**6. Conclusion**

398 Nitroxides – Theory, Experiment and Applications

100% of mice (Fig. 17).

generation of leukemia P388.

presumably, affect the biological activity of PNCs.

0

20

40

*ILS*

*n* **/** *ILS*

*o* **(%)**

60

80

<sup>100</sup> **Cisplatin**

develops 2.5-fold slower than that to cisplatin.

influence on their antitumor activity *in vivo*.

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**

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

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

**10a**

**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)

0 2 4 6 8 10 12

*n*

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, Recent studies (Wondrak, 2009; DeNicola et al., 2011) show that modulation of the redox state of cancerous cells could provide a new approach to suppression of tumor growth. Effects of nitroxyl radicals on the redox processes in normal cells and their cytotoxicity in tumor cells are documented in many examples (Gariboldi et al., 2000; Suy et al., 2005; Wilcox, 2010). Presumably, nitroxyls may affect the tumor cell viability through a redoxmediated signaling, which ultimately activate apoptosis.

On the other hand, the influence of nitroxyl radical on activity of anticancer agent, when they are used in combination or are covalently linked in one molecule, appears to depend on local concentration of radicals. At low concentrations of nitroxyls, which corresponds to rather low therapeutic doses of hybrid compounds, radicals are likely to impair the oxidative stress caused by tumor process and an anticancer agent itself. Published data show that *in vivo* the overall and/or specific toxicity may be reduced for hybrid compounds without significant loss of antitumor activity. On the other hand, high concentrations of nitroxyls, which can be implemented in combinations with anticancer agents, increase the intrinsic oxidative stress in tumor cells and exert a selective cytotoxic effect.

The known active anticancer complexes like cisplatin, oxaliplatin, and satraplatin bear in their structure redox-inert amino ligands. We synthesized structurally close analogs, i.e., platinum-nitroxyl complexes, amino ligands of which hold a wide spectrum of redox activity and are able to modulate biological properties of the new compounds. Their physicochemical properties, interaction with DNA, cytotoxicity *in vitro* and antitumor activity *in vivo* were studied. A limited correlation was found between the ability of new complexes to form adducts with DNA, thermal stability of these adducts and antitumor activity of the complexes. PNCs are characterized by features that distinguish them from platinum complexes with usual alkylamines. An antagonism of platinum and nitroxyl pharmacophore was observed in cell culture. As compared to cisplatin or satraplatin, structurally close nitroxyl derivatives are approximately 10 times less cytotoxic. This may be explained by a moderate inhibition of p53-dependent apoptosis due to the antioxidant properties of nitroxyl radicals. These *in vitro* findings do not correlate with *in vivo* data, and antitumor activities of some PNCs and cisplatin are approximately the same. The relatively slow development of resistance to PNCs and the presence of synergy for the combinations of PNCs and cisplatin imply the differences in the mechanism of antitumor action of cisplatin and PNCs. Due to antioxidant properties (Sen' et al., 2000), nitroxyls in PNCs may ameliorate side effects typical for cisplatin, such as nephrotoxicity and neurotoxicity. For example, nitroxyl derivative of daunorubicin, ruboxyl, has virtually no cardiotoxicity that limits therapeutical doses of the parental compound (Emanuel & Konovalova, 1992). Reduced side effects, in turn, contribute to the better survival and, as a result, the higher efficacy of tumor chemotherapy *in vivo*. Our and literature data show that nitroxyls are promising modulators of the activity of anticancer agents and, as such, could be approved for clinical use.

### **Author details**

Vasily D. Sen', Alexei A. Terentiev and Nina P. Konovalova *Institute of Problems of Chemical Physics, Russian Academy of Sciences, Chernogolovka, Russian Federation* 

## **Acknowledgement**

This work was partly supported by the Russian Foundation for Basic Research (Project No. 09-03-01187).
