Anticancer activity of 4′-phenyl-2,2′:6′,2″-terpyridines – behind the metal complexation

Journal Pre-proof Anticancer activity of 4′-phenyl-2,2':6′,2″-terpyridines – Behind the metal complexation Katarzyna Malarz, Dawid Zych, Michał Kuczak, Robert Musioł, Anna MrozekWilczkiewicz PII:

S0223-5234(20)30006-4

DOI:

https://doi.org/10.1016/j.ejmech.2020.112039

Reference:

EJMECH 112039

To appear in:

European Journal of Medicinal Chemistry

Received Date: 28 October 2019 Revised Date:

3 January 2020

Accepted Date: 4 January 2020

Please cite this article as: K. Malarz, D. Zych, Michał. Kuczak, R. Musioł, A. Mrozek-Wilczkiewicz, Anticancer activity of 4′-phenyl-2,2':6′,2″-terpyridines – Behind the metal complexation, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112039. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Anticancer activity of 4'-phenyl-2,2':6',2''-terpyridines – behind the metal complexation Katarzyna Malarz1*, Dawid Zych2, Michał Kuczak1,2, Robert Musioł2 and Anna MrozekWilczkiewicz1* 1

A. Chełkowski Institute of Physics and Silesian Centre for Education and Interdisciplinary

Research, University of Silesia, Chorzów, Poland 2

Institute of Chemistry, University of Silesia, Katowice, Poland

*[email protected] *[email protected]

Keywords: terpyridine, anticancer activity, metal chelators, reactive oxygen species, apoptosis, cell cycle inhibition

ABSTRACT Terpyridine complexes are known for their broad biological activities, of which their anticancer potency is the most extensively studied. Strikingly, free ligand activity has rarely been described in the literature. In this study, a lipophilic derivative of terpyridine 4'-(1-decyl2,3-triazol-4-yl)phenyl-2,2':6',2''-terpyridine (L) and its complexes were investigated to determine their mechanism of anticancer activity. Our results show that a free ligand expresses the same level of activity on a panel of cancer cells with a low toxicity towards normal fibroblasts as complexes with Cd, Zn or Cu. Breast cancer (MCF-7 cell line) was the most vulnerable for the tested compounds with the IC50 values in the nanomolar range (IC50 = 40 nM for L.) The addition of Cu(II) ions increased its activity even further, thus suggesting that ligand exchange and ROS production are the main components of its activity. A cell cycle analysis indicated its inhibition at the G0/G1 phase and the subsequent apoptosis as the cell death mode. A detailed analysis of the protein level that was involved in the aforementioned processes confirmed previous results. Furthermore, the reactive oxygen species generation and DNA intercalation confirmed its cleaving activity.

1

1. Introduction Terpyridines are heterocyclic compounds comprising three pyridine rings. Their specific structure makes them especially useful ligands for versatile complexes and more expanded molecules. Consequently, scientific interest is mainly focused on catalysis, supramolecular [1] or photovoltaic [2] applications and intelligent materials [3,4]. On the other hand, its ability to form complexes with metal ions along with its flat aromatic structure ensure activity towards biological systems. Among them, bipyridines and terpyridines as well as their complexes have been described as antibacterial and antifungal agents [5–7]. In these applications, however, the central metal atom has a significant share in the biological activity because free ligands are generally less active than complexes [8]. The most appealing field that has been explored is their anticancer activity and impact on mammalian cells. Strikingly, these activities have been almost solely studied on the metal complexes of terpyridines and their derivatives. Most typically a transition metal from the 8-11 groups such as platinum [9], ruthenium [10] or palladium [11] are used in anticancer complexes. These metals are generally recognised in medicinal chemistry for their cytotoxicity and their effect on anticancer agents (cisplatin). The mechanism of action for those Pt, Pd or Ru complexes is often described as being their DNA intercalation and non-specific cytotoxicity [12,13]. It should be noted, however, that a core metal atom is not crucial for those compounds to interact with DNA because they do not share a common mechanism of action with covalently binding cisplatin and related agents. In the case of the complexes that are built on the above-mentioned metals, this strong and nonspecific toxicity is often related to the metal-protein interactions such as in silver compounds, which bind to thiol groups. Moreover, several silver complexes of terpyridines have been described as having backbone binding and π-π groove stacking as their intercalation mode [14,15], which is crucial for anticancer activity. On the other hand, complexes that are built on metals such as Cu, Fe, Zn can be considered separately for the biological importance of their central atoms. These elements are crucial for metabolic balance and their concentrations are tightly regulated in cells and tissues. Cancer cells often tend to accumulate those metals and their complexes for that reason. Moreover, Cu and Fe are unique for their ability to change the oxidation state in complexes, which in turn may result in electron transfer [16]. This phenomenon is based on the production of reactive oxygen species (ROS) in a cell [17,18]. ROS are important for maintaining a normal metabolic balance as well as for the anticancer activity of large series of drugs [19,20]. The formation of ROS and a DNA-binding ability results in a “double-edged sword” effect in which a drug can act separately in the mitochondria and nucleus. Several terpyridine copper complexes have revealed a strong 2

DNA-cleavage ability [21–23]. This, however, strongly depends on the structural features such as molecule volume, because a highly aromatic derivative, that is substituted with naphthalene appears to be non-specific groove binder. What is particularly interesting is that a majority of Cu complexes exert their anti-proliferative effect through ligand exchange [24]. This supports the high activity level of free ligands, which can be observed for various compounds that have a common mechanism of action, e.g. thiosemicarbazones [25–28]. Surprisingly, to the best of our knowledge, this hypothesis has not yet been studied in terpyridines. Recently, we investigated a series of terpyridines as free ligands for their anticancer properties, among others [29]. Particularly interesting in terms of activity appeared to be triazole-substituted compounds, which was also observed in the literature [30,31]. It could be hypothesised that additional site of chelation in triazole moiety may plays a beneficial role. Indeed the activity of those terpyridines appeared to be on a similar level as in the complexes that reached promising sub-micromolar concentrations. Moreover the preference of lipophilic substituents on the triazole ring was observed. This study prompted us to investigate their mechanism of action more deeply on the basis of their impact on cellular metabolism, ROS production and intercalation of DNA. To ensure a good accumulation in a cell and to increase the DNA-binding properties, we designed a highly lipophilic derivative of terpyridine and prepared its complexes in order to verify the hypothesis of free-ligand potency.

2. Results and discussion 2.1. Chemistry The 4'-(1-Decyl-2,3-triazol-4-yl)phenyl-2,2':6',2''-terpyridine (L) was obtained in a Cu(I)catalysed 1,3-dipolar cycloaddition reaction of 4'-(4-ethynylphenyl)-2,2':6',2''-terpyridine with decyl azide, which was previously described for compounds whose structures are similar, which is presented in Scheme 1 [29]. The obtained trident ligand (L) was used in the synthesis of bis(terpyridine) complexes with Zn(II), (ZnL2), Cd(II) (CdL2) and Cu(II) (CuL2) by using a reaction between the appropriate metal chloride in dimethylformamide [32]. In the case of the Zn(II), the mono terpyridine complex was also obtained (ZnL). The structures of the compounds were confirmed using a 1H and

13

C NMR and HRMS analysis (Figures S1-

S11 supporting information).

3

C 10 H 21 N

N

N

C 10 H 21

H

N

N

N N

+

N

N

+

+

b

a

M N

N

+

+

N

+

N

N N

N

N

N

L N N

N C 10 H 21

Scheme 1. Synthesis of 4'-(1-decyl-2,3-triazol-4-yl)phenyl-2,2':6',2''-terpyridine (L) and its complexes with Zn(II) (ZnL2), Cd(II) (CdL2) and Cu(II) (CuL2). Reagents and reaction conditions: a) decyl azide, CuSO4*5H2O, sodium ascorbate, pyridine, EtOH, H2O, room temp., 24 h; b) ZnCl2/CuCl2/CdCl2, DMF, 90°C, 1.5 h.

2.2. Cytotoxicity studies The anticancer activity of five newly synthesised terpyridine derivatives (Fig. 1) was determined on a panel of cancer cell lines representing different types of human tumours. We selected breast (MCF-7), colorectal (HCT 116) and lung (A549) cancers because they are the most frequently diagnosed types worldwide. On the other hand, pancreatic (PANC-1) and glioblastoma (U-251) are characterised as being difficult with treatment that is often ineffective, which results in a typically poor prognosis and a low five-year survival rate. Additionally, the impact of the tested compounds on normal human fibroblast cells (NHDF) was also explored.

4

Figure 1. Structure of the tested terpyridine derivative – L (A) and its metal complexes (B).

As is presented in Table 1, most of the calculated IC50 values were below 1 µ M (shown in red), which indicates a high level of anticancer activity. There were some interesting relationships between the activity and the structure. We observed the influence of the central metal ions on the structure of the formed complexes and thus on the landscape of their cytotoxicity. The MCF-7 cell line was the most susceptible to the tested compounds. The lowest IC50 value was detected for a lone ligand – L on breast cancer (IC50 = 0.04 µ M), which indicates that the tested compound was more active than the reference drugs (ten times more active than doxorubicin and 26 times more active than oxaliplatin). In general, all of the tested compounds exhibited a high anticancer potency on the MCF-7 cell line with the IC50 values for the aforementioned of 0.04 (for L) to 0.22 µ M (for ZnL). A similar situation was observed for the PANC-1 cell line whose IC50 values were within the range of 0.13 (for ZnL2) to 0.59 µ M (for ZnL). The results that were obtained for these two cell lines indicate that all of the

5

tested compounds were more active than the two reference drugs. We also observed that the addition of a zinc ion, which was coordinated by the ligand and two chloride ions (ZnL compound), resulted in a decrease in the activity level for all of the cell lines that were tested. This observation seems to support the ligand exchange hypothesis that was mentioned earlier.

Table 1: The antiproliferative activity of the studied compounds against a panel of cancer cell lines and normal human fibroblast (NHDF) cells. Colour legend: red – IC50 < 1 µM; yellow – IC50 = 1 µM – 6.25 µM and grey – IC50 > 6.25 µM. Activity Cell line Comp. L

ZnL

ZnL2

CdL2

CuL2

doxorubicin

oxaliplatin

IC50 value [µM]

MCF-7

PANC-1

HCT 116

U-251

A549

NHDF

0.04 ±

0.44 ±

0.27 ±

0.14 ±

0.75 ±

20.83 ±

0.01

0.03

0.03

0.02

0.09

2.61

0.22 ±

0.59 ±

3.72 ±

2.23 ±

6.31 ±

0.02

0.05

0.27

1.11

1.12

0.15 ±

0.13 ±

0.98 ±

0.72 ±

1.56 ±

14.19 ±

0.04

0.04

0.05

0.12

0.43

4.19

0.09 ±

0.16 ±

0.19 ±

0.23 ±

0.59 ±

0.03

0.02

0.01

0.02

0.14

0.07 ±

0.19 ±

0.26 ±

0.24 ±

1.57 ±

14.66 ±

0.02

0.04

0.01

0.03

0.40

3.77

0.41 ±

0.73 ±

0.34 ±

0.05 ±

1.06 ±

0.14 ±

0.04

0.09

0.04

0.01

0.06

0.03

2.23 ±

2.16 ±

0.61

0.40

>25

>25

1.13 ± 0.28

>25

>25

>25

In general, ZnL was the least active compound and the impact of the two chloride ions on the lipophilicity seemed to be negative in the context of the pharmacokinetic. The addition of the second 4'-(1-Decyl-2,3-triazol-4-yl)phenyl-2,2':6',2''-terpyridine fragment to ZnL2 as well as changing the central metal ions to cadmium or copper (CdL2 and CuL2) significantly improved its ability to inhibit cancer cell proliferation. On the other hand, the differences in the IC50 values presented in Table 1 were not very high, which may suggest the degradation of the complex under cellular conditions. We did not observe any regularity about L – the ligand alone was the most active. Only in the case MCF-7 and U-251 cell lines did we see such a dependence. Apart from ZnL, for the rest of the compounds, the IC50 values for the 6

ligand alone and the complexes were similar. For the U-251 cells, the IC50 values were within the range 0.14 (for L) to 0.72 µ M (for ZnL2) and for the HCT 116 cell line from 0.19 (for CdL2) to 0.98 µ M (for ZnL2), which indicates a better activity than standard oxaliplatin. The lung A549 cell line was the most resistant to the tested compounds and both drugs as well. The IC50 values were in the range of 0.59 (for CdL2) to 6.31 µ M (for ZnL). We also determined the effect of the tested compounds on the normal cells. The results indicate that the NHDF cell line was resistant to the incubation with the tested terpyridine derivatives. Doxorubicin appeared to be the most toxic against normal cells, which is consistent with literature data [20]. The calculated selectivity indexes (SI) (Table S1) were within the range of 484.42 (for L) to 3.96 (for ZnL), which confirmed their nontoxicity towards healthy cells. When the SI values of almost all of the active derivatives (besides the moderately active complex ZnL) were compared with the SI values of the reference drugs, it was observed that all of them were much higher, which proves the high potential for the application of terpyridine compounds in clinical trials. Additionally, we tested the influence of the metal ions on the proliferation of the cancer cell line after incubation with ligand alone (L). Some of the metal chelators are known for their ionophoric activity; therefore, we tested the terpyridine derivatives in this context [20,26]. The results, which are presented in Fig. 2, indicate that supplementation with metal ions such as Fe2+, Fe3+, Cd2+, Zn2+ did not improve the anticancer activity of L. However, the addition of copper ions (Cu2+) caused a significant decrease in the proliferating fraction of cells. The IC50 values for the combination of L with the metal ions are presented in Table S2. In the case of the presence of metal ions such as Fe2+, Fe3+, Cd2+, Zn2+, the IC50 value of L fluctuated around 1 μM. In turn, after the addition of the copper ions, the IC50 value decreased to 0.039 μM in comparison to L ligand alone, for which it was 0.043 μM. The control cells that had been supplemented with a 20 µM solution of CuSO4, FeCl3, FeSO4, ZnCl2 or 5 µM CdSO4 did not have a toxic effect.

7

Figure 2. The effect of the terpyridine derivative (about two-fold of the IC50 concentration 0.1 μM) and a combination of L with the metal ions on the cellular proliferation of the MCF-7 cells. The data were analysed using a one-way ANOVA with a Bonferroni’s post-hoc test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to the L treatment alone.

2.3. Cell cycle assay After a preliminary examination of the cytotoxic effect of the ligand alone and its complexes with several metal ions, the next step was to evaluate the potential mechanism of their anticancer action. For the research, we selected the most active L derivative and we explored their impact on the cell cycle of the MCF-7 cell line. Additionally we studied the complex CuL2 in the context of the cell cycle inhibition (Fig. S12 supporting information). The results, which are presented in Fig. 3, indicate that the tested compound caused a significant increase in the G0/G1 phases in a concentration-dependent manner. The highest effect was observed for 5 µM of L. In this case, the fraction of the cells in the G0/G1 phases increased from 52.65% (for the untreated control cells) to 66.20% with a simultaneous decrease in the G2/M phases. These observations may indicate that there was a cell cycle inhibition in the G0/G1 phases after incubation with tested ligand. The similar results were observed for the complex with Cu (CuL2). As is presented in the Fig. S12 the cell cycle was inhibit in the G0/G1 phases, however we did not observed dose-dependent manner.

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Figure 3. Effect of L (1 µM, 2.5 µM and 5 µM) on regulating the cell cycle in the MCF-7 cells. The representative histograms show the distribution of the cells in the G0/G1, S and G2/M phases of the cell cycle for one of the experiments (A). Data were analysed using a one-way ANOVA with Bonferroni’s post-hoc test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to the control (B).

2.4. Annexin V binding assay In the next step, we explored the ability of L to induce apoptosis. In the flow cytometry experiments, we attempted to explain the high anticancer activity that had been reached in earlier studies. As is presented in Fig. 4A and B, the fraction of the apoptotic cells increased in a concentration-dependent manner. For the L used at a 5 µM concentration, the level of the apoptotic cells increased from 7.62% (control cells) to 29.33%. By contrast, the population of living cells decreased from 92.01% (control cells) to 55.27%. These results indicate that the tested compound induced apoptotic cell death. To confirm these observations, we performed ancillary imaging studies with Acridine Orange (AO)/Ethidium Bromide (EB) staining. As is presented in Fig. 4C, the cells that had been treated with the tested compound showed early apoptotic features (green and yellow cells; the green dots in the nuclei indicated chromatin condensation and nuclear fragmentation) and late apoptotic cells with condensed and fragmented nuclei that were stained orange. Additionally copper complex of terpyridine CuL2 was also evaluated (Fig. S13 supporting information). Pro-apoptotic properties were also confirmed in that case, however for the lower concentrations. This again is in agreement with literature hypothesis of ligand exchange. Namely ligand substitution in complex would result in exposing one molecule of free ligand thus increasing overall concentration of active molecules. 9

Figure 4. Evaluation of apoptosis induction in the MCF-7 cells after a 48-hour treatment with L at concentrations of 1 µM, 2.5 µM and 5 µM. The histograms show the percentage of early and late apoptosis for one of the experiments (A). Data were analysed using a one-way ANOVA with Bonferroni’s post-hoc test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to the control (B). The morphological changes in the MCF-7 cells that had been treated with L (5 µM). The cells were stained with AO/EB to indicate apoptosis. Scale bars = 20 µm (C).

2.5. Generation of reactive oxygen species (ROS) As was mentioned earlier, there are reports that describe anticancer metal complexes that disturb the proper cellular redox homeostasis [33,34]. An increased ROS level often leads to oxidative stress, thereby triggering apoptosis. For this reason, we performed kinetic experiments to measure the ROS concentration in the cells after incubation with the tested L ligand. As is presented in Fig. 5, we observed a growing trend after only 1 h, which reached the maximum level after 24 h of incubation, for which we recorded about a 140% increase in the ROS level compared to the control cells. These results proved that the tested compounds had an impact on ROS generation, thus disturbing the normal redox homeostasis. The consequences of this process may be a breakdown of the cellular defence, an inhibition of cell cycle progression and in the final phase, the induction of cell death via the apoptosis pathway 10

[35]. Additional experiments with the most active complex CuL2 confirmed generation of ROS for this entity (Fig. S14 supporting information).

Figure 5. Impact of the tested L (1 µM) on the ROS level in the MCF-7 cells. Data were normalised to the untreated control. The results are shown as the mean ± SD of four independent measurements. Data were analysed using the Student's t-test: *p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control group.

2.6. Subcellular localisation It is a commonly known fact that mitochondria are the main source of the ROS that are produced during the process of oxidative phosphorylation [17]. The specificity of the reactions that are triggered by ROS is a high reaction rate non-specificity. Therefore, the accumulation of the factors that are involved in ROS production results in a breakdown of the antioxidant system in a cell. To evaluate this statement, we performed experiments to determine the cellular localisation of the tested L ligand. We selected several dyes that stain specific organelles in a cell and as is shown in Fig. 6, we concluded that L was accumulated in the mitochondria. Pearson's Coefficient; r for these organelles equalled 0.833. We also tested the lysosomal tracker and also determined the moderate co-localisation (Pearson's Coefficient: r = 0.514). For the other organelles, we did not detect any significant penetration.

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Figure 6. Cellular localisation of the L compound in the MCF-7 cells. Scale bar represents 50 µm.

2.7. Immunoblotting In order to perform a detailed analysis of the impact of the tested compounds on the crucial cellular proteins, we performed Western Blot experiments. We selected several targets that are involved in the key cellular processes such as proliferation, cell cycle and apoptosis induction. The p21 protein is responsible for the cell-cycle progression. As is presented in Fig. 7, we observed a significant decrease in the p21 expression after its incubation with L.

Figure 7. Effect of the tested terpyridine derivative – L and DOX on the expression of the proteins that are associated with the cell cycle progression and the induction of apoptosis in

12

the MCF-7 cells (A). The densitometric analyses of p21, cytochrome c and AIF were normalised to the GAPDH. The results are the mean ± SD of four independent measurements. Statistical analysis was performed using a one-way ANOVA with Bonferroni’s post-hoc test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to the control – untreated cells (B).

This confirmed the earlier studies concerning the inhibition of the cell cycle in the G1 phase [36]. The three other analysed proteins were p53, cytochrome c and AIF. All of them were connected with apoptosis induction. In the performed experiments, we detected the release of the cytochrome c to the cytosol and an increased level of AIF. These results confirmed the apoptotic cell death was triggered by the tested compound. Surprisingly, we did not observe any p53 activation, which proved p53-independent apoptosis pathway.

2.8. Intercalation and cleavage DNA studies The anticancer activity of many of the complexes with transition metals can generally be related to their interactions with negatively charged DNA [37]. The terpyridine complexes with copper, cadmium, nickel or palladium are known for their binding modes such as a coordinative and electrostatic interaction, groove binding and intercalation to DNA [22,38,39]. These abilities result from the planarity of the 4′-phenyl-2,2′:6′,2″-terpyridine ligand and the presence of aromatic rings in its structure, which enhance the transference of electron [40]. Additionally, a disturbance of the redox homeostasis and the direct generation of ROS by terpyridine ligands may cause cleavage and may result in irreversible DNA damage. To explore these possibilities, we performed DNA-binding studies that rely on a spectroscopic analysis of the L ligand in the presence of calf thymus DNA. The spectroscopic data from the measurements are presented in Table 2 and the spectra information in Figure S15 (supporting information).

Table: 2 Spectroscopic properties of the L and DOX that bound to the CT-DNA. Changes in

%

absorbance

hypochromism

310

hypochromism

480

hypochromism

Compound

Absorption

L doxorubicina

∆ε M−1 cm−1

redshift*

18.2

2373.3

9

34.2

3235.6

10

*for the wavelengths of the maximum absorption for the individual and DNA-bound compounds. a

data from [20,41].

13

In general, the measure and strength of the interaction of the ligand with DNA as well as the shift in wavelength of the maximum absorption were induced by a hypo- or hyperchromic effect. A hypochromism mode is the result of a strong π–π stacking interaction between an aromatic chromophore and DNA bases. On the other hand, hyperchromism is usually connected with an electrostatic binding to the grooves or the partial uncoiling of the DNA helix. Compound L revealed a rather high hypochromism effect, which was 18.2% and had a strong redshift (bathochromism) at the absorption maximum. We reported a similarly strong interaction of terpyridine ligands in our previous studies [29]. On the one hand, the observed hypochromic effect was lower than that for doxorubicin, which is a classical strong intercalator. However, even this strength of the interaction with DNA may cause some damage in the DNA helix, which in turn leads to the launch of the repair system, the subsequent activation of the cell cycle checkpoints and ultimately to cell cycle arrest or apoptosis. In order to determine the direct influence of all of the tested compounds on the cleavage DNA, we performed experiments with supercoiled pUC19 DNA plasmid. As is presented in Figure 8, the cleavage reaction on the plasmid DNA was monitored using agarose gel electrophoresis. Three forms of plasmid, which migrated on the gel at different speeds, were generated by the cleavage reaction. The supercoiled form (SC, form I) migrated relatively quickly, but when a rupture occurred on one strand of a plasmid (nicking), the supercoil converted into a slower-moving open circular form (NC, form II). The last form of the plasmid was a linear form (LC, form III), which was generated because both strands of the DNA had been broken, and migrated between the SC and NC. In general, the L ligand and its complexes with metal ions exhibited a high ability to cleave plasmid at a 50 µM concentration. In all of the cases, the densitometric analysis showed an increase in the open circular and linear forms of the plasmids compare to the control – untreated plasmid. Interestingly, the highest effect was observed for the ZnL2 and CuL2 complexes. In the case of ZnL2, increase from 29.67% (control plasmid) to 45.12% for the NC form and an increase from 21.21% to 37.77% for the LC with a simultaneous reduction for supercoiled form were observed. For the copper complex (CuL2), we observed an increase in both forms (NC and LC) of the plasmid, but to a lesser extent. These observations may be related to the high anticancer activity of the complexes and may indicate a possible mechanism of action based on their interaction with DNA.

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Figure 8. DNA cleavage studies. Agarose gel electrophoresis with III forms of pUC19 DNA plasmid after treatment with terpyridine derivative – L and its metal complexes (A). Densitometric analysis of the III forms of plasmid that were generated after the treatment with the tested compounds (B).

3. Conclusions Terpyridines are well-known metal chelators and this property determines their biological activity, especially within an antiproliferative scope. However, in light of the available literature reports, it was unclear whether their activity is cause by a free ligand or metal complexes. Here, we investigated the role of the terpyridine moiety in anticancer activity against a panel of cancer cell lines. The free ligand was found to be similarly or even more active than the typical complexes and its activity decreased when it was incubated with metal ions. However, the exception was copper ions, which slightly increased the activity and 15

supported the oxidative damage caused by ROS as the mechanism of action. This was further confirmed by the mode of cell death and the metabolic response to the generation of oxidative stress. The DNA intercalation and cleavage by the free terpyridine ligand was also confirmed. To summarise, we conclude that 4'-phenyl-2,2':6',2''-terpyridines have a high level of anticancer activity, which is generated by a free ligand rather by its complexes. Their strong activity towards several cancer cell lines, and good selectivity indexes make them promising candidates for further research.

4. Experimental session 4.1. Equipment for the synthesis The NMR spectra were measured in deuterated chloroform or dimethyl sulfoxide using a Bruker Avance 400 MHz (1H and 13C NMR). The high-resolution mass spectrometry (HRMS) measurements were performed using a Mass Spectrometer Q-TOF, maXis impact (Bruker Daltonics) using the ESI-MS research method. The samples were weighed and dissolved in chloroform to achieve a sample concentration of 1 mg/mL. The samples were then diluted 1010 000 times with H2O:ACN 10:90 (v/v) solution to which 0.1% HCOOH had been added.

4.2. Synthesis of L The 4′-(4-ethynylphenyl)-2,2′:6′,2′′-terpyridine (0.10 g, 0.30 mM), decyl azide (0.066 g, 0.36 mM, ethanol (10 mL) and water (5 mL) were poured into a 50 mL round-bottom flask. The mixture was saturated with argon and then CuSO4·5H2O (0.090 g, 0.36 mM), sodium ascorbate (0.071 g, 0.36 mM) and pyridine (0.25 mL) were added. The mixture was stirred at room temperature for 24 h. Then, chloroform (20 mL) and a 5% ammonia solution (15 mL) were added and stirred for 10 min. The mixture was extracted with water (50 mL) and chloroform (2 x 50 mL). The crude product was purified using column chromatography (silica gel; CH2Cl2 followed by ethyl acetate). The product was obtained as a white solid in a 75% yield (0.116 g). 1H NMR (400 MHz, CDCl3) δ 8.66 (s, 2H), 8.64 – 8.59 (m, 2H), 8.54 (d, J = 8.0 Hz, 2H), 7.86 (q, J = 8.3 Hz, 4H), 7.76 – 7.68 (m, 3H), 7.24 – 7.17 (m, 2H), 4.22 (t, J = 7.2 Hz, 2H), 1.85 – 1.74 (m, 2H), 1.25 – 1.08 (m, 14H), 0.82 – 0.74 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 155.94, 155.78, 149.19, 148.93, 146.87, 137.58, 136.66, 131.42, 127.50, 125.98, 123.69, 121.16, 119.77, 118.26, 50.29, 31.72, 30.18, 29.34, 29.27, 29.13, 28.88, 26.37, 22.54, 14.01. HRMS (ESI): m/z calcd. for C33H37N6 [MH+] 517.3080; found 517.3100.

16

4.3. Synthesis of complexes The 4'-(1-decyl-2,3-triazol-4-yl)phenyl-2,2':6',2''-terpyridine (L) (0.075 g, 0.15 mM), appropriate chloride (0.18 mM/0.07 mM) and dimethylformamide (10 mL) were poured into a 25 mL round-bottom flask. The mixture was heated at 90°C for 1.5 hours. After that, the mixture was cooled and the obtained precipitate was filtered through a fritted funnel G3 and washed with water and diethyl ether.

ZnL - the product was obtained as a light yellow solid in a 74 % yield (34 mg). 1

H NMR (400 MHz, DMSO) δ 9.10 (s, 2H), 8.94 (d, J = 7.2 Hz, 2H), 8.81 (d, J = 22.2 Hz,

2H), 8.42 – 8.27 (m, 3H), 8.11 (d, J = 7.5 Hz, 2H), 7.87 (s, 2H), 7.77 – 7.65 (m, 1H), 7.57 (t, J = 7.4 Hz, 1H), 4.43 (t, J = 6.6 Hz, 2H), 1.90 (s, 2H), 1.27 (d, J = 25.8 Hz, 14H), 0.83 (d, J = 6.7 Hz, 3H). HRMS (ESI): m/z calcd. for C33H36ZnN6 [M]2+ 290.1141, found 290.1137. ZnL2 - the product was obtained as a yellow solid in a 63% yield (107 mg). 1

H NMR (400 MHz, DMSO) δ 9.13 (s, 4H), 8.97 (d, J = 6.7 Hz, 4H), 8.84 (s, 4H), 8.79 (s,

4H), 8.36 (d, J = 11.6 Hz, 6H), 8.12 (d, J = 7.1 Hz, 4H), 7.88 (s, 4H), 4.43 (t, J = 6.6 Hz, 4H), 1.94 – 1.83 (m, 4H), 1.23 (s, 28H), 0.83 (d, J = 6.8 Hz, 6H). HRMS (ESI): m/z calcd. for C66H72ZnN12 [M]2+ 548.2642, found 548.2634. CdL2 - the product was obtained as a light brown solid in a 41% yield (73 mg). 1

H NMR (400 MHz, DMSO) δ 9.12 (s, 4H), 9.02 (d, J = 6.6 Hz, 4H), 8.80 (s, 2H), 8.58 (s,

4H), 8.37 (d, J = 6.6 Hz, 4H), 8.30 (s, 4H), 8.14 (d, J = 7.8 Hz, 4H), 7.74 (s, 4H), 4.44 (t, J = 6.5 Hz, 4H), 1.90 (s, 4H), 1.26 (d, J = 25.4 Hz, 28H), 0.83 (d, J = 6.7 Hz, 6H). HRMS (ESI): m/z calcd. for C66H72CdN12 [M+4H]2+ 575.2669, found 575.3151. CuL2 - the product was obtained as a dark turquoise solid in a 48% yield (82 mg). 1

H NMR – no attribution of peaks due to the paramagnetic nature of the compound. HRMS

(ESI): m/z calcd. for C66H72CuN12 [M]2+ 547.7644, found 547.7636.

4.4. Cell culture conditions The human colon cancer cell lines HCT 116, the human breast carcinoma cell line MCF-7 and the human lung carcinoma cell line A549 were obtained from ATCC. The pancreatic cell line PANC-1 was purchased from Sigma-Aldrich. The glioblastoma cell line U-251 was kindly provided by prof. G. Kramer-Marek from the Institute Cancer Research in London, UK. The 17

normal human fibroblast cell lines NHDF were obtained from PromoCell. The cells were grown as monolayer cultures in Dulbecco’s modified Eagle’s medium with 1% v/v of penicillin/streptomycin (Gibco) in 75 cm2 flasks (Nunc). The DMEM for HCT 116, MCF-7, A549, U-251, PANC-1 were supplemented with 12% heat-inactivated foetal bovine serum and for the NHDF with 15% non-inactivated foetal bovine serum (all from Sigma-Aldrich). The cells were cultured under standard conditions at 37°C in a humidified atmosphere at 5% CO2. All of the cell lines were subjected to routine mycoplasma testing using the PCR technique with specific Mycoplasma primers in order to ensure that there was no contamination.

4.5. Cytotoxicity studies The cells were seeded in 96-well plates (Nunc) at a density of 5·103 cells/well (HCT 116, MCF-7, A549, U-251, PANC-1) and 4·103 cells/well (NHDF) and incubated at 37°C for 24 h. The assay was performed following a 72-h incubation with varying concentrations of the compounds that were being tested. Then, 20 µL of CellTiter 96®AQueous One Solution-MTS (Promega) was added to each well (with 100 µL DMEM without phenol red) and incubated for 1 h at 37°C. The optical densities of the samples were analysed at 490 nm using a multiplate reader (Synergy 4, Bio Tek). The results are expressed as a percentage of the control and were calculated as the inhibitory concentration (IC50) values (using GraphPad Prism 7). The IC50 parameter was defined as the compound concentration that was necessary to reduce the proliferation of cells to 50% of the untreated control. Each compound was tested in triplicate in a single experiment with each experiment being repeated three or four times.

4.6. Impact of the metal ions on cellular proliferation The MCF-7 cells were seeded in 96-well plates (Nunc) at a density of 5·103 cells/well and incubated at 37°C for 24 h. The MTS assay was performed following a 72-h incubation with varying concentrations of the tested compound – L. Additionally, a 20 µM solution of CuSO4, FeSO4, FeCl3, ZnCl2, or a 5 µM solution of CdSO4 was added into wells with the tested compounds. The controls that had been supplemented with these solutions did not affect cell viability (>95 %). The results are expressed as a percentage of the control and were calculated as the inhibitory concentration (IC50) values using GraphPad Prism 7. Each compound was tested in triplicate in a single experiment with each experiment being repeated three times.

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4.7. Cell cycle assay The MCF-7 cells were seeded in 3 cm Petri dishes (Nunc) at a density of 0.25·106 cells/well and incubated at 37°C for 24 h. Then, the medium was removed and freshly prepared solutions of the tested L and CuL2 at concentrations of 1 µM, 2.5 µM and 5 µM were added. After a 24-h treatment, the assays were performed using a Muse Cell-Cycle Kit (Millipore) according to the manufacturer's instructions. Briefly, the cells were collected, washed with cold PBS and centrifuged at 300g. Then, the cells were fixed in ice-cold 70% ethanol and stored at -20°C overnight. Afterwards, the cells were centrifuged and resuspended in 200 µL of Muse™ Cell Cycle Reagent and incubated for 30 min at room temperature in the dark. After staining, the cells were processed for cell cycle analysis using a Muse Cell Analyzer (Millipore). The experiments were performed at least three times.

4.8. Annexin V binding assay The MCF-7 cells were seeded in 3 cm Petri dishes (Nunc) at a density of 0.25·106 cells/well and incubated at 37°C for 24 h. Then, the medium was removed and freshly prepared solutions of the tested L and CuL2 at concentrations of 1 µM, 2.5 µM and 5 µM were added. After 48 h, the assays were performed using an Annexin V & Dead Cell Kit (Millipore) according to the manufacturer's instructions. Briefly, the detached and adherent cells were collected and centrifuged at 500 g for 5 min. Afterwards, the resuspended cells were incubated with 100 µL of Muse™ Annexin V & Dead Cell Reagent for 20 min at room temperature in the dark. After staining, the events for live, early and late apoptotic cells were counted using a Muse Cell Analyzer. The experiments were performed at least three times.

4.9. Apoptosis assay Morphological analysis of apoptosis using Acridine Orange (AO)/Ethidium Bromide (EB) fluorescent staining was performed. The MCF-7 cells were seeded on glass slides at a density of 1·105 cells/slide and incubated at 37° C for 24 h. Then, the medium was removed and a solution of L at a concentration of 5 µM was added. After 24 h, the cells were washed with PBS and 50 µL DMEM without phenol red and a 2 µL AO/EB (100 µg/mL in PBS, Sigma) solution was added. The cells were stained for 2-3 min, then washed with PBS and fixed with 3.7% paraformaldehyde for 15 minutes. The morphological changes in the cells were monitored under a Nikon epifluorescence microscope model Ni-U, Nikon Eclipse (Nikon, Poland) using the 485 nm excitation laser and a 520 nm emission filter. 4.10. Quantitative measurement of the level of ROS 19

The MCF-7 cells were seeded onto black 96-well plates (Corning) at a density of 9 ·103 cells/well and incubated at 37°C. After overnight incubation, the solutions of the tested L and CuL2 (both 1 µM) were added and incubated for 1, 3, 6, 9, 12 and 24 h in a kinetic

experiment. The generation of ROS was measured using a CellROX® Green Reagent (Molecular Probes). Additionally, the quantity of cells in each well was determined using Hoechst 33342 (Molecular Probes). The solutions of the tested compounds were removed and 100 µL of CellROX Green Reagent and Hoechst 33342 at a final concentration of 5 µM were added to each well. Then, the cells were incubated for 30 min at 37°C. The fluorescence was measured using a multi-plate reader (Synergy 4, Bio Tek) at a 485 nm excitation and a 520 nm emission for the CellROX Green Reagent and a 345 nm excitation laser and a 485 nm emission filter for Hoechst 33342. The experiments were performed three to four times. The levels of ROS are expressed as a percentage of the level of the control cells.

4.11. Subcellular localisation The MCF-7 cells were seeded onto glass slides at a density of 3·105 cells/slide and incubated at 37°C for 24 h. Then, the medium was removed and the solution of L at a concentration of 25 µM was added and further incubated for 2 h. Then, the cells were rinsed with PBS (pH 7.2) and a serum-free medium that contained MitoTracker® Orange (100 nM, 30 min incubation, Molecular Probes), LysoTracker® Red DND-99 (500 nM, 1 h incubation, Molecular Probes), ER-Tracker™ Red BODIPY® TR Glibenclamide (1 µM, 30 min incubation, Molecular Probes) or TO-PRO®-3 Iodide (642/661) (1 µM, 30 min incubation, Molecular Probes) was added. After staining with the organelle-specific trackers, the cells were washed three times with PBS and fixed by 3.7 % paraformaldehyde for 10 minutes. Subcellular localisation was observed using a Nikon Eclipse Ni-U microscope equipped with a Nikon Digital DS-Fi1-U3 camera with the corresponding software (Nikon, Tokyo, Japan). The analysis and processing of the images were performed using an ImageJ 1.41 (Wayne Rasband, National Institutes of Health, USA). The Pearson’s coefficient, which was used to show the co-localisation of L with the specific-organelle trackers, were calculated using the Image J plugin “JACoP”.

4.12. Immunoblotting The MCF-7 cells were seeded in 3 cm Petri dishes at the density of 0.5·106 cell/dish and incubated overnight. The next day, solutions of L and DOX (2.5 and 5 µM, respectively) were added and the cells were incubated for 24 h. Then, the cells were detached via trypsinisation, collected in tubes and centrifuged at 2000 rpm. Next, the cell pellets were suspended in 150 20

µL of a complete RIPA Buffer containing a Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail along with 0.5 M EDTA (all reagents from Thermo Scientific) and lysed on ice for 20 min on a rocking plate. Subsequently, the obtained lysates were sonicated and centrifuged at 10 000 rpm for 10 min at 4°C and the supernatants were collected for further analysis. The protein concentration was measured using a Micro BCA™ Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instruction. Equal amounts of the proteins (14 µg) were electrophoresed on SDS-Page gels and transferred onto nitrocellulose membranes. The membranes were blocked in 5% non-fat milk prepared in TPBS (PBS containing 0.1% Tween-20 for 1 h). After blocking, the membranes were incubated with the specific primary monoclonal antibodies at 1:1000 dilution: p53 (1C12), p21Waf1/Cip1 (12D1), cytochrome c (136F3), AIF (D39D2) and at 1:2000 dilution in the case of GAPDH (14C10) overnight at 4°C. The next day, the membranes were washed in TPBS and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. All of the monoclonal and secondary antibodies were purchased from CellSignaling. Lastly, the membranes were washed in TPBS and incubated with a SuperSignal™

West

Pico

Chemiluminescent

Substrate

(Thermo

Scientific).

The

chemiluminescence signals were captured using a ChemiDoc™ XRS+ System (Biorad). The experiments were performed at four times. The densitometric analysis was performed using ImageJ software.

4.13. Intercalation DNA For the DNA binding studies, Calf-thymus DNA (CT-DNA) was purchased from SigmaAldrich. The lyophilised CT-DNA was dissolved in 10 mM Tris-HCl, pH 7.9, mixed gently and left overnight at 4°C. The purity of the CT-DNA solution was determined by measuring the ratio of the UV absorbance at 260 and 280 nm. A ratio of more than 1.8 indicated that the DNA was sufficiently free from proteins. Then, the concentration of CT-DNA was determined from the absorbance at 260 nm using an extinction coefficient of 6600 M-1cm-1. The tested L and DOX were dissolved in DMSO to a concentration of 8.35 mM, which was then used as the stock solution to prepare the various concentrations (25, 12.5, 6 and 3 µM) in 1 mL in 10 mM of Tris-HCl (pH 7.9). Afterwards, 18 µM CT-DNA was added to the prepared solutions, which were incubated for 1.5 h at 37°C with occasional vortexing. The absorption spectra were measured using a Hitachi U-2900 spectrophotometer within the range of 200-500 nm. All of the absorption spectra were imported and compared in OriginPro 8.

21

4.14. DNA cleavage studies For the DNA cleavage studies, supercoiled pUC19 DNA plasmid was purchased from SigmaAldrich. The concentration of plasmid DNA was determined by measuring the absorbance of the solution containing the DNA at 260 nm. One optical unit corresponds to 50 mg/mL of double-stranded DNA. The interaction of L and its metal complexes (ZnL, ZnL2, CdL2, CuL2) with the pUC19 DNA plasmid was monitored using agarose gel electrophoresis. The tested compounds were dissolved in DMSO to a concentration of 8.35 mM, which was then used as the stock solution to prepare the various concentrations (25 µM, 50 µM and 100 µM) in 1 mL in 50 mM NaCl/5 mM Tris–HCl (pH 7.1). The cleavage reaction of the supercoiled pUC19 DNA by the tested compounds was investigated by incubating 260 ng of pUC19 in a 20 µL reaction mixture in NaCl/Tris-HCl buffer solution at 37°C, for 60 min. The reaction mixtures were vortexed from time to time. The reaction was terminated by a short centrifugation at 10 000 rpm and the addition of 5 µL of a loading buffer. The samples were subjected to electrophoresis on 1% agarose gel containing 1 µL of ethidium bromide (10 mg/mL concentration), which had been prepared in a 1X SB buffer. The electrophoresis was performed at a constant voltage (80 V) until the bromophenol blue had passed through 75% of the gel – about 2 h. The gels were viewed in a Molecular Imager® ChemiDoc™ XRS System and photographed using a CCD camera. The cleavage efficiency was measured by determining the ability of the complex to convert the supercoiled DNA (SC, form I) to a nicked circular form (NC, form II) and linear form (LC, form III). The analysis of images was performed using an ImageJ 1.41.

4.15. Statistical analysis The results are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis for the ROS measurements was performed using the two-tailed Student’s t-test. Statistical differences in the progression of the cell cycle, the Annexin V binding assay and immunoblotting were calculated using a one-way ANOVA with a Bonferroni post-hoc test. A p-value of 0.05 or less was considered to be statistically significant. GraphPad Prism 7 software was used for analysis.

Authors’ contributions

22

KM and AMW developed the research hypothesis and designed and performed the biological tests; MK performed the ROS measurements; DZ planned and performed the chemical syntheses; KM, AMW and RM wrote the manuscript.

Acknowledgements The financial support of the National Science Centre grants 2016/23/N/NZ7/00351 (K.M.) and 2018/31/B/NZ7/02122 (R.M.) is greatly appreciated.

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• • •

Complexes of 4'-phenyl-2,2':6',2''-terpyridine have been synthesized and tested for anticancer activity Anticancer activity depends on DNA intercalation and ROS generation Free ligand appeared to be equally active as its complexes

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: