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Synthesis, characterization, photochemical properties and cytotoxicity of the novel porphyrazine functionalized with nitroimidazolylbutylsulfanyl groups
Inorganic Chemistry Communications 29 (2013) 97–100
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Synthesis, characterization, photochemical properties and cytotoxicity of the novel porphyrazine functionalized with nitroimidazolylbutylsulfanyl groups Sebastian Lijewski a, Jaroslaw Piskorz b, Malgorzata Kucinska c, Marcin Wierzchowski a, Katarzyna Czerniak d, Hanna Billert d, Marek Murias c, Jadwiga Mielcarek b,⁎, Tomasz Goslinski a,⁎ a
Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30, 60-631 Poznan, Poland d Department of Experimental Anesthesiology, Poznan University of Medical Sciences, Sw Marii Magdaleny 14, 61-861 Poznan, Poland b c
a r t i c l e
i n f o
Article history: Received 23 November 2012 Accepted 6 January 2013 Available online 12 January 2013 Keywords: Nitroimidazole Porphyrazine Linstead macrocyclization Singlet oxygen generation Carcinoma
a b s t r a c t A novel porphyrazine analog possessing nitroimidazolylbutylsulfanyl groups was synthesized and characterized using UV–Vis, IR, MS MALDI and various NMR techniques. In addition, a computational model following the Density Functional Theory method (DFT) was applied to analyze the FT IR spectrum. Potential photosensitizing activity of the novel porphyrazine was evaluated by measuring its ability to generate singlet oxygen (ΦΔ), which was found to reach the value of 0.045 in DMF, and 0.035 in DMSO. A lower value of singlet oxygen generation in DMSO may result from the increased tendency to aggregate, which was studied in the UV–Vis and it was found to be stronger in DMSO than in DMF solutions. The investigation indicated no release of nitric oxide (NO) from porphyrazine functionalized with nitroimidazolylbutylsulfanyl groups. In vitro studies of the new compound were carried out to investigate photosensitizer-induced photocytotoxicity on two prostate human cancer cell lines, LNCaP, PC3, and one melanoma derived cell line, MeWo. © 2013 Elsevier B.V. All rights reserved.
Synthetic porphyrinoids such as phthalocyanines and porphyrazines (Pzs) exhibit numerous potential applications in technology and medicine, especially in photodynamic therapy (PDT) and photodynamic diagnosis (PDD) [1–11]. PDT is a relatively new method which has been successfully applied against tumors or dermatological lesions [12–14]. It has been described that nitroimidazoles and related compounds exhibit antibacterial and antiparasitic activity [15,16] and possess anticancer activity in deep hypoxic conditions, thus are often termed “oxygen-mimetic” radiosensitizers [17,18]. Nitroimidazole derivatives (pimonidazole, EF5) have been used for both immunohistochemical detection and non-invasive PET/SPECT imaging of hypoxia [19]. Radiosensitizing properties of nitroimidazoles have been well described for noninvasive in vivo mapping of brain hypoxia after stroke [20] and for imaging of hypoxic myocardium [21]. Moreover, many Technetium-99m labeled nitroimidazole derivatives have been tested for tumor hypoxia imaging [22–26]. Recently, some examples of porphyrinoids containing imidazole groups have been synthesized. Phthalocyanines bearing fused imidazole rings on the periphery have been synthesized and their spectroscopic and electrochemical properties have been studied [27–31]. Moreover, conjugates of 2-nitroimidazole and manganese porphyrin have been obtained and demonstrated selectivity towards tumor SCCVII cell lines implanted in C3H/He ⁎ Corresponding authors. Tel.: +48 61 854 66 33; fax: +48 61 854 66 39. E-mail addresses: [email protected] (J. Mielcarek), [email protected] (T. Goslinski). 1387-7003/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.inoche.2013.01.001
mice with good radiotherapeutic effect at low dose administrations and clear tumor magnetic resonance images [32]. In addition, cationic imidazolium substituted metalloporphyrins with different metal ions have been studied using human (HeLa) and mouse (CT26) cancer cell lines [33]. The promising photodynamic activity of new imidazole substituted pyropheophorbide-a derivatives have been found with A549 human lung adenocarcinoma cells [34]. Furthermore, the cyclodextrin-based nanoassembly consisting of tetraanionic porphyrin and [7-(adamantan-1-yloxy)heptyl]-[4-nitro-3-(trifluoromethyl)phenyl]amine induces high cancer cell mortality through a bimodal action based on the simultaneous photogeneration of nitric oxide (NO) and singlet oxygen (1O2) [35]. We report here the synthesis, photochemical properties, and in vitro cytotoxicity of the novel porphyrazine peripherally functionalized with nitroimidazolylbutylsulfanyl groups (5), carried out to determine its potential as a PDT photosensitizer. Its synthesis was accomplished in a three-step procedure. 4-Nitro-1-(4-bromobutyl)-1H-imidazole (2) was synthesized following the literature method [36] and used in alkylation reaction with commercially available dimercaptomaleonitrile disodium salt (3) to give substituted dimercaptomaleonitrile (4) [37]. Linstead macrocyclization reaction of 4 using magnesium n-butanolate in n-butanol resulted in porphyrazine 5 (Scheme 1) [38,39]. Structure of 5 was confirmed by 1H and 13C NMR studies, accompanied by two-dimensional techniques (1H1H COSY, 1H13C HSQC, 1H13C HMBC) to assist allocation. 1H1H COSY spectrum of 5 revealed connectivities between protons belonging to neighboring methylene groups of
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aliphatic chain. Noteworthy, in HMBC spectrum of 5 cross-peaks between alkyl chain protons (SCH2 at 4.33 ppm) and carbon atoms of macrocyclic core (140.6 ppm) were found. In addition, cross-peaks of CH2N protons at 4.07 ppm and imidazole ring carbon atoms (C5′ at 120.7 and C2′ at 136.8 ppm) were observed (Figs. 1S–4S, Table 1S, Supplementary). We next focused on calculations using the Density Functional Theory (DFT). The procedure consisted of conformational search and vibrational analysis [40–43]. The global minimum of 5 possesses a saddle-like structure of D2 symmetry (Fig. 1). By comparing the calculated molecular vibrational frequencies of 5 molecule with the experimental infrared spectra, the molecular motions that correspond to the measured bands on the basis of similar frequency magnitudes were assigned (Fig. 5S, Table 2S, Supplementary). The calculated and measured frequencies were in reasonable agreement and confirmed presence of nitroimidazole group, porphyrazine ring and aliphatic linker. Aromatic, stretching C–H band (exp. 3118 cm−1, calc. 3178 cm−1), asymmetric stretching NO2 band (exp. 1541 cm−1, calc. 1581 cm−1), imidazole ring stretching skeletal band (exp. 1488 cm−1, calc. 1488 cm−1), imidazole ring C–H breathing band (exp. 1286 cm−1, calc. 1281 cm−1), and imidazole out of plane C–H band (exp. 752 cm−1, calc. 724 cm−1) confirmed the presence of imidazole fragment in the analyzed structure. In addition, 1,4-butylene chain is represented by the following frequencies: asymmetrical and symmetrical stretching C–H (exp. 2919 cm−1, calc. 2945 cm−1 and exp. 2850 cm−1, calc. 2909 cm−1, respectively), scissoring C–H (exp 1403 cm−1, calc. 1444 cm−1), and rocking C–H (exp. 885 cm−1, calc. 876 cm−1). In the dactiloscopic region selected bands of the porphyrazine macrocycle resulting from breathing in plane (exp. 854 cm−1, calc. 867 cm−1) were found. In the UV–Vis spectrum of 5 (Fig. 6S, Supplementary) four distinct absorption bands were observed. Two of them are characteristic of tetrapyrrolic, macrocyclic ring with the Soret band occurring at 378 nm and a sharp, intense Q band at 670 nm. The third band at 292 nm being the result of 4–nitroimidazole ring absorption is consistent with the UV–Vis spectrum of maleonitrile precursor 4. Furthermore, a less intense C–T band, being a result of n→π⁎ transitions (electrondonor properties of sulfur atoms at peripheral positions) at 502 nm was observed [39]. The aggregation behavior of Pz 5 was studied in the UV–Vis in DMSO and DMF solutions (Figs. 2, 7S, Supplementary). Statistical analysis of the linear correlations between absorbance of the Q band and concentration of the Pz 5 revealed that the Beer–Lambert law is obeyed in DMF, but it is not obeyed in DMSO. These results indicate that the Pz 5 has a stronger tendency to aggregate in DMSO solutions [44].
HN
Fig. 1. Calculated and measured FT-IR spectra of Pz 5. Inset presents a saddle-like structure of D2 symmetry.
The potential photosensitizing activity of Pz 5 was evaluated by measuring its ability to generate singlet oxygen. The relative method, with zinc(II) phthalocyanine as a reference and DPBF (1,3-diphenylisobenzofuran) as a chemical quencher for singlet oxygen, was applied (Figs. 2, 8S, Supplementary) [45–47]. Solutions of 5 and DPBF in DMF or DMSO were irradiated with monochromatic light at wavelengths adjusted to the porphyrazine Q-band maximum absorption. The kinetics of subsequent oxidation of DPBF was used for calculation of singlet oxygen generation yield values (ΦΔ DMF =0.045, ΦΔ DMSO =0.035). Higher value of singlet oxygen generation in DMF may result from the above-described stronger tendency of 5 to aggregate in DMSO. The possibility of bimodal action based on the simultaneous photogeneration of nitric oxide (NO) and singlet oxygen (1O2) encouraged us to investigate the potential NO donating properties of this compound. The detection of nitric oxide, which can be generated upon irradiation was based on the reaction between reduced myoglobin and NO, that leads to the formation of myoglobin-NO species. The binding of photoreleased NO with myoglobin can be observed spectrophotometrically as the appearance of a new peak at 419 nm in the UV–Vis spectrum [48]. The intensity of light applied for the irradiation of the Pz 5 equaled N
+ -
N
(i) +
N
O
Br
N
Na S
N
+
+
O
N
O
-
N
R
(ii)
+ -
Na S
R
N
N
O
1
2
3
4 R
N R=
S
N
+
N O
O
N
-
N
N
R
N
R
R N
Mg
R
N
N
(iii)
R
R
N
R
5 Scheme 1. Reagents and conditions: (i) 1,4-dibromobutane, NaH (60% dispersion in mineral oil), DMF, rt to reflux, 2 h [36]; (ii) DMF, K2CO3, 50 °C, 24 h (60%); (iii) Mg(OnBu)2, nBuOH, reflux, 20 h (8%).
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generation (ΦΔ DMF =0.045, ΦΔ DMSO =0.035). A lower value of singlet oxygen generation in DMSO may result from the tendency to aggregation, which was studied in the UV–Vis and found to be stronger in DMSO than DMF solutions. Cytotoxic activity was tested using two prostate (LNCaP, PC3) and one melanoma (MeWo) human cancer derived cell lines. The tested compound revealed low cytotoxicity probably due to its higher tendency to aggregate and insufficient uptake by cells. Additionally, the cytotoxic effect appeared to be cell dependent. The possibility of bimodal action based on the simultaneous photogeneration of nitric oxide and singlet oxygen was excluded as the potential NO donating property of this compound was not observed. Acknowledgments The authors acknowledge financial support for the project from the Polish Ministry of Science and Higher Education (NN401 067238). Appendix A. Supplementary material The following publications refer to the experimental data contained in the Supporting Information [40–43,45–52]. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.inoche. 2013.01.001. References
Fig. 2. (A) UV–Vis spectra of Pz 5 dissolved in DMF. Inset presents a linear correlation between absorbance and concentration; (B) Changes in the UV–Vis spectra for DPBF and Pz 5 in DMF. Inset presents first-order plot for oxidation of the DPBF. Changes in the Q band absorption during the experiment did not exceed 1%.
2.9 mW/cm2 and the irradiation times were similar to that used in the below-described photocytotoxicity experiment. Nevertheless, the band at 419 nm was not observed, which indicated the lack of NO formation (see Supplementary). Biological activity of 5 was determined in vitro using MTT assay on two human prostate cancer cell lines, LNCaP, PC3, and one melanoma derived cell line, MeWo. Dark and light toxicity were evaluated in cells incubated with the tested compound at concentrations: 0.15–10 μM. At these concentrations, 5 in dark conditions did not reduce the viability of LNCaP, whereas PC3 cells were more sensitive and a decrease in the number of viable cells was observed at lower concentrations. Weak photocytotoxic effect of 5 on LNCaP and MeWo cells was observed after 10 and 20 min of irradiation. Surprisingly, no phototoxic effect was observed on PC3 cells after 10 min of irradiation, when proliferative effect was noticed, while significant phototoxicity appeared on the cells irradiated for 20 min (Fig. 9S, Supplementary). The low cytotoxicity at higher concentrations of 5 might be explained by its higher tendency to aggregate (Fig. 10, Supplementary) and insufficient uptake by cells. Summarizing, the new porphyrazine containing nitroimidazolylbutylsulfanyl groups was synthesized and characterized using UV–Vis, IR, MS MALDI and various NMR techniques. In addition, the computational model following the Density Functional Theory method (DFT) was applied to analyze the FT IR spectrum. The ability of novel porphyrazine to generate singlet oxygen, which plays an important role in the cytotoxic effect of PDT, was expressed by the quantum yield of singlet oxygen
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[39]
[40]
[41] [42] [43] [44]
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[49]
[50] [51] [52]
(300 MHz, DMSO-d6) δ 8.44 (s, 2H), 7.89 (s, 2H), 4.11 (t, 4H, 3J=6.9 Hz), 3.20 (t, 4H, 3J=7.2 Hz), 1.89 (p, 4H, 3J=7.8 Hz), 1.62 (p, 4H, 3J=7.2 Hz). 13C NMR (75 MHz, DMSO-d6) δ 147.0, 137.3, 121.5 (CN), 120.9, 112.3 (C=C), 46.6 (CH2N), 33.7 (SCH2), 28.6, 26.2. MS (ES) m/z 363 [M-4-NO2imid.]+, 499 [M+Na]+. Procedure for the synthesis of 2,3,7,8,12,13,17,18-Octakis-[4-(4-nitroimidazol-1-yl) butylthio]porphyrazinatomagnesium(II) (5): magnesium turnings (15 mg, 0.62 mmol), small crystal of iodine and n-butanol (30 mL) were refluxed for 5 h. After the mixture was cooled to room temperature it was transfered via syringe to flask containing dinitrile 4 (289 mg, 0.62 mmol). Reaction mixture was heated and refluxed for another 21 h. The solvent was evaporated with toluene and the residual mixture was dissolved in dichloromethane : pyridine (1:1 v/v) and celite filtered. Next flash column chromatography was performed (CH2Cl2 : MeOH; 35:1 to 10:1 v/v) to give a dark blue film of Pz 5 (23 mg, 8%): Rf (CH2Cl2 : MeOH, 10: 1 v/v)=0.47; UV–Vis (EtOAc : MeOH, 5:3 v/v)=292 (4.78), 378 (4.75), 670 (4.80). MS (MALDI) m/z 1929 [M–H]−; 1931 [M+H]+. 1H NMR (400 MHz, d5-pyridine) δ 8.21 (s, 8H, C5′imid.), 7.72 (s, 8H, C2′imid.), 4.33 (t, 16H, 8×SCH2), 4.07 (t, 16H, 8×CH2N), 2.25 (m, 16H, 8×CH2CH2N), 2.00 (m, 16H, 8×SCH2CH2). 13C NMR (100 MHz, d5-pyridine) 157.6, 148.0, 140.6, 136.8, 120.7, 47.6, 34.4, 29.6, 27.4. S. Michel, B.M. Hoffman, S. Baum, A.G.M. Barrett, Peripherally functionalized porphyrazines: novel metallomacrocycles with broad, untapped potential, in: K.D. Karlin (Ed.), Prog. Inorg. Chem., 50, J. Wiley & Sons, New York, 2001, pp. 473–590. Gaussian 09, Revision B.01. M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, Ö. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian, Inc., Wallingford CT, 2009. A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange, J. Chem. Phys. 98 (1993) 5648–5652. C. Lee, W. Yang, R.G. Parr, Development of the Colle–Salvetti correlation-energy formula into a functional of the electron density, Phys. Rev. B 37 (1988) 785–789. National Institute of Standards and Technology, Vibrational frequency scaling factors, http://cccbdb.nist.gov/vsf.asp, (access 25.10.2012). J. Piskorz, E. Tykarska, M. Gdaniec, T. Goslinski, J. Mielcarek, Synthesis, spectroscopic and photophysical properties of novel styryldiazepinoporphyrazine, Inorg. Chem. Commun. 20 (2012) 13–17. I. Seotsanyana-Mokhosi, N. Kuznetsova, T. Nyokong, Photochemical studies of tetra-2,3-pyridinoporphyrazines, J. Photochem. Photobiol. A 140 (2001) 215–222. T. Goslinski, T. Osmalek, J. Mielcarek, Photochemical and spectral characterization of peripherally modified porphyrazines, Polyhedron 28 (2009) 3839–3843. W. Spiller, H. Kliesch, D. Wöhrle, S. Hackbarth, B. Röder, G. Schnurpfeil, Singlet oxygen quantum yields of different photosensitizers in polar solvents and micellar solutions, J. Porphyr. Phthalocya. 2 (1998) 145–158. K. Ghosh, S. Kumar, R. Kumar, Synthesis and characterization of a novel ruthenium nitrosyl complex and studies on photolability of coordinated NO, Inorg. Chem. Commun. 14 (2011) 146–149. J. Coates, Interpretation of infrared spectra, A practical approach, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, John Wiley & Sons Ltd., Chichester, 2000, pp. 10815–10837. A.R. Katritzky, A.F. Pozharskii, Handbook of Heterocyclic chemistry, second ed. Pergamon, Oxford, UK, 2000. R.M. Silverstein, F.X. Webster, D.J. Kiemle, Spectrometric Identification of Organic Compounds, seventh ed., John Wiley & Sons, USA, 2005, pp. 72–126. M. Durmuş, V. Ahsen, T. Nyokong, Photophysical and photochemical studies of long chain-substituted zinc phthalocyanines, J. Photochem. Photobiol. A 186 (2007) 323–329.
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Inorganic Chemistry Communications journal homepage: www.elsevier.com/locate/inoche
Synthesis, characterization, photochemical properties and cytotoxicity of the novel porphyrazine functionalized with nitroimidazolylbutylsulfanyl groups Sebastian Lijewski a, Jaroslaw Piskorz b, Malgorzata Kucinska c, Marcin Wierzchowski a, Katarzyna Czerniak d, Hanna Billert d, Marek Murias c, Jadwiga Mielcarek b,⁎, Tomasz Goslinski a,⁎ a
Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30, 60-631 Poznan, Poland d Department of Experimental Anesthesiology, Poznan University of Medical Sciences, Sw Marii Magdaleny 14, 61-861 Poznan, Poland b c
a r t i c l e
i n f o
Article history: Received 23 November 2012 Accepted 6 January 2013 Available online 12 January 2013 Keywords: Nitroimidazole Porphyrazine Linstead macrocyclization Singlet oxygen generation Carcinoma
a b s t r a c t A novel porphyrazine analog possessing nitroimidazolylbutylsulfanyl groups was synthesized and characterized using UV–Vis, IR, MS MALDI and various NMR techniques. In addition, a computational model following the Density Functional Theory method (DFT) was applied to analyze the FT IR spectrum. Potential photosensitizing activity of the novel porphyrazine was evaluated by measuring its ability to generate singlet oxygen (ΦΔ), which was found to reach the value of 0.045 in DMF, and 0.035 in DMSO. A lower value of singlet oxygen generation in DMSO may result from the increased tendency to aggregate, which was studied in the UV–Vis and it was found to be stronger in DMSO than in DMF solutions. The investigation indicated no release of nitric oxide (NO) from porphyrazine functionalized with nitroimidazolylbutylsulfanyl groups. In vitro studies of the new compound were carried out to investigate photosensitizer-induced photocytotoxicity on two prostate human cancer cell lines, LNCaP, PC3, and one melanoma derived cell line, MeWo. © 2013 Elsevier B.V. All rights reserved.
Synthetic porphyrinoids such as phthalocyanines and porphyrazines (Pzs) exhibit numerous potential applications in technology and medicine, especially in photodynamic therapy (PDT) and photodynamic diagnosis (PDD) [1–11]. PDT is a relatively new method which has been successfully applied against tumors or dermatological lesions [12–14]. It has been described that nitroimidazoles and related compounds exhibit antibacterial and antiparasitic activity [15,16] and possess anticancer activity in deep hypoxic conditions, thus are often termed “oxygen-mimetic” radiosensitizers [17,18]. Nitroimidazole derivatives (pimonidazole, EF5) have been used for both immunohistochemical detection and non-invasive PET/SPECT imaging of hypoxia [19]. Radiosensitizing properties of nitroimidazoles have been well described for noninvasive in vivo mapping of brain hypoxia after stroke [20] and for imaging of hypoxic myocardium [21]. Moreover, many Technetium-99m labeled nitroimidazole derivatives have been tested for tumor hypoxia imaging [22–26]. Recently, some examples of porphyrinoids containing imidazole groups have been synthesized. Phthalocyanines bearing fused imidazole rings on the periphery have been synthesized and their spectroscopic and electrochemical properties have been studied [27–31]. Moreover, conjugates of 2-nitroimidazole and manganese porphyrin have been obtained and demonstrated selectivity towards tumor SCCVII cell lines implanted in C3H/He ⁎ Corresponding authors. Tel.: +48 61 854 66 33; fax: +48 61 854 66 39. E-mail addresses: [email protected] (J. Mielcarek), [email protected] (T. Goslinski). 1387-7003/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.inoche.2013.01.001
mice with good radiotherapeutic effect at low dose administrations and clear tumor magnetic resonance images [32]. In addition, cationic imidazolium substituted metalloporphyrins with different metal ions have been studied using human (HeLa) and mouse (CT26) cancer cell lines [33]. The promising photodynamic activity of new imidazole substituted pyropheophorbide-a derivatives have been found with A549 human lung adenocarcinoma cells [34]. Furthermore, the cyclodextrin-based nanoassembly consisting of tetraanionic porphyrin and [7-(adamantan-1-yloxy)heptyl]-[4-nitro-3-(trifluoromethyl)phenyl]amine induces high cancer cell mortality through a bimodal action based on the simultaneous photogeneration of nitric oxide (NO) and singlet oxygen (1O2) [35]. We report here the synthesis, photochemical properties, and in vitro cytotoxicity of the novel porphyrazine peripherally functionalized with nitroimidazolylbutylsulfanyl groups (5), carried out to determine its potential as a PDT photosensitizer. Its synthesis was accomplished in a three-step procedure. 4-Nitro-1-(4-bromobutyl)-1H-imidazole (2) was synthesized following the literature method [36] and used in alkylation reaction with commercially available dimercaptomaleonitrile disodium salt (3) to give substituted dimercaptomaleonitrile (4) [37]. Linstead macrocyclization reaction of 4 using magnesium n-butanolate in n-butanol resulted in porphyrazine 5 (Scheme 1) [38,39]. Structure of 5 was confirmed by 1H and 13C NMR studies, accompanied by two-dimensional techniques (1H1H COSY, 1H13C HSQC, 1H13C HMBC) to assist allocation. 1H1H COSY spectrum of 5 revealed connectivities between protons belonging to neighboring methylene groups of
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aliphatic chain. Noteworthy, in HMBC spectrum of 5 cross-peaks between alkyl chain protons (SCH2 at 4.33 ppm) and carbon atoms of macrocyclic core (140.6 ppm) were found. In addition, cross-peaks of CH2N protons at 4.07 ppm and imidazole ring carbon atoms (C5′ at 120.7 and C2′ at 136.8 ppm) were observed (Figs. 1S–4S, Table 1S, Supplementary). We next focused on calculations using the Density Functional Theory (DFT). The procedure consisted of conformational search and vibrational analysis [40–43]. The global minimum of 5 possesses a saddle-like structure of D2 symmetry (Fig. 1). By comparing the calculated molecular vibrational frequencies of 5 molecule with the experimental infrared spectra, the molecular motions that correspond to the measured bands on the basis of similar frequency magnitudes were assigned (Fig. 5S, Table 2S, Supplementary). The calculated and measured frequencies were in reasonable agreement and confirmed presence of nitroimidazole group, porphyrazine ring and aliphatic linker. Aromatic, stretching C–H band (exp. 3118 cm−1, calc. 3178 cm−1), asymmetric stretching NO2 band (exp. 1541 cm−1, calc. 1581 cm−1), imidazole ring stretching skeletal band (exp. 1488 cm−1, calc. 1488 cm−1), imidazole ring C–H breathing band (exp. 1286 cm−1, calc. 1281 cm−1), and imidazole out of plane C–H band (exp. 752 cm−1, calc. 724 cm−1) confirmed the presence of imidazole fragment in the analyzed structure. In addition, 1,4-butylene chain is represented by the following frequencies: asymmetrical and symmetrical stretching C–H (exp. 2919 cm−1, calc. 2945 cm−1 and exp. 2850 cm−1, calc. 2909 cm−1, respectively), scissoring C–H (exp 1403 cm−1, calc. 1444 cm−1), and rocking C–H (exp. 885 cm−1, calc. 876 cm−1). In the dactiloscopic region selected bands of the porphyrazine macrocycle resulting from breathing in plane (exp. 854 cm−1, calc. 867 cm−1) were found. In the UV–Vis spectrum of 5 (Fig. 6S, Supplementary) four distinct absorption bands were observed. Two of them are characteristic of tetrapyrrolic, macrocyclic ring with the Soret band occurring at 378 nm and a sharp, intense Q band at 670 nm. The third band at 292 nm being the result of 4–nitroimidazole ring absorption is consistent with the UV–Vis spectrum of maleonitrile precursor 4. Furthermore, a less intense C–T band, being a result of n→π⁎ transitions (electrondonor properties of sulfur atoms at peripheral positions) at 502 nm was observed [39]. The aggregation behavior of Pz 5 was studied in the UV–Vis in DMSO and DMF solutions (Figs. 2, 7S, Supplementary). Statistical analysis of the linear correlations between absorbance of the Q band and concentration of the Pz 5 revealed that the Beer–Lambert law is obeyed in DMF, but it is not obeyed in DMSO. These results indicate that the Pz 5 has a stronger tendency to aggregate in DMSO solutions [44].
HN
Fig. 1. Calculated and measured FT-IR spectra of Pz 5. Inset presents a saddle-like structure of D2 symmetry.
The potential photosensitizing activity of Pz 5 was evaluated by measuring its ability to generate singlet oxygen. The relative method, with zinc(II) phthalocyanine as a reference and DPBF (1,3-diphenylisobenzofuran) as a chemical quencher for singlet oxygen, was applied (Figs. 2, 8S, Supplementary) [45–47]. Solutions of 5 and DPBF in DMF or DMSO were irradiated with monochromatic light at wavelengths adjusted to the porphyrazine Q-band maximum absorption. The kinetics of subsequent oxidation of DPBF was used for calculation of singlet oxygen generation yield values (ΦΔ DMF =0.045, ΦΔ DMSO =0.035). Higher value of singlet oxygen generation in DMF may result from the above-described stronger tendency of 5 to aggregate in DMSO. The possibility of bimodal action based on the simultaneous photogeneration of nitric oxide (NO) and singlet oxygen (1O2) encouraged us to investigate the potential NO donating properties of this compound. The detection of nitric oxide, which can be generated upon irradiation was based on the reaction between reduced myoglobin and NO, that leads to the formation of myoglobin-NO species. The binding of photoreleased NO with myoglobin can be observed spectrophotometrically as the appearance of a new peak at 419 nm in the UV–Vis spectrum [48]. The intensity of light applied for the irradiation of the Pz 5 equaled N
+ -
N
(i) +
N
O
Br
N
Na S
N
+
+
O
N
O
-
N
R
(ii)
+ -
Na S
R
N
N
O
1
2
3
4 R
N R=
S
N
+
N O
O
N
-
N
N
R
N
R
R N
Mg
R
N
N
(iii)
R
R
N
R
5 Scheme 1. Reagents and conditions: (i) 1,4-dibromobutane, NaH (60% dispersion in mineral oil), DMF, rt to reflux, 2 h [36]; (ii) DMF, K2CO3, 50 °C, 24 h (60%); (iii) Mg(OnBu)2, nBuOH, reflux, 20 h (8%).
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generation (ΦΔ DMF =0.045, ΦΔ DMSO =0.035). A lower value of singlet oxygen generation in DMSO may result from the tendency to aggregation, which was studied in the UV–Vis and found to be stronger in DMSO than DMF solutions. Cytotoxic activity was tested using two prostate (LNCaP, PC3) and one melanoma (MeWo) human cancer derived cell lines. The tested compound revealed low cytotoxicity probably due to its higher tendency to aggregate and insufficient uptake by cells. Additionally, the cytotoxic effect appeared to be cell dependent. The possibility of bimodal action based on the simultaneous photogeneration of nitric oxide and singlet oxygen was excluded as the potential NO donating property of this compound was not observed. Acknowledgments The authors acknowledge financial support for the project from the Polish Ministry of Science and Higher Education (NN401 067238). Appendix A. Supplementary material The following publications refer to the experimental data contained in the Supporting Information [40–43,45–52]. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.inoche. 2013.01.001. References
Fig. 2. (A) UV–Vis spectra of Pz 5 dissolved in DMF. Inset presents a linear correlation between absorbance and concentration; (B) Changes in the UV–Vis spectra for DPBF and Pz 5 in DMF. Inset presents first-order plot for oxidation of the DPBF. Changes in the Q band absorption during the experiment did not exceed 1%.
2.9 mW/cm2 and the irradiation times were similar to that used in the below-described photocytotoxicity experiment. Nevertheless, the band at 419 nm was not observed, which indicated the lack of NO formation (see Supplementary). Biological activity of 5 was determined in vitro using MTT assay on two human prostate cancer cell lines, LNCaP, PC3, and one melanoma derived cell line, MeWo. Dark and light toxicity were evaluated in cells incubated with the tested compound at concentrations: 0.15–10 μM. At these concentrations, 5 in dark conditions did not reduce the viability of LNCaP, whereas PC3 cells were more sensitive and a decrease in the number of viable cells was observed at lower concentrations. Weak photocytotoxic effect of 5 on LNCaP and MeWo cells was observed after 10 and 20 min of irradiation. Surprisingly, no phototoxic effect was observed on PC3 cells after 10 min of irradiation, when proliferative effect was noticed, while significant phototoxicity appeared on the cells irradiated for 20 min (Fig. 9S, Supplementary). The low cytotoxicity at higher concentrations of 5 might be explained by its higher tendency to aggregate (Fig. 10, Supplementary) and insufficient uptake by cells. Summarizing, the new porphyrazine containing nitroimidazolylbutylsulfanyl groups was synthesized and characterized using UV–Vis, IR, MS MALDI and various NMR techniques. In addition, the computational model following the Density Functional Theory method (DFT) was applied to analyze the FT IR spectrum. The ability of novel porphyrazine to generate singlet oxygen, which plays an important role in the cytotoxic effect of PDT, was expressed by the quantum yield of singlet oxygen
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(300 MHz, DMSO-d6) δ 8.44 (s, 2H), 7.89 (s, 2H), 4.11 (t, 4H, 3J=6.9 Hz), 3.20 (t, 4H, 3J=7.2 Hz), 1.89 (p, 4H, 3J=7.8 Hz), 1.62 (p, 4H, 3J=7.2 Hz). 13C NMR (75 MHz, DMSO-d6) δ 147.0, 137.3, 121.5 (CN), 120.9, 112.3 (C=C), 46.6 (CH2N), 33.7 (SCH2), 28.6, 26.2. MS (ES) m/z 363 [M-4-NO2imid.]+, 499 [M+Na]+. Procedure for the synthesis of 2,3,7,8,12,13,17,18-Octakis-[4-(4-nitroimidazol-1-yl) butylthio]porphyrazinatomagnesium(II) (5): magnesium turnings (15 mg, 0.62 mmol), small crystal of iodine and n-butanol (30 mL) were refluxed for 5 h. After the mixture was cooled to room temperature it was transfered via syringe to flask containing dinitrile 4 (289 mg, 0.62 mmol). Reaction mixture was heated and refluxed for another 21 h. The solvent was evaporated with toluene and the residual mixture was dissolved in dichloromethane : pyridine (1:1 v/v) and celite filtered. Next flash column chromatography was performed (CH2Cl2 : MeOH; 35:1 to 10:1 v/v) to give a dark blue film of Pz 5 (23 mg, 8%): Rf (CH2Cl2 : MeOH, 10: 1 v/v)=0.47; UV–Vis (EtOAc : MeOH, 5:3 v/v)=292 (4.78), 378 (4.75), 670 (4.80). MS (MALDI) m/z 1929 [M–H]−; 1931 [M+H]+. 1H NMR (400 MHz, d5-pyridine) δ 8.21 (s, 8H, C5′imid.), 7.72 (s, 8H, C2′imid.), 4.33 (t, 16H, 8×SCH2), 4.07 (t, 16H, 8×CH2N), 2.25 (m, 16H, 8×CH2CH2N), 2.00 (m, 16H, 8×SCH2CH2). 13C NMR (100 MHz, d5-pyridine) 157.6, 148.0, 140.6, 136.8, 120.7, 47.6, 34.4, 29.6, 27.4. S. Michel, B.M. Hoffman, S. Baum, A.G.M. Barrett, Peripherally functionalized porphyrazines: novel metallomacrocycles with broad, untapped potential, in: K.D. Karlin (Ed.), Prog. Inorg. Chem., 50, J. Wiley & Sons, New York, 2001, pp. 473–590. Gaussian 09, Revision B.01. M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, Ö. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian, Inc., Wallingford CT, 2009. A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange, J. Chem. Phys. 98 (1993) 5648–5652. C. Lee, W. Yang, R.G. Parr, Development of the Colle–Salvetti correlation-energy formula into a functional of the electron density, Phys. Rev. B 37 (1988) 785–789. National Institute of Standards and Technology, Vibrational frequency scaling factors, http://cccbdb.nist.gov/vsf.asp, (access 25.10.2012). J. Piskorz, E. Tykarska, M. Gdaniec, T. Goslinski, J. Mielcarek, Synthesis, spectroscopic and photophysical properties of novel styryldiazepinoporphyrazine, Inorg. Chem. Commun. 20 (2012) 13–17. I. Seotsanyana-Mokhosi, N. Kuznetsova, T. Nyokong, Photochemical studies of tetra-2,3-pyridinoporphyrazines, J. Photochem. Photobiol. A 140 (2001) 215–222. T. Goslinski, T. Osmalek, J. Mielcarek, Photochemical and spectral characterization of peripherally modified porphyrazines, Polyhedron 28 (2009) 3839–3843. W. Spiller, H. Kliesch, D. Wöhrle, S. Hackbarth, B. Röder, G. Schnurpfeil, Singlet oxygen quantum yields of different photosensitizers in polar solvents and micellar solutions, J. Porphyr. Phthalocya. 2 (1998) 145–158. K. Ghosh, S. Kumar, R. Kumar, Synthesis and characterization of a novel ruthenium nitrosyl complex and studies on photolability of coordinated NO, Inorg. Chem. Commun. 14 (2011) 146–149. J. Coates, Interpretation of infrared spectra, A practical approach, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, John Wiley & Sons Ltd., Chichester, 2000, pp. 10815–10837. A.R. Katritzky, A.F. Pozharskii, Handbook of Heterocyclic chemistry, second ed. Pergamon, Oxford, UK, 2000. R.M. Silverstein, F.X. Webster, D.J. Kiemle, Spectrometric Identification of Organic Compounds, seventh ed., John Wiley & Sons, USA, 2005, pp. 72–126. M. Durmuş, V. Ahsen, T. Nyokong, Photophysical and photochemical studies of long chain-substituted zinc phthalocyanines, J. Photochem. Photobiol. A 186 (2007) 323–329.