Synthesis and Photophysical Properties of Benzotriazole-Derived Unnatural α-Amino Acids.

The synthesis of a new class of benzotriazole-derived α-amino acid is described using a highly efficient nucleophilic aromatic substitution of ortho-fluoronitrobenzenes with l-3-aminoalanine and a polymer-supported nitrite reagent-mediated diazotization and cyclization of the subsequent 1,2-aryldiamines as the key steps. Further functionalization of the benzotriazole unit by preparation of halogenated analogues and Suzuki-Miyaura cross-coupling with aryl boronic acids allowed the synthesis of α-amino acids with conjugated side chains. Analysis of the photophysical properties of these α-amino acids revealed that incorporation of electron-rich substituents results in charge-transfer-based, fluorescent compounds with MegaStokes shifts.

Stereoselective synthesis of 2,6-trans-4-oxopiperidines using an acid-mediated 6-endo-trig cyclisation.

An acid-mediated 6-endo-trig cyclisation of amine-substituted enones has been developed for the stereoselective synthesis of trans-6-alkyl-2-methyl-4-oxopiperidines. Performed under conditions that prevent removal of the Boc-protecting group or acetal formation, the key cyclisation was found to generate cleanly the 4-oxopiperidine products in high overall yields from a wide range of alkyl substituted enones. The synthetic utility of the trans-6-alkyl-2-methyl-4-oxopiperidines formed from this process was demonstrated with the total synthesis of the quinolizidine alkaloid, (+)-myrtine and the piperidine alkaloid, (-)-solenopsin A.

Structure- and Property-Based Optimization of Efficient Pan-Bromodomain and Extra Terminal Inhibitors to Identify Oral and Intravenous Candidate I-BET787.

The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.

Structure-Guided Design of a Domain-Selective Bromodomain and Extra Terminal N-Terminal Bromodomain Chemical Probe.

Small-molecule-mediated disruption of the protein-protein interactions between acetylated histone tails and the tandem bromodomains of the bromodomain and extra-terminal (BET) family of proteins is an important mechanism of action for the potential modulation of immuno-inflammatory and oncology disease. High-quality chemical probes have proven invaluable in elucidating profound BET bromodomain biology, with seminal publications of both pan- and domain-selective BET family bromodomain inhibitors enabling academic and industrial research. To enrich the toolbox of structurally differentiated N-terminal bromodomain (BD1) BET family chemical probes, this work describes an analysis of the GSK BRD4 bromodomain data set through a lipophilic efficiency lens, which enabled identification of a BD1 domain-biased benzimidazole series. Structure-guided growth targeting a key Asp/His BD1/BD2 switch enabled delivery of GSK023, a high-quality chemical probe with 300-1000-fold BET BD1 domain selectivity and a phenotypic cellular fingerprint consistent with BET bromodomain inhibition.

Investigation of Janus Kinase (JAK) Inhibitors for Lung Delivery and the Importance of Aldehyde Oxidase Metabolism.

The Janus family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) play an essential role in the receptor signaling of cytokines that have been implicated in the pathogenesis of severe asthma, and there is emerging interest in the development of small-molecule-inhaled JAK inhibitors as treatments. Here, we describe the optimization of a quinazoline series of JAK inhibitors and the results of mouse lung pharmacokinetic (PK) studies where only low concentrations of parent compound were observed. Subsequent investigations revealed that the low exposure was due to metabolism by aldehyde oxidase (AO), so we sought to identify quinazolines that were not metabolized by AO. We found that specific substituents at the quinazoline 2-position prevented AO metabolism and this was rationalized through computational docking studies in the AO binding site, but they compromised kinome selectivity. Results presented here highlight that AO metabolism is a potential issue in the lung.

Design, Synthesis, and Characterization of I-BET567, a Pan-Bromodomain and Extra Terminal (BET) Bromodomain Oral Candidate.

Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.

Conformationally rigid pyrazoloquinazoline α-amino acids: one- and two-photon induced fluorescence.

The synthesis and photophysical properties of a new class of α-amino acid bearing a rigid pyrazoloquinazoline chromophore are described. Confromational constraint of the amino acid side-chains resulted in high emission quantum yields, while the demonstration of two-photon-induced fluorescence via near-IR excitation signifies their potential for sensitive bioimaging applications.

Structure-Based Design of a Bromodomain and Extraterminal Domain (BET) Inhibitor Selective for the N-Terminal Bromodomains That Retains an Anti-inflammatory and Antiproliferative Phenotype.

The bromodomain and extraterminal domain (BET) family of epigenetic regulators comprises four proteins (BRD2, BRD3, BRD4, BRDT), each containing tandem bromodomains. To date, small molecule inhibitors of these proteins typically bind all eight bromodomains of the family with similar affinity, resulting in a diverse range of biological effects. To enable further understanding of the broad phenotype characteristic of pan-BET inhibition, the development of inhibitors selective for individual, or sets of, bromodomains within the family is required. In this regard, we report the discovery of a potent probe molecule possessing up to 150-fold selectivity for the N-terminal bromodomains (BD1s) over the C-terminal bromodomains (BD2s) of the BETs. Guided by structural information, a specific amino acid difference between BD1 and BD2 domains was targeted for selective interaction with chemical functionality appended to the previously developed I-BET151 scaffold. Data presented herein demonstrate that selective inhibition of BD1 domains is sufficient to drive anti-inflammatory and antiproliferative effects.

Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation.

The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.

Discovery of a Bromodomain and Extraterminal Inhibitor with a Low Predicted Human Dose through Synergistic Use of Encoded Library Technology and Fragment Screening.

The bromodomain and extraterminal (BET) family of bromodomain-containing proteins are important regulators of the epigenome through their ability to recognize N-acetyl lysine (KAc) post-translational modifications on histone tails. These interactions have been implicated in various disease states and, consequently, disruption of BET-KAc binding has emerged as an attractive therapeutic strategy with a number of small molecule inhibitors now under investigation in the clinic. However, until the utility of these advanced candidates is fully assessed by these trials, there remains scope for the discovery of inhibitors from new chemotypes with alternative physicochemical, pharmacokinetic, and pharmacodynamic profiles. Herein, we describe the discovery of a candidate-quality dimethylpyridone benzimidazole compound which originated from the hybridization of a dimethylphenol benzimidazole series, identified using encoded library technology, with an N-methyl pyridone series identified through fragment screening. Optimization via structure- and property-based design led to I-BET469, which possesses favorable oral pharmacokinetic properties, displays activity in vivo, and is projected to have a low human efficacious dose.

Click chemistry enables preclinical evaluation of targeted epigenetic therapies.

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.