Store-Operated Calcium Entry as a Therapeutic Target in Acute Pancreatitis: Discovery and Development of Drug-Like SOCE Inhibitors

Store-operated calcium entry (SOCE) is important in the maintenance of calcium homeostasis and alterations in this mechanism are responsible for several pathological conditions, including acute pancreatitis. Since the discovery of SOCE, many inhibitors have been identified and extensively used as chemical probes to better elucidate the role played by this cellular mechanism. Nevertheless, only a few have demonstrated drug-like properties so far. Here, we report a class of biphenyl triazoles among which stands out a lead compound, 34, that is endowed with an inhibitory activity at nanomolar concentrations, suitable pharmacokinetic properties, and in vivo efficacy in a mouse model of acute pancreatitis.

The crude product was used in the next step without further purification.
Step 2 Step 2: methyl 4-((trimethylsilyl)ethynyl)picolinate (173 mg, 0.74 mmol) was dissolved in THF (1.5 mL). The mixture was cooled at 0 °C and CH 3 COOH (50.9 µL, 0.89 mmol) and TBAF (0.89 mL, 0.89 mmol) were added. The reaction was stirred at 0 °C for 30 min. 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 6:4 and petroleum ether/ethyl acetate 5:5 as eluents to give compound 78 (111 mg, 0.69 mmol, 93%) as a brown solid. 1 H NMR (300 MHz, Biology Figure S1. Evaluation of the area under the curve (AUC), peak amplitude, and slope of the Ca 2+ -rise in in the absence or presence of the indicated compounds. Graph shows median and IQR of the AUC, peak amplitude and slope of the Ca 2+ -rise. Mann-Whitney U test of compound vs control (* p<0.0286, ** p<0.0038, *** p<0.0007, **** p<0.0001). Mann-Whitney U test. Mann-Whitney U test of compound vs Synta66 (# = 0.0485).

In vitro metabolism and pharmacokinetic.
A Thermo Scientific Q-Exactive Plus system equipped with a Thermo Scientific Vanquish UHPLC system with a binary pump VF-P10, a split sampler VF-A10, and a column compartment VH-C10 were used. Data were acquired and processed using Xcalibur ® software.
The operating conditions of the mass spectrometer were as follows: positive mode; sheath gas flow rate, 45 Auxiliary Units (A.U.), auxiliary gas flow rate, 10 A.U.; sweep gas flow rate, 2 A.U.; spray voltage, 3.50 kV; capillary temperature, 300 °C; auxiliary gas heater temperature, 300 °C. For metabolic stability assays, samples were acquired in positive full-MS and parallel reaction monitoring (PRM), using the parameters reported in Table S1 and monitoring the ions reported in the inclusion list S2. S16 Table S1. Parameters used for metabolic stability assays. Table S2. Ions monitored for metabolic stability assays.

1) LC-HRMS methods for metabolism studies.
For metabolite characterization of compound 34, samples were analysed in positive full-MS and dd-MS 2 (topN) modes using the parameters reported in Table S3. -Eluent: A: 0.1% formic acid in water.
-Analysis were performed in solvent and flow rate gradient elution (Table S4).  Table S5.

Purity of lead compounds and thermodynamic aqueous solubility.
A Shimadzu HPLC system (Shimadzu, Kyoto, Japan), consisting of two LC-10AD Vp module pumps, an SLC-10A Vp system controller, an SIL-10AD Vp autosampler, and a DGU-14-A on-line degasser were used for the analysis. The SPD-M10Avp photodiode array detector was used to detect the analytes. LC-Solution 1.24 software was used to process the chromatograms.

S19
In vivo PK evaluation of compound 34.