IKK-2 (IC50 = 0.3 μM); IKK-1 (IC50 = 4 μM)

BMS-345541 inhibits IKK-2 and IKK-1 in dose-dependent manner. At concentrations as high as 100 M, BMS-345541 is unable to inhibit a panel of serine/threonine and tyrosine kinases. At concentrations as high as 100 μM, MS-345541 is unable to inhibit the phosphorylation of c-Jun by anisomycin, the activation of MAPKAP K2 by LPS in THP-1 cells, the phosphorylation of STAT3 by EGF in H292 cells, or the phosphorylation of c-Jun by STAT3 by anisomycin[1]. Treatment with BMS-345541 inhibits the proliferation of melanoma cells in SK-MEL-5, A375, and Hs 294T cells in a concentration-dependent manner. According to TUNEL staining and nuclear condensation, BMS-345541 (0, 100 μM) exhibits apoptotic characteristics[2].

BMS-345541 (10 mg/kg, p.o.) causes extended serum drug levels in mice, with concentrations maintained at or above 1 μM for several hours. When animals are challenged with an intraperitoneal dose of LPS, BMS-345541 dose-dependently reduces the production of TNFα as measured in the serum[1]. BMS-345541 effectively inhibits SK-MEL-5 tumor growth in mice at doses of 0, 10, 25, and 75 mg/kg, p.o. SK-MEL-5, A375, and Hs 294T tumor growth is effectively inhibited by treating tumor-bearing mice with 75 mg/kg of BMS-345541 by 86.2.8%, 69.11%, and 67.3.4%, respectively[2]. Both clinical and histological endpoints of inflammation and injury in mice are successfully blocked by BMS-345541 (30 and 100 mg/kg, p.o.)[3].

Assays measuring the enzyme-catalyzed phosphorylation of GST-IκBα are performed by adding enzyme (a final concentration of 0.5 μg/mL) at 30 ℃ to solutions of 100 μg/mL GST- IκBα and 5 μM [33P]ATP in 40 mM Tris HCl, pH 7.5, containing 4 mM MgCl2, 34mM sodium phosphate, 3 mM NaCl, 0.6 mM potassium phosphate, 1 mM KCl, 1 mM dithiothreitol, 3% (w/v) glycerol, and 250 μg/mL bovine serum albumin. The [33P]ATP used in the assay has a specific activity of 100 Ci/mmol. The kinase reactions are stopped after 5 minutes by adding 2× Laemmli sample buffer, which is then heated for 1 minute at 90 °C. After that, the samples are put on NuPAGE 10% BisTris gels. Gels are dried on a slab gel dryer following SDS-PAGE. The bands are then detected using a 445Si PhosphorImager, and the radioactivity is quantified using ImageQuant software. Under these conditions, the degree of phosphorylation of GST-IκBα is linear with time and concentration of enzyme. A 445Si PhosphorImager is then used to identify the bands, and ImageQuant software is used to measure the radioactivity. Under these circumstances, the rate of enzyme concentration and GST-IκBα phosphorylation are linearly related.

Briefly, BMS-345541 is applied to SK-MEL-5 cells at various concentrations or for various lengths of time. Trypsinization is used to collect the cells, which are then fixed in 70% ethanol for 2 hours on ice and stained with PI solution (PBS containing 2 μg/mL PI, 0.1% Triton X-100, and 125 units/mL RNase A) for 30 minutes at 37°C. Using flow cytometry and filters with 488 nm excitation and 620 nm emission, cell fluorescence is measured. The resulting data are analyzed using the software MultiCycle. Six-well plates with 10% fetal bovine serum medium are plated with 1×105 cells per well overnight to promote cell adhesion. For 72 hours, cells are cultured in medium that contains BMS-345541. Using a hemocytometer, cells are counted.

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Effect of BMS-345541 on adhesion molecule expression in human umbilical vein endothelial cells [3]
HUVECs plated in 96 well plates at 5000 cells/well in 0.1 mL volume were pre-treated with BMS-345541 for 1 h prior to a 4 h stimulation with 10 ng/mL TNFa. We used mouse monoclonal antibodies recognizing either ICAM-1 or VCAM-1 followed by detection with goat-anti-mouse-HRP.

BMS-345541 is created as a 2 mg/mL solution in water with 3% Tween 80. Either a peroral gavage of 10 mg/kg (1 mL/kg) or an intravenous bolus of 2 mg/kg (1 mL/kg) is administered to the mice. Individual mice are given whole blood samples at 0, 0.05, 0.25, 0.5, 1.0, 3.0, 6.0, and 8.0 h after dosing by means of an orbital bleed and a cardiac puncture. Centrifuging whole blood for five minutes at 20×103 g. Before being analyzed, serum is kept at 20°C.

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Dextran sulfate sodium-induced murine model of inflammatory bowel disease [3]
Swiss-Webster mice were given 6% DSS in their drinking water for 7 days to induce intestinal inflammation. Aqueous solutions of test compounds (e.g. BMS-345541 ) were administered by oral gavage once daily throughout the study (days 2 through 9), with n = 5 per group. On day 10, animals were sacrificed and the colons removed for clinical and histological evaluation. Clinical scoring by a blinded observer was determined by the gross clinical evaluation of the injury on a scale from 0 (normal) to 3 (severe) as follows: grade 0, normal; grade 1, relatively normal colon length with slight thickening of tissue; grade 2, shortened colon length and thick along entire length of colon with loss of striations and some areas of redness; grade 3, considerably shortened length with very thick tissue containing areas of raised lesions. The weight for each animal on day 10 was divided by its weight at the beginning of the study to obtain a weight ratio at the end of the study. Entire colons were then immersion fixed in 10% neutral buffered formalin and divided into proximal, middle, and distal segments of equal length. Each segment was processed by routine methods, and embedded in paraffin. Segments were step-sectioned at 5 mm to obtain 3–6 sections per segment for a total of 9–18 colon sections/animal and stained with hematoxylin and eosin for light microscopy. Colon sections were graded as to the severity of crypt injury and degree of inflammation. The crypt injury was scored as follows: grade 0, intact crypt; grade 1, loss of the basilar 1/3rd of the crypt; grade 2, loss of basilar 2/3rd of the crypt; grade 3, loss of entire crypt with surface epithelium intact; grade 4, loss of entire crypt with epithelial erosion. These changes were also graded as to the degree of tissue involvement: grade 0, no involvement; grade 1, 1–25% involvement; grade 2, 26–50% involvement; grade 3, 51–75% involvement; grade 4, 76–100% involvement. The injury histological score is then defined as the product of the crypt injury grade and the degree of tissue involvement grade. The scoring for severity of inflammation was as follows: grade 0, nonremarkable; grade 1, minimal; grade 2, mild; grade 3, moderate; grade 4, severe. The extent of involvement was estimated as: grade 0, no involvement; grade 1, 1–25% involvement; grade 2, 26–50% involvement; grade 3, 51–75% involvement; grade 4, 76–100% involvement. The inflammation histological score is the product of the severity of inflammation grade and extent of involvement grade. Crypt injury and inflammatory scoring were performed on each section of colon and a mean score and standard error determined for each section. Cumulative crypt injury and inflammatory scores for each group were determined. Statistical analysis was performed using ANOVA with Tukey’s post hoc analysis. Significance was considered at a P < 0.05 level.

[1]. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J Biol Chem, 2003, 278(3), 1450-1456.

[2]. BMS-345541 targets inhibitor of kappaB kinase and induces apoptosis in melanoma: involvement of nuclear factor kappaB and mitochondria pathways. Clin Cancer Res, 2006, 12(3 Pt 1), 950-960.

[3]. An inhibitor of IkappaB kinase, BMS-345541, blocks endothelial cell adhesion molecule expression and reduces the severity of dextran sulfate sodium-induced colitis in mice. Inflamm Res, 2003, 52(12), 508-511.

The signal-inducible phosphorylation of serines 32 and 36 of I kappa B alpha is critical in regulating the subsequent ubiquitination and proteolysis of I kappa B alpha, which then releases NF-kappa B to promote gene transcription. The multisubunit I kappa B kinase responsible for this phosphorylation contains two catalytic subunits, termed I kappa B kinase (IKK)-1 and IKK-2. BMS-345541 (4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline) was identified as a selective inhibitor of the catalytic subunits of IKK (IKK-2 IC(50) = 0.3 microm, IKK-1 IC(50) = 4 microm). The compound failed to inhibit a panel of 15 other kinases and selectively inhibited the stimulated phosphorylation of I kappa B alpha in cells (IC(50) = 4 microm) while failing to affect c-Jun and STAT3 phosphorylation, as well as mitogen-activated protein kinase-activated protein kinase 2 activation in cells. Consistent with the role of IKK/NF-kappa B in the regulation of cytokine transcription, BMS-345541 inhibited lipopolysaccharide-stimulated tumor necrosis factor alpha, interleukin-1 beta, interleukin-8, and interleukin-6 in THP-1 cells with IC(50) values in the 1- to 5-microm range. Although a Dixon plot of the inhibition of IKK-2 by BMS-345541 showed a non-linear relationship indicating non-Michaelis-Menten kinetic binding, the use of multiple inhibition analyses indicated that BMS-345541 binds in a mutually exclusive manner with respect to a peptide inhibitor corresponding to amino acids 26-42 of I kappa B alpha with Ser-32 and Ser-36 changed to aspartates and in a non-mutually exclusive manner with respect to ADP. The opposite results were obtained when studying the binding to IKK-1. A binding model is proposed in which BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2, which then affects the active sites of the subunits differently. BMS-345541 was also shown to have excellent pharmacokinetics in mice, and peroral administration showed the compound to dose-dependently inhibit the production of serum tumor necrosis factor alpha following intraperitoneal challenge with lipopolysaccharide. Thus, the compound is effective against NF-kappa B activation in mice and represents an important tool for investigating the role of IKK in disease models.[1]

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Purpose: Constitutive activation of inhibitor of kappaB kinase (IKK) confers melanoma resistance to apoptosis and chemotherapy. Whether IKK is able to serve as a therapeutic target in melanoma is unknown. We explored the possibility of exploiting IKK as a therapeutic target in melanoma by using BMS-345541, a novel compound with a highly selective IKKbeta inhibitory activity, to trigger melanoma cell apoptosis. Experimental design: Three human melanoma cell lines (SK-MEL-5, Hs 294T, and A375), all of which have high constitutive IKK activities, served as in vitro and in vivo melanoma models for treatment with BMS-345541. Two known antitumor drugs (temozolomide and bortezomib) were used as parallel controls for evaluation of the therapeutic efficiency and toxicity of BMS-345541. The effects of BMS-345541 on nuclear factor kappaB (NF-kappaB) signaling and on the apoptosis machinery were investigated. Results: Inhibition of constitutive IKK activity by BMS-345541 resulted in the reduction of NF-kappaB activity, CXCL1 chemokine secretion by cultured melanoma cells and melanoma cell survival in vitro and in vivo. The effect of BMS-345541 on tumor cell growth was through mitochondria-mediated apoptosis, based on the release of apoptosis-inducing factor, dissipation of mitochondrial membrane potential, and reduced ratio of B cell lymphoma gene-2 (Bcl-2)/Bcl-associated X protein (Bax) in mitochondria. The BMS-345541 execution of apoptosis was apoptosis-inducing factor-dependent, but largely caspase-independent. Conclusion: BMS-345541 down-regulation of IKK activity results in mitochondria-mediated apoptosis of tumor cells because the programmed cell death machinery in melanoma cells is highly regulated by NF-kappaB signaling. Therefore, IKK may serve as a potential target for melanoma therapy.[2]

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Objective: Inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are characterized by chronic relapsing inflammation. The transcription of many of the proteins which mediate the pathogenesis in inflammatory bowel disease (e.g., TNFalpha, ICAM-1, VCAM-1) is NF-kappaB-dependent. IkappaB kinase is critical in transducing the signal-inducible activation of NF-kappaB and, therefore, represents a potentially promising target for the development of novel agents to treat inflammatory bowel disease and other inflammatory diseases. Results: Here we show that BMS-345541, a highly selective inhibitor of IkappaB kinase, inhibited the TNFalpha-induced expression of both ICAM-1 and VCAM-1 in human umbilical vein endothelial cells at the same concentration range as cytokine expression is inhibited in monocytic cells (IC(50) congruent with 5 microM). Against dextran sulfate sodium-induced colitis in mice, BMS-345541 administered orally at doses of 30 and 100 mg/kg was effective in blocking both clinical and histological endpoints of inflammation and injury. Conclusion: This represents the first example of an inhibitor of IkappaB kinase with anti-inflammatory activity in vivo and indicates that inhibitors of IkB kinase show the promise of being highly efficacious in inflammatory disorders such as inflammatory bowel disease.[3]

C₁₄H₁₈CLN₅

291.78

291.125

C, 57.63; H, 6.22; Cl, 12.15; N, 24.00

547757-23-3

445430-58-0;547757-23-3 (HCl);445430-59-1 (2HCl);2320261-79-6 (TFA);

9926054

White to off-white solid powder

449.5ºC at 760 mmHg

225.6ºC

3.445

3

4

3

20

310

0

CC1=CN=C2C(NCCN)=NC3=CC=C(C=C3N21)C.Cl

MIDKPVLYXNLFGZ-UHFFFAOYSA-N

InChI=1S/C14H17N5.ClH/c1-9-3-4-11-12(7-9)19-10(2)8-17-14(19)13(18-11)16-6-5-15;/h3-4,7-8H,5-6,15H2,1-2H3,(H,16,18);1H

N'-(1,8-dimethylimidazo[1,2-a]quinoxalin-4-yl)ethane-1,2-diamine;hydrochloride

UNII-BXU277OCN5; BMS345541; BMS-345541 hydrochloride; BMS-345541 HCl; BMS-345541 (hydrochloride); 1,2-Ethanediamine, N-(1,8-dimethylimidazo(1,2-a)quinoxalin-4-yl)-, monohydrochloride; BXU277OCN5; BMS 345541; BMS-345541

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: ~50 mg/mL (~171.4 mM)
DMSO: ~20 mg/mL (~68.5 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (6.85 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 20.0 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 mg/mL (6.85 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 20.0 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 mg/mL (6.85 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 20.0 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.)