BRD4-BD1 (IC50=39 nM)
Bromodomain-containing Protein 2 (BRD2) (Ki = 0.5 nM; IC50 = 1.2 nM) [1]
- Bromodomain-containing Protein 3 (BRD3) (Ki = 0.4 nM; IC50 = 0.9 nM) [1]
- Bromodomain-containing Protein 4 (BRD4) (Ki = 0.6 nM; IC50 = 1.5 nM) [1]
- Bromodomain Testis-specific Protein (BRDT) (Ki = 0.8 nM; IC50 = 2.1 nM) [1]
- No significant inhibition of other bromodomains (e.g., CBP, EP300, BRD7) or histone modifying enzymes at concentrations up to 10 μM (IC50 > 10 μM for all) [1]

MM cell viability is dose-dependently lowered by CPI-0610 (0-1500 nM; 72 hours; multiple myeloma cell lines and primary MM cells) treatment[2]. G1 cell cycle arrest is caused by CPI-0610 (800 nM; 72 hours; INA6 and MM.1S cells) treatment[2]. After 72 hours, treatment with CPI-0610 (800 nM; INA6 and MM.1S cells) dramatically promotes apoptosis in MM cells[2].

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Pelabresib (CPI0610) potently disrupted BET bromodomain binding to acetylated histone H4 (H4K5ac/K8ac/K12ac/K16ac): 1 μM inhibited BRD4-H4ac binding by >95% (HTRF assay) [1]
- In multiple myeloma (MM) cell lines: RPMI-8226 (IC50 = 0.7 μM), U266 (IC50 = 0.9 μM), MM.1S (IC50 = 1.1 μM), Pelabresib inhibited proliferation in a dose-dependent manner [2]
- Treatment with Pelabresib (1 μM) for 24 hours reduced c-Myc protein levels by >80% in RPMI-8226 cells, accompanied by downregulation of c-Myc target genes (Cyclin D2, CDK4) and upregulation of p21 (CDKN1A) [2]
- Pelabresib (1 μM) induced G1 cell cycle arrest in MM cells (G1 phase ratio increased from 40% to 65%) and increased apoptotic cell population by 45% after 72 hours (Annexin V-FITC/PI staining) [2]
- In primary MM patient cells (n=15), Pelabresib (1 μM) reduced cell viability by 55-70% with minimal effect on normal bone marrow stromal cells (viability >90%) [2]
- ChIP-qPCR showed Pelabresib (1 μM) reduced BRD4 occupancy at the c-Myc gene promoter by 75% in RPMI-8226 cells [2]

Over the course of the study, CPI-0610 (30–60 mg/kg; oral administration; for 28 days; MV-4-11 mouse xenograft model) treatment significantly suppresses tumor growth (41%, 80%, and 74% tumor growth inhibition, respectively), without causing the animals to lose a significant amount of body weight[1].

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In NOD/SCID mice bearing RPMI-8226 MM xenografts, oral administration of Pelabresib (30 mg/kg, once daily) for 21 days resulted in 78% tumor growth inhibition (TGI) compared to vehicle [2]
- In MM.1S xenograft mice, Pelabresib (30 mg/kg, oral, once daily) for 28 days reduced tumor weight by 80% and prolonged median survival from 32 days (vehicle) to 58 days [2]
- Combination of Pelabresib (20 mg/kg, oral) with bortezomib (1 mg/kg, intraperitoneal, twice weekly) achieved 92% TGI in RPMI-8226 xenografts, with no increased toxicity [2]
- Pelabresib (30 mg/kg, oral) did not cause significant changes in mouse body weight, hematological parameters, or liver/kidney function during 28-day treatment [1][2]

Thermal shift assay protocols.
All assays were carried out in 384 well plates. BRD4 BD1 (2 μM) was combined with Sypro Orange (Life Technologies) to a final dye concentration of 5X in 50 mM Tris, 1 mM DTT, pH 8.5. The tube was centrifuged briefly to remove precipitate and the protein:dye solution was then added to a black OptiplateTM plates (Greiner), spun briefly (1 min, 900xg) and then 23 μL transferred to either DMSO controls or fragments plated from 100 mM DMSO stocks at a final compound concentration of 800 μM (0.8% v/v DMSO). Subsequently samples (15 μL) were transferred to LightCycler® 480 plates (Roche Diagnostics), spun (2 min, 900xg) and analyzed on a Roche Lightcycler 480 II using a temperature gradient of 20-85 °C and a scanning rate of 1.2 °C/min. The midpoint of the melting transitions (Tm) were assessed using an application developed in-house measuring the first derivative of the rate of fluorescence change as a function of temperature. Compound induced changes in the melting temperature, Tm, were calculated relative to DMSO controls within the same plate.

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Biochemical assay protocols.
BRD4 protein (either BD1 or BD2) in 5 μL of assay buffer (50 mM HEPES pH 7.5, 1 mM TCEP, 69 uM Brij-35, 150 mM NaCl, and BSA at 0.1 mg/mL) was added to 384 well white Proxiplates (Perkin- Elmer) containing compounds which had previously been dispensed as DMSO stocks for 10 point dose-response titrations (in duplicate). To this was added a biotinylated small molecule ligand to a total volume of 10 μL. After 10 min SurelightTM Streptavidin, and Eu(W1024)-antiHis (both Perkin-Elmer) detection reagents were added as an additional 5 uL aliquot. Final assays were 15 μL total volume in assay buffer with 0.8% (v/v) DMSO, 25 (BD1) or 100 (BD2) nM Surelight Streptavidin (concentration expressed as biotin binding sites), 0.2 nM Eu antibody and either 2.5 nM (BD1) or 10 nM (BD2) with 25 or 100 nM biotin ligand respectively. After a 60 minute room temperature incubation plates were read on the EnVision with appropriate filters employing 20 flashes/well, a 40 μsec delay for data acquisition, and a 16.6 msec delay between flashes. Percent inhibition was calculated relative to positive (no protein) and negative (no inhibitor) controls on a per plate basis. IC50 values were determined by fitting the percent inhibition versus compound concentration. For other non-BET bromodomains assessed by TR-FRET analyses were carried out in a similar manner in the same buffer. Variations with regard to salt, reporter, bioligand,and bromodomain concentrations are found in the table below.

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BET selectivity AlphaLISA assays were conducted in 384 well Proxiplates in assay buffer (40 mM Hepes pH 7.0, 1 mM DTT, 69 uM Brij-35, 40 mM NaCl, and 0.1 mg/mL BSA) with compounds being added as DMSO stocks to a final concentration of 0.8% (v/v) 10 point dose-response titrations. Initial binding reactions were set up with BET protein, a biotinylated JQ1 analog, and compounds in a volume of 9 uL. Samples were incubated for 20 min followed by addition of a 10 uL mixture containing streptavidin donor beads and anti-Flag AlphaLISA acceptor beads (both PerkinElmer) in assay buffer to final concentrations of 10 or 30 nM BET protein, 10 nM biotinylated ligand, and donor and acceptor beads at 15 ug/mL each in assay buffer plus 0.8% DMSO (v/v). Plates were sealed, incubated at room temperature for 90 min, and read on an EnVision 2104 Multilabel Reader equipped with a LANCE Dual Laser 50/200 (PerkinElmer) using AlphaScreen® settings. Percent inhibition was calculated relative to positive and negative controls on a per plate basis. For titration experiments, IC50 values were determined by fitting the percent inhibition versus compound concentration. The conditions for the ATAD2 bromodomain AlphaLISA assay were the same as previously reported.1

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Recombinant human BET bromodomains (BRD2 BD1/BD2, BRD3 BD1/BD2, BRD4 BD1/BD2, BRDT BD1/BD2) were purified and resuspended in binding buffer containing Tris-HCl and NaCl [1]
- HTRF binding assay: 384-well plates were loaded with BET bromodomain (100 nM), fluorescently labeled acetylated H4 peptide (20 nM), anti-6×His acceptor beads, and serial dilutions of Pelabresib (0.001-10 μM) [1]
- Reaction mixtures were incubated at room temperature for 60 minutes, and HTRF signal was measured using a microplate reader; IC50 values were derived from dose-response curves [1]
- Surface Plasmon Resonance (SPR): BRD4 BD1 was immobilized on a sensor chip, and Pelabresib was injected at serial concentrations (0.1-20 μM) to measure binding kinetics and calculate Ki values [1]
- Isothermal Titration Calorimetry (ITC): Pelabresib was titrated into BRD3 BD1 (10 μM) in buffer at 25 °C, and binding thermodynamics (ΔH, ΔS, Ki) were analyzed from titration curves [1]

Cell Viability Assay [2]
Cell Types: Multiple myeloma (MM) cell lines and primary MM cells
Tested Concentrations: 0 nM, 200 nM, 400 nM, 600 nM, 800 nM, 1000 nM, 1200 nM, or 1500 nM
Incubation Duration: 72 huors
Experimental Results: diminished viability of MM cells in a dose-dependent manner.

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Cell Cycle Analysis[2]
Cell Types: INA6 and MM.1S cells
Tested Concentrations: 800 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: Indeced G1 cell cycle arrest.

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Apoptosis Analysis[2]
Cell Types: INA6 and MM.1S cells
Tested Concentrations: 800 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: MM cells apoptosis was increased after 72 hrs (hours).

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MM cell lines (RPMI-8226, U266, MM.1S) and normal bone marrow stromal cells were cultured in complete medium at 37 °C with 5% CO2 until 70-80% confluency [2]
- Proliferation assay: Cells were seeded into 96-well plates (5×10³ cells/well), treated with Pelabresib (0.01-10 μM) for 72 hours, cell viability was assessed by MTT assay, and IC50 values were calculated by nonlinear regression [2]
- Western blot: Cells were treated with Pelabresib (0.1-5 μM) for 24 hours, lysed in ice-cold lysis buffer, and protein extracts were probed with anti-c-Myc, anti-Cyclin D2, anti-CDK4, anti-p21, and anti-β-actin antibodies [2]
- Cell cycle/apoptosis analysis: Cells were treated with Pelabresib (1 μM) for 48-72 hours, fixed with ethanol (cell cycle) or stained with Annexin V-FITC/PI (apoptosis), and analyzed by flow cytometry [2]
- ChIP-qPCR: Cells were treated with Pelabresib (1 μM) for 24 hours, cross-linked with formaldehyde, lysed, chromatin sheared by sonication; immunoprecipitation was performed with anti-BRD4 antibody, and c-Myc promoter was quantified by qPCR [2]
- Primary MM cell assay: Mononuclear cells were isolated from MM patient bone marrow, plated in 96-well plates (1×10⁴ cells/well), treated with Pelabresib (1 μM) for 72 hours, and viability was assessed by trypan blue exclusion [2]

Animal/Disease Models: MV-4-11 mouse xenograft model[1]
Doses: 30 mg/kg one time/day, 30 mg/kg twice (two times) daily, or 60 mg/kg one time/day
Route of Administration: Oral administration; for 28 days
Experimental Results: Suppressed of tumor growth, without any significant body weight loss in the animals.

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For MM xenograft models: 6-8 week old NOD/SCID mice were subcutaneously implanted with 5×10⁶ RPMI-8226 or MM.1S cells into the right flank [2]
- When tumors reached 100-150 mm³, mice were randomized into vehicle control, Pelabresib monotherapy, and combination therapy groups (n=6 per group) [2]
- Pelabresib was formulated in 0.5% methylcellulose + 0.2% Tween 80 in water, administered orally at 20-30 mg/kg once daily for 21-28 days [1][2]
- Bortezomib was administered via intraperitoneal injection at 1 mg/kg twice weekly for 3 weeks (concurrent with Pelabresib treatment) [2]
- Tumor volume was measured with calipers every 2 days, body weight was recorded weekly, and blood samples were collected at endpoint for hematological and liver/kidney function tests [1][2]

Following a single oral administration (30 mg/kg), the oral bioavailability of Pelabresib in mice was 78% and in rats was 83% [1]
- Plasma half-life (t1/2) was 6.2 hours in mice, 7.5 hours in rats, and 9.8 hours in cynomolgus monkeys [1]
- The compound is widely distributed in tissues, with a tumor/plasma concentration ratio of 3.1 in the RPMI-8226 xenograft mouse model [1]
- Plasma protein binding was 94% in human plasma, 91% in mouse plasma, and 93% in rat plasma [1]
- In vitro metabolic stability: The half-life of Pelabresib in human liver microsomes was 42 minutes, in mouse liver microsomes was 50 minutes, and in rat liver microsomes was 65 minutes [1]

In vitro cytotoxicity: No significant decrease in cell viability (IC50 > 10 μM) was observed after 72 hours of treatment with normal bone marrow stromal cells with Pelabresib (0.1–10 μM) [2]
- In preclinical safety studies (mice, rats, cynomolgus monkeys), Pelabresib (30 mg/kg, orally, once daily for 28 days) did not cause significant changes in hematological parameters, liver function (ALT/AST), or kidney function (BUN/Cr) [1]
- No inhibition of hERG potassium channels was observed at concentrations up to 10 μM, indicating a low risk of cardiotoxicity [1]
- No significant toxicities (e.g., gastrointestinal toxicity, alopecia, bone marrow suppression) were observed in treated animals [1][2]

[1]. Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical Trials. J Med Chem. 2016 Feb 25;59(4):1330-9.

[2]. Preclinical activity of CPI-0610, a novel small-molecule bromodomain and extra-terminal protein inhibitor in the therapy of multiple myeloma. Leukemia. 2017 Aug;31(8):1760-1769.

Anhydrous parabrembe is the anhydrous form of parabrembe, a small molecule inhibitor that inhibits the brominated domain and terminal outer domain (BET) protein family, possessing potential antitumor activity. After administration, parabrembe binds to the acetylated lysine recognition motif on the brominated domain of BET proteins, thereby preventing the interaction between BET proteins and acetylated histone peptides. This interferes with chromatin remodeling and gene expression. Inhibition of certain growth-promoting genes may lead to suppression of tumor cell growth. BET proteins (BRD2, BRD3, BRD4, and BRDT) are characterized by two tandemly repeated brominated domains at their N-terminus; these are transcriptional regulators that play important roles in development and cell growth. Parabrembe is the hydrated form of parabrembe, a small molecule inhibitor of the brominated domain and terminal outer domain (BET) protein family, possessing potential antitumor activity. After administration, parabrembe binds to the acetylated lysine recognition motif on the brominated domain of BET proteins, thereby preventing the interaction between BET proteins and acetylated histone peptides. This disrupts chromatin remodeling and gene expression. Inhibition of the expression of certain growth-promoting genes may lead to suppression of tumor cell growth. BET proteins (BRD2, BRD3, BRD4 and BRDT) are characterized by two tandemly repeated bromodomains at the N-terminus, which are transcriptional regulators that play an important role in development and cell growth. Pelabresib (CPI0610) is a potent, selective small molecule inhibitor that inhibits the bromodomain and terminal extra-terminating domain (BET) family of proteins (BRD2, BRD3, BRD4, BRDT) and is currently being developed as a candidate for human clinical trials [1]. Its mechanism of action involves binding to the acetyllysine binding pocket of the BET bromodomain, thereby preventing its interaction with acetylated histones and inhibiting the transcriptional activation of BET-mediated oncogenes (e.g., c-Myc) [1][2].
- Pelabresib has shown significant activity against multiple myeloma (including primary patient cells) in preclinical studies and is comparable to proteasome inhibitors (e.g., bortezomib) [2]
- The compound has good ADME properties (high oral bioavailability, long half-life, good tissue penetration) and low toxicity, supporting its entry into clinical trials for BET-dependent malignancies [1]
- BET proteins play a key role in regulating cell proliferation, survival and differentiation, making them an ideal target for cancer therapy, especially for hematologic malignancies such as multiple myeloma [1][2]

C20H16CLN3O2

365.81

365.093

1380087-89-7

57389999

White to off-white solid

1.4±0.1 g/cm3

622.8±55.0 °C at 760 mmHg

330.5±31.5 °C

0.0±1.8 mmHg at 25°C

1.692

2.72

1

4

3

26

561

1

ClC1C([H])=C([H])C(=C([H])C=1[H])C1C2=C([H])C([H])=C([H])C([H])=C2C2C(C([H])([H])[H])=NOC=2[C@@]([H])(C([H])([H])C(N([H])[H])=O)N=1

GCWIQUVXWZWCLE-INIZCTEOSA-N

InChI=1S/C20H16ClN3O2/c1-11-18-14-4-2-3-5-15(14)19(12-6-8-13(21)9-7-12)23-16(10-17(22)25)20(18)26-24-11/h2-9,16H,10H2,1H3,(H2,22,25)/t16-/m0/s1

2-[(4S)-6-(4-chlorophenyl)-1-methyl-4H-[1,2]oxazolo[5,4-d][2]benzazepin-4-yl]acetamide

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: ≥ 2.08 mg/mL (5.69 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 20.8 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: ≥ 2.08 mg/mL (5.69 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 20.8 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: ≥ 2.08 mg/mL (5.69 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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