Drug Metabolism 1
20.201
S. R. Tannenbaum
September 2013
1
Basic Drug Metabolism
Drug Conjugation Excretion
Cytochrome P450 oxidation
Metabolic Conjugation Excretion
Product
Reactive Conjugation Excretion
Electrophile
Toxicity
2
Goals
• Review the types of P450-mediated
biotransformations
• Describe/postulate the mechanisms involved in
these reactions
• Understand how substituents can effect both
metabolic rate and metabolic pathway
• Predict both type of metabolism and where in the
molecule metabolism is most likely to occur
3
Liver Architecture: Form follows function
Bile
flow
Blood
flow
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4
Basic Histology by Junqueira, Carneiro, and Kelley; Appleton/Lange
Image of lobule of the liver removed due to copyright restrictions.
5
Schematic image of liver sinusoidal cells removed due to copyright restrictions.
6
Cytochromes P450
•P450s are a family of closely related enzymes (isoforms) that catalyze
the same reaction(s) on different substrates
•termed mixed function oxidases because they transfer one atom of molecular
oxygen (O2) to the substrate and the other undergoes a two electron reduction
and is converted to water
•functional enzyme consists of a membrane bound hemoprotein and a soluble
flavoenzyme, P450 reductase, which transfers electrons to P450
•catalyze the oxidation, reduction or hydrolysis of both endogenous
and exogenous substrates
•reactions are believed to proceed via free radical mechanisms (HAT vs SET)
•levels of these hemoproteins can be influenced by many chemicals
and the enzymes in turn are capable of metabolizing many compounds
7
Free Radicals in Enzyme Reactions
What is a free radical?
A free radical refers to any molecule that has an odd number of
electrons.
Free radicals generated from the cleavage of covalent bonds (e.g.,
homolytic bond breaking of C-H bonds) are symbolized with a
single dot which represents the unpaired electron.
H
R
C
H
H
R
H
C
+H
H
Abstraction of a single electron on either N, P or S results in the
formation of charged radical and is referred to as a cation radical
(•+).
CH
CH
R
N
3
CH3
R
N
3
CH3
8
Factors that influence where a radical will form in a molecule
upon reaction with Cytochrome P450
1. Electronics of the substrate which influence the “ease” of H
atom abstraction or electron transfer (i.e., electron withdrawing vs
electron donating properties)
2. Stabilization of the radical intermediate via resonance or
inductive effects (allyl ~ benzyl>3°> 2°>1°)
CH3
methyl
CH3CH2
1o
(CH3)2CH
(CH3)3C
2o
3o
CH2
CH2
benzylic
increasing stability
3. Steric considerations
9
Figure of absorbance vs wavelength (nm) removed due to copyright restrictions.
10
Image of horseradish peroxidase, chloroperoxidase, and catalase removed due to copyright restrictions.
11
Figure of cooxidation of xenobiotics (X) during the conversion of arachidonic acid
to PGH2 by prostaglandin H synthase removed due to copyright restrictions.
12
The P450 Cycle
SH (reversible substrate binding)
oxygen transfer to the most chemically
reactive site in substrate (other factors
being equal, which they rarely are)
P450-Fe(III)-SH (good e- acceptor)
P450-Fe(III)
SOH
ox
NADPH
Fl
e-1
Flrd
NADP+
Reductase (2 e- transfer)
P450-Fe(IV)-S
e-2
OH
kcat for 1st redn step ~100 min-1 if
substrate present; ~10 min-1 if not
P450-Fe(II)-SH
O2
oxygenation is v. fast; can
observe O2 adduct at low temp
P450-Fe(III)-SH
O-O
P450-Fe(IV)-SH
O
HAT
peroxidase
shunt
P450-Fe(III)-SH
P450-Fe(III)-SH
O-O
XO; ROOH
SET
NADH
Flox
b5rd
NAD+
Flrd
b5ox
X; ROH
P450-Fe(IV)-SH
P450-Fe(V)-SH
O
O
H2O
2H+
13
e-2
Major Human CYPs Responsible for
the Metabolism of Current Drugs
CYP2E1
CYP2D6
CYP1A2
CYP2C
CYP3A4
More than 90% of the therapeutics on the market are metabolized by these
P450s. Therefore, interaction with these CYPs could be clinically relevant!
14
General Properties of Human P450 Substrates
(Lewis, Biochem Pharmacol, 60, 291-306, 2000)
• 1A2: planar, moderately basic, medium volume
• 2A6: non-planar, medium volume
• 2C9: weakly acidic, lipophilic, 1 or 2 H-bond
donor/acceptor at 5-8 A from the site of metabolism
• 2C19: neutral or weakly basic, 2-3 H-bond
donor/acceptors at 4.5 A apart and 5-8 A from the site of
metabolism
• 2D6: basic, relatively hydrophilic, a H-bond
donor/acceptor at 5-7 A from site of metabolism
• 2E1: low volume, neutral, hydrophilic
• 3A: high volume, lipophilic, structurally diverse, 1-2 Hbond donors/acceptors at 5.5-7.5 A and 8-10 A from the
site of metabolism
15
Classes of Substrates for Cytochrome P450
I. Oxidation of C-H Centers
Functional Group
Product
OH
CH2 R'
R
R
aliphatic carbon
R
CH
alcohol
R'
R
aromatic carbon
OH
aromatic hydroxylation
OH
CH2 R
benzylic carbon
H
R
CH
benzylic hydroxylation
H
R'
C
C
alkene
R'
R
R
O
C
R'
C
epoxide
R'
16
I. Oxidation of C-H Centers cont’d
Functional Group
Product
OH
CH2 R'
allylic carbon
allylic alcohol
O
R
CH2 R'
C
acyl carbon
R'
CH
R
O
OH
C
CH
acyl alcohol
R'
II. Oxidation of Heteroatoms (dealkylation, oxygenation)
Functional Group
CH2 R'
X
R
X = N, O, S, halogen
R
X
Product
O
R
XH + HC
dealkylated product
O
R'
X = NR”, S
R'
R
X
R'
N or S oxide
17
Hydroxylation of Aliphatic Carbon Centers
H
C
C
H
C
C
H
C
C
H
H
C
C
C
H
H
C
H
H
18
Mechanism of Aliphatic Hydroxylation
(HAT)
(FeO)V HC
(FeOH)IV ·C
(Fe)III + HOC
(FeO)IV·+
•stepwise process-loss of stereochemistry
• hydrogen atom abstraction and carbon radical formation
• collapse of carbon radical perferric hydroxide radical pair
(radical recombination)
• large isotope effect (kH/kD >7) due to C-H bond breaking
19
Characteristics of Aliphatic Hydroxylation via P450
I. Regioselectivity
CH3
CH2
CH3
(99)
CH2
(<1)
H
*(5.6) H2C
CH2
CH2 (73.8) ω-1 position
(11.2)
CH3
CH2
P450
CF3CO3H
(1)
(0.2)
(25)
(89)
(8)
(3)
(43)
(3)
(23)
(4)
(9.5)
CH3
Relative reactivity order for H• abstraction of C-H groups
allyl ~ benzyl > 3° > 2° > 1°
*numbers in parenthesis indicate the percent of hydroxylation occurring that site
20
II. Substituent effects (electron donation vs electron withdrawal)
X
A
X
B
A-OH
B-OH
CH3
50
50
F
70
30
CF3
95
5
21
III. Subsequent Metabolism of Hydroxylated Hydrocarbons
R
OH
R
CH
CH2 CH3
R
CH2
CH2
R
CH2
CH
OH
CH3
OH
OH
R
C
OH
CH3
R
CH2
CH
OH
O
OH
R
C
R
CH3
CH2
OH
O
R
O
R
C
ketone
CH
CH2
C
O
CH3
R
CH2
C
acid
OH
22
Alkane Biotransformtion Pathways
OH
hydroxylation
CH2
CH2
CH
CH2
CH
H
C
dehydrogenation
CH2
C
H
+
H2O
• hydrogen atom abstraction and carbon radical formation
• oxygen rebound for hydroxylation
• removal of β-hydrogen for dehydrogenation
23
Aromatic Hydroxylation
R
D
Fe=O
D
R
b a, closure
D
R
O
a O
H
Fe
tetrahedral
intermediate
b, NIH shift
(hydride shift)
R
H
epoxide hydrolase,
GSH/GST
ring opening followed by deprotonation
R
H,D
D
re-aromatization
OH
O
overall rates are proportional to e- density
• when R is electron donating: para > ortho >meta hydroxylation
• when R is strongly electron withdrawing little to no aromatic hydroxylation occurs
24
Oxidation of Alkenes
Cl
Cl
C
homolytic bond breakage
C
H
Cl
O
N
N
Fe
N
N
nonconcerted process
Cl
C
H
N
N
N
Fe
N
O
Cl
H
H
Cl
O
C
Cl
C
C
N
C
Cl
Cl
C
N
Cl
Cl
1,2 Cl migration
H
C
O
N
N
Cl
C
Cl
H
N
Fe
Cl
C
Cl
O
N
Cl
epoxidation
Cl
O
N
Fe
N
Cl
HO
Cl
C
Cl
C
H
N
C
Cl
alkylation of heme nitrogen
25
Oxidation of Acetylenes
R
C
CH
P450
O
R
C
Wolfe rearrangement
CH
R
H
R
C
H 2O
C
OFe
oxirene formation
vs
bond breaking and migration of hydrogen
acid
H
C
C
O
ketene
intermediate
NuCVB
26
Heteroatom Dealkylation and Oxygenation Reactions
These processes are related conceptually by examination of
the site in the heteroatom-containing substrate to which
oxygen is transferred. In heteroatom oxygenation1,
oxidation occurs at the heteroatom, whereas in dealkylation
reactions2, oxygen is transferred to the carbon adjacent (α)
to the heteroatom.
O
1
α
CH3CH2CH2CH2X
O
OH
CH3CH2CH2CH2X
2
CH3CH2CH2CHX
-HX
CH3CH2CH2CH
27
Heteroatom Oxygenation vs Dealkylation
• Heteroatom oxygenation occurs via an initial abstraction of an electron from
the heteroatom (SET) resulting in the formation of a radical cation
intermediate
• Dealkylations are generally thought to undergo an initial H-atom abstraction
(HAT) resulting in the formation of a neutral carbon radical intermediate
• For carbon-oxygen systems (R-OCH3), heteroatom oxygenation is not
possible due to the high ionization potential of oxygen
• For carbon-nitrogen systems, N-dealkylation is favored when an α H+ is
present, whereas, N-oxygenation occurs in the absence of α H+ protons or if
the radical cation intermediate is stabilized via a nearby EDG
• For carbon-sulfur and carbon-phosphorous systems, heteroatom oxygenation
is favored due to formation of a stable radical cation
H
H
X
SET
CH
X
CH
(O)
O
H
X
CH
-H+
HAT
X
CH
(O)
O
OH
X
CH
X
CH
28
Oxidation of Carbon-Oxygen Systems
O-Dealkylation (HAT)
O
CH2CH3
1
O
CHCH3
OH
2
O CH CH3
3
1. hydrogen atom abstraction
2. oxygen rebound
3. disproportionation
CH3CH=O
OH
29
Substituent Effects on the Rate of O-Dealkylation by P450
O2N
OR
O2N
OH
p-Nitro Phenol Formation
R
KM
VMAX
V/K
CH3
16.7
143
0.68
Ethyl
0.68
72
1.05
Propyl
1.2
38
3.16
Iso Propyl
0.64
34
5.13
Butyl
0.42
8
1.9
30
Oxidation of Carbon-Nitrogen Systems
1. N-Dealkylation (αHs, HAT or SET)
SET
R2
N
R2
N
CH3
O
-H+
CH3
OH
HAT
R2
N
CH2
R2
N
CH2
R2NH
CH2
2. N-Oxidation (No αHs, SET)
NH2
NH2
NH
OH
31
Oxidation of Carbon-Sulfur Systems
1. S-Dealkylation
R
CH2 R'
S
R
S
CH2 R'
-H+
R
S
CH
R'
CH
OH
R
S
CH
O
R'
R
R'
SH
2. S-Oxidation
O
R
S
CH2 R'
R
S
CH2 R'
R
S
CH2 R'
3. Desulfuration (suggest a mechanism)
S
HN
O
R
O
NH
N
R
HN
O
O
R
NH
N
R
O
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20.201 Mechanisms of Drug Actions
Fall 2013
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