Experimental redox potentials of oxoammonium cation / nitroxide redox couple
Nitroxides are stable free radicals which have the >N-O moiety. In most cases, nitroxides have a ring structure. For example, imidazoline, isoindoline, piperidine and pyrrolidine ring nitroxides (Fig. 1) have been used as agents for spin labeling, imaging, and as antioxidants. These nitroxides have four substituents at the α-position; two substituents on each α-carbon. All four substituents are needed for avoiding the disproportionation reaction of nitroxides except for the case of a bridgehead at the α-position. Methyl groups have been chosen as simple and inert substituents. However, it has been reported that other types of substituents, especially ethyl groups, showed unique characteristics that were unlike those of the conventional methyl group.
In this chapter, we will introduce the conventional as well as the latest synthetic methods used to introduce the various substituents to the α-position. Also, we will describe the structure–reactivity relationships of α-substituted nitroxides.
2. Synthetic methods of α-substituted nitroxides
Imidazoline nitroxides have been synthesized from α-hydroxyaminoketone with carbonyl compounds (Scheme 1) (Volodarsky and Igor A, 1988). The R1 groups of α-hydroxyaminoketone and R2 groups of carbonyl compounds correspond to the α-position of the imidazoline ring. α-Hydroxyaminoketones are synthesized from appropriate olefines
R1 = Me; R2 = Me, Et, n-Bu, (CH2)4, (CH2)5
R1 = Et; R2 = Me, Et, (CH2)5, (CH2)2COONa
Isoindoline nitroxides have been prepared by the addition of a greater than fourfold excess of a single Grignard reagent to
R = Me, Et, n-Pr, n-Bu, Ph
Piperidine nitroxides have been synthesized by two main approaches. One is the synthesis from acetonin (2,2,4,4,6-pentamethyl-2,3,4,5-tetrahydropyrimidine) with carbonyl compounds (Schemes 3a and b). Murayama
The other main approach is a stepwise synthesis from the appropriate starting material (Schemes 3c, d and e). For example, Yoshioka
Recently, an alternative synthetic method has been developed (Scheme 3f) (Sakai et al., 2010). This method involves 2,2,6,6-tetramethyl-4-piperidone as a starting compound; this compound is more stable than acetonin and is available commercially. This compound with cyclohexanone directly gave the piperidone derivative having spirocyclohexyl groups at the 2,6-position under a mild reaction condition. Moreover, the reaction yield was increased by using 1,2,2,6,6-pentamethyl-4-piperidone and a base. With this starting compound, various substituents have been introduced to the α-position (Yamasaki et al., 2011; Yamasaki et al., 2010). From the investigation of the reaction mechanism, the nitrogen derived from ammonium chloride was introduced to the piperidone ring. Therefore, using 15N-labeled NH4Cl instead of 14NH4Cl, 15N-labeled 2,2,6,6-tetrasubstituted piperidin-4-one-1-oxyls can be produced with high (>98%) 15N content. Thus, the external NH4X compound seems to be the source of nitrogen during this reaction.
α-Substituted pyrrolidine nitroxides has been synthesized
R = Me, Et, n-Pr, n-Bu, Ph
3. Evaluation of α-substituted nitroxides
3.1. Common reactivity of nitroxides
Nitroxides have several potential advantages as spin probes (Kuppusamy et al., 2002; Yamada et al., 2006), spin labels (Borbat et al., 2001), contrast agents (Soule et al., 2007) and antioxidants (Wilcox and Pearlman, 2008). These applications are based on the complementary nature of the radical moieties in nitroxides; paramagnetism allows them to react with free radicals and interact with nuclear spin. For instance, these properties allow nitroxides to be used as contrast agents for magnetic resonance imaging (MRI) to give images of the morphological nature and redox imbalance in animal models of oxidative stress.
In biological systems, understanding of biophysical properties is helpful to promote effective utilization and control of the reactivity of nitroxides. Nitroxides are readily oxidized to oxoammonium cations or reduced to hydroxylamines by various
3.2. Reduction stability
As well as at β- or γ-positions, substituent groups at α-positions in a nitroxide ring can change their reactivity. For instance, phosphorylated pyrrolidinyl nitroxide showed moderate increase toward ascorbate reduction compared with the tetramethyl pyrrolidine nitroxide (Mathieu et al., 1997). On the other hands, the tetraethyl-substituted isoindoline (Marx et al., 2000), imidazoline (Kirilyuk et al., 2004), and imidazolidine (Kirilyuk et al., 2004) nitroxides showed high resistivity to ascorbate reduction than the corresponding tetramethyl compounds. Furthermore, Kirilyuk
Tetraethyl-nitroxides, having higher lipophilicity than tetramethyl compounds, have been reported to be less toxic to cells (Kinoshita et al., 2010) although the toxicity is reported to be correlated with the structure and lipophilicity of nitroxides (Ankel et al., 1987). Furthermore, single-dose administration of tetraethyl piperidine nitroxide has been shown to have lower blood pressure-lowering effects compared with that of Tempol (Kinoshita et al., 2010).
3.3. Electrochemical behaviours
The change in nitroxide reactivity due to the presence of tetraethyl substituents suggests that introduction of bulky alkyl groups at α-positions in a nitroxide ring are responsible for their reduction stability. Steric hindrance around the radical moiety is one of the most important factors inhibiting access to reductants. However, the ESR signal intensities of 7-Aza-3,11-dioxa-15-oxodispiro[18.104.22.168]hexadec-7-yl-7-oxyl (which has also bulky spirocyclohexyl rings at α-positions) decrease rapidly in the presence of ascorbate (Kinoshita et al., 2009). This suggests that the electronic environment around the N-O moiety also influences its reduction stability. In fact, the rate of reduction of β- or γ-substituted nitroxides by ascorbate has been reported to be primarily dependent upon their structure and correlation with E1/2 (Blinco et al., 2008; Kocherginsky and Swartz, 1995). The reactivity of α-substituted nitronyl nitroxides is also dependent upon the electronic properties of the substituent groups (Wu et al., 2006). The α-substitution of piperidine nitroxide has been reported to change dramatically their redox potentials for one-electron oxidation and reduction (Yamasaki et al., 2011). In the oxidation step, electron-donating substituents are likely to stabilize oxoammonium cations, and substituents with heteroatoms destabilize them because of the electron-withdrawing inductive effect (Fig. 4, Table 1). The redox potentials for one-electron reduction are listed in Table 2. The electron-withdrawing groups at the α-positions of the piperidine ring destabilize the reduced form of nitroxides, whereas electron-donating substituents stabilize them.
3.4. Structure–reactivity relationships
As described above, ascorbate can readily convert nitroxides into the corresponding hydroxylamines. The reduction rate is correlated with the inductive effects from the β-position in the piperidine ring and the γ-position in the pyrrolidine ring (Morris et al., 1991). Also, nitroxides with heteroatoms in their ring are unstable for the reduction (Couet et al., 1985).
Imidazole, isoindoline and piperidine nitroxides have a common feature: tetraethyl-nitroxides at α-positions adjacent to the radical moiety have high resistance to reduction by ascorbate compared with the widely used tetramethyl-nitroxides (see above). The rate of decay of the ESR signals of nitroxides seems to be inversely proportional to the number of ethyl groups (Yamasaki et al., 2010). Nitroxides containing four ethyl groups are more resistant to the reduction than those with two ethyl groups. The reduction rates of nitroxides which have heteroatoms in their spirocyclohexyl ring have been found to be higher than tetramethyl nitroxides. Electron-withdrawing groups at spirocyclohexyl rings decrease the electron density around the N-O moiety, thereby favoring the reduction reaction. The trend of redox potentials for nitroxide reduction from electrochemical experiments is likely to be exactly the same as that of the nitroxide reduction rate by ascorbate. The ESR signal decay rate and the electromotive force between nitroxide and ascorbate (ΔEN–A) or the change in Gibbs free energy (ΔG) demonstrates very good correlations with ΔG in the negative ΔG region (r2 = 0.988) (Fig. 5) (Yamasaki et al., 2010). This indicates that reduction of the nitroxide by ascorbate occurred spontaneously if the ΔG value is negative, and that the reduction is not spontaneous if the ΔG value is positive. The factors influencing the reduction process of the nitroxide are dependent not only upon steric hindrances but also on redox potentials. The α-substitutions of piperidine nitroxides would be an effective approach to control the reactivity of nitroxides as a function of their applications.
In vivoevaluation and imaging
Nitroxides are reduced to mainly the hydroxylamine form
In general, the stability of nitroxide is reflected by their type of ring, substituent groups, and lipophilicity. Piperidine-nitroxides show a short half-life compared with that of pyrroridine-nitroxides. A typical tetramethyl-piperidine nitroxide, Tempone (oxo-TEMPO), has a short life-time (2 min) in blood due to rapid reduction (Ishida et al., 1989; Schimmack et al., 1976). However, piperidine nitroxides with spirocyclohexyl groups show resistance to enzymatic reduction in mouse liver homogenates (Okazaki et al., 2007). Conversely, tetraethyl nitroxides show resistance to reduction by ascorbic acid and seem to be stable
A nitroxide with a long half-life can also be a candidate
Recently, various types of α-substituted nitroxides have been synthesized and their