Na+/Ca2+ exchanger (IC50 = 5.7±2.1 µM)
Inhibitor of reverse mode Na+/Ca2+ exchanger (NCXrev) in hippocampal neurons (IC50 = 5.7 ± 2.1 μM) [1]
Inhibitor of NMDA receptors in hippocampal neurons (IC50 = 13.4 ± 3.6 μM) [1]
Inhibitor of mitochondrial complex I in the respiratory chain (IC50 = 11.4 ± 2.4 μM for 2,4-DNP-stimulated respiration in neurons) [1]
Inhibitor of type 1 ryanodine receptor (RyR1) from mouse skeletal muscle (IC50 = 5.1 ± 0.9 μM for [3H]ryanodine binding) [2]
Inhibitor of type 2 ryanodine receptor (RyR2) from mouse cardiac muscle (IC50 = 13.4 ± 1.8 μM for [3H]ryanodine binding) [2]
Inhibitor of hERG potassium channels (IC50 = 88.6 ± 25.7 nM for hERG tail current) [3]
Inhibitor of native rabbit ventricular I_Kr (IC50 = 120.3 ± 27.5 nM) [3]
Inhibitor of reverse-mode NCX1 activity in prostate cancer cells [4]

KB-R7943 mesylate inhibits NMDA-induced cytosolic Ca2+ increase and blocks NMDAR-mediated ion currents with an IC50 value of 13.4±3.6 µM, but it speeds up calcium deregulation and mitochondrial depolarization in glutamate-treated neurons. The depolarizing effects of KB-R7943 on mitochondria are independent of Ca2+. With an IC50 of 11.4±2.4 µM, KB-R7943 prevents 2,4-dinitrophenol from stimulating the respiration of cultured neurons. KB-R7943 also reversibly and dose-dependently blocked NMDA-induced ion currents in addition to NCXrev. KB-R7943 confirms the inhibition of NMDA receptors seen in electrophysiological experiments[1] by inhibiting NMDA-induced increases in [Ca2+]c in a dose-dependent manner with an IC50 of 13.4±3.6 µM. Significantly reduced responses to caffeine were seen in wtRyR1-HEK 293 cells pretreated with KB-R7943 (10 μM, 10 min) dissolved in the bulk perfusion. Regarding this, KB-R7943 produced a more pronounced inhibition of caffeine-induced Ca2+ release elicited by 1 mM compared with 0.5 and 0.75 mM (60 versus 58 versus 37%, respectively, p<0.05)[2]. With IC50 values of ~89 and ~120 nM, respectively, for current tails at 40 mV after depolarizing voltage commands to +20 mV, KB-R7943 inhibits both IhERG and native IKr quickly on membrane depolarization. However, there is no preference for inactivated over activated channels when IhERG is inhibited by KB-R7943, which exhibits both time- and voltage-dependence[3].

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In cultured hippocampal neurons, 15 μM KB-R7943 accelerated delayed Ca2+ deregulation (DCD) induced by 25 μM glutamate, reducing the time to complete DCD from 817±27 s to 398±38 s. It also prevented recovery of [Ca2+] after glutamate removal and depolarized mitochondria. [1]
KB-R7943 inhibited gramicidin-induced increase in cytosolic Ca2+ concentration in hippocampal neurons with an IC50 of 5.7±2.1 μM. This effect was Na+-dependent. [1]
In electrophysiological patch-clamp experiments, 15 μM KB-R7943 inhibited whole-cell outward ion currents mediated by NCX in reverse mode (NCXrev) in cultured hippocampal neurons. [1]
KB-R7943 dose-dependently and reversibly blocked ion currents elicited by NMDA in hippocampal neurons. [1]
In cultured hippocampal neurons, KB-R7943 (15 μM) increased NAD(P)H fluorescence under resting conditions and suppressed glutamate-induced NAD(P)H oxidation, similar to the complex I inhibitor rotenone. [1]
In isolated brain mitochondria, KB-R7943 dose-dependently depolarized mitochondria supplied with complex I substrates (malate/glutamate) but not with succinate (a complex II substrate). It also inhibited respiration and Ca2+ uptake when mitochondria oxidized complex I substrates, but not succinate. [1]
In dissociated adult mouse flexor digitorum brevis (FDB) skeletal muscle fibers, 10 μM KB-R7943 reversibly attenuated electrically evoked Ca2+ transients, reducing the integrated peak value by 87.9±4.8% during 20-Hz stimulus trains. This effect was use-dependent. [2]
In HEK 293 cells stably expressing wild-type RyR1, 10 μM KB-R7943 significantly attenuated caffeine-induced Ca2+ release. [2]
In single-channel recordings, 10 μM KB-R7943 added to the cytoplasmic side reduced the open probability (Po) of reconstituted RyR1 channels by >11-fold (82±0.1% inhibition) and RyR2 channels by >10-fold (92±0.04% reduction) under activating Ca2+ conditions. [2]
KB-R7943 inhibited hERG current (IhERG) in HEK 293 cells with an IC50 of 88.6±25.7 nM. The inhibition was concentration-dependent, time- and voltage-dependent, and partially reversible. It slowed both the activation and deactivation time courses of IhERG. [3]
The inhibitory effect of KB-R7943 on IhERG was reduced by alanine mutations at Y652 (IC50 = 1.13±0.14 μM) and F656 (IC50 = 11.09±0.23 μM) in the hERG channel pore. The S624A mutation showed a modest effect (IC50 = 216.7±31 nM). [3]
KB-R7943 inhibited native I_Kr in rabbit ventricular myocytes with an IC50 of 120.3±27.5 nM. [3]
In prostate cancer PC3 and LNCaP cells, KB-R7943 at concentrations ≥10 μM dose-dependently decreased cell viability. Treatment with 30 μM KB-R7943 increased G1 phase cell cycle arrest, suppressed migration (in wound healing and transwell assays), and induced apoptosis. [4]
In PC3 and LNCaP cells, KB-R7943 increased LC3-II levels in a dose- (starting at 30 μM) and time-dependent (starting at 12 h) manner, and increased the number of eGFP-LC3 puncta and autophagosomes, indicating autophagosome accumulation. [4]
KB-R7943 (30 μM) blocked autophagic flux in PC3 cells, as combined treatment with chloroquine did not further increase LC3-II levels and P62 levels changed in a dose-dependent manner. [4]
KB-R7943 (30 μM) inhibited the PI3K/AKT/mTOR pathway (reduced p-AKT and p-mTOR) and activated the JNK pathway (increased p-JNK) in PC3 cells. The JNK inhibitor SP600125 reduced KB-R7943-induced LC3-II levels. [4]
Combined treatment of KB-R7943 (30 μM) with docetaxel (5 nM) or with autophagy inhibitors (chloroquine, wortmannin, 3-MA) reduced cell viability and increased apoptosis in prostate cancer cells compared to KB-R7943 alone. [4]

In a mouse xenograft model, daily intraperitoneal (IP) injection of KB-R7943 at 10 mg/kg significantly inhibited the growth of PC3 tumor xenografts. Immunohistochemistry of tumor tissues showed decreased Ki-67 levels and increased LC3 and cleaved caspase-3 levels. [4]

Mitochondrial respiratory rates were measured at 37°C under continuous stirring in a closed, thermostated chamber using a Clark-type oxygen electrode. The standard incubation medium contained KCl, HEPES, MgCl2, KH2PO4, and EGTA, supplemented with either pyruvate and malate or succinate and glutamate. Mitochondrial Ca2+ uptake was measured using a miniature Ca2+-selective electrode in a small chamber at 37°C under continuous stirring, with the incubation medium supplemented with BSA, ADP, and oligomycin. [1]
[3H]Ryanodine binding assays were performed by incubating microsomal membrane vesicles enriched in RyR1 or RyR2 with [3H]ryanodine in a solution containing HEPES, KCl, NaCl, and free Ca2+. The binding reaction was quenched by filtration through glass fiber filters, and non-specific binding was determined by adding excess unlabeled ryanodine. [2]
For single-channel recordings, RyR1 or RyR2 channels were reconstituted into planar lipid bilayers. Channel incorporation was induced by introducing sarcoplasmic reticulum vesicles to the cis chamber, which had a higher Cs+ concentration. Single-channel activity was measured using a patch-clamp amplifier at a holding potential applied to the trans chamber. Channel open probability, mean open times, and mean closed dwell times were calculated from the recordings. [2]
Whole-cell patch-clamp recordings of hERG current (IhERG) were conducted at room temperature. Fire-polished electrodes were fabricated from capillary glass. For IhERG measurement, the intracellular solution contained CsF, NaCl, EGTA, and HEPES; the external solution was Mg2+-free. For NCX-mediated currents, the electrode solution contained KCl, K-aspartate, tetraethylammonium-Cl, HEPES, K-EGTA, MgCl2, and Na-ATP; the external solution contained NaCl, CsCl, KCl, MgCl2, CaCl2, glucose, Na-HEPES, sucrose, nifedipine, ouabain, and TTX. A perfusion fast-step system was used to deliver drugs focally onto isolated neurons. [1]
Docking simulations of KB-R7943 into an open-state hERG pore homology model were performed using Flexidock software. The binding pocket comprised amino acid residues T623, S624, V625, G648, Y652, F656, and S660. Rotations of side chain bonds within these residues were allowed during docking. [3]

In Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 100 g/mL streptomycin, 100 U/mL penicillin, 1 mM sodium pyruvate, and 10% fetal bovine serum at 37°C under 5% CO2, EK 293 cells stably expressing the wtRyR1 (wtRyR1-HEK 293) are kept alive. wtRyR1-HEK 293 cells are loaded with 5 μM Fluo-4 acetoxymethyl ester at 37°C for 30 min to measure Ca2+ transients in an imaging buffer consisting of 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4, supplemented with 0.05% bovine serum albumin. Three imaging buffer washes are performed on the cells, and they are then left to sit at room temperature for an additional 20 minutes. An IX-71 microscope is equipped with a charge-coupled device camera with a 40× objective lens for imaging dye-loaded cells after they have been washed three times with imaging buffer. EasyRatioPro is used to record and keep track of the image sequence. AutoMate Scientific is used to focally apply caffeine that has been dissolved in the imaging buffer for 15 seconds. Caffeine is applied after KB-R7943 has been dissolved in the imaging buffer and incubated with wtRyR1-HEK 293 cells for 10 min[2].

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Primary cultures of hippocampal neurons were prepared from postnatal day 1 rat pups. For fluorescence imaging, neurons were co-loaded with Fura-2FF-AM and Rhodamine 123, then rinsed with a standard bath solution. Fluorescence imaging was performed using an inverted microscope with an EM-CCD camera. NAD(P)H autofluorescence was followed using excitation light at 360±20 nm and recorded at 460±25 nm. [1]
For cellular respirometry, cultured hippocampal neurons were grown in assay plates. The oxygen consumption rate (OCR) was measured using a Seahorse XF24 analyzer. The growth medium was replaced with a standard bath solution supplemented with glucose and pyruvate before measurement. Oligomycin, 2,4-dinitrophenol (DNP), and a combination of rotenone and antimycin A were applied to neurons as indicated. [1]
For photometric analysis of Ca2+ transients, isolated FDB fibers were loaded with Fluo-4 acetoxymethyl ester in normal Ringer's solution. Electrical field stimuli were applied, and fluorescence emission from individual fibers was measured using digital photometry. [2]
For Ca2+ imaging, HEK 293 cells stably expressing wild-type RyR1 were loaded with Fluo-4 acetoxymethyl ester. Cells were imaged with a CCD camera. Caffeine was locally applied, and KB-R7943 was incubated for 10 minutes before caffeine application. [2]
HEK 293 cells stably expressing wild-type or mutant hERG were used for IhERG measurements. Cells were passaged and plated. Patch pipettes were pulled and heat-polished. Series resistance was compensated. Measurements were made at 35-37°C. Action potential voltage clamp experiments employed a ventricular AP waveform. [3]
Prostate cancer cell viability was measured using the CCK-8 assay. Cells were seeded in 96-well plates, treated with compounds for 24h, and then CCK-8 solution was added. Absorbance at 450nm was measured. [4]
Cell cycle distribution was analyzed by flow cytometry using propidium iodide (PI) staining. PC3 cells were treated, fixed, and stained with PI, then analyzed. [4]
Apoptosis was detected by flow cytometry using an Annexin V-FITC kit. PC3 cells were treated, stained with Annexin V-FITC and PI, and then analyzed. [4]
For western blotting, total protein was extracted from cell lysates, separated by SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies (e.g., LC3A/B, p-mTOR, mTOR, P62, CyclinD1, p-JNK, JNK, etc.) overnight at 4°C, followed by secondary antibodies. Proteins were visualized using ECL. [4]
Monolayer wound healing assay: PC3 cells were scratched with a pipette tip to create a wound and treated with KB-R7943. Cell migration was photographed at 0, 24, and 48 hours. [4]
Matrigel invasion assay: PC3 cells were seeded into Transwell upper chambers with serum-free medium or KB-R7943. Medium with serum was added to the lower chamber. After incubation, invading cells were fixed, stained, and counted. [4]
For transmission electron microscopy, PC3 cells were treated with KB-R7943, then fixed in glutaraldehyde, treated with osmium tetroxide, dehydrated, embedded, and examined under a transmission electron microscope. [4]
Immunohistochemistry was performed on paraffin-embedded tissue sections. Sections were stained with primary antibodies (e.g., anti-LC3A/B, anti-caspase-3, anti-NCX1, anti-Ki67) and secondary antibody using the streptavidin-biotin peroxidase method. [4]

PC3 cells (1×10^6) were inoculated subcutaneously into the backs of male nude mice (4-5 weeks old). Fourteen days after tumor inoculation, mice were randomly divided into two groups (7 mice per group) and injected intraperitoneally (IP) daily with buffer only (control) or KB-R7943 at 10 mg/kg (dissolved in PBS). Tumor sizes and body weights were measured once a week. After 30 days of injections, animals were killed, and tumors were weighed and processed for western blotting or paraffin embedding. [4]

[1]. KB-R7943, an inhibitor of the reverse Na+ /Ca2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I. Br J Pharmacol. 2011 Jan;162(1):255-70.

[2]. The Na+/Ca2+ exchange inhibitor 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate(KB-R7943) also blocks ryanodine receptors type 1 (RyR1) and type 2 (RyR2) channels. Mol Pharmacol. 2009 Sep;76(3):560-8.

[3]. High potency inhibition of hERG potassium channels by the sodium-calcium exchange inhibitor KB-R7943. Br J Pharmacol. 2012 Apr;165(7):2260-73.

[4]. The reverse-mode NCX1 activity inhibitor KB-R7943 promotes prostate cancer cell death by activating the JNK pathway and blocking autophagic flux. Oncotarget. 2016;7(27):42059-70.

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) was introduced in 1996 as a selective inhibitor of NCX isoform 1 (NCX1) operating in the reverse mode. It remains the most widely used inhibitor of NCXrev. [1]
Besides NCX, KB-R7943 is known to have several off-target effects, including inhibition of L-type voltage-gated Ca2+ channels, store-operated Ca2+ influx, TRP channels, mitochondrial Ca2+ uniporter, and nicotinic acetylcholine receptors. [1, 2]
The compound's neuroprotection against glutamate excitotoxicity may be mediated by mild mitochondrial depolarization via complex I inhibition, which limits Ca2+ uptake by mitochondria and prevents Ca2+-induced damage. [1]
KB-R7943 is an activity-dependent blocker of RyR1 and RyR2, showing a greater reduction in channel open probability under conditions that enhance channel activity (e.g., higher cytoplasmic Ca2+ or higher stimulation frequency). [2]
Due to its potent hERG/I_Kr inhibition, the propensity of KB-R7943 to block these channels needs to be taken into account when interpreting data from cardiac preparations. The structurally related NCX inhibitor SN-6 was found to be ~100-fold less potent as a hERG inhibitor. [3]
KB-R7943 may be a potential treatment for prostate cancer, as it promotes cell death by activating the JNK signaling pathway and blocking autophagic flux. [4]

C17H21N3O6S2

427.49

427.087

C, 47.76; H, 4.95; N, 9.83; O, 22.46; S, 15.00

182004-65-5

182004-64-4;182004-65-5 (mesylate);

9823846

White to yellow solid powder

534.6ºC at 760mmHg

277.1ºC

1.67E-11mmHg at 25°C

5.251

3

8

7

28

479

0

S(/C(=N/[H])/N([H])[H])C([H])([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])[N+](=O)[O-].S(C([H])([H])[H])(=O)(=O)O[H]

WGIKEBHIKKWJLG-UHFFFAOYSA-N

InChI=1S/C16H17N3O3S.CH4O3S/c17-16(18)23-10-9-12-3-7-15(8-4-12)22-11-13-1-5-14(6-2-13)19(20)21;1-5(2,3)4/h1-8H,9-11H2,(H3,17,18);1H3,(H,2,3,4)

methanesulfonic acid;2-[4-[(4-nitrophenyl)methoxy]phenyl]ethyl carbamimidothioate

KB-R7943 mesylate; KB-R7943; KB-R 7943; KB R7943

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)

DMSO: 86~100 mg/mL (201.2~233.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.85 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.85 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.85 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.

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