Studies toward the synthesis of 4-(2-R-ethyl)amino-2,2,5,5-tetramethyl-3- imidazoline 1-oxyls. Nucleophilic substitution of bromide in the N-alkyl chain of the 1,2,4-oxadiazol-2-one precursor

Article abstract of DOI:10.1021/jo701803a

(Chemical Equation Presented) A synthetic approach to access the new nitroxides of the amidine type exhibiting pH-dependent EPR spectra through substitution of a halide in the exo-N-halogenoalkyl chain of 1-(2-bromoethyl)-6-oxyl-5,5,7,7-tetramethyltetrahy

Full text of DOI:10.1021/jo701803a

Studies toward the Synthesis of
4-(2-R-ethyl)amino-2,2,5,5-tetramethyl-3-imidazoline 1-Oxyls.
Nucleophilic Substitution of Bromide in the N-Alkyl Chain of the
1,2,4-Oxadiazol-2-one Precursor
Julya F. Polienko,† Thomas Schanding,‡ Yury V. Gatilov,† Igor A. Grigor’ev,† and
Maxim A. Voinov*,§,†
Institute of Organic Chemistry, AVe. akad. LaVrent’eVa 9, 630090, NoVosibirsk, Russia, Department of
Chemistry, North Carolina State UniVersity, Raleigh, North Carolina 27695-8204, and UniVersita¨t
Kaiserslautern, Erwin-Schro¨dinger-Strasse, D-67663 Kaiserslautern, Germany
mVoynoV@ncsu.edu
ReceiVed August 24, 2007
A synthetic approach to access the new nitroxides of the amidine type exhibiting pH-dependent EPR
spectra through substitution of a halide in the exo-N-halogenoalkyl chain of 1-(2-bromoethyl)-6-oxyl-
5,5,7,7-tetramethyltetrahydroimidazo[1,5-b][1,2,4]oxadiazol-2-one is reported. In this approach, an
oxycarbonyl moiety of the oxadiazolone heterocycle plays the role of a “protecting group” for the amidine
functionality. A nucleophilic cleavage of the oxadiazolone heterocycle under mild nonbasic conditions,
applicable to substrates bearing substituents vulnerable to attack by strong basic nucleophiles, is elaborated.
The approach allows for the new amidine nitroxides bearing various functional groups (e.g., such as CN,
N3, NH2, COOEt) to be synthesized. A series of nitroxides obtained through the Staudinger/intermolecular
aza-Wittig reaction of the azido derivative is also described. The nitroxides synthesized here were found
to have pH-dependent two-component EPR spectra indicative of a slow, on the EPR time scale, R• /
R•H+ chemical exchange, and pKa values ranging from 2.8 to 12.5 units. The guanidine derivatives
synthesized in this work show the highest pKa values (pKa ) 10.2 and 12.5, respectively) ever reported
for the nitroxide pH-probes of a “basic type”.
The ability of biological tissue to actively maintain constant
concentration of hydrogen ions is of fundamental importance
in living organisms: it plays a critical role in sustaining the
metabolic function of proteins and other macromolecules.1
Proton concentration, defined as pH, has been proven to change
significantly during myocardial ischemia,2 chronic heart failure,3
tumors,4 and inflammation.5 Therefore, a real-time pH assess-
ment is of considerable relevance for detecting pathophysi-
ological conditions. Since first reports in the early nineteen
eighties6 significant progress in EPR-based noninVasiVe methods
for pH monitoring has been made.7 The method is based on
(2) Bond, J. M.; Chacon, E.; Herman, B.; Lemasters, J. J. Am. J. Physiol.
Cell Physiol. 1993, 265, C129-C137.
(3) Scott, A. C.; Wensel, R.; Davos, C. H.; Georgiadou, P.; Kemp, M.;
Hooper, J.; Coats, A. J. S.; Piepoli, M. F. Circulation 2003, 107, 300-
306.
* Address correspondence to this author at North Carolina State University.
Phone: 1-919-515-3209. Fax: 1-919-513-7353.
† Institute of Organic Chemistry.
§ North Carolina State University.
‡ Universita¨t Kaiserslautern.
(1) Regulation of tissue pH in plants and animals: a reappraisal of
current techniques; Egginton, S.;, Taylor, E. W., Raven, J. A., Eds.;
Cambridge University Press: Cambridge, UK, 1999.
(4) Raghunand, N.; Gillies, R. J. Drug Resist. Update 2000, 3, 39-47.
(5) Issberner, U.; Reeh, P. W.; Steen, K. H. Neurosci. Lett. 1996, 208,
191-194.
10.1021/jo701803a CCC: $40.75 © 2008 American Chemical Society
Published on Web 12/18/2007
502
J. Org. Chem. 2008, 73, 502-510

Synthesis of 4-(2-R-ethyl)amino-3-imidazoline 1-Oxyls
SCHEME 1
dependence of magnetic parameters of the EPR spectra (i.e.,
isotropic nitrogen hyperfine coupling constant, aN, and g-factor)
of some stable nitroxides on the reversible protonation of
functional groups adjacent to the N-O moiety.6a,8 Owing to
recent developments of low-9 and high-frequency EPR spec-
troscopy10 and EPR-based techniques such as EPR imaging
(EPRI),11 longitudinally detected ESR (LODESR),12 and proton
electron double-resonance imaging (PEDRI),13 this method has
grown into a powerful tool with many biophysical and biomedi-
cal applications.7,9,11h,14,15 However, the potential of this method
is considered to be far beyond its current state. For the further
progress of the method, a series of new nitroxides with pH-
dependent EPR spectra and specific task-oriented properties
needs to be designed and synthesized.9c Recently, we described
an approach to synthesize new imidazoline nitroxides 1 (Scheme
1) having a protonatable N′,N′-disubstituted amidine functional-
ity.16 Various functional groups could be introduced into the
nitroxide molecule within this approach, thus allowing one to
manipulate the properties of the spin probe to a greater extent.
Over the last two decades the N′-monosubstituted 4-alkyl(aryl)-
amino-2,2,5,5-tetramethyl-3-imidazoline 1-oxyls 2 were among
the most widely used EPR pH-probes in biophysical studies
because they are easy to synthesize and have pKa values
matching the physiological range of pH.7 Amidines 2 could be
easily obtained through 1,3-dipolar cycloaddition of isocyanates
to the aldonitrone 2,2,5,5-tetramethyl-3-imidazoline 1-oxyl
followed by the alkaline cleavage of the oxadiazolone hetero-
cycle formed.17 The only obvious shortcoming of this synthesis
is that the structure of the alkyl(aryl) substituent in the exo-N-
alkyl chain in most cases is strictly predetermined by the
structure of the isocyanate used. Until now, chemical transfor-
mations did not allow for much diversity with respect to the
structure of the alkyl(aryl) group.17 We believe that introducing
functional groups into the exo-N-alkyl chain of 4-alkylamino-
2,2,5,5-tetramethyl-3-imidazoline 1-oxyls would provide ad-
ditional ways to tune the properties of pH spin probes. We
consider the substitution of halide in 4-(2-halogenoethyl)-amino-
2,2,5,5-tetramethyl-3-imidazoline 1-oxyl with nucleophiles to
be the most attractive way to modify the exo-N-alkyl chain.
Unfortunately, all the previous attempts to realize this approach
have failed. Specifically, under conditions of the nucleophilic
substitution of the chloride in 4-(2-chloroethyl)-amino-2,2,5,5-
tetramethyl-3-imidazoline 1-oxyl 3 with NaI, NaN3, NaOAc,
and potassium phthalimide the only product isolated was the
bicyclic amidine 2,3,4,5,6,7-hexahydro-6-oxyl-5,5,7,7-tetram-
ethyl-7H-imidazo[1,5-a]imidazole 4 (Scheme 1).17c This product
obviously results from the intramolecular nucleophilic attack
of the lone electron pair of the endo-cyclic nitrogen atom of
the amidine group on the exo-N-chloroethyl fragment.
Here we report the synthetic approach to substitute a halide
in the exo-N-halogenoalkyl chain in a way to overcome the
undesired reaction of intramolecular alkylation. We also report
on properties and chemical transformations of the resulting
compounds that allowed us to access new nitroxides exhibiting
pH-dependent EPR spectra. The key step in the synthesis is the
nucleophilic substitution of the bromide in the cycloadduct 1-(2-
bromoethyl)-6-oxyl-5,5,7,7-tetramethyltetrahydroimidazo[1,5-
b][1,2,4]oxadiazol-2-one 5. In our approach an oxycarbonyl
moiety of the oxadiazolone heterocycle plays the role of a
“protecting group” for the amidine functionality.
(6) (a) Khramtsov, V. V.; Weiner, L. M.; Grigor’ev, I. A.; Volodarsky,
L. B. Chem. Phys. Lett. 1982, 91 (1), 69-72. (b) Khramtsov, V. V.; Weiner,
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Ohno, K., Eds.; CRC Press: Boca Raton, FL, 1991; pp 65-72. (c) Foster,
M. A.; Grigor’ev, I. A.; Lurie, D. J.; Khramtsov, V. V.; McCallum, S.;
Panagiotelis, I.; Hutchison, J. M.S.; Koptioug, A.; Nicholson, I. Magn.
Reson. Med. 2003, 49, 558-567. (d) Khramtsov, V. V.; Grigor’ev, I. A.;
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2004, 18, 213-225. (e) Potapenko, D. I.; Foster, M. A.; Lurie, D. J.;
Kirilyuk, I. A.; Hutchison, J. M. S.; Grigor’ev, I. A.; Bagryanskaya, E. G.;
Khramtsov, V. V. J. Magn. Reson. 2006, 182 (1), 1-11.
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Publishers: New York, 2004; Vol. 22.
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Lurie, D. J. Br. J. Radiol. 2001, 74, 782-784. (h) Khramtsov, V. V.;
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Swartz, H. Magn. Reson. Med. 1996, 36, 694-697.
Results and Discussion
The oxadiazolone derivative 5a was obtained in a good yield
through 1,3-dipolar cycloaddition of the commercially available
(15) (a) Smirnov, A. I.; Ruuge, A.; Reznikov, V. A.; Voinov, M. A.;
Grigor’ev, I. A. J. Am. Chem. Soc. 2004, 126, 8872-8873. (b) Mo¨bius,
K.; Savitsky, A.; Wegener, C.; Plato, M.; Fuchs, M.; Schnegg, A.; Dubinskii,
A. A.; Grishin, Y. A.; Grigor’ev, I. A.; Ku¨hn, M.; Duche´, D.; Zimmermann,
H.; Steinhoff, H.-J. Magn. Reson. Chem. 2005, 43, S4-S19.
(16) Voinov, M. A.; Polienko, J. F.; Schanding, T.; Bobko, A. A.;
Khramtsov, V. V.; Gatilov, Y. V.; Rybalova, T. V.; Smirnov, A. I.;
Grigor’ev, I. A. J. Org. Chem. 2005, 70, 9702-9711.
(17) (a) Berezina, T. A.; Martin, V. V.; Volodarsky, L. B.; Khramtsov,
V. V.; Weiner, L. M. Bioorg. Khim. 1990, 16, 262-269. (b) Balakirev,
M.; Khramtsov, V. V.; Berezina, T. A.; Martin, V. V.; Volodarsky, L. B.
Synthesis 1992, 12, 1223-1225. (c) Berezina, T. A.; Reznikov, V. A.;
Volodarsky, L. B. Tetrahedron 1993, 49 (46), 10693-10704.
J. Org. Chem, Vol. 73, No. 2, 2008 503

Polienko et al.
SCHEME 2
110 °C the only product of the reaction was the amidine 10.
Compound 10 is identical with that obtained in the customary
manner by hydrolytic cleavage of the oxadiazolone 8 under
alkaline conditions (CH3ONa/CH3OH or NaOH/CH3OH). In all
cases a 2-fold molar excess of NaN3 was used. No product 4
resulting from the intramolecular alkylation was detected in the
reaction mixture. This observation provides evidence that the
nucleophilic substitution rate prevails over the oxadiazolone
heterocycle cleavage rate. For convenience, the experimental
conditions are summarized in Table S1 (Supporting Informa-
tion).
SCHEME 3
In principle, the cleavage of the oxadiazolone heterocycle
might occur under both thermolysis conditions18 and as a result
of attack by the nucleophile on the carbonyl carbon atom,17c,19
though the arguments in favor of nucleophilic pathway presented
seem to be quite questionable.
When compound 8 and reference compound 5b (Scheme 2)
were heated in DMF or DMSO at 110 °C without a nucleophile
present, amidines 10 and 2 (R ) Ph),17c respectively, were
obtained in good yields.
As we found, the reaction of 5a with 1 equiv of NaN3 in
aqueous DMSO at 55 °C affords in 4 h the azido cycloadduct
8 as the sole product. However, when 2 equiv of NaN3 was
applied at the same conditions, the mixture of 8 and 10 in 1:1.5
molar ratio was obtained in 4 h (reaction was interrupted at
this point). For more discussion and for a complete set of
conditions used for the oxadiazolone heterocycle cleavage, see
the Supporting Information.
SCHEME 4
We believe that the attack of a nucleophile on the carbonyl
carbon atom of the oxadiazolone heterocycle is the main reaction
pathway under mild conditions, when reaction temperature does
not exceed 50-55 °C. At more harsh conditions (100-110 °C)
the thermolytic pathway of the heterocycle cleavage most likely
operates along with the nucleophilic cleavage. At elevated
temperatures and in the absence of the nucleophile the ther-
molytic pathway seems to become a dominating one. Plausible
schemes for 1,2,4-oxadiazol-2-one conversion into an amidine
under both nucleophilic and thermolytic conditions are shown
in Schemes 5 and 6, respectively.
Thermal fragmentation of the oxadiazolone heterocycle is
presumably triggered by heterolysis of the weak N-O bond
(Scheme 6, Path A). In Path B, the transition state of the
fragmentation would take advantage of the stabilization provided
by the aromatic transition state. Both pathways seem to be
thermodynamically highly favorable, because they produce
stable CO2 and a compound with a CdN double bond.
The nucleophilic cleavage of the oxadiazolone heterocycle
under mild conditions, which we have discovered, would be
quite applicable to substrates bearing substituents vulnerable
to attack by strong basic nucleophiles like CH3ONa or NaOH.
To examine this hypothesis, the ester cycloadduct 5c was reacted
with 1 equiv of NaN3 in DMSO containing 10% of H2O at
55 °C. As a result, the ester amidine 11 was obtained in 74%
yield (Scheme 7; see the Supporting Information for more
details). The same reaction with KBr was interrupted after 75
h; the ester amidine 11 was isolated in 38% yield, and 27% of
2-bromoethyl isocyanate to the aldonitrone 2,2,5,5-tetramethyl-
3-imidazoline 1-oxyl 6 (Scheme 2). The bromoethyl derivative
5a was chosen as a starting compound in the synthesis for the
reason that bromide is a better leaving group than chloride.
Thus, under the phase-transfer catalysis conditions (CH2Cl2/
H2O/Bu4NBr, reflux) the nucleophilic substitution of the
bromide in the cycloadduct 5a with the cyanide ion gave 1-(2-
cyanoethyl)-6-oxyl-5,5,7,7-tetramethyltetrahydroimidazo[1,5-b]-
[1,2,4]oxadiazol-2-one 7 in 85% yield (Scheme 3). Under the
same conditions the substitution of the bromide with azide ion
afforded 1-(2-azidoethyl)-6-oxyl-5,5,7,7-tetramethyltetrahy-
droimidazo[1,5-b][1,2,4]oxadiazol-2-one 8 in 78% yield (Scheme
3).
We found that the composition of the reaction products in
substitution of the bromide with the azide ion varies depending
on conditions used. Thus, the substitution under the phase-
transfer conditions specified above gave solely azidoethyl
derivative 8, while using the high-boiling benzene instead of
dichloromethane (C6H6/H2O/Bu4NBr, reflux) resulted in the
formation of the mixture of products 8 and 10 in approximately
4:1 molar ratio (Scheme 4). When aqueous DMF or DMSO
(containing 10% of H2O) was used as a solvent for the
nucleophilic substitution and the temperature was increased to
(18) (a) Black, D. St. C.; Crozier, R. F.; Davis, V. C. Synthesis 1975, 4,
205-221. (b) Seidl, H.; Huisgen, R.; Grashey, R. Chem. Ber. 1969, 102,
926-930.
(19) (a) Goerdeler, J.; Schimpf, R. Chem. Ber. 1973, 106, 1496-1500.
(b) Wilkerson, C. J.; Green, F. D. J. Org. Chem. 1975, 40, 3112-3118. (c)
Hisano, T.; Yoshikawa, S.; Muraoka, K. Chem. Pharm. Bull. 1974, 22,
1611-1616.
504 J. Org. Chem., Vol. 73, No. 2, 2008

Synthesis of 4-(2-R-ethyl)amino-3-imidazoline 1-Oxyls
SCHEME 5
SCHEME 6
SCHEME 7
unreacted cycloadduct 5c was also recovered. This observation
explains, at least, why no detectable amount of azide amidine
10 was formed during the bromide substitution in 5a with NaN3
at 55 °C: the bromide anion released in the course of the
reaction seems to provide, in this condition, very little assistance
in the oxadiazolone heterocycle cleavage. To have the hetero-
cycle cleaved, one more equivalent of more reactive NaN3 must
be added. As we found, the outcome of the thermolysis is not
affected by the presence of water in DMF. It is likely that the
nucleophilicity of water is too low to affect this reaction
noticeably (see the Experimental Section and Table S2, Sup-
porting Information, for further details).
The Staudinger procedure seems to be the most effective way
to modify compounds containing an azido group thus far
developed. Typically, this reaction proceeds under mild condi-
tions, almost quantitatively, without noticeable formation of any
side products.20 The iminophosphorane formed is a rather
reactive intermediate and can be rapidly hydrolyzed to the
primary amine21 or reacted with almost any kind of electrophilic
reagent.20,21b,c Carbodiimide22 and isothiocyanate23,24 derivatives
are of general use for modification of proteins and chemically
modified nucleic acids. In this work we used the Staudinger/
intermolecular aza-Wittig reaction sequence to approach the
carbodiimido and isothiocyanato derivatives starting from
amidine nitroxide 10; such derivatives would be valuable pH-
sensitive spin labels for biophysical applications.
When the ether solution of azido derivative 10 was treated
with P(n-Bu)3 under argon, a vigorous nitrogen evolution was
observed. The reaction was over within 40 min (as determined
by TLC). PPh3 and PEt3 were also used in this reaction. For
PPh3, refluxing in THF was needed. Because of the high
reactivity, the intermediate iminophosphorane 12 was not
isolated as an individual compound and characterized. Further
reaction of 12a,b with carbon disulfide afforded a crystalline
solid. While elemental analysis of this solid was consistent with
isothiocyanato derivative 13 (Scheme 8), the IR spectrum did
(20) (a) Staudinger, H.; Meyer, J. HelV. Chim. Acta 1919, 2, 635-646.
(b) Staudinger, H.; Hauser, E. HelV. Chim. Acta 1921, 4, 861-886. (c)
Saxon, E.; Bertozzi, C. R. Science 2000, 287 (5460), 2007-2010. (d) Ko¨hn,
M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004, 43, 3106-3116.
(21) (a) Wallace, K. J.; Hanes, R.; Anslyn, E.; Morey, J.; Kilway, K.
V.; Siegel, J. Synthesis 2005, 12, 2080-2083. (b) Gololobov, Y. G.;
Zhmurova, I. N.; Kasukhin, L. F. Tetrahedron 1981, 37, 437-472. (c)
Gololobov, Y. G.; Kazukhin, L. F. Tetrahedron 1992, 48 (8), 1353-1406.
(22) (a) Beechey, R. B.; Roberton, A. M.; Holloway, C. T.; Knight, I.
G. Biochemistry 1967, 6, 3867-3879. (b) Musser, S. M.; Larsen, R. W.;
Chan, S. I. Biophys. J. 1993, 65, 2348-2359. (c) Hassinen, I. E.; Vuokila,
P. T. Biochim. Biophys. Acta 1993, 1144 (2), 107-124. (d) Kumarev, V.
P.; Knorre, D. G. Dokl. Akad. Nauk S.S.S.R. 1970, 193 (1), 103-105. (e)
Azzi, A.; Bragadin, M. A.; Tamburro, A. M.; Santato, M. J. Biol. Chem.
1973, 248, 5520-5526. (f) Girvin, M. E.; Fillingame, R. H. Biochemistry
1995, 34, 1635-1645.
(23) (a) Tonomura, Y.; Watanabe, S.; Morales, M. Biochemistry 1969,
8, 2171-2176. (b) Coock, R.; Morales, M. Biochemistry 1969, 8, 3188-
3194. (c) Schmidt, P. G.; Bertanowicz, M. S.; Rich, D. H. Biochemistry
1973, 21, 1830-1835. (d) Maeda, D. Y.; Berman, F.; Murray, T. F.; Aldrich,
J. V. J. Med. Chem. 2000, 43, 5044-5049.
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4535-4532.
not show absorptions in the region ν 2040-2220 cm-1
characteristic of the stretching vibrations of the heterocumulene
-NdCdS group.
,
The crystal structure of the product was resolved by a single-
crystal X-ray diffraction analysis (Figure 1) that allowed us to
describe it as 2′,2′,5′,5′-tetramethyl-1′-oxyl-4,5,2′,5′-tetrahydro-
3H,1′H-[1,4′]biimidazolyl-2-thione 14sthe product of intramo-
lecular cyclization of the isothiocyanato group on the exo-cyclic
nitrogen atom of the amidino group (Scheme 8).
Reaction of the iminophosphorane 12b,c with cyclohexyl
isothiocyanate performed in a similar manner yielded a yellow
crystalline solid with elemental analysis consistent with the
structure of carbodiimido derivative 15 (Scheme 8). No absorp-
tions near ν 2130 cm-1 characteristic of the -NdCdN- group
were observed in the IR spectrum of the compound, but rather
intense bands characteristic of CdN stretching vibration were
revealed at ν 1596 and 1661 cm-1. Similar to the reaction with
carbon disulfide, we assigned the resulting compound the
structure of guanidine derivative cyclohexyl(2′,2′,5′,5′-tetram-
(25) (a) Baldwin, J. E. J. Chem. Soc., Chem. Commun. 1976, 18, 734-
736. (b) Baldwin, J. E.; Cutting, J.; DuPont, W.; Kruse, L.; Silberman, L.;
Thomas, R. C. J. Chem. Soc., Chem. Commun. 1976, 18, 736-738.
(26) Voinov, M. A.; Martin, V. V.; Volodarskii, L. B. Russ. Chem. Bull.
1992, 41 (11), 2091-2095.
J. Org. Chem, Vol. 73, No. 2, 2008 505

Polienko et al.
SCHEME 8
SCHEME 9
SCHEME 10
FIGURE 1. ORTEP presentation of 14. Thermal ellipsoids are drawn
at the 50% probability level. Selected bond distances (Å) and angles
(deg): N(3)-C(4) 1.2789(19), C(4)-N(10) 1.3894(18), S(1)-C(11)
1.6606(16), N(10)-C(11) 1.3861(19), C(11)-N(12) 1.331(2), N(3)-
C(4)-N(10)-C(14) 171.44(15), C(4)-N(10)-C(11)-S(1) -3.2(3).
capacity. From the prospects of molecular recognition chemistry,
the guanidine 16 and corresponding guanidinium derivatives
17 and 18 could be of interest as nitroxide receptors for EPR
protein and DNA/RNA studies.
The iminophosphorane 12b was readily hydrolyzed by
aqueous ethanol to give amino derivative 19 in 75% yield
(Scheme 10). Reductive alkylation of 19 by an Eschweiler-
Clarke reaction afforded dimethylamino derivative 20 in a good
yield. A reduced reactivity of the amidino group in 20 toward
alkylating reagents16,26 enabled us to selectively alkylate the
amino group with dimethyl sulfate and afforded a quaternary
ammonium salt 21 in 61% yield (Scheme 10).
The X-band (9.5 GHz) EPR spectra of the amidine nitroxides
synthesized were studied as a function of pH. All new nitroxides,
with the exception of guanidinium salt 18, were found to show
a reversible effect of pH on EPR spectra. Irreversible character
of the titration curve for 18 is likely to arise from hydrolysis of
the guanidine group in a strong alkaline medium.31 Figure 3
shows a series of EPR spectra of the amino derivative 19 in
aqueous buffers with pH ranging from 1.52 to 6.37 units. The
FIGURE 2. ORTEP presentation of 17. Thermal ellipsoids are drawn
at the 50% probability level. Selected bond distances (Å) and angles
(deg): N(3)-C(4) 1.286(4), C(4)-N(10) 1.367(4), N(10)-C(11) 1.388-
(4), C(11)-N(16) 1.303(4), N(16)-C(17) 1.475(4), N(3)-C(4)-
N(10)-C(11) -5.6(11), C(4)-N(10)-C(11)-N(16) 1.8(10).
ethyl-1′-oxyl-4,5,2′,5′-tetrahydro-3H,1′H-[1,4′]biimidazolyl-2-
ylidene)amine 16. The assignment was confirmed by X-ray
diffraction analysis (Figure 2). For this purpose, the more readily
crystallizable N-methyl guanidinium methyl sulfate 17 was
prepared (Scheme 9). Further alkylation of 17 allowed us to
obtain the guanidinium salt 18. The regiochemical assignment
of the position of a new methyl group in 18 was made by
analogy to the literature data.27,28
To the author’s best knowledge, the nitroxides bearing
guanidine moiety have never been reported before. The literature
data indicate that the guanidinium group is the most popular
binding site motif in nature and in synthetic receptors for
oxoanions29 and peptides30 due to its exceptional binding
(29) (a) Mehrota, M. M.; Sternbach, D. D.; Rodriguez, M.; Charifson,
P.; Berman, J. Bioorg. Med. Chem. Lett. 1996, 6 (16), 1941-1946. (b)
Schug, K. A.; Lindner, W. Chem. ReV. 2005, 105, 67-113. (c) Orner, P.;
Hamilton, A. D. J. Inclusion Phenom. Macrocyclic Chem. 2001, 41, 141-
147. (d) Houk, R. J. T.; Tobey, S. L.; Anslyn, E. V. Top. Curr. Chem.
2005, 255, 199-229. (e) Fitzmaurice, R. J.; Kyne, G. M.; Douheret, D.;
Kilburn, J. D. J. Chem. Soc., Perkin Trans. 1 2002, 841-864.
(30) (a) Salvatella, X.; Peczuh, M. W.; Gairi, M.; Jain, R. K.; Sanchez-
Quesada, J.; Mendoza, J.; Hamilton, A. D.; Giralt, E. Chem. Commun. 2000,
15, 1399-1400. (b) Sanchez-Quesada, J.; Seel, C.; Prados, P.; Mendoza,
J. J. Am. Chem. Soc. 1996, 118, 277-278. (c) Peczuh, M. W.; Hamilton,
A. D.; Sanchez-Quesada, J.; Mendoza, J.; Haack, T.; Giralt, E. J. Am. Chem.
Soc. 1997, 119, 9327-9328. (d) Haack, M. W.; Peczuh, X.; Salvatella, J.;
Sanchez-Quesada, J.; Mendoza, J.; Hamilton, A. D.; Giralt, E. J. Am. Chem.
Soc. 1999, 121, 11813-11820.
(27) Xie, H.; Zhang, S.; Duan, H. Tetrahedron Lett. 2004, 45, 2013-
2015.
(28) Gao,Y.; Arritt, S. W.; Twamley, B.; Shreeve, J. M. Inorg. Chem.
2005, 44, 1704-1712.
(31) (a) Bell, J. J. Chem. Soc. 1926, 1213-1219. (b) Warner, R. C. J.
Biol. Chem. 1942, 142, 705-723. (c) Eloranta, J. Suomen Kem. 1961, 34,
107-110. (d) Homer, R. B.; Alwis, K. W. J. Chem. Soc., Perkin II 1976,
7, 781-784.
506 J. Org. Chem., Vol. 73, No. 2, 2008

Synthesis of 4-(2-R-ethyl)amino-3-imidazoline 1-Oxyls
TABLE 1. Magnetic Parameters (aNR•H+ and aNR•), ∆aN, and pKa
Values for Nitroxides 9-11, 14, 16, 17, and 19-21
aN, G
no.
R•H+
R•
∆aN, G
pKa
9
14.87 ( 0.03
14.85 ( 0.01
14.97 ( 0.01
14.81 ( 0.05
15.45 ( 0.01
15.44 ( 0.06
14.80 ( 0.01
14.83 ( 0.01
14.69 ( 0.01
15.73 ( 0.02
15.74 ( 0.01
15.87 ( 0.01
15.53 ( 0.03
15.60 ( 0.01
15.61 ( 0.05
15.61 ( 0.02
15.70 ( 0.02
15.63 ( 0.02
0.86 ( 0.036
0.89 ( 0.014
0.90 ( 0.014
0.72 ( 0.058
0.15 ( 0.014
0.17 ( 0.078
0.81 ( 0.022
0.87 ( 0.022
0.94 ( 0.022
5.06 ( 0.02
5.47 ( 0.02
4.76 ( 0.03
2.82 ( 0.05
10.2 ( 0.10
12.5 ( 0.10
4.11 ( 0.02
3.73 ( 0.02
3.80 ( 0.02
10
11
14
16
17
19
20
21
FIGURE 3. Representative room temperature 9.5 GHz (X-band) EPR
spectra of the amino nitroxide 19 taken in 1.5 mM buffer solutions at
various pH values (concentration of nitroxide ca. 0.5 mM).
SCHEME 11
FIGURE 4. Experimentally measured splitting between low-field and
central nitrogen hyperfine components of the nitroxide 19 as a function
of pH. Spectra were taken at room temperature in 1.5 mM buffer
solutions. The corresponding least-squares Henderson-Hasselbalch
titration curve is shown as a solid line.
two-component EPR spectra shown in Figure 3 are indicative
of a slow, on the EPR time scale, R• / R•H+ chemical
exchange6b and were observed for all nitroxides studied in this
work. Nitroxides 9-11 and 19-21 showed spectra with a
partially resolved high-field nitrogen hyperfine component
similar to that shown in Figure 3.
not satisfy the condition of slow exchange (3 < pKa < 11, ref
6b). Thus, the width of the high-field hyperfine component of
17 is most likely affected by both above-mentioned effects.
Though the EPR spectra of nitroxides described in this work
do not satisfy fast exchange conditions at X-band (9.5 GHz),
the experimentally observed splitting between the low-field and
the central nitrogen hyperfine components still could be taken
as an approximation of the weighted average of the isotropic
nitrogen hyperfine coupling constants.7,16 Figure 4 shows
experimentally measured splitting between these two compo-
nents of the first-derivative experimental EPR spectra, aN, for
the nitroxide 19, calibrated as a function of pH. Magnetic
parameters of the amidine nitroxides synthesized in this work
(aNR•H+, aNR•, and ∆aN) and corresponding observed pKa
values are summarized in Table 1.
It is worthwhile to note here that guanidine derivatives 16
and 17 synthesized in this work show the highest pKa values
(pKa ) 10.2 and 12.5, respectively) ever reported for nitroxide
probes of a “basic type” (cf. ref 7). Recently, lipophilic pH-
sensitive nitroxide probes of an “acidic type” with pKa values
in the range of 9-12 pH units have been described.32
For nitroxides 14, 16, and 17 even at pH close to pKa where
one would expect the existence of both protonated and non-
protonated nitroxide species, only broadening of the high-field
nitrogen hyperfine component was observed. For 14 (pKa )
2.82), this broadening is apparently attributed to the rate of
proton exchange being in an intermediate regime on the EPR
time scale (cf. refs 6b and 7). Line broadening of the high-field
component for nitroxides 16 and 17 is due to a small difference
between hyperfine splitting of protonated (aNR•H+) and non-
protonated (aNR•) forms of the nitroxide. For these nitroxides
the difference between aNR•H+ and aNR• was found to be only
∆aN ≈ 0.15 G, which is about one-sixth of that for nitroxides
9-11 and 19-21 (∆aN ≈ 0.9 G, see Table 1 and refs 7 and
16). The most likely reason for such a small ∆aN is that for
protonated forms of nitroxides 16 and 17 the positive charge is
delocalized over the guanidinium moiety of the molecule. This
effect noticeably diminishes the effective charge at the N-3 atom
of the imidazoline heterocycle. As a result, the nitrogen
hyperfine coupling constant of the nitroxide is only slightly
affected by the protonation. To illustrate this relationship it
would be appropriate to mention here that ∆aN gradually
decreases in the order 22 > 2 > 17 (Scheme 11).7 Moreover,
for nitroxide 17 (pKa ) 12.5), the rate of proton exchange does
Conclusion
We have synthesized a series of new nitroxides using the
approach based on the nucleophilic substitution of bromide in
(32) Reznikov, V. A.; Skuridin, N. G.; Khromovskikh, E. L.; Khramtsov,
V. V. Russ. Chem. Bull., Int. Ed. 2003, 52 (9), 2052-2056.
J. Org. Chem, Vol. 73, No. 2, 2008 507

Polienko et al.
was heated for 1 h in an oil bath at temperatures not exceeding 55
°C. After the reaction was completed, the mixture was diluted with
H2O and extracted with CHCl3 (3 × 5 mL). Combined organic
extracts were washed with brine (8 × 5 mL) and dried over MgSO4.
The solvent was removed under reduced pressure, and the residue
was separated by column chromatography (SiO2, CH2Cl2/EtOAc,
5:1). Yield: 0.14 g (0.56 mmol, 85%).
the exo-N-bromoalkyl chain of the cycloadduct, 1-(2-bromoet-
hyl)-6-oxyl-5,5,7,7-tetramethyltetrahydroimidazo[1,5-b][1,2,4]-
oxadiazol-2-one. In this approach an oxycarbonyl moiety of the
oxadiazolone heterocycle plays the role of “protecting group”
for the amidine functionality. The approach we report here
allows one to overcome the undesired intramolecular alkylation
of the amidino group with N-halogenoalkyl moiety that the
researchers have encountered earlier. A nucleophilic cleavage
of the oxadiazolone heterocycle under mild nonbasic conditions
is elaborated and its applicability to substrates bearing substit-
uents vulnerable to attack by strong basic nucleophiles (such
as CH3ONa or NaOH) is demonstrated. Within this approach,
new nitroxides bearing various functional groups (e.g., CN, N3,
NH2, and COOEt) were synthesized. A series of nitroxides
obtained through Staudinger/intermolecular aza-Wittig reaction
of the azido derivative is also described. The nitroxides
synthesized here were shown to have pH-dependent two-
component EPR spectra indicative of a slow, on the EPR time
scale, R• / R•H+ chemical exchange, and pKa values ranging
from 2.8 to 12.5 units. The guanidine nitroxides synthesized in
this work show the highest pKa values (pKa ) 10.2 and 12.5,
respectively) ever reported for nitroxide pH-probes of a “basic
type”. The synthetic approach to access nitroxides with pH-
dependent EPR spectra described here would facilitate further
progress in the application of the noninvasive EPR-based method
for pH monitoring in biophysical and biomedical studies.
1-(2-Azidoethyl)-6-oxyl-5,5,7,7-tetramethyl-tetrahydroimidazo-
[1,5-b][1,2,4]oxadiazol-2-one 8. Method A: A solution of NaN3
(0.13 g, 2 mmol) in H2O (3 mL) and Bu4NBr (0.05 g, 0.15 mmol)
were added to the solution of 5a (0.31 g, 1 mmol) in CH2Cl2 (5
mL). The resulting mixture was refluxed under vigorous stirring
for 4 h. The progress of the reaction was monitored by chroma-
tography on TLC plates (SiO2, CH2Cl2/EtOAc, 9:1). After the
reaction was completed, the organic layer was separated, and the
water phase was extracted with CH2Cl2 (2 × 5 mL). Organic
extracts were combined and dried over MgSO4, the solvent was
removed under reduced pressure, and the residue was separated by
column chromatography (SiO2, CH2Cl2/EtOAc, 5:1). The product
solidified after evaporation of solvent (0.21 g, 0.78 mmol, 78%).
Method B: NaN3 (0.21 g, 3.2 mmol) was added to the solution
of 5a (1.01 g, 3.3 mmol) in the mixture of DMSO or DMF (20
mL) and H2O (2 mL). The resulting mixture was heated in an oil
bath at temperatures not exceeding 55 °C. The progress of the
reaction was monitored by chromatography on TLC plates (SiO2,
hexane/THF, 10:1). After the reaction was over the mixture was
diluted with H2O and extracted with CHCl3 (5 × 5 mL). Combined
organic extract was washed with brine (8 × 5 mL) and dried over
MgSO4, the solvent was removed under reduced pressure, and the
residue was separated by column chromatography (SiO2, CH2Cl2/
EtOAc, 5:1). The product solidified after evaporation of solvent to
give 8 as yellow crystals (DMSO: 0.67 g, 2.5 mmol, 76%; DMF:
0.71 g, 2.6 mmol, 79%): mp 93-95 °C dec (hexane/EtOAc, 5:1);
IR (KBr, cm-1) 2100 (N3), 1757 (CdO). Anal. Calcd for
C10H17N6O3: C, 44.61; H, 6.36; N, 31.20. Found: C, 44.63; H,
6.38; N, 30.67.
Method C: NaN3 (0.86 g, 13.2 mmol) was added to the solution
of 5a (2.02 g, 6.6 mmol) in a mixture of DMSO (20 mL) and H2O
(2 mL). The resulting mixture was heated in an oil bath at the
temperature range of 50-56 °C. The progress of reaction was
monitored by chromatography on TLC plates (SiO2, hexane/THF,
10:1) and interrupted after 4 h. A workup procedure was performed
similar to that described in Method A. Yield of 8: 0.5 g, 1.86 mmol,
28%. Yield of 10: 0.65 g, 2.89 mmol, 44%.
Method D: A solution of NaN3 (0.21 g, 3.25 mmol) in H2O
(1.5 mL) was added to a mixture of benzene (5 mL), 5a (0.5 g,
1.63 mmol), and Bu4NBr (0.052 g, 0,16 mmol). The resulting
mixture was refluxed for 4 h. A workup procedure was performed
similar to that described in Method A. Yield of 8: 0.17 g, 0.63
mmol, 39%. Yield of 10: 0.04 g, 0.18 mmol, 11%.
4-[(2-Cyanoethyl)amino]-1-oxyl-2,2,5,5-tetramethyl-2,5-dihy-
dro-1H-imidazole 9. NaCN (0.02 g, 0.4 mmol) was added to a
solution of 7 (0.1 g, 0.4 mmol) in DMSO (5 mL). The resulting
mixture was heated for 8 h in an oil bath at temperatures not
exceeding 55 °C. The reaction mixture was diluted with H2O and
extracted with CHCl3 (3 × 5 mL). Combined organic extracts were
washed with brine (8 × 5 mL), an organic layer was dried over
Na2SO4, and the solvent was removed under reduced pressure. The
residue solidified after evaporation of solvent to give 9 as canary-
yellow crystals (0.036 g, 0.17 mmol, 44%): mp 209-211 °C dec
(hexane/EtOAc, 2:5); IR (KBr, cm-1) 3352, 1547 (N-H), 2251
(CN), 1626 (CdN). Anal. Calcd for C10H17N4O: C, 57.42; H, 8.13;
N, 26.79. Found: C, 57.56; H, 8.21; N, 26.70.
4-[(2-Azidoethyl)amino]-1-oxyl-2,2,5,5-tetramethyl-2,5-dihy-
dro-1H-imidazole 10. Method A: A solution of NaN3 (0.42 g,
6.6 mmol) in H2O (2 mL) was added to a solution of 5a (1.01 g,
3.3 mmol) in DMF or DMSO (20 mL). The resulting mixture was
heated in an oil bath at 110 °C for 5 h. The progress of the reaction
was monitored by chromatography on TLC plates (SiO2, CHCl3/
Experimental Section
Synthesis of 1-(2-R)-6-oxyl-5,5,7,7-tetramethyltetrahydroimi-
dazo[1,5-b][1,2,4]oxadiazol-2-one 5 (General Procedure). Com-
mercially available isocyanate (15 mmol) was added to the solution
of the aldonitrone 6 (10 mmol) in dry CH2Cl2 (10 mL). The reaction
mixture was allowed to stay at room temperature for 1-5 days.
The progress of reaction was monitored by chromatography on TLC
plates (SiO2, CHCl3/MeOH, 100:3). After the reaction was com-
pleted the solvent was removed under reduced pressure, and the
residue was separated by column chromatography (SiO2, CH2Cl2/
EtOAc, 5:1). 1-(2-Bromoethyl)-6-oxyl-5,5,7,7-tetramethyltetrahy-
droimidazo[1,5-b][1,2,4]oxadiazol-2-one 5a was obtained as
orange crystals (3.08 g, 9.98 mmol, 99%): mp 111-112 °C dec
(EtOAc/hexane, 1:3); IR (KBr, cm-1) 1744 (CdO). Anal. Calcd
for C10H17BrN3O3: C, 39.10; H, 5.58; N, 13.67. Found: C, 38.99;
H, 5.50; N, 13.40. 1-(2-Ethoxy-2-oxoethyl)-6-oxyl-5,5,7,7-tetram-
ethyltetrahydroimidazo[1,5-b][1,2,4]oxadiazol-2-one 5c was ob-
tained as orange crystals (2.43 g, 8.50 mmol, 85%): mp 91-92
°C dec (EtOAc/hexane, 1:2); IR (KBr, cm-1) 1771 (CdO), 1749
(COOEt). Anal. Calcd for C12H20N3O5: C, 50.35; H, 6.99; N, 14.69.
Found: C, 50.61; H, 7.15; N, 14.61.
1-(2-Cyanoethyl)-6-oxyl-5,5,7,7-tetramethyltetrahydroimidazo-
[1,5-b][1,2,4]oxadiazol-2-one 7. Method A: A solution of NaCN
(0.2 g, 4.1 mmol) in H2O (3 mL) and Bu4NBr (0.05 g, 0.15 mmol)
were added to the solution of 5a (0.5 g, 1.63 mmol) in CH2Cl2 (15
mL). The resulting mixture was refluxed under vigorous stirring
for 15 h. The progress of reaction was monitored by chromatog-
raphy on TLC plates (SiO2, CH2Cl2/EtOAc, 5:1). After the reaction
was completed, the organic layer was separated, and the water phase
was extracted with CH2Cl2 (2 × 5 mL). Combined organic extracts
were dried over MgSO4, the solvent was removed under reduced
pressure, and the residue was separated by column chromatography
(SiO2, CH2Cl2/EtOAc, 5:1). The product obtained after evaporation
of solvent solidified to give 7 as yellow crystals (0.35 g, 1.38 mmol,
85%): mp 161-162 °C dec (EtOAc/hexane, 3:2); IR (KBr, cm-1
)
2251 (CN), 1758 (CdO). Anal. Calcd for C11H17N4O3: C, 52.17;
H, 6.77; N, 22.11. Found: C, 52.42; H, 6.60; N, 21.94.
Method B: NaCN (0.03 g, 0.65 mmol) was added to the solution
of 5a (0.2 g, 0.65 mmol) in DMSO (7 mL). The resulting mixture
508 J. Org. Chem., Vol. 73, No. 2, 2008

Synthesis of 4-(2-R-ethyl)amino-3-imidazoline 1-Oxyls
methanol, 15:1). After the reaction was over the mixture was diluted
with H2O (20 mL) and extracted with CHCl3 (5 × 5 mL). Combined
organic extracts were washed with brine (4 × 5 mL) and dried
over MgSO4, and the residue obtained after removal of solvent
under reduced pressure was separated by column chromatography
(SiO2, CHCl3). The product solidified after evaporation of solvent
to give 10 as a yellow crystals (in DMF: 0.53 g, 2.35 mmol, 72%;
in DMSO: 0.52 g, 2.3 mmol, 70%): mp 86-88 °C dec (hexane/
EtOAc, 5:1); IR (KBr, cm-1) 3363, 1545 (N-H), 2105 (N3), 1627
(CdN). Anal. Calcd for C9H17N6O: C, 48.00; H, 7.64; N, 37.29.
Found: C, 47.54; H, 7.69; N, 37.21.
Method B: Compound 8 (0.27 g,1 mmol) was dissolved in 3
mL of 1 N CH3ONa/CH3OH and the solution was allowed to stay
at room temperature for 3 h, solvent was removed under reduced
pressure, then the residue was dissolved in 15 mL of H2O and
extracted with CHCl3. The organic solution was extracted with 3%
HCl, and the organic layer was discarded; the aqueous solution
was neutralized with Na2CO3 and extracted with CHCl3. Further
workup was performed similar to Method A. Yield: 0.2 g, 0.92
mmol, 92%.
Method C: A solution of 8 (0.37 mmol) in 5 mL of dry DMF
or DMSO was heated at 110 °C. The reaction times were as
follows: DMF, 5.5 h; DMSO, 12 h. The reaction mixture was
diluted with brine (5 mL) and extracted with EtOAc (5 × 5 mL).
Organic extract was thoroughly washed with brine (8 × 5 mL)
and dried over MgSO4, solvent was removed under reduced
pressure, and the residue was separated by column chromatography
(SiO2, CHCl3). Yield in DMF: 71%. Yield in DMSO: 68%.
Cleavage of the Reference Oxadiazolone 5b under Nucleo-
philic Conditions. NaN3 (0.12 g, 1.9 mmol) was added to a solution
of 5b (0.44 g, 1.9 mmol) in a mixture of DMSO (20 mL) and H2O
(2 mL). The resulting mixture was heated at 55 °C for 65 h. Further
workup was performed similar to that specified for 9. Analytical
data for 2, R ) Ph, are in a good agreement with those published
in the literature. The IR spectrum is identical with that of the
reference compound 2, R ) Ph, synthesized through the literature
procedure.17c 2, R ) Ph, was obtained as a yellow crystalline
compound in 95% yield.
Thermolysis of 1-Phenyl-6-oxyl-5,5,7,7-tetramethyltetrahy-
droimidazo[1,5-b][1,2,4]oxadiazol-2-one 5b. A solution of 5b
(0.37 mmol) in 5 mL of dry DMF or DMSO was heated at 110 °C.
The reaction times were as follows: DMF, 64 h; DMSO, 89 h.
The reaction mixture was diluted with brine (5 mL) and extracted
with EtOAc (5 × 5 mL). Organic extract was thoroughly washed
with brine (8 × 5 mL) and dried over MgSO4, solvent was removed
under reduced pressure, and the residue was separated by column
chromatography (SiO2, CHCl3) to give 2 (R ) Ph). Yield in DMF:
60%. Yield in DMSO: 62%.
CHCl3/EtOAc, 2:1). Further workup was performed similar to
Method A. Yield: 0.036 g, 0.149 mmol, 86%.
Method C: A solution of 5c (0.05 g, 0.175 mmol) in 2 mL of
dry DMSO was heated in an oil bath at 110 °C for 17 h. The course
of the reaction was monitored by TLC (SiO2, CHCl3/EtOAc, 2:1).
Further workup was performed similar to Method A. Yield: 0.032
g, 0.132 mmol, 75%.
Method D: A solution of 5c (0.05 g, 0.175 mmol) in 2 mL of
DMF (freshly distilled over P2O5) was heated in an oil bath at 110
°C for 31 h. The course of the reaction was monitored by TLC
(SiO2, CHCl3/EtOAc, 2:1). Further workup was performed similar
to Method A. Yield: 0.012 g, 0.049 mmol, 28%.
Method E: A solution of 5c (0.05 g, 0.175 mmol) in 2 mL of
DMF containing 5% v/v H2O was heated in an oil bath at 110 °C
for 31 h. The course of the reaction was monitored by TLC (SiO2,
CHCl3/EtOAc, 2:1). Further workup was performed similar to
Method A. Yield: 0.009 g, 0.037 mmol, 22%.
Method F: A solution of KBr (0.021 g, 0.175 mmol) was added
to a solution of 5c (0.05 g, 0.175 mmol) in 2 mL of dry DMF.
Water (0.075 mL) was added to facilitate the solubility of KBr in
DMF. The resulting mixture was heated in an oil bath at 110 °C
for 2 h. The progress of the reaction was monitored by TLC (SiO2,
CHCl3/EtOAc, 2:1). Further workup was performed similar to
Method A. Yield: 0.033 g, 0.137 mmol, 80%.
2′,2′,5′,5′-Tetramethyl-1′-oxyl-4,5,2′,5′-tetrahydro-3H,1′H-
[1,4′]biimidazolyl-2-thione 14. Method A: A solution of azide
10 (0.35 g, 1.56 mmol) in dry THF (10 mL) was placed into a
flask that was flushed with argon, and P(n-Bu)3 (0.4 mL, 1.59
mmol) was added under argon upon stirring. After the vigorous
N2 evolution had ceased, the reaction mixture was stirred for an
additional 30 min at room temperature and then heated to 50 °C
for 20 min. The reaction mixture was allowed to cool to the ambient
temperature and CS2 (3 mL, 50.75 mmol) was added dropwise.
The resulting mixture was allowed to stay under stirring for 12 h,
and the residue, obtained after evaporation of solvent, was separated
by chromatography (SiO2, CHCl3).
Method B: PPh3 (0.44 g, 1.7 mmol) was added under argon to
the stirred solution of 10 (0.25 g, 1.1 mmol) in dry THF (3 mL).
The reaction mixture was refluxed under stirring for 90 min, and
CS2 (3 mL, 50.75 mmol) was added dropwise. The resulting mixture
was refluxed for 2 h, the residue, obtained after evaporation of
solvent, was separated by chromatography (Kieselgel, CH2Cl2/CH3-
CN, 3:2) to give 14 as canary-colored crystals (0.28 g, 1.16 mmol,
75%, method A; 0.08 g, 0.32 mmol, 35%, method B): mp 140-
141 °C dec (hexane/EtOAc, 1:2); IR (KBr, cm-1) 3295, 1511 (NH),
1580 (CdN). Anal. Calcd for C10H17N4OS: C, 49.79; H, 7.10; N,
23.21. Found: C, 49.84; H, 7.04; N, 23.01.
Cyclohexyl(2′,2′,5′,5′-tetramethyl-1′-oxyl-4,5,2′,5′-tetrahydro-
3H,1′H-[1,4′]biimidazolyl-2-ylidene)amine 16. Method A: P(n-
Bu)3 (0.4 mL, 1.59 mmol) was added under argon to the stirred
solution of 10 (0.35 g, 1.56 mmol) in dry THF (10 mL). After the
vigorous N2 evolution had ceased, the reaction mixture was stirred
for an additional 30 min at room temperature and for 20 min at
50 °C. The reaction mixture was allowed to cool to ambient
temperature and cyclohexyl isothiocyanate (0.3 mL, 2.03 mmol)
was added dropwise. The resulting mixture was allowed to stay
under stirring at ambient temperature for 12 h. The residue, obtained
after evaporation of solvent, was separated by column chromatog-
raphy (Al2O3, CH3CN).
Method B: A 1 M solution of PEt3 (1.36 mL, 1.36 mmol) was
added under argon to the stirred solution of 10 (0.33 g, 1.33 mmol)
in dry THF (5 mL). After the vigorous N2 evolution had ceased,
the reaction mixture was stirred for an additional 40 min at room
temperature and for 20 min at 40 °C. The reaction mixture was
allowed to cool to ambient temperature and cyclohexyl isothiocy-
anate (0.24 g, 1.73 mmol) was added to the resulting mixture
dropwise. The reaction mixture was allowed to stay under stirring
at ambient temperature for 12 h. The residue, obtained after
evaporation of solvent, was separated by column chromatography
(1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-4-ylami-
no)acetic Acid Ethyl Ester 11. Method A: NaN3 (0.18 g, 2.8
mmol) was added to a solution of 5c (0.8 g, 2.8 mmol) in DMSO
(20 mL). The resulting mixture was heated in an oil bath at
temperatures not exceeding 55 °C for 14 h. The course of the
reaction was monitored by TLC (SiO2, CHCl3/methanol, 15:1). The
mixture was diluted with H2O and extracted with CHCl3 (3 × 5
mL). Combined organic extracts were washed with brine (7 × 5
mL), an organic layer was dried over MgSO4, the solvent was
removed under reduced pressure, and the residue was separated by
column chromatography (SiO2, CHCl3). The residue, obtained after
evaporation of solvent, solidified to give 11 as orange crystals (0.5
g, 2.1 mmol, 74%): mp 93-95 °C dec (hexane/EtOAc, 3:1); IR
(KBr, cm-1) 3331, 1565 (N-H), 1753 (COOEt), 1635 (CdN).
Anal. Calcd for C11H20N3O3: C, 54.56; H, 8.26; N, 17.36. Found:
C, 54.68; H, 8.28; N, 17.15.
Method B: A solution of NaN3 (0.011 g, 0.175 mmol) was added
to a solution of 5c (0.05 g, 0.175 mmol) in 2 mL of dry DMF.
Water (0.075 mL) was added to facilitate the solubility of NaN3 in
DMF. The resulting mixture was heated in an oil bath at 110 °C
for 2 h. The course of the reaction was monitored by TLC (SiO2,
J. Org. Chem, Vol. 73, No. 2, 2008 509

Polienko et al.
(Al2O3, CH3CN) to give 16 as a pale yellow crystals (0.3 g, 0.98
mmol, 63%, method A; 0.23 g, 0.75 mmol, 52%, method B): mp
140-142 °C dec (hexane/EtOAc, 4:1); IR (KBr, cm-1) 3256, 1535
(NH), 1596 (CdN amidine), 1661 (CdN guanidine). Anal. Calcd
for C16H28N5O‚0.25H2O: C, 62.03; H, 9.21; N, 22.62. Found: C,
61.58; H, 9.20; N, 22.49.
Cyclohexyl(3,2′,2′,5′,5′-pentamethyl-1′-oxyl-4,5,2′,5′-tetrahy-
dro-3H,1′H-[1,4′]biimidazolyl-2-ylidene)ammonium Methyl Sul-
fate 17. (CH3)2SO4 (0.05 mL, 0.53 mmol) was added to a solution
of 16 (0.11 g, 0.35 mmol) in dry ether (5 mL), and the mixture
was allowed to stay at ambient temperature for 8 h. The yellow
crystals of 17 formed (0.12 g, 0.28 mmol, 81%) were collected on
a filter: mp 165-168 °C dec (EtOAc); IR (KBr, cm-1) 1604 (Cd
N amidine), 1679 (CdN guanidine), 3550, 3486 (NH+). Anal. Calcd
for C18H34N5O5S‚H2O: C, 48.00; H, 8.00; N, 15.56. Found: C,
48.43; H, 7.93; N, 15.61.
added portionwise to an ice-cooled solution of amine 19 (0.5 g,
2.5 mmol) in a 35% solution of formaldehyde (5.5 mmol). The
resulting mixture was heated in an oil bath at 60 °C for 30 min,
diluted with water, basified (NaOH) to pH 12, and extracted with
ether (7 × 5 mL). An organic extract was dried over K2CO3, the
solvent was removed under reduced pressure, and 20 was obtained
as yellow crystals (0.4 g, 1.8 mmol, 70%): mp 89-90 °C dec
(hexane/EtOAc, 1:1); IR (KBr, cm-1) 3325 (N(CH3)2), 1627 (Cd
N), 1548 (NH). Anal. Calcd for C11H23N4O‚1/6H2O: C, 57.39; H,
10.14; N, 24.35. Found: C, 57.37; H, 9.94; N, 24.10. Mass spectrum
(M+): calcd for C11H23N4O 227.18718, found 227.18862.
[2-(1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-4-
ylamino)ethyl]trimethylammonium Methyl Sulfate 21. (CH3O)2-
SO2 (0.034 mL, 0.33 mmol) was added to a solution of 20 (0.075
g, 0.33 mmol) in dry ether (5 mL) and the resulting mixture was
allowed to stay at ambient temperature for a 6 h. The residue,
obtained after evaporation of solvent, solidified after triturating with
an ether/CH3CN mixture (20:1) to give 21 as pale yellow crystals
(0.071 g, 0.2 mmol, 61%): mp 115-117 °C dec (ether/CH3CN,
2:1); IR (KBr, cm-1) 3353 (NH), 1628 (CdN), 1543 (NH). Anal.
Calcd for C13H29N4O5S‚0.25H2O: C, 43.63; H, 8.25; N, 15.66.
Found: C, 43.53; H, 8.24; N, 15.39.
Cyclohexyl(3,2′,2′,5′,5′-pentamethyl-1′-oxyl-4,5,2′,5′-tetrahy-
dro-3H,1′H-[1,4′]biimidazolyl-2-ylidene)methylammonium Meth-
yl Sulfate 18. A solution of 17 (0.16 g, 0.37 mmol) in water (10
mL) was basified (NaOH) to pH 12 and extracted with ether (3 ×
5 mL), then the organic extract was dried over Na2SO4. The drying
agent was filtered off and (CH3)2SO4 (0.05 mL, 0.56 mmol) was
added to the ether solution. The reaction mixture was allowed to
stay at ambient temperature for 8 h. The ether layer was discarded,
and the oily residue formed on the bottom of the flask was treated
with dry hexane to give 18 as yellow crystals (0.12 g, 0.27 mmol,
X-Band EPR Titration of the Nitroxides. Nitroxides were
titrated in a buffer solution (10 mM citric acid, 10 mM sodium
phosphate, and 10 mM sodium borate). The pH value of nitroxide
solutions was adjusted with HCl or NaOH solutions. The pH
measurements were performed with a digital pH meter with
accuracy (0.05 pH unit. Hyperfine coupling constants, aN, were
measured as a distance between low-field and central nitrogen
hyperfine components of the EPR spectra.
73%): mp 157-159 °C dec (hexan/EtOAc, 1:3); IR (KBr, cm-1
)
1602 (CdN amidine), 1689 (CdN guanidine). Anal. Calcd for
C19H36N5O5S‚H2O: C, 49.13; H, 8.19; N, 15.09. Found: C, 49.00;
H, 7.87; N, 15.67.
4-(2-Aminoethylamino)-1-oxyl-2,2,5,5-tetramethyl-2,5-dihy-
dro-1H-imidazole 19. PBu3 (0.96 mL, 3.5 mmol) was added under
argon to a stirred solution of 10 (0.5 g, 2.25 mmol) in dry THF
(10 mL). After the vigorous N2 evolution had ceased, the reaction
mixture was stirred for an additional 30 min. The solvent was
removed under reduced pressure, 20 mL of the aqueous C2H5OH
solution (1:1) was added to the residue, and the reaction mixture
was allowed to stay under stirring at ambient temperature for 6 h.
C2H5OH was distilled off under reduced pressure, the water layer
was acidified with 3% HCl to pH 4 and extracted with CHCl3 (5
× 5 mL), and the organic layer was discarded. The water layer
was neutralized (NaHCO3) and extracted with CHCl3 (5 × 5 mL);
the organic layer was discarded again. The water layer was basified
(NaOH) to pH 12 and extracted with CHCl3 (7 × 5 mL), then the
organic layer was dried over K2CO3. Solvent was removed under
reduced pressure to give 19 as yellow crystals (0.33 g, 1.67 mmol,
Acknowledgment. The project was partially supported by
the DAAD Foundation through a grant made to T.S. and the
U.S. Civilian Research & Development Foundation (RUC1-
2635-NO-05). T.S. is also grateful to the University of Kaiser-
slautern for support of this work. We thank Dr. Andrey A.
Bobko and Denis A. Komarov (Novosibirsk) for technical
assistance in performing EPR experiments. The comments of
Dr. Alex I. Smirnov (Raleigh) are gratefully acknowledged.
Supporting Information Available: Experimental conditions
for the nucleophilic substitution of bromide in 5a and details of
the thermolytic and nucleophilic cleavage of the oxadiazolone
heterocycle; crystallographic data in CIF format, details of X-ray
data collection and structure determination, and crystal data and
experimental parameters for the crystal structure analyses of 14
and 17; discussion of the results of crystallographic analyses; general
experimental methods. This material is available free of charge via
the Internet at http://pubs.acs.org.
75%): mp 66-68 °C dec (hexane/EtOAc, 1:1); IR (KBr, cm-1
)
3365 (NH2), 1626 (CdN), 1549 (NH). Anal. Calcd for C9H19N4O‚
0.75H2O: C, 50.82; H, 9.65; N, 26.35. Found: C, 51.18; H, 9.39;
N, 26.09.
4-[(2-Dimethylaminoethyl)amino]-1-oxyl-2,2,5,5-tetramethyl-
2,5-dihydro-1H-imidazole 20. Formic acid (90%, 12.5 mmol) was
JO701803A
510 J. Org. Chem., Vol. 73, No. 2, 2008