Tuning the anticancer activity of maltol-derived ruthenium complexes by derivatization of the 3-hydroxy-4-pyrone moiety

Journal of Organometallic Chemistry 694 (2009) 922–929 Contents lists available at ScienceDirect Journal of Organometallic Chemistry journal homepage: www.elsevier.com/locate/jorganchem Tuning the anticancer activity of maltol-derived ruthenium complexes by derivatization of the 3-hydroxy-4-pyrone moiety Wolfgang Kandioller a, Christian G. Hartinger a,b,*, Alexey A. Nazarov a,b,*, Johanna Kasser a, Roland John a, Michael A. Jakupec a, Vladimir B. Arion a, Paul J. Dyson b, Bernhard K. Keppler a a b University of Vienna, Institute of Inorganic Chemistry, Waehringer Str. 42, A-1090 Vienna, Austria Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland a r t i c l e i n f o Article history: Received 12 September 2008 Received in revised form 9 October 2008 Accepted 10 October 2008 Available online 17 October 2008 Dedicated to Prof. Gérard Jaouen on the occasion of his 65th birthday. a b s t r a c t Organometallic ruthenium(II)-arene complexes coordinated to maltol-derived ligands were prepared and their anticancer activity against human tumor cell lines was studied. In addition, their hydrolysis behavior and reaction with 50 -GMP was tested and compared to the parent compound chlorido[2-methyl-3(oxo-jO)-pyran-4(1H)-onato-jO4](g6-p-cymene)ruthenium(II) (Ru-maltol). Improved stability and in vitro anticancer activity at maintained GMP binding capability were observed, in comparison to the Ru-maltol complex. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Antitumor agents Bioorganometallic chemistry Ruthenium(II)-arene complex Pyrone Hydrolysis 50 -GMP binding 1. Introduction Platinum complexes play an important role in the treatment of cancer and are included into ca. 50% of the therapeutic schemes [1]. Due to severe side effects, high toxicity, limited activity in common tumor types and tumor resistance, non-platinum complexes are undergoing intensive research as chemotherapeutics and also as radiodiagnostics [2–7]. Ruthenium compounds are the most widely developed alternatives, and (H2Ind) trans-[RuCl4(HInd)2] (KP1019, HInd = indazole; Fig. 1) and (H2Im) trans-[RuCl4(DMSO)(HIm)] (NAMI-A, HIm = imidazole) have entered clinical trials [4,8,9]. Both compounds exhibit only low side effects, which might be due to selective uptake via the transferrin cycle [10–15] and/or activation by reduction [16] in the reductive environment typical of neoplastic tissue. More recently stable organometallic compounds moved into the focus of interest [5]. Beside, for example, titanium, iron and * Corresponding authors. Address: University of Vienna, Institute of Inorganic Chemistry, Waehringer Str. 42, A-1090 Vienna, Austria. Tel.: +43 1 4277 52609; fax: +43 1 4277 52680 (C.G. Hartinger). E-mail addresses: christian.hartinger@univie.ac.at (C.G. Hartinger), alex.nazarov @univie.ac.at (A.A. Nazarov). 0022-328X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jorganchem.2008.10.016 gold compounds [17–19], some of which underwent clinical trials, Ru(II) species, such as RAPTA type complexes, have been observed to exhibit anticancer activity, tunable by careful selection of the ligand sphere [3,20–32]. These studies resulted in compounds with interesting modes of action, for example potentially targeting kinases [25,29,30], or drug resistance pathways [27,33] and light activated systems [34,35], and a number of encouraging in vivo studies have been reported [3,5,21,22,36,37]. More recently, dinuclear pyridinone-based Ru(II) complexes, such as PyrRu12 2 (Fig. 1), were observed to exhibit increased cytotoxicity due to jointly acting Ru(II)-arene moieties [31,32,38]. By varying the spacer length between the two ruthenium centers, compounds with mild to high cytotoxicity were obtained. Recent studies on their DNA binding properties revealed different modes of action for compounds only varying in the length of the spacer between the Ru(II)-arene fragments [31,32,38]. Other maltol-derived compounds have proven medicinal potential in the treatment of b-thalassemia and diabetes, and as radiodiagnostics [39] and, when coordinated to a Ga(III) center, in chemotherapy [2], or in form of a VO(maltolato) complex as a medication against diabetes type II [40,41]. The coordination ability of maltol-derived ligands with particular afﬁnities for divalent and trivalent metal ions allows very stable complexes to be prepared [31,32,42–45], which can easily be 923 W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929 HN NH N Cl Cl HN Ru Cl Cl N HN H N H N N Cl Cl Ru Cl Cl S O HN O Ru Cl O NAMI-A N ( ) n N KP1019 O O Cl Ru P Cl N N RAPTA-C Ru Cl Ru O Cl O N Fig. 2. 1H NMR of 3d in (a) CDCl3 and (b) D2O. PyrRu2 n (n = 6, 12) O Ru-maltol achieved in a similar fashion to the aldol reaction. Compound 1 was deprotonated with NaOH, and the anionic species obtained was reacted immediately with the corresponding aldehyde, yielding ligands 2a–e after addition of HCl and recrystallization (yield 64–90%). The reaction occurs only in sufﬁcient yield at pH 10.5. The air and light stable complexes (>1 year) 3a–e were obtained by deprotonation of the corresponding ligand with sodium methoxide and addition of the bis[dichlorido(g6-p-cymene)ruthenium(II)]. A slight excess of ligand (10%) was added in order to facilitate puriﬁcation, and all complexes were obtained in good yield (52–73%). The compounds were characterized by IR and 1D and 2D NMR spectroscopy, mass spectrometry and elemental analysis. All complexes possess two stereogenic centers and were isolated as mixtures of two diastereomers (R/R, S/S and R/S, S/R), as observed by NMR spectroscopy in aprotic solvents, where both forms are distinguishable. The separation of the diastereomers by fractional crystallization or chromatography was not successful. As a representative example, the 1H NMR of 3d in CDCl3 and in D2O is shown in Fig. 2. In CDCl3 two sets of signals are observed for the hydrogens of the coordinated arene ring and for the m-ﬂuoro substituted phenyl group. In D2O, the spectrum comprises a single Fig. 1. Structural formulae of the ruthenium anticancer drug candidates KP1019, NAMI-A, RAPTA-C, Ru-maltol and PyrRun2 . varied for modulation of important parameters such as solubility, hydrophobicity, etc. In the present work, the synthesis of mononuclear Ru(II)-arene complexes with novel maltol-derived ligands is reported. The substitution pattern of the aryl part of the ligand was found to be crucial for controlling both the in vitro anticancer activity and complex solubility. 2. Results and discussion 2.1. Synthesis and characterization of the Ru(II)-p-cymene complexes The synthesis of the precursor allomaltol 1 was performed in two steps, starting form kojic acid by reaction with thionylchloride followed by the reduction with zinc under acidic conditions (Scheme 1) [46]. Due to the higher reactivity of position 2 compared to 5 in the pyrone ring of 1, functionalization can easily be O O SOCl2 OH HO Cl O O Zn/HCl OH OH H 2O O O R1 O 1 R2 R3 O Ru Cl OH [RuCl2(cym)]2 O OH O OH O R1 R NaOH / H2 O O 1 R 3 R3 R 2a 2b 2c 2d 2e R1 = R2 = R3 = H R 1 = R 3 = H, R 2 = NO 2 R 1 = R 3 = H, R 2 = F R 1 = R 2 = H, R 3 = F R 1 = R 2 = R 3 = OCH3 2 R2 3a-e 2a-e Scheme 1. Synthesis of pyrone-based ligands and their ruthenium(II)-arene complexes. 924 W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929 set of signals, presumably due to fast inversion of the metal center, leading to epimerization of the diastereomers [47,48]. The 1H NMR signal of the OH proton was observed to be split in aprotic solvents into two doublets with Dd = 0.2 ppm, which may be due to the formation of a hydrogen bond between the aliphatic –OH group of the ligand and the coordinated alcoholate of the pyrone ring (Fig. 2). The IR spectra of the ligands 2a–e show C@O, C@C and C–O stretching bands at 1654–1120 cm1 [49]. The O–H stretching bands were observed as broad bands in the range 3420– 3280 cm1. The spectra of 3a–d contained four bands in the region of 1610–1460 cm1, which are typical for maltolato complexes [50,51]. Upon complexation the C@O stretching bands appear shifted by 40 cm1 to lower wavenumbers. In the case of 3e strong overlapping of the latter signals with those assignable to the methoxy groups was observed. In the spectra of the complexes the bands attributed to the OH stretching are in the range 3400– 3340 cm1 and are of signiﬁcantly lower intensity due to deprotonation and coordination of one of the OH groups to the Ru centers. ESI mass spectra of 3a–e were recorded in methanol in the positive ion mode. For all compounds the most abundant peaks were assigned to [MCl]+ ions, which had the appropriate ruthenium isotopic pattern. 2.2. Crystal structure determination The molecular structure of 3b was determined by X-ray diffraction analysis (Fig. 3), and the metal center was found to adopt the expected pseudo-octahedral ‘‘piano-stool” geometry. In the phenyl substituted maltolato complex 3b and PyrRu62 a short and a long Ru–O bond have been observed (Table 1), whereas in chlorido[3-(oxo-jO)-2-methyl-4-pyridinonato-jO4](g6-p-cymene)ruthenium(II) (Ru-maltol, Fig. 1) the two Ru–O bond lengths are almost identical. This observation might be explained by a lengthening of the Ru–O2 bond in the Ru-maltol complex due to the involvement of this oxygen donor atom in a strong intermolecular H-bond (donor–acceptor distance of 2.805(2) Å, and a donor–hydrogen–acceptor angle of 174°). The Ru–Cl bond length in 3b of 2.4310(4) Å is similar to that in Ru-maltol, whereas the bond observed for the pyridinone complex PyrRu62 is slightly shorter [31]. Although the Ru–Cl bond lenghts in the above mentioned structures are very different, the rate of hydrolysis is rapid in all cases (see below). 2.3. Hydrolysis, pKa and GMP binding Due to the low solubility of 3a–e in water (Table 2), all NMR experiments on the hydrolytic stability were performed in a 5% Table 1 Selected bond lengths (Å) and angles (°) of 3b and comparisons with the dinuclear complex PyrRu62 and the structurally related Ru-maltol complex chlorido[3-(oxo-jO)2-methyl-4-pyronato-jO4](g6-p-cymene)ruthenium(II). Ru–Cl Ru–O1 Ru–O2 O1–Ru–O2 O1–Ru–Cl Cl–Ru–O2 a b 3b PyrRu62 a Ru-maltolb 2.4310(4) 2.1179(10) 2.0766(10) 78.82(4) 83.35(3) 85.51(3) 2.4186(10) 2.101(3) 2.074(3) 79.63(11) 83.90(8) 85.11(8) 2.4329(5) 2.1035(13) 2.0901(13) 78.79(5) 83.42(4) 85.89(4) From Ref. [31]. From Ref. [28]. Table 2 Solubility in PBS and IC50 values of 3a–e and of Ru-maltol in the human cancer cell lines CH1, SW480 and A549. Compound Solubility (mg/mL) 3a 3b 3c 3d 3e Ru-maltol 0.25 Insoluble 0.1 0.1 1 >10 IC50 (lM) CH1 SW480 A549 50 ± 9 – 24 ± 4 29 ± 2 48± 6 >100 67 ± 10 – 44 ± 10 57 ± 8 84 ± 7 >100 172 ± 5 – 98 ± 4 138 ± 6 220 ± 14 – DMSO-d6/D2O solution. Coordination of DMSO to the ruthenium center was not observed (data not shown). NMR spectra of the complexes with and without addition of AgNO3 (used to remove the chlorido ligand) are identical, indicating that the hydrolysis proceeds immediately upon dissolving in water. The aqua complexes are stable in aqueous solution, whereas the Ru-maltol complex reacts to form a dimeric ruthenium compound [28], indicating that the hydroxyl-methyl-aryl substituent in position 2 of the pyrone ring inhibits the formation of dimeric species. The pKa values of the aqua complexes of 3a–e were estimated by titration with NaOD to afford the corresponding hydroxido compounds by monitoring the deprotonation process by 1H NMR spectroscopy (Table 3). To generate the aqua species, the complexes were dissolved in D2O containing 5% DMSO-d6. The chemical shifts of the Arcym-H2/H6 proton signal of the arene ring (e.g., from 5.75 ppm at pH 3.21 to 5.49 at pH 11.00 for 3c) were plotted against the pD value. The pK a values (in D2O) were determined from the inﬂection point of the sigmoid curve (see Fig. 4 for the titration curve of 3c) and corrected for the difference between D2O and water to yield the pKa (using Eq. (1), see Section 4). The pKa values of 3a–e were determined to be between 8.99 and 9.80, with the substitution of the aryl moiety of the ligand having only a minor inﬂuence. The pKa values are similar to those of the parent compound Ru-maltol (pKa 9.23) [28] and the dinuclear pyridinone-type ruthenium arene complexes (pKa 9.60–9.83) [31]. The reaction of 3a–e with the DNA model compound 50 -GMP was investigated by 1H and 31P NMR spectroscopy to evaluate the potential of DNA as an intracellular target, as reported for other Ru complexes and also the clinically established Pt compounds Table 3 pKa values of complexes 3a–e. Fig. 3. ORTEP plot of the molecular structure of 3b at 50% probability level. Compound pKa 3a 3b 3c 3d 3e 9.05 ± 0.02 9.80 ± 0.03 8.99 ± 0.01 9.56 ± 0.01 9.64 ± 0.02 W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929 925 Fig. 6. Concentration–effect curves of 3a and 3c–e in CH1 ovarian carcinoma cells. Fig. 4. Titration curve of 3c used to determine the pKa. [1,16,52,53]. Stepwise addition of 50 -GMP to the aqua complexes (in aqueous solutions) resulted in the formation of 50 -GMP adducts within seconds. After addition of an equimolar amount of the reagent, three new signals for the H8 of 50 -GMP were observed in the 1H NMR spectra between 7.7 and 7.9 ppm in a ratio of 1:1:2 (Fig. 5). The signals may be assigned to the four stereoisomers formed by the coordination of 50 -GMP to the ruthenium center. The change of the H8 signal in the 1H NMR spectrum from 8.15 ppm to approximately 7.80 ppm indicates coordination via the N7 atom of the guanine [53]. The 31P NMR spectrum contains 3 peaks between 3.7 and 3.1 ppm in 1:1:2 ratio in agreement with the 1H NMR spectra. 2.4. In vitro evaluation The in vitro anticancer activity of 3a and 3c–e was determined in SW480 (colon carcinoma), CH1 (ovarian carcinoma) and A549 (non-small cell lung carcinoma) human cancer cells using the colorimetric MTT assay, yielding IC50 values mostly in the 105 M range (Table 2). Due to the low solubility of 3b it was not possible to determine its anticancer activity. CH1 cells were found to be the most sensitive to all four ruthenium complexes, and in all cell lines 3c was the most active compound with an IC50 value of 24 lM in the ovarian cancer cell line (Fig. 6). In general, it appears as the solubility in medium/DMSO as a measure of lipophilicity determines the in vitro anticancer activity of the compounds (Table 2). Furthermore, the substituent on the aryl moiety of the ligand appears to inﬂuence the activity of the ruthenium complex in all tested cell lines: 3c and 3d with their electron withdrawing substituents exhibit a higher cytotoxic activity in CH1 cells than 3a. In contrast, electron-donating substituents such as the methoxy groups in 3e do not signiﬁcantly inﬂuence the activity in CH1 cells and are even disadvantageous in SW480 and A549 cells. In comparison to the parent compound Ru-maltol, the compounds bearing substituents at the 2-position are more cytotoxic, which may also be due to the increased lipophilicity of the compounds (Table 2). However, in order to establish deﬁnitive structure–activity relationships a higher number of compounds need to be studied. 3. Conclusions Ruthenium complexes have been established as potential drug candidates for treatment of cancer. Herein, ruthenium(II)-p-cymene complexes with 2-substituted 3-hydroxypyran-4(1H)-one ligands have been evaluated for potential anticancer activity. The complexes hydrolyze rapidly in aqueous solutions, in a process involving substitution of the chlorido ligand by an aqua ligand. Substitution of the 2-position of the pyrone moiety results in increased stability of the aqua complexes in aqueous solution in comparison to Ru-maltol which affords dimeric species. The reaction of the Ru moiety toward 50 -GMP is very fast, and binding occurs selectively at the N7 of the guanine. Complexes 3a and 3c–e exhibit moderate cytotoxicity against SW480 and CH1 human tumor cell lines, and poor activity against A549 cells, suggesting a certain degree of selectivity. The aryl moiety seems to be relevant for determining the in vitro activity of the compounds with electron withdrawing substituents at the phenyl moiety decreasing the IC50 value and electron donating groups having the opposite effect. There appears to be a correlation between the electronic effect of the substituents on the maltol-derived ligands, and the in vitro activity, but a larger library of compounds should be studied to conﬁrm this hypothesis. 4. Experimental 4.1. Materials and methods Fig. 5. 50 -GMP binding of 3d studied by 1H NMR spectroscopy after addition of (a) 0 eq, (b) 1 eq, and (c) an excess of 50 -GMP to a D2O solution of the Ru complex. All solvents were dried and distilled prior to use. Ruthenium(III) chloride (Johnson Matthey), kojic acid (Fluka), benzaldehyde (Fluka), p-nitrobenzaldehyde (Aldrich), p-ﬂuorobenzaldehyde (Fluka), m-ﬂuorobenzaldehyde (Fluka), 3,4,5-trimethoxybenzaldehyde (Aldrich) and sodium methoxide (Aldrich) were purchased and used without further puriﬁcation. Bis[dichlorido(g6-pcymene)ruthenium(II)], 2-chloromethyl-5-hydroxypyran-4(1H)one (chlorokojic acid), and 5-hydroxy-2-methyl-pyran-4(1H)-one 926 W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929 (allomaltol, 1) were synthesized as described elsewhere [46,54]. Melting points were determined with a Büchi B-540 apparatus and are uncorrected. Elemental analyses were carried out with a Perkin Elmer 2400 CHN Elemental Analyzer at the Microanalytical Laboratory of the University of Vienna. NMR spectra were recorded at 25 °C on a Bruker FT-NMR spectrometer Avance IIITM 500 MHz at 500.10 MHz (1H), 125.75 MHz (13C) and 202.44 MHz (31P) in DMSO-d6, D2O or CDCl3. The 2D NMR spectra were measured in a gradient-enhanced mode. An esquire3000 ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany), equipped with an orthogonal ESI ion source, was used for MS measurements. The solutions were introduced via ﬂow injection using a Cole-Parmer 74900 single-syringe infusion pump (Vernon Hills, IL). The ESIMS instrument was controlled by means of the ESQUIRECONTROL software (version 5.2), and all data were processed using DATAANALYSIS software (version 3.2) (both Bruker Daltonics). IR spectra were measured in KBr matrix (4000–400 cm1) with a Bruker Vertex 70 FT-IR spectrometer. Single crystals of 3b were grown from MeOH, and X-ray diffraction measurement was performed on a Bruker X8 APEXII CCD diffractometer at 100 K. The single crystal was positioned at 40 mm from the detector, and 2965 frames were measured, each for 3 s over 1° scan width. The data were processed using the SAINT software package [55]. Crystal data, data collection parameters, and structure reﬁnement details are given in Table 4. The structure was solved by direct methods and reﬁned by full-matrix leastsquares techniques. Non-hydrogen atoms were reﬁned with anisotropic displacement parameters. H atoms were inserted at calculated positions and reﬁned with a riding model. The following computer programs were used: structure solution, SHELXS-97 [56]; reﬁnement, SHELXL-97 [57]; molecular diagrams, ORTEP-3 [58]; computer, Pentium IV; scattering factors [59]. Table 4 Crystal data and details of data collection for 3b. Chemical formula M (g mol1) Temperature (K) Crystal size (mm) Crystal color, shape Crystal system Space group a (Å) b (Å) c (Å) a (°) b (°) c (°) V (Å3) Z Dc (g cm3) l (cm1) F(0 0 0) h Range for data collection (°) h range k range l range Number of reﬂections used in reﬁnement Number of parameters Rint R1a wR2b GOFc Residuals (e Å3) C23H24ClNO6Ru 546.95 100(2) 0.30 0.30 0.20 Orange, block Triclinic (No 2) P1 7.4276(4) 12.5755(5) 12.6091(6) 72.009(2) 87.811(3) 74.210(2) 1076.55(9) 2 1.687 8.94 556 2.04–30.07 10/10 17/17 17/17 6280 295 0.0354 0.0212 0.0532 1.007 0.592, 0.490 P P R1 = ||Fo| |Fc||/ |Fo|. P P 2 wR2 = { [w(Fo F2c)2]/w (F2o)2]}1/2. P c 2 2 2 GOF = { [w(Fo Fc ) ] /(n p)}1/2, where n is the number of reﬂections and p is the total number of parameters reﬁned. a 4.2. Synthesis 4.2.1. General procedure for the reaction of allomaltol with the aldehydes Allomaltol 1 (1 eq) and NaOH (1.1 eq) were dissolved in water and stirred for 5 min. Afterwards, the aldehyde (1.1 eq) was added dropwise to the reaction mixture. The solution was adjusted to pH 10.5 with 5 M NaOH solution and stirred at r.t. for 12 h. The reaction mixture was acidiﬁed to pH 1 with conc. HCl and the resulting precipitate was collected by ﬁltration. If no precipitation occurred, the reaction mixture was extracted with CH2Cl2 (3 20 mL). The combined organic layers were washed twice with saturated NaHCO3 (30 mL) and water (30 mL), dried over Na2SO4, ﬁltered and concentrated in vacuo. The crude product was puriﬁed by recrystallization. 4.2.1.1. 2-(Hydroxy-phenyl-methyl)-3-hydroxy-6-methyl-pyran-4(1H)one (2a). The reaction was performed according to the general procedure using 1 (2.0 g, 15.8 mmol) and benzaldehyde (1.8 mL, 17.5 mmol). The crude product was recrystallized from 2-propanol affording a white powder (3.3 g, 90%). M.p. 170–172 °C (decomp.); 1 H NMR (DMSO-d6) d: 2.18 (s, 3H, CH3), 5.99 (d, J = 4.5 Hz, 1H, CHOHPh), 6.14 (d, J = 5.0 Hz, 1H, CHOHPh), 6.19 (s, 1H, CH), 7.27 (t, J = 7.5 Hz, 1H, Ph-H40 ), 7.35 (t, J = 7.5 Hz, 2H, Ph-H30 /H50 ), 7.40 (d, J = 7.5 Hz, 2H, Ph-H20 /H60 ); 13C NMR (DMSO-d6) d: 19.7 (CH3), 66.3 (CHPhOH), 111.6 (CH3C@CH), 126.4 (Ph-C20 ), 127.8 (Ph-C40 ), 128.7 (Ph-C30 ) 141.0 (CH3C@CH), 141.8 (Ph-C10 ), 151.0 (HOCHC@COH), 165.0 (CHOH), 174.3 (C@O); IR (KBr, cm1, selected bands): 3323, 1654, 1607, 1562, 1256, 1209; Elemental Anal. Calc. for C13H12O4 0.2H2O: C, 66.20; H, 5.30. Found: C, 66.24; H, 5.33%. 4.2.1.2. 2-[(4-Nitrophenyl)-hydroxy-methyl]-3-hydroxy-6-methyl-pyran4(1H)-one (2b). The reaction was performed according to the general procedure using 1 (1.00 g, 7.9 mmol) and 4-nitrobenzaldehyde (1.32 g, 8.7 mmol, in 2 mL dioxane). The crude product was puriﬁed by recrystallization from 2-propanol affording yellow crystals (1.41 g, 64%). M.p. >200 °C decomp.; 1H NMR (DMSO-d6) d: 2.17 (s, 3H, CH3), 6.13 (s, 1H, CHOHAr), 6.21 (s, 1H, CH), 6.50 (brs, 1H, CHOHAr), 7.66 (d, J = 8.8 Hz, 2H, Ar-H20 /H60 ), 8.23 (d, J = 8.6 Hz, 2H, ArH30 /H50 ), 9.34 (s, 1H, COH); 13C NMR (DMSO-d6) d: 20.1 (CH3), 66.2 (CHOHAr), 112.1 (CH3C@CH), 124.4 (Ar-C20 /C60 ), 128.0 (Ar-C30 /C50 ), 141.9 (CH3C@CH), 147.7 (Ar-C10 ), 149.8 (Ar-C40 ), 150.2 (HOCHC@COH), 165,6 (CHOH), 174,7 (C@O); IR (KBr, cm1, selected bands): 3281, 1650, 1606, 1563, 1347, 1220; Elemental Anal. Calc. for C13H11NO6 0.1H2O: C, 55.95; H, 4.04; N, 5.01. Found: C, 55.90; H, 3.98; N, 4.98%. 4.2.1.3. 2-[(4-Fluorophenyl)-hydroxy-methyl]-3-hydroxy-6-methylpyran-4(1H)-one (2c). The reaction was performed according to the general procedure using 1 (1.00 g, 8.0 mmol) and 4-ﬂuorobenzaldehyde (1.08 g, 8.8 mmol, in 1 mL dioxane), affording colorless crystals (1.80 g, 91%). M.p. 158–160 °C; MS (ESI) m/z 249 [MH]; 1 H NMR (DMSO-d6) d: 2.19 (s, 3H, CH3), 5.99 (s, 1H, CHOHAr), 6.20 (s, 1H, CH), 6.19 (brs, 1H, CHOHAr), 7.18 (t, J = 9.0 Hz, 2H, Ar-H30 / H50 ), 7.43 (dd, J = 9.0 Hz, J = 3.0 Hz, Ar-H20 /H60 ), 9.10 (brs, 1H, COH); 13C NMR (DMSO-d6) d: 19.7 (CH3), 66.7 (CHOHAr), 111.6 (CH3C@CH), 115.5 (J = 21.5 Hz, Ar-C30 ), 128.4 (J = 8.1 Hz, Ar-C20 ), 138.0 (J = 3.0 Hz, Ar-C10 ), 141.0 (CH3C@CH), 150.7 (HOCHC@COH), 161.9 (J = 242.0 Hz, Ar-C40 ), 165.1 (CHOH), 174.3 (C@O); IR (KBr, cm1, selected bands): 3293, 1652, 1607, 1564, 1508, 1219; Elemental Anal. Calc. for C13H11FO4 1=4 H2O: C, 61.30; H, 4.55. Found: C, 61.56; H, 4.39%. b 4.2.1.4. 2-[(3-Fluorophenyl)-hydroxy-methyl]-3-hydroxy-6-methylpyran-4(1H)-one (2d). The reaction was performed according to W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929 the general procedure using 1 (0.50 g, 4.0 mmol) and 3-ﬂuorobenzaldehyde (0.54 g, 4.4 mmol, in 1 mL dioxane) affording colorless crystals (0.85 g, 85%). M.p. 170–172 °C; 1H NMR (DMSO-d6) d: 2.27 (s, 3H, CH3), 6.01 (s, 1H, CHOHAr), 6.20 (s, 1H, CH), 6.40 (brs, CHOHAr) 7.10 (m, Ar-H40 ), 7.18–7.21 (m, 2H, Ar-H20 /H60 ), 7.38 (q, J = 8.0 Hz, Ar-H50 ), 9.15 (brs, 1H, COH); 13C NMR (DMSOd6) d: 19.7 (CH3), 66.8 (CHOHAr), 111.7 (CH3C@CH) 113.0 (J = 22.1 Hz, Ar-C20 ), 114.6 (J = 21.3 Hz, Ar-C40 ), 122.4 (J = 3.0 Hz, Ar-C60 ), 130.7 (J = 8.2 Hz, Ar-C50 ) 141.2 (CH3C@CH), 144.8 (J = 8.3 Hz, Ar-C10 ), 150.4 (HOCHC@COH), 162.0 (J = 242.1 Hz, ArC30 ), 165.1 (CHOH), 174.3 (C@O); IR (KBr, cm1, selected bands): 3293, 1655, 1609, 1561, 1252, 1208; Elemental Anal. Calc. for C13H11FO4 1=4 H2O: C, 61.30; H, 4.55. Found: C, 61.30; H, 4.45%. 4.2.1.5. 2-[(3,4,5-Trimethoxyphenyl)-hydroxy-methyl]-3-hydroxy-6methyl-pyran-4(1H)-one (2e). The reaction was performed according to the general procedure using 1 (0.40 g, 3.2 mmol) and 3,4,5-trimethoxybenzaldehyde (0.68 g, 3.5 mmol, in 5 mL dioxane) affording a pale yellow solid (0.78 g, 69%). M.p. 182– 184 °C; 1H NMR (DMSO-d6) d: 2.22 (s, 3H, CH3), 3.64 (s, 3H, 40 OCH3), 3.76 (s, 6H, 30 /50 -OCH3), 5.95 (d, J = 5.2 Hz, 1H, CHOHAr), 6.15 (d, J = 5.2 Hz, 1H, CHOHAr), 6.20 (s, 1H, CH), 6.71 (s, 2H, ArH30 /H50 ), 9.05 (brs, 1H, COH); 13C NMR (DMSO-d6) d: 19.7 (CH3), 56.3 (30 /50 -OCH3), 60.5 (40 -OCH3) 66.4 (CHOHAr), 103.7 (Ar-C20 / C60 ), 111.6 (CH3C@CH), 137.2 (Ar-C10 ), 137.4 (Ar-C40 ), 140.0 (CH3C@CH), 150.8 (HOCHC@COH), 153.2 (Ar-C30 /C50 ), 165.0 (CHOH), 174.3 (C@O); IR (KBr, cm1, selected bands): 3416, 3293, 1646, 1600, 1558, 1226, 1126; Elemental Anal. Calc. for C16H18O7 1=4 H2O: C, 58.80; H, 5.71. Found: C, 58.84; H, 5.42%. 4.2.2. General procedure for the synthesis of the Ru(II) complexes The maltol-derived ligand (0.73 mmol) and sodium methoxide (43 mg, 0.80 mmol) were dissolved in methanol (15 mL) and stirred for 5 min under inert atmosphere to give a clear solution. After(200 mg, wards, bis[dichlorido(g6-p-cymene)ruthenium(II)] 0.33 mmol) was dissolved in CH2Cl2 (5 mL) and added dropwise to the reaction mixture which was stirred for further 5 (for 3a) or 18 h. The reaction mixture was concentrated in vacuo, and the residue was extracted with CH2Cl2 (3 15 mL). The combined organic layers were ﬁltered, and the solvent was removed. The crude product was puriﬁed by recrystallization or precipitation. 4.2.2.1. Chlorido[2-(hydroxy-phenyl-methyl)-6-methyl-3-(oxo-jO)(3a). The pyran-4(1H)-onato-jO4](g6-p-cymene)ruthenium(II) reaction was performed according to the general complexation protocol using 2a (168 mg, 0.73 mmol). The crude product was recrystallized from EtOAc/n-hexane affording a red crystalline solid (240 mg, 73%). M.p. 160–165 °C decomp.; MS (ESI+) m/z 391 [MCl]+; 1H NMR (CDCl3) d: 1.30–1.38 (m, 6H, CH3), 2.19 (s, 3H, CH3,pyr), 2.33 (s, 3H, CH3,cym), 2.93 (m, 1H, CH(CH3)2,cym), 5.28– 5.33 (m, 2H, Arcym-H3/H5), 5.52–5.57 (m, 2H, Arcym-H2/H6), 5.81 (s, 1H, CHOHPh), 5.86 (s, 1H, CHOHPh), 6.27 (s, 1H, CH), 7.30– 7.50 (m, 5H, Ph); 13C NMR (CDCl3) d: 18.6 (CH3), 19.8 (CH3cym), 22.3 (CH3,cym), 31.2 (CH(CH3)2), 72.9 (CHOHPh), 78.2 (Arcym-C3/ C5), 80.2 (Arcym-C2/C6), 95.8 (Arcym-C4), 99.5 (Arcym-C1), 109.4 (CH), 127.1 (Ph-C2), 128.0 (Ph), 128.5 (Ph), 141.5 (CH3C@CH), 152.9 (Ph-C1), 155.1 (HOCHC@COH), 164.2 (CHOH), 185.0 (C@O); IR (KBr, cm1, selected bands): 3394, 1604, 1561, 1503, 1476, 1259, 1206; Elemental Anal. Calc. for C23H25ClO4Ru: C, 55.03; H, 5.02. Found: C, 54.73; H, 5.02%. 4.2.2.2. Chlorido{2-[(4-nitrophenyl)-hydroxy-methyl)]-6-methyl3-(oxo-jO)-pyran-4-(1H)-onato-jO4}(g6-p-cymene)ruthenium(II) (3b). The reaction was performed according to the general complexation protocol using 2b (197 mg, 0.73 mmol). The crude product was recrystallized from EtOAc/diethyl ether/n-hexane 927 affording an orange powder (250 mg, 70%). M.p. 180–185 °C decomp.; MS (ESI+) m/z 512 [MCl]+; 1H NMR (CDCl3) d: 1.31–1.40 (m, 6H, CH3,cym), 2.19 (s, 3H, CH3,pyr), 2.34 (s, 3H, CH3,cym), 2.92 (m, 1H, CH(CH3)2), 5.29–5.35 (m, 2H, Arcym-H3/H5); 5.51–5.58 (m, 2H, Arcym-H2/H6), 5.93 (bs, 1H, CHOHAr), 5.99 (bs, 1H, CHOHAr), 6.30 (s, 1H, CH), 7.67–7.70 (m, 2H, Arcym-H2/H6), 8.20–8.25 (m, 2H, Arcym-H30 /H50 ); 13C NMR (CDCl3) d: 18.6 (CH3,pyr), 19.9 (CH3,cym), 22.5 (CH3,cym), 31.2 (CH(CH3)2), 71.7 (CHOHAr), 78.0 (Arcym-C3/C5), 80.3 (Arcym-C2/C6), 95.7 (Arcym-C4), 99.4 (Arcym-C1), 109.5 (CH), 123.6 (Ar-C2/C6), 127.5 (Ar-C3/C5), 148.0 (Ar-C1), 148.6 (Ar-C4), 151.0 (CH3C@CH), 155.5 (HOCHC@COH), 164.7 (CHOH), 185.2 (C@O); IR (KBr, cm1, selected bands): 3342, 1603, 1564, 1513, 1477, 1256, 1205; Elemental Anal. Calc. for C23H24ClNO6Ru: C, 50.51; H, 4.42; N, 2.56. Found: C, 50.35; H, 4.39; N, 2.51%. 4.2.2.3. Chlorido{2-[(4-ﬂuorophenyl)-hydroxy-methyl)]-6-methyl-3(oxo-jO)-pyran-4-(1H)-onato-jO4}(g6-p-cymene)ruthenium(II) (3c). The reaction was performed according to the general complexation protocol using 2c (183 mg, 0.73 mmol). The crude product was recrystallized from EtOAc/diethyl ether/n-hexane, affording an orange powder (240 mg, 71%). M.p. 90–95 °C decomp.; MS (ESI+) m/z 485 [MCl]+; 1H NMR (CDCl3) d: 1.30–1.38 (m, 6H, CH3,cym), 2.20 (s, 3H, CH3,pyr), 2.33 (s, 3H,CH3,cym), 2.92 (m, 1H, CH(CH3)2), 5.28–5.33 (m, 2H, Arcym-H3/H5), 5.53–5.57 (m, 2H, Arcym-H2/H6), 5.76 (bs, 1H, CHOHAr), 5.89 (bs, 1H, CHOHAr), 6.28 (s, 1H, CH), 6.99–7.07 (m, 2H, Ar-H3/H5), 7.46–7.48 (m, 2H, Ar-H2/H6); 13C NMR (CDCl3) d: 18.6 (CH3,pyr), 19.8 (CH3,cym), 22.4 (CH3,cym), 31.2 (CH(CH3)2), 72.2 (CHOHAr), 78.1 (Arcym-C3/C5), 80.2 (Arcym-C2/C6), 95.7 (Arcym-C4), 99.3 (Arcym-C1), 109.4 (CH), 115.3 (J = 22.3 Hz, Ar-C3/C5), 128.5 (J = 8.0 Hz, Ar-C2/C6), 137.3 (J = 4.1 Hz, Ar-C1), 152.2 (CH3C@CH), 155.2 (HOCHC@COH), 162.5 (J = 245.7 Hz, Ar-C4), 164.0 (CHOH), 185.0 (C@O); IR (KBr, cm1, selected bands): 3387, 1603, 1563, 1507, 1477, 1219; Elemental Anal. Calc. for C23H24ClFO4Ru: C, 53.13; H, 4.65. Found: C, 52.86; H, 4.62%. 4.2.2.4. Chlorido{2-[(3-ﬂuorophenyl)-hydroxy-methyl)]-6-methyl3-(oxo-jO)-pyran-4-(1H)-onato-jO4}(g6-p-cymene)ruthenium(II) (3d). The reaction was performed according to the general complexation protocol using 2d (183 mg, 0.73 mmol). The crude product was recrystallized from EtOAc/diethyl ether/n-hexane affording an orange powder (250 mg, 73%). M.p. 160–165 °C decomp.; MS (ESI+) m/z 485 [MCl]+; 1H NMR (CDCl3) d: 1.30–1.38 (m, 6H, CH3,cym), 2.20 (s, 3H, CH3,pyr), 2.33 (s, 3H, CH3,cym), 2.92 (m, 1H, CH(CH3)2), 5.30 (m, 2H, Arcym-H3/H5), 5.54 (m, 2H, Arcym-H2/H6), 5.83 (brs, 1H, CHOHAr), 6.01 (brs, 1H, CHOHAr), 6.28 (s, 1H, CH), 6.99 (m, 1H, Ar-H4), 7.22–7.33 (m, 3H, Ar-H2/H5/ H6); 13C NMR (CDCl3) d: 18.6 (CH3,pyr), 19.8 (CH3,cym), 22.2 (CH3,cym), 31.2 (CH(CH3)2), 72.4 (CHOHAr), 78.1 (Arcym-C3/C5), 80.2 (Arcym-C2/C6), 95.7 (Arcym-C4), 99.4 (Arcym-C1), 109.4 (CH), 113.9 (J = 22.3 Hz, Ar-C2), 114.7 (J = 22.2 Hz, Ar-C4), 122.3 (J = 3.0 Hz, Ar-C6), 129.9 (J = 7.4 Hz, Ar-C5), 144.0 (J = 7.2 Hz, ArC1), 152.1 (CH3C@CH), 155.2 (HOCHC@COH), 162.8 (J = 262.4 Hz, Ar-C3), 164.2 (CHOH), 185.0 (C@O); IR (KBr, cm1, selected bands): 3396, 1604, 1562, 1504, 1476, 1250, 1203; Elemental Anal. Calc. for C23H24ClFO4Ru: C, 53.13; H, 4.65. Found: C, 53.11; H, 4.65%. 4.2.2.5. Chlorido{2-[(3,4,5-trimethoxyphenyl)-hydroxy-methyl)]6-methyl-3-(oxo-jO)-pyran-4-(1H)-onato-jO4}(g6-p-cymene)ruthenium(II) (3e). The reaction was performed according to the general complexation protocol using 2e (234 mg, 0.73 mmol. The crude product was recrystallized from EtOAc/n-hexane, affording an orange powder (200 mg, 54%). M.p. 160–165 °C decomp.; MS (ESI+) m/z 557 [MCl]+; 1H NMR (CDCl3) d: 1.37–1.39 (m, 6H, CH3,cym), 2.22 (s, 3H, CH3,pyr), 2.32 (s, 3H, CH3,cym), 2.93 (m, 1H, CH(CH3)2), 928 W. Kandioller et al. / Journal of Organometallic Chemistry 694 (2009) 922–929 3.82 (s, 3H, 40 -OCH3), 3.89 (s, 6H, 30 /50 -OCH3) 5.29–5.31 (m, 2H, Arcym-H3/H5), 5.89 (m, 1H, CHOHAr), 6.30 (s, 1H, CH), 6.72 (s, 2H, Ar-H2/H6); 13C NMR (CDCl3) d: 18.6 (CH3,pyr), 19.8 (CH3,cym), 22.3 (CH3,cym), 31.2 (CH(CH3)2), 56.4 (30 /50 -OCH3), 60.7 (40 -OCH3), 71.8 (CHOHAr), 77.8 (Arcym-C3/C5), 80.5 (Arcym-C2/C6), 95.4 (Arcym-C4), 99.0 (Arcym-C1), 104.3 (Ar-C2/C6), 109.4 (CH), 136.8 (Ar-C1), 137.8 (Ar-C4), 152.8 (CH3C@CH), 153.3 (Ar-C3/C5), 155.2 (HOCHC@COH), 164.1 (CHOH), 185.0 (C@O); IR (KBr, cm1, selected bands): 3383, 1603, 1567, 1510, 1473, 1417, 1326, 1238, 1124; Elemental Anal. Calc. for C26H31ClO7Ru: C, 52.75; H, 5.28. Found: C, 52.80; H, 5.32%. 4.3. GMP binding Complexes 3a–e (1–2 mg/mL) were dissolved in D2O containing 5% DMSO-d6. The solution was titrated with a 50 -GMP solution (10 mg/mL) in 50 lL increments, and the reaction was monitored by 1H and 31P NMR spectroscopy until unreacted 50 -GMP was detected. DMSO were diluted in complete culture medium such that the maximum DMSO content did not exceed 1% (this procedure yielded opaque but colloidal solutions from which no precipitates could be separated by centrifugation). These dilutions were added in 200 lL aliquots to the microcultures after removal of the preincubation medium, and cells were exposed to the test compounds for 96 h. At the end of exposure, all media were replaced by 100 lL/ well RPMI1640 culture medium (supplemented with 10% heatinactivated fetal bovine serum and 4 mM L-glutamine) plus 20 lL/well MTT solution in phosphate-buffered saline (5 mg/mL). After incubation for 4 h, the supernatants were removed, and the formazan crystals formed by vital cells were dissolved in 150 lL DMSO per well. Optical densities at 550 nm were measured with a microplate reader (Tecan Spectra Classic), using a reference wavelength of 690 nm. The quantity of vital cells was expressed in terms of T/C values by comparison to untreated control microcultures, and 50% inhibitory concentrations (IC50) were calculated from concentration–effect curves by interpolation. Evaluation is based on means from three independent experiments, each comprising six replicates per concentration level. 4.4. pKa determination Complexes 3a–e were dissolved in D2O containing 5% DMSO-d6. The pH value was measured directly in the NMR tubes with an Eco Scan pH6 pH meter equipped with a glass-micro combination pH electrode (Orion 9826BN) and calibrated with standard buffer solutions of pH 4.00, 7.00 and 10.00. The pH titration was performed by addition of NaOD (0.4–0.0004% in D2O) and DNO3 (0.4–0.0004% in D2O). The observed shifts of the Arcym-H2/H6 protons of the arene ring in the 1H NMR spectra were plotted against the pH value, and the obtained curves were ﬁtted using the Henderson-Hasselbalch equation with Excel software (MicrosoftÒ Ofﬁce Excel 2003, SP3, Microsoft Corporation). The experimentally obtained pK a values were corrected with Eq. (1) [60], in order to convert the pK a in D2O to corresponding pKa values in aqueous solutions. pK a ¼ 0:929pK a þ 0:42 ð1Þ 4.5. Cytotoxicity in cancer cell lines 4.5.1. Cell lines and conditions CH1 cells originate from an ascites sample of a patient with a papillary cystadenocarcinoma of the ovary and were a generous gift from Lloyd R. Kelland, CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK. SW480 (adenocarcinoma of the colon) and A549 (non-small cell lung cancer) cells were kindly provided by Brigitte Marian (Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Austria). All cell culture reagents were obtained from Sigma–Aldrich Austria. Cells were grown in 75 cm2 culture ﬂasks (Iwaki) as adherent monolayer cultures in Minimal Essential Medium (MEM) supplemented with 10% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 4 mM L-glutamine and 1% non-essential amino acids (100). Cultures were maintained at 37 °C in a humidiﬁed atmosphere containing 5% CO2. 4.5.2. MTT assay conditions Cytotoxicity was determined by the colorimetric MTT (3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, purchased from Fluka) microculture assay. For this purpose, cells were harvested from culture ﬂasks by trypsinization and seeded into 96well microculture plates (Iwaki). Cell densities of 1.5 103 cells/ well (CH1), 2.5 103 cells/well (SW480) and 4 103 cells/well (A549) were chosen in order to ensure exponential growth throughout drug exposure. Cells were allowed to settle in drug-free complete culture medium for 24 h. Stocks of the test compounds in Acknowledgments We thank the University of Vienna, the Hochschuljubiläumsstiftung Vienna (H1556-2006), the Theodor-Körner-Fonds, the Austrian Council for Research and Technology Development, the FFG – Austrian Research Promotion Agency (Project FA 526003), the FWF – Austrian Science Fund (Schrödinger Fellowship J2613N19 [C.G.H.], project P18123–N11), the EPFL, and COST D39 for ﬁnancial support. We gratefully acknowledge Alexander Roller for collecting and reﬁning the X-ray diffraction data and Prof. Markus Galanski for recording the NMR spectra. Appendix A. Supplementary material CCDC 701753 contains the supplementary crystallographic data for 3b. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jorganchem.2008.10.016. References [1] M.A. Jakupec, M. Galanski, V.B. 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