Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-substituted tetrahydropyridine derivatives with potential anti-metastatic activity

Article abstract of DOI:10.1016/j.ejmech.2019.111854

Small Conductance Calcium (Ca²⁺)-activated potassium (K⁺) channels (SKCa) are now proved to be involved in many cancer cell behaviors such as proliferation or migration. The SK3 channel isoform was particularly described in breast cancer where it can be associated with the Orai1 Ca²⁺ channel to form a complex that regulates the Ca²⁺ homeostasis during tumor development and acts as a potent mediator of bone metastases development in vivo. Until now, very few specific blockers of Orai1 and/or SK3 have been developed as potential anti-metastatic compounds. In this study, we illustrated the synthesis of new families of lipophilic pyridine and tetrahydropyridine derivatives designed as potential modulators of SK3 channel. The toxicity of the newly synthesized compounds and their migration effects were evaluated on the breast cancer cell line MDA-MB-435s. Two molecules (7a and 10c) demonstrated a significant decrease in the SK3 channel-dependent migration as well as the SK3/Orai1-related Ca²⁺ entry. Current measurements showed that these compounds are more likely SK3-selective. Taken all together these results suggest that such molecules could be considered as promising anti-metastatic drugs in breast cancer.

Full text of DOI:10.1016/j.ejmech.2019.111854

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and
biological evaluation of 2-substituted tetrahydropyridine derivatives
with potential anti-metastatic activity
a
b
a
a
c
ꢀ
ꢀ
^
ꢁ
Sana Kouba , Julien Braire , Romain Felix , Aurelie Chantome , Paul-Alain Jaffres ,
Jacques Lebreton b, Didier Dubreuil b, Muriel Pipelier b, Xuexin Zhang d,
d
a
b, *, 1
ꢀ
Mohamed Trebak , Christophe Vandier , Monique Mathe-Allainmat
,
Marie Potier-Cartereau a
,
**, 1
a
ꢀ
University of Tours, Inserm U1069 Nutrition, Croissance et Cancer (N2C), Faculty of Medicine, 10 Boulevard Tonnelle, 37032, Tours Cedex, France
University of Nantes, CNRS, Faculty of Sciences, Chimie et Interdisciplinarite: Synthese, Analyse, Modelisation (CEISAM), UMR CNRS 6230, 2, Rue de La
b
ꢀ
ꢁ
ꢀ
ꢁ
Houssiniere, 44322, Nantes Cedex 3, France
c University of Brest, CNRS, CEMCA, UMR 6521, 6 Avenue Victor Le Gorgeu, 29238, Brest, France
d Pennylvania State University College of Medicine, Department of Cellular and Molecular Physiology, 700 HMC Crescent Road, Hershey, PA, 17033, USA
a r t i c l e i n f o
a b s t r a c t
Small Conductance Calcium (Ca2þ)-activated potassium (Kþ) channels (SKCa) are now proved to be
involved in many cancer cell behaviors such as proliferation or migration. The SK3 channel isoform was
particularly described in breast cancer where it can be associated with the Orai1 Ca2þ channel to form a
complex that regulates the Ca2þ homeostasis during tumor development and acts as a potent mediator of
bone metastases development in vivo. Until now, very few specific blockers of Orai1 and/or SK3 have
been developed as potential anti-metastatic compounds. In this study, we illustrated the synthesis of
new families of lipophilic pyridine and tetrahydropyridine derivatives designed as potential modulators
of SK3 channel. The toxicity of the newly synthesized compounds and their migration effects were
evaluated on the breast cancer cell line MDA-MB-435s. Two molecules (7a and 10c) demonstrated a
significant decrease in the SK3 channel-dependent migration as well as the SK3/Orai1-related Ca2þ entry.
Current measurements showed that these compounds are more likely SK3-selective. Taken all together
these results suggest that such molecules could be considered as promising anti-metastatic drugs in
breast cancer.
Article history:
Received 2 October 2019
Accepted 4 November 2019
Available online xxx
Keywords:
Ca2þ-activated Kþ channels
SK3
Breast cancer
Cell migration
Amphiphilic compounds
Tetrahydropyridine derivatives
© 2019 Published by Elsevier Masson SAS.
1. Introduction
elevate cytosolic Ca2þ was initially illustrated in red blood cells
more than 50 years ago and later then, pharmacological manipu-
Calcium (Ca2þ) is an ubiquitous second messenger and its
cytosolic concentration is highly regulated by a wide variety of Ca2þ
pumps, channels and transporters [1]. In a physiological context,
elevated concentrations of cytosolic Ca2þ are necessary to activate
several types of ion channels of which, the Ca2þ-activated Kþ
channels (KCa) [2]. The increased Kþ permeability consequent to
lation of cytosolic Ca2þ led to the identification of KCa channels in
molluscan neurons [3]. Consequently, a classification of the KCa
channels was made according to their single channel conductance,
molecular and pharmacological properties into three major sub-
families: the Big-conductance Ca2þ-dependent Kþ channels (BKCa
channels), the Intermediate-conductance Ca2þ-dependent Kþ
channels (IKCa channels), and the Small-conductance Ca2þ
-
dependent Kþ channels (SKCa channels). The SKCa channels have
small single-channel conductance (10e20 pS) and are thus named
“small-conductance” channels [4]. Four genes encoding the SKCa
channels have been identified: KCNN1 for SK1 (also named KCa1.1),
KCNN2 for SK2 (also named KCa2.1), KCNN3 for SK3 (also named
* Corresponding author.
** Corresponding author.
ꢀ
E-mail addresses: monique.mathe@univ-nantes.fr (M. Mathe-Allainmat), marie.
potier-cartereau@univ-tours.fr (M. Potier-Cartereau).
1
Both authors supervised equally the research work.
https://doi.org/10.1016/j.ejmech.2019.111854
0223-5234/© 2019 Published by Elsevier Masson SAS.
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

2
S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
KCa2.3) and KCNN4 for SK4 (also named KCa3.1) with highly
conserved sequences among species. Numerous studies focused on
the roles of the SKCa channels which are widely expressed in
excitable cells [5]. Their functional disruptions have been associ-
ated to many neurological diseases such as Parkinson's disease,
bipolar disorder and schizophrenia [6e8]. SKCa dysfunctions have
also been associated to cardiovascular pathologies such as cardiac
arrhythmia, atrial fibrillation and hypertension [9e12]. In addition
to their implication in neurological and cardiovascular diseases in
excitable cells, their role in non-excitable cells such as epithelial
cells was mostly described in cancer. The most compelling results
highlighting an evident connection between the SKCa channels
expression and cancer cell biology were mainly obtained from
studies on breast cancer [13].A work from our laboratory has shown
that the SK3 channel was abnormally expressed in the non-
excitable breast cancer cell line (MDA-MB-435s) and was found
to promote cancer cell migration in vitro and bone metastasis for-
limiting the bone resorption and fractures [15]. Because SK3 was
found to be implicated in the migration of breast cancer cells and
BMs development, it has become a challenge for scientists to
identify novel compounds to modulate its migratory activity.
Several natural compounds extracted from scorpions, sea
anemone or bee venome have already been described to act on
SKCa channels [16e18]. The most known compound is apamin, a
neurotoxic peptide found in bee venom. Apamin efficiently blocks
SKCa channels without having an effect on BKCa and IKCa channels
[19]. Despite its efficiency on SKCa channels, this molecule was not
selective between the SKCa isoforms. Consequently, during the two
last decades crystallographic [20] and molecular modelling [21]
studies have contributed to design and identify synthetic drugs as
positive or negative modulators of the SKCa channels with specific
sites of action. The anti-bacterial drug dequalinium chloride was
shown to be a SKCa channel blocker at the apamin allosteric
binding site [22]. Synthetic analogues of dequalinium salt, with a
constrained cyclic structure, such as ULC 1684, were identified to
present activities at low nanomolar range (Fig. 1) [23]. Extending
this SAR study to bis-charged tetraazacyclophanes bearing an
aliphatic bridging chain didn't give better result [24]. Comple-
mentary studies from a pharmacophore model based on apamin
and UCL1684 structures resulted in the identification of bis-
isoquinolinium derivatives [25] and tetrahydroisoquinolinium an-
alogues such as NMAG525E1 [26] as high affinity ligands for SK2/
SK3 channel subtypes (Fig. 1).
^
mation in vivo [14]. Chantome et al., demonstrated an association
between the SK3 channel and the Orai1 Ca2þ channel in distinct
nanodomains of the plasma membrane enriched with cholesterol
and sphingolipids known as lipid rafts. In association with Orai1,
the SK3 induced a hyperpolarization of the membrane leading to an
increase of the Ca2þ electrochemical driving force and a constitu-
tive Ca2þ entry via Orai1. This association between Orai1 and SK3
creates a positive loop since the Ca2þ influx via Orai1 not only ac-
tivates the Ca2þ-dependent migration of cancer cells, but it also
increases the activity of the SK3 channel (see Fig. S1 in SI).
If these compounds bearing permanent charges could be
regarded as apamin competitors, some small neutral molecules
were also proved to act as negative or positive modulators of SKCa
channels targeting both SK2 and SK3 types. As an example, the
amino pyrimidine derivative CyPPA [27] or the amino naph-
thothiazol SKA-31 and analogues [28]were characterized as posi-
tive modulators of the gating system binding at the channel/
In breast cancer, 60% of patients with advanced breast cancer
will eventually develop bone metastases (BM) during the course of
disease. One of the greatest challenges in breast cancer treatment is
then to limit the formation of BMs and until now, no treatment to
prevent this event has been proposed. The available therapies on
the market are mainly to improve the life quality of patients by
Fig. 1. Charged compounds identified as inhibitors of SK2/SK3 channels, targeting the Apamin binding site.
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
3
Fig. 2. Neutral compounds as SK2/SK3 channels positive or negative modulators.
calmoduline interface (Fig. 2). Whereas the N-alkyl-amino benz-
poor or without toxicity up to 1
m
M were selected and their effects
imidazole compound NS8593, with an additional tetrahydronaph-
thalene moiety, appeared to be a potent negative gating modulator,
rather than a pore blocker. Further investigations on the mode of
action of NS8593 have pointed out possible interactions of the
molecule at position deep within the inner pore vestibule [29].
As supra discussed, it is only recently that the involvement of
SK3 in cancer cell behaviors such as cell proliferation or cell
migration was highlighted. Consequently, charged phospholipidic
molecule such as edelfosine (Fig. 3) was found to disrupt SK3 ac-
tivity through a novel mechanism of action within the lipid rafts.
This amphiphilic compound perturbed the association of SK3 and
Orai1 at the plasma membrane to block cell migration [30]. How-
on the SK3-dependent migration, Ca2þ influx and current were
investigated.
2. Results and discussion
2.1. Chemistry
The novel amphiphilic compounds in series A and B were pre-
pared following a short and simple strategy we earlier published
[33] using commercially available 2-picoline 1, and selected alde-
hydes 2a-c prepared by oxidation of the corresponding alcohols
(Scheme 1). The length of the lipophilic chain was fixed at 16 to 18
methylene carbons close to that found in ohmline and edelfosine
compounds. The addition of the lithiated 2-picoline on the alde-
hydes 2a-c at low temperature afforded the hydroxy compounds
3a-c in 59e70% yields. These pyridine intermediates were qua-
ternarized to their corresponding NeMe-pyridinium salts 5a-c in
the presence of methyl paratoluene sulfonate in refluxing dichlo-
roethane. Moderate to good yields of compounds 5 were obtained
after a simple filtration of the precipitate formed in the reaction
medium (5a: 96%; 5b: 50%; 5c: 39%). Some assays were carried out
using methyl iodide as the N-alkylating agent but the purification
procedure of the iodide salts by simple precipitation appeared less
efficient. The pyridinium p-toluenesulfonate salts 5a-c were
reduced to NeMe-tetrahydropyridine derivatives 6a-c and 7a-c, by
treatment with an excess of sodium triacetoxyborohydride in a
mixture of DMSO/CH2Cl2 (Scheme 2). In these conditions, the
reduction of the pyridinium salts 5a-c proceeded with the forma-
tion of the sole 3,4-tetrahydropyridine regioisomers and a good
ever, its toxic effect at moderate concentration (3 mM) allowed the
chemists to design and synthesize sugar analogues such as neutral
glyco-glycero-ether lipids among which, ohmline appeared as the
most efficient member (Fig. 3) [31]. Ohmline inhibited selectively
the SK3 current and activity by decreasing the migration of breast
cancer cells in vitro at low concentrations (300 nM) [32] and also
reduced the development of BMs in a mouse model in vivo as
^
shown by Chantome et al. [14].
In this study we describe the synthesis of a novel amphiphilic
family of compounds bearing a pyridine or tetrahydropyridine
moiety, as found in some apamin competitors, and a saturated or
unsaturated fatty chain (Fig. 3, compounds A and B). The lipidic
chain was introduced starting from selected saturated (C18, C20) or
unsaturated (C18:1-cis-u9) fatty carboxaldehydes. A highly meta-
static breast cancer cell line (MDA-MB-435s) expressing the SK3
channel (shRD cells) or not (shSK3 cells) was chosen in order to
evaluate the toxicity of the novel compounds. The molecules with a
Fig. 3. Edelfosine and ohmline as amphiphilic hSK3 blockers and novel pyridine and tetrahydropyridine targeted compounds A and B.
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

4
S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
Scheme 1. Synthesis of the pyridine derivatives 3a-c and 4a-c.
Scheme 2. Synthesis of the tetrahydropyridine derivatives 6a-c: 7a-c and piperidine derivatives 8a and 9a.
diastereoselectivity in favor of the syn-( )-6a-c isomers over the
anti-( )-7a-c (4:1 ratio). Isomers 6a/7a and 6b/7b were formed in
60% and 62% yields respectively, whereas 6c/7c were formed in a
moderated 30% yield. Pure compounds 6 and 7 were isolated by
chromatography purification to be evaluated. To confirm the
structures of both diastereoisomers, syn-6a and anti-7a derivatives
were hydrogenated in the presence of Raney Nickel to avoid double
bond migration and consequently C2 epimerisation [34]. The cor-
responding syn-8a and anti-9a diastereoisomers were isolated in
good yields (Scheme 2).
Some modifications of the free hydroxyl group in compounds 3
and 6/7 were proposed. Indeed, O-methylation of the compounds
3a-c was achieved using NaH as the base in the presence of an
excess of MeI as alkylating agent (Scheme 1). The corresponding
compounds 4a-c were isolated after column chromatography pu-
rification with low yields (4a: 27%; 4b: 29% and 4c: 21%) due to the
formation of byproducts such as quaternarized pyridine de-
rivatives. Therefore, esterification of tetrahydropyridine com-
pounds 6a-c and 7a was performed with acetyl chloride in the
presence of diisopropylethylamine, leading to the corresponding
acetates 10a-c and 11a in good to excellent yields (10a: 55%; 10b:
83%, 10c: 99% and 11c: 98%) (Scheme 3).
2.2. Biological results and discussion
2.2.1. Cytotoxicity evaluations
As a preliminary biological study, the cytotoxicity of the novel
compounds 3a-c, 4a-c, 6a-c, 7a-c, 10a-c and 11a was evaluated to
exclude toxic effects on cell proliferation. We therefore treated the
MDA-MB-435s cells at different concentrations of selected ana-
logues using ohmline as reference (Table 1). Molecules showing
little or no toxic effect at 1
centration of 10 M whereas molecules having a toxic effect at 1
were further tested at maximum of 3 M. We observed that pyri-
dine derivatives (compounds 3 and 4) systematically showed a
toxicity at 1 M, except for compounds 3a and 4a which had no
toxic effect up to 10 M. It is also to be noticed that charged N-
mM were tested up to a maximal con-
m
mM
m
m
m
methylated compound such as compound 5a was more toxic than
its pyridine precursor 3a, perhaps due to the sulfonate counter ion
in this N-methyl pyridinium form.
For the tetrahydropyridine compounds (6, 7, 10 and 11) or for
piperidine analogues (8a and 9a), the cytotoxicity seemed lower
compared to the pyridine parents (Table 1) since most of them
showed no effect at 1 mM (Table 2). This behavior was systemati-
cally observed for all the compounds bearing the shorter lipophilic
chain (6a, 7a, 8a, 9a, 10a and 11a). For the compounds with a longer
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
5
Scheme 3. Synthesis of the O-acetyl derivatives 10a-c and 11a.
Table 1
Cytotoxic effects (24 h treatment) of pyridine derivatives (3a-c, 4a-c and 5a) on MDA-MB-435s cancer cells compared to ohmline.
Pyridine derivatives
% of mortality
1
0
m
M
3
m
M
10
0
mM
3a
NTa
3b
3c
20
20
20
25
NTa
NTa
4a
4b
4c
0
NTa
20
0
20
20
NTa
NTa
25
5a
30
0
NTa
80
0
Ohmline
0
a
NT for not tested.
saturated or unsaturated carbon chain, the O-acylated form (10b
and 10c) also appeared less toxic than the parent compounds (6b
and 6c). Furthermore, the tetrahydropyridine compounds 6a-c, 7a-
c, 10a-c and 11a were tested for their in vitro inhibition of cell
proliferation on a panel of seven human tumor cell lines (Huh7,
CaCo2, MDA-MB231, HCT116, PC3, NCIeH727 and MCF7) with the
normal diploid skin fibroblasts used as a control (Table 3). All novel
effect up to 25 mM on the seven cancer cell lines as well as on
fibroblasts.
2.2.2. Modulation of SK3-dependent cell migration
After performing the viability tests, we proceeded on studying
the effects of the molecules showing low or no toxicity, on the
migration of MDA-MB-435s breast cancer cells at a single dose of
derivatives presented a very poor toxicity, with an IC50 up to 6
mM,
1
mM. Results showed that compounds 7a and 10c significantly
particularly compounds 6a/7a and 6b/7b which have shown no
decreased the migration of MDA-MB-435 cells up to 60% and 40%
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

6
S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 2
Cytotoxic effects (24 h treatment) of the tetrahydropyridine derivatives (6a-c, 7a-c, 8a, 9a, 10a-c and 11a) and piperidine derivatives (8a and 9a) on MDA-MB-435 cells.
Tetrahydropyridine and piperidine derivatives
6a
% of mortality
1
0
m
M
3
m
M
10
80
mM
NTa
7a
6b
0
NTa
30
0
20
30
30
NTa
NTa
NTa
7b
6c
30
80
7c
30
5
40
NTa
70
10a
NTa
10b
10c
11a
0
0
0
NTa
NTa
NTa
20
70
20
8a
0
0
0
NTa
NTa
0
35
30
0
9a
Ohmline
a
NT for not tested.
Table 3
Antiproliferative activity of the tetrahydropyridine derivatives 6-a-b, 7a-b, 10a-c and 11a.
Cytotoxicity IC50
HuH7
(m
M)a
Compound
CaCo-2
MDA-MB-231
HCT116
PC3
NCIeH727
MCF7
Fibroblast
6a
6b
6c
>25
>25
10
21
>25
8
>25
>25
8
>25
>25
10
>25
>25
7
>25
>25
9
>25
>25
10
>25
>25
9
7a
7b
7c
10a
10b
10c
>25
>25
24
22
>25
10
16
14
6
9
17
9
>25
>25
14
21
>25
10
>25
>25
15
16
>25
9
>25
>25
19
15
>25
8
>25
>25
>25
>25
>25
19
>25
>25
18
19
>25
15
>25
>25
25
18
>25
13
11a
Doxorubicin
>25
0.03
9
0.17
22
0.02
24
0.12
26
0.12
>25
0.66
>25
0.16
24
0.03
a
IC50 was calculated after 48 h incubation with 6 concentrations of the compound (0.1e25
mM).
respectively (Fig. 4). In the same conditions Ohmline (1
decreased the migration of the cells around 35e40%.
m
M)
generated cells lacking the expression of the SK3 channel (MDA-
MB-435s shSK3). The experiments were performed and apamin
was used as a positive control at a concentration of 100 nM (Fig. 5).
At this concentration apamin is selective for SK3 and has no effect
on SK1 or SK2. The molecule 7a inhibited the migration of the
MDA-MB-435s shRD cells up to 60% at low concentrations (100 nM
and 300 nM) (Fig. 5A) and did not affect the migration of MDA-MB-
435 shSK3 cells (Fig. 5B). On the other hand, the molecule 10c
Based on these results, compound 7a and its congener com-
pound 10c with structural modulations on the lipophilic residue,
were selected for further biological evaluations. As a first approach,
their inhibitory impact on the SK3-dependent migration was
studied, followed by Ca2þ influx and currents measurements.
To determine if molecules 7a and 10c were SK3-selective, we
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
7
with around 40% inhibition of MDA-MD-435s cell migration at
300 nM, and appeared to be as efficient as edelfosine C18 having
60% of potency at the same concentration [31].
2.2.3. In vitro effect on Ca2þ influx
In order to investigate whether the behaviors of 7a and 10c on
the migration were SK3 and Ca2þ-dependent, we measured the
constitutive Ca2þ entry in MDA-MB-435s shRD vs. MDA-MB-435s
shSK3 transfected with a siRNA sequence directed against Orai1
(siOrai1) or not (siCTL) (note that, the experiments were performed
48 h after the transfection). The association between SK3 and the
Ca2þ channel Orai1 at the plasma membrane of MDA-MB-435s, has
already been described previously by our laboratory to induce a
constitutive Ca2þ entry necessary for the migration and bone
metastasis formation (see Fig. S1, SI) [14]. Consequently, one way to
explain the observed inhibition of cancer cell migration is to
measure the constitutive Ca2þ entry resulting from these channels
association. After treating the cells for 1 h with the compounds 7a
Fig. 4. Effect of the compounds at 1 mM on the migration of MDA-MB-435s breast
cancer cells. The normalized cell number corresponds to the ratio of the total number
of migrating cells in the presence of the compounds/the total number of migrating
cells in control experiments. Migration assays were realized for 24 h in three separate
experiments (in triplicate).
or 10c at 300 nM and 1 mM respectively, cells were resuspended in a
Ca2þ free solution for 20 s and the Ca2þ was added (2 mM solution).
Compound 7a inhibited the constitutive Ca2þ influx up to 40% in
MDA-MB-435s shRD expressing Orai1 (Fig. 6B) without any sig-
nificant effect on the cells after Orai1 knock-down (Fig. 6C). In cells
lacking the SK3 channel expression, the molecule 7a has no effect
on the constitutive Ca2þ influx in siCTL cells as well as in siOrai1
inhibited the migration of SK3-expressing cells in a dose dependent
manner up to 40% at 3 mM (Fig. 5C) with no significant impact on
the migration of the SK3-depleted MDA-MB-435 shSK3 cells
(Fig. 5D). These data suggested that 7a and 10c were most likely
SK3 selective. Furthermore, compound 7a can be compared to the
glyco-glycero lipid ohmline identified previously as a SK3 blocker
Fig. 5. Effect of 7a and 10c on the SK3-dependent migration of MDA-MB-435s breast cancer cells. (A) Dose-effect of compound 7a on the MDA-MB-435s shRD cells expressing the
SK3 channel. (B) Dose-effect of compound 7a on MDA-MB-435s shSK3 cells (C) Dose-effect of compound 10c on SK3-expressing cells and (D) Dose-effect of compound 10c on MDA-
MB-435s lacking the expression of SK3. Normalization corresponds to the ratio of the total number of migrating cells with the compounds/total number of migrating cells in control
experiments. Migration assays were realized for 24 h in three separate experiments (in triplicate). Histograms represent the mean SEM. NS means not significant.
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

8
S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 6. Effect of 7a and 10c on the SK3-dependent constitutive Ca2þ entry in the MDA-MB-435s breast cancer cell line. (A) Graph showing steady state fluorescence relative to Ca2þ
entry in the cells without and with 0.3 mM and 1 m
M of 7a and 10c respectively. (B) Inhibition of the constitutive entry of Ca2þ with 7a and 10c in MDA-MB-435s shRD-siCTL cells. (C)
Inhibition of the constitutive entry of Ca2þwith 7a and 10c in MDA-MB-435s shRD-siOrai1 cells. (D) and (E) No significant effect of the molecules was detected under the same
conditions on MDA-MB-435 shSK3. The results are obtained from three different experiments and each condition was performed in triplicate. Histograms represent the Mean SEM
of constitutive Ca2þ influx in presence of molecule normalized to control condition. NS means not significant.
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
9
cells (Fig. 6D and E). At a concentration of 1
m
M, compound 10c
silica-covered aluminum sheets (Kieselgel 60F254, MERCK). Eluted
TLC was revealed using UV radiation (
¼ 254 nm), or molybdate
solution. Flash column chromatography was performed on silica gel
showed similar behaviors as 7a (Fig. 6A, B, 6C, 6D and 6E). These
results suggest that both molecules 7a and 10c modulate the Ca2þ
entry in MDA-MB-435s cells only in the presence of both SK3 and
Orai1 meaning that they could potentially act on the SK3/Orai1
channel complex.
l
60 ACC 40e63 mm (SDS-CarloErba). NMR spectra were recorded on
a BRUKER AC300 (300 MHz for 1H) or on a BRUKER 400 (400 MHz
for 1H) at room temperature, on samples dissolved in an appro-
priate deuterated solvent. References of tetramethylsilane (TMS)
for 1H and deuterated solvent signal for 13C were used. Chemical
2.2.4. Effects on SK3 and CRAC currents
To follow the inhibitory role of compounds 7a and 10c on the
SK3 current, the SK3 stable-expressing HEK-293 cells were used to
displacement values (d) are expressed in parts per million (ppm),
and coupling constants (J) in Hertz (Hz). Low-resolution mass
spectra (MS) were recorded in the CEISAM laboratory on a Thermo-
Finnigan DSQII quadripolar at 70 eV (CI with NH3 gas). High-
Resolution Mass Spectrometry (HRMS in Da unit) analyses were
recorded on Xevo G2-XS Qtof apparatus (from Waters) in the lab-
oratory, or on a MALDI -TOF-TOF apparatus (Autoflex III from
Bruker) in the INRA center (BIBS platform, Nantes).
measure the SK3 current with acute addition of 7a (2
mM) and 10c
(10 M) (Fig. 7A). Both molecules significantly inhibited the SK3
m
current but 7a seemed to be more efficient with 90% inhibition of
the SK3 current (Fig. 7B and C) compared to 70% for 10c (Fig. 7D and
E). The 50% inhibition of SK3 current occurred within approxi-
mately 10 s for 7a compared to 100 s for 10c. These results sug-
gested that the compound 7a was highly efficient. It is also to be
noted that the addition of apamin induced an additive effect on the
current inhibition. This result is of great interest because apamin is
known to block the pore of the channel and the additive inhibitory
effect induced by 7a on the SK3 current suggests that this derivative
might not be a pore blocker.
Because 7a appeared to be a more potent modulator of SK3
current, we investigated whether 7a acts on the CRAC current (Ca2þ
Release Activated Ca2þ entry current or ICRAC), generated by the
Orai1 Ca2þ channel and its reticular regulator STIM1 (STromal
interaction molecule1). Indeed, the Orai Ca2þ channel interacts with
STIM1 and this interaction induces the Ca2þ entry via the Store
Operated Ca2þ entry which is independent of the SK3 in our model
of breast cancer. In order to investigate whether 7a might act on
Orai1 channel, ICRAC was measured in stable Orai1/STIM1-expressing
4.1.2. General synthetic procedure 1 for the synthesis of the
aldehydes (2)
To a solution of the alcohol (1 equiv) in anhydrous DCM was
added (diacetoxyiodo)benzene (1.1 equiv) followed by a catalytic
amount of TEMPO (0.05 equiv) and the mixture was stirred during
18 h. The reaction was hydrolyzed with a saturated aqueous
Na2S2O3 solution and the compound was extracted with CH2Cl2.
The organic layer was washed with a saturated aqueous NaHCO3
solution then with brine, dried over MgSO4, filtered and the solvent
was removed under reduced pressure. Purification of the residue by
column chromatography on silica gel (PE/CH2Cl2; 8:2) gave the
expected pure aldehyde.
HEK-293 cells. Acute application of 7a (2
mM) showed no effect on
4.1.2.1. Octadecanal (2a). Following general procedure 1 and
ICRAC (Fig. 8) suggesting that 7a is most likely specific to SK3 based
on the massive inhibitory effect observed on the SK3 current.
starting from commercial 1-octadecanol (15 g, 55 mmol), purifica-
tion by column chromatography on silica gel afforded aldehyde 2a
as a white powder (83% yield). 1H NMR (300 MHz, CDCl3):
d 9.75 (t,
3. Conclusion
J ¼ 1.8 Hz, 1H, CHO), 2.42 (dt, J ¼ 7.2, 1.8 Hz, 2H, CH2), 1.62 (quint,
J ¼ 7.2 Hz, 2H, CH2), 1.25 (m, 28H, 14 x CH2), 0.87 (t, J ¼ 6.6 Hz, 3H,
In this study we have prepared and evaluated novel series of
pyridine and tetrahydropyridine compounds bearing a lipophilic
chain and identified as new potential SK3 modulators. These mol-
ecules were efficiently prepared in three or four reaction steps
starting from commercial 2-picoline and lipophilic aldehydes. Most
of them revealed no intrinsic toxicity on a panel of cancer cell lines.
The most efficient and promising compounds evaluated as cell
migration blockers were obtained in tetrahydropyridine series.
Among them, compound 7a with a C17 saturated alkyl chain, and
10c featuring a mono-unsaturated lipid chain were tested as po-
tential SK3 channel modulators on MDA-MB-435s cells. The best
compound 7a gave efficient effect on breast cancer cell migration,
with 60% decrease of migration at 300 nM. This effect was shown to
be greatly dependent of the presence of the SK3/Orai1 complex in
the cells, but rather SK3 specific. Further complementary in-
vestigations are being carried out in our laboratory in order to
establish the action mode of these compounds prior to run in vivo
studies on xenograft mice models. The promising results observed
in this study from in vitro experiments suggest that these two
molecules 7a and 10c could be considered as a novel family of lipid
compounds aiming to modulate ion channels’ activity in cancer.
CH3). 13C NMR (75 MHz, CDCl3):
d 203.2 (CO), 44.2 (CH2CO),
32.3e22.4 (14 x CH2), 22.4 (CH2), 14.3 (CH3). MS (ESI) m/z 269.5
[MþH]þ.
4.1.2.2. Eicosanal (2b). Following general procedure 1 and starting
from commercial 1-eicosanol (5 g, 16.75 mmol) purification by
column chromatography on silica gel afforded aldehyde 2b as a
white powder (77% yield). 1H NMR (300 MHz, CDCl3):
d 9.76 (t,
J ¼ 1.8 Hz, 1H, CHO), 2.42 (dt, J ¼ 7.2, 1.8 Hz, 2H, COCH2), 1.62 (quint,
J ¼ 7.2 Hz, 2H, CH2), 1.24 (m, 32H, 16 x CH2), 0.87 (t, J ¼ 7.1 Hz, 3H,
CH3). 13C NMR (75 MHz, CDCl3):
d 203.1 (CO), 44.0 (CH2CO),
32.07e29.83-29.57e29.50-29.31e28.83 (16 x CH2), 22.23 (CH2),
14.26 (CH3). HRMS (ESI): calcd for C20H40ONa [MþNa]þ 319.2971,
found 319.2970.
4.1.2.3. Cis-9-octadecenal (2c). Following general procedure 1 and
starting from commercial oleyl alcohol (6.31 g, 20 mmol) purifica-
tion by column chromatography on silica gel afforded aldehyde 2c
as a colourless oil (77% yield). 1H NMR (300 MHz, CDCl3):
d 9.76 (t,
J ¼ 1.8 Hz, 1H, CHO), 5.41e5.28 (m, 2H, CH]CH), 2.42 (dt, J ¼ 7.2,
1.8 Hz, 2H, CH2), 2.08e1.91 (m, 4H, 2 x CH2), 1.61 (quint, J ¼ 7.2 Hz,
2H, CH2), 1.38e1.20 (m, 20H, 10 x CH2), 0.87 (t, J ¼ 6.6 Hz, 3H, CH3).
4. Experimental protocols
13C NMR (75 MHz, CDCl3):
d 202.81 (CO), 130.12 (CH]CH), 129.75
4.1. Chemistry
(CH]CH), 44.00 (COCH2), 32.00 (2
29.62e29.53-29.43e29.35-29.23-29.14-28.99e27.30 (10
x
CH2), 29.86e29.75-
CH2),
x
4.1.1. General experimental informations
22.78 (CH2), 22.16 (CH2), 14.19 (CH3). HRMS (ESI) calcd for
C
All solvents used were reagent grade and TLC was performed on
18H34ONa [MþNa]þ 289.2502, found 289.2506.
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

10
S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 7. Effect of 7a and 10c on the SK3 current in SK3 stable expressing HEK293 cells. (A) Representative current-voltage curves. The DMSO/Methanol mixture was used as vehicle
control (in black). Acute addition of 7a (2 M) with or without apamin (in gray and red respectively). (B and C) Time response inhibition of SK3 currents recorded at 0 mV with 2
m
mM
of 7a. (D and E) Time response inhibition of the SK3 current recorded at 0 mV with 10 mM of 10c. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
11
Fig. 8. The effect of 7a on the Ca2þ-Release Activated Current (CRAC current) in STIM1 and Orai1 stable expressing HEK293 cells. (A and B) Representative time-course traces of Icrac
recorded from STIM1 and Orai1 stable expressing HEK293 cells. 10 mM EGTA was included in the patch pipette for store depletion. DMSO/Methanol was used as vehicle control in
experiment (A) and 2 mM of 7a was used in the bath for pre-incubation in experiment (B). (C) Representative current-voltage curves were taken from where indicated by the color-
coded asterisks in A and B. (D) Quantification of current density for experiments A and B. (For interpretation of the references to color in this figure legend, the reader is referred to
the Web version of this article.)
4.1.3. General synthetic procedure 2 for the synthesis of the pyridine
derivatives (3)
25.66e22.68 (15 x CH2), 14.11 (CH3). HRMS (ESI) calcd for C24H44NO
[M þ H]þ 362.3423, found 362.3426.
To a solution of commercial 2-picoline (1.5 equiv) in anhydrous
THF cooled to ꢀ70 ꢁC was slowly added n-BuLi (2.5 M solution in
hexane, 1.5 equiv). The mixture was stirred at this temperature for
30 min and a solution of the aldehyde 2 in anhydrous THF (1equiv)
was added dropwise to the red mixture at ꢀ60 ꢁC. The reaction was
stirred at this temperature during 3 h and then hydrolyzed with a
saturated aqueous NH4Cl solution. The compound was extracted
with Et2O. The organic layer was washed with a saturated aqueous
NaHCO3 solution then with brine, dried over MgSO4, filtered and
the solvent was removed under reduced pressure. Purification of
the residue by column chromatography on silica gel (DCM/Et2O: 8/
2) gave the expected compound 3.
4.1.3.2. 1-(Pyridin-2-yl)henicosan-2-ol (3b). Following the general
procedure 2 and starting from eicosanal 2b (1.67 g, 5.5 mmol), after
purification, the expected compound 3b was obtained as a white
solid (yield 60%). 1H NMR (300 MHz, CDCl3):
d
8.48 (d, J ¼ 4. Hz, 1H,
CHar), 7.61 (dt, J ¼ 7.7, 1.8 Hz, 1H, CHar), 7.18e7.11 (m, 2H, 2 x CHar),
4.03 (m, 1H, CHOH), 2.95 (m, 2H, CH2), 1.63e1.25 (m, 34H, 17 x CH2),
0.88 (t, J ¼ 6.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 160.62 (Car),
148.75 (Car), 136.85 (Car), 123.83 (Car), 121.60 (Car), 71.18 (CH), 43.42
(CH2), 37.32 (CH2), 32.08e29.89-29.51e25.82-22.84- (17 x CH2),
14.28 (CH3). HRMS (ESI) calcd for C26H49NO [M þ H]þ 390.3736,
found 390.3718.
4.1.3.1. 1-(Pyridin-2-yl)nonadecan-2-ol (3a). Following the general
procedure 2 and starting from octadecanal 2a (1.67 g, 5.5 mmol),
after purification, the expected compound 3a was obtained as a
4.1.3.3. (Z)-1-(pyridin-2-yl)nonadec-10-en-2-ol (3c). Following the
general procedure
2 and starting from aldehyde 2c (3.5 g,
13.2 mmol), after purification, the expected compound 3b was
white solid (70% yield). 1H NMR (300 MHz, CDCl3):
d
8.49 (d,
obtained as a colorless oil (59% yield). 1H NMR (300 MHz, CDCl3):
J ¼ 4.8 Hz, 1H, CHar), 7.61 (dt, J ¼ 7.7, 1.8 Hz, 1H, CHar), 7.17e7.12 (m,
2H, 2 x CHar), 4.03 (m, 1H, CHOH), 2.95e2.78 (m, 2H, CH2),
1.56e1.24 (m, 32H, 16 x CH2), 0.87 (t, J ¼ 6.3 Hz, 3H, CH3). 13C NMR
d
8.47 (d, J ¼ 4.8 Hz, 1H, CHar), 7.58 (dt, J ¼ 7.7, 1.8 Hz, 1H, CHar),
7.14e7.10 (m, 2H, 2 x CHar), 5.34e5.27 (m, 2H, CH]CH), 5.07 (s, 1H,
OH), 4.03 (m, 1H, CHOH), 2.93e2.27 (m, 2H, CH2), 2.00 (m, 4H, 2 x
(75 MHz, CDCl3):
d
160.14 (Car), 148.02 (Car), 137.21 (Car), 123.95
CH2), 1.60e1.25 (m, 24H, 12 x CH2), 0.86 (t, J ¼ 6.3 Hz, 3H, CH3). 13
C
(Car), 121.60 (Car), 71.02 (CH), 43.09 (CH2), 37.19 (CH2), 31.91e29.69-
NMR (75 MHz, CDCl3): d 160.5039 (Car), 148.51 (Car), 136.59 (Car),
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

12
S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
129.83-129-78 (CH]CH), 123.61 (Car), 121.35 (Car), 70.91 (CH),
43.31 (CH2), 37.11 (CH2), 32.54 (CH2), 31.84 (CH2), 29.71e29.62-
4.1.5. General synthetic procedure 4 for the synthesis of the
tetrahydropyridine derivatives (6) and (7)
29.47e29.26-29.21-29.12-29.06-27.14-25.61e22.62 (11
x
CH2),
Methyl p-toluenesulfonate (1.5equiv) was added to a solution of
the pyridine derivative 3 (1equiv) in dry 1,2-dichloroethane
(150 mL). The mixture was refluxing during 18 h and a precipitate
felt down which was filtered and washed with petroleum ether to
afford pyridinium derivative 5, used without further purification.
The pyridinium salt 5 (1equiv) was then dissolved in a mixture of
DCM/DMSO (4: 1) and NaBH(OAc)3 (2.5 equiv) were added. The
reaction was stirred at room temperature for 2 days and then hy-
drolyzed with a saturated aqueous solution of NH4Cl. The com-
pound was extracted with DCM. The organic layer was washed with
saturated aqueous NaHCO3 then brine, dried (MgSO4), filtered and
the solvent was removed under reduced pressure. Purification of
the residue by column chromatography on alumina gel (DCM/
MeOH; 99.8:0.2) gave the expected pure diastereoisomers ( )-6
and ( )-7 as a racemic mixture.
14.24 (CH3). HRMS (ESI) calcd for C24H42NO [M þ H]þ 360.3266,
found 360.3272.
4.1.4. General synthetic procedure 3 for the synthesis of the pyridine
methoxy derivatives (4)
To a solution of the alcohol 3 (1 equiv) in THF (10 mL), was added
sodium hydride (60% dispersion in mineral oil, 1.5 equiv) at 0 ꢁC.
Methyl iodide (1.5 equiv) was introduced and the reaction was
stirred during 28 h. Water was added and the product extracted
with DCM. The organic layer was washed with brine, dried over
MgSO4 and concentrated under reduced pressure. The crude
product was purified by flash column chromatography on silica gel
(DCM/Et2O:7/3) to give the expected product 4.
4.1.4.1. 2-(2-methoxynonadecyl)pyridine (4a). Following the gen-
eral procedure 3 and starting from the alcohol 3a (510 mg,
1.38 mmol), after purification, the expected compound 4a was ob-
tained as white powder (27% yield). 1H NMR (300 MHz, CDCl3):
4.1.5.1. (R)-1-((R)-1-methyl-1,2,5,6-tetrahydropyridin-2-yl)non-
adecan-2-ol (6a) and (S)-1-((R)-1-methyl-1,2,5,6-tetrahydropyridin-
2-yl)nonadecan-2-ol (7a). Following the general procedure 4 and
starting from the alcohol 3a (5.2 g, 14.4 mmol), pyridinium deriv-
ative 5a was obtained as a white powder (96% yield). 1H NMR
d
8.50 (m, 1H, CHar), 7.54 (td, J ¼ 7.6, 1.8 Hz, 1H, CHar), 7.17 (d,
J ¼ 7.7 Hz, 1H, CHar), 7.07 (ddd, J ¼ 7.4, 4.9, 0.9 Hz, 1H, CHar), 3.59
(quint, J ¼ 5.3 Hz, 1H, CHOMe), 3.24 (s, 3H, OMe), 2.96 (dd, J ¼ 13.5,
6.9 Hz, 1H, A part of ABX system CHH0), 2.86 (dd, J ¼ 13.5, 5.7 Hz, 1H,
B part of ABX system CHH’), 1.50e1.22 (m, 32H, 16 x CH2), 0.85 (t,
(300 MHz, MeOD):
d
8.82 (d, J ¼ 5.8 Hz, 1H, CHar), 8.46 (t, J ¼ 7.1 Hz,
1H, CHar), 8.03 (d, J ¼ 7.5Hz, 1H, CHar), 7.88 (t, J ¼ 7.1Hz, 1H, CHar),
7.69 (d, J ¼ 8.0 Hz, 2H, CHar), 7.23 (d, J ¼ 8.0 Hz, 2H, CHar), 4.39 (s, 3H,
NMe), 3.99 (m, 1H, CHOH), 3.28e3.14 (m, 2H, CH2), 2.37 (s, 3H,
CH3Ph), 1.1.72e1.23 (m, 32H, 16 x CH2), 0.90 (t, J ¼ 6.3 Hz, 3H, CH3).
HRMS (ESI) calcd for C25H46NO, [M-Tos]þ 376.3579, found ¼ 376.
3582.
J ¼ 6.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 159.50 (Car), 149.11
(Car), 136.02 (Car), 124.06 (Car), 121.05 (Car), 81.10 (CHOMe), 56.94
(OMe), 42.89 (CH2), 33.84 (CH2), 31.86 (CH2), 29.64e29.53-29.30 12
x (CH2), 25.18 (CH2), 22.62 (CH2), 14.05 (CH3). HRMS (ESI) calcd for
The pyridinium salt 5a (2 g, 3.65 mmol) was reduced with
NaBH(OAc)3 to give after purification both isomers 6a and 7a
(global yield: 62%). Pure fractions of each diastereoisomer could be
obtained as an orange powder (6a 43% yield; 7a 5% yield).
C
25H46NO, [MþH]þ 376.3574, found 376. 3575.
4.1.4.2. 2-(2-methoxyhenicosyl)pyridine (4b). Following the general
procedure 3 and starting from the alcohol 3b (206 mg, 0.52 mmol),
after purification, the expected compound 4b was obtained as a
(6a): 1H NMR (300 MHz, CDCl3):
d 5.79 (m, 1H, CH]CH), 5.54
(dquint, J ¼ 10.5, 1.2 Hz, 1H, CH]CH), 3.76 (quint, J ¼ 6.6 Hz, 1H,
CHOH), 3.09 (m, 2H, NCH and A part of ABX systems NCHH0), 2.65
(dd, J ¼ 13.8, 5.7 Hz, 1H, B part of ABXX0 system NCHH) 2.47 (s, 3H,
NMe), 2.24 (m, 1H, A part of ABXX0 systems CH2CHH0), 1.79 (dt,
J ¼ 17.7, 4.8 Hz,1H, B part of ABXX0 systems CH2CHH’), 1.49e1.24 (m,
304H, 17 x CH2), 0.88 (t, J ¼ 6.3 Hz, 3H, CH3). 13C NMR (75 MHz,
white powder (29% yield). 1H NMR (300 MHz, CDCl3):
d 8.53 (d,
J ¼ 4.4 Hz, 1H, CHar), 7.58 (td, J ¼ 7.6, 1.8 Hz, 1H, CHar), 7.18 (d,
J ¼ 7.7 Hz, 1H, CHar), 7.11 (dd, J ¼ 6.7, 5.1 Hz, 1H, CHar), 3.62 (quint,
J ¼ 6 Hz, 1H, CHOMe), 3.27 (s, 3H, OMe), 2.99 (dd, J ¼ 13.6, 6.9 Hz,
1H, A part of ABX system CHH0), 2.86 (dd, J ¼ 13.6, 5.7 Hz, 1H, B part
of ABX system CHH’), 1.47e1.25 (m, 36H, 18 x CH2), 0.88 (t,
CDCl3):
d 127.83 (CH]CH), 124.74 (CH]CH), 72.81 (CHOH), 61.06
(NCH), 43.63 (NCH2), 40.83 (NMe), 38.61 (CH2), 38.23 (CH2), 31.92
(CH2), 29.78e29.69-29.36 (12 x CH2), 25.47 (CH2), 22.69 (CH2),
18.53 (CH2), 14.12 (CH3). HRMS (ESI) calcd for C25H50NO, [MþH]þ
380.3892, found 380.3886.
J ¼ 6.4 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 159.58 (Car), 149.11
(Car), 136.26 (Car), 124.13 (Car), 121.14 (Car), 81.18 (CHOMe), 57.04
(OMe), 42.97 (CH2), 33.91 (CH2), 31.91 (CH2), 29.75e29.69- 29.59
(14 x (CH2), 25.25 (CH2), 22.68 (CH2), 14.11 (CH3). HRMS (ESI) calcd
for C27H50NO [MþH]þ 404.3892; found: 404.3878.
(7a): 1H NMR (300 MHz, CDCl3):
d 5.898 (m, 1H, CH]CH), 5.423
(m, 1H, CH]CH), 3.85 (m, 1H, CHOH), 3.04 (ls, 1H, NCH), 2.86 (m,
1H, A part of ABX systems NCHH0), 2.46 (s, 3H, NMe), 2.33 (m, 2H, B
part of ABXX0 system NCHH0 and A part of ABXX0 systems CH2CHH0),
2.00e1.86 (m, 2H, B part of ABXX0 systems CH2CHH0 and A part of
ABXX0 systems CH2CHH0), 1.47e1.24 (m, 32H, 2 x B part of ABXX0
systems CH2CHH’ and 15 x CH2), 0.87 (t, J ¼ 6.3 Hz, 3H, CH3). 13C
4.1.4.3. (Z)-2-(2-methoxynonadec-10-en-1-yl)pyridine
(4c).
Following the general procedure 3 and starting from the alcohol 3c
(250 mg, 0.56 mmol), after purification, the expected compound 4c
was obtained as an oil (21% yield). 1H NMR (300 MHz, CDCl3):
d 8.52
(dd, J ¼ 4.8, 0.8 Hz, 1H, CHar), 7.57 (td, J ¼ 7.7, 1.8 Hz, 1H, CHar), 7.17
(d, J ¼ 7.8 Hz, 1H, CHar), 7.10 (ddd, J ¼ 7.3, 4.9, 0.8 Hz, 1H, CHar),
5.28e5.36 (m, 2H, CH]CH), 3.61 (quint, J ¼ 6 Hz, 1H, CHOMe), 3.27
(s, 3H, OMe), 3.99 (dd, J ¼ 13.5, 7.0 Hz, 1H, A part of ABX system
CHH0), 2.85 (dd, J ¼ 13.5, 5.9 Hz, 1H, B part of ABX system CHH’),
2.0e1.96 (m, 4H, 2 x CH2), 1.51e1.44 (m, 2H, CH2), 1.37e1.25 (m,
22H, 11 x CH2), 0.86 (t, J ¼ 6.4 Hz, 3H, CH3). 13C NMR (75 MHz,
NMR (75 MHz, CDCl3): d 128.57 (CH]CH), 126.47 (CH]CH), 69.28
(CH2OH), 61.75 (CHN), 52.11 (CH2N), 43.46 (NMe), 37.86 (CH2),
36.39 (CH2), 32.91 (CH2), 29.812e29.68-28.60e29.34 (12 x CH2),
25.63 (CH2), 25.36 (CH2), 22.67 (CH2), 14.09 (CH3). HRMS (ESI) calcd
for C25H50NO, [MþH]þ 380.3892, found 380.3900.
4.1.5.2. (R)-1-((R)-1-methyl-1,2,5,6-tetrahydropyridin-2-yl)henico-
san-2-ol (6b) and (S)-1-((R)-1-methyl-1,2,5,6-tetrahydropyridin-2-
yl)henicosan-2-ol (7b). Following the general procedure 4 and
starting from the alcohol 3b (1.45 g, 3.72 mmol), pyridinium de-
rivative 5b was obtained as a white powder (50% yield). 1H NMR
CDCl3):
d 160.04 (Car), 149.62 (Car), 136.46 (Car), 130.21e130.14
(CH]CH), 124.40 (Car), 121.39 (Car), 81.17 (CHOMe), 56.89 (OMe),
42.72 (CH2), 33.62 (CH2), 29.44e29.33-29.19-29.17-28.99e28.92 (9
x CH2), 26.87 (2 CH2), 24.90 (CH2), 22.31 (CH2), 13.69 (CH3). HRMS
(ESI) calcd for C25H44NO, [MþH]þ 374.3423, found 374.3432.
(300 MHz, CDCl3)
d
8.93 (d, J ¼ 5.8 Hz, 1H, CHar), 8.22 (t, J ¼ 7.1 Hz,
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
13
1H, CHar), 7.84 (d, J ¼ 7.5 Hz, 1H, CHar),7.74 (t, J ¼ 7.1 Hz, 1H, CHar),
7.64 (d, J ¼ 8.0 Hz, 2H, CHar), 7.1 (d, J ¼ 7.8Hz, 2H, CHar), 5.10 (sl, 1H,
OH), 4.48 (s, 3H, NMe), 4.08 (m, 1H, CHOH), 3.14 (m, 2H, CH2), 2.31
(s, 3H, CH3Ph), 1.93e0.98 (m, 36H, 18 x CH2), 0.87 (t, J ¼ 6.3 Hz, 3H,
CH3). The pyridinium salt 5b (1.37 g, 2.2 mmol) was reduced with
NaBH(OAc)3 to give after purification both isomers 6b and 7b
(global yield: 62%). Pure fractions of each diastereoisomer could be
obtained as a colored powder (6b 23% yield; 7b 4% yield).
2.89 (m, 1H, NCH), 2.42 (s, 3H, NMe), 2.37 (m, 2H, NCH2), 1.98e1.87
(m, 5H, 2 x CH2 and A part of ABXX0 system CH2CHH0), 1.42e1.24 (m,
35H, B part of ABXX0 system CH2CHH’ and 17 x CH2), 0.86 (t,
J ¼ 6.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 130.33e129.87-
126.6529 (2 x CH]CH), 68.79 (CHOH), 61.58 (NCH), 51.69 (NCH2),
43.14 (low resolution NMe), 37.83 (CH2), 36.68 (CH2), 32.59 (CH2),
31.88 (CH2), 29.76e29.64-29.53e29.50-29.30e29.25-29.16-29.10
(9 x CH2), 27.19 (CH2), 25.59 (CH2), 22.66 (CH2), 14.45 (CH3). HRMS
(ESI) calcd for C25H48NO, [MþH]þ 378.3730, found 378.3726.
(6b): 1H NMR (300 MHz, CDCl3):
d 5.76 (m, 1H, CH]CH), 5.53
(m, 1H, CH]CH), 3.77 (quint, J ¼ 6.6 Hz, 1H, CHOH), 3.09 (m, 1H, A
part of ABXX0 system NCHH0), 3.03 (m, 1H, NCH), 2.61 (dd, J ¼ 13.8,
5.7 Hz, 1H, B part of ABXX0 system NCHH0), 2.45 (s, 3H, NMe), 2.23
(m, 1H, A part of ABXX0 system CH2CHH0), 1.76 (dt, J ¼ 17.7, 4.8 Hz,
1H, B part of ABXX0 systems CH2CHH’), 1.47 (m, 4H, 2 x CH2), 1.24
(m, 34H, 17 x CH2), 0.86 (t, J ¼ 6.3 Hz, 3H, CH3). 13C NMR (75 MHz,
4.1.6. General synthetic procedure 5 for the synthesis of piperidine
derivatives (8a) and (9a)
Compounds 6 or 7 (0.4 mmol) in solution in MeOH (5 mL) were
hydrogenated under an atmosphere of H2 in the presence of a
catalytic amount of Raney Nickel (50% slurry solution in water).
After 22 h stirring at room temperature, the mixture was filtered
and the solvent evaporated in vacuo to afford the expected pure
piperidine compounds 8 or 9.
CDCl3):
d 128.34 (CH]CH), 125.08 (CH]CH), 73.26 (CHOH), 61.44
(NCH), 43.84 (NCH2), 41.30 (NMe), 38.95 (CH2), 38.58 (CH2), 32.25
(CH2), 30.12e29.62 (14 x CH2), 25.80 (CH2), 23.01 (CH2),18.85 (CH2),
14.44 (CH3). HRMS (ESI) calcd for C27H54NO, [MþH]þ 408.4200,
found 408.4195.
4.1.6.1. (R)-1-((S)-1-methylpiperidin-2-yl)nonadecan-2-ol
(8a).
(7b): 1H NMR (300 MHz, CDCl3):
d
5.88 (m, 1H, CH]CH), 5.42
The tetrahydropyridine 6a (0.16 g, 0.42 mmol) was hydrogenated
following the general procedure 5, to give pure piperidine com-
pound 8a after simple filtration (93% yield). 1H NMR (300 MHz,
(dt, J ¼ 9.9 Hz, 1H, CH]CH), 3.86 (m, 1H, CHOH), 3.11 (m, 1H, NCH),
2.89 (m, 1H, A part of ABXX0 system NCHH0), 2.42 (s, 3H, NMe), 2.36
(m, 2H, B part of ABXX0 system NCHH0 and A part of ABXX0 system
CH2CHH0), 2.02e1.85 (m, 2H, B part of ABXX0 system CH2CHH0 and A
part of ABXX0 system CH2CHH0), 1.42e1.24 (m, 35H, B part of ABXX0
system CH2CHH’ and 17 x CH2), 0.87 (t, J ¼ 6.3 Hz, 3H, CH3). 13C NMR
CDCl3):
d
3.76 (m, 1H, CHOH), 3.00 (m, 1H, A part of ABXX0 system
MeNCHH0), 2.66 (m, 1H, MeNH), 2.51 (m, 1H, B part of ABXX0 system
MeNCHH0), 2.42 (s, 3H, NMe), 1.87 (sex, 1H, A part of ABX systems
CH(OH)CHH0), 1.69e1.24 (m, 37H,
A
part of ABXX0 systems
(75 MHz, CDCl3):
d
128.41 (CH]CH), 126.50 (CH]CH), 69.16
CH2CHH0, 17 x CH2 and 2 x CH’H), 0.87 (t, J ¼ 6.3 Hz, 3H, CH3). 13
C
(CHOH), 61.62 (NCH), 52.01 (NCH2), 41.35 (NMe), 37.98 (CH2), 36.69
(CH2), 32.05 (CH2), 29.94e29.82-29.48 (14 x CH2), 25.77 (CH2),
25.06 (CH2), 22.81 (CH2), 14.24 (CH3). HRMS (ESI) calcd for
NMR (75 MHz, CDCl3): d 72.22 (CHOH), 60.86 (MeNCH), 51.58
(MeNCH2), 39.93 (NMe), 38.45 (CH2), 37.36 (CH2), 31.91 (CH2),
29.75e29.68-29.35 (12 x CH2), 26.04 (CH2), 25.57 (CH2), 22.67
(CH2), 22.57 (CH2), 20.69 (CH2), 14.10 (CH3). HRMS (ESI) calcd for
C
27H54NO, [MþH]þ 408.4200; found 408.4202.
C
25H52NO, [MþH]þ 382.4049, found 382.4062.
4.1.5.3. (R,Z)-1-((R)-1-methyl-1,2,5,6-tetrahydropyridin-2-yl)non-
adec-10-en-2-ol (6c) and (S,Z)-1-((R)-1-methyl-1,2,5,6-
tetrahydropyridin-2-yl)nonadec-10-en-2-ol (7c). Following the
general procedure 4 and starting from the alcohol 3c (2.9 g,
8.1 mmol), pyridinium derivative 5c was obtained as a white
4.1.6.2. (S)-1-((S)-1-methylpiperidin-2-yl)nonadecan-2-ol
(9a).
The tetrahydropyridine 7a (0.05 g, 0.13 mmol) was hydrogenated
following the general procedure 5, to give pure piperidine com-
pound 9a after simple filtration (91% yield). 1H NMR (300 MHz,
powder (yield 36%). 1H NMR (300 MHz, DMSO‑d6):
d
8.95 (d,
CDCl3): d
4.00 (m, 1H, CHOH), 2.88 (m, 1H, A part of ABXX0 system
J ¼ 5.8 Hz, 1H, CHar), 8.47 (t, J ¼ 7.8 Hz, 1H, CHar), 8.03 (d, J ¼ 7.8 Hz,
1H, CHar), 7.93 (t, J ¼ 6.4 Hz, 1H, CHar), 7.47 (d, J ¼ 7.8 Hz, 2H, 2 x
CHar), 7.1 (d, J ¼ 7.8 Hz, 2H, 2 x CHar), 5.33 (m, 2H, CH]CH), 5.02 (d,
1H, OH), 4.31 (s, 3H, NMe), 3.85 (m, 1H, CHOH), 3.36e3.03 (m, 2H,
CH2), 2.28 (s, 3H, CH3Ph), 2.98 (m, 4H, 2 x CH2), 1.52e1.25 (m, 24H,
12 x CH2), 0.84 (t, J ¼ 6.3 Hz, 3H, CH3). HRMS (ESI) calcd for
MeNCHH0), 2.32 (s, 3H, NMe) 2.17 (m,1H, MeNH),1.94 (m, 2H, B part
of ABXX0 systems MeNCHH0 and A part of ABX systems CH(OH)
CHH0), 1.75 (m, 2H, 2 x CHH0), 1.57e1.20 (m, 37H, 17 x CH2 and 3 x
CHH’), 0.87 (t, J ¼ 6.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 69.16
(CHOH), 62.92 (MeNCH), 57.42 (MeNCH2), 44.09 (NMe), 38.20
(CH2), 36.90 (CH2), 31.91 (CH2), 29.81e29.68-29.35 (13 x CH2),
25.85 (CH2), 25.65 (CH2), 24.51 (CH2), 22.67 (CH2), 14.10 (CH3).
HRMS (ESI) calcd for C25H52NO, [MþH]þ 382.4049, found 382.4051.
C
25H44NO, [M-Tos]þ 374.3423, found 374.3426.
The pyridinium salt 5c (1.2 g, 2.38 mmol) was reduced with
NaBH(OAc)3 to give after purification both isomers 6c and 7c
(global yield: 30%). Pure fractions of each diastereoisomer could be
obtained as a colored powder (6c 12% yield; 7c 2% yield).
4.1.7. General synthetic procedure 6 for the synthesis of the O-acetyl
compounds (10a-c) and (11a)
(6c): 1H NMR (300 MHz, CDCl3):
d
5.74 (m, 1H, CH]CH), 5.53
To a solution of tetrahydropyridine derivative (6 or 7) (1equiv)
in dry DCM (5 mL), acetyl chloride (1.5eq) was added dropwise at
0 ꢁC followed by the addition of diisopropylamine (1.5 equiv). The
solution was kept at 0 ꢁC for 1 h then stirred at room temperature
overnight. It was hydrolyzed with a saturated aqueous NaHCO3
solution, and the compound was extracted with DCM. The organic
layer was washed with brine, dried (MgSO4), filtered and the sol-
vent was removed under reduced pressure. Purification of the
residue by column chromatography on alumina gel (DCM/MeOH;
99.5:0.5) gave the expected compounds 10 or 11.
(dquint, J ¼ 10.5 Hz, 1.2 Hz, 1H, CH]CH), 5.31 (m, 2H, CH]CH), 3.76
(quint, J ¼ 6.6 Hz, 1H, CHOH), 3.10e3.02 (m, 2H, NCH and NCHH’),
2.62 (dd, J ¼ 13.8, 5.7 Hz, 1H, NCHH’), 2.45 (s, 3H, NMe), 2.22 (m, 1H,
A part of ABXX0 system CH2CHH), 1.97 (m, 4H, 2 x CH2), 1.75 (dt,
J ¼ 17.7, 4.8 Hz, 1H, B part of ABXX0 system CH2CHH’), 1.47 (m, 4H, 2
x CH2), 1.24 (m, 22H, 11 x CH2), 0.85 (t, J ¼ 6.3 Hz, 3H, CH3). 13C NMR
(75 MHz, CDCl3): d 130.89 (CH]CH), 130.17 (CH]CH), 128.18 (CH]
CH), 125.03 (CH]CH), 73.11 (CHOH), 61.35 (NCH), 43.91 (NCH2),
41.19 (NMe), 38.92 (CH2), 38.53 (CH2), 32.90 (CH2), 32.20 (CH2),
30.07e29.95-29.86e29.82-29.61- (8 x CH2), 27.50 (CH2), 25.76
(CH2), 22.97 (CH2), 18.85 (CH2), 14.40 (CH3). HRMS (ESI) calcd for
4.1.7.1. (R)-1-((R)-1-methyl-1,2,5,6-tetrahydropyridin-2-yl)non-
adecan-2-yl acetate (10a). Following the general procedure 6 and
starting from tetrahydropyridine derivative 6a (100 mg,
0.26 mmol), compound 10a was obtained as a brown oil (55% yield).
C
25H48NO, [MþH]þ 378.3730; found 378.3723.
(7c): 1H NMR (300 MHz, CDCl3):
d 5.86 (m, 1H, CH]CH), 5.40 (d,
J ¼ 9.9 Hz, 1H, CH]CH), 5.32 (m, 2H, CH]CH), 3.85 (m, 1H, CHOH),
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

14
S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
1H NMR (300 MHz, CDCl3):
d
5.75 (m, 1H, CH]CH), 5.65 (m, 1H,
C
27H52NO2, [MþH]þ 422.3998, found 422.4015.
CH]CH), 5.04 (m, 1H, CHOH), 2.47e2.38 (m, 1H, A part of ABX
systems NCHH0), 2.64 (m, 1H, NCH), 2.38 (m, 1H, B part of ABXX0
system NCHH0), 2.36 (s, 3H, NMe), 2.17 (m, 1H, A part of ABXX0
systems CH2CHH0), 2.00 (s, 3H, COCH3), 1.95 (m, 2H, B part of ABXX0
systems CH2CHH'and A part of ABXX0 systems CH(OH)CHH0),
1.63e1.50 (m, 3H, B part of ABXX0 systems CH(OH)CHH0) and CH2,
1.23 (m, 30H, 15 x CH2), 0.87 (t, J ¼ 6.0 Hz, 3H, CH3). 13C NMR
4.2. Biology
4.2.1. Cytotoxicity evaluation
4.2.1.1. Cell culture. Skin normal fibroblastic cells are purchased
from Lonza (Basel, Switzerland), HuH7, Caco-2, MDA-MB-231,
HCT116, PC3, MCF7 and NCIeH727 cancer cell lines were obtained
from the ECACC collection (Porton Down, UK). Cells are grown at
37 ꢁC, 5% CO2 in ECACC recommended media: DMEM for HuH7,
MDA-MB-231 and fibroblast, EMEM for MCF7 and CaCo-2, McCoy's
for HCT116 and RPMI for PC3 and NCIeH727. All culture media are
supplemented by 10% of FBS, 1% of penicillin-streptomycin and
2 mM glutamine.
(75 MHz, CDCl3): d 170.79 (CO), 128.46 (CH]CH), 125.04 (CH]CH),
71.34 (CHOH), 58.58 (NCH), 50.79 (NCH2), 42.94 (NMe), 37.69 (CH2),
35.20 (CH2), 31.91e29.68-29.54e29.49-29.35 (15 x CH2), 25.18
(CH2), 21.27 (COCH3), 14.11 (CH3). HRMS (ESI) calcd for C27H52NO2,
[MþH]þ 422.3998, found 422.4005.
4.1.7.2. (R)-1-((R)-1-methyl-1,2,5,6-tetrahydropyridin-2-yl)henico-
san-2-yl acetate (10b). Following the general procedure 6 and
starting from tetrahydropyridine derivative 6b (122 mg, 0.3 mmol),
compound 10b was obtained as a brown oil (83% yield). 1H NMR
4.2.1.2. Cytotoxic assay. Chemicals are solubilized in DMSO at a
concentration of 10 mM (stock solution) and diluted in culture
medium to the desired final concentrations. The dose effect cyto-
toxic assays (IC50 determination) is performed by increasing con-
centrations of each chemical (final well concentrations:
(300 MHz, CDCl3):
d 5.78 (m, 1H, CH]CH), 5.66 (m, 1H, CH]CH),
5.02 (m, 1H, CHOH), 2.96 (m, 1H, A part of ABXX0 system NCHH0),
2.88 (m, 1H, NCH), 2.58 (m, 1H, B part of ABXX0 system NCHH0), 2.44
(s, 3H, NMe), 2.28 (m, 1H, A part of ABXX0 system CH2CHH0),
2.10e2.04 (m, 2H, B part of ABXX0 systems CH2CHH'and A part of
ABXX0 system CH(OH)CHH0), 2.02 (s, 3H, COCH3), 1.63 (m, 1H, B part
of ABXX0 system CH(OH)CHH0), 1.53 (m, 2H, CH2), 1.23 (m, 34H, 17 x
0.1 mMe0.3 mM e 0.9 mMe3 mM e 9 mM - 25 mM). Cells are plated in
96 wells plates (4000 cells/well). Twenty-four hours after seeding,
cells are exposed to chemicals. After 48 h of treatment, cells are
washed in PBS and fixed in cooled 90% ethanol/5% acetic acid for
20 min and the nuclei are stained with Hoechst 33342 (B2261
Sigma). Image acquisition and analysis are performed using a Cel-
lomics ArrayScan VTI/HCS Reader (ThermoScientific). The survival
percentages are calculated as the percentage of cell number after
compound treatment over cell number after DMSO treatment. The
relative IC50 are calculated in Microsoft Excel.
CH2), 0.86 (t, J ¼ 6.0 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 171.15
(CO), 127.47 (CH]CH), 125.17 (CH]CH), 71.37 (CHOH), 58.65
(NCH), 50.24 (NCH2), 42.30 (NMe), 37.69 (CH2), 35.47 (CH2), 32.22
(CH2), 29.99e29.85-29.78e29.74-29.66 (15 x CH2), 25.50 (CH2),
23.81 (CH2), 21.53 (COCH3), 14.25 (CH3). HRMS (ESI) calcd for
C
29H56NO2, [MþH]þ 450.4311, found 450.4324.
4.2.2. Biological assays with MDA-MB-425s cell line
4.1.7.3. (R)-1-((R)-1-methyl-1,2,5,6-tetrahydropyridin-2-yl)henico-
san-2-yl acetate (10c). Following the general procedure 6 and
starting from tetrahydropyridine derivative 6c (100 mg,
0.26 mmol), compound 10c was obtained as a brown oil (98% yield).
4.2.2.1. Cell culture. The human breast cancer cell line MDA-MB-
435s was purchased from the American Type Culture Collection
(ATCC) and was grown in Alpha-MEM Medium supplemented with
5% Fetal Bovine Serum (FBS). This cell line was transduced with a
lentivector containing either an interfering short hairpin RNA
(shRNA) specific to SK3 (shSK3 cells) or a nontargeting shRNA
1H NMR (300 MHz, CDCl3):
d 5.78 (m, 1H, CH]CH), 5.65 (m, 1H,
CH]CH), 5.32 (m, 2H, CH]CH), 5.03 (m, 1H, CHOH), 2.90 (m, 1H, A
part of ABXX0 system NCHH’), 2.75 (m, 1H, NCH), 2.48 (m, 1H, B part
of ABXX0 system NCHH’), 2.39 (s, 3H, NMe), 2.25 (m, 1H, A part of
ABXX0 system CH2CHH), 2.10e1.85 (m, 9H, 3 x CH2 and COCH3), 1.62
(m, 1H, B part of ABXX0 system CH(OH)CHH’), 1.53 (m, 2H, CH2), 1.25
(m, 22H, 11 x CH2), 0.86 (t, J ¼ 6.0 Hz, 3H, CH3). 13C NMR (75 MHz,
^
(shRD cells) as previously described by Chantome et al.. The Human
Embryonic cell line HEK-293 was grown in Dulbecco's modified
Eagle's medium supplemented with 5% FBS. These cells were
transduced using lentivectors carrying the cDNA of SK3, Orai1 or
STIM1 to generate HEK-293 stable expressing cells. Cells were
grown at 37 ꢁC in a humidified atmosphere (95% air and 5% CO2).
CDCl3):
d 171.14 (CO), 130.73 (CH]CH), 130.57 (CH]CH), 128.11
(CH]CH), 125.13 (CH]CH), 71.51 (CHOH), 58.70 (NCH), 50.60
(NCH2), 42.70 (NMe), 37.82 (CH2), 35.50 (CH2), 32.92 (CH2), 32.21
(CH2), 30.08e30.00-29.97e29.83-29.80e29.75-29.63e29.50-29.36
(8 x CH2), 27.52 (CH2), 25.51 (CH2), 24.29 (CH2), 22.99 (CH2), 21.58
(COCH3), 14.36 (CH3). HRMS (ESI) calcd for C27H50NO2, [MþH]þ
420.3842, found 420.3860.
4.2.2.2. Transfection assay. 2 ꢂ 105 cells/well were seeded with
growth medium in a 6-well plate and were transfected with a free-
serum opti-MEM medium containing a mixture of Lipofectamine
RNAimax (Invitrogen) and 30 nM of siRNA. Transfected cells were
kept incubated at 37 ꢁC for 6 h. The medium was then removed and
replaced with a fresh growth medium.
4.1.7.4. (S)-1-((R)-1-methyl-1,2,5,6-tetrahydropyridin-2-yl)nano-
decan-2-yl acetate (11a). Following the general procedure 6 and
starting from tetrahydropyridine derivative (7a) (118 mg,
0.31 mmol), compound 11a was obtained as a brown oil (98% yield).
The siRNA sequence directed against Orai1 50-GCCATAA-
GACGGACCGACA [dT][dT]-30 as well as the control siRNA sequence
50-CUGUAUCGAAUGUUAUGAGCC [dT][dT]-30 were purchased from
sigma Aldrich.
1H NMR (300 MHz, CDCl3):
d 5.74 (m, 1H, CH]CH), 5.54 (m, 1H,
CH]CH), 5.03 (m, 1H, CHOH), 2.83 (m, 1H, A part of ABX systems
NCHH0), 2.68 (m, 1H, NCH), 2.43 (m, 1H, B part of ABXX0 system
NCHH’), 2.35 (s, 3H, NMe), 2.06 (m, 2H, CH2), 2.02 (s, 3H, COCH3),
1.72 (m, 2H, CH(OH)CH2), 1.53 (m, 2H, CH(OH)CH2), 1.24 (m, 28H, 14
x CH2), 0.87 (t, J ¼ 6.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
4.2.2.3. Viability assay. Cell viability was determined using the
tetrazolium salt reduction method (MTT). Approximately
4 ꢂ 104 cells were seeded in triplicate in a 24-well plate with
different concentrations of the molecules (100 nM, 300 nM, 1 mM,
3 mM and 10 m
M) and incubated at 37 ꢁC for 24 h. The medium was
d
170.84 (CO), 129.60 (CH]CH), 125.20 (CH]CH), 72.66 (CH2OH),
then removed, and cells were incubated for 45 min at 37 ꢁC with
the tetrazolium salt (3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyl
tetrazolium bromide). Metabolically active cells (viable cells)
reduced the dye into purple formazan. Formazan crystals were
59.13 (CHN), 49.86 (CH2N), 42.96 (NMe), 37.94 (CH2), 34.78 (CH2),
32.06 (CH2), 29.83e29.66-29.50 (12 x CH2), 25.29 (CH2), 23.76
(CH2), 22.82 (CH2), 21.46 (COCH3), 14.25 (CH3). HRMS (ESI) calcd for
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
15
dissolved with DMSO (Dimethylsulfoxide) and the absorbance was
4.2.4. Statistical analysis
measured at 570 nm.
All data were expressed as the means for a series of n exper-
iments SEM and analyzed by Mann Whitney test or One-way
ANOVA for multiple comparisons using GraphPad Prism 6.0 (San
Diego, CA). P-value < 0.05 was considered statistically significant,
NS stands for not significant.
4.2.2.4. Transwell migration assay. Cell migration was performed
using a hollow plastic chamber (Transwell cell culture insert
Corning), sealed at one end with an 8 mm pore size polyethylene
terephthalate membrane and suspended over a larger well. Briefly,
4 ꢂ 104 cells were seeded in the upper compartment containing
growth medium (5% FBS molecules), and allowed to migrate
through the pores to the other side of the membrane. The lower
compartment was also filled with growth medium (5% FBS mol-
ecules). 24 h after launching the experiment, non-migrated cells
were removed from the topside of the membrane, whereas
migrated cells in the bottom side of the inserts were fixed using ice-
cold methanol for 10 min. Nuclei of migrating cells were stained
with DAPI and automatically counted.
Acknowledgements
This work has been developed with technical (ImPACcell plat-
form, Rennes (F) R. Le Guevel) and financial supports from the
ꢀ
ꢀ
ꢀ
^
“Reseau Canaux Ioniques” of the “Canceropole Grand Ouest”. We
are also deeply grateful to the “Ligue Contre le Cancer” for financial
supports (interregional grants: CSIRGO 2015). This work was sup-
ꢀ
ported by “University of Tours”, “Region Centre Val de Loire”,
“INSERM”, and Sana Kouba held her fellowship from the “Ministere
de l'enseignement superieur et de la recherche”.
ꢁ
ꢀ
4.2.2.5. Intracellular Ca2þ measurements. Approximately 1 ꢂ 106
Appendix A. Supplementary data
Cells were seeded in Petri dishes and loaded with the ratiometric
dye Fura2-AM (1 m
M) for 45 min at 37 ꢁC. Cells were then collected
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111854.
and centrifuged at 700ꢂg for 5 min. After centrifugation, cells were
resuspended in a Ca2þ - free solution. 2 mM CaCl2 was then injected
and Fluorescence emission was measured at 510 nm after an exci-
tation at 340 and 380 nm wavelengths (Hitachi FL-2500).
References
[1] M.J. Berridge, M.D. Bootman, H.L. Roderick, Calcium signalling: dynamics,
4.2.3. Electrophysiology
homeostasis and remodelling, Nat. Rev. Mol. Cell Biol. 4 (7) (2003) 517e529.
€
[2] M. Kohler, B. Hirschberg, C.T. Bond, J.M. Kinzie, N.V. Marrion, J. Maylie,
4.2.3.1. ISK3 currents. Experiments were performed using the
whole-cell configuration patch-clamp technique. 2500 cells were
seeded into 35 mm Petri dishes. The extracellular solution con-
J.P. Adelman, Small-conductance, calcium-activated potassium channels from
mammalian brain, Science 273 (1996) 1709e1714.
[3] S.H. Thompson, Three pharmacologically distinct potassium channels in
molluscan neurones, J. Physiol. 265 (1977) 465e488.
tained 140 m
HEPES, 0.33 m
adjusted to pH 7.4 with NaOH. Patch pipettes were pulled to a
resistance of 2e4M from borosilicate glass capillary tubes in a
two-stage vertical puller (PP-830, Narishige, Tokyo, Japan) and
were filled with a pipette solution of 145 m KCl, 1 m MgCl2, 1 m
Mg-ATP, 0.7 m CaCl2, 1 m EGTA, and 10 m HEPES. The solution
was adjusted to pH 7.2 with KOH. The acute effects of 7a (2 M) and
10c (10
M) were tested on HEK-293 SK3þ cells using a ramp pro-
M
NaCl, 4 m
M KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM
[4] C.T. Bond, J. Maylie, J.P. Adelman, Small-conductance calcium-activated po-
tassium channels, Ann. N. Y. Acad. Sci. 868 (1999) 370e378.
M
NaH2PO4, and 11.5 mM D-glucose. The solution was
ꢀ ꢀ
[5] M.P. Burnham, R. Bychkov, M. Feletou, G.R. Richards, P.M. Vanhoutte,
A.H. Weston, G. Edwards, Characterization of an apamin-sensitive small-
sonductance Ca2þ-Activated Kþ channel in porcine coronary artery endothe-
lium: relevance to EDHF, Br. J. Pharmacol. 135 (5) (2002) 1133e1143.
[6] X.-K. Liu, G. Wang, S.-D. Chen, Modulation of the activity of dopaminergic
neurons by SK Channels: a potential target for the treatment of Parkinson's
disease? Neuroscience Bulletin 26 (2010) 265e271.
[7] K.G. Chandy, E. Fantino, O. Wittekindt, K. Kalman, L.-L. Tong, T.-H. Ho,
G.A. Gutman, M.-A. Crocq, R. Ganguli, V. Nimgaonkar, Morris-Rosendahl,
J.J. Gargus, Isolation of a novel potassium channel gene HSKCa3 containing a
polymorphic CAG repeat: a candidate for schizophrenia and bipolar disorder?
Mol. Psychiatry 3 (1998) 32e37.
[8] J.J. Gargus, E. Fantino, G.A. Gutman, A piece in the puzzle: an ion channel
candidate gene for schizophrenia, Mol. Med. Today 4 (1998) 518e524.
[9] J. Diness, J. Kirchhoff, M. Sheykhzade, T. Jespersen, M. Grunnet, Antiar-
rhythmic effect of either negative modulation or blockade of small conduc-
tance Ca2þ-activated Kþ channels on ventricular fibrillation in Guinea pig
Langendorff-perfused heart, J. Cardiovasc. Pharmacol. 66 (2015) 294e299.
[10] X.Y. Qi, J.G. Diness, B.J. Brundel, X.B. Zhou, P. Naud, C.T. Wu, H. Huang,
M. Harada, M. Aflaki, D. Dobrev, M. Grunnet, S. Nattel, Role of small-
conductance calcium-activated potassium channels in atrial electrophysi-
ology and fibrillation in the Dog, Circulation 129 (2014) 430e440.
U
M
M
M
M
M
M
m
m
tocol from ꢀ80 mV to þ80 mV with a holding potential of 0 mV
(500 ms duration, 4 s intervals) to inactivate endogenous potassium
currents. Potassium SK3 currents were analyzed at 0 mV to mini-
mize chloride currents (EClꢀ: 0 mV).
4.2.3.2. ICRAC currents. HEK293 cells with stable expression of
STIM1 and ORAI1 were seeded onto 35 mm round glass coverslips
in the 6-well plates 24 h before electrophysiological recordings.
Traditional whole-cell patch clamp recordings were performed
with an Axopatch 200B and Digidata 1440A (Molecular Devices,
San Jose, CA, USA) as previously published [35,36]. Pipettes were
pulled from borosilicate glass capillaries (World Precision In-
struments, Sarasota, FL, USA) with a P-1000 Flaming/Brown
micropipette puller (Sutter Instrument Company, Novato, CA, USA)
and polished using DMF1000 (World Precision Instruments, Sar-
[11] L. Skibsbye, C. Poulet, J.G. Diness, B.H. Bentzen, L. Yuan, U. Kappert,
K. Matschke, E. Wettwer, U. Ravens, M. Grunnet, T. Christ, T. Jespersen, Small-
conductance calcium-activated Ppotassium (SK) channels contribute to action
potential repolarization in human atria, Cardiovasc. Res. 103 (2014) 156e167.
[12] M.S. Taylor, A.D. Bonev, T.P. Gross, D.M. Eckman, J.E. Brayden, C.T. Bond,
J.P. Adelman, M.T. Nelson, Altered expression of small-conductance Ca2þ
-
activated Kþ (SK3) channels modulates arterial tone and blood pressure, Circ.
Res. 93 (2003) 124e131.
asota, FL, USA). Resistances of filled pipettes were 2e4 M
whole-cell configuration, only cells with tight seals (>16 G
U
U
. Under
) were
[13] M. Potier, V. Joulin, S. Roger, P. Besson, M.-L. Jourdan, J.-Y. LeGuennec,
P. Bougnoux, C. Vandier, Identification of SK3 channel as a new mediator of
breast cancer cell migration », Mol. Cancer Ther. 5 (2006) 2946e2953.
chosen to perform recordings. 250 ms voltage ramps from þ100
to ꢀ140 mV were run every 2 s for data acquisition. Clampfit 10.3
software was used for data analysis. 8 mM MgCl2 was included in
the pipette solution to inhibit TRPM7 currents.
Bath solution: 115 mM Na-methanesulfonate, 10 mM CsCl,
1.2 mM MgSO4, 10 mM Hepes, 20 mM CaCl2, and 10 mM glucose
(pH 7.4 with NaOH).
^
[14] A. Chantome, M. Potier-Cartereau, L. Clarysse, G. Fromont, S. Marionneau-
ꢀ
ꢁ
Lambot, M. Gueguinou, J.-C. Pages, C. Collin, T. Oullier, A. Girault, F. Arbion, J.-
ꢁ
P. Haelters, P.-A. Jaffres, M. Pinault, P. Besson, V. Joulin, P. Bougnoux,
C. Vandier, Pivotal role of the lipid raft SK3-Orai1 complex in human cancer
cell migration and bone metastases, Cancer Res. 73 (2013) 4852e4861.
[15] I.J. Diel, Bisphosphonates in breast cancer patients with bone metastases,
Breast Care 5 (2010) 306e311.
[16] J.P. Vincent, H. Schweitz, M. Lazdunski, Structure-function relationships and
site of action of apamin, a neurotoxic polypeptide of bee venom with an ac-
tion on the central nervous system, Biochemistry 14 (1975) 2521e2525.
Pipette solution: 135 mM Cs-methanesulfonate, 10 mM EGTA,
8 mM MgCl2, and 10 mM Hepes (pH 7.2 with CsOH).
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854

16
S. Kouba et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
ꢀ
ꢀ
[17] V. Quintero-Hernandez, J.M. Jimenez-Vargas, G.B. Gurrola, H.H.F. Valdivia,
L.D. Possani, Scorpion venom components that affect ion-channels function,
Toxicon 76 (2013) 328e342.
[27] C. Hougaard, B.L. Eriksen, S. Jørgensen, T.H. Johansen, T. Dyhring, L.S. Madsen,
D. Strøbæk, P. Christophersen, Selective positive modulation of the SK3 and
SK2 subtypes of small conductance Ca2þ-activated Kþ channels, Br. J. Phar-
macol. 151 (2007) 655e665.
[18] P.T.J. Tan, S. Ranganathan, V. Brusic, Deduction of functional peptide motifs in
scorpion toxins, J. Pept. Sci. 12 (2006) 420e427.
[19] M. Hugues, G. Romey, D. Duval, J.P. Vincent, M. Lazdunski, Apamin as a se-
lective blocker of the calcium-dependent potassium channel in neuroblas-
toma cells: voltage-clamp and biochemical characterization of the toxin
receptor, Proc. Natl. Acad. Sciences U.S.A. 79 (1982), 1308-1212.
ꢀ
[28] N. Coleman, B.M. Brown, A. Olivan-Viguera, V. Singh, M.M. Olmstead,
M.S. Valero, R. Kohler, H. Wulff, New positive Ca2þ-activated Kþ channel
€
gating modulators with selectivity for KCa3.1, Mol. Pharmacol. 86 (2014)
342e357.
[29] a) U.S. Sørensen, D. Strøbæk, P. Christophersen, C. Hougaard, M.L. Jensen,
E.Ø. Nielsen, D. Peters, L. Teuber, Synthesis and structure-activity relationship
studies of 2-(N-substituted)-aminobenzimidazoles as potent negative gating
modulators of small conductance Ca2þ-activated Kþ channels, J. Med. Chem.
51 (2008) 7625e7634;
€
[20] a) M.A. Schumacher, A.F. Rivard, H.P. Bachinger, J.P. Adelman, Structure of the
gating domain of
calmodulin, Nature 410 (2001) 1120e1124;
a
Ca2þ-activated Kþ channel complexed with Ca2þ
/
b) L.T.-Y. Cho, A.J. Alexandrou, R. Torella, J. Knafels, J. Hobbs, T. Taylor,
A. Loucif, A. Konopacka, S. Bell, E.B. Stevens, J. Pandit, R. Horst, J.M. Withka,
D.C. Pryde, S. Liu, G.T. Young, An intracellular allosteric modulator binding
pocket in SK2 ion channels is shared by multiple chemotypes, Structure 26
(2018) 1e12.
b) D.P. Jenkins, D. Strøbæk, C. Hougaard, M.L. Jensen, R. Hummel,
U.S. Sørensen, P. Christophersen, H. Wulff, Negative gating modulation by (R)-
N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) de-
pends on residues in the inner pore vestibule: pharmacological evidence of
deep-pore gating of KCa2 channels, Mol. Pharmacol. 79 (2011) 899e909.
[21] S. Dilly, A. Graulich, A. Farce, V. Seutin, J.-F. Liegeois, P. Chavatte, Identification
of a pharmacophore of SKCa channel blockers, J. Enzym. Inhib. Med. Chem. 20
(2005) 517e523.
^
[30] M. Potier, A. Chantome, V. Joulin, A. Girault, S. Roger, P. Besson, M.-L. Jourdan,
J.-Y. LeGuennec, P. Bougnoux, C. Vandier, The SK3/KCa2.3 potassium channel
[22] P.M. Dunn, Dequalinium, a selective blocker of the slow after hyperpolar-
ization in rat sympathetic neurones in culture, Eur. J. Pharmacol. 252 (1994)
189e194;
is a new cellular target for edelfosine, Br. J. Pharmacol. 162 (2011) 464e479.
^
[31] A. Girault, J.-P. Haelters, M. Potier, A. Chantome, M. Pinault, S. Marionneau-
ꢁ
ꢁ
Lambot, T. Oullier, G. Simon, H. Couthon-Gourves, P.-A. Jaffres, B. Corbel,
P. Bougnoux, V. Joulin, C. Vandier, New alkyl-lipid blockers of SK3 channels
reduce cancer cell migration and occurrence of metastasis, Curr. Cancer Drug
Targets 11 (2011) 1111e1125.
b) D. Galanakis, C.A. Davis, B. Del Rey Herrero, C.R. Ganellin, P.M. Dunn,
D.H. Jenkinson, Synthesis and structure-activity relationships of dequalinium
analogues as Kþ channel blockers. Investigations on the role of the charged
heterocycle, J. Med. Chem. 38 (1995) 595e606.
ꢁ
ꢁ
^
[32] P.-A. Jaffres, C. Gajate, A.M. Bouchet, H. Couthon-Gourves, A. Chantome,
M. Potier-Cartereau, P. Besson, P. Bougnoux, F. Mollinedo, C. Vandier, Alkyl
ether lipids, ion channels and lipid raft reorganization in cancer therapy,
Pharmacol. Ther. 165 (2016) 114e131.
[23] J. Campos Rosa, D. Galanakis, C. Robin Ganellin, P.M. Dunn, D.H. Jenkinson, Bis-
quinolinium cyclophanes
:
6,10-diaza-3(1,3),8(1,4)-dibenzena-1,5(1,4)-
diquinolinacyclodecaphane (UCL 1684), the first nanomolar, non-peptidic
blocker of the apamin-sensitive Ca2þ-activated Kþ channel, J. Med. Chem.
41 (1998) 2e5.
ꢁ
ꢀ
[33] A. Boussonniere, T. Ranaivondrambola, J. Lebreton, M. Mathe-Allainmat,
A rapid access to ( )-sedamine and some original N-benzyl unsaturated an-
alogues, Synthesis 14 (2010) 2456e2462.
[24] D. Yang, L. Arifhodzic, C. Robin Ganellin, D.H. Jenkinson, Further studies on
bis-charged tetraazacyclophanes as potent inhibitors of small conductance
Ca2þ-activated Kþ channels, Eur. J. Med. Chem. 63 (2013) 907e923;
b) J.-Q. Chen, D. Galanakis, C. Robin Ganellin, P.M. Dunn, D.H. Jenkinson, Bis-
[34] Notes: as We Earlier Observed (Ref. 33) Isomerisation at the C2 Position of the
Tetrahydropyridine Could Be Observed in the Presence of a Catalytic Amount
of Pd/C. In the Same Study We Have Published the RX Data of the 3,4-un-
saturated Analog of ( )-sedamine Obtained Following the Same Procedure
and So Confirmed the Syn-Diastereoselectivity of the Reduction Step with
NaBH(OAc)3.
[35] X. Zhang, T. Pathak, R. Yoast, S. Emrich, P. Xin, R.M. Nwokonko, M. Johnson,
S. Wu, S. Delierneux, M. Gueguinou, N. Hempel, J.W. Putney, D. Gill, M. Trebak,
A calcium/cAMP signaling loop at the ORAI1 mouth drives channel inactiva-
tion to shape NFAT induction, Nat. Commun. 10 (2019) 1971.
quinolinium
cyclophanes:
8,14-Diaza-1,7(1,4)-diquinolinacyclote-
tradecaphane (UCL 1848), a highly potent and selective, nonpeptidic blocker
of the apamin-sensitive Ca2þ-activated Kþ channel, J. Med. Chem. 43 (2000)
3478e3481.
ꢀ
[25] A. Graulich, S. Dilly, A. Farce, J. Scuve-Moreau, O. Waroux, C. Lamy, P. Chavatte,
ꢀ
V. Seutin, J.F. Liegeois, Synthesis and radioligand binding studies of bis-
isoquinolinium derivatives as small conductance Ca2þ-activated Kþ channel
blockers, J. Med. Chem. 50 (2007) 5070e5075.
[36] X. Zhang, W. Zhang, J.C. Gonzalez-Cobos, I. Jardin, C. Romanin, K. Matrougui,
M. Trebak, Complex role of STIM1 in the activation of store-independent
Orai1/3 channels, J. Gen. Physiol. 143 (2014) 345e359.
ꢀ
[26] J.-F.
Liegeois,
J.
Wouters,
V.
Seutin,
S.
Dilly,
Bis-(1,2,3,4-
tetrahydroisoquinolinium): a chiral scaffold for developing high-affinity li-
gands for SK channels, ChemMedChem 9 (2014) 737e740.
Please cite this article as: S. Kouba et al., Lipidic synthetic alkaloids as SK3 channel modulators. Synthesis and biological evaluation of 2-
substituted tetrahydropyridine derivatives with potential anti-metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/
10.1016/j.ejmech.2019.111854