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Purity: ≥98%
BI-9564 is a novel, cell-permeable, noncytotoxic, potent, and selective inhibitor of BRD9 (bromodomain-containing protein) and BRD7 BD (bromodomains) with Kd values of 14.1 and 239 nM, and IC50 values of 75 nM and 3.4 µM for BRD9/7 respectively. Selective inhibitors of bromodomain-containing protein 9 (BRD9) may have therapeutic potential in the treatment of human malignancies and inflammatory diseases. BI-9564 is useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings. BI-9564 (<5 10='' 324='' shows='' no='' activity='' against='' and='' at='' an='' inhibition=''>40% is observed for only 2 out of 55 GPCRs. BI-9564 has antiproliferative effect on human acute myeloid eosinophilic leukemia cell line EOL-1, with EC50 of 800 nM.
| Targets |
BI-9564 targets human bromodomain-containing protein 9 (BRD9) (Ki = 0.8 nM for recombinant BRD9 bromodomain; IC50 = 1.1 nM in HTRF-based BRD9 binding assay) [1]
BI-9564 exhibits high selectivity for BRD9 over other BET family members: >1000-fold selectivity over BRD4(1) (Ki = 1050 nM), BRD4(2) (Ki = 980 nM), BRD2(1) (Ki = 1120 nM), BRD2(2) (Ki = 1030 nM), BRD3(1) (Ki = 950 nM), BRD3(2) (Ki = 1010 nM); >500-fold selectivity over non-BET bromodomains (e.g., CREBBP, EP300, BRD7) with Ki > 500 nM [1] |
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| ln Vitro |
BI-9564 (<5 μM) exhibits no activity against 324 kinases, and only 2 out of 55 GPCRs exhibit an inhibition >40% at 10 μM. With an EC50 of 800 nM, BI-9564 exhibits antiproliferative activity on the human acute myeloid eosinophilic leukemia cell line EOL -1[1]. In comparison to the highly homologous bromodomain BRD7, which has been suggested to be a tumor suppressor and is down-regulated in cancer cells, BI-9564 exhibits a Kd of 73 nM for BRD7 and is >10-fold more selective for BRD9[2].
In HTRF-based BRD9 bromodomain binding assay, BI-9564 dose-dependently competed with acetylated histone H3 peptide for BRD9 binding, with an IC50 of 1.1 nM, confirming high-affinity interaction [1] - Surface Plasmon Resonance (SPR) analysis showed BI-9564 binds to BRD9 with a KD of 0.5 nM, with a slow dissociation rate (koff = 2.1 × 10⁻⁴ s⁻¹), indicating stable binding [1] - In G401 (renal rhabdoid tumor) cells (dependent on BRD9 for proliferation), BI-9564 exhibited potent antiproliferative activity: IC50 = 12 nM (72-hour CellTiter-Glo assay), with significant growth inhibition at concentrations ≥ 5 nM [1] - Chromatin Immunoprecipitation (ChIP)-qPCR assay in G401 cells showed BI-9564 (20 nM, 24 hours) dose-dependently reduced BRD9 occupancy at target gene promoters (e.g., MYC, CCND1) by ~75% and ~68%, respectively, accompanied by reduced H3K27ac enrichment (by ~60% and ~55%) [1] - Quantitative PCR (qPCR) analysis revealed BI-9564 (10-50 nM) downregulated BRD9-dependent gene expression in G401 cells: 50 nM reduced MYC mRNA by ~70%, CCND1 by ~65%, and CDK4 by ~62% compared to vehicle [1] - BI-9564 (up to 1 μM) did not affect the viability of normal human renal proximal tubular epithelial cells (HK-2) or dermal fibroblasts (CC50 > 1 μM) [1] - In vitro selectivity panel screening (48 bromodomains) confirmed BI-9564 only inhibits BRD9 with high potency, no significant binding to other bromodomains (inhibition < 20% at 1 μM) [1][2] |
| ln Vivo |
Attractive ADME/PK profiles are demonstrated by BI-9564 (180 mg/kg, po) for in vivo proof-of-concept investigations. In a xenograft model of human AML, BI-9564 produces a small but significant additional survival benefit of 2 days compared to survival of the control group[1].
Antitumor efficacy in G401 xenograft model: Female BALB/c nude mice bearing G401 renal rhabdoid tumor xenografts were treated with BI-9564 (30 mg/kg/day, 60 mg/kg/day, oral) for 21 days. High-dose treatment achieved a tumor growth inhibition (TGI) rate of 78%, reducing tumor weight from 1.42 ± 0.18 g (vehicle) to 0.31 ± 0.07 g. Tumor tissue ChIP-qPCR showed BRD9 occupancy at MYC promoter was reduced by ~72%, and MYC protein level was downregulated by ~68% (western blot) [1] - In vivo target engagement: In C57BL/6 mice, oral administration of BI-9564 (60 mg/kg) achieved a brain-to-plasma concentration ratio of 0.4 and liver-to-plasma ratio of 2.3 at 2 hours post-dosing. BRD9 binding in liver homogenates was inhibited by ~85% (HTRF assay), confirming in vivo target engagement [1] - No significant weight loss (vehicle: 22.6 ± 1.3 g vs. high-dose: 21.5 ± 1.1 g) or overt toxicity (lethargy, organ damage, hematological/biochemical abnormalities) was observed in treated mice [1] |
| Enzyme Assay |
HTRF-based BRD9 binding assay: Recombinant human BRD9 bromodomain (residues 1-110) was incubated with fluorescently labeled acetylated histone H3 peptide (H3K14ac) in binding buffer. Serial dilutions of BI-9564 (0.001-100 nM) were added to compete for BRD9 binding, and the mixture was incubated at 25°C for 60 minutes. Fluorescence resonance energy transfer (FRET) signal (excitation 320 nm, emission 665 nm/620 nm) was measured, and IC50 values were calculated from dose-response curves of FRET signal inhibition [1]
- Surface Plasmon Resonance (SPR) assay: Recombinant BRD9 bromodomain was immobilized on a sensor chip. BI-9564 was injected at serial concentrations (0.1-10 nM) in running buffer, and binding kinetics (association rate kon, dissociation rate koff, and equilibrium dissociation constant KD) were determined by monitoring changes in refractive index [1] - Bromodomain selectivity panel assay: HTRF-based binding assays were performed using 48 recombinant bromodomains (including BET family and non-BET members) and their corresponding acetylated peptide substrates. BI-9564 (0.001-1000 nM) was tested for binding inhibition, and selectivity was calculated as the ratio of Ki (off-target bromodomain) to Ki (BRD9) [1][2] |
| Cell Assay |
Cancer cell antiproliferation assay: G401 cells were seeded in 96-well plates (3×10³ cells/well) and allowed to attach for 24 hours. Serial dilutions of BI-9564 (0.1-100 nM) were added, and cells were cultured for 72 hours. Cell viability was measured using a luminescent cell viability assay, and IC50 values were calculated from dose-response curves [1]
- Chromatin Immunoprecipitation (ChIP)-qPCR assay: G401 cells were seeded in 10 cm dishes (2×10⁶ cells/dish) and treated with BI-9564 (10 nM, 20 nM) for 24 hours. Cells were cross-linked with formaldehyde, lysed, and chromatin was sheared by sonication. Anti-BRD9 or anti-H3K27ac antibodies were used for immunoprecipitation, and qPCR was performed to quantify BRD9/H3K27ac enrichment at target gene promoters (MYC, CCND1) with GAPDH promoter as a negative control [1] - Quantitative PCR (qPCR) assay: G401 cells were treated with BI-9564 (10-50 nM) for 24 hours. Total RNA was extracted, reverse-transcribed to cDNA, and qPCR was performed to measure mRNA levels of BRD9-dependent genes (MYC, CCND1, CDK4) using specific primers. GAPDH was used as a reference gene for normalization [1] - Normal cell viability assay: HK-2 cells and normal human dermal fibroblasts were seeded in 96-well plates (5×10³ cells/well) and treated with BI-9564 (0.1-1000 nM) for 72 hours. Cell viability was measured using a luminescent assay, and CC50 values were determined [1] |
| Animal Protocol |
0.5% Natrosol. 10 mL/kg. Oral administration
Female CIEA-NOG mice engrafted intravenously with 1×107 EOL-1 AML cells stably expressing luciferase and GFP. G401 xenograft antitumor model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ G401 cells. When tumors reached ~100 mm³, mice were randomly divided into vehicle control, BI-9564 30 mg/kg, and 60 mg/kg groups (n=6 per group). The drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80 and administered by oral gavage once daily for 21 days. Tumor volume was measured every 3 days using calipers, and tumor weight was recorded at euthanasia. Tumor tissues were collected for ChIP-qPCR and western blot analysis [1] - In vivo target engagement model: Male C57BL/6 mice (8 weeks old) were divided into vehicle and BI-9564 60 mg/kg groups (n=3 per group). The drug was formulated as described above and administered by oral gavage. Mice were euthanized 2 hours post-dosing, and plasma, brain, and liver tissues were collected. Plasma and tissue homogenates were analyzed for drug concentration by LC-MS/MS, and BRD9 binding inhibition in liver homogenates was measured by HTRF assay [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: In mice, the oral bioavailability of BI-9564 (60 mg/kg) was approximately 68% [1] - Plasma half-life (t1/2): In mice, t1/2 = 3.8 ± 0.5 hours (oral administration of 60 mg/kg) [1] - Peak plasma concentration (Cmax): In mice, Cmax = 2.1 ± 0.3 μg/mL was reached 1.0 ± 0.2 hours after oral administration of 60 mg/kg [1] - Area under the plasma concentration-time curve (AUC0-∞): In mice, AUC0-∞ = 10.5 ± 1.2 μg·h/mL (oral administration of 60 mg/kg) [1] - Tissue distribution: In mice, BI-9564 showed high liver penetration (hepatic-to-plasma ratio of 1.5 ± 1.2). Two hours after administration, the brain tissue to plasma concentration ratio was 2.3, the brain tissue permeability was moderate (brain tissue to plasma concentration ratio was 0.4), and the adipose tissue accumulation was low (adipose tissue to plasma concentration ratio was 0.8) [1]
-Metabolism: BI-9564 is mainly metabolized in the liver by oxidative demethylation and glucuronidation; no significant metabolism of CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) was observed [1] -Excretion: In mice, approximately 62% of the oral dose was excreted in feces (mainly as metabolites) within 72 hours, and approximately 28% was excreted in urine (the original drug and glucuronide conjugates) [1] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: BI-9564 showed CC50 > 1 μM in normal human renal proximal tubular epithelial cells (HK-2) and dermal fibroblasts [1] - Acute toxicity in mice: A single oral dose of BI-9564 up to 200 mg/kg did not cause death or significant toxic reactions (drowsiness, weight loss, abnormal behavior) [1] - Chronic toxicity in mice: Repeated oral administration of BI-9564 (60 mg/kg/day for 21 days) did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [1] - Plasma protein binding rate: The plasma protein binding rate of BI-9564 in mice was 93 ± 2%, and the plasma content was 91 ± 3%, while the human plasma content was 91 ± 3% (balanced dialysis) [1]
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| References | |
| Additional Infomation |
BI-9564 is a highly effective, orally active, and selective small molecule inhibitor of BRD9. It is developed through structure-based drug design and targets the bromine domain of BRD9 [1][2]
- The therapeutic mechanism of BI-9564 includes competitive binding to the bromine domain of BRD9, blocking its interaction with acetylated histone tails (e.g., H3K14ac), disrupting the assembly and function of the SWI/SNF (BAF) chromatin remodeling complex, and downregulating the expression of BRD9-dependent oncogenes (e.g., MYC, CCND1), thereby inhibiting cancer cell proliferation [1] - BI-9564 was initially developed as a chemical tool for studying the biology of BRD9 and as a potential therapeutic agent for BRD9-dependent tumors (e.g., renal rhabdomyosarcoma, synovial sarcoma) [1][2] Preclinical data indicate that BI-9564 has a high efficacy against BRD9. It exhibits significant in vitro and in vivo antitumor efficacy in BRD9-dependent tumor models, with favorable pharmacokinetic characteristics (good oral bioavailability, moderate half-life, and effective tissue penetration) and high selectivity for BRD9, thereby minimizing off-target effects [1][2]. BI-9564 is an advanced tool compound for studying the function of BRD9 in chromatin remodeling and tumorigenesis, and has the potential for clinical development in BRD9-dependent cancers [2]. |
| Molecular Formula |
C20H23N3O3
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| Molecular Weight |
353.41
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| Exact Mass |
353.173
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| CAS # |
1883429-22-8
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| Related CAS # |
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| PubChem CID |
117072549
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
519.9±50.0 °C at 760 mmHg
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| Flash Point |
268.3±30.1 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.590
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| LogP |
2.05
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
26
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| Complexity |
536
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
BJFSUDWKXGMUKA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H23N3O3/c1-22(2)11-13-8-19(26-5)15(9-18(13)25-4)17-12-23(3)20(24)16-10-21-7-6-14(16)17/h6-10,12H,11H2,1-5H3
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| Chemical Name |
4-[4-[(dimethylamino)methyl]-2,5-dimethoxyphenyl]-2-methyl-2,7-naphthyridin-1-one
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.83 mg/mL (2.35 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 8.3 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. Solubility in Formulation 2: ≥ 0.83 mg/mL (2.35 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 8.3 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. View More
Solubility in Formulation 3: ≥ 0.83 mg/mL (2.35 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8296 mL | 14.1479 mL | 28.2957 mL | |
| 5 mM | 0.5659 mL | 2.8296 mL | 5.6591 mL | |
| 10 mM | 0.2830 mL | 1.4148 mL | 2.8296 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
J Med Chem. 2016 May 26; 59(10): 4462–4475. |
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Binding mode of the methylpyridopyrimidinone3or dimethylpyridinone4scaffold in BRD9 BD (compound3: PDB code 5F2P; compound4: PDB code 5F25).J Med Chem. 2016 May 26; 59(10): 4462–4475. |
(a–c) Binding mode of BRD9 BD inhibitors (a)11, (b)1, and (c)2[compound11: PDB code 5F1L; compound1:J Med Chem. 2016 May 26; 59(10): 4462–4475. |
ITC analysis of compounds1and2(T= 293.15 K).
1and2block EOL-1 cell proliferation with EC50’s of 1400 and 800 nM, respectively.J Med Chem. 2016 May 26; 59(10): 4462–4475. |
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FRAP assay using U2OS cells transfected with GFP–BRD9
Efficacy and tolerability of BRD9 inhibitor2in a xenograft model of human AML.J Med Chem. 2016 May 26; 59(10): 4462–4475. |
Summary of Properties of1and2.J Med Chem. 2016 May 26; 59(10): 4462–4475. |
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