The design and synthesis of thiophene based phosphodiesterase 4 inhibitors
Creators: name: Neale, Diana Shaye
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: PDE4 inhibitors have been identified as therapeutic targets in a variety of conditions, particularly inflammatory diseases. Roflumilast (Daxas®, Nycomed) currently on the market in Europe and the USA for the treatment of COPD, is the most advanced of the PDE4 inhibitors, with other clinical candidates unable to show efficacy free from side effects. There has been recent interest in the development of novel chemotypes that may exhibit an improved therapeutic window. In addition, numerous structures have been obtained of PDE4-ligand co-crystals, providing the opportunity for structure-based optimisation. We serendipitously identified a novel class of PDE4 inhibitor, while screening for GPCR-ligands using a Cre-Luc reporter assay. This new class is centred around an amino tetrahydrobenzothiophene scaffold. This thesis explores the use of molecular docking for the design of PDE4 inhibitors, the synthesis of a small library of compounds and their pharmacological activity, and the generation of optically active amine compounds. A general molecular modelling study was carried out taking advantage of the large number of PDE4 and ligand co-crystal structures present in the PDB. Three different modelling methods were explored including molecular docking (Glide), shape similarity (ROCS) and pharmacophore modelling (Phase). The different techniques were evaluated for their ability to reproduce experimentally determined binding modes of 25 PDE4 inhibitors, identified by X-ray crystallography. Docking was found to correctly predict the experimental binding mode in 59% of cases. These results were subsequently published. As docking presented the best results of the modelling procedures, it was therefore used in the modelling experiments aiding in the design of PDE4 inhibitors in our study. Differing urea and amide moieties at the 6- and 2-position, respectively, of the amino tetrahydrobenzothiophene scaffold were explored using Glide in support of selection of candidates to synthesise and subsequently test in biological assays. A library of compounds to probe the PDE4 binding site was synthesised based on the lead compound, SVPTH764 (Nankervis et al), and the molecular docking studies. We endeavoured to determine the molecular features contributing to potency at PDE4. A range of inhibitors were explored around the amino tetrahydrobenzothiophene scaffold including 6-carbamates, 6-ureas, 6-amides and 2-amides. The activity of novel compounds at PDE4D displayed improved potencies to the lead compound (IC50 ~ 100 nM) including five compounds with IC50 values below 100 nM. A substituent in the 6-position was essential for activity and compounds containing an ethyl urea moiety exhibited consistently high potencies, while ethyl carbamate substitutions were also tolerated. The addition of substituted aromatic urea moieties in this position resulted in a loss of activity. Derivatisation with 6-amides maintained some activity and the addition of a tetrazole amide group to introduce polarity afforded potent activity at PDE4D. Aromatic amides substituted at the 2-position possessing smaller volumes than a phenyl or 2-thiophene moiety displayed increased activity which was in agreement with the modelling studies. When evaluated across all human PDE enzyme families, a number of compounds demonstrated selectivity for PDE4. An X-ray co-crystal structure of the catalytic domain of PDE4D2 in complex with compound 21 (IC50 = 28 nM) was acquired to a resolution of 1.98 Å. The ligand was bound in the nucleotide binding pocket possessing a similar binding mode to the previously published crystal structures (PDB codes 3SL4, 3SL5, 3SL6, 3SL8). The 6-urea group was located at the edge of the binding site exposed to the aqueous environment, while the thiophene acetamide group was buried deeply in the active site. The carbonyl of the ester group was involved in an interaction with the purine-scanning Gln369 confirming our SAR results and docking outcomes. The carbonyl of the thiophene acetamide moiety formed water mediated hydrogen bonds with the Tyr159 and Asp318 residues. Following the favourable pharmacological activity of the racemic PDE4 inhibitors, generating the optically active amines was undertaken. The main drive was determining if one enantiomer of a compound could be exploited for PDE4 subtype selectivity, and ascertaining any differences in activity between the two forms of the compound. Chiral resolution was investigated using chiral HPLC methods. Diastereoselective reductive amination using a chiral auxiliary ((R)-(+)-tert-butanesulfinamide) was also explored. The pharmacological evaluation of stereoisomers for two compounds at PDE4D exhibited similar results to the racemate. The research described in this thesis has effectively addressed all the aims by building up an SAR between amino tetrahydrobenzothiophenes and the PDE4 enzyme with the design of compounds assisted in part, by molecular modelling. The overall objective of seeking PDE4 subtype selective compounds may provide insight into the side effects associated with existing PDE4 inhibitors.