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University of Dundee New Synthetic Routes to Triazolo University of Dundee New synthetic routes to Triazolo-benzodiazepine analogues Baud, Matthias G. J.; Lin-Shiao, Enrique; Zengerle, Michael; Tallant, Cynthia; Ciulli, Alessio Published in: Journal of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.5b01135 Publication date: 2016 Document Version Publisher's PDF, also known as Version of record Link to publication in Discovery Research Portal Citation for published version (APA): Baud, M. G. J., Lin-Shiao, E., Zengerle, M., Tallant, C., & Ciulli, A. (2016). New synthetic routes to Triazolo- benzodiazepine analogues: expanding the scope of the bump-and-hole approach for selective Bromo and Extra- Terminal (BET) bromodomain inhibition. Journal of Medicinal Chemistry, 59(4), 1492-1500. DOI: 10.1021/acs.jmedchem.5b01135 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 17. Feb. 2017 This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Article pubs.acs.org/jmc New Synthetic Routes to Triazolo-benzodiazepine Analogues: Expanding the Scope of the Bump-and-Hole Approach for Selective Bromo and Extra-Terminal (BET) Bromodomain Inhibition † ‡ ∥ † ‡ ⊥ † ‡ § Matthias G. J. Baud, , , Enrique Lin-Shiao, , , Michael Zengerle, Cynthia Tallant, , † ‡ and Alessio Ciulli*, , † Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee DD1 5EH, U.K. ‡ Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K. *S Supporting Information ABSTRACT: We describe new synthetic routes developed toward a range of substituted analogues of bromo and extra- terminal (BET) bromodomain inhibitors I-BET762/JQ1 based on the triazolo-benzodiazepine scaffold. These new routes allow for the derivatization of the methoxyphenyl and chlorophenyl rings, in addition to the diazepine ternary center and the side chain methylene moiety. Substitution at the level of the side chain methylene afforded compounds targeting specifically and potently engineered BET bromodomains designed as part of a bump and hole approach. We further demonstrate that marked selectivity for the second over the first bromodomain can be achieved with an indole derivative that exploits differential interaction with an aspartate/histidine conservative substitution on the BC loop of BET bromodomains. ■ INTRODUCTION iophene) and bind to the acetyl-lysine (KAc) pocket of BET ffi − 11 The 1,4-benzodiazepine scaffold occupies a place of choice in bromodomains with high a nity (Kd of 1 =50 370 nM). These compounds display activity in vivo12 against a number of the toolbox of medicinal chemists and is often referred to as a 13 “privileged scaffold” in drug discovery. A large number of disease states, including NUT-midline carcinoma, multiple myeloma,14 mixed-lineage leukemia,15 and acute myeloid biologically active small molecules containing a 1,4-benzodia- 16,17 zepine scaffold have been approved by the FDA for the leukemia. Despite selectively targeting the BET bromodo- treatment of various disease states, although most of them are main family with high potency over other bromodomains, these 1 fi well-known for their psychotropic effects. Well known compounds are pan-BET selective thus do not signi cantly examples include diazepam, alprazolam or prazepam. The discriminate between individual bromodomains of the four therapeutic potential of 1,4-benzodiazepines has fueled the BET members. This lack of selectivity within the BET interest of synthetic chemists in developing new routes to a subfamily so far has prevented accurate deconvolution of the − range of substituted analogues for biological evaluation.2 4 biological function of individual BET proteins and of their Recently, this scaffold has attracted particular attention in the tandem bromodomains. To address this problem, we recently field of epigenetics, with the discovery of a class of potent small developed a chemical genetics approach to engineer the molecule inhibitors of the interaction between Bromo and selectivity of the BET bromodomain inhibitor I-BET762/JQ1 Extra-Terminal (BET) bromodomain proteins and their within the BET proteins family.11 This so-called “bump and acetylated histone substrates. BET proteins Brd2, Brd3, Brd4, hole” approach is based on the generation of orthogonal and and Brdt are key transcriptional co-regulators. Crucial to their high-affinity protein/ligand pairs and involves introducing a activity are paired and highly homologous bromodomains single point mutation (large to smaller amino acid, that is, the located in their amino-terminal regions. The individual function “hole”) onto the BET bromodomain of interest together with of the first bromodomain (e.g., Brd2(1)) versus second making a synthetic modification (bulky substituent, that is, the bromodomain (e.g., Brd2(2)) of BET proteins is however “bump”) onto the parent BET bromodomain binder to unclear. A number of BET bromodomain inhibitors are complement the newly created protein subpocket (Figure currently in clinical trials for the treatment of cancer,5 including representative molecules I-BET762 (1),6 JQ1 (2),7 Special Issue: Epigenetics GW841819X (3),8 OTX015 (4),9 and RVX-208 (5)10 (Figure 1A). In particular, compounds 1−4 are based on a triazolo- Received: July 20, 2015 aryldiazepine scaffold (aryl = methoxyphenyl or dimethylth- Published: September 14, 2015 © 2015 American Chemical Society 1492 DOI: 10.1021/acs.jmedchem.5b01135 J. Med. Chem. 2016, 59, 1492−1500 Journal of Medicinal Chemistry Article Figure 1. (A) Structures of BET bromodomain probes I-BET762 (1), JQ1 (2), GW841819X (3), OTX-015 (4), and RVX-208 (5), currently in clinical trials, (B) bump and hole approach to engineer the selectivity of BET bromodomain probes against individual BET bromodomains, (C) structure of ET (6), and (D) cocrystal structure of Brd2(2)L383A (blue, surface representation) in complex with ET (6) (stick representation, yellow carbons), PDB code 4QEW.11 The L/A mutation is shown in red. Figure 2. (A) Crystal structure of I-BET762 (1, yellow carbons) bound to Brd4(1) (PDB code 3P5O,2 surface representation; red indicates negative and blue positive electrostatic potential). W81, V87, and L94 are highlighted. (B, C) I-BET762 chemical structure and positions selected for derivatization to target the corresponding mutations. 1B). As a result, the bulky ligand is expected to bind with high the side chain methylene moiety, bound to leucine/alanine affinity to the mutated BET protein, while exhibiting weak to mutant BET bromodomains with low nanomolar affinity and no binding to wild-type (WT) proteins due to a steric clash displayed up to 540-fold and no less than 40-fold (160-fold on occurring between the “bump” and the naturally occurring average) selectivity relative to WT BET bromodomains across residue (Figure 1B). This approach was previously shown to aid the entire subfamily. This orthogonal bromodomain/ligand pair selective targeting of protein kinases through engineering of the was used within cancer cells to show that selective blockade of ATP binding site and ATP cofactor as well as ATP-competitive the first bromodomain of a given BET protein, Brd4, is inhibitors.18,19 In our study, we demonstrated for the first time sufficient to displace it from chromatin.11 The exquisite that the bump and hole approach can be used to selectively selectivity provided by ET for engineered bromodomains is disrupt protein−protein interactions within the BET family of currently exploited in our laboratory to probe the biology of proteins.11 Compound ET (6)(Figure 1C,D), a derivative of I- individual BET proteins through selective modulation of their BET762/JQ1 bearing an ethyl functional group at the level of interaction with their histone substrates. Selective modulation 1493 DOI: 10.1021/acs.jmedchem.5b01135 J. Med. Chem. 2016, 59, 1492−1500 Journal of Medicinal Chemistry Article a Scheme 1. Synthesis of 14 and Its Methylated Derivative 15 a fl fl fl ° Conditions: (a) SOCl2,CH2Cl2,re ux, 2.5 h; (b) benzophenone, CHCl3,re ux, 3 h; (c) Et3N, CHCl3,re ux, 16 h; (d) AcOH, 1,2-DCE, 60 C, 3 ’ fl · ° ° h; (e) Lawesson s reagent, toluene, re ux, 4 h; (f) hydrazine H2O, THF, 0 C, 5 h; (g) AcCl, Et3N, 0 C to rt, 16 h; (h) AcOH, rt, 2 days (R = H) or reflux, 3 h (R = Me). of individual BET bromodomains is important for accurate and tantly, all mutants retained competence to bind a tetra- reliable target validation in the different disease states that are acetylated H4 derived peptide27 as assessed by ITC albeit to associated with unbalanced activity of BET proteins. varying degrees (Table S1). Most mutants exhibited com- The ET−L/A orthogonal inhibitor−protein pair was parable peptide binding affinities relative to WT, while the V/A discovered and optimized within the framework of an extended proved the most disruptive mutation. study in which we explored several mutations (“holes”) and I- With three positions identified and corresponding mutants BET762 substitution patterns (“bumps”). In the current characterized, I-BET762 (1) was selected as the starting manuscript, we report the full journey that led to that scaffold because it is more synthetically tractable and better discovery. In doing so, we also describe our synthetic efforts suited to all required vectors than JQ1.
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