The discovery and initial optimisation of pyrrole-2-carboxamides as inhibitors of p38α MAP kinase

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 13, 2010, Pages 3936-3940

  Down, Kenneth ^a   Bamborough, Paul ^a   Alder, Catherine ^a   Campbell, Amanda ^a   Christopher, John A ^a   Gerelle, Maria ^a   Ludbrook, Steve ^a   Mallett, Dave ^a   Mellor, Geoff ^a   Miller, David D ^a   Pearson, Rosannah ^a   Ray, Keith ^a   Solanke, Yemisi ^a   Somers, Don ^a  

^a GLAXOSMITHKLINE   (United Kingdom)

Author keywords

p38 MAP kinase

Indexed keywords

DIHYDROQUINAZOLINONE; LOSMAPIMOD; MITOGEN ACTIVATED PROTEIN KINASE 14; PAMAPIMOD; PROTEIN SERINE THREONINE KINASE INHIBITOR; UNCLASSIFIED DRUG; VX 702;

ANIMAL EXPERIMENT; ARTICLE; CHEMICAL STRUCTURE; DRUG CLEARANCE; DRUG HALF LIFE; HUMAN; HUMAN TISSUE; HYDROGEN BOND; IC 50; IN VIVO STUDY; LUNG FIBROBLAST; NONHUMAN; RAT; SCREENING; SYNTHESIS;

AMIDES; DOSE-RESPONSE RELATIONSHIP, DRUG; DRUG DISCOVERY; HIGH-THROUGHPUT SCREENING ASSAYS; MOLECULAR STRUCTURE; P38 MITOGEN-ACTIVATED PROTEIN KINASES; PROTEIN KINASE INHIBITORS; PYRROLES; STEREOISOMERISM; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 77954309700     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.05.011     Document Type: Article

References (26)

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16. The pharmacophore was constructed manually from a crystal structure of a non-selective kinase inhibitor (unpublished results) complexed with cyclin-dependent kinase 2. Hydrogen-bonding and aromatic interactions between the inhibitor and conserved residues of the ATP-binding site acids were included in the pharmacophore query. The distance tolerances of the resulting model were set loosely in order to be suitable for multiple protein kinases. Searching was carried out using Catalyst (Accelrys Inc., www.accelrys.com ). All molecules identified from the external suppliers' database in this way were passed through proprietary in silico developability filters before being clustered and examined visually in order to make a final selection.
17. Compounds 1-6 were sourced from Bionet (Key Organics Ltd, www.keyorganics.net ).
18. 2 final in 12.5 mM HEPES buffer as above to a total volume of 6 μl. The reaction was incubated for 120 min at room temperature and then terminated by the addition of stop reagent (3 μl) containing 60 mM EDTA and detection reagents in buffer (40 mM HEPES, pH 7.4, 150 mM NaCl and 0.3% w/v BSA). Detection reagents comprise antiphosphothreonine-71ATF2 monoclonal antibody at 0.35 nM final concentration (Cell Signalling Technology, Beverly Massachusetts, USA) labelled with W-1024 europium chelate (Perkin-Elmer, Turku, Finland) and allophycocyanin-labelled streptavidin at 258 nM final concentration (Prozyme, San Leandro, California, USA). The reaction mixture (9 μl total volume) was further incubated for at least 60 min at room temperature. The degree of phosphorylation of ATF2 was measured using a suitable time-resolved fluorimeter such as a Rubystar (BMG, Aylesbury, Buckinghamshire, UK) or Envision (Perkin-Elmer Ltd, Seer Green, Beaconsfield, UK) as a ratio of specific 665 nm energy transfer signal to reference europium 620 nm signal.
19. Hsp27 phosphorylation in human lung fibroblast cells was measured as described in Ref. 10e.
20. An apo crystal of unphosphorylated human p38α (expressed, purified and crystallized as previously described, Ref. 10b) was soaked with 1 mM 1 for 40 h and cryoprotected as before. X-ray diffraction data were collected in-house on a Rigaku-MSC MicroMax007 rotating anode generator with a MarResearch Mar345 image plate detector mounted on a Mar-dtb. The data were processed and the structure solved as in Ref. 10b. The final R-factor and R-free achieved for the complex were 23.2% and 29.7%, respectively. Coordinates have been deposited in the PDB as entry 3MPT. Figures were produced using Pymol (DeLano, W.L., DeLano Scientific, Palo Alto CA, USA. http://www.pymol.org ).
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23. CYP450 inhibition was carried out as described in Ref. 10d.
24. Pharmacokinetic parameters in male CD rats were determined following intravenous (iv) and oral (po) administration at 1 mg/kg. Compound was administered as a solution in DMSO/PEG200/sterile water (1:7:2 v/v/v), 1 ml/kg (iv) and 4 ml/kg (po). Blood was collected over a 7-h time period. Plasma was prepared following centrifugation and compound extracted from 50 μl plasma using protein precipitation with acetonitrile. Samples were evaporated under nitrogen and re-suspended in 200 μl of 20:80 acetonitrile/water. Analysis was performed using LC-MS/MS on the API365 with a 3 min fast gradient comprising 0.1% formic acid in water and 0.1% formic acid in acetonitrile (mobile phases), 30 μl injection volume, flow rate 0.8 ml/min and Luna C18 column (50 × 2.1 mm, 5 μm). Pharmacokinetic data was generated using a non-compartmental approach.
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