Next Article in Journal
Detection Method and Common Characteristics of Waste Solvent from Semiconductor Industry
Next Article in Special Issue
3-Ferrocenyl-estra-1,3,5 (10)-triene-17-one: Synthesis, Crystal Structure, Hirshfeld Surface Analysis, DFT Studies, and Its Binding to Human Serum Albumin Studied through Fluorescence Quenching and In Silico Docking Studies
Previous Article in Journal
Chitosan and Its Carboxymethyl-Based Membranes Produced by Crosslinking with Magnesium Phytate
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 28
Issue 16
10.3390/molecules28165991
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Spin-Labeled Diclofenac: Synthesis and Interaction with Lipid Membranes

by
Denis S. Baranov
1,
Anna S. Kashnik
1,
Anastasiya N. Atnyukova
2 and
Sergei A. Dzuba
1,*
1
Voevodsky Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, 630090 Novosibirsk, Russia
2
Department of Physics, Novosibirsk State University, 630090 Novosibirsk, Russia
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(16), 5991; https://doi.org/10.3390/molecules28165991
Submission received: 21 July 2023 / Revised: 7 August 2023 / Accepted: 8 August 2023 / Published: 10 August 2023
(This article belongs to the Special Issue Organic Synthesis and Application of Bioactive Molecules)
Browse Figures
Versions Notes

Abstract

:
Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) from the group of phenylacetic acid derivatives, which has analgesic, anti-inflammatory and antipyretic properties. The interaction of non-steroidal anti-inflammatory drugs with cell membranes can affect their physicochemical properties, which, in turn, can cause a number of side effects in the use of these drugs. Electron paramagnetic resonance (EPR) spectroscopy could be used to study the interaction of diclofenac with a membrane, if its spin-labeled analogs existed. This paper describes the synthesis of spin-labeled diclofenac (diclofenac-SL), which consists of a simple sequence of transformations such as iodination, esterification, Sonogashira cross-coupling, oxidation and saponification. EPR spectra showed that diclofenac-SL binds to a lipid membrane composed of palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). 2H electron spin echo spectroscopy (ESEEM) was used to determine the position of the diclofenac-SL relative to the membrane surface. It was established that its average depth of immersion corresponds to the 5th position of the carbon atom in the lipid chain.

1. Introduction

Non-steroidal anti-inflammatory drugs are effective antipyretic and analgesic pharmacological agents [1], which are also used in the treatment of other diseases, including cancer [2], arthritis [3,4] and neurodegenerative diseases [5]. The most commonly used NSAIDs are ibuprofen, naproxen and diclofenac [6]. Due to their wide application, these compounds are found even in the environment in the form of human waste [7].
Like all NSAIDs, diclofenac works by inhibiting the activity of the enzyme cyclooxygenase (COX) to disrupt prostaglandin synthesis [8]. However, alternative mechanisms of action of NSAIDs associated with the membrane activity of drugs are known [9,10,11]. For example, changes in lipid composition and/or membrane structure can lead to the development of various cardiovascular pathologies, including hypertension, myocardial infarction and thrombosis [12]. Diclofenac has demonstrated the ability to chemically interact with gastrointestinal protective-barrier phospholipids, which may contribute to its gastrointestinal toxicity [13,14]. Therefore, understanding the mechanisms of interaction of diclofenac with the plasma membrane at the molecular level can be extremely useful for understanding the therapeutic effect of this drug and developing ways to limit the side effects.
Among the huge variety of experimental approaches aimed at studying the molecular mechanisms of the interaction of NSAIDs with membranes [6,9,10,11,13,14,15,16,17,18,19,20,21,22], one can also use electron paramagnetic resonance (EPR) spectroscopy of spin-labeled drug molecules [23]. The spin-label EPR method provides different possibilities for studying molecules and intermolecular interactions [24]: rotational motions on the ns-timescale, molecular librations (sub-ns), polarity and H-bonding, accessibility and proximity, intermolecular and intramolecular separations (the nm-scale) and others. Also, the large magnetic moment of the electron spin makes it possible to reduce the studied concentrations to tenths of a mole percent, which can be close to therapeutic doses.
This paper describes the synthesis of spin-labeled diclofenac and its interaction with a model membrane characterized by conventional and pulsed EPR. To introduce a nitroxide spin label into drugs that are derivatives of carboxylic acids, the use of transformations of carboxyl groups is the simplest way. Different variants of esterification and amidation reactions are well developed and widely used for the synthesis of esters and amides of many drugs [25,26,27,28]. For example, spin-labeled esters and amides of aspirin [29], ibuprofen [30], indomethacin [31] and diclofenac [32,33] were synthesized for their study using EPR. However, the loss of the carboxyl group in the original molecule of drugs is a significant drawback for their use as model objects of study. Indeed, the data from studies of the processes of action of NSAIDs [34], as well as the fact that the vast majority of them are derivatives of organic acids, convincingly indicate the exceptional importance of the carboxyl group.
Recently, we developed an alternative route to introduce the nitroxyl label into ibuprofen via the ethynylation of the benzene ring while retaining the carboxyl group and its associated molecular properties, particularly amphiphilicity [23]. Here, we report procedures for the synthesis of a novel diclofenac derivative in which the nitroxyl radical is linked to the benzene ring through an alkyne moiety distant from the carboxymethyl group.

2. Results and Discussion

2.1. Synthesis

The sequence of chemical transformation carried out in this work is shown in Scheme 1. To obtain spin-labeled diclofenac 1 (diclofenac-SL), we used a four-step synthesis, which is a new and simpler modification of our approach previously developed for ibuprofen [23]. Initially, iododiclofenac 2 was obtained in an 89% yield using the oxidative iodination of diclofenac with KI, NaIO4 and NaCl in acetic acid at room temperature. Then, iododiclofenac was converted into its methyl ester 3 in a 95% yield using an esterification reaction in boiling methanol with sulfuric acid. In the next step, a Pd/Cu-catalyzed Sonogashira coupling reaction of iodide 3 with 4-ethynyl-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine in the presence of Et3N at 75 °C produced precursor 4 (an 87% yield). Finally, the precursor was subjected to peroxidation in dioxane followed by saponification with aqueous sodium hydroxide to the target spin-labeled product 1 in a 34% yield.
Diclofenac has a tendency for complex degradation when interacting with hydrogen peroxide in the presence of catalysts, such as peroxidases [35,36]. The poor yield of the key product in the last step indicates that the Na2WO4 acts similarly to peroxidases. In addition, there is a gradual reduction in the compound during purification and storage, which is typical of many nitroxide radicals.
The structure and purity of synthesized compounds 1–4 were confirmed with 1H-NMR and 13C-NMR (see Supplementary Materials). According to the 1H-NMR spectra of the product, after a month of storage in the environment (see Figure S7), an accumulation of impurities in the region of 1.3 ppm (Me groups) was observed.

2.2. EPR Spectra: Interaction with the POPC Membrane

The CW EPR spectra at room temperature (25 °C) obtained for diclofenac-SL are shown in Figure 1. In Figure 1a, the spectrum is given for diclofenac-SL dissolved in toluene at a concentration of 5 mM. This spectrum is characteristic of nitroxide in an isotropic medium in a state of fast tumbling motion of a spin label in a solution [24]. The value of the g-factor for this spectrum is 2.0023 ± 0.0001 and the hyperfine interaction constant is a = 1.5 ± 0.02 mT.
Figure 1b shows the EPR spectra for diclofenac-SL in palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) multilamellar vesicles. Samples were prepared in two ways (see Section 3 below). In the first one, vesicles were prepared from the initial mixture of diclofenac-SL/POPC mixture (1:99 mol/mol). In the second, diclofenac dissolved in dimethyl sulfoxide (DMSO) was added to pure POPC vesicles. One can see from Figure 1b that the difference in the EPR spectra for the two different preparation methods is insignificant.
It is important that the spectra in Figure 1b show a significant decrease in the amplitude of the high-field component compared to the other two components (cf. Figure 1a). This decrease indicates a substantial retardation of the motion [24], which can take place only if the molecules are bound to the membrane. Thus, these data clearly indicate that diclofenac-SL is incorporated into the membrane.

2.3. Pulsed EPR: Location in the POPC Membrane

ESEEM spectroscopy is capable of providing information about the spatial position of the spin label [37,38,39,40]. The ESEEM effect is caused with an anisotropic hyperfine interaction of an unpaired electron of a spin label electron with neighboring nuclei. The ESEEM intensity depends on the distance r between the electron and nuclear spins as r−6, and disappears at distances greater than 1 nm [37,38,39,40]. The ESEEM frequencies are determined with the type of nucleus; therefore, if the membrane is hydrated with deuterium water, then 2H ESEEM allows you to distinguish the signal exclusively from water. Also, it depends on the number of the surrounding nuclei. Thus, 2H ESEEM makes it possible to determine the position of the spin label relative to the membrane surface: the larger the ESEEM signal, the closer the spin label is to the surface.
The 2H ESEEM time traces E(t) for diclofenac-SL in D2O-hydrated POPC bilayers were refined from the background spin relaxation by dividing by the mean echo decay, <E(t)>, as described previously [37,38,39,40]:
E n ( t ) = E ( t ) < E ( t ) > − 1
Figure 2a shows these refined ESEEM time traces for diclofenac-SL in D2O-hydrated POPC vesicles. Figure 2a also provides reference data on stearic acids, doxyl-spin-labeled (at the n-th carbon atom) positions along the carbon chain, n-DSA [23] and data [41] on 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho(tempo)choline (TEMPO-PC). In TEMPO-PC, the spin label is attached to the polar lipid head. The corresponding Fourier transforms are shown in Figure 2b.
It can be seen that the TEMPO-PC sample shows the highest amplitude, which is obviously due to the fact that the spin label is located directly in the water shell. For spin-labeled stearic acids, n-DSA, the signal amplitude becomes smaller with an increasing n, which corresponds to immersion inside the membrane. For diclofenac-SL, the ESEEM amplitude shows a close proximity to that of the 5-DSA sample. So, we may conclude that the spin label is embedded into the membrane interior and that its mean position corresponds to the fifth position of the carbon atom in the lipid chain.

3. Materials and Methods

3.1. Chemical Analysis

1H and 13C spectra were recorded with a Bruker AV-500 (500 (1H) and 126 (13C) MHz) spectrometer in CDCl3 or DMSO-D6 solvents, by using residual signals of undeuterated solvents (CHCl3: δ = 7.26 ppm for 1H and δ = 77.16 for 13C ppm. DMSO: δ = 2.50 ppm for 1H) as an internal standard. Melting points were measured with an Electrothermal MEL-TEMP 1101D apparatus. HRMS was recorded with a Thermo Scientific DFS high-resolution mass spectrometer (Thermo Electron Corp., Waltham, MA, USA). The IR spectra were obtained on a Shimadzu IRTracer-100 instrument with a GS10802-X Quest ATR ZnSe Accessory (Specac, Kyoto, Japan). Column chromatography was carried out using silica gel (70−230 mesh ASTM). All reagents and solvents were obtained from commercial sources and used without special purification. Diclofenac was purchased from Sigma-Aldrich (China).

3.2. Synthesis and Characterization

4-Ethynyl-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine was obtained according to the method reported in [42].
  • {2-[(2,6-Dichlorophenyl)amino]-5-iodophenyl}acetic acid (2). KI (1.8 g, 10.8 mmol) was added in several portions to a stirred mixture of diclofenac (3.2 g, 10.8 mmol), NaCl (1.26 g, 21.5 mmol), NaIO4 (2.3 g, 10.8 mmol) and H2O (10 mL) in AcOH (60 mL) at room temperature for 24 h. After the disappearance of the iodine color, the precipitate was filtered, washed with H2O and dried in air. The crude product was purified with flash chromatography on silica gel (eluent: ethyl acetate). Yield 4.0 g (88%), white solid, mp 191–193 °C (toluene). 1H-NMR (500 MHz, DMSO-D6) δ: 7.53 (m, 3H), 7.35 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.23 (m, 1H), 6.04 (d, J = 8.4 Hz, 1H), 3.68 (s, 2H) (see 1H-NMR in [43,44]). IR (film) cm−1: 3352 (NH), 3182, 3028, 2814 (OH), 1693 (C=O).
  • Methyl {2-[(2,6-dichlorophenyl)amino]-5-iodophenyl}acetate (3). A mixture of acid 2 (2.0 g, 4.75 mmol) and concentrated H2SO4 (1 mL) in methanol (45 mL) was stirred at reflux for 0.3 h. Then, the reaction mixture was cooled to room temperature; the precipitate was isolated using filtration, washed with methanol and dried in air. Yield 1.96 g (95%), white solid, mp 119–120 °C (methanol). 1H-NMR (500 MHz, CDCl3) δ: 7.53 (d, J = 1.5 Hz, 1H), 7.39 (dd, J = 8.5, 1.8 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.01 (t, J = 8.0 Hz, 1H), 6.94 (br.s, 1H), 6.28 (d, J = 8.5 Hz, 1H), 3.76 (s, 3H), 3.75 (s, 2H). 13C-NMR (126 MHz, CDCl3) δ: 172.24, 142.83, 139.44, 137.19, 136.95, 129.96, 129.08, 126.29, 124.78, 119.97, 84.13, 52.73, 38.16. IR (film) cm−1: 3321 (NH), 2949 (Me), 1714 (C=O). HRMS (ESI) m/z: [M]+ Calcd for C15H12Cl2INO2 434.9284. Found 434.9285.
  • Methyl {2-[(2,6-dichlorophenyl)amino]-5-[(2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridin-4-yl)ethynyl]phenyl}acetate (4). Under an argon atmosphere, a mixture of iodide 3 (500 mg, 1.15 mmol), PdCl2(PPh3)2 (20 mg, 0.03 mmol) and CuI (10 mg, 0.05 mmol) in toluene (20 mL) was stirred and heated to 55 °C. Then, Et3N (8 mL) and 4-ethynyl-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine (270 mg, 1.65 mmol) were added, and the reaction mixture was stirred at 75 °C for 16 h. After cooling, toluene (100 mL) was added and the mixture was filtered. The extract was washed with 6 M of aqueous ammonia (2 × 200 mL) and dried over MgSO4. The crude product was purified with column chromatography on silica gel (eluent: toluene/ethyl acetate, 4:1). Yield 470 mg (87%), white solid, mp 150–151 °C (ethyl acetate). 1H-NMR (500 MHz, CDCl3) δ: 7.34 (m, 3H, HAr), 7.19 (m, 1H), 7.11 (s, 1H), 7.01 (t, J = 8.0 Hz, 1H), 6.45 (d, J = 8.2 Hz, 1H), 6.05 (s, 1H), 3.76 (s, 2H), 3.75 (s, 3H), 2.87 (br.s, 1H), 2.07 (s, 2H), 1.25 (s, 6H), 1.20 (s, 6H). 13C-NMR (126 MHz, CDCl3) δ: 172.52, 142.71, 140.23, 137.06, 134.25, 131.34, 129.90, 129.04, 124.68, 123.59, 117.68, 116.46, 116.33, 89.61, 87.68, 52.70, 51.91, 49.71, 41.03, 38.46, 30.94, 29.75. IR (film) cm−1: 3348 (NH), 2961 (Me), 1745 (C=O). HRMS (ESI) m/z: [M]+ Calcd for C26H28Cl2N2O2 470.1522. Found 470.1521.
  • [4-{[3-(Carboxymethyl)-4-((2,6-dichlorophenyl)amino)phenyl]ethynyl}-2,2,6,6-tetramethyl-3,6-dihydropyridin-1(2H)-yl]oxidanyl (1). A mixture of compound 4 (120 mg, 0.25 mmol), Na2WO4·2H2O (13 mg, 0.04 mmol), EDTA disodium salt (13 mg, 0.04 mmol) and 30% H2O2 (0.3 mL) in 1,4-dioxane (3 mL) was stirred at the ambient temperature for 14 days. The suspension was filtered, and the solvent was evaporated under a vacuum condition. Then, 2 M of NaOH (3 mL) was added, and the reaction mixture was stirred at 80 °C for 0.5 h. Next, the resulting mixture was diluted with ethanol (3 mL) and stirred at reflux for 0.25 h. After cooling, H2O (5 mL) and CH2Cl2 (15 mL) were added. At 0 °C, 1 M of HCl was added dropwise to the stirred mixture until the pH was neutral. The organic layer was separated and dried over MgSO4. The solvent was evaporated under a vacuum condition. The crude product was purified with column chromatography on silica gel (eluent: toluene/ethyl acetate, 5:1). Yield 40 mg (34%), white solid, mp 190–192 °C (toluene). 1H-NMR (500 MHz, CDCl3) δ: 7.51 (m, 4H), 7.10 (s, 1H), 6.95 (s, 1H), 6.46 (s, 1H), 3.98 (s, 2H). 13C-NMR (126 MHz, CDCl3) δ: 176.065, 140.80, 135.85, 130.19, 128.73, 128.02, 127.33, 123.77, 122.90, 119.32, 117.51, 38.66. IR (film) cm−1: 3300 (NH), 3042 (OH), 2928, 2851 (Me), 2203 (C≡C), 1695 (C=O). HRMS (ESI) m/z: [M]+ Calcd for C25H25Cl2N2O3 471.1237. Found 471.1235.

3.3. Sample Preparations for EPR Investigation

For obtaining EPR spectra in the toluene solution, diclofenac-SL 1 was dissolved in toluene (Ekros-Analytica, St. Petersburg, Russia; distilled before use) at a concentration of 5 mM. Lipids’ POPC was from Avanti Polar Lipids (Birmingham, AL, USA).
The vesicle samples were prepared in two ways. First, POPC and diclofenac-SL were dissolved separately in chloroform, then two solutions were mixed, so that the diclofenac-SL/POPC molar ratio was 1:99. The solvent was removed in the nitrogen stream, with the subsequent storing of the mixture under a vacuum for 4 h. Then, phosphate-buffered saline (pH 7.0) was added to the resulting powder in a proportion of 10:1. The sample was stirred, then stored for 2 h, and the resulting vesicles were centrifuged to remove the excess solvent. Instead of ordinary water, deuterium-substituted water was used in some measurements.
For the second way of sample preparation, POPC vesicles were prepared in the same way but without diclofenac-SL, with the subsequent addition of a dimethylsulfoxide (DMSO) solution of diclofenac-SL (DMSO content was less than 10 vol% respective to the sample volume). The total diclofenac-SL mole content was 100 times smaller than the total lipid mole content.
The samples were placed in glass EPR tubes with an outer diameter of 3 mm and examined either at room temperature (25 °C) or at 80 K. In the latter case, the samples were quickly frozen using immersion in liquid nitrogen.

3.4. EPR Measurements

Conventional EPR spectra were obtained at room temperature with a Bruker ESP 380E spectrometer operating at a modulation amplitude of 0.01 mT, with the output microwave (MW) power of 100 mW, the MW attenuation set up to 25 dB and the sweep and constant times of 60 s and 46 ms, respectively. A Bruker ER 4118 X-MD-5 dielectric resonator was used. 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.
The 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 and the time delay τ was 204 ns, and 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 with a nitrogen flow stabilized by a Bruker ER4131VT temperature controller. The sample temperature was kept near 80 K.

4. Conclusions

This work shows that the NSAID diclofenac can be spin-labeled through a simple four-step sequence that includes iodination, esterification, Sonogashira cross-coupling, oxidation and saponification. The obtained CW EPR data indicate that diclofenac-SL binds to model lipid membranes. The ESEEM spectroscopy shows that diclofenac molecules are located under the polar heads of lipids with an average position near the fifth position of the carbon of the lipid chain.
Note that a standard problem with the labeling approach is that the label distorts the original molecule. In our case, the main structural properties of the diclofenac molecule are a polar carboxyl residue and a Cl-containing aromatic ring, and these properties are not violated in the suggested labeling scheme.
On the other hand, the advantages of spin-label EPR are that it makes it possible to reduce the studied concentrations to close to therapeutic doses and that it provides information of a different kind at the molecular level, such as molecular motions; polarity and H-bonding; accessibility and proximity; and nanoscale structure. With regard to the latter possibility, it has been recently shown that pulsed EPR in the version of double electron–electron resonance (DEER, also known as PELDOR) allows for obtaining data on the nanoscale clustering of mono-spin-labeled molecules [45,46,47,48].

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28165991/s1, Figure S1: 1H-NMR spectrum of compound 2 in DMSO-D6; Figures S2–S5: 1H-NMR spectrum of compounds 3–4 in CDCl3; Figure S6: 1H-NMR spectrum of compound 1 in CDCl3; Figure S7: 1H-NMR spectrum of compound 1 in CDCl3 after 1 month of storage in the environment; Figure S8: 13C-NMR spectrum of compound 1 in CDCl3.

Author Contributions

Methodology, synthesis and writing, D.S.B.; investigation, data curation and writing, A.S.K.; investigation, A.N.A.; conceptualization, writing—review and editing, S.A.D. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Russian Science Foundation, project # 21-13-00025.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Manrique-Moreno, M.; Heinbockel, L.; Suwalsky, M.; Garidel, P.; Brandenburg, K. Biophysical study of the non-steroidal anti-inflammatory drugs (NSAID) ibuprofen, naproxen and diclofenac with phosphatidylserine bilayer membranes. Biochim. Biophys. Acta 2016, 1858, 2123–2131. [Google Scholar] [CrossRef]
  2. Ulrich, C.M.; Bigler, J.; Potter, J.D. Non-steroidal anti-inflammatory drugs for cancer prevention: Promise, perils and pharmacogenetics. Nat. Rev. Cancer 2006, 6, 130–140. [Google Scholar] [CrossRef]
  3. Crofford, L.J. Use of NSAIDs in treating patients with arthritis. Arthritis Res. Ther. 2013, 15, S2. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. González-Gay, M.A.; González-Juanatey, C. NSAIDs and cardiovascular risk in arthritis. Nat. Rev. Cardiol. 2017, 14, 69–70. [Google Scholar] [CrossRef]
  5. Lleo, A.; Galea, E.; Sastre, M. Molecular targets of non-steroidal anti-inflammatory drugs in neurodegenerative diseases. Cell. Mol. Life Sci. 2007, 64, 1403–1418. [Google Scholar] [CrossRef] [PubMed]
  6. Moreno, M.M.; Garidel, P.; Suwalsky, M.; Howe, J.; Brandenburg, K. The membrane-activity of ibuprofen, diclofenac, and naproxen: A physico-chemical study with lecithin phospholipids. Biochim. Biophys. Acta Biomembr. 2009, 1788, 1296–1303. [Google Scholar] [CrossRef] [Green Version]
  7. Madikizela, L.M.; Chimuka, L. Determination of ibuprofen, naproxen and diclofenac in aqueous samples using a multi-template molecularly imprinted polymer as selective adsorbent for solid-phase extraction. J. Pharm. Biomed. Anal. 2016, 128, 210–215. [Google Scholar] [CrossRef] [PubMed]
  8. Warner, T.D.; Giuliano, F.; Vojnovic, I.; Bukasa, A.; Mitchell, J.A.; Vane, J.R. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: A full in vitro analysis. Proc. Natl. Acad. Sci. USA 1999, 96, 7563–7568. [Google Scholar] [CrossRef] [PubMed]
  9. Leite, C.P.; Nunes, C.; Reis, S. Interaction of nonsteroidal anti-inflammatory drugs with membranes: In vitro assessment and relevance for their biological actions. Prog. Lipid Res. 2013, 52, 571–584. [Google Scholar] [CrossRef]
  10. Zhou, Y.; Plowman, S.J.; Lichtenberger, L.M.; Hancock, J.F. The anti-inflammatory drug indomethacin alters Nanoclustering in synthetic and cell plasma membranes. J. Biol. Chem. 2010, 285, 35188–35195. [Google Scholar] [CrossRef] [Green Version]
  11. Seddon, A.M.; Casey, D.; Law, R.V.; Gee, A.; Templer, R.H.; Ces, O. Drug interactions with lipid membranes. Chem. Soc. Rev. 2009, 38, 2509–2519. [Google Scholar] [CrossRef] [PubMed]
  12. Wallace, J.L. Prostaglandins, NSAIDs, and gastric mucosal protection: Why doesn’t the stomach digest itself? Physiol. Rev. 2008, 88, 1547–1565. [Google Scholar] [CrossRef] [PubMed]
  13. Fernandes, E.; Soares, T.B.; Gonçalves, H.; Bernstorff, S.; Real Oliveira, M.E.C.D.; Lopes, C.M.; Lúcio, M. A Molecular Biophysical Approach to Diclofenac Topical Gastrointestinal Damage. Int. J. Mol. Sci. 2018, 19, 3411. [Google Scholar] [CrossRef] [Green Version]
  14. Lichtenberger, L.M.; Zhou, Y.; Dial, E.J.; Raphael, R.M. NSAID injury to the gastrointestinal tract: Evidence that NSAIDs interact with phospholipids to weaken the hydrophobic surface barrier and induce the formation of unstable pores in membranes. J Pharm. Pharmacol. 2006, 58, 1421–1428. [Google Scholar] [CrossRef] [PubMed]
  15. Alsop, R.J.; Armstrong, C.L.; Maqbool, A.; Toppozini, L.; Dies, H.; Rheinstädter, M.C. Cholesterol expels ibuprofen from the hydrophobic membrane core and stabilizes lamellar phases in lipid membranes containing ibuprofen. Soft Matter 2015, 11, 4756–4767. [Google Scholar] [CrossRef] [Green Version]
  16. Sharma, V.K.; Mamontov, E.; Tyagi, M. Effects of NSAIDs on the nanoscopic dynamics of lipid membrane. Biochim. Biophys. Acta Biomembr. 2020, 1862, 183100. [Google Scholar] [CrossRef]
  17. Yefimova, S.L.; Tkacheva, T.N.; Kasian, N.A. Study of the Combined Effect of Ibuprofen and Cholesterol on the Microviscosityand Ordering of Model Lipid Membranes by Timeresolved Measurement of Fluorescence Anisotropy Decay. J. Appl. Spectrosc. 2017, 84, 284–290. [Google Scholar] [CrossRef]
  18. Ramadurai, S.; Sarangi, N.K.; Maher, S.; Macconnell, N.; Bond, A.M.; McDaid, D.; Flynn, D.; Keyes, T.E. Microcavity-Supported Lipid Bilayers; Evaluation of Drug-Lipid Membrane Interactions by Electrochemical Impedance and Fluorescence Correlation Spectroscopy. Langmuir 2019, 35, 8095–8109. [Google Scholar] [CrossRef]
  19. Wood, M.; Morales, M.; Miller, E.; Braziel, S.; Giancaspro, J.; Scollan, P.; Rosario, J.; Gayapa, A.; Krmic, M.; Lee, S. Ibuprofen and the Phosphatidylcholine Bilayer: Membrane Water Permeability in the Presence and Absence of Cholesterol. Langmuir 2021, 37, 4468–4480. [Google Scholar] [CrossRef]
  20. Sun, S.; Sendecki, A.M.; Pullanchery, S.; Huang, D.; Yang, T.; Cremer, P.S. Multistep Interactions between Ibuprofen and Lipid Membranes. Langmuir 2018, 34, 10782–10792. [Google Scholar] [CrossRef]
  21. Kremkow, J.; Luck, M.; Huster, D.; Müller, P.; Scheidt, H.A. Membrane interaction of ibuprofen with cholesterol-containing lipid membranes. Biomolecules 2020, 10, 1384. [Google Scholar] [CrossRef] [PubMed]
  22. Aloi, E.; Rizzuti, B.; Guzzi, R.; Bartucci, R. Association of ibuprofen at the polar/apolar interface of lipid membranes. Arch. Biochem. Biophys. 2018, 654, 77–84. [Google Scholar] [CrossRef] [PubMed]
  23. Baranov, D.S.; Smorygina, A.S.; Dzuba, S.A. Synthesis of Spin-Labeled Ibuprofen and Its Interaction with Lipid Membranes. Molecules 2022, 27, 4127. [Google Scholar] [CrossRef] [PubMed]
  24. Marsh, D. Spin-Label Electron Paramagnetic Resonance Spectroscopy; CRC Press: Boca Raton, FL, USA, 2020. [Google Scholar] [CrossRef]
  25. Kozak, K.R.; Prusakiewicz, J.J.; Rowlinson, S.W.; Marnett, L.J. Enantiospecific, Selective Cyclooxygenase-2 Inhibitors. Bioorg. Med. Chem. Lett. 2002, 12, 1315–1318. [Google Scholar] [CrossRef] [PubMed]
  26. Jilani, J.; Idkaidek, N.; Alzoubi, K. Synthesis, In Vitro and In Vivo Evaluation of the N-Ethoxycarbonylmorpholine Ester of Diclofenac as a Prodrug. Pharmaceuticals 2014, 7, 453–463. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Tammara, V.K.; Narurkar, M.M.; Crider, A.M.; Khan, M.A. Synthesis and Evaluation of Morpholinoalkyl Ester Prodrugs of Indomethacin and Naproxen. Pharm. Res. 1993, 10, 1191–1199. [Google Scholar] [CrossRef]
  28. Gupta, K.; Kaub, C.J.; Carey, K.N.; Casillas, E.G.; Selinsky, B.S.; Loll, P.J. Manipulation of Kinetic Profiles in 2-Aryl Propionic Acid Cyclooxygenase Inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 667–671. [Google Scholar] [CrossRef]
  29. Akdogan, Y.; Emrullahoglu, M.; Tatlidil, D.; Ucuncu, M.; Cakan-Akdogan, G. EPR Studies of Intermolecular Interactions and Competitive Binding of Drugs in a Drug–BSA Binding Model. Phys. Chem. Chem. Phys. 2016, 18, 22531–22539. [Google Scholar] [CrossRef] [Green Version]
  30. Sasaki, K.; Ito, T.; Fujii, H.G.; Sato, S. Synthesis and Reduction Kinetics of Five Ibuprofen–Nitroxides for Ascorbic Acid and Methyl Radicals. Chem. Pharm. Bull. 2016, 64, 1509–1513. [Google Scholar] [CrossRef] [Green Version]
  31. Flores-Santana, W.; Moody, T.; Chen, W.; Gorczynski, M.J.; Shoman, M.E.; Velázquez, C.; Thetford, A.; Mitchell, J.B.; Cherukuri, M.K.; King, S.B.; et al. Nitroxide Derivatives of Non-Steroidal Anti-Inflammatory Drugs Exert Anti-Inflammatory and Superoxide Dismutase Scavenging Properties in A459 Cells. Br. J. Pharmacol. 2012, 165, 1058–1067. [Google Scholar] [CrossRef] [Green Version]
  32. Golubeva, E.N.; Chumakova, N.A.; Kuzin, S.V.; Grigoriev, I.A.; Kalai, T.; Korotkevich, A.A.; Bogorodsky, S.E.; Krotova, L.I.; Popov, V.K.; Lunin, V.V. Paramagnetic Bioactives Encapsulated in Poly(D,L-Lactide) Microparticules: Spatial Distribution and in Vitro Release Kinetics. J. Supercrit. Fluids 2020, 158, 104748. [Google Scholar] [CrossRef]
  33. Chumakova, N.A.; Golubeva, E.N.; Kuzin, S.V.; Ivanova, T.A.; Grigoriev, I.A.; Kostjuk, S.V.; Melnikov, M.Y. New Insight into the Mechanism of Drug Release from Poly(d,l-Lactide) Film by Electron Paramagnetic Resonance. Polymers 2020, 12, 3046. [Google Scholar] [CrossRef] [PubMed]
  34. Rainsford, K.D. (Ed.) Ibuprofen: Discovery, Development and Therapeutics; Wiley: Hoboken, NJ, USA, 2015. [Google Scholar] [CrossRef]
  35. Galati, G.; Tafazoli, S.; Sabzevari, O.; Chan, T.S.; O’Brien, P.J. Idiosyncratic NSAID Drug Induced Oxidative Stress. Chem. Biol. Interact. 2002, 142, 25–41. [Google Scholar] [CrossRef]
  36. Boelsterli, U. Diclofenac-Induced Liver Injury: A Paradigm of Idiosyncratic Drug Toxicity. Toxicol. Appl. Pharmacol. 2003, 192, 307–322. [Google Scholar] [CrossRef]
  37. Salnikov, E.S.; Erilov, D.A.; Milov, A.D.; Tsvetkov, Y.D.; Peggion, C.; Formaggio, F.; Toniolo, C.; Raap, J.; Dzuba, S.A. Location and aggregation of the spin-labeled peptide trichogin GA IV in a phospholipid membrane as revealed by pulsed EPR. Biophys. J. 2006, 91, 1532–1540. [Google Scholar] [CrossRef]
  38. Erilov, D.A.; Bartucci, R.; Guzzi, R.; Shubin, A.A.; Maryasov, A.G.; Marsh, D.; Dzuba, S.A.; Sportelli, L. Water concentration profiles in membranes measured by ESEEM of spin-labeled lipids. J. Phys. Chem. B 2005, 109, 12003–12013. [Google Scholar] [CrossRef]
  39. Milov, A.D.; Samoilova, R.I.; Shubin, A.A.; Grishin, Y.A.; Dzuba, S.A. ESEEM measurements of local water concentration in D2O-containing spin-labeled systems. Appl. Magn. Reson. 2008, 35, 73–94. [Google Scholar] [CrossRef]
  40. Dzuba, S.A.; Marsh, D. ESEEM of Spin Labels to Study Intermolecular Interactions, Molecular Assembly and Conformation. In A Specialist Periodic Report, Electron Paramagnetic Resonance; Gilbert, C., Chechik, V., Murphy, D.M., Eds.; RSC Publishing: Cambridge, UK, 2015; Volume 24, pp. 102–121. [Google Scholar]
  41. Konov, K.B.; Isaev, N.P.; Dzuba, S.A. Glycerol penetration profile in phospholipid bilayers measured by ESEEM of spin-labelled lipids. Mol. Phys. 2013, 111, 2882–28863. [Google Scholar] [CrossRef]
  42. Shapiro, A.B.; Skripnichenko, L.N.; Pavlikov, V.V.; Rozantsev, E.G. Synthesis of Nitroxyl Radicals Based on 4-Ethynyl-4-Hydroxy-2,2,6,6-Tetramethylpiperidine. Russ. Chem. Bull. 1979, 28, 140–148. [Google Scholar] [CrossRef]
  43. Tanwar, L.; Börgel, J.; Lehmann, J.; Ritter, T. Selective C-H Iodination of (Hetero)Arenes. Org. Lett. 2021, 23, 5024–5027. [Google Scholar] [CrossRef]
  44. Dalai, P.G.; Palit, K.; Panda, N. Generation of Dimethyl Sulfoxide Coordinated Thermally Stable Halogen Cation Pools for C−H Halogenation. Adv. Synth. Catal. 2022, 364, 1031–1038. [Google Scholar] [CrossRef]
  45. Smorygina, A.S.; Golysheva, E.A.; Dzuba, S.A. Clustering of Stearic Acids in Model Phospholipid Membranes Revealed by Double Electron−Electron Resonance. Langmuir 2021, 37, 13909–13916. [Google Scholar] [CrossRef] [PubMed]
  46. Unguryan, V.V.; Golysheva, E.A.; Dzuba, S.A. Double electron-electron resonance of spin-labeled cholestane in model membranes: Evidence for substructures inside the lipid rafts. J. Phys. Chem. B 2021, 125, 9557–9563. [Google Scholar] [CrossRef] [PubMed]
  47. Kashnik, A.S.; Baranov, D.S.; Dzuba, S.A. Ibuprofen in a lipid bilayer: Nanoscale spatial arrangement. Membranes 2022, 12, 1077. [Google Scholar] [CrossRef]
  48. Kashnik, A.S.; Syryamina, V.N.; Biondi, B.; Peggion, C.; Formaggio, F.; Dzuba, S.A. DEER/PELDOR Study of the Effect of Extremely Low Concentrations of the Antimicrobial Peptide Chalciporin A on the Membrane Lipid Organization. Appl. Magn. Reson. 2023, 54, 401–414. [Google Scholar] [CrossRef]
Molecules 28 05991 sch001
Scheme 1. Synthesis of diclofenac-SL 1.
Scheme 1. Synthesis of diclofenac-SL 1.
Molecules 28 05991 sch001
Molecules 28 05991 g001
Figure 1. EPR spectra at room temperature of diclofenac-SL for its 5 mM solution in toluene (a) and in the presence of POPC vesicles at its concentration of 1 mol% (b). In the latter case, the samples were prepared either by mixing diclofenac-SL and POPC prior to the sample preparation (solid line), or by adding the solution of diclofenac-SL in DMSO to the prepared vesicles (dashed line).
Figure 1. EPR spectra at room temperature of diclofenac-SL for its 5 mM solution in toluene (a) and in the presence of POPC vesicles at its concentration of 1 mol% (b). In the latter case, the samples were prepared either by mixing diclofenac-SL and POPC prior to the sample preparation (solid line), or by adding the solution of diclofenac-SL in DMSO to the prepared vesicles (dashed line).
Molecules 28 05991 g001
Molecules 28 05991 g002
Figure 2. (a) ESEEM time traces for diclofenac-SL in D2O-hydrated POPC bilayer. For comparison, the analogous data for TEMPO-PC and 5 (12,16)-DSA (see text) are given. The data are vertically shifted for convenience. (b) Their Fourier transforms.
Figure 2. (a) ESEEM time traces for diclofenac-SL in D2O-hydrated POPC bilayer. For comparison, the analogous data for TEMPO-PC and 5 (12,16)-DSA (see text) are given. The data are vertically shifted for convenience. (b) Their Fourier transforms.
Molecules 28 05991 g002
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Baranov, D.S.; Kashnik, A.S.; Atnyukova, A.N.; Dzuba, S.A. Spin-Labeled Diclofenac: Synthesis and Interaction with Lipid Membranes. Molecules 2023, 28, 5991. https://doi.org/10.3390/molecules28165991

AMA Style

Baranov DS, Kashnik AS, Atnyukova AN, Dzuba SA. Spin-Labeled Diclofenac: Synthesis and Interaction with Lipid Membranes. Molecules. 2023; 28(16):5991. https://doi.org/10.3390/molecules28165991

Chicago/Turabian Style

Baranov, Denis S., Anna S. Kashnik, Anastasiya N. Atnyukova, and Sergei A. Dzuba. 2023. "Spin-Labeled Diclofenac: Synthesis and Interaction with Lipid Membranes" Molecules 28, no. 16: 5991. https://doi.org/10.3390/molecules28165991

APA Style

Baranov, D. S., Kashnik, A. S., Atnyukova, A. N., & Dzuba, S. A. (2023). Spin-Labeled Diclofenac: Synthesis and Interaction with Lipid Membranes. Molecules, 28(16), 5991. https://doi.org/10.3390/molecules28165991

Article Metrics

Back to TopTop