Mechanistic insights into the bioactivation of phenacetin to reactive metabolites:

A DFT study
Nikhil Taxak
a
, K. Chaitanya Prasad
b
, Prasad V. Bharatam
a,b,
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar (Mohali), 160 062 Punjab, India
b
Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar (Mohali), 160 062 Punjab, India
a r t i c l e i n f o
Article history:
Received 1 September 2012
Received in revised form 6 November 2012
Accepted 9 November 2012
Available online 7 December 2012
Keywords:
Phenacetin
Carcinogenicity
Density functional theory
Reactive metabolites
Electrophilicity
Quinone imine
a b s t r a c t
The knowledge of biochemical mechanism of cancer induction and carcinogenicity by drugs is an essen-
tial requisite for drug metabolism and toxicity studies. Metabolic bioactivation of phenacetin leads to the
generation of several reactive metabolites (RMs) and intermediates, with varied toxicological conse-
quences. The carcinogenicity and mutagenecity of phenacetin have been known for several years; how-
ever, the molecular level details of the formation and reactivity of the RMs behind them are still elusive.
Quantum chemical analysis was carried out to identify the critical RMs generated via three different met-
abolic pathways of phenacetin. DFT-based descriptors were utilized to obtain the electrophilicity param-
eters of all the RMs involved in the bioactivation pathway. The three metabolic pathways studied are: (i)
O-dealkylation, (ii) N-hydroxylation, and (iii) N-deacetylation. It was observed that the O-dealkylation
process leading to the formation of acetaminophen is energetically (DG = 77.34 kcal/mol) more favor-
able than N-hydroxylation (20.13 kcal/mol) and deacetylation (3.51 kcal/mol) reactions. The activa-
tion barrier, calculated using B3LYP/6-311+G(d) basis set and implicit solvent effect, for O-dealkylation
(37.55 kcal/mol) was observed to be lower as compared to N-hydroxylation (42.38 kcal/mol) and N-
deacetylation (55.63 kcal/mol). The O-ethyl-N-acetyl-p-benzoquinone imine (OEt-NAPQI) was observed
to be the most critical and electrophilic metabolite (global electrophilicity; x= 19.43 eV), generated via
initial N-hydroxylation (Phase I) and subsequent Phase II metabolism. The higher electrophilicty of OEt-
NAPQI (compared to NAPQI; 4.23 eV) accounts for its easy binding with nucleophiles such as macromol-
ecules and DNA nucleotides, and higher reactivity (as compared to other RMs) leading to carcinogenicity.
Therefore, the knowledge of the RMs, especially OEt-NAPQI, involved in the carcinogenicity of phenac-
etin can be utilized in understanding the relevance of several crucial Phase I and II metabolic reactions.
2012 Elsevier B.V. All rights reserved.
1. Introduction
Phenacetin (N-(4-Ethoxyphenyl) acetamide) (Fig. 1) is a non-
opioid analgesic and antipyretic, which was once widely used in
the treatment of fever and related complications [13]. It is also
known as acetophenetidin, N-acetyl-p-phenetidine, aceto-4-phe-
netidine, acetophenetidine and p-ethoxyacetanilide [4]. The anal-
gesic effects were observed owing to the actions on the sensory
tracts of the spinal cord [1,5]. While, the antipyretic effect was ob-
served by its action on the brain via a decrease in the temperature
set point [1,6]. However, the long-term and chronic consumption
of phenacetin led to several toxicological complications ranging
from nephrotoxicity to carcinogenicity [712]. The carcinogenicity
is observed in the urinary tract and renal pelvis (transitional-cell
carcinoma) [913]. As a result of these severe complications, phen-
acetin and drugs containing phenacetin were withdrawn from the
market by the order of U.S. Food and Drug Administration in 1983
[14]. The other critical toxic effects of phenacetin identied were
renal papillary necrosis and tumors of the bladder in humans [9
13].
Various research groups have carried out in vitro and in vivo
studies to explore the carcinogenic and tumor inducing potential
of phenacetin [1522]. Several reactive metabolites (RMs) crucial
in these toxicological implications were identied in these studies.
It was observed that phenacetin undergoes biotransformation
reactions at various sites to generate the RMs, by different meta-
bolic pathways. Since, it is an aromatic amide, it can undergo sev-
eral metabolic reactions common to compounds containing this
group. CYP1A2 isoform catalyzes the metabolic reactions of phen-
acetin. One of the major metabolic reactions identied was O-deal-
kylation reaction (Pathway-1, Fig. 1), which leads to the formation
of acetaminophen (hepatotoxic at higher doses). The intermediate
N-acetyl-p-benzoquinoneimine (NAPQI) has been implicated to be
2210-271X/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.comptc.2012.11.018

Corresponding author at: Department of Medicinal Chemistry, National Insti-

tute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar
(Mohali), 160 062 Punjab, India. Tel.: +91 172 2292018; fax: +91 172 2214692.
E-mail address: pvbharatam@niper.ac.in (P.V. Bharatam).
Computational and Theoretical Chemistry 1007 (2013) 4856
Contents lists available at SciVerse ScienceDirect
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j our nal homepage: www. el sevi er . com/ l ocat e/ compt c
involved in the hepatotoxicity, via liver necrosis, by the reaction
with glutathione resulting in 3-(S-glutathionyl) acetaminophen
conjugate [21,22]. This pathway however, is not implicated to lead
to tumorigenic or carcinogenic effect in the human body. This
observation has been conrmed in a recent study by Walter et al.
[23], where no evidence regarding the relation between acetami-
nophen and incidence of malignancies or cancer complications
was obtained. Liu et al. [24] recently reported the application of
O-deethylation reaction of phenacetin as a marker reaction for
CYP1A2 activity. They utilized this reaction as a tool for the evalu-
ation of hepatic functional reserve in rats with chronic liver injury.
The other major metabolic reaction of phenacetin is the N-
hydroxylation (Pathway-2, Fig. 1) leading to N-hydroxy phenace-
tin (NOH phenacetin), which is also a well-known step in the acti-
vation of paracetamol and aromaticamide carcinogens such as 2-
acetylaminouorene [15,18,24,25]. Vaught et al. [19] have shown
that NOH phenacetin is carcinogenic and mutagenic to rat liver
microsomes. NOH phenacetin has been observed to undergo fur-
ther bioactivation reactions by Phase II metabolism, such as sulfate
and glucuronide conjugation reactions to form N,O-sulfate and
N,O-glucuronide conjugates. These conjugates can covalently bind
to proteins and result in toxicity. These conjugates have also been
proposed to undergo hydrolytic reactions in the acidic environ-
ment of urinary bladder, leading back to the formation of NOH
phenacetin, which can undergo further oxidation reactions
[16,17,19]. Nery [26] have shown the activation of phenacetin by
liver microsomes, and further covalent binding to nucleic acids
and proteins. Calder et al. [24] also suggested the involvement of
intermediates of high chemical reactivity in the toxicity of phenac-
etin by in vivo studies. They showed a rational pathway for NOH
phenacetin to p-benzoquinone and hydroquinone, both observed
to be nephrotoxic in rats. However, this group did not perform
the carcinogenic studies for these intermediates. Camus et al.
[27] implicated the role of NOH phenacetin and p-nitrosophene-
tole as the potent procarcinogens and mutagens [28].
N-deacetylation (Pathway-3, Fig. 1) is the other metabolic reac-
tion of phenacetin which results in the generation of phenitidine,
which after further oxidation reactions leads to the formation of
a RM, p-nitrosophenetole. It has been implicated in binding to
tRNA resulting in carcinogenic and mutagenic complications. Sev-
eral studies have shown the interaction of N-hydroxyphenitidine
(formed via deacetylation of NOH phenacetin) with nucleic acids
[29].
Therefore, several in vitro and in vivo studies have highlighted
the importance of bioactivation reactions of phenacetin in the car-
cinogenic and other toxicological complications. However, the
molecular level details and mechanistic insights for each metabolic
pathway have remained elusive till date and have not been ex-
plored in detail. The quantum chemical methods can be utilized
to obtain the molecular level details of these reaction pathways
N
C
O
OH
H
O
N
C
O
N
H C
O
OH
SG
N
H C
O
O
C
2
H
5
N
H C
O
O
C
2
H
5
N
C
O
O
OH
OC
2
H
5
N
C
O
N
H C
O
O
C
2
H
5
NH
2
O
C
2
H
5
N
O
C
2
H
5
O
Phenacetin Acetaminophen NAPQI Glutathione conjugate
of acetaminophen
Phenacetin N-hydroxy phenacetin O-Et-NAPQI
Phenacetin Phenitidine p-nitroso phenetole
Pathway-1
Pathway-2
Pathway-3
GSH
Phase II metabolism
Glucuronidation/sulfation
Hydrolysis
Oxidation
O-dealkylation
N-hydroxylation
C
2
H
5
N-deacetylation
N-hydroxylation
Oxidation
Fig. 1. Reaction pathways for the biotransformation of phenacetin to several reactive metabolites. (A) Pathway-1: O-dealkylation reaction; (B) Pathway-2: N-hydroxylation
reaction (oxidative metabolism); (C) Pathway-3: N-deacetylation reaction.
N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 4856 49
[30] and have proved to be useful tool for these types of metabo-
lism-related studies [31,32]. Other parameters such as hardness
and softness, electrophilicty and charge distribution have also been
reported to provide a wealth of information for metabolic reactions
[33,34]. Koyamans et al. [30] performed ab initio calculations using
STO-3G method to explore the metabolic pathway of phenacetin.
They suggested the oxidative metabolism of phenacetin to NOH
phenacetin; O-dealkylation to acetaminophen and NAPQI. How-
ever, the N-deacetylation reaction, reactivity and stability of RMs
were not studied and it is still not clear, which RM can be prefer-
entially formed leading to toxicity.
In this article, an attempt has been made to explore the mech-
anistic details of three metabolic pathways namely, O-dealkyla-
tion, N-hydroxylation and N-deacetylation, involved in the
toxicity of phenacetin. Density Functional Theory (DFT) was uti-
lized to investigate the 3D structures of all the intermediates and
RMs involved, transition states for key steps catalyzed by CYPs
such as hydroxylation, oxidation reactions, energetics involved in
these reactions, potential energy surface for the critical and rate
determining step of each metabolic pathway and electrophilicity
parameters of the RMs.
2. Computational methodology
Quantum chemical calculations for all the metabolic pathways
have been carried out using Gaussian03 suite of programs [35].
Density Functional Theory (DFT) was utilized to carry out geome-
try optimizations of phenacetin and its metabolites using B3LYP
functional with 6-31+G(d) basis set, denoted as BS1 [36]. The
B3LYP functional comprises of Beckes three parameter exchange
functional [37] with the correlation functional of Lee et al. [38].
This method and basis set have been reported to be satisfactory
and provide reasonable energy estimates, as seen in similar theo-
retical studies for metabolic reactions [31,32,39]. Single point cal-
culations for all the optimized geometries were carried out using
the higher basis set [6-311+G(d)], denoted as BS2 [36b]. The effect
of implicit solvent (BS3) was studied using Integral Equation For-
malism variant of Polarizable Continuum Model (IEFPCM) [40],
using solvent chlorobenzene (dielectric constant (e) = 5.7) to mimic
the bulk polarity effects of active site cavity of cytochrome P450.
Vibrational frequency calculations were carried out at the same le-
vel of geometry optimizations to characterize them as either min-
ima or transition states. The vibrational modes were examined for
each stationary point and transition states using GaussView 3.07
program. The transition state is characterized to be a rst order
saddle point, with one negative imaginary vibrational mode, on
the potential energy surface. A scaling factor of 0.9806 was utilized
for zero point energy corrections on the B3LYP-estimated energies
[41]. For all the reactions, enthalpies (DH = E + pDV) were calcu-
lated at the same level of theory. Transition state calculations were
performed for the cytochrome P450 catalyzed reactions (Phase I) of
all the three metabolic pathways, using model oxidant, hydrogen
peroxide (HOOH) [42]. The use of HOOH in modeling the oxidative
(metabolic) reactions has been reported in previous studies, re-
lated to metabolic reactions [42,31]. Gibbs free energy barrier for
the rst step of the three metabolic pathways were also evaluated
at 298 K and 1 atm, using thermal corrections to Gibbs free energy.
The product enthalpies (DH) and Gibbs free energies (DG) were
calculated using the model oxidants, HOOH and Cpd I [iron(IV)-
oxo heme-porphine radical cation, with SH

as the axial ligand]

to mimic the catalytic activity of CYP450. The B3LYP hybrid density
functional was used for the geometry optimizations of Cpd I and
related heme-porphyrin geometries, with LanL2DZ basis set on
iron atom [36a], and the 6-31+G(d) basis set for all the remaining
atoms [36b]. Cpd I has been reported to be an established and a
standard model to study CYP-mediated metabolism reactions
[32]. The multi-state reactivity (both doublet and quartet spin
states) of Cpd I was considered for this study. The reactivity of
the crucial metabolites was determined by the global electrophilic-
ity (x) parameter, calculated using standard equations (discussed
in Supporting information S1) [33]. The results have been dis-
cussed for three pathways using B3LYP/6-311+G(d) basis set and
inclusion of zero point corrections and implicit solvent effects, un-
less otherwise specied.
3. Results and discussion
Phenacetin is an aromatic amide, which undergoes biotransfor-
mation through three metabolic pathways:
(i) O-dealkylation pathway.
(ii) N-hydroxylation pathway.
(iii) N-deacetylation pathway.
The detailed study of these pathways was carried out to answer
several questions, related with the toxicity (carcinogenicity) of
phenacetin. The questions addressed are following: What are the
mechanisms for O-dealkylation, N-hydroxylation and N-deacetyla-
tion of phenacetin? Which metabolic pathway forms the most sta-
ble intermediate? What are the activation barriers for the key
Phase I metabolic reactions in these three pathways? What are
the enthalpies and Gibbs free energies of each step involved in
these pathways? Which intermediate is the most reactive, thermo-
dynamically more stable and majorly involved in the carcinogenic
implications of phenacetin? To answer these questions, model oxi-
dants, hydrogen peroxide and Cpd I (as described before) were uti-
lized in the study. Both Phase I and Phase II metabolic reactions of
phenacetin were studied. For calculating the enthalpies and Gibbs
free energies for Phase II reactions such as glucoronidation, glucu-
ronic acid was employed as the model oxidant. The energies of the
putative mechanistic intermediates were determined as a means of
identifying the most likely pathway for the metabolism of phenac-
etin, leading to its toxicological implications.
3.1. O-dealkylation pathway
As discussed before, O-dealkylation pathway is the major met-
abolic pathway of phenacetin. This pathway proceeds through an
initial step of methylene CH hydrogen abstraction (H-abstraction)
from the methylene carbon of O-ethyl linked to the phenyl ring in
phenacetin, followed by the reaction between the radicals to form
the hydroxylated phenacetin. The O-dealkylated product is acet-
aminophen (acetyl-p-aminophenol; APAP) as shown in Fig. 2, and
the side product is acetaldehyde. Fig. 2 shows the Gibbs free ener-
gies for the formation of product of all the independent steps in-
volved in this metabolic pathway. It was observed that the
formation of APAP is an exothermic reaction with the Gibbs free
energy of 77.34 kcal/mol at BS3, using HOOH as the model oxi-
dizing agent. However, with Cpd I as the oxidant (to mimic
CYP450), the formation of APAP was highly exothermic by
99.33 and 71.99 kcal/mol on the doublet and quartet spin states
respectively. In the subsequent steps of the metabolic process, it
has been reported that 8090% of APAP is excreted as conjugates
of O-glucuronide-APAP, O-sulfonyl-APAP and 3-OH-APAP (cate-
chol). This occurs due to the abundant amounts of uridine glucur-
onosyl transferases (UDG) and sulfonyl transferases present in the
liver cells, which assist in the excretion of large amounts of APAP.
O-glucuronidation and O-sulfonation reactions were observed to
be endothermic with Gibbs free energies of 8.53 and 19.53 kcal/
mol respectively. On the other hand, N-deacetylation reaction, by
50 N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 4856
the amidase enzyme, leading to the formation of p-amino phenol
(PAP) was marginally exothermic (DG = 3.48 kcal/mol). APAP
can also undergo oxidation at the third position leading to the for-
mation of 3-OH-APAP with the exothermicity of 60.29 kcal/mol,
using HOOH as the oxidant. This reaction was highly exothermic
with Cpd I also (DG = 82.28 kcal/mol, doublet and 54.94 kcal/
mol, quartet spin state).
However, there is an alternative metabolic pathway which
occurs in Phase I only, where, the hydroxylation process leads to
the formation of N-hydroxyl-APAP (NOH-APAP). This step re-
leases 20.73 kcal/mol of energy (with HOOH as the oxidant) and
is a thermodynamic process. The Gibbs free energy for the forma-
tion of NOH-APAP with Cpd I is observed to be 42.71 and
15.37 kcal/mol on the doublet and quartet spin states as shown
in Fig. 2.
This hydroxylated intermediate thereafter, undergoes dehydra-
tion reaction, leading to the formation of another intermediate,
N-acetyl-p-benzoquinone imine (NAPQI). This step occurs in the
hepatic cells, releasing about 22.75 kcal/mol of energy. This inter-
mediate has been reported to undergo conjugation reaction with
glutathione (GSH) present in hepatic cells (Pathway-1, Fig. 1)
[21,22]. The Gibbs free energy for the formation of GSH conjugated
product was 13.32 kcal/mol on this metabolic pathway of phen-
acetin. GSH is one of the most important endogenous compounds
(antioxidant) produced by the cells, and participates in the neutral-
ization of free radicals and reactive oxygen species (ROS) [43]. GSH
normally protects the cells against highly electrophilic metabolites,
via its free sulfhydryl group (acting as the nucleophile), to form
easily excretable GSH conjugates.
The role of GSH in the detoxication of NAPQI has been known
for several years, via the formation of 3-(S-glutathionyl)-APAP con-
jugate [21,22]. However, NAPQI reacts with the hepatic cellular
proteins upon depletion of GSH, thereby, leading to hepatotoxicity.
Since, NAPQI is electrophilic in nature, DFT-based descriptor;
global electrophilicty index (x) was determined. It was observed
that NAPQI has a high global electrophilicty of 4.23 eV, accounting
for its direct reaction (covalent binding) with the structural com-
ponents of hepatic cells resulting in hepatic necrosis. The forma-
tion of NAPQI by Phase I reaction in the preference to the
conjugates (via Phase II metabolism) occurs due to the higher
doses of phenacetin, and overproduction of APAP metabolite in
the body. This results in the over saturation of UDG and sulfonyl
transferases in the liver cells, which switches the conjugation of
APAP (Phase II) to N-hydroxylation (Phase I) of APAP.
Table 1 shows the enthalpies and Gibbs free energies of the all
the reactions (using HOOH, Cpd I and other model oxidants) in-
volved in the Pathway-1 of phenacetin metabolism.
3.2. N-hydroxylation metabolic pathway
N-hydroxylation of phenacetin is the most explored pathway
and has been reported to be the metabolic pathway leading to uri-
nary bladder carcinoma and nephrotoxicity. The pathway involves
initial N-oxidation step, where a direct oxygen transfer from the
model oxidant, HOOH takes place, followed by intramolecular
1,2-H shift to form N-hydroxylated phenacetin (NOH phenacetin).
The mechanism of N-oxidation is similar with Cpd I as the model
oxidant. The formation of NOH phenacetin is an exothermic reac-
tion, (DH = 22.56 kcal/mol and DG = 20.13 kcal/mol), with
HOOH as the oxidizing agent. The corresponding DG with Cpd I
is observed to be 42.12 and 14.78 kcal/mol on the doublet
and quartet spin states respectively. This hydroxylated intermedi-
ate undergoes Phase II biotransformations via glucoronidation and
sulfate conjugation to form NO-glucuronide (DG = 7.75 kcal/mol)
and NO-sulfonyl (DG = 15.60) conjugates respectively (Fig. 3).
These conjugates later enter into the systemic circulation and via
renal ltration reach the urinary bladder. These conjugated metab-
N
C
O
OH
H
O
N
C
O
N
H C
O
OH
SG
N
H C
O
O
C
2
H
5
Phenacetin Acetaminophen N-OH APAP NAPQI 3-GS-APAP
(APAP)
Pathway-1 O-dealkylation pathway
GSH
O-dealkylation
-77.34
(-99.32)
[-71.99]
N
C
O
OH
HO
-20.73
(-42.71)
[-15.37]
-22.75
Oxidation -HOH
-13.32
N
H
OH
H
N
C
O
OGlu
H
N
C
O
OSO
3
H
H
N
C
O
OH
H
OH
-60.29
(-82.28)
[-54.94]
-3.48 8.53
19.53
Oxidation N-deacetylation O-glucuronidation O-sulfonation
3-OH-APAP p-amino phenol O-Glu-APAP O-SO
3
H-APAP
Fig. 2. Gibbs free energies (DG) of each independent step associated with the various possible products on the O-dealkylation pathway (Pathway-1) of phenacetin, calculated
at BS3. The values are given in kcal/mol on a reaction path catalyzed by model oxidants. The values in italics and within parenthesis are enthalpies estimated using Cpd I
(doublet spin state). The values in square brackets are enthalpies estimated on the quartet spin state of Cpd I. All the 3D structures of model oxidants and products are given
in Supporting information (S2 and S3) respectively.
N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 4856 51
olites being lesser stable get hydrolyzed non-enzymatically, owing
to the acidic environment of the urinary bladder.
Upon hydrolysis, they generate back NOH phenacetin metabo-
lite. The NOH-phenacetin forms the ultimate carcinogen O-ethyl-
N-acetyl-p-benzoquinone imine (OEt-NAPQI) in the acidic envi-
ronment (Fig. 3) upon further oxidation. OEt-NAPQI binds cova-
lently with the transition cell epithelium macromolecules of
urinary bladder and nucleosides of DNA, thus, inducing urinary
bladder carcinogenicity. Table 2 shows the Gibbs free energies
and enthalpies of the all the reactions (using HOOH, Cpd I and
other model oxidants) involved in the Pathway-2 of phenacetin
metabolism.
3.2.1. Reactivity of OEt-NAPQI
OEt-NAPQI (Fig. 3) is the structural analogue of NAPQI. Similar
to NAPQI, it is also electrophilic in nature, and possesses the similar
Table 2
Schematic pathways for the reactions involved in the N-hydroxylation of phenacetin, along with the enzymes involved, product enthalpies and Gibbs free energies for each
independent step, using model oxidants, calculated at BS3.
Model reaction Enzyme involved in biochemical
reaction
Enthalpy (DH) (kcal/
mol)
Gibbs free energies (DG) (kcal/
mol)
Phenacetin + H
2
O
2
?NOH-Phenacetin + H
2
O CYP1A2 22.56 20.13
Phenacetin + Cpd I
2,4
?NOH-Phenacetin + heme-porphine
2,4
CYP1A2 42.73
(2)
, 14.83
(4)
42.12
(2)
, 14.78
(4)
NOH-Phenacetin + Glucuronic acid ?NO-Glu-
Phenacetin + H
2
UDG 5.03 7.75
NOH-Phenacetin + Sulfonic acid ?NOSO
3
H-
Phenacetin + H
2
Sulfotransferases 9.84 15.60
NO-Glu-Phenacetin + H
2
O ?NOH-
Phenacetin + Glucuronide
Non-enzymatic hydrolysis (acidic pH) 5.03 7.75
NOSO
3
H-Phenacetin + H
2
O ?NOH-Phenacetin + Sulfonyl
group
Non-enzymatic hydrolysis (acidic pH) 9.84 15.60
NOH-Phenacetin + H
+
?OEt-NAPQI + H
2
O Non-enzymatic hydrolysis (acidic pH) 252.92 263.16
N
C
O
O
HO
N
H C
O
O
C
2
H
5
Phenacetin N-OH phenacetin N-OH phenacetin O-Et-NAPQI
Pathway-2 N-hydroxylation pathway
Oxidation
-20.13
(-42.12)
[-14.78]
O-glucuronidation
O-sulfonation
C
2
H
5
N
C
O
O
GluO
C
2
H
5
N
C
O
O
HO
3
SO
C
2
H
5
N
C
O
O
HO
C
2
H
5
N C
O
O
C
2
H
5
N-O-Glu phenacetin
N-O-SO
3
H phenacetin
7.75
15.60
-15.60
-7.75
Fig. 3. Gibbs free energies (DG) of each independent step associated with the various possible products on the N-hydroxylation pathway (Pathway-2) of phenacetin,
calculated at BS3. The values are given in kcal/mol on a reaction path catalyzed by model oxidants. The values in italics and within parenthesis are enthalpies estimated using
Cpd I (doublet spin state). The values in square brackets are enthalpies estimated on the quartet spin state of Cpd I. All the 3D structures of products are given in Supporting
information (S4).
Table 1
Schematic pathways for each step involved in the O-dealkylation of phenacetin, along with the enzymes involved, product enthalpies and Gibbs free energies for each
independent step, using model oxidants, calculated at BS3.
Model reaction Enzyme involved in the
biochemical reaction
Enthalpy (DH)
(kcal/mol)
Gibbs free energies
(DG) (kcal/mol)
Phenacetin + H
2
O
2
?APAP + H
2
O CYP1A2 67.33 77.34
Phenacetin + Cpd I
2,4
?APAP + heme-porphine
2,4
CYP1A2 87.50
(2)
, 59.60
(4)
99.32
(2)
, 71.99
(4)
APAP + H
2
O
2
?3-OH-APAP + H
2
O CYP1A2, CYP1A1 62.24 60.29
APAP + Cpd I
2,4
?3-OH-APAP + heme-porphine
2,4
CYP1A2, CYP1A1 82.41
(2)
, 54.51
(4)
82.28
(2)
, 54.94
(4)
APAP + Glucuronic acid ?O-Glu-APAP + H
2
UDG 5.86 8.53
APAP + Sulfonic acid ?O-SO
3
H-APAP + H
2
Sulfotransferase 14.27 19.53
APAP + H
2
O ?PAP + CH
3
COOH Amidase 1.20 3.48
APAP + H
2
O
2
?NOH-APAP + H
2
O CYP1A2 22.22 20.73
APAP + Cpd I
2,4
?NOH-APAP + heme-porphine
2,4
CYP1A2 42.39
(2)
, 14.49
(4)
42.71
(2)
, 15.37
(4)
NOH-APAP ?NAPQI + H
2
O CYP1A2 13.40 22.75
NAPQI + GSH ?3-GS-APAP GSH transferases 25.12 13.32
52 N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 4856
tendency to bind covalently to the structural or functional units of
cells, thereby, leading to cellular dysfunction. NAPQI is generally
formed in liver and causes hepatotoxicity, whereas, the formation
of OEt-NAPQI occurs in the urinary bladder leading to carcinoma.
This is due to the availability of lesser potent microsomes rather
than the highly potent liver microsomes and GSH.
The DFT-based descriptors [33] were calculated to determine
the electrophilicty of NAPQI and OEt-NAPQI. Table 3 describes
various parameters calculated for both these intermediates. The
global electrophilicity index of OEt-NAPQI is observed to be
19.43 eV, indicating a very high electrophilicity as compared to
NAPQI (4.23 eV). This higher electrophilicity makes it the most
reactive intermediate, involved in the induction of urinary bladder
carcinogenicity. A lower hardness is observed for OEt-NAPQI
(2.48 eV) indicating that this intermediate is more susceptible
for the nucleophilic attack as compared to NAPQI (3.67 eV).
3.3. N-deacetylation metabolic pathway
N-deacetylation of phenacetin occurs through amidase en-
zymes present in the liver microsomes or cytosol. This involves
the hydrolysis of the amide bond, leading to the formation of an
amine (phenitidine) and acetic acid as the by-product, as shown
in Fig. 4. This reaction is slightly exothermic with DG of
3.51 kcal/mol.
Phenitidine is subsequently oxidized to form NOH phenitidine,
releasing 24.50 kcal/mol of the energy, with HOOH as the oxidant.
The exothermicity with Cpd I was observed to be 46.49 and
19.15 kcal/mol on the doublet and quartet spin states respec-
tively. It has been reported that NOH phenitidine has the ten-
dency to bind with DNA and cause mutagenicity; however, it
rapidly gets converted to p-nitrosophenetole, via two oxidation
reactions. First oxidation of NOH phenitidine generates N-dihy-
droxy phenitidine, with the Gibbs free energies of 32.20,
54.19 and 26.85 kcal/mol, using HOOH, Cpd I (doublet state)
and Cpd I (quartet state) respectively. Second oxidation reaction
leads to the formation of p-nitrosophenetole (DG = 31.81 kcal/
mol), which is also a highly reactive metabolite, similar to NAPQI
and OEt-NAPQI. However, it is detoxied by GSH in the body,
leading to the formation of glutathione conjugates. At acidic pH
and high amounts of GSH, it is semi-stable and leads to the forma-
tion of phenitidine. Table 4 shows the reaction enthalpies and
Gibbs free energies for each independent step involved in the N-
deacetylation pathway of phenacetin with various model oxidants,
calculated at BS3.
3.4. Transition states for the key metabolic reactions in the three
different metabolic pathways
To understand the generation of various reactive metabolites
from the three different metabolic pathways of phenacetin, the en-
ergy barriers for the metabolic reactions (Phase I), catalyzed by
CYP450, in these three metabolic pathways were calculated. HOOH
was utilized as the model oxidant to mimic the oxidative ability of
CYPs. Fig. 5 shows the transition state geometries for the initial
steps namely, O-dealkylation using HOOH as the model oxidant
Table 4
Schematic pathways for the reactions involved in the N-deacetylation of phenacetin, along with the enzymes involved, product enthalpies and Gibbs free energies of each
independent step, using model oxidants, calculated at BS3.
Model reaction Enzyme involved in
biochemical reaction
Enthalpy (DH)
(kcal/mol)
Gibbs free energies
(DG) (kcal/mol)
Phenacetin + H
2
O ?Phenetidine + CH
3
COOH Amidase 1.20 3.51
Phenetidine + H
2
O
2
?NOH-Phenetidine + H
2
O CYP1A2, CYP1A1 25.82 24.50
Phenitidine + Cpd I
2,4
?NOH-Phenitidine + heme-porphine
2,4
CYP1A2, CYP1A1 45.98
(2)
, 18.08
(4)
46.49
(2)
, 19.15
(4)
NOH-Phenetidine + H
2
O
2
?N-(OH,OH)-Phenetidine + H
2
O CYP1A2, CYP1A1 33.83 32.20
NOH-Phenitidine + Cpd I
2,4
?N-(OH,OH)-Phenitidine + heme-porphine
2,4
CYP1A2, CYP1A1 54.00
(2)
, 26.10
(4)
54.19
(2)
, 26.85
(4)
N-(OH,OH)-Phenetidine ?P-nitroso phenetole + H
2
O CYP1A2, CYP1A1 22.98 31.81
Table 3
Energy of the highest occupied molecular orbital (E
HOMO
, au), Lowest unoccupied molecular orbital (E
LUMO
, au), electronegativity (v, electron volt, eV), hardness (g, eV) and
electrophilicity index (x, eV) for NAPQI and OEt-NAPQI intermediates.
Molecule E
HOMO
(au) E
LUMO
(au) Electronegativity (v) eV Hardness (g) eV Electrophilicity index (x) eV
NAPQI 0.274 0.139 5.619 3.673 4.230
OEt-NAPQI 0.406 0.315 9.809 2.476 19.429
N
H
O
H
N
H C
O
O
C
2
H
5
Phenacetin Phenitidine N-OH phenitidine N-dihydroxy phenitidine p-nitroso phenetole
Pathway-3 N-deacetylation pathway
N-deacetylation
-3.51
C
2
H
5
Oxidation
-24.50
(-46.49)
[-19.15]
N
H
O
HO
C
2
H
5
-32.20
(-54.19)
[-26.85]
Oxidation
N
H
O
HO
C
2
H
5
N
O
C
2
H
5
O
-HOH
-31.81
Fig. 4. Gibbs free energies (DG) of each independent step associated with the various possible products on the N-deacetylation pathway (Pathway-3) of phenacetin,
calculated at BS3. The values are given in kcal/mol on a reaction path catalyzed by model oxidants. The values in italics and within parenthesis are enthalpies estimated using
Cpd I (doublet spin state). The values in square brackets are enthalpies estimated on the quartet spin state of Cpd I. All the 3D structures of products are given in Supporting
information (S5).
N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 4856 53
(TS-I), N-hydroxylation using HOOH as the model oxidant (TS-II)
and N-deacetylation reaction with HOH (TS-III) as the model for
hydrolysis step. The activation barrier for the rst step in all the
pathways was observed to be the highest on the potential energy
prole and thus, the rst step is the rate determining step on all
the three pathways. The transition state geometries for other key
steps have been shown in the Supporting information (S6). The
bond distances in and bond angles in degrees () for the transi-
tion state geometries are also shown in Fig. 5. In the O-dealkylation
step, it can be observed that the oxygen atom O1 of the oxidant,
HOOH abstracts the methylene hydrogen from phenacetin. The
O1H (methylene) bond distance was observed to be 1.13 , which
is accompanied by the elongation of methylene CH distance to
1.38 . The bond distance of O1O2 in HOOH was observed to in-
crease to 2.09 from 1.47 (usual O1O2 bond distance in
HOOH). The bond angle CHO1 was observed to be 165.33 in
TS-I, which is appropriate for the H-abstraction step. The by-prod-
uct water is formed in the reaction, which is indicated by the HO2
bond distance of 1.64 . The activation barrier for the O-dealkyla-
tion reaction of phenacetin was observed to be 37.55 kcal/mol. The
H-abstraction from the methyl carbon required a higher energy
barrier of 44.58 kcal/mol, therefore, indicating the preference of
H-abstraction from methylene carbon in correlation with previous
studies [30a,44]. APAP formed undergoes N-oxidation by HOOH,
leading to the formation of NOH APAP, with an activation barrier
of 42.50 kcal/mol. This further results in the formation of highly
electrophilic and reactive NAPQI, via water elimination step
(DE
a
= 47.96 kcal/mol). On the other hand, APAP can also lead to
the formation of 3-OH APAP, via hydroxylation at 3-position of
APAP (DE
a
= 35.05 kcal/mol), with respect to APAP and HOOH.
In the N-hydroxylation step, the initial step involved is the N-
oxidation, where-in a direct oxygen (O1) transfer from HOOH to
the nitrogen takes place. The NO1 distance was observed to be
1.91 , while, O1O2 distance was observed to be 2.00 . The bond
angle of 158.25 between NO1O2 was observed in TS-II, which
indicates that indeed, N-oxidation is taking place. A molecule of
water is eliminated in the reaction, as indicated by the HO2 dis-
tance of 1.34 . The N-oxidation step is followed by 1,2-H shift
leading to the formation of NOH phenacetin. The activation bar-
rier for the N-hydroxylation reaction of phenacetin was observed
to be 42.38 kcal/mol, higher than the O-dealkylation reaction
(37.55 kcal/mol) as shown in Table 5.
The N-deacetylation step involves hydrolysis, catalyzed by the
amidase enzyme. The mechanism involves the attack of water,
leading to the breakage of NC(O) bond resulting in the elimination
of acetic acid and formation of phenitidine (with free amino
group). The NH bond distance of 1.15 and OH distance of
1.40 was observed. The NC(O) bond in TS-III was observed to
be elongated to 1.63 from 1.36 (in phenacetin). The OHN
bond angle was observed to be 130.81 in TS-III. This step requires
a higher activation barrier of 55.63 kcal/mol. Phenitidine so formed
can undergo N-oxidation to form NOH phenitidine, requiring an
energy barrier of 26.47 kcal/mol with HOOH as the oxidant. An-
other N-oxidation (DE
a
= 26.20 kcal/mol) results in the formation
of N-dihydroxy phenitidine, which ultimately leads to p-nitrosoph-
enetole via water elimination.
Fig. 6 shows the potential energy prole for the initial metabolic
steps (rate determining steps) in the three pathways discussed
above.
The potential energy prole shows that the activation barriers
for the initial steps of three metabolic pathways are quite compara-
ble; E
a
for the O-dealkylation reaction was marginally lower
(37.55 kcal/mol) as compared to other pathways. Considering the
Gibbs free energy for the product formation, O-dealkylation
Fig. 5. 3D structures of the transition state geometries for the initial steps of O-dealkylation, N-hydroxylation and N-deacetylation metabolic pathways of phenacetin,
calculated at BS3. All the bond distances are in . All the bond angles are in degrees (). All the 3D structures of transition state geometries for other key steps are given in
Supporting information (S6).
Table 5
Energy barriers of activation (DE

) and Gibbs free energy barriers (DG

) in kcal/mol for the O-dealkylation, N-hydroxylation and N-deacetylation of phenacetin at BS1 (gas phase),
BS2 (gas phase at higher basis set) and BS3 (implicit solvent phase at higher basis set).
Metabolic pathway BS1 BS2 BS3
DE

(kcal/mol) DG

(kcal/mol) DE

(kcal/mol) DG

(kcal/mol) DE

(kcal/mol) DG

(kcal/mol)
O-dealkylation 38.74 48.81 38.19 48.26 37.55 47.62
N-hydroxylation 38.32 48.60 38.51 48.79 42.38 52.67
N-deacetylation 39.76 49.84 38.69 48.77 55.63 65.71
54 N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 4856
pathway was energetically most favorable (77.34 kcal/mol)
than N-hydroxylation (20.13 kcal/mol) and N-deacetylation
(3.51 kcal/mol).
It is important to note that the O-dealkylation pathway
branches into phase I and phase II metabolism in subsequent steps,
and only Phase I metabolism leads to the reactive metabolite NAP-
QI. On the other hand, N-hydroxylation pathway entirely leads to
the formation of OEt-NAPQI. Therefore, it can be concluded that
the formation of the reactive metabolites (NAPQI, OEt-NAPQI)
from these two pathways is approximately similar in quantity.
N-deacetylation pathway is energetically less favorable
(3.51 kcal/mol) and requires a larger activation barrier
(55.63 kcal/mol), thereby, indicating the relatively lower quantity
of the reactive metabolite (p-nitroso phenetole), as compared to
NAPQI and OEt-NAPQI.
4. Conclusions
Phenacetin is metabolized by three different biotransformation
pathways leading to the generation of reactive metabolites, which
cause carcinogenicity, hepatotoxicity and nephrotoxicity. The
three metabolic pathways studied are: (i) O-dealkylation leading
to the formation of acetaminophen and NAPQI, (ii) N-hydroxyl-
ation leading to the formation of OEt-NAPQI, and (iii) N-deacety-
lation leading to the formation of phenetidine and p-nitroso
phenetole. The molecular level details of these three pathways
were explored extensively, using various model oxidants for Phase
I and II metabolic reactions, by employing quantum chemical
methods, including implicit solvent effects (BS3). The reactivity
and electrophilicty parameters were also studied for several reac-
tive metabolites. The O-dealkylation reaction was observed to be
highly favorable (E
a
= 37.55 kcal/mol) over other two reactions,
owing to a lower activation barrier. The Gibbs free energy for the
formation of O-dealkylated product (acetaminophen) was ob-
served to be higher (77.34 kcal/mol) than N-hydroxy phenacetin
(20.13 kcal/mol) and phenitidine (3.51 kcal/mol). It was ob-
served that in accordance with the reported experimental studies,
the N-Hydroxylation of phenacetin (Phase-I) and its subsequent
Phase-II metabolism accounted for the formation of reactive
metabolite (OEt-NAPQI) in the acidic environment of urinary
bladder. This reactive metabolite was found to be more electro-
philic (19.43 eV) as compared to NAPQI (4.23 eV), therefore,
accounting for its high reactivity and easy binding with several
biological nucleophiles like macromolecules and DNA nucleotides
(GSH levels are very low in urinary bladder), ultimately leading
to urinary bladder carcinoma (carcinogenicity).
Acknowledgements
NT, INSPIRE Fellow and PVB thanks the Department of Science
and Technology (DST), New Delhi for nancial assistance.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.comptc.2012.11.
018.
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