Bromodomain (BRD)

Birabresib (OTX-015) (500 nM) exposure generates a substantial decrease of BRD2, BRD4 and c-MYC and rise of HEXIM1 proteins, whereas BRD3 expression is unaltered. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels do correlate however with viability following exposure to Birabresib (OTX-015)[2]. Birabresib (OTX-015) (0.1, 1, 5 μM) administration promotes HIV-1 full-length transcripts and viral outgrowth in resting CD4+ T cells from infected patients receiving suppressive antiretroviral therapy (ART), while exerting low toxicity and effects on T cell activation. Birabresib-mediated activation of HIV-1 involves an increase in CDK9 occupancy and RNAP II C-terminal domain (CTD) phosphorylation[3].

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Birabresib (OTX-015) was a potent inhibitor of BRD2, BRD3, and BRD4, with EC50 values from 10 to 19 nM. Binding of OTX015 to BRD2, BRD3, and BRD4 was inhibited by addition of OTX015 in a concentration-dependent manner, suggesting competitive inhibition. OTX015 inhibited the binding of BRD2, BRD3, and BRD4 to AcH4, with IC50 values from 92 to 112 nM. OTX015 inhibited the growth of a variety of human cancer cell lines; for most hematologic malignancies tested, GI50 values ranged from 60 to 200 nM.[1]

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Exposure to Birabresib (OTX-015) lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.[2]

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Birabresib (OTX-015) was assayed in three human TNBC-derived cell lines, HCC1937, MDA-MB-231 and MDA-MB-468, all showing antiproliferative activity after 72 h (GI50 = 75-650 nM). This was accompanied by cell cycle arrest and decreased expression of cancer stem cells markers. However, c-MYC protein and mRNA levels were only down-regulated in MDA-MB-468 cells. Gene set enrichment analysis showed up-regulation of genes involved in epigenetic control of transcription, chromatin and the cell cycle, and down-regulation of stemness-related genes. In vitro, combination with everolimus was additive in HCC1937 and MDA-MB-231 cells, but antagonistic in MDA-MB-468 cells [4].

Birabresib (OTX-015) (50 mg/kg) significantly (p?<0.05) reduces tumor burden in MDA-MB-231 murine xenografts when compared to mice given with a vehicle. More effective activity is seen by combining Birabresib (OTX-015) with 2 mg/kg RAD001 than by using Birabresib alone[4].

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In vivo experiments in MDA-MB-231 xenografts [4]
Based on in vitro combination studies, the MDA-MB-231 cell line was selected to generate murine xenografts to evaluate the in vivo effect of Birabresib (OTX-015) alone and in combination with everolimus. OTX015-treated mice showed a substantial reduction in tumor mass with respect to the control group (p < 0.05) from 7 days after treatment start (Figure 5B). The best T/C value was 41.3% recorded on day 23 (Table 2). On the other hand, everolimus alone did not substantially affect tumor growth with respect to the control group. In this experimental setting, the Birabresib (OTX-015)/everolimus combination was the most effective approach, showing a significant reduction in tumor mass with respect to vehicle-treated animals (p < 0.05) from day 4 after treatment start (Figure 5B) and a best T/C 20.7% on day 23 (Table 2). Furthermore, throughout the treatment period, mean tumor weights of the OTX015/everolimus combination group were significantly lower (p < 0.05) with respect to the everolimus and OTX015 single agents groups from day 7 and 21 onwards, respectively. More importantly, after treatment end, tumors of mice treated with the combination remained significantly smaller (p < 0.05) than those in animals treated with either of the single agents (Figure 5B). Of note, signs of toxicity were apparent in all mice in the everolimus-treated groups, as indicated by decreased mean body weight (Figure 5C). The everolimus schedule was therefore modified to once-a-week from day 14 after treatment start for both single agent and combination schedules.

Paclitaxel, which is widely used in the clinic to treat TNBC, was evaluated in a separate experiment, providing a clinical benchmark (Supplementary Figure S2). This drug showed activity with an optimal T/C of 28.9% on day 23 (Table 2). Comparison of the pharmacological efficacy of Birabresib (OTX-015), everolimus, the OTX015/everolimus combination and paclitaxel according to three parameters (T/C%, AGD and LCK), demonstrated clearly that the OTX015/everolimus combination is the most active treatment (Table 2).

At the end of the treatment period, half of the animals from the Birabresib (OTX-015) group were sacrificed 4 h after their last dose to determine drug levels in blood and tissues. Tumor and plasma presented equivalent OTX015 concentrations, which were significantly higher (p < 0.05) than the levels observed in peritumoral tissue (Figure 5D). These drug concentrations were ≈2 μM (in both plasma and tumor tissue), indicating that concentrations used in vitro are achievable in the tumor environment, which is coherent with a recent report from Gaudio et al. in lymphoma models. For the remaining animals, as mentioned above, tumor and body weight were monitored for a further 20 days.

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Expression of c-MYC, BETs and CSC markers in MDA-MB-231 xenografts [4]
Tumor tissue from xenograft mice sacrificed 4 weeks after treatment start was evaluated for changes in key genes (Figure 5E). None of the experimental groups showed variations in the expression of c-MYC, BRD2/3 mRNA with respect to the vehicle-treated animals. Nevertheless, mice treated with the Birabresib (OTX-015)/everolimus combination showed a significant reduction (p < 0.05) in mRNA levels of BRD4 in comparison with the vehicle control group. Likewise, as in the in vitro case, OTX015 induced a substantial increase in CD24 mRNA levels with concomitant reduction in CD44 expression (p < 0.05). However, the OTX015/everolimus combination resulted in only a marked increase (p < 0.05) in CD24, without affecting CD44 expression. On the other hand, everolimus induced a significant decrease (p < 0.05) in CD24 expression with no effect on CD44. Both OTX015 and the combination treatment down-regulated mRNA levels of the EpCAM, NANOG and OCT4 stemness markers (p < 0.05). In contrast, everolimus did not modify the EpCAM and OCT4 expression but significantly up-regulated NANOG mRNA levels (p < 0.05).

To assess binding of OTX015 to BRD2, BRD3, and BRD4, BRD-expressing CHO cell lysate (from CHO cells transfected with expression plasmids for Flag-tagged BRD2, BRD3, or BRD4 or vector alone), europium-conjugated anti-Flag antibody, XL-665-conjugated streptavidin, and biotinylated OTX015 were incubated at room temperature for 0.2 to 2h. Fluorescence was measured by TR-FRET using an EnVision 2103 Multilabel Reader and EC50 for binding was calculated by nonlinear regression using PRISM version 5.02. Using a similar system, the effect of OTX015 on binding of BRD2, BRD3, and BRD4 to acetylated histone H4 (AcH4) was evaluated by incubating biotin-conjugated -AcH4, BRD-expressing CHO cell lysate, europium-conjugated anti-Flag antibody, and XL-665-conjugated streptavidin. Fluorescence was measured by TR-FRET using an EnVision 2103 Multilabel Reader and percent binding was calculated by defining the value of the sample without biotin conjugate dAcH4 as 0% and the sample without OTX015 as 100%. The IC50 value was calculated by nonlinear regression using PRISM version 5.02. Effects of OTX015 on cancer cell proliferation were evaluated by incubating human tumor cells for 72 h with increasing concentrations of OTX015 and assessing proliferation using a tetrazolium salt (WST-8)-based colorimetric assay [1].

MTT assay, apoptosis assessment and cell cycle analysis [2]
For the MTT assay, cells were seeded in 24-well plates at 1×106 per well and treated with a range of Birabresib (OTX-015) concentrations 0.01nM-10μM for 72h. Cells were transferred to 96-well plates and incubated with 0.5mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in the dark at 37°C for 4h. Cells were then lysed with 25% sodium dodecyl sulfate (SDS) lysis buffer and absorbance was read at 570nm using a Promega Microplate Reader. Three independent experiments were run for each cell line and untreated cells were used as negative controls. The half maximal inhibitory concentration (IC50) values were calculated with Prism® v6 software.
For cell cycle analysis, 1×106 cells were treated with a range of Birabresib (OTX-015) concentrations 25-500nM for 48h then harvested, washed in PBS, and fixed in 70% ice cold ethanol. Cells were incubated with 100 μg/mL RNAse and stained with 25 μg/mL propidium iodide (PI; Becton Dickinson) for 30 minutes at 37°C.
For apoptosis analysis, 1×106 cells derived from patients or cell lines were resuspended in 1 ml culture medium and treated with Birabresib (OTX-015) for 72h. Apoptotic cells were detected using a FACSCalibur flow cytometer. Cells were stained with 5μg/mL PI and Annexin-V-FITC according to the manufacturer's instructions for 15 minutes at room temperature. Apoptotic cells were defined as Annexin V+ with or without PI uptake.

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MOLT-4/CCR5 outgrowth assay [3]
Purified resting CD4+ T cells (5 × 106) were treated with PMA plus ionomycin or Birabresib (OTX-015) for 18 h and washed with 1 ml sterile PBS to remove residual drug. Resting CD4+ T cells were then cultured with MOLT-4/CCR5 cells in 8 ml RPMI1640 medium plus 10% FBS in individual wells in six-well culture plates. After four days and seven days of culture, wells were resuspended and split 1:2 with the medium volume adjusted to 8 ml per well. After 14 days of culture, viral outgrowth was assessed using the HIV-1 p24 Antigen ELISA kit.

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Measurement of cell viability and detection of T cell activation markers and HIV-1 receptors/co-receptors [3]
PBMCs from healthy individuals were placed in 96-well plates and incubated with Birabresib (OTX-015) for 48 h. Cell viability was measured using the Cell Counting Kit-8 as described72. To measure changes in the cell activation status and presence of HIV-1 receptors/co-receptors, CD4+ lymphocytes isolated from healthy donors were incubated with prostratin, OTX015 or OTX015/prostratin for 48 h and immunostained with anti-CD25, anti-CD69, anti-HLA-DR, anti-CD4, anti-CCR5 or anti-CXCR4 antibodies for 20 min at 4 °C. Cells were fixed in 1% PFA and analyzed by flow cytometry.

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Drug combination studies [4]
Cells were seeded at a density of 20000 cells/ml in 96-well plates (100 μl/wells) and treated 24 h later with different concentrations of Birabresib (OTX-015) or everolimus, or combination of both drugs for 72 h. Cells were then incubated with 0.8 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) for 2–4 hours. Cell pellets were resuspended in 0.05 ml DMSO and absorbance was measured at 560 nm using an Infinte 200 microplate reade. OTX015 and everolimus EC50 values were determined as previously mentioned using Prism 5.00 software. Assays were performed in triplicate in at least three independent experiments. Results were further analyzed according to the Chou-Talalay algorithm with Compusyn software. Based on the calculated combination indexes values, < 0.90 reflects synergism, 0.9 to ≤ 1.10 indicates additive effects, and > 1.10 reflects antagonism.

To assess antiproliferative effects in vivo, BLAB/c-nu/nu mice bearing established Ty82 BRD-NUT midline carcinoma xenografts were given Birabresib (OTX-015) (0, 10, 30 or 100 mg/kg qd or 10 mg/kg bid) by oral gavage over 14 days. Animals were sacrificed on day 15 and tumors were extracted and weighed.[1]

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Mice were subcutaneously injected in the right flank with 10 × 106 MDA-MB-231 cells. When average tumor weight was ≈130 mg, mice were randomized (nine animals/group) to one of the following experimental groups: vehicle (for OTX15, water, twice daily, oral; for everolimus vehicle, 5% Tween-80/5% polyethylene glycol 400, thrice weekly, intraperitoneal); 50 mg/kg Birabresib (OTX-015), twice daily, oral; 2 mg/kg everolimus, thrice weekly, intraperitoneal; 50 mg/kg OTX015 + 2 mg/kg everolimus, according to the single agent dosing schedules. In the experiment with paclitaxel, mice were randomized (eight animals/group) to vehicle (cremophor:ethanol 1:1, then diluted 1:5 with saline; once weekly, intravenous) or 0.15 mg/kg paclitaxel, once weekly, intravenous. Mice were sacrified at the first sign of severe distress and tumors were collected. Tumor weight (1 mm3 = 1 mg) was determined using the formula d2 × D/2, where ‘d’ and ‘D’ are the minor and major diameters of the tumor in mm, respectively. [4]

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Birabresib (OTX-015) quantification in plasma and solid tissues: Mice were euthanized with CO2. Blood was collected by cardiac puncture and immediately heparinized. Plasma was separated by centrifugation at 2000 × g for 15 min at 4°C and stored at −80°C. Tumors and peritumoral tissues were rapidly frozen in dry ice and stored at −80°C. OTX015 concentrations were determined using a validated Acquity Ultra Performance Liquid Chromatography System coupled with a tandem mass spectrometry detection method (UPLC/MS/MS), as previously described.[4]

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0, 10, 30 or 100 mg/kg qd or 10 mg/kg bid; oral gavage

BLAB/c-nu/nu mice bearing established Ty82 BRD-NUT midline carcinoma xenografts.

[1]. Abstract C244: Development of the BET bromodomain inhibitor OTX015. Mol Cancer Ther November 2013 12; C244.

[2]. BET inhibitor OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cells. Oncotarget. 2015 Jul 10; 6(19): 17698–17712.

[3]. The BET inhibitor OTX015 reactivates latent HIV-1 through P-TEFb. Sci Rep. 2016 Apr 12;6:24100.

[4]. The bromodomain inhibitor OTX015 (MK-8628) exerts anti-tumor activity in triple-negative breast cancer models as single agent and in combination with RAD001. Oncotarget. 2017 Jan 31;8(5):7598-7613.

Birabresib (OTX-015) is a potent bromodomain (BRD2/3/4) inhibitor.
Birabresib is under investigation in clinical trial NCT02698176 (A Dose Exploration Study With MK-8628 in Participants With Selected Advanced Solid Tumors (MK-8628-006)).
Birabresib is a synthetic, small molecule inhibitor of the BET (Bromodomain and Extra-Terminal) family of bromodomain-containing proteins 2, 3 and 4 with potential antineoplastic activity. Upon administration, birabresib binds to the acetylated lysine recognition motifs on the bromodomain of BET proteins, thereby preventing the interaction between the BET proteins and acetylated histone peptides. This disrupts chromatin remodeling and gene expression. Prevention of the expression of certain growth-promoting genes, including c-Myc-dependent target genes, may lead to an inhibition of tumor cell growth. Characterized by a tandem repeat of bromodomain at the N-terminus, the BET proteins BRD2, BRD3, BRD4 are transcriptional regulators that play an important role in cellular growth.

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Introduction: The BET bromodomain proteins, including BRD2, BRD3, and BRD4, have emerged as major epigenetic regulators of proliferation and differentiation and also have been associated with predisposition to dyslipidemia or improper regulation of adipogenesis, elevated inflammatory profile, and increased susceptibility to autoimmune disease. OTX015, a novel thienotriazolodiazepine compound, was identified in a cell-based screen for inhibitors of cell adhesion. Subsequently, it was evaluated for inhibition of the binding between acetylated histone and BRD2, BRD3, and BRD4 and antiproliferative effects in both in vitro and in vivo tumor models. Material and Methods: To assess binding of OTX015 to BRD2, BRD3, and BRD4, BRD-expressing CHO cell lysate (from CHO cells transfected with expression plasmids for Flag-tagged BRD2, BRD3, or BRD4 or vector alone), europium-conjugated anti-Flag antibody, XL-665-conjugated streptavidin, and biotinylated OTX015 were incubated at room temperature for 0.2 to 2h. Fluorescence was measured by TR-FRET using an EnVision 2103 Multilabel Reader and EC50 for binding was calculated by nonlinear regression using PRISM version 5.02. Using a similar system, the effect of OTX015 on binding of BRD2, BRD3, and BRD4 to acetylated histone H4 (AcH4) was evaluated by incubating biotin-conjugated -AcH4, BRD-expressing CHO cell lysate, europium-conjugated anti-Flag antibody, and XL-665-conjugated streptavidin. Fluorescence was measured by TR-FRET using an EnVision 2103 Multilabel Reader and percent binding was calculated by defining the value of the sample without biotin conjugate dAcH4 as 0% and the sample without OTX015 as 100%. The IC50 value was calculated by nonlinear regression using PRISM version 5.02. Effects of OTX015 on cancer cell proliferation were evaluated by incubating human tumor cells for 72 h with increasing concentrations of OTX015 and assessing proliferation using a tetrazolium salt (WST-8)-based colorimetric assay. To assess antiproliferative effects in vivo, BLAB/c-nu/nu mice bearing established Ty82 BRD-NUT midline carcinoma xenografts were given OTX015 (0, 10, 30 or 100 mg/kg qd or 10 mg/kg bid) by oral gavage over 14 days. Animals were sacrificed on day 15 and tumors were extracted and weighed. Results: OTX015 was a potent inhibitor of BRD2, BRD3, and BRD4, with EC50 values from 10 to 19 nM. Binding of OTX015 to BRD2, BRD3, and BRD4 was inhibited by addition of OTX015 in a concentration-dependent manner, suggesting competitive inhibition. OTX015 inhibited the binding of BRD2, BRD3, and BRD4 to AcH4, with IC50 values from 92 to 112 nM. OTX015 inhibited the growth of a variety of human cancer cell lines; for most hematologic malignancies tested, GI50 values ranged from 60 to 200 nM. Oral administration of OTX015 significantly inhibited the growth of Ty82 BRD-NUT midline carcinoma tumors in nude mice, showing 79% TGI at 100 mg/kg qd and 61% TGI at 10 mg/kg bid. Conclusions: OTX015 is a potent inhibitor of BRD2, BRD3, and BRD4 and inhibits the binding of BRD2, BRD3, and BRD4 to AcH4. OTX015 showed significant anti-tumor activity both in vitro and in vivo tumor models. These findings encouraged the clinical development of OTX015, which is currently in Phase 1 studies in patients with advanced hematologic malignancies (ClinicalTrials.gov Identifier NCT01713582).[1]

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The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.[2]

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None of the currently used anti-HIV-1 agents can effectively eliminate latent HIV-1 reservoirs, which is a major hurdle to a complete cure for AIDS. We report here that a novel oral BET inhibitor OTX015, a thienotriazolodiazepine compound that has entered phase Ib clinical development for advanced hematologic malignancies, can effectively reactivate HIV-1 in different latency models with an EC50 value 1.95–4.34 times lower than JQ1, a known BET inhibitor that can reactivate HIV-1 latency. We also found that OTX015 was more potent when used in combination with prostratin. More importantly, OTX015 treatment induced HIV-1 full-length transcripts and viral outgrowth in resting CD4+ T cells from infected individuals receiving suppressive antiretroviral therapy (ART), while exerting minimal toxicity and effects on T cell activation. Finally, biochemical analysis showed that OTX015-mediated activation of HIV-1 involved an increase in CDK9 occupancy and RNAP II C-terminal domain (CTD) phosphorylation. Our results suggest that the BET inhibitor OTX015 may be a candidate for anti-HIV-1-latency therapies.[3]

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Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subgroup of breast tumors clinically defined by the lack of estrogen, progesterone and HER2 receptors, limiting the use of the targeted therapies employed in other breast malignancies. Recent evidence indicates that c-MYC is a key driver of TNBC. The BET-bromodomain inhibitor OTX015 (MK-8628) has potent antiproliferative activity accompanied by c-MYC down-regulation in several tumor types, and has demonstrated synergism with the mTOR inhibitor everolimus in different models. The aim of this study was to evaluate the anti-tumor activity of OTX015 as single agent and in combination with everolimus in TNBC models. OTX015 was assayed in three human TNBC-derived cell lines, HCC1937, MDA-MB-231 and MDA-MB-468, all showing antiproliferative activity after 72 h (GI50 = 75-650 nM). This was accompanied by cell cycle arrest and decreased expression of cancer stem cells markers. However, c-MYC protein and mRNA levels were only down-regulated in MDA-MB-468 cells. Gene set enrichment analysis showed up-regulation of genes involved in epigenetic control of transcription, chromatin and the cell cycle, and down-regulation of stemness-related genes. In vitro, combination with everolimus was additive in HCC1937 and MDA-MB-231 cells, but antagonistic in MDA-MB-468 cells. In MDA-MB-231 murine xenografts, tumor mass was significantly (p < 0.05) reduced by OTX015 with respect to vehicle-treated animals (best T/C = 40.7%). Although everolimus alone was not active, the combination was more effective than OTX015 alone (best T/C = 20.7%). This work supports current clinical trials with OTX015 in TNBC (NCT02259114).[4]

C25H22CLN5O2S

491.992482662201

491.118

C, 61.03; H, 4.51; Cl, 7.21; N, 14.23; O, 6.50; S, 6.52

1983196-25-3

Birabresib;202590-98-5; 204587-26-8 (dihydrate)

118704772

White to off-white solid powder

4.5

2

6

4

34

770

1

CC1=C(SC2=C1C(=N[C@@H](C3=NN=C(N32)C)CC(=O)NC4=CC=C(C=C4)O)C5=CC=C(C=C5)Cl)C

GNMUEVRJHCWKTO-HXUWFJFHSA-N

InChI=1S/C25H22ClN5O2S/c1-13-14(2)34-25-22(13)23(16-4-6-17(26)7-5-16)28-20(24-30-29-15(3)31(24)25)12-21(33)27-18-8-10-19(32)11-9-18/h4-11,20,32H,12H2,1-3H3,(H,27,33)/t20-/m1/s1

2-[(9R)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]-N-(4-hydroxyphenyl)acetamide

(R)-OTX-015; (R)-MK-8628; (R)-Birabresib; 1983196-25-3; 2-((6R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(4-hydroxyphenyl)acetamide; (R)-2-(4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(4-hydroxyphenyl)acetamide; 2-[(9R)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]-N-(4-hydroxyphenyl)acetamide; starbld0038411; SCHEMBL23728706;

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: 200 mg/mL (406.51 mM）

Solubility in Formulation 1: ≥ 5 mg/mL (10.16 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 50.0 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: ≥ 5 mg/mL (10.16 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 50.0 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: ≥ 5 mg/mL (10.16 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 50.0 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.)