An Introduction to the Principles Underlying Pharmacokinetics

Dr. Qosay Al-Balasqabalas@just.edu.jo

Absorption, Distribution, Metabolism and Excretion (ADME)

Absorption, Distribution, Metabolism and Excretion (ADME)

-

Medicinal Chemistry: is defined as the science that deals with the relationship between the chemical structure of the drug and its behavior in the biological systems, added to this the aspects of drug design and synthesis.

-

Absorption: process by which the drug is transported into the systemic circulation across biological membranes.

-

Distribution: process by which a drug is transported from systemic circulation across biological membranes.

-

Metabolism: chemical modification of the drug at different sites of the body in order to excrete it outside of the body.

-

Elimination: process by which the drug or modified drug is discarded outside body.

O

SHN

NH2

HOO

N

OOH

Biological

ActivityIn order for a drug to have biological activity, there are two

conditions:

1-

Reach to site of action:

Governed by absorption and distribution which in turn governed by physicochemical

properties of the drug.

2-

Interact with the site of action:

Governed by the chemical structure of the drug.

Physicochemical properties: measurable characters by which the chemical substance may interact with other systems. Such as: water solubility, lipid solubility, partitioning behavior, vapor

pressure, and pKa. These are the major factors affect the ADME

Pharmacodynamics

–

what the drug does to the body

Pharmacokinetics

–

what the body does to the drug

What are the concepts?What is the physiology?

What is the medicinal chemistry?How do we improve the delivery of drugs as medicines?

The [plasma]-time Curve After Drug Administration

The Processes Involved in ADME That Control the [plasma]-time Curve After Drug Administration

Drug Administered

Drug Absorbed

Pool of availableDrug in the plasma

Drug at the site of action

Metabolic inactivation

ExcretionPool of non-availableDrug in the tissues

Which route?Which formulation?•Injection –

aqueous or depot?is it stable?

•Tablet -

water solubility?site of release?pH stabilityenzyme stability

•Cream -

lipophilicity?•Aerosol -

lipophilicity/stability?

Which barriers to cross?Gut, skin, lungs?Stability at the site of absorption?

Plasma-protein binding?•Electrostatic chargeTissue-protein binding?Fat sequestration?•Lipophilicity[Volume of distribution]

Passive diffusion?Active transport?Blood-brain barrier penetration?

Rapid first-pass metabolism via Hepatic portal vein?Pro-drug activation

Can fast metabolism be blocked? How fast?

No metabolism required?

Physicochemical Properties of Drugs

Partition coefficientLipophilicity/hydrophilicity

Ionisation/dissociation constantStrong or weak acids/basesSalt formation

SolubilityWater-soluble saltsLipid soluble esters

Stability•Chemical degradation –

oxidation, hydrolysis, light

•Enzyme degradation –

esterases, amidases, cytochrome

P450

Why is Medicinal Chemistry Important in Drug ADME?

•Pharmacokinetics –

what the body does to the drug–How do you get it into the body?–How long does it stay in the body?–Where does it go to in the body?–Is it metabolised to another form?

•Pharmacodynamics –

what the drug does to the body–What is the therapeutic effect of the drug?–How does it exert its effect?–How does the drug interact with the target?–Can the effect be modified?

Absorption: Absorption via GIT

Factors affecting the absorption:

1-

pH: affect mainly the ionizability

and chemical stability

2-

Surface Area [SA]: intestine has villi

and microvilli

that increase the SA dramatically compared to other site of absorption

3-

Enzymes: might degrade the drugs before being absorbed

4-

Biomembranes: Lipid bilayer

composed of phospholipids, cholesterol and other components that make it lipophilic

Mechanism of Drug Absorption

-Passive Diffusion:

movement of drugs from the area of high concentration to the low concentration.

-Passive Diffusion:

depends mainly on lipophilicity (partitioning) and the concentration gradient.

-From GIT to the Blood, so the concentration in the blood is near

to zero, so the gradient is a continuous process.

-Active Transport:

used to transport endogenous compounds such as amino acids and neurotransmitters. The main factor of transport depends on the structure of the drug.

-Active transport:

is characterized by consuming energy and being a saturable

process.

-Ion-Pair Absorption

Barriers to Drug Absorption and Routes of Administration

Movement of Drugs Into, Around and Out of the Body: Ability to Diffuse Across/Partition Into

membranes

Drug Drug

Gutcontents Plasma

Drug Drug

PlasmaCell

Membranes have lipid character and act as barriers to the movement of drugs within the body

Lipophilicity/ Hydrophilicity

Functional groups will determine whether a drug will prefer to dissolve in water or diffuse into a membrane

O

OH

OH

OH

OH

CH2OH

Naphthalene Glucose

CH3

O

O_ +

Sodium acetate

Na

Hydrophilicity/ Water solubility

CH3

OH

OH

Donor

Acceptor

OH

O

H

H

Donor

Acceptor

Hydrophilicity/Water solubility

-

Water Solubility:

drug should have some water solubility because:

1-

Should be soluble in the gastric fluids2-

Blood which is the distribution vehicle

3-

Water is the reaction medium of the biological systems

-

Lipid Solubility:

-

Hydrophobic nature of membranes imposes some lipid solubility of the drugs to enable crossing. [charged molecules does not cross]-

Feature of the molecule that increase lipophilicity.

COOH

Ibuprofen

OHHN

HO

HO

Adrenaline/Epinephrine

3

F

Measurement of the Balance Between Hydrophilicity

and Lipophilicity is by

Determination of the Partition Coefficient:

Partitioning

P = [Co]/[Cw]

log P = log[Co/Cw]

Determined experimentally

log P > 2: highly lipophiliclog P < 0: hydrophilic

Common lipophilic and hydrophilic groupsAliphatic Aromatic Aliphatic Aromatic

-F -0.38 0.37 C6 H4 1.67 1.67

-Cl 0.06 0.94 -H 0.23 0.23

-Br 0.20 1.09 -NH- -2.15 -1.03

-I 0.59 1.35 -OH -1.64 -0.44

-NO2 -1.16 -0.03 -NH2 -1.54 -1.00

-O- -1.82 -0.61 -SH -0.23 0.62

-S- -0.79 0.03 -CONH- -2.71 -1.81

-CH3 0.89 0.89 -COOH -1.11 -0.03

-CH2 - 0.66 0.66 -CONH2 -2.18 -1.26

-CH< 0.43 0.43 -CN -1.27 -0.34

>C< 0.20 0.20 -CO- -1.90 -1.09

C6 H5 1.90 1.90 -CO2 - -1.49 -0.56

To Complicate Matters, Many Drugs are Weak Acids or Bases, Which Means They Can Ionise

- The ionized/charged forms of drugs (salts) tend to dissolve in water and they will not cross lipid membranes.

-

The unionized/uncharged forms (free acids or bases) tend to dissolve in organic solvents and will cross lipid membranes.

-

Drugs can therefore have both water soluble and fat soluble properties, which means they can be formulated to get into the body.

IONISED UNIONISED

Oral DrugAdmin.

IONISED

Drug absorptionDrug transportin plasma

What Determines the Ratio of the Ionized to the Unionized forms for a given drug?

1-

The dissociation constant, pKa

(which is fixed for a given drug)2-

The pH of the solution which the drug is in (which is variable)

We cannot change the ratio of the ionized to unionized species by changing the pKa

of the drug, but we need to take into account how

affects change.

For example, the pH in the stomach is 1-2, the small intestine pH varies from 6-8, whilst the plasma pH is 7.4.

0

stomachurine

gut

blood and mosttissues ca pH7.4

%dissociation

1 2 3 4 5 6 87 9 10

100

50

pH

O

Aspirin is undissociatedin the stomach

O

O

OH

O

O

O

O

H+

Aspirin is dissociatedin the small intestine,

plasma

pH Varies in Different Body Compartments

Compartment

pHPlasma

7.35 –

7.45

Buccal

cavity

6.2 –

7.2Stomach

1.0 –

3.0

Duodenum

4.8 –

8.2Jejunum & ileum

7.5 –

8.0

Colon

7.0 –

7.5

Drugs move between these compartments, so we needto know how they behave according to their ionizability

Ionisation & Dissociation-

ACIDS ARE PROTON DONORS

- Our definition of an acid

is a substance that can dissociate to

produce H+

and a negative ion (anion) which is called a conjugate base i.e.:

-The DISSOCIATION constant for the acid, HA, is given by Ka. Conversely, the reverse of the reaction would be the ASSOCIATION

constant for the conjugate base, A-, which we could refer to as Kb.

- In other words, acids are proton donors and their conjugate

bases are proton acceptors.

HA H + AKa

ACID CONJUGATE BASE

UNIONISED IONISED

UNDISSOCIATED DISSOCIATED

Kb

-

BASES ARE PROTON ACCEPTORS-

Bases can accept a proton to form the positively charged cation, referred to as the conjugate acid of the base.

-

The ASSOCIATION constant for the base (B) is referred to as Kb, Similarly, the DISSOCIATION constant for the conjugate acid of the base (BH+) is referred to as Ka.

Ionisation & Dissociation

BHB +Kb

BASE CONJUGATE ACID

UNIONISED IONISED

UNDISSOCIATEDDISSOCIATED

HKa

-

Consequently, we can say that a conjugate acid is a substance that can dissociate to produce H+

and a neutral molecule:

-

In this way, when we can quote Ka

values for both acids and bases (conjugate acids),

- Ka

values tell us how DISSOCIATED an acid or conjugate acid (base) is, which indicates how strong or weak the acid or conjugate acid is.

Ionisation & Dissociation

BH B +Kb

BASECONJUGATE ACID

UNIONISEDIONISED

UNDISSOCIATED DISSOCIATED

HKa

Ionisation & Dissociation

You can’t tell from the pKa

value whether the species in question is acidic or basic

To apply these rules, you must be able to recognise acidic and basic functional groups

Do not confuse pKa

with pH –

pH

is simply a measure of the [H+] concentration

pH = 1 is an acidic environmentpKa

= 1 DOES NOT

mean an acidic molecule

pH = 14 is a basic environmentpKa

= 1 DOES NOT

mean a basic molecule

Ionisation & Dissociation

Remember what pKa

is –

a dissociation constant and a measure of where the equilibrium lies

For acids: For acids: a high pka means the species is predominantly unionised, is a bad proton donor, and a weak acid

a low pka means the species is predominantly ionised, is a good proton donor, and a strong acid

For bases: For bases: a high pka means the species is predominantly ionised, is a good proton acceptor, and a strong base

a low pka means the species is predominantly unionised, is a bad proton acceptor, and a weak base

Ionisation & Dissociation

So Why Are pKa

Values for Acids and Bases Relevant and Important to Pharmaceutical

Scientists?

-

ONLY THE UNIONISED FORM OF A DRUG CAN PARTITION ACROSS BIOLOGICAL MEMBRANES (providing the unionized form is lipophilic)

-

[required for drug absorption into the body]

-

THE IONISED FORM TENDS TO BE MORE WATER SOLUBLE

-

[required fro drug administration and distribution in plasma]

Partitioning of Acids and Bases

•

The equilibrium in the aqueous layer is determined by the pKa

of the compound in

question, and the pH of the solution, as we have already demonstrated.

organiclayer

non-ionised

non-ionised ionisedaqueouslayer

Consider drugs that are acids, for example RCOOH, which has a pKa

of 4.0

•

If the pH shifts the balance towards the unionized/undissociated

form, the drug would be absorbed.

•

If the pH shifts the balance towards the ionized/dissociated form, the drug would not be absorbed.

•

Assume the pH of the stomach is 2.0 and the pH of the small intestine is 8.0. Where would you expect absorption to take place from?

BiologicalMembraneGut Contents

RCOOH

RCOO+H

RCOOH Drug Absorption

X No Drug Absorption

Partitioning of Acids and Bases

Now consider a drug that is basic, with a pKa

of 7.0

•

Where is the likely site of absorption?

BiologicalMembraneGut Contents

RNH2

RNH3

+H Drug Absorption

X No Drug Absorption

RNH2

Partitioning of Acids and Bases

•

When we were calculating where we would expect our acidic or basic drug to be absorbed from, we were assuming that the unionized from of the drug would partition into the lipid membrane.

•

However, this may not be the case: the unionized form of a drug

may still be hydrophilic. An unioinised

hydrophilic drug would therefore

not partition very readily into the body.

•

We therefore have to account for the partitioning properties of our unionized drug when determining whether we expect absorption to occur.

O

H

HO

H

HO

H

OHHH

NH3

O

H

OHO H

OHH H

H

HO

OH

O

H

HO

H

HO

H

OHHH

NH2

O

H

OHO H

OHH H

H

HO

OH

H

Partitioning of Acids and Bases

Similar to Drug absorption except the factor of pH as there is no difference between pH of blood and cell compartments.

-

Physicochemical properties that are responsible for the distribution.

-

Passive diffusion is the main process with little drugs transported by active transport.

Distribution

-

Drug will be fully distributed to all the blood within one minute, but the drug will not evenly distributed to the organs as this is dependent on the blood supply.

-

Drug escape the blood to tissues through capillaries [90-150 A], only the protein bound are not leaving.

-

Once reached the cell, the drug may act on the surface or should cross the cell membrane or even cross the nuclear membrane to reach the nucleic acids.

-

Lipophilicity of some drugs make the estimated dose is difficult to calculate as the drug partition to the fat tissues, e.g.

Barbiturates.

-

BBB is a fatty barrier lining capillaries preventing polar drugs cross toward brain except by pinocytosis

such as insulin

Distribution

Pharmacodynamics:

what the drug does to the body

N

N

How The Body WorksMain systems of the body are:

- CNS: - Sympathetic-

Parasympathetic

-

Signals move through [polarization-depolarization] mechanism

-

Liver:

-

Detoxification process-

Storage device

-

Hormones: -

From where they produced - How they work

To understand all of this you have to understand the molecular level of body organization

Both use neurotransmitters,

adrenaline, acetylcholine

Wikipedia: cell

Drug Targets-

Macromolecules:

1-

Lipids2-

Carbohydrates

3-

proteins4-

nucleic acids

-

For a drug to have an action, it should

interact

[bind] with one of these molecules at different ways.

-

The place of interaction

is called the binding site; which is a curvature or canyon at the surface of the enzyme. ***

-

Two types of interaction are of importance:1-

Irreversible -

covalent bonding (HOW)

2-

Reversible –

by different types of interactions

How Binding Takes Place-

Binding occur through points of attachment, e.g. mountain climbing.

-

For a chemical compound, the attachment pointsare the functional groups.

-

Functional groups use their electronic & shape charactersto perform their binding.

-

Bonds could be inter-molecular

or intra-molecular.

-

If we talk about reversible binding, binding of drug to receptor should be in equilibrium state.

drug has enough power to bind for certain period in order to deliver a task, then leaves to give chance for another drug

molecule to do the same or different task

Points of Attachment

Ligand

Protein

-

The receptor and drug are fully solvated

in water molecules, as the cell is full of water, so the drug should displace

water

before making any interactions with the receptor.

-

Displacement of water obey the second law of energy:

ΔG= ΔH-T ΔS

How Binding Takes place

-

For the interaction to take place ΔG value should be negative, and this is affected by enthalpy and entropy values; ΔH should be more negative [related to interaction energy], while ΔS should be positive to make ΔG value more negative [water molecules]

-

Much polar functional groups on the compound, more solvation

of the drug, more energy needed to desolvate

to allow interaction.

Another Consideration

- Why there is a lot of rings in most of the drugs?entropy issue

How Binding Takes place

Ordered water particles on hydrophobic surface

Bonding Forces-

Electrostatic or ionic forces:-

Happen between fully opposite charges

-

If more than one force are there, ionic is considered the main determinant

The main determinant factors for the strength are:-

As the distance increase, the strength decrease [other forces

are affected to higher extent with distance]

- It obeys coulomb law

-

Nature of the environment, better in hydrophobic media (WHY)

Bonding Forces-

In biological systems, it happen between residues have carboxylate

group; acidic (aspartic acid & glutamic acid) and

basic groups such as Histidine, Lysine and Arginine.

-

In biological systems, the pH is 7.4, while the pKa

of Lysine = 10.5, so the prominent form of this group is …………., pKa

of

aspartic acid is 3.86, so the prominent form is …………..

-

Hydrogen Bonds

-Should have H-Bond acceptor and H-Bond donor.

-The acceptor posses a partial………….charge.

-Mainly depend on electronegativity.

-An important factor is related to orbital interaction (sigma bond characters), so angle is important.

-Distance between 1.5-2.2 A

-NH2

, OH, form one or two H-bonds depending on the number of electron pairs

Bonding Forces

http://www.youtube.com/watch?v=58Vn1dldevE

-

Fluorine, very electronegative that hinders the electrons to be available for H-bond acceptor compared to O,N.

-

Increase electron density, better H-Bond acceptor, so anions better than neutral compounds e.g. aromatic and alkyl amines

-

H-Bond acceptors are better if the H is more electron deficient by attachment to more electron deficient atom such as quaternary ammonium compounds.

Bonding Forces

-

Dipole-

Dipole and Ion-Dipole interaction-

Formed between permanent dipoles on both drug and binding site.

-

Permanent dipoles occur in functional groups such as……………..-

Ion-dipole occur where one of the poles is an ion and the other is permanent dipole.

Bonding Forces

-

Van der

Waals

interactions-

Involve interaction of the hydrophobic

areas in molecules.

-

Formed through temporary

dipole formation, so this one induce others to be formed temporarily

Bonding Forces

-

Happen through formation of covalent bond.-

For covalent bond formation, there should be two poles; the electrophile

and the nucleophile.

-

Nucleophiles in biology have the following functional groups:-

Thiol

in the amino acid ………………

-

Hydroxyl in the amino acid…………..……..-

Amine in the amino acid……………..

-

Carboxylate

in the amino acid……….……..

-

Electrophiles-

Epoxide ring

-

Alkyl group attached to halogen-

Positively charged centre

-

Aziridinum

ion

Bonding Forces/ Irreversible

Bonding Forces/ Covalent

-

Small number of drugs act on lipid targets, mainly by disruption of the lipid structure of cell membranes.

-

Anesthetics, antifungal Amphotericin B

Drug Targets/Lipids

-

Polyhydroxyl

structures (polar), such as glucose, mannose, starch, etc…

-

Important role in cell recognition, regulation and growth.

-

Some are found bound to proteins, and so called glycoproteins

or proteoglycan.

-

For example: bacteria and viruses have to recognize host cells before attacking them, which occur via carbohydrates.

-

Designing a drug that binds to these sugars could block the invader to attack the cells.

Drug Targets/Carbohydrates

-

Primary structure of proteins is formed through linking amino acid through peptide bond.

-

Peptide bond is planar and non-rotatable

due to resonance effect of the amide bond.

Drug Targets/Proteins

-Secondary structure determine the ordered structures such as α- helix or β-turn due to the interaction of amino acid chain residues.

-Tertiary structure is the three dimensional structure of the protein and it is important in determining the function and interaction with drugs.

-Conformation of the protein is determined by the intramolecular forces between the parts of the protein.***

-Covalent bonds happen between two Cysteine residues (thiol

side chains) and called disulphide linkage [oxidation].

Drug Targets/Proteins

-

For the tertiary and quaternary structures, the most important bonding interactions is Van der

Wall ones, not in magnitude for

each bond but in number.

-

Polar residues oriented to the surface-

The inside of the active site usually hydrophobic in nature.

-

Vaspressin

and Oxytocin

contain disulphide linkage that is considered the most important bonding force. [Why?]

Bonding in Proteins

Proteins could be1-

Enzymes

2-

Receptors

3-

Carrier

4-

Structural

-

Carrier Proteins: used to transfer amino acids and neurotransmitters and other biological vital molecules.-

Recognition sites on the surface are necessary to select the

needed molecule to be transferred.-

Drugs that target these proteins such as cocaine, tricyclic

antidepressant.

Drug Targets/Proteins

-

Structural proteins:

such as tubulin

which has a role in cell proliferation, inhibited by Colchicine and cause depolymerization of microtubules.

Drug Targets/Proteins

-

Created for the purpose of performing reactions that are impossible to be done in vivo.

-

Normally reaction happen in equilibrium; reversible in nature.

-

Its role to reduce the activation energy of chemical reactions [How?].

Drug Targets/Enzymes

-

Provide surface for substrates to perform reactions-

Fixing in position

-

Bring reactants near to each other-

Increase the chance of meeting

-

Facilitate the formation of transition state

-

Weakening the bonds of reactants-

Force to change the conformation

-

Proper orientation

-

Participate in reaction-

The hydrophobic character of the active site help the reaction to take place.[ avoid water]

-

Certain amino acids could behave as catalytic or binding role

How enzymes work

-

Binding of substrate to the active site happens with the same bonding forces described before.

-

Structure of the active site and the substrate determine the type of bonding experienced

-

From the natural substrate or active drug structure you can have an idea about some details in the active site, as the relation is complementary one.

How enzymes work

- Old theory: Fischer’s Key and Lock:-

The enzyme and the substrates or [inhibitors] are rigid

structures.

-

There is one optimum substrate and the others are less effectively activated [Drawback, incorrect]

-

Koshland’s

theory of induced fit:- The substrate and more obvious the enzyme change their shapes in order to optimize the binding to some limit.

Binding in Enzymes

This theory succeeded to explain why there is a range of substrates and inhibitors that the enzyme could

accommodate.-

Moulding

process is performed to maximize the interaction

between the two which could cause weakening of substrate bonds and may break them

Lock and Key representation

Induced fit diagram

Acid-Base Chemistry in Enzymes--

Histidine amino acid contains imidazole ring.

-

Imidazole possesses pyrrole

and pyridine like nitrogens.

-

Pka= 6.0-7.0.

-

Dipole momentum is 3.7D in gas, but in solution differ by concentration due to presence of H-bonding.

-

Annular

Tautomeric

equilibrium

134.9

132.6

137.8

1.502

0.884

1.502

136.9

135.8

1.056

1.056

-

At physiological pH histidine is …………(% ionized)

-

Proton Bank, can take and donate protons as needed.

-

Activate water as nucleophile

Acid-Base Chemistry in Enzymes

N

N

H

His

N

NHHis

OH

H

NH H

N

S

Cys

Phe

AcpS R

O

N

N

H

His N

NHHis

OH

H

NH H N

S

CysPhe

O

Mal-ACPACP Acp

O

O

O

-

Serine, Threonine and Cysteine possess a hydroxyl [OH] and thiol

[SH] groups respectively and behave as nucleophiles

-

Considered stronger than water as nucleophiles, why?

-

Presence of neighboring groups help to activate them as nucleophiles by accumulating the charge on them.

-

Helix frying effect

increase the reactivity of thiols

and hydroxyl groups by donating more electrons.

-

Water could act as a nucleophile at later stage as liberating the product from the enzyme if the reaction is reversible.

-

Aspartic acid or glutamic acid could be nucleophiles?

Nucleophiles in Enzymes

Nucleophiles in Enzymes

Helix frying: accumulation of Partial negative charge on one sideof the helix and positive charge at the other side

-

Are non-peptide substances that are required to speed up reactions, could be metals [metal ion activator] such as Zn, Fe, Cu by forming coordinate bond or small organic groups such as NADH, NADPH [coenzymes].

-

Their attachment to the enzyme could be covalent [called prosthetic group] or attached by ionic and hydrogen bonding.

-

Usually are water soluble (look to structure).

-

NADH: used in ATP energy production reaction-

NADPH: mainly for redox

reactions

Cofactors

-

May be used by the enzyme to maintain its stability.

-

Less weakly metal binding enzymes is used for catalysis.

-

One role for metals is to act as electrophilic catalysts, stabilizing the increased electron density or negative charge that can develop during reactions

such as liver alcohol dehydrogenase

[Zn].

-

Another potential function of metal ions is to provide a powerful nucleophile at neutral pH. Coordination to a metal ion can increase the acidity of a nucleophile with an ionizable proton.

Cofactors/ metals

-

Enzymes should have a regulatory body to initiate, slow, stop their performance.

-

Allosteric

binding: product accumulation results in binding the product to different position of the enzyme that cause conformational changes that prevent the active site to perform its normal job.**

-

Why is the binding not in the same place?1-

Different molecule.

2-

competition at he same place between substrate and product -

as if it is inhibitor.

-

Regulation could be done externally such as nitrous oxide and then transferred to the cell via messengers

-

Phosphorylation

by protein kinases

could activate or inactivate enzymes depending on the nature of enzyme.

Regulation of Enzymes

-

More than one domain in enzymes could interact in different way[quaternary structure], then their properties are different such as kinetic properties [Km].

-

The change is due to subunit configuration, and the primary structure could be the same or different.

-

Lactate dehydrogenase

has isozyme

in the heart and muscles that is twice active in the later organ.

Isozymes

-

Competitive Reversible Inhibitors:

- Binding of the [inhibitor] to the enzyme should be stronger than the substrate to compete to the active site, so knowledge of the

amino acids in the active site could help to design stronger binder to the active site.

-

Increasing substrate concentration will reduce the effect of the inhibitor.

-

Sulphonamides

antibacterials

that bind reversibly to dihydropteroate

synthase, that is the folic acid line production

line.

-

Competitively bind to the active site where para-amino benzoic acid

binds.

-

Expected to have structural similarity to the substrate.

Enzyme Inhibitors

-

Non-competitive irreversible inhibitors:

-

Inhibitor that bind to the active site residues permanently and the strongest when the binding is covalent.

-

Penicillins bind to the bacterial enzymes irreversibly, bactericidals.

-

Aspirin binds to serine residue of COX enzyme.

--

Non-competitive reversible inhibitors [Allosteric]:

-

Bind to different site, so there is no necessary resemblance to the substrate and the high levels of the substrate not affect

the inhibitory effect.

-

[6-mercaptopurine], anticancer

Enzyme Inhibitors

-Transition state analogues inhibitors:

-

Design a drug that is similar to the transition state, so it is

bound irreversibly to the active site even without covalent bonding because the transition state found to be the most fixed structure compared to substrate or product.

-

Renin

inhibitors and viral protease inhibitors.

Enzyme Inhibitors

-

Special case of irreversible inhibitors as the normal substrate

will be converted in the active site to highly reactive species that alkylate the enzyme and finish it permanently.

-

Presence of an activating group in the structure cause this phenomena.

-

Has an advantage that they are selective and will not act as suicide inhibitors until reach the active site.

-

Clavulanic

acid act as a suicide substrate for bacteria.

-

5-fluorouracil, act as anticancer agent converted to reactive 5- flourodeoxyuracil monophosphate.

-

Presence of more isozymes

give the advantage to selectively inhibit one form rather than the other. e.g: COX1 and COX2.

Enzyme Inhibitors/ Suicide substrate

-

Mechaelis-

Menton

Equation

Enzyme Kinetics

Rate =

Km

is the concentration of substrate at which half of the active sites of theenzymes are filled.

Km

is the measure of how strong the substrate bind to the enzyme inversely.Km is dependent pH, temperature and ionic strength.

-

Lineweaver

Burk plots:

Enzyme Kinetics

reciprocals

Receptors-

Mostly membrane bound proteins, that selectively bind to small

molecules called ligands.

- Generally are integral

proteins.

- Composed of two parts, the recognition

and the amplification

sites.

- Ligand receptor interaction happen using the same bonding types that are used for drug-enzyme interaction.

-

Their presence is a method of communication between the different parts of the body (synapses, vasodilatation).

Receptors/ Communication Agents

-

Receptors receive endogenous chemical compounds such as neurotransmitters

(seratonin, acetylcholine) and hormones,

aminoacids, lipids (prostaglandins) and others.

-

The surface of receptors contain as in enzymes hollows, ravines that are considered the Binding Site.

-

Drug that act on these receptors could be either blocking the action of the receptor (Antagonist) or act as if they are the normal exciting agents (Agonist).

-

As in enzymes, ligand

binding induces conformational changes at receptor that in turn the message will be conveyed in different ways (ion flux, transfer a message to inside cell).

Agonist Design-

Required when there is a shortage

of the endogenous chemical

compound and we require to compensate for this shortage.

-

To design an agonist

you have to know:-

The geometry and topography of the active site and /or.

-

The chemical structure preferably (3D structure) of the normal substrate that act upon.

-

Thorough dissection for the Ligand

or the Binding site

will enable us to have an idea about:

-

The important

binding groups.-

The correct position of the binding groups that are related to their arrangement and distances.

-

The right size of the binding groups and sites (ISOSTERS)

-

Before enrolling in agonist design, you have to know the role of each functional group in terms of binding (Chemistry wise)

Functional Groups Binding Role-

Alcohols and phenols:-

Act as H-bond donor and acceptor with directional preference that is related to the tetrahedral geometry of the oxygen atom.

-

Their importance can be tested by methylation or esterification of the hydroxyl group which obviously remove their H-donor

properties and retard the H-

accepting characters (How?).

-

Esterification

will not necessarily give better H-accepting properties due to presence of two oxygen atoms instead of one. The reason is that the position of the new oxygen is not necessarily the same as the other oxygen, and the resonance effect will reduce the electron density over the other interacting oxygen.

Functional Groups Binding Role

-

Aromatic Ring and alkene:

-

Planar, hydrophobic and participate in van der

Wall interactions with hydrophobic areas within the active site.

-

Benzene importance is compared by cyclohexane

analogue which is v expected to be less effective binder due to:

-

Loss of planarity.-

The axial Hs will shield the cyclohexane

ring to be in proximity to

the hydrophobic region.-

Unable to bind in slots as what occur in benzene ring (Bulkier).

-

Incapable to form induced dipole interactions with ammonium ion.

Vs

-

Ketones and Aldehydes:

-

Planar hydrogen bond acceptor in which the oxygen is sp2

hybridized, so the lone pair of electrons are found in the same plane.

-

Participate in dipole-dipole or dipole-ion interactions.

-

Tested by its reduction to the corresponding alcohol (tetrahedral geometry).

-

Reduction will result in weakening of the binding properties, and if the oxygen is expected to have the same role as keton

(acceptor) then ether analogue could be studied.

Functional Groups Binding Role

-

Amines:

-

Could be H-donor and H-acceptor.

-

Aromatic and hetero-aromatic amines act as H-donor due the busyness of the lone pair of electrons in the aromatic system.

-

If ionized, it will be stronger H-donor with loss of H-accepting power.

-

Converting it to amides will rule out the possibility of H- accepting and ionic bonding character. (resonance will remove the

lone pair of electrons from N).

-

Secondary amide still have H, but due to steric factors it will hinder to be as H-donor.

Functional Groups Binding Role

-

Amides:

-

Bind active site through hydrogen bonding (carbonyl as acceptor and NH as only donor if the amide is primary or secondary).

-

Could be tested by N-methylated amide, primary/secondary amine, tertiary amine, ketone, alkene, carboxylic acids.

-

all except 1, 2 amine could test if H-

bond donor.

-

alkenes and amines check if H-

bond acceptor.

-

All the above groups except alkene

are not safe for testing amides as all could rotate in contrast to amides.

-

Alkenes are good tester as both H-donor and acceptor, but difficult to synthesize.

-

Lactams

could form intermolecular H-bonding.

Functional Groups Binding Role

-

Quaternary Ammonium Salts:

-

Ionized group that could form ionic bonding with carboxylates

or induced dipole interaction (Face of the ring is negative and positive at the edges).

- Tested by synthesizing a tertiary

amine instead (but could be protonated, so amides are better testers).

-

Acetylcholine contains quaternary ammonium group.

-

Carboxylic acids:-

Act as H-donor and acceptor, but could be as carboxylate

moiety

which in turn will be ionic bond pole or strong H-acceptor.

- Tested by synthesizing analogues such as ethers, alcohols and ketons.

Functional Groups Binding Role

Esters:-

4 sites as good H-acceptor, and could be tested by converting to

ethers.

-

Faced by the problem of esterases

in the body.

-

Esters could be protected from hydrolysis by electronic or steric factors.

-

Could be used deliberately to make pro-drugs.

-

Alkyl and Aryl Halides:-

Chemically reactive species and considered good leaving groups,

so

react with any nucleophile covalently.

-

Alkyl fluorides are not reactive as C-F is strong bond, and used to replace H because has the same size [used to protect from metabolism].

Functional Groups Binding Role

-

Aryl halides are not alkylating agents but affect the binding of the aromatic ring as they considered electron withdrawing groups.-

Tested by their counterparts that lack the halide moiety.

-

Thiols:

-

The SH group is considered a good ligand

for zinc ion, and is incorporated in drugs that target enzymes contain zinc as a cofactor.-

Tested by their counterpart alcohol which is less reactivity to

metals.

-

Heterocycles:

-

Very important as they form variety of bonding with the active site [mainly H-bonding]

Functional Groups Binding Role

-

The designed agonist

should contain all the necessary binding groups that the natural substrate posses in order to have the optimum response [Take in consideration the pharmacodynamic

properties].

-

In a series of agonists, there should be a great similarity

in structure between these molecules.

-

Chirality

is an important factor that should be taken in consideration as enantiomers

or diasteromers

do not bind in the

same way as receptors are enantiospecific.

Agonist Design

Agonist Design-

One of the enantiomers

will bind and the other will have no role in the

therapeutic activity or cause side effects.

-

Antagonist for the binding site:

-

They act by having some or all the binding groups but fail to induce an effect which could be achieved by:

-

inability to change the receptor conformation or,-

distort the receptor in wrong way.

-

Competitive antagonists

are not necessarily have great similarity to the substrate

or to each other.

-

These differences in structure are justified by the fact that antagonist is required to bind to sites near the active site that prevent physically the substrate to reach target.

-

In general, Antagonists are bulkier than agonists. And it is easier to make compound that block receptor site than molecule have specific interactions to induce conformational changes.

Antagonist Design

-

Addition of competitive antagonist shifts the curve to the right.

-

Allosteric

antagonist: bind in different site and could alter the shape of the active site (called non-competitive antagonist).

-

It is independent of the amount added of the substrate [Kd

not changing]

-

Umbrella effect: design of an competitive inhibitor that exploit the neighboring areas of binding sites which consequently will retard the binding of normal substrates.

-

The same bonding interactions could be used to for this design [ionic, van der

Wall, H-bonding].

Antagonist Design

Competitive Versus Noncompetitive Antagonist

-

A compound that cant be defined surly as agonist or antagonist. It act as agonist but not reach to the same level of substrate response.

-

It should bind to the same binding site with low ability to induce change.

-

It could bind partially to the binding site, but the other part will bind as antagonist.

-

Could be used to distinguish between different types of sub- receptor.

Partial Agonist

Buprenorphine

,used for opioid

addiction

Morphine

-

It is common to antagonist in that bind to the receptor binding site and prevent the normal substrate to act; however, it differs from the antagonist in that it exert an action that is opposite to the substrate.

-

It may act by preventing inherent activity to some receptors such as GABA and dihydropteridine.

-

Not necessarily bind to the same site of substrate and no need to have structural similarity to substrate.

-

Examples:-

Valium (diazepam): is an anticonvulsant while β-carboline

is an

inverse agonist and induce convulsion.

Inverse Agonist

http://images.google.jo/imgres?imgurl=http://upload.wikimedia.org/wikipedia/en/thumb/4/4b/Benzodiazepine_core_structure.png/255px-Benzodiazepine_core_structure.png&imgrefurl=http://www.nationmaster.com/encyclopedia/Benzodiazepine&usg=__MzkZ1TBFbe1R0LSvbnu4a3XsIoc=&h=316&w=255&sz=12&hl=en&start=4&um=1&tbnid=QNedo-ZAtdGP5M:&tbnh=117&tbnw=94&prev=/images%3Fq%3Dvalium%2Bstructure%26um%3D1%26hl%3Den

http://upload.wikimedia.org/wikipedia/commons/6/6d/Inverseagonist.gif

Desensitization & sensitization-

When a drug bind to receptor strongly it will act as agonist, then for this long time of binding the effect will be inversed and become

antagonist.

-

Phosphorylation

of the active site residues such as hydroxyl or phenol will change the conformation and inactivate it.

-

When ligand

leave, dephosphorylation

will occur.

-

If no leave of the ligand, the receptor will be enocytosed

and metabolized.

-

Desensitization will occur by reducing the receptors as the there is continuous activation by the tight binding.

-

Sensitization, occur when there is continuous use of antagonist,

so cell produce more receptors.

1991 -

Burger A.•

Compounds or groups that possess near-equal molecular shapes and volumes, approximately the same distribution of electrons, and which exhibit similar physical properties... .

Bioisosterism-

Isosteres: Atoms or group of atoms which have the same valency

(number of outer shell electrons) and have chemical or physical similarities.

-

Bioisoster: it include both classical and non-classical isosters, and it is a group that can be used to replace another group while retaining the desired biological activity.

-

Used to replace a functional group that is important for binding

but problematic in a way or another.

-

Used in drug design to vary the character of the molecule in rational way with respect to features such as size, polarity, electronic distribution and bonding.

-

Uses:-

Determine the importance of some binding groups

-

Investigate the type of binding some groups posses.

-

Example: propranolol

is a β-blocker that has an ether linkage; its replacement by CH=CH, SCH2

, CH2

CH2

, will eliminate activity. But replacement with NHCH2

retain activity.

-

F

is replaced by H

because they have similarsize properties but different electronic behavior.[no effect of size]

-

5-flourouracil is consumed by the substrate as it has the same size as Uracil, but the C-F

bond is strong and not broken as if it is H.

Bioisosterism

HN

NH

O

O

H(F)

O NHOH

Sultopride:

Dopamine antagonist was improved by replacing the amide by pyrrole

ring has led to increase activity and selectivity of D3

over D2.

NHON

OMe

EtO2S

NH

N

OMe

EtO2S

Bioisosterism

-

Transition state isosteres: are moieties that are used to mimic the crucial features of the transition state but which are stable.

-

Tetrazole

is a bioisoster of carboxylic acid, its replacement in the first compound has led to the discovery of losartan

(angiotensin

II

inhibitor).-

Planar, ionized, pKa= 4.7

-

More lipophilic resist metabolism

Bioisosterism

- Univalent isostere:

-

Bivalent isostere:

-

Trivalent isostere:

-

Ring equivalents:

Bioisosterism

N-

S OHN

-

- CH3, NH2, OH, F, Cl, SH- Br, i-Pr- I, t-Bu

-

Carbonyl group:

-

Carboxylic acid group:

Nonclassical

isosteres

OCNNC

OS S

OOS NO

O O

N

CN NOH NOCH3

O

OHS NO

O

H

RS OHO

OS NO

O

H

CN

NH

NH

OSO

OAr N

H

OSO

OAr

SNH

OSO

OAr

O

NO

NS

NO

NN

NH

X

N NN

HN

OOH OH

HO

OH

OO

OOH

NN

NOH

NNOH

N

NOH

N

NOH

OHFF

-

Amide:

-

Ester:

Nonclassical

isosteres

-

Hydroxyl group:

-

Catechol:

-

Halogens:

Nonclassical

isosteres

HO

HO

N

NH

X

O

HO

X= O, NR

NHO

O SHN

O

HO

-

Benzene:

-

Spacer:

Nonclassical

isosteres

Affinity, efficacy, and Potency

-

Affinity:

how strongly the drug bind to the receptor; depends on the molecular complementary of drug and receptor.

-

Measured by radio-ligand

labeling.

-

Efficacy: the maximum biological effect the drug can produce.

-

Potency: the amount of drug needed to achieve defined biological effect.

-

(Scatchard

plot, Schild

analysis ) refer to book.

-

An agonist is something which binds with both affinity and efficacy.

-

An antagonist is something which binds with affinity but no efficacy.

Nucleic Acids as Drug Targets

DNA Bases-

Purines:

Heterocyclic aromatic compounds that are composed of

pyrimidine

attached to imidazole.

-

Pyrimidines:

there are three bases for the DNA

and RNA?

Adenine Guanine

-

(Cytosine, Thymine, and Uracil)

-

Back to slide 1, you should be able to know the strength of the H- donors and acceptors and the reason behind this strength or

weakness.

DNA Bases

Drugs Act on DNA-

Drugs classified as Intercalating, alkylating and Chain cutters.

-

Intercalating:-

They must contain flat part of the molecule [Aromatic and Heteroaromatic].

-

Act by sliding between the DNA strands disturbing the helix structure and retard transcription and replication.

-

Examples: Proflavine, Dactinomycin, and Doxorubicin.

-

Intercalator

could be minor groove binder or major groove binder.

-

Proflavine

is an aminoacridine

derivative that possess yellow color, and used as antibacterial agent.

-

Used topically as it is toxic to the host cells systemically.

-

Actinomycin

D

(Dactinomycin):

naturally occurring antibiotic, posses flat phenoxazone

ring that bind to the minor groove.

-

Prefer the binding to G-C pairs and between two adjacent G-G units.

-

Form stable complex that prevent DNA-RNA dependent polymerase

to unwind the DNA which eventually cause cell apoptosis

and death.

Drugs Act on DNA

Pentapeptide

portion

-

Doxorubicin:-

Naturally occurring antibiotics group called Anthracyclines.

-

They are major groove binders and slide using the three flat cycles.

-

The positively ionized amine group interacts with the negatively phosphate groups.

-

They inhibit Topoisomerase

II. Very important enzyme for replication of DNA.

-

Hydroxyquinone

moiety chelates

iron, then produce reactive oxygen species which breakage of the DNA strands.

Drugs Act on DNA

-

Alkylating agents:-

Highly electrophilic compounds that react with nucleophiles in the DNA such as ………. to form covalent bonds.

-

Examples are : Nitrogen mustard (Chlormethine), Nitrosoureas (Lomustine), and Cisplatin.

Drugs Act on DNA

-

Chain Cutters:-

Cut the strands of the DNA, and prevent the DNA ligase

from

repairing the damage.

-

They act by forming oxygen radicals and peroxy

species.

-

Example: Calicheamicin

γ1, isolated from bacteria, binds to the minor groove and cuts the DNA by forming highly reactive species radical species.

Drugs Act on DNA

Enediyne

moiety

Trisulfide

-

Flouroquinolones:-

Form complex with DNA and with the enzyme topoisomerase

IV; and enzyme used to reduce tension through unwinding DNA for replication.

-

Nalidixic

acid the first agent of this group, enoxacin, ciprofloxacin.

-

They act by forming ternary complex [Quinolones, DNA, topoisomerase], so the cut DNA will not be sealed.

Drugs Act on DNA

Bin

ding

toEn

zym

e

Ciprofloxacin

Pharmacokinetics-Metabolism

Metabolism

-

Biochemical modification or degradation, usually through specialized enzymatic systems, often converts lipophilic chemical compounds into more readily excreted polar products.

-

Non-specific enzymes such as Cytochrome

P450

enzymes, add polar functional groups to wide variety of drugs.

-

Other group of enzymes unmask already polar groups such as methoxy

group that when demethylated

will give hydroxyl group.

-

Reactions are classified as Phase-I

or Phase-2; Phase-I

are Oxidation, reduction and hydrolysis. (functionalisations)

Metabolism

-

The main site of these reactions is the liver

but could be found in gut wall, blood plasma and other tissues.

- Oxidation mainly targeted groups such as N-methyl derivatives, aromatic rings, terminal position of alkyl groups and the least hindered position of alicyclic

rings (is an organic compound that is

both aliphatic and cyclic. They contain one or more all-carbon rings which may be either saturated or unsaturated, but do not have aromatic character).

-

Reduction

occur to groups such as Nitro, Azo, and Carbonyl

-

Amides and esters are prone to hydrolysis.

-

Drugs in general could be exposed to more than one reaction of metabolism.

-

Knowledge of the metabolic pathways enable the medicinal chemist to know the products expected to be formed.

-

Phase-II

reactions occur mainly in the liver, and most of them are conjugation reactions

–

polar conjugates are attached to polar

groups found in the drug. (conjugation reactions)

-

Both Phase-I, Phase-II are species specific, so what is found in vivo studies as metabolites in rats not necessarily the same as humans.

-

Also both of these reactions are regio-specific

and stereo-selective.

Metabolism

-

Cytochrome

P450 system: The most important enzymes in metabolism and are positioned in liver cells.

-

These are haemoproteins

(contain heme

and iron), act by catalyzing reactions that split molecular oxygen, one for the drug and the other as water molecule. [belong to enzymes called monooxygenases].

-

There are 33 enzymes of P450, classified to four groups CYP1 - CYP4.

-

Sub-groups classified by letter then by number such as CYP3A4.

-

Most drugs in current use are metabolized by five primary CYP enzymes (CYP3A, CYP2D6, CYP2C, CYP1A2, and CYP2E1).

-

The isozyme

CYP3A4 is particularly important in drug metabolism and is responsible of most drugs.

Metabolism

-

Oxidation

occurs to carbons that are exposed or activated; methyl groups of a carbon skeleton easily accessible to enzyme as they are exposed and form alcohol upon oxidation.

-

Long chains aliphatic compounds will have the last and the penultimate carbons will be highly exposed.

-

Aliphatic rings also will be oxidized to positions that are exposed.

-

Carbon atoms next to sp2

or sp

centers are activated and prone to oxidation. ***

-

Carbon atoms that are α-position to a heteroatom

which form unstable metabolite that immediately hydrolyzed and cause dealkylation

of amines, ethers and thioethers

or dehalogenation

of

alkyl halides. [Aldehyde]

Metabolism-Oxidation

-

Oxidation of unsaturated systems such as double bonds, triple bonds and aromatic systems. Alkenes for an epoxide then deactivated by epoxide hydrase

to form diol.

-

If the epoxide escaped the enzyme it will react with nucleophiles in the body and cause toxicity.

-

Aromatic rings in the same way they form epoxide intermediate that could be :

-

Rearrangement by which a hydride will be transferred to form a phenol, normally at para position.

-

Deactivated by epoxide hydrolase to form diol.-

React with glutathione S-transferase

[conjugation].

Metabolism-Oxidation

-

If the aromatic epoxide evaded the enzyme, it has proven to be alkylating agent and toxic. [electron rich aromatic rings are faster in metabolism than electron deficient].

-

Tertiary amines are oxidized to N-oxide, if there is no steric hindrance, then immediately converted to hydroxyl amines.

-

Aromatic primary amines oxidized to nitro, which are toxic.***due to the formation of highly electrophilic intermediate that alkylate

DNA

and proteins.

-

Primary and secondary amides are oxidized to hydroxyl amines that found related to toxicity and carcinogenicity.

-

Thiols

oxidized to disulfides or methylated to methyl sulfides then oxidized to sulfoxides

and sulphones.

Metabolism-Oxidation

--

“Oxidation of aromatic moieties (arenes) to their corresponding phenolic metabolites (arenols)”.

-

Most of the hydroxylation processes occur at para position.

-

Substituents nature affect the ease of hydroxylation process, in general, electron rich rings are hydroxylated

faster, while

deactivated rings attached to (COOH, Cl, N+R3, SO2

NHR) usually resistant or slowly hydroxylated.

-

If there is two aromatic rings, it is expected to hydroxylate

the electron rich ring.

Metabolism-Oxidation

-Examples:

-

Resistant compounds to hydroxylation processes.

Aromatic oxidation

-

More activated rings hydroxylated

first.

Aromatic oxidation

-

Polychlorinated dibenzo-1,4-dioxins are extremelytoxic such as (Sevesodioxin, TDCC-

2,3,7,8-

tetrachlorodibenzodioxin).-

TDCC: is a teratogenic

with LD50 in rats

45 μg/kg.-

Formed as a byproduct of commercial 2,4,5-trichlorophenol.

-

Forms epoxide that is more stable than the aromatic epoxide.-

Hydrolysis of the epoxide by hydration lead to trans 1,2 diols.

-

The following compounds form toxic epoxides.

Olifens

oxidation

Aflatoxin

-

“Carbon atoms attached to aromatic rings”.-

Oxidized to alcohol, aldehyde

(ketone) and then carboxylic acid.

-

Allylic

oxidation:

Benzylic/ Allylic

carbon oxidation

-(C=O) and imino

(C=N).

-

Oxidation of aliphatic and alicyclic

carbon atoms. (Omega and ω-1).-

Alicyclic: They contain one or more all-carbon rings which may be

either saturated or unsaturated, but do not have aromatic character

Oxidation of carbon at α

position to carbonyl & imines

Alicyclic

oxidation-

Mono substituted cyclohexyl group is usually hydroxylated

at

positions 3 and 4 with the possibility of cis and trans.

-

C-N, C-O, and C-S oxidation involve two main types.1-

Hydroxylation of the α-carbon atom directly attached to

heteroatom, to produce unstable intermediate which decomposes by cleavage the C-X bond.

2-

Hydroxylation of the heteroatom (N,S only) forming N- hydroxyl, N-oxide, sulphoxide

and sulphone.

Oxime, nitrone, nitroso, imino

Carbon-Heteroatom systems oxidation

-

Tertiary amines: oxidative removal of alky group (oxidative N- dealkylation) by P-450. Started by α-carbon hydroxylation to form

carbinol

amine intermediate, then cleavage of C-N bond to secondary amine and carbonyl moiety (Aldehyde

or keton).

-

Small alky groups are normally removed quickly, and the first is removed faster.

C-N system oxidation

Methadone to pyrrolidine

ring cyclization

-

Complete dealkylation

reactions will lead to oxidation of primary amine to carboxylic acid.

-

t-butyl moiety is not possible to be removed

because no alpha H to be hydroxylated

with the exception of t-butyl-norchlorcyclazine,

which occur through oxidation of terminal CH3

to carboxylic acid then decarboxylated

to produce H at alpha carbon.

-

Tertiary alicyclic

amines usually form Lactams

(nicotine).

C-N system oxidation

RX

H

RX

OH

R XH

O

ClN N

ClN N

OH

ClN N

COOHCl

N N

-

Secondary and primary amines:Undergo N-dealkylation, oxidative deamination, and N-oxidation reactions.

-

Carbinol

amine pathway is the same for tertiary amines, then produces primary amine.

-

Examples: propranolol, methamphetamine (dealkylation

to form keton

with same carbinol

amine intermediate).

-

Oxidative Deamination:

process by which a molecule loses the primary amine group by the same carbinol

intermediate.

-

Norketamine

does not undergo N-deamination. (why?)

-

In general, the first step is N-dealkylation, then deamination but there is exception such as propranolol

(aldehyde).

C-N system oxidation

-

Also some alicyclic

secondary

amines are transformed to their corresponding lactams

(phenmetrazine, methylphenidate).

-

N-oxidation

also happens but to less extent to form N- hydroxylamine

that is prone to form nitrone

derivative (N-

benzyl amphetamine, phenmetrzine).

-

Primary amines

normally undergo oxidative deamination or by N- oxidation. (endogenous compounds such as neurotransmitters

oxidized via monoamine oxidase [MAOs]).

C-N system oxidation

-

Phentermine

is dependent on the possibility of alpha

carbon oxidation (structural features of alpha hydrogen availability).

-

Decarboxylation

step could happen first then deamination occur (methyldopa).

-

N-hydroxylation

could occur first then converted to imine

by water loss, then converted to oxime

which will be converted to

ketone

(amphetamine).-

Primary aliphatic amines which are not possible to be oxidized at alpha position will be N-hydroxylated

and further oxidation

produced nitroso

and nitro

compounds. (phenteramine, amantadine).

C-N system oxidation

Aromatic amines and heterocyclic Nitrogen compounds

-

Tertiary aromatic amines

will undergo N-dealkylation

and N-oxide formation.

-

Secondary amines

undergo N-dealkylation

and

N-oxidation

to give N-hydroxyl amines which will oxidized again to give nitrone

derivatives, which they may hydrolyze to primary hydroxylamines.

-

Tertiary and secondary aromatic amines are not common in medicinal drugs while the primary amines are abundant

(from

enzymatic reduction of aromatic nitro compounds, reductive cleavage of azo

compounds and hydrolysis of aromatic amides).

-

Primary aromatic amines first produce hydroxyl derivative, then to nitroso.

C-N system oxidation

-

Aromatic N-oxidation

is considered a minor

constitute compared to N-acetylation

and aromatic hydroxylation.

-

Methemoglobinemia

is a common side effect of aromatic amines (dapsone) when converted to hydroxyl derivative. It oxidizes the Fe+2

to Fe+3

in hemoglobin which will prevent oxygen transport (suffocation).

-

Aromatic amines are considered carcinogenic: activated by N- oxidation to make them highly electrophilic and alkylated by DNA,

RNA.

C-N system oxidation

NN

NH

NN

NOH

NN

NOSO3

-

NN

N+

nitrenium ionN

N

N

GLG

-

N-oxidation

of N atoms inside heterocycle

occur less common to produce N-Oxide metabolite, (trimethoprim, cotinine

and

metronidazole).

C-N system oxidation

-

Oxidative C-N cleavage

(α-

carbon hydroxylation) and N- hydroxylation reactions.

-

Oxidative dealkylation

occur through carbinolamide

intermediate, unstable, fragmentation to form N-dealkylated

product (Diazepam).

-

Lactams, in the same way by forming carbinolamide

that lead to C-N breakage

(Cotinine).

-

Cyclophosphamide

has many metabolites, see book.

-

Aromatic amides, minor extent, toxicological importance, 2-acetyl aminoflourene

(AFF) [N-oxidation, sulfonation, then nitrinum

ion

production].

Amides

Diazepam Flurazepam

chlorpropamideCotinine Cyclophosphamide

- Acetaminophen:

Amides

HN CH3

O

OH

Renal excretion

HN CH3

O

ON-acetylamindoquinone

HN CH3

O

O

HN CH3

O

O

Glucuronide

SO3O-

Cause covalent binding with livercells, necrosis

-

Performed via microsomal

mixed function oxidases.

-

Oxidation involve α-oxidation to form hemiacetal

or hemiketal, followed by C-O bond breakage (phenol, alcohol) and (keton

or

aldehyde).

-

Small alkyl groups removed first (morphine).

-

Mescaline where the 3-O demethylation

is favored.

C-O System Oxidation

R1 H

OHOR2

R1 R3

OHOR2

-

Undergo S-dealkylation, desulfuration, and S-Oxidation. The first two involve C-S bond cleavage.

-

S-dealkylation

is proceeded in the same way as C-O and C-N dealkylation

by oxidizing the α-carbon.

-

Examples are 6-(methylthio)-purine produced 6-mercaptopurine.

-

Desulfuration: Conversion of thiono

(C=S) or (P=S) to (C=O) or P=O respectively.

-

Thiopental to pentobarbital, and parathion to paraoxon.

C-S System Oxidation

N

N NH

N

S

N

N NH

N

SH

HN

NH

S

O

O

S COOH

CF3

S

-

S-Oxidation reactions:-

Sulfide to sulfoxide

to sulphone.

C-S System Oxidation

-

Alcohol is produced from different metabolic pathways such as……

-

If the OH is not conjugated, it will further oxidized.

-

Primary alcohol and aldehydes

give facile oxidation to carboxylic acid.

-

Less important, secondary alcohol to keton, not that important as it may be reduced again to alcohol as it is easier to be conjugated.

-

The enzyme called alcohol dehydrogenase

perform reversible reaction that converts alcohol to aldehyde

and keton, using

NAD+ as a coenzyme.

-

Further oxidation of aldehyde

to COOH, is done by aldehyde oxidaze

and xanthine

oxidize.

Oxidation of Alcohols and Aldehydes

-

Oxidative aromatization, as in norgestrol.

-

Dehalogenation: halothane to trifluroacetic

acid with carbinol intermediate and HBr

elimination.

-

Chloroform produce phosgene (hepato

and nephrotoxicty).

-

Normally dehalogenation

reactions produce toxic acylhalides.

Other Oxidation Reactions

OH

HH

OH H

-

Have an important role in drugs contain carbonyl, nitro and azo functional groups, which usually followed by conjugation reactions.

-

Less common reduction reactions such as N oxides, sulphoxides, S-S and C-C cleavage reactions.

-

Aldehydes and ketones:-

Source:

-

From drugs.-

Oxidative deamination reactions.-

Aldehydes to primary alcohol

(but most of its reaction is oxidation to

COOH), rare case is the conversion of chloral hydrate to trichloroethanol

(then phase II).

-

Ketones to secondary alcohol then to conjugation reactions.

-

These reactions are normally performed by aldo-keto

reductase (NADPH) or oxidoreductase

enzyme (alcohol dehydrogenase).

Reduction Reactions

-

Propranolol

as major metabolite after deamination is COOH derivative and as minor is propranolol

glycol.

-

Chlorphenramine

first dealkylated, deaminated

then it could undergo oxidation or reduction.

-

Ketone reduction reactions are stereo-selective, which involve H transfer to the carbonyl group and then one steroisomer

will be

preferred over the other (Acetophenone, Warfarine).

-

Examples of compounds undergo oxidative deamination to ketone

and then reduction to alcohol (amphetamine, ephedrine).

Reduction Reactions

N

N

Cl

Chlorphenramine Acetophenone Warfarine Amphetamine Ephedrine

-

Nitro and Azo

compounds:

-

The end product is primary amines.-

Aromatic nitro to nitroso

to hydroxylamine to amine.

-

Aromatic azo

to hydrazo

to cleavage to two aromatic amines.-

Nitro reductase

and NADPH are needed for nitro reduction (7-

nitrobenzodiazepine [clonazepam, nitrazepam]).-

Prontosil

(azo) to the active metabolite sulfanilamide.

-

Tartrazine

and amaranth cleaved by intestinal bacteria. Sulphasalazine

is hydrolyzed to sulphapyridine

and 5-aminosalicylic acid.

Reduction reactions

PRONTOSIL SULPHASALAZINE

S NNO

OH2N

H2N

NH2 N N

SO

O

HN N

HOOC

HO

Clonazepam Nitrazepam

A.

(N-Oxide to tertiary amines).-

reduces the polarity of the tertiary amines, so reduce excretion.

B.

(Disulphide reduction).-

Disulfiram

is converted to N,N-dithylthiocarbamic

acid and

sulindac

(sulphoxide

to sulfide).

Other Reduction Reactions

N

SS

SS

NN

S SH

HO

O

F

SO

-

Esters and amides:-

Occur in various tissues and plasma.

-

The products are (COOH, alcohols, phenols, and amines). The result is more polar and easier to be conjugated.

-

Enzymes involved for esters are esterases

in liver, kidney and plasma and for amides amidases, esterases

and deacylases.

-

For drugs contain esters, hydrolysis is the

major

route because it is easily cleaved.

-

Examples (Aspirin, cocaine, ritaline), normally esters are prodrugs

that are activated inside the body such as clofibrate, diphenoxylate.

Hydrolytic Reactions

N

N

OO

-

Examples of using esters as prodrugs:”-

Clindamycin

& Chloramphenicol: as palmitate

ester to hide the bitter

taste of these drugs.

-

Geocillin, improve poor oral absorption of carbenicillin

[Indanyl

ester].

-

Prednisolone

hemisuccuinate

sodium salt for I.V injection.

-

Amides

hydrolyzed slowly compared to esters (procaine, procainamide).

-

Other examples: indomethacin

and prazocin.

Hydrolytic reactions

-

Other Hydrolysis reactions:

-

Hydrolysis of proteins and hormones at their terminal amino acid

groups by aminopeptidases; insulin, GH, prolactin, and PTH.

-

Hydrolysis of epoxides, and areneoxides.

-

Hydrolysis of phosphate esters, carbamate

esters, and cardiac glycosides.

Hydrolytic reactions

-

Aims to produce water soluble moiety, but not necessarily abolish parent compound activity.

-

Adds polar, small, endogenous and ionizable group to phase I metabolite or parent xenobiotics

such as (glucuronic acid, sulfate,

glycine, glutamine).

-

Generally, these metabolites are nontoxic and not active.

-

Other phase-II reactions such as acetylation

and methylation are not increasing water solubility, rather they act to terminate or

attenuate pharmacological activity.

-

The conjugated residues are first activated as coenzyme before transfer and attachment by transferase enzymes.

Phase II: conjugation reactions

-

The most common conjugation pathway due to:

-

Readily available D-glucuronic acid (from D-glucose).-

Many functional groups can be united with glucuronic acid.

-

Its ionized carboxylic acid and the polar OHs

increase water solubility to high extent.

-

β-glucuronides

formation involves two steps:-

Synthesis of activated coenzyme (UDPGA)

-

Transfer of glucuronyl

moiety to the xenbiotic

by UDP- glucuronyltransferases.

-

One step of glucuronation is sufficient to excrete a compound, so di-

process is not common.

-

Common features of the binding are :-

occur at C1 of the glucuronic acid.

-

The acceptor has the formula HXR, that the OH of glucuronic acid will leave.

Phase II: Glucuronic acid conjugation

-

There are many functional groups that can be glucuronated:-

O-

glucuronation: hydroxy and carboxy.

-

Hydroxy:

alcoholic or phenolic are the most common FGs

that undergo glucuronation.

-

Less common hydroxy groups undergo glucuronation are enols, N-hydroxyl amines, and N-hydroxyl amides.

-

Carboxy: Aryl acids prefer conjugation with glycine

but could be glucuronated.

-

N-

glucuronation: -

Occur occasionally with aromatic amines, amides and sulphonamide. Considered minor pathway compared to N-acetylation, or oxidative process.

-

Some compounds form quaternary ammonium glucuronide

metabolite.-

S-glucuronation:

-

With the thiol

group.-

C-

glucuronation:

-

Novel form of conjugation occur in little examples (Phenylbutazone).

Phase II: Glucuronic acid conjugation

-

Bile excretion of glucuronated endogenous compounds occur for compounds more than 300Da, which may be hydrolyzed by β-

glucuronidase.

-

In neonates and children, glucuronidation

is not fully mature, so some drugs and bilirubin

could be accumulated and cause serious

toxicity (gray baby syndrome).

Phase II: Glucuronic acid conjugation

-

Occur mainly with phenols, and to less extent alcohols, aromatic amines, and N-hydroxy compounds.

-

There is limited amount of sulphate, so it is limited reaction.

-

used extensively by the body to excrete endogenous compounds such as steroids, heparin, catecholamines.

-

The process involve formation of PAPS and then transfer process by sulfontransferase.

-

Lead to water soluble and inactive metabolite, but some O-sulfate and N-hydroxy compounds give toxic metabolites.

-

Low level of glucuronyltransferase

or undeveloped enzyme may cause acetaminophen to be mainly excreted as sulfate conjugate in neonates rather than glucuronyl

derivative as both process are

competitive.

Phase II: Sulphate

conjugation

-

O-sulfate ester conjugates and N-hydroxy compounds are important as they could cause reactive toxic intermediates.

-

Carcinogenic species such as 2-acetylaminofluorene mediate toxicity thorough O-sulfate esters that generate electrophilic niternium

species.

-

Phenacetin: is metabolized by N-hydroxylphenacetin

and then conjugated with sulfate which in turn binds to bind covalently to microsomal

protein causing hepato

and nephrotoxcity.

Phase II: Sulphate

conjugation

-

Glycine

and glutamine used to conjugate (COOH) aromatic acids and arylalkyl

acids.

-

This process is limited due to competition with glucuronic acid and limited supply of amino acids.

-

The process of conjugation is as follows:-

Activation of the carboxylic acid containing compound to form Acyl-CoA.

-

Acylation

of glycine

or glutamate by N-acyltransferase

which occur in mitochondria of liver and kidney.

-

Glutamine conjugation occur mainly for arylacetic

acids such as phenylacetic

acid and 3-indolylacetic acid.

Phase II: Aminoacids

conjugation

-

Important pathway for detoxifying chemically reactive electrophilic compounds.

-

sulfhydryl group is considered the important group that interact with electrophilic positions in the toxic compounds.

-

GSH is a tripeptide

(γ-glutamyl-cysteinyl

glycine) that found in many tissues which will be further biotransformed

to form S-

substituted N-acetylcysteine

called mercapturic

acid.

-

The enzyme involved in the GSH conjugation is called glutathione S-transferase, and the degradation step is performed by renal

and hepatic microsomal

enzymes.

-

No need to form activated coenzyme or substrate.

-

Compounds that react with GSH do this by two mechanisms:

-

Nucleophilic displacement at an electron deficient carbon or heteroatom.

-

Nucleophilic addition to an electron deficient double bond.

Phase II: GSH (mercapturic

acid conjugates)

-

Nucleophilic displacement at an electron deficient carbon or heteroatom.-

Aliphatic and aryl alkyl halides, sulphates

(OSO3-), sulphonates

(OSO2

R), and nitrates (NO2

) possess electron deficient carbon atoms that react with GSH to form GSH conjugates.

-

It is facilitated when the carbon atom is benzylic

or allylic

or when X is a good leaving group.

-

Examples are benzyl chloride, methyl-iodide, allyl-chloride, methylparathion.

-

Aromatic substitution occur when the ring is attached to strong electron withdrawing group.

-

Azathioprine

Phase II: GSH (mercapturic

acid conjugates)

H2C

R

GSHX GS CH2

R

HX

X: Br, Cl, OSO3, OSO2R, OPO(OR)2

-

Arene

and aliphatic epoxides

are detoxifies by GSH.

-

GSH conjugation involving substitution at heteroatoms such as O, found in organic nitrates. (nitroglycerine, isosorbiddinitrate). The

final product is glutathion

disulfide and alcohol.

Phase II: GSH (mercapturic

acid conjugates)

-

Nucleophilic addition to an electron deficient double bond.

-

Occur mainly on α,β-unsaturated systems (but not all)

that are electron deficient by being within resonance or conjugated with carbonyl group.

-

GSH adduct is formed by Michael addition reactions.

-

Oxidation reaction could produce α,β-unsaturated systems as in acetaminophen and 2-hydroxy estrogen.

Phase II: GSH (mercapturic

acid conjugates)

-

Some examples have showed that GSH could cause toxicity, as the GSH conjugates are themselves are electrophilic.

-

1,2 dichloroethane, react with GSH to produce S-(2- chloroethyl)glutathion, then the S will displace one of the chlorine

atoms to produce episulfonium

ion.

Phase II: GSH (mercapturic

acid conjugates)

-

Important pathway for drugs containing primary amino groups (ArNH2

, H2

NC6

H4

SO2

NHR, hydrazines

(-NHNH2

), hydrazides (CONHNH2

), and primary aliphatic amines).

-

The end product will be amides that are expected to be nontoxic and inactive.

-

No enhancement of water solubility, so it is expected to terminate activity or detoxification process (not always).

-

The source of acetyl group is acetyl-CoA, and then transferred by the enzyme N-acetyltransferase.

-

Examples of aromatic primary amines; procainamide, dapsone, Nitro derivatives; clonazepam, sulphonamides;

sulphamethoxazole,

sulfanilamide (crystallurea), hydrazine; hydralazine, hydrazide; INH, aliphatic amines (minor compared to oxidative deamination); histamine and mescaline

Phase II: Acetylation

.Phase II: Acetylation

-

Acetylation polymorphism: bimodal character in human relate to drug acetylation

process and classified as rapid or slow acetylator.

-

The variation is related to genetic characters that is associated with N-acetyltransferase

activity related to ethnic groups.

-

Eskimos and Asians are rapid acetylators, while Egyptians and some western European countries are slow acetylators, while other groups are intermediate between the two.

-

Rapid acetylators

will not show the expected results of the given dose, while the slow are expected to develop side effects.

-

INH is one example; in rapid acetylators

the t1/2 is 45-80 min, while in slow acetylators

is 140-200 min.

Phase II: Acetylation

-

Used for biosynthesis of endogenous compounds (ephedrine and melatonine).

-

Used also for inactivation of endogenous compounds (dopamine, seratonine).

-

Minor pathway for xenobiotic compounds.

-

Reduces water solubility except the case of creation quaternary ammonium compound.

-

S-adenosylmethionine

(SAM) is the coenzyme and then there is transferase enzymes that deliver this group.

-

Methyltransferase

enzymes are important such as catechol-O- methyltransferase

(COMT), phenol-O-methyltransferase.

-

COMT is important in performing O-methylation for neurotransmitters norepinephrine

and dopamine.

Phase II: Methylation

-

Catecholes

are metabolized by COMT, methyldopa and isoproterenol

to mono methylated (only C3

OH), terbutaline

is not O-methylated.

-

Little case where phenols were O-methylated (minor); Morphine to codeine.

-

N-methylation of xenobiotic compounds is very low such as amantadine.

-

N-methylation present at heteroatom such as nicotine and nicotinic acid to give quaternary ammonium products.

-

S-methylation for drugs contain thiols

(6-mercaprtopurine, propylthiouracil)

Phase II: Methylation

-

The process by which the activity of the metabolizing enzymes is increased, which is related mainly to increased amount of newly

synthesized enzyme.

-

Could lead to reduction of activity of some drugs as they will be metabolized faster.

-

Could cause Drug-Drug interaction (Phenobarbital-warfarin) and (Contraceptives with [rifampin

or phenobarbital]).

-

Drug-Side effects such as osteomalacia

when treatment with phenobarbital

and phenytoin

(vitamin D mertabolism).

-

By inducing glucuronyl

transferase to conjugate bilirubin

with glucuronic acid, so it may be used to treat hyperbilirubinemia.

-

Benzo[α] pyrene

a smoking byproduct is a strong enzyme inducer, so affect other drugs such as theophylline, phenacetin.

-

Exposure to pesticides and insecticides stimulates drug metabolism.

Enzyme Induction

-

Reduced metabolism cause accumulation of certain drugs so increase the duration of action and side effects.

-

This process occur by: substrate competition, interfere with protein synthesis, inactivate drug-metabolizing enzymes and hepatotoxicity.

-

Chloramphenicol

and disulfuram

and INH reduce the metabolism of phenytoin.

-

Grapefruit contain furanocumarines

and bioflavonoids

such as naringin

is weak CYP inhibitor.

Enzyme Inhibition

THE END

GOOD LUCK IN THE FINAL EXAM