Study on the Synthesis and in vitro Photodynamic Anti-cancer Activity of Tetra(trifluoroethoxy)Germanium Phthalocyanine①

WU Li-Rong HUANG Li-Ying LIN Zhen-Hua (College of Pharmay, Fujian Medil University, Fuzhou 350004, China) (Quanzhou Medil College, Quanzhou 362010, China) (2nd Affiliated Hospital, Fujian Medil University, Quanzhou 362000, China)

1 INTRODUCTION

Photodynamic therapy (PDT) has become one of the major therapies of cancers in developed countries along with the development of laser technique and optic fiber transmission, and is used widely in the treatment and prevention of cancers of head and neck, esophageal cancer, pancreatic cancer, skin cancer, lung cancer, prostatic cancer, brain tumors,cervical cancer, etc.[1]. The process of PDT is as follows[2]: Firstly, a photosensitizer is injected into veins, thus this photosensitizer would be enriched in cancer tissue, while it would be eliminated basically from the normal tissue after a certain time. Then, an optic fiber is introduced into the cancer tissue under the guidance of endoscope for irradiating the tumor using the laser of specific wavelength to activate the photosensitizer in the tumor so as to produce substances of anticancer activity and cause a series of photochemical reactions to kill the cancer cells.The therapeutic effect of PDT depends on 3 factors:photosensitizer, concentration of oxygen in tissues,and laser source[3]. An important use of phthalocyanine in the modern high-tech field lies in that it is used as photosensitizer of PDT for diagnosing and treating cancers. At present, several phthalocyanine complexes have been used tentatively in the clinical practice, for example, Russian Photosense (a sulfonated aluminum phthalocyanine photosensitizer),American Pc4 (a silicon phthalocyanine with axial quaternary amine group), and Swedish liposomewrapped zinc phthalocyanine[4].

A new phthalocyanine complex - tetra(trifluoroethoxy) germanium phthalocyanine (GePcF for short)was synthesized, with its structure shown in Fig. 1.The photodynamic anticancer activity of GePcF was researched in vitro. Based on the following three considerations, we used trifluoroethoxy group(CF3CH2O-) as circum-substituent and Ge4+as the central ion of this complex:

Fig. 1. Structure of GePcF

(1) Introducing fluorine atoms into the structure of photosensitizer may reinforce the activity of photosensitizer. Gao Lingdong et al.[5]reported a complex formed by tetra-(trifluoroethoxy) zinc phthalocya- nine and emulsifier Pluronic F68 and the anticancer activity of this complex as photosensitizer in vitro. Yslas EI et al.[6]reported their experiments on the activity of zinc phthalocyanine with trifluorophenylmethoxy group as substituent in vitro. Wesley M.Sharman et al.[7]synthesized phthalocyanine substituted by multiple fluorine atoms and resear- ched its photodynamic therapeutic activity on EMT-6 cells in emulsion polyoxyethylenated castor oil. All the above reports demonstrated that introducing polyfluoroalkoxy group into the phthalocyanine ring may reinforce their photodynamic anticancer activities in cells through improving their fat-solubility and inhibiting concurrently the aggregation of phthalocyanine molecules[8]. Aforesaid phthalocyanine complexes may be expected to be used as PDT photosensitizer of clinical value.

(2) Germanium is a member of family IVAin period IV in the periodic table of elements. Organic germanium has many bioactivities, such as anticancers, regulating immunity, and antiviruses, so it is applied increasingly in medicines, food additives,cosmetology and health care. Till now there are the following germanium compounds of anticancer activities under R&D[9]: Alkyl germanium compounds, spiro germanium compounds, sesquioxide/sulfur germanium compounds, and phosphoruscontaining organic germanium compounds, of which Ge-132 and spiro Ge have been used in clinical practice[10].

(3) After being coordinated with the hollow core of phthalocyanine, Ge4+may be coordinated also with other groups in the axial direction to increase the steric hindrance to inhibit effectively the aggregation of phthalocyanine complexes, so that this kind of germanium phthalocyanine may exist in the form of monomer of photosensitive activity[11].

2 EXPERIMENTAL

2.1 Instruments and reagents

DF-101S model heat collection-type magnetic stirrer (Zhengzhou Great Wall Scientific Industrial and Trade Co., Ltd.); WRS-1B model digital melting point meter (Shanghai Huayan Instruments Co.,Ltd.); LC-20A high performance liquid chromatograph (SHIMADZU Japan); Avatar 330 FT-IR(Thermo Nicolet, USA); TC-C18 chromatographic column (4.6×250 mm, 5 μm) (Agilent Technologies Inc.). MULTISKAN MK3 model microplate reader(US Thermo Inc.); UV-2450 model UV-vis spectrophotometer (SHIMADZU Japan); CaryEclipse model fluorescence spectrophotometer (Agilent Technologies Inc., USA); 2000 model Fouriertransform infrared spectrometer (US Perkin-Elmer Spertrum); DECAX-30000 LCQ Deca XP model ion trap mass spectrometer (Thermo-Finnigan Inc.).

4-Nitrophthalonitrile (Shijiazhuang AIFA Reagent Co., Ltd.); lithium hydroxide (Tianjin No. 3 Factory of Chemical Reagents); trifluoroethanol and germanium tetrachloride (Alfa Aesar Company),chromatographic silica gel (Branch of Qingdao Haiyang Chemical Factory); dry powder of RPMI1640 medium and high-sugar DMEM medium(GIBCO Inc., USA); Mycoplasma-free newborn calf serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.); mixed Penicillin-Streptomycin solution (Beijing Zoman Biotechnology Co., Ltd.);trypsin (US Difco Inc.); MTT(Lot: M2128) (US Sigma Co., Ltd.); dimethyl-sulfoxide (DMSO) (US BIOSHARP); aqueous solution of Pluronic F68(10%) (US Sigma Co., Ltd.); polyoxyethylenated castor oil (CrEL) (German BASF Inc.); N,N-dimethylformamide (DMF) and other reagents were analytically pure and produced by Sinopharm Chemical Reagent Co., Ltd.

2.2 Synthesis of 4-(trifluoroethoxy)phthalonitrile

According to the synthetic procedure[12](shown in Fig. 2), a solution of 4-nitrophthalonitrile (17.3 g,100 mmol), dried DMSO (300 mL) and trifluoroethanol (7.96 mL, 110 mmol, higher slightly than the theoretical value) was added into a 500 mL single-necked flask which was equipped with a drying tube filled with anhydrous calcium chloride.And 4.2 g of lithium hydroxide was added into this flask in four times within 2 h. The content was stirred at 25 ℃ for 72 h. The mixture was dripped slowly into ice water to get a yellow-brown turbid liquid and then frozen over night. The yellow crystals were filtered and recrystallized twice from methanol/water (v/v = 1:1). The solid was dried in vacuum to yield 16.28 g (72.0%).

2.3 Synthesis of tetra(trifluoroethoxy)germanium phthalocyanine (GePcF)

Tetra(trifluoroethoxy) germanium phthalocyanine(GePcF) was synthesized by referring to the method described in reference[13]and the synthetic route is depicted in Fig. 3. A solution of 4-(trifluoroethoxy)phthalonitrile (9.05 g, 40 mmol) and anhydrous germanium tetrachloride (1.20 mL, 11 mmol) in 10 mL dried chinoline was stirred at 200 ℃ for 4 h under nitrogen atmosphere. The reaction mixture was cooled to room temperature and precipitated with 50 mL of methanol/water (v/v= 1:2) and filtered. The product was purified by silica gel column chromatography with the eluent to be chloroform:methanol (30:1 v/v). The green band liquid was collected and the solutions were concentrated under reduced pressure. The product was freeze-dried into a dark-green solid in 5.449 g(yield rate: 52.0%).

Fig. 3. Synthesis of GePcF

2.4 Structure characterization

2.4.1 Measurement of the melting point

The sample was dried till its weight did not change, and then its melting point was measured using WRS digital melting point meter.

2.4.2 Infrared spectrum determination

The structures of 4-nitrophthalonitrile and all targeted products were tested with Fourier infrared spectrum instrument. The mixture of a little amount of the sample and proper amount of KBr powder was ground and made into thin slices by tablet compressing machine. Then these slices were scanned by IR spectrometer separately.

2.4.3 Conditions of the mass spectrometry

Atmospheric-pressure chemical ionization (APCI),anion detection (-), capillary voltage: 3500 V;flow-rate of dried nitrogen: 4.0 L/min, temperature of dried nitrogen: 350 ℃, the pressure of atomized gas: 60 Psi.

2.4.4 Absorption and fluorescence spectra of GePcF

The absorption and fluorescent properties of GePcF were measured according to the methods from literature[14]. The solution of GePcF in DMF was confected in the concentration of 10.0 μmol/L.The absorption spectrum was scanned at 300～800 nm on a UV-vis spectrophotometer. The fluorescence spectrum was scanned on a fluorescence spectrometer.

2.4.5 Analysis of the final product GePcF by high performance liquid chromatography

A method was developed for the determination of GePcF by reverse phase HPLC, using methanol/0.05% trifluoroacetic acid (85/15) as the mobile phase.

2.5 Researches on in vitro photodynamic activity

2.5.1 Cells and conditions of their culture

Human colonic adenocarcinoma SW480 cells were cultured in DMEM culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cervix adenocarcinoma HeLa cells were cultured in RPMI1640 culture medium supplemented with 10% fetal bovine serum and 1%penicillin-streptomycin. Cells were cultivated in medium at 37 ℃ in a humidified 95% air and 5%carbon dioxide (CO2) atmosphere.

2.5.2 Preparation of the samples

The phthalocyanine complexes are poorly watersoluble, but this may be enhanced by Pluronic F68[5].The saturated solution of GePcF in DMSO was firstly prepared, 0.50 mL of which was then dripped slowly into the Pluronic F68 solvent (9.50 mL)followed by vigorous stirring. After that, the mother liquor of the test solution contained 0.80 mmol/L GePcF (determined by UV-Vis). The mother liquor of reference solution was prepared as described for the test solution but using pure DMSO (0.50 mL)instead of the saturated solution of GePcF in DMSO to be examined. Finally, both the mother liquors were filtered through 0.45 μm micropore filtermembrane and then refrigerated and protected from light.

2.5.3 Cytotoxicity of GePcF for SW480 cells Phototoxicity

Based on the related reference[15], the cytotoxicity was measured using a standard MTT assay. The SW480 cells were grown in a 96-well cell-culture plate at 5 × 103per well and then incubated for 24 h at 37 ℃ under 5% CO2. After 24 h culturing, those 96-well plates were washed twice with PBS. The mother liquor was added to the culture medium at different concentration, with four parallel wells for each concentration (0, 5.00, 10.00, 20.00, 40.00, and 80.00 μmol/L). Those plates were allowed to incubate for another 24 h at 37 ℃ and then washed twice with PBS before irradiation. Those plates were irradiated by a 300 W iodine-tungsten lamp; the ray was cooled down when passing through a water tank and then filtered by 650 nm infrared filter before reaching these plates. After 20 min exposure of 650 nm light at 100 mw/cm2, the cells were allowed to incubate for an additional 48 h. To assess the effect of exposure to iodine-tungsten lamp on the cells with and without GePcF, the cell viability was measured by MTT assay. Typically, 20 μL of MTT solution (5 mg/mL MTT in PBS) was added to each well and incubated at 37 ℃ for 4 h. After removing the medium, the wells were washed by PBS, and then the intracellular formazan crystals were extracted into 150 μL of DMSO. The absorbance of cell lysate was recorded at 492 nm by a microtiter plate reader, and the cell viability could be calculated from the average value of four parallel wells.

The inhibition rate of carcinoma cells on each concentration could be calculated by the following formula:

In the above formula, A is the average absorbance value of the test groups and A0is the average absorbance value of control group.

Experiment on the dark toxicityThe experimental procedure was the same with that for phototoxicity test, and the only difference lies in that no irradiation was carried out in this experiment.

2.5.4 Cytotoxicity of GePcF for HeLa cells

The HeLa cells were grown in a 96-wellcellculture plate at 1 × 104per well and then incubated for 24 h at 37 ℃ under 5% CO2. Other procedures are the same with those for the experiments on SW480 cells.

3 RESULTS AND DISCUSSION

3.1 Selection of methods for synthesizing GePcF

We once tried to put 4-(trifluoroethoxy)phtha-

lonitrile, germanium tetrachloride and DBU (1,8-diazacyclo[5,4,0]undecen-7) as catalyst in n-pentanol by heating reflux method for 4～24 h to synthesize GePcF. However, only phthalocyanine was obtained with hollow core instead of germanium phthalocyanine. The reason may be that germanium is a member of family IVA in period IV in the periodic table of elements so it possesses both the natures of metals and nonmetals. Therefore, more energies may be needed to promote germanium to be inserted into the hollow site of phthalocyanine ring,while the quinoline with high melting point would be needed[16].

3.2 Structure characterization of4-(trifluoroethoxy) phthalonitrile

The melting point was 79.1～80.2 ℃ and its dissolving range was rather narrow, suggesting its high purity. Infrared spectrum of 4-nitro-phthalicnitrile is shown in Fig. 4: cyano-specific peak appeared at 2241 cm-1, and nitro-specific peaks occurred at 1355 and 1539 cm-1. Infrared spectrum of 4-(trifluoroethoxy) phthalonitrile is shown in Fig. 5:cyano-specific peak appeared at 2235 cm-1; while the original nitro-specific peaks at 1355 and 1539 cm-1disappeared. Instead, Ar-O-C-ether-bondspecific peaks are observed at 1259 and 1067 cm-1,and C-F-bond-specific peaks at 1170, 974 and 828 cm-1, demonstrating that 4-nitro-phthalicnitrile has been transformed into 4-(trifluoroethoxy) phthalonitrile.

Fig. 5. IR of 4-(trifluoroethoxy)phthalonitrile

thoxy)phthalonitrile. APCI-MS: m/z 453.4[2M+H]+. Results of elemental analyses: Calcd. (%):C, 53.11; H, 2.23; N, 12.39. Measured values (%): C,53.25; H, 2.39; N, 12.24.

3.3 Structure characterization of GePcF

The melting point of GePcF is ＞ 300 ℃. Infrared spectrum of GePcF (Fig. 7) shows the absorption peak specific to the phthalocyanine ring appeared at 1611, 1501, 1407 and 1348 cm-1; and that characteristic of Ar–O–C was found at 1241 and 1088 cm-1. The stretching vibration absorption peak specific to the C–F bond in the polyfluoroalkoxy group occurred at 1167 and 973 cm-1. Fig. 8 shows the MS-APCI (-) mass spectrum of GePcF(C40H20N8O4F12Cl2Ge). The peak of quasi-molecular ion [M-H]-formed by GePcF: m/z 1046.9,characteristic fragment peak: m/z 1012.6 and 1009.9,characteristic fragment peak: m/z 1012.6; peak of fragment [M-H-Cl]-formed by removing a chlorine from GePcF: 1009.9 (due to isotope). Mass spectrometry and elemental analyses demonstrated further that the synthesized product was just tetra-(trifluoroethoxy) germanium phthalocyanine(GePcF).

Fig. 6. MS of 4-(trifluoroethoxy)phthalonitrile

Fig. 7. IR of GePcF

The absorption spectrum of GePcF (Fig. 9) agreed with the absorption character of metal phthalocyanine; Q band λmaxappeared at 683 nm, and it was the absorption peak of monomer; B band λmaxis observed at 363 nm. In addition, a weak peak appeared at 612 nm, formed by the aggregation of phthalocyanine molecules[16]. The absorption spectrum of aggregated metal phthalocyanine complexes differed from that of separated metal phthalocyanine complexes, and this difference would act on the photosensitive activity of GePcF[17]. The peak of separated GePcF was higher noticeably than that of aggregated GePcF, indicating that the interaction of those complex molecules in DMF solvent was relatively weaker, and therefore GePcF is present in DMF mainly in the form of monomer with photosensitive activity.

Fig. 8. MS of GePcF

Fig. 10 shows the excitation and emission spectra of GePcF. Its excitation spectrum was very similar to its absorption spectrum, while its emission spectrum was basically like the mirror image of its absorption spectrum. The visible light emitted by phthalocyanine in human body after excitation helps to make the clinical diagnosis of tumors, and therefore researches on the fluorescent characteristics of phthalocyanine are of great significance.

Fig. 9. UV-Vis spectrum of GePcF in DMF (C = 10.0 μmol/L)

Fig. 10. Excitation and emission spectra of GePcF in DMF (C = 10.0 μmol/L)

Fig. 11. HPLC graph of GePcF

HPLC analyses of the GePcF isomer are shown in Fig. 11. Generally, synthetic phthalocyanine is present in the form of 4 isomers, with their theoretical proportion to be C4h:C2v:Cs:D2h=12.5:25:50:12.5[18]. Components 1, 2 and 3 were collected and scanned by UV-Vis, and the result demonstrated that the absorption spectrum of those 3 components was similar, suggesting that they were 3 isomers of the same molecule. The synthetic product reported in this article contained only 3 isomers,probably because the steric hindrance of trifluoroethoxy group prevented the formation of isomer D2h,thus isomers C4h, C2v, and Cswere observed.

3.4 Photodynamic action of GePcF on the SW480 and HeLa cells

Tables 1 and 2 show the experimental results on phototoxicity and dark toxicity of GePcF for SW480 and HeLa cells. It demonstrated that the inhibition action of GePcF on the growth of cancer cells was weak when irradiation or photosensitizer was absent;the inhibiting rate of cancer cells by GePcF would rise when irradiation was present, and the inhibiting rate would rise along with the increase of GePcF concentration. GePcF has shown noticeable phototoxicity for both SW480 and HeLa cells, and there was a lineal relationship between the phototoxicity intensity and GePcF concentration, as shown in Figs. 12 and 13. IC50 of GePcF was 36.53 μmol/L for SW480 cells, and 45.78 μmol/L for HeLa cells, suggesting that the anticancer activity of GePcF for HeLa cells was relatively weaker. Factors responsible for the difference of inhibiting rates included not only the structure and properties of photosensitizer itself, but also the cell types, location of photosensitizer in subcells and the excitation of photosensitizer in the definite site[19].

Fig. 13. Inhibiting rate of GePcF for the HeLa cells

Table 1. Comparison of Phototoxicity and Dark Toxicity of GePcF of Different Concentration to the SW480 Cells

Table 2. Comparison of Phototoxicity and Dark Toxicity of GePcF of Different Concentration to the HeLa Cells

4 CONCLUSION

A new metal phthalocyanine complex GePcF was synthesized, characterized and researched. Its maximal absorption peak was located at PDT window(600～800 nm) and it showed greater molar extinction coefficient and higher yield rate of singlet oxygen quanta, so it possessed photophysical properties necessary for ideal photosensitizers. Our research demonstrated that GePcF showed noticeable phototoxic action on SW480 and HeLa cells both,and the intensity of its phototoxic action depended lineally on its concentration. IC50of GePcF for SW480 and HeLa cells were 36.53 and 45.78 μmol/L, respectively. The dark toxicity of GePcF in experiments was weak, which was the priority for photosensitizer used in PDT. In summary, GePcF possessed basic properties necessary for the anticancer photosensitizer, and further researches on the mechanism responsible for the action of GePcF would be helpful for utilizing fully its potential in PDT.

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