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StructureActivity Relationship of Polypyridyl Ruthenium(II) Complexes as DNA Intercalators, DNA Photocleavage Reagents, and DNA Topoisomerase and RNA Polymerase Inhibitors by Xing Chen, Feng Gao*, Wei-Yan Yang, Zhu-Xin Zhou, Jin-Qiang Lin, and Liang-Nian Ji* MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, State Key Laboratory of Optoelectronic Materials and Technologies, MOE Key Laboratory of Gene Engineering, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, P. R. China (fax: þ 86-20-32412366; e-mail:
[email protected])
To investigate the relationship between the molecular structure and biological activity of polypyridyl RuII complexes, such as DNA binding, photocleavage ability, and DNA topoisomerase and RNA polymerase inhibition, six new [Ru(bpy)2(dppz)]2 þ (bpy ¼ 2,2’-bipyridine; dppz ¼ dipyrido[3,2-a:2,’,3’c]phenazine) analogs have been synthesized and characterized by means of 1H-NMR spectroscopy, mass spectrometry, and elemental analysis. Interestingly, the biological properties of these complexes have been identified to be quite different via a series of experimental methods, such as spectral titration, DNA thermal denaturation, viscosity, and gel electrophoresis. To explain the experimental regularity and reveal the underlying mechanism of biological activity, the properties of energy levels and population of frontier molecular orbitals and excited-state transitions of these complexes have been studied by densityfunctional theory (DFT) and time-depended DFT (TDDFT) calculations. The results suggest that DNA intercalative ligands with better planarity, greater hydrophobicity, and less steric hindrance are beneficial to the DNA intercalation and enzymatic inhibition of their complexes.
Introduction. – Since anticancer activity of cisplatin has been first reported in 1969 [1], Pt-based drugs have been the most potent drugs available in the cancer chemotherapy for certain solid tumors [2 – 8]. To overcome the limitations of anticancer Pt drugs, such as drug resistance and side-effects, nephrotoxicity, and neurotoxicity [9 – 11], diverse metal-coordinative and organometallic compounds with other transition metals, such as V, Fe, Cu, Au, and Ru, have been used to find new, more effective metal-based anticancer drugs [12 – 21]. Among these metal antitumor agents, Ru complexes have been considered as the most promising antitumor agents due to their hypotoxicity and some other attractive properties [22 – 24]. RuIII Complexes NAMI-A [25] and KP1019 [26] have entered clinical trials in recent years. Activation by reduction to reactive RuII species is hypothesized as their essential action mode [24]. Considering this, a series of RuIIarene complexes have been identified as potent anticancer agents, although the structureactivity relationship is fairly complex [27 – 29]. Recently, polypyridyl RuII complexes with notable antitumor activity have also been reported by us [30 – 33]. It is most likely that the strong DNA-binding ability of these complexes leads to the inhibition of two kinds of important enzymes in cancer cells, which are DNA topoisomerase II, modulating the topological structure of DNA, and T7 RNA polymerase, responsible for the transcription of DNA. 2013 Verlag Helvetica Chimica Acta AG, Zrich
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In fact, many antitumor drugs and antiviral agents, such as cisplatin, actinomycin D, anthracyclines, and ellipticines, act as inhibitors of DNA topoisomerase and DNA transcription activity, through their interaction with the substrate DNA, binding to the active site of enzymes, blocking the DNA/RNA channel, or targeting transcription factors [34 – 42]. Therefore, design of small-molecular DNA intercalators is significant for the exploration of novel enzymatic inhibitors and antitumor drugs. These findings provide the stage for the DNA-intercalative polypyridyl RuII complexes in the study on enzymatic inhibitors and antitumor drugs. Regretfully, the relationship between the structure of drugs and their biological activity is largely unknown. Quantum-chemistry calculations based on density-functional theory (DFT) and time-dependent density functional theory (TDDFT) have been successfully used to provide important informations on the geometric and electronic structures, the molecular orbital energies and distributions, as well as the spectral properties for the polypyridyl RuII complexes, which afforded reasonable explanations for the trend of their DNA-binding affinities [43 – 50]. In this work, to investigate the relationship between the experimental biological activities and their theoretical molecular structures, a series of polypyridyl RuII complexes (Fig. 1) were designed based on the classical DNA intercalator [Ru(bpy)2(dppz)]2 þ (bpy ¼ 2,2’-bipyridine, dppz ¼ dipyrido[3,2-a:2’,3’-c]phenazine), which has been proved to be an effective DNA topoisomerase II and RNA T7 polymerase inhibitor in our previous study. The DNA-binding ability, and DNA topoisomerase II and RNA T7 polymerase inhibition activity of the synthesized complexes were examined by experimental methods. The structure and quantumchemical properties of the complexes, such as planarity, aromatic conjugation area, and steric and electronic effects, were studied by DFT calculations. In addition, as an important aspect of the DNA-binding study of polypyridyl RuII complexes, the metalto-ligand charge transfer (MLCT) of the complexes were also discussed by both experimental spectral data and TDDFT calculations. Results and Discussions. – Synthesis, Characterization, and Spectral Properties. The key intermediate [Ru(bpy)2(phdo)](ClO4 )2 was synthesized by reaction of [Ru(bpy)2Cl2 ] · 2 H2O and 1,10-phenanthroline-5,6-dione (phdo). The RuII complexes, [Ru(bpy)2(L)](ClO4 )2 · 2 H2O, where L represent the dppz-like DNA intercalative ligands, have been prepared by condensation of different diamines with [Ru(bpy)2(phdo)](ClO4 )2 to give the desired substitution patterns in straightforward reactions. The complexes were well characterized by 1H-NMR (Fig. 2) and elemental analysis. The absorption spectra of these complexes below 300 nm are attributed to intraligand (IL) p – p* transitions; and the bands in the range of 400 – 500 nm are assigned to MLCT. To understand more details of the MLCT properties of the synthesized complexes, we first discuss the UV/VIS spectra of these complexes with the aid of TDDFT theoretical calculations. The experimental spectra of the complexes are in good accordance with the simulated electronic absorption spectra (Fig. 3). The computational spectral data and their comparison with corresponding experimental data are compiled in Table 1. There are three strong theoretical transitions (f > 0.05) in the range of MLCT band of 1 – 3 and 6, but four and two for 4 and 5, respectively. These strong transitions are all from the
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Fig. 1. Structures of the synthesized poly(bipyridyl) RuII complexes
three occupied d orbitals of central RuII-atom to the unoccupied p* orbitals of ligands, except for 4. One of the four strong transitions of 4 lies at 418 nm with IL character, because it mainly originates from pNO2-dppz ! p*NO2-dppz . Therefore, the experimental broad band with comparable intensity of 4 at 441 nm can be attributed to the MLCT band with partial IL character. DNA-Binding Properties. Binding of the complexes with CT-DNA was examined by absorption-spectral titration (Fig. 4), DNA thermal denaturation (Fig. 5), and viscosity experiments (Fig. 6). The experimental DNA-binding data of 1 – 6, together with the parent complex [Ru(bpy)2(dppz)]2 þ are collected in Table 2. [Ru(bpy)2(dppz)]2 þ is a potent DNA intercalator with a intrinsic binding constant (Kb ) of 1.2 106 m 1. In the presence of [Ru(bpy)2(dppz)]2 þ (10 mm), the melting
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Fig. 2. The 1H-NMR spectra of a) [Ru(bpy)2(dpqc)]2 þ (1), b) [Ru(bpy)2(Me-dppz)]2 þ (2), c) [Ru(bpy)2(F-dppz)]2 þ (3), d) [Ru(bpy)2(NO2-dppz)]2 þ (4), e) [Ru(bpy)2(dppn)]2 þ (5), and f) [Ru(bpy)2(dppb)]2 þ (6)
temperature (Tm ) of CT-DNA can increase 148. For 1, by changing the ligand dppz to dpqc, the value of Kb and the measured increase of DNA melting temperature (DTm ) decreased to 4.6 105 m 1 and 10.48, respectively. The less aromatic conjugation area and greater steric effect of cyclohexane compared with benzene ([Ru(bpy)2(dppz)]2 þ ) and other substituted benzenes (i.e., 2 – 4), have weakened the DNA-binding affinity of 1. The DNA-binding ability of 2 with electron-donating Me group is lower than that of [Ru(bpy)2(dppz)]2 þ , whereas the Kb values of 3 and 4 with electron-withdrawing F and NO2 groups, respectively, are obviously higher than those of [Ru(bpy)2(dppz)]2 þ . This
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Fig. 3. Calculated (calc.) and experimental (exp.) electronic absorption spectra of complexes a) 1, b) 2, c) 3, d) 4, e) 5, and f) 6 in aqueous solution. The oscillator strengths are calculated for excitations in aqueous solution (f > 0.05 in 1 MLCT band) by using TDDFT method at the level of B3LYP/LanL2DZ. The full width at half maximum (FWHM) of theoretical spectrum is set at 3000 cm 1.
finding suggests that the electronic effect of substitutent affects the DNA affinity of the complexes more greatly than the steric effect. On the other hand, 5 and 6 with greater planar area than dppz, which can stack with DNA base pairs more effectively, also have higher DNA affinities than [Ru(bpy)2(dppz)]2 þ . That the values of Kb and DTm both
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Table 1. Comparison between Experimental (lexp. ) and Computed (lcalc. ) Wavelengths of 1 MLCT Absorption Spectra of [ Ru(bpy)2(dpqc)]2 þ (1), [ Ru(bpy)2( Me-dppz)]2 þ (2), [ Ru(bpy)2( F-dppz)]2 þ (3), [ Ru(bpy)2( NO2-dppz)]2 þ (4), [ Ru(bpy)2(dppn)]2 þ (5), and [ Ru(bpy)2(dppb)]2 þ (6), together with the Calculated Excitation Energies ( DE ), Oscillator Strengths (f > 0.05), Major Orbital Transition Contributions ( > 10%; H, HOMO; L, LUMO), and Characters Using TDDFT Method at B3LYP/ LanL2DZ Level in Aqueous Solution Complex
lexp. [nm]
lcalc. [nm]
f
DE [eV]
Major contribution
Character
1
454
440
0.132
2.82
433
0.152
2.86
423
0.056
2.93
H-1 ! L þ 1 (41%), H-2 ! L þ 2 (30%), H-1 ! LUMO (18%) H-2 ! L þ 1 (49%), H-1 ! L þ 2 (33%), H-2 ! LUMO (10%) HOMO ! L þ 4 (74%),
* dRu ! p bpy * dRu ! p bpqc * dRu ! p bpy * dRu ! p bpy * dRu ! p bpqc * dRu ! p bpy * dRu ! p bpqc
479 432
0.086 0.146
2.58 2.87
430
0.142
2.88
H-2 ! L þ 2 (19%) H-1 ! LUMO (89%) H-2 ! L þ 2 (45%), H-1 ! L þ 3 (34%), H-2 ! L þ 1 (14%) H-2 ! L þ 3 (32%), H-1 ! L þ 2 (29%), H-1 ! L þ 1 (24%)
* dRu ! p bpqc * dRu ! p bpqc * -dppz dRu ! p Me * -dppz dRu ! p Me * dRu ! p bpqc * -dppz dRu ! p Me * -dppz dRu ! p Me * dRu ! p bpqc
433
0.119
2.86
431
0.153
2.88
423
0.077
2.93
H-1 ! L þ 2 (30%), H-2 ! L þ 3 (27%), H-1 ! L þ 1 (24%), HOMO ! L þ 4 (12%) H-2 ! L þ 2 (47%), H-1 ! L þ 3 (35%), H-2 ! L þ 1 (12%) HOMO ! L þ 4 (70%), H-2 ! L þ 3 (21%)
dRu ! p F*-dppz dRu ! p F*-dppz * dRu ! p bpqc dRu ! p F*-dppz dRu ! p F*-dppz dRu ! p F*-dppz * dRu ! p bpqc dRu ! p F*-dppz dRu ! p F*-dppz
465
0.063
2.67
432
0.170
2.87
429
0.154
2.89
H-1 ! L þ 1 (79%), H-1 ! L þ 2 (12%) H-2 ! L þ 4 (30%), H-1 ! L þ 2 (30%), H-1 ! L þ 3 (21%) H-2 ! L þ 3 (51%), H-1 ! L þ 4 (37%) H-3 ! LUMO(77%)
* dRu ! p bpqc dRu ! p*bpy * -dppz dRu ! p NO2 dRu ! p*bpy * -dppz dRu ! p NO2 * -dppz dRu ! p NO2 * -dppz dRu ! p NO2 * -dppz pNO2-dppz ! p NO2
H-3 ! L þ 3 H-2 ! L þ 2 H-2 ! L þ 1 H-3 ! L þ 2 H-2 ! L þ 3 H-3 ! L þ 1
* dRu ! p dppn * dRu ! p dppn * dRu ! p bpy * dRu ! p dppn * dRu ! p dppn * dRu ! p bpy
2
3
4
5
6
438
437
441
440
441
418
0.056
2.96
433
0.218
2.86
432
0.150
2.87
436
0.059
2.84
(36%), (33%), (22%) (44%), (35%), (15%)
H-1 ! L þ 3 (25%), HOMO ! L þ 2 (21%), H-1 ! L þ 1 (14%), H-2 ! L þ 4 (10%)
* pdppb ! p dppb * pdppb ! p dppb * pdppb ! p bpy * pdppb ! p dppb
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Table 1 (cont.) Complex
lexp. [nm]
lcalc. [nm]
f
DE [eV]
Major contribution
Character
430
0.154
2.88
429
0.154
2.89
H-2 ! L þ 3 (37%), H-1 ! L þ 4 (30%), H-2 ! L þ 2 (12%), H-2 ! L þ 1 (10%) HOMO ! L þ 2 (32%), H-2 ! L þ 4 (20%), H-1 ! L þ 1 (11%)
* pdppb ! p dppb * pdppb ! p dppb * pdppb ! p dppb * pdppb ! p bpy * pdppb ! p dppb * pdppb ! p dppb * pdppb ! p bpy
Table 2. DNA-Binding and Inhibitory Activity Data of Complexes [ Ru(bpy)2(dpqc)]2 þ (1), [ Ru(bpy)2( Me-dppz)]2 þ (2), [ Ru(bpy)2( F-dppz)]2 þ (3), [ Ru(bpy)2( NO2-dppz)]2 þ (4), [ Ru(bpy)2(dppn)]2 þ (5), [ Ru(bpy)2(dppb)]2 þ (6) and [Ru(bpy)2(dppz)]2 þ Complex
H [%] Dl [nm] Kb [105 m 1] s
DTm a ) [8] IC50 [mm] Topoisomerase T7 RNA IIa Polymerase
1 2 3 4 5 6 [ Ru(bpy)2(dppz)]2 þ b ) a
20 14 13 10 15 10 18
1 0 0 5 2 1 0
4.6 9.9 13 23 24 24 12
3.1 2.1 2.8 2.0 1.5 1.7 2.8
10 12 12 13 11 12 14
5.0 1.5 1.6 1.2 1.5 3.0 2.0
10 3.0 2.2 1.5 1.5 4.0 2.0
) Tm of CT-DNA was found to be 53.48 in 10 mm Na2HPO4/NaH2PO4 buffer (pH 7.0). b ) From [32].
follow the order of 1 < 2 < [Ru(bpy)2(dppz)]2 þ 3 < 4 < 5 6 indicates that an extended plane area and a small steric hindrance of the intercalative ligand, and the presence of electron-withdrawing substitutents will be beneficial to the DNA-binding of their complexes. It is known that DNA intercalators can lengthen the DNA double helix, leading to the increase of the relative specific viscosity of DNA. Complexes 1 – 6 also significantly increased the viscosity of CT-DNA (Fig. 6), indicating an intercalative DNA-binding mode of these complexes. The extent of viscosity increase follows an order of 1 < 2 < 3 < 4 < 5 < 6, well supporting the result of spectral titrition. DNA Molecular Light Switch. In the absence of DNA, complex 2 displayed no detectable luminescence in aqueous buffer. Upon addition of CT-DNA, the emission intensity of 2 increased remarkably with a slight red shift (Fig. 7, b). This implies the complex can strongly interact with DNA and be protected by DNA efficiently, since the hydrophobic environment inside the DNA helix reduces the accessibility of solvent H2O molecules to the complex, and the complex mobility is restricted at the binding site, leading to a decrease of the vibrational modes of relaxation. Complexes 3 – 6 also showed weak luminescence in aqueous buffer at ambient temperatures. However, upon addition of DNA, the luminescence was only partly
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Fig. 4. Absorption spectra of complexes a) 1, b) 2, c) 3, d) 4, e) 5, and f) 6 in 5 mm Tris · HCl and 50 mm NaCl buffer (pH 7.0) in the presence of increasing amounts of CT-DNA ([Ru] ¼ 10 mm, [DNA] ¼ 0 – 220 mm from top to bottom). Arrows indicate the change in absorbance upon increasing the DNA concentration.
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Fig. 5. The melting curves of CT-DNA (100 mm) at 260 nm in the absence and the presence (10 mm) of complexes 1 – 6
Fig. 6. Effect of increasing amounts of complexes 1 – 6 on the relative viscosity of CT-DNA at 28 0.18. The total concentration of DNA is 100 mm.
restored for 3 (Fig. 7, c) and nearly no changes were detected for 4 – 6. The fluorescence of complex 4, bearing the strong electron-withdrawing substituent NO2 , was easily quenched through alternative efficient quenching pathways [51]. Similarly, complex 3 containing F could also be quenched, but to a lower extent than its NO2 analog. The intercalative ligand of 5 and 6 have greater aromatic surface than dppz. Upon DNA
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Fig. 7. Luminescence spectra of complexes a) 1, b) 2, and c) 3 in 5 mm Tris · HCl and 50 mm NaCl buffer (pH 7.0) in absence and presence of increasing amounts of CT-DNA
intercalation, the N-atoms of phenazine on the dppn and dppb ligand are still exposed to H2O, thus the luminescence of the complex was quenched. For complex 1, although the luminescence showed remarkable enhancement upon addition of CT-DNA, the complex also emited luminescence alone and cannot be considered as molecular light switch for DNA. DNA Photocleavage. As an important feature of antitumor reagents and photodynamic therapy, DNA photocleavage that are activated by metal ions and complexes has attracted substantial and continuing interests. Therefore, DNA photocleavage experiments were carried out by gel-electrophoresis separation of pBR322 DNA after incubation with 1 – 6 and irradiation at 365 nm under aerobic conditions. No DNA cleavage was observed for the control in which RuII complex was absent, or incubation of the plasmid with different concentrations of complexes 1 – 4 and 6. However, complex 5 showed potent activity (Fig. 8). It is known that 1O2 is a reactive species
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Fig. 8. Photo-activated cleavage of pBR322 DNA in the presence of different concentrations of complex 5. Irradiation at 365 nm for 60 min.
Fig. 9. Energy diagram of the frontier molecular orbitals of complexes 1 – 6
responsible for the cleavage reaction of RuII complex [52] [53]. DFT and TDDFT calculations here show that the lowest singlet excited state of 5 is located on the dppn ligand and the energy level of HOMO is obviously higher than other similar complexes. The consequent smaller energy gap (Fig. 9) supports a more efficient 1O2 yield by the reaction of the excited state of 5 and 3O2 . Near-IR luminescence experiments revealed that the complex is an efficient 1O2 sensitizer with yields as high as 83% [54]. Topoisomerase II Inhibition. Topoisomerases are nuclear enzymes that regulate DNA topology. Topoisomerase II (Topo II) acts for transient breaks in both strands of one DNA molecule allowing the passage of another DNA duplex through the gap. DNA Intercalative RuII complexes can inhibit the activity of Topo II as catalytic inhibitors or poisons. The results of Topo II inhibition assay of 1 – 6 are shown in Fig. 10. In the absence of the RuII complexes, Topo II can render the negatively supercoiled plasmid DNA entirely relax. However, as the concentration of the complexes increased, the amount of the relaxed DNA decreased gradually. Under a certain complex concentration, Topo II could be completely inhibited, and the plasmid
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Fig. 10. Effects of different concentrations of complexes 1 – 6 on the activity of DNA topoisomerase IIa
retained exactly the initial negatively supercoiled topological state. The concentrations of the complexes that prevented 50% of the supercoiled DNA from being converted into relaxed DNA (IC50 values) are compiled in Table 2. All of the complexes 1 – 6 exhibited potent inhibitory activities, which are comparable with those of RuII complexes reported by us [30 – 33] [55] and significantly higher than some classical Topo II inhibitors used as antitumor drugs in clinic, such as novobiocin and etoposide [56] [57]. For 1, with poor planarity, the Topo II inhibitory activity was the lowest among all of the complexes. Surprisingly, 2 – 4, with substitutions of small groups on the intercalative ligands, showed slightly higher activities than that of [Ru(bpy)2(dppz)]2 þ , although the groups had different electronic characters. Complex 5 with more extended ligands plane also exhibited higher activity than [Ru(bpy)2(dppz)]2 þ . With evident steric bulk, the inhibitory activity of complex 6 decreased, although the hydrophobicity and DNA-binding ability was improved. These results suggest that the steric effect of intercalative ligand is more essential for the Topo II inhibition of this type of complexes rather than its electronic effect. Transcription Inhibition. The inhibition of transcription was examined by the amount of the imaged mRNA produced by T7 RNA polymerase during the transcription reaction of pGEM template DNA at different concentrations of
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Fig. 11. Inhibition on RNA production in transcription reaction by complexes 1 – 6 at different concentrations
complexes. As shown in Fig. 11, the produced mRNA decreased with the increase of the concentrations of complexes, relative to the control lane under the condition of absence of complexes. The concentrations of complexes required to inhibit 50% of the transcription (IC50) are collected in Table 2. For activated cisplatin, the IC50 value is 3.8 mm under similar experimental conditions. It was evident that the IC50 values of the synthesized complexes were quite comparable to that of activated cisplatin, and those of a series of DNA intercalative RhII complexes [58 – 60] and some polypyridyl RuII complexes recently reported by us [30 – 33]. The transcription-inhibition activity of 1 are apparently lower than that of [Ru(bpy)2(dppz)]2 þ by a factor of 5. The regularity of the RNA polymerase-inhibition activity of these complexes is quite accordant with their Topo II-inhibition activity. It is encouraging to see that the transcription-inhibition activity of the complexes parallels their Topo II-inhibition activity, and has close
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relationship with their DNA-binding ability. This conclusion is significant for the design of novel Topo II inhibitors and transcription inhibitors, because it is much easier for us to estimate the possible DNA-binding ability of a compound than its DNA topoisomerase and RNA polymerase inhibitory activity. Conclusions. – Both experimental and computational results revealed that the six designed polypyridyl RuII complexes are potent DNA intercalators and dual inhibitors of topoisomerase II and T7 RNA polymerase, however, their biological activities are quite different. Complexes 3 and 4 with electron-withdrawing groups (NO2 and F), and complex 5 with excellent planar conjugated structure have shown higher DNA-binding ability and enzym-inhibitory activity. On the other hand, the steric effects in complexes 1 and 6 have greatly hindered their DNA binding and enzymatic inhibition. In addition, complexes 2 and 5 were found to be effective DNA molecular light switch and DNA photocleavage reagent, respectively. These studies should be valuable in further understanding the efficiency of DNA binding and cleavage, and enzymatic inhibition by the metal complexes of dppz-based systems, as well as developing new useful DNA probes, topoisomerase, and transcription inhibitors, and antitumor agents. We thank the National Natural Science Foundation of China (No. 20801060, 20871122), the 973 Program of China, the Fundamental Research Funds for the Central Universities, and Foundation of Sun Yat-Sen University for financial support.
Experimental Part General. All materials were commercially available and of highest available purity. Calf-thymus DNA (CT-DNA) was obtained from Sigma. The compounds 1,10-phenanthroline-5,6-dione (phdo) [61] and [Ru(bpy)2(phdo)](ClO4 )2 [62] were synthesized according to the literature methods. UV/VIS Spectra: Perkin-Elmer Lambda 850 spectrophotometer. Emission Spectra: Perkin-Elmer L55 spectrofluorophotometer. 1H-NMR spectra: Varian-INOVA-500NB NMR spectrometer; (D6 )DMSO as solvent at r.t.; d in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS: LCQ system (Finnigan MAT, USA); MeCN as mobile phase, m/z values for the major peaks in the isotope distribution. Microanalyses (C, H, and N): Perkin-Elmer-240Q elemental analyzer. Bis(2,2’-bipyridine-kN1,kN1’)(9a,10,11,12,13,13a-hexahydrodipyrido[3,2-a:2’,3’-c]phenazine-kN4,kN5 )ruthenium(II) Diperchlorate Dihydrate ([Ru(bpy)2(dpqc)](ClO4 )2 · 2 H2O; 1). [Ru(bpy)2(phdo)](ClO4 )2 (0.1 mmol) was dissolved and refluxed in MeCN (5 ml) under Ar. A soln. of trans-cyclohexane-1,2-diamine (0.2 mmol) in EtOH (3 ml) was added. The mixture was stirred at 758 under Ar for 4 h to give a dark red soln. The soln. was evaporated under reduced pressure to afford a dark red solid. The crude product was purified by column chromatography (CC; Al2O3 ; EtOH/MeCN 1 : 1). The red product was then recrystallized from Et2O/MeCN and then dried under vacuum for 8 h: 0.0801g (89%). 1 H-NMR ((D6 )DMSO): 9.46 (d, J ¼ 8.3, 2 H); 8.86 (dd, J ¼ 14.2, 8.1, 4 H); 8.25 – 8.20 (m, 4 H); 8.13 (t, J ¼ 7.9, 2 H); 7.99 (dd, J ¼ 8.3, 5.4, 2 H); 7.84 (d, J ¼ 5.6, 2 H); 7.67 (d, J ¼ 5.6, 2 H); 7.60 (t, J ¼ 7.2, 2 H); 7.36 (t, J ¼ 7.2, 2 H); 3.29 (s, 4 H); 2.08 (s, 4 H). ES-MS (MeCN): 349.7 ([M – 2ClO4 ]2 þ ). Anal. calc. for C38H30Cl2N8O8Ru · 2 H2O: C 48.83, H 3.67, N 11.99; found: C 48.80, H 3.47, N 11.86. Bis(2,2’-bipyridine-kN1,kN1’)(11-methyldipyrido[3,2-a:2’,3’-c]phenazine-kN4,kN5 )ruthenium(II) Diperchlorate Dihydrate ([Ru(bpy)2(Me-dppz)](ClO4 )2 · 2 H2O; 2). This complex was synthesized as described for 1, with 4-methylbenzene-1,2-diamine in place of trans-cyclohexane-1,2-diamine. Yield: 0.0708 g (78%). 1H-NMR ((D6 )DMSO): 9.61 (d, J ¼ 8.2, 2 H); 8.88 (dd, J ¼ 12.0, 8.2, 4 H); 8.41 (d, J ¼ 8.7, 1 H); 8.30 (s, 1 H); 8.26 – 8.21 (m, 4 H); 8.14 (t, J ¼ 7.9, 2 H); 8.06 – 8.00 (m, 3 H); 7.84 (d, J ¼ 5.6, 2 H); 7.78 (t, J ¼ 5.1, 2 H); 7.61 (t, J ¼ 7.2, 2 H); 7.39 (t, J ¼ 6.9, 2 H); 2.75 (s, 3 H). ES-MS (MeCN): 354.8 ([M –
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2ClO4 ]2 þ ). Anal. calc. for C39H28Cl2N8O8Ru · 2 H2O: C 49.58, H 3.41, N 11.86; found: C 49.48, H 3.54, N 11.90. Bis(2,2’-bipyridine-kN1,kN1’)(11-fluorodipyrido[3,2-a:2’,3’-c]phenazine-kN4,kN5 )ruthenium(II) Diperchlorate Dihydrate ([Ru(bpy)2(F-dppz)](ClO4 )2 · 2 H2O; 3). This complex was synthesized as described for 1, with 4-fluorobenzene-1,2-diamine in place of trans-cyclohexane-1,2-diamine. Yield: 0.0690 g (76%). 1H-NMR ((D6 )DMSO): 9.61 (dd, J ¼ 7.6, 3.7, 2 H); 8.87 (dd, J ¼ 11.6, 8.1, 4 H); 8.63 (dd, J ¼ 9.4, 5.8, 1 H); 8.31 (dd, J ¼ 9.3, 2.8, 1 H); 8.28 – 8.21 (m, 4 H); 8.19 – 8.12 (m, 3 H); 8.06 – 8.02 (m, 2 H); 7.84 (d, J ¼ 4.9, 2 H); 7.78 (d, J ¼ 4.9, 2 H); 7.61 (t, J ¼ 7.3, 2 H); 7.40 (t, J ¼ 7.0, 2 H). ES-MS (MeCN): 356.5 ([M – 2ClO4 ]2 þ ). Anal. calc. for C38H25Cl2FN8O8Ru · 2 H2O: C 48.11, H 3.08, F 2.00, N 11. 81; found: C 48.04, H 3.15, F 2.11, N 11.74. Bis(2,2’-bipyridine-kN1,kN1’)(11-nitrodipyrido[3,2-a:2’,3’-c]phenazine-kN4,kN5 )ruthenium(II) Diperchlorate Dihydrate ([Ru(bpy)2(NO2-dppz)](ClO4 )2 · 2 H2O; 4). This complex was synthesized as described for 1, with 4-nitrobenzene-1,2-diamine in place of trans-cyclohexane-1,2-diamine. Yield: 0.0685 g (73%). 1H-NMR ((D6 )DMSO): 9.66 (d, J ¼ 7.9, 2 H); 9.31 (d, J ¼ 2.5, 1 H); 8.90 – 8.81 (m, 5 H); 8.76 – 8.73 (m, 1 H); 8.30 – 8.28 (m, 2 H); 8.24 (t, J ¼ 7.9, 2 H); 8.15 (t, J ¼ 7.9, 2 H); 8.09 – 8.02 (m, 2 H); 7.84 (d, J ¼ 5.6, 2 H); 7.78 (d, J ¼ 5.7, 2 H); 7.61 (t, J ¼ 7.2, 2 H); 7.41 (t, J ¼ 6.6, 2 H). ES-MS (MeCN): 370.1 ([M – 2ClO4 ]2 þ ). Anal. calc. for C38H25Cl2N9O10Ru · 2 H2O: C 46.78, H 3.00, N 12.92, O 19.68; found: C 46.47, H 2.92, N 13.01, O 19.74. (Benzo[i]dipyrido[3,2-a:2’,3’-c]phenazine-kN4,kN5 )bis(2,2’-bipyridine-kN1,kN1’)ruthenium(II) Diperchlorate Dihydrate ([Ru(bpy)2(dppn)](ClO4 )2 · 2 H2O; 5). This complex was synthesized as described for 1, with naphthalene-2,3-diamine in place of trans-cyclohexane-1,2-diamine. Yield: 0.0764 g (81%). 1H-NMR ((D6 )DMSO): 9.61 (d, J ¼ 8.2, 2 H); 9.24 (s, 2 H); 8.88 (t, J ¼ 9.0, 4 H); 8.45 (dd, J ¼ 6.5, 3.2, 2 H); 8.26 – 8.22 (m, 4 H); 8.16 (t, J ¼ 8.0, 2 H); 8.03 (dd, J ¼ 8.1, 5.4, 2 H); 7.84 (t, J ¼ 4.1, 4 H); 7.78 (dd, J ¼ 6.6, 3.2, 2 H); 7.61 (t, J ¼ 6.4, 2 H); 7.42 (t, J ¼ 6.4, 2 H). ES-MS (MeCN): 372.9 ([M – 2ClO4 ]2 þ ). Anal. calc. for C42H28Cl2N8O8Ru · 2 H2O: C 51.44, H 3.29, N 11.43; found: C 51.51, H 3.32, N 11.51. Bis(2,2’-bipyridine-kN1,kN1’)[(dipyrido[3,2-a:2’,3’-c]phenazin-11-yl-kN4,kN5 )(phenylmethanone)]ruthenium(II) Diperchlorate Dihydrate ([Ru(bpy)2(dppb)](ClO4 )2 · 2 H2O; 6). This complex was synthesized as described for 1, with 3,4-diaminobenzophenone in place of trans-cyclohexane-1,2-diamine. Yield: 0.0764 g (73%). 1H-NMR ((D6 )DMSO): 9.66 (dd, J ¼ 14.1, 8.8, 2 H); 8.88 (dd, J ¼ 10.5, 8.4, 4 H); 8.48 (dd, J ¼ 8.8, 1.8, 1 H); 8.29 – 8.21 (m, 4 H); 8.15 (t, J ¼ 7.9, 2 H); 8.08 – 8.00 (m, 2 H); 7.94 (dd, J ¼ 8.4, 1.4, 2 H); 7.84 (d, J ¼ 5.7, 2 H); 7.81 – 7.78 (m, 2 H); 7.69 (t, J ¼ 7.9, 2 H); 7.63 – 7.56 (m, 4 H); 7.50 (d, J ¼ 7.5, 1 H); 7.40 (t, J ¼ 5.9, 2 H). ES-MS (MeCN): 399.6 ([M – 2 ClO4 ]2 þ ). Anal. calc. for C45H30Cl2N8O9Ru · 2 H2O: C 52.23, H 3.31, N 10.83, O 17.01; found: C 52.19, H 3.39, N 10.87, O 17.13. DNA-Binding Experiments. The DNA-binding experiments of the RuII complexes, including absorption titration, calculation of intrinsic binding constants Kb , luminescence spectra, viscosity measurements, and thermal denaturation studies, were performed as described in [63 – 65]. DNA Photocleavage Experiments. The photo-induced DNA cleavage by RuII complexes was examined through gel-electrophoresis experiment. Negative supercoiled pBR322 DNA (0.1 mg) was treated with different concentrations of RuII complexes in the buffer (50 mm Tris · HCl, 18 mm NaCl, pH 7.2). The reaction soln. was then irradiated at 258 with a UV lamp (365 nm, 10 W, 60 min) and treated with DNA loading buffer (6 sterile soln. of 0.25% bromophenol blue and 40% (w/v) sucrose). The samples were then analyzed by electrophoresis for 1.5 h at 70 V on a 1% agarose gel in TBE buffer (89 mm Tris-borate acid, 2 mm Na2H2edta, pH 8.3). The gel was stained with 1 mg/ml ethidium bromide (EB) and photographed on an Alpha Innotech IS – 5500 fluorescence chemiluminescence and visible imaging system. Topoisomerase II-Inhibition Assay. The assay was performed as described previously with some modification [30 – 33]. DNA Topoisomerase IIa (Topo II) from Escherichia coli containing a clone of the human topoisomerase II gene was obtained from Affymetrix Incorporated. One unit of the enzyme was defined as the amount that completely relaxes 0.3 mg of negatively supercoiled pBR322 plasmid DNA in 15 min at 308 under the standard assay conditions. The mixtures (20 ml) containing 10 mm Tris · HCl (pH 7.9), 50 mm NaCl, 50 mm KCl, 5.0 mm MgCl2 , 0.1 mm Na2H2(edta), 15 mg/ml BSA, 1.0 mm ATP, 0.25 mg pBR322 DNA, 2 unit TopoII, and RuII complexes were incubated at 308 for 15 min. Reactions were stopped by addition of 4 ml of 5 stop soln. consisting of 0.25% bromophenol blue, 4.5% SDS, and
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45% glycerol. The samples were subjected to electrophoresis through 1.2% agarose in TBE buffer at 80 V for 1 h. The gel was stained and photographed as described above. Transcription-Inhibition Assay. The pGEM template DNA produces transcripts that are 1065 and 2346 bases in length with T7 RNA polymerase (Promega). The transcription reaction was conducted for 30 min at 378 HEPES buffer (pH 7.5; HEPES ¼ 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) in nuclease-free H2O in the presence of MgCl2 and NTPs. The inhibition of mRNA production by the RuII complexes and activated cisplatin were detected in vitro by the measurement of the mRNA generated upon addition of increasing amounts of metal complex to the assay. Reactions were stopped, processed, and subjected to gel electrophoresis as described above. Theoretical Calculations. DFT Calculations were carried out with the Gaussian03 quantumchemistry program package [66] using Beckes three-parameter hybrid functional (B3LYP) method [43] and LanL2DZ basis set (a double-zeta basis set containing effective core potential) [67]. The full geometry-optimization computations were carried out for the ground states (singlet state) of these complexes [68]. The stability of the optimized conformation of the complexes was confirmed by the frequency analysis, which shows no imaginary frequency for each energy minimum. TDDFT was used to calculate the 100 singlet excited state energies of the complexes to pattern the electronic absorption spectral characters. The conductor polarizable continuum model (CPCM) was applied to the solvent effect in aqueous soln. [69]. In addition, the optimized conformations and frontier molecular orbital plots of the complexes for the ground states were drawn with the Molden v3.7 program based on the computational results [70].
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