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| 10mg |
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| Targets |
The primary targets of NS309 are small- and intermediate-conductance calcium-activated potassium (KCa2/SK and KCa3.1/IK) channels. It acts as a potent positive modulator of these channels, increasing their calcium sensitivity and enhancing their activity. NS309 has EC50 values of 0.62 μM for SK2, 0.3 μM for SK3, approximately 20 nM for KCa3.1/SK4, approximately 600 nM for KCa2/SK channels, and 10 nM for hIK channels. The compound exhibits no activity at large-conductance calcium-activated potassium (BK) channels. By enhancing SK and IK channel activity, NS309 increases the amplitude of the afterhyperpolarization that follows action potentials, thereby modulating neuronal firing patterns and reducing neuronal excitability.
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| ln Vitro |
hSK3 and hIK channels are activated by NS309 (40 nM), with hIK channels experiencing the largest effects (an 8.5-fold increase in current and a 1.9-fold rise in hSK3 channel current) [2]. The whole-cell SK currents are greatly increased and the resting membrane potential of the detrusor smooth muscle (DSM) cell is hyperpolarized by NS309 (10 μM) [5]. In a concentration-dependent manner, NS309 suppresses the amplitude, force, frequency, duration, pitch, and spontaneous phasic contraction of isolated DSM strips [5].
In vitro, NS309 is a potent activator of SK and IK channels. It has EC50 values of 0.62 μM for SK2, 0.3 μM for SK3, approximately 20 nM for KCa3.1/SK4, approximately 600 nM for KCa2/SK channels, and 10 nM for hIK channels. The compound is 730-fold (SK channels) and 7,400-fold (IK channels) more potent than 1-EBIO. NS309 (10 μM) significantly increases whole-cell SK currents and hyperpolarizes the resting membrane potential of detrusor smooth muscle (DSM) cells. These effects are consistent with the compound's mechanism of action as a positive modulator of SK and IK channels, enhancing the calcium sensitivity of these channels and increasing their activity. |
| ln Vivo |
In rabbits, SCI/R is prevented by NS309 (2 mg/kg; intraperitoneal injection) [6].
In vivo, NS309 is used as a pharmacological tool to study the role of SK and IK channels in various physiological and pathological processes. By enhancing SK and IK channel activity, NS309 modulates neuronal firing patterns and reduces neuronal excitability. The compound's potent activity and selectivity for SK and IK channels over BK channels make it a valuable tool for dissecting the specific roles of these channel subtypes in neuronal function, smooth muscle contraction, and immune cell activation. While specific in vivo efficacy data for NS309 are not extensively detailed in the available literature, its mechanism of action suggests that it would be effective in modulating neuronal excitability and smooth muscle tone. |
| Enzyme Assay |
In vitro enzyme/receptor binding assays for NS309 are not conventional enzyme inhibition studies, as the compound is a positive modulator of ion channels rather than an enzyme inhibitor. Instead, the primary assays involve electrophysiological recordings using patch-clamp or two-electrode voltage clamp techniques in cells expressing SK or IK channels. In these assays, NS309 is applied to cells, and the amplitude of SK- or IK-mediated currents is measured in the presence of various concentrations of calcium. The EC50 values are determined from dose-response curves. Radioligand binding studies using channel-specific ligands can also be performed to assess the compound's binding affinity and to confirm its allosteric mechanism of action.
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| Cell Assay |
In vitro cellular assays for NS309 are conducted in cells expressing SK or IK channels, such as HEK293 cells, neuronal cell lines, or smooth muscle cells. Cells are treated with varying concentrations of NS309, and SK or IK channel activity is assessed using calcium imaging, fluorescent membrane potential dyes, or electrophysiological recordings. The compound's ability to enhance SK- or IK-mediated currents is quantified by measuring the increase in current amplitude or the shift in the calcium dose-response curve. These assays confirm that NS309 engages its target in a cellular context and produces the expected enhancement of SK and IK channel activity.
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| Animal Protocol |
Animal/Disease Models: Adult New Zealand White Rabbit (2.5-3.0 kg) [6]
Doses: 2 mg/kg Route of Administration: intraperitoneal (ip) injection Experimental Results: Neurological outcomes in SCI/R-challenged rabbits were Dramatically improved. In vivo animal studies for NS309 would typically be conducted in animal models of neurological or cardiovascular diseases where SK and IK channels are implicated. Animals would be administered the compound, and outcomes would be assessed based on the specific disease model. For example, in models of epilepsy, the compound's ability to reduce neuronal excitability could be assessed by measuring seizure frequency and severity. In models of hypertension, the compound's effects on vascular tone could be assessed by measuring blood pressure. These studies would confirm that NS309 is effective in vivo and provide information about its therapeutic potential. |
| ADME/Pharmacokinetics |
Pharmacokinetic properties of NS309 indicate that it has a molecular weight of 231.04 and a molecular formula of C8H4Cl2N2O2. The compound is soluble in DMSO at a concentration of ≥50 mg/mL, facilitating its use in in vitro assays and formulation for in vivo administration. For storage, the powder should be kept at -20°C for up to 3 years or at 4°C for up to 2 years. The compound's purity is typically ≥98%, ensuring high quality and reproducibility in experimental studies.
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| Toxicity/Toxicokinetics |
The toxicological profile of NS309 is primarily derived from its use as a research compound in preclinical studies. As a modulator of SK and IK channels, potential on-target effects could include changes in neuronal excitability, smooth muscle tone, and immune cell function, given the roles of these channels in these systems. Comprehensive toxicology studies would be required for therapeutic development, including assessments of neurological, cardiovascular, and immune function. However, NS309 is primarily used as a research tool and has not yet advanced to clinical development.
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| References |
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| Additional Infomation |
NS309 is a potent, nonselective activator of small- and intermediate-conductance calcium-activated potassium (SK and IK) channels. It has EC50 values of 0.62 μM for SK2, 0.3 μM for SK3, approximately 20 nM for KCa3.1/SK4, approximately 600 nM for KCa2/SK channels, and 10 nM for hIK channels. NS309 exhibits no activity at BK channels. The compound is 730-fold (SK channels) and 7,400-fold (IK channels) more potent than 1-EBIO. NS309 is often used in conjunction with CyPPA to distinguish between SK1 and SK3 subtypes. The compound is not approved for clinical use and is available from research chemical suppliers for preclinical studies.
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| Molecular Formula |
C8H4CL2N2O2
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| Molecular Weight |
231.032
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| Exact Mass |
229.964
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| CAS # |
18711-16-5
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| Related CAS # |
18711-16-5
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| PubChem CID |
135502903
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| Appearance |
White to off-white solid powder
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| Density |
1.8±0.1 g/cm3
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| Index of Refraction |
1.742
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| LogP |
2.66
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
14
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| Complexity |
241
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
CVOUSAVHMDXCKG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C8H4Cl2N2O2/c9-4-2-1-3-6(5(4)10)11-8(13)7(3)12-14/h1-2,14H,(H,11,12,13)
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| Chemical Name |
(Z)-6,7-dichloro-3-(hydroxyimino)indolin-2-one
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| Synonyms |
NS309 NS-309 NS 309.
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~432.83 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.3284 mL | 21.6422 mL | 43.2844 mL | |
| 5 mM | 0.8657 mL | 4.3284 mL | 8.6569 mL | |
| 10 mM | 0.4328 mL | 2.1642 mL | 4.3284 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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