Combinatorial Chemistry and High-Throughput Screening

1. Combinatorial Chemistry and High-Throughput Screening

2. Combinatorial Chemistry • Used extensively in relation with drug discovery • Principle of Combinatorial Chemistry • Generation of Compound Libraries from Molecular Building blocks

3. Combinatorial Chemistry • Establishment of Libraries • Unbiased libraries • Typically a common chemical core (starting point scaffold) • Large number og building blocks • Generating ”lead” structures • Directed libraries • Again a common chemical core • Limited number of building blocks • Directed towards a specific target • Used to optimize ”lead” structures

4. Solid Phase Synthesis • Product is Linked to a Solid Support • Easy removal of excess reagents through filtration • Dendrimer and poly ethylene glycol resins has been developed to improve the yield

5. Solution Phase Synthesis • Reaction proceeds in Solution • Faster validation times relative to solid phase synthesis • Standard analytical protocols can be used to characterize products between each reaction step

6. Advantages/Disadvantages • Solid phase synthesis + Easy purification • Low yield, Tagged at the point of attachment, Dificult to apply standard characterization methods on intermediates • Solution phase synthesis + Easy characterization of intermediates as well as end pruduct, No limitations in attachment point. - Difficult to drive the reaction towards the product, extensive purification is needed

7. Solution to Disadvantages • Polymer-supported reagents and scavangers • Hybrid between solid and solution phase synthesis • Reagents and scavangers are brougth to the reaction on solid supports

8. Preparation of Libraries • Parallel Synthesis • Each compund is prepared in a specific vessel • Array of reaction vessels • Automated control of reactions

9. Preparation of Libraries • Pool/Split Synthesis • Beads are split into different vessels • Then reacted, shuffled, and split again. • 1000 compund library prepared from 10 building blocks in each step  30 reaction steps. (1110 steps for parallel synthesis)

10. Keeping Track of the Reactions • Radio Frequency (RF) tagging • Transponder tags incase in porous glass beads with a loading capacity of 30-300 mg of resin beads • Nano tagging • One reacent development in the labeling of beads is the nano-reactors these are labled with 2D-barcodes making it possible to keep track of libraries with up to 100,000

11. Advantages/Disadvantages • Parallel synthesis + Easy to keep track of each compund, High yield - Large libraries takes manny reaction steps • Pool/Split + Large libraries are prepared through a limited number of reaction steps - Labelign are required to keep track of each compound

12. Extraction Techniques for Purification • Liquid-Liquid extraction • Extensivley used for solution-phase combinatorial synthesis. • Automated by frezing liquid phase. • Fails when; • Emulsions form • The impurities have the same solubility properties

13. ExtractionTechniquesforPurification • Fluorous phase technique • Attach a insoluble perfluorinated moiety to the compound. • Retain the molecules from fluorous solvent. • Solid-phase extraction • Based on adsorption to a suitable surfacesurface. • Impurities are washed away with a solvent where in the compound are insoluble.

14. Library Formats • Combinatorial Libraries vary in size, amount, purity and structual complexity • The libraries can be devided into 3 groups • 1: One-bead one-compound • 2: Preencoded libraries • 3: Spatially addressable libraries

15. Library Formats

16. Drug Discovery • 1991-2003; ~2500 libraries • ”Unbiased libraries”; 1-2 million compounds • Screening does not always result in hits. • ”Directed libraries” build on a privileged structure” • Libraries based on a modelling.

17. Lead Identification • By screening pool/split solid-phase library of 128 000 2-arylindoles (1) split split into 320 pools of 400 compounds and screened against16 G-protein coupled receptor targets • Some pools both active and selective • Compund 2 higly selective for Natural Killer Cell receptors, therefore viable lead for medical chemitsry

18. Lead Optimization Lead Identification vs. Lead Optimization • Lead identification libraries < 10 000 • Lead optimization libraries 1000-2000 • Lead optimization via focussed libraries based on a privileged structure • Both solution and solid-phase synthesis

19. Lead optimization • Solid phase synthesis with RF tagging • Screening of ~650 000 compounds • 28; active in a human erythropoietin (EPO) assay and have phosphodiesterase 3 activity • 32; treatment of anemia