Delivery of Floxuridine Derivatives to Cancer Cells by Water-Soluble

Article pubs.acs.org/bc

Delivery of Floxuridine Derivatives to Cancer Cells by Water-Soluble Organometallic Cages Jeong Wu Yi,† Nicolas P. E. Barry,‡ Mona A. Furrer,‡ Olivier Zava,§ Paul J. Dyson,§ Bruno Therrien,*,‡ and Byeang Hyean Kim*,† †

Department of Chemistry, Division of IBB, Pohang University of Science and Technology, Pohang 790-784, Korea Institut de Chimie, Université de Neuchâtel, 51 Ave de Bellevaux, 2000 Neuchâtel, Switzerland § Institut des Sciences et Ingénierie Chimique, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland ‡

ABSTRACT: The self-assembly of 2,4,6-tris(pyridin-4-yl)-1,3,5-triazine (tpt) triangular panels with p-cymene (pPriC6H4Me) ruthenium building blocks and 2,5dioxydo-1,4-benzoquinonato (dobq) or 5,8-dioxydo-1,4-naphthoquinonato (donq) bridges, in the presence of a pyrenyl-nucleoside derivatives (pyreneR), affords the triangular prismatic host−guest compounds [(pyrene-R)⊂Ru6(pPriC6H4Me)6(tpt) 2 (dobq) 3 ] 6+ ([(pyrene-R)⊂1] 6+ ) and [(pyrene-R)⊂Ru 6 (pPr i C 6 H 4 Me) 6 (tpt)2(donq)3]6+ ([(pyrene-R)⊂2]6+), respectively. The inclusion of six monosubstituted pyrenyl-nucleosides (pyrene-R1 = 5′-(1-pyrenyl butanoate)-2′-deoxyuridine, pyrene-R2 = 5-fluoro-5′-(1-pyrenyl butanoate)-2′-deoxyuridine, pyrene-R3 = 5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-glycyl}-2′-deoxyuridine, pyrene-R4 = 5-fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-glycyl}2′-deoxyuridine, pyrene-R5 = 5-fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2′-deoxyvuridine, pyrene-R6 = 5-fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2′-deoxyuridine) has been accomplished. The carceplex nature of [(pyrene-R)⊂1]6+ with the pyrenyl moiety firmly encapsulated in the hydrophobic cavity of the cage with the nucleoside groups pointing outward was confirmed by NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS), while the host−guest nature of [(pyrene-R)⊂2]6+ was studied in solution by NMR techniques. In contrast to the floxuridine compounds used in the clinic, the host−guest complexes are highly water-soluble. Consequently, the cytotoxicities of these water-soluble compounds have been established using human ovarian A2780 and A2780cisR cancer cells. All the host−guest systems are more cytotoxic than the empty cages alone [1][CF3SO3]6 (IC50 = 23 μM) and [2][CF3SO3]6 (IC50 = 10 μM), the most active compound [pyrene-R4⊂1][CF3SO3]6 being 2 orders of magnitude more cytotoxic (IC50 = 0.3 μM) on these human ovarian cancer cell lines (A2780 and A2780cisR).

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Chart 1

INTRODUCTION Synthetic nucleoside derivatives are commonly employed as antiviral and anticancer drugs. They include floxuridine, 5-fluorouracil, fludarabine, cladribine, zidovudine, as well as others (Chart 1).1 5-Fluorouracil is a widely used antimetabolite for the treatment of human solid tumors including colorectal and breast cancers.2 Similarly, 5-fluoro-2′-deoxyuridine (FdUrd, Floxuridine) is a 5-fluorouracil derivative known for its high antitumor activity against metastases of cancer.3,4 Floxuridine has been utilized to treat human solid tumors; however, its therapeutic effect is limited by the efficiency of cellular uptake and bioavailability of the drug. In order to overcome these limitations, various synthetic approaches have been envisaged to enhance the cell permeability and cytotoxicity of floxuridine derivatives as potent anticancer drugs.5−7 Pyrenyl-functionalized nucleic acids are popular conjugate molecules in biological chemistry.8 Indeed, the pyrenyl moiety fits perfectly between base pairs of DNA,9 possesses interesting fluorescence properties,10 and functionalization of pyrene is synthetically easy.11 Therefore, several derivatives of pyrenylfunctionalized nucleic acids have been prepared in recent years.12−17 However, despite such popularity and interest, low © 2012 American Chemical Society

Received: September 13, 2011 Revised: December 19, 2011 Published: January 22, 2012 461

dx.doi.org/10.1021/bc200472n | Bioconjugate Chem. 2012, 23, 461−471

Bioconjugate Chemistry

Article

Figure 1. Metalla-prisms [{Pt(acac)2}⊂Ru6(pPriC6H4Me)6(tpt)2(dobq)3]6+ ([Pt(acac)2⊂1]6+)15 and [guest⊂Ru6(pPriC6H4Me)6(tpt)2(donq)3]6+ ([guest⊂2]6+).36

of the nucleoside on the biological activity together with the ability of the metalla-prisms to deliver these hydrophobic molecules are discussed.

cell internalization remains one of the limitations of pyrenylfunctionalized nucleic acids for biological applications.18 Ruthenium-based anticancer agents have attracted much interest in recent years,19,20 and organometallic Ru(arene) compounds are the latest trend.21−23 By modification of the ligand sphere and linking more than one metal center, the properties of the complexes can be altered to obtain desired biological characteristics.24−26 Recently, new hybrid drug delivery systems built from arene ruthenium complexes have been developed.27−30 These water-soluble organometallic metalla-cages are able to encapsulate guest molecules. For example, the hexacationic prism [Ru6(pPriC6H4Me)6(tpt)2(dobq)3]6+ ([1]6+) (tpt =2,4,6-tris(pyridin-4-yl)-1,3,5-triazine; dobq = 2,5-dioxydo-1,4-benzoquinonato) was synthesized,31 which was capable of encapsulating planar Pt and Pd acetylacetonate complexes (Figure 1) as well as planar aromatic compounds of various sizes.32,33 Similarly, larger metalla-cages were synthesized such as [Ru6(pPriC6H4Me)6(tpt)2(donq)3]6+ ([2]6+) (tpt = 2,4,6-tris(pyridin-4-yl)-1,3,5-triazine; donq = 5,8-dioxydo-1,4-naphthoquinonato) in which the guest molecule was free to escape from the cavity of the host in solution, thus providing host−guest systems (Figure 1).34,35 In these complexes the encapsulated guest is stable, with the physical properties of the host being retained following encapsulation. The biological activity of these systems has been evaluated, and encouraging results were obtained. The metalla-prisms exhibit some activity which increases with the encapsulation of a guest, suggesting transport and leaching of the guest once inside the cell. Indeed, fluorescence experiments have been used to monitor the uptake of the fluorescentlabeled pyrenyl derivative 1-(4,6-dichloro-1,3,5-triazin-2-yl)pyrene (pyrene-X) to cancer cells demonstrating an uptake of the carceplex [pyrene-X⊂Ru6(pPriC6H4Me)6(tpt)2(dobq)3]6+ being 1 order of magnitude greater than that of pyrene-X alone.36 With a view to combine the drug delivery ability of the arene ruthenium metalla-prisms with the biological properties of pyrenylfunctionalized nucleic acids, a series of pyrenyl-nucleosides (pyrene-R) including derivatives analogous to floxuridine have been synthesized and encapsulated in the metalla-prisms [1]6+ and [2]6+. The in vitro anticancer activity of these water-soluble systems, [pyrene-R⊂1]6+ and [pyrene-R⊂2]6+, was evaluated against human ovarian A2780 and A2780cisR (acquired resistance to cisplatin) cancer cells. The implications

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RESULTS AND DISCUSSION The synthesis of the pyrenyl-nucleoside derivatives starts with conjugation of the butyryl pyrene derivatives, N-[1-oxo-4(1-pyrenyl)butyl]-glycine ethyl ester and N-[1-oxo-4(1-pyrenyl)butyl]-phenylalanine ethyl ester, with O-protected nucleosides. The butyryl pyrene derivatives conjugated with glycine and phenylalanine are obtained using typical peptidic coupling from 1-pyrene butyric acid. 3′-O-TBDMS-2′-deoxyuridine and 5-fluoro-3′-O-TBDMS-2′-deoxyuridine are prepared according to a published procedure37 from the corresponding unprotected nucleoside in two steps (Scheme 1). The pyrenyl modified nucleosides are synthesized through an esterification reaction between the butyl pyrenyl amino acids and deprotected silyl group via in situ reaction using tetrabutylammonium fluoride in THF (Scheme 2). These couplings give six pyrenyl-nucleoside derivatives which are characterized by 1 H and 13C{1H} NMR spectroscopy and mass spectrometry. The encapsulation of the pyrenyl-nucleosides (pyrene-R1−6) in the metalla-prism [1]6+ is performed in a two-step process, in which the p-cymene ruthenium dobq (2,5-dioxydo-1, 4-benzoquinonato) dinuclear complex is first reacted with silver triflate to produce a reactive intermediate, which is then combined with a 2:1 mixture of tpt (2,4,6-tris(pyridin-4-yl)1,3,5-triazine) and the functionalized pyrenyl derivative (Scheme 3), affording the corresponding carceplex systems [(pyrene-R1−6)⊂1][CF3SO3]6. These metalla-prisms are isolated in 69−73% yield and are characterized by 1H and 13C{1H} NMR spectroscopy and electrospray ionization mass spectrometry. The encapsulation of the pyrenyl-nucleosides (pyrene-R1−6) in the larger metalla-prism [2]6+ is achieved following the same strategy as for [(pyrene-R1−6)⊂1][CF3SO3]6 using the p-cymene ruthenium donq (5,8-dioxydo-1,4-naphthoquinonato) dinuclear complex (Scheme 4). The host−guest systems are isolated as their triflate salts [(pyrene-R1−6)⊂1][CF3SO3]6 in good yield. Under the electrospray ionization mass spectrometry (ESI-MS) conditions employed (see Experimental Section), all inclusion complexes are remarkably stable. The ESI mass spectra of all 462



Article

Scheme 1. Synthesis of Butyryl Pyrene Conjugated Amino Acids

Scheme 2. Synthesis of Modified Pyrenyl-Nucleoside Derivatives (Pyrene-R1-6)

Scheme 3. Synthesis of the Carceplex Systems [(Pyrene-R1-6)⊂1]6+

systems [pyrene-R⊂2][CF3SO3]6 show peaks corresponding to [pyrene-R + 2 + (CF3SO3)3]3+ at m/z 1182.8 (R1), 1188.1 (R2), 1201.5 (R3), 1207.5 R4), 1231.5 (R5), and 1236.5 (R6), respectively. These peaks are assigned unambiguously on the basis of

carceplex systems [pyrene-R⊂1][CF3SO3]6 show peaks corresponding to [pyrene-R + 1 + (CF3SO3)3]3+ at m/z 1132.1 (R1), 1137.8 (R2), 1151.5 (R3), 1157.5 (R4), 1181.5 (R5), and 1186.8 (R6), respectively. Similarly, the ESI mass spectra of the host−guest 463



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Scheme 4. Synthesis of the Host-Guest Systems [(Pyrene-R1-6)⊂2]6+

Figure 2. ESI mass of the peak envelopes corresponding to [pyrene-R + 1 + (CF3SO3)3]3+ and [pyrene-R + 2 + (CF3SO3)3]3+.

their characteristic Ru6 isotope pattern (Figure 2). In addition to these parent peaks [pyrene-R + 1 + (CF3SO3)3]3+ and [pyreneR + 2 + (CF3SO3)3]3+, a series of characteristic fragmentation peaks corresponding to the cages are observed. A typical spectrum is given in Figure 3, identifying the fragmentation peaks observed for [pyrene-R5⊂1][CF3SO3]6. This fragmentation pattern has been previously observed for related pyrene-R⊂cage systems.32,33 These systems are not only stable under ESI mass conditions, they are stable in solution at room and elevated temperature (45 °C) in D2O, as well as in biological media (aqueous solution containing RPMI 1640 medium with 10% fetal calf serum (FCS) and antibiotics), showing no leaching of the guest molecules or breaking up of the cages. Consequently, the antiproliferative activity of the empty metalla-cages [1]6+, [2]6+ and all the inclusion systems [pyrene-R1−6⊂1]6+ and [pyreneR1−6⊂2]6+ was evaluated against the human ovarian A2780 and A2780cisR (acquired resistance to cisplatin) cancer cell lines using the MTT assay, which measures mitochondrial

dehydrogenase activity as an indication of cell viability. The IC50 values (IC50 is the drug concentration necessary for 50% inhibition of cell viability) are listed in Table 1 and are reported together with that of cisplatin for comparison purposes. It was not possible to obtain IC50 values for the pyrene-R compounds, i.e., in the absence of the metalla-cage, due to solubility problems in biological media. All pyrene-R⊂cage complexes show higher cytotoxicity than their corresponding empty metalla-cages [1]6+ and [2]6+ on both A2780 and A2780cisR cancer cells. Moreover, they show activity comparable or superior to cisplatin with essentially the same level of activity on both nonresistant and cisplatin acquired resistance cell lines. In addition, as control experiments, the antiproliferative activity of the two butyryl pyrene derivatives N-[1-oxo-4-(1-pyrenyl)butyl]-glycine ethyl ester and N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanine ethyl ester was evaluated, and both show IC50 values >200 μM when tested alone, while the corresponding pyrenyl⊂cage systems possess IC50 values 464



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Figure 3. ESI mass spectrum of the [pyrene-R5⊂1][CF3SO3]6 system (clip = {Ru2(pPriC6H4Me)2(dobq)}2+).

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Table 1. IC50 Values for [1]6+, [2]6+, [(Pyrene-R1-6)⊂1]6+, and [(Pyrene-R1-6)⊂2]6+ Determined on Human Ovarian A2780 and A2780cisR Cancer Cell Lines

EXPERIMENTAL SECTION General. [Ru 2 (pPr i C 6 H 4 Me) 2 (dobq)Cl 2 ], 3 1 [Ru 2 (pPriC6H4Me)2(donq)Cl2],34 and 2,4,6-tris(4-pyridyl)-1,3,5triazine38,39 (tpt) were prepared according to published methods and all other reagents were commercially available (Sigma-Aldrich, S&T Pharma, TCI Europe) and used as received. The 1H, 13C{1H} NMR spectra were recorded either on a Bruker AvanceII 400 MHz (University of Neuchatel) or a FT-300 MHz Bruker Apect 3000 (POSTECH, Korea) spectrometers by using the residual protonated solvent as internal standard. Infrared spectra were recorded as KBr pellets on a Bruker FT-IR PS55+ spectrometer. Mass spectra (FAB) were obtained using a Jeol JMS700 high-resolution mass spectrometer at the Korea Basic Science Center, Daegu, Korea. The melting range of the solids was determined with the melting point Gallenkamp apparatus. Electrospray ionization mass spectra were obtained in positive-ion mode on a Bruker FTMS 4.7T BioAPEX II mass spectrometer. Synthesis of Butyryl Pyrene Conjugated Amino Acids. 1-Pyrene butyric acid (1.0 mmol), EDC·HCl (1.5 mmol), HOBT (1.5 mmol), and DMAP (1.5 mmol) in anhydrous DMF 10 mL were stirred at room temperature for 1 h. Amino acid ethyl ester (1.2 equiv) was added to the reaction mixture and stirred for an additional 2 h. The reaction mixture was concentrated in vacuo. The residue was diluted with dichloromethane (100 mL) and extracted with saturated NaHCO3 aqueous solution, water, and brine sequentially. The organic layer was dried over anhydrous MgSO4 and filtered. The filtrate was evaporated in vacuo, and the residue was recrystallized with EtOH to give pale-yellow solid. Synthesis of N-[1-oxo-4-(1-pyrenyl)butyl]-glycine ethyl ester was prepared according to the general procedure as mentioned above from glycine ethyl ester hydrochloride (1.14 g, 8.17 mmol). Yield 2.24 g (73%); M.p.: 146−147 °C; 1H NMR (300 MHz, CDCl3): δ 8.25 (d, J = 9.0 Hz, 1H), 8.14− 8.06 (m, 4H), 7.99−7.93 (m, 3H), 7.81 (d, J = 9.0 Hz, 1H), 5.94 (s, 1H, NH) 4.19 (q, J = 9.1 Hz, 2H, OCH2CH3), 4.00 (d, J = 6.0 Hz, 2H), 3.35 (t, J = 7.5 Hz, 2H, PyCH2CH2CH2−), 2.33 (t, J = 7.5 Hz, 2H, PyCH2CH2CH2−) 2.19 (m, 2H, PyCH2CH2CH2−), 1.25 (t, J = 9.1 Hz, 3H, OCH2CH3); 13 C{1H} NMR (75 MHz, CDCl3): δ 172.1, 159.1, 156.8, 156.6, 144.0, 143.9, 141.5, 138.6, 132.7, 132.5, 128.0, 127.3, 125.3, 172.8, 170.1, 135.8, 131.4, 130.9, 130.0, 128.8, 127.5, 127.4,

IC50/μM compound cisplatin [1]6+ [2]6+ [pyrene-R1⊂1]6+ [pyrene-R1⊂2]6+ [pyrene-R2⊂1]6+ [pyrene-R2⊂2]6+ [pyrene-R3⊂1]6+ [pyrene-R3⊂2]6+ [pyrene-R4⊂1]6+ [pyrene-R4⊂2]6+ [pyrene-R5⊂1]6+ [pyrene-R5⊂2]6+ [pyrene-R6⊂1]6+ [pyrene-R6⊂2]6+

A2780 1.6 23 6.0 2.1 1.3 0.4 0.4 5.6 1.3 0.3 0.2 5.9 1.7 1.2 1.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 2.1 1.2 0.14 0.05 0.03 0.10 0.9 0.3 0.03 0.02 0.3 0.06 0.03 0.18

A2780cisR

resistance factor (RF)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5.4 1.1 0.9 1.0 0.9 3.3 1.0 1.1 1.1 1.0 2.0 1.0 0.8 0.8 1.2

8.6 25 5.2 2.2 1.2 1.3 0.4 6.1 1.4 0.3 0.4 5.7 1.4 1.0 1.7

1.2 2.5 0.9 0.35 0.04 0.13 0.11 1.5 0.3 0.03 0.05 1.0 0.06 0.04 0.21

ranging from 2−5 μM. These observations are in agreement with results previously reported with other pyrenyl derivatives encapsulated in water-soluble arene ruthenium metallacages.29−33,35,36 On closer inspection of the IC50 data, it is apparent that the fluorinated compounds (pyrene-R2, pyreneR4, pyrene-R6) are more cytotoxic than their nonfluorinated counterparts (pyrene-R1, pyrene-R3, pyrene-R5). The high level of cytotoxicity of the pyrene-R4 derivatives of floxuridine, especially that involving the less cytotoxic metalla-cage, viz. [1]6+, are good candidates to improve the therapeutic efficiency of this clinically used compound.

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CONCLUSION

In this study, water-soluble arene ruthenium metalla-cages were used to deliver pyrenyl-nucleosides to cancer cells. The different floxuridine-metalla-cage combinations exhibit excellent antiproliferative effects on both A2780 ovarian cancer cells and their cisplatin resistant strains. Hence these molecules could be a good therapeutic alternative to floxuridine which suffers from poor cellular uptake and bioavailability. 465



Article

(m, 1H, C5′H), 2.54 (br s, 1H, exch D2O, OH), 2.31−2.27 (m, 2H, C2′H), 0.89 (s, 9H, C(CH3)3), 0.09 (s, 6H, Si(CH3)2); 13 C{1H} NMR (75 MHz, CDCl3): δ 163.2, 150.2, 141.1, 102.5, 87.6, 86.7, 71.4, 61.8, 40.9, 25.7, 18.0, −4.7, −4.9; IR (neat): 3447, 2963, 2930, 2857, 1699, 1559, 1472, 1274, 1082 cm−1; HRMS-FAB (m/z): calcd for C15H27N2O5Si+ [M + H]+, 343.1689; found, 343.1687. 5-Fluoro-3′-O-(tert-butyldimethylsilyl)-2′-deoxyuridine was prepared according to the general procedure as mentioned above from 5-fluoro-2′-deoxyuridine (1.11 g, 4.51 mmol). Yield 1.24 g (76%); M.p.: 140.6−142 °C; 1H NMR (300 MHz, CDCl3): δ 8.84 (br s, 1H, exch D2O, NH), 7.97 (d, 1H, J = 6.4 Hz, C6H), 6.23 (t, 1H, C1′H), 4.51 (m, 1H, C3′H), 4.00− 3.95 (m, 2H, C4′H and C5′H), 3.83−3.78 (m, 1H, C5′H), 2.35− 2.17 (2 m, 2H, C2′H), 0.90 (s, 9H, C(CH3)3), 0.09 (s, 6H, Si(CH3)2); 13C{1H} NMR (75 MHz, CDCl3): δ 156.9, 148.7, 142.0, 138.9, 125.2, 124.8, 87.6, 86.3, 71.4, 61.8, 41.2, 25.7, 18.0, 4.7, −4.9; IR (neat): 3446, 3193, 2955, 2929, 2857, 1716, 1699, 1559, 1404, 1260, 1097 cm−1; HRMS-FAB (m/z): calcd for C15H26FN2O5Si+ [M + H]+, 361.1595; found, 361.1597. Synthesis of Modified Nucleosides Conjugated Pyrenyl Derivatives. Butyryl pyrene conjugated amino acids (1.0 mmol) and lithium hydroxide monohydrate (3.0 mmol) was stirred in THF/H2O (4/1) at room temperature for 2−3 h. The resulting reaction mixture was neutralized with Dowex 50WX8−200 ionexchange resin, filtered, and concentrated in vacuo. The residue was coevaporated with pyridine three times and dried in vacuo. Without further purification, the residue was dissolved with 20 mL of anhydrous DMF. To the solution were added EDC·HCl (1.5 mmol), HOBT (1.5 mmol), and DMAP (1.5 mmol) and stirred at room temperature for 1 h. 3′-O-TBDMS nucleosides (1.0 mmol) was added to the reaction mixture. The resulting reaction mixture was stirred at ambient temperature for 6 h. The reaction mixture was concentrated in vacuo. The residue was extracted between dichloromethane (150 mL) and 1 N HCl aqueous solution (10 mL). The organic layer was washed with water three times, dried over anhydrous MgSO4, and filtered. The filtrate was evaporated in vacuo. The residue was dissolved with anhydrous THF and cooled at 0−5 °C. 1 M TBAF in THF (1.5 mL) was added slowly to the reaction mixture. The resulting reaction mixture was warmed up at room temperature and stirred for 6 h. Afterward, ethyl acetate was added to the mixture and the solution was washed with saturated NaHCO3 aqueous solution, water, and brine sequentially. The organic layer was dried over anhydrous MgSO4 and filtered. The filtrate was evaporated in vacuo, and the residue was purified by column chromatography to give a pale-yellow solid. 5′-(1-Pyrenyl butanoate)-2′-deoxyuridine (pyrene-R1) was prepared according to the general procedure as mentioned above from 3′-O-(tert-butyldimethylsilyl)-2′-deoxyuridine (212 mg, 0.62 mmol). Yield: 280 mg (91%); M.p.: 223−224 °C; 1 H NMR (300 MHz, acetone-d6): δ 10.2 (s, 1H), 8.41(d, J = 9.6 Hz, 1H), 8.24−8.18 (m, 6H), 8.01 (m, 1H), 7.92 (m, 1H), 7.73 (d, J = 8.1 Hz, 1H, C6H), 6.34 (t, J = 6.6 Hz, 1H, C1′H), 5.67 (d, J = 8.1 Hz, 1H, C5H), 4.48 (m, 1H, C4′H), 4.42−4.18 (m, 2H, C5′H), 4.17−4.15 (m, 1H, C3′H), 3.40 (t, J = 7.5 Hz, 2H, PyCH2CH2CH2), 2.56 (t, J = 7.2 Hz, 2H, PyCH2CH2CH2), 2.41−2.32 (m, 2H, PyCH2CH2CH2), 2.30− 2.08 (m, 2H, C2′H); 13C{1H} NMR (75 MHz, acetone-d6): δ 172.3, 162.5, 150.0, 139.6, 135.8, 131.1, 130.6, 129.6, 128.3, 127.1, 126.9, 126.2, 125.6, 124.6, 124.5, 124.4, 123.1, 101.6, 70.8, 63.5, 39.4, 32.9, 31.9, 26.5; IR (neat): 3421, 3044, 2947, 1698, 1653, 1460, 1383, 1272, 1248, 1089 cm−1; HRMS-FAB

126.7, 125.8, 125.1 125.0, 124.9, 124.8, 124.8, 123.4, 61.5, 41.4, 35.6, 32.7, 27.2, 14.1; IR (neat): 3310, 3037, 2937, 2867, 1739, 1651, 1537, 1198 cm−1; HRMS-FAB (m/z): calcd for C24H24NO3+ [M + H]+, 374.1756; found, 374.1751. Synthesis of N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanine ethyl ester was prepared according to the general procedure as mentioned above from phenylalanine ethyl ester hydrochloride (1.27 g, 5.53 mmol). Yield 2.08 g (81%); M.p.: 157− 158 °C; 1H NMR (300 MHz, CDCl3): δ 8.24 (d, J = 9.0 Hz, 1H), 8.13−8.03 (m, 4H), 7.98−7.94 (m, 3H), 7.78 (d, J = 9.0 Hz, 1H), 7.22−7.17 (m, 3H), 7.10 (m, 2H), 5.95 (d, J = 6.1 Hz, 1H), 4.90 (q, J = 6.1 Hz, 1H), 4.12 (q, J = 6.0 Hz, 2H), 3.29 (t, J = 9.1 Hz, 2H), 3.17−3.03 (m, 2H), 2.28−2.23 (m, 2H), 2.19−2.11 (m, 2H), 1.20 (t, J = 6.0 Hz, 3H); 13C{1H} NMR (75 MHz, CDCl3): δ 172.2, 171.8, 136.0, 135.8, 131.4, 130.9, 130.0, 129.3, 128.8, 128.6, 127.5, 127.4, 127.3, 127.1, 126.7, 125.9, 125.1, 125.0, 124.9, 124.8, 123.42, 61.6, 53.1, 38.0, 35.8, 32.7, 27.2, 14.2; IR (neat): 3308, 3289, 3098, 2947, 2865, 1709, 1660, 1557, 1451, 1198 cm−1; HRMS-FAB (m/z): calcd for C31H30NO3+ [M + H]+, 464.2226; found, 464.2224. Synthesis of 3′-O-Protected Nucleoside Derivatives. 3′-O-(tert-Butyldimethylsilyl)-2′-deoxynucleosides were prepared according to the published procedures.37 A solution of 2′-deoxynucleoside (1.0 mmol), 4,4′-dimethoxytrityl chloride (DMTrCl) (1.15 mmol), and anhydrous pyridine (5.0 mmol) in anhydrous DMF (10 mL) was stirred at room temperature for 5 h. To the reaction mixture was then added methanol (1 mL) for quenching, and the mixture was stirred for 30 min. The reaction mixture was partitioned between dichloromethane (250 mL) and saturated NaHCO3 aqueous solution (250 mL). The organic layer was washed with water two times and brine, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated and coevaporated with pyridine two times. The residue was dissolved in 10 mL of anhydrous DMF. To the solution was added imidazole (5.0 mmol) and the solution was stirred at room temperature for 30 min. After adding tertbutyldimethylsilyl chloride (1.5 mmol) the resulting reaction mixture was stirred overnight at room temperature. Then 100 mL of saturated NaHCO3 aqueous solution was added, and the mixture was extracted with dichloromethane two times. The combined organic layer was washed with water three times then brine, dried with Na2SO4, and filtered. The filtrate was concentrated in vacuo and coevaporated with toluene three times. The residue was dried over 6 h in vacuo and used to the next step without further purification. The concentrated residue was dissolved in 10 mL of anhydrous dichloromethane and stirred at 0−5 °C for 30 min. Then 10 mL of 5% trichloroacetic acid in dichloromethane was slowly added to the reaction mixture and the resulting reaction mixture was stirred at room temperature for 3 h. To the reaction mixture was then added 1 mL of methanol and 50 mL of saturated NaHCO3 aqueous solution was added to the mixture. The organic layer was collected, dried over anhydrous Na2SO4, and filtered. The filtrate was evaporated in vacuo, and the residue was purified by column chromatography to give a white solid. 3′-O-(tert-Butyldimethylsilyl)-2′-deoxyuridine was prepared according to the general procedure as mentioned above from 2′-deoxyuridine (1.04 g, 4.56 mmol). Yield 1.23 g (79%); M.p.: 206−207 °C; 1H NMR (300 MHz, CDCl3): δ 9.10 (br s, 1H, exch D2O, NH), 7.67 (d, 1H, J = 8.1 Hz, C6H), 6.18 (t, 1H, J = 6.6 Hz, C1′H), 5.75 (d, 1H, J = 8.1 Hz, C5H), 4.51− 4.47 (m, 1H, C3′H), 3.95−3.91 (m, 2H, C4′H and C5′H), 3.78 466



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(m/z): calcd for C29H27N2O6+ [M + H]+, 499.1869; found, 499.1866. 5-Fluoro-5′-(1-pyrenyl butanoate)-2′-deoxyuridine (pyrene-R2) was prepared according to the general procedure as mentioned above from 5-fluoro-3′-O-(tert-butyldimethylsilyl)-2′-deoxyuridine (209 mg, 0.58 mmol). Yield: 267 mg (89%); M.p.: 216− 218 °C; 1H NMR (300 MHz, CDCl3): δ 9.54 (s, 1H), 8.18 (d, 1H, J = 9.0 Hz), 8.08−7.95 (m, 7H), 7.75 (d, 1H), 7.51 (d, 1H, J = 5.7 Hz), 6.05 (t, 1H, C1′H), 4.30−4.26 (m, 1H, C4′H), 4.13− 4.02 (m, 3H, C5′H and C3′H), 3.31 (m, 2H, PyCH2CH2CH2), 2.43 (t, 2H, PyCH2CH2CH2), 2.28−2.16 (m, 2H, PyCH2CH2CH2), 1.88−1.83 (m, 2H, C2′H); 13C{1H} NMR (75 MHz, CDCl3): δ 173.1, 156.9, 148.6, 142.0, 138.9, 135.1, 131.3, 130.1, 130.0, 128.7, 127.5, 127.3, 126.8, 125.9, 125.0, 124.9, 124.8, 123.9, 123.4, 123.1, 85.5, 84.3, 80.7, 63.3, 40.3, 33.5, 32.4, 26.4; IR (neat): 3447, 3198, 3043, 2949, 1734, 1717, 1473, 1418, 1264, 1086 cm−1; HRMS-FAB (m/z): calcd for C29H26FN2O6+ [M + H]+, 517.1775; found, 517.1778. 5′-{N-[1-Oxo-4-(1-pyrenyl)butyl]-glycyl}-2′-deoxyuridine (pyrene-R3) was prepared according to the general procedure as mentioned above from 3′-O-(tert-butyldimethylsilyl)-2′deoxyuridine (224 mg, 0.65 mmol). Yield: 316 mg (87%); M.p. M.p.: 231−233 °C; 1H NMR (300 MHz, acetone-d6): δ 10.1 (s, 1H), 8.45(d, 1H, J = 9.0 Hz), 8.24−8.15 (m, 5H), 8.08− 7.93 (m, 3H), 7.75 (d, 1H, J = 8.1 Hz, C6H), 6.32 (t, 1H, J = 6.90 Hz, C1′H), 5.76 (d, 1H, J = 8.1 Hz, C5H), 4.72 (s, 1H), 4.50−4.48 (m, 1H, C4′H), 4.39−4.37 (m, 3H, C3′H and C5′H), 4.08 (d, 2H, J = 5.7 Hz, CαH), 3.42 (t, 2H, J = 7.5 Hz, PyCH2CH2CH2), 2.45 (t, 2H, 7.2 Hz, PyCH2CH2CH2), 2.34− 2.28 (m, 2H, PyCH2CH2CH2), 2.20−2.08 (m, 2H, C2′H); 13 C{1H} NMR (75 MHz, acetone-d6): δ 173.1, 169.9, 162.9, 150.4, 140.2, 136.7, 131.4, 131.0, 129.9, 128.7, 127.6, 127.3, 126.5, 125.9, 124.9, 124.8, 124.7, 123.7, 102.2, 84.9, 84.5, 71.1, 64.2, 41.1, 39.7, 34.9, 32.5, 27.6; IR (neat): 3333, 3046, 2940, 1753, 1696, 1540, 1460, 1271, 1191, 1088 cm−1; HRMS-FAB (m/z): calcd for C31H30N3O7+ [M + H]+, 556.2084; found, 556.2086. 5-Fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-glycyl}-2′-deoxyuridine (pyrene-R4) was prepared according to the general procedure as mentioned above from 5-fluoro-3′-O-(tertbutyldimethylsilyl)-2′-deoxyuridine (204 mg, 0.57 mmol). Yield: 276 mg (85%); M.p.: 227−229 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.39 (s, 1H), 8.36 (d, 1H, J = 9.3 Hz), 8.24−8.14 (m, 4H), 8.08 (s, 2H), 7.99 (t, 1H), 7.89 (t, 2H), 6.13 (t, 1H, J = 6.3 Hz, C1′H), 5.60 (br, 1H, NH), 4.30−4.25 (m, 3H, C4′H and C5′H), 3.96−3.89 (m, 3H, C3′H and CαH), 3.31 (t, 2H, J = 7.5 Hz, PyCH2CH2CH2), 2.32 (t, 2H, J = 7.2 Hz, PyCH2CH2CH2), 2.22−2.14 (m, 2H, PyCH2CH2CH2), 2.04−1.99 (m, 2H, C2′H); 13C{1H} NMR (75 MHz, acetone-d6): δ 173.6, 170.4, 157.8, 157.4, 149.5, 142.1, 139.0, 136.9, 131.3, 130.8, 129.7, 128.6, 128.0, 127.9, 127.7, 126.9, 126.6, 125.4, 125.3, 125.2, 125.0, 124.7, 124.6, 123.9, 85.1, 84.3, 79.4, 70.4, 64., 41.2, 3.1, 32.5, 27.9; IR (neat): 3429, 3027, 2941, 1696, 1664, 1559, 1194, 1027 cm−1; HRMS-FAB (m/z): calcd for C31H29FN3O7+ [M + H]+, 574.1990; found, 574.1986. 5-Fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2′deoxyuridine (pyrene-R5) was prepared according to the general procedure as mentioned above from 3′-O-(tert-butyldimethylsilyl)-2′-deoxyuridine (207 mg, 0.60 mmol). Yield: 316 mg (81%); M.p.: 238−239 °C; 1H NMR (300 MHz, acetone-d6): δ 10.2 (s, 1H), 8.42 (d, 1H), 8.26−8.00 (m, 7H), 7.90 (t, 1H), 7.71 (d, 1H, J = 8.1 Hz, C6H), 7.28−7.17 (m, 4H), 6.32 (t, 1H, J = 6.4 Hz, C1′H), 5.69 (d, 1H, J = 8.1 Hz, C5H), 4.78−4.72

(m, 1H, C4′H), 4.48−4.43 (m, 1H, CαH), 4.24−4.05 (m, 3H, C3′H and C5′H), 3.35−3.18 (m, 2H, PyCH2CH2CH2), 3.11−3.09 (m, 2H, phenyl), 2.40−2.29 (m, 4H, PyCH2CH2CH2), 2.14− 2.12 (m, 2H, C2′H); 13C{1H} NMR (75 MHz, acetone-d6): δ 172.7, 171.9, 162.8, 150.4, 140.3, 137.2, 136.7, 131.0, 129.9, 129.2, 128.7, 128.4, 127.6, 127.5, 127.3, 126.8, 126.5, 126.0, 124.9, 124.8, 123.7, 102.6, 84.9, 84.4, 71.5, 64.7, 54.5, 40.0, 37.3, 34.9, 32.4, 27.5; IR (neat): 3327, 3043, 2946, 1704, 1698, 1559, 1457, 1248, 1184, 1086 cm−1; HRMS-FAB (m/z): calcd for C38H36N3O7+ [M + H]+, 646.2553; found, 646.2550. 5-Fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2′deoxyuridine (pyrene-R6) was prepared according to the general procedure as mentioned above from 5-fluoro-3′-O-(tert-butyldimethylsilyl)-2′-deoxyuridine (224 mg, 0.62 mmol). Yield: 342 mg (83%); M.p.: 234−236 °C; 1H NMR (300 MHz, acetone-d6): δ 10.5 (s, 1H), 8.39 (d, 1H, J = 8.1 Hz), 8.24−8.13 (m, 6H), 8.04−7.99 (m, 3H), 7.67 (d, 1H, J = 7.5 Hz), 7.28−7.17 (m, 4H), 6.32 (t, 1H, J = 5.7 Hz, C1′H), 4.84−4.79 (m, 1H, C4′H), 4.55−4.51 (m, 1H, CαH), 4.37−4.13 (m, 3H, C3′H and C5′H), 3.33−3.22 (m, 2H, PyCH2CH2CH2), 3.20−3.16 (m, 2H, phenyl), 2.38−2.11 (m, 4H, PyCH2CH2CH2), 2.09−2.06 (m, 2H, C2′H); 13 C{1H} NMR (75 MHz, acetone-d6): δ 172.8, 171.7, 156.9, 149.0, 142.2, 139.1, 137.1, 136.6, 131.4, 130.0, 129.1, 128.7, 128.4, 127.5, 127.2, 126.7, 126.5, 126.0, 124.9, 124.8, 124.6, 124.5, 124.1, 123.7, 85.3, 84.7, 71.0, 64.5, 54.2, 39.6, 37.2, 34.9, 32.4, 27.5; IR (neat): 3402, 2964, 2929, 1717, 1700, 1653, 1540, 1473, 1361, 1264, 1079 cm−1; HRMS-FAB (m/z): calcd for C38H35FN3O7+ [M + H]+, 664.2459; found, 664.2463. Encapsulation of Modified Nucleosides Conjugated Pyrenyl Derivatives (Pyrene-R1−6) in Metalla-Cage [1]6+. A mixture of [Ru2(pPriC6H4Me)2(dobq)Cl2] (0.15 mmol), AgCF3SO3 (0.30 mmol), tpt (0.10 mmol), and pyrenyl derivatives (pyrene-R) (0.05 mmol) in MeOH (30 mL) was stirred at 60 °C for 15 h. The reaction mixture was kept in the dark. The resulting reaction mixture was filtered and concentrated in vacuo. The residue was dissolved in CH2Cl2 (2 mL), and diethyl ether (50 mL) was slowly added to precipitate the product, which was kept in the refrigerator for 2 h. The product was filtered and dried in vacuo. [Pyrene-R1⊂1][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R1 (27.1 mg, 0.05 mmol). Yield 136 mg (71%). 1H NMR (300 MHz, acetone-d6): δ 9.88 (s, 1H, N3H), 8.58 (s, 12H, Hα), 8.04 (br, 12H, Hβ), 7.87 (d, 1H, J = 8.1 Hz, C6H), 7.05 (d, 1H, J = 9.0 Hz, HPy), 6.89 (d, 1H, J = 15.6 Hz, HPy), 6.84 (d, 1H, J = 15.6 Hz, HPy), 6.57 (d, 1H, J = 9.3 Hz, HPy), 6.42 (t, 1H, J = 6.3, C1′H), 6.38 (d, 1H, J = 3.9 Hz, HPy), 6.24 (d, 12H, J = 6.0 Hz, Har), 6.19 (br, 8H, Hq and HPy), 6.02 (d, 12H, J = 6.0 Hz, Har), 5.99 (m, 1H, HPy) 5.85 (d, 1H, J = 7.5 Hz, HPy), 5.60 (d, 1H, J = 8.1 Hz, C5H), 4.68−4.58 (m, 3H, C3′H and C5′H), 4.44−4.43 (m, 1H, C4′H), 2.99 (sept, 6H, J = 6.9 Hz, CH), 2.57−2.55 (m, 6H, PyCH2CH2CH2 and C2′H), 2.23 (s, 18H, CH3), 1.72−1.69 (m, 2H, PyCH2CH2CH2), 1.42 (d, J = 6.90 Hz, 36H, CH3); 13C{1H} NMR (75 MHz, acetone-d6): δ 184.2, 172.8, 167.6, 162.7, 154.0, 150.5 143.6, 140.8, 135.5, 129.7, 129.1, 128.2, 127.5, 127.2, 126.9, 125.8, 124.9, 124.6, 124.4, 123.6, 122.8, 122.7, 122.2, 119.3, 104.1, 101.9, 99.2, 84.6, 83.8, 82.3, 71.1, 64.3, 38.7, 32.9, 31.8, 31.2, 26.4, 21.6, 17.2; IR (neat): 3446, 2964, 2927, 1717, 1521, 1376, 1258, 1030, 811 cm−1; ESI-MS (m/z): 1132.14 [pyrene-R1 + 1 + (CF3SO3)3]3+. [Pyrene-R2⊂1][CF3SO3]6. This complex was synthesized according to the procedure described above using compound 467



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pyrene-R2 (27.1 mg, 0.05 mmol). Yield 145 mg (72%). 1H NMR (300 MHz, acetone-d6): δ 10.4 (s, 1H, N3H), 8.57 (s, 12H, Hα), 8.04 (br, 12H, Hβ), 8.02 (d, 1H, J = 6.9 Hz), 7.08 (d, 1H, J = 8.7 Hz, HPy), 6.93 (d, 1H, J = 7.5 Hz, HPy), 6.87(d, 1H, J = 8.4 Hz, HPy), 6.58 (d, 1H, J = 9.3 Hz, HPy), 6.40−6.34 (m, 2H, HPy and C1′H), 6.21 (m, 19H, Har, Hq, and HPy), 6.01 (m, 13H, Har and HPy), 5.85 (d, 1H, J = 7.2 Hz, HPy), 4.76−4.58 (m, 3H, C3′H and C5′H), 4.45−4.43 (m, 1H, C4′H), 2.98 (sept, 1H, J = 6.9 Hz, CH), 2.57−2.52 (m, 6H, PyCH2CH2CH2 and C2′H), 2.22 (s, 18H, CH3), 1.73−1.68 (m, 2H, PyCH2CH2CH2), 1.42 (d, 36H, J = 6.9 Hz, CH3); 13C{1H} NMR (75 MHz, acetone-d6): δ 184.2, 172.8, 167.6, 153.8, 150.5 148.9, 143.6, 140.8, 135.4, 129.7, 129.1, 128.2, 127.9, 127.2, 127.0, 126.4, 125.8, 124.6, 124.2, 123.6, 122.9, 122.2, 119.3, 104.1, 101.9, 99.2, 85.2, 83.8, 82.3, 70.9, 66.7, 64.3, 38.7, 33.9, 31.2, 26.5, 21.6, 17.2; IR (neat): 3447, 2965, 2927, 1717, 1521, 1376, 1258, 1058, 1030, 811 cm−1; ESI-MS (m/z): 1137.78 [pyrene-R2 + 1 + (CF3SO3)3]3+. [Pyrene-R3⊂1][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R3 (27.6 mg, 0.05 mmol). Yield 134 mg (69%). 1H NMR (300 MHz, acetone-d6): δ 10.02 (s, 1H, N3H), 8.57 (s, 12H, Hα), 7.96 (br, 12H, Hβ), 7.81 (d, 1H, J = 8.1 Hz, C6H), 7.04 (d, 1H, J = 9.0 Hz, HPy), 6.91 (d, 1H, J = 7.8 Hz, HPy), 6.79 (d, 1H, J = 8.7 Hz, HPy), 6.56 (d, 1H, J = 9.0 Hz, HPy), 6.42 (t, 1H, J = 7.2 Hz, C1′H), 6.22 (m, 20H, Har, Hq, and HPy), 6.00 (d, 12H, J = 6.3 Hz, Har), 5.80 (m, 2H, HPy), 5.60 (d, 1H, J = 8.1 Hz, C5H), 4.60−4.52 (m, 3H, C3′H and C5′H), 4.26 (m, 3H, C4′H and CαH), 2.98 (sept, 6H, J = 6.9 Hz, CH), 2.56−2.55 (m, 2H, PyCH 2 CH 2 CH 2 ), 2.42 (m, 4H, PyCH2CH2CH2 and C2′H), 2.22 (s, 18H, CH3), 1.64 (m, 2H, PyCH2CH2CH2), 1.40 (d, 36H, J = 6.9 Hz, CH3); 13C{1H} NMR (75 MHz, acetone-d6): δ 184.2, 173.0, 170.1, 162.9, 153.8, 150.4, 140.3, 136.7, 131.4, 131.0, 129.9, 128.7, 127.6, 127.3, 126.5, 125.9, 125.5, 124.9, 124.8, 124.7, 124.2, 123.7, 104.1, 102.0, 102.2, 99.3, 84.9, 84.5, 83.9, 82.2, 71.1, 64.2, 41.1, 39.7, 34.9, 32.5, 31.2, 27.6, 21.6, 17.2; IR (neat): 3447, 2965, 2927, 1684, 1540, 1375, 1259, 1058, 811 cm−1; ESI-MS (m/z): 1151.47 [pyrene-R3 + 1 + (CF3SO3)3]3+. [Pyrene-R4⊂1][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R4 (28.7 mg, 0.05 mmol). Yield 143 mg (73%). 1H NMR (300 MHz, acetone-d6): δ 10.46 (s, 1H, N3H), 8.57 (s, 12H, Hα), 8.21−7.93 (br, d, 13H, Hβ and C6H), 7.04 (d, 1H, J = 9.0 Hz, HPy), 6.91 (d, 1H, J = 7.5 Hz, HPy), 6.78 (d, 1H, J = 9.0 Hz, HPy), 6.56 (d, 1H, J = 9.0 Hz, HPy), 6.39 (t, 1H, J = 7.8 Hz, C1′H), 6.21 (d, m, 18H, J = 6.0 Hz, Har and Hq), 6.12−5.81 (m, 3H, HPy), 6.00 (m, Har and HPy), 5.81 (d, 1H, J = 7.8 Hz, HPy) 4.65−4.48 (m, 3H, C3′H and C5′H), 4.28 (br, 3H, C4′H and CαH), 2.98 (sept, 6H, J = 6.9 Hz, CH), 2.58 (m, 2H, PyCH2CH2CH2), 2.43 (m, 4H, PyCH2CH2CH2 and C2′H), 2.22 (s, 18H, CH3), 1.62 (m, 2H, PyCH2CH2CH2), 1.40 (d, J = 6.90 Hz, 36H, CH3); 13C{1H} NMR (75 MHz, acetone-d6): δ 184.2, 173.5, 170.3, 167.6, 157.0, 153.8, 148.9, 143.3, 135.8, 129.7, 129.1, 128.1, 127.5, 127.2, 126.7, 126.2, 125.8, 125.4, 124.8, 124.5, 124.2, 123.5, 122.9, 122.7, 122.4, 119.3, 115.0, 104.1, 102.0, 99.2, 85.3, 84.8, 83.9, 82.2, 70.9, 64.5, 61.7, 41.1, 39.4, 35.1, 31.7, 31.2, 26.5, 21.6, 17.2; IR (neat): 3446, 2966, 2929, 1734, 1521, 1375, 1259, 1058, 811 cm−1; ESI-MS (m/z): 1157.47 [pyrene-R4 + 1 + (CF3SO3)3]3+. [Pyrene-R5⊂1][CF3SO3]6. This complex was synthesized according to the procedure described above using compound

pyrene-R5 (33.0 mg, 0.05 mmol). Yield 142 mg (70%). 1H NMR (300 MHz, acetone-d6): δ 10.15 (s, 1H, N3H), 8.57 (s, 12H, Hα), 8.30−7.91 (br, d, 13H, Hβ and C6H), 7.53 (m, 5H, Hph), 7.00 (d, 1H, J = 9.0 Hz, Hpy), 6.87 (t, 1H, J = 8.1 Hz, Hpy), 6.78 (m, 1H, Hpy), 6.55 (m, 1H, Hpy), 6.37−6.22 (m, 8H, C1′H, HPy and Hq), 6.20 (d, 12H, J = 6.3 Hz, Har), 6.08− 6.07 (m, 2H, HPy), 6.00 (m, 13H, Har and HPy), 5.94 (d, 1H, J = 8.1 Hz, Hpy), 5.62 (d, 1H, J = 8.1 Hz, C5H), 4.89−4.77 (m, 1H, C4′H), 4.58−4.51 (m, 1H, CαH), 4.47−4.12 (m, 3H, C3′H and C5′H), 3.42−3.33 (m, 2H, PyCH2CH2CH2), 3.00 (sept, 6H, J = 6.9 Hz, CH), 2.55−2.53 (m, 2H, PyCH2CH2CH2), 2.41−2.38 (m, 2H, C2′H), 2.22 (s, 18H, CH3), 1.62−1.60 (m, 2H, PyCH2CH2CH2), 1.41 (d, 36H, J = 6.9 Hz, CH3); 13C{1H} NMR (75 MHz, acetone-d6): δ 184.2, 173.3, 172.1, 167.6, 163.1, 162.8, 153.8, 150.4, 143.3, 140.7, 140.5, 137.6, 135.7, 129.7, 129.1, 128.8, 128.1, 127.8, 127.1, 126.9, 126.2, 125.8, 125.3, 124.7, 124.2, 123.5, 122.8, 122.3, 119.3, 115.0, 104.1, 102.0, 99.2, 84.8, 83.8, 82.3, 71.6, 65.1, 55.6, 39.8, 37.5, 35.0, 31.7, 31.2, 26.3, 21.6, 17.2; IR (neat): 3447, 2965, 2927, 1717, 1521, 1376, 1259, 1058, 1031, 811 cm−1; ESI-MS (m/z): 1181.47 [pyrene-R5 + 1 + (CF3SO3)3]3+. [Pyrene-R6⊂1][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R6 (33.1 mg, 0.05 mmol). Yield 142 mg (71%). 1H NMR (300 MHz, acetone-d6): δ 10.46 (s, 1H, N3H), 8.58 (s, 12H, Hα), 8.21−7.92 (br, d, 13H, Hβ and C6H), 7.55−7.38 (m, 5H, HPh), 7.02 (m, 1H, HPy), 6.95 (t, 1H, J = 6.3 Hz, HPy), 6.80 (d, 1H, J = 8.4 Hz, HPy), 6.77 (m, 1H, HPy), 6.54−6.22 (m, 8H, C1′H, HPy and Hq), 6.22 (d, 12H, J = 6.3 Hz, Har), 6.08− 6.02 (m, 3H, HPy), 6.01 (d, 12H, J = 6.0 Hz, Har), 5.99 (m, 1H, HPy), 4.88−4.68 (m, 1H, C4′H), 4.54−4.42 (m, 1H, CαH), 4.28−4.24 (m, 3H, C3′H and C5′H), 3.38−3.36 (m, 2H, PyCH2CH2CH2), 3.00 (sept, 6H, J = 6.9 Hz, CH), 2.55−2.53 (m, 2H, PyCH2CH2CH2), 2.41−2.38 (m, 2H, C2′H), 2.24 (s, 18H, CH3), 1.62−1.60 (m, 2H, PyCH2CH2CH2), 1.41 (d, 36H, J = 6.9 Hz, CH3); 13C{1H} NMR (75 MHz, acetone-d6): δ 184.2, 173.2, 172.1, 167.7, 153.9, 148.9, 143.3, 137.4, 129.7, 129.4, 129.1, 128.6, 128.1, 127.8, 127.1, 126.9, 126.7, 126.2, 125.8, 125.2, 124.7, 124.5, 124.2, 123.5, 122.8, 122.7, 122.3, 119.3, 115.0, 104.1, 102.0, 85.3, 84.6, 83.8, 82.3, 71.2, 64.8, 55.4 39.5, 37.2, 35.0, 31.6, 31.2, 26.3, 21.6, 17.2; IR (neat): 3447, 2966, 2926, 1716, 1521, 1377, 1258, 1057, 1030, 811 cm−1; ESI-MS (m/z): 1186.82 [pyrene-R6 + 1 + (CF3SO3)3]3+. Encapsulation of Modified Nucleosides Conjugated Pyrenyl Derivatives (Pyrene-R1−6) in Metalla-Cage [2]6+. A mixture of [Ru2(pPriC6H4Me)2(donq)Cl2] (0.17 mmol), AgCF3SO3 (0.34 mmol), tpt (0.11 mmol), and pyrenyl derivatives (pyrene-R) (0.055 mmol) in MeOH (30 mL) was stirred at 60 °C for 15 h. The reaction mixture was kept in the dark. The resulting reaction mixture was filtrated and concentrated in vacuo. The residue was dissolved in CH2Cl2 (2 mL), and diethyl ether (50 mL) was slowly added to precipitate the product, which was kept in the refrigerator for 2 h. The product was filtered and dried in vacuo. [Pyrene-R1⊂2][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R1 (28 mg, 0.055 mmol). Yield: 164 mg (75%); 1H NMR (300 MHz, acetone-d6): δ 9.89 (s, 1H, N3H), 8.64 (s, 12H, Hα), 7.80 (m, 13H, Hβ and C6H), 7.59 (s, 12H, Hq), 6.96 (m, 4H, HPy), 6.44 (m, 4H, HPy), 6.37 (m, 1H, C1′H), 6.01 (d, 12H, J = 5.7 Hz, Hcym), 5.78 (d, 12H, J = 5.7 Hz, Hcym), 5.50 (d, 1H, C5H), 5.12 (m, 1H, HPy), 4.65−4.40 (m, 4H, C3′H and C5′H), 3.40 (t, 2H, PyCH2CH2CH2), 2.94 (sept, 6H, J = 6.9, Hz, 468



Article

[Pyrene-R5⊂2][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R5 (31.0 mg, 0.048 mmol). Yield: 145 mg (73%); 1H NMR (300 MHz, acetone-d6): δ 10.04 (s, 1H, N3H), 8.62 (s, 13H, Hα and C6H) 8.25−7.73 (m, 14H, Hβ and HPh), 7.57 (s, 12H, Hq), 7.31 (m, 3H, HPh), 6.92 (br, 4H, HPy), 6.51 (m, 2H, HPy), 6.23 (m, 3H, HPy and C1′H), 5.99 (d, 12H, J = 6.0 Hz, Hcym), 5.79 (m, 1H, C5H), 5.76 (d, 12H, J = 6.0 Hz, Hcym), 5.57 (d, 1H, HPy), 4.90 (m, 1H, C4′H), 4.54 (m, 1H, CHα), 4.28−4.11 (m, 3H, C3′H and C5′H), 3.35 (m, 2H, PyCH2CH2CH2), 2.95 (sept, 6H, J = 6.9 Hz, CH(CH3)2), 2.32 (m, 4H, PyCH2CH2CH2 and C2′H), 2.11 (s, 18H, CH3), 1.62 (m, 2H, PyCH2CH2CH2), 1.37 (d, 36H, J = 6.9 Hz, CH(CH3)2); 13C{1H} NMR (75 MHz, acetone-d6): δ 173.4, 172.7, 171.9, 167.9, 162.8, 152.7, 150.4, 143.2, 134.2, 137.8, 129.5, 129.4, 128.8, 128.6, 127.8, 126.9, 126.0, 125.0, 124.1, 123.7, 123.6, 119.3, 115.0, 111.4, 103.7, 102.4, 100.1, 84.9, 83.1, 71.2, 64.5, 39.9, 37.3, 35.2, 31.8, 30.5, 26.6, 21.6, 16.4; IR (neat): 3446, 2966, 2925, 1717, 1534, 1275, 1030, 852 cm−1; ESI-MS (m/z): 1231.50 [pyrene-R5 + 2 + (CF3SO3)3]3+. [Pyrene-R6⊂2][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R6 (35.0 mg, 0.053 mmol). Yield: 162 mg (74%); 1H NMR (300 MHz, acetone-d6): δ 10.40 (s, 1H, N3H), 8.64 (s, 13H, Hα and C6H) 8.16−7.75 (m, 14H, Hβ and HPh), 7.56 (s, 12H, Hq), 7.31 (m, 3H, HPh), 6.84 (br, 4H, HPy), 6.47 (m, 2H, HPy), 6.27 (m, 3H, HPy and C1′H), 6.00 (d, 12H, J = 6.0 Hz, Hcym), 5.77 (d, 12H, J = 6.3 Hz, Hcym), 5.34 (d, 1H, HPy), 4.86−4.79 (m, 1H, C4′H), 4.56 (m, 1H, CHα), 4.30− 4.22 (m, 3H, C3′H and C5′H), 3.38 (m, 2H, PyCH2CH2CH2), 2.95 (sept, 6H, J = 6.9 Hz, CH(CH3)2), 2.32 (m, 4H, PyCH2CH2CH2 and C2′H), 2.14 (s, 18H, CH3), 1.42 (m, 2H, PyCH2CH2CH2), 1.39 (d, 36H, J = 6.9 HZ, CH(CH3)2); 13 C{1H} NMR (75 MHz, acetone-d6): δ 173.1, 172.1, 171.0, 168.0, 156.9, 152.8, 148.8, 143.3, 137.9, 129.8, 129.5, 128.8, 128.7, 127.8, 127.1, 126.9, 126.1, 124.9, 124.1, 123.7, 123.6, 123.0, 121.6, 119.3, 115.1, 111.4, 103.7, 100.1, 85.3, 84.9, 83.1, 71.2, 64.8, 54.8, 39.4, 37.3, 35.1, 31.7, 26.6, 21.6, 16.4; IR (neat): 3447, 2966, 2925, 1734, 1559, 1273, 1147, 1030, 852 cm−1; ESI-MS (m/z): 1236.48 [pyrene-R6 + 2 + (CF3SO3)3]3+. Cell Culture and Inhibition of Cell Growth. Human A2780 ovarian carcinoma cells were obtained from the European Centre of Cell Cultures (ECACC, Salisbury, UK) and maintained in culture as described by the provider. The cells were routinely grown in RPMI 1640 medium containing 10% fetal calf serum (FCS) and antibiotics at 37 °C and 6% CO2. For the evaluation of growth inhibition tests, the cells were seeded in 96-well plates and grown for 24 h in complete medium. Complexes were added to the required concentration and added to the cell culture for 72 h incubation. Solutions of the compounds were applied by diluting a freshly prepared stock solution of the corresponding compound in DMSO, with the final concentration of 0.05% in the medium. The MTT test was performed in the last 2 h without changing the culture medium. Following drug exposure, MTT (Sigma) was added to the cells at the final concentration of 0.2 mg/mL and incubated for 2 h, then the culture medium was aspirated and the violet formazan precipitate dissolved in DMSO. The optical density was quantified at 540 nm using a multiwell plate reader (iEMS Reader MF, Labsystems, US), and the percentage of surviving cells was calculated from the ratio of absorbance of treated to untreated cells. The IC50 values for the inhibition of cell growth were determined by fitting the plot of the percentage of surviving

CH(CH3)2), 2.38 (m, 6H, PyCH2CH2CH2 and C2′H), 2.12 (s, 18H, CH3), 1.35 (d, 36H, J = 6.9 Hz, CH(CH3)2); 13C{1H} NMR (75 MHz, acetone-d6): δ 172.8, 171.1, 167.8, 162.6, 152.7, 150.4, 143.1, 141.0, 137.9, 135.0, 129.8, 128.9, 128.4, 127.9, 127.2, 126.1, 125.3, 125.1, 124,2, 123,7, 123.6, 122.9, 121.7, 119.3, 103.7, 102.3, 100.2, 71.2, 64.5, 38.6, 33.6, 31.6, 30.6, 26.5, 21.6, 16.4; IR (neat): 3447, 3066, 2967, 1734, 1535, 1460, 1275, 853 cm−1; ESI-MS (m/z): 1182.80 [pyrene-R1 + 2 + (CF3SO3)3]3+. [Pyrene-R2⊂2][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R2 (29.0 mg, 0.06 mmol). Yield: 166 mg (74%); 1H NMR (300 MHz, acetone-d6): δ 9.89 (s, 1H, N3H), 8.64 (s, 12H, Hα), 7.99−7.74 (m, 13H, Hβ and C6H), 7.59 (s, 12H, Hq), 6.98 (m, 4H, HPy), 6.38 (m, 4H, HPy), 6.29 (t, 1H, C1′H), 6.01 (d, 12H, J = 5.7 Hz, Hcym), 5.78 (d, 12H, J = 5.7 Hz, Hcym), 5.12 (m, 1H, HPy), 4.68−4.43 (m, 4H, C3′H, C4′H and C5′H), 3.64 (t, 2H, PyCH2CH2CH2), 2.96 (sept, 6H, J = 7.2 Hz, CH(CH3)2), 2.38 (m, 4H, PyCH2CH2CH2 and C2′H), 2.11 (s, 18H, CH3), 1.37 (d, 36H, J = 7.2 Hz, CH(CH3)2); 13C{1H} NMR (75 MHz, acetone-d6): δ 172.8, 171.1, 167.8, 162.6, 156.7, 152.6, 150.3, 142.9, 141.0, 137.7, 135.1, 129.7, 128.9, 128.4, 127.9, 127.2, 126.1, 125.3, 125.0, 124,2, 123,7, 123.6, 123.0, 121.7, 119.4, 104.0, 100.2, 71.2, 64.4, 38.6, 33.5, 31.7, 30.6, 26.4, 21.5, 16.4; IR (neat): 3448, 3076, 2968, 1735, 1539, 1457, 1275, 1148, 853 cm−1; ESI-MS (m/z): 1188.13 [pyrene-R2 + 2 + (CF3SO3)3]3+. [Pyrene-R3⊂2][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R3 (27.4 mg, 0.05 mmol). Yield: 141 mg (71%); 1H NMR (300 MHz, acetone-d6): δ 8.64 (s, 13H, Hα and C6H), 8.16−7.75 (m, 12H, Hβ), 7.60 (s, 12H, Hq), 6.96 (m, 4H, HPy), 6.53 (m, 2H, HPy), 6.31 (m, 3H, HPy and C1′H), 6.00 (d, 12H, J = 6.0 Hz, Hcym), 5.77 (m, 13H, Hcym and C5H), 5.22 (d, 1H, HPy), 4.61−4.54(m, 4H, C3′H, C4′H and C5′H), 4.26 (m, 4H, PyCH2CH2CH2 and CH2α), 2.96 (sept, 6H, J = 6.9 Hz, CH(CH3)2), 2.35−2.20 (m, 6H, PyCH2CH2CH2 and C2′H), 2.12 (s, 18H, CH3), 1.38 (d, 36H, J = 6.9 Hz, CH(CH3)2); 13 C{1H} NMR (75 MHz, acetone-d6): δ 173.5, 171.1, 170.3, 168.0, 162.9, 152.7, 150.4, 143.1, 140.5, 137.9, 135.0, 129.8, 128.8, 128.3, 127.9, 127.2, 126.7, 126.1, 124.9, 124.1, 123.7, 123.6, 123.0, 122.9, 121.5, 119.3, 115.1, 111.4, 103.7, 102.4, 100.2, 84.9, 83.0, 71.1, 64.6, 41.3, 39.5, 35.2, 31.6, 30.3, 26.6, 21.5, 16.4; IR (neat): 3447, 3067, 2967, 1734, 1536, 1457, 1275, 1030, 853 cm−1; ESI-MS (m/z): 1201.46 [pyrene-R3 + 2 + (CF3SO3)3]3+. [Pyrene-R4⊂2][CF3SO3]6. This complex was synthesized according to the procedure described above using compound pyrene-R4 (27.3 mg, 0.05 mmol). Yield: 148 mg (76%); 1H NMR (300 MHz, acetone-d6): δ 8.64 (d, 13H, Hα and C6H), 8.16−7.75 (m, 12H, Hβ), 7.60 (s, 12H, Hq), 6.95 (m, 4H, HPy), 6.55 (m, 2H, HPy), 6.33 (m, 3H, HPy and C1′H), 6.00 (d, 12H, J = 6.3 Hz, Hcym), 5.77 (d, 12H, J = 6.0 Hz, Hcym), 5.24 (m, 1H, HPy), 4.61−4.54(m, 4H, C3′H, C4′H, and C5′H), 4.08 (m, 4H, PyCH2CH2CH2 and CH2α), 2.89 (sept, 6H, J = 6.9 Hz, CH(CH3)2), 2.38−2.20 (m, 6H, PyCH2CH2CH2 and C2′H), 2.12 (s, 18H, CH3), 1.32 (d, 36H, J = 6.9 Hz, CH(CH3)2); 13 C{1H} NMR (75 MHz, acetone-d6): δ 173.4, 171.1, 170.3, 168.0, 156.9, 156.3, 152.7, 148.9, 143.2, 142.1, 137.9, 129.8, 128.8, 127.8, 127.2, 126.7, 126.1, 124.8, 124.4, 123.7, 123.6, 123.0, 119.3, 115.0, 111.4, 103.7, 100.2, 85.2, 84.7, 83.0, 70.9, 64.5, 41.2, 39.3, 35.2, 31.7, 30.6, 26.6, 21.6, 16.4; IR (neat): 3447, 3066, 2967, 1735, 1539, 1457, 1275, 1057, 853 cm−1; ESI-MS (m/z): 1207.50 [pyrene-R4 + 2 + (CF3SO3)3]3+. 469



Article

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cells against the drug concentration using a sigmoidal function (Excel 2007, Microsoft Corporation).

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AUTHOR INFORMATION

Corresponding Author

*E-mails: [email protected], [email protected].

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ACKNOWLEDGMENTS Financial support of this work by the Strategic Korean-Swiss Cooperative program and a generous loan of ruthenium(III) chloride hydrate from Johnson Matthey Research Centre are gratefully acknowledged as well as the KNRRC program for modified nucleosides.

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REFERENCES

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