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

BMS-345541 dose-dependently inhibits the TNF-α-stimulated phosphorylation of IκBα in THP-1 monocytic cells with an IC50 of ~4 μM. Tumor necrosis factorα, interleukin-1β, interleukin-8, and interleukin-6 are all inhibited by BMS-345541 in THP-1 cells with IC50 values in the 1- to 5-μM range. BMS-345541 binds in a non-mutually exclusive manner to ADP and in a mutually exclusive manner to a peptide inhibitor corresponding to amino acids 26 to 42 of IB with Ser-32 and Ser-36 changed to aspartates. IKK-1 and IKK-2 have allosteric sites that are similar to each other when BMS-345541 binds to them, which alters how each subunit's active site functions.[1] BMS-345541 affects cytokinesis, prometaphase to anaphase progression, and a number of mitotic cell cycle transitions, including mitotic entry. BMS-345541 prevents the activation of Aurora A, B, and C, Cdk1 activation, and histone H3 phosphorylation in cells that have been released from arrest in G-phase. BMS-345541 treatment of mitotic cells causes premature cyclin B1 and securin degradation, flawed chromosome segregation, and improper cytokinesis. In cells imprisoned by nocodazole, BMS-345541 is also found to suppress the spindle checkpoint. These effects aren't primarily attributable to BMS-345541's direct inhibition of mitotic kinases like Cdk1, Aurora A or B, Plk1 or NEK2.[2] BMS-345541 (10 μM) inhibits metastatic melanoma cells (SK-MEL-5, A375, and Hs 294T) and normal human epidermal melanocytes' growth by 96% and 99%, respectively, at 72 hours. A caspase-independent and AIF-dependent mitochondrial-mediated process causes 87% of the SK-MEL-5 cell culture to undergo apoptosis after the addition of 100 μM of BMS-345541. Treatment with BMS-345541 (10 μM) decreases IKK and NF-kB activity as well as CXCL1 production by 76% and 95%, respectively.[3] BMS-345541 has an IC50 of 2–6 M and inhibits the growth of T-cell acute lymphoblastic leukemia (T-ALL) cell lines BE-13, RPMI–8402 and DND–41, all of which contain a Notch1 mutation, as well as T-ALL primary cells from pediatric patients. In BE-13 and DND-41 cells, 5 μM BMS-345541 causes a cell cycle arrest in the G2/M phase, and in RPMI-8402 cells, it causes a sub-G1 peak increase. Procaspase-8, Procaspase-3, and Poly (ADP-ribose) Polymerase (PARP) are cleaved in a time-dependent manner after being exposed to 5 μM BMS-345541 for 16 hours. This results in an increase in the number of apoptotic cells in all of these cells. I-B and p65 are dephosphorylated in a time-dependent manner by BMS-34554 (5 μM). BMS-345541 treatment of T-ALL cells results in nuclear translocation of FOXO3a and restoration of its functions, including regulation of p21Cip1 expression levels.[4] Human umbilical vein endothelial cells treated with BMS-345541 exhibit IC50 of 5 μM inhibition of TNF-induced expression of ICAM-1 and VCAM-1. [5]

BMS-345541 successfully slows the growth of melanoma tumors in a dose-dependent manner. When compared to control animals given just the vehicle, tumor-bearing mice treated with 75 mg/kg of BMS-345541 effectively inhibited the growth of the SK-MEL-5, A375, and Hs 294T tumors by 86%, 69%, and 67%, respectively. [3] With weight ratio, clinical scoring of the colons, mean injury score, and mean inflammation score of 0.86 (vs 0.77 of the vehicle group), 1.0 (vs 2.5 of the vehicle group), 5.66 (vs 8.52 of the vehicle group), and 6.82 (vs 12.33 of the vehicle group), respectively, BMS-345541 administered orally at doses of 100 mg/kg lessens the severity of dextran sulfate sodium-induced colitis in mice.[6] BMS-345541 (100 mg/kg), when administered by oral gavage in water once daily beginning at the time of the first collagen immunization, inhibits clinical signs of disease in the murine CIA model (0 vs ~8 of vehicle group), accompanied by reduced paw swelling. BMS-345541 (100 mg/kg) reduces cumulative arthritis injury score from 4.4 to 0, accompanied by lower degrade of tibiotarsal joints and severity of inflammation, synovial hyperplasia, bone resorption, and cartilage erosion. No discernible injury is observed in the joints of animals, which is histologically indistinguishable from those from age-matched, disease-free control animals. BMS-345541 dose-dependently inhibits IL-1β message, with animals in the 100 mg/kg dose group showing levels comparable with those of disease-free control animals.[7]

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 after SDS-PAGE is finished.

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 [6]
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.

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The IkappaB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFkappaB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKalpha also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541 , a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of Aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS-345541 function could be useful for cell cycle control studies and may provide valuable clues for the design of future therapeutics[2].

Mice: Groups of three 18-22 g female BALB/c mice receive BMS-345541 either intravenously through the tail vein or orally. 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. While awaiting analysis, serum is kept at -20°C.

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Dextran sulfate sodium-induced murine model of inflammatory bowel disease [6]
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]. J Biol Chem . 2003 Jan 17;278(3):1450-6.

[2]. Cell Cycle . 2007 Oct 15;6(20):2531-40.

[3]. Cell Cycle . 2012 Jul 1;11(13):2467-75.

[4]. J Biol Chem . 2003 Jan 17;278(3):1450-6.

[5]. Cell Cycle . 2012 Jul 1;11(13):2467-75.

[6]. Inflamm Res . 2003 Dec;52(12):508-11

[7]. Arthritis Rheum . 2003 Sep;48(9):2652-9.

N'-(1,8-dimethyl-4-imidazo[1,2-a]quinoxalinyl)ethane-1,2-diamine is a quinoxaline derivative.

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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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Several lines of evidence suggest that the IκB kinase (IKK)/nuclear factor-κB (NFκB) axis is required for viability of leukemic cells and is a predictor of relapse in T-cell acute lymphoblastic leukemia (T-ALL). Moreover, many anticancer agents induce NFκB nuclear translocation and activation of its target genes, which counteract cellular resistance to chemotherapeutic drugs. Therefore, the design and the study of IKK-specific drugs is crucial to inhibit tumor cell proliferation and to prevent cancer drug-resistance. Here, we report the anti-proliferative effects induced by BMS-345541 (a highly selective IKK inhibitor) in three Notch1-mutated T-ALL cell lines and in T-ALL primary cells from pediatric patients. BMS-345541 induced apoptosis and an accumulation of cells in the G 2/M phase of the cell cycle via inhibition of IKK/NFκB signaling. We also report that T-ALL cells treated with BMS-345541 displayed nuclear translocation of FOXO3a and restoration of its functions, including control of p21(Cip1) expression levels. We demonstrated that FOXO3a subcellular re-distribution is independent of AKT and ERK 1/2 signaling, speculating that in T-ALL the loss of FOXO3a tumor suppressor function could be due to deregulation of IKK, as has been previously demonstrated in other cancer types. It is well known that, differently from p53, FOXO3a mutations have not yet been found in human tumors, which makes therapeutics activating FOXO3a more appealing than others. For these features, BMS-345541 could be used alone or in combination with traditional therapies in the treatment of T-ALL.[3]

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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.[6]

C14H17N5

255.32

255.148

C, 65.86; H, 6.71; N, 27.43

445430-58-0

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

9813758

White to off-white solid powder

1.3±0.1 g/cm3

449.5±45.0 °C at 760 mmHg

225.6±28.7 °C

0.0±1.1 mmHg at 25°C

1.696

2.08

2

4

3

19

310

0

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

PSPFQEBFYXJZEV-UHFFFAOYSA-N

InChI=1S/C14H17N5/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)

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

BMS-345541; BMS345541; BMS-345541 free base; BMS345541; N1-(1,8-dimethylimidazo[1,2-a]quinoxalin-4-yl)ethane-1,2-diamine; IKK Inhibitor III, BMS-345541; IKK Inhibitor III; 1,2-Ethanediamine, N-(1,8-dimethylimidazo(1,2-a)quinoxalin-4-yl)-; BMS 345541; UNII-26SU0NEF5F; BMS-345541 free base

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: ≥ 1 mg/mL (3.92 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (3.92 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 10.0 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: ≥ 1 mg/mL (3.92 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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