TRPC3 (IC50 = 0.49 uM)

Pyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca2+ entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca2+ entry as compared with TRPC3-mediated Ca2+ entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation[1].
The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca2+ entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca2+ channels[1].

For characterization of Pyr6 and Pyr10 selectivity for TRPC channels, n-terminally GFP-tagged murine TRPC6, n-terminally YFP-tagged TRPC5 (Schindl et al., 2008) and c-terminally YFP-tagged TRPC4beta (Graziani et al., 2010) were used.

Electrophysiology[1] Patch pipettes were pulled from borosilicate glass capillaries (resistance 3–5 MΩ). Currents were recorded at room temperature using a List EPC7 patch-clamp amplifier. Signals were low-pass filtered at 3 and 10 kHz and digitized with 5 kHz. For HEK293 cells, voltage-clamp protocols (voltage ramps from −130 to +80 mV, holding potential 0 mV) were controlled by pClamp software. Extracellular solution (ECS) contained (in mM) 140 NaCl, 2 CaCl2, 2 MgCl2, 10 glucose, pH adjusted to 7.4 with NaOH. The pipette solution (ICS) contained (in mM) 120 caesium methanesulphonate, 20 CsCl, 15 HEPES, 5 MgCl2, 3 EGTA, pH adjusted to 7.3 with CsOH. To activate the TRPC3 channel, current cells were challenged with 100 μM carbachol or 100 μM 1-oleoyl-2-acetyl-sn-glycerol (OAG). For RBL-2H3 cells CRAC measurement, standard protocols and buffers were modified from Derler et al. (2009). In brief voltage ramps from −90 to +90 mV over 1 s (holding potential +30 mV) were applied and controlled by pClamp software. ECS contained (in mM) 130 NaCl, 5 CsCl, 1 MgCl2, 10 HEPES, 10 glucose, 20 CaCl2 at pH 7.4. ICS was comprised of 3.5 MgCl2, 145 caesium methanesulphonate, 8 NaCl, 10 HEPES, 20 EGTA at pH 7.2. Experiments in HEK-293 cells expressing STIM1 and Orai1 to reconstitute the CRAC pore were done as in Muik et al. (2008). ECS contained (in mM) 145 NaCl, 5 CsCl, 1 MgCl2, 10 HEPES, 10 Glucose, 10 CaCl2 at pH 7.4. ICS was comprised of 3.5 MgCl2, 145 caesium methanesulphonate, 8 NaCl, 10 HEPES, 20 EGTA at pH 7.2. For measuring the sodium currents in divalent-free conditions, protocols and buffers from Bergsmann et al. (2011) were used. If not mentioned otherwise for the experiments, cells were pre-incubated for 3 min and measured in the presence of either 3 μM Pyr2, Pyr3, Pyr6 or Pyr10.

[1]. Novel pyrazole compounds for pharmacological discrimination between receptor-operated and store-operated Ca(2+) entry pathways. Br J Pharmacol. 2012 Dec;167(8):1712-22.

The observed selectivity of Pyr6 and Pyr10 suggests that these compounds may be helpful in identifying and analyzing TRPC and Orai-mediated conductance in native tissues. Our results in RBL-2H3 mast cells and STIM1/Orai1-expressing HEK293 cells are consistent with the concept that in both cell systems, storage-operated Ca2+ influx occurs through the same channels characterized by significantly higher sensitivity to Pyr6 than to Pyr10. In contrast, the TRPC3 homologous pore structure is highly sensitive to Pyr10 but less sensitive to Pyr6. Importantly, the inhibitory effects of Pyr6 and Pyr10 on homologous channels of other TRPC isoforms (such as TRPC4, 5, and 6) were tested, showing low inhibitory potency (IC50 > 10 μM) on these channels, indicating significant TRPC isoform selectivity for Pyr10 (see Supplementary Infographic S3). [1] We considered the sensitivity of Pyr6 and Pyr10 to pyrazole to be characteristic of Orai1-mediated Ca2+ influx. Since RBL-2H3 cells express TRPC genes, including TRPC3 (Ma et al., 2008), our results may indicate that TRPC3 is not involved in storage-manipulated Ca2+ influx in mast cells. However, we cannot rule out the role of TRPC3-containing channel complexes in local Ca2+ signaling events. These local Ca2+ signaling events are not detectable by changes in global cellular Ca2+ but may be crucial to certain downstream signaling pathways, as recently reported with cardiac TRPC3 (Poteser et al., 2011). We found that NFAT translocations are highly sensitive to Pyr6 but insensitive to Pyr10, which is consistent with the recent view of Kar et al. (2011) that NFAT is mainly regulated in RBL-2H3 cells through the CRAC/Orai Ca2+ influx pathway. It is noteworthy that pyrazole compounds were initially considered effective inhibitors of NFAT signaling, although their mechanism of action remains unclear, as they are located downstream of Ca2+ signaling (Djuric et al., 2000; Trevillyan et al., 2001). This article reports that the inhibitory effects of Pyr6 and Pyr10 are closely related to their efficacy as CRAC/Orai1 inhibitors. Pyr6 showed a stronger inhibitory effect on mast cell degranulation than Pyr10, an observation that confirms the Orai channel is the main source of Ca2+ in RBL-2H3 cell exocytosis (Ma et al., 2008). Consistent with previous reports, Pyr6 has been found to effectively inhibit transcriptional activation and cytokine production in immune cells (Ishikawa et al., 2003; Birsan et al., 2004; Shirakawa et al., 2010; Law et al., 2011), highlighting the potential value of this chemical structure in the development of highly effective immunomodulators (Chen et al., 2002; Zitt et al., 2004) [1].

C17H9F7N4O

418.27

418.066

C, 48.82; H, 2.17; F, 31.79; N, 13.40; O, 3.82

245747-08-4

10598093

Light yellow to yellow solid powder

1.5±0.1 g/cm3

368.4±42.0 °C at 760 mmHg

176.6±27.9 °C

0.0±0.8 mmHg at 25°C

1.551

3.1

1

10

3

29

577

0

XZIQSOZOLJJMFN-UHFFFAOYSA-N

InChI=1S/C17H9F7N4O/c18-12-8-25-6-5-11(12)15(29)26-9-1-3-10(4-2-9)28-14(17(22,23)24)7-13(27-28)16(19,20)21/h1-8H,(H,26,29)

N-[4-[3,5-bis(trifluoromethyl)pyrazol-1-yl]phenyl]-3-fluoropyridine-4-carboxamide

Pyr 6; Pyr-6; N-(4-(3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-3-fluoroisonicotinamide; CHEMBL101896; N-[4-[3,5-bis(trifluoromethyl)pyrazol-1-yl]phenyl]-3-fluoropyridine-4-carboxamide; N-{4-[3,5-bis(trifluoromethyl)-1h-pyrazol-1-yl]phenyl}-3-fluoroisonicotinamide; N-{4-[3,5-bis(trifluoromethyl)pyrazol-1-yl]phenyl}-3-fluoropyridine-4-carboxamide; XZIQSOZOLJJMFN-UHFFFAOYSA-N; Pyr6

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~239.08 mM)

Solubility in Formulation 1: ≥ 10 mg/mL (23.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 100.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 10 mg/mL (23.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 100.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 10 mg/mL (23.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 100.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)