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].

---

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]

---

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]

---

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].

---

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.

---

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.

---

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.

---

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.

---

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]

---

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]

---

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]

---

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 being investigated in the clinical trial NCT02698176 (a dose-exploration study (MK-8628-006) in specific patients with advanced solid tumors). Birabresib is a synthetic small molecule inhibitor that inhibits bromodomain proteins 2, 3, and 4 of the BET (bromodomain and terminal domain) family, exhibiting potential antitumor activity. After administration, birabresib binds to the acetylated lysine recognition motif on the bromodomain 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, including c-Myc-dependent target genes, may suppress tumor cell growth. BET proteins BRD2, BRD3, and BRD4 are characterized by tandemly repeated bromodomains at their N-terminus; they are transcriptional regulators that play a crucial role in cell growth.

---

Introduction: BET bromodomain proteins (including BRD2, BRD3, and BRD4) have become major epigenetic regulators of proliferation and differentiation, and are associated with dyslipidemia or abnormal regulation of adipogenesis, elevated inflammation levels, and increased susceptibility to autoimmune diseases. OTX015, a novel thienotriazole diazapine compound, was discovered in a cell-based screening of cell adhesion inhibitors. We then evaluated the inhibitory effects of OTX015 on the binding of acetylated histones to BRD2, BRD3, and BRD4, and its antiproliferative effects in in vitro and in vivo tumor models. Materials and Methods: To evaluate the binding of OTX015 to BRD2, BRD3, and BRD4, we incubated BRD-expressing CHO cell lysates (from CHO cells transfected with Flag-tagged BRD2, BRD3, or BRD4 expression plasmids or empty vectors), europium-labeled anti-Flag antibody, XL-665-labeled streptavidin, and biotin-labeled OTX015 at room temperature for 0.2 to 2 hours. Fluorescence intensity was measured using TR-FRET on an EnVision 2103 multi-plate reader, and the EC50 value of binding was calculated using PRISM version 5.02 via nonlinear regression. Using a similar system, the effect of OTX015 on the binding of BRD2, BRD3, and BRD4 to AcH4 was evaluated by incubating biotin-labeled acetylated histone H4 (AcH4), BRD-expressing CHO cell lysates, europium-labeled anti-Flag antibody, and XL-665-labeled streptavidin. Fluorescence intensity was measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) method on an EnVision 2103 multi-plate reader. Samples without biotin-labeled AcH4 were defined as 0%, and samples without OTX015 were defined as 100%. The binding percentage was calculated. IC50 values were calculated using PRISM version 5.02 via nonlinear regression. The effect of OTX015 on cancer cell proliferation was evaluated by incubating human tumor cells with increasing concentrations of OTX015 for 72 hours and assessing cell proliferation using a tetrazolium salt-based (WST-8) colorimetric method. To assess in vivo antiproliferative effects, BLAB/c-nu/nu mice carrying established Ty82 BRD-NUT midline carcinoma xenografts were administered OTX015 (0, 10, 30, or 100 mg/kg once daily; or 10 mg/kg twice daily) by gavage for 14 days. Animals were sacrificed on day 15, and the tumors were removed and weighed. Results: OTX015 is a potent inhibitor of BRD2, BRD3, and BRD4, with EC50 values ranging from 10 to 19 nM. The binding of OTX015 to BRD2, BRD3, and BRD4 was inhibited in a concentration-dependent manner, suggesting competitive inhibition. OTX015 inhibited the binding of BRD2, BRD3, and BRD4 to AcH4, with IC50 values ranging from 92 to 112 nM. OTX015 inhibited the growth of various human cancer cell lines; for most of the tested hematologic malignancies, the GI50 values ranged from 60 to 200 nM. Oral administration of OTX015 significantly inhibited the growth of Ty82 BRD-NUT midline carcinoma in nude mice, with a tumor growth inhibition rate (TGI) of 79% in the once-daily 100 mg/kg dose group and 61% in the twice-daily 10 mg/kg dose group. Conclusion: OTX015 is a potent inhibitor of BRD2, BRD3 and BRD4, inhibiting the binding of BRD2, BRD3 and BRD4 to acetyltransferase 4 (AcH4). OTX015 showed significant antitumor activity in both in vitro and in vivo tumor models. These findings facilitated the clinical development of OTX015, which is currently undergoing a phase I clinical trial in patients with advanced hematologic malignancies (ClinicalTrials.gov registration number: NCT01713582). [1] Bromodomain (BRD) and terminal domain (BET) proteins, including BRD2, BRD3 and BRD4, have been identified as key targets for leukemia maintenance. A novel oral BRD2/3/4 inhibitor, thienotriazole diazazepine OTX015, is available for human use. This article reports its biological effects in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines and leukemia samples. At submicromolar concentrations, OTX015 inhibited the growth of acute leukemia cell lines and patient-derived leukemia cells, leading to cell cycle arrest and apoptosis, similar to the effects of the classic BET inhibitor JQ1. JQ1 and OTX015 treatments induced similar gene expression profiles in sensitive cell lines, including decreased c-MYC expression and increased HEXIM1 expression. OTX015 treatment also resulted in significantly decreased BRD2, BRD4, and c-MYC protein expression, increased HEXIM1 protein expression, while BRD3 expression remained unchanged. However, there was no correlation between the mRNA levels of c-MYC, BRD2, BRD3, BRD4, and HEXIM1 and cell viability after OTX015 treatment. Sequential combinations of OTX015 with other epigenetic modifiers, panobinostat, and azacitidine synergistically promoted the growth of the KASUMI cell line. Our results suggest that OTX015 and JQ1 have similar biological effects in leukemia cells, supporting the evaluation of OTX015 in a phase Ib clinical trial in patients with relapsed/refractory leukemia. [2]

---

Currently, no anti-HIV-1 drug can effectively clear the latent HIV-1 viral reservoir, which is a major obstacle to the complete cure of AIDS. We report here that a novel oral BET inhibitor, OTX015 (a thienotriazole diazepam compound that has entered phase Ib clinical development in advanced hematologic malignancies), can effectively activate HIV-1 in different latent models, with EC50 values 1.95–4.34 times lower than those of JQ1, a known BET inhibitor that can activate HIV-1 latency. We also found that OTX015 is more potent when used in combination with prostaglandins. More importantly, OTX015 treatment can induce the production of the full-length HIV-1 transcript and viral proliferation in resting CD4+ T cells of infected individuals receiving repressive antiretroviral therapy (ART), while having minimal effect on T cell activation. Finally, biochemical analysis showed that OTX015-mediated HIV-1 activation involved increased CDK9 occupancy and increased phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. Our results suggest that the BET inhibitor OTX015 may be a candidate drug for anti-HIV-1 latency therapy. [3]

---

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subtype of breast tumor clinically characterized by the lack of estrogen receptor, progesterone receptor and HER2 receptor, which limits the application of targeted therapies used in other breast malignancies. Recent evidence suggests that c-MYC is a key driver of triple-negative breast cancer (TNBC). The BET bromine domain inhibitor OTX015 (MK-8628) has demonstrated potent antiproliferative activity in various tumor types, accompanied by downregulation of c-MYC expression, and exhibits synergistic effects with the mTOR inhibitor everolimus in different models. This study aimed to evaluate the antitumor activity of OTX015 monotherapy and in combination with everolimus for the treatment of TNBC. We tested OTX015 using three human TNBC cell lines: HCC1937, MDA-MB-231, and MDA-MB-468. The results showed that all cell lines exhibited antiproliferative activity (GI50 = 75-650 nM) after 72 hours. This activity was accompanied by cell cycle arrest and decreased expression of cancer stem cell markers. However, downregulation of c-MYC protein and mRNA levels was observed only in MDA-MB-468 cells. Gene set enrichment analysis revealed upregulation of genes involved in transcriptional, chromatin, and cell cycle epigenetic regulation, while downregulation of stemness-related genes. In vitro experiments showed that OTX015 and everolimus had a synergistic effect in HCC1937 and MDA-MB-231 cells, but an antagonistic effect in MDA-MB-468 cells. In the MDA-MB-231 mouse xenograft model, OTX015 significantly reduced tumor volume compared with the vector control group (p < 0.05) (optimal T/C = 40.7%). Although everolimus monotherapy was ineffective, the efficacy of combination therapy was better than that of OTX015 monotherapy (optimal T/C = 20.7%). The results of this study support the ongoing clinical trial of OTX015 for the treatment of triple-negative breast cancer (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.

---

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.

---

View More

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.

---

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