Fluorinated Fe(III) Salophene Complexes: Optimization of Tumor Cell

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Fluorinated Fe(III) salophene complexes: Optimization of tumor cell specific activity and utilization of fluorine labelling for in vitro analysis Irene Würtenberger, Valeria Follia, Fanni Lerch, Christiane Cwickla, Nathalie Fahrner, Christina Kalchschmid, Brigitte Floegel, Brigitte Kircher, and Ronald Gust J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500986h • Publication Date (Web): 15 Dec 2014 Downloaded from http://pubs.acs.org on December 23, 2014

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Journal of Medicinal Chemistry

Fluorinated Fe(III) salophene complexes: Optimization of tumor cell specific activity and utilization of fluorine labelling for in vitro analysis Irene Würtenberger†1, Valeria Follia†1, Fanni Lerch†, Christiane Cwikla‡, Nathalie Fahrner†, Christina Kalchschmidt†, Brigitte Flögel§+, Brigitte Kircher§+, Ronald Gust†* †

Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular

Biosciences Innsbruck, Universität Innsbruck, CCB – Centrum for Chemistry and Biomedicine, Innrain 80/82, 6020 Innsbruck, Austria ‡

Freie Universität Berlin, Institut für Pharmazie, Pharmazeutische Chemie, Königin-Luise-Str. 2+4, 14195 Berlin, Germany

§

Immunobiology and Stem Cell Laboratory, Innsbruck Medical University, Department of Internal Medicine V (Hematology & Oncology), Anichstr. 35, 6020 Innsbruck, Austria +

Tyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck

KEYWORDS: iron salophene complexes, fluorine substituents, tumor cell specific activity, cytotoxicity, protein binding, cellular accumulation, DNA binding, apoptosis, high-resolution continuum source molecular absorption spectrometry

ACS Paragon Plus Environment

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ABSTRACT Fluorine substituted iron(III) salophene complexes (salophene = N,N’-bis(salicylidene)-1,2phenylenediamine) were synthesized and evaluated for biological activity. All complexes showed growth inhibitory effects with IC50 values ranging from 0.05 to 2.45 µM against HT-29 colon carcinoma as well as MCF-7 and MDA-MB-231 mammary carcinoma cells (Cisplatin: 5.75, 12.72, 5.81 µM, respectively). HR-CS MAS investigations revealed that the complexes were highly protein-bound, already after an incubation period of 10 min and accumulated more effectively in tumor cells than Cisplatin. Interestingly, the ligands were enriched in the cells, too, indicating that the salophene moiety acts as a carrier ligand and mediates the uptake of the complexes. Furthermore, induction of apoptosis proved to be dependent on the substitution pattern as well as on the tumor cell line, as evidenced from the Annexin V-FITC/PI assay. Most of the complexes, especially the highly active 5-Fe, showed tumor cell specific effects and no/less influence on the proliferation of T-cells generated from the peripheral blood of healthy individuals.


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INTRODUCTION The serendipitous discovery of the antitumor activity of Cisplatin in the late 1960s1 and its high effects in chemotherapy has stimulated the research on metal-based drugs. However, the therapeutic use of Cisplatin is limited by severe side effects such as nephrotoxicity, neurotoxicity, and myelosuppression, an unselective mode of action but also resistance phenomena.2-4 Hence, the development of novel metal-based anticancer agents is still a major challenge in medicinal chemistry.5-8 During the last decades non-platinum complexes came into the focus of scientists.9 Particularly the coordination of a variety of transition metals to substituted salene ligands led to complexes with interesting biological properties10,11 Dependent on the substituents at the salene scaffold, the complexes induce cell death via a mode of action different from Cisplatin.12 These positive results induced us to synthesize a series of saldach (i.e. N,N’-bis(salicylidene)-1,2-cyclohexanediamine) and salophene (i.e. N,N’-bis(salicylidene)-1,2-phenylenediamine) derivatives that were coordinated to several transition metals (i.e. Mn(II/III), Fe(II/III), Co(II), Ni(II), Cu(II), and Zn(II)).13 Among these derivatives, FeIII(salophene)Cl emerged as lead compound due to its excellent antitumor activity and was subsequently selected for further studies on the mode of action. It could be demonstrated that FeIII(salophene)Cl induces formation of reactive oxygen species (ROS) at nanomolar concentrations in MCF-7 and Jurkat cells, causes DNA double strand breaks via a non-intercalative mode of action, and induces apoptosis via the mitochondrial pathway indicating that cell death is caused by interference with more than one intracellular target.13,14 Moreover, FeIII(salophene)Cl proved to exhibit cytotoxic and apoptotic effects not only in solid tumor cell lines (MCF-7, MDA-MB-231, HT-29), but also in lymphoma (BJAB) and leukemia cells (primary leukemia cells taken from patients with relapsed childhood acute


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lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML)). The compound was able to overcome multiple drug resistance in Vincristine- and Daunorubicine-resistant leukemia cells (Nalm-6).14 In continuative studies, we investigated the influence of methoxy substituents in the salicylidene moieties on the biological activity.15 The results demonstrated a pronounced correlation between cytotoxicity and the position of the methoxy groups revealing the 3-methoxy-substituted derivative as the best compound in this series. For a further optimization, we decided to systematically incorporate fluorine into different positions

of

the

salicylidene

and

phenylenediamine

part

of

the

lead

compound

FeIII(salophene)Cl (see Scheme 1). The fluorination fulfills two objectives. On the one hand, it is well known that the exchange of a hydrogen by a fluorine changes the physicochemical properties of a bioactive molecule and thereby facilitates more desirable pharmacokinetic and pharmacodynamics properties, such as enhanced bioavailability, greater metabolic stability and improved receptor binding affinity.16-18 On the other hand, fluorine can be used as a probe in the molecule for in vitro studies. Normally, in vitro analysis of metal complexes is accomplished using atomic absorption spectrometry (AAS), which is a well-established technique for trace metal analysis. However, in the case of Fe(III) complexes, the pharmacological evaluation is hampered by the ubiquitous presence of iron inside the cells. Though, by applying a recently developed method for the determination of fluorine in biological matrices using high resolution continuum source molecular absorption spectrometry (HR-CS MAS)19 biological evaluation of this class of compounds becomes feasible. In analogy to detecting a metal atom in an organometallic


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compound by AAS, covalently bound fluorine can be used as a probe for in vitro characterization of fluorinated drug molecules by MAS. Using this technique, we were able to study the cellular accumulation profile as well as the DNA binding capability and the binding behavior to human serum albumin of the fluorinated FeIII(salophene)Cl derivatives. Moreover, the ability of the complexes 1-Fe to 14-Fe to reduce the growth of tumorigenic and normal cells was investigated, too.

Scheme 1. Fluorine substituents were incorporated into different positions of the salicylidene and phenylenediamine moieties of the lead compound FeIII(salophene)Cl. RESULTS AND DISCUSSION We synthesized a series of fluorine-substituted salophene derivatives (1 – 14) and their corresponding Fe(III) complexes (1-Fe – 14-Fe). Salophenes are a class of organic compounds bearing two Schiff’s bases that are connecting three aromatic moieties. In recent years, there has been growing interest in Schiff base ligands, since their synthesis is straightforward and they offer potent binding with transition metals. Moreover, it was shown that many of these complexes serve as models for biologically important species and can also be taken as a model for the metal coordination spheres of the antitumor drug Bleomycin.


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Synthesis. The Schiff bases 1 – 14 were obtained by reacting respectively substituted salicylaldehydes and phenylenediamines in ethanol according to a previously published procedure.20 The subsequent coordination to iron(III) was accomplished by reaction of the salicylidene derivatives with FeCl3 hexahydrate in analogy to Hille et al..15 The novel Fe(III) salophene complexes were characterized by infrared spectrometry, mass spectrometry, and elemental analysis (CHN). Characterization by NMR spectroscopy was impossible due to the marked paramagnetism of iron(III) complexes.

(i) EtOH, reflux, 1 – 2 h, (ii) FeCl3 · 6 H2O, EtOH, reflux, 1 – 2 h. Scheme 2. Synthetic pathway to the salophene ligands 1 – 14 and the corresponding Fe(III) salophene complexes 1-Fe – 14-Fe and schematic overview of the substitution pattern..


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Cytotoxic Activity Against Tumor Cells. Table 1. Antitumor activity of fluorinated Fe(III) salophene complexes in HT-29, MCF-7, and MDA-MB-231 cells. Compound

a

Growth inhibition IC50 [µM] HT-29

MCF-7

MDA-MB-231

1-Fe

3-F

0.92 ± 0.29

0.80 ± 0.21

0.60 ± 0.14

2-Fe

4-F

0.62 ± 0.29

0.66 ± 0.12

0.36 ± 0.01

3-Fe

5-F

0.27 ± 0.00

0.71 ± 0.08

0.23 ± 0.00

4-Fe

6-F

0.91 ± 0.18

0.50 ± 0.50

0.44 ± 0.13

5-Fe

3´-F

H

0.15 ± 0.05

0.05 ± 0.01

0.10 ± 0.03

6-Fe

3´-F

3-F

1.45 ± 0.24

2.45 ± 0.23

1.44 ± 0.39

7-Fe

3´-F

4-F

0.45 ± 0.27

0.68 ± 0.13

0.22 ± 0.13

8-Fe

3´-F

5-F

1.13 ± 0.13

0.66 ± 0.09

0.50 ± 0.21

9-Fe

3´-F

6-F

1.11 ± 0.59

0.77 ± 0.14

0.43 ± 0.01

10-Fe

4´-F

H

0.68 ± 0.10

0.60 ± 0.08

0.24 ± 0.18

11-Fe

4´-F

3-F

1.43 ± 0.06

0.69 ± 0.11

0.33 ± 0.06

12-Fe

4´-F

4-F

0.36 ± 0.20

0.58 ± 0.04

0.30 ± 0.24

13-Fe

4´-F

5-F

0.51 ± 0.33

0.78 ± 0.23

0.27 ± 0.05

14-Fe

4´-F

6-F

0.84 ± 0.18

0.67 ± 0.33

0.50 ± 0.09

FeIII(salophene)Cl

0.68 ± 0.01

0.83 ± 0.18

0.05 ± 0.01

Cisplatin

5.75 ± 0.23

12.72 ± 0.66

5.81 ± 0.35

IC50 values represent the concentration of the respective compound that results in a 50%

decrease in cell growth after 72 h of incubation. Results are the mean ± SD of at least two independent experiments.


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The antitumor effects of the fluorinated Fe(III) salophene complexes were analyzed in a panel of human cancer cell lines (i.e. colon carcinoma (HT-29), hormone-dependent (MCF-7) and hormone-independent mammary carcinoma (MDA-MB-231) cell lines). IC50 values were determined in a crystal violet staining assay after an incubation period of 72 h and are reported in Table 1. In the HT-29 cell line, all complexes exerted growth inhibitory effects with IC50 values < 1.5 µM, dependent on the position of fluorine substituent(s) in the aromatic rings. Fluorination at positions 3 (1-Fe) or 6 (4-Fe) of the salicylidene part led to complexes with comparable IC50 values of about 0.9 µM. Shifting the fluorine to position 4 (2-Fe) or 5 (3-Fe) increased the antitumor activity. Compound 3-Fe (IC50 = 0.27 µM) was about 20-fold more potent than Cisplatin. The most effective modification was the introduction of a 3´-F substituent in the phenylenediamine moiety (5-Fe; IC50 = 0.15 µM). Interestingly, the simultaneous presence of a fluorine substituent in the salicylidene rings distinctly reduced the cytotoxic activity. The 3’-F/4F substituted derivative 7-Fe was the only compound in this series that gave an IC50 < 0.5 µM. In contrast, when shifting the fluorine in the phenylenediamine ring from position 3’ to 4’, an additional fluorination in the salicylidene moieties did not result in a loss of activity. As listed in Table 1 MDA-MB-231 cells were in general more sensitive to Fe(III) salophene treatment than HT-29 and MCF-7 cells. All compounds exhibited highest cytotoxic efficacy against the MDA-MB-231 cell line resulting in IC50 values < 0.5 µM for most of the complexes. Only 5-Fe showed its highest efficacy against MCF-7 cells with an IC50 value of 0.05 µM, which is about 200-fold lower than that of Cisplatin. Interestingly, and in contrast to the results obtained for the HT-29 cell line, the substitution pattern had only a marginal influence on cytotoxic efficacy in the mammary carcinoma cell lines. With the exception of compounds 5-Fe


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(3´-F) and 6-Fe (3´-F/3-F), the IC50 values of all complexes ranged from 0.5 to 0.8 µM (MCF-7) and from 0.2 to 0.6 µM (MDA-MB-231), respectively. Importantly, all fluorinated Fe(III) salophene complexes were more active than Cisplatin in all cell lines under study. Furthermore, in the HT-29 and the MCF-7 cell line the antitumor activity was retained or even improved in comparison with the lead compound FeIII(salophene)Cl, whereas in MDA-MB-231 cells FeIII(salophene)Cl remains the most active compound. The same phenomenon has already been observed in a previous study, where methoxy substitution resulted in a loss of tumor cell selectivity, too.15 Additionally, six salophene ligands (i.e. compounds 1 – 4, 6, 11, see Figure S1, Supporting Information) were exemplarily tested for cytotoxicity and no growth inhibitory effects were detected up to a concentration of 20 µM, indicating that the chelated Fe(III) mediates antitumor activity. Hence, we assume a mode of action different from Cisplatin (i.e. DNA-metallation). It is very likely that the fluorinated Fe(III) salophene complexes primarily bind to DNA due to unspecific electrostatic interactions and damage the DNA by secondary effects, e.g. by generation of reactive oxygen species (ROS) as already demonstrated for FeIII(salophene)Cl13,21 and related methoxy-substituted derivatives.22 The significance of an iron center is also confirmed by the results of another study recently published by our group23, in which we investigated structurally related platinum(II) analogs of 1-Fe – 4-Fe. Only the Pt-analog of 1-Fe caused at the HT-29 cell line antiproliferative effects with an IC50 < 10 µM. Based on these results, compounds 1-Fe – 4-Fe were selected for further investigations, since in this series all compounds showed good cytotoxicity and antitumor effects proved to be dependent on the position of the fluorine substituent (IC50: 3-F ≈ 6-F > 4-F > 5-F) in HT-29 and


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MDA-MB-231 cells. Additionally, complexes 6-Fe and 11-Fe were included in these studies to elucidate the influence of fluorine substituents at the phenylenediamine moiety. Besides, these two complexes appeared to be interesting, as 6-Fe gave the lowest activity in all the tumor cells in this study and 11-Fe showed high selectivity for MDA-MB-231 cells. Binding Behavior to Human Serum Albumin. The degree of protein binding is a key pharmacokinetic parameter and it is well known that it alters the biological activity of metal complexes. Protein binding of metal-based compounds can be evaluated by different techniques, such as fluorescence and UV-VIS absorption spectrometry or atom absorption spectrometry.24 However, in this study we determined the binding behavior to human serum albumin for the first time via high resolution continuum source molecular absorption spectrometry (HR-CS MAS) technique. Here, the fluorine substituents in the ligands are used as a probe to detect the unbound fraction of the drug molecules.19 For this purpose, the complexes were incubated with HSA (40 mg mL-1) in PBS at a concentration of 3 µM. After appropriate incubation times, the protein was precipitated with ethanol and the supernatant was analyzed for the unbound fraction by HR-CS MAS. As depicted in Figure 1, all Fe(III) salophene complexes were highly protein bound (> 90%), already after an incubation period of 10 min. Cisplatin is known to have a high affinity for protein binding, too, although with different kinetic characteristics. The observed almost quantitative initial protein binding of the Fe(III) complexes might either result from their strong hydrophobic interactions with the proteins, or precipitation from aqueous solution upon ethanol addition. To exclude the latter, an analogous experiment was performed without HSA. In the resulting ethanolic PBS solution, the expected amount of the iron complexes (3 µM) could be


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detected, confirming that the observed effects can be attributed exclusively to protein binding processes.

Figure 1. Binding behavior to HSA of fluorinated Fe(III) salophene derivatives 1-Fe – 4-Fe, 6-Fe, and 11-Fe in comparison to Cisplatin. Cellular Accumulation (Cellular Uptake). A major requirement for drug molecules is the ability to cross the cell membrane in order to reach their intracellular targets. Hence, the degree of cellular uptake is an important parameter during drug development. Up to now, biological evaluation of Fe(III) complexes by AAS was hampered by the ubiquitous presence of iron in cells. However, by applying the recently developed method for determination of fluorine via HR-CS MAS, we were able to perform cellular uptake studies for this class of compounds utilizing covalently bound fluorine as a probe.


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For this experiment, MDA-MB-231 cells were incubated with the ligands 1 – 4, 6, and 11 as well as the corresponding Fe(III) complexes 1-Fe – 4-Fe, 6-Fe, and 11-Fe at a concentration of 5 µM. After appropriate incubation times (1 h and 6 h, respectively), the intracellular concentration of the respective compound was determined by HR-CS MAS. All compounds were accumulated in tumor cells more effectively than Cisplatin.

Figure 2. Time-dependent cellular uptake at a concentration of 5.0 µM into MDA-MB-231 cells: (a) Fe(III) salophene complexes 1-Fe – 4-Fe, 6-Fe, and 11-Fe, and (b) corresponding ligands 1 – 4, 6, and 11. Fe(III) salophene complexes with F-substituents at the salicylidene moiety showed a structuredependent uptake in the order 3-F < 4-F < 5-F < 6-F (see Figure 2a). If the phenylenediamine is F-substituted, the complexes (6-Fe and 11-Fe) were highly accumulated. The intracellular concentration of 11-Fe was almost identical to 3-Fe and superior to 1-Fe, 2-Fe, and 6-Fe. The


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highest accumulation grade (f = 15.6) was found for 4-Fe. Interestingly, the ligands proved to be enriched in the cells, too, although to a minor extent. A two- to three-fold accumulation was determined for 1 – 3, independent on the incubation time. For compounds 6 and 11 a significant lower cellular amount was determined, while 4 showed the highest accumulation grade with f = 7.5 (1 h). These findings might be attributed to the high lipophilicity of the compounds and it can be assumed that the salophene acts as carrier ligand and mediates the accumulation in tumor cells. DNA Binding. DNA is generally believed to be the major biological target of Cisplatin and other platinumcontaining compounds.25 In the case of Cisplatin, the cytotoxicity is attributed to the formation of 1,2-intrastrand crosslinks between the N7 atoms of two adjacent purine residues and the resulting inhibition of transcription.26,27 Interesting interactions with DNA were also observed for structurally related metal salen and salophene derivates (i.e. Fe(III) hydroxyl-salicylideneethylenediamines, Fe(III) salens, Mn(III) salen and salophene derivatives) and DNA cleaving efficiency was found to be highly dependent on both, the identity and position of the substituents.28-30 Concerning Fe(III) salophene complexes, the mode of action was already intensively investigated and it was demonstrated that cell death is caused by interference with more than one intracellular target.13-15,31 However, to date no information is available on the DNA binding properties of these complexes. Therefore, we were interested in analyzing the interactions of the novel Fe(III) complexes with isolated DNA. The DNA binding properties were analyzed by applying a method previously described32 with some modifications. Salmon testes DNA was incubated with the respective Fe(III) complex (conc. 40 µM) for 5 h. At specified time points aliquots were taken and the DNA was


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precipitated and washed with ethanol. The amount of DNA-bound compound was determined by HR-CS MAS and correlated with the DNA content, which was quantified by spectrophotometric analysis (260 nm). Interestingly, this study indicated with exception of 2-Fe for all compounds low binding affinity to DNA.

Figure 3. DNA binding properties of Fe(III) salophene complexes 1-Fe – 4-Fe, 6-Fe, and 11-Fe. As depicted in Figure 3, the reference compound Cisplatin showed time-dependent increase in DNA binding without reaching saturation. At the end of the experiment (300 min), 10.2 pmol compound/µg DNA were determined for Cisplatin. This value was exceeded by 2-Fe while all the other complexes showed a fast initial binding which remained constant during the whole experiment. For compound 2-Fe, a value of 17 pmol compound/µg DNA was measured after an incubation time of 60 min, which only slightly increased further till the end of the experiment.


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Induction of Apoptosis. Induction of apoptosis is known to play a major role in the mechanism of action of metalbased anticancer drugs. In earlier investigations we demonstrated that the lead compound FeIII(salophene)Cl causes programmed cell death in U-937 and K-562 cells31, and that apoptosis is induced in a concentration-dependent manner in BJAB cells. Moreover, a significant loss of the mitochondrial membrane potential has been observed in lymphoma cells indicating the involvement of the intrinsic mitochondrial pathway.14 Ansari et al. reported interesting apoptosis-inducing properties for a number of structurally related Fe(III) salen complexes, too, which did not correlate with DNA cleaving activity.33 To gain further insight into the mechanism of cytotoxicity induced by the fluorinated Fe(III) salophene complexes and to investigate whether cell death was due to apoptosis or unspecific cytotoxic effects, such as necrosis, their apoptosis-inducing potential was evaluated by flow cytometry. Apoptotic cells are characterized by a variety of morphological and biochemical changes such as loss of membrane integrity, cell shrinkage, chromatin condensation and DNA fragmentation. One of the earliest markers of apoptosis is the externalization of the membrane phospholipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane, which subsequently allows Annexin V binding to the cell surface. Thus, double staining with Annexin V fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI), which is excluded from cells with intact membrane, permits to discriminate between viable (Annexin V/PI-), early apoptotic (Annexin V+/PI-) and late apoptotic/necrotic (Annexin V+/PI+) cells. For apoptosis detection, HT-29, MCF-7, and MDA-MB-231 cells were incubated, respectively, for 24 h with the test compounds. Upon incubation with 2 µM, all complexes except 6-Fe, 8-Fe, and 13-Fe showed considerable apoptotic activity in MCF-7 and MDA-MB-


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231 cells (data not shown). Given these findings, we decided to investigate apoptosis induction at a lower concentration (1 µM) in order to obtain a more precise picture of the underlying processes. These results are graphically presented in Figure 4. For comparison, Cisplatin was tested at a concentration of 5 µM without induction of apoptotic effects.

Figure 4. Induction of early and late apoptosis in (a) HT-29, (b) MCF-7, and (c) MDA-MB-231 cells after an incubation of 24 h with fluorinated Fe(III) salophene complexes (1 µM), evaluated by flow cytometry. The Annexin V-FITC-positive and PI-negative cells (Annexin V+/PI-) represent the early apoptotic cells; the Annexin V-FITC-positive and PI-positive cells (Annexin V+/PI+) represent late apoptotic and necrotic cells.


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No apoptotic activity was observed in the HT-29 cell line, neither at a concentration of 1 µM nor at 2 µM. However, a relatively high proportion of apoptotic cells has already been detected in the control (cells without compounds), indicating that the cells are already damaged by the treatment with AccutaseTM and the two washing steps shortly before apoptosis measurement. On the contrary, the complexes caused pronounced apoptotic effects in the mammary carcinoma cell lines, with a clear preference for MCF-7 cells. In the MDA-MB-231 cell line, compounds 9-Fe > 14-Fe > 11-Fe > 5-Fe > 4-Fe and 1-Fe slightly induced apoptosis with early apoptotic cells ranging from 20.7 – 31.2% (see Table S1, Supporting Information). However, only marginal differences between the derivatives were observed. A completely different picture was obtained for the MCF-7 cell line. Compounds 10-Fe > 5-Fe > 14-Fe > 3-Fe induced apoptosis with fractions of early apoptotic cells as high as 54.8, 51.9, 33.0, and 28.4%, respectively, while all the other compounds were completely inactive. Figure 5 documents the morphological changes of MCF-7 cells incubated with 10-Fe for 12, 24, 48, and 72 h. As expected, the number of cells decreased after 72 h compared to the untreated control and cells treated with 10-Fe showed morphological changes pointing to apoptosis. Compound 5-Fe efficiently triggered apoptosis in MCF-7 and MDA-MB-231 cells, which is in agreement with the results obtained in the cytotoxicity studies, although no general correlation between cytotoxicity and apoptosis was discernible. Furthermore, it is worth noting that complexes 6-Fe, 8-Fe, and 13-Fe did not cause apoptosis in any cell line. Hence, it can be presumed that the incorporation of fluorine into the salophene ligand results in complexes that can – depending on the position of the fluorine substituent – either act merely cytostatic or specifically trigger apoptosis with high selectivity for mammary carcinoma cell lines, or display both activities (5-Fe).


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0 h incubation

12 h incubation with 10-Fe




72 h incubation without 10-Fe (untreated control)

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Figure 5. Time-dependent incubation of MCF-7 cells with complex 10-Fe.


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Activity Against Non-Tumorigenic Cells. Another point of interest is the influence of the fluorinated Fe(III) salophene complexes against non-tumorigenic cells. Therefore, in a first experiment fibroblast-like COS-7 cells were used in the crystal violet assay at concentrations of 0.25 to 1 µM. All complexes were less active compared to the other cell lines and reduced the cell growth if any only at a concentration of 1 µM (see Table S2). A reduction of the cell mass at 1 µM higher than 50% caused 2-Fe (74%), 3-Fe (55%), 7-Fe (69%), 10-Fe (53%), and 12-Fe (77%).

Proliferation Assay Fluorinated Fe(III) salophene complexes 140

% Proliferation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


120 0.25 µM 0.5 µM 1 µM 2.5 µM 5 µM 10 µM

100 80 60 40 20 0

Figure 6. Influence of Fe(III) salophene complexes on the proliferation of T-cells from healthy individuals In a second experiment, peripheral blood mononuclear cells isolated from the peripheral blood of four healthy persons were stimulated with the lectin phytohemagglutinin to generate T-cells and exposed to 0.25, 0.5, and 1 µM of the complexes. The incorporation of [3H]thymidine was quantified as a parameter of cellular proliferation. A concentration dependent inhibition of cell proliferation showed 1-Fe, 2-Fe, 10-Fe, and 12-Fe, while the complexes 3-Fe and 13-Fe were only active at 1 µM. All other complexes did not influence the proliferation of the T-cells (see Figure 6).


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These results clearly demonstrate that it might be possible to design tumor-specific Fe(III) salophene complexes. Especially the highly active complex 5-Fe did not influence COS-7 cells and T-cells from healthy individuals. The same mode of activity showed 4-Fe, 6-Fe, 8-Fe, 9-Fe, 11-Fe, and 14-Fe at the concentrations used, which documents the significance of the fluorine substituents on growth inhibitory activity and tumor cell specificity. CONCLUSIONS In this study, we could show that F-labeling of Fe(III) salophene complexes allows the use of HR-CS MAS for in vitro analysis. Compared to the lead compound FeIII(salophene)Cl, the growth inhibitory effects of the labeled complexes were retained or even improved in HT-29 and MCF-7 cells, whilst in MDA-MB-231 cells the fluorination led to a slight decrease in cytotoxicity compared to FeIII(salophene)Cl. The salophenes serve as carrier ligands and mediate the uptake of the Fe(III) complexes into the tumor cells. Investigations on the DNA binding did not give a clear indication that this target is involved in the mode of action. In this context, it is worth mentioning that the complexes induced apoptosis dependent on the used cell line. In HT-29 cells, no apoptotic effects were detected, while against breast cancer cells (MCF-7 and MDA-MB-231) the fluorination of the complexes proved to significantly influence the amount of living cells and the fraction of early apoptotic cells. It is very likely that the higher activity after fluorination of FeIII(salophene)Cl against MCF-7 cells is a result of an increased apoptosis rate. Furthermore, the complexes showed tumor cell selective effects dependent on the position and number of fluorine substituents. Especially the 3´-F substitution led to compounds which did not influence the proliferation of T-cells generated from the peripheral blood of healthy individuals.


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EXPERIMENTAL SECTION Material and Methods. All reagents and solvents were purchased from Merck, Sigma, and Fluka. Cisplatin was obtained from Serva (Heidelberg, Germany). Melting points were determined on a Büchi B-545 and are uncorrected. NMR spectra were obtained with an Avance/DPX 400 MHz spectrometer (Bruker, Karlsruhe) using TMS as internal standard. Electron impact mass spectra (70 eV) were measured on a CH-7A mass spectrometer (Varian MAT, Bremen). IR spectroscopy was performed on an ATI Mattson Genesis (Wigan, GB) or a Perkin-Elmer Spectrum 100 FTIR spectrometer. Samples were prepared as KBr disks. Elemental CHN analyses were carried out with a Vario EL elemental analyzer (Elementar, Hanau). Quantitative analysis of fluorine was achieved using a high-resolution continuum source atomic absorption spectrometer (HR-CSAAS) model contrAA700 (Analytik Jena, Jena, Germany) evaluating the molecular absorption of gallium monofluoride at the 211.2480 nm rotational line. All measurements were performed using the graphite furnace technique and pyrolytically coated IC standard graphite tubes (Analytik Jena, Jena, Germany) lined with tantalum foil (0.05 mm thickness, Plansee SE, Reutte, Austria) according to a method recently published by our group.19 Quantification was performed using matrix-matched calibration in a range from 0 – 50 µg L-1 F. The purity and structure of the newly synthesized compounds were verified by 1H NMR and IR spectroscopy, HRMS, and elemental analysis. Because of the paramagnetism of the Fe(III) complexes, a characterization by NMR spectroscopy was impossible. However, the coordination of the ligands to Fe(III) could be confirmed by infrared and mass spectroscopy. The elemental analysis values were found to be within 0.4% of the calculated values, indicating that the purity of all tested compounds was higher than 95%.


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General Procedure for the Synthesis of the Fe(III) Salophene Complexes. The respective ligand (0.30 mmol) was dissolved in 10 mL of ethanol and heated to reflux. One equivalent of iron(III) chloride hexahydrate dissolved in 5 mL of ethanol was added to the solution. The reaction mixture was refluxed for 1 – 2 h, concentrated under vacuum and stored for at least 12 h. Thereafter, the precipitate was filtered off, washed with ice-cold ethanol and dried over P2O5. [N,N´-Bis(3-fluorosalicylidene)-1,2-phenylenediamine]iron(III)

chloride

(1-Fe)

was

obtained from 1 (0.28 mmol, 98.7 mg) and iron(III) chloride hexahydrate (0.28 mmol, 75.7 mg). Yield: 15.2 mg (0.03 mmol, 11%) after recrystallization from DMF. Black powder. IR (KBr): 3063 w (νarom. C-H), 1612 s (νC=N), 1579 s, 1444 s, 1314 m (νC-O) cm-1. MS (EI, 230 °C): m/z (%) 441 (17) [M+], 406 (100) [M+-Cl], 56 (3) [Fe]. Anal. (C20H12F2N2O2FeCl x 0.3 DMF) calcd: C 54.15, H 3.07, N 6.95; found: C 54.45, H 3.40, N 7.30. [N,N´-Bis(4-fluorosalicylidene)-1,2-phenylenediamine]iron(III)

chloride

(2-Fe)

was

obtained from 2 (0.28 mmol, 98.7 mg) and iron(III) chloride hexahydrate (0.28 mmol, 75.7 mg). Yield: 66.6 mg (0.15 mmol, 54%). Black powder. IR (KBr): 3072 w (νarom. C-H), 1608 s (νC=N), 1581 s (νC=C), 1238 m, 1198 m (νC-O) cm-1. MS (EI, 230 °C): m/z (%) 441 (12) [M+], 406 (100) [M+-Cl], 56 (1) [Fe]. Anal. (C20H12F2N2O2FeCl) calcd: C 54.39, H 2.74, N 6.34; found: C 54.39, H 2.96, N 6.55. [N,N´-Bis(5-fluorosalicylidene)-1,2-phenylenediamine]iron(III)

chloride

(3-Fe)

was

obtained from 3 (0.28 mmol, 98.7 mg) and iron(III) chloride hexahydrate (0.28 mmol, 75.7 mg). Yield: 33.8 mg (0.08 mmol, 29%). Dark green powder. IR (KBr): 3061 w (νarom.

C-H),

1603 s

(νC=N), 1538 s, 1374 s (νC-N), 1283 m (νC-O) cm-1. MS (EI, 250 °C): m/z (%) 441 (10) [M+], 406


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(100) [M+-Cl], 56 (10) [Fe]. Anal. (C20H12F2N2O2FeCl) calcd: C 54.39, H 2.74, N 6.34; found: C 54.45, H 3.07, N 6.41. [N,N´-Bis(6-fluorosalicylidene)-1,2-phenylenediamine]iron(III)

chloride

(4-Fe)

was

obtained from 4 (0.28 mmol, 98.7 mg) and iron(III) chloride hexahydrate (0.28 mmol, 75.7 mg). Yield: 71.5 mg (0.16 mmol, 58%). Black powder. IR (KBr): 3073 w (νarom. C-H), 1619 s (νC=N), 1535 s, 1372 s (νC-N), 1242 m (νC-O) cm-1. MS (EI, 200 °C): m/z (%) 441 (12) [M+], 406 (100) [M+-Cl], 56 (12) [Fe]. Anal. (C20H12F2N2O2FeCl) calcd: C 54.39, H 2.74, N 6.34; found: C 54.64, H 2.96, N 6.40. [N,N´-Bis(salicylidene)-3´-fluoro-1,2-phenylenediamine]iron(III)

chloride

(5-Fe)

was

obtained from 5 (0.18 mmol, 60.2 mg) and iron(III) chloride hexahydrate (0.18 mmol, 48.7 mg). Yield: 11.0 mg (0.03 mmol, 14%). Black powder. IR (KBr): 3057 w (νarom. C-H), 1606 s (νC=N), 1534 s, 1385 s (νC-N), 1308 m (νC-O) cm-1. MS (EI, 200 °C): m/z (%) 423 (17) [M+], 388 (100) [M+-Cl], 56 (8) [Fe]. Anal. (C20H13FN2O2FeCl) calcd: C 56.70, H 3.09, N 6.61; found: C 56.92, H 3.08, N 6.34. [N,N´-Bis(3-fluorosalicylidene)-3´-fluoro-1,2-phenylenediamine]iron(III) chloride (6-Fe) was obtained from 6 (0.15 mmol, 55.5 mg) and iron(III) chloride hexahydrate (0.15 mmol, 40.5 mg). Yield: 15.0 mg (0.03 mmol, 22%). Black powder. IR (KBr): 3064 w (νarom. C-H), 1612 s (νC=N), 1549 s, 1391 m (νC-N), 1243 m (νC-O) cm-1. MS (EI, 200 °C): m/z (%) 459 (5) [M+], 424 (100) [M+-Cl], 56 (13) [Fe]. Anal. (C20H11F3N2O2FeCl x 0.5 EtOH) calcd: C 52.26, H 2.92, N 5.80; found: C 52.49, H 3.26, N 6.15. [N,N´-Bis(4-fluorosalicylidene)-3´-fluoro-1,2-phenylenediamine]iron(III) chloride (7-Fe) was obtained from 7 (0.13 mmol, 48.1 mg) and iron(III) chloride hexahydrate (0.13 mmol,


23


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35.1 mg). Yield: 23.0 mg (0.05 mmol, 38%). Black powder. IR (KBr): 3088 w (νarom. C-H), 1611 s (νC=N), 1536 s, 1387 s (νC-N), 1234 m (νC-O) cm-1. MS (EI, 175 °C): m/z (%) 459 (4) [M+], 424 (100) [M+-Cl], 56 (7) [Fe]. Anal. (C20H11F3N2O2FeCl x H2O) calcd: C 50.29, H 2.74, N 5.87; found: C 49.92, H 3.00, N 5.96. [N,N´-Bis(5-fluorosalicylidene)-3´-fluoro-1,2-phenylenediamine]iron(III) chloride (8-Fe) was obtained from 8 (0.15 mmol, 55.5 mg) and iron(III) chloride hexahydrate (0.15 mmol, 40.5 mg). Yield: 9.0 mg (0.02 mmol, 13%). Dark green powder. IR (KBr): 3060 w (νarom. C-H), 1606 m (νC=N), 1537 s, 1378 m (νC-N), 1296 m (νC-O) cm-1. MS (EI, 200 °C): m/z (%) 459 (9) [M+], 424 (100) [M+-Cl], 56 (6) [Fe]. Anal. (C20H11F3N2O2FeCl) calcd: C 52.27, H 2.41, N 6.10; found: C 52.44, H 2.39, N 6.33. [N,N´-Bis(6-fluorosalicylidene)-3´-fluoro-1,2-phenylenediamine]iron(III) chloride (9-Fe) was obtained from 9 (0.15 mmol, 55.5 mg) and iron(III) chloride hexahydrate (0.15 mmol, 40.5 mg). Yield: 40.0 mg (0.09 mmol, 58%). Black powder. IR (KBr): 3089 w (νarom. C-H), 1619 s (νC=N), 1534 s, 1372 s (νC-N), 1245 m (νC-O) cm-1. MS (EI, 150 °C): m/z (%) 459 (20) [M+], 424 (100) [M+-Cl], 56 (5) [Fe]. Anal. (C20H11F3N2O2FeCl) calcd: C 52.27, H 2.41, N 6.10; found: C 52.67, H 2.49, N 6.40. [N,N´-Bis(salicylidene)-4´-fluoro-1,2-phenylenediamine]iron(III) chloride (10-Fe) was obtained from 10 (0.30 mmol, 100.3 mg) and iron(III) chloride hexahydrate (0.30 mmol, 81.1 mg). Yield: 37.8 mg (0.09 mmol, 30%). Black powder. IR (KBr): 3065 w (νarom. C-H), 1604 s (νC=N), 1534 s, 1378 s (νC-N), 1275 m (νC-O) cm-1. MS (EI, 200 °C): m/z (%) 423 (1) [M+], 388 (100) [M+-Cl], 56 (3) [Fe]. Anal. (C20H13FN2O2FeCl) calcd: C 56.70, H 3.09, N 6.61; found: C 56.67, H 3.33, N 6.76.


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[N,N´-Bis(3-fluorosalicylidene)-4´-fluoro-1,2-phenylenediamine]iron(III) chloride (11-Fe) was obtained from 11 (0.27 mmol, 100.0 mg) and iron(III) chloride hexahydrate (0.27 mmol, 73.0 mg). Yield: 80.3 mg (0.17 mmol, 65%). Black powder. IR (KBr): 3069 w (νarom. C-H), 1610 s (νC=N), 1549 s, 1381 s (νC-N), 1245 m (νC-O) cm-1. MS (EI, 310 °C): m/z (%) 459 (58) [M+], 424 (100) [M+-Cl], 56 (6) [Fe]. Anal. (C20H11F3N2O2FeCl) calcd: C 52.27, H 2.41, N 6.10; found: C 52.53, H 2.79, N 6.32. [N,N´-Bis(4-fluorosalicylidene)-4´-fluoro-1,2-phenylenediamine]iron(III) chloride (12-Fe) was obtained from 12 (0.20 mmol, 74.1 mg) and iron(III) chloride hexahydrate (0.20 mmol, 54.1 mg). Yield: 64.9 mg (0.14 mmol, 71%). Metallic brown powder. IR (KBr): 3069 w (νarom. C-H), 1613 s (νC=N), 1538 s, 1381 s (νC-N), 1262 s (νC-O) cm-1. MS (EI, 310 °C): m/z (%) 459 (37) [M+], 424 (100) [M+-Cl], 56 (8) [Fe]. Anal. (C20H11F3N2O2FeCl) calcd: C 52.27, H 2.41, N 6.10; found: C 52.26, H 2.67, N 6.09. [N,N´-Bis(5-fluorosalicylidene)-4´-fluoro-1,2-phenylenediamine]iron(III) chloride (13-Fe) was obtained from 13 (0.27 mmol, 100.0 mg) and iron(III) chloride hexahydrate (0.27 mmol, 73.0 mg). Yield: 36.4 mg (0.08 mmol, 29%). Dark green powder. IR (KBr): 3090 w (νarom. C-H), 1608 s (νC=N), 1538 s, 1373 s (νC-N), 1297 m (νC-O) cm-1. MS (EI, 300 °C): m/z (%) 459 (57) [M+], 424 (100) [M+-Cl], 56 (14) [Fe]. Anal. (C20H11F3N2O2FeCl) calcd: C 52.27, H 2.41, N 6.10; found: C 52.03, H 2.80, N 6.39. [N,N´-Bis(6-fluorosalicylidene)-4´-fluoro-1,2-phenylenediamine]iron(III) chloride (14-Fe) was obtained from 14 (0.27 mmol, 100.0 mg) and iron(III) chloride hexahydrate (0.27 mmol, 73.0 mg). Yield: 69.4 mg (0.15 mmol, 56%). Black powder. IR (KBr): 3073 w (νarom. C-H), 1619 s (νC=N), 1534 s, 1370 s (νC-N), 1238 m (νC-O) cm-1. MS (EI, 250 °C): m/z (%) 459 (8) [M+], 424


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(100) [M+-Cl], 56 (4) [Fe]. Anal. (C20H11F3N2O2FeCl) calcd: C 52.27, H 2.41, N 6.10; found: C 52.45, H 2.49, N 6.12. Biological Methods. Cell Culture Conditions. The human MCF-7 and MDA-MB-231 breast cancer and HT-29 colon cancer as well as the COS-7 cell line were obtained from the American Type Culture Collection (ATCC). All cell lines were maintained as monolayer cultures in Dulbecco’s Modified Eagle’s Medium (DMEM) without phenol red, supplemented with sodium pyruvate (110 mg L-1), and fetal calf serum (FCS, 10%; all from PAA Laboratories, Pasching, Austria) using 75 cm2 flasks in a humidified atmosphere (5% CO2/95% air) at 37 °C. The cells were serially passaged twice a week. Mycoplasma contamination was routinely monitored, and only mycoplasma-free cultures were used. Drugs were prepared as stock solutions in DMSO and diluted with complete cell culture medium when used for the biological experiments (final DMSO concentration 0.1% (v/v)). Cisplatin was dissolved in DMF and diluted identically with complete medium. In vitro Chemosensitivity Assay. The in vitro testing of the complexes for growth inhibitory activity was carried out according to a protocol previously described.34,35 Briefly, exponentially growing cells were seeded in triplicate in 96-well microtiter plates at a density of 0.75 x 103 cells/well (MCF-7 and MDA-MB-231), 0.25 x 103 cells/well (HT-29), and 1.00 x 103 cells/well (COS-7), respectively, in 100 µL of complete cell culture medium. Plates were kept at 37°C for 72 h prior to the addition of 100 µL of complete medium containing the appropriate concentration (0.063 to 1 µM) of the respective compound or DMSO as control. After an incubation period of 72 h the medium was aspirated. Subsequently, the cells were fixed with a solution of 1% (v/v) glutaric dialdehyde in phosphate buffered saline (PBS) and stored at 4 °C.


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The number of adherent cells was measured by quantifying the absorbance of crystal violet staining at 590 nm using a microplate reader (Multiskan GO, Thermo Scientific). Cell viability is expressed as a percentage of the untreated control, which was set at 100%. Results are the mean ± SD of at least two independent experiments. The IC50 values were calculated with Prism (GraphPad, San Diego, CA) using non-linear regression and the log of the inhibitor versus variable slope response equation. The bottom constraint was set to 0%. Analysis of Normal Controls. Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of four healthy persons by density gradient centrifugation on Ficoll (Lymphocyte Separation Medium, PAA Laboratories, Pasching, Austria). The cells (50 x 103 cells/well) were incubated with phytohemagglutinin (PHA; 1 µg mL-1 final concentration, Sigma, St. Louis, USA), a lectin which specifically stimulates T-cells, and appropriate concentrations (0.25, 0.5, and 1.0 µM) of the compound in a U-bottomed microtiter plate for 72 h at 37 °C in humidified air with a CO2 content of 5%. For the last 12 – 16 h of incubation, cells were exposed to 2 µCi of [3H]thymidine (specific activity 74 GBq/mmol, Hartmann Analytic, Braunschweig, Germany), harvested using a semi-automated device, and the amount of radiolabeled [3H]thymidine incorporated into DNA expressed in counts per minute was measured in a liquid scintillation counter (Microbeta Trilux; Perkin-Elmer Life Sciences, Boston, MA). Binding Behavior to Human Serum Albumin (HSA). The experiments were performed according to a previously described procedure36 with some modifications. The compounds were incubated with HSA (40 mg mL-1) in PBS, pH 7.4, at a concentration of 3 µM. The samples were placed in a thermomixer (Eppendorf, Germany) at 400 rpm and 37 °C. After appropriate incubation periods (0, 10, 30, 60, 120, 180, 300 min), aliquots of 150 µL were taken and were 2-


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fold diluted with ice-cold ethanol. The samples were stored at -20 °C overnight to allow for protein precipitation prior to final centrifugation (4000 rpm, 10 min, 4 °C). 200 µL of the supernatant were removed and stored at -20 °C until further analysis. The amount of unbound compound was determined indirectly by quantifying the fluorine content in the supernatant applying a recently developed HR-CS MAS procedure19 using matrix-matched calibration. Values are the means of three independent experiments with two instrumental replicates each. The protein binding was obtained by subtracting the quantified unbound percentage from the total initial drug amount (3 µM, 100%). Cellular Uptake Studies. Exponentially growing MDA-MB-231 cells were seeded in 6-well microtiter plates at a density of 0.7 x 106 cells per well in 1 mL of complete cell culture medium. The plates were kept for 24 h at 37 °C to allow adherence of the cells. Thereafter, the medium was aspirated and 2 mL of serum-free RPMI 1640 medium containing the respective drug at a concentration of 5 µM were added in duplicate. After an incubation period of 1 and 6 h, respectively, the medium was removed. The cells were rinsed with ice-cold PBS and detached with Accutase™ (PAA Lavoratories, Pasching, Austria). Subsequently, the cells were harvested and the cell suspensions were centrifuged (2000 rpm, 10 min, 4 °C). The supernatant was discarded and the cell pellets were washed twice with ice-cold PBS and then stored at -20 °C until further analysis. Immediately after thawing, the cell pellets were re-suspended in 200 µL of H2O containing 0.2% (w/v) Triton-X-100 and lysed by sonification (20 s, 9 cycles, 80% power). Aliquots were removed and adequately diluted for protein determination by the method of Bradford37 using calibration standards containing HSA (25 – 500 µg mL-1) dissolved in 0.9% (w/v) NaCl solution. The remaining cell lysates were directly analyzed for fluorine content by HR-CS MAS. Calibrations were done with matrix-matched standards of the respective


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compounds. The cellular concentration of the compounds in the MDA-MB-231 cells was calculated as previously published.38 The results are expressed as accumulation factors (f), which is the ratio of the intracellular and extracellular fluorine concentration, and were calculated as the average of three independent experiments. DNA Binding Studies. Salmon testes DNA was dissolved in PBS, pH 7.4, and the respective compounds were added as stock solutions in DMSO or DMF. The final incubation mixture contained 40 µM of the respective drug and 250 µg mL-1 of salmon testes DNA. After being vortexed, the mixture was incubated in a thermomixer at 37 °C. After appropriate incubation periods (0, 10, 30, 60, 120, 180, 300 min), reactions were terminated by ethanol precipitation. For this, aliquots of 200 µL were taken and mixed with 100 µL of 0.9 M aqueous sodium acetate solution and 900 µL of ice-cold ethanol. Thereafter, samples were stored overnight at -20 °C. The precipitated DNA was isolated by centrifugation (4000 rpm, 5 min, 4 °C), and washed twice with 500 µL of ice-cold ethanol. The obtained DNA pellets were stored at -20 °C until further analysis. The samples were re-dissolved in 200 µL of water on the day of analysis. Aliquots were removed for DNA quantification by measuring the absorbance at 260 nm using UV 96-well plates (Greiner Bio-One, Germany) with a UV/VIS microplate reader (Multiskan GO, Thermo Scientific). The amount of drug bound to DNA was determined by analyzing the remaining DNA samples for fluorine content using HR-CS MAS methodology. Calibrations were performed with matrix-matched standards of the respective compounds. The results are expressed as pmol of compound per µg of DNA plotted vs. time, and were calculated as the average of three independent experiments. Analysis of Apoptosis. Cells were seeded in 96-well microtiter plates at a density of 0.5 x 105 cells/well in 100 µL of complete cell culture medium. Plates were kept at 37 °C for 24 h prior to


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the addition of 100 µL of a 2 µM solution of the respective compound in complete medium. After an incubation period of 24 h, the medium was removed and the cells were detached using Accutase™ (50 µL/well, 5 min, 37 °C). The cells were then transferred into flow cytometry tubes, washed once with PBS and for a second time with Annexin buffer and thereafter incubated with a solution containing 98 µL of Annexin Buffer, 1 µL of Annexin V-FITC, and 1 µL of propidium iodide for 25 min at 4 °C. Analysis was subsequently performed on a FACScan BD FACSCantoTM II Flow cytometer (Becton Dickinson, San Jose, CA) using DIVA 7.0 software. Detection of morphological changes. MCF-7 cells (0.3 x 106 cells in 1 mL) were placed in a 24-wells plate and incubated at 37 °C in a humidified 5% CO2/95% air atmosphere for 24 h to let the cells adhere. Thereafter, 1 µM compound was added and the cells were cultivated for another 72 h. Pictures were taken every hour (JuLi Live Fluoreszenz Cell Imaging; Digital Bio).

ASSOCIATED CONTENT Supporting Information. Experimental methods for the synthesis and characterization of compounds 1 – 14. Results of cytotoxicity studies of compounds 1 – 4, 6, 11, and tabular results of the Annexin V-FITC/PI apoptosis assay as well as the experiments using COS-7 and T-cells. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION 1

These authors contributed equally to this work.

Corresponding Author *Phone: +43 512 507 58200. Fax: +43 512 507 58299. E-mail: [email protected].


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ACKNOWLEDGMENT This work was supported by the Deutsche Forschungsgemeinschaft (DFG) within FOR 630 “Biological function of organometallic compounds”.

ABBREVIATIONS USED SD, standard deviation; TLC, thin layer chromatography; PBS, phosphate buffered saline; Annexin V-FITC, Annexin V fluorescein isothiocyanate; PI, propidium iodide.

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Table of Content Graphic


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Fluorinated Fe(III) Salophene Complexes: Optimization of Tumor Cell

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