Acyl-protected hydroxylamines as spin label generators for EPR brain imaging

Article abstract of DOI:10.1021/jm0105169

In a search for novel electron paramagnetic resonance (EPR) brain imaging agents, we have designed and synthesized the acyl-protected hydroxylamines 1-acetoxy-4-methoxycarbonyl-2,2,6,6-tetramethylpiperidine (AMCPe), 1-acetoxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (AMCPy), and 1-acetoxy-3-(acetoxymethoxy)carbonyl-2,2,5,5-tetramethylpyrrolidine (DACPy), in which both the ring size and the number of ester functions were varied. In all of them, the nitroxide was first reduced and the resultant hydroxylamine was then protected with an acetyl group. These compounds are lipophilic, which is a major prerequisite for blood-brain barrier penetration. Once in the brain, esterases and oxidants quickly convert these derivatives into ionic, water-soluble radicals and thus EPR detectable species that then reside in the central nervous system for periods of time sufficient for detection and imaging. The biological relevancy of these new compounds in mice has been assessed, and their biodistribution patterns have been compared. The five-membered ring derivative AMCPy emerged as a potent EPR brain imaging agent while the other two derivatives, AMCPe and DACPy, were quite ineffective.

Full text of DOI:10.1021/jm0105169

J . Med. Chem. 2002, 45, 2283-2288
2283
Acyl-P r otected Hyd r oxyla m in es a s Sp in La bel Gen er a tor s for EP R
Br a in Im a gin g
Alexander T. Yordanov,* Ken-ichi Yamada, Murali C. Krishna, Angelo Russo, J ohn Yoo, Sean English,
J ames B. Mitchell, and Martin W. Brechbiel
Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, and Radiation Biology Branch,
Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Received November 6, 2001
In a search for novel electron paramagnetic resonance (EPR) brain imaging agents, we have
designed and synthesized the acyl-protected hydroxylamines 1-acetoxy-4-methoxycarbonyl-
2,2,6,6-tetramethylpiperidine (AMCPe), 1-acetoxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyr-
rolidine (AMCPy), and 1-acetoxy-3-(acetoxymethoxy)carbonyl-2,2,5,5-tetramethylpyrrolidine
(DACPy), in which both the ring size and the number of ester functions were varied. In all of
them, the nitroxide was first reduced and the resultant hydroxylamine was then protected
with an acetyl group. These compounds are lipophilic, which is a major prerequisite for blood-
brain barrier penetration. Once in the brain, esterases and oxidants quickly convert these
derivatives into ionic, water-soluble radicals and thus EPR detectable species that then reside
in the central nervous system for periods of time sufficient for detection and imaging. The
biological relevancy of these new compounds in mice has been assessed, and their biodistribution
patterns have been compared. The five-membered ring derivative AMCPy emerged as a potent
EPR brain imaging agent while the other two derivatives, AMCPe and DACPy, were quite
ineffective.
Sch em e 1
In tr od u ction
Electron spin resonance (ESR) or electron paramag-
netic resonance (EPR) imaging operating in lower
frequency bands (less than 1 GHz) is an emerging
technique, which allows the observation of distribution
of exogenous free radicals, i.e., spin labels, in vivo.1-12
Initially, water-soluble free radicals such as TEMPOL
(4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) were
explored as spin labels for in vivo EPR imaging.
However, these otherwise in vitro stable nitroxides are
prone to bioreduction to the corresponding diamagnetic,
EPR silent hydroxylamines such as TEMPOL-H (Scheme
1). The oxidized (nitroxide) and the reduced (hydroxyl-
amine) species rapidly establish an equilibrium in vivo
that strongly favors the hydroxylamine form.13 Thus,
in vivo, the short half-life of nitroxides (t1/2 ≈ 3 min)
has limited the efficacy of the latter and has prevented
their development for a wide range of potential diag-
nostic and therapeutic applications. Recently, polyni-
troxyl-albumin (PNA) or polynitroxyl-dendrimers (PND),
in which 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)
moieties are attached to the macromolecule surface,
have been reported to be capable of reoxidizing TEM-
POL-H back to TEMPOL.14-16
of increasing interest, has proven to be a difficult task.
So far, all studies have focused only on five-membered
ring carbamoyl-PROXYL derivatives, and in all of them,
the lipophilicity has been increased by modifications of
only either the carbonyl (PCAM) or the hydroxylamine
(ACP) functional groups in the molecule (Figure 1), but
simultaneous modification of both of these functional
groups has hitherto been unexplored.23-26
Herein, we report the synthesis and the structure-
activity relationship (SAR) evaluation of the biodistri-
bution properties of three such doubly modified spin
label generating agents. Our approach includes not only
variation of the ring size of the label but also variation
in the number of the ester functions present in the
molecule. We have prepared 1-acetoxy-4-methoxycar-
bonyl-2,2,6,6-tetramethylpiperidine (3, AMCPe), a de-
rivative of the six-membered ring TEMPO radical, and
1-acetoxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrro-
lidine (7, AMCPy) and 1-acetoxy-3-(acetoxymethoxy)-
carbonyl-2,2,5,5-tetramethylpyrrolidine (10, DACPy);
both compounds are derivatives of the five-membered
ring PROXYL radical (Figure 1). In all three target
compounds, the lipid solubility was further enhanced
by the introduction of two (AMCPe and AMCPy) or three
(DACPy) ester functions in the molecules. The ester
functions were expected to be hydrolyzed by esterase
enzymes in the intracellular environment. This finding
suggests that 7 (AMCPy) is a novel compound possess-
Despite the fact that the half-lives of nitroxide radi-
cals have been increased to clinically relevant periods
of time and preliminary experiments with in vivo EPR
imaging in the past have successfully lead to the imag-
ing of rat tails, heads, kidneys, and hearts,14,15,17-20
ordinary spin labels do not readily penetrate the blood-
brain barrier (BBB).21,22 This is why the development
of nitroxides for EPR brain imaging, recently an area
* To whom correspondence should be addressed. Tel.: (301)496-
6494. Fax: (301)402-1923. E-mail: yordanov@mail.nih.gov.
10.1021/jm0105169 CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/01/2002

2284 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Yordanov et al.
of these acyl-protected hydroxylamine methyl esters was
purified by flash chromatography.
A somewhat different approach was used in the
preparation of 10 (DACPy). In this case, 4-carboxy-
PROXYL 4 was first reduced by hydrazine to 1-hydroxy-
3-carboxy-2,2,5,5-tetramethylpyrrolidine 8 (Scheme 4),
which was consequently acetylated to 1-acetoxy-3-
carboxy-2,2,5,5-tetramethylpyrrolidine 9. This acid was
then reacted with bromomethyl acetate in the presence
of TEA to yield DACPy, which was also purified by flash
chromatography.
All three target spin label generatorssAMCPe, AM-
CPy, and DACPysare stable nonradical compounds that
are not affected by oxidation. They show poor water
solubility and could not be dissolved without an organic
cosolvent such as dimethyl sulfoxide (DMSO) or ethanol,
which did not prevent their proper biological evaluation.
F igu r e 1.
Resu lts a n d Discu ssion
Sch em e 2
The originally used water-soluble spin labels in previ-
ous experiments did not penetrate the BBB.14,15,17,18,21,22
Thus, in all EPR imaging experiments of rat heads
achieved by use of carbamoyl-PROXYL, the brain was
imaged as a nitroxide-deficient area while the blood
vessels and/or the extracranium tissues were imaged
as a nitroxide-rich area. During periods when high
intensities of EPR signals were maintained, spots of
nitroxide accumulation were imaged at the central part
of the brain. However, those spots were assigned to the
moderate-sized blood vessels in the brain.22 Some brain
EPR images utilizing carbamoyl-PROXYL have also
been recently reported. In those studies, the label was
perfused by microdialysis. However, it has been difficult
to image cortex and striatum simultaneously. Further-
more, this administration method has taken a long time
(6 h) to perfuse the spin label.30 This inconvenience has
prompted investigators to look for new spin labels that
penetrate the BBB more efficiently or spin labels that
can be generated within the intracellular compartment
from stable, nonradical precursors.23-26 This has led to
some encouraging results in rat brain imaging.
We report, herein, the synthesis and the biodistribu-
tion SAR evaluation of three new in vivo spin label
generators, AMCPe (3), AMCPy (7), and DACPy (10).
AMCPe differs from AMCPy and DACPy by its six-
membered ring size while DACPy differs from the rest
by having three ester functions in its molecule. It was
expected that these three compounds would possess
higher lipophilicity for efficient BBB penetration than
the previously reported carbamoyl-PROXYL, PCAM,
and ACP (Figure 1). Compound 3, AMCPe, is a deriva-
tive of TEMPO free radical and contains a six-membered
ring and two ester functions. Both 7 (AMCPy) and 10
(DACPy) are derivatives of PROXYL free radical and
contain a five-membered ring. AMCPy and DACPy have
two and three ester functions, respectively. Upon enter-
ing the intracellular compartment, esterases should
hydrolyze the ester functional groups in the compounds
to produce ionic, water-soluble species, which cannot
penetrate back across the BBB and should be retained
in the brain intracellularly for longer periods of time
(Figure 2). In addition to that, oxidants in the intrac-
ellular environment (among them, free radicals) should
oxidize the formed hydroxylamines to nitroxides,31,32 the
ing the optimal lipophilicity and thus has a promising
role as a future spin label generator for EPR brain
imaging.
Ch em istr y
The design strategy for our target compounds is
shown in Schemes 2-4. All compounds were character-
ized by their exact fast atom bombardment (FAB) mass
spectra, 1H and 13C nuclear magnetic resonance (NMR)
spectra (if no free radical functional group was present
in the molecule), and elemental analysis, where ap-
plicable.
The six-membered ring nitroxide radical, 1-oxyl-4-
carboxy-2,2,6,6-tetramethylpiperidine 1 (4-carboxy-
TEMPO) was simultaneously reduced and esterified to
its hydroxylamine methyl ester 2 in a one step proce-
dure by bubbling dry HCl(g) through a methanol solu-
tion of the compound. This reaction was completed
within 30 min (Scheme 2). Under identical conditions,
however, the five-membered ring nitroxide radical,
1-oxyl-3-carboxy-2,2,5,5-tetramethylpyrrolidine 4 (3-car-
boxy-PROXYL) underwent esterification only, and no
reduction to its hydroxylamine was observed within the
short period of time employed for the synthesis of 2
(Scheme 3). However, if the HCl(g) saturated methanol
solution of the nitroxide methyl ester 5 is left at ambient
temperature for 24 h or longer, the reduction eventually
proceeds, and the hydroxylamine methyl ester 6 could
be isolated in quantitative yields. The reduction rate of
six-membered ring nitroxides is well-known to be greater
than that of five-membered rings both in vitro and in
vivo.27-29 Hydroxylamine methyl ester 6 could also be
prepared by the reduction of 5 with an excess of
hydrazine in methanol. Both hydroxylamines 2 and 6
were acetylated by using acetic anhydride in tetrahy-
drofuran (THF) in the presence of triethylamine (TEA)
to yield 3 (AMCPe) and 7 (AMCPy), respectively. Each

Acyl-Protected Hydroxylamines for EPR Brain Imaging
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2285
Sch em e 3
Sch em e 4
latter being the EPR detectable species. Our study
aimed not only to prove this hypothesis but also to
investigate whether there were any profound differences
in the biological behavior of the six- (3) and the five- (7
and 10) membered ring derivatives. A second aspect of
this study was to assess whether varying the number
of ester functions in the molecule would make any
difference in the biological behavior of these compounds.
To achieve these goals, a biodistribution study was
performed in mice. The concentration of the oxidized
form of the compounds (nitroxides) and the total con-
centration of both oxidized and reduced forms (nitrox-
ides + hydroxylamines) were monitored in brain, heart,
liver, kidney, spleen, and blood. The animals were
sacrificed 10 and 60 min after intraperitoneal injections
of each compound in DMSO solution. The organs were
immediately collected and homogenized in ice-cold
phosphate-buffered saline (PBS), and the nitroxide
concentration in them was measured by its EPR signal
intensity. The total (nitroxides + hydroxylamines)
concentration was measured afterward by EPR signal
intensity of the same sample when any free hydroxyl-
amine was oxidized to nitroxide by addition of excess
potassium ferricyanide (K3[Fe(CN)6]). Figure 3 displays
the biodistribution patterns of all three compounds in
F igu r e 2.
their nitroxide and total form after injection in mice. It
is clearly seen that AMCPy (7) penetrates the BBB
easily, as soon as 10 min postinjection, where it under-
goes hydrolysis by esterases to free hydroxylamine, part
of which is then oxidized by intracellular oxidants to
an EPR detectable nitroxide. After a clinically relevant
period of time (60 min), the nitroxide species can still
be observed in the brain in significant concentrations
as well as the heart, liver, kidney, spleen, and blood.
Thus, AMCPy is a useful spin label generator not only
for the purposes of brain imaging but also for the
imaging of other organs, especially kidney.
In comparison, the EPR signals after AMCPe (3)
injections showed very little intensity in all organs
collected. As a matter of fact, the signal from this
compound barely registers on the scale employed in
Figure 3. Because AMCPe is expected to be not any less
lipophilic and penetrate the BBB not any less easily

2286 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Yordanov et al.
F igu r e 3.
electron ionization (EI) mass spectra were obtained on a J EOL
J MS-SX102 instrument (Peabody, MA). Exact FAB mass
spectra were obtained on an Extrel 4000 (Pittsburgh, PA)
instrument in the positive ion detection mode. Elemental
analyses were performed by Galbraith Laboratories, Inc.
(Knoxville, TN). When the elemental analysis is not included,
the crude compounds were used in the next step without
further purification. Chromatographic separations were per-
formed on silica gel columns by flash (Silica Gel 60, 0.035-
0.070 mm; Fluka).
1-H yd r oxy-4-ca r b a m oyl-2,2,6,6-t e t r a m e t h ylp ip e r i-
d in e m eth yl ester (2). A sample of the commercially available
4-carboxy-TEMPO 1 (0.50 g, 2.5 mmol) was stirred in methanol
(50 mL) and placed in an ice bath. Dry HCl(g) was then
bubbled through the solution for 30 min. The solvent was
removed by evaporation, and the resulting oil was neutralized
with 10% NaHCO3. The product was then extracted with ethyl
acetate (3 × 100 mL). The organic layers were combined and
dried over Na2SO4, and the solvent was evaporated to give the
product as an oil; yield 0.51 g (94%). Exact FAB mass
spectrum: calcd for C11H21NO3 + H+, 216.1600; found, 216.1606.
1H NMR (CDCl3): δ 1.15 (s, 6H, CH3), 1.22 (s, 6H, CH3), 1.66
(m, 2H, CH2), 1.80 (m, 2H, CH2), 2.68 (t of t, 1H, CH), 3.68 (s,
3H, OCH3). 13C NMR (CDCl3): δ 7.18, 20.0, 22.07, 28.90, 40.37,
46.63, 163.00.
1-Acetoxy-4-m eth oxyca r bon yl-2,2,6,6-tetr a m eth ylp ip -
er id in e, AMCP e (3). Hydroxylamine 2 (0.51 g, 2.4 mmol) was
dissolved in a mixture of THF (40 mL) and TEA (5 mL). The
solution was placed in an ice bath, and then, acetic anhydride
(5 mL, 53 mmol) was added dropwise over 10 min. After 8 h
of stirring at ambient temperature, the excess of acetic
anhydride was carefully destroyed with water and the result-
ing solution was neutralized with aqueous 10% NaHCO3. The
THF was evaporated, and the product was then extracted with
ethyl acetate (3 × 100 mL) and dried over Na2SO4, and the
solvent was evaporated almost to dryness. Subsequent flash
chromatography (hexane:ethyl acetate ) 9:1) gave 3 as a
yellowish crystalline solid; yield 0.41 g (67%). Exact FAB mass
spectrum: calcd for C13H23NO4 + H+, 258.1705; found, 258.1711.
Elemental analysis calcd: C, 60.7%; H, 9.01%; N, 5.44%.
Found: C, 58.8%; H, 9.28%; N, 6.65%. 1H NMR (CDCl3): δ
1.02 (s, 6H, CH3), 1.11 (s, 6H, CH3), 1.76 (m, 4H, CH2), 2.02
(s, 3H, CH3), 2.64 (m, 1H, CH), 3.60 (s, 3H, OCH3). 13C NMR
(CDCl3): δ 16.63, 18.08, 29.13, 32.0, 38.44, 49.43, 56.88, 168.00.
than AMCPy, this difference is no doubt due to either
a slower rate of deacetylation for AMCPe to a free
hydroxylamine as compared to AMCPy or to a much
faster clearance of the compound from the body. Also,
previous reports have shown that the five-membered
ring radicals are much more stable in biological systems
than the six-membered ones.33 Therefore, the observa-
tion that AMCPe does not behave entirely as expected
is believed due to a combination of some or all of these
properties. The addition of a third ester group in DACPy
(10) has a negative effect on the BBB penetration ability
and the whole body retention of this spin label genera-
tor.
In conclusion, the most important finding of the
present investigation is that the five-membered ring
PROXYL derivative AMCPy (7) possesses the optimal
lipophilicity for BBB penetration where it is retained
in the brain for clinically relevant periods of time. Once
there, AMCPy is subsequently deacetylated and oxi-
dized into its nitroxide form, the latter being an EPR
detectable species. Clearly, AMCPy is a very promising
candidate for a spin label generating agent in EPR
imaging. Currently, murine models are under investiga-
tion to further evaluate and promote use of AMCPy in
EPR imaging techniques.
Exp er im en ta l Section
Ch em istr y. Reagents and anhydrous solvents were pur-
chased from Aldrich Chemical Co. (Milwaukee, WI) unless
otherwise stated, and all were used without further purifica-
tion. All spin label generators were stored at -20° C before
use. Anhydrous HCl(g) was supplied by Roberts Oxygen Co.
(Gaithersburg, MD). Proton and 13C NMR spectra were
recorded on a Varian Gemini 300 instrument (Palo Alto, CA).
NMR peak patterns were described by the following abbrevia-
tions: br ) broad, d ) doublet, t ) triplet, q ) quartet, m )
multiplet, and arom ) aromatic protons. Chemical shifts were
expressed as parts per million as referenced to tetramethyl-
silane (TMS) (CDCl3 as an internal standard for 13C) unless
otherwise noted. Low-resolution chemical ionization (CI) and

Acyl-Protected Hydroxylamines for EPR Brain Imaging
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2287
1-Oxyl-3-m eth oxyca r bon yl-2,2,5,5-tetr a m eth ylp yr r oli-
d in e (5). A sample of commercially available 3-carboxy-
PROXYL 4 (1.50 g, 8.05 mmol) was stirred in methanol (100
mL) and placed in an ice bath. Dry HCl(g) was then bubbled
through the solution for 30 min. The solvent was evaporated
quickly at ambient temperature, the resulting oil was neutral-
ized with 10% NaHCO3, and the product was extracted with
ethyl acetate (3 × 100 mL). The organic layers were combined
and dried over Na2SO4, and the solvent was evaporated to
complete dryness to produce 5 as an orange oil; yield 1.40 g
13C NMR (DMSO-d6): δ 18.94, 26.47, 27.65, 28.16, 38.27, 49.38,
60.71, 64.84, 175.05.
1-Acetoxy-3-car boxy-2,2,5,5-tetr am eth ylpyr r olidin e (9).
Hydroxylamine 8 (0.38 g, 2.0 mmol) was suspended in THF
(150 mL). The suspension was placed in an ice bath, and then,
acetic anhydride (8 mL, 84.8 mmol) was added dropwise over
10 min. The solution gradually cleared up, and after 18 h of
stirring at ambient temperature, the excess of acidic anhydride
was carefully destroyed with water. The THF was evaporated,
and the product was then extracted with ethyl acetate (3 ×
100 mL). The combined organic layers were extracted multiple
times with water (until neutral pH) and dried over Na2SO4,
and the solvent was evaporated almost to dryness. Subsequent
drying in vacuo gave the product as an oil, which solidified
upon standing; yield 0.40 g (80%). Exact FAB mass spectrum:
calcd for C11H19NO4 + H+, 230.1392; found, 230.1391. 1H NMR
(CDCl3): δ 1.10-1.28 (m, 9H, CH3), 1.32 (m, 3H, CH3), 1.91
(m, 1H, CH2), 2.12 (s, 3H, CH3), 2.25 (m, 1H, CH2), 2.95 (m,
1H, CH). 13C NMR (CDCl3): δ 17.44 (br), 19.26, 26.21 (br),
27.76 (br), 31.54 (br), 38.09 (br), 48.17 (br), 66.86 (br), 171.25,
177.64.
(87%). Exact FAB mass spectrum: calcd for
200.1287; found, 200.1283.
C10H18NO3,
1-Hyd r oxy-3-m eth oxyca r bon yl-2,2,5,5-tetr a m eth ylp yr -
r olid in e (6). Meth od I. Nitroxide 5 (1.40 g, 7.0 mmol) was
dissolved in methanol (100 mL). Anhydrous hydrazine (10 mL,
0.32 mol) was added dropwise to that solution. After 18 h of
stirring, the solvent and the unreacted hydrazine were evapo-
rated at room temperature almost to dryness. Toluene (15 mL)
was added, and the solvent was vacuum-evaporated to dryness
again (no heat!). This procedure, which removes any residual
hydrazine, was repeated as many times as necessary to give
6 as an oil; yield 1.30 g (92%).
1-Acet oxy-3-(a cet oxym et h oxy)ca r b on yl-2,2,5,5-t et r a -
m eth ylp yr r olid in e, DACP y (10). Acid 9 (1.4 g, 6.1 mmol)
was dissolved in dimethylformamide (DMF, 150 mL), and to
this solution bromomethyl acetate (4.0 g, 26.1 mmol) was
added, followed by TEA (15 mL). After 18 h of stirring at
ambient temperature, the solvent was evaporated under
reduced pressure and the residue was distributed between
CHCl3 and water. The organic phase was washed multiple
times with water and dried over Na2SO4, and the solvent was
evaporated almost to dryness. Subsequent flash chromatog-
raphy (hexane:ethyl acetate ) 7:1) gave the product as a
colorless oil; yield 1.2 g (65%). Exact FAB mass spectrum:
calcd for C14H23NO6 + H+, 302.1604; found, 302.1595. 1H NMR
(CDCl3): δ 1.09-1.30 (m, 12H, CH3), 1.85 (m, 1H, CH2), 2.11
(s, 6H, CH3), 2.27 (m, 1H, CH2), 2.94 (m, 1H, CH), 5.72 (d, 1H,
OCH2O), 5.80 (d, 1H, OCH2O). 13C NMR (CDCl3): δ 17.26 (br),
19.18, 20.70, 26.30 (br), 27.81 (br), 31.42 (br), 37.89 (br), 47.98
(br), 63.0 (br), 66.80 (br), 79.38, 169.55, 170.98.
In Vivo Biod istr ibu tion Exp er im en t. C3H HenMTV-
female mice were supplied by the National Cancer Institute
Animal Production Area. The animals were 6-8 weeks old and
weighed between 22 and 25 g. EPR experiments were per-
formed in accordance with the Guide for Care and Use of
Laboratory Animals prepared by the Institute of Laboratory
Animal Resources, National Research Council. The mice (three
per time point) received intraperitoneal injections of each
compound (500 µL of 40 mM in 20% ethanol in PBS). Ten and
sixty minutes after injection, the mice were euthanized, and
the brain, heart, liver, kidney, spleen, (CN)6], and blood were
immediately excised and homogenized separately in ice-cold
PBS. Each sample was split in halves. Potassium ferricyanide
(K3[Fe(CN)6], final concentration 1 mM) was added to the
second half of each sample in order to oxidize any residual
hydroxylamine to a nitroxide and thus measure the total
concentration of the oxidized and reduced form of the particu-
lar compound. The samples were stored on ice, and then, 50
µL of the solution was drawn into a gas permeable Teflon
capillary tube and placed into the EPR cavity. EPR spectra
were recorded on a Varian E9 X-band spectrometer with field
of 3370 G, modulation frequency of 100 kHz, and modulation
amplitude of 1 G.
Meth od II. A sample of commercially available 3-carboxy-
PROXYL 4 (1.50 g, 8.05 mmol) was stirred in methanol (100
mL) and placed in an ice bath. Dry HCl(g) was then bubbled
through the solution for 30 min. The HCl-saturated solution
was then stirred for 24 h at ambient temperature. The solvent
was evaporated, the residue was neutralized with 10%
NaHCO3, and the product was extracted with ethyl acetate (3
× 100 mL). The organic layers were combined and dried over
Na2SO4, and the solvent was evaporated to complete dryness
(no heat!) to give 6 as an oil; yield 1.40 g (86%).
Exact mass spectrum: calcd for C10H19NO3-H, 200.1287;
1
found, 200.1280. H NMR (CDCl3): δ 1.02 (s, 3H, CH3), 1.15
(s, 3H, CH3), 1.23 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.77 (m, 1H,
CH2), 2.13 (m, 1H, CH2), 2.74 (m, 1H, CH2), 3.70 (s, 3H, OCH3).
13C NMR (CDCl3): δ 14.17, 21.14, 21.78, 22.29, 32.25, 43.45,
46.52, 56.81, 60.77, 167.44.
1-Acetoxy-3-m eth oxyca r bon yl-2,2,5,5-tetr a m eth ylp yr -
r olid in e, AMCP y (7). Hydroxylamine 6 (9.4 g, 46.8 mmol)
was dissolved in a mixture of THF (250 mL) and TEA (80 mL).
The solution was placed in an ice bath, and then, acetic
anhydride (30 mL, 0.32 mol) was added dropwise over 10 min.
After 8 h of stirring at ambient temperature, the excess of
acidic anhydride was carefully destroyed with water and the
resulting solution was neutralized with aqueous 10% NaHCO3.
The THF was evaporated, and the product was then extracted
with ethyl acetate (3 × 100 mL) and dried over Na2SO4, and
the solvent was evaporated almost to dryness. Subsequent
flash chromatography (hexane:ethyl acetate ) 9:1) gave the
product as a colorless oil; yield 10.5 g (92%). Exact FAB mass
spectrum: calcd for C12H21NO4 + H+, 244.1549; found, 244.1547.
Elemental analysis calcd: C, 59.3%; H, 8.70%; N, 5.76%.
Found: C, 58.8%; H, 8.92%; N, 5.80%. 1H NMR (CDCl3): δ
1.06 (s, 3H, CH3), 1.20 (s, 6H, CH3), 1.28 (s, 3H, CH3), 1.81
(m, 1H, CH2), 2.09 (s, 3H, CH3), 2.25 (m, 1H, CH2), 2.91 (m,
1H, CH), 3.69 (s, 3H, OCH3). 13C NMR (CDCl3): δ 11.00 (br),
13.27, 19.96 (br), 21.43 (br), 25.06 (br), 31.97 (br), 41.79 (br),
46.03, 56.01 (br), 60.29 (br), 164.53, 166.12.
1-Hydr oxy-3-car boxy-2,2,5,5-tetr am eth ylpyr r olidin e (8).
A sample of commercially available 3-carboxy-PROXYL 4 (1.0
g, 5.4 mmol) was stirred in methanol (100 mL). Anhydrous
hydrazine (10 mL, 0.32 mol) was added dropwise to that
solution. After 18 h of stirring at ambient temperature, the
solvent and the unreacted hydrazine were removed by evapo-
ration. Toluene (15 mL) was added, and the solvent was
vacuum-evaporated to dryness again (no heat!). This proce-
dure, which removes any residual hydrazine, was repeated as
many times as necessary. The final residue was then dried
over 48 h in vacuo to give 8 as a white solid; yield 0.93 g (93%).
Exact FAB mass spectrum: calcd for C9H17NO3 + H+, 188.1287;
found, 188.1294. 1H NMR (DMSO-d6): δ 0.92 (s, 3H, CH3), 1.04
(s, 3H, CH3), 1.09 (s, 3H, CH3), 1.19 (s, 3H, CH3), 1.62 (m, 1H,
CH2), 1.97 (m, 1H, CH2), 2.52 (m, 1H, CH), 7.58 (br s, OH).
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