α-Helix proteomimetics represent an emerging class of ligands that can be used to inhibit an array of helix mediated protein-protein interactions. Within this class of inhibitor, aromatic oligobenzamide foldamers have been widely and succssefuly used. This manuscript describes alternative syntheses of these compounds that can be used to access mimetics that are challenging to synthesize using previously described methodologies, permiting access to compounds functionalized with multiple sensitive side chains and accelerated library assembly through late stage derivatisation

Adam Nelson

Alexander L. Breeze

Andrew J. Wilson

Arkin

Azzarito

Barnard

Burslem

Campbell

Cernak

Dames

Ernst

Freedman

George M. Burslem

Hannah F. Kyle

Henchey

Jung

Kyle

Lando

Long

Milroy

Moon

Murphy

Orner

Panchami Prabhakaran

Plante

Prabhakaran

Ravindranathan

Saraogi

Shaginian

Stuart L. Warriner

Thomas A. Edwards

Whitby

PubMed

New routes are developed to allow introduction of sensitive side-chains and synthesis of challenging proteomimetic sequences.</p

Crossref

Organic & Biomolecular Chemistry

Synthesis of highly functionalized oligobenzamide proteomimetic foldamers by late stage introduction of sensitive groups

α-Helix proteomimetics represent an emerging class of ligands that can be used to inhibit an array of helix mediated protein-protein interactions. Within this class of inhibitor, aromatic oligobenzamide foldamers have been widely and successfully used. This manuscript describes alternative syntheses of these compounds that can be used to access mimetics that are challenging to synthesize using previously described methodologies, permitting access to compounds functionalized with multiple sensitive side chains and accelerated library assembly through late stage derivatisation.</p

Burslem, George M.

Kyle, Hannah F.

Prabhakaran, Panchami

Breeze, Alexander L.

Edwards, Thomas A.

Warriner, Stuart L.

Nelson, Adam

Wilson, Andrew J.

University of Birmingham Research Portal

Burslem, GM

Kyle, HF

Prabhakaran, P

Breeze, AL

Edwards, TA

Warriner, SL

Nelson, A

Wilson, AJ

White Rose Research Online

Organic &Biomolecular ChemistryPAPERCite this: Org. Biomol. Chem., 2016,14, 3782Received 12th January 2016,Accepted 14th March 2016DOI: 10.1039/c6ob00078awww.rsc.org/obcSynthesis of highly functionalized oligobenzamideproteomimetic foldamers by late stage introductionof sensitive groups†George M. Burslem,‡a,b Hannah F. Kyle,b,c Panchami Prabhakaran,a,bAlexander L. Breeze,b,c,d Thomas A. Edwards,b,c Stuart L. Warriner,a,bAdam Nelsona,b and Andrew J. Wilson*a,bα-Helix proteomimetics represent an emerging class of ligands that can be used to inhibit an array of helixmediated protein–protein interactions. Within this class of inhibitor, aromatic oligobenzamide foldamershave been widely and successfully used. This manuscript describes alternative syntheses of these com-pounds that can be used to access mimetics that are challenging to synthesize using previously describedmethodologies, permitting access to compounds functionalized with multiple sensitive side chains andaccelerated library assembly through late stage derivatisation.IntroductionAn on-going challenge in chemical biology is the inhibition ofprotein–protein interactions (PPIs) using designed small mole-cules.1,2 An approach to the targeting of PPIs that has gainedattention is the use of secondary structure mimics3,4 whichhave been referred to as α-helix mimetics, proteomimetics,5topographical mimics and pharmacological chaperones.6 Oligo-benzamide helix mimetics (e.g. A–D Fig. 1) have been shownby us and others to act as effective and selective inhibitors of arange of PPIs.7–20 Our group has developed two generalapproaches for synthesis21 of these compounds (Scheme 1aand b): (i) iterative solution phase coupling of nitro-maskedmonomers with carboxy-protected terminal anilines, followedby unmasking of the latent amine10,12,13,22–24 and (ii) N-term-inal extension with Fmoc protected monomers followed by de-protection in solution or on solid-supported resin.7–9,11,25–28Herein, we report alternative complementary routes towardshighly functionalised compounds. These comprise (a) coup-ling of unfunctionalized monomers followed by subsequentintroduction of the amino acid side chain mimicking group(Scheme 1c) and (b) the preparation of compounds bearingside chain functionality through late stage functionalizationon the fully assembled scaffold (Scheme 1d).HIF-1 is an emerging, but not yet fully validated target forcancer chemotherapy; the interaction of HIF-1α with CREBbinding protein (CBP)/p300 plays a major role in regulatingthe hypoxic response and as such its inhibition represents anattractive approach to prevent development of new vasculatureby hypoxic tumours. The HIF-1α/p300 interaction is substan-tially mediated by the binding of HIF-1α around the p300 CH1domain and involves 3 regions which adopt a helical confor-mation on binding to p300 (Fig 2a).29,30 We recently reportedthat helix 3 plays the dominant role in mediating potency ofrecognition but that helix 2, either as a fusion with helix 1 or 3plays a role in binding (Fig. 2b).31 We also demonstrated thathelix 3 could be effectively mimicked using a 3-O-alkylated aro-Fig. 1 Structures of aromatic oligoamide helix mimetics A–D.†Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ob00078a‡Current Address: Yale University, New Haven, CT 06520, USA.aSchool of Chemistry, University of Leeds, Woodhouse Lane, Leeds, LS29JT, UK.E-mail: a.j.wilson@leeds.ac.ukbAstbury Centre for Structural Molecular Biology, University of Leeds, WoodhouseLane, Leeds, LS29JT, UKcSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, Universityof Leeds, Woodhouse Lane, Leeds LS2 9JT, UKdDiscovery Sciences, AstraZeneca R&D, Alderley Park, Cheshire, SK10 4TG, UK3782 | Org. Biomol. Chem., 2016, 14, 3782–3786 This journal is © The Royal Society of Chemistry 2016Open Access Article. Published on 14 March 2016. Downloaded on 26/05/2016 11:06:01.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineView Journal  | View Issuematic oligoamide helix mimetic (Fig 2c). Motivated by this, wesought to synthesise mimics of the helix-2 sequence andexplore the structure activity relationship around the helix-3 mimetic. Thus, using the HIF-1α/p300 interaction as amodel, the two new synthetic methods described below allowfurther analyses of α-helix mimicry of the p300 binding HIF-1αtransactivation sequence, but equally may be applied tofurther studies on any of the 2 or 3-O-alkylated helix mimeticsdescribed to date.Results and discussionHighly functionalised compounds – helix 2Initially, we sought to prepare mimetics capable of recapitulat-ing the side chains of a different region of the HIF-1α peptideto corroborate our observations from peptide binding studies.The helix 2 region of the HIF-1α CTAD contains two reported“hot-spot” residues which are also the sites of post-transla-tional modifications, Cys800 and Asn804 as shown in Fig. 2.33,34Initial attempts to prepare helix 2 mimics (Fig. 2c) usingmonomers designed to mimic Asn, Cys and Ser (see ESI,Scheme S2† for structures and synthesis) and either of ourestablished protocols were hampered by inefficient couplingbetween the respective amine and acid building blocks. Wetentatively suggest that the lack of coupling arises as a conse-quence of the extra steric bulk conferred by the large protect-ing groups on each monomer (see Table S1†) along with thepoor nucleophilicity of ortho-amino phenols.25,35 After screen-ing a range of amide bond forming conditions (see ESI,Table S1†), an alternative synthetic strategy was investigatedbuilding on the work of Ahn;35 this ligation approach exploitsthe efficiency of intramolecular acylation. The nitro acidmonomer 1 was reacted with methyl 4-amino-3-hydroxybenzo-ate 2 to give a 1 : 9 mixture of the amide 3 and the ester 4;O- to N-acyl transfer was then promoted by treating the mixtureScheme 1 General strategies for aromatic oligoamide helix mimetics synthesis (a) N-terminal elongation with nitro-functionalised monomers (b)solid-phase synthesis using Fmoc strategy (c) O–N-acyl-shift for post monomer coupling functional group introduction (d) late stagefunctionalization.Fig. 2 (a) Structure of the HIF-1α CTAD/p300 CH1 domain complex(PDB ID: 1L8C,32 p300 green, HIF-1α, blue) together with 3-O-alkylatedaromatic oligoamide helix mimetics for helix 2 and 3 of HIF-1α designedon the basis of (b) binding studies reported previously (regionsconfirmed to contribute affinity shown in purple and other regionsshown in blue) (c) Modelling of helix 2 mimetic (hot spot residues onhelix (in blue) are highlighted).Organic & Biomolecular Chemistry PaperThis journal is © The Royal Society of Chemistry 2016 Org. Biomol. Chem., 2016, 14, 3782–3786 | 3783Open Access Article. Published on 14 March 2016. Downloaded on 26/05/2016 11:06:01.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinewith caesium carbonate to afford the desired compound 3 in amoderate 44% yield. The second side chain was then intro-duced with 2-bromo-N-(triphenylmethyl)-acetamide beforefinal reduction, hydrolysis and deprotection to yield thedesired, densely functionalised dimer 5 as shown in Scheme 2.In the design of 5 an alcohol side chain was used on theN-terminal amino benzoic acid monomer in lieu of a thiol.36,37The O- to N-acyl synthetic method was also employed to syn-thesise the reverse side chain orientation of 5 (see ESI†). Inprinciple this synthetic method should be fully compatiblewith the solid-phase synthesis methodologies previouslyreported and facilitate the preparation of highly functionalisedhelix mimetics for use against other targets.Late stage derivatisation – helix 3Having demonstrated synthetic feasibility for late stage alkyl-ation of the phenolic position in the course of synthesisingdimer 5 (see above), we further adapted this approach topermit late stage derivatisation of the trimeric oligobenzamidescaffold (Fig. 1a) and rapid library generation for structureactivity relationship (SAR) studies around our initial helix 3inhibitors.38 Late stage derivatisation reduces the requirementto build and assemble large sets of monomers and shouldallow the incorporation of delicate functionalities (e.g.trifluoromethyl diazirines to support proteomics analyses andbinding site identification by cross-linking).Using the recently identified proteomimetic inhibitor (seeFig. 2a) of the HIF-1α/p300 interaction as inspiration,10 aseries of compounds with cleavable protecting groups was pre-pared, e.g. 6b. Phenolic allyl ethers are readily cleaved via theformation of a π-allyl complex with palladium(0) but are rela-tively inert to other conditions. This makes then ideal for usein the late stage derivatisation of oligobenzamides. Indeed, theallyl groups in 6a–c were readily cleaved with palladium(0)tetrakis(triphenylphosphine) employing toluene sulfinic acid asa scavenger (Scheme 3 for a representative example (6b→ 7b)).The phenols 7a–c could then be functionalised by employ-ing Mitsunobu chemistry or alkylation with an appropriatehalide yielding functionalized helix mimetics which, uponreduction of the aryl nitro group and ester hydrolysis in onepot, afford final compounds 8a–i (Table 1), following purifi-cation (Scheme 3 for a representative example (7b → 8e)). Thisapproach was thus employed to prepare a small library of com-pounds 8a–i with previously unreported side chain combi-nations. Alcohols and alkyl halides were selected to challengethe synthetic approach (focusing on tertiary amines) ratherthan a specific intention to develop optimised inhibitors.As a further illustrative example of the power of theapproach, we incorporated a trifluoromethyl diazirine func-tionality into the oligobenzamide for photo-crosslinkingexperiments. The fragile diazirine would not be stable to thereductive conditions required to reduce the nitro groups.Scheme 2 Synthesis of helix 2 mimetic by O–N-acyl shift and alkyl-ation sequence.Scheme 3 Late stage helix mimetic derivatisation (a) representativesynthetic route for 6b (b) structures of compounds 6a and 6b.Paper Organic & Biomolecular Chemistry3784 | Org. Biomol. Chem., 2016, 14, 3782–3786 This journal is © The Royal Society of Chemistry 2016Open Access Article. Published on 14 March 2016. Downloaded on 26/05/2016 11:06:01.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineHowever, using the late stage methodology described above,the aryl trifluoromethyl diazirine was incorporated follow-ing a slightly modified sequence following all the requiredreductions. By selectively, and irreversibly, deprotonating thephenol 7b with sodium hydride then treating with the iodide9, the desired functionality could be introduced regioselec-tively. Final ester hydrolysis was performed in situ by treatingthe reaction mixture with sodium hydroxide to yield the photo-crosslinking helix mimetic 8j. 8k was synthesized using asimilar sequence of steps from compound 6c (see ESI† fordetails) (Table 1).Biophysical analysesUsing a previously described fluorescence anisotropy competi-tive HIF-1α/p300 assay,10,31 the helix 2 mimetic 5 was shown tobe ineffective as a HIF-1α/p300 inhibitor; this is surprisinggiven that the group of Arora had previously shown a hydro-gen-bond surrogate constrained peptide derived from helix 2of HIF-1α was a good HIF-1α/p300 inhibitor39 but is consistentwith our observations that the helix 2 peptide alone is not auseful inhibitor.31 The compounds prepared by late stage deri-vatisation (7 and 8) were also screened for activity (Table 1);they showed reduced activity compared to the previouslyreported inhibitors by us and others.10,40 This is consistentwith the hydrophobicity of the binding groove. The diazarinebearing compound 8k also exhibits reduced binding whereas8j, was not active in our assay.In pursuit of preliminary cross-linking studies, compound 8kwas incubated with p300 and irradiated with 365 nMlight followed by LC-MS analysis. LC-MS analysis revealedalmost complete conversion of the diazarine moiety tocarbene quenching products but unfortunately no proteinlabelling was observed (see Fig. S3†). The quenched carbene pro-ducts and absence of cross-linking to protein suggest that thetrifluoromethyl diazarine is exposed to solvent when compound8k is bound to p300 rather than buried in the protein surface.Further experiments with other proteins are ongoing.ConclusionsIn summary, we have described methodology that providesaccess to challenging aromatic oligoamide sequences andcomplements current approaches for the synthesis of thesewidely used helix mimetics. This approach may be useful inbuilding up a picture of structure activity relationships (SAR)and is complementary to solution and SPS strategies for helixmimetic assembly useful for library production and initialscreening.8,18,25 To appreciate the power of this alternative syn-thetic approach, compounds 8b,d–e,g–i were prepared in atotal of 39 synthetic steps whereas by contrast, it would haverequired 51 synthetic steps to prepare this library using ourpreviously described methods.10,14,25 We have exemplified thiswith our preliminary results for inhibition of a key target intumour metabolism, HIF-1α/p300.AcknowledgementsWe thank AstraZeneca and EPSRC for PhD studentships(G.M.B. and H.F.K.) and the European Research Council[ERC-StG-240324, and ERC-PoC 632207] for support.Notes and references1 M. R. Arkin, Y. Tang and J. A. Wells, Chem. Biol., 2014, 21,1102–1114.2 L.-G. Milroy, T. N. Grossmann, S. Hennig, L. Brunsveld andC. Ottmann, Chem. Rev., 2014, 114, 4695–4748.3 V. Azzarito, K. 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Synthesis of Highly Functionalized Oligobenzamide Proteomimetic Foldamers by Late Stage Introduction of Sensitive Groups

Synthesis of Highly Functionalized Oligobenzamide Proteomimetic Foldamers by Late Stage Introduction of Sensitive Groups

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