Far-Red-Absorbing Cationic Phthalocyanine Photosensitizers

Jan 19, 2015 - develop novel water-soluble Pc's absorbing in the far-red region ..... cell membrane with no changes in the nucleus and cytoplasm along...
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Far-Red-Absorbing Cationic Phthalocyanine Photosensitizers: Synthesis and Evaluation of the Photodynamic Anticancer Activity and the Mode of Cell Death Induction Miloslav Machacek,† Antonin Cidlina,‡ Veronika Novakova,*,§ Jan Svec,‡ Emil Rudolf,∥ Miroslav Miletin,‡ Radim Kučera,‡ Tomas Simunek,† and Petr Zimcik*,‡

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Department of Biochemical Sciences, ‡Department of Pharmaceutical Chemistry and Drug Control, and §Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic ∥ Department of Medical Biology and Genetics, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove, Czech Republic S Supporting Information *

ABSTRACT: Novel zinc, magnesium, and metal-free octasubstituted phthalocyanine photosensitizers bearing [(triethylammonio)ethyl]sulfanyl substituents in the peripheral or nonperipheral positions were synthesized and investigated for their photophysical properties (ΦΔ value up to 0.91, λmax up to 750 nm) and photodynamic anticancer activity. The photodynamic treatment of 3T3, HeLa, SK-MEL-28, and HCT 116 cancer cells revealed that the magnesium complexes were not active (IC50 > 100 μM), whereas the IC50 values of the zinc complexes typically reached values in the submicromolar range with low toxicity in the dark (TC50 ≈ 1500 μM). The subcellular changes upon photodynamic treatment of the HeLa cells indicated that the studied photosensitizers induced damage primarily to the lysosomes, which was followed by a relocalization and damage to other organelles. The time-lapse morphological changes along with the flow cytometry and caspase activity measurements indicated a predominant involvement of necrosis-like cell death.

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INTRODUCTION Photodynamic therapy (PDT) is a well-established treatment modality of certain localized cancers and noncancerous conditions, with several photosensitizers (PSs) currently in clinical practice.1−3 The effect of PDT arises from the combination of the following three components: light, oxygen, and a PS. PSs absorb the light of a specific wavelength and efficiently transfer its energy to the surrounding oxygen, forming highly reactive species. Of the reactive species, singlet oxygen is the most aggressive, and it is formed by a process known as photoprocess type II.1 The active oxygen species destroy subcellular compartments, inducing apoptosis and/or necrosis of the affected cells.4 Phthalocyanines (Pc’s) are synthetic macrocyclic dyes with advantageous photophysical properties for PDT.5 Pc’s typically absorb light over 680 nm, and the absorption position can be tuned up to 800 nm or, as has been recently reported, even beyond 1000 nm.6 Pc’s are also characterized by a high singlet oxygen production (determined by the singlet oxygen quantum yields) when complexed with closed-shell cations.5 Therefore, the interest in the medical application of the Pc’s has increased, and numerous Pc’s have been synthesized and investigated as potential PSs in PDT over recent years.7−14 A disadvantage of © 2015 American Chemical Society

Pc’s is their typical low solubility in water and tendency to aggregate, which substantially decreases their photodynamic effect. Although a number of water-solubilizing substituents have been introduced into the Pc’s,15 only a small number of studies have reported the complete reduction of the aggregation in water.16−18 One of the limitations of PDT is the shallow penetration of light that becomes deeper with longer wavelengths, close to/ above 700 nm. Hence, the goal of the present study was to develop novel water-soluble Pc’s absorbing in the far-red region to investigate the relationships between their structure and activity and to understand the mode of the PDT-induced cell death. In addition, the aza analogue of the Pc’s that was previously found active against Hep2 cells using a different irradiation protocol19 was introduced into the present study to obtain the structure−activity relationships from additional viewpoints. The target compounds (Chart 1) were designed to contain eight cationic charges on the substituents that were bound to the Pc core in either the peripheral (β) positions Received: September 26, 2014 Published: January 19, 2015 1736

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Journal of Medicinal Chemistry Chart 1. Structures of the Investigated Compounds

Scheme 1. Syntheses of the Studied Compoundsa

a Reaction conditions: (a) (1) 2-(diethylamino)ethanethiol hydrochloride, anhyd K2CO3, DMSO, rt, 24 h; (2) diethyl ether, HCl gas, rt; (b) (1) NaOH; (2) Mg, I2, anhyd n-BuOH, reflux, 18−24 h; (c) 1% aq HCl, rt, 1 h; (d) anhyd Zn(CH3COO)2, pyridine, reflux, 1.5 h; (e) C2H5I, NMP, rt, 6 days; (f) TsCl, anhyd K2CO3, acetone, reflux, 2.5 h.

the nucleophilic substitution can be attributed to the better leaving properties of the leaving group. Compounds 4 and 6 were obtained and stored in the form of hydrochlorides (with nearly a quantitative conversion from the free base and back again) because their corresponding free bases were found to significantly decompose (darken) after a couple of days or weeks. The free base was released from the hydrochlorides immediately prior to the cyclotetramerization to the Pc. The peripherally and nonperipherally substituted Pc’s were obtained using the same procedure, starting from the synthesis of the magnesium complexes (1Mg and 2Mg) by magnesium butoxide-induced cyclotetramerization. The magnesium complexes were subsequently demetalated using 1% aq HCl to yield the metal-free derivatives 1H and 2H, which were complexed by a zinc cation using zinc acetate in pyridine (1Zn, 2Zn). All of the Pc’s were subsequently quaternized with ethyl iodide to yield the highly water-soluble derivatives. Absorption Spectra and Aggregation. The absorption spectra of all of the Pc derivatives in DMF had a sharp shape, which is typical for monomers of this type of compound, with the Q-band located above 650 nm (Table 1, Figure 1, and

(1M-Et) or nonperipheral (α) positions (2M-Et) or to an aza analogue of Pc (3Zn-Et). The position of the substituent on the Pc ring or the aza substitution has been reported to substantially influence the wavelength of the main absorption band.20,21 Cationic charges increase the water solubility of the molecules and are expected to reduce the aggregation via electrostatic repulsion forces.16 The effect of the central metal (M = Zn, Mg, and 2H) on the photophysical properties and its influence on cell survival were also studied.

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RESULTS AND DISCUSSION Chemistry. Synthesis of the Pc’s is typically performed by cyclotetramerization of the precursors, the suitably substituted phthalonitriles. In this study, the synthesis of precursors 4 and 6 was accomplished using nucleophilic substitution of the phthalonitriles substituted with the appropriate leaving groups (Scheme 1), commercially available 4,5-dichlorophthalonitrile and compound 5, respectively. Phthalonitrile 5 containing the tosyl leaving groups was obtained in high yield by tosylation of 3,6-dihydroxyphthalonitrile, which is similar to the published procedure.22 The higher yield of 4 (66%) than of 6 (22%) from 1737

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2Zn-Et and 3Zn-Et but differed considerably in the case of 1ZnEt. The addition of pyridine (up to 5%) to a water solution of 1Zn-Et restored the shape of the spectrum to the typical monomeric shape (Figure S3, Supporting Information). As expected, no significant changes (in addition to the solvatochromic shift of the Q-band) were observed upon addition of pyridine to the water solution of 2Zn-Et or 3Zn-Et (Figure S3). Determinations of fluorescence quantum yields (Table 1) also supported the presence of aggregates for 1Zn-Et and 1Mg-Et as the ΦF values in water were approximately one-third of those determined in DMF. The aggregation behavior of the derivatives 1Zn-Et, 2Zn-Et, and 3Zn-Et in water indicated that the aza substitution in the macrocycle ring and the movement of the substituents to the nonperipheral positions significantly aid in reducing the tendency to aggregate. The presence of eight charged substituents appears to be essential for the monomerization of the Pc’s in water because tetrasubstituted derivatives with the same cationic substituent were found significantly aggregated in the water, regardless of the position of the substituent on the ring.26 These relationships discussed for the zinc complexes can also be applied to the magnesium complexes or the metal-free derivatives. Photophysical Characterization. The fluorescence (ΦF) and singlet oxygen (ΦΔ) quantum yields were determined in DMF using a comparative method with unsubstituted zinc phthalocyanine (ZnPc) as the reference. The data are summarized in Table 1. All of the metal-free derivatives (1H, 2H, 1H-Et, and 2H-Et) produced singlet oxygen and fluorescence at a very low rate; therefore, they were excluded from the following biological evaluation. This result corresponded to previous observations of photophysics of metal-free Pc’s and may be related to the different relaxation channels of the excited states.23 For the metal complexes, the ΦΔ values were consistently higher for zinc than for the magnesium complexes (and vice versa for the ΦF values), regardless of the peripheral substitution because of the heavy-atom effect of the central cation.27 A comparison of the peripherally and nonperipherally substituted derivatives revealed that the singlet oxygen production was preferred for the latter. The compound 2Zn-Et appears to be of particular interest with a high ΦΔ value of 0.91. However, the fluorescence of the nonperipheral derivatives 2Zn-Et and 2Mg-Et was weak, but it remained sufficient for their visualization in the cellular studies using fluorescence microscopy. The ΦΔ and ΦF values for 3Zn were extremely low because of the photoinduced electron transfer (PET) between the peripheral tertiary amino substituents and the macrocyclic core.19 PET is known to efficiently deactivate the excited states of the aza analogues of the Pc’s.28 Alkylation of the amines blocked the PET; however, the quantum yields of 3Zn-Et remained relatively low. PET is apparently a less important pathway for the excited-state deactivation for the

Table 1. Absorption and Photophysical Data of the Studied Compounds in DMFa compd 1Mg 1Mg-Et 1H 1H-Et 1Zn 1Zn-Et 2Mg 2Mg-Et 2H 2H-Et 2Zn 2Zn-Et 3Znd 3Zn-Ete

λmax/nm (log ε) 707 702 705 700 707 704 785 749 819 751 783 756 655 656

(5.44) (5.32) (5.02), 729sh (5.30) (5.46) (5.45) (5.21) (5.11) (5.06) (4.87) (5.18) (5.20)

λem/nm

ΦΔb

ΦFb

714 708 711 710 714 712 803 762 822 787 803 771 661 661

0.23 0.28 0.06 0.22 0.59 0.68 0.44 0.25 0.04 0.08 0.68 0.91 0.04 0.20

0.24 0.17c 0.04 0.06 0.13 0.12c 0.02 0.016c 2000 192 ± 14 161 ± 11 226 ± 19

0.34 ± 0.088 >100 0.66 ± 0.108 >100 2.46 ± 0.465

0.32 ± 0.048 >100 0.22 ± 0.021 >100 2.365 ± 0.339

0.54 ± 0.090 >100 0.31 ± 0.121 >100 3.70 ± 0.192

0.69 ± 0.045 >100 1.41 ± 0.106 >100 4.09 ± 0.329

3035 n/a 619 n/a 61

Data are presented as TC50 or IC50 values ± standard deviations.

magnesium derivatives of the same ligand. However, the type of the macrocycle and the position of the substituents significantly affected the toxicity. The nonperipherally substituted 2Zn-Et was the most toxic followed by the aza analogue 3Zn-Et, whereas the peripherally substituted 1Zn-Et had more than 1 order of magnitude lower toxicity. To the best of our knowledge, 1Zn-Et may be considered as a PS with one of the lowest dark toxicity values reported in the literature. The photodynamic activities (referred to as phototoxicity, λ > 570 nm, 12.4 mW cm−2, 15 min, 11.2 J cm−2) of all of the compounds were determined on three malignant human cell lines, namely, cervical carcinoma (HeLa), melanoma (SK-MEL-28), and colorectal carcinoma (HCT 116) as well as on normal 3T3 cells. Figure 2b shows the dose-dependent decrease in the viability of the HeLa cells 24 h after irradiation. Similar results with the other cell lines are visualized as concentration− response curves (Figure S4, Supporting Information), and the half-maximum inhibitory concentration (IC50) values are listed in Table 2. For all of the compounds, the most susceptible cell line was SK-MEL-28 followed by HeLa and HCT 116. This order corresponds to the proliferation rates of these cancer cell lines. No substantial difference between normal (3T3) and cancer cells toward photodynamic treatment was observed. The magnesium complexes 1Mg-Et and 2Mg-Et did not exert any phototoxicity up to a 100 μM concentration, which is apparently associated with their low ΦΔ values. In the case of the zinc complexes, the IC50 values increased in the order 2Zn-Et < 1Zn-Et < 3Zn-Et for all of the cell lines. This result corresponded well with the ability of the complexes to produce singlet oxygen, as discussed above. Surprisingly, the photodynamic effect of 1Zn-Et was relatively high, even though the compound was found aggregated in the water solution. This observation is similar to that described in the literature in which

parent Pc derivatives (1Zn, 1Mg, 2Zn, and 2Mg) compared with their aza analogues, as deduced from their relatively high ΦΔ + ΦF values. However, even for the parent Pc derivatives, a partial increase in the quantum yields was observed after alkylation of the peripheral substituents (Table 1) because of the PET inhibition. Cytotoxicity Studies. These experiments were initiated with an evaluation of the rate of cellular uptake of 1Zn-Et and 3Zn-Et. Low fluorescence of 2Zn-Et did not allow determination of the uptake profile, but the similar structure (octacationic Pc) suggests comparable behavior. The cellular uptake (tested on a 10 μM solution) of 1Zn-Et in HeLa cells (human cervical carcinoma) was relatively fast within the first 2 h, reaching the plateau phase at approximately 12 h (Figure 2a). On the basis of this result, the incubation with dyes prior to the irradiation was set to 12 h. The initial toxicity experiments were performed on nonmalignant 3T3 mouse fibroblast cells and HeLa cells in the absence of light (referred to as dark toxicity). 3T3 cells represent the model of healthy cells that should not be affected by the inherent toxicity of the photosensitizers. The cells were exposed to increasing concentrations of 1Zn-Et, 1Mg-Et, 2Zn-Et, 2Mg-Et, and 3Zn-Et, up to 1500 μM, and the halfmaximum toxic concentration (TC50) values were calculated. The results are shown in Figure 2 and Figure S4 (Supporting Information) and listed in Table 2. The TC50 values obtained for HeLa and 3T3 cells for a particular derivative were comparable, indicating a limited difference in toxicity between normal and cancer cells. This is in good agreement with data obtained recently for these cell lines for water-soluble Pc bearing quaternized imidazoles on the periphery.16 Interestingly, the central cation did not significantly influence the dark toxicity; the TC50 values were similar for the zinc and 1739

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2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), a quencher of free radicals (e.g., HO•, RO•, NO2•, etc.31), which is reported to be insensitive to singlet oxygen.32 The HeLa cells were preincubated for 12 h with 1Zn-Et or 2Zn-Et at concentrations corresponding to their IC85 values and then loaded for 30 min with H2DCFDA in the dark and irradiated. The formation of the cellular ROS was detected as an increased fluorescence of the fluorescent product (dichlorofluorescein, DCF) over time (Figure 2c), which occurred only during the irradiation in the presence of the PSs. No significant secondary ROS production occurred after the end of the irradiation. The ROS production was more intense in the case of 2Zn-Et than in the case of 1Zn-Et. These results suggested involvement of photoprocess type I in photodynamic action, although its contribution may be less significant than that of photoprocess type II (singlet oxygen). Indeed, the high value of the sum of ΦΔ + ΦF, the two most important deactivation pathways of excited states, leaves little space for other photophysical processes. Subcellular Localization and Morphological Changes. Subcellular localization of any PS is crucial for the determination of the mode of induced cell death after a photodynamic treatment.4 Cationic Pc’s (and, generally, PSs) have been previously reported to localize primarily within the lysosomes16,17 or mitochondria.30 In this study, the localization of 1Zn-Et and 2Zn-Et based on their red fluorescence was evaluated using fluorescence microscopy of the HeLa cells using coincubations with the MitoTracker Green and LysoTracker Blue fluorescence probes to visualize the mitochondria and the lysosomes, respectively. Both of the studied Pc’s were primarily localized in the vesicles that colocalized well with the labeled lysosomes (Figure 4 (time 0 s); for a better resolution see Figures S5 and S6, Supporting Information). This finding is most likely a result of the mechanism of cell uptake, endocytosis of bulky and charged compounds, as has been previously reported for other cationic compounds.33 Subsequently, changes in the cellular morphology of the HeLa cells treated with 1Zn-Et (at a concentration corresponding to its IC50 value) and irradiated were monitored using microscopy using differential (Normanski) interference contrast (DIC) and fluorescence microscopy with LysoTracker Blue, MitoTracker Green, Hoechst 33342, and propidium iodide (PI) (Figures 4 and 5). The LED light source (with a Cy5 filter set) from the fluorescence microscope was used for irradiation in these experiments instead of a Xe lamp to directly visualize the changes in the specific fluorescence probes using fluorescence microscopy. The exposure times to activating light were shorter because the amount of light delivered from the LED source required for the fluorescence visualization of the PS caused relatively rapid changes in the (sub)cellular morphology. On the basis of the (sub)cellular morphology changes and the PI staining, the irradiation time of ∼240 s using the microscope LED source corresponded to 15 min of irradiation with the Xe lamp source used in our standard phototoxicity studies. The control and nonirradiated cells displayed punctate lysosomal staining as assessed with LysoTracker Blue, primarily within the perinuclear area and the rod-shaped mitochondria, with a vivid green fluorescence of MitoTracker Green (Figure 4, 0 s). Overall cellular morphology showed an intact cell membrane with no changes in the nucleus and cytoplasm along with a typical spindle-like cell shape (Figure 4, 0 min, DIC). Upon irradiation, a decrease and an eventual disappearance of the punctate blue fluorescence of the lysosomal probe

the octacationic pyridyloxy zinc phthalocyanine was highly toxic after irradiation even though being partially aggregated in water, and it attained IC50 values comparable to those of the fully nonaggregated octacationic silicon Pc’s.17 This result indicates that if the aggregation of the PS is not extensive, it may play a less significant role than its photophysical parameters, particularly ΦΔ. Furthermore, it has been previously found that [2,3,9,10,16,17,23,24-octakis[(2-(trimethylammonio)ethyl)sulfanyl]phthalocyaninato]zinc(II) octaiodide, a Pc structurally similar to 1Zn-Et, was approximately 3 times more phototoxic than 1Zn-Et (on HeLa cells) but at an approximately 4 times higher light dose (IC50(HepG2) = 0.20 μM, λ > 610 nm, 48 J cm−2).30 The concordance of both of these results on similar cell lines supports the validity of the obtained data. In addition to determination of IC50 values (i.e., constant fluence, varied concentrations), the photodynamic properties were also investigated as a function of the light dose. For this experiment, the concentration of the zinc complexes 1Zn-Et, 2Zn-Et, and 3Zn-Et was fixed (concentration corresponding to IC50 values from Table 2) and the illumination time was set to 5, 15, and 25 min, resulting in total fluence of 3.7, 11.2, and 18.7 J cm−2. As seen in Figure 3, the decrease of the cell viability was dependent on the light dose.

Figure 3. Viability of HeLa cells as a function of the light dose following the photodynamic treatment with 1Zn-Et (red), 2Zn-Et (blue), and 3Zn-Et (black) at concentrations corresponding to their IC50 values (see Table 2): full bar, 5 min of irradiation (total fluence 3.7 J cm−2); dashed bar, 15 min of irradiation (total fluence 11.2 J cm−2); open bar, 25 min of irradiation (total fluence 18.7 J cm−2). The experiments were performed in triplicate.

The determination of the TC50 (dark toxicity) and IC50 (light toxicity) values enabled the calculation of the TC50/IC50 index as a “therapeutic ratio” (Table 2). The TC50 and IC50 values for the HeLa cells, which stand at the middle of the tested cell lines (and were also used in further experiments as the model cancer cell line), were selected for the calculations. High therapeutic ratios for compounds 1Zn-Et and 2Zn-Et, reaching values over 3000 and 600, respectively, indicated that they are promising candidates for PDT; therefore, they were selected for further investigations on the mechanism of PDTinduced cell death. The aza analogue 3Zn-Et was characterized by a relatively low phototoxicity and, consequently, a low value for the therapeutic ratio (TC50/IC50 = 61); also considering its absorption only at 656 nm, it was not included in the following studies. In addition to singlet oxygen, other reactive oxygen species (ROS) (from photoprocess type I) have also been shown to initiate oxidative damage and cell death.1 The general ROS production during and after irradiation can be estimated using 1740

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Figure 4. Morphological changes in the HeLa cells treated with 1Zn-Et during irradiation with an LED source in the fluorescence microscope. The different subcellular compartments were visualized using specific probes. The arrows indicate important points discussed in the text. DIC = differential interference contrast.

were initially observed within the first 10 s of the treatment. The lysosomal damage was also apparent from the changes in the fluorescence localization of the studied PSs. The red fluorescence of 1Zn-Et was localized only within the lysosomes at the beginning of the irradiation (Figure 4, 0 s). The red signal started to leak from the lysosomes to the cytoplasm after 10 s of irradiation, and the red punctate lysosomal fluorescence gradually disappeared. Distribution throughout the cell was completed within 30 s of irradiation. The relocalization of the PS that remained activated by the light, most likely also supported by the release of low-mass redox-active iron and lytic enzymes (such as cathepsins), resulted in secondary harm to various cellular constituents, including the mitochondria, which are the key organelles regulating cellular death.34 Indeed, using the MitoTracker Green probe, the mitochondria were observed to lose their rod shape and to round up within 30 s of irradiation (Figure 4 and Figure S7, Supporting Information). The mitochondrion-specific fluorescence then decreased and, finally, completely disappeared (at 120 s of irradiation). Furthermore, the alteration of the cell shape, most likely due to damage of the cytoskeleton and the cell membrane, was observed using DIC microscopy (Figure 4). The initial slight stretching of the cells was followed by retraction of the filopodia, transition of the cells from spindle-like to saccular cytosolic appearance, and the eventual formation of cellular debris (30 s), typical for necrotic cell death. This sequence of events was also observed using dynamic recording (see the video file attached as Supporting Information). This recording shows rapid morphological changes occurring as early as a few

seconds after the start of irradiation in exposed cells. Conversely, in the same cells exposed to the same irradiation protocol but in the absence of photosensitizers, no apparent changes in cellular morphology were visible. Hoechst 33342, which stains the nuclei in all of the cells, and PI, which can only enter cells with a severely compromised plasma membrane, were employed to detect changes in the nucleus of the treated cells (Figure 5). Notably, Hoechst 33342 can also detect changes in the nuclear and chromatin morphology during cell death (particularly apoptosis), but none were observed in this study. Following the irradiation, as 1Zn-Et was spreading from the lysosomes throughout the cell, PI was able to pass through the damaged plasma membrane, which was detected as a slight PI fluorescence in the cytoplasm at 60 s of irradiation (Figure 5, PI). The PI fluorescence signal in the cytoplasm increased over time, and the cytoplasm was completely positive for PI at the end of the irradiation period (240 s), although remaining with a PI-negative nucleus. The first nuclei were stained with PI at 9 min postirradiation, and permeabilization of the nuclear membranes (PI positivity within the nuclei) was completed at 15 min postirradiation. This process was accompanied by nuclear staining by 1Zn-Et, most likely due to the interaction of the DNA with the cationic Pc. A strong interaction of the cationic PSs or the dyes in general (e.g., also PI) with DNA is known.14,35 Hence, these observations indicated that the photodynamic treatment with 1Zn-Et (and with 2Zn-Et, data not shown) induced primary damage to the PS-containing lysosomes, which was followed by a redistribution of the PS, with subsequent 1741

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Figure 5. Changes in the HeLa cells treated with 1Zn-Et during irradiation with the LED source in the fluorescence microscope and after irradiation. The nuclei were visualized with Hoechst 33342 (Hoechst) and propidium iodide (PI). The arrows indicate important details discussed in the text. DIC = differential interference contrast.

(apoptotic) and annexin V−FITC+/PI+ (late apoptotic/early necrotic) cells along with an increase in the debris (indicating severely damaged cells because of necrosis) were present 6 h after treatment. The cells treated with a higher dose of the PSs (IC85) manifested significant positivity for annexin V−FITC and PI positivity as soon as 2 h after treatment, and the changes further progressed over the subsequent hours (Figure 6). An increase in the debris fraction was significantly more intense than in the case of the IC15 groups. These data suggested that the necrotic and apoptotic pathways were activated during the photodynamic treatment with the studied PSs, with the former pathway being more significant, as deduced from the large increase in the severely damaged cells detected as debris. The apoptotic pathway was further studied in detail. The major signaling pathways of apoptosis are tightly regulated by a number of proapoptotic and antiapoptotic molecules and converge in a canonical model in the activation of important proteases (caspases). Caspases can be divided into two major groups, the initiator caspases (2, 8, 9, and 10), which activate the executioner caspases (3, 6, and 7).37 In this study, a chemiluminescence assay was used to detect the activities of caspases 3/7, 8, and 9 after irradiation over several periods (20 min, 2 h, 4 h, 6 h, and 24 h) with 1Zn-Et and 2Zn-Et at concentrations corresponding to the IC15 and IC85. In most of the cases, the investigated initiator caspases 8 (a key signaling molecule for the death receptor-mediated apoptotic pathway) and 9 (the mitochondrial pathway) displayed lower activities than the untreated control, except for the 6 h period in the case of 1Zn-Et at a concentration corresponding to the IC15 in

damage to the mitochondria and plasma membrane, as indicated by the cellular appearance. In the later stages, a complete destruction of the mitochondria and lysosomes was observed, with damage to the nucleus as the final step followed by an interaction of the Pc’s with the nuclear DNA. These morphological changes are typical of necrosis-type cell death.36 Determination of Cell Death. Following the photodynamic treatment, damaged cells have been previously reported to undergo cell death mainly via apoptotic or necrotic pathways.4 Apoptosis is a programmed cell death that requires ATP and can typically be morphologically characterized by cellular shrinkage, membrane blebbing, chromatin condensation, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. Necrosis is generally presented by vacuolization of the cytoplasm, swelling, and eventual rupture of the plasma membrane.4,37 In the present study, the mode of cell death of the HeLa cells after treatment with 1Zn-Et or 2Zn-Et was further investigated using several assays, including annexin V−FITC along with PI staining and caspase 3/7, 8, and 9 activity measurements. During apoptosis, the membrane phospholipid phosphatidylserine is translocated from the inner plasma membrane to the cell surface and can be detected by the specific binding of the green fluorescent annexin V−FITC conjugate using flow cytometry.38 As observed in Figure 6, following the photodynamic treatment with 1Zn-Et or 2Zn-Et at a dose corresponding to the IC15, most of the HeLa cells were negative for annexin V−FITC and PI (annexin V−FITC−/PI−) for up to 6 h. However, significant populations of the annexin V−FITC+/PI− 1742

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caspases 3 and/or 7 were comparable to or higher than those of the untreated control; particularly, their activity significantly increased approximately 4 h after irradiation (Figure 7). The lack of activation of the initiator (apical) caspases prior to the executioner caspases suggests that other mechanism(s) resulted in the activation of the latter. Given the demonstrated lysosomal damage, cathepsins and/or other proteases released into the cytoplasm appear to be the most plausible mechanism.39−42 Autophagy may play an important role during cell death either as a survival mechanism or as a contributor to the cell demise37 and has been previously reported in the context of PDT.43 Hence, in this study, fluorescence microscopy methods were used to evaluate whether autophagy was present during the damaging process inflicted by PDT with 1Zn-Et or 2Zn-Et. The presence of autophagosomes was detected using the fluorescence sensor monodansylcadaverine (MDC) and the Cyto-ID autophagy detection kit. Rapamycin, used as a positive control,44 resulted in large fluorescence-positive vacuoles throughout the cell (not only in the perinuclear region) (Figure S8, Supporting Information). Because no significant presence of autophagosomes was detected in the negative control or in the cells treated with 1Zn-Et or 2Zn-Et for 24 h after the irradiation period, it is possible that increased autophagy does not play a role in a response to the photodynamic treatment by these compounds using the irradiation protocol in this study.

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CONCLUSION In summary, several novel octasubstituted cationic Pc’s with different central cations and substituents attached to the peripheral or nonperipheral positions were prepared. These Pc’s were investigated with respect to their absorption and photophysical properties and were compared with the corresponding aza analogue. The position of the substituents on the macrocyclic core and the type of core were demonstrated to significantly influence the absorption and photophysical properties of the Pc’s. The cellular experiments revealed that the magnesium derivatives suffer from low photodynamic activity (IC50 > 100 μM) because of their low ΦΔ values. However, the zinc complexes were highly phototoxic in the order 2Zn-Et > 1Zn-Et > 3Zn-Et, which is proportional to their ability to produce singlet oxygen. Both photoprocess types I and II may contribute to the lethal effect as the production of ROS was also observed during irradiation. The peripherally substituted Pc’s were also characterized by an exceptionally low dark toxicity. With a TC50 value (HeLa) of >1600 μM, to the best of our knowledge, 1Zn-Et ranks among the least toxic PSs reported to date. The compounds with the best selectivity (determined by the TC50/IC50 ratio), i.e., 1Zn-Et and 2Zn-Et, were further investigated for their mechanism(s) of action. These compounds were

Figure 6. Flow cytometry assessment of the HeLa cell death mode for 1Zn-Et and 2Zn-Et analyzed at different times after irradiation at concentrations corresponding to the IC15 (a, b) and the IC85 (c, d), expressed as an increase in the debris against the control (a, c) or the cell count based on the annexin V−FITC or/and PI positivity (b, d). The experiments were performed in triplicate. Key: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

which a significant increase in the caspase 8 and 9 activities occurred (Figure 7). However, the activities of executioner

Figure 7. Activity of caspase 9 (a), caspase 8 (b), and caspase 3 + caspase 7 (c) after the photodynamic treatment with 1Zn-Et (red bars) and 2Zn-Et (blue bars) at concentrations corresponding to the IC15 and IC85. The data are expressed as percentages of the control (100%). The experiments were performed in triplicate. Key: *, p < 0.05. 1743

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Journal of Medicinal Chemistry

56 mmol) was sonicated in dimethyl sulfoxide (100 mL) for 30 min at rt, and 2-(diethylamino)ethanethiol hydrochloride (3.39 g, 20 mmol) was added. Six equal portions of 4,5-dichlorophthalonitrile (1.58 g, 8 mmol) were added over the next 2 h, and stirring was continued for 24 h at rt. The reaction mixture was poured into ice−water (700 mL). The precipitate was collected by filtration, washed thoroughly with water, and air-dried. The crude product was converted to hydrochloride 4 after dissolution in diethyl ether and bubbling with dry gaseous HCl. The precipitate was collected by filtration, washed with diethyl ether, and crystallized from iPrOH/EtOH (1:3 (v/v)): white solid; yield 2.46 g (66%); mp 239−244 °C dec; 1H NMR (300 MHz, D2O) δ = 7.94 (s, 2 H, ArH), 3.60−3.42 (m, 8 H, CH2), 3.31 (q, 8 H, J = 7.3 Hz, CH2), 1.31 (t, 12 H, J = 7.3 Hz, CH3); 13C NMR (75 MHz, D2O) δ = 142.9, 131.8, 116.3, 113.1, 50.4, 48.4, 27.1, 8.8. Anal. Calcd for C20H32Cl2N4S2: C, 51.82; H, 6.96; N, 12.09. Found: C, 51.55; H, 6.91; N, 12.25. This compound in the form of a free base has been previously reported in the literature.47,48 Preparation of 3,6-Bis{[2-(diethylamino)ethyl]sulfanyl}phthalonitrile Dihydrochloride (6). Potassium carbonate (6.9 g, 50 mmol) was sonicated in dimethyl sulfoxide (80 mL) for 30 min, and 2-(diethylamino)ethanethiol hydrochloride (4.24 g, 25 mmol) was added. The mixture was stirred for 10 min at rt, and then compound 5 (4.68 g, 10 mmol) was added in six equal portions over the next 2 h; stirring at rt continued for the next 24 h. The reaction mixture was poured into ice−water (700 mL) and extracted with ethyl acetate (2 × 50 mL). The organic layer was then extracted with dilute hydrochloric acid (1% (v/v)) (3 × 25 mL). The collected water phase was neutralized with 10% sodium hydroxide, and the product was extracted with ethyl acetate (3 × 25 mL). The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified via column chromatography on silica with acetone/methanol/triethylamine (100:10:1) as the eluent to yield 6 in the form of a free base: yellow solid; 0.89 g (23%); mp 62.7−63.7 °C; 1H NMR (300 MHz, CDCl3) δ = 7.53 (s, 2 H, ArH), 3.09 (t, 4 H, J = 7.1 Hz, SCH2), 2.70 (t, 4H, J = 7.1 Hz, NCH2), 2.52 (q, 8 H, J = 7.1 Hz, NCH2), 1.00 (t, 12 H, J = 7.1 Hz, CH3); 13C NMR (75 MHz, CDCl3) δ = 141.4, 132.2, 117.0, 113.8, 51.7, 46.9, 32.2, 11.8; IR (ATR) ν = 2966, 2932, 2871, 2808, 2221 (CN), 1524, 1429, 1382, 1295, 1275, 1242, 1193, 1178, 1145, 1108, 1067, 992 cm−1. Anal. Calcd for C20H30N4S2: C, 61.50; H, 7.74; N, 14.34. Found: C, 61.31; H, 7.65; N, 14.74. The whole product (0.89 g) was converted to its dihydrochloride (6) after dissolution in diethyl ether and bubbling with dry gaseous HCl. The precipitate was collected by filtration, washed with diethyl ether, and reprecipitated from MeOH/diethyl ether: yellow-white solid; yield 1.00 g (95%); mp 114.5−120.5 °C; 1H NMR (300 MHz, D2O) δ = 7.95 (s, 2 H, ArH), 3.60−3.39 (m, 8 H, CH2), 3.29 (q, 8 H, J = 7.1 Hz, NCH2), 1.29 (t, 12 H, J = 7.2 Hz, CH3); 13C NMR (75 MHz, D2O) δ = 140.1, 135.0, 118.0, 115.0, 50.8, 48.4, 27.9, 8.8; IR (ATR) ν = 3455, 3022, 2948, 2219 (CN), 1626, 1462, 1442, 1389, 1361, 1280, 1210, 1173, 1145, 1105, 1047, 1020, 999, 967, 843 cm−1. Anal. Calcd for C20H32Cl2N4S2· H2O: C, 49.88; H, 7.18; N, 11.63. Found: C, 50.22; H, 7.46; N, 11.94. General Procedure for the Synthesis of Magnesium Complexes 1Mg and 2Mg. The corresponding precursor (4 or 6, 1 equiv) was dissolved in water (100 mL) and converted to its free base using 10% NaOH. The suspension was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried (Na2SO4), and the solvent was removed under reduced pressure. Magnesium (∼7 equiv) and a small crystal of iodine were refluxed in dry butanol (∼40 mL) for 3 h. The corresponding precursor in the form of the free base was dissolved in butanol (∼10 mL) and added immediately in one portion. Reflux was continued for the next 24 h. The mixture was cooled, and the solvent was removed under reduced pressure. The dark green product was extracted from the residues of the magnesium butoxide using THF, and the solution was filtered and evaporated. The crude product was purified using column chromatography on aluminum oxide (the eluents are mentioned below). The product was dissolved in a small amount of CHCl3 and dropped into hexane. The fine precipitate was collected by filtration, washed with hexane, and dried.

shown to localize within the lysosomes and induced their rupture upon irradiation as the first step in a cascade leading to cell death. The results from the annexin V−FITC/PI staining and the measurements of the caspase activities along with the morphological observations suggested that classical apoptosis played a less significant role in cell death after photosensitization with 1Zn-Et and 2Zn-Et. The necrosis-like cell death most likely prevailed due to the release of the entire lysosomal content into the cytoplasm after rupture of the lysosomal membrane, which may have also led to secondary and nonspecific activation of the executional phase of apoptosis.42,45,46 In conclusion, 1Zn-Et and 2Zn-Et appeared to be highly efficient photosensitizers with a low dark toxicity. Particularly, the latter compound appears to be interesting for future development because it absorbs strongly at wavelengths over 750 nm, which are more permeable to activating light.

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EXPERIMENTAL SECTION

General Methods. All of the organic solvents used for the synthesis were of analytical grade. Anhydrous butanol for the cyclotetramerization was freshly distilled from magnesium. All of the chemicals for the synthesis were purchased from certified suppliers (Sigma-Aldrich, TCI Europe, Acros, Merck) and used as received. TLC was performed on Merck aluminum sheets coated with silica gel 60 F254. Merck Kieselgel 60 (0.040−0.063 mm) or neutral aluminum oxide 5/40 was used for the column chromatography. The melting points were measured on an Electrothermal IA9200-series digital melting point apparatus (Electrothermal Engineeering, Southend-onSea, Essex, Great Britain). The infrared spectra were obtained on a Nicolet 6700 spectrometer in the ATR mode. The 1H and 13C NMR spectra were recorded on a Varian Mercury Vx BB 300 NMR spectrometer or a VNMR S500 NMR spectrometer. The chemical shifts are given relative to Si(CH3)4 and were locked to the signal of the solvent. The elemental analyses were performed on an Automatic Microanalyzer EA1110CE (Fisons Instruments, Milan, Italy). The UV−vis spectra were recorded using a Shimadzu UV-2401PC spectrophotometer. The steady-state fluorescence spectra were measured using an AMINCO-Bowman series 2 luminescence spectrometer. The MALDI-TOF mass spectra were recorded in the positive reflectron mode on a Voyager-DE STR mass spectrometer (Applied Biosystems, Framingham, MA) in trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile as the matrix. The instrument was calibrated externally with a five-point calibration using a Peptide Calibration Mix1 kit (LaserBio Laboratories, SophiaAntipolis, France). All of the compounds gave satisfactory elemental analyses with a difference of ≤0.4% from the calculated values. The purity of 1Zn-Et, 1Mg-Et, 2Zn-Et, 2Mg-Et, and 3Zn-Et was further determined by HPLC and was found to be ≥95%. The LC 20A Prominence system (Shimadzu, Duisburg, Germany) consisting of a DGU-20A3 degasser, LC-20 AD pumps, an SIL-20AC autosampler, a CTO-20AC column oven, an SPD-M20A photodiode array detector, and a CBM-20AC communication module was used for the purity evaluation. The data were processed using LC solution software, version 1.21 SP1. The samples were separated on a polybutadienemodified zirconia stationary phase (150 × 4.6 mm; particle size 5 μm) from ZirChrom Separations, Inc. (Anoka, MN). Mobile phase A consisted of 25 mM hydrochloric acid with pH 2.1 adjusted by Tris, and mobile phase B was methanol. Chromatography was performed using the following gradient program: 0−10 min, 20% B; 10−15 min, from 20% to 65% B; 15−25 min, 65% B; 25−30 min, equilibration of the initial conditions. The other conditions were set as follows: temperature 40 °C and flow rate 0.5 mL·min−1. Compound 5 was prepared according to the literature22 and was further purified by crystallization from acetone/methanol to give white crystals (yield 84%). Compound 3Zn-Et was prepared as described in the literature.19 Preparation of 4,5-Bis{[2-(diethylamino)ethyl]sulfanyl}phthalonitrile Dihydrochloride (4). Potassium carbonate (7.7 g, 1744

DOI: 10.1021/jm5014852 J. Med. Chem. 2015, 58, 1736−1749

Article

Journal of Medicinal Chemistry

General Procedure for the Synthesis of the Zinc Complexes 1Zn and 2Zn. The corresponding metal-free Pc (1 equiv) was dissolved in pyridine (20 mL), and anhydrous zinc acetate (10 equiv) was added. The mixture was refluxed for 90 min. The resulting mixture was allowed to cool to room temperature and poured into water. The precipitate was collected by filtration and washed thoroughly with water. The product was dissolved in an aqueous solution of hydrochloric acid (1% (v/v)) and precipitated with a 10% solution of NaOH. The suspension was collected by filtration and washed with water and acetone. Preparation of [2,3,9,10,16,17,23,24-Octakis[(2-(diethylamino)ethyl)sulfanyl]phthalocyaninato]zinc(II) (1Zn). Compound 1Zn was prepared from 1H (590 mg, 3.2 mmol) following the general procedure for the synthesis of the zinc complexes: dark green solid; yield 530 mg (86%); λmax(DMF, 1 μM)/nm 707 (ε/dm3 mol−1 cm−1 289000), 634 (49800), 376 (91300); 1H NMR (300 MHz, CDCl3/C5D5N) δ = 8.80 (s, 8 H, ArH), 3.30 (t, 16 H, J = 7.4 Hz, SCH2), 2.83 (t, 16H, J = 7.4 Hz, NCH2), 2.48 (q, 32H, J = 7.1 Hz, NCH2), 0.88 (t, 48 H, J = 7.1 Hz; CH3); 13C NMR (75 MHz, CDCl3/C5D5N) δ = 151.6, 138.2, 135.2, 121.2, 50.8, 46.3, 30.5, 11.3; IR (ATR) ν = 2966, 2930, 2798, 1592, 1455, 1405, 1369, 1289, 1199, 1084, 1065, 992, 942, 871 cm−1; m/z (MALDI-TOF) 1624.6 [M]+. Anal. Calcd for C80H120N16S8Zn· 2H2O: C, 57.75; H, 7.51; N, 13.47. Found: C, 57.94; H, 7.49; N, 13.48. Synthesis of this compound has been previously reported in the literature using different approaches.47,48 Preparation of [1,4,8,11,15,18,22,25-Octakis[(2-(diethylamino)ethyl)sulfanyl]phthalocyaninato]zinc(II) (2Zn). Compound 2Zn was prepared from 2H (238 mg, 0.153 mmol) following the general procedure for the synthesis of the zinc complexes: dark purple solid; yield 240 mg (97%); λmax(DMF, 1 μM)/nm 783 (ε/dm3 mol−1 cm−1 152700), 705 (38200), 500 (9100), 343 (41500), 295 (101800); 1H NMR (300 MHz, CDCl3/C5D5N) δ = 7.62 (s, 8 H, ArH), 3.34−3.16 (m, 16 H, SCH2), 2.89−2.76 (m, 16 H, NCH2), 2.38 (q, 32 H, J = 7.1 Hz, NCH2), 0.81 (t, 48 H, J = 7.1 Hz, CH3); 13C NMR (75 MHz, CDCl3/C5D5N) δ = 152.5, 134.1, 131.2, 124.4, 51.3, 46.6, 29.2, 11.5; IR (ATR) ν = 2933, 1748, 1647, 1497, 1456, 1200, 1040, 867, 819 cm−1; m/z (MALDI-TOF) 1624.7 [M]+. Anal. Calcd for C80H120N16S8Zn·H2O: C, 58.38; H, 7.47; N, 13.62. Found: C, 58.46; H, 7.40; N, 13.61. General Procedure for Quaternization of the Pc’s. The corresponding Pc was dissolved in iodoethane (∼7 mL/0.1 mmol of Pc). The mixture was stirred at rt for 48 h, and during that time, a green solid precipitated. 1-Methyl-2-pyrrolidinone (the same volume as that of iodoethane) was added to the reaction to dissolve the precipitate, and stirring was continued for the next 120 h at rt. Subsequently, the resulting mixture was poured into diethyl ether. The crude product was collected by filtration and washed thoroughly with diethyl ether. Purification was performed by several precipitations of the product by diethyl ether after its dissolution in MeOH. The precipitate was collected by filtration, washed with diethyl ether, and air-dried. Preparation of [2,3,9,10,16,17,23,24-Octakis[(2-(triethylammonio)ethyl)sulfanyl]phthalocyaninato]magnesium(II) Octaiodide (1Mg-Et). Compound 1Mg-Et was prepared from 1Mg (110 mg, 0.07 mmol) following the general procedure for the quaternization of the Pc’s: dark green solid; yield 120 mg (61%); λmax(DMF, 1 μM)/nm 702 (ε/dm3 mol−1 cm−1 210000), 629 (36900), 377 (91400); λmax(H2O, 1 μM)/nm 697 (ε/dm3 mol−1 cm−1 68000), 662 (88600), 356 (77100), 225 (150900); 1H NMR (500 MHz, CD3OD) δ = 9.47−9.32 (br, 8 H, ArH), 4.66−3.38 (br, 80 H, SCH2 + NCH2), 1.66−1.09 (m, 72 H, CH3); 13C NMR (125 MHz, CD3OD) δ = 56.1, 53.7, 28.9, 7.4, the signals of aromatic carbons were not detected; IR (ATR) ν = 3433, 2977, 1595, 1471, 1454, 1402, 1371, 1283, 1186, 1155, 1111, 1067, 1022, 942 cm−1. Anal. Calcd for C96H160I8MgN16S8·6H2O: C, 39.18; H, 5.89; N, 7.62. Found: C, 39.21; H, 5.63; N, 7.71. Preparation of 2,3,9,10,16,17,23,24-Octakis[(2-(triethylammonio)ethyl)sulfanyl]phthalocyanine Octaiodide (1H-Et). Compound 1H-Et was prepared from 1H (141 mg, 0.091 mmol) following the general procedure for the quaternization of the Pc’s: dark green solid; yield 198 mg (77%); λmax(DMF, 1 μM)/nm 700 (ε/dm3 mol−1 cm−1 201500), 629 (37700), 371 (75900); λmax(H2O, 1 μM)/nm 668 (ε/dm3 mol−1 cm−1 76800), 431 (sh), 341 (60700), 224 (147100); 1H NMR (300 MHz,

Preparation of [2,3,9,10,16,17,23,24-Octakis[(2-(diethylamino)ethyl)sulfanyl]phthalocyaninato]magnesium(II) (1Mg). 1Mg was prepared from precursor 4 (2 g, 4 mmol) following the general procedure for the magnesium complexes: mobile phase chloroform/THF (10:1); green solid; yield 0.93 g (54%); λmax(DMF, 1 μM)/nm 707 (ε/dm3 mol−1 cm−1 278600), 635 (48100), 373 (109100); 1H NMR (300 MHz, CDCl3/C5D5N) δ = 8.46 (s, 8 H, ArCH2), 2.95 (br, 16 H, SCH2), 2.73−2.52 (m, 16 H, NCH2), 2.44−2.18 (m, 32 H, NCH2), 0.78 (t, 48 H, J = 7.0 Hz, CH3); 13C NMR (75 MHz, CDCl3/C5D5N) δ = 153.1, 138.0, 135.3, 120.7, 50.8, 46.3, 30.4, 11.0; IR (ATR) ν = 2966, 2930, 2799, 1592, 1453, 1403, 1367, 1288, 1198, 1062, 941, 871 cm−1; m/z (MALDI-TOF) 1584.8 [M]+. Anal. Calcd for C80H120MgN16S8·3H2O: C, 58.56; H, 7.74; N, 13.66. Found: C, 58.89; H, 8.00; N, 13.60. Preparation of [1,4,8,11,15,18,22,25-Octakis[(2-(diethylamino)ethyl)sulfanyl]phthalocyaninato]magnesium(II) (2Mg). Compound 2Mg was prepared from phthalonitrile 4 (1.35 g, 2.9 mmol) following the general procedure for the magnesium complexes: mobile phase chloroform/THF (1:1); dark purple solid; yield 0.51 g (44%); λmax(DMF, 1 μM)/nm 785 (ε/dm3 mol−1 cm−1 164800), 701 (40900), 508 (10400), 365 (53500), 295 (101800); 1H NMR (300 MHz, CDCl3/C5D5N) δ = 7.61 (s, 8 H, ArH), 3.30−3.20 (m, 16 H, SCH2), 2.89−2.77 (m, 16 H, NCH2), 2.39 (q, 32 H, J = 7.1 Hz, NCH2), 0.81 (t, 48 H, J = 7.1 Hz, CH3); 13C NMR (75 MHz, CDCl3/ C5D5N) δ = 152.4, 134.5, 131.2, 124.2, 51.3, 46.5, 29.1, 11.4; IR (ATR) ν = 2966, 2931, 2809, 1560, 1459, 1374, 1315, 1282, 1206, 1144, 1107, 1084 1063, 993, 920, 907, 885 cm−1; m/z (MALDI-TOF): 1584.7 [M]+. Anal. Calcd for C80H120MgN16S8·2H2O: C, 59.21; H, 7.70; N, 13.81. Found: C, 59.29; H, 7.63; N, 13.89. General Procedure for the Synthesis of Metal-Free Pc’s 1H and 2H. The magnesium complex 1Mg or 2Mg was dissolved in aqueous hydrochloric acid (1% (v/v), ∼100 mL) and stirred for 60 min at rt. Subsequently, the pH value of the solution was adjusted using 10% NaOH to 8. The precipitate was collected by filtration, washed thoroughly with water, and air-dried. The metal-free Pc was then dissolved in a small amount of chloroform and dropped into MeOH. The fine precipitate was collected by filtration, washed with MeOH, and dried. Preparation of 2,3,9,10,16,17,23,24-Octakis[(2-(diethylamino)ethyl)sulfanyl]phthalocyanine (1H). Compound 1H was prepared from 1Mg (610 mg, 0.38 mmol) following the general procedure for the synthesis of the metal-free Pc’s. The crude product was purified using column chromatography on aluminum oxide with chloroform/ MeOH (20:1) as the eluent before precipitation from MeOH: dark green solid; yield 527 mg (88%); λmax(DMF, 1 μM)/nm 729 (ε/dm3 mol−1 cm−1 105600), 705 (130300), 666 (sh), 636 (sh), 360 (70800), 331 (75200); 1H NMR (300 MHz, CDCl3/C5D5N) δ = 11.82 (br, 2 H, NH), 8.54 (s, 8 H, ArH), 3.21 (t, 16H, J = 7.0 Hz, SCH2), 2.84 (t, 16H, J = 7.0 Hz, NCH2), 2.50 (q, 32 H, J = 7.1 Hz; NCH2), 0.91 (t, 48 H, J = 7.1 Hz, CH3); 13C NMR (75 MHz, CDCl3/C5D5N) δ = 147.7, 139.5, 132.6, 120.7, 50.9, 46.4, 30.8, 11.4; IR (ATR) ν = 3294, 2966, 2929, 2799, 1590, 1501, 1458, 1399, 1469, 1323, 1290, 1200, 1124, 1070, 1011, 932, 904, 869 cm−1; m/z (MALDI-TOF) 1562.9 [M]+. Anal. Calcd for C80H122N16S8: C, 61.42; H, 7.86; N, 14.32. Found: C, 61.42; H, 8.25; N, 14.09. Preparation of 1,4,8,11,15,18,22,25-Octakis[(2-(diethylamino)ethyl)sulfanyl]phthalocyanine (2H). Compound 2H was prepared from 2Mg (440 mg, 0.2 mmol) following the general procedure for the synthesis of the metal-free Pc’s: dark purple solid; yield 420 mg (96%); λmax(DMF, 1 μM)/nm 819 (ε/dm3 mol−1 cm−1 114100), 350 (42100), 294 (98100); 1H NMR (300 MHz, CDCl3/C5D5N) δ = 7.53 (s, 8 H, ArH), 3.20−3.15 (m, 16 H, SCH2), 2.78−2.65 (m, 16 H, NCH2), 2.37 (q, 32 H, J = 7.1 Hz, NCH2), 0.80 (t, 48 H, J = 7.1 Hz, CH3); 13C NMR (75 MHz, CDCl3/C5D5N) δ = 131.9, 131.5, 126.7, 51.3, 46.5, 29.0, 11.5, one aromatic signal was not detected; IR (ATR) ν = 3294, 2965, 2929, 2797, 1560, 1464, 1382, 1368, 1279, 1224, 1201, 1180, 1185, 1140, 1090, 1064, 1034, 992, 907, 871 cm−1; m/z (MALDI-TOF) 1562.8 [M]+. Anal. Calcd for C80H122N16S8·H2O: C, 60.72; H, 7.90; N, 14.16. Found: C, 60.36; H, 7.80; N, 14.20. 1745

DOI: 10.1021/jm5014852 J. Med. Chem. 2015, 58, 1736−1749

Article

Journal of Medicinal Chemistry CD3OD) δ = 9.50 (s, 8 H, ArH), 4.41−3.96 (br, 16 H, SCH2), 3.94−3.79 (br, 16 H, NCH2), 3.78−3.47 (br, 48 H, NCH2), 1.54−1.14 (m, 72H, CH3); 13C NMR (75 MHz, CD3OD) δ = 56.1, 53.7, 7.4, one signal of aliphatic carbon and the signals of aromatic carbons were not detected; IR (ATR) ν = 3439, 3290, 2977, 1597, 1453, 1418, 1399, 1367, 1286, 1155, 1135, 1077, 1021, 935 cm−1. Anal. Calcd for C96H162I8N16S8·6H2O: C, 39.48; H, 6.01; N, 7.67. Found: C, 39.24; H, 5.63; N, 7.69. Preparation of [2,3,9,10,16,17,23,24-Octakis[(2-(triethylammonio)ethyl)sulfanyl]phthalocyaninato]zinc(II) Octaiodide (1Zn-Et). Compound 1Zn-Et was prepared from 1Zn (179 mg, 0.11 mmol) following the general procedure for the quaternization of the Pc’s: dark green solid; yield 256 mg (81%); λmax(H2O, 1 μM)/nm 699 (ε/dm3 mol−1 cm−1 sh), 660 (100000), 360 (80000), 226 (157200); λmax(DMF, 1 μM)/nm 704 (ε/dm3 mol−1 cm−1 280300), 631 (48000), 384 (103000); 1H NMR (500 MHz, CD3OD) δ = 9.37 (s, 8 H, ArH), 4.35−3.91 (br, 16 H, SCH2), 3.91−3.79 (br, 16 H, NCH2), 3.79−3.48 (br, 48 H, NCH2), 1.52−1.14 (m, 72 H, CH3); 13C NMR (125 MHz, CD3OD) δ = 57.3, 54.8, 29.0, 8.7, the signals of aromatic carbons were not detected; IR (ATR) ν = 3437, 2977, 1594, 1484, 1454, 1403, 1372, 1282, 1186, 1155, 1114, 1088, 1068, 943 cm−1. Anal. Calcd for C96H160I8N16S8Zn·4H2O: C, 39.12; H, 5.74; N, 7.60. Found: C, 39.09; H, 5.70; N, 7.63. Preparation of [1,4,8,11,15,18,22,25-Octakis[(2-(triethylammonio)ethyl)sulfanyl]phthalocyaninato]magnesium(II) Octaiodide (2Mg-Et). Compound 2Mg-Et was prepared from 2Mg (95 mg, 0.060 mmol) following the general procedure for the quaternization of the Pc’s: dark green solid; yield 110 mg (65%); λmax(DMF, 1 μM)/nm 749 (ε/dm3 mol−1 cm−1 128500), 677 (30700), 338 (33100), 287 (86100); λmax(H2O, 1 μM)/nm 742 (ε/dm3 mol−1 cm−1 104200), 663 (sh), 346 (39900), 285 (73000), 220 (155300); 1H NMR (500 MHz, CD3OD) δ = 8.16 (s, 8H, ArH), 4.00−3.80 (br, 16H, SCH2), 3.78−3.65(br, 16H, NCH2), 3.64−3.37 (br, 48H, NCH2), 1,43−1,09 (m, 72H, CH3); 13C NMR (125 MHz, CD3OD) δ = 57.7, 54.9, 26.5, 8.9, the signals of aromatic carbons were not detected; IR (ATR) ν = 3433, 2977, 1595, 1471, 1454, 1402, 1371, 1283, 1186, 1155, 1111, 1067. 1022, 942 cm−1. Anal. Calcd for C96H160I8MgN16S8·4H2O: C, 39.67; H, 5.83; N, 7.71. Found: C, 39.71; H, 5.81; N, 7.73. Preparation of 1,4,8,11,15,18,22,25-Octakis[(2-(triethylammonio)ethyl)sulfanyl]phthalocyanine Octaiodide (2H-Et). Compound 2H-Et was prepared from 1H (83 mg, 0.053 mmol) following the general procedure for the quaternization of the Pc’s: dark green solid; yield 89 mg (61%); λmax(DMF, 1 μM)/nm 751 (ε/dm3 mol−1 cm−1 74100), 671 (sh), 460 (20900), 326 (sh), 285 (80900); λmax(H2O, 1 μM)/nm 765 (ε/dm3 mol−1 cm−1 70400), 672 (sh), 356 (32200), 286 (72100), 225 (168000); 1H NMR (500 MHz, CD3OD/C5D5N) δ = 8.21 (br, 8 H, ArH), 4.07−3.88 (br, 16 H, SCH2), 3.86−3.70 (br, 16 H, NCH2), 3.69−3.48 (m, 48 H, NCH2), 1.42−1.20 (m, 72 H, CH3); 13C NMR (125 MHz, CD3OD/C5D5N) δ = 57.8, 55.0, 26.7, 9.0, the signals of aromatic carbons were not detected; IR (ATR) ν = 3439, 3290, 2977, 1597, 1453, 1418, 1399, 1367, 1286, 1155, 1135, 1077, 1021, 935 cm−1. Anal. Calcd for C96H162I8N16S8·6H2O: C, 39.48; H, 6.01; N, 7.67. Found: C, 39.46; H, 5.83; N, 7.51. Preparation of [1,4,8,11,15,18,22,25-Octakis[(2-(triethylammonio)ethyl)sulfanyl]phthalocyaninato]zinc(II) Octaiodide (2Zn-Et). Compound 2Zn-Et was prepared from 2Zn (61 mg, 0.037 mmol) following the general procedure for the quaternization of the Pc’s: dark purple solid; yield 90 mg (86%); λmax(DMF, 1 μM)/nm 756 (ε/dm3 mol −1 cm−1 163200), 678 (35900), 342 (36300), 291 (81600); λmax(H2O, 1 μM)/nm 739 (ε/dm3 mol −1 cm−1 142200), 666 (32900), 345 (46800), 286 (71000), 224 (170300); 1H NMR (300 MHz, CD3OD) δ = 7.98 (s, 8 H, ArH), 3.85−3.20 (br, 80 H, SCH2 + NCH2), 1.39−0.96 (m, 72 H, CH3); 13C NMR (75 MHz, CD3OD) δ = 57.6, 54.7, 26.7, 8.7, the signals of aromatic carbons were not detected; IR (ATR) ν = 3452, 2978, 1620, 1562, 1468, 1395, 1288, 1213, 1143, 1109, 1022, 918 cm−1. Anal. Calcd for C96H160I8N16S8Zn·5H2O: C, 38.88; H, 5.78; N, 7.56. Found: C, 38.77; H, 5.61; N, 7.48. Photophysical Measurements. The singlet oxygen quantum yields (ΦΔ) and the fluorescence quantum yields (ΦF) were determined in DMF using the published comparative methods with

unsubstituted ZnPc (obtained from Sigma-Aldrich) as the reference compound.49 The following values for ZnPc were used in the calculations: ΦΔ(DMF) = 0.5650 and ΦF(THF) = 0.32.23 The emission spectra were obtained after excitation of the sample and the reference at λexc = 630 nm and were not corrected. All of the determinations were performed in triplicate, and the data represent the means of the measurements. The estimated experimental error was ±10%. Cell Cultures and Compounds. The 3T3, HeLa, SK-MEL-28, and HCT 116 cell lines were purchased from the American Type Cell Culture Collection (ATCC; United States). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) without phenol red (Lonza, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Lonza), 1% penicillin/streptomycin solution (Lonza), 10 mM HEPES buffer (Sigma, Germany), and 4 mM 2 L-glutamine (Lonza). Each cell line was cultured in 75 cm tissue culture flasks (TPP, Switzerland) and maintained in an incubator at 37 °C in a humidified atmosphere of 5% CO2. The subconfluent cells were subcultured every 3−4 days. For the dark toxicity and the photodynamic therapy experiments, the cells were seeded in 96-well plates (TPP) at a density of 1 × 104 cells per well (3T3, SK-MEL-28) or 7.5 × 103 cells per well (HeLa, HCT 116) for 24 h prior to the addition of the studied compounds. The stock solutions of the investigated compounds were prepared in DMEM at a concentration of 1.5 mM (1Zn-Et, 1Mg-Et, and 3Zn-Et) or 0.5 mM (2Zn-Et and 2Mg-Et) and sterilized by filtration through 0.2 μm syringe filters. No change in the concentration was detected after passage through the filter on the basis of the absorption spectra measured before and after filtration. Uptake to the Cells. To establish the time profile of the intracellular accumulation of 1Zn-Et and 3Zn-Et, the HeLa cells were seeded in 6 cm Petri dishes (TPP) at a density of 4.5 × 104 cells per dish. The cells were left to attach for 24 h, the medium was removed, and 10 μM PSs was added to 5 mL of cultivation medium. The cells were washed three times with 5 mL of phosphate-buffered saline (PBS; Sigma) after 0.5, 1, 2, 4, 6, 8, and 12 h followed by the addition of 5 mL of medium. The cells were scraped and transferred to 15 mL centrifugation tubes (TPP) and centrifuged for 5 min at 70g. The supernatant was replaced with 2 mL of fresh medium, and the cell pellet was gently resuspended and centrifuged again. This process was repeated two times. After the last centrifugation, the medium was replaced with DMF. Lysis of the cells was performed overnight at −20 °C and for 30 min at rt in an ultrasound bath prior to the measurement. The fluorescence of 1Zn-Et (λem = 714 nm, λexc = 640 nm) or 3Zn-Et (λem = 662 nm, λexc = 392 nm) was measured and plotted against the incubation time. In the case of 3Zn-Et, 10 μL of 10% trifluoroacetic acid in DMF was added to eliminate a possible residual PET effect. The nonspecific fluorescence was excluded by the control experiments. The uptake experiments were performed in triplicate. Subcellular Localization. Approximately 6 × 104 HeLa cells were seeded on Petri dishes suitable for confocal microscopy (WillCo Wells, The Netherlands) in culture medium and incubated for 12 h with 10 μM 1Zn-Et or 2Zn-Et at 37 °C under a 5% CO2 atmosphere with constant humidity. The medium was removed, the cells were washed twice with PBS, and fresh medium was added. The cells were incubated with LysoTracker Blue DND-22 (Molecular Probes, 2 μM) and MitoTracker Green FM (Molecular Probes, 2 μM) and incubated for an additional 15 min. After incubation, the cells were rinsed twice with PBS and examined under a Nikon Eclipse Ti (Nikon, Japan) fluorescence microscope equipped with an Andor Zyla cooled digital sCMOS camera (Andor Technology, United Kingdom) and NIS Elements AR 4.20 software (Laboratory Imaging, Czech Republic). Cytotoxicity and Photodynamic Treatment. The inherent toxicities of the compounds without the presence of light (dark toxicity) were assayed over a wide concentration range after the 24 h incubations in the 3T3 and HeLa cells. The viabilities of the cells were determined using the neutral red (NR) uptake assay (Sigma) based on the ability of the living cells to incorporate NR into their intact lysosomes. The soluble NR was measured as its optical density at λ = 540 nm using a Tecan Infinite 200 M plate reader (Tecan, Austria). The viability of the experimental groups was expressed as the 1746

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Journal of Medicinal Chemistry

harvested by trypsinization and costained with PI (7.5 μM, Molecular Probes) and annexin V−FITC (1 vol %, Molecular Probes) for 15 min at room temperature. The cells that were negative or positive for red and/or green fluorescence were counted using an Accuri C6 flow cytometer (Accuri Cytometers Europe Ltd., United Kingdom) at 20, 120, 240, and 360 min after irradiation. Ten thousand events were collected per analysis. The experiments were performed in triplicate. Data Analysis. The statistical analysis was performed with the GraphPad Prism statistical program (version 6.04; GraphPad Software, Inc. San Diego, CA). A one-way ANOVA test with a Bonferroni’s multiple comparisons post hoc test was used. The results were compared with the control samples, and the means were considered significant if (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001.

percentage of the untreated controls incubated under the same conditions (100%). For the photodynamic treatment experiments, the 3T3, HeLa, SK-MEL-28, or HCT 116 cells were first loaded for 12 h with various concentrations of the studied compounds. After loading, the cells were washed with PBS, and fresh medium was added. Irradiation of the cells with light was performed using a 450 W ozone-free Xe lamp (Newport) with the intensity reduced to 400 W (λ > 570 nm, 12.4 mW cm−2, 15 min, 11.2 J cm−2) and with a long-pass filter (Newport OG570) and a water filter (8 cm) to eliminate the undesirable wavelengths and heat. After irradiation, the cells were incubated for an additional 24 h before their viability was assayed by NR as previously described. At least five independent experiments, each in quadruplicate, were performed. The concentrations of the tested compounds inducing a 50% viability decrease after treatment under the dark conditions (TC50 is the median toxic concentration) or after the photodynamic treatment (IC50 is the median inhibition concentration) were calculated using GraphPad Prism software (version 6.04; GraphPad Software, Inc., San Diego, CA) for each independent experiment. The data in Table 2 are presented as the means (± standard deviation) of these values. In another photodynamic experiment on HeLa cells, the concentration of selected PSs (1Zn-Et, 2Zn-Et, and 3Zn-Et) was fixed and the irradiation time was varied. The concentration of each PS corresponded to the IC50 value determined in the above-mentioned experiment (see also Table 2). Irradiation times of 5, 15, and 25 min resulted in fluence of 3.7, 11.2, and 18.7 J cm−2, respectively. Other conditions were used as described above. The experiments were performed in triplicate. ROS Production Assessment. For the time profile of the ROS production of 1Zn-Et and 2Zn-Et (at concentrations corresponding to their IC85 values), the HeLa cells were seeded in black 96-well plates with a transparent bottom (Corning, United States) at a density of 7.5 × 103 cells per well. The incubation and irradiation protocol was identical to that of the photodynamic treatment. The generation of the ROS was monitored by the intracellular conversion of the cellpermeant H2DCFDA (Molecular Probes) into a fluorescent product (DCF). The cells were incubated for 30 min in medium with 0.1 μM H2DCFDA. After incubation, the cells were washed two times with PBS, and fresh cultivation medium was added. The changes in the fluorescence intensity (λex = 485 nm and λem = 525 nm) were measured using a Tecan Infinite 200 M plate reader before (9, 6, 3, and 0 min), during (3, 6, 9, 12, and 15 min), and after (3−39 min in 3 min steps) irradiation under the same conditions as those in the photodynamic treatment experiments. Simultaneously, the experiments without an activating light (the dark control) and the experiments without the studied compounds in the presence of an activating light (the light control) were performed to exclude the nonspecific changes in the fluorescence (primarily via autoxidation). The experiments were performed in triplicate. Caspase Activity after the Photodynamic Treatment. The caspase activities were evaluated on the HeLa cells seeded on 96-well plates and treated as described above in the section on the photodynamic treatment with 1Zn-Et or 2Zn-Et at concentrations corresponding to their IC15 and IC85 values. After 20 min, 2 h, 4 h, 6 h, or 24 h, the cultivating medium was removed, and the cells were lysed by the addition of 200 μL of lysis buffer (100 mM HEPES, 10 mM CHAPS, 10 mM DTT, pH 7.4). The cells were lysed for 1 h and immediately frozen at −80 °C until they were used for the caspase activity assessment using the luminescent kits for caspases 3/7, 8, and 9 (Promega, United States). All of the experimental groups were corrected for the total protein concentration in each sample using the Bradford protein assay (Sigma) and were expressed as a percentage of the activities of the control group (100%). The experiments were performed in triplicate. Evaluation of Cell Death Using Flow Cytometry. The HeLa cells were seeded in 60 mm Petri dishes and incubated for 12 h with 1Zn-Et or 2Zn-Et at concentrations corresponding to their IC15 and IC85 values. The irradiation was performed under the same conditions as those for the photodynamic treatment. The cells were then

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ASSOCIATED CONTENT

S Supporting Information *

Absorption spectra of the studied compounds, phototoxicities and dark toxicities against the different cell lines (CSV), subcellular localization of 1Zn-Et and 2Zn-Et, photographs showing changes in the mitochondria, photographs from the autophagy assays, and video showing the morphological changes in cells upon irradiation in the absence and presence of photosensitizers (AVI). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +420 495067380. *E-mail: [email protected]. Phone: +420 495067257. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Jiři ́ Kuneš for NMR measurements and Juraj Lenčo for MALDI-TOF spectra. This work was supported by the Czech Science Foundation, Project No. 13-27761S, and Grant Agency of Charles University, Project No. 1916214. Publication was cofinanced by the European Social Fund and the state budget of the Czech Republic, Project No. CZ.1.07/2.3.00/ 30.0061.

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ABBREVIATIONS USED DCF, dichlorofluorescein; DIC, differential interference contrast; DMEM, Dulbecco’s modified Eagle’s medium; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; MDC, monodansyl cadaverine; NR, neutral red; Pc, phthalocyanine; PDT, photodynamic therapy; PET, photoinduced electron transfer; PI, propidium iodide; PS, photosensitizer; TC50, halfmaximum toxic concentration; ZnPc, unsubstituted zinc(II) phthalocyanine

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REFERENCES

(1) Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.; Piette, J.; Wilson, B. C.; Golab, J. Photodynamic therapy of cancer: an update. Ca-Cancer J. Clin. 2011, 61, 250−281. (2) Zimcik, P.; Miletin, M. Photodynamic therapy. In Dyes and Pigments: New Research; Lang, A. R., Ed.; Nova Science Publishers, Inc.: New York, 2009; pp 1−62. (3) Yano, S.; Hirohara, S.; Obata, M.; Hagiya, Y.; Ogura, S.-i.; Ikeda, A.; Kataoka, H.; Tanaka, M.; Joh, T. Current states and future views in photodynamic therapy. J. Photochem. Photobiol., C 2011, 12, 46−67.

1747

DOI: 10.1021/jm5014852 J. Med. Chem. 2015, 58, 1736−1749

Article

Journal of Medicinal Chemistry (4) Buytaert, E.; Dewaele, M.; Agostinis, P. Molecular effectors of multiple cell death pathways initiated by photodynamic therapy. Biochim. Biophys. Acta, Rev. Cancer 2007, 1776, 86−107. (5) Nyokong, T.; Antunes, E. Photochemical and photophysical properties of metallophthalocyanines. In Handbook of Porphyrin Science; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; World Scientific Publishing: Singapore, 2010; Vol. 7; pp 247−358. (6) Furuyama, T.; Satoh, K.; Kushiya, T.; Kobayashi, N. Design, synthesis, and properties of phthalocyanine complexes with maingroup elements showing main absorption and fluorescence beyond 1000 nm. J. Am. Chem. Soc. 2014, 136, 765−776. (7) Lourenco, L. M. O.; Pereira, P. M. R.; Maciel, E.; Valega, M.; Domingues, F. M. J.; Domingues, M. R. M.; Neves, M. G. P. M. S.; Cavaleiro, J. A. S.; Fernandes, R.; Tome, J. P. C. Amphiphilic phthalocyanine-cyclodextrin conjugates for cancer photodynamic therapy. Chem. Commun. 2014, 50, 8363−8366. (8) Liang, R.; Tian, R.; Ma, L.; Zhang, L.; Hu, Y.; Wang, J.; Wei, M.; Yan, D.; Evans, D. G.; Duan, X. A supermolecular photosensitizer with excellent anticancer performance in photodynamic therapy. Adv. Funct. Mater. 2014, 24, 3144−3151. (9) Lau, J. T. F.; Lo, P.-C.; Jiang, X.-J.; Wang, Q.; Ng, D. K. P. A dual activatable photosensitizer toward targeted photodynamic therapy. J. Med. Chem. 2014, 57, 4088−4097. (10) Jia, X.; Yang, F.-F.; Li, J.; Liu, J.-Y.; Xue, J.-P. Synthesis and in vitro photodynamic activity of oligomeric ethylene glycol−quinoline substituted zinc(II) phthalocyanine derivatives. J. Med. Chem. 2013, 56, 5797−5805. (11) Chen, Z.; Zhou, S.; Chen, J.; Li, L.; Hu, P.; Chen, S.; Huang, M. An effective zinc phthalocyanine derivative for photodynamic antimicrobial chemotherapy. J. Lumin. 2014, 152, 103−107. (12) He, H.; Lo, P.-C.; Ng, D. K. P. A glutathione-activated phthalocyanine-based photosensitizer for photodynamic therapy. Chem.Eur. J. 2014, 20, 6241−6245. (13) Ranyuk, E.; Cauchon, N.; Klarskov, K.; Guérin, B.; van Lier, J. E. Phthalocyanine−peptide conjugates: receptor-targeting bifunctional agents for imaging and photodynamic therapy. J. Med. Chem. 2013, 56, 1520−1534. (14) Manet, I.; Manoli, F.; Donzello, M. P.; Viola, E.; Masi, A.; Andreano, G.; Ricciardi, G.; Rosa, A.; Cellai, L.; Ercolani, C.; Monti, S. Pyrazinoporphyrazines with externally appended pyridine rings. 13. Structure, UV-visible spectral features, and noncovalent interaction with DNA of a positively charged binuclear (Zn-II/Pt-II) macrocycle with multimodal anticancer potentialities. Inorg. Chem. 2013, 52, 321− 328. (15) Dumoulin, F.; Durmus, M.; Ahsen, V.; Nyokong, T. Synthetic pathways to water-soluble phthalocyanines and close analogs. Coord. Chem. Rev. 2010, 254, 2792−2847. (16) Makhseed, S.; Machacek, M.; Alfadly, W.; Tuhl, A.; Vinodh, M.; Simunek, T.; Novakova, V.; Kubat, P.; Rudolf, E.; Zimcik, P. Watersoluble non-aggregating zinc phthalocyanine and in vitro studies for photodynamic therapy. Chem. Commun. 2013, 49, 11149−11151. (17) Li, H.; Jensen, T. J.; Fronczek, F. R.; Vicente, M. G. Syntheses and properties of a series of cationic water-soluble phthalocyanines. J. Med. Chem. 2008, 51, 502−511. (18) Jiang, X. J.; Yeung, S. L.; Lo, P. C.; Fong, W. P.; Ng, D. K. P. Phthalocyanine-polyamine conjugates as highly efficient photosensitizers for photodynamic therapy. J. Med. Chem. 2011, 54, 320−330. (19) Zimcik, P.; Miletin, M.; Radilova, H.; Novakova, V.; Kopecky, K.; Svec, J.; Rudolf, E. Synthesis, properties and in vitro photodynamic activity of water-soluble azaphthalocyanines and azanaphthalocyanines. Photochem. Photobiol. 2010, 86, 168−175. (20) Kostka, M.; Zimcik, P.; Miletin, M.; Klemera, P.; Kopecky, K.; Musil, Z. Comparison of aggregation properties and photodynamic activity of phthalocyanines and azaphthalocyanines. J. Photochem. Photobiol., A 2006, 178, 16−25. (21) Kobayashi, N.; Ogata, H.; Nonaka, N.; Luk’yanets, E. A. Effect of peripheral substitution on the electronic absorption and fluorescence spectra of metal-free and zinc phthalocyanines. Chem. Eur. J. 2003, 9, 5123−5134.

(22) Chauke, V.; Nyokong, T. Synthesis and electrochemical characterisation of new tantalum(V) alkythio phthalocyanines. Inorg. Chim. Acta 2010, 363, 3662−3669. (23) Zimcik, P.; Novakova, V.; Kopecky, K.; Miletin, M.; Uslu Kobak, R. Z.; Svandrlikova, E.; Vác hová, L.; Lang, K. Magnesium azaphthalocyanines: an emerging family of excellent red-emitting fluorophores. Inorg. Chem. 2012, 51, 4215−4223. (24) Topal, S. Z.; Isci, U.; Kumru, U.; Atilla, D.; Gurek, A. G.; Hirel, C.; Durmus, M.; Tommasino, J.-B.; Luneau, D.; Berber, S.; Dumoulin, F.; Ahsen, V. Modulation of the electronic and spectroscopic properties of Zn(ii) phthalocyanines by their substitution pattern. Dalton Trans. 2014, 43, 6897−6908. (25) Aydin Tekdas, D.; Kumru, U.; Gurek, A. G.; Durmus, M.; Ahsen, V.; Dumoulin, F. Towards near-infrared photosensitisation: a photosensitising hydrophilic non-peripherally octasulfanyl-substituted Zn phthalocyanine. Tetrahedron Lett. 2012, 53, 5227−5230. (26) Idowu, M.; Nyokong, T. Synthesis, photophysical and photochemical studies of water soluble cationic zinc phthalocyanine derivatives. Polyhedron 2009, 28, 416−424. (27) Tuhl, A.; Makhseed, S.; Zimcik, P.; Al-Awadi, N.; Novakova, V.; Samuel, J. Heavy metal effects on physicochemical properties of nonaggregated azaphthalocyanine derivatives. J. Porphyrins Phthalocyanines 2012, 16, 817−825. (28) Novakova, V.; Hladik, P.; Filandrova, T.; Zajicova, I.; Krepsova, V.; Miletin, M.; Lenco, J.; Zimcik, P. Structural factors influencing the intramolecular charge transfer and photoinduced electron transfer in tetrapyrazinoporphyrazines. Phys. Chem. Chem. Phys. 2014, 16, 5440− 5446. (29) Zimcik, P.; Miletin, M.; Musil, Z.; Kopecky, K.; Kubza, L.; Brault, D. Cationic azaphthalocyanines bearing aliphatic tertiary amino substituents-synthesis, singlet oxygen production and spectroscopic studies. J. Photochem. Photobiol., A 2006, 183, 59−69. (30) Duan, W. B.; Lo, P. C.; Duan, L.; Fong, W. P.; Ng, D. K. P. Preparation and in vitro photodynamic activity of amphiphilic zinc(II) phthalocyanines substituted with 2-(dimethylamino)ethylthio moieties and their N-alkylated derivatives. Bioorg. Med. Chem. 2010, 18, 2672− 2677. (31) Halliwell, B.; Gutteridge, J. Free Radicals in Biology and Medicine, 4th ed.; Oxford University Press: Oxford, U.K., 2007. (32) Bilski, P.; Belanger, A. G.; Chignell, C. F. Photosensitized oxidation of 2′,7′-dichlorofluorescin: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2′,7′dichlorofluorescein. Free Radical Biol. Med. 2002, 33, 938−946. (33) Kessel, D.; Luguya, R.; Vicente, M. G. H. Localization and photodynamic efficacy of two cationic porphyrins varying in charge distribution. Photochem. Photobiol. 2003, 78, 431−435. (34) Roberg, K. Relocalization of cathepsin D and cytochrome c early in apoptosis revealed by immunoelectron microscopy. Lab. Invest. 2001, 81, 149−158. (35) Manet, I.; Manoli, F.; Donzello, M. P.; Viola, E.; Andreano, G.; Masi, A.; Cellai, L.; Monti, S. A cationic Zn-II porphyrazine induces a stable parallel G-quadruplex conformation in human telomeric DNA. Org. Biomol. Chem. 2011, 9, 684−688. (36) Barros, L. F.; Kanaseki, T.; Sabirov, R.; Morishima, S.; Castro, J.; Bittner, C. X.; Maeno, E.; Ando-Akatsuka, Y.; Okada, Y. Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency. Cell Death Differ. 2003, 10, 687−697. (37) Duprez, L.; Wirawan, E.; Vanden Berghe, T.; Vandenabeele, P. Major cell death pathways at a glance. Microbes Infect. 2009, 11, 1050− 1062. (38) Vermes, I.; Haanen, C.; Steffensnakken, H.; Reutelingsperger, C. A novel assay for apoptosisflow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein-labeled annexin-V. J. Immunol. Methods 1995, 184, 39−51. (39) Albee, L.; Shi, B.; Perlman, H. Aspartic protease and caspase 3/7 activation are central for macrophage apoptosis following infection with Escherichia coli. J. Leukocyte Biol. 2007, 81, 229−237. 1748

DOI: 10.1021/jm5014852 J. Med. Chem. 2015, 58, 1736−1749

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Journal of Medicinal Chemistry (40) Ishisaka, R.; Utsumi, T.; Kanno, T.; Arita, K.; Katunuma, N.; Akiyama, J.; Utsumi, K. Participation of a cathepsin L-type protease in the activation of caspase-3. Cell Struct. Funct. 1999, 24, 465−470. (41) Hishita, T.; Tada-Oikawa, S.; Tohyama, K.; Miura, Y.; Nishihara, T.; Tohyama, Y.; Yoshida, Y.; Uchiyama, T.; Kawanishi, S. Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39. Cancer Res. 2001, 61, 2878−2884. (42) Guicciardi, M. E.; Leist, M.; Gores, G. J. Lysosomes in cell death. Oncogene 2004, 23, 2881−2890. (43) Reiners, J. J.; Agostinis, P.; Berg, K.; Oleinick, N. L.; Kessel, D. H. Assessing autophagy in the context of photodynamic therapy. Autophagy 2010, 6, 7−18. (44) He, C. C.; Klionsky, D. J. Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 2009, 43, 67−93. (45) Bursch, W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 2001, 8, 569−581. (46) Li, W.; Yuan, X.; Nordgren, G.; Dalen, H.; Dubowchik, G. M.; Firestone, R. A.; Brunk, U. T. Induction of cell death by the lysosomotropic detergent MSDH. FEBS Lett. 2000, 470, 35−39. (47) Bayir, Z. A. Synthesis and characterization of novel soluble octacationic phthalocyanines. Dyes Pigm. 2005, 65, 235−242. (48) Zhang, L.; Huang, J.; Ren, L.; Bai, M.; Wu, L.; Zhai, B.; Zhou, X. Synthesis and evaluation of cationic phthalocyanine derivatives as potential inhibitors of telomerase. Bioorg. Med. Chem. 2008, 16, 303− 312. (49) Novakova, V.; Miletin, M.; Filandrová, T.; Lenčo, J.; Růzǐ čka, A.; Zimcik, P. Role of steric hindrance in the Newman−Kwart rearrangement and in the synthesis and photophysical properties of arylsulfanyl tetrapyrazinoporphyrazines. J. Org. Chem. 2014, 79, 2082− 2093. (50) Michelsen, U.; Kliesch, H.; Schnurpfeil, G.; Sobbi, A. K.; Wöhrle, D. Unsymmetrically substituted benzonaphthoporphyrazines: a new class of cationic photosensitizers for the photodynamic therapy of cancer. Photochem. Photobiol. 1996, 64, 694−701.

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DOI: 10.1021/jm5014852 J. Med. Chem. 2015, 58, 1736−1749