TRPML3 Agonist Assays (AID 1448, AID 1526, AID 1562, AID 2510, AID 602128, and AID 602129)

The purpose of these assays is to identify test compounds that act as agonists of the TRPML3 cation channel. This assay employs a HEK293 cell line that stably expresses the human TRPML3-YFP cation channel. The cells are treated with test compounds followed by measurement of intracellular calcium as monitored by a fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as TRPML3 agonists will increase calcium mobilization, resulting in increased relative fluorescence of the indicator dye, and thus increase well fluorescence. Compounds were tested in singlicate (AID 1448) or triplicate (AID 1526) at a nominal dose of 3 μM, and in a 10-point, 1:3 dilution series starting at a nominal concentration of 30 μM (AID 1562, AID 2510, AID 602128, and AID 602129).

The TRPML3 HEK293 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Minimum Essential Medium with GlutaMAX and supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 800 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay 1500 cells in 3 μl of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95 % RH for 23 hours. Next, 2 μl of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer’s protocol) was added to each well. After a 1 hour incubation at 37 degrees C, 5% CO2, and 95 % RH followed by a 30 minute incubation at room temperature, the assay was started by performing a basal read of plate fluorescence (470–495 nm excitation and 515–575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of test compound (3 μM final nominal concentration) in DMSO, DMSO alone (0.3% final concentration), or the cholinergic agonist carbachol (87 μM final concentration) in DMSO were dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 120 seconds of the assay. A ratio for each well was calculated to normalize assay data, according to the following mathematical expression: Ratio = I_Max / I_Min. Where I_Max represents the maximum measured fluorescence emission intensity over the 125 second read and I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added. The percent activation was calculated from the median ratio as follows:

Where:

Test_Compound is defined as wells containing test compound.

High_Control is defined as wells with carbachol.

Low_Control is defined as wells with DMSO.

TRPN1 Counterscreen Assays (AID 1424, AID 1525, AID 1682, and AID 2583)

The purpose of this assay is to identify test compounds that act as agonists of the TRPN1 cation channel. This assay serves as a counterscreen to determine whether compounds are non-selective TRP agonists. This assay employs a HEK293 cell line that stably expresses the zebrafish TRPN1-YFP cation channel. The cells are treated with test compounds followed by measurement of intracellular calcium as monitored by a fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as TRPN1 agonists will increase calcium mobilization, resulting in increased relative fluorescence of the indicator dye, and thus increase well fluorescence. Compounds were tested in singlicate (AID 1424) or triplicate (AID 1525) at a nominal dose of 3 μM, and in a 10-point, 1:3 dilution series starting at a nominal concentration of 30 μM (AID 1682, and AID 2583).

The TRPN1 HEK293 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Minimum Essential Medium with GlutaMAX and supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 800 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay 1500 cells in 3 μl of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95 % RH for 23 hours. Next, 2 μl of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer’s protocol) was added to each well. After a 1 hour incubation at 37 degrees C, 5% CO2, and 95 % RH followed by a 30 minute incubation at room temperature, the assay was started by performing a basal read of plate fluorescence (470–495 nm excitation and 515–575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of test compound (3 μM final nominal concentration) in DMSO, DMSO alone (0.3% final concentration), or the cholinergic agonist carbachol (87 μM final concentration) in DMSO were dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 120 seconds of the assay. A ratio for each well was calculated to normalize assay data, according to the following mathematical expression: Ratio = I_Max / I_Min. Where I_Max represents the maximum measured fluorescence emission intensity over the 125 second read and I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added. The percent activation was calculated from the median ratio as follows:

Where:

Test_Compound is defined as wells containing test compound.

High_Control is defined as wells with carbachol.

Low_Control is defined as wells with DMSO.

Patch Clamp Assays (AID 2116, AID 2694, and AID 2692)

The purpose of these assays is to determine if test compounds can increase current recordings in TRPML3 ion channels (AID 2116 and AID 2694) or TRPN1 (AID 2692), along with controls employing the YFP-HEK parental background. Whole-cell currents were recorded with an Alembic Instruments VE-2 amplifier with 100% series resistance compensation, and acquired with JClamp software. The standard bath solution contained (in mM) 138 NaCl, 5.4 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 10 d-glucose, adjusted to pH 7.4 with NaOH. The standard pipette solution contained (in mM) 140 CsCl, 10 HEPES, 3 ATP-Na, 1 BAPTA, and 2 MgCl2, adjusted to pH 7.2. 100 μM 2-Aminoethyl-diphenyl borate was included in the bath solution to block gap junctions and had no effect on the expressed channels. Channel responses were plotted to 10 ms voltage steps (holding potential = +10 mV) between -200 mV and +100 mV in 20 mV incremental steps, normalized by cell capacitance (pF). Compounds were tested at 10 micromolar.

Fura-2 Calcium Influx Assays (AID 2116, AID 2719, and AID 2770)

The purpose of these assays is to determine whether compounds identified as probe candidates are able to increase whole cell Ca2+ influx in HEK293 cells transfected with human TRPML3, other human, or murine (m) TRP channels, or zebrafish TRPN1. In this assay cells transiently expressing channels or YFP control plasmid are perfused with test compound, followed by measurement of intracellular [Ca2+] for 2 minutes with the fluorescent indicator fura-2-AM. Compounds are added to cells 20–25 hours after transfection. Values are reported as mean values +/− SEM (n >= 3 independent experiments with 20–30 cells). The % activation values for TRPML2 in the SAR tables were calculated by normalizing the TRPML2 response ratios to TRPML3 response ratios. Compounds were tested at 10 micromolar.

Synthesis of 1

1-Naphthol (577 mg, 4 mmol, 1 equiv.) and potassium carbonate (1 g, 2 mmol) were ground together in a in a 10-mL round-bottomed flask until a fine powder was obtained. This mixture was then heated at 60 °C, and then diethyl sulfate (524 μL, 1 equiv.) was added under vigorous stirring. The mixture was heated at 60 °C until the reaction was complete as judged by TLC analysis. Water (10 mL) was added to the mixture and the aqueous layer was extracted three times with diethyl ether (10 mL). The combined organic layers were washed with 1N HCl, water and brine, and then dried over sodium sulfate. Solvent was removed under reduce pressure and compound 1 was obtained as a colorless oil (535 mg, 78 %) after purification by flash chromatography (silica gel, 100 % hexanes, R_f = 0.32).

¹H NMR (400 MHz, CDCl₃) δ 8.40-8.36 (1H, m), 7.87-7.84 (1H, m), 7.57-7.51 (2H, m), 7.49-7.40 (2H, m), 6.84 (1H, dd, J = 0.8 Hz, J = 7.6 Hz), 4.25 (2H, q, J = 7.1 Hz), 1.60 (3H, t, J = 7.0 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 154.7, 134.5, 127.4, 126.3, 125.9, 125.7, 125.0, 122.1, 120.0, 104.6, 63.6, 14.8.

Synthesis of 2

A solution of 1 (535 mg, 3.11 mmol, 1 equiv.) in chloroform (3.1 mL) in a 10-mL round-bottomed flask was cooled to −5 °C under an inert atmosphere. Chlorosulfonic acid (410 μL, 2 equiv.) was added dropwise to this solution. The stirred mixture was allowed to warm to room temperature and stirred for an additional 20 min. The mixture was then poured onto ice (13 g); chloroform (20 mL) was added along with a small amount of brine (5 mL). The heterogeneous solution was filtered through a short pad of Celite, the layers were separated and the organic layer was washed twice with water and once with brine. The organic phase was dried over sodium sulfate, filtered, and solvent was removed under reduce pressure to yield compound 2 as an off-white solid (511 mg, 61 %). This material was used in the next step without purification.

¹H NMR (400 MHz, CDCl₃) δ 8.71 (1H, ddd, J = 0.8 Hz, J = 0.8 Hz, J = 8.6 Hz), 8.41 (1H, ddd, J = 0.6 Hz, J = 1.2 Hz, J = 8.6 Hz), 8.28 (1H, d, J = 8.6 Hz), 7.78 (1H, ddd, J = 1.3 Hz, J = 6.9 Hz, J = 8.4 Hz), 7.62 (1H, ddd, J = 1.1 Hz, J = 6.9 Hz, J = 8.3 Hz), 6.77 (1H, d, J = 8.6 Hz), 4.28 (2H, q, J = 7.0 Hz), 1.59 (3H, t, J = 7.0 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 161.3, 132.0, 130.8, 129.7, 128.7, 126.7, 126.0, 123.7, 123.3, 102.2, 64.9, 14.4.

Synthesis of 3 (Probe 1, CID776924)

To a room temperature solution of sulfonyl chloride 2 (510 mg, 1.88 mmol, 1 equiv.) in dichloromethane (20 mL) were added triethylamine (525 μL, 2 equiv.) and azepane (318 μL, 1.5 equiv.). This mixture was stirred for 2 h. The mixture was poured into water (20 mL) and aqueous layer was extracted three times with dichloromethane. The combined organic layers were washed with 1N HCl, water and brine, and then dried over sodium sulfate. The mixture was filtered, solvent was removed under reduce pressure and compound 3 (493 mg, 79 %, white solid— mp = 103–104 °C) was obtained after purification by flash chromatography (silica gel, ethyl acetate-hexanes 20/80, R_f = 0.66).

Compound 3 (Probe 1, CID 776924): ¹H NMR (400 MHz, CDCl₃) δ 8.60 (1H, ddd, J = 0.9 Hz, J = 0.9 Hz, J = 8.5 Hz), 8.37 (1H, ddd, J = 0.8 Hz, J = 1.2 Hz, J = 8.4 Hz), 8.12 (1H, d, J = 8.3 Hz), 7.63 (1H, ddd, J = 1.4 Hz, J = 6.9 Hz, J = 8.4 Hz), 7.54 (1H, ddd, J = 1.1 Hz, J = 6.9 Hz, J = 8.3 Hz), 6.77 (1H, d, J = 8.4 Hz), 4.25 (2H, q, J = 7.0 Hz), 3.36 (4H, dd, J = 5.8 Hz, J = 6.0 Hz), 1.68-1.64 (4H, m), 1.59-1.55 (7H, m). ¹³C NMR (100 MHz, CDCl₃) δ 158.7, 131.5, 129.9, 128.1, 126.2, 126.1, 125.8, 124.9, 122.8, 102.3, 64.2, 47.7, 29.1, 26.9, 14.6. HRMS ([M+Na]⁺) expected: 356.1291, observed: 356.1307. IR (cm⁻¹): 2932, 2859, 1590, 1574, 1509, 1376, 1314, 1148, 1229, 1086, 1041, 961, 775, 709.

Synthesis of 4

3-(4-Chlorophenyl)-5-methylisoxazole-4-carboxaldehyde (1 g, 4.52 mmol, 1 equiv, purchased from Sigma-Aldrich, # 703281-1G) was dissolved in freshly distilled tetrahydrofuran (45 mL) under inert atmosphere in a flame-dried flask. The solution was cooled to −15 °C and then methylmagnesium bromide (3 M in diethyl ether, 2.26 mL, 1.5 equiv.) was slowly added. The mixture was allowed to warm up room temperature and was stirred for 1 h. The mixture was then poured into ice-water and 1N HCl was slowly added until the pH = 1. The aqueous layer was extracted with diethyl ether (3 × 100 mL) and the combined organic layers were washed with water (100 mL), brine (100 mL) and then dried over sodium sulfate. The solution was filtered, solvent was removed under reduced pressure to yield the corresponding secondary alcohol as a colorless oil (1.07 g, quant.) which was used in the following step without purification.

Chromic acid (904 mg, 9.04 mmol, 2 equiv.) and acetic anhydride (50 mL) were mixed with stirring in a 100-mL round-bottomed flask until homogeneous. A solution of secondary alcohol from the previous step (1.07 g, 4.52 mmol, 1 equiv.) in acetic anhydride (20 mL) was then added dropwise via syringe pump (10 min). The resulting mixture was stirred for 30 min, then was extracted with diethyl ether (4×50 mL). The combined organic layers were washed several times with saturated aqueous ammonium chloride solution. The organic layer was dried over sodium sulfate, filtered and then solvent was removed under reduced pressure. The crude methyl ketone was purified by flash chromatography over silica gel (ethyl acetate-hexanes 20/80, R_f = 0.28) to yield 4 (1.07 g, 99 % over two steps) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 7.45 (4H, s), 2.70 (3H, s), 2.13 (3H, s). ¹³C NMR (100 MHz, CDCl₃) δ 192.7, 174.7, 161.0, 136.3, 130.4, 128.9, 127.3, 117.2, 30.6, 13.7.

Synthesis of 5

A solution of 4 (530 mg, 2.25 mmol, 1 equiv.) in dry toluene (7.5 mL) in a flame-dried sealed tube was treated with tert-butoxy bis(dimethylamino)methane (511 μL, 1.1 equiv.) under an inert atmosphere. The tube was sealed and the mixture was heated at 110 °C overnight. Toluene was then removed under reduce pressure to give the crude enamino-ketone intermediate as a pale yellow oil (641 mg, 98%) which was used in the next step without further purification.

The enamino-ketone from the preceding experiment (360 mg, 1.24 mmol, 1 equiv.) was dissolved in acetic acid (2 mL) in a 5-mL microwave-vial, and anhydrous hydrazine (43 μL, 1.1 equiv.) was added. The mixture was submitted to microwave irradiations for 1 h at 120 °C. Water (5 mL) and dichloromethane (5 mL) were added and then the resulting mixture was transferred to a separatory funnel. Saturated aqueous sodium hydrogenocarbonate solution was added until pH = 4–5, then the aqueous layer was extracted with dichloromethane (3 × 15 mL). The combined organic layers were washed with saturated aqueous ammonium chloride solution (30 mL), brine (30 mL) and dried over sodium sulfate. The solution was filtered and solvent was removed under reduce pressure. Pyrazole 5 was obtained as a pale yellow solid (300 mg, 91 %, 2 steps) after purification by flash chromatography (silica gel, ethyl acetate-hexanes 40/60, R_f = 0.65).

¹H NMR (400 MHz, CDCl₃) δ 10.31 (1H, br s), 7.51 (1H, d, J = 2.2 Hz), 7.46 (2H, d, J = 8.5 Hz), 7.30 (2H, d, J = 8.5 Hz), 6.16 (1H, d, J = 2.1 Hz), 2.50 (3H, s). ¹³C NMR (100 MHz, CDCl₃) δ 168.4, 160.3, 139.7, 135.8, 131.5, 129.7, 128.8, 127.5, 108.1, 106.0, 12.0.

Synthesis of Probe 2 (6a, CID 53239838) and Its Regioisomer 6b

Method A: Pyrazole 5 (115 mg, 0.44 mmol, 1 equiv.) and p-tosyl chloride (84 mg, 1 equiv.) were dissolved in dry dichloromethane (4.4 mL) under inert atmosphere. Freshly distilled triethylamine (124 μL, 2 equiv.) was added and the mixture was stirred at room temperature for 24 h. At this time water (10 mL) and aqueous 1N HCl solution (10 mL) were added. The aqueous layer was extracted with dichloromethane (3 × 15 mL) The combined organic layers were washed with water (15 mL) and brine (15 mL) and dried over sodium sulfate. The solution was filtered, then solvent was removed under reduce pressure to give a 3:1 mixture of regioisomers 6a and 6b. The major regioisomer 6a (104 mg, white solid—mp = 133–134 °C) and the minor isomer 6b (34 mg, pale yellow solid) were separated by flash chromatography over silica gel (ethyl acetate/hexanes 20/80, R_f6a = 0.26, R_f6b= 0.14) with an overall yield of 76 %. The minor regioisomer6bhas the structure originally listed for Probe 2 the commercial supplier.

Method B: Pyrazole 5 (20 mg, 0.08 mmol, 1 equiv.), p-tosyl chloride (14.7 mg, 1 equiv.) and 97 % sodium hydride (3.8 mg, 2 equiv.) were weighed into a flame-dried 10-mL round-bottomed flask inside an inert atmosphere glovebox. The flask was sealed under inert atmosphere and the solids were dissolved in freshly distilled tetrahydrofuran (0.8 mL). The mixture was stirred at room temperature for 24 h. At this time water (5 mL) and aqueous 1N HCl solution (5 mL) were added with precaution. The aqueous layer was separated and extracted with dichloromethane (3 × 10 mL) The combined organic layers were washed with water (10 mL) and brine (10 mL) and dried over sodium sulfate. The solution was filtered, and solvent was removed under reduce pressure yielding regioisomers 6a (minor regioisomer 6b was not observed by 1H NMR analysis of the crude product). The major regioisomer 6a (26 mg, 82 %, white solid— mp = 133–134 °C) was purified by flash chromatography over silica gel (ethyl acetate/hexanes 20/80, R_f6a= 0.26).

Major regioisomer 6a (Probe 2, CID 53239838): ¹H NMR (400 MHz, CDCl₃) δ 8.08 (1H, d, J = 2.8 Hz), 7.87 (2H, d, J = 8.4 Hz), 7.37-7.33 (4H, m), 7.24 (2H, d, J = 8.7 Hz), 6.12 (1H, d, J = 2.7 Hz), 2.50 (3H, s), 2.46 (3H, s). ¹³C NMR (100 MHz, CDCl₃) δ 169.3, 160.3, 147.7, 146.1, 135.6, 133.7, 131.8, 130.0, 129.9, 128.6, 128.2, 127.2, 108.8, 107.7, 21.7, 12.2. MS ([M+H]+): 414.1. IR (cm⁻¹): 3142, 3126, 2921, 1641, 1475, 1370, 1178, 1157, 1093, 1048, 929, 778, 669.

Minor regioisomer 6b (with the structure listed for CID 2745583): ¹H NMR (400 MHz, CDCl₃) δ 7.83 (1H, d, J = 1.6 Hz), 7.48 (2H, d, J = 8.4 Hz), 7.15-7.14 (4H, m), 7.10 (2H, d, J = 8.1 Hz), 6. 04 (1H, d, J = 1.6 Hz), 2.34 (3H, s), 2.29 (3H, s). ¹³C NMR (100 MHz, CDCl₃) δ 170.3, 159.6, 146.0, 143.5, 135.8, 134.8, 133.9, 129.7, 128.7, 128.4, 128.0, 127.1, 113.2, 103.9, 21.6, 11.7. MS ([M+H]+): 414.1. IR (cm⁻¹): 3128, 2924, 2853, 1640, 1596, 1452, 1418, 1389, 1269, 1194, 1172, 1124, 1089, 914, 844, 809, 678, 661.

Determination of the Structures of Regioisomers 6a and 6b

It is expected that sulfonylation of pyrazole 5 should lead selectively to regioisomer 6a. Indeed, isomer 6a was obtained exclusively when the sulfonylation was performed according to Method B. Fortunately, regioisomer 6b was obtained as the minor component of a 3 : 1 mixture from the sulfonylation performed as described in method A. Therefore, samples of both compounds were subjected to testing in the TRPML3 assay, from which it was determined that regioisomer 6a was the active probe compound.

Further evidence that regioisomer 6a is the correct structure of Probe 2 was provided as follows. TLC analysis demonstrated conclusively that the synthetic compound 6a is the same as a sample of Probe 2 obtained from a commercial supplier. However, compound 6b—which is inactive in the TRPML3 assay and which is different from commercial samples of Probe 2 by TLC analysis—has the structure as originally listed for Probe 2 in PubChem (CID 2745583) and in the commercial vendor’s catalogs. Therefore, we suspected that the structure of the compound is incorrect in the vendor catalogs, and that this error has been duplicated in the PubChem database.

We have performed NMR analysis of a commercial sample of the probe compound, obtained from Maybridge Ltd (the supplier ID is SPB04991), and have verified that synthetic probe 6a is IDENTICAL to the material—written with the wrong structure—in the vendor catalog.

Therefore, the following NMR experiments were performed in order to clarify the structures of regioisomers 6a and 6b. The 1H nOe summarized in the following figures are highly instructive. The nOe data in Figure 1, obtained by irradiation of the ortho hydrogen atoms of the toluenesulfonyl group, resulted in a strong enhancement of the indicated ortho pyrazoles ring hydrogen in structure 6a, but only a weak enhancement in 6b. Conversely, this nOe study (Figure 4) led to a stronger enhancement of the oxazoles methyl group in 6b than in 6a. These data are fully consistent with the assigned structures. This conclusion is enhanced by the 1H nOe study summarized in Figure 5, in which the ortho pyrazole hydrogen was irradiated in the two regioisomers. This resulted in a strong enhancement of the ortho hydrogen atoms of the toluenesulfonyl group in regioisomer 6a but only a weak enhancement in 6b.

Figure 4

1H nOe enhancements from irradiation of 6a at 7.87 ppm and 6b at 7.48 ppm.

Figure 5

1H nOe enhancements from irradiation of 6a at 8.08 ppm and 6b at 7.83 ppm.

Therefore, based on these studies, the structure of Probe 2 must be reassigned from the original structure (listed in PubChem as CID 2745583, as in isomer 6b) to that of compound 6a (CID 53239838).