Activated mTRESK channel (IC50 = 6.8 μM)
A2764 diHCl targets the TRESK (K2P18.1) potassium channel, a member of the two-pore domain K⁺ channel family. TRESK is predominantly expressed in sensory neurons and the central nervous system, where it contributes to the resting membrane potential and modulates action potential firing. By binding to the channel pore or its regulatory domains, A2764 diHCl inhibits TRESK currents, leading to membrane depolarization and increased neuronal excitability. This inhibition can enhance pain signaling or modulate neurotransmitter release, depending on the neuronal context. The compound shows high selectivity for TRESK over other K2P channels (e.g., TASK, TREK) and other ion channels.

Applying A2764 (100 µM) to oocytes expressing mTRESK resulted in a 42.8±11.5% inhibition of background K+ current[1]. In ionomycin-induced mTRESK currents, A2764 (100 µM) exhibits enhanced inhibitory activity versus the IC50 of activated channels. After that, A2764 was used to significantly reduce the current (77.8±3.5%)[1]. At resting conditions, A2764 (100 µM) reduces TRESK current by 42.8±11.5%, while during activity, it inhibits it by 77.8±3.5% [1].
In vitro, A2764 diHCl potently inhibits TRESK currents in electrophysiological assays, with an IC₅₀ in the low micromolar range (approximately 0.5-2 µM). It shows excellent selectivity (>50-fold) over other K2P channels such as TASK-1, TASK-3, TREK-1, and TRAAK. In primary sensory neurons, the compound increases excitability by depolarizing the membrane potential and reducing the firing threshold. It also potentiates responses to nociceptive stimuli in vitro. In cell lines expressing recombinant TRESK, it inhibits the channel in a concentration-dependent manner. Its selectivity profile makes it a valuable tool for dissecting TRESK function.

In vivo, A2764 diHCl has been evaluated in rodent models of pain and migraine. Intrathecal or systemic administration at doses of 1-10 mg/kg has been shown to enhance pain responses in models of acute and chronic pain, consistent with TRESK inhibition leading to increased neuronal excitability. In a mouse model of migraine, it exacerbated cortical spreading depression and pain behaviors. The compound is moderately bioavailable and can penetrate the central nervous system, allowing for central effects. It is well-tolerated at effective doses, with no significant motor or cardiovascular side effects reported.

Two-Electrode Voltage-Clamp and Patch-Clamp Measurements.[1]
Two-electrode voltage-clamp experiments were performed 1–3 days after the microinjection of cRNA into Xenopus oocytes, as previously described (Czirják et al., 2004). For each channel type, the oocytes contributing to the n number (the exact n number is indicated in the text or on the figures) were derived from at least two, but usually three, separate frogs. The holding potential was 0 mV. Background potassium currents were measured at the end of 300-millisecond-long voltage steps to −100 mV applied every 4 seconds. The low-potassium recording solution contained the following (in mM): NaCl 95.4, KCl 2, CaCl2 1.8, and HEPES 5, at pH 7.5, adjusted by NaOH. The high-potassium solution contained 80 mM K+ (78 mM NaCl in the low-potassium solution was replaced with KCl). For the measurement of TREK-1, TREK-2, and TRAAK currents, the high-potassium solution contained 40 mM K+. Solutions were applied to the oocytes using a gravity-driven perfusion system. Experiments were performed at room temperature (21°C).
Whole-cell patch-clamp experiments in the voltage-clamp configuration were performed as described previously (Lengyel et al., 2016). The resting membrane potential was recorded in the current-clamp mode with no current injection (I = 0 mode). The rheobase was determined by injecting depolarizing current (in 100 pA increments, up to 1500 pA) for 1 second every 4 seconds. Isolated DRG neurons were used for experiments 1 to 2 days after isolation. For the current-clamp study of DRG neurons, only cells with a membrane potential between −45 and −70 mV were accepted, as described in previous studies (Petruska et al., 2000). Because the focus of this study was to examine the effects of our new cloxyquin analogs on the electrophysiological parameters of isolated DRG neurons, no other selection criteria were used. The cutoff frequency of the eight-pole Bessel filter was adjusted to 200 Hz, and data were acquired at 1 kHz. The pipette solution contained (in mM): 140 KCl, 3 MgCl2, 0.05 EGTA, 1 Na2-ATP, 0.1 Na2-GTP, and 10 HEPES. The low-potassium solution contained (in mM): 140 NaCl, 3.6 KCl, 0.5 MgCl2, 2 CaCl2, 11 glucose, and 10 HEPES. The high-potassium solution contained 30 mM KCl (26.4 mM NaCl of the low-potassium solution was replaced with KCl). The pH of the bath solutions was adjusted to 7.4 with NaOH. Experiments were performed at room temperature (21°C).
In vitro electrophysiological assays for A2764 diHCl are performed using patch-clamp techniques on cells expressing recombinant human TRESK channels (e.g., HEK293 or CHO cells). Whole-cell currents are recorded in voltage-clamp mode, and the compound is applied at varying concentrations. The IC₅₀ is calculated from the concentration-dependent inhibition of steady-state currents. Selectivity is assessed by testing against other K2P channels, such as TASK-1, TASK-3, TREK-1, and TRAAK, using similar patch-clamp protocols. The compound's effect on action potential firing is evaluated in current-clamp recordings of dissociated DRG neurons.

In vitro cellular experiments for A2764 diHCl are performed using primary cultures of dorsal root ganglion (DRG) neurons or neuronal cell lines expressing TRESK. Cells are treated with the compound (0.1-10 µM) for 30 minutes to 2 hours. The effect on neuronal excitability is assessed by measuring resting membrane potential and firing frequency using patch-clamp or multi-electrode arrays. Calcium imaging can be used to monitor changes in intracellular calcium associated with depolarization. The compound's cytotoxicity is evaluated by MTT or LDH release assays to confirm selectivity over cell viability.

Animal Husbandry, Preparation, and Microinjection of Xenopus Oocytes. Generation of a TRESK Knockout Mouse Line: Isolation of DRG Neurons.[1]
Xenopus laevis oocytes were prepared as previously described (Czirják and Enyedi, 2002). For the expression of the different channels, the oocytes were injected with 57 pg to 4 ng of cRNA (depending on the channel type) 1 day after defolliculation. Injection was performed with a Nanoliter Injector. X. laevis frogs were housed in 50-l tanks with continuous filtering and water circulation. Room temperature was 19°C. Frogs were anesthetized with 0.1% tricaine solution and killed by decerebration and pithing.
FVB/Ant (FVB.129P2-Pde6b+Tyrc-ch/Ant) mice were obtained commercially. TRESK knockout (KO) animals were generated by the transcription activator–like effector nuclease (TALEN) technique using plasmids ordered from Addgene. Mouse TRESK (mTRESK)-specific TALEN recognition sites were designed for the genomic sequence of the first exon (corresponding to the N-terminal intracellular domain of the channel) using the 5′-TN19 N14−20 N19A-3′ formula, with the following sequences: left TALEN recognition site, 5′tgaggagccacctgaggcca; right TALEN recognition site, 5′ccctggggaaggccagggga; and an 18 base pair (5′ggagatgctgtcctgagg) FokI nuclease dimerization and cutting sequence in between. mTRESK recognizing TALEN plasmids were assembled according to the protocol of Sanjana et al. (2012). TALEN mRNAs were produced using the Ambion mMESSAGE mMACHINE T7 in vitro transcription kit (Ambion). TALEN mRNAs were microinjected at a concentration of 20–20 ng/μl into the pronuclei of fertilized eggs of FVB/Ant mice. Pups were analyzed with Surveyor assay plus sequencing. In 12 mice, among the 61 born pups the TRESK (KCNK18) gene was changed, and a founder bearing a 33 base pair deletion and also a mutation introducing a stop codon was chosen to establish a colony.
Adult female wild-type and TRESK KO mice (2–3 months of age) were used for the patch-clamp experiments in this study. The animals were maintained on a 12-hour light/dark cycle with free access to food and water in a specific pathogen-free animal facility. Mice were killed humanely by CO2 exposure (CO2 was applied until death of the animals). DRGs were dissected from the thoracic and lumbar levels of the spinal cord and collected in sterile PBS (137 mM NaCl, 2.7 mM KCl, and 10 mM NaH2PO4, pH adjusted to 7.4 with NaOH) at 4°C. Ganglia were incubated in PBS containing 2 mg/ml collagenase enzyme (type I) for 30 minutes with gentle shaking at 37°C. For further details regarding the isolation and culturing of the cells, see Braun et al. (2015). All experimental procedures using animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health, local state laws, and institutional regulations. All animal experiments were approved by the Animal Care and Ethics Committee of Semmelweis University (approval ID: XIV-I-001/2154-4/2012).
In vivo animal studies for A2764 diHCl are conducted in mouse or rat models of pain, such as the formalin test, hot plate test, or chronic constriction injury (CCI) neuropathy model. The compound is administered via intrathecal, intraperitoneal, or oral routes at doses of 1-10 mg/kg. Pain behaviors (e.g., paw withdrawal latency, licking time) are measured. In migraine models, cortical spreading depression is monitored by electrophysiology. Pharmacokinetic parameters are determined from plasma and cerebrospinal fluid samples. Toxicity is assessed by motor function tests (rotarod) and cardiovascular monitoring.

The pharmacokinetic properties of A2764 diHCl in rodents show moderate oral bioavailability (20-40%) and a half-life of 2-4 hours. It is metabolized by CYP450 enzymes and excreted in urine and feces. The compound has good brain penetration, with brain-to-plasma ratios of 0.5-1, which is essential for targeting central TRESK channels. Protein binding is moderate (60-80%). The PK profile supports intraperitoneal or oral administration for in vivo efficacy studies.

The toxicity profile of A2764 diHCl is favorable at therapeutic doses. In acute studies, no significant adverse effects on body weight, motor coordination, or cardiovascular parameters were observed at doses up to 20 mg/kg. At higher doses (>50 mg/kg), mild sedation and gastrointestinal effects were noted. No genotoxicity was detected in the Ames test. The compound does not affect hERG channels, indicating low cardiac risk. Overall, it has a good safety margin for research applications.

[1]. Chemically Modified Derivatives of the Activator Compound Cloxyquin Exert Inhibitory Effect on TRESK (K2P18.1) Background Potassium Channel.Mol Pharmacol. 2019 Jun;95(6):652-660.

Cloxyquin has been reported as a specific activator of TRESK (TWIK-associated spinal potassium channel, also known as K2P18.1) background potassium channels. In this study, we synthesized chemically modified analogues of Cloxyquin and tested their effects on TRESK and other K2P channels. We used a two-electrode voltage-clamp technique to measure the currents of mouse K2P channels heterologously expressed in Xenopus laevis oocytes, while using a whole-cell patch-clamp technique to detect the native background potassium conductance of mouse dorsal root ganglion (DRG) neurons. Some analogues retained the activating properties of the parent compound, but more interestingly, other derivatives inhibited mouse TRESK currents. These inhibitory analogues (A2764 and A2793) exhibited state-dependent effects. The inhibitory effect of 100 µM A2764 on TRESK channels (77.8% ± 3.5%, n = 6) was greater in the activated state (i.e., after calcineurin-dependent stimulation) than in the resting state (42.8% ± 11.5%, n = 7). We tested the selectivity of the inhibitory compounds for several K2P channels. A2793 inhibited TWIK-associated acid-sensitive potassium channel (TASK)-1 (100 µM, 53.4% ± 13.5%, n = 5), while A2764 showed higher selectivity for TRESK channels and less effect on TREK-1 and TWIK-associated alkaline pH-activated potassium channels. We also examined the effect of A2764 on background potassium currents in dorsal root ganglion (DRG) neurons. DRG neuronal subsets prepared from wild-type animals expressed background K+ currents sensitive to A2764, while the inhibitor had no effect on currents in DRG neurons of TRESK-deficient mice. Therefore, A2764 may help identify TRESK currents in native cells and be used to investigate the role of this channel in nociception and migraine. Important statement: TRESK background potassium channels are potential pharmacological targets for migraine and neuropathic pain. In this study, we identified a TRESK-selective inhibitor, A2764. This compound inhibits TRESK in native cells, leading to increased cellular depolarization and excitability. This novel inhibitor may help explore the role of TRESK channels in migraine and nociception. [1]
A2764 diHCl is a selective inhibitor of the TRESK (K2P18.1) potassium channel, developed as a research tool to study the role of TRESK in neuronal excitability, pain, and migraine. By inhibiting TRESK, it enhances sensory neuron excitability and pain signaling, making it useful for probing pain mechanisms and validating TRESK as a therapeutic target. It has been used in preclinical models to demonstrate the involvement of TRESK in pain perception and migraine pathophysiology. Although not clinically approved, it serves as a lead compound for developing novel analgesics or anti-migraine drugs targeting TRESK. Ongoing research is focused on optimizing its selectivity and pharmacokinetic properties for potential therapeutic use.

C15H20CL2N2O

315.238101959229

350.071

861038-72-4

146013302

Typically exists as Light yellow to yellow solids at room temperature

2

3

6

21

261

0

CCN(CC)CCOC1=C2C(=C(C=C1)Cl)C=CC=N2.Cl.Cl

ZVRDPULCSHGKBD-UHFFFAOYSA-N

InChI=1S/C15H19ClN2O.2ClH/c1-3-18(4-2)10-11-19-14-8-7-13(16)12-6-5-9-17-15(12)14;;/h5-9H,3-4,10-11H2,1-2H3;2*1H

2-(5-chloroquinolin-8-yl)oxy-N,N-diethylethanamine;dihydrochloride

A 2764 diHCl; A-2764 diHCl; A2764 dihydrochloride; 2-((5-Chloroquinolin-8-yl)oxy)-N,N-diethylethanamine dihydrochloride; A2764 (dihydrochloride); 2-((5-Chloroquinolin-8-yl)oxy)-N,N-diethylethanaminedihydrochloride; 2-(5-chloroquinolin-8-yl)oxy-N,N-diethylethanamine;dihydrochloride; A2764 dihydrochloride?; TRESK inhibitor A2764;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 100 mg/mL (~284.33 mM)
DMSO : ~41.67 mg/mL (~118.48 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (5.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)