PCI - BPH4 - METABOLISM chapter pdf simple

METABOLISM
MR. PRATIP K. CHASKAR (M. PHARM.)
ASST. PROFESSOR, D Y PATIL SCHOOL OF PHARMACY

• Drug Metabolism
• Phase I Reactions
• Phase II Reactions
• Factors Affecting Metabolism
• Importance of Metabolism
• Stereochemical Aspects
SYLLABUS

• Sum total of all the enzyme – catalysed reactions that
occur in an organism
• Main purpose
• Conversion of food/fuel to energy to run cellular
processes.
• Conversion of food/fuel to building blocks for
proteins, lipids, nucleic acids, and some
carbohydrates
• Elimination of nitrogenous wastes.
DRUG METABOLISM

• Metabolic Stages
DRUG METABOLISM
ANABOLIC
Synthesis of
complex
molecules in
living organisms
from simpler
ones
Constructive
metabolism
• E.g. Breaking down of glucose to
pyruvate by cellular respiration
CH2OH
CH2OH
OH
H
H
HO
OH
H
OH
H
COO
CH3
O

• Metabolic Stages
DRUG METABOLISM
CATABOLIC
Synthesis of
simple molecules
in living
organisms from
complex ones
Destructive
metabolism
• E.g. Synthesis of glucose from
pyruvate
CH2OH
CH2OH
OH
H
H
HO
OH
H
OH
H
COO
CH3
O

DRUG METABOLISM
Catabolism Anabolism
Catabolism breaks down big complex molecules into
smaller, easier to absorb molecules.
Anabolism builds molecules required for the body’s
functionality.
The process of catabolism releases energy. Anabolic processes require energy.
Hormones involved in the processes are adrenaline,
cytokine, glucagon, and cortisol.
Hormones involved in the process are estrogen,
testosterone, growth hormones and insulin.
Examples of catabolic processes are proteins
becoming amino acids, glycogen breaking down into
glucose and triglycerides breaking up into fatty acids.
Examples include the formation of polypeptides from
amino acids, glucose forming glycogen and fatty
acids forming triglycerides.
In catabolism, potential energy is changed into
kinetic energy.
In anabolism, kinetic energy is converted into
potential energy.
It is required to perform different activities in living
entities.
It is required for maintenance, growth, and storage.

• Pharmacokinetic process – renders lipid soluble and
non-polar compounds to water soluble and polar
compounds so that they are excreted by various
processes.
• Metabolism is a necessary biological process that limits
the life of a substance in the body.
DRUG METABOLISM

• Conversion of active drug to inactive or less active
metabolite – pharmacological inactivation.
• Conversion of active drug to more active metabolite –
bioactivation or toxicological activation.
FUNCTIONS
HN
N
H
Ph
O
O
O
HN
N
H
Ph-p-OH
O
O
O
Phenobarbitone p-Hydroxyphenobarbitone
O
N
CH3
H3CO
HO
Codeine
O
N
CH3
HO
HO
Morphine

• Conversion of inactive drug to more active toxic
metabolite – lethal synthesis
• Conversion of inactive drug (pro-drug) to active
metabolite – pharmacological activation
FUNCTIONS
Fluoroacetate
Fluorocitrate
F
O
O
O OH
O
F
O
OH
O OH
Phenacetin Paracetamol
O
H
N
O
HO
H
N
O

• Conversion of active drug to equally active metabolite
(no change in activity)
• Conversion of active drug to active metabolite having
entirely different pharmacological activity (change in
activity)
FUNCTIONS
Digitoxin
O
O
O
O
O
O
O
HO
OH
OH
O
HO
HO
Digoxin
O
O
O
O
O
O
O
HO
OH
OH
O
HO
HO
OH
Iproniazid
N
N
H
O
H
N
Isoniazid
N
N
H
O
NH2

• Major site – liver (microsomal enzyme systems of
hepatocytes)
• Secondary organs of biotransformation
• Kidney (proximal tubule)
• Lungs (type II cells)
• Testes (Sertoli cells)
• Skin (epithelial cells); plasma. nervous tissue (brain);
intestines
SITE OR ORGANS

Liver
• Relatively rich in large variety of metabolizing enzymes.
• Metabolism by other organs (extra-hepatic metabolism)
– less importance – low level of metabolizing enzymes.
• Drug metabolising activity is found in the smooth
endoplasmic reticulum and the cytosol.
• Drug metabolism can also occur in mitochondria,
nuclear envelope and plasma membrane.
SITE OR ORGANS

Liver
• Few drugs are also metabolised by non-enzymatic
means – non-enzymatic metabolism.
• E.g. Atracurium, a neuromuscular blocking drug, is
inactivated in plasma by spontaneous non-enzymatic
degradation (Hoffman elimination) in addition to that by
pseudocholinesterase enzyme.
SITE OR ORGANS

Endoplasmic Reticulum (Microsomes)
• Primary location for the metabolizing enzymes.
• Phase I – cytochrome P450, flavin-containing
monooxygenase, aldehyde oxidase, carboxylesterase,
epoxide hydrolase, prostaglandin synthase, esterase.
• Phase II – uridine diphosphate-glucuronosyltransferase,
glutathione-S-transferase, amino acid conjugating
enzymes.
SITE OR ORGANS

Cytosol
• Many water-soluble enzymes.
• Phase I – alcohol dehydrogenase, aldehyde reductase,
aldehyde dehydrogenase, epoxide hydrolase, esterase.
• Phase II – sulfotransferase, glutathione S-transferase, N-
acetyl transferase, catechol-O-methyl transferase, amino
acid conjugating enzymes.
Lysosomes
• Phase I – peptidase
SITE OR ORGANS

Mitochondria
• Phase I – monoamine oxidase, aldehyde dehydrogenase,
cytochrome P450.
• Phase II – N-acetyl transferase, amino acid conjugating
enzymes.
Nucleus
• Phase II – uridine diphosphate glucuronosyltransferase
SITE OR ORGANS

• Some drugs gets metabolised before reaching the
circulation – reduces bioavailability – e.g. Propranolol,
Lignocaine and Nitroglycerine
• Intestinal first-pass effect – metabolised in GIT by
enzymes present in either gut mucosa or gut lumen
• Hepatic first-pass effect – absorbed across GIT and enter
portal circulation, but rapidly metabolised during the
passage through liver.
FIRST PASS EFFECT

PHASES OF METABOLISM
Metabolism
Phase I
Functionalization
Phase II
Conjugation
Oxidation
• Aromatic moieties, Olefins
• Benzylic & allylic C atoms and
a-C of C=O and C=N
• At aliphatic and alicyclic C
• C-Heteroatom system
• C-N (N-dealkylation, N-oxide
formation, N-hydroxylation)
• C-O (O-dealkylation)
• S-dealkylation
• S-oxidation, desulfuration
• Oxidation of alcohols and
aldehydes,
• Miscellaneous
• Glucuronic acid
• Sulfate, Glycine and other AA
• Glutathione or mercapturic acid
• Acetylation, Methylation
Hydrolysis
• Esters, amides, epoxides
and arene oxides by
epoxide hydrase
Reduction
• Aldehydes and ketones
• Nitro and azo
• Miscellaneous

DRUGS
Highly Lipophilic
Metabolically Stable
Lipophilic Polar Hydrophilic
Storage in Body
Tissues
Phase I
Metabolism
Phase II
Metabolism
Elimination
Polar
Hydrophilic

Hepatic microsomal enzymes
(oxidation, conjugation)
Extra-hepatic microsomal enzymes
(oxidation, conjugation)
Hepatic non-microsomal enzymes
(acetylation, sulfation, GSH,
alcohol/aldehyde dehydrogenase,
hydrolysis, oxidation, reduction)

• Result in inactivation, activation or no change in
pharmacological potency or activity – Non-synthetic or
Functionalization reactions
PHASE I REACTIONS
OXIDATION
REDUCTION
HYDROLYSIS

• Add one atom of molecular oxygen to a drug substrate,
another to protons to form water.
• Linked by NADPH-cytochrome P450 reductase (NADPH
cytochrome c reductase) to the oxidation of NADPH to
NADP+
• Have relatively low substrate selectivity – drugs may be
metabolized by more than one CYP – common feature is
lipophilicity
MIXED FUNCTION OXIDASES (MONO-
OXYGENASES) CYTOCHROME P450 OR CYP

CYP Nomenclature
• Families – CYP + arabic numeral (e.g. CYP1)
• Subfamily – CYP + arabic numeral + alphabet (e.g.
CYP1A)
• Subfamily – CYP + arabic numeral + alphabet +
additional numeral for more than 1 subfamily; e.g.
CYP1A2

• NADPH + H+ + O2 + Drug → NADP+ + H2O + Oxidized Drug
• CO binds to reduced Fe(II) heme and absorbs at 450 nm (origin of
enzyme family name)
• CYP monooxygenase enzyme family is major catalyst of drug and
endogenous compound oxidations in liver, kidney, GIT, skin, lungs
• Oxidative reactions require the CYP heme protein, reductase,
NADPH, phosphatidylcholine and molecular oxygen
• The reductase serves as the electron source for the oxidative
reaction cycle

• Multiple CYP gene families have been identified in humans, and
the categories are based upon protein sequence homology
• Most of the drug metabolizing enzymes are in CYP 1, 2, & 3
families.
• Frequently, two or more enzymes can catalyze the same type of
oxidation, indicating redundant and broad substrate specificity.
• CYP3A4 is very common to the metabolism of many drugs; its
presence in the GI tract is responsible for poor oral availabilty of
many drugs

CYP Fe3+
Drug
CO
2H+
H2O
2
3
4
CYP Fe3+ Drug
CYP Fe3+ Drug
CYP Fe3+ Drug
O2
CYP Fe3+ Drug
CO
CYP Fe3+
Drug
O2
CYP Fe3+ Drug
O
Cytochrome
P450 Reductase
NADPH
NADP+
e-
O2
Cytochrome
P450 Reductase
NADPH
NADP+ e-
5
1
6
Drug OH
Oxidized Product
Activated Oxygen Species

• Initial step of this catalytic reaction cycle starts with the binding
of the substrate to the oxidized (Fe3+) resting state of CYP to form
a P450-substrate complex.
• The next step involves the transfer of one electron from NADPH-
dependent CYP reductase to the P450-substrate complex. This
one-electron transfer reduces Fe3+ to Fe2+. It is this reduced (Fe2+)
P450-substrate complex that is capable of binding dioxygen (O2).
• The dioxygen–P450-substrate complex that is formed then
undergoes another one-electron reduction (by CYP reductase-
NADPH and/or cytochrome b5 reductase-NADH) to yield a
peroxide dianion–P450 (Fe3+)-substrate complex.

• Water (containing one of the oxygen atoms from the original
dioxygen molecule) is released from the latter intermediate to
form an activated oxygen–P450-substrate complex.
• Activated oxygen [FeO]3+ in this complex is highly electron
deficient and a potent oxidizing agent – transferred to the
substrate (R-H), and the oxidized substrate product (R-OH) is
released from the enzyme complex to regenerate the oxidized
form of CYP.
• The key sequence of events appears to center around the
alteration of a dioxygen–P450-substrate complex to an activated
oxygen–P450-substrate complex, which can then effect the critical
transfer of oxygen from P450 to the substrate.

• Refers to mixed function oxidation of aromatic
compounds (arenes) to their corresponding phenolic
metabolites (arenols).
• Almost all aromatic hydroxylation reactions are believed
to proceed initially through an epoxide intermediate
called an “arene oxide,” which rearranges rapidly and
spontaneously to the arenol product.
OXIDATION OF AROMATIC MOIETIES
R R
O
R
OH

• E.g.
O NH
OH
Propranolol
NH
HN
O
O
O
Phenobarbitone
HN NH
Ph
O
Phenytoin
O
O O
OH
O
Warfarin HO
OH
C CH
17-Ethinylestradiol

• Substituents attached to the aromatic ring – Influence
the ease of hydroxylation.
• Reactions proceeds more readily in activated (electron-
rich) rings, whereas deactivated aromatic rings (e.g.,
those containing electron-withdrawing groups Cl, -N+R3,
COOH, SO2NHR) are slow or resistant to hydroxylation.
Clonidine
Slow
N
H
H
N
N
Cl
Cl O2S N
HOOC
Probenecid
No oxidation

• In compounds with two aromatic rings, hydroxylation
occurs preferentially in the more electron-rich ring.
• E. g.
Diazepam
Chlorpromazine
N
H
N
O
Cl N
S
Cl
N

• Arene oxide intermediates are formed
when a double bond in aromatic
moieties is epoxidized.
• Arene oxides are of toxicologic concern –
bcoz electrophilic and chemically
reactive - bcoz of strained epoxide ring)
– detoxified to arenols
• If not effectively detoxified – bind
covalently with nucleophilic groups
present on proteins, DNA & RNA –
leading to serious cellular damage.
R
O
R
M
OH
M = DNA, RNA
or protein

• Metabolite formed – oxide ring opens in the direction
that generates the most resonance-stabilized
carbocation (positive charge on C-3 carbon is resonance
stabilized by the OCH3 group).
• The zwitterionic species (positive C-3 and negative
oxygen) undergoes a NIH shift to form the dienone &
then to 3-deuterio-4-hydroxyanisole with the loss of a
proton because of the weaker bond energy of the C–H
bond (compared with the C–D bond).

• Conversion of oxides to arenols – accompanied by novel
intramolecular hydride migration – NIH shift. E.g.
O
D
4-deuterioanisole
O
D O
H
Arene oxide
O
D
O
H
Zwitterionic species
O
O
H
D
NIH Shift
Dienone
O
OH
3-Deutero-4-hydroxyanisole
Tautomerism
D

• NIH shift are illustrated with the mixed function
aromatic oxidation of 4-deuterioanisole to 3-deuterio-4-
hydroxyanisole.
• Zwitterionic species may undergo direct loss of D+ to
generate 4-hydroxyanisole, in which there is no
retention of deuterium – more favorable than the NIH
shift in some aromatic oxidation reactions.

• Two enzymatic reactions also aid in neutralizing
reactivity of arene oxides.
tra n s - D ih y d r o d io ls
R
O
H 2 O
R
O H
O H
R
G S H
O H
G lu ta th io n e a d d u c ts
G S H

• Hydration i.e. nucleophilic attack of water on epoxide to
yield inactive trans-dihydrodiol metabolites – catalyzed
by microsomal enzymes called epoxide hydrases.
• Nucleophilic ring opening of the arene oxide by the
sulfhydryl (SH) group present in GSH to yield the
corresponding trans-1,2-dihydro-1-S-glutathionyl-2-
hydroxy adduct, or GSH adduct – catalyzed by various
GSH S-transferases.

• Benzo[α]pyrene – aromatic hydroxylation can occur at
several positions e.g. 4,5-oxide; 7,8-oxide; 9,10-oxide
Benzo[a]pyrene
7,8-oxide
O 7
8
HO
OH 7,8-trans-dihydrodiol
4,5-epoxide O
4
5
4,5-trans-dihydrodiol
OH
O 10
9
9,10-epoxide
HO
9,10-trans-dihydrodiol
OH
OH

• Initial epoxidation reaction to 7,8-oxide then converted
by epoxide hydrase to 7R,8R-dihydroxy-7,8-
dihydrobenzo[α]pyrene.
• The two-step enzymatic formation of this trans-
dihydrodiol is stereospecific.
• Subsequent epoxidation at the 9,10-double bond of the
latter metabolite generates predominantly 9,10-epoxide
– reacts with DNA to form many covalently bound
adducts.

• Metabolic oxidation of olefinic carbon–carbon double
bonds leads to the corresponding epoxide (or oxirane).
• Epoxides derived from olefins – more stable than
epoxides of arene oxides.
• Epoxides are susceptible to enzymatic hydration by
epoxide hydrase to form trans-1,2-dihydrodiols – also
called 1,2-diols or 1,2-dihydroxy compounds.
OXIDATION OF OLEFINS
O HO OH

COOH
O
Cl
Alcofenac
COOH
O
Cl
Alcofenac epoxide
O
COOH
O
Cl
Dihydroxy alcofenac
HO
OH

• Some olefins undergo metabolic epoxidation – epoxide
metabolites may be the reactive species responsible for
the cellular toxicity.
Diethylstilbestrol
Diethylstilbestrol epoxide
O
OH
HO
HO
OH

• An interesting group of olefin-containing compounds
causes the destruction of CYP.
• E.g. allylisopropylacetamide, secobarbital, fluroxene.
• Olefinic moiety present in these compounds – activated
metabolically by CYP to form a reactive intermediate
that covalently binds to the heme portion of CYP – N-
alkylated protoporphyrins - N-alkyl moiety is derived
directly from the olefin.

• Benzylic C-H bonds are weaker than sp3 hybridized C-H –
because radical formed from homolysis is resonance
stabilized.
• Benzylic carbon – Carbon attached to benzene
• Benzylic hydrogen – Hydrogen attached to benzylic carbon
OXIDATION AT BENZYLIC CARBON
CH3
H
H
Benzylic Carbon
Benzylic Hydrogen

• Resonance stabilization of the benzylic radical
CH3
H
CH3
H
CH3
H
CH3
H

CH3
H
H
CH3
OH
H
• Because of weak C-H bonds, benzylic hydrogens can form
benzylic halides.
CH2Cl2
O
OH
+ MnO2
O
O
+ Mn(OH)2
(4-Methoxyphenyl)methanol 4-Methoxybenzaldehyde
• Benzylic alcohols are oxidized selectively by a suspension
of activated manganese (IV) dioxide, MnO2.

• Allylic carbon – Carbon next to C=C
• Allylic hydrogen – Hydrogen attached to allylic carbon
OXIDATION AT ALLYLIC CARBON
Allylic Carbon
Allylic Hydrogen
CH2
C
H
H
H
H
H3C
H H
H
H3C
OH
(E)-pent-2-en-1-ol
(E)-pent-2-ene

• Allylic alcohols are oxidized selectively by a suspension of
activated manganese (IV) dioxide, MnO2.
• Primary allylic alcohols are oxidized to aldehydes and
secondary allylic alcohols are oxidized to ketones.
CH2Cl2
+ MnO2 + Mn(OH)2
H
H
H3C
OH H
H
H3C
O
(E)-pent-2-enal
(E)-pent-2-en-1-ol
CH2Cl2
+ MnO2 + Mn(OH)2
H
H
H3C
OH H
H
H3C
O
H3C H3C
(E)-hex-3-en-2-ol (E)-hex-3-en-2-one

N
NH
O
O
CH3
O
H3C
N
NH
O
O
CH3
O
H3C
HO
Hexobarbital 3-Hydroxyhexobarbital

• Treatment of alkylbenzenes with strong oxidizing agents
under vigorous conditions converts alkyl side chain into a
carboxylic acid group.
• Oxidants used Na2Cr2O7 or CrO3, KMnO4 or O2, etc.
OXIDATION AT ALKYLBENZENES
100°C, 3 - 4 h
+ KMnO4
100°C, 48 h
+ CrO3
CH3
Cl
COOH
Cl
o-chlorotoluene o-chlorobenzoic acid
COOH
CH3
1-propylbenzene benzoic acid
40% H2SO4

• Oxidation of alkyl side chains requires the presence of a
benzylic hydrogen – tert-butylbenzene – resistant to
benzylic oxidation – no benzylic hydrogen.
• Reaction conditions – vigorous – e.g. 1-phenylethanol is
oxidized to acetophenone under mild conditions & to
benzoic acid under vigorous conditions.
OXIDATION AT ALKYLBENZENES
Vigourous
CrO3
CH3
OH
Mild
CrO3
1-phenylethanol
CH3
O
OH
O
acetophenone
benzoic acid
Vigourous CrO3

• First carbon to the C=O or C=N is called as  carbon.
OXIDATION AT CARBON ATOMS  TO
CARBONYL AND IMINES
N
N
O
H3C
Cl N
N
O
H3C
Cl
OH
Diazepam 3-Hydroxydiazepam

• Oxidation at terminal methyl group – -oxidation
• Oxidation of second last carbon - -1-oxidation
R-CH3  R-CH2-OH
OXIDATION AT ALIPHATIC CARBON
O OH
Valproic acid
O OH
OH
5-Hydroxy valproic acid
O OH
OH
4-Hydroxy valproic acid
-1 oxidation
 oxidation

• Cyclohexyl group – susceptible to mixed function oxidation
– alicyclic hydroxylation
• Enzymatic introduction of –OH group in monosubstituted
cyclohexane ring – occurs at C-3 or C-4 – lead to cis and
trans conformational stereoisomers
OXIDATION AT ALICYCLIC CARBON
N
N
N
H2N NH2
O
N
N
N
H2N NH2
O
OH

• Metabolism of nitrogen functionalities (e.g. amines,
amides)
• Nitrogen containing compounds divided in 3 classes:
• Aliphatic (primary, secondary, and tertiary) and alicyclic
(secondary and tertiary) amines
• Aromatic and heterocyclic nitrogen compounds
• Amides
OXIDATION INVOLVING CARBON–
HETEROATOM SYSTEMS

• Carbon-Nitrogen systems – N-Dealkylation
• Loss of alkyl group attached to N
R-NH-CH3  [R-NH-CH2-OH]  R-NH2 + HCHO
HETEROATOM SYSTEMS
N
N
CH3
CH3
Imipramine
N
N
CH3
H
Desimipramine

• Carbon-Nitrogen systems – Oxidative deamination
HETEROATOM SYSTEMS
CH3
R
NH2
CH3
R
NH2
H OH
R CH3
O
+ NH3
NH2
CH3
O
CH3
+ NH3
Amphetamine Phenylacetone

• Carbon-Nitrogen systems – N-oxide formation
(CH3)3N  [(CH3)3N-OH]+  (CH3)2N-O
HETEROATOM SYSTEMS
N
O
O
C2H5
CH3
N
O
O
C2H5
O
Meperidine Meperidine N-oxide

• Carbon-Nitrogen systems – N-hydroxylation
R-NH-R  R-N(OH)-R
HETEROATOM SYSTEMS
N
H
O
CH3
N
OH
O
CH3
2-Acetylaminofluorene N-hydroxy-2-acetylaminofluorene

• Carbon-Sulphur systems – S-dealkylation
• Loss of alkyl group attached to S
R-S-CH3  [R-S-CH2-OH]  R-SH + HCHO
HETEROATOM SYSTEMS
N
N
H
N
N
S
H3C
6-Methylthiopurine
N
N
H
N
N
S
H
6-Mercaptopurine

• Carbon-Sulphur systems – Desulfuration
R3P=S  R3P=O
HETEROATOM SYSTEMS
O2N
O
P
O
O
S
C2H5
C2H5
Parathion
O2N
O
P
O
O
O
C2H5
C2H5
Paraoxon

• Carbon-Sulphur systems – S-oxidation
HETEROATOM SYSTEMS
Chlorpromazine
N
S
Cl
N
H3C
CH3 Chlorpromazine sulfoxide
N
S
Cl
N
H3C
CH3
O
S R
R S R
R
OH
R
S
R
O

• Carbon-Oxygen systems – O-dealkylation
• Loss of alkyl group attached to O
R-O-CH3  [R-O-CH2-OH]  R-OH + HCHO
HETEROATOM SYSTEMS
O
N
CH3
O OH
H3C O
N
CH3
HO OH
Codeine Morphine

• Alcohols are further oxidized to aldehydes (if primary
alcohols) or to ketones (if secondary alcohols).
• Aldehyde metabolites resulting from oxidation of primary
alcohols or from oxidative deamination of primary
aliphatic amines often undergo facile oxidation to generate
polar carboxylic acid derivatives.
• Primary alcoholic groups and aldehyde functionalities are
quite vulnerable to oxidation.
OXIDATION OF ALCOHOLS & ALDEHYDES

R-CH2OH + NAD+  R-CHO + NADH + H+
R-CHO + NAD+  R-COOH + NADH + H+
OXIDATION OF ALCOHOLS & ALDEHYDES
Mefanamic acid
NH
COOH
CH3
CH2OH
NH
COOH
CH3
CHO
NH
COOH
CH3
COOH

OXIDATIVE REACTIONS – SUMMARY
Aromatic
Hydroxylation
Olefinic carbon
Benzylic carbon
Allylic carbon
R R
O
R
OH
O HO OH
CH3
H
H
CH3
OH
H
CH2Cl2
MnO2
H
H
H3C
OH H
H
H3C
O

Alkylbenzenes
Carbon atom  to
carbonyl and
imines
Aliphatic carbon
Alicyclic carbon
CrO3
COOH
CH3
40% H2SO4
R
R
O
R
R
O
OH
R
N
R
N
OH
R
R
H H
O OH
O OH
OH
O OH
OH
 -1
R N R N OH
H

N-Dealkylation
N-Oxidation
Oxidative
Deamination
N-oxide formation
N-hydroxylation
R
H
N
CH3
R
H
N
H2C
O
+
H
R
H
N
R
H
N
OH
R1
N
R1
N
R2
R2
OH
H
H
R
NH2
R
NH2
OH
R
O
+ NH3
N CH3 N O
R N
H
O
CH3
R N
OH
O
CH3

S-Oxidation
S-dealkylation
Desulfuration
O-Dealkylation
Alcohols &
Aldehydes
R1
S
R1
S
R2
R2
O
R
S
H3C
R
S
H
R
P
R
R
S
R
P
R
R
O
R
O
H3C
R
HO
R
R
CH2OH
R
R
CHO
R
R
COOH

• Reduction of aldehydes and ketones
• Reduction of nitro compounds
• Reduction of azo compounds
• Miscellaneous reduction reactions
REDUCTION

• Aldehydes are more readily oxidized to carboxylic acids
then reduced to alcohols
• Ketones are resistant to oxidation & are mainly reduced
to secondary alcohols
• Aldo-keto reductase enzymes
• Bioreduction of ketones often leads to the creation of
asymmetric centre thus 2 possible stereoisomeric
alcohols produced
REDUCTION OF ALDEHYDES & KETONES

REDUCTION OF ALDEHYDES & KETONES
O H
Cl
Cl
Cl
Chloral hydrate Trichloroethanol
HO H
Cl
Cl
Cl
CH3
HO
CH3
O H
CH3
HO
H
acetophenone (R)-1-phenylethanol (S)-1-phenylethanol

• Reduction of aromatic nitro molecules leads to aromatic
primary amine metabolites.
• Aromatic nitro compounds are reduced initially to the
nitroso and hydroxylamine intermediates
REDUCTION OF NITRO COMPOUNDS
Ar N
O
O Ar N
O
Ar NH
OH
Ar NH2
HO
N
H
HO
O
Cl
Cl
NO2
Chloramphenicol
HO
N
H
HO
O
Cl
Cl
NH2
Arylamine

• Reduction of aromatic azo molecules leads to aromatic
primary amine metabolites
• Azo reduction proceeds via formation of hydrazo
intermediate (-NH-NH-) that subsequently is cleaved
reductively to yield the corresponding aromatic amines
REDUCTION OF AZO COMPOUNDS
Ar N
N
Ar
Ar NH
NH
Ar
Ar NH2
N
N
SO2NH2
H2N
NH2
NH2
H2N
NH2
H2N
SO2NH2
+
1,2,4-triaminobenzene
Prontosil Sulfanilamide

Reduction of N-oxides
• Yields corresponding tertiary amines – drug elimination
of tertiary amine is delayed.
MISCELLANEOUS REDUCTION REACTIONS
R
N
R
R
Tertiary amine
R
N
R
N-oxide
O

Reduction of sulphur containing functional groups
• Disulfide moieties e.g. Reductive cleavage of disulfide
bond in disulfiram (Antabuse) yields N,N-
diethyldithiocarbamic acid as a major metabolite in
humans.
N S
S N
S
S
N SH
S
N,N-diethyldithiocarbamic acid
Disulfiram

Reduction of sulphur containing functional groups
• Sulfoxide functionalities are oxidized mainly to sulfones
(-SO2-), they sometimes undergo reduction to sulphides
e.g. Sulindac metabolised to sulphide metabolite.
F CH2COOH
CH3
S
O
H3C
F CH2COOH
CH3
S
H3C
Sulindac sulfide metabolite
Sulindac

REDUCTION
Aldehydes &
Ketones
Nitro Compounds
Azo Compounds
N-oxides
Disulfide moieties
Sulfones &
Sulfoxides
O R
R
HO R
R
Ar N
O
O Ar N
O
Ar NH
OH
Ar NH2
Ar N
N
Ar
Ar NH
NH
Ar
Ar NH2
R
N
R
R
R
N
R
O
R
S
S
R
R
SH
R
S
O
H3C
R
S
H3C

• Hydrolysis of Esters
• Hydrolysis of Amides
HYDROLYSIS

• Metabolism of esters is catalyzed by hydrolytic enzymes
present in various tissues and in plasma.
• Metabolic products formed (carboxylic acids, alcohols,
phenols, and amines) generally are polar and
functionally more susceptible to conjugation and
excretion than the parent ester or amide drugs.
• Enzymes carrying out ester hydrolysis include several
nonspecific esterases found in the liver, kidney, and
intestine as well as the pseudocholinesterases present in
plasma.
HYDROLYSIS OF ESTERS

R-COO-R  R-COO-H + R-OH
HYDROLYSIS OF ESTERS
N
H
COOC2H5
CH3
N
O
COOH
N
H
COOH
CH3
N
O
COOH
Enalaprit Enalapril

• Metabolism of amides is catalyzed by hydrolytic
enzymes present in various tissues and in plasma.
• Amide hydrolysis appears to be mediated by liver
microsomal amidases, esterases, and deacylases.
• Amides are hydrolyzed slowly in comparison to esters.
HYDROLYSIS OF AMIDES

R-CO-NRR  R-COO-H + HNRR
HYDROLYSIS OF AMIDES
O
H
N
N CH3
NH2
CH3
O OH
NH2
H2N
N CH3
CH3
4-aminobenzoic acid
N1
,N1
-diethylethane-1,2-diamine
Procainamide

• Hydrolysis of recombinant human peptide drugs and
hormones at the N- or C-terminal amino acids by
carboxypeptidase and aminopeptidase and proteases –
e.g. human insulin, growth hormone (GH), prolactin,
parathyroid hormone (PTH), and atrial natriuretic factor
(ANF).
• Hydrolysis of phosphate esters – e.g. sulfonylureas,
cardiac glycosides, carbamate esters, and
organophosphate compounds.
MISCELLANEOUS HYDROLYTIC REACTIONS

• Glucuronide and sulfate conjugates also can undergo
hydrolytic cleavage by -glucuronidase and sulfatase
enzymes.
• Hydration or hydrolytic cleavage of epoxides and arene
oxides by epoxide hydrase.
MISCELLANEOUS HYDROLYTIC REACTIONS

HYDROLYSIS
Esters
Amides
Miscellaneous
Peptide drugs and hormones
Phosphate esters
Glucuronide and sulfate conjugates
Cleavage of epoxides and arene oxides
R O
O
R
R O
O
H
H
O
R
+
R N
H
O
R O
O
H H2N
R
+
R

• A molecule endogenous to the body donates a portion of
itself to the foreign molecule – Conjugation reactions
PHASE II REACTIONS
GLUCURONIDATION
SULFATE CONJUGATION
GLYCINE CONJUGATION
ACETYLATION
METHYLATION
TRANSULFURATION
GLUTAMINE
CONJUGATION
MERCAPTURIC ACID
SYNTHESIS

• Most common conjugative pathway
• Greatly enhances water solubility
• Numerous functional groups can combine with it
• Readily available in body
• Has polar carboxyl and hydroxyl groups
• Products are called glucuronides
• RN-G; RO-G; RCOO-G; RS-G; RC-G glucuronides could
form at C1 atom of β-glucuronide
• Phenolic & alcoholic hydroxyls are most common
functional groups metabolized
• Glucuronidation is not fully developed in infants and
children
GLUCURONIDATION

• Microsomal enzyme glucuronyl transferase – donation of
glucuronic acid from endogenously synthesized Uridine-
5-diphospho--D-glucuronic acid (UDPGA) to various
substrates to form glucuronide conjugates – e.g.
morphine & acetaminophen.
GLUCURONIDATION
N
HN
O
OH
HO
O
P
O
O
O
O
O
P
O
O
O
O
OH
HO
COOH
OH
Uridine-5-diphospho--D-glucuronic Acid

GLUCURONIDATION
N
HN
O
OH
HO
O
P
O
O
O
O
O
P
O
O
O
O
OH
HO
COOH
OH
Uridine-5-diphospho--D-glucuronic Acid
HO
O
benzoic acid
O
OH
HO
COOH
OH O
O
Benzoyl glucuronide

O-Glucuridation
SUBSTRATES FORMING GLUCURONIDES
O
N
H
OH
H3C
Hydroxyl phenol
Acetaminophen
OH
HN
Cl
Cl O
O2N
OH
Alcohol
Chloramphenicol
O
Carboxy group
Fenoprofen
CH3
OH
O

N-Glucuridation
Carbamate
Meprobamate
CH3
H3C
O
H2N O
O
NH2
O
Sulfonamide
Sulfadimethoxine
SO2
N
H
N N
NH2
OCH3
H3CO
Amine
Desipramine
NHCH3

S-Glucuridation
Disulfide
Disulfiram
SH
N
S
C2H5
C2H5
Sulfahydryl group
Methimazole
N
N
H3C
SH
C-Glucuridation
Phenylbutazone
Ph
Ph
O
O CH3

• Conducted by the soluble enzyme sulfotransferase
• Endogenous donor molecule to conjugation is 3-
phosphoadenosine-5-phosphosulfate (PAPS)
SULFATE CONJUGATION
N
O
OH
O
O
P
O
O
O
S
O
O
O
3-Phosphoadenosine-5-phosphosulfate
N
N
N
NH2
HO3P

• Conjugates are ethereal in character & non-inducible
• Cytosolic enzyme sulfotransferase – donation of sulfate
from endogenously synthesized PAPS to substrates –
form sulfate conjugates – e.g. acetaminophen.
• Occurs primarily with phenols and occasionally with
alcohols, aromatic amines and N-hydroxy compounds
• Sulfate amount in body is limited
• Leads to water soluble and inactive metabolites
• Glucuronidation of phenols is a competing reaction and
may predominate
SULFATE CONJUGATION

SULFATE CONJUGATION
N
O
OH
O
O
P
O
O
O
S
O
O
O
3-Phosphoadenosine-5-phosphosulfate
N
N
N
NH2
HO3P
H3C
O
H
N
OH
p-Hydroxyacetanilide
H3C
O
H
N
O S
OH
O
O
p-Hydroxyacetanilide sulfate

• Genetic variation occurs
• Some individuals are fast acetylators
• Some individuals are slow acetylators
• Acetyl coenzyme A is the endogenous donor molecule
• Aromatic amines (Ar-NH2), Hydrazine (-NH-NH2),
sulfonamides (H2N-C6H4-SO2-NH-R), hydrazides (-CO-NH-
NH2) & primary aliphatic amines
• Gives inactive and nontoxic metabolites but does not
enhance water solubility
ACETYLATION

• Various acetylases – e.g. choline acetylase & N-acetyl
transferase , all soluble enzymes, conduct the transfer of the
acetyl group of acetyl CoA to various substrates.
• Important metabolic route for drugs containing primary
amino groups – e.g. Isoniazid
ACETYLATION
N
O
OH
O
O
P
O
O
O
P
O
O
O
Acetyl coenzyme A
N
N
N
NH2
HO3P
CH3
CH3
HO
HN
O
HN
O
S
O
CH3

• Role in biosynthesis of many endogenous compounds and
inactivation of numerous active biogenic amines
• Minor pathway for conjugation of drugs and xenobiotics
• Does not give polar, water soluble metabolites but
pharmacologically inactive products
• Catechols, phenols, amines and N-heterocyclic and thiol
compounds
• Substrates undergoing O-methylation by COMT must contain
an aromatic 1,2-dihydroxy group
METHYLATION

• Cytosolic enzymes – catechol-O-methyl transferase (COMT)
& phenylethanolamine-N-methyl transferase (PNMT) –
donates methyl group from endogenously synthesized S-
adenosylmethionine to various substrates to form
methylated conjugates.
• Norepinephrine is N-methylated by PNMT to form
epinephrine.
• Norepinephrine, epinephrine, dopamine, and L-DOPA are O-
methylated by COMT.
METHYLATION

• A family of soluble enzymes that conducts
• N-methylation; N-CH3
• O-methylation; O-CH3
• S-methylation; S-CH3
• S-adenosylmethionine (SAM) is the endogenous donor
molecule – demethylated to S-adenosylhomocysteine
METHYLATION
N
O
OH
HO
S
S-adenosylmethionine
N
N
N
NH2
CH3
HOOC
H2N

METHYLATION
N
O
OH
HO
S
S-adenosylmethionine
N
N
N
NH2
CH3
HOOC
H2N
HO
OH
NH2
HO
Norepinephrine
HO
OH
NH
HO
Epinephrine
OH
OH
Catechol
R
OH
O
O-Methylated Catechol
R
CH3
PNMT
COMT

• Mediated by -mercaptopyruvate sulphur transferase
• Mediated by mitochondrial thiosulfate sulphur transferase
TRANSULFURATION
COOH
O
HS
H3C COOH
O
-mercaptopyruvic acid Acetic acid
Cyanide Thiocyanate
CNS
CN-

• Amino acids – glycine & glutamine – conjugate carboxylic
acids, aromatic acids & arylalkyl acids
• Carboxylic acid substrate is activated with ATP & coenzyme
A (CoA) to form an acyl-CoA complex
• Limited amount of amino acids in body is available so few
conjugation reactions occur
• Competes with conjugation with glucuronic acid
• Polar and water soluble metabolites
AMINO ACID CONJUGATION

Glutamine Conjugation
• Glutamine conjugation occurs with
arylacetic acids
N
O
Diphenhydramine
COOH
O O
O
HN
O
COOH
H2N
Glutamine conjugate
NH2
O
H2N
O
OH
Glutamine

Glycine Conjugation
• Glycine conjugation occurs with
aromatic acids and arylalkyl acids
Salicylic acid
Salicyl CoA
C
OH
ATP
Co-A
C
OH
O
CoA
O
OH
Salicyluric acid
C
OH
O
N
H
O
OH
H2N
O
OH
Glycine

• Also called glutathione (GSH) conjugation
• Conjugation of substrate to glutathione by the enzyme
glutathione transferase
• Hydrolytic removal of glutamic acid by glutamyl
transpeptidase
MERCAPTURIC ACID SYNTHESIS
HOOC
N
H
H
N COOH
NH2 O
SH
O
Glutathione

• Hydrolytic removal of glycine by cysteinyl glycinase
• Acetylation of the cysteinyl substrate by N-acetyltransferase
to form the N-acetylated cysteinyl conjugate of substrate;
substrate referred to as a mercapturate
• Important pathway for detoxifying chemically reactive
electrophilic compounds
• Covalent interaction of metabolically generated electrophilic
intermediates with cellular nucleophiles leads to drug
toxicity

• GSH protects cellular constituents by bonding to metabolites
via –SH group
• Molecules conjugated with GSH usually are not excreted as
such but undergo further biotransformation to give S-
substituted N-acetylcysteine products called mercapturic
acid
• Nucleophilic GSH reacts with electrophilic substrates as –
• Nucleophilic displacement at an electron deficient carbon
or heteroatom
• Nucleophilic addition to an electron deficient double bond

• Aliphatic and arylalkyl halides (Cl, Br, I), sulfates (-O-SO3
-),
sulfonates (-O-SO2-R),
• Nitro compounds (-NO2), and organophosphates (-O-P[OR]2)
have electron deficient carbon atoms to conjugate with GSH
• If not sufficiently electron deficient (not enough electron
withdrawing groups) GSH conjugation does not take place

Naphthalene
O
Epoxide
OH
S
H
N
HOOC N
H
COOH
O
O
NH2
OH
S
H2N
N
H
COOH
O
OH
S
H2N
OH
O
OH
S
H
N
OH
O
H3C
O
S
H
N
OH
O
H3C
O
1-Naphthyl mercapturic
acid

FACTORS AFFECTING METABOLISM
AGE DIFFERENCES
ENZYME INDUCTION
SEX DIFFERENCES
ENZYME INHIBITION
SPECIES AND STRAIN
DIFFERENCES
HEREDITARY OR GENETIC
FACTORS
MISCELLANEOUS

Age Differences
• Apparent in the newborn – Fetal and newborn animals,
undeveloped or deficient oxidative and conjugative
enzymes – responsible for reduced metabolic capability.
• Oxidative and conjugative (e.g. glucuronidation) of
newborns are low compared with those of adult humans
– e.g. CYP metabolism of tolbutamide is lower in
newborn (half-life > 40 hrs) compared to adults (8 hrs).
• Poor glucuronidating ability – because of deficiency in
glucuronyltransferase activity.

Age Differences
• Drug metabolism diminishes with old age.
• Depends on
• Prolonged plasma half-lives of drugs that are
metabolized totally or mainly by hepatic microsomal
enzymes (e.g. antipyrine, phenobarbital, etc.).
• Loss of enzymatic activity with aging
• Effect of diseased liver from hepatitis, cirrhosis, etc.
• Decreased renal function.

Species and Strain Differences
• Species dependent – Different animal species may
biotransform a molecule by similar or different
metabolic pathways.
• Within the same species – individual variations (strain
differences) may result in significant differences in a
specific metabolic pathway.
• Species variation has been observed in many oxidative
biotransformation reactions – e.g. metabolism of
amphetamine occurs by two main pathways – oxidative
deamination or aromatic hydroxylation.

Species and Strain Differences
• Human, rabbit & guinea pig – oxidative deamination is
predominant while in rat, aromatic hydroxylation is
more important route.
• Species differences in conjugation reactions – caused by
the presence or absence of transferase enzymes involved
in conjugative process – e.g. cats lack
glucuronyltransferase enzymes, hence phenolic
molecules are conjugated by sulfation

Hereditary or Genetic Factors
• Genetic or hereditary factors are responsible for large
differences in the rate of metabolism of drugs.
• E.g. – biotransformation of isoniazid by acylation.
• Response to codeine and codeine analogs – CYP2D6
isozyme does not readily O-demethylate codeine to form
morphine.

Sex Differences
• Rate of metabolism varies according to gender – e.g.
adult male rats show N-demethylation of aminopyrine,
hexobarbital oxidation, etc. at a much faster rate than
female rats
• Also depends on the substrate, because some molecules
are metabolized at the same rate in both female and
male rats.
• Sex differences can also be species dependent e.g.
rabbits and mice do not show significant sex difference
in drug metabolism.

Sex Differences
• Nicotine and aspirin are metabolized differently in
women and men.
• Gender differences can become significant in terms of
drug–drug interactions based on the drug’s metabolism
– e.g. antibiotic rifampin, CYP3A4 inducer, can shorten
the half-life of oral contraceptives.

Enzyme Induction
• Activity of hepatic microsomal enzymes (e.g. CYP mixed-
function oxidases) can be increased by exposure to
drugs, pesticides, etc. – enzyme induction.
• Enzyme induction – increases rate of drug metabolism &
decreases duration of drug action.
• Inducing agents may increase the rate of their own
metabolism as well as those of other unrelated drugs or
foreign compounds

Enzyme Induction
• Concomitant administration of two or more drugs may
lead to serious drug interactions
• E.g. Induction of microsomal enzymes by phenobarbital
increases metabolism of warfarin – decreases
anticoagulant effect.

Enzyme Inhibition
• Several drugs, other xenobiotics, other foods, etc. can
inhibit drug metabolism
• Decreased metabolism – drug accumulates leading to
prolonged drug action & serious adverse effects.
• Occurs by diverse mechanisms – e.g. substrate
competition, interference with protein synthesis,
inactivation of drug metabolizing enzymes, etc. leading
to impairment of enzyme activity.

Enzyme Inhibition
• Concomitant administration of two or more drugs may
lead to serious drug interactions
• E.g. Phenylbutazone stereoselectively inhibits
metabolism of more potent (S)(–) enantiomer of
warfarin – increased anticoagulant effect – causing
haemorrhage.
• Grapefruit drug interaction – caused by bioflavonoids or
the furanocoumarins.

Miscellaneous
• Dietary factors (e.g. protein-to-carbohydrate ratio)
• Indoles present in vegetables stimulate metabolism
• Vitamins, minerals, starvation, malnutrition also
influence drug metabolism.
• Physiological factors (pathological state of liver,
pregnancy, hormonal disturbances, etc. may affect drug
metabolism.

• Many drugs – e.g. warfarin, propranolol, hexobarbital,
etc. are administered as racemic mixtures in humans.
• The two enantiomers may differ in pharmacological
activity – One enantiomer tends to be much more active
than the other e.g. (S)(-) enantiomer of warfarin is 5
times more potent as an oral anticoagulant than the
(R)(+) enantiomer.
STEREOCHEMICAL ASPECTS

• The two enantiomers may have totally different
pharmacological activities e.g. (+)--propoxyphene is
analgesic, whereas (-)--propoxyphene is antitussive.
Levopropoxyphene
Antitussive
Dextropropoxyphene
Analgesic

• Stereochemical factors have a dramatic influence on how
the drug molecule interacts with the target receptors to
elicit its pharmacological response.
• Difference in interaction of stereoisomers with drug
metabolizing enzymes may lead to differences in
metabolism for the two enantiomers of a racemic
mixture e.g. less active (+)-enantiomer of propranolol
undergoes rapid metabolism than the corresponding (-)-
enantiomer.

• Individual enantiomers of a racemic mixture also may be
metabolized by different pathways e.g. the more active
(S)(-)-isomer of warfarin is 7-hydroxylated (aromatic
hydroxylation), whereas (R)(+)-isomer undergoes keto
reduction to yield primarily the (R,S) warfarin alcohol as
the major plasma metabolite.

• Metabolism often lead to creation of a new asymmetric
center in the metabolite (i.e. stereoisomeric or
enantiomeric products). The preferential metabolic
stereoisomeric product is called product
stereoselectivity – e.g. bioreduction of ketones produces
predominantly one stereoisomeric alcohol

• Oxidative biotransformations display product
stereoselectivity – e.g. phenytoin contains two phenyl
rings – p-hydroxylation occurs preferentially (90%) at
the pro-(S)-phenyl ring to give primarily (S)(-)-5-(4-
hydroxyphenyl)-5-phenylhydantoin. The other phenyl
ring also is p-hydroxylated to a minor extent (10%).

• The term regioselectivity – denotes selective metabolism
of two or more similar functional groups (e.g. OCH3, OH,
NO2) or two or more similar atoms that are positioned in
different regions of a molecule – e.g. of the four methoxy
groups present in papaverine, the 4-OCH3 group is
regioselectively O-demethylated

N
N
OH
CH3
NO2
N
I
I
OH
N
H3C
CH3
NH
S NH
O
N
N N
N
H
O
O
H3C
CH3
N
O
N CH3
CH3
NH2
CH3

PCI - BPH4 - METABOLISM chapter pdf simple

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PCI - BPH4 - METABOLISM chapter pdf simple