¹H NMR (500 MHz, methanol-D₄) (top) and ¹³C NMR (100 MHz, methanol-D₄) (bottom) spectra of 2,6-difluoro-N-(1-{[4-hydroxy-2-(trifluoromethyl)phenyl]methyl}-1H-pyrazol-3-yl)benzamide (9).

1H NMR (500 MHz, methanol-D4) (top) and 13C NMR (100 MHz, methanol-D4) (bottom) spectra of 2,6-difluoro-N-(1-{[4-hydroxy-2-(trifluoromethyl)phenyl]methyl}-1H-pyrazol-3-yl)benzamide (9).</p

¹H NMR (500 MHz, methanol-D₄) (top) and ¹³C NMR (100 MHz, methanol-D₄) (bottom) spectra of 3-trifluoromethyl-4-(hydroxymethyl)phenol (18).

1H NMR (500 MHz, methanol-D4) (top) and 13C NMR (100 MHz, methanol-D4) (bottom) spectra of 3-trifluoromethyl-4-(hydroxymethyl)phenol (18).</p

¹H NMR (400 MHz, CDCl₃) (top) and ¹³C NMR (100 MHz, CDCl₃) (bottom) spectra of 4,6-dimethoxypyridin-3-yl)boronic acid (30).

1H NMR (400 MHz, CDCl3) (top) and 13C NMR (100 MHz, CDCl3) (bottom) spectra of 4,6-dimethoxypyridin-3-yl)boronic acid (30).</p

S18 Fig -

Example fluorescence over time graph (A) and IC50 (B) for AnCoA4 following 30 minute preincubation and example fluorescence over time graph (C) and IC50 (D) for AnCoA4 following 90 minute preincubation. (TIF)</p

¹H NMR (400 MHz, DMSO-D₆) (top) and ¹³C NMR (100 MHz, DMSO-D₆) (bottom) spectra of 2,6-difluoro-<i>N</i>-(1<i>H</i>-pyrazol-3-yl)benzamide (21).

1H NMR (400 MHz, DMSO-D6) (top) and 13C NMR (100 MHz, DMSO-D6) (bottom) spectra of 2,6-difluoro-N-(1H-pyrazol-3-yl)benzamide (21).</p

Chemical structures of the small molecule SOCE inhibitors examined in this study and their common names.

Chemical structures of the small molecule SOCE inhibitors examined in this study and their common names.</p

Synthesis of GSK5503A (8) from intermediate 21.

Calcium (Ca2+) is a key second messenger in eukaryotes, with store-operated Ca2+ entry (SOCE) being the main source of Ca2+ influx into non-excitable cells. ORAI1 is a highly Ca2+-selective plasma membrane channel that encodes SOCE. It is ubiquitously expressed in mammals and has been implicated in numerous diseases, including cardiovascular disease and cancer. A number of small molecules have been identified as inhibitors of SOCE with a variety of potential therapeutic uses proposed and validated in vitro and in vivo. These encompass both nonselective Ca2+ channel inhibitors and targeted selective inhibitors of SOCE. Inhibition of SOCE can be quantified both directly and indirectly with a variety of assay setups, making an accurate comparison of the activity of different SOCE inhibitors challenging. We have used a fluorescence based Ca2+ addback assay in native HEK293 cells to generate dose-response data for many published SOCE inhibitors. We were able to directly compare potency. Most compounds were validated with only minor and expected variations in potency, but some were not. This could be due to differences in assay setup relating to the mechanism of action of the inhibitors and highlights the value of a singular approach to compare these compounds, as well as the general need for biorthogonal validation of novel bioactive compounds. The compounds observed to be the most potent against SOCE in our study were: 7-azaindole 14d (12), JPIII (17), Synta-66 (6), Pyr 3 (5), GSK5503A (8), CM4620 (14) and RO2959 (7). These represent the most promising candidates for future development of SOCE inhibitors for therapeutic use.</div

¹H NMR (500 MHz, methanol-D₄) (top) and ¹³C NMR (125 MHz, methanol-D₄) (bottom) spectra of 2,6‐difluoro‐<i>N</i>‐{1‐[(2‐phenoxyphenyl)methyl]‐1<i>H</i>‐pyrazol‐3‐yl}benzamide (8).

1H NMR (500 MHz, methanol-D4) (top) and 13C NMR (125 MHz, methanol-D4) (bottom) spectra of 2,6‐difluoro‐N‐{1‐[(2‐phenoxyphenyl)methyl]‐1H‐pyrazol‐3‐yl}benzamide (8).</p

Synthetic route to 7-azaindole (12).

Calcium (Ca2+) is a key second messenger in eukaryotes, with store-operated Ca2+ entry (SOCE) being the main source of Ca2+ influx into non-excitable cells. ORAI1 is a highly Ca2+-selective plasma membrane channel that encodes SOCE. It is ubiquitously expressed in mammals and has been implicated in numerous diseases, including cardiovascular disease and cancer. A number of small molecules have been identified as inhibitors of SOCE with a variety of potential therapeutic uses proposed and validated in vitro and in vivo. These encompass both nonselective Ca2+ channel inhibitors and targeted selective inhibitors of SOCE. Inhibition of SOCE can be quantified both directly and indirectly with a variety of assay setups, making an accurate comparison of the activity of different SOCE inhibitors challenging. We have used a fluorescence based Ca2+ addback assay in native HEK293 cells to generate dose-response data for many published SOCE inhibitors. We were able to directly compare potency. Most compounds were validated with only minor and expected variations in potency, but some were not. This could be due to differences in assay setup relating to the mechanism of action of the inhibitors and highlights the value of a singular approach to compare these compounds, as well as the general need for biorthogonal validation of novel bioactive compounds. The compounds observed to be the most potent against SOCE in our study were: 7-azaindole 14d (12), JPIII (17), Synta-66 (6), Pyr 3 (5), GSK5503A (8), CM4620 (14) and RO2959 (7). These represent the most promising candidates for future development of SOCE inhibitors for therapeutic use.</div

Raw data used to calculate IC₅₀ values.

Baseline corrected Δ340/380 ratios from three independent biological replicates (Plate A, B and C) which each included three technical replicates for each condition tested. (XLSX)</p