Pyrazolopyridines as a novel structural class of potent and selective PDE4 inhibitors

Bioorganic and Medicinal Chemistry Letters

Volumn 18, Issue 14, 2008, Pages 4237-4241

  Hamblin, J Nicole ^a   Angell, Tony D R ^a   Ballantine, Stuart P ^a   Cook, Caroline M ^a   Cooper, Anthony W J ^a   Dawson, John ^a   Delves, Christopher J ^a   Jones, Paul S ^a   Lindvall, Mika ^a   Lucas, Fiona S ^a   Mitchell, Charlotte J ^a   Neu, Margarete Y ^a   Ranshaw, Lisa E ^a   Solanke, Yemisi E ^a   Somers, Don O ^a   Wiseman, Joanne O ^a  

^a GLAXOSMITHKLINE   (United Kingdom)

Author keywords

Crystallography; PDE4 inhibitors; Pyrazolopyridine; SAR

Indexed keywords

LIPOPOLYSACCHARIDE; PHOSPHODIESTERASE IV; PHOSPHODIESTERASE IV INHIBITOR; PYRAZOLE DERIVATIVE; PYRAZOLO[3,4 B]PYRIDINE 5 CARBOXAMIDE DERIVATIVE; PYRIDINE DERIVATIVE; ROFLUMILAST; ROLIPRAM; TUMOR NECROSIS FACTOR ALPHA;

ARTICLE; CELL ISOLATION; CRYSTAL STRUCTURE; CYTOKINE PRODUCTION; DRUG CONFORMATION; DRUG POTENCY; DRUG SELECTIVITY; ENZYME ACTIVE SITE; HIGH THROUGHPUT SCREENING; HUMAN; HUMAN CELL; IC 50; PERIPHERAL BLOOD MONONUCLEAR CELL; STRUCTURE ACTIVITY RELATION; X RAY CRYSTALLOGRAPHY;

AMINOPYRIDINES; ANIMALS; BENZAMIDES; CHEMISTRY, PHARMACEUTICAL; CRYSTALLOGRAPHY, X-RAY; CYCLIC NUCLEOTIDE PHOSPHODIESTERASES, TYPE 4; CYCLOPROPANES; DRUG DESIGN; HUMANS; LEUKOCYTES, MONONUCLEAR; MODELS, BIOLOGICAL; MODELS, CHEMICAL; PYRAZOLES; PYRIDINES; PYRROLIDINONES; RATS; ROLIPRAM; TUMOR NECROSIS FACTOR-ALPHA;

RATTUS;

EID: 47149103616     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2008.05.052     Document Type: Article

References (17)

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16. 2 for a further 20 h. The supernatant was then assayed for TNF-α using a commercially available ELISA (Pharmingen).
17. PDE4B2B protein (152-503) was derived from PDE4B2B (152-528) by the action of endogenous protease activity during extraction and purification procedures from insect cells. Purification was performed essentially as described in Rocque et al. (Protein Expr. Purif. 1997, 9, 191) but with some modifications. During cell breakage and extraction of Sf9 insect cells expressing human PDE4B2B (152-528) protease inhibitors were not included to promote cleavage. After clarification by centrifugation and filtration PDE4B2 protein was purified on Q-Sepharose FF and Cibacron Blue Sepharose FF as previously described. The eluate was applied to a 10 ml Q-Sepharose HP column and eluted with a NaCL gradient to separate cleaved from uncleaved protein. The cleaved protein was further purified by size exclusion on a Superdex 200 column (26 × 60) and finally dialysed into 10 mM Hepes, pH 7.0, 20 mM NaCL, 0.1 mM EDTA for crystallography. The site of cleavage was confirmed by LC-MS/MS on the C-terminal peptide from a trypsin digest using an LCQ Deca XP Plus ion trap mass spectrophotometer. Purified protein was concentrated to 16-20 mg/ml in 10 mM Hepes, pH 7.0, 20 mM NaCl, 0.1 mM EDTA. Diffraction quality crystals were obtained using a modified condition to that described by Xu, et al. (Science 2000, 288, 1822). The crystals were grown at 4 °C by vapour diffusion using an equal volume of protein to well solution (14-18% PEG 3000, 10% glycerol, 50 mM sodium cacodylate, pH 6.5, 100 mM sodium acetate, 1 M sodium chloride, 1% DMF and 5 mM DTT). Crystals typically grew up to 0.5 mm in length within 1-4 weeks. The complex structure was obtained from a crystal soaked with 1 mM inhibitor (compound 20a) for approximately 3 days in well solution containing 18% PEG 3000. X-ray diffraction data were collected at 100 K at wavelength 0.933 Å using beamline ID14.4 with an ADSC Quantum 4 CCD detector at the European Synchrotron Radiation Facility. The data were processed with HKL data processing package (Otwinowski. Z., Minor, W. Methods Enzymol. 1997, 276: Macromolecular Crystallography, part A, p. 307) and the structure solved using molecular replacement starting from the native pde4b coordinates (molecule A of PDB entry 1f0j) as the search model in Amore (Navaza J. Acta Cryst. 1994, A50, 157) followed by refinement by REFMAC (Murshudov, et al. J. Acta Cryst. 1997, D53, 240). Three surface cysteines in the crystal structure have been modelled with an associated Arsenic atom as they appear to be chemically modified (at least partially) possibly due to cacodylate in the crystallisation buffer (Maignan et al. JMB 1998, 282, 359). The final R-factor achieved for the complex was 19.9% (R-free = 24.1%) and the coordinates deposited in the PDB as entry 3D3P.