Phase II or Conjugation Reactions
– Conjugation reactions link an endogenous moiety
(an endocon) to the original drug (if polar functions
are already present) or to the Phase I metabolite.
 They are catalyzed by enzymes known as transeferases.
 They involve a cofactor which binds to the enzyme in
the close proximity of the substrate and carries the
endogenous molecule or moiety to be transferred.
 Except for methylations and acetylations, this endocon
is highly polar and its size is comparable to that of the
substrate.
Phase II Reactions Include:
1. Glucuronidation
2. Sulfate Conjugation
3. Acetylation
4. Amino Acids (GLYCINE) Conjugation
5. Methylation
6. Glutathione Conjugation
1. Glucuronidation
 The mechanism of glucuronidation is one of nucleophilic
substitution with inversion of configuration, -D-glucuronic
acid in UDPGA forming -D-glucuronides.
OH
gluconic acid
OH
COOH
OH
OH
HO
OH
O OH
COOH
OH
HO
glucose
O OH
OH
OH
HO
glucuronic acid
OH
Glucuronidation Pathway and Enterohepatic
Recirculation
 The functional groups able to undergo glucuronidation are
classified as O-, N-, S- and C-glucuronides.
• O-Glucuronidation
includes
phenolic
xenobiotics
or
metabolites generally at high doses (O-sulfation is
predominating at low doses).
OH
R
O
GLU
R
GLU = glucuronic acid
– Alcohols are another major substrates, may primary,
secondary or tertiary ones.
Estriol glucuronide
 An interesting example is that of
morphine, which is conjugated on its
phenolic and secondary alcohol groups to
form the 3-O-glucuronide and the 6-Oglucuronide respectively.
N
CH 3
N
1
1
3
3
ULG
O
CH 3
O
3-O-glucuronide
6
OH
HO
O
6-O-glucuronide
6
O
GLU
The second important after O-Glucuronides are the
N-glucuronides
formed
from
carboxamides,
sulfonamides, and various amines. N-Glucuronidation
of aromatic and aliphatic amines in addition to
pyridine-type nitrogens has been observed in only a
R1
few cases.
R1
N H
R2
O O
R1 S
N H
R1
N
GLU
R2
O O
R1 S
N GLU
R2
R2
R1
N CH3
CH3
N
R2
R2
GLU
R
R
N
N GLU
S-Glucuronides are formed from aliphatic thiols,
aromatic thiols and dithiocarboxylic acids.
R
SH
R
S
GLU
C-Glucuronidation is seen in humans for 1,3-dicarbonyl
drugs such as sulfinpyrazone
.
C6H5
C6H5
N
O
C6H5
N
N
O
H
C6H5
HOOC
CH 2CH 2SC6H5
O
O
O
HO
HO
OH
N
O
CH 2CH 2SC6H5
O
 Beside xenobiotics, a number of endogenous
substrates, notably bilirubin and steroids are
eliminated as glucuronide conjugates.
 In neonates and children, glucuronidation
processes often are not fully developed. In such
subjects, drugs and endogenous compounds (e.g.
bilirubin) that are normally metabolized by
glucuronidation may accumulate and cause serious
toxicity.
 For example, neonatal hyperbilirubinemia and
gray baby syndrome, result from accumulation of
toxic levels of the free chloramphenicol.
2. Sulfate conjugation
In reactions of sulfoconjugation, a sulfate molecule
is
transferred
from
the
cofactor
(3’phosphoadenosine 5’-phosphosulfate, PAPS) to the
substrate by cytosolic enzymes known as
sulfotransferases.
NH2
N
N
R X H
N
O
HO O2 S
O
P
O
O
O
N
R X SO3H
N
+
O
HO
OH
H
NH2
P
N
O
O
OH
O
P
OH
H
OH
OH
O
O
P
OH
OH
OH
N
N
 Phenols compose the main group of substrates
undergoing sulfate conjugation. Thus, drugs
containing phenolic moieties often susceptible to
sulfate formation.
O
O
O
NHCCH 3
NHCCH 3
NHCCH 3
+
OH
OC6H9O6
O-Glucuronide
Conjugate
OSO3O-Sulfate
conjugate
The sulfate conjugation of N-hydroxylamines and
N-hydroxyamides takes place occasionally as well.
O-Sulfate conjugates of N-hydroxy compounds are
of considerable toxicologic concern because they
can lead to reactive intermediates that are
responsible for cellular toxicity.
O
O
O3SO
HO
NHCCH 3
N
CCH 3
OC2H5
OC2H5
O
-
N
CCH 3
OC2H5
3. Amino acid conjugation
This is a major route for many xenobiotic acids,
involving the formation of an amide bond between the
xenobiotic acyl-CoA and the amino acid.
O
ATP
PPi
O
OH
CoASH
AMP
O
AMP
SCoA
Amino acid
N-acyltransferase
H 2N
CoASH
O
COOH
C R
N
H
H
Amino acid conjugate
COOH
C R
H
Amino acid
Glycine is the amino acid most frequently used for
conjugation.
The xenobiotic acids undergoing amino acid
conjugation are mainly benzoic acids such as benzoic
acid itself and salicylic acid, forming hippuric acid and
salicyluric acid respectively.
R
R
O
O
O
OH
Bnzoic acid, R=H
Salicylic acid, R=OH
NHCH2COH
Hippuric acid, R=H
Salicyluric acid, R=OH
4. Conjugation with glutathione (GSH)
– Glutathione (GSH, -glutamylcysteinylglycine) is a
thiol-containing tripeptide.
– GSH conjugation is an important pathway by which
chemical reactive electrophilic compounds are
detoxified.
– Many serious drug toxicities may be explainable also
in terms of covalent interaction of metabolically
generated electrophilic intermediates with cellular
nucleophiles.
– GSH protects vital cellular constituents against
chemically reactive species by virtue of its
nucleophilic sulfhydryl (SH) group.
NH2
H
N
HS
C
C
E
S
H
H
GSH S-Transferase
N
H
COOH
O
C
O
C
C
O
+
Electrophilic substrate
H
N
COOH
H
H
E
NH2
C
COOH
O
N
H
COOH
GSH
Adduct or Conjugate
GSH
Amino acid (AA)
Glutamyl
Transpeptidase
O
Glutamyl AA
C
H3C
NH
E
S
C
H
CoASH
C
O
OH
Mercapturic Acid Derivative
Acetyl
CoA
N-Acetylase
E
S
NH2
C
H
Glycine
E
NH2
C
H
C
C
O
OH
S-Substituted
Cysteine Derivative
S
Cysteinyl Glycinase
O
N
H
COOH
 Unlike other conjugative phase II reactions, GSH
conjugation does not require the initial formation of
an activated enzyme or substrate.
–Two general mechanisms are known for the reaction
of compounds with GSH:
1. Nucleophilic displacement at an electron-deficient
carbon or heteroatom.
GSH

CH2

X
SG
R
R = Alkyl, Aryl, Benzylic, Allylic
X = Br, Cl, I, OSO3-, OSO2R, OPO(OR)2
CH2
R
+
HX
2- Nucleophilic addition to an electron-double bond.
O-
O
C

C

C
C
C
GSH
C
C
Michael
Addition
+
C
O-
O
C
C CH C
SG
SG
Unsaturated system
Glutathione Adducts
– The major reactions of glutathione are summarized
in the following scheme:
OH
H
R
O
OH
SG
O
SG
H
OH
OH
R1
H
C
C
R2
R
R1
C
X
X
X
C
C
H
O
SG
CH2
R2
R
X
C
R
R
C
X
R
HNO2
ONO2
GSH
R
OSG
SG
R
SG
(X= halogen)
R
C
SG
X
C
X
O
C
Cl
X
C
R
X
X
(X= CO-R2, CN, ...)
X
SG
OH
O
GSSG
R
OH
– In most instances, GSH conjugation
regarded as a detoxifying pathway
is
– In a few cases, GSH conjugation has been
implicated in causing toxicity e.g. 1,2-dichloroethane.
Cl
H2C
CH2 Cl
GSH
Cl
H2C
CH2
Cellular
S
SG
Macromolecule
G
S-(2-chloroethyl)glutathion
Episulfonium ion
Toxicity
5- Acetylation Reactions
–The most common reactions are acetylations of
xenobiotics containing a primary amino group.
–The major reactions of N-acetylation
summarized in the following scheme.
NH2
NH-COCH3
R1
R1
N-CO-CH3
N-H
R
R
R2
R2
R
NH-NH2
R
NH-NH-CO-CH3
NHOH
NH-OCOCH3
R
R
OH
N
R
COCH3
are
O
Ar NH 2
O
CoA S
R NH 2
R OH
R
Ar N
H
+
Acetyl transferase
SH
O
R O
CH3
CH3
O
O
R N
H
CH3
R
S
Examples: Procainamide, isoniazid, sulfanilimide,
histamine
N-acetyl transferase enzyme is found in many
tissues, including liver
CH3
–Acetylation result in decreased water solubility.
– Genetic differences exist in some animal species
and in humans, where one distinguishes between
slow and fast acetylators.
– Examples of drugs exhibiting acetylation
polymorphism is the antituberculosis drug isoniazid
(INH)
–The drugs metabolized by acetylation may become
ineffective in fast acetylators and cause toxicity in
slow acetylators.This liver toxicity may be
attributed to the formation of chemically reactive
acylating intermediates as shown in the following
scheme:
O
NHNH2
O
NHNHCOCH 3
O
OH
O
Hydrolysis
CH3CNHNH2
+
N
N
N
N-Oxidation
Liver
Damage
Covalent
Binding
O
O
CH3C
+ CH3C
Reactive intermediates
Procainamide
O
Unchanged
in Urine, 59%
H2 N
N
H
24% Fast
17% Slow
H
N
Unchanged
in Urine, 85%
N
O
3%
O
NAPA
N
H
N
1%
0.3%
H
N
O
O
N
H
H
N
O
H2 N
N
H
H
N
Procainamide
O
H2 N
N
N
H
trace metabolite
HO
H
N
O
N
N
H
non-enzymatic
O
O N
N
H
N
Lupus?
6- Methylation Reactions
Methylation is an important reaction in the
biosynthesis of endogenous compounds such as
adrenaline, and in the inactivation of biogenic amines
such as catecholamines.
Reactions of methylation imply the transfer of a
methyl group from the onium-type cofactor Sadenosylmethionine (SAM) to the substrate by means
of a methyltransferase
H2N
H2N
CO2H
H
H
N
R
X
NH2
O
O
N
N
Methyltransferase
R
X
CH3
OH
N
+
HO
HO
NH2
S
S
H
N
N
N
H3C
CO2H
OH
N
• Methyltransferases having particular importance in the
metabolism of foreign compounds include catechol-Omethyltransferase (COMT), phenol-O-methyltransferase and
nonspecific N-methyltransferase and S-methyltransferase.
• Examples of drugs undergoing methylation are given
in the following scheme.
SH
OH
HO
3
CH3
CH3
COOH
HO
NH2
NH2
N
N
Nicotine
N
N
CH3
CH3
N
CH3
N
H
6-Mercaptopurine
Norephedrine
S(-)--Methyldopa
N
N
FACTORS AFFECTING DRUG METABOLISM
– Many factors may affect drug metabolism,
these include:
1. AGE
2. DIET
3. STATE OF HEALTH
4. GENDER
5. GENETIC VARIATION
6. SPECIES VARIATION
7. SUBSTRATE COMPETITION
8. ENZYME INDUCTION
9. ROUTES OF DRUG ADMINISTRATION
AGE
 Age – related differences in drug metabolism are generally
quite apparent in newborn.
 Undeveloped or deficient oxidative and conjugative
enzymes are chiefly responsible for the reduced capability.
Examples:
• Undeveloped glucuronidating processes in
neonates and children are responsible for
neonatal hyperbilirubinemia and gray baby
syndrome as dissussed before.
• The oxidative metabolism of tolbutamide is
significantly lower in infants (t½ more than 40 hrs)
compared with adults (t½ ~ 8 hrs).
SPECIES VARIATION
– The metabolism of many drugs and xenobiotics is often
species dependent.
Examples:
HN
OH
O
N
H
O
S(-)-p-Hydroxyphenytoin
(Man)
OH
HN
O
N
H
O
Phenytoin
HN
O
N
H
O
R(+)-m-Hydroxyphenytoin
(Dog)
O
Oxid.
Oxid. deamination
OH
O
Benzoic acid
(Man, Rabbit, Guinea pig)
NH2
Amphetamine
Aromatic hydroxylation
NH2
HO
p-Hydroxyamphetamine
(Rat)
 Phenylbutazone half-life is 3 h in rabbit, ~6 h in rat,
guinea pig and dog and 3 days in humans.
Other notable species differences include:
– Dogs: deficient in N-acetyl transferase (cannot acetylate
aromatic amines)
– Guinea-pigs: deficient in sulfotransferase activity; no Nhydroxylation
– Cats: poor UDPGlucuronosyltransferase activity (unable to
glucuronidate phenols)
– Rats: often very rapid metabolizers; CYP2C is the major
family in the liver with significant gender differences
– Cynomolgus monkeys: reported to have low CYP1A2
activity
GENETIC VARIATION
• Marked individual differences in the metabolism of
several drugs exists in humans.
• Many of these genetic or hereditary factors are
responsible for the large differences seen in the rate of
metabolism of these drugs.
•Example: Isoniazid - N-acetyltransferase
O
NHNH2
O
NHNHCOCH 3
O
OH
O
Hydrolysis
CH3CNHNH2
+
N
N
N
N-Oxidation
Liver
Damage
Covalent
Binding
O
O
CH3C
+ CH3C
Reactive intermediates
• First line drug in the treatment of TB; normally
given at a does of 5 mg/kg, max. 300 mg/day for
period of 9 months.
• Rapid and slow acetylators first seen in TB patients;
t1/2 for fast is 70 min; t1/2 for slow is 180 min.
•
N-acetyltransferase (NAT2 isoform) is in liver, gut
• Peripheral neuropathy (about 2% patients; higher
doses produce effects in 10-20%) seen in slow
acetylators.
•
Hepatotoxicity also seen, esp. in older patients.
Genetic Polymorphisms in Drug Metabolizing Enzymes
GENDER
• Male Rats metabolize many drugs faster than
females through oxidative pathways, glucuronide and
GSH conjugation of certain substances,
• In Humans this difference also existed, though very
limited and usually clinically not important.
• e.g.The clearance of benzodiazepines eliminated by
metabolic conjugation is significantly smaller in
women than in man.
ENZYME INDUCTION
• Certain drug substrates of CYP, upon repeated
administration, “induce” or elevate the amounts
of specific forms of the enzyme.
• Induction results in accelerated metabolism of
the drug inducer and any other drugs
metabolized by the induced enzyme.
Classic Enzyme Inducers
•
•
•
•
•
Chronic ethanol
Phenobarbital
Tobacco (benzo[a]pyrene)
PCBs (Poly chlorinated biphenyls), dioxin (environmental)
Glucocorticoids
ENZYME INHIBITION
• Some
drugs
are
capable
of
inhibiting
metabolizing enzymes.
• This is usually only important if a patient on a
stable drug regimen starts taking a second drug
metabolized by the same enzyme.
• Examples: cimetidine, ethanol
Drugs that Inhibit Drug Metabolism by Forming
Complexes with CYPs
Amphetamine
Cimetidine
Dapsone
2,5-Dimethoxy-4methylamphetamine
Diphenylhydramine
Erythromycin
Fenfluramine
Itraconazole
Ketoconazole
Methadone
Methamphetamin
eNortriptyline
SKF 525A
Sulfanilamide
Non-nitrogenous Substances that Effect Drug Metabolism
by Forming Complexes with CYPs
• Grapefruit juice - CYP 3A4 inhibitor; highly
variable effects; unknown constituents
• Isosafrole, safrole - CYP1A1, CYP1A2 inhibitor;
found in root pear, perfume
• Piperonyl butoxide & alcohol -CYP1A1, CYP1A2
inducer; insecticide constituent
Effect of Grapefruit Juice on Felodipine Plasma Concentration
Cl
H
CH 3 O 2 C
CH 3
N
H
Cl
CO 2 CH 3
CH 3
Cl
3A4
Cl
CO 2 CH 3
CH 3 O 2 C
CH 3
N
CH 3
5mg tablet
with juice
without
STATE OF HEALTH
•
•
•
•
Liver disease
Kidney disease
Endocrine diseases
Reduced blood flow
Sample biotransformation problem
H
N
O
C
OCH3
CH3
N
O
A.
O
C
OCH3
C
O
CH3
H3C
N
O
O
B.
C
C
cocaine
OCH3
O
C.
O
CH3
N
O
O
C
CH3
N
OCH3
D.
OH
O
C
OCH3
O
A………………………………..
B………………………………...
C…………………………………
D………………………………….
C
C CH3
O
Optimising Pharmacokinetic
Properties
7. Designing Prodrugs and Bioprecursors
Prodrug is any compound that undergoes
biotransformation prior to exhibiting its
pharmacological effects.
Prodrugs can be divided into two classes:
7.1. The Carrier-prodrugs :
Result from a temporary linkage of the active
molecule with a transport moiety that is
frequently of lipophilic nature.
Prodrugs to mask toxicity and side effects
• Mask groups responsible for toxicity/side effects
• Used when groups are important for activity
Example:
Aspirin for salicylic acid
O
OH
H3C
CO2H
O
CO2H
Salicylic acid
Aspirin
•
Analgesic, but causes stomach
ulcers due to phenol group
•
•
Phenol masked by ester
Hydrolysed in body
Drug
Barrier to
delivery of
drug
Pro-moiety Drug
Barrier to
delivery of
drug
Pro-moiety
Drug
Biotransformation
Pro-moiety
Non-toxic &
rapidly excreted
+
+
Drug
Efficacy
7.1.1 Practical applications of carrier-prodrug design
1. Improvement of the lipophilicity
OH
O
O
O
CH3
NH2
OH
Esterase
HO
Cl
Cl
O
CH3
NH2
+
OH
HO
Epinephrine HCl
Dipivefrin HCl
O
Pivalic acid
Candoxatril for Candoxatrilat (protease inhibitor)
OMe
OMe
O
O
H
N
HO
O
O
Candoxatrilat
•
•
O
CO2H
O
Candoxatril
5-indanyl group
Varying the ester varies the rate of hydrolysis
Electron withdrawing groups increase rate of hydrolysis
(e.g. 5-indanyl)
•
H
N
O
Leaving group (5-indanol) is non toxic
CO2H
2. Enhancement of water solubility
Prodrugs to increase water solubility
•
•
•
Often used for i.v. drugs
Allows higher concentration and smaller dose volume
May decrease pain at site of injection
Example:
Succinate ester of chloramphenicol (antibiotic)
HO
O
Succinate ester
O
O
H H
N
H
O2N
O
OH
OH
H H
N
Cl
Cl
Esterase
H
Cl
Cl
O
OH
O2N
Chloramphenicol
Prodrugs to increase water solubility
Example:
Phosphate ester of clindamycin (antibacterial)
CH3CH2CH2
Me
N
H
H
O
C
Cl
H
C
CH3
N C
H
O H
HO
OH H
H
SCH3
H
•
H
OPO32-
Less painful on injection
Prodrugs to increase water solubility
Example:
Lysine ester of oestrone
Me
H
NH2
H
O
H
H
H
H2N
O
O
Prodrug
•
•
•
•
Me
H2N
H
NH2
H
H
+
O
Lysine
O
OH HO
Oestrone
Lysine ester of oestrone is better absorbed orally than oestrone
Increased water solubility prevents formation of fat globules in gut
Better interaction with the gut wall
Hydrolysis in blood releases oestrone and a non toxic amino acid
3. Prodrugs to prolong activity
Add hydrophobic groups
Increase the duration of Pharmacological effects
Example:
Hydrophobic esters of fluphenazine (antipsychotic)
fatty ester
N
N
(CH2)8CH3
O
O
H
N
CF3
S
•
•
•
•
Given by intramuscular injection
Concentrated in fatty tissue
Slowly released into the blood supply
Rapidly hydrolysed in the blood supply
Mask polar groups
•
Reduces rate of excretion
Example:
Azathioprine for 6-mercaptopurine
O2N
N
SH
S
N
Me
N
N
N
N
N
N
H
N
N
H
6-Mercaptopurine
Azathioprine
(suppresses immune response)
•
Short lifetime - eliminated too quickly
•
•
Slow conversion to 6-mercaptopurine
Longer lifetime
4. Improvement of patient acceptance
– Used to reduce solubility of foul tasting orally active drugs
– Less soluble on tongue
– Less revolting taste
Example:
Palmitate ester of chloramphenicol (antibiotic)
OH NHCOCHCl2
O2N
CH CH CH2 O CO(CH2)14CH3
Palmitate ester
O
O
H H
N
H
O2N
O
OH
OH
H H
N
Cl
Esterase
Cl
H
Cl
Cl
O
OH
O2N
Chloramphenicol
5. Prodrugs used to target drugs
Example:
Hexamine
N
N
N
N
•
•
•
•
Stable and inactive at pH>5
Stable at blood pH
Used for urinary infections where pH<5
Degrades at pH<5 to form formaldehyde (antibacterial agent)
6. Increased site-specificity.
Two targeting possibilities can be considered:
6.1. Site-directed drug delivery
OH
CO2H
H
H
O
N
DRUG
(CH2)n O
H
H
OH
H
Bile acids for liver specific targeting
6.2. Site-specific drug release
HO
NH2
HO2C
O
HO
N N
S NH
N
azoreductase
Amino Salicylic acid
+
O
HO2C
O
Sulfasalazine
H2N
N
S NH
O
Sulfapyridine
Chemical Delivery Systems: Pharmacologically inactive
molecules that require several steps of chemical and/or
enzymatic conversion to the active drug and enhance
drug delivery to particular organs or sites. Example:
Brain-specific chemical delivery system
7.2. The Bioprecursors :
Result from a molecular modification of the active
principle itself. This modification generates a new
compound, able to be a substrate for the metabolizing
enzymes (often, oxidation and reduction). The
metabolite being the expected active principle.
Example for bioprecursor design based on oxidative
bioactivation is the cytotoxic agent cyclophosphamid.
Cyclophosphoramide for phosphoramide mustard
(anticancer agent)
NH
O
Phosphoramidase
Cl
(liver)
H2N
HO
N
Cl
P
P
O
O
N
Cl
Cl
Cyclophosphoramide
Phosphoramide mustard
•
•
•
Non toxic
Orally active
Alkylating agent
Sulindac (NSAID) is a representative example
for bioprecursor bioactivated by reduction.
CO2H
CO2H
F
F
CH3
CH3
In vivo
Reduction
O
S
S
CH3
CH3
Inactive
Active
Soft Drugs
Biologically
active,
therapeutically
useful
chemical molecules (drugs) characterized by a
predictable and controllable in vivo deactivation,
after achieving the therapeutic objective, to
nontoxic, inactive compounds.
Soft Drugs Examples:
O
O
Cl
N
O
OH
Acetylcholine Estrase
+
N
HO
OH
O
N
Cl
O
Succinylcholine Chloride
O
Succinic acid
Choline Chloride
Cl
Hard drugs: these can be defined as drugs that
are biologically active and non metabolizable in
vivo eg: enalaprilat, lisinopril, DDT
Insecticide
DDT
chlorophenyl)ethane)
(1,1,1-trichloro-2,2-bis(p-