Nitroxides with two pK values - Useful spin probes for pH monitoring within a broad range

Article abstract of DOI:10.1039/b418707e

A series of 4-dialkylamino-2,5-dihydroimidazole nitroxides with pyridine-4-yl, 4-dimethylaminophenyl or 4-hydroxyphenyl groups in position 2 of the imidazole ring were prepared using the reaction of RMgBr with corresponding 5-dialkylamino-4,4-dimethyl-4ff

Full text of DOI:10.1039/b418707e

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A R T I C L E
Nitroxides with two pK values—useful spin probes for pH
monitoring within a broad range
Igor A. Kirilyuk,*a Andrey A. Bobko,b Valery V. Khramtsovb,c and Igor A. Grigor’eva
a N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Academician Lavrent’ev 9,
Novosibirsk, 630090, Russia. E-mail: kirilyuk@nioch.nsc.ru; Fax: +7-3832-309752;
Tel: +7-3832-307387
b Institute of Chemical Kinetics & Combustion, Novosibirsk, 630090, Russia.
E-mail: bobko@kinetics.nsc.ru; Fax: +7-3832-308196; Tel: +7-3832-332294
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 13th December 2004, Accepted 7th February 2005
First published as an Advance Article on the web 28th February 2005
A series of 4-dialkylamino-2,5-dihydroimidazole nitroxides with pyridine-4-yl, 4-dimethylaminophenyl or
4-hydroxyphenyl groups in position 2 of the imidazole ring were prepared using the reaction of RMgBr with
corresponding 5-dialkylamino-4,4-dimethyl-4H-imidazole 3-oxides. The EPR spectra of the nitroxides were shown to
be pH-sensitive due to consecutive protonation of the amidino moiety and the basic group(s) at position 2 of the
imidazole ring. The 5,5-dimethyl-4-(dimethylamino)-2-ethyl-2-pyridine-4-yl-2,5-dihydro-1H-imidazol-1-oxyl showed
a monotonic increase in the isotropic nitrogen hyperfine (hfi) coupling constant aN of 1 .4 G over a pH range from 2
to 6.5. Such a broad range of pH-sensitivity could be useful for many biophysical and biomedical applications,
including pH-monitoring in the stomach.
are considered to be the most promising pH-sensitive spin probes
for EPR studies in vivo due to the relatively large effect of pH
Introduction
The measurement of pH is probably the most widely performed
test in the biochemical laboratory, reflecting its critical role in
the physiology and pathophysiology of living organisms. Most
non-invasive pH measurements, particularly those conducted in
on their EPR spectra (DaN varies from 0.7 to 1.0 G) and the
appropriate pK values in the range from 4.5 to 7.4.5,7,8
It is well established that nitroxides with ionizable (basic or
acidic) groups in the side chain have rather moderate EPR
vivo, rely on endogenous and/or exogenous molecular probes.
spectral responses to pH changes.5 However, nitroxides with
The absorption of fluorescent pH-sensitive dyes was found
several basic or acidic groups (intracyclic or exocyclic) may
particularly effective for pH study on the cellular and subcellular
undergo consequent protonation of the two basic centers to
levels,1 while magnetic resonance approaches based on EPR and
produce complementary effects on the hfi splitting constant
NMR spectroscopies have advantages for in vivo applications in
(aN) of the nitroxide. Very few examples of such nitroxides are
known, e.g., 1 (Fig. 1.). The EPR spectrum of this nitroxide
animals and humans.2 EPR has a crucial advantage over NMR
in that it is more than three orders of magnitude more sensitive.
undergoes two consequent transitions at pH 0–2 and 10.5–12.
However, the low depth of microwave penetration into aqueous
Here we report a further development of this approach to the
samples and the absence of endogenous paramagnetic probes
molecular design of new pH-sensitive spin probes: specifically,
significantly limit the application of EPR to biological systems.
Despite these formidable problems, recently developed low-field
we synthesized a series of 4-amino-2,5-dihydro-1H-imidazole
spin probes with 4-dimethylaminophenyl, 4-hydroxyphenyl and
EPR-based techniques, in combination with a wide variety of
4-pyridyl groups at the position 2 of the imidazole ring and
spin pH probes, offer another unique opportunity for non-
studied the sensitivity of their EPR spectra to pH.
invasive pH measurements (for recent reviews see ref. 3 and 4).
Among various pH-sensitive spin probes described to date,5,6
EPR spectra of perhydroimidazole-derived nitroxides (e.g.,
HMI, Fig. 1) have the highest sensitivity to pH, DaN ca. 1.3
G. However, the utility of these nitroxides in biomedical studies
is somewhat limited by low pK values (ca. 4.5). The nitroxides of
the 4-amino-2,5-dihydro-1H-imidazole series (e.g., ATI, Fig. 1)
Results and discussion
Synthesis
The nitroxides 8a–e and 9 were prepared using a recently
developed method7 (Scheme 1), via organometallic reagent
addition to 4H-imidazol-3-oxides.
The 2,5-dihydro-1H-imidazoles 3a–c were synthesized by
condensation of 3-(hydroxyamino)-3-methylbutan-2-one 2 with
ammonia and corresponding aldehydes, in a similar way to
the procedure for 3a described earlier.9 A mild oxidation of
3a–c with lead dioxide in methylene chloride yielded 4,4,5-
trimethyl-4H-imidazole 3-oxides 4a–c. The nitrosation of 4b–c
was performed using the i-PrONO–i-PrONa system, while for
4a, a better yield of the 5-hydroximinomethyl derivative 5a was
achieved using the i-PrONO–Et3N system. Treatment of the
oximes 5a–c with TsCl–Et3N yielded carbonitriles 6a–c, the key
compounds which were involved in the reaction with amines to
Fig. 1
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 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 6 9 – 1 2 7 4
1 2 6 9
©

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Titrations
The EPR spectra of all the nitroxides synthesized were found to
be pH-dependent (See Table 1). The hydroxyphenyl derivative
9 showed hfi changes in two distinct pH ranges, apparently
corresponding to deprotonation of the phenoxy group (pK 9.8)
and to protonation of the amidino moiety (pK 5.9). The hfi
constants aN of the nitroxides 8a–d and 8f undergo monotonic
changes upon titration due to consecutive protonation of the
amidino moiety and the basic groups at position 2 of imidazoline
ring with close pK values (Fig. 2 and 3). The nitroxides 8b, 8d and
8f showed the highest sensitivity of EPR spectra to pH; these
spin probes cover a range over 4 units of pH with anoverall
DaN of ca.1.4 G. It is interesting to note that the introduction
of the basic groups, 4-dimethylaminopenyl and pyridine-4-yl, at
position 2 of the imidazole ring in 8f lead neither to an increase in
the overall DaN nor to an expansion of the range of pH sensitivity
in comparison to the nitroxide 8d. Titration data of this nitroxide
may be fitted to three-pK base eqn. (3) as well as to eqn. (2), see
Fig. 3. It is not clear whether the protonation of both adjacent
pyridine and dimethylaminophenyl basic centers occurs within
the pH range studied.
Recently we reported on the synthesis of nitroxides 10 and 11
(Fig. 4),7 which have similar basic groups at position 2 of the
imidazole ring. The titration of these nitroxides gave a simple
Scheme 1 Reagents: (i) RCHO, NH3, EtOH–H2O; (ii) PbO2–CH2Cl2;
(iii) i-PrONO, Et3N–CHCl3 or i-PrONO, i-PrONa–i-PrOH; (iv) TsCl,
Et3N–CHCl3; (v) NHR1R2–CH2Cl2; (vi) R3MgBr, THF; then H2O,
MnO2; for 3–6 R = p-Me2NC6H4 (a), 4-Py (b) and p-BnOC6H4 (c);
for details of R, R1, R2 and R3 for 7 and 8 see Table 1.
give 4H-imidazoles 7a–e. The 4H-imidazole 3-oxides 7a–e were
treated with an excess of organometallic reagent (EtMgBr or
p-Me2NC6H4MgBr). After quenching the reaction mixture with
water and then a consequent oxidation with manganese dioxide,
the nitroxides 8a–f were isolated. To prepare the nitroxide 9
the benzyl group in 8e was removed using hydrogenolysis on
Pd/C, and the hydroxylamine formed was reoxidized with MnO2
(Scheme 2).
Fig. 2 The pH dependence of nitrogen hyperfine splitting (aN) mea-
sured as a distance between low- and central-field components of the
EPR spectra of the nitroxides 8c (᭺), and 8d (ꢀ). The solid line is a
nonlinear least-squares fit of the data to eqn. (2), see Experimental.
Scheme 2
Table 1 Parameters of the new nitroxide spin probes: pK values, changes in hfi splitting (DaN) between protonated and unprotonated forms and
partition coefficients (Kp) measured in octanol–(0.1 N NaOH) mixtures
R
R1
R2
R3
pK
DaN, G
Kp
8a
8b
8c
8d
8f
p-Me2NC6H4
4-Py
(CH2)5
(CH2)5
Me
Et
6.1
3.4
4.8
2.8
6.25
3.50
5.08
2.86
4.76a
2.38a
0.83
0.35
0.8
>300
>300
100
25
Et
0.6
p-Me2NC6H4
4-Py
Me
Me
Me
Et
0.77
0.26
0.81
0.53
0.61a
0.68a
0.61b
0.82
0.12
Me
Et
4-Py
Me
p-Me2NC6H4
5.03b
3.95b
2.29b
0.44b
0.24b
64
9
p-HOC6H4
Me
Me
Et
5.9
9.8
0.033
a Calculated for two pK. b Calculated for three pK.
1 2 7 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 6 9 – 1 2 7 4

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quartz capillary. The 3-hydroxyamino-3-methylbutan-2-one 2
was prepared according to the procedure described previously.10
Titration of the nitroxides 8 and 9 was performed similarly
to the procedure described in.11 The nitroxides were dissolved in
1 mM phosphate buffer, pH = 6.03, to the final concentration of
the nitroxide of ca. 0.1 mM. The poorly-soluble nitroxides were
first dissolved in 0.1 mL of acetone or DMSO, and then added
to the buffer solution. The resulting solutions were titrated with
KOH or HCl solutions to the required pH. The pH was measured
using a digital pH-meter equipped with a glass electrode. The
accuracy of the measurements was estimated to be 0.02 pH units.
The hfi splittings were measured as the distance between the low
field and the central lines of the nitroxide EPR spectra and
are accurate within 0.02 G. To obtain the pK values of the
compounds, the experimental dependence of aN on pH was fitted
to one of the conventional titration equations: eqn, (1) for 8e,
eqn. (2) for 8a–d and 9:
p1 + p2 × 10pK−pH
aN(pH) =
(1)
1 + 10pK−pH
Fig. 3 The pH dependence of hyperfine splitting (aN) of the EPR
spectra of the nitroxide 8f. The experimental data were fitted to eqn.
(3) (3-pK, solid line) and eqn. (2) (2-pK, dashed line). The pK and DaN
values calculated are listed in Table 1.
−pH
+pK −2×pH
2
(p1 + p2 × 10pK
+ p3 × 10pK
)
1
1
aN(pH) =
(2)
−pH
+pK −2×pH
2
1 + 10pK
+ 10pK
1
1
where p1–p3 represent nitrogen hfi splittings of the nitroxide in
different ionization states. For the nitroxide 8f both eqn. (2) and
(3) were used; the error of pK determination was 0.1.
aN(pH)
1−pH
1+pK2−2×pH
1+pK2+pK3−3×pH
(p1 + p2 × 10pK
+ p3 × 10pK
+ p4 × 10pK
)
=
1 + 10pK
+ 10pK
+ 10pK
1−pH
1+pK2−2×pH
1+pK2+pK3−3×pH
(3)
where p1–p4 represent nitrogen hfi splittings of the nitroxide in
different ionization states.
Fig. 4
The partition coefficients of the nitroxides 8 and 9 were
determined using a previously described procedure.7 A sample
of the nitroxide (ca. 2 lmol) was placed in a tube containing
octanol (5 mL) and 0.1 M NaOH solution (5 mL). The mixture
was shaken vigorously and allowed to stand until separation
of the phases was complete. The Kp were determined from the
difference in integral intensity of the EPR spectra of the nitroxide
in water and in octanol. Accuracy of the measurements was up
to 5%.
titration curve with a DaN value of 0.8 G, corresponding to
protonation of the amidine moiety. The absence of a second pK
in the titration curve of 10 could be due inefficiency of the meta-
position of amino group in the phenyl ring for the transmission
of electronic effect of the substituent. For nitroxide 11, both the
amidine group and the pyridine ring nitrogen are likely to be
engaged in coordination with protons. This may also account
for the relatively high pK (5.4) of the nitroxide.
The partition coefficients of the nitroxides 8a, 8c, 8d, 8f
and 9 were measured in 0.1 M NaOH–octanol mixtures and
represent the relative lipophilicities of the unprotonated forms
of the nitroxides. At lower pH values the lipophilicities decrease
because of an equilibrium with highly hydrophilic protonated
forms. Among all the nitroxides studied, compound 8d seems to
be the most promising spin probe, because of a relatively high
solubility in water and a broad range of sensitivity to pH (Fig 2).
This probe is particularly suitable for pH monitoring in stomach
using non-invasive low-field EPR techniques.3
4,5,5-Trimethyl-2,5-dihydro-1H-imidazole-1-ols 3b,c, general
procedure
Aldehyde (20 mmol) was added to a stirred solution of 3-
hydroxyamino-3-methyl-butan-2-one (2) (3 g, 20 mmol) in a
mixture of methanol (15 mL) and aqueous ammonia (10 mL).
◦
The reaction mixture was stirred for 5 h at 25 C and allowed
to stand overnight at −5 ◦C. The crystalline precipitate was
filtered off and washed with cold EtOH 50% and with cold
water to give dihydroimidazoles 3b and 3c. Compound 3b, yield
4.1 g, (70%) colorless crystals, mp 119–122 ◦C (EtOAc). Found:
C, 62.94; H, 7.79; N, 19.56. Calc. for C11H15N3O 1/2 H2O: C,
62.54; H, 7.47; N, 19.89. mmax(KBr)/cm−1 1633, 1607, 1568, 1501,
1416, 1384, 1358, 1295, 1250, 1240, 1033, 1022, 1007 and 803;
kmax(EtOH)/nm 258 (lg e 3.21); dH(400 MHz; CDCl3-CD3OD 1 :
1) 1.27 (3 H, s, 2-CH3), 1.32 (3H, s, 2-CH3), 2.06 (3 H, d, J 3.0, 4-
CH3), 5.43 (1 H, quartet, J 3.0, CH), 7.55 and 8.52 (4 H, AAꢀBBꢀ,
J 9.0, Py); dC(100 MHz; CDCl3-CD3OD 1 : 1) 16.37, 16.41 (2-
CH3), 24.38 (4-CH3), 73.11 (C5), 90.21 (C2), 124.06 (C3, Py),
Experimental
The IR spectra were recorded on a Bruker Vector 22 FT-
IR spectrometer in KBr pellets (concentration 0.25%, pellet
thickness 1 mm). The UV spectra were measured on a HP
Agilent 8453 spectrometer in EtOH. The 1H NMR spectra
were recorded on a Bruker AC-200 (200.132 MHz) spectrometer
for 5–10% solutions using the signal of the solvent as the
standard. The 13C NMR spectra were recorded on a Bruker
AC-200 (50.323 MHz) and a 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 based on analysis of intensities,
on the spectra measured in J-modulation mode, and using
the data reported previously.7,8 EPR spectra were recorded
with a Bruker ER-200D-SRC spectrometer using a 100 lL
2
i
=
149.56 (C , Py), 151.09 (C , Py) and 183.13 (C N). Compound
3c, yield 5.3 g, (85%) colorless crystals, mp 144–146 ◦C (EtOAc).
Found:C, 73.97;H, 7.27; N, 8.98. Calc. for C19H22N2O2: C, 73.52;
H, 7.14; N, 9.03. mmax(KBr)/cm−1 2965, 2864, 1643, 1612, 1587,
1511, 1461, 1425, 1381, 1300, 1285, 1232, 1171, 1022, 874, 829
and 750; kmax(EtOH)/nm 226 (lg e 4.20); dH(200 MHz; CDCl3)
0.94 (3 H, s, 2-CH3), 1.05 (3H, s, 2-CH3), 1.92 (3 H, d, J 3.0,
4-CH3), 5.07 (2 H, s, CH2), 5.29 (1 H, quartet, J 3.0, CH), 6.32
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 6 9 – 1 2 7 4
1 2 7 1

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(1 H, s, OH), 6.95 and 7.25 (4 H, AAꢀBBꢀ, J 8.5, C6H4O), and
7.38 (5 H, m, Ph); dC(50 MHz; CDCl3) 15.86, 16.02 (2-CH3),
23.90 (4-CH3), 70.04 (CH2), 71.71 (C5), 91.01 (C2), 114.72 (Cm,
C6H4O), 127.22 (Co, C6H4O), 132.61 (Ci, C6H4O), 158.72 (C–
60 ◦C until all Na was dissolved. The solution was allowed
to cool to room temperature to form a suspension of i-PrONa.
Isopropyl nitrite (3.5 mL, 39 mmol) and a solution of 4b or 4c
(16 mmol) in 20 mL of isopropanol were added subsequently
to the stirred suspension of i-PrONa in isopropanol and the
mixture was allowed to stand for 1–8 h. After the reaction
was complete (TLC, Silufol UV-254, eluent EtOAc) the mixture
was acidified with AcOH to pH 6–7 and isopropanol was
removed in vacuum. A saturated solution of NaCl (20 mL)
was added to the residue and the precipitate of 5b or 5c was
filtered off and recrystallized from EtOAc. Compound 5b, yield
(80%), mp 240–244 ◦C (EtOAc). Found: C, 57.11; H, 5.29;
N, 24.03. Calc. for C11H12N4O2: C, 56.89; H, 5.21; N 24.12.
mmax(KBr)/cm−1 1604, 1559, 1525, 1472, 1414, 1364, 1319, 1204,
1059, 1005, 834 and 812; kmax(EtOH)/nm 356 (lg e 3.68), 259
(4.11); dH(200 MHz; (CD3)2SO + t-BuOK 5%) 1.62 (6H, s, 4-
O, C6H4O), 127.69 (Cp, Ph), 128.38 (Cm, Ph), 128.90 (Co, Ph),
i
=
137.16 (C , Ph), and 178.42 (C N).
4H-Imidazole 3-oxides 4a–c, general procedure
A suspension of 3a–c (10 mmol), and PbO2 (4.78 g, 20 mmol)
in CH2Cl2 (30 mL) was stirred for 1–3 h. After the reaction
was complete (monitored by TLC analysis, Silufol, eluent Et2O–
methanol 25 : 1, development with I2 vapour) the lead oxides
were filtered off and the solvent was removed in vacuum.
Compound 4a, yield 90%, yellow crystals, mp 135–137 ◦C
(EtOAc–hexane 1 : 1), Found: C, 68.69; H, 7.48; N, 17.12.
Calc. for C14H19N3O: C, 68.45; H, 7.79; N 17.10. mmax(KBr)/cm−1
1605, 1547, 1508, 1452, 1435, 1390, 1322, 1294, 1218, 1192,
1127, 1106, 1071, 945 and 835; kmax(EtOH)/nm 385 (lg e 4.25),
295 (4.15); dH(200 MHz; (CD3)2CO) 1.39 (6H,s, 4-Me), 2.30
(3H, s, 5-Me), 3.02 (6H, s, NMe2), 6.80, 8.58 (2H each, AAꢀBBꢀ,
J 9 Hz, Ar); dC(50 MHz; (CD3)2SO) 16.71 (5-Me), 21.52 (4-Me),
36.20 (NMe2), 80.39 (C4), 144.78 (C2), 181.92 (C5), Ar: 114.81
(Ci), 128.79 (Co), 111.22 (Cm), 151.29 (◦C–N). Compound 4b,
yield 90%, yellow crystals, mp 201–204 C (EtOAc–hexane 1 :
1). Found: C, 65.36; H, 6.38; N, 20.58. Calc. for C11H13N3O:
C, 65.01; H, 6.45; N, 20.68. mmax(KBr)/cm−1 1598, 1547, 1523,
1475, 1464, 1428, 1416, 1398, 1376, 1322, 1304, 1233, 1220, 1196,
1066, 990 and 833; kmax(EtOH)/nm 334 (lg e 3.93), 280 (3.40);
dH(200 MHz; CDCl3) 1.47 (6H, s, 4-Me), 2.32 (3H, s, 5-Me),
8.41, 8.72 (2H each, AAꢀBBꢀ, J 6.3 Hz, Py); dC(50 MHz; CDCl3)
16.48 (5-Me), 21.59 (4-Me), 82.59 (C4), 144.00 (C2), 180.52 (C5),
Py: 133.38 (Ci), 120.09 (C3), 150.09 (C2). Compound 4c, yield
90%, yellow crystals, mp 201–204 ◦C (EtOAc–hexane 1 : 1).
Found: C, 74.11; H, 6.64; N, 9.03. Calc. for C19H20N2O2: C,
74.00; H, 6.54; N 9.08. mmax(KBr)/cm−1 1602, 1587, 1543, 1497,
1421, 1394, 1371, 1302, 1245, 1205, 1174, 1116, 997, 841 and
754; kmax(EtOH)/nm 339 (lg e 3.97), 268 (4.38); dH(200 MHz;
CDCl3) 1.43 (6H, s, 4-Me), 2.26 (3H, s, 5-Me), 5.06 (2H, s,
CH2), 7.03, 8.65 (2H each, AAꢀBBꢀ, J 9 Hz, C6H4), 7.33 (5H,
m, Ph); dC(50 MHz; CDCl3) 16.36 (5-Me), 21.59 (4-Me), 69.86
(CH2), 80.93 (C4), 144.90 (C2), 180.52 (C5), C6H4: 136.45 (Ci),
129.43 (Co), 114.61 (Cm), 160.18 (C–O), Ph: 120.52 (Ci), 127.76
(Cp), 127.18, 128.32 (Co, Cm).
ꢀ
ꢀ
=
Me), 7.96 (1H, s, HC N–O), 8.39, 8.67 (2H each, AA BB J
6 Hz, 4-Py); dC(50 MHz; (CD3)2SO + t-BuOK 5%) 25.35 (4-
4
2
5
=
Me), 81.04 (C ), 144.05 (C ), 177.46 (C ), 144.19 (HC NO),
Py: 134.40 (Ci), 120.27 (C3), 150.22 (C2). Compound 5c, yield
(80%), mp 211–215 ◦C (EtOAc). Found: C, 67.77; H, 5.71;
N, 12.17. Calc. for C19H19N3O3: C, 67.64; H, 5.68; N 12.46.
mmax(KBr)/cm−1 1604, 1537, 1469, 1450, 1429, 1387, 1343, 1315,
1297, 1256, 1183, 1024, 836 and 731; kmax(EtOH)/nm 379 (lg
e 3.63), 291 (4.54); dH(200 MHz; CDCl3 + CD3OD (10%))
=
1.66 (6H, s, 4-Me), 5.15 (2H, s, CH2), 7.99 (1H, s, HC N–
O), 7.11, 8.61 (2H each, AAꢀBBꢀ, J 9 Hz, C6H4), 7.40 (5H, m,
Ph); dC(50 MHz; CDCl3 + CD3OD (10%)) 23.75 (4-Me), 70.48
4
2
5
=
(CH2), 81.70 (C ), 147.73 (C ), 174.89 (C ), 144.06 (HC NO),
C6H4: 136.82 (Ci), 130.27 (Co), 115.25 (Cm), 161.34 (C–O), Ph:
120.31 (Ci), 128.31 (Cp), 127.68, 128.82 (Co, Cm).
4H-Imidazole-5-carbonitrile 3-oxides 6a–c, general procedure
TsCl (9.5 g, 50 mmol) was added portionwise to a stirred solution
of oxime 5a–c (50 mmol) of 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 in vacuum and the residue was separated
by a column chromatography (Kieselgel 60, Merck, eluent-
chloroform) to give 6a–c. Compound 6a, yield 90%, yellow
crystals, mp 200–203 ◦C (hexane) (Found: C, 65.52; H, 6.31;
N, 21.99. Calc. for C14H16N4O: C, 65.61; H, 6.29; N, 21.86);
mmax (KBr)/cm−1 2982, 2914, 2820, 2223, 1613, 1524, 1488, 1466,
1440, 1391, 1376, 1293, 1235, 1212, 1110, 1066, 946, 820 and
741; kmax(EtOH)/nm 469 (lg e 3.61), 346 (4.45), 330 (4,38);
dH(200 MHz; (CD3)2CO) 1.59 (6H,s, 4-Me), 3.06 (6H, s, NMe2),
6.84, 8.42 (2H each, AAꢀBBꢀ, J 9 Hz, Ar); dC(50 MHz; CDCl3-
CCl4 1 : 1) 21.54 (4-Me), 39.83 (N–Me), 82.14 (C4), 111.61
(C≡N), 147.20 (C2), 149.96 (C5), Ar: 113.80 (Ci), 128.76 (Co),
111.10 (Cm), 151.78 (C–N). Compound 6b, yield 80%, yellow
crystals, mp 153–156 ◦C (hexane). Found: C, 61.57; H, 4.46;
N, 26.39. Calc. for C11H10N4O: C, 61.67; H, 4.71; N 26.15.
mmax (KBr)/cm−1 3044, 2987, 2228, 1598, 1556, 1524, 1508,
1469, 1402, 1386, 1328, 1297, 1201, 993, 829, 789 and 724; kmax
(EtOH)/nm 368 (lg e 3.84), 299 (4.02), 229 (4.26); dH(200 MHz;
CDCl3) 1.57 (6H, s, 4-Me), 8.22, 8.70 (2H each, AAꢀBBꢀ J 6 Hz,
4-Py); dC(50 MHz; CDCl3) 21.58 (4-Me), 84.88 (C4), 111.00
(C≡N), 145.37 (C2), 149.76 (C5), Py: 132.22 (Ci), 119.48 (C3),
150.46 (C2). Compound 6c, yield 90%, yellow crystals, mp 153–
4,4-Dimethyl-2-(4-dimethylaminophenyl)-4H-imidazole-
5-carbaldehyde oxime 3-oxide (5a)
Isopropyl nitrite (3 mL, 34 mmol) was added to a solution of
4H-imidazole 4a (2.2 g, 9 mmol) in a mixture of chloroform (5
mL) and triethylamine (2 mL) and the mixture was allowed to
stand at 22 ◦C for 24 h. The crystalline precipitate was filtered off
and washed with a mixture tert-butylmethylether–iso-propanol
(2 : 1) to yield 1.7 g (70%) of oxime 5a, red crystals, mp 233–
236 ◦C (dec.) (EtOAc). Found: C, 61.24; H, 6.90; N, 20.39. Calc.
for C14H18N4O2: C, 61.30; H, 6.61; N 20.42. mmax(KBr)/cm−1
1607, 1559, 1539, 1513, 1492, 1439, 1414, 1366, 1345, 1293,
1267, 1201, 1026, 945, 865 and 821; kmax(EtOH)/nm 415 (lg e
3.82), 336 (4.55); dH(200 MHz; (CD3)2SO) 1.51 (6H, s, 4-Me),
=
3.06 (6H, s, NMe2), 8.00 (1H, s, HC N–O), 6.80, 8.42 (2H each,
◦
AAꢀBBꢀ J 9 Hz, Ar), 12.60 (1H, s, OH); dC(50 MHz; (CD3)2SO +
155 C (hexane). Found: C, 71.40; H, 5.20; N, 13.07. Calc. for
t-BuOK 5%) 25.00 (4-Me), 39.51 (NMe2), 78.23 (C4), 145.45
C19H17N3O2: C, 71.46; H, 5.37; N 13.16). mmax(KBr)/cm−1 3089,
3065, 2987, 2924, 2225, 1605, 1521, 1469, 1451, 1427, 1380, 1303,
1294, 1257, 1203, 1176, 1041, 1030, 842 and 745; kmax(EtOH)/nm
269 (lg e 4.25); dH(400 MHz; CDCl3) 1.57 (6H, s, 4-Me), 5.11
(2H, s, CH2), 7.07, 8.53 (2H each, AAꢀBBꢀ, J 9 Hz, C6H4), 7.39
(5H, m, Ph); dC(100 MHz; CDCl3) 21.49 (4-Me), 69.87 (CH2),
83.04 (C4), 146.59 (C2), 149.86 (C5), 111.44 (C≡N), C6H4: 136.10
(Ci), 128.96 (Co), 114.86 (Cm), 160.83 (C–O), Ph: 119.07 (Ci),
127.95 (Cp), 127.25, 128.43 (Co, Cm).
2
5
i
=
(C ), 178.12 (C ), 143.62 (HC NO), Ar: 115.81 (C ), 128.66
(Co), 110.81 (Cm), 150.80 (C–N).
4,4-Dimethyl-4H-imidazole-5-carbaldehyde oxime 3-oxides 5b
and 5c, general procedure
Na (1 g, 41 mmol) was dissolved in isopropanol (30 mL);
after the reaction slowed down the mixture was heated to
1 2 7 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 6 9 – 1 2 7 4

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5-Dialkylamino-4H-imidazole 3-oxides 7a–e, general procedure
dropwise under vigorous stirring, the mixture was diluted with
t-BuOMe (20 mL) and MnO2 (3 g, 34.5 mmol) was added. The
mixture was stirred vigorously for 2 h, the oxidant was filtered off
and the filtrate was dried over Na2CO3. The solvent was removed
in vacuum and the nitroxides 8a–f were isolated from the residue
by column chromatography on Al2O3, eluent CHCl3. Compound
8a, yield 80%, orange crystals, mp 90–92 ◦C (hexane). Found: C,
70.16; H, 9.15; N, 16.05. Calc. for C20H31N4O: C, 69.93; H, 9.10;
N, 16.31. mmax(KBr)/cm−1 2979, 2936, 2849, 2800, 1594, 1561,
1518, 1469, 1443, 1414, 1373, 1346, 1284, 1217, 1189, 1170, 1130,
1025, 946, 923, 894, 860 and 815; kmax(EtOH)/nm 256 (lg e 4.26),
229 (4.24). Compound 8b, yield 80%, orange crystals, mp 110–
The amine (piperidine (7a or 7b) or liquid dimethylamine (7c–e),
3 mmol) was added to a solution of 6a–c (2.5 mmol) in CH2Cl2
(2 mL). The reaction mixture was allowed to stand overnight
at room temperature. The reaction mixture was diluted with
CHCl3 (50 mL), washed with saturated solution of NaCl (10 mL)
and dried over K2CO3. The solvent was removed in vacuum
and the residue was triturated with Et2O and the precipitate
was filtered off to give 7a–e. Compound 7a, yield 90%, yellow
crystals, mp 194–196 ◦C (THF–t-BuOMe). Found: C, 68.39;
H, 8.65; N, 17.82. Calc. for C18H26N4O: C, 68.76; H, 8.33; N,
17.82. mmax(KBr)/cm−1 2939, 2918, 2854, 1592, 1536, 1514, 1458,
1433, 1420, 1392, 1369, 1294, 1239, 1225, 1197, 1185, 1118,
1020, 945, 924, 897, 873, 854 and 738; kmax(EtOH)/nm 329 (lg
e 4.40), 274 (3.47), 239 (3,70); dH(200 MHz; CDCl3) 1.63 (6H,s,
4-Me), 1.69 (6H, br. m, C–CH2–C), 2.99 (6H, s, NMe2), 3.64
(4H, br. m, N–CH2), 6.69, 8.57 (2H each, AAꢀBBꢀ, J 9 Hz, Ar);
dC(50 MHz; CDCl3) 21.88 (4-Me), 39.91 (N–Me), 73.51 (C4),
147.66 (C2), 172.41 (C5), Piperidine: 23.59, 25.37 and 46.21, Ar:
115.73 (Ci), 130.16 (Co), 110.88 (Cm), 151.44 (C–N). Compound
7b, yield 90%, yellow crystals, mp 148–158 ◦C dec. (THF).
Found: C, 66.17; H, 7.52; N, 20.79. Calc. for C15H20N4O: C,
66.15; H, 7.40; N, 20.57. mmax(KBr)/cm−1 2986, 2935, 2855, 1601,
1544, 1434, 1361, 1313, 1295, 1235, 1179, 1106, 912, 772 and
703; kmax(EtOH)/nm 394 (lg e 3.76), 266 (4.29); dH(200 MHz;
CDCl3) 1.64 (6H, s, 4-Me), 1.69 (6H, br. m, C–CH2–C), 3.64
(4H, br. m, N–CH2), 8.44, 8.67 (2H each, AAꢀBBꢀ J 6 Hz, 4-Py);
dC(50 MHz; CDCl3) 21.95 (4-Me), 75.49 (C4), 145.00 (C2), 171.41
(C5), Piperidine: 23.83, 25.65 and 46.61, Py: 134.25 (Ci), 121.22
(C3), 149.84 (C2). 7c, yield 90%, yellow crystals, mp 154–156 ◦C
(THF). Found: C, 65.18; H, 8.09; N, 20.37. Calc. for C15H22N4O:
C, 65.67; H, 8.08; N, 20.42). mmax(KBr)/cm−1 2986, 2930, 2813,
1599, 1540, 1515, 1472, 1437, 1391, 1367, 1280, 1230, 1200, 1184,
1114, 945 and 833; kmax(EtOH)/nm 332 (lg e 4.46), 270 (3.77), 238
(4,01); dH(400 MHz; CDCl3) 1.63 (6H,s, 4-Me), 3.02 (6H, s, Ar–
◦
112 C (hexane). Found: C, 67.89; H, 8.50; N, 18.30. Calc. for
C17H25N4O: C, 67.74; H, 8.36; N, 18.59. mmax(KBr)/cm−1 2973,
2936, 2854, 1587, 1478, 1462, 1452, 1429, 1409, 1371, 1325, 1291,
1260, 1233, 1218, 1207, 1172, 1138, 1124, 1073, 1023, 993, 954,
893, 853 and 811; kmax(EtOH)/nm 226 (lg e 4.24). Compound
8c, yield 90%, orange crystals, mp 109–111 ◦C (hexane). Found:
C, 67.56; H, 8.83; N, 18.54. Calc. for C17H27N4O: C, 67.29; H,
8.97; N, 18.46. mmax(KBr)/cm−1 2979, 2937, 2920, 2889, 2812,
1601, 1559, 1520, 1479, 1443, 1416, 1401, 1350, 1321, 1227,
1206, 1192, 1165, 1136, 1120, 1062, 960, 941, 932, 908, 826
and 810; kmax(EtOH)/nm 256 (lg e 4.24). Compound 8d, yield
◦
70%, orange crystals, mp 94–95 C (hexane). Found: C, 64.43;
H, 7.78; N, 21.45. Calc. for C14H21N4O: C, 64.34; H, 8.10;
N, 21.44. mmax(KBr)/cm−1 2966, 2937, 2875, 1600, 1590, 1497,
1467, 1409, 1403, 1366, 1327, 1291, 1274, 1233, 1139, 1120,
1067, 993, 959, 941, 912, 835 and 819; kmax(EtOH)/nm 220
(lg e 4.19). Compound 8e, yield 70%, orange crystals, mp 91–
93 ◦C (hexane). Found: C, 71.79; H, 7.72; N, 11.37. Calc. for
C22H28N3O2: C, 72.10; H, 7.70; N, 11.47. mmax(KBr)/cm−1 2972,
2933, 2875, 1603, 1580, 1505, 1497, 1469, 1456, 1400, 1381, 1240,
1172, 1138, 1114, 1012, 827 and 759; kmax(EtOH)/nm 265 (lg e
3.25), 227 (4.37); pK = 5.7, DaN = 0.87 G.
5,5-Dimethyl-4-(dimethylamino)-2-(4-dimethylaminophenyl)-
2-pyridine-4-yl-2,5-dihydro-1H-imidazol-1-oxyl (8f)
ꢀ
ꢀ
=
NMe2), 3.14 (6H, s, N C–NMe2), 6.68, 8.58 (2H each, AA BB ,
=
J 9 Hz, Ar); dC(100 MHz; CDCl3) 21.27 (4-Me), 39.88 (N C–N–
Me), 39.88 (Ar–N–Me), 73.74 (C4), 147.25 (C2), 173.27 (C5), Ar:
115.84 (Ci), 130.07 (Co), 110.92 (Cm), 151.43 (C–N). Compound
7d, yield 90%, yellow crystals, mp 148–152 ◦C (THF). Found: C,
62.06; H, 6.96; N, 24.37. Calc. for C12H16N4O: C, 62.05; H, 6.94;
N, 24.12. mmax(KBr)/cm−1 3047, 2941, 2898, 1600, 1556, 1514,
1474, 1443, 1406, 1371, 1315, 1226, 1219, 1225, 1197, 1125,
1066, 991, 934, 873, 827, 779 and 714; kmax(EtOH)/nm 390 (lg
e 3.77), 263 (4.30); dH(200 MHz; CDCl3) 1.64 (6H, s, 4-Me),
3.17 (6H, s, NMe2), 8.43, 8.67 (2H each, AAꢀBBꢀ J 6 Hz, 4-Py);
dC(50 MHz; CDCl3) 21.34 (4-Me), 37.99 (N–Me), 75.75 (C4),
144.89 (C2), 172.43 (C5), Py: 134.22 (Ci), 121.20 (C3), 149.91
(C2). Compound 7e, yield 90%, yellow crystals, mp 240–245 ◦C
(THF). Found: C, 70.92; H, 6.87; N, 12.48. Calc. for C20H23N3O2:
C, 71.19; H, 6.87; N, 12.45. mmax(KBr)/cm−1 3063, 3003, 2978,
2932, 2885, 1598, 1535, 1502, 1474, 1415, 1391, 1368, 1301,
1279, 1248, 1190, 1170, 1115, 1014, 937, 873, 844, 772 and 746;
kmax(EtOH)/nm 361 (lg e 3.76), 283 (4.51); dH(200 MHz; CDCl3-
CD3OD) 1.81 (6H, s, 4-Me), 3.34 (6H, s, NMe2), 5.26 (2H, s,
CH2), 7.22, 8.81 (2H each, AAꢀBBꢀ, J 9 Hz, C6H4), 7.52 (5H, m,
Ph); dC(100 MHz; CDCl3) 21.07 (4-Me), 38.35 (N–Me), 70.28
(CH2), 74.52 (C4), 149.35 (C2), 174.73 (C5), C6H4: 136.81 (Ci),
131.37 (Co), 114.77 (Cm), 161.18 (C–O), Ph: 120.74 (Ci), 128.91
(Cp), 127.62, 128.72 (Co, Cm).
A solution of Grignard reagent was prepared from Mg (0.2 g,
8.3 mmol) and 4-bromo-N,N-dimethylaniline (1.5 g, 7.5 mmol)
in THF (10 mL) was added dropwise to a stirred solution of 7c
(0.58 g, 2.5 mmol) in THF (10 mL). The reaction mixture was
stirred for 0.5 h. Then water (1–3 mL) was added drop-
wise under vigorous stirring, the mixture was diluted with
t-BuOMe (20 mL) and MnO2 (3 g, 34.5 mmol) was added. The
mixture was stirred vigorously for 2 h, the oxidant was filtered
off and the filtrate was dried over Na2CO3. The solvent was
removed in vacuum and the residue was purified by column
chromatography on Al2O3, eluent CHCl3 to give nitroxide 8f,
0.5 g (66%), orange crystals, mp 171–174 ◦C (hexane–t-BuOMe
1 : 1). Found: C, 68.22; H, 7.85; N, 19.80. Calc. for C20H26N5O: C,
68.15; H, 7.44; N, 19.87. mmax(KBr)/cm−1 3075, 2981, 2936, 2892,
2809, 1592, 1560, 1519, 1490, 1447, 1407, 1366, 1279, 1233, 1179,
1163, 1127, 1067, 951, 919, 852, 829 and 807; kmax(EtOH)/nm
260 (lg e 4.38); 8f–H dH(200 MHz; D2O–N2D4 1 : 10) 1.33 (3H, s,
5-Me), 1.44 (3H, s, 5-Me), 3.22 (9H, s, NMe2), 3.34 (3H, s,
NMe2), 7.59, 7.63 (2H each, AAꢀBBꢀ, J 9 Hz, Ar), 8.11, 8.77 (2H
each, AAꢀBBꢀ J 6 Hz, 4-Py).
4-Dimethylamino-2-ethyl-2-(4-hydroxy-phenyl)-5,5-dimethyl-
2,5-dihydro-1H-imidazol-1-oxyl (9)
Palladium catalyst (Pd/C, 10% Pd, 0.2 g) was added to a solution
of 8e (1 g, 2.7 mmol) in methanol (20 mL), the air in the flask
was replaced with H2 and the suspension was vigorously stirred
at 25 ◦C until absorption of H2 finished (ca. 90 mL, 4.05 mmol,
of H2 absorbed). The catalyst was filtered off, the methanol was
removed in vacuum, the residue was dissolved in CHCl3 (20 mL),
MnO2 (1 g, 11 mmol) was added and the mixture was stirred for
1 h. The oxidant was filtered off and CHCl3 was removed under
reduced pressure to give yellow crystalline residue of 9, yield:
2,5-Dihydroimidazole-1-oxyls 8a–e, general procedure
A solution of EtMgBr (1 M) in THF was added dropwise to a
stirred solution or a suspension of 7a–e (2 mmol) in THF (10
mL). The reaction was controlled by TLC (Al2O3 Polygram Alox
N/UV 254, Macherey-Nagel, eluent CHCl3–methanol 50 : 1–
2). Usually 3–5 mL of the organometallic reagent solution was
sufficient for the reaction to be complete. The reaction mixture
was allowed to stand for 0.5 h. Then water (1–3 mL) was added
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 6 9 – 1 2 7 4
1 2 7 3

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90%, mp 85–95 ◦C (hexane–EtOAc 3 : 1). Found: C, 61.23; H,
8.37; N, 14.07. Calc. for C15H22N3O2 H2O: C, 61.20; H, 8.22; N,
14.27. mmax(KBr)/cm−1 1603, 1512, 1455, 1412, 1337, 1279, 1232,
1171, 1140, 1118 and 829; kmax(EtOH)/nm 268 (lg e 3.40), 226
(4.32).
Raghunand, S. Zhang, A. D. Sherry and R. J. Gillies, Acad. Radiol.,
2002, 9(Suppl 2), S481–3; V. V. Khramtsov, I. A. Grigor’ev, M. A.
Foster, D. J. Lurie, J. L. Zweier and P. Kuppusamy, Spectroscopy,
2004, 18(Suppl 2), 213–225; M. A. Foster, I. A. Grigor’ev, D. J. Lurie,
V. V. Khramtsov, S. McCallum, I. Panagiotelis, J. M. Hutchison, A.
Koptioug and I. Nicholson, Magn. Reson. Med., 2003, 49(3), 558–67;
C. Kroll, W. Hermann, R. Stosser, H. H. Borchert and K. Mader,
Pharm. Res., 2001, 18(4), 525–30.
3 V. V. Khramtsov, I. A. Grigor’ev, M. A. Foster, D. J. Lurie and I.
Nicholson, Cell. Mol. Biol., 2000, 46, 1361–1374.
4 V. V. Khramtsov, I. A. Grigor’ev, M. A. Foster and D. J. Lurie,
Antioxid. Redox Signaling, 2004, 6, 667–676.
5 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.
6 V. V. Khramtsov, and L. M. Weiner, in Imidazoline Nitroxides, vol. 2,
ed. L. B. Volodarsky, CRC Press, Boca Raton, 1988, p. 37–80.
7 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.
Acknowledgements
The authors thank Ekaterina Khromovskih and Nikita Skuridin
for technical assistance. This work was partly supported by grant
NIH KO1 EB 03519 and grants RFBR 04-03-32299 and 01-03-
32452.
References
1 N. F. Sheppard, A. Guiseppi-Elie, in The Measurement, Instru-
mentation and Sensors Handbook, Ch. 10: pH Measurement, ed.
J. G. Webster, CRC Press and IEEE Press, Boca Raton, Florida,
1999; G. Miesenbock, D. A. De Angelis and J. E. Rothman, Nature,
1998, 394(6689), 192–5; H. J. Lin, P. Herman and J. R. Lakowicz,
Cytometry, 2003, 52A(2), 77–89; C. C. Overly, K. D. Lee, E.
Berthiaume and P. J. Hollenbeck, Proc. Natl. Acad. Sci. U. S. A., 1995,
92(8), 3156–60; D. A. Russell, R. H. Pottier and D. P. Valenzeno,
J. Photochem. Photobiol., B, 1995, 29(1), 17–22.
2 R. J. Gillies, N. Raghunand, M. L. Garcia-Martin and R. A. Gatenby,
IEEE Eng. Med. Biol. Mag., 2004, 23(5), 57–64; P. B. Barker, E. J.
Butterworth, M. D. Boska, J. Nelson and K. M. Welch, Magn. Reson.
Med., 1999, 41(2), 400–6; J. Zhou, J. F. Payen, D. A. Wilson, R. J.
Traystman and P. C. van Zijl, Nat. Med., 2003, 9(8), 1085–90; N.
8 I. A. Kirilyuk, A. A. Bobko, I. A. Grigor’ev and V. V. Khramtsov,
Org. Biomol. Chem., 2004, 2, 1025–1030.
9 I. A. Kirilyuk, I. A. Grigor’ev and L. B. Volodarsky, Izv. Sib. Otd.
Akad. Nauk. SSSR, Ser. Khim. Nauk., 1989, 2, 99; I. A. Kirilyuk, I. A.
Grigor’ev and L. B. Volodarsky, Chem. Abs., 1990, 111, 232670.
10 L. B. Volodarskii and T. K. Sevastyanova, Zh. Org. Khim., 1971, 7(8),
1687–1692; L. B. Volodarskii and T. K. Sevastyanova, Chem. Abs.,
1971, 75(8), 140425–1692.
11 M. Balakirev, V. Khramtsov, T. Berezina, V. Martin and L. Volo-
darsky, Synthesis, 1992, (12), 1223.
1 2 7 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 2 6 9 – 1 2 7 4