Investigation of Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) Porphyrazine for Application as Photosensitizer in Photodynamic Therapy of Cancer

The phthalocyanine analogue containing nonperipheral long alkyl-substituted benzenoid rings and pyridine rings, zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine, was synthesized. Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine reacted with dimethyl sulfate and monochloroacetic acid to produce their quaternized products and diethyl sulfate to produce the sulfo-substituted products. All quaternized and sulfo-substituted showed amphiphilic character. Identical peaks in cyclic voltammograms appeared for these products before and after quaternization. During the evaluation of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine for its photodynamic therapy of cancer (PDT) efficacy by cancer cell culture, the light exposed dimethyl sulfate quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines in IU-002 cells produce cell disruption that can be detected as a decrease in fluorescence.


INTRODUCTION
Phthalocyanine derivatives have attracted attention as functional chromophores for applications, especially organic charge carriers in photocopiers, as laser light absorbers in data storage systems, as photoconductors in photovoltaic cells, and in electrochromic displays [1][2][3]. Moreover, phthalocyanine derivatives can be utilized as sensitizers in photodynamic therapy of cancer (PDT).
Sensitizers for PDT require high photostability, high selectivity to tumors, no dark cytotoxity, strong absorption in the region between 600 and 800 nm where penetration of tissue is good, a long triplet lifetime, and satisfactory photosensitization of singlet oxygen. Phthalocyanine derivatives are known to satisfy the aforementioned conditions [3][4][5][6][7][8].
We previously synthesized the nonperipherally substituted phthalocyanine derivatives, zinc alkylbenzopyridopor-phyrazines, which possessed didecylbenzenoid and pyridinoid moieties in the molecule and described regio isomer separation of one of the alkylbenzopyridoporphyrazines [9]. We reported a fundamental study on PDT by measuring for the triplet state lifetime of the alkylbenzopyridoporphyrazins and regio isomers [10,11]. As alkylbenzopyridoporphyrazine exhibited solubility in organic solvents and was expected to have a higher tumor affinity, quaternation of the pyridine nitrogen in the alkylbenzopyridoporphyrazine was done to give solubility in anaqueous media, and to have bioavailability and in vivo distribution [12]. Then, Nyokong et al. reported that phthalocyanine analogues, tetra-2, 3-pyridoporphyrazine and its quaternized compounds have excellent properties compared to zinc phthalocyanine-type photosensitizer [13]. The amphiphilic phthalocyanine derivatives were concluded the best compound for a new generation of photosensitizers for PDT   [12]. In our previous papers [9][10][11][12], the reported zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido) porphyrazine and its regio isomers were prepared by 1 : 1 mixture of 3,6didecylphthalonitrile and 3,4-pyridine dicarbonitrile.
The synthesized zinc bis(1,4-didecylbenzo)-bis(2,3pyrido) porphyrazine was anticipated to be a mixture of products, with different numbers of pyridine rings in the molecule. However, the target compound comprised only the proposed constituent as confirmed by thin layer chromatography (TLC). As the target compound had been purified by TLC using benzene as eluent, only one blue-colored constituent was obtained. It is thought that the desired compound was obtained in accordance with the mole ratio of the raw materials used. The same phenomenon has been observed in the case of synthesis of zinc bis(1,4-didecylbenzo)-bis(3,4-pyrido) porphyrazine [9][10][11][12].
The respective products obtained were greenish-bluecolored powders of which the yields were 24, 21, and 25% for MCAA, DES, and DMS, respectively (see Figure 3). Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine was dissolved in toluene, chloroform, pyridine, and methanol but not in water. After reacting with quaternizing agents, the products were also soluble in water.  In the cases of MCAA and DMS, analysis revealed that the structures of the products were in good agreement with those having N-CH 2 COOH and N-CH 3 groups, respectively. Whereas when DES was used as a quaternizing agent, no N-CH 2 CH 3 singlet peak was present in the 1 H-NMR spectrum, S=O stretching in the IR spectrum was observed. Therefore, sulfonation but not quaternization was achieved [12,15].
After reaction with the quaternizing agents, all compounds possessed amphiphilic properties.
The quarternized derivatives of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines showed strongest absorption at 676, 687, and 687 nm in water after reaction with DMS, DES, and MCAA, respectively (Table 1); these Q bands were bathochromic compared to the nonquaternized parent compound. As the UV-Vis spectra of the quaternized compounds in water showed very broad peaks, the amphiphilic compounds had excellent molecular association tendency.
Zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine and its quaternized compounds fluoresced on exposure to ultraviolet light. Although fluorescence spectra generally were known to be mirror images of UV-Vis spectra at the longer wavelengths, the Q bands nearly overlapped with the wavelengths at which fluorescence occurs in the case of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine and its quaternized compounds, thus, the differences between λ max of UV-Vis and the F max of fluorescence spectra, called the Storkes shift, were very small between 10 to 20 nm. These observations are similar to that seen with the phthalocyanines zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine and its quaternized derivatives. These compounds are molecules with high planarity which cannot change their configuration after quaternization.
The important parameters of a cyclic voltammetry (CV) are the reduction (E pc ) and oxidation (E pa ) potential, the difference between reduction and oxidation potentials, (ΔE p ), and formal reduction potential (E • ) of the observed waves.
The potential difference in CVs between the reduction and oxidation correspond to the HOMO-LUMO energy gaps of the compound [16]. Just as chemical reactions occur during the electron transfer between HOMO and LUMO energy levels, photochemical reactions are also based on similar phenomena of energy transfer. Before and after the quaternization, the HOMO-LUMO energy gap of zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazine was unchanged. The shapes of CVs clearly showed that quaternized zinc bis(1,4-didecylbenzo)-bis(2,3-pyrido) porphyrazines had increased electron responsibility.
Cell rupture can be detected. Intact cells selectively concentrated fluorescence. After exposure to halogen light for 10 minutes showed damage and loss of fluorescence although fluorescence in cells occurred in perinuclear area (see Figure 4).

Equipment
IR spectra were recorded on a Shimadzu FT-IR 8100A spectrometer using potassium bromide (KBr) pellets. UV-Vis spectra were measured on a Shimadzu UV-2400PC spectrometer; each sample was prepared at 5.0 × 10 −5 mol dm −3 in pyridine, toluene, and water. Fluorescence spectra were recorded in toluene, pyridine, and water using either a Hitachi F-4500 fluorescence spectrometer or a Jasco (Nihon Bunko) FP-6600 spectrofluorometer. 1 H-NMR spectra were measured at 400 MHz on a Bruker Avance 400S in benzened 6 (C 6 H 6 -d 6 ) or chloroform-d (CHCl 3 -d) using tetramethylsilane (TMS) as an internal standard. Elemental analyses were carried out using a Perkin-Elmer 2400CHN instrument. Samples for elemental analysis were purified by repeated sublimation; the instrument was calibrated with copper phthalocyanine. CVs were recorded on a BAS CV-50 W voltammetric analyzer at room temperature in 1 × 10 −3 mol dm −3 acetonitrile solution containing a 0.01 mol dm −3 tetrabutylammonium perchlorate (TBAP). CVs were recorded by scanning the potential at a rate of 50 mV s −1 . The working and counter electrodes were platinum wires and the reference electrode was a silver/silver chloride-(Ag/AgCl) saturated sodium chloride electrode. The area of the working electrode was 2.0 × 10 −2 cm 2 .

Materials
TLC was performed using Merck 60 F 254 silica gel on aluminium sheets. Merck Silica gel 60, particle size 0.063-0.200 nm 7734 grade was used in chromatographic separations.

Cell culture
IU-002 cells were maintained in MEM medium supplemented 5% fetal calf serum.
Cells seeded into 96-well tissue culture plates and incubated to allow attachment to the plates. The sensitizer was added to the medium at concentration ranging from 0 to 2mgcm 3 . Cells were incubated for 3 hours. The medium was removed, the cells were washed with phosphate-buffered saline (PBS), and fresh medium was added. Cells were exposed halogen light for 10 minutes. Appearance of cells was observed used a fluorescence microscope.

ACKNOWLEDGMENTS
The assistance of Miss T. Komoriya in the taking of data for part of cell culture is greatly appreciated. The authors would like to thank Professor Kohono for his helpful advice given to them regarding their paper. This research work wassupported financially by Advanced Research Center for Life Science and Human Environment, Graduate School of Industrial Technology, Nihon University (Narashino-shi, Japan), which was adopted by a project for the promotion of high technology within The Ministry of Education and Science, Japan's Academic Frontier Project.