key: cord-0891254-tuza2iad authors: AIT-RAMDANE-TERBOUCHE, Chafia; ABDELDJEBAR, Hasnia; TERBOUCHE, Achour; LAKHDARI, Houria; BACHARI, Khaldoun; ROISNEL, Thierry; HAUCHARD, Didier title: Crystal structure, chemical reactivity, kinetic and thermodynamic studies of new ligand derived from 4-hydroxycoumarin: Interaction with SARS-CoV-2 date: 2020-07-16 journal: J Mol Struct DOI: 10.1016/j.molstruc.2020.128918 sha: 24443a94cb2e4182e5ed78570235abc0f3d19f59 doc_id: 891254 cord_uid: tuza2iad Currently, Covid-19 pandemic infects staggering number of people around the globe and causes a high rate of mortality. In order to fight this disease, a new coumarin derivative ligand (4-[(pyridin-3-ylmethyl) amino]-2H-chromen-2-one) (L(TA)) has been synthesized and characterized by single-crystal X-ray diffraction, NMR, ATR, UV-Visible and cyclic voltammetry. Chemical reactivity, kinetic and thermodynamic studies were investigated using DFT method. The possible binding mode between L(TA) and Main protease (Mpro) of SARS-CoV-2 and their reactivity were studied using molecular docking simulation. Single crystal X-ray diffraction showed that L(TA) crystallizes in a monoclinic system with P2(1) space group. The reactivity descriptors such as nucleophilic index confirm that L(TA) is more nucleophile, inducing complexation with binding species like biomolecules. The kinetic and thermodynamic parameters showed that the mechanism of crystal formation is moderately exothermic. The binding energy of the SARS-CoV-2/Mpro-L(TA) complex and the calculated inhibition constant using docking simulation showed that the active L(TA) molecule has the ability to inhibit SARS-CoV-2. Coumarins (2H-chromen-2-ones) from natural sources are non-toxic and non-inhibitory against the biotransforming organism. These compounds have been identified in a large number of plants such as rutaceae, umbelliferae, legumes and orchids [1] [2] [3] . Many of the naturally and synthetic derivatives of coumarin have shown broad-spectrum of pharmacological, biological and physiological properties, including antibacterial, antifungal, antioxidant, anti-inflammatory, anti-allergic, antiviral, hepatoprotective, anti-tumor, anticoagulant, anti-HIV and anti-carcinogenic agents [4] [5] [6] [7] [8] [9] [10] [11] [12] . Recently, this family of compounds has been used to prepare new drugs with very low toxicity [13, 14] , especially for skin and autoimmune diseases [15] . 4 -hydroxycoumarins are important precursors in the preparation of antifungal, bacteriostatic, anti-coagulant, potentially anti-HIV, spasmolytic drugs and herbicidal agents. This category of products presents photochemical and photophysical characteristics and play a major role in modern optoelectronic [16] [17] [18] [19] . Coumarins-fused pyridine hybrids are the most important type of pharmaceutical intermediates, and are defined in the literature as potential anti-osteoporotic agents [20] . They have a high bio-compatibility due to the nitrogen atom that can form hydrogen bonds with biological molecules [21] . The complexes formed with coumarin-pyridine derivatives have attracted more attention from chemists because of their diverse structural design and scientific applications [22] [23] [24] . Since the listed positive effects of coumarin derivatives, we wondered if an alternative new synthetic molecule derived from coumarin might be designed to fight against infectious diseases caused by more virulent viruses. In perspective, this would prepare a drug against coronaviruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The coronavirus disease 2019 (COVID-19) causes severe respiratory infection and is known to be very contagious [25] . Currently, there are no treatments available for SARS-CoV-2, therefore, treatment is focused on symptoms which may include dry cough, fever and pneumonia [26] . According to the work of Lima de Oliveira et al., the identification of small molecules (ligands) which specifically target the sensitive sites of viruses plays a very important role in antiviral drugs discovery [27] . Herein we wish to report on our preliminary studies towards this objective. In this work, the new 4-[(pyridin-3-ylmethyl)amino]-2H-chromen-2-one ligand (L TA ) was synthesized and characterized by different techniques such as single-crystal X-ray diffraction, NMR, ATR, UV-Visible and cyclic voltammetry. Theoretical studies using DFT with B3LYP combined with the standard base set 6-31G+(d) were investigated to understand the chemical reactivity and the formation mechanism of L TA . The aim of this study is also to evaluate the mode of interaction of L TA with SARS-CoV-2/Main protease (Mpro), whose inhibition of this enzyme would prevent the virus from replication. Diffraction data were collected at 150 K on a D8 Venture Bruker AXS diffractometer equipped with a (CMOS) PHOTON 100 detector, Mo-Kα radiation (λ = 0.71073 Å, multilayer monochromator). Crystallographic data for the structural analysis have been deposited at the Cambridge Crystallographic Data Center, CCDC No 1981639. These data can be obtained via https://www.ccdc.cam.ac.uk, e-mail: deposit_reply@ccdc.cam.ac.uk. The attenuated total reflectance (ATR) spectrum was carried out on a BRUKER LPHA-T Spectrometer. All nuclear magnetic resonance measurements ( 1 H-NMR, 13 C-NMR and 2D-NMR) were performed at 25 °C with a Bruker Avance III Spectrometer. The electronic spectra were recorded on a SECORD plus Spectrophotometer in the range 200-500 nm. The electrochemical measurements were carried out using Potentiostat/Galvanostat Metrohm Autolab 302N. The theoretical calculations were performed using DFT/B3LYP/6-31G+ (d) level of theory. All reagents and solvents used to prepare the measurement solutions were of the highest purity and analytical grade from Sigma-Aldrich or Fluka chemicals Company. The ligand (4-[(pyridin-3-ylmethyl)amino]-2H-chromen-2-one: L TA ) was synthesized by adding 1.08 mL of 3-picolylamine (10 mmol) to 10 mmol (1.62 g) of 4-hydroxycoumarin dissolved in 10 mL of absolute ethanol. The mixture was stirred under reflex at 70º C for 48h. The bright incolor crystals of the product were grown by slow evaporation technique in ethanol for two weeks. [28] . Indeed, synthesis of L TA is carried out via nucleophilic attack of NH 2 group of 3-picolylamine on the C10 carbonyl of 2, 4chromandione with elimination of water molecule [29, 30] , according to the following detailed mechanism: . Scheme 1. Synthesis mechanism of ligand (L TA ). The crystal data, the conditions of data collection, isotropic and anisotropic displacement parameters, bond lengths (Å), bond angles (°) and torsion angles (°) are summarized in Table 1, 2 and Tables S1, S2. There are three inequivalent hydrogen bonds in the crystal ( Fig. 1b and Table S3 ). The first and the second hydrogen bonds (N12-H12···O21 and N32-H32···O1) are short, their hydrogen bond distance are H12···O21 = 1.99 Å and H32···O1= 1.97 Å, respectively. The third hydrogen bond, C39-H39···N18 (not shown in Fig. 1b) should be weaker because it corresponds a large distance H39···N18 = 2.61 Å. In order to obtain stable structure, an optimization of the geometrical parameters of ligand L TA has been carried out at DFT levels of theory [31] using the Becke's three parameter hybrid exchange functional in combination with Lee-Yang-Parr correlation functional (B3LYP) [32] at 6-31+G(d) basis sets. All calculations were obtained with the Gaussian09 program package [33] . The optimized parameters, namely, bond lengths and angles and Dihedral angles calculated by B3LYP/6-31+G(d,) and found by XRD are reported in Table 2 , according to the numbering atoms given in scheme 1 and Fig. 2 . The quantitative-chemical calculations of the two systems (L TA(C1-C19) ) and L TA(C21-C39) ) performed theoretically give similar results for bond lengths and angles, which are in agreement with the X-ray diffraction data. The slight differences found between the experimentally and theoretically calculated dihedral angles are due to the molecular interactions not taken into account by this chosen theoretical method, and to the effects of the solvent which have been neglected (gas phase). However, the analysis of the total energy shows the same energy for: L TA(C1-C19) (-838.80986151 a.u) and L TA(C21-C39) (-838.80986137 a.u) with a slight difference of around 10 -7 . For this, L TA(C1-C19) structure was chosen for the rest of the theoretical study. The ATR spectrum was recorded to investigate the vibration modes of the functional groups and thus provide information on the structural dynamics of the ligand. In this study, the experimental spectrum of L TA was superposed on that obtained theoretically (Fig. 3) . The assignments of the various stretching and bending vibrations of the ligand were shown in Table S4 . The functional groups present in L TA correspond to the frequencies at which absorption are located. The characteristic vibration frequencies for the studied compound are given as follows: In the experimental spectrum of L TA , N-H stretching vibration band is observed at 3286 cm -1 and its deformation vibration band is located at 1574 cm -1 . However, the theoretical spectrum showed N-H stretching and deformation vibrations at 3282 cm -1 and 1578 cm -1 , respectively. The symmetric and asymmetric C-H stretching vibrations of CH 2 group can be found between 2850 and 3050 cm -1 [34, 35] . For this ligand, C-H stretching vibrations are observed theoretically and experimentally in the range 2900-3050 cm -1 , and the deformation bands are found around 1468 cm -1 in both theoretically and experimental spectra. The other bands of C-H at 770-997 cm -1 are assigned to the out-of-plane deformation vibrations. C-N stretching vibrations are located at 1350 cm -1 , whereas these calculated with B3LYP program are given at 1335 cm -1 . In the ATR spectrum, C=O stretching modes are observed at 1672 cm -1 associated theoretically to the peak appeared at 1760 cm -1 [36] . However, the peaks found in the range 1270-1300 cm -1 and at 711 cm -1 are assigned to the stretching and deformation vibrations of the C-O groups, respectively. NMR spectroscopy is one of the most powerful instruments for organic and inorganic structural identifications. The chemical shift displacements were calculated by the GIAO model (Gauge Including Atomic Orbital) [37, 38] in DMSO using the PCM model. 1 H-NMR and 13 C-NMR spectra of the ligand were recorded in deuterated DMSO solution and were reported in Fig. 4a , b. HSQC-NMR and HMBC-NMR spectra are shown in Figs. S1a, b. The theoretical and experimental chemical shifts in proton and carbon NMR of L TA molecule noted δ Cal and δ Exp , respectively, are shown in Table S5 . The chemical shift between 8.28 and 8.38 ppm in the experimental proton NMR spectrum is attributed to H12 proton bound to N12 nitrogen. The same proton presents a chemical shift of 4.83 ppm in the theoretical spectrum. The atomic charge on H12 is found to be large positive ( Fig. 2 ) as electronegative N atom is attached with this hydrogen. Therefore, H12 is observed on higher ppm scale [39] . The proton signals on the aromatic ring are observed in the region 7.32-8.12 ppm and at 7.11-7.51 ppm in the experimental and theoretical spectrum, respectively. The protons on pyridine ring are seen experimentally between 7.36 and 8.76 ppm and theoretically between 7.14 and 8.49 ppm. The junction between the cycles shows proton resonances between 4.58-4.60 ppm in the experimental spectrum and between 4.49-4.37 ppm in the theoretical spectrum. Correlation between experimental and calculated chemical shifts of 1 H-NMR and 13 C-NMR are given in Figs. S1 c, d. 13 C-NMR calibration plot showed a good linear relationship between the experimental and theoretical chemical shifts, with correlation coefficient The lower correlation coefficient (R 2 =0.80) obtained from 1 H-NMR calibration plot is due to the chemical shift of the proton at position 12 found experimentally in the range 8. 28-8.38 ppm and theoretically at 4.83 ppm. The electrochemical behavior of the ligand was studied by cyclic voltammetry in DMSO and tetrabutylammonium hexafluorophosphate (TBAPF 6 ) at a potential scan rate of 20 mV.s -1 Platinum, glassy carbon and saturated Ag/AgCl were used as working electrode, a counter electrode and a reference electrode of platinum, respectively. To assess the stability of the ligand, CVs were recorded up to 25 cycles at scanning rate of 20 mV/s (Fig. 5a) . The results obtained showed good reproducibility of the voltammograms, indicating the stability of this product (L TA ) at room temperature. The recorded voltammogram of the ligand (Fig. 5b) gives a cathodic peak at Ep c =-0.56 V assigned to the reduction of C=O to OH group which in turn oxidizes at Ep a =-0.41V. In the anodic part, the second peak at Ep a = 0.30V is due to the oxidation of N-H to imine group (C=N) [42] . This result reveals the presence of two tautomeric forms of the ligand in solution (Scheme 2). Tautomeric equilibrium of ligand (L TA ). Functional density theory (DFT) in computational chemistry is a highly dynamic tool used in several research projects to predict and determine structural properties, chemoselectivity, stereoselectivity, regioselectivity, reaction mechanisms, energy profiles and various spectroscopic characterizations. Lowest Unoccupied Molecular Orbital (LUMO) are important quantum chemistry parameters. Indeed, the knowledge of the frontier orbitals allows determining the molecular properties [43] . The difference in energy between LUMO and HOMO, corresponding to energy gap (ΔE), serves to characterize the chemical reactivity and the kinetic stability of the molecule. The highest occupied molecular orbitals and lowest unoccupied molecular orbitals of the ligand have been studied and the results were given in Fig. 6 . The HOMO density plot of the stable ligand showed that the electron density is observed essentially on the 4-amino-2H-chromen-2one ring and on the nitrogen of the pyridine ring, whereas the LUMO showed the localization of electron density just on the 4-amino-2H-chromen-2-one ring. This result predicts the localization of the electrophilic and nucleophilic attack sites of L TA . On the other hand, we also note that the value of the energy gap of this ligand is significant (4.596 eV), this indicates that L TA is more stable due to its hard character. This result was confirmed by the analysis of the global reactivity indices (section 3. 3. 5). MEP is a very useful method to investigate the electrophilic and nucleophilic attack sites. It is directly related to the electron density, as well as hydrogen bonding interactions [44, 45] . Molecular electrostatic potential was evaluated using the B3LYP/6-31+G(d) method. According to Fig. 7a , we observe different regions: those colored in red and yellow correspond to electron-rich sites (maximum negative potential), the blue region due to electron-poor sites and the green regions correspond to neutral sites (region of zero potential). For L TA , the most negative regions (red and yellow) cover the carbonyl group of coumarin and the intracyclic nitrogen of 3-picolylamine. The high electronegativity of these groups (C=O and N) presents the most reactive part (the most suitable sites for electrophilic attack) of the ligand. Whereas, the blue region represents the site of highest reactivity to nucleophilic attacks. In addition, the contour MEP is a two-dimensional (2D) representation of the areas where the relative electron density values are within a specific margin at 0010. For our compound, the contoured MEP for the positive and negative potentials is given in Fig. 7b . The found results allow giving information about the sites of intermolecular attacks (electrophilic and nucleophilic attacks) and the MEP map. The atomic charges play an important role in the application of quantum chemical calculations to molecular systems [46] because they affect dipole moment, molecular polarizability and electronic structure [43] . Atomic charges can be used to describe the processes of electronegativity equalization and predicting the charge transfer in chemical reactions [47, 48] . The distribution of the net charges on the main L TA atoms and the calculations were made using NBO theory. result, we conclude that the O1, O3, N12 and N18 sites are more favored for electrophilic attacks, but the H12 site is more favored for nucleophilic attacks. This result is in agreement with that found using molecular electrostatic potential (MEP). The Natural Bond Orbital (NBO) method provides information on the intramolecular and intermolecular interactions that can form within the molecule. Indeed, NBO determines the stabilization energies of a given compound using second-order perturbation theory [37] . For each donor NBO (i) and acceptor NBO (j), the stabilization energy E(2) associated with electron delocalization between donor and acceptor is determined as follows [49, 50] : With E(2) is the stabilization (delocalization) energy (kJ/mol), (ξ j -ξ i ) is the difference in energy (a.u) between the donor (i) and acceptor (j) NBO orbitals, (F i, j ) is the Fock matrix elements (a.u) between i and j NBO orbitals and qi is the orbital occupancy. The results of the second-order perturbation theory of the Fock Matrix [51] are presented in Table 3 . The two largest values of energy (E(2)) are assigned to Lp(1)N12π*C10-C11 (45.86 kcal. mol -1 ) and Lp(2)O1π*C2-O3 (40.29 kcal. mol -1 ) interactions, this means that the two atoms N12 and O1 participate in the donor mesomeric relocation to stabilize the system. Other hyperactive-conjugative energy of LP(2)O1π*C9-C4 (32.64 kcal. mol -1 ) and LP(2)O1π*C2-O1 (34.06 kcal. mol -1 ) and the resonance in the aromatic ring of the 4amino-2H-chromen-2-one fragment and in pyridine ring also plays an important role in stabilization of ligand structure. The global reactivity descriptors are calculated according to the Koopmans theorem [52] [53] [54] [55] by using HOMO and LUMO energy values of the geometry found by DFT calculations. These descriptors (electronic affinity (A), electronic ionization (I), chemical potential (µ), electronegativity (X), chemical hardness (η), softness (S), electrophilicity index (ω) and nucleophilie index (N)) are used to estimate the capacity of the molecule to exchange electrons with the external medium. These parameters are listed with formulas in Table 4 . The global reactivity descriptors are calculated in order to confirm the nature of this crystal (electrophilic or nucleophilic). The obtained results indicate that the ligand present a hard character (chemical hardness) and the nucleophilic index value confirm that L TA is more nucleophile, which promotes its complexation with biomolecules and metal ions. Therefore, the low value of chemical potential (μ), which is the additive inverse of the electronegativity (χ), may be explained by the fact that the tendency of the molecule to attract electrons is low. 2H-chromen-2-one. The geometries and energies of all stationary points: reagents, transition states (TS) and product (L TA ) have been completely optimized and the vibration frequencies [56] have been calculated using B3LYP/6-31G+(d) model, implemented in Gaussian09 [57] . In order to study the kinetic and thermodynamic characteristics of the reaction mechanism of L TA crystal formation given in the previous scheme 1, calculations were performed and the results were reported in Table 5 and Fig. 8 . We note that enthalpy and free enthalpy values are very low, indicating that the reaction is moderately exothermic. In addition, the formation mechanism of L TA gives a high activation enthalpy value (59.58 kcal.mol -1 ). This significant value is explained by the fact that the formation reaction of the crystal is slow at 70 °C (crystals are formed after two weeks of evaporation). We also note that the entropy variation of this chemical reaction is positive (ΔS°=0.0011255), indicating an irreversible reaction. These results showed that the mechanism is well concerted (a single-step reaction) and the obtained product (L TA ) is thermodynamically stable. The thermodynamic parameters were calculated using the DFT/B3LYP/6-31+G(d) level at temperature of 298.15 K and in solution phase (ethanol) of L TA using PCM model. The computed parameters were tabulated in Table 5 . The total energy of ligand was reported as -915.2642448 Hartrees. The sums of zero-point correction and thermal correlations to energies, enthalpies and Gibbs free energies with this calculated total energy value have been given sum of electronic and zero-point energy (-914.999196 Hartree/particle), sum of electronic and thermal energies (-914.979869 Hartree/particle), sum of electronic and thermal enthalpies (-914.978925 Hartree/particle) and sum of electronic and thermal free energies with -915.052821 Hartree/particle value. The Zero-point vibrational energy was calculated as 166.32076 kcal.mol -1 . The calculated total value of entropy is 0.155528 kcal.mol -1 . The molecular docking of SARS-CoV-2/Mpro-L TA binding mode was simulated using AutoDock Tools Vina 1.5.6. This method allows explaining of the most energetically favorable binding sites of (4-[(pyridin-3-ylmethyl) amino]-2H-chromen-2-one) to SARS-CoV-2/Mpro receptor in terms of the binding energy and inhibition constant. The 3D Main protease (Mpro)-L TA structure visualization was recorded using Discovery Studio 2020 and PyMOL software. The aim of this study is to investigate the mode of interaction of L TA with Main protease (Mpro), whose inhibition of this enzyme would prevent the virus from replication and therefore constitutes one of the potential anti-coronaviral strategies [57] , which gives further clues to the prospect of developing SARS-CoV-2 drugs. Target protein (SARS-CoV-2/Mpro: (Fig. 9b) . The interpolated charge (electrostatic charge) contour map (Fig. 10c) shows significant increase of negative charge in the red contour region, leading to increased activity with L TA . The SARS-CoV-2/Mpro also displayed few hydrophobic contacts mediated by the aliphatic amino acids (Fig. 10e) , and it is moderately basic (in blue) (Fig. 10f) . The 3D structure visualizations (Fig. 10a, b-f) Comparing the results found in this work with those reported in the literature, we find that both molecules chloroquine and hydroxychloroquine present lower binding energy (chloroquine: -5.9 kcal. mol -1 ; hydroxychloroquine: -6.0 kcal. mol -1 ) and inhibition constants (chloroquine: 30.8 μM; hydroxychloroquine: 22.9 μM) [27] . In the present work, synthesis, spectroscopic characterization (ATR, NMR and UV-Visible), The results of single crystal X-ray diffraction analysis show that this compound crystallized in monoclinic system with space group P 2 1 (Z=4). The natural bond order (NBO) analysis and the molecular MEP map showed that the O1, O3, N12 and N18 sites are more favored for electrophilic attacks, but the H12 site is more favored for nucleophilic attacks. The stabilization energy E(2) showed that N12 and O1 participate to stabilize L TA . The global reactivity descriptors obtained indicate that the ligand is more nucleophile and presents a hard character. The kinetic and thermodynamic parameters of L TA showed that the formation mechanism of the crystal is moderately exothermic with a high activation enthalpy. These results indicate that the synthetic product (L TA ) is thermodynamically stable. The interaction of (4-[(pyridin-3-ylmethyl) amino]-2H-chromen-2-one) with SARS-CoV- Houria LAKHDARI: Substantial contribution to ligand synthesis, Analysis and interpretation of data. Khaldoun BACHARI: The acquisition, Interpretation of data, Reviewing. Thierry ROISNEL: X-ray analysis, Refinement structure and representation of crystallographic results. Didier HAUCHARD: Substantial contribution to ligand analysis, Methodology, Interpretation of the electrochemical results. Table 1 . Crystal data and structure refinement for L TA . and DFT/B3LYP/6-31 + G(d) for the ligand L TA . Table 3 . Results of second-order perturbation calculations of the Fock matrix on a NBO basis corresponding to the intramolecular interaction of ligand L TA . Table 4 . Calculated energy gap |∆E| and global reactive descriptors (eV) for L TA using B3LYP/6-31+G(d). (ξ j -ξ i ): Energy difference (a.u) between donor (i) an acceptor (j) NBO orbitals. (F i, j ): Fock matrix elements (a.u) between i and j NBO orbitals. El-din Coumarin derivative as antiviral agent, pharmaceutical composition thereof, its preparation and use (International Publication Number Patent: WO 2016/156888 Al; International Application Number Patent: PCT/HR20 16/000005) Toxicological and Pharmacological Investigations of Newly Synthesized Derivatives of 4-hydroxycoumarin China coronavirus: cases surge as official admits human to human transmission I. j. o. d. i. I. p. o. s. t. I. S. f. I. D. Zumla "The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health-The latest 2019 novel coronavirus outbreak in Wuhan Comparative Computational Study of SARS-CoV-2 Receptors Antagonists from Already Approved Drugs Gaussian 09, Revision A.1 The Handbook oh Infrared and Raman Characeristic Frequencies of Organic Molecules Introduction to the resonating-valence-bond theory of superconductivity: Crest superconductor and through superconductors Theoretical Model of Chemical Bonding Charge Distribution and Chemical Effets Neutral blue-shifting and blue-shifted hydrogen bonds SARS-CoV-2 main protease with unliganded active site (2019-nCoV The authors would like to acknowledge the MESRS Algerian Ministry and Directorate-General for Scientific Research and Technological Development (Algeria) for supporting the present research.