Synthesis of Spin-Labeled Ibuprofen and Its Interaction with Lipid Membranes

Ibuprofen is a non-steroidal anti-inflammatory drug possessing analgesic and antipyretic activity. Electron paramagnetic resonance (EPR) spectroscopy could be applied to study its interaction with biological membranes and proteins if its spin-labeled analogs were synthesized. Here, a simple sequence of ibuprofen transformations—nitration, esterification, reduction, Sandmeyer reaction, Sonogashira cross-coupling, oxidation and saponification—was developed to attain this goal. The synthesis resulted in spin-labeled ibuprofen (ibuprofen-SL) in which the spin label TEMPOL is attached to the benzene ring. EPR spectra confirmed interaction of ibuprofen-SL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. Using 2H electron spin echo envelope modulation (ESEEM) spectroscopy, ibuprofen-SL was found to be embedded into the hydrophobic bilayer interior.


Introduction
Ibuprofen, along with aspirin and paracetamol, is the most significant non-steroidal anti-inflammatory drug, and is widely used for analgesic and antipyretic purposes. The over-the-counter status of these drugs are based on years of extensive research into their efficacy and safety [1][2][3]. In particular, the general pattern of blocking prostaglandin synthesis via inhibition of membrane enzyme cyclo-oxygenase (COX) by ibuprofen, as well as the parallel side effects caused by this inhibition, mainly in the gastrointestinal tract, has been studied in detail [4]. The other beneficial properties of ibuprofen, such as slowing down neurodegenerative diseases [5][6][7] and cancer [8][9][10][11] have also been shown. Improvement of ibuprofen-containing drugs is carried out in several directions: increasing its bioavailability [12,13], its use in combination with other drugs [14,15], reducing the negative impact on the gastrointestinal tract [16], and others. In this regard, new data on the processes of interaction of ibuprofen with lipid membranes and with proteins, previously inaccessible for detection and description, could be extremely useful. Different methods, such as X-ray diffraction/scattering [17,18], differential scanning calorimetry [19], quasielastic neutron scattering [20], fluorescence [21], neutron spin echo [22], electrochemical impedance [23], Raman microspectroscopy [24], vibrational sum frequency spectroscopy [25], powder NMR [26] and electron paramagnetic resonance (EPR) spectroscopy [27,28] are used to study ibuprofen-mediated changes in model lipid membranes.
Spin-label EPR spectroscopy [29] allows one to obtain structural and dynamical information at the molecular level, of the kind that is accessible only with this technique. In particular, a pulsed EPR version of electron spin echo envelope modulation (ESEEM) spectroscopy probes the position of molecules in the membranes [30][31][32][33], while double electron-electron resonance (DEER, also known as PELDOR) spectroscopy probes molecular self-assembling in membranes and other biological systems [34][35][36][37][38].
For EPR applications, employment of spin-labeled ibuprofen would be desirable. Usually the nitroxyl label is introduced into ibuprofen through the transformation of the carboxyl group: condensation reactions of ibuprofen with amino or hydroxyl derivatives of nitroxides under the action of dehydrating agents (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or N,N -dicyclohexylcarbodiimide/N,N-dimethyl-4-aminopyridine) were used to synthesize esters [39] and amides [40], respectively. A significant disadvantage of this approach is the replacement of an important functional group in the molecule, namely the carboxyl group, with an ester or amide group. In such cases, there is a change in the rate and efficiency of drug-target interactions, since the inhibition of COX is achieved, among other ways, through the formation of critical bonds of its active site with the carboxyl group of ibuprofen [41,42]. In addition, the carboxyl group is the hydrophilic "anchor" of ibuprofen, playing a key role in the drug's interaction with the lipid membranes and its components [43,44].
Therefore, introduction of a substituent in the benzene ring to preserve basic properties of the ibuprofen (the most important is amphiphilicity of the molecule) would be reasonable. Here, we report a six-step synthesis of a new ibuprofen spin-labeled derivative (ibuprofen-SL), containing a nitroxyl radical 2,2,6,6-tetramethylpiperidin-1-yl (TEMPOL) in the meta position relative to the 1-carboxyethyl group. Also, interaction of ibuprofen-SL with a model membrane of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was characterized by conventional and pulsed EPR.

Synthesis
The route of synthesis is presented in Scheme 1. We used the Sonogashira crosscoupling of 4-ethynyl-2,2,6,6-tetramethylpiperidin-4-ol with iodo-ibuprofen methyl ester 2 to assemble a precursor 3, which, after oxidation with hydrogen peroxide followed by saponification with aqueous sodium hydroxide solution in one pot, gave the target product 1. This order of transformations is a good alternative to the use of ethynyl-nitroxides in cross-coupling, which is usually difficult due to the side processes resulting in low yields of spin-labeled products [45][46][47]. Standard procedures for the oxidative iodination (I 2 /HIO 3 /AcOH) of ibuprofen have resulted in an inseparable mixture of 2-and 3-iodoibuprofen ( Figure S1). Therefore, the necessary compound 2 was obtained in several steps. Initially, ibuprofen was carefully nitrated with nitric acid in sulfuric acid at −5 • C in 89% yield. Esterification of the nitro compound 4 with methanol in the presence of sulfuric acid gave the ester 5 (98% yield). The amine 6 was obtained in 92% yield by reduction with iron filings in aqueous methanol at 65 • C. In the next step, the amine 6 was diazotized with sodium nitrite in hydrochloric acid and subjected to Sandmeyer reaction with potassium iodide in water, to prepare the iodo-ibuprofen methyl ester 2 (51% yield). The structure and purity of the obtained compounds 1-6 were confirmed by elemental analysis, IR, 1 H-NMR, 13 C-NMR and mass spectrometry (see Supplementary Materials).

Conventional EPR Spectra: Interaction with the POPC Membrane
EPR spectrum of ibuprofen-SL dissolved in toluene at concentration of 1 mM was recorded at room temperature (25 • C)-see Figure 1. This spectrum is typical for the nitroxide rapidly tumbling in a solution. The hyperfine interaction a constant directly obtained from the spectrum is 1.5 ± 0.02 mT.  Figure 1 also shows room-temperature EPR spectra for ibuprofen-SL in the presence of POPC bilayers, for the samples obtained in two ways. First, bilayers were prepared from an initial ibuprofen-SL/POPC mixture (1:99 mol/mol). Secondly, a dimethyl sulfoxide (DMSO) solution of ibuprofen-SL was added to pure POPC bilayers. One can see from Figure 1 that both methods of sample preparation result in very similar EPR spectra. A slight difference between them is the presence of a small admixture of a narrow triplet for the second kind of the sample. This narrow triplet obviously belongs to ibuprofen-SL which did not enter the membrane but remains in the water phase. Also, a slight narrowing of the EPR lines is here seen; as DMSO is less viscous than the lipid bilayer, this narrowing can be readily explained by the plasticization effect of DMSO.
The EPR spectra in the POPC bilayer show that molecular motion is hampered remarkably, as compared with the freely rotating case of toluene solution (see Figure 1). This retardation is typical for EPR spectra of spin-labeled molecules in room-temperature POPC membrane-see, e.g., [48]. Therefore, we may conclude that in our samples the ibuprofen-SL molecules interact with the membranes.

Pulsed EPR: Location in the POPC Membrane
Information about location of the ibuprofen-SL molecule in the membrane can be obtained using pulsed EPR. The approach used [30][31][32][33] employs ESEEM spectroscopy.
The ESEEM effect appears because of an anisotropic hyperfine interaction of the unpaired electron of the spin label with the nearby nuclei; its magnitude is determined by the number of nuclei located in the immediate proximity of the spin label-at distances less than 1 nm [30][31][32][33]. 2 H ESEEM of D 2 O-hydrated membranes allows one to determine the location of the spin label respectively to the membrane surface.
The ESEEM signal time trace for ibuprofen-SL in D 2 O-hydrated POPC bilayers, obtained as a function of the delay t (see Experimental), E(t), was refined from the influence of the background spin relaxation by normalizing to the mean echo decay, <E(t)>, [30][31][32][33]: The result is shown in Figure 2a. Also in Figure 2a are the analogously obtained reference data on stearic acids, doxyl-spin-labeled at the n-th carbon atom positions along the carbon chain, n-DSA, and the analogous data [49] on 2-oleoyl-1-palmitoyl-sn-glycero-3phospho(TEMPO)choline (denoted as TEMPO-PC), in which the spin label is attached to the polar lipid head. The corresponding Fourier transforms (see [32]) are given in Figure 2b. One can see from the data presented in Figure 2 that ESEEM amplitudes for all the samples are different. The largest amplitude is for the TEMPO-PC sample which can be readily understood because the spin label here is directly exposed to the water shell. For the spin-labeled stearic acids, n-DSA, the signal amplitude becomes subsequently smaller with the n increase, which corresponds to increasing the distance from the membrane surface.
The ESEEM amplitude for ibuprofen-SL in Figure 2 demonstrates its closeness to that of the 5-DSA sample. It is known from molecular dynamics (MD) simulations [50], that the carbon atom positions in the spin-labeled stearic acids resemble approximately those for the host bilayer lipid (with some minor deviation). Note that in the 5-DSA molecule the location of the nitroxide is fixed by its covalent attachment to the alkane chain, whereas in ibuprofen-SL there could be a broader distribution including molecules buried deep into the membrane and others near the surface. Nevertheless, this closeness allows one to state that the spin label in the ibuprofen-SL molecule is located mostly in the membrane interior.
This statement is to be compared with the results of MD simulations for ibuprofen embedded in lipid bilayers [28,51,52] which indicate that ibuprofen molecules prefer to be located in the hydrophobic interior. This is not surprising because, although a hydrophilic carboxylate group of ibuprofen interacts with lipid hydrophilic heads, its large non-polar residue is indeed expected to be located in the hydrophobic interior. Therefore, the presence of a spin label does not remarkably change the hydrophobicity of the backbone of the parent ibuprofen molecule, which might lead to its excursion from the membrane interior. Therefore ibuprofen-SL molecules can adequately mimic the parent ibuprofen molecules embedded into the biological membrane.

General Information
Elemental analysis was performed with a CHN-analyzer (Model 1106, "Carlo Erba", Italy). NMR spectra were recorded on a Bruker AV-400 (400 ( 1 H) and 101 ( 13 C) MHz) or AV-500 (500 ( 1 H) and 126 ( 13 C) MHz) spectrometer in the CDCl 3 . Chemical shifts (δ) are given in ppm with reference to the residual signals of CDCl 3 (δ = 7.26 ppm for 1 H and δ = 77.16 for 13 C ppm). Melting points were determined with a 1101D Mel-Temp Digital Melting Point apparatus (Electrothermal). HRMS were measured on a Thermo Electron Corporation DFS mass spectrometer (70 eV), using direct injection, the temperature of the ionization chamber was 220-270 • C. The IR spectra were recorded on a Shimadzu IRTracer-100 instrument with GS10802-X Quest ATR ZnSe Accessory (Specac). Column chromatography was performed on silica gel (0.063-0.200 mm). Merck 60 WF254 plates were used for TLC analysis. All reagents and solvents were obtained from commercial sources and used directly after purchased without special purification. Racemic ibuprofen was purchased from Sigma-Aldrich.

Sample Preparations for EPR Investigation
To obtain EPR spectra in toluene solution, ibuprofen-SL was dissolved in toluene (Ekros-Analytica, St. Petersburg, Russia, distilled before the use) at concentration of 1 mM.
The bilayer samples with ibuprofen-SL were prepared in two ways. For the first method, POPC and ibuprofen-SL were dissolved separately in chloroform, then the two solutions were mixed, so that the ibuprofen-SL/POPC molar ratio in the mixture was 1:99. Then the solvent was removed in a nitrogen stream and the mixture was stored under vacuum for 4 h. Then phosphate-buffered saline (pH 7.0) was added to the resulting powder in a proportion of 10:1 (v/v), the sample was stirred and then stored for 2 h. Upon this procedure, multilamellar vesicles (MLVs) are formed [54]. Then the mixture was centrifuged to remove excess solvent. For some experiments, deuterium-substituted water has been used instead of ordinary water. For the second method, MLVs were similarly prepared, but without ibuprofen-SL; then, a dimethylsulfoxide (DMSO) solution of ibuprofen-SL was subsequently added. DMSO content was less than 10 vol% respectively to the sample volume; the total ibuprofen-SL quantity was 100 times smaller than the total lipid quantity (mol/mol).
For comparative purposes, POPC bilayers containing 1 mol% of doxyl-spin-labeled stearic acids n-DSA (n = 5, 12 or 16) were also prepared, in a manner similar to the first method described above.
The prepared samples were put into EPR glass tubes of 3 mm o.d. and studied either at room temperature or at 80 K. In the latter case, the samples were quickly frozen by immersion into liquid nitrogen.

EPR Measurements
Conventional EPR spectra were obtained at room temperature with an X-band EPR benchtop SPINSCAN-X spectrometer (ADANI, Minsk, Belorussia), operating at modulation amplitude of 0.01 mT, with the output microwave (MW) power of 100 mW, the MW attenuation set to −25 dB, and with sweep and constant times of 60 s and 46 ms, respectively.
In pulsed EPR studies, an X-band Bruker ELEXSYS E580 EPR spectrometer was used equipped with a split-ring Bruker ER 4118 X-MS-3 resonator and an Oxford Instruments CF-935 cryostat. A three-pulse ESEEM sequence (π/2)-τ-(π/2)-t-(π/2)-τ-echo was employed, with excitation at the maximum of the echo-detected EPR spectrum. The pulse lengths were 16 ns, time delay τ was 204 ns, the time delay t was scanned from 300 ns to 10 µs, with a 12 or 16 ns time step. The resonator was cooled with a stream of cold nitrogen gas. The temperature was controlled by a nitrogen flow stabilized by a Bruker ER4131VT temperature controller, the sample temperature was kept near 80 K.

Conclusions
It is shown in this work that ibuprofen-a non-steroidal anti-inflammatory drug possessing analgesic and antipyretic activity-can be labeled by a TEMPOL spin label via a simple six-step sequence of nitration, esterification, reduction, Sandmeyer reaction, Sonogashira cross-coupling, oxidation and saponification. The TEMPOL spin label in ibuprofen is coupled to the benzene core of the molecule via a triple bond.
EPR data show that spin-labeled ibuprofen interacts with lipid membranes. The immersion depth into the membrane was found with pulsed EPR ( 2 H ESEEM spectroscopy) in D 2 O-hydrated bilayers, the results indicate that spin label is buried into the membrane interior, which is in agreement with literature MD data on the immersion of the ibuprofen molecule. So, the proposed method of synthesis allows one to obtain spin-labeled ibuprofen that can adequately mimic the parent ibuprofen molecules interacting with the biological membrane.