Development of pyrene-based fluorescent ether lipid as inhibitor of SK3 ion channels

DOI: 10.1016/j.ejmech.2020.112894

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European Journal of Medicinal Chemistry (2021)

Update date:2022-08-10

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Authors:

Bauduin, Alicia

Papin, Marion

Chant?me, Aurélie

Couthon, Hélène

Deschamps, Laure

Requejo-Isidro, Jose

Vandier, Christophe

Jaffrès, Paul-Alain

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Article abstract of DOI:10.1016/j.ejmech.2020.112894

We report the synthesis of three bioactive pyrene-based fluorescent analogues of Ohmline which is the most efficient and selective inhibitor of SK3 ion channel. The interaction of these Ohmline-pyrene (OP1-3) with liposomes of different composition reveals that only OP2 and OP3 are readily integrated into liposomes. Fluorescence measurements indicate that, depending on their concentration, OP2 and OP3 exist either as monomer or as a mixture of monomer and excimers within the liposome bilayer. Among the three Ohmline Pyrene compounds (OP1-3) only OP2 is able to reduce SK3 currents and is the first efficient fluorescent modulator of SK3 channel as revealed by patch clamp measurements (- 71.3 ± 13.3% at 10 μM) and by its inhibition of SK3-dependent cancer cell migration at (?32.5% ± 4.8% at 1 μM). We also report the first fluorescence study on living breast cancer cells (MDA-MB-231) showing that OP2 is rapidly integrated in bio-membranes followed by cell internalization.

Full text of DOI:10.1016/j.ejmech.2020.112894

European Journal of Medicinal Chemistry 209 (2021) 1 12894

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Development of pyrene-based ﬂuorescent ether lipid as inhibitor of

SK3 ion channels

ꢀ

ꢀ ꢁ

Alicia Bauduin , Marion Papin , Aurelie Chantome , Helene Couthon ,

a, *

ꢁ

Laure Deschamps , Jose Requejo-Isidro , Christophe Vandier , Paul-Alain Jaffres

^aUniv Brest, CEMCA UMR CNRS 6521, 6 Avenue Victor Le Gorgeu, F-29238, Brest, France

ꢀ

Univ Tours, Inserm U1069 Nutrition, Croissance et Cancer (N2C), Faculte de pharmacie, 10 Boulevard Tonnelle, 37032, Tours Cedex, France

^cCentro Nacional de Biotecnología (CNB), CSIC, Madrid, Spain; Unidad de Nanobiotecnología, CNB-CSIC-IMDEA Nanociencia Associated Unit, Madrid, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:

We report the synthesis of three bioactive pyrene-based ﬂuorescent analogues of Ohmline which is the

most efﬁcient and selective inhibitor of SK3 ion channel. The interaction of these Ohmline-pyrene (OP1-

3) with liposomes of different composition reveals that only OP2 and OP3 are readily integrated into

liposomes. Fluorescence measurements indicate that, depending on their concentration, OP2 and OP3

exist either as monomer or as a mixture of monomer and excimers within the liposome bilayer. Among

the three Ohmline Pyrene compounds (OP1-3) only OP2 is able to reduce SK3 currents and is the ﬁrst

efﬁcient ﬂuorescent modulator of SK3 channel as revealed by patch clamp measurements (- 71.3 13.3%

Received 22 June 2020

Received in revised form

4 September 2020

Accepted 24 September 2020

Available online 6 October 2020

Keywords:

at 10

M) and by its inhibition of SK3-dependent cancer cell migration at (ꢀ32.5% 4.8% at 1

mM). We

Glyco-glycerolipid

Ether lipid

Liposome

Fluorescence

Amphiphiles

SK3

also report the ﬁrst ﬂuorescence study on living breast cancer cells (MDA-MB-231) showing that OP2 is

rapidly integrated in bio-membranes followed by cell internalization.

Ion channel

1. Introduction

cancer cells (e.g. breast MDA-MB-435s cells) and are associated to

cancer-cell migration [12,13]. Ohmline (Fig. 1) is one of the most

The involvement of ion channels in cancer development [1e3],

cancer cell migration [4,5] or cancer chemotherapeutic resistance

[6,7] emerged over the last 20 years leading to the identiﬁcation of

novel targets and possibly new cancer treatments. Moreover, the

modiﬁcation of the expression of some ion channels is correlated to

the aggressiveness of cancers and are thus studied as potential

cancer biomarkers [8,9]. To the best of our knowledge, none of the

ion channel modulators (mostly small molecules) currently on the

market (e.g. type II diabetes, hypertension, pain, anxiety) [10] are

used for cancer therapy except for the treatment of the side effects

of chemotherapy (treatment of nausea via an antagonist effect on

5HT3 receptors). In our laboratories, we have previously reported

for the ﬁrst time that amphiphilic compounds belonging to the

class of glyco-glycero ether lipids were able to modulate SK3

channels. SK3 channels (KCa2.3; small conductance Kþ channel

activated by cytosolic calcium) [1 1] are overexpressed in some

efﬁcient compounds [14] that selectively inhibits SK3 (73% of

reduction of SK3 current according to patch clamp measurements

at 10

mM) and that reduces SK3-dependent cancer-cell migration

(50% according to a chronic test at 0.3

mM for 24 h). These results

were conﬁrmed in vivo with a human cancer cell xenograft model

in NMRI/nude mice that developed bone metastasis after 8-16

weeks. The treatment of these mice with Ohmline abolished the

formation of bone metastasis without any visible side effects [15].

Subsequently, other analogues of Ohmline featuring different polar

head groups (phospho-saccharide [16], digalactoside [1 7], other

disaccharides [18] or more recently tetrahydropyrine [19]) were

synthesized and were also able to inhibit SK3 ion channels but with

a lesser efﬁciency or a lower selectivity. Opposite to tetraethy-

lammonium (TEA) [20] or apamin [21] that inhibit SK3 respectively

by a cork effect at the pore channel and via an allosteric mecha-

nism, Ohmline proceeds differently because it is not able to displace

apamin from SKCa channels [1 1]. The mechanism of action of

Ohmline starts by its integration in membranes as conﬁrmed by ²H

NMR studies in membrane models using deuterated Ohmline [22].

* Corresponding author.

ꢁ

E-mail address: pjaffres@univ-brest.fr (P.-A. Jaffres).

https://doi.org/10.1016/j.ejmech.2020.1 12894

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

After integration in membranes, Ohmline reduced the order pa-

rameters of rigid membranes (e.g. rich in cholesterol- 30%; deter-

mined by ²H NMR of membranes that include perdeuterated lipids

as probe) whereas it has almost no effect on ﬂuid membrane [22].

According to molecular dynamic studies, the integration of Ohm-

line in cholesterol-containing membranes induced a modiﬁcation

of the equilibrium position of cholesterol embedded in the mem-

brane [22]. Altogether, our previous studies suggest that Ohmline is

able to modify the biophysical properties of cholesterol-rich

membranes and consequently the function of SK3 ion channels

embedded in nanodomains enriched with cholesterol also named

lipid rafts. We have also shown that Ohmline was able to dissociate

Orai1-SK3 channel complexes that are localized in cholesterol-rich

domains of plasma membranes of some cancer cells [15] and to

modulate the action anti-EGFR monoclonal antibodies [23]. Alto-

gether, different hypotheses exist to explain the effect of Ohmline.

1-Ohmline, after its integration in plasma membrane, could

interact directly with SK3 channels and modulate SK3 currents via

allosteric mechanism of action. However, we reported that the

chirality at the sn-2 position of the glycerol has no effect on the

efﬁcacy of Ohmline [1 1]. This observation reduces the pertinence of

this hypothesis since direct protein-antagonist interactions are

frequently very sensitive to the absolute conﬁguration of the

antagonist. 2- Ohmline by modifying the ﬂuidity of rigid mem-

branes could destabilize SK3 channels (functional SK3 channels are

constituted by the assembly - homo or hetero-tetramers- of four

SK3 protein and are sensitive to cholesterol) [23]. 3- We cannot

exclude either that the integration of Ohmline in the membrane

could trigger endocytosis processes by acting on membrane cur-

vature [24] or by other unidentiﬁed mechanism that could reduce

the expression of SK3 channels at the plasma membrane. To

investigate further the distribution of Ohmline at a cellular scale

that could contribute to better understand its mechanism of action,

the preparation of a ﬂuorescent analogue of Ohmline was required.

In this article, we report the synthesis and the ﬂuorescence prop-

erties of three analogues of Ohmline featuring a pyrene moiety

attached at the extremity of a short lipid chain (OP1-3, Fig. 1). Then,

we report their action on SK3 ion channels and ﬁnally the kinetic of

distribution of OP2 at a cellular scale.

2. Results and discussion

2.1. Synthesis

The introduction of a ﬂuorescent probe on a bioactive com-

pound presents the risk of reducing or abolishing its activity. For

this reason, the structure of the ﬂuorescent probe and its location

on the molecular structure is of primary importance. In previous

studies, we have shown the importance of the structure of the polar

head group of the amphiphilic derivatives on their ability to

modulate SK3 channels. This polar head group can be either a

mono/di-saccharide unit or a phosphocholine moiety but in the

latter case (e.g. Edelfosine) [25] higher toxicity was revealed. For

the saccharide moiety, we have shown with Ohmline that the ste-

reochemistry of the glycosylated link between the lactose and the

glycerol unit was essential (only the stereoisomer

b is active [18]).

These results suggest that structural modiﬁcations on the polar

head group could impair its capacity to modulate SK3 channels.

Regarding the sn-2 position of the glycerol unit, we have ﬁrst re-

ported that the conﬁguration of the carbon atom bearing the

methoxy group had no effect on the capacity of Ohmline to

modulate SK3 [1 1]. The presence of an unsubstituted alcohol in sn-2

position of a compound possessing a phosphocholine polar head

group in sn-3 position produced a compound exempt of any activity

on SK3. Accordingly, we would like to keep unchanged the methoxy

group present on the sn-2 position of glycerol. Regarding the

structure of the hydrophobic moiety, a straight and saturated lipid

tail with a minimum of 16 carbon atoms is suitable. We suppose

that this lipid chain acts as an anchor of the molecule in mem-

branes. Altogether, we hypothesized that the best location for the

ﬂuorescent probe could be on the lipid chain. Previously, the

functionalization of the lipid chain of Edelfosine was achieved by a

polyene [26], polyene-yne or BODIPY [27] ﬂuorescent moiety but

the stability of some of these chromophores limited their use.

Accordingly, we have selected pyrene, which was largely employed

to label lipids to study membranes and cell biology [28]. Pyrene is a

hydrophobic ﬂuorescent probe that readily forms excimers

featuring an intense ﬂuorescence (emission at 470 nm) [29]. To

determine the best position of the pyrene moiety in the structure of

Fig. 1. Molecular structure of Ohmline and the ﬂuorescent analogues OP1-3.

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

the lipid chain, we considered that its introduction as a ramiﬁcation

of a straight lipid chain could induced important modiﬁcation of

the supramolecular interactions as previously observed with

branched amphiphiles [30]. On the other hand, the proximity of the

pyrene group to the glycerol would require a di-substituted pyrene

(one to connect pyrene to glyceryl and a second to place a lipid

chain) which is a more difﬁcult option from a synthesis point of

view [31]. Hence, we have selected to place the pyrene moiety at

the extremity of the lipid chain. First, we used pyrene methanol as

commercial precursor because it was previously used to prepare

pyrene conjugates [32,33]. Accordingly (Figs. S1e1), solketal was

alkylated with 11-Bromoundecanol to produce the lipid alcohol 1

which was converted in bromo derivative 2 by using Appel’s re-

action. Pyrene methanol was then used as nucleophile to produce

the pyrene-lipid intermediate 3. However, the deprotection of the

acetal function of 3 with either HCl/methanol or acetic acid (80%)

produced a mixture of compound likely due to the reactive ether

position close to the pyrene moiety. The cleavage of part of this

ether position is likely due to its benzylic position that after pro-

tonation promotes its cleavage. In consequence, we modiﬁed the

synthesis scheme to avoid the incorporation of heteroatom in the

hydrophobic moiety. As shown in Fig. 2, we ﬁrst prepared the

pyrene derivatives 4a-c that feature a straight alkyl chain sepa-

rating the pyrene moiety to the bromine atom.

The constitution of this alkyl chain, that can inﬂuence the

modulation effect on SK3, contains either 4, 11 or 16 methylene

units. 4a was prepared from pyrene-butanol as previously reported

[34] and 4b-c were prepared by adapting reported procedures [35]

using pyrene as substrate (Figs. S2e1). Then, solketal (racemic) was

deprotonated by NaH in toluene and reacted with 4a-c in presence

of NaI to produce 5a-c. The deprotection of the acetal 5b in pres-

ence of 12 M HCl (procedure used for the preparation of Ohmline)

failed in the present case. Accordingly, we used acetic acid to pro-

duce 6a-c in excellent yield. Then, the protection of the primary

alcohol was attempted with trityl protecting group but we faced

low and unreproducible yields. Accordingly, we used t-butyldime-

thylchlorosilane and imidazole in dichloromethane [36] to protect

the more reactive alcohol to produce 7a-c in nearly quantitative

yield without any trace of migration of silyl group at the sn-2 po-

sition as it was previously reported in different reaction conditions

[37]. Of note, a second protocol was developed for the preparation

of 7a by reaction of pyrenebutanol (Figs. S3e1) with silylated gly-

cidol but the yield was low (21%). Then, we applied a methylation

with silver oxide and iodomethane [38] that produced 8a-c in

nearly quantitative yields. The methylation protocol used for the

synthesis of Ohmline (NaH, THF and then methyliodide) was not

efﬁcient. The deprotection of the primary alcohol in presence of n-

tetrabutylammonium ﬂuoride (TBAF) produced 9a-c in 51e93%

yield. The glycosylation of 9a-c was achieved with the same con-

dition than for Ohmline (trichloroacetimidate derivative of per-

acetylated lactose in presence of BF₃) produced 10a-c in 42e58%

yield. Finally, the deprotection of the hydroxyl groups present on

the lactose unit was achieved with a catalytic amount of potassium

carbonate in methanol. After neutralization with Amberlite IR120H

acid resin Ohmline-Pyrene 1e3 (OP1-3) were isolated in 71e93%

yield. As previously reported for the glycosylation reactions

NMR spectra of compounds 5be10b and ﬁnal compounds are

presented in Fig. S4.

2.2. Fluorescence and interaction with liposomes

Then, we have recorded the ﬂuorescence (Fig. 3; gain of the

detector either 600 or 800) of compounds OP1-3 (excitation at

331 nm) in water at different concentration (0.1, 0.2 and 0.4 mM). It

is observed that the ﬂuorescence of the monomers is characterized

by emission ranging from 360 to 440 nm whereas the excimer

emission is characterized by a broad band from 425 to 600 nm

[41e43]. It is clearly observed that OP1, which features the shortest

alkyl spacer (4 methylene units), only exists as monomer at these

concentrations. The ﬂuorescence of OP2 features only the signature

of the monomer at 0.1

mM but at higher concentration, both

monomer and excimer are detected. The presence of the excimer is

attributed to its higher hydrophobicity (the spacer contains 11

methylene units) that promotes its self-assembly in water. For OP3,

which possesses the longest spacer (16 methylene units), the

excimer is predominant even at the lowest concentration sug-

gesting that at 0.1 mM this compound forms aggregates [35].

Then, we investigated the ﬂuorescent properties of compounds

OP1-3 placed in a lipid environment. For that study, we used li-

posomes of different compositions as a model of plasma membrane

and we used two strategies to incorporate OP1-3 in the liposomes.

A ﬁrst protocol (pre-insertion) involved mixing one of the OP

compounds with the other lipids (e.g. DOPC, DOPC/

Cholesterol ꢀ70/30 mol % or Egg-PC) before the formation of the

liposomes. The second protocol involved adding one of the OP

compounds on the liposomes already formed (post-insertion). Ac-

cording to the ﬁrst protocol, one of the OP1-3 compounds was

mixed with either DOPC, DOPC/Cholesterol (70/30 mol %), Egg-PC

or Egg-PC/Cholesterol (70/30 mol %) to form a lipid ﬁlm that pro-

duced liposomal solutions after hydration and sonication. In these

formulations, the ﬁnal concentration of OP compounds was 0.2

and the concentration of lipid either 8 M, 24 M and 40 mM.

Accordingly, the lipid/OP molar ratios are 40, 120 and 200. For the

second protocol (post-insertion) one of the OP compounds, dis-

solved in a few

mL of methanol, is added on liposomal solution

(3 mL) at the same composition and concentration than those used

for the pre-insertion protocol. The pre-insertion protocol aims to

mimic the homogeneous distribution of OP compounds in the li-

posomes whereas the post-insertion protocol allows taking into

account the migration process of OP1-3 compounds from the so-

lution to the liposomes.

For the pre-insertion procedure (Fig. 4, left column, gain of the

detector 750) with compounds OP2 and OP3 the ﬂuorescence in-

tensity of the monomer increases when the lipid/OP ratios increase

from 40 to 200 whereas the ﬂuorescence of the excimer decreases.

This shows that both compounds, whose ﬂuorescence in water are

very low (Fig. 3), are inserted in the liposomes and that they only

exist as monomer for highest dilution (high lipid/OP ratio of 200).

When the lipid/OP ratio decreases the ﬂuorescence of both

monomers and excimers are observed in the liposomes suggesting

some proximity of the pyrene-based compounds.

For OP1 the result is completely different. Only the ﬂuorescence

of the monomer is observed irrespective of the two lipid/OP1 ratios

and the intensity of ﬂuorescence is almost identical in both cases.

These results suggest that either OP1 is integrated in the liposomes

but it is fully disperse thus rendering the proximity of the pyrene

probe unlikely and the excimer absent or, more likely, OP1 is, at this

involving per-acetylated lactose, only the

b-stereoisomer was

formed likely due to the assistance of the adjacent acetyl group

[39]. This stereochemistry was determined on OP1-3 by ¹³Ce¹H

NMR or “gated decoupling” experiment. The value of 160 Hz for the

¹JC₁-H₁coupling constant indicates an axial proton i.e the

b-ste-

reoisomer. Moreover, the chemical shifts (¹³C NMR) of the anomeric

concentration (0.2 mM), fully soluble in water. Therefore, OP1 es-

carbons are between 103.8 and 102.9 ppm for OP-1, OP-2 and OP-3,

capes from the liposomes to be present in water.

which are characteristics of

tween 99 and 101 ppm is an indication of an

-linkage whereas chemical shift be-

-linkage [40]. All the

For the post-inserted procedure (Fig. 4, right column), the

ﬂuorescence intensity of the monomer is, lower than for the pre-

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

Fig. 2. Synthesis of the ﬂuorescent Ohmline-Pyrene OP1-3.

inserted procedure (for the ratio egg-PC/OP 200) for OP2 and OP3.

This expected result is explained by the fact that according to the

post-insertion protocol, a full distribution within the two leaﬂets

requires ﬂip-ﬂop processes that likely take a longer time. It repre-

sents almost 65% of the signal obtained for the pre-insertion

method for OP2 and 35% for OP3. Since the ﬂuorescent intensity

of the monomer of OP2 and OP3 in water is very low (Fig. 3B-C),

this result suggests that OP2-3 compounds are integrated, at least

partly, in the liposome bilayer. The evolution of the ratio of OP2 or

OP3 existing as monomer or excimer in liposomes can be evaluated

by measuring the ratio of intensity I₃₇₆/I₄₈₄. For lipid/OP ratio equal

to 200 (highest dilution), the ratio of intensity I₃₇₆/I₄₈₄is equal to 21

for the pre-insertion procedure for both compounds and to 8.5 for

OP2 and 4 for OP3 for the post-insertion protocol. This result in-

dicates that: 1- the post-insertion protocol produces a more het-

erogeneous distribution of OP2 and OP3 within the lipid bilayer

since the signal of the excimer is more intense when compared to

the pre-insertion protocol that promotes homogeneous distribu-

tion of the OP compounds within the liposome bilayer. 2- The more

hydrophobic nature of OP3 suggests that in water OP3 is more

aggregated and consequently could integrate the liposomes with a

higher aggregation state or feature lower mobility within the

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

Fig. 3. Fluorescence spectra of compounds OP1-3 after excitation at 331 nm and at different concentrations in water: 0.1

OP1 (gain of the detector 600); B) OP2 (gain of the detector 800); C) OP3 (gain of the detector 800).

mM (solid line), 0.2 mM (dashed line) 0.4 mM (dotted line) A)

bilayer thus producing a relative lower value for the ratio I₃₇₆/I₄₈₄

when compared to OP2. To evaluate this second hypothesis, we

recorded the size of the nano-objects by dynamic light scattering

(DLS) for the three samples OP1-3 in water at 4 different concen-

suggesting that the composition of the liposomal formulation has a

weak effect on the incorporation of these amphiphiles irrespective

the protocol used (pre or post insertion). Altogether, the ﬂuores-

cence analysis of OP1-3 in different liposomal compositions in-

dicates that OP1 is likely not integrated in the liposomes. On the

contrary, OP2 and OP3 are able to integrate in the liposomes irre-

spective to both the protocol used (pre or post-insertion) and the

composition of the liposomes. However, the integration rates of

OP2 and OP3 are slightly higher with the pre-insertion protocol.

trations (40

mM, 10

mM and 1

mM; Table S1). We found that only

OP2 and OP3, at the concentration of 40

M and 10 M, produced

nano-objects with a repeatable size (Table S1). We observed that at

these two concentrations OP2 produced nano-objects featuring a

size close to 50 nm whereas OP3 produced larger nano-objects

(close to 160 nm). These results are therefore consistent with the

interpretation of the ﬂuorescence data.

For OP1, the post-insertion procedure produced similar ﬂuo-

rescence spectra than with the pre-insertion procedure. In that case

also, the lipid/OP1 ratio has no effect on the ﬂuorescence spectra.

These results suggest that OP1 is likely not integrated in the lipo-

somes and the absence of excimer suggests that at this concen-

2.3. Effect of OP1-3 on SK3

Then, compounds OP1-3 were evaluated for their capacity to

modulate the activity of SK3 channels using the whole cell

conﬁguration of the patch clamp technique. Using this technique

HEK293 cells (cells that overexpressed SK3 channel) were placed

tration (0.2 mM) OP1 is not aggregated in water.

under acute condition (high concentration of OP2 10 mM, and 1 min

Similar experiments were carried out with different liposomal

formulations. For OP1, which seems to be mainly localized in water

(Fig. 4A), the same conclusions were obtained with Egg-PC/

cholesterol (70/30 mol ratio) liposomal solution. Only the signa-

ture of the monomer was detected and the intensity ratio were

identical when using the pre-insertion or post-insertion protocols

(Figs. S5e1). For OP2 and OP3, we tested EggPC/Cholesterol (70/

30), DOPC and DOPC/Cholesterol 70/30 (Figs. S6e1 and Figs. S7e1).

Similar trends than those reported in Fig. 4B and C were observed

until the steady state reached). Fig. 5A shows typical currents

recorded at membrane potentials varying from ꢀ100 to þ100 mV

for 500 ms in control condition, after application of OP2 com-

pounds, and after application of apamin at the end of experiment.

Apamin was used to inhibit SK3 residual current which was not

sensitive to OP2. As illustrated OP2 reduced most of the amplitude

of SK3 currents but not totally as shown by the additional inhibition

of residual SK3 currents by apamin. Fig. 5B shows that OP2 (10

mM)

reduced SK3 currents by ꢀ71.3% ( 13.3%; N ¼ 6) and with a

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

Fig. 4. Fluorescence spectra (excitation at 331 nm) of OP1-3 (0.2 mM) integrated in egg-PC- based liposomes at two concentrations 8 mM and 40 mM producing ﬁnal ratios egg-PC/

OP ¼ 40 (solid line) and 200 (dashed line); gain of the detector: 750. Two protocols were used: pre-insertion (left column) and post-insertion (right column) A) OP1, B) OP2, C) OP3.

maximal effect of OP2 compound attempted in 1 min. In contrast to

OP2, OP1 and OP3 had no effect on SK3 currents (Fig. S8). These

results emphasize the importance of the length of the linker be-

tween the pyrene moiety and the glycerol backbone since only OP2,

featuring 11 methylene units in the spacer, is efﬁcient. The impor-

tance of the aliphatic chain length between the pyrene and the

glycerol unit on the modulation of SK3 could be explained as

follow: 1- in the case of a short aliphatic chain (OP1), the hydro-

phobic domain is too short to localized OP1 in the bilayer as

demonstrated by ﬂuorescence. Accordingly, OP1 do not modify the

lipid-bilayer biophysics and therefore has no effect on SK3 function.

2- in the case of a long aliphatic chain (OP3), it is likely that the

hydrophobic domain length (aliphatic chain and pyrene) is longer

than the average of the lipid chain of phospholipid or sphingosine

derivatives present in bio-membranes. In consequence, the hy-

drophobic chain of OP3 must adopt either a U-shape conformation

to keep its hydrophobic chain within one leaﬂet or has a hydro-

phobic domain that cross over the inter-leaﬂet area; in both cases

the effect on bio-membrane biophysics will be different to OP2,

which has a hydrophobic chain length almost similar to those of the

phospholipid present in the membrane.

(Fig. 5C). OP2 signiﬁcantly reduces the migration of SK3þ cells at

M (ꢀ32.5% 4.8%) but not at 0.3 M in contrast to Ohmline,

which is effective at 0.3 M. Interestingly, OP2 has no effect on the

migration of SK3- cells demonstrating that this compounds inhibits

SK3-dependent cancer cell migration as already observed with

Ohmline [14]. In addition, the non-toxicity of the compounds OP1-

3 was conﬁrmed at 1 mM by MTT assays (Fig. S9). Altogether, these

results show that although having a slightly decrease efﬁciency in

SK3 currents and SK3-dependent cancer cell migration compare to

Ohmline, OP2 is a molecule that opens major opportunities to

investigate the cellular distribution of Ohmline and is the ﬁrst

ﬂuorescent analogue of Ohmline reported so far.

2.4. In vitro ﬂuorescence imaging

To further understand OP2 effects on cells, we imaged OP2

excimers in breast cancer cells (MDA-MB-231) that had been

treated with OP2. Two types of experiments were performed: (1)

cells were imaged following addition of OP2 to the medium and, (2)

cells were left in an OP2-containing medium for 24 h before im-

aging. In the former case, the serum supplemented medium used

for cell culture was replaced with serum-free medium for OP2

Then, we studied the effect of OP2 on the migration of MDA-

MB-435s that expressed (SK3þ) or not SK3 channel (SK3-)

treatment (1 mM OP2 total concentration); in the latter, OP2 was

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

Fig. 5. Effects of OP2 in SK3 currents and in SK3-dependent cancer cell migration. A) Representative whole-cell SK3 currents recorded at membrane potentials varying

from þ100 mV to ꢀ100 mV for 500 msec from a constant holding potential of 0 mV at pCa 6, before and after application of 10 M OP2, then with the addition of 0.1 M apamin

(SK3 peptide blocker) in HEK293T cells expressing recombinant rat SK3. B) Analyses of OP2 effect on SK3 currents at 0 mV at 10 M, a membrane potential where the current is only

carried by SK3 channels. Left, graphs showing a representative time course of inhibitory effect of OP2 on SK3 currents at 0 mV. Right, histograms showing the % of SK3 currents

block by OP2 when the steady-state inhibition was reached at I ¼ 0 mV. Lines indicate the median, each point represents the % of SK3 currents sensitive to OP2 of each cell analyzed

(N ¼ 6, **p < 0.01, Wilcoxon signed rank test). C) Effect of OP2 in SK3-dependent cell migration. Results are relative to control condition and are expressed as mean S.E.M. Left,

histogram showing the effect of OP2 and Ohmline in the cell migration of MDA-MB-435s expressing endogenous SK3 channels (SK3þ). N ¼ 3, n ¼ 6 (CTL vs Ohmline 0.3

**p < 0.01, CTL vs OP2 1

mM *p < 0.05, Dunn’s multiple comparison test). Right, histogram showing no effect of OP2 in the cell migration of MDA-MB-435s not expressing SK3

channel (SK3-). N ¼ 3, n ¼ 9 (p > 0.05, Mann-Whitney test).

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

added in serum supplemented medium and cells were incubated

the design of amphiphiles labelled with a pyrene moiety. To this

for a further 24 h to test cellular viability (10

mM OP2 total con-

goal, we have incorporated a pyrene moiety at the u-position of the

centration). OP2 incorporated into the cells within 45 min

following treatment (Fig. 6a) and with an abundant distribution in

the plasma membrane. At that time, background ﬂuorescence could

still be detected in the medium, indicating that OP2 uptake was not

lipid chain bonded via an ether linkage to glycerol. The number of

methylene units separating glycerol from the pyrene moiety was

either 4, 11 or 16 leading respectively, after a seven-steps sequence,

to the compounds Ohmline-Pyrene 1e3 (OP1-3). Then, we report

the interaction of these compounds with liposomes as a mimic of

plasma membrane. Thanks to the singular ﬂuorescent properties of

pyrene derivatives that informed on its aggregation state (isolated

species versus excimers) and based on two types of protocols for

the incorporation of the ﬂuorescent compounds in the liposomes

(pre or post-insertion), we found that OP1 (compound with the

shortest lipid chain) is not integrated in the liposome. On the

opposite, OP2 and OP3, following the post-insertion protocol,

integrate within liposomes with some aggregation. We also found

that OP2 integrates in the liposomes more efﬁciently thus

emphasizing the effect of the length of the tether between the

glycerol and the pyrene moiety on the supramolecular properties of

this series of compounds. The three compounds OP1-3 were tested

as modulators of SK3. We found that only OP2 (possessing 11

yet complete. 24 h after OP2 treatment cells (Fig. 6b; OP2 at 10 mM)

the population of ﬂuorescent cells had increased, indicating cells

were healthy and viable, and no ﬂuorescence background emission

from the excimers could be observed from the medium. We note

that the concurrent presence of OP2 excimers and monomers in

solution was already observed at a concentration 25 times lower

(0.4 mM, Fig. 3b, dotted line) and that the proportion of excimers to

monomers increases with compound concentration. This, together

with the large excimer-emission background observed at short

times after addition of OP2 at 10 mM, strongly suggests that most of

the compound is in excimers form at this concentration.

To further understand the dynamics of OP2 cellular uptake, we

imaged the same region of the culture dish every 15 min, starting

1 h after treatment (Fig. 6cek). Background excimer-emission was

negligible 1.5 h after treatment (Fig. 6e), suggesting complete

incorporation into the cells. OP2 accumulation at the periphery of

the nucleus was visible 2 h after treatment (Fig. 6g, yellow arrows)

while the nuclei were not ﬂuorescent. This accumulation was still

observed 24 h after treatment (Fig. 6b, yellow arrows). Altogether,

this study shows that OP2 is rapidly incorporated in the lipid

bilayer, since we clearly observed cell internalization within 45 min

methylene units) is able to modulate SK3 (ꢀ71.3% 13.3 at 10

and to reduce SK3 dependent cancer cell migration (ꢀ32.5% 4.8%

at 1 M). OP2 is therefore the ﬁrst ﬂuorescent analogue of Ohmline,

mM)

which is able to modulate SK3 channel. In the last part of this study,

we used ﬂuorescent microscopy to track OP2 over time. We found

that, in agreement with patch clamp measurements, OP2 is readily

integrated in the plasma membranes and, after 2 h, cell internali-

zation occurred. The exact location of OP2 into the cell will need

further study but opens a new perspective for the modulation the

SK3 channels localized in intercellular compartments.

after addition of OP2 even at the lowest concentration (1 mM). This

rapid integration of OP2 in the plasma membrane is consistent

with the patch clamp measurements. A second point is that OP2,

after after ﬁrst incorporating into the plasma membrane (within 2

hours) , is then internalized close to the nucleus. However, it is

difﬁcult to accurately identify the cellular region where OP2 ac-

cumulates. In a previous study focused on ﬂuorescent analogues of

Edelfosine, Mollinedo et al. reported that these ﬂuorescent ana-

logues of Edelfosine localized in mitochondria and that the inter-

nalization was mediated by lipid rafts [27]. In the case of Ohmline

and OP2, further studies will be needed to establish their exact

location within the cell, but taking into account that SK3 has been

recently identiﬁed in mitochondria [44], the development of drugs

4. Experimental protocols

4.1. Chemistry

4.1.1. General experimental protocols

Solvents were dried with a solvent puriﬁcation system MBraun-

SPS (toluene, CH₂Cl₂, THF) or freshly distilled on appropriate driers.

All compounds were fully characterized by ¹H (500.133, 399.922 or

300.131 MHz) and ¹³C NMR (125.803 or 75.474 MHz) NMR spec-

troscopy (Bruker AMX-3 300, Avance 400, Avance 500 and DRX 500

that could modulate intracellular target represents

potential.

a great

Spectrometers). Chemical shifts

d are given in ppm. Coupling con-

In order to test if the decrease of the activity of SK3 channel by

OP2 may be due to a decrease in the expression of SK3 protein at

the plasma membrane, we used the cell-surface protein expression

with proteinase K cleavage assay as we described previously [25]. In

the absence of proteinase K, SK3 ran at a molecular mass of 75 kDa,

consistent with the full-length SK3 protein (Fig. S10). The presence

of proteinase K lead to a cleavage of SK3 protein located at the

plasma membrane resulting to an apparent molecular mass of

55 KDa. Ohmline and OP2 treatments both revealed a 55 KDa

cleaved SK3 band in the presence of proteinase K demonstrating

that these compounds did not change the expression of SK3 protein

at the plasma membrane.

stants J are given in hertz. The following abbreviations were used: s

for singlet, d doublet, t triplet, q quadruplet, qt quintuplet, m

multiplet, dd doublet of doublet, dt doublet of triplets. When

needed, ¹³C Heteronuclear HMQC and HMBC were used to unam-

biguously establish molecular structures. Mass spectrometry ana-

lyses were performed by Bruker Autoﬂex MALDI TOF-TOF (Brest

University) Matrix used UTL HCCA. The size of the nano-objects

were determined at 25 ^ꢁC in water by DLS with a NanoZS equip-

ment (Malvern). The melting points were measured by a koﬂer

bench. Molecular sieve (5 Å) was used when indicated; Lactose

trichloroacetimidate was prepared following reported procedure

[45]. The pyrene derivative 4a was prepared following a reported

procedure [35].

3. Conclusion

4.1.2. General procedure for the insertion of the 2,2-dimethyl-1,3-

dioxolane (compounds 5a-c)

Ohmline is a neutral amphiphilic compound belonging to ether-

lipids, which was found, at a non-toxic concentration, to inhibit SK3

channel and consequently to reduce cancer cell mobility. Previous

studies have shown that this compound was integrated in mem-

branes and increased the ﬂuidity of rigid membranes. To get further

information relative to the distribution of this compound at a

cellular scale, we report herein the synthesis of ﬂuorescent ana-

logues of Ohmline and also propose a general synthesis scheme for

NaH (5.5 eq.) was placed under nitrogen in a three-neck-round

bottom ﬂask ﬁtted with a reﬂux condenser and an additional fun-

nel. 10 mL of toluene were added. Solketal (5 eq.) was added and

the mixture was reﬂuxed for 1 h. At room temperature, 4a-c (1eq.)

was slowly added without solvent. NaI was equally added without

solvent (1 eq.) and the mixture was then reﬂuxed for 1 days. The

reaction was quenched by few mL of MeOH and the solution was

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

Fig. 6. OP2 cellular uptake. OP2 is internalized in MDA-MB-231 cells following its addition to the culture medium. (a) 45 min after addition of OP2 at 1

Upon internalization, OP2 distributes in the plasma membrane and in the cytoplasm, but it is not internalized in the cell nucleus. (b) Cells imaged 24 h after treatment with OP2

(10 M total concentration), and ﬁnishing at (j)

M), conﬁrming cellular viability. (cej) Cells were imaged every 15 min starting at (c) t₀¼ 60 min following addition of OP2 (1

m. Fluorescence

mM (total concentration):

t_f¼ 165 min after treatment. OP2 accumulates in patches around the nucleus that start developing 2 h after treatment (g, yellow arrows). (aej) scale bar is 50

excitation at 340 nm and emission recorded from 468 to 552 nm. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the Web version of this

article.)

extracted with Et₂O. The organic phase was washed with water,

dried over MgSO₄, ﬁltered and concentrated to give the crude

compounds 5a-c. The product was puriﬁed by chromatography on

silica gel (Eluent: toluene/Et₂O (9/1)) to give the pure compounds

5a-c between 32% and 88% yield as a yellow oil.

fatty chain); 1.40 (s, 3H, CH₃solketal); 1.34 (s, 3H, CH₃solketal); ¹³

NMR (CDCl₃, 75.474 MHz): 136.9 (C_q); 131.60 (C_q); 131.09 (C_q);

129.92 (C_q); 128.78 (C_q); 127.67e125.94 (CH pyrene); 125.25 (C_q);

124.98e123.58 (CH pyrene); 109.52 (C_qsolketal); 74.89 (CH sn-2);

72.09 (CH₂sn-1); 71.73 (CH₂

a O); 67.04 (CH₂sn-3); 33.44 (CH₂a

pyrene); 29.75 (CH₂b pyrene); 28.48 (CH₂fatty chain); 26.93 (CH₃

acetal); 25.57 (CH₃acetal); MALDI TOF m/z calculated 388.499

found M^þꢂ388.313.

4.1.2.1. 2,2-Dimethyl-4-(((4-(pyren-1-yl)butyl)oxy)methyl)-1,3-

dioxolane (5a). Using the aforementioned general procedure, the

compound 5a was prepared in 32% yield (480 mg). Rf (Eluent:

toluene/Et₂O (95/5)): 0.47; ¹H NMR (CDCl₃, 300.131 MHz):

8.27e8.25 (d, 1H, H_ar, J_HH¼ 8 Hz); 8.16e7.96 (m, 7H, H_ar); 7.86e7.84

(d, 1H, H_ar, J_HH¼ 8 Hz); 4.27e4.21 (qt, 1H, J_HH¼ 12 Hz, J_HH2¼ 6 Hz,

CH sn-2); 4.04e4.01 (dd, 1H, J_HH¼ 6 Hz, J_HH¼ 8 Hz, H_aCH₂sn-3);

3.71e3.68 (dd, 1H, J_HH¼ 6 Hz, J_HH¼ 8 Hz, H_bCH₂sn-3); 3.57e3.38

(m, 4H, CH₂sn-1 þ CH₂a-solketal); 3.35 (t, 2H, J_HH¼ 7.7 Hz, CH₂a

pyrene); 1.96e1.88 (m, 2H, CH₂fatty chain); 1.79e1.72 (m, 2H, CH₂

4.1.2.2. 2,2-Dimethyl-4-(((11-(pyren-1-yl)undecyl)oxy)methyl)-1,3-

dioxolane (5b). Using the aforementioned general procedure, the

compound 5b was prepared in 88% yield (5.2 g). Rf (Eluent: toluene/

Et₂O (9/1)): 0.37; ¹H NMR (CDCl₃, 399.922 MHz): 8.3 (d, 1H, H_ar,

J_HH¼ 9.2 Hz); 8.17e7.86 5 (m, 8H, H_ar); 4.28 (qt,1H, J_HH¼ 6.4 Hz, CH

sn-2); 4.07 (dd,1H, J_HH¼ 6.4 Hz, J_HH¼ 8.4 Hz, H_aCH₂sn-3); 3.74 (dd,

1H, J_HH¼ 6.4 Hz, J_HH¼ 8.4 Hz, H_bCH₂sn-3); 3.65 (t, 2H, J_HH¼ 6.8 Hz,

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

CH₂

-OH); 3.53e3.39 (m, 4H, CH₂sn-1 þ CH₂

-solketal); 3.34 (t,

pyrene); 72.24 (CH₂sn-1); 71.68 (CH₂

solketal); 70.41 (CH sn-2);

pyrene);

2H, J_HH¼ 7.6 Hz, CH₂

pyrene); 1.85 (m, 2H, CH₂fatty chain); 1.6

64.08(CH₂sn-3); 33.47 (CH₂pyrene); 31.81 (CH₂b

(m, 2H, CH₂fatty chain); 1.5 (m, 2H, CH₂fatty chain); 1.45 (s, 3H,

CH₃solketal); 1.39 (s, 3H, CH₃solketal); 1.29 (m, 14H, CH₂fatty

chain); ¹³C NMR (CDCl₃, 75.474 MHz): 137.3 (C_q); 131.38 (C_q); 130.9

(C_q); 129.63 (C_q); 127.56 (C_q); 127.49e124.69 (CH pyrene); 125.03

(C_q); 124.55e123.48 (CH pyrene); 109.31 (C_qsolketal); 74.73 (CH sn-

2); 71.8 (CH₂sn-1); 71.77 (CH₂a O); 66.90 (CH₂sn-3); 32.57 (CH₂a

pyrene); 31.91 (CH₂b pyrene); 29.79e29.41 (CH₂fatty chain); 26.75

(CH₃acetal); 26.01 (CH₂fatty chain); 25.40 (CH₃acetal); MALDI TOF

m/z calculated 486.313 found [MþH]^þ487.350.

29.69e29.31 (CH₂fatty chain); 25.92 (CH₂fatty chain); MALDI TOF

m/z calculated 446.282 found M^þC446.454.

4.1.3.3. 3-((16-(Pyren-1-yl)hexadecyl)oxy)propane-1,2-diol

(6c).

Using the aforementioned general procedure, the compound 6c

was prepared in 95% yield (4.4 g). Rf TLC (Eluent: toluene/Et₂O (3/

7)): 0.4; Melting point: 90 ^ꢁC; ¹H NMR (CDCl₃, 300.131 MHz): 8.3 (d,

1H, H_ar, J_HH¼ 9.0 Hz); 8.17e7.97 (m, 7H, H_ar); 7.87 (d, 1H,

J_HH¼ 7.6 Hz, H_ar); 3.87 (m, 1H, CH sn-2); 3.85e3.61 (m, 2H, CH₂sn-

3); 3.54e3.43 (m, 4H, CH₂sn-1 þ CH₂

a-solketal); 3.33 (t, 2H,

4.1.2.3. 2,2-Dimethyl-4-(((16-(pyren-1-yl)hexadecyl)oxy)methyl)-

1,3-dioxolane (5c). Using the aforementioned general procedure,

the compound 5c was prepared in 89% yield (5 g). Rf (Eluent:

toluene/Et₂O (95/5)): 0.53; ¹H NMR (CDCl₃, 300.131 MHz): 8.3 (d,

1H, H_ar, J_HH¼ 12 Hz); 8.17e7.86 (m, 8H, H_ar); 4.25 (qt,1H, J_HH¼ 7 Hz,

CH sn-2); 4.07 (dd, 1H, J_HH¼ 6.4 Hz, J_HH¼ 8.4 Hz, H_aCH₂sn-3); 3.74

(dd, 1H, J_HH¼ 6.4 Hz, J_HH¼ 8.4 Hz, H_bCH₂sn-3); 3.53e3.39 (m, 4H,

CH₂sn-1 þ CH₂a-solketal); 3.34 (t, 2H, J_HH¼ 7.6 Hz, CH₂a pyrene);

1.86 (m, 2H, CH₂fatty chain); 1.6 (m, 2H, CH₂fatty chain); 1.5 (m,

2H, CH₂fatty chain); 1.45 (s, 3H, CH₃solketal); 1.39 (s, 3H, CH₃

solketal); 1.26 (s, 21H, CH₂fatty chain); ¹³C NMR (CDCl₃,

75.474 MHz): 137.5 (C_q); 131.61 (C_q); 131.07 (C_q); 129.83 (C_q);

J_HH¼ 8 Hz, CH₂a pyrene); 2.09 (s, 2H, OH); 1.87 (m, 2H, CH₂fatty

chain); 1.56 (m, 2H, CH₂fatty chain); 1.47 (m, 2H, CH₂fatty chain);

1.29 (m, 21H, CH₂fatty chain); ¹³C NMR (CDCl₃, 75.474 MHz):

137.40 (C_q); 131.52 (C_q); 131.01 (C_q); 129.73 (C_q); 128.66 (C_q);

127.59e125.79 (CH pyrene); 125.14 (C_q); 124.82e123.58 (CH pyr-

ene); 72.41 (CH₂sn-1); 71.90 (CH₂

solketal); 70.64 (CH sn-2);

pyrene);

64.24 (CH₂sn-3); 33.68 (CH₂pyrene); 32.02 (CH₂

29.92e29.56 (CH₂fatty chain); 26.13 (CH₂fatty chain); MALDI TOF

m/z calculated 516.360 found M^þC516.476.

4.1.4. General procedure for the primary alcohol protection with

tert-butyldimethylsilyle chloride (compounds 7a, b and c)

128.80(C_q);

124.87e123.65 (CH pyrene); 109.47 (C_qsolketal); 74.88 (CH sn-2);

72.01 (CH₂sn-1); 71.94 (CH₂O); 67.06 (CH₂sn-3); 33.75 (CH₂

pyrene); 32.08 (CH₂pyrene); 29.96e29.59 (CH₂fatty chain);

127.65e125.85

(CH

pyrene);

125.28

(C_q);

A solution of tert-Butyl(chloro)dimethylsilane (1.3 eq.) and

imidazole (2.5 eq.) previously mixed in anhydrous CH₂Cl₂(20 mL)

were added in solution of 6a-c (1eq.) in CH₂Cl₂(20 mL). The

mixture was stirred for 2 h at 40 ^ꢁC. The mixture was poured into

water (300 mL) and extracted with Et₂O. The combined ether ex-

tracts were washed with brine, dried over Na₂SO₄, ﬁltered and

concentrated to give the crude compounds 7a-c as yellow wax

between 96% and quantitative yield. These compounds were used

in the next step without further puriﬁcation.

26.90 (CH₃acetal); 26.18 (CH₂fatty chain); 25.56 (CH₃acetal);

MALDI TOF m/z calculated 556.392 found M^þꢂ556.559.

4.1.3. General procedure for the acetal function deprotection

(compounds 6a-c)

Acetic acid 80% (60 mL) and 15 mL of H₂O were added to 5a-c (1

eq.). The mixture was stirred for 3 h at 50 ^ꢁC. Acetic acid and water

were distilled under vacuum to produce compounds 6a-c between

88 and 100% yield as yellow solid. These compounds were used in

the next step without further puriﬁcation.

4.1.4.1. 1-((tert-Butyldimethylsilyl)oxy)-3-((4-(pyren-1-yl)butyl)oxy)

propan-2-ol (7a). Using the aforementioned general procedure, the

compound 7a was prepared in 96% yield (0.480 g). Rf TLC (Eluent:

toluene/ethyl acetate (4/1)): 0.6; ¹H NMR (CDCl₃, 399.922 MHz):

8.28e8.26 (d, 1H, H_ar, J_HH¼ 9.2 Hz); 8.16e7.96 (m, 7H, H_ar);

7.87e7.84 (d,1H, J_HH¼ 7.8 Hz, H_ar); 3.83e3.78 (qt,1H, J_HH¼ 10.9 Hz,

J_HH¼ 5.3 Hz, CH sn-2); 3.68e3.41 (m, 6H, CH₂sn-3 þ CH₂sn-1 þ CH₂

4.1.3.1. 3-((4-(Pyren-1-yl)butyl)oxy)propane-1,2-diol

(6a).

Using the aforementioned general procedure, the compound 6a

was prepared in 88% yield (0.380 g). Rf TLC (Eluent: toluene/Et₂O

(3/7)): 0.2; ¹H NMR (CDCl₃, 399.922 MHz): 8.27.8.25 (d, 1H, H_ar,

J_HH¼ 9.0 Hz); 8.18e7.97 (m, 7H, H_ar); 7.86e7.84 (d, 1H, J_HH¼ 7.7 Hz,

H_ar); 3.88e3.83 (qt, 1H, J_HH¼ 5.6 Hz, CH sn-2); 3.71e3.32 (m, 8H,

CH₂sn-3 þCH₂sn-1 þ CH₂a-solketal þ CH₂a pyrene); 2.09 (s, 2H,

OH); 1.94e1.87 (m, 2H, CH₂fatty chain); 1.77e1.71 (m, 2H, CH₂fatty

chain); ¹³C NMR (CDCl₃, 75.474 MHz): 136.63 (C_q); 131.50 (C_q);

130.96 (C_q); 129.89 (C_q); 128.67 (C_q); 127.58e125.88 (CH pyrene);

125.15 (C_q); 124.94e123.40 (CH pyrene); 72.39 (CH₂sn-1); 72.39

(CH₂a O); 71.56 (CH₂a solketal); 70.68 (CH sn-2); 64.14 (CH₂sn-3);

33.27 (CH₂a pyrene); 29.59 (CH₂b pyrene); 28.28 (CH₂fatty chain);

-solketal); 3.36 (t, 2H, J_HH¼ 7.8 Hz, CH₂a pyrene); 1.96e7.88 (m,

2H, CH₂

b pyrene); 1.79e1.73 (m, 2H, CH₂b solketal); 0.87 (m, 9H,

CH₃Si-t-Bu); 0.04 (m, 6H, CH₃Si); ¹³C NMR (CDCl₃, 75.474 MHz):

136.72 (C_q); 131.45 (C_q); 130.93 (C_q); 129.81 (C_q); 128.62 (C_q);

127.56e125.83 (CH pyrene); 125.11 (C_q); 124.85e123.41 (CH pyr-

ene); 71.64 (CH₂sn-1); 71.39 (CH₂a solketal); 70.73 (CH sn-2); 64.13

(CH₂sn-3); 33.28 (CH₂

a pyrene); 29.69 (CH₂fatty chain); 28.34

(CH₂fatty chain); 25.93 (CH₃Si-t-Bu); ꢀ5.36 (CH₃Si); MALDI TOF

m/z calculated 462.259 found M^þC462.344 and [MþNa]^þ485.335,

theoretical value for [MþNa]^þ485.248.

4.1.4.2. 1-((tert-Butyldimethylsilyl)oxy)-3-((11-(pyren-1-yl)undecyl)

oxy)propan-2-ol (7b). Using the aforementioned general proced-

ure, the compound 7b was prepared in 99% yield (4.5 g). Rf TLC

(Eluent: toluene/ethyl acetate (4/1)): 0.57; ¹H NMR (CDCl₃,

399.922 MHz): 8.3 (d, 1H, H_ar, J_HH¼ 9.2 Hz); 8.17e7.97 (m, 7H, H_ar);

7.87 (d, 1H, J_HH¼ 7.6 Hz, H_ar); 3.81 (m, 1H, CH sn-2); 3.64 (m, 2H,

CH₂sn-3); 3.46e3.42 (m, 4H, CH₂sn-1 þ CH₂a-solketal); 3.34 (t, 2H,

4.1.3.2. 3-((11-(Pyren-1-yl)undecyl)oxy)propane-1,2-diol

(6b).

Using the aforementioned general procedure, the compound 6b

was prepared in quantitative yield (5.5 g). Rf TLC (Eluent: toluene/

Et₂O (1/1)): 0.15; Melting point: 56 ^ꢁC; ¹H NMR (CDCl₃,

399.922 MHz): 8.3 (d, 1H, H_ar, J_HH¼ 9.2 Hz); 8.17e7.97 (m, 7H, H_ar);

7.87 (d, 1H, J_HH¼ 7.6 Hz, H_ar); 3.86 (m, 1H, CH sn-2); 4.07 (m, 2H,

CH₂sn-3); 3.50e3.41 (m, 5H, CH₂sn-1 þ CH₂a-solketal); 3.34 (t, 2H,

J_HH¼ 8 Hz, CH₂

a pyrene); 2.36 (s, 2H, OH); 1.85 (m, 2H, CH₂b

J_HH¼ 8 Hz, CH₂

pyrene); 2.32 (s, 2H, OH); 1.85 (m, 2H, CH₂

pyrene); 1.56 (m, 2H, CH₂b solketal); 1.54 (m, 2H, CH₂g solketal);

1.36 (m, 14H, CH₂fatty chain); 0.89 (m, 9H, CH₃Si-t-Bu); 0.069 (m,

6H, CH₃Si); ¹³C NMR (CDCl₃, 75.474 MHz): 136.97 (C_q); 131.25 (C_q);

130.74 (C_q); 129.48 (C_q); 128.85 (C_q); 128.38e125.46 (CH pyrene);

125.14 (C_q); 124.89e123.23 (CH pyrene); 71.41 (CH₂sn-1); 71.31

Solketal); 1.5 (m, 2H, CH₂

pyrene); 1.45 (m, 2H, CH₂g Solketal);

1.38e1.29 (m, 12H, CH₂fatty chain); ¹³C NMR (CDCl₃, 75.474 MHz):

137.18 (C_q); 131.31 (C_q); 130.80 (C_q); 129.54 (C_q); 129.33 (C_q);

128.91e125.18 (CH pyrene); 124.94 (C_q); 124.60e123.35 (CH

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

(CH₂

solketal); 70.52 (CH sn-2); 63.97(CH₂sn-3); 33.37 (CH₂

131.33 (C_q); 130.82 (C_q); 129.56 (C_q); 128.92 (C_q); 128.47e125.20

(CH pyrene); 124.97 (C_q); 124.62e123.36 (CH pyrene); 80.99 (CH

pyrene); 31.71 (CH₂b pyrene); 29.66e29.34 (CH₂fatty chain); 25.97

(CH₂fatty chain); 25.77 (CH₃Si-t-Bu); ꢀ5.55 (CH₃Si); MALDI TOF

m/z calculated 560.369 found [MþNa]^þ583.515, Theoretical value

for [MþNa]^þ583.358.

sn-2); 71.58 (CH₂sn-1); 70.02 (CH₂

solketal); 62.40 (CH₂sn-3);

pyrene);

57.97 (CH₃eOMe); 33.48 (CH₂pyrene); 31.82 (CH₂

29.74e29.41 (CH₂fatty chain); 26.05 (CH₂fatty chain); 25.83 (CH₃

Si-t-Bu); ꢀ5.50 (CH₃Si); MALDI TOF m/z calculated 574.384 found

[MþNa]^þ597.488; Theoretical value for [MþNa]^þ597.373.

4.1.4.3. 1-((tert-Butyldimethylsilyl)oxy)-3-((16-(pyren-1-yl)hex-

adecyl)oxy)propan-2-ol (7c). Using the aforementioned general

procedure, the compound 7c was prepared in quantitative yield

(5.2 g). Rf TLC (Eluent: toluene/Et₂O (9:1)): 0.52; ¹H NMR (CDCl₃,

300.131 MHz): 8.31 (d, 1H, H_ar, J_HH¼ 9.2 Hz); 8.2e7.96 (m, 7H, H_ar);

7.88 (d, 1H, J_HH¼ 7.6 Hz, H_ar); 3.84 (m, 1H, CH sn-2); 3.67 (m, 2H,

CH₂sn-3); 3.52e3.44 (m, 4H, CH₂sn-1 þ CH₂a-solketal); 3.34 (t, 2H,

4.1.5.3. tert-Butyl(2-methoxy-3-((16-(pyren-1-yl)hexadecyl)oxy)pro-

poxy)dimethylsilane (8c). Using the aforementioned general pro-

cedure, the compound 8c was prepared in quantitative yield (2 g).

Rf TLC (Eluent: toluene/ethyl acetate (4/1)): 0.33; ¹H NMR (CDCl₃,

300.131 MHz): 8.3 (d, 1H, H_ar, J_HH¼ 9.0 Hz); 8.17e7.95 (m, 7H, H_ar);

7.87 (d, 1H, J_HH¼ 7.5 Hz, H_ar); 3.71 (m, 2H, CH sn-2 þ CH_asn-3); 3.55

(m, 1H, CH_bsn-3); 3.48e3.38 (m, 7H, CH₃eOMe þ CH₂sn-1 þ CH₂

J_HH¼ 8 Hz, CH₂

a pyrene); 2.17 (s, 1H, OH); 1.87 (m, 2H, CH₂b

pyrene); 1.59 (m, 2H, CH₂b solketal); 1.50 (m, 2H, CH₂a solketal);

1.28 (m, 22H, CH₂fatty chain); 0.94 (m, 9H, CH₃Si-t-Bu); 0.10 (m,

6H, CH₃Si); ¹³C NMR (CDCl₃, 75.474 MHz): 137.45 (C_q); 131.55 (C_q);

131.05 (C_q); 129.77 (C_q); 128.70 (C_q); 127.63e125.82 (CH pyrene);

125.18 (C_q); 124.85e123.62 (CH pyrene); 71.76 (CH₂sn-1); 71.52

-solketal); 3.33 (t, 2H, J_HH¼ 8 Hz, CH₂a pyrene); 1.86 (m, 2H, CH₂b

pyrene); 1.57 (m, 2H, CH₂b solketal); 1.50 (m, 2H, CH₂g Solketal);

1.27 (m, 23H, CH₂fatty chain); 0.93 (m, 9H, CH₃Si-t-Bu); 0.10 (m,

6H, CH₃Si); 13C NMR (CDCl₃, 75.474 MHz): 137.47 (C_q); 131.58 (C_q);

131.07 (C_q); 129.80 (C_q); 128.72 (C_q); 127.66e125.85 (CH pyrene);

125.21 (C_q); 124.88e123.65 (CH pyrene); 81.22 (CH sn-2); 71.85

(CH₂

solketal); 70.76 (CH sn-2); 64.18 (CH₂sn-3); 33.72 (CH₂

pyrene); 32.06 (CH₂

pyrene); 29.95e29.60 (CH₂fatty chain);

26.23 (CH₂fatty chain); 25.99 (CH₃Si-t-Bu); ꢀ5.30 (CH₃Si); MALDI

TOF m/z calculated 630.447 found [MþNa]^þ653.592, Theoretical

value for [MþNa]^þ653.436.

(CH₂sn-1); 70.27 (CH₂

solketal); 62.61 (CH₂sn-3); 58.24 (CH₃

pyrene); 29.98e29.65

eOMe); 33.75 (CH₂pyrene); 32.09 (CH₂

(CH₂fatty chain); 26.28 (CH₂fatty chain); 26.05 (CH₃Si-t-

Bu); ꢀ5.23 (CH₃Si); MALDI TOF m/z calculated 644.462 found

[MþNa]^þ667.619; Theoretical value for [MþNa]^þ667.452.

4.1.5. General procedure for the methylation reaction (compounds

8a, b and c)

To a stirred solution of 7a-c (1eq.) previously diluted in anhy-

drous CH₂Cl₂(20 mL) and placed under nitrogen atmosphere, were

added silver oxide (5 eq.) and molecular sieves (~2 g). Iodomethane

(10 eq.) was added dropwise. The mixture was reﬂuxed for 2 days

and ﬁltered on Celite. The celite pad was washed with dichloro-

methane. The ﬁltrate was concentrated to produce 8a-c as a dark

yellow wax in quantitative yield. These compounds were used in

the next step without further puriﬁcation.

4.1.6. General procedure for the deprotection of the tert-butyl-

dimethylsilyle protecting group (compound 9a-c)

Tetra-n-butylammonium ﬂuoride (5 eq.) was added to a stirred

solution of 8a-c (1 eq.) diluted in dry THF (15 mL) previously placed

under nitrogen atmosphere. The reaction was stirred at room

temperature overnight. The solution was quenched by addition of

water (10 mL) and extracted with Et₂O (3 ꢃ 30 mL). The organic

layer was washed with brine, dried over Na₂SO₄, ﬁltered and

concentrated to give the crude compound 9a-c. The product was

puriﬁed by chromatography on silica gel (Eluent: pentane/ethyl

acetate (3/7)) to give the pure compound 9a-c as a yellow wax or

yellow solid between 51% and 93% yield.

4.1.5.1. tert-Butyl(2-methoxy-3-((4-(pyren-1-yl)butyl)oxy)propoxy)

dimethylsilane (8a). Using the aforementioned general procedure,

the compound 8a was prepared in quantitative yield (470 mg). Rf

TLC (Eluent: toluene/ethyl acetate (4/1)): 0.4; ¹H NMR (CDCl₃,

399.922 MHz): 8.17 (d, 1H, H_ar, J_HH¼ 7.6 Hz); 8.15e7.98 (m, 7H, H_ar);

7.87 (d, 1H, J_HH¼ 7.6 Hz, H_ar); 3.72 (m, 2H, CH sn-2 þ CH_asn-3);

4.1.6.1. 2-Methoxy-3-((4-(pyren-1-yl)butyl)oxy)propan-1-ol

9a.

Using the aforementioned general procedure, the compound 9a

was prepared in 51% yield (185 mg). Rf TLC (Eluent: pentane/ethyl

acetate (3/7)): 0.66; Melting point ¼ 82 ^ꢁC; ¹H NMR (CDCl₃,

399.922 MHz): 8.28e8.25 (m, 1H, H_ar); 8.17e7.15 (m, 2H, H_ar);

8.11e8.08 (m, 2H, H_ar); 8.05e7.95 (m, 3H, H_ar); 7.87e7.84 (m, 1H,

H_ar); 3.76e3.71 (m, 1H, CH_asn-3); 3.65e3.60 (m, 1H, CH_bsn-3);

3.61e3.42 (m, 8H, CH_bsn-3 þ CH₃eOMe þ CH₂sn-1 þ CH₂

a-sol-

ketal); 3.38 (t, 2H, J_HH¼ 8 Hz, CH₂a pyrene); 1.98e1.92 (m, 2H, CH₂

pyrene); 1.88e1.78 (m, 2H, CH₂b solketal); 0.94 (m, 9H, CH₃Si-t-

Bu); 0.10 (m, 6H, CH₃Si); ¹³C NMR (CDCl₃, 75.474 MHz): 136.89 (C_q);

131.50 (C_q); 130.98 (C_q); 129.82 (C_q); 128.68 (C_q); 127.57e126.57

(CH pyrene); 125.80 (C_q); 125.14e123.65 (CH pyrene); 81.17 (CH sn-

2); 71.49 (CH₂sn-1); 70.38 (CH₂a solketal); 62.56 (CH₂sn-3); 58.19

(CH₃eOMe); 33.35 (CH₂a pyrene); 29.77 þ 28.45 (CH₂fatty chain);

25.98 (CH₃Si-t-Bu); ꢀ5.30 (CH₃Si); MALDI TOF m/z calculated

476.275 found [MþNa]^þ499.366; Theoretical value for [MþNa]^þ

499.264.

3.57e3.38 (m, 9H, CH sn-2 þ CH₂sn-1 þ CH₂

eOMe); 3.36 (m, CH₂pyrene); 1.96e1.87 (m, 2H, CH₂

1.80e1.72 (m, 3H, CH₂

solketal); ¹³C NMR (CDCl₃, 75.474 MHz):

-solketal þ CH₃

pyrene);

136.72 (C_q); 131.46 (C_q); 130.94 (C_q); 129.82 (C_q); 128.64 (C_q);

127.55e125.81 (CH pyrene); 125.11 (C_q); 124.87e123.42 (CH pyr-

ene); 80.01 (CH sn-2); 71.60 (CH₂

a solketal); 70.53 (CH₂sn-1);

62.55 (CH₂sn-3); 57.80 (CH₃eOMe); 33.28 þ 29.64 þ 28.32 (CH₂

4.1.5.2. tert-Butyl(2-methoxy-3-((11-(pyren-1-yl)undecyl)oxy)pro-

poxy)dimethylsilane (8b). Using the aforementioned general pro-

cedure, the compound 8b was prepared in quantitative yield (2 g).

Rf TLC (Eluent: toluene/Et₂O (9/1)): 0.21; ¹H NMR (CDCl₃,

500.133 MHz): 8.3 (d, 1H, H_ar, J_HH¼ 9.0 Hz); 8.17e7.96 (m, 7H, H_ar);

7.87 (d, 1H, J_HH¼ 7.5 Hz, H_ar); 3.67 (m, 2H, CH sn-2 þ CH_asn-3); 3.55

(m, 1H, CH_bsn-3); 3.44 (s, 3H, CH₃eOMe); 3.43e3.36 (m, 4H, CH₂

sn-1 þ CH₂a-solketal); 3.34 (t, 2H, J_HH¼ 8 Hz, CH₂a pyrene); 1.85

(m, 2H, CH₂b pyrene); 1.48 (m, 2H, CH₂b solketal); 1.37 (m, 2H, CH₂

pyrene); MALDI TOF m/z calculated 362.188 found M^þC362.287.

4.1.6.2. 2-Methoxy-3-((11-(pyren-1-yl)undecyl)oxy)propan-1-ol 9b.

Using the aforementioned general procedure, the compound 9b

was prepared in 93% yield (1 g). Rf TLC (Eluent: toluene/ethyl ac-

etate (7/3)): 0.23; ¹H NMR (CDCl₃, 399.922 MHz): 8.3 (d, 1H, H_ar,

J_HH¼ 9.2 Hz); 8.17e7.98 (m, 7H, H_ar); 7.88 (d, 1H, J_HH¼ 7.5 Hz, H_ar);

3.78e3.74 (m, 1H, CH_asn-3); 3.67e3.62 (m, 1H, CH_bsn-3);

3.57e3.41 (m, 9H, CH sn-2 þ CH₂sn-1 þ CH₂

eOMe); 3.34 (t, 2H, J_HH¼ 8 Hz, CH₂pyrene); 1.85 (m, 3H, CH₂

pyrene); 1.56 (m, 3H, CH₂b solketal); 1.48 (m, 3H, CH₂g Solketal);

-solketal þ CH₃

Solketal); 1.27 (m,10H, CH₂fatty chain); 0.89 (m, 9H, CH₃Si-t-Bu);

0.071 (m, 6H, CH₃Si); ¹³C NMR (CDCl₃, 75.474 MHz): 137.13 (C_q);

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

1.27 (m, 11H, CH₂fatty chain); ¹³C NMR (CDCl₃, 75.474 MHz): 137.26

(C_q); 131.37 (C_q); 130.86 (C_q); 129.59 (C_q); 128.52 (C_q);

127.46e125.65 (CH pyrene); 125.0 (C_q); 124.69e123.44 (CH pyr-

pyrene); 20.67 þ 20.48 þ 20.38 (C eOAc Lactose); 14.05 (ethyl ac-

etate); MALDI TOF m/z calculated 980.368 found [MþNa]^þ

1003.318, theoretical value for [MþNa]^þ1003.357.

ene); 79.81 (CH sn-2); 71.82 (CH₂sn-1); 70.50 (CH₂

a solketal);

62.57 (CH₂sn-3); 57.67 (CH₃eOMe); 33.53 (CH₂pyrene); 31.87

4.1.7.2. 1-O-Undecylpyrene-2-O-methyl-rac-glycer-3-yl-2,3,4,6-

(CH₂

pyrene); 29.75e29.37 (CH₂fatty chain); 25.99 (CH₂fatty

tetra-O-acetyl-b-D-galactopyranosyl-(1e4)-2,3,6-tri-O-acetyl-b-D-

chain); MALDI TOF m/z calculated 460.298 found M^þC460.474.

glucopyranoside (10b). Using the aforementioned general proced-

ure, the compound 10b was prepared in 58% yield (1.0 g). Rf TLC

(Eluent: pentane/ethyl acetate (3/2)): 0.32; ¹H NMR (CDCl₃,

500.133 MHz): 8.22 (d, 1H, H_ar, J_HH¼ 9.0 Hz); 8.11e7.91 (m, 7H, H_ar);

7.81 (d, 1H, J_HH¼ 8.0 Hz, H_ar); 5.32 (m, 1H, H_4’); 5.18e5.14 (m, 1H,

H₃); 5.11e5.07 (m, 1H, H_2’); 4.96e4.93 (m, 1H, H_3’); 4.91e4.86 (m,

4.1.6.3. 2-Methoxy-3-((16-(pyren-1-yl)hexadecyl)oxy)propan-1-ol

9c. Using the aforementioned general procedure, the compound 9c

was prepared in 51% yield (840 mg). Rf TLC (Eluent: pentane/ethyl

acetate (1/1)): 0.77; ¹H NMR (CDCl₃, 399.922 MHz): 8.3 (d, 1H, H_ar,

J_HH¼ 9.2 Hz); 8.16e7.95 (m, 7H, H_ar); 7.87 (d, 1H, J_HH¼ 7.5 Hz, H_ar);

3.77e3.73 (m, 1H, CH_asn-3); 3.66e3.62 (m, 1H, CH_bsn-3);

1H, H₂); 4.48e4.43 (m, 3H, H_6aþ H₁þ H_1’); 4.1e4.02 (m, 3H, H_6’a

H_6’bþ H_6b); 3.88e3.79 (m, 2H, sn-3_bþ H_5’); 3.75 (t, 1H, H₄);

3.58e3.50 (m, 2H, sn-3_aþ H₅); 3.44e3.33 (m, 9H, eOCH₃þ CH sn-

3.57e3.39 (m, 9H, CH sn-2 þ CH₂sn-1 þ CH₂

-solketal þ CH₃

eOMe); 3.33 (t, 2H, J_HH¼ 8 Hz, CH₂

pyrene); 1.85 (m, 3H, CH₂

2 þ CH₂sn-1 þ CH₂

Solketal); 3.27 (t, 2H, J_HH¼ 7.8 Hz, CH₂

pyrene); 1.56 (m, 3H, CH₂b solketal); 1.48 (m, 3H, CH₂g Solketal);

1.27 (m, 22H, CH₂fatty chain); ¹³C NMR (CDCl₃, 75.474 MHz):

137.43 (C_q); 131.50 (C_q); 130.99 (C_q); 129.71 (C_q); 128.64 (C_q);

127.57e125.77 (CH pyrene); 125.12 (C_q); 124.79e123.58 (CH pyr-

pyrene); 2.13e1.95 (m, 21H, CH₃-OAc Lactose); 1.82e1.76 (m, 2H,

CH₂b pyrene); 1.53e1.49 (m, 2H, CH₂b solketal); 1.45e1.39 (m, 2H,

CH₂

g pyrene); 1.32 (m, 2H, CH₂g solketal); 1.23 (m, 10H, fatty

chain); ¹³C NMR (CDCl₃, 125.771 MHz): 170.12 (CO); 170.09 (CO);

169.92 (CO); 169.81 (CO); 169.54 (CO); 169.31 (CO); 168.87 (CO);

137.10 (C_q); 131.21(C_q); 130.70 (C_q); 129.43 (C_q); 128.35 (C_q);

127.32e125.53(CH pyrene); 124.82 (C_q); 124.55e123.30 (CH pyr-

ene); 79.85 (CH sn-2); 71.97 (CH₂sn-1); 70.67 (CH₂

62.75 (CH₂sn-3); 57.80 (CH₃eOMe); 33.66 (CH₂pyrene); 32.0

a solketal);

(CH₂

b pyrene); 29.88e29.49 (CH₂fatty chain); 26.11 (CH₂fatty

chain); MALDI TOF m/z calculated 530.376 found M^þC530.469.

ene); 100.83 (C_1’); 100.69 þ 100.58 (C₁

(C_sn-2e two diastereoisomers); 76.10 (C₄); 72.61 (C₃); 72.39 (C₅);

71.56 (C Solketal); 71.49 (C_5’); 70.78 (C₂); 70.44 (C_3’); 70.08 (C sn-

3_a- two diastereoisomers); 69.72 þ 69.62 (C sn-1 - two di-

astereoisomers); 68.93 (C2⁰); 68.52 (C sn-3_b

two di-

astereoisomers); 66.47 (C_4’); 61.87 (C₆); 60.66 (C_6’); 58.00 þ 57.66

(C eOMe); 33.35 (CH₂pyrene); 31.70 (CH₂pyrene);

anomere); 79.14 þ 78.77

4.1.7. General procedure for the glycosylation reaction (compounds

10a-c)

Peracetylated lactose trichloroacetimidate (1.2 eq.) solubilized

in dry CH₂Cl₂(20 mL) was added dropwise to a solution of 9a-c (1

eq.) in CH₂Cl₂(15 mL). The mixture was stirred with molecular

sieves (~2 g) during 1 h under nitrogen atmosphere. At 0 ^ꢁC, boron

triﬂuoride diethyl etherate (0.8 eq.) was added dropwise and the

mixture was stirred overnight at RT under inert atmosphere. The

mixture was quenched by addition of water (20 mL). The organic

layer was washed with aqueous saturated NaHCO₃solution and

brine. The organic layer was dried over MgSO₄, ﬁltered, concen-

trated to give the crude compounds 10a-c. The product was puri-

ﬁed by chromatography on silica gel (Eluent: pentane/ethyl acetate

(3/2)) to give the pure compounds 10a-c as white foam between

42% and 58% yield.

29.58 þ 29.36 þ 29.23 (CH₂fatty chain); 25.85 (CH₂fatty chain);

20.60 þ 20.40 þ 20.29 (C eOAc Lactose); MALDI TOF m/z calculated

1078.477 found [MþNa]^þ1101.513, theoretical value for [MþNa]^þ

1101.467.

4.1.7.3. 1-O-Hexadecylpyrene-2-O-methyl-rac-glycer-3-yl-2,3,4,6-

tetra-O-acetyl-

D-galactopyranosyl-(1e4)-2,3,6-tri-O-acetyl-b-D-

glucopyranoside (10c). Using the aforementioned general proced-

ure, the compound 10c was prepared in 44% yield (805 mg). Rf TLC

(Eluent: pentane/ethyl acetate (7/3)): 0.14; ¹H NMR (CDCl₃,

399.922 MHz): 8.24 (d,1H, H_ar, J_HH¼ 9.6 Hz); 8.11e7.91 (m, 7H, H_ar);

7.82 (d, 1H, J_HH¼ 7.6 Hz, H_ar); 5.31 (m, 1H, H_4’); 5.19e5.14 (m, 1H,

H₃); 5.10e5.06 (m, 1H, H_2’); 4.94e4.90 (m, 1H, H_3’); 4.88e4.84 (m,

4.1.7.1. 1-O-Butylpyrene-2-O-methyl-rac-glycer-3-yl-2,3,4,6-tetra-O-

acetyl-b-D-galactopyranosyl-(1e4)-2,3,6-tri-O-acetyl-b-D-glucopyr-

anoside (10a). Using the aforementioned general procedure, the

compound 10a was prepared in 42% yield (410 mg). Rf TLC (Eluent:

pentane/ethyl acetate (3/7)): 0.7; ¹H NMR (CDCl₃, 500.133 MHz):

8.24 (d, 1H, H_ar, J_HH¼ 9.6 Hz); 8.14e7.85 (m, 7H, H_ar); 7.84 (d, 1H,

J_HH¼ 7.6 Hz, H_ar); 5.33 (m, 1H, H_4’); 5.16e5.13 (m, 1H, H₃);

5.10e5.07 (m, 1H, H_2’); 4.94e4.84 (m, 2H, H_3’þ H₂); 4.45e4.38 (m,

3H, H_6aþ H₁þ H_1’); 4.1e3.99 (m, 4H, H_6’aþ H_6’bþ H_6bþ ethyl

acetate); 3.88e3.79 (m, 2H, sn-3_bþ H_5’); 3.73e3.68 (m, 1H, H₄);

3.59e3.28 (m, 12H, sn-3_aþ H₅þ eOCH₃þ CH sn-2 þ CH₂sn-

1H, H₂); 4.49e4.43 (m, 3H, H_6aþ H₁þ H_1’); 4.1e4.01 (m, 3H, H_6’aþ

H_6’bþ H_6b); 3.88e3.79 (m, 2H, sn-3_bþ H_5’); 3.75 (t,1H, J_HH¼ 9.2 Hz,

H₄); 3.59e3.50 (m, 2H, sn-3_aþ H₅); 3.45e3.38 (m, 9H, eOCH₃þ CH

sn-2 þ CH₂sn-1 þ CH₂a Solketal); 3.28 (t, 2H, J_HH¼ 8.0 Hz, CH₂a

pyrene); 2.13e1.93 (m, 21H, CH₃-OAc Lactose); 1.82e1.76 (m, 2H,

CH₂b pyrene); 1.53e1.49 (m, 2H, CH₂b solketal); 1.45e1.39 (m, 2H,

CH₂

g pyrene); 1.32 (m, 2H, CH₂g solketal); 1.19 (m, 24H, fatty

chain); ¹³C NMR (CDCl₃, 75.474 MHz): 170.36 (CO); 170.16 (CO);

170.06 (CO); 169.78 (CO); 169.56 (CO); 169.10 (CO); 137.37 (C_q);

131.47 (C_q); 130.96 (C_q); 129.69 (C_q); 128.61 (C_q); 127.56e125.77

(CH pyrene); 125.08 (C_q); 124.79e123.55 (CH pyrene); 101.09 (C_1’);

1 þ CH₂

Solketal þ CH₂

pyrene); 2.14e1.95 (m, 21H, CH₃-OAc

pyrene); 1.77e1.69 (m, 2H, CH₂sol-

Lactose); 1.89 (m, 2H, CH₂

ketal); 1.24 (t, 1.5H, ethyl acetate); ¹³C NMR (CDCl₃, 125.803 MHz):

171.0 (CO); 170.20 (CO); 170.02 (CO); 169.93 (CO); 169.62 (CO);

169.42 (CO); 168.95 (CO); 136.64 (C_q); 131.28 (C_q); 130.76 (C_q);

129.64 (C_q); 128.46 (C_q); 127.39e125.69 (CH pyrene); 124.92 (C_q);

100.95 þ 100.85 (C₁

anomere); 79.14 þ 79.03 (C_sn-2e two di-

astereoisomers); 76.34 (C₄); 72.85 (C₃); 72.65 (C₅); 71.83(C

Sol-

ketal); 71.74 (C_5’); 71.03 (C₂); 70.70 (C_3’); 70.36 (C sn-3_a- two

diastereoisomers); 69.99 þ 69.88 (C sn-1 - two diastereoisomers);

69.17 (C2⁰); 68.79 (C sn-3_b- two diastereoisomers); 66.68 (C_4’);

62.09 (C₆); 60.87(C_6’); 60.38 (ethyl acetate); 58.27 þ 57.93 (C

124.71e123.30 (CH pyrene); 100.91 (C_1’); 100.71 þ 100.66 (C₁

anomere); 79.19 þ 78.83 (C_sn-2e two diastereoisomers); 76.13 (C₄);

72.61 (C₃); 72.39 (C₅); 71.52 (C_5’); 71.36 (C Solketal); 70.85 (C₂);

eOMe); 33.62 (CH₂

pyrene); 31.97 (CH₂

pyrene);

70.50 (C_3’); 70.1 (C sn-3_a- two diastereoisomers); 69.83 þ 69.71 (C

sn-1 - two diastereoisomers); 68.95 (C2⁰); 68.54 (C sn-3_b- two di-

astereoisomers); 66.48 (C_4’); 61.8 (C₆); 60.67 (C_6’); 60.38 (ethyl

acetate); 58.11 þ 57.76 (C eOMe); 33.12 þ 29.46 þ28.21 (CH₂

29.85 þ 29.69 þ 29.51 (CH₂fatty chain); 26.11 (CH₂fatty chain);

21.04 þ 20.84 þ 20.64 þ 20.53 (C eOAc Lactose); 14.22 (ethyl ac-

etate); MALDI TOF m/z calculated 1148.556 found [MþNa]^þ

1171.381 and [MþK]^þ1187.343, theoretical value for

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

[MþNa]^þ1171.545, theoretical value for [MþK]^þ1187.519.

71% yield (420 mg). ¹H NMR (DMSO, 500.133 MHz): 8.33 (d, 1H, H_ar,

J_HH¼ 9.5 Hz); 8.26e8.04 (m, 7H, H_ar); 7.93 (d, 1H, J_HH¼ 8.0 Hz, H_ar);

5.1e5.07 (m, 2H, OH); 4.77e4.49 (m, 5H, OH); 4.19e4.18 (m, 2H,

H₁þ H_1’); 3.76e3.73 (m, 2H, H_6aþ CH₂sn-3_a); 3.60e3.29 (m, 25H,

H₃þ H₄þ H₅þ H_6bþ H_2’þ H_3’þ H_4’þ H_5’þ H_6’aþ H_6’bþ CH₃e

OMe (two diastereoisomers) þ CH₂a Solketal þ CH₂a pyrene þ CH

sn-2 þ CH₂sn-3_bþ CH₂sn-1); 2.99 (t, 1H, J_HH¼ 7.8 Hz, H₂);

4.1.8. General procedure for ﬁnal deprotection of peracetylated

lactose (compounds OP1-3)

Potassium carbonate (0.5 eq) was added to a stirred solution of

OP1-3 (1 eq.) diluted in anhydrous MeOH (15 mL). The mixture was

stirred for 6 h at RT. The mixture is quenched by addition of a little

amount of Amberlite (IR120H) (~300 mg) and the solution was

stirred for 30 min. Then, the solution was ﬁltered on a sintered

funnel and the solvent was evaporated to produce compounds

OP1-3 between 71% and 93% yield as a white powder.

1.78e1.75 (m, 2H, CH₂

b pyrene); 1.44e1.41 (m, 4H, CH₂b

solketal þ CH₂g pyrene); 1.33e1.32 (m, 2H, CH₂g Solketal); 1.19 (m,

20H, CH₂fatty chain); ¹³C NMR (DMSO, 125.771 MHz): 137.07 (C_q);

130.88 (C_q); 130.39 (C_q); 129.15 (C_q); 127.99 (C_q); 127.43e123.44

(CH pyrene); 124.15 (C_q); 123.44 (CH pyrene); 103.84 (C_1’);

4.1.8.1. 1-O-Butylpyrene-2-O-methyl-rac-glycer-3-yl-

-galactopyr-

102.93 þ 102.86 (C₁

(C₄); 78.72 78.67 (C sn-2

75.49 þ 74.96 þ 74.82 þ 73.09 (C₃þ C₅þ C_2’þ C_3’); 73.21 (C₂); 70.56

(C Solketal); 70.51 (C_5’); 69.84 (C sn-1 - two diastereoisomers);

68.66 þ 68.42 (C sn-3 - two diastereoisomers); 68.11 (C_4’); 60.48

(C₆); 60.36 (C_6’); 57.11 þ 57.02 (C eOMe); 32.61 (CH₂pyrene);

31.49 (CH₂

chain); 25.60 (CH₂fatty chain); MALDI TOF m/z calculated 854.482

found [MþNa]^þ877.475, theoretical value for [MþNa]^þ877.471,

found [MþK]^þ893.437, theoretical value for [MþK]^þ893.445.

anomere e two diastereoisomers); 80.73

anosyl-(1e4)- -glucopyranoside (OP1). Using the aforemen-

-D

two diastereoisomers);

tioned general procedure, the compound OP1 was prepared in 73%

yield (102 mg). ¹H NMR (DMSO, 500.133 MHz): 8.36 (d, 1H, H_ar,

J_HH¼ 9.2 Hz); 8.29e8.21 (m, 4H, H_ar); 8.14e8.11 (m, 2H, H_ar);

8.07e8.05 (m, 1H, H_ar); 7.96e7.95 (m, 1H, H_ar); 4.20e4.19 (m, 2H,

H₁þ H_1’); 3.79e3.72 (m, 2H, H_6aþ CH₂sn-3_a); 3.61e3.16 (m, 25H,

H₃þ H₄þ H₅þ H_6bþ H_2’þ H_3’þ H_4’þ H_5’þ H_6’aþ H_6’bþ CH₃e

OMe (two diastereoisomers) þ CH₂a Solketal þ CH₂a pyrene þ CH

sn-2 þ CH₂sn-3_bþ CH₂sn-1); 3.01 (t, 1H, J_HH¼ 8 Hz, H₂); 1.84e1.81

(m, 2H, CH₂b pyrene); 1.67e1.65 (m, 2H, CH₂b solketal); ¹³C NMR

(DMSO, 125.771 MHz): 136.97 (C_q); 130.88 (C_q); 130.41 (C_q); 129.20

(C_q); 128.05 (C_q); 127.46e124.76 (CH pyrene); 124.21 (C_q); 124.15

pyrene); 29.16 þ 28.97 þ 28.86 þ 28.83 (CH₂fatty

4.2. Fluorescence and liposomal formulations

(Cq); 123.50 (CH pyrene); 103.85 (C_1’); 102.95 þ 102.89 (C₁

Fluorescence was recorded on Agilent Cary Eclipse Fluorescence

Spectrophotometer. Samples were excited at 331 nm in quartz cells.

Stock solutions of OP compounds were prepared in methanol at

50 mM. EggPC, DOPC and cholesterol were purchased from Sigma-

Aldrich. EggPC (ref 840051) is a mixture of PC whose molecular

weight is 770 g/mol. It contains mainly C_16:0(32.7%), C_18:1(32%),

anomere e two diastereoisomers); 80.73 (C₄); 78.73 þ 78.67 (C sn-2

e two diastereoisomers); 75.50 þ 74.97 þ 74.84 þ 73.22 þ 73.10

(C₃þ C₅þ C_2’þ C_3’þ C₂); 70.52 (C_5’); 70.37 (C

Solketal); 69.83 (C

sn-1 - two diastereoisomers); 68.63 þ 68.41 (C sn-3 - two di-

astereoisomers); 68.12 (C_4’); 60.47 (C₆); 60.38 (C_6’); 57.11 þ 57.02 (C

eOMe); 32.34 þ 29.09 þ 28.18 (CH₂fatty chain); MALDI TOF m/z

calculated 686.294 found [MþNa]^þ709.346, theoretical value for

[MþNa]^þ709.283, found [MþK]^þ725.308, theoretical value for

[MþK]^þ725.257.

_18:2(17.1%), C_18:0(12.3%), C _20:4(2.7%), C _16:1(1.1%). Stock solutions

of EggPC, DOPC and cholesterol were prepared in chloroforme at

1 mmol/L. For mixtures of EggPC or DOPC with cholesterol, stock

solutions of the mixture were prepared containing 30% of choles-

terol and 70% of either EggPC or DOPC (mol/mol). An aliquot of

these lipids solutions was introduced in a glass tube and solvent

was evaporated under vacuum. After solvent removal, pure water

was added and let at 4 ^ꢁC for 5 nights. The samples were then

warmed, vortexed, sonicated for 10 min. After 3 h they were vor-

texed again and ﬂuorescence was measured. The ﬁnal concentra-

4.1.8.2. 1-O-Undecylpyrene-2-O-methyl-rac-glycer-3-yl-

b-D-gal-

actopyranosyl-(1e4)- -glucopyranoside (OP2). Using the afore-

b-D

mentioned general procedure, the compound OP2 was prepared in

93% yield (530 mg). ¹H NMR (DMSO, 500.253 MHz): 8.33 (d, 1H, H_ar,

J_HH¼ 9.5 Hz); 8.26e8.02 (m, 7H, H_ar); 7.93 (d, 1H, J_HH¼ 8.0 Hz, H_ar);

5.08 (m, 2H, OH); 4.66e4.52 (m, 5H, OH); 4.20e4.17 (m, 2H,

H₁þ H_1’); 3.77e3.72 (m, 2H, H_6aþ CH₂sn-3_a); 3.60e3.26 (m, 25H,

H₃þ H₄þ H₅þ H_6bþ H_2’þ H_3’þ H_4’þ H_5’þ H_6’aþ H_6’bþ CH₃e

OMe (two diastereoisomers) þ CH₂a Solketal þ CH₂a pyrene þ CH

sn-2 þ CH₂sn-3_bþ CH₂sn-1); 3.0 (t, 1H, J_HH¼ 7.8 Hz, H₂); 1.79e1.73

tion of lipids is either 8

For pre-insertion experiments, stock solutions of one of the OP

compounds (10 L) were added before evaporation of the solvent.

For post-insertion they were added after sonication. The ﬁnal

mM or 40 mM.

concentration of OP 1, 2 or 3 was always 0.2

ratios ¼ 40 or 200).

mM (lipid/OP

(m, 2H, CH₂

pyrene); 1.34e1.28 (m, 2H, CH2

pyrene); 1.46e1.38 (m, 4H, CH₂

solketal þ CH₂

Solketal); 0.83 (m, 10H, CH₂fatty

chain); ¹³C NMR (DMSO, 125.804 MHz): 137.07 (C_q); 130.86 (C_q);

130.38 (C_q); 129.13 (C_q); 127.98 (C_q); 127.42e124.19 (CH pyrene);

124.18 (C_q); 124.14 (C_q); 123.44 (CH pyrene); 103.83 (C_1’);

4.3. Cell culture

MDA-MB-435s and HEK cell line that sur-expressed SK3 chan-

nels were cultured as already described [14]. All cell lines were

obtained from the American Type Culture Collection (ATCC, LGC

Promochem, Molsheim, France).

102.92 þ 102.85 (C₁

(C₄); 78.73 78.71 (C sn-2

75.48 þ 74.95 þ 74.79 þ 73.08 (C₃þ C₅þ C_2’þ C_3’); 73.2 (C₂); 70.54

(C Solketal); 70.50 (C_5’); 69.81 (C sn-1 - two diastereoisomers);

68.66 þ 68.41 (C sn-3 - two diastereoisomers); 68.09 (C_4’); 60.47

pyrene);

anomere e two diastereoisomers); 80.73

two diastereoisomers);

4.4. Cell migration

(C₆); 60.35 (C_6’); 57.10 þ 57.01 (C eOMe); 32.60 (CH₂

31.49 (CH₂b pyrene); 29.13 þ 28.96 þ 28.92 þ 28.90 þ 28.80 (CH₂

fatty chain); 25.58 (CH₂fatty chain); MALDI TOF m/z calculated

784.403 found [MþNa]^þ807.443, theoretical value for [MþNa]^þ

807.393.

Cell migration assays were analyzed in 24-well plates receiving

8 mm pore size cell culture inserts (Becton Dickinson, France), as

described [14]. Compounds OP1-3 were solubilized in DMSO to

prepare stock solution at 10 mM.

4.1.8.3. 1-O-Hexadecylpyrene-2-O-methyl-rac-glycer-3-yl-

actopyranosyl-(1e4)- -glucopyranoside (OP3). Using the afore-

mentioned general procedure, the compound OP3 was prepared in

-gal-

4.5. Cell viability

b-D

Cell viability assays were determined using MTT assay as already

A. Bauduin, M. Papin, A. Chantome et al.

European Journal of Medicinal Chemistry 209 (2021) 112894

ꢀ

[2] N. Comes, A. Serrano-Albarras, J. Capera, C. Serrano-Novillo, E. Condom,

described [14]. Cells were seeded on 24-well plates and grown for

24 h. Compounds OP1-3 were then added for 48 h at 1 mM.

S.R. Cajal, J.C. Ferreres, A. Felipe, Involvement of potassium channels in the

progression of cancer to a more malignant phenotype, Biochim. Biophys. Acta

1848 (2015) 2477e2492.

ꢀ

[3] N. Deliot, B. Constantin, Plasma membrane calcium channels in cancer: al-

4.6. Electrophysiology

terations and consequences for cell proliferation and migration, Biochim.

Biophys. Acta 1848 (2015) 2512e2522.

Whole-cell SK3 currents were captured using 1322-A Digidata

converter (Axon Instruments, USA) and pClamp 8.1 software (Axon

Instruments, USA) as described [14]. Compounds OP1-3 were sol-

ubilized in DMSO to prepare stock solution at 10 mM.

[4] K. Morishita, K. Watanabe, H. Ichijo, Cell volume regulation in cancer cell

migration driven by osmotic water ﬂ ow, Canc. Sci. 110 (2019) 2337e2347.

[5] S. Martial, Am. J. Physiol. Cell Physiol. 310 (2016) C710eC727.

[6] R. Peruzzo, I. Szabo, Contribution of mitochondrial ion channels to chemo-

resistance in cancer cells, Cancers 11 (2019) 761.

[7] P. Kischel, A. Girault, L. Rodat-Despoix, M. Chamlali, S. Radoslavova, H. Abou

Daya, T. Lefebvre, A. Foulon, P. Rybarczyk, F. Hague, I. Dhennin-Duthille,

M. Gautier, H. Ouadid-Ahidouch, Ion channels: new actors playing in

chemotherapeutic resistance, Cancers 11 (2019) 376.

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Cartereau, KCa and Ca2 þ channels: the complex thought, Biochim. Biophys.

Acta 1843 (2014) 2322e2333.

4.7. Proteinase K digestion and western blot

ꢀ

Proteinase K digestion was performed as described previously

[25]. Brieﬂy, cells were incubated with or without 200 mg/ml pro-

teinase K (Sigma-Aldrich, France) at 37 ^ꢁC for 30 min and the

digestion was quenched by adding ice-cold PBS containing 6 mM

phenylmethylsulfonyl ﬂuoride and 25 mM EDTA. SK3 bands were

revealed using Western blot experiments as already described [25].

[9] E. Lastraioli, J. Iorio, A. Arcangeli, Ion channel expression as promising cancer

biomarker, Biochem. Biophys. Acta 1848 (2015) 2685e2702.

[10] H. Wulff, P. Christophersen, P. Colussi, K.G. Chandy, V. Yarov-Yarovoy, Anti-

bodies and venom peptides: new modalities for ion channels, Nat. Rev. Drug

Discov. 18 (2019) 339e357.

ꢁ

[11] A. Girault, J.P. Haelters, M. Potier-Cartereau, A. Chantome, P.A. Jaffres,

P. Bougnoux, V. Joulin, C. Vandier, Targeting SKCa channels in cancer: po-

tential new therapeutic approaches, Curr. Med. Chem. 19 (2012) 697e713.

[12] A. Chantome, A. Girault, M. Potier, C. Collin, P. Vaudin, J.C. Pages, C. Vandier,

V. Joulin, KCa2.3 channel-dependent hyperpolarization increases melanoma

cell motility, Exp. Cell Res. 315 (2009) 3620e3630.

4.8. Imaging

100.000 MDA-MB-231 cells were seeded in 35 mm dishes in

1 mL serum-supplemented DMEM. Cells were treated with OP2

24 h after seeding. The stock solution of OP2 was prepared in

DMSO/EtOH (60/40) at 1 mM. This solution was diluted either 1000

ꢁ

[13] 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.

or 100 times for the experiments at 1e10

mM. For observation

[14] A. Girault, J.P. Haelters, M. Potier, A. Chantome, M. Pineau, S. Marionneau-

immediately after treatment, the serum-containing culture me-

dium was replaced with serum-free DMEM before adding OP2 at

the required concentration; for observation 24 h after treatment,

OP2 was added to the serum-supplemented DMEM.

Cells were imaged on a wide-ﬁeld Leica microscope endowed

with an EM-CCD (Andor DU8285) through an oil immersion 20ꢃ

microscope objective (HC PL APO 20/0.70 IMM). OP2 ﬂuorescence

was excited at 340 nm and emission was imaged from 468 to

552 nm (the emission range of pyrene excimer).

ꢁ

Lambot, T. Oullier, G. Simon, H. Couthon-Gourves, P.A. Jaffres, V. Joulin,

B. Corbel, P. Bougnoux, C. Vandier, New alkyl-lipid blockers of SK3 channels

reduce cancer cell migration and occurrence of metastasis, Curr. Cancer Drug

Targets 11 (2011) 1111e1125.

[15] 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, Canc. Res. 73 (2013) 4852e4861.

ꢁ

[16] C.M. Sevrain, J.P. Haelters, A. Chantome, H. Couthon-Gourves, A. Girault,

ꢁ

C. Vandier, P.A. Jaffres, Glyco-Phospho-Glycero Ether Lipids (GPGEL): syn-

thesis and evaluation as small conductance Ca2 þ -activated Kþ channel (SK3)

inhibitors, MedChemComm 3 (2012) 1471e1478.

Declaration of competing interest

ꢁ

[17] C.M. Sevrain, J.P. Haelters, A. Chantome, H. Couthon-Gourves, M. Gueguinou,

ꢁ

M. Potier-Cartereau, C. Vandier, P.A. Jaffres, DiGalactosyl-Glycero-Ether Lipid:

synthetic approaches and evaluation as SK3 channel inhibitor, Org. Biomol.

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Chem. 11 (2013) 4479e4487.

[18] W. Berthe, C.M. Sevrain, A. Chantome, A.M. Bouchet, M. Gueguinou,

ꢁ

Y. Fourbon, M. Potier-Cartereau, J.P. Haelters, H. Couthon-Gourves, C. Vandier,

ꢁ

P.A. Jaffres, New disaccharide-based ether lipids as SK3 ion channel inhibitors,

ChemMedChem 11 (2016) 1531e1539.

[19] S. Kouba, J. Braire, R. Felix, A. Chantome, P.A. Jaffres, J. Lebreton, D. Dubreuil,

Acknowledgements

ꢀ

ꢁ

ꢀ

M. Pipelier, X. Zhang, M. Trebak, C. Vandier, M. Mathe-Allainmat, M. Potier-

Cartereau, Lipidic alkaloids as SK3 channel modulators. Synthesis and bio-

logical evaluation of 2-substituted tetrahydropyridine derivatives with po-

tential anti-metastatic activity, Eur. J. Med. Chem. 186 (2020) 111854.

[20] M.J. Lenaeus, M. Vamvouka, P.J. Focia, A. Gross, Structural basis of TEA

blockade in a model potassium channel, Nat. Struct. Mol. Biol. 12 (2005)

454e459.

[21] C. Hanselmann, S. Grissmer, Characterization of apamin-sensitive Ca(2 þ )-

activated potassium channels in human leukaemic T lymphocytes, J. Physiol.

496 (1996) 627e637.

We are grateful to Ana M. Bouchet for constructive scientiﬁc

discussions and critical assistance for OP2 cellular internalization

experiments and Isabelle Domingo for technical assistance. This

study was funded by Institut National du Cancer (INCA N^ꢁ2018-

ꢀ

151), the University of Brest, the University of Tours, the Region

ꢀ

Bretagne and Region Centre-Val de Loire, INSERM, CNRS, Cancer-

opole Grand Ouest, La Ligue Nationale Contre le Cancer and the

ꢁ

ꢀ

[22] F. Herrera, C.M. Sevrain, P.A. Jaffres, H. Couthon-Gourves, A. Grelard,

CANCEN Association. AB is grateful for a PhD fellowship from “La

E.J. Dufourc, A. Chantome, M. Potier-Cartereau, C. Vandier, A. Bouchet, Sin-

ꢀ

Ligue contre le Cancer” and “region Bretagne” and for a mobility

gular interaction between an antimetastatic agent and the lipid bilayer: the

ohmline case, ACS Omega 2 (2017) 6361e6637.

grant from the university of Brest. We thank the platforms (NMR

and mass spectrometry) from the University of Brest.

ꢀ

[23] M. Gueguinou, T. Harnois, D. Crottes, N. Deliot, A. Gambade, A. Chantome,

ꢁ

J.P. Haelters, P.A. Jaffres, M.L. Jourdan, G. Weber, P. Bougnoux, O. Mignen,

N. Bourmeyster, B. Constantin, T. Lecomte, C. Vandier, M. Potier-Cartereau,

SK3/TRPC1/Orai1 complex regulates SOCE-dependent colon cancer cell

migration: a novel opportunity to modulate anti-EGFR mAb action by the

alkyl-lipid Ohmline, Oncotarget 7 (2016) 36168e36184.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.1 12894.

€

[24] T. Schubert, W. Romer, How synthetic membrane systems contribute to the

understanding of lipid-driven endocytosis, Biochem. Biophys. Acta 1853

(2015) 2992e3005.

[25] M. Potier, A. Chantome, V. Joulin, A. Girault, S. Roger, P. Besson, M.L. Jourdan,

J.Y. Le Guennec, P. Bougnoux, C. Vandier, The SK3/KCa2.3 potassium channel is

a new cellular target for edelfosine, Br. J. Pharmacol. 162 (2011) 464e479.

[26] E. Quesada, J. Delgado, C. Gajate, F. Mollinedo, A.U. Acuna, F. Amat-Guerri,

Fluorescent phenylpolyene analogues of the ether phospholipid edelfosine for

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