Molecular docking and computational assessment of spectroscopic analysis of ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate as a potential antibacterial agent†

Wissam Habibi^a, Saadia Ouizat^b, Mohamed Chellegui*^cd, Bushra Shakoor^e, Marwa Alaqarbeh^f, Mohamed Adel Sayed^g, Mostafa Khouili^a, Abdessamad Tounsi^b, Haydar A. Mohammad-Salim^hi and Mohamed Anouar Harrad^j
aMolecular chemistry, Materials and Catalysis laboratory, Faculty of Sciences and technology, Sultan Moulay Slimane University, Beni-Mellal 23000, Morocco
^bEnvironmental, Ecological, and Agro-Industrial Engineering Laboratory, Sultan Moulay Slimane University, Beni-mellal 23000, Morocco
^cLaboratory of Organic Chemistry (LR17ES08), Faculty of Sciences, University of Sfax, 3038 Sfax, Tunisia. E-mail: mohamed.chellegui.etud@fss.usf.tn
^dNamur Institute of Structured Matter, University of Namur, Rue de Bruxelles, 61, B-5000 Namur, Belgium
^eSynthetic and Natural Products Drug Discovery Lab., Department of Chemistry, Government College Women University, Faisalabad, 38000, Pakistan
^fDepartment of Chemistry, Faculty of Science, Applied Science Private University, Amman, 11931, Jordan
^gPharmaceutical Chemistry Department, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo 11829, Egypt
^hDepartment of Chemistry, Faculty of Science, University of Zakho, Zakho 42002, Kurdistan Region, Iraq
ⁱTCCG Lab, Scientific Research Center, University of Zakho, Zakho 42002, Kurdistan Region, Iraq
^jInterdisciplinary Research Laboratory in Bioresources, Environment and Materials (LIRBEM) - École Normale Supérieure - Cadi Ayyad University, Morocco

First published on 11th August 2025

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1. Introduction

Chemical compounds known as enaminones consist of an amino group linked to a carbonyl group by a CC double bond. Enamine chemistry is an emerging area of organic synthesis. These ethylenes are push–pull in nature, with the amino group pushing the electron density because it is nucleophilic and the carbonyl attracting it because it is electrophilic. Heterogeneous catalysis, a method within green chemistry, facilitates the synthesis of enamines, which serve as versatile chemical intermediates.¹ The use of these molecules as building blocks in chemical synthesis has attracted attention in recent years.^2,3

These compounds demonstrate considerable reactivity in diverse reduction, oxidation, photochemical processes, and nucleophilic and electrophilic substitutions.^4,5 Moreover, they have served as precursors for numerous pharmaceutical compounds exhibiting anti-epileptic,⁶ antibacterial,⁷ anticonvulsant,⁸ anticancer,⁹ and antiparasitic activities,¹⁰ along with physiologically and therapeutically active compounds.^11,12

Minimal research has been conducted on quantum chemical calculations and their correlation with the experimental properties of enamines, as indicated by the bibliographic study.^13–18 Enaminones, including α,β-unsaturated esters bearing an amino substituent at the β-position such as ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate, represent a valuable class of compounds with diverse applications in heterocyclic chemistry and drug design.^19–21 This work details the synthesis and characterization of an enaminoester. The attributes of the synthesized compound's molecular structure were examined using theoretical calculations based on density functional theory (DFT). DFT calculations have proven to be powerful tools for probing molecular structures, energetics, and reactivity patterns in various chemical systems. These methods provide valuable insights that complement experimental findings.^22,23 Frontier molecular orbitals (FMO), including the highest occupied molecular orbital (HOMO)–lowest unoccupied molecular orbital (LUMO) energy gap, were also calculated using the DFT framework. The relative electrophilicity and nucleophilicity of the investigated compound was evaluated using the condensed Fukui function and the molecular electrostatic potential (MEP) surface. Molecular docking analysis was used to investigate the biological activities of the compound. The study concentrated on the active bending residues linked to hydrogen bonds and the bending energy of a specific chemical that demonstrates bacterial activity. Molecular docking simulations are widely employed to investigate non-covalent interactions and to predict the preferred binding orientation of small molecules within host systems or active sites. These approaches are particularly useful for exploring recognition processes and complement quantum chemical methods in studying supramolecular assemblies.^24,25 The physico-chemical properties and ADME-Tox predictions of the prospective bioactive compound were assessed. We also find inspiration in the recent developments in the application of enaminones and β-enamino esters in drug discovery, specifically antimicrobial activity. Previous studies have demonstrated the biological relevance of structurally congruent scaffolds to antibacterial, anticancer, and anti-inflammatory applications.^19–21 Taking advantage of these findings, we synthesized and characterized a novel β-enamino ester and employed a combined theoretical and molecular docking approach for the investigation of its chemical reactivity and antibacterial properties. This integrated approach via DFT, Fukui, MEP, and docking methods is a comprehensive assessment of the reactivity and biological relevance of the compound and is consistent with more recent literature on computational drug design.^22–26

2. Experimental methods

2.1. Materials

Analytical grade compounds, such as ethyl 3-oxo-3-phenylpropanoate and 4-chloro-2-methyl-aniline, were acquired from Janssen Chemica and Sigma-Aldrich.

2.2. Instrumentation

A Bruker Advanced 300 spectrometer at 300 MHz and 75 MHz in CdCl₃ solvent was used to obtain ¹H and ¹³C NMR spectra. Banc Kofler apparatus was used to determine the melting points. Fourier-transform infrared (FTIR) spectra were obtained utilizing a Nicolet is 5 Thermo scientific spectrometer. Pellets were composed of 100 mg of finely powdered KBr and 2 mg of the sample.

2.3. Synthesis of the β-enaminoester

15 mg of ZnAl₂O₄@ZnO was added to a magnetically stirred mixture of ethyl 3-oxo-3-phenylpropanoate (0.32 g; 1.7 mmol) and 4-chloro-2-methyl-aniline (0.23 g; 1.7 mmol) and the resulting solution was agitated under ambient conditions for 20 minutes. Upon completion of the reaction, 10 mL of distilled water was introduced to the residue and subsequently extracted with diethyl ether (3 × 25 mL). The organic layer was dehydrated with Na₂SO₄, and the solvent was evaporated at reduced pressure. The pure β-enamino ester was isolated using column chromatography on silica gel, employing a hexane/ethyl acetate solvent system. With a yield of 82%, the purified product was obtained as a bright colourless powder.

M.p. 116–118 °C. ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 1.13 (t, J = 6.6 Hz, 3H), 1.16–1.22 (m, 6H), 3.07–3.17 (m, 3H), 3.98 (q, J = 6.6 Hz, 2H), 4.53 (s, 1H), 7.01–7.15 (m, 2H, Ar), 9.73 (br s, 1H, NH). ¹³C NMR (75 MHz, CDCl₃): δ 12.31, 15.64, 17.65, 56.25, 83.12, 123.56, 133.74, 133.49, 133.73, 135.39, 157.26, 168.21.

3. Computational details

3.1. Density functional theory (DFT) calculations

The quantum calculations were carried out using density functional theory (DFT) with the Gaussian 16 software.²⁷ The geometric optimization of the studied compound was carried out with the hybrid functional B3LYP, which combines the three-parameter exchange potential of Becke and the correlation functional of Lee, Yang and Parr,²⁸ using the basic set 6-311++G(d,p). The D3 correction was also included to account for dispersion correction energies.²⁹ The vibrational frequencies were calculated at the same level of calculation to verify the nature of the minimum obtained. Under the same level of calculation, the frontier molecular orbitals (FMO), in particular the highest occupied molecular orbital (HOMO) and the lowest unoccupied orbital (LUMO), as well as the molecular electrostatic potential (MEP), were calculated and identified.

Following the approach of conceptual DFT (CDFT),^30–32 the global chemical reactivity indices, such as the global hardness (η),³³ the global softness (S), the absolute electronegativity (χ), the global electrophilicity (ω)³⁴ and the nucleophilicity (N),³⁵ have been calculated from the energies of the E_HOMO and E_LUMO boundary orbitals obtained, using the equations:

Hardness

η = ELUMO − EHOMO/2	(1)

Softness

S = 1/η	(2)

Electronegativity

χ = −EHOMO − ELUMO/2	(3)

Electrophilicity index

ω = χ2/2η	(4)

Nucleophilicity index

N = EmoleculeHOMO − ETCEHOMO	(5)

TCE is the tetracyano-ethylene reference.

In addition, the electrophilic P⁺_k(r) and nucleophilic P⁻_k(r) Parr functions^35,36 were calculated by analyzing the Mulliken atomic spin density for the anionic and cationic forms of the compound studied, following the method proposed by Domingo.³⁷ The local reactivity indices derived from these functions make it possible to identify the atoms or the molecular sites likely to behave as donors or acceptors of electrons.³⁸ These parameters provide essential information to better understand the chemical reactivity of the different regions of the molecule.

Nuclear magnetic resonance chemical shifts have been calculated with the gauge including the atomic orbital (GIAO) approach and compared with experimental spectra to determine the chemically significant area.

Gauss view 6.0 software³⁹ is used to obtain the highest occupied and lowest unoccupied molecular orbital maps (HOMO–LUMO), band energy gap and molecular electrostatic potential map (MEP) for identifying the potential region.

Non-covalent interaction (NCI) analysis, electron localization function (ELF), localized orbital locator (LOL), and Fukui function were carried out by Multiwfn,⁴⁰ which is a multifunctional wave function analysis program and all isosurface maps were rendered by the VMD program.⁴¹

3.2. Molecular docking simulations

Molecular operating environment (MOE) 2019.0102^42,43 was used for the preparation of both protein and ligands, molecular docking, and evaluation of ligand–protein interaction through visualization of poses and scoring function. The docking process was performed via the Amber10 protocol using docking placement: triangular matcher, rescoring: London dG, forcefield and refinement: affinity dG.

Copper-dependent laccase (CotA) [PDB id: 1of0], DNA gyrase B [PDB id: 6kzv] and wild-type Staph. aureus penicillin binding protein 4 (PBP4) [PDB id: 5tw8], are considered as important bacterial proteins in Bacillus subtilis, E. coli and Staph. aureus that play crucial roles in antimicrobial drug design through evolving research.^44,45 These isozymes were retrieved from the protein data bank (https://www.rcsb.org) with Bacillus subtilis [PDB id: 1of0], E. coli [PDB id: 6kzv] and Staph. aureus [PDB id: 5tw8]. The protein structure was checked and repaired through automatic correction and fixation order of MOE. Hydrogen atoms were added to the structure during the protonation step. The co-crystalized ligands were used to determine the binding sites. Then, the co-crystallized water molecules and bound ligands were removed. After docking completion, observation and filtration of the results through scoring values and visualized poses were performed.

MOE was used to prepare a 3D model library from the active and selective target compound (A). This compound was subjected to an energy minimization process and automatic calculation of the partial charges. Finally, this prepared library was saved in the form of an MDB file to be used in the docking calculations with target isozymes.

Validation of the docking protocol is performed via calculation of the root mean square deviation (RMSD). The RMSD is predicted via redocking of the co-crystalized ligand on its target enzyme then superimposing the redocked co-crystalized ligand on its original co-crystallized bound conformation. During this study, the RMSD of copper-dependent laccase (CotA) [PDB id: 1of0], DNA gyrase B [PDB id: 6kzv] and wild-type Staph. aureus penicillin binding protein 4 (PBP4) [PDB id: 5tw8], appeared in the acceptable range with values equal to 1.7295, 1.6839 and 1.2139 Å, respectively.

3.3. Molecular dynamic simulations

Compound A/(PBP4) of Staph. aureus complex was subjected to molecular dynamic analysis study. The MD simulations were carried out by iMod server (iMODS) (https://imods.chaconlab.org/) which gives a convenient interface for this enhanced normal mode analysis (NMA) methodology in inner coordinates⁴⁶ at 300 K constant temperature and 1 atm constant pressure. Finally, 50 ns molecular dynamic simulation was carried out for the target complex.

3.4. ADMET LAB 2.0 – drug likeness and ADME studies

ADMET LAB 2.0 platform (https://admetmesh.scbdd.com/) is a freely accessible web tool that has gathered the most relevant computational techniques to provide a global estimation of the pharmacokinetics profile of small molecules. Their methodologies were chosen by the web tool creators for their robustness, as well as their ease of interpretation, to allow effective translation to medicinal chemistry. Some of these approaches were updated by web tool designers utilising open-source algorithms, while others were unmodified versions of the original authors’ methods.⁴⁷ The molecular structure of the target compound A was uploaded into the ADMET LAB 2.0 web tool section using the simplified molecular-input line-entry specification (SMILES) nomenclature technique using Marvin sketch software 19.19 and then the result report was generated.

4. Results and discussion

The β-enaminoester was synthesized by condensation reaction of an amine with a dicarbonyl in the presence of ZnAl₂O₄@ZnO as a catalyst according to Scheme 1. The reaction was carried out solvent-free at room temperature with good yield. The compound obtained underwent analysis using NMR.

4.1. Synthesis of the title compound

The synthesis of the title compound was carried out in accordance with the current study concentrated on the theoretical and structural analysis of ethyl 3-(4-chloro-2-methylanilino)-3-phenylpropanoate 3. The title compound was synthesized according to the experimental procedures outlined in Scheme 1. Briefly, a stoichiometric quantity of ethyl 3-oxo-3-phenylpropanoate 1 and 2,4,6-trimethyl-phenylamine 2 was mixed in the presence of ZnAl₂O₄@ZnO at room temperature. The reaction completion was followed by TLC. At the end of the reaction followed by workup steps and purification by column chromatography, the desired product was isolated as a colourless powder with a yield of 82%.

Scheme attempts to illustrate the proposed mechanism for this reaction. According to this hypothesis, the reaction begins with amines bearing electron-donating substituents and possessing nucleophilic properties. Initially, the amine stabilizes the cationic intermediate, which is then converted into an intermediate, continuing until the final regeneration of the catalyst (Scheme 2).

4.2. Spectroscopic studies

Nuclear magnetic resonance (NMR) spectroscopy is a physicochemical technique for determining the structural properties of an organic compound. DFT calculations have been demonstrated to be a useful method for predicting NMR spectra and investigating the relationship between molecular structure and chemical shifts (Table 1). As a result, the use of experimental and theoretical methods allows for the evaluation of molecule structure.

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The experimental ¹H NMR spectrum of the enamino ester (Scheme 3) shows aromatic protons between 6.15 and 7.33 ppm, calculated in the range of 6.03–7.62 ppm in chloroform (Fig. S1, ESI†). We also note the presence of a signal in the form of a singlet at 5 ppm, due to the proton of the methylene group, and a quadruplet at 4.20–4.13 ppm, due to the (O–CH₂–CH₃) protons. In addition, a singlet at 10.15 ppm is assigned to the (N–H) proton of the isoxazoline ring (Fig. S1, ESI†). The calculated chemical shift values of the (H1–N– (10.29 ppm)), (O–CH₂–CH₃ (3.59–4.91 ppm)) and (CCH (4.88 ppm)) protons were obtained using the DFT method and were in good agreement with the experimental values (Table 1).

For ¹³C NMR (Fig. S2 and S3, ESI†), the chemical shifts calculated by the DFT method show excellent agreement with the experimental values. The theoretical spectrum of the aromatic carbons is observed between 126 and 142 ppm, while in the experimental spectrum it is detected between 124 and 135 ppm. Chemical shifts for the CH carbon of the isoxazoline ring were observed at 91 ppm, which can be explained by the attractive effect of the oxygen in the isoxazoline ring. Furthermore, the signal at 59 ppm corresponds to the CH₂ group, whereas the calculated value is 61 ppm. These results are in good agreement with the experimental results. We also noted the presence of two signals at 156 ppm and 177 ppm, which indicate that they correspond to the CN carbon of the isoxazoline ring and to the carbonyl (CO), while the calculated values are observed at 164 ppm and 174 ppm, respectively. The FT-IR spectrum of ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3, presented in Fig. S4 (ESI†), exhibits a broad absorption band of weak intensity at approximately 3300 cm⁻¹, which can be assigned to the N–H stretching vibration. A strong absorption band at 1541 cm⁻¹ is attributed to the stretching vibration of the CC bond. The aromatic C–H stretching modes are observed at 3050 cm⁻¹, while the N–C stretching vibration is identified at 1294 cm⁻¹.

4.3. DFT-based computational chemistry approach

4.4. Molecular docking simulations

The target ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3 was selected as a hit effective antibacterial inhibitor that revealed predicted inhibition for Bacillus subtilis, E. coli and Staph. aureus via docking against an important isozyme, named copper-dependent laccase (CotA) [PDB id: 1of0], DNA gyrase B [PDB id: 6kzv] and wild-type Staph. aureus penicillin binding protein 4 (PBP4) [PDB id: 5tw8], respectively. Molecular operating environment 2019.0102. was used for the preparation steps of either the enzymes or target compound and the docking process of the previously mentioned compound into the active site of the microbial proteins.

Concerning Bacillus subtilis isozyme, compound 3 revealed a probable effective antibacterial inhibition for the copper-dependent laccase. It showed a binding score equal to −4.5689 kcal mol⁻¹ in addition to an interaction pose that includes three hydrophobic interactions with the key amino acid residues of the enzyme His419, Gly417 and Gly323 (Fig. 9).

Regarding E. coli's DNA gyrase B isozyme, compound 3 generally escalated as a potent antibacterial hit. It showed better binding score (−6.4421 kcal mol⁻¹) to the target protein (Table 3). Moreover, it succeeded to bind Glu50, Asp73 and Asn46 via three strong hydrogen bonds in addition to two hydrophobic interactions with Thr165 and Asn46, key residues of the target protein, respectively (Fig. 10 and Table 3).

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Finally, regarding Staph. aureus, ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3 revealed a good binding score of −6.3545 kcal mol⁻¹ via hydrogen bonding and hydrophobic interactions with the key amino acid residues Ser116, Ala182 and Ser263 (Fig. 11 and Table 3).

Fig. 12 displays the co-crystallized and docked ligand conformations with an RMSD value range from 1.60 to 1.80 Å. The very low RMSD values indicate that the binding pose prediction is accurate and supports the docking results obtained for compound 3 with its bacterial target.

4.5. Molecular dynamic simulations

The outcomes of the molecular dynamics (MD) simulation and normal mode analysis (NMA) for the docked complex of the wild-type Staphylococcus aureus penicillin-binding protein 4 (PBP4) isozyme with antibacterial ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3, performed using the iMOD server (iMODS; https://imods.chaconlab.org), are presented in Fig. 13. The simulation aimed to evaluate the flexibility of protein residues in response to binding with ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3. The main-chain deformability graph (Fig. 13A) highlights regions with higher peaks, representing areas of increased protein deformability. These peaks suggest flexibility and the repositioning of amino acid residues to enhance their interaction with ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3. The experimental B-factor analysis (Fig. 13B) identifies the precise positions of atoms and accounts for crystal irregularities. Higher B-factor values correspond to greater atomic mobility and flexibility. The B-factor graph illustrates significant fluctuations in protein residues across the atomic index range of 0–2500. Notably, the ligand-enzyme complex of ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3 exhibited a low eigenvalue of 8.684631 × 10⁻⁴ (Fig. 13-C), indicating enhanced deformability and substantial protein flexibility. Additionally, the covariance map (Fig. 13-D) reveals strong correlations between ligand and enzyme residues. Correlated motions are denoted in red, uncorrelated motions in white, and anti-correlated motions in blue, emphasizing the dynamic interplay between the protein and ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3.

4.6. ADMET LAB 2.0 – drug likeness and ADME studies

The predictions of ADMET properties for ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3 were calculated using ADMET LAB 2.0 web tool (https://admetlab3.scbdd.com/). Compound 3 was characterized by better human intestinal absorption (HIA) results, which was supported by being accepted by the Lipinski rule. In addition, these hit compounds succeeded in passing through the blood brain barrier (BBB) indicating their suitability to treat brain bacterial infections. They did not exhibit any PAINS structural toxicity alerts. Concerning metabolism, ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3 exhibited an inhibitory effect only for CYP1A2, CYP2C19 and CYP2C9. Moreover, it acts as a substrate for CYP1A2, CYP2C19 and CYP3A4 (Table 4). Regarding the toxicity profile, ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3 showed low tendency to act as a hERG blocker with no effects on the respiratory system or ability to induce any drug-neurotoxicity. The hit compound 3 failed to show a complete safety profile because of exhibiting drug-induced liver injury (DILI) ability in addition to a carcinogenicity alert, which necessitates future drug structure modification (Table 5). Finally, studying clearance and excretion of the hit molecule showed that compound 3 exhibited a clearance value equal to 3.478 mL min⁻¹ kg⁻¹ which is considered as a moderate value.

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5. Conclusion

The synthesis and characterisation of an enamino ester, ethyl 3-(4-chloro-2-methylphenylamino)-3-phenylacrylate 3, has been detailed in this paper. The molecular structure of the synthesized compound was investigated using density functional theory (DFT) calculations at the B3LYP-D3/6-311++G(d,p) level of approximation. DFT has proven to be a reliable tool for predicting NMR spectra and exploring the relationship between molecular structure and chemical shifts. The theoretical results show good agreement with the experimental data, supporting the accuracy of the computational approach.

Additionally, the DFT framework was employed to compute molecular orbitals, including the LOMO–HUMO energy gap. The condensed Fukui function and the molecular electrostatic potential (MEP) surface were employed to assess the relative electrophilicity and nucleophilicity of the molecule being studied. The results obtained demonstrate the current molecule's ability to engage in chemical reactions while preserving significant stability, positioning it as a potentially intriguing candidate for applications where electronic interaction plays a crucial role.

The biological activities of the compound were investigated using molecular docking analysis. The study concentrated on the active bending residues linked to hydrogen bonds and the bending energy of a specific chemical that demonstrates bacterial activity. Regarding Bacillus subtilis isozyme, compound 3 demonstrated a likely effective antibacterial inhibition for the copper-dependent laccase. Concerning the compounds targeting the DNA gyrase B isozyme of E. coli, there has been a notable increase in their potency as antibacterial agents. In conclusion, Staph. aureus compound 3 demonstrated a favorable binding score through hydrogen bonding and hydrophobic interactions with the critical amino acid residues Ser116, Ala182, and Ser263.

The assessment covered the physico-chemical characteristics and the ADME-Tox forecasts of the prospective bioactive compound. In terms of the toxicity profile, compound 3 demonstrated a minimal inclination to function as an hERG blocker, exhibiting no impact on the respiratory system or capacity to induce any drug-related neurotoxicity. Ultimately, the investigation into the clearance and excretion of the target molecule revealed that compound 3 demonstrated a clearance value that is regarded as moderate.

Author contributions

Wissam Habibi, Saadia Ouizat, Mohamed Chellegui, Bushra Shakoor, Marwa Alaqarbeh, Mohamed Adel Sayed, and Mostafa Khouili: writing, investigation, validation, methodology; Abdessamad Tounsi, Haydar A. Mohammad-Salim and Mohamed Anouar Harrad: editing, reviewing, and supervision. All authors have read and approved the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the ESI.†

Acknowledgements

M. C. is grateful to UNamur for granting him the status of scientific collaborator. M. C. gratefully acknowledges the F. R. S.-FNRS for financially supporting his research stay at UNamur.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5nj01851j

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