Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels

The concentration of free cytosolic Ca2+ and the voltage across the plasma membrane are major determinants of cell function. Ca2+-permeable non-selective cationic channels are known to regulate these parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca2+-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145.

After 1h, the mixture was diluted with EtOAc (70 mL) and washed with brine (50 mL). The layers were separated and the organic slurry was filtered and the cake was washed with icecold ethanol leading to 3 (1.61 g, 65 %) as a white solid. Analytical data were in agreement with data reported in patent WO 2014143799.
Compound 5: To a solution of compound 3 (500 mg, 1.35 mmol, 1 equiv.) in DMF (6 mL) in a 10 mL conical flask was added 3-(trifluoromethoxy)phenol 4 (262 μL, 361 mg, 2.03 mmol, 1.5 equiv.) followed by K2CO3 (802 mg,5.81 mmol,4.3 equiv.). The flask was sealed and the resulting mixture was heated at 80 °C overnight (the flask was entirely immersed in the heating bath). The mixture was then partitioned between water (50 mL) and EtOAc (80 mL). The aqueous phase was extracted with EtOAc (2 x 80 mL) and the combined organic layers were dried (Na2SO4) and concentrated in vacuo. The resulting white solid was washed with ethanol in a sinter leading to 5 (360 mg, 57 %) as a white solid. Analytical data were in agreement with data reported in patent WO 2014143799.
Compound 7: To a solution of compound 5 (300 mg, 0.643 mmol, 1 equiv.) in DMF (3.75 mL) in a 10 mL conical flask was added 2-(3-bromopropoxy)tetrahydro-2H-pyran 6 (215 mg, 0.964 mmol, 1.5 equiv.) followed by K2CO3 (267 mg, 1.929 mmol, 3 equiv.). The flask was sealed and the resulting mixture was heated at 80 °C overnight (the flask was entirely immersed in the heating bath). The mixture was then partitioned between water (20 mL) and EtOAc (20 mL). The aqueous phase was extracted with EtOAc (2x20 mL) and the combined organic layers were dried (Na2SO4) and concentrated in vacuo leading to compound 7 as a white solid, which was immediately used in the next reaction.
Compound 8 (C31): All of compound 7 was dissolved in EtOH (5.0 mL) in a 100 mL round bottom flask and concentrated HCl (1 mL) was added. The mixture was stirred overnight at 80 °C in a sealed flask. The reaction was quenched with saturated aqueous NaHCO3 (20 mL) and the aqueous phase was extracted with EtOAc (3x20 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The residue was purified using flash chromatography (pentane/EtOAc, 1:1) to give 8 (C31) (256 mg, 76 % over 2 steps). Analytical data were in agreement with data reported in patent WO 2014143799 (see below).
Compound 10: To a solution of compound 9 (800 mg, 2.14 mmol) in DMF (10 mL) was added Cs2CO3 (2.1 g, 6.42 mmol) and 3-(trifluoromethoxy)phenol 4 (0.27 mL, 2.1 mmol). The mixture was heated at 80 o C overnight. The mixture was cooled and aqueous LiCl solution (10%, 1 mL) was added and the mixture was partitioned between EtOAc (10) and water (10 mL). The organic layer was washed with brine (10 mL), dried (MgSO4), filtered and evaporated to dryness. The crude product was triturated with EtOH to afford white solid product 10 which was used without further purification (760 mg, 76%). Analytical data were in agreement with data reported in patent WO 2014143799.
Compound 11: To a solution of compound 10 (660 mg, 1.4 mmol) in DMF (5 mL) was added 2-(3-bromopropoxy)tetrahydro-2H-pyran 6 (370 mg, 1.9 mmol) and Cs2CO3 (1.37 g, 4.2 mmol).The reaction was heated at 50 o C for 3 hours. The mixture was cooled, partitioned between EtOAc (10 mL) and water (10 mL). The organic layer was dried (MgSO4), filtered and concentrated to give a crude product which was purified by flash column chromatography by eluting with 0-100% EtOAc in Pet. Ether. The product fractions were combined and the solvent was evaporated to afford 11 as a white solid (820 mg, 95%). Analytical data were in agreement with data reported in patent WO 2014143799.
Compound 12: To a solution of compound 11 (800 mg, 1.3 mmol) in ethanol (10 mL) was added 37% concentrated HCl (1.8 mL) and the reaction was refluxed for 24 hours until all starting material was converted to product. Upon completion, the reaction mixture was concentrated in vacuo to give a crude product, which was purified using flash column chromatography by eluting with 0-100% EtOAc in Pet.Ether. The product fractions were combined and the solvent was evaporated to afford 12 as a white solid (300 mg, 58%).
Analytical data were in agreement with data reported in patent WO 2014143799.
Compound 15: Compound 14 (4.15 g, 8.08 mmol) was suspended in EtOH (70 mL) and concentrated aqueous HCl (10 mL) was added. The mixture was heated at reflux for 1.5 h (all solid dissolves). After cooling, the reaction mixture was concentrated to dryness in vacuo. The crude product was purified by flash column chromatography using 0-40% EtOAc in DCM, affording 15 as a white solid (2.45 g, 71%). Analytical data were in agreement with data reported in patent WO 2014143799.

Comparison of three different batches of C31
1 H NMR showed that all three batches of C31 were analytically pure (Fig S2-S4).
Stock solutions of the three different batches of C31 (10 mM in DMSO) were prepared by two different investigators. The concentrations of these DMSO stock solutions were compared by ESI-LC-MS. For this, DMSO stock solutions were diluted to 1 mM with DMSO. All three batches were then analysed by calcium recording in HEK293 cells conditionally expressing TRPC4-C1 concatemeric channels. Data are shown below. All three batches of C31 fully inhibited (-)-Englerin A-activated TRPC4-TRPC1 concatemeric channels at a concentration of 1 nM.