Discovery of Benzotriazolo[4,3-d][1,4]diazepines as Orally Active Inhibitors of BET Bromodomains

Abstract

Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo [b]isoxazolo [4,5-d] T he bromodomain and extra terminal (BET) family is made up of BRD2, BRD3, BRD4, and BRDT, each of which contains tandem bromodomains (BD-1 and BD-2) that recognize specific acetylated lysine residues in the N-terminal tails of histones. 1 Members of the BET family modulate gene expression by recruiting transcriptional regulators to specific genomic locations. 2 In 2009, the Mitsubishi Tanabe Pharma Corporation reported the first small molecule inhibitors of this family of chromatin adaptors, 3 and publications followed on the use of BET inhibitors to demonstrate antitumor activity 4 and modulate inflammation. 5 Experiments applying genetic ablation or specific inhibitor treatment have established the role of BET family members in regulating genes crucial for normal cell growth. 6−8 Subsequent studies 9,10 have defined a mechanistic link between potency of BET inhibition and activity against hematologic malignancies and demonstrated that BET inhibition regulates the MYC oncogene, resulting in cell cycle arrest and apoptosis. In vivo BET bromodomain inhibition leads to efficacy in xenograft models representing multiple cancer types, and BET bromodomains are also implicated in nononcology indications, i.e., human immunodeficiency virus and human t-lymphotrophic viral gene expression, DNA damage response, and cardiac hypertrophy. Importantly, BET inhibitors have been shown to selectively regulate a subset of multiple inflammatory cytokines, including interleukins 6, 1β, 17, 21, and 23, and granulocyte macrophage colony-stimulating factor 11,12 and have demonstrated efficacy in several in vivo models of inflammation, including endotoxic shock, experimental autoimmune encephalomyelitis, and arthritis. 5,11 Because of the potential therapeutic application of BET inhibition, we initiated a drug discovery program and recently reported on a fragment-based effort that resulted in a series of isoxazolo [5,4-c]thieno[2,3-e]azepine BET inhibitors exemplified by CPI-3 (Scheme 1). 13 Concurrent with that effort we were interested in exploring a different connectivity within the seven-membered ring that would provide a unique basis for modulation of the physicochemical properties and BET potency of the resultant compounds. Specifically, we hypothesized that changing the cyclic imine to a substituted aniline would retain the concave shape important for specific binding to the bromodomain while providing a different metabolic profile for the series. We were also interested in evaluating a modified linker in combination with the reported triazole binding element 4,5 and in finding a replacement for the thiophene group of CPI-3 because of its potential metabolic instability. Here we describe the resulting benzo [b]isoxazolo- [4,5-d]azepi...

Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein‐Detected NMR Spectroscopy

“…These findings were consistent with previous observations that the relatively large and hydrophobic acetyl‐lysine binding pockets of BET bromodomains are highly druggable compared to other bromodomains . Indeed, many screening efforts have successfully identified small molecules for BET bromodomain acetyl‐lysine binding pockets . In summary, screening BRD4(BD2) against our chemical library identified acetaminophen, a compound known to interact with bromodomains, and demonstrated the feasibility and success of our approach in identifying compounds that interact with target proteins for further characterization.…”

“…Baseplane noise was not shown to contributes ignificantly to DIA values( Figures S2 and S4). Manual inspection of spectral overlays for these ten samples verified that the seven samples with the highest positivei ntensityv alues (1,24,31,39,40,45, and 50) derived from distinct CSPs. By contrast, the three samples with the lowestp ositive intensity values above the + +1 s threshold (23, 53, and 54) did not give rise to substantial CSPs and were classified as false positives.…”

“…[28] Indeed, many screening efforts have successfully identified smallm olecules for BET bromodomain acetyl-lysineb inding pockets. [29][30][31] In summary,s creeningB RD4(BD2) against our chemical library identified acetaminophen, ac ompound known to interact with bromodomains, and demonstrated the feasibility and success of our approach in identifying compounds that interactw ith target proteins for furthercharacterization.…”

“…To determine the spectralb asis of these variances, we manually inspected 2D spectralo verlays of all datasets. Three of the eight NMR samples (31,40, and 45) contained unambiguous CSPs. These three samples were also found in ac luster distinct from the remaining five samples (20-22, 42, and 48).…”

“…For CXCL12, DIA identified ten NMR samples that gave positive intensities above the + +1 s threshold (1,23,24,31,39,40,45, 50, 53, and 54) and thirteen samples that gave negative intensities below the À1 s threshold (1,20,21,22,31,32,39,40,41,42,45,48,and 50,Figure2C). Six of these samples (1,31,39,40,45, and 50) exceeded both thresholds and gave rise to the largest positive intensities,w hich is expected from ligand binding, as ap erturbation that shifts ac rosspeakf rom the DMSO controlw ill result in equal intensity positive and negative peaks in the 2D difference spectrum.W ea ssigned all ten samples exceeding the + +1 s threshold as potential hits. Baseplane noise was not shown to contributes ignificantly to DIA values( Figures S2 and S4).…”