Medicinal chemistry on metabolism(Phase I & Phase II Reactions)

General Metabolic
Pathways
 Glucuronic acid conjugation
 Sulfate Conjugation
 Glycine and otherAA
 Glutathion or mercapturic acid
 Acetylation
 Methylation
Reduction
 Aldehydes and ketones
 Nitroand azo
 Reductive Dehalogenation
Oxidation
 Aromatic moieties
 Olefins
 Benzylic & allylic C atoms and
-C of C=O and C=N
 Ataliphaticand alicyclic C
 C-Heteroatomsystem
N.(, N-oxide formation, N-
hydroxylation, N-
dealkylation)
O. (O-dealkylation)
C-S (S-dealkylation, S-oxidation,
desulfuration)
 Oxidationof alcohols and
aldehydes
 Miscellaneous
Phase II -
Conjugation
Phase I -
Functionalization
Drug
Metabolism
Hydrolytic Reactions
 Estersand amides
Mr. Mote G.D

Phase I Reaction
⚫Polarfunctional groupsareeither
⚫ introduced into the molecule
⚫ or modified by oxidation, reduction or hydrolysis.
molecules by
ketones and
⚫ or convert lipophilic molecules into more polar
introducing orexposing polar functional groups.
⚫Aromatic and aliphatic hydroxylation or reduction of
aldehydes toalcohols.
⚫Phase I reactions may increase or decrease or leave unaltered the
pharmacological activityof thedrugs
⚫Objectives
⚫ Increasein hydrophilicity
⚫ Reduction in stability
⚫ Facilitationof conjugation
Mr. Mote G.D

Phase I Reaction
⚫Reactions in Phase I Metabolism
⚫Oxidation reaction
⚫Reduction
⚫Hydrolysis
⚫Hydration
⚫Isomerisation
⚫Miscellaneous
Mr. Mote G.D

Phase I Reaction
⚫Enzymes catalyzing phase I reactions
include
⚫cytochrome P-450
⚫aldehyde and alcohol dehydrogenase
⚫deaminases
⚫esterases
⚫amidases
⚫epoxide hydratases
⚫Location of these enzymes
⚫numerous tissues
⚫some are present in plasma.
⚫Subcellular locations include
⚫cytosol
⚫mitochondria
⚫endoplasmic reticulum
Mr. Mote G.D

Cytochrome P 450 Monooxygenase
⚫ General features
⚫ A large number of families (at least 18 in
mammals) of cytochrome P-450 (abbreviated
“CYP”) enzymes exists
⚫ each member of which catalyzes the
biotransformation of a unique spectrum of drugs.
⚫ some overlap in the substrate specificities.
⚫ This enzyme system is the one most frequently
involved in phase I reactions.
Mr. Mote G.D

Cytochrome P 450 Monooxygenase
⚫General features
⚫ The cytochrome P-450 families are referred to using an arabic
numeral, e.g., CYP1, CYP2, etc.
⚫ Each family has a numberof subfamilies denoted by an upper
case letter, e.g., CYP2A, CYP2B, etc.
⚫ The individual enzymes within each subfamily are denoted
byanotherarabic numeral, e.g., CYP3A1, CYP3A2, etc.
⚫ Cytochrome P-450 catalyzes numerous reactions, including:
⚫ aromatic and aliphatic hydroxylations
⚫ dealkylations at nitrogen, sulfur, and oxygen atoms
⚫ heteroatom oxidations at nitrogenand sulfuratoms
⚫ reductions at nitrogenatoms
⚫ esterand amide hydrolysis
Mr. Mote G.D

Cytochrome P 450
Monooxygenase
⚫ General features
⚫ The CYP3A subfamily is:
⚫ responsible forup to half of the total cytochrome P-450 in the
liver
⚫ accounts forapproximately 50% of the metabolism of
clinically importantdrugs.
⚫ CYP3A4 is a particularlyabundantenzyme.
Mr. Mote G.D

Cytochrome P 450
Monooxygenase
⚫Representative P450 isozymes
Mr. Mote G.D

Cytochrome P 450
Monooxygenase
⚫Cytochrome P450
⚫ The primary location of cytochrome P-450 is the liver,
⚫ Othertissues, including:
⚫ theadrenals
⚫ ovariesand testis
⚫ tissues involved in steroidogenesisand steroid metabolism.
⚫ Theenzyme's subcellularlocation is the endoplasmic reticulum.
Mr. Mote G.D

Cytochrome P 450
Monooxygenase
CH3
L CH3
HOOC
N N
CH2 Fe+3
N N
CH3
HOOC
CH3 CH2
O
H R
Substrate binding site
⚫Cytochrome P450
⚫ Name based on its lightabsorptionat 450 nm when complexed with
carbon monoxide
⚫ Hemoprotein (heme-thiolate) containing an iron atom which can
alternate between the ferrous (Fe++) and ferric (Fe+++) states
⚫ Electronacceptor
⚫ Servesas terminal oxidase
Heme portion
with activated
Oxygen
Mr. Mote G.D

Cytochrome P 450
Monooxygenase
⚫ Mechanism of reaction
⚫ In theoverall reaction:
⚫ thedrug is oxidized
⚫ oxygen is reduced towater.
⚫ Reducing equivalents are provided by nicotinamide
dinucleotide phosphate (NADPH), and generation
adenine
of this
cofactoris coupled to cytochrome P-450 reductase.
Mr. Mote G.D

Phase I Metabolism
A. Oxidation
B. Reduction
C. Hydrolysis
Mr. Mote G.D

Oxidation Reaction
13
⚫ Addition of oxygen or removal of hydrogen.
⚫ Normally the first and most common step involved in
the drug metabolism
⚫ Majority of oxidation occurs in the liver and it is possible to
occur in intestinal mucosa, lungs and kidney.
⚫ Most important enzyme involved in this type of oxidation is
cytochrome P450
⚫ Increased polarity of the oxidized products (metabolites)
increases their water solubility and reduces their tubular
reabsorption, leading to their excretion in urine.
⚫ These metabolites are more polar than their parent
compounds and might undergo further metabolism by
phase II pathways Mr. Mote G.D

Oxidation Reaction
Catalysed by microsomal enzymes
Require both molecular oxygen and reducing agent
NADPH, they are referred to as mixed function oxidases
(MFO).
⚫MFO consists of 3 components
⚫ Cytochrome P450- heme protein, transferof oxygen atom
⚫ Flavoprotein- NADPH dependent, acts as electron carrier, catalyze
the reductionof Cytochrome P450
⚫ Phosphatidylcholine- facilitate electrontransfer
RH + O2 + NADPH + H+ ROH + H2O + NADP+
14
Mr. Mote G.D

A. Oxidation
HN C
O
CH3
OH
HN C
O
CH3
Oxidation
Cyt p450 c Oxidase
Acetanilide
Paracetamol
Mr. Mote G.D

Oxidation reaction
a) Oxidation of aromaticcarbon atoms
b) Oxidation of olefins (C=C)
c) Oxidation of Allylic carbon atom
d) Oxidation of Alicyclic carbon atom
e) Oxidation of Carbon- hetero atom
f) Oxidation of Carbon – Nitrogen system
⚫N- dealkylation
⚫Oxidative deamination
⚫N- oxide formation
⚫N- hydroxylation
Mr. Mote G.D

a) Oxidation of aromatic carbon atoms
NH
C
N
H
O
O
NH
C
N
H
O
O
HO
Phenytoin
p-hydroxy Phenytoin
CYP2C19
Mr. Mote G.D

b) Oxidation of olefins (C=C)
N
C
O
NH2
CYP3A4
N
C
O
NH2
O
N
C
O
NH2
OH
OH
Epoxide Hydroxylase
Carbamazepine
(Inactive)
Carbamazepine
epoxide
(Active)
Carbamazepine 10,11 trans diol
(Inactive)
Mr. Mote G.D

c) Oxidation of Allylic carbon atom
N NH
O
O
H3C
H3C
O
N NH
O
O
H3C
H3C
O
Hydroxylation
OH
Hexobarbital
3-hydroxy hexobarbital
Mr. Mote G.D

d. Oxidation of Alicyclic carbon atom
N
N
H2N N
NH2
R
N
N
H2N N
NH2
Minoxidil
OH
O O
4-Hydroxy Minoxidil
Hydroxylation
Mr. Mote G.D

e. Oxidation of Carbon- hetero atom
I. N-oxidation
II. N-hydroxylation
III. N-dealkylation
IV. O-dealkylation
V. S-Oxidation
VI. S-dealkylation
VII.Oxidative deamination
VIII.Oxidative desulphuration
Mr. Mote G.D

I) N-oxidation(Alicyclic chain)
N
N
CH3
Nicotine N
N
CH3
Nicotine N-Oxide
O
Mr. Mote G.D

I. N-oxide(Aliphatic chain)
N
N
H3C
H3C
N
N
H3C
H3C
O
Imipramine
(Antidepressant)
N-imipramine N-oxide
Mr. Mote G.D

II. N-hydroxylation
CH3
H3C
HN
C
N
C2H5
C2H5
O
Lidocaine
(Local anesthetic)
CH3
H3C
N
C
N
C2H5
C2H5
O
Hydroxylation
HO
N-hydroxy lidocaine
Mr. Mote G.D

III. N-dealkylation
C
N
N
H3C
O
Cl
C
N
N
H
O
Cl
H C
O
H
Formaldehyde
Desmethyl Diazepam
Diazepam(Sedative)
Benzodiazepines
+
Mr. Mote G.D

O-Dealkylation
H3CO
H3C
O
N
N NH2
O
H2
C
OH
NH2
H3CO
H3CO
N
N NH2
OH
NH2
Trimethoprim
(antibacterial-UTI)
Mr. Mote G.D

S-Dealkylation
N
N
N
H
N
S
CH3
6-(Methyl thio)-Purine
Anticancer agent (Purine Antagonist)
N
N
N
H
N
S
H
6-Mercaptopurine
H-CHO
O
Mr. Mote G.D

S-Oxidation
N
S
N
CH3
S
CH3
N
S
N
CH3
S
CH3
Thioridazone(Antipsychotics)
(Phenothiazine) Sulphoridazone
O
O
O2
Mr. Mote G.D

Oxidative deamination
CH3
NH2
Amphetamine
(Adrenergic aginist)
CH3
O
Phenyl acetone
+ NH3
Mr. Mote G.D

Oxidative Desulphuration
N
H
NH
S
O
H3C
H3
C
O
Thiopental
(Sedative-Hypnotics)
N
H
NH
O
O
H3C
H3C
O
Pentobarbital
Mr. Mote G.D

Reductive Reactions
⚫Bioreduction of C=O (aldehyde and keton) generates
alcohol (aldehyde → 1o alcohol; ketone → 2o alcohol)
⚫Nitroand azo reductions lead toamino derivatives
⚫Reduction of N-oxides to their corresponding 3o amines
and reduction of sulfoxides to sulfides are less frequent
⚫Reductive cleavage of disulfide (-S-S-) linkages and
reduction of C=C are minor pathways in drug
metabolism
⚫Reductive dehalogenation is a minor reaction primarily
differ from oxidative dehalogenation is that the adjacent
carbon does not have to have a replaceable hydrogen
and generally removes one halogen from a group of two
or three
Mr. Mote G.D

Reductive Reactions
■ Reduction of Carbonyls
■ C=O moiety, esp. the ketone, is frequently
encountered in drugs and additionally, ketones
and aldehydes arise from deamination
■ Ketones tend to be converted to alcohols which
can then be glucuronidated. Aldehydes can also
be converted to alcohols, but have the additional
pathway of oxidation to carboxylic acids
■ Reduction of ketones often leads to the creation
of an asymmetric center and thus two
stereoisomeric alcohols are possible
Mr. Mote G.D

Reductive Reactions
• Reduction of Carbonyls
• Reduction of α, β,–unsaturated ketones found in steroidal
drugs results not only in the reduction of the ketone but
also of the C=C
• Aldo–keto oxidoreductases carry out bioreductions of
aldehydes and ketones. Alcohol dehydrogenase is a
NAD+ dependent oxidoreductase that oxidizes alcohols
but in the presence of NADH or NADPH, the same
enzyme can reduce carbonyl compounds to alcohols
H
R C OH
H
R C O
H
Aldehyde 1 alcohol
H
R1 C OH
R C O
R2
Ketone
R2
2 alcohol
Mr. Mote G.D

B. Reduction
N N
S
O
O
H2N
H2N
NH2
Prontosil(Prodrug)
S
O
O
H2N
H2N
NH2
Sulphanilamide
H2N
NH2
triamino benzene
+
Mr. Mote G.D

B. Reduction
a) Reduction of Aldehydes/ketones
b) Reduction of Nitro compounds
c) Reductive Dehalogenation
Mr. Mote G.D

I a) Reduction of Aldehyde
C
Cl
Cl
Cl
C
O
H .H2O
C
Cl
Cl
Cl
C
H
OH
H
Chloral Hydrate
CNS Depressants, Sedative hypnotics
Trichloro ethanol
Mr. Mote G.D

I. b)Reduction of Ketone
O
CH3 C
H
OH
CH3
Acetophenone
General Anesthetic, Hypnotics MethylPhenyl Carbinol
Mr. Mote G.D

II. Reduction of Nitro group
N
H
N
O2N
O
N
H
N
H2N
O
NADPH2 + O2
Nitrazepam(Benzodiazepine)
Sedative-Hypnotics
7-Amino metabolite
(active)
Mr. Mote G.D

Reductive dehalogenation
F3C C
Br
Cl
H
Halothane
(General Anesthetics)
NADPH2
F3C C
H
H
H
1,1,1 trifluro ethane
Mr. Mote G.D

⚫Hydrolyzes (adds water to) esters and amides and their
isosteres; the OH from water ends up on the carboxylic
acid (or its isostere) and the H in the hydroxy oramine
⚫Differs from oxidative and reductive reactions in 3
respects
⚫Does not involve change in the state of oxidation of
substrate
⚫Results in large chemical change in substrate, loss of
large fragment
⚫Hydrolytic enzymes acts on endogenous substrate
⚫Hydrolysis of esters and ethers
⚫Hydrolysis of amides
Mr. Mote G.D

■ Enzymes: Non-microsomal hydrolases;
however, amide hydrolysis appears to be
mediated by liver microsomal amidases,
esterases, and deacylases
■ Electrophilicity of the carbonyl carbon,
Nature of the heteroatom, substituents
on the
substituents
carbonyl carbon, and
on the heteroatom
influnce the rateof hydrolysis
■ In addition, Nucleophilicity of
attacking species, Electronic charge,
and Nature of nucleophile and its steric
factors also influence the rate of
hydrolysis
Naming carbonyl - heteroatom groups

O
R1 C R2
+
R1 R2 Name Susceptibility
to Hydrolysis
C O Ester Highest
C S Thioester
O O Carbonate
C N Amide
O N Carbamate
N N Ureide Lowest
Mr. Mote G.D

C. Hydrolysis
C
O
OH
O C
O
CH3 + H2O
C
O
OH
OH
Aspirin
Salicylic acid
+ CH3 C
O
OH
Acetic acid
Mr. Mote G.D

C. Hydrolysis
a) Hydrolysis of esters and ethers
b) Hydrolysis of amides
Mr. Mote G.D

O
H
R1 C N R2
O
R1 C OH H2N R2
O
R1 O C O R2
O
R1 OH HO C O R2
O
HO C OH
HO R2 O C O O H
H
+
+
+
Carbonate Carbonic acid derivative Carbonic acid
Carbonate hydrolysis
Ester hydrolysis
O
R1 C O R2
Amide hydrolysis(slower)
O
R1 C OH HO R2
Mr. Mote G.D

C. i) Hydrolysis of ester
C
O
OH
+ H2O
O C CH3
C
O
OH
OH
+
O
CH3 C
O
OH
Asprin(Ester)
Analgesic Salicylic acid
Mr. Mote G.D

C ii)Hydrolysis of Amide
CH3
CH3
H
N
N
O
Lidocaine
(Local Anasthetic)
CH3
CH3
NH2
N
O
OH
H2O
2,6-dimethylbenzenamine
2-(diethylamino)acetic acid
+
Mr. Mote G.D

Phase II Metabolism
A. Conjugation with glucuronic acid
B. Conjugation with sulphate moieties
C. Conjugation with alpha amino acids
D. Conjugation with glutathione
E. Acetylation reaction
F. Methylation reaction
Mr. Mote G.D

Phase II reactions
• Phase II metabolites are
• Attachment of small polar endogenous molecules such as glucuronic acid,
sulfate and amino acids to Phase Imetabolites or parentdrugs
• Products are more water-soluble and easilyexcretable
• Trapping highly electrophilic molecules with endogenous nucleophiles
such as glutathione prevent damage to important macromolecules (DNA,
RNA, proteins)
• Regarded as true detoxifying pathway (with few exceptions)
• In general, appropriate transferase enzymes activate the transferring group
(glucuronate, sulphate, methyl, acetyl) in a coenzyme form
Mr. Mote G.D

Phase II reactions
Characteristics of conjugation reactions
• Initial activation step
Drug is activated
or
Conjugating reagent activated
• Capacity limited reaction
a. Limited amount of conjugating reagent
b. Limited amount of enzyme
Order of capacity limited reaction
Glucuronidation > Amino acid conjugation > Sulphation and Glutathione conjugation
Mr. Mote G.D

Phase II reactions
Phase II reactions and their characteristics
Conjugation
reaction
Conjugating
Agent
Enzyme
involved
Activated
intermediate
Functional
groups
Glucuronidation Glucuronic Acid UDP-glucuronyl
transferase
UDPGA -OH, -COOH,
-NH2, -SH
Sulphation Sulphate Sulpho
transferase
PAPS -OH, -NH2
Amino Acid Glycine Acyl
transferase
Acyl CoA -COOH, NH2
Glutathione Glutathione Glutathione-S
transferase
- Alkyl halide,
alkyl nitrate,
alkyl epoxide,
etc
Acetylation Acetyl CoA N-Acetyl
Transferase
Acetyl CoA Hydrazines, -
NH2, -SO2NH2
Methylation L-Methionine Methyl
Transferase
S-adenosyl
Methionine
-OH,
-NH2, -SH
Mr. Mote G.D

• Glucuronidation is the most common conjugation pathway
• The coenzyme, UDP glucuronic acid is synthesized from the corresponding
phosphate
• UDP-glucuronic acid contains D-glucuronic acid in the a-configuration at the
anomeric center, but glucuronate conjugates are b-glycoside, meaning inversion
of stereochemistry is involved in the glucuronidation
• Glucuronides are highly hydrophilic and watersoluble
• UDP glucuronosyl transferase is closely associated with Cyp450 so that Phase I
products of drugs are efficientlyconjugated
• Four general classes of glucuronides: O-, N-, S-, and C-
• Neonates have undeveloped liver UDP-glucuronosyl transferase activity, and
may exhibit metabolic problem. For example, chloramphenicol (Chloroptic)
leads neonates to “gray baby syndrome”
• Neonatal jaundice may be attributable to their inability to conjugate bilirubin
with glucuronicacid
Conjugation with glucuronic acid
Mr. Mote G.D

1. Catalysed by microsomal enzymes.
2. Dominates at high substrate
concentration
3. Most common phase II reaction. Why?
Mr. Mote G.D

Steps in glucuronidation formation
1. Synthesis of activated coenzyme UDPGA
2. Transfer of glucuronyl moiety from UDPGA to substrate RXH
pyrophosphorylase
-D-Glucose-1-phosphate +UTP UDPG + PPi
UDPG-dehydrogenase
UDPG +2NAD+ + H2O UDPGA+ 2NADH +2H+
UDP-glucuronyl transferase
UDPGA +RXH RX-glucuronic acid + UDP
Where, X = O, COO, NH or S
Mr. Mote G.D

Mr. Mote G.D

A. Conjugation with glucuronic acid
OH
HN C
O
CH3
Paracetamol
(Active)
+
O
C
O
HO
OH
OH
OH
O
CH3
O
HN C
O
CH3
C
O
O
H3CO
OH
OH
OH
Glucoronide
(Inactive)
Non Polar
Water Soluble
Excretable
Mr. Mote G.D

1.O-glucuronide conjugation
a. OH compounds
Ex. Alcohol:- Chloramphenicol
Phenol:- Morphine, paracetamol
b. COOH
compounds Ex.
Salicylic acid
1. N-glucuronide conjugation
Ex. Desimipramine, Meprobamate
3. S-glucuronide conjugation
Ex. Thiophenol
4. C-glucuronide conjugation
Ex. Phenylbutazone
Mr. Mote G.D

• O-Glucuronide: Phenols
Acetaminophen Morphine
OH
CH3
O
H
N
HO OH
N
O
CH3
Mr. Mote G.D

• O-Glucuronide: Alcohols
Chloramphenicol Propranolol
O2N
Cl
Cl
H
N
OH
O
OH CH3
CH3
OH H
N
O
Mr. Mote G.D

O O
• O-Glucuronide: Enols
Hydroxycoumarine
OH
Mr. Mote G.D

• O-Glucuronide: N-hydroxyamines/amides
N-hydroxydapsone N-Hydroxy-2-
acetylaminoflourene
H2N S NHOH
O2
CH3
N
OH
Mr. Mote G.D

• O-Glucuronide:
Aryl
acids
Salicylic
Acid
Fenoprofe
n
O
OH
Arylalkyl
Acids
CH3
O
OH
COOH
Mr. Mote G.D

• N-Glucuronide:
Arylamine
N
H
N
NH2
O2N
7-Amino-5-nitroindazole
Alkylamines
Desipramine
N
H
CH3
N
Mr. Mote G.D

• N-Glucuronide:
Sulphonamides
Sulfisoxazo
le
H2N
N
H
O
O
S
CH3
N
CH
3
O
H3C
O
NH2
NH2
H3C O
O
Amides
O
Meprobamat
e
Mr. Mote G.D

• N-Glucuronide:
Amitryptiline
3o Amines
Cyproheptadine
CH3
N
3o Amines
Mr. Mote G.D

Nicotinine
• N-Glucuronide:
Aromatic Amine
Mr. Mote G.D

• S-Glucuronide:
Sulfhydryl
Methimazole
Carbodithioic acid
Disulfirum
(reduced form)
HS
N
CH3
N
H3C
H3C
S
SH
N
Mr. Mote G.D

• C-Glucuronide:
Phenylbutazone
CH3
O
O
N
N
Mr. Mote G.D

Example: Benzoic
acid
Water Soluble,
Excreted
Mr. Mote G.D

Conjugation with sulphate moiety
• Occurs less frequently than does glucuronidation
presumably due to fewer number of inorganic
sulfates in mammals and fewer number of
functional groups (phenols, alcohols, arylamines and
N-hydroxy compounds)
1. Catalysed by nonmicrosomal enzymes
2. Less common
3. Dominates at low substrateconcentration
Mr. Mote G.D

Steps in Sulphate Conjugation
1. Synthesis of activated coenzyme PAPS
2. Transfer of sulphate moiety from PAPS to substrate
RXH
ATP-sulfurylase/Mg++
SO4
2
-
ATP + APS + PPi
APS-phosphokinase/Mg++
APS +ATP PAPS+ ADP
Sulfotransferase
PAPS +RXH RX-SO3 + PAP
Where, X = O, NH
Mr. Mote G.D

Three enzyme-catalyzed reactions are involved in sulfate
conjugation
O
S
O
-O
ATP PPi
O-
Mg+2
ATP sulfurylase
Sulfate
O O
S O P O O
O-
-O
O
Ad
HO OH
Adenosine-5'-
phosphosulfate (APS)
Mg
+2
APS phosphokinase
O O
S O P O O
O-
-O
O
Ad
-2O3PO OH
ATP ADP
3'-phosphoadenosine-5'-
phosphosulfate (PAPS)
PAP
RXH
-O
O
S XR
O
Sulfate
conjugate
Sulfotransferase
(soluble)
Mr. Mote G.D

• Phenolic sulfation predominates
• Phenolic O-glucuonidation competes favorably with sulfation due to
limited sulfate availability
• Sulfate conjugates can be hydrolyzed back to the parent compound by
various sulfatases
• Sulfoconjugation plays an important role in the hepatotoxicity and
carcinogenecity of N-hydroxyarylamides
• In infants and young children where glucuronyltransferase activity is
not well developed, have predominating O-sulfate conjugation
• Examples include: a-methyldopa, albuterol, terbutaline,
acetaminophen, phenacetin, phenol (salbutamol, paracetamol),
amines (aniline)
Mr. Mote G.D

B. Conjugation with sulphate moieties
N
N
H2N N
NH2
O
+ S
O
OH
O
N
N
H2N N
NH2
O
S
O
O
HO
Minoxidil Sulphate
Inactive, Water soluble, Non Polar
Minoxidil N-Oxide
(Phase I Metabolite)
Active, Polar, Ionised
Mr. Mote G.D

• Examples
COOH
H3C
H
H
N
HO
HO HO
HO
CH3
CH3
CH3
OH H
N
-Methyldopa
OH
OH H
N CH3
CH3
CH3
HO
Albuterol Terbutaline
Mr. Mote G.D

Conjugation with alpha amino acids
• The first mammalian drug metabolite isolated, hippuric acid, wasthe
product of glycine conjugation of benzoicacid
• Amino acid conjugation of a variety of caroxylic acids, such as
aromatic, arylacetic, and heterocyclic carboxylic acids leads to amide
bond formation
• Glycine conjugates are the mostcommon
• Taurine, arginine, asparagine, histidine, lysine, glutamate, aspartate,
alanine, and serine conjugates have also beenfound
R O
COH
Benzoic Acid, R = H
Salicylic Acid, R = OH
R O O
CONHCH2COH
Hippuric Acid, R = H
Salicyluric Acid, R = OH
Mr. Mote G.D

Less common due to limited number of amino
acids
Steps:
1. Activation of carboxylic acid drug
substrate
2. Acetylation of amino acid
Acyl synthetase
RCOOH + ATP RCOAMP + H2O + PPi
Acyl CoA transferase
RCOAMP + CoASH
RCOSCoA + AMP
N-acyl transferase
RCOSCoA + H2N-R’ -COOH RXH RCONH-R’-COOH +
CoASH
Where, R’ = -CH2 If glycine, CH-CH2-CH2-NH2 if
glutamine
Mr. Mote G.D

Mechanism of Amino Acid conjugation
An Acyl-CoA
Intermediate
Glycine Conjugate R = H
Glutamine Conjugate R =
CH2CH2CONH2
Examples:
1. Aryl acid: Salicylic acid
2. Heterocyclic aryl acid: Nicotinic acid
Drug-
COOH
Mr. Mote G.D

C. Conjugation with alpha amino acids
N
Br
C
O
OH
Brom pheniramine Phase I Metabolite
+
N
H
H2
C
C
O
OH
Glycine
N
Br
C
O
NH
H
-H2O
H2
C
C
O
OH
Glycine Conjugate
Mr. Mote G.D

Conjugation with glutathione
Structure of glutathione
• Glutathione is a tripeptide (Glu-Cys-Gly) – found virtually in all
mammalian tissues
• Its thiol functions as scavenger of harmful electrophilic parent drugs
or their metabolites
• Examples include SN2 reaction, SNAr reaction, and Michael addition
Mr. Mote G.D

Conjugation with glutathione
Mechanisms of GSH conjugation
1.Nucleophilic substitution
RX + GSH R-S-G + H+ +
Ex. Sulphates, Organophosphates
2.Nucleophilic addition
x-
R-CH=CH-R’ + GSH RCH-S-G-CH2-R’
Ex. Paracetamol
Mr. Mote G.D

D. Conjugation with glutathione
HO
O
C
H2
H
N
O
SH
N
H
O
CH2
H2
C
NH2
O
OH
+
H2C
ONO2
HC
CH2
ONO2
O NO2
HO
O
C
H2
H
N
O
S
N
H
O
CH2
H2
C
NH2
O
OH
O
H2
C C
H
H2
C ONO2
O2NO
Glutathione Conjugate
Glutathione Nitroglycerine
Mr. Mote G.D

• Metabolism for drugs containing a primary amino group,
(aliphatic and aromatic amines), amino acids, sulfonamides,
hydrazines, and hydrazides
• The function of acetylation is to deactivate the drug, although
N-acetylprocainamide is as potent as the parent antiarrhythmic
drug procainamide (Procanbid) or more toxic than the parent
drug, e.g., N-acetylisoniazid
• Examples:
Aliphatic amines: Histamine
Aromatic amines: PAS,PABA, Dapsone
Sulphonamide: Sulphanilamide, Sulphapyridine
Hydrazides: Isoniazide
Mr. Mote G.D

• Acetylation is two-step, covalent catalytic process
involving N- acetyl transferase
3
H C SCoA
O
CoASH
3
H C X
O
H2N R
X-
3
O
H C NHR
X-
N-Acetylation of amines
Mr. Mote G.D

C
OH
O
OH
NH2
+ CH3 C
O
SCOA
C
OH
O
OH
HN
+
C
O
CH3
N-Acetylated PAS
Para Amino Salicylic acid
(PAS)
Acetyl Coenzyme A
Mr. Mote G.D

F. Methyl conjugation
Characteristics:
1. Metabolites formed are not polar
2. Drug and metabolite having equal pharmacological
activity
3. Less important for xenobiotics
4. Important in biosynthesis of endogenous amines, ex.
Adrenaline, while as inactivation of endogenous
amines, ex. NA, 5-HT, Histamine
Mr. Mote G.D

• Minor conjugation pathway, important in biosynthesis of
epinephrine and melatonin; in the catabolism of
norepinephrine, dopamine, serotonin, and histamine; and in
modulating the activities of macromolecules (proteins and
nucleic acids)
• Except for the formation of quarternary ammonium salts,
methylation of an amine reduces the polarity and hydrophilicity
of the substrates
• A variety of methyl transferase, such as COMT (catechol O-
methyl transferase), phenol-O-methyltransferase, N-methyl
transferase, S- methyltransferase etc are responsible for
catalyzing the transfer of methyl group from SAM to RXH
Mr. Mote G.D

Steps in methyl conjugation
1. Synthesis of an activated coenzyme SAM
2. Transfer of methyl group from SAM to substrate
Mr. Mote G.D

Methyl conjugation
Examples
1.O-methylation: Morphine, L-DOPA
2.N-methylation: Normorphine, Nicotine
3.S-methylation: Propylthiouracil, 6-
mercaptopurine
Mr. Mote G.D

F. Methylation
H2C CH NH2
COOH
OH
HO
+
O
HO
OH
S
H2N C
O
OH
H3C
S-Adenosyl methionine
H2C C
H
NH2
COOH
OCH3
HO
+
O
HO
OH
S
H2N C
O
OH
S-Adenosyl
homocysteine
Cystine
Methylated Metabolite
Mr. Mote G.D

Aspirin Metabolism
C
O
OH
+ H2O
O C CH3
C
O
OH
OH
+
O
CH3 C
O
OH
Asprin(Ester)
Analgesic Salicylic acid
Phase-I Metabolism
Mr. Mote G.D

Aspirin Metabolism
O
C
O
HO
OH
OH
OH
O
CH3
O C
O
O
H3CO
O
H
OH
OH
Glucoronide
C
O
OH
OH
Salicylic acid
Phase II Metabolism
Glucoronide Conjugate
Mr. Mote G.D

Phenytoin Metabolism
C
N
H
NH
O O C
N
H
NH
O O
O2
Cyt c oxidase
HO
Oxidation
Phase -I Metabolism
Mr. Mote G.D

Phenytoin Metabolism
C
N
H
NH
O O
HO
O
C
O
HO
OH
OH
OH
O
CH3
Glucoronide
+
Phase II Metabolism
C
N
H
NH
O O
O
O
C
O
HO
HO
HO
O
H3C
Mr. Mote G.D

Content
• Factors affecting biotransformation of
drugs
• Physicochemical properties of drug
• Chemical factors
• Induction of drug metabolising enzymes
• Inhibition of drug metabolising enzymes
• Environmental factors chemicals
• Biological factors
• Species differences
• Strain differences
• Sex differences
• Age
• Diet
Mr. Mote G.D

Content
• Factors affecting biotransformation of
drugs
• Altered physiological factors
• Pregnancy
• Hormonal imbalance
• Diseased states
• Temporal factors
• Circadian rhythm
9
5
Mr. Mote G.D

Factors affecting
biotransformation of drug
• Molecular size and shape
• pKa
• Acidity/ Basicity
• Liphophilicity
• Steric and electronic characteristics
• The chemical structure (the absence or presence of
certain functional groups) of the drug determines its
metabolic pathways.
Mr. Mote G.D

Factors affecting biotransformation of drug
• Steric and electronic characteristics
Mr. Mote G.D

Factors affecting biotransformation
of drugs
• Enzyme induction
• Increased drug metabolising ability of the enzymes by
drug
• Properties of enzyme inducers
1.Lipophilic
2. Long half life
• Mechanism of enzyme induction
• Increased liver size and blood flow
• Increased total and microsomal protein content
• Increased stability of enzymes
• Increased synthesis of cytochrome P450
Mr. Mote G.D

of drugs
• Classification of enzyme inducers
1. Phenobarbital type
inducers Ex. Phenytoin,
warfarin etc.
2. Polycyclic hydrocarbon type inducers
Ex. Cigarette smoking can cause increased
metabolism and elimination of theophylline.
• Autoinduction
Ex. Carbamazepine, Meprobamate, Rifampicin
Mr. Mote G.D

of drugs
• Consequences of enzyme induction
• Increased rate of metabolism
• Decrease in drug plasma concentration
• Enhanced oral first pass metabolism
• Reduced bioavailability
• If metabolite is active or reactive, increased
drug effects or toxicity
Mr. Mote G.D

of drugs
Mr. Mote G.D

of drugs
• Enzyme inhibition
• Decrease in the drug metabolising ability
• Classification of enzyme inhibition
• Direct inhibition
• Competitive inhibition
• Non-Competitive inhibition
• Product inhibition
• Indirect Inhibition
• Repression
• Altered physiology
Mr. Mote G.D

of drugs
• Direct inhibition
• Competitive inhibition
Ex. Methacholine inhibit metabolism of Ach for choline
esterase
• Non-Competitive inhibition
Ex. INH inhibit metabolism of Phenytoin
• Product inhibition
Ex. Xanthine oxidase inhibitor such as allopurionol
• Indirect Inhibition
• Repression
Ex. Disulphiram
• Altered physiology
Due to nutritional deficiency or hormonal imbalance
Mr. Mote G.D

of drugs
• Consequences of Enzyme Inhibition
• Increase in the plasma concentration of parent
drug
• Reduction in metabolite concentration
• Exaggerated and prolonged pharmacological
effects
• Increased liklihood of drug-induced toxicity
Mr. Mote G.D

of drugs
“Enzyme inhibition is more important clinically than enzyme
induction for drugs having narrow therapeutic index.”
Mr. Mote G.D

Factors affecting biotransformation of drugs
• Biological Factors
• Sex differences
• Age
• Diet
• Pregnancy
• Disease states
• Temporal factor
Mr. Mote G.D

of drugs
Mr. Mote G.D

• Phenylbutazone half-life is 3h in rabbit, ~6 h in rat,
guinea pig, and dog and 3days in humans.
• Isoniazid is known to be acetylated by N-acetyltransferase
into inactive metabolite.
• The rate of acetylation in asian people is higher or faster
than that in eurpoean or north american people. Fast
acetylators are more prone to hepatoxicity than slow
acetylator.
• Isoniazide (fast (Whites) and slow (Eskimos) acetylators)
Mr. Mote G.D

• Sex differences
• Responsiveness women to certain drugs
is different for men and
• Males shows greater metabolism than female
• Hormonal changes during development have a
profound effect
on drug metabolism
• Metabolism of Diazepam, caffiene, and paracetamol
is faster in females than in males while oxidative
metabolism of lidocaine, chordiazepoxide are faster
in men than in females Mr. Mote G.D

• Age
• Neonates: (0-2 months)
• Slow biotransformation
• Drug eliminated as unchanged
• Caffene- Half life 4 d
• Chloramphenicol- Gray baby syndrome or cyanosis
• Sulphonamides- Renal toxicity
• Paracetamol - Hepatotoxicity
• Infants: (2 months to one year)
• Same as neonates with improved enzyme activity.
Mr. Mote G.D

• Age
• Children : (1-12 years)
• Fast biotransformation
• Require large dose of drug
• Elderly person:
• Liver size reduced
• Blood flow decreased
• Enzyme activity decreased
• Drug conjugation not affected
Mr. Mote G.D

Biological Factors
Diet
•Low protein diet decreases drug metabolising ability.
•High protein diet increases drug metabolising ability.
•High protein-carbohydrate ratio increases MFO activity.
•Fat free diet decreases CYP 450 levels.
•Vitamin deficiency of A,C,E, and B can result in a
decrease of oxidativepathway incase of vitamin deficiency,
while vitamin E deficiency decreases dealkylation and
hydroxylation.
•Ca, Mg, Zn deficiencies decreases drug
metabolism capacity whereas Fedeficiency increases it.
Mr. Mote G.D

• Diet
• Essential fatty acid (esp. Linoleic acid) deficiency
reduce the metabolism of ethyl morphine and
hexobarbital by decreasing certain drug-metabolizing
enzymes.
• Grapefruit juice decrease metabolism of many drugs.
• Starvation- decreases amount of glucuronide
formation.
• Malnutrition in women- increases metabolism of
sex hormone.
• Alcohol ingestion results in short term decrease
followed by increase in enzyme activity.
Mr. Mote G.D

of drugs
• Pregnancy
• Metabolising ability decreased- high levels of
steroid hormons
• Metabolism of promazine and pethidine
reduced.
• Metabolism of anticonvulsants increased-
induction by circulating progesterone.
• Enzyme induction or inhibition
Mr. Mote G.D

• Disease states
• Liver- Primary site for metabolism, Diseases like
hepatic carcinoma, hepatitis, cirrhosis, obstructive
jaundice decreases metabolism.
• Kidney: Glycine conjugation of salicylates,
oxidation of vitamin D,
hydroysis of procaine impaired in renal disease.
• CCF and MI: decrease blood flow to liver- decrease
metabolism of high extraction ratio drugs-
Propranolol, lidocaine
• Diabetes: decreased glucuronidation
Mr. Mote G.D

• Temporal factor
• Diurnal variations or variations in enzyme
activity with light cycle
• Enzyme activity maximum in early morning.
• Enzyme activity minimum in late afternoon.
• Ex: aminopyrine, hexobarbital, imipamine
Mr. Mote G.D

• Genetic Variation
• Wide variability in the response todrugs between
individuals
• consequences of such variation may be therapeutic
failure oran adverse drug reaction
• Genetic diversity is the rule rather than the exception with
all proteins, including drug metabolizing enzymes
• allelic variants with different catalytic activities from that
of the wild-type form have been identified
• inheritance leads to subpopulations (genetic
polymorphisms) with different drug metabolizing abilities
lack of activity reduction in catalytic ability enhanced
activity Mr. Mote G.D

• frequency of the polymorphism often varies according
tothe ethnic ancestry of the individual
• CYP2D6 is extensively studied, the gene for CYP2D6 is
highly polymorphic
• It’s expression leads to 3 phenotypes (phenotype is the
expression of genetic make-up)
• Extensive metabolizers (EMs) have functional enzyme activity
• Intermediate metabolizers (IMs) have diminished enzyme
activity
• Poor metabolizers (PMs) have little or no activity
• 5-10% of Caucasians and 1-2% of Asians exhibit the PM
phenotype Mr. Mote G.D

• Debrisoquine, formerly used in the treatment
of hypertension, is metabolized by CYP2D6 to
4- hydroxydebrisoquine
• Remarkable inter individual variation in
pharmacological effect of the drug
• Urine of volunteers given debrisoquine was
examinedfor presence of 4- hydroxy debrisoquine
• One subject had a very low conversion of parent drug
to metabolite
• was very sensitive to the antihypertensive effects of
debrisoquine
Mr. Mote G.D

• Drug dosing
• 1- An increase in drug dosage would increase
drug concentration and may saturate certain
metabolic enzymes.
• 2- when metabolic pathway becomes
saturated, an alternative pathway may be
pursued.
Mr. Mote G.D

• Route of administration
• Orally administered drugs are absorbed from the GIT and
transported to the liver before entering the systemic
circulation. Thus the drug is subjected to hepatic
metabolism (first pass effect) before reaching the site of
action.
•Sublingually and rectally administered drugs take longer
time to
be metabolized than orally taken drugs. Nitroglycerine is
ineffective when taken orally due to hepatic metabolism.
•IV administration avoid first pass effect. Because
drug is the delivered directly to the blood stream.
Mr. Mote G.D

References
• Brahmankar DM , Jaiswal SB. Biopharmaceutics and
Pharmacokinetics A Treatise. 2nd edition. Vallabh Prakashan;New
Delhi, 2009 Page No. 139- 169.
• Wilson and Gisvold’s Textbook of Organic
Medicinal and Pharmaceutical Chemistry 11th ed.
• Lippincott, Williams & Wilkins ed.
• Foye’s Principles of Medicinal Chemistry
Mr. Mote G.D

Medicinal chemistry on metabolism(Phase I & Phase II Reactions)

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Medicinal chemistry on metabolism(Phase I & Phase II Reactions)

Similar to Medicinal chemistry on metabolism(Phase I & Phase II Reactions) (20)

More from Ganesh Mote

More from Ganesh Mote (20)

Recently uploaded

Recently uploaded (20)

Medicinal chemistry on metabolism(Phase I & Phase II Reactions)