Electronic Coupling in 1,2,3-Triazole Bridged Ferrocenes and Its Impact on Reactive Oxygen Species Generation and Deleterious Activity in Cancer Cells

Mixed-valence (MV) binuclear ferrocenyl compounds have long been studied as models for testing theories of electron transfer and in attempts to design molecular-scale electronic devices (e.g., molecular wires). In contrary to that, far less attention has been paid to MV binuclear ferrocenes as anticancer agents. Herein, we discuss the synthesis of six 1,2,3-triazole ferrocenyl compounds for combined (spectro)electrochemical, electron paramagnetic resonance (EPR), computational, and anticancer activity studies. Our synthetic approach was based on the copper-catalyzed 1,3-dipolar azide–alkyne cycloaddition reaction and enabled us to obtain in one step compounds bearing either one, two, or three ferrocenyl entities linked to the common 1,2,3-triazole core. Thus, two series of complexes were obtained, which pertain to derivatives of 3′-azido-3′-deoxythymidine (AZT) and 3-azidopropionylferrocene, respectively. Based on the experimental and theoretical data, the two mono-oxidized species corresponding to binuclear AZT and trinuclear 3-azidopropionylferrocene complexes have been categorized as class II mixed-valence according to the classification proposed by Robin and Day. Of importance is the observation that these two compounds are more active against human A549 and H1975 non-small-cell lung cancer cells than their congeners, which do not show MV characteristics. Moreover, the anticancer activity of MV species competes or surpasses, dependent on the cell line, the activity of reference anticancer drugs such as cisplatin, tamoxifen, and 5-fluorouracil. The most active from the entire series of compounds was the binuclear thymidine derivative with the lowest IC50 value of 5 ± 2 μM against lung H1975 cancer cells. The major mechanism of antiproliferative activity for the investigated MV compounds is based on reactive oxygen species generation in cancer cells. This hypothesis was substantiated by EPR spin-trapping experiments and the observation of decreased anticancer activity in the presence of N-acetyl cysteine (NAC) free-radical scavenger.


■ INTRODUCTION
Mixed-valence (MV) species derived from d-transition-metal complexes are fascinating objects for chemical and spectroscopic studies. In particular, they are attractive from the perspective of basic studies on electron transfer processes as well as investigation of magnetic exchange interaction phenomena. 1−9 Moreover, MV compounds are considered to be a source of components and devices for the emerging field of molecular electronics. 7,8,10−13 The rate of electron delocalization (electronic coupling or communication) in MV species can be examined by a variety of analytical techniques including electrochemistry, ultraviolet/visible (UV−vis) spectroscopy, near-infrared (NIR) spectroscopy, electron paramagnetic resonance (EPR), and Mossbauer spectroscopy. 14−16 Each of them operates in different time scale. Therefore, to accurately assess the extent of electron delocalization, a combination of slower (EPR and Mossbauer) and faster (UV−vis/NIR) techniques is desirable. Accessible with electrochemical measurements, half-wave potential splitting (ΔE 1/2 ) often provides a misleading approximation of the amount of electronic coupling in MV compounds. 14 A much more reliable measure of electron coupling in MV systems is provided by the electronic coupling matrix element H ab (V ab ). H ab can be determined from the intervalence charge transfer (IVCT) band and using Hush's two-state model according to eq 1S (see the Supporting Information (SI)). 17,18 According to the classification of Robin and Day, there are three classes of MV compounds. 19 Class I comprises valencetrapped systems, class II comprises weakly coupled systems, and class III comprises valence delocalized systems. In fully delocalized class III systems, the electronic coupling matrix element H ab is half the energy at the IVCT band maximum, whereas in class I compounds, the IVCT band is not present.
Reported in 1951, ferrocene (FcH = Fe(η 5 -C 5 H 5 ) 2 ) has become a cornerstone of modern organometallic chemistry. 20,21 In the last 71 years, ferrocenyl (Fc) compounds have found many applications in catalysis, biology, materials chemistry, and so forth. 22−35 One of the reasons behind this success is due to the electrochemical properties of ferrocene and its derivatives. The Fc/[Fc] + redox couple is usually characterized by superb chemical reversibility combined with great thermal stability. 36 Thus, compounds containing Fc groups linked by aromatic or π-electron cyclic or acyclic bridges have been recognized as a source of MV species that are nicely suited for electronic communication studies. 37 In this respect, bridges such as benzene, 38,39 pyridine, 40 1,3,5triazine, 40 pyrrole, 41−43 thiophene, 44−48 selenophene, 49 thiadiazole, 48 thiazole, 50 phosphole, 51,52 and silole, 53 to name just a few, have been studied. The Fc/[Fc] + redox couple has also found numerous applications in biology. It can be tentatively categorized as analytical and therapeutic. Regarding the former, adequately designed ferrocenylated DNA oligomers have been applied for single-base mismatches 54 and viral DNA 55 electrochemical detection as well as for redox coding of nucleobases and their ratiometric sensing. 56 The role of redox chemistry in therapeutic applications of ferrocene derivatives is exemplified by a family of ferrocifen drugs. 33,57 The mechanism of action of these remarkably anticancer-active compounds begins with single oxidation of the Fc entity, which is embedded in the "ferrocenyl-ene-phenol" structural motif.
A high concentration of reactive oxygen species (ROS) in cancer cells is a well-established phenomenon 58 that is utilized for activation of aminoferrocene-based antitumor prodrugs. 59,60 In brief, their mechanism of action includes the initial ROS-activated cleavage of the phenylboronic acid "cap" from the prodrug, which then enables fragmentation of the thus-obtained molecule to form organic quinone methide (QM) and ferrocenium ion products. 59 Ferrocenium ions themselves or liberated from them Fe 2+/3+ ions react with endogenous ROS to further elevate oxidative stress (OS) in cancer cells, which finally leads to deleterious effects. Yet another relevant example of redox-activated anticancer-active ferrocenes pertains to ferrocene-(vinyl)Ru(CO)Cl(P i Pr 3 ) 2 compounds A and B (Figure 1). 16,61 These compounds differ from ferrocifenes and aminoferrocene prodrugs as their molecular structure features two nonequivalent metal redox centers. Combined (spectro)electrochemical, EPR, and Mossbauer studies on B revealed that it belongs to class II MV systems. 16 Interestingly, compound B showed high anticancer activity in HT-29 colon carcinoma and MCF-7 breast cancer cells in vitro. 61 Its activity exceeded that of A, and it was much better in terms of activity than the corresponding mononuclear ferrocenyl and ruthenium complexes used as references in the same study. 61 Remarkable biological activity of A and B has stimulated our interest in the development of new mixed-valence ferrocenyl systems as anticancer agents.
Inorganic Chemistry pubs.acs.org/IC Article the source material for compounds 1a−c was motivated by the biological significance of deoxythymidine nucleoside and general importance of CuAAC reactions in nucleic acid chemistry and biology. 64,68 Taking into account the above motivation, compounds 1a and 2a as well as their mononuclear analogues 1c and 2c were used to study their anticancer activity in human A549 and H1975 non-small-cell lung cancer (NSCLC) cells and nonmalignant bronchial epithelium BEAS-2B cells. Anticancer activity assays have been also performed in the presence of free-radical scavenger N-acetyl cysteine (NAC) to investigate the impact of ROS on compounds' activity.

■ RESULTS AND DISCUSSION
Synthesis. Compounds 1a and 2a belong to 5-alkynyl-1,2,3-triazoles, a subclass of highly substituted 1,2,3-triazole derivatives with great potential for synthetic chemistry. A literature survey shows several synthetic approaches giving an access to this class of compounds. 69−73 One of them relies on the palladium-catalyzed Sonogashira cross-coupling reaction of 5-iodo-1,2,3-triazoles with terminal alkynes. 65,73 Due to apparent simplicity, we have chosen this approach for the synthesis of compounds 1a and 2a. In the first step, we attempted to obtain the 5-iodo-1,2,3-triazole 1b and 2b intermediates. Their syntheses were carried out by the reaction of AZT or 3-azidopropionylferrocene (C) with ethynylferrocene (D), N-bromosuccinimide (NBS), and N,N-diisopropylethylamine (DIPEA) according to Scheme 1. 74 As expected, the respective reactions afforded 5-iodo-1,2,3triazole 1b and 2b in 9 and 24% yields, respectively. Besides this and to our satisfaction, reactions also afforded the desired compounds 1a and 2a in 39 and 22% yields, respectively. Furthermore, 4-ferrocenyl-1,2,3-triazole derivatives 1c and 2c were obtained, although in low yields of 6 and 15%, respectively. We have found that simple modifications of the reaction conditions (e.g., increase of either the reaction time and/or temperature) only resulted in a decrease of compounds 1a and 2a yield. Also, any attempt to transform 1b or 2b into corresponding compounds 1a and 2a by the Sonogashira crosscoupling reaction with ethynylferrocene (D) failed. On the contrary, the yields of compounds 1c and 2c were easily increased using the classical CuAAC reaction conditions according to Scheme 2.
Formation of 5-iodo-1,2,3-triazole 1b and 2b can be explained by the mechanism proposed by Zhang. 74 However, the observation of other reaction products suggests that further mechanism(s) can be also operational in the course of the reaction. Their investigation was out of our interest as the effort was entirely focused on electronic coupling and anticancer activity studies. After completion of the reaction and purification, compounds 1a and 2a−c were isolated as orange crystalline solids, whereas 1b and 1c were isolated as yellow crystalline solids. Characterization of all complexes was carried out with 1 H and 13 C NMR and IR spectroscopy, mass spectrometry, and elemental analyses. The 1 H and 31 C NMR spectra of 1a−c and 2a−c are shown in Figures S1−S12 (see the SI). Furthermore, the structures of 1a, 2a, and 2c in the solid state were determined by single-crystal X-ray structural analysis. Crystallographic Studies. Single-crystals of 1a, 2a, and 2c suitable for X-ray diffraction (XRD) analysis were obtained by diffusion of n-hexane in a solution of the respective complex in dichloromethane at room temperature. The crystal and structure refinement data are presented in Table S1 (see the SI). The molecular structures of 1a, 2a, and 2c with the atomlabeling scheme and selected geometrical parameters are provided in Figures 2−4, respectively. The bond distances (Å) and valence and torsion angles (deg) are given in Tables S2−S10 (see the SI). Compounds 1a and 2c both crystallized in the orthorhombic space group, P2 1 (1a) and Cc (2c). Compound 2a crystallized in the monoclinic space group P2 1 / c. In the crystals of 1a and 2a, two crystallographically independent molecules (A and B) are observed.
Crystallographic analysis confirmed the postulated structures of examined complexes and indicate their conformational flexibility (two different conformers for 1a and 2a in the crystal lattices). Particularly, for 1a and 2a, the molecular architecture in which the ferrocenyl and the ethynylferrocenyl entities are bonded to a 1,2,3-triazole scaffold in a 4,5-substitution pattern was unambiguously confirmed. The through space distance between the Fe atoms in 1a was 8.402(2) and 8.075(2) Å in conformers A and B, respectively. The analogous distance for compound 2a was 8.548(3) Å (conformer A) and 6.770(3) Å (conformer B). The sandwich Fc groups adopt intermediate conformations between the staggered and the eclipsed form. 75 Table S11 (see the SI) provides the geometrical details for  Inorganic Chemistry pubs.acs.org/IC Article these conformations. The geometry of the thymine nucleobase in 1a does not show significant differences with similar species reported in the literature. 76 Furthermore, structural analysis confirmed that the absolute configuration of the deoxyribosyl moiety present in two independent molecules of 1a in the crystal can be assigned as D (D-ribose). Of notice is, however, that the sugar conformations are different in each independent molecule. The puckering of the deoxyribosyl moiety within conformer A adopts an envelope C2′-endo conformation, whereas in conformer B, a twist C2′-endo−C3′-exo conformation is characteristic. 77,78 The numerical data for both conformations are given in Table S12 (see the SI).
(Spectro)electrochemistry. Electrochemical studies of compounds 1a, 1c, 2a, and 2c were carried out using cyclic voltammetry (CV) and square-wave voltammetry (SWV) ( Table 1 80,81 The voltammetry experiments were performed at 25°C. All potentials are referenced to the FcH/[FcH] + (Fc = Fe(η 5 -C 5 H 4 )(η 5 -C 5 H 5 )) redox couple (E°′ = 0 mV). 82 The cyclic voltammogram of 1a shows two separated reversible redox events at 80 and 280 mV, while 2a with its further FcC(O)CH 2 CH 2 unit features in total three redox processes at 45, 280, and 365 mV vs FcH/[FcH] + , as expected ( Figure 5 and Table 1). To assign the appropriate redox waves, compounds 1c and 2c were measured under identical conditions. It was found that the ferrocenyl-based redox event of 1c appears at 60 mV and the ones of 2c appear at 20 and 330 mV (Table 1 and Figure S13, see the SI). Comparing these values leads to the conclusion that the first oxidation occurs at the Fc moiety directly bonded to the 1,2,3-triazole core. Such an assignment is consistent with data obtained for other ferrocenyl-1,2,3-triazole systems 83−85 and supported by DFT calculations (see the SI). In the following electrochemical process, the respective FcCC unit is oxidized. The potentials confirm that compound 2a is more electron-rich than 1a and hence is easier to be oxidized, whereas the follow-up redox event occurs at the same potential. The difference between the formal potentials is 200 mV for 1a and 235 mV for 2a (Table  1), pointing to the fact that monocationic [2a] + should be a somewhat more stable mixed-valent species than [1a] + (vide supra). The formal potential of the FcC(O)CH 2 CH 2 terminal group can be found at 330 (2c) and 365 mV (2a) due to the influence of the previously introduced positive charges.
The in situ electrochemical behavior of 1a ( Figure 6) and 2a ( Figure 7) was investigated by spectroelectrochemical UV− vis/NIR measurements within an optically transparent thinlayer electrochemical (OTTLE 86 ) cell with SiO 2 windows in tetrahydrofuran solutions of the analyte, containing [NBu 4 ]-[B(C 6 F 5 ) 4 ] (0.1 mol·L −1 ) as the supporting electrolyte. 87,88 In the course of the measurements, the applied cell potential was increased stepwise (step width: 25, 50, or 100 mV). At the end of each measurement, the analyte was reduced at −500 mV vs Ag/AgCl for 30 min, and an additional spectrum was recorded to prove the reversibility of the oxidation. The spectroelectrochemical UV−vis/NIR data of 1a in tetrahydrofuran display weak absorptions in the NIR region between 0 and 250 mV vs Ag/AgCl upon formation of the mixed-valent species [1a] + ( Figure 6). A further increase of the potential leads to the generation of dicationic [1a] 2+ (250−500 mV vs Ag/AgCl).
80 (60) 4 ] as the supporting electrolyte at 25°C. b E°′ = formal potential. c ΔE p = difference between the cathodic and anodic peak potentials |E pc − E pa |. d K C = comproportionation constant K C = exp(nF/RT)ΔE 1/2 , F = Faraday constant, R = gas constant, T = temperature, ΔE 1/2 = difference of half-wave potentials, n = number of transferred electrons.      Figure  S14, see the SI). Based on these values, 89 the electronic matrix coupling element V ab (H ab ) (eq 1S, see the SI) can be calculated and results in 100 cm −1 for 1a and 127 cm −1 for 2a, confirming the weak nature of their electronic coupling.
In the example of 1a, spectroelectro-IR studies were carried out applying an OTTLE cell with CaF 2 windows under identical measurement conditions (vide infra). Oxidation of neutral 1a to monocationic [1a] + leads to higher intensities of the triple bond vibrational band, which is accompanied by a shift from 2214 to 2210 cm −1 (Figure 8). Smaller wavenumbers imply that the carbon−carbon triple bond comprises more electron density in [1a] + , proposing that electron transfer between the ferrocenic species passes through the carbon− carbon triple bond, making this a "through-bond" electron transfer process. A further increase of potential leads to the generation of [1a] 2+ , which is followed by a characteristic shift of the band from 2210 to 2216 cm −1 . This observation is the result of decreased electron density due to both ferrocenyl systems featuring Fe 3+ ions. Therefore, electron delocalization between the Fc and FcCC units via the 1,2,3-triazole connectivity is reduced compared to [1a] + .
DFT Calculations. To gain more detailed insight into the electronic structures of the examined compounds, calculations were carried out at the BLYP/6-31+G(d)/LanL2DZ level of DFT theory 90 utilizing the Gaussian 16 code. 91 Details on structural optimization and calculations are provided in the Experimental Section and the SI. According to DFT calculations, the highest occupied molecular orbital (HOMO) orbital of 1a, 1c, 2a, and 2c is localized at the ferrocenyl group directly bonded to the 1,2,3-triazolyl moiety ( Figure S15). Upon first oxidation, one electron (a β spin state) is removed from the 3d xy orbital of the ferrocene ring. The 3d xy orbital becomes the singly occupied molecular orbital (SOMO) for the α-electron and the lowest unoccupied molecular orbital (LUMO) in the β-electron configuration in the oxidized species. In the case of [1a] + and [2a] + , the spin density is not located on one ferrocenyl group but expands over the ca. 11 Å ferrocenyl-1,2,3-triazolyl-ethynylferrocenyl part of the molecule (Figure 9). This feature provides additional evidence for the possibility of electron communication between the two Fc moieties in [1a] + and [2a] + . However, the spin density is not uniformly distributed over the 1,2,3-triazolyl bridge. Its highest contribution is on the two carbon (formally CC bond) and the middle nitrogen atom of the 1,2,3-triazolyl core.
DFT calculations were found very useful with respect to spectroelectro-IR study result interpretation. Accordingly, an excellent agreement between experimental and calculated C C bond stretching frequencies was obtained (Table S13, see the SI). This further validates our theoretical approach and supports the experimental evidence of the electron transfer between the two ferrocenyl moieties in [1a] + . In the dicationic species [1a] 2+ , however, the "through-bond" electron transfer was lost, as both ferrocenyl units exist in the Fe 3+ form. According to calculations, the ground state of [1a] 2+ was found to be a triplet state (rather than a single state) with the two singly occupied MOs ( Figure S16, see the SI). Interestingly, the relative increase in the CC stretching frequency ([1a] + < 1a < [1a] 2+ ) correlates well with the calculated CC bond length in the respective series (Table S13, see the SI): with an increase in frequency, the bond becomes shorter. The relative change is small but indicative. This also supports the involvement of the CC bond in Fe 2+ −Fe 3+ delocalization in [1a] + on the intrinsic IR time scale and the lack of the corresponding communication between the two ferrocenyl entities in [1a] 2+ .
Electron Paramagnetic Resonance (EPR) Spectroscopic Study. With the purpose of gaining better insights into the charge delocalization in one-electron oxidized compounds, we performed in situ EPR spectroelectrochemical measurements for compounds 1a and 2a. While the organic radical could be obtained at room temperature, an anisotropic signal of the ferrocenium ion is only detectable at low temperature (below 77 K) due to fast spin-lattice relaxation. The EPR spectra of electrochemically generated [1a] + and [2a] + show no signals at 298 and 85 K. The absence of any signals during the first redox event under specified conditions indicates that the oxidation process in the compounds is predominantly located on the ferrocenyl moiety at the EPR time scale, substantiating the presence of the weakly coupled class II MV system according to Robin and Day. Thus, further information about the electronic coupling between the  Instead, the EPR spin-trapping technique was employed to detect short-lived free radicals (reactive oxygen species; ROS) generated in dimethylformamide (DMF) solutions of ferrocene compounds in the presence of molecular oxygen. Free radicals are key cell-damage causative agents that are often generated by ferrocenium species inside cancer cells. 27,31,59−61 It was therefore justified to check whether our compounds are also capable of free-radical generation. In this regard, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a spin trap. The EPR spectra measured in air-saturated DMF solutions selected for measurement compounds of 1a, 1c, and 2a show a mixture of DMPO adducts, indicating the production of several free radicals ( Figure 10).
On the basis of the hyperfine splitting constants of DMPO adducts, 92 the main radicals formed in the systems are oxygencentered ones (superoxide radical anion O 2 •− and its protonated form hydroperoxyl radical • OOH). The simulated spectra fit very well with the experimental ones ( Figure S17, see the SI). EPR parameters of the spin trap adducts obtained from simulations of experimental spectra are presented in Table 2.
Under O 2 -saturated conditions, the signal of the superoxide radical anion adduct of DMPO is significantly broadened due to the high concentration of radicals in the solution (Figures 10b and S18a). All of these observations are the confirmation of a single-electron-transfer reaction between a ferrocenyl group and molecular oxygen, resulting in the formation of superoxide anion radicals. It should be also noted that the concentration of the radicals formed in the system with 1c is much lower than that with 1a and 2a. It indicates that the binuclear compounds containing ferrocenyl and ethynylferrocenyl moieties are more effective ROS generators. In an inert (N 2 ) atmosphere, carbon-centered (alkyl) radicals are mainly formed (Figures 10b and S18b, see the SI). Radicals • CH 3 and • CH 2 N(CH 3 )CHO have been earlier found as a result of ultrasound-induced pyrolysis of DMF. 93 The main DMPO adducts obtained under an inert atmosphere can be assigned to DMPO/ • CH 3 and DMPO/ • CH 2 N(CH 3 )CHO. The alkyl radicals of DMF are also present in small amounts in airand O 2 -saturated solutions.
Antiproliferative Activity. Our first reports on anticanceractive MV ferrocenyl compounds occurred over a decade ago. 16,61 Recently, they were followed by another report on anticancer-active electronically coupled ferrocene systems. 94 Herein, the antiproliferative activity of 1a, 1c, 2a, and 2c is examined in human NSCLC A549 and H1975 cells as well as against nonmalignant human bronchial epithelium BEAS-2B cells. The calculated IC 50 concentrations after 72 h of compound incubation with the cells are shown in Table 3 (cell survival curves related to IC 50 values are provided in Figures S19−S27).
The most active complexes among ferrocenyl compounds tested were 1a and 2a. Noticeably, compound 1a was more active against H1975 cells than tamoxifen and 5-fluorouracil and almost equally active as cisplatin (5 ± 2 (1a) vs 4 ± 0.1 μM(cisPt)). Furthermore, it was found that 1a was more active  In other words, the most anticancer-active compound, 1a had an IC 50 value of 469 ± 10 μM against BEAS-2B cells, respectively, which is about 156-, 52-, and 78-times higher values than the IC 50 values for cisplatin, tamoxifen, and 5fluorouracil (3 ± 0.1, 9 ± 0.2, and 6 ± 0.1 μM), respectively, against the same BEAS-2B cells. Antiproliferative activity assays showed that cancer cells rich in ROS 58,95 are more susceptible to 1a and 2a in comparison to normal BEAS-2B cells. Likewise, mononuclear compounds 1c and 2c showed negligible activity in either cancer or noncancerous cells. For anticancer activity, the presence of two electronically connected ferrocenyl groups is required. However, of 1a and 2a compounds, the latter had one ferrocenyl entity more than the former but it shows a lower anticancer effect. This observation indicates that also the nucleotide thymidynyl entity contributes to the anticancer effect as well as the fact that a simple increase of the number of redox-active ferrocenyl centers in a given scaffold does not immediately lead to the improved anticancer effect. In general, antiproliferative activity studies are in agreement with our earlier observation of the high anticancer activity of MV ferrocenyl compounds. 16,61 Oxidative stress (OS) resulting from ROS production is an important factor that takes part in the anticancer activity of organometallic compounds. 27,31,59,60 Concerning that, the aim of the following studies was to examine whether studied compounds generate ROS in cancer cells and how the viability of the treated cells changes in the presence of N-acetyl cysteine (NAC) free-radical scavenger. 96  Compounds 1a and 1c were more effective ROS generators than cisplatin, tamoxifen, and 5-fluorouracil in both cancer cell types. Of the two ferrocene compounds, the most effective ROS generator was binuclear complex 1a. It generated about 1.6 and 2.5 times more ROS than 1c in H1975 and A549 cells. Furthermore, 1a was about 2 and 2.5 times more potent in ROS generation than reference drugs in H1975 and A549 cells. The addition of NAC had almost no effect on ROS generation by cisplatin, tamoxifen, and 5-fluorouracil. Oppositely, the ROS amount produced by 1a in NAC-treated A549 and H1975 cells was approximatively between 0.4 and 0.8 times lower compared to A549 and H1975 NAC nontreated cells. This definitely pin points a key role of ROS in the mechanism of the anticancer action of 1a and corroborates with EPR study results (see the Electron Paramagnetic Resonance (EPR) Spectroscopic Study section). Further support for the pivotal role of ROS in inducing compound 1a anticancer activity was provided by the viability assays (Figures 12, S29, and S30, see the SI).
Cells treated with NAC were partially protected from the deleterious influence of compound 1a. Accordingly, the viability of H1975 cells treated with NAC and compound 1a increased approximately to 20% compared to cells treated only with compound 1a ( Figure 12) and an analogous increase was also observed for A549 and BEAS-2B cells (Figures S29 and  S30, see the SI). These results once again pinpoint the induction of OS/ROS as a key factor responsible for the antiproliferative activity of 1a.    19 The EPR study shows that 1a and 2a are better ROS generators than mononuclear complex 1c. Importantly, 1a and 2a showed higher anticancer activity toward A549 and H1975 NSCLC cells than their non-mixed-valence generating counterparts 1c and 2c. Their anticancer efficacy was similar to the efficacy of well-established anticancer drugs such as cisplatin, tamoxifen, and 5-fluorouracil. Of note, 1a and 2a are also characterized by very low toxicity against normal BEAS-2B cells. Observed with EPR studies, the ability for ROS generation of compounds 1a and 2a was further observed in vitro in A549 and H1975 cancer cells. Obtained data allow concluding that the highly deleterious effects of 1a and 2a in investigated cancer cells are primarily due to the ROS and oxidative stress generation. However, the increased ability for ROS generation is not the only mechanism through which these compounds work. This supposition is corroborated by the fact that thymidine derivative 1a has higher anticancer activity than triferrocenyl compound 2a, but of the two compounds, the latter one (2a) is more electron-rich and thus is more susceptible to oxidation in cancer cells. This observation underlines that the thymine portion of compound 1a has also contributed to the anticancer effect. This might be a valuable starting point for the design of new ferrocenyl mixed-valence systems conjugated to nucleic acid components such as nucleosides or nucleotides.

■ EXPERIMENTAL SECTION
General Considerations. All preparations were carried out using standard Schlenk techniques. Chromatographic separations were performed using silica gel 60 (Merck, 230−400 mesh ASTM). Azidothymidine (AZT) and ethynylferrocene were purchased from a commercial supplier and used without prior purification. Solvents were of reagent grade and also used without prior purification. 3-Azidopropanoylferrocene was synthesized according to the literature guidelines. 97 1 H NMR (600 MHz) and 13 C{H} NMR (150 MHz) spectra were recorded with a Bruker ARX 600 spectrometer operating at 298 K in Fourier transform mode. Chemical shifts are given in δ units (ppm) using residual dimethyl sulfoxide (DMSO) ( 1 H δ 2.50 ppm, 13 C δ 39.5 ppm) or CDCl 3 ( 1 H δ 7.26 ppm, 13 C δ 77.0 ppm) peaks as a reference. All of the mass spectra were recorded using a Synapt G2-Si mass spectrometer (Waters) equipped with an electrospray ionization (ESI) source and a quadrupole time-of-flight (quadrupole-TOF) mass analyzer. The mass spectrometer was operated in the positive ion detection mode. The measurements were performed with the capillary voltage set to 2.7 kV and the sampling cone voltage set to 20 V. The source temperature was 110°C . To ensure the accuracy of mass measurements, data were collected in the centroid mode and mass was corrected during acquisition using leucine enkephalin solution as an external reference (Lock-Spray). The results of the measurements were processed using MassLynx 4.1 software (Waters) incorporated with the instrument. The IR spectra were recorded on a Fourier transform infrared (FTIR) Nexus Nicolet apparatus. Microanalyses were performed by Analytical Services of the Polish Academy of the Sciences (Łodz).
Synthesis of 1a−c. A Schlenk tube charged with AZT (120 mg, 0.45 mmol, 1.0 equiv), ethynylferrocene (189 mg, 0.90 mmol, 2.0 equiv), CuI (120 mg, 0.63 mmol, 1.4 equiv), and N-bromosuccinimide (96 mg, 0.54 mmol, 1.2 equiv) was flushed with argon. Then, anhydrous THF (6 mL) and N,N-diisopropylethylamine (0.08 mL, 0.45 mmol, 1.0 equiv) were added. The resulting reaction mixture was protected against light and stirred at ambient temperature for 24 h. Then, 50 mL of 2% aqueous solution of hydrogen chloride was added and the mixture was extracted with dichloromethane (2 × 25 mL). The organic layer was separated, dried over anhydrous Na 2 SO 4 , and transferred to a round-bottomed flask, and all volatiles were evaporated under reduced pressure. After evaporation, the remaining oil was subjected to column chromatography on SiO 2 (ethyl acetate/ chloroform/methanol 35:30:3 v/v/v). Three fractions were collected. The first fraction contained compound 1a, the second contained compound 1b, and the third contained compound 1c. Chromatographically purified compounds were crystallized from a mixture of dichloromethane/n-hexane to afford analytically pure samples. Compound 1a was obtained as an orange crystalline solid in 39% (120 mg) yield, compound 1b was obtained as a yellow crystalline solid in 9% (25 mg) yield, and compound 1c was obtained as a yellow crystalline solid in 6% (12 mg) yield.
X-ray Structure Analysis. Good-quality single crystals of 1a, 2a, and 2c were selected for the X-ray diffraction experiments at T = 100(2) K. Diffraction data were collected on an Agilent Technologies SuperNova Dual Source diffractometer with CuKα radiation (λ = 1.54184 Å) using CrysAlis RED software. 98 Analytical absorption correction using a multifaceted crystal model based on expressions derived by Clark and Reid (1a and 2c) and numerical absorption correction based on Gaussian integration over a multifaceted crystal model (2a) were applied. 98,99 The structural determination procedure was carried out using the SHELX package. 100 The structures were solved with an intrinsic phasing method, and then, successive leastsquares refinement was carried out based on the full-matrix leastsquares method on F 2 using the SHELXL program. 100 All H-atoms were positioned geometrically with C−H bond lengths equal to 0.93, 0.96, 0.97, and 0.98 Å for the aromatic, methyl, methylene, and methine H-atoms, respectively, and constrained to ride on their parent atoms with U iso (H) = xU eq (C), where x = 1.2 for the aromatic, methylene, and methine and x = 1.5 for the methyl H-atoms. In the case of 1a, the N−H and O−H bond lengths were equal to 0.86 and 0.82 Å for the amine and hydroxyl H-atoms, respectively, and constrained to ride on their parent atoms with U iso (H) = xU eq (N,O), where x = 1.2 for the amine and 1.5 for the hydroxyl H-atoms, respectively. Nine out of twelve cyclopentadienyl rings in 2a were subject to RIGU restraints, whereas on the N1A, N2A, N2B, C4B, and C26B atoms, ISOR restraints were additionally applied. These types of restraints were also used during refinement of 1a. RIGU was applied to restrain cyclopentadienyl moiety defined by atoms C20A− C24A, while atoms C19A−C24A, C13B, and C20B were subject to ISOR restraints. In the case of 1a, a few distinct peaks on the difference Fourier map are indicating the presence of disordered solvent molecules. All attempts to model disordered solvents used for crystallization failed. Therefore, solvent contribution has been removed by applying the appropriate MASK procedure in the Olex2 program. 101 The calculated void volume was approximately 947.9 Å 3 occupied by 187.0 electrons per unit cell. The figures for this publication were prepared using the Olex2 program. 101 Electrochemistry. Measurements on 1.0 mmol·L −1 solutions of analytes 1a, 1c, 2a, and 2c in anhydrous dichloromethane solutions, containing 0.1 mol·L −1 [NBu 4 ][B(C 6 F 5 ) 4 ] as the supporting electrolyte, were conducted under an atmosphere of argon at 25°C. A threeelectrode cell, which utilized a Pt auxiliary electrode, a glassy carbon working electrode (surface area 0.031 cm 2 ), and an Ag/Ag + (0.01 mol·L −1 AgNO 3 ) reference electrode, was used as described in refs 82 and 102−104. Successive experiments under the same experimental conditions showed that all formal potentials were reproducible within ±5 mV. Experimental potentials were referenced against an Ag/Ag + reference electrode, but results presented are referenced against the ferrocene [FcH/FcH + couple = 220 mV vs Ag/Ag + , ΔE p = 61 mV; FcH = Fe(η 5 -C 5 H 5 ) 2 ] as an internal standard. 82 When decamethylferrocene [Fc* = Fe(η 5 -C 5 Me 5 ) 2 ] was used as an internal standard, the experimentally measured potentials were converted into E vs FcH/FcH + (under our conditions, the Fc*/Fc* + couple was at −614 mV vs FcH/FcH + , ΔE p = 60 mV).
Spectroelectrochemistry. The spectroelectrochemical measurements of 1a and 2a in anhydrous tetrahydrofuran containing [NBu 4 ][B(C 6 F 5 ) 4 ] (0.1 mol·L −1 ) as the supporting electrolyte were performed at 25°C in an optically transparent thin-layer electrochemistry (OTTLE) cell 87 with quartz windows (UV−vis/NIR, compounds 1a and 2a) by a Varian Cary 5000 spectrophotometer or CaF 2 windows (IR, 1a) with a Nicolet IR200 spectrometer (Thermo Fisher). Between the spectroscopic measurements, the applied potentials were increased stepwise using step heights of 25, 50, or 100 mV. At the end of the measurements, the analyte was reduced at −500 mV vs Ag/AgCl for 30 min, and an additional spectrum was recorded to prove the reversibility of the oxidations.
Computational Details. Structures of 1a, 1c, 2a, and 2c (oxidized/reduced forms) were optimized using the gradient corrected pure functional BLYP, with an effective core potential (ECP) basis set from the Los Alamos National Laboratory, LANL2DZ, 90 on Fe atoms and with 6-31+G(d) basis set on other elements. All computational experiments were conducted using Gaussian 16 software. 91 The search for conformers was performed by molecular modeling software PCMODEL 10.0 (using the MMX force field). 105 Frequency calculations were performed to calculate thermal corrections to Gibbs free energies (at 298.15 K). Implicit solvation was modeled using the SCRF = SMD continuum solvation method at the (U)BLYP/6-31+G(d)/LANL2DZ level in dichloromethane (ε = 8.93) as a model solvent. 106 EPR Measurements. EPR measurements were performed using a CW X-band EMXplus spectrometer with a PremiumX microwave bridge and a high-sensitivity resonator (Bruker, Germany). The EPR spectra were registered at 100 kHz modulation and a microwave power of 5 mW at room temperature. An NMR teslameter (Bruker, Germany) was used for precise g value determination. For in situ EPR spectroelectrochemical experiments, a three-electrode EPR flat cell was used. A laminated gold mesh (Goodfellow, U.K.) as the working electrode, an AgCl-coated silver wire as the pseudoreference electrode, and a platinum wire as the counter electrode were used in spectroelectrochemical experiments. The 0.1 M [N(Bu) 4 ][B-(C 6 F 5 ) 4 ] in THF (anhydrous, ≥99.9%, inhibitor-free, Sigma-Aldrich) was used as the supporting electrolyte. Cell assembling and the measurements were performed under an inert (nitrogen) atmosphere. In the spin-trapping experiments, dimethylformamide (DMF, anhydrous, ≥99.8%, Sigma-Aldrich) solutions were bubbled with air, oxygen, or nitrogen for 2 h. 50 mM spin trap 5,5-dimethyl-1pyrroline N-oxide (DMPO, ≥99.0% (GC), Dojindo, Japan) and 1.5 mM ferrocene compound were added to the solution one after another.
Biological Assays. Cells. Human non-small-cell lung cancer cell lines A549 and H1975 and the human bronchial epithelial BEAS-2B cell line were purchased from ATCC (Manassas, VA). Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 media supplemented with 10% v/v fetal bovine serum, 100 U·mL −1 penicillin, and 100 μg·mL −1 streptomycin. Cells were grown in a humidified atmosphere at 37°C and 5% CO 2 .
Reactive Oxygen Species (ROS) Generation. Cells were incubated for 1 h in a fresh medium or in a medium containing 20 μM of compounds 1a and 1c and tamoxifen, 5-fluorouracil, and cisplatin, alone or together with 50 μM N-acetyl cysteine (NAC). Then, detached cells were resuspended in 0.5 mL of phosphatebuffered saline (PBS) containing 10 μM·L −1 fluorescent probe 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate-acetyl ester (CM-H2DCFDA) and incubated for 15 min at 37°C. Afterward, the incubation cells were centrifuged at 13,000 rpm for 30 s and resuspended in 0.5 mL of PBS. The fluorescence of each sample (index of ROS levels) was read at 488 nm (λ excitation ) and 520 nm (λ emission ). The results were expressed as DCF fluorescence per mg cell proteins normalized vs control.
Cell Viability with the Crystal Violet Assay. Crystal violet staining was used to assess cell viability. Cells were seeded in a 24-well plate and incubated with 20 μM concentration of compounds 1a, 1c, 2a, and 2c and tamoxifen, 5-fluorouracil, and cisplatin, with or without 50 μM NAC. After 72 h, the medium was discarded and cells were stained for 30 min with 5% w/v crystal violet solution in 66% v/ v methanol, 200 μL per well. After staining, the crystal violet solution was removed, and the 24-well plate was washed with water to eliminate the excess solution. When dried, the plates were photographed. Quantitation of crystal violet staining was performed after solubilizing the dye in 10% acetic acid, 400 μL per well, and reading the absorbance of each well at 540 nm (HT Synergy 96-well microplate reader, Bio-Tek Instruments, Winooski, VT). The relative absorbance of untreated cells was considered as 100% viability; results were expressed as a percentage of viable cells vs untreated cells. To calculate IC 50 , cells were incubated 72 h with increasing concentrations (1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM) of compounds 1a, 1c, 2a, and 2c and tamoxifen, 5-fluorouracil, and cisplatin. IC 50 was defined as the concentration of each compound that reduced the cell viability to 50% compared to untreated cells, producing 50% cell death (GraphPad Prism, version 5).
Spectra ( 1 H/ 13 C/NMR), EPR spectra, crystal data, and biological data (PDF) Accession Codes CCDC 2158730−2158732 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Inorganic Chemistry pubs.acs.org/IC Article