Said Tighadouini

Smaail Radi
email: s.radi@ump.ac.ma

Farid Abrigach
email: abrigach.farid@live.fr

Redouane Benabbes

Driss Eddike

Monique Tillard

Novel -keto-enol Pyrazolic Compounds as Potent Antifungal Agents. Design, Synthesis, Crystal Structure, DFT, Homology Modeling and Docking Studies

Keywords: , Synthesis, Crystal Structure, DFT, Homology Modeling, and Docking Studies Synthesis, Keto-enols, Pyrazole, Crystal structure, DFT calculations, Homology modelling, Molecular docking, Antifungal activity

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Introduction

Compounds bearing -keto-enol functionality are well established in the world of medicinal chemistry as important biologically effective drugs. [START_REF] Egbertson | HIV Integrase Inhibitors: From Diketo Acids to Heterocyclic Templates: History of HIV Integrase Medicinal Chemistry at Merck West Point and Merck Rome (IRBM) Leading to Discovery of Raltegravir. In HIV-1 Integrase: Mechanism and Inhibitor Design[END_REF][START_REF] Pommier | Integrase Inhibitors to Treat HIV/AIDS[END_REF] Extensive research over the last decade indicates that this compound's family possesses potent anti-HIV properties such as: the S-1360 (designed by Shionogi, Ltd) [START_REF] Goldgur | Structure of the HIV-1 Integrase Catalytic Domain Complexed with an Inhibitor: A Platform For Antiviral Drug Design[END_REF] and the L-708,906 (designed by Merck Research Laboratories) [START_REF] Hazuda | Inhibitors of Strand Transfer That Prevent Integration and Inhibit HIV-1 Replication in Cells[END_REF] (Figure 1). This kind of compounds also inhibits the proliferation of cancer cells, such as the 5-CITEP,which designed by the National Cancer Institute, [START_REF] Goldgur | Structure of the HIV-1 Integrase Catalytic Domain Complexed with an Inhibitor: A Platform For Antiviral Drug Design[END_REF] and of the influenza virus. [START_REF] Ishikawa | Binding Mode Prediction and Inhibitor Design of Anti-Influenza Virus Diketo Acids Targeting Metalloenzyme RNA Polymerase by Molecular Docking[END_REF] It should be noted that the natural product, the curcumin (Figure 1), and its derivatives, with similar -keto-enol pharmacophore site, is of high importance regarding its biological properties. Actually, this natural product presents the main active ingredient of the plant Curcuma longa. [START_REF] Chattopadhyay | Turmeric and Curcumin: Biological Actions and Medicinal Applications[END_REF] Curcumin has been reported to possess strong antioxidant activity, [START_REF] Anand | Biological Activities of Curcumin and Its Analogues (Congeners) Made by Man and Mother Nature[END_REF] it acts on several reactive oxygen and nitrogen species including hydroxyl radicals, superoxide anion radicals as well as nitrogen dioxide radicals. [START_REF] Furlan | Inverse Molecular Docking as a Novel Approach to Study Anticarcinogenic and Anti-Neuroinflammatory Effects of Curcumin[END_REF] Curcumin had also shown interesting anticancer activity in various cell lines and animal models, due to its capacity to bind to different proteins and to acts by different mechanisms. [START_REF] Brglez Mojzer | Polyphenols: Extraction Methods, Antioxidative Action, Bioavailability and Anticarcinogenic Effects[END_REF][START_REF] Kohyama | Structure-Activity Relationships of the Antitumor C5-Curcuminoid GO-Y030[END_REF][START_REF] Tan | Synthesis and Evaluation of Bisbenzylidenedioxotetrahydrothiopranones as Activators of Endoplasmic Reticulum (ER) Stress Signaling Pathways and Apoptotic Cell Death in Acute Promyelocytic Leukemic Cells[END_REF][START_REF] Liang | Exploration and Synthesis of Curcumin Analogues with Improved Structural Stability Both In vitro and In vivo as Cytotoxic Agents[END_REF][START_REF] Adams | A Novel Synthetic Curcumin Analog, Induces Apoptosis in Cancer Cells via a Redox-Dependent Mechanism[END_REF] Additionally, It exerts antiangiogenic, antimalarial, anti-HIV, anti-inflammatory and anti-tubercular activities. [START_REF] Minassi | Dissecting the Pharmacophore of Curcumin. Which Structural Element Is Critical for Which Action?[END_REF][START_REF] Liang | Synthesis and Anti-Inflammatory Activities of Mono-carbonyl Analogues of Curcumin[END_REF][START_REF] Agrawal | Curcumin and its Analogues: Potential Anticancer Agents[END_REF] The main advantage of these molecules to be active against all virus genotypes and drugresistant variants is due essentially to the presence of a high potential β-keto-enol pharmacophore site. The biological responses of this moiety may be explained by its facility to penetrate into the vessel walls and plasma cell membranes, its strong interaction with the amino acid residues of active sites, its capability to chelate with metals in biological processes, and also its reaction with oxygen or with cell macromolecules resulting in oxidative stress, in complex immune responses to hapten-conjugate adducts and in modulation of genes expression. 17 Recently, several analogous compounds containing the -keto-enol pharmacophore, were synthetized , such as the keto-enol tetrazoles and triazoles as anti-HCV agents, [START_REF] Song | Tetrazole and Triazole as Bioisosteres of Carboxylic Acid: Discovery of Diketo Tetrazoles and Diketo Triazoles as Anti-HCV Agents[END_REF] the triazolylketo-enol calix [4]arene as potent integrase strand transfer inhibitory activity, [START_REF] Luo | Synthesis and Anti-Integrase Evaluation of Novel Calix[4]arene Derivatives Containing the Triazolyl 1,3-diketo Moiety[END_REF] and coumarinyl chalcones as high selective agents for the breast cancer cell lines, [START_REF] Patel | Synthesis of Some Coumarinyl Chalcones and Their Antiproliferative Activity Against Breast Cancer Cell Lines[END_REF] etc.

On the other part, various computational approaches have been developed in the last years and used across the whole drug discovery process in order to reduce the cost and the time needed to develop and discovery a new drug. Particularly, molecular docking study becomes one of the most popular theoretical tools used to screen and compare wide libraries of compounds especially in the field of medicinal chemistry. The main objective of this technique is to provide an atomistic insight into molecular recognition by predicting the ability of a molecule to bind to the active site of a protein. Based on computational methods, it models the conformation and the orientation of a molecule interacting with a receptor site. The method has experienced rapid growth and many improvements over the last two decades and the number of publications associated with molecular docking studies has increased enormously. [START_REF] Prieto-Martínez | Molecular Docking: Current Advances and Challenges[END_REF] In continuation of our recent works in the field of the synthesis of novel -keto-enol heterocyclic derivatives through efficient and simple routes and of the study of their biological activities, [START_REF] Radi | Synthesis of Novel β-Keto-Enol Derivatives Tethered Pyrazole, Pyridine and Furan as New Potential Antifungal and Anti-Breast Cancer Agents[END_REF][START_REF] Radi | Crystal Structure of (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3-(4-methoxyphenyl)prop-2-en-1-one, C15H16N2O3[END_REF][START_REF] Radi | Crystal Structure of (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3-phenylprop-2-en-1-one, C14H14N2O2[END_REF][START_REF] Radi | Crystal Structure of (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-(4-ethoxyphenyl)-3-hydroxyprop-2-en-1-one, C16H18N2O3[END_REF][START_REF] Radi | Crystal Structure of (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3-(p-toly)prop-2-en-1-one, C15H16N2O2[END_REF][START_REF] Radi | Crystal Structure of (Z)-3-hydroxy-3-(4-methoxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one, C15H13NO3[END_REF][START_REF] Radi | β-Keto-enol Tethered Pyridine and Thiophene: Synthesis, Crystal Structure Determination and Its Organic Immobilization on Silica for Efficient Solid-Liquid Extraction of Heavy Metals[END_REF][START_REF] Tighadouini | Removal Efficiency of Pb (II), Zn (II), Cd (II) and Cu (II) from Aqueous Solution and Natural Water by Ketoenol-Pyrazole Receptor Functionalized Silica Hybrid Adsorbent[END_REF][START_REF] Radi | New Hybrid Material Based on a Silica-Immobilised Conjugated β-Ketoenol-bipyridine Receptor and Its Excellent Cu(II) Adsorption Capacity[END_REF][START_REF] Radi | Thermodynamics and Kinetics of Heavy Metals Adsorption on Silica Particles Chemically Modified by Conjugated β-Ketoenol Furan[END_REF][START_REF] Radi | Quantitative Removal of Zn(II) From Aqueous Solution and Natural Water Using New Silica-Immobilized Ketoenol-Pyridine Receptor[END_REF] we report in the present investigation on the synthesis and the properties of a novel series of pyrazoles with the -keto-enol functionality as powerful moieties especially in fungal activity. To the best of our knowledge, this is the first real study aiming to develop diversified structures of keto-enol pyrazoles.

Materials and Methods

General Information

All the chemical reagents used in this study were of analytical grade (Aldrich, purity > 99%).

Melting points were measured with a BUCHÏ 510 m.p. apparatus. [START_REF] Egbertson | HIV Integrase Inhibitors: From Diketo Acids to Heterocyclic Templates: History of HIV Integrase Medicinal Chemistry at Merck West Point and Merck Rome (IRBM) Leading to Discovery of Raltegravir. In HIV-1 Integrase: Mechanism and Inhibitor Design[END_REF] H and [START_REF] Adams | A Novel Synthetic Curcumin Analog, Induces Apoptosis in Cancer Cells via a Redox-Dependent Mechanism[END_REF] C NMR spectra were recorded on a Bruker AC 300 spectrometer operating at 300 MHz for proton and 75.47 for carbon nuclei. Molecular weights were determined using a JEOL JMS DX-300 mass spectrometer. Elemental analysis was performed by Microanalysis Central Service (CNRS).

Infrared (IR) spectra were acquired on a Shimadzu infrared spectrophotometer using the KBr disc technique. X-ray diffraction data collection was carried out on the four-circle Oxford Xcalibur diffractometer (Mo-Kα radiation, λ = 0.71073 Å). The in vitro antibacterial and antifungal activity was tested by the agar diffusion technique.

Synthesis of the β-keto-enol pyrazole derivatives

The β-keto-enol pyrazole derivatives studied in this paper were prepared according to the experimental procedure described in our previous work. [START_REF] Radi | Synthesis of Novel β-Keto-Enol Derivatives Tethered Pyrazole, Pyridine and Furan as New Potential Antifungal and Anti-Breast Cancer Agents[END_REF] briefly, a 12.01 mmol of pyrazole carboxylate dissolved in 25 mL of toluene was slowly added to a suspension of sodium (15.21 mmol) in 20 mL of toluene; then a toluene solution (10 mL) of aryl methyl ketones (12.01 mmol) was added at 0 °C. After stirring at room temperature for 2 days, the precipitate formed was filtered off, washed with toluene, dissolved in water and neutralized with acetic acid to pH 5. The organic layer obtained after an extraction with CH2Cl2, was dried over anhydrous sodium sulfate and concentrated in vacuo. Filtration of the obtained crude through silica using CH2Cl2/MeOH as eluent followed by recrystallization from methanol (95%) afforded the desired products 1-10 as solids in acceptable yields.

(Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3(2-methoxyphenyl)prop-2-en-1-one (1).

Yellow crystal; yield: 26%; M.p. 80°C; Rf = 0.87 (CH2Cl2/MeOH 9/1)/ silica. IR (KBr, cm -1 ): Yellow crystal; yield: 32; M.p. 106 o C; Rf: 0.27 (CH2Cl2/MeOH 9/1)/ silica, IR (KBr, cm -1 ): ν(OH) =3430cm -1 ; ν (C=O) = 1676 cm -1 ; ν (C=C enolic) = 1529 cm -1 . [START_REF] Egbertson | HIV Integrase Inhibitors: From Diketo Acids to Heterocyclic Templates: History of HIV Integrase Medicinal Chemistry at Merck West Point and Merck Rome (IRBM) Leading to Discovery of Raltegravir. In HIV-1 Integrase: Mechanism and Inhibitor Design[END_REF]

ν(OH) =3433cm -1 ; ν (C=O) = 1675 cm -1 ; ν (C=C enolic) = 1530 cm -1 ; 1 H NMR (CDCl3):  2.48

(Z)-3-(4-bromophenyl)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxyprop-2-en-1-one (5).

Brown powder; yield: 32%; M.p. 150°C; Rf: 0.93 (CH2Cl2/MeOH 9/1)/ silica. IR (KBr, cm -1 ): ν(OH) = 3448; ν (C=O) = 1611; ν (enolic C=C) = 1565; 1 H NMR (CDCl3): δ 2.30(s, 3H, Pz-CH3); 3.63 (s, 3H, CH3-N): 3.76 (s, 0.1H, keto, CH2); 6.52 (s, 1H, Pz-H); 6.69 (s, 0.9H, enol, C-H); 7.72 (m, 2H, Ar-H3,5); 7.85 (m, 2H, Ar-H2,6). [START_REF] Adams | A Novel Synthetic Curcumin Analog, Induces Apoptosis in Cancer Cells via a Redox-Dependent Mechanism[END_REF]

X-ray diffraction analysis

Crystals with fairly regular shape were selected using a stereomicroscope equipped with a polarizing filter. The CrysAlis software 32 has been used for data reduction and for unit cell determination by least-squares from the 5522 (including symmetry equivalent and redundant) collected reflections at 173K within the complete diffraction sphere (Mo-Kα radiation, λ = 0.71073 Å). Full-matrix least-squares refinements on F 2 used a set of 3076 unique reflections of which 2393 are observed according to the criterion I > 2σ(I). The diffracted intensities were corrected for Lorentz and polarization effects. Structure has been solved and refined with SHELX-2013 program packages. [START_REF] Sheldrick | A Program for Crystal Structures Solution; SHELXL. A Program for Refining Crystal Structures[END_REF] Positional and anisotropic displacement parameters were refined for all non-H atoms. The H-atoms at keto-enol group, detected in the final Fourier difference, were freely refined while other H-atoms were considered with an isotropic displacement parameter equal to 1.2 times (1.5 for terminal -CH3) the Ueq of the parent atom.

The molecular representation is drawn with ORTEP-3 for windows. [START_REF] Farrugia | WinGX and ORTEP for Windows: An Update[END_REF]

Biological evaluation 2.4.1. Antifungal assay

The in vitro antifungal potential of the prepared compounds against the pathogen fungus Fusarium oxysporum f.sp albedinis (FAO) was determined by the agar diffusion technique. [START_REF] Neri | Control of Penicillium Expansum by Plant Volatile Compounds[END_REF][START_REF] Garrod | Antibiotic and Chemotherapy[END_REF] Briefly, potato dextrose agar (PDA) medium was mixed with different volumes (50, 200 and 500 µL) of a DMSO solution of the tested compounds to prepare Petri plates with different concentrations. Thereafter, discs of 6 mm diameter of the microorganism (FAO) were placed into the middle of these Petri plates. After an incubation at 28 °C for 7 days, inhibition percentages were calculated and the half-maximal inhibitory concentration (IC50) was determined using a non-linear regression algorithm of the dose -inhibition percentage graph.

Benomyl was used as the standard drug (positive control).

Antibacterial Test

In accordance with the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. [START_REF]Performance Standards for Antimicrobial Disk Susceptibility Tests -Sixth Edition; Approved Standard[END_REF] The antibiotic effect of target compounds 1-10 was assessed against one gram-negative bacterium viz. Escherichia coli and two gram-positive strains viz. Bacillus subtilis and Micrococcus luteus. For assays, test compounds were dissolved in dimethyl sulfoxide (DMSO). Sterile WHATMAN paper discs (6 mm in diameter) were impregnated with different volumes of each compound and then placed in the middle of Petri plates containing the culture media (Muller-Hinton agar) previously inoculated with overnight cultures of the target strains. After 24 hours of incubation at 37 °C, diameter of inhibition zones around each disc was measured. Gentamicin (1 mg. mL -1 used at 10 µL and 20 µL) was used as standard drug.

Computational studies 2.5.1. Homology modeling

Due to the lack of a 3D experimental crystal structure of Fusarium oxysporum Guanine nucleotide-binding protein beta (Fgb1) in the Protein Data Bank (PDB, www.rcsb.org), a homology modeling study was carried out to build a suitable 3D structural model for this protein prior to use in our docking study. This model was constructed as it has been described in our previous paper. [START_REF] Abrigach | In Vitro Screening, Homology Modeling and Molecular Docking Studies of Some Pyrazole and Imidazole Derivatives[END_REF]

DFT calculations and Molecular docking

The structures of benomyl and compound 9 (9t and 9p) were drawn in ACD/ChemSketch software. [START_REF]ACD/ChemSketch version 12.01[END_REF] Their geometries were optimized using density functional theory (DFT) method, at B3LYP level of theory with a 6-31 G (d,p) basis set, provided in the Gaussian 09 package. [START_REF] Frisch | [END_REF] The Molecular Electrostatic Potential (MEP) was calculated for the optimized molecules and maps have been generated utilizing the GaussView 05 software. SwissDock server which is a free service was used to perform the docking studies against the generated model of FGB1

protein. [START_REF] Grosdidier | A Protein-Small Molecule Docking Web Service Based on EADock DSS[END_REF] The docking results were then analyzed by the UCSF Chimera molecular viewer. [START_REF] Pettersen | UCSF Chimera-A Visualization System for Exploratory Research and Analysis[END_REF] The protonated form of compound 9 (9p) was determined using "Marvin Sketch version 17.1.30'' software. [START_REF]Marvin Sketch was used to determine the protonation state[END_REF]

Results and Discussion

Compounds chemistry

The main route for the preparation of compounds in this work is given below (Scheme 1). The synthesis of ten target compounds characterized by -keto-enol group tethered pyrazole was developed using our method which has been recently described elsewhere. [START_REF] Radi | Synthesis of Novel β-Keto-Enol Derivatives Tethered Pyrazole, Pyridine and Furan as New Potential Antifungal and Anti-Breast Cancer Agents[END_REF] Indeed, the synthesis is governed by a one-pot in-situ condensation under mild conditions (room temperature, two days). The reaction takes place through stirring a mixture of ketone derivatives and ethyl pyrazole-2-carboxylate using toluene as the solvent and sodium metal as the base.

After neutralization to pH = 5 of the generated keto-enolate salts using acetic acid, and filtering them through silica using CH2Cl2/MeOH as eluent, the title products were obtained in acceptable yields. We would like to emphasize that the desired products have been almost exclusively obtained in the -keto-enol tautomer form (> 90%). In fact, the -keto-enol form is greatly favored over the -diketone form due to the conjugation effect of the enol with the carbonyl group, and the increase in stability resulting from a strong six-centered intramolecular hydrogen bond. Indeed, the -keto-enol interconversion rate (> 90%) was determined from the NMR spectra by integration of the signals associated to the enol =C-H and the ketone CH2. We noted the presence of the keto form only as traces visible around 4 ppm. In addition, a very small negative signal from CH2 was observed on the DEPT-135 spectra. Finally, crystals were isolated by slow evaporation from methanol for majority of the -keto-enol compounds.

Two driving forces are responsible for the formation of the favored keto-enol isomer in solution, these forces have been postulated by Plessis et al. [START_REF] Du Plessis | Cyclic Voltammetry of Ferrocene-containing Β-diketones as a Tool to Obtain Group Electronegativities[END_REF] The first one is that related to the electronic driving force in which the formation of the preferred enol isomer is controlled by the group electronegativity (χR) of the substituents R and R′ fixed on the β-diketone R′COCH2COR.

However, this driving force was not found to apply to the β-keto-enol obtained form. The second force for determining which keto-enol isomer will be favored is the resonance driving force which annuls the electronic driving force when one or the two substituents R and R' are aromatic.

Accordingly, our previously prepared products were exclusively obtained in the -keto-enol tautomer form, governed by the resonance driving force, as confirmed by XRD. [START_REF] Radi | Crystal Structure of (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3-(4-methoxyphenyl)prop-2-en-1-one, C15H16N2O3[END_REF][START_REF] Radi | Crystal Structure of (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3-phenylprop-2-en-1-one, C14H14N2O2[END_REF][START_REF] Radi | Crystal Structure of (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-(4-ethoxyphenyl)-3-hydroxyprop-2-en-1-one, C16H18N2O3[END_REF][START_REF] Radi | Crystal Structure of (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3-(p-toly)prop-2-en-1-one, C15H16N2O2[END_REF][START_REF] Radi | Crystal Structure of (Z)-3-hydroxy-3-(4-methoxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one, C15H13NO3[END_REF][START_REF] Radi | β-Keto-enol Tethered Pyridine and Thiophene: Synthesis, Crystal Structure Determination and Its Organic Immobilization on Silica for Efficient Solid-Liquid Extraction of Heavy Metals[END_REF] Herein, as proved by its crystal structure determined from the X-ray diffraction study, compound 10 was also found to exist with the -keto-enol tautomer form.

X-ray Crystal Structure Description

The single crystal structure of C18H16N2O2 (compound 10) has been determined from X-ray diffraction data. The main crystal data and refinement parameters are reported in The molecular unit C18H16N2O2 is built with three fragments, a dimethyl pyrazole ring connected through a -keto-enol unit to a naphthalene ring (Figure 2). The monoclinic lattice contains two symmetry dependent molecules. In this structure, each fragment is planar as indicated by the RMS deviations of the fitted atoms that are respectively 0.006, 0.008 and 0.019.

Yet, the entire molecule deviates to flatness as reflected by the value of 6.8° for the dihedral angle between keto-enol and pyrazole fragments and that of 14.8° between keto-enol and naphthalene groups. The bonds lengths and angles in the molecule are comparable to those found in previous works for other similar compounds. Molecular packing in the monoclinic lattice is such that molecules superimpose along the a and baxes as shown by the projections given in

Pharmacology

All the newly synthesized -keto-enol pyrazoles were evaluated for their antibacterial activity against three bacterial strains namely Echerichia coli, Bacillus subtilis, and Micrococcus luteus and their antifungal activity against one fungal strain (Fusarium oxysporum f.sp albedinis ) by the agar diffusion method.

Surprisingly, the antimicrobial assay revealed that all the screened compounds possessed very weak or no antibacterial effect against all used strains. They showed inhibition zones less than 7 mm when it's compared to that of the standard drug gentamicin (inhibition zone equal or greater than 20 mm). These findings can be possibly interpreted by the absence of pharmacophore sites which can act as potential and specific features to inhibit the growth of bacteria. [START_REF] Abrigach | Synthesis, Biological Screening, POM, and 3D-QSAR Analyses of Some Novel Pyrazolic Compounds[END_REF][START_REF] Hatzade | Antimicrobial/Antioxidant Activity and POM Analyses of Novel 7-ODglucopyranosyloxy-3-(4, 5-disubstituted imidazol-2-yl)-4H-chromen-4-ones[END_REF] In contrast, most of these molecules exhibit potent antifungal activity against FOA in a manner dependent on the dose as indicated in Table 3. Their activity strongly depends on the structure activity relationships (SARs) and shows an interesting influence of the substitution pattern. When looking at the influence of the substituent R, we found that compound 8 with the 3-thiophene group and compound 10 with the 2-naphtalene group lead to the same inhibition percentage of 94% as the benomyl fungicide. Interestingly, compounds 2 and 9 respectively containing the m-OMe-C6H4 and the 2-pyridine groups even display a slightly better activity of 96%. The inhibitory concentration (IC50) is lower for these active compounds 2, 8, 9 and 10 with IC50 values of 71.00, 48.00, 14.80 and 53.00 μM, respectively than the other compounds.

Compared to our previous works in this era, these findings present the best obtained results for all the already tested analog products of this family against FOA fungus.

In the context of a marked biological interest for heterocyclic compounds, this suggests that the nature of the R substituent should be further exploited to modify the properties and to determine the structure activity relationship for this novel class of antifungal agents.

Computational studies 3.4.1. DFT calculations

Calculation of the electronic parameters of a molecule is a well-established tool for the interpretation and the prediction of its reactivity in several types of reactions. The molecular frontier orbital descriptors HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) are of great importance in the determination of the reactivity of a molecule. They respectively depict its electron donating and receiving ability. Also, the energy gap (ΔE = ELUMO -EHOMO) is an important parameter which measures the intramolecular charge transfer and the kinetic stability; it has been extensively used to explain the biological activity results. [START_REF] Demir | Molecular Structure, Spectroscopic Characterization, HOMO and lUMO analysis af PU and PCL Grafted Onto PEMA-co-PHEMA with DFT Quantum Chemical Calculations[END_REF][START_REF] Mabkhot | Novel Enaminone Derived from Thieno [2,3-b]thiene: Synthesis, X-Ray Crystal Structure, HOMO, LUMO, NBO Analyses and Biological Activity[END_REF] Generally, molecules with a large energy gap are associated with less chemical reactivity, high kinetic stability, while those with a small ΔE are more reactive and less stable. [START_REF] Khoutoul | Liquid-Liquid Extraction of Metal Ions, DFT and TD-DFT Analysis for Some Pyrane Derivatives with High Selectivity For Fe(II) and Pb(II)[END_REF] In our case, the HOMO and LUMO energies of the most active antifungal compound i.e. compound 9 and of the standard drug i.e. benomyl were calculated at the DFT/B3LYP/6-31 G (d,p) level of theory to compare and understand their reactivity. The HOMO calculated energy (EHOMO) is comparable for compound 9 and benomyl. In contrast, their LUMO energies markedly differ by -1.036 eV. The ELUMO energies are -1.555 eV for compound 9 and -0.519 eV for benomyl, leading to values of 4.218 and 5.303 eV for the calculated energy gaps, which means that benomyl is more stable (therefore less active) than compound 9. In conclusion, it was observed that the LUMO energy level has a critical effect on the increase in reactivity of compound 9 compared to benomyl. These findings are in good agreement with our experimental results. The shape and the location of LUMO orbital should also be considered, it can be affected by the presence (or not) of electron-donating or withdrawing groups in the molecule.

Many investigations revealed that the placement of the LUMO orbitals, along with their energy, can together influence the biological activity. [START_REF] Kumar | Design, Synthesis, DFT, Docking Studies and ADME Prediction of Some New Coumarinyl Linked Pyrazolylthiazoles: Potential Standalone or Adjuvant Antimicrobial Agents[END_REF] Here, the representation of the frontier molecular orbital shapes for both molecules (Figure 4) shows that the occupied orbital (HOMO) is mainly localized on the pyridine ring and the -keto-enol pharmacophore for compound 9 and on the benzimidazole moiety and the ester function for benomyl. On the contrary, the empty LUMO orbital is delocalized over the entire molecule in both cases. Additionally, many other quantum chemical parameters can be used as measures of the reactivity of molecules. Their values are generally calculated from the HOMO and LUMO energies. The hardness of a molecule is an important concept to evaluate the stability and reactivity of a species, it defines the resistance towards deformation or polarization of the electron density distribution of a chemical system in an electric field. [START_REF] Kaya | A New Equation for Calculation of Chemical Hardness of Groups and Molecules[END_REF] This concept can be expressed as chemical hardness (η) and softness (σ). [START_REF] Gázquez | Hardness and Softness in Density Functional Theory[END_REF] The electronegativity (χ) is a measure of the power of a molecule to attract electrons whereas the chemical potential (μ) evaluates the tendency of an electron to escape from the molecule. The ability of a species to accept electrons is quantified by the electrophilicity index. [START_REF] Parr | Electrophilicity Index[END_REF] As a consequence, high chemical hardness values characterize stable compounds with low reactivity while high values for softness, electronegativity, chemical potential and electrophilicity denote less stable hence more reactive compounds. All these indices (Table 4) have been calculated from the computed HOMO and LUMO energies, using the appropriate equations.

The analysis of these parameters clearly reveals a high reactivity for compound 9. In fact, it shows a weaker chemical hardness than benomyl, this means that benomyl is more resistant to the charge transfer and thus less reactive than compound 9. On the other hand, the values of σ, χ, μ and ω also denote a reactivity for compound 9 higher than for benomyl, which nicely correlates with the experimental antifungal findings. Finally, a molecular electrostatic potential (MEP) investigation was carried out in order to identify and predict the reactive sites for nucleophilic and electrophilic attacks. MEP is commonly used to understand the biological recognition processes and hydrogen bonding interactions. [START_REF] Panicker | HOMO-LUMO, NBO, MEP Analysis and Molecular Docking Study of 3-methyl-4-{(E)-[4-(methylsulfanyl)benzylidene]amino}1H-1,2,4-triazole-5(4H)-thione[END_REF] The MEP plots have been generated for the DFT optimized geometries of compound 9 and benomyl, and they are given in Figure 5. The electrostatic potential at the surface increases in the order red < orange < yellow < green < blue (see in Figure 5). It varies from negative regions (red, orange and yellow) related to electrophilic reactivity towards positive regions (green and blue) which represent the nucleophilic reactivity. For compound 9, the negative electrostatic potential regions are mainly centered over the oxygen atoms of the -keto-enol pharmacophore and the nitrogen atom of pyridine with values varying from -0.598 to -1.850 eV, while the positive regions are localized on the CH3 groups of the pyrazole ring. For benomyl, the map shows negative values from -0.952 to -1.387 eV mainly centered over the oxygen atoms of carbonyl groups (C=O) and the unsubstituted nitrogen atom of the benzimidazole ring while the positive values are focused over the NH groups.

Homology modeling

As described previously, all the prepared compounds containing -keto-enol pharmacophore site and important azole therapeutic moiety (pyrazole). And as is known, these groups act on the cell membrane of microorganisms. This fact encourages us to postulate that our compounds can interact with specific targets located on the membrane of the studied fungal. While, it is well established that fungal cell membranes and cell walls are of particular importance in the development of antifungal drugs. [START_REF] Rautenbach | Antifungal Peptides: To Be or Not to Be Membrane Active[END_REF] based on these considerations, Fusarium oxysporum Guanine nucleotide-binding protein beta (Fgb1) was chosen as a target in the present work.

Actually, this protein is one of the most important membrane proteins and it is implicated in several biological processes of F. oxysporum fungal, which make it a potential target to develop new inhibitors. [START_REF] Lengeler | Signal Transduction Cascades Regulating Fungal Development and Virulence[END_REF][START_REF] Soundararajan | Macromolecular Docking Simulation to Identify Binding Site of FGB1 for Antifungal Compounds[END_REF] Unfortunately, till now there is no experimental structure of this protein in the Protein Data Bank. For these reasons, a homology study was performed to build a threedimensional structure of this protein based on an already known structure following the procedure described in our previous work. [START_REF] Abrigach | In Vitro Screening, Homology Modeling and Molecular Docking Studies of Some Pyrazole and Imidazole Derivatives[END_REF] Briefly, the Fgb1 primary residues sequence (accession number Q96VA6) was used as a target. A BLASTp search was carried out to find a suitable template with already known 3D structure for this amino acid sequence. With a similarity of 67% to Fgb1 sequence, the 3SN6_chain B (Rattus norvegicus Guanine nucleotidebinding protein G(I)/G(S)/G(T) subunit beta-1) was identified as the best template. Sequence alignment performed between the target and the template, revealing a total conservation of 63.23% of residues (227 residues), strong similarity of 15.04% (54 residues) and weak identity of 5.30% (19 residues) (Supporting Information (SI), Figure S1). While, 59 amino acids (16.43%) of the FGB1 were found non-matching those of the selected template. based on the obtained aligned sequence; a 3D model was generated for the Fgb1 protein (SI, Figure S2).

Then the PROCHECK suite was utilized to check the quality of this model. Superimposing the generated model on the template backbone (3SN6_ chain B experimental structure) results in a very low RMS deviation of 0.257 Å between their main chain atoms. The Ramachandran plot was used to evaluate the structure quality by visualizing the energetically allowed regions, it provides the amount of residues belonging to the favored (>90%), additional allowed, generously allowed and disallowed regions. As can be seen from Figure 6, the majority of the amino acid residues are located in the most favored regions (red color) of the phi-psi distribution with a percentage of 90.9%. While 7.2%, 1.3% and 0.7% of residues were found to locate the additional allowed (yellow color), generously allowed (light yellow color) and the disallowed regions (white color) respectively which is very comparable with the values obtained with the experimental structure of the used template (92.5% of residues are in the most favored regions, 6.7% for additional allowed regions, 0.7% for generously allowed regions and 0.0% for the disallowed regions). Additionally, the mean G-factor takes respectively the values of -0.21 and 0.11 for the generated model and the template, both placed in the optimal region (values below -0.5 are unusual). In conclusion, all these parameters attest reliability and good quality for the generated model of the Fgb1 protein.

Molecular docking

In the present work, to rationalize the antifungal results obtained and to investigate the most probable binding mode, the modeled Fgb1 protein was targeted to perform a docking study of benomyl and of the most active compound 9 of the β-keto-enol pyrazolic series. The binding energy ΔGbinding, the H-bonds and their lengths have been used to evaluate the binding affinity of each molecule to Fgb1 protein. The most negative value of ΔGbinding corresponds to the most stable ligand-receptor complex, giving valuable information about the most active compound.

The calculated parameters have been reported in Table 5. In addition, the influence of protonation states and tautomeric forms on virtual screening results has been well established and an extensive research was made in this field. [START_REF] Martin | Let's Not Forget Tautomers[END_REF][START_REF] Knox | Considerations in compound database preparation--"hidden" impact on virtual screening results[END_REF][START_REF] Kim | Effects of Histidine Protonation and Rotameric States on Virtual Screening of M. tuberculosis RmlC[END_REF][START_REF] Brink | Influence of protonation, tautomeric, and stereoisomeric states on protein-ligand docking results[END_REF][START_REF] Kalliokoski | The effect of ligand-based tautomer and protomer prediction on structure-based virtual screening[END_REF] It can influence the hydrogen bonding ability of the ligand to the receptor. In this context, and in order to investigate the possible effects of both these phenomena during our docking simulation, the tautomeric form (9t) and the protonated state (9p) at the physiological pH (pH = 7-8) of compound 9 were docked against the same Fgb1 protein model used previously. The docking results are summarized and displayed in Table S1 and Figure S3 in Supporting Information.

With a binding score of -6.780 kcal/mol, the best pose of the tautomeric form (9t) was found to be linked to the active pocket of Fgb1 by three bonds involving the amino acid residues Ser206, Thr118 and Cys165 with distances of 2.33 Å, 2.74 Å and 2.76 Å, respectively. While, in protonated form (9p) docking result (Figure S3B) indicates that Asn340 and Gln251 formed hydrogen bonds with O -(C-O -) and N2(pyrazole) respectively. The distances were found to be 2.23 Å with Asn340 and 2.43 Å with Gln251. Furthermore, through comparing the binding energies of the three forms, it was found that the enol form (9) has slightly better binding score value in comparison with the keto isomer (9t) and the protonated form (9p) due to the presence of more polar properties and more interaction with protein in the case of the enol tautomeric. [START_REF] Kumar | In silico Inhibition Studies of NF-κB p50 Subunit by Curcumin and its Natural Derivatives[END_REF] However, it is important to mention that the calculated binding energies do not always reflect the biological response, [START_REF] Méndez-Cuesta | Homology Modeling, Docking and Molecular Dynamics of the Leishmania Mexicana Arginase: A Description of the Catalytic Site Useful for Drug Design[END_REF] and also none of the compounds 9t and 9p has tested in vitro against the studied fungal.

Finally, these important observations open up the way for investigation into various structural and chemical modifications in order to make our compounds even more efficient.

Furthermore, within the general context of development of new anti-Fusarium agents targeting proteins and specially Fgb1, additional suitable experimental assays are required to confirm the good capacity of these active compounds.

Conclusion

This manuscript described the first synthesis of novel -keto-enol embedded with pyrazolic moiety compounds, and evaluated their in vitro antifungal activities. Some of the prepared compounds show better antifungal activity than the reference. Interestingly, compounds 2 and 9 display 96% antifungal activity which is even better than the benomyl fungicide reference.

DFT calculations were performed to determine the reactivity of the most active compound i.e.

compound 9 and of benomyl and to demonstrate the correlation with our experimental results.

Furthermore, molecular docking study was carried out to determine the binding mode of compound 9 and benomyl into the Fgb1 protein pocket. The possible effects of ligand protonation and tautomeric states on the docking simulation results were also investigated for the compound 9. Hopefully, the obtained results in this study will present a guideline to facilitate a better understanding of the mode of action of ligand-enzyme complex and give a clear insight to design new molecules in the future. However, appropriate in vitro experiments on the inhibition of Fgb1 with the novel -keto-enol pyrazolic compounds should be performed in the future and their interaction should be identified by others performing methods such as molecular dynamics simulations and free-energy calculations.

Figure 1 .

 1 Figure 1. Structures of some drugs contain keto-enol functionality.

  (s, 3H, Pz-CH3); 3.62 (s, 3H, CH3-N): 3.89 (s, 3H, O-CH3);4.63 (s, 0.1H, keto, CH2); 6.50 (s, 1H, Pz-H); 6.60 (s, 0.9H, enol, C-H); 6.99 (m, 2H, Ar-H3); 7.07 (m, 2H, Ar-H5);7.47 (m, 2H, Ar-H4) 7.60 (m, 2H, Ar-H6).[START_REF] Adams | A Novel Synthetic Curcumin Analog, Induces Apoptosis in Cancer Cells via a Redox-Dependent Mechanism[END_REF] C NMR (CDCl3): δ 11.14 (1C, Pz-CH3); 37.30 (1C,CH3-N); 49.29 (1C, keto CH2); 56.30 (1C, o-OCH3-Ar); 98.50 (1C, enol C-H); 106.34 (1C,= CH-Pz);112.87 (1C, Ar-C1); 120.47 (1C, Ar-C3); 121.07 (1C, Ar-C5); 121.76 (1C, Ar-C6); 158.51 (1C, Ar-C2); 184.07 (1C, C-OH); 190.17 (1C, C=O). Anal. calcd for C15H16N2O3 : C 66.16, H 5.92, N 10.29. Found C 66.12, H 5.90, N 10.35; m/z: 273.06 (M+H) + . (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3(3-methoxyphenyl)prop-2-en-1-one (2).

Scheme 1 .

 1 Scheme 1. Main route of the synthesis of compounds 1-10.

Figure 2 .

 2 Figure 2. Ortep molecular representation of C18H16N2O2 (10).

Figure 3 .

 3 The shorter intermolecular contacts are found at atoms N1 and O2 that are involved in weak interactions N1••••H15 i C15 i (2.743 Å, angle 130.63°), O2••••H5 ii C5 ii (2.630 Å, angle 137.17°) and O2••••H7 ii C7 ii (2.596 Å, angle 151.59°) with two neighboring molecules (i: 1-x, ½+y, 1-z; ii: -x, -½+y, -z).

Figure 3 .

 3 Figure 3. Structural representation of 10 viewed along (left) a axis and (right) baxis of the monoclinic lattice.

Figure 4 .

 4 Figure 4. HOMO and LUMO plots of compound 9 and benomyl. Positive and negative phases are shown in red and green colors, respectively.

Figure 5 .

 5 Figure 5. Molecular electrostatic potential (MEP) maps of compound 9 (left) and benomyl (right).

Figure 6 .

 6 Figure 6. Ramachandran plot analysis of (A) the generated Fgb1 model and (B) the template model 3SN6_chain B.

Å), Thr212 ( 2 .

 2 35 Å) and Gln251 (2.46 Å) residues through respectively the two carbonyl groups and the N3 atom of the benzimidazole ring. Interestingly, a good correlation is found between our docking results and the electronic calculations discussed above. In fact, the interactions between the studied molecules and the amino acid residues of the receptor Fgb1, occur at the high electron density regions of the molecule identified previously by the MEP maps. However, more theoretical calculations and/or experiment investigations are required to confirm the validity of these docking results.

Figure 7 .

 7 Figure 7. Binding mode of compound 9 (A) and benomyl (B) into the active pocket of the Fgb1 protein.

-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3-(thiophen-2-yl)prop-2-en-1-one (6).

	C NMR (CDCl3): δ 11.38 (1C, Pz-CH3);
	37.04 (1C, CH3-N); 48.78 (1C, keto CH2); 94.56 (1C, enol C-H); 106.45 (1C,= CH-Pz);121.94
	(1C, Ar-C4); 126.09 (2C, Ar-C3,5); 129.22 (2C, Ar-C2,6); 180.54 (1C, C-OH); 183.59 (1C, C=O).
	Anal. calcd for C14H13BrN2O2 : C 52.36, H 4.08, N 8.72. Found C 52.27, H 4.03, N 8.81; m/z:
	321 (M+H) + .
	(Z)Brown powder; yield: 30%; M.p. 110°C; Rf: 0.42 (CH2Cl2/MeOH 9/1)/ silica. IR (KBr, cm -1 ):
	ν(OH) =3434; ν (C=O) = 1672; ν (C=C enolic) = 1531; 1 H NMR (DMSO-d6): 2.30 (s, 3H, Pz-
	CH3); 3.84 (s, 3H, CH3-N); 4.56 (s, 0.1H, keto, CH2); 6.58(s, 1H, Pz-H); 6.80 (s, 0.9H, enol,
	C-H); 7.13 (m, 1H, Th-Hβ); 7.60 (d, 1H, Th-Hγ); 7.79 (d, 1H, Th-Hᾳ). 13 C NMR (DMSO-d6):
	11.41 (1C, Pz-CH3); 37.00 (1C,CH3-N); 46.76 (1C, keto CH2); 92.89 (1C, enol C-H); 106.11
	(1C, = CH-Pz); 128.32 (1C, Th-Hγ); 130.20 (1C, Th-Cβ); 132.16 (1C, Th-Cᾳ); 140.46 (1C, Th-
	Cε); 177.87 (1C, C-OH); 180.96(1C, C=O). Anal. Calcd. for C12H12N2O2S: C 58.05, H 4.87, N
	11.28. Found: C 58.02, H 4.99, N 11.31. m/z: 249.06 (M+H) + .
	(Z)

-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3-(thiophen-3-yl)prop-2-en-1-one

	(8).
	White powder; yield 25, M.p. 176 o C; Rf: 0.22 (CH2Cl2/MeOH 9/1)/ silica. IR (KBr, cm -1 ):
	ν(OH) =3434; ν (C=O) = 1672; ν (C=C enolic) = 1531; 1 H NMR (DMSO-d6): 2.49 (s, 3H, Pz-
	CH3); 3.36 (s, 0.1H, keto, CH2); 3.74 (s, 3H, CH3-N); 6.43(s, 1H, Pz-H); 7.16 (s, 0.9H, enol,
	C-H); 7.36 (d, 1H, Th-Hᾳ); 7.48 (m, 1H, Th-Hβ); 7.83 (d, 1H, Th-Hγ). 13 C NMR (DMSO-d6):
	11.04 (1C, Pz-CH3); 37.04 (1C,CH3-N); 47.76 (1C, keto CH2); 96.23 (1C, enol C-H); 108.14
	(1C, =CH-Pz); 123.32 (1C, Th-Cβ); 127.46 (1C, Th-Cε); 128.16 (1C, Th-Cᾳ); 141.20 (1C, Th-
	Hγ); 176.97 (1C, C-OH); 179.25 (1C, C=O). Anal. Calcd. for C12H12N2O2S: C 58.05, H 4.87,
	N 11.28. Found: C 57.95, H 4.92, N 11.35. m/z: 249.06 (M+H) + .
	(Z)

-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-hydroxy-3-(pyridine-2-yl)prop-2-en-1-one

	(9). Brown powder; yield 41%; M.p. 189 o C; Rf: 0.47 (CH2Cl2 /MeOH 9/1) / silica. IR (KBr, cm -1 ): ν(OH) =3431; ν (C=O) = 1672; ν (C=C enolic) = 1529; 1 H NMR (DMSO-d6): 2.29 (s, 3H, Pz-1 ): ν(OH) =3433; ν (C=O) = 1675; ν (C=C enolic) = 1534; 1 H NMR (DMSO-d6): 2.34 (s, 3H, Pz-CH3); 3.89 (s, 3H, CH3-N); 3.50 (s, 0.1H, keto, CH2); 6.66(s, 1H, Pz-H); 7.16 (s, 0.9H, CH3); 3.85 (s, 3H, CH3-N); 3.72 (s, 0.Pale-yellow crystal, yield 27%, M.p. 159°C; Rf: 0.91 (CH2Cl2/MeOH 9/1)/ silica. IR (KBr, cm -enol, C-H); 7.56 (m, 1H, Ar-H3); 7.89 (m, 2H, Ar-H2,7);7.97 (m, 2H, Ar-H1,6); 8.04 (m, 1H, Ar-

1H, keto, CH2); 6.66(s, 1H, Pz-H); 7.53 (s, 0.9H, enol, C-H);7.39 (t, 1H, Py-Hβ); 7.82 (m, 1H, Py-Hγ); 8.07 (t, 1H, Py-Hδ), 8.68 (d, 1H, Py-Hᾳ).

[START_REF] Adams | A Novel Synthetic Curcumin Analog, Induces Apoptosis in Cancer Cells via a Redox-Dependent Mechanism[END_REF]

C NMR (DMSO-d6): 11.39 (1C, Pz-CH3); 37.04 (1C,CH3-N); 48.78 (1C, keto CH2); 94.56 (1C, enol C-H); 106.45 (1C,= CH-Pz); 121.94 (1C, Py-Cδ); 126.10 (1C, Py-Cβ); 137.02(1C, Py-Cγ); 149.47 (1C, Py-Cᾳ); 152.40 (1C, Py-Cε); 180.04 (1C, C-OH), 183.78 (1C, C=O). Anal. Calcd. for C13H13N3O2: C 64.19, H 5.39, N 17.27. Found: C 64.08, H 5.27, N 17.38. m/z: 244.10

(M+H) + . (Z)-1-(1,5-dimethyl-1H-pyrazol-3-yl)-3-(naphthalene-1-yl)prop-2-en-1-one (10). H4); 8.56 (m, 1H, Ar-H5). 13 C NMR (DMSO-d6): 11.45 (1C, Pz-CH3); 37.04(1C,CH3-N); 46.76 (1C, keto CH2); 93.70 (1C, enol C-H); 106.36 (1C,= CH-Pz); 123.44 (1C, Ar-C1); 126.78 (1C, Ar-C10); 127.87 (1C, Ar-C4); 128.04 (1C, Ar-C9); 128.17 (1C, Ar-C5); 128.40 (1C, Ar-C4); 128.63 (1C, Ar-C3); 129.45 (1C, Ar-C6); 132.38 (1C, Ar-C1); 138.39 (1C, Ar-C1); 182.03 (1C, C-OH); 183.25 (1C, C=O). Anal. Calcd. for C18H16N2O2: C 73.95, H 5.52, N 9.58. Found: C 73.91, H 5.40, N 9.72. m/z: 293.12 (M+H) + .

Table 1

 1 and atom positional and equivalent displacement parameters are given in

Table 2 .

 2 Supplementaryinformation can be obtained free of charge (CCDC 1874191) from the CCDC.[START_REF] Groom | The Cambridge Structural Database[END_REF]

Table 1 .

 1 Crystal data and structure refinement for C18H16N2O2 (compound 10).

	Formula, M, Z	C18H16N2O2, 292.33, 2
	Temperature	173(2) K
	Space group	Monoclinic, P 21 (N° 4)
	Lattice	a = 761.0(1), b = 581.1(1), c = 1634.3(3) pm
		α = 90, β = 93.02(1), γ = 90°
	Crystal	0.26 × 0.10 × 0.07 mm
	θ range	2.50 to 29.23°
	Reflections	5522 collected / 3076 unique [Rint = 0.0396]
	Completeness to θ=25.242°	99.9 %
	Data / parameters	3076 / 209
	Final R indices [I>2 σ(I)]	R1 = 0.0515, wR2 = 0.0940
	R indices (all data)	R1 = 0.0745, wR2 = 0.1041
	Δρ Fourier residuals	0.168 / -0.170 e.Å -3

Table 2 .

 2 Atomic coordinates (×10[START_REF] Hazuda | Inhibitors of Strand Transfer That Prevent Integration and Inhibit HIV-1 Replication in Cells[END_REF] ) and equivalent displacement parameters (Å Ueq is defined as one third of the trace of the orthogonalized Uij tensor.

	2 ×10	3 ) for C18H16N2O2.

Table 3 .

 3 Structures and anti-Fusarium activities of synthetized pyrazole β-keto-enols.

	Comp.	Structure	Volume withdrawn (µL)	Diameter of the strain (cm)*	Inhibition (%)	IC50 μM (μg. mL -1 )
			50	3.5	30
	1		200	2.1	58	-
			500	1.8	64
			50	4.2	16
	2		200	2.8	44	71.00 (260.74)
			500	0.2	96
			50	5	0
	3 17		200	3.6	28	150.00 (550.86)
			500	2.5	50
			50	5	0
	4 49		200	3.8	24	-
			500	2.7	46
			50	4.0	20
	5		200	3.2	36	-
			500	2.6	48
			50	3.6	28
	6		200	2.4	52	-
			500	1.3	74
			50	3.5	30
	7 46		200	2.1	58	-
			500	1.2	76
			50	3.2	36
	8		200	1.8	64	48.00 (193.31)
			500	0.3	94
			50	2.3	54
	9		200	1.2	76	14.80 (60.84)
			500	0.2	96
			50	3.2	36
	10		200	2.1	58	53.00 (181.30)
			500	0.3	94
			50	2.3	54
	Benomyl	-	200	1.1	78	-
			500	0.3	94
	* Diameter of the FOA strain in the presence of the tested compound (cm)

Table 4 .

 4 Calculated quantum chemical parameters for compound 9 and benomyl.

	Chemical reactivity indices (eV)	9	Benomyl
	EHOMO	-5.768	-5.822
	ELUMO	-1.550	-0.519
	ΔE = ELUMO -EHOMO	4.218	5.303
	Chemical hardness ( η = (ELUMO -EHOMO)/2)	2.109	2.651
	Softness ( σ = 1/η)	0.474	0.377
	Electronegativity ( χ = -(ELUMO + EHOMO)/2 )	3.659	3.170
	Chemical potential ( μ = -χ )	-3.659	-3.170
	Electrophilicity index ( ω = μ 2 /2η )	3.174	2.525

Table 5 .

 5 Intermolecular interactions between the ligands docked with Fgb1.

	Compound ΔGbinding (kcal/mol)	Hydrogen bond	Bond length (Å )
			N1(pyrazole) -Arg167	1.73
	9	-7.161	N1(pyrazole) -Arg167 O(OH) -Gln251	2.82 2.21
			O(C=O) -Val338	2.58
			O(C=O) -Leu209	2.17
	Benomyl	-6.985	O(C=O) -Thr212	2.35
			N3(benzimidazole) -Gln251	2.46
	According to in silico protein-ligand calculations, with a binding score of -7.461 kcal/mol,
	the compound 9 is found better complexed with the Fgb1 receptor than benomyl (binding score
	of -6.985 kcal/mol). Based on this finding, compound 9 seems more active than benomyl, which
	is in total accordance with the experimental antifungal measurements.
	As depicted in

Figure 7, compound 9 and

  benomyl can both form many hydrogen bonds with the active site residues of the Fgb1 protein. Compound 9 makes four H-bonds with the protein: two bonds of 1.73 and 2.82 Å between the N1 atom of pyrazole and the amino acid residue Arg167, one bond of 2.21 Å between the O atom of the alcohol function and the Gln251 residue and one bond of 2.58 Å between the O atom of the carbonyl group (C=O) and the Val338 residue. The molecule of benomyl (standard drug) shows interactions with the Leu209 (2.17

Acknowledgments

The authors extend their appreciation to the PPR2-MESRSFC-CNRST-P10 project (Morocco) for supporting this work.

Author Information

Corresponding Authors *E-mail: (S. Radi) s.radi@ump.ac.ma *E-mail: (F . Abrigach) : abrigach.farid@live.fr

ORCID:

Smaail Radi : 0000-0002-5062-6904 Farid Abrigach : 0000-0001-8062-7572 Monique Tillard : 0000-0002-0609-7224

Notes

The authors declare no competing financial interest.

Supporting Information

The supporting data contains further information related to the homology modeling (Sequence alignment between Fgb1 (F. oxysporum) and the template (3SN6_ chain B) (Figure S1), the 3D structure of the generated homology model (Figure S2)) and the docking simulation (results of docking study and the 3D representation of the interactions between compounds 9t and 9p and the active pocked residues of Fgb1 protein). This information is available free of charge via the Internet at http://pubs.acs.org


Laurent Chavatte

Milène Juan

Sandra Mounicou

Emmanuelle Leblanc Noblesse

Karl Pays

Carine Nizard

Anne-Laure Bulteau
email: abulteau@research.lvmh-pc.com

Lvmh

Emmanuelle Leblanc Noblesse

Elemental and molecular imaging of human full thickness skin after exposure to heavy metals

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Heavy metals contamination is a major environmental concern particularly in China [START_REF] Cheng | Heavy metal pollution in China: origin, pattern and control[END_REF] . They are found in various media including soil, water air and food 2, 3

. Heavy metals are known to be persistent in the human body, with excretion half-lives that last for decades 4 . At the interface between the body and the environment, the skin is directly exposed to chemical oxidants, pollutants, all of which are potent inducers of reactive oxygen species (ROS). Although, epidemiological and clinical studies highlight the adverse effects of heavy metals on human health, very little research is available to date regarding cutaneous effects [START_REF] Wang | Soil contamination and plant uptake of heavy metals at polluted sites in China[END_REF] . A major mechanism by which ambient heavy metals such as cadmium, nickel exert their detrimental effects is through the generation of oxidative stress which is an important contributor to skin aging 5, 6 . They can cause several genotoxic effects on cells, including oxidative stress, DNA breakage and protein modification [START_REF] Caicedo | Analysis of metal ion-induced DNA damage, apoptosis, and necrosis in human (Jurkat) T-cells demonstrates Ni2+ and V3+ are more toxic than other metals: Al3+, Be2+, Co2+, Cr3+, Cu2+, Fe3+, Mo5+, Nb5+, Zr2+[END_REF][START_REF] Orhan | Application of lipid peroxidation and protein oxidation biomarkers for oxidative damage in mammalian cells. A comparison with two fluorescent probes[END_REF][START_REF] Rana | Metals and apoptosis: recent developments[END_REF] . Moreover, they can, especially nickel, induce the overexpression of collagenases enzymes leading to the weakening of the extracellular matrix resulting in a loss of fitness and elasticity of the skin 10

. Besides their high level of toxicity, they can also induce contact allergies and other serious skin diseases 11 . They also may affect cell growth and proliferation of keratinocytes 12 . Studies of heavy metals have analyzed the effects of one single metal. However, we must consider that people are exposed to a cocktail of metals and a synergic effect of each pollutant cannot be excluded. Therefore, in our study, we considered the joint exposure to pollutants. The exact mechanisms by which heavy metals can cause skin damage has yet to be elucidated. Based on current evidence, there may be two potential mechanisms (i) generation of free radicals, (ii) induction of inflammatory response and disruption of the skin barrier 11 . Ex vivo experiments based on excised epidermis model could help to provide information on heavy metals induced biological processes, skin penetration and their rates. Permeability of metals through the skin is highly variable since they can penetrate the epidermis through different routes. Furthermore, metals are retained in the skin which could lead to a reservoir and extended exposure even after their removal from the outer surface of the skin 13 . Dermal exposure can lead to systemic inflammation [START_REF] Thyssen | Metal allergy--a review on exposures, penetration, genetics, prevalence, and clinical implications[END_REF] . Therefore, it is important to localize and measure the amount of each type of metals in the skin. Imaging mass spectrometry is based on the measurement of the abundance of elemental or molecular ions generated locally by means of a laser and offers a typical resolution of 20-50m. Elemental imaging by laser ablation (LA ICP-MS) allows a sensitive imaging of metals and thus an ultimate way of tracking the penetration of heavy metals into the skin Please do not adjust margins

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The current study was undertaken to characterize the effects of heavy metals (lead, mercury, nickel and cadmium) exposure on skin and to assess how alterations in skin integrity may contribute to oxidative stress.

Figure 1. Effect of heavy metals exposure on skin explants. Metals exposure protocol. Skin full thickness models were cultured for 4 days in a survival medium and at day 4 a mixture of 4 heavy metals was deposited on a filter paper and applied on explants. Control samples received only filter paper. 24 hours after the explants were taken and prepared for histology, LA-ICP MS or immunostaining.

Results

Heavy metals induced changes in skin morphology and an increase of skin oxidative damages.

Skin explants were exposed to the metals cocktail (nickel, lead, mercury and cadmium) for 4 days (Figure 1). 24 hours after the last exposure, we first analyzed the skin morphology in the samples (Figure 2A). Light transmission microscopy visualization of Masson's Trichrome 5-µm thick sections showed that each skin piece used as control maintained good morphology throughout the experiments as assessed by the constant presence of the stratum corneum layer (SC), a compact viable epidermis and a collagen-filled dermis. No macroscopic modification of the skin architecture was observed in PBS-exposed specimens, indicating that the protocols used from explants to fixation did not damage the skin. However, we found a significant alteration in skin morphology and structure in the explants exposed to metals compared to untreated controls (Figure 2A). We found slight defects underlying cell death in the specimens exposed to pollution. The anomalies observed included spongiosis, pyknotic nuclei, and even epidermal detachment. Dermis was also altered with a decrease in collagen density. To better understand the exact nature of the cell death observed in the explants exposed to pollution, we investigated quantification of the Malonaldehyde (MDA) which is a measure of skin oxidative damages (Figure 2B). As reported by other authors, the devastating effect of heavy metals on skin viability is mostly owning to an increase in reactive oxygen species (ROS) in the cells 8, 15 . Interestingly, exposure to pollutants induced a marked increase of MDA in the medium of explants indicating that heavy metals have induced an oxidative stress in the skin.

Heavy metals compromised skin barrier.

To further investigate the effects of the pollutants on epidermal function, we examined the expression of differentiation markers and desmosomal proteins by immunostaining (Figure 3). As expected, we observed a dramatic reduction in the expression levels of all the differentiation markers between control and treated explants. Significant differences in the amount of early, (desmoglein 1) and late (loricrin) differentiation markers were observed in skin from heavy metals-exposed samples suggesting that skin barrier was disrupted.

Heavy metals compromised cellular antioxidant systems in cells and induced DNA damage.

In response to stressing agents including metals and oxidative stress, cells are able to induce the metal binding-proteins metallothionein [START_REF] Thirumoorthy | A review of metallothionein isoforms and their role in pathophysiology[END_REF] . As expected metallothionein expression was induced by exposure to heavy metals (Figure 3). These proteins are small protein composed of 80 amino acids and contain up to 15 cysteine residues that can chelate metals and to avoid any toxic effect of the free metal in the cytosol. Glutathione peroxidase (Gpx) are involved in detoxification of hydrogen peroxidde and lipid peroxidation products and methionine sulfoxide reductase (MSR) are involved in the repair of oxidatively modified proteins in the skin [START_REF] Labunskyy | Selenoproteins: molecular pathways and physiological roles[END_REF] . To determine how these enzymes are involved in skin protection after exposure to pollutants, these proteins were detected by immunostaining in skin biopsies. Surprisingly, GPX2 and MSR labelling were less pronounced in the heavy metals-treated epidermis compared to the control ones (Figure 3). To determine if metals exposure could induce DNA damage, we looked at phosphorylation of H2AX which is used to quantify accumulation of DNA damage. Immunofluorescence against histone H2AX phosphorylated at Ser-139 revealed that metals exposure induced an increase in DNA damage in keratinocytes (Figure 3). This result indicates that heavy metals exposure can be associated with antioxidant enzymes downregulation, that may explain the accumulation of oxidative damages in the skin as shown in Figure 2B.

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Metals localization inside the skin

One goal of this study was the localization of metals in order to check their penetration in the skin. The nickel containing signature of the detected compounds is asserted by the cluster due to the two major isotopes of the nickel, 58

Ni and, 60 Ni, [START_REF] Franken | In Vitro Permeation of Metals through Human Skin: A Review and Recommendations[END_REF] C is used as a control. Nickel accumulates into the dermis and was absorbed by the skin (Figure 4). Pb) is found both in the dermis and the epidermis.

In conclusion, we demonstrated in this study that exposure to pollution decreased cell viability and antioxidant enzymes activities in the ex vivo human skin model. These anomalies are confirmed at the histological level, by the presence of cell necrosis features including spongiosis, pyknotic nuclei and dermo epidermal detachment. This may be explained by the fact that heavy metals could penetrate the epidermis up to the dermis and are retained. This journal is © The Royal Society of Chemistry 20xx

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Discussion

This study evaluated the effects of heavy metals (nickel, cadmium, lead and mercury) exposure on skin. This is the first study directly exposing human skin explants to such a cocktail. In vitro models are a promising approach to characterize the impact of pollution on skin. Interaction between metals and cell membranes play a key role in metals-induced toxicity by inducing ROS production 15 . This ROS production can damage proteins, DNA of the cells but also can trigger inflammation by activation of cytokines and might be involved in the initiation or pathogenesis of allergic or non-allergic cutaneous inflammation 18, 19 . We have shown that exposure to metals lead to DNA damage that may be explained mainly by ROS production (Figure 3). We found that exposure to metals resulted in an increase of lipid peroxidation due to ROS production (Figure 2B).

ARTICLE

Please do not adjust margins Please do not adjust margins Malondialdehyde is an indirect mediator of oxidative stress, formed by lipid peroxidation of polyunsaturated fatty acids in, for example, cholesterol, phospholipids and triglycerides 20 . It is highly reactive and able to readily modify the sulfhydryl group of cysteine residues and the imidazole group of histidine residues leading to protein oxidation. Various types of protein oxidative modifications are directly or indirectly induced by ROS by reactions with secondary products of oxidative stress 21 . Cysteine and methionine residues are particularly prone to oxidative modifications, but they might not be directly linked to protein damage, since they also participate in cellular signaling events 22 . We have shown that glutathione peroxidase and methionine sulfoxide reductase were inhibited upon pollutant exposure suggesting that they may be targets of oxidative stress (Figure 3). Our results suggest that ROS and lipid peroxidation products may react directly with these enzymes which active site contains a cysteine. Decrease in the antioxidant enzymes capacities of the skin will result in an increase in oxidative stress and to oxidative damage leading to intrinsic aging of the skin [START_REF] Rinnerthaler | Oxidative stress in aging human skin[END_REF] .

We also wanted to investigate if the heavy metals can permeate the skin and thus lead to a storage compartment of these pollutants in the skin. Because metals can penetrate through diverse routed and that surface modification with diverse ligand could modify hydrophobicity of the skin and thus metals speciation, penetration of each metals is specific 13 . Moreover, thinner facial skin is more permeable than abdominal skin 24 . Skin permeation is influenced by age, vascular and collagen composition 25 . We found that all the four metals through intact human skin under physiological conditions (Figure 4). Based on our LA-ICP MS analysis, the order of permeability is Pb>Ni>Hg>Cd. It was known that lead was easily absorbed into the skin through sweat glands and hair follicles . We found that the dermal absorption of lead is important that may contribute to the total body burden. We found that nickel is capable of permeating through the skin confirming a study done human volunteers exposed to metallic nickel for 30 minutes 27 . As filaggrin is a very rich in histidine which has huge affinity for nickel, it is most likely that nickel is bound to filaggrin in the stratum corneum and that the damage to skin barrier due to the cocktail of metals lead to a better penetration of nickel into dermis [START_REF] Irvine | Filaggrin mutations associated with skin and allergic diseases[END_REF] . Another dangerous effect caused by nickel is the induction of matrixmetalloproteinases (MMP) especially the one that can degrade collagen leading to a loss of elasticity and fitness of the skin 10 . Because nickel is retained in the dermis it can lead to a permanent increased expression of these enzymes. Regarding mercury and cadmium, they penetrate only in the epidermis as it was shown before that their solubility into the SC is a rate limiting process 13 . The concentration applied to the skin can therefore influence their permeability. The high concentration of cadmium in the epidermis may explain the induction of metallothionein, as this metal is by far the best activator of metallothionein expression. These in vitro studies indicate large variations in the results of metals permeability through the skin. It is evident that permeation of metals may be influenced by pH and speciation of the metals [START_REF] Franken | In Vitro Permeation of Metals through Human Skin: A Review and Recommendations[END_REF] .

Our results demonstrate that two metals, nickel and lead can permeate skin whereas mercury and cadmium are localized in the epidermis leading to disruption of skin barrier. Our results also demonstrated that exposure of keratinocytes to pollutants induced a significant decline in antioxidants enzymes. This was accompanied by DNA damage. We also observed an increase in lipid peroxidation products malondialdehyde (MDA) which is involved in the formation of oxidatively modified proteins. Taken together, these findings suggest that antioxidant systems may be particularly vulnerable to inactivation in conditions associated with metals exposure resulting in accumulation of oxidatively modified proteins, mitochondrial dysfunction and cell death.

Experimental Chemicals and antibodies

All chemicals were purchased from Sigma-Aldrich (Saint Quentin Falavier, France). The antibodies used in the study were rabbit antibodies against, Desmoglein (Life technology, 326000), Gamma H 2 AX (Upstate, 07627), Loricrin (Covance, PRB-145-P100), GPX2 (Abcam, ab64322) and MSR, a gift from Dr Bertand Friguet, (Sorbonne University, Paris,

30

) and mouse antibodies metallothionein (Dako M063901-2)

Skin preparation

12 mm diameter explants were prepared from a 36 years old woman abdominoplasty and placed in a survival medium at 37°C in a humid atmosphere enriched with 5 percent CO 2 .

Please do not adjust margins Please do not adjust margins With respect to the ethical permissions, for studies using skin biopsies, the principle requirements of the Declaration of Helsinki were considered to protect the rights, safety and wellbeing of subjects participating in the study. Before initiating the studies, the investigator had obtained written consent from the participants and full approval from the Freiburg Ethics Commission International for the protocol, protocol amendment(s), if applicable. All participants who provided their skin biopsies for this research provided their written informed consent to participate in this study and for their data to be used for research purposes.

Exposure to heavy metals.

On day four, the survival medium was renewed and heavy metals were deposited on a patch applied on explants at these concentrations; Hg 2.5 g/mL, Ni 2.5 g/mL, Pb 2.5 g/mL, Cd 2.5 g/mL (ICP multi-element standard V, Merck, Germany). On day 5 (24 hours after exposure), explants were taken and prepared for histology, biochemistry or immunostaining or LA-ICP-MS imaging. Three explants per conditions were prepared.

Histology

After 24 hours in buffered formalin, samples were dried and impregnated in paraffin, 5 µm sections were made and mounted on slides for histological studies. 3 explants per conditions were prepared and stained using Masson's trichrome staining. The general morphology was evaluated by microscopy analysis (Leica optical microscope type DMLB) at the magnification of 40. 5 pictures per explants were taken.

LA-ICP MS imaging

Biopsies of human skin were embedded in a Tissue-Tek OCT (Sakura Finetek USA, Inc.), frozen in liquid nitrogen and stored at -80°C. Sections of 10 µm were cut on a cryostat, mounted on a glass slide and desiccated. A 213 nm nanosecond laser (NewWave, UP-213) was directly coupled with ICP MS (Agilent 7700cs) using a 60 cm Tygon tube (5.0 mm i.d.) as described in [START_REF] Bianga | Complementarity of MALDI and LA ICP mass spectrometry for platinum anticancer imaging in human tumor[END_REF] .

The scanning was performed at the repetition rate of 20 Hz, fluence of 14 J/cm 2 , spot size of 55 μm, and scan speed of 40 μm/s. Each LA ICP MS image is composed of between 20 and 40 laser scans transects (125 µm between each line) depending on the skin thin section dimensions. Ablated material was carried to ICPMS by 500 mL/min flow of He mixed just before the torch entrance with 760 mL/min of Ar gas. ICP MS was used in the collision cell mode using He (10 mL/min) as collision/reaction gas. A 1.5 mm i.d. injector torch and Pt cones were used. Working conditions were optimized in terms of detection limits and speed by the ablation of a raw tissue partially impregnated with heavy metals. The signal intensity heatmaps were obtained using a labwritten application for Matlab R2009b (Mathworks, Natick, MA).

Immunofluorescence assays

Biopsies of human skin were embedded in a Tissue-Tek OCT (Sakura Finetek USA, Inc.), frozen in liquid nitrogen and stored at -80°C. Sections of 10 µm were cut on a cryostat, placed on Superfrost+, mounted on slides (Dako, Trappes, France) and fixed in acetone at -20°C for 10 min. Then, they were rinsed with phosphate-buffered saline (PBS) and incubated for 30 min with PBS containing BSA 1% at room temperature. After three times washing with PBS, slides were incubated with primary antibodies, overnight at 4°C in a dark humid chamber. After another cycle of washing, they were incubated with the appropriate secondary Alexa Fluor 568 or 488 labeled antibody at 1/200 dilution (Molecular Probes) for 1 h at room temperature. Nuclei were stained with DAPI (300 M, Sigma-Aldrich, St.Louis, MO, USA). Negative controls were realized without primary antibodies (data not shown). After a final rinsing with PBS, the slides were mounted with the Dako fluorescent mounting system and stored at 4°C, protected from light, until analyzing with a SP5 Leica confocal microscope (magnification: 6630). Quantification analysis was performed using the LeicaQWin Software. Quantifications of all the skin markers were standardized to the epidermis surface.

MDA assay

MDA was determined in the survival medium on day 5 using the lipid peroxidation kit determination from Abcam (Cambridge, MA, USA).

Statistical analysis

Results were expressed as mean ± SEM and analyzed using GraphPad Prism 5 Software. The Mann-Whitney and one-way ANOVA tests were used to compare data sets. Statistical significance was set at P < 0.05.

Conclusions

Finally, and although more experiments are needed to completely understand the penetration mechanisms of metals, this study represents a proof of principle and provides a breakthrough in the field of skin absorption which allows us to envisage potential toxicological risks of metals. Further investigations using Nano-SIMS technology will help us to localize the metals at the level of the organites to decipher better their effects on cell physiology

Figure 2 .

 2 Figure 2. Effect of heavy metals exposure on skin structure and oxidative. A. Masson's trichrome staining of the explants at Day 5 . Two representative pictures are shown for each conditions, control and heavy metals treated (HM). B. Quantification of Lipid peroxidation. MDA was determined in the survival media at 24 hours after heavy metals exposure using a kit as described in the methods.

Figure 3 .

 3 Figure 3. Heavy metals induce changes in skin proteins expression at day 5. Immunofluorescence staining of 5 proteins in epidermis using specific antibodies. Metallothionein (response to metals), Gamma H2AX which is a measure of DNA damage, Desmoglein and Loricrin (proteins from the skin barrier) and antioxidant proteins (Gpx2 and PMSR) proteins in ex vivo human skin. Representative pictures of skin slides of non-stressed (Control) and heavy metals stressed (HM) are shown. Following confocal laser microscopy acquisition, pictures were analyzed, and relative expression of the different proteins was quantified and standardized to the epidermis area (LeicaQWin software).

Figure 4 .

 4 Figure 4.Figure 4. Penetration of heavy metals in human full thickness skin at day 5. A. Pictures of skin slices (E, epidermis and D, dermis). B. LA ICP -MS qualitative images. C. Heat map of metals (nickel, mercury, cadmium, lead). The signal intensity heatmaps were obtained using a lab -written application for Matlab (Mathworks, Natick, MA) as described in

Figure 4 .

 4 Figure 4.Figure 4. Penetration of heavy metals in human full thickness skin at day 5. A. Pictures of skin slices (E, epidermis and D, dermis). B. LA ICP -MS qualitative images. C. Heat map of metals (nickel, mercury, cadmium, lead). The signal intensity heatmaps were obtained using a lab -written application for Matlab (Mathworks, Natick, MA) as described in 31 .

  with 0.1 s as integration time except 13 C (0.01s). Acquisition time per skin thin section was approximately 40 min.

29 .

 29 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins Notes and references

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

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This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

Conflicts of interest

There are no conflicts to declare.


Abderrahim Titi

Ismail Warad

Monique Tillard

Rachid Touzani

Mouslim Messali

Mohamed El Kodadi

Driss Eddike

Abdelkader Zarrouk

Intermolecular Interactions in [C6H10N3]2[CoCl4] complex; Synthesis, XRD/HSA relation, spectral and Catecholase catalytic analysis

Keywords: Cobalt(II), Crystal structure, HSA, Catecholase activity

Complex of bis(6-amino-2,4-dimethylpyrimidin-1-ium) tetrachloridecobaltate(II)

[C6H10N3]2[CoCl4] was synthesized and characterized using several physicochemical surveys as FT-IR, 1H & 13C-NMR, Hirshfeld surface (HSA) and XRD-diffraction analysis. The XRD and HSA quantified a significant contribution of non-covalent H-bonds interactions in lattice to build an ionic form of the [C6H10N3]2[CoCl4] complex. The molecular arrangement is qualified for the desired complex as an alternation of inorganic and organic species, the organic C6H10N3 cationic ligand and inorganic [CoCl4] 2-tetrahedral anionic metal center connected together via several short interactions like -HN-H…Cl, Nring-H+…Cl and Cring-H…Cl Hbonds interactions. The complex was tested as a catalyst for the oxidation reaction in methanol from 3,5-ditertiarybutylcatechol to 3,5 ditertiarybutylquinone. The [C6H10N3]2[CoCl4] complex displays a good catalytic activity in aerobic condition, with a reaction rate Vmax = 10.11×10-4 M.L -1 .h -1 and key kinetics parameters KM = 0.12 M and kcat = 84.08 M -1 .h -1 .

Introduction

Organic and inorganic hybrids material are highly wanted compounds due to their broad range of applicable properties, their exceptional structural and amazing composition diversity [1]. Therefore, the structures of the inorganic anion and organic cations reflected several unique properties like optical, electrical, electroluminescence and magnetic [1][2][3][4][5][6]. Currently, hybrid organic-inorganic crystalline solids containing anionic metal complexes and organic cations have been developed for their hydrogen bonding like N-H….X connections with halides of the metal center to build and design the crystal structure lattice of hybrid material for structural analysis [7][8][9]. However, studies dilling with such kind of material is still limited.

Cobalt-organic hybrid material complexes are one of the most significant materials in inorganic chemistry, its advantages include good tunable bandgap, electric mobility, thermal stability, magnetic properties, and structural varieties served it to be very good catalysts [1][2][3][4][5][6].

Even after the discovery of the famous hydroformylation catalytic process by Otto Roelen, many other catalytic applications have been promoted [START_REF] Falbe | Carbon Monoxide in Organic Synthesis[END_REF][START_REF] Hebrard | [END_REF][12][13][14][15]. Design of cobalt-organic hybrid material that can activate molecular oxygen stile a challenge to predict super-oxidation catalysts (SOC) [16]. Synthetic model studies on the reactivity of cobalt complexes towards Catecholase oxidation processes, both implicate structural and electronic parts as being accountable for the catalytic activity [16][17][18].

Considering the attributes and attractive the of tetrachlorocobaltate(II), herein, we report on the preparation of a new [C6H10N3]2[CoCl4] complex based on cobalt(II) and (6-amino-2,4dimethylpyrimidin-1-ium) ligand, the intermolecular interactions in the structure was compared by XRD and HAS, physicochemical and oxidation properties of the complex were also figure out under RT conditions.

Experimental

Materials and physical measurements

All the chemicals reagents and solvents were commercial available and used as received. IR were recorded at RT using a Perkin Elmer FT-IR spectrometer in the 4000-500 cm -1 frequency range. 1 H and 13 C NMR were recorded on a BRUKER equipment at 400 MHz for 1 H and 100 MHz for 13 C. Spin resonances are presented as chemical shifts (δ) in ppm and referenced as follow to the residual peak of the solvent employed as an internal standard: DMSO-d6 2.50 ppm ( 1 H NMR), 39.9 ppm ( 13 C NMR). The UV -Vis spectra were recorded on an UV-1280 Shimadzu spectrometer. The Hirschfeld surfaces have been computed using the Crystal Explorer 3.1 program [START_REF] Wolff | Crystal explorer 2.1[END_REF].

Synthesis of [C12H20N6CoCl4]

A volume of 10 mL of 2,6-dimethylpyrimidin-4-amine (50 mg, 0.41 mmol) in solution in ethanol) was treated with 10 mL of solution of CoCl2.6H2O (96.5 mg, 0.41 mmol) dissolved in ethanol, (35.6 mg, 0.82 mmol) of HCl was added to the reaction mixture (Scheme 1).

Precipitation of a blue powder occurred while stirring at room temperature for 24h. The blue powder was filtered and washed with EtOH (30mL), followed by Et2O (30mL) to give the blue solid (1.3g, 70%). Recrystallization of the crude product from a solvent pair (CH3)2CO/Et2O gave blue crystals (1.2g,64.6%). 1 H NMR (DMSO-d6, 400MHz): δ = 2.13 (s, CH3), 2.25 (s, CH3), 6.06 (s, C-H), 6.63 (s, NH2). 13 Scheme1: Synthesis of the complex CoCl4LH.

X-ray crystal structure

A blue platelet was selected under a stereo microscope with a polarizing filter to be a single crystal suitable for diffracted intensities recording. Data were measured at room temperature on a Bruker D8 Venture 4-circle diffractometer equipped with a Incoatec IµS 3.0 Mo microsource (110µm beam, Kα radiation λ = 0.71073 Å) and a Photon II CPAD detector. Cell refinement, data reduction and Lorentz-polarization corrections were performed using the Apex software suite [START_REF]APEX3. Version 2017[END_REF] and reflections were corrected for absorption effects (multi-scan SADABS).

The structure was solved and refined by full-matrix least-squares on F 2 using the SHELX programs [START_REF] Sheldrick | [END_REF]22]. Positional and anisotropic displacement parameters were refined for all non-H atoms. The H atoms were detected in the final Fourier difference and, with the exception of the freely refined amine and ammonium H atoms, were treated using AFIX instructions with displacement parameters equal to 1.2 times (1.5 for CH3) the Ueq of the parent atom. The main crystal data and refinement parameters are given in Table 1. Largest diff. peak and hole (e.Å -3 ) 0.621 / -0.667

Results and discussion

Synthesis, CHN-EA, IR, UV-vis. and NMR analysis

The prepared of bis(6-amino-2,4-dimethylpyrimidin-1-ium) tetrachloridecobaltate(IV) The 1 H and 13 C-NMR spectra of [C6H10N3]2[CoCl4] complex dissolved in DMSO-d6 reflected the complex as paramagnetic compound since the Co is with +2, therefore the electronic configuration of the tetrahedral Co center will be 4s 0 3d 7 with eg 4 and t1g 3 d-splitting.

Two types of protons consistent with the structure formula detected by XRD were recorded as in Fig. 3. 1H NMR two signal belong to 6H of the CH3 at δ 2.13 and 2.25 ppm, one signal at 6.06 ppm cited to Cring-H and the broad signal at δ 8.3 cited to 2H of NH2 (Fig. 3a). The N-H+ protons signals did not appear due to D-exchanged with the D2O. The extinction conditions suggested centrosymmetry and space group Pnma. The structure contains anionic (-2) tetrachlorocobalt and 2 cationic (+1) 6-amino-2,4dimethylpyrimidin-1-ium units represented with ORTEP (Fig. 4a). In the crystal lattice, four types of non-covalent bonds shorter than 3 Å was detected by XRD measurements, since two polar H atoms together with three types of N atoms quoted in the complex structure, therefore, 4 types of H-bond linkages were recorded experimentally in the packed lattice of the desired complex as two types of -HN-H…..Cl with 2.307 Å and --HN-H…..Cl….. H-NH with 2.463 Å as illustrated in Fig. 5a. one longer H-bonds were detected as -Nring-H…..Cl….. H-Nring with 2.385 Å (Fig. 5b). Furthermore, 2 longer H-bonds were recorded as -Cring-H…..Cl with 2.888 Å (Fig. 5c), no other interaction types were recorded.

HSA

To obtain additional information on the role played by intermolecular interactions within the crystal, a Hirshfeld surface analysis was performed [29][30][31][32]. The results are illustrated in Fig. 3 with representations of the Hirshfeld three dimensional surface mapped over the normalized dnorm function (Fig. 6) and shape index (Fig. 6b) established in the range 0.654 to 1.768 a. u. The colors are related to the distance from the nearest neighbors and illustrate the strength of the intermolecular contacts. Large red zones are visible close to the chlorine atoms and the polar hydrogen atoms, six red spots were found on the surface computed for the molecular structure, they correspond to the presence of three short contacts or hydrogen type bonds. Two N-H…..Cl bonds with a distance of 2.38 Å, two NH2…..Cl bonds with a distance of 2.31 Å and two Cring-H….Cl with a distance of 3.17 Å was counted in good agreement with packing in the crystal structure.

The 2D fingerprint plots represented in Fig. 6b has been built from the 3D Hirshfeld surface by considering inside and outside closest neighbors. These integrated views on intermolecular contacts are useful to visualize the contributions of either polar or non-polar interactions to the crystal packing forces. It is clear that the H…H intermolecular contacts represent the larger part (28.9%), which is due to the high number of H-atoms and their spatial position in the molecule of the complex. In contrast, the H….N interactions are found with the lowest and almost zero ratios. The other atom…atom intermolecular contacts appear in the order of importance H….Cl (10.6%) > H….N (2.2%) > H….C(1.2%).

Catecholase studies

catecholase activity via UV-Vis study

The catecholase activity of the complex was observed by treating 10 -4 M solution of complex with 100 equivalents of 3,5-DtBC (10 -2 M) solution under aerobic fitness at room temperature in MeOH, as seen in Scheme 2. To properly establish the catalytic activity of the 3,5-DtBC on the production kinetics of 3,5-DtBQ, a volume of 0.15 mL of complex (1×10 -4 M) dissolved in methanol was added to 2 ml of 3,5-DtBC (1×10 -1 M), and then the spectral variations were quantified using the progression of the absorbance band characteristic of the 3,5-DtB-quinone registered every 5 min. The kinetic experiments were realized at room temperature.

Conclusion

During this work, we synthesized a The complex displays a good catalytic behavior, under ambient conditions, estimate to the oxidation of 3,5-DtBC to 3,5-DtBQ using atmospheric oxygen as oxidant in methanol. Overall the catalytic activity of the [C6H10N3]2[CoCl4] complex is found very similar to that of some other cobalt complexes.

  C NMR (DMSO-d6, 100 MHz): δ = 23.71 (CH3), 25.72 (CH3), 100.53 (C-H), 164.29 (C), 164.38 (C), 166.99 (C). Selected IR peaks of the desired complex v = 3350 (N-H), 2934 (C-H), 1678 (C=N), 1490 (C=C), 1436 (C-H), 1358 (N=N), 1018(C-N), 720(C-H), 520 (Co-Cl) cm -1 .

[C6H10N3] 2 [

 2 CoCl4] Complex was made available by mixing of [1:1] CoCl2.4H2O with of 6amino-2,4-dimethylpyrimidin-1-ium in the presence of HCl dissolved in ethanol. The blue color precipitate indicated the formation of the desired complex with good yield after one day of stirring under open RT condition. The complex is water-soluble, poor insolubility in ROH and insoluble in hexane or ethers which supported the complex [C6H10N3]2[CoCl4] salty nature. The composition of the complex was confirmed using CHN-elemental analysis, the calculated atomic percentage of the C, 32.09; H, 4.49 and N, 13.12% from C12H20Cl4CoN6 formula and found experimentally as C, 32.02; H, 4.20 and N, 13.16% which is with agree with the general [C6H10N3]2[CoCl4] formula. The reaction of CoCl2.4H2O with 6-amino-2,4-dimethylpyrimidin-1-ium ligand to produce [C6H10N3]2[CoCl4] complex was monitored by IR. IR spectra of the ligand and its complex are illustrated in Fig. 1.

Fig. 1 .

 1 Fig. 1. FT-IR spectra of free ligand (a) and the [C6H10N3]2[CoCl4] complex (b).

Fig. 2 .

 2 Fig. 2. UV-Visible of [C6H10N3]2[CoCl4] complex

13C-

  NMR spectrum reflected δ's 23.71 and 25.72 belong to 2C of CH3 groups and three signals with δ's 164.29, 164.38, 166.99 ppm belong to the 3C of the aromatic ring (Fig.3b).

Fig. 3 . 3 . 2 .

 332 Fig. 3. (a) 1 H NMR and (b) 13 C NMR spectra in DMSO-d6

Fig. 4 .

 4 Fig. 4. ORTEP of [C6H10N3]2[CoCl4] with two molecular units CoCl4 2-& C6H10N3 + and (b) the orthorhombic molecular packing lattice of C12H20N6CoCl4

Fig. 5 .

 5 Fig. 5. H-bonds interaction: a) -HN-H…..Cl & -HN-H…..Cl….. H-NH, b) -Nring-H…..Cl…..

Fig. 6 .

 6 Fig.6. Hirshfeld surface mapped over dnorm (a), shape index (b) and inside/outside 2-D fingerprint plots (c).

Scheme 2 .

 2 Scheme 2. Catalytic oxidation of 3.5-DtBC to 3,5-DtBQ.The above oxidation process was monitored by UV-vis., the Spectrophotometric data have been noted by generating absorbance versus wavelength (λ= 400 nm scan) plots at a regular time intervals of 5 min in the range 325-625 nm (Fig.7a). The 3,5-DtBQ product band appeared at 400 nm which graduately increases in intensity by time reflected the completeness of the reaction, No absorbance at this band was observed in the absent of [C6H10N3]2[CoCl4] or even in the presence of CoCl2.6H2O using the 3,5-DtBC substrate (Fig.7b) , therefore, it is clearly observed that [C6H10N3]2[CoCl4] acted as a mild condition catalyst in absence of complex as indicated by the black curve. It clearly appears that the complex drives the oxidation reaction of 3,5-DtBC to 3,5-DtBQ under mild conditions.

Fig. 7 .

 7 Fig. 7. (a) Wavelength scan and (b) Abs. vs. time (at λ = 400 nm) plots for the oxidation of 3,5-DtBC to 3,5-DtBQ in the absence and presence of the [C6H10N3]2[CoCl4] complex.

Fig. 8 .

 8 Fig. 8. a) Enzymatic Michaelis-Menten Kinitic plot (rate vs. [substrate]) and b) Lineweaver-Burk plot (1/rate vs.1/[substrate] for the complex in MeOH.

  [C6H10N3]2[CoCl4] complex designed by 6-amino-2,4dimethylpyrimidin-1-ium ligand and CoCl2.6H2O salt. The complex is described by various spectroscopic techniques as 1 H & 13 C NMR, UV-vis., FT-IR, CHN-EA and HSA. The structure of [C12H20N6CoCl4] was proved by XRD-diffraction. The XRD reflected the tetrachloridecobaltate(II) anion as tetrahedral geometry around the cobalt center. The HSA and XRD analysis supported the presence of many H-bond like of -HN-H…..Cl with 2.307 Å and --HN-H…..Cl….. H-NH with 2.463 Å,-Nring-H…..Cl….. H-Nring with 2.385 Å and -Cring-H…..Cl with 2.888 Å distances.

Table 1 .

 1 Crystal data and refinement parameters of C12H20N6CoCl4

	Chemical formula	C12H20N6CoCl4
	CCDC	1987738
	Mr	449.07
	Crystal system, space group	Orthorhombic, Pnma
	Absorption coefficient (mm -1 )	1.421
	Theta range (°)	2.648 to 29.152

Table 2 .

 2 Main bond lengths [Å] in C12H20N6CoCl4

	No.	Bond type	Å	No.	Angle type	( o )
	1	Cl1 Co	2.2544	1	Cl1 Co Cl2	107.32
	2	Cl2 Co	2.309	2	Cl1 Co Cl3	109.58
	3	Cl3 Co	2.283	3	Cl1 Co Cl1	113.01
	4	Co	Cl1	2.2544	4	Cl2 Co Cl3	109.98
	5	N1	C2	1.346(3)	5	Cl2 Co Cl1	107.32
	6	N1	C7	1.361(3)	6	Cl3 Co Cl1	109.58
	7	C2	N3	1.313(3)	7	C2 N1	C7 122.2(2)
	8	C2	C9	1.488(4)	8	N1 C2	N3 122.2(2)
	9	N5 H5A	0.98(5)	9	N1 C2	C9 117.6(2)
	10	N5	C4	1.333(3)	10	N3 C2	C9 120.2(2)
	11	N5 H5B	0.89(3)	11	H5A N5	C4	121(2)
	12	N3	C4	1.366(3)	12	H5A N5 H5B 118(3)
	13	C8	C7	1.485(4)	13	C4 N5 H5B 121(2)
	14	C4	C6	1.404(3)	14	C2 N3	C4 117.3(2)
	15	C7	C6	1.351(3)	15	N5 C4	N3 116.6(2)