DEFINITION The automated synthesis of a large number of compounds in a short time period using a defined reaction route and a large variety of reactantsThe automated synthesis of a large number of compounds in a short time period using a defined reaction route and a large variety of reactants Normally carried out on small scale using solid phase synthesis and automated synthetic machinesNormally carried out on small scale using solid phase synthesis and automated synthetic machines Parallel synthesis Single product formed in each reaction vesselSingle product formed in each reaction vessel Useful for SAR and drug optimisationUseful for SAR and drug optimisation Synthesis of mixtures Mixtures of compounds formed in each reaction vesselMixtures of compounds formed in each reaction vessel Useful for finding lead compoundsUseful for finding lead compounds

Starting material, reagents and solvent Swelling Linkers SOLID PHASE TECHNIQUES Beads must be able to swell in the solvent used, and remainBeads must be able to swell in the solvent used, and remainstable Most reactions occur in the bead interiorMost reactions occur in the bead interior

Merrifield resin for peptide synthesis (chloromethyl group) Linker Peptide Release from solid support

Linking functional group Wang resin Linker BeadLinker

Wang resin Carboxylicacid Carboxylicacid piperidine deprotection

Each tea bag contains beads and is labelledEach tea bag contains beads and is labelled Separate reactions are carried out on each tea bagSeparate reactions are carried out on each tea bag Combine tea bags for common reactions or work up proceduresCombine tea bags for common reactions or work up procedures A single product is synthesised within each teabagA single product is synthesised within each teabag Different products are formed in different teabagsDifferent products are formed in different teabags Economy of effort - e.g. combining tea bags for workupsEconomy of effort - e.g. combining tea bags for workups Cheap and possible for any labCheap and possible for any lab Manual procedure and is not suitable for producing large quantities of different productsManual procedure and is not suitable for producing large quantities of different products Parallel Synthesis Houghton’s Tea Bag Procedure 22

AUTOMATED SYNTHETIC MACHINES Parallel Synthesis Automated parallel synthesis

ETC Parallel Synthesis Automated parallel synthesis of all 27 tripeptides from 3 amino acids

27 TRIPEPTIDES 27 VIALS Parallel Synthesis Automated parallel synthesis of all 27 tripeptides from 3 amino acids

Synthesis of all possible tripeptides using 3 amino acids 4. Mixed Combinatorial Synthesis The Mix and Split Method

4. Mixed Combinatorial Synthesis The Mix and Split Method

4. Mixed Combinatorial Synthesis The Mix and Split Method

MIX 4. Mixed Combinatorial Synthesis The Mix and Split Method

SPLIT 4. Mixed Combinatorial Synthesis The Mix and Split Method

4. Mixed Combinatorial Synthesis The Mix and Split Method

4. Mixed Combinatorial Synthesis The Mix and Split Method

4. Mixed Combinatorial Synthesis The Mix and Split Method

MIX 4. Mixed Combinatorial Synthesis The Mix and Split Method

SPLIT 4. Mixed Combinatorial Synthesis The Mix and Split Method

4. Mixed Combinatorial Synthesis The Mix and Split Method

4. Mixed Combinatorial Synthesis The Mix and Split Method

4. Mixed Combinatorial Synthesis The Mix and Split Method

No. of Tripeptides Mixed Combinatorial Synthesis The Mix and Split Method

No. of Tripeptides Tripeptides 3 Vials 4. Mixed Combinatorial Synthesis The Mix and Split Method

TEST MIXTURES FOR ACTIVITY 4. Mixed Combinatorial Synthesis The Mix and Split Method

Synthesise each tripeptide and test 4. Mixed Combinatorial Synthesis The Mix and Split Method

5.1 Recursive Deconvolution Method of identifying the active component in a mixtureMethod of identifying the active component in a mixture Quicker than separately synthesising all possible componentsQuicker than separately synthesising all possible components Need to retain samples before each mix and split stageNeed to retain samples before each mix and split stageExample Consider all 27 tripeptides synthesised by the mix and split strategy from glycine, alanine and valine 5. Identification of structures from mixed combinatorial synthesis

Gly AlaVal Mix and Split All possible dipeptides in three vessels Retain a sample from each vessel

Gly AlaVal Mix and Split All possible tripeptides in three vessels

5. Identification of structures from mixed combinatorial synthesis Mixture Inactive Mixture Inactive Mixture Active 9 Possible tripeptides in active mixture9 Possible tripeptides in active mixture All end in valineAll end in valine Add valine to the three retained dipeptide mixturesAdd valine to the three retained dipeptide mixtures 5.1 Recursive Deconvolution

Active component narrowed down to one of three possible tripeptidesActive component narrowed down to one of three possible tripeptides Synthesise each tripeptide and testSynthesise each tripeptide and test 5. Identification of structures from mixed combinatorial synthesis 5.1 Recursive Deconvolution

Lysine Tryptophan 5.2 Tagging SCAL = Safety CAtch Linker 5. Identification of structures from mixed combinatorial synthesis

5.2 Tagging Example

NHXNHXNHXNHXNHX NHXNHXNHXNHXNHXNHX NHXNHXNHXNHXNHXNHX 6. Identification of structures from combinatorial synthesis 6.2 Photolithography - example LIGHT NHXNHXNHXNHX NHXNHXNHXNHXNHXNHX NHX CO 2 H coupling NHX NHX NHX NHXNHX NHX NHX NHXNHXNH 2 NH 2 NH 2 NHXNHXNHXNH 2 NH 2 NHX NHXNHXNHXNHXNHXNHX Deprotection

YY Y 6. Identification of structures from combinatorial synthesis 6.2 Photolithography - example Y amino acids OMe OMe O O 2 N O X= Nitroveratryloxycarbonyl fluorescent tag Target receptor Y

7. Combinatorial synthesis Heterocyclic synthesis - 1,4-benzodiazepines Drawback: Final product must contain X= OH or CO 2 H

7. Combinatorial synthesis Heterocyclic synthesis - improved synthesis of benzodiazepines Functional group released from the resin takes part in the final cyclisationFunctional group released from the resin takes part in the final cyclisation Does not remain as an extra, possibly redundant groupDoes not remain as an extra, possibly redundant group

8. Planning a Combinatorial Synthesis 8.1 Aims To generate a large number of compoundsTo generate a large number of compounds To generate a diverse range of compoundsTo generate a diverse range of compounds Increase chances of finding a lead compound to fit a binding siteIncrease chances of finding a lead compound to fit a binding site Synthesis based on producing a molecular core or scaffold with functionality attachedSynthesis based on producing a molecular core or scaffold with functionality attached

8. Planning a Combinatorial Syntheses 8.1 Aims Target molecules should obey Lipinski’s ‘Rule of Five’ for oral activity a molecular weight less than 500a molecular weight less than 500 a calculated log P value less than +5a calculated log P value less than +5 no more than 5 H-bond donating groupsno more than 5 H-bond donating groups no more than 10 H-bond accepting groupsno more than 10 H-bond accepting groups

8.2 Scaffolds Molecular weight of scaffold should be low to allow variation of functionality, without getting products with a MWt > Planning a Combinatorial Syntheses ‘Spider’ scaffolds preferable for exploring conformational space‘Spider’ scaffolds preferable for exploring conformational space Allows variation of functional groups around whole molecule to increase chances of finding suitable binding interactionsAllows variation of functional groups around whole molecule to increase chances of finding suitable binding interactions

8.2 Scaffolds Tadpole scaffolds - variation restricted to a specific region round the molecule - less chance of favourable interactions with a binding site Privileged scaffolds - scaffolds which are common in medicinal chemistry and which are associated with a diverse range of activities - benzodiazepines, hydantoins, benzenesulphonamide etc 8. Planning a Combinatorial Syntheses

8.2 Scaffolds - examples Benzodiazepines Hydantoins  -Lactams Pyridines Good scaffoldsGood scaffolds Spider likeSpider like Low molecular weightLow molecular weight Variety of synthetic routes availableVariety of synthetic routes available Dipeptides 8. Planning a Combinatorial Syntheses

8.2 Scaffolds - poor examples Glucose Spider like and small molecular weight - good points But multiple OH groups Difficult to vary R 1 -R 5 independently Steroid M.Wt. relatively high Restricts no. of functional groups to keep MWt.< 500 Relatively few positions where substituents easily added Indole Tadpole like scaffold Restricted region of variability 8. Planning a Combinatorial Syntheses

Example - Ligands for carbonic anhydrase 9. Dynamic combinatorial chemistry Reaction - reversible formation of iminesReaction - reversible formation of imines Reaction carried out in presence of carbonic anhydraseReaction carried out in presence of carbonic anhydrase Three aldehydes and four amines present as building blocksThree aldehydes and four amines present as building blocks Sodium cyanoborohydride added to ‘freeze’ the mixtureSodium cyanoborohydride added to ‘freeze’ the mixture Products quantified and identifiedProducts quantified and identified Experiment repeated in absence of target to identify amplified product(s)Experiment repeated in absence of target to identify amplified product(s) Amplified product is not necessarily present in greatest amountsAmplified product is not necessarily present in greatest amounts

Example - Ligands for carbonic anhydrase 9. Dynamic combinatorial chemistry Building blocksBuilding blocks Amplified productAmplified product