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Article Cite This: J. Med. Chem. 2018, 61, 9756−9783

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

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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*,†,∥ †

Department of Pharmaceutical Sciences, Università del Piemonte Orientale, Novara 28100, Italy Department of Chemical, Biological, Pharmaceutical, and Enviromental Sciences, Università di Messina, Messina 98166, Italy § Department of Molecular Biophysics, Saarland University CIPMM, Homburg 66421, Germany ∥ ChemICare Srl, Enne3, Novara 28100, Italy ‡

S Supporting Information *

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 with a number of disorders, spanning from immune disorders to acute pancreatitis and have been suggested to be druggable targets. 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 1,4disubstituted 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.

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INTRODUCTION During evolution, Ca2+ ions were excluded from the cytosol to protect phosphate from precipitating.1 The very low concentration of Ca2+ in the cytosol, together with the massive gradient across membranes (Ca2+ is 105 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 second messenger. Ca2+ 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 death. A Ca2+ 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 © 2018 American Chemical Society

each cell type has a unique set of proteins that are chosen for specific tasks (the calcium toolkit, as defined by one of the fathers of modern calcium signaling).2 Given the importance of fine-tuning Ca2+ signals to generate highly specific signals, it is not surprising that a vast array of disorders is thought to have calcium dysregulation as an underlying cause. As mentioned above, high concentrations of calcium are present in intracellular organelles (in particular in the ER/SR) and in the extracellular space and Ca2+ fluxes occur through channels located on the plasma membrane or on the membrane of intracellular organelles. Given that Ca2+ pumps and Received: September 28, 2018 Published: October 22, 2018 9756

DOI: 10.1021/acs.jmedchem.8b01512 J. Med. Chem. 2018, 61, 9756−9783

Journal of Medicinal Chemistry

Article

exchangers are located on both membranes to extrude Ca2+ 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 that allows entry through the plasma membrane to refill the depleted organelles.3 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,

This simple, yet powerful, experimental approach remains valid to unmask the phenomenon in screenings. It is now thought that the principal components of SOCE are a Ca2+ sensor on the ER membrane (STIM proteins) and a plasma membrane Ca2+ channel (Orai channels).4 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 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 Capacitative calcium entry, mediating the ISOC current,6 is not solely a replenishment mechanism 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 consolidated role played by capacitative calcium entry in autoimmunity8 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 in cancer9 to acute pancreatitis.10 Last, genetic defects of STIM and Orai proteins have been described that give rise to loss- and gain-of-function genetic disorders mainly affecting the immune and skeletal systems, respectively.11 Specific pharmacological manipulation of SOCE has been challenging. For a long time, nonselective agents such as 2APB,12 CAI,13 and SKF-9636514 have been the primary

Figure 1. Store-operated calcium entry (SOCE).

emptying of the ER/SR store leads to opening of a plasma membrane channel through which Ca2+ can flow back in the cell, and these two phenomena can be dissected by adding Ca2+ to the extracellular solution after the intracellular stores depletion.

Figure 2. Chemical structures of SOCE modulators. 9757



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Scheme 1. Click Approach for the Synthesis of Pyrtriazoles

Scheme 2. Preparation of Azide 1a

a

Reagents and conditions: (a) DMF, reflux, 2 h, 96%; (b) H2, 5% Pd/C, EtOAc, rt, 2 h, 94%; (c) NaNO2, NaN3, HCl, H2O, rt, 1 h, 78%.

current depending on concentrations,24 while MDEB, another borinate compound, has only a potentiating ability on ISOC.25 We now describe a new family of compounds, that we refer to as pyrtriazoles,26 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.

experimental tools to modulate SOCE (Figure 2). Over the years, much interest has been directed at a series of 3,5bistrifluoromethyl pyrazole derivatives, disclosed by Abbott in 2000.15 Specifically, BTP2 (Pyr2, YM-58483) is a potent inhibitor, but it has pleiotropic effects on both Orai and TRPC channels.16 Subsequently, other pyrazoles, identified as Pyrs, have been reported. Among them, Pyr3, a previously suggested selective inhibitor of TRPC3,17 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 While the initial interest was mainly the discovery of experimental tools to gain insights into the role played by SOCE, drug development programs in this direction are currently underway.19 In this context, second-generation agents have been described with different characteristics in terms of mechanism of action and selectivity profile, including Synta66,20 RO2959,21 and GSK-7975A.22 Four SOCE inhibitors have entered clinical trials (CM2489 for psoriasis, CM4620 for acute pancreatitis, RP3128 for asthma, and RP4010 for nonHodgkin’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 repurposed 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

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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 of our extensive experience in click chemistry27 and its exploitation in drug discovery,28 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 (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. 9758



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Scheme 3. Preparation of Azide 2a

a

Reagents and conditions: (a) DMF, reflux, 2 h, 95%; (b) 37% aqueous HCl, EtOH, reflux, 72 h, 87%; (c) H2, 5% Pd/C, EtOAc, rt, 2 h, 95%; (d) NaNO2, NaN3, HCl, H2O, rt, 1 h, 85%.

Scheme 4. Preparation of Alkyne 3a

a

Reagents and conditions: (a) DMF, reflux, 2 h, 87%; (b) DIPEA, CuI, Pd(PPh3)2Cl2, trimethylsilylacetylene, toluene, reflux, 2 h, 79%; (c) TBAF, THF, 0 °C, 30 min, 79%.

Scheme 5. Preparation of Alkyne 4a

a Reagents and conditions: (a) conc H2SO4, EtOH, reflux, 48 h, 90%; (b) DIPEA, CuI, Pd(PPh3)2Cl2, ethynyltrimethylsilane, toluene, reflux, 2 h, 98%; (c) TBAF, THF, 0 °C, 30 min, 81%.

store-operated calcium entry in BV-2 cells, a mouse microglial cell line, using Fluo-4 as a fluorimetric dye (Table 1). In brief, a multiplate 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 readded after 600 s to the extracellular medium and intracellular levels determined in a protocol similar to the one depicted in Figure 1. The use of crude compounds was possible as copper salts (CuI and CuII) 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 23 crude compounds that showed at least 20% difference of SOCE compared to control were resynthesized, purified by column chromatography, and retested by fluorescence microscopy using the ratiometric dye Fura-2 in BV-2 cells. Twelve compounds, 10 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 R3 group, despite being absent in the previously reported Pyr compounds, is favorable for the modulation of SOCE within this new class of compounds, as demonstrated by the presence of this

To this aim, two pyrazolic azides 1 and 2 were prepared according to Schemes 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 Schemes 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 21 different alkynes A−U and, similarly, 3 and 4 were coupled with 21 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 enhances its catalytic activity and favors the cycloaddition.29 Four reactions were not successful, even when TBTA was used. Over the 84 performed reactions, 79 products precipitated, were filtered, washed with water and heptane, characterized at this stage only by mass analysis, and screened for activity on 9759



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Table 1. Data Obtained from the Preliminary Screening in BV-2 Cells at 10 μMc

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Table 1. continued

Alkyne (1 equiv), azide (1 equiv), sodium ascorbate (0.1 equiv), CuSO4·5H2O (0.01 equiv), t-BuOH, H2O. bAlkyne (1 equiv), azide (1 equiv), TBTA (0.05 equiv), Cu(OAc)2 (0.05 equiv), sodium ascorbate (0.1 equiv), THF, H2O. cThe table shows the yield of the click chemistry reactions and the percentage of residual SOCE in BV-2 cells after administration of crude compounds at 10 μM.

a

functional group in seven compounds over the 12 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 modulating activity on SOCE, while the other three series (1, 2, 3) comprise both

negative and positive modulators, depending on the substituents R1 and R2 on the pyrazole ring, the R3 group, and the relative position A or B of the triazole ring. Regarding R3, nonsubstituted phenyl ring and 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, 9761



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Table 2. Effect of Pyr3, Pyr6, and the Purified Selected 12 Compounds on Store-Operated Calcium Entry in Hek Cells at 10 μM

Characterization of the Inhibitors. We then performed concentration−response curves of 1S compared to Pyr3 in the three cell lines (see Supporting Information). The IC50 for inhibition in Hek cells for 1S was 0.6 ± 0.1 μM, in Jurkat cells 0.6 ± 0.1 μM, and in BV-2 cells 4.9 ± 1.8 μM, compared to 0.5 ± 0.1, 0.3 ± 0.1, and 2.9 ± 0.8 μM, respectively, for Pyr3. Both 1S and Pyr3 were full inhibitors as 100 μM treatment led to a near

according to the other functionalities featured by the compound. Curiously, the presence of a chlorine in the 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. 9762



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Figure 3. Effect of Pyr3, Pyr6, or 1S on store-operated calcium entry. (A) Average Ca2+-traces of SOCE in the presence or absence of Pyr3, Pyr6, or 1S in Hek cells. Traces are the average of 720 cells. (B) SOCE in wild-type Hek cells and in cells transfected with shTRPC1. The inset shows the relative abundance of the transcript as measured by RT-PCR. Traces are average of 360 cells from 12 plates on three separate experimental days. (C) RT-PCR of wild-type and Stim1−/−/Stim2−/− MEF cells. Values are mean ± SEM of five determinations. (D) SOCE in wild-type or Stim1−/−/Stim2−/− MEF cells in the presence or absence of 1S. Traces are average of 360 cells from 12 plates on three separate experimental days. (E) ISOC currents recorded in the presence or absence of 1S.

evaluating viability of cells after 24 h treatment. Viability of cells treated with Pyr3 10 μM was 28.6 ± 0.5% compared to 84.0 ± 1.0% of 1S. We also tested Pyr6, which displayed an IC50 in Hek cells for SOCE inhibition of 0.3 ± 0.1 μM and a 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 100fold the IC50 for SOCE inhibition. Given that our cellular models also expressed TRPC1, we decided to investigate in Hek cells the contribution of TRPC1 in

abolishment of SOCE. This was true also for the other nonselected 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 composition of Orai and STIM, as well as by a different Orai1/STIM1 ratio30 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 IC50s, differences were apparent when 9763



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Figure 4. Effect of 2T and 3G on store-operated calcium entry. (A) Traces of SOCE in the presence or absence of 2T or 3G. Traces are the average of 720 cells. (B) Calculated parameters of SOCE in the presence or absence of 2T or 3G. Values are the mean of 720 determinations. 2T and 3G were used at a concentration of 10 μM. * p < 0.05 as compared to control. (C) Dose−response curve of 2T or 3G applied simultaneously to Ca2+ after store depletion. Values are the mean of 180 determinations from six plates in three separate experimental days. Unlike for inhibitors, IC50s curves could not be fitted for the absence of a plateau phase. (D) ISOC currents recorded in the presence or absence of 2T or 3G.

the observed Ca2+ 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 wildtype or Stim1−/−/Stim2−/− mice.31 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 in calcium in Stim1−/−/Stim2−/− MEFs contrasts to what reported previously in these cells31 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 that 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−/− 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). 9764



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Figure 5. Effect of 2T on store-operated calcium entry in wild-type and shTRPC1 Hek cells (A,B) and in wild-type Stim1−/−/Stim2−/− MEF cells (C). (A,B) Traces of SOCE in the presence or absence of 2T. Traces are average of 360 cells from 12 plates on three separate experimental days. (C) Effect of 2T on wild-type and Stim1−/−/Stim2−/− MEF cells. Traces are average of 360 cells from 12 plates on three separate experimental days.

Characterization of the Activators 2T and 3G. 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, IC50s of these compounds could not be calculated within the applied range of concentrations. To further investigate the mechanism of action, we performed electrophysiological recordings on Hek cells stably overexpressing Orai1 and transiently expressing STIM1. Both 2T and 3G were able to significantly potentiate ISOC measured in

these cells using strong buffering conditions of the internal solution (20 mM BAPTA). Buffering the internal solution to contain 150 nM Ca2+ 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 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 that 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 9765



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Figure 6. Effect on voltage-operated channels VOCC (A), TRPV1 (B), and TRPM8 (C). (A) Histograms show the effect of the indicated compounds on the peak amplitude in primary cultures of mouse cerebellar granule cells in the presence of 50 mM KCl. Values are mean ± SEM of 300 cells from 15 plates on four separate experimental days. *** p < 0.001 as compared to KCl. (B,C) Histograms show the effect of the compounds on the peak amplitude in Hek cells overexpressing TRPV1 in the presence of capsaicin (100 nM) (B) or overexpressing TRPM8 in the presence of menthol (100 μM) (C). Values are mean ± SEM of 600 cells from 30 plates on four separate experimental days. *** p < 0.001 as compared to capsaicin (B) or menthol (C).

shown previously to be affected by SOCE inhibitors. For voltage-operated Ca2+ 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 3G did not influence voltagedependent calcium entry, while Pyr3 and Pyr6 reduced entry by about 50%. 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, and Pyr3 had no effect on the response, while Pyr6 significantly reduced menthol-mediated calcium rise. Synthesis of Analogues of Compound 1S and Characterization. 1S 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 (Tables 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

channel opening by these compounds. At 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. We also investigated whether 2T and 3G were use-dependent, i.e., required the triggered opening of the Orai channel to elicit their effects. To do this, we monitored intracellular Ca2+ in resting BV-2, Hek, or Jurkat cells in the presence of extracellular Ca2+. While 3G 10 μM did not elicit any significant calcium entry compared to control in the 800 s of observation in any of the three cell lines, 2T-treated cells displayed a slow rise of Ca2+, 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 differently as a SOCE enhancer in Orai1- and Orai3-expressing cells,32 and it will be therefore of interest to test these compounds in similar conditions. 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 voltageoperated channels, TRPV1 and TRPM8 channels that have been 9766



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Table 3. Effect of 1S Analogues on Store-Operated Calcium Entry, IC50 Values, and Cytotoxicity in Hek Cellsa

a Values at 10 μM refer to the percentage of SOCE activity obtained with purified compounds. Residual substrates are determined in mouse plasma after 30 min incubation.

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). 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 hydrolyzed 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 ester of intermediate 8 was reduced in the presence of LiAlH4, converted into azide 45, and clicked 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 hydrolyzed, 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 Tables 3 and 4. These included cell viability via the MTT assay at 10 μM and determination of the IC50 for the inhibition of SOCE for 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 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 (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 can be seen (Tables 3, 4, and Supporting Information for full data), the short linear esters (Pyr3, 1S, and 30) and the (benzyloxy)9767



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Table 4. Effect of 1S Analogues on Store-Operated Calcium Entry, IC50 Values, and Cytotoxicity in Hek Cellsa

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Table 4. continued

a Values at 10 μM refer to the percentage of SOCE activity obtained with purified compounds. Residual substrates are determined in mouse plasma after 30 min incubation.

Scheme 6. Modification of the Triazole Moiety of Compound 1Sa

a

Reagents and conditions: (a) K2CO3, benzyl bromide, dry DMF, rt, 16 h, 47%; (b) EDCI, DIPEA, DMAP, dry CH2Cl2, rt, 16 h, 84−94%; (c) H2, 5% Pd/C, MeOH, rt, 1 h, 55−72%; (d) TEA, benzyl bromide, DMF, MeOH, H2O, rt−100 °C, 16 h, 36%; (e) Cp*RuCl(cod), microwaves, DMF, 90 °C, 10 min, 39%.

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 min 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 h 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 in the presence of NADPH. Alternatively, given the presence of a carboxyl acid moiety, the formation of 9769



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Scheme 7. Modification of the Ester Moiety of Compound 1Sa

a

Reagents and conditions: (a) conc H2SO4, MeOH, or 2-propanol, reflux, 32 h, 81−90%; (b) H2, 5% Pd/C, EtOAc, rt, 2 h, 91−94%; (c) NaNO2, NaN3, HCl, H2O, rt, 3 h, 62−87%; (d) sodium ascorbate, CuSO4·5H2O, t-BuOH, H2O, rt, 16 h, 53−87%; (e) NaOH, H2O, acetone, rt, 3 h, 60%; (f) EDCI, DMAP, TEA, dry CH2Cl2, rt, 16 h, 47−68%; (g) LiAlH4, dry THF, rt, 6 h, 80%; (h) isopropylamine, EDCI, HOBt, TEA, dry CH2Cl2, rt, 16 h, 59%.

Scheme 8. Synthesis of Compounds 49, 50, and 51a

Reagents and conditions: (a) conc NH4OH, 90 °C, 3 h, 63%; (b) NaOH, H2O, acetone, 55 °C, 2 h, 78%; (c) methansulfonamide, EDCI, TEA, DMAP, dry CH2Cl2, rt, 16 h, 71%. a

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 (

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