NEPHAR 305

Pharmaceutical Chemistry I

Drug Metabolism:
Phase I

Assist.Prof.Dr. Banu Keşanlı

 Drug Metabolism
¾ Drug’s biochemical modification or degradation, usually through
specialized enzymatic systems
¾ Xenobiotic: a chemical which is found in an organism but which is
not normally produced or expected to be present in it
¾ Drug metabolism often converts lipophilic chemical compounds
into more readily excreted polar products
¾ Duration and intensity of the pharmacological action of drugs is important
¾ Drug metabolism can result in toxication if the metabolite of a compound is more
toxic than the parent drug or chemical
¾ or detoxication (process of preventing toxic entities from entering the body in the
first place) by the activation or deactivation of the chemical

A prodrug is a pharmacological substance (drug) that is administered in an inactive

(or significantly less active) form. Once administered, the prodrug is
metabolised in vivo into an active metabolite.
 Importance of Drug Metabolism

Basic premise:

Lipophilic Drugs Hydrophilic Metabolites

(Not excreted) (Excreted)

Water soluble increased renal excretion

and
Decreased tubular re-absorption of lipophilics
Importance of Drug Metabolism

Metbolism Termination of Drug

- Bioinactivation
- Detoxification
- Elimination

Metabolism Bioactivation
- Active Metabolites
- Prodrugs
- Toxification
 Phase I or Functionalization Reactions
Oxidative Reactions
• Oxidation of aromatic moieties
• Oxidation of olefins
• Oxidation at benzylic, allylic carbon, carbon atoms α to carbonyl and imines
• Oxidation at aliphatic and alicyclic carbon
• Oxidation involving carbon-heteroatom systems:
Carbon-nitrogen systems (aliphatic and aromatic amines; N-dealkylation,
oxidative deamination, N-oxide formation, N-hydroxylation)
Carbon-oxygen systems (O-dealkylation)
Carbon-sulfur systems (S-dealkylation, S-oxidation, and desulfuration)
• Oxidation of alcohols and aldehydes
• Other miscellaneous oxidative reactions

Reductive Reactions
• Reduction of aldehydes and ketones
• Reduction of nitro and azo compounds
• Miscellaneous reductive reactions

Hydrolytic Reactions
• Hydrolysis of esters and amides
• Hydration of epoxides and arene oxides by epoxide hydrase
 Phase II or Conjugation Reactions

• Glucuronic acid conjugation

• Sulfate conjugation
• Conjugation with glycine, glutamine, and other amino acids
• Glutathione or mercapturic acid conjugation
• Acetylation
• Methylation
 Transformation of Xenobiotics by Biological Systems
¾ Phase I and Phase II reactions are biotransformations of chemicals that occur
during drug metabolism
¾ Oxidative biotransformations require both molecular oxygen and the reducing
agent NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)
¾ One atom of molecular oxygen (O2) is introduced into the substrate R-H to form
R-OH and the other oxygen atom is incorporated into H2O

OH

O2

NADPH
 Aromatic Hydroxylation
¾ Major route of metabolism for many drugs containing a phenyl group
(and aromatic)
¾ Proceeds initially with an “arene oxide” intermediate
¾ Hydroxylation occurs at para position
Activated rings – electron donating substituents
Deactivated rings don’t get oxidized – toxic
¾ Dihydrol metabolite formation is possible
¾ Undergoes further conversions to polar water soluble glucuronide or
sulfate conjugates, which are readily excreted in the urine

(a)
Tetrahedral
Tethedral ara ürün H H
intermediate
O
(b)

H
O OH
 Oxidation Involving Carbon-Heteroatom Systems
¾ Hydroxylation of α-carbon atom attached to the heteroatom (N, O, S)
results in unstable intermediate which decomposes via cleavage of carbon –
heteroatom bond

¾ Hydroxylation or oxidation of heteroatom (N, O, S) could fall under these

mechanisms: N-hydoxylation, N-oxide formation, sulfoxide, sulfone formation

¾ Structural factors determine the metabolic pathway – complicated

¾ Nitrogen functionalities such as amines, amides are found in natural products

(morphine, nicotine etc) and in numerous drugs (antihistamines, phenothiazine etc)
Tertiary aliphatic and Alicyclic amines

Oxidative N-dealkylation: Oxidative removal of alkyl groups

Small groups such as methyl, ethyl, isopropyl removed rapidly, t-butyl is
impossible H
H O O

R1 N C R1 N C R1 NH + C
α α
R2 R2 R2
Secondary Carbonyl
Tertiary amine Carbinolamine amine (aldehyde
or ketone)

Antiestrogenic agent Tamoxifen (Nolvadex)

H3C H3C
NCH2CH2O O
HNH2 CH2CO
H3C +
C
CH2CH3
CH2CH3 H H

Tamoxifen
 Secondary and Primary Amines
• Oxidative N-dealkylation
• Oxidative deamination
• N-oxidation

9 Carbinolamine pathway to give corresponding primary amine metabolite through

N-dealkylation

9 Which then is susceptible to oxidative deamination

• initial α-C hydroxylation followed by C-N bond cleavage to give carbonyl
metabolite and ammonia
¾ Metabolism of Propranolol both by direct deamination and deamination of its primary
amine metabolite
OH OH OH
H O
O CH O CH O O CH
C CH2 C CH C C
H2 H2 H2
HN HN CH3 H
CH3
C C
H H

Propranolol CH3 CH3

Aldehyde metabolite
NH2
Direct oxidative Carbinolamine
deamination

OH NH3
OH oxidative
O CH deamination
C CH2 through primary amine
O CH
H2
C CH2
HN CH3 H2
C NH2
O H
CH3
O
Primary amine metabolite
C (Desisopropyl Propranolol)
Carbinolamine
H 3C CH3
 N-Dealkylation

Oxidative Deamination
¾ Metabolic N-oxidation of secondary amine leads to N-oxygenated products,
N-hydroxylamines which are susceptible to further oxidation giving nitrone
metabolites OH -
O
+
NH N N

CH3 CH3 CH2

Secondary
Hydroxylamine Nitrone
amine

CF3 CH3 CF3 CH3 CF3 CH3

HN CH3 N CH3 -
N+ CH3
OH O

Hydroxylation Nitrone

¾ Secondary amines undergo oxidative dealkylation and deamination more than

N-oxidation
¾ Primary aliphatic amines are biotransformed by oxidative deamination or
by N-oxidation
¾ Monoamine oxidase (MAO) enzymes are responsible for oxidative deamination
¾ Structural features, e.g. α-substituents determine whether C or N oxidation
will occur
• If no hydrogen atom on α-C then α-C hydroxylation is impossible
 Aromatic Amines and Heterocyclic Nitrogen Compounds
¾ Tertiary aromatic amines undergo oxidative N-dealkylation and N-oxide formation
¾ Secondary aromatic amines undergo N-dealkylation and N-oxide formation
¾ Primary amines undergo N-oxidation generating N-hydroxylamine metabolite
¾ Oxidation of hydroxylamine derivative to nitroso derivative is possible
¾ N-oxidation is minor compared to other biotransformations such as N-acetylation
aromatic hydroxylation
¾ Conjugation pathways are observed (e.g. sulfate conjugation)

CH3
a Ar NHCH3
Ar N
CH3 CH2OH
Ar N
CH3 b
CH3
+
-
Ar N O
CH3
 Oxidation of Alcohols and Aldehydes
¾ Primary alcohols are oxidized to aldehydes which often undergo facile oxidation
to generate polar carboxylic acid derivatives

¾ Secondary alcohols are oxidized to ketones – more likely to form conjugates

or be reduced back to alcohol form

¾ Catalyzed by soluble alcohol dehydrogenase present in liver and other tissues

+ +
RCH2OH + NAD RCHO + NADH + H

+ RCOOH + NADH
RCHO + NAD

COOH

NH

H 3C CH2 OH

R=COH Mefenamic acid

R=COOH
 Reductive Reactions

¾ Plays an important role in the metabolism of many compounds containing

carbonyl, nitro and azo groups
¾ Bioreduction of carbonyl compounds generates alcohol derivatives
¾ Nitro and azo reductions lead to amino derivatives
¾ Hydroxyl and amino moieties of the metabolites are more susceptible to
conjugation than the functional groups of the parent compounds
¾ Facilitate drug elimination

Reduction of Aldehydes and Ketones

¾ Carbonyl containing drugs and metabolites are common

¾ Aldehydes are more readily oxidized to carboxylic acids then reduced to alcohols
¾ Ketones are resistant to oxidation, mainly reduced to secondary alcohols
¾ Aldo-keto reductase enzymes are responsible for reduction
¾Bioreduction of ketones often leads to the creation of asymmetric center thus
2 possible stereoisomeric alcohols
 O
CH3
CHCH2CH2 N CHCH2CH
CH3
Cl CH2 Cl CH2

Chlorpheniramine
Klorfeniramin Aldehit
Aldehydeyapısındaki metabolit
metabolite

oxidation
Oksidasyon reduction
Redüksiyon

CHCH2COOH CHCH2CH2OH
Cl CH2 Cl CH2
 O HO H H OH
C C C
CH3 CH3 CH3
+

Acetophenone
A f S-(-)-Methyl R-(+)-Methyl
Phenyl Carbinol Phenyl Carbinol

O OH H H OH
C C C
CH3 CH3 CH3
OH CH2 OH CH2 OH CH2
C C6H 5 C C6H5 C C6H5
+
H H H
O O O O O O

R(+)-Warfarin R,S-(+)-Alcohol R,R-(+)-Alcohol

Major diastereomer Minor diastereomer
 Hydrolytic Reactions

Hydrolysis of Esters and Amides

¾Metabolism of ester and amide linkages in many drugs is catalyzed by hydrolytic
enzymes in tissues and plasma
¾ Metabolic products formed (carboxylic acids, alcohols, phenols, amides) are polar
¾ Functionally more susceptible to conjugation and excretion than the parent ester
or amide
¾ Hydrolysis is a major biotransformation pathway for ester functionality

¾ Many parent drugs have been chemically modified or derivatized to generate

prodrugs to overcome some undesirable property (e.g. bitter taste, poor absorption,
poor solubility, irritation at site of injection)
¾ Ester derivatives are ideal prodrug candidates
¾ Amides are hydrolyzed slower compared to esters
 COOH O COOH
O C CH 3 OH
+ CH3COOH

Acetic acid
Aspirin Salicylic acid
(Acetylsalicylic acid)
O
O O
C OCH3
C OH C OCH 3
CH3 N H
O CH 3 N H CH 3 N H
C O OH
H C +
O
H O H

Cocaine Benzoylecgonine Methylecgonine

O
H2N C NHCH2CH2N(C2H5)2 Slow hydrolysis
Yavaş hidroliz
Prokainamit O
Procainamide H2N C OH
O
H2N C OCH2CH2N(C2H5)2 Hızlı hidroliz
Fast hydrolysis
Prokain
Procaine
 NEPHAR 305
Pharmaceutical Chemistry I

Drug Metabolism
Phase II

Prof. Dr. Hakkı Erdoğan

Assist.Prof. Banu Keşanlı
PHASE II REACTIONS

1. Glucuronidation
2. Sulfate Conjugation
3. Acetylation
4. Amino Acid Conjugation
5. Methylation
6. Glutathione Conjugation
 Glucuronic Acid Conjugation
Uridine-5’-α-D-glucuronic Acid
C-1

¾ The microsomal enzyme glucuronyl transferase conducts the donation of

glucuronic acid from the endogenously synthesized UDPGA to various substrates
to form glucuronide conjugates.
¾ Examples of such substrates are morphine and acetaminophen.
 Glucuronic Acid Conjugation

¾ Most common conjugative pathway

¾ Greatly enhances water solubility
¾ Numerous functional groups can combine with it
¾ Readily available in body
¾ Has polar carboxyl and hydroxyl groups
¾ Products are called glucuronides
RN-G; RO-G; RCOO-G; RS-G; RC-G glucuronides could form at
C1 atom of β-glucuronide

¾ Phenolic and alcoholic hydroxyls are most common functional groups metabolized
¾ Glucuronidation is not fully developed in infants and children

C-1
 Table 5.10. Substrates forming Glucuronides
(R. B. Silverman “The Organic Chemistry of Drug Design and Drug Action” 1992, s.331)
Type Compound Formula
a) O-Glucuridation
Hydroxyl (ether glucuronide)
Acetaminophen CH 3CONH OH
phenol
OH H
O2N N CHCl 2
Alcohol Chloramphenicol O
OH

C6H 5O CH 3

Carboxyl (ester glucuronide) Fenoprofen OH

O

b) N- Glucuridation

Amine Desipramine
NHCH 3

Amide OCONH2

Carbamate Meprobamate H 3C CH3

O C NH2
O

OCH 3
Sulfonamide Sulfadimethoxine N
H 2N SO 2NH N

OCH 3
c) S- Glucuridation N
Sulfahydryl Methimazole SH
N
CH3

(C2H 5)2N SH
Karboditiyonik asit Disulfiram S

(reduced metabolite)
d) C- Glucuridation O
C6H5 N
Phenilbutazone CH 3
C6H5 N
O
 Glucuronidation of Benzoic Acid

UGT= UDP-α-D-Glucuronsyltransferase
Glucuronidation of Aniline
 Morphine Metabolism
Morphine → Morphine -6-glucuronide (active metabolite)
Morphine → Morphine -3-glucuronide (inactive metabolite)

4 5

Morphine -3-glucuronide is the major metabolite

A small amount of morphine undergoes N-demethylation
 Sulfate Conjugation

3’-Phosphoadenosine-5’-phosphosulfate (PAPS)

¾The cytosolic enzyme sulfotransferase conducts the donation of sulfate from

the endogenously synthesized PAPS to various substrates to form sulfate
conjugates.
¾An example of such substrate is acetaminophen.
 Sulfate Conjugation

¾ Occurs primarily with phenols and occasionally with alcohols,

aromatic amines and N-hydroxy compounds
¾ Sulfate amount in body is limited
¾ Leads to water soluble and inactive metabolites
¾ Glucuronidation of phenols is a competing reaction and
may predominate
 Sulfate Conjugation

PAP: 3’-phosphoadenosine- 5’-phosphate

OH

HO R = H Albuterol
R = SO3H Metabolite
RO NHC(CH3)3
 Acetylation
N-Acetyltransferase
• A soluble enzyme
• Isoniazid is a substrate
• Genetic variation occurs
– Some individuals are fast acetylators
– Some individuals are slow acetylators
• Acetyl coenzyme A is the endogenous donor molecule

¾ Important metabolic route for drugs containing primary amino groups

¾ Aromatic amines (ArNH2), sulfonamides (H2NC6H4SO2NHR),
¾ Hydrazine (-NHNH2), hydrazides (-CONHNH2) and primary aliphatic
amines
¾ Gives inactive and nontoxic metabolites but does not enhance
water solubility much
 Acetyl CoA

•Various acetylases, for examples, choline acetylase and N-acetyl transferase, all
soluble enzymes, conduct the transfer of the acetyl group of acetyl CoA to
various substrates.
•For example, N-acetylation of isoniazid. Genetic polymorphism occurs with N-
acetyltransferase.
 N-Acetyltransferase

N4

N1

(Antibacterial)

H O H O H O
N N N
O
7 7 7
O2N N H 2N N CH3 NH N
Cl Cl Cl

Klonazepam
Clonazepam 7-Amino metaboliti 7-Asetamino metaboliti
7-amino metabolite 7-acetylamino metabolite
(anticonvulsant,
muscle relaxant)
 Methylation
S-Adenosylmethionine (SAM)

9 Cytosolic enzymes such as catechol-O-methyl transferase (COMT) and

phenylethanolamine-N-methyl transferase (PNMT) conducts the donation of the
methyl group from the endogenously synthesized SAM to various substrates to
form methylated conjugates.
9 Norepinephrine is N-methylated by PNMT to form epinephrine.
Norepinephrine, epinephrine, dopamine, and L-DOPA are O-methylated by
COMT.
 Methyltransferases

• 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. It is demethylated to S-adenosylhomocysteine
 Methylation
9 Methylation reactions play an important role in biosynthesis of many
endogenous compounds and inactivation of numerous active
biogenic amines
9 Minor pathway for conjugation of drugs and xenobiotics
9 Does not give polar, water soluble metabolites but pharmacologically
inactive products
9 Catechols, phenols, amines and N-heterocyclic and thiol compounds
9 Substrates undergoing O-methylation by COMT must contain an
aromatic1,2-dihydroxy group
 N-Methyltransferases
PNMT- Phenylethanolamine-N-methyltransferase

Norepinephrine PNMT Epinephrine

SAM

(Neurotransmitters)
 O-Methylation Of Catecholamines

COMT- catechol-O-methyltransferase
 O-Methylation of Norepinephrine

COMT- catechol-O-methyltransferase
 S-Methylation of 6-Mercaptopurine

(Immunosuppressive)

TPMT - thiopurinemethyltransferase; some individuals are deficient in this

enzyme that is critically important for the metabolism of this agent
 AMINO ACID CONJUGATION
9 Amino acids, glycine and glutamine are used to conjugate carboxylic acids,
particularly aromatic acids and arylalkyl acids
9 Carboxylic acid substrate is activated with ATP and coenzyme A (CoA) to form
an acyl-CoA complex
9 Limited amount of amino acids in body is available so few conjugation
reactions occur
9 Competes with conjugation with glucuronic acid
9 Polar and water soluble metabolites
9 Glycine conjugation occurs with aromatic acids and arylalkyl acids
9 Glutamine conjugation occurs with arylacetic acids

Glycine Glutamine
AMINO ACID CONJUGATION

(mitochondria)
 Salicyluric Acid is the Glycine Conjugate of Aspirin
9 Multiple Metabolic Pathways Exist for Aspirin’s Metabolism

• Hydrolysis produces
salicylic acid metabolite

Amide linkage

Salicylic acid

¾ Salicyluric acid, the glycine conjugate of salicylic acid, is the main

metabolite of aspirin
¾ Approximately 76% of aspirin is metabolized through amino acid
conjugation.
 Glutamine Conjugation

Glutamine
Glutamin
N-acyltransferase
N-açiltransferaz COOH
O O O CH 2CONHCHCH 2CH 2CONH 2
COOH
N(CH 3)2

Difenhidramin
Diphenhydramine Glutamin konjügatı
Glutamine conjugate
 Glutathione (GSH) Conjugation
¾ Important pathway for detoxifying chemically reactive electrophilic compounds
¾ Covalent interaction of metabolically generated electrophilic intermediates with
cellular nucleophiles leads to drug toxicity
¾ GSH protects cellular constituents by bonding to metabolites via –SH group

Glutathione

¾ Xenobiotics conjugated with GSH usually are not excreted as such but undergo
further biotransformation to give S-substituted N-acetylcysteine products called
mercapturic acid
¾ Nucleophilic GSH reacts with electrophilic substrates
* nucleophilic displacement at an electron deficient carbon or heteroatom
* nucleophilic addition to an electron deficient double bond
¾ Aliphatic and arylalkyl halides (Cl, Br, I), sulfates (OSO3-),
sulfonates (OSO2R),
¾ Nitro compounds (NO2), and organophosphates (OP[OR]2) have
electron deficient carbon atoms to conjugate with GSH
¾ If not sufficiently electron deficient (not enough electron
withdrawing groups) GSH conjugation does not take place

δ+ δ−
GSH + CH2 X GS-CH2 + HX

R R

R= Alkyl, aryl, benzyl, allylic

X = Br, Cl, I, OSO3-, OSO2R, OPO(OR)2
 ¾ Paracetamol is metabolised primarily in the liver, into non-toxic products

acetaminophen
Bioactivation of Acetaminophen
 Stereochemical Aspects of Drug Metabolism

¾ Many drugs often are administered as racemic mixtures in humans

e.g. warfarin, propranolol, hexobarbital, ibuprofen, glutethimide
¾ May differ in pharmacological activity
¾ Individual enantiomers of a racemic drug often are metabolized at different rates
and could be metabolized by different pathways
¾ Substrate stereoselectivity: a preference for one stereoisomer as a substrate for a
¾ Metabolizing enzyme or metabolic process
¾ Biotransformations could lead to new asymmetric center e.g. bioreduction of
ketone xenobiotics
 Metabolism of Warfarin Enantiomers:
9 More active (S)(-) isomer is 7-hydroxylated (aromatic hydroxylation)
9 (R)(+) isomer undergoes keto reduction to yield (R,S) warfarin alcohol
as the major plasma metbolite HO H
C
OH H2C CH3

C C6H5

H5
O
O O
C
R(+) enantiomer
OH H2C CH3
R, S(+)-Alcohol
C H

C6H5
O
O O
S(-) C
Warfarin
OH H2C CH3

(Anticoagulant) C H

C6H5

HO O O

7-Hydroxywarfarin
 *local anesthetic

• hydrolysis
• Conjugation
*non-steroidal anti-inflammatory drug

• Sulfoxide to sulfide reduction

 *antidepressant

• N-demethylation
Meperidine (ethyl 1-methyl-4-phenylpiperidine-4-carboxylate) is a narcotic
analgesic drug which undergoes phase 1 and phase 2 reactions. Show its
metabolites forming from possible metbolism pathways.

C2H5 O

N
CH3