CHEMMEDCHEM FULL PAPERS DOI: 10.1002/cmdc.201402001

Structure–Activity Relationship of TrifluoromethylContaining Metallocenes: Electrochemistry, Lipophilicity, Cytotoxicity, and ROS Production Marcus Maschke,[a] Hamed Alborzinia,[b] Max Lieb,[a] Stefan Wçlfl,[b] and Nils Metzler-Nolte*[a] We report the synthesis of trifluoromethylated metallocenes (M = Fe, Ru) and related metal-free compounds for comparison of their biological properties with the aim to establish structure–activity relationships toward the anti-proliferative activity of this compound class. All new compounds were comprehensively characterized by NMR spectroscopy (1H, 13C, 19F), mass spectrometry, IR spectroscopy, and elemental analysis. A single-crystal X-ray structure was obtained on the Ru derivative, 1-(1-hydroxy-1-hexafluoromethylethyl)ruthenocene (3). The cytotoxicity of all compounds was tested on MCF-7, HT-29, and PT-45 cells, and IC50 values as low as 12 mm were observed. Both the metallocene moiety and the hydroxy function are crucial for cytotoxicity. In addition, the activity decreased sharply even if only one trifluoromethyl group was replaced with a methyl group. Electrochemical investigations by cyclic voltammetry revealed that all CF3-containing compounds are

harder to oxidize than the unsubstituted metallocenes. Moreover, log P determination by RP-HPLC showed the fluorinated derivatives to have higher lipophilicity, with log P values up to 4.6. At the same time, the generation of reactive oxygen species (ROS) in Jurkat cells by these compounds was investigated by flow cytometry. Strong ROS production was shown exclusively for the bis-CF3 derivative 1-(1-hydroxy-1-hexafluoromethylethyl)ferrocene (1) after 6 and 24 h. Also on the Jurkat cell line, only compound 1 strongly induces necrosis after 24 and 48 h, as shown by annexin V/propidium iodide staining. No induction of apoptosis was observed. We propose that compound 1 is more efficiently incorporated into cancer cells relative to all other derivatives, causing significant induction of oxidative stress within the cell, which ultimately leads to cell death.

Introduction Chemotherapeutic agents and antibiotics based on metallocenes, especially ferrocene derivatives, have attracted considerable attention over the past decades.[1] Among these compounds, fluorinated species are nearly unknown.[2] On the other hand, fluorinated compounds such as fludarabine and ciprofloxacin are among the most successful commercially available chemotherapeutic agents and antibiotics.[3] Many reactions that are designed for the incorporation of fluorinated groups involve the use of strongly acidic and/or oxidizing conditions.[4] Therefore, most of these reactions cannot be used on metallocene systems such as ferrocene or ruthenocene, which are prone to protonation and decomposition by oxidation of the metal center. Accordingly, reaction schemes without oxidizing or acidic conditions must be chosen. Two suitable reactions are the direct attack of electrophiles [a] M. Maschke, Dr. M. Lieb, Prof. Dr. N. Metzler-Nolte Lehrstuhl fr Anorganische Chemie I—Bioanorganische Chemie Fakultt fr Chemie und Biochemie, Ruhr-Universitt Bochum Universittsstraße 150, 44801 Bochum (Germany) Fax: (+ 49) (0) 234 3214378 E-mail: [email protected] [b] Dr. H. Alborzinia, Prof. Dr. S. Wçlfl Institut fr Pharmazie und Molekulare Biotechnologie Ruprecht-Karls-Universitt Heidelberg Im Neuenheimer Feld 364, 69120 Heidelberg (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201402001.

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(pseudo-Friedel–Crafts acylation) and lithiation at low temperature followed by attack of fluorinated electrophiles. Both reaction types are well established, high yielding, and reliable. Moreover, the reaction conditions are mild and non-oxidizing relative to the use of classical fluorinating agents such as sulfur tetrafluoride. Herein we report the synthesis, characterization, and biological evaluation of a series of fluorine-substituted metallocenes. In a previous work we described the synthesis and biological evaluation of novel trifluoromethylated metallocene triazoles.[2b] These compounds exhibit higher resistance toward oxidation and greater lipophilicity than the unsubstituted metallocene scaffold. Moreover, we observed only moderate cytotoxicity with these compounds, but noted the importance of the metal center. To investigate which part of these molecules is primarily responsible for the anti-proliferative effect, we have now synthesized compounds 1–4 with trifluoromethyl groups bound directly to the metallocene scaffold (Scheme 1), starting from lead compound 1. Compound 1 results from addition of hexafluoroacetone (HFA) to ferrocene. To determine the importance of the hydroxy group for cytotoxic activity, it was protected with a methyl group (compound 2). We also investigated the dependence of the cytotoxic effect on the presence of CF3 groups by substituting them stepwise by methyl groups (4 and 5) and lastly by two hydrogen atoms (compound 6). To elaborate on the influence of the metal atom we synthesized ChemMedChem 2014, 9, 1188 – 1194

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Scheme 1. Compounds prepared in this work for structure–activity relationship studies.

the ruthenium-containing compound 3 as well as two metalfree compounds, 7 and 8. All compounds were tested for cytotoxicity on various cancer cell lines, the effect of the electronwithdrawing substituents on the metal center was revealed by cyclic voltammetry, and the lipophilicity was determined by reversed-phase high-performance liquid chromatography (RPHPLC).[5] Redox stress has previously been implicated as a possible cause for the toxicity of ferrocene derivatives.[6] Hence, we investigated whether the formation of intracellular reactive oxygen species (ROS) plays an important role in the mode of action. Finally, because apoptosis has often been shown to be the mechanism of cell death in anti-proliferative metallocene derivatives, this was also investigated in this study. This work delineates, for the first time, a structure–activity relationship (SAR) for CF3-substituted metallocenes.

www.chemmedchem.org The reaction of 1 with sodium hydride and methyl iodide gave 2 in 81 % yield. The aromatic protons of the substituted Cp ring appear as one signal at 4.39 ppm in the 1H NMR spectrum. The three protons of the methyl group gave a singlet at 3.38 ppm. The reaction of ruthenocene with hexafluoroacetone tris(hydrate) in a microwave reactor at 120 8C gave 1-(1-hydroxy-1-hexafluoromethylethyl)ruthenocene (3) in 66 % yield after workup. Characterization by 1H NMR showed two of the four aromatic protons of the substituted Cp ring as a singlet. For the second pair of these protons and those of the unsubstituted Cp ring a broad signal (4.72–4.67 ppm) with an integral of seven protons was observed. Suitable crystals of 3 for a single-crystal X-ray structure determination were obtained by slow evaporation of a chloroform solution. The ORTEP plot of 3 shows that this compound possesses the classical “sandwich”-like structure of metallocenes (Figure 1). The average

Results Synthesis and characterization As outlined in Scheme 1, compounds 1–6 all contain a metallocene unit (M = Fe, Ru). All synthesized compounds were characterized by NMR, elemental analysis, IR, and mass spectrometry. The HFA-containing compounds 1 und 3 were obtained by microwave-assisted synthesis. The reaction conditions by Woodward and co-workers for the synthesis of 1 were adapted to microwave conditions.[7] With our improved conditions, the reaction time decreased from five days to 30 min with no loss of yield or purity. In addition to this, the synthesis of the previously unreported 1-(1-hydroxy-1-hexafluoromethylethyl)ruthenocene (3) became possible with our new conditions. Applying the original conditions by Woodward et al., no product formation was observed. Instead, a black, insoluble solid was obtained. This might be due to the irreversible redox behavior of the ruthenium atom. Reaction of ferrocene with hexafluoroacetone tris(hydrate) in a microwave reactor at 120 8C gave 1-(1-hydroxy-1-hexafluoromethylethyl)ferrocene (1) in 72 % yield. Characterization by 1 H NMR spectroscopy showed the aromatic protons of the substituted cyclopentadienyl (Cp) ring as two slightly broadened singlets. Moreover, the OH signal at 3.35 ppm disappeared upon addition of CD3OD. In the 19F NMR spectrum a singlet for the symmetric CF3 groups was observed at 75 ppm.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. ORTEP plot of 3. Selected bond lengths []: Ru(1) Ccentroid : 1.812, Ru(1) Caverage : 2.175, C Faverage : 1.317, C(11) O(1): 1.401.

Ru C bond length is 2.175 , which is in accordance with published data for ruthenocene.[8] Additionally, the compounds 1,1,1-trifluoro-2-ferrocenylpropan-2-ol (4) and 1-(propan-2-ol)ferrocene (5) were synthesized via lithiation and subsequent reaction with the corresponding carbonyl compound (compound 4: trifluoroacetone (TFA); compound 5: acetone). Potassium tert-butoxide (KOtBu) was used in the reaction to create a Schlosser base in situ, which is known to deprotonate ferrocene readily and cleanly.[9] Only compound 4 shows two separate signals for C2 and C5 in the 13 C NMR spectra due to the chiral Ca atom. Mass spectrometry (EI) of compound 5 shows the mass of 5 without the hydroxy group. This effect is well described in the literature. Many years ago this function and its propensity for loss of the OH group was used to create a carbocation for rapid reactions with nucleophiles.[10] The resulting cation is stabilized by the electrondonating effect of the adjacent CH3 groups. In the case of 1, this is not possible due to the strong destabilizing electronwithdrawing effect of both CF3 groups.[11] Ionization of 5 with ChemMedChem 2014, 9, 1188 – 1194

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softer methods such as electrospray ionization (ESI) does not lead to detectable loss of the OH function.

Electrochemical studies The redox behavior of all metal-containing complexes was investigated by cyclic voltammetry; Figure 2 shows representative plots of the obtained voltammograms. The cyclic voltammograms of all substituted ferrocene derivatives revealed oneelectron waves with quasi-reversible Nernst behavior (DEp = 109–120 mV) and peak current ratios Ipox/Ipred of ~ 1 (Table 1,

tributed to the exchange of one CF3 group by a methyl group. As expected, the smaller electron-withdrawing effect leads to a lower half-wave potential than 1 and 2. Along the same reasoning, for compound 5 a half-wave potential of 20 mV was observed, which is ~ 120 mV lower than that of 4. This is in line with the previously discussed half-wave potential for 4. Indeed, replacing one CF3 group by a CH3 group leads to a decrease of ~ 100 mV (1: DE0f = 214, 4: 101, 5: 20 mV). The half-wave potentials of 5 and 6 are nearly the same. These values reveal that the electron-donating effect of the methyl groups has no influence on the iron center at all (Table 1).

Cytotoxicity

Figure 2. Cyclic voltammograms of 1, 2, 4, 5, and 6 (1 mm) in CH3CN with TBAPF6 as supporting electrolyte (0.1 m) with a scan rate of 250 mV s 1. The experiments were undertaken with ferrocene (FcH) as external reference (1 mm). An electrochemical micro volume cell with a stationary glassy carbon working electrode (Ø = 3 mm), Ag/AgCl in aqueous KCl (3 m) as reference electrode, and a platinum wire (Ø = 2 mm) as counter electrode were used.

Table 1. Cyclic voltammetry data for compounds 1, 2, 4, 5, and 6.[a] Compd

DE0f [mV] vs. Fc0/ +

DEp [mV]

Ipox [mA]

Ipred [mA]

Ipox/Ipred

1 2 4 5 6

214 232 101 20 19

110 114 114 109 120

19.2 19.8 20.2 26.4 30.2

18.2 19.6 16.8 22.6 28.8

1.05 1.01 1.20 1.17 1.05

[a] Compound concentration 1 mm in CH3CN with TBAPF6 as the supporting electrolyte (0.1 m), and ferrocene (1 mm) as standard; scan rate: 250 mV s 1.

Before starting biological experiments, adequate stability, particularly of the metal compound 1, under physiological conditions was established by dissolving the compound in a small amount of dimethyl sulfoxide (DMSO), diluting with cell culture buffer (DMEM), and monitoring the HPLC trace at regular intervals. A solution of 1 was found basically unchanged after 74 h, suggesting excellent stability under the conditions required for cytotoxicity assays, for example (see Supporting Information Figures S1 and S2). The cytotoxicity of the substituted metallocenes 1–6 was tested against MCF-7 (breast), HT-29 (colon), and PT-45 (pancreatic) cancer cells. To assess the importance of the metallocene moiety for the cytotoxic effect, the metal-free compounds 7 and 8 were also tested (Scheme 1). In line with previous findings, the unsubstituted metallocenes (ferrocene and ruthenocene) have no cytotoxic effect on cancer cells at all. Metallocene 1 is moderately active against all three cancer cell lines (Table 2). Protection of the OH function with a methyl group (compound 2) leads to complete loss of cytotoxicity against cancer cells up to 100 mm. This result shows that the hydroxy function is crucial for the cytotoxic behavior of the compound. Interestingly, the ruthenium compound 3 showed nearly twice the cytotoxic activity against MCF-7 and PT-45 cells as that of 1, but 3 seems a bit less active than 1 against the colon cancer cell line HT-29 (Table 2). Substitution of one or two CF3 groups by CH3 groups or even hydrogen atoms leads to a complete loss of anti-proliferative activity. This result underscores the importance of the CF3 groups.

Table 2. IC50 values of compounds 1–8.

MCF-7

IC50 [mm][a] HT-29

PT-45

28  0.8 89  5 12  0.5 inact inact inact inact inact

26  3 96  1 36  4 inact inact inact inact inact

59  13 100  0.2 26  0.4 inact inact inact inact inact

Compd

Figure 2). Compound 1 has a half-wave potential DE0f of about + 214 mV. The half-wave potential is an indication of the sensitivity of a compound toward oxidation. A DE0f value higher than 0 mV (ferrocene is set at 0 mV) shows that 1 is harder to oxidize than ferrocene. The half-wave potential of 2 is 18 mV higher than the DE0f value of 1. This shift can clearly be attributed to the methyl-protected OH group, which is the only structural difference between 1 and 2. Metallocene 4 can be oxidized more easily than 1 and 2, as reflected by the lower half-wave potential of 101 mV. This is at 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

1 2 3 4 5 6 7 8

[a] Determined by the crystal violet assay (see Experimental Section for details); inact: inactive up to 100 mm.

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The IC50 values of 7 and 8 indicate that only the combination of a metallocene unit, the two trifluoromethyl groups, and the free OH function must have some crucial function in the cytotoxicity toward cancer cells (Table 2). All non-metal-containing compounds show no effect up to 100 mm against the three tested cancer cell lines. Lipophilicity Lipophilicity is one of four key properties of potential drugs according to Lipinski’s rule of five, and the improved version thereof from Wendoloski and co-workers.[12] Moreover, the log P value should stay in a range between 0.4 and 5.4, but should not significantly exceed a value of 5.[12] Although the ferrocene moiety is already rather lipophilic, incorporation of trifluoromethyl groups in the metallocene scaffold is likely to shift the log P to even higher values.[2b] The enhanced lipophilicity will likely improve cell membrane permeability. To test this expectation, the water/octanol partition coefficients (log P values) of 1, 3, 4, 5, 6, and the unsubstituted metallocenes (ferrocene and ruthenocene) were determined by the RP-HPLC method of Minick et al.[13] The results, as listed in Table 3, indicate an increased lipophilicity for all fluorine-containing metallocenes relative to their non-fluorinated analogues. The log P value increases by ~ 1.3 units relative to ferrocene for compound 1. Comparison of 1 and the similar structures of 4 and 5 reveals a decrease in lipophilicity due to the exchange of the CF3 groups with CH3 groups. In brief, lipophilicity rises with increasing fluorine content, as expected. The log P values for compounds without CF3 groups are in the same range (5 and 6). The ruthenium-containing compounds (ruthenocene and 3) have similar values compared with the iron species (ferrocene and 1). In other words, the lipophilicity is not sensitive toward the nature of the metal atom. ROS production The production of ROS has previously been implicated as an important contribution toward the mode of action, particularly for redox-active metal complexes.[1c, 6, 14] Therefore, the amount of ROS produced by the compounds described herein was determined by flow cytometry at two concentrations (50 and 100 mm) after 6 and 24 h (Figure 3). For this experiment, Jurkat cells (an immortalized leukemia cell line) had to be used which can be grown in solution. As shown in Figure 3, only compound 1 shows strong ROS production at the lower concentration even after 6 h. It should be

Table 3. Octanol/water partition coefficients of unsubstituted metallocenes and compounds 1, 3, 4, 5, and 6 as determined indirectly by RPHPLC (see Experimental Section for details). Compd ferrocene 1 4 5 6 ruthenocene 3

log kW

log P

R2

2.69 3.72 2.87 2.26 3.53 2.94 3.53

3.37 4.64 3.59 2.86 2.25 3.67 4.40

0.997 0.998 0.999 0.996 0.999 1.000 0.995

noted that 1 is also the most toxic of all compounds against Jurkat cells, and therefore less ROS are observed at higher concentrations due to the fact that many cells are dead upon extended exposure to 1. An independently performed cytotoxicity assay confirms that most Jurkat cells are indeed dead after 72 h exposure to 1 at 100 mm (MTT assay, see Supporting Information Figure S3 and S4). This finding corresponds qualitatively with the more accurate IC50 values given above, which were determined on adherently growing cells. Some ROS are also produced by compound 4, but only at higher concentrations and upon longer exposure. The Ru-containing compound 3 shows only small induction of ROS even at high concentrations, although it is also somewhat cytotoxic on Jurkat cells.

Induction of apoptosis/necrosis If investigated at all for metal complexes with anti-proliferative activity, apoptosis is usually the main mechanism of cell death for such compounds, and this is normally linked to ROS production.[20] Accordingly, we have studied the mechanism of cell death by staining with annexin V and propidium iodide as markers for apoptosis and necrosis, respectively. To ensure coherence with the above ROS experiments, Jurkat cells were chosen for this experiment, and cell numbers were counted by flow cytometry. Notably, only compound 1 is active and indu-

Figure 3. ROS level after 6 and 24 h incubation with compounds 1–4 and 7 at 50 and 100 mm; NT: untreated control.

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Figure 4. Induction of apoptosis/necrosis in Jurkat cells after 24 h incubation with compounds 1–4 and 7 at 100 mm; NT: untreated control; Camptothecin (CMPT): positive control.

ces almost exclusively necrosis after 24 h (Figure 4) and 48 h (Figure S5, Supporting Information). This is unusual, as apoptosis has been observed as the primary mechanism of cell death for metallocene derivatives. Moreover, even after 48 h neither apoptosis nor necrosis is induced significantly by compound 4, although a decrease in cell number is clearly observed.

Conclusions We report the synthesis, characterization, and biological evaluation of metallocenes 1–6 derived from hexafluoroacetone and derivatives thereof. Two reaction types (pseudo-Friedel–Crafts acylation and lithiation/substitution) were used to create a number of substituted metallocenes with the aim to establish a structure–activity relationship. Through microwave-assisted synthesis, the preparation of 3 was possible for the first time. In addition to this, using the optimized microwave procedure, all HFA-containing compounds could be synthesized within 30 min as opposed to five days. The electrochemical behavior of compounds 1, 2, 4, 5, and 6 was investigated by cyclic voltammetry. All fluorine-containing compounds showed a higher resistance toward oxidation than ferrocene. The introduction of one CF3 group results in a shift of the half-wave potential by + 100 mV per group. Determination of log P by RP-HPLC revealed a higher lipophilicity for metallocenes with increasing fluorine content. Moreover, it was shown that the lipophilicity is not sensitive toward the nature of the metal atom. Cytotoxicity was tested on MCF-7, HT-29, and PT-45 cells, and IC50  12 mm were found for all compounds. These tests revealed that the metallocene moiety, both CF3 groups, and the free OH function are all crucial contributions for the cytotoxic effect. In addition, it is a striking fact that in the ferrocene series the two most effective compounds also showed the highest lipophilicity and half-wave potential. More important, the ruthenium-containing compound 3 was nearly twice as potent toward MCF-7 and PT-45 cells, despite its lack of electrochemically reversible oxidation  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org chemistry. This fact corresponds to an increased antibacterial activity found by our group for ruthenocene versus ferrocenesubstituted antibacterial peptides,[15] but it does not correspond to ROS production in the Jurkat cell line, which was only observed for the ferrocene derivatives 1 and 4. Interestingly as well, the cytotoxicity profile of the various metals on adherent cell lines differs from that in leukemia cells, against which the ferrocene derivatives were the most active. This fact is not easily explained and merits further investigations. As one initial lead, necrosis was found to be the cause of cell death with 1, at least for the leukemia cells in suspension. This is remarkable and might provide an initial clue, because apoptosis, and not necrosis, is often observed as the mechanism of cell death in metal-containing anti-proliferative drug candidates. In any case, these results do underscore that careful control experiments are warranted when using different cell lines for different tests, which is sometimes an inevitable experimental constraint.

Experimental Section General Unless noted otherwise, all syntheses were carried out under an inert atmosphere of argon or N2 using standard Schlenk techniques. All reagents and anhydrous solvents were purchased from commercial sources and used as received unless noted otherwise. Hydroxymethylferrocene (6) and hexafluoro-2-propanol (8) were purchased from Sigma–Aldrich and used as received. 1,1,1,3,3,3Hexafluoro-2-phenyl-2-propanol (7) was obtained from Alfa Aesar and used as received. NMR spectra were recorded at room temperature on Bruker DPX 200 and DPX 250 spectrometers (1H = 250 MHz, 19F = 235 MHz, and 13C = 63 MHz). Chemical shifts (d) are reported in parts per million (ppm); coupling constants (J) are quoted in Hz. Br stands for broad, sh for shoulder. The numbering of carbon atoms follows the suggestion of Braun et al.[16] IR spectra were recorded on a Bruker Tensor 27 spectrometer with an ATR unit as solid samples, wavenumbers are given in cm 1. Electrospray ionization mass spectra (ESI-MS) were recorded on a Bruker Esquire 6000 spectrometer. GC–MS spectra were recorded on a Shimadzu GCMS-QP2010 instrument. Elemental analyses of ruthenium-containing compounds were carried out at the laboratory for microanalytics and thermal analyses, University of Duisburg-Essen (Inorganic Chemistry Department); all others were carried out at the RUBiospek Biospectroscopy Department, Ruhr-Universitt Bochum. X-ray structure determination of compound 3: Single crystals were obtained by slow evaporation of a CHCl3 solution. A crystal (colorless needle) was placed on a glass capillary in perfluorinated oil. The intensity data were measured with a Rigaku Mercury 375 R/M CCD (XtaLAB mini) diffractometer. Structure solution was performed with direct methods (SHELXS 97[17]), and refined against F2 with all measured reflections (SHELXL 97[17] and Platon/Squeeze[18]). The compound crystallized in the monoclinic space group C2/c with unit cell dimensions a = 16.697(3) , b = 17.179(3) , c = 11.022(2) , b = 124.29(3)8. 7939 unique reflections were refined to final R indices R1 = 0.0501 (I > 2 sI) and wR2 = 0.1564 (all data). Additional crystallographic data can be found in the Supporting Information (Table S1). CCDC 983945 (for 3) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. ChemMedChem 2014, 9, 1188 – 1194

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CHEMMEDCHEM FULL PAPERS Synthesis of HFA-substituted compounds 1 and 3: A microwave tube was charged with a stir bar, corresponding metallocene (1 equiv), and hexafluoroacetone·3 H2O (5.4 equiv). The contents were irradiated in a CEM Discovery microwave reactor for 30 min at 120 8C at 250 W. Then, CH2Cl2 (20 mL) was added to the mixture. Afterward, the organic layer was washed with water twice, dried (MgSO4), and the solvent was evaporated in vacuo. The crude product was purified by column chromatography (silica Merck 60) using first hexanes and then a mixture of hexanes/EtOAc. 1-(1-hydroxy-1-hexafluoromethylethyl)ferrocene (1): Column chromatography: hexanes/EtOAc 6:1. Yellow–gold sparkling powder (1.36 g, 3.86 mmol, 72 %); Rf = 0.86 (hexanes/EtOAc 4:1). 1 H NMR (CDCl3): d = 4.47 (br, s, 2 H, CpH), 4.38 (br, s, 2 H, CpH), 4.31 (s, 5 H, CpH), 3.35 ppm (s, 1 H, OH); 19F NMR (CDCl3): d = 75 ppm (s, 6F, 2  CF3); 13C NMR (CDCl3): d = 122.5 (q, 1JCF = 288.5 Hz, CF3), 82.7 (br, s, C1), 75.6 (septet, 2JCF = 30.4 Hz, Ca), 69.5 (s, C3,4), 69.4 (s, C1’–5’), 67.9 ppm (m, C2,5); IR (solid): 3516 n(O H), 1413 n(C Caromatic), 1204 cm 1 n(C F); MS (EI): m/z = 352 [M] + , 186 [Fc] + , 121 [CpFe] + , 56 [Fe] + ; Anal. calcd for C13H10F6FeO: C 44.35, H 2.86, found: C 44.68, H 2.84 %. 1-(1-methoxy-1-hexafluoromethylethyl)ferrocene (2): Under N2, 1 (0.2 g, 0.57 mmol, 1 equiv) was dissolved in DMF (20 mL) at 0 8C. NaH (0.027 g, 1.13 mmol, 2 equiv) was added, and the reaction mixture was stirred vigorously at 0 8C for 1 h. CH3I (0.05 mL, 0.85 mmol, 1.5 equiv) was added, and the mixture was stirred for 1 h at 0 8C and then for 12 h at room temperature. CH2Cl2 (15 mL) and H2O (10 mL) were added. The organic layer was separated and washed with water twice, dried (MgSO4), and the solvent was evaporated in vacuo. The crude product was purified by column chromatography (silica Merck 60) using a mixture of hexanes/ EtOAc (4:1). Drying under reduced pressure yielded an orange solid (0.21 g, 0.57 mmol, 81 %); Rf = 0.63 (hexanes/EtOAc, 4:1); 1 H NMR (CDCl3): d = 4.39 (s, 4 H, CpH), 4.30 (s, 5 H, CpH), 3.38 ppm (s, 3 H, CH3); 19F NMR (CDCl3) d = 71 ppm (s, 6F, 2  CF3); 13C NMR (CDCl3): d = 122.8 (q, 1JCF = 291.3 Hz, CF3), 81.6 (septet, 2JCF = 28.5 Hz, Ca), 76.5 (br, s, C1), 70.5 (s, C1’–5’), 69.7 (s, C3,4), 69.2 (br, s, C2,5), 54.5 ppm (s, CH3); IR (solid): 2956 n(C H), 1463 n(C Caromatic), 1202 cm 1 n(C F); MS (EI): m/z = 366 [M] + , 185 [Fc H] + , 121 [CpFe] + , 56 [Fe] + . 1-(1-hydroxy-1-hexafluoromethylethyl)ruthenocene (3): Column chromatography: hexanes/EtOAc 12:1. Beige solid (0.15 g, 0.40 mmol, 66 %); Rf = 0.45 (hexanes/EtOAc, 10:1); 1H NMR (CDCl3): d = 4.92 (s, 2 H, CpH), 4.72–4.67 (m, 7 H, CpH), 2.85 ppm (s, 1 H, OH); 19F NMR (CDCl3): d = 74 ppm (s, 6F, 2  CF3); 13C NMR (CDCl3): d = 122.1 (q, 1JCF = 287.4 Hz, CF3), 87.9 (br, s, C1), 74.1 (septet, 2JCF = 30.4 Hz, Ca), 71.9 (s, C1’–5’), 71.7 (s, C3,4) 70.7 ppm (q, 4JCF = 1.8 Hz, C2,5); IR (solid): 3414, 2918 n(C H), 1709, 1414 n(C Caromatic), 1192 cm 1 n(C F); MS (EI): m/z = 398 [M] + , 329 [M CF3] + , 259 [M 2xCF3] + , 232 [RuCp2] + , 167 [CpRu] + ; Anal. calcd for C13H10F6RuO: C 39.30, H 2.54, found: C 41.30, H 2.90 %. 1,1,1-trifluoro-2-ferrocenylpropan-2-ol (4): A 250 mL threenecked Schlenk flask was equipped with a stir bar, a septum, a low-temperature thermometer, and a gas bubbler. Ferrocene (1 g, 5.38 mmol, 1 equiv) and KOtBu (0.15 g, 1.34 mmol, 0.25 equiv) were dissolved in dry THF (40 mL). The reaction mixture was cooled to 78 8C (CO2, acetone) and tert-butyllithium (1.6 m in n-hexane, 6.66 mL, 2 equiv) was slowly added, ensuring that the internal temperature did not rise above 65 8C. Afterward, the reaction mixture was warmed to 0 8C with an ice bath, and freshly distilled trifluoroacetone (1.2 mL, 2 equiv) was added rapidly to the stirred solution. The solvent was evaporated in vacuo, and H2O

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www.chemmedchem.org (10 mL) and CH2Cl2 (15 mL) were added. The organic layer was separated and washed with water twice, dried (MgSO4), and the solvent was evaporated in vacuo. The crude product was purified by column chromatography (silica Merck 60) using first hexanes and then a mixture of hexanes/EtOAc (10:1). Drying under reduced pressure yielded a red solid (0.40 g, 1.35 mmol, 25 %). 1H NMR (CDCl3): d = 4.39 (s, 1 H, CpH), 4.27 (s, 8 H, CpH), 2.59 (s, 1 H, OH), 1.67 ppm (s, 3 H, CH3); 19F NMR (CDCl3): d = 81 ppm (s, 3F, CF3); 13 C NMR (CDCl3): d = 125.4 (q, 1JCF = 285.6 Hz, CF3), 90.1 (d, 3JCF = 1 Hz, C1), 72.4 (q, 2JCF = 29.4 Hz, Ca), 68.9 (sh, C1’–5’), 68.7 (C3,4), 68.0 (br, s, C5), 66.5 (q, JCF = 1.6 Hz, C2), 23.0 ppm (q, 3JCF = 1.4 Hz, CH3); MS (EI): m/z = 298 [M] + , 186 [Fc] + , 121 [CpFe] + . 1-(propan-2-ol)ferrocene (5): Same procedure as that outlined for compound 4 was used; acetone (0.8 mL, 2 equiv) was used instead of trifluoroacetone. Experimental data are in line with published values.[16, 19] 1H NMR (CDCl3): d = 4.41 (s, 2 H, CpH), 4.23 (s, 7 H, CpH), 1.49 ppm (s, 6 H, 2  CH3); IR (solid): 3265, 2975 n(C H), 1460 n(C Caromatic), 1357 cm 1; MS (EI): m/z = 226 [M OH] + , 186 [Fc] + , 121 [CpFe] + ; ESI-MS (+ , 70 eV): m/z = 243.83 ([M] + , exact mass of C13H16FeO: 244.06); Anal. calcd for C13H16FeO: C 63.96, H 6.61, found: C 64.02, H 6.72 %.

Electrochemistry and bioassays Electrochemical measurements: An electrochemical micro volume cell with a stationary glassy carbon working electrode (Ø = 3 mm), Ag/AgCl in aqueous KCl (3 m) as reference electrode and a platinum wire (Ø = 2 mm) as counter electrode were used. All electrochemical measurements were undertaken with 5 mL 1 mm solutions of the respective metallocene in acetonitrile. CV measurements of ferrocene were performed in the same electrolyte solution, and the electrochemical half-wave potential of the redox couple ferrocene/ ferrocenium FcH0/ + vs. Ag/AgCl (E1/2) was set to 0 mV as the reference potential for all measurements. Ferrocene was used as an internal reference for the scan rate at 50 mV s 1 and used as an external reference for all other scan rates to avoid an overlap of the corresponding redox processes (Figure 2). The electrochemical behavior of compounds 1, 2, 4, 5, and 6 was examined by cyclic voltammetry (CV) at five different scan rates (50, 100, 250, 500, and 1000 mV s 1). Determination of log P values by RP-HPLC: The RP-HPLC method of Minick et al. was used for log P determinations.[13] All values were determined on a Reprosil-Par C18-AQ, 5 mm  250 mm  4.6 mm column. The water layer was saturated with octanol. As eluents, 3-(N-morpholino)propanesulfonic acid buffer (MOPS, 0.02 m, pH 7.4) with n-decylamine (0.15 % v/v) and MeOH with n-octanol (0.25 % v/v) were used. For calibration, 4-methoxyaniline, 4-bromoaniline, naphthalene and tert-butylbenzene were eluted at six isocratic eluent concentrations (60–85 % MeOH in 5 % increments). The retention times, together with the dead times obtained from uracil, were used to calculate the corresponding capacity factors k’ H2O/MeOH ratio. The extrapolation to 100 % H2O (0 % MeOH) gave the log kw value for each standard. All four log kw values of the reference compounds were plotted against the corresponding published log P values to obtain a linear function that correlates both parameters. This function was used to determine all log P values from their log kw values, which were measured in the same way as reported for the references. Cytotoxicity assays: MCF-7, HT-29, and PT-45 cells were cultured in DMEM supplemented with 10 % fetal calf serum (FCS), 2 mm l-glutamine, penicillin (100 U mL 1), and streptomycin (100 mg mL 1) in a 5 % CO2 atmosphere. Crystal violet assays were applied to deterChemMedChem 2014, 9, 1188 – 1194

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CHEMMEDCHEM FULL PAPERS mine the absolute cell numbers. All cells were seeded in 96-well cell-culture-treated microtiter plates (MTP) and grown for 24 h under standard conditions. All tested compounds were dissolved in cell culture medium with 0.5 % DMSO and applied to the cells at 1, 5, 20, 50, 100, 500 mm concentrations for 72 h. Cells were fixed with 4 % glutaraldehyde in 2 % water for 25 min at room temperature. Membranes were permeabilized by Triton X-100 (0.1 %) in PBS for 10 min, then aqueous crystal violet solution (0.04 %) was added to the cells. After 30 min of mechanically shaking, the cells were washed five times with water and the crystal violet was eluted with 70 % EtOH for 3 h. Absorbance was detected at 570 nm (Tecan Sapphire 2 microplate reader). The cell mass was plotted against the concentration. IC50 values were calculated from the sigmoidal function. FACS measurements (determination of ROS levels)[20]: Jurkat cells (human acute lymphoblastic leukemia; ~ 106 cells per mL) cultured in RPMI supplemented with 10 % FCS (Gibco) were treated with indicated concentrations (50 and 100 mm) for 6, 24, and 48 h. After treatment, cells were collected, washed, and re-suspended (2.5  105 cells per 0.5 mL) in FACS buffer [D-PBS (Gibco) with 1 % FCS]. Next, a solution of dihydroethidium (D1168, Molecular Probes, Invitrogen; 2 mL, 5 mm) was added to each sample followed by 15 min incubation at room temperature in the dark. Signal intensity was analyzed with a FACS Calibur flow cytometer (Becton Dickinson). Excitation and emission settings were 488 and 564–606 nm (FL2 filter), respectively. Results are presented as a percentage of cells that showed physiological levels of ROS (low ROS) and a percentage of cells with a high level of ROS. MTT assays for Jurkat cells: Approximately 10 000 cells per well were seeded in a 96-well plate, 200 mL per well, using RPMI-1640 medium supplemented with 10 % FCS (Gibco). After 72 h treatment with 1, 2, 3, 4, and 7, cells were collected and centrifuged (200 g, 5 min). Carefully, 150 mL supernatant were removed and replaced with 150 mL RMPI-1640 medium supplemented with 2 % FCS containing 0.5 mg mL 1 tetrazolium salt (MTT, Sigma–Aldrich). Cells were incubated for 2 h in a standard tissue culture incubator. After 2 h incubation, cells were collected and centrifuged (200 g, 5 min). Again, 150 mL medium were removed gently and 150 mL DMSO were added in each well and after 15 min shaking, the plate absorbance was measured at 595 nm using a Tecan Ultra microplate reader (Tecan). Annexin V/propidium iodide (PI) assay for apoptosis/necrosis: Approximately 5  105 Jurkat cells were seeded in 2 mL RPMI medium in six-well plates. Cells were treated with each compound at 100 mm; 24 and 48 h later, cells were collected and washed once with 1 mL PBS. The pellet was washed with 1 mL annexin V buffer. After spinning down cells, the pellet was again re-suspended in 50 mL annexin V buffer, and 5 mL annexin V-FITC (eBioscience) were added. The cells were incubated for 30 min at room temperature while shaking according to the manufacturer’s instructions. After 30 min, 450 mL buffer were added and mixed with PI (100 mg mL 1 final concentration). Incubation was continued for another 10 min at room temperature. The cell suspensions were transferred to FACS tubes. FACS analyses were performed on a FACS Calibur flow cytometer (Becton Dickinson).

Acknowledgements This work is supported by the DFG-funded Research Unit FOR630 “Biological Function of Organometallic Compounds” (www.rub.de/for630). M.M. thanks the Ruhr University Research  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org School. The authors are grateful to Dr. Klaus Merz (Ruhr University Bochum) for help with measuring the data and solving the X-ray crystal structures, and David Bulfield (Ruhr University Bochum) for providing compound 4. We also thank Annegret Knfer (Ruhr University Bochum) for performing the cytotoxicity tests. Keywords: bioinorganic chemistry · cytotoxicity · lipophilicity · metallocenes · structure–activity relationships [1] a) G. Gasser, I. Ott, N. Metzler-Nolte, J. Med. Chem. 2011, 54, 3 – 25; b) D. R. van Staveren, N. Metzler-Nolte, Chem. Rev. 2004, 104, 5931 – 5986; c) A. Nguyen, A. Vessires, E. A. Hillard, S. Top, P. Pigeon, G. Jaouen, Chimia 2007, 61, 716 – 724; d) M. Salmain, N. Metzler-Nolte in Ferrocenes (Ed.: P. Stepnicka), Wiley, Chichester, 2008, pp. 499 – 639; e) L. Delhaes, C. Biot, L. Berry, P. Delcourt, L. A. Maciejewski, D. Camus, J. S. Brocard, D. Dive, ChemBioChem 2002, 3, 418 – 423; f) C. Biot, G. Glorian, L. A. Maciejewski, J. S. Brocard, O. Domarle, G. Blampain, P. Millet, A. J. Georges, H. Abessolo, D. Dive, J. Lebibi, J. Med. Chem. 1997, 40, 3715 – 3718. [2] a) C. Rhode, J. Lemke, M. Lieb, N. Metzler-Nolte, Synthesis 2009, 12, 2015 – 2018; b) M. Maschke, M. Lieb, N. Metzler-Nolte, Eur. J. Inorg. Chem. 2012, 5953 – 5959. [3] a) J. J. Kavanagh, I. H. Krakoff, G. P. Bodey, Eur. J. Cancer 1985, 21, 1009 – 1011; b) A. M. Tsimberidou, W. G. Wierda, S. Wen, W. Plunkett, S. O’Brien, T. J. Kipps, J. A. Jones, X. Badoux, H. Kantarjian, M. J. Keating, Clin. Lymphoma Myeloma Leuk. 2013, 13, 568 – 574; c) A. Bauernfeind, C. Petermller, Eur. J. Clin. Microbiol. 1983, 2, 111 – 115; d) K. L. Kirk, J. Fluorine Chem. 2006, 127, 1013 – 1029; e) C. Isanbor, D. O’Hagan, J. Fluorine Chem. 2006, 127, 303 – 319. [4] A. E. Feiring, J. Fluorine Chem. 1977, 10, 375 – 386. [5] a) A. L. C. Hansch, Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley, New York, 1979; b) S. P. Gubin, S. A. Smirnova, L. I. Denisovich, A. A. Lubovich, J. Organomet. Chem. 1971, 30, 243 – 255. [6] A. Vessires, C. Corbet, J. M. Heldt, N. Lories, N. Jouy, I. Laı¨os, G. Leclercq, G. Jaouen, R.-A. Toillon, J. Inorg. Biochem. 2010, 104, 503 – 511. [7] V. Albrow, A. J. Blake, A. Chapron, C. Wilson, S. Woodward, Inorg. Chim. Acta 2006, 359, 1731 – 1742. [8] A. O. Borissova, M. Y. Antipin, D. S. Perekalin, K. A. Lyssenko, CrystEngComm 2008, 10, 827 – 832. [9] M. Schlosser, Pure Appl. Chem. 1988, 60, 1627 – 1634. [10] R. Herrmann, I. Ugi, Tetrahedron 1981, 37, 1001 – 1009. [11] P. G. Gassman, T. T. Tidwell, Acc. Chem. Res. 1983, 16, 279 – 285. [12] a) C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug Delivery Rev. 1997, 23, 3 – 25; b) A. K. Ghose, V. N. Viswanadhan, J. J. Wendoloski, J. Comb. Chem. 1999, 1, 55 – 68. [13] D. J. Minick, J. H. Frenz, M. A. Patrick, D. A. Brent, J. Med. Chem. 1988, 31, 1923 – 1933. [14] U. Schatzschneider, N. Metzler-Nolte, Angew. Chem. Int. Ed. 2006, 45, 1504 – 1507; Angew. Chem. 2006, 118, 1534 – 1537. [15] H. B. Albada, A.-I. Chiriac, M. Wenzel, M. Penkova, J. E. Bandow, H.-G. Sahl, N. Metzler-Nolte, Beilstein J. Org. Chem. 2012, 8, 1753 – 1764. [16] S. Braun, T. S. Abram, W. E. Watts, J. Organomet. Chem. 1975, 97, 429 – 441. [17] G. M. Sheldrick, SHELXTL 97, University of Gçttingen, Germany, 1997. [18] P. van der Sluis, A. L. Spek, Acta Crystallogr. Sect. A 1990, 46, 194 – 201. [19] a) W. H. Horspool, R. G. Sutherland, J. R. Sutton, Can. J. Chem. 1969, 47, 3085 – 3088; b) K. Gonsalves, L. Zhan-Ru, R. W. Lenz, M. D. Rausch, J. Polym. Sci. Polym. Chem. Ed. 1985, 23, 1707 – 1722; c) L. Zhan-Ru, K. Gonsalves, R. W. Lenz, M. D. Rausch, J. Polym. Sci. A 1986, 24, 347 – 357. [20] A. Hille, I. Ott, A. Kitanovic, I. Kitanovic, H. Alborzinia, E. Lederer, S. Wçlfl, N. Metzler-Nolte, S. Schfer, W. Sheldrick, C. Bischof, U. Schatzschneider, R. Gust, J. Biol. Inorg. Chem. 2009, 14, 711 – 725. Received: February 1, 2014 Revised: April 16, 2014 Published online on May 18, 2014

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