Publications by authors named "Robert J Sheppard"

21 Publications

  • Page 1 of 1

Optimization of Potent ATAD2 and CECR2 Bromodomain Inhibitors with an Atypical Binding Mode.

J Med Chem 2020 05 6;63(10):5212-5241. Epub 2020 May 6.

WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, United Kingdom.

Most bromodomain inhibitors mimic the interactions of the natural acetylated lysine (KAc) histone substrate through key interactions with conserved asparagine and tyrosine residues within the binding pocket. Herein we report the optimization of a series of phenyl sulfonamides that exhibit a novel mode of binding to non-bromodomain and extra terminal domain (non-BET) bromodomains through displacement of a normally conserved network of four water molecules. Starting from an initial hit molecule, we report its divergent optimization toward the ATPase family AAA domain containing 2 (ATAD2) and cat eye syndrome chromosome region, candidate 2 (CECR2) domains. This work concludes with the identification of (GSK232), a highly selective, cellularly penetrant CECR2 inhibitor with excellent physicochemical properties.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00021DOI Listing
May 2020

A Qualified Success: Discovery of a New Series of ATAD2 Bromodomain Inhibitors with a Novel Binding Mode Using High-Throughput Screening and Hit Qualification.

J Med Chem 2019 08 9;62(16):7506-7525. Epub 2019 Aug 9.

GlaxoSmithKline Tres Cantos , 28760 Tres Cantos , Madrid , Spain.

The bromodomain of ATAD2 has proved to be one of the least-tractable proteins within this target class. Here, we describe the discovery of a new class of inhibitors by high-throughput screening and show how the difficulties encountered in establishing a screening triage capable of finding progressible hits were overcome by data-driven optimization. Despite the prevalence of nonspecific hits and an exceptionally low progressible hit rate (0.001%), our optimized hit qualification strategy employing orthogonal biophysical methods enabled us to identify a single active series. The compounds have a novel ATAD2 binding mode with noncanonical features including the displacement of all conserved water molecules within the active site and a halogen-bonding interaction. In addition to reporting this new series and preliminary structure-activity relationship, we demonstrate the value of diversity screening to complement the knowledge-based approach used in our previous ATAD2 work. We also exemplify tactics that can increase the chance of success when seeking new chemical starting points for novel and less-tractable targets.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00673DOI Listing
August 2019

Route to Prolonged Residence Time at the Histamine H Receptor: Growing from Desloratadine to Rupatadine.

J Med Chem 2019 07 5;62(14):6630-6644. Epub 2019 Jul 5.

Amsterdam Institute for Molecules, Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science , VU University Amsterdam , De Boelelaan 1083 , 1081 HV Amsterdam , The Netherlands.

Drug-target binding kinetics are an important predictor of in vivo drug efficacy, yet the relationship between ligand structures and their binding kinetics is often poorly understood. We show that both rupatadine () and desloratadine () have a long residence time at the histamine H receptor (HR). Through development of a [H]levocetirizine radiolabel, we find that the residence time of exceeds that of more than 10-fold. This was further explored with 22 synthesized rupatadine and desloratadine analogues. Methylene-linked cycloaliphatic or β-branched substitutions of desloratadine increase the residence time at the HR, conveying a longer duration of receptor antagonism. However, cycloaliphatic substituents directly attached to the piperidine amine (i.e., lacking the spacer) have decreased binding affinity and residence time compared to their methylene-linked structural analogues. Guided by docking studies, steric constraints within the binding pocket are hypothesized to explain the observed differences in affinity and binding kinetics between analogues.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750840PMC
July 2019

Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor.

Sci Rep 2019 05 27;9(1):7906. Epub 2019 May 27.

Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ, Amsterdam, The Netherlands.

Drug-target binding kinetics are suggested to be important parameters for the prediction of in vivo drug-efficacy. For G protein-coupled receptors (GPCRs), the binding kinetics of ligands are typically determined using association binding experiments in competition with radiolabelled probes, followed by analysis with the widely used competitive binding kinetics theory developed by Motulsky and Mahan. Despite this, the influence of the radioligand binding kinetics on the kinetic parameters derived for the ligands tested is often overlooked. To address this, binding rate constants for a series of histamine H receptor (HR) antagonists were determined using radioligands with either slow (low k) or fast (high k) dissociation characteristics. A correlation was observed between the probe-specific datasets for the kinetic binding affinities, association rate constants and dissociation rate constants. However, the magnitude and accuracy of the binding rate constant-values was highly dependent on the used radioligand probe. Further analysis using recently developed fluorescent binding methods corroborates the finding that the Motulsky-Mahan methodology is limited by the employed assay conditions. The presented data suggest that kinetic parameters of GPCR ligands depend largely on the characteristics of the probe used and results should therefore be viewed within the experimental context and limitations of the applied methodology.
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http://dx.doi.org/10.1038/s41598-019-44025-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536503PMC
May 2019

Aiming to Miss a Moving Target: Bromo and Extra Terminal Domain (BET) Selectivity in Constrained ATAD2 Inhibitors.

J Med Chem 2018 09 18;61(18):8321-8336. Epub 2018 Sep 18.

Molecular Discovery Research, Cellzome GmbH , GlaxoSmithKline , Meyerhofstrasse 1 , 69117 Heidelberg , Germany.

ATAD2 is a cancer-associated protein whose bromodomain has been described as among the least druggable of its class. In our recent disclosure of the first chemical probe against this bromodomain, GSK8814 (6), we described the use of a conformationally constrained methoxy piperidine to gain selectivity over the BET bromodomains. Here we describe an orthogonal conformational restriction strategy of the piperidine ring to give potent and selective tropane inhibitors and show structural insights into why this was more challenging than expected. Greater understanding of why different rational approaches succeeded or failed should help in the future design of selectivity in the bromodomain family.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00862DOI Listing
September 2018

Structure-Affinity Relationships and Structure-Kinetic Relationships of 1,2-Diarylimidazol-4-carboxamide Derivatives as Human Cannabinoid 1 Receptor Antagonists.

J Med Chem 2017 12 21;60(23):9545-9564. Epub 2017 Nov 21.

Division of Medicinal Chemistry, LACDR, Leiden University , 2300RA Leiden, The Netherlands.

We report on the synthesis and biological evaluation of a series of 1,2-diarylimidazol-4-carboxamide derivatives developed as CB receptor antagonists. These were evaluated in a radioligand displacement binding assay, a [S]GTPγS binding assay, and in a competition association assay that enables the relatively fast kinetic screening of multiple compounds. The compounds show high affinities and a diverse range of kinetic profiles at the CB receptor and their structure-kinetic relationships (SKRs) were established. Using the recently resolved hCB receptor crystal structures, we also performed a modeling study that sheds light on the crucial interactions for both the affinity and dissociation kinetics of this family of ligands. We provide evidence that, next to affinity, additional knowledge of binding kinetics is useful for selecting new hCB receptor antagonists in the early phases of drug discovery.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5734604PMC
December 2017

Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics.

Mol Pharmacol 2017 Jan 1;91(1):25-38. Epub 2016 Nov 1.

AstraZeneca, Discovery Sciences, Alderley Park, United Kingdom (J.D.H., S.S.); AstraZeneca, Oncology, Cambridge, United Kingdom (R.J.S.); AstraZeneca, Discovery Sciences, Mölndal, Sweden (H.C.); AstraZeneca, Discovery Sciences, Cambridge Science Park, United Kingdom (I.L.D., P.B.R.); AstraZeneca, Oncology, Alderley Park, United Kingdom (M.J.W.); Leiden Academic Centre for Drug Research, Division of Medicinal Chemistry, The Netherlands (D.G., W.K., A.P.I.J.); and University of Nottingham, School of Life Sciences, United Kingdom (S.J.H.)

The duration of action of adenosine A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.
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http://dx.doi.org/10.1124/mol.116.105551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198511PMC
January 2017

A Chemical Probe for the ATAD2 Bromodomain.

Angew Chem Int Ed Engl 2016 09 17;55(38):11382-6. Epub 2016 Aug 17.

GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, UK.

ATAD2 is a cancer-associated protein whose bromodomain has been described as among the least druggable of that target class. Starting from a potent lead, permeability and selectivity were improved through a dual approach: 1) using CF2 as a sulfone bio-isostere to exploit the unique properties of fluorine, and 2) using 1,3-interactions to control the conformation of a piperidine ring. This resulted in the first reported low-nanomolar, selective and cell permeable chemical probe for ATAD2.
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http://dx.doi.org/10.1002/anie.201603928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314595PMC
September 2016

GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain.

ACS Med Chem Lett 2016 Jun 9;7(6):552-7. Epub 2016 May 9.

Epinova Discovery Performance Unit, Quantitative Pharmacology, Experimental Medicine Unit, Flexible Discovery Unit, and Platform Technology and Science, GlaxoSmithKline , Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.

The BRPF (Bromodomain and PHD Finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. A selective benzimidazolone BRPF1 inhibitor showing micromolar activity in a cellular target engagement assay was recently described. Herein, we report the optimization of this series leading to the identification of a superior BRPF1 inhibitor suitable for in vivo studies.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904261PMC
June 2016

Interrogating the Druggability of the 2-Oxoglutarate-Dependent Dioxygenase Target Class by Chemical Proteomics.

ACS Chem Biol 2016 07 19;11(7):2002-10. Epub 2016 May 19.

Cellzome GmbH, a GlaxoSmithKline company , Meyerhofstrasse 1, Heidelberg, Germany.

The 2-oxoglutarate-dependent dioxygenase target class comprises around 60 enzymes including several subfamilies with relevance to human disease, such as the prolyl hydroxylases and the Jumonji-type lysine demethylases. Current drug discovery approaches are largely based on small molecule inhibitors targeting the iron/2-oxoglutarate cofactor binding site. We have devised a chemoproteomics approach based on a combination of unselective active-site ligands tethered to beads, enabling affinity capturing of around 40 different dioxygenase enzymes from human cells. Mass-spectrometry-based quantification of bead-bound enzymes using a free-ligand competition-binding format enabled the comprehensive determination of affinities for the cosubstrate 2-oxoglutarate and for oncometabolites such as 2-hydroxyglutarate. We also profiled a set of representative drug-like inhibitor compounds. The results indicate that intracellular competition by endogenous cofactors and high active site similarity present substantial challenges for drug discovery for this target class.
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http://dx.doi.org/10.1021/acschembio.6b00080DOI Listing
July 2016

Cell Penetrant Inhibitors of the KDM4 and KDM5 Families of Histone Lysine Demethylases. 1. 3-Amino-4-pyridine Carboxylate Derivatives.

J Med Chem 2016 Feb 15;59(4):1357-69. Epub 2016 Jan 15.

Epinova Discovery Performance Unit, Medicines Research Centre, GlaxoSmithKline R&D , Stevenage SG1 2NY, U.K.

Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥ 50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6-8 μM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100-125 nM).
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http://dx.doi.org/10.1021/acs.jmedchem.5b01537DOI Listing
February 2016

Structure-Based Optimization of Naphthyridones into Potent ATAD2 Bromodomain Inhibitors.

J Med Chem 2015 Aug 31;58(15):6151-78. Epub 2015 Jul 31.

∥Cellzome GmbH, Molecular Discovery Research, GlaxoSmithKline, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

ATAD2 is a bromodomain-containing protein whose overexpression is linked to poor outcomes in a number of different cancer types. To date, no potent and selective inhibitors of the bromodomain have been reported. This article describes the structure-based optimization of a series of naphthyridones from micromolar leads with no selectivity over the BET bromodomains to inhibitors with sub-100 nM ATAD2 potency and 100-fold BET selectivity.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00773DOI Listing
August 2015

Fragment-Based Discovery of Low-Micromolar ATAD2 Bromodomain Inhibitors.

J Med Chem 2015 Jul 9;58(14):5649-73. Epub 2015 Jul 9.

∥Drug Metabolism and Pharmacokinetics (DMPK), GlaxoSmithKline, Park Road, Ware, Hertfordshire SG12 0DP, United Kingdom.

Overexpression of ATAD2 (ATPase family, AAA domain containing 2) has been linked to disease severity and progression in a wide range of cancers, and is implicated in the regulation of several drivers of cancer growth. Little is known of the dependence of these effects upon the ATAD2 bromodomain, which has been categorized as among the least tractable of its class. The absence of any potent, selective inhibitors limits clear understanding of the therapeutic potential of the bromodomain. Here, we describe the discovery of a hit from a fragment-based targeted array. Optimization of this produced the first known micromolar inhibitors of the ATAD2 bromodomain.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00772DOI Listing
July 2015

Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation.

Nat Chem Biol 2015 Mar 26;11(3):189-91. Epub 2015 Jan 26.

1] EpiNova DPU, Immuno-Inflammation Therapy Area, GlaxoSmithKline,Medicines Research Centre, Stevenage, Hertfordshire, UK. [2] AstraZeneca, Oncology iMed, Cambridge Science Park, Cambridge, UK (R.J.S. and D.M.W.); Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA (D.J.S.); University of Massachussetts Medical School, Worcester, Massachusetts, USA (P.R.T.).

PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
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http://dx.doi.org/10.1038/nchembio.1735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397581PMC
March 2015

1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain.

ACS Med Chem Lett 2014 Nov 10;5(11):1190-5. Epub 2014 Sep 10.

Epinova Discovery Performance Unit and Molecular Discovery Research, GlaxoSmithKline , Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.

The BRPF (bromodomain and PHD finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. Here, we report the discovery, binding mode, and structure-activity relationship (SAR) of the first potent, selective series of inhibitors of the BRPF1 bromodomain.
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http://dx.doi.org/10.1021/ml5002932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233354PMC
November 2014

[2,3]-Sigmatropic rearrangement of allylic selenimides: strategy for the synthesis of peptides, peptidomimetics, and N-aryl vinyl glycines.

J Org Chem 2014 May 10;79(9):3895-907. Epub 2014 Apr 10.

Department of Chemistry, Imperial College London , South Kensington, London SW7 2AZ, U.K.

The scope of the NCS-mediated amination/[2,3]-sigmatropic rearrangement of enantioenriched allylic selenides has been expanded to provide access to three new product classes. The use of N-protected amino acid amides provides a novel strategy for accessing peptide chains containing unnatural vinyl glycine amino acid residues. Also reported is the use of amino acid esters, allowing the diastereoselective synthesis of N,N-dicarboxymethylamines, a motif found in a number of pharmaceuticals. Furthermore, use of a range of N-aromatic and N-heteroaromatic amines allows the formation of enantioenriched N-arylamino acids, a motif found in a number of synthetically and biologically interesting compounds.
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http://dx.doi.org/10.1021/jo500341eDOI Listing
May 2014

A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response.

Nature 2012 Aug;488(7411):404-8

Epinova DPU, Immuno-Inflammation Therapy Area, GlaxoSmithKline R&D, Medicines Research Centre, Stevenage SG1 2NY, UK.

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.
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http://dx.doi.org/10.1038/nature11262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691848PMC
August 2012

Discovery and optimisation of potent, selective, ethanolamine inhibitors of bacterial phenylalanyl tRNA synthetase.

Bioorg Med Chem Lett 2005 May;15(9):2305-9

GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.

High throughput screening of Staphylococcus aureus phenylalanyl tRNA synthetase (FRS) identified ethanolamine 1 as a sub-micromolar hit. Optimisation studies led to the enantiospecific lead 64, a single-figure nanomolar inhibitor. The inhibitor series shows selectivity with respect to the mammalian enzyme and the potential for broad spectrum bacterial FRS inhibition.
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http://dx.doi.org/10.1016/j.bmcl.2005.03.003DOI Listing
May 2005

Optimisation of aryl substitution leading to potent methionyl tRNA synthetase inhibitors with excellent gram-positive antibacterial activity.

Bioorg Med Chem Lett 2003 Feb;13(4):665-8

GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.

Optimisation of the left-hand-side aryl moiety of a file compound screening hit against Staphylococcus aureus methionyl tRNA synthetase led to the identification of a series of potent nanomolar inhibitors. The best compounds showed excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics.
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http://dx.doi.org/10.1016/s0960-894x(02)01027-2DOI Listing
February 2003

Nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase with potent antibacterial activity against gram-positive pathogens.

J Med Chem 2002 May;45(10):1959-62

Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics. Compound 11 demonstrated in vivo efficacy in an S. aureus rat abscess infection model.
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http://dx.doi.org/10.1021/jm025502xDOI Listing
May 2002