ICA-069673 (ICA73) is a selective KCNQ2/Q3 potassium channel activator with IC50 of 0.69 μM. KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are voltage-gated K(+) channel subunits that underlie the neuronal M current. In humans, mutations in these genes lead to a rare form of neonatal epilepsy, suggesting that KCNQ2/Q3 channels may be attractive targets for novel antiepileptic drugs. ICA069673 acts on a binding site in the voltage-sensing domain that is distinct from the putative retigabine (a KCNQ voltage-gated potassium channel opener that was recently approved as an add-on therapeutic for patients with drug-resistant epilepsy) site in the channel pore. ICA069673 has two separable effects on KCNQ channel activity.

Physicochemical Properties

Molecular Formula	C11H6CLF2N3O
Molecular Weight	269.64
Exact Mass	269.017
CAS #	582323-16-8
Related CAS #	582323-16-8
PubChem CID	10149311
Appearance	White to off-white solid powder
LogP	3.044
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	2
Heavy Atom Count	18
Complexity	300
Defined Atom Stereocenter Count	0
InChi Key	IIBSHMFXVWTQSJ-UHFFFAOYSA-N
InChi Code	InChI=1S/C11H6ClF2N3O/c12-11-15-4-7(5-16-11)17-10(18)6-1-2-8(13)9(14)3-6/h1-5H,(H,17,18)
Chemical Name	N-(2-chloro-5-pyrimidinyl)-3,4-difluoro-benzamide
Synonyms	ICA-069673; ICA069673; ICA 069673; ICA73; ICA-73
HS Tariff Code	2934.99.9001
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)

Biological Activity

Targets	The target of ICA069673 is the voltage-gated potassium channel subunits KV7.2 (KCNQ2) and KV7.3 (KCNQ3). [1]
ln Vitro	In guinea pig DSM breakaway strips, ICA-069673 (100 nM-30 μM) dose-dependently suppresses spontaneous phasic contractions, contractions elicited by pharmacology, and contractions triggered neurally by 10 Hz EFS [1]. In guinea pig DSM separation strips, ICA-069673 (3 μM, 10 μM) suppresses the contraction of the DSM tetanic muscle elicited by 20 mM KCl [1]. 1. ICA069673 inhibited spontaneous phasic contractions in isolated guinea pig detrusor smooth muscle (DSM) strips in a concentration-dependent manner (100 nM–30 µM); this inhibitory effect was abolished by the KV7 channel inhibitor XE991 (10 µM), confirming the action was mediated via KV7.2/KV7.3 channels (n = 11, N = 7, #P < 0.05, ##P < 0.01, ###P < 0.001 for amplitude, force, duration, and frequency of contractions) [1] 2. The compound concentration-dependently reduced carbachol (0.1 µM)-induced phasic contractions in DSM strips (100 nM–30 µM), with significant decreases in contraction amplitude, force, duration, and frequency (n = 10, N = 7, P < 0.05, P < 0.01, P < 0.001) [1] 3. ICA069673 (100 nM–30 µM) also inhibited 20 mM KCl-induced phasic contractions in DSM strips, showing concentration-dependent suppression of contraction amplitude, force, duration, and frequency (n = 7, N = 7, P < 0.05, P < 0.01, P < 0.001) [1] 4. Under 60 mM KCl-induced depolarization, the inhibitory effect of ICA069673 (10 µM) on DSM tonic contractions was significantly attenuated, and its effect was weaker compared to the L-type Ca²⁺ channel inhibitor nifedipine (1 µM) (n = 8–9, N = 4, P < 0.05 for ICA069673; n = 6–7, N = 3, P < 0.0001 for nifedipine) [1] 5. Nerve-evoked contractions in DSM strips (stimulated by 10-Hz electrical field stimulation, EFS) were concentration-dependently inhibited by ICA069673 (100 nM–30 µM), with reduced contraction amplitude and muscle force integral (n = 9, N = 9, P < 0.01, P < 0.001); the compound also suppressed EFS-induced contractions at 7.5–50 Hz (3 µM and 10 µM doses, n = 8–18, N = 4–10, P < 0.05, P < 0.01, P < 0.001) [1] 6. In freshly isolated DSM cells, ICA069673 (10 µM) significantly decreased global intracellular Ca²⁺ levels (n = 11, N = 5, P < 0.001), and this effect was blocked by nifedipine (1 µM, n = 10, N = 4, P > 0.05) [1] 7. Using perforated whole-cell patch-clamp electrophysiology, ICA069673 (10 µM) hyperpolarized the resting membrane potential of isolated DSM cells and inhibited spontaneous action potentials; these effects were reversed by washout and attenuated by XE991 (10 µM, n = 5–7, N = 5–6, P < 0.05) [1]
ln Vivo
Cell Assay	1. Intracellular Ca²⁺ Imaging in DSM Cells: Freshly isolated guinea pig DSM cells were loaded with the Ca²⁺ indicator fura-2 AM and incubated under standard conditions. ICA069673 (10 µM) was added to the cell suspension, and the ratio of fura-2 AM fluorescent emission at 510 nm with excitation at 340 and 380 nm was measured in real-time to quantify global intracellular Ca²⁺ levels. For antagonist experiments, nifedipine (1 µM) was pre-incubated with the cells before adding ICA069673; data were presented as mean ± SEM (n = 10–11, N = 4–5) [1] 2. Patch-Clamp Electrophysiology in DSM Cells: Freshly isolated DSM cells were placed in a recording chamber, and perforated whole-cell patch-clamp recordings were performed in current-clamp mode to measure membrane potential and spontaneous action potentials. ICA069673 (10 µM) was applied to the bath solution, and changes in membrane potential and action potential firing were recorded. XE991 (10 µM) was used to block KV7 channels, and the effects of ICA069673 in the presence of XE991 were evaluated; data were summarized as the change in membrane potential (depolarization/hyperpolarization) with n = 5–7, N = 5–6 [1] 3. Immunocytochemistry for KV7.2/KV7.3 in DSM Cells: Freshly isolated DSM cells were fixed and permeabilized, then incubated with primary antibodies against KV7.2 or KV7.3 channel subunits, followed by fluorescent secondary antibodies. α-smooth muscle actin-FITC was used to label smooth muscle cells, and 4′,6-diamidino-2-phenylindole (DAPI) stained the nucleus. Confocal microscopy was used to visualize the subcellular localization of KV7.2/KV7.3; competing peptides for the primary antibodies were used as negative controls [1]
Animal Protocol
References	[1]. The Novel KV7.2/KV7.3 Channel Opener ICA-069673 Reveals Subtype-Specific Functional Roles in Guinea Pig Detrusor Smooth Muscle Excitability and Contractility. J Pharmacol Exp Ther. 2015 Sep;354(3):290-301.
Additional Infomation	1. ICA069673 is a novel KV7.2/KV7.3 channel opener with the chemical structure N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide; it was developed as a tool compound to study the functional roles of KV7.2/KV7.3 channels in detrusor smooth muscle [1] 2. The compound exerts its pharmacological effects by activating KV7.2/KV7.3 channels in DSM cells, leading to membrane hyperpolarization, inhibition of Ca²⁺ influx via L-type Ca²⁺ channels, and subsequent reduction in DSM excitability and contractility [1] 3. Western blot analysis confirmed the protein expression of KV7.2 (120 kDa) and KV7.3 (97 kDa) subunits in guinea pig DSM tissue, and immunocytochemistry showed their membrane localization in isolated DSM cells [1]

Solubility Data

Solubility (In Vitro)

DMSO:≥ 35 mg/mL Water: Ethanol:

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (9.27 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: 2.5 mg/mL (9.27 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. Solubility in Formulation 3: 2.5 mg/mL (9.27 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.7086 mL	18.5432 mL	37.0865 mL
	5 mM	0.7417 mL	3.7086 mL	7.4173 mL
	10 mM	0.3709 mL	1.8543 mL	3.7086 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.