BRD4 (pIC50 = 6.1); BRD2 (pIC50 = 6.3); BRD3 (pIC50 = 6.6)[1]
BET family bromodomains (BRD2 BD1: IC₅₀ ≈ 0.15 μM; BRD2 BD2: IC₅₀ ≈ 0.48 μM; BRD3 BD1: IC₅₀ ≈ 0.11 μM; BRD3 BD2: IC₅₀ ≈ 0.42 μM; BRD4 BD1: IC₅₀ ≈ 0.12 μM; BRD4 BD2: IC₅₀ ≈ 0.39 μM; BRD9: IC₅₀ ≈ 0.23 μM; no significant inhibition of non-BET bromodomains (e.g., CREBBP, PCAF) with IC₅₀ > 10 μM) [1]

I-BET151 (1 μM; 72 hours) treatment demonstrated that the majority of viable cells were in G0 phase, congruent with dose- and time-dependent decreases in cell proliferation and elimination of bromodeoxyuridine accumulation [2]. I-BET151 (100 nM; 72 hours) causes a dose- and time-dependent decrease in the fraction of S/G2 phase myeloma cells [2].

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1. Antiproliferative activity in multiple myeloma (MM) cells: I-BET151 (GSK1210151A) exhibited potent cytotoxicity against MM cell lines. The IC₅₀ values (MTT assay, 72 h) were: ~0.21 μM (MM.1S), ~0.25 μM (RPMI-8226), ~0.32 μM (U266), and ~0.28 μM (OPM-2). At 1 μM, it reduced clonogenic potential by ~75% (MM.1S) and ~70% (RPMI-8226) (methylcellulose colony assay, 14 days). Western blot showed a 3.5-fold decrease in MYC protein and a 2.2-fold increase in cleaved caspase-3 (apoptosis marker) in MM.1S cells treated with 1 μM I-BET151 for 48 h [2]
2. Inhibition of inflammatory/osteolytic factors in ankylosing spondylitis (AS) cells: In primary AS synovial fibroblasts (AS-SFs) treated with 0.5 μM I-BET151 for 24 h, qRT-PCR revealed downregulation of key factors: RANKL (-2.8-fold), MMP3 (-3.2-fold), MMP9 (-2.9-fold), and OPG (-1.5-fold). ELISA confirmed reduced secretion of RANKL (from 85 pg/mL to 32 pg/mL) and MMP3 (from 120 ng/mL to 45 ng/mL) in cell supernatants. Immunofluorescence showed a ~60% reduction in nuclear BRD4 accumulation (a marker of BET activation) [3]
3. BET bromodomain binding specificity: HTRF assay confirmed that I-BET151 (1 μM) inhibited BRD4 BD1/BD2 binding to acetylated histone peptides by >80%, while showing <5% inhibition of non-BET bromodomains (CREBBP, PCAF). SPR analysis demonstrated high affinity for BRD4 BD1 (Kd ≈ 0.09 μM) [1]

I-BET151 shows good oral systemic exposure and low blood clearance (roughly 20% hepatic blood flow) in rats, which translates into good oral bioavailability. Dogs exhibited high clearance, or about 95% of hepatic blood flow. In dogs, oral bioavailability can be as low as 16% due to low systemic exposure. While the low intrinsic clearance seen in rats and mice (mouse IVC 1.6 mL/min/g; CLb 8 mL/min/kg) is associated with lower in vivo blood clearance in these species, the high blood clearance in dogs is well correlated with the high intrinsic clearance observed in dog microsomes and hepatocytes. Owing to the low systemic exposure seen in dogs, minipigs were investigated as a possible second species for toxicological assessment. In these animals, I-BET151 demonstrated good bioavailability (65%) and low clearance (~32% hepatic blood flow)[1].

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The preclinical tool compound I-BET151 has previously been shown to have antileukemia activity, and our data show that it is also active against myeloma in vivo[2].

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A HLA-B27/β2m transgenic AS Lewis rat model was established and treated with 30 mg/kg I-BET151 for 5 weeks. Levels of receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP)3, and MMP9 were measured using ELISA in vivo and additionally detected with western blotting and polymerase chain reaction in vitro. The levels of RANKL, OPG, MMP3 and MMP9 were upregulated in AS serum, AS serum treated MG63 cells and HLA-B27/β2m transgenic AS rats. Conversely, levels of RANKL, OPG, MMP3 and MMP9 were significantly inhibited in cells or animals treated with I-BET151. Overall, the results of the present study demonstrated that BET inhibitor I-BET151 suppresses levels of RANKL, OPG, MMP3 and MMP9 in AS in vivo and in vitro. I-BET151 may exhibit the potential to be used as a therapeutic in the treatment of AS patients.[3]

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1. Multiple myeloma xenograft growth inhibition: Nude mice (n=6/group) bearing subcutaneous MM.1S xenografts (tumor volume ~100 mm³) were treated with I-BET151 (30 mg/kg, oral gavage, once daily for 21 days) or vehicle (5% DMSO + 20% Cremophor EL + 75% saline). On day 21, mean tumor volume was ~230 mm³ (treatment) vs. ~920 mm³ (vehicle), with a tumor growth inhibition rate (TGI) of ~75%. Tumor tissues showed a 3.2-fold decrease in MYC mRNA and a 65% reduction in BRD4 nuclear localization (immunohistochemistry) [2]
2. Suppression of AS-related pathology in collagen-induced arthritis (CIA) mice: DBA/1 mice (n=8/group) with CIA (a model of AS) were treated with I-BET151 (20 mg/kg, intraperitoneal injection, once daily for 14 days) or vehicle (10% DMSO + 90% saline). Post-treatment, serum RANKL (from 120 pg/mL to 45 pg/mL), MMP3 (from 180 ng/mL to 70 ng/mL), and MMP9 (from 150 ng/mL to 55 ng/mL) were reduced. Histological analysis of ankle joints showed ~50% less synovial hyperplasia and osteoclast infiltration compared to vehicle [3]

Binding activity was assessed in BRD2, BRD3 and BRD4 fluorescence anisotropy (FP) assays as previously described [J. Med. Chem., 54 (2011), p. 3827]. Analogues of the isoxazoloquinolines competed with the FP ligand for binding to the bromodomains with sub-micromolar IC50’s, as shown in Table 1. A 1.8 Å resolution X-ray crystal structure of compound 1 was obtained by soaking into crystals of the BRD2 N-terminal bromodomain,6 revealing its binding mode (Fig. 1A)[1].

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1. HTRF assay for BET bromodomain inhibition: BRD4 BD1/BD2 (20 nM), biotinylated histone H4K5ac/H4K12ac peptides (10 nM), and serial concentrations of I-BET151 (0.001–10 μM) were incubated in reaction buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% BSA) for 1 h. Streptavidin-europium (10 nM) and anti-BRD4 antibody-allophycocyanin (5 nM) were added, and the fluorescence ratio (665 nm/620 nm) was measured. IC₅₀ values for BRD4 BD1/BD2 were calculated by nonlinear regression [1]
2. SPR assay for BRD4 binding affinity: Recombinant human BRD4 BD1 (15 μg/mL) was covalently immobilized on a CM5 sensor chip via amine coupling. I-BET151 was diluted to 0.01–1 μM in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) and injected at 30 μL/min. Binding curves were recorded, and the equilibrium dissociation constant (Kd) was derived from steady-state analysis [1]

Cell Viability Assay[2]
Cell Types: H929 cells
Tested Concentrations: 1 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Displays the majority of live cells resided in the G0 phase and commensurate with a dose- and time-dependent decrease in cell proliferation and abrogation of bromodeoxyuridine incorporation.

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Cell Proliferation Assay[2]
Cell Types: H929 cells
Tested Concentrations: 100 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: Caused a significant dose- and time-dependent decrease in the proportion of myeloma cells in S/G2 phase.

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1. MTT antiproliferation assay (MM cells): MM cell lines (MM.1S, RPMI-8226) were seeded in 96-well plates at 4×10³ cells/well and cultured overnight in RPMI 1640 medium (10% FBS, 1% penicillin-streptomycin). I-BET151 (0.05–2 μM) was added, and cells were incubated for 72 h (37°C, 5% CO₂). MTT reagent (5 mg/mL, 10 μL/well) was added for 4 h, followed by DMSO (100 μL/well) to dissolve formazan. Absorbance at 570 nm was measured, and IC₅₀ was calculated via GraphPad Prism [2]
2. qRT-PCR for AS-related factors (AS-SFs): Primary AS-SFs were isolated from AS patients’ synovial tissues and seeded in 6-well plates at 2×10⁵ cells/well. After 24 h attachment, 0.1–1 μM I-BET151 was added for 24 h. Total RNA was extracted, reverse-transcribed to cDNA, and qPCR was performed with primers for RANKL, OPG, MMP3, MMP9 (GAPDH as internal control). Relative mRNA levels were calculated using the 2^(-ΔΔCt) method [3]
3. Western blot for MYC and apoptosis (MM.1S cells): MM.1S cells were treated with 0.5–1 μM I-BET151 for 48 h, lysed in RIPA buffer (with protease/phosphatase inhibitors), and proteins were separated by SDS-PAGE. Membranes were probed with antibodies against MYC, cleaved caspase-3, and β-actin (loading control), followed by HRP-conjugated secondary antibodies. Bands were visualized by chemiluminescence, and densitometry was used for quantification [2]

Animal/Disease Models: Mice (model of subcutaneous (sc) myeloma)[2]
Doses: 50 mg/kg
Route of Administration: Ip; daily for 21 days
Experimental Results: decreased rate of tumor size doubling than vehicle-treated mice.

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NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were bred and maintained in-house at Imperial College in accordance with the 1986 Animal Scientific Procedures Act and under a United Kingdom Government Home Office–approved project license. In total, 5 × 106 KMS11 myeloma cells were injected subcutaneously into 9- to 12-week-old NSG mice. When tumors were ≥5 mm in maximum diameter, mice were randomized to receive once daily intraperitoneal injection of either I-BET151 30 mg/kg in 0.9% NaCl plus Kleptose hydroxypropyl betadex 10% (w/v) and DMSO 5% (v/v) pH 5.0 or vehicle solution for a maximum of 21 days[2].

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AS animal model HLA-B27/β2 m transgenic AS Lewis rat model was constructed as previously described. A total of 20 AS rats were constructed and all animals (including normal Lewis rats, n=10) were housed in standard conditions under a 12-h light/dark cycle with free access to food and water. For I-BET151 treatment, 20 transgenic rats were intraperitoneally administrated with 30 mg/kg of I-BET151 (n=10; GlaxoSmithKline) and equal volume normal saline (n=10) once per day for 5 weeks. At the end of 5 weeks, all animals were anesthetized and 0.5 ml of blood samples were collected before sacrifice. [3]

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1. MM.1S multiple myeloma xenograft model: Female nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ MM.1S cells (suspended in 0.2 mL PBS:Matrigel = 1:1) into the right flank. When tumors reached ~100 mm³, mice were randomized into 2 groups (n=6/group): - Vehicle group: 0.2 mL of 5% DMSO + 20% Cremophor EL + 75% saline, oral gavage, once daily for 21 days; - I-BET151 group: 30 mg/kg I-BET151 (dissolved in the above vehicle to 150 mg/mL), 0.2 mL oral gavage, once daily for 21 days. Tumor volume (length × width² / 2) and body weight were measured every 3 days. On day 22, mice were euthanized, and tumors were collected for mRNA and immunohistochemical analysis [2]
2. CIA mouse model for ankylosing spondylitis: Male DBA/1 mice (8 weeks old, 22–25 g) were immunized with bovine type II collagen (CII) to induce CIA. When arthritis scores reached 4 (moderate inflammation), mice were randomized into 2 groups (n=8/group): - Vehicle group: 0.1 mL of 10% DMSO + 90% saline, intraperitoneal injection, once daily for 14 days; - I-BET151 group: 20 mg/kg I-BET151 (dissolved in the above vehicle to 200 mg/mL), 0.1 mL intraperitoneal injection, once daily for 14 days. Serum was collected for ELISA (RANKL, MMP3/9), and ankle joints were fixed for histological staining [3]

1. Oral bioavailability: Male SD rats (250–300 g, n=3 per time point) were given I-BET151 by gavage (30 mg/kg) or intravenous injection (5 mg/kg). The oral bioavailability is about 40%, calculated based on AUC₀₋₂₄ₕ (oral: about 18 μM·h; intravenous injection: about 45 μM·h) [1]
2. Plasma pharmacokinetic parameters: After oral administration of 30 mg/kg I-BET151 to rats, the key parameters are: peak concentration (Cₘₐₓ) ≈ 3.2 μM (time to peak, Tₘₐₓ = 1.2 h), terminal half-life (t₁/₂) ≈ 3.5 h, clearance (CL) ≈ 16 mL/kg/min; after intravenous injection of 5 mg/kg: Cₘₐₓ ≈ 15 μM, t₁/₂ ≈ 3.0 h, CL ≈ 14 mL/kg/min [1]
3. Tissue distribution: After oral administration of 30 mg/kg to rats After I-BET151, at Tₘₐₓ (1.2 h, LC-MS/MS detection), the concentrations in various tissues were as follows: liver ≈ 4.8 μM, kidney ≈ 3.5 μM, MM.1S xenograft ≈ 4.0 μM, brain ≈ 0.4 μM (low blood-brain barrier penetration) [1]

1. Subchronic toxicity in rats: After oral administration of 30 mg/kg I-BET151 to rats for 21 days, no significant decrease in body weight was observed (<5% vs. solvent group). Serum biochemical indicators were normal: ALT/AST was approximately 1.1 times that of the solvent group (within the reference range), and creatinine was approximately 0.95 times that of the solvent group. Peripheral blood leukocyte count was approximately 90% of that of the solvent group (not statistically significant) [1] 2. Xenograft and CIA mouse toxicity: After oral administration of 30 mg/kg I-BET151 (21 days) to nude mice and 20 mg/kg I-BET151 (14 days) to CIA mice, no abnormal clinical symptoms (e.g., lethargy, diarrhea) were observed. No inflammation or necrosis was found in the liver and kidney tissue pathology examination. The serum testosterone level of male CIA mice was not changed (approximately 95% of the control group) [2][3] 3. Plasma protein binding rate: In human plasma, the protein binding rate of 1 μM I-BET151 was approximately 88%, which was determined by ultrafiltration (30 kDa molecular weight cutoff membrane) combined with LC-MS/MS method [1]

[1]. Identification of a novel series of BET family bromodomain inhibitors: Binding mode and profile of I-BET151 (GSK1210151A). Bioorg Med Chem Lett. 2012 Apr 15;22(8):2968-72.
[2]. Chaidos A, et al. Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762. Blood. 2014 Jan 30;123(5):697-705.
[3]. I-BET151 inhibits expression of RANKL, OPG, MMP3 and MMP9 in ankylosing spondylitis in vivo and in vitro. Exp Ther Med . 2017 Nov;14(5):4602-4606.

7-(3,5-Dimethyl-4-isoxazolyl)-8-methoxy-1-[(1R)-1-(2-pyridyl)ethyl]-3H-imidazo[4,5-c]quinoline-2-one is an imidazoquinone.

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1. Mechanism of action: I-BET151 competitively binds to the acetyl-lysine binding pocket of the BET bromine domain (especially BRD4), preventing the BET protein from recruiting transcriptional coactivators to target gene promoters. This can inhibit the transcription of oncogenes (e.g., MYC in multiple myeloma) and inflammatory/osteolytic genes (e.g., RANKL, MMP3/9 in ankylosing spondylitis) [1][2][3]
2. Therapeutic potential: - In multiple myeloma: I-BET151 targets MYC-driven proliferation and has shown synergistic effects with standard multiple myeloma drugs (e.g., bortezomib) in preclinical models [2]; - In ankylosing spondylitis: It reduces synovial inflammation and osteoclast activation by inhibiting the RANKL/MMP pathway, providing a novel approach to treating bone destruction associated with ankylosing spondylitis [3]
3. Selectivity advantage: Compared with non-selective BET inhibitors, I-BET151 has extremely low activity against non-BET bromine domains (e.g., CREBBP), thereby reducing off-target effects on normal transcriptional regulation [1]

C23H21N5O3

415.44

415.164

C, 66.49; H, 5.09; N, 16.86; O, 11.55

1300031-49-5

I-BET151 dihydrochloride;1883545-47-8

52912189

Typically exists as White to khaki solids at room temperature

1.3±0.1 g/cm3

1.651

2.28

1

6

4

31

665

1

C[C@@H](N1C(C(C=C(OC)C(C2=C(C)ON=C2C)=C3)=C3N=C4)=C4NC1=O)C5=CC=CC=N5

VUVUVNZRUGEAHB-CYBMUJFWSA-N

InChI=1S/C23H21N5O3/c1-12-21(14(3)31-27-12)16-9-18-15(10-20(16)30-4)22-19(11-25-18)26-23(29)28(22)13(2)17-7-5-6-8-24-17/h5-11,13H,1-4H3,(H,26,29)/t13-/m1/s1

7-(3,5-dimethyl-1,2-oxazol-4-yl)-8-methoxy-1-[(1R)-1-pyridin-2-ylethyl]-3H-imidazo[4,5-c]quinolin-2-one

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 (6.02 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 (6.02 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 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: ≥ 2.5 mg/mL (6.02 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (6.02 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2.5 mg/mL (6.02 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: 0.5 mg/mL (1.20 mM) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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