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DEFINITIONDEFINITION
• The automated synthesis of a large number of compounds in a The automated synthesis of a large number of compounds in a short time period using a defined reaction route and a large short time period using a defined reaction route and a large variety of reactantsvariety of reactants
• Normally carried out on small scale using solid phase synthesis Normally carried out on small scale using solid phase synthesis and automated synthetic machinesand automated synthetic machines
Parallel synthesisParallel 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 mixturesSynthesis 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 TECHNIQUESSOLID PHASE TECHNIQUES
• Beads must be able to swell in the solvent used, and remain Beads must be able to swell in the solvent used, and remain stablestable
• Most reactions occur in the bead interiorMost reactions occur in the bead interior
Resin bead
Deprotection
O
aa1aa2aa3
O
aan NH2
Merrifield resin for peptide synthesis (chloromethyl group)Merrifield resin for peptide synthesis (chloromethyl group)
O
O
R
NHBoc
H
O
O
R
NH2
H
HO2C NHBoc
R2H
coupling
O
O
R
NH
H
O
NHBoc
R2H
= resin bead
Cl HO2C NHBoc
R H+
Linker
HF
OH
aa1aa2aa3 aanHO2C NH2
Peptide
Release from solid support
Linking functional groupO
OH
Wang Resin
Wang resinWang resin
Linker
OH
Bead Linker
peptidesynthesis
O Caa1aa2aa3
O
aan NH2
TFAcleavage
OH
aa1aa2aa3 aan NH2HO2C
Fmoc =
O
O
Wang resinWang resin
OH
Carboxylic Carboxylic acidacid
Carboxylic Carboxylic acidacid
HO2C NH(Fmoc)
R H
+ O C NH(Fmoc)
R H
O
O C NH2
R H
O
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 Combine tea bags for common reactions or work up proceduresprocedures
• 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 Manual procedure and is not suitable for producing large quantities of different productsquantities of different products
Parallel SynthesisParallel SynthesisHoughton’s Tea Bag ProcedureHoughton’s Tea Bag Procedure
22
AUTOMATED SYNTHETIC MACHINES
Parallel SynthesisParallel SynthesisAutomated parallel synthesisAutomated parallel synthesis
ETC
Parallel SynthesisParallel SynthesisAutomated parallel synthesis of all 27 tripeptides from 3 amino acidsAutomated parallel synthesis of all 27 tripeptides from 3 amino acids
27 TRIPEPTIDES
27 VIALS
Parallel SynthesisParallel SynthesisAutomated parallel synthesis of all 27 tripeptides from 3 amino acidsAutomated parallel synthesis of all 27 tripeptides from 3 amino acids
Synthesis of all possible tripeptides using 3 amino acidsSynthesis of all possible tripeptides using 3 amino acids
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
MIXMIX
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
SPLITSPLIT
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
MIXMIX
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
SPLITSPLIT
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
No. of Tripeptides
9 9 9
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
No. of Tripeptides
9 9 9
27 Tripeptides 3 Vials
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
TEST MIXTURES FOR ACTIVITYTEST MIXTURES FOR ACTIVITY
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
Synthesise each tripeptide and testSynthesise each tripeptide and test
4. Mixed Combinatorial Synthesis4. Mixed Combinatorial SynthesisThe Mix and Split MethodThe Mix and Split Method
5.1 Recursive Deconvolution5.1 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 stage
Example Example Consider all 27 tripeptides synthesised by the mix and split strategy Consider all 27 tripeptides synthesised by the mix and split strategy from glycine, alanine and valinefrom glycine, alanine and valine
5. Identification of structures from mixed 5. Identification of structures from mixed combinatorial synthesiscombinatorial synthesis
Gly
Ala
Val
Gly
Ala
Val
Gly
AlaVal Gly
AlaVal Gly
AlaVal
Gly Ala Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Mix and Split
All possible dipeptides in three vesselsAll possible dipeptides in three vesselsRetain a sample from each vesselRetain a sample from each vessel
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly Ala Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Val
Val
Val
Val
Val
Val
Val
Val
Val
Mix andSplit
All possible tripeptides in three vesselsAll possible tripeptides in three vessels
5. Identification of structures from mixed 5. Identification of structures from mixed combinatorial synthesiscombinatorial synthesis
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Ala
Val
Gly
Gly
Gly
Gly
Ala
Val
Ala
Ala
Ala
Gly
Ala
Val
Val
Val
Val
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Val
Val
Val
Val
Val
Val
Val
Val
Val
MixtureInactive
MixtureInactive
MixtureActive
• 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 Deconvolution5.1 Recursive Deconvolution
Gly
Ala
Val
Gly
Ala
Val
Gly
Ala
Val
Gly
Gly
Gly
Ala
Ala
Ala
Val
Val
Val
Val Val Val
Gly
Ala
Val
Gly
Ala
Val
Gly
Ala
Val
Gly
Gly
Gly
Ala
Ala
Ala
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Active
• Active component narrowed down to one of three possible Active component narrowed down to one of three possible tripeptidestripeptides
• Synthesise each tripeptide and testSynthesise each tripeptide and test
5. Identification of structures from mixed 5. Identification of structures from mixed combinatorial synthesiscombinatorial synthesis5.1 Recursive Deconvolution5.1 Recursive Deconvolution
NH
O NH2
HN
O
O
HN
MeOS
SOMe
O
H2N
HN
H
H
Lysine
Tryptophan
NH2
NH2
5.2 Tagging5.2 Tagging
SCAL = Safety CAtch LinkerSCAL = Safety CAtch Linker
5. Identification of structures from mixed 5. Identification of structures from mixed combinatorial synthesiscombinatorial synthesis
NH2
NH2
RCHBrCO2H
Step 1
NH2
NH
O
R
Br
amino acid(aa 1)
Tag 1
HN
NH
O
R
Br
aa1
NH2
R'NH2
Step 2
HN
NH
O
R
NHR'
aa1
NH2
amino acid(aa 2)
Tag 2
HN
NH
O
R
NHR'
aa1
aa2
NH2
R"COCl
Step 3
HN
NH
O
R
NR'COR"
aa1
aa2
NH2
amino acid(aa 3)
Tag 3
HN
NH
O
R
NR'COR"
aa1
aa2
aa3 NH2
5.2 Tagging5.2 Tagging
ExampleExample
NHX NHX NHX NHX NHX
NHXNHXNHXNHXNHXNHX
NHXNHXNHXNHXNHXNHX
6. Identification of structures from combinatorial 6. Identification of structures from combinatorial synthesissynthesis6.2 Photolithography - example6.2 Photolithography - example
NHXNHXNHXNHXNHXNHX
MASK 1
Mask
LIGHT
LIGHT
NHXNHXNHXNHX
NHXNHXNHXNHXNHXNHX
NHX
CO2H
coupling
NHX NHX
NHX NHX NHX
NHX NHXNHX NHX NH2 NH2 NH2
NHXNHXNHXNH2NH2NHX
NHXNHXNHXNHXNHXNHX
Deprotection
Y Y Y
repeat
6. Identification of structures from combinatorial 6. Identification of structures from combinatorial synthesissynthesis6.2 Photolithography - example6.2 Photolithography - example
Y
amino acids
OMe
O MeO
O2N
O
X= Nitroveratryloxycarbonyl
fluorescent tag
Target receptor
Y
7. Combinatorial synthesis7. Combinatorial synthesisHeterocyclic synthesis - 1,4-benzodiazepinesHeterocyclic synthesis - 1,4-benzodiazepines
O
Ar
NHFmocR
X
X=OH or CO2HFmoc=protecting group
NHFmoc
R'
O
F
1. Base2. R"I
Alkylation
TFA/H2O/Me2S
Cleavage
R
X
N
N
Ar
R'
OR"
Tentagelresin O
Ar
NHFmocR
Piperidine
deprotectionO
Ar
NH2
R
O
Ar
NHR
ONHFmoc
R'
Piperidine
deprotection O
Ar
NHR
ONH2
R'
AcOH
Cyclisation
R
N
N
Ar
R'
OH
R
N
N
Ar
R'
OR"
Drawback:Final product must contain X= OH or CO2H
7. Combinatorial synthesis7. Combinatorial synthesisHeterocyclic synthesis - improved synthesis of benzodiazepinesHeterocyclic synthesis - improved synthesis of benzodiazepines
• Functional group released from the resin takes part in the final Functional group released from the resin takes part in the final cyclisationcyclisation
• Does not remain as an extra, possibly redundant groupDoes not remain as an extra, possibly redundant group
NH
R3
R4
R2
NH
Imines
+
O
OR1
NH2
Amino acid
Cl(CH2)2Cl
O
OR1
N
R2
NHR4
R3
Adducts
TFA
N
N
R2
R1
OR4
R3
8. Planning a Combinatorial Synthesis8. Planning a Combinatorial Synthesis8.1 Aims8.1 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 Increase chances of finding a lead compound to fit a binding sitesite
• Synthesis based on producing a molecular core or scaffold with Synthesis based on producing a molecular core or scaffold with functionality attachedfunctionality attached
Centroid or scaffold
Substituent'arms' Binding groups
8. Planning a Combinatorial Syntheses8. Planning a Combinatorial Syntheses8.1 Aims8.1 Aims
Target molecules should obey Lipinski’s ‘Rule of Five’ for oral Target molecules should obey Lipinski’s ‘Rule of Five’ for oral activityactivity
• a molecular weight less than 500a molecular weight less than 500• a calculated log a calculated log PP value less than +5 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 Scaffolds8.2 Scaffolds
Molecular weight of scaffold should be low to allow variation of Molecular weight of scaffold should be low to allow variation of functionality, without getting products with a MWt > 500functionality, without getting products with a MWt > 500
8. Planning a Combinatorial Syntheses8. Planning a Combinatorial Syntheses
• ‘‘Spider’ scaffolds preferable for exploring conformational Spider’ scaffolds preferable for exploring conformational spacespace
• Allows variation of functional groups around whole molecule Allows variation of functional groups around whole molecule to increase chances of finding suitable binding interactionsto increase chances of finding suitable binding interactions
RECEPTORBINDINGSITE
Binding regions
Screen compoundlibrary
8.2 Scaffolds8.2 ScaffoldsTadpole scaffoldsTadpole scaffolds
- variation restricted to a specific region round the molecule- variation restricted to a specific region round the molecule- less chance of favourable interactions with a binding site- less chance of favourable interactions with a binding site
Privileged scaffolds Privileged scaffolds - scaffolds which are common in medicinal chemistry and - scaffolds which are common in medicinal chemistry and which are associated with a diverse range of activitieswhich are associated with a diverse range of activities- benzodiazepines, hydantoins, benzenesulphonamide etc - benzodiazepines, hydantoins, benzenesulphonamide etc
8. Planning a Combinatorial Syntheses8. Planning a Combinatorial Syntheses
'Spider' Scaffold with 'dispersed' substituents
'Tadpole' scaffold with 'restricted' substituents
8.2 Scaffolds - examples8.2 Scaffolds - examples
BenzodiazepinesBenzodiazepines
R
X
N
N
Ar
R'
O
R"
HydantoinsHydantoins
N
N
O
O
R4
R3R1 R2
-Lactams-Lactams
N
O R5
R4R3R1
R2
PyridinesPyridines
N
R
HO2C
Me
R2
O
R3
• Good scaffoldsGood scaffolds• Spider likeSpider like• Low molecular weightLow molecular weight• Variety of synthetic routes availableVariety of synthetic routes available
DipeptidesDipeptides
O
R2
C
O
CN
N
O
R4
R1
R3
R5
R6
8. Planning a Combinatorial Syntheses8. Planning a Combinatorial Syntheses
8.2 Scaffolds - poor examples8.2 Scaffolds - poor examples
O
OR2
OR1
OR5
R4O
R3O
GlucoseGlucose
Spider like and small molecular weight - good pointsSpider like and small molecular weight - good pointsBut multiple OH groupsBut multiple OH groupsDifficult to vary RDifficult to vary R11-R-R55 independently independently
R1CO
Me
Me
R2
SteroidSteroid
M.Wt. relatively highM.Wt. relatively highRestricts no. of functional groups to keep MWt.< 500Restricts no. of functional groups to keep MWt.< 500Relatively few positions where substituents easily Relatively few positions where substituents easily addedadded
H2N
O
N
R3
R2
R
IndoleIndole
Tadpole like scaffoldTadpole like scaffoldRestricted region of Restricted region of variabilityvariability
8. Planning a Combinatorial Syntheses8. Planning a Combinatorial Syntheses
Example - Ligands for carbonic anhydraseExample - Ligands for carbonic anhydrase9. Dynamic combinatorial chemistry9. Dynamic combinatorial chemistry
• Reaction - reversible formation of iminesReaction - reversible formation of imines
C OH
R+ H2N R'
Aldehyde Primary amine
C NH
R
R'
Imine
• 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 mixture Sodium cyanoborohydride added to ‘freeze’ the mixture
C NH
R
R'Imine
NaCNBH3HC NHH
R
R'Secondary amine
• Products quantified and identifiedProducts quantified and identified• Experiment repeated in absence of target to identify amplified Experiment repeated in absence of target to identify amplified
product(s)product(s)• Amplified product is not necessarily present in greatest Amplified product is not necessarily present in greatest
amountsamounts
Example - Ligands for carbonic anhydraseExample - Ligands for carbonic anhydrase9. Dynamic combinatorial chemistry9. Dynamic combinatorial chemistry
• Building blocks Building blocks
OCHOHO3S
CHO
HO2C
CHOS
Aldehydes
O
O
H2N
H2NNH2
O
H2NNH
O
HO2CH2N
H2NNH
OO
Amines
• Amplified productAmplified product
SNO
O
H2N
Active compound
NaBH3CN
SHNO
O
H2N
Derived secondary amine