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Stereoselective Metabolism of Omeprazole by Cytochrome P-450 2C19 and 3A4: Mechanistic Insights from DFT Study Kalyanashis Jana, Tusar Bandyopadhyay, and Bishwajit Ganguly J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b01179 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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The Journal of Physical Chemistry

Stereoselective Metabolism of Omeprazole by Cytochrome P-450 2C19 and 3A4: Mechanistic Insights from DFT Study Kalyanashis Janaa,b, Tusar Bandyopadhyayc, and Bishwajit Gangulya,b,* a

Computation and Simulation Unit (Analytical Discipline and Centralized Instrument Facility), CSIR–Central Salt

and Marine Chemicals Research Institute, Bhavnagar–364002, Gujarat, India b

c

Academy of Scientific and Innovative Research, CSIR–CSMCRI, Bhavnagar–364002, Gujarat, India

Theoretical Chemistry Section, Chemistry Group MOD LAB, Bhabha Atomic Research Centre, Trombay,

Mumbai 400 085, India * To whom correspondence should be addressed. E-mail: [email protected] and [email protected]

Abstract The efficacy of S-omeprazole as proton pump inhibitor compared to its enantiomer R-omeprazole is studied using density functional theoretical calculations. The pharmacokinetic studies suggest that the efficacy of S-omeprazole presumably depends on metabolic pathway and excretion from the human body. The DFT calculations at SMDWater-B3LYP-D3/6-311+G(d,p)/LANL2DZ//B3LYP/6-31G(d)/LANL2DZ with triradicaloid model active species, [Por•+FeIV(SH)O], of the CYP2C19 enzyme with high spin quartet and low spin doublet states demonstrate C-H bond activation mechanism through a two-state rebound process for hydroxylation of R-omeprazole and S-omeprazole. The calculated activation free energy barrier for the hydrogen abstraction is 15.7 kcal/mol and 17.5 for R-omeprazole and Someprazole, respectively. The hydroxylation of R-omeprazole and S-omeprazole is thermodynamically favored; however, the hydroxylated intermediate of S-omeprazole further disintegrates to metabolite 5O-desmethylomeprazole with a higher kinetic barrier. We have examined the sulfoxidation of the Someprazole to omeprazole sulfone metabolite by CYP3A4, and the observed activation free energy barrier is 9.9 kcal/mol. The computational results reveal that CYP2C19 exclusively metabolizes the R1

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omeprazole to hydroxyomeprazole, which is hydrophilic and can easily excrete, whereas CYP3A4 metabolizes S-omeprazole to lipophilic sulfone, hence the excretion of this metabolite would be relatively slower from the body. The spin density analysis and MO analysis performed using biorthogonalization calculations indicate that R-omeprazole favors high spin pathway for metabolism process, however, S-omeprazole prefers the low spin pathway.

Introduction Gastroesophageal reflux disease (GERD) and peptic ulcer (PU) are most common pathological condition observed globally because of the inappropriate levels of gastric acid in stomach and intestine. Heartburn arises due to the GERD, and pain in the stomach and small intestine is the symptom of a peptic ulcer.1–3 The first initial medical target was identified as histamine-2 receptor. Subsequently, the second target was gastric proton pump, the gastric H+,K+-ATPase. The proton transfer by the gastric H+,K+-ATPase is the final step in the process, inhibitors of this type would be effective for acid secretion.4 Timoprazole was developed to inhibit gastric acid secretion; however, studies on timoprazole revealed enlargement of the thyroid gland due to inhibition of iodine uptake as well as atrophy of the thymus gland.4 Therefore, the substituted timoprazole started to appear for their efficacy to inhibit gastric acid secretion. Experiments performed with these proton pump inhibitors (PPIs) showed that they were able to inhibit H+,K+-ATPase activity only under acidic conditions. Further, the studies also revealed that there is a lag between administration of the drugs and the action of inhibition of such inhibitors. Therefore, PPIs are considered as prodrugs, which were activated with acid to function as PPIs. PPIs are the commonly used clinical as well as pharmacological approach to regulate the acid secretion since PPI can inhibit acid secretion independently.4 Omeprazole, the first clinically used gastric proton pump H+,K+-ATPase’s inhibitor, has been employed for the treatment of acid-related diseases since its discovery in 1989. Omeprazole exists as a racemic mixture of its two optical isomers, S-omeprazole (esomeprazole) and R-

2


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omeprazole; however, the pharmacological studies revealed that the esomeprazole is more efficacious compared to the R-omeprazole.5–7 Experimental studies performed with PPI reveal that such inhibitors accumulate in the parietal cell due to the protonation, which helps to bind the pump. Further, the inhibitor is activated by the second protonation on the surface of the protein for disulfide formation.8 The disulfide formation between the PPIs and cysteine residue of H+,K+-ATPase has been examined computationally.9 The computational results further reveal that both the R-omeprazole and S-omeprazole are acid activated in the acidic environment of the parietal cell and their activated form participates in disulfide formation which are energetically comparable.8,9 The activation free energy barrier for the acid inhibition process of Romeprazole and S-omeprazole are 32.4 and 31.9 kcal/mol at M062X-SMDaqueous/6-31++G(d,p), respectively and the acid inhibition process is thermodynamically favorable for both the R-omeprazole and S-omeprazole. Therefore, it appears that the relative efficacy of R-omeprazole and S-omeprazole presumably depends on the metabolic pathway and excretion from the human body. The metabolism studies have demonstrated stereoselective metabolisms of the R-omeprazole and Someprazole by several known cytochrome P450 (CYP) family’s enzymes, e.g., CYP2C19, CYP2C9, CYP3A4, CYP2A6, and CYP2D6.10,11 The CYP2C19 primarily metabolizes R-omeprazole and Someprazole through hydroxylation to hydroxyomeprazole and 5-O-desmethylomeprazole, respectively, along with the other proton pump inhibitors.10 On the other hand, CYP3A4 metabolizes S-omeprazole through oxidation to the lipophilic omeprazole sulfone.

3



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Scheme 1. Metabolism of R-omeprazole and S-omeprazole by cytochrome P450 family enzyme. a) S-omeprazole is metabolized by CYP3A4 and CYP2C19 to metabolite omeprazole sulfone and 5-O-desmethylomeprazole, respectively. b) CYP2C19 metabolizes R-omeprazole to hydroxyomeprazole.

The principal metabolites of omeprazole found in plasma are hydroxyomeprazole and omeprazole sulfone (Scheme 1). A minor metabolite 5-O-desmethylomeprazole of S-omeprazole was identified in human liver microsomal incubations. Metabolism studies revealed that esomeprazole is metabolized to a lesser degree compared to the R-omeprazole by CYP2C19. The oxidation of S-omeprazole by the CYP3A4 enzyme contributes less toward omeprazole excretion from human body compared to the hydroxylation pathway. The formation intrinsic clearance (CLint) of the omeprazole sulfone is four times lower than that of hydroxyomeprazole in human liver microsomes. The CLint values show that 5O-desmethylomeprazole and omeprazole sulfone metabolites of S-omeprazole are equally important for its elimination, however, the hydroxy metabolite dominates the elimination of R-omeprazole. Metabolism studies revealed the sum of the formation CLint of all three metabolites is 14.6 ml/min/mg of protein for S-omeprazole and 42.5 ml/min/mg of protein for R-omeprazole.10 Consequently, the metabolism studies reveal the stereoselectivity in the metabolism of these two optical isomers. However, there is no report available in the literature on the mechanistic details of the R-omeprazole and Someprazole metabolism by CYP2C19 to hydroxyomeprazole and 5-O-desmethylomeprazole, 4


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respectively, and omeprazole sulfone metabolite formation by the CYP3A4 enzyme. A few reports speculated the mechanism and reported the possible orientation of the drug molecules in the active site of the CYP2C19 and CYP3A4.12,13 On the other hand, many reports are available in the literature where hydroxylation of alkane and oxidation of sulfides by CYP family enzymes have been studied extensively.14–23

Scheme 2. Schematic mechanism of rebound process of alkane R-H.

It has been reported that the hydroxylation of alkane (RH) and endogenous molecules by cytochrome P450 takes place through C-H bond activation.17 Earlier reports show that the hydroxylation occurs through a multi-step reaction via C-H activation process (Scheme 2). In the first step, alkane molecule forms the alkane-porphyrin complex (2) with the catalytic site of cytochrome P450, known as Compound I, and initial hydrogen abstraction from the alkane (RH) occurs by the active ferryloxene species (Compound I, Por•+FeIV-O).24 In the next step, the alkane radical (R•), which formed due to the hydrogen abstraction, rebounds on the ferryl-hydroxy intermediate, 3 to generate the ferric-alcohol complex 4. The complex 4 then releases the alcohol (ROH) and restores to the resting state 5. The rebound mechanism has been widely considered as the oxidation mechanism of the alkane to alcohol. 5



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To the best of our knowledge, the mechanism of the stereoselective metabolism of the optical isomers of proton pump inhibitors have not been examined experimentally and computationally to date. In this article, we have considered the first clinically used proton pump inhibitor omeprazole to study the metabolism pathway for the first time at the molecular level. We have explored the mechanistic pathway of the hydroxylation of the R-omeprazole and S-omeprazole by CYP2C19 using DFT calculations. Furthermore, we have examined the metabolism pathway of S-omeprazole to omeprazole sulfone by the CYP3A4 enzyme to compare the kinetics as well as the thermodynamics of the metabolism of Romeprazole and S-omeprazole. To examine hydroxylation and oxidation of omeprazole, we have also analyzed the orbital occupancy and Mulliken spin density using optimized geometries. We have considered the catalytic moiety, [Por•+FeIV(SH)O], of the CYP2C19 and CYP3A4 from PDB IDs: 4GQS and 4K9W, respectively.25,26 Computational Details Thereafter, we have optimized all the geometries using UB3LYP DFT functional in conjunction with LANL2DZ basis set, which encompasses a double-ζ quality basis set with the Los Alamos effective core potential, for Fe and 6-31G(d) basis set for C, N, O, H and S in the gas phase.27–31 The UB3LYP DFT functional is one of the best functional, which is widely used for the transition metal chemistry as well as radical chemistry. The C-H bond activation and hydroxylation of alkane by heme moiety using UB3LYP DFT functional and 6-31G(d) basis set for C, N, O, H and S, have been reproted.17,20,23,32 Further, we have performed frequency calculations for all stationary points to confirm them as local minima with no imaginary frequency whereas transition structures are confirmed with only one imaginary frequency. We have also carried out single point energy calculations at SMDWater-B3LYPD3/6-311+G(d,p)/LANL2DZ level of theory in aqueous phase (ε=78.8) using B3LYP/631G(d)/LANL2DZ level of theory optimized geometries. The dispersion corrections were computed with the Grimme’s dispersion correction (DFT-D3) to account the London dispersion energy.33 The aqueous phase calculations were performed with Self Consistent Reaction Field (SCRF) method using 6


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SMD solvation model.34,35 We have calculated the aqueous phase free energy using the following equation: = + , ………2 where, is the aqueous phase free energy, is the aqueous phase energy, and , is the free energy correction value of the gas phase. The aqueous phase free energy differences have been calculated for the oxidation of R-omeprazole and S-omeprazole by CYP enzymes as: ΔG = G − G ……………1 where G is the free energy of the intermediate or transition state and G is the free energy of initial molecules, and ΔG is the difference in the free energies. Further, we have performed the biorthogonalization and spin natural orbital calculations at the B3LYP/6-31G(d)/LANL2DZ level of theory. All quantum chemical calculations were performed using the Gaussian 09 package (G09).36

Results and Discussion Toxic molecules, as well as endogenous molecules comprising with alkyl groups and sulfide groups, can undergo metabolism by cytochrome P450 through multi-step reaction processes.17 Earlier reports have shown the hydroxylation mechanism of alkane molecule as well as oxidation of sulfide by CYP series enzymes using QM and QM/MM calculations.15–20,37–41 The hydroxylation of methane as a model system for alkanes by iron-porphyrin complex and the importance of rebound mechanism was also studied.17 Other groups also examined such processes and manifested the orbital occupancies of the iron-porphyrin complexes.42–45 The metabolic pathways for R-omeprazole and S-omeprazole with CYP series of enzymes also involve the hydroxylation of alkyl groups and oxidation of sulfoxide groups in such PPIs.10 We have examined the metabolism process of R-omeprazole and S-omeprazole with CYP2C19 and CYP3A4 because of their major contribution to this process. We have considered whole catalytic moiety, 7



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[Por•+FeIV(SH)O], of the cytochrome enzyme except cysteine amino acid residue ligated to iron. We replaced the cysteine amino acid moiety with -S-H group for computational simplicity. It is reported in the literature that the -S-H group mimics the cysteine amino acid residue and produce reasonable results.17 The iron-porphyrin complex can have two possible spin states, i.e., low spin doublet and high spin quartet, depending upon the distribution of electrons in the molecular orbitals (Scheme 3). We have considered both low spin (doublet) and high spin (quartet) states of porphyrin complex to examine the rebound process of the proton pump inhibitor omeprazole.

Scheme 3. Orbital Occupancies of the doublet and quartet spin state.

8


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Scheme 4. Schematic mechanism of R-Omeprazole metabolism by [Por•+FeIV(SH)O]-CYP2C19.

R-omeprazole metabolism by CYP2C19. We have carried out the C-H and hydroxylation of the Romeprazole by CYP2C19 at the SMDWater-B3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory (Scheme 4, Figure 1, and Figure S2). The mechanistic pathway for the hydroxylation process of the R9



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omeprazole follows rebound mechanism of alkane hydroxylation (Scheme 2). The activation free energy barrier for the hydrogen abstraction process is 16.0 kcal/mol and 15.7 kcal/mol for the doublet (2ROme-TS1) and quartet (4R-Ome-TS1) ferryloxene species, respectively. Similar free energy activation barriers have been obtained with the previously studied model systems at B3LYP/ECP+LACVP-631G(d,p) level of theory.17 Further, hydroxo radical

2

R-Ome-2 intermediates formed due to the

hydrogen abstraction process were found to be unstable by 1.7 kcal/mol compared to the 2R-Ome-1, whereas and 4R-Ome-2 intermediates is stable by 4.5 kcal/mol than that of 4R-Ome-1. The hydrogen abstraction process has also generated a radical of R-omeprazole which further forms the rebound complex (2,4R-Ome-3), which are marginally stable compared to intermediate

2,4

R-Ome-2 (Figure 1).

The rebound complex, R-Ome-3, generates the hydroxyomeprazole-CYP2C19 complex through the transition state R-Ome-TS2 (Figure 1). The formation of 2R-Ome-TS2 in the aqueous phase is barrierless and stable by 3.8 kcal/mol compared to the preceding rebound complex, 2R-Ome-3, and the hydroxylated product 2R-Ome-4 is stable by 48.8 kcal/mol with respect to 2R-Ome-1. On the other hand, the formation of the hydroxyomeprazole from high spin state seems to be barrierless and the product complex, 4R-Ome-4, is stable by 46.2 kcal/mol compared to the 4R-Ome-1. The DFT calculations at the SMDWater-B3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory have demonstrated that formation of 2,4R-Ome-TS1 are the rate determining step for the formation of hydroxy omeprazole by CYP2C19. In the previous reports, it has been reported that the hydroxylation of the methane proceeds through a barrierless process for the low spin state; however, the transition state structure was determined of high spin state complex.17 The hydroxylation of the R-omeprazole by the CYP2C19 prefers the high spin quartet pathway compared to that of the low spin doublet process at the SMDWaterB3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory. The influence of polarization function for hydrogens on the geometries in this high spin hydroxylation mechanism has also been examined using B3LYP/6-31G(d,p)/LANL2DZ level of theory. The optimized geometries compared with B3LYP/6-

10


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31G(d)/LANL2DZ level of theory and B3LYP/6-31G(d,p)/LANL2DZ level of theory showed no significant changes in the geometries of reactant, and saddle points. (Figure S1).

Figure 1. Free energy surface of the R-Omeprazole metabolism pathway by CYP2C19 in aqueous phase at the SMDWater-B3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory.

The geometrical analyses performed using the B3LYP/6-31G(d)/LANL2DZ level of theory optimized geometries suggest that substituted 5-methyl group of the pyridine ring interacts with the ferryl oxygen of the Compound I where C-H….O distances are 2.75 Å, and the 4-substituted methoxy hydrogen is 2.93 Å in R-Ome-1 (Scheme 4). The benzimidazole nitrogen forms hydrogen bond (~ 1.93 Å) with the carboxyl group of the porphyrin ring in the 2,4R-Ome-TS1 and pyrimidine ring aligns almost parallel to the porphyrin ring which facilitates the π-π interaction (Figure 2). It is worth to mention here that the hydrogen of •CH2 of omeprazole radical interacts with the ferryl-hydroxy group in an almost collinear arrangement of the O-H-C moiety (Figure 2). The B3LYP/6-31G(d)/LANL2DZ level of theory calculated O….H distances is ~1.20 Å and H…..C distance is ~1.35 Å in R-Ome-TS1. We have

11



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observed similar geometrical orientation in 2R-Ome-TS2 as found in the 2R-Ome-TS1. The O….C distance in the 2R-Ome-TS1 is 2.98 Å, and the perceived H-bond distance between the benzimidazole nitrogen and carboxyl group is 1.89Å (Figure 2). The orientation of R-omeprazole with active site of [Por•+FeIV(SH)O] chosen for the DFT study has been corroborated by the docked study. The docked Romeprazole in the active site of the 4GQS CYP enzymes using Autodock 4.2 program also showed that the pyridine moiety of the R-omeprazole interacts with the oxygen of the active ferryloxene species.46 The earlier docking study performed for the same system also showed the similar orientation of the pyridine moiety of the R-omeprazole.13

Figure 2. The transition state geometries have been shown along with the key distances in Å. The distances are shown in square bracket corresponds to the quartet transition state. Fe: Deep Grey, N: Blue, H: White, C: Grey, O: Red, and S: Yellowish Brown.

The spin density is one of the diagnostic probes used for metal-porphyrin systems to examine the changes in the electronic structures of such systems. We have analyzed the Mulliken spin density for all the geometries of hydroxylation process of the R-omeprazole to explore the spin distribution on key elements (Figure S3) We have observed significant charge transfer from methane to the ferryl-oxene moiety in the 2,4R-Ome-TS1 species. The Mulliken spin densities reveal that the charge transfer is more pronounced in the low-spin 2R-Ome-TS1 compared to the high-spin species, 2R-Ome-TS1. The B3LYP/6-31G(d)/ LANL2DZ level of theory optimized transition states exhibit positive spin densities on the iron whereas a negative spin density on the migrating hydrogen atom (Figure S3). Earlier reports suggest that this spin polarization is typical of transition states of a hydrogen abstraction process by a 12


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radical.17 The spin densities on the rebound ferryl-oxene moiety of the 2R-Ome-3 species locate two units of spin on the FeO fragment, while 4R-Ome-3 species place one unit of spin on the FeO fragment. The spin density of iron is ~ 1 for the low spin state, however, the spin density for high spin state gradually increases from reaction to product complex in order to conserve the total quartet spin (Figure S3).

Figure 3. The spin natural orbitals of 2R-Ome-1. A) alpha-MO 259; dxz of iron and px of the oxygen. B) alpha-MO 260; dyz of iron and py of the oxygen and C) beta-MO 259; pz of sulfur and a2u orbital porphyrin. Fe: light purple, N: Magenta, H: Cyan, C: Yellow, O: Red, and S: Yellowish black.

Molecular orbital (MO) occupancies have also been analyzed to understand the observed Mulliken spin densities of the porphyrin. We have carried out the biorthogonalization calculations at the 13



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B3LYP/6-31G(d)/ LANL2DZ level of theory to get the partition of electron populations over natural atomic orbitals (NAOs) in the MOs. The electronic populations in the different MOs have been given in Table S1. In the 2R-Ome-1, iron, oxygen, and sulfur have 1.23, 0.84, and -0.57 spin densities, respectively. The set of biorthogonalized orbital analyses revealed that two d-orbitals have 0.56 and 0.55, i.e., ~ 1.1 spin comes from these two orbitals. The spin natural orbital (SNO) analysis has also been performed to characterize the orbitals at the same level of theory. The SNO analyses demonstrate that the d-orbitals are dxz and dyz and px and py orbitals of oxygen of ferryl-oxene moiety are occupied. The beta spin centered on the pz orbital of the sulfur and the occupancy is 0.51 which corroborates the observed spin density. However, the SNO analysis suggests that the beta spin is also located in the a2u orbital (Figure 3). In the case of quartet state, the three unpaired electrons are in alpha molecular orbital. MO analyses also show that the d-orbitals of the iron have major occupancies than that of other orbitals as observed with spin density calculations that the spin on the iron center gradually increases from reactant to product in the high spin complexes (Table S1). The complex 4R-Ome-4 has the spin contribution of 2.48 on the iron and MO analyses suggest that the dxz, dyz, and dz2 have the major occupancies (Table S1). The spin density and the MO analyses are in good agreement with each other.

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•+

IV

Scheme 5. Schematic mechanism of S-omeprazole metabolism by [Por Fe (SH)O]-CYP2C19

S-omeprazole metabolism by CYP2C19. To examine the higher efficacy of enantiomers of omeprazole, we have continued to explore the metabolism of S-omeprazole by CYP2C19. It is 15



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speculated that the 5-methoxy group of the benzimidazole moiety of S-omeprazole comes closer to the ferryl oxygen since S-omeprazole is the enantiomer of the R-omeprazole and further docking study corroborates the conjecture.12,13 The DFT calculations demonstrated that S-omeprazole forms a complex (S-Ome-1) with Compound I as it was observed with R-omeprazole, however, the orientation of the benzimidazole and pyridine aromatic rings are opposite in S-Ome-1 to that of R-Ome-1 complex (Scheme 4 and 5). Further, the hydrogen migration (S-Ome-TS1), hydroxo radical intermediate (SOme-2), radical rebound intermediate (S-Ome-3), and finally hydroxylated complex (S-Ome-4) formation has followed similar alkane hydroxylation and rebound mechanism (Scheme 4 and 5) as observed in R-omeprazole hydroxylation. The intermolecular oxidation of R′-OCH3 (S-omeprazole) to R′-OCH2OH is catalyzed by the CYP2C19 and R′-OCH2OH disintegrates to R-OH and HCHO in the presence of a water molecule. The oxidation of R′-OCH3 to R′-OCH2OH is rare in organic reaction, however, a recent report supports our predicted mechanism.47 In the metabolic process of S-omeprazole, the hydrogen abstraction process is the rate determining step and the activation free energy barriers for this step is 17.5 and 19.3 kcal/mol for doublet and quartet species, respectively (Figure 4 and Figure S5).

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Figure 4. Free energy surface of the S-omeprazole metabolism pathway by CYP2C19 in aqueous phase at the SMDWater-B3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory. Decomposition of the hydroxylated S-omeprazole to 5-O-desmethylomeprazole has been calculated at the same level of theory.

Geometrical analyses suggest that the

2,4

S-Ome-TS1 is stable by the π-π interaction between the

benzimidazole moiety and porphyrin ring (Figure 5). The hydroxo radical intermediate 2S-Ome-2 is unstable compared to the corresponding transition state, which implies that the 2S-Ome-2 does not exist on the aqueous phase free energy surface (FES) though it subsists on gas phase FES. We could not locate the rebound complex for the low spin state, however, the high spin radical rebound (4S-Ome-3) complex is stable by ~12 kcal/mol compared to the 4S-Ome-1.

Both high spin and low spin

intermediate product complexes 2S-Ome-4 and 4S-Ome-4 are stable by 52.7 and 55.8 kcal/mol, respectively, compared to that of 2S-Ome-1 and 4S-Ome-1. The S-omeprazole prefers the low spin doublet metabolism pathway by CYP2C19 enzyme compared to that of high spin quartet pathway.

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The hydrogen abstraction process is the rate determining step for both R-omeprazole and Someprazole with SMDWater-B3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory, where the former is energetically favored by ~1.5 kcal/mol compared to the later in the aqueous phase (Figure 1 and 4). Interestingly, the radical intermediate R-Ome-2 is relatively more stable compared to that of the corresponding intermediate S-Ome-2, which facilitates the formation of hydroxyomeprazole. The stability of R-Ome-2 is achieved due to the stabilization of radical in the benzyl moiety, which is however absent in S-Ome-2. Therefore, the formation of minor metabolite 5-O-desmethylomeprazole in the metabolic process of S-omeprazole is less favourable and consequently, the presence of prodrug will be relatively in higher concentration to inhibit the H+,K+-ATPase. We have further carried out spin density analysis and observed similar trend as it has been seen with R-omeprazole metabolism by ironporphyrin complex (Figure S6). The MO occupancies have also been analyzed to understand the observed Mulliken spin densities which also corroborates the spin density analysis (Table S2). The earlier report suggested that there is intermolecular oxidation of R′-OCH3 (Per-O-methylated βCyclodextrin dimer) to R′-OCH2OH, which is disintegrated to ROH and HCHO and such oxidation is rare in organic reaction.47 We have examined similar oxidation process of S-Ome-5 at the SMDWaterB3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory. The intermediate S-Ome-5 decomposes to 5-Odesmethylomeprazole through a water assisted process. The 5-O-desmethylomeprazole formation is thermodynamically unfavored by 0.6 kcal/mole compared to the intermediate S-Ome-5 (Scheme 5 and Figure 4). The formation of 5-O-desmethylomeprazole from hydroxylated S-omeprazole proceeds via higher energetic barrier (25.4 kcal/mol), hence the formation of later product is less likely in this case (Figure 4).

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Figure 5. The transition state geometries have been shown along with the distances in Å. The distances are given in square bracket corresponding to the quartet transition state. Fe: Deep Grey, N: Blue, H: White, C: Grey, O: Red, and S: Yellowish Brown.

S-omeprazole metabolism by CYP3A4. The pharmacokinetic study revealed that the intrinsic clearance (CLint) of the formation of the hydroxy metabolite from S-omeprazole was 10-fold lower than that from R-omeprazole.10 The sum of the CLint of the formation of all three metabolites, i.e., hydroxyomeprazole, 5-O-desmethylomeprazole, and omeprazole sulfone, was 14.6 and 42.5 ml/min/mg protein for S- and R-omeprazole, respectively as reported in the literature.10 These results suggest that Someprazole is cleared more slowly than R-omeprazole in vivo. Importantly, one main metabolite of Someprazole is omeprazole sulfone which is oxidized (57 %) by CYP3A4, a sister enzyme of CYP2C19. Therefore, we have computationally examined the sulfoxidation of S-omeprazole by CYP3A4 using SMDWater-B3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory (Scheme 6). The calculated free energy of activation is 9.9 kcal/mole for low spin complex (Figure 6 and Figure S7). The geometrical analysis reveals that the benzimidazole moiety of the S-omeprazole and porphyrin ring are coplanar and stabilizes by C-H-π (3.56 Å and 3.52 Å) and π-π (3.72 Å) interactions in the 2S-Ome-TS1-CYP3A4. However, the high spin free energy activation barrier is 17.3 kcal/mol, and the 4S-Ome-TS1-CYP3A4 is 19



Page 20 of 29

stabilized by C-H-π and π-π interactions as well. The product complex,

2,4

S-Ome-2-CYP3A4, is stable

by -42.4 kcal/mol for doublet state and -48.5 kcal/mol for the quartet state. Furthermore, the Mulliken spin density and the MO analyses support the trends observed for the R-omeprazole and S-omeprazole hydroxylation mechanism (Figure S8 and Table S3).

•+

IV

Scheme 6. Schematic mechanism of S-Omeprazole metabolism by [Por Fe (SH)O]-CYP3A4

The DFT calculations demonstrate that the oxidation of S-omeprazole to omeprazole sulfone metabolite by CYP3A4 is kinetically favored compared to the formation of 5-O-desmethylomeprazole by CYP2C19. It has been reported that the CYP3A4 enzyme contributes 57 % of CLint and ~40 % by the CYP2C19 enzyme. Our computational observation also corroborates the exclusive metabolism of the Romeprazole to hydroxyomeprazole by the CYP2C19 enzyme. The CYP3A4 enzyme metabolizes Someprazole to omeprazole sulfone metabolite is kinetically favored than the minor metabolites 5-Odesmethylomeprazole formation by the CYP2C19 enzyme. The omeprazole sulfone metabolite is lipophilic in nature, and hence the excretion of this metabolite would be slower from the body.

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Figure 6. Free energy surface of the S-omeprazole metabolism pathway by CYP3A4 to sulfone at SMDWaterB3LYP-D3/6-311+G(d,p)/LANL2DZ. In the TS structure, benzimidazole moiety is coplanar to the porphyrin ring which facilitates the π-π and C-H-π interactions. The all the distances have been given in Å. The distances corresponding to the quartet state are in square bracket.

Conclusion In summary, we have investigated the metabolism of the R-omeprazole and S-omeprazole with CYP2C19 and CYP3A4 enzymes computationally. This study considered the catalytic moiety, [Por•+FeIV(SH)O], of the active site of cytochrome P450 having triradicaloid iron-porphyrin (Compound I) complex. The high spin quartet state and low spin doublet state of the catalytic moiety have been considered for the hydroxylation of R-omeprazole and S-omeprazole using SMDWater-B3LYPD3/6-311+G(d,p)/LANL2DZ level of theory. The calculated results reveal that R-omeprazole and Someprazole metabolism by CYP2C19 follow the rebound mechanism of alkane hydroxylation process. Omeprazole molecule forms a stable complex with the Compound I and then the ferryloxene species abstract the hydrogen to form a ferryl-hydroxy radical intermediate. Further, the ferryl-hydroxy radical intermediate undergoes rebound process to generate the hydroxylated omeprazole. The DFT calculations show that the hydroxylated S-omeprazole further disintegrates to 5-O-desmethylomeprazole and formaldehyde. The oxidation of R′-O-CH3 to R′-O-CH2OH as observed in the case of S-omeprazole is rare in literature. The B3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory calculations in aqueous 21



Page 22 of 29

phase have also demonstrated that the hydrogen abstraction process, the rate determining step, of the Romeprazole favored by ~1.5 kcal/mol compared to S-omeprazole (Figure 1 and 4). On the other hand, the radical intermediate R-Ome-2 is more stable compared to that of the radical intermediate S-Ome-2 because of the resonance stabilization, which however, is absent in the later one. Therefore, the SMDWater-B3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory calculated results reveal that 5-Odesmethylomeprazole is the minor metabolite in the metabolic process of S-omeprazole and one would expect that the prodrug will be present in sufficient concentration to inhibit the H+,K+-ATPase. The CYP3A4 enzyme is responsible for the metabolism of S-omeprazole to omeprazole sulfone. The calculated free energy of activation for the sulfoxidation of the S-omeprazole to the omeprazole sulfone is 9.9 kcal/mol using SMDWater-B3LYP-D3/6-311+G(d,p)/LANL2DZ level of theory, and kinetically favored compared to the hydroxylation of the S-omeprazole by CYP2C19 (free energy activation is 17.5 kcal/mol). This result corroborates the pharmacokinetics studies reported for the metabolic pathways of omeprazole. This study sheds light on the molecular level mechanism of the metabolic pathways of Romeprazole and S-omeprazole and their efficacy to inhibit H+,K+-ATPase. Importantly, the Romeprazole favors the high spin metabolism pathway, and S-omeprazole prefers low spin pathway. The spin density calculation and further MO analysis report the radical characteristics of the intermediates as well as orbital occupancies of the catalytic moiety of CYP2C19 and CYP3A4. DFT results show that CYP2C19 exclusively metabolizes the R-omeprazole to hydroxyomeprazole and CYP3A4 metabolizes S-omeprazole to sulfone. The lipophilic omeprazole sulfone does not easily excrete from the body, and hence the efficacy of S-omeprazole is higher compared to the R-omeprazole to treat the patient suffering from gastric acid related diseases. This computational study sheds light on the molecular level mechanism of the metabolic pathways of R-omeprazole and S-omeprazole and their efficacy to inhibit H+,K+-ATPase, which would help to understand the metabolism of the analogous proton pump inhibitors.

Associated Contents: 22


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Supporting Information Spin density distribution, biorthogonalization data, and B3LYP/6-31G(d)/LANL2DZ level of theory optimized Cartesian coordinates have been given in SI. The B3LYP/6-31G(d,p)/LANL2DZ level of theory optimized geometries for high spin state S-omeprazole metabolism by CYP2C19 have been compared with the B3LYP/6-31G(d)/LANL2DZ level of theory optimized geometries. This information is available free of charge via the Internet at

Author Information: Corresponding Author *Fax: (+91)-278-2567562. Telephone: +91-278-2567760, ext 6770. E-mail: [email protected] and [email protected]

Acknowledgments CSMCRI communication no:. K. J. is thankful to UGC, New Delhi, India, for awarding a senior research fellowship. K. J. acknowledges to AcSIR for his Ph.D. registration. This work is supported by Department of Atomic Energy, Government of India, Board of Research in Nuclear Sciences (DAEBRNS) under grant no. 2013/37C/54/BRNS/2278. B.G. thanks DST, and DBT, New Delhi for financial support. K. J. would like to thank Dr. Rabindranath Lo for helpful discussions and valuable suggestions to design this project. K. J. would also acknowledge Dr. Dibyendu Mallik and Mr. Arvind Singh Chandel for helpful scientific discussion.

We thank the anonymous reviewers’ for their valuable

suggestions and comments that have helped us to improve the paper.

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