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Cite this: J. Med. Chem. 2020, 63, 17, 9070–9092

Publication Date:July 21, 2020

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Supporting Info (2)»Supporting Information

The bromodomain and extra terminal domain (BET) family of proteins consists of four isoforms: BRD2, BRD3, BRD4, and BRDT. Each of these BET proteins contain two bromodomains, known as bromodomain 1 (N-terminus, BD1) and bromodomain 2 (C-terminus, BD2). The profound anti-proliferative and anti-inflammatory effects of pan-BET inhibitors, acting at both BD1 and BD2 of all four BET proteins, have now been well documented.(1−22) Indeed, a number of pan-BET inhibitors are progressing through phase I to II clinical trials for oncology indications.(23,24) However, associated pharmacology-driven toxicities with pan-BET inhibitors have also been reported.(24−26) Our strategy was therefore to target the rational design of selective BET inhibitors in order to elucidate the functional contribution of each domain to the phenotype observed with pan-BET inhibitors with the ambition of improving on the adverse event profile observed in humans. Due to the high homology across the four bromodomain-containing proteins, it was envisaged that obtaining a single isoform-selective inhibitor would be complex. However, the homology between the BD2 and the BD1 domains is lower with key residues close to the acetylated lysine (AcK) recognition pocket altered (see Figure S2), which, it was hypothesized, would give the opportunity to deliver pan BD2 selectivity.(27−32) Herein, we describe our approach to identify a tool molecule with second bromodomain (BD2) pIC50 > 7 at BRD2, 3, 4, and T combined with selectivity over the first domain (BD1) of at least 100-fold. In addition, we wanted to explore the opportunity of using BD2-selective molecules as tools to understand the in vivo capabilities and wider developability of our series.

At the time this work was carried out, there were no highly potent BD2-selective inhibitors known. The best-studied molecule reported to show bias in favor of BD2 was RVX-208 (Figure 1), which has a Kd of 8.9 μM against BD1 and 300 nM against BD2 by ITC, a selectivity of approximately 30-fold.(33) Independent ITC measurements showed greater potency but lower selectivity (Kd of 1.1 μM for BRD4 BD1 and 135 nM for BRD4 BD2).(27) We have subsequently reported a series of quinoxaline-based inhibitors such as GSK340, which show greater potency and selectivity (Kd in the Discoverx BROMOscan assays of 1.5 μM against BRD4 BD1 and 7 nM against BRD4 BD2).(29) Very recently, ABBV-744 was reported as a nanomolar BD2 inhibitor and clinical candidate with >290-fold selectivity over the BD1 domains of BRD2/3/4.(34,35)