Novel 1H-Pyrrolo[3,2-c]quinoline Based 5-HT6 Receptor Antagonists with Potential Application for the Treatment of Cognitive Disorders Associated with Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.6b00090

Modulators of the serotonin 5-HT₆ receptor (5-HT₆R) offer a promising strategy for the treatment of the cognitive deficits that are associated with dementia and Alzheimer's disease. Herein, we report the design, synthesis, and characterization of a novel class of 5-HT₆R antagonists that is based on the 1H-pyrrolo[3,2-c]quinoline core. The most active compounds exhibited comparable binding affinity to the reference compound, SB-742457, and markedly improved selectivity. Lead optimization led to the identification of (S)-1-[(3-chlorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-pyrrolo[3,2-c]quinoline (14) (K_i = 3 nM and K_b = 0.41 nM). Pharmacological characterization of the 5-HT₆R's constitutive activity at Gs signaling revealed that 14 behaved as a neutral antagonist, while SB-742457 was classified as an inverse agonist. Both compounds 14 and SB-742457 reversed phencyclidine-induced memory deficits and displayed distinct procognitive properties in cognitively unimpaired animals (3 mg/kg) in NOR tasks. Compounds 14 and SB-742457 were also active in the Vogel test, yet the anxiolytic effect of 14 was 2-fold higher (MED = 3 mg/kg). Moreover, 14 produced, in a 3-fold higher dose (MED = 10 mg/kg), antidepressant-like effects that were similar to those produced by SB-742457 (MED = 3 mg/kg). Together, these data suggest that the 4-(pyrrolidine-3-yl-amino)-1H-pyrrolo[3,2-c]quinoline scaffold is an attractive molecular framework for the development of procognitive agents. The results are promising enough to warrant further detailed mechanistic studies on the therapeutic potential of 5-HT₆R antagonists and inverse agonists for the treatment of cognitive decline and depression/anxiety symptoms that are comorbidities of Alzheimer's disease.

Full text of DOI:10.1021/acschemneuro.6b00090

Research Article
pubs.acs.org/chemneuro
Novel 1H‑Pyrrolo[3,2‑c]quinoline Based 5‑HT6 Receptor Antagonists
with Potential Application for the Treatment of Cognitive Disorders
Associated with Alzheimer’s Disease
Katarzyna Grychowska,† Grzegorz Satała,§ Tomasz Kos,∥ Anna Partyka,‡ Evelina Colacino,⊥
Severine Chaumont-Dubel,# Xavier Bantreil,⊥ Anna Wesołowska,‡ Maciej Pawłowski,† Jean Martinez,∇
Philippe Marin,# Gilles Subra,∇ Andrzej J. Bojarski,§ Frederic Lamaty,⊥ Piotr Popik,∥ and Paweł Zajdel*,†
́
́
‡
†Department of Medicinal Chemistry, and Department of Clinical Pharmacy, Jagiellonian University Medical College, 9 Medyczna
Street, 30-688 Krakow, Poland
∥
§Department of Medicinal Chemistry, and Department of Behavioral Neuroscience and Drug Development, Institute of
Pharmacology, Polish Academy of Sciences, 12 Smętna Street, 31-343 Krakow, Poland
⊥Institut des Biomolec
́
ules Max Mousseron (IBMM), UMR 5247, CNRS, Universite
Montpellier Campus Triolet Place Eugene Bataillon, 34095 CEDEX 5 Montpellier, France
#Institut de Gen
omique Fonctionnelle, CNRS UMR 5203, INSERM U661, Universite de Montpellier, Montpellier 34094, France
∇Institut des Biomolec
ules Max Mousseron (IBMM) UMR 5247, CNRS, Universite de Montpellier, ENSCM, Faculte de Pharmacie
́ ́
de Montpellier, ENSCM, Universite de
̀
́
́
́
́
́
15, avenue Charles Flahault BP14491, 34093 CEDEX 5 Montpellier, France
S
* Supporting Information
ABSTRACT: Modulators of the serotonin 5-HT6 receptor (5-HT6R) offer a promising strategy for the treatment of the cognitive
deficits that are associated with dementia and Alzheimer’s disease. Herein, we report the design, synthesis, and characterization of
a novel class of 5-HT6R antagonists that is based on the 1H-pyrrolo[3,2-c]quinoline core. The most active compounds exhibited
comparable binding affinity to the reference compound, SB-742457, and markedly improved selectivity. Lead optimization led to
the identification of (S)-1-[(3-chlorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-pyrrolo[3,2-c]quinoline (14) (Ki = 3 nM and
Kb = 0.41 nM). Pharmacological characterization of the 5-HT6R’s constitutive activity at Gs signaling revealed that 14 behaved
as a neutral antagonist, while SB-742457 was classified as an inverse agonist. Both compounds 14 and SB-742457 reversed
phencyclidine-induced memory deficits and displayed distinct procognitive properties in cognitively unimpaired animals (3 mg/kg)
in NOR tasks. Compounds 14 and SB-742457 were also active in the Vogel test, yet the anxiolytic effect of 14 was 2-fold higher
(MED = 3 mg/kg). Moreover, 14 produced, in a 3-fold higher dose (MED = 10 mg/kg), antidepressant-like effects that were
similar to those produced by SB-742457 (MED = 3 mg/kg). Together, these data suggest that the 4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline scaffold is an attractive molecular framework for the development of procognitive agents. The results are
promising enough to warrant further detailed mechanistic studies on the therapeutic potential of 5-HT6R antagonists and inverse
agonists for the treatment of cognitive decline and depression/anxiety symptoms that are comorbidities of Alzheimer’s disease.
KEYWORDS: 5-HT6R antagonist, 5-HT6R inverse agonist, pyrrolo[3,2-c]quinoline, SB-742457, cognition, memory,
novel object recognition test, forced swim test, Vogel test, Alzheimer’s disease
symptomatic treatment without affecting the neurodegenera-
lzheimer’s disease (AD), an irreversible neurodegenerative
Adisorder, is the most common form of dementia world- tion process.1
In recent years, the serotonin 5-HT6 receptor (5-HT6R) has
emerged as a promising molecular target for the treatment of
wide. AD is characterized by the progressive deterioration of
various cognitive domains, including a decline in memory
and thinking. More than 35 million people are suffering from
AD. Currently available therapeutic options for the treat-
ment of AD, based on acetylcholinesterase inhibitors and
NMDA receptor antagonist, provide modest efficacy and
Received: March 23, 2016
Accepted: April 21, 2016
© XXXX American Chemical Society
A
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cognitive deficits in AD.2 The 5-HT6R belongs to the family
of G-protein coupled receptors, which are positively coupled
with adenylyl cyclase. Additionally, 5-HT6Rs engage extracellular
signal-regulated kinase (ERK)1/2 pathway via the Src family
tyrosine kinase Fyn,3 as well as cyclin-dependent kinase (Cdk)5,4
and mammalian Target Of Rapamycin (mTOR) pathways.5
Moreover, nonphysiological mTOR activation in prefrontal
cortex, under the control of 5-HT6R, underlies deficits in social
cognition and episodic memory in two neurodevelopmental
models of schizophrenia in the rat, neonatal administration of the
NMDA receptor antagonist phencyclidine and rearing in social
isolation after the weaning.5 5-HT6R displays high constitutive
activity at various signaling cascades, including Gs adenylyl
cyclase,6 and Cdk5 pathways.4 Accordingly, the deleterious
influence of 5-HT6R in AD may reflect an alteration in receptor
expression as well as a deregulation of its activity. The 5-HT6R
is almost exclusively expressed in the CNS, with high levels in
the prefrontal cortex, hippocampus, and striatum, the brain
regions that are predominantly involved in the learning and
memory processes.7 It has also been demonstrated that 5-HT6R
antagonists modify the transmission of acetylcholine, glutamate,
dopamine, and norepinephrine.8,9
Figure 2. Structures of 5-HT6R antagonists evaluated in phase 3 clinical
trials.
produced procognitive and/or antiamnesic effects in other
neuropsychiatric disorders, i.e. schizophrenia, and Parkinson’s
disease, and might open therapeutic opportunity for autism
treatment.5,18
Continuing our efforts to generate selective 5-HT6R ligands,
we applied a scaffold-hopping approach to fuse a pyrrole ring
onto the c edge of the quinoline fragment of 6-nitroquipazine
(selective serotonin-reuptake inhibitor) and then replaced the
piperazine moiety with its 3-aminopyrrolidine bioisostere.
Although the resulting N-4-(pyrrolidin-3-yl-amino)-1H-pyrrolo-
[3,2-c]quinoline I, displayed a low affinity for the 5-HT6R,
further functionalization of I, introducing the phenylsulfonyl
moiety on the nitrogen atom of pyrrole (II), fulfilled the
structural requirements of a 5-HT6R antagonist and significantly
increased the compound’s affinity for the 5-HT6R. This result
prompted us to further investigate the substitution pattern of the
arylsulfonyl fragment of derivative II to develop potent 5-HT6R
antagonists (Figure 3).
The progress in development of 5-HT6R modulators has
recently been comprehensively reviewed by Holenz et al.,10
Glennon et al.,11 and Benhamu et al.12 Important research in the
field led to the discovery of several classes of indole and indole-
like derivatives (e.g., benzofurans, indazoles, pyrazolopyrimidines,
indolines) that preferentially bind to the 5-HT6R and behave as
antagonists at these sites (Figure 1).13
Figure 3. Strategy to develop a new group of 5-HT6R antagonists.
Herein, we report on a small series of arylsulfonyl derivatives of
a novel molecular framework that is based on N-4-(pyrrolidin-3-
yl-amino)-1H-pyrrolo[3,2-c]quinoline as potent and selective
5-HT6R antagonists. The most promising derivatives were
evaluated for their ability to reverse drug-induced memory
deficits and for their procognitive properties in novel object
recognition test in rats. Because noncognitive traits are often
associated with dementia and Alzheimer’s disease, the most
promising derivatives were also evaluated in animal models of
anxiety and depression.
Figure 1. Representatives of 5-HT6R antagonists from various
chemotypes.
As a consequence, the structural requirements for 5-HT6R
antagonists have been described, and pharmacophore models for
5-HT6R antagonists have been developed.14
CHEMISTRY
■
It is worth noting that 5-HT6R antagonists displayed
procognitive effects in several learning and memory in vivo
paradigms in rodents, and their efficacy was confirmed in phase II
clinical trials when used in combination with donepezil
(AChE inhibitor).15,16 Specifically, SB-742457 and idalopirdine
(Lu-AE58054) have been evaluated in phase III clinical trials as
an adjunctive therapy to donepezil in patients with mild to
moderate AD (Figure 2).17 Furthermore, 5-HT6R antagonists
The designed compounds were synthesized according to a multi-
step procedure (Scheme 1).
The starting nitrobenzaldehyde, tosylamine, and methyl
acrylate reacted in the presence of DABCO and titanium
isopropoxide, in isopropanol, under the aza-Bayliss−Hillman
reaction conditions, to yield the unsaturated β-aminoester 1.19
Subsequently, compound 1 was treated with allyl bromide in a
biphasic system to give diene 2. The latter was submitted to a
B
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a
Scheme 1
a
Reagents and conditions: (i) DABCO, Ti(iOPr)4, molecular sieves, isopropanol, RT, o/n; (ii) allyl bromide, K2CO3, DMF, RT, 6 h; (iii) Grubbs
second-generation catalyst (3 mmol %), CH2Cl2, 36 °C MW, 30 min; (iv) NaOt-Bu, DMF, RT, 2 h; (v) H2, Pd/C, MeOH, RT, 2 h; (vi) AcOH,
sec-BuOH, 60 °C, 3 h; (vii) POCl3, 105 °C, 4 h; (viii) 1-Boc-3-aminopyrrolidine, MeCN, 140 °C, MW, 5 h; (ix) arylsulfonyl chloride, BTPP,
CH2Cl2, 0 °C 3 h; (x) 4 N, HCl/dioxane.
ring-closing metathesis reaction, using Grubbs second-gener-
ation catalyst, in refluxing dichloromethane for 30 min, to yield
pyrroline 3. This process was promoted by microwave irradia-
tion in the open-vessel mode. Next, the tosyl group of 3 was
eliminated in the presence of sodium tert-butoxide (NaOt-Bu).20
Such an approach allowed for the simultaneous aromatization of
the pyrroline moiety and generation of the pyrrole derivative 4.
Compound 4 was then reduced using palladium on activated
charcoal, in methanol, under hydrogen atmosphere. Intra-
molecular cyclization of aromatic amine 5, upon treatment
with acetic acid, provided the lactam derivative 6, which was
subsequently submitted to oxidative chlorination to yield
4-chloro-1H-pyrrolo[3,2-c]quinoline (7). Nucleophilic substitu-
tion with 1-Boc-3-aminopyrrolidine (either a racemic mixture or
pure enantiomers) was carried out under microwave-assisted
conditions and gave compounds 8a−8c. Coupling 8a−8c with
selected arylsulfonyl chlorides in the presence of a strong
phosphazene base, P1-t-Bu-tris(tetramethylene) (BTPP), pro-
vided sulfonyl derivatives of Boc-protected N-4-(pyrrolidin-3-yl-
amino)-1H-pyrrolo[3,2-c]quinolines.21 These compounds fur-
nished the final products 9−27, as hydrochloride salts, upon
removal of the Boc group in acidic conditions.
D2 receptors (Table 1). The initially identified compound 9,
with the phenylsulfonyl group, displayed high affinity for the
5-HT6R (Ki = 10 nM). First, the lead optimization focused on the
arylsulfonyl moiety.
Introduction of electron-withdrawing substituents, such as
fluorine, chlorine (10, 13) or the bulky trifluoromethyl group
(16), in the meta position of the phenyl ring slightly increased
the affinity for the 5-HT6R. A shift of the fluorine atom from the
meta to the para position of the phenylsulfonyl fragment induced
a 2-fold decrease in the affinity (19 with Ki = 18 nM vs 10 with
Ki = 9 nM). Introduction of an additional fluorine atom in the
ortho position of the phenyl ring further decreased the affinity,
as compared with the monofluorinated analogue (20 vs 10).
Interestingly, replacement of the electron-withdrawing substitu-
ent in the meta position of the phenyl ring with electron-donating
methoxy (21, Ki = 7 nM) or methyl (22, Ki = 8 nM) groups
maintained the high affinity for the 5-HT6R.
Conversely, introduction of sterically hindered tert-butyl or
isopropyl groups in the para position (23, 24) significantly
decreased the affinity, suggesting that bulky substituents at this
position were unfavorable for binding to the 5-HT6R (23 vs 9, 24
vs 9). Compounds23 and 24, with bulky t-Bu and i-Pr substituents,
were the least potent ligands among the evaluated series, with Ki
of 101 nM and 99 nM, respectively.
To further reveal the impact of the size of substituents in
the arylsulfonyl fragment on 5-HT6R affinity, bicyclic aromatic
systems were introduced. Replacement of the phenyl moiety
with 1-naphthyl (25, Ki = 19 nM) slightly reduced affinity for
the 5-HT6R, whereas the 8-quinolinyl fragment (26, Ki = 4 nM)
was better accommodated. Furthermore, it was found that
the sterically encumbered 5-Me-benzothien-2-yl moiety caused
a 10-fold decrease in the affinity for 5-HT6 sites (27 with
Ki = 94 nM vs9 with Ki = 10 nM) and, thus, showedthe limitations
of the binding pocket.
RESULTS AND DISCUSSION
■
Pharmacological in Vitro Evaluation and Structure−
Activity Relationship Studies. Pyrroloquinolines have been
previously explored as privileged structures for the synthesis of
biologically active compounds with potential anticancer, anti-
malarial, antiobesity, and analgesic properties.22−25 In the present
study, the 1H-pyrrolo[3,2-c]quinoline scaffold was originally
proposed as a promising core motif for the development of a new
class of 5-HT6R antagonists.
The synthesized compounds 9−27 displayed high-to-moderate
affinity for the 5-HT6R (Ki = 3−101 nM) and generally did not
bind to serotonergic 5-HT1A, 5-HT2A, 5-HT7, and dopaminergic
C
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Table 1. Binding Data of Compounds 9−27 for the 5-HT6R and Their Functional Activity for 5-HT6R, and Binding Data for
5-HT1A, 5-HT2A, 5-HT7, and D2Rs for the Selected Compounds
a
b
Ki [nM]
Ki [nM]
5-HT2A
c
d
compd
Ar
enantiomer
5-HT6
agonist effect
antagonist effect
5-HT1A
5-HT7
D2
9
phenyl
R/S
R/S
S
10
9
2
1
1
3
1
1
2
1
1
1
2
6.5
9.6
9.8
0.3
2
68
81
83
72
90
93
86
80
81
78
52
NT
69
67
NT
NT
60
70
NT
9148
3006
3198
2898
3026
2351
2675
5876
5291
6233
5832
NT
1984
2262
2349
2190
2768
1281
4192
2778
4566
1345
6754
NT
7890
9594
9770
9367
2133
4989
3943
3816
2083
699
1133
900
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
3-F-phenyl
3-F-phenyl
7
873
3-F-phenyl
R
12
5
907
3-Cl-phenyl
R/S
S
1437
1012
754
3-Cl-phenyl
3
9.6
0.8
10.8
9.6
6.7
4.8
3-Cl-phenyl
R
7
3-CF3-phenyl
3-CF3-phenyl
3-CF3-phenyl
4-F-phenyl
R/S
S
7
1282
1553
942
6
R
9
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
R/S
18
6877
NT
664
e
2,5-diF-phenyl
3-MeO-phenyl
3-Me-phenyl
4-tert-Bu-phenyl
4-iso-Pr-phenyl
1-naphthyl
94 15
NT
NT
7
8
2
2
6.5
NT
2387
3421
NT
3816
3256
NT
1282
1765
NT
5.2
NT
101 17
99 16
NT
NT
10.6
16.4
NT
NT
NT
NT
NT
19
4
2
1
5330
15230
NT
5789
8765
NT
1421
18470
NT
2260
6400
NT
quinolin-8-yl
5-Me-benzothien-2-yl
94 14
NT
a
b
Mean Ki values, based on three independent binding experiments. Mean Ki values (SEM ≤ 36%), based on at least two independent binding
c
d
experiments. Percent activity of agonist control response at 10−6 M; performed at Eurofins Cerep. Percent inhibition of control agonist response at
e
10−6 M; performed at Eurofins Cerep NT: not tested.
Because the stereochemical properties of molecules might
influence their binding in the receptor pocket, three pairs of
enantiomers were investigated. A slight preference for the
S enantiomers (11, 14, 17) over their R counterparts (12, 15, 18)
was observed with respect to their 5-HT6 receptor affinity and
selectivity. Compound 14, an (S)-enantiomer bearing a chloro
substituent in the meta position of the phenyl ring, displayed the
highest affinity for the 5-HT6R (Ki = 3 nM).
Table 2. Antagonist Activity of the Selected Compounds 11,
12, 14, 15, and SB-742457 for the 5-HT6R
The most potent compounds (Ki < 20 nM) were further
evaluated for their functional activity at the 5-HT6R in cAMP
cellular assays. In the screening procedure carried out in Cerep,
compounds were initially classified as 5-HT6R antagonists
(Table 1). Subsequently, two pairs of enantiomers, 11, 12, 14,
and 15, displaying the highest affinity for the 5-HT6R and the
highest receptor-antagonist activity (% inhibition of control
agonist), were evaluated in the cellular functional assay to inhibit
5-CT-induced cAMP production (Table 2). Compounds bearing
a chlorine atom in the meta position of the arylsulfonyl displayed
stronger antagonist properties than their fluoro counterparts.
Among the tested enantiomers, the S isomers (11, 14) were the
most potent antagonists and showed a greater antagonistic effect
at the 5-HT6 sites than those exhibited by SB-742457 with Kb
values 0.83 nM and 0.41 nM for 11 and 14, respectively.
5-HT6
a
b
compd
Ar
enantiomer
Ki [nM]
Kb [nM]
11
12
14
15
3-F-phenyl
3-F-phenyl
3-Cl-phenyl
3-Cl-phenyl
S
7
12
3
1
3
1
2
0.83
12
R
S
0.41
6.1
R
7
c
SB-742457
1.4 0.2
1.3
a
Mean Ki values, based on three independent binding experiments.
Mean Kb values (SEM ≤ 22%), based on three independent binding
b
c
experiments. For selectivity panel see ref 13.
observed,6 expressing the 5-HT6R strongly activates cAMP pro-
duction in NG108−15 cells, and the level of constitutive activity
represented approximately 60% of the activity of a maximally
effective concentration of the full agonist WAY181187 (Figure 4A).
The constitutive activity was reduced in a concentration-dependent
manner by the 5-HT6R antagonist SB-742457, which behaved as
an inverse agonist in this model, with pEC50 of 7.67 (Figure 4A
and 4B). In contrast, neither 14 nor 15 significantly affected
receptor-elicited cAMP production (Figure 4B), indicating that
these compounds behaved as neutral antagonists.
Furthermore, to look for potential inverse agonist activity, two
5-HT6R antagonists, 14 and 15, and model 5-HT6R antagonist
SB-742457, were evaluated in an NG108−15 neuroblastoma cell
line that was transiently expressing the 5-HT6R. The antagonists’
effects on the receptor’s constitutive activity at Gs signaling was
assessed by BRET using the cAMP sensor CAMYEL. As previously
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memory decline or time-dependent natural forgetting in rats
in a variety of preclinical behavioral paradigms (e.g., NOR, ASST,
5CCT).26−29 The precise impact of 5-HT6R antagonists on
learning and memory depends on the age of animals and the
task used.30−33
Based on rodents’ natural preference for novelty, the NOR
test is regarded as an etiologically relevant paradigm for study-
ing visual episodic memory.34 This paradigm is based on the
spontaneous exploration of novel and familiar objects, and the
NOR test has been used for investigating drug-induced (e.g.,
phencyclidine, scopolamine) cognitive deficits in psychiatric
disorders, including schizophrenia and Alzheimer’s disease.35,36
Furthermore, as the use of long intertrial intervals (24 h) in
cognitively unimpaired animals abolishes object discrimination,
the NOR test is also a screening method for evaluating potential
cognitive enhancers. In such conditions requiring longer retention
of information, animals display poor memory that closely
resembles natural forgetting. This test allows for determination
of the procognitive properties of the evaluated compounds.
The procognitiveeffects of compounds 14 and 15 were assessed
in the NOR task in rats. Following a single administration, 14 and
15 significantly reversed phencyclidine-induced episodic memory
decline at doses of 1−3 mg/kg (ip) (Figure 5). The effects were
comparable to that produced by SB-742457, which was used as an
active comparator. It is worth noting that 15, which is the R
enantiomer, produced a slightly stronger effect than its S congener.
The same compounds were further evaluated in the NOR
test to determine their memory-enhancing effects 24 h after
their administration in cognitively unimpaired rats (Figure 6).
In contrast to PCP-impaired conditions, compounds 14 and
Figure 4. Influence of compounds 14, 15, and SB-742457 on the
5-HT6R constitutive activity at Gs signaling in NG108−15 cells. (A)
The 5-HT6 receptor constitutively activates Gs-cAMP signaling in
NG108−15 cells. Cells were transfected with an empty vector (control)
or the plasmid encoding the 5-HT6 receptor and were treated with
vehicle (5-HT6), WAY181187, or SB-742457 (1 μM each) for 5 min.
(B) The NG108-15 cells expressing the 5-HT6 receptor were exposed to
incremental concentrations of either SB-742457 or 14 or 15 for 5 min.
Cyclic AMP levels were estimated by BRET using the cAMP sensor
CAMYEL. Data are the means SEM of the values obtained in three
independent experiments that were performed in quadruplicate using
different sets of cultured cells. ***p < 0.001 vs vehicle (ANOVA
followed by Student−Newman−Keuls test).
It was further demonstrated that the selected compounds
14 and 15 did not bind to adrenergic α1A (<18% at 1 μM),
histaminergic H1 (<17% at 1 μM), muscarinic M1 (<9% at
1 μM), and serotonin 5-HT2C and 5-HT2B receptors (<21% at
1 μM), and they did not show affinity for the serotonin transporter
(SERT) (<12% at 1 μM). Of note, this distinguishes the reported
compounds 14 and 15 from the well-known 5-HT6R agents; for
example, SB-742457 displays a high affinity for 5-HT2A (99% at
1 μM) and 5-HT2BR (99% at 1 μM),13 and idalopirdine binds at
α1AR (Ki = 21 nM) and displays moderate affinity for the 5-HT2AR
(Ki = 83 nM).26
Figure 6. Effects of the selected compounds 14 (3 mg/kg, ip), 15
(3 mg/kg, ip), and SB-742457 (1 mg/kg and 3 mg/kg, ip) in the novel-
object recognition test performed in rats in cognitively unimpaired
conditions. Data are presented as the mean standard error of the
mean of N = 9−10 animals per group. (A) T2, **p < 0.01 vs familiar,
***p < 0.001 vs familiar (paired Student’s t test). (B) Discrimination
index, *p < 0.05 vs vehicle, ***p < 0.001 vs vehicle (one-way ANOVA
followed by Dunnett’s post hoc test).
Pharmacological in Vivo Evaluation. Several preclinical
studies have shown that 5-HT6R antagonists reverse drug-induced
Figure 5. Effects of the selected compounds, 14, 15, and SB-742457, in the novel-object recognition test in rats performed in PCP-impaired conditions.
Data are presented as the mean standard error of the mean of N = 7−10 animals per group. (A) T2, **p < 0.01 vs familiar, ***p < 0.001 vs familiar
(paired Student’s t test). (B) Discrimination index, *p < 0.05 vs vehicle, **p < 0.01 vs vehicle, ***p < 0.001 vs vehicle, ###p < 0.001 vs PCP (one-way
ANOVA followed by Newman−Keuls post hoc test).
E
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SB-742457 (3 mg/kg, ip) strongly affected the ability of rats to
distinguish between novel and familiar objects, indicating distinct
procognitive effects.
The results suggest that the mechanism of antidepressant
action of the tested compounds is associated with noradrenergic,
rather than serotonergic, neurotransmission,39,40 which is in
agreement with literature data for 5-HT6 receptor antagonists.41
The antidepressant-like activity of compounds 14, 15, and SB-
742457 seems to be specific because they did not affect the rats’
locomotor activity in the open-field paradigm (data not shown).
Because anxiety and depression are common comorbidities
of AD, the improvement of these behavioral symptoms may be
considered a valuable therapeutic attribute in the treatment of
patients with AD. Thus, to further extend the pharmacological
evaluation of the newly designed compounds, their anxiolytic
and antidepressant properties were evaluated in Wistar rats in
commonly used screening procedures: the Vogel conflict drinking
test37 and the modified forced swim test.38,39 SB-742457 was used
as a reference. Compound 14, given at a dose of 3 mg/kg (ip),
demonstrated anticonflict activity in Vogel test by significantly
increasing the number of shocks that were accepted by rats. This
effect was almost 2-fold higher than that observed for SB-742457
(340% vs 178%) (Figure 7). Moreover, compounds 14 and 15
CONCLUSION
■
As part of ongoing efforts to develop novel procognitive
therapies, we demonstrated that the scaffold-hopping approach
could be efficiently applied to identify a structurally novel class of
5-HT6R antagonists in a group of 1H-pyrrolo[3,2-c]quinolines.
Initial lead optimization around the arylsulfonyl fragment
afforded compound 14, a more selective and potent 5-HT6R
antagonist than the reference compound SB-742457. Further
evaluation of the 5-HT6R’s constitutive activity showed that 14
was classified as a neutral antagonist, while SB-742457 behaved
as an inverse agonist. The high potency and different functional
properties of 14 and SB-742457 that were demonstrated in
in vitro tests translated well into in vivo procognitive properties
either in PCP-induced memory decline (MED = 3 mg/kg) or in
the natural forgetting model in NOR tasks (MED = 3 mg/kg).
Compound 14 has additionally demonstrated a higher anxiolytic
effect than SB-742457 in Vogel test (MED for 14 = 3 mg/kg)
and showed similar antidepressant-like properties in 3-fold
higher dose than that observed for SB-742457 in the forced
swim test. The study evidenced comparable behavioral effects of
14 (5-HT6R antagonist) and SB-742457 (5-HT6R inverse agonist).
These results support the therapeutic potential of 5-HT6R
antagonists and/or inverse agonists for the treatment of cognitive
decline and other symptoms associated with Alzheimer’s disease.
It seems that more detailed biochemical studies would provide
further information about the action of 5-HT6R ligands. Likewise,
their therapeutic potential in other neuropsychiatric disorders like
autism or Parkinson’s disease certainly warrants further exploration.
Figure 7. Anxiolytic-like effects of 14, 15, and SB-742457 in the Vogel
conflict drinking test in rats. Values represent the mean standard error
of the mean. *p < 0.05 vs vehicle, **p < 0.01 vs vehicle, ****p < 0.0001
vs vehicle (one-way ANOVA followed by Bonferroni’s post hoc test);
N = 7−8.
were both active at doses of 10 mg/kg (ip), a dose of this level
increased the number of shocks that rats accepted by 787% and
729%, respectively.
In the forced swim test, the derivatives 14 (10 mg/kg, ip)
and 15 (3 mg/kg, ip) and SB-742457 (3 mg/kg, ip) exerted
significant antidepressant-like properties, with similar potencies;
the effects were assessed according to the decreased immobility
time and increased climbing time of the animals (Figure 8).
METHODS
■
General Methods. The synthesis was carried out at ambient
temperature, unless indicated otherwise. Organic solvents (from Aldrich
and Chempur) were of reagent grade and were used without purifica-
tion. The reagents were purchased from Sigma-Aldrich, Apeiron
Synthesis, Chembridge, and Fluorochem.
1H NMR and 13C NMR spectra were obtained in a Varian BB 200
spectrometer and were recorded at 300 and 75 MHz, respectively;
J values are in hertz (Hz), and splitting patterns are designated as
follows: s (singlet), d (doublet), t (triplet), and m (multiplet).
UPLC/MS were carried out on a system consisting of a Waters
Acquity UPLC, coupled to a Waters TQD mass spectrometer. All the
analyses were carried out using an Acquity UPLC BEH C18, 100 ×
2.1 mm column, at 40 °C. A flow rate of 0.3 mL/min and a gradient of
(0−100)% B over 10 min was used. Eluent A, water/0.1% HCO2H;
eluent B, acetonitrile/0.1% HCO2H. Retention times tR were given
in minutes. The UPLC/MS purity of all the test compounds and key
intermediates was determined to be >99%.
HRMS analyses were performed at The Laboratory for Physical
Measurements of the Universite
(Waters) with ESI ionization mode.
́
de Montpellier, on a Synapt G2-S
Melting points were determined with a Buchi apparatus and are
̈
uncorrected.
Figure 8. Antidepressant-like properties of 14, 15 and SB-742457 in the
modified forced swim test in rats. Values represent the mean standard
error of the mean. *p < 0.05 vs vehicle, **p < 0.01 vs vehicle (ANOVA is
followed by the Bonferroni’s post hoc test); N = 7−8.
Optical rotation was measured on a Jasco polarimeter (model P-2000,
Eastan) using a sodium lamp, emitting light at a wavelength of 589 nm
and 10 cm polarimetric tube. Measurements were performed in thermo-
static conditions at 20 °C.
F
DOI: 10.1021/acschemneuro.6b00090
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ACS Chemical Neuroscience
Research Article
Elemental analyses for C, H, and N were carried out using the
elemental Vario EI III elemental analyzer (Hanau, Germany). Elemental
analyses were found within 0.4% of the theoretical values.
with a saturated solution of NaHCO3, three times with water, and once
with brine. The organic layer was dried over Na2SO4 and evaporated.
The obtained crude product was purified on silica gel with AcOEt/Hex
(4/6) as a developing solvent.
Synthetic Procedures. Methyl 2-[(2-Nitrophenyl)(p-toluenesul-
fonylamino)-methyl]acrylate (1). In a dried flask, p-toluenesulfonamide
(18 g, 105 mmol, 1 equiv) and 1,4-diazabicyclo[2.2.2]octane (DABCO)
(1.78 g, 15.7 mmol, 0.15 equiv) were mixed together with the previously
activated molecular sieves (4 Å, 21 g). The mixture was suspended
in isopropanol (300 mL), followed by addition of nitrobenzaldehyde
(15.8 g, 105 mmol, 1 equiv) and methyl acrylate (10.7 mL, 115 mmol,
1.1 equiv). Subsequently, titanium isopropoxide (Ti(iOPr)4) was added
as a freshly prepared solution in isopropanol (0.6 mL, 2.1 mmol,
0.02 equiv). The flask was filled with nitrogen and the mixture was stirred
at room temperature overnight. Then, a mixture was filtered through
Celite, which was rinsed with CH2Cl2. The solvent was evaporated, and
the remaining crude was dissolved in AcOEt (400 mL), washed three
times with 1 M KHSO4, once with saturated NaHCO3, water, and brine,
and dried over Na2SO4. Evaporation of the solvent gave a yellow oil
which was subsequently dissolved in AcOEt (300 mL) and precipitated
upon portionwise addition of n-hexane (300 mL). The appearing white
precipitate was filtered, rinsed with diethyl ether and dried under vacuum.
White solid, 63% yield, tR = 6.30, Mp 109−111 °C, C18H18N2O6S,
MW 390.41. 1H NMR (300 MHz, CDCl3) δ (ppm) 2.40 (s, 3H), 3.57
(s, 3H), 5.68 (d, J = 0.77 Hz, 1H), 5.84 (d, J = 8.72 Hz, 1H), 6.08 (d, J =
8.46 Hz, 1H), 6.21 (s, 1H), 7.20−7.26 (m, 2H), 7.40 (m, J = 7.76, 1.41
Hz, 1H), 7.54 (m, J = 7.69, 1.28 Hz, 1H), 7.66−7.71 (m, 3H), 7.82 (dd,
J = 8.08, 1.41 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ (ppm) 21.1, 52.1,
54.4, 124.5, 127.2, 128.8, 129.1, 129.6, 130.1, 133.1, 133.4, 137.1, 137.4,
143.6, 148.0, 165.4. Monoisotopic mass 390.09, [M + H]+ 391.4.
Methyl 2-[(N-Allyl-N-tosylamino)(2-nitrophenyl)-methyl]acrylate
(2). β-Aminoester 1 (10 g, 25.6 mmol, 1 equiv) was dissolved in DMF
(100 mL), followed by addition of K2CO3 (10.5 g, 76.5 mmol, 3 equiv).
Subsequently, allyl bromide (4.42 mL, 51.2 mmol, 2 equiv) was added
dropwise. The reaction was stirred at room temperature for 6 h. The
mixture was diluted with ethyl acetate (300 mL) and washed 5× with
water and brine, and dried over Na2SO4. The organic phase was filtered
and hexane was added to precipitate a white solid, which was filtered and
dried under vacuum.
White solid, 95% yield, tR = 7.48, Mp 83−85 °C, C21H22N2O6S, MW
430.47. 1H NMR (300 MHz, CDCl3) δ (ppm) 2.41 (s, 3H), 3.49−3.55
(m, 3H), 3.92−4.01 (m, 1H), 4.15−4.25 (m, 1H), 4.92−5.03 (m, 2H),
5.43−5.59 (m, 2H), 6.44 (s, 1H), 6.79 (s, 1H), 7.23−7.27 (m, 2H),
7.42−7.49 (m, 1H), 7.61−7.68 (m, 3H), 7.78 (d, J = 7.69 Hz, 1H), 7.93
(dd, J = 8.21 Hz, 1.28 Hz, 1H). Monoisotopic mass 430.12, [M + H]+
430.8. HRMS calcd for C21H23N2O6S, 431.1277; found, 431.1277.
Methyl 2,5-Dihydro-2-(2-nitrophenyl)-1-tosyl-1H-pyrrole-3-
carboxylate (3). The β-aminoester 2 (5 g, 11.6 mmol, 1 equiv) was
dissolved in CH2Cl2 (30 mL) in a round-bottom flask, and the Grubbs II
catalyst (297 mg, 0.35 mmol, 3 mol %) was added. The reaction was
stirred at 36 °C for 30 min under microwave irradiation in the open
vessel mode. DMSO (1.22 mL, 17.4 mmol, 1.5 equiv) was added and the
mixture stirred at room temperature for 20 h. After this time, silica gel
was added into the solution and it was stirred for 5 min. The mixture was
diluted with CH2Cl2 and filtered through a layer of silica gel. The filtrate
was evaporated and the obtained residue was treated with diethyl ether
giving a white precipitate which was filtered and dried under vacuum.
White solid, 80% yield, Mp 125−127 °C, tR = 6.83, C19H18N2O6S,
MW 402.42.1H NMR (300 MHz, CDCl3) δ (ppm) 2.36 (s, 3H), 3.45 (s,
3H), 4.29 (m, 1H), 4.54 (m, 1H), 6.61−6.64 (m, 1H), 6.68 (t, J = 2.0 Hz,
1H), 7.27 (d, J = 8.27 Hz, 2H), 7.31−7.36 (m, 1H), 7.48−7.50 (m, 2H),
7.74 (d, J = 8.27 Hz, 2H), 7.83 (d, J = 8.22 Hz 1H). 13C NMR (75 MHz,
CDCl3) δ (ppm) 21.56, 52.03, 55.73, 62.29, 124.54, 127.75, 128.62,
129.64, 130.04, 133.16, 133.38, 134.91, 135.68, 136.76, 144.25, 148.70,
161.57. Monoisotopic mass 402.09, [M + H]+ 403.3. HRMS calcd for
C19H19N2O6S, 403.0964; found, 403.0967.
Yellow oil, 85% yield, tR = 5.72, C12H10N2O4, MW 246.22. 1H NMR
(300 MHz, CDCl3) δ (ppm) 3.62 (s, 3H), 6.64 (t, J = 2.80 Hz, 1H), 6.72
(t, J = 2.95 Hz, 1H) 6.82−6.85 (m, 1H) 7.44−7.48 (m, 1H) 7.51−7.58
(m, 1H) 7.60−7.66 (m, 1H) 8.02 (dd, J = 8.21, 1.28 Hz, 1H). 13C NMR
(75 MHz, CDCl3) δ (ppm) 51.02, 111.45, 113.60, 118.61, 124.37,
127.29, 129.49, 132.54, 149.57, 164.67. Monoisotopic mass 246.06,
[M + H]+ 247.3.
Methyl 2-(2-Aminophenyl)-1H-pyrrole-3-carboxylate (5). The
nitro derivative 4 (2.5 g, 10.2 mmol, 1 equiv) was dissolved in methanol,
and 10% Pd/C (180 mg, 7 weigh%) and acetic acid (0.26 mL, 4.6 mmol,
0.45 equiv) were subsequently added. The mixture was stirred under
a hydrogen atmosphere at room temperature for 2 h. Then, the mixture
was filtered though Celite, which was rinsed with methanol. The sub-
sequent evaporation yielded an orange oil.
Orange oil, 95% yield, tR = 4.84, C12H12N2O2, MW 216.24. 1H NMR
(300 MHz, CDCl3/methanol-d4) δ (ppm) 3.60−3.62 (m, 3H) 6.59
(d, J = 3.08 Hz, 1H) 6.67−6.70 (m, 2H) 6.71−6.73 (m, 1H) 7.04−7.11
(m, 2H). Monoisotopic mass 216.09, [M + H]+ 217.0.
1H-Pyrrolo[3,2-c]quinolin-4(5H)-one (6). Compound 5 (2.5 g,
11.6 mmol, 1 equiv) was suspended in sec-butanol (50 mL), and acetic
acid (0.5 mL, 8.7 mmol, 0.75 equiv) was added. The reaction mixture
was stirred at 70 °C for 3 h. The solvent was evaporated under vacuum
giving an orange oil.
Orange oil, 99% yield, tR = 2.97, C11H8N2O, MW 184.19. 1H NMR
(300 MHz, methanol-d4) δ (ppm) 6.81 (d, J = 3.08 Hz, 1H), 7.19 (d,
J = 3.08 Hz, 1H), 7.23−7.30 (m, 1H), 7.36−7.45 (m, 2H), 7.97 (d, J =
7.69 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ (ppm) 105.53, 113.45,
115.20, 116.25, 121.81, 123.02, 127.51, 134.83, 136.53, 160.20,
172.54. Monoisotopic mass 184.06, [M + H]+ 185.0. HRMS calcd for
C11H9N2O ,185.0715; found, 185.0714.
4-Chloro-1H-pyrrolo[3,2-c]quinoline (7). 80 mL of POCl3 was
added to the lactam 6 (2 g, 10.8 mmol, 1 equiv), and the mixture was
heated at 105 °C for 4 h. Then, the mixture was evaporated under
vacuum and 50 g of ice was added. After melting, the solution was
neutralized with ammonia solution. The product was extracted three
times with CH2Cl2. The organic layer was dried over Na2SO4, filtered,
and evaporated, yielding a white solid.
White solid, 85% yield, Mp 253−255 °C, tR = 5.12, C11H7ClN2, MW
202.64. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 6.71−6.73 (m, 1H),
7.60−7.66 (m, 1H), 7.93−7.98 (m, 3H), 8.36−8.41 (m, 1H), 12.82 (s,
1H). 13C NMR (75 MHz, DMSO-d6) δ (ppm) ppm 102.72, 118.01,
119.18, 121.70, 126.05, 126.67, 127.63, 128.77, 136.45, 142.94, 144.47.
Monoisotopic mass 202.03, [M + H]+ 203.1, 205.1. HRMS calcd for
C11H8ClN2, 203.0376; found, 203.0377.
General Procedure for Preparation of Compounds 8a−8c.
Compound 7 (0.35 g, 1.7 mmol, 1 equiv) was suspended in 12 mL of
MeCN followed by addition of (R,S)-1-Boc-3-aminopyrrolidine (8a),
(R)-1-Boc-3-aminopyrrolidine (8b) or (S)-1-Boc-3-aminopyrrolidine
(8c) (1.3 g, 6.9 mmol, 4 equiv). The reaction was performed in a Biotage
Microwave and stirred at 140 °C for 5h. The solvent was subsequently
evaporated and the mixture was purified on silica gel with CH2Cl2/
MeOH 9/1.5 as a developing solvent.
(R,S)-4-(1-tert-Butoxycarbonyl-pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline (8a). Orange oil, 60% yield, tR = 4.38,
1
C20H24N4O2, MW 352.43. H NMR (300 MHz, DMSO-d6) δ (ppm)
1.40 (s, 9H), 1.98 (bs, 1H), 2.30 (bs, 1 H), 3.27−3.56 (m, 4H),
3.71−3.81 (m, 1 H), 4.93 (bs, 1H), 6.55 (d, J = 3.08 Hz, 1H), 7.13 (d,
J = 3.08 Hz, 1H), 7.16−7.23 (m, 1H), 7.25−7.27 (m, 1H), 7.33−7.34 (t,
J = 7.31 Hz, 1H), 7.71−7.81 (d, J = 8.46 Hz, 1H), 7.86 (dd, J = 7.95,
1.28 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ (ppm) 28.65, 44.59,
44.75, 50.56, 52.05, 78.65, 102.14, 111.10, 115.93, 120.63, 121.24,
122.72, 126.41, 134.96, 144.33, 152.01, 154.11. Monoisotopic mass
352.19, [M + H]+ 353.2. HRMS calcd for C20H25N4O2, 353.1978;
found, 353.1978.
Methyl 2-(2-Nitrophenyl)-1H-pyrrole-3-carboxylate (4). To a
solution of 2,5-dihydropyrrole 3 (2.5 g, 6.2 mmol, 1 equiv) in DMF
(40 mL), sodium tert-butoxide (2.09 g, 18.7 mmol, 3 equiv) was added.
The reaction was stirred for 2 h and monitored by TLC. The mixture
was diluted with ethyl acetate, neutralized with 1 M KHSO4, and washed
(R)-4-(1-tert-Butoxycarbonyl-pyrrolidine-3-yl-amino)-1H-pyrrolo-
[3,2-c]quinoline (8b). Orange oil, 62% yield, tR = 4.38, C20H24N4O2,
G
DOI: 10.1021/acschemneuro.6b00090
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ACS Chemical Neuroscience
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1
MW 352.43. H NMR (300 MHz, DMSO-d6) δ (ppm) 1.37−1.50
(R,S)-1-[(3-Chlorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (13). White solid, 90% yield,
tR = 4.13, Mp 220−221 °C, Anal. (C21H21Cl3N4O2S·H2O) C, H, N. MW
517.85. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 2.20−2.25 (m, 1H),
2.36−2.43 (m, 1H), 3.25−3.36 (m, 1H), 3.49−3.57 (m, 4H), 5.24 (s,
1H), 7.36−7.46 (m, 1H), 7.63 (t, J = 7.96 Hz, 2H), 7.81−7.83 (m, 1H),
7.88−8.08 (m, 2H), 8.19−8.20 (m, 1H), 8.30−8.33 (m, 1H), 8.63−8.66
(m, 1H), 9.46 (s, 2H). Monoisotopic mass 426.09, [M + H]+ 427.2.
HRMS calcd for C21H20ClN4O2S, 427.0995; found, 427.0996.
(S)-1-[(3-Chlorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (14). White solid, 92% yield,
tR = 4.18, Mp 219−221 °C, Anal. (C21H21Cl3N4O2S·H2O) C, H, N.
MW 517.85. 1H NMR (300 MHz, CDCl3/methanol-d4) δ (ppm) 2.25−
2.38 (m, 1H), 2.54−2.68 (m, 1H), 3.23−3.35 (m, 1H), 3.57−3.76 (m,
4H), 5.50−5.64 (m, 1H), 7.35−7.45 (m, 2H), 7.48−7.61 (m, 3H),
7.68−7.72 (m, 1H), 7.84−7.95 (m, 2H), 8.46 (d, J = 8.46 Hz, 1H), 8.75
(dd, J = 8.59, 1.15 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ ppm
31.16, 44.21, 49.11, 52.70, 108.85, 112.93, 115.91, 120.30, 123.69,
125.56, 126.41, 127.18, 130.19, 130.67, 132.77, 134.42, 135.29, 136.14,
138.49, 148.45. [α]2D0 = +2.93° (0.40, EtOH/H2O 9/1). Monoisotopic
mass 426.09, [M + H]+ 427.2. HRMS calcd for C21H20ClN4O2S,
427.0995; found, 427.0992.
(R)-1-[(3-Chlorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (15). White solid, 91% yield,
tR = 4.18, Mp 221−223 °C, Anal. (C21H21Cl3N4O2S·H2O) C, H, N.
MW 517.85. 1H NMR (300 MHz, CDCl3/methanol-d4) δ (ppm) 2.24−
2.40 (m, 1H), 2.52−2.69 (m, 1H), 3.27−3.44 (m, 2H), 3.56−3.79 (m,
3H), 5.46−5.68 (m, 1H), 7.35−7.46 (m, 1H), 7.52−7.65 (m, 2H),
7.75−7.85 (m, 2H), 7.92−8.02 (m, 2H), 8.05−8.20 (bs, 1H), 8.48 (d,
J = 8.25 Hz, 1H), 8.76 (dd, J = 8.53, J = 1.10 Hz, 1H). 13C NMR
(75 MHz, DMSO-d6) δ ppm 31.16, 44.21, 49.11, 52.72, 108.85, 112.92,
115.90, 120.33, 123.69, 125.57, 126.41, 127.18, 130.20, 130.67, 132.77,
134.42, 135.29, 136.14, 138.48, 148.44. [α]2D0 = −5.65° (0.48, EtOH/
H2O 9/1). Monoisotopic mass 426.09, [M + H]+ 427.2. HRMS calcd for
C21H20ClN4O2S, 427.0995; found, 427.0998.
(R,S)-1-{[3-(Trifluoromethyl)phenyl]sulfonyl}-4-(pyrrolidine-3-yl-
amino)-1H-pyrrolo[3,2-c]quinoline dihydrochloride (16). White solid,
90% yield, tR = 4.68, Mp 223−225 °C, Anal. (C22H21Cl2F3N4O2S·H2O)
C, H, N. MW 551.41. 1H NMR (300 MHz, CDCl3/methanol-d4)
δ (ppm) 2.22−2.44 (m, 1H), 2.53−2.74 (m, 1H), 3.32 (s, 2H), 3.66
(d, J = 10.18 Hz, 3H), 5.50−5.71 (m, 1H), 7.43 (d, J = 7.70 Hz, 1H),
7.51−7.68 (m, 2H), 7.81 (d, J = 7.70 Hz, 2H), 7.90−8.03 (m, 2H), 8.12
(s, 1H), 8.49 (d, J = 8.25 Hz, 1H), 8.79 (dd, J = 8.53, J = 0.83 Hz, 1H).
Monoisotopic mass 460.12, [M + H]+ 461.2. HRMS calcd for
C22H20N4OF3S, 461.1259, found, 461.1259.
(S)-1-{[3-(Trifluoromethyl)phenyl]sulfonyl}-4-(pyrrolidine-3-yl-
amino)-1H-pyrrolo[3,2-c]quinoline dihydrochloride (17). White solid,
91% yield, tR = 4.67, Mp 219−221 °C, Anal. (C22H21Cl2F3N4O2S·H2O)
C, H, N, MW 551.41. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 2.21 (d,
J = 5.28 Hz, 1H), 2.33−2.44 (m, 1H), 3.48 (dd, J = 11.58, 5.72 Hz, 2H),
3.62−3.68 (m, 3H), 5.29 (bs, 1H), 7.46 (t, J = 7.48 Hz, 1H), 7.64 (t, J =
7.77 Hz, 1H), 7.79−7.89 (m, 1H), 8.01−8.20 (m, 2H), 8.25 (d, J =
3.52 Hz, 1H), 8.39 (bs, 2H), 8.67 (d, J = 7.92 Hz, 1H), 9.51 (bs, 2H).
13C NMR (75 MHz, DMSO-d6) δ ppm 31.08, 44.22, 49.17, 52.47,
108.66, 113.03, 114.85, 115.20, 115.91, 120.49, 123.34, 123.73, 124.01,
125.46, 130.14, 130.58, 133.41, 134.47, 138.66, 148.62, 160.54, 163.87.
[α]2D0 = +3.50° (0.49, EtOH/H2O 9/1). Monoisotopic mass 460.12,
[M + H]+ 461.2. HRMS calcd for C22H20N4OF3S, 461.1259, found,
461.1258.
(m, 9H), 2.00 (bs, 1H), 2.30 (bs, 1H), 3.28−3.56 (m, 4H), 3.70−3.85
(m, 1H), 4.92 (bs, 1H), 6.50−5.58 (d, J = 3.08 Hz, 1H), 7.10−7.15
(d, J = 3.08 Hz, 1H), 7.17−7.24 (m, 1H), 7.25−7.28 (m, 1H), 7.34−7.43
(t, J = 7.18 Hz, 1H), 7.72−7.82 (d, J = 7.95 Hz, 1H), 7.83−7.90 (dd,
J = 7.95, J = 1.03 Hz, 1H). Monoisotopic mass 352.19, [M + H]+ 353.2.
HRMS calcd for C20H25N4O2, 353.1978; found, 353.1978.
(S)-4-(1-tert-Butoxycarbonyl-pyrrolidine-3-yl-amino)-1H-pyrrolo-
[3,2-c]quinoline (8c). Orange oil, 60% yield, tR = 4.38, C20H24N4O2,
MW 352.43. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 1.35−1.49 (m,
9H) 1.98 (bs, 1H) 2.30 (bs, 1H) 3.27−3.56 (m, 4H) 3.71−3.81 (m, 1H)
4.93 (bs, 1H) 6.55 (d, J = 3.08 Hz, 1H) 7.13 (d, J = 3.08 Hz, 1H)
7.16−7.23 (m, 1H) 7.25−7.27 (m, 1H) 7.33−7.34 (t, J = 7.31 Hz, 1H,)
7.71−7.81 (d, J = 8.46 Hz, 1H) 7.86 (dd, J = 7.95, 1.28 Hz, 1H).
Monoisotopic mass 352.19, [M + H]+ 353.2. HRMS calcd for
C20H25N4O2, 353.1978, found, 353.1978.
General Procedure for Preparation of Final Compounds 9−27.
Compounds 8a−8c (100 mg, 0.28 mmol, 1 equiv) were dissolved in
CH2Cl2 (5 mL), and BTPP (171 μL, 0.56 mmol, 2 equiv) was added.
The mixture was placed in an ice-bath, sulfonyl chloride (1.8 equiv) was
added, and the reaction mixture was stirred for 3 h. Subsequently, the
mixture was evaporated and the remaining yellow crude product was
purified on silica gel. The Boc-protected derivatives were treated with
4 N HCl solution in dioxane to give the final products as HCl salts of
secondary amines.
(R,S)-1-(Phenylsulfonyl)-N-(pyrrolidin-3-yl)-1H-pyrrolo[3,2-c]-
quinolin-4-amine dihydrochloride (9). White solid, 80% yield, tR
=
4.78, Mp 220−222 °C, Anal. (C21H22Cl2N4O2S·H2O) C, H, N. MW
483.41. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 2.21 (d, J = 4.98 Hz,
1H), 2.31−2.45 (m, 1H), 3.56 (d, J = 4.98 Hz, 5H), 5.28 (bs, 1H), 7.44
(d, J = 7.04 Hz, 1H), 7.55−7.65 (m, 3H), 7.68−7.77 (m, 1H), 7.92 (d,
J = 7.62 Hz, 2H), 8.19 (d, J = 3.52 Hz, 1H), 8.65 (d, J = 7.92 Hz, 1H),
9.50 (bs, 2H). Monoisotopic mass: 392.13, [M + H]+ 393.1. HRMS
calcd for C21H21N4O2S, 393.1385, found, 393.1389.
(R,S)-1-[(3-Fluorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (10). White solid, 87% yield,
tR = 3.96, Mp 207−209 °C, Anal. (C21H21Cl2FN4O2S·H2O) C, H, N.
MW 501.40. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 2.15−2.26 (m,
1 H), 2.25−2.40 (m, 1H), 3.12−3.18 (m, 1H), 3.20−3.35 (m, 1H),
3.45−3.70 (m, 3H), 5.29 (bs, 1H), 7.41−7.46 (m, 1H), 7.58−7.71 (m,
3H), 7.78 (d, J = 7.06 Hz, 1H), 7.93 (d, J = 7.92 Hz, 1H), 8.06 (bs, 1H),
8.19 (d, J = 3.52 Hz, 1H), 8.39 (bs, 1H), 8.63 (d, J = 8.21 Hz, 1 H), 9.52
(bs, 2H). Monoisotopic mass 410.12, [M + H]+ 411.3. HRMS calcd for
C21H20FN4O2S, 411.1291; found, 411.1291.
(S)-1-[(3-Fluorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (11). White solid, 85% yield,
tR = 3.95, Mp 197−199 °C, Anal. (C21H21Cl2FN4O2S·H2O) C, H, N,
1
MW 501.40. H NMR (300 MHz, DMSO-d6) δ (ppm) 2.21 (d, J =
4.69 Hz, 1H), 2.37−2.42 (m, 1H), 3.23−3.34 (m, 2 H), 3.52−3.59
(m, 3H), 5.28 (bs, 1H), 7.43−7.47 (m, 1H), 7.58−7.69 (m, 2H), 7.77
(d, J = 7.04 Hz, 1H), 7.94 (d, J = 7.92 Hz, 1H), 8.05 (bs, 1H), 8.18
(d, J = 3.52 Hz, 1H), 8.39 (bs, 1H), 8.63 (d, J = 8.21 Hz, 1 H), 9.51
(bs, 2H). 13C NMR (75 MHz, DMSO-d6) δ (ppm) 31.07, 44.22,
49.18, 52.49, 108.61, 113.01, 114.85, 115.20, 115.89, 120.49, 123.73,
124.02, 125.46, 130.41, 130.58, 133.41, 134.47, 138.65, 148.62, 160.56,
163.88. Monoisotopic mass 410.12, [M + H]+ 411.3. HRMS calcd for
C21H20FN4O2S, 411.1291; found, 411.1292.
(R)-1-[(3-Fluorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (12). White solid, 90% yield,
tR = 3.76, Mp 220−221 °C, Anal. (C21H21Cl2FN4O2S·H2O) C, H, N.
MW 501.40. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 2.14−2.27 (m,
1H), 2.33−2.45 (m, 1H), 3.20−3.35 (m, 2H), 3.50−3.67 (m, 3H), 5.30
(bs, 1H), 7.42−7.46 (m, 1H), 7.56−7.70 (m, 3H), 7.73−7.80 (m, 1H),
7.94 (d, J = 7.92 Hz, 1H), 8.07 (bs, 1H), 8.19 (d, J = 3.81 Hz, 1H), 8.40
(bs, 1H), 8.63 (dd, J = 8.50, 1.17 Hz, 1H), 9.52 (bs, 2H). 13C NMR
(75 MHz, DMSO-d6) δ ppm 31.18, 44.19, 49.10, 52.73, 108.80, 112.94,
114.86 115.20, 115.89, 120.26, 123.73, 124.03, 125.56, 130.16, 130.63,
133.42, 134.44, 138.63, 148.47, 160.53, 163.86. Monoisotopic mass
410.12, [M + H]+ 411.3. HRMS calcd for C21H20FN4O2S, 411.1291;
found, 411.1291.
(R)-1-{[3-(Trifluoromethyl)phenyl]sulfonyl}-4-(pyrrolidine-3-yl-
amino)-1H-pyrrolo[3,2-c]quinoline dihydrochloride (18). White solid,
88% yield, tR = 4.67, Mp 230−232 °C, Anal. (C22H21Cl2F3N4O2S·H2O)
C, H, N. MW 551.41. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 2.11−
2.27 (m, 1H), 2.30−2.45 (m, 1H), 3.20−3.35 (m, 1H), 3.40−3.52 (m,
1H), 3.60−3.82 (m, 3H), 5.27 (bs, 1H), 7.42−7.46 (m, 1H), 7.63 (t,
J = 7.04 Hz, 1H), 7.79−7.87 (m, 1H), 8.01−8.20 (m, 2H), 8.25 (d, J =
2.93 Hz, 1H), 8.39 (bs, 2H), 8.67 (d, J = 8.50 Hz, 1H), 9.47 (bs, 2H).
13C NMR (75 MHz, DMSO-d6) δ ppm 31.18, 44.19, 49.10, 52.70,
108.80, 112.94, 114.86, 115.20, 115.89, 120.26, 123.35, 123.73, 124.02,
125.56, 130.16, 130.63, 133.42, 134.44, 138.61, 148.47, 160.53, 163.86.
H
DOI: 10.1021/acschemneuro.6b00090
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ACS Chemical Neuroscience
Research Article
[α]2D0 = −2.57° (0.48, EtOH/H2O 9/1). Monoisotopic mass 460.12.
[M + H]+ 461.3. HRMS calcd for C22H20N4OF3S, 461.1259; found,
461.1263.
8.05−8.10 (m, 2H), 8.10−8.48 (d, J = 8.50 Hz, 4H), 8.49−8.52 (m, 1H),
9.51 (bs, 2H). Monoisotopic mass 442.15, [M + H]+ 443.2. HRMS calcd
for C25H23N4O2S, 443.1542; found, 443.1541.
(R,S)-1-(Quinolin-8-ylsulfonyl)-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (26). White solid, 86% yield,
tR = 5.11, Mp 209−211 °C, Anal. (C24H23Cl2N5O2S·H2O) C, H, N.
MW 534.46. 1H NMR (300 MHz, CDCl3/methanol-d4) δ (ppm)
2.17−2.33 (m, 1H), 2.48−2.63 (m, 1H), 3.27−3.40 (m, 2H), 3.50−
3.57 (m, 3H), 5.39−5.55 (m, 1H), 7.25−7.40 (m, 1H), 7.55−7.70
(m, 4H), 8.10−8.23 (m, 2H), 8.22−8.39 (m, 4H), 8.50−8.80 (m, 3H).
Monoisotopic mass 443.14, [M + H]+ 444.2. HRMS calcd for
C24H22N5O2S, 444.1494; found, 444.1491.
(R,S)-1-[(4-Fluorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (19). White solid, 90% yield,
tR = 3.76, Mp 220−221 °C, Anal. (C21H21Cl2FN4O2S·H2O) C, H, N.
MW 501.40. 1H NMR (300 MHz, CDCl3/methanol-d4) δ (ppm) 2.07−
2.33 (m, 1H), 2.10−2.29 (m, 1H), 2.41−2.58 (m, 1H), 3.21−3.35 (m,
1H), 3.43−3.68 (m, 3H), 5.36 (bs, 1H), 7.03 (t, J = 7.95 Hz, 2H), 7.27−
7.38 (m, 1H), 7.47 (t, J = 7.82 Hz, 1H), 7.59−7.72 (m, 2H), 7.72−7.80
(m, 1H), 7.80−7.88 (m, 1H), 8.27 (d, J = 8.46 Hz, 1H), 8.70 (d, J =
8.72 Hz, 1H). Monoisotopic mass 410.12, [M + H]+ 411.3. HRMS calcd
for C21H20FN4O2S, 411.1291; found, 411.1292.
(R,S)-1-[(5-Methylbenzo[b]thiophen-2-yl)sulfonyl]-4-(pyrrolidine-
3-yl-amino)-1H-pyrrolo[3,2-c]quinoline dihydrochloride (27). White
solid, 65% yield, tR = 5.23, Mp 221−223 °C, Anal. (C24H24Cl2N4O2S2·
H2O) C, H, N. MW 553.52. 1H NMR (300 MHz, DMSO-d6) δ (ppm)
2.20 (d, J = 4.10 Hz, 1H), 2.36 (s, 4H), 3.25−3.32 (m, 2H), 3.50−3.56
(m, 2H), 5.25 (bs, 1H), 7.39 (dd, J = 8.65, 1.32 Hz, 1H), 7.50 (bs, 1H),
7.63 (t, J = 7.18 Hz, 1H), 7.79 (s, 1H), 7.92 (d, J = 8.50 Hz, 1H),
8.12−8.18 (m, 1H), 8.31 (d, J = 17.59 Hz, 1H), 8.57 (s, 1H), 8.86 (d,
J = 8.50 Hz, 1H), 9.50 (bs, 2H). Monoisotopic mass 462.12, [M + H]+
463.0. HRMS calcd for C24H23N4O2S2, 463.1262; found, 463.1258.
In Vitro Pharmacology. Cell Culture and Preparation of Cell
Membranes for Radioligand Binding Assays. HEK293 cells with
stable expression of human 5-HT1A, 5-HT2A, 5-HT6, 5-HT7b, and D2L
receptors (prepared with the use of Lipofectamine 2000) or CHO-K1
cells with plasmid containing the sequence coding for the human
serotonin 5-HT2A receptor (PerkinElmer) were maintained at 37 °C in a
humidified atmosphere with 5% CO2 and grown in Dulbecco’s modified
Eagle’s medium containing 10% dialyzed fetal bovine serum and
500 mg/mL G418 sulfate. For membrane preparation, cells were
subcultured in 150 cm2 diameter dishes, grown to 90% confluence,
washed twice with prewarmed to 37 °C phosphate buffered saline (PBS)
and pelleted by centrifugation (200 g) in PBS containing 0.1 mM EDTA
and 1 mM dithiothreitol. Prior to membrane preparation, pellets were
stored at −80 °C.
Radioligand Binding Assays. Cell pellets were thawed and homo-
genized in 10 volumes of assay buffer using an Ultra Turrax tissue
homogenizer, and centrifuged twice at 35 000g for 15 min at 4 °C, with
incubation for 15 min at 37 °C in between. Composition of the assay
buffers was as follows. for 5-HT1AR: 50 mM Tris HCl, 0.1 mM EDTA,
4 mM MgCl2, 10 mM pargyline and 0.1% ascorbate. 5-HT2AR: 50 mM
Tris HCl, 0.1 mM EDTA, 4 mM MgCl2 and 0.1% ascorbate. 5-HT6R:
50 mM Tris HCl, 0.5 mM EDTA and 4 mM MgCl2. 5- HT7bR: 50 mM
Tris HCl, 4 mM MgCl2, 10 mM pargyline and 0.1% ascorbate.
Dopamine D2LR: 50 mM Tris HCl, 1 mM EDTA, 4 mM MgCl2,
120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2 and 0.1% ascorbate. All assays
were incubated in a total volume of 200 μL in 96-well microtiter
plates for 1 h at 37 °C, except for 5-HT1AR and 5-HT2AR, which were
incubated at room temperature and 27 °C, respectively. 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 (PerkinElmer). For displacement
studies, the assay samples contained as radioligands (PerkinElmer):
2.5 nM [3H]-8-OH-DPAT (135.2 Ci/mmol) for 5-HT1AR; 1 nM
[3H]-ketanserin (53.4 Ci/mmol) for 5-HT2AR; 2 nM [3H]-LSD
(83.6 Ci/mmol) for 5-HT6R; 0.8 nM [3H]-5-CT (39.2 Ci/mmol) for
5-HT7R or 2.5 nM [3H]-raclopride (76.0 Ci/mmol) for D2LR.
Nonspecific binding was defined with 10 μM of 5-HT in 5-HT1AR
and 5-HT7R binding experiments, whereas 20 μM of mianserin, 10 μM
of methiothepine or 10 μM of haloperidol were used in 5-HT2AR,
5-HT6R and D2LR assays, respectively. Each compound was tested in
triplicate at 7 concentrations (10−10−10−4 M). The inhibition constants
(Ki) were calculated from the Cheng-Prusoff equation.42 Results were
expressed as means of at least two separate experiments.
(R,S)-1-[(2,5-Difluorophenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-
1H-pyrrolo[3,2-c]quinoline dihydrochloride (20). White solid, 79%
yield, tR = 4.63, Mp 191−193 °C, Anal. (C21H20Cl2F2N4O2S·H2O) C, H,
1
N. MW 519.39. H NMR (300 MHz, DMSO-d6) δ (ppm) 2.22 (d,
J = 5.28 Hz, 1H), 2.34−2.45 (m, 1H), 3.12−3.35 (m, 2H), 3.50−3.68
(m, 3H), 5.27 (bs, 1H), 7.45 (bs, 1H), 7.51−7.66 (m, 3H), 7.70−7.79
(m, 1H), 7.90−8.07 (m, 1H), 8.14 (bs, 1H), 8.35 (bs, 1H), 8.54
(d, J = 8.80 Hz, 1H), 9.49 (bs, 2H). 13C NMR (75 MHz, DMSO-d6)
δ (ppm) 30.75, 44.33, 49.11, 51.88, 106.91, 112.82, 114.60, 116.63,
116.99, 119.47, 119.58, 119.89, 123.56, 125.61, 130.28, 130.62, 134.99,
147.82. Monoisotopic mass: 428.11, [M + H]+ 429.0. HRMS calcd for
C21H19F2N4O2S, 429.1197; found, 429.1201.
(R,S)-1-[(3-Methoxyphenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-
1H-pyrrolo[3,2-c]quinoline dihydrochloride (21). White solid, 95%
yield, tR = 3.92, Mp 210−212 °C, Anal. (C22H24Cl2N4O3S·H2O) C, H,
1
N. MW 513.43. H NMR (300 MHz, CDCl3/methanol-d4) δ (ppm)
2.41 (s, 3H), 2.62−2.72 (m, 3H), 3.67−3.78 (m, 4H), 6.89 (d, J =
3.85 Hz, 1H), 7.07 (m, 1H), 7.29−7.44 (m, 2H), 7.57−7.75 (m, 3H),
7.81 (dd, J = 8.46, 0.77 Hz, 1H), 7.87−7.93 (m, 1H), 8.48 (dd, J = 8.46,
J = 0.77 Hz, 1H), 8.56−8.63 (m, 1H). Monoisotopic mass 422.14.
[M + H]+ 423.2. HRMS calcd for C22H23N4O3S, 423.1491; found,
423.1494.
(R,S)-1-[(3-Methylphenyl)sulfonyl]-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (22). White solid, 90% yield,
tR = 3.88, Mp 200−202 °C, Anal. (C22H24Cl2N4O2S·H2O) C, H, N. MW
1
497.44. H NMR (300 MHz, CDCl3/methanol-d4) δ (ppm) 2.47 (s,
3H), 2.25−2.38 (m, 1H), 2.57−2.67(m, 1H), 3.26−3.35 (m, 1H),
3.40−3.47 (m, 1H), 3.59−3.75 (m, 3H), 5.52−5.64 (m, 1H), 7.29−7.61
(m, 5H), 7.70 (t, J = 1.80 Hz, 1H), 7.84−7.99 (m, 2H), 8.49 (d, J =
8.46 Hz, 1H), 8.78 (dd, J = 8.59, J = 1.15 Hz, 1H). Monoisotopic mass
406.15. [M + H]+ = 407.2. HRMS calcd for C22H23N4O2S, 407.1541;
found, 407.1542.
(R,S)-1-{[4-(tert-Butyl)phenyl]sulfonyl}-4-(pyrrolidine-3-yl-amino)-
1H-pyrrolo[3,2-c]quinoline dihydrochloride (23). White solid, 78%
yield, tR = 5.02, Mp 196−198 °C, Anal. (C25H30Cl2N4O2S·H2O) C, H,
1
N. MW 539.52. H NMR (300 MHz, CDCl3/methanol-d4) δ (ppm)
1.17 (s, 9H) 2.23−2.39 (m, 1H), 2.53−2.70 (m, 1H), 3.20−3.32 (m,
2H), 3.55−3.65 (m, 3H), 5.49−5.64 (m, 1H), 7.32−7.46 (m, 3H),
7.49−7.58 (m, 1H), 7.58−7.77 (m, 2H), 7.78−7.92 (m, 2H), 8.36−8.49
(m, 1H), 8.84 (d, J = 8.53 Hz, 1H). Monoisotopic mass 448.19,
[M + H]+ 449.3. HRMS calcd for C25H29N4O2S, 449.2011; found,
449.2007.
(R,S)-1-{[4-(iso-Propyl)phenyl]sulfonyl}-4-(pyrrolidine-3-yl-
amino)-1H-pyrrolo[3,2-c]quinoline dihydrochloride (24). White solid,
75% yield, tR = 5.00, Mp 190−192 °C, Anal. (C24H28Cl2N4O2S·H2O) C,
H, N. MW 525.49. 1H NMR (300 MHz, CDCl3/methanol-d4) δ (ppm)
1.12 (s, 6H) 2.20−2.35 (m, 1H), 2.50−2.69 (m, 1H), 3.18−3.30 (m,
2H), 3.55−3.66 (m, 3H), 5.51−5.66 (m, 1H), 7.27 (s, 1H), 7.32−7.45
(m, 2H), 7.46−7.70 (m, 3H), 7.78−7.90 (m, 2H), 8.36−8.50 (m, 2H),
8.84 (d, J = 8.53 Hz, 1H). Monoisotopic mass 434.18, [M + H]+ 435.2.
HRMS calcd for C24H27N4O2S, 435.1854; found, 435.1855.
(R,S)-1-(Naphthalen-1-ylsulfonyl)-4-(pyrrolidine-3-yl-amino)-1H-
pyrrolo[3,2-c]quinoline dihydrochloride (25). White solid, 85% yield,
tR = 4.30, Mp 223−225 °C, Anal. (C25H25Cl2N4O2S·H2O) C, H, N. MW
533.47. 1H NMR (300 MHz, CDCl3/methanol-d4) δ (ppm) 2.14−2.27
(m, 1H), 2.32−2.45 (m, 1H), 3.20−3.33 (m, 2H), 3.54−3.63 (m, 3H),
5.28 (bs, 1H), 7.33,(t, J = 7.33 Hz, 1H), 7.53−7.81 (m, 4H),
Preliminary Agonist/Antagonist Screening. The functional activity
of compounds on intracellular cAMP levels, studied in CHO cells stably
expressing the human 5-HT6 receptor, was determined at CEREP.
cAMP concentration was measured by the HTRF method. Adenylate
cyclase activity was expressed as the percentage of the maximal effect
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DOI: 10.1021/acschemneuro.6b00090
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obtained with 300 nM serotonin. Compounds were tested at a 10−6
M
of the objects was replaced by a novel one (A = familiar and B = novel).
Both trials lasted 3 min, and animals were returned to their home cage
after T1. The objects used were the glass beakers filled with the gravel
and the plastic bottles filled with the sand. The heights of the objects
were comparable (∼12 cm) and the objects were heavy enough not to
be displaced by the animals. The sequence of presentations and the
location of the objects was randomly assigned to each rat. The animals
explored the objects by looking, licking, sniffing or touching the object
while sniffing, but not when leaning against, standing or sitting on the
object. Any rat spending less than 5 s exploring the two objects within
3 min of T1 or T2 was eliminated from the study. Exploration time of the
objects and the distance traveled were measured using the Any-maze
video tracking system. Based on exploration time (E) of two objects
during T2, discrimination index (DI) was calculated according to the
formula: DI = (EB − EA)/(EA + AB). Phencyclidine (PCP), used
to attenuate learning, was administered at the dose of 5 mg/kg (ip)
45 min before familiarization phase (T1). The compounds 14 or 15
were administrated ip 30 min before PCP injection. In nondisturbed
paradigm, memory improvement, compounds were administrated ip
30 min before familiarization phase (T1).
Vogel Conflict Drinking Test. The testing procedure was based on a
method of Vogel et al.37 as a conditional model where a noxious stimulus
is applied. Anxiety Monitoring System Vogel test produced by TSE
Systems (Germany) was used. It was consisted of polycarbonate
cages (dimensions 26.5 × 15 × 42 cm3), equipped with a grid floor made
from stainless steel bars and drinking bottles containing tap water.
Experimental chambers were connected to PC software by control
chassis and electric shocks’ generator. On the first day of the experiment,
the rats were adapted to the test chambers and drink water from the
bottle spout for 10 min. Afterward, the rats were returned to their home
cages and were given 30 min free access to water followed by a 24 h water
deprivation period. The adaptation session and water deprivation
protocols were repeated on the second day of the experiment. On the
third day, the rats were placed again in the test chambers 60 min after the
compounds administration and were given free access to the drinking
tube. Recording data started immediately after the first lick and rats were
punished with an electric shock (0.5 mA, lasting 1 s) delivered to the
metal drinking tube every 20 licks. The number of licks and the number
of shocks received during a 5 min experimental session were recorded
automatically.
concentration. Serotonin was used as a reference compound for deter-
mination of the agonist effect and methiothepin for the antagonistic
effect. Experimental conditions are described online at www.cerep.fr.
Functional cAMP Assay Protocol. The antagonistic properties
of compounds at the 5-HT6R were evaluated, as their ability to
inhibit cAMP production induced by the agonist 5-CT (100 nM) in
HEK293 cells overexpressing 5 HT6R. Each compound was tested in
triplicate at 8 concentrations (10−11− 10−4 M). Cells (prepared with the
use of Lipofectamine 2000) were maintained at 37 °C in a humidified
atmosphere with 5% CO2 and were grown in Dulbeco’s modified Eagle’s
medium containing 10% dialyzed fetal bovine serum and 500 mg/mL
G418 sulfate. For functional experiments, cells were subcultured
in 25 cm2 diameter dishes, grown to 90% confluence, washed twice
with prewarmed (37 °C) phosphate buffered saline (PBS) and were
centrifuged for 5 min (160g). The supernatant was aspirated, and the cell
pellet was resuspended in stimulation buffer (1 × HBSS, 5 mM HEPES,
0.5 mM IBMX, 0.1% BSA). Total cAMP was measured using the
LANCE cAMP detection kit (PerkinElmer), according to the manufac-
turer instructions. For cAMP levels quantification, cells (5 μL) were
incubated with compounds (5 μL) for 30 min at room temperature, in a
384-well white opaque microtiter plate. After incubation, the reaction
was stopped and cells were lysed by addition of 10 μL working solution
(5 μL Eu-cAMP and 5 μL ULight-anti-cAMP). The assay plate was
incubated for 1 h at room temperature. Time-resolved fluorescence
resonance energy transfer (TR-FRET) was detected by an Infinite
M1000 Pro (Tecan) using instrument settings from LANCE cAMP
detection kit manual. Kb values were calculated from Cheng−Prusoff
equation42 specific for the analysis of functional inhibition curves:
Kb = IC50/(1 + A/EC50) where A is the agonist concentration, IC50 is the
antagonist concentration producing a 50% reduction in the response to
agonist, and EC50 is the agonist concentration, which causes a half of the
maximal response.
Determination of cAMP Production as 5-HT6R Constitutive Activity.
cAMP measurement was performed in NG108-15 cells transiently
expressing 5-HT6 receptor using the bioluminescence resonance energy
transfer (BRET) sensor for cAMP, CAMYEL (cAMP sensor using
YFP-Epac-RLuc).43 NG108-15 cells were cotransfected in suspension
with 5-HT6 receptor and CAMYEL constructs, using Lipofectamine
2000, according to the manufacturer protocol, and plated in white
96-well plates (Greiner), at a density of 80 000 cells per well. At 24 h
after transfection, cells were washed with PBS containing calcium
and magnesium. Coelanterazine H (Molecular Probes) was added at a
final concentration of 5 μM, and left at room temperature for 5 min.
BRET was measured using a Mithras LB 940 plate reader (Berthold
Technologies).
In Vivo Pharmacology. Novel Object Recognition Protocol.
Procedures are based on earlier studies of Popik et al.44 The experiments
were conducted in accordance with the NIH Guide for the Care and Use
of Laboratory Animals and were approved by the Ethics Committee for
Animal Experiments, Institute of Pharmacology. Male Sprague−Dawley
rats (Charles River, Germany) weighing ∼250 g at the arrival were
housed in the standard laboratory cages, under standard colony
A/C controlled conditions: room temperature 21 2 °C, humidity
(40−50%), 12 h light/dark cycle (lights on at 06:00) with ad libitum
access to food and water. Rats were allowed to acclimatize for at least
7 days before the start of the experimental procedure. During this week
animals were handled for at least 3 times. Behavioral testing was carried
out during the light phase of the light/dark cycle. At least 1 h before the
start of the experiment, rats were transferred to the experimental room
for acclimation. Rats were tested in a dimly lit (25 lx) “open field”
apparatus made of a dull gray plastic (66 × 56 × 30 cm3). After each
measurement, the floor was cleaned and dried. Procedure consisted of
habituation to the arena (without any objects) for 5 min, 24 h before the
test and test session comprised of two trials separated by an intertrial
interval (ITI). For PCP-induced memory impairment paradigm, 1 h ITI
was chosen. For memory improvement paradigm, 24 h ITI was chosen.
During the first trial (familiarization, T1) two identical objects (A1 and
A2) were presented in opposite corners, approximately 10 cm from
the walls of the open field. In the second trial (recognition, T2) one
The experiments were performed on male Wistar rats (230−260 g).
The animals were housed in polycarbonate Makrolon type 3 cages
(dimensions 26.5 × 15 × 42 cm3) in an environmentally controlled
room (ambient temperature 21 2 °C; relative humidity 50−60%;
12:12 light/dark cycle, lights on at 08:00), in groups of four rats.
Standard laboratory food (LSM-B) and filtered water were freely
available. Animals were assigned randomly to treatment groups. Each
experimental group consisted of 6−8 animals/dose, and the animals
were used only once in each test. All the experiments were performed by
three observers unaware of the treatment applied between 9:00 and
14:00 on separate groups of animals. All the experimental procedures
were approved by the First Local Ethical Committee on Animal Testing
́
at the Jagiellonian University in Krakow. Compounds 14, 15, and SB-
722457 were dissolved in distilled water and injected intraperitoneally
(ip) at a volume of 2 mL/kg 60 min before the test procedures. Control
animals received a vehicle injection according to the same schedule.
Modified Forced Swim Test in Rats. The experiment was carried out
according to the method of Porsolt et al. modified by Detke et al.38,39
On the first day of an experiment, the animals were gently individually
placed in Plexiglas cylinders (50 cm high, 18 cm in diameter) containing
30 cm of water maintained at 23−25 °C for 15 min. On removal from
water, the rats were placed for 30 min in a Plexiglas box under a 60-W
bulb to dry. On the following day (24 h later), the rats were replaced
in the cylinder and the total duration of immobility, swimming,
and climbing was recorded during the whole 5 min test period. The
swimming behavior entailed active swimming motions, for example,
moving horizontally around in the cylinder. Climbing activity consisted
of upward directed movements of the forepaws along the side of the swim
chamber, and immobility was assigned when no additional activity was
observed other than that necessary to keep the rat’s head above the
J
DOI: 10.1021/acschemneuro.6b00090
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
water.41 Fresh water was used for each animal. Compounds 14, 15, and
SB-742457 were dissolved in distilled water and injected intraperitoneally
(ip) 60 min before the test.
Open field test in rats was performed using Motor Monitor System
(Campden Instruments, Ltd., UK) consisting of two Smart Frame Open
Field (OF) stations (40 × 40 × 38 cm3) with 16 × 16 beams, located in
sound attenuating chambers and connected to PC software by control
chassis. Individual vehicle- or drug-injected animals were gently placed
in the center of the station. An automated Motor Monitor System the
total distance covered by a rat for 5 min.
The experiments were performed on male Wistar rats (230−260 g).
The animals were housed in polycarbonate Makrolon type 3 cages
(dimensions 26.5 × 15 × 42 cm3) in an environmentally controlled
room (ambient temperature 21 2 °C; relative humidity 50−60%;
12:12 light/dark cycle, lights on at 08:00), in groups of four rats.
Standard laboratory food (LSM-B) and filtered water were freely
available. Animals were assigned randomly to treatment groups. Each
experimental group consisted of 6−8 animals/dose, and the animals
were used only once in each test. All the experiments were performed by
three observers unaware of the treatment applied between 9:00 and
14:00 on separate groups of animals. All the experimental procedures
were approved by the First Local Ethical Committee on Animal Testing
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́
at the Jagiellonian University in Krakow.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acschemneuro.6b00090.
■
S
1H NMR and 13C NMR spectra for representative
compounds (PDF)
(12) Benhamu, B., Martin-Fontecha, M., Vazquez-Villa, H., Pardo, L.,
and Lopez-Rodriguez, M. L. (2014) Serotonin 5-HT6 receptor
antagonists for the treatment of cognitive deficiency in Alzheimer’s
disease. J. Med. Chem. 57, 7160−7181.
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5-HT6R ligands: A comprehensive insight into their selectivity and
activity. Curr. Bioact. Compd. 9, 64−100.
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Pardo, L., and Campillo, M. (2005) A three-dimensional pharmaco-
phore model for 5-hydroxytryptamine 6 (5-HT6) receptor antagonists. J.
Med. Chem. 48, 4216−4219.
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drugs targeting 5-HT6 receptors for the tretament of Alzheimer’s
disease. Expert Opin. Invest. Drugs 24, 1515−1528.
(16) Ramirez, M. J. (2013) 5-HT6 receptors and Alzheimer’s disease.
Alzheimers Res. Ther. 5, 15.
AUTHOR INFORMATION
Corresponding Author
*E-mail: pawel.zajdel@uj.edu.pl. Tel.: +48 126205450. Fax:
+48 126205405.
■
Funding
The study was financially supported by the Polish Ministry of
Science and Higher Education (MNiSW), Grant No. N N405
671540, the project “Prokog”, UDA-POIG.01.03.01-12-063/09-00,
cofinanced by the European Union from the European Fund
of Regional Development (EFRD), and by the University
Montpellier, CNRS. F.L. and P.M. were supported by a grant
from Institut Carnot Chimie Balard and Pole BioSante Rabelais.
̂ ́
K.G. thanks the French Embassy in Warsaw for the cotutelle
fellowship.
(17) Galimberti, D., and Scarpini, E. (2015) Idalopirdine as a treatment
for Alzheimer’s disease. Expert Opin. Invest. Drugs 24, 981−987.
(18) Meneses, A. (2015) Serotonin, neural markers, and memory.
Front. Pharmacol. 6, 1−22.
Notes
The authors declare no competing financial interest.
́
(19) Benakki, H., Colacino, E., Andre, C., Guenoun, F., Martinez, J.,
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