2-Phenyl-1 H-pyrrole-3-carboxamide as a New Scaffold for Developing 5-HT6Receptor Inverse Agonists with Cognition-Enhancing Activity

Article abstract of DOI:10.1021/acschemneuro.1c00061

Serotonin type 6 receptor (5-HT6R) has gained particular interest as a promising target for treating cognitive deficits, given the positive effects of its antagonists in a wide range of memory impairment paradigms. Herein, we report on degradation of the

Full text of DOI:10.1021/acschemneuro.1c00061

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Research Article
2‑Phenyl‑1H‑pyrrole-3-carboxamide as a New Scaffold for
Developing 5‑HT6 Receptor Inverse Agonists with Cognition-
Enhancing Activity
Marcin Drop, Vittorio Canale, Séverine Chaumont-Dubel, Rafał Kurczab, Grzegorz Satała,
Xavier Bantreil, Maria Walczak, Paulina Koczurkiewicz-Adamczyk, Gniewomir Latacz, Anna Gwizdak,
Martyna Krawczyk, Joanna Gołębiowska, Katarzyna Grychowska, Andrzej J. Bojarski,
Agnieszka Nikiforuk, Gilles Subra, Jean Martinez, Maciej Pawłowski, Piotr Popik, Philippe Marin,
Frédéric Lamaty, and Paweł Zajdel*
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ABSTRACT: Serotonin type 6 receptor (5-HT6R) has gained
particular interest as a promising target for treating cognitive
deficits, given the positive effects of its antagonists in a wide range
of memory impairment paradigms. Herein, we report on
degradation of the 1H-pyrrolo[3,2-c]quinoline scaffold to provide
the 2-phenyl-1H-pyrrole-3-carboxamide, which is devoid of
canonical indole-like skeleton and retains recognition of 5-HT6R.
This modification has changed the compound’s activity at 5-HT6R-
operated signaling pathways from neutral antagonism to inverse
agonism. The study identified compound 27 that behaves as an inverse agonist of the 5-HT6R at the Gs and Cdk5 signaling
pathways. Compound 27 showed high selectivity and metabolic stability and was brain penetrant. Finally, 27 reversed scopolamine-
induced memory decline in the novel object recognition test and exhibited procognitive properties in the attentional set-shifting task
in rats. In light of these findings, 27 might be considered for further evaluation as a new cognition-enhancing agent, while 2-phenyl-
1H-pyrrole-3-carboxamide might be used as a template for designing 5-HT6R inverse agonists.
KEYWORDS: Cognition, 5-HT6 receptor, constitutive activity, inverse agonism, Cdk5 signaling, 2-phenyl-1H-pyrrole-3-carboxamide,
novel object recognition test, attentional set shifting task
mTOR activity under the control of the 5-HT6R contributes
to cognitive deficits associated with schizophrenia8 and cannabis
abuse during adolescence.9 Activation of Cdk5 signaling by the
5-HT6R plays a crucial role in the migration of cortical neurons
and the initiation of neurite growth.10
Another important feature of the 5-HT6R is represented by a
high level of constitutive activity. This particular property
corresponds to the ability of the receptor to be spontaneously
active in the absence of an agonist.11 The 5-HT6R constitutive
activity was established for recombinant receptors expressed in
cell lines12 and subsequently confirmed for native receptors in
primary cultured neurons and mouse brain.13
1. INTRODUCTION
Cognitive decline and mental retardation are associated with
several neurological and psychiatric disorders such as
Alzheimer’s disease, Parkinson’s disease, schizophrenia, depres-
sion, Down syndrome, and autism spectrum disorders.1,2 The
complexity and progressive nature of these diseases and the
limited efficacy of the currently used drugs indicate the
paramount need to develop novel therapeutic approaches.
Among the different molecular targets, the 5-HT6 receptor
(5-HT6R) has emerged as a particularly promising target to
alleviate cognitive impairments.3,4
The 5-HT6R belongs to the family of G-protein-coupled
receptors (GPCRs), which are canonically coupled to the
adenylyl cyclase pathway.5 Recent studies have identified 125
candidate receptor partners, making the 5-HT6R one of the
GPCRs with the most extensively characterized interactome.6,7
In addition to the canonical Gs-adenylyl cyclase signaling
pathway, the 5-HT6R has been linked to cellular signaling
cascades involved in cognitive processes and neurogenesis, such
as mammalian target of rapamycin (mTOR) and cyclin-
dependent kinase 5 (Cdk5) pathways. Indeed, enhanced
Received: February 1, 2021
Accepted: February 24, 2021
Published: March 11, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. Replacement of 1H-pyrrolo[3,2-c]quinoline scaffold with 2-phenyl-1H-pyrrole-3-carboxamide and structural functionalization providing the
target compounds.
Scheme 1. General Synthetic Route for the Preparation of Final Compounds 7−32
The highest expression of 5-HT6Rs is found in the central
nervous system regions involved in mnemonic functions such as
the hippocampus, striatum, nucleus accumbens, and prefrontal
cortex. Further lines of evidence demonstrated the expression of
the 5-HT6R in the primary cilium, highlighting the involvement
of the 5-HT6R in neuronal morphology.14,15 The cognitive-
enhancing properties result from the blockade of 5-HT6Rs
located on GABAergic neurons and promotion of corticolimbic
release of acetylcholine and glutamate.16
Our recent interest in 5-HT6R ligands has culminated in the
identification of (S)-1-[(3-chlorophenyl)sulfonyl]-4-(pyrroli-
dine-3-yl-amino)-1H-pyrrolo[3,2-c]quinoline (CPPQ), a pyr-
roloquinoline-derived potent and selective 5-HT6R neutral
antagonist.17,18 Inspired by the activity of CPPQ,9,13 we applied
the scaffold-hopping approach to replace the planar 1H-
pyrrolo[3,2-c]quinoline skeleton with a more flexible 2-
phenyl-1H-pyrrole-3-carboxamide in order to investigate the
effect of a central core retraction on 5-HT6R affinity and
functional activity. The chemical diversity around the new
framework involved (i) introduction of a fluorine atom at the 2-
phenyl fragment (R1), (ii) functionalization of N1 pyrrole with
aryl- or heteroarylsulfonyl moieties (R2), and (iii) changing
configuration at the 3-aminopyrrolidinyl moiety at the 3-
carboxamide fragment (Figure 1).
conditions and whether it could facilitate cognitive flexibility
in the attentional set-shifting task (ASST) in rats.
2. CHEMISTRY
The general synthetic route used to prepare final compounds 7−
32 is summarized in Scheme 1. By use of our previously
optimized procedures,19,20 intermediates 1−4 were synthesized
via a four-step sequence involving (i) aza-Baylis−Hillman
reaction, (ii) N-allylation, (iii) ring-closing metathesis, and
(iv) removal of the tosyl group with simultaneous aromatization.
Next, saponification of the esters 4a−c in a refluxing aqueous
solution of NaOH furnished the carboxylic acids 5a−c. Amide
coupling with (R) or (S) 1-Boc-3-aminopyrrolidine was then
performed using 1-hydroxybenzotriazole (HOBt) and benzo-
triazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP) as a carboxyl group activating agents. The
obtained 2-aryl-1H-pyrrole-3-carboxamides 6a−f were subse-
quently reacted with various arylsulfonyl chlorides in the
presence of a phosphazene base P1-t-Bu-tris(tetramethylene)
(BTPP). Finally, Boc-deprotection using methanolic solution of
HCl resulted in target compounds 7−32 as corresponding
hydrochloride salts.
3. RESULTS AND DISCUSSION
The present manuscript reports on the identification of a
promising arylsulfonamide of 2-phenylpyrrole-3-carboxamide
analogue (27) which displays inverse agonist properties at
5-HT6R-operated Gs and Cdk5 signaling pathways. We also
examined whether 27 is distributed to the brain and could
reverse cognitive impairments in the novel object recognition
(NOR) test under scopolamine-induced memory decline
3.1. Structure−Activity Relationship (SAR) Studies. To
reveal common pharmacophore features for 5-HT6R antago-
nists,21 2-phenyl-1H-pyrrole-3-carboxamide was used as an
aromatic ring hydrophobic site instead of the previously used
1H-pyrrolo[3,2-c]quinoline scaffold. The applied new frame-
work opened the possibility of introducing the second
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Figure 2. (A) Comparison of the binding mode of 18 (yellow) with 28 (orange) and CPPQ (green). (B) Comparison of the binding mode of 24
(magenta), 27 (cyan), and 28 (orange).
hydrophobic site linked to the central core by a sulfonyl group at
the N1 position of pyrrole as a hydrogen bond acceptor and an
alicyclic amine in the 3-carboxamide fragment providing a
positively ionizable atom.
Table 1. Binding Data of Synthesized Compounds 7−32 and
Reference for the 5-HT6R
Final compounds were evaluated in the [3H]-LSD binding
assay using HEK293 cells with stable expression of human
5-HT6R. The SAR studies initially revealed that the degradation
of the 1H-pyrrolo[3,2-c]quinoline central core to 2-phenyl-1H-
pyrrole-3-carboxamide significantly decreased the affinity of new
derivatives 7 and 8 for 5-HT6R (I, Ki = 10 nM vs 7, Ki = 208 nM;
8, Ki = 106 nM) (Figure 1). Because the fluorine atom might
affect the affinity at target protein as well as physicochemical and
pharmacokinetic properties of compound,22 fluorine substitu-
tion at the 2-phenyl ring was applied. The introduction of
fluorine in the C4 position (R1) maintained (17 vs 27; 18 vs 28)
or slightly increased (7 vs 24; 12 vs 26) the affinity for 5-HT6R,
while the C3 position was less preferable (21 vs 17 and 27). The
analysis of the binding mode of 18 and 28 (Figure 2A)
confirmed that the 2-phenyl moiety is placed in the hydrophobic
cavity between transmembrane domains (TMs) 4−6.
a
compd
R1
R2
R/S Ki [nM], 5-HT6R
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3-F
3-F
3-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
Ph
Ph
2-CH3-Ph
2-Cl-Ph
3-CH3-Ph
3-F-Ph
R
S
208
106
324
179
96
88
159
38
52
31
35
25
R
R
R
R
R
S
R
S
R
S
S
S
R
S
R
R
R
R
R
S
R
S
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
3-CF3-Ph
3-CF3-Ph
3-OCF3-Ph
3-OCF3-Ph
3-Cl-Ph
3-Cl-Ph
thien-2-yl
Considering our previously reported data,17,23,24 which
indicate that structural functionalization of the arylsulfonamide
fragment (R2) highly impacts the affinity for the 5-HT6R, diverse
substitution, i.e., halogen atom, alkyl, or alkoxyl moieties, was
investigated. Generally, substitution in the C3 position at the
phenylsulfonyl ring was beneficial for interaction with the
5-HT6R. Introduction of electron-withdrawing substituents
such as fluorine, chlorine, trifluoromethyl, or the bulky
trifluoromethoxy group significantly increased the affinity for
5-HT6R (24 vs 26; 8 vs 18; 8 vs 14; 8 vs 16; respectively).
Among electron-withdrawing substituents, chlorine was the
most privileged one, increasing the affinity for the 5-HT6R up to
6-fold (7 vs 17; 8 vs 18). This may result from the fact that
halogen atoms stabilize the ligand−receptor complex by forming
additional interactions and thus improve the affinity for 5-
HT6Rs.25 Indeed, the analysis of the binding modes (Figure 2B)
revealed that chlorine atom in C3 position at the phenylsulfonyl
ring may form halogen bonding contacts with the carbonyl
oxygen of A4.56 and S4.57, which were not detected for
unsubstituted derivatives (27 vs 24). Interestingly, these amino
acids were also indicated as halogen bonding hot spots in other
classes of 5-HT6R ligands.26,27 On the other hand, the
introduction of electron-donating substituents was less favorable
for interaction with the 5-HT6R (24 vs 25), while a small methyl
substituent was tolerated (11, Ki = 96 nM). The shifting of a
chlorine atom or a methyl group from the C3 to C2 position
decreased the affinity for the 5-HT6R (10 vs 17; 9 vs 11).
162
1463
98
1-methyl-1H-pyrazol-4-yl
3-Cl-Ph
3-Cl-Ph
4-F-Ph
Ph
28
853
102
350
56
30
22
2577
638
391
1210
10
3-OCH3-Ph
3-F-Ph
3-Cl-Ph
3-Cl-Ph
4-F-Ph
4-F-Ph
naphth-1-yl
quinol-8-yl
S
b
I
b
CPPQ
3
a
Mean Ki values (SEM 19%) based on three independent binding
experiments. Data taken from ref 17, where I is encoded as 9, and
b
CPPQ is encoded as 14.
Surprisingly, a loss of affinity for the 5-HT6R was observed when
the fluorine atom was moved from the C3 to C4 position (26 vs
29). Finally, the replacement of the phenyl fragment with five-
membered heterocyclic moieties, namely, thien-2-yl (19) and 1-
methyl-1H-pyrazol-4-yl (20) or bicyclic naphth-1-yl (31) and
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quinol-8-yl (32), was not suitable for interaction with the 5-
HT6R (Table 1).
Table 3. Antagonist Property of Selected Compounds in
1321N1 Cells and Their Functional Activity at 5-HT6R-
Dependent Gs Signaling in NG108-5 Cells
Because the stereochemical properties of compounds might
modify the affinity for the 5-HT6R, both enantiomers of the
pyrrolidin-3-yl fragment were investigated. With regard to their
binding with target protein, a preference for the S enantiomers
over its R counterparts was observed (Table 1). A comparison of
the binding mode of 27 and 28 (Figure 2B) demonstrated
modest differences in the orientation of the basic group, while
both enantiomers created a strong salt bridge with D3.32.
Molecular docking was also used to study the binding mode of
newly synthesized 2-phenyl-1H-pyrrole-3-carboxamides using
CPPQ as a reference compound.17 Generally, the binding mode
was coherent and showed a similar interaction pattern to CPPQ;
i.e., the protonated fragment containing basic nitrogen formed a
salt bridge with D3.32, the aromatic scaffold created CH−π
interaction with F6.52/F6.51, and the terminal substituted
phenyl ring expanded into a hydrophobic cavity formed by TMs
3, 4, and 5 and the extracellular loop 2 (ECL2). Comparison of
the binding modes of 18 and 28 with CPPQ (Figure 2A)
indicates that the phenyl moiety of the central core is twisted
compared to the pyrroloquinoline system of CPPQ so that
interaction with the aromatic cluster F6.52/F6.51 is further
stabilized.
5-HT6R
5-HT6R
Gs signaling
c
a
b
compd
18
22
Ki [nM]
Kb [nM]
IC50 [nM]
functional profile
25
28
30
22
3
35.0
6.0
11.4
7.6
170
306
265
140
inverse agonist
inverse agonist
inverse agonist
inverse agonist
neutral antagonist
inverse agonist
27
28
CPPQ
d
0.41
1.95
e
e
SB-271046
1.2
98
a
Mean Ki values (SEM 19%) based on three independent binding
experiments. Mean Kb values (SEM
independent experiments in 1321N1 cells. Mean IC50 values (SEM
18%) obtained in three independent experiments in NG108-15 cells.
b
15%) obtained in three
c
d
e
Data taken from ref 17, where CPPQ is encoded as 14. Data taken
from ref 31.
operated Gs signaling. Experiments were performed in NG108-
15 cells transiently expressing recombinant receptors, a cellular
model exhibiting constitutively active 5-HT6R. In contrast to
CPPQ, a reference neutral antagonist,13,17,32 all the tested
compounds decreased basal cAMP level in a concentration-
dependent manner and behaved as 5-HT6R inverse agonists
(Figure 3). Additionally, the evaluated compounds showed
3.2. Selectivity Profiles of Selected Compounds. The
most potent compounds (Ki ≤ 30 nM) exhibiting structural (18,
22, 28) and enantiomeric (27, 28) diversity were further
evaluated for their selectivity over serotonin receptors
(5-HT1AR, 5-HT2AR, 5-HT7R) and dopamine D2 receptor
(D2R) in radioligand binding assays. The tested compounds
displayed high selectivity over 5-HT1A, 5-HT2A, 5-HT7, and D2
receptors (Table 2). In contrast to the reference, intepirdine, the
Table 2. Binding Data of Selected Compounds 18, 22, 27, 28
and Intepirdine for 5-HT6, 5-HT1A, 5-HT2A, 5-HT7, and D2
Receptors
Figure 3. Impact of compounds 18, 22, 27, and 28 and SB-271046 on
basal cAMP production in NG108-15 cells transiently expressing 5-
HT6Rs. Data are the mean SEM of the values obtained in three
independent experiments performed in quadruplicate in different sets
of cultured cells: ***P < 0.001 vs vehicle (ANOVA followed by
Student−Newman−Keuls test).
a
Ki [nM]
compd
5-HT6R
5-HT1A
R
5-HT2A
R
5-HT7R
D2R
18
22
27
28
25
28
30
22
1.4
1366
65680
53670
61490
2370
14610
15180
13470
8565
26
65520
7063
42000
3528
6843
6659
7080
7336
997
b
intepirdine
14230
potency similar to that of SB-271046, the reference 5-HT6R
inverse agonist (Table 3). Thus, it may be concluded that the
replacement of the 1H-pyrrolo[3,2-c]quinoline scaffold with the
2-phenyl-1H-pyrrole-3-carboxamide moiety shifted the func-
tional activity of compounds at the Gs signaling pathway from
neutral antagonism to inverse agonism and allowed us to target
different active states of the 5-HT6R.
In addition to the canonical Gs-adenylyl cyclase pathway, the
5-HT6R activates Cdk5 signaling in an agonist-independent
manner. Preventing this activation with inverse agonists inhibits
neurite growth.10 Compound 28 which possesses the most
potent inverse agonist properties at Gs signaling and its
enantiomer 27 were examined to assess their effect on Cdk5-
dependent neurite growth. As shown in Figure 4, transfection of
the 5-HT6R in NG108-15 cells results in a significant increase in
neurite length as compared to green fluorescent protein (GFP)
transfected cells. Both compounds 27 and 28, applied at a
concentration of 10 nM, significantly reduced neurite length in
NG108-15 cells in the same way as our reference SB-271046
(with decreases of 36.9%, 57.5%, and 49.6%, respectively). Thus,
a
Mean Ki values (SEM 27%) based on three independent binding
b
experiments. Data taken from ref 30.
selected compounds did not show any significant affinity for 5-
HT2AR. Thus, they might be devoid of the side effects associated
with the modulation of 5-HT2AR, such as hallucinations,
psychosis, fear, hypotension, headache, and dizziness.28,29
3.3. Functional Profiles of Selected Compounds. The
effect of compounds 18, 22, 27, and 28 on adenylate cyclase was
examined in 1321N1 cells expressing the 5-HT6R. Regardless of
the type of substituent at the 2-phenyl-1H-pyrrole moiety and
spatial isomerism, all of the tested compounds inhibited the 5-
carboxamidotryptamine (5-CT)-stimulated cAMP accumula-
tion and thus were classified as 5-HT6R antagonists (Kb = 6−35
nM) (Table 3).
As the high level of 5-HT6R constitutive activity was observed
in in vitro and in vivo settings,12,13 we subsequently evaluated
whether the representatives of 2-phenyl-1H-pyrrole-3-carbox-
amides (18, 22, 27, 28) modulate agonist-independent 5-HT6R-
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Subsequently, the pharmacokinetic profile of 27 and 28 was
assessed in male Wistar rats after single intragastric admin-
istration at the dose of 10 mg/kg. The studied compounds were
rapidly absorbed and crossed the blood−brain barrier, reaching
the Cmax in 5 min in both plasma and brain. Compound 27
showed higher distribution in the systemic circulation, with the
brain/plasma ratio of 0.63 (Table 4). Although 28, the S
enantiomer of 27, showed slightly higher affinity and
antagonism at Gs signaling for the 5-HT6R in in vitro assays,
the in vivo pharmacokinetic profile precluded its further
development.
3.5. Extended in Vitro Off-Target Selectivity and
Safety Profile Assessment for Compound 27. Compound
27 was subsequently evaluated for its affinity toward several off-
targets. It was found that 27 did not bind to α1A adrenoreceptor
(15% at 1 μM), M1 muscarinic (0% at 1 μM), H1 histaminic (9%
at 1 μM), D3 dopamine (17% at 1 μM), and serotonin 5-HT2C
(10% at 1 μM) and 5-HT3 (2% at 1 μM) receptors; it also did
not exhibit affinity for the serotonin transporter (SERT) (10% at
1 μM) and the human ether-a-go-go-related gene (hERG)
channel (1% at 1 μM). Therefore, compound 27 should not
induce adverse effects associated with the above targets, i.e.,
convulsions, anxiety, psychosis, hypotension, cardiac arrhyth-
mia, nausea, and vomiting.
Considering a potential drug−drug interaction, 27 was further
evaluated for its inhibitory activity on human cytochrome P450
(CYP450). Although compound 27 decreased the activity of
CYP3A4 subtype (IC50 = 69 nM), it did not significantly
influence CYP2D6. Since drug-induced liver injuries constitute a
clinical issue, compound 27 was tested in the human
hepatocellular carcinoma (HepG2) model to exclude its
hepatotoxicity. Compound 27 did not impair the metabolic
activity of cells assessed in the MTT test and did not affect the
cell membrane integrity as determined in the LDH test in a wide
range of concentrations (0.1−25 μM) (Figure 5). Finally,
compound 27 was subjected to the Ames test to assess its
potential to induce mutation in genes involved in histidine
synthesis. Compound 27 was found to be non-mutagenic using
Salmonella typhimurium TA10.
Figure 4. Impact of compounds 27 and 28 on Cdk5 signaling pathway.
NG108-15 cells were transfected with a plasmid encoding a GFP-
tagged 5-HT6 receptor or GFP alone. Cells expressing the receptor were
exposed to DMSO (control), compounds 27 and 28, and SB-271046
(10−8 M) for 24 h. The histogram shows the mean SEM of neurite
length for each experimental condition measured from three
independent experiments: ***P < 0.001 vs cells expressing GFP;
ANOVA followed by Student−Newman−Keuls test.
these compounds might be regarded as full inverse agonists at
agonist-independent 5-HT6R-mediated Cdk5 signaling. Of
note, compound 27 displayed more significant impact on
Cdk5 signaling compared to 28 and SB-271046 (Figure 4).
3.4. Pharmacokinetics Evaluation. Preliminary ADME
and pharmacokinetics assessment play a crucial role in the
optimization process of a preclinical lead compound. Thus,
compounds 27 and 28 were further subjected to biotransforma-
tion studies using rat liver microsomes (RLMs). The tested
compounds showed a low value of intrinsic clearance (0.82 μL/
min/mg and 1.03 μL/min/mg for 27 and 28, respectively),
indicating high metabolic stability.
a
Table 4. Pharmacokinetic Parameters for 27 and 28
27
28
parameter
plasma
brain
plasma
brain
AUC0→t [ng·min/mL]
MRT [min]
77251
163.3
117.5
1039.12
5
48727
180.9
250.9
427.1
5
203547
200.8
297
3498.7
5
17335
266
t
C
t
0.5 [min]
max [ng/mL][ng/g]
max [min]
b
51.7
5
a
Measured after p.o. administration of dose 10 mg/kg: t0.5, terminal
3.6. Behavioral Evaluation of the Procognitive Effects
of Compound 27. 5-HT6R antagonists, which behave as
neutral antagonists or inverse agonists, enhanced cognitive
performance in animal models.17,33,34 Thus, the effect of
half-life; AUC, area under the curve; MRT, mean residence time; Cmax
maximum concentration; tmax, time to reach the maximum
b
concentration. Concentration in brain. N = 64.
Figure 5. Effect of compound 27 on hepatotoxicity in HepG2 cellular model. Cells were cultured in the presence of compound 27 for 24 h.
Doxorubicin (DOX) was used as reference standard. (A) MTT assay was performed to measure cellular viability (mitochondria metabolism rate). The
log dose−response curves were generated in GraphPad Prism 7. Data represent the mean SD of three repeats. (B) LDH assay was used to study the
cytotoxicity of 27. Graph represents (percent of cells cytotoxicity) − (% of LDH release to culture medium compared to control condition) incubated
with 27 in concentrations 0.1−150 μM. Data represent the mean SD of two repeats: *P < 0.05 (Mann−Whitney U test).
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compound 27 on short-term memory was investigated in the
NOR test in rats treated with scopolamine.35−37 As expected,
scopolamine-treated but not vehicle-treated rats spent signifi-
cantly less time exploring the novel object than the familiar one,
indicating that scopolamine abolished the ability to discriminate
novel and familiar objects. Administration of scopolamine
blocks muscarinic acetylcholine receptors and thus serves as a
pharmacological model of cognitive decline. Compound 27
given p.o. at the dose of 6, but not 3 mg/kg, prevented
scopolamine-induced cognitive deficits in the NOR test (Figure
6).
(6 mg/kg) was effective only during the Rev2 phase (Figure 7,
upper panel). Additionally, compound 27 did not affect the
mean time to complete the trial during any of the discrimination
stages (Figure 7, lower panel).
Figure 6. Effects of compound 27 (3 and 6 mg/kg, p.o.) on
scopolamine-induced cognitive impairment in the novel object
recognition test in rats. Data are presented as the mean SEM of
discrimination index (DI). For vehicle, scopolamine, 27 (3 mg/kg) +
scopolamine, and 27 (6 mg/kg) + scopolamine, we used 7, 7, 6, and 8
rats, respectively, summing to 28 animals: *P < 0.05; ***P < 0.001 vs
vehicle; #P < 0.05 vs scopolamine (Tukey’s multiple comparisons post-
hoc test, following one-way ANOVA, F(3,24) = 7.30, P = 0.0012).
Figure 7. Effects of compound 27 (6 and 9 mg/kg, p.o.) in attentional
set shifting test (ASST). Data are presented as the mean SEM of the
trials to criterion (upper panel) and time to complete the trial during
discrimination stages (lower panel). For 27 used at the doses of 0, 6, and
9 mg/kg, there were 11, 10, and 10 rats, respectively, per group,
summing to 31 animals: *P < 0.05 vs respective vehicle; #P < 0.05 vs
vehicle at the intradimensional (ID) stage (Newman−Keuls post-hoc
test, following significant two-way ANOVA’s stage × treatment
interaction: F(12,168) = 1.95, P = 0.031). For the time to complete
the trial during discrimination stages data (lower panel) two-way
ANOVA’s stage × treatment interaction yielded insignificant results:
F(12,168) = 0.945, NS.
Neuropsychiatric and neurodegenerative disorders are
associated with cognitive impairments, including cognitive
inflexibility, resulting in deficient adaptation to the changing
conditions. These impairments are often reported in schizo-
phrenic patients and in subjects with lesions of the prefrontal
cortex.38 The effects of compound 27 were further examined in
the ASST. This test consists of a series of two-choice
discriminations, in which one of the two stimulus dimensions
(e.g., the material covering the bait) is relevant and the other
dimension (e.g., an odor applied to the bait container) is
irrelevant for successful discrimination. The animals are
required to learn the currently relevant rule and must maintain
the application of that specific rule to a novel set of stimuli. This,
in turn, leads to the formation of an attentional set that can be
seen as “locking” the subject within an initially relevant
dimension. In the crucial extra-dimensional (ED) stage, the
animals must switch their attention to previously irrelevant
stimulus dimension; for instance, they have to discriminate
between the odors instead of the materials covering the food
reward. The animal’s performance during the ED stage is
regarded as an index of cognitive flexibility. Additionally, reversal
learning stages examine rats’ ability to adjust responses following
a change in the stimuli signifying food reward. Reduction in the
number of trials to criterion at the reversal trials and at the ED
stage in particular suggests an improvement in cognitive
flexibility.39 Compound 27 administered at the dose of 9 mg/
kg p.o. enhanced cognitive flexibility as indicated by a decreased
number of trials to criterion during the ED and three reversal
stages (Rev1, Rev2, and Rev3). The lower dose of compound 27
4. CONCLUSIONS
The scaffold-hopping approach around pyrroloquinoline
derivatives was applied to design 2-phenyl-1H-pyrrole-3-
carboxamides as a new structural framework for developing
5-HT6R antagonists. SAR studies revealed the subsequent
structural requirements for high affinity for the 5-HT6R, i.e., a
fluorine atom in the C4 position at the 2-phenylpyrrole fragment,
a 3-chlorophenylsulfonyl moiety in position N1, and pyrrolidine
as a basic center. The in vitro functional activity evaluation for
5-HT6R-operated Gs and Cdk5 signaling allowed classification
of the identified compounds as 5-HT6R inverse agonists. This
observation is in sharp contrast to the prototypic pyrroloquino-
line-based compound CPPQ, which behaved as a neutral
antagonist. Selected compound 27 showed good brain
distribution, no cytotoxicity, and no mutagenic activity.
Therefore, it might be placed in low-risk safety space. The
cognition-enhancing properties of 27 were subsequently
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5.1.5. Characterization Data for Selected Final Compounds.
5.1.5.1. (S)-2-Phenyl-1-[(3-chlorophenyl)sulfonyl]-N-(pyrrolidin-3-
yl)-1H-pyrrole-3-carboxamide (18). Boc-derivative: colorless oil,
0.12 g (yield 64%) after chromatographic purification over silica gel
with EtOAc/Hex (7/3, v/v); UPLC/MS purity 98%, tR = 7.74,
C26H28ClN3O5S, MW 530.04, monoisotopic mass 529.14, [M + H]+
530.1.
Hydrochloride: white solid, 0.07 g (yield 66%); UPLC/MS purity
100%, tR = 4.80, C21H21Cl2N3O3S, MW 466.38. 1H NMR (500 MHz,
CD3OD) δ 1.72−1.81 (m, 1H), 2.09−2.18 (m, 1H), 3.02 (dd, J = 12.3,
4.9 Hz, 1H), 3.16−3.27 (m, 2H), 3.36 (dd, J = 12.3, 7.2 Hz, 1H), 4.25−
4.31 (m, 1H), 6.75 (d, J = 3.4 Hz, 1H), 7.01−7.05 (m, 2H), 7.12 (t, J =
2.0 Hz, 1H), 7.28−7.34 (m, 3H), 7.39−7.46 (m, 2H), 7.57 (d, J = 3.4
Hz, 1H), 7.60−7.64 (m, 1H). 13C NMR (126 MHz, CD3OD) δ 29.5,
44.3, 48.9, 49.5, 110.5, 122.5, 122.9, 125.6, 127.3, 127.4, 129.0, 129.2,
130.9, 131.9, 134.4, 134.8, 135.5, 139.5, 165.1, monoisotopic mass
429.09, [M + H]+ 430.1; HRMS calculated for C21H20ClN3O3S
429.0914; found 430.1000.
demonstrated in the NOR test (6 mg/kg, p.o.) in scopolamine-
induced memory decline conditions. Compound 27 also
increased the cognitive flexibility mediated by the prefrontal
cortex in the ASST in rats (9 mg/kg, p.o.). In view of the above
findings, compound 27 might be regarded as a probe to study
the contribution of agonist-independent 5-HT6R activity to
neurological and neurodegenerative disorders.
5. EXPERIMENTAL METHODS
5.1. Chemistry. 5.1.1. 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 purification. All reagents (Sigma-Aldrich,
Fluorochem, Across, and TCI) were of the highest purity. Column
chromatography was performed using silica gel Merck 60 (70−230
mesh ASTM).
Mass spectra were recorded on a UPLC−MS/MS system consisting
of a Waters ACQUITY UPLC (Waters Corporation, Milford, MA,
USA) coupled to a Waters TQD mass spectrometer. All the analyses
were carried out using an Acquity UPLC BEH C18 100 × 2.1 mm2
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% HCOOH; eluent
B, acetonitrile/0.1% HCOOH. Retention times, tR, were given in
minutes. The UPLC/MS purity of all the test compounds and key
intermediates was determined to be >98%. HRMS analyses were
performed on an UPLC Acquity H-class from Waters hyphenated to a
Synapt G2-S mass spectrometer with a dual ESI source from Waters.
1H NMR and 13C NMR spectra were recorded at 300 and 75 MHz
(Varian BB 200), 400 and 101 MHz (Bruker Avance III), or 500 and
126 MHz (JEOL JNM-ECZR500 RS1) using CDCl3 or CD3OD as
solvents. Chemical shifts are given in ppm. The J values are reported in
hertz (Hz), and the splitting patterns are designated as follows: br s
(broad singlet), s (singlet), d (doublet), t (triplet), q (quartet), dd
(doublet of doublets), dt (doublet of triplets), td (triplet of doublets),
ddd (doublet of doublets of doublets), m (multiplet).
5.1.5.2. (S)-2-(3-Fluorophenyl)-1-[(3-chlorophenyl)sulfonyl)-N-
(pyrrolidin-3-yl)-1H-pyrrole-3-carboxamide (22). Boc-derivative: col-
orless oil, 0.10 g (yield 68%) after chromatographic purification over
silica gel with EtOAc/Hex (6/4, v/v); UPLC/MS purity 99%, tR = 7.97,
C26H27ClFN3O5S, MW 548.03, monoisotopic mass 547.13, [M + H]+
548.1.
Hydrochloride: white solid, 0.18 g (yield 76%), UPLC/MS purity
100%, tR = 5.06, C21H20Cl2FN3O3S, MW 484.37. 1H NMR (500 MHz,
CD3OD) δ 1.84−1.92 (m, 1H), 2.15−2.24 (m, 1H), 3.08 (dd, J = 12.2,
5.0 Hz, 1H), 3.19−3.27 (m, 1H), 3.31−3.42 (m, 2H), 4.26−4.33 (m,
1H), 6.74−6.79 (m, 2H), 6.82 (dt, J = 7.6, 1.1 Hz, 1H), 7.15−7.21 (m,
1H), 7.22 (t, J = 1.9 Hz, 1H), 7.31 (td, J = 7.9, 6.0 Hz, 1H), 7.35−7.38
(m, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.60 (d, J = 3.7 Hz, 1H), 7.64−7.68
(m, 1H). 13C NMR (126 MHz, CD3OD) δ 29.4, 44.4, 49.0, 49.5, 110.3,
115.9 (d, J = 21.7 Hz), 118.8 (d, J = 22.9 Hz), 122.8, 123.0, 125.6, 127.3,
127.8 (d, J = 3.0 Hz), 128.9 (d, J = 8.5 Hz), 131.0, 131.2, 134.1, 134.5,
134.9, 139.4, 161.8 (d, J = 245.1 Hz), 164.8, monoisotopic mass 447.08,
[M + H]+ 448.1; HRMS calculated for C21H19ClFN3O3S 448.0820;
found 448.0898.
The synthetic procedures for intermediates 5 and 6 and final
compounds (7−32) as well as characterization data for selected final
compounds (18, 22, 27, 28) are presented below. The synthesis of
intermediates 1−4 was performed according to the previously
described procedures19,20 and is reported in the Supporting
Information together with spectroscopic data for all intermediates
and remaining final compounds.
5.1.2. General Procedure for Ester Hydrolysis (5a−c). The
appropriate methyl ester derivative 4 (1 equiv) was heated under
reflux with the excess of 10% aqueous solution of NaOH for 3h. Then,
10% aqueous solution of HCl was added portionwise to acidic pH. The
mixture was diluted with EtOAc and washed three times with water and
once with brine. The organic layer was dried with Na2SO4, evaporated,
and dried under vacuum.
5.1.3. General Procedure for Amide Coupling (6a−f). The
carboxylic acid 5 (1 equiv) was added to a solution of 1-
hydroxybenzotriazole (HOBT) (1.2 equiv) and benzotriazole-1-yl-
oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP)
(1.2 equiv) in DMF, and the mixture was stirred for 30 min with
triethylamine (3 equiv). Then, (R) or (S) enantiomer of 1-Boc-3-
aminopyrrolidine (1.2 equiv) was added and left to react overnight. The
mixture was diluted with EtOAc and washed with H2O and brine, dried
(Na2SO4), concentrated, and purified over silica gel.
5.1.4. General Procedure for the Synthesis of Final Compounds
7−32. The synthesized amide 6 (1 equiv) was dissolved in CH2Cl2, and
phosphazene base P1-t-Bu-tris(tetramethylene) (BTPP) (1.2 equiv)
was added. The mixture was cooled down (ice bath), appropriate
sulfonyl chloride (1.2 equiv) was added, and the reaction mixture was
stirred for 3 h. Subsequently, the mixture was evaporated and the
remaining crude product was purified on silica gel. Then, the free bases
were dissolved in anhydrous ethanol and treated with 1.25 M
methanolic HCl to give the final products as hydrochloride salts after
evaporation.
5.1.5.3 (R)-2-(4-Fluorophenyl)-1-[(3-chlorophenyl)sulfonyl]-N-
(pyrrolidin-3-yl)-1H-pyrrole-3-carboxamide (27). Boc-derivative: col-
orless oil, 0.12 g (yield 77%) after chromatographic purification over
silica gel with EtOAc/Hex (6/4, v/v); UPLC/MS purity 100%, tR =
7.92, C26H27ClFN3O5S, MW 548.03, monoisotopic mass 547.13, [M +
H]+ 548.1.
Hydrochloride: white solid, 0.12 g (yield 80%), UPLC/MS purity
99%, tR = 4.93, C21H20Cl2FN3O3S, MW 484.37. 1H NMR (500 MHz,
CD3OD) δ 1.85−1.94 (m, 1H), 2.17−2.26 (m, 1H), 3.11 (dd, J = 12.2,
5.0 Hz, 1H), 3.22−3.29 (m, 1H), 3.33−3.44 (m, 2H), 4.30−4.38 (m,
1H), 6.81 (d, J = 3.7 Hz, 1H), 7.06 (d, J = 7.2 Hz, 4H), 7.18 (t, J = 1.7
Hz, 1H), 7.36−7.39 (m, 1H), 7.47 (t, J = 8.2 Hz, 1H), 7.59 (d, J = 3.4
Hz, 1H), 7.67 (ddd, J = 8.1, 2.1, 1.0 Hz, 1H). 13C NMR (126 MHz,
CD3OD) δ 29.5, 44.3, 48.9, 49.5, 110.4, 114.2 (d, J = 21.7 Hz), 122.6,
123.0, 125.1 (d, J = 3.6 Hz), 125.5, 127.3, 131.0, 134.0 (d, J = 9.0 Hz),
134.5, 134.6, 134.9, 139.4, 163.4 (d, J = 250.5 Hz, 164.9, monoisotopic
mass 447.08, [M + H]+ 448.1; HRMS calculated for C21H19ClFN3O3S
448.0820; found 448.0896.
5.1.5.4. (S)-2-(4-Fluorophenyl)-1-[(3-chlorophenyl)sulfonyl]-N-
(pyrrolidin-3-yl)-1H-pyrrole-3-carboxamide (28). Boc-derivative: col-
orless oil, 0.451 g (yield 61%) after chromatographic purification over
silica gel with EtOAc/Hex (6/4, v/v); UPLC/MS purity 100%, tR =
7.91, C26H27ClFN3O5S, MW 548.03, monoisotopic mass 547.13, [M +
H]+ 548.1.
Hydrochloride: white solid, 0.321 g (yield 83%), UPLC/MS purity
100%, tR = 4.93, C21H20Cl2FN3O3S, MW 484.37. 1H NMR (500 MHz,
CD3OD) δ 1.85−1.93 (m, 1H), 2.17−2.26 (m, 1H), 3.11 (dd, J = 12.2,
5.0 Hz, 1H), 3.22−3.29 (m, 1H), 3.33−3.44 (m, 2H), 4.31−4.37 (m,
1H), 6.81 (d, J = 3.7 Hz, 1H), 7.07 (d, J = 7.2 Hz, 4H), 7.19 (t, J = 2.0
Hz, 1H), 7.36−7.39 (m, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.60 (d, J = 3.4
Hz, 1H), 7.67 (ddd, J = 8.0, 3.4, 0.9 Hz, 1H). 13C NMR (126 MHz,
CD3OD) δ 29.5, 44.3, 48.9, 49.5, 110.4, 114.2 (d, J = 22.3 Hz), 122.60,
123.0, 125.1 (d, J = 3.6 Hz), 125.6, 127.3, 131.0, 134.0 (d, J = 8.4 Hz),
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134.5, 134.6, 134.9, 139.4, 163.4 (d, J = 249.9 Hz), 164.9, monoisotopic
mass 447.08, [M + H]+ 448.1; HRMS calculated for C21H19ClFN3O3S
448.0820; found 448.0898.
5.2. Molecular Docking. The 5-HT6R homology models were
obtained according to the procedure described before on the β2
receptor template (PDB code 4LDE) and successfully used in our
earlier studies of different groups of 5-HT6R ligands simulations.24,40
The three-dimensional structures of the ligands were prepared using
with a mixture of compounds (5 μL) for 30 min at room temperature in
384-well white opaque microtiter plate. After incubation, the reaction
was stopped and cells were lysed by the addition of 10 μL of working
solution (5 μL of Eu-cAMP and 5 μL of ULight-anti-cAMP) 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 equation specific for the
analysis of functional inhibition curves: Kb = IC50/(1 + A/EC50) where
A represents agonist concentration, IC50 the concentration of
antagonist producing a 50% reduction in the response to agonist, and
EC50 the agonist concentration which causes a half of the maximal
response.44
5.3.3. Determination of cAMP Production in NG108-15 Cells.
NG108-15 cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% dialyzed fetal calf serum, 2%
hypoxanthine/aminopterin/thymidine (Life Technologies), and anti-
biotics. cAMP measurement was performed in cells transiently
expressing 5-HT6R using the bioluminescence resonance energy
transfer (BRET) sensor for cAMP, CAMYEL (cAMP sensor using
YFP-Epac-RLuc).45 NG108-15 cells were cotransfected in suspension
with 5-HT6R (0.5 μg DNA/million cells) and CAMYEL constructs (1
μg DNA/million cells), using Lipofectamine 2000 according to the
manufacturer’s protocol, and plated in white 96-well plates (Greiner) at
a density of 50 000 cells per well. After 24 h of transfection, cells were
washed with PBS containing calcium and magnesium. To test the
inverse agonist properties of compounds 18, 22, 27, 28 and SB-271046,
cells were treated with vehicle or with the tested compound at a
concentration ranging from 0.1 nM to 10 μM. 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). Expression of 5-HT6R in
NG108-15 cells induced a strong decrease in CAMYEL BRET signal,
compared with cells transfected with an empty vector instead of the
plasmid encoding the 5-HT6R. This decrease in CAMYEL BRET signal
was thus used as an index of 5-HT6R constitutive activity at Gs
signaling.
5.3.4. Impact of Compounds on Neurite Growth. NG108-15 cells
were transfected with plasmids encoding either cytosolic GFP or a
GFP-tagged 5-HT6R in suspension using Lipofectamine 2000 (Life
Technologies) and plated on glass coverslips. Six hours after
transfection, cells were treated with either DMSO (control), 27, and
28 or SB-271046 (10−8 M) for 24 h. Cells were fixed in 4%
paraformaldehyde (PFA) supplemented with 4% sucrose for 10 min.
PFA fluorescence was quenched by incubating the cells in PBS
containing 0.1 M glycine, prior to mounting in Prolong Gold antifade
reagent (Thermo Fisher Scientific). Cells were imaged using an
AxioImagerZ1 microscope equipped with epifluorescence (Zeiss),
using a 20× objective for cultured cells, and neurite length was assessed
using the Neuron J plugin of the ImageJ software (NIH).
5.4. Pharmacokinetics Evaluation. 5.4.1. In Vitro Metabolic
Stability Study. Metabolic stability of compounds 27 and 28 was
analyzed using incubation systems composed of tested compound (10
μM), rat liver microsomes (RLMs, microsomes from rat male liver,
pooled; 0.4 mg/mL; Sigma-Aldrich), NADPH-regenerating system
(NADP+, glucose-6-phosphate, and glucose-6-phosphate dehydrogen-
ase in 100 mM potassium buffer, pH 7.4; all from Sigma-Aldrich) and
potassium buffer, pH 7.4. Stock solution of tested compounds was
prepared in methanol (the final methanol concentration in incubation
mixture does not exceed 0,1%). First, all samples containing incubation
mixture (without NADPH-regenerating system) were preincubated in
thermoblock at 37 °C for 10 min. Then reaction was initiated by the
addition of NADPH-regenerating system. In control samples NADPH-
regenerating system was replaced with potassium buffer. Probes were
incubated for 30 and 60 min at 37 °C. After addition of internal
standard (pentoxifylline, 10 μM) biotransformation process was
stopped by addition of perchloric acid. Next, samples were centrifuged
and supernatants were analyzed using UPLC/MS (Waters Corpo-
ration, Milford, MA). All experiments were run in duplicates. Half-life
time was evaluated using nonlinear regression model using GraphPad
̈
̈
LigPrep version 3.6 [Schrodinger Release 2020-4: LigPrep, Schro-
dinger, LLC, New York, NY, 2020], and the appropriate ionization
states at pH = 7.0
0.5 were assigned using Epik version 3.4
̈
̈
[Schrodinger Release 2020-4: Epik, Schrodinger, LLC, New York, NY,
2020]. Protein Preparation Wizard was used to assign bond orders and
appropriate amino acid ionization states and to check for steric clashes.
The receptor grid was generated by centering the grid box with a size of
12 Å on D3.32. Automated flexible docking was performed using Glide
̈
version 7.0 at the SP level [Schrodinger Release 2020-4: Glide,
̈
Schrodinger, LLC, New York, NY, 2020].
5.3. In Vitro Pharmacological Evaluation. 5.3.1. Radioligand
Binding Assays. All experiments were carried out according to the
previously published procedures.41−43 HEK293 cells stably expressing
human 5-HT1A, 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
containing 5% CO2 and grown in Dulbecco’s modified Eagle’s medium
containing 10% dialyzed fetal bovine serum and 500 μg/mL G418
sulfate. For membrane preparation, cells were cultured in 150 cm2
flasks, grown to 90% confluence, washed twice with prewarmed to 37
°C phosphate buffered saline (PBS), and centrifuged (200g) in PBS
containing 0.1 mM EDTA and 1 mM dithiothreitol. Prior to membrane
preparation, pellets were stored at −80 °C. Cell pellets were thawed and
homogenized 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 between the centrifugations. The
composition of the assay buffers was experimentally selected to achieve
the maximum signal window (more details in Supporting Information).
All assays were carried out in a total volume of 200 μL in 96-well plates
for 1 h at 37 °C except 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, USA). For displacement studies
the assay samples contained the following as radioligands (PerkinElm-
er, USA): 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 in the presence of 10 μM 5-HT in
5-HT1AR and 5-HT7R binding experiments, whereas 20 μM mianserin,
10 μM methiothepine, and 10 μM haloperidol were used in 5-HT2AR,
5-HT6R, and D2LR assays, respectively. Each compound was tested in
triplicate at seven concentrations (10−10−10−4 M). The inhibition
constants (Ki) were calculated from the Cheng−Prusoff equation.44
Additionally, the affinity of compound 27 at the adrenergic α1A,
muscarinic M1, histaminergic H1, dopaminergic D3, serotoninergic 5-
HT2C, 5-HT3 receptors, serotonin transporter (SERT), and the human
ether-a-go-go-related gene (hERG) channel were evaluated in Eurofins
Cerep. The results were expressed as the % inhibition at 1 μM according
to experimental protocols described online at https://www.eurofins.
com/.
5.3.2. Determination of cAMP Production in 1321N1 Cells. The
properties of compounds 18, 22, 27, 28 to inhibit cAMP production
induced by 5-CT (1000 nM), a 5-HT6R agonist, were evaluated.
Compounds were tested in triplicate at eight concentrations (10−11
−
10−4 M). The level of cAMP was measured using frozen recombinant
1321N1 cells expressing the human serotonin 5-HT6R (PerkinElmer).
Total cAMP was measured using the LANCE cAMP detection kit
(PerkinElmer), according to the manufacturer’s recommendations. For
quantification of cAMP levels, 2000 cells/well (5 μL) were incubated
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Prism software, and intrinsic clearance was calculated from the equation
Cytochrome P450: CYP3A4 and CYP2D6. The luminescent CYP3A4
P450-Glo and CYP2D6 P450-Glo assays and protocols were provided
by Promega (Madison, WI, USA). The P450-Glo systems based on the
conversion proluciferins, derivatives of beetle luciferin [(4S)-4,5-
dihydro-2-(6′-hydroxy-2′-benzothiazolyl)-4-thiazolecarboxylic acid]
into D-luciferin were catalyzed by respective CYP isoform. D-Luciferin
is formed and detected in a second reaction with the luciferin detection
reagent (LDR). The amount of light produced in the second reaction is
proportional to CYP activity.
Clint = (volume of incubation [μL]/protein in the incubation
46
[mg])0.693/t1/2
.
5.4.2. In Vivo Pharmacokinetic Study. 5.4.2.1. Instrumentation.
For LC−MS/MS analyses, an HPLC Nexera system (Shimadzu, Kyoto,
Japan) combined with the triple quadrupole API 3200 mass
spectrometer (SCIEX, Framingham, MA, USA) interfaced via an
electrospray source and controlled by Analyst software version 1.5.2
(SCIEX, Framingham, MA, USA) was applied.
5.4.2.2. LC/MS/MS Analyses. The chromatographic separation was
performed on Acclaim Polar Advantage II (3.0 mm × 74 mm, 3 μm,
120A, Dionex, USA) analytical column using the mobile phase
composed of eluent A, HPLC grade acetonitrile acidified with 0.1%
(v/v) formic acid, and eluent B, HPLC grade water with 0.1% (v/v)
formic acid. The elution gradient started with 90% of eluent B,
increasing to 90% of eluent A over 5 min, returned to 90% of eluent B
over 5 min, and maintained at 90% of eluent B for 5 min. The mobile
phase flow rate was set at 400 μL/min. The injection volume was 20 μL,
and the total time of analysis was 15 min. The temperatures of the
column thermostat and the autosampler were set at 40 and 10 °C,
respectively.
For increased sensitivity and selectivity, the MS/MS data acquisition
was performed in the selected reaction monitoring (SRM) mode. The
ions measured were m/z 585.1 (Q1) and m/z 516.2 (Q3) for
compounds 27 and 28 and m/z 305 (Q1) and m/z 248 (Q3) for IS.
The quantification was done via peak area ratio.
The stock solutions (10 mM) of the references ketoconazole (KE),
quinidine (QD), and examined compounds were performed in DMSO.
The 4× concentrated dilutions were prepared before the assays. The
enzymatic reactions were conducted in white polystyrene, flat-bottom
Nunc MicroWell 96-well microplates (Thermo Scientific, Waltham,
MA USA).
The CYPs, proluciferin, and examined compounds (25 μL/well)
were preincubated first for 5 min, and next the NADPH regeneration
system was added (25 μL) to start the reaction. The final
concentrations of KE and examined compounds were in range from
0.01 μM to 25 μM. The final concentrations of QD were from 0.001 to
10 μM. The control reactions for measuring the 100% of CYPs activity
and minus-CYP negative control reactions for measure background
luminescence were also prepared. The microplate was incubated in
room temperature for 30 min (CYP3A4) or 45 min (CYP2D6). Finally,
LDR was added (50 μL/well), and after 20 min of incubation in room
temperature the luminescence signal was measured with a microplate
reader (EnSpire, PerkinElmer) in luminescence mode. The signal
produced by CYPs without the presence of compounds was considered
as 100% of CYP activity. The IC50 values and were calculated using
GraphPad Prism 5 software.
The optimized MS/MS experimental conditions were as follows: ion
spray voltage, 5500 V; source temperature, 300 °C; gas 1 pressure, 20
psi; gas 2 pressure, 20 psi; curtain gas pressure, 20 psi; collision gas
pressure, 12 psi.
5.5.2. Assessment of Hepatotoxic Activity. 5.5.2.1. Cell Culture.
Human hepatocellular carcinoma cells (HepG2) were cultured using
standard procedures (protocol from ATCC). Cells were cultured in
Eagle’s minimum essential medium (EMEM) in flasks with an area of
25 cm2 (Falcon), supplemented with 10% of fetal bovine serum (FBS,
Life Technologies) with the addition of 100 IU/mL penicillin (Sigma-
Aldrich) and 100 μg/mL streptomycin (Sigma-Aldrich) and incubated
at 37 °C in a humidified atmosphere with 5% CO2.
The developed method was validated according to validation
procedures, parameters, and acceptance criteria based on FDA and
EMA guidelines for bioanalytical method validation.47,48
5.4.2.3. Sample Preparation. Protein precipitation with acetonitrile
was used for purification of plasma and brain homogenates. The whole
brain was homogenized using an electric tissue homogenizer in an
appropriate amount of phosphate buffer (pH 7.4) in 1:2.5 ratio.
Thereafter, a volume of 100 μL of plasma or brain homogenate was
transferred to 2 mL Eppendorf tubes, and a 5 μL aliquot of the internal
standard (IS, PH002437, Merck, Darmstadt, Germany) in working
solution (5 μg/mL) was added and vortex-mixed for 10 s. Thereafter,
200 μL of acetonitrile was added and mixed for 20 min, followed by
centrifugation (28 672g) for 10 min at 4 °C. The supernatant was
transferred to chromatographic vials, and 20 μL was injected into the
analytical column.
5.4.2.4. Animals and Ethical Statement. A group of 64 male, 8-
week-old, Wistar strain outbred rats weighing between 200 and 220 g
each were purchased from the Animal House at the Faculty of
Pharmacy, Jagiellonian University Medical College, Krakow (Poland)
and housed in standard polycarbonate cages, in groups of four animals
per cage. Environmental conditions during the study were constant:
relative humidity 50−60%, temperature 22 2 °C, normal 12 h light−
dark cycle (7 a.m. to 7 p.m. light). Standard rodent chow and water were
available ad libitum. Compounds 27 and 28 were dissolved in saline and
administered by intragastric gavage at a dose of 10 mg/kg, and the
animals were sacrificed at specific time-points: 0 min, predose (n = 4), 5
min (n = 4), 15 min (n = 4), 30 min (n = 4), 60 min (n = 4), 120 min (n
= 4), 240 min (n = 4), and 480 min (n = 4) after administration.
Animals were deeply anaesthetized by ip injections of 50 mg/kg
ketamine plus 8 mg/kg xylazine before sacrifice. First, the blood was
collected into heparinized tubes, and the blood samples were
centrifuged at 1000g for 10 min to obtain plasma. Following the
animals’ euthanasia, the whole brain was collected. The plasma and
brain samples were immediately frozen at −80 °C for future analysis. All
experimental procedures were carried out in accordance with EU
Directive 2010/63/EU and approved by the I Local Ethics Committee
for Experiments on Animals of the Jagiellonian University in Krakow,
Poland (No. 83/2018).
5.5.2.2. LDH Assay. Cells were seeded at density 2 × 104 cells/per
well in 95-well plates. After 24 h, compound 27 and reference standard
DOX (highly cytotoxic agent) were added to final concentrations of
0.1−150 μM. After 24 h incubation, plates were centrifuged (200g, 2
min) and 50 μL of the supernatant was transferred into the
corresponding 96-well plate. Subsequently, 50 μL of LDH-reaction
mixture prepared according to the manufacturer’s instructions
(Invitrogen) was added to each well. Incubation was conducted in
darkness for 30 min at room temperature. Next, stop solution was
added and absorbance was measured at 490 nm (A490) using a plate
reader (Spectra Max iD3, Molecular Devices). Cytotoxicity was
determined as follows: cytotoxicity (%) = [(compound LDH activity
− spontaneous LDH activity)/(maximum LDH activity − spontaneous
LDH activity)] × 100. The maximum LDH activity was prepared by
treating cells with lysis buffer. The medium used in the LDH assay
contained 1% FBS. Two independent experiments were performed for
each condition.
5.5.2.3. MTT Assay. The MTT assay was used to determine the
viability of HepG2 cells incubated in the presence of compound 27 and
DOX. Cells were seeded at a density of 2 × 104 in 96-well plates.
Following overnight culture, the cells were treated with with 27 and
DOX in concentration range 0.1−150 μM for 24 h. Following cell
exposure to each compound 10 μL of MTT reagent (Sigma-Aldrich)
was added to each well. After 4 h of incubation (37 °C, 5% CO2) the
culture medium was aspirated and formazan crystals were dissolved in
100 μL of DMSO. Then, optical density (OD) at 570 nm was
determined on a plate reader (Spectra Max iD3, Molecular Devices).
Each individual experiment was repeated at least three times.
5.5.3. Evaluation of the Mutagenic Potential: AMES Test. Ames
microplate fluctuation protocol (MPF) assay was performed with
Salmonella typhimurium strain TA100, enabling the detection base
substitution mutations. Bacterial strain and exposure and indicator
5.5. Extended in Vitro Off-Target Selectivity and Safety
Profile Assessment for Compound 27. 5.5.1. Inhibition of
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medium were purchased from Xenometrix AG (Allschwil, Switzerland).
The mutagenic potential of tested structures was evaluated by
incubation of bacteria, incapable of producing histidine, with particular
concentration of test compounds for 90 min in exposure medium,
containing limited amount of histidine. The occurrence of reversion
events to histidine prototrophy was observed as a growth of bacteria in
the indicator medium without histidine after 48 h of incubation in room
temperature. Bacterial growth in 384-well plates was visualized by color
change of medium from violet to yellow due to addition of pH indicator
dye. Compound was classified as mutagenic, if the fold increase in
number of positive wells over the medium control baseline was greater
than 2.0. The solvent control baseline was defined as the mean number
of positive wells in the negative control sample, increased by one
standard deviation. 1% DMSO in media was used as a negative and 4-
nitroquinoline N-oxide (NQNO) as positive control. The experiment
was performed in triplicate. Fraction S9 was not added.
5.6. In Vivo Pharmacological Evaluation of the Cognitive
Effects of the Compound 27: Animals and the Ethical
Statement. The experiments were conducted in accordance with
the NIH Guide for the Care and Use of Laboratory Animals and were
approved by the II Local Ethics Committee for Animal Experiments,
Maj Institute of Pharmacology. Male inbred 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, 06:00) with ad libitum access to food (unless
stated otherwise) 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 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.
5.6.1. Novel Object Recognition (NOR) Test under Scopolamine-
Induced Memory Decline. The experiments were performed according
to the previously reported procedures.35−37 Twenty-eight rats were
tested in a dimly lit (25 Lx) “open field” apparatus made of a dull gray
plastic measuring (66 cm × 56 cm × 30 cm). After each measurement,
the floor was cleaned and dried.
5.6.1.1. Drug Treatment in the NOR Test. Scopolamine hydro-
bromide used to attenuate learning was purchased from Sigma-Aldrich
(Germany). Scopolamine and the compound 27 were solubilized in
distilled water and then administered at the dose of 1.25 mg/kg (ip) and
3−6 mg/kg (po) 30 and 120 min before familiarization phase (T1),
respectively. The choice of scopolamine dose was based on our earlier
report;37 the doses of the 27 compound were selected based on the PK
study.
5.6.1.2. NOR Test Experimental Procedure and Statistics. The
procedure consisted of habituation to the arena (without any objects)
for 5 min, 24 h before the test, and test session comprised two trials
separated by an inter trial interval (ITI). For scopolamine-induced
memory impairment paradigm, 1 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 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 were 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. On the basis of exploration time (E) of two objects during T2,
discrimination index (DI) was calculated according to the formula: DI
= (EB − EA)/(EA + AB).
Data were analyzed using one-way ANOVA with treatment as
between-subject factor. As a post-hoc, we used Tukey’s multiple
comparisons post-hoc test. Statistical significance was set at P < 0.05.
Statistics was performed with Prism 9.0 for Mac.
5.6.2. Attentional Set Shifting Task (ASST). Thirty-two male
Sprague-Dawley rats were group housed with a mild food restriction
(17 g of food pellets per day) for at least 1 week prior to training.
5.6.2.1. ASST Apparatus. Testing was conducted in a Plexiglas
apparatus (length × width × height: 38 cm × 38 cm × 17 cm) with the
grid floor and wall dividing half of the length of the cage into two
sections. During testing, one ceramic digging pot (internal diameter of
10.5 cm and a depth of 4 cm) was placed in each section. Each pot was
defined by a pair of cues along with two stimulus dimensions. To mark
each pot with a distinct odor, 5 μL of a flavoring essence (Dr Oetker,
Poland, or The Body Shop, U.K.) was applied to a piece of blotting
paper fixed to the external rim of the pot immediately prior to use. A
different pot was used for each combination of digging medium and
odor; only one odor was ever applied to a given pot. The bait (one-half
of a Honey Nut Cheerios, Nestle) was placed at the bottom of the
“positive” pot and buried in the digging medium. A small amount of
powdered Cheerios was added to the digging media to prevent the rat
from trying to detect the buried reward by its smell.
5.6.2.2. ASST Procedure. As described previously,49 the procedure
lasted 3 days for each rat.
Day 1, Habituation. Rats were habituated to the testing area and
trained to dig in the pots filled with sawdust to retrieve the food reward.
Rats were transported from the housing facility to the testing room
where they were presented with one unscented pot (filled with several
pieces of Cheerios) in their home cages. After the rats had eaten the
Cheerios from the home cage pot, they were placed in the apparatus and
given three trials to retrieve the reward from both of the sawdust-filled
baited pots. With each exposure, the bait was covered with an increasing
amount of sawdust. Animals that did not dig for a food reward were
subjected again to the training on the next day. If a rat did not start to
dig in three daily sessions, it was excluded from an experiment.
Day 2, Training. Rats were trained on a series of simple
discriminations (SDs) to a criterion of six consecutive correct trials.
For these trials, rats had to learn to associate the food reward with an
odor cue (e.g., arrack vs orange, both pots filled with sawdust) and/or a
digging medium (e.g., plastic balls vs pebbles, no odor). All rats were
trained using the same pairs of stimuli. The positive and negative cues
for each rat were presented randomly and equally. These training
stimuli were not used again in later testing trials.
Day 3, Testing. Rats performed a series of discriminations in a single
test session. An incorrect choice was recorded as an error. Digging was
defined as any distinct displacement of the digging media with either
the paw or the nose; the rat could investigate a digging pot by sniffing or
touching without displacing material. Experiments were performed by
an experimenter blinded to the treatment group. Testing was continued
at each phase until the rat reached the criterion of six consecutive
correct trials, after which testing proceeded to the next phase. If a rat
does not make either a correct or an incorrect response during any trials
of the ASST within 5 min, the trial was reinitiated after a 10 min break. If
the rat was still not responding, the test was discontinued and the rat
was excluded from the data analysis.
In the simple discrimination involving only one stimulus dimension,
the pots differed along one of two dimensions (e.g., digging medium).
For the compound discrimination (CD), the second (irrelevant)
dimension (i.e., odor) was introduced but the correct and incorrect
exemplars of the relevant dimension remained constant. For the
reversal of this discrimination (Rev1), the exemplars and relevant
dimension were unchanged, but the previously correct exemplar was
now incorrect and vice versa. The ID shift was then presented,
comprising new exemplars of both the relevant and irrelevant
dimensions with the relevant dimension remaining the same as
previously. The ID discrimination was then reversed (Rev2) so that the
formerly positive exemplar became the negative one. For the ED shift, a
new pair of exemplars was again introduced, but this time a relevant
1237
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Research Article
dimension was also changed. Finally, the last phase was the reversal
(Rev3) of the ED discrimination.
Xavier Bantreil − IBMM, Université de Montpellier, CNRS,
ENSCM, 34095 Montpellier, France; orcid.org/0000-
0002-2676-6851
Maria Walczak − Faculty of Pharmacy, Jagiellonian University
Medical College, 30-688 Kraków, Poland; orcid.org/0000-
0002-5670-9866
Paulina Koczurkiewicz-Adamczyk − Faculty of Pharmacy,
Jagiellonian University Medical College, 30-688 Kraków,
Poland; orcid.org/0000-0003-2939-224X
Gniewomir Latacz − Faculty of Pharmacy, Jagiellonian
University Medical College, 30-688 Kraków, Poland;
orcid.org/0000-0001-9247-2598
Anna Gwizdak − Faculty of Pharmacy, Jagiellonian University
Medical College, 30-688 Kraków, Poland; Institut de
Génomique Fonctionelle, Université de Montpellier, CNRS,
INSERM, 34094 Montpellier, France
Martyna Krawczyk − Maj Institute of Pharmacology, Polish
Academy of Sciences, 31-343 Kraków, Poland
Joanna Gołębiowska − Maj Institute of Pharmacology, Polish
Academy of Sciences, 31-343 Kraków, Poland
Katarzyna Grychowska − Faculty of Pharmacy, Jagiellonian
University Medical College, 30-688 Kraków, Poland;
orcid.org/0000-0002-2264-251X
Andrzej J. Bojarski − Maj Institute of Pharmacology, Polish
Academy of Sciences, 31-343 Kraków, Poland; orcid.org/
0000-0003-1417-6333
The exemplars were always presented in pairs and varied so that only
one animal within each treatment group received the same
combination. The assignment of each exemplar in a pair as being
positive or negative at a given phase, and the left−right positioning of
the pots in the test apparatus on each trial were randomized.
5.6.2.3. Drugs for ASST. Compound 27 was dissolved in distilled
water and was given 120 min before ED phase (i.e., 90 min before first
stage of ASST test at the doses of 0, 6, and 9 mg/kg, po). The choice of
27 compound doses was based on the PK study. The drug or vehicle
(distilled water) was administered at a volume of 1 mL/kg of body
weight.
5.6.2.4. Statistics for ASST. As the main cognitive measure, the
number of trials required to achieve the criterion of six consecutive
correct responses (i.e., trials to criterion, TTC) was recorded for each
rat and for each discrimination phase of the ASST. Additionally, we
analyzed the mean time to complete a single trial in a given
discrimination stage to examine nonspecific effects of the compound
27.
Data were analyzed using mixed design ANOVAs with treatment as
between-subject factor and discrimination phase (SD, CD, Rev1, etc.)
as a repeated measure. As a post-hoc, we used Newman−Keuls test.
Statistical significance was set at P < 0.05. The statistical analyses were
performed using Statistica 12.0 for Windows.
5.6.2.5. Study Limitations. In the experiments examining the
cognitive effects of 27 compound, we used only two doses (3 and 6 mg/
kg in the NOR test and 6 and 9 mg/kg in the ASST test).
Agnieszka Nikiforuk − Maj Institute of Pharmacology, Polish
Academy of Sciences, 31-343 Kraków, Poland
Gilles Subra − IBMM, Université de Montpellier, CNRS,
ENSCM, 34095 Montpellier, France; orcid.org/0000-
0003-4857-4049
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.1c00061.
Jean Martinez − IBMM, Université de Montpellier, CNRS,
ENSCM, 34095 Montpellier, France; orcid.org/0000-
0002-9267-4621
Maciej Pawłowski − Faculty of Pharmacy, Jagiellonian
University Medical College, 30-688 Kraków, Poland
Piotr Popik − Maj Institute of Pharmacology, Polish Academy of
Sciences, 31-343 Kraków, Poland; orcid.org/0000-0003-
0722-1263
Synthetic procedures for intermediates 1−4; character-
ization data for all intermediates and final compounds
excluded from the main manuscript; evaluation of
CYP450 inhibition and mutagenic properties (Ames
test) (PDF)
AUTHOR INFORMATION
■
Philippe Marin − Institut de Génomique Fonctionelle,
Université de Montpellier, CNRS, INSERM, 34094
Montpellier, France; orcid.org/0000-0002-5977-7274
Frédéric Lamaty − IBMM, Université de Montpellier, CNRS,
ENSCM, 34095 Montpellier, France; orcid.org/0000-
0003-2213-9276
Corresponding Author
Paweł Zajdel − Faculty of Pharmacy, Jagiellonian University
Medical College, 30-688 Kraków, Poland; orcid.org/0000-
0002-6192-8721; Phone: +48 126205500;
Email: pawel.zajdel@uj.edu.pl
Authors
Complete contact information is available at:
Marcin Drop − Faculty of Pharmacy, Jagiellonian University
Medical College, 30-688 Kraków, Poland; IBMM, Université
de Montpellier, CNRS, ENSCM, 34095 Montpellier, France;
orcid.org/0000-0001-5307-700X
Vittorio Canale − Faculty of Pharmacy, Jagiellonian University
Medical College, 30-688 Kraków, Poland; orcid.org/0000-
0001-7940-9500
Séverine Chaumont-Dubel − Institut de Génomique
Fonctionelle, Université de Montpellier, CNRS, INSERM,
34094 Montpellier, France; orcid.org/0000-0001-6860-
6891
Rafał Kurczab − Maj Institute of Pharmacology, Polish
Academy of Sciences, 31-343 Kraków, Poland; orcid.org/
0000-0002-9555-3905
https://pubs.acs.org/10.1021/acschemneuro.1c00061
Author Contributions
Experimental work was conducted by M.D., V.C., S.C.-D., R.K.,
G.S., M.K., M.W., P.K.-A., G.L., A.G., and J.G. Data analysis and
interpretation were conducted by M.D., V.C., R.K., S.C.-D., X.B.,
M.W., P.K.-A., K.G., A.J.B., A.N., and G.S. Writing, review, and/
or revision of the manuscript was conducted by M.D., V.C., S.C.-
D., M.W., J.M., M.P., P.P., F.L., and P.Z. Research design and
supervision were conducted by P.Z.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Grzegorz Satała − Maj Institute of Pharmacology, Polish
Academy of Sciences, 31-343 Kraków, Poland; orcid.org/
0000-0002-0756-7232
■
The authors acknowledge the financial support from the
National Science Centre, Poland (Grant 2016/21/B/NZ7/
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Research Article
Serotonin 5-HT6 Receptor Promotes Receptor Constitutive Activity.
Proc. Natl. Acad. Sci. U. S. A. 113, 12310−12315.
01742), Priority Research Area qLife under the program
“Excellence InitiativeResearch University” at the Jagiellonian
University in Krakow, statutory activity of Jagiellonian
University Medical College and Maj Institute of Pharmacology,
Polish Academy of Sciences, PHC Polonium Programme,
Université de Montpellier and Centre National de la Recherche
Scientifique (CNRS). S.C.-D. and P.M. were supported by
grants from CNRS, INSERM, Montpellier University of
Excellence (iSITE MUSE), the French Foundation for Medical
Research (FRM) and ANR (Grants ANR-17-CE16-0013-01
and ANR-17-CE16-0010-01). M.D. thanks French Embassy in
Poland for the French Government Scholarships. A.G. thanks
Erasmus+ Programme for scholarship. The graphical abstract
was created with BioRender.com.
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