**5. Antitumor activity of PNCs in animal experimental tumors**

Data on cytotoxicity revealed from cell culture studies and antitumor activity observed in animal tumor models do not correlate for platinum complexes. A striking example of this phenomenon is carboplatin, the known antitumor chemotherapy drug possessing negligible cytotoxicity *in vitro*. Carboplatin has been approved for clinical use because of favorable efficiency to toxicity ratio found in animal tumor models (Jakupec et al., 2008).

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

Toxicity and biological activity of Pt(II) diamino complexes depend on the structure of both the carrier amino ligands and the leaving groups, the latter being replaced during metabolism and target binding (Ho et al., 2003). The biradical complexes **1** containing two bulky amino ligands poorly bind to DNA *in vitro* (see above) and exhibit low toxicity and antitumor activity *in vivo* (Table 2).

Complexes **2** and **3** bearing one bulky substituent are close in their properties to cisplatin. Their *LD*50 values are only 1.5 to 2.5-fold higher than that of cisplatin. They efficiently platinate DNA and exhibit antitumour activity comparable with cisplatin. Influence of the nitroxyl structure on the complex activity can be seen when comparing complexes **2b** and **30b** that differ only in the size of nitroxyl cycle. Compound **30b** is both more toxic and more active against leukemia P388. For complexes **4**, the correlation between the rate of leaving ligands


Platinum Complexes with Bioactive Nitroxyl Radicals: Synthesis and Antitumor Properties 397

**Table 2.** Toxicity (*LD*50) and antileukemic (P388) activity (*ILS*) of PNCs. *LD*50 is a dose which is lethal to 50% of healthy mice. Increase in the life span *ILS* = [100(*T*/*C* – 1)], where *T* and *C* the average life-time (days) of treated and control animals, respectively. The numbers of animals survived for more than 60 days in the group of six animals are given in brackets.

hydrolysis and the toxicity was established (Sen' et al., 1996; Shugalii et al., 1998). Readily hydrolyzable **4c** has the highest toxicity whereas **4e**, the most slowly hydrolyzable compound among complexes **4**, exhibits the lowest toxicity, but, like carboplatin, it possesses good antitumor activity only at high doses. Compound **4d**, the structural analogue of oxaliplatin, is approximately 2-fold less toxic compared to the latter. The observed decrease in toxicity might be due to the influence of the nitroxyl group. The data presented demonstrate that, among Pt(II) complexes with amino nitroxyl radicals, high antitumor activity *in vivo* is characteristic for those that contain no more than one bulky amino ligand, platinate DNA with high efficiency, and cause moderate destabilization of DNA duplex (Fig. 9, Table 2).

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

396 Nitroxides – Theory, Experiment and Applications

**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

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

Data on cytotoxicity revealed from cell culture studies and antitumor activity observed in animal tumor models do not correlate for platinum complexes. A striking example of this phenomenon is carboplatin, the known antitumor chemotherapy drug possessing negligible cytotoxicity *in vitro*. Carboplatin has been approved for clinical use because of favorable

Toxicity and biological activity of Pt(II) diamino complexes depend on the structure of both the carrier amino ligands and the leaving groups, the latter being replaced during metabolism and target binding (Ho et al., 2003). The biradical complexes **1** containing two bulky amino ligands poorly bind to DNA *in vitro* (see above) and exhibit low toxicity and

Complexes **2** and **3** bearing one bulky substituent are close in their properties to cisplatin. Their *LD*50 values are only 1.5 to 2.5-fold higher than that of cisplatin. They efficiently platinate DNA and exhibit antitumour activity comparable with cisplatin. Influence of the nitroxyl structure on the complex activity can be seen when comparing complexes **2b** and **30b** that differ only in the size of nitroxyl cycle. Compound **30b** is both more toxic and more active against leukemia P388. For complexes **4**, the correlation between the rate of leaving ligands

harboring wild-type p53 gene, to our knowledge, was observed for the first time.

**5. Antitumor activity of PNCs in animal experimental tumors** 

efficiency to toxicity ratio found in animal tumor models (Jakupec et al., 2008).

in the control (C) and after 6 h exposure to CP and complex **10d**.

**5.1. Pt(II) complexes** 

antitumor activity *in vivo* (Table 2).

Toxicity of Pt(IV) complexes **9** and **10** varies in the wide range depending on the structure of axial ligands. Compounds **9a,b** and **10a,b** are 1.6 to 3-fold less toxic compared to the corresponding Pt (II) analogues, **2** and **3** (Table 2). As in the case of divalent complexes, the piperidine oxyl derivatives **10** are both more toxic and more active against leukemia P388 than pyrrolidine oxyl derivatives **9**.

An important feature of PNCs is found in comparative study of development of tumor resistance in leukemia P388 to complex **10a** and cisplatin (Sen' et al., 2003; Goncharova et al., 2011). The development of resistance was induced by sequential inoculation of tumor cells from animals treated with equitoxic doses of drugs. The tumor acquired resistance (≤ 20% of the sensitivity of the parent tumor) to cisplatin at the 4th and to complex **10a** at the 10th generation of tumor (Fig. 16). This data demonstrate that the resistance to complex **10a** develops 2.5-fold slower than that to cisplatin.

Interesting results were observed when PNCs and cisplatin were used in combination at low doses (1/10 to 1/20 of *LD*50) for leukemia P388 treatment. Individual compounds in the same doses caused low *ILS*–indices with no cured animals, but their combinations cured up to 100% of mice (Fig. 17).

Complexes **10a** and **10b** containing piperidinoxyl moiety exhibit higher antitumor activity compared to that of complex **9b**, both as single agents and in combination with cisplatin. Thus, small difference in the structure of nitroxyl radicals in these PNCs has a significant influence on their antitumor activity *in vivo*.

**Figure 16.** Development of resistance to cisplatin and complex **10a** in a series of successive transplant generations (*n*). *ILS*<sup>0</sup> is the increase in the life span of treated animals bearing the sensitive (parent) generation of leukemia P388.

It is known that reduction potentials *E*1/2(>N+=O/>N─O•) of nitroxyls of piperidine series are, on the average, approximately 0.1 V lower than that of radicals of pyrrolidine series (Goldstein et al., 2006; Manda et al., 2007; Sen' & Golubev, 2009). Therefore, piperidinoxyls are oxidized by HO2• radical more readily (Fig. 10), and they are more efficient superoxide dismutase mimetics compared to pyrrolidinoxyls (Goldstein et al., 2006). At the same time, *in vivo*, pyrrolidinoxyls undergo reduction to corresponding hydroxylamines about tenfold slower than piperidinoxyls (Komarov et al., 1994). Along with possible differences in pharmacokinetics of the complexes, these features of the redox properties of nitroxyls, presumably, affect the biological activity of PNCs.

**Figure 17.** Synergy for the antitumor effect of cisplatin (0.6 mg/kg) combined with complexes **9b** (2,3 mg/kg), **10a** (1,4 mg/kg) or **10b** (5,0 mg/kg) against leukemia P388 (days of treatment 1, 3, 5, 7).
