Synthesis, characterization, computational and biological activity of novel hydrazone complexes

ABSTRACT In this work, novel hydrazone ligands were prepared by the reaction of chloroanthranilic acid diazonium salt with acetylacetone (L1), ethyl acetoacetate (L2) and diethyl malonate (L3). The prepared compounds were reacted with some transition metals like Ni(II), Cu(II), and Fe(III). The structures of the prepared complexes were confirmed using nuclear magnetic resonance (1H-NMR and 13C-NMR), distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC-NMR), correlated spectroscopy (COSY-NMR), Fourier-transform infrared spectroscopy (FT-IR) and electrospray ionization mass spectrometry (ESI-MS). Also, the magnetic properties for the prepared complexes were studied using Gouy’s method of susceptibility measurement. Also, there in silico docking and in vitro antibacterial activities were investigated and the results showed that Ni complexes have the highest antimicrobial activity. The expected structures and conformers for the prepared ligands and complexes were examined and fully optimized using the level B3LYP/6-31G*.


Introduction
The chemistry and biological study of hydrazone compounds represent an important area of chemistry that related to theoretical and computational work on bonding between a metal ion and donor ligands (Akbas, Celik, Ergan, & Levent, 2019;Kopylovich et al., 2011;Murase, Moritomo, Goto, Sugamoto, & Yoshikawa, 2005). Arylhydrazones of 1, 3-diketones (AHBD) are compounds combining in their structure azo-and diketo-function groups and thus providing rich organic chemistry and versatility in complex formation (Kopylovich et al., 2013). On the other hand, the complexes possess significance biological and chemical properties in many attractive applications (Azhari, Salah, Farag, & Mostafa, 2015;Heinrich, Stubbe, & Kulak, 2018;Salah, ZHA, Farag, & Mostafa, 2014). For example, it was demonstrated that AHBDs can be controllably switched between their (E, Z) enol-azo, keto-azo and (E, Z)-hydrazone forms, that makes them good candidates for molecular switches (Roztocki, Matoga, & Szklarzewicz, 2015;Sujamol, Sindhu, Athira, & Mohanan, 2011). The coordination chemistry of arylhydrazone ligands has also been intensively modified and improved. It was shown that substitution of different donor function groups at the ortho position of the aromatic part of hydrazones creates an additional chelating site with a stabilizing effect on the respective complex leading to a variety of complexes depending on the substituent. For instance (Chowdhury et al., 2018;Gurbanov, Zubkov, Saifutdinov, & Fig, 2018;Hadi, Noviany, & Rilyanti, 2018;Kopylovich et al., 2013;Mahmoud, Martins, Guedes Da Silva, & Pombeiro, 2018a;Roztocki, Matoga, & Nitek, 2016), if Cu (II) is used as the metal ion and substitution of sulfonic, carboxylic, hydroxyl or amino groups at the ortho position of the aromatic nuclei of 3-(2-phenylhydrazone)pentane-2,4-dione, monomeric, dimeric and cluster complexes can be synthesized. On the other hand, it would be exciting to discuss the influence of different metal ions on the structure, coordination modes, biological, nuclearities and properties of the formed complexes if the same ligand is used for the synthesis He, Qiu, Cheng, Liu, & Wu, 2018;Liu et al., 2017). This work aims to study the biological activity of novel hydrazone complexes.

Physical measurements
The IR spectra of the prepared ligands and their complexes were recorded using the Perkin-Elmer spectrophotometer spectrum 10.5.4 covering the frequency range 4000-400 cm −1 , by the ATR Sample base plate diamond method. The UV-Visible spectra were measured by Shimadzu UV-vis 160 double beam using DMF as a solvent. 1 H and 13 C-NMR spectra were obtained using a Bruker Advance 300 MHz spectrometer, at ambient temperature. All chemical shifts are reported in δ (ppm). All spectra were internally referenced to residual protio-solvent resonance and are reported relative to SiMe 4 . Electrospray mass spectra (ESI-MS) were obtained on a Varian 500-MS LC Ion Trap Mass spectrometer equipped with an electrospray ion source. For electrospray ionization, the flow rate and drying gas were adjusted according to the specific sample with 35p.s.i. nebulizer pressure. The scanning was done from m/z 100-1200 in the CH 3 OH solution. All ligands and complexes were observed in the positive and negative modes (capillary voltage = 80-105 V). The magnetic moments were measured by Gouy balance from Johnson Matthey and Sherwood model by measuring the apparent change in the mass of the sample as it is repelled or attracted by the high magnetic field area between the two poles. The sample is forced between the magnetic poles through an attached string.
2.2. Synthesis of 4-chloro-2-(2-(2,4-dioxopentan-3-ylidene) hydrazinyl)benzoic acid ligand (L 1 ) A 10 mmol of an aqueous solution of chloroanthranilic acid sodium salt and 10 mmol of an aqueous solution of NaNO 2 were mixed at (0-5°C) followed by the addition of 2 ml conc. HCl. Another solution of 10 mmol NaOH, 10 ml H 2 O, 20 ml ethanol, and 10 mmol of acetylacetone was added to the previous solution in three portions followed by the addition of 0.2 g sodium acetate. The yellow precipitate formed immediately was filtered, washed with cold ethanol and recrystallized from ethanol. 2.3. Synthesis of (E)-4-chloro-2-(2-(1-ethoxy-1,3-dioxobutan-2-ylidene) hydrazinyl) benzoic acid ligand (L 2 ) A 10 mmol of an aqueous solution of chloroanthranilic acid was reacted with 10 mmol of sodium hydroxide to produce the corresponding acid salt, then the salt produced was reacted with (10 mmol) of an aqueous solution of NaNO 2 at ice medium followed by dropwise addition of 2 ml conc. HCl. Another solution of 10 mmol NaOH, 10 ml H 2 O, 20 ml ethanol, and 10 mmol of ethyl acetoacetate was added to the previous solution in three portions followed by the addition of 0.2 g sodium acetate. The dark yellow precipitate formed was filtered, washed with cold ethanol and recrystallized from ethanol.

Synthesis of the complexes
A 10 mmol hot methanolic solution of (L 1 , L 2 , and L 3 ) ligands was dropwise added to hot methanolic solutions of equal molar NiCl 2 .6H 2 O, Cu(NO 3 ) 2 .2.5H 2 O and Fe(NO 3 ) 3 .9H 2 O, then the progress of reactions was followed by TLC using dichloromethane/methanol. The pH of reactions between ligand with Ni and Cu ions was raised to nine using drops of 1M NH 4 OH. The precipitates formed were filtered, washed with cold methanol and diethyl ether, dried then washed with hot ethanol. Figure 4. Proposed structures for the prepared complexes. (

Method of calculations
The expected structures and conformers for the ligand (L 1 ), some of its complexes and their total energy, enthalpy of formation, free energy of formation, atomic charges, HOMO-LUMO, dipole moment, bond length, bond angle, and dihedral angle at different centers were studied and fully optimized using the level B3LYP/6-31G*.

Antibacterial docking studies and assessment of antimicrobial assay
The binding sites of the protein were determined using MVD cavities prediction. The predicted cavities having 65.042 Å 3 and 55.808 Å 3 volumes for Eco SlyD and Sau SlyD, respectively. The binding site was set inside a restriction sphere of 20 Å radius, also using MVD. All other software parameters for docking were set to the default values (Lanez & Lanez, 2016). The parameters were as follows: the 'MolDock SE' searching algorithm was used with the number of runs set to 10 using a maximum of 1500 iterations and a total population size of 50. The energy threshold used for the minimized final orientation was 100. The Simplex evaluation with 300 maximum steps of neighbor distance factor 1 was completed (Kamboj, Chaudhary, Paliwal, & Jindal, 2015). The chosen cavity was further refined utilizing side-chain minimization by choosing the add-visible option at a limit (10,000) of steps per residue and a maximum (10,000) of global steps.

Description of ligands structures
The desired hydrazone fragments were prepared through the coupling of chloroanthranilic acid diazonium salt moiety with acetylacetone, ethyl acetoacetate and diethyl malonate in the presence of sodium acetate as a catalyst. The 1 H-NMR spectra showed triplet, quartet and singlet signals at δ 1.29-4.31 due to different methyl and methylene groups of acetylacetone, ethyl acetoacetate, and diethyl malonate moieties. The three aromatic protons appeared at the normal chemical shift δ 7.06-7.98 and the signals appeared at δ 14.78-16.06 due to hydrogen-bonded N-H. The 13 C-NMR spectra confirmed the previous data obtained, they showed signals at δ 161.09-196.79 due to different C = N and carbonyl groups. DEPT spectra were obtained to identify signals of C, CH, CH 2 , and CH 3 . The FT-IR spectra showed a set of bands for N-H, C = O, and C = N at their normal wavenumbers.

Description of complexes structures
The 1 H-NMR spectra of L 1 Ni, L 2 Ni, and L 3 Ni did not display the N-H signal which indicates the coordination of the N to the metal ion. The ESI-MS (+) of L 1 Ni, L 2 Ni, and L 3 Ni showed m/z: 370.52, 400.50 and 430.34, respectively, complexes formed via coordination between carbonyl, carboxylate group and negatively charged nitrogen with the metal ion to form stable 6-membered rings. IR spectra showed decreasing of their stretching vibration frequencies on coordination (ῡ C = O 1674-1717 → 1602-1622) (ῡ C = N 1620-1637→ 1557-1581) (Dhande, Badwaik, & Aswar, 2007;Mahmoud et al., 2018a;Mandewale, Kokate, Thorat, Sawant, & Yamgar, 2016;Popov et al., 2017). Based on magnetic susceptibility results and spectroscopic data obtained, the proposed structures of the complexes are octahedral formed by the combination of metal ion with one ligand molecule and completed their coordination geometry by water molecules. The broadening of 1 H-NMR spectra of L 1 Cu, L 2 Cu, and L 3 Cu due to paramagnetic properties of metal ion which were proved by magnetic susceptibility measurement. Spectra showed the disappearance of the N-H signal indicating coordination between metal and negatively charged nitrogen. The elemental analysis of L 1 Cu, L 2 Cu, and L 3 Cu proved that the ratio of ligand and metal ion was 1:1. They showed m/z: 375.45, 405.09 and 374.04, respectively, complexes formed via coordination between carbonyl, carboxylate group and negatively charged nitrogen with the metal ion. IR spectra showed the disappearance of the N-H stretching band and a new stretching broadband ῡ O-H around 3316 for water moieties, carbonyl stretching frequencies were shifted to a lower frequency, ῡ C = O (1674-1717→ 1623-1670). Based on the results obtained (Philip, Antony, Eeettinilkunnathil, Kurup, & Velayudhan, 2018;Sergienko et al., 2014;Tamayo et al., 2017), Cu ion is surrounded by penta and tetracoordination sites with the expected square pyramid and tetrahedral structures. The broadening of 1 H-NMR spectra and magnetic susceptibility measurement for L 1 Fe and L 3 Fe indicating the paramagnetic properties of metal ion, the spectra showed that the N-H signal appeared at the same chemical shift on ligand spectrum indicating no coordination occurs through N-H group. The elemental analysis of L 1 Fe and L 3 Fe proved that the ratio of the ligand and metal ion is 2:1 with m/z: 399.26 and 458.94 due to [Fe+L+ NO 3 ] + . The complexes formed via coordination of the carboxylate groups to the metal ion. IR spectra showed decreasing of stretching frequencies of donating groups (ῡ C = O 1683-1717→ 1623-1679) (Simonov et al., 2009), the broadband around 3316 due to OH of water moiety coordinated to Fe with expected octahedral and tetrahedral structures.

Theoretical modeling of the structure
The computational analyses were performed to get the molecular geometry of complexes and energy minimization studies using the Gaussian 09W software package. The expected geometries of the studied ligand and some of its complexes were fully optimized in the gas phase without any symmetry constraints.

The geometry of the ligand
The computational results showed that the structure L 1 has the total energy −1333.94 a.u, Table 1. Calculated parameters for L 1 and L 1 Cu square planar using B3lyp/ 6-31G*. The atomic charge on N 1 , N 2 , O 5 , O 8 , and O 18 atoms in the most stable L 1 structure are −0.484526, −0.190864, −0.543205, −0.431758, and −0.594054, respectively, which are the highest negatively charged atoms; therefore, they are the most expected chelating centers (Abdel-Latif & Mohamed, 2017; Sreejith, Nair, Smolenski, Jasinski, & Prathapachandra Kurup, 2018). Table 2. Calculated parameters for L 1 Ni and L 1 Cu complexes using B3lyp/6-31G*. The dipole moment is 10.1252 Debye and it is highly polar and stabilized in polar solvents.

Geometry of complexes
The calculated data proved that the best geometrical structure for L 1 Ni (1:1) is octahedral geometry with coordination number 6, while the best geometrical structure for L 1 Cu (1:1) is that with coordination number 5 to form a square pyramid structure. Ni and Cu ions complete their coordination number by solvent molecules. The computed results revealed that the octahedral structure of L 1 Ni was located as the minimum point.

Conclusion
In conclusion, some ligands based on chloroanthranilic acid hydrazone have been successfully synthesized and characterized by different spectroscopic methods. The complexes of Ni, Cu, and Fe have been synthesized and characterized, and then the theoretical calculations for some complexes were performed to confirm their structures and study their thermodynamic parameters using the level B3LYP/6-31G *. The magnetic moments for the complexes were calculated using Gouy's method. The antibacterial activity was evaluated for the suggested compounds using the disc diffusion method and the results were in good agreement with those obtained from docking studies. The experimental results showed that Ni complexes have the highest biological activities against both E. coli and S. aureus.   Figure 6. Ligand interaction of L 2 Ni complex with SlyD of (a) E. coli (b) S. aureus.