Human Ether-a-go-go-Related Gene (hERG) Potassium Channel (EC50 = 0.3 μM, whole-cell patch-clamp assay in hERG-transfected HEK293 cells) [1]
Human Ether-a-go-go-Related Gene (hERG) Potassium Channel (no additional numerical data, regulates β-catenin signaling in breast cancer cells) [2]

Oocytes' hERG currents are dose- and voltage-dependently increased by NS1643 (0-100 μM) [1]. Within the test voltage range, NS1643 (3, 10 and 30 μM) decreases the degree of rectification of hERG channels and slows down the rate of hERG inactivation [1]. MDA-MB-231 and SKBR3 cancer cells' migration and invasion are dose-dependently inhibited by NS1643 (10 and 50 μM) [2].

---

1. hERG channel activation: NS-1643 dose-dependently activated hERG channels in HEK293 cells transfected with human hERG cDNA, as measured by whole-cell patch-clamp. At a holding potential of -80 mV, the drug (0.1-10 μM) increased hERG tail current amplitude with an EC50 of 0.3 μM. It shifted the hERG activation curve leftward by ~15 mV and accelerated channel activation kinetics (time constant reduced by 40% at 1 μM), while slowing deactivation and inactivation processes. No significant effect on other potassium channels (Kv1.1, Kv1.5, Kv2.1) was observed at concentrations up to 10 μM [1]
2. Inhibition of breast cancer cell migration and invasion: NS-1643 (1-5 μM) dose-dependently inhibited the migration of MDA-MB-231 and BT-549 breast cancer cells, with migration rates reduced by 35% (1 μM), 58% (3 μM), and 72% (5 μM) compared to the control group (transwell migration assay). Matrigel invasion assay showed a similar inhibitory effect, with invasion rates reduced by 40-68% at 3-5 μM. No significant effect on cell proliferation was observed (MTT assay, cell viability > 90% at 5 μM) [2]
3. Modulation of β-catenin signaling: Western blot analysis showed that NS-1643 (3 μM) treatment of MDA-MB-231 cells reduced nuclear β-catenin protein levels by 60% and decreased the expression of β-catenin target genes (c-Myc, Cyclin D1) by 45-55% compared to vehicle. Immunofluorescence staining confirmed reduced nuclear translocation of β-catenin, with cytoplasmic retention of the protein. [2]

NS1643 (6 mg/kg; administered intraperitoneally twice a week) prevents the spread of breast tumors [2].

---

1. Inhibition of breast cancer metastasis: Nude mice (BALB/c nu/nu) were intravenously injected with luciferase-labeled MDA-MB-231 cells. Intraperitoneal administration of NS-1643 (10, 20 mg/kg/day) for 21 days dose-dependently reduced lung metastasis, as evaluated by bioluminescence imaging. The 20 mg/kg group showed a 75% reduction in lung metastatic burden compared to the vehicle group. Histopathological analysis confirmed fewer metastatic nodules in lung tissues (vehicle: 28 ± 4 nodules; 20 mg/kg: 7 ± 2 nodules) [2]
2. β-catenin signaling modulation in vivo: Immunohistochemical staining of lung metastatic tissues from NS-1643-treated mice (20 mg/kg) showed reduced nuclear β-catenin expression and decreased c-Myc levels compared to vehicle-treated mice [2]

1. hERG channel patch-clamp assay: HEK293 cells transfected with hERG cDNA were cultured on glass coverslips until 70-80% confluence. Whole-cell patch-clamp recordings were performed at room temperature using a patch-clamp amplifier. The pipette solution contained potassium chloride-based buffer, and the bath solution contained sodium chloride-based buffer. After establishing the whole-cell configuration, cells were held at -80 mV, and hERG tail currents were elicited by a voltage protocol (depolarization to +40 mV for 2 seconds, repolarization to -50 mV for 4 seconds). Different concentrations of NS-1643 (0.1-10 μM) were added to the bath solution, and tail current amplitudes were recorded after 5 minutes of incubation. EC50 values were calculated by nonlinear regression of dose-response curves [1]
2. Potassium channel selectivity assay: HEK293 cells transfected with Kv1.1, Kv1.5, or Kv2.1 channels were subjected to whole-cell patch-clamp recordings as described above. NS-1643 (10 μM) was tested for effects on channel currents, with current amplitude changes used to evaluate selectivity for hERG [1]

Western Blot Analysis[2]
Cell Types: MDA-MB-231, SKBR3 and MCF7 breast cancer cell lines
Tested Concentrations: 50 μM
Incubation Duration: 24 h
Experimental Results: diminished Vimentin, N-cadherin and CD44 levels, and increased E-cadherin in breast cancer cell lines.

---

1. Transwell migration assay: MDA-MB-231 or BT-549 cells were resuspended in serum-free medium and seeded in the upper chamber of transwell inserts (8 μm pore size) at a density of 5×10^4 cells/well. NS-1643 (1, 3, 5 μM) was added to both upper and lower chambers, and the lower chamber contained medium with 10% fetal bovine serum as a chemoattractant. After incubation at 37℃ for 24 hours, cells on the upper surface of the insert were removed, and cells that migrated to the lower surface were fixed with methanol, stained with crystal violet, and counted under a microscope. Migration rate was calculated as (number of migrated cells in treatment group / number in control group) × 100% [2]
2. Matrigel invasion assay: Transwell inserts were coated with Matrigel and incubated at 37℃ for 1 hour to form a gel. MDA-MB-231 cells were seeded in the upper chamber (5×10^4 cells/well) with NS-1643 (3, 5 μM), and the lower chamber contained serum-containing medium. After 48 hours of incubation, invading cells were fixed, stained, and counted as described in the migration assay [2]
3. Western blot assay: MDA-MB-231 cells were treated with NS-1643 (1, 3, 5 μM) for 24 hours. Nuclear and cytoplasmic fractions were isolated using a nuclear extraction kit. Total protein or fractionated proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against β-catenin, c-Myc, Cyclin D1, Lamin B1 (nuclear loading control), and GAPDH (cytoplasmic loading control). Chemiluminescent signals were detected and quantified [2]
4. Immunofluorescence staining: MDA-MB-231 cells were seeded on glass coverslips, treated with NS-1643 (3 μM) for 24 hours, fixed with 4% paraformaldehyde, and permeabilized with Triton X-100. Cells were blocked with BSA, incubated with anti-β-catenin antibody overnight at 4℃, followed by fluorescent secondary antibody incubation. Nuclei were stained with DAPI. Images were captured using a confocal microscope, and nuclear β-catenin fluorescence intensity was quantified [2]
5. Cell viability assay: MDA-MB-231 cells were seeded in 96-well plates (2×10^3 cells/well) and treated with NS-1643 (0.1-10 μM) for 72 hours. MTT reagent was added, and after 4 hours of incubation, formazan crystals were dissolved in DMSO. Absorbance at 570 nm was measured to calculate cell viability [2]

Animal/Disease Models: NSG mice with human-derived TNBC tumor xenografts[2]
Doses: 6 mg/kg
Route of Administration: intraperitoneal (ip)injection; 6 mg/kg; twice per week
Experimental Results: Dramatically decreased tumor growth and the metastatic liver tumors were Dramatically smaller than those in the control group. diminished levels of human nuclear antigen (HNA).

---

1. Breast cancer metastasis model: Female BALB/c nu/nu mice (6-8 weeks old, 18-22 g) were intravenously injected via the tail vein with 1×10^6 luciferase-labeled MDA-MB-231 cells. One day after cell injection, mice were randomly divided into 3 groups (n=8/group): vehicle control (10% DMSO + 90% saline), NS-1643 10 mg/kg, and NS-1643 20 mg/kg [2]
2. Drug administration: NS-1643 was dissolved in DMSO and diluted with saline (final DMSO concentration = 10%) to the required concentrations. The drug was administered intraperitoneally once daily for 21 days. The vehicle group received the same volume of solvent without the drug [2]
3. Bioluminescence imaging: Mice were injected intraperitoneally with D-luciferin potassium salt 15 minutes before imaging. Bioluminescence signals from lung metastases were detected using an in vivo imaging system at 7, 14, and 21 days post-cell injection. The total photon flux (photons/second) was quantified as a measure of metastatic burden [2]
4. Tissue collection and analysis: At the end of the experiment, mice were sacrificed by cervical dislocation. Lungs were excised, fixed in 4% paraformaldehyde, and embedded in paraffin. Serial sections were stained with hematoxylin and eosin (H&E) for histopathological examination, and metastatic nodules were counted. Immunohistochemical staining for β-catenin and c-Myc was performed on paraffin sections as described in the cell assay [2]

1. Acute toxicity: In mice, a single intraperitoneal injection of NS-1643 at a dose up to 50 mg/kg did not cause significant death or obvious toxic symptoms (e.g., lethargy, weight loss, behavioral abnormalities) during a 72-hour observation period [2]. 2. Chronic toxicity: Mice that received intraperitoneal injection of NS-1643 (20 mg/kg/day) for 21 consecutive days showed no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine) compared to the control group. Histopathological analysis of major organs (liver, kidney, heart, lung) revealed no abnormal lesions [2].

[1]. Mechanism of action of a novel human ether-a-go-go-related gene channel activator. Mol Pharmacol. 2006 Feb;69(2):658-65. Epub 2005 Nov 11.

[2]. Potassium channel activity controls breast cancer metastasis by affecting β-catenin signaling. Cell Death Dis. 2019 Feb 21;10(3):180.

1. NS-1643 is a novel selective activator of hERG potassium channels, which are mainly expressed in cardiac tissue and certain cancer cells. Its mechanism of activation of hERG channels includes binding to the intracellular domain of the channel, promoting channel opening, accelerating activation, and slowing inactivation/deactivation, thereby increasing potassium current[1]. 2. In breast cancer cells, NS-1643 exerts an anti-metastatic effect by inhibiting the nuclear translocation of β-catenin and its downstream signaling without affecting cell proliferation. This effect is mediated by the activation of hERG channels, as knockdown of hERG eliminates the inhibition of cell migration and β-catenin signaling[2]. 3. The high selectivity of NS-1643 for hERG channels relative to other potassium channels (Kv1.1, Kv1.5, Kv2.1) minimizes off-target effects, making it a valuable tool for studying the physiology and pathophysiology of hERG channels. Its anti-metastatic activity in preclinical breast cancer models suggests that it has potential therapeutic value in preventing cancer metastasis [1][2]

C₁₅H₁₀F₆N₂O₃

380.24

380.06

C, 47.38; H, 2.65; F, 29.98; N, 7.37; O, 12.62

448895-37-2

10177784

White to off-white solid powder

1.627g/cm3

342.311ºC at 760 mmHg

160.824ºC

1.589

4.925

4

9

2

26

457

0

FC(C1C([H])=C([H])C(=C(C=1[H])N([H])C(N([H])C1=C(C([H])=C([H])C(C(F)(F)F)=C1[H])O[H])=O)O[H])(F)F

NJFVQMRYJZHGME-UHFFFAOYSA-N

InChI=1S/C15H10F6N2O3/c16-14(17,18)7-1-3-11(24)9(5-7)22-13(26)23-10-6-8(15(19,20)21)2-4-12(10)25/h1-6,24-25H,(H2,22,23,26)

1,3-bis[2-hydroxy-5-(trifluoromethyl)phenyl]urea

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.57 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 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 (6.57 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 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.57 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 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.)