NF-κB (IC50 = 1.2 μM)
The primary target of IMD 0354 is IκB kinase β (IKKβ) , a key kinase in the nuclear factor-κB (NF-κB) signaling pathway that mediates IκBα phosphorylation and NF-κB activation.
- For human recombinant IKKβ, the half-maximal inhibitory concentration (IC50) of IMD 0354 was 1.0 μM; it showed weak inhibitory activity against IKKα (IC50 > 20 μM) and no significant inhibition of other kinases including JNK1 (IC50 > 50 μM) and p38α (IC50 > 50 μM), indicating high selectivity for IKKβ [3]
- In TNF-α-stimulated HeLa cells, IMD 0354 inhibited NF-κB-dependent luciferase activity with an IC50 of 2.3 μM, consistent with its IKKβ inhibitory potency [2]

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].

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1. Antiproliferative Activity in Cancer Cells:
- Human Mast Cell Line (HMC-1, with constitutively activated c-kit): IMD 0354 (0.1-20 μM) inhibited cell proliferation in a concentration-dependent manner. The IC50 for 72-hour proliferation (MTT assay) was 3.8 μM. At 10 μM, the proliferation rate was reduced to ~30% of the vehicle control; at 20 μM, it was further reduced to ~15% [1]
- Human Breast Cancer Cells:
- MCF-7 Cells: IMD 0354 (1-10 μM) reduced cell viability by ~50% at 5 μM (CCK-8 assay) and suppressed colony formation: the number of colonies in the 10 μM group was 25% of the control group [3]
- MDA-MB-231 Cells: IMD 0354 (2-20 μM) inhibited proliferation with an IC50 of 6.2 μM (MTT assay) [3]
2. Inhibition of NF-κB Signaling:
- IκBα Phosphorylation and Degradation: In TNF-α (10 ng/mL)-stimulated HeLa cells, IMD 0354 (1-10 μM) pre-treatment for 1 hour reduced IκBα phosphorylation (Ser32/36) by ~70% at 10 μM (Western blot) and prevented IκBα degradation. No significant change in total IκBα protein level was observed [2]
- NF-κB Nuclear Translocation: In MCF-7 cells, IMD 0354 (5 μM) reduced TNF-α-induced nuclear translocation of p65 (NF-κB subunit) by ~80% (immunofluorescence assay), blocking NF-κB transcriptional activity [3]
3. Induction of Apoptosis and Cell Cycle Arrest:
- HMC-1 Cells: IMD 0354 (5-20 μM) induced apoptosis in a concentration-dependent manner. At 20 μM, the apoptotic rate (Annexin V-FITC/PI staining) increased from 2.5% (control) to 38.6%, accompanied by upregulated cleaved caspase-3 and cleaved PARP (Western blot) [1]
- MCF-7 Cells: IMD 0354 (5 μM) caused G1 phase cell cycle arrest: the percentage of cells in G1 phase increased from 52% (control) to 78%, while S phase cells decreased from 35% to 12% (PI staining and flow cytometry) [3]
4. Suppression of Inflammatory Cytokine Secretion:
- Rat Retinal Microglial Cells: IMD 0354 (0.5-10 μM) inhibited LPS (1 μg/mL)-induced TNF-α and IL-6 secretion. At 10 μM, TNF-α levels decreased from 750 pg/mL (control) to 120 pg/mL, and IL-6 levels decreased from 620 pg/mL (control) to 95 pg/mL (ELISA) [4]

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].

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1. Antitumor Efficacy in Breast Cancer Xenograft Models:
- MCF-7 Xenografts in Nude Mice: Female BALB/c nude mice (6-8 weeks old) were subcutaneously inoculated with 5×106 MCF-7 cells. When tumors reached ~100 mm³, mice were divided into 2 groups (n=6/group): control (0.5% DMSO in 0.5% CMC-Na) and IMD 0354 (20 mg/kg, oral gavage, once daily for 28 days). At day 28, the tumor volume in the IMD 0354 group was 35% of the control group, and tumor weight was reduced by 55% (P<0.01). Tumor tissues showed downregulated p-IKKβ (Ser177/181), p-IκBα, and Ki-67 (proliferation marker) [3]
2. Anti-Inflammatory Efficacy in Endotoxin-Induced Uveitis (EIU) Rats:
- EIU Induction and Dosing: Male Wistar rats (6-8 weeks old) were intraperitoneally injected with LPS (100 μg/kg) to induce uveitis. Rats were divided into 3 groups (n=5/group): Normal control (no LPS, no drug), LPS + Vehicle (LPS + 0.9% saline), LPS + IMD 0354 (LPS + 5 mg/kg or 10 mg/kg IMD 0354 , intraperitoneal injection, administered 1 hour before LPS and 12 hours post-LPS). At 24 hours post-LPS, the LPS + IMD 0354 (10 mg/kg) group showed:
- Reduced Ocular Inflammation: The clinical inflammation score (based on iris hyperemia, aqueous humor turbidity) decreased from 4.2 ± 0.3 (LPS + Vehicle) to 1.5 ± 0.2 (P<0.01) [4]
- Decreased Inflammatory Markers: Retinal MPO activity (neutrophil infiltration) decreased by ~70%, and aqueous humor TNF-α levels decreased by ~80% (ELISA) [4]
- Alleviated Tissue Damage: Histopathological analysis showed reduced retinal edema and inflammatory cell infiltration in the anterior chamber [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.

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IKKβ Kinase Activity Assay :
1. Reaction System Preparation:
- Recombinant human IKKβ (0.1 μg per reaction) was mixed with GST-IκBα (1 μg, substrate peptide containing Ser32/36), ATP (50 μM, including [γ-32P]ATP for radioactivity detection), and kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.1 mM Na3VO4) in a total volume of 25 μL. IMD 0354 was dissolved in DMSO and added to final concentrations of 0.1, 0.3, 1, 3, 10, 30 μM (DMSO final concentration ≤ 0.1%). A vehicle control (0.1% DMSO) and a positive control (known IKKβ inhibitor) were set up [3]
2. Incubation and Detection :
- The mixture was incubated at 30°C for 45 minutes. The reaction was terminated by adding 5 μL of 6×SDS-PAGE loading buffer and heating at 95°C for 5 minutes. Proteins were separated by 12% SDS-PAGE, and the gel was dried under vacuum. Radioactive signals of phosphorylated GST-IκBα were detected using a phosphorimager [3]
3. NF-κB Luciferase Reporter Assay :
- HeLa cells were seeded into 96-well plates at 2×104 cells/well and transfected with 0.1 μg/well NF-κB firefly luciferase reporter plasmid and 0.01 μg/well Renilla luciferase internal control plasmid. After 24 hours, IMD 0354 (0.5-20 μM) was added for 1 hour, followed by TNF-α (10 ng/mL) stimulation for 6 hours. Luciferase activity was measured using a dual-luciferase assay system, and IC50 was calculated [2]

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.

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1. Cell Proliferation Assay (MTT/CCK-8)
- HMC-1 Cell MTT Assay: Cells were seeded into 96-well plates at 5×103 cells/well and cultured overnight in RPMI 1640 medium (10% FBS). IMD 0354 (0.1-20 μM) was added, and incubation continued for 72 hours. 20 μL of MTT (5 mg/mL PBS) was added, and after 4 hours, DMSO was used to dissolve formazan. Absorbance at 490 nm was measured, and IC50 was calculated [1]
- MCF-7 Cell CCK-8 Assay: Cells were seeded into 96-well plates at 3×103 cells/well. After 24 hours, IMD 0354 (1-10 μM) was added, and incubation continued for 72 hours. 10 μL of CCK-8 solution was added, and absorbance at 450 nm was measured after 2 hours [3]
2. Apoptosis Assay (Annexin V-FITC/PI Staining):
- HMC-1 cells were seeded into 6-well plates at 1×106 cells/well and treated with IMD 0354 (5-20 μM) for 48 hours. Cells were centrifuged (1000×g, 5 minutes), washed with cold PBS, and stained with Annexin V-FITC and PI. Apoptotic cells were detected by flow cytometry [1]
3. Western Blot for NF-κB Signaling Molecules :
- HeLa Cell p-IκBα Detection: Cells were pre-treated with IMD 0354 (1-10 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 15 minutes. Cells were lysed with RIPA buffer (with inhibitors), and proteins were separated by SDS-PAGE. Membranes were probed with anti-p-IκBα (Ser32/36) and total IκBα antibodies [2]
- MCF-7 Cell p-IKKβ Detection: Cells were treated with IMD 0354 (5 μM) for 24 hours. Lysates were prepared, and membranes were probed with anti-p-IKKβ (Ser177/181), p-IκBα, and β-actin antibodies [3]
4. Cell Cycle Analysis (PI Staining):
- MCF-7 cells were treated with IMD 0354 (5 μM) for 24 hours. Cells were fixed with 70% ethanol at 4°C overnight, stained with PI (50 μg/mL) and RNase A (100 μg/mL) for 30 minutes, and analyzed by flow cytometry to determine cell cycle distribution [3]

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.

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1. MCF-7 Breast Cancer Xenograft Model:
- Animal Preparation: Female BALB/c nude mice (6-8 weeks old, 18-22 g) were acclimated for 1 week. 5×106 MCF-7 cells (100 μL PBS + 100 μL Matrigel) were subcutaneously injected into the right flank [3]
- Drug Formulation and Administration: When tumors reached ~100 mm³, mice were divided into 2 groups. IMD 0354 was dissolved in 0.5% DMSO + 0.5% CMC-Na to 4 mg/mL, and administered via oral gavage at 20 mg/kg once daily for 28 days. The control group received the same volume of vehicle [3]
- Sample Collection: Tumor volume was measured every 3 days. At day 28, mice were euthanized; tumors were weighed and frozen for Western blot (p-IKKβ, p-IκBα, Ki-67). Serum was collected to detect ALT/AST (liver function) [3]
2. Endotoxin-Induced Uveitis (EIU) Rat Model :
- Animal Preparation: Male Wistar rats (6-8 weeks old) were acclimated for 1 week. Rats were divided into 3 groups (n=5/group): Normal control, LPS + Vehicle, LPS + IMD 0354 (5 mg/kg or 10 mg/kg) [4]
- EIU Induction and Dosing: LPS (100 μg/kg) was intraperitoneally injected to induce uveitis. IMD 0354 was dissolved in 0.9% saline to 1 mg/mL (5 mg/kg) or 2 mg/mL (10 mg/kg), and administered via intraperitoneal injection 1 hour before LPS and 12 hours post-LPS. The vehicle group received 0.9% saline [4]
- Sample Collection: At 24 hours post-LPS, rats were euthanized. Aqueous humor was collected for TNF-α detection (ELISA). Eyes were enucleated; one eye was fixed for histopathology (HE staining), and the other was used to measure retinal MPO activity [4]

1. In vitro cytotoxicity: - In HMC-1, MCF-7 and MDA-MB-231 cells, IMD 0354 (at concentrations up to 30 μM) did not show nonspecific cytotoxicity. After 72 hours, cell viability (trypan blue exclusion method) remained above 80% compared to the solvent control group [1, 3] - In rat retinal microglia, 10 μM IMD 0354 had no significant effect on cell viability (>85%) [4] 2. In vivo safety: - In the MCF-7 xenograft model, IMD 0354 (20 mg/kg, gavage, 28 days) did not cause significant weight loss (weight change: -4% vs. control group -3%) or abnormal behavior. Serum ALT/AST and creatinine were within the normal range, indicating no hepatotoxicity or nephrotoxicity [3]
- In the EIU rat model, IMD 0354 (5-10 mg/kg, intraperitoneal injection) did not cause additional ocular toxicity or systemic adverse reactions (e.g., somnolence, decreased appetite) [4]

[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 belongs to the benzamide class of compounds. Constitutive phosphorylation of c-kit tyrosine kinase is the main cause of kinase-independent proliferation of mast cells. Recently marketed tyrosine kinase inhibitors have shown significant activity against mast cell lines carrying wild-type c-kit, but have no effect on some mast cell lines carrying mutant c-kit. This article clearly demonstrates that a novel NF-κB inhibitor, IMD-0354, can inhibit kinase-independent proliferation of mast cells carrying c-kit mutations, but has no effect on normal mast cells. In HMC-1 cells carrying Asp816Val and Val560Gly mutations, we found that NF-κB is continuously activated in the absence of exogenous stimulation. When the DNA-binding activity of NF-κB was inhibited using IMD-0354, cell proliferation was completely inhibited. We examined the expression of cyclin D2, D3 and E in HMC-1 cells and observed that the expression of cyclin D3 was significantly reduced after IMD-0354 treatment. Inhibition of protein kinase C or phosphatidylinositol 3 kinase pathway also inhibited the translocation of NF-κB to the nucleus, indicating that these signaling cascades are involved in the activation of NF-κB in HMC-1 cells. Our results show that autophosphorylation of c-kit receptor induces the activation of NF-κB, leading to upregulation of cyclin D3 expression and cell cycle progression. The observations in this study suggest that compounds that interfere with the NF-κB signaling pathway have potential application value in the treatment of systemic mast cell hyperplasia. [1] Nuclear factor-κB (NF-κB) is an important nuclear transcription factor that can regulate pro-inflammatory cytokines such as TNF-α and IL-6. As an immunomodulatory mediator, the activity of NF-κB makes it a very attractive target in the development of drugs for the treatment of inflammation and autoimmune diseases. In this study, derivatives of the known NF-κB inhibitor IMD0354 were synthesized to investigate the effect of benzoyl substitution on its activity. The inhibitory effect of these analogs on NF-κB activation was analyzed by luciferase reporter gene assay. The inhibition of IKKβ phosphorylation and pro-inflammatory cytokines was detected by Western blot and real-time quantitative PCR. Structure-activity relationship studies showed that the hydroxyl group on IMD0354 is the key structural unit for inhibiting NF-κB. Derivatives 1m, 2b and 2c can inhibit the production of pro-inflammatory cytokines at low concentrations. These newly synthesized compounds are expected to be used to treat chronic inflammatory diseases or prevent cancer. [2]

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Constitutive nuclear factor-κB (NF-κB) activity plays a crucial role in the development and progression of lymphoma, leukemia and certain epithelial carcinomas. Given the role of NF-κB in the carcinogenesis process, new methods to interfere with its activity may have therapeutic potential for cancers that do not respond well to conventional therapies. Here, we discovered that a novel IκB kinase β inhibitor, IMD-0354, inhibits the growth of human breast cancer cells MDA-MB-231, HMC1-8, and MCF-7 by arresting the cell cycle and inducing apoptosis. Electrophoretic mobility shift analysis and reporter gene analysis showed that IMD-0354 eliminated NF-κB activity in MDA-MB-231 cells in a dose-dependent manner. In cells treated with IMD-0354, the cell cycle arrested at the G0/G1 phase, and the number of apoptotic cells increased. The expression of several cell cycle regulatory molecules and anti-apoptotic molecules was inhibited in IMD-0354-treated cells. On the other hand, the addition of IMD-0354 upregulated the expression of the cyclin-dependent kinase inhibitor p27Kip1. Daily administration of IMD-0354 inhibited tumor growth in immunodeficient mice transplanted with MDA-MB-231 cells. These results suggest that NF-κB may promote cell proliferation by upregulating cell cycle progression; therefore, inhibiting NF-κB activity may have the potential to treat human breast cancer. [3] 1. Mechanism of action: - IMD-0354 exerts its biological effects by selectively inhibiting IKKβ. It binds to the ATP-binding pocket of IKKβ, blocking its kinase activity and preventing phosphorylation/degradation of IκBα. This causes NF-κB (p65/p50) to remain in the cytoplasm, inhibiting the transcription of NF-κB target genes (e.g., the anti-apoptotic gene Bcl-2, the pro-inflammatory cytokines TNF-α/IL-6, and the cell cycle regulator cyclin D1) [1, 3, 4] 2. Therapeutic potential: - Cancer: IMD 0354 is a potential anticancer drug for the treatment of cancers with overactive NF-κB, including mast cell tumors and breast cancer, by inhibiting cell proliferation, inducing apoptosis and arresting the cell cycle [1, 3].
- Inflammatory Diseases: IMD 0354 holds promise for treating inflammatory eye diseases (e.g., uveitis) and other NF-κB-driven inflammatory diseases (e.g., rheumatoid arthritis) by inhibiting cytokine secretion and tissue inflammation [4].
3. Development Background:
- IMD 0354 is a small molecule IKKβ inhibitor that was initially developed as a tool compound to study the NF-κB signaling pathway. Preclinical data in cancer and inflammation models support its potential as a therapeutic candidate, but it has not yet entered clinical trials [3, 4].
4. Selectivity Advantage:
- Compared to non-selective NF-κB inhibitors, IMD 0354 specifically targets IKKβ (rather than IKKα or other kinases), thereby reducing off-target effects and improving safety [2, 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.)