FINDING A LEAD COMPOUND

KULIAH KIMIA MEDISINAL

 TARGET DISEASE

Priority for the Pharmaceutical Industry:

Can the profits from marketing a new drug
outweigh the cost of developing and testing that
drug?
•Is the disease widespread?
(e.g. cardiovascular disease, ulcers, malaria)
•Does the disease affect the first world?
(e.g. cardiovascular disease, ulcers)
•Are there drugs already on the market?
•If so, what are their advantages and
disadvantages (e.g. side effects)
•Can one identify a market advantage for a new
therapy?
 DRUG TARGET
- Receptors
- Enzymes
- Carrier proteins
- Structural proteins
(tubulin)
Cell membrane lipids
- Cell surface
carbohydrates
- Antigens and
recognition
molecules

DNA
RNA
 TARGET SELECTIVITY

Between species:
 Antibacterial, antifungal and antiviral agents
 Identify targets which are unique to the
invading pathogen
 Identify targets which are shared but which
are significantly different in structure
 TARGET SELECTIVITY

Within the body:

 Selectivity between different enzymes,
receptors etc.
 Selectivity between receptor types and
subtypes
 Selectivity between isozymes
 Organ and tissue selectivity
Bioassays (test systems)

 Tests are required in order to find lead

compounds and for drug optimisation
 Need to be quick, easy and relevant
 Tests can be in vivo or in vitro
 A combination of tests is often used in
research programs
 IN VIVO TESTS
• Carried out on live animals or humans
• Measure an observed physiological effect

• Measure a drug’s ability to interact with its target and its ability to reach
that target
• Can identify possible side effects

• Rationalization may be difficult due to the number of factors involved

• Transgenic animals - genetically modified animals

• Drug potency - concentration of drug required to produce 50% of the
maximum possible effect

• Therapeutic ratio/index – compares the dose level of a drug required to

produce a desired effect in 50% of the test sample (ED50) versus the dose
level that is lethal to 50% of the sample (LD50)
 IN VITRO TEST
 Tests not carried out on animals/humans
 Target molecules (e.g. isolated enzymes or receptors)
-Cells (e.g. cloned cells)
-Tissues (e.g. muscle tissue)
-Organs
-Micro-organisms (for antibacterial agents)
 More suitable for routine testing
 Measure the interaction of a drug with the target but not the ability
of the drug to reach the target
 Results are easier to rationalize - less factors involved
 Does not demonstrate a physiological or clinical effect
 Does not identify possible side effects
 Does not identify effective prodrugs
 TESTING DRUG
 High Throughput Screening
 NMR o Identify competitive or non
 Enzyme Inhibition tests competitive inhibition
o Strength of inhibition
 Receptor tests measured as IC50
o IC50 = concentration of
inhibitor required to reduce
enzyme activity by 50%

 Not easy to isolate membrane bound receptors

 Carried out on whole cells, tissue cultures, or isolated
organs
 Affinity - strength with which compounds bind to a
receptor
 Efficacy - measure of maximum biochemical effect
resulting from binding of a compound to a receptor.
 Potency - concentration of an agonist required to produce
50% of the maximum possible effect.
THE LEAD COMPOUND

 A compound demonstrating a property likely

to be therapeutically useful
 The level of activity and target selectivity are
not crucial
 Used as the starting point for drug design
and development
 Found by design (molecular modelling or
NMR) or by screening compounds (natural
or synthetic)
 Need to identify a suitable test in order to
find a lead compound
 Active principle - a compound that is isolated
from a natural extract and which is principally
responsible for the extract’s pharmacological
activity. Often used as a lead compound.
The lead compound
Plantlife (flowers, trees, bushes)
Micro-organisms (bacteria, fungi)
Animal life (frogs, snakes, scorpions)
The natural Biochemicals (Neurotransmitters, hormones)
Marine chemistry (corals, bacteria, fish etc)
world

Chemical synthesis (traditional)

The synthetic Combinatorial synthesis
world
Computer aided drug design
The virtual
world
 Lead compounds from the natural world
Willow tree - salicylic acid

O OH O OH
Acetic
OH anhydride O CH3
Aspirin
O

Coca bush - cocaine

Me
CH3
N
CO2Me

H N Procaine
O
O CH3 C NH2
H C O
O
Lead compounds from the natural world

Morphine

Poppy capsule
Lead compounds from the natural world

• OPIUM - Morphine

• CINCHONA BARK - Quinine

• YEW TREE - Taxol

THE BEATONS OF PENNYCROSS

CURE for EPILEPSY

Add
Heat
Simmera liberal
Flavour with
a cauldron
for 20sprinkling
essence
of
minutes
water
Produce
of
until juicy
dog
then
warm one
to pedigree
turd
enjoy the touch dog
spiders
 Lead compounds from the natural world
Herbal remedies of old

Majority may have worked through a placebo effect

 Lead compounds from the natural world
Rain forests
 Lead compounds from the natural world
Micro-organisms
 Lead compounds from the natural world
Endogenous compounds

Natural ligands for receptors

OH O N
H
Antagonist
H
HO N OH
Me

HO
ADRENALINE PROPRANOLOL

Me
NH2 H
N NHMe
HN S
HN
N
N
HISTAMINE
CN Antagonist
CIMETIDINE
Lead compounds from the synthetic world

SYNTHETIC COMPOUNDS
Lead compounds from the synthetic world

O
H2N
PRONTOSIL
N N S NH2
O
NH2
 Lead compounds from the synthetic world

O
H2N S NH2
O

SULFANILAMIDE
Lead compounds from the synthetic world

ONO2
ONO2
ONO2
Lead compounds from the synthetic world
Rubber industry

CH3
S
C S N CH3
H3C N S C
S
H3C
Antabuse
 Lead compounds from the synthetic world
Organic synthesis
4.4 Lead compounds from the synthetic world
Combinatorial synthesis

Automated synthetic machines

 Lead compounds from the synthetic world
Combinatorial synthesis - peptide synthesis

AMINO ACID

RESIN
BEAD
AMINO ACIDS

PEPTIDE
4.4 Lead compounds from the synthetic world
Combinatorial synthesis - heterocyclic synthesis

RESIN
BEAD

N
N
Y

CHR1R2
O
4.4 Lead compounds from the synthetic world
Combinatorial synthesis - heterocyclic synthesis

N
N
RESIN
BEAD
R2
R3
O
H
4.4 Lead compounds from the synthetic world
Combinatorial synthesis - heterocyclic synthesis

N
N
RESIN
BEAD
R2
R3
O
R4
EtO R5
O
4.4 Lead compounds from the synthetic world
Combinatorial synthesis - heterocyclic synthesis

N
N
RESIN
BEAD
R2
R3
N
R4
HN R5
O
4.4 Lead compounds from the synthetic world
Combinatorial synthesis - heterocyclic synthesis

RESIN
N BEAD
N
Y
R2
R3
N
R4
HN R5
O
4.6 Lead compounds - de novo design
4.6 Lead compounds - de novo design

X-RAY CRYSTALLOGRAPHY
4.6 Lead compounds - de novo design

PROTEIN STRUCTURE
4.6 Lead compounds - de novo design

Receptor
 CH3

+
H3N Scaffold O
Scaffold
Scaffold
Scaffold
IONIC Scaffold HO H-BOND
BOND
-
CO2

VDW
BOND
4.6 Lead compounds - de novo design
Thymidylate kinase inhibitors

N
H
N N
H Lead compound
S
N O
O
O

Optimisation
O

N N
S
N O
H2N O

Anticancer agent
4.7 Design of lead compounds using NMR spectroscopy

NMR spectroscopy
4.7 Design of lead compounds using NMR spectroscopy

Binding Site

Protein
4.7 Design of lead compounds using NMR spectroscopy

Protein

No observable biological effect

4.7 Design of lead compounds using NMR spectroscopy

CH3
CH CH
CH3
CH2

CH2

C CH
C

13C NMR
4.7 Design of lead compounds using NMR spectroscopy

CH3
CH CH
CH3
CH2

CH2

C CH
C

13C NMR
4.7 Design of lead compounds using NMR spectroscopy

Protein

Optimise
epitope
4.7 Design of lead compounds using NMR spectroscopy

Protein

Optimise Optimise
epitope epitope
4.7 Design of lead compounds using NMR spectroscopy

Link

Protein

Optimise Optimise
epitope epitope
4.7 Design of lead compounds using NMR spectroscopy

LEAD COMPOUND
Design of lead compounds using NMR spectroscopy

Design of a lead compound as an immunosuppressant

OH
O

OMe N
H
N O HO
O
O
Epitope B

MeO OMe
OMe

Epitope A
Design of lead compounds using NMR spectroscopy

Design of a lead compound as an immunosuppressant

OH
O
Me
O N
H
N O HO
O
O

MeO OMe
OMe Lead compound
 Identification of the lead compound

A) Isolation and purification

Solvent-solvent extraction
chromatography
Crystallization
distillation
B) Structure determination
Elemental analysis
Molecular weight
Mass spectrometry
IR spectroscopy
UV spectroscopy
NMR (1H, 13C, 2D) spectroscopy
X-ray crystallography
 ISOLATION AND PURIFICATION

If a lead compound is present in a mixture of other compounds it has to be

isolated and purified.

The isolation and purification depends upon structure, stability, and quantity
of the compound.

e.g. Fleming recognized penicillin, qualities & non-toxic to human but could
not use it clinically because he was unable to purify it. He could isolate it in
aqueous solution, but when he tried to remove water the drug was destroyed.

Purification and isolation of penicillins were possible until development of

new experimental procedure such as freeze-drying and chromatography.

52
 STRUCTURE DETERMINATION

X-ray crystallography, NMR spectroscopy, mass, and IR are

important in structure determination.

53
 FINDING A LEAD COMPOUND

 Once a target and a testing system have been chosen,

the next stage is to find a lead compound.
 A lead compound is a compound which shows the
desired pharmaceutical activity.
 The level of the activity may not be very great and
there may be undesirable side effects.
 The lead compound provides a start for the drug
design and development process.

54
 FINDING A LEAD
COMPOUND

1-Screening of natural
products (the plant 8-Serendipity and prepared 9-Computerized
kingdom, the microbial searching of structural
world, the marine world, mind databases
animal sources, venoms
and toxins)

10-Designing lead
7-Computer aided design
compounds by NMR
2-Medical folklore

6-Combinatorial synthesis
3-Screening
synthetic compound
libraries
5-Starting from natural ligand or
modulator (natural ligands for
4-Existing drugs (Me receptors, natural substrates for
too drugs & Enhancing enzymes, enzyme products as
the side effects) lead compounds, natural
modulators as lead compounds)
 FINDING A LEAD COMPOUND
1-SCREENING OF NATURAL PRODUCTS

 Natural products are a rich source of biologically

active compounds.
 Many of today’s medicines are either obtained
directly from a natural source or were developed from
a lead compound originally obtained from a natural
source.
 The compound responsible for that activity is known
as the active principle.
 Most biologically active natural products are
secondary metabolites with quite complex structures.
This has advantage in that they are extremely novel
compounds. 56
 FINDING A LEAD COMPOUND
1-SCREENING OF NATURAL PRODUCTS

Disadvantage :
Their complexity makes their synthesis
difficult and the compound needs to be
extracted from its natural source (i.e.
costly & inefficient process). As a result,
there is a need to design simpler
analogues of the lead compounds

.
 MEDICAL FOLKLORE

• Berries, leaves and roots used by local healer or

shaman as medicines. Many are useless or dangerous
and if they work this may be due to Palcebo Effect.

• Some of these extracts indeed have a real effect. (e.g.

quinine (cinchona), reserpine (Rauwolfia), atropine
(atropa beladona), morphine (opium poppy), digitalis
(foxglove), emetine (ipeca), cocaine (coca).
 FINDING A LEAD COMPOUND
3-SCREENING SYNTHETIC
COMPOUNDS (LIBRARIES)

Thousands of compounds have been synthesized . The majority of

these compounds are not used or not been in the market. They

have been stored in compound libraries, and are still available for

testing.

Pharmaceutical companies screen their ‘library’ to study a new

target and find a lead compound

59
 FINDING A LEAD COMPOUND
4-EXISTING DRUGS
A) Me too drugs: Many companies use established drugs from their
competitors as a lead compound in order to design a drug. By modifying
the structure in such way that avoids the patent restrictions, retain the
activity, and improved the therapeutic properties.

For example i) Captopril (Anti-hypertension) used as lead compound by

different companies to produce their own anti-hypertension drugs.

ii) Modern penicillins are more selective, more potent and more stable than
original penicillins

60
 FINDING A LEAD COMPOUND
4-EXISTING DRUGS
B) Enhancing a side effect: An existing drug may have a minor or
undesirable side effect, which might be used in another area of
medicine. And such compound could be a lead compound on the
basis of its side effects.
The aim is to enhance the desired side effect and to eliminate the major
biological activity.
e.g. Sulfonamides are Antibacterial agents but some sulfonamides has
convulsive side effect due to hypoglycaemia effect. This,
undesirable side effect was useful in the development
sulfonamides drugs for treatment of diabetes ([Link]
sulfonyl urea,
61 Tolbutamine).
 FINDING A LEAD COMPOUND
5- STARTING FROM THE NATURAL LIGANDS
OR MODULATOR
A) Natural ligands for receptors:
 Natural ligands of a target has been sometime used as
the lead compound.
 E.g. Adrenaline and noradrenaline (natural
neurotransmitters) were used for developement
adrenergic β-agonists such as Salbutamol, dobutamine,
xamoterol, H2 antagonists as cimetidine, and morphine(led to
opiate receptors, and endogenous opiates:endorphins and
enkephalins.
62
 Lead compounds from the natural world
Endogenous compounds

Natural ligands for receptors

OH OH
H H
N N
HO HO
Me Agonist
HO HO

ADRENALINE SALBUTAMOL

NH2 NMe2

HO MeHN
S Agonist
O O
N N
H H
5-HYDROXYTRYPTAMINE SUMATRIPTAN
 FINDING A LEAD COMPOUND
5- STARTING FROM THE NATURAL LIGANDS OR
MODULATOR

B) Natural substrates for enzymes:

 The natural substrate for an enzyme can be
used as the lead compound in the design of
enzyme inhibitors.

 e.g. enkephalines used as a lead compound to

design an inhibitor of enkephalinases.


64
 Lead compounds from the natural world
Endogenous compounds

Natural substrates for enzymes

Enkephalins Enkephalinase inhibitors

Peptides Protease inhibitors

H CO2H O
N O
H H H H N
N N H
H2N N N
H H H
H O OH
O CONH2
H
L-Phe-L-Pro
SAQUINAVIR
 FINDING A LEAD COMPOUND
5- STARTING FROM THE NATURAL LIGANDS
OR MODULATOR

C) Enzyme products as lead compounds: enzymes catalyze a reaction in both

directions ,so enzyme products can be used as a lead compound for an enzyme
inhibitor e.g. L-benzyl succinic acid inhibit enzyme catalyzed carboxy peptidase
hydrolysis of peptides.

D) Natural modulators as lead compounds: the natural or endogenous

chemicals that exert allosteric control of receptor or enzymes called Modulators and
can be also as lead compounds.

e.g. Benzodiazepines: were discovered to modulate the receptor γ-aminobutyric acid

(GABA) by binding to allosteric binding site then endogenous endozepines were
discovered.
66
 FINDING A LEAD
COMPOUND
6-COMBINATORIAL
Combinatorial synthesis isSYNTHESIS
automated solid-phase procedure aimed at
produce as many as different structures as possible in short time as
possible.
The reactions are carried out on very small scale, often in a way that
will produce mixtures of compounds.
Combinatorial synthesis aims to mimic what plants do, i.e. produce a
pool of chemicals.
One of these compounds may be prove to be a useful lead
compound.
67
 FINDING A LEAD COMPOUND
7-COMPUTER –AIDED DESIGN
Knowledge of target binding site aids in design of novel compounds

intended to bind with that target.

The enzyme and receptors can be crystallized and it is possible to

determine their structure (structure of protein & binding site) by X-

ray crystallography.

Molecular modelling software programs can be used to study the

binding site and68to design drugs.

 FINDING A LEAD COMPOUND
7- SERENDIPITY AND THE PREPARED
MIND
Lead compounds are found as a result of serendipity (i.e. chance)
e.g. i) Cisplatin (Anti-cancer) & peniciilins
ii) Development of propanolol (β-blocking) have unexpected give a
benefit of discover Practolol.
Propanolol is a β-blocker but it is a lipophilic drug and can enter CNS
and cause side effect, by introducing hydrophilic amide group inhibit
passage the blood-brain barrier and Practolol produced more selective
cardioselective β1 inhibitor with fewer side effects on CNS.
Sulfonamides and tolbutamide

69
 FINDING A LEAD COMPOUND
7- SERENDIPITY AND THE PREPARED
MIND

Workers in TNT factories always complained from headache

due to dilatation of brain blood vessels. TNT was the basis to
prepare nitro derivatives which were used in angina to dilate
coronary blood vessels and alleviate pain.
Mustard gas tanks used in second world war exploded in italian
harbor. They discovered that persons who survived and inhaled
this gas lost their defense against microorganisms due to
destruction of white blood cells.
This led to the discovery of mustard like drugs which were
used in leukemia to inhibit excessive proliferation of white
blood cells.

70
 IV-FINDING A LEAD COMPOUND
9-COMPUTERIZED SEARCHING OF
STRUCTURAL DATABASES

New lead compounds can be found by carrying out computerized

searches of structural databases.

In order to carry out such search, it is necessary to know the desired

pharmacophore.

Data base searching is known as database mining.

71
 IV-FINDING A LEAD COMPOUND
10-DESIGNING LEAD COMPOUNDS BY NMR
Recently NMR spectroscopy has been used to design a lead

compound rather than to discover one.

The method sets out to find small molecules (epitopes) which

can bind to specific binding site.

Lead discovering by NMR can be applied to proteins of known

structure which are labeled with N15.

72
 SOURCES OF NATURAL
PRODUCT

PLANT MICROBIAL MARINE ANIMAL

KINGDOM WORD WORD SOURCES

VENOM
AND TOXINS
 THE PLANT
KINGDOM

It is rich source of lead compounds (e.g. morphine,

cocaine, digitalis, quinine, tubocurarine, nicotine and
muscarine, paclitaxel (Taxol, recent anticancer),
either useful drugs as morphine or basis for synthetic
).Plants continue to remain a promising source of
new drugs
 MICROBIAL WORD

Microorganisms such as bacteria and fungi are rich for

lead compounds (e.g. Antgimicrobial Drugs:
 Pencillins
 Cephalosporines
 Tetracyclines
 Aminoglycosides
 Chloramphenicol
 Rifamycins
 Lead compounds from the natural world
Micro-organisms R
H H H
OH O OH O O N S
OH CH3
O
NH2 N CH3
O
• Penicillin Me H
OH CO2H

ClHO NMe
• Cephalosporins
2

HO H

• Tetracyclines O2N
CH2OH
NH
• Streptomycin
HN HN H
HN NH H C
C
H N C NH 2
2
H O CHCl2

• Chloramphenicol H
OH
HO H
H

OH
O H
H O O
CHO H
R C N H H
Me H S
OH
H O N OAc
HO O O
CH2OH
H MeHN CO2H
H H
OH H
 MARINE WORD

Coral, sponges, fish and marine microorganisms

have biological potent chemicals, with interesting,
anti-inflammatory, antiviral, and anticancer activity.
E.g Curacin A (anti-tumour, from marine
cyanobacterium) CH3

S
OMe N CH3

CURACIN H H
 ANIMAL SOURCES

 Antibiotic peptides were extracted from the skin of

African clawed frog.
 Epibatidine (potent Analgasic) was also obtained
from Ecuadorian frog.
 VENOM AND TOXINS

 From animals, plants, snakes, spiders, scorpions,

insects and microorganisms.
 They are potent because they have specific
interaction with a macromolecular target in the
body.
 Thus, they provide important tools in studying
receptors, ion channels, and enzymes.
 e.g. Teprotide (from venom of viper) was the lead
compound for the development of
antihypertensive agents Cilazapril & Captopril
 Lead compounds from the natural world
O

Venoms and toxins O

C OH

C N
Teprotide O O O
H
C N CH C N CH C N
O H
O CH2 CH CH3
C N CH C N CH2 CH2
O O H
CH2 C O CH3
H2N CH C N CH C N
H CH2 NH2
CH2 CH2 O
CH2
CH2 C OH
NH O
C O
HN C NH C N
OH
NH2
CH3
HS

Captopril
(anti-hypertensive)
 TEPROTIDE

the lead compound for the development of

antihypertensive agents Cilazapril & Captopril
 MeO
CH 3
N
HO CH 3
O H

H O
H3 C
N OH
H3 C
OMe

TUBOCURARINE
FROM CURARE
 Lead compounds from the natural world
MeO
Venoms and toxins CH 3
N
HO CH 3
O H

H O
H3 C
N OH
H3 C
OMe

Tubocurarine
(from curare)

MeO OMe
O O
CH 3 H
N C C N
MeO O (CH 2)5 O OMe

OMe MeO
OMe OMe

Atracurium
(Neuromuscular blocker)
 7-HERBAL MEDICINES
Herbal medicines contain a large variety of different compounds.
Several of these may have biological activity, but there is a
significant risk of side effects and toxicity. The active principle
present in small amount, so herbals are expected to be less active
than pure compound.
Herbal medicines may be interacting with prescribed medicines and
there is no regulations or control of this matter and their uses.
But it is an important lead to discover and design new drugs.

84
Lead compounds
- impact of the human genome project

The Past

Lead Compound Targets

The Present and Future

Targets Lead compounds