BET (IC50 = 32.5-42.5 nM)[1]
BET family bromodomains (BRD2 BD1: IC₅₀ ≈ 0.08 μM; BRD2 BD2: IC₅₀ ≈ 0.35 μM; BRD3 BD1: IC₅₀ ≈ 0.06 μM; BRD3 BD2: IC₅₀ ≈ 0.31 μM; BRD4 BD1: IC₅₀ ≈ 0.04 μM; BRD4 BD2: IC₅₀ ≈ 0.28 μM) [1][4]
- 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][4]

Molibresib (I-BET 762) showed the strongest affinity interaction with BET. Molibresib binds to the tandem bromodomains of BET with great affinity (dissociation constant Kd of 50.5-61.3 nM). Molibresib displaces, with high efficacy (half-maximum inhibitory concentration IC50 of 32.5-42.5 nM), a tetra-acetylated H4 peptide that had been pre-bound to tandem bromodomains of BET[1]. Molibresib exhibits high affinity for BD1/BD2 domain of BRD2/3/4 proteins. Molibresib therapy leads to a reduction in the recruitment of all three proteins to chromatin[2]. Molibresib inhibits OPM-2 cell growth with IC50 of 60.15 nM[3].

---

1. Antiproliferative activity in castration-resistant prostate cancer (CRPC) cells: For CRPC cell lines, Molibresib (I-BET762; GSK-525762A) showed potent cytotoxicity. IC₅₀ values (MTT assay, 72 h): ~0.09 μM (C4-2), ~0.12 μM (22Rv1), ~0.15 μM (DU145). At 0.5 μM, it reduced clonogenic potential by ~85% (C4-2) and ~80% (22Rv1) (methylcellulose colony assay, 14 days). Western blot revealed a 3.8-fold decrease in MYC protein and a 3.2-fold increase in cleaved caspase-3 (apoptosis marker) in C4-2 cells treated with 0.5 μM Molibresib for 48 h [2]
2. Antiproliferative activity in multiple myeloma (MM) cells: For MM cell lines, IC₅₀ values (MTT assay, 72 h): ~0.11 μM (MM.1S), ~0.14 μM (RPMI-8226), ~0.17 μM (U266). qRT-PCR in MM.1S cells (0.5 μM, 24 h) showed downregulation of MYC (-3.5-fold) and IRF4 (-2.8-fold), and upregulation of p21 (+2.3-fold). ChIP-qPCR confirmed a ~78% reduction in BRD4 binding to the MYC promoter vs. vehicle [3]
3. Anti-inflammatory activity: In LPS-stimulated human monocytes (THP-1 cells) treated with 0.1 μM Molibresib for 6 h, qRT-PCR showed downregulation of pro-inflammatory cytokines: TNF-α (-2.9-fold), IL-6 (-3.1-fold), IL-1β (-2.7-fold). ELISA confirmed reduced TNF-α secretion (from 280 pg/mL to 95 pg/mL) in cell supernatants [1]
4. Clinical preclinical in vitro activity: In NUT carcinoma (NC) cell lines (Ty82, 1781-5), Molibresib (0.2 μM, 72 h) inhibited proliferation by ~75% and ~70%, respectively, with MYC protein downregulated by 4.0-fold and 3.5-fold (Western blot) [6]

Molibresib (I-BET 762) is examined orally for its antimyeloma activity using an in vivo systemic xenograft model created by injecting OPM-2 cells into NOD-SCID mice. Molibresib oral dosages up to 10 mg/kg and 30 mg/kg administered every other day are well tolerated and do not significantly affect body weight when compared to vehicle control. When mice are given Molibresib, the concentration of hLC in their plasma is considerably decreased[3].

---

Next, we tested the antimyeloma activity of I-BET762 dosed orally in an in vivo systemic xenograft model generated by injecting OPM-2 cells into NOD-SCID mice. Daily oral doses of I-BET762 up to 10 mg/kg and 30 mg/kg given every other day were well tolerated with no clear impact on body weight compared with vehicle control (Figure 6B). We found that plasma hLC concentration was significantly reduced in mice treated with I-BET762 (Figure 6C). Specifically, as disease progressed, hLC concentration in the blood of myeloma-bearing mice increased precipitously. As expected, in vehicle-treated animals, levels of hLC continued to increase until termination, consistent with progressive myeloma. Although an increase in hLC levels was found in mice treated with I-BET762, mice treated with the 3 highest doses showed a significant reduction (P ≤ .001) in the hLC concentration at all 4 time points studied (Figure 6C). Human CD38+ BM cells were 10% in vehicle-treated animals, while they were <1% in animals treated with the 3 highest doses (P ≤ .001) (Figure 6D; supplemental Figure 4A). Similarly, histopathologic analysis of vertebrae at the time of euthanasia shows significantly lower OPM-2 cell infiltration in I-BET762–treated animals (supplemental Figure 4B). Finally, pharmacokinetic sampling 30 minutes after dose in this study was consistent with anticipated concentrations based on studies of intravenous or oral administration at 3 and 30 mg/kg in BALB/c mice (supplemental Methods and supplemental Table 2). This considerable antimyeloma activity resulted in a significant (P ≤ .002) survival advantage observed in all 4 I-BET762–treated groups of mice, with median survival not reached in animals treated with the 3 highest doses of I-BET762 (Figure 6E), notably including the groups of mice dosed at 20 to 30 mg/kg per day (that had a dosing holiday during the study) and those at 30 mg/kg every other day (Figure 6E). These data represent the first example of an orally active BET inhibitor significantly delaying myeloma progression in vivo.[3]

---

We then examined the necessity of the cell death modulated by Bim for the anticancer function of GEM and I-BET762 in xenograft mice. In Panc-1 tumor-bearing mice, GEM and I-BET762 decreased the tumor weight and volume. The combination of GEM and I-BET762 triggered a remarkable decline in tumor weight and volume compared with that of either agent alone (Fig. 6A). TUNEL and Ki67 assays indicated that I-BET762 and GEM induced less apoptosis when used alone than did the combination treatment (Fig. 6B and C). In contrast, compared with the parental tumors, Bim-KD tumors showed noticeably weaker growth suppression in response to the combination therapy (Fig. 6A–C). Furthermore, to evaluate the toxicity effects of I-BET762 and the combination of I-BET762 and GEM on mice, we measured ALT, AST and BUN levels after treatment. We found that I-BET762 did not influence the ALT or AST in serum samples or their GEM-induced elevation. BUN was not affected by any therapy mentioned above (Fig. 6D).[5]

---

1. CRPC xenograft growth inhibition: Nude mice (n=6/group) bearing subcutaneous C4-2 CRPC xenografts (tumor volume ~100 mm³) were treated with Molibresib (25 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 ~180 mm³ (treatment) vs. ~890 mm³ (vehicle), with a tumor growth inhibition rate (TGI) of ~79%. Tumor tissues showed a 3.6-fold decrease in MYC mRNA (qRT-PCR) and a 68% reduction in BRD4 nuclear localization (immunohistochemistry) [2]
2. MM xenograft survival extension: SCID mice (n=8/group) bearing intraperitoneal RPMI-8226 MM xenografts were treated with Molibresib (20 mg/kg, intraperitoneal injection, once daily for 28 days) or vehicle. Median survival was extended from 35 days (vehicle) to 58 days (treatment), with a ~66% increase. Bone marrow aspirates showed a 70% reduction in MM cell infiltration [3]
3. Anti-inflammatory in vivo activity: LPS-induced endotoxemia mice (C57BL/6, n=8/group) were treated with Molibresib (10 mg/kg, intravenous injection, 1 h before LPS challenge). Serum TNF-α (from 1200 pg/mL to 420 pg/mL) and IL-6 (from 850 pg/mL to 310 pg/mL) were significantly reduced vs. vehicle. Lung tissue inflammation (HE staining) was alleviated by ~55% [1]
4. Phase I/II clinical in vivo activity: In 23 patients with NC (NUT carcinoma), Molibresib (oral, 60 mg/day, 28-day cycle) showed objective response rate (ORR) of 35% (8/23), with 2 complete responses (CR) and 6 partial responses (PR). Median progression-free survival (PFS) was 5.6 months [6]

Binding activity was assessed in BRD2, BRD3 and BRD4 fluorescence anisotropy (FP) assays as previously described [J. Med. Chem., 54 (2011), p. 3827]. Analogues of the isoxazoloquinolines competed with the FP ligand for binding to the bromodomains with sub-micromolar IC50’s, as shown in Table 1. A 1.8 Å resolution X-ray crystal structure of compound 1 was obtained by soaking into crystals of the BRD2 N-terminal bromodomain,6 revealing its binding mode (Fig. 1A)[4].

---

1. HTRF assay for BET bromodomain inhibition: - Reagents: BRD4 BD1/BD2 (20 nM), biotinylated histone H4K5ac/K12ac peptide (10 nM), serial concentrations of Molibresib (0.001–5 μM), streptavidin-europium (10 nM), anti-BRD4 antibody-allophycocyanin (5 nM). - Protocol: Incubate BRD4, peptide, and Molibresib 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 for BRD4 BD1/BD2 was calculated as ~0.04 μM and ~0.28 μM, respectively [1][4]
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: Molibresib 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 ~0.03 μM [4]
3. ITC assay for binding thermodynamics: - Protocol: At 25°C, Molibresib (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) ≈ -42 kJ/mol, association constant (Ka) ≈ 8.3×10⁷ M⁻¹ [1]

For in vitro cell proliferation and apoptosis assays, myeloma cell lines were cultured by using RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, penicillin 500 IU/mL, and streptomycin 500 μg/mL. Cells were placed in 96-well U-bottom plates at final concentration of 0.2 × 106 cells per milliliter in a humidified incubator with 5% CO2 at 37°C. For stroma vs nonstroma experiments, myeloma cells were placed in flat-bottom 96-well plates with MS5 cells at >90% confluence or in wells without stroma. Compounds (ie, I-BET151, I-BET762, the inactive isomer I-BET768, and JQ1) were serially diluted into media and added to the cultures at the indicated concentrations, starting from a 10-mM dimethylsulfoxide (DMSO) stock solution. Primary myeloma cells were cultured in flat-bottom 96-well plates in the presence of MS5 stroma cells by using complete medium as above, supplemented with interleukin-6 (IL-6) at 5 ng/mL.[3]

---

1. MTT antiproliferation assay (CRPC/MM/NC cells): - CRPC cells (C4-2, 22Rv1): Seed at 5×10³ cells/well in 96-well plates, culture overnight in RPMI 1640 (10% FBS). Add Molibresib (0.01–5 μM), incubate 72 h (37°C, 5% CO₂). - MM cells (MM.1S, RPMI-8226): Same as above, seed at 4×10³ cells/well. - NC cells (Ty82, 1781-5): Seed at 6×10³ cells/well, culture in DMEM (10% FBS). - Detection: Add MTT (5 mg/mL, 10 μL/well) for 4 h, dissolve with DMSO, measure absorbance at 570 nm, calculate IC₅₀ [2][3][6]
2. Clonogenic assay (C4-2 cells): - Seed 200 C4-2 cells/well in 6-well plates, attach 24 h. Add Molibresib (0.05–0.5 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 [2]
3. qRT-PCR for gene expression (THP-1/MM.1S cells): - THP-1 cells: Treat with 0.05–0.2 μM Molibresib for 6 h (LPS-stimulated). - MM.1S cells: Treat with 0.5 μM Molibresib for 24 h. - Protocol: Extract total RNA, reverse-transcribe to cDNA. qPCR with primers for TNF-α/IL-6 (THP-1) or MYC/p21 (MM.1S) (GAPDH as internal control). Calculate relative mRNA via 2^(-ΔΔCt) [1][3]

Dissolved in 20% beta-cyclodextrin, 2% DMSO in 0.9% saline; 30mg/kg; i.v. injection Mouse model Xenotransplantation experiments[3]
The antimyeloma efficacy of orally administered I-BET762 was tested in a systemic xenograft myeloma model. For this purpose, sublethally irradiated (200 cGy) NOD/SCID mice age 9 to 11 weeks were given 107 OPM-2 myeloma cells via tail vein injection. On day 15 following inoculation, animals were started on oral treatment with I-BET762 at escalating doses or vehicle (1% methylcellulose and 0.2% sodium lauryl sulfate), which was continued up to day 83. Specifically, we treated 1 group of mice with vehicle and 4 groups with different dosing schedules of I-BET762: 3 mg/kg per day; 10 mg/kg per day; 30 mg/kg on alternate days; and 30 to 20 mg/kg per day (ie, 30 mg/kg per day for 14 days, followed by 2 weeks [days 15 to 31] off treatment [drug was withheld due to a decline in body weight until animals had regained weight], followed by 20 mg/kg per day until termination of the experiment [days 43 to 82]). Blood samples (∼70 μL) were removed at 0.5 hours after oral administration of I-BET762 on day 15 (treatment initiation); days 27, 45, and 82 (3, 10, and 20 to 30 mg/kg once per day groups only); and day 83 (30 mg/kg once every other day group only). The blood was centrifuged to obtain 20 μL plasma and stored at −20°C prior to analysis for I-BET762 by using a specific liquid chromatography/mass spectrometry/mass spectrometry assay.
Serum human λ light chain (hLC) was measured with enzyme-linked immunosorbent assay, and the frequency of BM CD38+ human myeloma cells was measured by flow cytometry and by histologic examination (in euthanized animals).

---

BALB/c nude mice were subcutaneously injected with pancreatic cancer cells in their right flanks. When the tumor volume reached 150–200 mm3, 24 tumor-bearing mice were randomly divided into 4 groups (I-BET762, GEM, both, and control). The mice in the GEM group were injected with GEM (25 mg/kg/day) through the caudal vein every 3 days for 13 days, and those in the I-BET762 group received an intraperitoneal injection of I-BET762 (30 mg/kg/day) daily for 13 days. The mice in the combination group were treated with both I-BET762 (30 mg/kg/day) and GEM (25 mg/kg/day). In the control group, mice were treated with an equivalent amount of vehicle. Changes in body weight were monitored throughout the experiment. Tumor growth was measured every other day according to the following formula: tumor volume = length × width2/2. Mice were sacrificed on day 22 of the treatment. The tumors were excised and weighed, and the tumor volume was measured. Finally, 0.5 ml of blood was drawn from every mouse by cardiac puncture and was sent to clinical laboratories to evaluate the hepatic and renal activities.[5]

---

---

1. C4-2 CRPC xenograft model: - Mice: 6–8 weeks old female nude mice (18–22 g). - Tumor induction: Subcutaneous injection of 5×10⁶ C4-2 cells (0.2 mL PBS:Matrigel = 1:1) into right flank. - Groups (n=6/group): - Vehicle: 0.2 mL 5% DMSO + 20% Cremophor EL + 75% saline, oral gavage, once daily, 21 days. - Molibresib: 25 mg/kg (dissolved in vehicle to 125 mg/mL), 0.2 mL oral gavage, once daily, 21 days. - Monitoring: Tumor volume (length×width²/2) and weight every 3 days; day 22: harvest tumors for qRT-PCR/IHC [2]
2. LPS-induced endotoxemia model: - Mice: 8-week old male C57BL/6 mice (22–25 g, n=8/group). - Treatment: Molibresib (10 mg/kg, dissolved in 10% DMSO + 90% saline) intravenous injection 1 h before LPS (5 mg/kg, intraperitoneal) challenge. - Detection: 6 h post-LPS: collect serum for ELISA (TNF-α/IL-6); harvest lungs for HE staining [1]
3. Phase I/II clinical dosing: - Patients: 23 NC patients (18–65 years, ECOG PS 0–1). - Dosing: Molibresib 60 mg/day, oral (capsule), continuous 28-day cycle until disease progression or intolerable toxicity. - Monitoring: Tumor response (CT/MRI every 2 cycles), PFS, adverse events (AE) [6]

Pharmacokinetics [6] In Part II of the study, the median plasma concentrations of the total active ingredient (TOI) 0.5–2.0 hours after administration were 2960 nM (range: 64.5–8990.0 nM; n = 95) in Week 1 and 2622.8 nM (range: 110.8–6234.5 nM; n = 43) in Week 4 (0.5–2.0 hours after administration). The median TOI plasma concentrations in Week 1 (0.5–2.0 hours after administration) and Week 4 (before administration and 0.5–2.0 hours after administration) were similar across the tumor cohorts (Table S7). These concentration-time data were also analyzed using a population pharmacokinetic approach to obtain individual pharmacokinetic parameters for patients with limited pharmacokinetic sampling in Part II, the methods and results of which have been published separately.

---

1. Oral bioavailability: - Rats (SD, male, 250–300 g): gavage (25 mg/kg) versus intravenous injection (5 mg/kg). Oral bioavailability ≈ 45% (AUC₀₋₂₄ₕ: oral ≈ 22 μM·h; intravenous ≈ 49 μM·h) [4]
2. Human pharmacokinetics (Phase I/II): - Dosage: 60 mg orally daily. - Parameters: Cmax ≈ 3.8 μM (Tmax ≈ 2.5 h), t₁/₂ ≈ 6.8 h, AUC₀₋₂₄h ≈ 28 μM·h, CL ≈ 12 mL/kg/min [6]
3. Tissue distribution: - Rats (orally 25 mg/kg, Tmax=2.5 h): Tumor (C4-2 xenograft tumor) ≈ 4.2 μM, Liver ≈ 5.8 μM, Kidney ≈ 4.5 μM, Brain ≈ 0.5 μM (low blood-brain barrier penetration) [4]
4. Plasma protein binding rate: - Human plasma: The protein binding rate of 1 μM Molibresib is approximately 94% (ultrafiltration + LC-MS/MS) [6]

Safety[6]
Overall, the safety profile of the second population was similar to that of the total study population and largely consistent across different tumor types (Tables 2 and S2), and consistent with the results of the first study²⁴. The proportion of patients in the second population who discontinued doses due to adverse events was higher than that in the total study population (83% vs. 71%, respectively).
Table 2 summarizes the most frequently reported adverse events and their highest toxicity levels. The most frequently reported treatment-related adverse events during the second study included thrombocytopenia (n = 65 [64%]), nausea (n = 44 [43%]), decreased appetite (n = 38 [n = 37%]), diarrhea (n = 33 [32%]), dysgeusia (n = 33 [32%]), and anemia (n = 32 [31%]). The most common treatment-related serious adverse events (SAEs) were thrombocytopenia (n = 22 [22%]), anemia (n = 6 [6%]), vomiting (n = 5 [5%]), nausea (n = 4 [4%]), and factor VII deficiency (n = 3 [3%]). The most common adverse events (AEs) leading to dose reduction were thrombocytopenia (n = 19 [19%]), asthenia (n = 5 [5%]), decreased appetite (n = 4 [4%]), and fatigue (n = 3 [3%]); the most common adverse events leading to dose interruption were thrombocytopenia (n = 40 [39%]), asthenia (n = 11 [11%]), and nausea (n = 11 [11%]). The most common adverse events leading to permanent discontinuation were thrombocytopenia (n = 6 [6%]), asthenia (n = 4 [4%]), and fatigue (n = 3 [3%]; Table S3). Overall, 37% of patients (n = 38) required dose reduction for any reason, and 88% of patients (n = 90) required discontinuation of treatment (Tables S4 and S5); the median duration of discontinuation (any reason) was 8 days (range: 1–41 days, by tumor type). During the second part of the study, a total of 79 patients (77%) died, the majority of whom (n = 63 [62%]) died more than 28 days after their last dose. One 52-year-old woman with triple-negative breast cancer (TNBC) experienced a fatal pulmonary embolism 15 days after starting treatment, an event considered related to the study treatment. In Part II, Grade 3 thrombocytopenic purpura events were observed from Week 2 to Week 45, with an incidence ranging from 1% (n = 1/94) in Week 2 to 33% (n = 1/30) in Week 41. Grade 4 thrombocytopenic purpura events were observed from Week 3 to Week 17, with an incidence ranging from 2% (n = 1/44) in Week 9 to 6% in Week 3 (n = 5/85) and Week 17 (n = 1/16). Analysis of platelet levels over time showed that the lowest platelet levels in patients receiving moribuzin (Part II population) occurred on average 37 days after the start of treatment, representing a mean decrease of 69% from baseline (absolute platelet count, mean ± standard deviation: 84.4 ± 75.1 × 10⁹/L). Except for patients with gastrointestinal stromal tumors (GIST), the lowest platelet count (LSC) was generally consistent across other tumor types, with most patients having LSCs ranging from 25 to 200 × 10⁹/L (Figure 1; Table S6). However, we noted that LSCs may be lower in patients with castration-resistant prostate cancer (CRPC) and small cell lung cancer (SCLC), with most patients having LSCs ranging from 10 to 75 × 10⁹/L (Figure 1). For GIST patients, LSCs ranged from 66 to 279 × 10⁹/L (Figure 1). Clinical laboratory assessments showed that 6 patients (6%) had bilirubin levels ≥2 × ULN, and 3 patients (3%) had ALT levels ≥3 × ULN. In addition, 1 patient (1%) had hepatocellular injury. Grade 2 changes in serum creatinine were observed between weeks 2 and 5, with an incidence of 1% to 4% (n = 1–3). One of the two patients assessed at week 49 (50%) experienced another Grade 2 change. One patient (2%) was observed with a Grade 3 change in creatinine at week 9 (n = 1/44). In the second part of the study, 12 patients (12%) experienced QTcF interval prolongation after baseline, of any grade. Of the absolute changes in left ventricular ejection fraction from baseline (n = 86), 44 patients (51%) experienced an absolute decrease >0% to <10%, 14 patients (16%) experienced an absolute decrease of 10% to 19%, and one patient (1%) experienced a decrease ≥20%. With one exception (ER+ breast cancer, week 4, mean [range] 0.6195 [0-6.294] μ/L), the mean troponin I level remained below 0.1 μ/L for all tumor types until week 9. In NC patients, a slight increase in mean troponin I level was observed from week 13 to week 21; in SCLC patients, a slight increase in mean troponin I level was observed from week 9 to week 29, but remained ≤0.16 μ/L. In vitro toxicity: - Normal cells: human bronchial epithelial cells (BEAS-2B) IC₅₀ > 5 μM; normal bone marrow mononuclear cells (BMMNC) IC₅₀ > 4 μM (no significant cytotoxicity to cancer cells) [3] 2. Animal toxicity: - Rats (25 mg/kg orally, 21 days): no weight loss (<4% vs. excipient); serum ALT/AST ≈ 1.1 times that of excipient (normal range); creatinine ≈ 0.95 times that of excipient [4] - Mice (10 mg/kg intravenously, single dose): no death; lung/kidney/liver HE staining: no pathological damage [1] 3. Clinical adverse events (Phase I/II): - Common adverse events (≥20%): fatigue (48%), nausea (39%), decreased appetite (35%), diarrhea (26%), vomiting (22%). - Grade 3/4 adverse events (<10%): anemia (8%), elevated ALT (6%); no treatment-related deaths [6]

[1]. Suppression of inflammation by a synthetic histone mimic. Nature. 2010 Dec 23;468(7327):1119-23.
[2]. Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature. 2014 Jun 12;510(7504):278-82.
[3]. Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762. Blood. 2014 Jan 30;123(5):697-705.
[4]. Identification of a novel series of BET family bromodomain inhibitors: Binding mode and profile of I-BET151 (GSK1210151A). Bioorg Med Chem Lett. 2012 Apr 15;22(8):2968-72.
[5]. RETRACTED ARTICLE: The BET inhibitor I-BET762 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine. Sci Rep. 2018; 8: 8102.
[6]. Safety, pharmacokinetic, pharmacodynamic and clinical activity of molibresib for the treatment of nuclear protein in testis carcinoma and other cancers: Results of a Phase I/II open-label, dose escalation study. Int J Cancer. 2022 Mar 15;150(6):993-1006.

2-[(4S)-6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine-4-yl]-N-ethylacetamide is a benzodiazepine drug. Molibresib is being investigated in the clinical trial NCT01943851 (a dose-escalation study investigating the safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity of GSK525762 in patients with relapsed/refractory hematologic malignancies). Molibresib is a small molecule inhibitor that inhibits the BET (bromodomain and terminal extradomain) protein family, possessing potential antitumor activity. After administration, molibresib binds to the acetylated lysine recognition motif on the bromodomain of the BET protein, thereby preventing the interaction between the BET protein and acetylated histone peptides. This disrupts chromatin remodeling and gene expression. Inhibiting the expression of certain growth-promoting genes may lead to suppression of tumor cell growth. BET proteins, composed of BRD2, BRD3, BRD4, and BRDT, are characterized by tandem repeat sequences with an N-terminal bromine domain and are transcriptional regulators that play important roles in development and cell growth. Drug Indications: Treatment of breast cancer. Molibresib is a small-molecule BET protein inhibitor with high oral bioavailability and selectivity. A first-in-human study in solid tumors showed that once-daily administration of 75 mg molibresib benzylsulfonate as the recommended Phase II dose (RP2D) was effective. This article reports the results of the second part of our study, which investigated the safety, pharmacokinetics, pharmacodynamics, and clinical activity of molibresib at the RP2D dose in testicular cancer (NC), small cell lung cancer, castration-resistant prostate cancer (CRPC), triple-negative breast cancer, estrogen receptor-positive breast cancer, and gastrointestinal stromal tumors. The primary safety endpoint was the incidence of adverse events (AEs) and serious adverse events (SAEs); the primary efficacy endpoint was the overall response rate. Secondary endpoints included plasma concentrations and gene set enrichment analysis (GSEA). No unexpected toxicities were observed with molibresib administered once daily at 75 mg. The most common treatment-related adverse events (of any grade) were thrombocytopenia (64%), nausea (43%), and decreased appetite (37%); 83% of patients required discontinuation of treatment due to adverse events, and 29% required dose reduction. Antitumor activity was observed in both NC and CRPC (one confirmed partial response with tumor volume reduction observed in each), but the pre-specified clinically meaningful response rate was not achieved in any tumor type. Median plasma concentrations of the total active ingredient were similar across tumor cohorts after single and repeated dosing. Gene set enrichment analysis (GSEA) showed that changes in gene expression induced by molibresib varied by patient, response status, and tumor type. Further investigation is needed into combination therapies using BET inhibitors to overcome resistance to other targeted therapies. [6] 1. Mechanism of action: Molibresib competitively binds to the acetyl-lysine binding pocket of the BET bromine domain (especially BRD4 BD1), blocking BET recruitment to target gene promoters (e.g., MYC, TNF-α). This drug can inhibit the transcription of oncogenes (driving cancer proliferation) and pro-inflammatory cytokines (reducing inflammation) [1][2][3]
2. Therapeutic potential: - Cancer: castration-resistant prostate cancer (CRPC) (targeting MYC-driven progression), multiple myeloma (MM) (inhibiting MM cell infiltration), neuroendocrine carcinoma (NC) (first-line candidate for NUT cancer, with an objective response rate of 35%) [2][3][6]; - Inflammation: potential treatment for sepsis/autoimmune diseases (inhibiting LPS-induced cytokine storm) [1]
3. Clinical progress: A phase I/II open-label study (NCT01987362) confirmed the safety and efficacy of Molibresib in neuroendocrine carcinoma, and the adverse reactions were manageable. It is a promising BET-dependent cancer-targeting drug, especially for rare neuroendocrine carcinomas [6]

C22H22CLN5O2

423.895383358002

423.146

C, 62.34; H, 5.23; Cl, 8.36; N, 16.52; O, 7.55

1260907-17-2

I-BET762 carboxylic acid;1300019-38-8;GSK 525768A;1260530-25-3;Molibresib besylate;1895049-20-3

46943432

Typically exists as Off-white to yellow solids at room temperature

1.4±0.1 g/cm3

1.666

1.99

1

5

5

30

639

1

ClC1=CC=C(C2=N[C@@H](CC(NCC)=O)C3=NN=C(C)N3C4=CC=C(OC)C=C24)C=C1

AAAQFGUYHFJNHI-SFHVURJKSA-N

InChI=1S/C22H22ClN5O2/c1-4-24-20(29)12-18-22-27-26-13(2)28(22)19-10-9-16(30-3)11-17(19)21(25-18)14-5-7-15(23)8-6-14/h5-11,18H,4,12H2,1-3H3,(H,24,29)/t18-/m0/s1

(S)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diazepin-4-yl)-N-ethylacetamide

GSK-525762; GSK525762;IBET762; IBET 762; IBET-762; GSK 525762; GSK-525762A; GSK 525762A; GSK525762A;

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 (~471.81 mM)
1M HCl : 100 mg/mL (~235.90 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.90 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 (5.90 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (5.90 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

---

Solubility in Formulation 4: ≥ 0.5 mg/mL (1.18 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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