[4] drug discovery-lect-1

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DRUG DISCOVERY:FINDING A LEAD

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Drug discovery: Finding a lead

When a pharmaceutical company or

university research group initiates a

new medicinal chemistry project

through to the identification of a lead

compound, they will consider the

following steps in order:

1-Choosing the disease

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2-Choosing a drug target Drug targets Discovering drug targets Target specificity and selectivity

between species Target specificity and selectivity

within the body Targeting drugs to specific organs

and tissues Pitfalls

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3-Identifying a bioassay Choice of bioassay In vitro test In vivo tests Test validity High-through screening Screening by NMR Affinity screening Surface Plasmon resonance Scintillation proximity assay

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4-Finding a lead compound Screening of natural products (the

plant kingdom, the microbial world, the marine world, animal sources, venoms and toxins)

Medical folklore Screening synthetic compound “

libraries” Existing drugs Starting from natural ligand or

modulator (natural ligands for receptors, natural substrates for enzymes, enzyme products as lead compounds, natural modulators as lead compounds)

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4-Finding a lead compound Combinatorial synthesis Computer aided design Serendipity and prepared mind Computerized searching of

structural databases Designing lead compounds by

NMR

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5-Isolation and purification 6-Structural determination 7-Herbal medicine

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I- Choosing the disease

Pharmaceutical companies tend to concentrate on developing drugs for diseases which are prevalent in developed countries, and aim to produce compounds with better properties than existing drugs.

Pharmaceutical companies have to consider economic factors as well as medical ones when they decide which disease to target when designing a new drug.

A huge investment has to be made towards the research and development of a new drug.

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I- Choosing the disease

Therefore, companies must ensure that they get a good financial return for their investment.

As a result, research projects tend to focus on diseases that are important in the developed world, because it is the best market for new drugs.

Thus, research is carried out on ailments such as migraine, depression, ulcers, obesity, flu, cancer and cardiovascular disease.

Less is carried out on the tropical diseases of the developed world. Only when such diseases start to make an impact in richer countries, the pharmaceutical companies sit up and take notice.

Example: research in antimalarial drugs has increased due to increase in tourism to more exotic countries and the spread of malaria into southern states of US.

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II- Choosing a drug target

Choosing which disease to tackle is usually a matter for company’s market strategists. The science becomes important at the next stage.

A molecular target is chosen which is believed to influence a particular disease when affected by a drug.

The greater the selectivity that can be achieved, the less chance of side effects.

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II- Choosing a drug target 1- Drug targets

Once a therapeutic area has been identified the next stage is to

identify a suitable drug target (e.g. receptor, enzyme or nucleic acid)

Understanding which biomacromolecules are involved in a particular

disease state is very important.

This will allow the medicinal chemist whether agonist or antagonist

to be designed for a particular receptor or whether inhibitors should

be designed for a particular enzyme.

For example, tricyclic antidepressants such as Desipramine are

known to inhibit the uptake of NA from nerve synapses. However,

these drugs also inhibit uptake of serotonin, so the possibility arose

that inhibiting serotonin uptake might be beneficial. A search for

selective serotonin uptake inhibitors has led to the discovery of

Fluoxetine, the best selling antidepressant.

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II- Choosing a drug target 2- Discovering drug targets

If a drug or a poison produces a biological effect, there must be a

molecular target for that agent in the body.

In the past, the discovery of drug targets depends on finding the drug

first. Then, natural chemical messengers started to be discovered.

But many targets still stay hidden (orphan receptors i.e,novel

receptors whose endogenous ligand is unknown ) and their

chemical messengers are also unknown. The challenge is to find a chemical that will interact with these targets

in order to find their function and whether they will be suitable as drug targets. This is one of the main driving forces behind the rapidly expanding area of Combinatorial synthesis (synthesis of a large number of compounds in a short period of time using different reagents and starting material and are tested for activity.)

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II- Choosing a drug target 3- Target specificity and selectivity between species

Target specificity and selectivity is a crucial factor in

modern medicinal chemistry research

The more the selective a drug is for its target, the

less chance that it will interact with different targets

and have less undesirable side effects.

For example, penicillin target an enzyme involved in

bacterial cell wall biosynthesis. Mammalian cells

does not have a cell wall, so this enzyme is absent in

human cells and penicillin has few side effects.

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II- Choosing a drug target4-Target specificity and selectivity within the body

Selectivity is also important for drug acting on targets within the body

Enzyme inhibitors should only inhibit the target enzyme and not some

other enzyme.

Receptors agonist/ antagonist should ideally interact with a specific

kind of receptor (adrenergic receptor) rather than a variety of different

receptors, or even a particular receptor type ( such as β- receptor) or

even a particular receptor subtype β2- receptor.

Ideally, enzyme inhibitors should show selectivity between the various

isozymes of an enzyme.

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II- Choosing a drug target5-Targeting drugs to specific organs and tissues

Targeting drugs against specific receptor subtypes often allows

drugs to be targeted against specific organ or against specific

areas of brain.

This is because the various receptor subtypes are not

uniformly distributed around the body, but are often

concentrated in particular tissues. For example, adrenergic

receptors in the heart are predominantly β1 while those in the

lungs are β2. If a drug acts on either, less side effects would be

observed.

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II- Choosing a drug target6-Pitfalls

The body is a highly complex system. It is

possible to identify whether a particular enzyme

or receptor plays a role in a particular aliments.

For any given function, there are usually

several messengers, receptors, and enzymes

involved in the process

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II- Choosing a drug target6-Pitfalls

For example, there is no one simple cause for hypertension, there are variety of receptors and enzymes which can be targeted in its treatment. These include β1-adrenoceptors, calcium ion channels, angiotessin-converting enzyme (ACE), and potassium ion channels.

Sometimes, more than one target may need to be addressed for a particular ailment. For example, most of the current therapies for asthma involve a combination of bronchodilator (β2 agonist) and an anti-inflammatory agent such as a corticosteroid

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III-Identifying a bioassay1-Choice of bioassay

Choosing the right bioassay or test system is crucial to the success of a drug research program.

The test should be simple, quick and relevant as there are usually a large number of compounds to be analyzed.

Human testing is not possible at such early stage, so the test has to be done in vitro first. Because in vitro tests are cheaper, easier to carry out, less controversial and can be automated than in vivo one.

In vivo tests needed to check the drugs interaction with specific target and to monitor their pharmacokinetics properties.

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III-Identifying a bioassay2-In vitro tests

They do not involve live animals. Instead, specific

tissues, cells, or enzymes are isolated and used.

Enzyme inhibitors can be tested on pure enzyme in

solution.

Receptor agonist and antagonists can be tested on

isolated tissues or cells.

Antibacterial drugs are tested in vitro by measuring how

effectively they inhibit or kill bacterial cells in culture

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III-Identifying a bioassay3-In vivo tests

In vivo tests on animals often involve inducing a

clinical condition in the animal to produce

observable symptoms.

The animal is then treated to see whether the drug

alleviates the problem by eliminating the observable

symptoms. For example, the development of non-

steroidal inflammatory drugs was carried out by

inducing inflammation on test animals.

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The animals used may be transgenic i.e,some

mouse genes are replaced by human genes so the

mouse produces the human receptor or enzyme. Or

the mouse’s gene may be altered to be susceptible

for some disease such as breast cancer.

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There are several problems associated with in vivo

testing. It is slow and it also causes animal suffering.

There are also many problems of pharmacokinetics

and the result obtained may be misleading. For

example, penicillin methyl ester is hydrolyzed in

mice into active penicillin, while it is not hydrolyzed

in humans or rabbits. Also, thalidomide is

teratogenic in rabbits and humans while it is not in

mice.

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III-Identifying a bioassay4-Test validity

Sometimes the validity of testing procedure is easy

and clear. For example, the antibacterial drug can

be tested by its effect on killing bacteria. Local

anaesthetics are tested by their effect on blocking

action potential in isolated nerve.

In other cases, the testing procedure is more

difficult. For example, there is no animal model for

antipsychotic drug.

Thus, validity of the test should be carried out.

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III-Identifying a bioassay 5-High throughput screening (HTS)

HTS involves the miniaturization and automation of in

vitro tests such that a large number of tests can be

carried out in a short period of time.

It involves testing of large number of compounds versus

a large number of targets. The test should produce

easily measurable effect. This effect may be cell growth,

an enzyme catalyzed reaction which produces a color

change (may be a dye) or displacement of radioactive

labelled ligand from its receptors.

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III-Identifying a bioassay 6-Screening by NMR

NMR was used as a tool for determining the molecular

structures of compounds

Recently, compounds can be tested or screened for their

affinity to a macromolecular target by NMR spectroscopy. The

relaxation times of ligands bound to a macromolecule are

shorter than when they are unbound (can’t be detected).

In NMR spectroscopy the compound is radiated with a short

pulse of energy which excites the nuclei of specific atoms

(H,N,C) afterwards, the excited nuclei slowly relax back to the

ground state giving off energy as they so.

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There are, several advantages in using NMR as a detection system:

1-It is possible to screen 1000 small molecular weight compounds a day with one machine.

2-The method can detect weak binding which would be missed by conventional screening methods.

3-It can identify the binding of small molecules to different regions of binding site.

4-It is complementary to HTS. The later may give false-positive results, but these can be checked by NMR to ensure that the compounds concerned are binding in the correct binding site.

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5-The identification of weakly binding molecules

allows the possibility of using them as building

blocks for the construction of larger molecules that

bind more strongly.

6-Screening can be done on a new protein without

needing to know its function.

NMR screening also has limitations, the main one

being that at least 200 mg of the protein required.

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III-Identifying a bioassay 7-Surface Plasmon resonance (SPR) & scintillation proximity assay (SPA)

SPR (change in refractive index)& SPR

(reduction of emission of light) are two visual

methods of detecting whether ligands bind to

macromolecular targets .

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IV-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.

There are various ways in which a lead compound might

be discovered. However, the following are the ways of

discovering the lead compound: 1-Screening of natural products (the plant kingdom,

the microbial world, the marine world, animal sources, venoms and toxins)

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IV-Finding a lead compound 2-Medical folklore 3-Screening synthetic compound “ libraries” 4-Existing drugs (Me too drugs & Enhancing the side

effects) 5-Starting from natural ligand or modulator (natural

ligands for receptors, natural substrates for enzymes, enzyme products as lead compounds, natural modulators as lead compounds)

6-Combinatorial synthesis 7-Computer aided design 8-Serendipity and prepared mind 9-Computerized searching of structural databases 10-Designing lead compounds by NMR

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IV-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.

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But the disadvantage of 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 .

Natural products can be obtained from different sources such as:

1-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.

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2-The microbial world: microorganisms such as bacteria and

fungi are rich for lead compounds (e.g. Antgimicrobial Drugs:

pencillins, cephalosporines, tetracyclines, aminoglycosides,

chloramphenicol, rifamycins).

3-The marine world: 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)

4-Animal sources: antibiotic peptides were extracted from the

skin of African clawed frog.

Epibatidine (potent Analgasic) was also obtained from

Ecuadorian frog.

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5-Venoms 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

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IV-Finding a lead compound 2- 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).

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IV-Finding a lead compound3-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

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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

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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 (e.g.antidiabetic sulfonyl urea,

Tolbutamine).

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IV-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.

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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.

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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.

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6-Combinatorial synthesis Combinatorial synthesis is 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.

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IV-Finding a lead compound7-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 and to design drugs.

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IV-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

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IV-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.

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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.

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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.

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V-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.

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6-Structure determination

X-ray crystallography, NMR spectroscopy,

mass, and IR are important in structure

deterimination.

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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.

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A lead compound is a structure which shows a useful pharmacological activity and can act as the starting point for drug design.

Natural products are a rich source of lead compounds. The agent responsible for biological activity of a natural extract is known as the active principle.

Lead compound have been isolated from plants, trees, microorganisms, animals, venoms, and toxin. A study of medical folklore indicates plants and herbs which may contain novel lead compounds.

Lead compounds can be found by screening synthetic compounds obtained from combinatorial syntheses and other sources.

Existing drugs can be used as a lead compounds for design of novel structures in the same therapeutic area. Alternatively, the side effects of an existing drug can be enhanced to design novel drugs in a different therapeutic area.

Summary

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Summary The natural ligand, substrate, product, or modulator

for a particular target can act as a lead compound. The ability to crystallize a molecular target allows the

use of X-ray crystallography and molecular modeling to design lead compounds which will fit the relevant binding site.

Serendipity has played a role in the discovery of new lead compounds.

Knowledge of an existing drug’s pharmacophore allows the computerized searching of structural databases to identify possible new lead compounds which share the pharmacophore.

NMR spectroscopy can be used to identify whether small molecules (epitopes) bind to specific region of a binding site. Epitopes can be optimized then linked together to give a lead compound.

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Summary If a lead compound is present in a natural extract or a

combinatorial synthetic mixture, it has to be isolated and purified such that its structure can be determined. X- ray crystallography and NMR spectroscopy are particular important in structure determination.

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