Drug discovery: and Synthesis

Chapter 4

Drug chemical substances that are used to prevent or cure diseases in humans, animals and plants Activity pharmaceutical/pharmacological effect on the subject, e.g. analgesic or -blocker Potency the quantitative nature of the effect

 Drug is any substance presented for treating, curing or preventing disease in human beings or in animals. It may also be used for making a medical diagnosis or for restoring, correcting, or modifying physiological functions.

Receptors are macromolecules involved in chemical signaling between and within cells; they may be located on the cell surface membrane or within the cytoplasm. Receptors get information from drugs or enzymes or hormones etc.

Ligands Molecules (eg. drugs, hormones,

neurotransmitters) that bind to a receptor are called ligands. A ligand may activate or inactivate a receptor; activation may increase or decrease a particular cell function

Agonists : Agonist drugs activate receptors to

produce the desired response. Conventional agonists increase the proportion of activated receptors.

Drugs + Receptor

Desire effect or positive response

Many hormones, neurotransmitters (eg, acetylcholine, histamine, norepinephrine), and drugs (eg, morphine ,phenylephrine , isoproterenol , act as agonists. Increased binding = increased effect Decrease binding = decrease effect
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Antagonists :

Agonist drugs deactivate receptors to produce the undesired desired response. Antagonists: pharmacological efficacy = zero Produce no effect on receptor Dont change activity state of receptor

Prodrugs

Primary objective-design and discovery of new compounds that are suitable for use as drugs.
A team of workers chemistry, biology, biochemistry, pharmacology, mathematics, medicine and computing, amongst others Requires of drug discovery or designsynthesis of the drug, a method of administration, the development of tests and procedures to establish how it operates in the body, and a safety assessment
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Drug discovery:

1-Choosing the disease (like Cancer, HIV, Asthma, Brain tumor).

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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Drug discovery: Finding a lead

2-Finding a lead compound

Combinatorial synthesis Computer aided design Computerized searching of structural databases Designing lead compounds by NMR

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What is mean of Lead compound ?. Prototype having desired activity but also other undesirable characteristics, e.g., toxicity, other activities, insolubility, metabolism problems, oral bioavailability

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 extracts pharmacological activity. Often used as a lead compound.

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Lead Compounds from the Natural World

PLANT EXTRACTS

OPIUM

- Morphine

CINCHONA BARK - Quinine YEW TREE - Taxol

Lead Compounds from the Natural World

PLANTS AND ANCIENT RECORDS
CH3 H H3 C O H O CH3
ARTEMISININ

O O

H O

That is, suitable quantity to cure or excess to be poisonous! E.g. aspirin, paracetamol can be toxic if excesses.

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Side effect unwanted effect usually; however, they are not always non-beneficial For example, the drowsiness side effect of antihistamine may help sleep.

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Drug discovery: Finding a lead

Isolation and purification Structural determination Lead compound Lead modified by synthesis

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Sources of Lead Compounds

Nature is still an excellent source of new drugs (or precursors of new drugs). Of the 20 leading drugs in 1999, 9 were derived from natural products. From 1983-1994 almost 40% of the 520 new drugs approved were natural products or derived from natural products. 60% of anti-tumor and anti-infective drugs are natural products or derived from natural products.
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Sources of Lead Compounds

A) The Natural World
Plant life (flowers, trees, bushes) Micro-organisms (bacteria, fungi) Animal life (frogs, snakes, scorpions) Biochemicals (Neurotransmitters, hormones) Marine chemistry (corals, bacteria, fish etc)

B) The Synthetic World

Chemical synthesis (traditional) Combinatorial synthesis Computer aided drug design

C) The Virtual World

Drug discovery:
2-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

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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. 22

Finding a lead compound

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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Finding a lead compound 1-Screening of natural products

Natural products are a rich source of biologically active compounds. Many of todays 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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Finding a lead compound 1-Screening of natural products

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.
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Finding a lead compound 1-Screening of natural products

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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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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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-Computerized searching of structural databases 9-Designing lead compounds by NMR

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ANALYSIS OF DRUGS
ANALYSIS OF DRUGS by NMR, IR, UV, EI-MS, HREIMS, HMBC, HSQC, COSY, NOEY

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Drug discovery: Synthesis 3-Identifying a bioassay

Choice of bioassay In vitro test In vivo tests Test validity High-through screening A combination of tests is often used in research programs

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in vivo Tests
Carried out on live animals or humans Measure an observed physiological effect Measure a drugs ability to interact with its target and its ability to reach that target Can identify possible side effects Transgenic animals - genetically modified animals Drug potency-concentration of drug required to produce 50% of the maximum possible effect

in vitro Tests
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 Used in high throughput screening Measure the interaction of a drug with the target but not the ability of the drug to reach the target Results are easier to rationalise - less factors involved Does not demonstrate a physiological or clinical effect Does not identify possible side effects

III-Identifying a bioassay 4-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 6-Screening by NMR

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 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).

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III-Identifying a bioassay 6-Screening by NMR

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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Drug discovery: Finding a lead 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

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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.

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3. DRUG TARGETS
A) LIPIDS

Cell Membrane Lipids

B) PROTEINS

Receptors Enzymes Carrier Proteins Structural Proteins (tubulin)

C) NUCLEIC ACIDS

DNA RNA
D) CARBOHYDRATES

Cell surface carbohydrates Antigens and recognition molecules

2. DRUG TARGETS
TARGET SELECTIVITY

Between species
Antibacterial and antiviral agents Identify targets which are unique to the invading pathogen.

Within the body

Selectivity between different enzymes, receptors etc. Selectivity between receptor types and subtypes Selectivity between isozymes Organ selectivity

Enzyme Inhibition Tests

Identify competitive or non competitive inhibition
Strength of inhibition measured as IC50 IC50 = concentration of inhibitor required to reduce enzyme activity by 50%

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II- Choosing a drug target

Choosing which disease to tackle is usually a matter for companys 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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Choosing a drug target

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

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II- Choosing a drug target 4-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 - receptor) or even a particular receptor subtype 2- receptor. Ideally, enzyme inhibitors should show selectivity between the various isozymes of an enzyme.

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Choosing a drug target

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 1 while those in the lungs are 2. If a drug acts on either, less side effects would be observed.
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Choosing a drug target

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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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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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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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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Drug Synthesis
Retrosynthesis

Synthesis of cyclopropane

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Synthesis of Aspirin

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Diketone synthesis

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Summary
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.
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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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Summary

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