Pyrtriazoles, a Novel Class of Store-Operated Calcium Entry Modulators: Discovery, Biological Profiling, and in Vivo Proof-of-Concept Efficacy in Acute Pancreatitis

Article abstract of DOI:10.1021/acs.jmedchem.8b01512

In recent years, channels that mediate store-operated calcium entry (SOCE, i.e., the ability of cells to sense a decrease in endoplasmic reticulum luminal calcium and induce calcium entry across the plasma membrane) have been associated with a number of d

Full text of DOI:10.1021/acs.jmedchem.8b01512

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Article
Pyrtriazoles, a Novel Class of Store-Operated Calcium
Entry Modulators: Discovery, Biological Profiling and
In Vivo Proof-of-Concept Efficacy in Acute Pancreatitis
Beatrice Riva, Alessia Griglio, Marta Serafini, Celia Cordero Sanchez, Silvio Aprile, Rosanna Di Paola, Enrico
Gugliandolo, Dalia Alansary, Isabella Biocotino, Dmitry Lim, Giorgio Grosa, Ubaldina Galli, Barbara Niemeyer,
Giovanni Sorba, Pier Luigi Canonico, salvatore cuzzocrea, Armando A. Genazzani, and Tracey Pirali
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01512 • Publication Date (Web): 22 Oct 2018
Downloaded from http://pubs.acs.org on October 23, 2018
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Pyrtriazoles, a Novel Class of Store-Operated
Calcium Entry Modulators: Discovery, Biological
Profiling and In Vivo Proof-of-Concept Efficacy in
Acute Pancreatitis
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Beatrice Riva1,4#, Alessia Griglio , Marta Serafini , Celia Cordero-Sanchez , Silvio Aprile ,
1#
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Rosanna Di Paola , Enrico Gugliandolo , Dalia Alansary , Isabella Biocotino , Dmitry Lim ,
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Giorgio Grosa , Ubaldina Galli , Barbara Niemeyer , Giovanni Sorba , Pier Luigi Canonico ,
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1,4 @
Salvatore Cuzzocrea , Armando A. Genazzani *, Tracey Pirali *
1
Department of Pharmaceutical Sciences; Università del Piemonte Orientale; Novara, 28100;
Italy; 2 Department of Chemical, Biological, Pharmaceutical and Enviromental Sciences;
3
Università di Messina; Messina, 98166; Italy; Department of Molecular Biophysics; Saarland
4
University CIPMM; Homburg, 66421; Germany; ChemICare S.r.l.; Enne3; Novara, 28100; Italy.
# contributed equally
*
contributed equally
@ Corresponding Author:
Prof. Tracey Pirali
Dept. of Pharmaceutical Sciences
Università del Piemonte Orientale
Largo Donegani, 2 – 28100 Novara
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Phone: 0039-0321-375852
Fax: 0039-0321-375821
e-mail: tracey.pirali@uniupo.it
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ABSTRACT
In recent years, channels that mediate Store-Operated Calcium Entry (SOCE, i.e. the ability of
cells to sense a decrease in endoplasmic reticulum luminal calcium and induce calcium entry across
the plasma membrane) have been associated to a number of disorders, spanning from immune
disorders to acute pancreatitis and have been suggested to be druggable targets.
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In the present contribution, we exploited the click chemistry approach to synthesize a class of
SOCE modulators where the arylamide substructure, that characterizes most inhibitors so far
described, is substituted by a 1,4-disubstituted 1,2,3-triazole ring.
Within this series, inhibitors of SOCE were identified and the best compound proved effective in
an animal model of acute pancreatitis, a disease characterized by a hyperactivation of SOCE.
Strikingly, two enhancers of the process were discovered, affording invaluable research tools to
further explore the (patho)physiological role of capacitative calcium entry.
Introduction
During evolution, Ca2+ ions were excluded from the cytosol to protect phosphate from
precipitating. The very low concentration of Ca2+ in the cytosol, together with the massive
1
2
+
5
gradient across membranes (Ca is 10 times more abundant in organelles and in the extracellular
medium compared to the cytosol), became thereafter an opportunity to use this ion as a specific
2
+
second messenger. Ca -signals, defined as a transient rise of this ion in the cytosol, are now
widespread transducers of signals, mediating various processes ranging from fertilization to cell
2
+
death. A Ca -signal encodes a message through its amplitude, the duration and the frequency of
its rises and the exact spatial localization in the cell. It has grown to such specialization that
hundreds of proteins govern this process and each cell type has a unique set of proteins that are
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chosen for specific tasks (the calcium toolkit, as defined by one of the fathers of modern calcium
2
2+
signalling). Given the importance of fine-tuning Ca -signals to generate highly specific signals,
it is not surprising that a vast array of disorders is thought to have calcium dys-regulation as an
underlying cause.
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As mentioned above, high concentrations of calcium are present in intracellular organelles (in
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particular in the ER/SR) and in the extracellular space and Ca -fluxes occur through channels
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+
located on the plasma membrane or on the membrane of intracellular organelles. Given that Ca -
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pumps and exchangers are located on both membranes to extrude Ca from the cytosol, it would
be expected that the intracellular organelle pool would be soon depleted. It was already back in
1986 that Jim Putney, a pioneer of the cross-talk between the ER and the plasma membrane,
formalized the hypothesis that a phenomenon known as “capacitative calcium entry” would exist
3
that allows entry through the plasma membrane to refill the depleted organelles. The key
experiment to exemplify capacitative calcium entry, or Store-Operated Calcium Entry (SOCE), as
it is referred to nowadays, is depicted in Figure 1. In brief, emptying of the ER/SR store leads to
2
+
opening of a plasma membrane channel through which Ca can flow back in the cell and these
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+
two phenomena can be dissected by adding Ca to the extracellular solution after the intracellular
stores depletion. This simple, yet powerful, experimental approach remains valid to unmask the
phenomenon in screenings.
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+
It is now thought that the principal components of SOCE are a Ca -sensor on the ER membrane
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+
4
(STIM proteins) and a plasma membrane Ca -channel (Orai channels). STIM proteins are single-
span membrane proteins, highly conserved across species. Two members of the family have been
described, STIM1 and STIM2, of which the former appears more expressed. Orai channels reside
on the plasma membrane channel and three members of the family (Orai1, Orai2, and Orai3) have
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been described, with Orai1 being the most abundant. It is important to stress that other crucial
proteins participate in the SOCE process, including TRPC channels.5
6
Capacitative calcium entry, mediating the ISOC current, is not solely a replenishment mechanism,
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but also encodes cellular signals per se, regulating cytokine production, gene expression, cell
growth, proliferation, differentiation and even cell death. Therefore, dysfunctions in this cellular
event are implicated in a vast range of channelopathies, pointing to SOCE as a druggable target.7
The most important reason to pursue the search for specific SOCE inhibitors is the historically
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consolidated role played by capacitative calcium entry in autoimmunity and the paucity of well-
tolerated immunosuppressant drugs. Nevertheless, aberrant activity of SOCE is involved in a
number of pathological states not limited to the immune system, ranging from neo-angiogenesis
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10
in cancer to acute pancreatitis. Last, genetic defects of STIM and Orai proteins have been
described that give rise to loss- and gain-of-function genetic disordersmainly affecting the immune
and skeletal systems, respectively.11
Specific pharmacological manipulation of SOCE has been challenging. For a long time, non-
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selective agents such as 2-APB, CAI and SKF-96365 have been the primary experimental
tools to modulate SOCE (Figure 2). Over the years, much interest has been directed at a series of
1
5
3
,5-bistrifluoromethyl pyrazole derivatives, disclosed by Abbott in 2000. Specifically, BTP2
(Pyr2, YM-58483) is a potent inhibitor, but it has pleiotropic effects on both Orai and TRPC
1
6
channels. Subsequently, other pyrazoles, identified as Pyrs, have been reported. Among them,
1
7
Pyr3, a previously suggested selective inhibitor of TRPC3, was shown to inhibit both TRPC3-
and Orai1-mediated calcium entry. By contrast, two compounds, Pyr6 and Pyr10, are able to
distinguish to a certain degree Orai and TRPC-mediated calcium entry.18
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While the initial interest was mainly the discovery of experimental tools to gain insights into the
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role played by SOCE, drug development programs in this direction are currently underway. In
this context, second-generation agents have been described with different characteristics in terms
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of mechanism of action and selectivity profile, including Synta66, RO2959, and GSK-7975A.
Four SOCE inhibitors have entered clinical trials (CM2489 for psoriasis, CM4620 for acute
pancreatitis, RP3128 for asthma and RP4010 for non-Hodgkin’s lymphoma), although none has
reached regulatory approval and the advent of novel modulators would certainly increase the
chances of clinical benefit. Interestingly, in silico screening of an FDA-approved drug library has
recently led to the discovery of five compounds that can be either re-purposed or might serve as
leads for future development of SOCE inhibitors.23
Besides negative modulators, SOCE potentiating agents have been reported. 2-APB is a well-
known, though not so selective, effector of SOCE, but it may either enhance or inhibit the current
2
4
depending on concentrations, while MDEB, another borinate compound, has only a potentiating
ability on ISOC.25
26
We now describe a new family of compounds, that we refer to as Pyrtriazoles, that is able to fine-
tune SOCE and includes both inhibitors and enhancers. While the focus of our characterization is
on the inhibitors, as these have immediate therapeutic applications, the positive SOCE modulators
are to our knowledge the first SOCE enhancers that do not display a borinate substructure.
Results
Synthesis and rapid screening of compounds
The work described in this paper stems from the observation that the arylamide substructure
appears to be an essential pharmacophoric feature for SOCE modulation and almost all the
inhibitors reported in the literature share this structural redundancy (Figure 2). Taking advantage
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of our extensive experience in click chemistry and its exploitation in drug discovery, we
wondered whether the arylamide could be isosterically replaced by the 1,4-disubstituted 1,2,3-
triazole ring. To this aim, we started from the structure of known pyrazole derivatives, namely
BTP2 and Pyr compounds,15-18 and isosterically substituted the amide moiety with the triazole ring
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(
Scheme 1). The trifluoromethyl groups in the C3 and C5 positions of the pyrazole ring or,
alternatively, the ethyl carboxylate group in the C4 position are necessary for the inhibitory activity
and were kept fixed, while the portion beared by the triazole ring, which appears to contribute to
selectivity, was extensively varied.
To this aim, two pyrazolic azides 1 and 2 were prepared according to Scheme 2 and 3. The
synthetic protocol was straightforward and afforded the two precursors in three steps:
condensation, reduction of the aromatic nitro group to amine and diazotation-azidation. In the case
of 2, the pyrazole was resistant to elimination of water and treatment with hydrochloric acid in
boiling ethanol was necessary for dehydration/aromatization.
Similarly, two pyrazolic alkynes 3 and 4 were prepared according to Scheme 4 and 5. The
condensation reaction is followed by Sonogashira coupling with trimethylsilylacetylene and
deprotection of the silyl group in the presence of TBAF.
1
and 2 were clicked with twenty-one different alkynes A-U and, similarly, 3 and 4 were coupled
with twenty-one azides A-U (Table 1). The click reactions were performed under classical
conditions on a 50 mg scale. In general, the reactivity of the two alkynes 3 and 4 was lower than
the two corresponding azides 1 and 2, as demonstrated by a general decrease in the yields. Six
reactions worked only in the presence of TBTA, a powerful stabilizing ligand for copper (I) that
2
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enhances its catalytic activity and favors the cycloaddition. Four reactions were not successful,
even when TBTA was used.
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Over the eighty-four performed reactions, seventy-nine products precipitated, were filtered,
washed with water and heptane, characterized at this stage only by mass analysis and screened for
activity on Store-Operated Calcium Entry in BV-2 cells, a mouse microglial cell line, using Fluo-
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as a fluorimetric dye (Table 1). In brief, a multi-plate reader approach was employed in which
intracellular stores of BV-2 cells were emptied with tBhQ (50 µM) in the presence of the
compounds (at 10 µM). Calcium was then re-added after 600 sec to the extracellular medium and
intracellular levels determined in a protocol similar to the one depicted in Figure 1. The use of
I
II
crude compounds was possible as copper salts (Cu and Cu ) up to 10 μM were devoid of any
activity on SOCE (not shown) and it is therefore a time-efficient strategy to identify rapidly hit
compounds. While false positives would be picked up in the characterization of purified
compounds, we do acknowledge that it is possible, albeit unlikely, that false negatives might occur.
Therefore, the twenty-three crude compounds that showed at least 20% difference of SOCE
compared to control were re-synthesized, purified by column chromatography and re-tested by
fluorescence microscopy using the ratiometric dye Fura-2 in BV-2 cells. Twelve compounds, ten
that decreased SOCE and two that enhanced SOCE, that showed at least a 30% difference
compared to control of SOCE were identified and tested also in Hek and Jurkat cells (Table 2).
It is interesting to note that a carboxylic moiety in the R group, despite being absent in the
3
previously reported Pyr compounds, is favourable for the modulation of SOCE within this new
class of compounds, as demonstrated by the presence of this functional group in seven compounds
over the twelve selected for their activity. On the other hand, substructures typically displayed by
Pyrs (e.g. 3-fluoropyridine in Pyr6, Figure 2) do not confer a significant modulating activity on
SOCE (2J, 4J, Table 1), corroborating that Pyrtriazoles are characterized by SARs substantially
different from Pyrs. Overall, we can state that series 4 is the only one that does not display
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modulating activity on SOCE, while the other three series (1, 2, 3) comprise both negative and
positive modulators, depending on the substituents R and R on the pyrazole ring, the R group
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and the relative position A or B of the triazole ring. Regarding R , non-substituted phenyl ring and
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most of the exploited functionalized aromatic rings do not confer significant modulating activities.
On the other hand, 4-(-3-carboxypropyl)phenyl (3D), 3-aminophenyl (1H, 3H), 4-hydroxy-3-
methoxyphenyl (1N) and 3-carboxyphenyl (1S, 2S, 3S) might yield an inhibitory character,
according to the other functionalities featured by the compound. Curiously, the presence of a
chlorine in para position (3G) confers a positive modulator character, while the carboxyl group in
the same position might lead to a positive (2T) or a negative modulation (1T, 3T), depending on
the substitution pattern displayed by the molecule.
Characterization of the inhibitors
We then performed concentration-response curves of 1S compared to Pyr3 in the three cell lines
(see Supporting Information). The IC for inhibition in Hek cells for 1S was 0.6 ± 0.1 μM, in
50
Jurkat cells 0.6 ± 0.1 μM, in BV-2 cells 4.9 ± 1.8 μM, compared to 0.5 ± 0.1, 0.3 ± 0.1 and 2.9 ±
.8 μM, respectively, for Pyr3. Both 1S and Pyr3 were full inhibitors as 100 μM treatment led to
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a near abolishment of SOCE. This was true also for the other non-selected compounds that fully
inhibited SOCE at 100 μM (not shown). The lower sensitivity of BV-2 cells (about 10-fold) may
be accounted by the different source of cells (murine versus human), by the different isoform
3
0
composition of Orai and STIM, as well as by a different Orai1/STIM1 ratio, or by a different
level of Orai1 (indeed BV-2 appears to have 10-fold less Orai mRNA; not shown), although this
was not investigated further. While the effect of Pyr3 and 1S at 10 µM on SOCE was similar as
were the IC s, differences were apparent when evaluating viability of cells after 24 h treatment.
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also tested Pyr6, which displayed an IC in Hek cells for SOCE inhibition of 0.3 ± 0.1 μM and a
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cell viability at 10 μM of 70.3 ± 5.8%. Therefore, it would appear that Pyr3 is the most cytotoxic,
but some cytotoxicity is also displayed by its parent compound Pyr6 and by 1S, albeit at
concentration 100-fold the IC for SOCE inhibition.
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Given that our cellular models also expressed TRPC1, we decided to investigate in Hek cells the
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contribution of TRPC1 in the observed Ca influx. For this, we proceeded via shRNA experiments
and were able to reduce mRNA expression of TRPC1 by 65 ± 13%. Partial silencing of TRPC1 in
Hek cells slightly reduced SOCE by 11% (NS, p = 0.48; Figure 3B). The fact that 1S inhibited
SOCE by about 90% in Hek cells (Table 2), together with the limited contribution of TRPC1 to
SOCE, suggests that TRPC1 is not a target for our compounds. To further substantiate Orai/STIM
as the target, we next used murine embryonic fibroblast (MEF) cells derived from wild-type or
-/-/
-/-
31
Stim1 Stim2 mice. Wild-type cells had a similar expression profile of TRPC and Orai subtypes
-/-/
-/-
compared to Stim1 Stim2 except for STIM expression (Figure 3C). As it can be observed in
Figure 3D, tBhQ-triggered SOCE was low in the two cell lines, with some differences. Indeed, the
-/-/
-/-
peak of calcium entry was slightly higher in Stim1 Stim2 MEFs. The presence of an initial rise
-/-/
-/-
31
in calcium in Stim1 Stim2 MEFs contrasts to what reported previously in these cells and is
unlikely to be attributable to SOCE, as it does not respond to 1S or Pyr3, unlike the initial rise
observed in wild-type cells. It should be noted that the extent of calcium entry is small compared
-/-/
-
to what observed in all other cell types. Nonetheless, the biggest difference between Stim1 Stim2
/
-
and wild-type MEFs was represented by the significant decrease in the plateau of calcium entry
-/-/
-/-
in Stim1 Stim2 MEFs. As expected, and in line with data in Figure 3A, in wild-type cells 1S
-/-/
-/-
significantly affected both the peak amplitude and the plateau. In contrast, in Stim1 Stim2
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MEFs, the peak amplitude was unaffected, while the prolonged phase was affected although to a
lesser degree compared to wild-type.
In line with SOCE measurements, ISOC measured in Hek cells co-overexpressing both Orai1 and
STIM1 showed an inhibition of ~40% of current in cells treated with 10 µM 1S suggesting that
the effects measured in SOCE are indeed mediated by Orai1 and STIM1 (Figure 3E).
Characterization of the activators 2T and 3G
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To our great surprise, two of the synthesized compounds (2T and 3G) were able to enhance SOCE
(Figure 4A, Table 2). Both compounds, tested at a concentration of 10 µM, significantly increased
the AUC of calcium entry, the peak amplitude and the slope (Figure 4B) in Hek cells. A similar
effect was also observed in BV-2 and Jurkat cells (not shown). The minor differences observed
between the two compounds were probably attributable to the fact that 2T was more potent or
efficacious. The effect of 2T was evident at concentrations above 0.3 µM and the effect of 3G was
evident above 3 µM increased up to the highest concentration tested (100 µM; Figure 4C for Hek
cells; see Supporting Information for concentration-response curves of 2T and 3G on all three cell
lines). For this reason, IC s of these compounds could not be calculated within the applied range
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of concentrations.
To further investigate the mechanism of action, we performed electrophysiological recordings on
Hek cells stably over-expressing Orai1 and transiently expressing STIM1. Both 2T and 3G were
able to significantly potentiate ISOC measured in these cells using strong buffering conditions of
2
+
the internal solution (20 mM BAPTA). Buffering the internal solution to contain 150 nM Ca led
to the abolishment of the potentiation of the current. However, in the latter conditions the kinetics
of activation indicate that treatment with either 2T or 3G leads to faster activation of ISOC, as
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indicated by the significantly reduced time constants of activation (Figure 4D). These data show
that SOCE is the target of these compounds.
In a similar manner to what described above, we decided to investigate whether the effect observed
with 2T was attributable to Orai/STIM or to the potentiation of TRPC. In cells silenced for TRPC1,
the potentiating effect observed by 2T was still evident, suggesting that Orai/STIM were the target
of the agent (Figure 5A for wild type and 5B for shTRPC1). It should be noted that while AUC
and peak amplitude were significantly different between control and 2T-treated cells, the slope
was not modified in shTRPC1 (see Supporting Information). A direct or indirect effect on TRPC1
activity cannot therefore be excluded, but it does not appear to be the main target. We next used
the MEF system described above to verify the need of STIM1/2 for channel opening by these
compounds. At it can be observed in Figure 5C, 2T strongly potentiated/induced calcium entry in
both systems, suggesting that STIM is dispensable for this effect and the putative molecular target
could be an Orai protein.
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We also investigated whether 2T and 3G were use-dependent, i.e. required the triggered opening
2
+
of the Orai channel to elicit their effects. To do this, we monitored intracellular Ca in resting BV-
2
+
2
, Hek or Jurkat cells in the presence of extracellular Ca . While 3G 10 µM did not elicit any
significant calcium entry compared to control in the 800 seconds of observation in any of the three
2
+
cell lines, 2T-treated cells displayed a slow rise of Ca , which started shortly after the addition of
the compound (see Supporting Information). These data suggest that 2T is an activator/channel
opener while 3G is a potentiator/enhancer, but further characterizations will be required in the
future to fully elucidate the mechanism of 2T and 3G. It should be noticed that 2-APB behaves
3
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differently as a SOCE enhancer in Orai1- and Orai3-expressing cells and it will be therefore of
interest to test these compounds in similar conditions.
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Selectivity of 1S, 2T and 3G
It has been reported that Pyr compounds may also affect other channels.15-18 In this respect, we
tested the activity of Pyr3, Pyr6, 1S, 2T and 3G on voltage-operated channels, TRPV1 and TRPM8
channels that have been shown previously to be affected by SOCE inhibitors. For voltage-operated
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Ca -channels we used primary cultures of mouse cerebellar granule cells challenged with 50 mM
KCl in the presence or absence of the compounds. As shown in Figure 6A, at 10 µM 1S, 2T and
3
G did not influence voltage-dependent calcium entry, while Pyr3 and Pyr6 reduced entry by about
5
0%. When testing TRPV1 in TRPV1-overexpressing Hek cells challenged with capsaicin (100
nM), Pyr3 had no effect, while 1S and Pyr6 significantly affected the response (Figure 6B). Finally,
we tested TRPM8 inhibition by using TRPM8-overexpressing Hek cells challenged with the
known agonist menthol (100 µM). As shown in Figure 6C, 1S, 2T, 3G, Pyr3 had no effect on the
response, while Pyr6 significantly reduced menthol-mediated calcium rise.
Synthesis of analogues of compound 1S and characterization
1
S appears like an inhibitor of SOCE with different peculiarities compared to Pyr compounds. In
particular, it does not have an effect on voltage-dependent channels and is less cytotoxic, although
it inhibits TRPV1 channels. We therefore next focused our efforts on performing a preliminary
SAR study of this compound (Table 3 and 4). First of all, we investigated whether the triazole ring
behaved merely as an isostere of the amide group or played a role in the interaction with the SOCE
machinery (Table 3). To this aim, 1S analogues displaying both the direct (19) and inverse (22)
amide were synthesized, according to Scheme 6. The amine and the carboxylic acid were coupled,
while protecting and deprotecting the additional carboxylic group. We also synthesized the
corresponding 1,5-disubstituted triazole (23) using a ruthenium-based catalyst (Scheme 6).
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Then, the ethyl ester portion of 1S was modified (Table 4). In particular, intermediate 7 was
transesterified (24, 25), reduced to amine (26, 27), converted into azide (28, 29) and clicked with
alkyne S to give the final methyl ester 30 and isopropyl ester 31 (Scheme 7). Alternatively,
intermediate 1 was hydrolized to 32, coupled with different alcohols (33-38) and clicked with
alkyne S, leading to a series of structurally different esters (39-44) (Scheme 7). Moreover, the ethyl
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ester of intermediate 8 was reduced in the presence of LiAlH , converted into azide 45 and clicked
4
with alkyne S to give the final primary alcohol 46 (Scheme 7). Intermediate 32 was coupled with
isopropylamine and then clicked with alkyne S, yielding the secondary amide 48 (Scheme 7). The
ethyl ester of compound 1S was reacted with aqueous ammonia to give the primary amide 49 and
hydrolized leading to the corresponding carboxylic acid 50 (Scheme 8).
Last, we investigated whether the carboxylic moiety of compound 1S was tolerant to substitution
with acyl methansulfonamide 51 (Scheme 8), methyl ester 58, methyl ketone 59, primary amide
60, primary alcohol 61, sulfonamide 62 and hydroxamic acid 63 (Scheme 9). To this aim, the
corresponding alkynes 52-56 were prepared by a Sonogashira coupling and deprotection, while
alkyne 57 was synthesized by coupling reaction using O-(trimethylsilyl)hydroxylamine and
deprotection (see Supporting Information).
The synthesized analogues were screened in the same manner as described above after purification
by column chromatography and the results are displayed in Table 3 and 4. These included cell
viability via the MTT assay at 10 μM and determination of the IC for the inhibition of SOCE for
50
the most active compounds (see Supporting Information for concentration-response curves of 31
and 39). From these data, the first observation is that the triazole ring, the key novelty added in
this contribution, does not merely behave as an isostere of the amide group, but plays a pivotal
role in the modulation of SOCE. Indeed, when substituted with both its direct and the inverse
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amide (19 and 22), a drop in activity surprisingly occurs. Second, the 1,4-disubstituted triazole
ring cannot be replaced with the 1,5-disubstituted analogue 23. Third, the methyl ester 30 and the
isopropyl ester 31 are more active compared to the ethyl ester 1S, the pentan-2-yl ester 39 inhibits
SOCE to the same extent, while the other synthesized esters (40-44) lose the ability to act on
SOCE. Furthermore, the ethyl ester cannot be replaced by a primary alcohol (46) or by an amide
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(48, 49). Finally, the carboxylic group moiety is not tolerant to the substitution with other
functionalities (51, 58-62), except for the hydroxamic acid 63, which is endowed with remarkable
activity, but significantly affects cell viability at 10 µM.
Given that the ester group in 1S might be susceptible to hydrolysis, we decided to investigate
whether the corresponding carboxylic acid retained activity. As it can be seen, 50 is not active,
demonstrating that the ester group is necessary for the inhibition of SOCE. Therefore, we decided
to investigate the hydrolytic stability of 1S and the esters selected according to their activity on
SOCE (30, 31, 39, 43) in mouse plasma. As it can be seen (Table 3, 4 and Supporting Information
for full data) the short linear esters (Pyr3, 1S and 30) and the (benzyloxy)carbonyl 43 underwent
fast hydrolysis, suggesting that they might not be good hit compounds for systemic use.
Conversely, the branched esters 31 and 39 showed an overall good stability in plasma, with a
residual substrate after 30 minutes of 87.7 and 97.8%, respectively. Given that the plasma stability
of Pyr3 was poor (20.4% residual substrate after 30 min), 31 and 39 appeared as major
improvements in this respect.
We therefore decided to evaluate their metabolism in mouse liver homogenates. In mouse liver S9
fractions (MLS9), their hydrolytic stability was confirmed with the residual substrate after 1 hour
being 81.3% and 73.7% for 31 and 39, respectively (see Supporting Information). We next
investigated the in vitro oxidative metabolism of the two compounds by performing incubations
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in the presence of NADPH. Alternatively, given the presence of a carboxyl acid moiety, the
formation of acylglucuronide metabolites was assessed by incubating the compounds in mouse
liver microsomes (MLM) activated by UDPGA (see Supporting Information). Overall, hydrolysis
of the ester function was the main metabolic pathway in liver metabolism, as no significant
differences in residual substrate were observed in the presence or absence of NADPH or UDPGA.
However, minute amounts of other metabolites (<1%) were detected and characterized by mass
spectrometry analysis. Compound 39 underwent oxidative hydroxylation at the pentan-2-yl chain,
giving the metabolite 39-OH. Moreover, both the carboxyl functions of compounds 31 and 39
were susceptible to the formation of acylglucuronide metabolites (31-G and 39-G; Scheme 10).
Finally, no traces of glucuronide conjugates of metabolite 50 were detected in the incubations.
Last, we evaluated the selectivity of 31 and 39 in respect to voltage-operated calcium entry,
TRPV1 and TRPM8 channels. Gratifyingly, as it can be observed in Figure 7, neither 31 or 39 had
an effect on these channels. This therefore makes these two compounds stand out both compared
to Pyr3 and Pyr6, the latter in terms of selectivity and the former in terms of selectivity, stability
and cytotoxicity.
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In vivo evaluation of 39
We next opted to verify the potential use in vivo of 39.
First, to verify its tolerability, we investigated its toxicity after a single dose (40 mg/Kg) for 14
days, monitoring mortality, body weight, clinical signs of distress and histological alterations of
major organs (brain, heart, liver, lung, kidney, lung, stomach and ileum) in accordance to the
OECD guideline for acute toxicity. None of the 6 treated animals showed any alterations.
We next performed a pharmacokinetic analysis to further characterize the compound. The half-life
of 39 was 1.3 hours, with a Cmax of 640 μg/L after 1 hour. The clearance was 17 L/h/Kg, with a
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volume of distribution of 32 L/Kg. Despite the surprisingly high volume of distribution, the plasma
concentrations reached (approx. 1.2 µM) led us to believe that it might have exerted a therapeutic
effect and decided therefore to perform proof-of-principle in vivo experiment. Gerasimenko et al.
have shown that SOCE inhibition in vitro protects against POAEE-induced necrosis of pancreatic
acinar cells10a and this was followed by the demonstration that GSK-7975A and CM128 were
protective in three separate models of chemically-induced acute pancreatitis,10b including the
model that uses cerulein. Indeed, a clinical trial has been initiated on CM4620, another SOCE
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inhibitor. Given that this is an acute disorder, we reasoned that 39, with a half-life of hours, might
have been suitable. 39 was given twice, 30 and 150 minutes after the first cerulein injection. As
shown in Figure 8, the histological scores were significantly ameliorated by treatment with 39.
This was evident in the haematoxylin/eosin stainings, when evaluating oedema and inflammation
and when determining neutrophil infiltration via MPO staining.
Discussion and Conclusions
Structural information on Orai and STIM proteins is relatively scarce. Only the crystal structure
of Orai from Drosophila melanogaster, which shares 73% sequence identity with human Orai1
3
4
within its transmembrane region, has been described at 3.35 Å resolution, while there are not yet
any solved structures of STIM bound to Orai. Thus, a rational design of modulators is at present
complicated and we decided to pursue a click chemistry approach starting from Pyr compounds.
In the present contribution, we show that click chemistry is an effective tool to develop novel
modulators of this mechanism and describe a novel class of agents, which we call Pyrtriazoles,
that are able to modulate Store-Operated Calcium Entry. While almost all the SOCE inhibitors
reported to date share an arylamide moiety, the modulators described by us display a 1,4-diaryl-
1H-1,2,3-triazole ring. An extensive screening of compounds displaying different R groups
3
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suggested that the presence of a carboxylic group on the terminal aryl ring is favourable for the
modulation of SOCE (Table 1 and 2). It must be stressed that the triazole ring does not merely
behave as an isostere of the amide group displayed by Pyr compounds, but plays a pivotal role in
the interaction with SOCE machinery. Indeed, when substituted with both its direct and inverse
amide in compound 1S, a decrease in activity occurred in all cases (Table 3), validating this series
of compounds as an entirely novel class of SOCE modulators. This is confirmed by the fact that
subtructures featured by Pyrs, such as 3-fluoropyridine, do not confer significant activity on SOCE
in the concomitant presence of the triazole ring.
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Among the synthesized compounds, we identified both negative and positive modulators: yet,
Pytriazoles are able to fine-tune SOCE, according to the substitution pattern displayed by each
compound.
By a preliminary screening, we identified ten inhibitors that represent therefore the first
participants of the new class of Pyrtriazoles. We have characterized further one of the inhibitors,
1
S, and have shown that it was able to reduce SOCE in cells that expressed wild-type Orai and
STIM. The hydrolytic instability of 1S, while on one hand dampening its use as a systemic agent,
on the other hand offers an opportunity to exploit it as a soft drug for topical use. For example,
3
5
36
SOCE has been implicated in psoriasis and UV-induced melanogenesis and this could therefore
be its correct collocation. It should be stressed that the fact that 1S is also a TRPV1 antagonist
provides a further putative advantage, as these channels are implicated in the same skin conditions
and we have recently shown that selective soft drugs acting on TRPV1 are effective in in vivo
37
models of pruritus. 1S could therefore be the founder of a new class of soft Pyrtriazoles for skin
disorders endowed with dual action.
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Starting from compound 1S, we performed a preliminary SAR study and selected two structural
analogues (31 and 39), according to SOCE inhibition, cytotoxicity and extent of hydrolysis, for
further investigations on metabolic stability and selectivity (Table 4). These analogues display
IC s in the low micromolar range and are specific on SOCE compared to the channels evaluated
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in the present study (voltage-operated, TRPV1 and TRPM8). Moreover, these compounds
appeared suitable as lead compounds for systemic use as they appeared relatively stable. Indeed,
slow hydrolysis of the ester function was the main metabolic pathway both in mouse plasma and
liver, with minute formation of the oxidative (31) or glucuronidated (31 and 39) metabolites. The
in vivo study showed that the half-life of 39 was 1.3 hours and a twice-administration of the
compound significantly ameliorated cerulein-induced pancreatitis, in the absence of signs of
toxicity. Yet the high volume of distribution and the short half-life in vivo might suggest that
further refinements are necessary. Also we acknowledge that we have investigated selectivity of
the compounds over the main channels that have been reported to be targets of Pyrs with the
exception of TRPC family members, which have also been reported as targets for BTP2 and Pyr3.
Serendipitously, we have also identified two compounds, 2T and 3G, which are able to potentiate
Store-Operated Calcium Entry, representing the first non-borinate enhancers of SOCE. While we
have not fully investigated the selectivity of the two compounds reported here, initial data indicate
that these may be better experimental tools to manipulate SOCE. In particular, unlike 2-APB and
analogues, they do not have a dual effect depending on concentration and, unlike MDEB, it act at
lower micromolar concentrations.25
As for other SOCE modulators, the exact mechanism of action of Pyrtriazoles remains elusive.
Given that TRPC1 knock-down does not affect significantly the action of the reported compounds,
we hypothesize that the target will either be STIM or Orai. Furthermore, the two enhancers display
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–/–/
–/–
their effect also in Stim1 Stim2 cells, suggesting that these compounds most likely directly bind
to Orai, although we can only postulate that positive and negative modulators share the same target.
Overall, the described compounds hold great promise to both get insights into the biological role
of SOCE and pave the way for the treatment of human disorders arising from imbalanced calcium
homeostasis.
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EXPERIMENTAL SECTION
Chemistry
General Experimental Methods. Commercially available reagents and solvents were used as
purchased without further purification. When needed, solvents were distilled and stored on
molecular sieves. Column chromatography was performed on silica gel Merck Kieselgel 70-230
mesh ASTM. Thin layer chromatography (TLC) was carried out on 5 cm × 20 cm plates with a
layer thickness of 0.25 mm (Merck silica gel 60 F254). When necessary, TLC plates were
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visualized with aqueous KMnO or with aqueous Pancaldi solution. Melting points were
4
determined in open glass capillary with a Stuart scientific SMP3 apparatus. All the target
1
13
compounds were checked by IR (FT-IR Thermo-Nicolet Avatar), H- and C-NMR (Jeol ECP
3
00 MHz), and mass spectrometry (Thermo Finningan LCQ-deca XP-plus) equipped with an ESI
source and an ion trap detector. Chemical shifts are reported in parts per million (ppm). The purity
of compounds was determined by high performance liquid chromatography (HPLC) using the
instrumentation and methods reported in Supporting Information. Purity of all final compounds
was 95% or higher. Azides and alkynes (except for azide K, alkyne J and compound 52-57, see
Supporting Information) are commercially available or were synthesized following procedures
already reported in the literature.
Ethyl 1-(4-azidophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate, (azide 1).
To a suspension of compound 8 (2.60 g, 8.70 mmol) in water (40.7 mL) 37% HCl aqueous solution
(3.3 mL) was added. The resulting mixture was cooled at 0 °C and a solution of NaNO (0.57 g,
2
8
.26 mmol) in water (3.9 mL) was added. After 10 min a solution of NaN (0.64 g, 9.85 mmol) in
3
water (3.9 mL) was added and the reaction was left to reach room temperature and stirred for 1 h.
The mixture was worked up by dilution with ethyl acetate and washing with water (x1). The
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organic layer was dried over sodium sulfate and evaporated yielding compound 1 (2.20 g, 6.77
1
mmol, 78%) as a pale red oil. H NMR (300 MHz, CDCl ): δ = 8.08 (s, 1H), 7.39 (d, J = 8.5 Hz,
3
1
3
2H), 7.11 (d, J = 8.5 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H). C NMR (75
1
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2
2
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MHz, CDCl ): δ = 160.9, 142.6, 141.9, 135.9, 133.6 (q, J = 39.0 Hz), 127.4, 119.6, 119.1 (q, J =
3
2
1
1
69.1 Hz), 116.9, 61.4, 14.1. IR (KBr) ṽ = 3406, 3221, 2987, 2415, 2096, 1713, 1560, 1510, 1355,
-1
286, 1148, 971, 848, 558, 404 cm .
-(4-Azidophenyl)-3,5-bis(trifluoromethyl)-1H-pyrazole, (azide 2).
The title compound was synthesized as previously reported for the preparation of compound 1,
starting from compound 12 (4.16 g, 14.10 mmol). The crude material was purified by column
chromatography using petroleum ether/ethyl acetate 95:5 as eluent, yielding compound 2 (3.84 g,
1
1
7
1.96 mmol, 85%) as a pale red oil. H NMR (300 MHz, CDCl ): δ = 7.47 (d, J = 8.5 Hz, 2H),
3
1
3
.14 (d, J = 8.5 Hz, 2H), 7.0 (s, 1H). C NMR (75 MHz, CDCl ): δ = 143.0 (q, J = 39.5 Hz),
3
142.3, 134.7, 133.8 (q, J = 40.0 Hz), 127.3, 123.9 (q, J = 266.8 Hz), 119.6, 116.8 (q, J = 266.8
Hz), 107.2. IR (KBr) ṽ = 3732, 3217, 3059, 2853, 2700, 2412, 2263, 2141, 1900, 1562, 1286,
-1
1
234, 1142, 1019, 838, 763, 535, 465 cm .
Ethyl 1-(4-ethynylphenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate, (alkyne 3).
Step 1: To a solution of ethyl 1-(4-bromophenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate
(1.90 g, 5.23 mmol) in toluene (30 mL) DIPEA (3.6 mL, 20.9 mmol), CuI (0.18 g, 0.95 mmol),
Pd(PPh ) Cl (0.22 g, 0.31 mmol) and ethynyltrimethylsilane (3 mL, 20.9 mmol) were added in a
3 2
2
Schlenk apparatus. The reaction was stirred at reflux for 2 h under nitrogen atmosphere. Then,
ethyl acetate was added and the organic phase was washed with water (x1), dried over sodium
sulfate and evaporated. The crude material was purified by column chromatography using
petroleum ether/ethyl acetate 95:5 as eluent to give ethyl 5-(trifluoromethyl)-1-(4-
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(
(trimethylsilyl)ethynyl)phenyl)-1H-pyrazole-4-carboxylate as a brown solid (1.57 g, 4.13 mmol,
1
7
9%). H NMR (300 MHz; CDCl ): δ = 8.10 (s, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.2 Hz,
3
2H), 4.36 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H), 0.26 (s, 9H). MS (ESI): m/z: 381 [M + H]+.
Step 2: Ethyl 5-(trifluoromethyl)-1-(4-((trimethylsilyl)ethynyl)phenyl)-1H-pyrazole-4-
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carboxylate (1.30 g, 3.42 mmol) was dissolved in THF (13 mL). The mixture was cooled at 0 °C
and a solution of 1M TBAF in THF (4.10 mL, 4.10 mmol) was added. The reaction was stirred for
additional 30 min at 0 °C. The volatile was removed under vacuum, ethyl acetate was added and
the organic layer was washed with water (x1). After drying over sodium sulfate and evaporation
of the solvent, the crude material was purified by column chromatography using petroleum
ether/ethyl acetate 95:5 as eluent to give compound 3 (0.83 g, 2.70 mmol, 79%) as a yellow solid.
1
H NMR (300 MHz, CDCl ): δ = 8.11 (s, 1H), 7.60 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H),
3
1
3
4
.36 (q, J = 7.1 Hz, 2H), 3.19 (s, 1H), 1.37 (t, J = 7.1 Hz, 3H). C NMR (75 MHz, CDCl ): δ =
3
160.5, 142.5, 139.3, 132.7, 132.3 (q, J = 39.2 Hz), 127.4, 125.8, 124.0, 119.1 (q, J = 269.1 Hz),
1
7
1
17.0, 79.7, 61.1, 13.9. IR (KBr) ṽ = 3266, 3129, 2982, 2112, 11717, 1565, 1511, 1298, 1039, 847,
-1
+
86 cm . MS (ESI): m/z: 309 [M + H] .
-(4-Ethynylphenyl)-3,5-bis(trifluoromethyl)-1H-pyrazole, (alkyne 4).
Step
1:
3,5-Bis(trifluoromethyl)-1-(4-((trimethylsilyl)ethynyl)phenyl)-1H-pyrazole
was
synthesized as previously reported for the preparation of compound 3 in step 1, starting from
compound 15 (1.70 g, 4.73 mmol). The crude material was subjected to chromatography column
using petroleum ether and petroleum ether/ethyl acetate 95:5 as eluent, yielding 3,5-
bis(trifluoromethyl)-1-(4-((trimethylsilyl)ethynyl)phenyl)-1H-pyrazole (1.75 g, 4.64 mmol, 98%)
1
as a yellow oil. H NMR (300 MHz; CDCl ): δ = 7.65 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.3 Hz,
3
2H), 7.06 (s, 1H), 0.27 (s, 9H). MS (ESI): m/z: 377 [M + H]+.
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Step 2: The title compound was synthesized as previously reported for the preparation of
compound
3
in
step
2,
starting
from
3,5-bis(trifluoromethyl)-1-(4-
((trimethylsilyl)ethynyl)phenyl)-1H-pyrazole (4.45 g, 11.84 mmol). The crude material was
1
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purified by column chromatography using petroleum ether and petroleum ether/ethyl acetate 95:5
1
as eluent to give compound 4 (2.92 g, 9.59 mmol, 81%) as a brown oil. H NMR (300 MHz,
1
3
CDCl ): δ = 7.56 (d, J 6.9 Hz, 2H), 7.44 (d, J = 6.9 Hz, 2H), 7.08 (s, 1H), 3.17 (s, 1H). C NMR
3
(
75 MHz, CDCl ): δ = 143.2 (q, J = 38.9 Hz), 138.2, 133.7 (q, J = 40.1 Hz), 132.5 (2C), 125.5,
3
1
24.5, 120.5 (q, J = 267.4 Hz), 119.0 (q, J = 267.4 Hz), 107.4, 79.5. IR (KBr) ṽ = 3917, 3290,
-1
+
3
161, 1914, 1296, 1241, 993, 672, 466 cm . MS (ESI): m/z 305 [M + H] .
General procedure A
To a suspension of azide (0.29 mmol, 1 eq) in water (570 µL) and t-BuOH (570 µL) alkyne (0.29
mmol, 1 eq) was added. Then, 30 µL of aqueous solution of sodium ascorbate 1M and copper
sulfate pentahydrate (0.0029 mmol, 0.01 eq) were added and the mixture was maintained under
vigorous stirring overnight. Ice was added and the precipitate was filtered and rinsed with water
and heptane. Compounds 1A, 1B, 1C, 1D, 1F, 1G, 1H, 1J, 1K, 1L, 1M, 1N, 1O, 1Q, 1R, 1S, 1T,
1U, 2A, 2B, 2C, 2D, 2F, 2G, 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, 2P, 2Q, 2R, 2S, 2T, 2U, 3A, 3B,
3C, 3D, 3E, 3F, 3G, 3H, 3I, 3L, 3M, 30, 3P, 3Q, 3S, 3T, 3U, 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H,
4I, 4J, 4K, 4L, 4M, 4O, 4P, 4Q, 4S and 4U were synthesized following this procedure.
All the crude products were then screened and the selected molecules (1H, 1N, 1S, 1T, 2S, 2T,
3D, 3G, 3H, 3S and 3T) were re-synthesized and subjected to chromatography column.
General procedure B
To a solution of Cu(OAc) (0.0145 mmol, 0.05 eq) in THF (26 µL) TBTA (0.0077 mmol, 0.05 eq)
2
was added and the resulting mixture was stirred at room temperature for 30 min. A solution of
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azide (0.29 mmol, 1 eq) in THF (0.05 mL), a solution of alkyne (0.29 mmol, 1 eq) in THF (0.05
mL) and a solution of sodium ascorbate (0.03 mmol, 0.1 eq) solubilized in the minimum amount
of water were added. The reaction was stirred at room temperature overnight. The precipitate was
filtered and rinsed with water (x2) and diethyl ether (x2) to give a solid. Compounds 1E, 1I, 1P,
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E, 3J, 3K were synthesized following this procedure.
All the crude products were then screened and the selected molecule 3J was re-synthesized and
subjected to chromatography column.
Ethyl
1-(4-(4-(3-aminophenyl)-1H-1,2,3-triazol-1-yl)phenyl)-5-(trifluoromethyl)-1H-
pyrazole-4-carboxylate, (1H).
1
Pale yellow solid; yield 83% (106 mg, 0.24 mmol); mp 152-153 °C. H NMR (300 MHz, DMSO-
d6): δ = 9.27 (s, 1H), 8.36 (s, 1H), 8.18 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.1 Hz, 2H), 7.22 (s, 1H),
7
.13 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 7.8 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H), 5.25 (br s, 2H), 4.35
1
3
(q, J = 6.9 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). C NMR (75 MHz, DMSO-d ): δ = 160.9, 149.7,
6
1
2
1
48.9, 143.0, 139.0, 138.2, 132.3 (q, J = 39.5 Hz), 131.1, 130.0, 128.5, 121.3, 120.0, 119.5 (q, J =
69.1 Hz), 117.0, 114.7, 113.9, 111.2, 61.7, 14.5. IR (KBr): ṽ = 3374, 2987, 2636, 1723, 1555,
-1
+
493, 1418, 1248, 1182, 1077, 810, 690, 581 cm . MS (ESI): m/z: 443 [M + H]
Ethyl
1-(4-(4-(4-hydroxy-3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)phenyl)-5-
(trifluoromethyl)-1H-pyrazole-4-carboxylate, (1N).
1
Dark yellow solid; yield 38% (52 mg, 0.11 mmol); mp 129-130 °C. H NMR (300 MHz, DMSO-
d6): δ = 9.28 (s, 1H), 8.35 (s, 1H), 8.17 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H), 7.50 (s, 1H),
7
.38 (d, J = 8.0 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 3.87 (s, 3H), 1.30 (t,
1
3
J = 7.1 Hz, 3H). C NMR (75 MHz, DMSO-d ): δ = 160.9, 148.7, 148.6, 147.6, 143.0, 139.0,
6
138.2, 132.3 (q, J = 39.5 Hz), 128.5, 121.9, 121.0, 119.5 (q, J = 268.8 Hz), 119.2, 118.9, 117.0,
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16.5, 110.1, 61.7, 56.2, 14.5. IR (KBr): ṽ = 3113, 2986, 2933, 1727, 1519, 1297, 1150, 1039, 847,
-1
+
79 cm . MS (ESI): m/z: 474 [M + H] .
3-(1-(4-(4-(Ethoxycarbonyl)-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-1H-1,2,3-triazol-
-yl)benzoic acid, (1S).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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8
9
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4
1
Yellowish solid; yield 52% (71 mg, 0.15 mmol); mp 215-216 °C. H NMR (300 MHz, DMSO-
d6): δ = 9.58 (s, 1H), 8.55 (s, 1H), 8.37 (s, 1H), 8.22 (d, J = 8.7 Hz, 2H), 8.16 (d, J = 7.6 Hz, 1H),
7
2
1
.97 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.63 (t, J = 7.7 Hz, 1H), 4.34 (q, J = 7.1 Hz,
1
3
H), 1.32 (t, J = 7.1 Hz, 3H). C NMR (75 MHz, CDCl ): δ = 167.7, 160.8, 147.4, 142.9, 139.1,
3
38.0, 132.4, 132.2 (q, J = 39.2 Hz), 131.0, 129.9, 129.6, 128.4, 126.7, 121.1 (2C), 120.9, 119.5
(q, J = 269.3 Hz), 117.0, 61.6, 14.4. IR (KBr): ṽ = 3083, 2829, 2671, 1736, 1688, 1524, 1300,
-1
+
1
150, 1041, 845, 759, 682 cm . MS (ESI): m/z: 472 [M + H] .
4
-(1-(4-(4-(Ethoxycarbonyl)-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-1H-1,2,3-triazol-
4-yl)benzoic acid, (1T).
1
Yellowish solid; yield 52% (72 mg, 0.15 mmol); mp 291-292 °C. H NMR (300 MHz, DMSO-
d6): δ = 9.56 (s, 1H), 8.36 (s, 1H), 8.19 (d, J = 7.4 Hz, 2H), 8.14-8.03 (m, 4H), 7.87 (d, J = 7.4 Hz,
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3
2
1
H), 4.34 (q, J = 6.6 Hz, 2H), 1.32 (t, J = 6.3 Hz, 3H). C NMR (75 MHz, DMSO- d ): δ = 167.2,
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60.8, 147.3, 143.0, 139.2, 137.9, 134.6, 132.2 (q, J = 38.9 Hz), 128.4 (2C), 128.0, 121.5, 121.2
(2C), 120.2 (q, J = 268.6 Hz), 117.0, 61.6, 14.4. IR (KBr): ṽ = 3107, 2984, 2671, 2545, 1379, 1677,
-1
+
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524, 1246, 1038, 971 cm . MS (ESI): m/z: 472 [M + H] .
3-(1-(4-(3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-1H-1,2,3-triazol-4-yl)benzoic
acid, (2S).
1
Yellow solid; yield 62% (84 mg, 0.18 mmol); mp 214-215 °C dec. H NMR (300 MHz, DMSO-
d6): δ = 9.1 (s, 1H), 8.55 (s, 1H), 8.25 (d, J = 8.8 Hz, 2H), 8.20 (s, 1H), 8.10-7.86 (m, 4H), 7.66 (t,
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Journal of Medicinal Chemistry
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J = 7.7, 1H). C NMR (75 MHz, DMSO-d ): δ = 167.6, 147.4, 142.3 (q, J = 38.4 Hz), 138.4,
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37.8, 134.4 (q, J = 39.5 Hz), 132.2, 130.9, 129.7, 128.5 (2C), 126.7, 121.3 (2C), 121.0 (q, J =
267.4 Hz), 120.9, 119.3 (q, J = 268.0 Hz), 106.8. IR (KBr): ṽ = 3158, 2822, 2670, 1696, 1526,
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245, 1136, 994, 757, 559 cm . MS (ESI): m/z 468 [M + H] .
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-(1-(4-(3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-1H-1,2,3-triazol-4-yl)benzoic
acid, (2T).
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Yellow solid; yield 48% (65 mg, 0.14 mmol); mp 279-280 °C dec. H NMR (300 MHz, DMSO-
d6): δ = 9.59 (s, 1H), 8.23 (d, J = 8.1 Hz, 2H), 8.18-8.03 (m, 4H), 7.95 (d, J = 8.1 Hz, 2H), 7.91
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(
s, 1H). C NMR (75 MHz, DMSO-d ): δ = 167.1, 146.8, 141.9 (q, J = 39.0 Hz), 137.8, 137.4,
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134.1, 133.8 (q, J = 39.2 Hz), 132.0, 130.3, 128.0, 125.5, 121.5 (q, J = 266.8 Hz), 121.0 (2C),
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19.3 (q, J = 268.0 Hz), 108.5. IR (KBr): ṽ = 3106, 2854, 2362, 1678, 1527, 1240, 1142, 1031,
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+
50, 775 cm . MS (ESI): m/z 468 [M + H] .
4-(4-(4-(4-(Ethoxycarbonyl)-5-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-1H-1,2,3-triazol-
-yl)butanoic acid, (3D).
White solid; yield 69% (87 mg, 0.20 mmol). mp 151-152 °C. H NMR (300 MHz, DMSO-d ): δ
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8.74 (s, 1H), 8.32 (s, 1H), 8.05 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 4.47 (t, J = 6.9 Hz,
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H), 4.33 (q, J = 6.9 Hz, 2H), 2.30 (t, J = 6.9 Hz, 2H), 2.11 (quint, J = 6.9 Hz, 2H), 1.31 (t, J =
1
3
.1 Hz, 3H). C NMR (75 MHz, DMSO-d ): δ = 174.2, 160.9, 145.7, 142.8, 138.6, 133.1, 132.0
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(
q, J = 39.2 Hz), 127.3, 126.3, 122.9, 119.5 (q, J = 269.1 Hz), 116.8, 61.6, 49.5, 30.9, 25.7, 14.4.
-1
IR (KBr): ṽ = 3117, 2915, 2360, 1906, 1731, 1562, 1300, 1134, 972, 781 cm . MS (ESI): m/z 438
M + H]+.
[
Ethyl
1-(4-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)phenyl)-5-(trifluoromethyl)-1H-
pyrazole-4-carboxylate, (3G).
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Yellow solid; yield 59% (79 mg, 0.17 mmol); mp 208-209 °C. H NMR (300 MHz, CDCl ) δ =
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.26 (s, 1H), 8.14 (s, 1H), 8.05 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 8.7 Hz, 2H), 7.61-7.50 (m, 4H),
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4.39 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H). C NMR (75 MHz, DMSO-d ): δ = 160.9, 146.8,
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42.8, 139.1, 136.0, 133.8, 132.5, 132.1 (q, J = 38.9 Hz), 130.5, 127.4, 126.6, 122.4, 121.3, 119.5
(
q, J = 269.1 Hz), 116.8, 61.6, 14.5. IR (KBr): ṽ = 3154, 2983, 2365, 1917, 1731, 1497, 1233,
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043, 811, 525, 492 cm . MS (ESI): m/z 462 [M + H] .
1-(4-(1-(3-aminophenyl)-1H-1,2,3-triazol-4-yl)phenyl)-5-(trifluoromethyl)-1H-
pyrazole-4-carboxylate, (3H).
Ethyl
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Dark yellow solid; yield 69% (88 mg, 0.20 mmol); mp 141-142 °C. H NMR (300 MHz, DMSO-
d6): δ = 9.30 (s, 1H), 8.33 (s, 1H), 8.14 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 7.27 (t, J =
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.7 Hz, 1H), 7.16 (s, 1H), 6.99 (d, J = 7.7 Hz, 1H), 5.59 (br s, 2H), 4.33 (q, J = 7.0 Hz, 2H), 1.32
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(
t, J = 7.1 Hz, 3H). C NMR (75 MHz, DMSO-d ): δ = 160.0, 150.7, 146.3, 142.8, 138.9, 138.0,
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132.7, 132.1 (q, J = 39.5 Hz), 130.8, 127.4, 126.5, 121.0, 119.6 (q, J = 260.1 Hz), 116.8, 114.7,
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07.5, 105.5, 61.6, 14.5 IR (KBr): ṽ = 3444, 3343, 3121, 2902, 2365, 1718, 1554, 1160, 1045, 971,
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+
81, 558 cm . MS (ESI): m/z 443 [M + H] .
Ethyl 1-(4-(1-(3-fluoropyridin-4-yl)-1H-1,2,3-triazol-4-yl)phenyl)-5-(trifluoromethyl)-1H-
pyrazole-4-carboxylate, (3J).
1
Yellow solid; yield 41% (54 mg, 0.12 mmol); mp 162-163 °C. H NMR (300 MHz, DMSO-d ): δ
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9.39 (s, 1H), 8.99 (d, J = 5.8 Hz, 1H), 8.71 (d, J = 5.8 Hz, 1H), 8.34 (s, 1H), 8.21 (d, J = 8.3
Hz, 2H), 8.13 (t, J = 5.8 Hz, 1H), 7.71 (d, J = 8.3 Hz, 2H), 4.33 (q, J = 7.0 Hz, 2H), 1.32 (t, J =
13
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.0 Hz, 3H). C NMR (75 MHz, DMSO-d ): δ = 160.9, 151.6, 148.0, 146.9, 142.8, 141.1, 140.8,
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39.3, 131.8, 131.6 (q, J = 47.5 Hz), 127.4, 126.9, 123.9, 119.5 (q, J = 269.1 Hz), 118.2, 116.9,
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