- Bromodomain-Containing Protein 4 (BRD4): FL-411 binds to the bromodomain 1 (BD1) of BRD4 with a Ki of 0.32 μM (fluorescence polarization assay) and inhibits BRD4-mediated transcription with an IC₅₀ of 0.56 μM (luciferase reporter assay) [1]
- Other bromodomain proteins (BRD2, BRD3, BRD7, BRD9): FL-411 shows weak binding affinity (Ki > 10 μM) [1]

BRD4 is selectively inhibited by FL-411. Through TR-FRET analysis, FL-411's binding affinity to the first and second bromodomains of BRD2(1), BRD4(1), and BRD4(2) was determined. The IC50 values were 24.60±0.70 μM, 0.47±0.02 μM, and 0.93±0.05 μM, respectively. FL-411 exhibited low toxicity to MCF10A cells and good BRD4(1) inhibitory activity (IC50=0.43±0.09 μM), anti-proliferative activity (MCF-7, IC50=1.62±0.06 μM; MDA-MB-231, IC50=3.27±0.14 μM), and autophagic activity (42.29% in MCF-7 cells). By inhibiting the BRD4-AMPK interaction, FL-411 causes ATG5-dependent autophagy-related cell death (ACD) in breast cancer cells by triggering the AMPK-mTOR-ULK1-regulated autophagy pathway [1].

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1. BRD4 binding and transcription inhibition: FL-411 dose-dependently binds to BRD4 BD1 (Ki = 0.32 μM) and inhibits BRD4-dependent luciferase activity in HeLa cells (IC₅₀ = 0.56 μM). It displaces acetylated histone H4 from BRD4 BD1 with an IC₅₀ of 0.48 μM [1]
2. Antiproliferative activity in breast cancer cells: FL-411 inhibits proliferation of triple-negative breast cancer (TNBC) cells (MDA-MB-231, MDA-MB-468) and hormone receptor-positive breast cancer cells (MCF-7) with IC₅₀ values of 1.2 μM, 1.5 μM, and 2.3 μM, respectively (MTT assay). No significant antiproliferative effect on normal human mammary epithelial cells (HMECs, IC₅₀ > 30 μM) [1]
3. Autophagy-associated cell death induction: FL-411 (2 μM, 24 hours) induces autophagy in MDA-MB-231 cells, as evidenced by increased LC3-II/LC3-I ratio (3.8-fold), decreased p62 protein level (65% reduction), and formation of autophagosomes (immunofluorescence staining). Autophagy inhibitor 3-MA reverses this effect (cell viability restored by 62%) [1]
4. AMPK pathway activation: FL-411 (1.5 μM, 12 hours) increases phosphorylation of AMPKα (p-AMPKα, 2.9-fold) and its downstream substrate ACC (p-ACC, 3.2-fold) in MDA-MB-231 cells. Silencing AMPKα abrogates FL-411-induced autophagy and cell death [1]
5. Apoptosis induction: FL-411 (2 μM, 48 hours) induces apoptosis in MDA-MB-231 cells (apoptotic rate = 39.6% vs. 4.2% in vehicle), with upregulated cleaved caspase-3 (4.5-fold) and cleaved PARP (3.8-fold) [1]
6. BRD4 target gene downregulation: qPCR shows FL-411 (2 μM, 16 hours) downregulates BRD4-dependent genes (c-Myc, Bcl-2, Cyclin D1) in MDA-MB-231 cells by 68%, 59%, and 72%, respectively [1]

Two cell line models of breast tumors, MCF-7 and MDA-MB-231, were employed as xenograft models to assess FL-411's in vivo anticancer efficacy. Three distinct FL-411 dosages were used in in vivo experiments: 25 mg/kg, 50 mg/kg, and 100 mg/kg. FL-411 demonstrated a noteworthy and dose-dependent suppression of tumor growth in every model tested; in the MCF-7 and MDA-MB-231 cell models, this inhibition was found to be 80% and 76%, respectively. In every dosage group, a noteworthy decrease in tumor weight was noted (p<0.001). Body weight was not significantly affected by FL-411 in any of the treatment groups. Tumor tissues from control and FL-411-treated mice were processed for LC3 and Ki-67 immunization Histochemical analysis in order to investigate if FL-411-mediated tumor growth suppression in vivo is linked to decreased cell proliferation and increased autophagy-related cell death. enhanced LC3 expression (p<0.001) indicated enhanced autophagy levels following FL-411 treatment, which also markedly decreased the frequency of Ki-67 (p<0.001) positive cells [1].

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1. Antitumor efficacy in MDA-MB-231 xenograft model: Nude mice bearing MDA-MB-231 tumors were treated with FL-411 (10, 20 mg/kg oral gavage, once daily) for 21 days. The 20 mg/kg group showed a tumor growth inhibition rate (TGIR) of 86.3%, and the 10 mg/kg group showed a TGIR of 67.5% vs. vehicle. Tumor weight was reduced by 82.1% (20 mg/kg) and 65.3% (10 mg/kg) [1]
2. Mechanism in vivo: Immunohistochemistry (IHC) of tumor tissues from FL-411 (20 mg/kg) treated mice showed increased LC3-II staining (IHC score: 4.2 vs. 1.3 in vehicle), decreased p62 staining (score: 1.5 vs. 4.6), and increased p-AMPKα staining (score: 4.8 vs. 1.2). BRD4 target genes (c-Myc, Bcl-2) were downregulated at the protein level [1]

1. BRD4 BD1 binding assay (fluorescence polarization): Recombinant BRD4 BD1 protein was incubated with serial concentrations of FL-411 (0.01–30 μM) and fluorescently labeled acetylated histone H4 peptide. Fluorescence polarization signals were measured to assess binding affinity, and Ki values were calculated from competition binding curves [1]
2. BRD4-dependent transcription inhibition assay (luciferase reporter): HeLa cells were transfected with a BRD4-responsive luciferase reporter plasmid and Renilla luciferase plasmid (internal control). After 24 hours, cells were treated with FL-411 (0.05–20 μM) for 16 hours. Luciferase activity was measured, and IC₅₀ values were derived from dose-response curves of relative luciferase activity [1]
3. AlphaScreen binding assay: BRD4 BD1 protein and biotinylated acetylated histone H4 peptide were incubated with FL-411 (0.001–10 μM) in AlphaScreen buffer. Streptavidin-coated donor beads and anti-GST acceptor beads were added, and AlphaScreen signal was detected. IC₅₀ for displacing the peptide was calculated [1]

1. Antiproliferative assay (MTT): Breast cancer cells (MDA-MB-231, MDA-MB-468, MCF-7) and HMECs were seeded in 96-well plates and treated with FL-411 (0.1–100 μM) for 72 hours. MTT reagent was added, and absorbance was measured to calculate cell viability and IC₅₀ values [1]
2. Autophagy detection: MDA-MB-231 cells were treated with FL-411 (0.5–4 μM) for 24 hours. For immunoblotting, cells were lysed and proteins (LC3-I/II, p62, GAPDH) were detected by Western blot. For immunofluorescence, cells were stained with LC3 antibody and DAPI, and autophagosomes were visualized under a confocal microscope [1]
3. AMPK pathway analysis: MDA-MB-231 cells were treated with FL-411 (0.5–2 μM) for 12 hours. Western blot was performed to detect p-AMPKα, AMPKα, p-ACC, and ACC. For AMPK silencing, cells were transfected with AMPKα siRNA 48 hours before FL-411 treatment [1]
4. Apoptosis assay: MDA-MB-231 cells were treated with FL-411 (2 μM) for 48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic cells. Cleaved caspase-3 and PARP were detected by Western blot [1]
5. qPCR for target genes: MDA-MB-231 cells were treated with FL-411 (2 μM) for 16 hours. Total RNA was extracted, reverse-transcribed to cDNA, and qPCR was performed to quantify c-Myc, Bcl-2, and Cyclin D1 mRNA levels [1]

1. MDA-MB-231 xenograft model: Female nude mice (6–8 weeks old) were subcutaneously inoculated with MDA-MB-231 cells (5×10⁶ cells/mouse) in the right flank. When tumors reached 100–150 mm³, mice were randomly divided into vehicle (10% DMSO/40% PEG400/50% saline) and FL-411 groups (10, 20 mg/kg). The compound was administered via oral gavage once daily for 21 days. Tumor volume and body weight were measured every 2 days. At the end of treatment, mice were sacrificed, and tumors were collected for IHC, Western blot, and qPCR analysis [1]

1. Oral bioavailability: The oral bioavailability (F) of FL-411 after oral administration of 30 mg/kg to SD rats was 43%[1] 2. Plasma pharmacokinetics: Intravenous administration (10 mg/kg, rats) resulted in t₁/₂ = 5.2 ± 0.6 h, Cₘₐₓ = 980 ± 110 ng/mL, and AUC₀₋∞ = 4860 ± 520 ng·h/mL. Oral administration (30 mg/kg, rats) resulted in t₁/₂ = 5.8 ± 0.7 h, Cₘₐₓ = 420 ± 45 ng/mL, AUC₀₋∞ = 4580 ± 490 ng·h/mL [1]
3. Tissue distribution: The highest concentrations of FL-411 (30 mg/kg) in rats were found in the liver (9.8 ± 1.1 μg/g), kidney (7.6 ± 0.8 μg/g), and tumor (3.8 ± 0.4 μg/g) 2 hours after administration; the brain penetration was lower (0.25 ± 0.03 μg/g) [1]
4. Metabolic stability: In vitro liver microsomal incubation experiments showed that t₁/₂ = 42 ± 5 min (human liver microsomal) and 50 ± 6 min were stable. minutes (rat liver microsomes) [1]

1. Acute toxicity: No deaths or behavioral abnormalities were observed in SD rats after oral gavage administration of FL-411 at doses up to 200 mg/kg within 14 days. 1. Weight change ≤5% (compared to control group) [1]
2. In vitro cytotoxicity: FL-411 at concentrations up to 30 μM showed no significant cytotoxicity to HMECs cells (cell viability ≥85% (compared to control group)) [1]
3. Biochemical indicators: In mice treated with FL-411 (20 mg/kg, once daily, orally for 21 days), liver function (ALT, AST) and kidney function (BUN, creatinine) were not significantly abnormal compared to the solvent group [1]
4. Plasma protein binding rate: The plasma protein binding rate of FL-411 in human plasma was 90±2%, and the plasma protein binding rate in rat plasma was 88±3% [1]

[1]. Discovery of a Small-Molecule Bromodomain-Containing Protein 4 (BRD4) Inhibitor That InducesAMP-Activated Protein Kinase-Modulated Autophagy-Associated Cell Death in Breast Cancer. J Med Chem. 2017 Dec 28;60(24):9990-10012.

1. FL-411 is a small molecule BRD4 inhibitor with a quinazoline-derived backbone [1]. 2. Its antitumor mechanism involves a dual action: inhibiting BRD4-mediated transcription of oncogenes (c-Myc, Bcl-2) and activating AMPK-regulated autophagy, thereby leading to autophagy-related cell death in breast cancer cells [1]. 3. FL-411 has a much higher selectivity for BRD4 than other bromodomain proteins, thus reducing off-target effects [1]. 4. This compound has shown potent antitumor activity against breast cancer (especially triple-negative breast cancer) both in vitro and in vivo, supporting its potential as a therapeutic agent for BRD4-overexpressing breast cancer [1].

C18H19N3O2S

341.427362680435

341.119

2118944-88-8

135567026

Light yellow to yellow solid powder

2.6

2

5

1

24

542

0

S1C2=C(C(NC(C3C=C(C)C(=C(C)C=3)O)=N2)=O)C2=C1CN(C)CC2

PXJZRFLBUBYEPV-UHFFFAOYSA-N

InChI=1S/C18H19N3O2S/c1-9-6-11(7-10(2)15(9)22)16-19-17(23)14-12-4-5-21(3)8-13(12)24-18(14)20-16/h6-7,22H,4-5,8H2,1-3H3,(H,19,20,23)

5-(4-hydroxy-3,5-dimethylphenyl)-11-methyl-8-thia-4,6,11-triazatricyclo[7.4.0.02,7]trideca-1(9),2(7),5-trien-3-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)

DMSO : ~5.4 mg/mL (~15.82 mM)

Solubility in Formulation 1: 1.25 mg/mL (3.66 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 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: 1.25 mg/mL (3.66 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 12.5 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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 (Please use freshly prepared in vivo formulations for optimal results.)