TRPML3 Agonist Assays (PubChem AIDs 1448, 1526, 1562, and 2116)

The purpose of these assays is to identify compounds that act as agonists of the TRPML3 cation channel. These assays employ 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 the fluorescent, cell permeable calcium indicator dye Fluo-8. 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) and in triplicate (AID 1526) at a nominal test concentration of 3 μM, and in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 29.9 micromolar (AID 1562 and 2116).

TRPN1 Agonist Assays (PubChem AIDs 1424, 1525, 1682, and 2116)

The purpose of these assays is to identify test compounds that act as agonists of the TRPN1 cation channel. These assays also serve as counterscreen to identify compounds that are nonselective TRP agonists due to activation of TRPN1. These assays employ 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 the fluorescent, cell permeable calcium indicator dye Fluo-8. 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) and in triplicate (AID 1525) at a nominal test concentration of 3 μM, and in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 29.9 micromolar (AID 1682 and 2116).

Patch Clamp Assays (PubChem AID 2116)

The purpose of these assays is to determine if test compounds can increase current recordings in TRPML3 ion channels (Assay 3) or the YFP-HEK parental background (Assay 4). 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. These assays were performed by the assay provider.

Fura-2 Assays (PubChem AID 2116)

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 are tested at 10 micromolar. These assays were performed by the assay provider.

Probe chemical structure including stereochemistry. Separation of diastereomers (if necessary)

The structures of probes ML122 and ML123 are shown below. Because the stereochemistry of ML123 was not clearly defined in the information provided from commercial suppliers, we synthesized the stereoisomers of ML123 and determined that the most active isomer is the trans-S,S-isomer of ML123, as discussed in this report.

Probe 1 (ML123)

Secondary Arylsulfonamide Scaffold

(S, S)-trans isomer, CID1167615

racemate, SR-1984

original (Commercial), CID2911646

Probe 2 (ML122)

Sulfonarylpiperazine Scaffold CID701237

Structure verification with 1H NMR and LCMS results. See next section.

Solubility. The solubility of the probes was measured in phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4) at room temperature (23°C). The solubility of probe ML122 and ML123 and the associated stereomers are shown below.

Stability. The stability of the probes was measured at room temperature (23°C) in PBS (no antioxidants or other protectants; DMSO concentration below 0.1%). The stability, represented by the half-life, was found to be > 48 hours. Below is a graph showing loss of compound with time over a 48 hour period with a minimum of 6 time points. The table indicates the percent of compound remaining at the end of the 48 hours.

Stability of ML123 (trans racemate SR3-1984)

Stability of ML123 (cis racemate SR3-1985)

Stability of ML123 (R enantiomer of trans isomer SR3-2020)

Stability of ML123 (S enantiomer of trans isomer SR3-2021)

Stability of Probe ML122 (Resynthesis SR-03000002103)

The probes were measured for their ability to form glutathione adducts. At concentrations of 100 μM reduced GSH, 10 μM of the probes do not appear to react with glutathione. These compounds do not have functionality that would render them to be Michael acceptors [11, 12].

Synthesis of ML123 and Identification of the Stereochemistry of the Probe

Stereochemistry of the probe, ML123, was not specified by the original commercial supplier of samples identified from the HTS. Four possible stereoisomers exist: the racemic trans diastereomer (4) and the racemic cis diastereomer (10). Samples of the racemic 4 and 10 (both as 1:1 mixtures of enantiomers) were synthesized by the routes summarized below.

Synthesis of Racemic 4 (SR-03000001984)—Racemic Trans isomer. This synthesis involved ring-opening of cyclohexene oxide 1 with morpholine to give 2. Activation of the hydroxyl group of 2 as a mesylate and then displacement with ammonium hydroxide—via an azidinium ion—provided 3. Acylation of racemic 3 with p-chlorobenzenesulfonyl chloride then provided racemic 4 (SR-03000001984).

(i) morpholine, EtOH, reflux, 12h, 98%. (ii) mesyl chloride, Et₃N, 2h, then NH₄OH, 7h, 79%. (iii) 4-chlorophenylsulfonyl chloride, Et₃N, DCM, 1h, 74%.

Synthesis of Racemic 10 (SR-03000001985)—Racemic Cis isomer. The synthesis of racemic 10 (SR-03000001985) was accomplished starting with the reaction of cyclohexene oxide 1 with benzylamine to give 5. Protection of the secondary amine as the Boc carbamate 6 and then oxidation of the hydroxyl group gave ketone 7. Reductive amination of 7 with morpholine gave the cis-diamine derivative 8. Deprotection of the Boc and N-benzyl groups gave the cis diamine derivative 9, which was then acylated with p-chlorobenzenesulfonyl chloride which provided racemic 10 (SR-03000001985).

(i) Benzylamine 3% Zn(OTf)₂, rt, 24h. (ii) (Boc)₂O, DCM, rt, 11h, 85% (2 steps), (iii) PCC, DCM, rt, 24h, 96%. (iv) morpholine, 6% p-toluenesulfonic acid, benzene, 110 °C, 36h, then H₂, Pd/C, THF, rt, 48 h, 53%. (v) TFA, 1h, then H₂, Pd/C MeOH, 3h. (vi) 4-chlorophenylsulfonyl chloride, Et₃N, DCM, 1h, 11% over 2 steps.

Because several of the TRPML3/2 agonist probe analogs—such as analogs 1–5—are ortho-substituted aniline sulfonamide derivatives, and because the racemic trans diastereomer 4 of ML123 (but not the cis diastereomer 10) maps best onto the structures of analogs 1–5, we surmised that the ML123 probe would have the trans stereochemistry. This assumption was verified by the results of testing of 4 and 10, which demonstrated that 4 is about 10-fold more active than 10 in the TRPML3/2 assay. Therefore, we proceeded to determine the structure of the most active enantiomer by the synthesis summarized below.

Synthesis of Single Trans Enantiomers 16 (SR-03000002020) and 18 (SR-03000002021). This synthesis was accomplished by treatment of cyclohexene oxide 1 with (R)-a-methyl-benzylamine. Conversion of 11 to the aziridine 12 set the stage for ring opening of the aziridine with morpholine. This reaction gave a ca. 3:2 mixture of the diastereomeric diamines 13 and 14 which were separated chromatographically. Debenzylation of 13 gave the (R,R)-15, whereas debenzylation of 14 gave the enantiomeric diamine, (S,S)-17. Acylation of 15 and 17 with p-chlorobenzenesulfonyl chloride then provided (R,R)-(−)-16 (SR-03000002020) and (S,S)-(+)-18 (SR-03000002021), respectively.

(i) (R)-alpha-methyl-benzylamine_, 5% Zn(OTf)₂, neat, rt, 24h.

(ii) DIAD, PPh₃, DCM, 0 °C to rt, 8h, 73% (2 steps).

(iii) morpholine, 5% Zn(OTf)₂, MeCN, 80 °C, 12h, 86% (ca. 3/2 ratio of 13/14).

(iv) H₂, 20% Pd(OH)₂/C, AcOH, rt, 1h.

(v) 4-chlorophenylsulfonyl chloride, Et₃N, DCM, rt, 30 min. 59% (2 steps).

(vi) H₂, 20% Pd(OH)₂/C, MeOH/AcOH (9/1), rt, 24h.

(vii) 4-chlorophenylsulfonyl chloride, Et₃N, DCM, rt, 30 min. 62% (2 steps).

Finally, the absolute stereochemistry of the primary amine intermediates 15 and 17 was verified by conversion of each to the corresponding Cbz derivatives (R,R)-(−)-19 and (S,S)-(+)-20, respectively. The optical rotation obtained for (S,S)-(+)-20 ([α]_D²⁸ = +37.9 (c = 1.0 in CHCl₃)), but not for (R,R)-(−)-20 ([α]_D²⁸ = −35.7 (c = 1.0 in CHCl₃)), was in close agreement with the value reported in the literature for this compound ([α]_D²⁸ = +45.7 (c = 1.0 in CHCl₃); [13]

(i) CbzCI, Et₃N, DCM, 0 °C to rt, 1h

Detailed experimental procedures for synthesis of Probe ML123 and Stereoisomers

trans-2-Morpholinocyclohexanol (2). To a solution of cyclohexene oxide (5.0 mL, 49.5 mmol) in anhydrous ethanol (25 mL) was added morpholine (7.0 mL, 79.3 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 12 hours. After being cooled to ambient temperature, the solvent was removed under reduced pressure. Purification of the residue by flash chromatography eluting with a step gradient ranging from 0% to 20% MeOH-EtOAc provided 9.0 g (48.6 mmol, 98%) of 2 as a viscous oil. ¹H NMR (400 MHz, CDCl₃) δ 4.02 (d, J = 2.4 Hz, 1H), 3.60-3.52 (m, 4H), 3.38-3.31 (m, 1H), 2.63-2.40 (m, 4H), 2.13-2.07 (m, 1H), 1.91-1.87 (m, 1H), 1.74-1.66 (m, 2H), 1.60-1.58 (m, 1H), 1.19-1.07 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 69.29, 68.20, 66.77, 48.72, 33.91, 24.89, 23.74, 23.22; MS (ESI) 186 m/z [M+H]⁺.

trans-2-Morpholinocyclohexanamine (3). To a solution of 2 (2.3 g, 12.5 mmol) and triethylamine (2.6 mL, 18.6 mmol) in dry ether (50 mL) was added methanesulfonyl chloride (1.2 mL, 15.4 mmol) dropwise at 0 °C. A white precipitate formed, and the reaction mixture was stirred at room temperature for 2 hours. After completion of the mesylation step (monitored by LCMS for the absence of 186 m/z [M+H]⁺), NH₄OH (50 mL) was added. The reaction mixture was vigorously stirred at ambient temperature for additional 7 hours. The reaction mixture was then diluted with ether (50 mL) and washed with saturated aq. NaHCO₃ (30 mL) and brine (30 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification of the crude product by flash chromatography using a SNAP KP-NH Cartridge by eluting with a step gradient ranging from 12% to 100% EtOAc-hexane provided 1.8 g (9.92 mmol, 79%) of 3 as a viscous oil. ¹H NMR (400 MHz, CDCl₃) δ 3.70-3.60 (m, 4H), 2.67-2.55 (m, 3H), 2.39-2.34 (m, 2H), 2.01-1.91 (m, 2H), 1.79-1.74 (m, 2H), 1.65-1.60 (m, 3H), 1.22-1.02 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 70.98, 67.90, 50.65, 49.01, 35.30, 25.98, 25.12, 22.85; MS (ESI) 185 m/z [M+H]⁺.

trans-4-Chloro-N-(trans-2-morpholinocyclohexyl)benzenesulfonamide (racemic-4, ML123, SR-0300001984-1). To a solution of 3 (0.11g, 0.60 mmol) and triethylamine (0.12 mL, 0.90 mmol) in dry CH₂Cl₂ (10 mL) was added 4-chlorophenylsulfonyl chloride (0.15 g, 0.72 mmol) at room temperature. The reaction mixture was stirred for 1 hour at ambient temperature. The solvent was removed under reduced pressure, and the crude product was diluted with ethyl acetate (50 mL). The organic layer was washed with saturated aq. NaHCO₃ (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification of the crude product by flash chromatography eluting with a step gradient ranging from 12% to 100% EtOAc-hexane provided a viscous oil. The oil crystallized at 0°C to yield 0.16 g (0.449 mmol, 74%) of 4; purity >98% by LCMS; m. p. = 110 °C; IR (neat) 3236, 2942, 2861, 2817, 1588, 1479, 1451, 1333, 1166, 1070, 1013, 910, 827, 754 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.79 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 5.97 (s, 1H), 3.54-3.45 (m, 4H), 2.72 (td, J = 10.4, 4.0 Hz, 1H), 2.35-2.31 (m, 1H), 2.19-2.09 (m, 5H), 1.80-1.71 (m, 2H), 1.62-1.60 (m, 1H), 1.24-0.98 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 139.15, 138.80, 129.39, 128.72, 67.28, 66.92, 53.33, 48.13, 32.95, 25.24, 24.19, 22.93; MS (ESI) 359 m/z [M+H]⁺; HRMS (ESI) 359.1203 m/z [M+H]⁺, 381.1027 m/z [M+Na]⁺; calc. 359.1191 [M+H]⁺, 381.1010 [M+Na]⁺.

trans-2-(Benzylamino)cyclohexanol (5). To cyclohexene oxide (3 mL, 29.6 mmol) were added zinc trifluoromethanesulfonate (0.33 g, 3 mol%) and benzylamine (3 mL, 27.5 mmol) at room temperature. The reaction mixture was stirred neat at ambient temperature for 24 hour. The reaction mixture solidified, and the un-reacted reagents were removed under reduced pressure overnight. The crude solid product 5 (6.3 g, 30. 6 mmol) was directly used in the next step without further purification; MS (ESI) 206 m/z [M+H]⁺.

trans-tert-Butyl benzyl(trans-2-hydroxycyclohexyl)carbamate (6). To a solution of crude 5 (6.3 g, 30.6 mmol) in dry CH₂Cl₂ (50 mL) was added di-tert-butyl dicarbonate (8.8 g, 40.2 mmol) at room temperature. The reaction mixture was stirred for 11 hours at ambient temperature. The mixture was diluted with CH₂Cl₂ (100 mL) and washed with water (30 mL) and brine (30 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification of the crude product by flash chromatography eluting with a step gradient ranging from 12% to 100% EtOAc-hexane provided 7.1 g (23.4 mmol, 85% over 2 steps) of 6 as a viscous oil; ¹H NMR (400 MHz, DMSO-d₆, Temp = 60 °C) δ 7.30-7.18 (m, 5H), 4.44-4.35 (m, 2H), 4.23 (d, J = 5.2 Hz, 1H), 3.60 (septet, J = 4.8 Hz, 1H), 3.48 (br, 1H), 1.94-1.91 (m, 1H), 1.57-1.53 (m, 3H), 1.36 (s, 10H), 1.24-1.09 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 157.09, 140.00, 128.41, 126.86, 80.17, 71.15, 62.07, 47.43, 35.14, 29.76, 28.36, 25.39, 24.38; MS (ESI) 206 (Boc decomposition) m/z [M+H]⁺.

tert-butyl Benzyl(2-oxocyclohexyl)carbamate (7). To a solution of 6 (7.1 g, 23.4 mmol) in dry CH₂Cl₂ (100 mL) was added pyridinium chlorochromate (PCC) (8.1 g, 37.6 mmol), and the reaction mixture was stirred at room temperature for 24 hours. After completion of the reaction (monitored by TLC), the mixture was diluted with ether and filtered through a pad of Celite. The solvent was removed under reduced pressure. Purification of the crude product by flash chromatography eluting with a step gradient ranging from 6% to 50% EtOAc-hexane provided 6.8 g (22.4 mmol, 96%) of 7 as a viscous oil; ¹H NMR (400 MHz, DMSO-d₆) δ 7.32-7.19 (m, 5H), 4.57-4.47 (m, 1H), 4.36 (q, J = 1.6 Hz, 1H), 4.12-4.02 (m, 1H), 2.41-2.29 (m, 2H), 1.99-1.53 (m, 6H), 1.31 (d, J = 41.2 Hz, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 207.37, 156.30, 140.29, 128.38, 126.75, 126.59, 80.49, 77.43, 64.73, 49.67, 41.69, 32.49, 31.09, 28.43, 26.58, 25.07; MS (ESI) 204 (Boc decomposition) m/z [M+H]⁺.

tert-butyl Benzyl(cis-2-morpholinocyclohexyl)carbamate (8). To a solution of 7 (0.41 g, 1.34 mmol) in dry benzene (10 mL) was added p-toluenesulfonic acid (17 mg, 0.089 mmol) at room temperature. The reaction mixture was stirred at 50 °C until all of acid had dissolved. Morpholine (0.15 mL, 1.73 mmol) was added to the reaction mixture, and the reaction mixture was refluxed at 100 °C for 3 hours. The solvent was removed by distillation. Additional morpholine (0.15 mL) and dry benzene (10 mL) was added to the reaction mixture, which was refluxed at 110 °C for 14 hours. The solvent was distilled at atmospheric pressure, and excess morpholine was removed under reduced pressure with a vacuum pump to give the crude enamine intermediate; MS (ESI) 373 m/z [M+H]⁺. The crude enamine was dissolved in dry THF (10 mL), and 10% Pd/C (78.5 mg) was added. After removing air in the flask using a vacuum pump, hydrogen gas was introduced using a balloon. The reaction mixture was stirred for 48 hours at ambient temperature. Palladium on carbon was removed by filtration. The filtrate was evaporated to give a crude viscous oil, which was purified by flash chromatography using SNAP KP-NH Cartridge by eluting with a step gradient ranging from 6% to 50% EtOAc-hexane to yield 8 (0.27 g, 0.712 mmol, 53%) as a viscous oil; MS (ESI) 375 m/z [M+H]⁺.

cis-2-Morpholinocyclohexanamine (9): To a solution of 8 (0.27 g, 0.712 mmol) in CH₂Cl₂ (4 mL) was added trifluoroacetic acid (4 mL) at room temperature. The reaction mixture was stirred at ambient temperature and monitored by mass spectrometer. The Boc-deprotection was complete after one hour, and the solvent and trifluoroacetic acid were removed under reduced pressure; MS (ESI) 275 m/z [M+H]⁺. To the crude intermediate were added MeOH (10 mL) and 10% Pd/C (ca. 0.1 g), and the reaction mixture was stirred at ambient temperature. Air in the flask was removed using a vacuum pump, and hydrogen gas was introduced using a balloon. The reaction was monitored by mass spectrometer and after 3 hours, benzyl deprotection was complete. Palladium on carbon was removed by filtration. The filtrate evaporated to give a crude viscous oil 9, which was directly used in the next step without further purification; MS (ESI) 185 m/z [M+H]⁺.

cis-4-Chloro-N-(cis-2-morpholinocyclohexyl)benzenesulfonamide (10, SR-0300001985-1). Racemic cis-isomer 10 (29 mg, 80.7 μmol, 11% over 2 steps) was obtained as a white solid by following the procedure described for the synthesis of 4; m. p. = 140 °C; IR (neat) 3171, 2928, 2867, 2806, 1584, 1449, 1392, 1370, 1329, 1170, 1113, 1093, 995, 754 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.80 (dt, J = 8.8, 2.0 Hz, 2H), 7.46 (dt, J = 8.8, 2.0 Hz, 2H), 5.34 (s, 1H), 3.49 (t, J = 4.8, 4H), 3.35 (d, J = 3.2, 1H), 2.27-2.23 (m, 3H), 2.09-2.02 (m, 3H), 1.74 (d, J = 10.8, 2H), 1.46 (qd, J = 13.2, 3.2 Hz, 1H), 1.34-1.30 (m, 1H), 1.24-1.09 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 148.56, 133.38, 132.76, 132.03, 127.26, 119.31, 51.80, 47.44, 47.01, 25.73, 24.00, 20.84, 17.75; MS (ESI) 359 m/z [M+H]⁺; HRMS (ESI) 359.1199 m/z [M+H]⁺, 381.1019 m/z [M+Na]⁺; calc. 359.1191 [M+H]⁺, 381.1010 [M+Na]⁺.

trans-2-(((R)-1-Phenylethyl)amino)cyclohexanol (11). Compound 11 (7.3 g, 33.5 mmol) was obtained by following the procedure described for the synthesis of 5 using 1 (3 mL, 29.7 mmol), (R)-alpha-methylbenzylamine (4 mL, 31.4 mmol), and zinc trifluoromethanesulfonate (0.32 g, 3 mol%); MS (ESI) 220 m/z [M+H]⁺.

7-((R)-1-Phenylethyl)-7-azabicyclo[4.1.0]heptanes (12). To a solution of 11 (ca. 30 mmol) in dry CH₂Cl₂ (30 mL) was added PPh₃ (9.5 g, 36.0 mmol) at room temperature. The reaction mixture was stirred until PPh₃ completely dissolved, then was cooled to 0 °C. Diisopropyl azodicarboxylate (DIAD, 7 mL, 36.0 mmol) was added dropwise to the reaction mixture. The mixture was warmed to ambient temperature and stirred for 8 hours. The solvent was removed under reduced pressure. The crude product was diluted with ethyl acetate (90 mL) and was washed with water (20 mL) and brine (20 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification of the crude product by flash chromatography eluting with a step gradient ranging from 0% to 50% EtOAc-hexane provided 4.4 g (22.0 mmol, 73% over 2 steps) of 12 as a viscous oil; ¹H NMR (400 MHz, CDCl₃) δ 7.38 (d, J = 7.2 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.21 (t, J = 7.2 Hz, 1H), 2.43 (q, J = 6.8, 1H), 1.90-1.68 (m, 3H), 1.65-1.57 (m, 2H), 1.48-1.38 (m, 3H), 1.36 (d, J = 6.8, 3H), 1.22-1.10 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 145.93, 128.29, 126.88, 126.67, 70.04, 38.34, 38.05, 25.12, 24.82, 23.81, 20.94, 20.80; MS (ESI) 202 m/z [M+H]⁺.

(1R,2R)-2-Morpholino-N-((R)-1-phenylethyl)cyclohexanamine (13) and (1S,2S)-2-Morpholino-N-((R)-1-phenylethyl)cyclohexanamine (14). To a solution of 12 (1.5 g, 7.64 mmol) in dry CH₃CN were added zinc trifluoromethanesulfonate (0.13g, 5 mol%) and morpholine (0.8 mL, 9.25 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 12 hours. The mixture was cooled to ambient temperature, then the solvent was removed by evaporation. The reaction mixture was diluted with ethyl acetate (80 mL) and washed with water (20 mL) and brine (20 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification of the crude product by flash chromatography using a SNAP KP-NH Cartridge by eluting with a step gradient ranging from 0% to 50% EtOAc-hexane provided 1.2 g (4.29 mmol, 56%) of 13 as a white solid (R_f = 0.51 at 25% EtOAc/Hexane on KP-NH TLC plate) and 0.66 g (2.29 mmol, 30%) of 14 as a viscous oil (R_f = 0.39 at 25% EtOAc/Hexane on KP-NH TLC plate)

Data for 13: ¹H NMR (400 MHz, CDCl₃) δ 7.36 (d, J = 7.2 Hz, 2H), 7.29 (t, J = 7.2 Hz, 2H), 7.22-7.17 (m, 1H), 3.77-3.65 (m, 5H), 2.90 (s, 1H), 2.69-2.64 (m, 2H), 2.52-2.40 (m, 3H), 2.27-2.20 (m, 1H), 1.86-1.82 (m, 1H), 1.74-1.66 (m, 2H), 1.53-1.49 (m, 1H), 1.36 (d, J = 6.8 Hz, 3H), 1.23-1.00 (m, 3H), 0.93-0.87 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 147.73, 128.23, 126.54, 126.50, 67.82, 67.77, 57.62, 57.49, 48.60, 33.58, 25.60, 24.64, 24.41, 22.96; MS (ESI) 289 m/z [M+H]⁺.

Data for 14: ¹H NMR (400 MHz, CDCl₃) δ 7.32-7.28 (m, 2H), 7.24-7.20 (m, 3H), 3.77 (q, J = 6.8 Hz, 1H), 3.70-3.58 (m, 4H), 3.09 (br, 1H), 2.24-2.13 (m, 6H), 2.03 (td, J = 10.4, 4.4 Hz, 1H), 1.77-1.70 (m, 2H), 1.66-1.62 (m,1H), 1.43 (d, J = 6.8 Hz, 3H), 1.19-0.93 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 146.33, 128.44, 126.96, 126.47, 67.87, 67.81, 53.95, 53.26, 48.67, 31.88, 25.73, 24.56, 24.53, 22.68; MS (ESI) 289 m/z [M+H]⁺.

(1R,2R)-2-Morpholinocyclohexanamine (15). To a solution of 13 (16 mg, 56.5 μmol) in AcOH (10 mL) was added 20% Pd(OH)₂/C (9.6 mg) at room temperature. After removing air in the flask using a vacuum pump, hydrogen gas was introduced using a balloon. The mixture was stirred for 5 minutes, the balloon was removed, and the reaction mixture was stirred for 1 hour at ambient temperature. Palladium on carbon was removed by filtration through a Celite pad. The filtrate was evaporated to give a crude viscous oil 15, which was directly used in the next step without further purification; MS (ESI) 185 m/z [M+H]⁺.

4-chloro-N-((1R,2R)-2-Morpholinocyclohexyl)benzenesulfonamide (16, CID1167614, SID104223082, SR-0300002020). Compound 16 (12 mg, 33.4 μmol, 59% over 2 steps) was obtained as a white solid by following the procedure described for synthesis of 4. The spectroscopic data (¹H, ¹³C, and MS) of 16 are same to those of 4; m.p. = 168 °C; [α]_D²⁸ = −72.2 (c = 1.0 in CHCl₃). The purity of 16 was >98% based on LCMS.

(1S,2S)-2-Morpholinocyclohexanamine (17). To a solution of 14 (0.20 g, 0.695 mmol) in MeOH/AcOH (50 uL/5 mL) was added 20% Pd(OH)₂/C (38 mg) at room temperature. After removing air in the flask using a vacuum pump, hydrogen gas was introduced using a balloon. The reaction mixture was stirred for 24 hour at ambient temperature. Palladium on carbon was removed by filtration through a Celite pad. The filtrate was collected and evaporated to give a crude viscous oil, which was directly used for the next step without further purification. The spectra data of 17 (¹H and MS) are same to those of 3.

4-Chloro-N-((1S,2S)-2-morpholinocyclohexyl)benzenesulfonamide (18, CID1167615, SID104223083, SR-0300002021). Compound 18 (0.15 g, 0.428 mmol, 62% over 2 steps) was obtained as a white solid by following the procedure described for synthesis of 4. The spectroscopic data (¹H, ¹³C, and MS) for 18 are same to those of 4; m.p. = 173 °C; [α]_D²⁸ = +79.9 (c = 1.0 in CHCl₃). The purity of 18 was >98% based on LCMS.

Benzyl ((1R, 2R)-2-Morpholinocyclohexyl)carbamate (19). To a solution of 15 (20 mg, 0.107 mmol) and triethylamine (0.1 mL) in dry CH₂Cl₂ (5 ml) was added benzyl chloroformate (0.02 mL) dropwise at 0 °C. After being stirred 15 minutes, the reaction mixture was warmed to room temperature and stirred for additional 1 hour. The reaction mixture was diluted with dichloromethane (40 mL) and washed with saturated aq. NaHCO₃ (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification of the crude product by flash chromatography eluting with a step gradient ranging from 12% to 100% EtOAc-hexane provided 24 mg (77 μmol, 72%) of 19 as a viscous oil: [α]_D²⁸ = −35.7 (c = 1.0 in CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.35-7.28 (m, 5H), 5.36 (br, 1H), 5.08 (s, 2H), 3.66-3.55 (m, 4H), 3.31 (septet, J = 4.4 Hz, 1H), 2.65-2.60 (m, 2H), 2.43-2.41 (m, 1H), 2.35-2.30 (m, 2H), 2.15 (td, J = 10.8, 3.2 Hz, 1H), 1.88-1.77 (m, 2H), 1.68-1.62 (m, 1H), 1.28-1.03 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 156.77, 137.08, 128.71, 128.23, 128.22, 67.88, 67.69, 66.56, 51.47, 48.58, 33.28, 25.61, 24.79, 23.34; MS (ESI) 319 m/z [M+H]⁺.

Benzyl ((1S, 2S)-2-Morpholinocyclohexyl)carbamate (20). Compound 20 (23 mg, 73 μmol, 75%) was obtained as a viscous oil by following the procedure described for synthesis of 19: [α]_D²⁸ = +37.9 (c = 1.0 in CHCl₃), (ref. [α]_D²⁸ = +45.7 (c = 1.0 in CHCl₃) [13]; MS (ESI) 319 m/z [M+H]⁺.

Fura-2 Assays

These assays employ the calcium indicator dye fura-2-AM to detect increases in intracellular calcium in TRPML3-transfected HEK cells. The results of these assays for the two probes are shown below.

Probe 1 (ML123): Secondary Arylsulfonamide Scaffold

SID 24801657/CID2911646

Probe 2 (ML122): Sulfonarylpiperazine Scaffold

SID 24787221/CID701237

Patch Clamp Assays

The purpose of these assays is to determine if test compounds can increase current recordings in TRPML3 and TRPN1 ion channels. 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.