NF-κB (IC50 = 1.2 μM)

IMD-0354 completely suppresses the growth factor-independent proliferation of mast cells in HMC-1 cells by impairing NF-κB activity. Cell proliferation is completely suppressed when the DNA-binding activity of NF-κB is inhibited by treatment with IMD-0354. IMD-0354 or STI571 are added to HMC-1 cells in escalating concentrations for 24, 48, and 72 hours; the number of cells and their viability are then assessed using the dye exclusion test and the MTT assay. Cell proliferation is inhibited by IMD-0354 in a time- and dose-dependent manner. When compared to STI571, IMD-0354 has a remarkable inhibitory effect, even at lower concentrations[1]. IMD0354 has an IC50 of 1.2±0.3 uM and inhibits TNF-αinduced NF-κB transcription activity[2].

In nude mice implanted with MDA-MB-231 tumors, a daily dose of 5 mg/kg IMD-0354 significantly inhibits tumor growth. Tumor progression is slowed down in mice receiving IMD-0354 treatment[3]. In rats treated with 30, 10, 3, or 0 mg/kg of IMD-0354, the number of infiltrating cells in the aqueous humor was 53.6±9.8×105, 72.5±17.0×105, 127.25±32.0×105, and 132.0±25.0×105 cells/mL, respectively. Rats treated with 30, 10, 3, and 0 mg/kg of IMD-0354, respectively, had total protein concentrations of 92.6±3.1 mg/mL, 101.5±6.8 mg/mL, 112.6±1.9 mg/mL, and 117.331.8 mg/mL in their aqueous humor[4].

IMD-0354 inhibits TNF-α induced NF-κB transcription activity with an IC50 of 1.2±0.3 uM.
Electrophoretic mobility shift assay[1]
After they were incubated with indicated concentrations of IMD-0354 or with each signal inhibitor for 24 hours, nuclear extractions were prepared from 107 cells with the use of NE-PER nuclear and cytoplasmic extraction reagents according to manufacturer's instructions. A biotin-labeled double-strand DNA probe containing the consensus DNA-binding sequence for NF-κB was synthesized by incubating sense and antisense oligonucleotides (sense, 5′-AGTTGAGGGGACTTTCCCAGGC-3′; antisense, 5′-GCCTGGGAAAGTCCCCTCAACT-3′) in Tris-EDTA (Tris-ethylenediaminetetraacetic acid) buffer for 2 minutes at 85°C, for 15 minutes at 65°C, for 15 minutes at 37°C, for 15 minutes at room temperature, and for 15 minutes on ice. With a LightShift Chemiluminescence electrophoretic mobility shift assay (EMSA) kit, 0.02 pmol biotin-labeled DNA probe was incubated with 5 μg nuclear extraction for 20 minutes at room temperature. The conjugate, mixed with 5 × loading buffer and 20 μL mixture containing 4 μg nuclear protein, was applied to each lane of 6% DNA-polyacrylamide gel electrophoresis (DNA-PAGE) mini-gel. Electrophoresis was performed in Tris-boric acid-EDTA buffer, and the separated proteins were transferred to Hybond-N+ membrane. After ultraviolet (UV) cross-linking, the membrane was blocked and was incubated with LightShift stabilized streptavidin-HRP conjugate (Pierce) for 60 minutes. Positive reactions were visualized by incubating the membrane in LightShift Luminol/Enhancer solution. All procedures were performed according to the manufacturer's instructions except as indicated. For competition assays, unlabeled NF-κB consensus oligonucleotides and mutant oligonucleotides with a single-base substitution were used. For supershift assays, 4 μg anti-p65, anti-cRel, or anti-p50 subunit antibodies in each reaction was added.

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Chemiluminescence transcription factor assay[1]
CBhCMCs (106 cells per each condition) were incubated with or without various concentrations of IMD-0354 for 24 hours at 37°C. After centrifugation, nuclear extractions were prepared with the use of NE-per nuclear and cytoplasmic extraction reagents, as described in “Electrophoretic mobility shift assay.” Reactivity of the p65 subunit of NF-κB in nucleus was analyzed with the use of an EZ-Detect NF-κB p65 transcription factor kit according to manufacturer's instructions. Chemiluminescence signals were detected with the use of a luminometer.

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Luciferase assay[1]
With the use of an Effectene transfection reagent kit, 200 ng pNF-κB-TA-Luc plasmid was introduced into 2 × 106 HMC-1 cells according to manufacturer's instructions. Forty-eight hours later, cells were treated with various concentrations of IMD-0354 in α-MEM containing 10% FCS and were further incubated for 24, 48, and 72 hours. A luciferase activity in supernatants of cell lysates was measured with a Bright-Glo Luciferase Assay system as a substrate.

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Electrophoretic mobility shift assay.[3]
After incubation with the indicated concentrations of IMD-0354 or each signal inhibitor for 24 hours, nuclear extractions were prepared from 107 cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) according to the manufacturer's instructions. A biotin-labeled double-stranded DNA probe containing the consensus DNA binding sequence for NF-κB was synthesized by incubating sense and antisense oligonucleotides (sense, 5′-AGTTGAGGGGACTTTCCCAGGC-3′; antisense, 5′-GCCTGGGAAAGTCCCCTCAACT-3′) in Tris-EDTA buffer for 2 minutes at 85°C, for 15 minutes at 65°C, for 15 minutes at 37°C, for 15 minutes at room temperature, and for 15 minutes on ice. With a LightShift chemiluminescent electrophoretic mobility shift assay kit, 0.02 pmol of the biotin-labeled DNA probe was incubated with 5 μg of the nuclear extraction for 20 minutes at room temperature. The conjugate was mixed with 5× loading buffer and 20 μL of the mixture containing 4 μg nuclear protein was applied onto each lane of 6% DNA-PAGE mini gel. Electrophoresis was done in Tris–boric acid–EDTA buffer, and the separated proteins were transferred to Hybond-N+ membrane. After UV cross-linking, the membrane was blocked, incubated with LightShift stabilized streptavidin-horseradish peroxidase conjugate for 60 minutes. Positive reactions were visualized by incubating the membrane in LightShift luminol/enhancer solution. All procedures were done according to the manufacturer's instructions except where indicated. For competition assays, unlabeled NF-κB consensus oligonucleotides and mutant oligonucleotides with a single base substitution were used. For supershift assays, 4 μg of anti-p65, anti-cRel, or anti-p50 subunit antibodies in each reaction was added.

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Luciferase assay. [3]
Using an Effectene transfection reagent kit, 200 ng of pNF-κB-TA-Luc plasmid was introduced into MDA-MB-231 cells according to the manufacturer's instructions. We used β-galactosidase expression vector (pCMVβ) to control transfection efficiency. Twenty-four hours later, cells were washed and treated with various concentrations of IMD-0354 in DMEM containing 10% FCS and further incubated for 24 hours. Luciferase activity in supernatants of cell lysates was measured with a Bright-Glo luciferase assay system as a substrate. Results were normalized to the luciferase activity of mock TA-Luc plasmid.

For the indicated hours, HMC-1 cells (2×105 cells/mL) are incubated with varying concentrations of IMD-0354 (0.1, 0.5, 1, 5, and 10 uM), STI571, or pyrrolidine dithiocarbamate (PDTC), and viable cell counts are determined using the trypan blue dye exclusion test at each time point. Cells (2×105 cells/mL) are incubated in phenol red-free -MEM containing 10% FCS (for HMC-1 and IC-2 cells) or 5% FCS (for CBhCMCs), antibiotics with or without varying concentrations of IMD-0354 (0.1, 0.5, 1, 5, and 10 uM), STI571, or PDTC. Recombinant rat or recombinant human SCF, 100 ng/mL, is added to the medium and is incubated with IC-2WT cells and CBhCMCs. Each well of 96-well culture plates receives 100 microliters of cell suspension, which is then left there for 24, 48, and 72 hours of incubation. 10 μL of 5 mg/mL MTT dissolved in PBS are added to each well prior to 4 hours from the culture's end. Once 100 L of 10% SDS in 0.01 N HCl are added, the reaction is stopped. ImmunoMini NJ-2300[1] is used to measure absorbance at 577 nm.

Mice: Female BALB/c nude mice are injected s.c. with MDA-MB-231 cells suspended in PBS (5×106 cells/100 L mouse) when they are 4 to 5 weeks old. Following growth, the tumor is surgically removed, and at the age of 4 weeks, under ether anesthesia, 100 mg of each established tumor is transplanted to the back of additional female nude mice. Each mouse receives an intraperitoneal injection of 5 mg/kg body weight of IMD-0354 (suspended in 100 L/mouse) once daily for 28 days following the implantation. As a control, saline is injected into naked mice. Calculations are made for the estimated tumor weight (mg) and volume (mm3).
Rats: Lewis rats (180–220 g) that are eight weeks old are used. Endotoxin-induced uveitis (EIU) is brought on by injecting 200 μg of diluted Escherichia coli LPS in 200 l of PBS subcutaneously. IMD-0354, diluted in 500 μL of 0.5% CMC, is injected intraperitoneally into the rats at the same time in doses of 30, 10, or 3. 500 μL of CMC alone is intraperitoneally administered to control EIU rats. Control rats are naive rats. Five animals are used in each group for each experiment, which is carried out in triplicate.

[1]. A novel NF-kappaB inhibitor, IMD-0354, suppresses neoplastic proliferation of human mast cells with constitutively activated c-kit receptors. Blood. 2005 Mar 15;105(6):2324-31.

[2]. Study of the inhibitory effects on TNF-α-induced NF-κB activation of IMD0354 analogs. Chem Biol Drug Des. 2017 Dec;90(6):1307-1311.

[3]. A new IkappaB kinase beta inhibitor prevents human breast cancer progression through negative regulation of cell cycle transition. Cancer Res. 2006 Jan 1;66(1):419-26

[4]. Amelioration of endotoxin-induced uveitis treated with an IκB kinase β inhibitor in rats. Mol Vis. 2012;18:2586-97.

N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide is a member of benzamides.

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Constitutive phosphorylation of c-kit tyrosine kinase is the major cause of factor-independent proliferation of mast cells. Recently available tyrosine kinase inhibitors have shown marked activity against mast cell lines that carry wild-type c-kit, and some, but not others, carry mutant c-kit. Here we clearly demonstrated that a novel NF-kappaB inhibitor, IMD-0354, restrained factor-independent proliferation of mast cells with c-kit mutations but not of normal mast cells. In HMC-1 cells with the Asp816Val and Val560Gly mutations, we found that NF-kappaB was constitutively activated without exogenous stimulation. When the DNA-binding activity of NF-kappaB was inhibited by treatment with IMD-0354, cell proliferation was completely suppressed. We detected the expression of cyclin D2, D3, and E in HMC-1 cells and observed that cyclin D3 expression was dramatically decreased by treatment with IMD-0354. Abolishing protein kinase C or phosphatidylinositol 3 kinase pathways also inhibited NF-kappaB translocation to the nucleus, indicating the involvement of these signaling cascades in NF-kappaB activation in HMC-1 cells. Our findings indicated that autophosphorylated c-kit receptors induced NF-kappaB activation, resulting in the up-regulation of cyclin D3 expression and cell cycle progression. The observations from the current study suggest a therapeutic potential, in systemic mastocytosis, for compounds that interfere with NF-kappaB signaling.[1]

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Nuclear factor-κB (NF-κB) is an important nuclear transcription factor which regulates pro-inflammatory cytokines such as TNF-α, IL-6. Its role as immunoregulatory mediator makes it an attractive target in the development of treatments for inflammatory and autoimmune diseases. In this study, we synthesized derivatives of IMD0354, a known inhibitor for NF-κB, in attempt to understand the effect of benzanilide substitutions on its activity. The inhibition of these analogs on NF-κB activation was analyzed by luciferase assay. The inhibition of IKKβ phosphorylation and pro-inflammatory cytokines was determined by Western blot and real-time PCR. The structure activity relationships showed that the hydroxyl group on IMD0354 is a critical moiety that resulting in the inhibition of NF-κB. Derivatives 1m, 2b, and 2c were shown to inhibit pro-inflammatory cytokine production at low concentration. These newly synthesized compounds may be useful for the treatment of chronic inflammatory disorders or for cancer prevention. [2]

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Constitutive nuclear factor-kappaB (NF-kappaB) activity plays a crucial role in the development and progression of lymphoma, leukemia, and some epithelial cancers. Given the contribution of NF-kappaB in carcinogenesis, a novel approach that interferes with its activity might have therapeutic potential against cancers that respond poorly to conventional treatments. Here, we have shown that a new IkappaB kinase beta inhibitor, IMD-0354, suppressed the growth of human breast cancer cells, MDA-MB-231, HMC1-8, and MCF-7, by arresting cell cycle and inducing apoptosis. In an electrophoretic mobility shift assay and a reporter assay, IMD-0354 abolished the NF-kappaB activity in MDA-MB-231 cells in a dose-dependent manner. In the cells incubated with IMD-0354, cell cycle arrested at the G0-G1 phase and apoptotic cells were increased. The expression of some cell cycle regulatory molecules and antiapoptotic molecules was suppressed in cells treated with IMD-0354. On the other hand, cyclin-dependent kinase suppressor p27Kip1 was up-regulated by the addition of IMD-0354. Daily administration of IMD-0354 inhibited tumor expansion in immunodeficient mice into which MDA-MB-231 cells were transplanted. These results indicate that NF-kappaB may contribute to cell proliferation through up-regulation of cell cycle progression; accordingly, inhibition of NF-kappaB activity might have a therapeutic ability in the treatment of human breast cancers.[3]

C15H8CLF6NO2

383.67

383.015

C, 46.96; H, 2.10; Cl, 9.24; F, 29.71; N, 3.65; O, 8.34

978-62-1

5081913

White to off-white solid powder

1.561g/cm3

323.1ºC at 760 mmHg

149.2ºC

1.543

5.408

2

8

2

25

462

0

O=C(C1C(O)=CC=C(Cl)C=1)NC1C=C(C(F)(F)F)C=C(C(F)(F)F)C=1

CHILCFMQWMQVAL-UHFFFAOYSA-N

InChI=1S/C15H8ClF6NO2/c16-9-1-2-12(24)11(6-9)13(25)23-10-4-7(14(17,18)19)3-8(5-10)15(20,21)22/h1-6,24H,(H,23,25)

N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide

IKK2 Inhibitor V; IMD0354; N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide; IKK-2 Inhibitor V; IMD0354; N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide; TCMDC-125465; IMD 0354; IMD-0354

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 (6.52 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 (6.52 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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% DMSO+5% Tween 80+0.5% CMC Na: 3 mg/mL

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