2-Butyl-2-tert-butyl-5,5-diethylpyrrolidine-1-oxyls: Synthesis and Properties

Nitroxides are broadly used as molecular probes and labels in biophysics, structural biology, and biomedical research. Resistance of a nitroxide group bearing an unpaired electron to chemical reduction with low-molecular-weight antioxidants and enzymatic systems is of critical importance for these applications. The redox properties of nitroxides are known to depend on the ring size (for cyclic nitroxides) and electronic and steric effects of the substituents. Here, two highly strained nitroxides, 5-(tert-butyl)-5-butyl-2,2-diethyl-3-hydroxypyrrolidin-1-oxyl (4) and 2-(tert-butyl)-2-butyl-5,5-diethyl-3,4-bis(hydroxymethyl)pyrrolidin-1-oxyl (5), were prepared via a reaction of the corresponding 2-tert-butyl-1-pyrroline 1-oxides with butyllithium. Thermal stability and kinetics of reduction of the new nitroxides by ascorbic acid were studied. Nitroxide 5 showed the highest resistance to reduction.


Introduction
Nitroxides are broadly used as molecular probes and labels in biophysics, structural biology, and biomedical research [1][2][3][4][5][6]. Modern trends in these research fields, such as in-cell electron paramagnetic resonance (EPR)/pulsed electron-electron double resonance (PELDOR) experiments [7,8] and in vivo NMR and EPR imaging [9,10], require stable paramagnetic agents that can retain the radical center in live cells or tissues long enough for the measurements. Resistance of a nitroxide group bearing an unpaired electron to chemical reduction with low-molecular-weight antioxidants and enzymatic systems is crucial for these applications. The redox properties of nitroxides depend on the ring size (for cyclic nitroxides) and electronic and steric effects of the substituents [11,12]. Five-membered-ring nitroxides of the pyrrolidine series show the highest resistance to reduction [13,14]. Introduction of bulkier alkyl substituents instead of methyls at the carbon atom adjacent to the N-O· group can increase nitroxide stability in model systems and in biological samples [15][16][17]. 2,2,5,5-Tetraethylpyrrolidine-1-oxyls 1 and 2 are the most resistant to reduction among all currently known nitroxides (Chart 1) [18][19][20]. One can assume that a further increase in the steric demand of
The general procedure for the synthesis of similar 1-pyrroline 1-oxides has been developed by Reznikov et al. [25]. Using a procedure similar to the one described in the literature [26], 1-hydroxy-5,5-diethyl-2,2,4-trimethyl-2,5-dihydroimidazole 7 [27] was treated with an excess of lithium diisopropylamide to produce dianion intermediate 8, which reacts with ethyl pivalate to form 9 (Scheme 2). Quenching of the reaction mixture in aerobic conditions leads to partial oxidation into the corresponding nitroxide; therefore, the mixture was treated with manganese dioxide to finalize the conversion. Nitroxide 10 was isolated as an orange paramagnetic crystalline solid with spectral parameters close to those of known 4-(2-oxoalkylidene)-imidazolidines: strong absorption at 298 nm in the UV spectrum indicated formation of an enaminoketone conjugated system, IR-spectra of 10 showed characteristic strong vibration bands of the enaminoketone moiety at 1635 and 1561 cm −1 (cf. [26]). Hydrogenation of 10 on a palladium catalyst afforded hydroxylamine 9. The structure of 9 was confirmed by a 1 H NMR spectrum: a methine proton signal at 5.01 ppm and a broad line of NH at 5.35 ppm indicate that 9 predominantly exists in 4-(2-oxoalkylidene)-imidazolidine form in CDCl3.
Treatment of enaminoketone 9 with conc. hydrochloric acid for 82 h resulted in hydrolysis of the heterocycle, and subsequent neutralization of the reaction mixture to pH 7-8 led to recyclization with the formation of 6 (Scheme 2). IR, UV, and NMR spectra of 6 are close to those provided in the Chart 1. Structures of nitroxides 1-5.
nitroxide of the 2,5-dihydroimidazole series, 3, with tert-butyl and n-butyl substituents at position 5 of the imidazole ring via the reaction of corresponding cyclic α-tert-butylnitrone with n-butyllithium (n-BuLi). Regretfully, this nitroxide appears to be thermally unstable, rapidly decomposing at 80 °C via tert-butyl radical abstraction [21]. Here we used the same strategy to synthesize highly strained nitroxides of the pyrrolidine series. Two nitroxides, 4 and 5, were prepared via a reaction of corresponding 2-tert-butyl-1-pyrroline-1-oxides with BuLi. Thermal stability and redox properties of the new nitroxides were studied. The new nitroxides are very lipophilic compounds with poor solubility in water; however, their hydroxyl groups may be employed for chemical binding to a delivery vehicle. The utility of lipophilic nitroxides with large hydrophobic groups for the design of lipid nanoparticles or micelles for MRI contrast and MRI-controlled drug delivery has been demonstrated by various research groups [22][23][24].

Scheme 1. Synthetic strategy for nitroxide 4.
The general procedure for the synthesis of similar 1-pyrroline 1-oxides has been developed by Reznikov et al. [25]. Using a procedure similar to the one described in the literature [26], 1-hydroxy-5,5-diethyl-2,2,4-trimethyl-2,5-dihydroimidazole 7 [27] was treated with an excess of lithium diisopropylamide to produce dianion intermediate 8, which reacts with ethyl pivalate to form 9 (Scheme 2). Quenching of the reaction mixture in aerobic conditions leads to partial oxidation into the corresponding nitroxide; therefore, the mixture was treated with manganese dioxide to finalize the conversion. Nitroxide 10 was isolated as an orange paramagnetic crystalline solid with spectral parameters close to those of known 4-(2-oxoalkylidene)-imidazolidines: strong absorption at 298 nm in the UV spectrum indicated formation of an enaminoketone conjugated system, IR-spectra of 10 showed characteristic strong vibration bands of the enaminoketone moiety at 1635 and 1561 cm −1 (cf. [26]). Hydrogenation of 10 on a palladium catalyst afforded hydroxylamine 9. The structure of 9 was confirmed by a 1 H NMR spectrum: a methine proton signal at 5.01 ppm and a broad line of NH at 5.35 ppm indicate that 9 predominantly exists in 4-(2-oxoalkylidene)-imidazolidine form in CDCl3. The general procedure for the synthesis of similar 1-pyrroline 1-oxides has been developed by Reznikov et al. [25]. Using a procedure similar to the one described in the literature [26], 1-hydroxy-5,5-diethyl-2,2,4-trimethyl-2,5-dihydroimidazole 7 [27] was treated with an excess of lithium diisopropylamide to produce dianion intermediate 8, which reacts with ethyl pivalate to form 9 (Scheme 2). Quenching of the reaction mixture in aerobic conditions leads to partial oxidation into the corresponding nitroxide; therefore, the mixture was treated with manganese dioxide to finalize the conversion. Nitroxide 10 was isolated as an orange paramagnetic crystalline solid with spectral parameters close to those of known 4-(2-oxoalkylidene)-imidazolidines: strong absorption at 298 nm in the UV spectrum indicated formation of an enaminoketone conjugated system, IR-spectra of 10 showed characteristic strong vibration bands of the enaminoketone moiety at 1635 and 1561 cm −1 (cf. [26]). Hydrogenation of 10 on a palladium catalyst afforded hydroxylamine 9. The structure of 9 was confirmed by a 1 H NMR spectrum: a methine proton signal at 5.01 ppm and a broad line of NH at 5.35 ppm indicate that 9 predominantly exists in 4-(2-oxoalkylidene)-imidazolidine form in CDCl 3 .
3). Complete conversion to dimer 12 can be achieved via oxidation of 6 with manganese dioxide. The samples of similar dimers are known to have quintet EPR spectra, initially attributed to a contribution of the biradical form [28]. It has been demonstrated later that the signal belongs to paramagnetic impurities [29], presumably to radical ions of the dinitrones [30]. Exchange with these radical ions may account for the strong broadening of some signals in 13 C NMR spectra of 12 (see Supplementary Materials). Single-crystal X-ray analysis of 12 uncovered a dihedral angle of 60° between nearly planar pyrrolinone rings (Figure 1; deviation within ±0.032 Å) (cf. [29]). Scheme 2. Synthesis of pyrroline N-oxide 6.
Treatment of enaminoketone 9 with conc. hydrochloric acid for 82 h resulted in hydrolysis of the heterocycle, and subsequent neutralization of the reaction mixture to pH 7-8 led to recyclization with the formation of 6 (Scheme 2). IR, UV, and NMR spectra of 6 are close to those provided in the literature for similar compounds [25]. According to NMR data, 11 exists in CDCl 3 as a mixture of tautomeric forms (6A and 6B) in a 4:7 ratio (Scheme 2) (cf. [25]). The presence of the N-hydroxyenamine form 6B makes 3-oxo-3,4-dihydro-2H-pyrrole 1-oxides susceptible to oxidation to vinylnitroxide radicals, which in turn are prone to dimerization [28]. Indeed, similarly to other known 3-oxo-3,4-dihydro-2H-pyrrole 1-oxides, 6 undergoes oxidation by air oxygen in solutions, thus yielding the emergence of deep violet coloring due to ethylene-type dimer formation (Scheme 3). Complete conversion to dimer 12 can be achieved via oxidation of 6 with manganese dioxide. The samples of similar dimers are known to have quintet EPR spectra, initially attributed to a contribution of the biradical form [28]. It has been demonstrated later that the signal belongs to paramagnetic impurities [29], presumably to radical ions of the dinitrones [30]. Exchange with these radical ions may account for the strong broadening of some signals in 13 C NMR spectra of 12 (see Supplementary Materials). Single-crystal X-ray analysis of 12 uncovered a dihedral angle of 60 • between nearly planar pyrrolinone rings (Figure 1; deviation within ±0.032 Å) (cf. [29]).
Of note, earlier processing of the reaction mixture in the hydrolysis of 9 leads to the formation of byproducts and decreases the yield of 6. If the reaction mixture was processed, after 40 h, another compound (13, yield 15%) was isolated along with 6 (Scheme 2). Obviously, enaminoketone 9 hydrolysis proceeds via the consecutive formation of acyclic enaminoketone 14 and β-diketone 15 intermediates, both present in the reaction mixture, and formation of 13 is a result of cyclization of the former. Formation of similar compounds has been previously observed in a recyclization reaction of imidazolidine enaminoketones with an acceptor group at the exo-methylene carbon atom [31].
It is known that treatment of 3-oxo-3,4-dihydro-2H-pyrrole 1-oxides with organometallic reagents leads to the formation of enolate salts, which nevertheless remain capable of reacting with the second equivalent of a Grignard reagent or an organolithium compound via nucleophilic addition onto a nitrone carbon. A number of pyrrolidine nitroxides have been prepared via this reaction, including 5-(tert-butyl)-2,2-dimethyl-1-oxo-5-phenylpyrrolidin-1-oxyl [34]. It is noteworthy that the addition of EtMgBr to 6 did not occur either at ambient temperature or upon heating. After heating to reflux in THF with a large excess of EtMgBr for 15 h, the nitrone was almost quantitatively recovered from the reaction mixture. In contrast, 6 readily reacted with BuLi within 1.5 h, and quenching of the reaction mixture with water under aerobic conditions afforded nitroxide 18 with a 60% yield (Scheme 3).

Scheme 3. Synthesis of nitroxide 4.
Nitroxide 18 is unstable, slowly decomposing at room temperature or upon heating, with deep crimson coloring typical for 3-oxo-3,4-dihydro-2H-pyrrole 1-oxide dimers. We already mentioned Of note, earlier processing of the reaction mixture in the hydrolysis of 9 leads to the formation of byproducts and decreases the yield of 6. If the reaction mixture was processed, after 40 h, another compound (13, yield 15%) was isolated along with 6 (Scheme 2). Obviously, enaminoketone 9 hydrolysis proceeds via the consecutive formation of acyclic enaminoketone 14 and β-diketone 15 intermediates, both present in the reaction mixture, and formation of 13 is a result of cyclization of the former. Formation of similar compounds has been previously observed in a recyclization reaction of imidazolidine enaminoketones with an acceptor group at the exo-methylene carbon atom [31].
It is known that treatment of 3-oxo-3,4-dihydro-2H-pyrrole 1-oxides with organometallic reagents leads to the formation of enolate salts, which nevertheless remain capable of reacting with the second equivalent of a Grignard reagent or an organolithium compound via nucleophilic addition onto a nitrone carbon. A number of pyrrolidine nitroxides have been prepared via this reaction, including 5-(tert-butyl)-2,2-dimethyl-1-oxo-5-phenylpyrrolidin-1-oxyl [34]. It is noteworthy that the addition of EtMgBr to 6 did not occur either at ambient temperature or upon heating. After heating to reflux in THF with a large excess of EtMgBr for 15 h, the nitrone was almost quantitatively recovered from the reaction mixture. In contrast, 6 readily reacted with BuLi within 1.5 h, and quenching of the reaction mixture with water under aerobic conditions afforded nitroxide 18 with a 60% yield (Scheme 3).
Reduction of 18 with sodium borohydride gave nitroxide 4 as a single diastereomer (racemic mixture) according to HPLC (Figure SI67). Single-crystal X-ray analysis ( Figure 1) revealed that the hydroxyl group is at cis-position to the tert-butyl group, apparently indicating kinetic control of the reduction reaction in which the reagent approaches the C=O bond from the less hindered side.

Synthesis of Nitroxide 5
This synthesis is based on a recently developed procedure for assembling sterically hindered pyrrolidines via a three-component domino process [20]. A mixture of tert-leucine, dimethyl fumarate, 2-pentanone, and DMF in toluene was heated to reflux with a Dean-Stark apparatus, thereby affording a diastereomeric mixture of pyrrolidine-3,4-diesters 23a,b. A portion of the mixture was subjected to column chromatography to separate the isomers for structure confirmation. After exhaustive reduction of the ester groups with LiAlH4, the mixture was oxidized with a H2O2/Na2WO4 system in aqueous methanol to give single nitrone 24 (Scheme 5). Spectral parameters of 24 are close to those of its 2,5,5-triethyl analog [20]. Addition of the nitrone to a solution of EtMgBr in THF leads to precipitation, presumably due to magnesium bis-alkoxide formation. Subsequent stirring of the reaction mixture with an excess of EtMgBr at room temperature or upon heating to reflux did not lead to Grignard reagent addition, and nitrone 24 was almost quantitatively recovered after the processing of the reaction mixture. To prevent precipitation of magnesium salts, the hydroxy groups were protected via treatment with trimethylsilyl chloride and triethylamine in dry THF (Scheme 6). Silylated nitrone 25 also showed no reactivity toward EtMgBr but reacted with n-BuLi. The reaction was complete after stirring at ambient temperature for 72 h, affording a mixture of nitroxides (presumably, diastereomers) with major isomer content exceeding 90%. The major isomer (racemate) was isolated via crystallization with the yield 65%, and the structure was determined by single-crystal X-ray analysis (Figure 1). Reduction of 18 with sodium borohydride gave nitroxide 4 as a single diastereomer (racemic mixture) according to HPLC ( Figure SI67). Single-crystal X-ray analysis (Figure 1) revealed that the hydroxyl group is at cis-position to the tert-butyl group, apparently indicating kinetic control of the reduction reaction in which the reagent approaches the C=O bond from the less hindered side.

Synthesis of Nitroxide 5
This synthesis is based on a recently developed procedure for assembling sterically hindered pyrrolidines via a three-component domino process [20]. A mixture of tert-leucine, dimethyl fumarate, 2-pentanone, and DMF in toluene was heated to reflux with a Dean-Stark apparatus, thereby affording a diastereomeric mixture of pyrrolidine-3,4-diesters 23a,b. A portion of the mixture was subjected to column chromatography to separate the isomers for structure confirmation. After exhaustive reduction of the ester groups with LiAlH 4 , the mixture was oxidized with a H 2 O 2 /Na 2 WO 4 system in aqueous methanol to give single nitrone 24 (Scheme 5). Spectral parameters of 24 are close to those of its 2,5,5-triethyl analog [20].
Reduction of 18 with sodium borohydride gave nitroxide 4 as a single diastereomer (racemic mixture) according to HPLC (Figure SI67). Single-crystal X-ray analysis (Figure 1) revealed that the hydroxyl group is at cis-position to the tert-butyl group, apparently indicating kinetic control of the reduction reaction in which the reagent approaches the C=O bond from the less hindered side.

Synthesis of Nitroxide 5
This synthesis is based on a recently developed procedure for assembling sterically hindered pyrrolidines via a three-component domino process [20]. A mixture of tert-leucine, dimethyl fumarate, 2-pentanone, and DMF in toluene was heated to reflux with a Dean-Stark apparatus, thereby affording a diastereomeric mixture of pyrrolidine-3,4-diesters 23a,b. A portion of the mixture was subjected to column chromatography to separate the isomers for structure confirmation. After exhaustive reduction of the ester groups with LiAlH4, the mixture was oxidized with a H2O2/Na2WO4 system in aqueous methanol to give single nitrone 24 (Scheme 5). Spectral parameters of 24 are close to those of its 2,5,5-triethyl analog [20]. Addition of the nitrone to a solution of EtMgBr in THF leads to precipitation, presumably due to magnesium bis-alkoxide formation. Subsequent stirring of the reaction mixture with an excess of EtMgBr at room temperature or upon heating to reflux did not lead to Grignard reagent addition, and nitrone 24 was almost quantitatively recovered after the processing of the reaction mixture. To prevent precipitation of magnesium salts, the hydroxy groups were protected via treatment with trimethylsilyl chloride and triethylamine in dry THF (Scheme 6). Silylated nitrone 25 also showed no reactivity toward EtMgBr but reacted with n-BuLi. The reaction was complete after stirring at ambient temperature for 72 h, affording a mixture of nitroxides (presumably, diastereomers) with major isomer content exceeding 90%. The major isomer (racemate) was isolated via crystallization with the yield 65%, and the structure was determined by single-crystal X-ray analysis (Figure 1).

Scheme 5. Synthesis of nitrone 24.
Addition of the nitrone to a solution of EtMgBr in THF leads to precipitation, presumably due to magnesium bis-alkoxide formation. Subsequent stirring of the reaction mixture with an excess of EtMgBr at room temperature or upon heating to reflux did not lead to Grignard reagent addition, and nitrone 24 was almost quantitatively recovered after the processing of the reaction mixture. To prevent precipitation of magnesium salts, the hydroxy groups were protected via treatment with trimethylsilyl chloride and triethylamine in dry THF (Scheme 6). Silylated nitrone 25 also showed no reactivity toward EtMgBr but reacted with n-BuLi. The reaction was complete after stirring at ambient temperature for 72 h, affording a mixture of nitroxides (presumably, diastereomers) with major isomer content exceeding 90%. The major isomer (racemate) was isolated via crystallization with the yield 65%, and the structure was determined by single-crystal X-ray analysis (Figure 1). Along with nitroxide 5, colorless oily compound 26 was isolated from the reaction mixture using chromatography. Analysis of 1 H and 13 C NMR spectra with 1 H-1 H and 1 H- 13  Along with nitroxide 5, colorless oily compound 26 was isolated from the reaction mixture using chromatography.

Properties of the New Nitroxides
EPR spectra of the synthesized nitroxides are presented in Figure 2. Parameters of the EPR spectra of new nitroxides 4 and 5 are given in Table 1, and spectral parameters of 1 and 2 recorded under the same conditions are provided for comparison. The spectra of both new nitroxides were characterized by a triplet of doublets with hyperfine coupling (hfc) on nitrogen and hydrogen nuclei. This behavior is typical for 3-substituted or 3,4-disubstituted pyrrolidine nitroxides with geminal ethyl groups at position 2 or 5 [18,20]. The origin of this additional doublet splitting has been previously investigated for imidazolidine nitroxides, which have similar geometry [35]. It was shown that a repulsion with a substituent at a neighboring sp 3 -hybridized asymmetric center makes geminal ethyls favor a conformation with high spin density at one of four methylene hydrogens. Replacement of the ethyl with any other group or removal of the neighboring substituent may change conformational behavior with disappearance of the large hfc.
Molecules 2020, 25, 845 7 of 17 the reaction of 1, which is in the anionic form at pH 7.4, with ascorbate anion is complicated by Coulomb's repulsion, which is more important in a solvent with lower polarity. The presented data indicate that nitroxide 5 is the most resistant to reduction.    Unlike 18, nitroxides 4 and 5 showed remarkable thermal stability. Heating of the samples of 4 and 5 at 100 • C for 80 min didn't lead to significant decay of the nitroxides EPR signal (See Figure SI78). Nitroxides 4 and 5 are rather lipophilic (their octanol-water partition coefficients are 1000 and 850, respectively). Their solubility allowed to register the EPR spectra, but the signal-to-noise ratio was not sufficient to follow the kinetics of the reduction. Moreover, dissolving them in an ultrasonic bath yielded unreproducible results, presumably due to the formation of unstable micellar solutions. For this reason, redox properties of 4 and 5 were investigated in 50% methanol, where stable 0.1 mM solutions can be prepared. The kinetics of nitroxide decay in the presence of 0.5 M ascorbate at pH 7.4 are shown in Figure 3. Second-order reduction rate constants of the nitroxides are listed in Table 1. The rate constants were obtained from the initial part of the kinetics when accumulation of dehydroascorbate anion was negligible [36]. Reduction of 1 and 2 under the same conditions was evaluated for comparison. Reduction of 1 and 2 proceeded much slower in 50% methanol than in aqueous PBS solution, showing a decrease in the rate constant by the factor of 20.8 and 3.2, respectively. It is known that a solvent can significantly affect the rate constant of chemical reactions [37]. The basic mechanism is the influence of solvent polarity on the stabilization energy of initial compounds as well as the energy level of a transition state. An increase in solvent polarity accelerates the rates of reactions where a charge develops in the activated complex, starting from a neutral or slightly charged reactant. The solvent effect is well known in the chemistry of reversible homolyses of alkoxyamines, where one of the reaction partners is a nitroxide radical [38].

General Information
2,2,5,5-Tetraethyl-3,4-bis(hydroxymethyl)pyrrolidin-1-oxyl (2) was prepared according to a  Table 1. The kinetics of reduction in 50% methanol were very similar among 1, 2, and 4, whereas in water, the reduction of 1 proceeded significantly faster than that of 2 (see Figure SI77). Presumably, this difference can be attributed to a difference in solvation of the nitroxide, ascorbate anion, and the transition state. While the observed difference for 2 follows the pattern of an ion-dipole interaction, the reaction of 1, which is in the anionic form at pH 7.4, with ascorbate anion is complicated by Coulomb's repulsion, which is more important in a solvent with lower polarity. The presented data indicate that nitroxide 5 is the most resistant to reduction.
The structures of compounds 4, 5, and 12 were determined by single-crystal X-ray analysis (Table S2). X-ray diffraction data were obtained on a Bruker P4 for 12 and on a Bruker Kappa Apex II CCD diffractometer for 4 and 5 with Mo Kα radiation (λ = 0.71073 Å) and a graphite monochromator. Absorption corrections were applied empirically using SADABS programs [40] for 4 and 5 and by the integration method for 12. The structures were solved by direct methods and refined by the full-matrix least-squares method against all F 2 in anisotropic approximation (besides the H atoms) using the SHELXL2014/7 software suite (Dept. of Structural Chemistry, University of Göttingen, Göttingen, Germany [41]). The H atoms' positions were processed via the riding model, except for the positions of hydroxyl groups in 5 (refined independently). There are two independent molecules in the asymmetric unit of the cell for 4. Datasets CCDC 1972133-1972135 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk.
HPLC analyses were performed with an Agilent 1100 liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with a quaternary pump, online degasser, autosampler, and diode array detector. Chromatographic separations were carried out on a ZORBAX SB-C18 column (150 mm × 4.6 mm, 5.0 µm). The flow rate was 0.6 mL/min. Detection was performed at 238 nm. Acetonitrile, methanol and water were used to prepare the mobile phase.
The EPR spectra in water were recorded on a Bruker ER-200D-SRC spectrometer in 100 µL quartz capillary for 0.1 mM solutions degassed via bubbling with argon. Spectrometer settings: frequency, 9.87 GHz; microwave power, 5.0 mW; modulation amplitude, 0.05 mT; time constant, 50 ms; and conversion time, 5.12 ms. The EPR spectra in water-methanol solutions and in toluene were recorded with an Elexsys E540 X-band spectrometer (Bruker, Billerica, MA, USA) in a 100 µL quartz capillary for 0.1-0.3 mM solutions, with the following spectrometer settings: field center, 351.600 mT; sweep range, 10 mT; modulation amplitude, 0.15 mT; microwave power, 2 mW; time constant, 10.24 ms; and scan time, 41 ms. The EasySpin software (Version 5.2.28, easyspin.org, [42]) was employed for simulation of spectra. For kinetic measurements, the EPR spectra were acquired at the same instrument settings but with a greater modulation amplitude (0.2 mT) to optimize the signal-to-noise ratio. Thermal stability of nitroxides 4 and 5 was studied in 1 mM solutions in toluene within a sealed 100 µL glass capillary using a double sample resonator (Bruker, ER 4105DR). One of the two samples with the nitroxide was placed into a water bath (95-100 • C) for 80 min incubation, and nitroxide decay was followed via comparison of intensities. Partition coefficients in a water-octanol mixture were estimated from the amplitudes of the EPR spectra of a nitroxide in water after extensive shaking with different portions of added octanol and separation.
For kinetic measurements in water, stock solutions of the nitroxide, ascorbic acid, and of glutathione in phosphate buffer (1 mM, pH 7.4) were prepared, and pH was adjusted to 7.4 with NaHCO 3 . All the solutions were deoxygenated with argon, carefully and quickly mixed in a small tube to attain final concentrations (nitroxide, 0.1-0.3 mM; GSH, 2 mM; and ascorbate, 166.7 mM) and were placed into an EPR capillary (50 µL). The capillary was sealed on both sides and placed into the EPR resonator. Alternatively, all the reagents were dissolved in a MeOH-H 2 O mixture (1:1), pH of the stock solutions was adjusted to 7.4 with NaHCO 3 , and the solutions were mixed in a similar manner to obtain final concentrations: nitroxide, 0.1 mM; GSH, 2 mM; and ascorbate, 500 mM. The decay of amplitude of the low-field component of the EPR spectrum was followed to obtain the kinetics. Initial part of the decay curves (up to 200 min) was used for fitting. Kinetics of the decay was fitted to a monoexponential function to calculate the first-order rate constants. Then, these constants were divided by the concentration of ascorbic acid to calculate the second-order reaction constants.

(Z)-4-(3,3-Dimethyl-2-oxobutylidene)-5,5-diethyl-2,2-dimethylimidazolidin-1-oxyl (10)
A solution of 4.3 g (23.4 mmol) of imidazoline 6 in 30 mL of dry diethyl ether was added dropwise to 35 mL of a 2 M solution of lithium diisopropylamide in an argon atmosphere. After 50 min of stirring, ethyl pivalate (12 mL, 78.5 mmol) was added, and the reaction mixture was heated to reflux for 48 h. Then, the mixture was cooled to 5 • C, and 100 mL of water was added carefully. The organic layer was separated, and the aqueous layer was extracted with diethyl ether (30 mL × 3 times). The combined extract was agitated with 10 g of manganese dioxide for 1 h and dried over magnesium sulfate. The precipitate was filtered off, and the filtrate was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel with gradient elution (from hexane to a diethyl ether-hexane 1:4 mixture) to give 10 (5.5 g, 88% yield) as a yellow crystalline solid, m.p. 84-86 • C (hexane). 3.2.5. 3,3'-Bis(2-tert-butyl-5,5-diethyl-4-oxopyrrolinylidene) 1,1'-dioxide (12) Manganese dioxide 0.75 g (8.63 mmol) was added to a solution of 0.15 g (0.71 mmol) of 6 in chloroform (3 mL), and the reaction mixture was stirred vigorously for 7 days. The precipitate was filtered off, and the filtrate was evaporated under reduced pressure. The residue was recrystallized from ethyl acetate to give 0.145 g (95%) of dimer 12 as a dark violet crystalline solid, m.p. 157-160 • C (ethyl acetate). Elemental analysis: found: C, 68.91; H, 9.27 3.2.6. 5-(tert-Butyl)-5-butyl-2,2-diethyl-3-oxopyrrolidin-1-oxyl (18) A warm solution of 0.5 g (2.37 mmol) of pyrroline 6 in dry benzene (30 mL) was added dropwise to a 2.6 M toluene solution of BuLi (14 mL) in an argon atmosphere. The reaction mixture was heated at 60 • C for 1 h and then kept at room temperature for 24 h. After that, the mixture was cooled to 0-5 • C and quenched with 30 mL of water. The organic layer was separated, and the aqueous layer was extracted with benzene. The combined extract was dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel with gradient elution (from tetrachloromethane to a chloroform-tetrachloromethane 1:5 mixture) to isolate 18 (0.39 g, 61%) as a yellow crystalline solid, m.p. 42

Thermal Decomposition of Nitroxide 18 in the Presence of TEMPO
A mixture of 0.3 g (1.12 mmol) of nitroxide 18 and 1.6 g (10.3 mmol) of TEMPO was placed into a Schlenk flask and heated at 70-80 • C in vacuum for 7 days. The mixture was separated by column chromatography on silica gel with gradient elution (from hexane to a diethyl ether-hexane 1:2 mixture) to give 20 (0.09 g, 38%) and 21 (0.2 g, 85%).

Conclusions
Here, we again demonstrated that the addition of organolithium compounds to cyclic α-tert-butyl nitrones is an efficient method of the synthesis of highly strained nitroxides. The new 2-butyl-2-tert-butyl pyrrolidine-1-oxyls were prepared in good yields. The data on reduction of the new nitroxides mean that a further increase in the steric demand of the substituents will make the nitroxides more resistant to reduction. Modification of the hydroxy groups may enable incorporation of these nitroxides into macromolecular or supramolecular nanostructures that can serve as organic radical contrast agents for in vivo MRI. Examples of similar application of highly lipophilic nitroxides were mentioned in the introduction. The use of 5 and similar structures with exceptionally high resistance to reduction can ensure long lifetime of paramagnetic centers in live tissues and a prolonged enhancement for MRI.