1,2,4-Oxadiazole-Bearing Pyrazoles as Metabolically Stable Modulators of Store-Operated Calcium Entry

Article abstract of DOI:10.1021/acsmedchemlett.1c00034

Store-operated calcium entry (SOCE) is a pivotal mechanism in calcium homeostasis, and, despite still being under investigation, its dysregulation is known to be associated with severe human disorders. SOCE modulators are therefore needed both as chemical probes and as therapeutic agents. While many small molecules have been described so far, their poor properties in terms of drug-likeness have limited their translation into the clinical practice. In this work, we describe the bioisosteric replacement of the ester moiety in pyrazole derivatives with a 1,2,4-oxadiazole ring as a means to afford a class of modulators with high metabolic stability. Moreover, among our derivatives, a compound able to increase the calcium entry was identified, further enriching the library of available SOCE activators.

Full text of DOI:10.1021/acsmedchemlett.1c00034

pubs.acs.org/acsmedchemlett
Letter
1
,2,4-Oxadiazole-Bearing Pyrazoles as Metabolically Stable
Modulators of Store-Operated Calcium Entry
Silvio Aprile, Beatrice Riva, Irene Preet Bhela, Celia Cordero-Sanchez, Giulia Avino,
Armando A. Genazzani, Marta Serafini,* and Tracey Pirali
Cite This: ACS Med. Chem. Lett. 2021, 12, 640−646
Read Online
ACCESS
Metrics & More
Article Recommendations
*
sı Supporting Information
ABSTRACT: Store-operated calcium entry (SOCE) is a pivotal mechanism
in calcium homeostasis, and, despite still being under investigation, its
dysregulation is known to be associated with severe human disorders. SOCE
modulators are therefore needed both as chemical probes and as therapeutic
agents. While many small molecules have been described so far, their poor
properties in terms of drug-likeness have limited their translation into the
clinical practice. In this work, we describe the bioisosteric replacement of the
ester moiety in pyrazole derivatives with a 1,2,4-oxadiazole ring as a means to
afford a class of modulators with high metabolic stability. Moreover, among
our derivatives, a compound able to increase the calcium entry was identified,
further enriching the library of available SOCE activators.
KEYWORDS: Drug discovery and development, Store-Operated Calcium Entry, 1,2,4-Oxadiazole, Bioisosteric replacement,
CRAC channels
1
2
13,14
he ubiquitous second messenger calcium is a central
regulator of a plethora of physiological processes,
allergy, and acute pancreatitis.
Therefore, it is not
T
surprising that this cellular pathway has become a promising
target for the therapeutic treatment of several pathological
conditions.
spanning from fertilization of oocytes to cell death, mobility,
secretion, and gene expression. Infinitesimal oscillations of this
ion concentration in the different cell compartments control a
multitude of processes. This is the reason why every cell
maintains a huge gradient between the plasma membrane or
the intracellular calcium repository and the cytoplasm, with
various pumps and exchangers in charge of finely regulating its
15−18
Over the years, many small molecules
able to modulate
SOCE have been reported. The earliest modulators are SKF-
1
9,20
21,22
96365,
2-APB and its derivatives,
and bis-
(trifluoromethyl)pyrazoles compounds, initially named BTP
BTP1 (1), BTP2 (2), Figure 1), followed by Pyr’s (Pyr3
3), Pyr6 (4), Pyr10 (5), Figure 1).
975A (6, Figure 1), RO2959 (7, Figure 1), and Synta66
23
(
(
7
1
,2
24,25
levels.
A central mechanism in calcium homeostasis is
Among others, GSK-
26
27
represented by the store-operated calcium entry (SOCE), i.e.,
the influx of calcium across the plasma membrane activated by
11,12
(
8, Figure 1)
have been extensively used as chemical
3
the depletion of this ion in the endoplasmic reticulum (ER).
probes. More recently, two compounds have entered clinical
development, CM4620 (9, Figure 1) for acute pancreatitis
phase II) and PRCL-02 for psoriasis (phase II), whose
structure has not been disclosed. These are not the only SOCE
inhibitors that are in human use. Indeed, a virtual screening
performed on FDA-approved drugs recently unveiled that two
approved drugs are also SOCE inhibitors with relevant activity
SOCE is mediated mainly, although not exclusively, by two
protein families: STIM and Orai. STIM1 and its homologue
STIM2 are single-span membrane proteins residing on the ER
membrane and act as sensors of calcium levels. Orai are a
family of proteins (Orai1, Orai2 and Orai3) that form the ion
28
29
(
4
−6
channels on the plasma membrane.
When a depletion of
calcium from the ER stores occurs, STIM undergoes a
conformational change leading to self-association into puncta
and migration near the plasma membrane. There, interaction
with Orai promotes the opening of the channel and the
subsequent influx of calcium. The empty ER stores are then
refilled by the sarco-endoplasmic reticulum calcium ATPase
30
at therapeutic doses, the prodrug leflunomide (10, Figure
Received: January 18, 2021
Accepted: March 8, 2021
Published: March 10, 2021
7
,8
(
SERCA) pump. Due to its prominent role in encoding
calcium signals, SOCE is implicated in several human
9
,10
11
disorders, including cancer,
inflammatory bowel disease,
©
2021 The Authors. Published by
American Chemical Society
https://dx.doi.org/10.1021/acsmedchemlett.1c00034
6
40
ACS Med. Chem. Lett. 2021, 12, 640−646

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
a
Scheme 1. Preparation of Amine 17
a
Reagents and conditions: (a) DMF, reflux, 2 h, 96%. (b) H , 5% Pd/
2
C, EtOAc, rt, 2 h, 94%. (c) NaH 60%, N-hydroxyacetamidine, dry
THF, 0 °C to rt, 4 h, 76%.
Then, the ethyl ester reacted with N-hydroxyacetamidine to
afford amine 17.
Starting from this amine, a structure−activity relationship
SAR) study was undertaken. To this aim, eight coupling
(
reactions were performed (19−23, 32−33, and 36, Scheme 2)
Scheme 2. Preparation of Oxadiazole-Bearing Pyrazoles
Figure 1. Structures of SOCE inhibitors reported in the literature.
a
1
9−41
3
1,32
33,34
1
)
and its active form teriflunomide (11, Figure 1).
The two compounds are approved for the treatment of
rheumatoid arthritis and multiple sclerosis, respectively.
Teriflunomide is a dihydroorotate dehydrogenase
(
DHODH) inhibitor, while leflunomide is devoid of in vitro
activity on this enzyme.
Our group of research has contributed to the development
of druglike SOCE modulators by reporting pyrtriazoles, among
which the most promising candidate is CIC-37 (12, Figure
3
5
1
).
In contrast to other SOCE modulators, CIC-37 is a
promising molecule as it displays selectivity over other calcium
channels, such as TRPV1, TRPM8, and voltage-operated
3
5
calcium channels. However, the molecule is affected by poor
metabolic stability, and this is attributable to the ester group,
which is shared by pyrtriazoles and some of the Pyr’s (3, Figure
1
). Yet, this soft spot represents a fundamental portion in the
pharmacophore of pyrtriazoles as its removal leads to a drop in
the inhibitory activity, as previously described by our group.
35
Therefore, in the present contribution, starting from the
structure of CIC-37 (12, Figure 1), we replaced the ester
moiety with a hydrolytically stable isostere, a 1,2,4-
3
6,37
oxadiazole.
Moreover, in the design of this novel class,
a
Reagents and conditions: (a) PyBOP, DIPEA, dry CH Cl , rt, 16−
2
2
we substituted the 1,4-disubstituted 1,2,3-triazole ring of
compound 12 with an amide group (Figure 2). Indeed, the
4
2 h, 40−97%. (b) NaOH, H O, THF, rt, 5 h, 98%.
2
based on the prototype substructures displayed by reported
SOCE inhibitors, including BTP1 (1, Figure 1), BTP2 (2,
Figure 1), Pyr10 (5, Figure 1), GSK-7975A (6, Figure 1),
Synta66 (8, Figure 1), CM4620 (9, Figure 1), leflunomide
(
10), and CIC-37 (12, Figure 1). Compound 42, whose
Figure 2. Design of 1,2,4-oxadiazole-bearing pyrazoles.
substructure mimics Pyr10 (5, Figure 1), was synthesized
according to Scheme 3, from amine 17 and tosyl chloride
(TsCl). Moreover, 15 additional coupling reactions were
performed (24−31, 34−35, and 37−41, Scheme 2) exploiting
different carboxylic acids and further expanding the SAR study.
With this approach, 24 oxadiazole-bearing pyrazoles were
synthesized and tested in human embryonic kidney (HEK)
cells for SOCE inhibition. To this aim, intracellular Ca stores
of HEK cells were emptied with t-butylhydroquinone (tBhQ,
50 μM) in the presence of the compounds at 10 μM
development of 1,2,3-triazoles is hampered by several issues,
including poor aqueous solubility, limitations in the scale-up
process due to the explosive nature of azides and safety
38
concerns associated with copper catalysts.
The class of oxadiazole-bearing pyrazoles was prepared from
amine 17 (Scheme 1). The synthetic route consists of three
steps: after a condensation between 13 and 14 and a reduction
of the aromatic nitro group, intermediate 16 was obtained.
2
+
6
41
https://dx.doi.org/10.1021/acsmedchemlett.1c00034
ACS Med. Chem. Lett. 2021, 12, 640−646

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
a
Scheme 3. Synthesis of compound 42
under these conditions. According to both activity and
cytotoxicity, for the selected five compounds, the representa-
tive traces are depicted in Figure 3.
Moreover, for these selected compounds, IC50 values were
calculated and are reported in Table 1. The most potent
molecule was 22, with an IC50 value of 3.1 μM.
Surprisingly, compound 37, in which the side chain is
represented by a linear aliphatic substructure, afforded an
increase in calcium entry, with a percentage of SOCE activity
of 146.2% compared to the control. More in detail, the
compound, tested at the concentration of 10 μM, significantly
increased the area under the curve (AUC) of calcium entry and
the peak amplitude, without affecting the slope (Figure 4). We
also investigated whether 37 required the triggered opening of
the Orai channel to elicit its effect. To do this, we monitored
a
Reagents and conditions: (a) TsCl, pyridine, dry CH Cl , 0 °C to rt,
2
2
5
h, 45%.
concentration. After 600 s, Ca2+ was added and intracellular
levels were measured in fluorescence microscopy with the
calcium dye Fura-2. As shown in Table 1, 8 compounds out of
2
4 were able to reduce calcium entry by more than 50%
2
+
intracellular Ca in resting cells in the presence of extracellular
compared to control. In particular, the substructures that
provided the highest inhibitory activity were those derived
from GSK-7975A (22, % of SOCE residual activity: 38.6%),
BTP1 (23, 40.8%), leflunomide (32, 33.2%), Pyr2 (33,
4
(
group in para position on the aromatic ring, or 31 (47.4%) in
which the aromatic ring is fused with an imidazole, afforded a
good inhibitory activity. For these compounds, the cell viability
was therefore evaluated. An MTT assay was performed and the
compounds that affected cell viability by more than 25% at 10
μM were discarded (23, 31, and 33). Notably, the remaining
five compounds (22, 27, 29, 32, and 42) were not cytotoxic
2
+
Ca and we observed that the compound did not elicit any
significant calcium entry at 10 μM compared to control in the
3
00 s of observation, suggesting that 37 is a potentiator/
enhancer of SOCE. The same effect was also evident at 3
118.2%) and 1 μM (116.7%) but was not detected at
6.5%), and Pyr10 (42, 47.6%). Also, compounds 27
40.8%) and 29 (49.6%), displaying an electron-withdrawing
(
concentrations above 10. In particular, at 30 and 100 μM, 37
acts as weak SOCE inhibitor, with a percentage of residual
activity of about 70%.
The identification of a SOCE enhancer among a class of
inhibitors has been reported in three previously published
classes of modulators, with two of them described by us and
represented by pyrtriazoles (AL-2T (43), NM-3G (44), Figure
a
Table 1. Oxadiazole-Bearing Pyrazoles and Their Activity on SOCE, IC50 Values and Cytotoxicity in HEK Cells
b
c
compd, yield (%)
% SOCE residual activity (10 μM)
% viability (10 μM)
IC50 (μM)
MLS9 /MLM stability (residual substrate)
b
Pyr3 (3)
Synta66 (8)
CIC-37 (12)
8.9 ± 0.6
9.2 ± 1.7
28.6 ± 0.5
75.8 ± 8.0
93.6 ± 4.6
0.5 ± 0.1
0.2 ± 0.3
4.4 ± 1.2
43%
15%
74%
c
b
17.5 ± 1.6
77.2 ± 3.7
53.6 ± 0.9
56.7 ± 1.3
38.6 ± 4.3
40.8 ± 3.6
121.1 ± 2.2
110.0 ± 5.9
109.0 ± 6.5
40.8 ± 6.9
107.9 ± 3.9
49.6 ± 6.5
53.2 ± 3.9
47.4 ± 1.0
33.2 ± 1.9
46.5 ± 2.8
61.1 ± 3.9
69.8 ± 2.9
59.9 ± 4.2
146.2 ± 4.5
53.8 ± 4.4
95.9 ± 6.2
87.5 ± 3.8
63.6 ± 3.9
47.6 ± 4.2
19, 69%
20, 97%
21, 50%
22, 66%
23, 53%
24, 86%
25, 53%
26, 48%
27, 52%
28, 54%
29, 56%
30, 60%
31, 58%
32, 80%
33, 80%
34, 54%
35, 55%
36, 66%
37, 56%
38, 55%
39, 40%
40, 42%
41, 61%
42, 45%
b
95.6 ± 8.5
72.9 ± 11.7
3.1 ± 1.4
>99%
b
80.2 ± 7.4
5.5 ± 0.8
9.7 ± 2.3
>99%
b
100.8 ± 6.7
73%
56.5 ± 2.5
77.7 ± 4.4
59.1 ± 6.3
b
9.6 ± 2.5
99%
b
88. Five ±2.9
91%
b
79.4 ± 4.2
9.5 ± 1.7
76%
a
b
Biological data were derived for three independent experiments, and numbers represent mean ± standard error of mean (SEM). Residual
c
substrates were determined in the MLS9 fraction after 1 h of incubation. Residual substrates were determined in mouse liver microsomes (MLM)
as previously described.
39
6
42
https://dx.doi.org/10.1021/acsmedchemlett.1c00034
ACS Med. Chem. Lett. 2021, 12, 640−646

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Figure 4. Effect of 37 on SOCE. (A) Traces of SOCE in the presence
or absence of 37 at a concentration of 10 μM. Traces are the average
of 200 cells from three independent experiments. (B) Evaluation of
2
+
peak amplitude, AUC, and slope of the Ca -rise in the absence or
presence of 37. The graph shows the median and IQR of the peak
2
+
amplitude, AUC, and slope of the Ca -rise. Mann−Whitney U test of
compound vs control (**p ≤ 0.002).
Figure 3. Effect of compounds 22, 27, 29, 32, and 42 on SOCE. (A)
Traces of SOCE in the presence or absence of compounds 22, 27, and
Figure 5. Structures of positive modulators of SOCE reported in the
literature.
29 (10 μM). Traces are the average of 200 cells from three
independent experiments (top panel). Evaluation of peak amplitude,
2
+
area under the curve (AUC), and slope of the Ca -rise in the absence
or presence of 22, 27, and 29. The graph shows the median and
interquartile range (IQR) of the peak amplitude, AUC, and slope of
in vitro hepatic metabolic stability. To this aim, the candidates
were incubated in MLS9 fraction supplied with NADPH and
the residual substrate was measured after 1 h. Under these
conditions, the oxadiazole-bearing pyrazoles resulted overall
metabolically stable, with only two compounds affording a
residual substrate lower than 80% and the remaining molecules
higher than 90% after incubation (Table 1 and Supporting
Information). In contrast, Pyr3 and CIC-37, which suffered
from hydrolysis of the ester function, provided a residual
2+
the Ca -rise. Mann−Whitney U test of compound vs control (** p ≤
0
.0043; **** p ≤ 0.0001; bottom panel). (B) Traces of SOCE in the
presence or absence of compounds 32 and 42 (10 μM). Traces are
the average of 200 cells from three independent experiments (top
2
+
panel). Evaluation of peak amplitude, AUC, and slope of the Ca -rise
in the absence or presence of 32 and 42. The graph shows the median
2
+
and IQR of the peak amplitude, AUC, and slope of the Ca -rise.
35
substrate of 43 and 74%, respectively (Table 1). Similarly,
the residual substrate of Synta66 in MLM is of 15%, due to an
important hydrolysis of the amide group, absent in our class
Mann−Whitney U test of compound vs control (**p ≤ 0.0069;
*
***p ≤ 0.0001; bottom panel).
39
(
Table 1).
We have previously reported CIC-37 that had as its main
3
5
39
5
), and biphenyl triazoles (compound 45, Figure 5) and
40
one reported in the literature (IA65 (46), Figure 5).
A
advantage over other modulators the absence of cytotoxicity
but had metabolic stability as its Achille’s heel. The most
potent compound in the present contribution, 22, is similarly
not cytotoxic but shows a major improvement in in vitro
metabolic stability (Table 1). Compared to the reference
compounds of the literature (Pyr3 and Synta66), therefore, our
newest compound does not affect cellular viability, displays a
similar efficacy, but shows a significantly improved metabolic
stability (Table 1).
In our SAR study, a SOCE enhancer has been identified,
adding this compound to the previously reported pool of
positive modulators. Due to poor available crystallographic
data, the binding partner of the reported SOCE modulators
has not been conclusively ascertained, but the presence of
similar behavior is also shared by 2-APB (47), a well-known
inhibitor of IP3 receptors and TRP channels. The compound is
a SOCE modifier in Orai1- and Orai3-expressing cells, acting
as SOCE enhancer at low concentrations, while high
concentrations induce a transient increase followed by
41
complete inhibition. Although a precise rationale can not
be extrapolated from the scaffolds of the enhancers, in our
experience it often occurs that a minimal structural
modification in a class of compounds designed to negatively
modulate SOCE is capable of turning an inhibitor into a
molecule able to increase calcium entry.
The five selected inhibitors (22, 27, 29, 32, and 42) and the
identified SOCE enhancer (37) were then evaluated for their
6
43
https://dx.doi.org/10.1021/acsmedchemlett.1c00034
ACS Med. Chem. Lett. 2021, 12, 640−646

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
activators in our series strongly suggests that it is the Orai
channel. The interaction of SOCE modulators with the ion
channel is also supported by a recently published article in
which, through computational approaches, a docking pose of
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
42
Synta66 in the Orai channel has been proposed. Moreover,
azopyrazole-derived SOCE inhibitors have been recently
reported as the first photoswitchable SOCE modulators able
to induce the activation of Orai using light, further supporting
the hypothesis that our compounds interact directly with this
Notes
The authors declare the following competing financial
interest(s): B.R. and T.P. are co-founders of ChemICare
S.r.l., a start-up that develops SOCE modulators. The other
authors declare that they do not have competing interests.
4
3
ion channel.
ACKNOWLEDGMENTS
On one hand, SOCE inhibitors are currently being
investigated for their potential therapeutic applications and
have progressed in clinical trials; on the other hand, activators
are still in their infancy, representing both chemical probes to
better elucidate SOCE biology and hit compounds for the
development of agents able to boost the immune system in
loss-of-function mutations associated with severe combined
■
Marta Serafini is supported by Fondazione AIRC (Associa-
zione Italiana per la Ricerca sul Cancro) fellowship for Abroad
(Rif. 25278). This work was partly supported by a Telethon
grant to Armando A. Genazzani (GGP19110).
ABBREVIATIONS
■
44,45
immunodeficiency (SCID)-like disorders.
AUC, area under the curve; DHODH, dihydroorotate
dehydrogenase; DIPEA, N,N-diisopropylethylamine; DMF,
N,N-dimethylformamide; ER, endoplasmic reticulum; EtOAc,
ethyl acetate; HEK, human embryonic kidney; IQR,
interquartile range; MLM, mouse liver microsomes; MLS9,
mouse liver S9; PyBOP, benzotriazol-1-yloxytripyrrolidino-
phosphonium hexafluorophosphate; SAR, structure−activity
relationship; SEM, standard error of mean; SERCA, sarco-
endoplasmic reticulum calcium ATPase; SCID, severe
combined immunodeficiency; SOCE, Store-Operated Calcium
Entry; THF, tetrahydrofuran; TsCl, tosyl chloride; tBhQ, t-
butylhydroquinone
ASSOCIATED CONTENT
■
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00034.
Synthesis and characterization of compounds; in vitro
metabolism and purity of lead compounds; biology
methods (cell cultures, MTT assay, and calcium
imaging) (PDF)
REFERENCES
AUTHOR INFORMATION
■
■
(
1) Carafoli, E.; Santella, L.; Branca, D.; Brini, M. Generation,
Corresponding Author
control, and processing of cellular calcium signals. Crit. Rev. Biochem.
Mol. Biol. 2001, 36 (2), 107−260.
Marta Serafini − Department of Pharmaceutical Sciences,
Universita
̀
degli Studi del Piemonte Orientale, 28100 Novara,
(2) Berridge, M. J.; Bootman, M. D.; Roderick, H. L. Calcium
signalling: dynamics, homeostasis and remodeling. Nat. Rev. Mol. Cell
Biol. 2003, 4 (7), 517−529.
Italy; orcid.org/0000-0002-5305-8359; Phone: +39
495338810; Email: marta.serafini@uniupo.it
3
(
3) Putney, J. W., Jr A model for receptor-regulated calcium entry.
Authors
Silvio Aprile − Department of Pharmaceutical Sciences,
Universita degli Studi del Piemonte Orientale, 28100 Novara,
Cell Calcium 1986, 7, 1−12.
4) Putney, J. W. Origins of the concept of store-operated calcium
entry. Front. Biosci., Scholar Ed. 2011, S3, 980−984.
5) Putney, J. W. The physiological function of store-operated
calcium entry. Neurochem. Res. 2011, 36 (7), 1157−1165.
6) Berna-Erro, A.; Woodard, G. E.; Rosado, J. A. Orais and STIMs:
(
̀
(
Italy; orcid.org/0000-0003-4804-9543
Beatrice Riva − Department of Pharmaceutical Sciences,
(
Universita degli Studi del Piemonte Orientale, 28100 Novara,
̀
physiological mechanisms and disease. J. Cell. Mol. Med. 2012, 16 (3),
Italy; ChemICare S.r.l., Enne3, 28100 Novara, Italy
407−424.
Irene Preet Bhela − Department of Pharmaceutical Sciences,
(7) Lewis, R. S. The molecular choreography of a store-operated
calcium channel. Nature 2007, 446 (7133), 284−287.
Universita
Italy
̀
degli Studi del Piemonte Orientale, 28100 Novara,
(8) Darbellay, B.; Arnaudeau, S.; König, S.; Jousset, H.; Bader, C.;
Demaurex, N.; Bernheim, L. STIM1- and Orai1-dependent store-
operated calcium entry regulates human myoblast differentiation. J.
Biol. Chem. 2009, 284 (8), 5370−5380.
Celia Cordero-Sanchez − Department of Pharmaceutical
Sciences, Universita degli Studi del Piemonte Orientale,
8100 Novara, Italy
Giulia Avino − Department of Pharmaceutical Sciences,
Universita degli Studi di Trieste, 34127 Trieste, Italy
Armando A. Genazzani − Department of Pharmaceutical
Sciences, Universita degli Studi del Piemonte Orientale,
8100 Novara, Italy; orcid.org/0000-0003-1923-7430
Tracey Pirali − Department of Pharmaceutical Sciences,
Universita degli Studi del Piemonte Orientale, 28100 Novara,
̀
2
(9) Xie, J.; Pan, H.; Yao, J.; Zhou, Y.; Han, W. SOCE and cancer:
recent progress and new perspectives. Int. J. Cancer 2016, 138 (9),
067−2077.
10) Guéguinou, M.; Harnois, T.; Crottes, D.; Uguen, A.; Deliot, N.;
Gambade, A.; Chantome, A.; Haelters, J. P.; Jaffres, P. A.; Jourdan, M.
̀
2
(
̀
̂
̀
2
L.; Weber, G.; Soriani, O.; Bougnoux, P.; Mignen, O.; Bourmeyster,
N.; Constantin, B.; Lecomte, T.; Vandier, C.; Potier-Cartereau, M.
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 2016, 7 (24),
̀
Italy; ChemICare S.r.l., Enne3, 28100 Novara, Italy;
orcid.org/0000-0003-3936-4787
3
6168−36184.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00034
(11) Di Sabatino, A.; Rovedatti, L.; Kaur, R.; Spencer, J. P.; Brown, J.
T.; Morisset, V. D.; Biancheri, P.; Leakey, N. A. B.; Wilde, J. I.; Scott,
6
44
https://dx.doi.org/10.1021/acsmedchemlett.1c00034
ACS Med. Chem. Lett. 2021, 12, 640−646

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
L.; Corazza, G. R.; Lee, K.; Sengupta, N.; Knowles, C. H.; Gunthorpe,
M. J.; McLean, P. G.; MacDonald, T. T.; Kruidenier, L. Targeting gut
T cell Ca2+ release-activated Ca channels inhibits T cell cytokine
production and T-box transcription factor T-bet in inflammatory
bowel disease. J. Immunol. 2009, 183 (5), 3454−3462.
channel blockers is affected by the Orai pore geometry. Cell Calcium
2013, 53 (2), 139−151.
2+
(27) Chen, G.; Panicker, S.; Lau, K. Y.; Apparsundaram, S.; Patel, V.
A.; Chen, S. L.; Soto, R.; Jung, J. K. C.; Ravindran, P.; Okuhara, D.;
Bohnert, G.; Che, Q.; Rao, P. E.; Allard, J. D.; Badi, L.; Bitter, H. M.;
Nunn, P. A.; Narula, S. K.; DeMartino, J. A. Characterization of a
novel CRAC inhibitor that potently blocks human T cell activation
and effector functions. Mol. Immunol. 2013, 54 (3−4), 355−367.
(28) (a) CM4620 injectable emulsion versus supportive care in
patients with acute pancreatitis and SIRS. Status: completed.
ClinicalTrials.gov Identifier: NCT03401190. https://www.
clinicaltrials.gov/ct2/show/NCT03401190?term=NCT03401190=2=
(
12) Ng, S. W.; di Capite, J.; Singaravelu, K.; Parekh, A. B. Sustained
activation of the tyrosine kinase Syk by antigen in mast cells requires
local Ca2+ influx through Ca release-activated Ca channels. J. Biol.
2+
2+
Chem. 2008, 283 (46), 31348−31355.
(13) Wen, L.; Voronina, S.; Javed, M. A.; Awais, M.; Szatmary, P.;
Latawiec, D.; Chvanov, M.; Collier, D.; Huang, W.; Barrett, J.; Begg,
M.; Stauderman, K.; Roos, J.; Grigoryev, S.; Ramos, S.; Rogers, E.;
Whitten, J.; Velicelebi, G.; Dunn, M.; Tepikin, A. V.; Criddle, D. N.;
Sutton, R. Inhibitors of ORAI1 prevent cytosolic calcium-associated
injury of human pancreatic acinar cells and acute pancreatitis in 3
mouse models. Gastroenterology 2015, 149 (2), 481−492.
1
. Last update: May 3, 2019 (accessed 2020-11-04). (b) Study of
CM4620 to reduce the severity of pancreatitis due to asparaginase.
Status: recruiting. ClinicalTrials.gov Identifier: NCT04195347.
https://www.clinicaltrials.gov/ct2/show/NCT04195347?term=
NCT04195347=2=1. Last update: September 10, 2020 (accessed
(14) Gerasimenko, J. V.; Gryshchenko, O.; Ferdek, P. E.; Stapleton,
E.; Hébert, T. O. G.; Bychkova, S.; Peng, S.; Begg, M.; Gerasimenko,
2
(
020-11-04).
2
+
2+
O. V.; Petersen, O. H. Ca release-activated Ca channel blockade as
a potential tool in antipancreatitis therapy. Proc. Natl. Acad. Sci. U. S.
A. 2013, 110 (32), 13186−13191.
29) A study of PRCL-02 in moderate to severe chronic plaque
psoriasis. Status: completed, has results. ClinicalTrials.gov Identifier:
NCT03614078. https://www.clinicaltrials.gov/ct2/show/
NCT03614078?term=NCT03614078=2=1. Last update: March 30,
(15) Stauderman, K. A. CRAC channels as targets for drug discovery
and development. Cell Calcium 2018, 74, 147−159.
16) Tian, C.; Du, L.; Zhou, Y.; Li, M. Store-operated CRAC
channel inhibitors: opportunities and challenges. Future Med. Chem.
016, 8 (7), 817−832.
17) Pevarello, P.; Cainarca, S.; Liberati, C.; Tarroni, P.; Piscitelli, F.;
2
(
020 (accessed 2020-11-04).
30) Rahman, S.; Rahman, R. Unveiling some FDA-approved drugs
(
2+
as inhibitors of the store-operated Ca entry pathway. Sci. Rep. 2017,
(1), 12881−12893.
31) Breedveld, F. C.; Dayer, J. M. Leflunomide: mode of action in
the treatment of rheumatoid arthritis. Ann. Rheum. Dis. 2000, 59,
41−849.
32) Schattenkirchner, M. The use of leflunomide in the treatment
2
7
(
(
2+
2+
Severi, E. Ca release-activated Ca channel inhibitors. Pharm. Pat.
Anal. 2014, 3 (2), 171−182.
8
(
(18) Sweeney, Z. K.; Minatti, A.; Button, D. C.; Patrick, S. Small-
molecule inhibitors of store-operated calcium entry. ChemMedChem
009, 4 (5), 706−718.
19) Singh, A.; Hildebrand, M. E.; Garcia, E.; Snutch, T. P. The
of rheumatoid arthritis: an experimental and clinical review.
2
(
Immunopharmacology 2000, 47 (2−3), 291−298.
(33) Palmer, A. M. Teriflunomide, an inhibitor of dihydroorotate
transient receptor potential channel antagonist SKF96365 is a potent
blocker of low-voltage-activated T-type calcium channels. Br. J.
Pharmacol. 2010, 160 (6), 1464−1475.
dehydrogenase for the potential oral treatment of multiple sclerosis.
Curr. Opin. Invest. Drugs 2010, 11 (11), 1313−1323.
(34) Davis, J. P.; Cain, G. A.; Pitts, W. J.; Magolda, R. L.; Copeland,
(20) Merritt, J. E.; Armstrong, W. P.; Benham, C. D.; Hallam, T. J.;
R. A. The immunosuppressive metabolite of leflunomide is a potent
inhibitor of human dihydroorotate dehydrogenase. Biochemistry 1996,
Jacob, R.; Jaxa-Chamiec, A.; Leigh, B. K.; McCarthy, S. A.; Moores, K.
E.; Rink, T. J. SK&F 96365, a novel inhibitor of receptor-mediated
calcium entry. Biochem. J. 1990, 271 (2), 515−522.
3
(
5 (4), 1270−1273.
35) (a) Riva, B.; Griglio, A.; Serafini, M.; Cordero-Sanchez, C.;
(21) Bootman, M. D.; Collins, T. J.; Mackenzie, L.; Roderick, H. L.;
Aprile, S.; Di Paola, R.; Gugliandolo, E.; Alansary, D.; Biocotino, I.;
Lim, D.; Grosa, G.; Galli, U.; Niemeyer, B.; Sorba, G.; Canonico, P.
L.; Cuzzocrea, S.; Genazzani, A. A.; Pirali, T. Pyrtriazoles, a novel class
of store-operated calcium entry modulators: discovery, biological
profiling, and in vivo proof-of-concept efficacy in acute pancreatitis. J.
Med. Chem. 2018, 61 (21), 9756−9783. (b) Pirali, T.; Riva, B.;
Genazzani, A. A. Modulators of SOCE, compositions and use thereof.
W.O. Patent WO212,414, December 14, 2017.
Berridge, M. J.; Peppiatt, C. M. 2-aminoethoxydiphenyl borate (2-
2
+
APB) is a reliable blocker of store-operated Ca entry but an
2
+
inconsistent inhibitor of InsP3-induced Ca release. FASEB J. 2002,
6 (10), 1145−1150.
22) Goto, J.; Suzuki, A. Z.; Ozaki, S.; Matsumoto, N.; Nakamura,
1
(
T.; Ebisui, E.; Fleig, A.; Penner, R.; Mikoshiba, K. Two novel 2-
aminoethyl diphenylborinate (2-APB) analogues differentially activate
2
+
and inhibit store-operated Ca entry via STIM proteins. Cell Calcium
010, 47 (1), 1−10.
23) Djuric, S. W.; BaMaung, N. Y.; Basha, A.; Liu, H.; Luly, J. R.;
(
36) Boström, J.; Hogner, A.; Llinas
Oxadiazoles in medicinal chemistry. J. Med. Chem. 2012, 55 (5),
817−1830.
37) Goldberg, K.; Groombridge, S.; Hudson, J.; Leach, A. G.;
̀
, A.; Wellner, E.; Plowright, A. T.
2
(
1
(
Madar, D. J.; Sciotti, R. J.; Tu, N. P.; Wagenaar, F. L.; Wiedeman, P.
E.; Zhou, X.; Ballaron, S.; Bauch, J.; Chen, Y. W.; Chiou, X. G.; Fey,
T.; Gauvin, D.; Gubbins, E.; Hsieh, G. C.; Marsh, K. C.; Mollison, K.
W.; Pong, M.; Shaughnessy, T. K.; Sheets, M. P.; Smith, M.;
Trevillyan, J. M.; Warrior, U.; Wegner, C. D.; Carter, G. W. 3,5-
Bis(trifluoromethyl)pyrazoles: a novel class of NFAT transcription
factor regulator. J. Med. Chem. 2000, 43 (16), 2975−2981.
MacFaul, P. A.; Pickup, A.; Poultney, R.; Scott, J. S.; Svensson, P. H.;
Sweeney, J. Oxadiazole isomers: all bioisosteres are not created equal.
MedChemComm 2012, 3, 600−604.
(38) Serafini, M.; Pirali, T.; Tron, G. C. Click 1,2,3-triazoles in drug
discovery and development: from the flask to the clinic? Adv.
Heterocycl. Chem. 2020, DOI: 10.1016/bs.aihch.2020.10.001..
(24) Schleifer, H.; Doleschal, B.; Lichtenegger, M.; Oppenrieder, R.;
(39) Serafini, M.; Cordero-Sanchez, C.; Di Paola, R.; Bhela, I. P.;
Derler, I.; Frischauf, I.; Glasnov, T. N.; Kappe, C. O.; Romanin, C.;
Groschner, K. Novel pyrazole compounds for pharmacological
discrimination between receptor-operated and store-operated Ca2
entry pathways. Br. J. Pharmacol. 2012, 167 (8), 1712−1722.
Aprile, S.; Purghe, B.; Fusco, F.; Cuzzocrea, S.; Genazzani, A. A.; Riva,
̀
+
B.; Pirali, T. Store-operated calcium entry as a therapeutic target in
acute pancreatitis: discovery and development of drug-dike SOCE
inhibitors. J. Med. Chem. 2020, 63 (23), 14761−14779.
(25) Law, M.; Morales, J. L.; Mottram, L. F.; Iyer, A.; Peterson, B.
R.; August, A. Structural requirements for the inhibition of calcium
mobilization and mast cell activation by the pyrazole derivative BTP2.
Int. J. Biochem. Cell Biol. 2011, 43 (8), 1228−1239.
(40) Azimi, I.; Stevenson, R. J.; Zhang, X.; Meizoso-Huesca, A.; Xin,
P.; Johnson, M.; Flanagan, J. U.; Chalmers, S. B.; Yoast, R. E.; Kapure,
J. S.; Ross, B. P.; Vetter, I.; Ashton, M. R.; Launikonis, B. S.; Denny,
W. A.; Trebak, M.; Monteith, G. R. A new selective pharmacological
(
26) Derler, I.; Schindl, R.; Fritsch, R.; Heftberger, P.; Riedl, M. C.;
2
+
Begg, M.; House, D.; Romanin, C. The action of selective CRAC
enhancer of the Orai1 Ca channel reveals roles for Orai1 in smooth
6
45
https://dx.doi.org/10.1021/acsmedchemlett.1c00034
ACS Med. Chem. Lett. 2021, 12, 640−646

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
and skeletal muscle functions. ACS Pharmacol. Transl. Sci. 2020, 3 (1),
1
(
35−147.
41) Motiani, R. K.; Abdullaev, I. F.; Trebak, M. A novel native
store-operated calcium channel encoded by Orai3: selective require-
ment of Orai3 versus Orai1 in estrogen receptor-positive versus
estrogen receptor-negative breast cancer cells. J. Biol. Chem. 2010, 285
(
25), 19173−19183.
42) Waldherr, L.; Tiffner, A.; Mishra, D.; Sallinger, M.; Schober, R.;
Frischauf, I.; Schmidt, T.; Handl, V.; Sagmeister, P.; Köckinger, M.;
(
̈
Derler, I.; Uc a̧ l, M.; Bonhenry, D.; Patz, S.; Schindl, R. Blockage of
2
+
Store-Operated Ca
Influx by Synta66 is Mediated by Direct
2
+
Inhibition of the Ca Selective Orai1 Pore. Cancers 2020, 12 (10),
876−2895.
43) Yang, X.; Ma, G.; Zheng, S.; Qin, X.; Li, X.; Du, L.; Wang, Y.;
2
(
Zhou, Y.; Li, M. Optical control of CRAC channels using
photoswitchable azopyrazoles. J. Am. Chem. Soc. 2020, 142 (20),
9
(
460−9470.
44) McCarl, C. A.; Picard, C.; Khalil, S.; Kawasaki, T.; Rother, J.;
Papolos, A.; Kutok, J.; Hivroz, C.; Ledeist, F.; Plogmann, K.; Ehl, S.;
Notheis, G.; Albert, M. H.; Belohradsky, B. H.; Kirschner, J.; Rao, A.;
Fischer, A.; Feske, S. ORAI1 deficiency and lack of store-operated
2+
Ca entry cause immunodeficiency, myopathy, and ectodermal
dysplasia. J. Allergy Clin. Immunol. 2009, 124, 1311−1318.
(45) Picard, C.; McCarl, C. A.; Papolos, A.; Khalil, S.; Luthy, K.;
Hivroz, C.; LeDeist, F.; Rieux-Laucat, F.; Rechavi, G.; Rao, A.;
Fischer, A.; Feske, S. STIM1 mutation associated with a syndrome of
immunodeficiency and autoimmunity. N. Engl. J. Med. 2009, 360,
1971−1980.
6
46
https://dx.doi.org/10.1021/acsmedchemlett.1c00034
ACS Med. Chem. Lett. 2021, 12, 640−646