A. Patel et al. / Bioorg. Med. Chem. 19 (2011) 7643–7652
7651
0
(1
CH
2 2
), 62.00 (4aCH
), 83.25 ( C), 134.13 ( CH), 169.07 (5bC). Anal.
(215.24): C 55.80, H 7.96, N 6.51; found: C
5.75, H 7.93, N, 6.56. HRMS calcd for C10
5
2
17 4 1
Calcd. for C10H O N
5
N
17
H O
4
N
1
[C10
H
17
O
4
+
a
+H] : 216.24. Found: 216.24.
1
5
.1.3. 4-Hydroxymethyl-5-methyl-5-propoxycarbonyl-pyrroline
N-oxide (HMPPO)
2 2 2 2
Column chromatography: gradient CH Cl ? CH Cl /MeOH
(
R
(
v/v = 99:1), color reaction with ninhydrine reagent: green.
1
f
= 0.26 (CH
2
Cl
2
/MeOH, v/v = 9:1), colorless oil. H NMR: d 0.94
0
0
3
5a
2
3H, t, CH
3
), 1.60 (3H, s, CH
3
), 1.70–1.76 (2H, m, CH
2
), 2.42–
b
c
3
3
2
.56 (1H, m, CH
2
), 2.76–2.91 (1H, m, CH
2
), 2.93–3.06 (1H, m,
0
4
4a
1
CH), 3.64–3.80 (2H, m, CH
2
3
), 4.10–4.21 (2H, m, CH
2
), 6.92 (1H,
0
0
2
13
5a
2
t, CH). C NMR: d 10.61 ( CH
3
), 14.65 ( CH
3
), 21.88 ( CH
2
),
0
3
4
4a
1
5
2
1
5
9.80 ( CH
2
), 44.11 ( CH), 61.45 ( CH
2
), 68.01 ( CH
2
), 81.31 ( C),
(215.24): C
2
5b
35.01 ( CH), 170.36 ( C). Anal. Calcd. for C10
H
17
O
4
N
1
5.80, H 7.96, N 6.51; found: C 55.79, H 7.95, N 6.52. HRMS calcd
+
17 4 1 17 4 1
for C10H O N [C10H O N + H] : 216.24. Found: 216.24.
5
.1.4. 4-Hydroxymethyl-5-methyl-5-iso-propoxycarbonyl-
pyrroline N-oxide (HMiPPO)
Column chromatography: gradient CH
v = 99:1), color reaction with ninhydrine reagent: green, R
2
Cl
2
? CH
2
Cl
2
/MeOH (v/
= 0.26
f
1
(
CH
2
Cl
2
/MeOH, v/v = 9:1), colorless oil. H NMR: d 1.22 (6H, d,
0
00
2
,2
5a
3
CH
3
), 1.58 (3H, s, CH
3
), 2.42–3.53 (1H, m, CH
2
), 2.73–2.87
3
4
4a
(
1H, m, CH
2
), 2.87–2.95 (1H, m, CH), 3.56–3.72 (2H, m, CH
2
),
d
1
0
2
13
4
.96–5.08 (1H, m, CH), 6.91 (1H, t, CH). C NMR: d 14.54
0
00
5
a
2 ,2
3
4
4a
(
3
CH ), 21.47 (
CH
3
), 29.95 ( CH
2
), 43.81 ( CH), 61.12 ( CH
2
),
0
1
5
2
5b
7
0.15 ( CH), 81.09 ( C), 135.92 ( CH), 169.34 ( C). Anal. Calcd.
for C10
H 7.98, N 6.50. HRMS calcd for C10
16.24. Found: 216.23.
17 4 1
H O N (215.24): C 55.80, H 7.96, N 6.51; found: C 55.76,
2
0 G
+
17 4 1 17 4 1
H O N [C10H O N +H] :
2
Figure 9. Formation of hydrogen spin adducts (reduction products) from EHMPO,
EEHPO, HMPPO and HMiPPO and KBH .a) EHMPO (50 mM, initial concentration)
was incubated with approximately 1 mg KBH for 1 min, then EDTA (2 mM) was
added, the solution was diluted 1:1 with phosphate buffer (300 mM, pH 7.4,
containing 20 mM DTPA), a few drops of a K Fe(CN) solution were added until the
4
4
Acknowledgments
3
6
The authors thank Christian-Doppler Society for financial sup-
port of this investigation, Dr. Andreas Hofinger for NMR measure-
ments, and Mr. Philipp Jodl for skilful technical assistance in
synthesis, purification, and characterization of the spin traps.
mixture remained slightly yellow and the spectrum was recorded using the
following spectrometer settings: sweep width, 120 G; modulation amplitude, 0.1 G;
3
microwave power, 20 mW; time constant, 0.08 s; receiver gain, 5 ꢁ 10 ; scan rate,
8
1
6 G/min, 1024 data points. (b) same as in a), except that EEHPO was used (gain
4
ꢁ 10 , modulation amplitude, 0.2 G). (c) same as in a), except that HMPPO was
4
used (gain 1 ꢁ 10 , modulation amplitude, 0.2 G). (d) same as in a), except that
4
HMiPPO was used (gain 1 ꢁ 10 , modulation amplitude, 0.2 G).
Supplementary data
1
Supplementary data (experimental procedures and spectra ( H,
5
.1.1. 5-Ethoxycarbonyl-4-hydroxymethyl-5-methyl-pyrroline
1
3
N-oxide (EHMPO)
2 2 2 2
Column chromatography: gradient CH Cl ? CH Cl /MeOH
(
v/v = 99:1), colorless oil, color reaction with ninhydrine reagent:
green. R
f
= 0.20 (CH
2
Cl
2
/MeOH, v/v = 9:1). 1H NMR: d 1.29 (3H, t,
0
5a
3
2
CH
3
), 1.64 (3H, s, CH
3
), 2.45–3.51 (1H, ddd, CH
2
), 2.79–2.87
References and notes
3
4
4a
(
1H, ddd, CH
2
), 3.04 (1H, m, CH), 3.66–3.78 (2H, m, CH
2
),
2
), 6.92 (1H, t, CH). C NMR: d 14.00
0
1
2
13
1. Fréjaville, C.; Karoui, H.; Tuccio, B.; Le Moigne, F.; Culcasi, M.; Pietri, S.;
4
.19–4.32 (2H, m, CH
0
Lauricella, R.; Tordo, P. J. Med. Chem. 1995, 38, 258.
Stolze, K.; Udilova, N.; Nohl, H. Free Radical Biol. Med. 2000, 29, 1005.
3. Stolze, K.; Udilova, N.; Nohl, H. Biol. Chem. 2002, 383, 813.
2
5a
3
4
1
(
CH
3
), 14.85 ( CH
3
), 29.78 ( CH
2
), 43.79 ( CH), 61.34 ( CH
2
),
), 81.17 ( C), 134.83 ( CH), 170.07 ( C). Anal. Calcd.
9 15 4 1
H O N (201.22): C 53.72, H 7.51, N 6.96; found: C 53.70,
2
.
4
a
5
2
5b
6
2.61 ( CH
2
4
5
6
7
8
9
.
.
.
.
.
.
Stolze, K.; Udilova, N.; Rosenau, T.; Hofinger, A.; Nohl, H. Biol. Chem. 2003, 384,
93.
for C
H 7.52, N, 690. HRMS calcd for C
2
4
+
9 15 4 1 9 15 4 1
H O N [C H O N +H] :
Stolze, K.; Rohr-Udilova, N.; Rosenau, T.; Stadtmüller, R.; Nohl, H. Biochem.
Pharmacol. 2005, 69, 1351.
Stolze, K.; Rohr-Udilova, N.; Rosenau, T.; Hofinger, A.; Kolarich, D.; Nohl, H.
Bioorg. Med. Chem. 2006, 14, 3368.
Stolze, K.; Rohr-Udilova, N.; Rosenau, T.; Hofinger, A.; Nohl, H. Bioorg. Med.
Chem. 2007, 15, 2827.
Hardy, M.; Chalier, F.; Ouari, O.; Finet, J.-P.; Rockenbauer, A.; Tordo, P. Chem.
Commun. 2007, 1083.
02.22. Found: 202.22.
5
.1.2. 5-Ethoxycarbonyl-5-ethyl-4-hydroxymethyl-pyrroline N-
oxide (EEHPO)
Column chromatography: gradient CH
2
Cl
2
? CH
2
Cl
2
/MeOH (v/
= 0.24
v = 99:1), color reaction with ninhydrine reagent: green. R
f
Chalier, F.; Hardy, M.; Ouari, O.; Rockenbauer, A.; Tordo, P. J. Org. Chem. 2007,
72, 7886.
1
(
2
CH
2
Cl
2
/MeOH, v/v = 9:1), colorless oil. H NMR: d 1.29 (3H, t,
0
5a
3
CH
3
), 1.64 (3H, s, CH
3
), 2.45–3.51 (1H, ddd, CH
), 3.04 (1H, m, CH), 3.66–3.78 (2H, m, CH
.19–4.32 (2H, m, CH ), 6.92 (1H, t, CH). C NMR: 8.22 ( CH
2
), 2.79–2.87
10. Hardy, M.; Rockenbauer, A.; Vasquez-Vivar, J.; Felix, C.; Lopez, M.; Srinivasan,
S.; Avadhani, N.; Tordo, Paul.; Kalyanaraman, B. Chem. Res. Toxicol. 2007, 20,
1053.
3
4
4a
(
1H, ddd, CH
2
2
),
),
0
0
5 a
1
2
13
4
1
2
5a
3
1
1. Stolze, K.; Rohr-Udilova, N.; Patel, A.; Rosenau, T. Bioorg. Med. Chem. 2011, 19,
0
2
3
4
3 2
3.19 ( CH ), 22.00 ( CH ), 29.98 ( CH
2
), 43.51 ( CH), 60.00
985–993.