DOI: 10.1002/chem.201400001
Communication
& Photosensitizers
A Glutathione-Activated Phthalocyanine-Based Photosensitizer for Photodynamic Therapy Hui He, Pui-Chi Lo,* and Dennis K. P. Ng*[a] Abstract: A zinc(II) phthalocyanine substituted with a 2,4dinitrobenzenesulfonate group has been prepared. Its fluorescence emission and reactive oxygen species generation can be greatly enhanced by glutathione in phosphate-buffered saline and inside MCF-7 cells. This compound thus functions as a highly efficient molecularbased activatable photosensitizer.
Photodynamic therapy (PDT) has emerged as a clinical modality for treatment of certain localized and superficial cancers.[1] It requires a photosensitive drug, light of a specific wavelength, and molecular oxygen to induce a series of photochemical reactions, resulting in the formation of reactive oxygen species (ROS), in particular singlet oxygen, which is the major cytotoxic agent. These ROS destroy tumors by multiple mechanisms, including killing malignant cells by apoptosis and/or necrosis, shutting down the tumor vasculature, and stimulating the host immune system.[2] With a view to improving the therapeutic outcome, various strategies have been explored to enhance the ROS generation efficiency and tumor selectivity of the photosensitizers.[3] As an alternative approach, “smart” photosensitizers that can be activated by tumor-associated stimuli have received considerable attention.[4] These systems generally contain a photosensitizing unit and a quencher. Their fluorescence and photosensitizing properties are quenched mainly by photoinduced electron transfer (PET)[5] or fluorescence resonance energy transfer.[6] Self-quenched molecular[7] and polymeric[8] systems have also been reported. Upon interactions with acids or thiols, which mimic the acidic and reducing environment of tumors, cancer-related proteases, or nucleic acids, which have high tumor specificity, these systems can be activated by changing the nature of the quencher, detaching it from the photosensitizer, or disaggregating the photosensitizing units, resulting in restoration of their fluorescence and photosensitizing properties. This approach can therefore minimize the damage to normal tissues.
[a] Dr. H. He, Prof. P.-C. Lo, Prof. D. K. P. Ng Department of Chemistry The Chinese University of Hong Kong Shatin, N.T., Hong Kong (P. R. China) Fax: (+ 852) 2603 5057 E-mail: [email protected] [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201400001. Chem. Eur. J. 2014, 20, 6241 – 6245
As part of our continuing effort in the development of novel and efficient photosensitizers for PDT,[9] we have recently prepared several phthalocyanine-based photosensitizers that can be activated by acids[5b, c, 7c] or dithiothreitol.[10] The activation effects have been demonstrated in solutions and in vitro. As an extension of these studies, we report herein a simple yet very efficient photosensitizer based on zinc(II) phthalocyanine, which can be activated by glutathione (GSH). GSH is the most abundant cellular thiol and a major reducing agent in various biochemical processes.[11] The intracellular GSH concentration (ca. 10 mm) is known to be substantially higher than the extracellular levels (ca. 2 mm), which provides a mechanism for selective intracellular drug release.[12] The GSH levels are also often elevated in tumor tissues as compared with normal tissues.[13] This characteristic can also be utilized for selective release and activation of drugs in tumor. To date, a large number of GSH-responsive drug delivery and release systems[14] and fluorescent probes[15] have been reported. However, GSH-activated photosensitizers remain very rare. To our knowledge, only two examples have been reported so far in which chlorin e6 is conjugated to a nanosized graphene oxide[16] or a hyaluronic acid backbone[8f] via a disulfide linker. The compound described below can be regarded as the first molecular GSH-activated photosensitizer. This compound was prepared readily by treating 2-hydroxyphthalocyaninatozinc(II) (1)[17] with 2,4-dinitrobenzenesulfonyl chloride in the presence of Et3N in THF (Scheme 1). The synthesis followed the general method for sulfonates and had a reasonably high yield (70 %). Generally, substituted phthalocyanines are prepared by cyclization of the corresponding substituted phthalonitriles. This post-cyclization modification approach clearly has an advantage as it can prevent undesirable cleavage of the sulfonate during base-promoted cyclization. The strongly electron-withdrawing 2,4-dinitrobenzenesulfonyl group has been widely used to quench the fluorescence of various fluorophores by PET[18] and is believed to serve as an effective quencher for phthalocyanines as well. The electronic absorption spectrum of 2 was recorded in phosphate buffered saline (PBS) in the presence of 0.15 % Tween 80 (Figure S1 in Supporting Information). It showed a Soret band at 345 nm, a vibronic band at 607 nm, and an intense and sharp Q band at 676 nm, which strictly followed the Lambert–Beer law. The result indicated that the compound, after being formulated with Tween 80, was essentially nonaggregated in PBS. The spectrum was very similar to that of compound 1 (Figure S2 in Supporting Information), indicating that the 2,4-dinitrobenzenesulfonyl moiety had little effect on the
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Scheme 1. Synthesis of GSH-responsive photosensitizer 2.
ground-state property of the phthalocyanine core. Upon excitation at 610 nm, compound 2 exhibited a very weak fluorescence emission at 687 nm with a fluorescence quantum yield (FF) of 0.01 relative to the unsubstituted zinc(II) phthalocyanine (ZnPc) in DMF (FF = 0.28).[19] Compound 1, however, showed a much stronger fluorescence (Figure S3 in Supporting Information). Its fluorescence quantum yield was 0.20, which was 20-fold higher than that of compound 2. The result clearly indicated that the fluorescence of phthalocyanine was effectively quenched by the 2,4-dinitrobenzenesulfonyl moiety. To demonstrate that compound 2 is responsive toward biological reducing stimuli, we recorded the fluorescence spectra of 2 (4 mm) in the presence of GSH (1 mm) over 5 h (Figure S4 in Supporting Information). The fluorescence intensity increased gradually, particularly during the first 2 h. Figure 1 shows the change in fluorescence spectrum of 2 with the concentration of GSH in PBS after stirring the mixtures for 2 h. Similarly, the intensity increases gradually with increasing concentrations of GSH. This can be attributed to the separation of the 2,4-dinitrobenzenesulfonyl quencher from the phthalocyanine core as a result of cleavage of the sulfonate ester by GSH. The final intensity was close to the fluorescence intensity of 1 under the same conditions. The cleavage rate seemed to be comparable with that observed for other systems containing
Figure 1. Change in fluorescence spectrum of 2 (4 mm) upon addition of GSH (0-1 mm) in PBS with 0.15 % Tween 80. The spectra were recorded after stirring the mixtures for 2 h. The inset plots the fluorescence intensity at 687 nm versus the concentration of GSH. Chem. Eur. J. 2014, 20, 6241 – 6245
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the disulfide bond, which is a very common thiol-cleavable linker.[14d, 15d, 20] The singlet oxygen generation efficiency of 1 and 2 in PBS was also measured and compared using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) as the probe, which can be oxidized by singlet oxygen to yield the corresponding endoperoxide.[21] The efficiency was reflected by the rate of decay of ABMDMA measured by monitoring the decrease in absorbance at 400 nm. As shown in Figure 2, compound 2 is the least efficient singlet
Figure 2. Comparison of the rate of decay of ABMDMA (initial concentration = 90 mm) in PBS with 0.15 % Tween 80 using 1, 2, and ZnPc as the photosensitizers (all at 4 mm). For the data points with GSH (1 mm), the mixture was stirred at ambient temperature for 2 h before the addition of ABMDMA and recording of data.
oxygen generator, which can be attributed to the presence of 2,4-dinitrobenzenesulfonyl quencher. However, upon addition of GSH (1 mm), the singlet oxygen generation efficiency of 2 was greatly enhanced due to the cleavage of the sulfonate ester and removal of the quencher. The resulting efficiency was comparable with that of 1 and ZnPc used as the reference. To study the activation of 2 by GSH at the cellular level, the intracellular fluorescence of 1 and 2 was first examined and compared. Since GSH cannot be internalized by cells due to its anionic nature, glutathione monoester (GSH-OEt) was used as an external stimulus. This compound can be readily internalized by cells and rapidly hydrolyzed to generate GSH.[14a, 22] For this part of study, MCF-7 human breast cancer cells were first treated with 0 or 10 mm GSH-OEt for 30 min, followed by incubation with 1 or 2 for 2 h. As shown in Figure 3, the cells incubated with 2 without the pretreatment with GSH-OEt show a weak intracellular fluorescence. In contrast, the fluorescence intensity was greatly enhanced (by ca. 4-fold) when the cells were pretreated with GSH-OEt. The intensity was comparable with that for 1, which was not affected by the pretreatment. 6242
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Figure 4. ROS production induced by 1 or 2 (4 mm) in MCF-7 cells with or without pretreatment with GSH-OEt (10 mm) in the absence or presence of light (l > 610 nm, 40 mW cm 2, 48 J cm 2). Data are expressed as the mean standard error of the mean (SEM) of three independent experiments, each performed in quadruplicate.
Figure 3. a) Bright field and confocal fluorescence images of MCF-7 cells after incubation with 0 or 10 mm GSH-OEt for 30 min, followed with 1 or 2 (2 mm) for 2 h. b) Comparison of the relative intracellular fluorescence intensity of 1 and 2 without or with the pretreatment with GSH-OEt. Data are expressed as the mean standard deviation (number of cells = 25).
The intracellular ROS production by these compounds was also studied using 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) as the quencher.[23] In this experiment, MCF-7 cells were first treated with GSH-OEt (10 mm) for 2 h, followed by incubation with 1 or 2 (4 mm) for a further 2 h. Both 1 and 2 could not generate ROS in the dark, but upon illumination, they could sensitize the ROS production in the cells (Figure 4). The efficiency was significantly higher for 1 compared with 2. For the case of preincubation with GSH, the intracellular ROS production by 2 was greatly enhanced and the efficiency was comparable with that of 1, for which the effect of GSH was negligible. These results clearly showed that both the fluorescence and ROS generation efficiency of 2 could also be enhanced by GSH in tumor cells. The photocytotoxicity of 1 and 2 was also briefly examined against MCF-7 cells. The dark toxicity of these compounds was not significant. However, both compounds were highly photocytotoxic with an IC50 value, defined as the dye concentration required to kill 50 % of the cells, of 0.13 mm (for 1) or 0.19 mm (for 2) (Figure S5 in Supporting Information). For this assay, the response of these two compounds was similar. It is likely that 2 was activated by intracellular GSH and even a small amount of ROS generated could effectively trigger the oxidative damage of the cells. Finally, the activation of 2 was demonstrated in vivo. Nude mice bearing a HT29 human colorectal carcinoma were treated Chem. Eur. J. 2014, 20, 6241 – 6245
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with an intratumoral dose of 2 (1 mmol per kg body weight). Their whole-body fluorescence images were then monitored and quantified continuously for 24 h. As shown in Figure 5, the fluorescence of 2 was gradually increased in the first 8 h and was slightly diffused afterward till 24 h. This observation clearly showed that the fluorescence of 2 was restored inside the tumor, likely due to the detachment of the quencher. As a control, another group of nude mice were treated with the non-responsive analogue 1 similarly. The intratumoral fluorescence intensity after injection for 1 h was already noticeable, which increased slightly and dropped again over 24 h (Figure S6 in Supporting Information). The results were quite consistent with an “always-on” situation. In summary, we have developed a simple yet very efficient phthalocyanine-based activatable photosensitizer. Its fluores-
Figure 5. Change in fluorescence intensity per unit area of the tumor with time. The inset shows the fluorescence images of the tumor-bearing nude mice before and after intratumoral injection of 2 (1 mmol per kg body weight) over 24 h (n = 5).
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Communication cence emission and singlet oxygen (or ROS) generation can be activated by GSH in PBS and inside MCF-7 cells owing to the separation of the photosensitizing unit and the quencher as a result of cleavage of the sulfonate ester linker by GSH. The fluorescence activation has also been demonstrated in vivo. On the basis that the intracellular GSH level is in the mM range and tumor tissues generally have an elevated GSH concentration compared with normal tissues, this molecular-based GSHactivated system can serve as a promising tumor-selective fluorescent probe and photosensitizer for targeted PDT.
Experimental Section Preparation of 2 A solution of 2,4-dinitrobenzenesulfonyl chloride (40 mg, 0.15 mol) in THF (2 mL) was added dropwise into a mixture of 2-hydroxyphthalocyaninatozinc(II) (1) (50 mg, 84 mmol) and Et3N (0.1 mL) in THF (10 mL) at 0 8C. The mixture was stirred at ambient temperature for 1 h. It was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography using THF/ hexane (2:3, v/v) as the eluent. The bluish green fraction was collected and concentrated. The crude product was further purified by recrystallization from a mixture of THF and hexane to afford a bluish green solid (49 mg, 70 %). 1H NMR (400 MHz, [D6]DMSO with a trace amount of [D5]pyridine): d = 9.18–9.29 (m, 8 H, Pc-Ha), 9.04 (br s, 1 H, ArH), 8.64 (dd, J = 2.4, 8.8 Hz, 1 H, ArH), 8.47 (d, J = 8.8 Hz, 1 H, ArH), 8.19–8.25 (m, 6 H, Pc-Hb), 7.82 ppm (dd, J = 2.0, 8.4 Hz, 1 H, Pc-Hb) (Figure S7 in Supporting Information); 13C{1H} NMR (100.6 MHz, [D6]DMSO with a trace amount of [D5]pyridine): d = 153.9, 153.6, 152.5, 152.3, 151.8, 150.1, 149.9, 148.7, 148.5, 138.7, 138.2, 138.1, 138.0, 136.5, 134.2, 130.9, 129.5, 129.3, 129.2, 127.7, 122.4, 122.3, 122.2, 122.1, 121.3, 115.4 ppm (some of the signals were overlapped); MS (ESI): an isotopic cluster peaking at m/z 823 (100 %) [M+H] + ; HRMS (ESI): m/z calcd for C38H18N10O7SZn: 823.0445 [M+H] + ; found 823.0444. The experimental details for studying the GSH-responsive properties, photocytotoxicity, and in vivo imaging are given in Supporting Information.
Acknowledgements We thank Prof. W.-P. Fong and S.-L. Yeung for technical support in the biological studies. This work was supported by a grant from the Research Grant Council of the Hong Kong Special Administrative Region (project no. 402211). Keywords: glutathione · photodynamic therapy photosensitizers · phthalocyanines · singlet oxygen
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[1] a) D. E. J. G. J. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev. Cancer 2003, 3, 380 – 387; b) S. B. Brown, E. A. Brown, I. Walker, Lancet Oncol. 2004, 5, 497 – 508. [2] a) A. P. Castano, P. Mroz, M. R. Hamblin, Nat. Rev. Cancer 2006, 6, 535 – 545; b) L. M. Sanabria, M. E. Rodrguez, I. S. Cogno, N. B. R. Vittar, M. F. Pansa, M. J. Lamberti, V. A. Rivarola, Biochim. Biophys. Acta 2013, 1835, 36 – 45. [3] a) A. M. Bugaj, Photochem. Photobiol. Sci. 2011, 10, 1097 – 1109; b) F. Schmitt, L. Juillerat-Jeanneret, Anti-Cancer Agents Med. Chem. 2012, 12, 500 – 525. Chem. Eur. J. 2014, 20, 6241 – 6245
www.chemeurj.org
[4] a) J. F. Lovell, T. W. B. Liu, J. Chen, G. Zheng, Chem. Rev. 2010, 110, 2839 – 2857; b) M. Verhille, P. Couleaud, R. Vanderess, D. Brault, M. BarberiHeyob, C. Frochot, Curr. Med. Chem. 2010, 17, 3925 – 3943. [5] a) S. O. McDonnell, M. J. Hall, L. T. Allen, A. Byrne, W. M. Gallagher, D. F. O’Shea, J. Am. Chem. Soc. 2005, 127, 16360 – 16361; b) X.-J. Jiang, P.-C. Lo, Y.-M. Tsang, S.-L. Yeung, W.-P. Fong, D. K. P. Ng, Chem. Eur. J. 2010, 16, 4777 – 4783; c) X.-J. Jiang, P.-C. Lo, S.-L. Yeung, W.-P. Fong, D. K. P. Ng, Chem. Commun. 2010, 46, 3188 – 3190. [6] a) P.-C. Lo, J. Chen, K. Stefflova, M. S. Warren, R. Navab, B. Bandarchi, S. Mullins, M. Tsao, J. D. Cheng, G. Zheng, J. Med. Chem. 2009, 52, 358 – 368; b) J. Chen, T. W. B. Liu, P.-C. Lo, B. C. Wilson, G. Zheng, Bioconjugate Chem. 2009, 20, 1836 – 1842; c) Z. Tang, Z. Zhu, P. Mallikaratchy, R. Yang, K. Sefah, W. Tan, Chem. Asian J. 2010, 5, 783 – 786; d) M. Verhille, H. Benachour, A. Ibrahim, M. Achard, P. Arnoux, M. Barberi-Heyob, J.-C. Andre, X. Allonas, F. Baros, R. Vanderesse, C. Frochot, Curr. Med. Chem. 2012, 19, 5580 – 5594. [7] a) X. Zheng, U. W. Sallum, S. Verma, H. Athar, C. L. Evans, T. Hasan, Angew. Chem. 2009, 121, 2182 – 2185; Angew. Chem. Int. Ed. 2009, 48, 2148 – 2151; b) Y. Gao, G. Qiao, L. Zhuo, N. Li, Y. Liu, B. Tang, Chem. Commun. 2011, 47, 5316 – 5318; c) M.-R. Ke, D. K. P. Ng, P.-C. Lo, Chem. Commun. 2012, 48, 9065 – 9067. [8] a) Y. Choi, R. Weissleder, C.-H. Tung, Cancer Res. 2006, 66, 7225 – 7229; b) B.-c. Bae, K. Na, Biomaterials 2010, 31, 6325 – 6335; c) H. Koo, H. Lee, S. Lee, K. H. Min, M. S. Kim, D. S. Lee, Y. Choi, I. C. Kwon, K. Kim, S. Y. Jeong, Chem. Commun. 2010, 46, 5668 – 5670; d) S. Y. Park, H. J. Baik, Y. T. Oh, K. T. Oh, Y. S. Youn, E. S. Lee, Angew. Chem. 2011, 123, 1682 – 1685; Angew. Chem. Int. Ed. 2011, 50, 1644 – 1647; e) S. J. Lee, H. Koo, D.-E. Lee, S. Min, S. Lee, X. Chen, Y. Choi, J. F. Leary, K. Park, S. Y. Jeong, I. C. Kwon, K. Kim, K. Choi, Biomaterials 2011, 32, 4021 – 4029; f) H. Kim, S. Mun, Y. Choi, J. Mater. Chem. B 2013, 1, 429 – 431. [9] For some recent examples, see a) X.-J. Jiang, S.-L. Yeung, P.-C. Lo, W.-P. Fong, D. K. P. Ng, J. Med. Chem. 2011, 54, 320 – 330; b) J. T. F. Lau, P.-C. Lo, W.-P. Fong, D. K. P. Ng, Chem. Eur. J. 2011, 17, 7569 – 7577; c) J. T. F. Lau, P.-C. Lo, Y.-M. Tsang, W.-P. Fong, D. K. P. Ng, Chem. Commun. 2011, 47, 9657 – 9659; d) M.-R. Ke, S.-L. Yeung, W.-P. Fong, D. K. P. Ng, P.-C. Lo, Chem. Eur. J. 2012, 18, 4225 – 4233; e) J. T. F. Lau, P.-C. Lo, W.-P. Fong, D. K. P. Ng, J. Med. Chem. 2012, 55, 5446 – 5454; f) M.-R. Ke, S.-L. Yeung, D. K. P. Ng, W.-P. Fong, P.-C. Lo, J. Med. Chem. 2013, 56, 8475 – 8483. [10] J. T. F. Lau, X.-J. Jiang, D. K. P. Ng, P.-C. Lo, Chem. Commun. 2013, 49, 4274 – 4276. [11] A. Meister, M. E. Anderson, Ann. Rev. Biochem. 1983, 52, 711 – 760. [12] R. Cheng, F. Feng, F. Meng, C. Deng, J. Feijen, Z. Zhong, J. Controlled Release 2011, 152, 2 – 12. [13] M. P. Gamcsik, M. S. Kasibhatla, S. D. Teeter, O. M. Colvin, Biomarkers 2012, 17, 671 – 691. [14] a) R. Hong, G. Han, J. M. Fernndez, B.-j. Kim, N. S. Forbes, V. M. Rotello, J. Am. Chem. Soc. 2006, 128, 1078 – 1079; b) J. Chen, S. Chen, X. Zhao, L. V. Kuznetsova, S. S. Wong, I. Ojima, J. Am. Chem. Soc. 2008, 130, 16778 – 16785; c) A. N. Koo, H. J. Lee, S. E. Kim, J. H. Chang, C. Park, C. Kim, J. H. Park, S. C. Lee, Chem. Commun. 2008, 6570 – 6572; d) M. H. Lee, J. Y. Kim, J. H. Han, S. Bhuniya, J. L. Sessler, C. Kang, J. S. Kim, J. Am. Chem. Soc. 2012, 134, 12668 – 12674; e) W. Cao, Y. Li, Y. Yi, S. Ji, L. Zeng, Z. Sun, H. Xu, Chem. Sci. 2012, 3, 3403 – 3408; f) L. Wang, M. Kim, Q. Fang, J. Min, W. I. Jeon, S. Y. Lee, S. J. Son, S.-W. Joo, S. B. Lee, Chem. Commun. 2013, 49, 3194 – 3196; g) J. Lai, B. P. Shah, E. Garfunkel, K.-B. Lee, ACS Nano 2013, 7, 2741 – 2750. [15] a) N. Shao, J. Jin, H. Wang, J. Zheng, R. Yang, W. Chan, Z. Abliz, J. Am. Chem. Soc. 2010, 132, 725 – 736; b) J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han, B. R. Cho, J. Am. Chem. Soc. 2010, 132, 1216 – 1217; c) B. Zhu, X. Zhang, Y. Li, P. Wang, H. Zhang, X. Zhuang, Chem. Commun. 2010, 46, 5710 – 5712; d) M. H. Lee, J. H. Han, P.-S. Kwon, S. Bhuniya, J. Y. Kim, J. L. Sessler, C. Kang, J. S. Kim, J. Am. Chem. Soc. 2012, 134, 1316 – 1322; e) L.-Y. Niu, Y.-S. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, J. Am. Chem. Soc. 2012, 134, 18928 – 18931; f) K. Xu, M. Qiang, W. Gao, R. Su, N. Li, Y. Gao, Y. Xie, F. Kong, B. Tang, Chem. Sci. 2013, 4, 1079 – 1086; g) Z. Lou, P. Li, X. Sun, S. Yang, B. Wang, K. Han, Chem. Commun. 2013, 49, 391 – 393; h) G. Li, Y. Chen, J. Wu, L. Ji, H. Chao, Chem. Commun. 2013, 49, 2040 – 2042; i) M. Isik, T. Ozdemir, I. S. Turan, S. Kolemen, E. U. Akkaya, Org. Lett. 2013, 15, 216 – 219. [16] Y. Cho, Y. Choi, Chem. Commun. 2012, 48, 9912 – 9914.
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Communication [17] M. Hu, N. Brasseur, S. Z. Yildiz, J. E. van Lier, C. C. Leznoff, J. Med. Chem. 1998, 41, 1789 – 1802. [18] a) J. Bhaumik, R. Weissleder, J. R. McCarthy, J. Org. Chem. 2009, 74, 5894 – 5901; b) J. Shao, H. Sun, H. Guo, S. Ji, J. Zhao, W. Wu, X. Yuan, C. Zhang, T. D. James, Chem. Sci. 2012, 3, 1049 – 1061. [19] I. Scalise, E. N. Durantini, Bioorg. Med. Chem. 2005, 13, 3037 – 3045. [20] S. Santra, C. Kaittanis, O. J. Santiesteban, J. M. Perez, J. Am. Chem. Soc. 2011, 133, 16680 – 16688. [21] N. A. Kuznetsova, N. S. Gretsova, O. A. Yuzhakova, V. M. Negrimovskii, O. L. Kaliya, E. A. Luk’yanets, Russ. J. Gen. Chem. 2001, 71, 36 – 41.
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[22] M. E. Anderson, A. Meister, Anal. Biochem. 1989, 183, 16 – 20. [23] H. M. Shen, C. Y. Shi, Y. Shen, C. N. Ong, Free Radical Biol. Med. 1996, 21, 139 – 146.
Received: January 1, 2014 Revised: March 18, 2014 Published online on April 15, 2014
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& Photosensitizers
A Glutathione-Activated Phthalocyanine-Based Photosensitizer for Photodynamic Therapy Hui He, Pui-Chi Lo,* and Dennis K. P. Ng*[a] Abstract: A zinc(II) phthalocyanine substituted with a 2,4dinitrobenzenesulfonate group has been prepared. Its fluorescence emission and reactive oxygen species generation can be greatly enhanced by glutathione in phosphate-buffered saline and inside MCF-7 cells. This compound thus functions as a highly efficient molecularbased activatable photosensitizer.
Photodynamic therapy (PDT) has emerged as a clinical modality for treatment of certain localized and superficial cancers.[1] It requires a photosensitive drug, light of a specific wavelength, and molecular oxygen to induce a series of photochemical reactions, resulting in the formation of reactive oxygen species (ROS), in particular singlet oxygen, which is the major cytotoxic agent. These ROS destroy tumors by multiple mechanisms, including killing malignant cells by apoptosis and/or necrosis, shutting down the tumor vasculature, and stimulating the host immune system.[2] With a view to improving the therapeutic outcome, various strategies have been explored to enhance the ROS generation efficiency and tumor selectivity of the photosensitizers.[3] As an alternative approach, “smart” photosensitizers that can be activated by tumor-associated stimuli have received considerable attention.[4] These systems generally contain a photosensitizing unit and a quencher. Their fluorescence and photosensitizing properties are quenched mainly by photoinduced electron transfer (PET)[5] or fluorescence resonance energy transfer.[6] Self-quenched molecular[7] and polymeric[8] systems have also been reported. Upon interactions with acids or thiols, which mimic the acidic and reducing environment of tumors, cancer-related proteases, or nucleic acids, which have high tumor specificity, these systems can be activated by changing the nature of the quencher, detaching it from the photosensitizer, or disaggregating the photosensitizing units, resulting in restoration of their fluorescence and photosensitizing properties. This approach can therefore minimize the damage to normal tissues.
[a] Dr. H. He, Prof. P.-C. Lo, Prof. D. K. P. Ng Department of Chemistry The Chinese University of Hong Kong Shatin, N.T., Hong Kong (P. R. China) Fax: (+ 852) 2603 5057 E-mail: [email protected] [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201400001. Chem. Eur. J. 2014, 20, 6241 – 6245
As part of our continuing effort in the development of novel and efficient photosensitizers for PDT,[9] we have recently prepared several phthalocyanine-based photosensitizers that can be activated by acids[5b, c, 7c] or dithiothreitol.[10] The activation effects have been demonstrated in solutions and in vitro. As an extension of these studies, we report herein a simple yet very efficient photosensitizer based on zinc(II) phthalocyanine, which can be activated by glutathione (GSH). GSH is the most abundant cellular thiol and a major reducing agent in various biochemical processes.[11] The intracellular GSH concentration (ca. 10 mm) is known to be substantially higher than the extracellular levels (ca. 2 mm), which provides a mechanism for selective intracellular drug release.[12] The GSH levels are also often elevated in tumor tissues as compared with normal tissues.[13] This characteristic can also be utilized for selective release and activation of drugs in tumor. To date, a large number of GSH-responsive drug delivery and release systems[14] and fluorescent probes[15] have been reported. However, GSH-activated photosensitizers remain very rare. To our knowledge, only two examples have been reported so far in which chlorin e6 is conjugated to a nanosized graphene oxide[16] or a hyaluronic acid backbone[8f] via a disulfide linker. The compound described below can be regarded as the first molecular GSH-activated photosensitizer. This compound was prepared readily by treating 2-hydroxyphthalocyaninatozinc(II) (1)[17] with 2,4-dinitrobenzenesulfonyl chloride in the presence of Et3N in THF (Scheme 1). The synthesis followed the general method for sulfonates and had a reasonably high yield (70 %). Generally, substituted phthalocyanines are prepared by cyclization of the corresponding substituted phthalonitriles. This post-cyclization modification approach clearly has an advantage as it can prevent undesirable cleavage of the sulfonate during base-promoted cyclization. The strongly electron-withdrawing 2,4-dinitrobenzenesulfonyl group has been widely used to quench the fluorescence of various fluorophores by PET[18] and is believed to serve as an effective quencher for phthalocyanines as well. The electronic absorption spectrum of 2 was recorded in phosphate buffered saline (PBS) in the presence of 0.15 % Tween 80 (Figure S1 in Supporting Information). It showed a Soret band at 345 nm, a vibronic band at 607 nm, and an intense and sharp Q band at 676 nm, which strictly followed the Lambert–Beer law. The result indicated that the compound, after being formulated with Tween 80, was essentially nonaggregated in PBS. The spectrum was very similar to that of compound 1 (Figure S2 in Supporting Information), indicating that the 2,4-dinitrobenzenesulfonyl moiety had little effect on the
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Scheme 1. Synthesis of GSH-responsive photosensitizer 2.
ground-state property of the phthalocyanine core. Upon excitation at 610 nm, compound 2 exhibited a very weak fluorescence emission at 687 nm with a fluorescence quantum yield (FF) of 0.01 relative to the unsubstituted zinc(II) phthalocyanine (ZnPc) in DMF (FF = 0.28).[19] Compound 1, however, showed a much stronger fluorescence (Figure S3 in Supporting Information). Its fluorescence quantum yield was 0.20, which was 20-fold higher than that of compound 2. The result clearly indicated that the fluorescence of phthalocyanine was effectively quenched by the 2,4-dinitrobenzenesulfonyl moiety. To demonstrate that compound 2 is responsive toward biological reducing stimuli, we recorded the fluorescence spectra of 2 (4 mm) in the presence of GSH (1 mm) over 5 h (Figure S4 in Supporting Information). The fluorescence intensity increased gradually, particularly during the first 2 h. Figure 1 shows the change in fluorescence spectrum of 2 with the concentration of GSH in PBS after stirring the mixtures for 2 h. Similarly, the intensity increases gradually with increasing concentrations of GSH. This can be attributed to the separation of the 2,4-dinitrobenzenesulfonyl quencher from the phthalocyanine core as a result of cleavage of the sulfonate ester by GSH. The final intensity was close to the fluorescence intensity of 1 under the same conditions. The cleavage rate seemed to be comparable with that observed for other systems containing
Figure 1. Change in fluorescence spectrum of 2 (4 mm) upon addition of GSH (0-1 mm) in PBS with 0.15 % Tween 80. The spectra were recorded after stirring the mixtures for 2 h. The inset plots the fluorescence intensity at 687 nm versus the concentration of GSH. Chem. Eur. J. 2014, 20, 6241 – 6245
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the disulfide bond, which is a very common thiol-cleavable linker.[14d, 15d, 20] The singlet oxygen generation efficiency of 1 and 2 in PBS was also measured and compared using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) as the probe, which can be oxidized by singlet oxygen to yield the corresponding endoperoxide.[21] The efficiency was reflected by the rate of decay of ABMDMA measured by monitoring the decrease in absorbance at 400 nm. As shown in Figure 2, compound 2 is the least efficient singlet
Figure 2. Comparison of the rate of decay of ABMDMA (initial concentration = 90 mm) in PBS with 0.15 % Tween 80 using 1, 2, and ZnPc as the photosensitizers (all at 4 mm). For the data points with GSH (1 mm), the mixture was stirred at ambient temperature for 2 h before the addition of ABMDMA and recording of data.
oxygen generator, which can be attributed to the presence of 2,4-dinitrobenzenesulfonyl quencher. However, upon addition of GSH (1 mm), the singlet oxygen generation efficiency of 2 was greatly enhanced due to the cleavage of the sulfonate ester and removal of the quencher. The resulting efficiency was comparable with that of 1 and ZnPc used as the reference. To study the activation of 2 by GSH at the cellular level, the intracellular fluorescence of 1 and 2 was first examined and compared. Since GSH cannot be internalized by cells due to its anionic nature, glutathione monoester (GSH-OEt) was used as an external stimulus. This compound can be readily internalized by cells and rapidly hydrolyzed to generate GSH.[14a, 22] For this part of study, MCF-7 human breast cancer cells were first treated with 0 or 10 mm GSH-OEt for 30 min, followed by incubation with 1 or 2 for 2 h. As shown in Figure 3, the cells incubated with 2 without the pretreatment with GSH-OEt show a weak intracellular fluorescence. In contrast, the fluorescence intensity was greatly enhanced (by ca. 4-fold) when the cells were pretreated with GSH-OEt. The intensity was comparable with that for 1, which was not affected by the pretreatment. 6242
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Figure 4. ROS production induced by 1 or 2 (4 mm) in MCF-7 cells with or without pretreatment with GSH-OEt (10 mm) in the absence or presence of light (l > 610 nm, 40 mW cm 2, 48 J cm 2). Data are expressed as the mean standard error of the mean (SEM) of three independent experiments, each performed in quadruplicate.
Figure 3. a) Bright field and confocal fluorescence images of MCF-7 cells after incubation with 0 or 10 mm GSH-OEt for 30 min, followed with 1 or 2 (2 mm) for 2 h. b) Comparison of the relative intracellular fluorescence intensity of 1 and 2 without or with the pretreatment with GSH-OEt. Data are expressed as the mean standard deviation (number of cells = 25).
The intracellular ROS production by these compounds was also studied using 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) as the quencher.[23] In this experiment, MCF-7 cells were first treated with GSH-OEt (10 mm) for 2 h, followed by incubation with 1 or 2 (4 mm) for a further 2 h. Both 1 and 2 could not generate ROS in the dark, but upon illumination, they could sensitize the ROS production in the cells (Figure 4). The efficiency was significantly higher for 1 compared with 2. For the case of preincubation with GSH, the intracellular ROS production by 2 was greatly enhanced and the efficiency was comparable with that of 1, for which the effect of GSH was negligible. These results clearly showed that both the fluorescence and ROS generation efficiency of 2 could also be enhanced by GSH in tumor cells. The photocytotoxicity of 1 and 2 was also briefly examined against MCF-7 cells. The dark toxicity of these compounds was not significant. However, both compounds were highly photocytotoxic with an IC50 value, defined as the dye concentration required to kill 50 % of the cells, of 0.13 mm (for 1) or 0.19 mm (for 2) (Figure S5 in Supporting Information). For this assay, the response of these two compounds was similar. It is likely that 2 was activated by intracellular GSH and even a small amount of ROS generated could effectively trigger the oxidative damage of the cells. Finally, the activation of 2 was demonstrated in vivo. Nude mice bearing a HT29 human colorectal carcinoma were treated Chem. Eur. J. 2014, 20, 6241 – 6245
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with an intratumoral dose of 2 (1 mmol per kg body weight). Their whole-body fluorescence images were then monitored and quantified continuously for 24 h. As shown in Figure 5, the fluorescence of 2 was gradually increased in the first 8 h and was slightly diffused afterward till 24 h. This observation clearly showed that the fluorescence of 2 was restored inside the tumor, likely due to the detachment of the quencher. As a control, another group of nude mice were treated with the non-responsive analogue 1 similarly. The intratumoral fluorescence intensity after injection for 1 h was already noticeable, which increased slightly and dropped again over 24 h (Figure S6 in Supporting Information). The results were quite consistent with an “always-on” situation. In summary, we have developed a simple yet very efficient phthalocyanine-based activatable photosensitizer. Its fluores-
Figure 5. Change in fluorescence intensity per unit area of the tumor with time. The inset shows the fluorescence images of the tumor-bearing nude mice before and after intratumoral injection of 2 (1 mmol per kg body weight) over 24 h (n = 5).
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Communication cence emission and singlet oxygen (or ROS) generation can be activated by GSH in PBS and inside MCF-7 cells owing to the separation of the photosensitizing unit and the quencher as a result of cleavage of the sulfonate ester linker by GSH. The fluorescence activation has also been demonstrated in vivo. On the basis that the intracellular GSH level is in the mM range and tumor tissues generally have an elevated GSH concentration compared with normal tissues, this molecular-based GSHactivated system can serve as a promising tumor-selective fluorescent probe and photosensitizer for targeted PDT.
Experimental Section Preparation of 2 A solution of 2,4-dinitrobenzenesulfonyl chloride (40 mg, 0.15 mol) in THF (2 mL) was added dropwise into a mixture of 2-hydroxyphthalocyaninatozinc(II) (1) (50 mg, 84 mmol) and Et3N (0.1 mL) in THF (10 mL) at 0 8C. The mixture was stirred at ambient temperature for 1 h. It was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography using THF/ hexane (2:3, v/v) as the eluent. The bluish green fraction was collected and concentrated. The crude product was further purified by recrystallization from a mixture of THF and hexane to afford a bluish green solid (49 mg, 70 %). 1H NMR (400 MHz, [D6]DMSO with a trace amount of [D5]pyridine): d = 9.18–9.29 (m, 8 H, Pc-Ha), 9.04 (br s, 1 H, ArH), 8.64 (dd, J = 2.4, 8.8 Hz, 1 H, ArH), 8.47 (d, J = 8.8 Hz, 1 H, ArH), 8.19–8.25 (m, 6 H, Pc-Hb), 7.82 ppm (dd, J = 2.0, 8.4 Hz, 1 H, Pc-Hb) (Figure S7 in Supporting Information); 13C{1H} NMR (100.6 MHz, [D6]DMSO with a trace amount of [D5]pyridine): d = 153.9, 153.6, 152.5, 152.3, 151.8, 150.1, 149.9, 148.7, 148.5, 138.7, 138.2, 138.1, 138.0, 136.5, 134.2, 130.9, 129.5, 129.3, 129.2, 127.7, 122.4, 122.3, 122.2, 122.1, 121.3, 115.4 ppm (some of the signals were overlapped); MS (ESI): an isotopic cluster peaking at m/z 823 (100 %) [M+H] + ; HRMS (ESI): m/z calcd for C38H18N10O7SZn: 823.0445 [M+H] + ; found 823.0444. The experimental details for studying the GSH-responsive properties, photocytotoxicity, and in vivo imaging are given in Supporting Information.
Acknowledgements We thank Prof. W.-P. Fong and S.-L. Yeung for technical support in the biological studies. This work was supported by a grant from the Research Grant Council of the Hong Kong Special Administrative Region (project no. 402211). Keywords: glutathione · photodynamic therapy photosensitizers · phthalocyanines · singlet oxygen
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[1] a) D. E. J. G. J. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev. Cancer 2003, 3, 380 – 387; b) S. B. Brown, E. A. Brown, I. Walker, Lancet Oncol. 2004, 5, 497 – 508. [2] a) A. P. Castano, P. Mroz, M. R. Hamblin, Nat. Rev. Cancer 2006, 6, 535 – 545; b) L. M. Sanabria, M. E. Rodrguez, I. S. Cogno, N. B. R. Vittar, M. F. Pansa, M. J. Lamberti, V. A. Rivarola, Biochim. Biophys. Acta 2013, 1835, 36 – 45. [3] a) A. M. Bugaj, Photochem. Photobiol. Sci. 2011, 10, 1097 – 1109; b) F. Schmitt, L. Juillerat-Jeanneret, Anti-Cancer Agents Med. Chem. 2012, 12, 500 – 525. Chem. Eur. J. 2014, 20, 6241 – 6245
www.chemeurj.org
[4] a) J. F. Lovell, T. W. B. Liu, J. Chen, G. Zheng, Chem. Rev. 2010, 110, 2839 – 2857; b) M. Verhille, P. Couleaud, R. Vanderess, D. Brault, M. BarberiHeyob, C. Frochot, Curr. Med. Chem. 2010, 17, 3925 – 3943. [5] a) S. O. McDonnell, M. J. Hall, L. T. Allen, A. Byrne, W. M. Gallagher, D. F. O’Shea, J. Am. Chem. Soc. 2005, 127, 16360 – 16361; b) X.-J. Jiang, P.-C. Lo, Y.-M. Tsang, S.-L. Yeung, W.-P. Fong, D. K. P. Ng, Chem. Eur. J. 2010, 16, 4777 – 4783; c) X.-J. Jiang, P.-C. Lo, S.-L. Yeung, W.-P. Fong, D. K. P. Ng, Chem. Commun. 2010, 46, 3188 – 3190. [6] a) P.-C. Lo, J. Chen, K. Stefflova, M. S. Warren, R. Navab, B. Bandarchi, S. Mullins, M. Tsao, J. D. Cheng, G. Zheng, J. Med. Chem. 2009, 52, 358 – 368; b) J. Chen, T. W. B. Liu, P.-C. Lo, B. C. Wilson, G. Zheng, Bioconjugate Chem. 2009, 20, 1836 – 1842; c) Z. Tang, Z. Zhu, P. Mallikaratchy, R. Yang, K. Sefah, W. Tan, Chem. Asian J. 2010, 5, 783 – 786; d) M. Verhille, H. Benachour, A. Ibrahim, M. Achard, P. Arnoux, M. Barberi-Heyob, J.-C. Andre, X. Allonas, F. Baros, R. Vanderesse, C. Frochot, Curr. Med. Chem. 2012, 19, 5580 – 5594. [7] a) X. Zheng, U. W. Sallum, S. Verma, H. Athar, C. L. Evans, T. Hasan, Angew. Chem. 2009, 121, 2182 – 2185; Angew. Chem. Int. Ed. 2009, 48, 2148 – 2151; b) Y. Gao, G. Qiao, L. Zhuo, N. Li, Y. Liu, B. Tang, Chem. Commun. 2011, 47, 5316 – 5318; c) M.-R. Ke, D. K. P. Ng, P.-C. Lo, Chem. Commun. 2012, 48, 9065 – 9067. [8] a) Y. Choi, R. Weissleder, C.-H. Tung, Cancer Res. 2006, 66, 7225 – 7229; b) B.-c. Bae, K. Na, Biomaterials 2010, 31, 6325 – 6335; c) H. Koo, H. Lee, S. Lee, K. H. Min, M. S. Kim, D. S. Lee, Y. Choi, I. C. Kwon, K. Kim, S. Y. Jeong, Chem. Commun. 2010, 46, 5668 – 5670; d) S. Y. Park, H. J. Baik, Y. T. Oh, K. T. Oh, Y. S. Youn, E. S. Lee, Angew. Chem. 2011, 123, 1682 – 1685; Angew. Chem. Int. Ed. 2011, 50, 1644 – 1647; e) S. J. Lee, H. Koo, D.-E. Lee, S. Min, S. Lee, X. Chen, Y. Choi, J. F. Leary, K. Park, S. Y. Jeong, I. C. Kwon, K. Kim, K. Choi, Biomaterials 2011, 32, 4021 – 4029; f) H. Kim, S. Mun, Y. Choi, J. Mater. Chem. B 2013, 1, 429 – 431. [9] For some recent examples, see a) X.-J. Jiang, S.-L. Yeung, P.-C. Lo, W.-P. Fong, D. K. P. Ng, J. Med. Chem. 2011, 54, 320 – 330; b) J. T. F. Lau, P.-C. Lo, W.-P. Fong, D. K. P. Ng, Chem. Eur. J. 2011, 17, 7569 – 7577; c) J. T. F. Lau, P.-C. Lo, Y.-M. Tsang, W.-P. Fong, D. K. P. Ng, Chem. Commun. 2011, 47, 9657 – 9659; d) M.-R. Ke, S.-L. Yeung, W.-P. Fong, D. K. P. Ng, P.-C. Lo, Chem. Eur. J. 2012, 18, 4225 – 4233; e) J. T. F. Lau, P.-C. Lo, W.-P. Fong, D. K. P. Ng, J. Med. Chem. 2012, 55, 5446 – 5454; f) M.-R. Ke, S.-L. Yeung, D. K. P. Ng, W.-P. Fong, P.-C. Lo, J. Med. Chem. 2013, 56, 8475 – 8483. [10] J. T. F. Lau, X.-J. Jiang, D. K. P. Ng, P.-C. Lo, Chem. Commun. 2013, 49, 4274 – 4276. [11] A. Meister, M. E. Anderson, Ann. Rev. Biochem. 1983, 52, 711 – 760. [12] R. Cheng, F. Feng, F. Meng, C. Deng, J. Feijen, Z. Zhong, J. Controlled Release 2011, 152, 2 – 12. [13] M. P. Gamcsik, M. S. Kasibhatla, S. D. Teeter, O. M. Colvin, Biomarkers 2012, 17, 671 – 691. [14] a) R. Hong, G. Han, J. M. Fernndez, B.-j. Kim, N. S. Forbes, V. M. Rotello, J. Am. Chem. Soc. 2006, 128, 1078 – 1079; b) J. Chen, S. Chen, X. Zhao, L. V. Kuznetsova, S. S. Wong, I. Ojima, J. Am. Chem. Soc. 2008, 130, 16778 – 16785; c) A. N. Koo, H. J. Lee, S. E. Kim, J. H. Chang, C. Park, C. Kim, J. H. Park, S. C. Lee, Chem. Commun. 2008, 6570 – 6572; d) M. H. Lee, J. Y. Kim, J. H. Han, S. Bhuniya, J. L. Sessler, C. Kang, J. S. Kim, J. Am. Chem. Soc. 2012, 134, 12668 – 12674; e) W. Cao, Y. Li, Y. Yi, S. Ji, L. Zeng, Z. Sun, H. Xu, Chem. Sci. 2012, 3, 3403 – 3408; f) L. Wang, M. Kim, Q. Fang, J. Min, W. I. Jeon, S. Y. Lee, S. J. Son, S.-W. Joo, S. B. Lee, Chem. Commun. 2013, 49, 3194 – 3196; g) J. Lai, B. P. Shah, E. Garfunkel, K.-B. Lee, ACS Nano 2013, 7, 2741 – 2750. [15] a) N. Shao, J. Jin, H. Wang, J. Zheng, R. Yang, W. Chan, Z. Abliz, J. Am. Chem. Soc. 2010, 132, 725 – 736; b) J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han, B. R. Cho, J. Am. Chem. Soc. 2010, 132, 1216 – 1217; c) B. Zhu, X. Zhang, Y. Li, P. Wang, H. Zhang, X. Zhuang, Chem. Commun. 2010, 46, 5710 – 5712; d) M. H. Lee, J. H. Han, P.-S. Kwon, S. Bhuniya, J. Y. Kim, J. L. Sessler, C. Kang, J. S. Kim, J. Am. Chem. Soc. 2012, 134, 1316 – 1322; e) L.-Y. Niu, Y.-S. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, J. Am. Chem. Soc. 2012, 134, 18928 – 18931; f) K. Xu, M. Qiang, W. Gao, R. Su, N. Li, Y. Gao, Y. Xie, F. Kong, B. Tang, Chem. Sci. 2013, 4, 1079 – 1086; g) Z. Lou, P. Li, X. Sun, S. Yang, B. Wang, K. Han, Chem. Commun. 2013, 49, 391 – 393; h) G. Li, Y. Chen, J. Wu, L. Ji, H. Chao, Chem. Commun. 2013, 49, 2040 – 2042; i) M. Isik, T. Ozdemir, I. S. Turan, S. Kolemen, E. U. Akkaya, Org. Lett. 2013, 15, 216 – 219. [16] Y. Cho, Y. Choi, Chem. Commun. 2012, 48, 9912 – 9914.
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Communication [17] M. Hu, N. Brasseur, S. Z. Yildiz, J. E. van Lier, C. C. Leznoff, J. Med. Chem. 1998, 41, 1789 – 1802. [18] a) J. Bhaumik, R. Weissleder, J. R. McCarthy, J. Org. Chem. 2009, 74, 5894 – 5901; b) J. Shao, H. Sun, H. Guo, S. Ji, J. Zhao, W. Wu, X. Yuan, C. Zhang, T. D. James, Chem. Sci. 2012, 3, 1049 – 1061. [19] I. Scalise, E. N. Durantini, Bioorg. Med. Chem. 2005, 13, 3037 – 3045. [20] S. Santra, C. Kaittanis, O. J. Santiesteban, J. M. Perez, J. Am. Chem. Soc. 2011, 133, 16680 – 16688. [21] N. A. Kuznetsova, N. S. Gretsova, O. A. Yuzhakova, V. M. Negrimovskii, O. L. Kaliya, E. A. Luk’yanets, Russ. J. Gen. Chem. 2001, 71, 36 – 41.
Chem. Eur. J. 2014, 20, 6241 – 6245
www.chemeurj.org
[22] M. E. Anderson, A. Meister, Anal. Biochem. 1989, 183, 16 – 20. [23] H. M. Shen, C. Y. Shi, Y. Shen, C. N. Ong, Free Radical Biol. Med. 1996, 21, 139 – 146.
Received: January 1, 2014 Revised: March 18, 2014 Published online on April 15, 2014
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