Spin trapping experiments with different carbamoyl-substituted EMPO derivatives

Article abstract of DOI:10.1016/j.bmc.2008.07.057

The spin trapping behavior of five carbamoyl-substituted EMPO derivatives, 5-aminocarbonyl-5-methyl-pyrroline N-oxide (CAMPO (AMPO)), 5-aminocarbonyl-5-ethyl-pyrroline N-oxide (CAEPO), 5-aminocarbonyl-5-propyl-pyrroline N-oxide (CAPPO), 5-aminocarbonyl-5-n-butyl-pyrroline N-oxide (CABPO), and 5-aminocarbonyl-5-n-pentyl-pyrroline N-oxide (CAPtPO), toward different oxygen- and carbon-centered radicals is described, the stabilities of the superoxide adducts ranging from about 8 to 17 min.

Full text of DOI:10.1016/j.bmc.2008.07.057

Bioorganic & Medicinal Chemistry 16 (2008) 8082–8089
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Spin trapping experiments with different carbamoyl-substituted
EMPO derivatives
a,*
b
c
c
Klaus Stolze , Natascha Rohr-Udilova , Andreas Hofinger , Thomas Rosenau
a
Molecular Pharmacology and Toxicology Unit, Department of Biomedical Sciences, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210 Vienna, Austria
Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
Department of Chemistry, University of Natural Resources and Applied Life Sciences (BOKU), Muthgasse 18, A-1190 Vienna, Austria
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The spin trapping behavior of five carbamoyl-substituted EMPO derivatives, 5-aminocarbonyl-5-methyl-
pyrroline N-oxide (CAMPO (AMPO)), 5-aminocarbonyl-5-ethyl-pyrroline N-oxide (CAEPO), 5-aminocar-
bonyl-5-propyl-pyrroline N-oxide (CAPPO), 5-aminocarbonyl-5-n-butyl-pyrroline N-oxide (CABPO), and
Received 18 February 2008
Revised 17 July 2008
Accepted 22 July 2008
Available online 25 July 2008
5-aminocarbonyl-5-n-pentyl-pyrroline N-oxide (CAPtPO), toward different oxygen- and carbon-centered
radicals is described, the stabilities of the superoxide adducts ranging from about 8 to 17 min.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
EPR
Spin trapping
Superoxide
EMPO/AMPO derivatives
1
. Introduction
5-methyl-1-pyrroline-N-oxide (EMPO), 5-ethoxycarbonyl-5-ethyl-
1
-pyrroline-N-oxide (EEPO), 5-ethoxycarbonyl-5-propyl-1-pyrro-
Derivatives of the spin trap EMPO have previously been shown
line-N-oxide (EPPO), 5-ethoxycarbonyl-5-n-butyl-1-pyrroline-N-
oxide (EBPO), and 5-ethoxycarbonyl-5-n-pentyl-1-pyrroline-N-
oxide (EPtPO), in which the ethoxycarbonyl substituent has been
to possess increased superoxide adduct stabilities if an ethyl or
methyl substituent is present in position 3 or 4 of the pyrroline
1
–3
ring.
The diastereomers in these compounds exhibited—due to
2
replaced by a CONH group. Although the compound 5-carbam-
the presence of two asymmetric carbon centers—significantly dif-
ferent spin trapping properties and spin adduct stabilities (e.g.,
cis- and trans-3,5-EDPO/ OOH: t1/2 = 11 and 45 min, respectively).
Recent publications by other groups suggested that these differ-
ences can also be used to distinguish between the formation path-
ways of spin adducts, for example, whether the hydroxyl radical
adduct has been formed by the trapping of hydroxyl radicals or
by reduction of initially formed superoxide adducts.4
oyl-5-methyl-1-pyrroline-N-oxide has recently been referred to
as ‘AMPO ’ we would like to use the abbreviations CAMPO, CAEPO,
5
Å
CAPPO, CABPO, and CAPtPO, respectively, for these compounds
(CA = 5-carbamoyl–) in order to avoid confusion with the respec-
tive 5-alkoxycarbonyl-5-methyl-1-pyrroline N-oxides, also re-
7
ferred to as ‘AMPO ’ (for structures, see Fig. 1A). A general
synthetic scheme is given in Figure 1B.
Structural identity of the novel spin traps was confirmed by 13C
1
Furthermore, it has been suggested that a better spin trapping per-
formance can also be achieved if the ethoxycarbonyl group of EMPO is
NMR (Table 1), H NMR (Table 2), ESI Q-TOF MS analysis (Table 3),
FTIR spectroscopy (Table 4), and UV–vis spectroscopy (Table 5).
5,6
1
13
replaced by an aminocarbonylgroup. Wetherefore investigated the
effect of different 5-alkyl substituents on the spin trapping perfor-
mance of several 5-carbamoyl-substituted EMPO derivatives.
A complete set of H, H–H correlated, C, HMQC, and HMBC
spectra was recorded for each compound, which allowed for a
1
13
complete signal assignment in both the H and C domains. Car-
bon NMR confirmed the presence of a heterocyclic pyrroline ring.
The resonances for C-2 were found around 135.4–138.2 ppm,
mostly down-field shifted by about 1.1 ppm compared to the
respective esters (134.9–136.3 ppm), those for C-3 (22.5–
2
2
. Results
.1. Structure of the spin traps
2
(
8.2 ppm) varied considerably more compared to the esters
25.4–26.3 ppm), a significant up-field shift being detectable for
CABPO (ꢀ3.5 ppm) and CAPtPO (ꢀ3 ppm), those for C-4 (25.1–
0.6 ppm) were up-field shifted by about 2 ppm compared to the
All spin traps synthesized and investigated within this study
can be considered as derivatives of the series 5-ethoxycarbonyl-
3
esters (27.4–31.9 ppm), and those for C-5 ranged between 79.2
and 81.9 ppm, comparable to the esters (78.5–82.1 ppm).
*
Corresponding author. Tel.: +43 1 25077 4406; fax: +43 1 25077 4490.
E-mail address: Klaus.Stolze@vu-wien.ac.at (K. Stolze).
0
968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.057

K. Stolze et al. / Bioorg. Med. Chem. 16 (2008) 8082–8089
8083
R
1
R
3
R
4
R
5
The shift of the aminocarbonyl group was down-field shifted by
about 2.5 ppm to values between 170.5 and 174.7 ppm compared
to the ethoxycarbonyl group of the esters (169.3–170.3 ppm).
DMPO
EMPO
3
4
CAMPO
CAEPO
CAPPO
CABPO
CAPtPO
CH3
COOC H
,5-EDPO COOC H CH3
,5-EDPO COOC H
H
H
H
H
H
CH3
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
R
4
R3
2
5
0
0
In contrast to this structure, the resonances of the 1 C of the 5-
2
5
5
R
1
H
H
H
H
H
H
2
alkyl substituents were much more dependent on the substituent.
The proton spectra also showed typical resonance patterns. H-2
exhibited coupling constants of around 2.5 Hz and appeared as a
triplet. The triplet pattern of H-2—not a doublet of doublets—indi-
cated similar coupling constants to the geminal H-3 protons. Due
to the diastereotopic properties of the amide group (restricted
rotation around the C-N bond as a consequence of its partial double
CONH2
CONH2
CONH2
CONH2
R
5
N
C H
2
5
7
C H
3
-
O
C H
4
9
CONH2
C H
5
11
O
O
i
ii
2
bond character due to p-conjugation), the NH protons appeared as
O
R
O
R
two separate resonances, their difference steadily increasing from
CAMPO (AMPO) (7.16 and 7.38 ppm) to CAPtPO (5.81 and
8.40 ppm). It should be noted that such comparisons are only pos-
sible if the spectra were recorded under strictly similar conditions
with regard to concentration, temperature, and solvent. The reso-
nances of the 5-alkyl substituents were comparable in the carbam-
oyl and the ester derivatives.
The diastereotopic protons of the methylene groups in the pyr-
roline ring at C-3 and C-4 of the carbamoyl compounds appeared in
the same range as in the respective esters, but their diastereotopic
splitting was, however, somewhat more pronounced.
Br
NO2
R = H, Me, Et, n-Prop, n-Bu
O
O
O
O
O
O
H N
2
iii
+
iv
+
N
N
R
+
O
O
R
R
N
O
O
R = H, CAMPO
R = Me, CAEPO
R = Et, CAPPO
i = NaNO , 1,3,5-Ph(OH) , DMF;
2
3
ii = H C=CH-CHO, MeCN, NEt ;
iii = NH Cl / Zn, H O/MeOH (6:4);
2
3
R =
R =
n
n
-Prop, CABPO
-Bu, CAPtPO
The major absorption peaks in the respective FTIR spectra were
4
2
ꢀ
1
ꢀ1
ꢀ
ꢀ1
iv = NH3 (aq.)
around 1680 cm (C@O), 1580 cm (C@N), and 1205 cm (N–
1
ꢀ1
O). The absorbances around 3150 cm and 3350 cm (N–H va-
lence bands) are not always well resolved and, in addition, very
Figure 1. (a) General structure of the spin traps and (b) general synthetic scheme.
Table 1
1
3C NMR data (ppm) of the spin traps
0
0
00
00
00
00
5
2C
3C
4C
5C
CO
1
C
2
C
3
C
4
C
C
EMPO12
134.9
135.6
25.4
25.3
25.0
28.2
24.7
22.5
23.2
31.9
30.6
30.5
26.1
26.9
25.4
25.1
78.5
79.2
79.1
81.5
81.9
81.8
80.2
169.3
174.7
172.9
171.5
172.4
172.3
170.5
20.3
22.5
24.2
24.6
38.5
36.2
34.8
—
—
—
—
—
—
—
14.0
24.9
29.8
—
—
—
—
—
—
—
—
—
—
12.2
CAMPO (Lit.)5
137.4
CAEPO
CAPPO
CABPO
CAPtPO
136.6
138.2
137.2
135.4
7.5
16.8
26.9
—
a
a
13.7
21.3b
20.6b
a,b
Tentative assignment (reversed order also possible).
Table 2
1
H NMR data (ppm) of the spin traps
0
0
00
00
00
00
5
2CH
3CH
4CH
4CH
2.60m
2.58–2.62m
3.14–3.20m
2.49–2.58m
2.82–2.89m
2.90–3.00m
2.91–2.99m
NH
NH
1
CH
2
CH
3
CH
x
4
CH
x
CH
3
2
A
B
A
B
x
x
EMPO12
6.97t
6.97t
2.75m
2.46–2.53m
2.79m
2.44–2.49m
2.49–2.58m
2.55–2.66m
2.51–2.68m
2.16m
4.26ma
7.16s
5.85b
7.38b
6.16b
5.95b
5.81b
1.31tb
7.38s
8.46b
7.85b
8.26b
8.41b
8.40b
1.72s
1.73s
1.90s
1.83–1.92m
1.88–2.05m
1.99–2.12m
1.96–2.12m
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
CAMPO (Lit.)5
1.96–2.03m
2.27–2.34m
2.04–2.09m
2.08–2.15m
2.13–2.23m
2.14–2.22m
7.19t
CAEPO
CAPPO
CABPO
CAPtPO
7.09t
6.99t
7.03t
7.02t
0.81t
1.18–1.40m
1.26–1.37m
1.28–1.36m
0.90t
1.26–1.37m
1.28–1.36m
0.89t
1.28–1.36m
0.89t
Abbreviations: s, singlet; b, broad singlet; t, triplet; m, multiplet.
0
a
1
From ethoxy group CH
2
.
.
0
b
From ethoxy group 2 CH
3

8
084
K. Stolze et al. / Bioorg. Med. Chem. 16 (2008) 8082–8089
Table 3
Table 5
ESI Q-TOF HR-MS analysis of the spin traps
Half-life of the superoxide adducts, n-octanol/buffer partition coefficients, and UV–vis
spectroscopic data of the spin traps
Sample Acquired Calcd
Error
(ppm)
mDa Acquired
Calcd
[MNa]
Error
(ppm)
mDa
+
+
+
+
[
MH]
[MH]
[MNa]
Compound
Apparent t1/2
min) Xa/XOD
Partition coefficient
n-octanol/phosphate
buffer (100 mM, pH 7.0)
UV data kmax and
molar absorptivity e
(
CAMPO 143.08543 143.0820 23.97
CAEPO 157.10249 157.0976 31.13
CAPPO 171.11634 171.1132 18.35
CABPO 185.13436 185.1288 30.03
CAPtPO 199.14548 199.1444 5.42
3.43 165.06934 165.0640 32.35
4.89 179.0852 179.0796 31.27
3.14 193.09918 193.0952 20.61
5.56 207.11692 207.1108 29.55
1.08 221.12958 221.1264 14.38
5.34
5.60
3.98
6.12
3.18
EMPO12
CAMPO
CAEPO
CAPPO
CABPO
CAPtPO
8.612
0.1512
231 nm, 7417 ± 46
232 nm, 7652 ± 43
233 nm, 7906 ± 28
233 nm, 7508 ± 14
233 nm, 7741 ± 55
234 nm, 6885 ± 39
8.58 ± 0.86
16.76 ± 0.36
10.40 ± 0.25
10.65 ± 0.25
11.13 ± 0.21
0.016 ± 0.002
0.056 ± 0.001
0.211 ± 0.003
0.668 ± 0.004
2.72 ± 0.05
sensitive to traces of humidity. They are, therefore, not listed in Ta-
ble 4.
2
.2. Superoxide radical adducts
When CAEPO (20 mM) was incubated in the presence of 0.2 mM
hypoxanthine/500 mU/ml xanthine oxidase in 20 mM oxygenated
phosphate buffer (pH 7.4, 0.4 mM DTPA), an ESR spectrum consist-
ing of a mixture of at least two different species was observed from
the beginning, whose intensity increased steadily for about 10 min.
At this point, 250 U/ml SOD was added and the spectrum recorded
immediately thereafter (Fig. 2a). In order to obtain the spectra of
individual components, the spectrum was recorded again after
a
b
3
0 min (Fig. 2b), resulting in a slightly different ratio of the compo-
nents. The difference spectrum shown in Figure 2c (spectrum ‘‘a”
subtracted from spectrum ‘‘b”) represents the CAEPO superoxide
adduct, which (assuming a mixture of two different isomers with-
4
out taking into account a possible dynamic exchange ) can best be
simulated using the following set of parameters (Fig. 2d):
a
N
= 13.18 G, a
H
= 9.95 G, 53%, and a
N
H
= 13.18 G, a = 12.25 G, 47%;
D
H = 1.75 G, ratio Gauss/Lorentz 1:1).
A typical exponential decay curve (CAEPO/ OOH, t1/2 = 16.95 min,
Å
2
R = 0.998) together with the respective exponential fit can be seen
in Figure 3, obtained from intensity measurements of the low-field
peak every 1.5 min, and after subtraction of the respective contribu-
tion of the secondary product mentioned below.
c
d
From the other spin traps investigated similar results were ob-
tained (HFS parameters, see Table 6), except that secondary prod-
ucts were formed much faster, see Figure 4a for the secondary
product formed from CAMPO (AMPO) and Figure 4b for its com-
puter simulation (a
N H N
= 14.43 G, a = 19.34 G, and a = 2.17 G,
D
H = 1.50 G, ratio Gauss/Lorentz 1:2). The formation of the second-
ary amino adduct was superoxide-dependent (no adduct formed
when SOD was added prior to xanthine oxidase), and an identical
secondary species was also detected in a system containing azide,
hydrogen peroxide, and HRP. The expected azide radical adduct
was barely visible and decayed rapidly (experiment not shown),
whereas the relative intensity of the secondary amino adduct in-
creased with time. Since these secondary products were formed
under oxidizing conditions, we assume that the second amino
group is located in position 5, formed according to a Hofmann deg-
radation reaction.
2
0 G
Figure 2. Mixture of radicals formed from the spin trap CAEPO incubated with
hypoxanthine/xanthine oxidase. (a) CAEPO (20 mM), catalase (250 U/mL), hypo-
xanthine (0.2 mM), and xanthine oxidase (500 mU/mL) in oxygenated phosphate
buffer (20 mM, pH 7.4, containing 0.4 mM DTPA) were incubated for 10 min, the
reaction stopped upon addition of SOD (250 U/ml) and measured using the
following EPR parameters: sweep width, 80 G; modulation amplitude, 0.5 G;
An additional control experiment, where CAMPO (500 mM) was
incubated with a sodium hypochlorite solution (2.5%) for 2 min,
followed by 1:12 dilution with 150 mM phosphate buffer (pH
5
microwave power, 20 mW; time constant, 0.08 s; receiver gain, 1 ꢁ 10 ; scan rate,
5
7 G/min. (b) Spectrum recorded for 30 min after (a), (c) difference (a ꢀ b), showing
the CAEPO superoxide adduct (d); and simulation of the spectrum.
Table 4
IR data (cmꢀ1) of the spin traps
EMPO12 2985 2940 2874 1741
CAMPO 3047 2939 2784 1679 1630 1587 1475 1446 1396 1379 1298 1218 1199 1115 1097 1029 998 953 885 843 744 700 656 611
—
1582 1464 1446 1377 1341 1288 1236 1182 1107
—
1024
—
950 926 862 796
—
—
—
CAEPO
CAPPO
CABPO
2974 2940 2883 1680
2963 2930 2875 1680 1614 1579 1464
2957 2930 2870 1681 1614 1577 1462
—
1580 1463
—
—
—
—
1386
—
1288 1208 1157 1124 1037
—
985 940
—
—
—
—
—
—
—
—
798
796
—
—
694 623
693 624
1383 1350 1293 1205 1181 1122 1042 1020 981 941
1381
1381
—
—
1294 1202
—
1124
—
—
1024
1011
—
—
949
954
792 696 624 603
796 696 628 602
CAPtPO 2956 2927 2862 1682 1613 1555 1460
1286 1221 1200 1123
Intensities: strong (1741), medium (1464), weak (950).

K. Stolze et al. / Bioorg. Med. Chem. 16 (2008) 8082–8089
8085
5
4
3
2
1
0000
tions. In principle, two different types of radical adducts can be
formed from alcohols: oxygen-centered alkoxyl radical adducts
and carbon-centered radical adducts. Alkoxyl radical adducts were
synthesized by nucleophilic addition of methanol or ethanol, using
.
[a.u.]
2
CAEPO / OOH
t
1/2 = 16.95 min
R
= 0.998
0000
10
a model system previously described by Dikalov et al. and al-
1
–3,11,12
ready tested with other spin traps.
As shown in Figure 6a,
0000
0000
0000
0
Å
CAPPO/ OCH
3
was obtained from methanol and iron-(III) chloride,
the mixture of which was diluted 1:50 after 2 min with 300 mM
phosphate buffer containing 20 mM DTPA. In the same manner,
Å
CAPPO/ OC
2
H
5
was obtained from ethanol and iron (III) chloride
Å
(
(
(
Fig. 6b), CAPtPO/ OCH
3
from methanol and iron-(III) chloride
from ethanol and iron-(III) chloride
Fig. 6d). All alkoxyl radical adducts remained only detectable for
Å
2 5
Fig. 6c), and CAPtPO/ OC H
a short period of time (1–5 min), after which the more stable car-
bon-centered adducts became predominant.
0
5
10
15
20
25
30 [min]
Å
Figure 3. Decay kinetics of the CAEPO/ OOH radical adduct ESR spectra of the
CAEPO/ OOH adduct (see above, Fig. 2a) were recorded at 1.5 min intervals for
0 min, the secondary product formed subtracted from each individual spectrum
and the intensity of the low-field peak measured (in arbitrary units). From the
exponential fit shown above (apparent first order) t1/2 = 16.95 min and r2 = of 0.998
were calculated.
Å
2.5. Carbon-centered radical adducts from methanol and
ethanol
3
In order to detect radical adducts of alcohol-derived carbon-
centered radicals (i.e., the hydroxymethyl and the a-hydroxyethyl
1
3
radicals), a Fenton-type system was chosen consisting of the spin
trap (40 mM), hydrogen peroxide (0.2%), EDTA (2 mM), and iron-
(II) sulfate (1 mM) in a mixture of methanol (or ethanol, respec-
tively) with water (20/80, v/v). After 10 s, the reaction was stopped
upon 1:1 dilution with phosphate buffer (300 mM, pH 7.4) contain-
ing DTPA (20 mM). In Figure 7a, the hydroxymethyl radical adduct
of CAPPO is shown, consisting of two different species
7
.4, containing 10 mM DTPA), also resulted in the formation of the
same secondary species (not shown). From these observations it
can be concluded that the second nitrogen splitting from this sec-
ondary product stems from an amino group attached to position 5
(
formed upon oxidative degradation of the carbamoyl group to an
amino group). The structure is in agreement with the HFS param-
eters of the DMPO amino adduct previously reported by Chignell
(a
N
= 14.53 G; a
H N H
= 23.08 G; 63%, and a = 14.70 G; a = 20.82 G;
et al.8 (a
= 15.9 G, a
= 19.3 G, a
= 1.6 G) and by Kirino et al.
9
N
H
N
H
a = 1.20 G; 37%), which can clearly be distinguished from the
(
(
a
N
= 15.86 G, a
H
= 19.04 G, a
N
= 1.72 G). Without isotope labeling
respective methoxyl radical adduct (a
N
= 13.50 G; a
H
= 9.69 G;
1
5
15
14 14
Å
using 50% N/ N plus 50% N/ N and looking for the formation
100%). In the same way, the adducts CAPPO/ CH(CH
3
)OH (Fig. 7b),
1
4
15
Å
Å
of N/ N products) we cannot, however, rule out an intermolec-
ular reaction pathway, leading to 2-amino substituted products.
From the other spin traps, less intensive ESR spectra were ob-
tained from the respective secondary products, some of which
were formed as mixtures of two stereoisomers, for example, from
CAPtPO/ CH
2
OH (Fig. 7c), and CAPtPO/ CH(CH
3
)OH (Fig. 7d) were
obtained.
2.6. Carbon-centered radical adducts from formate
CABPO (Fig. 4c; a
N
= 14.30 G, a
H
= 19.75 G, a
N
= 2.40 G, 53%; and
In order to detect the respective radical adducts of the carbon
1
3
a
N
= 14.05 G, a = 16.30 G, a
H
N
= 2.70 G, 47%), obtained by computer
dioxide anion radical, a Fenton-type system was chosen consist-
ing of the spin trap CAMPO (40 mM), hydrogen peroxide (0.2%),
EDTA (2 mM), and iron-(II) sulfate (1 mM) in aqueous sodium for-
mate solution (200 mM). After 10 s, the reaction was stopped upon
1:1 dilution with phosphate buffer (300 mM, pH 7.4) containing
simulation (Fig. 4d). All data are summarized in Table 6.
Half-lives of the superoxide adducts were determined from a
series of repetitive scans (every 90 s for 30 min) using a first-order
exponential decay approximation (Table 5, Pearson correlation
2
coefficient R > 0.97). In all cases, secondary products were sub-
DTPA (20 mM) (a
a = 18.30 G; 42%; Fig. 8a). Similarly, the adducts CAEPO= CO
N H N
= 14.46 G; a = 16.62 G; 58%; and a = 14.20 G;
Å
ꢀ
tracted from each individual spectrum before the decay kinetics
was calculated. In addition to a small contribution of the respective
hydroxyl radical adducts probably formed due to partial hydrolysis
of the spin adducts, increasing amounts of amino adducts were ob-
served, especially with CAMPO (AMPO), where the observation of a
neat superoxide adduct was virtually impossible.
H
2
Å
ꢀ
(Fig. 8b; a
N
= 14.04 G; a
H
= 19.31 G; 100%), CAPPO= CO (Fig. 8c),
2
Å
ꢀ
Å
ꢀ
CABPO= CO (Fig. 8d), and CAPtPO= CO (Fig. 8e) were obtained.
2
2
In the same way, the respective methyl adducts were formed,
except that DMSO (10%) was used instead of formate (spectrum
not shown). For detection of the H adducts, the respective spin
traps were incubated in the presence of a small amount of KBH
(ca. 0.5 mg/500 l) for 1 min, after which the pH was readjusted
to pH 7.4 by dilution with phosphate buffer (300 mM, pH 7.4).
4
2
.3. Hydroxyl radical adducts
l
Incubation of CAMPO (AMPO) in the presence of a Fenton sys-
An overview of the spin adducts of all investigated spin traps
and the comparison with the respective EMPO adducts is given
in Table 6.
tem resulted in the detection of the respective hydroxyl radical ad-
duct (Fig. 5a). The spectrum consists of two different stereoisomers
in the ratio 81/19, and was identified according to data recently
5
presented by Villamena et al. Similar results were obtained from
CAEPO (Fig. 5b), CAPPO (Fig. 5c), CABPO (Fig. 5d), and CAPtPO
3. Discussion
(Fig. 5e). Except for the latter two, the spectra of the hydroxyl ad-
ducts remained detectable for more than 30 min.
Four novel amino derivatives of the spin trap EMPO were syn-
thesized in this study and compared with a fifth, CAMPO (AMPO),
the synthesis and properties of which have recently been described
2
.4. Alkoxyl adducts from methanol and ethanol
5
,6
elsewhere.
The structure of the compounds was comprehen-
1
13
Methanol and ethanol were chosen as model compounds for
metabolites, for example, obtained from lipids after oxidation reac-
sively characterized by full NMR assignment ( H and C), ESI Q-
TOF MS, FTIR, and UV–vis spectroscopy (Tables 1–5).

8
086
K. Stolze et al. / Bioorg. Med. Chem. 16 (2008) 8082–8089
Table 6
Comparison of the EPR parameters of different radical adducts of various EMPO amino derivatives
*
Radical
ÅOOH
HFS (G) EMPO
CAMPO
CAEPO
CAPPO
CABPO
CAPtPO
*
*
**
trans
cis
x
(94%)
(6%)
13.25
10.15
(50%)
13.19
9.89
(50%)
13.19
12.09
(53%)
13.18
9.95
(47%) (50%)
13.18 13.25
12.25 9.50
(50%) (50%)
13.25 13.15
11.90 9.71
(50%) (50%)
13.15 13.23
12.01 9.80
(50%)
13.23
12.00
a
N
a
N
a
H
13.27 13.25
12.37 9.30
(55.5%/44.5% of trans ) 1.50
x
(
80%)5
(20%)5
13.1
12.5
a
N
a
H
a
H
13.0
10.8
—
1.75
**
**
trans
cis
ÅOH
(76%)
14.11
12.80
0.63
(24%)
14.18
15.27
0.62
(81%)
13.97
13.64
—
(19%)
13.95
12.45
—
(68%)
14.00
14.85
—
(32%) (67%)
13.80 14.06
11.65 14.71
(33%) (50%)
13.80 13.52
11.67 13.75
(50%) (74%)
14.70 14.00
22.00 14.95
(26%)
13.63
12.05
—
a
N
a
H
a
H
a
H
a
H
a
H
—
—
—
—
—
—
0.43
0.50
0.29
0.07
(69%)5
14.0
(31%)5
14.0
(
(
3)
2)
(3)
0.21
0.13
a
a
N
(2)
H
13.5
12.5
ÅH
a
N
a
N
a
H
a
H
(100%)
15.52
22.21
20.82
(100%)
15.30
22.23
20.00
(100%)
15.16
23.12
19.35
(100%)
15.21
23.05
19.37
(100%)
15.17
23.09
19.39
(100%)
15.20
23.03
19.39
ÅCH
(100%)
15.42
22.30
—
(100%)
15.15
21.43
—
(54%)
15.06
23.63
—
(46%) (53%)
15.06 15.06
20.43 23.53
(47%) (53%)
15.06 15.06
20.53 23.51
(47%) (50%)
15.06 14.97
20.51 21.71
(50%)
14.47
22.21
—
3
a
N
a
H
a
H
—
—
—
—
—
—
—
ÅOCH
(50%)
13.74
10.87
—
(50%)
13.74
7.81
—
(74%)
14.37
19.14
2.13
(26%)
14.63
19.81
—
(100%)
13.45
9.90
—
(100%)
13.50
9.69
—
(100%)
13.52
9.64
—
(100%)
13.50
10.25
3
a
N
a
H
a
H
–
ÅCH
(100%)
14.95
21.25
—
(100%)
14.75
21.20
—
(63%)
14.50
23.00
—
(37%) (63%)
14.70 14.53
21.30 23.08
(37%) (69%)
14.70 14.53
20.82 23.04
(31%) (60%)
14.72 14.54
20.63 23.11
(40%)
14.74
20.69
1.20
2
OH
a
N
a
H
a
H
1.45
—
1.20
—
1.20
—
ÅOC
H
5
(50%)
13.75
10.88
—
(50%)
13.75
8.08
—
(74%)
14.35
19.10
2.13
(26%)
14.63
19.88
—
(100%)
13.70
10.70
—
(100%)
13.60
10.00
—
(100%)
13.64
10.00
—
(100%)
13.55
10.50
—
2
a
N
a
H
a
H
ÅCH(OH)CH
(67%)
14.94
20.82
(33%)
15.00
22.40
(100%)
14.77
21.34
(50%)
14.90
21.96
(50%) (50%)
14.30 14.85
22.56 22.01
(50%) (50%)
14.35 14.85
22.51 21.93
(50%) (50%)
14.35 14.95
22.43 21.75
(50%)
14.45
22.25
3
a
a
N
H
ÅCO
2ꢀ
(100%)
14.74
17.16
(58/53 %)
5
(42/47 %)
5
(100%)
(100%)
14.06
19.15
(100%)
14.07
19.15
(100%)
14.05
19.24
5
a
a
N
14.46/14.535 14.20/14.25 14.04
16.62/16.48 18.30/18.155 19.31
5
H
5
Villamena et al.
NH
2
adduct (sec. product)
—
—
—
—
(100%)
14.43
19.34
2.17
(56%)
14.38
19.93
2.18
(44%) (100%)
14.10 14.30
16.60 19.78
(53%)
14.30
19.75
2.40
(47%) (54%)
14.05 14.30
16.30 19.60
(46%)
14.05
16.50
2.70
a
N
a
H
a
N
2.85
2.15
2.70
2.40
*Data from Stolze et al.12
*
*
4
x
5
5
;
d
a
t
a
f
r
o
m
C
u
l
c
a
s
i
e
t
a
l
.
(
m
i
x
t
u
r
e
o
f
r
o
t
a
m
e
r
s
)
;
d
a
t
a
f
r
o
m
V
i
l
l
a
m
e
n
a
e
t
a
l
.
.
Recent findings have shown that secondary products formed
from decaying superoxide adducts can also be used as indicators
of superoxide formation, especially when the relative ratios of dia-
stereomers formed (cis and trans forms as well as rotamers) are
of the amino adduct was superoxide-dependent in the xanthine
oxidase system (no adduct formed in the presence of SOD), it can-
not be regarded as specific for superoxide, since it was also de-
tected as a secondary product in other oxidizing systems, such as
mixtures containing the spin trap and hypochlorite or incubations
containing azide, hydrogen peroxide, and HRP (experiment not
shown).
The stability of the hydroxyl radical adducts varied consider-
ably. While the hydroxyl adducts of CAMPO (AMPO), CAEPO, and
CAPPO were stable for 30 min or more, the respective adducts of
CABPO and CAPtPO were stable only for a couple of minutes, form-
ing as yet unknown secondary products, possibly due to attack at
the alkyl side chain, followed by trapping by a second molecule
of the spin trap, leading to broad, unresolved ESR lines. The relative
change of the ratio between the different diastereomers does not
seem to depend solely on the effect of steric hindrance of the 5-al-
1
4–18
considered.
In order to draw unequivocal conclusions, however, well re-
solved ESR lines are a prerequisite, as well as the absence of sec-
ondary products formed in other side reactions.
We did not determine the rate constants of the superoxide trap-
ping reactions, especially because of excessive formation of 5-ami-
no adducts observed with all spin traps. Since the formation of the
amino adducts was still observed after additional purification
steps, we assume that the amino group is introduced by oxidative
degradation of the 5-carbamoyl group to a 5-amino group (Hof-
mann degradation) and does not stem from impurities due to
hydrolytic degradation of the amide group. Although the detection

K. Stolze et al. / Bioorg. Med. Chem. 16 (2008) 8082–8089
8087
a
b
c
d
Figure 5. Formation of hydroxyl radical adducts of the spin traps CAMPO (AMPO),
CAEPO, CAPPO, CABPO, and CAPtPO (a) CAMPO (AMPO) (40 mM, initial concentra-
tion) was incubated with a Fenton system containing FeSO
4
(1 mM), EDTA (2 mM),
and H (0.2%). The reaction was stopped after 10 s by 1:1 dilution with phosphate
2 2
O
buffer (300 mM, pH 7.4, containing 20 mM DTPA) and the spectrum was recorded
using the following spectrometer settings: sweep width, 80 G; modulation ampli-
2
0 G
tude, 0.21 G; microwave power, 20 mW; time constant, 0.08 s; receiver gain,
4
2
ꢁ 10 ; and scan rate, 57 G/min; (b) incubation as in (a), except that CAEPO
(
40 mM, init. conc.) was used; (c) incubation as in (a), except that CAPPO (40 mM,
Figure 4. Amino adducts formed as secondary products from the hydroperoxide
adducts of the spin traps CAMPO (AMPO) and CABPO. (a) CAMPO (AMPO) (20 mM),
catalase (250 U/mL), xanthine (0.2 mM), and xanthine oxidase (500 mU/mL) in
oxygenated phosphate buffer (20 mM, pH 7.4, containing 0.4 mM DTPA) were
incubated for 10 min and measured using the following EPR parameters: sweep
width, 80 G; modulation amplitude, 0.5 G; microwave power, 20 mW; time
init. conc.) was used; (b) incubation as in (a), except that CABPO (40 mM, init. conc.)
was used; and (b) incubation as in (a), except that CAPtPO (40 mM, init. conc.) was
used.
4
constant, 0.08 s; receiver gain, 5 ꢁ 10 ; and scan rate, 57 G/min. The spectrum
Å
cidate their different pathways of formation (as those observed
with azide/HRP).
shown is the secondary CAMPO (AMPO)/ NH
2
adduct seen after the decay of the
Å
initially formed CAMPO (AMPO)/ OOH adduct; (b) simulation of the EPR spectrum
shown in Figure 2a. (c) Same as in (a), except that CABPO (20 mM) was used; and
(
d) Simulation of the EPR spectrum shown in Figure 2c.
5
5
. Experimental
.1. Chemicals
kyl chain, since there is no gradual change from the ratio from the
methyl to the pentyl derivatives, as can be seen in Table 6. Instead,
a combination of steric and electronic factors seems to be respon-
sible for the different stabilities of the diastereomers.
Acrolein and triethylamine were commercially available from
Fluka, all other chemicals from VWR.
Methoxyl and ethoxyl radical adducts were rather unstable and
difficult to detect, whereas carbon-centered adducts from metha-
nol, ethanol, or formate were readily detectable.
5
.2. Syntheses
Synthesis and characterization of the compounds were per-
1
–3
formed in analogy to those reported previously
of EMPO and its derivatives
ven below.
for the synthesis
1
1,12,19–22
4
. Conclusion
with minor adaptations as gi-
In conclusion, the four novel EMPO-derived carbamoyl deriva-
tives are forming superoxide adducts which were slightly more
5.2.1. Synthesis of the ethyl 2-nitroalkanoates
11,12,19–22
stable compared to the parent compound EMPO,
the most
The respective ethyl 2-bromoalkanoate (-propionate for CAM-
PO, -butanoate for CAEPO, -pentanoate for CAPPO, -hexanoate for
CABPO, or -heptanoate for CAPtPO) was added under stirring to a
solution of sodium nitrite (7.2 g, 104 mmol) and phloroglucinol
(6.6 g, 52 mmol) in dry N,N-dimethyl formamide (120 ml) at room
temperature. The solution was stirred overnight, poured into ice
water (240 ml), and extracted four times with ethyl acetate
Å
23
stable adduct (CAEPO/ OOH) being comparable to DEPMPO. In
addition, secondary products (amino adducts) are formed under
oxidizing conditions which interfere with the EPR spectra of the
initially formed adducts. In the case of CAMPO (AMPO), the inten-
sity of the amino compound (SOD-sensitive) is very high for more
than 30 min, so that further studies might be useful in order to elu-

8
088
K. Stolze et al. / Bioorg. Med. Chem. 16 (2008) 8082–8089
Figure 6. Formation of alkoxyl spin adducts from CAPPO and CAPtPO in the
presence of methanol or ethanol. (a) After a 10 s incubation of CAPPO (1 M, init.
conc. in methanol) with FeCl
:20 dilution with phosphate buffer (0.3 M, pH 7.4, containing 20 mM DTPA), and
the spectrum was recorded with the following spectrometer settings: sweep width,
3
(2 mM in methanol), the reaction was stopped by
1
Figure 7. Iron-dependent formation of carbon-centered spin adducts from CAPPO
and CAPtPO in the presence of methanol or ethanol. (a) CAPPO (40 mM, initial
8
0
0 G; modulation amplitude, 1.48 G; microwave power, 20 mW; time constant,
.02 s; receiver gain, 2 ꢁ 10 ; and scan rate, 229 G/min; (b) same as in (a), except
5
concentration) was incubated with a Fenton system containing FeSO
4
(1 mM), EDTA
that CAPPO (1 M, init. conc. in ethanol) was used; (c) same as in (a), except that
CAPtPO (1 M, init. conc. in methanol) was used; and (d) same as in (a), except that
CAPtPO (1 M, init. conc. in ethanol) was used.
(
2 mM), H (0.2%) in 10% methanol. The reaction was stopped after 10 s by 1:1
2 2
O
dilution with phosphate buffer (300 mM, pH 7.4, containing 20 mM DTPA) and the
spectrum was recorded using the following spectrometer settings: sweep width,
8
0
0 G; modulation amplitude, 0.21 G; microwave power, 20 mW; time constant,
.08 s; receiver gain, 4 ꢁ 10 ; scan rate, 57 G/min; (b) same as in (a), except that
4
ethanol was used; (c) same as in (a), except that CAPtPO was used; and (d) same as
in (a), except that CAPtPO and ethanol were used.
(
100 ml). The combined extracts were treated with 100 ml of a
NaHCO /Na CO solution and dried over MgSO . After removal of
3
2
3
4
the solids by filtration, the solvent was evaporated in vacuo. Dee-
ply colored products were extracted with water. After removal of
the solvent from the organic phase, the pale yellow product was
used without further purification the aqueous phase (containing
the major part of the colored impurities) was discarded. Average
yield: 85% (crude product).
added within 30 min, the mixture was carefully kept at room tem-
perature. The mixture was stirred for 4.5 h at room temperature,
the white precipitate and the remaining zinc powder were re-
moved by filtration, and the residue was washed five times with
methanol (30 ml). The liquid phase was concentrated to about
1
0 ml and extracted four times with 60 ml CH
2 2
Cl . The organic
5
.2.2. Ethyl 2-alkyl-2-nitro-4-oxobutanoate
phase was dried with MgSO , filtered, and concentrated. Column
4
The respective ethyl 2-nitroalkanoate was dissolved in a mix-
ture of acetonitrile (10 g, 244 mmol) and triethylamine (0.2 g,
mmol). Acrolein (2 g, 38 mmol) was slowly added at 0 °C. The
solution was kept at 10 °C for 1.5 h and then poured into a solution
chromatography on silica gel with a petroleum ether/ethanol gra-
dient allowed the separation from the majority of side products
and provided a dark yellow or light brown product. Additional
purification was done on a 1 ml solid phase extraction column
using a Chromabond C-18 100 mg column obtained from Mache-
rey-Nagel (Düren, Germany), using a water/methanol gradient as
the eluant. Colored solutions were subjected to the same proce-
dure until the solution containing the N-oxides remained colorless.
2
of ice-cold HCl (5 ml of concentrated HCl in 150 ml of water). The
2 2
solution was extracted three times with CH Cl and dried over
MgSO . After filtration, the mixture was distilled under reduced
4
pressure, and the purity of the remaining product was assessed
by thin layer chromatography and IR spectroscopy. Average yield:
2 2
The products were extracted three times with CH Cl , the solvent
8
4% (crude product).
was removed, and the identity checked by TLC, IR, and UV–vis
spectroscopy. Average yield: 46% (purified product).
5
.2.3. Synthesis of the nitrones
Synthesis of the nitrones was performed according to the proce-
5.2.4. Synthesis of the 5-alkyl-5-carbamoylpyrroline N-oxides
Synthesis of the 5-alkyl-5-carbamoylpyrroline N-oxides was
performed according to Villamena et al. Briefly, the respective
nitrones (EMPO, EEPO, EPPO, EBPO, and EPtPO) were dissolved in
aqueous ammonia solution and stirred for several days until the
spot (TLC) or peak (HPLC) of the original N-oxide had completely
1
–
dure described recently for the synthesis of EMPO derivatives.
3
,11,12
5,6
To a concentrated solution of 25 mmol of the respective
ethyl-2-alkyl-2-nitro-4-oxobutanoate in H O/CH OH (v/v = 6:4),
2
3
an aqueous solution of ammonium chloride (1.87 g in 8 ml of
water) was added. While zinc dust (8.5 g, 130 mmol) was slowly

K. Stolze et al. / Bioorg. Med. Chem. 16 (2008) 8082–8089
8089
HMBC (heteronuclear multiple bond connectivity) spectra. All
chemical shift data are given in ppm units (Tables 1 and 2).
Mass spectra were obtained as follows (Table 3): Samples were
diluted in the ratio 1:10,000 in 70% methanol containing 0.1% for-
mic acid and injected offline to ESI Q-TOF MS on a Waters Micro-
mass Q-TOF Ultima Global at a flow rate of 5 ll/min. Capillary
voltage was adjusted bewteen 1.2 and 3.0 kV. Data analysis was
performed with MassLynx 4.0 SP4 Software (Waters Micromass).
IR spectra were recorded as film on an ATI Mattson Genesis Ser-
ies FTIR spectrometer (see also Table 4).
UV–vis spectra were recorded on Hitachi 150-20 and U-3300
spectrophotometers in double-beam mode against a blank of the
respective solvent (Table 5). Determination of the concentrations
was done measuring the absorption maxima in the range between
2
5
00 and 300 nm. For determination of the partition coefficients,
00 l of n-octanol was added to 500 l of a solution of the respec-
l
l
tive spin trap (100 mM or 5–10 mg, respectively) in 100 mM phos-
phate buffer, pH 7.4. The mixture was vortexed for 2 min at room
temperature. If necessary, the procedure was repeated several
times, until equilibration between the two phases was achieved.
After careful separation of the phases, the absorbance was read
at the maximum around 235 nm after dilution with methanol.
For EPR experiments, Bruker spectrometers (ESP300E and EMX)
were used, operating at 9.7 GHz with 100 kHz modulation fre-
quency, equipped with a rectangular TE102 or a TM110 microwave
cavity. All calculations for spectral simulation were done using
the SinFonia Program by Bruker (Table 6).
Acknowledgments
The authors thank P. Jodl for skilful technical assistance in syn-
thesis, purification, and characterization of the spin traps.
Figure 8. Iron-dependent formation of carbon-centered spin adducts from CAMPO,
CAEPO, CAPPO, CABPO, and CAPtPO in the presence of formate. (a) CAMPO (40 mM,
init. conc.) was incubated with a Fenton system containing FeSO
4
(1 mM), EDTA
(0.2%), and sodium formate (200 mM). The reaction was stopped after
0 s by 1:1 dilution with phosphate buffer (300 mM, pH 7.4, containing 20 mM
References and notes
(
2 2
2 mM), H O
1
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DTPA), and the spectrum was recorded using the following spectrometer settings:
sweep width, 80 G; modulation amplitude, 0.2 G; microwave power, 20 mW; time
4
constant, 0.08 s; receiver gain, 5 ꢁ 10 ; scan rate, 57 G/min; (b) same as in (a),
3.
4.
5.
6.
7.
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except that CAEPO was used; (c) same as in (a), except that CAPPO was used; (d)
same as in (a), except that CABPO was used; and (e) same as in (a), except that
CAPtPO was used.
disappeared. If dark colored secondary products were formed in
larger amounts (e.g., during the synthesis of CAMPO), the incu-
bation was stopped earlier. After removal of the aqueous ammo-
nia solution, the crude products were subjected to column
chromatography on silica gel using methylene chloride/ethanol
mixtures as the eluants. After purification on 1 ml solid phase
extraction columns as described above (Chromabond C-18
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0. Dikalov, S. I.; Mason, R. P. Free Radic. Biol. Med. 2001, 30, 187.
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4
1
00 mg columns), the compounds were obtained as white crys-
1
tals or colorless oils that crystallized in the refrigerator after sev-
eral days. The purity of the obtained products was assessed by
2
7
TLC and UV–vis spectroscopy. Final identification of the purified
products was performed by 1H NMR, 13C NMR, and IR spectros-
1
1
1
1
2
6. Karoui, H.; Clément, J.-L.; Rockenbauer, A.; Siri, D.; Tordo, P. Tetrahedron Lett.
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copy. Purity was confirmed by HRMS (see Tables 1–4). Average
yield: 29% (purified product).
5
.3. Instruments
NMR spectra were recorded on a Bruker Avance at 400 MHz for
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P.; Tordo, P.; Kalyanaraman, B. FEBS Lett. 2000, 473, 58.
1
H, and 100 MHz for 13C at 22 °C. CDCl
3
was used as the solvent
throughout, TMS (tetramethylsilane) as the internal standard. Con-
centrations were set to 10 mg sample/0.6 ml solvent. C peaks
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2001, 31, 599.
1
3
2
2
2. Weaver, J.; Tsai, P.; Pou, S.; Rosen, G. M. J. Org. Chem. 2004, 69, 8423.
3. Fréjaville, C.; Karoui, H.; Tuccio, B.; Le Moigne, F.; Culcasi, M.; Pietri, S.;
Lauricella, R.; Tordo, P. J. Med. Chem. 1995, 38, 258.
were assigned by means of APT (attached proton test), HMQC
1
(
H-detected heteronuclear multiple-quantum coherence), and