Synthesis of the tetraethyl substituted pH-sensitive nitroxides of imidazole series with enhanced stability towards reduction

Article abstract of DOI:10.1039/b400252k

The synthesis of 2,2,5,5-tetraethylimidazole nitroxides from 3-ethylpent-2-ene is described. The newly synthesized nitroxides, namely 4-methyl-2,2,5,5-tetraethyl-2,5-dihydro-1H-imidazol-1-yloxy (1), 3,4-dimethyl-2,2,5,5-tetra-ethylperhydroimidazol-1-yloxy (2) and 2,2,5,5-tetraethyl-4-pyrrolidin-1-yl-2,5-dihydro-1H-imidazol-1-oxyl (3), were found to be pH sensitive spin probes, with pK values of 1.2, 4.95 and 7.4, respectively. The most important finding was the fact that these new nitroxides were 20-30 times more stable in the presence of ascorbate and had significantly longer halflifes in rat blood as compared to 2,2,5,5-tetramethyl analogs. The latter observation provides a unique advantage for the application of tetraethyl substituted imidazole nitroxides as functional EPR probes.

Full text of DOI:10.1039/b400252k

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Synthesis of the tetraethyl substituted pH-sensitive nitroxides of
imidazole series with enhanced stability towards reduction
Igor A. Kirilyuk,*a Andrey A. Bobko,b Igor A. Grigor’eva and Valery V. Khramtsovb,c
a N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, pr. Acad. Lavrentjeva 9,
Novosibirsk 630090, Russia. E-mail: kirilyuk@nioch.nsc.ru; Fax: 007 3832 344752;
Tel: 007 3832 342387
b Institute of Chemical Kinetics & Combustion, Novosibirsk 630090, Russia.
E-mail: bobko@ns.kinetics. nsc.ru; Fax: 007 3832 342350; Tel: 007 3832 332294
c Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University, Columbus,
OH 43210, USA. E-mail: khramtsov-1@medctr.osu.edu; Fax: 1 614 2934799;
Tel: 1 614 6883664
Received 9th January 2004, Accepted 12th February 2004
First published as an Advance Article on the web 3rd March 2004
The synthesis of 2,2,5,5-tetraethylimidazole nitroxides from 3-ethylpent-2-ene is described. The newly synthesized
nitroxides, namely 4-methyl-2,2,5,5-tetraethyl-2,5-dihydro-1H-imidazol-1-yloxy (1), 3,4-dimethyl-2,2,5,5-tetra-
ethylperhydroimidazol-1-yloxy (2) and 2,2,5,5-tetraethyl-4-pyrrolidin-1-yl-2,5-dihydro-1H-imidazol-1-oxyl (3),
were found to be pH sensitive spin probes, with pK values of 1.2, 4.95 and 7.4, respectively. The most important
finding was the fact that these new nitroxides were 20–30 times more stable in the presence of ascorbate and had
significantly longer halflifes in rat blood as compared to 2,2,5,5-tetramethyl analogs. The latter observation
provides a unique advantage for the application of tetraethyl substituted imidazole nitroxides as functional
EPR probes.
Introduction
The range of applications of stable nitroxides is immense
and involves living polymerisation,1 magnetic materials,2 syn-
thetic tools,3 probes for measurement of oxygen4 and pH5,6 in
living tissues, structural studies of biological macromolecules,7
therapeutic agents,8 radical scavengers,9 MRI contrast
agents,10,11 and other fields. The unique properties of the
nitroxides originate from the presence of an unpaired electron
in their structure. However instability of the paramagnetic
ؒ
N–O fragment towards chemical reduction in many systems
is an unavoidable factor that significantly limits the use of
nitroxides in many applications, particularly in biological
systems.
The reduction rates of nitroxides vary significantly depend-
ing on their structure and microenvironment.10,12–16 The 5-
membered nitroxides14 including imidazoline nitroxides15 were
Scheme 1
found to be among the most stable.
Results and discussion
Recently it has been shown that 1,1,3,3-tetraethylisoindoline
nitroxides have significantly increased stability towards reduc-
tion compared to the 1,1,3,3-tetramethyl analogs.17 Introduc-
tion of bulky alkyl substituents into neighboring positions of
the nitroxide group might be a useful approach to increase
stability of other classes of nitroxides as well. The imidazoline
radicals have an additional advantage over other nitroxide
classes as functional EPR probes due to their use for measure-
ment of pH and thiols, including in vivo.5,6,18,19 Therefore
synthesis of new imidazoline radicals with enhanced stability
towards reduction may have a significant impact on the
applications of nitroxides as functional EPR probes as well
as beyond this field.
In the present work we describe the synthesis of tetraethyl
substituted pH-sensitive nitroxides of imidazoline and imid-
azolidine series, 1–3 (Scheme 1). Stability of the new nitroxides
towards reduction and sensitivity of their EPR spectra to pH
were studied and compared to corresponding data for their
analogs 4–7.
Synthesis
Common synthetic routes for obtaining basic structures, 4, 5,
and 7 of imidazoline and imidazolidine radicals were described
earlier.20,21 Recently we employed organometallic reagent addi-
tion to 4H-imidazol-3-oxides for the synthesis of 4-amino-3-
imidazoline-1-oxyls,22 e.g. the nitroxide 6. The latter approach
significantly increases the number of available structures. In
current studies we modified the above approaches to synthesize
tetraethyl substituted pH-sensitive nitroxides 1–3. The nitrox-
ides 1–3 were prepared from 3-ethylpent-2-ene 8 (Scheme 2).
The 3-ethylpent-2-ene nitrosochloride 9 was prepared using the
procedure described for preparation of 2-nitroso-3-chloro-3-
methylbutane dimer.20,23 Following treatment with hydroxyl-
amine acetate and hydrolysis (cf ref. 24) gave the key compound
3-hydroxyamino-3-ethylpentan-2-one hydrochloride 11. The
condensation of 11 with pentan-3-one and ammonium acetate
was performed according to a modified procedure described
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 2 5 – 1 0 3 0
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Scheme 2
in.25 A good yield of 4-methyl-2,2,5,5-tetraethyl-2,5-dihydro-
position of the spectra of protonated, 6H؉, and unprotonated,
1H-imidazole-1-ol 12 was obtained when the reaction was
carried out under argon to avoid in situ oxidation. Pure imid-
azole 12 was converted into 4-methyl-2,2,5,5-tetraethyl-2,5-
dihydro-1H-imidazol-1-yloxy, 1, using careful oxidation with
MnO2. Alkylation of 1 with dimethylsulfate gives a highly
hygroscopic imidazolinium salt 13 which was immediately,
without isolation, converted into nitroxide 2 (cf ref. 21).
The nitroxide 3 was prepared from hydroxyaminoketone
11 using the procedures for the synthesis of 6 as described in
reference 22.
6, forms (Scheme 3), which differ in their values of hyperfine
splitting, aN, and g-factor. This is in agreement with R RHϩ
chemical exchange being slow on an EPR timescale.
Effect of pH on EPR spectra of the nitroxides 1–7
Scheme 3
The EPR spectra of imidazoline and imidazolidine nitroxides
are known to be highly sensitive to the pH of the medium due
to reversible protonation of the N-3 atom of the heterocycle.26
The range of sensitivity of the EPR spectrum of the particular
nitroxide to pH is within about 3 pH units centered on the pK
value for its protonation. A significant effect of the substitution
at carbons C-2 and C-4 of the heterocycle on the pK value was
found. As a consequence, a wide set of pH sensitive nitroxides,
valid for various ranges of pH assessment, was proposed.18
However a lack of useful spin pH probes with a pK value about
7.0–7.4 was also recognized.5
The dependence of aN on pH allows its approximation with
a conventional titration curve yielding a pK value of 7.3 (see
Fig. 2). An even higher pK value, 7.4, has been found for nitrox-
ide 3 (see Table 1 for spectral parameters). A similar effect of
methyl group substitution for the ethyl group on pK values was
observed for the imidazolidine nitroxides (4.7 and 4.95 for the
Fig. 1 demonstrates the EPR spectra of the nitroxide 6 at
various pH values. At pH 7.0 the EPR spectrum shows super-
Fig. 2 The pH dependence of hyperfine splitting, aN, measured as a
distance between low- and central-field components of the EPR spectra
of the nitroxide 6. The solid line is a nonlinear least-squares fit of the
data to a conventional titration curve (eqn 1, see Experimental) yielding
aN(6) = 15.5 G, aN(6H؉) = 14.5 G, pK = 7.3.
Fig. 1 EPR spectra of the nitroxide 6 (pK = 7.3) at various pH values.
The spectrometer settings were as follows: modulation amplitude,
0.5 G; microwave power, 20 mW; time constant, 100 ms; sweep time,
100 s.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 2 5 – 1 0 3 0
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Table 1 The differences in hfi splitting constants (∆aN) and g-factors (∆g) between protonated and unprotonated forms of the radicals 1–7a, their
pK values, bimolecular rate constants for the reduction by ascorbic acid (k), and initial rates of the reduction in the rat blood (V0)
Nitroxides
1
2
3
4
5
6
7
∆aN/G
∆g
0.9
0.0003
1.2
0.5 0.3
0.033
1.3
0.0003
4.95
0.04 0.003
<0.01
0.9
0.0003
7.4
0.74 0.07
1.0
0.9
0.0002
1.3
5.6 0.3
nd
1.3
0.0003
4.7
0.85 0.05
0.22
1.0
0.0003
7.3
13.4 0.9
1.8
0.8
0.0002
6.1
22.5 1.6
2.4
pK
k/MϪ1 sϪ1
V0/µM minϪ1
a ∆aN, ∆g, and pK values for the radicals 4–7 are taken from refs. 18, 22.
Fig. 3 Left. The ratio of current, [R], and initial, [R]0 concentrations of the imidazoline nitroxides 3, 6, and 7 measured by EPR in 0.1 M
Na-phosphate buffer, pH 7.5, in the presence of 1 mM ascorbic acid plotted versus time. Initial concentration of the nitroxides was 0.1 mM.
The experimental data support exponential decay of the EPR signal upon the reduction according to the equation: [R] = [R]0exp(Ϫkobst); Right. The
dependence of pseudo-first order rate constant of reduction, kobs, of nitroxides 3, 6, and 7 on the concentration of ascorbate. Solid lines represent
linear extrapolation yielding the bimolecular rate constants (see Table 1).
radicals 5 and 2, correspondingly). The increase of the pK
makes the new spin pH probes 2 and 3 more appropriate for
application in the physiological pH range.
The comparative study of the nitroxides reduction with ascorbic
acid
Ascorbic acid is a principal reducing agent responsible for the
nitroxides reduction in biological fluids.12 The reduction of the
nitroxides 1–7 with ascorbic acid in phosphate buffer at pH 7.5
was studied. The typical kinetics of the reduction of the imid-
azoline nitroxides 3, 6 and 7 with various numbers of ethyl
groups in the vicinity of the radical fragment are shown in Fig.
3. The bimolecular rate constants for the reduction of the
nitroxides 1–7 with ascorbate are given in the Table 1. Introduc-
tion of bulky ethyl groups surrounding the nitroxide moiety,
ؒ
N–O , results in slowing down the reduction of the nitroxide.
The 2,2,5,5-tetraethyl substituted nitroxides were found to be
Fig. 4 The kinetics of the reduction of the radicals 1–3, and 5–7 in rat
20–30 times more stable towards the reduction by ascorbate
as compared to that of their tetramethyl substituted analogs
(cf. the rate constants for imidazoline nitroxides 3 and 7, and
imidazolidine nitroxides 2 and 5, Table 1).
Note also that the observed higher resistance against the
reduction of the imidazolidine nitroxides compared to that for
imidazoline nitroxides is in agreement with earlier reported
data.15
blood measured by EPR.
absence of a quantitative correlation between the reduction of
the nitroxides by ascorbate and their reduction by the blood
(see Table 1) denotes a complex mechanism for the nitroxides
reduction in the blood. This may imply penetration of the
nitroxides through the erythrocyte membrane and eventual
intracellular reduction by thiols.12,27
The study of the reduction of the nitroxides in the rat blood
Conclusion
The 2,2,5,5-tetraethyl substituted nitroxides 1–3 showed excel-
lent stability in rat blood. Typical decay kinetics of the nitrox-
ides 1–3 and 5–7 are shown in the Fig. 4. Table 1 lists the initial
rates of the reduction. The data demonstrate the beneficial
effect of the ethyl groups introduced in the vicinity of the N–O
fragment on their stability towards reduction, the most stable
Introduction of ethyl groups instead of methyl groups to the
α-carbon of the imidazoline and imidazolidine nitroxides
results in significant protection of the nitroxyl group against
reduction in aqueous solutions of ascorbic acid or in blood
samples, presumably due to steric hindrance of the radical
fragment. In addition, this substitution shifts the pK values of
ؒ
being the tetraethyl substituted imidazolidine nitroxide 2. The
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 2 5 – 1 0 3 0
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the imidazoline spin pH probes to the favorable region of
neutral pH, making them particularly useful for biological
applications.
3-Hydroxyamino-3-ethylpentan-2-one hydrochloride (11)
A solution of oxime 10 (50 g, 0.31 mol) in concd. hydrochloric
acid (100 ml) was saturated with gaseous HCl and the resulting
suspension of hydroxylamine hydrochloride was allowed to
stand at 0 ЊC for 24 h. The hydroxylamine hydrochloride was
filtered off and the hydrochloric acid was removed under
reduced pressure (40 ЊC). The crystalline residue was stirred
with dry acetonitrile (250 ml), the precipitate of hydroxylamine
hydrochloride was filtered off and the solution was diluted with
diethyl ether (250 ml) and allowed to stand at Ϫ5 ЊC for 24 h.
The hydrochloride 11 (colorless crystals) was filtered off and
washed with dry THF. Yield 34 g, 60%, mp 113–117 ЊC (repre-
cipitated from acetonitrile with diethyl ether) (Found: C, 46.14;
H, 8.54; N, 7.69, Cl 19.20. Calc. for C7H16ClNO2: C, 46.28; H,
8.88; N, 7.71, Cl 19.52%); νmax(KBr)/cmϪ1 1711, 3244, 1579,
1466, 1435, 1370, 1297, 1224, 1136, 883 and 762; δH(200 MHz;
(CD3)2SO) 0.81 (6 H, t, J 8, 2 × CH3, Et), 1.86 (4 H, quartet, J 8,
2 × CH2, Et) and 2.25 (3 H, s, CH3); δC(50 MHz; (CD3)2SO)
Experimental
The IR spectra were recorded on Bruker Vector 22 FT-IR
spectrometer in KBr pellets (the concentration was 0.25%; the
pellet thickness was 1 mm). The UV spectra were measured on
HP Agilent 8453 spectrometer in EtOH. The 1H NMR spectra
were recorded on a Bruker AC-200 (200.132 MHz) spectro-
meter for 5% solutions using the signal of the solvent as the
standard. The 13C NMR spectra were recorded on Bruker
AC-200 (50.323 MHz) and Bruker AM-400 (100.614 MHz)
spectrometers for 5–10% solutions at 300 K using the signal of
the solvent as the standard. The assignment of the signals in the
13C NMR spectra was made based on analysis of intensities, on
the spectra measured in J-modulation mode, and using the data
reported previously.22,28 EPR spectra were recorded on a Bruker
ER-200D-SRC spectrometer using 100 µl quartz capillary or
200 µl EPR flat cells.
7.71 (CH , Et), 23.61 (CH , Et), 26.08 (CH –C᎐O), 76.50
᎐
3
2
3
(C(Et) ) and 205.45 (C᎐O).
᎐
2
4-Methyl-2,2,5,5-tetraethyl-2,5-dihydro-1H-imidazole-1-ol (12)
3-Chloro-3-ethyl 2-nitrosopentane dimer (9)
A suspension of hydroxyaminoketone 11 (3 g, 16.5 mmol) and
ammonium acetate (4.5 g, 60 mmol) in a mixture of pentan-3-
one (6 ml, 57 mmol) and methanol (5 ml) was stirred under
argon for 3 weeks. The resulting solution was extracted with
diethyl ether (3 × 10 ml), the ether extract was thoroughly
washed with water (5 × 10 ml) and with saturated KHCO3
solution and dried over Na2CO3. The ether was removed under
reduced pressure and the residue was triturated with hexane, the
crystalline precipitate of 12 was filtered off and washed with
hexane, yield 2 g, 57%, colorless crystals, mp 119–121 (hexane)
(Found: C, 67.93; H, 11.54; N, 13.37. Calc. for C12H24N2O: C,
67.88; H, 11.39; N, 13.19%); νmax(KBr)/cmϪ1 2966, 2937, 2879,
1660, 1460, 1432, 1378, 1038, 938 and 898; δH(200 MHz;
CDCl3) 0.90 (6 H, br m, 2 × CH3, Et), 0.96 (6 H, br m, 2 × CH3,
A suspension of NaNO2 (105 g, 1.5 mol) in a solution of 3-
ethylpent-2-ene 9 (115 ml 84 g, 0.85 mol) in methanol (450 ml)
was cooled to Ϫ15–Ϫ5 ЊC, and 36% hydrochloric acid (270 ml,
3.2 mol) was added dropwise within 2 h to the stirred suspen-
sion keeping the temperature of the reaction mixture below
Ϫ5 ЊC. The reaction mixture was stirred for 3 h and then
poured into cold water (2 L). Precipitate of nitrosochloride 9
was filtered off, washed with cold water and dried at 25 ЊC.
Yield: 130 g, 93%, colorless crystals, mp 67–71 ЊC (hexane)
(Found: C, 51.24; H, 8.64; N, 8.69, Cl 20.50. Calc. for
C7H14ClNO: C, 51.38; H, 8.62; N, 8.56, Cl 21.66%); νmax(KBr)/
cmϪ1 2979, 2947, 1453, 1386, 1373, 1448, 1294, 1197, 1122,
1109, 1009, 938 and 841; δH(200 MHz; CDCl3) 1.00 (12 H,
t, J 7.5, 4 × CH3, Et), 1.44 (6 H, d, J 7.0, 2 × CH3, CH–CH3),
2.00 (8 H, m, 4 × CH2, Et) and 6.02 (2 H, quartet, J 7.0, 2 × CH,
CH–CH3); signals of nitroso monomer (∼20%) 0.94 (6 H,
t, J 7.5, 2 × CH3, Et), 1.41 (3 H, d, J 7.0, CH3, CH–CH3), 2.00
(4 H, m, 2 × CH2, Et) and 5.93 (2 H, quartet, J 7.0, CH, CH–
CH3).
Et), 1.70 (8 H, br m, 4 × CH , Et), 1.91 (3 H, s, CH C᎐N) and
᎐
3
2
4.65 (1 H, br s, OH); δC(50 MHz; CDCl3) 7.56, 8.33 (CH3, Et),
26.35, 27.56 (CH , Et), 15.90 (CH –C᎐N), 77.39 (C5), 93.15 (C2)
᎐
2
3
and 171.79 (C᎐N).
᎐
4-Methyl-2,2,5,5-tetraethyl-2,5-dihydro-1H-imidazol-1-yloxy
(1)
3-Hydroxyamino-3-ethylpentan-2-one oxime (10)
Manganese dioxide (2 g) was added to a stirred solution of 12
(1 g, 4.7 mmol) in chloroform (20 ml). The suspension was
stirred for 0.5 h, manganese oxides were filtered off and the
filtrate was evaporated under reduced pressure to leave an
orange oil, which was purified by column chromatography on
silica (Kieselgel 60, Merck) using diethyl ether–hexane 1 : 20
mixture as eluent to give nitroxide 1 (1.9 g, 95%) as an orange
oil (Found: C, 68.14, H, 10.95; N, 13.28. Calc. for C12H23N2O:
C, 68.20; H, 10.97; N, 13.26%); νmax(neat)/cmϪ1 2971, 2940,
2881, 1640, 1461, 1428, 1383, 1278, 1151, 957, 931, 901 and 831.
A solution of hydroxylamine hydrochloride (120 g, 1.71 mol) in
water (235 ml) was added to a solution of NaOAc (140 g, 1.71
mol) in water (235 ml). The resulting solution was diluted with
ethanol (1 L) and the precipitate of NaCl was filtered off.
Nitrosochloride 9 (130 g) was added to the filtrate and the solu-
tion was heated under reflux for 3 h and allowed to stand over-
night. The ethanol was removed under reduced pressure, and
the residue was shaken with water (0.8 L), saturated NaCl solu-
tion (0.5 L), acetic acid (50 ml) and hexane (200 ml). The
organic layer was separated, and the water layer was extracted
with hexane (200 ml). Combined organic phase was washed
with a mixture of saturated NaCl solution (50 ml) and acetic
acid (5 ml). Solid KHCO3 was added portionwise to a stirred
combined water solution to pH 9 and the precipitate of oxime
10 was filtered off, washed with water and dried at 25 ЊC. Yield:
77 g, 60%, colorless crystals, mp 115–117 ЊC (hexane–ethyl
acetate 1 : 1) (Found: C, 52.25; H, 9.71; N, 17.39. Calc. for
C7H16N2O2: C, 52.48; H, 10.07; N, 17.48%); νmax(KBr)/cmϪ1
3302, 3244, 2978, 2944, 2847, 1489, 1460, 1430, 1385, 1294,
1183, 1083, 1031, 999, 976, 945, 925 and 868; δH(200 MHz;
(CD3)2SO) 0.67 (6 H, t, J 8, 2 × CH3, Et), 1.44 (4 H, quartet, J 8,
2 × CH2, Et), 1.69 (3 H, s, CH3), 4.10 (1 H, br s, NH), 6.80 (1 H,
3,4-Dimethyl-2,2,5,5-tetraethylperhydroimidazol-1-yloxy (2)
Dimethyl sulfate (0.5 g, 4 mmol) was added to a solution of 5
(0.5 g, 2.4 mmol) in dry diethyl ether (3 ml). The solution was
allowed to stand for 0.5 h at 25 ЊC, filtered and diethyl ether was
removed under reduced pressure. The residue was heated to
50 ЊC for 30 min under reduced pressure and then triturated
with dry diethyl ether to form a highly hygroscopic yellow crys-
talline precipitate of quaternary salt 13. The precipitate was
filtered off (without drying), washed with dry diethyl ether and
immediately dissolved in ethanol (5 ml). NaBH4 (150 mg,
4 mmol) was added to the solution and the mixture was stirred
for 1 h. Ethanol was removed under reduced pressure, the resi-
due was dissolved in water (2 ml) and extracted with diethyl
ether (3 × 2 ml). The extract was dried over Na2CO3, diethyl
br s, OH) and 10.40 (1 H, br s, C᎐NOH); δ (50 MHz;
᎐
C
(CD ) SO) 7.61 (CH , Et), 23.26 (CH , Et), 9.77 (CH –C᎐N),
᎐
3
2
3
2
3
66.74 (C(Et) ) and 158.58 (C᎐N).
᎐
2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 2 5 – 1 0 3 0
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ether was removed under reduced pressure to leave a yellow oil,
which was purified by column chromatography on silica
(Kieselgel 60, Merck), using a mixture of diethyl ether–hexane
1 : 20 as eluent to give the nitroxide 2 (320 mg, 60%) as a yellow
oil (mp ca. 15 ЊC) (Found: C, 69.04, H, 11.95; N, 12.78. Calc.
for C13H27N2O: C, 68.67; H, 11.97; N, 12.32%); νmax(neat)/cmϪ1
2971, 2938, 2881, 2792, 1462, 1383, 1337, 1243, 1222, 972, 938
and 830.
δH(200 MHz; (CDCl3) 0.62 (6H, t, J 7.5, 2 × CH3, 4-Et), 1.25
(3H, t, J 7.5, CH3, 2-Et), 2.02 (4H, m, 2 × CH2, 4-Et) and 2.79
(2H, quartet, J 7.5, CH2, 2-Et); δC(50 MHz; (CD3)2CO–CCl4
1 : 5) 4.21 (CH3, 4-Et), 6.87 (CH3, 2-Et), 16.03 (CH2, 2-Et),
4
2
᎐
25.78 (CH , 4-Et), 87.66 (C ), 108.91 (C᎐N), 143.70 (C ) and
᎐
2
152.33 (C᎐N).
᎐
5-Pyrrolidin-1-yl-2,4,4-triethyl-4H-imidazol 3-oxide (17)
Pyrrolidine (1 ml, 12 mmol) was added to a solution of nitrile
16 (1 g, 5.2 mmol) in CH2Cl2 (2 mL) and the mixture was
allowed to stand overnight. The solution was evaporated under
reduced poressure to leave brown oil, which was dissolved in a
mixture of diethyl ether (20 ml) and water (20 ml). The mixture
was shaken vigorously, organic layer was separated and
extracted with water 5 × 10 ml. The combined water extracts
were saturated with NaCl, and basified with Na2CO3 (1 g), and
then pyrrolidinoimidazole 17 was extracted back with CHCl3
(3 × 5 ml). The CHCl3 extract was dried over Na2CO3, CHCl3
was removed in vacuo and the residue was separated by column
chromatography on aluminium oxide (neutral) using chloro-
form as eluent to give pyrrolidinoimidazole 17 (0.7 g, 57%) as
an orange oil; νmax(neat)/cmϪ1 2973, 2937, 2878, 1607, 1558,
1459, 1215, 1177, 1081, 1057, 713 cmϪ1; δH(200 MHz; (CDCl3)
0.65 (6 H, t, J 7.5, 2 × CH3, 4-Et), 1.22 (3 H, t, J 7.5, CH3, 2-Et),
1.90 (8 H, br m, CH2, 4-Et and 2 × CH2 pyrrolidin-1-yl), 2.68
(2 H, quartet, J 7.5, CH2, 2-Et), 3.58 (4 H, br m, 2 × CH2
pyrrolidin-1-yl); the compound was used for preparation of the
nitroxide 3 without further purification and characterized as
hydrochloride, 2,4,4-triethyl-5-pyrrolidin-1-yl-4H-imidazol 3-
oxide hydrochloride 17 × HCl. A solution of pyrrolidinoimid-
azole 17 (0.7 g, 6.8 mmol) in ethanol (10 mL) was gently acid-
ified with concd. hydrochloric acid to pH 3 and evaporated
under reduced pressure, the residue was dried in vacuum, dis-
solved in propan-2-ol (10 ml) and diluted with diethyl ether
(20 mL). The crystalline brownish precipitate of hydrochloride
17 × HCl was filtered off, washed with THF and dried at 25 ЊC,
mp 148–151 ЊC (Found: C, 56.78, H, 8.77, N, 15.08, Cl 12.80.
Calc. for C13H24N3OCl: C, 57.03, H, 8.84; N, 15.35, Cl,
12.95%); νmax(KBr)/cmϪ1 1633, 1572, 1462, 1422, 1383, 1346,
1192, 1173, 1065, 948 and 907; λmax(EtOH)/nm 312 (log ε 4.01);
δH(200 MHz; (CDCl3) 0.67 (6 H, br t, J 6.5, 2 × CH3, 4-Et), 1.20
(3 H, t, J 7.5, CH3, 2-Et), 2.02 (8 H, br m, CH2, 4-Et and 2 ×
CH2 pyrrolidin-1-yl), 2.76 (2 H, quartet, J 7.5, CH2, 2-Et), 3.69
(4 H, br m, 2 × CH2 pyrrolidin-1-yl) and 12.44 (1 H, br s, HCl);
δC(50 MHz; (CDCl3) 7.66 (CH3, 4-Et), 9.62 (CH3, 2-Et), 20.77
(CH2, 2-Et), 24.61 (CH2, 4-Et), 23.05, 25.94, 47.38, 50.19
(pyrrolidin-1-yl), 80.35 (C4), 176.66 (C2) and 177.63 (C5).
4-Methyl-2,5,5-triethyl-2,5-dihydro-1H-imidazole-1-ol (14)
A
mixture of propanal (2.8 mL, 38 mmol), hydroxy-
aminoketone 11 (6 g, 33 mmol), ethanol (5 mL) and of 25%
aqueous ammonia (10 mL) was stirred for 2 h at 25 ЊC and
allowed to stand at Ϫ5 ЊC overnight. The crystalline precipitate
was filtered off, washed with cold 50% EtOH to give di-
hydroimidazole 14 (5.6 g, 90%) as colorless crystals, mp 114–
115 ЊC (hexane) (Found: C, 64.94; H, 11.35; N, 15.11. Calc. for
C10H20N2O: C, 65.18; H, 10.94; N, 15.20%); νmax(KBr)/cmϪ1
2962, 2926, 2868, 1650, 1456, 1430, 1386, 1033, 1017 and 903;
δH(200 MHz; (CD3)2CO) 0.78 (3 H, t, J 8.0, CH3, Et), 0.84 (3H,
t, J 8.0, CH3, Et), 0.95 (3 H, t, J 8.0, CH3, Et), 1.36 (1 H, m,
CH2, Et), 1.49 (2 H, m, CH2, Et), 1,53 (1H, m, CH2, Et), 1.67
(1 H, m, CH2, Et), 2.02 (1 H, m, CH2, Et), 1.82 (3 H, d, J 2,
CH C᎐N), 4.50 (1 H, m, CH) and 7.15 (1 H, s, OH). δ (100
᎐
3
C
MHz; (CD3)2CO) 8.86, 9.66, 10.04 (CH3, Et), 26.54, 29.15,
30.17 (CH , Et), 17.03 (CH –C᎐N), 79.09 (C5), 94.11 (C2) and
᎐
2
3
174.86 (C᎐N).
᎐
2,4,4-Triethyl-4H-imidazole-5-carbaldehyde oxime 3-oxide (15)
Isopropyl nitrite (4 mL, 45 mmol) was added portionwise to a
solution of dihydroimidazole 14 (5 g, 27 mmol) in CH2Cl2 (10
ml), CCl4 (10 ml) and triethylamine (1 mL) during 3 h. Then
another portion of isopropyl nitrite (8 ml, 90 mmol) was added
and the solution was allowed to stand for 5 h. The mixture was
evaporated under reduced pressure to leave an orange oil, which
was dissolved in a mixture of diethyl ether (100 ml) and water
(100 ml). The mixture was shaken vigorously, the organic layer
was separated and extracted with 1% water solution of NaOH,
5 × 100 ml. Combined water extracts were saturated with NaCl,
acidified with HOAc to pH 5–6 and oxime 15 was extracted
back with CHCl3 (3 × 50 ml). The CHCl3 extract was dried over
MgSO4, CHCl3 was removed under reduced pressure and the
residue was triturated with EtOAc, the precipitate was filtered
off and recrystallized from t-BuOMe to yield oxime 15 (4.5 g,
80%) as yellow crystals, mp 113–115 ЊC (t-BuOMe) (Found: C,
56.55; H, 8.45; N, 19.40. Calc. for C10H17N3O2: C, 56.85; H,
8.11; N, 19.89%); νmax(KBr)/cmϪ1 1544, 1459, 1365, 1330, 1218,
1025, 1002, 882 and 734; λmax(EtOH)/nm 367 (log ε 3.84), 275
(3.44) and 229 (4.07); δH(200 MHz; (CD3)2CO) 0.51 (6 H, t,
J 7.5, 2 × CH3, 4-Et), 1.28 (3 H, t, J 7.5, CH3, 2-Et), 2.15 (4 H,
quartet, J 7.5, 2 × CH2, 4-Et), 2.83 (3 H, quartet, J 7.5, CH2,
2,2,5,5-Tetraethyl-4-pyrrolidin-1-yl-2,5-dihydro-1H-imidazol-
1-oxyl (3)
A 1 M solution of EtMgBr in THF (3 mL) was added dropwise
to a stirred solution of pyrrolidinoimidazole 17 (0.5 g, 2.1
mmol) in THF (5 mL). A drop of the reaction mixture was
quenched with water, and absence of the starting compound in
the reaction mixture was verified by TLC (Al2O3 Polygram Alox
N/UV 254, Macherey-Nagel) using a CHCl3–methanol mixture
50 : 1–2 as eluent. After the reaction was complete (usually
0.5 h), the reaction mixture was quenched with water (1–3 mL)
under vigorous stirring and diluted with t-BuOMe (20 mL).
Then MnO2 (3 g, 34.5 mmol) was added and the mixture was
stirred vigorously for 2 h, the oxidant was filtered off and the
filtrate was dried over Na2CO3. The solvent was removed under
reduced pressure to leave an orange oil, which was purified by
column chromatography on aluminium oxide (neutral) using
chloroform as eluent to give the nitroxide 3 (250 mg, 45%) as
orange oil (Found: C, 67.73; H, 10.76; N, 15.63. Calc. for
C15H28N3O: C, 67.63, H, 10.59; N, 15.77%); νmax(neat)/cmϪ1
2969, 2939, 2877, 1593, 1458, 1417, 1377, 1345, 1320, 1275,
1225, 1191, 1166, 962, 930 and 820.
2-Et), 7.97 (1 H, s, CH᎐N) and 12.17 (1 H, s, OH); δ (50 MHz;
᎐
C
(CD3)2CO) 6.55 (CH3, 4-Et), 9.02 (CH3, 2-Et), 18.49 (CH2,
2-Et), 29.95 (CH , 4-Et), 88.19 (C4), 144.12 (CH᎐N), 155.41
᎐
2
(C2) and 170.05 (C᎐N).
᎐
4,4-Trimethyl-4H-imidazole-5-carbonitrile 3-oxide (16)
TsCl (9.5 g, 50 mmol) was added portionwise to a stirred solu-
tion of oxime 15 (10.55 g 50 mmol) in a mixture of CHCl3
(75 mL) and triethylamine (16 mL, 110 mmol). The resulting
solution was stirred for 1 h, washed with water and dried over
MgSO4. The CHCl3 was removed under reduced pressure to
leave an orange oil, which was purified by column chromato-
graphy (Kieselgel 60, Merck), using dichloromethane as eluent
to give nitrile 16 (4.8 g, 85%), yellow crystals, mp 25–30 ЊC
(pentane) (Found: C, 61.19; H, 7.71; N, 21.03. Calc. for
C10H15N3O: C, 62.15; H, 7.82; N, 21.74%); νmax(neat, 30 ЊC)/
cmϪ1 2977, 2939, 2882, 2217, 1532, 1478, 1456, 1399, 1306,
1179, 1057, 854, 799 and 707; λmax(EtOH)/nm 375 (log ε 4.08);
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 2 5 – 1 0 3 0
1029

View Article Online
2 K. Itoh and M. Kinoshita, Molecular magnetism: new magnetic
Determination of ionization constants for nitroxides 1–3
materials, Kodansha, Tokyo, Gordon & Breach, Amsterdam, 2000;
M. Deumal, J. Cirujeda, J. Veciana and J. J. Novoa, Chem. Eur. J.,
1999, 5, 2250–2260.
A solution of the nitroxides (0.5 mM) in a mixture of 2.5 mM
sodium phosphate and 2.5 mM sodium citrate buffers contain-
ing DTPA (0.1 mM) were titrated with solutions of HCl or
NaOH to the required pH. The pH was measured using a pH
meter OP-205/1 (Hungary) to 0.05 pH units. EPR spectra of
the samples were recorded in a 200 µl quartz EPR flat cell. Hfi
splitting constants (aN) were measured as a distance between
low-field and central-field components of the EPR spectra. To
obtain the pK values of the compounds, the experimental
dependence of aN on pH was fitted to the conventional titration
equation:
3 L. B. Volodarsky, V. A. Reznikov and V. I. Ovcharenko, Synthetic
chemistry of stable nitroxides, CRC Press, Boca Raton, 1994; H. G.
Aurich, in Nitrones, Nitronates and Nitroxides, ed. S. Patai and
Z. Rappoport,. John Wiley & Sons, Chichester, New York, Brisbane,
Toronto, Singapore, 1989, pp. 313–370; A. E. J. de Nooy, A. C.
Besemer and H. van Bekkum, Synthesis, 1996(10), 1153–1174.
4 H. M. Swartz and R. B. Clarkson, Phys. Med. Biol., 1998, 43, 1957–
1975; S. S. Velan, R. G. S. Spencer, J. L. Zweier and P. Kuppusamy,
Magn. Reson. Med., 2000, 43, 804–809.
5 V. V. Khramtsov, I. A. Grigor’ev, M. A Foster, D. J. Lurie and
I. Nicholson, Cell. Mol. Biol., 2000, 46, 1361–1374.
6 M. A. Foster, I. A. Grigor’ev, D. J. Lurie, V. V. Khramtsov,
S. McCallum, I. Panagiotelis, J. M. S. Hutchison, A. Koptioug and
I. Nicholson, Magn. Reson. Med., 2003, 49, 558–567.
7 Biological Magnetic Resonance, Volume 14: Spin Labeling: The Next
Millennium, ed. L. J. Berlinery, Plenum Press, New York and
London, 1998.
8 H. Li, L. Ma, C. J. Hsia, J. L. Zweier and P. Kuppusamy,
Free Radical Biol. Med, 2002, 32, 712–719; N. Kocherginsky, H. M.
Swartz and M. Sentjure, in Nitroxide spin labels, reactions in biology
and chemistry, ed N. Kocherginsky and H. M. Swartz, CRC Press,
Boca Raton, 1995, 175–198; R. Zhang, E. Shohami, E. Beit-Yannai,
R. Bass, V. Trembovler and A. Samuni, Free Radical Biol. Med.,
1998, 24, 332–340.
(1)
where p1 and p2 are experimentally measured hfi splittings at
pHӷpK and pHӶpK, correspondingly.
Determination of the rate constant of nitroxides reduction by
ascorbic acid
A solution of the nitroxides (1, 4, and 6, 0.05 mM; 2, 3, 5, and 7,
0.1 mM) in 0.1 M sodium phosphate buffer, pH = 7.5, contain-
ing 0.1 mM DTPA, were prepared. Ascorbic acid (Sigma) was
dissolved in the same buffer, and the following concenrations
were used: 33, 66, and 133 mM for 1; 250, 375, and 500 mM for
2; 10.4, 20.8, and 41.7 mM for 3; 0.83, 1.67, and 2.5 mM for 4;
4.2, 8.3, 16.7, and 33.3 mM for 5; 1, 2, and 4 mM for 6; 0.5, 1,
1.5, and 2 mM for 7. The solutions of the nitroxides and of
ascorbic acid were bubbled with argon for 20 min, and mixed
afterwards. Then 0.1 ml aliquots were immediatelly placed into
100 µl quartz capillary tubes, transferred into the cavity of EPR
spectrometer, and the spectra were recorded at 23–25 ЊC. The
kinetics of decreasing the peak intensity of the low field spec-
tral component was measured and fitted to exponential decay,
yielding the rate constant, kobs. The experiment for each con-
centration of ascorbic acid was repeated three times, and the
bimolecular rate constants were calculated.
9 A. Samuni and M. C. Krishna, in Handbook of synthetic
antioxidants, ed L. Packer and E. Cadenas, Marcel Dekker, New
York, 1997, pp. 351–373; K. Takeshita, K. Saito, J.-i. Ueda,
K. Anzai and T. Ozawa, Biochim. Biophys. Acta, 2002, 1573, 156–
164.
10 J. F. W. Keana, S. Pow and G. M. Rosen, Magn. Reson. Med., 1987,
3,
83–88.
11 C. S. Winalski, S. Shortkroff, R. V. Mulkern, E. Schneider and G. M.
Rosen, Magn. Reson. Med., 2002, 48, 965–972.
12 N. Kocherginsky and H. M. Swartz, Nitroxide spin labels, reactions
in biology and chemistry, Boca Raton, CRC Press, 1995, pp. 1–265.
13 S. Morris, G. Sosnovsky, B. Hui, C. O. Huber, N. U. M. Rao and
H. M. Swartz, J. Pharm. Sci., 1991, 80, 149–152.
14 J. F. W. Keana and F. L. Van Nice, Physiol. Chem. Phys. Med. NMR,
1984, 16, 477–480.
15 V. I. Yelinova, A. G. Krainev, A. Savelov and I. A. Grigor’ev,
J. Chem. Soc., Perkin Trans. 2, 1993(11), 2053–2055.
16 K. Takeshita, A. Hamada and H. Utsumi, Free Radic. Biol. Med.,
1999, 26, 1951–1960; R. J. Mehlhorn, J. Biol. Chem., 1991, 266,
2724–2731.
EPR measurements of the nitroxides reduction in the rat blood
17 L. Marx, R. Chiarelli, T. Guiberteau and A. Rassat, J. Chem. Soc.,
Perkin Trans. 1, 2000, 1181–1182.
A WAG rat (AL Bacharach, Glaxo Ltd 1924 from Wistar stock,
female, 1 year old) was kindly donated by N. G. Kolosova
(Institute of Cytology and Genetics SB RAS, Novosibirsk,
Russia) and treated in compliance with Russian Legislation.
The rat was decapitated and blood was collected. A 20 µl ali-
quot of the nitroxides 1–3, and 5–7 in 0.1 M sodium phosphate
buffer, pH =7.5, containing 0.1 mM DTPA, was mixed with
180 µl of whole rat blood (final concentration of the nitroxide
50 µM). The sample was placed in 100 µl quartz capillary and
transferred into the EPR cavity immediately afterwards, and
the kinetics of the nitroxide decay was measured by following
the decrease in the peak intensity of the low field spectral
component.
18 V. V. Khramtsov and L. B. Volodarsky, in Biological Magnetic
Resonance, Volume 14: Spin Labeling: The Next Millennium, ed L. J.
Berliner, Plenum Press, New York and London, 1998, 109–180.
19 V. V. Khramtsov, I. A. Grigor’ev, M. A. Foster and D. J. Lurie,
Antioxid. Redox Signal., 2004, in press; K. Mäder, B. Gallez,
K. J. Liu and H. M. Swartz, Biomaterials, 1996, 17, 457–461.
20 L. B. Volodarsky and I. A. Grigor’ev, in Imidazoline nitroxides,
ed. L. B. Volodarsky, CRC Press, Boca Raton, 1988, vol 1, pp. 5–23.
21 L. B. Volodarsky, V. A. Reznikov and I. A. Grigor’ev, in Imidazoline
nitroxides, ed. L. B. Volodarsky, CRC Press, Boca Raton, 1988,
vol 1, pp. 29–76.
22 I. A. Kirilyuk, T. G. Shevelev, D. A. Morozov, E. L. Khromovskih,
N. G. Skuridin, V. V. Khramtsov and I. A. Grigor’ev, Synthesis,
2003(6), 871–878.
23 E. G. Lubenets, L. D. Nikulina, F. S. Morozova, L. B. Volodarsky,
A. G. Khmelnitsky and V. S. Kobrin, Belgian Patent, 1983, 896,093.
24 L. B. Volodarskii and T. K. Sevastyanova, Zh. Org. Khim., 1971, 8,
1971 (Chem. Abstr., 1971, 75, 140425).
25 V. A. Reznikov and L. B. Volodarsky, Russ. Chem. Bull., 1997, 46,
1577–1581.
26 V. V. Khramtsov, L. M. Weiner, I. A. Grigor’ev and L. B.
Volodarsky, Chem. Phys. Lett., 1982, 91, 69–72; V. V. Khramtsov,
L. M. Weiner, S. I. Eremenko, O. I. Belchenko, P. V. Schastnev, I. A.
Grigor’ev and V. A. Reznikov, J. Magn. Reson., 1985, 61, 397–408.
27 O. Saphier, T. Silberstein, A. I. Shames, G. I. Likhtenshtein,
E. Maimon, D. Mankuta, M. Mazor, M. Katz, D. Meyerstein and
N. Meyerstein, Free Radical Res., 2003, 37, 301–308.
Acknowledgements
This work was supported by RFBR (grants 99-04049921,
01-03-32452 and 02-04-48374), INTAS (grants 99-1766 and
99-1086), NHLB 53333 and KO1 EB03519. The authors are
grateful to RFBR for access to the STN International data-
bases (grant 00-03-40142) via STN Center at the Novosibirsk
Institute of Organic Chemistry SB RAS, 630090 Novosibirsk,
Russia.
28 I. A. Grigor’ev, I. A. Kirilyuk and L. B. Volodarskii, Chem.
Heterocycl. Compd. (Engl.Transl.), 1988, 24, 1355; I. A. Grigor’ev,
G. I. Shchukin, V. V. Martin and V. I. Mamatyuk, Chem. Heterocycl.
Compd. (Engl.Transl.), 1985, 21, 205.
References
1 Controlled Radical Polymerization, ed. K. Matyjaszewski, American
Chemical Society, Washington DC, 1998, pp. 170–257.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 2 5 – 1 0 3 0
1030