Synthesis, Spectral Analysis and Preliminary in Vitro Evaluation of Some Tetrapyrrolic Complexes with 3d Metal Ions

In this paper, two tetrapyrrolic complexes, Zn(II)-5-(3-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin and Cu(II)-5-(3-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin were synthesized, and characterized from a spectral and biological point of view. The study provided data concerning the behavior of identical external substituents vs. two different core insertions. Some of the properties of the proposed tetrapyrrolic structures were highlighted, having photodynamic therapy of cancer as a targeted biomedical application. Elemental analysis, NMR, FTIR and UV-Vis data in various solvents were provided. A preliminary in vitro study on normal and cancer cultured cells was carried out for biocompatibility assessment in dark conditions. The preliminary in vitro study performed on human peripheral mononuclear cells exposed to tetrapyrrolic compounds (2 µM) showed that the proposed compounds had a convenient cytotoxic profile on human normal peripheral blood mononuclear cells under dark conditions. Meanwhile, the investigated compounds reduced the number of metabolically active breast tumor MCF-7 cells, with the exception of Zn(II) complex-containing a symmetrical ligand. Accordingly, preliminary in vitro data suggest that the proposed tetrapyrrolic compounds are good candidates for PDT, as they limit tumor expansion even under dark conditions, whilst sparing normal cells.

performed on human peripheral mononuclear cells exposed to tetrapyrrolic compounds (2 µM) showed that the proposed compounds had a convenient cytotoxic profile on human normal peripheral blood mononuclear cells under dark conditions. Meanwhile, the investigated compounds reduced the number of metabolically active breast tumor MCF-7 cells, with the exception of Zn(II) complex-containing a symmetrical ligand. Accordingly, preliminary in vitro data suggest that the proposed tetrapyrrolic compounds are good candidates for PDT, as they limit tumor expansion even under dark conditions, whilst sparing normal cells.

Introduction
Tetrapyrrolic compounds, especially porphyrins and metalloporphyrins, are still a spearhead in the studies related to diagnosis and photodynamic therapy (PDT) of malignant tumours [1][2][3][4][5][6][7][8][9]. The therapeutic effect of tetrapyrrolic compounds results from their ability to accumulate preferentially in the malignant tissue and to generate, following light-mediated activation in the presence of molecular oxygen, reactive oxygen species responsible for cell death [10][11][12]. The use of porphyrinic compounds as fluorescent markers in the detection of various cancer types is based on their photophysical characteristics, such as emissions in the spectral range 600-800 nm, large Stokes shift and high fluorescence lifetime [2,3].
In diagnostic and therapeutic applications the efficiency of tetrapyrrolic compounds depends on their structural and physical-chemical characteristics [13]. High absorption coefficients in the 600-680 nm spectral range, high singlet oxygen generation quantum yield, acceptable solubility in biologic fluids, selectivity for diseased tissues and superior uptake values, photostability and lack of toxicity in the absence of light excitation are the most important parameters that dictate the efficiency of porphyrins as photosensitizers [1][2][3].
Current mainstream research approaches include more and more complex long-chain studies of both porphyrins and metalloporphyrins configured as third generation photosensitizers [14,15]. The quest for optimal or at least most improved structures, remains active, despite the already clinical approved porphyrin-base drugs [16]. Among metalloporphyrins, some forms with Zn(II) have been recently studied as starting points for future efficient photodynamic agents, mostly with a focus on asymmetrically substituted bases; meanwhile, copper porphyrins are less present in this type of studies, for instance as compounds involved in the side-effects of phototherapy [17][18][19][20].

Chemistry
The syntheses presented in this study have been successfully repeated several times with identical results. The dry media synthesis procedure was performed in three stages: irradiation followed by cooling and with UV-Vis analytical monitoring of samples. After synthesis and purification, the porphyrinic complexes were characterized by elemental analysis, FTIR, 1 H-and 13 C-NMR and UV-Vis spectrometry.
The infrared spectral data obtained for the synthesized metalloporphyrins revealed the presence of typical vibration modes of both the tetrapyrrolic macrocycles and phenyl substituents. This is generally in agreement with results previously reported for similar porphyrinic structures [25][26][27][28][29][30]. The large band registered at about 3410 cm −1 can be assigned to the O-H stretching vibration of the -OH functional group, near the typical 3423 cm −1 of the N-H vibration. In the higher wavenumber region at ~2920 cm −1 a medium band was registered, corresponding to C-H vibrations of the phenyl groups. Around 2850 cm −1 the band corresponding to C-H vibration frequencies of the -O-CH3 group was detected. For both synthesized metalloporphyrins the IR signals at 1709 cm −1 can be assigned to the C=O stretching vibration, while the absorption band evidenced at 1104 cm −1 was attributed to the C-O bond vibrations. In the infrared spectra of P1 and P2, the bands corresponding the C=N and C-N stretching vibrations were highlighted in the spectral range of 1490-1510 cm −1 and 1600 cm −1 , respectively. Several signals belonging to the pyrrole ring are also present at 975 cm −1 for δC-H and 798 cm −1 for γC-N. The general shape of the spectra remains similar for both complexes even below 1600 cm −1 ; in this region several medium intensity signals attributed to C-C vibrations as 1397, 1349, 1326 cm −1 or γC-C at 880 and 846 cm −1 are present.

Absorption and Emission Spectra
The complexes were studied by UV-Vis spectroscopy to confirm their structure and to establish their profiles in environments with different polarities. Due to solvent dependence of the position of the Soret band and all the other bands, measurements were concentrated around organic solvents with impact in biomedical studies. Due to the presence of -OH groups at the periphery of the tetrapyrrolic complex, the solubility was increased, but remained an issue for systems containing water.
Parameters of the solvents from the polarity to the donor-acceptor process often cause significant changes in the band energy values leading to shifts in the spectral profiles. As expected, in our particular case the Soret band shifted in a narrow range of 4 nm for P1 but doubled in the case of P2. The relative intensities were sensitive to the nature of the solvent, but within narrow limits as can be seen in Table 1 and Figure 2. Table 1. Absorption and emission data of the porphyrinic complexes in various solvents (c = 2.5 × 10 −6 M, λex = 420 nm).  The insertion of an ionic metal in the porphyrinic core led to a decrease of the vibrational modes evidenced by the reduced number of Q bands. In this context, P1 displayed two Q bands, Qy(0,0) and Qx(1,0), respectively. Meanwhile, in the case of P2, the spectrum contained only one Q band. Figures 3 and 4 show the quenching influence of the more polar solvents both on Soret band and on the Q bands. The general tendencies are the same for P1 and P2, despite the small differences between these compounds, given in part by the different complexation power of the two metals. The absence of P2 emission is in agreement with data reported in the literature [31][32][33]. P1 on the other hand had two distinctive signals. The differences in terms of peak maximum values remained constant and independent of the solvent. Dichloromethane proved to have the most significant hypsochromic influence, suggesting that less polar solvents seem to be more suitable for further biological tests.

The Effect of Porphyrinic Compounds on Human Tumor MCF-7 Cells
Results presented in Figure 5 show that all the tested compounds at 2 µM concentration did not affect the viability of normal PBMC (P, P2, P3, P4) or even tended increase the MTS reduction (P1, P5). Meanwhile, most of the investigated compounds reduced the number of metabolically active tumor cells (P, P1, P2, and P3, P5). This cytotoxic effect was more pronounced in the case of compounds P and P5. In turn, the Zn(II)-containing complex (P4) exerted no major effect on tumor cells. Compounds P and P5 proved to be promising candidates for cancer therapy by drastically inhibiting the growth of tumor cells, whilst having no cytotoxic action on normal PBMC. We emphasize that the Zn(II)-containing compound (P4) had almost no impact on normal and cancer cells

General Information
Commercially available chemicals and solvents from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Whitehouse Station, NJ, USA) were used for chemical synthesis. For the microwave-assisted synthesis a MWG775H type temperature-controlled microwave oven (Clatronic, Kempen, Germany) was used. The elemental analysis of C, H and N was performed with an automatic 1108 analyzer (Carlo Erba, Milan, Italy). IR spectra were recorded with a FT-IR Tensor 27 spectrophotometer (Bruker, Fremont, CA, USA). The UV-Vis spectra of the porphyrinic complexes were recorded with a Lambda 35 spectrophotometer (Perkin-Elmer, Waltham, MA, USA) and fluorescence spectra were recorded with a FP 6500 spectrofluorimeter (Jasco, Tokyo, Japan). The metalloporphyrin solutions in different organic solvents (ethanol, isopropyl alcohol, dimethylformamide, dichloromethane, dimethyl sulfoxide) were freshly prepared in the spectrally pure solvents at the concentration 2.5 × 10 −6 M and kept in dark until the measurement to prevent photodegradation. The NMR spectra of the porphyrinic complex were recorded with a 400 MHz NMR spectrometer (Bruker).

Preparation of Tetrapyrrolic Compounds for the in Vitro Study
Tetrapyrrolic compounds were dissolved initially in DMSO and were further diluted in culture medium before the experiments to obtain working concentrations of 2 μM. The final DMSO concentration in cell cultures was 0.12%.

Cells
The human breast carcinoma MCF-7 cell line was purchased from ATCC (ATCC ® HTB-22™) and was cultivated in DMEM (Biochrome, Berlin, Germany) culture medium supplemented with 10% fetal bovine serum (FBS, Biochrome) and antibiotic-antimycotic solution (Sigma-Aldrich). Passage of cells was performed at 48 h by trypsinization of adherent MCF-7 cells using 0.25%/0.02% Trypsin/EDTA (Gibco, Life Technologies, Carlsbad, CA, USA). For experiments, MCF-7 cells were plated in 96 well culture plates, at a density of 40,000 cells/cm 2 . Human peripheral mononuclear cells (PBMC) were isolated from blood samples of 6 healthy volunteers, based on their informed consent. Briefly, 3 mL blood collected in Li-heparin coated vacutainers was layered on Biocoll solution (Biochrome), and was centrifuged for 20 min at 2000 rpm, at room temperature. Mononuclear cells were collected and were washed twice in RPMI 1640 culture medium (Biochrome) by centrifugation for 10 min at 1500 rpm, 4 °C. Cells were finally suspended in RPMI 1640 medium supplemented with 10% FBS (Biochrome) and antibiotic-antimycotic solution (Sigma-Aldrich). Cells were counted in a Burker-Turk hemocytometer. Cellular viability was assessed by the trypan blue exclusion test. Only cell suspensions with viability >95% were used for experiments.

Cell Cultures
For experiments, MCF-7 cells were plated in 96 well culture plates, 7000 cells/well (21,000 cell/cm 2 ), and were cultivated for 24 h to reach adherence. PBMC isolated from 6 healthy volunteers were mixed for obtaining activated PBMC by mixed lymphocyte reaction. PBMC were thereafter plated in 96 well culture plates, 10 5 cells/well. Cells were treated with the test substances (2 μM concentration). Non-treated cells and cells treated with the solvent (DMSO) were used as controls. Samples containing culture medium and dilutions of the test substances were used for background assessment. Final sample volume in experiments was 100 μL. All samples were plated in triplicate. Cell cultivation was performed at 37 °C, in 5% CO2 atmosphere.

The MTS Reduction Test
The MTS reduction test was used for assessing the number of metabolically active cells in culture, using the CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Cells were cultivated as described above, and treated with test substances for 48 h. At the end of the treatment time, 20 μL of the kit reagent were added to each well of the culture plate. Cells were further incubated for 3 h at 37 °C, in 5% CO2 atmosphere. The optical density (OD) of samples was measured at 490 nm against 620 nm reference, using a Sunrise ELISA reader (Tecan, Männedorf, Schweiz). Final OD in cellular samples was calculated by subtracting the OD of corresponding background samples. Data were processed as percentage of test sample OD relative to DMSO-corresponding OD, and were presented as mean ± standard error of the mean (SEM) for independent experiments.

Conclusions
The spectral data confirmed the expected general profiles of the studied tetrapyrrolic complexes, and placed them in suitable position for the next step in our quest to identify convenient photosensitizers. The preliminary in vitro study indicated that the new porphyrinic complexes did not display dark condition cytotoxic effects on normal PBMC, and generally reduced the number of metabolically active tumor MCF-7 cells. Taking into account that an appropriate PS candidate for efficient and safe PDT in tumors should minimally affect normal and diseased cells under dark conditions, the Zn(II)-containing compounds proved to be more convenient for potential biomedical use than Cu(II)-containing ones.