Theoretical Study of Ibuprofen Phototoxicity - ACS Publications

The photochemical properties and degradation of the common nonsteroid anti-inflammatory drug ibuprofen is studied by means of hybrid density functiona...
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J. Phys. Chem. B 2007, 111, 13345-13352

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Theoretical Study of Ibuprofen Phototoxicity Klefah A.K. Musa and Leif A. Eriksson* Department of Natural Sciences and O ¨ rebro Life Science Center, O ¨ rebro UniVersity, 701 82 O ¨ rebro, Sweden ReceiVed: August 15, 2007; In Final Form: September 10, 2007

The photochemical properties and degradation of the common nonsteroid anti-inflammatory drug ibuprofen is studied by means of hybrid density functional theory. Computed energies and properties of various species show that the deprotonated form dominates at physiological pH, and that the species will not be able to decarboxylate from a singlet excited state. Instead, decarboxylation will occur, with very high efficiency, provided the deprotonated compound can undergo intersystem crossing from an excited singlet to its excited triplet state. In the triplet state, the C-C bond connecting the carboxyl group is elongated, and the CO2 moiety detaches with a free energy barrier of less than 0.5 kcal/mol. Depending on the local environment, the decarboxylated product can then either be quenched through intersystem crossing (involving the possible formation of singlet oxygen) and protonation, or serve as an efficient source for superoxide anions and the formation of a peroxyl radical that will initiate lipid peroxidation.

I. Introduction Nonsteroid anti-inflammatory drugs (NSAIDs), including compounds used as analgesics, constitute one of the most important groups of pharmaceuticals worldwide, with an estimated annual production of several kilotons. As several of these drugs are also available for over-the-counter (OTC) purchase without prescription, the actual consumption is substantially higher. As a result of the high consumption as well as the drugs’ pharmacokinetics (half-life, urinary and fecal excretion, metabolism, etc.), analgesics and anti-inflammatory drugs reach readily detectable concentrations in the environment.1 NSAIDs are a heterogeneous group of compounds that exhibit favorable anti-inflammatory, analgesic, and antipyretic properties, for which the major clinical application is their action as anti-inflammatory agents in muscle skeletal diseases. NSAIDs act by reducing prostaglandin biosynthesis through the inhibition of cyclo-oxygenase (COX), which exists as two major isoforms (COX-1 and COX-2). The prostinoids produced by the COX-1 isoenzyme protect the gastric mucosa, regulate renal blood flow, and induce platelet aggregation. NSAID-induced gastrointestinal toxicity, for example, is generally believed to occur by blocking COX-1 activity, whereas the anti-inflammatory effects of NSAIDs are thought to occur primarily through inhibition of the inducible isoform, COX-2. Some of the NSAIDs might also inhibit the lipo-oxygenase pathway by acting on hydroperoxy fatty acid peroxidase.2-4 Most of the anti-inflammatory drugs are carboxylic acids, of which 2-aryl-propionic acids form a very large group. Ibuprofen, or 2-(4-isobutylphenyl) propionic acid (IBU), is a well-known NSAID extensively used to clinically treat rheumatoid arthritis, and is a promising drug with anti-atherosclerotic properties.2,5-7 It is available for OTC sale, and non-prescription sales of IBU have more than tripled since it was approved as an OTC drug in 1984.8 Because of its low pKa value (4.91), IBU exists predominantly in its deprotonated (acidic) form at physiological pH.9 It is used to treat minor aches and pains associated with common colds, headache, toothache, muscular aches, backache, * Corresponding author. E-mail: [email protected].

minor arthritic pain, and menstrual cramps, reduce fevers, and provide pain relief in women with primary dysmenorrhea.10,11 The main structural features of IBU may be considered in terms of three basic units: (i) the acidic side chain, (ii) the central aryl moiety, and (iii) a hydrophobic terminal residue (Figure 1). The propionic side chain is an important factor for the antiinflammatory activity, as is the deprotonation energy (pKa value). In particular, the deprotonation energy and the lipophilicity have been proposed as being important factors for the biological activity,12 and the smaller the deprotonation energy, the larger the anti-inflammatory activity. IBU was first introduced in the market in 1974 as a racemic mixture. Focus from the pharmaceutical industries subsequently shifted to the production of (S)-(+)-ibuprofen in 1989, when it was reported that this form more rapidly enhances the effect of analgesia in animals (including humans), than does its racemate. The production of optically pure (S)-ibuprofen acid is important to avoid the consumption of the unwanted (R)-enantiomer, which is toxic to human health.13 Besides its positive effects on inflammation and pain, several animal studies have shown that ibuprofen also inhibits tumor initiation and proliferation, although the molecular mechanisms are not fully understood. Both (S)- and (R)-ibuprofen show similar antiproliferative effects in human colon carcinoma cell lines, irrespective of their COX-inhibiting potencies.5,14 This suggests that, in the case of tumor inhibition, both ibuprofen enantiomers are more or less equally effective. Interestingly, measurements of intracellular IBU concentrations have revealed that there are clear differences in intracellular drug uptake by different cells, which, in turn, might have an impact on the efficacy of IBU in these cells. Thus, further studies are needed to evaluate the molecular mechanisms responsible for the different intracellular drug concentrations of the two enantiomers.14 A common feature of many NSAID compounds is their sensitivity toward radiation-induced degradation: their phototoxicity. Each year, millions of people are overexposed to the sun resulting in photodamage of the skin. Photosensitive skin reactions (phototoxicity or the less common photoallergy) occur

10.1021/jp076553e CCC: $37.00 © 2007 American Chemical Society Published on Web 10/25/2007

13346 J. Phys. Chem. B, Vol. 111, No. 46, 2007

Musa and Eriksson SCHEME 1: Proposed Mechanism of IBU Photodegradation

Figure 1. Ibuprofen and the atomic numbering scheme used throughout the study.

when human skin reacts abnormally to ultraviolet (UV) radiation or visible light. Several hundred chemical substances and drugs are known to act as photoactive agents, and may thus invoke phototoxic or photoallergic reactions. In order to avoid photosensitive reactions, the photosensitizing properties of a chemical should be carefully investigated before it is introduced in therapy or made available on the market. In recent years, much research has focused on identifying what photoreactive agents can be formed in products and how to prevent or control photosensitivity disorders. In a phototoxic reaction, the photoactivated chemical causes direct cellular damage; no sensitization period is required, and the mechanism is nonimmunologic, meaning that it can manifest itself already during initial exposure. The reaction is dependent upon the amount of the compound, the level of activating radiation, and the quantity of other chromophores in the skin. Absorption of UV radiation produces an excited-state chemical or metabolite, which may in turn follow one of two pathways that lead to photosensitization, involving either the generation of a free radical or the generation of singlet oxygen. These will, in turn, result in the oxidation of biomolecules, damaging critical cellular components and initiating the release of erythrogenic mediators.15,16 Studies of the phototoxicity of IBU have revealed that, despite its relatively high photostability (in comparison with, e.g., the closely related ketoprofen), the formation of several products resulting from decarboxylation, including highly cytotoxic alcohols and (less toxic) aromatic ketones, occurs rather efficiently.17 This is consistent with the finding that IBU readily penetrates into lipid bilayers6 and that it has a high lipid peroxidation rate, manifested as malondialdehyde yieldsa highly mutagenic degradation product from lipid peroxidation reactions.18 However, because of its action as an inhibitor of prostaglandin synthesis, IBU in general also provides photoprotection against UV radiation-induced edema. Edema is a direct result of the activation of the immune system and a cascading production of cytokines, prostaglandins, and arachidonic acid. As a NSAID, IBU can interrupt these cascading events and reduce the subsequent swelling of the skin. Therefore, the earlier the drug is applied to the skin post-UV exposure, the earlier this cascade can be halted. In addition, UV exposure to skin also induces the production of reactive oxygen species (ROS) in a two-pronged process: either directly from UV exposure of skin components (mainly lipids), or arising from the activated immune system. While the application of IBU is likely able to reduce the immune system production of ROS, the direct production of ROS (leading to epidermal lipid damage) is unaffected.15 The UV spectrum of IBU has been reported in several publications. It appears to be clear that the UV spectrum at physiological pH, at which the acid is deprotonated, displays a small, yet distinct peak at 222-224 nm.6,19 However, a different spectrum of IBU has also been reported displaying one small peak with a maximum at λ ) 265 nm, and a much larger and very broad peak with a maximum at around 225 nm.20

With the increasing usage of topical applications of NSAIDcontaining gels (usually containing ibuprofen, ketoprofen, or diclofenac) against, for example, aching joints or muscle pain, combined with the increased human exposure to UV radiation, it is important to explore in more detail the molecular aspects of the photodegradation of NSAIDs. Deeper insight into the physicochemical properties of these compounds will in turn assist in the design of new drugs with less harmful side effects. To this end, theoretical studies are a valuable tool in understanding the action mechanism of the compound. We have, in the current work, explored the possible photoinduced decarboxylation of ibuprofen, subsequent reactions leading to the generation of ROS, and initiators of lipid peroxidation reactions. II. Methodology All geometries of IBU, its radical anion, radical cation, anionic form, and decarboxylated forms were optimized at the hybrid Hartree-Fock density functional theory (DFT) B3LYP/ 6-31G(d,p) level. Solvent effects were taken into consideration implicitly, through single point calculations on the optimized geometries at the same level of theory, including the integral equation formulation of the polarized continuum model (IEFPCM).21-23 Water was used as solvent, through the value 78.31 for the dielectric constant in the IEFPCM calculations. Frequency calculations were performed on the optimized geometries at the same level of theory, to ensure that the systems were local minima (no imaginary vibration frequencies) and to extract zero-point vibrational energies (ZPE) and thermal corrections to the Gibbs free energies at 298 K. Excitation spectra were calculated using the time-dependent formalism (TD-DFT) at the same level of theory.24-26 The numbering scheme of the atoms used throughout the study is given in Figure 1. All calculations were performed with the Gaussian 03 program package.27 On the basis of our previous detailed account of the photodegradation mechanism of ketoprofen,28 a proposed mechanism for IBU photodegradation as shown in Scheme 1 has been explored herein; each step involved is discussed in detail below.

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Figure 2. B3LYP/6-31G(d,p) optimized structures of (a) neutral ground-state IBU (A), (b) radical anion (A•-), (c) radical cation (A•+), and (d) the deprotonated form (A-).

TABLE 1: B3LYP/6-31G(d,p) ZPE-Corrected Electronic Energies in Gas Phase, and IEFPCM- B3LYP/6-31G(d,p) Gibbs Free Energies in Aqueous Solution system

E(ZPE)a

A (singlet) A•- (doublet) A•+ (doublet) A (triplet) A- (singlet) A- (triplet)

-656.449121 -656.412609 -656.158628 -656.321898 -655.889949 -655.781120

B•- (triplet) B•- (singlet) B (singlet) B• (doublet)

-467.206299 -467.272254 -467.895399 -467.262028

∆Gaq298 a

∆∆Gaq298 b

µaqc

0 22.9 182.3 79.8 350.9 419.2

-656.508605 -656.536863 -656.283770 -656.383701 -656.035176 -655.911899

0 -17.7 141.1 78.4 297.1 374.4

1.9 7.5 2.6 2.0 18.9 15.0

0 -41.4 -432.4 -35.0

-467.327440 -467.385928 -467.939219 -467.307626

0 -36.7 -383.9 12.4

4.5 8.8 0.2 0.3

-617.606372

C• (doublet) a

∆E(ZPE)b

b

-617.659754

4.1

c

Absolute energies in a.u. Relative energies in kcal/mol. Dipole moments µ (debye) in aqueous solution.

III. Results and Discussion A. Redox Chemistry of Ibuprofen. We begin by investigating the redox properties of the ibuprofen parent compound A. In Figure 2 we display the optimized structures of IBU, its radical anion and radical cation (A•- and A•+), and its deprotonated acid (A-). The main difference in their optimized structures is a change in the C1-C2 bond length, which binds the carboxylic group, from 1.523 Å in the parent compound to 1.618 Å in the deprotonated form. However, very little change in this bond length is seen for either the radical anion or the radical cation, compared with the neutral structure. From the calculated energies we obtain the electron affinity (EA) and ionization potential (IP) as -22.9 and 182.3 kcal/mol, respectively, in gas phase. The negative EA of the anionic form implies that it, in this case, is unstable. Applying bulk solvation through the IEFPCM method, we instead obtain values of 17.7 and 141 kcal/mol for EA and IP, respectively. In Table 1 we list the absolute and relative ZPE-corrected energies in gas phase, free energies in aqueous solution, and the dipole moments obtained in aqueous solution. Of the different forms investigated, we note that the anionic species A•- in aqueous solution is the most stable species. Solvent stabilization accounts for about 40.6 kcal/mol, and is similar to previous results for ketoprofen for which solvent stabilization of the reduced species was 45 kcal/mol.28 The adiabatic IP in

vacuo is 182 kcal/mol, whereas the aqueous solution stabilizes the cation, thereby reducing the Gibbs free energy to 141 kcal/ mol. The free energy difference between the neutral and deprotonated species in the gas phase is 350 kcal/mol, which is reduced to 297 kcal/mol in aqueous solution. Again, the data are highly similar to within a few kcal/mol to those obtained for the related ketoprofen compound.28 The small changes in the computed dipole moments of most of the molecules listed in Table 1 are explained through small changes in the delocalized π-cloud of the aromatic entity. However, comparing the dipole moment of the parent compound and its deprotonated counterpart, we note a very large increase in dipole moment (more than 16 D). This is due to the charge localization in this case, on the carboxylic moiety. The total charges of the carboxylic moiety O14-C1-O15(-H16), of the neutral molecule, radical anion, radical cation, and deprotonated form, are -0.050, -0.335, 0.074, and -0.675 e-, respectively (not shown). This correlates well with the different dipole moments of the different compounds, as discussed above, in that the deprotonated species with large charge on the carboxylic group also has a large dipole moment. For the radical anion, the charge is intermediate, and it also displays a larger dipole moment than the remaining two species. The unpaired electron density of the radical anion and radical cation (Table 2) is localized on atoms C4 and C7 of the phenyl

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Musa and Eriksson

TABLE 2: Atomic Spin Densities (B3LYP/6-31G(d,p) Level) on Selected Atoms for the Radical Species of IBUa system A•- (doublet) A•+ (doublet) A (triplet) A- (triplet) B•- (triplet) B• (doublet)

C2

C4

-0.049 0.119

0.252 0.377 0.652 0.405

0.727 0.758

-0.215 -0.197

C5

C6

C7

0.576 0.526

-0.244 -0.217

0.287 0.342 0.649 0.516

C8

C9

0.556 0.384

-0.240 -0.100

0.584 -0.235

0.100 -0.129

0.112 0.259

0.350 -0.129

0.412 0.237

0.108

C• b (doublet) a

For atomic labeling, see Figure 1. b Spin densities in the peroxyl group Oinner)0.302 and Oouter) 0.691.

ring that connects to the other substituents (C4 is connected to the propionic acid, and C7 is connected to the isobutyl moiety). In the radical anion (radical cation), the spin densities at these atoms account for 0.252 e (0.377 e) and 0.287 e (0.342 e), respectively. In the case of either the neutral or the deprotonated form in the triplet state, the spin densities corresponding to both of the unpaired electrons are localized all over the phenyl ring and with a small residue also on the C2 of the propionic acid moiety. For the decarboxylated moiety 3B•- the main portion of the spin density is localized to the C2 atom that is connected to the carboxylic moiety in the parent compound. The second radical electron in the decarboxylated triplet is delocalized over the aromatic ring. Optimization of the different species mentioned above show very small overall changes in the structures (Figure 2). We note, however, that there is a considerable elongation in the C1-C2 bond length for the deprotonated species, from 1.523 Å in neutral form to 1.618 Å. This may have implications for the ease for the species to decarboxylate. In the related 2-arylpropionic acid ketoprofen, a very similar elongation in C1-C2 distance upon deprotonation was observed.28 In order to provide a setting for the photochemistry of ibuprofen, we show in Figure 3 the computed highest occupied and lowest unoccupied molecular orbitals (HOMOs and LUMOs) of the neutral and deprotonated species. The HOMO of the protonated form is localized mainly on the phenyl ring and, to a lesser extent, on the propionic acid moiety and the C10 of the isobutyl moiety. HOMO-1 is localized entirely on the phenyl ring, whereas HOMO-2 is found on the propionic acid moiety. The LUMO and LUMO+2 are both delocalized over the phenyl and propionic acid moieties, whereas LUMO+1 is localized only on the phenyl ring. The deprotonated form of IBU, on the other hand, displays a different orbital pattern than the neutral form. All three HOMOs are highly localized to the carboxylic moiety of the propionic acid, the LUMO and LUMO+1 are localized on the phenyl ring, and LUMO+2 is localized on the isobutyl moiety. The difference in MOs between the neutral and deprotonated acid is reflected in the Mulliken atomic charge distribution on the carboxylic group, which in the neutral form is only -0.050 e-, but in the deprotonated species it is -0.675 e-. Such a different MO distribution will also have a considerable impact on the photochemical behavior of the neutral versus acidic form of IBU. B. Excitation of Ibuprofen and Its Deprotonated Species. The initial step in the photodegradation of IBU is the excitation of A in its neutral or deprotonated form to the first excited singlet state S1 followed by intersystem crossing (ISC) to the first excited triplet state. Photodegradation from A- is more likely, considering both the pKa value of the acid and the structural changes upon deprotonation discussed above. The computed UV spectra of A and A- are displayed in Figure 4; as seen, and relating to the orbitals of Figure 3, the two spectra are markedly different.

For the neutral (protonated) species, the first vertical S1 excitation (HOMO f LUMO) occurs at λ ) 236 nm, in the UV regime of the spectrum, with oscillator strength f ) 0.013, indicative of low probability. This is consistent with the orbitals depicted in Figure 3 showing that the excitation is of π-π* nature. The first peak with significant oscillator strength (f ) 0.120) is found at λ ) 224 nm, with additional peaks noted in the computed spectrum at shorter wavelengths (λ ) 211 nm, f ) 0.061; λ ) 177 nm, f ) 0.600). These are, however, at too high energy to be photochemically relevant. For the deprotonated species, excitation to the S1 state is found already at λ ) 347 nm with oscillator strength f ) 0.016. The peak with highest probability is noted at λ ) 319 nm and has a computed oscillator strength f ) 0.083. Relating to experimental data with one absorption at approximately 220 nm, there is a distinct (yet relatively weak) peak occurring at 208 nm (which, in fact, consists of two closely related absorptions).

Figure 3. B3LYP/6-31G(d,p) computed orbitals for neutral (left column) and deprotonated (right column) forms of IBU.

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Figure 4. Computed absorption spectra in the 200-400 nm range of the neutral (solid) and deprotonated (dashed) forms of IBU, and the decarboxylated product 3B- (dot-dashed), obtained at the TD-B3LYP/ 6-31G(d,p) level.

The somewhat too high calculated excitation energy (ca. 0.2 eV) is highly consistent with the accuracy of the method. Again, several excitations with large oscillator strengths are also noted at λ < 200 nm. With IBU being a substituted arylpropionic acid that exists predominately in the deprotonated form at physiological pH,9 photodegradation is expected to occur from this form of the species, with decarboxylation as the dominant initial process and where radical formation after decarboxylation is also expected. The singlet excited systems in either the neutral or deprotonated form (1A,1A-) will upon ISC lead to formation of the triplet state of IBU. The optimized triplet state of the deprotonated form (3A-) lies 68.3 kcal/mol above the optimized ground state A-; for the protonated species, the corresponding free energy difference is 79.8 kcal/mol. The values are affected very little by the inclusion of bulk solvation. The energies of the optimized deprotonated and neutral triplets agree well with the vertical T1 energies obtained from TD-DFT calculations of the deprotonated and neutral species (73.8 kcal/mol and 84.8 kcal/ mol, respectively), indicating that there is relatively little structural relaxation of the T1 state. In Figure 5 we display the optimized structures of triplet states of the neutral and deprotonated species. For the protonated form, very small changes in geometry are noted, compared to the singlet state. In the deprotonated species, the main structural change is a considerable elongation of the C1-C2 bond length (corresponding to decarboxylation) from 1.618 Å in the singlet ground state to 1.765 Å in the triplet state. The situation is hence in this case rather different from the case of ketoprofen, in that its deprotonated triplet state undergoes spontaneous decarboxylation.28 A negative charge of approximately -0.501 e- is located on the carboxyl moiety of IBU in the optimized deprotonated triplet, while only -0.044 e- is located on the same group in the neutral case. This can also be rationalized from the difference between the HOMO and LUMO of the two forms (neutral and deprotonated). Comparing with the ground-state acid, a charge of -0.675 e- was observed on the carboxylic moiety. Upon excitation from HOMO to LUMO, more of the electron distribution is moved from the carboxylic group into the phenylic rings (see Figure 3)shence the slightly reduced charge. Energetically, the initial S0 f S1 excitation of the deprotonated system requires 82.3 kcal/mol. Following ISC to the T1 state, this will relax to a free energy level located 68.3 kcal/

Figure 5. Optimized structures (B3LYP/6-31G(d,p) level) of (a) neutral and (b) deprotonated T1 states of IBU.

Figure 6. Energy curves for decarboxylation of deprotonated IBU, including the ground state (gs) and several of the lowest excited singlet (es) states.

mol above S0. For the neutral system, the S0 f S1 excitation requires 121 kcal/mol, which is considerably higher than that for the deprotonated acid; following ISC, the relaxed triplet lies ca. 85 kcal/mol above S0. For the neutral triplet to deprotonate requires 296.1 kcal/mol, which is very similar energy to that of the singlet-state deprotonation (297.1 kcal/mol). We may hence conclude that, in the case where the neutral system is excited, the pKa of the triplet state is similar to that of the ground state, and will thus most likely also result in the formation of 3A-. This behavior is similar to that obtained in our previous study of ketoprofen. In order to investigate whether decarboxylation can occur from an excited singlet state of the deprotonated form, the C1C2 bond was scanned outward from the optimized value (1.618 Å) in steps of 0.1 Å. In each new point, the structures were re-optimized, and the vertical excitation energies were calculated. The resulting energy curves, obtained at the TD-B3LYP/ 6-31G(d,p) level, are displayed in Figure 6, including several of the lowest singlet excited states. The ground and most of the lowest excited singlet state surfaces are strictly endothermic through the scan of the C1-C2 bond, and hence show no sign of decarboxylation. The exception is the S4 state (and S7, which, however, lies far too high in energy to be of relevance), which displays an apparent transition barrier with a maximum at a

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Musa and Eriksson -CO2

O2

A- 98 3B- 98 2B• + O2-

3

2 •

O2

B 98 2C•

Figure 7. Energy barrier for decarboxylation from the first excited triplet state of deprotonated IBU.

Figure 8. Reaction path for the formation of peroxyl radical 2C• from 2B• and molecular oxygen.

C1- C2 distance of ∼2.4 Å. The barrier to decarboxylation is rather high (around 20 kcal/mol), and it can thus be concluded that decarboxylation is not likely to occur from the excited singlet states of ibuprofen. As for the triplet structures, this situation is also markedly different from the case of ketoprofen, for which several of the singlet excited states of the deprotonated form would lead to decarboxylation by overcoming a barrier of only a few kilocalories per mole.28 Because of the elongation of the C1-C2 bond length by 0.15 Å in the optimized triplet state of the deprotonated form (Figure 5) compared to the S1 state, the energy barrier for decarboxylation of deprotonated IBU in the triplet state was conducted in the same manner as described above. The resulting energy curve is depicted in Figure 7, and shows the presence of an energy barrier of only 0.3 kcal/mol for decarboxylation to take place in the triplet state. The C1- C2 distance of the transition state is approximately 1.97 Å. This can be compared again with the corresponding process in KP, for which decarboxylation from the triplet state occurred spontaneously upon formation. C. The Fate of Decarboxylated Ibuprofen. Given the high photosensitivity of 1A- and the very low barrier to decarboxylation from 3A-, we conclude that, upon excitation, the yield of the decarboxylated species 3B- should be significant. Hence, we do not expect 3A- in itself to be able to serve as any substantial source for singlet oxygen formation resulting from de-excitation. The subsequent reaction pathways of the photoinduced degradation of IBU depicted in Scheme 1 can be summarized as follows (reactions 1-3). -

-CO2

A 98

3

3 - ISC, H+ B 9 8 O2

B + O2 1

(1)

(2) (3)

Reaction 1 starts from the photodegradation process of IBU 3A- leading to the formation of the p-ethylisobutylbenzene anion 3B-. Optimization of this species in its singlet and triplet forms shows that the triplet state lies about 41.4 kcal/mol above the singlet state, a difference that is reduced to 36.7 kcal/mol when the calculations are done in bulk solvation. The triplet state has a spin density of -0.727 e localized mainly on C2, with the second unpaired electron delocalized to the phenyl ring. Protonation of the radical anion to generate the singlet form of p-ethylisobutylbenzene B will involve ISC and protonation. During this process, the singlet oxygen formation can be expected in the ISC process. The excitation energy of groundstate molecular oxygen leading to the formation of singlet oxygen is 22.5 kcal/mol29 compared with the 37 kcal/mol energy difference between the triplet and singlet forms of B-. 1B- has a very high proton affinity, around 347 kcal/mol in aqueous solution, and is hence expected to protonate more or less instantaneously. The absorption spectra of the decarboxylated species 3B- were also calculated, and are displayed Figure 4. Apart from a couple of transitions with very low intensity in the visible and UV-A region (not likely to be detectable), there are two dominant absorptions at 260 and 240 nm. This is in line with the spectrum reported in some studies (two peaks at approximately 265 and 225 nm, with the latter of higher intensity than the former20) and might suggest that the compound in those cases has undergone a first photodegrading step. These absorptions of 3B- are markedly different from and much more intense than those of the carboxylated parent compound. Another possible fate of 3B- is for the species to undergo electron transfer to molecular oxygen (reaction 2), which will generate superoxide radical anions. Because of the large stabilization of the charged species by the polar solvent, the anion is 12.4 kcal/mol more stable than the radical form 2B• (Table 1). This should be compared with the adiabatic EA of molecular oxygen to generate superoxide in solution, estimated to be 90.2 kcal/mol (3.91 eV),30 which implies that the reaction will be exergonic by approximately 77 kcal/mol. In the case of ketoprofen, the corresponding process was exothermic by only 27 kcal/mol.28 Irreversible quenching of the triplet anion by oxygen-dependent electron transfer to form the p-ethylisobutylbenzyl radical 2B• and superoxide is thus likely to occur under aerobic conditions in polar media. The addition of molecular oxygen to the p-ethylisobutylbenzyl radical 2B• (reaction 3) to generate the peroxyl radical 2C• will not only give the various oxygenated derivatives mentioned above, but also initiate lipid peroxidation reactions. If embedded in a biological membrane, the peroxyl radical 2C• can abstract a hydrogen atom from a lipid molecule, which, through the addition of molecular oxygen to the thus generated lipid radical site L•, creates a propagating radical damage (reactions 4-7). 2 •

B + O2 f 2BOO• ()2C•)

(4)

BOO• + LH f B(OOH) + L•

(5)

2

•

L + O2 f LOO

•

LOO• + LH f LOOH + L•

(6) (7)

Once initiated, the chain reactions 6 and 7 will repeat until terminated by, for example, radical-radical addition or the action of antioxidants such as vitamin E. In 2B•, the main fraction

Ibuprofen Phototoxicity of the unpaired spin (0.76 e) is, as expected, located on carbon C2. The addition reaction with molecular oxygen in its 3Σ ground state to form 2C• was explored by scanning the C2-OO distance in steps of 0.1 Å; the energy diagram for this process is displayed in Figure 8. The energy difference between the peroxyl radical product 2C• and that where the two reactants are separated by 3.5 Å, is 20.4 kcal/mol. The reaction is strictly exothermic with a clear change in slope at a C-O distance around 2.2 Å. The generation of the peroxyl radical is hence also strictly exergonic under aerobic conditions; in the presence of molecular oxygen, reaction 4 will occur spontaneously and without any barrier. IV. Conclusions The photochemical degradation mechanism of the NSAID ibuprofen is explored using computational chemistry. Calculated structures for the neutral species and the deprotonated acid show that deprotonation slightly elongates the C-C bond attaching the carboxylic unit, whereas, for the first excited triplet state of the deprotonated species, this bond is extended further (to 1.77 Å). The deprotonation free energy in bulk solvation (297 kcal/ mol) agrees well with the experimental pKa value of 4.91, showing that the compound will be deprotonated under physiological conditions. The computed absorption spectra for the neutral and deprotonated species reveal clear differences between the two forms. The calculated peak at 210 nm for the deprotonated species agrees with the spectra recorded between 200 and 300 nm at pH 7. The decarboxylation process occurring upon excitation was investigated by scanning the C1-C2 distance for both excited singlet and triplet states. It is concluded that for decarboxylation to occur from an excited singlet, a barrier of approximately 20 kcal/mol must be overcome. In addition, decarboxylation from most of the low-lying singlets is strictly endergonic, and will thus not occur. The computed data explains the observation that the molecule is more photostable than its related compound ketoprofen, and that this is primarily caused by the low reactivity of the excited singlet states. In order for IBU to undergo decarboxylation, it is required that ISC takes place to reach the first excited triplet. However, since the system is in the lowestlying triplet state, the decarboxylation reaction will be very efficient, as only a very small barrier (0.3 kcal/mol) must be overcome. Decarboxylation will thus be strongly dependent on the quantum yield for ISC versus the rate of de-excitation from the excited singlet. Having formed the decarboxylated product (p-ethylisobutylbenzene anion, 3B-), this can readily generate singlet oxygen through triplet-triplet excitation energy transfer in the quenching and protonation process, or generate superoxide through electron transfer and then form a peroxyl radical through O2 addition at the decarboxylated radical site. This, in turn, will initiate lipid peroxidation very efficiently. We furthermore predict that, in a basic and oxygen-free environment (to avoid protonation or superoxide/peroxide generation), detection and identification of 3B- should be possible, because of its strong absorption spectrum. The current study provides a detailed outline of the photodegradation mechanism of one of the most common NSAIDs, and provides valuable insight into key factors required to be addressed in order to design new compounds with similarly high biological activities yet reduced photochemical side effects. Acknowledgment. The MENA programme (KAKM) and the Swedish Science Research Council (LAE) are gratefully

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