Novel cis-Pt(II) Complexes with Alkylpyrazole Ligands: Synthesis, Characterization, and Unusual Mode of Anticancer Action

One concept of improving anticancer effects of conventional platinum-based antitumor drugs consists of conjugating these compounds with other biologically (antitumor) active agents, acting by a different mechanism. Here, we present synthesis, physicochemical characterization, biological effects, and mechanisms of action of four new analogs of conventional cisplatin, namely, cis-Pt(II) complexes containing either methyl or ethyl pyrazole N-donor ligands and chlorido or iodido ligands. It is noteworthy that while chlorido complexes display activity in a variety of cancer cell lines comparable to cisplatin, iodido complexes are considerably more potent due to their enhanced hydrophobicity and consequently enhanced cellular accumulation. Moreover, all of the studied Pt(II) alkylpyrazole complexes display a higher selectivity for tumor cells and effectively overcome the acquired resistance to cisplatin. Further results focused on the mechanism of action of the studied complexes and showed that in contrast to cisplatin and several platinum-based antitumor drugs, DNA damage by the investigated Pt(II)-alkylpyrazole complexes does not play a major role in their mechanism of action. Our findings demonstrate that inhibition of the tubulin kinesin Eg5, which is essential for forming a functional mitotic spindle, plays an important role in their mechanism of antiproliferative action.


Introduction
Cisplatin, carboplatin, and oxaliplatin are the only worldwide approved metal-based antitumor drugs [1][2][3][4]. For decades, the search for new antitumor Pt drugs followed the rules established based on knowledge of the mechanism of action of cisplatin and its direct analogs. However, this search is now shifting to the greater structural diversity of platinum complexes, including nonclassical structures [5][6][7][8][9][10][11][12][13][14][15][16]. Cis-Pt(II) iodido complexes have long been predominantly used as intermediates in synthesizing target platinum chlorides and carboxylates, particularly by the Dhara method or its modifications [17,18]. However, later several studies have shown that the iodido ligand may have unique chemical, physical, and biological properties, with a mechanism of action different from that of Pt(II) chlorido analogs [19][20][21][22]. For example, cis-Pt(II) diiodides containing 7-azaindole and its derivatives were significantly more active than cisplatin in several cancer cell lines [19]. e use of heterocyclic ligands instead of simple amines makes it possible to tune the activity of Pt complexes. In addition, the large surface area allows more efficient and stronger interactions with DNA bases by π-π-stacking or altered interactions with DNA; also, the presence of a hindered group can prevent the inactivation of complexes by reactions with sulfur-containing ligands [22][23][24][25][26]. Pioneering work on the synthesis of Pt(II) complexes by Jan Reedijk and his coworkers in the 1970s provided 1H-imidazole, 1-methyl-1H-imidazole, and 1H-pyrazole complexes, which were investigated by a wide range of physicochemical methods [27,28]. Later, asymmetric cis-Pt(II) complexes with isopropylamine, 1methyl-1H-pyrazole, or 1-methyl-1H-imidazole ligands were also synthesized, and their in vitro antiproliferative activity and interactions with DNA bases were investigated by the Navarro-Ranninger group [29]. is article reports the preparation and physicochemical characterization of new cis-Pt(II) complexes with 1-alkyl-1Hpyrazole ligands containing the iodido or chlorido leaving groups. We also turned our attention to investigating antiproliferative activity in cancer cells and some unique aspects of the mechanism of action in further pursuit of novel platinum cancer drug candidates.

General Procedure for the Synthesis of Pt Diiodido
Complexes. K 2 [PtCl 4 ] (0.5 mmol) was dissolved in 10 mL of deionized water at room temperature, and the 24 equivalents of KI (12 mmol) were added to the red solution, which turned dark brown over 30 min of stirring in the dark. en, desired pyrazole was added in one portion, and the reaction mixture was stirred overnight at room temperature. e formed brown solid was removed by filtration and washed with deionized water (3 × 5 mL), methanol (3 × 2 mL), and diethyl ether (3 × 5 mL). e products were dried under a vacuum.

General Procedure for the Synthesis of Pt Dichlorido
Complexes. Suspension of 1a or 1b (0.5 mmol) in 5 mL of deionized water was treated with AgNO 3 (0.95 mmol), and the reaction mixture was stirred overnight at room temperature in the dark. e precipitate of silver iodide was filtered off, and 20 equivalents of KCl (10 mmol) were added and stirred for 24 h in the dark. e obtained yellow solid was removed by filtration and washed with deionized water (3 × 3 mL), acetone (3 × 1 mL), and diethyl ether (3 × 5 mL e A2780, A2780cisR, and RD cells were grown in RPMI 1640 medium (Biosera, Boussens, France) supplemented with gentamycin (50 mg/mL, Serva, Heidelberg, Germany) and 10% heat-inactivated fetal bovine serum (PAA, Pasching, Austria). e acquired resistance of A2780cisR cells was maintained by supplementing the medium with 1 µM cisplatin repeatedly every second passage. e HCT116, MDA-MB-231, MRC5 pd30, CHO-K1, and MMC-2 cells were grown in DMEM medium (Dulbecco's Modified Eagle's Medium, high glucose (4.5 gL −1 , PAA) supplemented with gentamycin (50 mgmL −1 , Serva) and 10% heat-inactivated fetal bovine serum (PAA). e cells were cultured in an incubator at 37°C in a humidified 5% CO 2 atmosphere and subcultured 2-3 times a week.
2.6. Antiproliferative Activity. Effects of the platinum complexes on cell proliferation were evaluated using commonly used MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] as already described [30]. Briefly, the cells were seeded in 96-well tissue culture plates at a density of 1 × 10 4 A2780/cisR cells/well, 3 × 10 3 MDA-MB-231, RD, CHO-K1, or MMC-2 cells/well, 1 × 10 4 MRC-5 pd30 cells/well, 1.5 × 10 3 HCT-116 cells/well, in 100 μL of medium and kept overnight at 37°C in a 5% CO 2 humidified atmosphere. en, the cells were treated with the tested compounds in the range of 0 to 100 µM in a final volume of 200 µL/well. After 72 h of treatment, a freshly diluted MTT solution (20 µL, 1.25 mg/mL in PBS) was added to the wells, and the plates were further incubated for 4 h. Subsequently, the medium was removed, and the formazan product was dissolved in 100 μL of DMSO. e number of living cells was evaluated by measuring the absorbance at 570 nm (reference 620 nm) using an absorbance reader (Spark, TECAN). e reading values were converted to the percentage of control and IC 50 values calculated from the curves constructed by plotting the number of living cells (%) versus drug concentration (μM) (IC 50 = concentration of the compound inhibiting cell growth by 50%). Concentrations of Pt complexes present in the medium during treatment were always verified by flameless atomic absorption spectrometry (FAAS).

Cellular
Uptake. Cellular accumulations of studied complexes were determined as already described earlier [31]. Briefly, MDA-MB-231 cells were seeded on 100 mm Petri dishes (1 × 10 6 cells per dish). After overnight preincubation, the cells were exposed to platinum compounds for 5 or 24 h.
After the treatment, the cells were detached by trypsinization, exhaustively washed with ice-cold PBS, counted using cell counter, and collected by centrifugation. Finally, the cell pellets were digested using a microwave acid (HCl) digestion system (CEM Mars). e quantity of metal taken up by the cells was determined by ICP-MS.

Quantification of Platinum
Bound to DNA Isolated from the Cells. MDA-MB-231 cells were cultured, treated with platinum complexes, and collected as described above. e cell pellets were lysed in DNAzol (DNAzol, MRC) supplemented with RNase A (100 µgmL −1 ). Genomic DNA was precipitated from the lysate by ethanol (100%), washed twice with 75% ethanol, and resuspended in 8 mM NaOH. e DNA content in each sample was determined by UV spectrophotometry. To avoid the interference of high DNA concentration on the detection of platinum in the samples, the DNA samples were digested in the presence of 30% hydrochloric acid (Suprapur, Merck Millipore). e amount of metal bound to nucleic acids was quantified by ICP-MS.

Measurement of the Partition Coefficients.
e partition coefficients (P) of the investigated platinum complexes were determined by the "shake-flask" method. e complexes were dissolved in octanol-saturated water (OSW) containing either 10 mM KI (compounds 1a,b) or 10 mM KCl (compounds 2a,b) and mixed with water-saturated octanol (WSO). After 30 min of mixing at room temperature, centrifugation was done at 3000 g for 5 min to separate the two phases. e layers were separated carefully using a finetip pipette, and the water fraction was analyzed for metal content by flameless atomic absorption spectroscopy (FAAS). e partition coefficients were calculated as the ratio of the concentration of the compound in the octanol layer to that in the aqueous layer: log P = log

DNA Binding in a Cell-Free Medium.
Calf thymus DNA (64 μgmL −1 ) was incubated with platinum complexes at their 20 µM concentration in 10 mM NaClO 4 at 37°C for 24 h. After that, the free (unbound) platinum in these samples was removed by gel filtration using Sephadex G-25 columns, and the samples were redissolved in double-distilled water. UV and flameless atomic absorption spectrophotometry were used to determine DNA concentration and platinum contents in these samples, respectively.

Reaction with GSH.
e interaction of platinum complexes to GSH was followed by recording UV absorption, as already described [32,33]. e absorbance at 260 nm was monitored as a function of time using a Beckman 7400 DU spectrophotometer equipped with a thermoelectrically controlled cell holder. Reactions were carried out in the dark at 37°C. e experimental procedure was as already outlined by Dabrowiak et al. [34]. To establish the rate of the initial reaction, the curve was fitted by nonlinear regression to the equation , where A1, A2, b1, b2; C are constants, and t is the time of the reaction, by using GraphPad Prism software. 2.14. Imaging of Spindle Organization. MDA-MB-231 cells (1.5 × 10 5 cells) were seeded on glass coverslips (Marienfeld Superior cover glasses 22 × 22 mm, thickness No1, Paul Marienfeld GmbH & Co. KG, Germany) coated with 0.1% gelatin from bovine skin Type B (Sigma, Prague) placed in 6-well culture dishes and grown overnight. e cells were then treated with the tested compounds for 24 h. Following the treatment, the cells were washed and fixed with p-formaldehyde (4% in PBS; 10 min) and permeabilized with 0.1% Triton X-100 for 20 min. Following the permeabilization, the blocking solution was applied (5% goat serum in PBST) for 1 h. As a next step, the primary antibody (anti-β I Tubulin antibody Abcam, dilution 1 : 500) was added and incubated at room temperature for 2 h. After four washes with PBST, the secondary antibody was applied (Goat Anti-Rabit-Alexa Fluor 488, Abcam, dilution 1 : 200) and incubated for 1 h. After exhaustive washing (at least five times) with PBST, the cells were mounted with ProLong TM Diamond Antifade with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, Invitrogen, ermo Fisher Scientific, Prague, Czech Republic). e images were taken with the laser scanning confocal microscopy Leica SP-8 (Leica Microsystems GmbH, Wetzlar, Germany). DAPI was excited by blue laser (405 nm) and Alexa Fluor 488 by WLL (488 nm). Sequential detection at adequate channels (450-470 nm and 540-560 nm, respectively) was employed.
e structure of all complexes was characterized by 1 H, 13 C, and 195 Pt NMR spectroscopy and purity by HRMS. In 1 H NMR spectra of 1b, 2a, and 2b, we observed three signals of pyrazole ring (C 3 H, C 4 H, C 5 H). Interestingly, we found only two indistinguishable multiples from the pyrazole ring for 1a in both DMF-d 7 and acetone-d 6 . Coordination to the platinum center resulted in a lower field shift for pyrazole protons compared to the free ligands.
In 195 Pt NMR spectra, we found resonance at δ ca. −2000 ppm for the chlorido complexes and for iodides at ca. −3200 ppm, which confirmed the complexes' cis-geometry. In the HR-ESI+ mass spectra for complexes 1a,b and 2a,b (Figures S1A−S1D), the most abounded peak was identified as [M + Na] + or [M + H] + ; for the 1a,b and 2a,b, the peak identified as [M − Hal] + was also present in the spectra.

Stability.
e stability of complexes 1a,b, and 2a,b in the presence of water was examined by 1 H NMR (40% DMF-d 7 / 60% D 2 O). We found that the chlorido complexes 2a,b are stable in the used water-containing solvent mixture because no new signals were shown in the 1 H NMR spectra ( Figure S2). e characteristic signal of the C 4 -H pyrazole ring was detected at 6.44 ppm (for 2a) and 6.47 ppm (for 2b). In contrast, the new 1 H NMR signals were detected in the spectra of both iodido complexes 1a,b under the aqueous conditions. For example, the characteristic C 4 -H signal, which was detected at 6.45 ppm and 6.49 ppm for the parent complexes 1a and 1b, respectively, was also observed at 6.30 ppm in the spectra of both complexes ( Figure S2). is signal (6.30 ppm) is attributable to the released pyrazole derivatives mepz (for 1a) and etpz (for 1b) because the signal positions correlate well with those of free pyrazoles (studied for comparative purposes). For 1a, ca. 15% and 30% (t = 5 and 24 h, respectively) of the initial complex released its Although the nature of the processes connected with the ligand release is not the same for cisplatin (i.e., Pt-Cl bond hydrolysis) and 1a,b (N-donor ligand release), both lead to the activated species capable of interacting with various biomolecules. However, the kinetics of these processes seems to be different. In particular, the cisplatin hydrolysis with t 1/2 ≈ 2 h [36][37][38] is a much faster process than the release of pyrazole derivatives mepz and etpz from complexes 1a,b (t 1/2 > 24 h). It is worth noting that a release of N-donor ligand was recently reported for Pt(II) diiodido complexes of different structural types, specifically for trans-diiodidoplatinum complexes [21,39] and unsymmetrical cis-ammine-diiodidoplatinum [40] complexes.
In general, chlorido derivatives of cisplatin are usually less hydrolytically stable than their iodido analogs [20,41], which contradicts the results describing the stability of cis-Pt(II) alkylpyrazole complexes investigated in this work ( Figure S2). Additionally, a mildly acidic environment (close to the situation in cancer cells) was shown to destabilize the ammonia ligands in cis-[PtI 2 (NH 3 ) 2 ] in contrast to cisplatin and thus favors ammonia release over iodide release [20].

Antiproliferative Activity.
e activity of the four complexes with alkylpyrazole ligands (1a,b, and 2a,b) was determined against human ovarian carcinoma cell lines A2780 and A2780cisR (sensitive and cisplatin-resistant), human breast adenocarcinoma cells MDA-MB-231, human colon carcinoma cell HCT116, and human rhabdomyosarcoma RD cells, commonly used to test the cytotoxic activity of cisplatin derivatives and other antitumor metallodrugs. In addition, human lung fibroblasts MRC5 pd30 were also included in the analysis as a model of noncancerous, healthy cells. e cell lines were incubated with platinum complexes for 72 h, and the number of viable cells in the cultures was evaluated by MTT assay as described in the experimental section (Materials and Methods). e resulting IC 50 values are summarized in Table 1.
e data revealed that both iodido complexes 1a and 1b were significantly more effective than their chlorido counterparts 2a and 2b. Importantly, they were also significantly more effective than clinically used cisplatin against all inherently cisplatin-resistant cell lines tested. In contrast, chlorido derivatives showed activity comparable to cisplatin in most of the cells tested (with one exception in cisplatin-sensitive A2780 cells). Notably, all alkylpyrazole complexes were effective in overcoming acquired cisplatin resistance; iodido derivatives 1a and 1b are even more effective compared to chlorido complexes 2a,b.
is suggests that the mechanism underlying the biological action of the new alkylpyrazole Pt(II) complexes is likely to be somewhat different from that of cisplatin, allowing the compounds to successfully overcome the resistance mechanisms that work with cisplatin. Notably, the antiproliferative activity of Pt(II) alkylpyrazole complexes was considerably higher in tumor cells compared to nontumorigenic normal MRC5 pd30 cells (Table 1).

Intracellular Accumulation of
Platinum. An essential step for the biological effect of platinum drugs is the efficient uptake of these drugs through the plasma membrane and the subsequent accumulation in cells.
erefore, the intracellular platinum concentration in the cells treated with the investigated complexes was determined to estimate the possible impact of the cellular uptake of these compounds on their efficacy against cancer cells.
Cell platinum levels were measured after 5 h and 24 h of exposure of MDA-MB-231 to 1a, 1b, 2a, 2b and cisplatin at their equimolar (10 μM) concentrations by ICP-MS analysis. It was verified that the cell viability was at least 85% under the conditions of these experiments, as measured by the trypan blue exclusion assay. us, total cell uptake was not significantly affected by the dying/dead cell membrane permeability impairment. e results expressed as ng Pt per 10 6 cells are shown in Figure 3 and Table S1. Consistent with the antiproliferative potency, the cellular Pt content increased in the order of cisplatin < 2a ∼ 2b < 1a ∼ 1b and correlated with their lipophilicity (Table S1). e most lipophilic complex contains the ethyl group at pyrazole ring and iodido ligands and changes to chlorido ligands, or the methyl group decreases lipophilicity. Interestingly, the results in Figure 3 show that incubating the cells for more than 5 h increased the accumulation of platinum from the complexes only relatively slightly.
is result can be interpreted to mean that the incubation of the cells for 5 h was sufficient for almost total platinum accumulation.
However, the correlation between cytotoxic efficacy and intracellular Pt content is not straightforward. e data presented in Table S1 and Figure 3 also show that the accumulation of Pt from complexes 2a,b in MDA-MB-231 cells significantly exceeded the accumulation from cisplatin, although the antiproliferative activity of the three compounds was approximately the same (Table 1). Similarly, complexes 1a,b were about 5-fold more active in MDA-MB-231 cells than cisplatin, although the intracellular level of Pt from 1a,b was about 90-fold higher. us, these estimates suggest that significantly higher levels of Pt(II) alkylpyrazole complexes are required to achieve a certain level of biological activity compared to cisplatin. Furthermore, this suggests that the mechanism underlying the activity of Pt(II) alkylpyrazole complexes differs from the mechanism of the antitumor effect of cisplatin. Bioinorganic Chemistry and Applications 3.5. DNA Interactions. Nuclear DNA has been shown to be a major pharmacological target of cisplatin, so the anticancer activity of conventional platinum anticancer drugs is attributed to their binding to DNA [1,3]. As Pt(II) complexes with alkylpyrazole ligands can be considered cisplatin analogs, we hypothesized that DNA could also play an important role in the activity of these compounds. erefore, further experiments were focused on DNA binding in the cell-free medium and subsequently in the cellular environment. We first focused on quantifying the binding of cis-Pt(II) alkylpyrazole complexes to mammalian (calf thymus) double-stranded DNA in a cell-free medium, as described in Materials and Methods. e amount of platinum bound to DNA after 24 h of incubation in 10 mM NaClO 4 is plotted in Figure 4. As expected, cisplatin binds DNA quantitatively after 24 h, consistent with previously published data [42]. However, in contrast to cisplatin, the DNA binding of all Pt(II) alkylpyrazole complexes was much less efficient. In particular, chloride derivatives 2a and 2b bound DNA poorly so that the level of platinum associated with DNA was almost below the detection limit of FAAS. Iodide analogs 1a and 1b were able to bind DNA under experimental conditions but to a much lesser extent than cisplatin. ese results correlate and can be related to the above observation that chlorido complexes 2a,b do not hydrolyze at all under the aqueous conditions, and the N-donor ligand (i.e., mepz and etpz) release from the iodido complexes is slower than the hydrolysis reported for cisplatin ( Figure S2). us, this experiment revealed that the ability of Pt(II) alkypyrazole complexes to bind to DNA in a cell-free medium is significantly reduced compared to cisplatin. However, the situation in living cells may differ from extracellular conditions, and hydrolytic processes may vary significantly due to the more complex intracellular environment. erefore, DNA platination in living cells was also determined.
As shown in Figure 5(a) and Table S2, the level of platinum associated with nuclear DNA in the treated cells was significantly higher for the iodido complexes 1a and 1b than in the chlorido analogs 2a and 2b, as well as for cisplatin. us, the trend in intracellular DNA platination of the investigated complexes roughly follows their cellular uptake and accumulation. However, evaluation of the efficiency of intracellular DNA platination (the amount of platinum bound to DNA versus the total amount of platinum in the cells) showed remarkable results ( Figure 5(b)). Of the total Pt inside MDA-MB-231 cells, a significantly higher fraction of platinum from cisplatin was bound to DNA than 1a,b, and 2a,b. ese fractions represent 1.3% (cisplatin), 0.25% (1a), 0.21% (1b), 0.15% (2a), and 0.16% (2b) of the total Pt accumulated in cells. In aggregate, the yield of platination of DNA by Pt(II) alkylpyrazole complexes was significantly (5-9-fold) lower than that of cisplatin. ese results correlate with a reduced   ability of new Pt-alkylpyrazole complexes to bind DNA found in a cell-free medium.

Influence of DNA Nucleotide Excision Repair on Biological Activity.
e results demonstrating DNA platination by the investigated Pt-alkylpyrazole complexes in the cell-free medium or cells (Figures 4 and 5) suggest that, in contrast to antitumor effects of cisplatin, DNA may not be such an important target for the Pt(II) complexes investigated in this study. A generally accepted criterion that proves that DNA is the target is based on the observation that the investigated drug exhibits higher toxicity in cells that are deficient in DNA repair [1] because the persistence and toxicity of DNA lesions depend on the ability of the cells to repair the damage. erefore, the pair of Chinese hamster ovary cells CHO-K1 line (wild-type) and its isogenic mutant line MMC-2 (nucleotide excision repair (NER) deficient, carrying the ERCC3/XPB mutation) was used to distinguish whether DNA damage is involved in the mechanism of action of the new Pt(II) alkylpyrazole complexes. e data in Table 2 show that DNA repair-deficient MMC-2 cells were significantly more sensitive (11-fold) to cisplatin than wild-type CHO-K1 cells, consistent with previously published results [30]. However, when the cells were treated with Pt(II) alkylpyrazole complexes investigated in this work, the difference in the sensitivity of repair-deficient cells and repair-proficient cells decreased markedly, suggesting that unrepaired DNA lesions formed by the investigated Pt-alkylpyrazole complexes contribute markedly less to their cytotoxicity than those formed by cisplatin. us, DNA damage is unlikely to be a decisive factor responsible for the activity of the investigated Pt(II)-alkylpyrazole complexes.

Interaction with GSH.
Before platinum drugs reach the DNA in the nucleus of tumor cells, they can interact with various sulfur-containing molecules because Pt(II) compounds show a strong thermodynamic preference for binding to sulfur donor ligands [43]. ese interactions are generally thought to play a role in the mechanisms underlying the activity (inactivation) of Pt drugs [43,44]. e study of the interactions of platinum antitumor complexes with sulfur-containing compounds of biological importance may help elucidate other aspects of the action of the new Pt compounds. So the reactions of cis-Pt(II) alkylpyrazole complexes with GSH were investigated using the methods already described [34].
Complexes 1a,b, 2a,b or cisplatin were incubated with GSH in a thiol to drug ratio of 500:1, which is a physiologically relevant value [34]. Figure 6 shows the UV absorbance (at 260 nm) of the platinum complexes and GSH as a function of time, subtracting the absorbance of the platinum complex  itself. To determine the rate of initial reaction with respect to the platinum complex, each difference curve was fitted by nonlinear regression (GraphPad Prism) to the following equation: and C are constants and t is the reaction time. e initial slope (S in ) was calculated as A1b1 + A2b2 [34]. e values of S in values of (7.6 ± 0.9) × 10 −4 , (8.4 ± 0.9) × 10 −4 , (7.1 ± 0.5) × 10 −4 , (8.6 ± 0.4) × 10 −4 , and (7.9 ± 0.5) × 10 −4 were calculated for the reaction of GSH with cisplatin, 1a, 1b, 2a, and 2b, respectively.
Remarkably, we have found that the pattern of GSH interaction of iodido complexes 1a and 1b was almost superimposable to that of cisplatin. e interaction rates of complexes 2a and 2b appear to be slightly faster, but these differences are not statistically significant ( Figure 6). As the absorbance at 260 nm reflects the presence of platinumsulfur and disulfide bonds [34,45], the observations can be interpreted to mean that under the conditions of our experiment, all four investigated Pt-alkylpyrazole complexes react with GSH at a similar initial rate. It implies that differences in the biological activity of these complexes are unlikely to be related to their different inactivation by GSH.

e Effect on the Organization of the Mitotic Spindle and the Cell Cycle.
e results mentioned above revealed that DNA damage, although contributing to overall activity, is unlikely to be a predominant factor underlying the biological activity of the investigated Pt-alkylpyrazole complexes.
erefore, further steps have been taken to further elucidate the mechanism of action of the compounds. Impedance monitoring of cellular responses was used for this purpose. It has been shown [46][47][48] that biologically active compounds produce time-dependent cell response profiles (TCRPs), which can be predictive of the mechanism of action of the investigated molecules.
e TCRPs of cisplatin and Ptalkylpyrazole complexes 1a, b and 2a, b are displayed in Figure 7. e TCRP obtained for cisplatin at the concentration close to the IC 50 value was characterized by an initial slight increase in the cell index (CI) in comparison with the control, followed by a time-dependent decrease in the CI below the control level (reflecting cytotoxic responses) without any indication of recovery of CI (see the red curve in Figure 7(a) or blue curve in Figure 7(f )).
us, the TCRP of cisplatin coclustered with the TCRPs of compounds interfering with DNA synthesis and replication, transcription, and translation [46], which are also known to induce cell cycle arrest followed by the induction of cell death.
In contrast, treatment with the investigated Pt-alkylpyrazole complexes (Figures 7(b)-7(e)) resulted in TCRPs different from those typical for DNA-damaging agents, including cisplatin. e TCRPs obtained for 1a, 1b, 2a, and 2b also at the concentrations close to their IC 50 Figure 7(f )). Interestingly, these profiles can be coclustered with those displayed by nontubulin-targeting mitotic inhibitors, such as monastrol and S-tritylcysteine, which inhibit the mitotic kinesin Eg5 [46]. TCRPs obtained for all investigated complexes at considerably higher concentrations (Figures 7(a)-7(e) caused the complete killing of adherent cells at the longest times of cell growth. e finding that TCRPs obtained for Pt-alkylpyrazole complexes can be coclustered with those displayed by nontubulin-targeting mitotic inhibitors which inhibit the mitotic kinesin Eg5 was surprising, as no antitumor Pt complexes have been reported to act via this mechanism.  However, a search in the literature has yielded several references suggesting that pyrazoles and their derivatives, such as dihydropyrazole, are widely recognized as potent inhibitors of Eg5 [49][50][51][52].
us, the TRCPs resembling Eg5 inhibition could potentially result from the activity of alkylpyrazole ligands or their metabolic products. Further experiments were therefore performed to confirm or refute this hypothesis.
Kinesin Eg5 is an essential spindle motor protein. Its important role is to assemble and maintain the bipolar spindle during mitosis, making it an attractive therapeutic target that could promote tumor growth regression [53][54][55][56][57]. Kinesin Eg5 inhibition stops the migration of centrosome to the polar region resulting in the formation of monoastral spindle [58,59]. is atypical phenotype plays an essential role in activating the mitotic spindle assembly checkpoint, resulting in mitotic arrest [53,58,60].
e following experiments, therefore, focused on evaluating the effect of the investigated Pt-alkylpyrazole complexes on the organization of the mitotic spindle and the cell cycle. Figure 8 shows representative images of mitotic MDA-MB-231 cells immunostained for β-tubulin (green) and DNA (blue). As indicated, control untreated cells form uniformly bipolar spindles of normal morphology  found for 1a, 1b, 2a, 2b and cisplatin at their equitoxic concentrations (IC 50,72h ). e vertical lines indicate the start of the treatment after allowing the cells to adhere to microelectrodes and grow for 24 h. Cell indices were normalized to account for differences in cell counts that existed across the wells before the treatment. Incubations were performed in triplicate with 3000 cells per well. (Figure 8(a)). However, complexes 1a, b and 2a, b used at equitoxic concentrations caused monopolar (monoastral) spindles formation in MDA-MB-231 (Figures 8(b)-8(e)), similar to monastrol and other EG5 inhibitors. is effect is consistent with weak-binding motors being unable to resist inward-directed spindle forces [61]. Interestingly, all mitotic cells in the investigated samples showed solely monoastral (symmetrical or asymmetrical) spindles. ese data validate our results obtained from the TCRPs assay, indicating that these compounds are indeed effective inhibitors of Eg5 activity.
To evaluate whether Pt-alkylpyrazole compounds can disrupt normal cell cycle progression, the effect of the compounds on MDA-MB-231 cells was analyzed by flow cytometry. e results showed (Figures 9 and S3) that after treatment, all compounds 1a, b and 2a, b caused the cell cycle arrest at the G2/M phase, consistent with their mitotic inhibitory action predicted by TCRPs. is effect was both concentration-and time-dependent (Figures 9 and S3). In contrast, cisplatin arrests the MDA-MB-231 cell cycle in the way that cells cannot proceed to the G2/M phase according to its DNA-damaging mode of action and in accordance with already published data [62].

Conclusions
Four new analogs of conventional cisplatin have been prepared and characterized by physicochemical methods, namely, cis-Pt(II) complexes containing either methyl or ethyl pyrazole N-donor ligands and chlorido or iodido ligands. While complexes with chlorido ligands are stable for 48 h in the aqueous medium, complexes with iodido ligands undergo hydrolysis in the aqueous medium, releasing the alkylpyrazole ligand(s). e investigated Pt(II) complexes with alkylpyrazole ligands show interesting antiproliferative activity in tumor cells (Table 1). Chlorido complexes display activity comparable to cisplatin. In contrast, iodido complexes are considerably more potent, which is most likely related to their enhanced transport into cells and cellular accumulation ( Figure 3) due to their greater lipophilicity (Table S1). However, all of the studied Pt(II) alkylpyrazole complexes are less active in nontumor healthy cells than the clinically used cisplatin, thus displaying a higher selectivity for tumor cells. Also importantly, they very effectively overcome the acquired resistance to cisplatin. ese initial results allowed us to suggest that the investigated Pt(II) alkylpyrazole complexes affect tumor cells by a mechanism different from that of cisplatin. is also implies that the resistance pathways that the tumor cells have developed against cisplatin were less effective if the tumor cells were treated with the investigated Pt(II) alkylpyrazole complexes. e studied Pt(II) alkylpyrazole complexes were unable to bind DNA in cell-free media either at all (chlorido derivatives) or bound only very little (iodido derivatives). However, a considerable DNA binding of platinum was observed in the cells treated with both iodido and chlorido complexes ( Figure 5(a)), probably due to the action of the cellular environment (containing enzymes and other bioactive molecules which may interact with the complexes accumulated inside cells) and consequently may activate these complexes. However, the frequency of DNA adducts formed by Pt(II) alkylpyrazole complexes in MDA-MB-231 was significantly lower than that formed by cisplatin ( Figure 5(b)). In addition, a comparison of the amount of Pt per DNA with the antiproliferative activity of the platinum complexes showed that the adducts formed by alkylpyrazole complexes are much less effective in inhibiting the viability of MDA-MB-231 cells than those formed by cisplatin; much fewer DNA adducts of cisplatin were sufficient to elicit the same biological effect as DNA adducts of Pt(II) alkylpyrazole complexes. ese observations, along with the markedly smaller cytotoxic contribution of the unrepaired DNA lesions formed by the investigated Pt(II)alkylpyrazole complexes ( Table 2), implicate that DNA damage by the investigated Pt(II) alkylpyrazole complexes does not play a major role in their mechanism of action, although it may contribute to a small extent.
In contrast, our results ( Figure 8) have clearly shown, for the first time, that inhibition of the tubulin kinesin Eg5, which is essential for the formation of a functional mitotic spindle, plays an important role in the mechanism of antiproliferative action of platinum-based antitumor drugs. As a result, tumor cell division is inhibited. is mechanism may also explain the greater selectivity to tumor cells found for the investigated Pt(II) alkylpyrazole complexes compared to cisplatin because healthy cells do not divide or divide significantly more slowly. Eg5 is becoming an increasingly attractive therapeutic target for the treatment of cancer by chemotherapeutics [57]. Hence, the introduction of cis-Pt(II) alkylpyrazole complexes in this work may be an incentive to search for new anticancer chemotherapeutics derived from platinum complexes with this mechanism of action.

Data Availability
All data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.