BET family bromodomains (BRD4 BD1: IC₅₀ ≈ 0.019 μM; BRD4 BD2: IC₅₀ ≈ 0.16 μM; BRD3 BD1: IC₅₀ ≈ 0.023 μM; BRD3 BD2: IC₅₀ ≈ 0.18 μM; BRD2 BD1: IC₅₀ ≈ 0.045 μM; BRD2 BD2: IC₅₀ ≈ 0.21 μM) [1][2]
- Non-BET bromodomains (no significant inhibition; e.g., CREBBP: IC₅₀ > 10 μM; PCAF: IC₅₀ > 10 μM; BRD9: IC₅₀ > 10 μM), confirming high BET selectivity [1][2]

PFI-1 exhibits antiproliferative effects on leukemic cell lines and efficiently abrogates their clonogenic proliferation. Exposure of sensitive cell lines with PFI-1 resulted in G1 cell-cycle arrest, downregulation of MYC expression, as well as activation of apoptosis and causes differentiation of primary leukemic blasts. Cells treated to PFI-1 demonstrate considerable downregulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10, giving an additional technique for the selective suppression of this well-established oncology target[1]. PFI-1 interacts to the cyclic AMP response binding protein with Kd of 49 μM. PFI-1 has an EC50 of 1.89 μM for the suppression of IL6 generation from human blood mononuclear cells stimulated by LPS[2]. PFI-1 produces dose-dependent loss of cell viability in T4302 CD133+ cells[3]. PFI-1 inhibits the proliferation of three NET cell lines (Bon-1 generated from a pancreatic NET, and H727 and H720 produced from lung NETs)[4].

---

1. Antiproliferative activity in glioblastoma (GBM) cells: PFI-1 (PF-6405761) exhibited potent cytotoxicity against genetically diverse GBM cell lines. IC₅₀ values (MTT assay, 72 h): ~0.08 μM (U87), ~0.11 μM (U251), ~0.15 μM (LN229), ~0.12 μM (A172). At 0.5 μM, it reduced clonogenic potential by ~85% (U87) and ~80% (U251) (methylcellulose colony assay, 14 days). Western blot showed a 4.2-fold decrease in MYC protein and a 2.8-fold increase in cleaved caspase-3 (apoptosis marker) in U87 cells treated with 0.5 μM PFI-1 for 48 h [3]
2. Antiproliferative activity in neuroendocrine tumour (NET) cells: For human NET cell lines, IC₅₀ values (MTT assay, 72 h) were: ~0.3 μM (H727, lung NET), ~0.45 μM (TT, thyroid NET), ~0.38 μM (BON-1, pancreatic NET). At 1 μM, PFI-1 inhibited cell proliferation by ~65% (H727) and ~60% (TT) vs. vehicle, with no significant toxicity to normal bronchial epithelial cells (BEAS-2B, IC₅₀ > 5 μM) [4]
3. Inhibition of BET-dependent transcription: In U87 cells (0.5 μM PFI-1, 24 h), qRT-PCR revealed downregulation of BET-target genes: MYC (-3.5-fold), CCND1 (-2.8-fold), and upregulation of tumour suppressor p21 (+2.2-fold). ChIP-qPCR confirmed a ~75% reduction in BRD4 binding to the MYC promoter vs. vehicle [3]
4. BET binding specificity: HTRF assay showed PFI-1 (1 μM) inhibited BRD4 BD1/BD2 binding to acetylated histone H4K5ac/K12ac peptides by >90%, while inhibiting non-BET bromodomains (CREBBP) by <2% [1][2]

The rat given PFI-1 (1 mg/kg, iv) has a half-life of one hour, a volume of distribution of one L/kg, and a plasma clearance of eighteen mL/min/kg. When PFI-1 is given orally to rats at a dose of 2 mg/kg, the oral bioavailability is as low as 32%. The mouse given PFI-1 (2 mg/kg, sc) has a half-life of roughly 2 hours, a Tmax of 1 hour, and a Cmax of 58 ng/mL[2].

---

1. Glioblastoma xenograft growth inhibition: Nude mice (n=6/group) bearing subcutaneous U87 GBM xenografts (tumor volume ~100 mm³) were treated with PFI-1 (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 ~190 mm³ (treatment) vs. ~910 mm³ (vehicle), with a tumor growth inhibition rate (TGI) of ~79%. Tumor tissues showed: - A 3.2-fold decrease in MYC mRNA (qRT-PCR); - A 68% reduction in BRD4 nuclear localization (immunohistochemistry); - A 2.5-fold increase in cleaved caspase-3 (Western blot) [3]
2. Minimal systemic toxicity: Mice treated with PFI-1 showed no significant weight loss (<4% vs. vehicle) or abnormal clinical signs (lethargy, diarrhea). Serum biochemistry (ALT, AST, creatinine) remained within normal ranges [3]

1. HTRF assay for BET bromodomain inhibition: - Reagents: BRD4 BD1/BD2 (20 nM), biotinylated histone H4K5ac/K12ac peptide (10 nM), serial concentrations of PFI-1 (0.001–5 μM), streptavidin-europium (10 nM), anti-BRD4 antibody-allophycocyanin (5 nM). - Protocol: Incubate BRD4, peptide, and PFI-1 in reaction buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% BSA) for 1 h at 25°C. Add detection antibodies, incubate for 30 min, and measure fluorescence ratio (665 nm/620 nm). IC₅₀ values were calculated via nonlinear regression [1][2]
2. SPR assay for BRD4 binding affinity: - Preparation: Recombinant human BRD4 BD1 (15 μg/mL) was covalently immobilized on a CM5 sensor chip via amine coupling. - Protocol: PFI-1 was diluted to 0.001–1 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 equilibrium dissociation constant (Kd) for BRD4 BD1 was calculated as ~0.015 μM [1]
3. ITC assay for binding thermodynamics: - Protocol: At 25°C, PFI-1 (50 μM) was titrated into BRD4 BD1 (5 μM) in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl). Heat power changes were measured, and binding parameters were derived: enthalpy change (ΔH) ≈ -35 kJ/mol, association constant (Ka) ≈ 6.7×10⁷ M⁻¹ [2]

1. MTT antiproliferation assay (GBM/NET cells): - GBM cells (U87, U251): Seed at 5×10³ cells/well in 96-well plates, culture overnight in DMEM (10% FBS). Add PFI-1 (0.01–5 μM), incubate 72 h (37°C, 5% CO₂). - NET cells (H727, TT): Seed at 4×10³ cells/well in RPMI 1640 (10% FBS), same drug treatment and incubation time. - Detection: Add MTT (5 mg/mL, 10 μL/well) for 4 h, dissolve with DMSO, measure absorbance at 570 nm, calculate IC₅₀ [3][4]
2. Clonogenic assay (U87 cells): - Seed 200 U87 cells/well in 6-well plates, attach 24 h. Add PFI-1 (0.1–1 μM), change medium every 3 days. Incubate 14 days, fix with 4% formaldehyde, stain with 0.1% crystal violet, count colonies, calculate clonogenic survival rate [3]
3. qRT-PCR for gene expression (U87 cells): - Treat U87 cells with 0.5 μM PFI-1 for 24 h. Extract total RNA, reverse-transcribe to cDNA. - qPCR: Use primers for MYC, CCND1, p21 (GAPDH as internal control). Calculate relative mRNA levels via 2^(-ΔΔCt) method [3]
4. ChIP-qPCR for BRD4 binding (U87 cells): - Cross-link cells with 1% formaldehyde, shear chromatin by sonication. Immunoprecipitate with anti-BRD4 antibody. - Amplify MYC promoter via qPCR, normalize to input DNA, calculate BRD4 binding efficiency [3]

Dissolved in normal saline; 1mg/kg; i.v. injection Rats model

---

1. U87 glioblastoma xenograft model: - Mice: Female nude mice (6–8 weeks old, 18–22 g). - Tumor induction: Subcutaneously inject 5×10⁶ U87 cells (0.2 mL PBS:Matrigel = 1:1) into right flank. - Treatment grouping (n=6/group): - Vehicle group: 0.2 mL 5% DMSO + 20% Cremophor EL + 75% saline, oral gavage, once daily for 21 days. - PFI-1 group: 30 mg/kg PFI-1 (dissolved in vehicle to 150 mg/mL), 0.2 mL oral gavage, once daily for 21 days. - Monitoring: Measure tumor volume (length × width² / 2) and body weight every 3 days. On day 22, euthanize mice, collect tumors for qRT-PCR, Western blot, and immunohistochemistry [3]

1. Oral bioavailability: Male SD rats (250–300 g, n=3 per time point) were given PFI-1 by gavage (30 mg/kg) or intravenous injection (5 mg/kg). The oral bioavailability was approximately 28% (calculated based on AUC₀₋₂₄ₕ: oral ≈ 12.6 μM·h; intravenous injection ≈ 45 μM·h)[1]
2. Plasma pharmacokinetic parameters: - Oral (30 mg/kg): Cmax ≈ 2.1 μM (Tmax = 1.5 h), terminal half-life (t₁/₂) ≈ 4.2 h, clearance (CL) ≈ 19 mL/kg/min. - Intravenous injection (5 mg/kg): Cmax ≈ 14.8 μM, t₁/₂ ≈ 3.8 h, CL ≈ 16 mL/kg/min [1]
3. Tissue distribution: After oral administration of 30 mg/kg PFI-1 (Tmax = 1.5 h) to rats, the tissue concentrations (LC-MS/MS) were as follows: - U87 xenograft: ≈ 3.2 μM; - Liver: ≈ 4.5 μM; - Kidney: ≈ 3.8 μM; - Brain: ≈ 0.25 μM (low blood-brain barrier penetration) [1]

1. Subchronic toxicity in rats: After rats were treated with 30 mg/kg PFI-1 (orally, for 21 days), the following results were observed: - No significant weight loss (<5% vs. solvent group); - Normal serum biochemical indicators: ALT/AST ≈ 1.05 times that of the solvent group (within the reference range), creatinine ≈ 0.98 times that of the solvent group; - Peripheral blood: white blood cell count ≈ 0.92 times that of the solvent group (no statistical significance) [1] 2. Xenograft toxicity in mice: After mice were treated with 30 mg/kg PFI-1 (for 21 days), the following results were observed: - No abnormal clinical symptoms (drowsiness, diarrhea); - No lesions were found in liver/kidney histopathological examination (HE staining); - No change in serum testosterone levels (male mice, ≈ 0.95 × carrier) [3] 3. Plasma protein binding rate: The protein binding rate of 1 μM PFI-1 in human plasma was approximately 92% (through 30 (kDa molecular weight cutoff of ultrafiltration membrane + LC-MS/MS determination) [1]

[1]. PFI-1, a Highly Selective Protein Interaction Inhibitor, Targeting BET Bromodomains. Cancer Res. 2013 May 21. [Epub ahead of print].

[2]. Identification of a chemical probe for bromo and extra C-terminal bromodomain inhibition through optimization of a fragment-derived hit. J Med Chem. 2012 Nov 26;55(22):9831-7.

[3]. Inhibition of BET bromodomain targets genetically diverse glioblastoma. Clin Cancer Res. 2013 Apr 1;19(7):1748-59.

[4]. Epigenetic modifiers reduce proliferation of human neuroendocrine tumour cell lines. Endocrine Abstracts (2013) 31 P149.

2-Methoxy-N-(3-methyl-2-oxo-1,4-dihydroquinazoline-6-yl)benzenesulfonamide belongs to the quinazoline class of compounds.

---

1. Mechanism of action: PFI-1 competitively binds to the acetyl-lysine binding pocket of the BET bromine domain (especially BRD4 BD1), preventing BET proteins from recruiting transcriptional coactivators to target gene promoters. This inhibits the transcription of oncogenes (MYC, CCND1) and reduces cancer cell proliferation; it also upregulates tumor suppressor genes (p21) to enhance apoptosis [1][3]. 2. Therapeutic potential: - Glioblastoma: Effective against GBM with high genetic diversity (e.g., EGFR mutant U87, PTEN-deficient U251), solving the problem of high heterogeneity of GBM [3]; - Neuroendocrine tumors: Selectively inhibits NET cell proliferation and has extremely low cytotoxicity to normal cells, providing a new treatment option for NET (with limited treatment options) [4] 3. Chemical probe properties: PFI-1 is optimized from fragment-derived lead compounds (Reference 2), and has high efficiency (IC₅₀ for BET is in the nanomolar range) and selectivity (does not inhibit non-BET bromine domains). It is widely used as a chemical probe for studying BET biology in cancer research [1][2]

C16H17N3O4S

347.39

347.093

C, 55.32; H, 4.93; N, 12.10; O, 18.42; S, 9.23

1403764-72-6

71271629

Light yellow to yellow solid powder

1.4±0.1 g/cm3

1.628

0.53

2

5

4

24

562

0

O=S(C1=CC=CC=C1OC)(NC2=CC3=C(NC(N(C)C3)=O)C=C2)=O

TXZPMHLMPKIUGK-UHFFFAOYSA-N

InChI=1S/C16H17N3O4S/c1-19-10-11-9-12(7-8-13(11)17-16(19)20)18-24(21,22)15-6-4-3-5-14(15)23-2/h3-9,18H,10H2,1-2H3,(H,17,20)

2-methoxy-N-(3-methyl-2-oxo-1,2,3,4-tetrahydroquinazolin-6-yl)benzenesulfonamide

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 (7.20 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (7.20 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.

---

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