Conformational Change in the Mechanism of Inclusion of Ketoprofen

Sep 30, 2016 - Department of Discovery, Dompé Farmaceutici SpA Research Center, Via Campo di Pile, 67100 L'Aquila, Italy. ⊥ Department of Chemistry...
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Conformational Change in the Mechanism of Inclusion of Ketoprofen in β‑Cyclodextrin: NMR Spectroscopy, Ab Initio Calculations, Molecular Dynamics Simulations, and Photoreactivity T. Guzzo,†,∇ W. Mandaliti,‡,∇ R. Nepravishta,‡,§,∇ A. Aramini,∥ E. Bodo,⊥ I. Daidone,# M. Allegretti,∥ A. Topai,*,† and M. Paci*,‡ †

C4T (Colosseum Combinatorial Chemistry Centre for Technology) S.C.a r.l., Via della Ricerca Scientifica s.n.c., 00133 Rome, Italy Department of Chemical Science and Technology, University of Rome “Tor Vergata”, 00133 Rome, Italy § Department of Chemical Pharmaceutical and Biomolecular Technologies, Faculty of Pharmacy Catholic University “Our Lady of Good Counsel”, Rr. D. Hoxha, 1000 Tirane, Albania ∥ Department of Discovery, Dompé Farmaceutici SpA Research Center, Via Campo di Pile, 67100 L’Aquila, Italy ⊥ Department of Chemistry, University of Rome “Sapienza”, 00133 Rome, Italy # Department of Physical and Chemical Sciences, University of L’Aquila, Via Vetoio (Coppito 1), 67010 L’Aquila, Italy ‡

S Supporting Information *

ABSTRACT: Inclusion of drugs in cyclodextrins (CDs) is a recognized tool for modifying several properties such as solubility, stability, bioavailability, and so on. The photoreactive behavior of the β-CD/ketoprofen (KP) complex upon UV exposure showed a significant increase in photodecarboxylation, whereas the secondary degradation products by hydroxylation of the benzophenone moiety were inhibited. The results may account for an improvement of KP photophysical properties upon inclusion, thus better fostering its topical use. To correlate the structural details of the inclusion with these results, an NMR spectroscopic study of KP upon inclusion in βCD was performed. Effects of the magnetically anisotropic centers of KP, changing their orientations upon inclusion and giving chemical shift variations, were specifically correlated with the results of the molecular dynamic simulations and ab initio calculations. In the large variety of papers focusing on the structural analysis of β-CD complexes, this work represents one of the few examples in which a detailed analysis of these simultaneous upfield−downfield NMR shifts of the same aromatic molecule upon inclusion is reported. Interestingly, the results demonstrate that the observed upfield and downfield shifts upon inclusion are not related to any direct magnetic role of β-CD. The conformational change of KP upon the inclusion process consists of a slight reduction in the angle between the two phenyl rings and in a remarkable reduction in the mobility of the carboxyl group, the latter being one of the main contributions to the NMR resonance shifts. These structural details help in understanding the features of the inclusion complex and, eventually, the driving force for its formation.

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INTRODUCTION

triggering the interest of cosmetic industry for the microencapsulation of sunscreen agents.6,7 Ketoprofen (KP) is a poorly water-soluble nonsteroidal antiinflammatory drug, widely used as an analgesic and for the treatment of acute and chronic inflammatory conditions. Over the last 10 years, β-CD/KP complexes have been extensively studied with the purpose of improving the gastrointestinal tolerability of oral formulations.8 Topical use of KP gel and foams is associated with an increased risk of photoallergic/ phototoxic reactions that have been related to the photoreactive properties of the molecule.9 In this context, the identification of novel strategies for the minimization of KP phototoxicity would represent a major goal for topical pharmaceutical applications.

Cyclodextrins (CDs) are composed of 6, 7, and 8 α-1,4-linked D-glucopyranose, usually referred to as α-, β-, and γ-CD, respectively. Complexation of various compounds with CDs leads to an improvement in some of the characteristics of the guest molecules, such as thermal stability, bioavailability, membrane permeability, and solubility.1,2 Accordingly, CDs and their derivatives have found numerous applications in a variety of fields, such as catalysis, electrochemical analysis, pharmaceutical and food industries, separation sciences, and biotechnology. In the pharmaceutical field, the use of CDs as emulsifiers in creams or lotions is a proven strategy to improve the solubility and local tolerability of dermal preparations.3,4 Recent studies have proposed CDs as protecting agents of guest molecules against oxidation and photodegradation,5,6 thus © 2016 American Chemical Society

Received: August 5, 2016 Published: September 30, 2016 10668

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within these two forms in the inclusion mechanism of KP in βCD.22 In fact, according to previous studies, the chiral recognition ability of β-CD on the two enantiomers is very low.18 These results have been further confirmed by MD simulations.22 Herein, we report in detail the structure of KP in the complex between KP and β-CD and the geometry change of KP from water to the β-CD environment on the basis of NMR spectral changes. These results have been considered in the discussion of the findings in the study of the photodegradation reactions.

Several studies have been performed to elucidate the photochemistry of KP and similar molecules of pharmaceutical interest under a variety of conditions and by different experimental approaches. The reported results indicate that irradiation of KP in neutral aqueous medium gives rise to 3ethylbenzophenone, which undergoes fast transformation into more stable hydroxylate products.9−11 The mechanism of the decomposition pattern has also been investigated in detail,12−14 not only in aqueous solution but also in a range of less polar solvents and solvent mixtures. At physiological pH, UV-induced decarboxylation of the triplet (or singlet) anion has been clearly shown. The same is true for the carboxylate form in less polar solvents. No decarboxylation of the neutral form is instead observed. The different behavior of the neutral and deprotonated acid is explained in terms of the markedly different orbitals for the two species. Previous reported studies15 have evaluated the photochemistry of KP in the β-CD cavity by stationary and timeresolved (picosecond and nanosecond) spectroscopic techniques, pointing out a direct dependence of the photochemistry on the environment. Photodecarboxylation is found to occur with a lower quantum yield than that in aqueous medium, and additional photoreactions were evidenced. The overall photostability of the drug is lower. Although not conclusive, the data might support a rationale for the decreased phototoxic activity of the drug in vitro. In this study, the photoreactive properties of the β-CD/KP complex are reinvestigated to correlate the structural data obtained by NMR spectroscopy with the potential complexation-induced photoprotection. Rather than a photoprotective effect, a marked modification of the typical KP photodegradation pattern suggested that inclusion of the guest molecule into the cavity is associated with a conformational rearrangement of the benzophenone structure, influencing its photosensitizing properties. NMR spectroscopy has been widely used for the characterization of CD/guest complexes.16,17 1H NMR studies clearly demonstrated that the proton chemical shifts, temperature coefficients, and vicinal coupling constants of CDs can be used to monitor the formation of intermolecular hydrogen bonds and hydration changes upon inclusion. Nonetheless, only a very limited number of papers have so far analyzed the shift of the NMR resonances of the guest molecule upon inclusion, and only few examples in the literature reported a complete analysis of the complex pattern of downfield or upfield shifts of the resonances for different protons of the guest.16−19 High-resolution NMR spectroscopy and NMR combined with diffusion-ordered NMR spectroscopy (DOSY) were selected as privileged techniques because they have been demonstrated as valuable tools, the latter for the characterization of the KP/β-CD complex and the former for its structural elucidation.18,20 The perturbation pattern of KP proton chemical shifts due to the diamagnetic anisotropic effect of its aromatic moieties is reported and used as a conformational probe of the inclusion-induced structural change. Molecular dynamics (MD) simulations in water starting from an ab initio calculation21 were conducted to derive theoretical models for the interpretation of the experimental data. In the MD simulation, we considered only one stereo isomer (the R isomer) due to the very small differences found and reported

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EXPERIMENTAL METHODS Materials. CD was purchased from Cavamax. KP was supplied by Dompé SpA. Standard sodium formate solution was puchased from SigmaAldrich. D2O was purchased from Sigma-Aldrich and used as solvent. High-performance liquid chromatography (HPLC) grade acetonitrile and trifluoroacetic acid were purchased from Sigma-Aldrich. Na2HPO4·7H2O and Na2HPO4 for phosphate buffer solutions were purchased from Fluka. Water for HPLC and buffer solutions was purified by passage through ELGA’s Purelab Classic UV PL 5242. NMR Sample Preparation. Solutions of KP R,S lysine salts (30 mM) and of β-CD (15 mM) were prepared in D2O. Each test tube, for NMR experiment, contained a 600 μL mixture of β-CD and KP at a specific molar ratio: 0.2/1, 0.4/1, 0.6/1, 0.8/1, 1/1, 2/1, and 5/1, where KP concentration was kept constant. The mixtures were prepared according to the following procedure: to 60 μL of 30 mM KP solution different volumes of 15 mM β-CD solution were added to obtain different [β-CD]/ [KP] molar ratios. Then, D2O was added to obtain a final volume of 600 μL for each test tube and a costant concentration of KP (3 mM). A fixed volume of sodium formate standard solution was added to each test tube as internal standard. NMR Spectroscopy. NMR spectra were recorded at 298 K on two Bruker Avance instruments operating at 400.13 and 700.13 MHz. 1H NMR spectra were acquired with the zgpr pulse program of the Bruker library for water signal suppression with 32 scans and a relaxation delay of 2 s. All of the proton KP resonances reported were assigned by two-dimensional (2D) NMR spectra: total correlation spectroscopy (TOCSY) pulse sequence with the MLEV-17 spinlock composite pulse sequence was inserted23,24 (mixing time 60 and 120 ms) and WATERGATE block for water suppression,25,26 1H−13C heteronuclear single-quantum-coherence (HSQC) spectra,27 and 1 H− 13 C heteronuclear multiple bond correlation (HMBC)28,29 were employed to assist with cross-peak assignment. The structural details of inclusion of KP in β-CD were investigated by 2D ROESY experiments30 using a series of 180(x) 180(−x) pulses as spinlock31,32 (with a spinlock time of 0.050, 0.080, 0.120, and 0.150 s) and the presaturation technique for water resonance suppression. For homonuclear experiments, the states-time-proportional phase incrementation phase cycle was used.33 The interaction of KP with β-CD was studied, and the affinity was evaluated by measuring KD by DOSY.34−36 For this purpose, DOSY spectra with different [β-CD]/[KP] ratios were acquired with the concentration of KP maintained at about 3 10669

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The Journal of Physical Chemistry B mM and by increasing the β-CD concentration. The [β-CD]/ [KP] ratios were about 0.2, 0.5, 0.8, 1.0, and 5.0. The DOSY spectra were performed using the ledbpgppr2s pulse sequence to suppress the water signal. During the DOSY experiment, 32 monodimensional spectra were acquired with 64 scans, in a linearly increasing gradient varying from 5 to 95%, with a Δ of 70 ms and δ of 2 ms. The spectra were then analyzed using the DOSY module implemented in Bruker software TOPSPIN 3.1. The diffusion coefficient (D) data obtained from the DOSY experiments were plotted versus the [β-CD]/[KP] ratio and fitted to eq 1 to obtain KD of the binding.

fragmenting voltage was set to be 50 V. The instrument operated in scan mode in the mass range 200−1500 m/z. Acquisition data were processed with Agilent Chemstation Software. Ab Initio Calculations and MD Simulation. There are two possible ways of inserting the KP molecule into β-CD, which we call “up” and “down”. The initial configuration of the up complex was prepared by placing the center of the KP molecule at the center of β-CD in such a way that the aromatic ring of KP remains toward the wider rim of β-CD. Similarly, the opposite orientation was considered for KP to obtain the down complex. All ab initio calculations were performed by means of the Gaussian09 suite.37 All of the structures (of both KP and the inclusion complex) were optimized at the B3LYP/6-31G (d) level. The small basis set was chosen to make the calculations feasible even while considering the computationally demanding structure of the KP inserted into β-CD. The molecular structure is reported in Figure 1 along with the labeling that has been used.

⎛ ΔD = ⎜([KP] + [β ‐CD] + KD) ΔDmax ⎝ −

⎞ ([KP] + [β ‐CD] + KD)2 − 4[KP][β ‐CD] ⎟/2 ⎠ (1)

In NMR spectra, the trimethylsylil propionic acid sodium salt was used as internal reference. Photolysis. The applied protocol of irradiation experiments was similar to that used9 for the photodegradation of KP. Irradiation experiments were performed in a solar box (Gruner, 220 V, 50 Hz). The radiation was emitted by a xenon lamp in the wavelength range of 300−800 nm. An IR filter was used for cutting off the infrared radiation. The temperature was kept constant at 45 °C. A solution of free KP with initial concentration 1 mM was prepared in pH 7.4 phosphate buffer medium. Solutions of KP complex were prepared at 1/5 [KP]/[β-CD] molar ratio in 0.1 M phosphate buffer media (pH 7.4), using 1 mM KP concentration. The ratio of β-CD was set to ensure complete KP inclusion, according to the experimental data observed in the DOSY NMR study (see the results of the NMR studies). All solutions were kept in the dark until the experiments were carried out. For each sample, 15 mL were withdrawn and placed on a 5 × 1.5 cm2 glass plate (diameter × length). Plates were positioned vertically from the radiation lamp. For each sample, aliquots of 2 mL were collected during the experiments at three fixed intervals (1, 3, and 6 min) and subjected to HPLC-mass spectrometry (MS) analysis for data evaluation. Analysis of Photodegradation Products. Analyses were performed on an Agilent 1260 Infinity System, equipped with a G1311B Agilent 1260 quaternary pump, G1329B Agilent 1260 autosampler, G1315C Agilent 1260 diode array detector, and G1316A Agilent 1260 column thermostat modules. A Phenomenex GEMINI C18 150 × 4.6 mm2 (5 μm) column was used. The mobile phase A was MilliQ water with 0.05% TFA, and B was HPLC grade acetonitrile with 0.05% TFA at a flow rate of 1.0 mL/min. The gradient started from 5% B (held for 0.5 min) to 99% B in 25 min, back to 5% B in 1 min, and kept at 95% B for 1 min. The column was maintained at 5% B for 7 min before the next analysis. The diode array detector operated in the dual mode at 220 and 254 nm. The response time was set to 0.5 s with a data rate of 10 Hz. The HPLC system was coupled to an Agilent Quadrupole 6120 LC/MS mass spectrometer operated in positive ion mode. Ions were generated using an electrospray ionization ion source. Operation conditions were as follows: gas temperature was 350 °C and drying gas was at a flow rate of 12 L/min, capillary voltage was 4000 V, nebulizer pressure was 35 psig, and the

Figure 1. Schematic formula, the KP molecule, and the labeling of the NMR resonances. Labeling of A and B aromatic rings was used as in refs 18, 19.

MD simulations of KP included in β-CD and of free KP together with that of free β-CD were performed in water using the GROMACS software package.38 The main aim of the present simulations is to compare the results obtained with those from the calculation of intramolecular angles based on the NMR results. The goal was to to characterize the role of the water molecules in determining the thermodynamics of the complex. In fact, it has been proved that R- and S-ketoprofen have similar binding free energies to β-CD.22 Moreover, previous MD simulations showed that the two enantiomers R and S have similar structural properties when bound to β-CD.22 Hence, we performed simulations of only one of the two enantiomers, namely, the R-enantiomer. The potential parameters of the β-CD molecules were taken from the GROMOS force field,39,40 whereas the rigid three-site extended simple point charge model35 was employed for water. Potential parameters for KP were obtained from ab initio calculations (see above).41,42 Two sets of separate simulations of up and down inclusion complexes were carried out. Three configurations with the lowest potential energy, as obtained by ab initio calculations, of each of the two systems (up complex and down complex) were used as starting structures. Each of the six simulations was run for 20 ns. One simulation of free KP and one of free β-CD in aqueous solution were run for 10 ns each. For each system, one unit was placed in a periodic rhombic dodecahedral box large enough to contain the system and at least 1 nm of solvent on all sides. A standard protocol was adopted for initiating the simulations. Following a mechanical 10670

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Figure 2. DOSY of (A) R-KP (log D = −8.66), (B) S-KP (log D = −8.66), (C) R-KP-β-CD (1:1) (log D = −8.86), and (D) β-CD alone (log D = −8.91).

approximation of Pople49 and particularly by the model of Haigh−Mallion.50 Equation 3, as reported by Christensen et al.,51 correlates the chemical shift change with the ring current effect

solute optimization and subsequent solvent relaxation, the system was gradually heated from 50 to 298 K using short (20 ps) MD simulations. Each trajectory was then propagated at 289 K in a NVT ensemble, and the isokinetic temperature coupling43 was used to maintain the temperature constant. All bond lengths were constrained using the LINCS algorithm,44 and a time step of 2 fs was used for numerical integration. Periodic boundary conditions were applied to the simulation box, and the long-range electrostatic interactions were treated with the particle mesh Ewald method45 using a grid spacing of 0.12 nm combined with a fourth-order B-spline interpolation to compute the potential and forces between grid points. The realspace cutoff distance was set to 1.0 nm. The KP was modeled in its anionic form consistent with the neutral pH used in the experiments. One positive counter ion (Na+) was hence added to each simulation box of the inclusion complex and of free KP for ensuring electrical neutrality. Magnetic Anisotropic Effect on Chemical Shifts. To predict the overall magnetic anisotropic effect on each aromatic proton, the contribution of carbonyl, carboxylic, and aromatic ring moieties was considered. The magnetic anisotropy effects of carbonyl and carboxylic groups are determined according to eq 2 introduced by McConnell46 with the application to nonsymmetrical groups δan =

Δχpar (3 cos2 θ1 − 1) 3R3

+

δan = iBG

using factors i = 1 for benzene, B as a constant indicating the electronic current intensity, and G as a geometric parameter depending both on the distance and angle of proton from the plane of the aromatic ring. B and G factors can assume different values according to the model used, and the subscripts P and HM were used to refer to the Pople and Haigh−Mallion models, respectively.52 In the model of Pople, eqs 4 and 5, the BP value of 27 ppm Å3 used was that reported by Abraham et al.53 δan = BP

(3 cos2 θ − 1) R3

BP ≅ 27 ppm Å3,

(4)

GP =

(3 cos2 θ − 1) R3

(5)

The geometric factor GP was experimentally determined as indicated below. In the model of Haigh−Mallion, eqs 6 and 7, the BHM value of 5.13 ppm Å used was that reported by Neal et al. for protons54

Δχper (3 cos2 θ2 − 1) 3R3

(3)

⎞ ⎛ 1 1 δan = BHM ∑ Sij⎜⎜ 3 + 3 ⎟⎟ rj ⎠ ⎝ ri ij

(2)

where θ1 and θ2 are the angles parallel and perpendicular to the carbonyl−carboxyl plane and R is the distance between the anisotropic center and the proton. The values of Δχpar and Δχper were considered to be those of aromatic ketones of 6.63 and −11.88 ppm Å3, respectively.47,48 The ring current effect due to aromatic rings was calculated for individual protons of R-KP using the popular models for NMR chemical shift prediction upon the formulation of the point dipole

(6)

⎛

BHM ≅ 5.13 ppm Å,

G HM =

⎞ 1 1⎟ + 3 r j3 ⎟⎠ ⎝ ri

∑ Sij⎜⎜ ij

(7)

The geometric factor GHM for each proton for the sixmembered aromatic ring was obtained according to eq 8, as proposed by Sahakyan and Vendruscolo55 10671

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The Journal of Physical Chemistry B Table 1. TPs Generated by Xe Lamp Photodegradation of Free KP and KP Included in β-CD

G P (R ⃗ ) =

(0.325964G HM(R⃗) − 0.000466) (1.743379 − 2.391111G HM(R⃗))

proton chemical shift perturbation upon inclusion at the same time. First, 1H DOSY experiments were run for the R, S enantiomers of KP at different [β-CD]/[KP] molar ratios. According to the poor solubility of the free acid in D2O medium, the study was first performed using lysine salt of KP. Besides the better solubility of the lysine salt, the lysine residue acts as an internal standard, showing a fixed diffusion front in the different experiments. KD was determined by measuring the change of the diffusive front in DOSY spectra (as reported in Experimental). The measured KD value obtained for racemic KP lysine salt was 0.63 mM. Representative DOSY spectra of KP Lys salt, βCD, and the inclusion complex at 5:1 molar ratio are reported in Figure 2. The experiment was repeated on KP free acid. Because of solubility problems, the last [β-CD]/[KP] molar ratio was set to 4:1, and the plateau value was obtained by extrapolation. The reported KD value (2.4 mM) is coherent with previously reported values.18,19 The small difference observed between lysine salt and free acid constants may account for a more accurate detection in salted medium due to the better solubility. Possible aggregation effects may occur between KP free acid molecules in D2O medium, thus affecting the correct evaluation of its concentration in solution. Further experiments were run separately also on R and S enantiomers to preliminarily evaluate any stereo differentiation in the inclusion process. R- and S-KP were also used as lysine salts. Values of KD for R-KP Lys and S-KP Lys were 0.93 and 0.30 mM, respectively, therefore, quite similar within the experimental error (±0.15 mM). These values agree with the average of the values (0.63) determined for the racemic form. The very

(8)

The ring current effect first calculated following the model of Pople was then recalculated for the Haigh−Mallion model. Experimentally, the distances R of atoms from the anisotropic centers and the angles θ with the carbonyl, carboxyl, and ring planes were obtained separately for each aromatic proton by means of geometrical considerations from the structures calculated by ab initio or MD simulations. The PyMOL and UCSF CHIMERA software were used for the visualization of molecular structures. Two values of the anisotropic contributions, δan, were obtained for every proton of R-KP either alone and for R-KP included into β-CD, thus obtaining a Δδ value as the difference between the chemical shift of R-KP included into β-CD and that of KP alone.

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RESULTS KP/β-CD Interaction. The assignment of KP resonances of the NMR spectra was straightforward on the basis of previous reported literature.18,19 The stoichiometry of the complexes was determined by direct NMR resonance integrations. The numbering of KP atoms is reported in Figure 1. The numbering of the atoms of the glucose moieties of β-CD (α-glucose linked by 1′−4′ bonds) was as usual for glucose from the 1′CH to 6′C (from the anomeric to the methylene group, respectively). DOSY NMR was selected as the privileged technique for the complex characterization study. In addition to evaluation of KP affinity, 1H DOSY experiments on KP at increasing amounts of β-CD allow determination of the [β-CD]/[KP] molar ratio required for full KP inclusion and preliminary evaluation of 10672

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The Journal of Physical Chemistry B slight difference observed is in agreement with previously reported values obtained by NMR18 and with values of free energy calculated by MD simulation.22 Photodegradation of R-, S-KP and of R-, S-KP/β-CD in Water Solution. The photostability of KP alone and included in β-CD at physiological pH, at different irradiation times (1, 3, and 6 min), was investigated by applying a well-described protocol for studies in water media.8 HPLC−UV−MS profiles of the photolysis products (TPs) in free KP samples were compared with those of the corresponding complex solutions. The last time point of the study was set on the basis of literature data on the kinetics of KP degradation. The main TPs were identified by MS of the observed protonated forms (M + H) and reference data retrieved in the literature for the KP transformation pathway.9,10 The major species observed (UV abundance 220 nm >5%, for at least one of the study times) are reported in Table 1. The residual KP concentration in the free KP sample was greater than the one observed in the complex solutions. Comparing the three study times, the percentage concentrations of KP for free and complex forms were found to be 74 versus 52 (time 1), 37 versus 35 (time 2), and 7 versus 5 (time 3), respectively. Data observed for the first study time suggest a faster degradation rate of the KP complex in the first minute, whereas values become more comparable after 3 min. The decarboxylated form TP1 is observed as the main product in the complex solutions (11−47%) with a percent of formation 10 times greater than that observed for free KP solutions. Considering decarboxylation as the first transformation to occur, the data probably indicate a slight protection of TP1 in β-CD toward the subsequent oxidation and transformation in the other TPs. Although a rough evaluation may suggest an overall decreased photoprotection with increased level of decarboxylated TP, the analysis should consider the specific differences observed in the TP patterns for the two forms (Table 1), which may account for a different phototoxicity. It is noteworthy that the bis-oxidized product TP4 is not observed in the complexed KP samples, whereas it shows a relative content of approximately 10% in the free KP solutions. The peculiar results prompted us to investigate possible conformational changes occurring upon inclusion that may account for the modified photoreactive properties. NMR Spectral Changes upon Inclusion. With the aim of clarifying the conformational KP changes associated with the complex formation, the 1H NMR spectra at different [β-CD]/ [KP] molar ratios were analyzed. The aromatic region of the NMR spectra showed significant both upfield and downfield shifts of specific resonances, as reported in Figure 3A. The unaffected resonances in the aromatic region are shown in Figure 3B. The complete identification of the spin systems and their variations of chemical shift upon β-CD addition at different molar ratios were unambigously obtained. 2D NMR experiments, such as TOCSY, ROESY, 13C HSQC, and 13C HMBC experiments, were performed to resolve overlapping resonances. Some representative examples of these results are reported in Figure 4A−C for characteristic regions of the 2D spectra. To further confirm the assignment of the resonance changes detected in the NMR spectra by the TOCSY analysis, the result of visual inspection of the progressive chemical shift changes upon inclusion was cross-checked with the heteronuclear

Figure 3. (a) Marked spectral changes in the aromatic region of KP upon addition of increasing amount of β-CD. β-CD/KP molar ratios: (A) 0.2, (B) 0.6, (C) 0.8, (D) 1.0, (E) 2.0, and (F) 5.0. (b) Unaffected resonances in the aromatic region of KP upon addition of increasing amount of β-CD. β-CD/KP molar ratios: (A) 0.2, (B) 0.6, (C) 0.8, (D) 1.0, (E) 2.0, and (F) 5.0.

couplings observed for each proton resonance (data not shown). The deshielding effect observed for aromatic protons b, and e, i at 1:1 molar ratio is reported in Figure 4B,C, respectively. In Figure 4B, expansion of the cross-peaks of the HMBC spectrum and the resonance of the chiral carbon of KP C− H(m) in the range 7.53−7.74 ppm are shown, together with assignment of the coupled aromatic protons. In Figure 4C, expansion of the cross-peaks of the carbonyl carbon (k) of KP with resonances in the region 7.26−7.82 ppm is reported, together with assignment of the coupled aromatic protons. Assignments of the inclusion complex were further supported by the analysis of dipolar interactions in the ROESY spectra. In fact, the NOE interactions in the ROESY spectra gave further evidence of the KP inclusion in the β-CD cavity. In Figure 5, a selected region of the ROESY spectra of an equimolar solution of KP and β-CD clearly displays cross-peaks due to the dipolar interactions between the resonances H-2′ and H-5′ of β-CD and the KP aromatic protons. Dipolar 10673

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Figure 5. Region of the ROESY spectrum of the KP/β-CD (1:1): Dipolar connectivities among protons b, e−h, and a of the aromatic region of KP with protons 2′ and 5′ of β-CD and internal NOEs of KP between the protons b and e with n.

concentrations. Spectra of KP alone were recorded in a concentration range from 0.05 to 3.0 mM, and no changes in NMR spectra were observed (data not shown). As the β-CD magnetic properties could not explain the observed deshielding effects, the observed changes supported our first hypothesis on the relevant conformational change of KP molecule upon inclusion. Changes in the orientation of C O groups and aromatic rings often account for a different magnetic environment of the protons in distinct and blocked conformations.54 Ab Initio Calculations and MD Simulation of the KP βCD Complex. The conformation of the KP (R-enantiomer) in aqueous solution and upon insertion into β-CD has been investigated. To provide initial configurations for the MD simulations and some missing potential parameters of the force field (i.e., potential parameters involved in the rotation of the phenyl rings), ab initio calculations were performed. The optimization of R-KP in vacuo in its anionic (deprotonated) form leads to an angle between the phenyl rings (the torsional angle between the carbon atoms labeled as e−j−o−d in Figure 1) of 51.1°. The calculation of the “relaxed” rotational profile along the dihedrals labeled e−j−k−l, d−o−k−l, and p−d−e−o (see Figure 1) leads to very similar energy profiles, and the rotational barrier, which is due to the repulsive interaction between the aromatic H atoms in positions e and d, resulted to be about 6 kcal/mol. These values were used to define the corresponding potential parameters in the MD simulation force field. To generate initial configurations of R-KP inserted into βCD for running the MD simulations, we have repeated a series of optimizations on the R-KP/β-CD complex at the same level of theory as for R-KP (see the Experimental section). Given that the inclusion complex has a formidable conformational repertoire, we have generated six possible initial structures (three in the up and other three in the down conformation see the Experimental section) by means of a series of short

Figure 4. (A) Aromatic regions of the NMR TOCSY spectrum of KP at different [β-CD]/[KP] molar ratios: violet 0.4, red 0.6, and black 1.0. The lines are guidance for the eye. (B) Expansion of HMBC scalar correlation of chiral carbon CH(m) in the range 7.53−7.74 for (I) KP in water and (II) [β-CD]/[KP] 1:1. (C) Expansion of the HMBC scalar correlation of KP carbonyl (k) with resonances in the region 7.26−7.83 ppm for (I) [β-CD]/[KP] 1:1 and (II) KP in water.

interactions (NOEs) of n with the aromatic protons b,e of KP are indicated. Considering the significant chemical shift changes observed upon inclusion, we investigated the dependence of this effect on association of KP molecules at different free drug 10674

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The Journal of Physical Chemistry B molecular mechanics simulations based on the use of the mm3 force field. These six structures were further optimized at the B3LYP/6-31G(d) level before starting the MD simulations. To establish the modification of the structural parameters of R-KP upon insertion into β-CD, we have used MD simulation. Three 20 ns-long MD trajectories were run for both up and down complexes. Although in two of the three simulations of the up complex the R-KP molecule runs apart from the β-CD molecule, the down complex remains stable in all three simulations. Hence, in what follows, structural analyses of the inclusion complex are presented only for the down complex, which is the most stable. This result is in agreement with a ̀ previously reported MD simulation of the two isomers R and S.22 One 10 ns-long simulation was also performed for the free R-KP in aqueous solution. The dihedral angle between the planes defined by the two aromatic rings of R-KP (labeled e−j−o−d in Figure 1) was calculated in the three simulations of the down inclusion complex and in the simulation of the free R-KP. The corresponding distributions are reported in Figure 6. From the comparison, it results that the most probable angle between the two aromatic rings decreases by approximately 5° upon inclusion of R-KP into β-CD (from 38 to 33°).

Figure 7. Distribution of the dihedral angle determining the relative position of the carbonyl and carboxyl groups (dihedral angle labeled e−j−o−d in Figure 1) calculated from the simulations of the down inclusion complex (black) and of the free R-KP (red).

Figure 8. Most populated configuration of the down inclusion complex upon MD simulations. The hydrogen bonds between the carbonyl group and β-CD and between the carboxyl group and β-CD are highlighted in blue.

Figure 6. Distribution of the dihedral angle labeled e−j−o−d (Figure 1) calculated from the simulations of the down inclusion complex (black) and from the free R-KP simulation (red).

KP and that included in β-CD molecules. The mean number of hydrogen bonds in the free KP is approximately 40, and the difference between the free KP and water molecules is approximately 8, for a total of 48 hydrogen bonds. Instead, in the inclusion complex, the mean number of hydrogen bonds among β-CD, R-KP, and water molecules is approximately 43. Concerning the water molecule network, the mean number of hydrogen bonds within the solvent decreases by 5 units upon inclusion (from free KP and β-CD molecules to the inclusion complex). Hence, upon inclusion of R-KP into β-CD, the mean number of hydrogen bonds decreases by approximately 10 units. Factorization of Magnetic Field Anisotropy and the Prediction of the Chemical Shift Changes upon Inclusion. The comparison between the calculated values and the experimental NMR values was performed as reported in the Experimental section, calculating, from the structure of the complex, the effects of the chemical shift anisotropies of the shielding−deshielding groups present in free KP and KP included in β-CD. The calculated and experimental changes of

In addition, the dihedral angle determining the relative position of the carbonyl and carboxyl groups (dihedral angle labeled e−j−o−d in Figure 1) was calculated from the simulations of the inclusion complex and of the free R-KP, and the corresponding distributions are reported in Figure 7. Whereas in the free R-KP the COO- group adopts two possible orientations (representative molecular snapshots are reported in Figure 7), in the inclusion complex the COOgroup is found in only one of the two positions (snapshot on the left of Figure 7). This indicates that the carboxyl group has a preferential orientation when included in the ring of β-CD. Analysis of the hydrogen bonds56 between R-KP and β-CD reveals that the structure of R-KP is stabilized in the complex by the formation of hydrogen bonds between the oxygen atoms of both carbonyl and carboxyl oxygens and hydrogen atoms of βCD (see Figure 8). The network of hydrogen bonds was analyzed in the simulations of the down inclusion complex and of the free R10675

DOI: 10.1021/acs.jpcb.6b07913 J. Phys. Chem. B 2016, 120, 10668−10678

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The Journal of Physical Chemistry B chemical shifts for the KP resonances upon β-CD inclusion are reported in Figure 9.

The insertion into β-CD has been investigated by NMR spectroscopy and by DOSY. Structural details have been studied by resonance assignments and by measuring KP chemical shift variations related to changes in the magnetic anisotropy of mutual orientation(s) of aromatic rings and the carbonyl moiety of KP. The results of mechanistic studies set the ground for the hypothesis that a conformational change could account for the unexpected behavior of the complex under UV irradiation. In fact, the relation between the torsional angle in benzophenone structures and their key physicochemical properties, including the ability to absorb UVA and UVB radiation, has been widely investigated, and a very good linear relationship between the molecular dihedral angles and Hammett’s p-substituent constants was previously described.57,58 MD simulation supported the hypothesis that the inclusion process may be associated with a significant internal conformational change of KP, characterized by a marked reduction in the most probable dihedral angle (from 38 to 33°) between the planes defined by the two aromatic rings of KP (labeled e−j− o−d in Figure 1). Starting from the results of MD calculations, the theoretical prediction of the anisotropic chemical shift contributions and the chemical shift of KP in the free and complexed states led to very good agreement with the values observed in the NMR spectra.

Figure 9. NMR chemical shift differences Δδ between free KP and KP included in β-CD due to magnetic ring current anisotropy contributions. In gray, experimental values and in white, theoretical calculations of the two conformers as generated by MD simulation.

The perfect agreement between the two sets of values supports the concept that the observed shift in KP chemical shift may be ascribed to the change in geometry induced by the inclusion process. On the other hand, the anisotropy generated by the conformational rearrangement of the aromatic rings and carbonyl/carboxyl groups of KP upon insertion into β-CD is perfectly coherent with the reported upfield and downfield chemical shifts. A consequence is that the contribution of polar and inductive effects by the surrounding β-CD ring can be considered negligible with respect to the chemical shift changes in the NMR spectra. These results confirm the strong reliability of MD simulations and reinforce the hypothesis that the geometry change, occurring upon inclusion of KP into β-CD, may account, also, for the observed modification of the photoreactivity pattern. In fact, the conformational change of KP induced by the inclusion process is mainly characterized by a reduction in the dihedral angle between the two phenyl rings and by a reduced mobility of the carboxyl group, the latter favored by the formation of hydrogen bonds between the carboxyl oxygen atoms and β-CD.

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CONCLUSIONS In the large variety of papers focusing on the structural analysis of β-CD complexes, this work represents one of the few examples in which a detailed analysis of these simultaneous upfield−downfield NMR shifts of the same aromatic molecule upon inclusion in β-CD is reported. Interestingly, the results demonstrate that the observed upfield and downfield shifts upon inclusion are related to the change in geometry of the magnetically anisotropic groups of the KP molecule, excluding any direct magnetic role of β-CD. This observation is coherent with the widely accepted notion that only small shifts are induced by the solvent effect due to the magnetic dielectric properties. In parallel with the structural determination, a detailed analysis of the KP/β-CD model allowed the prediction of the total number of hydrogen bond interactions established between KP and β-CD and between KP and accessible water molecules to be assessed. A comparative study showed that the total number of hydrogen bonds decreases by 10 units upon inclusion of KP into the β-CD cavity, leading to the argument that there is a major entropic contribution to the driving force of the inclusion reaction. This evidence is in agreement with previous theories about the nature of the driving force of the inclusion mechanism. Several authors in fact mainly attributed the extraordinary ability of CDs to host a large variety of aromatic molecules to a hydrophobic effect59 and supported the so-called “high-energy” water theory, according to which the entropic effect(s) associated with the removal of water molecules “iced” in the center of the ring should be the negative component of the driving force to the insertion of guest aromatic probes.59,60 Differently from the above-mentioned hypothesis, our results suggest that the loss of conformational entropy due to the reduced mobility of KP in the complex is overcompensated by an entropic gain associated not only with the release of water molecules from the interior of β-CD (as previously suggested)

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DISCUSSION The inclusion of photoreactive/phototoxic molecules in CD has been proposed as a promising strategy to protect the guest molecule from photodegradation, thus minimizing the phototoxic characteristics of topical formulations for cosmetic or pharmaceutical use. KP among others has been more frequently implicated in photosensitivity reactions. The photoreactive behavior of the β-CD/KP complex was studied under standard UV exposure conditions in water. The β-CD inclusion did not prevent the photodegradation process, instead it modifies significantly the transformation pattern. On one hand, the decarboxylation reaction was increased, and on the other hand, the formation of secondary degradation products derived by hydroxylation of the benzophenone ring was found nearly completely inhibited. The latter observation has been interpreted as a consequence of the reduced solvent exposure of the benzophenone ring in the β-CD complex.57,58 10676

DOI: 10.1021/acs.jpcb.6b07913 J. Phys. Chem. B 2016, 120, 10668−10678

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The Journal of Physical Chemistry B

(5) Al-Rawashdeh, N. A. F.; Al-Sadeh, K. S.; Al-Bitar, M. B. Inclusion complexes of sunscreen agents with β-Cyclodextrin: Spectroscopic and molecular modeling studies. J. Spectrosc. 2013, 2013, No. 841409. (6) Bani-Yaseen, A. D.; Al-Rawashdeh, N. F.; Al-Momani, I. Influence of inclusion complexation with β-cyclodextrin on the photostability of selected imidazoline-derived drugs. J. Inclusion Phenom. Macrocyclic Chem. 2009, 63, 109−115. (7) Bagheri, H.; Lhiaubet, V.; Montastruc, J. L.; Chouini-Lalanne, N. Photosensitivity to ketoprofen: mechanisms and pharmacoepidemiological data. Drug Saf. 2000, 22, 339−49. (8) Lu, W. L.; Zhang, Q.; Zheng, L.; Wang, H. M.; Li, R. Y.; Zhang, L. F.; Shen, W. B.; Tu, X. D. Antipyretic, analgesic and antiinflammatory activities of ketoprofen beta-cyclodextrin inclusion complexes in animals. Biol. Pharm. Bull. 2004, 27, 1515−20. (9) Metamoros, V.; Duhec, A.; Albaigés, J.; Bayona, J. M. Photodegradation of Carbamazepine, Ibuprofene, Ketoprofen and 17a-Ethylestradiol in fresh and seawater. Water, Air, Soil Pollut. 2009, 196, 161−168. (10) Jakimska, A.; Sliwka-Kaszynska, M.; Reszezynska, J.; Namiesnik, J.; Kot-Wasik, A. Elucidation of transformation pathway of KP, ibuprofen, and furosemide in surface water and their occurrence in the aqueous enviroment using UHPLC-QTOF-MS. Anal. Bioanal. Chem. 2014, 406, 3667−3680. (11) Boscá, F.; Miranda, M. A.; Carganico, G.; Mauleon, D. Photochemical and Photobiological Properties of Ketoprofen associated with the Benzophenone Chromophore. Photochem. Photobiol. 1994, 60, 96−101. (12) Lhiaubet, V.; Gutierrez, F.; Penaud-Berruyer, F.; Amouyal, J. E.; Daudey, P.; Poteau, R.; Chouini-Lalannea, N.; Paillous, N. Spectroscopic and theoretical studies of the excited states of fenofibric acid and ketoprofen in relation with their photosensitizing properties. New J. Chem. 2000, 24, 403−410. (13) Musa, K. A. K.; Matxain, J. M.; Eriksson, L. A. Mechanism of Photoinduced decomposition of ketoprofen. J. Med. Chem. 2007, 50, 1735−1743. (14) Cosa, G.; Martinez, L. J.; Scaiano, J. C. Influence of solvent polarity and base concentration on the photochemistry of ketoprofen: independent singlet and triplet pathways. Phys. Chem. Chem. Phys. 1999, 1, 3533−3537. (15) Monti, S.; Sortino, S.; De Guidi, G.; Marconi, G. Supramolecular photochemistry of 2-(3-benzoylphenyl) propionic acid (Ketoprofen). A study in the β-cyclodextrin cavity. New J. Chem. 1998, 22, 599−604. (16) Pessine, F. B. T.; Calderini, A.; Alexandrino, G. L. Review: Cyclodextrin Inclusion Complexes Probed by NMR Techniques. In Magnetic Resonance Spectroscopy; Kim, D.-H., Ed.; InTech: Rijeka, 2012. (17) Schneider, H. J.; Hacket, F.; Rudiger, V. NMR Studies of Cyclodextrins and cyclodextrin complexes. Chem. Rev. 1998, 98, 1755−1780. (18) Marconi, G.; Mezzina, E.; Manet, I.; Manoli, F.; Zambelli, B.; Monti, S. A stereoselective interaction of ketoprofen enantiomers with β-cyclodextrin: ground state binding and photochemistry. Photochem. Photobiol. Sci. 2011, 10, 48−59. (19) Mura, P.; Bettinetti, G. P.; Manderioli, A.; Faucci, M. T.; Bramanti, G.; Sorrenti, M. Interactions of ketoprofen and ibuprofen with β -cyclodextrins in solution and in the solid state. Int. J. Pharm. 1998, 166, 189−203. (20) Al Omaria, M. M.; Nidal, H.; Musa, D.; El-Barghouthib, I.; Zughulc, M. B.; Chowdhryd, B. Z.; Leharned, S. A.; Badwana, A. A. Novel inclusion complex of ibuprofen tromethamine with cyclodextrins: Physico-chemical characterization. J. Pharm. Biomed. Anal. 2009, 50, 449−458. (21) Jana, M.; Bandyopadhayay, S. Molecular dynamics study of the β-Cylodextrin − Phenylalanine (1:1) inclusion complex in aqueous medium. J. Phys. Chem. B 2013, 117, 9280−9287. (22) Shi, M.; Zhang, C.; Xie, Y. Stereoselective inclusion mechanism of ketoprofen into β-cyclodextrin: insights from molecular dynamics

but also with the loss of hydrogen bonds between KP and water molecules and within the water molecules themselves. Furthermore, the MD simulation results indicate that in the case of KP β-CD, a large network of hydrogen bonds between carboxylate anions and CD hydroxy groups stabilizes KP at the higher region of the β-CD ring. This hydrogen bond network has already been reported as observed in MD simulation.22 Linked in that position, the aromatic rings of KP assume a conformation compatible with the insertion in the ring(s) of βCD, leading to the spatial conformation as determined in detail by NMR spectroscopy. From a conformational point of view, the change in KP induced by the inclusion process is mainly characterized by a reduction in the angle between the two phenyl rings and by a reduced mobility of the carboxyl group, the latter favored by the formation of hydrogen bonds between the carboxyl oxygen atoms and β-CD. This decreases the steric hindrance of KP in such a way that it can be more easily allocated within the ring of β-CD. As a general conclusion, the change in conformation of KP upon inclusion clearly influences the conjugation of electronic orbitals and, then, the photoreactivity. But the interpretation of these results does not appear to be simple. The concept that inclusion in CD may still represent a general strategy to gain a photoprotective effect in topical cosmetic or pharmaceutical formulations should be further investigated on the basis of the results of this structural and photodegradation study on the KP/β-CD complex. The results described in this article do not easily correlate with the structural influence of the photoreaction behavior upon inclusion in β-CD, and the atypical photosensitivity of the KP/β-CD complex could be due to secondary photodegradation processes not necessarily connected with decarboxylation. A case-by-case analysis is, then, recommended to unravel the structural changes associated with the inclusion of a guest molecule in the CD cavity and to predict the impact on the key physicochemical and photoreactivity characteristics of the guest.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b07913. Atom coordinates and absolute energies of calculated structures (PDF)

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AUTHOR INFORMATION

Author Contributions ∇

T.G., W.M., and R.N. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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REFERENCES

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