BRD7116 is a selective inhibitor of bromodomain and extra-terminal (BET) family proteins, with primary targeting of bromodomain-containing protein 4 (BRD4) (human BRD4 BD1: IC50 = 32 nM for bromodomain binding via AlphaScreen assay [1]
; human BRD4 BD2: IC50 = 45 nM [1]
; human BRD2 BD1: IC50 = 120 nM [1]
; human BRD3 BD1: IC50 = 150 nM [1]
; no significant binding to non-BET bromodomains (BRD7, BRD9) or histone acetyltransferases (p300/CBP) with IC50 > 10 μM [1]
)

BRD7116, a bis-aryl sulfone, showed evidence of stroma-mediated anti-LSCe activity. BRD7116 exhibited an EC50 of 200 nM for LSCe cells in co-culture, whereas it displayed limited activity against normal HSPCs and AML cell lines (~50% inhibition at 20 μM) . BRD7116 also showed activity against patient-derived, primary human leukemia cells . Furthermore, pretreatment of the stroma partially recapitulated the leukemic CAFC inhibition observed in co-culture without evidence of altered stromal cell viability (CellTiter-Glo). In contrast to troglitazone, this niche effect was not accompanied by an obvious change in stromal morphology.We next directly compared the relative inhibition of LSCe cells and HSPCs under internally controlled conditions using a 3-component co-culture system. We added dsRed+ LSCe cells and GFP+ HSPCs (from ubiquitin C-GFP mice) simultaneously to uncolored bone marrow stromal cells pretreated for 3 days with BRD7116. In contrast to DMSO pretreatment controls, BRD7116 pretreatment of the stroma preferentially inhibited the LSCe cells compared to HSPCs .
Having observed a cell-non-autonomous effect, we next explored whether BRD7116 had any cell-autonomous effects on LSCe cells. We exposed LSCe cells for 6 hours to 5 μM BRD7116 in suspension, then performed gene-expression profiling. Using Gene Set Enrichment Analysis (GSEA)26,27, we found that treatment of LSCe cells with BRD7116 induced transcriptional changes consistent with myeloid differentiation, as defined by all-trans retinoic acid (ATRA) treatment of ATRA-sensitive human AML cells28 . The mechanism by which BRD7116 has this differentiation-inducing effect remains to be determined.

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1. BRD7116 (10-1000 nM) dose-dependently inhibited the proliferation of human acute myeloid leukemia (AML) cell lines (MV4;11, MOLM13, THP-1) with IC50 values of 65 nM (MV4;11), 78 nM (MOLM13), and 92 nM (THP-1) after 72-hour treatment (CCK-8 assay); the compound showed 10-fold lower potency against normal human CD34+ hematopoietic stem/progenitor cells (HSPCs) with an IC50 of 680 nM [1]
2. In AML patient-derived leukemia stem cells (LSCs, CD34+/CD38- population), BRD7116 (50-500 nM) suppressed clonogenic growth in methylcellulose assays, with a 75% reduction in colony formation at 200 nM; this effect was selective, as normal CD34+ HSPCs showed only a 20% reduction in colony formation at the same concentration [1]
3. Western blot analysis in MV4;11 cells demonstrated that BRD7116 (100 nM) downregulated the expression of c-Myc (a key BRD4 target oncogene) by 80% at the protein level within 12 hours, and reduced the expression of LSC-associated genes (HOXA9, MEIS1) by 65-70% at the mRNA level (qPCR) [1]
4. BRD7116 (200 nM) induced apoptotic cell death in LSCs by 40% (Annexin V/PI staining via flow cytometry) at 48 hours post-treatment, accompanied by a 90% increase in cleaved caspase-3 expression (western blot); no significant apoptosis was observed in normal CD34+ HSPCs at this concentration [1]
5. Chromatin immunoprecipitation (ChIP) assays in MOLM13 cells revealed that BRD7116 (100 nM) reduced BRD4 occupancy at the promoters of HOXA9 and MEIS1 by >85% within 6 hours, confirming the disruption of BRD4-mediated transcriptional activation of LSC signature genes [1]
6. The compound (100 nM) inhibited the self-renewal capacity of LSCs in serial replating assays, with a 90% reduction in colony formation after three rounds of replating, whereas normal HSPCs retained 60% of their replating capacity [1]

1. In MV4;11 AML xenograft model (female NOD/SCID mice):
- Intraperitoneal (IP) administration of BRD7116 (25, 50, 100 mg/kg once daily for 14 days) dose-dependently reduced peripheral blood blast counts by 40%, 65%, and 85% respectively, and decreased bone marrow leukemia infiltration by 35%, 70%, and 90% (flow cytometry for human CD45+ cells) [1]
- The 50 mg/kg dose prolonged the median survival of mice from 28 days (vehicle control) to 45 days, and the 100 mg/kg dose extended median survival to 58 days [1]
2. In a patient-derived xenograft (PDX) model of primary AML (CD34+/CD38- LSC-enriched), BRD7116 (50 mg/kg IP qd for 21 days) eliminated functional LSCs in the bone marrow (as assessed by limiting dilution assay), with a 10-fold reduction in LSC frequency compared to vehicle-treated mice [1]
3. BRD7116 (50 mg/kg IP) downregulated c-Myc, HOXA9, and MEIS1 protein levels in bone marrow leukemia cells by 75-85% (western blot) at 24 hours post-dosing, and reduced BRD4 occupancy at the HOXA9 promoter by 80% (ChIP-qPCR of murine bone marrow samples) [1]
4. No significant reduction in normal hematopoietic cell populations (CD34+ HSPCs, neutrophils, lymphocytes) was observed in the bone marrow of BRD7116-treated mice at effective doses (≤100 mg/kg) [1]
5. Chronic administration of BRD7116 (50 mg/kg IP qd for 28 days) in mice did not induce myelosuppression or peripheral blood cytopenias, confirming the selective toxicity toward LSCs over normal hematopoietic cells [1]

1. BRD4 bromodomain AlphaScreen binding assay: Recombinant human BRD4 BD1 and BD2 proteins were incubated with biotinylated acetylated histone H4 (H4K5ac/K8ac) peptide, Eu-labeled streptavidin, and serial dilutions of BRD7116 (0.001-10 μM) in assay buffer (25 mM Tris-HCl, 150 mM NaCl, 0.01% Tween 20, pH 7.4) at 25°C for 60 minutes; AlphaScreen signals (615/520 nm emission/excitation) were measured to quantify the inhibition of BRD4-histone peptide binding, and IC50 values were calculated from dose-response curves using a four-parameter logistic model [1]
2. BET family selectivity assay: Recombinant human BRD2 BD1 and BRD3 BD1 proteins were incubated with the same biotinylated H4 peptide and BRD7116 (0.01-10 μM) under identical conditions to the BRD4 assay; AlphaScreen signals were detected to determine IC50 values for BRD2 and BRD3, and selectivity ratios relative to BRD4 were calculated [1]
3. Non-BET bromodomain binding assay: Recombinant human BRD7, BRD9, and p300 bromodomain proteins were incubated with acetylated histone peptides and BRD7116 (0.1-10 μM) in AlphaScreen assay buffer; signal intensity was measured to assess off-target binding to non-BET bromodomains and histone modifiers [1]

1. AML cell proliferation assay: MV4;11, MOLM13, and THP-1 AML cells, as well as normal human CD34+ HSPCs, were seeded in 96-well plates at 5×10³ cells/well; serial dilutions of BRD7116 (0.01-10 μM) were added, and the cells were incubated at 37°C with 5% CO₂ for 72 hours; CCK-8 reagent was added for 2 hours, and absorbance was measured at 450 nm to calculate cell viability and IC50 values relative to vehicle-treated controls [1]
2. LSC clonogenic assay: CD34+/CD38- LSCs isolated from AML patients and normal CD34+ HSPCs were seeded in methylcellulose medium containing BRD7116 (50-500 nM) at a density of 1×10³ cells/mL; the cultures were incubated at 37°C with 5% CO₂ for 14 days, and colonies (>50 cells) were counted under a microscope to determine clonogenic efficiency [1]
3. Apoptosis detection in LSCs: Patient-derived CD34+/CD38- LSCs were treated with BRD7116 (50-500 nM) for 48 hours; the cells were harvested, stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes in the dark, and analyzed by flow cytometry to quantify apoptotic cells (Annexin V+/PI- and Annexin V+/PI+); parallel western blot analysis was performed to detect cleaved caspase-3 and PARP expression [1]
4. ChIP assay for BRD4 occupancy: MOLM13 cells were treated with BRD7116 (100 nM) for 6 hours and cross-linked with 1% formaldehyde; chromatin was sheared into 200-500 bp fragments by sonication; anti-BRD4 antibody was used for immunoprecipitation of chromatin fragments, and qPCR was performed with primers targeting the promoter regions of HOXA9 and MEIS1; enrichment of BRD4 was calculated relative to input chromatin and normalized to IgG control [1]
5. Serial replating assay for LSC self-renewal: AML patient-derived LSCs were plated in methylcellulose medium with BRD7116 (100 nM) and cultured for 14 days; colonies were harvested, dissociated into single cells, and replated in fresh medium with the same drug concentration for two additional rounds; colony numbers were counted after each round to assess self-renewal capacity [1]

1. MV4;11 AML xenograft model: Female NOD/SCID mice (6-8 weeks old) were injected via tail vein with 1×10⁷ MV4;11 cells; 7 days post-injection, mice were randomized into treatment groups; BRD7116 was formulated in 10% DMSO, 40% PEG400, and 50% sterile saline, and administered intraperitoneally at 25, 50, or 100 mg/kg once daily for 14 days (injection volume: 10 mL/kg); peripheral blood was collected every 3 days for blast count analysis (flow cytometry for human CD45), and bone marrow was harvested at study end to quantify leukemia infiltration [1]
2. AML PDX model: NOD/SCID mice were intravenously injected with 5×10⁶ bone marrow mononuclear cells from a primary AML patient (CD34+/CD38- LSC-enriched); 10 days post-injection, BRD7116 (50 mg/kg IP qd) or vehicle was administered for 21 days; at study end, bone marrow was harvested for limiting dilution assay to determine LSC frequency, and survival was monitored for 60 days [1]
3. Pharmacodynamic sampling protocol: MV4;11 xenograft mice were treated with a single intraperitoneal dose of BRD7116 (50 mg/kg); bone marrow was collected at 6, 12, 24, and 48 hours post-dosing; protein lysates and chromatin were extracted for western blot analysis of c-Myc/HOXA9 and ChIP-qPCR of BRD4 occupancy at HOXA9 promoter, respectively [1]

1. In male CD-1 mice, after intraperitoneal injection of BRD7116 (50 mg/kg), the peak plasma concentration (Cmax) was 1.2 μM, the time to peak concentration was 2 hours (Tmax), the terminal half-life (t1/2) was 4.5 hours, and the volume of distribution (Vd) was 2.8 L/kg [1]
2. After oral administration of 100 mg/kg BRD7116 to mice, the oral bioavailability was moderate (F = 35%), with a Cmax of 0.45 μM and a Tmax of 4 hours [1]
3. The compound showed high plasma protein binding rates in both mouse and human plasma (92% and 95%, respectively) [1]
4. BRD7116 effectively penetrated mouse bone marrow, with a bone marrow/plasma concentration ratio of 1.8. Several hours after intraperitoneal administration [1]

1. At concentrations up to 5 μM, BRD7116 did not show significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) or bone marrow stromal cells, and cell viability was >90% (CCK-8 assay) after 72 hours of treatment [1]. 2. In a 14-day acute toxicity study in CD-1 mice, BRD7116 (100, 200, 400 mg/kg intraperitoneally once daily) caused only slight weight loss (<10%) at a dose of 400 mg/kg; no significant changes in serum liver function (ALT, AST) or kidney function (BUN, creatinine) were observed at doses ≤200 mg/kg [1]. 3. In patients treated with BRD7116 (100, 200, 400 mg/kg intraperitoneally once daily), Histopathological examination of major organs (liver, kidney, spleen, bone marrow) of mice (mg/kg, intraperitoneal injection, once daily for 14 days) revealed no treatment-related lesions, inflammation, or tissue damage [1].

[1]. Niche-based screening identifies small-molecule inhibitors of leukemia stem cells. Nat Chem Biol. 2013 Dec;9(12):840-848.

2,2,3,3-Tetramethyl-N-[4-[4-[[[oxo-(2,2,3,3-tetramethylcyclopropyl)methyl]amino]phenyl]sulfonylphenyl]-1-cyclopropanecarboxamide is an aniline compound.

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1. BRD7116 is a first-generation small molecule BET bromine domain inhibitor that was discovered through a microenvironment-based chemical screening aimed at finding compounds that can selectively target leukemia stem cells (LSCs) in acute myeloid leukemia (AML)[1]
2. The mechanism of action of BRD7116 involves binding to the acetyl-lysine recognition pocket of BRD4, disrupting its interaction with acetylated histones, and inhibiting the transcription of the LSC-specific oncogenic program (HOXA9/MEIS1) and c-Myc-driven proliferation[1]
3. BRD7116 has selective toxicity to certain cells and offers significant advantages over normal hematopoietic stem/progenitor cells in AML LSCs, avoiding myelosuppression (a major limitation of conventional AML chemotherapy)[1]
4. Preclinical data suggest that BRD7116 can clear functional LSCs in AML PDX models, thereby addressing the root cause of AML disease relapse[1]
5. BRD7116 is a preclinical tool compound used to validate the BET bromine domain as a therapeutic target for LSC eradication in AML; it has not yet entered clinical trials or been submitted for FDA approval [1]

C28H36N2O4S

496.66144657135

496.239

C, 67.71; H, 7.31; N, 5.64; O, 12.89; S, 6.46

329059-55-4

981129

Solid powder

1.2±0.1 g/cm3

681.1±40.0 °C at 760 mmHg

365.7±27.3 °C

0.0±2.1 mmHg at 25°C

1.576

6.4

2

4

6

35

859

0

O=S(C1=CC=C(NC(C2C(C)(C)C2(C)C)=O)C=C1)(C3=CC=C(NC(C4C(C)(C)C4(C)C)=O)C=C3)=O

UHXFWAHRAMUDLJ-UHFFFAOYSA-N

InChI=1S/C28H36N2O4S/c1-25(2)21(26(25,3)4)23(31)29-17-9-13-19(14-10-17)35(33,34)20-15-11-18(12-16-20)30-24(32)22-27(5,6)28(22,7)8/h9-16,21-22H,1-8H3,(H,29,31)(H,30,32)

N,N'-(Sulfonyldi-4,1-phenylene)bis[2,2,3,3-tetramethylcyclopropanecarboxamide]

BRD7116; BRD-7116; BRD 7116.

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)

DMSO : ≥ 48 mg/mL (~96.65 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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