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.

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.

[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.

C14H17N5

255.32

255.15

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);

Solid powder

CC1=CC2=C(C=C1)N=C(C3=NC=C(N23)C)NCCN

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; 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)