**2. Synthesis of PNCs**

### **2.1. Pt(II) complexes**

386 Nitroxides – Theory, Experiment and Applications

et al., 2010).

Sosnovsky & Li, 1985a), cyclophosphamide (Tsui et al., 1982), actinomycin D (Sinha et al., 1979), ethylenimino triazines (Emanuel & Konovalova, 1992), nitrosoureas (Raikov et al., 1985; Sosnovsky & Li, 1985b; Emanuel et al., 1986; Sen', 1993), 5-fluorouracil (Emanuel et al., 1985; Sen' et al., 1989), daunorubicin (Emanuel et al., 1982) were synthesized and studied. In comparison with the parent compounds, nitroxyl derivatives of the cytostatic drugs possess lower overall toxicity in animal studies and higher values of the *half inhibitory* concentrations IC50 in cell cultures. At the same time, they exhibit higher chemotherapeutic indexes (are effective in a wider range of doses) and, in the cases studied, are characterized by fewer side effects. For example, ruboxyl, a nitroxyl derivative of daunorubicin, is 8-fold less toxic to mice than the parent compound. At optimal doses, ruboxyl is more effective in experimental animal tumors and has no cardiotoxicity (Emanuel et al., 1982, 1992). After a successful phase II clinical trials (1991), its further study was interrupted due to financial problems.

Over the past 30 years, platinum complexes occupy leading positions among drugs for cancer chemotherapy. The antitumor activity of cisplatin (CP) was discovered in 1960s, and in 1978 it was approved for clinical use (Kelland, 2007). The subsequent search for improved cisplatin analogues resulted in introduction of carboplatin (1989) and oxaliplatin (2002) into clinical practice. About 15 other complexes, for various reasons, have been rejected in clinical trials. Currently, JM216 (satraplatin), picoplatin, and nanopolymer ProLindac, bearing the oxaliplatin moiety, are subject to clinical trials (Wheate et al., 2010) (Fig. 1).

**Figure 1.** Platinum anticancer drugs which are in clinical use and undergoing clinical trials.

Cisplatin and other bivalent platinum complexes are effective against a number of human tumors. They are used in almost half of the treatment regimes in combinations with other anticancer drugs (Wheate et al., 2010). Complexes of bivalent platinum are highly reactive and, therefore, they are highly toxic drugs. To avoid acute toxicity, cisplatin is administered by continuous infusion of a very dilute solution (Blokhin & Perevodchikova, 1984). Another disadvantage of cisplatin is a rapid development of tumor resistance to this drug (Koeberle

Complexes of Pt(IV), being chemically more inert than Pt(II) complexes, are characterized by moderate toxicity, and are suitable for oral administration. Complexes like satraplatin can pass through the digestive tract where they are absorbed into the bloodstream. With the bloodstream they reach organs and tissues, interact with cellular targets, and thus provide an antitumor effect (Kelland, 1999). Complexes of Pt(IV) are prodrugs (drug precursors) that, after entering into the cell or on the way to it, are reduced to corresponding active Pt(II) derivatives causing cytotoxic effect. At the same time, Pt(IV) complexes are potent inhibitors Most of platinum complexes with high antitumor activity are non-ionic compounds with the *cis*-arrangement of the amino ligands (Hambley, 1997) (Fig. 1). Synthesis of complexes with two identical amino ligands or one diamino ligand is similar to the synthesis of cisplatin and is generally outlined in Fig. 2.

**Figure 2.** Synthesis of Pt(II) *cis*-diamino complexes.

To achieve good yields, diiodo complexes cis-[Pt(RNH2)2I2] were prepared in the first step of the synthesis. The diiodo complexes were then converted to the target complexes by exchange reaction via water soluble dinitrato complexes (Fig. 2) (Dhara, 1970).

Complexes with two bulky amino ligands, such as compounds **1** (see Fig. 4), poorly bind to the DNA target (Section 3). Presumably, this is the main cause of their weak antitumor activity. The search for the active complexes led to the development of synthesis of mixedligand *cis*-diamino complexes containing only one bulky aminoligand (Giandomenico et al., 1995). The key intermediate in this synthesis is the salt K[Pt(NH3)Cl3], which can be obtained in several ways (Oksanen & Leskela, 1994; Oksanen, 1997; Potucka et al., 2011). The described method (Giandomenico et al., 1995) was modified by us for the preparation of PNCs with general formulae **2** and **3** (Fig. 3, 4).

**Figure 3.** Synthesis of mixed amine complexes of platinum (II) (R – nitroxyl radical).

**Figure 4.** Platinum(II) amino nitroxyl complexes.

The formulae of complexes obtained are shown in Fig. 4 (Sen' et al., 1996, 1998, 2000). Two biradical complexes of type **1** were synthesized previously (Mathew et al., 1979; Claycamp et al., 1986). For the preparation of compounds of general formula **4**, which are structural analogues of oxaliplatin, we synthesized the first nitroxyl with two vicinal amino groups, *trans*-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl (Sen', 1989). Binuclear complex **5b** was also obtained based on this radical.

### **2.2. Pt(IV) complexes**

Complexes of Pt(IV) with mixed amino ligands can be obtained only by oxidation of the Pt(II) precursors. According to the published method (Giandomenico et al., 1995), the starting Pt(II) complexes **6** are oxidized with an excess of H2O2 under relatively harsh conditions (70 °C, ≥ 2 h). Under these conditions, the oxidation of Pt(II)-nitroxyl complexes leads to the formation of significant amount of by-products, probably as a result of oxidation of nitroxyl radical by Pt(IV) at elevated temperature. We found that catalytic amounts of salts of tungstic acid strongly accelerate the reaction so that preparative oxidation under mild conditions (0 – 20 °C) is limited only by the rate of dissolution of the starting complex and takes from 0.5 to 2.5 hours.

**Figure 5.** Synthesis of platinum(IV) complexes, R - nitroxyl radical, R '- carboxylic acid residue. O

This significantly increases the reaction selectivity and the yield of the target products. *trans*-Dihydroxo complexes **7** resulted from the oxidation are of interest themselves. Their acylation with organic acid anhydrides leads to the *trans*-dicarboxylate derivatives **8** (Fig. 5) (Sen' et al., 2003, 2006).

The method described allows one to introduce different amines and exchange the so-called leaving X-ligands at the step of preparation of Pt(II) complexes (Fig. 3) and incorporate various carboxylate ligands with the alkyl residue R' of different length at the final step (Fig. 5). Thus, we can obtain the amino complexes of Pt(IV), which differ in chemical activity, solubility in water and aqueous-lipid distribution. The formulae of Pt(IV) complexes are shown in Fig. 6.

**Figure 6.** Platinum(IV) amino nitroxyl complexes.

388 Nitroxides – Theory, Experiment and Applications

2) RNH2 1) KI

Cl Cl

> H3N Pt

RNH2

H3N Pt H3N

**Figure 4.** Platinum(II) amino nitroxyl complexes.

was also obtained based on this radical.

starting complex and takes from 0.5 to 2.5 hours.

**2.2. Pt(IV) complexes** 

**Figure 3.** Synthesis of mixed amine complexes of platinum (II) (R – nitroxyl radical).

–AgCl

Pt Cl

Cl Cl

> H3N Pt

2AgNO3 2KCl

RNH2

– KCl H3N

ONO2 ONO2

–AgI Cl

K+

Pt Cl

> H3N Pt

RNH2

Cl Cl –

Cl

Et4NCl H3N

Cl I

H+

The formulae of complexes obtained are shown in Fig. 4 (Sen' et al., 1996, 1998, 2000). Two biradical complexes of type **1** were synthesized previously (Mathew et al., 1979; Claycamp et al., 1986). For the preparation of compounds of general formula **4**, which are structural analogues of oxaliplatin, we synthesized the first nitroxyl with two vicinal amino groups, *trans*-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl (Sen', 1989). Binuclear complex **5b**

Complexes of Pt(IV) with mixed amino ligands can be obtained only by oxidation of the Pt(II) precursors. According to the published method (Giandomenico et al., 1995), the starting Pt(II) complexes **6** are oxidized with an excess of H2O2 under relatively harsh conditions (70 °C, ≥ 2 h). Under these conditions, the oxidation of Pt(II)-nitroxyl complexes leads to the formation of significant amount of by-products, probably as a result of oxidation of nitroxyl radical by Pt(IV) at elevated temperature. We found that catalytic amounts of salts of tungstic acid strongly accelerate the reaction so that preparative oxidation under mild conditions (0 – 20 °C) is limited only by the rate of dissolution of the The structure of PNCs was proved by elemental analysis and spectroscopic data (Sen' et al., 1996, 1998, 2000, 2003, 2006). For complexes **2b**, **4d**, and **10a**, the structures are determined by X-ray crystallography (Sen' et al., 2000, 2003; Chekhlov, 2005).

### **3. Interaction of PNCs with DNA**

Reactivity of Pt(II) diamine complexes **4** depends strongly on the nature of leaving Xligands. Pseudo-monomolecular rate constants for X-ligands hydrolysis in complexes **4c**, **4d**, cisplatin, and **4e** at 25 °C in 0.08 *M* NaOH are > 10–2, 1.2·10–4, 1.9·10–5, and 2.9·10–7 s–1, respectively, *i.e*., differ by five orders of magnitude (Shugalii et al., 1998). Therefore, the reaction of complexes with S- or N-donor groups can proceed either through the step of preliminary hydrolysis with the formation of an active intermediate aqua complex (Fig. 7, path *A*), or by the direct substitution of the X-ligands (path *B*).

**Figure 7.** Reaction of Pt(II) complexes with nucleophilic atom Nu of a target molecule.

For relatively easily hydrolyzable complexes, including cisplatin, the reaction proceeds through the path *A* (Alderden et al., 2006), but for complexes of type **4e** (X + X' = cyclobutane dicarboxylate) there is an evidence for direct substitution of the X-ligands by N-donor groups (Frey, 1993).

It is known that cisplatin and its analogues bind mainly to the guanine and adenine bases of DNA with the formation of cross-links, thus perturbing the structure of DNA (Kelland, 2007; Wheate et al., 2010). Analysis of the EPR spectra of DNA modified with PNCs, together with hydrolytic determination of platinated DNA bases, showed (Shugalii et al., 1998) that complexes **30b** and **4d** form predominantly ( 95%) bidentate intrastrand adducts with DNA. In adducts formed by both complexes, the rotation of the nitroxyl radicals is equally slow (Fig. 8) (correlation time τ ~ 10–8 s–1).

**Figure 8.** EPR spectra of free **4d** in water (a) and ethanol (d), and DNA modified by **4d** (b, e), and **30b** (c, f) in aqueous 0.01 M NaHCO3 at Pt-to-nucleotide ratio *r* =0.16, 37C, 24 h. Spectra were recorded at magnetic modulation 0.32 mT, microwave power 5 (20C) and 0.5 mW (–196C), field scan 20 mT, scan time 8 min, time constant 1 s.

This result can be explained by immobilization of the radical moiety in the major DNA groove for the complex **30b** adducts, and by the immobilization and/or by rigid structure of the double bound radical moiety in adducts formed by complex **4d**. Adducts formed by complexes **31b** and **32c** whose radical moiety is separated from the Pt atom by the methylene or ethylene bridge, are characterized by an order of magnitude lower values of the parameter τ. Presumably, this phenomenon is related to partial release of nitroxyl moiety from relatively shallow major groove of DNA, which increases its rotational mobility (Sen', 2002).

390 Nitroxides – Theory, Experiment and Applications

groups (Frey, 1993).

time 8 min, time constant 1 s.

equally slow (Fig. 8) (correlation time τ ~ 10–8 s–1).

**Figure 7.** Reaction of Pt(II) complexes with nucleophilic atom Nu of a target molecule.

For relatively easily hydrolyzable complexes, including cisplatin, the reaction proceeds through the path *A* (Alderden et al., 2006), but for complexes of type **4e** (X + X' = cyclobutane dicarboxylate) there is an evidence for direct substitution of the X-ligands by N-donor

It is known that cisplatin and its analogues bind mainly to the guanine and adenine bases of DNA with the formation of cross-links, thus perturbing the structure of DNA (Kelland, 2007; Wheate et al., 2010). Analysis of the EPR spectra of DNA modified with PNCs, together with hydrolytic determination of platinated DNA bases, showed (Shugalii et al., 1998) that complexes **30b** and **4d** form predominantly ( 95%) bidentate intrastrand adducts with DNA. In adducts formed by both complexes, the rotation of the nitroxyl radicals is

**Figure 8.** EPR spectra of free **4d** in water (a) and ethanol (d), and DNA modified by **4d** (b, e), and **30b** (c, f) in aqueous 0.01 M NaHCO3 at Pt-to-nucleotide ratio *r* =0.16, 37C, 24 h. Spectra were recorded at magnetic modulation 0.32 mT, microwave power 5 (20C) and 0.5 mW (–196C), field scan 20 mT, scan

Exciting opportunities for the instrumental use of PNCs were shown by Dunham et al., 1998. An adduct of **30a** complex with synthetic DNA fragment containing 11 base pairs was synthesized. Its structure in solution was determined by NMR based on the dependence between paramagnetic broadenings of protons of DNA bases and distances between bases and nitroxyl radical. Formation of the adduct was proved to result in the bending of DNA molecule that forms the angle ~80° with respect to the major groove, whereas the minor groove becomes strongly broaden.

The ability of complexes **1** – **5** to bind to the isolated DNA *in vitro* was determined under standard conditions and characterized by a parameter *r*, which is equal to the number of linked labels per one nucleotide. In the series of complexes with the same amino ligand **4c e**, parameter *r* grows with the increase in the rate of hydrolysis of X-ligands. Platinating activity of compounds with different amino ligands depends on the total volume of these ligands and/or their linear sizes. Bulky biradical complex **1c** and complexes **31b** and **32c**, whose sizes are enlarged due to a methylene or ethylene bridge, bind to DNA 5 to 10 times less efficiently than cisplatin or complexes **4c**, **d** (Fig. 9).

**Figure 9.** Relationship between the platinating activity (*r*) and specific destabilization of the DNA duplex (*T*m) for PNCs. The platination of DNA was carried out in 0.01 *M* NaHCO3 over a period of 24 h at 37 C at the initial molar ratio *r*in = 0.1.

Ordinates in Fig. 9 are the values of specific destabilization of the DNA duplex, *Tm*, that corresponds to the decrease in the DNA melting temperature due to formation of one adduct per 100 nucleotides. These values were calculated according to the formula

$$\delta T\_{\text{w}} = (T\_{\text{m}}\,' - T\_{\text{m}}) / 100r\_{\text{r}}$$

where *Tm* and *Tm′* are the melting points for unmodified and platinated DNA, respectively. The data in Fig. 9 show that the low-activity complexes (with low *r*–values) cause the greatest disorder in the DNA duplex. Presumably, adducts of these complexes are readily recognized by the repair machinery. This is in agreement with the data on low antitumor activity of such complexes (see below). The binuclear complex **5b** stabilizes DNA due to a predominant (~70%) formation of the interstrand crosslinks interfering with the thermal dissociation of the DNA duplex. It is interesting that bi- and trinuclear platinum amino complexes exhibit cytotoxic properties different from that of mononuclear ones, in particular, the former are active against cisplatin-resistant cells (Farrell et al., 1999).

### **4. Cytotoxicity of PNCs in tumor cell cultures**

A simplified mechanism of cytotoxic effect of cisplatin and its analogs includes the transport of the complexes into the cell, their activation by the hydrolysis of leaving ligands (Cl—, carboxylates), penetration into the nucleus, and formation of adducts with DNA (Kelland, 2007; Wheate et al., 2010). The DNA lesions are either repaired, or initiate a complex process of programmed cell death, *i.e*., apoptosis. In addition, it is known that cisplatin, directly or indirectly, causes the generation of reactive oxygen species. This process is important for the initiation of apoptosis (Miyajima et al., 1997; Bragado et al., 2007) and may also be responsible for side effects, *e.g.* nephrotoxicity (Tsutsumishita et al., 1998).

Nitroxyl radicals are antioxidants, which can react with active radicals not only stoichiometrically, but also act as catalysts of redox reactions and mimetics of enzymatic systems. For example, in aqueous medium they perform superoxide dismutation through the reduction of radical HO2• by nitroxyl radical and the oxidation of radical O2•─ by oxoammonium cation (Sen' et al., 1976, 2009; Goldstein et al., 2003) (Fig. 10).

**Figure 10.** Superoxide dismutase-like activity of nitroxyl radicals

Interestingly, the nitroxyl based catalysis of dismutation of HO2• radical, generated in organic compounds undergoing oxidation, is carried out by the pair of nitroxyl radical/hydroxylamine (Denisov, 1996) (Fig. 11) Thermodynamic data are presented in support of the latter mechanism in organic medium. The measured constants of forward and reverse reactions at 50 C are equal to 104 – 105 M–1·s–1 (Denisov, 1996). Existence of two mechanisms for different media is not excluded. It looks reasonable that in an aqueous

**Figure 11.** Catalytic disproportionation of radical HO2• in organic medium

particular, the former are active against cisplatin-resistant cells (Farrell et al., 1999).

responsible for side effects, *e.g.* nephrotoxicity (Tsutsumishita et al., 1998).

oxoammonium cation (Sen' et al., 1976, 2009; Goldstein et al., 2003) (Fig. 10).

HO2 <sup>+</sup> H+ •

**Figure 10.** Superoxide dismutase-like activity of nitroxyl radicals

N O**•**

A simplified mechanism of cytotoxic effect of cisplatin and its analogs includes the transport of the complexes into the cell, their activation by the hydrolysis of leaving ligands (Cl—, carboxylates), penetration into the nucleus, and formation of adducts with DNA (Kelland, 2007; Wheate et al., 2010). The DNA lesions are either repaired, or initiate a complex process of programmed cell death, *i.e*., apoptosis. In addition, it is known that cisplatin, directly or indirectly, causes the generation of reactive oxygen species. This process is important for the initiation of apoptosis (Miyajima et al., 1997; Bragado et al., 2007) and may also be

Nitroxyl radicals are antioxidants, which can react with active radicals not only stoichiometrically, but also act as catalysts of redox reactions and mimetics of enzymatic systems. For example, in aqueous medium they perform superoxide dismutation through the reduction of radical HO2• by nitroxyl radical and the oxidation of radical O2•─ by

*k*<sup>+</sup> = 5•10<sup>5</sup> M–1s–1

N+ O

– <sup>+</sup> H+

•–

pH 7

HO2 H2O2 •– O2

Interestingly, the nitroxyl based catalysis of dismutation of HO2• radical, generated in organic compounds undergoing oxidation, is carried out by the pair of nitroxyl radical/hydroxylamine (Denisov, 1996) (Fig. 11) Thermodynamic data are presented in support of the latter mechanism in organic medium. The measured constants of forward and reverse reactions at 50 C are equal to 104 – 105 M–1·s–1 (Denisov, 1996). Existence of two mechanisms for different media is not excluded. It looks reasonable that in an aqueous

*k*– > 10<sup>9</sup> M–1s–1

O2 O2

**4. Cytotoxicity of PNCs in tumor cell cultures** 

*Tm=(Tm′ – Tm)/100r,*  where *Tm* and *Tm′* are the melting points for unmodified and platinated DNA, respectively. The data in Fig. 9 show that the low-activity complexes (with low *r*–values) cause the greatest disorder in the DNA duplex. Presumably, adducts of these complexes are readily recognized by the repair machinery. This is in agreement with the data on low antitumor activity of such complexes (see below). The binuclear complex **5b** stabilizes DNA due to a predominant (~70%) formation of the interstrand crosslinks interfering with the thermal dissociation of the DNA duplex. It is interesting that bi- and trinuclear platinum amino complexes exhibit cytotoxic properties different from that of mononuclear ones, in

> medium the preferred process is an electron transfer followed by a thermodynamically favorable hydration of oxoammonium cation (Fig. 10), while in an organic medium more typical reactions are the redox processes involving a hydrogen atom transfer (Fig. 11). Therefore, in biphasic aqueous-organic systems present in biological objects, both mechanisms are possible.

> Like other antioxidants, under certain conditions, nitroxyls may exhibit pro-oxidant activity. The structure and concentration of nitroxyls, the medium properties, and other hard-toidentify factors can determine their anti– or pro-oxidant effect. At submillimolar concentrations, nitroxyls, as a rule, exhibit antioxidant properties and protect cells from apoptosis (Wilcox, 2010). At millimolar concentrations, nitroxyls are cytotoxic toward cultured tumor cells (Gariboldi et al., 1998, 2000, 2003, 2006; Suy et al., 2005) and are active against model animal tumors (Konovalova et al., 1964; Suy et al., 2005). Nitroxyl radicals cause cell death both in the wild type and p53 mutant cells (Suy et al., 2005). The study of interplay of platinum and nitroxyl pharmacophores combined in one molecule is of interest also in connection with the recent discussions on application of antioxidants and redoxactive agents in tumor chemotherapy (Seifried, 2003; Wondrak, 2009).

> To elucidate the interaction between platinum and nitroxyl pharmacophores, we studied the effect of 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino-TEMPO) on the toxicity of cisplatin to HeLa cells. According to the published approach (Reynolds & Maurer, 2005; Chou, 2006), the dose-response relationships were determined for individual agents and their mixtures at a fixed concentrations ratio. These data were transformed into the combination index (CI) – dead cells fraction (*f*a) dependence (Fig. 12). In a wide range of fraction affected *f*a, values of log (CI) are positive, which indicates antagonism of the agents. In the range of relatively high fraction affected, corresponding to millimolar concentrations of 4-amino-TEMPO, a transition to negative values of log (CI) takes place which means the mutual reinforcement of cytotoxicity, i.e., synergy. These results are consistent with the antioxidant properties of nitroxyls at low and their pro-oxidant properties at high concentrations.

> Data on the cytotoxicity of PNCs also reflects the antagonism of platinum and nitroxyl pharmacophores. Complexes of platinum (II) **2b** and **30b**, containing nitroxyl radicals of different structure, are significantly less toxic to tumor cells compared to cisplatin (Table 1).

**Figure 12.** Combination index [log (CI)] versus fraction affected (*f*a) of HeLa cells for combination of 4 amino-2,2,6,6-tetramethylpiperidine-1-oxyl and cisplatin.


**Table 1.** 50% Inhibitory concentrations for PNCs in HeLa and H1299 cells; MTT assay, 24 h of exposure. \*Evaluation of IC50 is limited by low solubility of complexes.

Platinum(IV)-nitroxyl complexes **9b** and **10b**, being structurally close to JM-216, are also considerably less toxic to cells compared to the latter. Elongation of axial ligands Y increases both lipophilicity and cytotoxicity (complexes **9c** and **10c-f**), which is obviously due to the increased accumulation of complexes in cells (Fig. 13).

**Figure 13.** Relationship between lipophilicity (log POW) and cytotoxicity (IC50) of complexes **10b-f** in HeLa () and H1299 () cell lines; *n* means the total number of carbon atoms in carboxylic residues Y.

The H1299 cells are less sensitive to platinum complexes. Unlike the HeLa cells, H1299 cells do not contain p53 protein because of mutations of p53 gene in both alleles (Mitsudomi et al., 1992). Since p53 protein plays a key role in the process of apoptosis in response to DNA damages (Vousden & Prives, 2009), the observed lower sensitivity of H1299 cells to platinum complexes compared to HeLa cells can be related to lack of p53 function.

394 Nitroxides – Theory, Experiment and Applications

amino-2,2,6,6-tetramethylpiperidine-1-oxyl and cisplatin.

\*Evaluation of IC50 is limited by low solubility of complexes.

increased accumulation of complexes in cells (Fig. 13).

*n* =4

0,1

1

10

IC50, M

100

Cell line

Cis-

**Figure 12.** Combination index [log (CI)] versus fraction affected (*f*a) of HeLa cells for combination of 4-

HeLa 14.8 125 112 14.4 >200\* 13.4 200 4.18 2.45 0.23 0.09 H1299 66.7 >150\* >150\* 38.8 >200\* 25.4 220 24.6 16.6 1.36 0.69 **Table 1.** 50% Inhibitory concentrations for PNCs in HeLa and H1299 cells; MTT assay, 24 h of exposure.

Platinum(IV)-nitroxyl complexes **9b** and **10b**, being structurally close to JM-216, are also considerably less toxic to cells compared to the latter. Elongation of axial ligands Y increases both lipophilicity and cytotoxicity (complexes **9c** and **10c-f**), which is obviously due to the

**Figure 13.** Relationship between lipophilicity (log POW) and cytotoxicity (IC50) of complexes **10b-f** in HeLa () and H1299 () cell lines; *n* means the total number of carbon atoms in carboxylic residues Y.

log Pow

024

*n* =10

*n* =8

*n*

=12

O

<sup>N</sup> NH2 •

**5b-f**

*n* 

=16

Pt H3N Cl

OY

OY

Cl

IC50, µM

platin **2b 30b** JM216 **9b 9c 10b 10c 10d 10e 10f** 

Our further study was focused on complex **10d** since it combined high cytotoxicity with sufficient solubility in water. The effect of complex **10d** and cisplatin on the cell cycle of HeLa and H1299 cells was studied (Fig. 14). According to flow cytofluorimetry data, both complexes cause approximately fivefold increase in the number of HeLa cells in the subG1 fraction, thus indicating induction of cell death. Accumulation of HeLa cells in early S phase was also observed, which suggests that cell death is induced after cell cycle arrest during DNA synthesis. For H1299 cells, some increase in the S phase population and two-fold decrease in G2/M phases population was demonstrated, which shows cytostatic activity of the complexes without significant cell death.

**Figure 14.** Cell cycle analysis of HeLa and H1299 cells treated with cisplatin and complex **10d** (concentration IC50, 24 h).

Cell death found in flow cytofluorimetry experiments was shown to be apoptotic. Both cisplatin and complex **10d** cause in HeLa cells characteristic for apoptosis morphological changes of cell nuclei and internucleosomal cleavage of DNA leading to electrophoretic DNA laddering (Fig. 15a-d).

As it was discussed above, cisplatin and its analogues form adducts with DNA that, when are not repaired, trigger the tumor suppressor protein p53 (Alderden et al., 2006; Kelland, 2007; Wheate et al., 2010). Unlike cisplatin, the **10d** complex does not cause increase of the p53 protein expression in MCF7 cells containing wild-type p53 gene (Fig. 15e). This finding indicates differences in the mechanism of cytotoxic action of these two complexes. Interestingly, on the rat glioma C6 cells, the simple nitroxyl Tempol was shown to cause apoptosis without elevation of p53 protein levels (Gariboldi et al., 2003). Many compounds

**Figure 15.** The mechanism of cytotoxicity of platinum complexes. *a*—*c* DAPI staining of DNA in HeLa cells in the control (a) and after 24 h exposure to cisplatin (CP) (b) or complex **10d** (c); arrows indicate the fragmented nuclei of the apoptotic cells. *d* Agarose gel electrophoresis of HeLa cells DNA after 12 h and 24 h exposure to CP and complex **10d**. *e* Immunoblotting of MCF7 cell lysates with antibody to p53 in the control (C) and after 6 h exposure to CP and complex **10d**.

including nitroxyls (Sui at al., 2005) and platinum complexes (Gorczyca et al., 1993; Kalimutho et al., 2011; Roubalová et al., 2010) induce apoptosis both in cells with wild-type p53 gene and in p53-deficient cells. However, p53-independent apoptosis of tumor cells harboring wild-type p53 gene, to our knowledge, was observed for the first time.
