Novel type ketone-substituted metallophthalocyanines: synthesis, spectral, structural, computational and anticancer studies

This work reports on the synthesis and characterization of phthalocyanines (M 1⁄4 Cu(II) (2), Zn(II) (3) In(III) (4) and Co(II) (5)) peripherally tetra-substituted with 1-(4-hydroxyphenyl)propan-1-one. Confirmation of the synthesized phthalocyanine structures are performed with a combination of elemental analysis, FTIR, HNMR, C-NMR, UV-vis and MALDI-MS SEM and spectral data. Their aggregation properties were examined in THF by UV-vis. Spectral and photophysical (fluorescence quantum yield) properties of complexes (2–4) were reported in THF (tetrahydrofuran). These results suggest that the metal in the core of the phthalocyanine plays an important role in the fluorescence quantum yields FF of the synthesized complexes (2–4). Also, the anticancer activities of complexes were studied on MCF-7, MG63, and L929 cell lines. Finally, all synthesized phthalocyanines were investigated by quantum chemical studies. Chemical reactivity parameters such as EHOMO, ELUMO, DE (HOMO–LUMO energy gap) were calculated by Gaussian software.


Introduction
Cancer is a multi-step process in which cells undergo metabolic and behavioral changes leading to excessive and timeless proliferation. Cancer begins with rapid proliferation of cells and loss of apoptosis functions. According to the International Cancer Research Institute (IARC), an average of 14 million people are diagnosed with cancer each year and 8.2 million people die. It is estimated that 12 million people will lose their lives due to cancer in 2030. There are three basic treatment methods for cancer treatment, namely surgery, chemotherapy and radiotherapy. These three treatment methods are applied to cancer patients in combination with each other, depending on the type of cancer, its stage, its location, the general health of the patient, the sex of the patient, and some other factors. 1 Phthalocyanines which are synthetic analogues of porphyrin compounds such as hemoglobin and chlorophyll, have visible optical properties and good thermal stability because of having an 18-p aromatic macrocycle. 2 Since the discovery of phthalocyanines (Pcs), many scientists have aimed to synthesize/design optimal molecules for a variety of applications such as chemical sensors, display devices, catalysts, biological imaging, and photosensitizers for photodynamic therapy (PDT). [3][4][5][6][7] They have been explored to be effective photosensitizers for PDT owing to the strong absorption in the red visible region and high efficiency in producing reactive oxygen species (ROS), e.g. singlet oxygen ( 1 O 2 ), leading to destruction of cancer cells. The variety of metal ion that is inserted to the inner core of phthalocyanines strongly inuences the ROS production. 8 Especially, scientists have aimed to synthesize the phthalocyanines (Pc) that have diamagnetic ions (Zn 2+ , Si 4+ , In 3+ , Ga 3+ , etc.) because of high ROS yields for PDT 9 such as gallium phthalocyanines (GaPcs) 10 and indium phthalocyanines (InPcs). 11 The enhancement of the solubility in organic solvents is very indispensable in the applications of phthalocyanine complexes. 12 For this, it is necessary to attach some functional groups at the peripheral regions of phthalocyanine ring and/or insert some metal ions to the inner core of phthalocyanines [such as Mn(III), In(III) and Ga(III)]. The features like solubility, aggregation, absorption, photophysical and photochemical strongly depend on the functional groupsat the peripheral and/or nonperipheral regions of phthalocyanines. 13,14 George and et al. conjugated Rubus-phthalocyanine apply to MCF-7 cell line for 24 h. Decreased cell viability was observed from 90% to 60%. 15 Horne et al. the novel metallophthalocyanine was apply to MCF-7 cell line. They were found overall morphology averaging a viability of >85%. 16 Ogbodu et al. studied new zinc phthalocyanine-spermine-single walled carbon nanotube conjugate on MCF-7 breast cancer cell line.
When dosed at 40 mM cell viability decreased from 100% to 75%. 17 Aliphatic and aromatic ketones are the compounds having reactive carbonyl group. They have characteristic avors, aromas and therapeutic properties. Many synthetic and naturally formed ketones are used in cosmetics, perfumes, food additives, and are important in a variety of medical and biological materials such as antimicrobial activity. 14,18,19 Pharmaceutical industries spend billions of dollars for the development of effective agents in the diagnosis and treatment of cancer each year. Therefore, many organic based cytotoxic agents have been discovered, and they are extensively applied for treatment of cancer. 20 In this paper, we have rstly submitted the synthesis and characterization of phthalocyanines (M ¼ Cu(II) (2), Zn(II) (3), In(III) (4) and Co(II) (5)) substituted with 1-(4-hydroxyphenyl) propan-1-one at the non-peripheral regions to compare the effect of different metals and the functional group "aromatic ketone" in a variety of medical and biological materials such as new agent for cancer therapeutic. Also, spectral and photophysical (uorescence quantum yield) properties of phthalocyanine complexes (2)(3)(4) were reported in THF (tetrahydrofuran). Additionally, the synthesized phthalocyanines were investigated by quantum chemical studies such as E HOMO , E LUMO , DE (HOMO-LUMO energy gap). Finally, the synthesized phthalocyanines were evaluated for anticancer activity on MCF-7, MG63, and L929 cancer cell lines.

Synthesis
2.2.1. The synthesis of 3-(4-propionylphenoxy)phthalonitrile (1). Under N 2 atmosphere, 1-(4-hydroxyphenyl)propan-1one (0.95 g, 6.35 mmol) were dissolved in dry DMF (20 mL) and then 3-nitrophthalonitrile (1 g, 5.78 mmol) was put to the solution. Aer 10 min, anhydrous K 2 CO 3 (5.36 g, 38.88 mmol) was put by stirring efficiently for 2 h. The reaction mixture was stirred under N 2 at 45 C for 3 days. Then the solution was poured into ice-water (200 mL). The obtained product was ltered off, rstly cleaned with water and then with diethyl ether and nally puried by chromatography over a silica gel column by using a eluent that is a mixture of CH 2 Cl 2 /ethanol.
The product was soluble in CHCl 3 , CH 2 Cl 2 , THF, DMF and DMSO. Yield of (  [5.4.0]undec-7ene) in n-hexanol (2 mL) was heated at 150 C in a sealed glass tube for 8 hours. Aer cooling to room temperature, the product was cooled and precipitated by adding hexane and ltered. Aer being cleaned with methanol and then with diethylether several times. Finally, the product puried by chromatography over a silica gel column by chloroform-methanol solvent system. Finally and dried and vacuo.

Cell lines
Human breast adenocarcinoma (MCF-7), human bone osteosarcoma (MG63), and mouse broblast (L929) cell lines were supplied by the American Type Culture Collection (ATCC). Cell culture studies of human breast adenocarcinoma cell line (MCF-7), human bone osteosarcoma (MG63), and mouse broblast (L929) appropriate conditions and materials required in order to reproduce broth must be provided. In our study 10% fetal bovine serum (FBS), 1% L-glutamine, 100 IU per mL penicillin and 10 mg mL À1 streptomycin in DMEM (low glucose) were used. Cancer cell lines in DMEM medium was produced in 95% humidity and 5% CO 2 incubator at 37 C.

Cytotoxic assay of the synthesized compounds
XTT test which was monitor azo aldehyde thioureas derivatives toxicity was applied on cancer cells. Firstly, the cancer cells were plated in 96-well plate (10 Â 10 4 cells in each well) and the compounds were tested by 4-fold diluting the starting mixture at four different concentration ranging from (12.5; 50; 75; 100 mM) 24 and 48 h. Secondly, for determination of living cells 50 mL XTT reagents were added to each well. Aer 4 h, at 450 nm micro plate reader was used for absorbance measurements. Finally, the percentage of cell viability was calculated.
Anticancer activity of 6 azo chromophore containing new compounds were evaluated in breast cancer cell line and broblast cell line was used as control. Cells were exposed to four (12.5; 25; 50; 100 mM) concentration range. XTT protocol was applied aer 24 and 48 h and compared with control cell lines. The cell viability of control has been accepted as 100% in order to determined cell viability of compounds.

Statistical analysis
Data were expressed in the form of arithmetic mean AE standard deviation (x AE SD). One way ANOVA and post hoc Tukey analyses were used to reveal the relationships between groups. The differences were accepted as signicant for p < 0.05. Statistical analysis were performed with SPSS for windows 22.0 package. All determinations were computed three times.
2.6. Computational methodology 2.6.1. Quantum chemical calculation method. Density functional theory is absolutely most widely used methodology for the prediction of chemical reactivity of molecules. Input les of complexes were created using Gauss view version 5.0.8. 21 All the entry les of calculations were made by Gauss 09 Revision C.01 in TÜBİTAK-TR Grid. 22 All calculations were fully optimized by using Density Functional Theory (DFT)/B3LYP 23 and Hartree-Fock (HF) 24 method. In calculations, Gen keyword was used LANL2DZ for metal atom and SDD, 6-311G and 3-21G basis set in gas phase for rest atoms were selected as basis sets in vacuum.
With the help of Density Functional Theory, chemical reactivity indices such as chemical hardness (h), electronegativity (c), chemical potential (m) are dened as derivatives of the electronic energy (E) and a constant external potential n(r). [25][26][27][28][29][30][31] In the conceptual this theory, mentioned chemical properties are given as 32,33 In the conceptual Koopman's theorem, 34 the negative of the highest occupied molecular orbital energy and negative of the lowest unoccupied molecular orbital energy corresponds to electron affinity and ionization energy (ÀE LUMO ¼ A and ÀE HOMO ¼ I). As a result of Koopman's theorem, chemical potential and chemical hardness can be explained as: 35 According to Pearson, the global soness is dened as the inverse of the global hardness and this quantity is given as: 36,37 Parr et al. by which the global electrophilicity index (u) introduced are dened via eqn (6). With the help of this index, electrophilic power of a chemical compound is associated with its electronegativity and chemical hardness. Nucleophilicity (3) which is the inverse of the electrophilicity, is given via eqn (7).
3. Results and discussion

Aggregation studies
The aggregation of phthalocyanines affects their application properties such as photophysical, electrochemical and spectroscopic properties. So, the investigation of aggregation properties of phthalocyanine are important. Aggregation is dependent on the nature of the solvent, concentration, nature of substituents, complex metal ions and temperature. 38 In this study, the aggregation behaviors of (2-5) were examined in THF (Fig. 4). All synthesized phthalocyanines did not show any aggregation behaviors in THF. When the concentrations of the Pc increased, the intensity of absorption of the Q-band also increased (the Lambert-Beer law was obeyed), and new blue or red shied band formation were not observed.

Fluorescence measurements
Fluorescence quantum yields were calculated using eqn (8) where The uorescence behaviors of (2-4) were studied in THF. The absorption, uorescence emission and excitation spectra for (2)(3)(4) were shown in Fig. 5. Fluorescence emission peaks were observed at 705 nm for (2), 704 nm for (3) and 721 nm for (4) in THF, respectively. The Q bands of the non-peripheral substituted phthalocyanines' luminescent spectra are bathochromic shied when compared to the corresponding peripheral substituted phthalocyanines. The red shis observed in emission maxima are 40-60 nm in comparison with peripheral substituted phthalocyanines. 40 Fluorescence excitation peaks were observed at 694 nm for (2), 687 nm for (3) and 698 nm for (4) in THF, respectively. Absorption spectra of (2-4) indicated characteristic one absorption band at Q band region that was at 687 nm for (2), 689 nm for (3) and 702 nm for (4) in THF, respectively. Absorption and uorescence excitation spectra of (2-4) are very similar and mirror images of their emission spectrum in the solvent of THF. The observed Stokes shis for (2)(3)(4) were within the region 15-19 nm in THF which was typical for phthalocyanines. 41,42 Absorption, uorescence excitation and emission peaks for (2)(3)(4) in THF were listed in Table 1.
Considering different metal effects on F F values, the highest F F value (0.115) was obtained for the complex (3) containing closed shell diamagnetic ions, such as Zn 2+ which is important for PDT. The metal of Zn 2+ have been proved as highly promising photosensitizers for PDT (photodynamic therapy) due to their intense absorption in the red region of the visible light. 43,44 Fig. 1 The FT-IR spectra of the synthesized phthalocyanines (2)(3)(4)(5).

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The other obtained F F values were quite low (0.006) for (2) and (0.045) for (4) due to the heavy atom effect. 45 The F F values of the studied phthalocyanine compounds (2-4) were lower than those of unsubstituted zinc(II) phthalocyanine (ZnPc) in THF. The decrease in the F F values is because of the substituents. This implies that the presence of non-peripheral 1-(4-hydroxyphenyl) propan-1-one substituents caused some uorescence quenching of the parent (2-4).

Anticancer activity
In this work, anticancer activities of compounds of phthalocyanine were studied on MCF-7, MG63, and L929 cell lines. Anticancer activity of the synthesized compounds were investigated and results are presented in Fig. 6 Table 2). The compounds were applied to three different types of cell lines on a 24 h incubation. Complex (5) acted well on MCF-7 and MG63 cell lines. It is not suitable for application to cancer cell lines due to its complex toxicity.
Complex (5) showed anticancer effect on MCF-7 and MG63 cell lines. The complex (4) is not suitable for application to cancer cell lines from the toxic nature of the compound on L929 cell lines. Complex (3) both MCF-7 and MG63 cell lines were anticancer effective; the compound is most likely to have more of an anticancer effect on the MCF-7 cell lines. Complex (2) does not have anticancer effect for MG63 cell lines, whereas MCF-7 cell lines has anticancer effect.

Computational results
3.5.1. Quantum chemical calculations. All synthesized phthalocyanines were investigated by quantum chemical studies. The obtained result in this study are given in detail below by Gaussian So Ware. Chemical reactivity parameter such as E HOMO , E LUMO , DE (HOMO-LUMO energy gap), chemical hardness, soness, electronegativity, proton affinity, electrophilicity and nucleophilicity are important and useful tools to compare activity of molecules. The molecular reactivity of the studied molecules in this paper was investigated and compared with to analysis of Frontier molecular orbitals. The energy of HOMO is associated with the electron donating ability of molecule. High energy value of HOMO state show prone to donate electrons to appropriate acceptor molecules having low energy and empty molecular orbital. In the light of this information, HOMO energy level is an indicator of electron donating abilities of molecules. Chemical hardness is dened the resistance against electron cloud polarization or deformation of chemical species. 46 Hard and So Acid-Basis (HSAB) 47,48 and Maximum Hardness Principles (MHP) 49 based on chemical hardness concept that are very benecial in terms of estimating This journal is © The Royal Society of Chemistry 2017 the directions of chemical reactions. As can be understood from this denition chemical hardness, soness and DE that are quantum chemical parameters in theoretical chemistry, are associated with each other. 50 As a result of Koopman's theorem, both soness and hardness are given based on HOMO and LUMO orbital energies. Hard molecules that have high HOMO-LUMO energy gap, cannot act as good active molecule. On the other hand, so molecules that have low HOMO-LUMO energy gap, are good active molecule. 51,52 For the reason that they can very easy give electron to metals. As a result of calculated HOMO and LUMO energy levels given in Tables 3 and 4 for studied compounds. Furthermore, the order of increasing reactivity is: 5    In quantum chemical studies, the electrophilicity index (u) is a very important parameter that indicates the tendency of the active molecule to accept the electrons. As it is well known that this quantity is constantly used in the analysis of chemical reactivity of molecules. Nucleophilicity (3) is a physically the inverse of electrophilicity (1/u). For the reason that is should be stated that a molecule have large electrophilicity value that is ineffective of activity of molecule although a molecule that have large nucleophilicity value is a good activity of molecule. As it is well known that molecular electrophilicity are based on both molecular electronegativity and molecular hardness.
The Gibbs free energy (DG) was calculated using below equation: where E M-L complex is the energy of the metal complex, E ligand is the energy of the ligand and E M 2+ is energy of the metal ion. 53 As it is well known three possibilities for Gibbs free energy.
First, if DG is negative for a process then the process is spontaneous and it will occur in the forward direction. Second, if DG is zero for a process then the process is in equilibrium state. The reaction will not take place. Third, if DG is positive for a process then the reaction doesn't proceed in the forward direction. It may occur in the reverse direction. We see in Table 5. All of metal-complexes is calculated the Gibbs free energy (DG) in the Table 5. The reaction with the negative high Gibbs free energy value is easier to achieve than the others. The order in which the reactions are spontaneous is as follows: It is clear known that the gure of molecular electrostatic potential (ESP) of six molecules givens an indication of the total charge distribution (electron + nuclei) of the molecule and correlates with dipole moments, electronegativity, partial charges and chemical reactivity of six molecules. It provides that a visual method to understand the relative polarity of the molecules. An electron density isosurface of six molecules mapped with electrostatic potential surface the size, shape, charge density and site of chemical reactivity of molecules. 30 The different value of the electrostatic potential represented by different colors: red represents the region of the most negative electrostatic potential, blue represent the regions of the most positive electrostatic potential and green represents the region of zero potential. The potential increases in the order red < orange < yellow < green < blue. From the light of the result given in the mapped have been plotted for title molecules in 6-311++G** basis set using the computer soware gauss view (Fig. 9).

Conclusion
In this paper, we have synthesized and characterized the phthalocyanines (M ¼ Cu(II) (2), Zn(II) (3), In(III) (4) and Co(II) (5)) which is nonperipherally tetra-substituted with 1-(4-s hydroxyphenyl)propan-1-one. In THF, the uorescence quantum yields F F for complexes (2)(3)(4) were found to be lower than unsubstituted zinc(II) phthalocyanine (ZnPc) in THF. This implies that the presence of non-peripheral 1-(4-hydroxyphenyl) propan-1-one substituents caused some uorescence quenching of the parent (2)(3)(4). The most suitable compound for the MCF-7 cell line from the four compounds is the complex (3); the anticancer effect of the complex (5) in the MG63 cell line is good. The synthesized azo chromophore containing compounds might be potentially useful in the eld of cancer treatment. Major problem that is chemotherapeutic agents are side-effect, which kill both cancerous and normal cells since they are not selective. So there is new agent for cancer therapeutic. Our studied showed anticancer effect on MCF-7 and MG63 cell line. Complex (3) both MCF-7 and MG63 cell lines were anticancer effective. Complex (3) was applied to MCF-7 cell line and cell viability decreased from 100% to 60%.

Conflicts of interest
There are no conicts to declare.