Synthesis, characterization and interactions with 9-methylguanine of ruthenium(II) η6-arene complexes with aromatic diimines

Accepted Manuscript Synthesis, characterization and interactions with 9-methylguanine of ruthenium(II) η 6-arene complexes with aromatic diimines Theodoros Tsolis, Konstantinos Ypsilantis, Andreas Kourtellaris, Achilleas Garoufis PII: DOI: Reference:

S0277-5387(18)30202-X https://doi.org/10.1016/j.poly.2018.04.025 POLY 13127

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Polyhedron

Received Date: Accepted Date:

16 March 2018 20 April 2018

Please cite this article as: T. Tsolis, K. Ypsilantis, A. Kourtellaris, A. Garoufis, Synthesis, characterization and interactions with 9-methylguanine of ruthenium(II) η 6-arene complexes with aromatic diimines, Polyhedron (2018), doi: https://doi.org/10.1016/j.poly.2018.04.025

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Synthesis, characterization and interactions with 9-methylguanine of ruthenium(II) η6-arene complexes with aromatic diimines. Theodoros Tsolis a, Konstantinos Ypsilantis a, Andreas Kourtellaris b and Achilleas Garoufis a * a b

Department of Chemistry, University of Ioannina, GR-45110 Ioannina, Greece. Department of Chemistry, University of Cyprus, P.O. Box 20537, CY-1678 Nicosia, Cyprus

ABSTRACT The complexes of the formula [(η6-arene)Ru(L)Cl]PF6, where arene is benzene (bz) or p-cymene (cym) and L is 2,(2΄-pyridyl)quinoline (pqn), were synthesized and characterized by means of NMR spectroscopic techniques, HR-ESI mass spectrometry and, in the case of [(η6-cym)Ru(pqn)Cl]PF6, by X-ray single crystal diffraction. Their resistance in hydrolysis was also studied. A comparative NMR study of their 9methylguanine (9-MeG) complexes, [(η6-arene)Ru(pqn)(9-MeG)](PF6)2, with similar diimine complexes revealed that the unimpeded rotation of 9-MeG is hindered by interactions between the 9-MeGO6 and the p-cymene aromatic proton H2 and, by the bulky shape of the pqn. This conformation forces the 9-MeGH8 to be in close proximity to the aromatic ring system of pqn. NMR spectroscopic techniques lead to the conclusion that the strong shielding effect on 9-MeGH8 depends on the extension of the aromatic system of the ligand. Also, we conclude that the strong deshielding on the 9-MeGNH1 is influenced by both the N7 ruthenation of 9-MeG and the addendum electron density in the 9-MeG ring system, due to the proximity to the aromatic ring system of pqn. Keywords: η6-arene ruthenium; 9-methylguanine; cancer chemotherapy; NMR; mass spectrometry

* Corresponding author: AG, [email protected]

1. Introduction During the last decades, ruthenium complexes have attracted considerable research attention, due to their potential application in cancer chemotherapy. This interest was encouraged since the complexes NAMI-A [1] and KP1019 [2] have entered clinical trials, exhibiting decreases of the metastasis speed and high cytotoxicity against primary tumors, respectively. The discovery of the cytotoxic properties of the complex [(η6-C6H6)RuCl2(metronidazole)] (metronidazole is 1-β-hydroxyethyl-2-methyl-5-nitroimidazole) [3] has inspired the efforts of many research groups in a new class of arene-based organometallic ruthenium compounds. Among them two classes of promising compounds stand out; these with general formula [(η6arene)Ru(en)X]+, en = 1,2-ethylenediamine, X = halide, (monofunctional) [4] and [(η6-arene)Ru(pta)X2], pta = 1,3,5-triaza-7-phospha-tricyclo-[3.3.1.1]decane, X = halide, (bifunctional) [5]. In the chromatin of cancer cells, [(η6-arene)Ru(pta)X2], primarily associated with the protein component, not the DNA. In contrast [(η 6arene)Ru(en)X]+ does preferentially target the DNA component of chromatin. This is probably the reason of the highly cytotoxic activity of the later and the inhibition of the cancer metastasis or other therapeutic activity, of the former [5c]. The compounds with ethylenediamine target the guanine of the DNA after being hydrolyzed [6], forming stable adducts. The binding is furthermore stabilized through hydrogenbonding between the carbonyl group of guanine and the –NH2 group of the ethylenediamine [7]. Also, it has been suggested that an alkyl substitution on the ethylenediamine NH group can lead to the loss of cytotoxicity of the complex [8], indicating that the additional hydrogen bonding may be responsible for the cytotoxic activity. On the other hand, there are enough paradigms of similar compounds, where the ethylenediamine is replaced by an aromatic diimine, exhibiting noteworthy cytotoxicity, depending on the nature of diimine. (e.g. some recent reports [9]). Complexes with 2,2΄-bipyridine [10] or 2,2΄-bipyrimidine [11] are rather inactive, while the one with bathophenthroline (4,7-diphenyl-1,10-phenanthroline) is highly cytotoxic exhibiting IC50 < 0.5 μΜ against the A2780 human ovarian cancer cell line [11]. Many factors were supposed to be responsible for the cytotoxicity of these compounds, such as the ligand’s lipophilicity and cellular uptake [12], the exchange rate of the halide ligand and the pKa values of the aquated species [13], the N7 guanine coordinative binding [6] and the additional binding to DNA of the η6-coordinated arene [7] or the binding of the diimine ligand [14]. In our previous work, we observed that complexes of the general formula [(η6-arene)Ru(L)Cl]PF6 (η6-arene = bz or cym and L = 2-(2΄-pyridyl)quinoxaline (pqx) and 2-(2΄-pyridyl)benzo[g]quinoxaline (pbqx)), exhibit remarkable cytotoxicity against several cancer cell lines, with IC50 values less than 1μΜ [15]. Moreover, we found that the complexes of the type [(η6-cym)Ru(L)Cl]PF6 bind to 9-MeG through N7. Based on NMR spectroscopic studies, we suggested that the imidazole of the 9-MeG is oriented towards the ligand L, causing significant shielding on the 9-MeGH8 [15]. X-ray crystal structures of similar complexes such as [(η6-cym)Ru(bpm)(9-EtG)]2+ [11] and [(η6-tha)Ru(bpy(OH)O)(9-EtG)]2+ (tha = tetrahydroanthracene and bpy(OH)O = deprotonated 2,2΄-bipyridine-3,3΄-diol) [10] confirm this type of orientation. However, an almost overturn placing of guanine was observed at the case of [(η6- bip)Ru(en)(9-EtG)]2+ (9-EtG = 9ethylguanine, bip = biphenyl) [7] . With a view to contribute the knowledge about the orientation of the guanine in such complexes, herein we report on the synthesis and the characterization of the [(η6-arene)Ru(pqn)Cl]PF6, where arene is benzene (bz) or p-cymene (cym) and L is 2,(2΄-pyridyl)quinoline (pqn) (Fig.1), as well as their complexes with 9-methylguanine (9-MeG). (PLEASE INSERT FIGURE 1)

2. Experimental 2.1. Materials and methods All solvents were of analytical grade and were used without further purification. Hydrated ruthenium trichloride, RuCl3∙3H2O, was purchased from Pressure Chemical Company (Pittsburgh, USA). The ligands 2,(2′-pyridyl)quinoxaline, [16] 2-(2′-pyridyl)quinoline [17] and the complexes [(η6-bz)Ru(μ-Cl)Cl]2 [18], [(η6-cym)Ru(μ-Cl)Cl]2 [19], , [(η6-bz)Ru(pqx)Cl](PF6) [15], [(η6-bz)Ru(bpy)Cl](PF6) [20] and [(η6cym)Ru(en)Cl](PF6) [4], were synthesized according to the literature methods. The transformation of the [PF6]- salts of the complexes to the corresponding [Cl]- was achieved as described in [14]. C, H and N determinations were performed on a Perkin-Elmer 2400 Series II analyzer. NMR spectra were recorded on Bruker Avance spectrometers operating at 1H frequencies of 400.13, 500.13 MHz and 13C at 100 MHz and processed using Topspin 1.3 (Bruker Analytik GmbH). TOCSY and HMQC experiments were used to assist the assignments of 1H and 13C signals. Two-dimensional NOESY (nuclear Overhauser effect spectroscopy) experiments were performed with mixing times of 500 and 700 ms. High resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained on an Agilent Technology LC/MSD trap SL instrument and Thermo Scientific, LTQ Orbitrap XL™ system. 2.2. X-Ray crystallography X-ray diffraction data were collected (ω-scans) with an Oxford Diffraction Xcalibur-3 diffractometer under a flow of nitrogen gas at 100(2) K using MoKα radiation (λ = 0.71069 Å). Data were collected and processed using the CRYSALIS CCD and RED software [21] respectively. Empirical absorption corrections (multiscan based on symmetry related measurements) were applied using CrysAlis RED software. The structure was solved by direct methods using SIR2014 [22] and refined on F2 using full-matrix least-squares with the latest version of SHELXL [23]. All non-H atoms were refined anisotropically and carbon-bound Hatoms were introduced at calculated positions and allowed to ride on their parent atoms. Geometric/crystallographic calculations were carried out using PLATON [24] and WINGX [25] packages; graphics were prepared with X-Seed [26]. The CCDC file 1828018 contains the supplementary crystallographic data for (2)PF6. Selected crystal data of the compound are given in Table 1. Table 1. Selected crystal data of the compound (2)PF6. (2)PF6 Chemical formula C24H24ClF6N2PRu Formula Mass 621.94 Crystal system monoclinic Crystal dimensions/mm 0.03 x 0.05 x 0.07 a/Å, b/Å, c/Å 12.6477(9), 13.4349(8), 13.9028(7) a, b, g /deg 90, 94.996(5), 90 3 Unit cell volume/Å 2353.4(2) Temperature/K 100 Space group P21/n Radiation type MoKa 0.71073 No. of reflections measured 11623 No. of independent reflections 5251 Rint 0.051 R, wR2, S 0.0588, 0.1240, 1.01

2.3. Synthesis of the complexes [(η6-bz)Ru(pqn)Cl](PF6), (1)PF6 50 mg (0.1 mmol) of [(η6-bz)Ru(μ-Cl)Cl]2 were suspended in 40 mL of a mixture CH3OH:CH3CN, 1:3 and a solution containing 51 mg (0.25 mmol) of pqn in 10 mL of CH3OH, was added. The mixture was stirred at ambient temperature overnight, filtered from the unreacted material and concentrated to dryness under reduced pressure. The light yellow crude product was dissolved in 3 mL of H2O and a few drops of an aqueous saturated solution of NH4PF6 were added. After cooling the solution overnight at 7 °C, a microcrystalline yellow precipitate appeared which was collected by filtration, washed with water (2 × 5 mL), methanol (2 × 5 mL) diethyl ether (2 × 5 mL) and dried under vacuum over CaCl2. Yield 75 % (80 mg, 0.15 mmol). C20H16ClF6N2PRu (565.83); calc. C, 42.45; H, 2.85; N, 4.95. Found C, 42.30; H, 2.91; N, 4.78. HRESI-MS, calc. for [C20H16ClN2102Ru]+ m/z = 421.0040, found m/z = 421.0012, corresponding to [(η6bz)Ru(pqn)Cl]+. 1H NMR (dmso-d6, 298 K, δ in ppm, 400 MHz), H1;(bz) 6.18(s), H3; 8.91(d); H4; 8.73(d), H5; 8.25(d), H6; 7.94(t), H7; 8.11(t), H8; 8.89(d), H3’; 8.83(d), H4’; 8.37(t), H5’; 7.88(t), H6’; 9.67 (d). 13C NMR (dmso-d6, 298 K, δ in ppm, 100 MHz), C1(bz); 87.5, C2; 155.9, C3; 140.9, C4; 119.3, C4a; 129.2, C5; 129.0, C6; 129.4, C7; 130.0, C8; 125.2, C8a; 144.9, C2’; 148.5, C3’; 132.7, C4’; 140.1, C5’; 28.0, C6’; 156.4. [(η6-cym)Ru(pqn)Cl](PF6), (2)PF6 60 mg (0.1 mmol) of [(η6-cym)Ru(μ-Cl)Cl]2 were suspended in 20 mL of CH3OH and a solution containing 50 mg (0.25 mmol) of pqn in 10 mL CH 3OH was added. The mixture was stirred at ambient temperature overnight and the clear solution was concentrated to dryness under reduced pressure. The red-orange crude product was dissolved in 3 mL of H2O and a few drops of an aqueous saturated solution of NH4PF6 were added. After cooling the solution overnight at 7 °C, a microcrystalline yellow precipitate appeared which was collected by filtration, washed with water (2 × 5 mL), methanol (2 × 5 mL), diethyl ether (2 × 5 mL), and dried under vacuum over CaCl2. Yield 80 % (100 mg, 0.16 mmol). C 24H24ClF6N2PRu (621.9); calc. C, 46.35; H, 3.89; N, 4.50. Found C, 46.28; H, 3.96; N, 4.48. HR-ESI-MS, calc. for [C24H24ClN2102Ru]+ m/z = 477.0666, found m/z = 477.0636, corresponding to [(η6-cym)Ru(pqn)Cl]+. 1H NMR (dmso-d6, 298 K, δ in ppm, 400 MHz), H2(cym); 6.05(d), H3(cym); 5.91(d), H5(cym); 5.96(d), H6(cym); 6.08(d), H7(cym); 2.20(s), H8(cym); 2.15(m), H9(cym); 0.76(d), H10(cym); 0.71(d), H3; 8.85(d); H4; 8.66(d), H5; 8.23(d), H6; 7.92(t), H7; 8.10(t), H8; 8.73(d), H4’; 8.77(t), H5’; 7.85(t), H6’; 9.50 (d). 13C NMR (dmso-d6, 298 K, δ in ppm, 100 MHz), C1(cym); 105.3, C2(cym); 85.0, C3(cym); 86.7, C4(cym); 104.2, C5(cym); 84.3, C6(cym); 87.6, C7(cym); 18.7, C8(cym); 30.7, C9(cym); 21.9, C10(cym); 21.4, C2; 156.3, C3; 141.4, C4; 119.5, C4a; 129.7, C5; 129.4, C6; 130.0, C7; 130.1, C8; 125.6, C8a; 155.0, C2’; 148.9, C3’; 133.2, C4’; 140.6, C5’; 128.6, C6’; 156.6. Red-orange crystals, suitable for x-ray diffraction analysis, were obtained by slow vapor diffusion of diethyl ether to a solution of (2)PF6 in acetone. [(η6-bz)Ru(pqn)(9-MeG)](PF6)2, (3)(PF6)2 To a solution containing 63 mg (0.1 mmol) of [(η6-bz)Ru(pqn)Cl](PF6) in 30 mL CH3COCH3:H2O, 1:1, 15 mg (0.09 mmol) of AgNO3 which was dissolved in 1mL of H 2O were added. The mixture was heated at reflux for 24 h in the dark and the precipitate was removed with centrifugation. To the clear yellow solution, 50 mg (0.3 mmol) of 9-MeG were added and the mixture was left to react at 40 °C for 72 h. The solution was evaporated to dryness, and the crude product was dissolved in 3 mL of dry hot acetone and filtered from the insoluble materials. After the addition of a few drops of a saturated solution of NH4PF6 in acetone and 5 mL of (CH3CH2)2O, the solution was kept at -20 °C overnight. A yellow microcrystalline precipitate appeared which was collected by filtration, washed with diethyl ether (2 × 5 mL) and dried under vacuum. Yield 60 % (50 mg, 0.06 mmol). C26H23F12N7OP2Ru (840.5); calc. C, 37.15; H, 2.76; N, 11.67. Found C, 37.32; H, 2.88; N, 11.52. HR-ESI-MS, calc. for [C26H23N7O102Ru]2+ m/z = 275.5498, found m/z = 275.5487,

corresponding to [(η6-bz)Ru(pqn)(9-MeG)]2+. 1H NMR (dmso-d6, 298 K, δ in ppm, 400 MHz), H1(bz); 6.32(s), H3; 9.07(d) ; H4; 8.87(d), H5; 8.39(d), H6; 8.04(t), H7; 8.19(t), H8; 8.69(d), H3’; 8.82(d), H4’; 8.36(t), H5’; 7.89(t), H6’; 9. 83(d); NH1(9-MeG); 11.27(s), NH2(9-MeG); 6.96(b), H8(9-MeG); 6.75(s), CH3(9-MeG); 3.34(s). [(η6-cym)Ru(pqn)(9-MeG)](PF6)2, (4)(PF6)2 The synthetic procedure for complex (4) was the same as that of (3), differing only in the amount of starting material and in the final yield. Yield 70 % (63 mg, 0.07 mmol). C30H31F12N7OP2Ru (896.6); calc. C, 40.19; H, 3.09; N, 11.13. Found C, 40.08; H, 3.03; N, 11.08. HR-ESI-MS, calc. for [C30H30N7O102Ru]2+ m/z = 303.5811, found m/z = 303.5817, corresponding to [(η6-cym)Ru(pqn)(9-MeG)]2+. 1H NMR (dmso-d6, 298 K, δ in ppm, 400 MHz), H2(cym); 6.35(d), H3(cym); 6.02(d), H5(cym); 6.21(d), H6(cym); 6.60(d), H7(cym); 2.04(s), H8(cym); 2.47(m), H9(cym); 0.81(d), H10(cym); 0.55(d), H3; 9.10(d); H4; 8.69(d), H5; 8.43(d), H6; 8.08(t), H7; 8.29(t), H8; 8.81(d), H4’; 8.37(t), H5’; 7.91(t), H6’; 9.97 (d), NH1(9-MeG); 11.22(s), NH2(9-MeG); 6.94(b), H8(9-MeG); 6.88(s), CH3(9MeG); 3.23(s). [(η6-bz)Ru(pqx)(9-MeG)](PF6)2, (5)(PF6)2 The synthetic procedure for complex (5) was the same as that of (3), differing only in the amount of starting material and in the final yield. Yield 70 % (58 mg, 0.07 mmol). C25H22F12N8OP2Ru (841.5); calc. C, 35.68; H, 2.64; N, 13.32. Found C, 35.81; H, 2.68; N, 13.40. HR-ESI-MS, calc. for [C25H22N8O102Ru]2+ m/z = 276.0475, found m/z = 276.0475, corresponding to [(η6-cym)Ru(pqn)(9-MeG)]2+. 1H NMR (dmso-d6, 298 K, δ in ppm, 400 MHz), H1(bz); 6.38(s), H3; 10.17(s), H5; 8.48(m), H6; 8.27(m), H7; 8.27(m), H8; 8.67(m), H3’; 9.08(d), H4’; 8.44(t), H5’; 7.94(t), H6’; 9.86 (d), NH1(9-MeG); 11.21(s), NH2(9-MeG); 6.92(b), H8(9-MeG); 6.74(s), CH3(9-MeG); 3.21(s). [(η6-cym)Ru(en)(9-MeG)](PF6)2 (6)(PF6)2 This complex was prepared in a similar manner to that of [(η6-bph)Ru(bpm)(9-EtG)]2+ [7]. Yield 80 % (67 mg, 0.08 mmol). C18H29F12N7OP2Ru (750.5); calc. C, 28.81; H, 3.89; N, 13.06. Found C, 28.86; H, 3.81; N, 13.02. HR-ESI-MS, calc. for [C18H29N7O102Ru]+ m/z = 230.5733 found m/z = 230.5722, corresponding to [(η6cym)Ru(bpm)(9-MeG)]2+. 1H NMR (dmso-d6, 298 K, δ in ppm, 400 MHz), H2/H6(cym); 5.71(d), H3/H5(cym); 5,65 (d), H7(cym); 2.10(s), H8(cym); 2.58(m), H9(cym); 1.10(d), H10(cym); 1.12(d), NH2(en); 6.41(a), NH2(en); 7.92 (b), CH2(en); 2.28(m) and 2.17(m), NH1(9-MeG); 11.31(s), NH2(9-MeG); 6.91(b), H8(9-MeG); 8.31(s), CH3(9-MeG); 3.63(s). [(η6-bz)Ru(bpy)(9-MeG)](PF6)2, (7)(PF6)2 This complex was prepared in similar manner to that of [(η6-cym)Ru(bpy)(9-MeG)]2+ [15] Yield 80 % (65 mg, 0.08 mmol). C22H21F12N7OP2Ru (790.4); calc. C, 33.43; H, 2.68; N, 12.40. Found C, 33.61; H, 2.76; N, 12.32. HR-ESI-MS, calc. for [C22H21N7O102Ru]2+ m/z = 250.5420, found m/z = 250.5415, corresponding to [(η6-bz)Ru(bpy)(9-MeG)]2+. 1H NMR (dmso-d6, 298 K, δ in ppm, 400 MHz), H1(bz); 6.30(s), H3H3’; 8.59(d), H4H4’; 8.32(t), H5H5’; 7.87(t), H6H6’; 9.91(d), NH1(9-MeG); 11.03(s), NH2(9-MeG); 6.84(b), H8(9-MeG); 7.17(s), CH3(9-MeG); 3.39(s).

3. Results and discussion 3.1. Synthesis The complexes (1) and (2) were prepared by the reaction of the corresponding dimers [(η6arene)RuCl2]2, where arene is benzene (bz) or p-cymene (cym), with the diimine 2-(2’-pyridyl)quinoline (pqn) in methanol and were isolated as [PF6]- salts after the addition of NH4PF6. In order to study their aqueous chemistry, they were transformed to their water-soluble chloride salts [14]. The compounds (3)(PF6)2-(7)(PF6)2 were synthesized by the subtraction of the coordinated chloride with AgNO3 from the compounds (1)PF6, (2)PF6, [(η6-bz)Ru(pqx)Cl](PF6) [15], [(η6-bz)Ru(bpy)Cl](PF6) [20] and [(η6cym)Ru(en)Cl](PF6) [4], followed by the reaction of the resulting solution with excess of 9-MeG. In summary the synthetic procedure is presented in Fig. 2. (PLEASE INSERT FIGURE 2) 3.2. Characterization of the complexes (1) and (2) The synthesized compounds were characterized by 1H and 13C NMR spectroscopy (Fig. S1-S5), HRESI-MS and, in the case of (2)PF6, by single crystal X-ray diffraction methods. The molecular structure of the hexafluorophosphate salt of complex (2) is presented in Fig. 3. The ruthenium centre adopts a pseudo-octahedral geometry, surrounded by the π-bonded p-cymene, two σbonded nitrogen atoms from the chelating ligand 2,(2’-pyridyl)quinoline and the Cl. The value of Ru-pcymene (centroid) bond distance is similar to other complexes containing N,N’-chelating ligands [11, 27]. However, it is smaller than this of the similar complex [(η6-cym)Ru(pqx)Cl]+ (1.703 Å). The Ru-Cl bond distance is within the values of analogous complexes [11, 28]. Both Ru-N(1) and Ru-N(2) bond distances are typical for chelating N,N’ aromatic diimines [11, 28], although they are slightly shorter than those of the complex [(η6-cym)Ru(pqx)Cl]+ (2.115 and 2.081 Å respectively), reflecting the strong donor character of the ligand 2,(2’-pyridyl)quinoline. (PLEASE INSERT FIGURE 3) The HR-ESI mass spectra of (1)PF6 and (2)PF6 in acetone show one cluster peak each, corresponding to the single charged cations [(η6-bz)Ru(pqn)Cl]+ and [(η6-cym)Ru(pqn)Cl]+. The 1H NMR spectra of (1) and (2) were recorded in acetone-d6 and in dmso-d6, in order to ensure that dmso does not replace any ligand from the ruthenium coordination sphere [29]. Since the obtained spectra were almost identical, solubility and comparability reasons imposed the use of dmso-d6. In the 1H NMR spectrum of (1) in dmso-d6, distinct signals for each proton of the ligand pqn were observed. The signals of H6΄ and H8 shifted downfield by about 0.89 and 0.86 ppm respectively, compared to the free ligand, indicating that pqn coordinates through N1΄ and N1. However, these shifts are larger than those in the similar complex, (2) where they shifted by about 0.72 and 0.70 ppm. This observation is consistent with the weaker donor character of benzene, compared to p-cymene, which influences the strength binding of the chelating ligand [30]. In the case of complex (2), it will be useful to accurately assign the four aromatic protons of the pcymene (H2, H3, H5 and H6), since four separate signals were observed due to the asymmetric nature of the chelating ligand pqn. Based on the NOESY map of (2), two NOE crosspeaks between the cym–CH3 of the isopropyl group and the signals at 6.08 and 5.95 ppm were observed, indicating that these signals correspond to H2 and H6. Similarly, two NOE crosspeaks between the cym–CH3 of the methyl group and the signals at 6.06 and 5.90 ppm were observed, indicating that these protons are located at both sides of C4 and correspond to H3 and H5. The above observations were also confirmed by the COSY spectrum of the

compound. Τhe discrimination between the protons H2/H6 and H3/H5 is obtained from the relative position of cym to the chelating ligand pqn. Thus, the observed NOE crosspeaks between pqnH8 and cym– CH3 of the methyl group, together with these of the pqnH6’ and cym–CH3 of isopropyl group, indicate the orientation of the p-cymene shown in Fig. 4A. Taking into account this conformation, the NOE crosspeaks between pqnH8 and the signals at 6.06 and 5.90 ppm and those between the pqn H6’ and the signals at 6.08 and 5.95 (Fig. 3B), can easily lead to the following assignment of the cym protons: H5, 6.06 ppm; H6, 5.90 ppm; H2, 6.08 ppm and H3, 5.95 ppm. (PLEASE INSERT FIGURE 4) 3.4. Aqueous chemistry of (1)Cl and (2)Cl In order to investigate the hydrolysis of the complexes (1) and (2), a solution of 20 mM in D2O was kept at 298K for several days. During this period and at standard time intervals, the 1H NMR and the ESI-MS spectra of the complexes were recorded. The 1H NMR spectrum of (2) remained unchanged, and the ESI mass spectrum shows only a cluster peak (m/z = 477.0637) assigned to the single charged cation [C24H24ClN2102Ru]+. However, in the case of complex (1) both 1H NMR and ESI mass spectra show the presence of hydrolyzed products. More particularly, in the ESI mass spectrum of (1), apart from the cluster peaks of the [(η6-bz)Ru(pqn)Cl]+, low intensity peaks of the double charged cation [(η6-bz)Ru(pqn)]2+ and the single charged [(η6-bz)Ru(pqn)(OH)]+ were observed. In parallel, in the 1H NMR spectrum, new signals of the hydrolyzed product appeared, remaining unchanged after several days and amounting to less than 5% of the total complex concentration (Fig. 5). Regarding the origin of these cations, [(η6-arene)RuL]2+, it has been suggested that similar cations originated from the aqua complexes after dissociation of the water molecule in MS contitions. [14, 31] (PLEASE INSERT FIGURE 5) On the other hand, the hydroxo species were formed from the aqua species, after the dissociation of H , due to the stronger acidic character of the η6-benzene complexes compared to the similar of pcymene (e.g. pKa values for ([(η6-bz)Ru(bpy)(H2O)]2+ = 6.9, ([(η6-cym)Ru(bpy)(H2O)]2+ = 7.2 at 293K [32]. Considering that the chloride counter ion inhibits the hydrolysis reaction, an amount equivalent to one AgNO3 was added to the samples in order to precipitate the chloride as AgCl. Indeed, upon addition of AgNO3 the hydrolysis reaction begins and the 1H NMR spectrum changes, in slow kinetics at 293 K, towards hydrolyzed species. Both complexes replace slowly the Cl, reaching equilibrium after few hours. At this point, one more equivalent of AgNO3 was added, shifting the equilibrium towards the product, as observed from 1H NMR spectra of the compounds, where only one set of signals appeared (Fig. S6, S7). Since the acid-base equilibrium is in fast kinetic in the NMR time scale, the simultaneous presence of hydroxo and aqua species can not be excluded. Indeed, in the ESI-MS of the fully hydrolyzed complexes the cation [(η6bz)Ru(pqn)(OH)]+ and [(η6-bz)Ru(pqn)-H]+ were observed. +

3.2. Characterization of 9-MeG complexes (3)-(7). The HR-ESI mass spectra of (3)-(7) show cluster peaks corresponding to the double charged cations of the complexes and some minor additional peaks that were assigned to products which were formed in the gas phase. Particularly, in the case of complexes (3), (5) and (7), peaks assigned to [Ru(L)(9-MeG)]2+ and [(η6-bz)Ru(L)]2+ (L = pqn, pqx, bpy) were more intense. Comparing the 1H NMR spectra of the complexes (3)-(7) with those of their precursors, four new signals belonging to 9-MeG appeared. The new signals were found to be significantly shifted in comparison to those of free 9-MeG. In general, coordination of 9-MeG through N7, withdrawing electron density from

the nitrogen atom and the neighboring H8 shifted downfield. However this is not observed in the cases of the complexes (3)-(5) and (7). Large upfield shifts were observed at about 0.3-0.8 ppm, depending on the nature of the chelating ligand. 3.2.1 The shielding effect on 9-MeGH8 In order to clarify this phenomenon, a comparison of complexes (3) and (4) with similar complexes was attempted. Fig. 6 presents the induced upfield shifts of 9-MeGH8 for similar ruthenium complexes under the same conditions. (PLEASE INSERT FIGURE 6) It is readily apparent from Fig. 6 that, in the complex with ethylenediamine (en), a downfield shift for 9-MeGH8 was observed as normally expected. However, in the complexes with aromatic diimines (pqn, pqx, bpy) upfield shifts were observed, depending on the length of the aromatic ring system. Ligands with one six-membered aromatic ring (bpy) shield less the 9-MeGH8 compared to those with two fused rings (pqx, pqn). Comparing the Δδ values of the benzene complexes with those of p-cymene, it can be concluded that the η6- coordinated arene has marginally influenced the 9-MeGH8 shielding. However, systematically slightly higher values were observed in all cases of the benzene complexes, reflecting the differences of donor character between these two arenes [30]. Other chelating ligands, such as the acetylacetonate anion, are also causing upfield shifts to the 9EtGH8 (0.15 ppm) in the complex [Ru(η6-cym)(acac)(9-EtG)]2+ [32]. In this case, a hydrogen bond between the enolo C6OH and the oxygen atom of the chelating acetylacetonate ligand was suggested, which may be responsible for the observed shielding of 9-EtGH8. The influence of the ancillary ligands in the chemical shift of 9-EtGH8 was studied for the complexes [Ru(η6-cym)(NH3)(9-EtG)Cl]+ and [Ru(η6-cym)(NH3)(9EtG)2]2+. In both complexes, typical downfield shifts were observed - more particularly, at about 0.12 ppm for the complex [Ru(η6-cym)(NH3)(9-EtG)Cl]+, while the corresponding diguanine adduct was shifted at about 0.3 ppm [33]. A strong shielding (0.88 ppm) of 9-MeGH8 was observed in the case of [Ru(η6cym)(LH)(9-MeG)]2+ (L = 2-(2’-pyridyl)benzimidazole) [34]. In the crystal structure of the complex, hydrogen bonds between the 9-MeGO6 and (i) the benzimidazole aromatic proton H6’, (ii) the p-cymene H5, stabilize the 9-MeG in a way, so that the 9-MeGH8 is orientated towards the benzimidazole ring system. In the NOESY spectra of the complexes (3) and (4), apart from the intra-ligand crosspeaks, crosspeaks between the 9-MeGH8 and the chelating ligand protons were observed (Fig. 7). (PLEASE INSERT FIGURE 7) Inter-ligand NOE crosspeaks between the pqnH8 and 9MeGH8 were detected in the cases of (3) and (4). Such inter-ligand NOE’s (ligandH8 and 9-MeGH8) have also been observed in the complexes (5) and [(η6-cym)Ru(pqx)(9-MeG)]2+ [15], as well as in the case of 9-EtG complex [η6-cym)Ru(bpm)(9-EtG)]2+ (bpm = 2,2’-bipyrimidine) (bpm H6’ and 9EtGH8) [35].The above observations indicate that the purine ring system of guanine is oriented in such a way, that the imidazole H8 faces the aromatic ring of the diimine ligands. In this orientation, the free rotation of the 9-MeG around the Ru-N7 bond should be excluded, hindered mainly by the bulky shape of the ligand. The hindrance of the methyl and isopropyl group of cym is rather marginal, since in the η6-benzene complexes (3) and (5) similar crosspeaks were observed. 3.2.2. The deshielding effect on 9-MeGN1H In all studied complexes (3)-(7), large (+0.5 to +0.8 ppm) downfield shifts were observed for the 9MeGN1H (Table 2). These significant downfield shifts indicate reduction in the electron density of the N1-H

nucleus and a weaker bond between the N1 and H. Metallation of 9-substituted 6-oxopurines through N7 causes an increase by 1.2 - 2.0 log units in the value of the dissociation constant K of the N1-H bond [36]. However, this should be independent of the nature of the chelating ligand, which is not the case. By inspecting carefully the Table 2, it is observed that the magnitude of the downfield shift of N1H follows the upfield shift of H8. Since the H8 shift is related to the proximity of 9-MeGH8 to the aromatic ring system of the chelate ligands, it can be concluded that the shielding on H8 contributes, probably implicitly, to the withdrawal of electron density of N1H. Alternatively, the orientation of 9-MeG is such, so that 9MeGO6 may participate in hydrogen bonding. Interactions of this type withdraw electron density from the bond of N1-H and have been reported several times in the literature. For example, in the complex [(η6cym)Ru(en)(9-EtG)]2+, a strong hydrogen bond ((en)NH…O6(9-EtG) = 1.9 Å) between the protons of the ethylenediamine amino group and the O6 of the carbonyl group of 9-EtG was observed [6]. In order to investigate the influence of this interaction in the chemical shift of N1H, we synthesize the complex (6) and we record the 1H NMR spectrum under the same conditions as the studied complexes. Indeed, the proton signal of the N1H shifted downfield by about 0.80 ppm, confirming that the participation of O6 in hydrogen bonding causes significant deshielding on N1H. In the case of the complexes (4) and [(η6-cym)Ru(pqx)(9-MeG)]2+, the cymH2 shifted significantly downfield upon coordination of 9-MeG (Fig. 7). Also, the pyridine H6’ of the ligands shifted significantly downfield, while the rest of the protons of the ring shifted marginally (Table 2). Considering the magnitude of these downfield shifts (+0.52 to +0.30 ppm), we can suppose that H2 and H6’ may take part in interactions with 9-MeGO6. Similarly, in the case of complex [(η6-cym)Ru(bpy)(9-MeG)]2+ [15] the respective protons, H2 and H6’, show the most downfield shifts (0.18 to 0.26 ppm). However, these are significantly smaller, indicating weaker interactions. (PLEASE INSERT FIGURE 8) On the other hand, in the complexes (3), (5) and (7), the benzene proton signals do not show any noteworthy downfield shift in comparison with their precursors (Table 2). Moreover, in both symmetric and asymmetric ligands, only one singlet peak appears, indicating that all the benzene protons are equal. In other words, the benzene protons do not participate in interactions with the 9-MeGO6. However, the magnitude of the N1H downfield shift is similar to that of the p-cymene analogues, and only in the case of complex (7) the bpyH6’ shifted similarly downfield. Table 2. Differences (Δδ) of 1H chemical shifts for selected protons between the complexes (1), (2), [Ru(η6bz)(pqx)Cl]+ [17], [Ru(η6-cym)(pqx)Cl]+ [17], [Ru(η6-bz)(bpy)Cl]+ [22], [Ru(η6-cym)(bpy)Cl]+ [22], [Ru(η6cym)(en)Cl]+ [4] and (3), (4), (5), [Ru(η6-cym)(pqx)(9-MeG)]2+ [17], (7), [Ru(η6-cym)(bpy)(9-MeG)]+ [17], (6) respectively. The Δδ values for 9-MeG denote the differences between the free and the coordinated 9MeG. Downfield shifts are indicated as (+) changes, whereas upfield as (-). For all complexes the spectra were recorded under the same conditions of temperature (298 K), solvent (dmso-d6) and 1H resonance frequency (400 MHz). Protons

H2 H3 H5 H6 H7

6

6

6

6

6

6

6

[Ru(η -cym) [Ru(η -cym) [Ru(η -cym) [Ru(η -cym) [Ru(η -bz) [Ru(η -bz) [Ru(η -bz) 2+ 2+ 2+ 2+ 2+ 2+ 2+ (pqn)(9-MeG)] (pqx)(9-MeG)] (bpy)(9-MeG)] (en)(9-MeG)] (pqn)(9-MeG)] (pqx)(9-MeG)] (bpy)(9-MeG)] Cymene benzene +0.52 +0.35 +0.23 +0.09 +0.14 +0.10 +0.08 +0.26 +0.08 +0.15 +0.17 +0.30 +0.19 +0.15 +0.17 +0.12 -0.01 +0.23 +0.09 -0.12 -0.17 -0.33 -0.06

Ligand H3 H4 H5 H6 H7 H8 H3’ H4’ H5’ H6’

+0.22 +0.15 +0.19 +0.17 +0.19 -0.02 +0.08 +0.04 +0.05 +0.45

+0.02 +0.18 +0.13 +0.13 -0.03 -0.07 -0.02 +0.01 +0.30

-0.03 +0.01 +0.04 +0.26 -0.03 +0.01 +0.04 +0.26

NH1 H8

+0.71 -0.77

+0.72 -0.83

+0.53 -0.37

9-MeG +0.80 +0.68

-0.02 -0.06 +0.14 +0.10 +0.08 -0.20 -0.01 -0.06 -0.01 +0.16

+0.08 +0.14 +0.08 +0.06 -0.12 +0.01 +0.01 0 +0.12

-0.03 +0.01 +0.04 +0.26 -0.03 +0.01 +0.04 +0.26

+0.76 -0.87

+0.70 -0.88

+0.52 -0.45

4. Conclusions In conclusion, we have demonstrated that coordination of asymmetric chelating aromatic diimine ligands in the complexes of the type [(η6-cym)Ru(diimine)Cl]+ eradicate the chemical equivalence of the pcymene aromatic protons, and their separate 1H NMR signals were assigned with accuracy based on NOE spectroscopy. This allowed us to identify a large downfield shift of cymH2 in [(η6-cym)Ru(pqn)(9-MeG)]2+ and to conclude that it may be involved in interactions with 9MeGO6. Also, the large downfield shift of pqnH6’ upon coordination of 9-MeG may also indicate its involvement in a similar interaction. These interactions, together with the bulky shape of pqn, hinder the free rotation of 9-MeG, forcing the 9-MeGH8 in a close proximity to the pqn aromatic ring system. We support this conclusion by NOE spectroscopy, where crosspeaks between the 9MeGH8 and pqxH8 were observed for both (3) and (4). The influence of the above 9-MeG conformation on the chemical shift of 9-MeGH8 was comparatively studied with similar complexes and we found that it depends on the extension of the aromatic system of the ligand. Also, we found that the magnitude of the strong downfield shift of the 9-MeGNH1 follows the strong upfield shift of 9-MeGH8, leading to the conclusion that this phenomenon is influenced from both the N7 ruthenation of 9MeG and the addendum electron density in the 9-MeG ring system, due to its proximity to the aromatic ring system of pqn. Appendix A. Supplementary data CCDC 1828018 contains the supplementary crystallographic data for the compound (2)PF6. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: [email protected]. Acknowledgements We would like to thank Prof. A. J. Tasiopoulos at the University of Cyprus for facilitating the use of the X-ray diffractometer. Also, we acknowledge the Unit of Environmental, Organic and Biochemical high resolution analysis-ORBITRAP-LC-MS and the NMR Center of the University of Ioannina for providing access to the facilities. 5. References 1. (a) E. Alessio, Eur. J. Inorg. Chem. (2017) 1549. (b) S. Leijen, S.A. Burgers, P. Baas, D. Pluim, M. Tibben, E. van Werkhoven, E. Alessio, G. Sava, J.H. Beijnen, Invest. New Drugs 33 (2014) 201.

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5 6

4 3 2

7

2'

3' 4'

8 5' 6'

Figure 1. The structure of 2,(2΄-pyridyl)quinoline (pqn) with atom numbering

R2

R2

N = pqn Cl

Cl

Cl

N

Ru

Ru

R1

R2

Ru

R1

CH3OH, CH3CN, NH4PF6

Cl

N

Cl N R1 R1 , R2 = H; benzene (bz) R1 = Me, R2 = Pri; = p-cymene (cym)

AgNO3 CH3OH

(1)-(2)

9-MeG R2

2+

NH4PF6

[(η6-bz)Ru(pqx)Cl](PF6)

-20 oC

[(η6 -bz)Ru(bpy)Cl](PF6) [(η6 -cym)Ru(en)Cl](PF6)

-AgCl R1

N Ru O N

N

N

H2N

N

N

CH3 (3)-(7)

Figure 2. Synthetic procedure for the complexes (1)-(7)

+

Figure 3. ORTEP diagram of (2) at 50% probability level. The [PF6]- anion was omitted for clarity. Selected bond distances (Å) and angles (o): Ru-arene(centroid), 1.682(5); Ru-Cl(1), 2.4019(13); Ru-N(1), 2.105(4); RuN(2) 2.063(4); arene(centroid)-Ru-Cl(1), 130.58(14); arene(centroid)-Ru-N(1), 128.68(12); arene(centroid)Ru-N(2), 131.27(11); Cl(1)-Ru-N(1), 85.17(11); Cl(1)-Ru-N(2) 85.61(12); N(1)-Ru-N(2), 77.22(16) .

2

3

7

9 8 10

5

6 6' 5' 8

7 3'

4'

6 5

4

A

3

B

Figure 4. (A) The proposed structure of complex (2) showing the NOE contacts: (i) between the cym protons themselves (red lines), (ii) between the cym aliphatic protons and the ligand pqn (blue arrows) and (iii) between the cym aromatic protons and the ligand pqn (green arrows). (B) Part of the NOESY spectrum of the complex (2) (dmso-d6, 500 MHz, tm = 700 ms, 298 K) showing, in the opposite to diagonal phase, the NOE crosspeaks between the aromatic protons of cymene and the ligand pqn.

Figure 5. (A) Part of the 1H NMR spectrum of (1)Cl after 7 days showing the hydrolyzed product. (B) HR-ESIMS of the same complex.

Figure 6. Differences (Δδ in ppm) in H8 chemical shift for complexes of the type [(η6-arene)Ru(L)(9-MeG)]2+, arene = p-cymene (cym) and benzene (bz), L = 2,2΄-bipyridine (bpy), 2,(2΄-pyridyl)quinoxaline (pqx) and 2,(2΄-pyridyl)quinoline (pqn). Chemical shifts for the complexes, [(η6-cym)Ru(pqx)(9-MeG)](PF6)2 and [(η6cym)Ru(bpy)(9-MeG)](PF6)2 were taken from the reference [15], otherwise the 1HNMR spectra were recorded in the present study.

Figure 7. NOESY spectra of the complexes (A) [(η6-bz)Ru(9-MeG)(pqn)](PF6)2 and (B) [(η6-cym)Ru(9MeG)(pqn)](PF6)2 in dmso-d6, 298 K, 500 MHz and mixing time 700 ms.

Figure 8. Part of 1H NMR spectra of complexes (2) and (4) showing the downfield shifts of the p-cymene aromatic protons upon coordination of 9-MeG.

Complexes of the general formula [(η6-arene)Ru(L)Cl]PF6, were synthesized and characterized by means of spectroscopic and analytical techniques. A comparative NMR study of their 9-methylguanine (9-MeG) complexes with similar diimine complexes revealed that the unimpeded rotation of 9-MeG is hindered by interactions between the 9-MeGO6 and the p-cymene aromatic proton H2 and, by the bulky shape of the chelating ligand.