Natural product inspired optimization of a selective TRPV6 calcium channel inhibitor

Natural product derived analogues were surveyed, and an oxygenated analog was identified as a potent and selective TRPV6 inhibitor, with high microsomal stability and low off-target effects.


4-hydroxy-4-(3-(trifluoromethyl)phenyl)cyclohexan-1-one (37).
A dry three-neck round-bottom flask equipped with a condenser was charged with Mg (349 mg, 1.4 eq.) and dry THF (1.0 mL) under Argon atmosphere. To this suspension, a solution of 1-bromo-3-trifluormethyl-benzene (33, 1.7 mL, 1.2 eq.) in dry THF (0.4 mL) was added dropwise using an addition funnel. The reaction mixture was stirred under reflux and Argon atmosphere for 30 min. The obtained Grignard-reagent was used in situ, without purification. After cooling the previous mixture to r.t., a solution of 1,4-dioxaspiro [4.5]decan-8-one (1.6 g, 1 eq.) in dry THF (1 mL) was added dropwise and the reaction was stirred for another 30 min, under reflux and Argon atmosphere. The reaction was quenched by addition of aqueous saturated solution of NH4Cl (6 mL) and was extracted with Et2O (3 x 50 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The obtained residue column chromatographed (hexanes:EtOAc, 95:05 to 75:25) to yield the intermediate 35 used in the next step without further purification. In a round-bottom flask containing a solution of 35 (2.7 g, 1 eq.) was dissolved in a mixture of acetone and H2O (1:1, 160 mL). To this solution, PPTS (4.4 g, 2.0 eq.) was added and the mixture was stirred at 60 °C for 6 h. The organic solvent was removed under vacuum and the resulting aqueous phase was extracted with EtOAc (3 x 100 mL). The collected organic phase was dried over Na2SO4 and evaporated. The crude was column S9 chromatographed (hexanes:EtOAc, 9:1) to yield the desired ketone as a white powder (1.9 g, 71 %). 1 -1-one (38). A dry three-neck round-bottom flask equipped with a condenser was charged with Mg (342 mg, 1.4 eq.) and dry THF (1.0 mL) under Argon atmosphere. To this suspension, a solution of 1-bromo-2-trifluormethyl-benzene (34, 1.7 mL, 1.2 eq.) in dry THF (0.4 mL) was added dropwise using an addition funnel. The reaction mixture was stirred under reflux and Argon atmosphere for 30 min. The obtained Grignard-reagent was used in situ, without purification. After cooling the previous mixture to r.t., a solution of 1,4-dioxaspiro [4.5]decan-8-one (1.6 g, 1 eq.) in dry THF (1 mL) was added dropwise and the reaction was stirred for another 30 min, under reflux and Argon atmosphere. The reaction was quenched by addition of aqueous saturated solution of NH4Cl (6 mL) and was extracted with Et2O (3 x 50 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The obtained residue column chromatographed (hexanes:EtOAc, 95:05 to 75:25) to yield the intermediate 36 used in the next step without further purification. In a round-bottom flask containing a solution of 36 (471 mg, 1 eq.) was dissolved in a mixture of acetone and H2O (1:1, 20 mL). To this solution, PPTS (1 g, 2.0 eq.) was added and the mixture was stirred at 60 °C for 6 h. The organic solvent was removed under vacuum and the resulting aqueous phase was extracted with EtOAc (3 x 50 mL). The collected organic phase was dried over Na2SO4 and evaporated. The crude was column chromatographed (hexanes:EtOAc, 9:1) to yield the desired ketone as a white powder (292 mg, 38 %). 1 (39, 3OG). In a round-bottom flask containing a solution of 37 (1.9 g, 1 eq.) and benzylpiperazine (1.5 mL, 1.2 eq.) in dry DCE (72 mL), was added AcOH (400 µL,). After 15 min, NaBH(OAc)3 (2.1 g, 1.4 eq.) was added and the mixture was stirred for 24 h at r.t. The solvent was removed under vacuum and the crude was purified by column chromatography (hexanes:EtOAc, 7:3 + 0.5 % Et3N). The obtained intermediate was dissolved in Et2O and was precipitated by the addition of a methanol solution of HCl (3.0 M). The collected solids were washed with Et2O and hexanes and were used in the next step without further purification. The previously obtained compound was dissolved in a mixture of methanol (65 mL) and AcOH (210 µL, 2 eq.). To this stirring solution, Pd/C (55 mg, 0.3 eq.) was added and the reactional mixture was stirred for 16 h, at r.t., under H2 atmosphere. The reaction mixture was filtered through a pad of Celite to remove the catalyst (washed with MeOH, 3 x 100 mL). The desired compound was obtained as white powder (540 mg, 18 %). 1

TRPV6 FLIPR assay
Calcium-5 was bought from Molecular Devices LLC. All other chemicals were purchased from Sigma-Aldrich.
hTRPV6 activity was measured using the HEK293 cell line stably overexpressing human TRPV6 as previously reported. 1,4 Stable cells were trypsinized and plated at 7.5 x 10 4 cells/well onto poly-D-lysine coated 96-well black plates with clear bottom using 100 μl DMEM supplemented with 10% FBS and 2 mM glutamine without antibiotics or phenol-red. After 16 h the medium was replaced with 90 μL of nominally calcium-free (NCF) loading buffer (modified Krebs buffer containing 117 mM NaCl, 4.8 mM KCl, 1 mM MgCl 2, 5mM D-glucose, 10 mM HEPES, and calcium-5 fluorescence dye (50 μL/mL loading buffer)). Cells were incubated in the NCF-loading buffer at 37 °C for 1 h. Fluorescence Cd 2+ measurements were carried out using FLIPR TETRA high throughput (Molecular Devices, LLC), fluorescence microplate reader. Cells were excited using a 470-495 nm LED module, and the emitted fluorescence signal was filtered with a 515-575 nm emission filter (manufacturer's guidelines). Stable Ca 2+ -free baselines were established for 50 seconds before 10 μL of a 10X compound was added to the cells. Cells were incubated at 37 °C and fluorescence was monitored in the presence of compound for an additional 5 minutes before administration of 100 μL of CdCl2 (final concentration: 50 μM). The activity of TPRV6 was measured by calculating the area under the curve of the Cd 2+ entry traces.
Screening experiments were done with 3 to 6 repeats per group at 10 µM (6-9, 11-14, 16-17, 19-20, 24-26, 30-32, and 39-40) or 50 µM (41-46). Fluorescence signals were analyzed using the ScreenWorks 3.1.1.8 software (Molecular Devices). Dose-response curves were generated (9-point curve, 6 repeats/concentration, 2-fold serial dilution starting at 10 µM), and the IC50 values were extrapolated from these plots for each compound (GraphPad ® Prism, v. 5.0, San Diego, CA, US). Inhibition curves were obtained by non-linear regression using the built-in log(inhibitor) vs. response-variable slope function (four parameters). S61 7. TRPV1 FLIPR assay hTRPV1 activity was measured using HEK293T cells transiently overexpressing TRPV1 as previously reported. 6 Briefly, HEK293 cell were trypsinized and plated at 1.5 x104 cells/well onto Corning® 96-well black polystyrene clear bottom microplates (CLS3603 Sigma-Aldrich) coated with 100 μg/mL poly-D-lysine (P6407 Sigma-Aldrich) using 100 μL phenol-red free DMEM with 10% FBS and 2mM glutamine without antibiotics. Cells were incubated at 37 °C for 24 h. On the following day, transfection was performed using 200 ng of pcDNA 3.1 hTRPV1 and 0.6 μL Lipofectamine 2000 reagent/well. 24 h after the transfection, the medium was replaced with 90 µL of loading buffer (modified Krebs buffer containing 117 mM NaCl, 4.8 mM KCl, 1 mM MgCl2, 5 mM D-glucose, 10 mM HEPES, 1.8 mM CaCl2 and calcium-5 fluorescence dye (50 µL/mL loading buffer)). Cells were incubated in the loading buffer at 37 °C for 1 h in dark. Fluorescence Ca 2+ measurements were carried out using FLIPR TETRA high-throughput, fluorescence microplate reader as described before. Stable baselines were established for 50 s before 10 µL of a 10X solution of compound 39 or the TRPV1-inhibitor capsazepine (CPZ) prepared in 1.8 mM CaCl2-containing Krebs buffer was robotically administered to the cells. Cells were incubated and fluorescence was monitored in the presence of compound for an additional 5 min before administration of the agonist in 1.8 mM CaCl2containing Krebs buffer (final concentration of capsaicin in the assay plate was 100 nM). The activity of TRPV1 was measured by quantifying the area under the curve (AUC) of the fluorescence intensity, following administration of the agonist.
The current HEK-hTRPV1 method employs a constant concentration of Ca 2+ (1.8 mM) in the assay buffer during the whole experiment. 6 We observed that addition of compound 39 at 10.0 µM did not activated the channel, as the recorded baseline remained comparable to DMSO treatment. Upon addition of capsaicin (100 nM) the channel was activated and a high influx of Ca 2+ was immediately recorded. The activation of the TRPV1 channel is supposed to occur through displacement of lipids in the transmembrane domain. This allosteric effect was investigated in depth through functional assays, by cocrystallization/cryo-EM methods, molecular dynamics simulations and mutagenesis. [7][8][9] The Omethylcathecol head and the amide linker of capsaicin are the pharmacophoric features responsible for its binding to TRPV1. The long hydrophobic tail mediates Van der Waals interactions with apolar residues, resembling the interactions found for the phospholipids. 10 We believe that the replacement of the Omethylcathecol to pyridine/pyridone and the amide to piperazine in compounds 1 and 39 explains their lack of activity on TRPV1.

SOCE FLIPR assay
MDA-MB-231 cells were trypsinized and plated at 6 x 10 4 cells/well onto 96-well black plates with clear bottom using 100 μl phenol-red free RPMI medium supplemented with 10% FBS. SOCE activity was measured 16 h later using the previously described FLIPR assay. 11 After 16 h the medium was replaced with 50 μL of NCF loading buffer and the cells were incubated at 37 °C for 40 min, following which 50 µL of 2X drugs were manually applied (GSK-7975A, 1 or 39) and the cells were incubated for another 20 min. The SOCE inhibitor GSK-7975A (cat. no. AOB4124-1) was purchased from Aobious, Gloucester, MA, USA. Stable Ca 2+ -free baselines were established for 50 seconds before 50 μL of a 3X thapsigargin (Tg) was robotically administered to the cells. Cells were incubated at 37 °C and fluorescence was monitored in the presence of Tg for an additional 10 minutes before administration of 50 μL of 4X CaCl2. The activity of SOCE was measured by calculating the area under the curve of the Ca 2+ entry traces.

Confocal microscopy
HEK-hTRPV6 were trypsinized and plated at 1 x 104 cells/well onto poly-D-lysine coated Nunc Lab-Tek II 8-well chambered coverglass plates (Faust Laborbedarf AG, Schaffhausen) using 100 μl DMEM supplemented with 10% FBS and 2 mM glutamine without antibiotics or phenol-red. After 24 h of incubation at 37 °C, the medium was replaced by 200 µL/well of a mixture of Leadmiun Green (final concentration: 5 ng/µL), Hoechst 33258 (final concentration: 1 ng/µL) and wheat germ agglutinin Alexa Fluor® 594 conjugate (final concentration: 5 ng/µL) in NCF buffer. The chamber was covered with aluminum foil and was incubated at 37 °C for 30 min. Then, the buffer was removed, cells were washed twice with fresh NCF buffer (2 x 200 µL) and another 190 µL of NCF containing 10X of 39 was added. For control cells, 200 µL of NCF was added after washing. The chamber was mounted in the confocal microscope and 10 µL of CdCl2 (final concentration: 50 μM) was added right after the start of imaging (total duration: 30 min).
The cells were imaged at 100X lenses with a confocal, laser scanning microscope setup using a Nikon Eclipse TE2000-E fully automatized inverted, epifluorescence microscope outfitted with Nikon D-Eclipse C1 laser confocal optics. The system equipped with a violet-diode (405 nm) and a multiline Argon (457-515 nm) from Melles Griot, and a Helium/Neon (594 nm) lasers from JDS Uniphase. Nikon EZ-C1 3.6 confocal imaging software installed on an HP xw4400 workstation was used for image acquisition. Brightness and contrast were adjusted with ImageJ. Fig S3. Time-course of Cd 2+ uptake in HEK-hTRPV6 cells with Leadmiun Green (LG), wheat germ agglutinin Alexa Fluor® 594 conjugate (AF), and Hoechst 33258 (H) along 30 min. Images were collected using confocal microscopy (Nikon Eclipse TE2000-E, 100X). HEK-hTRPV6 cells were incubated with fluorescent dyes for 30 min at 37 °C. To these cells DMSO (A) or 39 (10 µM, C) was applied followed by a solution of Cd 2+ (50 µM). The total fluorescence intensity for each channel was plotted in graphs (B) and (D), respectively for DMSO and compound 39. Images were collected with excitation for LG at λex = 488 nm and emission at λem = 520 nm, H at λex = 352 nm and emission at λem = 461 nm, and AF at λex = 590 nm and emission at λem = 617 nm. White bars denote 20 µm.
The XTT assay was used to evaluate cell proliferation. Cells were plated at a density of 5 x 103 cells/well in 96-well plates and were incubated at 37 °C for 24 h. On the following day, the medium was carefully aspirated and replaced with 100 µL of a 39 or 1 at concentrations ranging from 100 µM to 0.4 μM. Doxorubicin (10 µM) and DMSO (0.01 %) were used as positive and negative controls, respectively. All the treatments used the original cell medium (RPMI or DMEM), depending on the cell line. Cells were incubated for a total of 6 days at 37 °C with the medium (treatment and controls) being replaced by a freshly prepared solution every 48 h. Then, 25 μL of XTT (with 1.25 % of PMS) was added to each well and incubated at 37 °C for 2 h. Subsequently, the absorbance was read at 650 nm and subtracted from the absorbance of 450 nm by spectrophotometry (Vmax Kinetic Microplate Reader, Molecular Devices LLC). The resulting subtracted absorbance for each compound concentration (At) was expressed as a percentage of viable cells relative to the negative control (Ab), and the percentage of viable cells was plotted and the inhibition curves obtained by non-linear regression using the built-in log(inhibitor) vs. response-variable slope function (GraphPad® Prism, v. 5.0, San Diego, CA, US).