depression, anxiety and psychosis. BPSD, also called neuropsychiatric symptoms
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(NPS) , pose severe problems to patients, their families and caregivers, in many cases more
unbearable than the core cognitive decline.3,4 The most troublesome non-cognitive aspects
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are addressed by antidepressant, anxiolytic and antipsychotic drugs often administered
off-label.4–6 Furthermore, antipsychotics have modest effectiveness in the treatment of agitation,
aggression, and psychosis associated with dementia and increase risk of cerebrovascular adverse
events, cognitive deterioration and mortality.7,8 Consequently, there is still a need
for considerably more research on well-tolerated and effective therapy of BPSD.9
In the past years, the “one-target, one-drug” paradigm has been dominant in the drug
discovery processes. This approach relies on finding a single compound that acts on a particular
10
molecular target. For instance, so called cognitive enhancers frequently used in the treatment
of memory and learning deficits mostly in dementia, act by inhibiting a single target, namely
the enzyme acetylcholinesterase and thus increase both the level and duration of action
11
of the neurotransmitter acetylcholine. They improve or delay cognitive decline and reduce
behavioral changes, particularly apathy, depression and aberrant motor behavior, being
inefficient in the remaining symptoms though.12,13 Consequently, to improve
the pharmacotherapy of BPSD, atypical antipsychotics are often used in the combination
5
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with other drugs such as antidepressants (SSRIs) or anxiolytics (benzodiazepines). As a result,
patients receive a pharmacological cocktail made up of several single drugs with potentially
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different and mutually intertwined bioavailability, pharmacokinetics and metabolism, as well
as multiplied side-effects.14–16 Moreover, diversified dosing regimen in polypharmacy might
be confusing and often cause drug-drug interactions, particularly in the case of elderly patients
with coexisting diseases.17,18
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Literature data suggests that it is possible to overcome those limitations by designing
a multifunctional molecule which simultaneously modulates several targets and therefore being
beneficial in the treatment of various psychiatric diseases.14,19–21 In our search for versatile
molecule, addressing the most common neuropsychiatric symptoms typical for dementia,
we have recognized molecules that had been proven effective in the given single symptoms
to inspire design of multiple ligands.
Atypical antipsychotics have been extensively used to treat dementia patients,
and the selected ones have been reported to improve neuropsychiatric symptoms. The American
Psychiatric Association even recommended the use of antipsychotic medications, though only
in emergency settings for the treatment of agitation or psychosis when symptoms are severe
2
2
and dangerous. One of them, that was positively rated in terms of efficacy in the treatment
of agitation, anxiety, and depression as well as safety and tolerance in Alzheimer’s Disease
patients is aripiprazole.23–25 A molecular target that has gained our considerable attention
is therefore dopamine D
an atypical antipsychotic touted as a dopamine system stabilizer that acts primarily as D
receptor partial agonist, possess also antidepressant and anxiolytic activity, which might result
2
receptor, particularly effects of its partial activation. Aripiprazol,
2
from its substantial affinity for serotonin 5-HT1A and 5-HT
7
receptors, as well as dopamine D
3
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receptors. Furthermore, dopamine D
2
receptor partial agonists demonstrated synergic mood
modulating activity when administered together with selective serotonin reuptake inhibitors
SSRIs) in late-life depression.27,28
(
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Serotonin transporter (SERT) inhibition itself is an attractive target for designing a
molecule of therapeutic value in neuropsychiatric symptoms. Besides the clinically-proven
efficacy of SSRIs in major depressive disorder and other affective disorders, their favorable safety
profile has been proven in dementia patients. Moreover, SSRIs show ability to reduce psychotic
episodes and behavioral disturbances in patients with dementia and also depression.29,30
An example of single drug acting on multiple targets worth considering in the context of
BPSD is vortioxetine, serotonin modulator and stimulator approved for the treatment of major
depressive disorder.31–33 Its antidepressant and additional anxiolytic effect result from
the concomitant inhibition of SERT and modulation a wide range of serotonergic receptors, such
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as; 5-HT1A (agonist), 5-HT1B (partial agonist) as well as 5-HT1D, 5-HT
3
and 5-HT (antagonist). It
7
should be highlighted that the unique mechanism of action of vortioxetine, comparing to
the currently available SSRIs, has largely contributed to the improvement of therapy of
treatment-resistant patients.35 Another multifunctional ligand combining activity towards
serotonin receptors and its transporter is lumateperone, a phase III investigational atypical
antipsychotic capable of reducing negative symptoms of schizophrenia. It acts as 5-HT2A receptor
3
6
antagonist and D
none of the aforementioned drugs significantly influence 5-HT
Degenerative changes in the CNS of dementia patients result in dysregulation of
2
receptor partial agonist, having also SERT inhibitory properties. Interestingly
6
receptors.
3
7
serotonergic system, which is in turn a source of cognitive dysfunction. A potential novel
therapy of BPSD should include the effect on cognition. Undisputedly, cognitive functions mustn’t
be disturbed to a greater extent in dementia patients.38 Given that the blockade of 5-HT
receptors induces acetylcholine release, it became reasonable to consider that 5-HT receptor
antagonism could be a promising approach for improving cognitive abilities. A large amount of
scientific data describes the research on 5-HT receptor ligands as effective procognitive agents
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in preclinical studies.39–41 Two antagonists, intepirdine and idalopirdine have been successfully
evaluated in phase II clinical trials, which revealed their potential as drug candidates for the
symptomatic treatment of AD.42–45
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The aforementioned facts have encouraged us to design multi-target ligands that will be
able to precisely modulate the activity of several monoaminergic receptors and serotonin
transporter, expecting that this multifunctional profile will contribute positively to
the therapeutic potential in dementia patients. We have identified D
2
and 5HT receptors as well
6
as SERT as crucial molecular targets in the development of new potential anti-BPSD agents, since
drugs acting via these targets proved to be effective in the treatment of psychotic symptoms and
mood disorders in patients with dementia. The pharmacological profile of the newly designed
molecules is presumed to be improved comparing to aripiprazole and enhanced with
procognitive activity. Herein we present computer-aided design, synthesis and in vitro activity
characterization of a series of arylsulfonamide-based derivatives as multimodal agents. The most
promising molecule of the whole series was further characterized in extended pharmacological
studies.
RESULTS AND DISCUSSION
Design
In the course of discovery of novel designed multiple ligands (DML) targeting behavioral
and psychological symptoms of dementia (BPSD), we have previously described a series of
arylsulfonamide-based hybrid molecules.38,46 As a result of those studies, substantially supported
by molecular modeling, we designed and pharmacologically characterized a group of compounds
being the 5-HT
6
/5-HT
7
/5-HT2A and D
2
receptor antagonists, that displayed pronounced
antipsychotic activity in the absence of cognitive or motor impairment. The design of those
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compounds was inspired by investigation into the binding mode of 5-HT
7
receptor antagonists.47
receptor
receptor as well, with the 3-(piperidin-
Based on this, we constructed a series of multifunctional ligands by combining the 5-HT
blocking arylsulfonamide moiety, preferred by the 5-HT
7
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4-yl)-1,2-benzoxazole or 3-(piperazin-4-yl)-1,2-benzothiazole fragments, responsible for the
receptors.38,48
antagonism of the 5-HT2A and D
2
In the present study we followed the same logic, but instead of the abovementioned
arylamine fragments we used the appropriately substituted 3-(1,2,3,6-tetrahydropyridin-4-yl)-
1H-indole (THPI), a fragment that had previously been proven to introduce SERT inhibition (Figure
4
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1
A,B and supporting information figure S1, S2). This in turn might have implemented additional
antidepressant activity, besides the expected antipsychotic effect from the D
2
receptors (partial
receptors. Such
agonism) and procognitive effect resulting from antagonism of the 5-HT
6
pharmacological profile would have enhanced the favorable activity, not causing excessive
sedation or EPS resulting from D receptor blockade. The initially constructed hybrid molecule
Figure 1C, compound 14) was docked to the homology models of the main therapeutic targets,
affinity for which we intended to incorporate in the novel multimodal ligands. Complementarity
of interactions between the prototype compound and the active sites of 5-HT , D as well as
-HT1A/7 receptors, which was the main assumption of the design rationale, was therefore
2
(
6
2
5
verified. The putative binding modes in the given receptor sites provided the basis to assess
probability of binding to the given biological targets (Figure 1D, for details see supporting
information figures S3-S5). The binding patterns of the prototype hybrid in complexes with the
examined targets were encouraging enough to proceed with analysis of its potential interactions
with the main anti-targets. These included the M cholinergic receptor and the hERG potassium
1
channel, blockade of which could lead to adverse effects, such as memory disruption or cardiac
arrhythmia, respectively. As expected, based on the experiences with the previously described
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arylsulfonamide-based hybrids sharing the same general structure, also in the case of the
3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole derivatives the relevant affinity for both
the anti-targets occurred to be unlikely due to excessive volume of the molecule or lack of
interaction complementarity (Figure 1E, supporting information figure S6). Subsequently, a first
series of 18 hybrid molecules, varying in the substitution of the 3-(1,2,3,6-tetrahydropyridin-4-
yl)-1H-indole moiety, length of the alkyl linker as well as structure of the arylsulfonamide portion
was designed and synthesized (Table 1). Having early in vitro results pointing out superior activity
of the derivatives containing 5-chloro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole moiety, a
further 18 modifications preserving the latter fragment were proposed in the structure
optimization stage (Table 2). The presented project resulted in 36 original compounds (Figure 1F)
which were scheduled for in-depth pharmacological characterization.
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Figure 1. Design of multifunctional ligands. Proposed binding mode of the prototype compound
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4, arylsulfonamide fragment of which mimics interactions of (1R)-3,N-dimethyl-N-[1-methyl-3-
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(
4-methylpiperidin-1-yl)propyl]benzenesulfonamide (SB-258719) in the 5-HT receptor site (A)
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and the 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole moiety is responsible for SERT blocking
properties (B). Structure of compound 14 (C) proved to satisfy steric and electrostatic
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requirements in docking studies to the homology models of the 5-HT
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, 5-HT1A and D
2
receptors
(
D). Furthermore, the molecule was incompatible with structural representations of hERG
channel and M receptor (E). The design stage resulted in a series of 36 multiple ligands
1
characterized by potentially high affinity for the desirable biological targets and low risk of
binding to the off-targets (F). Amino acid residues engaged in ligand binding (within 4 Å from the
ligand atoms) are displayed as sticks, whereas those forming H-bonds (dotted yellow lines), π-π
stacking (dotted blue lines) or cation-π (dotted green lines) are represented as thick sticks. M
1
receptor structure displayed with antagonist tiotropium, since docking of 14 resulted in no valid
poses (E). For the sake of clarity, extracellular loop 2 (ECL2) of the given GPCRs was hidden (A and
D). The detailed complexes are shown in Supporting info figures S1-S6.
Synthesis
The synthesis of the designed molecules 13-48 is outlined in Scheme 1. In the first step,
the appropriately substituted 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole 1-3 was reacted with
the corresponding 2-(bromoalkyl)-1H-isoindoline-1,3(2H)-dione in the presence of potassium
o
carbonate, catalytic amount of potassium iodide in acetonitryle at 70 C, to give key intermediates
4
-12. In the next step, deprotection of phthalimides 4-12 with 40% solution of methylamine
afforded free amines, which were reacted immediately with diverse commercially available
sulfonyl chlorides to provide the final sulfonamides 13-48. The reaction was carried out in
tetrahydrofuran, in the presence of 1M KOH solution. The structures of the final molecules are
presented in Scheme 1.
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Scheme 1. The synthesis of the final compounds 13-48.
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Structure-activity relationships
All the synthesized compounds were subjected for in vitro radioligand binding assays to
determine affinity for the serotonin 5-HT
6
, 5-HT
7
, 5-HT1A, dopamine D receptors and SERT.
2
Series I
Firstly, potent SERT-binding capabilities of the molecules containing the 3-(1,2,3,6-
tetrahydropyridin-4-yl)-1H-indole fragment was proven. This conclusion is true for both 5-F (K
range 1.7 ± 0.9 nM – 8.5 ± 0.9 nM) and 5-Cl derivatives (2.7 ± 0.8 nM – 6.2 ± 2.2 nM). On the other
i
hand,
the
2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-5-chloro-1H-indoles
displayed
considerably lower affinity for the transporter (K
i
between 12 ± 4.6 nM and 40 ± 7.1 nM).
The aromatic ring at the opposite end of the molecule proved to play a secondary role in binding
to this target. Compounds containing 2-naphthalen, particularly those with a possibly short
(2-unit) linker, performed slightly better than those having 3-methylphenyl, which in turn
preferred a longer (4-unit) alkyl chain.
Analysis of the affinity for the serotonin and dopamine receptors revealed poor
performance of the 2-methyl-indole derivatives (K range 99 ± 5.6 nM – 610 ± 42 nM). This result
i
was more surprising in that the analysis of the molecular surface of the given receptor models
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pointed to the possibility of introducing small alkyl group in position 2 of indole ring.
Nevertheless, further development of methylated analogs was abandoned. Of the remaining
combinations, the 3-(1,2,3,6-tetrahydropyridin-4-yl)-5-chloro-1H-indoles displayed far better
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
affinity for the tested serotonin receptors (K
i
≥ 0.6 ± 0.2 nM) than fluorine analogs (K
i
≥ 10 ± 1.6
nM), still maintaining reasonable activity towards dopamine receptor (K
i
≥ 2.5 ± 0.8 nM vs. K ≥
i
0.3 ± 0.1, respectively). In this group, compound 16 was characterized by the most favorable in
vitro profile, having one-digit nanomolar affinity for all the desired molecular targets (Table 1).
The structure of this compound reflected preliminary requirements for potent DMLs of assumed
activity – single-ring arylsulfonamide and short ethylene spacer were privileged in the binding
sites of the receptors of interest. To explore in-depth the allowed chemical space around the
most promising 3-(1,2,3,6-tetrahydropyridin-4-yl)-5-chloro-1H-indole fragment, pharmacological
characterization of a new series was performed.
Table 1. Structure and receptor binding profile of compounds 13−30.
D
2
R
5-HT
6
R
5-HT
7
R
5-HT1A
R
SERT
Ar
R1
R2
n
Cmpd
K
i
[nM] ± SEMa
180±13 110±8.0 8.5±0.9
-F
-H
-H
-H
-H
0
1
2
0
13
14
15
16
20±3.9
17±2.4
3.0±0.4
3.3±1.2
34±3.8
60±7.3
250±18
6.4±0.7
-
F
95±5.7
66±9.3
96±4.9
1.8±0.2
5.4±1.2
2.3±1.4
5.0±0.4
-F
Cl
120±10
-
0.6±0.2
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9
-
Cl
-Cl
Cl
-Cl
Cl
-F
-H
1
2
0
1
2
0
1
2
0
1
2
0
1
2
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.5±1.4
2.5±0.8
350±29
67±4.4
56±2.9
14±3.0
0.7±0.2
0.3±0.1
44±6.5
3.7±0.5
2.6±0.2
180±17
100±11
99±5.6
36±5.4
210±15
5.0±0.0
70±9.3
5.0±0.3
6.0±1.3
70±11
6.2±2.2
3.9±2.3
32±11
-H
-
-CH3
-CH3
-CH3
-H
130±8.4 24±6.8
100±4.2 150±18
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
110±13
170±23
36±5.4
27±6.1
10±1.6
18±2.2
70±4.7
7.0±1.0
280±15
520±36
470±43
40±7.1
18±2.3
1.7±0.9
2.6±0.3
4.3±0.7
2.7±0.8
3.8±0.4
5.2±1.2
12±4.6
16±3.8
25±2.7
-
360±31
67±9.0
250±13
380±22
80±11
32±7.2
-
F
-H
-F
-H
200±8.0 240±16
-
-
-
-
-
-
Cl
-H
20±3
69±8.2
15±1.3
Cl
Cl
Cl
Cl
Cl
-H
220±16
-H
100±7.0 110±13
-CH3
-CH3
-CH3
180±11
350±25
250±28
520±39
150±27
610±42
a
Data expressed as the mean ± SEM of three independent experiments carried out in duplicate.
Affinities of reference compounds are as follows: 5-HT R K = 1.0 ± 0.1 nM and 5-HT R K = 1.0 ±
.1 nM (methiothepin), 5-HT1AR K = 1.3 ± 0.1 nM (serotonin), D R K = 1.4 ± 0.4 nM (haloperidol),
SERT K = 1.0 nM (imipramine).
6
i
7
i
0
i
2
i
i
Series II
In the follow-up series of the 3-(1,2,3,6-tetrahydropyridin-4-yl)-5-chloro-1H-indoles, the
arylsulfonamide moiety and linker length were differentiated to optimize in vitro binding
properties in the given chemical space. In the case of the 5-HT receptor affinity, superior
6
properties were observed for derivatives possessing 2-unit linkers and 1-ring aryl ring. Amongst
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
them, m-CH3 derivative from series I subsided only the one containing m-OH (compound 40 of
top K = 3.5 ± 0.6 nM). There are few examples of 3-unit spacer derivatives having moderate
i
affinity, always inferior to those possessing shorter ones. The compounds having 4-unit spacer
were able to compete only when coupled to the more extensive lipophilic rings (preferably
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
-naphthyl – compound 27 of series I K
i
= 100 ± 7 nM). Similar relationships were observed for
the 5-HT receptor, noting that propylene-linker derivatives had a slightly higher affinity than
7
ethylene-based analogs (again preferred were m-CH3- and m-OH-phenyl, K
0.3 nM, respectively), and definitely higher than those of butylene linker (K
i
i
= 0.6 ± 0.2 and 1.0 ±
over 61 ± 9.3 nM).
The relationships for the D
linkers. Nevertheless, there were also examples of excellent binders within ethylene spacer
derivatives (e.g. m-CH3- and m-OH-phenyl, K = 3.3 ± 1.2 and 1.5 ± 0.6 nM, respectively).
2
receptor were reversed, favoring compounds with longer (3-4 unit)
i
Analysis of the 5-HT1A and SERT affinity suggested that there’s no certain linker length preferred.
The compounds displayed very high affinities of usually one-digit nanomolar value, no matter
which arylsulfonamide fragment was applied (Table 2).
Table 2. Structure and receptor binding profile of compounds 31−48.
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55
56
57
58
59
60
D
2
R
5-HT
6
R
5-HT
7
R
5-HT1A
R
SERT
Ar
n
Cmpd
[nM] ± SEMa
K
i
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
37±4.1
2.8±0.4
2.3±0.5
2.4±0.2
1.7±0.2
1.8±0.7
23±4.9
5.6±1.2
1.0±0.3
1.5±0.6
2.6±0.5
2.5±0.8
10±1.9
3.5±0.6
4.4±1.0
11±2.3
2.7±0.4
4.5±0.8
46±4.0
280±13
130±7.2
10±2.1
200±15
320±28
10±3.7
120±8.0
110±6.9
3.5±0.6
30±3.2
330±21
9.2±1.5
92±5.3
300±32
35±4.0
180±12
150±10
29±8.0
18±2.1
140±18
2.0±0.1
14±3.0
91±12
4.0±0.9
5.0±0.9
4.0±0.5
1.0±0.3
10±3.6
3.0±2.0
7.4±0.8
11±2.4
1.7±0.6
6.1±1.8
4.0±0.5
10±3.1
1.0±0.1
12±1.3
21±2.9
16±5.6
9.0±1.3
10±3.2
2.7±0.4
4.8±0.5
5.1±1.2
3.6±1.0
1.1±0.8
3.2±0.1
3.3±1.3
2.3±1.4
2.8±0.8
2.2±0.9
3.5±0.4
4.2±1.7
5.3±0.9
4.1±2.1
5.7±0.5
6.0±0.3
4.6±1.5
6.1±1.2
17±4.1
12±1.1
65±7.9
1.0±0.3
2.0±0.7
61±9.3
9.0±0.4
10±1.8
80±14
51±6.6
6.0±0.5
90±12
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Data expressed as the mean ± SEM of three independent experiments carried out in duplicate.
Affinities of reference compounds are as follows: 5-HT R K = 1.0 ± 0.1 nM and 5-HT R K = 1.0 ±
.1 nM (methiothepin), 5-HT1AR K = 1.3 ± 0.1 nM (serotonin), D R K = 1.4 ± 0.4 nM (haloperidol),
SERT K = 1.0 nM (imipramine).
6
i
7
i
0
i
2
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
i
The novel ligands proved to have a wide safety window, showing an estimated affinity for
the main antitargets of over 1 µM. The values were lower than 6.7% and 25.1% inhibition of
control specific binding at the concentration of 1.0E-06 M for M and hERG, respectively (Table
). Screening for adverse targets was carried out for the most promising compounds of highest
1
3
affinity for the main targets.
Table 3. Antitarget activity data for the selected compounds.
M
1
Ra
hERGb
Cmpd
%
activity at 1.0E-06 M
1
1
1
3
3
3
4
4
4
5
6
8
1
4
7
0
3
5
3.2
0.7
nd
25.1
8.6
19.0
6.2
0.0
6.2
6.7
3.7
0.0
1.1
6.4
11.9
7.8
0.0
10.9
2.6
46
12.1
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a
b
% inhibition of control specific binding at the concentration of 1.0E-06 M. % inhibition of hERG-
mediated potassium currents at the concentration of 1.0E-06 M. Assays carried out in duplicate
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(n=2). nd – not determined. Affinity of reference pirenzepine for M
1
R K = 20 nM and inhibition
i
of hERG with E-4031 - 96% at the concentration of 1.0E-06 M.
In vitro functional activity
Compounds of outstanding affinity were tested in functional in vitro assays towards the
receptors of interest to pick up molecule of possibly most suitable pharmacological profile.
Furthermore, serotonin (5-HT) uptake reduction was determined. According to design
assumptions, to exert the desirable pharmacological action, a molecule should act as a partial
agonist at the D
antagonist at the 5-HT
efficiently block 5-HT uptake (antidepressant activity) and show agonistic properties for the 5-
HT1A receptor (anxiolytic effect), while the 5-HT receptors should be antagonized. The tested
compounds proved to be efficient DMLs, which displayed partial agonist activity for the D
receptor in the vast majority (up to 42% of agonist effect), and presented a prominent antagonist
effect at the 5-HT receptor (92% inhibition of control response). The 5-HT uptake functional
assay showed robust SERT inhibiting activity (IC50 ≤ 17 nM ± 1.7 nM). Additionally, the compounds
demonstrated pronounced antagonist efficacy at the 5-HT receptor (even 91% inhibition of
2
receptor (antipsychotic/antidepressant action resembling aripiprazole) and as
6
receptor (procognitive effect). Moreover, a molecule is supposed to
7
2
6
7
control agonist response) and were almost full agonists of the 5-HT1A receptor – reaching 100%
of agonist control response (Table 4).
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. Functional data for the selected compounds.
5-HT
D
2
R
5-HT
6
R
5-HT
7
R
5-HT1AR
Cmpd
uptake
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
%AGOa %ANTb %AGOa %ANTb %AGOa %ANTb %AGOa %ANTb IC50 [nM]
1
1
1
3
3
3
4
4
4
4
5
6
8
1
4
7
0
3
5
6
8
84
106
94
26
60
28
100
73
92
44
96
nd
2
nd
92
nd
nd
87
63
86
76
7
nd
1
nd
36
30
nd
25
25
91
50
16
18
24
92
102
98
90
40
92
96
99
73
91
51
-6
nd
42
36
15
5
17 ± 1.7
nd
nd
nd
1
-1
nd
1
-37
-34
-6
nd
10 ± 2.4
11 ± 0.7
16 ± 2.1
12 ± 2.2
nd
1
1
0
57
-8
10
12
28
14
49
3
1
0
-2
0
-22
-33
-13
0
1
3
50
40
1
nd
ARI
0
0
43
nd
a
b
Percent activity of agonist control response at 1.0E-06 M; Percent inhibition of control agonist
response at 1.0E-06 M; Assay carried out in duplicate (n=2); ARI – aripiprazole; nd – not
determined. 5-HT uptake for the reference imipramine IC50 = 61 nM.
Compound 16 demonstrated the highest agonist response towards the D receptor (42%
2
in the concentration of 1.0E-06 M), and thus functional profile of activity in multiple
concentrations was determined to verify its performance indicated in screening tests. The studies
confirmed a partial agonist function of compound 16 in the D receptor (EC50 = 56 ± 9.3 nM; Emax
2
=
56 ± 2.9%; K
b
= 2.1 ± 0.042 nM) as well as robust antagonist effect in the 5-HT
6
receptor (K =
B
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3
5
4
.6 ± 3.2 nM). Moreover, the functional 5-HT uptake assay resulted in favorable IC50 = 17 ± 1.7
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nM. The above data satisfied the expected design criteria and together with other advantageous
pharmacological data, authorized its choice as a lead compound for in vivo behavioral studies.
For safety reasons, concentration-response curve of hERG binding of compound 16 was
evaluated in 3 independent experiments as well, resulting in IC50 > 1 µM each time.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In vivo pharmacology
A set of behavioral tests were selected to challenge pharmacological effects of compound
1
6 against reference compounds. The compound was tested in mice for antipsychotic-like activity
in the MK-801-induced hyperlocomotion test, anxiolytic-like effect in the four-plate test and
antidepressant-like capabilities in the forced swim test. Possible procognitive activity was
assessed in the step-through passive avoidance task. Moreover, its potential adverse effects, i.e.
the risk of causing catalepsy and sedation were tested in the bar test and the spontaneous
locomotor activity test, respectively (table 5).50,51
The main goal of the in vivo studies was to obtain an animal level proof-of-concept for
the multimodal ligand activity in direct comparison to the reference compounds. Since all of the
results for the reference drugs were previously obtained after intraperitoneal (ip) administration,
we chose the same route of administration for the innovative compounds. Indeed, whilst testing
the compounds by oral administration would provide an additional information, notably by
suggesting favorable bioavailability, some potential confounds may prevent clear conclusions to
be drawn. For example, the effects of the parent compound might be interfered by the effects of
its metabolites (first pass effect), which would be undesired at this proof-of-concept level.
Considering the above, at this stage, we chose the ip administration for the innovative
compounds in all the in vivo studies.
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Characterization of compound 16 in behavioral in vivo pharmacological tests in
comparison to reference drugs. Tests carried out on CD-1 mice (tests 1, 3, 6 and 7) and Swiss
albino mice (tests 2, 4, 5) after intraperitoneal (ip) administration. ED50 – median effective dose;
MED – minimum effective dose; MSD – minimum sedative dose.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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Test procedures
Cmpd 16
Aripiprazole Diazepam Imipramine Citalopram
1
2
3
4
5
6
. Catalepsy (bar test)
ED50 [mg/kg]
> 100
1.881
(1-10)
(dose range [mg/kg])
(10-100)
. Spontaneous locomotor
activity MSD [mg/kg]
> 10
1.0
2.5
>30
>10
(dose range [mg/kg])
(0.625-10)
(0.5-5)
(1.25-5)
(10-30)
(2.5-10)
. MK-801-induced hyper-
locomotion MED [mg/kg]
1.25
0.5
(dose range [mg/kg])
(1-10)
(0.125-0.5)
. Four-plate test
MED [mg/kg]
0.625
> 0.5
1.25
30
5
(dose range [mg/kg])
(0.3125-10)
(0.06-0.5)
(0.625-5)
(10-30)
(2.5-40)
. Forced swim test
MED [mg/kg]
1.25
> 0.5
10
2.5
(dose range [mg/kg])
(0.3125-5)
(0.01-0.5)
(5-20)
(1.25-5)
. Passive avoidance (PA)
MED [mg/kg]
0.3125
> 2.0
(dose range [mg/kg])
(0.15-0.625)
(0.25-2)
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. Scopolamine effect reversal
in PA MED [mg/kg]
dose range [mg/kg])
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0.3125
> 2.0
(
(0.15-0.625)
(0.25-2)
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Compound 16 displayed antipsychotic-like activity, significantly reversing MK-801-
induced hyperactivity at minimum effective dose (MED) 1.25 mg/kg. Moreover, in contrast to
aripiprazole, it was also found to be active in classic models of antidepressant- and anxiolytic-like
activity in the similar dose range (0.625-1.25 mg/kg). In fact, compound 16 was even more potent
than the standard antidepressants imipramine and citalopram as well as the anxiolytic drug
diazepam. It should be noted that, while being active in models of psychosis and mood deficits,
compound 16 did not elicit catalepsy (ED50 > 100 mg/kg) nor inhibited spontaneous locomotor
activity (up to 10 mg/kg). Both the features related to motor impairments beneficially
differentiated its activity profile from aripiprazole, which elicited sedation and catalepsy in the
same experimental paradigm, presenting modest dose separation from the antipsychotic-like
activity. Considering the rather benign safety profile of aripiprazole in clinic, it testifies for a
particular safety of compound 16 in this respect.27,28
The effect of compound 16 on learning and memory was evaluated in the step-through
passive avoidance test (a hippocampus-dependent memory task). It was shown that compound
1
6 administered at 0.3125 mg/kg displayed memory-enhancing properties in naïve mice, as it
significantly increased the latency time in the retention, but not the acquisition trial. Moreover,
the same dose of the compound 16 ameliorated memory deficits induced by the anticholinergic
agent scopolamine, which is of particular importance in the view of the cholinergic deficit in
dementia patients. It is worth noting that the reference antipsychotic aripiprazole did not
improve memory or ameliorate memory deficits caused by scopolamine. Similarly, our previous
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experiments demonstrated that the antidepressant fluoxetine (a selective serotonin reuptake
5
2
inhibitor; 5-20 mg/kg) was also inactive in this test. Those results are even more significant,
considering that most of the antipsychotic drugs elicit cognitive deficits in passive avoidance
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paradigm. Our previous efforts to develop designed multiple ligands with antipsychotic activity
resulted in potent molecules with benign effect on cognition, however not able to reverse the
cholinergic deficits.38,46 This work constitutes therefore a step forward in search for novel
candidates for treatment of both non-cognitive and cognitive symptoms associated with
dementia.
CONCLUSIONS
In search for new possibilities of dealing with symptoms of dementia, we designed
multifunctional ligands of promising pharmacological activity, which address the main
bothersome aspects: cognitive deficits, as well as non-cognitive symptoms e.g. psychosis,
depression and anxiety. Their therapeutic-like activity resulted from the unique structural
features incorporated within the hybrid molecule, combining scaffolds of
the 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole
-yl)propyl]benzenesulfonamide. Such a combination of pharmacophore fragments allowed for
specific interactions in nanomolar range with essentially matched biological targets, resulting in
an adequate functional activity. These included partial agonist efficacy at the D dopamine
receptor, which was supposed to exert antipsychotic/antidepressant effect resembling the one
of aripiprazole, combined with antagonism of the 5-HT serotonin receptor, responsible for
(THPI)
and
(4-methylpiperidin-
1
2
6
procognitive effect (vide intepirdine) and blockade of serotonin transporter, bringing
antidepressant activity characteristic for SSRIs, e.g. escitalopram. The 5-HT1A receptors were
considered secondary targets able to mediate anxiolytic effect upon their activation (like in the
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case of buspiron). Ultimately, the 5-HT
antidepressant effect. Along with optimizing affinity for the relevant receptor/transporter
proteins, avoiding binding to antitargets – the M muscarinic receptor and hERG channel - was
7
receptors blockade have been anticipated to strengthen
4
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a major concern in order to unmask cholinomimetic action from the 5-HT
adverse cardiac effects, respectively.
6
receptors and avoid
The compound selected upon in vitro testing cascade – N-{2-[4-(5-chloro-1H-indol-3-yl)-
,2,3,6-tetrahydropyridin-1-yl]ethyl}-3-methylbenzene-1-sulfonamide (16) proved to have a
1
broad spectrum of antipsychotic-, antidepressant- and anxiolytic-like activity in mice.
Importantly, in the active dose range it did not elicit catalepsy nor inhibit spontaneous locomotor
activity, both features related to potential motor impairments. Most interestingly, it was shown
that compound 16 possessed memory-enhancing properties in naïve mice as well as it
ameliorated memory disruption induced by cholinergic deficit. In the given experimental
paradigm, compound 16 outperformed the most significant comparators, including aripiprazole.
The overall effectiveness and expected safety margin implies therapeutic-like potential of the
presented THPI-derivatives and warrants their further evaluation as potential symptomatic
treatment of dementia.
EXPERIMENTAL SECTION
Molecular modeling
Computer-aided ligand design consisted in ligand-receptor complex analysis resulting from
docking to the previously developed homology models or refined crystal structures of the D
2
,
5
-HT1A, 5-HT and 5-HT receptors, as well as SERT. Protein optimization stage, common for all
6
7
the evaluated structures, was based on induced-fit docking (IFD) from Schrödinger. General
procedure for building ligand-optimized models of high predictive value has been characterized
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4
55
previously. Models of SERT and M receptor were derived from crystal structures - 5I73 and
1
5
6
5CXV , respectively. Structure refinement stage using Protein Preparation Wizard, was followed
by docking (IFD ) a reference inhibitor - s-citalopram to SERT model, and antagonist - tiotropium
0
1
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in the case of M receptor. Selection of the best complex was based on scoring function and visual
1
analysis of binding mode. Protein models optimized that way served as grid for docking studies.
The homology model of the hERG potassium channel and its induced fit structure applied for
docking studies were acquired from http://www.schrodinger.com.57
For each of the remaining receptor types, the most appropriate structural template for homology
modeling was chosen, based on sequence identity and other structural characteristics, e.g.
common construction of loops forming binding site. Hhsearch tool via GeneSilico Metaserver58
was applied for sequence identity prediction. A template that got the highest score and sequence
identity index of all the crystal structures accessible (at the time of the given model preparation)
was taken for homology modeling. Thus, the human 5-HT
-adrenergic receptor (2RH1),38,59 the D
receptor model was constructed on the D
structure (3PBL),60,61 while the 5-HT
receptor model was based on the relatively novel 5-HT1B
7
receptor model was built using
β
2
2
3
receptor
6
crystal structure 4IAR,62,63 similarly to the model of 5-HT1A receptor, which is presented for the
first time herein. The alignment of amino acid sequences (UniProt database ID: P08908) of the
5
-HT1A and 5-HT2B receptors was implemented from hhsearch model, which established the
overall amino acid sequence identity for 38%. Crude homology model was obtained via
64
SwissModel platform . The artificial fragments replacing the third intracellular loop (ICL3) in the
protein crystal structure were removed and short loops were created by joining Arg217 and
Thr346. Then it was validated by processing in Protein Preparation Wizard. Ligand-based
optimization of the binding site, which was performed using IFD protocol, as well as crossdocking-
based (Glide XP) validation were carried out using various chemical classes of high affinity
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4
-HT1AR ligands. The described procedure resulted in a variety of conformational models that
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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served as molecular targets in docking studies. The binding mode of compound 2 (Figure 1 in
design section and S4 in the supporting information) was presented in a model optimized using
structure of agonist befiradol (3-chloro-4-fluorophenyl-[4-fluoro-4-([(5-methylpyridin-2-
yl)methylamino]methyl)piperidin-1-yl]methanone.65 Ligand structures were optimized using
LigPrep tool. Glide XP flexible docking procedure was carried out using OPLS3 force field and
0
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default parameters. H-bond constraint, as well as centroid of a grid box for docking to 5-HT
6
receptor were located on Asp3.32. Glide, induced fit docking, LigPrep and Protein Preparation
Wizard were implemented in Small-Molecule Drug Discovery Suite (Schrödinger, Inc.), which was
licensed for Jagiellonian University Medical College.
Synthesis
General chemistry information
Unless otherwise indicated, all the starting materials were obtained from commercial suppliers
and were used without further purification. Analytical thin-layer chromatography (TLC) was
performed on Merck Kieselgel 60 F254 (0.25 mm) pre-coated aluminum sheets (Merck, Darmstadt,
Germany). Visualization was performed with a 254 nm UV lamp. Column chromatography was
performed using silica gel (particle size 0.063–0.200mm; 70–230 Mesh ATM) purchased from
Merck. The UPLC-MS or UPLC-MS/MS analyzes were run on UPLC-MS/MS system comprising
®
®
Waters ACQUITY UPLC (Waters Corporation, Milford, MA, USA) coupled with Waters TQD mass
spectrometer (electrospray ionization mode ESI with tandem quadrupole). Chromatographic
separations were carried out using the ACQUITY UPLC BEH (bridged ethyl hybrid) C18 column: 2.1
×
100 mm and 1.7 µm particle size. The column was maintained at 40 °C and eluted under
gradient conditions using 95% to 0% of eluent A over 10 min, at a flow rate of 0.3 ml/min. Eluent
A: water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid (0.1%, v/v). A total of 10 µl of
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each sample were injected, and chromatograms were recorded using Waters eλ PDA detector.
The spectra were analyzed in the range of 200–700 nm with 1.2 nm resolution and at a sampling
rate of 20 points/s. MS detection settings of Waters TQD mass spectrometer were as follows:
source temperature 150 °C, desolvation temperature 350 °C, desolvation gas flow rate 600 l/h,
cone gas flow 100 l/h, capillary potential 3.00 kV, and cone potential 20 V. Nitrogen was used for
both nebulizing and drying. The data were obtained in a scan mode ranging from 50 to 1000 m/z
at 0.5 s intervals; 8 scans were summed up to obtain the final spectrum. Collision activated
dissociation (CAD) analyzes were carried out with the energy of 20 eV, and all the fragmentations
were observed in the source. Consequently, the ion spectra were obtained in the range from 50
to 500 m/z. MassLynx V 4.1 software (Waters) was used for data acquisition. Standard solutions
0
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(1 mg/ml) of each compound were prepared in a mixture comprising analytical grade
acetonitrile/water (1/1, v/v). The UPLC/MS purity of all the test compounds and key
1
13
19
intermediates was determined to be >95%. H NMR, C NMR, and F NMR spectra were
obtained in a Varian Mercury spectrometer (Varian Inc., Palo Alto, CA, USA), in CDCl3, CD OD,
3
1
13
19
DMSO or acetone operating at 300 MHz ( H NMR), 75 MHz ( C NMR), and 282 MHz ( F NMR).
1
Chemical shifts are reported in terms of δ values (ppm) relative to TMS δ = 0 ( H) as internal
standard. The J values are expressed in Hertz. Signal multiplicities are represented by the
following abbreviations: s (singlet), br.s (broad singlet), d (doublet), dd (doublet of doublets), dt
(
doublet of triplets), dtd (doublet of triplet of doublets), t (triplet), td (triplet of doublets), tdd
triplet of doublet of doublets), q (quartet), quin (quintet), m (multiplet). Melting points (Mp)
(
were determined with a Büchi Melting Point B-540 apparatus using open glass capillaries and are
uncorrected.
Synthetic procedures
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General procedure for the synthesis of phtalimide derivatives 4-12
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A mixture of 11.1 mmol (1.1 equiv) of appropriate tetrahydropyridinindole derivative 1-3 and 10
mmol (1 equiv) of appropriate 2-(bromoalkyl)-1H-isoindoline-1,3(2H)-dione, 20 mmol (2 equiv)
0
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of potassium carbonate, catalytic amount of potassium iodide and 25 mL of CH CN was stirred at
3
o
7
0 C for 12h. After that time, the solid was filtrated and the solvent was evaporated under the
reduced pressure. Next, the reaction mixture was purified by column chromatography over silica
gel using chloroform/methanol 98:2 as eluent.
2
-{2-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}-2,3-dihydro-1H-
isoindole-1,3-dione (4)
The title compound was prepared starting from 5-fluoro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-
indole 1 (11.1 mmol, 1.1 equiv, 2.38 g) and 2-(2-bromoethyl)-2,3-dihydro-1H-isoindole-1,3-dione
1
(
10 mmol, 1 equiv, 2.52 g). Yield: 70%, yellowish solid, H NMR (300 MHz, DMSO-d
6
): δ 11.19 (br.
s., 1 H), 7.72 - 7.90 (m, 4 H), 7.47 (dd, J = 2.30, 10.80 Hz, 1 H), 7.42 (d, J = 2.31 Hz, 1 H), 7.33 (dd,
J = 4.74, 8.85 Hz, 1 H), 6.92 (dt, J = 2.44, 9.04 Hz, 1 H), 6.00 (br. s., 1 H), 3.75 (t, J = 6.28 Hz, 2 H),
3
.14 (br. s., 2 H), 2.56 - 2.73 (m, 4 H), 2.41 (br. s., 2 H); LC-MS (ESI) calc for C23
H20FN
3
2
O 390.16
+
+
[
M+H ], found 390.32 [M+H ]
2
-{3-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]propyl}-2,3-dihydro-1H-
isoindole-1,3-dione (5)
The title compound was prepared starting from 5-fluoro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-
indole 1 (11.1 mmol, 1.1 equiv, 2.38 g), 2-(3-bromopropyl)-2,3-dihydro-1H-isoindole-1,3-dione
1
(
10 mmol, 1 equiv, 2.68 g). Yield: 61%, yellowish solid, H NMR (300 MHz, CDCl
3
): δ 8.50 (br. s.,
1
H), 7.75 - 7.89 (m, 2 H), 7.58 - 7.67 (m, 2 H), 7.42 (dd, J = 2.18, 10.39 Hz, 1 H), 7.22 - 7.32 (m, 1
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H), 7.11 (s, 1 H), 6.92 (dt, J = 2.31, 8.98 Hz, 1 H), 5.99 (br. s., 1 H), 3.81 (t, J = 6.92 Hz, 2 H), 3.20
(br. s., 2 H), 2.68 - 2.78 (m, 2 H), 2.62 (t, J = 7.18 Hz, 2 H), 2.48 (br. s., 2 H), 2.00 (t, J = 7.05 Hz, 2
+
+
H); LC-MS (ESI) calc for C24
H22FN
3
O 404.18 [M+H ], found 404.34 [M+H ]
2
0
1
2
3
4
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3
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2
-{4-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]butyl}-2,3-dihydro-1H-
isoindole-1,3-dione (6)
The title compound was prepared starting from 5-fluoro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-
indole 1 (11.1 mmol, 1.1 equiv, 2.38 g), 2-(4-bromobutyl)-2,3-dihydro-1H-isoindole-1,3-dione (10
1
mmol, 1 equiv, 2.82 g). Yield: 64%, yellowish solid, H NMR (300 MHz, CDCl
3
): δ 8.72 (br. s., 1 H),
7.78 - 7.89 (m, 2 H), 7.64 - 7.75 (m, 2 H), 7.45 (dd, J = 2.18, 10.39 Hz, 1 H), 7.25 - 7.34 (m, 1 H),
7
.07 - 7.18 (m, 1 H), 6.91 (dt, J = 2.31, 8.98 Hz, 1 H), 6.03 (br. s., 1 H), 3.74 (t, J = 6.67 Hz, 2 H),
.30 (br. s., 2 H), 2.81 (d, J = 11.28 Hz, 2 H), 2.50 - 2.70 (m, 4 H), 1.60 - 1.85 (m, 4 H); LC-MS (ESI)
3
+
+
calcd for C25
H24FN
3
O 418.19 [M+H ], found 418.37 [M+H ]
2
2
-(2-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)isoindoline-1,3-dione (7)
The title compound was prepared starting from 5-chloro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-
indole 2 (11.1 mmol, 1.1 equiv, 2.56 g) and 2-(2-bromoethyl)isoindoline-1,3-dione (10 mmol, 1
1
equiv, 2.52 g). Yield: 65%, yellowish solid, H NMR (300MHz, DMSO-d
6
): δ 11.28 (br. s., 1 H), 7.87
-
7.77 (m, 4 H), 7.73 (d, J = 2.1 Hz, 1 H), 7.42 (d, J = 2.6 Hz, 1 H), 7.38 - 7.32 (m, 1 H), 7.07 (dd, J =
.1, 8.5 Hz, 1 H), 6.02 (s, 1 H), 3.76 (t, J = 6.4 Hz, 2 H), 3.15 (d, J = 2.8 Hz, 2 H), 2.72 - 2.60 (m, 4 H),
2
2
2
+
+
.41 (m, 2 H); LC-MS (ESI) calc for C23
H20ClN
3
O
2
406.12 [M+H ], found 406.33 [M+H ]
-(3-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)isoindoline-1,3-dione (8)
The title compound was prepared starting from 5-chloro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-
indole 2 (11.1 mmol, 1.1 equiv, 2.56 g) and 2-(3-bromopropyl)isoindoline-1,3-dione (10 mmol, 1
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Journal of Medicinal Chemistry
1
2
3
1
equiv, 2.68 g). Yield: 81%, yellowish solid, H NMR (300MHz, DMSO-d
6
): δ 11.27 (br. s., 1 H), 7.86
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
- 7.77 (m, 2 H), 7.75 - 7.66 (m, 3 H), 7.40 - 7.32 (m, 2 H), 7.08 (dd, J = 2.1, 8.5 Hz, 1 H), 5.96 (s, 1
H), 3.65 (t, J = 6.9 Hz, 2 H), 3.02 (d, J = 2.8 Hz, 2 H), 2.59 - 2.49 (m, 2 H), 2.42 (t, J = 6.7 Hz, 2 H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.36 (br. s., 2 H), 1.80 (quin, J = 6.9 Hz, 2 H); LC-MS (ESI) calc for C24
H22ClN
3
O
420.14 [M+H+],
2
found 420.32 [M+H+]
2
-(4-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)butyl)isoindoline-1,3-dione (9)
The title compound was prepared starting from 5-chloro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-
indole 2 (11.1 mmol, 1.1 equiv, 2.56 g) and 2-(4-bromobutyl)isoindoline-1,3-dione (10 mmol, 1
1
equiv, 2.82 g). Yield: 65%, yellowish solid, H NMR (300MHz, DMSO-d
6
): δ 11.29 (br. s., 1 H), 7.88
-
7.78 (m, 4 H), 7.74 (d, J = 2.1 Hz, 1 H), 7.45 - 7.33 (m, 2 H), 7.08 (dd, J = 2.1, 8.7 Hz, 1 H), 6.02
br. s., 1 H), 3.59 (t, J = 6.9 Hz, 2 H), 3.04 (d, J = 2.8 Hz, 2 H), 2.60 - 2.52 (m, 2 H), 2.46 - 2.33 (m, 4
(
H), 1.70 - 1.56 (m, 2 H), 1.54 - 1.41 (m, 2 H); LC-MS (ESI) calc for C25
found 434.39 [M+H+].
H24ClN
3
O
434.16 [M+H+],
2
2
1
-{2-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}-2,3-dihydro-
H-isoindole-1,3-dione (10)
The title compound was prepared starting from 5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-
4
1
-yl)-1H-indole 3 (5.5 mmol, 1.1 equiv, 1.357 g) and 2-(2-bromoethyl)-2,3-dihydro-1H-isoindole-
1
,3-dione (5 mmol, 1 equiv, 1.270 g). Yield: 40%, yellowish solid, H NMR (300 MHz, CDCl
3
) δ 7.99
(br. s., 1H), 7.82 (dd, J = 2.95, 5.51 Hz, 2H), 7.68 (dd, J = 3.08, 5.64 Hz, 2H), 7.45 (d, J = 1.80 Hz,
1
2
C
H), 7.09 - 7.17 (m, 1H), 6.98 - 7.06 (m, 1H), 5.66 (br. s., 1H), 3.93 (t, J = 6.80 Hz, 2H), 3.30 (d, J =
.82 Hz, 2H), 2.77 - 2.89 (m, 4H), 2.50 (d, J = 1.54 Hz, 2H), 2.36 (s, 3H); LC-Ms (ESI) calc for
+
+
24
H22ClN
3
O 420.15 [M+H ], found 420.37 [M+H ].
2
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