Toxicity Originating from Thiophene Containing Drugs: Exploring the

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Toxicity Originating from Thiophene Containing Drugs: Exploring the Mechanism using Quantum Chemical Methods Chaitanya K Jaladanki, Nikhil Taxak, Rohith Anand Varikoti, and Prasad V. Bharatam Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00364 • Publication Date (Web): 17 Nov 2015 Downloaded from http://pubs.acs.org on November 25, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Chemical Research in Toxicology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Toxicity Originating from Thiophene Containing Drugs: Exploring the Mechanism using Quantum Chemical Methods Chaitanya K. Jaladanki,a Nikhil Taxak,a Rohith A. Varikoti,b Prasad V. Bharatama,* a

Department of Medicinal Chemistry, bDepartment of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector - 67, S. A. S. Nagar (Mohali), 160 062 Punjab, India.

*Corresponding Author Prof. Prasad V. Bharatam Department of Medicinal Chemistry National Institute of Pharmaceutical Education and Research (NIPER), Sector – 67, S. A. S. Nagar (Mohali), 160 062 Punjab, India Telephone: +91 172 2292018; Fax: +91 172 2214692. E-mail: [email protected]

ACS Paragon Plus1 Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Entry for Table of Contents


Page 2 of 40

Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Abstract Drug metabolism of thiophene containing substrates by cytochrome P450s (CYP450) leads to toxic side effects, for example, nephrotoxicity (suprofen, ticlopidine), hepatotoxicity (tienilic acid), thrombotic thrombocytopenic purpura (clopidogrel), aplastic anemia (ticlopidine). The origin of toxicity in these cases has been attributed to two different CYP450 mediated metabolic reactions – S-oxidation and epoxidation. In this work, the molecular level details of the bioinorganic chemistry associated with the generation of these competitive reactions are reported. Density Functional Theory was utilized (i) to explore the molecular mechanism for S-oxidation and epoxidation using the radical cationic centre Cpd I [(iron (IV)-oxo-heme porphine system with SH- as the axial ligand, to mimic CYP450s] as the model oxidant, (ii) to establish the 3D structures of the reactants, transition states and products on both the metabolic pathways, and (iii) to examine the potential energy (PE) profile for both the pathways to determine the energetically preferred toxic metabolite formation. The energy barrier required for S-oxidation was observed to be 14.75 kcal/mol as compared to that of epoxidation reaction (13.23 kcal/mol) on the doublet PE surface of Cpd

I. The formation of epoxide metabolite was found to be highly exothermic (-23.24 kcal/mol), as compared to S-oxidation (-8.08 kcal/mol). Hence, on a relative scale epoxidation process was observed to be thermodynamically and kinetically more favorable. The energy profiles associated with the reactions of the S-oxide and epoxide toxic metabolites were also explored. This study helps in understanding the CYP450-catalyzed toxic reactions of drugs containing thiophene ring at the atomic level. Keywords: Thiophene, S-oxidation, epoxidation, Density Functional Theory, metabolites, cytochrome.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 40

INTRODUCTION Metabolism of xenobiotics is a biochemical process, catalyzed by cytochromes P450 (CYPs/CYP450), often it leads to the formation of toxic metabolites (TMs).1–3 Several metabolic pathways are involved in such reactions, including alkyl / aryl / alkene / N- / Soxidation, epoxidation, reduction, dealkylation, etc.4–6 Majority of drugs contain several heterocyclic rings, biotransformation of which leads to toxic products. The heterocyclic rings of concern are furan, thiophene, indole, benzimidazole, benzoxazole,1,2 among these, thiophene ring was declared as a structural alert.7–10 Several thiophene-containing drugs (Figure 1) were withdrawn (or in the process of being withdrawn) from the market, such compounds include suprofen (NSAID),11–15 tienilic acid

diuretic),12,16–19

(loop

(anti-platelet),20–22

ticlopidine

(NSAID),17,23

tenoxicam

methapyrilene (anti-histaminic),8,24–28 thenalidine (anti-puritic).29 Thiophene based toxic effects were reported for the block buster drug clopidogrel also.21,22,30–38 The toxic end points which prompt the drug withdrawals of thiophene containing drugs generally are hepatotoxicity, renal toxicity, blood dyscrasias, aplastic anemia, etc. O

O

OH O

S

S Cl

O

OH

O Cl

Suprofen

Tienilic Acid

O O S N OH

S

O

S

S

S

Cl

Cl Ticlopidine

N

N

HO N S

N

Methapyrilene

Thenalidine

Zileuton

Figure 1. Important drug molecules carrying the thiophene ring.


NH2 O

S

N

N Tenoxicam

N

N

Clopidogrel

N

O HN

O

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Two different metabolic pathways (Figure 2) were reported for drugs containing thiophene ring (1), (i) S-oxidation, leading to the formation of toxic sulfoxide metabolite (TM1) and (ii) epoxidation leading to the formation of toxic thiophene epoxide (TM2). Most of the drugs containing thiophene ring undergo CYP450-catalyzed oxidation to form unstable and reactive thiophene-S-oxide metabolite (TM1).39,40 They also undergo hydroxylation at the carbon atoms adjacent to the sulfur atom and get converted to 2- or 5-hydroxythiophenes presumably via an initial epoxide formation (TM2).41 These 2- or 5-hydroxy metabolites could result from an isomerization of either a thiophene-S-oxide intermediate or an epoxide intermediate.2,40 Rademacher et al. expressed doubts regarding the conversion of thiophene-

S-oxide to the 2-hydroxy thiophene.42 The toxic metabolites TM1 and TM2 are highly electrophilic and react with nucleophilic amino acids in biochemical conditions, eventually leading to unwanted covalent bond formation, disturbing the balance of several biochemical reactions leading to toxicity, via mechanism based inhibition (MBI).

Figure 2. Metabolic reactions of thiophene under cytochrome conditions.

Several in vitro and in vivo studies were reported on the metabolic pathways of thiophene ring. Recently, Dolenc and coworkers reviewed the bioactivation potential of thiophene containing drugs, the toxic reactive metabolites of thiophene ring and their reactions.7 Mansuy and coworkers reported the formation of dihydrothiophene sulfoxide mercapaturic acid supporting the S-oxidation metabolite.40 They also studied the oxidative ACS Paragon Plus5 Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 40

metabolism of 3-aroylthiophene and determined that it forms four compounds, two of which are S-oxides and two are epoxides.10,39 They further studied the oxidative metabolism of tienilic acid (TA) by CYP2C9 and shown that this isoenzyme is mainly responsible for the generation of toxic reactive metabolites, which react with the nucleophilic amino acids in the active site of CYP2C9, resulting in the inactivation of the enzyme.17,19,43,44 Melet et al. reported TA S-oxide forms covalent bond with Serine 365 in the active site of CYP2C9.18 Mansuy et al. studied the chemistry of toxicity originating from TA and its isomer (TAI) and proposed that both the species form different metabolites – TA prefers to form Soxide metabolite whereas, TAI prefers to form epoxide metabolite.39,45,46 Rademacher et al. confirmed these differential biotransformation reactions using biophysical methods.42,47 Koenigs et al. reported the mechanism based inactivation of the CYP2C9 by TA using highperformance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESIMS).43 O’Donnell et al. reported that suprofen causes the MBI of CYP2C9.13 Hutzler et al. compared the CYP2C9 inactivation efficiency of suprofen and TA via MBI.

12

Medower et

al. studied the formation of electrophilic S-oxide intermediate from the anticancer drug OSI930.48 Dansette et al. provided the first evidence that CYP450s may catalyses both Soxidation and epoxidation of thiophene.23 Lu et al. reported the MBI of CYP1A2 by zileuton.49 Joshi et al. studied oxidative metabolism of zileuton by human liver incubations, reported that formation of reactive 2-acetylbenzothiophene-S-oxide metabolite (triggered by cytochrome dependent metabolism) is responsible for the hepatotoxicity.9 The hepatotoxicity by methapyrilene depends on CYP450 oxidative metabolism of the thiophene ring.24–28 The reactive metabolite formation from the anti-platelet aggregation agent clopidogrel was also attributed to the thiophene ring.31–33,38,50 Similarly, both ticlopidine-S-oxide and ticlopidine epoxide are reported to be associated with aplastic anemia.20


Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Computer aided analysis of drug metabolism provides many clues regarding the chemistry occurring on drugs in the active site cavity of cytochromes. Molecular docking, computational site of Metabolism (SOM) prediction, toxicocophore/metabolophore mapping, etc. methods are being used to predict the most appropriate metabolite formation.42,51 Molecular docking studies are being employed to understand the role of the amino acids in active site cavity of CYP450s in selectively causing the epoxidation or S-oxidation.42 Quantum chemical calculations are being employed to explain the energy profile of TM formation from drugs51–69 recently a report on the bioinorganic chemistry of furan ring exploring all possible biotransformations was reported.59 . It is pertinent to note that the quantum chemical methods are generally performed on model systems ignoring the amino acid details, also they do not consider the dynamical state of the enzyme inhibition complexes. Even with these limitations, the information emerging from the quantum chemical analysis provides sufficient clarity on the reaction mechanism. As pointed out earlier, several experimental studies provided evidence for the formation of S-oxide and epoxide TMs from thiophene ring, however, the molecular level details of the mechanism for these metabolic pathways were not elucidated till date. In this article, an initial molecular docking analysis was carried out to determine the probability of occurrence of S-oxidation and epoxidation reactions in thiophene. Later, Density Functional Theory (DFT) was utilized (i) to explore the CYP450 catalysed mechanisms of S-oxidation and epoxidation reactions, (ii) to investigate the 3D structures of all the transition states and product complexes on the reaction paths, (iii) to estimate the free energy changes (∆G) associated with these reactions, (iv) to generate potential energy surface on the two metabolic pathways and (v) to distinguish the reaction pathways which can cause MBI among many possibilities



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

COMPUTATIONAL METHODOLOGY Molecular Docking Methodology. The 3D structures of a library of thiophene containing drugs were built using Maestro 9.3 molecular modeling package.70 Minimization and preparation of the compounds were performed using LigPrep module71 of Maestro (Maestro 9.3, Schrödinger, LLC, New York) interface with OPLS_2005 force field from the generated database. Finally, a database of 90 ligands was created in Maestro. For the purpose of molecular docking, crystal structures of the CYP3A4 (PDB ID: 1TQN),72 CYP2C9 (PDB ID: 1R9O),73 CYP2D6 (PDB ID: 3TBG)74 and CYP2C19 (PDB ID : 4GQS)75 were utilized. Protein Preparation wizard tool incorporated in Maestro 9.3 interface76 was used to refine the structure of CYP isoforms. Hydrogen atoms were added and assigned the right bond orders. The carboxylic acid side chains of all the Glutamic acid (Glu) and Aspartic acid (Asp) residues were deprotonated and the side chains of the basic amino acid residues (Arginine (Arg) and Lysine (Lys)) were protonated to mimic the ionization state of the enzyme at physiological pH. Oxygen atom is added to Fe centre for active state (i.e. Fe(IV) state at heme center) and appropriate charges added to Fe, O, N and S atoms. Receptor Grid Generation module of Glide was used for grid generation for the docking purpose, the outer grid was extended up to 15 Å in the vicinity of Fe-porphyrin (Active site of CYP’s), while maintaining the inner box size to the default value (10 x 10 x 10 Å3). Molecular docking of the prepared library of compounds was performed using Glide 5.8 module77 incorporated in maestro 9.3 package. Multiple ligand docking was carried out, 30 poses per ligand were generated which were organized according to their Gscore scoring function. Highest ranked poses were visually inspected and analyzed for their interactions with active site residues as well as with the Fe=O centre of heme.


Page 8 of 40

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Quantum Chemical Methodology. Quantum chemical calculations were carried out using Gaussian09 suite78 of programs for studying the metabolic pathways of thiophene ring. Density Functional Theory (DFT)79–81 was utilized for geometry optimizations of all the structures involved in the metabolic pathways of thiophene ring using B3LYP functional and 6-31+G(d) basis set (BS1).82–84 Cpd

I [Fe(IV)-oxo heme-porphine radical cation, with SH- as the axial ligand] was employed as the model oxidant to mimic the catalytic domain 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,85 and the BS1 for all the remaining atoms.

Cpd I has been reported to be an established and a standard model to study CYP-mediated metabolism reactions and Lanl2DZ was found to be quite useful in defining the iron centre in

Cpd I.35 The multi-state reactivity (both doublet and quartet spin states) of Cpd I was considered for this study. This method and basis set have been reported to be satisfactory and provide reasonable energy estimates, as seen in similar theoretical studies on drug metabolism reactions.26,51-63 Vibrational frequency calculations were carried out at the same level of geometry optimizations to characterize them as either minima or transition states. Single point energy calculations for all the optimized geometries were carried out using the higher basis set [6-311+G(d)],86 denoted as BS2 in the manuscript. The effect of implicit solvent was included in the study using Integral Equation Formalism variant of Polarizable Continuum Model (IEFPCM),87 using solvent chlorobenzene (dielectric constant (ε) =5.7) to mimic the bulk polarity effects of active site cavity of cytochrome P450 (this option is denoted as BS3 in the manuscript). Natural Bond Orbital (NBO) method88 was used to estimate partial atomic charges and to evaluate second order interactions and spin densities. The electrophilicity analysis89 was carried out to estimate the global electrophilicity indices of all important reactive metabolites considered.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 40

RESULTS AND DISCUSSION Molecular Docking. Molecular docking studies on a

A

B

library of 90 thiophene ring containing ligands were performed to find out the preferred site of metabolism of these compounds, screened against 4 isoforms of

metabolizing

CYP2C9,

enzymes

CYP2C19

CYP3A4,

and

CYP2D6.

Among 90 ligands chosen, the docking poses of only ~60 substrates have showed

Figure 3. Representation of the docking poses of clopidogrel (A) shows the C1 centre of thiophene head towards heme centre favoring epoxidation in CYP2C9 (B) shows the S of thiophene ring towards heme centre favoring S-oxidation in CYP3A4.

SOM at thiophene ring at any of the two sites i.e., S-oxidation and epoxidation within the top three poses (Table 1). A few of the ligands showed SOM at sulfur atom and others at unsaturated center of thiophene ring, but ligands like clopidogrel, suprofen, tenilic acid and others showed SOM at both centers of the thiophene ring in their top five poses within the active site cavity of CYP’s.

Table 1. The number of substrates taken and selectivity of the substrates towards the cytochromes (as per the 1st ranked molecular docking pose). CYP CYP3A4

Total no. of substrates 90

S-oxidation Predicted (1st ranked pose/top3 poses) 24/34

Epoxidation Predicted (1st ranked pose/top3 poses) 35/39

CYP2C9

90

26/35

34/40

CYP2D6

90

21/31

20/30

CYP2C19

90

25/34

29/36

10Environment ACS Paragon Plus

Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In CYP3A4, the hydrophobic groups of the dome region (mostly phenylalanines) direct the thiophene ring towards the heme Fe=O centre. Alternatively in the case of CYP2C9, the hydrophilic residues arginine and lysine control the docking pose, directing the thiophene ring towards the heme prosthetic group. For example, clopidogrel (antiplatelet aggregation drug), is known to produce epoxidation preferentially in CYP2C9 isoform, whereas it is known to show to S-oxidation preferentially in CYP3A4 isoform. The molecular docking results (Figure 3) support this argument. In the first ranked pose of clopidogrel in the active site of CYP2C9 isoform, the carbon atom of thiophene ring is pointed towards the Fe=O centre. Alternatively in the active site of CYP3A4 isoform, the sulfur atom is pointed towards the Fe=O centre. Subtle changes in orientation of clopidogrel are clearly visible from Figure 3. The differences are quite small, hence both the reactions (S-oxidation and epoxidation) are equally possible. Even the experimental literature is fuzzy in pinpointing the major metabolite produced. The first ranked pose of tienilic acid in CYP2C9 isoform shows preference towards S-oxidation reaction (with Gscore -6.95) whereas fourth ranked pose (Gscore -6.57) shows a preference towards epoxidation. Molecular docking analysis helps in identifying which residues of the enzyme control the orientation of the ligand inside the active site rather than nailing down the actual SOM because both the possible SOMs are adjacent to each other.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 40

QUANTUM CHEMICAL ANALYSIS S-oxidation of thiophene with Cpd I. Sulfoxidation reactions are common reactions in drug metabolism as well as in substrate detoxification processes in the liver. The reaction mechanism studies on substrate sulfoxidation reactions have been carried out using experimental as well as theoretical studies and a concerted SN2 mechanism has been suggested. Quantum chemical studies have been extensively carried out to understand the S-oxidation process from different model oxidants like

H2O2,55,90,91

HOONO,55,92–95

and

C4a-hydroperoxyflavin,55,96

cytochrome

P450,35,52,62,97,98 etc., using dimethyl sulfide (DMS),98,99 thiazolidinedione,52,55 etc. as model substrates. Bach et al. reported a two-step SN2 oxidation mechanism of dimethyl sulfide by H2O2.90,100 Trout et al.101 suggested the importance of explicit water molecules (2 or 3) in the transfer of oxygen to organic sulfides. Shaik et al.35,62,97,102–104 and de Visser67 investigated a range of mainly aliphatic sulfides that differ in the length of the alkyl chain and range from a hydrogen atom to an ethyl group. S-oxidation of thiazolidinediones was reported by Bharatam et al.52,55,105 From the above studies, it has become clear that the radical character of the catalyst Cpd I (model system of cytochromes) is responsible for the reduction in the barrier. Also, the doublet state of the Cpd I is generally involved in the reaction mechanism (rather than the quartet state), hence the following discussion is mostly based on the doublet PE surface. S-oxidation of thiophene leads to the generation of the thiophene-S-oxide (TM1). This process is explored in this work using model oxidant Cpd I on doublet and quartet potential energy surfaces. The mechanism involved a direct transfer of oxygen atom from Cpd I to sulphur centre of thiophene via the transition state TS1 (Figure 4). Initially, a reactive complex (RC1) is formed in which the sulfur weakly interacts with the oxygen atom of Cpd

I. In the doublet state, of RC1, the S-O distance turned out to be 4.11 Å (4.21 Å in quartet


Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Geometries of reactive complex (RC1), transition state (TS1) and product complex (PC1). All the distance are in Å units and angles in degrees and energies in kcal/mol.

state). The main difference between the geometries of RC1 doublet and quartet spin states originates from the Fe-O-S angle, which is 126.2° in doublet state and 136.2° in the quartet state. The reaction complex (RC1) is marginally stable in comparison to the sum of energies of thiophene and Cpd I by about 3.58 kcal/mol in the doublet state (3.21 kcal/mol in the quartet state). Molecular orbital analysis revealed that the observed differences between the 2

RC1 and 4RC1 can be traced to the electronic interactions in these two complexes. In the

doublet state, the donation of the electron density preferentially takes places from the p type of lone pair electrons of sulfur to the π* orbital of Fe-O bond whereas in the quartet state the donation takes place to the σ*z2 orbital of the Fe-O bond (Figure 5). The above noted difference between doublet and quartet structures continues to be persistent in the transition state TS1 also. In the TS1, the Fe-O bond gets marginally elongated. The S-O distance greatly reduced to 2.21 Å and Fe-O-S angle marginally reduced on the doublet potential energy surface. In the quartet state, on the other hand, the Fe-O-S angle increased from 136.2° to 140.1°. All these changes are consistent with the observed molecular orbital interactions. The energy barrier for the oxygen transfer reaction is estimated to be about 17.01 kcal/mol on the potential energy surface (~35.26 kcal/mol on quartet potential energy surface). In the product complex

2,4

PC1 the Fe-O bond is almost broken,

however, continues to show some interaction. In 2,4PC1 the sulfur atom of the thiophene ring adopts strong pyramidalization (sum of angles around sulfur ~310.6°). The product complex



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. The participation of orbitals in the electron transfer during sulfoxidation pathway (a) cartoon of orbital overlap of sulfur lone pair with π∗xz of heme in doublet spin state, (b) cartoon of orbital overlap of sulfur lone pair with σ∗z2 of heme in quartet spin state. 2,4

PC1 is 8.07 kcal/mol exergonic on the doublet spin surface of Cpd I (4.69 kcal/mol

exergonic on quartet spin surface). The above discussion clearly established the electron transfer from the sulfur center to the π*-orbital of Cpd I on the doublet potential energy surface is an energetically favorable process and facilitates the transfer of oxygen to thiophene ring. This analysis was repeated using basis sets BS2 and BS3 which estimated the barrier to be 14.74 kcal/mol and 15.67 kcal/mol respectively. For comparison purpose, the Soxidation process at the thiophene sulfur using hydrogen peroxide (HOOH) as the oxidizing agent was also studied, the barrier for the oxygen transfer is estimated to be 37.07 kcal/mol (using basis set BS1). This clearly indicates the S-oxidation with the help of cytochromes is a very favorable process, mainly originating from the radical character of the catalytic center in cytochromes.

Epoxidation of thiophene with Cpd I. Epoxidation is an important metabolic step for the drugs containing unsaturated functional units like alkenes, furan, thiophene, phenyl, pyridine, etc. Several quantum chemical studies on epoxidation on functional groups like alkene, propene, furan, etc. were reported. The epoxidation reaction of the aliphatic and alicyclic unsaturated centres using density functional theory (DFT) has been reported by Shaik and coworkers.106–108 It was proposed that epoxidation reaction involves a two-step reaction mechanism which is initiated


Page 14 of 40

Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Geometries of reactive complex (RC2), Transition State (TS2), Intermediate (I) and Product Complex (PC2) in both doublet and quartet states. All the distance are in Å units and angles in degrees.

by a C=C bond activation step followed by the oxygen rebound phase and subsequent ring closure (epoxide) resulting in the generation of the heme-epoxide complex. The influence of the electronic factors which affect the epoxidation of propene reaction with Cpd I has been explained by de Visser et al.109 and revealed that the epoxidation is more favorable at lower spin state of Cpd I. Kumar et al.107 reported the essential factors for the epoxidation reaction by Cpd I and non-heme systems (enzymes) in substrates like ethene, propene, 1-butene, etc. They explained the role of the ionization energy (IE) of the substrate in epoxidation process. Stare et al.110 assessed several features of the epoxidation reaction using propene by hydrogen peroxide (HOOH) as an oxidant, and elucidated some of the influential factors governing its mechanism. In our previous studies, we explored several aspects of epoxidation of furan using oxidant Cpd I and elucidated some of the important factors governing its mechanism and also explored the mechanism based inhibition of epoxide metabolites.59 In this section, quantum chemical analysis of epoxidation of thiophene is reported. Figure 6 shows the 2D structures on the potential energy surface of epoxide formation on simple thiophene. Initially, a reactive complex (2,4RC2) is formed in which the π cloud weakly interacts with the oxygen atom of Cpd I in both doublet and quartet states. The reaction complex (2,4RC2) is marginally stable compared to the sum of energies of thiophene and Cpd I by about 3.21 kcal/mol in the doublet state and 3.09 kcal/mol in the quartet state. Epoxidation involved two steps; (i) initial C1=C2 activation for the formation of radical intermediate (2,4I) with C1-O bond, followed by (ii) ring closure generating the epoxide metabolite of the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

thiophene (2,4PC2, a complex of thiophene epoxide and heme-porphine (Figure 6). In the doublet state, the donation of the electron density preferentially takes place from the π cloud of unsaturated bond (C=C) to the π*-orbital of Fe-O bond. In 2TS2, the Fe-O bond gets marginally elongated, the C---O distance gets reduced to 2.06 Å, angle is 128.7° in comparison to 2RC2. The initial step of epoxidation process of the thiophene is preferred at C1 centre rather than at C2 centre. This observation is supported by the greater nucleophilicity (1.38 eV) at C1 as compared to the same at C2 centre (0.34 eV). The overall energy barrier for the reaction is 15.36 kcal/mol on the low spin doublet potential energy surface. Using higher basis sets BS2 and BS3, the barrier values are found to be 13.29 and 14.23 kcal/mol respectively. The structural features of 2I indicate that the Fe---O interaction (2.52 Å) continues to exist. 2PC2 is characterized by a weak Fe---O electrostatic interaction and is stable by -23.24 kcal/mol on the potential energy surface. For comparison, epoxidation reaction at thiophene requires a barrier of 34.22 kcal/mol using hydrogen peroxide as oxidizing agent. The reduction in the energy barrier from 34.22 (HOOH) to 15.36 kcal/mol (Cpd I) clearly establishes that the heme centre of CYPs significantly facilitates the oxygen transfer process due to its radical nature. Also, to compare with different substrates, the energy barrier for the epoxidation of ethane by Cpd I was reported to be 14.9 kcal/mol108 and epoxidation of furan was reported to be 12.33 kcal/mol using BS1.59 This comparison establishes that thiophene ring epoxidation is relatively more energy demanding (15.36 kcal/mol). Comparison of energy barriers for S-oxidation and epoxidation reactions of 1, (Figure 7) indicates that the epoxidation process is marginally more preferred (by 1.44 kcal/mol) as per kinetic factors. However, since the barriers are quite comparable, both the processes are observed experimentally. The epoxidation product is clearly more stable in comparison with the S-oxide product, hence thermodynamical factors favor the epoxidation reaction.


Page 16 of 40

Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Energy profile comparision between epoxidation and S-oxidation of thiophene in presence of Cpd I, on the doublet spin surface of Cpd I.

Reactions of Thiophene-S-oxide metabolite. Thiophene-S-oxide metabolite (TM1) can undergo two important reactions, (i) dimerization leading to the formation of a Diels-Alder adduct (D1), and (ii) nucleophilic attack by nucleophiles (Figure 8).

Ea=35.49 ∆G=-12.63

S O

D1

S O

MeO− ( MeOH)

Dimerization

3 4 MeNH2

Ea=43.81 ∆G=-8.53

S O

N H

MI1-NH

2 S O

S O

O

Ea=5.48 (54.06) ∆G=-44.43 (-4.73)

MI1-OH

1

TM1

MeS− (MeSH) S O

S

Ea=7.83 (59.93) ∆G=-26.43 (-3.86)

MI1-SH

Figure 8.Various reactions of thiophene-S-oxide (TM1) metabolite under biochemical conditions and free energies. The Ea and ∆G values are given in kcal/mol.

Dimerization: The dimerization of thiophene-S-oxide was reported with the help of mass spectral evidence.23,45 Two molecules of TM1 can undergo Diels-Alder reaction leading to the formation of S-oxide dimer (D1) (Figure 8). The C1 and C2 centres of the dienophilic TM1 attacks at the C1 and C4 centres of diene TM1, leading to the generation of Diels-Alder 17Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 40

adduct (D1). In the transition state TS3, bond lengths of the newly forming C---C bonds are 2.17 Å and 2.50 Å, confirming that the reaction follows a concerted, almost synchronous [4+2] cycloaddition path. The activation energy required for dimerization is 35.49 kcal/mol (TS3) (Figure 9), the dimer product is exergonic by 12.63 kcal/mol. Interestingly, this type of dimerization is energetically forbidden in thiophene (Ea=61.77 kcal/mol), also in furan (Ea=51.11 kcal/mol). The barrier via TS3 (35.49 kcal/mol) is comparable to that of simplest 1,3-diene and ethylene (34.60 kcal/mol). Nucleophilic Attack: The

electrophilicity

of

thiophene

increases

upon

S-oxidation.

The

global

electrophilicity (ω) of TM1 is 1.5 eV, much larger than that of thiophene 0.61 eV. Nucleophilic attack is one of the major reactions possible. Many nucleophilic amino acids are present in the active sites of cytochromes (threonine, serine, cysteine and NH2 centres). A few nucleophilic amino acids, which are proposed to be responsible for the covalent adduct formation, are present in the active sites of cytochromes leading to the inactivation of cytochromes, through MBI. Analysis of crystal structures of various isoforms of cytochromes (collected from the protein data bank), showed the presence of nucleophilic amino acids serine, threonine, cysteine, arginine and lysine in the vicinity (within 5 Å distance) of heme centre (Table S2, supporting information). Threonine is the most common amino acid in the cavity, the attack of nucleophilic amino acids at C1/C2 centres of TM1 can lead to covalent bond formation and thus cause MBI. To understand the structural and energetic factors associated with this reaction, quantum chemical analysis has been carried out, with model nucleophiles MeOH, MeSH and MeNH2.


Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. 3D structures of transition states (TS3, TS4-OH, TS4-SH, TS4-NH, TS4-O¯, TS4-S¯) in biochemical transformation of thiophene-S-oxide (TM1). All the bond lengths are in Angstroms (Å) and bond angles are in Degrees (°).

Experimentally the nucleophilic addition process at the thiophene-S-oxide was reported on C1 centre rather than at C2 centre. This observation is supported by the greater electrophilicity (ωk+=0.40 eV) at C1 centre in comparison the electrophilicity at C2 centre (ωk+= 0.19 eV) in TM1. Figure 9 shows the 3D structures of transition states (TS4-OH, SH,

NH) and covalent adducts for nucleophilic attack by MeOH, MeSH and MeNH2. The ∆G for the reaction between TM1 and methanol to give MI1-OH is found to be -4.73 kcal/mol energetically favorable. But the energy barrier for this reaction is found to be very high 54.06 kcal/mol. Similarly the ∆G for the reaction between TM1 and methanethiol to give MI1-SH is exergonic by 3.86 kcal/mol, with an energy barrier of 59.93 kcal/mol. This indicates that threonine/cys attack may not take place in their neutral forms. Under physiological conditions, base mediated threonine-O- ion formation is easy and thus attack by RO¯/RS¯ can be envisaged. The energy barrier for the reaction between TM1 and MeO¯ is estimated to be 5.48 kcal/mol. Similarly, the energy barrier for the reaction between TM1 and MeS¯ is 7.83 kcal/mol. Thus, it is clear that the neutral amino acids may not cause covalent bond formation 19Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 40

with thiophene-S-oxide metabolites. Ser/thr/cys can cause MBI using acid-base enzyme catalysis process, which generates their anions. When the S-oxide metabolite diffuses out of the cytochrome, it may be attacked by glutathione in its anionic state leading to glutathione adduct formation. The energy barrier required for the attack of methamine at the C1 centre of TM1 is 43.81 kcal/mol, leading to the formation of covalent product MI1-NH, which is exergonic by 8.53 kcal/mol. The NH2 groups are in the active sites of CYPs are not expected to cause MBI, because of high barrier.

Reactions of Thiophene epoxide metabolite. Ring opening of epoxides has been a subject of interest to experimental and theoretical chemists.59,111–116 Ethylene oxide or oxirane is a three-membered oxygen-containing ring, which is highly strained, electrophilic and easily undergoes ring opening. The epoxide ring opening reactions of furan epoxide using model nucleophilies and water leading to covalent adduct formation was studied by Taxak and Bharatam using quantum chemical methods.59 Various possible pathways have been proposed in the literature for the inactivation of thiophene epoxide by cytochrome,7,117 but the mechanisms involved are vaguely described and the covalent adducts involved in the MBI are not understood with atomic level details. The thiophene epoxide TM2 can undergo many varieties of reactions – isomerization, hydrolysis and nucleophilic attack. The MBI and associated toxicities can occur from isomers / products. In this section, the isomerization of

TM2 and its consequences will be discussed first. Subsequently, the hydrolysis and nucleophilic reaction on TM2 will be presented, in that order.


Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


OMe Ea=10.50 (28.38) G=-42.79 (0.74)

MeOH)

TI2

S OH MI2-OH

Ea=14.57 G=-3.17

O

MeS (MeSH)

S MI2-SH

S O

E=-6.98

SMe Ea=16.11 (26.72) G=-37.70 (-1.48)

O

S MeO

TM1

OH S

NHMe

H2O

TI1 Ring Opening

S MI2-NH

OH

E=-7.70

S

1,3-H shift

S

E=-28.05 1,2-H shift

TI3 E=-25.37

Ea=51.44

OH SH

G=-34.43

MI4

4 S

Ea=12.97 G=-24.12

S O O HOH TM3

S-oxidation

O

MeS (MeSH)

S

1

S MI5-SH

TM2

OH 1,5-H shift S E=-11.03 TI5 1,3-H shift

Ea=9.58 (35.42) G=-29.21(-12.84)

OH O

Ea=15.98(36.36) G=-8.68(-11.72)

OH

MeCOOH

NH S MI5-NH

E=-14.74 S TI7

S HO

O S MI5-OH

2

1,2-H shift

MI3

O OH

MeO MeOH)

3

Ea=36.06 G=-10.69

OH

MeNH2

TI4

S OH MI5-H2O

E=16.70

Ea=14.57 G=-11.36

MeNH2

OH

O

O

OH

Ea=42.89 G=-14.54

OH S

Ea=14.53 G=-11.45 O COMe

MI6

S O TI6

Figure 10. Various reactions of thiophene epoxide (TM2) metabolites under biochemical conditions and free energies. The Ea and ∆G values are given in kcal/mol. 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Isomers:

TM1 is an isomer of TM2, it is less stable than TM2 by 16.70 kcal/mol, TM2 has many more structural isomers and tautomers TI1-TI7 (Figure 10), most of them are reported to be relevant in toxicity and metabolism studies.7 Initially, it is worth establishing the relative energies of all the isomers. Quantum chemical analysis showed that the isomers TI1-

TI7 are more stable than TM2, the relative energy values (∆E) are given in Figure 10. Rearrangement of thiophene epoxide TM2 due to the opening of both the five membered and three membered rings, leads to the generation of the isomer TI1 (cis-2-butene1-al-4-thione). TI1 formation has been reported in a few carcinogenicity experiments.25,28,45,52 This can happen without the involvement of cytochrome. TM2 to TI1 isomerization is an exergonic process by 11.36 kcal/mol, involves a barrier of 14.57 kcal/mol (via TS5). The anomeric effect (negative hyper conjugation) originating from the lone pairs of electrons of the bridging oxygen atom of TM2 (breaking of S-C1 bond) may be the trigger for this rearrangement. TI1 is highly electrophilic in nature, with high global electrophilicity index (ω = 4.85), much larger than that of TM2 (ω = 1.23). Nucleophilic residues or glutathione can attack at the C2 position of TI1 and produce C2 adducts, because local electrophilicity at C2 position is maximum (ωc+=0.40) in comparison to that in other positions. Similar to reactions on TM1, the energy barriers for the nucleophilic attack reactions on TI1 can also be estimated using model nucleophiles MeOH, MeSH, MeO¯, MeS¯ and MeNH2. In this case also the reactions happening with neutral nucleophiles (MeOH and MeSH) require very high barriers hence they should not be expected. Reactions happening under acid-base catalysis conditions (i.e. with RX¯) are quite favourable. Figure 10 shows that these reactions are exergonic and barriers are small (10.50, 16.11 and 14.57 kcal/mol for MeO¯, MeS¯ and MeNH2 respectively). Figure 11 shows the 3D structures of transition states and covalent adducts. Isomer TI2 adopts trans arrangement across the central alkene unit in TI1, it is about


Page 22 of 40

Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7 kcal/mol more stable than TI1. TI2 and its reactions are not yet noticed under experimental conditions, presumably due to the high energy of the rotation across central alkene unit. Also, nucleophilic attack at C2 in TI2 cannot lead to cyclization as noticed. The isomers TI3 and TI4 are also more stable than TM2 but never considered in previous studies. Formation of TI3 from TM2 can be envisaged through an acid catalyzed 1,2-H shift process, which can break the C2-O bond. TI3 is an alcohol, which is more stable than TM2 by 28.05 kcal/mol, it can tautomerize to a more stable (35.75 kcal/mol) keto isomer TI4. TI5 is an isomer similar to TI3, which can also be formed from TM2 in an acid catalyzed 1,2-H shift process. TI6 is a keto isomer of the enol TI5, it is more stable by 11.03 kcal/mol. TI7 is another isomer of TM2, which is the most stable isomer (-40.10 kcal/mol) among all. It can be formed by a 1,5-hydrogen shift in TI5 (with an energy difference of 14.73 kcal/mol). TI7 can undergo hydrolysis directly or upon initial S-oxidation.7,21,33 The free energy change associated with the hydrolysis of TI7 is 34.43 kcal/mol (exergonic) with an energy barrier of 51.40 kcal/mol (via TS7). Upon initial S-oxidation, the same reaction is exergonic by 24.12 kcal/mol, with low energy barrier of 12.97 kcal/mol (via TS8). Hydrolysis processes similar to these reactions are reported to yield disulphide metabolites.7,21,31,33,50 The reactions of the isomers TI4 and TI6 are also expected to be quite similar to that of TI7, the energy profiles of which are provided in the supporting information. Experimentally, the reaction products of TI4 are not reported, it is worth exploring the same.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 11. 3D structures of various transition states involves in biochemecal metabolism of thiophene epoxide (TM2). Distances are in angstroms (Å) units and angles are in degree (°).


Page 24 of 40

Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hydrolysis: Thiophene epoxide metabolite (TM2) can directly undergo hydrolysis reaction by water molecule(s) in the active site. This hydrolysis results in the formation of vicinal diols, which are generally non-toxic in nature. This reaction may occur due to the direct attack of the water molecule on the C1/C2 centres of epoxide ring. The electrophilicity calculations reveal that the C1 carbon center is more electrophilic than the C2 carbon center of TM2, and hence, hydrolysis at C1 is expected to be relatively more easily feasible. The energy barrier for the hydrolysis of TM2 by water is estimated to be 36.06 kcal/mol. In comparison, the energy barrier for a similar model reaction on oxirane requires a barrier of 39.24 kcal/mol, indicating that the thiophene ring in TM2, is marginally reducing the barrier for direct epoxide ring opening by water. The thiophene diol product (MI5-H2O) formation is exergonic by -10.69 kcal/mol, but the barrier is very high (36.06 kcal/mol) and hence, the reaction needs to be assisted by the residues in the cavity of an enzyme. It may be concluded that direct hydrolysis by water cannot be expected in TM2. Theoretical studies on the oxirane ring opening by a model epoxidase environment suggested that the barrier gets reduced to 10.4 kcal/mol due to participation of tyrosine group.113 Nucleophilic Attack: Inside the cavity of cytochromes, several nucleophilic residues are present which may attack the epoxide ring either in their neutral/ionic states. The free energy change associated with the reaction between TM2 and MeOH is found to be exergonic by 12.84 kcal/mol, but the energy barrier for this reaction is found to be high 35.42 kcal/mol (via TS9-OH). This indicates that serine/threonine attack may not take place in their neutral form. Under biological conditions, base mediated serine-O¯/threonine-O¯ ion formation is easy. The free energy change associated with the reaction of TM2 with MeO¯ is exergonic process by 29.21 kcal/mol and the corresponding activation energy is low (9.58 kcal/mol via TS9-O¯). This



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

indicates that the serine/threonine of CYP in their ionic state can react easily with the thiophene epoxide metabolite, forming O-C covalent bond. The nucleophilic attack may takes place immediately after the formation of the epoxide, while the oxygen of epoxide continues to hold onto the Fe centre of the heme porphyrin moiety. Hence, the quantum chemical analysis of TM2 with MeOH and MeO¯ was carried out, while keeping the Cpd I environment intact. When TM2 is coordinated to the Fe centre via O---Fe interaction, the activation barrier for MeO¯ attack is further reduced to 6.41 kcal/mol. Hence, it can be concluded that inside the cavity of the enzyme, the thiophene epoxide metabolite can react with serine-O¯/threonine-O¯ centre very easily producing O-C covalent bond formation, thus leading to MBI. Barriers for the reaction of TM2 with methylamine and methanethiol are very high (21.89, 33.36 kcal/mol respectively). The barrier for the reaction between TM2 and MeS¯ is reduced to 15.98 kcal/mol (via TS9-S¯). This analysis confirms that the nucleophilic attack by cys/glutathione on thiophene epoxide might be taking place in the corresponding ionic states. The amine groups in the active sites are probably not involved in the covalent bond formation because of high barriers. TM2 may also undergoes nucleophilic attack by COO¯ group of amino acids. This may happen with the help of additional hydrogen donor centre at the oxygen of the epoxide ring as in acid catalysis of oxirane.111 Quantum chemical analysis suggested that the energy barrier for the MeCOO¯ attack on TM2 is estimated to be 14.53 kcal/mol (in the presence of H-donor MeCOOH, TS10), the reaction is exergonic by 11.45 kcal/mol such a reaction can also lead to MBI.


Page 26 of 40

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CONCLUSIONS Quantum chemical analysis was carried out to elucidate the mechanistic details of the metabolic pathways of thiophene ring using simplest thiophene ring as a model. The biochemical mechanisms of S-oxidation and epoxidation involved direct oxygen transfer (least motion path) to the nucleophilic S-atom of the thiophene ring and to the double bond center respectively. The epoxidation process is more favorable (barrier of 13.23 kcal/mol) than the S oxidation process (barrier of 14.75 kcal/mol). Moreover, the epoxide metabolite was observed to be relatively more stable (-23.24 kcal/mol) than S-oxide metabolite (−8.08 kcal/mol) indicating that epoxidation is more preferred as compared to S-oxidation on thermodynamic as well as kinetic counts. The S-oxide metabolite can undergo dimerization (Ea= 35.49 kcal/mol). The nucleophilic attack on the S-oxide metabolite takes place preferably at the C1 centre. This attack by a nucleophile in its neutral state requires high barrier, and hence, it is not expected. Anionic nucleophiles reduce the barrier. Hence, the nucleophilic attack on the S-oxide metabolite by ser/thr in their anionic states is expected to facilitate MBI formation. The epoxide metabolite can isomerize to produce many isomers, which are more stable and very reactive. The isomer TI1 is the most electrophilic species, can lead to the toxic effects by forming mechanism based inhibitor with the help of ionic amino acid complex with lower energy barriers. The thiolactone isomers which may be formed via the initial generation of hydroxy thiophene are highly reactive, leading to more stable thioles/sulfenic acids via hydrolysis (requires ~12-13 kcal/mol), eventually leading to disulfide adducts and thus leading to MBI. The epoxide metabolite can also undergo direct covalent bond formation through a nucleophilic addition reaction. The barriers for the same are very high when neutral nucleophiles are employed. However, this nucleophilic addition reaction becomes highly



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

favorable when the anionic states of the nucleophiles (MeO¯ and MeS¯) are considered. The association of the epoxide ring with the heme porphine ring further facilitates the nucleophilic attack. This work helps in tracing the energy profiles, the S-oxidation vs epoxidation reactions of thiophene ring and also helped in tracing the reaction profiles of the reactive metabolites. This work also helped in quantitatively estimating the reaction profiles leading to MBI formation.


Page 28 of 40

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABBREVATIONS TM, Toxic metabolite; CYP450, Cytochrome P450; MBI, Mechanism Based Inhibition; TA, Tienilic Acid; DFT, Density Functional Theory; BS, Basis Set; Cpd I, Compound I; PE, Potential Energy, NSAID, Nonsteroidal Anti-Inflammatory Drug; B3LYP, Becke, threeparameter, Lee-Yang-Parr; IEFPCM, Integral Equation Formalism Polarizable Continuum Model; NBO, Natural Bond Analysis; SOM, Site Of Metabolism; RC Reaction Complex; TS, Transition State; PC, Product Complex; MI, Mechanism based Inhibitory Complex; TI, Thiophene epoxide Isomer.

ACKNOWLEDGEMENT CJ and PVB thank the Department of Biotechnology (DBT), New Delhi for financial support. NT thanks the Department of Science and Technology (DST), New Delhi for providing Inspire fellowship.

SUPPORTING INFORMATION Complete details of electrophilicity index of thiophene epoxide isomers, nucleophilic residues present in the active site of in CYP 3A4, 2C9 and 2D6, docking poses of tienilic acid, 3D structure of transition states of dimerization, hydrolysis of TI6, TI7 and TM3. Cartesian coordinates, Gibbs free energies and docking details, structures of drugs/experimental leads used for the docking for all the structures discussed in the text. This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author * Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER),

Sector – 67, S. A. S. Nagar (Mohali), 160 062 Punjab, India

Telephone: +91 172 2292018; Fax: +91 172 2214692. E-mail: [email protected] 29Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Funding We gratefully acknowledge the financial support of the Department of Biotechnology, New Delhi.

Notes The authors declare no competing financial interest


Page 30 of 40

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Kalgutkar, A. S., and Dalvie, D. (2015) Predicting Toxicities of Reactive Metabolite– Positive Drug Candidates. Annu. Rev. Pharmacol. Toxicol. 55, 35–54. (2) Orr, S. T. M., Ripp, S. L., Ballard, T. E., Henderson, J. L., Scott, D. O., Obach, R. S., Sun, H., and Kalgutkar, A. S. (2012) Mechanism-based inactivation (MBI) of cytochrome P450 enzymes: structure-activity relationships and discovery strategies to mitigate drug-drug interaction risks. J. Med. Chem. 55, 4896–933. (3) Stachulski, A. V., Baillie, T. A., Kevin Park, B., Scott Obach, R., Dalvie, D. K., Williams, D. P., Srivastava, A., Regan, S. L., Antoine, D. J., Goldring, C. E. P., Chia, A. J. L., Kitteringham, N. R., Randle, L. E., Callan, H., Castrejon, J. L., Farrell, J., Naisbitt, D. J., Lennard, M. S., Park, B. K., Obach, R. S., Dalvie, D. K., Williams, D. P., Srivastava, A., Regan, S. L., Antoine, D. J., Goldring, C. E. P., Chia, A. J. L., Kitteringham, N. R., Randle, L. E., Callan, H., Castrejon, J. L., Farrell, J., Naisbitt, D. J., and Lennard, M. S. (2013) The Generation, Detection, and Effects of Reactive Drug Metabolites. Med. Res. Rev. 33, 985– 1080. (4) Guengerich, F. P., and MacDonald, J. S. (2007) Applying mechanisms of chemical toxicity to predict drug safety. Chem. Res. Toxicol. 20, 344–369. (5) Walsh, J. S., and Miwa, G. T. (2011) Bioactivation of drugs: risk and drug design. Annu. Rev. Pharmacol. Toxicol. 51, 145–167. (6) Park, B. K., Boobis, A., Clarke, S., Goldring, C. E. P., Jones, D., Kenna, J. G., Lambert, C., Laverty, H. G., Naisbitt, D. J., Nelson, S., Nicoll-Griffith, D., Obach, R. S., Routledge, P., Smith, D. A., Tweedie, D. J., Vermeulen, N., Williams, D. P., Wilson, I. D., and Baillie, T. A. (2011) Managing the challenge of chemically reactive metabolites in drug development. Nat. Rev. Drug Discov. 10, 292–306. (7) Gramec, D., Maši, L. P., and Dolenc, M. S. (2014) Bioactivation Potential of Thiophene Containing Drugs. Chem. Res. Toxicol. 27, 1344–1358. (8) Chen, W., Caceres-Cortes, J., Zhang, H., Zhang, D., Humphreys, W. G., and Gan, J. (2011) Bioactivation of substituted thiophenes including α-chlorothiophene-containing compounds in human liver microsomes. Chem. Res. Toxicol. 24, 663–669. (9) Joshi, E. M., Heasley, B. H., Chordia, M. D., and Macdonald, T. L. (2004) In Vitro Metabolism of 2-Acetylbenzothiophene: Relevance to Zileuton Hepatotoxicity. Chem. Res. Toxicol. 17, 137–143. (10) Valadon, P., Dansette, P. M., Girault, J. P., Amar, C., and Mansuy, D. (1996) Thiophene Sulfoxides as Reactive Metabolites: Formation upon Microsomal Oxidation of a 3Aroylthiophene and Fate in the Presence of Nucleophiles in Vitro and in Vivo. Chem. Res. Toxicol. 9, 1403–1413. (11) Wang, M., Brand-schieber, E., Zand, B., Nguyen, X., Falck, J. R., Balu, N., and Schwartzman, M. L. (1998) Cytochrome P450-Derived Arachidonic Acid Metabolism in the Rat Kidney : Characterization of Selective Inhibitors. Pharmacology 284, 966 –973.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(12) Hutzler, J. M., Balogh, L. M., Zientek, M., Kumar, V., and Tracy, T. S. (2009) Mechanism-based inactivation of cytochrome P450 2C9 by tienilic acid and (+/-)-suprofen: A comparison of kinetics and probe substrate selection. Drug Metab. Dispos. 37, 59–65. (13) O’Donnell, J. P., Dalvie, D. K., Kalgutkar, A. S., and Obach, R. S. (2003) Mechanismbased inactivation of human recombinant P4502C9 by the nonsteroidal anti-inflammatory drug suprofen. Drug Metab. Dispos. 31, 1369–1377. (14) Hart, D., Ward, M., and Lifschitz, M. D. (1987) Suprofen-Related NephrotoxicityA Distinct Clinical Syndrome. Ann. Intern. Med. 106, 235–238. (15) Wolfe. S. M. (1987) Suprofen-Induced Transient Flank Pain and Renal Failure. N. Engl. J. Med. 316, 1025–1026. (16) Bonierbale, E., Valadon, P., Pons, C., Desfosses, B., Dansette, P. M., and Mansuy, D. (1999) Opposite behaviors of reactive metabolites of tienilic acid and its isomer toward liver proteins: Use of specific anti-tienilic acid-protein adduct antibodies and the possible relationship with different hepatotoxic effects of the two compounds. Chem. Res. Toxicol. 12, 286–296. (17) Mancy, A., Broto, P., Dijols, S., Dansette, P. M., and Mansuy, D. (1995) The substrate binding site of human liver cytochrome P450 2C9: An approach using designed tienilic acid derivatives and molecular modeling. Biochemistry 34, 10365–10375. (18) Melet, A., Assrir, N., Jean, P., Pilar Lopez-Garcia, M., Marques-Soares, C., Jaouen, M., Dansette, P. M., Sari, M. A., and Mansuy, D. (2003) Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid. Arch. Biochem. Biophys. 409, 80–91. (19) Lecoeur, S., Bonierbale, E., Challine, D., Gautier, J., Valadon, P., Dansette, P. M., Catinot, R., Ballet, F., Mansuy, D., and Beaune, P. H. (1994) Specificity of in Vitro Covalent Binding of Tienilic Acid Metabolites to Human Liver Microsomes in Relationship to the Type of Hepatotoxicity: Comparison with Two Directly Hepatotoxic Drugs. Chem. Res. Toxicol. 7, 434–442. (20) Shimizu, S., Atsumi, R., Nakazawa, T., Fujimaki, Y., Sudo, K., and Okazaki, O. (2009) Metabolism of ticlopidine in rats: Identification of the main biliary metabolite as a glutathione conjugate of ticlopidine S-oxide. Drug Metab. Dispos. 37, 1904–1915. (21) Tantry, U. S., Kereiakes, D. J., and Gurbel, P. A. (2011) Clopidogrel and proton pump inhibitors: influence of pharmacological interactions on clinical outcomes and mechanistic explanations. Cardiovasc. Interv. 4, 365–80. (22) Walgren, J. L., Mitchell, M. D., and Thompson, D. C. (2005) Role of metabolism in drug-induced idiosyncratic hepatotoxicity. Crit. Rev. Toxicol. 35, 325–361. (23) Dansette, P. M., Bertho, G., and Mansuy, D. (2005) First evidence that cytochrome P450 may catalyze both S-oxidation and epoxidation of thiophene derivatives. Biochem. Biophys. Res. Commun. 338, 450–455. (24) Mercer, A. E., Regan, S. L., Hirst, C. M., Graham, E. E., Antoine, D. J., Benson, C. A., 32Environment ACS Paragon Plus

Page 32 of 40

Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Williams, D. P., Foster, J., Kenna, J. G., and Park, B. K. (2009) Functional and toxicological consequences of metabolic bioactivation of methapyrilene via thiophene S-oxidation: Induction of cell defence, apoptosis and hepatic necrosis. Toxicol. Appl. Pharmacol. 239, 297–305. (25) Graham, E. E., Walsh, R. J., Hirst, C. M., Maggs, J. L., Martin, S., Wild, M. J., Wilson, I. D., Harding, J. R., Kenna, J. G., Peter, R. M., Williams, D. P., and Park, B. K. (2008) Identification of the thiophene ring of methapyrilene as a novel bioactivation-dependent hepatic toxicophore. J. Pharmacol. Exp. Ther. 326, 657–671. (26) Ratra, G. S., Powell, C. J., Park, B. K., Maggs, J. L., and Cottrell, S. (2000) Methapyrilene hepatotoxicity is associated with increased hepatic glutathione, the formation of glucuronide conjugates, and enterohepatic recirculation. Chem. Biol. Interact. 129, 279– 295. (27) Ratra, G. S., Morgan, W. A., Mullervy, J., Powell, C. J., and Wright, M. C. (1998) Methapyrilene hepatotoxicity is associated with oxidative stress, mitochondrial disfunction and is prevented by the Ca2+ channel blocker verapamil. Toxicology 130, 79–93. (28) Hamadeh, H. K., Knight, B. L., Haugen, A. C., Sieber, S., Amin, R. P., Bushel, P. R., Stoll, R., Blanchard, K., Jayadev, S., Tennant, R. W., Cunningham, M. L., Afshari, C. A., and Paules, R. S. (2002) Methapyrilene toxicity: anchorage of pathologic observations to gene expression alterations. Toxicol. Pathol. 30, 470–482. (29) Pondence, C., Adams, D. A., and Perry, S. (1958) Sandosten. Br. Med. J. 2, 636. (30) Boulenc, X., Djebli, N., Shi, J., Perrin, L., Brian, W., and Horn, R. Van. (2012) Effects of Omeprazole and Genetic Polymorphism of CYP2C19 on the Clopidogrel Active Metabolite. Drug Metab. Dispos. 40, 187–197. (31) Pereillo, J. M., Maftouh, M., Andrieu, A., Uzabiaga, M. F., Fedeli, O., Savi, P., Pascal, M., Herbert, J. M., Maffrand, J. P., and Picard, C. (2002) Structure and stereochemistry of the active metabolite of clopidogrel. Drug Metab. Dispos. 30, 1288–1295. (32) Dansette, P. M., Libraire, J., Bertho, G., and Mansuy, D. (2009) Metabolic Oxidative Cleavage of Thioesters: Evidence for the Formation of Sulfenic Acid Intermediates in the Bioactivation of the Antithrombotic Prodrugs Ticlopidine and Clopidogrel. Chem. Res. Toxicol. 22, 369–373. (33) Dansette, P. M., Rosi, J., Bertho, G., and Mansuy, D. (2012) Cytochromes P450 catalyze both steps of the major pathway of clopidogrel bioactivation, whereas paraoxonase catalyzes the formation of a minor thiol metabolite isomer. Chem. Res. Toxicol. 25, 348–356. (34) Angiolillo, D. J., Gibson, C. M., Cheng, S., Ollier, C., Nicolas, O., Bergougnan, L., Perrin, L., LaCreta, F. P., Hurbin, F., and Dubar, M. (2011) Differential effects of omeprazole and pantoprazole on the pharmacodynamics and pharmacokinetics of clopidogrel in healthy subjects: randomized, placebo-controlled, crossover comparison studies. Clin. Pharmacol. Ther. 89, 65–74. (35) Meunier, B., de Visser, S. P., and Shaik, S. (2004) Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes. Chem. Rev. 104, 3947–3980.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(36) Potashman, M. H., and Duggan, M. E. (2009) Covalent Modifiers : An Orthogonal Approach to Drug Design. J. Med. Chem. 52, 1231–1246. (37) Tang, D., Zhu, L., and Hu, C. (2012) Elucidating active species and mechanism of the direct oxidation of benzene to phenol with hydrogen peroxide catalyzed by vanadium-based catalysts using DFT calculations. RSC Adv. 2, 2329. (38) Zhu, Y., Zhou, J., and Jiao, B. (2013) Deuterated clopidogrel analogues as a new generation of antiplatelet agents. ACS Med. Chem. Lett. 4, 349–352. (39) Mansuy, D., Valadon, P., Erdelmeier, I., Lopez-Garcia, P., Amar, C., Girault, J. P., and Dansette, P. M. (1991) Thiophene S-oxides as new reactive metabolites: formation by cytochrome P-450 dependent oxidation and reaction with nucleophiles. J. Am. Chem. Soc. 113, 7825–7826. (40) Dansette, P. M., Thang, D. C., and Mansuy, D. (1992) Evidence for thiophene-s-oxide as a primary reactive metabolite of thiophene in vivo: Formation of a dihydrothiophene sulfoxide mercapturic acid. Biochem. Biophys. Res. Commun. 186, 1624–1630. (41) Kalgutkar, A. S., Gardner, I., Obach, R. S., Shaffer, C. L., Callegari, E., Henne, K. R., Mutlib, A. E., Dalvie, D. K., Lee, J. S., Nakai, Y., O’Donnell, J. P., Boer, J., and Harriman, S. P. (2005) A Comprehensive Listing of Bioactivation Pathways of Organic Functional Groups. Curr. Drug Metab. 6, 161–225. (42) Rademacher, P. M., Woods, C. M., Huang, Q., Szklarz, G. D., and Nelson, S. D. (2012) Differential oxidation of two thiophene-containing regioisomers to reactive metabolites by cytochrome P450 2C9. Chem. Res. Toxicol. 25, 895–903. (43) Koenigs, L. L., Peter, R. M., Hunter, A. P., Haining, R. L., Rettie, A. E., Friedberg, T., Pritchard, M. P., Shou, M., Rushmore, T. H., and Trager, W. F. (1999) Electrospray Ionization Mass Spectrometric Analysis of Intact Cytochrome P450: Identification of Tienilic Acid Adducts to P450 2C9. Biochemistry 38, 2312–2319. (44) López-Garcia, M. P., Dansette, P. M., and Mansuy, D. (1994) Thiophene derivatives as new mechanism-based inhibitors of cytochromes P-450: inactivation of yeast-expressed human liver cytochrome P-450 2C9 by tienilic acid. Biochemistry 33, 166–175. (45) Treiber, A., Dansette, P. M., El Amri, H., Girault, J. P., Ginderow, D., Mornon, J. P., and Mansuy, D. (1997) Chemical and Biological Oxidation of Thiophene: Preparation and Complete Characterization of Thiophene S-Oxide Dimers and Evidence for Thiophene SOxide as an Intermediate in Thiophene Metabolism in Vivo and in Vitro. J. Am. Chem. Soc. 119, 1565–1571. (46) Belghazi, M., Jean, P., Poli, S., Schmitter, J. M., Mansuy, D., and Dansette, M. P. (2001) Use of Isotopes and LC-MS-ESI-TOF for Mechanistic Studies of Tienilic Acid Metabolic Activation, in Biological Reactive Intermediates VI SE - 17 (Dansette, P., Snyder, R., Delaforge, M., Gibson, G. G., Greim, H., Jollow, D., Monks, T., and Sipes, I. G., Eds.), pp 139–144. Springer US. (47) Coen, M., Rademacher, P. M., Zou, W., Scott, M., Ganey, P. E., Roth, R., and Nelson, S. D. (2012) Comparative NMR-based metabonomic investigation of the metabolic phenotype


Page 34 of 40

Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


associated with tienilic acid and tienilic acid isomer. Chem. Res. Toxicol. 25, 2412–2422. (48) Medower, C., Wen, L., and Johnson, W. W. (2008) Cytochrome P450 Oxidation of the Thiophene-Containing Anticancer Drug 3-[(Quinolin-4-ylmethyl)-amino]-thiophene-2carboxylic Acid (4-Trifluoromethoxy-phenyl)-amide to an Electrophilic Intermediate. Chem. Res. Toxicol. 21, 1570–1577. (49) Lu, P., Schrag, M. L., Slaughter, D. E., Raab, C. E., Shou, M., and Rodrigues, D. (2003) Mechanism-based inhibition of human liver microsomal cytochrome P450 1A2 by zileuton, A 5-lipoxygenase inhibitor. Drug Metab. Dispos. 31, 1352–1360. (50) Zhu, Y., and Zhou, J. (2013) In vitro biotransformation studies of 2-oxo-clopidogrel: Multiple thiolactone ring-opening pathways further attenuate prodrug activation. Chem. Res. Toxicol. 26, 179–190. (51) Braga, R. C., Alves, V. M., Fraga, C. M., Barreiro, E. J., de Oliveira, V., and Andrade, C. H. (2012) Combination of docking, molecular dynamics and quantum mechanical calculations for metabolism prediction of 3,4-methylenedioxybenzoyl-2-thienylhydrazone. J. Mol. Model. 18, 2065–2078. (52) Taxak, N., Dixit, V. A., and Bharatam, P. V. (2012) Density Functional Study on the Cytochrome-Mediated S-Oxidation: Identification of Crucial Reactive Intermediate on the Metabolic Path of Thiazolidinediones. J. Phys. Chem. A 116, 10441–10450. (53) Taxak, N., Patel, B., and Bharatam, P. V. (2013) Carbene generation by cytochromes and electronic structure of heme-iron-porphyrin-carbene complex: A quantum chemical study. Inorg. Chem. 52, 5097–5109. (54) Taxak, N., Chaitanya Prasad, K., and Bharatam, P. V. (2013) Mechanistic insights into the bioactivation of phenacetin to reactive metabolites: A DFT study. Comput. Theor. Chem. 1007, 48–56. (55) Taxak, N., Parmar, V., Patel, D. S., Kotasthane, A., and Bharatam, P. V. (2011) Soxidation of thiazolidinedione with hydrogen peroxide, peroxynitrous acid, and C4ahydroperoxyflavin: a theoretical study. J. Phys. Chem. A 115, 891–898. (56) Taxak, N., Desai, P. V, Patel, B., Mohutsky, M., Klimkowski, V. J., Gombar, V., and Bharatam, P. V. (2012) Metabolic-intermediate complex formation with cytochrome P450: theoretical studies in elucidating the reaction pathway for the generation of reactive nitroso intermediate. J. Comput. Chem. 33, 1740–1747. (57) Dixit, V. A., and Bharatam, P. V. (2011) Toxic metabolite formation from Troglitazone (TGZ): new insights from a DFT study. Chem. Res. Toxicol. 24, 1113–1122. (58) Arfeen, M., Patel, D. S., Abbat, S., Taxak, N., and Bharatam, P. V. (2014) Importance of cytochromes in cyclization reactions: Quantum chemical study on a model reaction of proguanil to cycloguanil. J. Comput. Chem. 35, 2047–2055. (59) Taxak, N., Kalra, S., and Bharatam, P. V. (2013) Mechanism-based inactivation of cytochromes by furan epoxide: unraveling the molecular mechanism. Inorg. Chem. 52, 13496–13508.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(60) Hirao, H., Chuanprasit, P., Cheong, Y. Y., and Wang, X. (2013) How is a metabolic intermediate formed in the mechanism-based inactivation of cytochrome P450 by using 1,1dimethylhydrazine: hydrogen abstraction or nitrogen oxidation? Chem. A Eur. J. 19, 7361– 7369. (61) Hirao, H., Cheong, Z. H., and Wang, X. (2012) Pivotal role of water in terminating enzymatic function: a density functional theory study of the mechanism-based inactivation of cytochromes P450. J. Phys. Chem. B 116, 7787–7794. (62) Li, C., Zhang, L., Zhang, C., Hirao, H., Wu, W., and Shaik, S. (2007) Which oxidant is really responsible for sulfur oxidation by cytochrome P450? Angew. Chem. Int. Ed. Engl. 46, 8168–8170. (63) Balding, P. R., Porro, C. S., McLean, K. J., Sutcliffe, M. J., Maréchal, J.-D., Munro, A. W., and de Visser, S. P. (2008) How do azoles inhibit cytochrome P450 enzymes? A density functional study. J. Phys. Chem. A 112, 12911–12918. (64) de Visser, S. P., Valentine, J. S., and Nam, W. (2010) A biomimetic ferric hydroperoxo porphyrin intermediate. Angew. Chem. Int. Ed. Engl. 49, 2099–2101. (65) de Visser, S. P., Kumar, D., Cohen, S., Shacham, R., and Shaik, S. (2004) A predictive pattern of computed barriers for C-H hydroxylation by compound I of cytochrome p450. J. Am. Chem. Soc. 126, 8362–8363. (66) de Visser, S. P., and Shaik, S. (2003) A proton-shuttle mechanism mediated by the porphyrin in benzene hydroxylation by cytochrome p450 enzymes. J. Am. Chem. Soc. 125, 7413–7424. (67) Kumar, D., Sastry, G. N., and de Visser, S. P. (2011) Effect of the axial ligand on substrate sulfoxidation mediated by iron(IV)-oxo porphyrin cation radical oxidants. Chemistry 17, 6196–6205. (68) Kumar, D., Tahsini, L., de Visser, S. P., Kang, H. Y., Kim, S. J., and Nam, W. (2009) Effect of porphyrin ligands on the regioselective dehydrogenation versus epoxidation of olefins by oxoiron(IV) mimics of cytochrome P450. J. Phys. Chem. A 113, 11713–11722. (69) Kirchmair, J., Williamson, M. J., Tyzack, J. D., Tan, L., Bond, P. J., Bender, A., and Glen, R. C. (2012) Computational prediction of metabolism: sites, products, SAR, P450 enzyme dynamics, and mechanisms. J. Chem. Inf. Model. 52, 617–648. (70) Maestro, version 9.3, Schrödinger, LLC, New York, NY, 2012. (71) LigPrep, version 2.5, Schrödinger, LLC, New York, NY, 2012. (72) Yano, J. K., Wester, M. R., Schoch, G. A., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2004) The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-Å resolution. J. Biol. Chem. 279, 38091–38094. (73) Wester, M. R., Yano, J. K., Schoch, G. A., Yang, C., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2004) The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-Å resolution. J. Biol. Chem. 279, 35630–35637. (74) Wang, A., Stout, C. D., Zhang, Q., and Johnson, E. F. (2015) Contributions of Ionic


Page 36 of 40

Page 37 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Interactions and Protein Dynamics to Cytochrome P450 2D6 (CYP2D6) Substrate and Inhibitor Binding. J. Biol. Chem. 290 , 5092–5104. (75) Reynald, R. L., Sansen, S., Stout, C. D., and Johnson, E. F. (2012) Structural characterization of human cytochrome P450 2C19: Active site differences between P450s 2C8, 2C9, and 2C19. J. Biol. Chem. 287, 44581–44591. (76) Schrödinger Suite 2013 Protein Preparation Wizard; Epik version 2.4, Schrödinger, LLC, New York, NY, 2013; Impact version 5.9, Schrödinger, LLC, New York, NY, 2013; Prime version 3.2, Schrödinger, LLC, New York, NY, 2013. Schrödinger Suite 2013 Protein Preparation Wizard; Epik version 2.4, Schrödinger, LLC, New York, NY, 2013; Impact version 5.9, Schrödinger, LLC, New York, NY, 2013; Prime version 3.2, Schrödinger, LLC, New York, NY, 2013. (77) Glide, version 5.8, Schrödinger, LLC, New York, NY, 2012. (78) Gaussian09, B.01, R., M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. S., M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. M., G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. H., A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. H., M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. N., Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, J., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. B., K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. N., K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. T., M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. C., V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. S., O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. O., R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. V., P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. D., O. Farkas, J. B. Foresman, J. V. Ortiz, J. C., and Fox, and D. J. (2010) Gaussian 09, Revision B.01. Gaussian, Inc., Wallingford CT. (79) Lee, C., Yang, W., and Parr, R. G. (1988) Development of the Colle-Salvetti correlationenergy formula into a functional of the electron density. Phys. Rev. B 37, 785–789. (80) Becke, A. D. (1993) Density functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652. (81) Becke, A. D. (1988) Density functional exchange energy approximation with correct asymptotic behavior. Phys. Rev. A 38, 3098–3100. (82) Hehre, W. J., Ditchfield, R., and Pople, J. A. (1972) Self Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian-Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules. J. Chem. Phys. 56, 2257. (83) Francl, M. M., Pietro, W. J., Hehre, W. J., Binkley, J. S., Gordon, M. S., DeFrees, D. J., and Pople, J. A. (1982) Self consistent molecular orbital methods. XXIII. A polarization type basis set for second row elements. J. Chem. Phys. 77, 3654–3665. (84) Scott, A. P., and Radom, L. (1996) Harmonic Vibrational Frequencies: An Evaluation of Hartree−Fock, Møller−Plesset, Quadratic Configuration Interaction, Density Functional Theory, and Semiempirical Scale Factors. J. Phys. Chem. 100, 16502–16513. (85) Schafer, A., Horn, H., and Ahlrichs, R. (1992) Fully Optimized Contracted GaussianBasis Sets for Atoms Li to Kr. J. Chem. Phys. 97, 2571–2577.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(86) Krishnan, R., Binkley, J. S., Seeger, R., and Pople, J. A. (1980) Self consistent molecular orbital methods. XX. A basis set for correlated wave functions. J. Chem. Phys. 72, 650–654. (87) Tomasi, J., Mennucci, B., and Cammi, R. (2005) Quantum mechanical continuum solvation models. Chem. Rev. 105, 2999–3093. (88) Reed, A. E., Weinstock, R. B., and Weinhold, F. (1985) Natural population analysis. J. Chem. Phys. 83, 735–746. (89) Chattaraj, P. K., Chattaraj, P. K., Sarkar, U., Sarkar, U., and Roy, D. R. (2006) Electrophilicity Index. Chem. Rev. 106, 2065–2091. (90) Bach, R. D., McDouall, J. J. W., Owensby, A. L., and Schlegel, H. B. (1990) Theoretical study of oxygen atom transfer. The role of electron correlation. J. Am. Chem. Soc. 112, 7065– 7067. (91) Bach, R. D., Owensby, A. L., Gonzalez, C., Schlegel, H. B., and McDouall, J. J. W. (1991) Nature of the transition structure for oxygen atom transfer from a hydroperoxide. Theoretical comparison between water oxide and ammonia oxide. J. Am. Chem. Soc. 113, 6001–6011. (92) Geletii, Y. V, Musaev, D. G., Khavrutskii, L., and Hill, C. L. (2003) Peroxynitrite Reactions with Dimethylsulfide and Dimethylselenide: An Experimental Study. J. Phys. Chem. A 108, 289–294. (93) Musaev, D. G., Geletii, Y. V, and Hill, C. L. (2003) Theoretical Studies of the Reaction Mechanisms of Dimethylsulfide and Dimethylselenide with Peroxynitrite. J. Phys. Chem. A 107, 5862–5873. (94) Bach, R. D., Dmitrenko, O., and Estévez, C. M. (2003) Theoretical Analysis of Peroxynitrous Acid: Characterization of Its Elusive Biradicaloid (HO···ONO) Singlet States. J. Am. Chem. Soc. 125, 16204–16205. (95) Bach, R. D., Dmitrenko, O., and Estévez, C. M. (2005) Chemical Behavior of the Biradicaloid (HO···ONO) Singlet States of Peroxynitrous Acid. The Oxidation of Hydrocarbons, Sulfides, and Selenides. J. Am. Chem. Soc. 127, 3140–3155. (96) Canepa, C., Bach, R. D., and Dmitrenko, O. (2002) Neutral versus Charged Species in Enzyme Catalysis. Classical and Free Energy Barriers for Oxygen Atom Transfer from C4aHydroperoxyflavin to Dimethyl Sulfide. J. Org. Chem. 67, 8653–8661. (97) Shaik, S., Kumar, D., de Visser, S. P., Altun, A., and Thiel, W. (2005) Theoretical Perspective on the Structure and Mechanism of Cytochrome P450 Enzymes. Chem. Rev. 105, 2279–2328. (98) Bach, R. D., Glukhovtsev, M. N., and Canepa, C. (1998) Oxidation of Alkenes, Sulfides, Amines, and Phosphines with Peroxynitrous Acid: Comparison with Other Oxidants Such as Peroxyformic Acid and Dimethyldioxirane. J. Am. Chem. Soc. 120, 775–783. (99) Bach, R. D., and Dmitrenko, O. (2003) Electronic Requirements for Oxygen Atom Transfer from Alkyl Hydroperoxides. Model Studies on Multisubstrate Flavin-Containing Monooxygenases. J. Phys. Chem. B 107, 12851–12861.


Page 38 of 40

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(100) Bach, R. D. (1994) Inductive versus Coulombic Effects on the Barriers to Oxygen Atom Transfer from Alkyl Hydroperoxides. Model Studies. J. Am. Chem. Soc. 116, 5392– 5399. (101) Chu, J.-W., and Trout, B. L. (2003) On the Mechanisms of Oxidation of Organic Sulfides by H2O2 in Aqueous Solutions. J. Am. Chem. Soc. 126, 900–908. (102) Shaik, S., Wang, Y., Chen, H., Song, J., and Meir, R. (2010) Valence bond modelling and density functional theory calculations of reactivity and mechanism of cytochrome P450 enzymes: thioether sulfoxidation. Faraday Discuss. 145, 49–70. (103) Kumar, D., de Visser, S. P., Sharma, P. K., Hirao, H., and Shaik, S. (2005) Sulfoxidation mechanisms catalyzed by cytochrome P450 and horseradish peroxidase models: spin selection induced by the ligand. Biochemistry 44, 8148–8158. (104) Sharma, P. K., de Visser, S. P., and Shaik, S. (2003) Can a single oxidant with two spin states masquerade as two different oxidants? A study of the sulfoxidation mechanism by cytochrome p450. J. Am. Chem. Soc. 125, 8698–8699. (105) Bharatam, P. V., and Khanna, S. (2004) Rapid Racemization in Thiazolidinediones: A Quantum Chemical Study. J. Phys. Chem. A 108, 3784–3788. (106) Kumar, D., Karamzadeh, B., Sastry, G. N., and de Visser, S. P. (2010) What Factors Influence the Rate Constant of Substrate Epoxidation by Compound I of Cytochrome P450 and Analogous Iron(IV) -Oxo Oxidants? J. Am. Chem. Soc. 132, 7656–7667. (107) Shaik, S., Cohen, S., Wang, Y., Chen, H., Kumar, D., and Thiel, W. (2010) P450 enzymes: their structure, reactivity, and selectivity-modeled by QM/MM calculations. Chem. Rev. 110, 949–1017. (108) Shaik, S., de Visser, S. P., Ogliaro, F., Schwarz, H., and Schröder, D. (2002) Two-state reactivity mechanisms of hydroxylation and epoxidation by cytochrome P-450 revealed by theory. Curr. Opin. Chem. Biol. 6, 556–567. (109) de Visser, S. P., Ogliaro, F., Sharma, P. K., and Shaik, S. (2002) What Factors Affect the Regioselectivity of Oxidation by Cytochrome P450? A DFT Study of Allylic Hydroxylation and Double Bond Epoxidation in a Model Reaction. J. Am. Chem. Soc. 124, 11809–11826. (110) Stare, J., Henson, N. J., and Eckert, J. (2009) Mechanistic aspects of propene epoxidation by hydrogen peroxide. Catalytic role of water molecules, external electric field, and zeolite framework of TS-1. J. Chem. Inf. Model. 49, 833–846. (111) Laitinen, T., Rouvinen, J., and Peräkylä, M. (1998) Ab initio quantum mechanical and density functional theory calculations on nucleophile- and nucleophile and acid-catalyzed opening of an epoxide ring: A model for the covalent binding of epoxyalkyl inhibitors to the active site of glycosidases. J. Org. Chem. 63, 8157–8162. (112) Dixit, V. A., Rathi, P. C., and Bharatam, P. V. (2010) Intramolecular dihydrogen bond: A new perspective in Lewis acid catalyzed nucleophilic epoxide ring opening reaction. J. Mol. Struct. 962, 97–100.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(113) Lau, E. Y., Newby, Z. E., and Bruice, T. C. (2001) A theoretical examination of the acid-catalyzed and noncatalyzed ring-opening reaction of an oxirane by nucleophilic addition of acetate. Implications to epoxide hydrolases. J. Am. Chem. Soc. 123, 3350–3357. (114) Kóna, J. (2008) Theoretical study on the mechanism of a ring-opening reaction of oxirane by the active-site aspartic dyad of HIV-1 protease. Org. Biomol. Chem. 6, 359–865. (115) Filho, R. C. D. M., de Sousa, S. A. A., Pereira, F. D. S., Ferreira, M. M. C., Sousa, S. A. A. de, Pereira, F. D. S., and Ferreira, M. M. C. (2010) Theoretical Study of AcidCatalyzed Hydrolysis of Epoxides. J. Phys. Chem. A 114, 5187–5194. (116) Brönsted, J. N., Kilpatrick, M. M., and Kilpatrick, M. M. (1929) Kinetic Studies On Ethylene Oxides. J. Am. Chem. Soc. 51, 428–461. (117) Dalvie, D. K., Kalgutkar, A. S., Khojasteh-Bakht, S. C., Obach, R. S., and O’Donnell, J. P. (2002) Biotransformation Reactions of Five-Membered Aromatic Heterocyclic Rings. Chem. Res. Toxicol. 15, 269–299.


Page 40 of 40

Toxicity Originating from Thiophene Containing Drugs: Exploring the

Recommend Documents