Formulations of synthesized phthalocyanines and starting material are shown in Fig.1. 2-Nitrophenol, 4-nitrophenol, and piperonyl alcohol as the stoichiometric was reacted under nitrogen in the dimethylformamide to form 4-(2-(benzo[d] [1,3] dioxol-5-ylmethoxy) phenoxy) phthalonitrile. Zinc and cobalt phthalocyanines were then synthesized from this starting material. The reaction of zinc chloride and cobalt chloride salts with 4-(2-(benzo[d] [1,3] dioxol-5-ylmethoxy) phenoxy) phthalonitrile at 210 ˚C yielded zinc and cobalt phthalocyanines.
Spectral measurements of the compounds support the expected structure. Vibration of aromatic C - H peak in the IR spectrum of compound number 4 is 3086 cm-1, CH2 vibration 2926 cm-1, C≡N vibration 2233 cm-1, Ar - O - Ar vibration 1249 cm-1, C = C vibration peaks were observed at 1564 and 1500 cm-1. The vibration peaks observed here are compatible with the expected structure. The most prominent change is observed in nitrile peaks after the conversion of this compound to zinc and cobalt phthalocyanine compounds. The nitrile peak is expected to disappear completely. The nitrile peak observed in this study is also not seen in phthalocyanine compounds. Vibration peaks for phthalocyanine 5 were observed as aromatic C - H at 3084 cm-1, CH2 at 2926 cm-1, C = C at 1598 cm-1, and Ar-O-Ar at 1234 cm-1. Finally, the vibrations of phthalocyanine 6 were observed at aromatic CH peak 3018 cm-1, CH2 2970 cm-1, C = C 1598 cm-1, Ar - O - Ar peak at 1238 cm-1 peaks appear to be in good harmony.
As expected in the 1H NMR spectrum of compound 4 in DMSO-d6, aromatic protons were observed as multiplets in the 8.18-6.93 ppm range. CH2 protons located between oxygen and oxygen among the aliphatic protons were observed at 6.01 ppm, while the CH2 peak bound to single oxygen was observed at 5.14 ppm. The 13C NMR spectrum of the compound in DMSO-d6 also gives the peaks of the expected aromatic and aliphatic carbon atoms of the compound. Here, the carbon peaks of nitrile were observed at 116.68-116.15 ppm, the aromatic carbon peaks at 162.09-106.50 ppm, the carbon peak of the CH2 atom between oxygen and oxygen at 101.59 ppm, and the carbon peak of single oxygen-bonded CH2 at 70.92 ppm. These observed peaks are in good agreement with the structure of the compound. 1H NMR and 13C NMR spectra of this compound are given in Fig. 2-3. For zinc phthalocyanine compound 5, 1H NMR protons in DMSO-d6 are observed as multiplets between 7.24-6.07 ppm, while aliphatic CH2 protons are observed at 6.00 ppm and 5.54 ppm. Due to its paramagnetic nature, 1H NMR measurements of compound 6 were not made.
Mass spectra of compounds (4-6) confirmed the proposed structure with the molecular ion being identified at 393.08 [M+Na]+, 1545.31 [M+H]+, and 1540.31 [M+H]+, respectively.
Another spectral device related to the structure of phthalocyanines is UV spectroscopy. It gives absorbance value for Q and B bands which are characteristic for phthalocyanines . It also indicates that the phthalocyanine compound does not bind the metal bond . For the phthalocyanines 5 and 6, the Q band absorption values in the THF solvent were measured as 676 and 668 nm respectively. The B band of compound 5 was found to be 348 nm. These values are consistent with the tetra peripheral substitute structure. The absorption values of these compounds measured at different concentrations in THF are given in Fig. 4 and 5. As can be seen from these diagrams, these phthalocyanine compounds exist as monomers in the measured concentration range. This shows that it may be useful for some areas that wish to be non-aggregated (such as photodynamic therapy).
One of the foremost applications of phthalocyanine compounds is their use as a sensor agent in photodynamic therapy. For this application, the compound should exhibit fluorescence. The data obtained from fluorescence absorption, excitation, and emission spectra reveal fluorescence properties. Fluorescence absorption, excitation, and emission spectra of the zinc phthalocyanine compound observed is shown in Fig. 6. Stokes shifts are within the expected range for the phthalocyanine compound. The Stokes shift detected in tetrahydrofuran for this phthalocyanine is 10 nm. The fluorescence emission was measured at 694 nm, while the excitation value was 682. The quantum yield of this compound (ΦF) was calculated as 0.30. This value is higher than the unsubstituted zinc phthalocyanine value . These spectral values are consistent with the studies given in the literature [20, 21]. These data strengthen the possibility that the compound can be used as a sensor.
In addition to the fluorescence properties and fluorescence quantum efficiencies of the photosensors considered for photodynamic therapy, they can show an effective photosensor effect with their ability to produce singlet oxygen. In this study, a chemical method was used for the singlet oxygen production test to determine the photosensory ability of the zinc phthalocyanine compound. The singlet oxygen quantum yield (ΦΔ) of phthalocyanines 5 was determined in DMSO by using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen quencher. During the measurements, the Q bands of phthalocyanine compound 5 did not change, while a decrease in DPBF absorbance was observed due to the reaction of singlet oxygen with DPBF (at 417 nm). ( Fig.7). The ΦΔ value of compound 5 (ΦΔ = 0.57) was found to be lower than Std-ZnPc . Here, one of the factors that can affect singlet oxygen production is substituted (2- (benzo [d] [1,3] dioxol-5-ylmethoxy) phenoxy) groups. The other is the electronic structure of the selected metal atom.
Quantum chemical calculation instructions
DFT calculations   used in phthalocyanine compounds were carried out using the method coded 6-31G and LanL2DZ base set of this method in the Gaussian 09 program . Within the scope of this study, optimization of metallo phthalocyanines in the gas phase and ground state was performed. Molecular orbital energies were calculated especially in phthalocyanine minimum energy optimization. The log and chk extension files obtained from the program were visualized with the program named Gauss View 6 and numerical data were transferred to tables.
Structure Details and Analysis
The molecular structure optimized by the two methods of the zinc phthalocyanine compound is shown in Fig. 8, together with the bond length values. The optimized structural parameters calculated with the DFT/ B3LYP 6-31G – LanL2DZ basis set are determined. Here the planar structure of the nucleus of the phthalocyanine compound is obtained. The bond lengths and angles between the optimized molecular atoms were compared with each other using the two basis sets for MPc’s, given in Table 1. When comparing the two methods, the bond lengths of the M - N atoms in phthalocyanine nuclei were calculated as 2.00 Å in Zn-N24 / B3LYP / 6-31G and 1.95 Å in Co-N24 / B3LYP / LanL2DZ, respectively. In the second method, bond lengths were shorter. The small differences between them are due to the sensitivity of the methods and the metal atoms' radii. In the optimization, the phthalocyanine nucleus has a planar structure and the metal atom is positioned at an angle of about 90. Dihedral angles of Zn-Pc were calculated as N18-Zn41-N24-C23 179.5°, and N5-Zn41-N24-C20 -178.43° with 6-31G. Dihedral angles of Co-Pc were calculated as N5-Co169-N24-C20 0.98° and N18-Co169-N24-C23 179.73° with LanL2DZ basis sets. Both DFT methods with the basis sets demonstrate that the phthalocyanine compound is planar in accordance with the dihedral angles of about 180°.
HOMO and LUMO analysis
Quantum chemical calculations are widely used to calculate basic electronic parameters related to orbitals in the molecule. As frontier orbital molecules, the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) are important. However, by using these values, the activity parameters and energy deficit of the molecule are calculated and the chemical character of the molecule is determined. Perturbation MO theory is based on the energy difference between HOMO and LUMO[28-30]. The closer the energy levels of the interacting MO's to each other, the stronger the interaction. That is, the smaller the HOMO - LUMO energy difference, the stronger the interaction of the reactants and the easier the reaction.
The calculated results and their comparison with the two methods are presented in Table 2. Fig. 9-10 is the density orbital representation of the HOMO and LUMO of the Zn-Pc and Co-Pc compounds in both methods. LUMO+1 and HOMO-1 graphs of the compound were also taken. Frontiers orbitals of Zn-Pc, HOMO -4.78 eV LUMO -2.60 eV value at DFT / B3LYP / 6-31G level of the molecule and for Co-Pc, HOMO -4.96 eV LUMO -2.77 eV values at DFT / B3LYP / LanL2DZ level were calculated. The HOMO and LUMO, frontiers orbitals also help determine the degree of activity and interaction of the molecule with other species [32-34]. HOMO, LUMO, and the chemical reactivity descriptors calculated in the two methods were correlated and shown in Fig. 11. This is due to the calculation accuracy of these two methods from different parameters.
Nonlinear Optics (NLO) analysis
The dipole moment, which is the specialty of energy, is applied intramolecular[35-37]. The dipole moment consists of Van der Waals-type intermolecular interactions and intermolecular attraction is strong. The electronic dipole moment and total dipole moment are listed in Table 3.
The dipole moment, molecular polarization and hyperpolarization values should be calculated to determine Nonlinear Optical (NLO) properties[29, 38-40]. In the Zn-Pc / 6-31G method / B3LYP basis set, the parameters are respectively μ = 8.0 D, α = 593.6 au, β = 5.03x10-31 esu. Also, in the Co-Pc / LanL2DZ basic set, the parameters are respectively μ = 8.4 D, α = 593.5 au, β = 5.08x10-31 esu. According to these values, the molecule has parametric quantities that can be considered as NLO materials.
Molecular electrostatic potential (MEP)
MEP maps provide information about the electronic charge distribution of a molecule. The density of the electron distribution on the molecule is useful for illuminating bonds with descriptors such as polarity, electronegativity. The electronic structure and molecular reactivity of complex molecules can exhibit rich topographic properties [28, 38, 40-42].
In this study, electrophilic potential (MEP) maps of three phthalocyanine molecules were obtained. As shown in Fig. 7 they are visualized with MEP maps at the DFT / B3LYP method with 6-31G and LanL2DZ basis set using the GaussView 6.0 software. The MEP maps show that the region characterized by the blue color around the Zn and Co atoms have positive values. The red regions on the map indicate the region rich in electrons. The aromatic ring region shows an almost neutral potential, most of which is represented by a yellow-green color. Contour maps of phthalocyanines confirm negative and positive potential parameters in accordance with the electrostatic potential map (ESP). The phthalocyanine nucleus in the structures shows the delocalized structure and high stabilization with green-yellow colors in color. Moreover, in the MEP maps, the negative potentials are mainly found in, for example, O-107 and O-118, while the potential occurring around the H-147 atom is the most positive. In terms of electronegativity, oxygen atoms can be interpreted higher than other atoms.
Molecular docking analysis
The protein crystal structure of different enzymes was selected for the phthalonitrile compound in the Protein Data Bank (http://www.rcsb.org). From these complexes, PDB codes were selected and retrieved from the Protein Data Bank. As protein receptors, human carbonic anhydrase isozyme II (PDB ID: 6R6F), acetylcholinesterase (PDB ID: 4RVK), and butyrylcholinesterase (PDB ID: 6SAM) enzymes were used. The Docking studies were performed using the commercial software Schrodinger suite version 10.2 Maestro..
Ligand preparation and protein preparation via LigPrep, and Protein preparation modules were used in accordance with the previous studies[44-46]. In addition, enzyme inhibition in-silico study of phthalonitrile compound was conducted in this study. Enzyme binding affinity was found as -5.24 and 7.67 kcal and gave well results. The best docking poses were selected for analysis of interactions, and protein-ligand interaction was presented with Discovery Studio Client 2017 software.
Computational ADME modeling is a very mature but still-developing field. In silico ADME tools are routinely applied to drug design . The synthesized phthalonitrile molecule also complied with the drug similarity rules and displayed acceptable predicted ADME properties. In the analysis made on SwissADME: Drug similarity was evaluated a free web tool to evaluate. In this analysis, it was found to be compatible compared to the Lipinski (Pfizer) filter. The online servers SwissADME (http://www.swissadme.ch/index.php) were employed to check the chemo-informatics and biological properties of this ligand molecule.
The results in Table 4 show that the compounds are compatible with MW 370.36 g / mol (<500), LogP values according to the Lipinski rule with 1.9 (<5), and HBA 6 (<10). Topological PSA 84.50 <140 A2, and ABS is 79.85%.
With (6SAM BChE) phthalonitrile the compound AChE had the most effective coupling score (-7.69) and good binding affinity was obtained (Fig. 12). Here, when the ligand interactions with protein residues are evaluated, the strongest bindings were line up as TRP82 3.09 Å conventional hydrogen bond, TRP430 3.05 Å conventional hydrogen bond, GLY116 4.03 Å Amide-pi Stacked. TRP82-4.30 Å residues and nitrile groups on the ligand were observed to frame the H-bond side chain.
In docking of the compound with another enzyme (6R6F AC II), the glide score was calculated to be -6.219. TRP5 2.11 Å traditional hydrogen bond, HIS4 2.42 Å conventional hydrogen bond, GLN92 2.59 Å traditional hydrogen bond, HIS64 4.83 Å pi-pi T-shaped, HIS94 4.86 Å pi-pi T-shaped, ALA65 4.89 Å pi- alkyl, LEU198 4.91 Å pi-alkyl, VAL121 4.54 Å formed pi-alkyl bonds.
Finally, in another docking with receptor 4RVK AChE, a docking score of -5.24 was obtained. This docking complex, respectively ASP148 exhibited interactions with 4.84 Å pi-anion, GLU91 4.47 Å pi-anion, VAL23 4.30 Å pi-alkyl, LEU137 5.26 Å and 5.37 Å pi-alkyl, LEU15 4.05 Å pi-alkyl, and TYR86 3.08 Å conventional hydrogen bonds (Fig. 13). Here, it was determined that the phthalonitrile compound on the three enzyme proteins exhibited good performance in enzyme inhibition. The ligand-receptor binding score in the docking study also correlated well with the published data[49, 50].