Medicinal chemistry: Drug metabolism, Phase I ,Phase II Reaction

1. DRUG METABOLISM
Mrs. Asha Suryawanshi
Asst. Professor
M. Pharm(Pharmaceutical Chemistry)
SDDVCOP & RC New Panvel
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2.  Introduction
 History
 Definition
 Significance
 Drug metabolism principles
 Phase I reactions
•Oxidation
•Reduction
•Hydrolysis
 Phase II reactions
•Conjugation reactions
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INTRODUCTION
Drug metabolism is a complex and important part of biochemical
pharmacology.
The pharmacological activity of many drugs is reduced or
nullified by enzymatic processes, and drug metabolism is one of
the main mechanisms by which drugs may be inactivated.
Metabolism, or the biotransformation of a drug, is the process
whereby living organisms effect chemical changes to a molecule .
The product of such a chemical change is called a “metabolite”.
In practice, all xenobiotics undergo transformations in living
organisms.

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HISTORY
The first human study of drug metabolism occurred in
1841 when Ure noted that Hippuric acid could be isolated
from the urine after the ingestion of benzoic acid, and the
first metabolic interaction between drugs was reported by
Hoffmann in 1877 who found that quinine could decrease
the formation of Hippuric acid from benzoic acid.
Richard Tecwyn Williams was
a Welsh biochemist who founded the systematic study
of xenobiotics metabolism with the publication of his
book Detoxication mechanisms in 1947.
He has been referred to as the founder of the field of
drug metabolism.
Richard Tecwyn Williams
(20 /02/1909–29/12/1979)

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DEFINITION
Drug metabolism may be defined as the biochemical
modification of one chemical form to another, occurring usually
through specialized enzymatic systems
Biotransformation is the process by which a substance
changes from one chemical to another (transformed) by a
chemical reaction within the body
A prodrug is a medication or compound that, after
administration, is metabolized into a pharmacologically active
drug.

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SIGNIFICANCE
•Pharmacological inactivation of drugs:
• Here, the metabolite formed has little or absolutely no
pharmacological activity.
Eg.The metabolite of phenytoin (antiepileptic), p-hydroxy phenytoin
has no pharmacological activity.
• No effect on pharmacological activity:
Here, the metabolites Genetic polymorphism has appeared to be the
common phenomenon, leading to variations in metabolic process in
humans.
biotransformation processes have equal and similar activity to that of
the original drug. Nortriptyline possesses equal activity as that of the
parent drug, amitriptyline (antidepressant).
• Toxicological activation of drugs:
Here, the metabolites formed after biotransformation process have a
high tissue reactivity, thus causing toxicity, which may include
necrosis, hepatitis, etc. Nacetyl p-benzoquinoneimine which is a
metabolite of paracetamol causes hepatotoxicity.

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• Pharmacological activation of drugs:
By this process, prodrugs (inactive) are metabolized to
highly active drugs.
These are used to improve patient acceptability, improve
absorption, alter metabolism, biodistribution and
elimination. Ampicillin (antibiotic) is the active form of
pivampicillin.
• Changed pharmacological activity:
Here, the therapeutic activity displayed by the
metabolite formed is different from that of the original
drug. Isoniazid (antitubercular) is the metabolite of
Iproniazid (antidepressant)
SIGNIFICANCE (Cont…..)

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DRUG METABOLISM PRINCIPLES
As a rule, metabolism converts relatively lipid soluble
drugs into more water-soluble, inactive metabolites. In
some cases, however, metabolism produces an active
metabolite.
The liver is the richest source of metabolic enzymes and
the cytochrome p450 system is the most important group
of metabolic enzymes.
Cytochrome p3A4 alone metabolizes around 45% of
prescription medicines and accounts for about 60% and
70% of the liver’s and intestine’s ability to metabolize
drugs respectively.

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PHASE METABOLISM
Phase I
-Functionalization reaction
-Convert the parent drug to a more polar metabolite by
introducing or unmasking a functional group(OH,SH,NH2)
-Phase II
-Conjugation reaction
-Subsequence reaction in which a covalent bond formation
between phase I metabolite and endogenous substances
like amino acid or between functional group and parent
molecule

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Phase I reactions are broadly grouped into three categories
Oxidation
Reduction
Hydrolysis.
Phase I

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The enzymes involved in Phase I reactions are primarily
located in the endoplasmic reticulum of the liver cell, they
are called microsomal enzymes
The enzyme-catalyzed reactions of Phase I metabolism bind
oxygen, hydrogen, water, or amino acids to the lipophilic drug
molecule to expose or introduce a hydroxyl (-OH), amino (-NH2),
sulfhydryl (-SH), or carboxyl (-COOH) polar functional group, and
thus, result in a modest increase in the parent drug's water
solubility.

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OXIDATION
Reactions resulting in the addition of oxygen and/or the removal of
hydrogen:
•R-H ⇒ R-OH (hydroxylation): Conversion of a hydrogen to a
hydroxyl group.
•R-C-OH ⇒ R-C=O (dehydrogenation): Conversion of a hydroxyl
group to a carbonyl group.
•R-C=O ⇒ R-COOH (carboxylation): Conversion of a carbonyl
group to a carboxyl group.
•R-C-NH2⇒R-C=O (deamination): Conversion of an amino
group to a carbonyl group.
•R-CH3⇒ R-H (demethylation): Conversion of a methyl group to
a hydrogen.
An example of an oxidation reaction is the hydroxylation of
amphetamine to 4-hydroxyamphetamine and norephedrine.
Another example is hydroxylation of delta-9-THC to 11-OH-delta-9-
THC.
The enzymes of oxidation include mixed-function oxidases,
monooxygenases, and cytochrome P450 enzymes.

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Cytochromes P450 (CYPs) are a superfamily
of enzymes containing heme as a cofactor that function
as monooxygenases In mammals, these proteins
oxidize steroids, fatty acids, and xenobiotics, and are
important for the clearance of various compounds, as well as
for hormone synthesis and breakdown. In plants, these
proteins are important for the biosynthesis of defensive
compounds fatty acids, and
CYPs are, in general, the terminal oxidase enzymes in electron
transfer chains, broadly categorized as P450-containing
systems. hormones
Cytochromes P450 (CYPs)

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Example of Phase I reaction

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Reactions resulting in the addition of hydrogen
and/or the removal of oxygen:
•R-OH ⇒ R-H (de-hydroxylation)
•R-C=O ⇒ R-C-OH (hydrogenation)
•R-COOH ⇒ R-C=O (decarboxylation)
•R-NO2⇒ R-NH2 (amination)
•R-C-H ⇒ R-CH3 (methylation)
An example of a reduction reaction is the
inactivation of warfarin by the transformation of a
ketone group to a hydroxyl group (hydrogenation).
Enzymes involved in reduction reactions are called
reductases.
REDUCTION

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Cont….
Cytochromes P450 reductase, also known as
NADPH: ferrihemoprotein oxidoreductase,
NADPH:hemoprotein oxidoreductase,
NADPH:P450 oxidoreductase, P450 reductase, POR, CPR,
CYPOR, is a membrane-bound enzyme required for
electron transfer to Cytochrome P450 in the microsome of
the eukaryotic cell from a FAD- and FMN-containing
enzyme NADPH: cytochrome P450 reductase The general
scheme of electron flow in the POR/P450 system is:
NADPH → FAD → FMN → P450 → O2
During reduction reactions, a chemical can enter futile
cycling, in which it gains a free-radical electron, then
promptly loses it to oxygen (to form a superoxide anion)

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HYDROLYSIS
In a reaction with water, a bond in the compound is broken,
resulting in two compounds. At the same time the water molecule
splits in two, with a hydrogen transferring to one of the
compounds and a hydroxide to the other compound.
R-COO-R' + H2O ⇒ R-COOH + R'-OH
R-CO-NH-R' + H2O ⇒ R-COOH + R'-NH2
The conversion of cocaine to benzoylecognine and ecognine
methyl ester are examples of hydrolysis reactions.
The enzymes of hydrolysis reactions include esterases,
peptidases, and amidases.

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Phase II
Phase II reactions are also known as conjugation reactions.
Conjugation reactions are anabolic processes which, in general,
form compounds of higher molecular weight with greatly
reduced biological activity, increased water solubility and
usually reduced mobility.

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Phase II Reactions
Conjugation with Glucuronic acid
Conjugation with sulphate moieties
Conjugation with alpha amino acids
Conjugation with glutathione and mercapturic acid formation
Acetylation reaction
Methylation reaction
Miscellaneous reaction

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Phase II reactions and their characteristics

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Glucuronidation consists of transfer of the glucuronic acid component
of uridin diphosphate glucuronic acid to a substrate by any of several
types of UDP-glucuronosyl transferase.
DP-Glucuronic acid (Glucuronic acid linked via a glycosidic bond
to uridine diphosphate) is an intermediate in the process and is
formed in the liver. One example is the N-glucuronidation of
an aromatic amine, 4-aminobiphenyl, by UGT1A4 or UGT1A9 from
human, rat, or mouse liver.
The substances resulting from glucuronidation are known
as glucuronides (or glucuronosides) and are typically much
more water-soluble than the non-glucuronic acid-containing
substances from which they were originally synthesized.
CONJUGATION WITH GLUCURONIC ACID

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Conjugation with sulphate moieties
Sulphation is similar to glucuronidation but it is catalyzed
by nonmicrosomal enzymes and occurs less commonly as
the moiety that transfers sulphate to the substrate is easily
depleted.
This process is thus, easily saturable in comparison to
glucuronidation.
Sulphation is dominant at low substrate concentration,
whereasGlucuronidation is dominant at high substrate
concentration.

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Sulphation occurs in 2 steps:
1) Synthesis of an activated coenzyme 3'phosphoadenosine-5'-
phosphosulphate (PAPS) which acts as a donor of sulphate
to the substrate
2) Transfer of sulphate group from PAPS to the substrate RXH
in presence of enzyme sulphotransferase (sulphokinase) and
subsequent liberation of 3'-phosphoadenosine-5'-phosphate
(PAP).
Examples of compounds undergoing sulphation are:
Phenols e.g. paracetamol, salbutamol
Alcohols e.g. aliphatic alcohols C-1 to C-5
Aryl amines e.g. aniline.

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compounds undergoing sulphation

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Conjugation with alpha amino acids
This reaction also occurs to a limited extent because of limited
availability of amino acids.
The reaction occurs in two steps:
1 Activation of carboxylic acid drug substrate with ATP and
coenzyme A (CoA) to form an acyl CoA intermediate. Thus,
the reaction is a contrast of glucuronidation and
sulphation where the donor coenzyme is activated and not the
substrate.
2. Acylation of the -amino acid by the acyl CoA in presence of
enzyme N-acyl transferase.
Examples of drugs forming glycine or glutamine conjugates are:
Aliphatic acids e.g. isopropoxyacetic acid
Aryl acids e.g. salicylic acid
Heterocyclic aryl acids e.g. nicotinic acid.

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Conjugation occurs commonly with glycine.
Glutamine conjugation occurs to a lesser extent. Conjugation
with other amino acids like aspartic acid, serine and taurine is
still uncommon. The substrate is generally an acid (aromatic in
particular) and the reaction product is an amide.
where R’ = -CH2- (if glycine) or >CH-CH2-CH2-
CONH2 (if glutamine)

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Conjugation with glutathione and mercapturic acid formation
Glutathione ( γ-glutamyl cysteinyl glycine or GSH) is a
tripeptide with a strongly nucleophilic character due to the
presence of a -SH (thiol) group in its structure.
GSH conjugation differs from other conjugation reactions in
that the process does not require initial activation of the coenzyme
or the substrate since the GSH, which is a nucleophile itself, is
highly reactive towards an electrophilic substrate.

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The interaction between the substrate and the GSH is catalyzed by
enzyme glutathione-S-transferase to form S-substituted glutathione
conjugate.
This conjugate is not excreted as such in the urine but undergoes
cleavage, first by the enzyme -glutamyl transpeptidase to release the
free glutamyl residue and cysteinyl glycine derivative; the latter is
cleaved by enzyme cysteinyl glycinase to produce free glycine and
the cysteine conjugate.
Finally, N-acetylation of this conjugate by the enzyme N-acetylase
yields S-substituted-N-acetyl cysteine products called
as mercapturic acids.

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This reaction differs from general characteristics of phase II reactions in
several ways:
The metabolites formed are not polar or water-soluble.
The metabolites, in a number of instances, have equal or greater
pharmacological activity than the parent drug, e.g. morphine formed from
normorphine.
The reaction is of lesser importance in metabolism of xenobiotics.
It is more important in the biosynthesis
Methylation can be considered as intermediate of phase I and phase II
reactions.
It can be called as a phase I reaction as it is reverse of demethylation
reaction and can be classed as a phase II reaction because of its
mechanism.
METHYLATION

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Methylation of substrates proceeds in two steps:
1. Synthesis of an activated coenzyme S-adenosyl
methionine (SAM), the donor of methyl group, from L-
methionine and ATP.
2. Transfer of the methyl group from SAM to the substrate
in presence of nonmicrosomal enzyme methyl transferase.
Examples of substrates undergoing Methylation are:
O-Methylation
Phenols e.g. morphine
Catechols e.g. -methyl dopa, L-DOPA, isoprenaline
S-Methylation
Thiols e.g. propylthiouracil, 6-mercaptopurine
METHYLATION

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ACETYLATION REACTION
This reaction is basically an acylation reaction and thus similar to
conjugation with α-amino acids.
The analogy also lies in the fact that both reactions yield amide
products.
Acetylation however differs from α-amino acid conjugation in that
the substrates are exogenous amines (and not carboxylic acids)
and the acylating agent is endogenous acetyl CoA (CH3COSCoA).
The general sequence of reaction is similar to that for α-amino acid
conjugation. The enzyme involved is the nonmicrosomal N-acetyl
transferase.

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Examples of drugs undergoing acetylation are
Primary aliphatic amines e.g. histamine, mescaline.
Primary aromatic amines e.g. procainamide, PAS, PABA, Dapsone.
Sulphonamides e.g. sulphanilamide, sulphapyridine.
Hydrazines/hydrazides e.g. hydralazine, isoniazid, phenelzine.
Acetyl derivatives of some sulphonamides

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MISCELLANEOUS REACTION
Conjugation of Cyanide
The toxicity of cyanide ion is due to its ability to arrest
enzymes involved in cellular respiration
and convert hemoglobin to cyanomethemoglobin which
lacks the ability to transport oxygen to tissues.
Conjugation of cyanide ion involves transfer of sulphur
atom from thiosulphate to the cyanide ion
in presence of enzyme rhodanese to form inactive
thiocyanate.
Some of the rare conjugation reactions are mentioned below.

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Conjugation with Ribose
Endogenous Purine and Pyrimidine bases conjugate with
ribose to form nucleotides.
Conjugation with Taurine
Taurine, a β-amino Sulphonic acid, conjugates the
endogenous bile acids to produce components of bile.

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