BET bromodomain[1]
ABBV-744 is a potent and highly selective inhibitor of the second bromodomain (BD2) of the Bromodomain and Extra-Terminal (BET) family proteins (BRD2, BRD3, BRD4, BRDT). [1]
Selectivity data: In TR-FRET assays, ABBV-744 showed >290-fold selectivity for BD2 over BD1 of BRD2, BRD3, and BRD4, and >95-fold selectivity over BD1 of BRDT. [1]
Binding Affinity: For BRD4, SPR-derived Kd values were 3,300 nM for BD1 and 2.1 nM for BD2. NanoBRET-derived IC50 values were 20,700 nM for BD1 and 27.5 nM for BD2. [1]
It lacked significant activity against 75 kinases and 22 other bromodomain-containing proteins. [1]

KLK2 and MYC gene expression is downregulated by ABBV-744 (90 nM; 0~24 h; LNCaP cells) [1]. Senescence is induced by ABBV-744 (90 nM; 0~72 h) on LNCaP cells, which causes cell cycle arrest in the G1 phase [1].

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ABBV-744 demonstrated potent antiproliferative activity (IC50 < 100 nM) predominantly in acute myeloid leukemia (AML) cells and a subset of prostate cancer cell lines expressing the full-length androgen receptor (AR), but not in AR-V7 expressing or AR-negative prostate cancer lines. For example, IC50 values in prostate cancer cell lines were: LNCaP: 11 nM; MDA-PCa-2b: 38.3 nM; 22RV1: 467 nM; VCaP: 354 nM; PC3: >1000 nM; DU-145: 706.9 nM. [1]
In AR-positive, ABBV-744-sensitive LNCaP cells, treatment (90 nM, a BD2-selective concentration) induced cell cycle arrest in G1 phase followed by senescence after prolonged treatment (12 days), similar to the effects of the dual BET inhibitor ABBV-075 (60 nM) and the AR antagonist enzalutamide. [1]
RNA-sequencing in LNCaP cells stimulated with dihydrotestosterone (DHT) revealed that ABBV-744 (90 nM) elicited far fewer gene expression changes compared to ABBV-075 (60 nM) at concentrations that similarly inhibited BD2 of BRD4. ABBV-744 downregulated a small, specific set of genes (e.g., ACPP, MYC), which were highly enriched for DHT-responsive genes, but did not affect genes like HEXIM1, SPDEF, and ZG16B that were responsive to ABBV-075. At a high concentration (6 µM, likely engaging both BD1 and BD2), ABBV-744 recapitulated the broad transcriptional profile of ABBV-075. [1]
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) in LNCaP cells showed that ABBV-744 (90 nM) displaced BRD4 from chromatin with a globally weaker but similar pattern compared to ABBV-075 (60 nM). It preferentially displaced BRD4 from AR-containing super-enhancers and downregulated genes associated with these super-enhancers (e.g., KLK2, ACPP), suggesting inhibition of AR-dependent transcription. [1]
Co-immunoprecipitation experiments in LNCaP cells showed that the DHT- and acetylation-dependent interaction between BRD4 and AR was disrupted by ABBV-744 (90 nM), similar to ABBV-075. In contrast, interactions of BRD4 with CDK9, GATA2, or the CDK9/cyclin T1 complex with HEXIM1 were not affected, indicating BD2-dependency for the BRD4-AR interaction. [1]
ABBV-744 demonstrated limited potency in viability assays using mouse megakaryocyte colony-forming units (Mk-CFU) and rat intestinal epithelial IEC-6 cells, which are potential surrogates for platelet toxicity and gastrointestinal toxicity, respectively, compared to the dual inhibitor ABBV-075. [1]

In comparison to ABBV-075 [1], ABBV-744 (4.7 mg/kg; interfacial gavage; 28 days) shown comparable or superior anti-tumor effectiveness and retarded the formation of tumors. /kg; 14) has strong anti-tumor properties. By 20%, ABBV-744 (30 mg/kg) suppresses interference [1].

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In a mouse xenograft model using LNCaP cells, treatment with 4.7 mg kg−1 ABBV-744 (1/16 of the maximum tolerated dose (MTD)) caused a delay in tumour growth that was equivalent to ABBV-075 treatment at the MTD dose of 1 mg kg−1 (Fig. 4a). Comparing efficacious exposure levels of ABBV-744 in LNCaP tumour-bearing mice (4.7 mg kg−1; area under the curve, 1.1 μg h ml−1) and MTD (75 mg kg−1; area under the curve, 13.1 μg h ml−1) demonstrated that ABBV-744 was able to produce significant antitumour activity at 1/12 of the highest tolerable exposure of ABBV-744 (Extended Data Fig. 8a). The activity exhibited by ABBV-744 at 1/16 of the MTD of ABBV-744 was superior to the activities achieved using JQ1 and iBET at their respective MTDs or, in the case of RVX-208, at the highest feasible dose in this model (Extended Data Fig. 8b, c). Similarly, ABBV-744 at 1/16 MTD also displayed equivalent or better antitumour activity compared with ABBV-075 at MTD in the enzalutamide-resistant MDA-PCa-2b xenograft model (Fig. 4b). As a control, lowering the dose of ABBV-075 to 1/2 of the MTD resulted in a significant reduction in antitumour activity to 42% tumour growth inhibition in the LNCaP xenograft model. Even in the xenograft model using OPM2 cells, one of the most sensitive models to DbBi, ABBV-075 at 1/4 of the MTD of ABBV-075 (0.25 mg kg−1) had only marginal antitumour efficacy[1].

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In toxicity studies in rats, ABBV-075 at 3 mg kg−1 (3× the efficacious exposure in the LNCaP mouse xenograft model), caused a 59% reduction in platelets, a decrease in Alcian blue staining of the mucosa and the loss of goblet cells. By contrast, ABBV-744 at 30 mg kg−1 (25× the efficacious exposure) triggered a reduction in platelets of only 20%, and at 60 mg kg−1 (47× the efficacious exposure) did not cause loss of goblet cells or other gross intestinal defects (Fig. 4c and Extended Data Fig. 8a). Similarly, 2.5 mg kg−1 ABBV-075 caused germ cell degeneration in the testes, whereas no microscopic changes in the testes were observed with 25 mg kg−1 ABBV-744. These efficacy and tolerability results collectively suggest that selectively targeting BD2 can induce antitumour activity in some cancer settings while mitigating key tolerability issues of DbBi. These findings support the advancement of ABBV-744 for clinical evaluation (ClinicalTrials.gov identifier NCT03360006) and call for further investigation of BD2-dependent transcription programs to reveal additional therapeutic opportunities.

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In a mouse xenograft model using AR-positive LNCaP prostate cancer cells, daily oral administration of ABBV-744 at 4.7 mg/kg (1/16 of its maximum tolerated dose, MTD) significantly delayed tumor growth. This efficacy was equivalent to treatment with the dual BET inhibitor ABBV-075 at its MTD (1 mg/kg). [1]
In an enzalutamide-resistant MDA-PCa-2b prostate cancer xenograft model, ABBV-744 at 1/16 MTD (4.7 mg/kg) displayed equivalent or better antitumor activity compared to ABBV-075 at its MTD (1 mg/kg). [1]
In toxicity studies in rats, daily oral administration of ABBV-744 at 30 mg/kg (25x the efficacious exposure in the LNCaP mouse model) for 14 days resulted in only a 20% reduction in platelet counts. In contrast, ABBV-075 at 3 mg/kg (3x its efficacious exposure) caused a 59% platelet reduction. Furthermore, ABBV-744 at 60 mg/kg did not cause the significant loss of goblet cells or reduction in Alcian blue staining of the intestinal mucosa that was observed with ABBV-075 at 3 mg/kg. [1]

TR-FRET binding assays. [1]
Alexa647-labeled MS417 was used as the fluorescent probe in assay buffer (20 mM sodium phosphate, pH 6.0, 50 mM NaCl, 1 mM ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-dithiothreitol) containing His-tagged bromodomain, europium-conjugated anti-His antibody and Alexa-647- conjugated probe. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multi-label plate reader.[1]

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SPR binding experiment. [1]
The binding kinetics of compounds were assayed via Surface Plasmon Resonance (SPR) using a Biacore T200 instrument and manufacturer provided software. Brd4 BD1(h)(57-168) and BD2(h)(352-457) coupling solution was prepared by diluting proteins to 50 µg/mL in 10 mM phosphate solution (pH 6.5). Carboxyl groups on the dextran layer of the chip were activated by injecting a 1:1 mixture of 0.4 M N-ethyl-N-(3- dimethylaminopropyl)carbodiimide and 0.1 M N-hydroxysuccinimide for 7 min. The proteins coupling solution was injected over the activated chip surface for 800 sec at 10 l/min to achieve an immobilization level of 3452 and 1640 resonance units (RU) of Brd4 BD1 and BD2 respectively. Remaining free activated carboxyl groups were blocked by injecting a solution 1 M ethanolamine for 7 min. A blank surface was treated similarly but without any protein solution and used as a reference surface during binding assays. Briefly, the running buffer during immobilization was HSB-P+ Buffer (10 mM Hepes, 150 mM NaCl, 0.05% (vol/vol) surfactant P20, pH 7.4) and the coupling procedure was run at a flow rate of 5 l/min. Binding affinity measurements were performed at flow rate of 80 l/min using 10 mM Hepes, 150 mM NaCl, 0.05% (vol/vol) surfactant P20, pH 7.4, containing 15 mM DTT and 1 % DMSO. Compounds were assayed using singlecycle kinetics mode as provided by the Biacore T200 control software and were recorded at a frequency of 10 Hz. The compounds were diluted in the running buffer and injected in a series of increasing concentrations for contact time of 260 sec each and dissociation was monitored for up to 10000 sec. Sensorgrams were processed and analyzed using Biacore T200 evaluation software and the binding curves were fit to determine the equilibrium dissociation constant (Kd).

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TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assays were used to determine the inhibitory potency and selectivity of ABBV-744 for the BD1 and BD2 domains of BET proteins (BRD2, BRD3, BRD4, BRDT). The assay measured the displacement of a fluorescent tracer from the bromodomain. [1]
Surface Plasmon Resonance (SPR) experiments were performed to determine the binding kinetics and affinity (Kd) of ABBV-744 for the BD1 and BD2 domains of BRD4. For ABBV-744 binding to BD1, very fast on/off kinetics were observed, requiring a steady-state fit to equilibrium responses. For BD2, dissociation was very slow, so binding was profiled using the single-cycle kinetics method. [1]
NanoBRET (NanoLuc Bioluminescence Resonance Energy Transfer) target engagement assays in cells were used to determine the cellular IC50 values for ABBV-744 inhibition of BRD4 BD1 and BD2 binding to chromatin. [1]

Western Blot Analysis[1]
Cell Types: LNCaP cells
Tested Concentrations: 90 nM
Incubation Duration: 0~24 hrs (hours)
Experimental Results: Downregulated the expression of KLK2 and MYC genes.

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Cell Cycle Analysis[1]
Cell Types: LNCaP cells
Tested Concentrations: 90 nM
Incubation Duration: 0~72 hrs (hours)
Experimental Results: Induced cell cycle arrest in G1 followed by senescence.

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Antiproliferative activity was assessed across a panel of cancer cell lines. Cells were treated with ABBV-744 for 5 days, and cell viability was measured to determine IC50 values. [1]
For cell cycle and senescence analysis, LNCaP cells were treated with ABBV-744 (90 nM) or control compounds. Cell cycle distribution was analyzed by flow cytometry after 72 hours. Senescence was assessed after 12 days of treatment by β-galactosidase staining. [1]
For gene expression analysis (RNA-seq), LNCaP cells were treated with DHT and ABBV-744 (90 nM), ABBV-075 (60 nM), or vehicle for 24 hours. RNA was extracted, sequenced, and differentially expressed genes were identified. Gene set enrichment analysis was performed. [1]
For chromatin profiling (ChIP-seq), LNCaP cells treated with DHT and ABBV-744 (90 nM), ABBV-075 (60 nM), or vehicle for 6 hours were cross-linked. Chromatin was immunoprecipitated using antibodies against BRD4, AR, or H3K27ac. Sequencing libraries were prepared and analyzed to map protein-DNA interactions and histone marks. [1]
Co-immunoprecipitation (co-IP) assays were performed to study protein-protein interactions. Nuclear extracts from treated LNCaP cells were immunoprecipitated with antibodies against AR, CDK9, or cyclin T1. Co-precipitated proteins (e.g., BRD4, GATA2, HEXIM1) were detected by western blotting. [1]

Animal/Disease Models: Mouse
Doses: 4.7 mg/kg (pharmacokinetic/PK/PK analysis) Dosing: po (oral gavage); 28-day
Experimental Results: compared with ABBV-075, caused tumor growth delay and demonstrated the same or better anti- tumor activity.

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Animal/Disease Models: SD (SD (Sprague-Dawley)) rat
Doses: 30 mg/kg (pharmacokinetic/PK/PK analysis) Dosing time: 14 days
Experimental Results: Produced significant anti-tumor activity.

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LNCaP Xenograft Efficacy Study: Male NSG or Fox Chase SCID mice (6-8 weeks old) were implanted subcutaneously with LNCaP prostate cancer cells. When tumors reached a measurable size, mice were randomized into treatment groups. ABBV-744 was administered orally via gavage once daily at a dose of 4.7 mg/kg for the duration of the study (e.g., 21-28 days). Tumor volumes and body weights were measured regularly. [1]
MDA-PCa-2b Xenograft Efficacy Study: Mice bearing enzalutamide-resistant MDA-PCa-2b tumors were treated similarly, with daily oral gavage of ABBV-744 at 4.7 mg/kg. [1]
Rat Toxicology Study: Male Sprague-Dawley rats (56-58 days old) were randomized into groups. ABBV-744 was administered orally via gavage once daily at doses of 30 mg/kg or 60 mg/kg for 14 days. Control groups received vehicle. Blood samples were collected for hematology (e.g., platelet counts). At study end, animals were euthanized, and tissues (including intestine) were collected for histopathological examination (e.g., hematoxylin and eosin staining, Alcian blue staining for goblet cells). [1]

ABBV-744 is primarily metabolized by the cytochrome P450 enzyme CYP3A4. [1] ABBV-744 has oral bioavailability and its efficacy and tolerability can be studied in vivo by oral administration. [1] Pharmacokinetic parameters in mice are shown in the extended data. In tumor-bearing mice, the effective exposure (area under the curve, AUC) of ABBV-744 at a dose of 4.7 mg/kg was 11.3 µg·h/mL. [1]

In a 14-day rat toxicology study, daily oral administration of 30 mg/kg of ABBV-744 resulted in a 20% decrease in platelet count. At a dose of 60 mg/kg, ABBV-744 did not cause significant changes in colorectal histopathology (e.g., loss of goblet cells, decreased mucosal staining), while these changes were observed at much lower doses (3 mg/kg) with the dual BET inhibitor ABBV-075. [1]
Compared to ABBV-075, ABBV-744 exhibited limited antiproliferative activity against normal mouse megakaryocyte progenitor cells (Mk-CFU assay) and rat intestinal epithelial cells (IEC-6), suggesting that its hematological and gastrointestinal toxicities may be improved. [1]

[1]. Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer. Nature. 2020;578(7794):306-310.

ABBV-744, a BET inhibitor, is an orally bioavailable inhibitor of the bromodomain and terminal domain (BET) protein family with potential antitumor activity. After oral administration, ABBV-744 preferentially binds to the second bromodomain (BD2) of the BET protein, thereby preventing the interaction between the BET protein and acetylated histones. This interferes with chromatin remodeling and gene expression. Inhibition of certain growth-promoting genes may lead to the suppression of the proliferation of BET-overexpressing tumor cells. The BET protein, composed of BRD2, BRD3, BRD4, and BRDT, is a class of transcriptional regulators containing two homologous bromodomains, namely the BD1 and BD2 domains. They play important roles in development and cell growth.
See also: Abesinol tatocilizumab (note moved to).

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ABBV-744 was discovered in a medicinal chemistry study aimed at developing a highly selective BD2 inhibitor with drug-like properties, which aimed to address the limitations of dibromodomain BET inhibitors (DbBi), which have shown dose-limiting thrombocytopenia and gastrointestinal toxicity in clinical practice. [1]
The BD2 selectivity of this compound can be structurally explained by its binding mode. The acetamide moiety utilizes the channel formed by His433, Tyr386 and Pro430 in BD2, which is absent in BD1. The 2,6-dimethylphenyl ether moiety binds to the smaller Val435 in BD2 better than to the larger Ile162 in BD1, thus optimizing the binding to BD2, but resulting in poorer binding to BD1. [1]
Unlike DbBi, which has a broader range of activities, its antitumor activity is mainly limited to cancers that rely on the full-length AR signaling pathway (e.g., some prostate cancers) and some AML models. This selective activity is related to its ability to disrupt the binding of BRD4 to AR-containing superenhancers and to suppress AR-dependent transcription with a smaller overall transcriptional effect. [1]
Compared to DbBi (such as ABBV-075), ABBV-744 has better tolerability (reduced platelet and gastrointestinal toxicity), suggesting that selective BD2 inhibition may separate the therapeutic effect from the targeted toxicity associated with pan-BET inhibition. [1]

C28H30FN3O4

491.5539

491.22203

C, 68.42; H, 6.15; F, 3.86; N, 8.55; O, 13.02

2138861-99-9

132010322

White to light yellow solid powder

3.9

3

5

6

36

852

0

FC1C=C(C)C(=C(C)C=1)OC1C=CC(=CC=1C1=CN(C)C(C2=C1C=C(C(NCC)=O)N2)=O)C(C)(C)O

OEDSFMUSNZDJFD-UHFFFAOYSA-N

InChI=1S/C28H30FN3O4/c1-7-30-26(33)22-13-20-21(14-32(6)27(34)24(20)31-22)19-12-17(28(4,5)35)8-9-23(19)36-25-15(2)10-18(29)11-16(25)3/h8-14,31,35H,7H2,1-6H3,(H,30,33)

N-ethyl-4-(2-(4-fluoro-2,6-dimethylphenoxy)-5-(2-hydroxypropan-2-yl)phenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide

ABBV744; ABBV 744; 9MX546E2SF; UNII-9MX546E2SF; ABBV-744.

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 : ~100 mg/mL (~203.44 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.09 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 (5.09 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 25.0 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: ≥ 2.5 mg/mL (5.09 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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Solubility in Formulation 4: ≥ 2 mg/mL (4.07 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.

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Solubility in Formulation 5: ≥ 2 mg/mL (4.07 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.

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Solubility in Formulation 6: 2.5 mg/mL (5.09 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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