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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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Development of a Selective Chemical Inhibitor for the Two-Pore Potassium Channel, KCNK9

, , , , , , , , , , , , , and .

Author Information

Received: ; Last Update: February 28, 2013.

ML308 was identified as a novel inhibitor of the potassium channel, subfamily K, member 9 (KCNK9) two-pore domain potassium channel. Two-pore domain potassium channels provide a background leak conductance that is selectively permeable to potassium. These channels regulate cell membrane potential and excitability and thereby modulate a variety of processes including hormone secretion, proliferation, and central nervous system (CNS) function. A high throughput fluorescent screen measuring thallium influx through KCNK9 channels was used to identify bisamide and thiotriazole classes of inhibitors. Chemical modification of the thiotriazole scaffold yielded ML308 which displayed a potent block of KCNK9 channels in a thallium influx fluorescent assay (IC50 = 130 nM) and in an automated electrophysiology assay (IC50 = 413 nM). ML308 afforded >50-fold selectivity for block of KCNK9 over the closely-related, two-pore domain potassium channel, KCNK3, in fluorescent assays and displayed little or no block at 10 μM of the more distantly related potassium channels, Kir2.1, potassium voltage-gated channel, KQT-like subfamily, member 2 (KCNQ2), and human ether-a go-go-related gene (HERG). The potency and selectivity profile of ML308 makes it a useful pharmacological probe for in vitro studies of KCNK9 function and in further studies aimed at therapeutic intervention.

Assigned Assay Grant #: R03 MH090849-01

Screening Center Name & PI: Johns Hopkins Ion Channel Center, Min Li

Chemistry Center Name & PI: University of Kansas Specialized Chemistry Center, Jeffrey Aubé

Assay Submitter & Institution: Meng Wu, Ph.D., Johns Hopkins University, School of Medicine

PubChem Summary Bioassay Identifier (AID): 488964

Probe Structure & Characteristics

Image ml308fu1
CID/ML#Target NameThallium flux assay IC50 (nM) [SID 134418982, AID 623887]Anti-target NamesAnti-target percent inhibition at 10 μMAnti-target thallium assay IC50 (μM) [SID 134418982, AID 623894]Thallium assay flux (FDSS)
Fold Selectivity*
KCNK9 Qpatch: IC50 (nM) [SID 134418982, AID 623898]KCNK3 Qpatch: IC50 (nM) [SID 134418982, AID 623915]Qpatch Fold Selectivity
CID 56928204

KCNK9130 nMKir2.120.55NDNA41332007.7
KCNK9 non-induced (parental)6.94NDNA

Selectivity = anti-target IC50/KCNK9 IC50

ND = not determined due to insufficient inhibition to generate a valid IC50 value

NA = not applicable

1. Recommendations for Scientific Use of the Probe

ML308 is a pharmacological probe that can be used to examine the role of KCNK9 channels in a diverse set of biological functions. Two-pore domain potassium channels are involved in regulating a variety of physiological processes including chemosensation, hormone secretion, immune function, proliferation, and cardiac and neuronal excitability.1,2 The related TASK channels KCNK9 and KCNK3 are inhibited by extracellular protons. Biophysical experiments and studies with knockout animals suggest a specific role of these channels in oxygen sensing in carotid body glomus cells, aldosterone secretion from adrenal glomerulosa cells in response to angiotensin II and potassium, and carcinoma cell proliferation.3 Investigations into the physiological functions of these channels using genetic manipulations can be limited by species differences in expression patterns and by compensatory and other changes that may occur in knockout animals. For instance, in KCNK3−/− mice, efforts to identify the specific role for these channels in aldosterone secretion from adrenal glomerulosa cells is complicated by changes in the expression pattern of aldosterone synthase in the adrenal gland.4 Specific pharmacological modulation of KCNK9 and KCNK3 (TASK1 and TASK3 respectively) will provide important information on the roles of these channels in aldosterone secretion and salt and fluid balance. ML308 will also be used for in vitro studies of the pharmacological properties of leak potassium currents and their specific roles in aldosterone secretion using a combination of electrophysiological analysis and biochemical measures of hormone secretion.

KCNK9 knockout mice display alterations in sleep patterns and sensitivity to antidepressant drugs.5 The development of ML308 provides a pharmacological tool to investigate the cellular basis of these behavioral changes using in vitro electrophysiological techniques. ML308 can also be used to investigate the roles of KCNK9 channels in chemosensory control of respiration via central chemosensory neurons and carotid body cells and also in the responses of pulmonary vascular cells to hypoxia. In vitro studies with ML308 may support mechanistic studies of the cellular basis of these processes. Genetic studies using a dominant negative mutant of KCNK9 implicate a role for these channels in tumor growth.3 ML308 will be a valuable tool for in vitro studies of the roles of these channels in growth of a wider variety of tumor cell types.

2. Materials and Methods

  1. Cell Lines: KCNK9-expressing HEK293 Cells, KCNK3-expressing CHO cells (provided by D. Bayliss, University of Virginia), hERG-expressing CHO cells, KCNQ2-expressing CHO cells, Kir2.1-expressing HEK293 cells
  2. Cell Culture Media: Dulbecco’s Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Mediatech, Cat#10-013-CV), DMEM/F12 50/50 (Mediatech, Cat#15-090-CV), Hams F-12 Nutrient Mixture (1X), liquid w/L-Glut (Mediatech, Cat#10-080-CV)
  3. Fetal Bovine Serum: Gibco Cat #26140, Gemini, Cat#100-106
  4. L-Glutamine (Invitrogen, Cat#25030081)
  5. 100× Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
  6. CellStripper (Mediatech, Cat#25-056-Cl)
  7. Antibiotics: Blasticidin S (Research Products International Corp., Cat#B12150), Hygromycin (Mediatech, Cat#30-240-CR), Geneticin: (Gibco, Cat#11811-031)
  8. Compounds: SID 17386958 (Chembridge, Cat#8926102), XE-991 (Tocris, Cat#2000), Dofetilide (Fisher, Cat#NC9753685), Chlorpromazine hydrochloride (Sigma, Cat#C8138)
  9. HEPES (Sigma, Cat#H4034)
  10. 10XHBSS (Invitrogen, Cat#14065056)
  11. Tetracycline (Sigma, Cat# T7660)
  12. QPlate16 (Sophion Bioscience, Ballerup, Denmark)
  13. FluxOR detection kit (Invitrogen, Cat #F10017)
  14. Triple-layer flask (VWR, Cat #62407-082)
  15. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)
  16. Detachin (Genlantis, Cat#T100110)

2.1. Assays

  1. Summary of probe development for inhibitors of the two-pore domain potassium channel KCNK9: Summary AID 488964.
  2. Primary cell-based screen for identification of compounds that inhibit the two-pore domain potassium channel KCNK9: AID 488922.
  3. Confirmatory assays for the identification of compounds that inhibit the two-pore domain potassium channel KCNK9: AID 492997, AID 492992.
  4. Selectivity assays for the identification of selective inhibitors of KCNK9 using parental cells: AID 540324, AID 540324. AID 588759, AID 623884
  5. Selectivity assay for the identification of compounds that inhibit the two-pore domain potassium channel KCNK9 using KCNK3 expressing cells: AID 588776, AID 623894, AID 623912, AID 623915
  6. Selectivity assays for the identification of compounds that inhibit the two-pore domain potassium channel KCNK9 using cells expressing non-K2P potassium channels: AID 492993, AID 540323, AID 540321, AID 504920, AID 504922, AID 588741, AID 588760, AID 588761, AID 588800, AID 588798, AID 623883, AID 623881, AID 623886, AID 623914, AID 623913
  7. SAR assays for compounds that inhibit the two-pore domain potassium channel KCNK9: AID 540322, AID 504846, AID 504902, AID 588724, AID 623887, AID 588724
  8. KCNK9 validation assays using Automated Electrophysiology: AID 623898
  9. Manual Patch Clamp test for KCNK9 inhibitors: AID 624121

2.2. Probe Chemical Characterization

Probe compound name, structure and physiochemical data

The IUPAC name of the probe ML308 is 4-((S)-1-((1R, 2R, 4S)-bicyclo[2.2.1]heptan-2-yl)ethyl)-5-(furan-2-yl)-4H-1,2,4-triazole-3-thiol.

Image ml308fu2

Structure Verification and Purity: 1H NMR, 13C NMR, LCMS, and HRMS Data

Proton and carbon NMR data for ML308, SID 134418982, CID 56928204: Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental proton and carbon data are represented below for SID 134418982.

Proton NMR Data for ML308, SID 134418982, CID 56928204: 1H NMR (400 MHz, CDCl3) δ 10.90 (s, 1H), 7.63 (s, 1H), 6.87 (s, 1H), 6.57 (apparent dd, J = 1.8, 3.4 Hz, 1H), 4.76 (br. s, 1H), 4.08 (br. s, 0.5H), 3.24 (br. s, 0.5H), 2.23 (s, 1H), 1.70–1.50 (m, 4H), 1.45–1.29 (m, 4H), 1.20–1.11 (m, 1H), 1.09–0.97 (m, 2H), 0.81–0.63 (m, 1H).

Carbon NMR Data for ML308, SID 134418982, CID 56928204: 13C NMR (126 MHz, CDCl3) δ 169.24, 144.81, 143.47, 140.15, 114.87, 112.07, 57.74, 46.05, 38.57, 37.24, 36.83, 36.02, 29.60, 28.65, 16.79.

LCMS and HRMS Data for ML308, SID 134418982, CID 56928204: Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental LCMS and HRMS data are represented as follows: LCMS retention time: 3.320 min. LCMS purity at 214 nm: 91.8%. HRMS: m/z calcd for C15H20N3OS (M + H+) 290.1322, found 290.1321.

Optical rotation was determined for ML308 as a single diastereomer: [α]D24 = +10.2 (c 0.173, CHCl3).

Aqueous solubility

Solubility was measured in phosphate buffered saline (PBS) at room temperature (23 °C). PBS by definition is 137 μM NaCl, 2.7 μM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4.6 Probe ML308 was found to have a solubility measurement of 9.5 mg/mL, or 31.8 mM, under these conditions. Solubility was also assessed in thallium flux (FDSS) assay media (1× HBSS pH 7.4) and in stimulus buffer (2.8 mM thallium and 10 mM potassium sulfate). Probe ML308 was determined to have a thallium flux (FDSS) assay media solubility of 4.7 μg/mL, or 16.2 μM, and a stimulus buffer solubility of 6.4 μg/mL, or 22.1 μM. Aqueous solubility in pION buffer was determined at three pH levels: pH 5.0/6.2/7.4, and ML308 solubility under these conditions was found to be 2.1/2.4/9.8 μg/mL, respectively. In each of the three tested media, the solubility of ML308 was determined to be significantly higher than the IC50 value of the probe on channels for which it was active.


Aqueous Stability: Stability was measured under two distinct conditions with ML308 (CID 56928204, SID 134418982, Figure 1). Aqueous stability, represented by closed circles, was assessed at room temperature (23 °C) in PBS (no antioxidants or other protectants and DMSO concentration below 0.1%). Aqueous stability, illustrated with closed triangles in the graph, was also assessed with 50% acetonitrile added to account for challenges with solubility of the compound in PBS alone. Stability data in each case is depicted as a graph showing the loss of compound with time over a 48 hr period with a minimum of 6 time points and providing the percent remaining compound at the end of the 48 hr.6 With no additives (closed circles), 58.38% of ML308 remains after 48 hours; however, this data is dependent on and misleading due to the solubility limitations in PBS buffer. With the addition of 50% acetonitrile to account for solubility (closed triangles), 100% of ML308 remained after 48 hours.

Figure 1. Graph depicting stability of ML308 after 48 h in PBS and with PBS/ACN.

Figure 1

Graph depicting stability of ML308 after 48 h in PBS and with PBS/ACN.

Chemical Stability: ML308 was also evaluated for susceptibility to nucleophilic addition and formation of conjugates by treatment with glutathione (GSH) or dithiothreitol (DTT). Figure 2 represents the time course experiment with ML308 under various conditions. ML308 was dissolved at 10 μM in PBS at pH 7.4 (1% DMSO) and independently incubated at room temperature with no nucleophile present, 50 μM glutathione (GSH), or 50 μM dithiothreitol (DTT). The test reactions were sampled every hour for eight hours and analyzed by LCMS. The analytical LCMS system utilized for the analysis was a Waters Acquity system with UV-detection and mass-detection (Waters LCT Premier). The analytical method conditions included a Waters Acquity HSS T3 C18 column (2.1 × 50mm, 1.8 μm) and elution with a linear gradient of 1% water to 100% CH3CN at 0.6 mL/min flow rate. Peaks on the 214 nm chromatographs were integrated using the Waters OpenLynx software. Absolute areas under the curve were compared at each time point to determine relative percent parent remaining. The masses of potential adducts and dimers of ML308 were searched for in the final samples to determine if any detectable adduct formed or dimerization had occurred. All samples were prepared in duplicate. Ethacrynic acid, a known Michael acceptor, was used as a positive control. In the case of ML308, no adducts or dimers were detected at any time point using LCMS detection.7

Figure 2. Chemical stability of ML308 over 8 h in the presence of 5-fold glutathione or dithiothreitol.

Figure 2

Chemical stability of ML308 over 8 h in the presence of 5-fold glutathione or dithiothreitol.

Table 1 summarizes the percent remaining of ML308 at the endpoints of each run in each experiment. It was noted that the addition of nucleophile generally led to a greater percentage of ML308 remaining at the end of the 8-hour experiment, as compared to controls lacking any nucleophile. It is unclear at this time if this is due to standard deviation in measurement, solubility differences in the experiments, or other contributors.7

Table 1. Percent remaining of ML308 at the conclusion of the experiment (8h).

Table 1

Percent remaining of ML308 at the conclusion of the experiment (8h).

In separate experiments, ML308 was treated with a range of equivalents of L-glutathione (GSH) in DMSO for 72 h at 37 °C. The three experiments were monitored by LCMS at each of the following time points: 1 h, 2 h, 4 h, 24 h, 48 h, and 72 h. Procedure: To a solution of ML308 (1.0 mg, 1 eq) in DMSO (1.0 mL) was added:

  1. L-glutathione (1.3 mg, 4.15 μmol, 1.2 eq) and the mixture stirred at 37 °C for 72 h
  2. L-glutathione (2.1 mg, 6.92 μmol, 2.0 eq) and the mixture stirred at 37 °C for 72 h
  3. L-glutathione (3.2 mg, 10.38 μmol, 3.0 eq) and the mixture stirred at 37 °C for 72 h

LCMS analysis of each reaction vial, taken after time (t) = 1 h, 2 h, 4 h, 24 h, 48 h, and 72 h, showed only the presence of starting material. No glutathione conjugate or other peaks were observed. These results suggest that the compound is not generally electrophilic or susceptible to protein-derived nucleophiles. No dimerization of ML308 was detected.7

ML308 Synthetic Route

The probe ML308 was synthesized by the method shown (Figure 3). An enantioselective Diels-Alder reaction8,9 was employed to produce, after olefin reduction, a mixture of endo/exo cycloadducts 1 and 2 in a ratio of 95:5 as determined by 1H NMR. These products were found to have optical rotations nearly identical to reported values.9,10ab Conversion of the mixture to the Weinreb amide, followed by methyl Grignard addition afforded the corresponding mixture of methyl ketones, 3 and 4. Reductive amination of combined 3 and 4, followed by subsequent Fmoc protection generated four diastereomeric amides (5a–b, 6a–b) which were separated by chiral HPLC. FMOC protection was used to assist with handling a volatile amine during optimization of chemistry, to provide a suitable chromophore for separation, and for aiding in crystallization. Amide 6b was isolated as 15% of the mixture of four diastereomers, 5a–b and 6a–b. Desired amide 6b, whose stereochemistry was established by X-ray crystallography, was treated with a piperazine resin to remove the Fmoc protecting group, and the resulting amine was converted to an HBr salt for ease of handling, as the free amine is unexpectedly volatile. Treatment of salt 7 with thiophosgene generated isothiocyanate 8,11 which was heated with the preferred acyl hydrazide to generate, after reaction with base and acidic workup,12 the desired probe, ML308. The probe was isolated in 5% overall yield, as a single pure diastereomer as determined by LCMS and 1H NMR. Specific experimental details for the probe are detailed in section 2.3, entitled, “Probe Preparation”.

Figure 3. General synthetic route to probe ML308.

Figure 3

General synthetic route to probe ML308.

2.3. Probe Preparation

General experimental and analytical details:1H and 13C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating at 400 and 101 MHz respectively) or a Bruker AVIII spectrometer (operating at 500 and 126 MHz respectively) in CDCl3 with 0.03% TMS as an internal standard or DMSO-d6. The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet and m = multiplet. The LCMS analysis was performed on an Agilent 1200 RRL chromatograph with photodiode array UV detection and an Agilent 6224 TOF mass spectrometer. The chromatographic method utilized the following parameters: a Waters Acquity BEH C-18 2.1 × 50mm, 1.7 μm column; UV detection wavelength = 214 nm; flow rate = 0.4ml/min; gradient = 5 − 100% acetonitrile over 3 minutes with a hold of 0.8 minutes at 100% acetonitrile; the aqueous mobile phase contained 0.15% ammonium hydroxide (v/v). The mass spectrometer utilized the following parameters: an Agilent multimode source which simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and hexakis(1H, 1H, 3H-tetrafluoropropoxy) phosphazine; and a make-up solvent of 90:10:0.1 MeOH:Water:Formic Acid which was introduced to the LC flow prior to the source to assist ionization. Melting points were determined on a Stanford Research Systems OptiMelt apparatus. Optical rotations were measured on a Rudolph Research Analytical Autopol IV polarimeter.

The probe was prepared using the following protocols:

Image ml308fu3

3-((1R,2R,4R)-Bicyclo[2.2.1]hept-5-ene-2-carbonyl)oxazolidin-2-one (endo); 3-((1S,2R,4S)-Bicyclo[2.2.1]hept-5-ene-2-carbonyl)oxazolidin-2-one (exo). To an argon cooled, flame dried vial containing activated 4 Å molecular sieves was added (4R,4′R)-2,2′-(propane-2,2-diyl)bis(4-(tert-butyl)-4,5-dihydrooxazole, D-L Chiral Chemicals LLC, Chengdu, China (cat # WK05-1005)) (0.19 g, 0.64 mmol) and Cu(CF3SO3)2 (0.209 g, 0.58 mmol). The vial was evacuated with argon 3 times and diluted with anhydrous CH2Cl2 (15 mL). The resulting green-colored reaction was stirred for 1 h at rt. The 3-acryloyloxazolidin-2-one (0.82 g, 5.8 mmol) was then added, and the reaction was cooled to −72 °C. Freshly cracked cyclopentadiene (1.14 g, 17.3 mmol) was added and the reaction stirred for 1 h at −72 °C, warmed to −50 °C and stirred for 17 h at this temperature, and then warmed to 0 °C and stirred for 2 h until completion was determined, as monitored by NMR. The reaction was diluted with 1:1 EtOAc:Hex, filtered through silica and concentrated to produce a mixture of endo, 3-((1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl)oxazolidin-2-one and exo, 3-((1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl)oxazolidin-2-one (0.79 g, 3.8 mmol, 66% yield). 1H NMR (400 MHz, CDCl3, 95:5 endo:exo by NMR):δ 6.26 (dd, J1 = 5.7 Hz, J2 = 3.1 Hz, 1H, endo), 6.18 (s, 0.1H, exo), 5.88 (dd, J1 = 5.7 Hz, J2 = 3.1 Hz, 1H, endo), 4.45–4.35 (m, 2.2H), 4.06–3.90 (m, 3.22 H), 3.31 (br s, 1H, endo), 3.28 (br d, J = 8.8 Hz, 0.05H, exo), 3.01 (s, 0.05, exo), 2.94 (br s, 1H, endo), 1.99–1.92 (m, 1H), 1.56–1.39 (m, 3.20H).

Image ml308fu4

3-((1S,2R,4R)-Bicyclo[2.2.1]heptane-2-carbonyl)oxazolidin-2-one (endo); 3-((1R,2R,4S)-Bicyclo[2.2.1]heptane-2-carbonyl)oxazolidin-2-one (exo). To a vial was added the mixture of endo, 3-((1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl)oxazolidin-2-one and exo, 3-((1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl)oxazolidin-2-one and diluted with EtOAc (15 mL). The cyclohexene (2.20 g, 26.8 mmol) and Pd/C (0.16 g, 1.5 mmol) was added and the reaction stirred at 70 °C for 18 h. The reaction was filtered through Celite and concentrated to produce 3-((1S,2R,4R)-bicyclo[2.2.1]heptane-2-carbonyl) oxazolidin-2-one (endo) and 3-((1R,2R,4S)-bicyclo[2.2.1]heptane-2-carbonyl)oxazolidin-2-one (exo) (0.79 g, 3.8 mmol, 99% yield). (400 MHz, CDCl3): δ 4.39 (t, J = 8.0 Hz, 2H), 4.10 – 3.96 (m, 2H), 3.86 – 3.81 (m, 1H), 2.70 (brs, 1H), 2.43 (br s, 0.05H), 2.34 (t, J = 6.7 Hz, 0.2H), 2.29 (br s, 1H), 1.83–1.78 (m, 1.05H), 1.64–1.48 (m, 3.3H), 1.39–1.30 (m, 3.1H), 1.23–1.17 (m, 1.05H).

Image ml308fu5

(1S,2R,4R)-N-Methoxy-N-methylbicyclo[2.2.1]heptane-2-carboxamide (endo); (1R,2R,4S)-N-Methoxy-N-methylbicyclo[2.2.1]heptane-2-carboxamide (exo). To a vial was added the N,O-dimethylhydroxylamine hydrochloride (0.88 g, 9.0 mmol) and dry THF (10 mL). The reaction was lowered to −10 °C and 2.0 M trimethylaluminum in toluene (4.52 mL, 9.0 mmol) was added dropwise. After addition of trimethylaluminum the reaction was warmed to rt and stirred for 30 minutes. The reaction was cooled again to −10 °C and the mixture of 3-((1S,2R,4R)-bicyclo[2.2.1]heptane-2-carbonyl) oxazolidin-2-one and 3-((1R,2R,4S)-bicyclo[2.2.1]heptane-2-carbonyl)oxazolidin-2-one (0.79 g, 3.8 mmol) in dry THF (10 mL) was added dropwise. The reaction then warmed to 0 °C and stirred for 4 h after which 1 N HCl (10 mL) was added to the mixture and continued stirring for 1 h. The mixture was then extracted with CH2Cl2 (3 × 15 mL) and the organic layer was separated from the aqueous layer, dried with MgSO4, filtered, adsorbed to silica and purified by ISCO Combiflash flash chromatography (0–40% EtOAc:Hex) to produce a mixture of (1S,2R,4R)-N-methoxy-N-methylbicyclo [2.2.1]heptane-2-carboxamide (endo); (1R,2R,4S)-N-methoxy-N-methylbicyclo[2.2.1]heptane-2-carboxamide (exo) (0.67 g, 3.7 mmol, 97%). (400 MHz, CDCl3): δ 4.46 (t, J = 7.8 Hz, 0.5H), 4.35 (t, J = 7.8 Hz, 0.07H), 3.79 (t, J = 7.8 Hz, 0.08H), 3.68 (s, 3H), 3.64 (t, J = 7.8 Hz, 0.5H), 3.59 (s, 0.28H), 3.19 (s, 3.0H), 3.18 (s, 0.25H), 3.02 (br s, 1H), 2.62 (s, 0.30H), 2.55, (br s, 1H), 2.25 (br s, 1H), 1.80–1.75 (m, 1.07H), 1.60–1.33 (m, 6.50H).

Image ml308fu6

1-((1S,2R,4R)-Bicyclo[2.2.1]heptan-2-yl)ethanone (endo); 1-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-yl)ethanone (exo). Methylmagnesium bromide (3.0 M in ether, 1.83 mL, 5.48 mmol, 1.5 equiv) was added dropwise to a −10 °C mixture of (1S,2R,4R)-N-methoxy-N-methylbicyclo[2.2.1]heptane-2-carboxamide and (1S,2R,4R)-N-methoxy-N-methylbicyclo[2.2.1]heptane-2-carboxamide (669 mg, 3.65 mmol, 1 equiv) in THF (6 mL). The mixture was stirred at −10 °C for 3 h then allowed to warm to room temp. TLC (10% EtOAc/Hexanes) showed consumption of starting material and a single spot (Rf = 0.65, KMnO4 stain) as the product. The reaction was quenched by addition of sat. aq. NH4Cl, and the resulting mixture was extracted with EtOAc. The organic layers were combined, dried (Na2SO4) and concentrated to give a mixture of 1-((1S,2R,4R)-bicyclo[2.2.1]heptan-2-yl)ethanone (endo); 1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethanone (exo) (443 mg, 3.21 mmol, 88 % yield) as a colorless oil.

Image ml308fu7

1-((1S,2R,4R)-Bicyclo[2.2.1]heptan-2-yl)ethanamine (endo) and 1-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-yl)ethanamine (exo). Ammonium acetate (2.47 g, 32.1 mmol, 10 equiv) was added to a mixture of 1-((1S,2R,4R)-bicyclo[2.2.1]heptan-2-yl)ethanone, 1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethanone (443 mg, 3.21 mmol) and sodium cyanoborohydride (604 mg, 9.62 mmol, 3 equiv) in MeOH (12 mL) and the mixture stirred for 16 h at room temperature. IR showed consumption of the starting ketone. The reaction mixture was acidified to pH 2 with 12N HCl. The solution was then basified to pH 10 with solid KOH. The aqueous layer was extracted with CH2Cl2 (5 × 10 mL) and the combined organic layer dried (Na2SO4) and concentrated to give a mixture of 1-((1S,2R,4R)-bicyclo[2.2.1]heptan-2-yl)ethanamine (endo) and 1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethanamine (exo) (378 mg, 2.71 mmol, 85% yield. 1H NMR (400 MHz, CDCl3) δ 2.66–1.85 (m, 5H), 1.68–0.46 (m, 12H).

Image ml308fu8

(9H-Fluoren-9-yl)methyl (1-((1S,2R,4R)-bicyclo[2.2.1]heptan-2-yl)ethyl)carbamate (endo) and (9H-Fluoren-9-yl)methyl (1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethyl)carbamate (exo). To a vial containing a mixture of 1-((1S,2R,4R)-bicyclo[2.2.1]heptan-2-yl)ethanamine and 1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethanamine (378 mg, 2.71 mmol, 1 equiv) and N-ethyl-N-isopropylpropan-2-amine (1.18 mL, 6.79 mmol, 2.5 equiv) in dry THF (5 mL) at 0 °C was added a solution of (9H-fluoren-9-yl)methyl carbonochloridate (1756 mg, 6.79 mmol, 2.5 equiv) in dry THF (5 mL) dropwise. Once the addition was completed the mixture was warmed to room temperature and then heated at 60 °C for 16 h. The mixture was diluted with EtOAc and washed sequentially with NaHCO3 (3 × 10 mL), 1N HCl (2 × 10 mL), water (10 mL), and brine (10 mL). The organic extract was separated, dried (Na2SO4), and filtered. The solvent was evaporated, and the residue subjected to reversed-phase Combiflash (0–100% ACN-H2O) to yield a mixture of (9H-fluoren-9-yl)methyl (1-((1S,2R,4R)-bicyclo[2.2.1]heptan-2-yl)ethyl)carbamate (endo) and (9H-fluoren-9-yl)methyl (1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethyl)carbamate (exo) (637 mg, 1.762 mmol, 64.9% yield) as a white foam. 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.6 Hz, 2H), 7.57 (apparent dd, J = 2.4, 7.3 Hz, 2H), 7.38 (apparent t, J = 7.5 Hz, 2H), 7.29 (apparent td, J = 1.1, 7.4 Hz, 2H), 4.60–4.14 (m, 4H), 3.50 (br s, 0.4H), 3.42 – 3.25 (m, 0.4H), 3.12 (br s, 0.2H), 2.26–2.04 (m, 2H), 1.78–1.41 (m, 3H), 1.41–0.61 (m, 9H).

Image ml308fu9

(9H-Fluoren-9-yl)methyl((S)-1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethyl)carbamate. The mixture of (9H-fluoren-9-yl)methyl (1-((1S,2R,4R)-bicyclo[2.2.1]heptan-2-yl)ethyl)carbamate (endo) and (9H-fluoren-9-yl)methyl (1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethyl)carbamate (exo) was dissolved in 100% isopropanol (100 mg/mL). The mixture was injected into the preparatory chiral OD-H HPLC system (1000 μL, 100 mg loading) and separated with 3% isopropanol in hexanes. The desired product eluted at 27.70 min and was collected and concentrated to yield pure (9H-Fluoren-9-yl)methyl((S)-1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethyl)carbamate which is carried directly into the next reaction. 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.4 Hz, 2H), 7.38 (apparent t, J = 7.5 Hz, 2H), 7.30 (apparent t, J = 7.4 Hz, 2H), 4.54 (d, J = 8.5 Hz, 1H), 4.48 – 4.34 (m, 2H), 4.21 (t, J = 6.7 Hz, 1H), 3.37–3.26 (m, 1H), 3.14 (br s, 0.1H), 2.22 (s, 1H), 2.12 (s, 1H), 1.41–0.92 (m, 12H). Stereochemical assignment was unambiguously confirmed by X-ray crystallography. Crystals were obtained by dissolving purified compound in 100% isopropanol (approximately 10 mg per 0.10 mL), and gently heating to dissolve the compound. The solution was allowed to cool to room temperature and rest, undisturbed. X-ray quality crystals were obtained after 24h.

Image ml308fu10

(S)-1-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-yl)ethanaminium bromide: To a vial was added (9H-Fluoren-9-yl)methyl (1-(-bicyclo[2.2.1]heptan-2-yl)ethyl)carbamate (94 mg, 0.260 mmol, 1 equiv) and dioxane (1 mL). Piperazine (224 mg, 2.60 mmol, 10 equiv) on resin (Aldrich #547549, 0.520 g, 5 mmol/g loading) was then added, and the mixture stirred at 60 °C overnight. The reaction mixture was diluted with MeOH and filtered. The filtrate was treated with HBr in acetic acid (30% w/v, 91 μL, 0.520 mmol, 2 equiv) and concentrated. The residue was triturated with ether/hexane (1:1) and dried under vacuum to afford (S)-1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethanaminium bromide (41 mg, 0.186 mmol, 72% yield) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.08 (br s, 2H), 3.06–2.93 (m, 1H), 2.55 (d, J = 3.6 Hz, 1H), 2.25 (s, 1H), 1.80–1.68 (m, 1H), 1.60–1.31 (m, 6H), 1.30–1.01 (m, 5H).

Image ml308fu11

(1R,2R,4S)-2-((S)-1-Isothiocyanatoethyl)bicyclo[2.2.1]heptane: A saturated solution of aqueous NaHCO3 (2.2 mL) was added to a solution of (S)-1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethanaminium bromide (41.2 mg, 0.187 mmol, 1 equiv) in CH2Cl2 (2.2 mL) at 0 °C. The mixture was stirred for 10 minutes, stirring stopped, and thiophosgene (0.021 mL, 0.197 mmol, 1.05 equiv) was added to the CH2Cl2 layer in one portion via syringe. The mixture was stirred (~500 rpm) for 30 minutes at 0 °C. The reaction mixture was then diluted with CH2Cl2, and the organic layer was separated. The aqueous layer was extracted with CH2Cl2 and the combined organic extracts were dried (Na2SO4). Following filtration, the solvent was removed in vacuo to afford (1R,2R,4S)-2-((S)-1-isothiocyanatoethyl)-bicyclo[2.2.1]heptane (33 mg, 0.182 mmol, 97% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 3.34–3.26 (m, 1H), 2.34 (d, J = 3.7, 1H), 2.23 (s, 1H), 1.61–1.43 (m, 3H), 1.42–1.31 (m, 1H), 1.27–1.20 (m, 4H), 1.19–1.08 (m, 3H), 0.99–0.91 (m, 1H).

Image ml308fu12

4-((S)-1-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-yl)ethyl)-5-(furan-2-yl)-4H-1,2,4-triazole-3-thiol: ML308; SID 134418982; CID 56928204. To a solution of furan-2-carbohydrazide (10.16 mg, 0.081 mmol, 1 equiv) in ethanol (2.0 mL) was added (1R,2R,4S)-2-((S)-1-isothiocyanatoethyl)bicyclo[2.2.1]heptane (14.6 mg, 0.081 mmol, 1 equiv). The mixture was heated at 90 °C for 16 h and the solvent evaporated in vacuo. Sodium hydroxide (2N, 0.403 mL, 0.805 mmol, 10 equiv) was added to the resulting solid and the solution heated at 95 °C for 16 h. The solution was cooled to room temperature, acidified to pH 3 with 6N HCl, extracted with EtOAc (4 × 10 mL), and washed with brine (10 mL). The organic extracts were separated, dried over Na2SO4, filtered and concentrated to afford a residue. The residue was chromatographed using RP-MPLC (0–100% ACN-H2O) to give 4-((S)-1-((1R,2R,4S)-bicyclo[2.2.1]heptan-2-yl)ethyl)-5-(furan-2-yl)-4H-1,2,4-triazole-3-thiol (11.2 mg, 0.039 mmol, 48% yield) as an off-white solid. Mp: 206.8–209.8 °C; [α]D24 = +10.2 (c 0.173, CHCl3). 1H NMR (400 MHz, CDCl3) δ 10.90 (s, 1H), 7.63 (s, 1H), 6.87 (s, 1H), 6.57 (apparent dd, J = 1.8, 3.4 Hz, 1H), 4.76 (br s, 1H), 4.08 (br s, 0.5H), 3.24 (br s, 0.5H), 2.23 (s, 1H), 1.70–1.50 (m, 4H), 1.45–1.29 (m, 4H), 1.20–1.11 (m, 1H), 1.09–0.97 (m, 2H), 0.81–0.63 (m, 1H). 13C NMR (126 MHz, CDCl3) δ 169.24, 144.81, 143.47, 140.15, 114.87, 112.07, 57.74, 46.05, 38.57, 37.24, 36.83, 36.02, 29.60, 28.65, 16.79. Purity: 91.8%. LCMS retention time: 3.320 min. HRMS m/z calculated for C15H20N3OS [M++H]: 290.1322, found 290.1321.

3. Results

3.1. Dose Response Curves for Probe

The effect of KCNK9 inhibitors were titrated in a thallium flux assay (AID 504846, AID 540322, AID 588724, AID 623887) similar to that used in the primary high-throughput screen (AID 488922) and further examined in automated electrophysiology experiments (AID 623898).

Fluorescent assay

The primary HTS assay utilized a thallium sensitive dye to measure potassium channel activity in a HEK293 cell line expressing KCNK9 (AID 492992). An extracellular solution containing both thallium and potassium was added triggering thallium influx into the cells. The electrochemical gradient drives the net inflow of thallium down its concentration gradient and the accumulation of intracellular thallium increases the fluorescence of the FluxOR™ dye (Invitrogen). Figure 4 illustrates the typical titratable inhibition of KCNK9-mediated thallium influx by ML308. After normalization to buffer controls (0% inhibition) and KCNK9-independent thallium signal (100% inhibition) the resulting dose depended curve was fit with an IC50 value of 0.128 μM. In replicate experiments (n=4), the mean ± SD of the determined IC50 values were 0.128 ± 0.040 μM.

Figure 4. Inhibition of thallium signal in KCNK9-expressing cells by ML308.

Figure 4

Inhibition of thallium signal in KCNK9-expressing cells by ML308. Left panel (A) displays fluorescent signals in individual wells in the presence of different concentrations of ML308. Concentration response curve normalized to controls showing inhibition (more...)

Automated Electrophysiological assay

To further validate the compound effect on KCNK9 channels, an electrophysiological assay was developed on the Qpatch 16× automated electrophysiology instrument (Sophion) (AID 623898). Whole cell voltage clamp recordings were made using the same KCNK9 expressing HEK293 cells used in the primary screen.

Concentration response curves were generated from data collected at −30 mV and normalized to 100% inhibition determined through the application of 2 mM barium in 140 mM potassium solution at pH 5.8. This protocol was used to evaluate select compounds and the data has been collected. ML308 effect on the KCNK9 channel was evaluated using this method. ML308 inhibited KCNK9 currents with an IC50 of 0.41 ± 0.05 μM in multiple experiments (n=8).

3.2. Cellular Activity

The primary HTS assay and all secondary assays are cell-based assays, indicating that ML308 can gain access to its molecular target when applied to cells. The compound did not exhibit acute toxicity in cell-based assays at concentrations up to 30 μM.

3.3. Profiling Assays

ML308 (CID 3745727) is a new chemical entity and has not yet been tested in other MLPCN assays as a pure diastereomer. To more fully characterize this novel KCNK9 inhibitor, ML308 was tested on Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen which includes a binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM. ML308 was found to displace radioligand binding, leading to > 50% inhibition on 4 of the 68 targets assessed (Table 2). Included in the Ricerca screening panel are a number of ion channels (Calcium Channel, L-Type and N-Type; Potassium channel [KATP]; Potassium channel [hERG]) showing the selectivity profile for ML308.

Table 2. Targets from Ricerca profiling that showed > 50% inhibition.

Table 2

Targets from Ricerca profiling that showed > 50% inhibition.

ML308 was determined to inhibit endothelin (ETA), melatonin, and kappa opioid receptors, all members of the G-protein superfamily (66%, 64%. and 67% inhibition, respectively). Of the ion channels screened against, only significant inhibition was determined against the calcium channel L-Type (79% inhibition). The significance of these will be assessed by determining individual IC50 values in future work.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Compounds with known KCNK9 inhibitory activity at the start and for the better duration of this MLPCN project were lacking in significant KCNK9 potency and/or practical selectivity that limits them as tools for independently investigating the role of KCNK9 across an array of pharmacological processes. The development of ML308 represents a significant improvement over these compounds as it possesses submicromolar potency for KCNK9 and demonstrates selectivity over hERG, Kir2.1, KCNQ2, and the related channel, KCNK3. ML308 is a best in class probe in this context.

In the late stages of this MLPCN project, two articles were published that described compounds with reported KCNK9 and KCNK3 inhibition data. The Streit, et al. (2011) compound, A1899,13 possessed striking similarity to compounds appearing in a Sanofi-Aventis patent14 describing voltage-gated potassium channel inhibitors and was determined to have 10-fold selectivity for KCNK3 over KCNK9. The Coburn/Merck compound, C23, was published in January 2012 and had a reported KCNK9 potency of 35 nM, representing a 10-fold selectivity over KCNK3.15 The reported data for this compound was obtained using a different automated electrophysiology assay system at Merck. These two compounds were synthesized by the KU SCC for comparison to the probe ML308 described herein. Using a fluorescent thallium influx assay for KCNK9 (human) activity, A1899 exhibited an IC50 value of 3.2 μM while the measured IC50 for C23 was 190 nM. Selectivity versus the closely related two-pore-domain potassium channel, KCNK3 (rat), was also determined using a thallium influx assay. A1899 and C23 inhibited KCNK3 channels with observed IC50 values of 200 nM and 4 nM, respectively. Thus, both A1899 and C23 preferentially blocked KCNK3, rather than KCNK9 channels. In contrast, ML308 preferentially blocked KCNK9 channels, inhibited KCNK9 with potency similar to C23 and displayed more than 20-fold better IC50 for KCNK9 than what is observed for A1899.

ML308 is a small molecule with submicromolar potency for KCNK9, ~ 8-fold selectivity over KCNK3, and with little-to-no liability on Kir2.1, hERG, and KCNQ2. Moreover, the SAR effort has revealed some intriguing promise for future analogs: (1) simplification of the ethyl norbornyl system is acceptable (e.g. (R)-methyl-CH2-cyclohexyl), thus permitting a more streamlined synthesis; (2) of the diastereomerically-pure, norbornyl amines surveyed so far, the (R)-exo, (S)-methyl CH2-norbornyl system has emerged as an important moiety in tuning the selectivity between KCNK9 and KCNK3 without engaging other channels. Late isolated, related, diasteromerically-pure norbornyl amines have been generated and are being assessed for their effects; (3)N-alkylated thiotriazoles may represent a powerful combination of obtaining the desired KCNK9 profile and eliminating the potential liabilities of a thiol-containing thiotriazole.

5. References

Bayliss DA, Barrett PQ. Emerging roles for two-pore-domain potassium channels and their potential therapeutic impact. Trends Pharmacol Sci. 2008;29:566–75. [PMC free article: PMC2777628] [PubMed: 18823665]
Es-Salah-Lamoureux Z, Steele DF, Fedida D. Research into the therapeutic roles of two-pore-domain potassium channels. Trends Pharmacol Sci. 2010;12:587–95. [PubMed: 20951446]
Pei L, Wiser O, Slavin A, Mu D, Powers S, Jan LY, Hoey T. Oncogenic potential of TASK3 (KCNK9) depends on K+ channel function. Proc Natl Acad Sci USA. 2003;100:7803–7. [PMC free article: PMC164668] [PubMed: 12782791]
Heitzmann D, Derand R, Jungbauer S, Bandulik S, Sterner C, Schweda F, El Wakil A, Lalli E, Guy N, Mengual R, Reichold M, Tegtmeier I, Bendahhou S, Gomez-Sanchez CE, Aller MI, Wisden W, Weber A, Lesage F, Warth R, Barhanin J. Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis. EMBO J. 2008;27:179–187. [PMC free article: PMC2206116] [PubMed: 18034154]
Gotter AL, Santarelli VP, Doran SM, Tannenbaum PL, Kraus RL, Rosahl TW, Meziane H, Montial M, Reiss DR, Wessner K, McCampbell A, Stevens J, Brunner J, Fox SV, Uebele VN, Bayliss DA, Winrow CJ, Renger JJ. TASK-3 as a potential antidepressant target. Brain Res. 2011;1416:69–79. [PMC free article: PMC3179828] [PubMed: 21885038]
Experiments performed by and data obtained from Sanford Burnham, Layton Smith’s laboratory.
Experiments performed by and data obtained from the KU SCC.
Desimoni G, Faita G, Guala M, Pratelli C. An efficient catalyst for highly enantioselective exo-Diels-Alder reaction between alkenoyl-1,3-oxazolidin-2-ones and cyclopentadiene. Tetrahedron. 2002;58:2929–2935.
Evans David A, Miller Scott J, Lectka Thomas, von Matt Peter. Chiral Bis(oxazoline)copper(II) Complexes as Lewis Acid Catalysts for the Enantioselective Diels–Alder Reaction. JACS. 1999;121:7559–7573.
Optical rotation was determined for the endo cycloadduct: [α]D20 = 153.2 (c = 0.83 in CHCl3); the optical rotation of the enantiomer has been reported: [α]D20 = −160 (c = 0.83 in CHCl3.
Optical rotation was determined for the exo cycloadduct: [α]D20 = −17.8 (c = 0.54 in CHCl3), Lit. [α]D25 = −17.3 (c = 0.54 in CHCl3).
Fuerst Douglas E, Jacobsen Eric N. J. Am. Chem. Soc. 2005;127:8964–8965. [PMC free article: PMC3166885] [PubMed: 15969569]
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Streit AK, Netter MF, Kempf F, Walecki M, Rinne S, Bollepalli MK, Renigunta V, Daut T, Baukrowitz T, Sansom MSP, Stansfeld PJ, Decher N. A Specific Two-pore Domain Potassium Channel Blocker Defines the Structure of the TASK-1 Open Pore. J Biol Chem. 2011;286:13977–13994. [PMC free article: PMC3077598] [PubMed: 21362619]
Brendel J, Goegelein H, Wirth K, Kamm W. Inhibitors of the TASK1 and TASK3 Ion Channel. US20090149496. 2009 (Sanofi-Aventis)
Coburn CA, Luo Y, Cui M, Wang J, Soll R, Dong J, Hu B, Lyon MA, Santarelli VP, Kraus RL, Gregan Y, Wang Y, Fox SV, Binns J, Doran SM, Reiss DR, Tannenbaum PL, Gotter AL, Meinke PT, Renger JJ. Discovery of a pharmacologically active antagonist of the two-pore-domain potassium channel K2P9.1 (TASK-3) ChemMedChem. 2012;7:123–133. [PubMed: 21916012]


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