1,2,4-Oxadiazole-Bearing Pyrazoles as Metabolically
Stable Modulators of Store-Operated Calcium Entry
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.
<!>Preparation of Amine 17<!><!>Preparation of Oxadiazole-Bearing
Pyrazoles 19–41<!>Synthesis of compound 42<!><!>Author Contributions<!>
<p>The ubiquitous second messenger
calcium is a central regulator of a plethora of physiological processes,
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 levels.1,2 A central mechanism in calcium homeostasis
is represented by the store-operated calcium entry (SOCE), i.e., the influx of calcium across the plasma membrane activated
by the depletion of this ion in the endoplasmic reticulum (ER).3 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 channels on the plasma membrane.4−6 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 (SERCA) pump.7,8 Due to its prominent
role in encoding calcium signals, SOCE is implicated in several human
disorders, including cancer,9,10 inflammatory bowel
disease,11 allergy,12 and acute pancreatitis.13,14 Therefore,
it is not surprising that this cellular pathway has become a promising
target for the therapeutic treatment of several pathological conditions.</p><p>Over the years, many small molecules15−18 able to modulate SOCE have been
reported. The earliest modulators are SKF-96365,19,20 2-APB and its derivatives,21,22 and bis(trifluoromethyl)pyrazoles
compounds, initially named BTP (BTP1 (1), BTP2 (2), Figure 1),23 followed by Pyr's (Pyr3 (3), Pyr6 (4), Pyr10 (5), Figure 1).24,25 Among others, GSK-7975A (6, Figure 1),26 RO2959 (7, Figure 1),27 and Synta66 (8, Figure 1)11,12 have been extensively used as chemical probes. More recently, two
compounds have entered clinical development, CM4620 (9, Figure 1) for acute
pancreatitis (phase II)28 and PRCL-02 for
psoriasis (phase II),29 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 at therapeutic doses,30 the prodrug leflunomide (10, Figure 1)31,32 and its active form
teriflunomide (11, Figure 1).33,34 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.</p><p>Structures of SOCE inhibitors reported in the
literature.</p><p>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 1).35</p><p>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 calcium channels.35 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-oxadiazole.36,37 Moreover, in the design of this novel class, we substituted the
1,4-disubstituted 1,2,3-triazole ring of compound 12 with
an amide group (Figure 2). Indeed, the 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 concerns
associated with copper catalysts.38</p><p>Design of 1,2,4-oxadiazole-bearing
pyrazoles.</p><p>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. Then,
the ethyl ester reacted with N-hydroxyacetamidine
to afford amine 17.</p><!><p>Reagents and conditions: (a)
DMF, reflux, 2 h, 96%. (b) H2, 5% Pd/C, EtOAc, rt, 2 h,
94%. (c) NaH 60%, N-hydroxyacetamidine, dry THF,
0 °C to rt, 4 h, 76%.</p><!><p>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) 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 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.</p><!><p>Reagents
and conditions: (a)
PyBOP, DIPEA, dry CH2Cl2, rt, 16–42 h,
40–97%. (b) NaOH, H2O, THF, rt, 5 h, 98%.</p><!><p>Reagents
and conditions: (a)
TsCl, pyridine, dry CH2Cl2, 0 °C to rt,
5 h, 45%.</p><!><p>With this approach, 24 oxadiazole-bearing
pyrazoles were synthesized
and tested in human embryonic kidney (HEK) cells for SOCE inhibition.
To this aim, intracellular Ca2+ stores of HEK cells were
emptied with t-butylhydroquinone (tBhQ, 50 Î¼M) in the presence of the compounds at 10 Î¼M
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 24 were able to reduce calcium entry
by more than 50% 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, 46.5%), and Pyr10 (42, 47.6%). Also,
compounds 27 (40.8%) and 29 (49.6%), displaying
an electron-withdrawing 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 Î¼M were discarded (23, 31, and 33). Notably, the remaining five compounds (22, 27, 29, 32, and 42) were not cytotoxic under these conditions. According to both activity
and cytotoxicity, for the selected five compounds, the representative
traces are depicted in Figure 3.</p><p>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 29 (10 Î¼M). Traces are the average of 200
cells from three independent experiments (top panel). Evaluation of
peak amplitude, area under the curve (AUC), and slope of the Ca2+-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 the Ca2+-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 Î¼M). Traces are the average
of 200 cells from three independent experiments (top panel). Evaluation
of peak amplitude, AUC, and slope of the Ca2+-rise in the
absence or presence of 32 and 42. The graph
shows the median and IQR of the peak amplitude, AUC, and slope of
the Ca2+-rise. Mann–Whitney U test of compound vs
control (**p ≤ 0.0069; ****p ≤ 0.0001; bottom panel).</p><p>Biological data
were derived for
three independent experiments, and numbers represent mean ± standard
error of mean (SEM).</p><p>Residual substrates were determined
in the MLS9 fraction after 1 h of incubation.</p><p>Residual substrates were determined
in mouse liver microsomes (MLM) as previously described.39</p><p>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 Î¼M.</p><p>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 Î¼M, 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
intracellular Ca2+ in resting cells in the presence of
extracellular Ca2+ and we observed that the compound did
not elicit any significant calcium entry at 10 Î¼M compared to
control in the 300 s of observation, suggesting that 37 is a potentiator/enhancer of SOCE. The same effect was also evident
at 3 (118.2%) and 1 Î¼M (116.7%) but was not detected at concentrations
above 10. In particular, at 30 and 100 Î¼M, 37 acts
as weak SOCE inhibitor, with a percentage of residual activity of
about 70%.</p><p>Effect of 37 on SOCE. (A) Traces of SOCE in the presence
or absence of 37 at a concentration of 10 Î¼M. Traces
are the average of 200 cells from three independent experiments. (B)
Evaluation of peak amplitude, AUC, and slope of the Ca2+-rise in the absence or presence of 37. The graph shows
the median and IQR of the peak amplitude, AUC, and slope of the Ca2+-rise. Mann–Whitney U test of compound vs control
(**p ≤ 0.002).</p><p>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 5),35 and biphenyl
triazoles (compound 45, Figure 5)39 and one reported
in the literature (IA65 (46), Figure 5).40 A 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 complete inhibition.41 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.</p><p>Structures of positive modulators of SOCE reported in
the literature.</p><p>The five selected inhibitors (22, 27, 29, 32, and 42) and the identified
SOCE enhancer (37) were then evaluated for their 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 substrate of 43 and 74%, respectively
(Table 1).35 Similarly, the residual substrate of Synta66
in MLM is of 15%, due to an important hydrolysis of the amide group,
absent in our class (Table 1).39</p><p>We have previously reported
CIC-37 that had as its main 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).</p><p>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 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 Synta66 in the
Orai channel has been proposed.42 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 ion channel.43</p><p>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 immunodeficiency (SCID)-like disorders.44,45</p><p>Synthesis and
characterization of compounds; in vitro
metabolism and purity of lead compounds; biology methods (cell cultures,
MTT assay, and calcium imaging) (PDF)</p><p>ml1c00034_si_001.pdf</p><!><p>The manuscript
was written through contributions of all authors. All authors have
given approval to the final version of the manuscript.</p><!><p>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.</p>