COMBINATORIAL CHEMISTRY :

IN THE DRUG DESIGNING PROCESS

Vaishali Gupta (1311056, SCS)

Shweta Rai (1311043, SCS)

Sapuru Vinay Kumar (1311037, SBS)

What is Combinatorial
Chemistry ?
Combinatorial chemistry is a new technique developed in pharmaceutical
industry, which involves synthesis of compounds in mass, which are
screened as a whole mixture for particular biological activity.
It has reduced the time and cost to produce effective and marketable new
drugs.

Combinatorial Libraries
Collection of structurally related compounds (peptides, oligonucleotides,
oligosaccharides, organic molecules) obtainable by chemical or biological
means simultaneously as a mixture and screened for activity as a mixture of
compounds.
Size : Depends on the number of building blocks used per reaction and the
number of steps where a new building block is introduced.

Combinatorial libraries can be structurally related by :

A central core (scaffold), or

e.g. Benzodiazepines aminobenzophenones,
amino acids, alkyl groups
A common backbone
e.g. Peptides amino acids

Combinatorial Chemistry Workflow

Evaluate
and refine
library

Target
compounds
for synthesis

Determine
synthetic
routes
Determine
available
reagents

Identify
active
compounds
Screen and
collect
bioassay data

Build library

Select/store
building
blocks

Parallel Synthesis
Each synthetic reaction is
started in a different reaction
vessel and all the necessary
operations are executed in
parallel.
Solid support as well as solution
chemistry is possible.

Parallel Synthesis
Advantages :
Each compound is substantially pure in its location.
Defined location provides the structure of a certain compounds.
Easier biological evaluation
Disadvantages :
Applicable only for medium libraries.

Split and Pool Synthesis

Carried out on beads made of a polymeric
material.
After each synthesis, beads are combined,
washed, de-protected and then split into
batches for further synthesis.
Library size increases exponentially.

Split and Pool Synthesis

Advantages :
Only few reaction vessels required.
Method of choice for large libraries

Disadvantages :
More amount of resin beads required.
Only little amounts of synthetic compounds available.

Screening
Identification of biologically active compound among all the
compounds synthesized.
Virtual Screening : Using computers to predict whether a
compound will show the desired activity based on the 2-D and
3-D structures.
High Throughput Screening (HTS)

High Throughput Screening

Finding an active compound against a chosen target using
robotics, data processing and control software, liquid handling
devices and sensitive detectors.
Millions of tests can be conducted very quickly.
Results of these experiments provide leads for the drug design.

Biological methods for Library generation

Mainly limited to peptide and oligonucleotide library.
For peptide libraries, methods are based on the construction of
clones each one expressing a different peptide on its surface.
The peptides are fused to proteins normally expressed on the
surface of microorganism used.
Phage display libraries are most commonly used.

Screening of the Phage display libraries

Amplification is
infection of E.Coli.

done

by

This process increases both the

number of active phages and
stringency of selection.

Combinatorial Chemistry:
Synthetic methods

Shweta Rai
School of Chemical Sciences
NISER
1302343

Outline
Methods in combinatorial libraries :
Solid phase synthesis
Parallel synthesis
Mix & split technique
Tea bag synthesis
Multi-pin synthesis
Light-directed synthesis on silica wafers (based on semiconductor
photolithography
Solution phase synthesis
Encoding and Decoding of combinatorial libraries

Solid Phase Synthesis

In this method, the reaction is carried out on a solid support such as resin
beads.
The solid phase synthesis of peptides was pioneered by Merrifield-synthesis
of peptides.
The bead is treated with different starting materials which bound together.
Then it is mixed with another reagent to get the product which is bound to
solid support.

The excess reagent or by product can be easily removed by washing with

appropriate solvent.

Schematic Representation Of the Resin Bead

Solid Support and Linkers

Polystyrene resins, Tentagel resins, Pepsin, PEGA, Glass and ceramics.

The linker is the molecule that sits between the compound and the support.
The linker moves the point of the attachment of the substrate away from the surface of the bead.
Different linkers are used depending on whether the functional group is present on the substrate, or the
functional group is desired on the final product once it is released.
Types of linkers:
Wang Resin : Linker suitable for release of carboxylic acids.
Merrifield Resin : Linker suitable for peptide products.
Rink Resin : Linker suitable for attachment & release of carboxamide.

Protecting Group
Protecting groups are important for the blocking & regenerating
certain functional groups in a reaction sequence.
Some examples of protecting groups are :
FMOC ( Fluoro methoxy carbonyl)
TBOC ( Tertiary butyloxy carbonyl)

Merifield Synthesis Of Peptides

Tea Bag Synthesis

Tea bag is a type of reaction vessels consisting of porous mesh bag that
encloses resin & it allows passage of reagents & solvents.
Several such teabags are immersed in a given reagent & then manipulated
one reagent to next to generate combinatorial libraries.
Advantages:
Greater Quality of each compounds is available at once (structural
characterization )
Labeling of the tea bag : Easier identification of each compound.

Multi-Pin Synthesis
The reaction vessels consists of brush like arrays of pins, at the end of its
consists of bead(lollypop) with suitable linker. Here the synthesis takes place.
It is inserted in to the plates where the reagents & the solvents kept

Light-Directed Synthesis on Silica

Wafers (Based on Semiconductor
Photolithography)
Each set of building blocks contains a photo
labile protecting group.
Only the building blocks which have exposed to
light can be coupled with another building block.

Pattern of masks and the sequence of protecting

define the final structure of the compound
synthesized.
Each member of the library is synthesized at a
specific location on the functionalized silica
wafer.
Libraries : 50000 compounds located in a 50
micrometer square site.

Solution Phase Combinatorial Chemistry

It is the modified reaction to accommodate a solid support reaction.
Solution phase combinatorial chemistry often leads to formation of mixture
of products.
Problems:
Difficulty of removing unwanted material.
Purification at each step is necessary
Other practical problems.

Encoding
Encoding should provide a fast and simple way to identify the structure of all
library members
Classification :
Positional Encoding: In this method the re-synthesis and rescreening is
carried out to know the identity of the active compound.
Chemical encoding: Every reaction used in the library synthesis is recorded
on the solid support by the chemical attachment of a tag.
Electronic encoding: Radio frequency memory chip attached to the solid
support records and emits coded information.

Encoding in Split-pool Synthesis

Decoding
Every bead has tags that provide information, once cleaved, about
the chemical history of that bead

Combinatorial Chemistry:
Applications in drug discovery

By : Vaishali Gupta (SCS, 1311056)

S. Vinay Kumar (SBS, 1311037)
Shweta Rai (SCS, 1311042)

Will combinatorial chemistry deliver real

medicines ?
Chemical part of the drug discovery process cannot stand alone; the integration of synthesis and
biological assays is fundamental to the combinatorial approach.

Because of the long time it takes to develop pharmaceutical drugs there are few examples of
marketed drugs discovered by combinatorial methods.
Whether combinatorial approaches can also be adapted to deal with all the other requirements of
a successful pharmaceutical (lack of toxicity, bioavailability etc.) is open to question but there are
already examples of potential structures obtained from it.

Lets be optimistic !

Applications in drug discovery

Examples of applications of combinatorial chemistry in lead optimization and drug discovery:
Synthesis of peptoids
Generation of a benzodiazepine library
Lead optimization of histamine H3 receptor antagonist.

Peptoids
Poly-N-substituted glycines, are a class of peptide mimetics whose side chains are appended to the
nitrogen atom of the peptide backbone, rather than to the -carbons.
Conformationally unstable, due to the flexibility of the main-chain methylene groups and the
absence of stabilizing hydrogen bond interactions along the backbone.
With appropriate side chains it is possible to form specific steric or electronic interactions that
favour the formation of stable secondary structures

Why peptoids ?
Completely resistant to proteolysis.
Since secondary structure in peptoids does not involve hydrogen bonding, it is not
typically denatured by solvent, temperature, or chemical denaturants such as urea.
Many useful polypeptides sorted from polypeptide libraries have low bioavailability and
unfavourable pharmacokinetic properties.
Therefore focus has shifted to synthetic peptide-mimetics like peptoides.

Synthesis of peptoids
N-substituted glycine units as monomers. (Similar to natural -amino acid side chain.)
Polymerization reaction
Full monomer oligomer synthesis
Sub monomer oligomer synthesis

Functions established for specific peptoids

The first demonstration of the use of peptoids was in screening a combinatorial library of diverse
peptoids which yielded novel high-affinity ligands for transmembrane G-protein-couple receptors.
Inhibitor of -amylase.
Inhibitor of hepatitis A virus 3C protease.

Antagonists to 1 adrenergic receptor.

Some bind to HIV RNA.

Construction of benzodiazepine library

Created in the early 1990s by Ellman and colleagues.

Become a classic contribution in library construction.

Why was such a library necessary ?
Small organic molecules can be powerful tools for impacting biology and medicine, functioning as both
therapeutics and as probes that help to illuminate the macromolecules regulating biological processes.

Prepared a total of 192 members

with different points of diversity,
including:
Amide
Acid
Amine
Phenol and
Indole functionalities

by combining :
2-aminobenzophenones
Amino acids, and
Alkylating agents.
Points of
diversities

Histamine H3 receptors
Expressed in the CNS and to a lesser extent the PNS, acts as autoreceptors in presynaptic histaminergic neurons, and also controls histamine turnover by feedback inhibition of
histamine synthesis and release.
Is a G-protein coupled receptor. The H3 receptor is coupled to the Gi G-protein, so it leads to
inhibition of the formation of cAMP and hence consequently reduces the release of neurotransmitters down the pathway.
NOTE :

An auto-receptor is a type of receptor located in the membranes of presynaptic nerve cells. It serves
as part of a negative feedback loop in signal transduction. It is only sensitive to
the neurotransmitters or hormones released by the neuron on which the auto-receptor sits.
Gi alpha subunit is a heterotrimeric G protein subunit that inhibits the production of cAMP from ATP.

Combinatorial lead optimization of Histamine

H3 receptor antagonist
Blockade of this receptors leads to increased level
of histamine and other neurotransmitters
throughout the brain.
Wide distribution of H3 receptor makes its
physiological role and therefore therapeutic
potential evident.
Targeted for Alzheimers disease, epilepsy,
Parkinsons disease as well as metabolic disorders
such as obesity.
A series of biaryl derivatives have been
developed as H3 receptor antagonists.

Ongoing venture. .
Combinatorial lead optimisation of dihydrofolate reductase
inhibitor.
For development of effective antimicrobial agents to treat
infections caused by multi-antibiotic resistant S.aureus and
S.pneumoniae.
A library of 1392 compounds has been already synthesised
and is under evaluation.

Conclusion
Combinatorial approaches have had tremendous impact of the identification of new leads.
Many active compounds have been selected to-date, following combinatorial methodologies, and a
considerable number of those have progressed into clinical trials.
Offers higher productivity at lower expense.

Application in conjunction with computational chemistry and molecular modelling techniques are
coming into picture.

Thank you !