In vitro, BAY 1238097 showed strong inhibitory activity (IC50 < 100 nM) in a TR-FRET assay using BET BRD4 bromodomain 1 and an acetylated peptide derived from histone H4. In the NanoBRET assay, the interaction between BRD4, BRD3 or BRD2 and H4 was inhibited with IC50 values of 63 nM, 609 nM and 2430 nM, showing selectivity of the compound for BRD4. A strong reduction of c-Myc transcript and protein levels was observed in treated MOLM-13 (AML) and MOLP-8 (MM) cell lines. ChIP experiments performed in these models additionally revealed that BAY 1238097 prevented binding of BRD4 to c-Myc regulatory regions.

In vitro, BAY 1238097 showed strong inhibitory activity (IC50 < 100 nM) in a TR-FRET assay using BET BRD4 bromodomain 1 and an acetylated peptide derived from histone H4. In the NanoBRET assay, the interaction between BRD4, BRD3 or BRD2 and H4 was inhibited with IC50 values of 63 nM, 609 nM and 2430 nM, showing selectivity of the compound for BRD4. A strong reduction of c-Myc transcript and protein levels was observed in treated MOLM-13 (AML) and MOLP-8 (MM) cell lines. ChIP experiments performed in these models additionally revealed that BAY 1238097 prevented binding of BRD4 to c-Myc regulatory regions.

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(R)-BAY1238097 exhibited anti-proliferative activity in a panel of 51 lymphoma-derived cell lines, with a median 50% inhibitory concentration (IC50) of 231 nmol/L (95% CI: 161–280 nmol/L). The activity was higher in B-cell lymphoma models than in T-cell lymphoma models. [2]
The anti-tumor activity of (R)-BAY1238097 was mainly cytostatic. Treatment (500 nmol/L for 72 hours) induced cell cycle arrest in the G1 phase and decreased the G2/M phase in several diffuse large B-cell lymphoma (DLBCL) cell lines. [2]
Time-dependent apoptosis was observed in eight cell lines treated with 500 nmol/L of (R)-BAY1238097 for 72 or 96 hours. [2]
Gene expression profiling (GEP) in a germinal center B-cell (GCB) DLBCL cell line (DOHH-2) exposed to 500 nmol/L (R)-BAY1238097 for 8, 12, or 24 hours showed downregulation of target genes of MYC, NOTCH, E2F, and members of the NF-κB/MYD88 and mTOR/AKT signaling pathways. Upregulated transcripts were mainly represented by histones. [2]
(R)-BAY1238097 synergized in vitro with the EZH2 inhibitors DZNep and GSK126, particularly in EZH2-mutated GCB-DLBCL cell lines. [2]
(R)-BAY1238097 synergized in vitro with the mTOR inhibitor everolimus in 7 out of 8 DLBCL cell lines tested. [2]
(R)-BAY1238097 synergized in vitro with the BTK inhibitor ibrutinib in 2 out of 2 ABC-DLBCL cell lines harboring the MYD88 L265P mutation, but no benefit was observed in MYD88 wild-type ABC-DLBCL cells. [2]
Treatment with (R)-BAY1238097 downregulated EZH2 protein levels and the histone modification H3K27me3 (a marker of EZH2 activity) in GCB-DLBCL cell lines. The combination with GSK126 led to a stronger downregulation of H3K27me3. [2]
Treatment with (R)-BAY1238097 (500 nmol/L) downregulated phospho-AKT (pAKT) levels in two GCB-DLBCL cell lines. [2]
Chromatin immunoprecipitation (ChIP) assay in a DLBCL cell line showed that treatment with 500 nmol/L (R)-BAY1238097 for 3 hours decreased BRD4 binding to the upstream regulatory region of the EZH2 gene. [2]
The presence of somatic mutations in the EZH2 gene was associated with higher sensitivity to (R)-BAY1238097 in GCB-DLBCL cell lines. The MYD88 L265P mutation was associated with higher sensitivity in ABC-DLBCL cell lines. [2]

In vivo, BAY 1238097 showed strong efficacy in the AML models THP-1, MOLM-13 and KG-1, with T/C between 13 and 20%. Overall, the compound was well tolerated at MTD, with body weight losses of 5-9% at nadir. BAY 1238097 was also active in MM models. Efficacy was observed against a human IGH-cyclin D1 translocated MOLP-8 model with a T/C of 3%, whereas the standard-of-care agents bortezomib and lenalidomide were inactive or poorly active. In this model, BAY 1238097 was well tolerated at 10 mg/kg applied over 14 days, with no body weight loss measured. BAY 1238097 was also active against the FGFR/MMSET translocated model NCIH929, with 19% T/C versus 49% T/C for the standard-of-care lenalidomide. The compound was well tolerated applied at 12 mg/kg for 9 days (maximum body weight loss 6%).
Gene expression profiling performed in liver, blood and duodenum of rats treated daily with 2 mg/kg BAY 1238097 for 14 days demonstrated substantial effects on genes involved in cell proliferation and in the immune response in vivo.

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In a SU-DHL-8 (GCB-DLBCL) xenograft model established in SCID mice, oral administration of (R)-BAY1238097 at its maximum tolerated dose (MTD) of 15 mg/kg once daily for 12 days significantly inhibited tumor growth compared to the vehicle control. The treated/control (T/C) tumor volume ratio was 15% on day 14 post-inoculation. [2]
In an OCI-LY-3 (ABC-DLBCL) xenograft model established in SCID mice, oral administration of (R)-BAY1238097 at its MTD of 45 mg/kg twice weekly for 4 weeks significantly inhibited tumor growth. The T/C ratio was 23% on day 48 post-inoculation. [2]

The anti-proliferative activity of (R)-BAY1238097 was assessed using a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Lymphoma cell lines were exposed to various concentrations of the compound for 72 hours. The absorbance was measured, and the IC50 and LC50 values were calculated from the dose-response curves. [2]
Apoptosis was evaluated by staining cells with Annexin V and 7-aminoactinomycin D (7-AAD) after treatment with (R)-BAY1238097. Cells were analyzed by flow cytometry to quantify the percentage of apoptotic cells. [2]
Cell cycle distribution was analyzed by flow cytometry after treatment with (R)-BAY1238097. Cells were fixed, permeabilized, stained with a DNA-binding dye (e.g., propidium iodide), and the DNA content was measured to determine the percentage of cells in different phases of the cell cycle (G1, S, G2/M). [2]
For western blotting, cells were lysed, and proteins were extracted. Proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific primary antibodies (e.g., against BRD4, MYC, EZH2, H3K27me3, histone H3, phospho-AKT, AKT, phospho-mTOR, GAPDH). After incubation with appropriate secondary antibodies conjugated to horseradish peroxidase, signals were detected using chemiluminescence. [2]
Chromatin immunoprecipitation (ChIP) was performed to assess BRD4 binding to the EZH2 promoter region. Cells were treated with (R)-BAY1238097 or DMSO, cross-linked with formaldehyde, and lysed. Chromatin was sonicated to shear DNA. BRD4-bound DNA fragments were immunoprecipitated using a specific anti-BRD4 antibody. After reversing cross-links, the purified DNA was quantified by quantitative real-time PCR using primers specific for the EZH2 upstream regulatory region. [2]
Gene expression profiling was performed using microarray technology (HumanHT-12 v4 Expression BeadChip). Total RNA was extracted from cells treated with (R)-BAY1238097 or DMSO control, labeled, and hybridized to the microarray chips. Fluorescence signals were scanned, and data were analyzed to identify differentially expressed genes. Gene set enrichment analysis (GSEA) was performed to identify enriched biological pathways. [2]
Drug combination studies were performed by exposing cells to serial dilutions of (R)-BAY1238097 and a second drug (e.g., GSK126, everolimus, ibrutinib) alone and in combination. Cell viability was measured after 72 hours. Synergism, additivity, or antagonism was determined by calculating the Combination Index (CI) using the Chou-Talalay method. A CI < 0.9 indicated synergism, CI between 0.9 and 1.1 indicated additivity, and CI > 1.1 indicated no benefit/antagonism. [2]

For in vivo efficacy studies, severe combined immunodeficiency (SCID) female mice (9–12 weeks old) were used.
In the SU-DHL-8 (GCB-DLBCL) model, mice were inoculated subcutaneously with 5 × 10^6 SU-DHL-8 cells suspended in Matrigel. Treatment began on day 4 post-inoculation. (R)-BAY1238097 was formulated as a solution in 0.9% NaCl in water, adjusted to pH 4. It was administered orally (p.o.) at a dose volume of 10 ml/kg. The treatment group received (R)-BAY1238097 at its maximum tolerated dose (MTD) of 15 mg/kg once daily for 12 consecutive days. The control group received the vehicle solution. Tumor volumes were measured twice a week. [2]
In the OCI-LY-3 (ABC-DLBCL) model, mice were inoculated subcutaneously with OCI-LY-3 tumor fragments. Treatment started on day 21 post-inoculation when tumors reached a median size. (R)-BAY1238097 was formulated as above and administered orally (p.o.) at its MTD of 45 mg/kg using an intermittent schedule (twice a week) for 4 weeks. The control group received the vehicle solution. Tumor volumes were measured twice a week. [2]
Body weight was monitored daily as an indicator of toxicity. [2]

In SCID mice carrying lymphoma xenografts, the maximum tolerated dose (MTD) of (R)-BAY1238097 was determined to be 15 mg/kg daily, or 45 mg/kg twice weekly. [2] In the SU-DHL-8 model, a dose of 15 mg/kg daily resulted in a maximum mean weight loss of 6%. In the OCI-LY-3 model, a dose of 45 mg/kg twice weekly resulted in a maximum mean weight loss of 9%. A weight loss of ≥20% was predefined as excessive toxicity, but this was not observed at the MTD doses used. [2]

[1]. (2015) Abstract 3524: BAY 1238097, a novel BET inhibitor with strong efficacy in hematological tumor models. Cancer Research, 75(15 Suppl), 884.

[2]. Preclinical evaluation of the BET bromodomain inhibitor BAY 1238097 for the treatment of lymphoma. Br J Haematol. 2017 Sep;178(6):936-948.

(R)-BAY1238097 is a novel BET bromodomain inhibitor with good preclinical anti-lymphoma activity. [2]
Its mechanism of action is to inhibit the binding of BET proteins (such as BRD4) to acetylated histones, thereby interfering with the transcription of key oncogenes and signaling pathways (such as MYC, NF-κB, and mTOR). [2]
Sensitivity to (R)-BAY1238097 is associated with specific genetic traits, such as the EZH2 mutation in GCB-DLBCL and the MYD88 L265P mutation in ABC-DLBCL, suggesting that these mutations may serve as potential predictive biomarkers. [2]
Based on the synergistic effects and common signaling pathway interference observed in vitro, this study proposes a rational combination therapy strategy for (R)-BAY1238097 with EZH2, mTOR, and BTK inhibitors. [2]

C25H33N5O3

451.56

451.258

1564269-85-7

BAY1238097;1564268-08-1

90041345

Light yellow to yellow solid powder

2.4

1

6

4

33

688

1

C1CN(C)CCN1C1=CC=C(C=C1)C1=NN(C(=O)NC)[C@@H](CC2C1=CC(=C(C=2)OC)OC)C

CJIPEACKIJJYED-KRWDZBQOSA-N

InChI=1R/C25H33N5O3/c1-17-14-19-15-22(32-4)23(33-5)16-21(19)24(27-30(17)25(31)26-2)18-6-8-20(9-7-18)29-12-10-28(3)11-13-29/h6-9,15-17H,10-14H2,1-5H3,(H,26,31)/t17-/m0/s1

(R)-7,8-dimethoxy-N,4-dimethyl-1-(4-(4-methylpiperazin-1-yl)phenyl)-4,5-dihydro-3H-benzo[d][1,2]diazepine-3-carboxamide

R-isomer of BAY-1238097; BAY1238097; BAY-1238097; BAY 1238097; BAY 12-38097; BAY 123; BAY-123; BAY12-38097; BAY-12-38097

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~150 mg/mL (~332.18 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.54 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.54 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.

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