Preliminary structure-activity relationship (SAR) of a novel series of pyrazole SKF-96365 analogues as potential store-operated calcium entry (SOCE) inhibitors

Article abstract of DOI:10.3390/ijms19030856

From a series of (1R, 1S)-1[β-(phenylalkoxy)-(phenetyl)]-1H-pyrazolium hydrochloride as new analogues of SKF-96365, one has an interesting effect for endoplasmic reticulum (ER) Ca²⁺ release and store-operated Ca²⁺ entry (SOCE) (IC

Full text of DOI:10.3390/ijms19030856

International Journal of
Molecular Sciences
Article
Preliminary Structure-Activity Relationship (SAR) of
a Novel Series of Pyrazole SKF-96365 Analogues as
Potential Store-Operated Calcium Entry
(SOCE) Inhibitors
Camille D. Dago 1 , Paul Le Maux , Thierry Roisnel
,2
1
3
ID
, Christophe Brigaudeau 4,5,
2
4,5, ID
and Jean-Pierre Bazureau 1,6,
Yves-Alain Bekro , Olivier Mignen
*
*
1
Institut des Sciences Chimiques de Rennes (ISCR), UMR CNRS 6226, Groupe CORINT,
Université de Rennes 1 (UR1), Campus de Beaulieu, Bât. 10A, 263 Avesnue du Général Leclerc,
CS 74205, 35042 Rennes CEDEX, France; deliko.dago@univ-rennes1.fr (C.D.D.);
paul.lemaux@free.fr (P.L.M.)
Laboratoire de Chimie Bio Organique et de Substances Naturelles (LCBOSN),
Université Nangui Abrogoua (UNA), Abidjan BP 802, Côte d’Ivoire; yvesalainb2014@gmail.com
Institut des Sciences Chimiques de Rennes (ISCR), UMR CNRS 6226, Centre de Diffractométrie X (cdifx),
Université de Rennes 1 (UR1), Campus de Beaulieu, Bât. 10B, 263 Avenue du Général Leclerc, CS 74205,
2
3
3
5042 Rennes CEDEX, France; thierry.roisnel@univ-rennes1.fr
Laboratoire Canalopathies & Signalisation Calcique, Inserm U1227, Université de Bretagne Occidentale (UBO),
2 Avenue Camille Desmoulins, 29200 Brest CEDEX, France; christophe.brigaudeau@univ-brest.fr
CalciScreen Platform, Université de Bretagne Occidentale (UBO), 22 Avenue Camille Desmoulins,
9200 Brest CEDEX, France
S2Wave Platform, ScanMAT UMS 2001 CNRS, Université de Rennes 1 (UR1), Campus de Beaulieu, Bât. 10A,
63 Avenue du Général Leclerc, CS 74205, 35042 Rennes CEDEX, France
4
5
6
2
2
2
*
Correspondence: olivier.mignen@univ-brest.fr (O.M.); jean-pierre.bazureau@univ-rennes1.fr (J.-P.B.);
Tel.: +33-223-236-603 (J.-P.B.)
Received: 15 January 2018; Accepted: 8 March 2018; Published: 14 March 2018
Abstract: From a series of (1R, 1S)-1[ -(phenylalkoxy)-(phenetyl)]-1H-pyrazolium hydrochloride as
β
2+
new analogues of SKF-96365, one has an interesting effect for endoplasmic reticulum (ER) Ca release
and store-operated Ca2 entry (SOCE) (IC50 25
+
µM) on the PLP-B lymphocyte cell line. A successful
resolution of (
±
) 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol has been developed by using the method of
“
half-concentration” in the presence of (+)-(1S)- or (−)-(1R)-CSA.
Keywords: pyrazole; SKF-96365; analogues; resolution; half-quantities; SOCE; B lymphocyte cell;
SOCE inhibitor; Ca2 signalling
+
1
. Introduction
Cytoplasmic Ca2+ is very important for fundamental biological processes; this includes cell
+
proliferation, apoptosis, migration, and gene expression [
concentration following many plasma membrane receptors is connected to the release of stored Ca2
within the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) associated to the influx of
1
]. The increase of cytoplasmic Ca2
+
extra-cellular Ca2 across the plasma membrane. Store-operated Ca entry (SOCE) is the typical
mechanism to generate Ca signals that combines the intracellular and extra-cellular processes and
represents one of the most common and ubiquitous Ca2+ influx routes in non-excitable cells [
].
+
2+
2+
2–4
SOC entry is mediated by calcium selective channels such as the archetypal calcium-release-activated
calcium (CRAC) channels in lymphocytes, which are typically supported by Orai1 proteins and
regulated by STIM1 (Stromal Interacting Molecule). STIM1 is a trans-membrane protein mainly
Int. J. Mol. Sci. 2018, 19, 856; doi:10.3390/ijms19030856
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018, 19, 856
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located in the ER membrane acting as a calcium-sensing protein, whereas Orai1 is located in the
plasmalemma forming the calcium-selective pore of the CRAC channel [ ]. STIM and Orai are
expressed in all tissues and therefore are of first important in the numerous cellular functions.
Abnormal SOC channels activities cause several human diseases, such as breast cancer [ ],
inflammatory bowel diseases [ ], thrombosis [10], and severe combined immunodeficiency (SCID)
5
6–8
9
disorders [11], which leads to an increasing interest in developing small molecule compounds to
regulate aberrant SOC and especially CRAC channels function [12]. The therapeutic potential of
inhibiting CRAC currents (named ICRAC) has been established by the clinical use of calcineurin
inhibitors (cyclosporine A and tacrolinus) to prevent rejection of organ transplants. Examination
of literature showed that the first identified inhibitor of the CRAC channel was SKF-96365 or
1 β-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride [13,14] in 1990
-{
2+
(
Figure 1) and is still used as a tool for the probing of receptor-mediated Ca entry processes [15] in
non-excitable cells [16], but it is interesting to note that the synthesis of SKF-96365 was not detailed
in academic literature and also not patented. Recent studies demonstrated that it strongly inhibits
voltage-gated sodium current (INa) in rat ventricular myocytes using the whole-cell patch voltage-clamp
technique [17]. SKF-96365 SOCE inhibitor exhibited potent anti-neoplastic activity by inducing cell-cycle
arrest and apoptosis in colorectal cancer cells (HCT-116 and HT29 cells) [18]. Effect of SKF-96365 was
also observed on hERG current in HEK 293 cells in a concentration-dependent manner. These blocking
properties were similar to those observed previously for hERG channels by the calmodulin inhibitor
W-7 [19]. All together, these results suggest a low specificity of this compound for CRAC channels.
A variety of new small molecules blocking the CRAC channels have been identified and
developed, i.e., curcumin and caffeic acid phenethyl ester (CAPE) in ORAI1/STIM-co-expressing HEK
0
2
93 cells [20], 4 -[(trifluoromethyl)pyrazol-1-yl]carboxanilides exhibiting high selectivity for the CRAC
2+
channel over the voltage-operated Ca (VOC) channels [21], 2-APB with a concentration-dependent
effect [22], GSK-7975A by altering the Orai pore geometry [23], Synta 66 with good selectivity for
CRAC channels, and no effect for Ca pumps and K channels and no interference with STIM1
aggregation [24].
2+
+
Cl
N
O
O
O
O
O
O
HO
HO
N
O
O
,
HCl
O
S
O
NH2
HO
OH
O
N
H
caffeic acid phenethyl ester (CAPE)
SKF-96365
W-7
curcumin
HO
F
Me
O
N
O
variable chain lenght
R2
H
N
R4
N
H2N
B
N
N
H
F
O
S
F3C
N
N
O
*
N
N
lipophilic
group (CF3)
or not
F3C
N
n
GSK-7975A
O
2-APB
polar group
or not
R
R3
R1
4
'-[(trifluoromethyl)pyrazol-1-yl]carboxanilides
R = CF3, H, Me, 4-MeC6H5, Cl, Br, CN
, HCl
chiral
center
O
O
F
initial skeleton
pyrazole SKF-96365
analogs
N
H
N
Synta 66
Figure 1. Structure of pharmacological inhibitors of calcium channels and structure–activity
relationship (SAR) parameters of the pyrazole SKF-96365 analogues.
In this context [25], we decided to examine the synthesis of SKF-96365 analogues bearing
substituted (or not) pyrazole platforms and the other parameters for this structure-activity relationship
(SAR) study are respectively the length of the chain in position Cβ and the presence (or not) of a
methoxy polar group in para-position on the phenylethyl skeleton and on the phenylalkyloxy side
chain. The generic phenylethyl skeleton is maintained for this SAR study. Effects of these analogues
are also examined for endoplasmic reticulum (ER) Ca2+ release and SOCE on a B lymphocyte cell
line [26].

Int. J. Mol. Sci. 2018, 19, 856
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2
. Results and Discussion
2
.1. Chemistry
These pyrazole analogues of SKF-96365 were prepared as shown in Scheme 1 using a modified
method described in literature [27]. The first step involved reaction of 2-bromoacetophenone
or 2-bromo-1-(4-methoxyphenyl)ethan-1-one 1b with various pyrazoles substituted by
one or two trifluoromethyl groups (2a pyrazole, 2b 3-trifluoromethylpyrazole, and 2c
,5-bis-trifluoromethylpyrazole). The reaction was conducted with potassium carbonate in acetonitrile
at room temperature. After work-up, the four desired N-substituted pyrazoles 3a were obtained
was realized
1
a
2
:
:
:
3
–
d
in yields ranging from 53% to 69% (Table 1). Reduction of the ketone function of 3a
in methanol solution at 25 C using sodium tetrahyborohydride during 5–7 h. The desired hydroxyl
–
d
◦
compounds 4a–b were obtained after elimination of volatile compounds in vacuo, and simple
treatment of the crude reaction mixture with deionized water afforded 4a–d in good yields (82–98%)
after crystallization.
Table 1.
Results for the preparation of 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-one
1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol 4a and )-(1R, 1S)
-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium hydrochloride 7a–e from phenylalkyl halides 5a–d.
3
1
a
–
d
,
–d
(±
Overall Yield (%) b
Compound
R 1
R 2
R 3
n
R 4
Yield (%) a
3
a
H
H
H
CF3
CF3
H
H
H
H
CF3
H
H
H
CF3
H
H
H
H
H
H
H
H
H
CF3
CF3
CF3
-
-
-
-
-
-
-
-
0
2
0
2
2
2
0
2
2
0
2
2
-
-
-
-
-
-
-
-
53
66
68
69
82
86
94
98
10
21
33
18
33
0
0
0
0
0
0
0
-
-
-
3
b
MeO
MeO
MeO
H
MeO
MeO
MeO
H
3
c
3
d
-
4
a
43
57
64
68
4
4b
H
4
c
CF3
CF3
H
H
H
H
H
H
CF3
CF3
CF3
CF3
CF3
CF3
4d
7
a
MeO
MeO
MeO
MeO
H
7
b
H
9
7
c
MeO
MeO
MeO
H
MeO
MeO
MeO
MeO
MeO
MeO
19
10
17
-
-
-
-
-
-
-
7d
7
e
c
7
f
H
d
d
d
7
g
MeO
MeO
H
MeO
MeO
H
7
7
7
h
i
jd
d
7
7
k
l d
Br
Cl
Br
Br
O
O
O
5
a
5b
Isolated yield after purification; b Overall yield calculated from compound
68 h; d No crystallization after 336 h.
5c
5d
3
; c No crystallization observed after
a
1
In the next step, for the introduction of molecular diversity on C
we used a series of alkyl halides carrying various chains (n = 0 or 2) and methoxy group in para-position
of the phenyl moiety (5a 4-methoxybenzyl chloride; 5b: 1-(2-bromoethyl)-4-methoxybenzene;
: 1-(3-bromopropyl)-4-methoxybenzene; 5d: 3-phenylpropyl bromide). For optimization of the
reaction condition parameters, we developed a set of experiments which are the following: (i) the
β hydroxyl group of 4 by O-alkylation,
5
:
5c
choice of the solvent (DMF, DMSO, MeCN, etc.); (ii) the choice of appropriate base (DIPEA, Et N,
3
KOH); (iii) the number of equivalents for the base; (iv) the reaction time; and (v) appropriate reaction
temperature. We obtained good reproducibility (Table 2 for each product) when we applied a reaction
time of 48 or 72 h (monitored by thin layer chromatography on silica with appropriate eluent for
◦
evaluation of consumption of the starting reagents), 2–5 equivalents of KOH, heating at 50 C (during

Int. J. Mol. Sci. 2018, 19, 856
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4
8 or 72 h) after addition of alkyl halide
5
to produce the intermediate
6
which was not isolated (initial
attempts to isolate it by flash chromatography on silica gel failed). After the addition of saturated brine
to the crude reaction mixture (to solubilize the N-alkylated pyrazolium by-product), the intermediate
was extracted with dry diethyl ether and the collected extracts were directly treated with a commercial
solution of 1 M HCl in ether. The desired salt was collected by simple filtration after complete
crystallization. It should be noted that this protocol yielded compounds 7a
6
7
–
–
e
l
in 10–33% (Table 1),
failed in spite of long
on the contrary, attempts to prepare the lipophilic trifluoromethyl derivatives 7f
◦
crystallization times (168 and 336 h) at 4 C and modification of the solvent for reaction (THF, dioxane).
R2
R2
X
O
O
N
N
OH
N
N
n
Br
R2
R4
N
K CO
NaBH4
5a-d
2
3
HN
R3
R3
R1
MeCN, 25¡C,
-24 h
R1
MeOH, 25¡C,
5-7 h
R1
KOH,
8
DMSO, 50¡C,
48-72 h
R3
1a,b
2a-c
3a-d
4a-d
R4
R4
R2
R2
O
N
N
1M HCl, Et O
O
n
N
n
2
N
, HCl
25¡C,
R3
72-336 h
R3
R1
R1
6
a-n
7a-e
Scheme 1. Synthesis of (1R, 1S) 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium hydrochloride 7a–e.
Table 2. Optimization of the reaction conditions for the preparation of the intermediates 6a–n.
Starting
Compound 4
Reagent 5 for
O-alkylation
KOH (Number
of Equiv.)
Reaction
Time (h)
Entry
Conversion for 6 (%) a
Cl
0
1
2
2
2
48
48
6a
6
O
5
a
Br
4a
6m
-
OH
N
N
O
5
b
Br
3
4
3
2
48
48
6b
6f
76
50
O
5
c
Br
5
d
Cl
5
6
5
5
72
72
6c
68
-
O
5
a
Br
4
b
OH
N
N
6n
O
5
b
Br
Br
Br
7
8
O
5
5
72
72
6d
6e
43
42
O
5
c
Br
5
5
5
d
Cl
9
5
5
5
72
72
72
6g
6h
6i
86
80
74
O
CF3
5a
4
c
OH
N
N
1
0
1
O
5c
O
Br
1
d
Cl
12
13
14
5
5
5
72
72
72
6j
6k
6l
78
70
67
CF3
O
5
a
4d
OH
N
N
O
CF3
5c
O
Br
d
a
1
Conversion determined by H NMR in DMSO-d
6
solution after monitoring of the reaction mixture by TLC on
0.2 mm precoated plates of silica gel 60 F-254 (Merck KGaA, Darmstadt, Germany).

Int. J. Mol. Sci. 2018, 19, 856
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For this structure-activity relationship (SAR) study, we were also interested to evaluate
the potential impact of chirality’s of the compounds on SOC channels activities because our
present protocol for preparation of these pyrazole SKF-96365 analogues produced a ( ) racemic
mixture. Based on this finding, our attention was attracted by the method of half-quantities [28
for resolution of our ( )-(1R, 1S)-1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b mixture
Scheme 2). To find a simple and cost effective resolution procedure for (1R)-4b and (1S)-4b
we used commercial (+)-(1S)- and ( )-(1R)-10-camphorsulfonic acid (CSA). Treatment of ( )-(1R,
S)-1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b with 0.5 equivalent of ( )-(1R)-CSA in
dry acetone caused the formation of a fine suspension for the first fraction of diastereomer
)-(1S)-4b/( )-(1R)-CSA (>85% de), which was recrystallized in dry acetone (52% isolated yield,
97% de). Then, a solution of this salt [( )-(1S)-4b/( )-(1R)-CSA] in dry acetone was submitted
to diasteromeric enrichment by addition of 0.02 equivalent of ( ) racemic 4b. After work-up the
pure ( )-(1S)-4b/( )-(1R)-CSA was obtained in 48% yield (>99% de) as white powder with [ D] =
6.0 (c 1.0, MeOH) (Table 3). This protocol was also applied to (+)-(1S)-CSA for resolution of the (
racemic 4b, and after diastereomeric enrichment, the other diastereomer [(+)-(1R)-4b/(+)-(1S)-CSA,
>99% de)] was obtained in 51% yield with [ D] = +6.0 (c 1.0, MeOH). Crystal structures of the salt
diastereomers were determined by X-ray crystal structure analysis, as example the diastereomer
+)-(1R)-4b/(+)-(1S)-CSA is shown in Figure 2.
7
±
]
±
(
,
−
±
1
−
(
>
−
−
−
−
±
−
−
α
−
±
)
(
α
(
Table
3.
Products
issued
from
resolution
of
(
±
)-(1R,
1S)
1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b with
(
−
)-(1R)-CSA and (+)-(1S)-CSA
using the method of “half-quantities”.
Product
[αD] a
−6
Retention Time tR (min.) b
Yield (%) e
OH
SO3
O
N
N
H
34.0 c
(
−)-(1S)-4b/(−)-(1R)-CSA
48
O
OH
(
+)-(1R)-4b/(+)-(1S)-CSA
N
N
+6
31.5 c
52.6 d
51
38
H
O
O
O3S
OH
N
N
(
−)-(1S)-4b
−10
O
CF3
4d
OH
N
N
63.5 d
(
+)-(1R)-4b
+10
41
CF3
O
a
b
From: c 1.0, MeOH; Retention time obtained by chiral HPLC analysis using Chiracel OJ-H column (250 × 4.60 mm
)
and UV detector at 220 nm; c hexane/i-PrOH 94:6 v/v as eluent with flow rate = 0.8 mL/min; d hexane/i-PrOH 96:4
v/v as eluent with flow rate = 0.6 mL/min; e Isolated yield after purification.
OH
N
N
H
O
O3S
O
(
+)-(1R)-4b/(+)-(1S)-CSA
Figure 2.
Ortep diagram of (+)-(1R) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol
4
b/(+)-(1S)-10-camphorsulfonic acid obtained by X-ray diffraction.

Int. J. Mol. Sci. 2018, 19, 856
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Next, access to the free enantiomer (1S)-4b and (1R)-4b was operated by a simple neutralization
of the respective diastereomers ( )-(1S)-4b/( )-(1R)-CSA or (+)-(1R)-4b/(+)-(1S)-CSA using exactly
equivalent of MeONa in dry MeOH. After mixing during 12 h followed by elimination of volatile
compounds in vacuo, the crude reaction mixture was treated with deionized water and the resulting
insoluble enantiomer ( )-(1S)-4b or (+)-(1R)-4b precipitated and was collected by classical filtration.
As can be seen in Table 3, pure enantiomers ( )-(1S)-4b and (+)-(1R)-4b were obtained respectively
in isolated yields of 38 and 41% with [ D] = 10.0 (c 1.0, MeOH) for ( )-(1S)-4b (>99% ee) and
α ] = +10.0 (c 1.0, MeOH) for (+)-(1R)-4b (>99% ee).
−
−
1
−
−
α
−
−
[
D
During the resolution of the (
)-(1R)-CSA or (+)-(1S)-CSA and neutralization of the two diastereomers, all these compounds were
submitted to chiral HPLC analysis with a Chiracel OJ-H column (250 4.6 i.d. mm) and a UV detector
at 220 nm using hexane/i-PrOH as mobile phase with appropriate composition and flow rate.
±
) racemic mixture 4b by the “half-quantities” protocol with
(
−
×
(
+)-CSA
or
OH
OH
OH
(
+)-CSA or (-)-CSA
(-)-CSA
N
N
N
N
N
0.5 equiv.
N
H
MeONa 1 equiv.
MeOH, 25¡C,
O
O
O
12 h
(
1R, 1S)-4b
diasteroisomer [(1R)-4b, (1S)-CSA]
or [(1S)-4b, (1R)-CSA]
enantiomer (+)-(1R)-4b
or (-)-(1S)-4b
filtrate
>
99% de
> 99% ee
(
+)-CSA
OH
N
OH
or
OH
(
+)-CSA or (-)-CSA
0.5 equiv.
(-)-CSA
N
N
H
N
N
N
MeONa 1 equiv.
MeOH, 25¡C,
O
O
O
12 h
enantiomer (+)-(1R)-4b
or (-)-(1S)-4b
diasteroisomer [(1R)-4b, (1S)-CSA]
or [(1S)-4b, (1R)-CSA]
enantiomer (+)-(1R)-4b
or (-)-(1S)-4b
>
99% de
> 99% ee
Scheme 2. Resolution of (
±
)-(1R, 1S) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b from
(1R)-(−)-10-camphorsulfonic acid and (1S)-(+)-10-camphorsulfonic acid.
For the ( ) racemic mixture 4b, we obtained only two peaks, the retention time of the first
±
and second eluted enantiomers were measured respectively as 65.9 and 71.4 min (hexane/i-PrOH
6/4 v/v, flow rate = 0.6 mL/min). The quality of the results obtained by the “half-quantities” and
neutralization methods were confirmed by the presence of only one peak for the two diastereomers
9
(
−
)-(1S)-4b/(
−
)-(1R)-CSA, (+)-(1R)-4b/(+)-(1S)-CSA and the two enantiomers ( )-(1S)-4b and
−
(
+)-(1R)-4b. These data were summarized in Table 3 (the corresponding chromatograms were reported
in Supplementary Materials).
With the two pure enantiomers (
to prepare potentially the compounds (
−
)-(1S)-4b, (+)-(1R)-4b on hand, we were stimulated
)-(1S)-7d and (+)-(1R)-7d with the alkyl halide
-(3-bromopropyl)-4-methoxybenzene 5c according to Scheme 2. We applied the protocol used initially
−
1
◦
for the preparation of racemic (1R, 1S)-7d using 5 equivalents of KOH in solution of DMSO at 50 C
during 72 h from (
−
)-(1S)-4b or (+)-(1R)-4b and 4-(3-bromopropyl)-4-methoxybenzene 5c (Scheme 3).
1
Progress of the transformation and consumption of the starting reagents were monitored by H NMR
and also by thin layer chromatography on silica plates. After extraction with dry Et O and treatment
2
of the collected extracts with a solution of 1 M HCl, to our surprise, we did not observe precipitation
of the desired ( )-(1S)-7d and (+)-(1R)-7d. We tried to change the solvent for extraction (dioxane as
−
example) but this modification was not well suited for crystallization at room temperature even for an
◦
additional day at 4 C.
2
.2. Biology
The synthesized hydrochloride compounds 7a
ability on endoplasmic reticulum (ER) Ca2+ release and SOCE using the PLP-B lymphocyte cell line.
–c as described above were evaluated for their
For comparison, commercial SKF-96365 hydrochloride was used as reference. All compounds 7 and
SKF-96365 were added 3 min before recording intracellular Ca2+ level variation in the presence
of Fura-2 dye loaded on PLP-B lymphocyte cell. Fluorescence measurements were realized in

Int. J. Mol. Sci. 2018, 19, 856
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Flex Station
™ 3 microplate reader. Fura-2 loaded PLP-B lymphocyte cells were stimulated with
2+
2
µM Thapsigargin (Tg) for 30 min and SOCE was measured after addition of 1.8 mM Ca in the
extracellular medium. Results are respectively summarized in Table 4 and also in Figure 3A–E. For this
structure–activity relationship (SAR) study, we examined mainly the effect of MeO substituent on the
para-phenyl group of the phenethyl-1H-pyrazolium skeleton and also on the phenylalkoxy chain of
Cβ compared to the SKF-96365 reference.
O
O
OH
O
N
N
O
2
N
N
2
N
, HCl
N
Br
4
2%
1M HCl, Et2O
2
O
O
25¡C,
72 h
O
O
(-)-(1S)-6d
O
(-)-(1S)-7d
(
-)-(1S)-4b
5
c
KOH,
DMSO, 50¡C,
2 h
O
O
OH
N
1M HCl, Et O
2
7
N
73%
O
N
N
25¡C,
O
2
N
N
2
,
HCl
7
2 h
(
+)-(1R)-4b
O
(+)-(1R)-6d
O
(+)-(1R)-7d
Scheme 3. Synthesis of 1-[β-((4-methoxyphenyl)-(3-(4-methoxyphenyl)propoxyethyl)]-1H-pyrazole
(
−)-(1S)-6d, (+)-(1R)-6d and assays to their hydrochloride derivatives.
Examination of Table 1 and also dose response curves (Figure 3E) showed that compound 7d
IC50 25 M) was a better potent SOCE inhibitor than SKF-96365 (IC50 60 M). This means that a
slight modification of the heterocyclic platform, particularly the position of the second nitrogen atom
N-3 position in imidazole for SKF-96365 and N-2 position for 7d), has a direct impact on SOCE activity.
The effect of the length for the C -(4-methoxyphenylalkoxy) side chain appeared to be important
in Figure 3C when we compared compounds 7c and 7d: for the same concentration of , the curve
obtained for compound 7d gave higher effect on SOCE inhibition level (%). Again, the presence
or absence of MeO group in para-position for the phenethyl-1H-pyrazolium skeleton of and for
the C -phenylpropoxy side chain afforded variation of IC , i.e., comparison of 7b (IC50 34 M)
and 7d (IC50 25 M) showed that absence of MeO group in the skeleton of 7b has a lower effect on
SOCE IC . On the other hand, absence of MeO group of 7c (IC50 48 M) on the side chain led to
(
µ
µ
(
β
7
7
β
µ
50
µ
µ
50
a more important effect and the IC50 decreased. Similar observations were also made when a more
physiological stimulation of SOCE activation was implemented (Figure 4). When B cells are stimulated
with an antigen (M Immunoglobulin: IgM), B cell receptor (BCR) activation leads to an increase of
2+
Ca concentration mainly due SOCE. However, inhibition by 7c and 7d compounds is quite identical
and less than what was observed with SKF-96365.
Here, we have reported the racemic synthesis of pyrazole SKF-96365 analogues in four steps
without substituent (CF group) on the pyrazole platform in moderate to good yields. For the ( )
±
3
hydroxyl intermediate
using the method of “half-concentration” with commercial (+)-(1S)- and (
acid (CSA) followed by neutralization of diastereomers with MeONa in dry MeOH solution. With the
pure enantiomers ( )-(1S)-4b and (+)-(1R)-4b, initial attempts to obtain the crystallized ( )-(1S)-7d
and (+)-(1R)-7d after treatment of intermediate 6d with a solution of 1 M HCl (for precipitation of
hydrochloride salt 7d). Effects of compounds 7a
on endoplasmic reticulum (ER) Ca2+ and SOCE
4, we have developed a successful approach for separation of enantiomers
−
)-(1R)-10-camphorsulfonic
−
−
–
d
were evaluated on PLP-B lymphocyte cell line, and 7d was identified as a better SOCE inhibitor than
SKF-96365. However, the inhibitory effects of 7c and 7d compounds on SOCE are inferior to what
was observed for SKF-96365 when evaluated with BCR stimulation. This preliminary SAR study
showed that the MeO group in para-position of the phenethyl-1H-pyrazolium skeleton or for the
Cβ-phenylpropoxy side chain of 7 influenced the SOCE activity. These results offered possibilities
to increase molecular diversity for a complete SAR study, and we are also currently exploring the
potential of this synthetic methodology.

Int. J. Mol. Sci. 2018, 19, 856
8 of 24
Figure 3. Cont.

Int. J. Mol. Sci. 2018, 19, 856
9 of 24
Figure 3. Effect of compounds 7c, A–D) showed typical representative
7d, and SKF-96365 compounds on ER Ca2+ release and store-operated Ca2+ entry (SOCE). (
traces of intracellular Ca2+ concentration variations measured with Fura-2 dye in PLP-B lymphocyte cell line when SOCE activation protocol is implemented. Tested
2
+
compounds 7c
(
,
7d, and SKF-96365 were added 3 min before recording intracellular Ca level variation with the FlexStation 3
™
plate reader. Endoplasmic reticulum
ER) Ca2+ content is first totally depleted in Ca free medium by injection of 2
2+
µ
M Thapsigargin (Tg) and SOCE is next measured by addition of 1.8 mM Ca in the
2+
extracellular medium; (
compounds (100 and 1
inhibition dose-response curves: points represented the mean
D
µ
) Representative traces of the SOCE Ca2+ signals recorded after ER Ca2+ stores releases with Tg in cells treated with 7c
M). SOCE amplitudes in the presence of the different drugs are normalized to vehicle control; ( 7c 7d, and SKF-936365 compounds
SEM of three different experiments and expressed as the percentage of SOCE Ca entry amplitude
inhibition on PLP-B Lymphocyte cell line normalized to vehicle control (100%).
, 7d, and SKF-96365
E
)
,
2+
±

Int. J. Mol. Sci. 2018, 19, 856
10 of 24
A
B
C
2
+
Figure 4. Effect of 7c
,
7d, and SKF-96365 compounds on BCR (B Cell Receptor) induced Ca responses in B cells stimulated with IgM in the presence of extracellular
2
+
2+
Ca . (A
) histograms represent the average inhibition of IgM-induced Ca transients in PLP cells treated with five different concentrations of 7c
,
7d and SKF-96363
compounds (100, 50, 10, 1 and 0.1
µ
M). Data are normalized to vehicle control (100%) and represented as the mean
±
SEM of 3 different experiments; ( ) panels
B,C
2
+
show typical representative traces of intracellular Ca concentration variations measured with Fura-2 dye in PLP cells with BCR activation stimulation by IgM in the
presence or absence of the different tested drugs. Tested compounds 7c
BCR-induced Ca2+ responses are measured in 10 mM Ca extracellular medium in cells stimulated with 10
7d, and SKF-96365 were added 3 min before recording intracellular Ca2 level variation.
+
,
2+
µg/mL IgM; (
B) Representative traces obtained in control
(
−
) conditions or in the presence of SKF-696365 (100
µM); (C
) Representative traces normalized to control conditions in cells treated with 7c,
7d, and SKF-96365
compounds (100 and 1 µM).

Int. J. Mol. Sci. 2018, 19, 856
11 of 24
Table 4.
Results for effects of ( )-(1R, 1S) 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium
±
2
+
2+
hydrochloride 7a–e on store-operated Ca entry with PLP-B lymphocyte. Store-operated Ca
2
+
2+
entry was induced by depletion of Endoplasmic Reticulum Ca stores (ER Ca stores) with
thapsigargin (Tg) in a Ca2+-free medium and measured following the addition of 1.8 mM CaCl2
in the extracellular medium.
Compound 7
Concentration of 7 (µM)
00
Effect of 7 on Amplitude of SOCE Peak a
IC50 (µM) b
1
−44.4
−29.0
−14.5
−12.0
−24.5
O
N
N
50
10
1
O
102
7a, HCl
0
.1
1
50
10
1
0
00
−83.0
−65.0
−14.8
−9.3
O
N
N
34
O
7
b, HCl
.1
−5.6
1
50
10
1
0
00
−53.1
−46.0
−2.2
−7.1
7.6
O
N
N
O
60
25
48
60
7
c, HCl
O
.1
1
50
10
1
0
00
−87.5
−81.8
−0.3
O
N
N
O
7d, HCl
−10.7
−0.3
O
.1
1
50
1
1
00
−81.7
−51.1
−5.4
O
N
N
0
−3.6
7
e, HCl
O
0
.1
−11.3
1
5
1
1
00
0
0
−58.7
−44.3
−15.4
−26.4
−3.4
O
N
N
O
,
HCl
O
SKF-96365
0
.1
a
) or activation. Controlled with DMSO 100%; b IC50 expressed in
%
of inhibition (
assays, ±0.5 µM.
−
µ
M are the average of three
3
. Materials and Methods
3
.1. Chemistry Section
General Information. Preparative chromatography was realized on a Combi Flash R 200 psi UV
f
ref. 208K20284 (Serlabo Technologies, Entraigues-sur-la-Sorgue, France) using pre-packed column
of alumina gel 60 F 254 Merck equipped with a DAD UV/Vis 200–360 nm detector. Thin-layer
chromatography (TLC) was accomplished on 0.2 mm precoated plates of silica gel 60 F-254 (Merck)
with appropriate eluent. Visualization was made with ultraviolet light (254 and 365 nm) or with
a fluorescence indicator. Solvents were evaporated with a BUCHI rotary evaporator (New Castle,
PA, USA). All reagents and solvents were purchased from Acros Fisher (Illkirch, France), Sigma-Aldrich
Chimie (St. Quentin Fallavier, France), and Fluka Chimie (Paris, France) and were used without further
1
13
purification. H NMR spectra were recorded on Bruker AC 300 P (300 MHz) spectrometer and
NMR spectra on Bruker AC 300 P (75 MHz) spectrometer. Chemical shifts are expressed in parts per
million downfield. Data are given in the following order: value, multiplicity (s, singlet; d, doublet; t,
C
δ
triplet; q, quartet; quint: quintuplet, m, multiplet; br, broad), number of protons, coupling constants J
is given in Hertz. The high-resolution mass spectra (HRMS) were recorded in positive mode using
direct Electrospray infusion, respectively on Waters Q-TOF 2 or on Thermo Fisher Scientific Q-Exactive

Int. J. Mol. Sci. 2018, 19, 856
12 of 24
spectrometers at the “Centre Régional de Mesures Physiques de l’Ouest” platform (CRMPO platform,
ScanMAT UMS 2001 CNRS, Rennes, France). Melting points were determined on a Kofler melting
point apparatus and were uncorrected. Optical rotations [
α ] were measured on a Perkin-Elmer
D
◦
−3 −1 −1
dm (c in
2
14 polarimeter at room temperature (25 C) and are recorded in units of deg cm
g
−
3
g cm in MeOH) with a 1.0 cm cell. The ee- and de-values were determined by chiral HPLC analysis
using Chiracel OJ-H column (250 4.60 mm) with UV detector at 220 nm using hexane/i-PrOH as
mobile phase with appropriate composition and flow rate.
×
1
-Phenyl-2-(1H-pyrazol-1-yl)ethan-1-one (3a). To a solution of 2-bromoacetophenone 1a (4 g, 20.1 mmol)
in 20.8 mL of acetonitrile, pyrazole 2a (1.45 g, 21.3 mmol, 1.06 equiv.) was added in small portions
under vigorous magnetic stirring (550 rpm) at room temperature, and mixing was pursued until
complete dissolution of the reagents. To this homogeneous solution, K CO (2.92 g, 21.1 mmol,
2
3
◦
1.05 equiv.) was poured and the resulting suspension was stirred for 8 h at 25 C and monitored by
thin layer chromatography on 0.2 mm plates of silica gel 60 F-254 (Merck) using cyclohexane/AcOEt
(
1:1 v/v) as eluent. The reaction mixture was diluted with 20 mL of AcOEt, and the resulting solution
◦
was filtered on a Büchner funnel (porosity N 4) and the residual precipitate was washed with AcOEt
(
2
×
10 mL). The collected filtrate was transferred into a separating funnel. The organic phase was
washed successively with deionized water (3 80 mL), brine (3 80 mL), and dried over magnesium
sulfate. After filtration on a filter paper, the filtrate was concentrated in a rotary evaporator under
reduced pressure and the oily residue was submitted to purification by preparative chromatography
×
×
(
Combi Flash R 200 psi apparatus with a DAD 200/360 nm detector) on pre-packed column of silica
f
gel 60 F-254 (Merck) using a stepwise gradient of cyclohexane/AcOEt (0–50%) for elution. Pooling for
6
3
0 min and elimination of the solvent in vacuo gave 3.74 g (53% yield) of the pure desired compound
◦
a as yellowish powder. Mp = 96–97 C. H NMR (DMSO-d ) δ = 5.84 (s, 2H, CH , H-2), 6.31 (dd,
6 2
1
0
0
J = 2.3, 1.9 Hz, 1H, H-4 ), 7.48 (dd, 1H, J = 1.9, 0.7 Hz, H-3 ), 7.52–7.63 (m, 2H, H-3”, H-5”, Ar), 7.66–7.77
0
13
0
(
m, 2H, H-4”, H-5 , Ar), 7.97–8.09 (m, 2H, H-2”, H-6”). C NMR (DMSO-d6)
δ
= 57.7 (C-2), 105.6 (C-4 ),
0
0
128.1 (C-3”, C-5”), 129.0 (C-2”, C-6”), 131.6 (C-5 ), 134.0 (C-4”), 134.6 (C-1”), 139.0 (C-3 ), 193.7 (C=O).
+
+
ES HRMS, m/z = 209.0689 found (calculated for C H N ONa [M + Na] requires 209.0691).
11
10
2
1-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-one
(
3b).
To
a
solution
of
2-bromo-1-(4-methoxyphenyl)ethan-1-one 1b (4.6 g, 20.1 mmol) in 20.8 mL of acetonitrile,
pyrazole 2a (2.74 g, 40.2 mmol, 2 equiv.) was added in small portions under vigorous magnetic
stirring (550 rpm) at room temperature, and mixing was pursued until complete dissolution of the
reagents. To this homogeneous solution, K CO (5.56 g, 40.2 mmol, 2 equiv.) was poured and the
2
3
◦
resulting suspension was stirred for 12 h at 25 C and monitored by thin layer chromatography on
.2 mm plates of silica gel 60 F-254 (Merck) using cyclohexane/AcOEt (3:7 v/v) as eluent. The reaction
0
mixture was diluted with 20 mL of AcOEt, and the resulting suspension was filtered on a Büchner
◦
funnel (porosity N 4) and the residual precipitate was washed with AcOEt (2
×
10 mL). The collected
filtrate was transferred into a separating funnel. The organic phase was washed successively with
deionized water (3 80 mL), brine (3 80 mL), and dried over magnesium sulfate. After filtration on
a filter paper, the filtrate was concentrated in a rotary evaporator under reduced pressure and the
×
×
oily residue was submitted to purification by preparative chromatography (Combi Flash R 200 psi
f
apparatus with a DAD 200/360 nm detector) on pre-packed column of silica gel 60 F-254 (Merck) using
a stepwise gradient of cyclohexane/AcOEt (0–70%) for elution. Pooling for 60 min and elimination of
the solvent in vacuo gave 4.35 g (66% yield) of the pure desired compound 3b as yellowish needles.
◦
0
1
Mp = 102–103 C. H NMR (DMSO-d )
δ = 3.86 (s, 3H, OCH ), 5.77 (s, 2H, CH , H-2), 6.30 (t, 1H,
3 2
6
0
J = 2.1 Hz, H-4 ), 7.09 (d, 2H, J = 8.9 Hz, H-3”, H-5”, Ar), 7.47 (dd, 1H, J = 1.9, 0.7 Hz, H-3 ), 7.72 (dd,
0
13
1
5
H, J = 2.3, 0.7 Hz, H-5 ), 8.01 (d, 2H, J = 8.9 Hz, H-2”, H-6”, Ar). C NMR (DMSO-d6)
7.3 (C-2), 105.5 (C-4 ), 114.2 (C-3”, C-5”), 127.4 (C-5 ), 130.4 (C-2”, C-6”), 131.6 (C-1”), 138.8 (C-3 ),
63.6 (C-4”), 191.9 (C=O). ES HRMS, m/z = 239.0798 found (calculated for C H N O Na [M + Na]
δ
= 55.7 (OCH ),
3
0
0
0
+
+
1
12 12 2 2
requires 239.0797).

Int. J. Mol. Sci. 2018, 19, 856
13 of 24
2
2
3
-(3-Trifluoromethyl-1H-pyrazol-1-yl)-1-(4-methoxyphenyl)ethan-1-one
-bromo-1-(4-methoxyphenyl)ethan-1-one 1b (0.5 g, 2.18 mmol) in 2.25 mL of acetonitrile,
-trifluoromethylpyrazole 2b (0.89 g, 6.54 mmol, 3 equiv.) was added in small portions under vigorous
(
3c).
To
a
solution of
magnetic stirring (550 rpm) at room temperature, and mixing was pursued until complete dissolution
of the reagents. To this homogeneous solution, K CO (0.905 g, 6.54 mmol, 3 equiv.) was poured and
2
◦
3
the resulting suspension was stirred for 12 h at 25 C and monitored by thin layer chromatography on
.2 mm plates of silica gel 60 F-254 (Merck) using cyclohexane/AcOEt (1:1 v/v) as eluent. The reaction
0
mixture was diluted with 5 mL of AcOEt, and the resulting solution was filtered on a Büchner funnel
◦
(
porosity N 4) and the residual precipitate was washed with 5 mL of AcOEt. The collected filtrate
was transferred into a separating funnel. The organic phase was washed successively with deionized
water (3 20 mL), brine (3 20 mL), and dried over magnesium sulfate. After filtration on a filter
×
×
paper, the filtrate was concentrated in a rotary evaporator under reduced pressure and gave a solid
residue. After addition of 40 mL of hexane and mixing for 4 h, the solid was filtered on a Büchner
◦
◦
funnel (porosity N 4) then dried at 60 C for 3 h and gave 0.62 g (68% yield) of the desired compound
◦
1
3
c
as white powder. Mp = 146–147 C. H NMR (DMSO-d )
δ
= 3.87 (s, 3H, OCH ), 5.94 (s, 2H, CH ,
6
3
2
0
H-2), 6.78 (dd, 1H, J = 2.4, 0.7 Hz, H-4 , Ar), 7.12 (d, 2H, J = 8.9 Hz, H-3”, H-5”, Ar), 7.95 (dq, 1H,
0
13
J = 2.1, 1.0 Hz, H-5 , Ar), 8.02 (d, 2H, J = 8.9 Hz, H-2”, H-6”, Ar). C NMR (DMSO-d6)
δ
= 55.8 (OCH3),
0
0
0
5
8.1 (C-2), 104.4 (C-4 ), 104.4 (C-4 ), 114.2 (C-2”, C-6”), 114.3 (C-2”, C-6”), 127.1 (C-1”), 130.4 (C-3”,
0
+
C-5”), 130.5 (C-3”, C-5”), 134.2 (C-5 ), 134.3 (C-5 ), 163.8 (C-4”), 191.0 (C-1). ES HRMS, m/z = 307.0671
found (calculated for C H N O F Na [M + Na]+ requires 307.0670); 285.0862 found (calculated for
1
3
11
2
2 3
+
C H N O F [M + H] requires 285.0851).
13
12
2
2 3
2
2
3
-(3,5-Bis-trifluoromethyl-1H-pyrazol-1-yl)-1-(4-methoxyphenyl)ethan-1-one (3d).
-bromo-1-(4-methoxyphenyl)ethan-1-one 1b (0.5 g, 2.18 mmol) in 2.25 mL of acetonitrile,
,5-bis-trifluoromethylpyrazole 2c (1.778 g, 8.72 mmol, 4 equiv.) was added in small portions under
To a solution of
vigorous magnetic stirring (550 rpm) at room temperature, and mixing was pursued until complete
dissolution of the reagents. To this homogeneous solution, K CO (1.205 g, 8.72 mmol, 4 equiv.)
2
3
◦
was poured and the resulting suspension was stirred for 24 h at 25 C and monitored by thin layer
chromatography on 0.2 mm plates of silica gel 60 F-254 (Merck) using cyclohexane/AcOEt (1:1 v/v)
as eluent. The reaction mixture was diluted with 5 mL of AcOEt, and the resulting solution was
◦
filtered on a Büchner funnel (porosity N 4) and the residual precipitate was washed with 5 mL of
AcOEt. The collected filtrate was transferred into a separating funnel. The organic phase was washed
successively with deionized water (3
After filtration on a filter paper, the filtrate was concentrated in a rotary evaporator under reduced
pressure and gave a solid residue which was dried under high vacuum (10 Torr) at 25 C for 2 h.
×
20 mL), brine (3
×
20 mL), and dried over magnesium sulfate.
−
2
◦
◦
1
The desired compound 3d was obtained in 69% yield as white powder. Mp = 114–115 C. H NMR
DMSO-d6) = 3.88 (s, 3H, OCH ), 6.19 (s, 2H, CH , H-2), 7.12 (d, 2H, J = 8.9 Hz, H-3”, H-5”, Ar),
(
7
5
δ
3
2
0
13
.69 (s, 1H, H-4 , Ar), 8.05 (d, 1H, J = 8.9 Hz, H-2”, H-6”, Ar). C NMR (DMSO-d6)
8.5 (C-2), 107.5 (C-4 ), 114.3 (C-3”, C-5”), 126.3 (C-1”), 130.8 (C-2”, C-6”), 164.2 (C-4”), 189.9 (C=O). ES
δ
= 55.8 (OCH ),
3
0
+
+
HRMS, m/z = 375.0544 found (calculated for C H N O F Na [M + Na] requires 375.0544); 353.0734
14
10
2
2 6
+
found (calculated for C H N O F [M + H] requires 353.0734).
14
11
2
2 3
3
.1.1. General Procedure for Reduction of 1-Phenyl-2-(1H-pyrazol-1-yl)ethan-1-one (3a–c) into
1
-Phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol (4a–c)
To a solution of 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-one
3 (5 mmol) in an appropriate volume of
anhydrous methanol (9–40 mL), sodium borohydride NaBH (0.189 g, 5 mmol, 1 equiv.) was added in
4
◦
small portions at 0 C (ice bath) under magnetic stirring (300 rpm). The resulting mixture was stirred
(500 rpm) for an appropriate reaction time (5–7 h) at room temperature, and the reaction solution
was monitored by thin layer chromatography (TLC) on 0.2 mm plates of silica gel 60 F-254 (Merck)
using an appropriate mixture of solvents as eluent. The reaction mixture was concentrated in a rotary
evaporator under reduced pressure and the resulting solid residue was washed with deionized water

Int. J. Mol. Sci. 2018, 19, 856
14 of 24
◦
(
3
×
45–3
×
47 mL) on a Büchner funnel. Then, the desired compounds 3a
–
c
were dried at 60 C
for 3 h.
1-Phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol (4a). According to the standard procedure, the compound
4
a
was prepared from 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-one 3a (0.93 g, 5 mmol) in 9.3 mL of
anhydrous methanol after a reaction time of 5 h using CH Cl /MeOH 9:1 v/v as eluent for thin layer
2
2
chromatography. Washing work-up was realized with 3
g (82% yield) of the desired compound 4a as white powder. Mp = 132–134 C. H NMR (DMSO-d )
×
45 mL of deionized water and gave 0.94
◦
1
6
δ = 4.14–4.30 (m, 2H, CH , H-2), 4.83–5.01 (m, 1H, CH, H-1), 5.65 (br d, 1H, J = 4.7 Hz, OH), 6.18 (t,
2
0
1
H, J = 2.0 Hz, H-4 ), 7.19–7.39 (m, 5H, H-2”, H-3”, H-4”, H-5”, H-6”, Ar), 7.43 (dd, 1H, J = 1.9, 0.7 Hz,
H-3 ), 7.58 (dd, 1H, J = 2.3, 0.7 Hz, H-5 ). C NMR (DMSO-d )
26.1 (C-3”, C-5”), 127.4 (C-4”), 128.2 (C-2”, C-6”), 130.7 (C-5 ), 138.5 (C-1”), 142.8 (C-3 ). ES HRMS,
m/z = 211.0848 found (calculated for C H N O Na [M + Na] requires 211.0847).
0
0
13
0
δ
= 58.7 (C-2), 71.8 (C-1), 104.7 (C-4 ),
6
0
0
+
1
+
1
1
12
2
2
1
-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b).
the compound 4b was prepared from 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-one 3b (1.08 g,
mmol) in 14.4 mL of anhydrous methanol after a reaction time of 7 h using CH Cl /MeOH 8:2
According to the standard procedure,
5
2
2
v/v as eluent for thin layer chromatography. Washing work-up was realized with 3
deionized water and gave 1.091 g (86% yield) of the desired compound 4b as white crystals with
de = 50% (hexane/i-PrOH 96:4 v/v as eluent, flow rate = 0.6 mL/min.), retention times t (1) = 65.92
×
47 mL of
R
◦
1
and tR(2) = 71.38 min. [
α
] = 0.0 (c 1.0, MeOH). Mp = 116–118 C. H NMR (DMSO-d )
δ
= 3.73 (s, 3H,
D
6
OCH ), 4.10–4.27 (m, 2H, CH , H-2), 4.87 (dt, 1H, J = 7.5, 5.1 Hz, CH, H-1), 5.54 (br d, 1H, J = 4.6 Hz
OH), 6.17 (dd, 1H, J = 1.8 Hz, H-4 ), 6.88 (d, 2H, J = 8.7 Hz, H-3”, H-5”, Ar), 7.15–7.29 (m, 2H, H-2”,
,
3
2
0
0
0
13
H-6”, Ar), 7.42 (dd, 1H, J = 1.8, 0.7 Hz, H-3 ), 7.57 (dd, 1H, J = 2.2, 0.8 Hz, H-5 ). C NMR (DMSO-d6)
0
δ
= 55.0 (OCH ), 58.7 (C-2), 71.4 (C-1), 104.6 (C-4 ), 113.5 (C-3”, C-5”), 127.2 (C-2”, C-6”), 130.5 (C-1”),
3
0 0
34.8 (C-5 ), 138.4 (C-3 ), 158.5 (C-4”). ES HRMS, m/z = 241.0954 found (calculated for C H N O Na
12 14 2 2
+
1
[
+
M + Na] requires 241.0953).
-(4-Methoxyphenyl)-2-(3-trifluoromethyl-1H-pyrazol-1-yl)ethan-1-ol
to the standard procedure, the compound
-(4-methoxyphenyl)-2-(3-trifluoromethyl-1H-pyrazol-1-yl)ethan-1-one 3c (1.42 g, 5 mmol) in
9 mL of anhydrous methanol after a reaction time of 7 h using hexane/AcOEt 1:1 v/v as eluent
for thin layer chromatography. Washing work-up was realized with 3 47 mL of deionized water
and gave 1.431 g (94% yield) of the desired compound 4c as white powder. Mp = 110–112 C. H
NMR (DMSO-d6) = 3.74 (s, 3H, OCH ), 4.27 (d, 2H, J = 6.5 Hz, CH , H-2), 4.90 (t, 1H, J = 6.5 Hz, CH,
H-1), 5.63 (br s, 1H, OH), 6.66 (d, 1H, J = 2.3 Hz, H-4 , Ar), 6.90 (d, 2H, J = 8.6 Hz, H-3”, H-5”, Ar),
1
(
4c).
was
According
prepared from
4c
1
3
×
◦
1
δ
3
2
0
0
13
7
5
.28 (d, 2H, J = 8.3 Hz, H-2”, H-6”, Ar), 7.85 (s, 1H, H-5 , Ar). C NMR (DMSO-d6)
5.1 (OCH3), 59.3 (C-2), 71.0 (C-1), 103.7, (C-4 ), 103.7 (C-4 ), 113.6 (C-2”, C-6”), 113.6 (C-2”, C-6”),
0 0
27.2 (C-3”, C-5”), 127.3 (C-3”, C-5”), 133.1 (C-5 ), 133.2 (C-5 ), 134.2 (C-1”), 140.13 (q, J = 37.1 Hz, CF ),
3
δ
= 55.0 (OCH3),
0
0
1
1
3
+
+
58.7 (C-4”). ES HRMS, m/z = 309.0824 found (calculated for C H N O F Na [M + Na] requires
13
12
2
+
2 3
09.0827); 269.0902 found (calculated for C H N OF [M − H O + H] requires 309.0827).
1
3
11
2
3
2
2
2
3
-(3,5-Bis-trifluoromethyl-1H-pyrazol-1-yl)-1-(4-methoxyphenyl)ethan-1-ol (4d).
-(3,5-bis-trifluoromethyl-1H-pyrazol-1-yl)-1-(4-methoxyphenyl)ethan-1-one 3d (1.76 g, 5 mmol) in
To a solution of
1 mL of anhydrous methanol, sodium borohydride NaBH (0.378 g, 10 mmol, 2 equiv.) was added
4
◦
in small portions at 0 C (ice bath) under magnetic stirring (300 rpm). The resulting mixture was
stirred (500 rpm) for 7 h at room temperature and the reaction solution was monitored by thin layer
chromatography on 0.2 mm plates of silica gel 60 F-254 (Merck) using hexane/AcOEt 1:1 v/v as eluent.
After concentration of the reaction mixture in vacuo, 44 mL of deionized water was added in the
crude oily residue, and extraction was conducted with AcOEt (3
The combined extracts were washed with 44 mL of brine and dried over magnesium sulfate. After
filtration on a filter paper, the filtrate was concentrated in a rotary evaporator under reduced pressure
×
44 mL) in a separating funnel.

Int. J. Mol. Sci. 2018, 19, 856
15 of 24
and gave an oily residue which was dried under high vacuum (10 2 Torr) at 25 C for 2 h. The desired
−
◦
1
compound 4d (1.771 g) was obtained as yellowish mobile oil in 98% yield. H NMR (DMSO-d6)
δ = 3.74 (s, 3H, OCH ), 4.29 (dd, 1H, J = 13.9, 8.9 Hz, CH, H-2), 4.40 (dd, 1H, J = 13.9, 4.4 Hz, CH, H-2),
3
5
.01 (dt, 1H, J = 8.9, 4.4 Hz, CH, H-1), 5.73 (dd, 1H, J = 4.6, 0.7 Hz, OH), 6.92 (d, 2H, J = 8.7 Hz, H-3”,
0
13
H-5”, Ar), 7.28 (d, 2H, J = 8.4 Hz, H-2”, H-6”, Ar), 7.50–7.54 (m, 1H, H-4 , Ar). C NMR (DMSO-d6)
δ = 55.1 (OCH3), 58.8 (C-2), 70.6 (C-1), 106.8 (C-4 ), 113.7 (C-3”, C-5”), 127.2 (C-2”, C-6”), 133.5 (C-1”),
0
+
+
158.9 (C-4”). ES HRMS, m/z = 377.0700 found (calculated for C H N O F Na [M + Na] requires
14 12 2 2 6
+
3
77.0701); 337.0763 found (calculated for C H N OF [M − H O + H] requires 337.0776).
14
11
2
6
2
3
.1.2. General Procedure for the Synthesis of (1R, 1S) 1-[
β-(Phenylalkoxy)-phenethyl]-1H-pyrazolium
Hydrochloride (7a–e)
Pellets of potassium hydroxide KOH (0.112 g, 2 mmol, 2 equiv.) were added to a solution of
-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol (1 mmol, 1 equiv.) in 3.76 mL of dry dimethylsulfoxide pa at
room temperature. The resulting mixture was stirred vigorously (550 rpm) for 30 min until complete
1
4
dissolution of KOH, then commercial arylalkyl halide
5
(1 mmol, 1 equiv.) was added in one portion
◦
in the reaction mixture. The reaction mixture was heated at 50 C under magnetic stirring (300 rpm)
for 48 h. After cooling down to room temperature, 18.7 mL of saturated brine was added to the
reaction mixture and the resulting solution was transferred into a separating funnel. Extraction was
conducted with 18.7 mL of diethyl ether Et O, then the organic phase was dried over magnesium
2
sulphate and filtered on a filter paper. The filtrate was concentrated in a rotary evaporator under
reduced pressure. The oily crude residue containing1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazole 6
was dissolved in appropriate volume of diethyl ether Et O pa under a stream of argon with magnetic
2
stirring (200 rpm). To this homogeneous solution, a commercial solution of 1 M HCl (1 equiv.) in
ether was added dropwise rapidly. During mixing at room temperature, the initial pale yellow oil
crystallized progressively on the circumference of the round flask and mixing (from 24 h to 5 days) was
pursued until complete crystallization of the desired 1-[β-(phenylalkoxy)-phenethyl]-1H-pyrazolium
hydrochloride
7. The resulting precipitate was collected by filtration on a Büchner funnel (porosity
◦
N 4) and washed with dry diethyl ether pa. The desired compound
for 4 h and stored in a dessicator.
7 was dried at room temperature
(
1R, 1S) 1-[
procedure from 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol 4a (0.188 g, 1 mmol) and 4-methoxybenzyl
chloride 5a (0.157 g, 1 mmol, 1 equiv.), the 1-[ -((4-methoxybenzyl)oxy)-phenethyl]-1H-pyrazole
(0.199 g, 0.65 mmol) was dissolved in 4.6 mL of dry diethyl ether pa under a stream of argon.
β-((4-Methoxybenzyl)oxy)-phenethyl]-1H-pyrazolium hydrochloride (7a). Using the standard
β
6
a
The addition of 0.65 mL of 1M HCl (0.65 mmol, 1 equiv.) produced a viscous gum on the circumference
of the round flask after mixing at room temperature for 5 days. Then, this reaction mixture was
◦
stored in a refrigerator at 4 C for 2 weeks and produced a compact gum. This gum was then
manually and carefully triturated in 4 mL of dry hexane and progressively produced a powder.
The resulting suspension was stirred (200 rpm) at room temperature for 5 days, and produced a
completely divided powder. The desired salt 7a was collected by filtration on a Büchner funnel
◦
◦
(
porosity N 4) and washed with 0.5 mL of dry diethyl ether pa. After drying at 25 C for 4 h,
the 1-[ -((4-methoxybenzyl)oxy)-phenethyl]-1H-pyrazolium hydrochloride 7a (35.72 mg, 10% yield)
β
◦
1
was obtained as white powder. Mp = 62–65 C. H NMR (DMSO-d )
δ
= 3.72 (s, 3H, OCH ), 4.09 (d,
6
3
0
0
1
H, J = 11.6 Hz, CH, H-1”), 4.19–4.32 (m, 2H, CH, H-1”, H-1 ), 4.40 (dd, 1H, J = 13.9, 8.5 Hz, CH, H-1 ),
.78 (dd, 1H, J = 8.4, 4.4 Hz, CH, H-2 ), 6.22 (t, 1H, J = 2.0 Hz, H-4, Ar), 6.82 (d, 2H, J = 8.6 Hz, H-3b,
0
4
H-5b, Ar), 6.99 (d, 2H, J = 8.6 Hz, H-2b, H-6b, Ar), 7.27–7.44 (m, 5H, H-2a, H-3a, H-4a, H-5a, H-6a),
1
3
7
5
1
.46 (d, 1H, J = 1.8 Hz, H-3, Ar), 7.62 (d, 1H, J = 2.2 Hz, H-5, Ar). C NMR (DMSO-d6)
δ
= 55.1 (OCH3),
7.0 (C-1 ), 69.7 (C-1”), 79.4 (C-2 ), 104.9 (C-4), 113.6 (C-3a, C-5a), 126.8 (C-2a, C-6a), 128.1 (C-4a),
28.6 (C-2a, C-6a), 128.8 (C-3a, C-5a), 129.9 (C-1b), 130.9 (C-5), 138.6 (C-3), 139.0 (C-1a), 158.6 (C-4b).
0
0
+
+
ES HRMS, m/z = 331.1418 found (calculated for C H N O Na [M + Na] requires 331.1417).
19
20
2
2

Int. J. Mol. Sci. 2018, 19, 856
16 of 24
(
1R, 1S) 1-[
Using the standard procedure from 1-phenyl-2-(1H-pyrazol-1-yl)ethan-1-ol 4a (0.188 g,
mmol) and 1-(3-bromopropyl)-4-methoxybenzene 5c (0.229 g, mmol, equiv.),
the 1-[ -((3-phenylpropyl)oxy)-phenethyl]-1H-pyrazole 6b (0.151 g, 0.45 mmol) was dissolved
in 3 mL of dry diethyl ether pa under a stream of argon. The addition of 0.45 mL of 1M HCl
0.45 mmol, 1 equiv.) produced a pale yellow viscous oil that crystallized progressively on
β
-((3-(4-Methoxyphenyl)propyl)oxy)-phenethyl]-1H-pyrazolium hydrochloride
(7b).
1
1
1
β
(
the circumference of the round flask, and mixing (200 rpm, 72 h) was pursued until complete
crystallization of the oily gum. The desired salt 7b was collected by filtration on a Büchner funnel
◦
◦
0.5 mL of dry diethyl ether pa. After drying at 25 C for 4 h,
(
porosity N 4) and washed with 3
×
the 1-[ -((3-(4-methoxyphenyl)propyl)oxy)-phenethyl]-1H-pyrazolium hydrochloride 7b (78.3 mg,
1% yield) was obtained as white powder. Mp = 76–80 C. H NMR (DMSO-d )
β
◦
1
2
6
3
δ
= 1.62 (dt, 2H, J = 8.2
,
6
.2 Hz, CH , H-2”), 2.36 (dd, 2H, J = 8.4, 6.7 Hz, CH , H-3”), 3.05 (dt, 1H, J = 9.5, 6.1 Hz, CH, H-1”),
2
2
0
.22 (dt, 1H, J = 9.5, 6.0 Hz, CH, H-1”), 3.69 (s, OCH ), 4.23 (dd, 1H, J = 13.9, 4.4 Hz, CH, H-1 ), 4.37 (dd,
H, J = 13.9, 8.7 Hz, CH, H-1 ), 4.69 (dd, 1H, J = 8.7, 4.2 Hz, CH, H-2 ), 6.23 (t, 1H, J = 2.1 Hz, H-4,
3
0
0
1
Ar), 6.77 (d, 2H, J = 8.6 Hz, H-3b, H-5b, Ar), 6.94 (d, 2H, J = 8.6 Hz, H-2b, H-6b, Ar), 7.27–7.44 (m, 5H,
13
H-2a, H-3a, H-4a, H-5a, H-6a, Ar), 7.46–7.50 (m, 1H, H-3), 7.68 (dd, 1H, J = 2.3, 0.7 Hz, H-5). C NMR
0
0
(DMSO-d6)
δ
= 30.4 (C-3”), 31.1 (C-2”), 54.9 (OCH3), 57.2 (C-1 ), 67.4 (C-1”), 80.1 (C-2 ), 104.9 (C-4),
1
1
13.6 (C-3b, C-5b), 126.7 (C-2a, C-6a), 128.0 (C-4a), 128.5 (C-3a, C-5a), 129.2 (C-2b, C-6b), 131.0 (C-5),
+
33.4 (C-1b), 138.4 (C-3), 139.3 (C-1a), 157.3 (C-4b). ES HRMS, m/z = 359.1733 found (calculated for
+
+
C H N O Na [M + Na] requires 359.1735); 337.1906 found (calculated for C H N O [M + H]
21
24
2
2
21 25
2
2
requires 337.1916).
(
1R, 1S) 1-[ -((4-Methoxybenzyl)oxy)-(4-methoxyphenethyl]-1H-pyrazolium hydrochloride
β
(
7c).
Using the standard procedure from 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol
(0.218 g, 1 mmol) and 4-methoxybenzyl chloride 5a (0.157 g, 1 mmol, 1 equiv.),
the 1-[ -((4-methoxybenzyl)oxy)-(4-methoxyphenethyl]-1H-pyrazole 6c (0.231 g, 0.68 mmol)
4
b
β
was dissolved in 4.57 mL of dry diethyl ether pa under a stream of argon. The addition of 0.68 mL
of 1M HCl (0.68 mmol, 1 equiv.) produced a pale pink viscous oil that crystallized rapidly (30 min.)
on the circumference of the round flask, and mixing (200 rpm, 72 h) was pursued until complete
crystallization of the viscous gum into divided powder. The desired salt 7c was collected by filtration
◦
on a Büchner funnel (porosity N 4) and washed with 3
×
0.5 mL of dry diethyl ether pa. After
◦
drying at 25 C for 4 h, the 1-[
β
-((4-methoxybenzyl)oxy)-(4-methoxyphenethyl]-1H-pyrazolium
◦
1
hydrochloride 7c (123.7 mg, 33% yield) was obtained as white powder. Mp = 112–113 C. H NMR
(
(
DMSO-d6) δ = 3.72 (s, 3H, OCH ), 3.76 (s, 3H, OCH ), 4.06 (d, 1H, J = 11.6 Hz, CH, H-1”), 4.16–4.30
3 3
0
0
m, 2H, CH, H-1 , H-1”), 4.40 (dd, 1H, J = 13.8, 8.5 Hz, CH, H-1 ), 4.71 (dd, 1H, J = 8.4, 4.5 Hz, CH,
0
H-2 ), 6.22 (t, 1H, J = 2.0 Hz, H-4, Ar), 6.82 (d, 2H, J = 8.6 Hz, H-3a, H-5a, Ar), 6.89–7.03 (m, 4H, H-2a
H-6a, H-3b, H-5b, Ar), 7.26 (d, 2H, J = 8.6 Hz, H-2b, H-6b, Ar), 7.47 (d, 1H, J = 1.9 Hz, H-3, Ar), 7.62 (d,
1
7
5
1
3
0
H, J = 2.2 Hz, H-5, Ar). C NMR (DMSO-d6)
δ
= 55.1 (OCH ), 55.1 (OCH ), 57.0 (C-1 ), 69.3 (C-1”),
3
3
0
8.9 (C-2 ), 104.9 (C-4), 113.6 (C-3a, C-5a), 114.0 (C-3b, C-5b), 128.2 (C-2a C-6a), 128.8 (C-2b, C-6b), 130.0
(C-1a), 130.8 (C-1b), 130.9 (C-5), 138.5 (C-3), 158.6 (C-4a), 159.1 (C-4b). ES HRMS, m/z = 361.1526
+
+
found (calculated for C H N O Na [M + Na] requires 361.1528); 339.1715 found (calculated for
C H N O [M + H] requires 339.1709).
2
0
22
2
3
+
20
23
2
3
(
1R, 1S) 1-[
Using the standard procedure from 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b
0.218 g, 1 mmol) and 1-(3-bromopropyl)-4-methoxybenzene 5c (0.229 g, 1 mmol, 1 equiv.),
the 1-[ -((4-methoxyphenyl)-(3-(4-methoxyphenyl)propoxyethyl)]-1H-pyrazole 6d (0.156 g, 0.42 mmol)
β-((4-Methoxyphenyl)-(3-(4-methoxyphenyl)propoxyethyl)]-1H-pyrazolium hydrochloride (7d).
(
β
was dissolved in 2.84 mL of dry diethyl ether pa under a stream of argon. The addition of 0.42 mL
of 1M HCl (0.42 mmol, 1 equiv.) produced a pale pink viscous oil that crystallized (12 h) on
the circumference of the round flask, and mixing (200 rpm, 72 h) was pursued until complete
crystallization of the viscous gum. The desired salt 7d was collected by filtration on a Büchner

Int. J. Mol. Sci. 2018, 19, 856
17 of 24
◦
◦
0.5 mL of dry diethyl ether pa. After drying at 25 C for 4 h,
funnel (porosity N 4) and washed with 3
×
the 1-[β-((4-methoxyphenyl)-(3-(4-methoxyphenyl)propoxyethyl)]-1H-pyrazolium hydrochloride 7d
72.53 mg, 18% yield) was obtained as white powder. Mp = 92–94 C. H NMR (DMSO-d ) δ = 1.47–1.70
◦
1
(
6
(
m, 2H, CH , H-2”), 2.36 (t, 2H, J = 7.5 Hz, CH , H-3”), 3.03 (dt, 1H, J = 9.6, 6.2 Hz, CH, H-1”), 3.19 (dt,
2
2
1
H, J = 9.5, 6.0 Hz, CH, H-1”), 3.69 (s, 3H, OCH ), 3.75 (s, 3H, OCH ), 4.18 (dd, 1H, J = 13.8, 4.3 Hz,
3
0
3
0
0
CH, H-1 ), 4.35 (dd, 1H, J = 13.8, 8.7 Hz, CH, H-1 ), 4.62 (dd, 1H, J = 8.7, 4.2 Hz, CH, H-2 ), 6.22 (t, 1H,
J = 2.0 Hz, H-4, Ar), 6.77 (d, 2H, J = 8.6 Hz, H-3b, H-5b, Ar), 6.86–7.02 (m, 4H, H-3a, H-5a, H-2b, H-6b,
Ar), 7.26 (d, 2H, J = 8.6 Hz, H-2a, H-6a, Ar), 7.47 (d, 1H, J = 1.8 Hz, H-3, Ar), 7.67 (d, 1H, J = 2.2 Hz, H-5,
Ar). 13C NMR (DMSO-d )
7
δ
0
= 30.4 (C-3”), 31.1 (C-2”), 54.9 (OCH ), 55.1 (OCH ), 57.2 (C-1 ), 67.0 (C-1”),
3 3
6
0
9.7 (C-2 ), 104.9 (C-4), 113.6 (C-3b, C-5b), 113.9 (C-3a, C-5a), 128.0 (C-2a, C-6a), 129.2 (C-2b, C-6b),
+
1
30.8 (C-3), 131.1 (C-1a), 133.4 (C-1b), 138.5 (C-5), 157.3 (C-4b), 159.0 (C-4a). ES HRMS, m/z = 389.1838
+
found (calculated for C H N O Na [M + Na] requires 389.1841).
22
26
2
3
(
1R,
Using the standard procedure from 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol
(0.218 g, 1 mmol) and 1-bromo-3-phenylpropane 5d (0.199 g, 1 mmol, 1 equiv.),
the 1-[ -((4-methoxyphenyl)-(3-phenylpropoxyethyl)]-1H-pyrazole 6e (0.124 g, 0.37 mmol)
1S)
1-[
β
-((4-Methoxyphenyl)-(3-phenylpropoxyethyl)]-1H-pyrazolium
hydrochloride
(7e).
4
b
β
was dissolved in 2.5 mL of dry diethyl ether pa under a stream of argon. The addition of 0.37 mL
of 1 M HCl (0.37 mmol, 1 equiv.) produced a translucent colourless viscous oil that crystallized
progressively (24 h) on the circumference of the round flask, and mixing (200 rpm, 72 h) was pursued
until complete crystallization in divided powder. The desired salt 7e was collected by filtration on a
◦
Büchner funnel (porosity N 4) and washed with 3
×
0.5 mL of dry diethyl ether pa. After drying at
◦
2
5 C for 4 h, the 1-[
β
-((4-methoxyphenyl)-(3-phenylpropoxyethyl)]-1H-pyrazolium hydrochloride
◦
d (82.04 mg, 22% yield) was obtained as white powder. Mp = 87–89 C. H NMR (DMSO-d ) δ =
6
1
7
1
3
4
.54–1.76 (m, 2H, CH , H-2”), 2.37–2.46 (m, 2H, CH , H-3”), 3.04 (dt, 1H, J = 9.6, 6.1 Hz, CH, H-1”),
2
2
0
.21 (dt, 1H, J = 9.6, 6.0 Hz, CH, H-1”), 3.75 (s, 3H, OCH ), 4.18 (dd, 1H, J = 13.8, 4.3 Hz, CH, H-1 ),
3
0
0
.35 (dd, 1H, J = 13.8, 8.7 Hz, CH, H-1 ), 4.63 (dd, 1H, J = 8.7, 4.3 Hz, CH, H-2 ), 6.21 (t, 1H, J = 2.1 Hz,
H-4, Ar), 6.92 (d, 2H, J = 8.7 Hz, H-3a, H-5a, Ar), 6.99–7.06 (m, 2H, H-2b, H-6b, Ar), 7.08–7.17 (m, 1H,
H-4b, Ar), 7.17–7.31 (m, 4H, H-2a H-6a, H-3b, H-5b, Ar), 7.46 (dd, 1H, J = 1.9, 0.7 Hz, H-3, Ar), 7.66 (dd,
1
3
0
1
6
H, J = 2.3, 0.7 Hz, H-5, Ar). C NMR (DMSO-d6)
δ
= 30.9 (C-2”), 31.4 (C-3”), 55.1 (OCH ), 57.2 (C-1 ),
3
0
7.1 (C-1”), 79.7 (C-2 ), 104.9 (C-4), 113.9 (C-3a, C-5a), 125.7 (C-4b), 128.1 (C-2a, C-6a), 128.2 (C-3b,
C-5b), 128.4 (C-2b, C-6b), 130.8 (C-5), 131.2 (C-1a), 138.6 (C-3), 141.7 (C-1b), 159.1 (C-4a). ES HRMS,
m/z = 359.1736 found (calculated for C H N O Na [M + Na] requires 359.1736).
+
+
21
24
2
2
3
.1.3. Resolution of (±)-(1R, 1S) 1-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b) with
(
−)-(1R)-10-Camphorsulfonic Acid and (+)-(1S)-10-Camphorsulfonic Acid by Two Convergent
Methods of Half-Quantities
Method 1 from (−)-(1R)-CSA:
(
1
−
)-(1S) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b): To a stirred solution of (
-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (0.5 g, 2.29 mmol) in 5 mL of dry acetone,
a solution of ( )-(1R)-10-camphorsulfonic acid (0.266 g, 1.145 mmol) in 2 mL of dry acetone was
added dropwise at room temperature for 5 min. Stirring (300 rpm) was pursued for 12 h at 25 C.
It is interesting to note that during the addition of the solution of ( )-(1R)-CSA, the formation of
±
)
−
◦
−
a white fine suspension of diastereomer appeared in the reaction mixture. This first fraction of
diastereomer salt was collected by filtration on a Büchner funnel (porosity N 4), washed thoroughly
◦
with 4
×
0.3 mL of dry acetone, and dried in vacuum to give 0.363 g of the desired the salt
)-(1S)-4b/( )-(1R)-CSA (>85% de). This salt was recrystallized in dry acetone and afforded 0.271 g
52% isolated yield) of salt ( )-(1S)-4b/( )-(1R)-CSA (>97% de). This salt was dissolved in 0.6 mL
(
(
−
−
−
−
of dry acetone under magnetic stirring (300 rpm), and to this suspension, 0.02 equiv. of racemic
) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b was added for diastereomeric enrichment.
(
±

Int. J. Mol. Sci. 2018, 19, 856
18 of 24
This suspension was stirred for 18 h at room temperature. The enriched salt was successively filtered
on a Büchner funnel, washed with 0.5 mL of dry acetone, dried in vacuum, and recrystallized in dry
acetone that produced 0.251 g (48% yield) of (
αD] = −6,0 (c 1.0, MeOH).
−
)-(1S)-4b/( )-(1R)-CSA (>99% de) as white powder;
−
[
Starting from 200 mg of the pure salt (
−
)-(1S)-4b/( )-(1R)-CSA, a mixed suspension was
−
prepared in 2.5 mL of dry methanol and 1 equiv. of commercial MeONa was added in one portion.
The resulting reaction mixture was stirred at room temperature for 12 h and then concentrated in
a rotary evaporator under reduced pressure. The crude solid residue was washed thoroughly with
4
× 2 mL of deionized water. The insoluble compound was collected by filtration on a Büchner funnel
◦
(porosity N 4) and dried in vacuum, which produced 0.096 g (38% yield) of the desired (
−
)-(1S)
1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (99% ee); [ D
α
] =
−
10,0 (c 1.0, MeOH) and
◦
Mp = 113–114 C.
Recovery of the (+)-(1R) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b: The filtrate of the first
fraction was submitted to concentration in a rotary evaporator under reduced pressure and afforded
0
.345 g of a crude residue. To this solid residue, 14.5 mL of dry toluene was added and the resulting
suspension was submitted to magnetic stirring for 4 h. This suspension was filtered through a Büchner
◦
funnel (porosity N 4) and the filtrate was concentrated in a rotary evaporator under reduced pressure
and produced 0.190 g (0.87 mmol) of a white powder of (+)-(1R)-4b (65% ee). This white solid was
dissolved in 3.8 mL of dry acetone under magnetic stirring and to the resulting solution, a solution
of (+)-(1S)-CSA (0.2 g, 0.87 mmol, 1 equiv.) in 1.52 mL of dry acetone was added dropwise. During
the addition of the (+)-(1S)-CSA solution, a fine suspension appeared in the mixture, which was
stirred at room temperature for 12 h. The new diastereomer salt was collected by filtration then
washed with 4
+)-(1R)-4b/(+)-(1S)-CSA salt (96% de) as white powder. From this salt, diastereomeric enrichment
was conducted with 0.04 equiv. of racemic ( ) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b
×
0.25 mL of dry acetone, dried in vacuum, which gave 0.249 g (63% yield) of the
(
±
.
The enriched suspension was stirred for 18 h at room temperature and the resulting enriched salt
was successively submitted to filtration on a Büchner funnel, washing with 0.5 mL of dry acetone,
drying in vacuum, and finally recrystallisation in dry acetone which gave 0.202 mg (51% yield) of
(
+)-(1R)-4b/(+)-(1S)-CSA (>99% de) as white needles; [α ] = +6,0 (c 1.0, MeOH).
D
Starting from 200 mg of the pure salt (+)-(1R)-4b/(+)-(1S)-CSA, a mixed suspension was
prepared in 2.5 mL of dry methanol, and 1 equiv. of commercial MeONa was added in one portion.
The resulting reaction mixture was stirred at room temperature for 12 h and then concentrated in
a rotary evaporator under reduced pressure. The crude solid residue was washed thoroughly with
4
×
2 mL of deionized water. The insoluble compound was collected by filtration on a Büchner
◦
funnel (porosity N 4) and dried in vacuum which gave 0.078 g (41% yield) of the desired (+)-(1R)
1
-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (99% ee); [αD] = +10,0 (c 1.0, MeOH) and
◦
Mp = 110–111 C.
Method 2 with (+)-(1S)-CSA:
(
1
+)-(1R) 1-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b): To a stirred solution of ( )
-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (0.5 g, 2.29 mmol) in 5 mL of dry acetone,
±
a solution of (+)-(1S)-10-camphorsulfonic acid (0.266 g, 1.145 mmol) in 2 mL of dry acetone was
◦
added dropwise at room temperature for 5 min. Stirring (300 rpm) was pursued for 12 h at 25 C.
It is interesting to note that during the addition of the solution of (+)-(1S)-CSA, the formation of
a white fine suspension of diastereomer appeared in the reaction mixture. This first fraction of
◦
diastereomer salt was collected by filtration on a Büchner funnel (porosity N 4), washed thoroughly
with 4
×
0.3 mL of dry acetone, and dried in vacuum to give 0.358 g of the desired the salt
(+)-(1R)-4b/(+)-(1S)-CSA (>87% de). This salt was recrystallized in dry acetonitrile and afforded

Int. J. Mol. Sci. 2018, 19, 856
19 of 24
0
0
.280 g (54% isolated yield) of salt (+)-(1R)-4b/(+)-(1S)-CSA (>96% de). This salt was dissolved in
.6 mL of dry acetone under magnetic stirring (300 rpm) and to this suspension was added 0.02 equiv.
) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b for diastereomeric enrichment.
of racemic (
±
This suspension was stirred for 18 h at room temperature. The enriched salt was successively filtered
on a Büchner funnel, washed with 0.5 mL of dry acetone, dried in vacuum, and recrystallized in
dry acetone that gave 0.229 g (44% yield) of (+)-(1R)-4b/(+)-(1S)-CSA (>99% de) as white powder;
[
αD] = +6,0 (c 1.0, MeOH).
Starting from 200 mg of the pure salt (+)-(1R)-4b/(+)-(1S)-CSA, a mixed suspension was
prepared in 2.5 mL of dry methanol, and 1 equiv. of commercial MeONa was added in one portion.
The resulting reaction mixture was stirred at room temperature for 12 h and then concentrated in
a rotary evaporator under reduced pressure. The crude solid residue was washed thoroughly with
4
×
2 mL of deionized water. The insoluble compound was collected by filtration on a Büchner
◦
funnel (porosity N 4) and dried in vacuum, which gave 0.089 g (35% yield) of the desired (+)-(1R)
1
1
-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (99% ee); [
10–111 C.
α
D] = +10,0 (c 1.0, MeOH) and Mp =
◦
Recovery of the (
fraction was submitted to concentration in a rotary evaporator under reduced pressure and afforded
.366 g of a crude residue. To this solid residue, 14.5 mL of dry toluene was added and the resulting
−
)-(1S) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b: The filtrate of the first
0
suspension was submitted to magnetic stirring for 4 h. This suspension was filtered through a Büchner
◦
funnel (porosity N 4) and the filtrate was concentrated in a rotary evaporator under reduced pressure
and produced 0.207 g (0.95 mmol) of a white powder of (
dissolved in 4.2 mL of dry acetone under magnetic stirring and to the resulting solution, a solution of
)-(1R)-CSA (0.219 g, 0.95 mmol, 1 equiv.) in 1.66 mL of dry acetone was added dropwise. During
the addition of the ( )-(1R)-CSA solution, a fine suspension appeared in the mixture, which was
stirred at room temperature for 12 h. The new diastereomer salt was collected by filtration then
washed with 4 0.25 mL of dry acetone and dried in vacuum, which gave 0.285 g (66% yield) of the
)-(1S)-4b/( )-(1R)-CSA salt (98% de) as white powder. From this salt, diastereomeric enrichment
was conducted with 0.01 equiv. of racemic ( ) 1-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b
−
)-(1S)-4b (74% ee). This white solid was
(
−
−
×
(
−
−
±
.
The enriched suspension was stirred for 18 h at room temperature and the resulting enriched salt
was successively submitted to filtration on a Büchner funnel, washing with 0.5 mL of dry acetone,
drying in vacuum, and finally recrystallisation in dry acetone which gave 0.258 mg (60% yield) of
(
−)-(1S)-4b/(−)-(1R)-CSA (>99% de) as white needles; [α ] = −6,0 (c 1.0, MeOH).
D
Starting from 200 mg of the pure salt (
−
)-(1S)-4b/( )-(1R)-CSA, a mixed suspension was
−
prepared in 2.5 mL of dry methanol, and 1 equiv. of commercial MeONa was added in one portion.
The resulting reaction mixture was stirred at room temperature for 12 h and then concentrated in
a rotary evaporator under reduced pressure. The crude solid residue was washed thoroughly with
4
×
2 mL of deionized water. The insoluble compound was collected by filtration on a Büchner
◦
funnel (porosity N 4) and dried in vacuum which gave 0.090 g (43% yield) of the desired (
−
)-(1S)
1
-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol 4b (>99% ee); [
α
D] =
−
10,0 (c 1.0, MeOH) and Mp
◦
=
113-114 C.
◦
(
−
)-(1S)-4b/(
−
)-(1R)-CSA salt: Yield = 48%. White powder, Mp = 168–170 C. [
α
D] =
−
6.0 (c 1.0,
MeOH) and >99% de (retention time t = 34.01 min. using hexane/i-PrOH 94:6 v/v as eluent,
R
1
flow rate = 0.8 mL/min.). H NMR (DMSO-d6)
δ = 0.75 (s, 3H, C-7”CH ), 1.04 (s, 3H, C-7”CH ),
3 3
1
1
2
.19–1.40 (m, 2H, CH , H-2”, H-5”), 1.81 (d, 1H, J = 18.1 Hz, CH, H-4”), 1.80–1.92 (m, 1H, CH, H-5”),
2
.95 (t, 1H, J = 4.5 Hz, CH, H-4”), 2.16–2.34 (m, 1H, CH, H-3”), 2.46 (d, 1H, J = 14.7 Hz, CH, CH SO H),
2
3
.55–2.76 (m, 1H, CH, H-2”), 2.93 (d, 1H, J = 14.7 Hz, CH, CH SO H), 3.73 (s, 3H, OCH ), 4.10–4.32
2
3
3
0
(m, 2H, CH , H-2), 4.87 (dd, 1H, J = 7.3, 5.5 Hz, CH, H-1), 6.20 (t, 1H, J = 2.1 Hz, H-4 , Ar), 6.87 (d, 2H,
2
0
J = 8.7 Hz, H-3a, H-5a, Ar), 7.22 (d, 2H, J = 8.5 Hz, H-2a, H-6a, Ar), 7.47 (dd, 1H, J = 1.9, 0.7 Hz, H-3 ,

Int. J. Mol. Sci. 2018, 19, 856
20 of 24
Ar), 7.61 (dd, 1H, J = 2.3, 0.7 Hz, H-5 , Ar). 13C NMR (DMSO-d6)
0
δ
= 19.5 (C-7”CH ), 20.0 (C-7”CH ),
3
3
2
4.2 (C-2”), 26.4 (C-5”), 42.1 (C-3”), 42.2 (C-4”), 46.8 (CH SO H), 47.1 (C-7”), 55.0 (OCH3), 58.1 (C-6”),
2 3
0
0
5
1
8.6 (C-2), 71.3 (C-1), 104.8 (C-4 ), 113.5 (C-3a, C-5a), 127.2 (C-2a, C-6a), 130.9 (C-5 ), 134.7 (C-1a),
0
38.1 (C-3 ), 158.5 (C-4a), 216.0 (C=O). ES HRMS, m/z = 241.0946 found (calculated for C H N O Na
12 14 2 2
+
+
[M – H + Na] requires 241.0947).
◦
(
+)-(1R)-4b/(+)-(1S)-CSA salt: Yield = 51%. White powder, Mp = 169-171 C. [αD] = +6.0 (c 1.0, MeOH)
and >99% de (retention time t = 35.5 min. using hexane/i-PrOH 94:6 v/v as eluent, flow rate =
R
1
0
2
.8 mL/min.). H NMR (DMSO-d )
δ
= 0.75 (s, 3H, C-7”CH ), 1.04 (s, 3H, C-7”CH3), 1.17–1.42 (m,
6
3
H, CH , H-2”, H-5”), 1.81 (d, 1H, J = 18.1 Hz, CH, H-4”), 1.78–1.93 (m, 1H, CH, H-5”), 1.95 (t, 1H,
2
J = 4.5 Hz, CH, H-4”), 2.25 (dt, 1H, J = 18.0, 4.0 Hz, CH, H-3”), 2.45 (d, 1H, J = 14.7 Hz, CH, CH SO H),
2
3
2
.54-2.76 (m, 1H, CH, H-2”), 2.93 (d, 1H, J = 14.7 Hz, CH, CH SO H), 3.73 (s, 3H, OCH ), 4.08–4.32
2 3 3
0
(m, 2H, CH , H-2), 4.87 (dd, 1H, J = 7.3, 5.5 Hz, CH, H-1), 6.20 (t, 1H, J = 2.1 Hz, H-4 , Ar), 6.87 (d,
2
0
2
7
2
5
H, J = 8.7 Hz, H-3a, H-5a, Ar), 7.22 (d, 2H, J = 8.5 Hz, H-2a, H-6a, Ar), 7.43-7.41 (m, 1H, H-3 , Ar),
.60 (dd, 1H, J = 2.3, 0.7 Hz, H-5 , Ar). C NMR (DMSO-d6)
4.2 (C-2”), 26.4 (C-5”), 42.1 (C-3”), 42.2 (C-4”), 46.8 (CH SO H), 47.1 (C-7”), 55.0 (OCH ), 58.1 (C-6”),
8.6 (C-2), 71.3 (C-1), 104.8 (C-4 ), 113.5 (C-3a, C-5a), 127.2 (C-2a, C-6a), 130.9 (C-5 ), 134.7 (C-1a), 138.1
0
13
δ = 19.5 (C-7”CH ), 20.0 (C-7”CH ),
3 3
2
3
3
0
0
0
+
(C-3 ), 158.5 (C-4a), 216.0 (C=O). ES HRMS, m/z = 241.0946 found (calculated for C H N O Na
12
14
2
2
+
[M − H + Na] requires 241.0947).
X-ray crystallographic data for (+)-(1R)-4b/(+)-(1S)-CSA salt: (C H N O , C H O S);
12
15
2
2
10 15
4
M = 450.54. APEXII, Bruker-AXS diffractometer, Mo-K
α
radiation (
λ
= 0.71073 Å), T = 150(2) K;
3
orthorhombic P 21 21 21 (I.T.#19), a = 7.2781(5), b = 9.5344(5), c = 32.8134(19) Å, V = 2277.0(2) Å . Z
− −1
3
=
4, d = 1.314 g
·
cm
, µ = 0.182 mm . The structure was solved by direct methods using the SIR97
program [29], and then refined with full-matrix least-square methods based on F2 (SHELXL-97) [30
]
with the aid of the WINGX [31] program. All non-hydrogen atoms were refined with anisotropic
atomic displacement parameters. Except oxygen- and nitrogen-linked hydrogen atoms that were
introduced in the structural model through Fourier difference maps analysis, H atoms were finally
2
included in their calculated positions. A final refinement on F with 4854 unique intensities and
2
289 parameters converged at
ω
R(F ) = 0.0932 (R(F) = 0.0415) for 4347 observed reflections with I
> 2σ(I). Crystallographic data for the structure of (+)-(1R)-4b/(+)-(1S)-CSA salt in this paper have
been deposited in the Cambridge Crystallographic Data Centre as supplementary publication number
CCDC 1580544. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB21EZ, UK (fax: +44(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
(
=
−
)-(1S) 1-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b): Yield = 38%. White powder, Mp
◦
113–114 C. [
α
D] =
−
10.0 (c 1.0, MeOH) and >99% de (retention time t = 52.6 min. using
R
1
hexane/i-PrOH 96:4 v/v as eluent, flow rate = 0.6 mL/min.). H NMR (DMSO-d )
δ = 3.73 (s, 3H,
6
OCH ), 4.06–4.30 (m, 2H, H-2, CH , H-2), 4.87 (q, 1H, J = 5.6 Hz, CH, H-1), 5.50 (br d, 1H, J = 4.5 Hz
,
3
2
0
OH), 6.16 (t, 1H, J = 2.0 Hz, H-4 , Ar), 6.87 (d, 2H, J = 8.6 Hz, H-3a, H-5a, Ar), 7.21 (d, 2H, J = 8.6 Hz
,
0
0
13
H-2a, H-6a, Ar), 7.41 (d, 1H, J = 1.8 Hz, H-3 , Ar), 7.56 (d, 1H, J = 2.2 Hz, H-5 , Ar). C NMR (DMSO-d )
6
0 0
= 55.0 (OCH ), 58.7 (C-2), 71.4 (C-1), 104.6 (C-4 ), 113.5 (C-3a, C-5a), 127.2 (C-2a, C-6a), 130.5 C-5 ),
3
δ
0
34.8 (C-1a), 138.4 (C-3 ), 158.5 (C-4a). ES HRMS, m/z = 241.0950 found (calculated for C H N O Na
12 14 2 2
+
1
+
[M – H + Na] requires 241.0953).
(
1
9
+)-(1R) 1-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)ethan-1-ol (4b): Yield = 41%. White powder, Mp =
◦
10–111 C. [
α
] = +10.0 (c 1.0, MeOH) and >99% de (retention time t = 63.5 min. using hexane/i-PrOH
D
R
1
6:4 v/v as eluent, flow rate = 0.6 mL/min.). H NMR (DMSO-d )
δ
= 3.73 (s, 3H, OCH ), 4.09–4.29
6
3
(
1
m, 2H, H-2, CH , H-2), 4.87 (q, 1H, J = 5.6 Hz, CH, H-1), 5.50 (br d, 1H, J = 4.5 Hz, OH), 6.16 (t,
2
0
H, J = 2.0 Hz, H-4 , Ar), 6.87 (d, 2H, J = 8.6 Hz, H-3a, H-5a, Ar), 7.22 (d, 2H, J = 8.6 Hz, H-2a, H-6a,
0
0
13
Ar), 7.41 (d, 1H, J = 1.8 Hz, H-3 , Ar), 7.56 (d, 1H, J = 2.2 Hz, H-5 , Ar). C NMR (DMSO-d6)
=
δ
0
0
55.0 (OCH ), 58.7 (C-2), 71.4 (C-1), 104.6 (C-4 ), 113.5 (C-3a, C-5a), 127.2 (C-2a, C-6a), 130.5 C-5 ),
3

Int. J. Mol. Sci. 2018, 19, 856
21 of 24
0
+
1
[
34.8 (C-1a), 138.4 (C-3 ), 158.5 (C-4a). ES HRMS, m/z = 241.0950 found (calculated for C H N O Na
12
14
2
2
+
M − H + Na] requires 241.0953).
3
.2. Biology Section
3
.2.1. Reagents
Dimethyl Sulfoxyde (DMSO-Ref D2438) and Thapsigargin (Tg-Ref: T9033) was purchased from
®
Sigma-Aldrich. Corning Cell-Tak
™
was purchased from Beckton Dickinson (Le Pont de Claix, France)
(
(
Ref: 354241). Fura-2 QBT™ Calcium Kit was purchased from Molecular Devices (Berkshire, UK)
Ref: R8198).
3
.2.2. Cell Culture
The B-cell leukemia cell line PLP [32], which possesses the phenotypic characteristics of B-CLL
cells [33], were maintained in RPMI-1640 containing 10% fetal calf serum and antibiotics (1% of
penicillin/streptomycin).
3
.2.3. Cytosolic Ca2+ Protocol
For Ca2+ experiments, cells were seeded overnight into four wells plate (Nunc multidisches
TM
5
4
wells flat-bottom-Ref: 176740) at 7
×
10 cells/mL in 1 mL culture medium. Cell suspension was then
transfered into 1.5 mL Microtube, pelleted, and loaded with Fura-2 acetoxymethyl ester (Fura-2 QBT
fluorochrome according to the manufacturer’s protocol. Cell suspension was dispensed onto Corning
™)
®
Cell-Tak
™
pre-coated 96 wells black clear bottom plate (80
µ
L per wells, density approximatively
4
◦
5
×
10 cells per well) and incubated 1 h at 37 C, 5% CO . The Fura-2 QBT
™
was aspirated and
2
2+
replaced by an equal volume of free Ca Hepes-buffered solution containing (in mM): 135 NaCl,
5
KCl, 1 MgCl , 1 EGTA, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH. The tested compounds
2
◦
or DMSO control solvent solutions were added in wells (4
evaluating calcium level on FlexStation 3™ Instrument.
µL) and incubated for 5 min at 37 C until
3
.2.4. Measurement of Intracellular Calcium Levels
Intracellular calcium levels were monitored by using the FlexStation 3
™
(Molecular Devices,
2+
Berkshire, UK), a fluorescence plate reader measuring time–resolved intracellular Ca concentration in
a 96-well format with the dual-wavelength fluorescent calcium-sensitive dye Fura-2AM. Dual excitation
wavelength capability permits ratiometric measurements of Fura-2AM peak emissions (510 nm) after
2+
excitations at 340 (bound to calcium) and 380 nm (unbound to Ca ). Modifications in the 340/380 ratio
2
+
reflect changes in intracellular-free Ca concentrations. The FlexStation 3
™
temperature was setting
◦
2+
at 37 C during data acquisition. Thapsigargin (2
µM) solution and Hepes-buffered solution with Ca
containing (in mM): 135 NaCl, 5 KCl, 1 MgCl , 2 CaCl , 10 Hepes, 10 glucose, pH adjusted at 7.45 with
2
2
NaOH were added from a 96-well reservoir plate during Calcium Mobilization Assay running at
00 and 750 s, respectively. Experimental setup parameters were optimized (pipette heights, volumes
1
and rate of additions) to minimize disturbance of the cells while ensuring rapid mixing. The data
were stored for later analysis by using SoftmaxPro (Molecular Devices), Excel and Graph Pad Prism
software (Graph Pad Software, La Jolla, CA, USA).
3
.2.5. Measurement of Store-Operated Calcium (SOC) Entry
2
+
Intracellular Ca stores were depleted with 2
µ
M Thapsigargin an inhibitor of ER SERCA pumps
2+
2+
under Ca -free conditions to determine the magnitude of intracellular Ca release. Next, cells were
returned to Ca -containing Hepes-buffered solution to measure SOCE. The magnitude of SOCE was
estimated as the maximal values of normalized F340/F340 ratio following Ca2+ re-addition.
2+

Int. J. Mol. Sci. 2018, 19, 856
22 of 24
3
.2.6. Calculation and Data Analysis
Results are expressed as mean
±
SEM for each group. The number of experiments for each group
is represented as n. Data analysis for functional assays was performed using Softmax Pro and Graph
Pad Prism. (Graph Pad): Ca2 concentration variations are estimated using the ratio of peak RFU at
+
340 and 380 nm (F340/F380) and for each measurement F340/F380 ratio values are normalized to
the initial basal ratio before TG addition. Results are expressed in terms of percentage of response
or percentage of inhibition compared to the maximum response on control condition. Inhibition is
estimated as follow: % Inhibition = (100
−
% response). The EC values were estimated from the
50
2+
dose/response curves (log(inhibitor) vs. amplitude of the Ca response) using a regression model
(
LogIC50−X)×HillSlope
) with variable slope (Prism5 GraphPad software).
(
Y = Min + (Max − Min)/(1 + 10
Min and Max are the minimal and maximal inhibition obtained. EC50 is defined as the concentration
of drug required to obtain 50% of inhibitory effect.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/3/
8
56/s1.
Acknowledgments: Camille D. Dago wishes to thank the Fondation Benianh International and the Ministère
de l’Enseignement Supérieur et de la Recherche Scientifique de Côte d’Ivoire for their grants. Financial support
of this program carried out under the French National Cancer Institute, Cancéropôle Grand Ouest, by contract
“
Ion Channel-Network”, is gratefully acknowledged. The authors are grateful to the assistance of the staff of the
CRMPO analytical chemistry core facility for HRMS analysis (CRMPO platform, ScanMAT UMS 2001 CNRS,
Université de Rennes 1, Bât. 11A, Campus de Beaulieu, Rennes, France).
Author Contributions: Camille D. Dago realized the organic synthesis; Paul Le Maux performed the chiral HPLC
analysis and taught this method of analysis to Camille D. Dago; Thierry Roisnel performed X-ray diffraction;
Christophe Brigaudeau realized measurements of calcium in tumoral cells; Yves-Alain Bekro and Jean-Pierre
Bazureau conceived and designed the chemical experiments; Olivier Mignen conceived and designed experiments
in biology; Jean-Pierre Bazureau wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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