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Extending the Excitation Wavelength of Potential Photosensitizers via Appendage of a Kinetically Stable Terbium(III) Macrocyclic Complex for Applications in Photodynamic Therapy Phuc Ung,*,†,‡ Michèle Clerc,† Huaiyi Huang,†,§ Kangqiang Qiu,§ Hui Chao,§ Michael Seitz,∥ Ben Boyd,‡,⊥ Bim Graham,# and Gilles Gasser*,g †

Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, P. R. China ∥ Institute of Inorganic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany ‡ Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, ⊥ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, and #Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia g Laboratory for Inorganic Chemical Biology, Chimie ParisTech, PSL Research University, F-75005 Paris, France §

S Supporting Information *

ABSTRACT: The development of viable photodynamic therapy protocols is often hindered by photosensitizers that require high-energy UV irradiation that has limited potential for clinical use due to its low tissue penetration. Herein, we report a strategy for extending the excitation wavelength of potential photosensitizers via the covalent attachment of a terbium(III)-1,4,7,10tetraazacyclododecane-1,4,7-triacetate complex (DO3A-Tb). The method was systematically demonstrated with a series of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene, anthracene, pyrene, and fluoranthene) to prepare six new complexes (Tb1−Tb6) with bathochromic shifts that extended into the visible region. Determination of their quantum yields for singlet oxygen (1O2) production at 350 and 420 nm showed significant enhancements from the parent molecule in all cases. Cell viability studies on cervical cancer cells (HeLa) and noncancerous MRC-5 cells showed no measurable cytotoxicity for all complexes prior to light irradiation. However, after irradiation at 420 nm (20 min, 9.27 J cm−2), Tb3−Tb6 were phototoxic to HeLa cells with IC50 values between 14.3−32.3 μM. Cell morphological studies and fluorescence microscopy with live/dead cell stains confirmed these findings. In addition, these complexes were highly stable in human blood plasma, with no significant degradation observed after 96 h at 37 °C. This excellent phototoxicity profile and high stability in blood plasma, coupled with the moderately lipophilic nature of the complexes, favorably indicate the potential of DO3A-Tb as a heavy atom-bearing moiety for modification of potential photosensitizers into ideal phototherapeutic drug candidates with longer excitation wavelengths for in vivo application.

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INTRODUCTION Photodynamic therapy (PDT) is a localized treatment modality that is currently used for the treatment of various diseases and infections including cancers, dermatological diseases, fungal infections, age-related macular degeneration, and pathological myopia.1,2 During PDT, a patient is administered a nontoxic photosensitizer (PS). The PS is activated by light, which mediates the conversion of molecular oxygen (3O2) to various © 2017 American Chemical Society

reactive oxygen species (ROS) that subsequently causes cell death. As such, PDT offers minimal invasiveness and precise spatiotemporal control. Ten PSs have already been approved for clinical use, including the recently approved Tookad Soluble for Received: March 21, 2017 Published: July 6, 2017 7960

DOI: 10.1021/acs.inorgchem.7b00677 Inorg. Chem. 2017, 56, 7960−7974

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Inorganic Chemistry the treatment of prostate cancer, and at least seven more are currently in clinical trials.1,3−5 Despite considerable success, PDT has yet to gain clinical acceptance as a first-line treatment option for cancer and is usually restricted to adjunctive therapy after surgical resection due to the lack of an ideal PS. While porphyrin and its structurally related analogues (chlorins, porphycenes, and pthalocyanines) have made enormous contributions and comprise all of the approved drugs (e.g., Photofrin, Levulan, Foscan, etc.), their tedious synthesis, poor bioavailability, and prolonged patient photosensitivity has limited their application. Thus, there is rising interest in the investigation of alternative structural motifs to develop next-generation PSs, with notable examples including various phenothiazinium derivatives,6 anthracyclines and anthraquinones (e.g., hypericin and doxorubicin),7−9 xanthenes,10,11 cyanine12,13 and BODIPY dyes,14 and ruthenium,15−22 rhodium,16 rhenium,23−25 and lanthanide complexes.26−42 Metal complexes are particularly suitable for this task, as the inclusion of a metal into the PS takes advantage of the heavy atom effect43 to populate the excited triplet state of the PS (3PS*), which in turn facilitates generation of ROS and, ultimately, cell death. The application of metals in cancer diagnosis and treatment is well-established, with prominent examples including Pt(II) for use as chemotherapeutic drugs (e.g., cisdichlorodiammineplatinum(II) (cisplatin)) and Gd(III) for use in magnetic resonance imaging (MRI) contrasting agents (e.g., Magnevist, Dotarem, etc.).44 Only more recently have the unique physical and chemical properties of the lanthanides begun to be exploited in the development of PDT agents.44 Indeed, lanthanide-substituted porphyrins and porphyrin analogues make promising PSs for use in PDT, and the number of reports in this area has steadily increased.34−42 The most successful of these include the diamagnetic lutetium(III) texaphyrin complex (MLu, Lutrin, Figure 1), a remarkable compound that can be

As such, there is still significant scope in the development of small nonporphyrinic lanthanide complexes as PSs for PDT. Herein, we report the employment of an alternative lanthanidebearing moiety, DO3A-Tb (Figure 1), for derivatization of chromophoric units into PSs with improved photophysical properties for PDT. Although photoactive Tb(III) complexes have been reported before,30,31,33,52 this strategy represents the first demonstration using a macrocylic, encapsulating ligand that offers the possibility for variation of the conjugated PS. The synthesis and characterization of six new Tb(III) complexes is reported from the conjugation of DO3A-Tb to a series of polycyclic aromatic hydrocarbons (PAHs) as models for potential PSs. The series include naphthalene (Tb1), phenanthrene (Tb2), anthracene (Tb3), pyrene (Tb4), and fluoranthene (Tb5 and Tb6, Figure 2). The PAHs used in this study

Figure 2. Structures of the Tb(III) complexes studied in this work.

offer unique advantages over porphyrinic molecules due to their low molecular weight, ease of derivatization, commercial availability, and capacity to introduce a moderate degree of lipophilicity to the complexes. In contrast, the synthesis and purification of porphyrinic molecules is often tedious and yields highly insoluble products. The photophysical properties of Tb1−Tb6, including 1O2 formation quantum yields, were investigated using UV/vis spectroscopy, and the complexes’ distribution coefficients, human blood plasma stability, and toxicity toward human cervical carcinoma cells (HeLa) and noncancerous MRC-5 cells measured to determine their viability as new PSs for PDT. Conjugation of DO3A-Tb is shown to be a useful strategy for the optimization and development of the next generation of phototherapeutic drugs.

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RESULTS AND DISCUSSION Synthesis and Characterization. The synthesis of the Tb(III) complexes commenced with the preparation of bromoacetyl derivatives of the parent PAHs (P1−P6; Scheme 1). While P1 and P4 can be obtained commercially, P2, P3, P5, and P6 were prepared in-house. P2 and P3 were synthesized according to a literature procedure, with analytical data consistent with that reported previously.53−55 The synthesis of P5 and P6 began with Friedel−Crafts acylation of fluoranthene using acetyl chloride and AlCl3 in dry dichloromethane (DCM) under Schlenk conditions, to afford both the 3-acetyl (1) and 8acetyl (2) fluoranthene derivatives.56 Because of the chemical similarity of the two products, separation was performed by column chromatography using very fine grade silica (10−14 μm, LC35A Davisil, Grace Davison) to fully resolve the products. The products were eluted using a mixture of 1:2 hexane−DCM, with 1 eluting first followed immediately by 2. Interestingly, under 365

Figure 1. Structures of Lu(III) texaphyrin (MLu, Lutrin) and DO3ATb.

excited at 732 nm and is currently undergoing clinical trials for breast cancer and photoangioplasty.45−48 Others have implemented lanthanides in doped nanoparticles to overcome issues with stability of the PS and for tumor-targeted drug delivery.1 In particular, lanthanide-doped upconverting nanoparticles (UCNPs) have gained immense interest for near-IR-triggered PDT.44,49,50 However, nanoparticles in general suffer poor translation to clinical application due to difficulties in the modulation of their biodistribution, which results in poor net delivery to solid tumors (100 g/L). The log D values of Tb2 and Tb3 are similar, while those of Tb4, Tb5, and Tb6 are nearly zero, clearly demonstrating at least a moderately lipophilic character of these compounds (water solubility = 53.3, 8.61, 1.82, 1.52, 1.68 g/L, respectively). Next, a fluorometric cell viability study (Resazurin, Promocell GmbH) was performed on two different cell lines (cancerous HeLa cells and noncancerous MRC-5 cells) to determine the feasibility of these complexes to act as PSs for PDT. The aqueous solubility of the complexes allowed the experiments to be conducted at very high concentrations (Tb1−Tb3 ≤ 500 μM, Tb4−Tb6 ≤ 300 μM), before precipitation was observed in the culture media. Interestingly, no cytotoxicity was observed for any

Figures S30−S34). The stability was further confirmed by MS analysis after 96 h of incubation, which revealed m/z signals and isotopic patterns consistent with the intact complexes (see Supporting Information, Figures S35−S40). Distribution Coefficients and Phototoxicity. The distribution coefficients (log D) of the Tb(III) complexes were measured to speculate on their cellular uptake efficiencies, since Barton et al.67 have demonstrated that increasing the lipophilicity of a transition-metal complex can increase its cellular uptake. (Note, however, that similar lanthanide complexes have been shown to enter cells through active endocytosis, and not through a passive diffusion mechanism, which is usually limited to small, neutral, and hydrophobic molecules).68 The log D values for Tb1−Tb6 were determined using the “shake-flask” procedure17 with octanol and PBS (pH 7.01). The values are summarized in Table 3, revealing a correlation between log D and the relative 7965

DOI: 10.1021/acs.inorgchem.7b00677 Inorg. Chem. 2017, 56, 7960−7974

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Figure 6. RP-UPLC traces of Tb4 after incubation in human blood plasma at 37 °C and extraction in methanol, using caffeine as an internal standard, recorded at 275 nm. Traces are offset in time for clarity.

Table 3. IC50 Values for Tb1−Tb6 in Cancerous HeLa Cells and Noncancerous MRC-5 Cells

Tb1 Tb2 Tb3 Tb4 Tb5 Tb6 cisplatin 5-ALA

MRC-5 [μM] (48 h, dark)

HeLa [μM] (48 h, dark)

HeLa [μM] (4 h, dark)

HeLaa [μM] (4 h, 350 nm)

HeLab [μM] (4 h, 420 nm)

>500 >500 >500 >300 >300 >300 12.5 ± 2.5 >50

>500 >500 >500 >300 >300 >300 11.8 ± 1.8 >50

>500 >500 >500 >300 >300 >300 11.4 ± 0.1 >50

>500 >500 14.6 ± 1.0 12.8 ± 3.1 10.6 ± 3.0 11.9 ± 2.0 11.3 ± 0.1 >50

>500 >500 32.3 ± 7.1 14.3 ± 1.9 25.5 ± 3.6 25.3 ± 4.9 11.2 ± 0.1 >50

PIc (420 nm)

log D

>15.5 >21.0 >11.8 >11.9

−1.83 −0.50 −0.37 −0.02 0.04 0.04

350 nm centered irradiation for 10 min, light dose = 2.58 J cm−2. b420 nm centered irradiation for 20 min, light dose = 9.27 J cm−2. cPI = IC50 (dark)/IC50 (after 420 nm irradiation). a

of the complexes at these high concentrations, even after 48 h of incubation, suggesting that regular cellular homeostasis remains unperturbed. However, of more interest was the phototoxicity of these compounds, in view of their ability to photosensitize molecular oxygen to produce 1O2. Therefore, HeLa cells were incubated with Tb1−Tb6 for 4 h, before replacing the media with fresh complex-free media and irradiating with 350 (10 min, 2.58 J cm−2) or 420 nm (20 min, 9.27 J cm−2) light. Control experiments with the same light exposures were performed in all cases to confirm that the cells were unaffected by these conditions in the absence of treatment with complexes. Positive controls were also performed with cisplatin and 5-ALA (protoporphyrin, Levulan). The calculated IC50 values from these experiments are summarized in Table 3. These clearly show that Tb3−Tb6 are phototoxic. Their phototoxicities were found to be higher under 350 nm irradiation (IC50 = 10.6−14.6 μM), which is most likely due to the higher molar absorptivity of the PSs at this wavelength. However, 420 nm irradiation is more biologically relevant due to its deeper tissue penetration and lower toxicity compared to UV radiation, and at this wavelength,

Tb3−Tb6 showed significant phototoxicity, with the most exemplary compound being Tb4 with an IC50 of 14.3 μM and a phototoxicity index (PI, defined by the ratio of cytotoxicity in the dark divided by the phototoxicity) > 21. This phototoxicity is comparable to the dark cytotoxicity of the common chemotherapeutic drug cisplatin and higher than that of the clinically approved PDT drug 5-ALA under our experimental conditions. Tb1 and Tb2 did not show any photocytotoxic effects, which may be a consequence of their low lipophilicity (log D = −1.83 and −0.50, respectively) and, therefore, low cellular uptake. To further investigate the phototoxicity of the complexes, bright-field microscopy was used to check for cell morphological changes indicative of oxidative damage from the photosensitization process.69 As such, HeLa cells were incubated with 20 μM Tb1−Tb6 for 4 h in the dark, then fresh medium for 24 h, before bright-field images were acquired (Figure 5). Under these conditions, no changes in HeLa cell morphology were observed. However, when the cells were irradiated at 420 nm (20 min, 9.27 J cm−2) after replacement of the medium, bright-field images acquired after the same incubation time showed a 7966

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Figure 7. Morphology of HeLa cells treated with Tb1−Tb6 (20 μM, 4 h) in the dark or after 420 nm irradiation (20 min, 9.27 J cm−2).

Figure 8. Calcein-AM (λex/em = 488/550 nm, Live) and EthD-1 (λex/em = 488/630 nm, Dead) dual staining of HeLa cells treated with Tb1−Tb6 (20 μM, 4 h) in the dark or after 420 nm irradiation (20 min, 9.27 J cm−2).

24 h from the red fluorescence of the EthD-1 dye. As before, Tb1 and Tb2 did not induce any cell death (absence of red fluorescence), while Tb4 proved to be the most phototoxic of the complexes. Overall, the observed variation in phototoxicity between the complexes can be ascribed to variations in their 1O2 production quantum yields, absorbance profile, molar absorptivity, and cellular uptake efficiency.

significant amount of cell blebbing, cell debris, and shrinkage for samples treated with Tb4 (Figure 7). Cell shrinkage was also observed for Tb3, Tb5, and Tb6 to varying degrees, but Tb4 appeared to be the most phototoxic of the complexes upon irradiation with 420 nm light. The relative phototoxicities of the complexes were confirmed using a two-color fluorescent cell viability assay (LIVE/DEAD Viability/Cytotoxicity Kit, Life Technologies) to stain live and dead cells based on their physical or biochemical properties.70 In this assay, calcein-AM dye is used to stain live cells with green fluorescence (λex/em = 495/515 nm) as a result of ubiquitous intracellular esterase activity, while dead cells are stained with the nucleus dye EthD-1 to give red fluorescence (λex/em = 488/630 nm). As shown in Figure 8, no significant cell death occurred when HeLa cells were treated with 20 μM of Tb1−Tb6 in the dark for 4 h, then allowed to recover for 24 h in fresh medium. Under these conditions, all the samples emitted strong green fluorescence. However, when the cells were treated with Tb3− Tb6 for 4 h and then irradiated at 420 nm (20 min, 9.27 J cm−2) in fresh medium, large numbers of dead cells were evident after

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CONCLUSIONS Research into the development of stable nonporphyrinic macrocylic lanthanide complexes as new PSs for PDT is scarce, despite Lu(III) texaphyrin (Lutrin) having emerged as a highly successful lanthanide-based PDT agent. Here, we have shown that the highly stable macrocylic Tb(III) chelate DO3A-Tb can be appended to several PAHs to render their photophysical properties more suitable for application as PSs for PDT. Longer wavelength excitation and excellent 1O2 production quantum yields were observed for all of the complexes prepared in this study (Tb1−Tb6). Four of the complexes (Tb3−Tb6) were 7967

DOI: 10.1021/acs.inorgchem.7b00677 Inorg. Chem. 2017, 56, 7960−7974

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(0.982 g, 7.36 mmol) and fluoranthene (1.49 g, 7.36 mmol) in dry distilled DCM (150 mL) was added acetyl chloride (0.581 g, 0.528 mL, 7.36 mmol) dropwise over 1 h at −5 °C under a N2 atm. The resulting solution was stirred at −5 °C for 3 h and allowed to warm to room temperature overnight. After this time, the mixture was poured into 500 mL of iced water, and the DCM layer was extracted, dried with anhydrous MgSO4, filtered, and evaporated. The residue was redissolved in 200 mL of Et2O and extracted with 2 × 200 mL of water. The organic layer was collected and dried with anhydrous MgSO4, filtered, and evaporated. The crude residue was purified using silica gel chromatography eluting with a 1:2 mixture of hexane−DCM to obtain the 3-Ac derivative, followed by the 8-Ac derivative, as yellow and orange solids after evaporation of the combined fractions. Yield: 0.478 g, 26% and 0.454 g, 25% respectively. 3-Acetyl-fluoranthene: ES-MS (m/z): 245.0 [M + H]+. Analytical RPHPLC (chiral, 250 nm): Rt = 26.81 min. 1H NMR (CDCl3, 400 MHz) δ: 2.76 (s, 3H, CH3), 7.32−7.44 (m, 2H, CH), 7.66 (t, 1H, J = 7.7 Hz, CH), 7.81−7.90 (m, 4H, CH), 8.12 (d, 1H, J = 7.3 Hz, CH), 8.76 (d, 1H, J = 8.6 Hz, CH); 13C NMR (CDCl3, 101 MHz) δ: 29.15 (CH3), 118.60 (CH), 120.71 (CH), 121.64 (CH), 122.36 (CH), 127.21 (CH), 127.83 (CH), 127.90 (C), 128.98 (CH), 130.32 (CH), 132.16 (CH), 133.04 (C), 133.72 (C), 136.98 (C), 138.16 (C), 140.70 (C), 141.98 (C), 200.52 (CO). 8-Acetyl-fluoranthene: ES-MS (m/z): 245.0 [M + H]+. Analytical RPHPLC (chiral, 250 nm): Rt = 26.62 min. 1H NMR (CDCl3, 400 MHz) δ: 2.70 (s, 3H, CH3), 7.67 (t, 2H, J = 7.7 Hz, CH), 7.86−7.95 (m, 3H, CH), 7.99 (t, 3H, J = 7.2 Hz, CH), 8.48 (s, 1H, CH); 13C NMR (CDCl3, 101 MHz) δ: 26.94 (CH3), 120.89 (CH), 121.29 (2xCH), 121.54 (CH), 127.30 (CH), 128.00 (CH), 128.19 (CH), 128.33 (CH), 128.84 (CH), 130.11 (C), 133.25 (C), 135.79 (C), 136.10 (C), 136.37 (C), 139.70 (C), 143.72 (C), 128.01 (CO). General Procedure for the Synthesis of P2, P3, P5, and P6. The procedure for this reaction was adapted from Shamsa et al.53 A mixture of the acetyl−PAH (1 equiv) and CuBr2 (2 equiv) was refluxed in a 1:1 mixture of EtOAc/CHCl3 for 6 h then stirred at room temperature overnight. After this time the solution was filtered, and the filtrate was collected and evaporated. The residue was then redissolved in DCM and extracted with 2 × 200 mL of water. After this, the organic layer was collected and dried over anhydrous MgSO4, filtered, and evaporated to dryness to obtain the product in quantitative yield. 3-(Bromoacetyl)phenanthrene (P2). 3-Acetyl-phenanthrene (0.402 g, 1.83 mmol), CuBr2 (0.818 g, 3.66 mmol), CHCl3/EtOAc (100 mL). The product was obtained as a yellow oil with analytical data consistent with literature.53,55 Analytical RP-HPLC (C18, 250 nm): Rt = 20.81 min. 1 H NMR (CDCl3, 400 MHz) δ: 4.80 (s, 2H, CH2), 7.78 (d, 1H, CH), 7.85−7.89 (m, 2H, CH), 7.94 (t, 2H, J = 7.8 Hz, CH), 8.03−8.09 (m, 1H, CH), 8.18 (d, 1H, J = 8.3 Hz, CH), 8.76 (d, 1H, J = 7.3 Hz, CH), 9.28 (s, 1H, CH); 13C NMR (CDCl3, 101 MHz) δ: 31.59 (CH2), 122.27 (CH), 124.07 (CH), 125.07 (CH), 125.74 (CH), 127.02 (CH), 127.07 (CH), 128.50 (CH), 128.58 (CH), 129.18 (C), 129.80 (CH), 129.95 (C), 130.92 (C), 131.68 (C), 134.84 (C), 190.71 (CO). 2-(Bromoacetyl)anthracene (P3). 2-Acetyl-anthracene (0.219 g, 0.994 mmol), CuBr2 (0.445 g, 1.99 mmol), CHCl3/EtOAc (100 mL). The product was obtained as a yellow solid with analytical data consistent with literature.54 Analytical RP-HPLC (C18, 250 nm): Rt = 20.99 min. 1H NMR (CDCl3, 400 MHz) δ: 4.60 (s, 2H, CH2), 7.50− 7.58 (m, 2H, CH), 7.96 (dd, 1H, J = 9.0/0.7 Hz, H3), 8.00−8.06 (m, 3H, CH), 8.43 (s, 1H, CH), 8.57 (d, 1H, J = 0.5 Hz, CH), 8.68 (q, 1H, J = 1.6/0.8 Hz, H1); 13C NMR (CDCl3, 101 MHz) δ: 30.89 (CH2), 122.92 (CH), 126.33 (CH), 126.54 (CH), 127.18 (CH), 128.37 (CH), 128.64 (CH), 129.32 (CH), 129.50 (CH), 130.23 (C), 130.98 (C), 132.31 (C), 132.69 (CH), 132.83 (C), 133.70 (C), 191.29 (CO). 3-Bromoacetyl fluoranthene (P5). 3-Acetyl-fluoranthene (0.309 g, 1.26 mmol), CuBr2 (0.565 g, 2.53 mmol), EtOAc/CHCl3 (100 mL). The product was obtained as a yellow solid. ES-MS (m/z): 323.1 [M + H]+; HR-MS (m/z): calcd for C18H11BrO [M + H]+ 323.006 60, found: 323.006 74. IR (neat, 4000−800 cm−1): vmax = 3056, 2942, 1666, 1609. Analytical RP-HPLC (C18, 250 nm): Rt = 21.77 min. 1H NMR (CDCl3, 400 MHz) δ: 4.57 (s, 2H, CH2), 7.29−7.43 (m, 2H, CH), 7.63 (t, 1H, J = 7.7 Hz, CH), 7.75−7.90 (m, 4H, CH), 8.10 (d, 1H, J = 7.3 Hz, CH), 8.65

shown to exhibit significant phototoxic effects on HeLa cells upon light irradiation at 420 nm (IC50 = 14.3−32.3 μM) as can be seen by the cell morphological changes and through fluorescence microscopy experiments, while remaining completely nontoxic in the dark. In addition, these complexes showed no significant degradation in human blood plasma for over 96 h at 37 °C. These results demonstrate the usefulness of DO3A-Tb as a generic heavy atom-bearing building block that may be exploited in the development of new PSs for PDT, as it overcomes the drawbacks associated with porphyrin-based PSs (synthetic accessibility and amenability, purification, and poor tissue elimination arising from low water solubility). Moreover, the Tb(III) ion can be easily substituted for another lanthanide or similar transition metal to introduce new properties to the PS. For example, a Gd(III) ion could be used to prepare a bimodal MRI and PDT agent, or the radioisotope 177Lu could be substituted for simultaneous radionuclide therapy and PDT.

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EXPERIMENTAL SECTION

Materials and Reagents. All starting materials, reagents, and solvents were obtained from commercial suppliers and were of general reagent grade or analytical grade and used without further purification. All solvents were used as received. Dry DCM was prepared by predrying with CaCl2 and distilled over CaH2. Standard Schlenk techniques were used for reactions requiring inert conditions. Instrumentation and Methods. 1H NMR spectra were recorded at 400 MHz, and 13C NMR spectra were recorded at 101 MHz in deuterated solvents at room temperature on a Bruker Avance 400 NMR spectrometer. The chemical shifts (δ) are reported in units of parts per million (ppm) and referenced internally from the residual proteosolvent resonance. Multiplicities for NMR resonances are abbreviated for reporting; s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), br (broad), Ar (aromatic). ESI-MS were measured with a Bruker Daltonics HCT 6000 mass spectrometer. HR-MS were performed on a Bruker maXis QTOF high-resolution mass spectrometer (Bruker Daltonics, Bremen, Germany). UPLC-MS were measured with a Waters Acquity UPLC system equipped with a PDA detector, an autosampler, and a Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm) coupled to the Bruker Daltonics HCT 6000 mass spectrometer. The elution method was 0.6 mL/min with a linear gradient of buffer A (double distilled water with 0.1% v/v formic acid) and buffer B (CH3CN, Sigma-Aldrich HPLC grade): t = 0−0.5 min (5% B), t = 4 min (100% B), t = 5 min (100% B). RP-UPLC analyses were performed on a VWR Hitachi Chromaster UPLC instrument equipped with a Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm). The runs were performed at a flow rate of 0.6 mL min−1 using a linear gradient of buffer C (distilled water containing 0.1% v/v TFA) and buffer B in two different methods; Method A: t = 0−0.5 min (5% B), t = 1.5 min (100% B), t = 2 min (100% B); Method B: t = 0−0.5 min (5% B), t = 9.5 min (100% B). Analytical RP-HPLC was performed on a VWR Hitachi Chromaster HPLC system (Chromaster HPLC 5430 diode array detector, Chromaster HPLC 5310 column oven, Chromaster HPLC 5210 autosampler and a Chromaster HPLC 5110 pump) with a Macherey Nagel EC 250/3 Nucleosil 100−5 C18 column. The elution method was 0.75 mL/min with a linear gradient of buffer C and buffer B: t = 0−3 min (5% B), t = 20 min (80% B), t = 22 min (100% B). Chiral analytical RP-HPLC analyses were performed to characterize the structural isomers −1 and 2, using the same system and elution method with a Daicel Chiralpak IC column (5 μm, 4.6 × 250 mm). Preparative RP-HPLC purifications were performed on a Varian Prostar HPLC system using an Agilent Zorbax 300 SB-C18 prep column (5 μm, 150 × 21.2 mm). IR absorption spectra were obtained on a SpectrumTwo FTIR Spectrometer (Perkin−Elmer) equipped with a Specac Golden Gate attenuated total reflection (ATR) accessory. Elemental microanalyses were performed on a LecoCHNS-932 elemental analyzer. Syntheses. 3-Acetyl-fluoranthene (1) and 8-Acetyl-fluoranthene (2). These compounds were synthesized according to a literature procedure.56 To a 250 mL Schlenk flask containing anhydrous AlCl3 7968

DOI: 10.1021/acs.inorgchem.7b00677 Inorg. Chem. 2017, 56, 7960−7974

Article

Inorganic Chemistry (d, 1H, J = 8.5 Hz, CH); 13C NMR (CDCl3, 101 MHz) δ: 33.00 (CH2), 118.39 (CH), 120.99 (CH), 121.70 (CH), 122.54 (CH), 126.84 (CH), 127.96 (CH), 128.33 (C), 129.31 (CH), 130.29 (C), 130.61 (CH), 132.30 (CH), 133.08 (C), 137.13 (C), 137.95 (C), 140.68 (C), 142.88 (C), 193.05 (CO). 8-Bromoacetyl fluoranthene (P6). 8-Acetyl-fluoranthene (0.374 g, 1.53 mmol), CuBr2 (0.685 g, 3.06 mmol), EtOAc/CHCl3 (100 mL). The product was obtained as a yellow solid. ES-MS (m/z): 323.1 [M + H]+; HR-MS (m/z): calcd for C18H11BrO [M + H]+ 323.006 60, found: 323.006 75. IR (neat, 4000−800 cm−1): vmax = 3046, 2941, 1687, 1670, 1608. Analytical RP-HPLC (C18, 250 nm): Rt = 21.72 min. 1H NMR (CDCl3, 400 MHz) δ: 4.47 (s, 2H, CH2), 7.55−7.63 (m, 2H, CH), 7.76 (d, 1H, J = 7.9 Hz, CH), 7.80−7.90 (m, 5H, CH), 8.31 (s, 1H, CH); 13C NMR (CDCl3, 101 MHz) δ: 31.47 (CH2), 120.97 (CH), 121.27 (CH), 121.65 (CH), 121.72 (CH), 127.37 (CH), 128.12 (CH), 128.19 (CH), 128.23 (CH), 128.62 (CH), 129.93 (C), 132.78 (C), 133.06 (C), 135.30 (C), 135.53 (C), 139.63 (C), 144.22 (C), 190.91 (CO). Tri-tert-butyl 2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (DO3tBu). This compound was synthesized according to a literature procedure.57 To a stirred mixture of cyclen (51.7 g, 0.300 mol) and NaHCO3 (75.6 g, 0.900 mol) in CH3CN (250 mL) at 0 °C was added tert-butyl-bromoacetate (175 g, 133 mL, 0.900 mol) dropwise over 2 h. Stirring was continued at 0 °C, and the resulting mixture was allowed to warm to room temperature overnight. The mixture was then evaporated to dryness, redissolved in 500 mL of DCM, and extracted with 2 × 250 mL of H2O. The organic layer was dried with anhydrous MgSO4, filtered, and evaporated to dryness. The product was then purified via silica gel chromatography using DCM with a gradient of MeOH (0−10%), and the fractions containing the product were combined and evaporated to obtain the product as a white solid. Yield: 59.5 g, 39%. ES-MS (m/z): 515.5 [M + H]+. 1H NMR (CDCl3, 400 MHz) δ: 1.28 (br m, 27H, CH3), 1.82−2.48 (br m, 2H, 2.48−3.00 (br m, 14H, CH2), 3.00−3.46 (br m, 6H, CH2), 9.69 (br s, NH); 13C NMR (CDCl3, 101 MHz) δ: 27.59 (CH3), 27.89 (CH3), 47.17 (CH2), 48.50 (CH2), 48.80 (CH2), 50.94 (CH2), 55.33 (CH2), 57.72 (CH2), 81.22 (CCH3), 81.76 (CCH3), 169.38 (CO), 170.27 (CO). General Procedure for the Synthesis of 3−8. To a solution of DO3tBu (1 equiv) in CH3CN was added the (bromoacetyl)polyaromatic hydrocarbon (1 equiv), K2CO3 (2 equiv), and KI (0.2 equiv), and the solution was stirred overnight at room temperature. After this time, the mixture was filtered, and the filtrate concentrated in vacuo. The crude material was then purified via silica gel chromatography using DCM with a gradient of MeOH (0−10%), and the fractions containing the product were combined and evaporated to dryness. Tri-tert-butyl 2,2′,2″-(10-(2-(naphthalen-2-yl)-2-oxoethyl)1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (3). DO3tBu (0.104 g, 0.20 mmol), 2-(bromoacetyl)naphthalene (0.050 g, 0.20 mmol), K2CO3 (0.055 g, 0.40 mmol), KI (0.007 g, 0.040 mmol), CH3CN (50 mL). The product was obtained as a yellow oil with analytical data consistent with literature.55 Yield: 0.068 g, 50%. ES-MS (m/z): 515.3 [M-3tBu+H]+, 571.3 [M-2tBu+H]+, 627.5 [M-tBu+H]+, 683.5 [M + H]+, 705.5 [M + Na]+. Analytical RP-HPLC (C18, 250 nm): Rt = 19.06 min. 1H NMR (CDCl3, 400 MHz) δ: 0.92−1.65 (br m, 27H, CH3), 1.90−3.94 (br m, 22H, CH2), 4.16 (br s, 2H, CH2), 7.51−7.63 (m, 2H, CH), 7.83−7.92 (m, 3H, CH), 7.99 (d, 1H, CH), 8.49 (s, 1H, CH); 13C NMR (CDCl3, 101 MHz) δ: 27.88 (CH3), 27.91 (CH3), 48.78 (br, CH2), 52.77 (br, CH2) 55.70 (CH2), 55.74 (CH2), 60.39 (CH2), 82.03 (CCH3), 82.10 (CCH3), 123.19 (CH), 127.05 (CH), 127.79 (CH), 128.61 (CH), 128.83 (CH), 129.67 (CH), 129.76 (CH), 132.48 (C), 133.04 (C), 135.85 (C), 172.87 (CO), 173.00 (CO), 199.75 (CO). Tri-tert-butyl 2,2′,2″-(10-(2-oxo-2-(phenanthren-3-yl)ethyl)1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (4). DO3tBu (0.309 g, 0.60 mmol), 3-(bromoacetyl)phenanthrene (0.179 g, 0.60 mmol), K2CO3 (0.166 g, 1.2 mmol), KI (0.020 g, 0.12 mmol), CH3CN (200 mL). The product was obtained as a yellow oil with analytical data consistent with literature.55 Yield: 0.158 g, 37%. ES-MS (m/z): 565.3 [M-3tBu+H]+, 621.4 [M-2tBu+H]+, 677.5 [M-tBu+H]+, 733.5 [M + H]+, 755.5 [M + Na]+; HR-MS (m/z): calcd for C42H60N4O7 [M + H]+ 733.453 48, found: 733.453 04. IR (neat, 4000−800 cm−1): vmax = 2974,

2901, 1724, 1681. Analytical RP-HPLC (C18, 250 nm): Rt = 20.00 min. 1 H NMR (CDCl3, 400 MHz) δ: 1.38 (s, 18H, CH3), 1.40 (s, 9H, CH3), 1.86−3.51 (m, 22H, CH2), 4.17 (br s, 2H, CH2), 7.59 (t, 1H, J = 7.4 Hz, CH), 7.66 (d, 1H, J = 7.5 Hz, CH), 7.70 (d, 1H, J = 8.9 Hz, CH), 7.81 (d, 1H, J = 8.9 Hz, CH), 7.87 (t, 2H, J = 8.4 Hz, CH), 7.99 (d, 1H, J = 8.4 Hz, CH), 8.68 (d, 1H, J = 8.3 Hz, CH), 9.21 (s, 1H, CH); 13C NMR (CDCl3, 101 MHz) δ: 27.84 (CH3), 27.88 (CH3), 48.52 (br, CH2), 52.58 (br, CH2), 55.60 (CH2), 55.76 (CH2), 60.44 (CH2), 81.93 (CCH3), 81.97 (CCH3), 122.55 (CH), 123.16 (CH), 124.53 (CH), 126.23 (CH), 127.33 (CH), 127.38 (CH), 128.88 (CH), 129.06 (CH), 129.70 (C), 129.95 (CH), 130.48 (C), 132.15 (C), 133.37 (C), 135.28 (C), 172.77 (CO), 172.86 (CO), 199.72 (CO). Tri-tert-butyl 2,2′,2″-(10-(2-(anthracen-2-yl)-2-oxoethyl)-1,4,7,10tetraazacyclododecane-1,4,7-triyl)triacetate (5). DO3tBu (0.252 g, 0.49 mmol), 2-(bromoacetyl)anthracene (0.146 g, 0.49 mmol), K2CO3 (0.135 g, 0.98 mmol), KI (0.016 g, 0.098 mmol), CH3CN (200 mL). The product was obtained as a yellow oil. Yield: 0.135 g, 38%. ES-MS (m/z): 565.3 [M-3tBu+H]+, 621.4 [M-2tBu+H]+, 677.5 [M-tBu+H]+, 733.5 [M + H]+, 755.5 [M + Na]+; HR-MS (m/z): calcd for C42H60N4O7 [M + H]+ 733.45348, found: 733.453 95. IR (neat, 4000−800 cm−1): vmax = 2975, 2932, 2826, 1723, 1677, 1625. Analytical RP-HPLC (C18, 250 nm): Rt = 20.00 min. 1H NMR (CDCl3, 400 MHz) δ: 0.93−1.46 (m, 27H, CH3), 1.87−3.59 (m, 22H, CH2), 4.23 (br s, 2H, CH2), 7.48−7.56 (m, 2H, CH), 7.85 (dd, 1H, J = 8.9/1.3 Hz, CH), 8.01 (d, 2H, J = 8.9 Hz, CH), 8.06 (d, 1H, J = 7.5 Hz, CH), 8.41 (s, 1H, CH), 8.64 (s, 1H, CH), 8.73 (s, 1H, CH); 13C NMR (CDCl3, 101 MHz) δ: 27.89 (CH3), 27.93 (CH3), 48.61 (br, CH2), 52.40 (br, CH2), 55.63 (CH2), 55.67 (CH2), 60.32 (CH2), 81.90 (CCH3), 81.96 (CCH3), 82.03 (CCH3), 121.97 (CH), 124.39 (C), 126.07 (CH), 126.20 (CH), 126.92 (CH), 128.13 (CH), 128.84 (CH), 129.35 (CH), 130.16 (C), 131.14 (CH), 132.13 (C), 132.44 (CH), 132.71 (C), 133.38 (C), 172.78 (CO), 172.94 (CO), 199.50 (CO). Tri-tert-butyl 2,2′,2″-(10-(2-oxo-2-(pyren-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (6). DO3tBu (0.152 g, 0.29 mmol), 1-(bromoacetyl)pyrene (0.095 g, 0.29 mmol), K2CO3 (0.802 g, 0.58 mmol), KI (0.010 g, 0.058 mmol), CH3CN (50 mL). The product was obtained as a yellow solid. Yield: 0.135 g, 62%. ES-MS (m/ z): 701.5 [M-tBu+H]+, 757.5 [M + H]+, 779.5 [M + Na]+; HR-MS (m/ z): calcd for C44H60N4O7 [M + H]+ 757.453 48, found: 757.453 16. IR (neat, 4000−800 cm−1): vmax = 2975, 2933, 2827, 1722, 1678. Analytical RP-HPLC (C18, 250 nm): Rt = 20.47 min. 1H NMR (CDCl3, 400 MHz) δ: 0.85−1.52 (br m, 27H, CH3), 1.95−3.67 (br m, 22H, CH2), 4.18 (s, 2H, CH2), 8.03 (t, 3H, J = 9.2 Hz, CH), 8.16 (t, 2H, J = 8.6 Hz, CH), 8.21 (d, 2H, J = 7.4 Hz, CH), 8.37 (d, 1H, J = 8.1 Hz, CH), 8.91 (d, 1H, J = 9.4 Hz, CH); 13C NMR (CDCl3, 101 MHz) δ: 27.71 (CH3), 27.84 (CH3), 27.92 (CH3), 48.52 (br, CH2), 52.83 (br, CH2), 55.61 (CH2), 55.86 (CH2), 62.75 (CH2), 82.01 (CCH3), 82.09 (CCH3), 124.07 (C), 124.22 (CH), 124.97 (C), 125.05 (CH), 126.10 (CH), 126.28 (CH), 126.57 (CH), 126.61 (CH), 127.15 (CH), 129.46 (CH), 129.66 (C), 129.96 (CH), 130.05 (C), 130.35 (C), 131.01 (C), 134.41 (C), 172.89 (CO), 173.16 (CO), 203.33 (CO). Tri-tert-butyl 2,2′,2″-(10-(2-(fluoranthen-3-yl)-2-oxoethyl)1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (7). DO3tBu (0.227 g, 0.44 mmol), 3-(bromoacetyl)fluoranthene (0.143 g, 0.44 mmol), K2CO3 (0.122 g, 0.88 mmol), KI (0.014 g, 0.088 mmol), CH3CN (200 mL). The product was obtained as a yellow glassy solid. Yield: 0.154 g, 46%. ES-MS (m/z): 379.3 [M+2H]2+, 757.5 [M + H]+; HR-MS (m/z): calcd for C44H60N4O7 [M + H]+ 757.453 48, found: 757.453 06. IR (neat, 4000−800 cm−1): vmax = 2976, 2933, 2849, 1724, 1678. Analytical RP-HPLC (C18, 250 nm): Rt = 20.59 min. 1H NMR (CDCl3, 400 MHz) δ: 1.34−1.74 (br m, 27H, CH3), 1.89−3.81 (br m, 22H, CH2), 4.14 (s, 2H, CH2), 7.33−7.44 (m, 2H, CH), 7.50 (t, 1H, J = 7.7 Hz, CH), 7.81−7.96 (m, 4H, CH), 8.21 (d, 1H, J = 7.3 Hz, CH), 8.53 (d, 1H, J = 8.6 Hz, CH); 13C NMR (CDCl3, 101 MHz) δ: 27.99 (CH3), 28.03 (CH3), 47.64 (CH2), 48.86 (br, CH2), 51.43 (CH2), 52.85 (br, CH2), 55.72 (CH2), 56.07 (br, CH2), 58.29 (CH2), 61.84 (CH2), 81.99 (CCH3), 82.08 (CCH3), 82.17 (CCH3), 118.87 (CH), 120.72 (CH), 121.69 (CH), 122.67 (CH), 127.02 (CH), 127.72 (C), 128.08 (CH), 129.20 (CH), 129.75 (CH), 130.94 (CH), 132.53 (C), 133.03 (C), 7969

DOI: 10.1021/acs.inorgchem.7b00677 Inorg. Chem. 2017, 56, 7960−7974

Article

Inorganic Chemistry

δ: 47.73 (br, CH2), 48.87 (CH2), 51.05 (br, CH2), 51.30 (br, CH2), 53.91 (CH2), 55.55 (CH2), 56.61 (CH2), 122.75, 123.38, 123.74, 124.03, 125.89, 126.19, 126.58, 128.20, 128.34, 129.13, 129.43, 129.92, 133.40, 169.67 (br, CO), 177.93 (CO). 2,2′,2″-(10-(2-(Fluoranthen-3-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (13). Compound 7 (0.154 g, 0.20 mmol), TFA (4 mL). General procedure B. Yield: 0.085 g, 70%. ESMS (m/z): 295.1 [M+2H]2+, 589.3 [M + H]+, 611.3 [M + Na]+; HR-MS (m/z): calcd for C32H36N4O7 [M + H]+ 589.265 68, found: 589.265 69. IR (neat, 4000−800 cm−1): vmax = 3403, 3086, 2970, 1729, 1668, 1613. Analytical RP-HPLC (C18, 250 nm): Rt = 14.19 min. 1H NMR (MeOD, 400 MHz) δ: 2.67−3.71 (br m, 18H, CH2), 3.71−4.31 (br m, 6H, CH2), 7.30−7.52 (m, 2H, CH), 7.71 (t, 1H, J = 7.3 Hz, CH), 7.91 (d, 1H, J = 7.1 Hz, CH), 7.96 (t, 2H, J = 7.0 Hz, CH), 8.02−8.07 (m, 1H, CH), 8.37 (d, 1H, J = 6.9 Hz, CH), 8.77 (s, 1H, CH); 13C NMR (MeOD, 101 MHz) δ: 52.95 (br, CH2), 54.80 (br, CH2), 61.02 (br, CH2), 64.41 (CH2), 73.86 (CH2), 113.74, 116.64, 119.55, 119.75, 121.92, 122.65, 123.64, 128.28, 129.16, 129.28, 130.51, 131.73, 133.81, 138.17, 139.04, 141.75, 169.10 (br, CO), 174.68 (br, CO). 2,2′,2″-(10-(2-(Fluoranthen-8-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (14). Compound 8 (0.099 g, 0.13 mmol), TFA (4 mL). General procedure B. Yield: 0.053 g, 69%. ESMS (m/z): 295.1 [M+2H]2+, 589.3 [M + H]+, 611.3 [M + Na]+; HR-MS (m/z): calcd for C32H36N4O7 [M + H]+ 589.265 68, found: 589.265 49. IR (neat, 4000−800 cm−1): vmax = 3418, 3213, 2857, 1730, 1669, 1611. Analytical RP-HPLC (C18, 250 nm): Rt = 14.48 min. 1H NMR (MeOD, 400 MHz) δ: 2.81−3.73 (br m, 18H, CH2), 3.73−4.23 (br m, 6H, CH2), 7.70 (t, 2H, J = 7.6 Hz, CH), 7.91−7.99 (m, 2H, CH), 8.04 (s, 2H, CH), 8.07−8.13 (m, 2H, CH), 8.55 (s, 1H, CH); 13C NMR (MeOD, 101 MHz) δ: 51.60 (br, CH2), 55.01 (br, CH2), 60.49 (br, CH2), 64.42 (CH2), 73.86 (CH2), 113.81, 116.72, 119.63, 122.12, 122.31, 122.50, 123.06, 128.60, 129.21, 129.43, 131.48, 134.15, 136.50, 136.74, 140.77, 145.78, 170.45 (br, CO), 173.38 (br, CO). General Procedure for Preparation of Complex Tb1−Tb6. The triacetic acid macrocycles (1 equiv) were dissolved in H2O (5 mL), and Tb(OTf)3 (1.2 equiv) was added. The pH was then adjusted to 6, and the solution was allowed to stir for 30 min before readjusting the pH to 6. After this time, the product was purified via preparative RP-HPLC, and the fractions were combined and lyophilized to obtain a fluffy white solid. Tb1. Compound 9 (0.010 g, 0.020 mmol), Tb(OTf)3 (0.015 g, 0.024 mmol). General procedure C. Yield: 0.012 g, 90%. ES-MS (m/z): 671.2 [M + H]+; HR-MS (m/z): calcd for C26H31N4O7Tb [M + H]+ 671.151 89, found: 671.151 83. IR (neat, 4000−800 cm−1): vmax = 3384, 2984, 2872, 1615, 1594. Analytical RP-HPLC (C18, 250 nm): Rt = 11.83 min. Anal. Calcd for C26H31N4O7Tb(CF3CO2H)(H2O)3 (%): C, 40.11; H, 4.57; N, 6.68; found: C, 40.43; H, 4.52; N, 6.56. Tb2. Compound 10 (0.045 g, 0.080 mmol), Tb(OTf)3 (0.058 g, 0.096 mmol). General procedure C. Yield: 0.049 g, 85%. ES-MS (m/z): 721.2 [M + H]+; HR-MS (m/z): calcd for C30H33N4O7Tb [M + H]+ 721.167 54, found: 721.167 37. IR (neat, 4000−800 cm−1): vmax = 3392, 3069, 2983, 2871, 1632, 1607. Analytical RP-HPLC (C18, 250 nm): Rt = 14.11 min. Anal. Calcd for C30H33N4O7Tb(CF3CO2H)1.5(H2O)2.5 (%): C, 42.32; H, 4.25; N, 5.98; found: C, 42.39; H, 4.39; N, 5.74. Tb3. Compound 11 (0.031 g, 0.055 mmol), Tb(OTf)3 (0.040 g, 0.066 mmol). General procedure C. Yield: 0.035 g, 89%. ES-MS (m/z): 721.2 [M + H]+; HR-MS (m/z): calcd for C30H33N4O7Tb [M + H]+ 721.167 54, found: 721.167 49. IR (neat, 4000−800 cm−1): vmax = 3370, 2990, 2915, 2876, 1605. Analytical RP-HPLC (C18, 250 nm): Rt = 14.39 min. Anal. Calcd for C30H33N4O7Tb(CF3CO2H)1.5(H2O)2.5 (%): C, 42.32; H, 4.25; N, 5.98; found: C, 42.68; H, 4.44; N, 5.79. Tb4. Compound 12 (0.031 g, 0.053 mmol), Tb(OTf)3 (0.039 g, 0.064 mmol). General procedure C. Yield: 0.031 g, 80%. ES-MS (m/z): 745.2 [M + H]+; HR-MS (m/z): calcd for C32H33N4O7Tb [M + H]+ 745.167 54, found: 745.167 24. IR (neat, 4000−800 cm−1): vmax = 3391, 2916, 2850, 1613, 1591. Analytical RP-HPLC (C18, 250 nm): Rt = 14.79 min. Anal. Calcd for C32H33N4O7Tb(CF3CO2H)(H2O)3 (%): C, 44.75; H, 4.42; N, 6.14; found: C, 44.39; H, 4.33; N, 6.06. Tb5. Compound 13 (0.053 g, 0.090 mmol), Tb(OTf)3 (0.065 g, 0.108 mmol). General procedure C. Yield: 0.059 g, 88%. ES-MS (m/z):

137.14 (C), 138.12 (C), 140.49 (C), 142.36 (C), 170.66 (CO), 172.99 (CO), 202.22 (CO). Tri-tert-butyl 2,2′,2″-(10-(2-(fluoranthen-8-yl)-2-oxoethyl)1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (8). DO3tBu (0.165 g, 0.32 mmol), 8-(bromoacetyl)fluoranthene (0.104 g, 0.32 mmol), K2CO3 (0.089 g, 0.64 mmol), KI (0.011 g, 0.064 mmol), CH3CN (200 mL). The product was obtained as a yellow glassy solid. Yield: 0.099 g, 41%. ES-MS (m/z): 589.3 [M-3tBu+H]+, 645.4 [M-2tBu +H]+, 701.5 [M-tBu+H]+, 757.5 [M + H]+, 779.5 [M + Na]+; HR-MS (m/z): calcd for C44H60N4O7 [M + H]+ 757.45348, found: 757.453 26. IR (neat, 4000−800 cm−1): vmax = 2975, 2928, 2828, 1726, 1681. Analytical RP-HPLC (C18, 250 nm): Rt = 20.55 min. 1H NMR (CDCl3, 400 MHz) δ: 1.36−1.73 (br m, 27H, CH3), 2.03−3.72 (br m, 22H, CH2), 4.19 (br s, 2H, CH2), 7.71 (t, 2H, J = 7.5 Hz, CH), 7.89−8.01 (m, 4H, CH), 8.04−8.10 (m, 2H, CH), 8.51 (s, 1H, CH); 13C NMR (CDCl3, 101 MHz) δ: 27.99 (CH3), 28.09 (CH3), 49.12 (br, CH2), 52.95 (br, CH2), 53.56 (CH2), 55.82 (CH2), 56.02 (CH2), 60.75 (CH2), 82.03 (CCH3), 82.13 (CCH3), 120.97 (CH), 121.17 (CH), 121.54 (CH), 121.80 (CH), 127.52 (CH), 127.57 (CH), 128.25 (CH), 128.32 (CH), 128.50 (CH), 130.18 (C), 133.23 (C), 134.94 (C), 135.73 (C), 135.97 (C), 139.75 (C), 144.17 (C), 172.93 (CO), 173.20 (CO), 199.55 (CO). General Procedure for the Synthesis of 9−14. The tert-butyl ester protected compound (1 equiv) was treated with 4 mL of TFA and stirred at room temperature for 4 h. After this time, the TFA was evaporated using a gentle stream of N2. The crude residue was redissolved in MeOH and purified using preparative RP-HPLC. Fractions containing the product were combined and lyophilized to obtain the product as a fluffy white solid. 2,2′,2″-(10-(2-(Naphthalen-2-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9). Compound 3 (0.128 g, 0.19 mmol), TFA (4 mL). General procedure B. Yield: 0.031 g, 32%. Analytical data were consistent with those of literature.55 ES-MS (m/z): 515.3 [M + H]+, 537.3 [M + Na]+. Analytical RP-HPLC (C18, 250 nm): Rt = 12.00 min. 1H NMR (D2O, 400 MHz) δ: 2.83−4.39 (br m, 22H, CH2), 5.16 (br s, 2H, CH2), 7.66 (t, 1H, J = 7.1 Hz, CH), 7.73 (t, 1H, J = 7.3 Hz, CH), 7.89−8.05 (m, 3H, CH), 8.07 (d, 1H, J = 8.0 Hz, CH), 8.54 (s, 1H, CH). 2,2′,2″-(10-(2-Oxo-2-(phenanthren-3-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (10). Compound 5 (0.091 g, 0.12 mmol), TFA (4 mL). General procedure B. Yield: 0.052 g, 78%. Analytical data were consistent with those of literature.55 ES-MS (m/z): 565.3 [M + H]+; HR-MS (m/z): calcd for C30H36N4O7 [M + H]+ 565.265 68, found: 565.266 06. IR (neat, 4000−800 cm−1): vmax = 3438, 3085, 2855, 1727, 1681, 1614. Analytical RP-HPLC (C18, 250 nm): Rt = 13.57 min. 1H NMR (D2O, 400 MHz) δ: 1.97−4.27 (br m, 24H, CH2), 6.90−7.72 (br m, 7H, CH), 8.34 (s, 1H, CH), 8.57 (s, 1H, CH); 13C NMR (D2O, 101 MHz) δ: 48.13 (br, CH2), 51.27 (br, CH2), 54.75 (CH2), 122.56, 124.46, 125.91, 127.56, 128.67, 129.65, 131.44, 134.95, 169.67 (br, CO), 173.35 (br, CO). 2,2′,2″-(10-(2-(Anthracen-2-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (11). Compound 4 (0.061 g, 0.08 mmol), TFA (4 mL). General procedure B. Yield: 0.039 g, 88%. ESMS (m/z): 565.3 [M + H]+, 587.3 [M + Na]+; HR-MS (m/z): calcd for C30H36N4O7 [M + H]+ 565.265 68, found: 565.265 93. IR (neat, 4000− 800 cm−1): vmax = 3421, 3090, 2967, 1730, 1671. Analytical RP-HPLC (C18, 250 nm): Rt = 13.77 min. 1H NMR (D2O, 400 MHz) δ: 2.27− 4.33 (br m, 24H, CH2), 6.95−7.78 (m, 7H, CH), 8.01 (s, 1H, CH), 8.13 (s, 1H, CH); 13C NMR (D2O, 101 MHz) δ: 48.62 (br, CH2), 51.52 (br, CH2), 53.58 (br, CH2), 58.09 (br, CH2), 121.22, 125.83, 126.04, 126.55, 126.97, 127.75, 128.26, 128.64, 129.06, 129.26, 131.31, 131.87, 132.68, 134.59, 169.29 (br, CO), 173.58 (br, CO). 2,2′,2″-(10-(2-Oxo-2-(pyren-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (12). Compound 6 (0.135 g, 0.18 mmol), TFA (4 mL). General procedure B. Yield: 0.096 g, 91%. ES-MS (m/z): 589.3 [M + H]+, 611.3 [M + Na]+; HR-MS (m/z): calcd for C32H36N4O7 [M + H]+ 589.265 68, found: 589.265 92. IR (neat, 4000− 800 cm−1): vmax = 3377, 2916, 2849, 1726, 1669, 1593. Analytical RPHPLC (C18, 250 nm): Rt = 14.19 min. 1H NMR (MeOD, 400 MHz) δ: 2.66−4.37 (br m, 24H, CH2), 8.00−8.34 (m, 8H, CH), 8.54 (s, 1H, CH), 9.13 (br s, 1H, OH); 13C NMR (D2O, 50 mM NaOH, 101 MHz) 7970

DOI: 10.1021/acs.inorgchem.7b00677 Inorg. Chem. 2017, 56, 7960−7974

Article

Inorganic Chemistry 373.1 [M+2H]2+, 745.2 [M + H]+; HR-MS (m/z): calcd for C32H33N4O7Tb [M + H]+ 745.16754, found: 745.16776. IR (neat, 4000−800 cm−1): vmax = 3396, 3100, 2983, 2915, 2848, 1725, 1668, 1610. Analytical RP-HPLC (C18, 250 nm): Rt = 14.98 min. Anal. Calcd for C32H33N4O7Tb(CF3CO2H)(H2O)3 (%): C, 44.75; H, 4.42; N, 6.25; found: C, 44.81; H, 4.59; N, 5.92. Tb6. Compound 14 (0.045 g, 0.076 mmol), Tb(OTf)3 (0.055 g, 0.091 mmol). General procedure C. Yield: 0.051 g, 89%. ES-MS (m/z): 373.1 [M+2H]2+, 745.3 [M + H]+; HR-MS (m/z): calcd for C32H33N4O7Tb [M + H]+ 745.16754, found: 745.16744. IR (neat, 4000−800 cm−1): vmax = 3342, 2987, 2865, 1681, 1596, 1557. Analytical RP-HPLC (C18, 250 nm): Rt = 15.18 min. Anal. Calcd for C32H33N4O7Tb(CF3CO2H)(H2O)3 (%): C, 44.75; H, 4.42; N, 6.25; found: C, 44.48; H, 4.59; N, 6.12. Spectroscopic Studies. UV/vis absorbance measurements were performed on an Agilent Cary 8454 UV/vis spectrophotometer. Steadystate emission spectra were acquired on a Fluorolog-3 DF spectrofluorimeter (Horiba) with the excitation light source being a 450 W continuous xenon lamp. Emission after excitation at 350 nm was monitored at 90° using an R2658P detector module operated at −1500 V. Spectral selection was achieved by double grating DFX/DFM monochromators (excitation: 1200 grooves/mm, 330 nm blaze; emission: 1200 grooves/mm, 500 nm blaze). Low-temperature emission spectra were recorded on frozen glasses (MeOH/EtOH 1:1, v/v) using a dewar cuvette filled with liquid N2 (T = 77 K). Irradiation experiments were conducted using a Rayonet RPR-200 photochemical reactor equipped with six RPR-3500A or RPR-4190A lamps (14 W each) for 350 or 420 nm irradiation, respectively. Samples were irradiated in a fluorescence quartz cuvette (width 10 mm). Electron Paramagnetic Resonance Assay. EPR measurements were performed at room temperature on a Bruker Model A300 EPR spectrometer equipped with a Bruker ER 4122 SHQ resonator (X-band frequencies ≈ 9.88 GHz). A 450 W xenon lamp with optical filters was used to generate the desired wavelength for irradiation. The spectra were recorded with Tb1−Tb6 (25 μM) and the spin trap 2,2,6,6tetramethylpiperidine (TEMP, 20 mM) in oxygen-saturated ACN. The parameters for the measurement are 20 dB microwave attenuation (nonsaturating 2 mW microwave power); modulation amplitude = 0.2 mT peak-to-peak; time constant = 1.28 ms; receiver gain = 50−200; sweep time = 30 s; and conversion time = 5.12 ms. Distribution Coefficients (log Doct/PBS). The distribution coefficients were measured using the previously reported “shake-flask” procedure.17 Each complex was added into 1 mL of n-octanol to give a final concentration of 50 μM. To this solution was added 1 mL of phosphate buffer (10 mM, pH 7.01), and the mixture was shaken 100 times and allowed to equilibrate for 4.5 h. Aliquots from the n-octanol phase and the aqueous phase were extracted and analyzed using RPUPLC to determine their relative concentrations in each phase. The measurements were repeated three times for each complex. Water Solubility. Standard solutions of the complexes in MeOH are prepared at 0.2, 0.4, 0.6, 0.8, and 1.0 mM and analyzed using RP-UPLC. The area of the absorbance under curve is integrated and plotted against the concentration to obtain a calibration curve. The water solubility of the complexes was measured by preparing a saturated (i.e., cloudy) solution of the complex in 200 μL of water, with sonication for 30 min. The solution is then allowed to equilibrate overnight before filtration through a 0.2 μm membrane filter (Whatman Puradisc 30). The filtrate is diluted by a known factor and analyzed using RP-UPLC. The area of the absorbance under curve is integrated, and the concentration of the complex is calculated from the calibration curve taking into consideration the dilution factor. Stability in Human Plasma. The stability of the compounds in human plasma at 37 °C was evaluated using a modified literature procedure.17 The human plasma was provided by the Blutspendezentrum, Zürich, Switzerland. Caffeine was used instead of diazepam, as it has already been established to be a suitable internal reference for human plasma.66 Stock solutions of the complexes (20 mM) and caffeine (0.15 M) were prepared in dimethyl sulfoxide (DMSO). For each experiment, a final volume of 1 mL was achieved by addition of the stock solutions and DMSO to 975 μL of human plasma to final concentrations of 0.5

mM of the complex and 1 mM of caffeine. The resulting solution was incubated at 37 °C for the respective time intervals with continuous shaking (ca. 600 rpm). The experiment was stopped after the incubation time by addition of 2 mL of methanol, and the mixture was centrifuged for 45 min at 650 g and 4 °C. The methanolic solution was extracted, filtered through a 0.2 μm membrane filter (Whatman Puradisc 30), and analyzed using RP-UPLC and UPLC-MS. Stability in PBS. Aliquots from 20 mM stock solutions of the compounds were diluted into PBS to obtain a final concentration of 0.4 mM. The resulting solution was incubated at 37 °C with continuous shaking (ca. 600 rpm). The solution was then analyzed using RP-UPLC at different time intervals. Stability from Titration with Na2HPO4. Aqueous solutions were prepared to obtain a final concentration of 0.4 mM complex and 10, 20, 30, 50, or 100 mM Na2HPO4. Analysis of the solutions was performed using RP-UPLC. Singlet Oxygen Measurements. Quantum yields for singlet oxygen formation (ϕ) were determined in both acetonitrile and PBS using a comparative method with phenalenone as reference (ϕref = 95%). For measurements in acetonitrile, a 1 mL solution containing the complex (at a concentration with OD = 0.1 at the irradiation wavelength), p-nitrosodimethyl aniline (RNO; 24 μM), and imidazole (12 mM) was prepared in a luminescence quartz cuvette. The solution was then irradiated in a Rayonet RPR-200 photochemical reactor (Southern New England Ultraviolet Co.) for different time intervals. For measurements in PBS, a 1 mL solution containing the complex (OD = 0.1 at the irradiation wavelength), RNO (20 μM), and histidine (10 mM) was used instead. After each irradiation interval, the absorbances were recorded at 420 nm for acetonitrile and 440 nm for PBS, and these data were used to create plots of the variations in absorbance (A0 − A, where A0 is the absorbance at time zero) over irradiation time. The slope of the linear regression (Ssample) for the plot was calculated and compared against the slope of the linear regression of the reference compound (Sref) with inclusion of the absorbance correction factors (Iref/Isample) to determine the quantum yield of singlet oxygen formation for the sample (ϕsample). The following equations were used for the calculations:

ϕsample = ϕref ×

Ssample Sref

×

Iref Isample

where:

I = I0 × (1 − 1 × 10−Aλ) I0 = light intensity of the irradiation source in the irradiation interval, and Aλ = absorbance of sample at λ. Cell Culture. Human cervical carcinoma cell line (HeLa) was cultured in DMEM medium (Gibco) supplemented with 5% fetal calf serum (FCS, Gibco), penicillin (100 U mL−1), and streptomycin (100 μg mL−1) in a humidified atmosphere at 37 °C and 5% CO2. Human fibroblast cell line (MRC-5) was cultured in F-10 medium (Gibco) supplemented with 10% FCS (Gibco), penicillin (100 U mL−1), and streptomycin (100 μg mL−1) in a humidified atmosphere at 37 °C and 5% CO2. Cytotoxicity Studies. The cytotoxicities of the terbium complexes, cisplatin, and ALA were measured using a fluorometric cell viability assay containing the fluorescent probe resazurin (Promocell GmbH). The cells were seeded in triplicate at a density of ∼4 × 103 (HeLa) or 7.5 × 103 (MRC-5) cells per well in 100 μL of medium in a 96-well plate and incubated for 24 h at 37 °C with 5% CO2. After incubation, the cells were treated with serially diluted concentrations of the compounds. For the phototoxicity experiments, the cells were treated for only 4 h, and after this time, the medium was replaced with fresh medium before irradiation with 350 nm (10 min, 2.58 J cm−2) or 420 nm (20 min, 9.27 J cm−2) light. Light doses were measured using a Gigahertz Optic X1−1 optometer. After irradiation, the cells were incubated for a further 44 h, before the medium was replaced with 100 μL of fresh medium containing resazurin (0.2 mg mL−1). The cells were incubated for a further 4 h before quantification of the fluorescence (λexc = 540 nm, λem = 590 nm) using a SpectraMax M5 microplate reader. The 4 h dark 7971

DOI: 10.1021/acs.inorgchem.7b00677 Inorg. Chem. 2017, 56, 7960−7974

Inorganic Chemistry

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control experiments were incubated with the compounds for 4 h before replacement of the medium with fresh medium and incubating for a further 44 h. The 48 h dark control experiments were incubated with the compounds for the full 48 h. After this time the medium was replaced with 100 μL of fresh medium containing resazurin (0.2 mg mL−1), incubated for a further 4 h, and the fluorescence was quantified as before. Cell Morphology Observation. HeLa cells, at a concentration of 4 × 103 cells per well, were seeded in triplicate in a 96-well plate and incubated for 24 h for attachment. The cells were then exposed to Tb1− Tb6 (20 μM) for 4 h. For dark treatment, the cells were washed with sterile PBS twice and incubated in fresh DMEM medium for 24 h. For the phototoxicity experiments, the cells were washed with sterile PBS twice, and fresh DMEM medium added. The cells were then irradiated with 420 nm light (20 min, 9.27 J cm−2) and incubated for a further 24 h. After this time, bright-field images were obtained directly using an inverted fluorescence microscope (Carl Zeiss, Göttingen, Germany) with a 20× objective.19 Fluorescence Microscopy to Determine Cell Viability. The cell viability assay was performed using the LIVE/DEAD Viability/ Cytotoxicity Kit for mammalian cells (Life Technologies). HeLa cells at a density of ∼4 × 103 cells per well were seeded in triplicate in a 96well plate and incubated for 24 h for attachment. The cells were then treated with 20 μM of Tb1−Tb6 for 4 h. For dark treatment, the cells were washed with sterile PBS twice and incubated in fresh DMEM medium for 24 h. For the phototoxicity experiments, the cells were washed with sterile PBS twice, and fresh DMEM medium was added, before irradiation with 420 nm light (20 min, 9.27 J cm−2). After irradiation, the cells were incubated for 24 h, treated with calcein-AM (2 μM) and EthD-1 (4 μM) for 30 min, and imaged using an inverted fluorescence microscope (Carl Zeiss, Göttingen, Germany) with a 10× objective. A GFP channel (λex = 488 nm, λem = 550 ± 20 nm) was used to measure calcein-AM fluorescence, while a rhodamine channel (λex = 488 nm, λem = 630 ± 20 nm) was used for EthD-1.71

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b00677. NMR data for novel compounds, IR absorption spectra, steady-state emission spectra at 77 K, and additional RPUPLC traces and UPLC-MS traces for Tb1−Tb6 stability measurements (PDF)

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (P.U.) *E-mail: [email protected]. Website: www. gassergroup.com. (G.G.) ORCID

Hui Chao: 0000-0003-4153-5303 Ben Boyd: 0000-0001-5434-590X Gilles Gasser: 0000-0002-4244-5097 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financed by the Swiss National Science Foundation (Professorships PP00P2_133568 and PP00P2_157545 for G.G.), the Univ. of Zurich (G.G.), and the Victorian Government (Victorian Postdoctoral Research Fellowship for P.U.). This work has received support under the program Investissements d’Avenir launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). 7972

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