Computational and Theoretical Chemistry 1007 (2013) 48–56

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Computational and Theoretical Chemistry

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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, Indiab 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 a b s t r a c t

Article history:Received 1 September 2012Received in revised form 6 November 2012Accepted 9 November 2012Available online 7 December 2012

Keywords:PhenacetinCarcinogenicityDensity functional theoryReactive metabolitesElectrophilicityQuinone imine

2210-271X/$ - see front matter � 2012 Elsevier B.V.http://dx.doi.org/10.1016/j.comptc.2012.11.018

⇑ Corresponding author at: Department of Medicintute of Pharmaceutical Education and Research (NI(Mohali), 160 062 Punjab, India. Tel.: +91 172 229201

E-mail address: pvbharatam@niper.ac.in (P.V. Bha

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 thegeneration 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-dealkylationprocess 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 (O–Et-NAPQI) was observedto be the most critical and electrophilic metabolite (global electrophilicity; x = 19.43 eV), generated viainitial N-hydroxylation (Phase I) and subsequent Phase II metabolism. The higher electrophilicty of O–Et-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 O–Et-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 inthe treatment of fever and related complications [1–3]. It is alsoknown 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 sensorytracts of the spinal cord [1,5]. While, the antipyretic effect was ob-served by its action on the brain via a decrease in the temperatureset point [1,6]. However, the long-term and chronic consumptionof phenacetin led to several toxicological complications rangingfrom nephrotoxicity to carcinogenicity [7–12]. The carcinogenicityis observed in the urinary tract and renal pelvis (transitional-cell

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al Chemistry, National Insti-PER), Sector-67, S.A.S. Nagar8; fax: +91 172 2214692.

ratam).

carcinoma) [9–13]. As a result of these severe complications, phen-acetin and drugs containing phenacetin were withdrawn from themarket by the order of U.S. Food and Drug Administration in 1983[14]. The other critical toxic effects of phenacetin identified wererenal papillary necrosis and tumors of the bladder in humans [9–13].

Various research groups have carried out in vitro and in vivostudies to explore the carcinogenic and tumor inducing potentialof phenacetin [15–22]. Several reactive metabolites (RMs) crucialin these toxicological implications were identified in these studies.It was observed that phenacetin undergoes biotransformationreactions 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 thisgroup. CYP1A2 isoform catalyzes the metabolic reactions of phen-acetin. One of the major metabolic reactions identified was O-deal-kylation reaction (Pathway-1, Fig. 1), which leads to the formationof acetaminophen (hepatotoxic at higher doses). The intermediateN-acetyl-p-benzoquinoneimine (NAPQI) has been implicated to be

NCO

OH

H

O

NCO

NH C

O

OHSG

NH C

O

OC2H5

NH C

O

OC2H5

NCO

O

OH

OC2H5

NCO

NH C

O

OC2H5

NH2

OC2H5

N

OC2H5

O

Phenacetin Acetaminophen NAPQI Glutathione conjugateof acetaminophen

Phenacetin N-hydroxy phenacetin O-Et-NAPQI

Phenacetin Phenitidine p-nitroso phenetole

Pathway-1

Pathway-2

Pathway-3

GSH

Phase II metabolism

Glucuronidation/sulfationHydrolysis

Oxidation

O-dealkylation

N-hydroxylation

C2H5

N-deacetylation N-hydroxylationOxidation

Fig. 1. Reaction pathways for the biotransformation of phenacetin to several reactive metabolites. (A) Pathway-1: O-dealkylation reaction; (B) Pathway-2: N-hydroxylationreaction (oxidative metabolism); (C) Pathway-3: N-deacetylation reaction.

N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 48–56 49

involved in the hepatotoxicity, via liver necrosis, by the reactionwith glutathione resulting in 3-(S-glutathionyl) acetaminophenconjugate [21,22]. This pathway however, is not implicated to leadto tumorigenic or carcinogenic effect in the human body. Thisobservation has been confirmed in a recent study by Walter et al.[23], where no evidence regarding the relation between acetami-nophen and incidence of malignancies or cancer complicationswas obtained. Liu et al. [24] recently reported the application ofO-deethylation reaction of phenacetin as a marker reaction forCYP1A2 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 (N–OH phenacetin), which is also a well-known step in the acti-vation of paracetamol and aromaticamide carcinogens such as 2-acetylaminofluorene [15,18,24,25]. Vaught et al. [19] have shownthat N–OH phenacetin is carcinogenic and mutagenic to rat livermicrosomes. N–OH phenacetin has been observed to undergo fur-ther bioactivation reactions by Phase II metabolism, such as sulfateand glucuronide conjugation reactions to form N,O-sulfate andN,O-glucuronide conjugates. These conjugates can covalently bindto proteins and result in toxicity. These conjugates have also beenproposed to undergo hydrolytic reactions in the acidic environ-ment of urinary bladder, leading back to the formation of N–OHphenacetin, which can undergo further oxidation reactions

[16,17,19]. Nery [26] have shown the activation of phenacetin byliver microsomes, and further covalent binding to nucleic acidsand proteins. Calder et al. [24] also suggested the involvement ofintermediates of high chemical reactivity in the toxicity of phenac-etin by in vivo studies. They showed a rational pathway for N–OHphenacetin to p-benzoquinone and hydroquinone, both observedto be nephrotoxic in rats. However, this group did not performthe carcinogenic studies for these intermediates. Camus et al.[27] implicated the role of N–OH 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 ofa RM, p-nitrosophenetole. It has been implicated in binding totRNA resulting in carcinogenic and mutagenic complications. Sev-eral studies have shown the interaction of N-hydroxyphenitidine(formed via deacetylation of N–OH phenacetin) with nucleic acids[29].

Therefore, several in vitro and in vivo studies have highlightedthe importance of bioactivation reactions of phenacetin in the car-cinogenic and other toxicological complications. However, themolecular level details and mechanistic insights for each metabolicpathway have remained elusive till date and have not been ex-plored in detail. The quantum chemical methods can be utilizedto obtain the molecular level details of these reaction pathways

50 N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 48–56

[30] and have proved to be useful tool for these types of metabo-lism-related studies [31,32]. Other parameters such as hardnessand softness, electrophilicty and charge distribution have also beenreported to provide a wealth of information for metabolic reactions[33,34]. Koyamans et al. [30] performed ab initio calculations usingSTO-3G method to explore the metabolic pathway of phenacetin.They suggested the oxidative metabolism of phenacetin to N–OHphenacetin; O-dealkylation to acetaminophen and NAPQI. How-ever, the N-deacetylation reaction, reactivity and stability of RMswere 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 thetoxicity of phenacetin. Density Functional Theory (DFT) was uti-lized to investigate the 3D structures of all the intermediates andRMs involved, transition states for key steps catalyzed by CYPssuch as hydroxylation, oxidation reactions, energetics involved inthese reactions, potential energy surface for the critical and ratedetermining step of each metabolic pathway and electrophilicityparameters of the RMs.

2. Computational methodology

Quantum chemical calculations for all the metabolic pathwayshave 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 B3LYPfunctional with 6-31+G(d) basis set, denoted as BS1 [36]. TheB3LYP functional comprises of Becke’s three parameter exchangefunctional [37] with the correlation functional of Lee et al. [38].This method and basis set have been reported to be satisfactoryand 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 usingthe higher basis set [6-311+G(d)], denoted as BS2 [36b]. The effectof 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 mimicthe 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 foreach stationary point and transition states using GaussView 3.07program. The transition state is characterized to be a first ordersaddle point, with one negative imaginary vibrational mode, onthe potential energy surface. A scaling factor of 0.9806 was utilizedfor 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 wereperformed for the cytochrome P450 catalyzed reactions (Phase I) ofall the three metabolic pathways, using model oxidant, hydrogenperoxide (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 forthe first step of the three metabolic pathways were also evaluatedat 298 K and 1 atm, using thermal corrections to Gibbs free energy.The product enthalpies (DH) and Gibbs free energies (DG) werecalculated 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 densityfunctional was used for the geometry optimizations of Cpd I andrelated heme-porphyrin geometries, with LanL2DZ basis set oniron atom [36a], and the 6-31+G(d) basis set for all the remainingatoms [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 spinstates) of Cpd I was considered for this study. The reactivity ofthe crucial metabolites was determined by the global electrophilic-ity (x) parameter, calculated using standard equations (discussedin Supporting information S1) [33]. The results have been dis-cussed for three pathways using B3LYP/6-311+G(d) basis set andinclusion of zero point corrections and implicit solvent effects, un-less otherwise specified.

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 answerseveral questions, related with the toxicity (carcinogenicity) ofphenacetin. The questions addressed are following: What are themechanisms 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 keyPhase I metabolic reactions in these three pathways? What arethe enthalpies and Gibbs free energies of each step involved inthese pathways? Which intermediate is the most reactive, thermo-dynamically more stable and majorly involved in the carcinogenicimplications 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 ofphenacetin were studied. For calculating the enthalpies and Gibbsfree energies for Phase II reactions such as glucoronidation, glucu-ronic acid was employed as the model oxidant. The energies of theputative mechanistic intermediates were determined as a means ofidentifying 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 aninitial step of methylene C–H hydrogen abstraction (H-abstraction)from the methylene carbon of O-ethyl linked to the phenyl ring inphenacetin, followed by the reaction between the radicals to formthe hydroxylated phenacetin. The O-dealkylated product is acet-aminophen (acetyl-p-aminophenol; APAP) as shown in Fig. 2, andthe 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 theformation of APAP is an exothermic reaction with the Gibbs freeenergy of �77.34 kcal/mol at BS3, using HOOH as the model oxi-dizing agent. However, with Cpd I as the oxidant (to mimicCYP450), the formation of APAP was highly exothermic by�99.33 and�71.99 kcal/mol on the doublet and quartet spin statesrespectively. In the subsequent steps of the metabolic process, ithas been reported that 80–90% of APAP is excreted as conjugatesof 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 theliver cells, which assist in the excretion of large amounts of APAP.O-glucuronidation and O-sulfonation reactions were observed tobe endothermic with Gibbs free energies of 8.53 and 19.53 kcal/mol respectively. On the other hand, N-deacetylation reaction, by

NCO

OH

H

O

NCO

NH C

O

OHSG

NH C

O

OC2H5

Phenacetin Acetaminophen N-OH APAP NAPQI 3-GS-APAP(APAP)

Pathway-1 O-dealkylation pathway

GSHO-dealkylation

-77.34(-99.32)[-71.99]

NCO

OH

HO

-20.73(-42.71)[-15.37]

-22.75

Oxidation -HOH

-13.32

NH

OH

H NCO

OGlu

HNCO

OSO3H

HNCO

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-SO3H-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, calculatedat 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 givenin Supporting information (S2 and S3) respectively.

N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 48–56 51

the amidase enzyme, leading to the formation of p-amino phenol(PAP) was marginally exothermic (DG = �3.48 kcal/mol). APAPcan 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 exothermicwith Cpd I also (DG = �82.28 kcal/mol, doublet and �54.94 kcal/mol, quartet spin state).

However, there is an alternative metabolic pathway whichoccurs in Phase I only, where, the hydroxylation process leads tothe formation of N-hydroxyl-APAP (N–OH-APAP). This step re-leases 20.73 kcal/mol of energy (with HOOH as the oxidant) andis a thermodynamic process. The Gibbs free energy for the forma-tion of N–OH-APAP with Cpd I is observed to be �42.71 and�15.37 kcal/mol on the doublet and quartet spin states as shownin 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 thehepatic cells, releasing about 22.75 kcal/mol of energy. This inter-mediate has been reported to undergo conjugation reaction withglutathione (GSH) present in hepatic cells (Pathway-1, Fig. 1)[21,22]. The Gibbs free energy for the formation of GSH conjugatedproduct 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]. GSHnormally protects the cells against highly electrophilic metabolites,via its free sulfhydryl group (acting as the nucleophile), to formeasily excretable GSH conjugates.

The role of GSH in the detoxification of NAPQI has been knownfor several years, via the formation of 3-(S-glutathionyl)-APAP con-jugate [21,22]. However, NAPQI reacts with the hepatic cellularproteins 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 observedthat NAPQI has a high global electrophilicty of 4.23 eV, accountingfor 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 theconjugates (via Phase II metabolism) occurs due to the higherdoses of phenacetin, and overproduction of APAP metabolite inthe body. This results in the over saturation of UDG and sulfonyltransferases in the liver cells, which switches the conjugation ofAPAP (Phase II) to N-hydroxylation (Phase I) of APAP.

Table 1 shows the enthalpies and Gibbs free energies of the allthe 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 pathwayand has been reported to be the metabolic pathway leading to uri-nary bladder carcinoma and nephrotoxicity. The pathway involvesinitial N-oxidation step, where a direct oxygen transfer from themodel oxidant, HOOH takes place, followed by intramolecular1,2-H shift to form N-hydroxylated phenacetin (N–OH phenacetin).The mechanism of N-oxidation is similar with Cpd I as the modeloxidant. The formation of N–OH phenacetin is an exothermic reac-tion, (DH = �22.56 kcal/mol and DG = �20.13 kcal/mol), withHOOH as the oxidizing agent. The corresponding DG with Cpd Iis observed to be �42.12 and �14.78 kcal/mol on the doubletand quartet spin states respectively. This hydroxylated intermedi-ate undergoes Phase II biotransformations via glucoronidation andsulfate conjugation to form N–O-glucuronide (DG = 7.75 kcal/mol)and N–O-sulfonyl (DG = 15.60) conjugates respectively (Fig. 3).These conjugates later enter into the systemic circulation and viarenal filtration reach the urinary bladder. These conjugated metab-

NCO

O

HON

H CO

OC2H5

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

C2H5

NCO

O

GluO

C2H5

NCO

O

HO3SO

C2H5

NCO

O

HO

C2H5

N CO

OC2H5

N-O-Glu phenacetin

N-O-SO3H 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 usingCpd 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 Supportinginformation (S4).

Table 1Schematic pathways for each step involved in the O-dealkylation of phenacetin, along with the enzymes involved, product enthalpies and Gibbs free energies for eachindependent step, using model oxidants, calculated at BS3.

Model reaction Enzyme involved in thebiochemical reaction

Enthalpy (DH)(kcal/mol)

Gibbs free energies(DG) (kcal/mol)

Phenacetin + H2O2 ? APAP + H2O CYP1A2 �67.33 �77.34Phenacetin + Cpd I2,4 ? APAP + heme-porphine2,4 CYP1A2 �87.50(2), �59.60(4) �99.32(2), �71.99(4)

APAP + H2O2 ? 3-OH-APAP + H2O CYP1A2, CYP1A1 �62.24 �60.29APAP + Cpd I2,4 ? 3-OH-APAP + heme-porphine2,4 CYP1A2, CYP1A1 �82.41(2), �54.51(4) �82.28(2), �54.94(4)

APAP + Glucuronic acid ? O-Glu-APAP + H2 UDG 5.86 8.53APAP + Sulfonic acid ? O-SO3H-APAP + H2 Sulfotransferase 14.27 19.53APAP + H2O ? PAP + CH3COOH Amidase �1.20 �3.48APAP + H2O2 ? N–OH-APAP + H2O CYP1A2 �22.22 �20.73APAP + Cpd I2,4 ? N–OH-APAP + heme-porphine2,4 CYP1A2 �42.39(2), �14.49(4) �42.71(2), �15.37(4)

N–OH-APAP ? NAPQI + H2O CYP1A2 �13.40 �22.75NAPQI + GSH ? 3-GS-APAP GSH transferases �25.12 �13.32

52 N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 48–56

olites being lesser stable get hydrolyzed non-enzymatically, owingto the acidic environment of the urinary bladder.

Upon hydrolysis, they generate back N–OH phenacetin metabo-lite. The N–OH-phenacetin forms the ultimate carcinogen O-ethyl-N-acetyl-p-benzoquinone imine (O–Et-NAPQI) in the acidic envi-ronment (Fig. 3) upon further oxidation. O–Et-NAPQI binds cova-lently with the transition cell epithelium macromolecules ofurinary bladder and nucleosides of DNA, thus, inducing urinary

Table 2Schematic pathways for the reactions involved in the N-hydroxylation of phenacetin, aloindependent step, using model oxidants, calculated at BS3.

Model reaction Enzyme involved inreaction

Phenacetin + H2O2 ? N–OH-Phenacetin + H2O CYP1A2Phenacetin + Cpd I2,4 ? N–OH-Phenacetin + heme-porphine2,4 CYP1A2N–OH-Phenacetin + Glucuronic acid ? N–O-Glu-

Phenacetin + H2

UDG

N–OH-Phenacetin + Sulfonic acid ? N–O–SO3H-Phenacetin + H2

Sulfotransferases

N–O-Glu-Phenacetin + H2O ? N–OH-Phenacetin + Glucuronide

Non-enzymatic hydr

N–O–SO3H-Phenacetin + H2O ? N–OH-Phenacetin + Sulfonylgroup

Non-enzymatic hydr

N–OH-Phenacetin + H+ ? O–Et-NAPQI + H2O Non-enzymatic hydr

bladder carcinogenicity. Table 2 shows the Gibbs free energiesand enthalpies of the all the reactions (using HOOH, Cpd I andother model oxidants) involved in the Pathway-2 of phenacetinmetabolism.

3.2.1. Reactivity of O–Et-NAPQIO–Et-NAPQI (Fig. 3) is the structural analogue of NAPQI. Similar

to NAPQI, it is also electrophilic in nature, and possesses the similar

ng with the enzymes involved, product enthalpies and Gibbs free energies for each

biochemical Enthalpy (DH) (kcal/mol)

Gibbs free energies (DG) (kcal/mol)

�22.56 �20.13�42.73(2), �14.83(4) �42.12(2), �14.78(4)

5.03 7.75

9.84 15.60

olysis (acidic pH) �5.03 �7.75

olysis (acidic pH) �9.84 �15.60

olysis (acidic pH) �252.92 �263.16

N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 48–56 53

tendency to bind covalently to the structural or functional units ofcells, thereby, leading to cellular dysfunction. NAPQI is generallyformed in liver and causes hepatotoxicity, whereas, the formationof O–Et-NAPQI occurs in the urinary bladder leading to carcinoma.This is due to the availability of lesser potent microsomes ratherthan the highly potent liver microsomes and GSH.

The DFT-based descriptors [33] were calculated to determinethe electrophilicty of NAPQI and O–Et-NAPQI. Table 3 describesvarious parameters calculated for both these intermediates. Theglobal electrophilicity index of O–Et-NAPQI is observed to be19.43 eV, indicating a very high electrophilicity as compared toNAPQI (4.23 eV). This higher electrophilicity makes it the mostreactive intermediate, involved in the induction of urinary bladdercarcinogenicity. A lower hardness is observed for O–Et-NAPQI(�2.48 eV) indicating that this intermediate is more susceptiblefor 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 involvesthe hydrolysis of the amide bond, leading to the formation of anamine (phenitidine) and acetic acid as the by-product, as shownin Fig. 4. This reaction is slightly exothermic with DG of�3.51 kcal/mol.

Phenitidine is subsequently oxidized to form N–OH 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

Table 4Schematic pathways for the reactions involved in the N-deacetylation of phenacetin, aloindependent step, using model oxidants, calculated at BS3.

Model reaction Eb

Phenacetin + H2O ? Phenetidine + CH3COOH APhenetidine + H2O2 ? N–OH-Phenetidine + H2O CPhenitidine + Cpd I2,4 ? N–OH-Phenitidine + heme-porphine2,4 CN–OH-Phenetidine + H2O2 ? N-(OH,OH)-Phenetidine + H2O CN–OH-Phenitidine + Cpd I2,4 ? N-(OH,OH)-Phenitidine + heme-porphine2,4 CN-(OH,OH)-Phenetidine ? P-nitroso phenetole + H2O C

Table 3Energy of the highest occupied molecular orbital (EHOMO, au), Lowest unoccupied molecuelectrophilicity index (x, eV) for NAPQI and O–Et-NAPQI intermediates.

Molecule EHOMO (au) ELUMO (au) Electronegativ

NAPQI �0.274 �0.139 5.619O–Et-NAPQI �0.406 �0.315 9.809

NH

O

HN

H CO

OC2H5

Phenacetin Phenitidine N-OH phenitidin

Pathway-3 N-deacetylation pathway

N-deacetylation

-3.51

C2H5

Oxidation

-24.50(-46.49)[-19.15]

NH

O

HO

C2

Fig. 4. Gibbs free energies (DG) of each independent step associated with the varioucalculated at BS3. The values are given in kcal/mol on a reaction path catalyzed by modelCpd I (doublet spin state). The values in square brackets are enthalpies estimated on theinformation (S5).

�19.15 kcal/mol on the doublet and quartet spin states respec-tively. It has been reported that N–OH phenitidine has the ten-dency to bind with DNA and cause mutagenicity; however, itrapidly gets converted to p-nitrosophenetole, via two oxidationreactions. First oxidation of N–OH 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 reactionleads to the formation of p-nitrosophenetole (DG = �31.81 kcal/mol), which is also a highly reactive metabolite, similar to NAPQIand O–Et-NAPQI. However, it is detoxified by GSH in the body,leading to the formation of glutathione conjugates. At acidic pHand high amounts of GSH, it is semi-stable and leads to the forma-tion of phenitidine. Table 4 shows the reaction enthalpies andGibbs 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 threedifferent metabolic pathways

To understand the generation of various reactive metabolitesfrom the three different metabolic pathways of phenacetin, the en-ergy barriers for the metabolic reactions (Phase I), catalyzed byCYP450, in these three metabolic pathways were calculated. HOOHwas utilized as the model oxidant to mimic the oxidative ability ofCYPs. Fig. 5 shows the transition state geometries for the initialsteps namely, O-dealkylation using HOOH as the model oxidant

ng with the enzymes involved, product enthalpies and Gibbs free energies of each

nzyme involved iniochemical reaction

Enthalpy (DH)(kcal/mol)

Gibbs free energies(DG) (kcal/mol)

midase �1.20 �3.51YP1A2, CYP1A1 �25.82 �24.50YP1A2, CYP1A1 �45.98(2), �18.08(4) �46.49(2), �19.15(4)

YP1A2, CYP1A1 �33.83 �32.20YP1A2, CYP1A1 �54.00(2), �26.10(4) �54.19(2), �26.85(4)

YP1A2, CYP1A1 �22.98 �31.81

lar orbital (ELUMO, au), electronegativity (v, electron volt, eV), hardness (g, eV) and

ity (v) eV Hardness (g) eV Electrophilicity index (x) eV

3.673 4.2302.476 19.429

e N-dihydroxy phenitidine p-nitroso phenetole

H5

-32.20(-54.19)[-26.85]

OxidationNH

O

HO

C2H5

N

OC2H5

O

-HOH

-31.81

s possible products on the N-deacetylation pathway (Pathway-3) of phenacetin,oxidants. The values in italics and within parenthesis are enthalpies estimated usingquartet spin state of Cpd I. All the 3D structures of products are given in Supporting

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 Å. All the bond angles are in degrees (�). All the 3D structures of transition state geometries for other key steps are given inSupporting information (S6).

54 N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 48–56

(TS-I), N-hydroxylation using HOOH as the model oxidant (TS-II)and N-deacetylation reaction with HOH (TS-III) as the model forhydrolysis step. The activation barrier for the first step in all thepathways was observed to be the highest on the potential energyprofile and thus, the first step is the rate determining step on allthe three pathways. The transition state geometries for other keysteps have been shown in the Supporting information (S6). Thebond distances in Å and bond angles in degrees (�) for the transi-tion state geometries are also shown in Fig. 5. In the O-dealkylationstep, it can be observed that the oxygen atom O1 of the oxidant,HOOH abstracts the methylene hydrogen from phenacetin. TheO1–H (methylene) bond distance was observed to be 1.13 Å, whichis accompanied by the elongation of methylene C–H distance to1.38 Å. The bond distance of O1–O2 in HOOH was observed to in-crease to 2.09 Å from 1.47 Å (usual O1–O2 bond distance inHOOH). The bond angle C–H–O1 was observed to be 165.33� inTS-I, which is appropriate for the H-abstraction step. The by-prod-uct water is formed in the reaction, which is indicated by the H–O2bond distance of 1.64 Å. The activation barrier for the O-dealkyla-tion reaction of phenacetin was observed to be 37.55 kcal/mol. TheH-abstraction from the methyl carbon required a higher energybarrier of 44.58 kcal/mol, therefore, indicating the preference ofH-abstraction from methylene carbon in correlation with previousstudies [30a,44]. APAP formed undergoes N-oxidation by HOOH,leading to the formation of N–OH APAP, with an activation barrierof 42.50 kcal/mol. This further results in the formation of highlyelectrophilic and reactive NAPQI, via water elimination step(DEa = 47.96 kcal/mol). On the other hand, APAP can also lead tothe formation of 3-OH APAP, via hydroxylation at 3-position ofAPAP (DEa = 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

Table 5Energy barriers of activation (DE�) and Gibbs free energy barriers (DG�) in kcal/mol for the OBS2 (gas phase at higher basis set) and BS3 (implicit solvent phase at higher basis set).

Metabolic pathway BS1 BS2

DE� (kcal/mol) DG� (kcal/mol) DE� (kc

O-dealkylation 38.74 48.81 38.19N-hydroxylation 38.32 48.60 38.51N-deacetylation 39.76 49.84 38.69

the nitrogen takes place. The N–O1 distance was observed to be1.91 Å, while, O1–O2 distance was observed to be 2.00 Å. The bondangle of 158.25� between N–O1–O2 was observed in TS-II, whichindicates that indeed, N-oxidation is taking place. A molecule ofwater is eliminated in the reaction, as indicated by the H–O2 dis-tance of 1.34 Å. The N-oxidation step is followed by 1,2-H shiftleading to the formation of N–OH phenacetin. The activation bar-rier for the N-hydroxylation reaction of phenacetin was observedto 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 theamidase enzyme. The mechanism involves the attack of water,leading to the breakage of N–C(O) bond resulting in the eliminationof acetic acid and formation of phenitidine (with free aminogroup). The N–H bond distance of 1.15 Å and O–H distance of1.40 Å was observed. The N–C(O) bond in TS-III was observed tobe elongated to 1.63 Å from 1.36 Å (in phenacetin). The O–H–Nbond angle was observed to be 130.81� in TS-III. This step requiresa higher activation barrier of 55.63 kcal/mol. Phenitidine so formedcan undergo N-oxidation to form N–OH phenitidine, requiring anenergy barrier of 26.47 kcal/mol with HOOH as the oxidant. An-other N-oxidation (DEa = 26.20 kcal/mol) results in the formationof N-dihydroxy phenitidine, which ultimately leads to p-nitrosoph-enetole via water elimination.

Fig. 6 shows the potential energy profile for the initial metabolicsteps (rate determining steps) in the three pathways discussedabove.

The potential energy profile shows that the activation barriersfor the initial steps of three metabolic pathways are quite compara-ble; Ea for the O-dealkylation reaction was marginally lower(37.55 kcal/mol) as compared to other pathways. Considering theGibbs free energy for the product formation, O-dealkylation

-dealkylation, N-hydroxylation and N-deacetylation of phenacetin at BS1 (gas phase),

BS3

al/mol) DG� (kcal/mol) DE� (kcal/mol) DG� (kcal/mol)

48.26 37.55 47.6248.79 42.38 52.6748.77 55.63 65.71

Fig. 6. Potential energy profile for the initial metabolic steps (rate determining) in the three different biotransformation pathways of phenacetin, calculated at BS3. All thevalues are in kcal/mol. R represents the reactants; phenacetin and HOOH for O-dealkylation and N-hydroxylation pathway; phenacetin and water for N-deacetylationpathway. TS represents the transition states. P represents the products; acetaminophen and water for O-dealkylation pathway; N-hydroxy phenacetin and water for N-hydroxylation pathway; phenitidine and acetic acid for N-deacetylation pathway.

N. Taxak et al. / Computational and Theoretical Chemistry 1007 (2013) 48–56 55

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 pathwaybranches 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 tothe formation of O–Et-NAPQI. Therefore, it can be concluded thatthe formation of the reactive metabolites (NAPQI, O–Et-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 quantityof the reactive metabolite (p-nitroso phenetole), as compared toNAPQI and O–Et-NAPQI.

4. Conclusions

Phenacetin is metabolized by three different biotransformationpathways leading to the generation of reactive metabolites, whichcause carcinogenicity, hepatotoxicity and nephrotoxicity. Thethree metabolic pathways studied are: (i) O-dealkylation leadingto the formation of acetaminophen and NAPQI, (ii) N-hydroxyl-ation leading to the formation of O–Et-NAPQI, and (iii) N-deacety-lation leading to the formation of phenetidine and p-nitrosophenetole. The molecular level details of these three pathwayswere explored extensively, using various model oxidants for PhaseI and II metabolic reactions, by employing quantum chemicalmethods, including implicit solvent effects (BS3). The reactivityand electrophilicty parameters were also studied for several reac-tive metabolites. The O-dealkylation reaction was observed to behighly favorable (Ea = 37.55 kcal/mol) over other two reactions,owing to a lower activation barrier. The Gibbs free energy for theformation 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 subsequentPhase-II metabolism accounted for the formation of reactivemetabolite (O–Et-NAPQI) in the acidic environment of urinarybladder. 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 severalbiological nucleophiles like macromolecules and DNA nucleotides

(GSH levels are very low in urinary bladder), ultimately leadingto urinary bladder carcinoma (carcinogenicity).

Acknowledgements

NT, INSPIRE Fellow and PVB thanks the Department of Scienceand Technology (DST), New Delhi for financial assistance.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.comptc.2012.11.018.

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