Combinatorial Chemistry and

High-Throughput Screening

Used extensively in relation with drug discovery

Principle of Combinatorial Chemistry

◦ Generation of Compound Libraries from Molecular Building blocks

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

Combinatorial Chemistry

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

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

Solution Phase Synthesis

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

Advantages/Disadvantages

Polymer-supported reagents and scavangers

◦ Hybrid between solid and solution phase synthesis

◦ Reagents and scavangers are brougth to the reaction on solid supports

Solution to Disadvantages

Parallel Synthesis

◦ Each compund is prepared in a specific vessel

◦ Array of reaction vessels

◦ Automated control of reactions

Preparation of Libraries

Pool/Split Synthesis

◦ Beads are split into differentvessels

◦ 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)

Preparation of Libraries

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

Keeping Track of the Reactions

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

Advantages/Disadvantages

Liquid-Liquid extraction◦ Extensivley used for solution-phase

combinatorial synthesis. ◦ Automated by frezing liquid phase.

Extraction Techniques for Purification

Fails when; ◦ Emulsions form◦ The impurities have the same solubility

properties

Extraction Techniques for Purification

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.

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

Library Formats

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.

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

Lead OptimizationLead 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

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