by

j.naresh
m.pharm (chemistry)
 Introduction:
•Combinatorial Chemistry is a new method developed by academics
and researchers to reduce the time and cost of producing effective,
marketable and competitive new drugs.

• Scientists use Combinatorial Chemistry to create large numbers of

molecules that can be detected efficiently.

•This technique has captured the attention of many areas such as

Pharmaceutical chemistry, Biotechnology and Agro chemistry.
Definition:
•Combinatorial chemistry is a technique by which large numbers of
different but structurally similar molecules are produced rapidly and
submitted for pharmacological assay.

•This technique uses the same reaction conditions with the same
reaction vessels to produce a large range of analogues.

• Technique invented in the late 1980s and early 1990s to enable

tasks to be applied to many molecules simultaneously
 Application:
Applications of combinatorial chemistry are very wide
Scientists use combinatorial chemistry to create large
populations of molecules that can be screened efficiently.
By producing larger, more diverse compound libraries,
companies increase the probability that they will find
novel compounds of significant therapeutic and
commercial value.
 Provides a stimulus for robot-controlled and
immobilization strategies that allow high-thrughput and
multiple parallel approaches to drug discovery
Advantages:
Fast
Combinatorial approach can give rise to million of compound in
same time as it will take to produce one compound by
traditional method of synthesis .
Economical
A negative result of mixture saves the effort
of synthesis, purification & identification of each compound
Easy
Isolation purification & identification of active molecule from
combinatorial library is relatively easy.
Drug Discovery
Mixed Combinatorial synthesis produces chemical pool.
Probability of finding a molecule in a random screening
process is proportional to the number of molecules subjected
to the screening process
Drug Optimization
Parallel synthesis produces analogues with slight differences
Disadvantages:
Efficiency is highly affected by compound's size,
solubility and function group.

Compounds produced tend to be Achiral of Racemic

 Combinatorial Chemistry within drug design (
Impact at lead discovery

• traditionally lead drugs were found from

• natural products
• synthetic custom crafted organic molecules made in
small numbers
• analogues of known actives (analogue me-toos)

• High Throughput screening (HTS) requires large numbers of

compounds to fuel the discovery process

• As an alternative to traditional synthesis many compounds rapidly

constructed was needed
 Tools:
1. Solid Phase Techniques
2.1. Advantages
2.2. Requirements
2.3. Examples of Solid Supports (2 slides)
2.4. Anchor or linker
2.4.1. Merrifield resin for peptide synthesis (chloromethyl
group)
2.4.2. Wang resin (2 slides)
2.4.3. Rink resin (2 slides)
2.4.4. Dihydropyran resin (2 slides)
2. Parallel Synthesis
3.1. Houghton’s Tea Bag Procedure
3.2. Automated parallel synthesis (2 slides)
3.3. Automated parallel synthesis of all 27 tripeptides from 3 amino
acids (2 slides)
3. Mixed Combinatorial Synthesis
4. Solution phase synthesis
1. SOLID PHASE TECHNIQUES
• Reactants are bound to a polymeric surface and modified whilst still
attached. Final product is released at the end of the synthesis

Advantages
• Specific reactants can be bound to specific beads
• Beads can be mixed and reacted in the same reaction vessel
• Products formed are distinctive for each bead and physically distinct
• Excess reagents can be used to drive reactions to completion
• Excess reagents and by products are easily removed
• Reaction intermediates are attached to bead and do not need to be isolated
and purified
• Individual beads can be separated to isolate individual products
• Polymeric support can be regenerated and re-used after cleaving the
product
• Automation is possible
 1. SOLID PHASE TECHNIQUES

Requirements
• A resin bead or a functionalised surface to act as a solid support
• An anchor or linker
• A bond linking the substrate to the linker. The bond must be
stable to the reaction conditions used in the synthesis
• A means of cleaving the product from the linker at the end
• Protecting groups for functional groups not involved in the
synthesis
 1. SOLID PHASE TECHNIQUES

Examples of Solid Supports

• Partially cross-linked polystyrene beads hydrophobic in nature
causes problems in peptide synthesis due to peptide folding

• Sheppard’s polyamide resin - more polar

• Tentagel resin - similar environment to ether or THF

• Beads, pins and functionalised glass surfaces

1. SOLID PHASE TECHNIQUES

• Beads must be able to swell in the solvent used, and remain

stable
• Most reactions occur in the bead interior

Swelling Starting material,

Resin bead reagents and solvent
Linkers
1. SOLID PHASE TECHNIQUES
Anchor or linker
• A molecular moiety which is covalently attached to the solid
support, and which contains a reactive functional group
• Allows attachment of the first reactant
• The link must be stable to the reaction conditions in the synthesis
but easily cleaved to release the final compound
• Different linkers are available depending on the functional group to
be attached and the desired functional group on the product
• Resins are named to define the linker e.g.
Merrifield, Wang, Rink
Solid phase synthesis: protecting groups
 A few protecting groups used in solid phase synthesis.

 For amines.
 Boc ( t-butoxycarbonyl )
 Fmoc (9-fluorenylmetoxy carbonyl)

Tmsec (2 [ trimethylsilyl ] ethoxycarbonyl)

 For carboxylic acids.

 Tert Bu ester(t-butyl ester)
 Fm ester(9-fluronyl methyl ester)
 Tmse ester(2 [trimethylsilyl] ethyl)
17
17
ield resin for peptide synthesis (chloromethyl group)
= resin bead

Cl HO2C NHBoc O Deprotection

+ O
R H
NHBoc

Linker R H

HO2C NHBoc

O O
R2 H
O O O
NH2 coupling NH NHBoc
R H R H H
R2

O HF
O
Release from OH
aa1aa2aa3 aan NH 2
solid support
HO2C aa1aa2aa3 aan NH2

Peptide
 2. Parallel Synthesis
Aims:
• To use a standard synthetic route to produce a range of analogues,
with a different analogue in each reaction vessel, tube or well

• The identity of each structure is known

• Useful for producing a range of analogues for SAR or drug

optimisation
2. Parallel Synthesis
2.1 Houghton’s Tea Bag Procedure
• Each tea bag contains beads and is labelled 22

• Separate reactions are carried out on each tea bag

• Combine tea bags for common reactions or work up procedures
• A single product is synthesised within each teabag
• Different products are formed in different teabags
• Economy of effort - e.g. combining tea bags for workups
• Cheap and possible for any lab
• Manual procedure and is not suitable for producing large
quantities of different products
2. Parallel Synthesis
Automated parallel synthesis

Wells

• Automated synthesisers are available with 42, 96 or 144 reaction

vessels or wells
• Use beads or pins for solid phase support
• Reactions and work ups are carried out automatically
• Same synthetic route used for each vessel, but different reagents
• Different product obtained per vessel
3. Parallel Synthesis
Automated parallel synthesis of all 27 tri peptides from 3 amino
acids

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

27 TRIPEPTIDES

27 VIALS
2. Parallel Synthesis
2.2 Automated parallel synthesis

AUTOMATED SYNTHETIC MACHINES

3. Mixed Combinatorial Synthesis
Aims
• To use a standard synthetic route to produce a large variety of
different analogues where each reaction vessel or tube contains a
mixture of products
• The identities of the structures in each vessel are not known with
certainty
• Useful for finding a lead compound
• Capable of synthesising large numbers of compounds quickly
• Each mixture is tested for activity as the mixture
• Inactive mixtures are stored in combinatorial libraries
• Active mixtures are studied further to identify active component
3. Mixed Combinatorial Synthesis
The Mix and Split Method
Example
- Synthesis of all possible dipeptides using 5 amino acids

•Standard methods would involve 25 separate syntheses

Glycine (Gly) 25 separate Gly-Gly Ala-Gly Phe-Gly Val-Gly Ser-Gly

Alanine (Ala) experiments Gly-Ala Ala-Ala Phe-Ala Val-Ala Ser-Ala
Phenylalanine (Phe) Gly-Phe Ala-Phe Phe-Phe Val-Phe Ser-Phe
Valine (Val) Gly-Val Ala-Val Phe-Val Val-Val Ser-Val
Serine (Ser) Gly-Ser Ala-Ser Phe-Ser Val-Ser Ser-Ser

Combinatorial procedure involves five separate syntheses using a

mix and split strategy
 + Gly Gly

+ Ala Ala Ala

Phe
+ Phe Phe combine
Val
+ Val Val
Ser
+ Ser Ser Gly

Split

Ala Ala Ala Ala Ala

Phe Phe Phe Phe Phe
Val Val Val Val Val

Ser Ser Ser Ser Ser

Gly Gly Gly Gly Gly

Gly Ala Phe Val Ser

Ala Gly Ala Ala Ala Phe Ala Val Ala Ser
Phe Gly Phe Ala Phe Phe Phe Val Phe Ser
Val Gly Val Ala Val Phe Val Val Val Ser

Ser Gly Ser Ala Ser Phe Ser Val Ser Ser
Gly Gly Gly Ala Gly Phe Gly Val Gly Ser
3. Mixed Combinatorial Synthesis
The Mix and Split Method
Synthesis of all possible tri peptides using 3 amino acids
3. Mixed Combinatorial Synthesis
The Mix and Split Method
3. Mixed Combinatorial Synthesis
The Mix and Split Method
3. Mixed Combinatorial Synthesis
The Mix and Split Method

MIX
3. Mixed Combinatorial Synthesis
The Mix and Split Method

SPLIT
3. Mixed Combinatorial Synthesis
The Mix and Split Method
3. Mixed Combinatorial Synthesis
The Mix and Split Method
3. Mixed Combinatorial Synthesis
The Mix and Split Method
3. Mixed Combinatorial Synthesis
The Mix and Split Method

MIX
3. Mixed Combinatorial Synthesis
The Mix and Split Method

SPLIT
3. Mixed Combinatorial Synthesis
The Mix and Split Method
3. Mixed Combinatorial Synthesis
The Mix and Split Method
3. Mixed Combinatorial Synthesis
The Mix and Split Method
 3. Mixed Combinatorial Synthesis
The Mix and Split Method

No. of 9 9 9
Tripeptides
 3. Mixed Combinatorial Synthesis
The Mix and Split Method

No. of 9 9 9
Tripeptides

27 Tripeptides
3 Vials
3. Mixed Combinatorial Synthesis
The Mix and Split Method

TEST MIXTURES FOR ACTIVITY

3. Mixed Combinatorial Synthesis
The Mix and Split Method

Synthesise each tripeptide and test

3. Mixed Combinatorial Synthesis
The Mix and Split Method

20 AMINO ACIDS HEXAPEPTIDES

etc.
34 MILLION PRODUCTS
(1,889,568 hexapeptides / vial)
5. Identification of structures from mixed
combinatorial synthesis
5.1 Recursive Deconvolution:
• Method of identifying the active component in a mixture
• Quicker than separately synthesising all possible components
• Need to retain samples before each mix and split stage

Example:
Consider all 27 tripeptides synthesised by the mix and split strategy
from glycine, alanine and valine
 Gly
Gly

Ala
Ala

Val
Val

Mix and Split

Gly Val Gly Val Gly Val

Ala Ala Ala

Gly Ala Val

Gly Gly Gly Ala Gly Val

Ala Gly Ala Ala Ala Val

Val Gly Val Val

Val Ala

All possible di peptides in three vessels

Retain a sample from each vessel
 Gly Gly Gly Gly Gly Gly Gly Gly
Ala Gly
Ala Gly Ala Gly Ala Gly
Val Gly Val Gly Val Gly Val Gly

Gly Ala Gly Ala Gly Ala Gly Ala

Ala Ala
Ala Ala Ala Ala Ala Ala
Val Ala Val Ala Val Ala Val Ala

Gly Val Gly Val Gly Val Gly Val

Ala Val
Ala Val Ala Val Ala Val
Val Val Val Val Val Val Val Val

Mix and Gly Ala Val

Split
Gly Gly Gly Gly Gly Ala Gly Gly Val

Ala Gly Gly Ala Gly Ala Ala Gly Val

Val Gly Gly Val Gly Ala Val Gly Val

Gly Ala Gly Gly Ala Ala Gly Ala Val

Ala Ala Gly Ala Ala Ala Ala Ala Val

Val Ala Gly Val Ala Ala Val Ala Val

Gly Val Gly Gly Val Ala Gly Val Val

Ala Val Gly Ala Val Ala Ala Val Val

Val Val Gly Val Val Ala Val Val Val

All possible tripeptides in three vessels

 5. Identification of structures from mixed
combinatorial synthesis
Recursive Deconvolution
Gly Gly Gly Gly Gly Ala Gly Gly Val

Ala Gly Gly Ala Gly Ala Ala Gly Val

Val Gly Gly Val Gly Ala Val Gly Val

Gly Ala Gly Gly Ala Ala Gly Ala Val

Ala Ala Gly Ala Ala Ala Ala Ala Val

Val Ala Gly Val Ala Ala Val Ala Val

Gly Val Gly Gly Val Ala Gly Val Val

Ala Val Gly Ala Val Ala Ala Val Val

Val Val Gly Val Val Ala Val Val Val

Mixture Mixture Mixture

Inactive Inactive Active

• 9 Possible tripeptides in active mixture

• All end in valine
• Add valine to the three retained dipeptide mixtures
 5. Identification of structures from mixed
combinatorial synthesis
Recursive Deconvolution
Gly Gly Gly Ala Gly Val

Ala Gly Ala Ala Ala Val

Val Gly Val Val

Val Ala

Val Val Val

Gly Ala Val Gly Val Val

Gly Gly Val

Ala Gly Val Ala Ala Val Ala Val Val

Val Gly Val Val Val Val

Val Ala Val

Active

• Active component narrowed down to one of three possible

tripeptides
• Synthesise each tripeptide and test
5. Identification of structures from mixed
combinatorial synthesis
5.2 Tagging:
SCAL = Safety CAtch Linker H
N

H2N H

O NH2 HN

MeOS O

NH H

O Tryptophan
SOMe

HN

Lysine
NH2

NH2
5.2 Tagging
Example NH2
NH2 NH2
RCHBrCO2H amino acid(aa 1) aa1
NH2 HN R'NH2
NH R
Step 1 Tag 1 NH R
Step 2
O Br
O Br

NH2 NH2
NH2 aa2 aa2

aa1 aa1 aa1

HN HN HN
amino acid(aa 2) R"COCl
NH R NH R NH R
Tag 2 Step 3
O NHR' O NHR' O NR'COR"

aa3 NH2
aa2

amino acid(aa 3) aa1

HN

Tag 3 NH R

O NR'COR"
6. Identification of structures LIGHT from
combinatorial synthesis LIGHT

6.2 Photolithography - example

NHX NHX NHX NHX NHX

NHX NHX NHX NHX NHX NHX

MASK 1
Mask
NHX NHX NHX NHX NHX NHX NHX NHX NHX NHX NHX NHX

NHX NHX NHX

NHX NHX NHX

NHX NHX NH2 NH2 NH2 NHX NHX

Deprotection CO2H
NHX NH2 NH2 NHX NHX NHX NHX NHX NHX NHX

coupling
NHX NHX NHX NHX NHX NHX NHX NHX NHX NHX NHX NHX
6. Identification of structures from
combinatorial synthesis
6.2 Photolithography - example Y
Y Y Y

repeat

O O Me
Y fluorescent tag

O 2N OMe
Target receptor
amino acids X= Nitroveratryloxycarbonyl
7. Planning a Combinatorial Synthesis
7.1 Aims
• To generate a large number of compounds
• To generate a diverse range of compounds
• Increase chances of finding a lead compound to fit a binding site
• Synthesis based on producing a molecular core or scaffold with
functionality attached
Centroid
or scaffold

Substituent
'arms' Binding groups
7. Planning a Combinatorial Syntheses
7.1 Aims
Target molecules should obey Lipinski’s ‘Rule of Five’ for oral
activity

• a molecular weight less than 500

• a calculated log P value less than +5
• no more than 5 H-bond donating groups
• no more than 10 H-bond accepting groups
7. Planning a Combinatorial Syntheses
7.2 Scaffolds
• ‘Spider’ scaffolds preferable for exploring conformational space
• Allows variation of functional groups around whole molecule to
increase chances of finding suitable binding interactions
Binding
regions

Screen compound
library
RECEPTOR
BINDING
SITE

Molecular weight of scaffold should be low to allow variation of

functionality, without getting products with a MWt > 500
7. Planning a Combinatorial Syntheses
7.2 Scaffolds
Tadpole scaffolds
- variation restricted to a specific region round the molecule
- less chance of favourable interactions with a binding site

'Spider' Scaffold with 'Tadpole' scaffold with

'dispersed' substituents 'restricted' substituents

Privileged scaffolds
- scaffolds which are common in medicinal chemistry and
which are associated with a diverse range of activities
- benzodiazepines, hydantoins, benzenesulphonamide etc
7. Planning a Combinatorial Syntheses
7.2 Scaffolds - examples
R"
R O Me O
R4
N
O R2 R3 HO2C
N R1 R4
R' R2
O
N
N N N
X Ar R1 R2 O R5 R3
R3 R

Benzodiazepines Hydantoins β -Lactams Pyridines

R3 R4

O
R5
• Good scaffolds
•
C N
R1 O C N
R6
Spider like
O
• Low molecular weight
R2 • Variety of synthetic routes available
Dipeptides
7. Planning a Combinatorial Syntheses
7.2 Scaffolds - poor examples
OR5
Spider like and small molecular weight - good
R4O

R3O
O
OR1
points
OR2 But multiple OH groups
Glucose Difficult to vary R1-R5 independently
Me

Me
M.Wt. relatively high
Restricts no. of functional groups to keep MWt.<
R1CO 500
R2
Relatively few positions where substituents
Steroid easily added
O
R3
Tadpole like scaffold
Restricted region of
H 2N
R2
N

R
variability
Indole
References:
An introduction to Medicinal chemistry,3rd edition,
Graham L.Patric
web.centre.edu/muzyka/articles/ch14slides.ppt
www.authorstream.com/.../hariteja43-1369711-2003-
combinatorial-chemistry.
www.slideshare.net/.../combinatorial-chemistry-
hts-and-its-applications.
