Synthesis, Crystallographic Structure, Hirshfeld Surface Analysis, Drug-likeness Properties and Molecular Docking Studies of New Oxime-pyridine Compounds

A detailed description of the two new pyridine ligands, (2E , 3Z )- 3-[2-(3-chloropyridin-2-yl)hydrazinylidene]-N-hy-droxybutan-2-imine and 3-chloro-2-{(2Z)-2-[1-(4 nitrophenyl)ethylidene]hydrazinyl}, is reported. The synthesized compounds were characterized by spectroscopic studies, spectral features were performed by TD-DFT calculations. New-generation pyridine ligand of HL 2 was also determinate by single-crystal X-ray diffraction and Hirshfeld surface analysis with two-dimensional fingerprint plots was used to analyze intermolecular interactions in crystals. Molecu-lar-docking was performed to investigate the binding areas of chemical compounds, and the results showed the inhib-itory activity of the studied HL 1 and HL 2 against E. coli . The results of the current study revealed the drug-likeness and bioactive properties of the ligands. A detailed description of the two new pyridine ligands, (2E , 3Z )- 3-[2-(3-chloropyridin-2-yl)hydrazinylidene]-N-hy-droxybutan-2-imine and 3-chloro-2-{(2Z)-2-[1-(4 nitrophenyl)ethylidene]hydrazinyl}, is reported. The synthesized compounds were characterized by spectroscopic studies, spectral features were performed by TD-DFT calculations. New-generation pyridine ligand of HL 2 was also determinate by single-crystal X-ray diffraction and Hirshfeld surface analysis with two-dimensional fingerprint plots was used to analyze intermolecular interactions in crystals. Molecu-lar-docking was performed to investigate the binding areas of chemical compounds, and the results showed the inhib-itory activity of the studied HL 1 and HL 2 against E. coli . The results of the current study revealed the drug-likeness and bioactive properties of the ligands.


Introduction
For biological activities, pyridine compounds are widely used as antibacterial, antifungal, and anticancer agents. [1][2][3] Several studies have been conducted on biological compounds in health-related journals and books. Accordingly, pyridine derivatives cause interactions with high binding capacity by targeting enzymes, proteins, and deoxyribonucleic acid (DNA) that create biological problems. 4 With the discovery of new compounds, several studies were performed in the last decade to inhibit antibacterial drug resistance and reduce associated adverse effects on human health. 5 It is clear that new types of viruses and bacteria affect the lives of humans worldwide in a variety of ways. Accordingly, this places immense responsibility on researchers and chemists who work to develop new materials to decrease the effects of viruses and bacteria as well as biologists and physicians who test the new compounds on animals and humans. Requiring extensive research, conducting in vitro studies is costly and time-consuming; accordingly, one of the most important advantages of the current study was the contribution to perform in silico studies inactivating viruses, bacteria, and cancer cells by the production of ligands with medicinal potential. Pyridine and oxime compounds have a high interference of hydrogen bond in electrostatic potential capacity. Intramolecular and intermolecular hydrogen bonds play a major role in the interacting and binding of biological molecules. In addition, pyridine and oxime compounds have been selected particularly for their ability to easily transfer the electrons of nitrogen atoms participating in the aromatic ring and C=N groups into the donor-acceptor system. [6][7][8][9][10][11] Due to their high levels of antibacterial properties and bioactive multizones, nitrogenous organic or inorganic compounds are reported to have positive effects on Escherichia coli. 12 The reason for the investigation of E. coli pathogens in molecular-docking studies is this bacterium's resistance against some medications and its high binding capacity. 5 Furthermore, E. coli, also known as the most-common human pathogen, causes different types of infections, such as kidney, gallbladder, skin, and respiratory infections in addition to meningitis in neonates. 13,14 The physicochemical properties of compounds affect bioavailability, including electrostatic potential, molar absorptivity, stability, solubility, structure, intracellular absorption, hydrogen bonds, and bonding energy. 15,16 The theoretical method such as Density Functional Theory (DFT) in the computational chemistry is important tool to predict the assignment of specific electronic transitions in the UV-Vis spectra. In the present study, oxime and pyridine derivative compounds were synthesized and characterized by X-ray and spectroscopic methods. In addition, the surface analysis was performed to analyze their chemical properties, and molecular electrostatic potential was calculated to determine the nucleophilic and electrophilic zones. Molecular-docking was conducted to investigate the hydrogen-binding interactions of E. coli DNA gyrase subunit B (GyrB) and E. coli beta-ketoacyl-acyl carrier protein synthase III (FabH) and determined pharmacokinetic and pharmacological properties.

1. Materials and Physical Measurements
The chemicals and solvents: methanol, acetonitrile, 3-chloro-2-hydrazinopyridine, 2,3 butanedione monoxime, 4´-nitroacetophenone were obtained from Sigma-Aldrich. BRUKER BIOSPIN NMR AVANCE Spectrometer III 400MHz model spectrometer was used for 1 H-NMR and 13 C-NMR analysis. Elemental analysis were determined using a Costech Elemental analysis device ECS 4010 Model analyzer. IR spectrums were recorded in the 400-4000 cm -1 on Perkin Elmer FTIR-Spectrometer Spectrum Two Model and Mass Spectra (ESI) on TSQ Fortis™ Triple Quadrupole Mass Spectrometer. Melting points of ligands were determined by Stuart SMP10. The mains water was passed through the Thermo Scientific Smart2pure device to make it pure water. Absorption spectra was carried out using Shimadzu UV-1800 UV-VIS spectrophotometer. Single-crystal X-ray structure was determined using an Agilent SuperNova Dual CCD detector diffractometer eguipped with graphite-monochromated MoKα radiation (λ = 0.71073 °A) at room temperature.

3. X-ray Crystallography Analysis
Orange crystal of the C 13 H 11 ClN 4 O 2 compound was obtained in acetonitrile solution through slow evaporation for 5 days at room temperature. The data set of reflections were collected using an Agilent SuperNova X-Ray diffractometer with MoKα (λ = 0.71073) at 293 K. The data reduction and data correction were performed by Olex2 software (version 1.3). 19,20 Refinements were obtained by the Full-Matrix method on F 2 using the Olex2 software, and crystal packing diagrams were created by Mercury 4.3.0 software. All the nonhydrogen atoms were anisotropically refined using the riding model approximation. 21

1. Description of the Crystal Structures and Hydrogen Bonding
Slow evaporation technique was used to make the sample suitable for X-ray structure analysis. The HL 2 crys-
Basically 3 pairs of hydrogen bond interactions are observed in the structure, these are C-H···O, C-H···N and C-H···Cl intramolecular and intermolecular hydrogen bonds. 29,30 Selected hydrogen bond distances and angles are listed in Table 3. In HL 2 , carbon atom C10 acts as a donor to N1 atom at C(10)-H(10A)···N(1) (x,1.5-y,-1/2+z), developing the capped stick style and two-dimensional chain along the crystallographic axis as depicted in Fig. 3a.
As illustrated in Fig. 3b, in HL 2 , C-H···O intermolecular hydrogen bonding interactions were used to generate a three-dimensional (3D) supramolecular network along the c axis. All the figures were drawn in Mercury software. Other hidrogen bonds were C(10)-H(10A)···Cl (11) Fig. 4. 1 H-NMR spectrum of the HL 1 ligand oxime (N-OH) group of the proton (H13) was observed at 9.83 ppm as a singlet peak. The NH proton (H8) generated a signal at 8.45 ppm. The spectrum of the (2E,3Z)-3-[2-(3-chloropyridin-2-yl)hydrazinylidene]-N-hydroxybutan-2-imine ligand observed at singlet peaks 2.23 ppm (H14A, H14B, H14C) and 2.34 ppm (H15A, H15B, H15C) methylene group of protons. While H1 proton signal appeared at d 8.37 ppm, the signals at d 7.62 and d 6.84 ppm were due to H3 and H2 of pyridinium moiety, respectively. 32 In HL 2 , the NH proton (H7) generated a sharp signal at 8.48 ppm, as a singlet peak. The peaks were observed at range 8.30-6.84 ppm were assignable to the protons of aromatic rings as multiplet peaks. The spectrum of the 3-chloro ligand observed at singlet peaks 2.38 ppm (H10A, H10B, H10C) methylene group of protons. The signal at d 8.30 ppm was attributed at to (H6) aromatic proton of pyridine. The proton signal appearing at d 8.20 and d 8.01 ppm were due to (H13, H17, H16, H14) of aromatic moiety respectively. Aromatic protons of pyridine moiety produced a broad signal at d 7.61 and t 6.84 ppm (H5, H4). 11,18,33 It was observed that the obtained results were exactly compatible with the structure. 13 C-NMR spectrum of HL 1 is shown in Fig. 5. 13 C-NMR spectrum HL 1 observed a single resonance at 157.39 and 157.32 ppm, respectively which showed that the oxime (C=NOH) and hydrazone (-NHN=CH) (C11,C10) carbon atoms. All the signals were assigned to the aromatic carbons (C1-C5) of the pyridine at the range of 150.00-115. 35

4. UV-Vis Absorption Spectra and TD-DFT Calculations
UV-Vis calculations were performed by TD-DFT/ B3LYP method with 6-31G basis set using Gaussian 09 program. 34,35 The electronic absorption spectra of the ligands, along with the molar extinction coefficient, were obtained in a 5 × 10 -5 mol L -1 chloroform solution in the wavelength zone (240-480 nm) using the spectroscopic method. 23 The electron transition possibilities of different compounds were compared using ultraviolet-visible spectroscopy. For HL 1 and HL 2, slightly different absorption peaks centered at 322, 285 nm and 362, 312 nm (ε = 42800, 41100 and 34520, 26300 mol -1 L cm -1 ), respectively. The experimental UV-Vis spectra of the HL 1 compund and corresponding theoretical calculations are plotted in Fig. 6. The-oretical calculations predicted two peaks at 307, 287 nm and 364, 331 nm which indicated formation of the HL 1 and HL 2 . 36 The calculated excitation energy, excitation wavelength, oscillator strength with the aid of TD-DFT/B3LYP method are given in Table 4. The electronic absorption spectra of the ligands were defined with two sharp absorption bands. These two bands were observed at 285-312 and 322-362 nm indicative of the π-π* and n-π* transitions, respectively. 37 While the π-π* transitions of the ligands originated from the electrons of the pyridine ring, the n-π* band occurred due to hydrazone groups (-NHN=CH) of atoms. 38

5. Mass and FT-IR Spectra
Mass spectral datas of ligands were obtained by electrospray ionization (ESI) method. The mass exhibited the molecular ion at m/z 226.66284 [M+1] + and 290.70558 [M+1] + which indicated formation of the HL 1 and HL 2 (Fig. S1) . The moleculer peak of both ligands have a 100% relative abundance. 32 Generally compunds are characterized by three IR absorption bands such as ν(O-H), ν(C=N) and ν(N-O) stretching vibrations. FT-IR spectrum of the HL 1 ligand showed (C=N) imine (C=N) oxime peaks at 1596 cm −1 and 1516 cm −1 (Fig. S2). 39 The bands 3361 cm −1 and 3377 cm −1 were due to (N-H) vibrations for HL 1 and HL 2 respectively. Also (O-H) band of oxime group peak was observed at 3111 cm -1 . 33 But this peak was not seen at HL 2 ligand. At the same time, the bands at 935 cm −1 and 1392 cm −1 assignable to (N-O) and (NO 2 ) vibrations, respectively. The FT-IR spectrum of ligands displayed bands at 2981 cm −1 and 2972 cm −1 which assignable to (C-H Ar ) protons. The medium bands observed at 553 cm −1 and 546 cm −1 assigned to pyridyl rings. For HL 1 and HL 2, the bands 1454-1451 cm −1 , 1394-1392 cm −1 , 1044-1032 cm −1 and 1012-1032 cm −1 assignable to the aromatic pyridine ring. 40 FT-IR analysis give us the preliminary information about whether this structure is formed or not. Our datas are in agreement with similar oxime and pyridine ligands in the literature. 41

6. Hirshfeld Surface Analysis
The surface analysis method is the best way to identify crystal packing and intermolecular interacting in a structure. For this reason, the Hirshfeld surface analysis was performed using CrystalExplorer software (version 17.5). 42,43 Accordingly, the close correlations between the fragments were quantitatively analyzed. Furthermore, it is one of the computer calculation programs to investigate the mechanism of molecular interactions in proteins and with which intermolecular interactions they could bond to a receptor. In addition, it helps to identify the intermolecular hydrogen bond as well as π-π, C-H···X (X=halogens) interactions with great importance in crystal package arrangement and stabilization of the molecule. Therefore, C(17)-H(17)···O (20) and C(13)-H(13)···Cl(11) interactions are illustrated in Fig. 7.

7. Molecular Electrostatic Potential Analysis
Molecular electrostatic potential mapping is a method for the observation of the interactions of molecules with each other based on their charge distribution on 3D diagrams. This is an auxiliary method that estimates electrophilic and nucleophilic reactive sites of ligands leading to investigate protein binding and medicine developing by defining hydrogen bond interactions. 4,45 Electrostatic po- tential (3D) diagrams of ligands were mapped using the Hartree-Fock theory Slater type orbital-3G base set of Hirshfeld surface analysis by CrystalExplorer software as depicted in Fig. 9. The input file of the geometry was obtained using Tonto. 46,47 Surface qualification values were set -0,025-0,025 au and high resolution was selected.
When it was performed, the molecule was taken to transparency mode and atoms were made clear. The red color represents the negative electrostatic potential regions and acceptor hydrogen bonds. Moreover, the blue color represents the positive electrostatic potential regions and donor hydrogen bonds. 48,49 In addition, blue-colored zones are the preferred regions for the nucleophilic attack, while red-colored negative zones are susceptible to electrophilic attack. 11 Furthermore, red-colored regions have electron-rich atoms or atom groups; therefore, they can easily interact with amino acid residues. Based on the findings of molecular docking studies, hydrogen bond interactions between donor-acceptor confirmed the results of the electrostatic potential analysis as depicted in Fig. 11.

8. Molecular Docking Studies
In silico docking calculations are of great importance in drug design and medical chemistry fields. Molecular docking is commonly used in the studies carried out on target medicine designing by estimating the binding mechanisms of small molecules on target biologic proteins. 50,51 Donor-acceptor binding mechanisms create complexations with hydrophobic hydrogen bond and electrostatic interactions. 52 The current study examined the ligands creating intramolecular hydrogen bonds by targeting the active zones of E. coli DNA GyrB (PDB Code:4WUB) and E. coli FabH (PDB Code:1HNJ) amino acid residues. The protein-related data were obtained from Research Collaboratory for Structural Bioinformatics Protein Data Bank https://www.pdb.org. Molecular docking studies analyzed binding energy, hydrogen bonding, and interactions between the ligands and bacteria. [53][54][55][56] Furthermore, by the calculation of the ligands' lowest binding energy to aminoacids residues, it was determined that which structure has stronger hydrogen bonding and higher binding energy score. 57 Ligands SMILES formats were created at page https://www.cheminfo.org; it is C/C(=N\ O)/C(C)=N/Nc1ncccc1Cl for HL 1 and C/C(=N/Nc1nc-ccc1Cl)c2ccc(N(=O)=O)cc2 for HL 2 .
Molecular docking studies were performed Autodock vina program (https://www.vina.scripps.edu). Ligands were converted into mol2 format and prepared for molecular docking at Chimera software program with receptors (E.coli FabH and GyrB) (https://www.cgl.ucsf.edu/chimera/). 58 In the Dock Prep method, firstly, all nonstandarts and solvents were selected and refined from receptors. Then, protein models were added by the selection of all hydrogen atoms (also considered H-bonds) and Gasteiger charges. Docking studies were conducted by targeting ligand receptors and binding the most convenient coordinates. 59 While defining an approximate donor and acceptor binding zone in in-silico studies, docking parameters are of great importance. Different types of grid box values were applied for the best and most accurate binding. The application of target protein binding zones (as a great scale) in a cubic box resulted in the best binding; however, this method requires a long computer calculation. 60,61 The grid box values of ligands applied to E. coli GyrB receptor were 10, 25, -10 at the center with a grad spacing of 0.375 Å including default the sizes of 40, 40, 40 for HL 1 and HL 2 . The same method was employed to E. coli FabH receptor reporting the values of 30, 15, 30 at the center with the sizes of 30, 30, 30 for HL 1 and HL 2 . In addition, default values were used for other parameters. Root-mean-square deviation were selected as minumum value. The binding energy values of -7.9 and -8.8 kcal/mol were applied by HL 1 and HL 2 ligands to the GyrB receptor, respectively. Fig. 10 illustrates the hydrophobicity surface area of HL 1 inside GyrB and FabH proteins. The binding energy values of -6.1 and -6.8 kcal/mol were applied by HL 1 and HL 2 ligands to the FabH receptor, respectively. The binding interactions and docking poses are depicted in Fig. 11. Since the ligands bind to the GyrB receptor with higher binding energy than that reported for the FabH receptor, there was a better docking to the ligands-GyrB complex. While there were two hydrogen bonds in the complex that ligands made with 4WUB protein, 1HNJ protein had one hydrogen bond. This finding proved that this effect increases the binding energy value. As the binding energy value enhances, the binding score increases indicating a better docking. 41,62 Therefore, the binding energy of HL 2 was higher than that reported for HL 1 which is considered a better docking. The other reason behind that is believed to be the electron density and unpaired electron couples of two oxygen atoms that bond to the nitrogen atom of HL 2 . 63 In a study carried out by Fathima et al. (2018), docking results were observed to be -8.4 and -8.5 kcal/mol for 2AB-P2C-1HNJ and 2ABHB-1HNJ, respectively. 54 The HL 1 created a hydrogen bond to GLY 117 and GLN 335.A HE22 amino acid residues of 4WUB protein with N12 and O13 atoms and binding lengths were observed as 1.992 Å and 1.838 Å, respectively. The HL 2 bonded to GLY 119 and VAL 120 amino acid residues of 4WUB proteins with O19 and O20 atoms and binding lengths were observed as 2.252 Å and 2.404 Å. In another study conducted by Metelytsia et al. (2020), the binding of ligands to the regions of amino acid residues were reported as GLY 119 and His 116. This finding is similar to the results of docking investigations in the current study in terms of attachment regions. 52 The HL 1 and HL 2 created a hydrogen bond to GLY 209, ARG 36.A HH22 amino acid residues of 1HNJ receptor with N8 and O19 atoms and binding lengths were observed as 2.481 Å and 2.171 Å, respectively. Donor and acceptor hydrogen binding interactions are shown as Table 5. 64 The findings of docking studies confirmed that the results and interactions of molecular electrostatic potential generally occur in red regions. The

9 Drug-Likeness and Biological Activity
Drug-likeness and bioactivity of ligands were obtained from molinspiration (https://www.molinspiration. com/cgi-bin/properties). Although the parameters of druglikeness cannot estimate the biological activity of each compound, it is one of the most successful and efficient methods for the analysis of compounds with medicinal potential through the determination of pharmacokinetic characteristics. Based on Table 6, the parameters of drug-likeness and biologic activity for the compounds in the present study (miLogP, TPSA, nAtoms, MW, nON, nOHNH, nviolations, and rotb) and (enzyme inhibitor, protease inhibitor, nuclear receptor ligand, kinase inhibitor, ion channel modulator, and GPRC ligand). The aforementioned findings have been the first obtained data regarding the medicinal potential of the compounds. 66 The values of drug-likeness were determined in this study, and the ligands were examined regarding midicinal potential with the consideration of these values. In this sense, the miLogP parameter, which is the capacity of penetrating the cell membrane, is expected to be under 5. In this regard, the values of miLogP parameter were reported as 1.81 and 3.37 for HL 1 and HL 2 , respectively. The polar surface area (TPSA) represents the hydrogen bonding potential of a compound. Accordingly, the TPSA values were observed as 69.88 and 83.11 A 2 for HL 1 and HL 2 , respectively. These values were below the 160 A 2 limit defined for TPSA and at a good performance. [67][68][69][70] Although miLogP and TPSA are not sufficient criteria for the investigation of druglikeness, they are two important parameters to represent oral absorption in cells. 71 The number of acceptor hydrogen bonds was set to nON ≤ 10, and the number of donor hydrogen bonds was set to nOHNH ≤ 5. In this study, the obtained results were below the aforementioned values. 72 Low molecule weight is important in terms of easy transport, diffusion, and absorption of the molecule. The values of molecular weight are expected to be < 500 Da. In this study, the molecular weights of the ligands were lower than the aforementioned value. The ligands were reported with successful results according to Lipinski's rule of five. Based on Fig. 12, the red column shows Lipinski's rule of five, and the green and red columns depict the druglikeness score of the ligands. 68 If the value of violations equals 0, it shows that crystallized compounds can easily bond to the receptor. This value was reported as 0 for the compounds of the present study. The number of rotatable bonds is a simple topological value and measurement of flexibility. 73 If the bioactivity results of the compounds are > 0, -5.0-0.0, and < -5.0, they are considered active, medium active, and not active, respectively. All the results of biological activity parameters were within the range of -5.0-0.0; therefore, the ligandswere regarded as medium active. 74 As a result, it was concluded that the ligands of the current study obtained satisfactory druglikeness scores and properties to be considered medicine potential agents.