Annalisa Di Stazio
email: annalisa.distazio@cnrs-orleans.fr

Christian Chauveau

Guillaume Dayma

Philippe Dagaut

Combustion in micro-channels with a controlled temperature gradient

Keywords: Micro combustion, Micro combustor, Premixed combustion, Transient flame behaviors, Wall temperature

published or not. The documents may come

Combustion in micro-channels with a controlled temperature gradient

Introduction

Nowadays the growing need for low fuel consumption systems makes very attractive the development and the application of MicroElectroMechanical (MEMS) devices, which may produce more energy than the modern batteries by using hydrocarbon fuels [START_REF] Fernandez-Pello | Micropower generation using combustion: Issues and approaches[END_REF][START_REF] Kyritsis | Mesoscale combustion: a first step towards liquid fueled batteries[END_REF]. This concept is coupled with that of "excess-enthalpy" combustion [START_REF] Weinberg | Combustion Temperatures: The Future?[END_REF], which led to the design of a new device, called the Swiss Roll combustors [START_REF] Lloyd | A burner for mixtures of very low heat content[END_REF] consisting of a micro reactor where the reactants are preheated by burned gas, with the combustion zone at the center of a pair of spiral channels. Recently channels with an inner diameter lower than the quenching diameter have been used for this burner [START_REF] Kim | Flame stabilization and emission of small Swiss-roll combustors as heaters[END_REF][START_REF] Kim | Development and scale effects of small Swiss-roll combustors[END_REF], and the combustion stabilization in such devices is a far from trivial problem [START_REF] Sánchez-Sanz | Premixed flame extinction in narrow channels with and without heat recirculation[END_REF][START_REF] Vican | Development of a microreactor as a thermal source for microelectromechanical systems power generation[END_REF]. In order to deepen and widen knowledge on micro-scale combustion, many studies have been performed [START_REF] Ju | Microscale combustion: Technology development and fundamental research[END_REF][START_REF] Kaisare | A review on microcombustion: Fundamentals, devices and applications[END_REF]. With the reduction of the combustor's size, the difficulty to sustain a stable combustion increases, due to the larger surface to volume ratio. The effects of quenching, thermal and radical, both increase. However, with the continuous heating of the solid phase it is possible to sustain combustion in a tube having an internal diameter smaller than the ordinary quenching diameter and in lean conditions. Nevertheless, the management of the combustor flow and thermal conditions is required to reduce the wall effects [START_REF] Egolfopoulos | Wall effects on the propagation and extinction of steady, strained, laminar premixed flames[END_REF]; the miniaturization enhances the heat losses [START_REF] Li | Experimental investigation on heat loss and combustion in methane/oxygen micro-tube combustor[END_REF] and, as shown in the previous studies, all the combustor design parameters influence the flame stabilization [START_REF] Veeraragavan | Flame speed predictions in planar micro/mesoscale combustors with conjugate heat transfer[END_REF]. As observed by Popp and Baum [START_REF] Popp | Analysis of wall heat fluxes, reaction mechanisms, and unburnt hydrocarbons during the head-on quenching of a laminar methane flame[END_REF], the wall heat flux increases with increasing wall temperature and the interaction between the flame and the wall is very fast: the higher the wall temperature is, the higher is the fuel consumption and the behavior of the reactive mixture changes, with an increase in concentration of H and OH species. High temperatures enhance the Soret effect, particularly for light species, resulting in a reduction of burning velocity [START_REF] Bongers | The effect of simplified transport modeling on the burning velocity of laminar premixed flames[END_REF][START_REF] Ern | Thermal diffusion effects in hydrogen-air and methane-air flames[END_REF]. As described by Maruta et al. [START_REF] Maruta | Characteristics of combustion in a narrow channel with a temperature gradient[END_REF], three distinct flame behaviors can be observed in narrow channels: bright and stable flames for high flow velocity, flames with repetitive extinction and ignition (FREI) in the intermediate flow rate range and weak flames at low flow velocity. The flame luminosity decreases with the reduction of the flow rate. The flame is stabilized in a certain position where balance between the flame temperature, wall heat losses and flow rate occurs.

The objective of this work is to provide detailed information on methane/air mixture flame behavior's in narrow channels. To this end, experiments at different conditions (flow velocity and equivalence ratio) were performed and instabilities analysis was conducted.

Experimental set-up

The experimental setup was designed to investigate the combustion in narrow channels with a premixed methane/air blend at atmospheric pressure. The micro reactor used here consists of a cylindrical quartz tube, inside which the gaseous mixture flows. This tube is heated from outside thanks to three blowtorches around the tube, separated from each other by 120°. This system ensures a stationary, homogeneous and Gaussian temperature profile starting from ambient temperature at the upstream side (gas inlet) to a maximum of 1600K at downstream (gas outlet). This temperature slope allows the flame to auto-ignite, overcoming quenching issues. The three external heat sources are fed by a hydrogen/oxygen mixture. The heating flames are then free of CH* species, which allows a better visualization of CH* chemiluminescence from the flame studied inside the microchannel.

The methane/air mixture is premixed in a small tank and six thermal mass flowmeters Brooks GF40 (error less than 1%) are used to feed the reactor. Each gas line (CH 4 , O 2 , N 2 ) has 2 flowmeters with overlapping flow rate ranges, allowing a good exploration of all flame regimes.

The temperature distribution along the outer side of the tube is measured by a FLIR A655sc thermal camera with a 640×480 pixels matrix, with a measurement uncertainty of ±3%. The operating range is selected between 300 and 2000° C. The temperature measurements are taken in real time at a frequency of 10 Hz, which ensures a continuous recording of the temperature imposed to the micro reactor. An emissivity correction is carried out because in the operating spectral range of the camera (7.5-14μm), fused silica emissivity varies with temperature, as shown by Sova et al. [START_REF] Sova | High-temperature infrared properties of sapphire, AlON, fused silica, yttria, and spinel[END_REF].

A Princeton Instrument spectroscopy EMCCD camera (ProEM 1600), equipped with a 105-mm Nikkor lens (f/2.8D), is used to detect the CH * signal at 430 nm. The CCD camera operates in a 1600×200 pixels matrix with a 16-bit output digitization. A band-pass filter (20BPF10-430) is used to observe the flame chemiluminescence through the quartz tube. In order to increase sensitivity and frame rate, binning operation and adapted exposure time are performed. The spatial resolution is 62 pixels/mm. For instable flames the maximum recording frequency of 4260 Hz is used.

A schematic representation of the experimental apparatus is showed in Fig. 1. The temperature profile along the tube is displayed in Fig. 2. The origin of the horizontal axis is set at the maximum of the temperature profile. Using this set-up, experiments were conducted over a range of flow rates from 0.03 m/s to 1 m/s in a 2.15 mm inner diameter tube and compared to those obtained in previous studies. Then experiments were performed in a 1.85mm inner diameter tube over a range of equivalence ratios from 0.5 to 1.5. Finally, to further examine the unstable regime, a study on FREI frequencies was carried out and the results are discussed.

Results

Flame visualization

After auto-ignition in the side downstream, the flame propagates upstream and finally stabilizes at a stable position in the temperature gradient where balance between the flame propagation velocity and the fresh gases flow rate is obtained. As observed in previous experiments by Maruta et al. [START_REF] Maruta | Characteristics of combustion in a narrow channel with a temperature gradient[END_REF], we also detected three dynamic flame behaviors depending on the inlet velocity: stable flames, Flames with Repetitive Extinction and Ignition (FREI) and weak flames.

Stable flames, set at a given position in the temperature gradient, show curvature that depends on the flow rate, as shown in Fig. 3. When the fresh gas flow rate is increased the flame moves towards the maximum of the imposed temperature profile. Due to increased heat losses, the flame temperature and flame speed become lower [START_REF] Chao | Analytical and experimental study of premixed methane-air flame propagation in narrow channels[END_REF] and the shape of flame is driven by the incoming fresh gases velocity profile, as described in [START_REF] Daou | Influence of conductive heat-losses on the propagation of premixed flames in channels[END_REF]. We have also observed unstable oscillating flames, in the transition between stable flames and FREI regime. The flame ignites downstream, propagates upstream and oscillates before extinction. This phenomenon, reported previously by Tsuboi et al. [START_REF] Tsuboi | Study on ignition and weak flame in heated meso-scale channel[END_REF] and called 'oscillating FREI', is highlighted on Figs. 4 and5. This regime has been simulated numerically by Minaev et al. [START_REF] Minaev | Nonlinear dynamics of flame in a narrow channel with a temperature gradient[END_REF] using a 1D thermo-diffusive model and by Jackson et al. [START_REF] Jackson | Flames in narrow circular tubes[END_REF] using a fixed Poiseuille flow-field.

Flow Flow

Temporal evolution of the CH* signal is displayed on Fig. 5. One can notice on the Fig. 5a the cyclic trace of the signal before the extinction. For example, at fresh gases velocity of 0.34 m/s the number of oscillations is variable, whereas at 0.33 m/s, two oscillations before extinction are observed. At 0.32 m/s, the FREI regime without oscillation is observed as shown in Fig. 5b. The FREI regime occurs for fresh gases velocities down to 0.05 m/s. One can notice that the flame luminosity is increased artificially because the flame is oscillating at the same position. Finally, weak flames, typical of mild combustion [START_REF] Cavaliere | Mild Combustion[END_REF], are observed for velocities lower than 0.05 m/s. These weak flames have very low luminosity and, as shown in previous studies, the behavior of reactive mixture can completely change [START_REF] De Joannon | Analysis of process parameters for steady operations in methane mild combustion technology[END_REF][START_REF] Sabia | Modeling Negative Temperature Coefficient region in methane oxidation[END_REF] resulting in a weak temperature increase during the combustion process. At 0.04 m/s oscillating weak flames are visible; this regime has been predicted numerically by Jackson et al. [START_REF] Jackson | Flames in narrow circular tubes[END_REF] and referred in [START_REF] Tsuboi | Study on ignition and weak flame in heated meso-scale channel[END_REF] as a 'combination' flame with small amplitude. The weak flame becomes stable at 0.03 m/s, as shown in Fig. 6b. If we compare our results with those obtained by Tsuboi et al. [START_REF] Tsuboi | Study on ignition and weak flame in heated meso-scale channel[END_REF][START_REF] Tsuboi | Lower limit of weak flame in a heated channel[END_REF] one can see that the same trends are observed, but significant differences appear in terms of temperature readings. We believe that these differences are mainly due to differences in experimental setup. Primarily, the temperature gradient of the present study is steeper and micro blowtorches distribute heat evenly around the tube whereas in the experiments of Tsuboi et al. [START_REF] Tsuboi | Study on ignition and weak flame in heated meso-scale channel[END_REF], the tube is heated from the below by a flat flame burner. Furthermore, the flame in the tube has an impact on the wall temperature. The temperature profile is measured continuously by the infrared camera in our experiments showing that heat released by the flame inside the tube cannot be neglected, as can be seen in Fig. 8. To better understand the differences between the present results and those of Tsuboi et al. [START_REF] Tsuboi | Study on ignition and weak flame in heated meso-scale channel[END_REF], experiments with two heat sources were carried out. The top burner was removed to simulate the heating from below used by Tsuboi et al. [START_REF] Tsuboi | Study on ignition and weak flame in heated meso-scale channel[END_REF]. The results are shown in Fig. 9. It can be noticed that the shape of the flame is affected by the non-symmetric distribution of heat sources. With two heating sources, the flames brightness is less intense and they are angled, which brings uncertainty in the detection of the flame position and consequently uncertainty in the determination of temperature at the flame position.

Effect of flow velocity

Let us examine the trends in Fig 7, according to the different regimes observed. Firstly in the stable regime, the evolution of the stabilization temperature is quasi-linear for the high velocity regime of 0.6 to 1 m/s (not shown in Fig 7). This area corresponds to the convex shape of the flame front. Then the dependency on flow velocity is emphasized, stabilization temperature is reduced with the speed reduction (0.6 to 0.35 m/s). This is the region where the flame front becomes concave (directed from the burned toward the unburned gas, Fig. 3). That makes it less sensitive to heat loss, as demonstrated by Daou and Matalon [START_REF] Daou | Influence of conductive heat-losses on the propagation of premixed flames in channels[END_REF].

In the FREI regime, the dependence of the ignition and extinction points according to the inlet velocity is examined in more details. A weak dependency is observed for the ignition data points, except for very low velocity regimes where the ignition occurs at lower temperatures. The experimental data points obtained by Tsuboi et al. [START_REF] Tsuboi | Study on ignition and weak flame in heated meso-scale channel[END_REF] are also very close to ours at low speed. Regarding extinction, a net temperature dependence of quenching is observed according to the flow velocity. Lower the inlet velocity is, lower the extinction temperature is. This phenomenon has been numerically investigated by Norton and Vlachos [START_REF] Norton | Combustion characteristics and flame stability at the microscale: a CFD study of premixed methane/air mixtures[END_REF] and Minaev et al. [START_REF] Minaev | Nonlinear dynamics of flame in a narrow channel with a temperature gradient[END_REF]. From a phenomenological point of view the following explanation can be proposed: in the case of flames at moderate inlet velocity (0.2 to 0.34 m/s) the residence time is low, the convective time scale becomes short compared to the axial conductive time scale. This has a twofold effect. First, the flame loses little heat to the wall and more power is generated, resulting in a high temperature, and a short intense reaction zone. This is evidenced by the time tracking of CH* signal which increases dramatically to form a narrow peak (see Fig. 5). Secondly, by reducing of heat transfer to the wall, it takes a longer distance to preheat the reacting mixture which will lead to the extinction of the flame by thermal quenching.

For low inlet velocities (0.05 to 0.2 m/s), the residence time is higher, the convective time scale becomes long. For slow flows, axial conduction through the gas and the solid dominates, spreading the heat, leading to nearly isothermal conditions. The wall temperature remains high while the power generated by the reaction decreases, resulting in an increase in heat lost relative to heat generated, causing extinction.

But, as shown in [START_REF] Aghalayam | The role of radical wall quenching in flame stability and wall heat flux: hydrogen-air mixtures[END_REF] for relatively weak flames the radicals recombination is exothermic and its released heat can stabilize the flame. At the same time the quenching properties change with materials [START_REF] Miesse | Submillimeter-scale combustion[END_REF]: the fused silica has a very good chemical inertness but its thermal conductivity depends on temperature [START_REF] Wray | Thermal Conductivity of Clear Fused Silica at High Temperatures[END_REF]. As seen in reference [START_REF] Raimondeau | Modeling of high-temperature microburners[END_REF], significant temperature gradients appear along the tube radius when the heat transfer coefficient increases, resulting in lack of flame propagation: by decreasing the tube diameter, gradients become weaker and the interaction between the gas and the wall is faster, leading to the increase of radical depletion effect. Furthermore, radical quenching is promoted with insulating materials, which do not strongly adsorb radicals: this could explain why at lower inlet velocity the flame extinguishes at lower temperature.

Regarding the most pronounced differences with the results of Tsuboi et al. [START_REF] Tsuboi | Study on ignition and weak flame in heated meso-scale channel[END_REF] concerning the FREI extinction temperatures, the gap can be assigned to the non-uniformity of the wall temperature, the difference between top and bottom, causing an earlier thermal quenching.

Equivalence ratio effect

Experiments were conducted over a range of equivalence ratios from 0.5 to 1.5 in a tube of 1.85 mm of internal diameter. The results are shown in Fig. 10. One can notice that for an equivalence ratio of 1.1, we have the lowest temperatures of stabilization (i.e. stable regime) and extinction (FREI regime). The flames obtained with a different equivalence ratio, lean or rich, stabilize at a higher temperature for stable regime, and for FREI regime, extinction temperatures follow the same trend. This is due to the flame temperature [START_REF] Bakali | Experimental and modeling study of the oxidation of natural gas in a premixed flame, shock tube, and jet-stirred reactor[END_REF] and the flame speed [START_REF] Chao | Analytical and experimental study of premixed methane-air flame propagation in narrow channels[END_REF] which are higher near the stoichiometric condition. The higher temperature increases the heat transfer towards the wall and the convective exchange to fresh gas. However, as the flame velocity is itself increased, convective exchanges are favored over conduction. This allows the flame to stabilize at a lower wall temperature.

It can be noticed that the transition between stable and FREI regimes is different for the lean and the rich side (Fig. 10). For lean mixtures, this transition is effective for lower speeds when equivalence ratio decreases: 0.4 m/s for φ = 1.1 while it is 0.3 m/s for φ = 0.5. Conversely, the stable to FREI transition speed is slightly higher for rich mixture, and equal to 0.44 for  = 1.5.

For φ <1, FREI ignition points are omitted because the flames were less bright and ignition was difficult to precisely localize. Flow Fig. 10. The effect of equivalence ratio on flame positions.

FREI frequencies

The FREI regime is a specific regime where the flame ignites, spread on and extinguishes repeatedly with remarkable regularity over time. This feature allows us to perform a frequency analysis of the measured CH* signal. Using a Fourier Transform analysis of the spectral cross-section of the CH* signal (Fig. 11), the characteristic frequency of flames in FREI regime was determined. This characteristic frequency was determined for each flow rate and for each equivalence ratio considered here. Fig. 12 summarizes the results. The first point concerns the linear evolution of the frequency versus the flow velocity. The second concerns the minimization of the frequency for an equivalence ratio near stoichiometry, while the frequency increases as the equivalence ratio deviates from this optimum, either to the rich or to the lean side.

To explain the variation of frequency as a function of inlet velocity, we determined the characteristic time between ignition and extinction. The time between ignition and extinction of the flame (τ IE ) and the time between the extinction and ignition (τ EI ) are shown in Fig. 11.

The evolutions of these characteristic times depend on the velocity as shown in Fig. 13 for a fixed equivalence ratio. It can be noted that the time required to transport the fresh gas to the ignition point decreases with increasing flow rate, while τ IE does not change significantly. The FREI frequency being the inverse of the sum of these two characteristic times, implies that frequency increases with the increase of the inlet velocity. Similarly to Richecoeur et al. [START_REF] Richecoeur | Experimental study of flame stabilization in low Reynolds and Dean number flows in curved mesoscale ducts[END_REF] who brought the Strouhal number to characterize the effect of diameter on the frequency, we have used the Strouhal number to characterize the effect of the equivalence ratio on the frequency. The Strouhal number is defined by St = fd / v, where f is the characteristic FREI frequency, d is the inner diameter of the tube and v is the inlet velocity. Figure 14 shows the evolution of the Strouhal number for different equivalent ratios. We observed a minimum of St near the stoichiometry (1 ≤ φ ≤ 1.1) and equal to St = 0.3. It is interesting to note that this value is identical to that indicated by Richecoeur et al. [START_REF] Richecoeur | Experimental study of flame stabilization in low Reynolds and Dean number flows in curved mesoscale ducts[END_REF] for curved channel, and by Crow and Champagne [START_REF] Crow | Orderly structure in jet turbulence[END_REF] as a preferred mode of vibration for incompressible jets. One can notice on Fig. 14 that the points are well superimposed to the minimum, while in rich and lean conditions there is a greater dispersion. One should note though that at very high frequency, there may be a greater experimental uncertainty.

Conclusions

In this study, the combustion characteristics of CH 4 /air mixtures in a micro channel were studied under various experimental conditions where we varied the inlet flow velocity and the equivalence ratio. The new experimental device is original by his heating method that ensures uniform radial temperature distribution, and by the diagnostics based on infrared thermography which allows a temporal tracking of the longitudinal temperature profile. The different regimes (stable, FREI, weak) observed in pioneering studies of Maruta et al. [START_REF] Maruta | Characteristics of combustion in a narrow channel with a temperature gradient[END_REF] were confirmed in this work. This work validates the reliability of the experimental setup. A systematic exploration of different equivalent ratios has allowed characterizing the dynamics of the flame according to this parameter. A detailed frequency analysis of the FREI regime has identified that the minimum frequency corresponds to a Strouhal number of 0.3, and it correspond to a stoichiometric equivalent ratio.

Fig. 1 .Fig. 2 .

 12 Fig. 1. The experimental set-up

Fig. 3 .

 3 Fig. 3. Images of stable flames for stoichiometric CH 4 /air mixtures in a 2.15 mm inner diameter tube. Flow rates: (a) 0.37 m/s, (b) 0.5 m/s, (c) 1 m/s.

Fig. 4 .

 4 Fig. 4. Images of instable flames for a stoichiometric CH 4 /air mixture and 2.15 mm inner diameter tube: the ignition occurs close to the blowtorches position (at left) while extinction is upstream. Flow rates: (a) 0.32 m/s, (b) 0.33 m/s, (c) 0.34 m/s.

Fig. 5 .

 5 Fig. 5. Temporal evolution of FREI at (a) 0.34 m/s and (b) 0.32 m/s. At v = 0.34 m/s the flame oscillates over several cycles before extinction.

Fig. 6 .

 6 Fig. 6. Images of weak flames for a stoichiometric CH 4 /air mixture in a 2.15 mm inner diameter tube: (a) at 0.04 m/s where the flame oscillates, (b) at 0.03 m/s where the flame is stable.

Figure 7 Fig. 7 .

 77 Fig. 7. Flame position as a function of wall temperature and mixture velocity.

Fig. 8 .

 8 Fig. 8. Flame effect on the wall temperature profiles recorded (a) in absence of flow and (b) with flow of 0.35 m/s.

Fig. 9 .

 9 Fig. 9. Flames obtained with three and two heat sources at different flow velocities in a 2.15 mm diameter tube.3.3 Equivalence ratio effectExperiments were conducted over a range of equivalence ratios from 0.5 to 1.5 in a tube of 1.85 mm of internal diameter. The results are shown in Fig.10. One can notice that for an equivalence ratio of 1.1, we have the lowest temperatures of stabilization (i.e. stable regime) and extinction (FREI regime). The flames obtained with a different equivalence ratio, lean or rich, stabilize at a higher temperature for stable regime, and for FREI regime, extinction temperatures follow the same trend. This is due to the flame temperature[START_REF] Bakali | Experimental and modeling study of the oxidation of natural gas in a premixed flame, shock tube, and jet-stirred reactor[END_REF] and the flame speed[START_REF] Chao | Analytical and experimental study of premixed methane-air flame propagation in narrow channels[END_REF] which are higher near the stoichiometric condition. The higher temperature increases the heat transfer towards the wall and the convective exchange to fresh gas. However, as the flame velocity is itself increased, convective exchanges are favored over conduction. This allows the flame to stabilize at a lower wall temperature.It can be noticed that the transition between stable and FREI regimes is different for the lean and the rich side (Fig.10). For lean mixtures, this transition is effective for lower speeds when equivalence ratio decreases: 0.4 m/s for φ = 1.1 while it is 0.3 m/s for φ = 0.5. Conversely, the stable to FREI transition speed is slightly higher for rich mixture, and equal to 0.44 for  = 1.5.For φ <1, FREI ignition points are omitted because the flames were less bright and ignition was difficult to precisely localize.

Fig. 11 .Fig. 12 .

 1112 Fig. 11. Spectral cross-section CH* signal.

Fig. 13 . 1 Fig. 14 .

 13114 Fig. 13. Comparison between τ EI and τ IE for 2.15 mm diameter tube,  = 1

Acknowledgments

The research leading to these results has received funding from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement n° 291049 -2G-CSafe.


Ferdaousse Rhoufal

Salaheddine Guesmi

Mostafa El

Ketatni

Laurent Jouffret

El Kebir Hlil

Nicolas Sergent

Saïd Obbade

Fouad Bentiss

First divalent copper complex of a

Keywords: Thiosemicarbazido-pyrazoline, Cu II -ligand radical complex, Crystal structure, Hirshfeld surface analysis, Magnetic properties, Antimicrobial activity

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Introduction

The chemistry of copper complexes bis(thiosemicarbazone) based ligands has been extensively studied in recent years. The interest of these compounds has increased when their properties have been demonstrated in the medical field. Indeed, several studies have shown that they are potentially interesting as chemotherapeutic [1][2][3][4], antibacterial [5] or even as radiopharmaceutical agents for hypoxia imaging [6][7][8] and for evaluation of Cerebral and Myocardial Blood Flow with positron emission tomography (PET) [9,10]. The bis(thiosemicarbazone) derivative of 2,4-pentanedione acting as tetradentate (H 2 L 1 , N 2 S 2 ) (Scheme 1a) leads, with the divalent transition metals, after a double deprotonation of the ligand, to mononuclear complexes [M II L 1 ] of square planar geometry (Scheme 2) [11]. The latter exposed to the air undergo oxidation of the central carbon of the methylene group and lead to [M II L 1 O] type complexes [11][12][13]. Bilyj et al. explained that this reaction should probably proceed via an intermediary M II -ligand radical complex [13]. The same authors have described a comparative study between copper and nickel complexes bis(thiosemicarbazone) based ligands [13]. Indeed, the authors have shown that with Cu II , only the symmetrical [M II L 1 ] type complex is formed which is sensitive to oxygen and turns into a [M II L 1 O] type complex (Scheme 2). Whereas with Ni II , they have in addition an asymmetric stable complex even in air [13,14]. Spectro-electrochemical oxidation of crystalline [M II LO] leads to a product of oxidation with two possibilities, Cu III complex or Cu II ligand centred radical [14]. Other studies have shown that this type of ligands may be seen as redox non-innocent ligands [14][15][16]. Others complexes with 2,4-pentadione bis(thiosemicarbazone) ligand in its mono anionic form were also obtained [3].

The oxidation at the metal level and the stabilization of unusual high oxidation states such as

Fe IV and Cu III , is generally done with pentane-2,4-dione bis(S-alkyl-isothiosemicarbazone) (N 2 N 2 )

ligands [17][18][19]]. However, an example of N 2 S 2 chelation system with bis(S-benzyldithiocarbazate) derivative, stabilizing Cu III , was cited by Akbar Ali et al. [20]. The pro-ligand used to access these complexes ([M II L 1 ] and [M II L 1 O]), whose the structure has been controversial for a certain time [21][22][23]; is in fact the derivative of thiosemicarbazido-pyrazoline (H 2 L 2 , Scheme 1b) which in contact with the metal opens to coordinate in the tetradentate acyclic form, bis(thiosemicarbazone) (Scheme 2) [11][12][13].

Scheme 2. Oxidative formation of [M II L 1 O] type complexes.

The coordination complexes of the ligand in its pyrazolinic form were reported [19,24] with their equivalents dithiocarbazatopyrazoline, but their number remains very limited. Indeed, only two examples have been described: the first one is a terdentate dithiocarbazatopyrazoline technetium (V) complex [24] and the second one is with manganese as a divalent metal with neutral dithiocarbazatopyrazoline ligand, which can be seen as the result of an oxidation after deprotonation of the initial ligand [19]. This property is well known with this kind of ligands and can be explained

[H 2 L 1 ] [M II L 1 ] [M II L 1 O]
by the reaction given in the Scheme 3 [19]. However, no complex with the ligand in the pyrazolinic form has been isolated from bis(thiosemicarbazone) ligand (H 2 L 1 ) to our knowledge. Scheme 3. "Redox non-innocent" behaviour of isothiosemicarbazido-pyrazoline leading to a neutral ligand.

In order to isolate new Cu III complexes based on bis(thiosemicarbazone) with interesting properties, we thought to use the thiocyanate ion (coordinating and anionic ligand) as a coligand which might have more beneficial biological properties. If this pseudohalide leads to complexes with activities in the electrochemical [25][26][27] or magnetic field [28][29][30], the presence of thiocyanate ions has been detected in human biological fluids, saliva and cerebrospinal and in oral cavities in case of stress [31][32][33][34][35][36]. Its role has also been demonstrated in the biological field as a detoxifier [37].

In this work, we confirm the pyrazolinic form of the ligand via a crystallographic study. Moreover, we investigate the reaction of the divalent copper acetates with the pyrazoline ligand (H 2 L 2 ) in the presence of SCN -, which unexpectedly gives the first copper complex [(L 2• )Cu(SCN)] with terdentate carbamothioyldiazenyl-pyrazoline (SNS) and thiocyanate ion as coligand, whose metal is in tetrahedral geometry. This complex can be described as the result of a double deprotonation coupled with a monoelectronic oxidation and it may be regarded as a Cu II -ligand radical type complex. Hirshfeld surface analysis, spectroscopic characterisations (IR, UV-Visible, NMR and Raman) and thermal studies of (H 2 L 2 ) and [(L 2• )Cu(SCN)] were performed. The magnetic properties of the title copper complex were also studied. Indeed, an attention is paid to DFT calculations to investigate both electronic and magnetic structures using FLAPW method. In addition, we have evaluated the activity of the ligand H 2 L 2 and its complex [(L 2• )Cu(SCN)] against several fungal and bacterial diseases affecting crops, including Verticillium dahliae, Fusarium

N N NH S C H 3 CH 3 NH N NH 2 S CH 3 C H 3 N N NH S C H 3 CH 3 N N NH S CH 3 C H 3 -2H + , -2e
oxysporum fsp. melonis, Pseudomonas syringae pv. syringae, P. syringae pv. tabaci and Agrobacterium tumefaciens. To the best of our knowledge, the activity of thiosemicarbazidepyrazoline derivatives against phytopathogens has never been reported.

Experimental

Materials

All solvents and reagents for synthesis were purchased from Aldrich and Merck and used as received without further recrystallization or distillation.

Synthesis of thiosemicarbazido-pyrazoline (H 2 L 2 )

The thiosemicarbazido-pyrazoline ligand (H 2 L 2 ) has been synthesized using the method previously reported [21]. Anal.

Synthesis of [(L 2• )Cu(SCN)]

To a solution of H 2 L 2 (0.1 mmol, 24.6 mg) dissolved in acetone (25 mL), under magnetic stirring at room temperature, was added successively an aqueous solution (5 mL) of Cu(CH

Crystal X-ray data collection

Single crystals were selected for the X-ray diffraction (XRD) data collection and mounted with cryoloops on a Bruker APEX II diffractometer with a MoK α graphite-monochromated radiation ( = 0.71073 Å). The diffractometer is equipped with a CCD bidimensional detector and an OXFORD CRYOSTREAM 700 system to cool the sample to 173 K [38]. The ligand thiosemicarbazido-pyrazoline (H 2 L 2 ) crystals all presented twinning along the (0 0 1) axis, thus requiring longer data acquisition than the copper containing crystals. SAINT+ 6.02 program was used for extraction and integration of diffraction intensities [39] and SADABS program was carried out for correction of absorption effect [40]. The structures were solved by direct methods using SHELXL [41] and refined (by weighted full matrix least-square on F 2 techniques) to convergence using the SHELXL program. All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were positioned geometrically and refined using a riding model.

Hirshfeld surface analysis

The Hirshfeld surfaces calculated for H 2 L 2 ligand and [(L

Physical measurements

UV-Visible absorption spectra in dimethylformamide (DMF) solvent were recorded in the range 250-800 nm using a Perkin Elmer Lambda 365 spectrophotometer.

FTIR spectra were recorded on a SHIMADZU FT-IR 8400S spectrometer with a Smart iTR attachment and diamond attenuated total reflectance (ATR) crystal in the range 500-4000 cm -1 .

The 1 H and 13 C NMR spectra of H 2 L 2 were recorded with the Bruker AVANCE 300 at 25 °C. All chemical shifts 1 H and 13 C are given in ppm using dimethyl sulfoxide-d 6 (DMSO-d 6 ) as solvent.

Element analyses (C, H, N and S) were performed on a VARIO-ELEMENTAR analyser.

Raman spectra have been acquired with a Renishaw InVia spectrometer in micro-Raman configuration (objective  50) equipped with a Peltier cooled CCD detector. The 785 nm excitation line of a LASER diode have been used at a power less than 3 mW onto the sample in order to avoid local heating.

Thermogravimetric analysis (TGA) was carried out on a SDT-Q600 thermal analyser and platinum crucibles, in the range of room temperature to 800°C under air flow (99 ml/min) with 5 °C min -1 fixed heating rate. The TGA curve has been normalized with respect to the sample weight.

Magnetic measurements were carried out using the commercial Physical Properties

Measurements System device (Quantum Design, PPMS), and magnetization M(T) was measured versus temperature ranging from 2 to 400 K, under an external magnetic field of 0.05 T. The magnetic field dependence of the magnetization M(H) was also measured at 2 K by varying the applied magnetic field between -6 and 6 T.

Electronic structure calculations

We used FLAPW method which performs DFT calculations using the local density approximation [START_REF] Blaha | WIEN2k, An Augmented Plane Wave plus Local Orbitals Program for Calculating Crystal Properties[END_REF]. The Kohn-Sham equation and energy functional were evaluated consistently using the Full Potential Linearized Augmented Plane Wave (FLAPW) method. For this method, the space was divided into the interstitial and the non-overlapping muffin tin spheres centred on the atomic site. The employed basis function inside each atomic sphere was a linear expansion of the radial solution of a spherically potential multiplied by spherical harmonics. In the interstitial region the wave function was taken as an expansion of plane waves and no shape approximation for the potential was introduced in this region which is consistent with the full potential method. The core electrons were described by atomic wave functions which were solved relativistically using the current spherical part; the valence electrons were also treated relativistically in our case. These FLAPW calculations were performed with our crystal structure parameters. Here, polarized spin as well as the ferromagnetic state were considered for all atoms of our compound.

In vitro antimicrobial activity

Antifungal screening was performed against three strain of Verticillium dahliae (SE, SH and SJ), previously isolated from olive trees in different region in Morocco and one isolate of Fusarium oxysporum fsp. melonis (FOM). They were sub-cultured on potato dextrose agar (PDA) plates at 27°C in the dark. H 2 L 2 and [(L 2• )Cu(SCN)] were dissolved in 0.2% DMSO and added at concentrations of 50, 100 and 200 µg mL -1 to the PDA medium before being autoclaved and cooled.

The amended media was poured into Petri dishes and inoculated using 5 mm plugs of agar and mycelium taken from actively growing cultures of fungus on PDA. Petri plates were incubated in the dark at 27°C. The mycelial growth was assessed by measuring 2 orthogonal diameters of each colony after 7 days of incubation. Mycelial growth was compared with growth on PDA amended with 0.2% DMSO (control) and the percentage of inhibition growth was expressed [START_REF] Smaili | [END_REF] relative to the control. The regression line between colony diameter of the pathogen and log concentration of products was determined. Sensitivity was then measured using effective IC50 values. The experiment was repeated 3 times.

The in vitro antibacterial screening of the two compounds were performed against the following phytopathogenic bacteria: Agrobacterium tumefaciens (Ach5, A281 and C58 strains), Pseudomonas syringae pv. tabaci (strain CFBP2106) and Pseudomonas syringae pv. syringae (strain CFBP692). A. tumefaciens strains were sub-cultivated on LB solid medium while P. syringae pv. syringae and P. syringae pv. tabaci were cultured on King B (KB) medium at 28°C.

Antibacterial activities of the two compounds were investigated by the agar disc diffusion method as described previously [47]. Bacteria that were grown overnight were diluted to a concentration of 3 × 10 8 CFU. mL -1 . Sterile cellulose discs of 6 mm diameter were impregnated with the products (200 µg mL -1 in 0.2% DMSO) and discs were placed on either LB or KB solid medium previously amended with 3 × 10 8 CFU mL -1 of bacteria. DMSO (0.2%) was used as a negative control. The inoculated plates were incubated at 28°C for 48 h. The antimicrobial activity was evaluated by measuring the clearance zone around the discs which indicates a positive antibacterial activity of the respective products. Each experiment was repeated 3 times.

All data were expressed as the mean and confidence intervals. Data were subjected to ANOVA, and means were compared using Tukey's test and p values of 0.05 were considered to be significantly different.

Results and discussion

Crystal structure determination and description

The title complex [(L 2• )Cu(SCN)] was obtained by reaction, at room temperature, of copper acetate with H 2 L 2 in the presence of the pseudohalide SCN -as illustrated in Scheme 4. The organic ligand obtained from acetylacetone and thiosemicarbazide [21] did not lead to 2,4-pentanedione bis(thiosemicarbazone) but to its cyclic correspondent H 2 L 2 (Scheme 1). The pyrazoline form of the ligand has been suggested by its spectroscopic properties and confirmed by single crystal X-ray diffraction. Arion having proposed the structure of H 2 L 2 [16] without further structural details, therefore we decided to determine the crystallographic structure of this ligand in order to be able to make comparison with its copper complex. The structural study of the new title copper complex reveals that it is an unexpectedly copper complex pyrazoline terdentate ligand doubly deprotonated with thiocyanate ion (SCN -) coordinated via nitrogen atom. The oxidation state of the metal is formally Cu III where the pyrazolinic ligand should be in dianionic form (L 2 ) 2-, but the ligand being potentially redox-active (non-innocent), the possibility of a Cu II ligand-radical with the organic ligand in monoanionic form (L For both compounds, the crystal structures were determined in monoclinic symmetry, where the H 2 L 2 ligand and its copper complex [(L The crystal structure views of the asymmetric units of ligand and its complex are shown in 2 and3. The H 2 L 2 crystal structure is further stabilized by sulfur, carbon and nitrogen (N-H⋯A with A = S, C, N) hydrogen interactions, where each unit H 2 L 2 shares with neighbouring molecules, four sulfur N-H⋯S with distances range from 2.521(6) to 2.849(6) Å, with "H⋯A" length bonds of 2.630(6) Å in N1-H1A⋯C5 and 2.495(6) Å in N2-H2A⋯N5, as presented in Fig. 3 and Table 2. It is noteworthy to point that, aside from the hydrogen bonds D-H⋯A (D = C, N and A = S, C, N), the S⋯S intermolecular interaction is also another useful organizing force in this supramolecular network. Further analysis of the crystal structure of [(L 2• )Cu(SCN)] reveals short S⋯S contacts between SCN thiocyanate ligands of two neighbouring molecules, with a sulfur bond length S3⋯S3 about 3.51 Å, which is less than the van der Waals radius of two sulfur atoms, leading to the formation three-dimensional network, as indicated in Fig. 5. This structural feature with weak sulfur interactions is typical for related compounds [52][53][54]. It is also notable that the N7-C8 distance in the NCS thiocyanate ligand is extremely short (N7-C8 = 1.145(3) Å), presumably due to a chemical pressure caused by the S3⋯S3 contacts.

Hirschfeld surface analysis

A Hirschfeld surface analysis was performed to give insights into the important intermolecular interactions. These are normalized by van der Waals radii through a red-white-blue colour scheme, where the red spots denote close contacts of molecules. Two views of the Hirschfeld surface mapped over three dimensional d norm of the H 2 L 2 ligand and [(L 2• )Cu(SCN)] complex are shown in Fig. 6. The red points represent closer contacts and negative d norm values on the surface corresponding to the N-H⋯S interactions mentioned above (N-H⋯S1/S2 for H 2 L 2 and N-H⋯S3

for [(L 2• )Cu(SCN)], while the other short intermolecular contacts appear as light-red spots (N-H⋯N and N-H⋯C for the Ligand and N-H⋯S1 and S3⋯S3 for the complex). In the view Hirshfeld surface mapped over the electrostatic potential in Fig. 7, the red and blue regions around the electronegative and electropositive atoms result from the polarization of charges about the acceptors and donors, respectively. In H 2 L 2 ligand, the most negative sites are associated with the S atoms which undergoes electrophilic reactivity while the positive region over the protons of amine group indicates that these sites are susceptible for nucleophilic attack. The electrostatic potential calculated for the complex shows that the potential energy is positive over the organic cation and around the NH 2 group while the negative potential energy described by red region around the sulphur atom of the thiocyanate group, resulting a global electrostatic attraction between N-H and the S atoms of (NCS) group.

The two dimensional fingerprint plots in Figs. 8 and9 for H 2 L 2 and [(L 2• )Cu(SCN)],

respectively, showed the intermolecular contacts and their percentage distributions on the Hirschfeld surface. All the relative percentage contributions to the overall Hirschfeld surface for ligand and complex are summarized in Table S7. The large red regions in Figs. 6 and7 represent H⋯S/S⋯H contacts, which are relevant in the d norm maps for ligand and complex. The H⋯S/S⋯H contacts are similar for ligand and complex, with highest contributions of 31.6% and 32.6%, respectively, of the total Hirschfeld surface area. In both ligand and complex, the S⋯H (20.1 and 21.4%, respectively) interactions make a larger contribution than the H⋯S (11.5 and 11.2%, respectively) interactions, indicating amine H-atoms interacting with S atoms for the formation of the supramolecular assembly, is represented by the spikes in the bottom right and left region, d e + d i  2.35 Å (Fig. 8c and9b). For the ligand, the H⋯H interactions appear in the middle of the scattered points in the fingerprint plots with a highest percentage contribution (53.6%) (Fig. 8b), while their contribution in [(L 2• )Cu(SCN)] are 27.2% (Fig. 9c), which underlines the importance of van der Waals interactions. For H 2 L 2 , the small red spots represent N⋯H/H⋯N and C⋯H/H⋯C contacts attributed to N-H⋯N and N-H⋯C hydrogen bonds, which can also be seen in the fingerprint as a spikes at d e + d i  2.45 Å (Fig. 8d) and d e + d i  2.60 Å (Fig. 8e), respectively. The C⋯H/H⋯C and N⋯H/H⋯N interactions account for 13.5% and 11.9% in complex, respectively (Fig. 9d and9e). These contacts stabilize the structure of complex. The S⋯S contacts have a contribution to the Hirschfeld surfaces of 2.9% in complex (Fig. 9h), revealing the S3⋯S3 interactions between the layers, with S-S distance 3.507 Å which is less than the van der Waals,

Spectroscopy studies

Ultraviolet-visible spectroscopy

The electronic spectra of H 2 L 2 ligand and [(L 2• )Cu(SCN)] complex have been measured in the range 250-800 nm and are shown in Fig. S1. The absorption maxima with the corresponding extinction coefficients are given in Table 4. In the UV region the presence of the organic ligand is confirmed by a very intense peak at λ max , nm (ε max , M -1 cm -1 ) 268(25039) nm with a shoulder at 280(14856) nm, comparable to those of the free ligand at 266(24322) and 276(22052) nm and may be assigned to intra-ligand transitions π-π* and n-π* respectively [55]. Moreover, for the copper complex, the spectrum reveals two broad lower intensity transitions at 376 nm (2831) and 465nm (2366). These bands could be attributed to a combination of charge transfer absorptions in the Cu II complex [12,19,55]. In addition, the spectrum shows a weak broad absorption attributable to d-d transition at 717 (1822 M -1 cm -1 ) nm, for a Cu(II) ion d 9 in tetrahedral geometry [56].

1 H and 13 C NMR analysis

The 1 H and 13 C NMR spectra of free ligand H 2 L 2 were recorded in DMSO-d 6 are given in Indeed, the two methyl groups are clearly distinguishable as two singlets at 1.71 and 1.94 ppm and the signal appearing as a quadruplet at 2.81 ppm, is assigned to AB system of methylene protons. The hydrazinic protons (HN-NH) are exhibited as two singlets at 6. to indicate that the latter becomes paramagnetic once in solution. Indeed, the 1 H and 13 C NMR analyses of this copper complex performed in DMSO do not give the expected spectra for a diamagnetic complex. This behaviour was reported by Fox et al. [57] in a study of transition metalthiolate complexes which clearly showed that the investigated complex is diamagnetic as crystalline solid and becomes paramagnetic when dissolved in solvent.

FTIR spectroscopy

The FTIR spectra of H 2 L 2 ligand and its complex are shown in Fig. S2 and the corresponding data are summarised in Table 5. The FTIR spectrum of the free ligand shows five broad bands between 3143 and 3423 cm -1 , assignable to different N-H vibrations (υ(NH 2 ) and υ(HN-NH)) and an intense band at 1579 cm -1 assigned to υ (C=N) pyrazoline cycle [58,59]. The FTIR spectrum doesn't display any band in the region 2550-2600 cm -1 range due to the S-H group, which indicates that ligand exists in thione form in solid state. In the FTIR spectrum of the title complex (Fig. S2), only 3 bands and one shoulder were observed for the N-H vibrations, which means that ligand has been deprotonated and is coordinated in anionic form. Comparison between the spectra of H 2 L 2 ligand and [(L 2• )Cu(SCN)] complex showed also a new sharp and strong signal at 2115 cm -1 attributed to the stretching vibration υ(CN) of thiocyanate group; a similar frequency was observed in a Cu complexes with SCN -ion coordinated to the metal via N atom [60,61].

Table 5

FTIR data (cm -1 ) of H 2 L 2 ligand and its complex [(L

Raman spectroscopy

Fig. S3 shows the Raman spectra of H 2 L 2 ligand and its complex [(L 2• )Cu(SCN)]. The spectra of H 2 L 2 and its complex are clearly very different suggesting a strong interaction/link between Cu and the ligand, leading to a strong perturbation of all the ligand bonds. Some interesting features can be extracted. In the free ligand spectrum, the band at 1054 cm -1 can be attributed to the C-S vibration [62]. In the complex spectrum, it can be suggested that this band is shifted down to 978 cm -1 due to the S-Cu link. The very weak band at 2110 cm -1 observed in the complex spectrum can be attributed to NCS -anions [START_REF] Nakamoto | Infrared and raman spectra of inorganic and coordination compounds: Part B: Applications in coordination[END_REF].

Thermal decomposition study

The thermogravimetric study was carried out under air from room temperature up to 800°C, in order to examine the thermal behaviour of the ligand and its copper complex and the obtained thermograms of H 2 L 2 ligand and its complex [(L stages starting with dissociation of the hydrazinic N2-N3 bond (Fig. 1a) and release of the cyclic moiety. At the end, no more noticeable weight loss is detected from 568 °C in agreement with the formation of carbon as a residual product (observed 4.50 %; calcd. 4.87 %).

Magnetic properties and Electronic structure calculations

A preliminary magnetic study of [(L 2• )Cu(SCN)] complex has been carried under an applied magnetic field of 500 Oe in temperature range 2-400 K and presented in Fig. 12. As shown in Fig. 12a, the thermal evolution of [(L 2• )Cu(SCN)] magnetization, indicates that the complex adopts a diamagnetic comportment, which may be explain by a strong antiferromagnetic interaction between unpaired electrons due to a large overlap between magnetic orbitals and/or by the effect of the presence of the non-innocent ligand (L 2• ) -.

In addition, the plot of magnetization versus applied field in the range ± 6T presented in Fig. 12b, indicates that there is neither magnetic hysteresis loop nor saturation of magnetization up to 6

T a linear relationship between applied field and magnetization with a positive slope and very small value about 0.0045  B /Cu under H = 6T which is far below the expected saturation magnetisation of   , assuming g = 2, confirms a diamagnetic behaviour for Cu complex. To try finding explanation this magnetic evolution of studied complex, copper electronic structure have been evaluated using Density-functional theory (DFT) calculations. Thus, the total Density of State (DOS) of our compound deduced from electronic structure calculations is reported in Fig. 13. Here, the Fermi level is taken as reference for energy. As seen, this DOS is perfectly symmetrical with respect to energy axis, pointing out that the system is not magnetically ordered. The total DOS is dominated by the resolved picks taking place in both occupied states at negative energies and unoccupied states localized at positive energies. Such features reveal the molecular character of the compound. The l-decomposed DOS of d and s likestates of Cu have been performed as well. Further analysis gives evidence that the DOS of Cu is mainly from the 3d band. In addition, it should be noticed that the DOS analysis points out to the absence of contributions from the s band of Cu. The charge transfer of atoms is computed as well. Particularly, it reveals that the copper is in Cu 2+ state loosing 2 electrons. Precisely, one electron is lost from 3d band whereas the second one is lost from 4s band leading to the electronic configuration transition from Cu(3d 10 4s 1 ) to Cu(3d 9 4s 0 ). Obviously the empty 4s band does not contribute to the magnetism. Moreover, we underline that the odd number of electrons in the 3d band does not induce a Cu magnetic state; since electron computation shows 4.5 electrons in 3d up-spin band and 4.5 electrons in 3d down-spin band. For all such reasons, we conclude that the Cu atom is in a diamagnetic state. In addition, no magnetic moment is observed on the remaining atoms and only a weak magnetic moment of 0.0044 B/f.u. is revealed as contribution from the interstitial magnetism. In addition, copper magnetic moment about 0.0044 B /Cu, deduced from electronic structure calculations, should be confirmed by a second magnetic measurement M(H), with Ms about 0.0045 B /Cu at maximum magnetic field of 6T. Similar behaviour has already been observed in different complexes as Cu(hfac) 2 (NITmNO 2 ) [START_REF] Zhang | [END_REF], Cu(hfac) 2 (NITPh) 2 [65] and Cu(hfac) 2 (TMPNO) [66], where hfac = hexafluoroacetylacetonate, NITmNO

Antimicrobial activities

The effect of H 2 L 2 and its complex [(L 2• )Cu(SCN)] was assessed through the mycelial growth inhibition of 3 strains of V. dahliae and one strain of Fusarium oxysporum fsp. melonis. V. dahliae and F. oxysporum are the causal agent of wilt that affects several crops of economic interest [73,74]. Their effect on radial growth (Table 6, Fig. 14) showed that H 2 L 2 ligand did not exhibit any significant inhibition of mycelial growth whatever the concentration used and the fungal strain or species. However, its coordination with copper metal enhanced its activity against the strains SE and SJ of V. dahliae, given rise to IC 50 values of 231 and 31 µg mL -1 , respectively. The positive control thiophanate methyl caused a drastic inhibition of all the strains used, except for the strain SJ, which suggest that it is resistant to the thiophanate methyl fungicide. The elevated activity of [(L 2• )Cu(SCN)] complex may be due to the synergetic effect of the ligand and the copper cation.

The differential sensitivity of these fungal strains to the investigated complex could be explained by their different genetic background [75]. Pyrazole derivatives possess a wide range of biological activities, including antibacterial activity [76]. Their activities rely on the inhibition of DNA gyrase and topoisomerase [77].

However, their efficacy has been proved against Gram+ bacteria since they have only an outer peptidoglycan, which is not an effective permeability barrier conversely to Gram-bacteria possessing an outer phospholipidic membrane and lipopolysaccharide components. In this study we evaluated the effect of these pyrazolic derivatives against several phytopathogenic bacteria of

SE SJ

economic interest such as A. tumefaciens, which is the causal agent of crown gall disease [78]. They showed moderate inhibition of the strains A281, C58 and Ach5 and no significant difference was observed between the ligand and its complex when compared to the standard chloramphenicol (Fig. 15). Similar tendency was observed against two different Pseudomonas syringae pathovars, which are taxonomically related, but differ in pathology, virulence and symptoms development in various crops [79]. Among them, P. syringae pv. tabaci causes wild fire disease of tobacco, and P. syringae pv. syringae, a common epiphytic bacterium that causes diseases of various plants [80]. In this study, the growth of P. syringae pv. syringae was moderately inhibited by both H 2 L 2 and [(L 2• )Cu(SCN)] conversely to P. syringae pv. tabaci, which was completely insensitive to both of them. The reason for different sensitivities between tested bacteria could be ascribed to the morphological difference between their exopolysaccharides (EPS) polymers, which form a capsule or slime layer around bacterial cells that may act as an effective permeability barrier [81].

Conclusions

The title complex [(L 2• )Cu(SCN)] has been prepared by reaction of H 2 L 2 ligand and thiocyanate salt with copper acetates and has been fully characterized. Single-crystal X-ray diffraction reveals that the complex is a neutral monomer with copper in distorted tetrahedral environment and would be the result of a double deprotonation coupled with a monoelectronic oxidation. The ligand is mono anionic and remains in cyclic form and is coordinated as terdentate ligand (SNS). According to the magnetic study, the Cu atom in the complex is in a diamagnetic state. This behaviour can be explained by a strong antiferromagnetic coupling between the metal ion and the radical ligand, who suggests that [(L 2• )Cu(SCN)] can be seen as a Cu II -ligand radical type complex with (L 2• ) -in mono anionic form. This study reinforces the notion of the noninnocence of the organic ligand. The [(L 2• )Cu(SCN)] complex showed also elevated antifungal activity against various strains of phytopathogenic fungus suggesting its possible use as a fungicide in crop protection.

First divalent copper complex of a terdentate thiosemicarbazido-pyrazoline derived from 2 , 4 -

 24 pentanedione bis(thiosemicarbazone): Synthesis, structural characterisation and antimicrobial in vitro activity assessment Ferdaousse Rhoufal, Salaheddine Guesmi, El Mostafa Ketatni, Laurent Jouffret, El-Kebir Hlil, Nicolas Sergent, Saïd Obbade, Fouad Bentiss To cite this version: Ferdaousse Rhoufal, Salaheddine Guesmi, El Mostafa Ketatni, Laurent Jouffret, El-Kebir Hlil, et al.. First divalent copper complex of a terdentate thiosemicarbazido-pyrazoline derived from 2,4pentanedione bis(thiosemicarbazone): Synthesis, structural characterisation and antimicrobial in vitro activity assessment. Polyhedron, 2021, 195, pp.114992. 10.1016/j.poly.2020.114992. hal-03087598

2 Scheme 1 .

 21 Scheme 1. Two isomer forms a) bis(thiosemicarbazone) (H 2 L 1 ) and b) thiosemicarbazido-pyrazoline (H 2 L 2 ), where R = H in this work.

Scheme 4 .

 4 Scheme 4. Proposed mechanism of the formation reaction of copper complex [(L 2• )Cu(SCN)].

Fig. 1a and

  Fig. 1a and 1b, respectively. Thus, we can observe that during the reaction of the ligand H 2 L 2 in the aqueous solution of Cu(CH 3 COO) 2 and KSCN, the formation of [(L 2• )Cu(SCN)] resulted from the two protons loss of nitrogen atoms N2 and N3 of the ligand H 2 L 2 , following the complexation

Fig. 1 .

 1 Fig. 1. Molecular structure of (a) ligand H 2 L 2 and (b) copper complex [(L 2• )Cu(SCN)].

Fig. 2 .

 2 Fig. 2. Crystal structure view of (a) ligand H 2 L 2 in the (b, c) plan and (b) of copper complex [(L 2• )Cu(SCN)] along c axis.

Fig. 3 .

 3 Fig. 3. D-H⋯A (D = C, N and A = S, C, N) hydrogen-interactions in crystal structure of H 2 L 2 ligand.

Fig. 4 .

 4 Fig. 4. Formation of the three-dimensional network involving D-H⋯A (D = C, N and A = S, C, N) hydrogen-interactions in crystal structure of [(L 2• )Cu(SCN)].

Fig. 5 .

 5 Fig. 5. View of the three-dimensional network involving S⋯S interactions in crystal structure of [(L 2• )Cu(SCN)].

  forming three-dimensional supramolecular network.The Hirshfeld surfaces in the distortion coordination geometry formed by the N 2 S 2 about the copper (II) centre in [(L 2• )Cu(SCN)] molecule that has been mapped to a normal surface are shown in Fig.10. The dark red regions on the d norm surface for Cu show the contact between N-Cu and Cu⋯S. The Hirshfeld surfaces of the Cu 2+ ion is much more concave towards the bond directions defined by the N atom of the thiocyanate group than those associated with coordination towards the N and S atoms (Figs.10a and 10b). Although the covalent bonds with the sulfur and nitrogen atoms are dominant. The Cu atom has up to 49.7% of the contacts from the N-Cu interaction on the Hirshfeld surface (the pair of aligned red points at different inclinations from d e + d i  2.0 Å) followed by 29.6% of the contacts from the S-Cu coordinates (d e + d i  2.2 Å).The Hirshfeld surface analysis confirms the importance of H atoms contacts in estabilishing the packing. The large number of H⋯H, S⋯H/H⋯S, N⋯H/H⋯N and C⋯H/H⋯C interactions suggest that van der Waals interactions and hydrogen bonding play dominant role in stabilizing the lattice of the ligand and complex structures, revealing also different packing modes. The HS of the metal centre differentiates relatively strong bonds from weaker contacts taking place within the interaction sphere.

Fig. 6 .

 6 Fig. 6. Hirshfeld surface of H 2 L 2 ligand mapped over d norm , showing short interatomic N-H⋯S, N-H⋯N and C-H⋯N contacts by dashed lines and Hirshfeld surface of [(L 2• )Cu(SCN)] complex mapped over d norm , showing short interatomic N-H⋯S and S⋯S contacts by dashed lines.

Fig. 7 .Fig. 8 .Fig. 9 .Fig. 10 .

 78910 Fig. 7. Electrostatic potential mapped over the Hirshfeld surfaces of the (a) H 2 L 2 ligand and (b) [(L 2• )Cu(SCN)] complex.

Fig. 11 .

 11 Fig. 11. The 1 H NMR spectrum presents two groups of signals for each group of the predicted linear

Fig. 11 .

 11 Fig. 11. 1 H and 13 C NMR spectra of H 2 L 2 recorded in DMSO-d 6 .

49 and 8 .

 8 05 ppm and, for thiocarbamoyl and thiosemicarbazide NH 2 protons; the spectrum shows 3 singlets (1H, 1H, 2H) between 7.43 and 7.80 ppm. These values are in the range of those found for analogous pyrazoline [13,22,23]. The 13 C NMR spectrum in Fig. 11 displays 7 signals instead of the expected four for the symmetrical bis(thiosemicarbazone). The two carbons of methyl groups C6 and C5 appear at 16.33 and 23.92 ppm respectively, the methylene carbon C3 at 47.29 ppm and the signals at 84, 88 and 155, 56 ppm are attributed to C2 and C4 respectively. The two signals at 175.13 and 183.10 ppm are assigned to the carbons of C=S groups. The comparison of these data with those previously cited [12,23], suggests the pyrazoline form of the prepared ligand. NMR of [(L 2• )Cu(SCN)] seems

Fig. 12 .

 12 Fig. 12. (a) Temperature dependence of magnetic moment under 500 Oe and (b) Magnetization as a function of applied field at 2K, of [(L 2• )Cu(SCN)] complex.

Fig. 13 .

 13 Fig. 13. Total DOS and d like-states of Cu of [(L 2• )Cu(SCN)] from FLAPW calculations.

2 = 2 -

 22 (3-nitrophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3oxide, NITPh = 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and TMPNO = 2,2,6,6tetramethylpiperidine-N-oxyl. It is worth also mentioning that copper(II)-diamagnetic metal ion complexes may be interesting to explore the correlation of d-d band position with structural parameters, as previously proposed by Kanda et al.[67] and explored later in other works[68][69][70][71][72].

  Data are means and confidence intervals from three replicates (= 5%). Percentage of inhibition growth was determined at 7 days after incubation. Letters indicate significant differences within each treatment group according to Tukey HSD test (P < 0.55). Thiophanate methyl was used as standard fungicide.

Fig. 14 .

 14 Fig. 14. Photos showing antifungal activity of [(L 2• )Cu(SCN)] complex against the strains SE and SJ of Verticillium dahliae.

Fig. 15 .

 15 Fig. 15. Antibacterial activity of H 2 L 2 and [(L2• )Cu(SCN)] against the strains Ach5, A281 and C58 of Agrobacterium tumefaciens, Pseudomonas syringaepv. syringae (PSS) and Pseudomonas syringaepv. tabaci (PST). Data are means and confidence intervals from three replicates ( = 5%). Inhibition diameter was recorded 48 h after incubation at 28 °C with sterile cellulose discs impregnated with 200 µg ml -1 of the two products or with 50 µg ml -1 of chloramphenicol antibiotic (Cm) used as standard. Letters indicate significant differences according to Tukey HSD test (P < 0.55).

H 2 L 2 (L 2 •

 22 )Cu(SCN) Cm

  2• ) ˉ cannot be excluded as shown in scheme 4. The mechanism that we propose starts with a mono deprotonated intermediate (complex A in Scheme 4), which has been suggested by Bylij et al. but with an acetate in place of the thiocyanate and leads to classical complexes with the open form of the ligand [14].

  1 /c and C 2/c, with four and eight formula per unit cell, respectively. The crystal structures determination details of both compounds are given in Table1, with atomic positions, isotropic thermal displacement factors, selected bond lengths and angles given in Tables S1-S6 of supplementary information file. Visualization of both structures were made with the Diamond 3.1

	program [48].

2• )Cu(SCN)] crystallize in the centrosymmetric space SCN SCN SCN groups P 2

Table 1

 1 Single crystal and structure refinement data for H 2 L 2 ligand and [(L 2• )Cu(SCN)] complex.

	Chemical formula	H 2 L 2 : C 7 H 14 N 6 S 2	[(L 2• )Cu(SCN)] : C 8 H 12 CuN 7 S 3
	Formula weight (g/mol)	246.36		365.97
	Temperature (K)	173(2)		173(2)
	Wavelength (Å)	0.71073		0.71073
	Crystal size (mm)	0.134 × 0.168 × 0.250	0.140 × 0.168 × 0.257
	Crystal system	monoclinic		monoclinic
	Space group	P 2 1 /c		C 2/c
	Unit cell dimensions (Å)	a = 8.7329(10)	a = 15.684(3)
		b = 8.9424(9) , β = 105.802(4)°	b = 10.9895(18) , β = 90.236(5)°
		c = 15.7709(18)	c = 16.758(3)
	Volume (Å 3 )	1185.1(2)		2888.4(9)
	Z	4		8
	Density (calculated) (g/cm 3 )	1.381		1.683
	Absorption coefficient (mm -1 )	0.429		1.942
	F(000)	520		1488
	Data collection and structure refinement
	Theta range for data collection (°)	2.42 to 30.51	3.32 to 31.51
	Index ranges	-12<=h<=12, -12<=k<=12, -22<=l<=22 -23<=h<=23, -16<=k<=16, -
				24<=l<=24
	Reflections collected	46262		56787
	Independent reflections	3608 [R (int) = 0.0507]	4809 [R (int) = 0.0611]
	T max /T min	0.945 / 0.900	0.7730 /0.6350
	Resolution technique	Direct methods	Direct methods
	Refinement method	Full-matrix least-squares on F 2	Full-matrix least-squares on F 2
	Refinement program	SHELXL-2018/3	SHELXL-2018/3
	Function minimized	Σ w(F o 2 -F c	2 ) 2	Σ w(F o 2 -F c	2 ) 2
	Data/restraints/parameters	3608 / 0 / 144	4809 / 0 / 174
	Goodness-of-fit on F 2	1.231		1.028
	Final R indices	3191 data; I > 2σ(I) R 1 = 0.0792,	3159 data; I > 2σ(I) R 1 = 0.0331,
		wR 2 = 0.2049	wR 2 = 0.0753
		All data, R 1 = 0.0865, wR 2 = 0.2081	All data, R 1 = 0.0645, wR 2 = 0.0873
	Weighting scheme	W = 1/[σ 2 (F o 2 ) + 10.2460 P] where P = (F o 2 + 2F c 2 )/3	2 ) + (0.0352 P) 2 + 1.3732 P] where P = (F o W = 1/[σ 2 (F o 2 + 2F c	2 )/3
	Largest diff. peak and hole (eÅ -3 )	0.568 and -0.546	0.350 and -0.411
	R.M.S. dev. from mean (eÅ -3 )	0.145		0.062

Table 2

 2 H⋯H and D-H⋯A hydrogen-bond geometry of H 2 L 2 (D = C, N and A = S, C, N).

	D-H⋯A	H⋯A (Å)	D⋯A (Å)	D-H⋯A (°)
	N6-H6A⋯S1	2.630(6)	3.318(5)	135.8(5)
	N6-H6B⋯S1	2.590(6)	3.451(5)	166.1(5)
	N3-H3A⋯S2	2.849(6)	3.415(4)	126.5(5)
	N1-H1B⋯S2	2.521(6)	3.388(6)	168.7(5)
	N1-H1A⋯C5	2.630(6)	3.272(6)	126.8(5)
	N2-H2A⋯N5	2.495(6)	3.381(5)	153.5(5)
	N-H + ⋯ + H-D	H⋯H
	N1-H1B⋯H6B-N6	2.345(4)
	N1-H1A⋯H52-C5	2.272(9)

Table 3 S⋯S

 3

	D-H⋯A	H⋯A (Å)	D⋯A (Å)	D-H⋯A (°)
	N6-H6B⋯S3	2.571(1)	3.379(3)	174.9(1)
	N1-H1B⋯S3	2.574(1)	3.417(2)	160.6(1)
	N1-H1A⋯S3	2.678(1)	3.526(2)	162.2(1)
	C5-H51⋯S3	2.998(1)	3.919(3)	157.2(1)
	N6-H6A⋯S1	2.873(1)	3.703(2)	157.9(1)
	C3-H32⋯N6	2.707(2)	3.687(3)	170.9(1)
	S⋯S	S⋯S (Å)
	S3⋯S3	3.507(1)

and D-H⋯A hydrogen-bond geometry of [(L 2• )Cu(SCN)] (D = C, N and A = S, C, N).

Table 4

 4 Assignments of the UV-Vis bands of H 2 L 2 (10 -4 M) and [(L 2• )Cu(SCN)] complex (1.5 10 -4 M) in DMF.

			λ max , nm (ε max , M -1 cm -1 )
	Compound	π-π*	n-π*	Charge transfer	d-d
	H 2 L 2	266(24322)	276(22052)	-	-
	[(L 2• )Cu(SCN)]	268(25039)	280(14856)	376(2831); 465(2366)	717(1822)

  2• )Cu(SCN)] are given in Figs. S4-S5. As shown in Fig.S4, the TG curve of [(L 2• )Cu(SCN)] indicates that the copper complex is not decomposed up to 190°C and then gradual decomposition occurs in three successive steps within the temperature range 191°C -741 °C. The three stages with a total mass loss of 73.31% are attributed to the successive decomposition of the organic ligand and the thiocyanate ion from the copper complex.

	The first weight loss (observed 30.90 %; calcd. 31.46 %), between 191 and 386°C, is associated
	with the release of the pyrazole cycle part linked to the aminothiocarbonyl group, after dissociation
	of C4-C3 and C2-N4 bonds (Fig. 1b). The second step (386 °C -488 °C) is complying with the
	loss (observed 31.19 %; calcd. 30.92 %) of the remaining entity of pyrazole cycle (without NH 2
	group) and the third step (observed 11.22 %; calcd. 11.75 %), between 618 and 741°C, can be
	attributed to the degradation of thiocyanate and removal of the last part of the organic ligand with
	probably the formation of CuS as the final residue at 741 °C (observed 26.69 %; calcd. 26.12 %). In
	the case of the free H 2 L 2 ligand, the inspection of the obtained thermogram, given in Fig. S5, clearly
	indicated that, between 117 and 568 °C, globally two big stages of decomposition (observed 69.54
	% and 24.60 %) can be observed. The comparison with the theoretical values of the decomposition
	(calcd. 69.51 % and 25.61 %), indicates that the total degradation of the free ligand is done in two

Table 6

 6 In vitro inhibition of mycelial growth of Fusarium oxysporum fsp melonis and the strains SH, SE and SJ of Verticillium dahliae.

	Compound	Concentration	Vd SH (%)	Vd SE (%)	Vd SJ (%)	FOM (%)
		(µg mL -1 )
	H 2 L 2	50	9±1 a	23±3 a	14±9 a	6±3 a
		100	14±3 a	27±3 a	18±6 a	10±4 a
		200	16±3 a	29±4 a	25±10 a	13±3 a
	[(L 2• )Cu(SCN)] 50	14±2 a
		100	15±3 a
		200

Acknowledgements

The authors are greatly thankful to the CUR CA2D of Chouaib Doukkali University for its support and UCA-PARTNER for the use of the Bruker SC diffractometer. We thank Prof. V.B.

Arion for giving us the H 2 L 1 ligand and Prof. M. Faize for his biologic assessment contribution.

Supplementary material

CCDC 2022916 and CCDC 2022923 contain the supplementary crystallographic data for H 2 L 2 ligand and its complex [(L 2• )Cu(SCN)]. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam. ac.uk, or by contacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.