Synthesis of 5-(alkylphenylphosphoryl)-5-methyl-3,4-dihydro-2H-pyrroline N-oxide as a new spin trapping reagent

Article abstract of DOI:10.1246/cl.2003.604

Novel DMPO analogues attached to a phenyl group at the phosphorus atom were synthesized and examined for their ability in the spin trapping of oxygen-centered radicals. The lipophilicities of these analogues were higher than those of DMPO.

Full text of DOI:10.1246/cl.2003.604

604
Chemistry Letters Vol.32, No.7 (2003)
Synthesis of 5-(Alkylphenylphosphoryl)-5-methyl-3,4-dihydro-2H-pyrroline N-Oxide as a New
Spin Trapping Reagent
Kosei Shioji,ꢀ Shigeyuki Tsukimoto, Hidehiko Tanaka, and Kentaro Okuma
Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 814-0180
(Received March 25, 2003;CL-030253)
Novel DMPO analogues attached to a phenyl group at the
phosphorus atom were synthesized and examined for their abil-
ity in the spin trapping of oxygen-centered radicals. The lipo-
philicities of these analogues were higher than those of DMPO.
Table 1. These signals were inhibited by the presence of cata-
lase (50 U/mL).
O
O
P
Me
P
OEt
RMgBr
H
H
THF, at -20 °C
Spin trapping studies have been applied to the detection of
oxygen-centered radicals in biological processes.1 5,5-Di-
methyl-3,4-dihydro-2H-pyrroline N-oxide (DMPO) is widely
used as a spin trapping reagent,2 but has some limitations.3
The reaction rate with superoxide is low and the spin adduct de-
composes rapidly.4 The partition coefficient of DMPO between
1-octanol and water has been found to be only 0.02–0.1.5 Many
researchers have reported the improved synthesis of DMPO for
biological uses and more lipophilic derivatives.6 One of these
reports revealed that phosphoryl-DMPOs, such as 5-(diethoxy
phosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), have
longer life times than those of the original DMPO. Moreover,
the phenyl group attached to the pyrroline ring enhanced the
lipophilicity of phosphoryl-DMPO derivatives.7 However, there
is no report on the synthesis and properties of modified DMPO,
which contains non-alkoxy substituents at the phosphorus atom.
We report herein the synthesis of a new class of phosphoryl-
DMPO containing a phenyl group at the phosphorus atom,
and the substitution effect toward solubility and electronic prop-
erties.
1b
1a
O
P
R
O
Cl
+ NH3
P
R
H
O
N
H
EtOH, at 50 °C
2
1
O
P
R
m-CPBA
N
O
CHCl3, at -10 °C
3
3a; R = Me, 3b; R = OEt
Scheme 1.
O
P
R
O
P
R
OH
OH
N
O
N
O
3-OH
3
Scheme 2.
Secondary phosphine oxide 1a was prepared by the reaction
of ethyl phenylphosphinate 1b with methylmagnesium bromide
(90% yield). Pyrroline N-oxide 3 was synthesized according to
a method described by Tordo.8 Pyrrolidine 2 was obtained by
bubbling ammonia into ethanol solution of 5-chloropentane-2-
one and phosphine oxide 1. The oxidation of 2 with m-CPBA
was the most sensitive step in the synthetic pathway and was
carried out at ꢁ10 ꢂC to give 3a and 3b as a mixture of four dia-
stereoisomers in 43% and 49% yields, respectively (Scheme 1).
The ratio of the major 3a to the minor product was 5:1, whereas
that of 3b was 1:1. The major isomer of the methyl derivative
3a was able to be separated by column chromatography for use
as a spin trap.9
The trapping of hydroxyl radicals with pyrroline N-oxides 3
in the Fenton reaction was carried out under the following con-
ditions (Scheme 2): spin trap (10 mol dmꢁ3, 200 mL), 30%
H2O2(100 mL), K4[Fe(CN6)] (10 mmol dmꢁ3, 100 mL). The
ESR measurements were performed with the following settings:
microwave power 10 mW, field 336:4 ꢃ 5 mT, modulation
0.1 mT, time constant 0.3 sec, sweep time 8 min. Under these
conditions, the ESR signal of 3a, b showed a doublet of doub-
lets of triplets pattern which is split by a large phosphorus cou-
pling (Figure 1). The hyperfine splitting constants calculated
from the spectra are summarized in Entries 1 and 2 of
Figure 1. ESR Spectra of spin adducts obtained by
the reaction with superoxide radicals (a) 3a-OH
(pH 7.4); 3b-OH (pH 7.4).
The trapping of superoxide radicals with pyrroline N-oxides
3 was also examined using an aqueous reaction (phosphate buf-
fer, pH 7.4) of xanthine (0.4 mmol dmꢁ3, 800 mL) and xanthine
oxidase (0.5 U/mL, 800 mL) in the presence of 200 mL
(10 mol dmꢁ3) of a spin trap (Scheme 3). The ESR measure-
ments were made with the settings: microwave power 10 mW,
field 335 ꢃ 5 mT, modulation 0.1 mT, time constant 0.1 sec,
and sweep time 8 min. The ESR signal of the spin adduct ob-
ꢄ
ꢁ
tained in the reaction of 3a and O2 is shown in Figure 2.
Copyright Ó 2003 The Chemical Society of Japan

Chemistry Letters Vol.32, No.7 (2003)
605
The signal disappeared with the addition of superoxide dismu-
tase (320 U/mL). Hfsc’s of 3-OH and 3-OOH are listed in En-
tries 3 and 4 of Table 1.
found that the partition coefficient of the methyl derivative 3a
attained 0.7.
We succeeded in synthesizing lipophilic phosphoryl DMPO
analogues. These analogues are suitable for spin trapping ex-
periments that involve interfacially-generated (between the cell
and the outer sphere) or intracellularly-generated free radicals.
O
P
R
O
P
R
OOH
O2
N
O
N
O
References and Notes
3
3-OOH
1
a) ‘‘Oxidative Stress,’’ ed. by H. Sies, Academic Press, Lon-
don (1985). b) B. Halliwell and J. M. C. Gutteridge, ‘‘Free
Radicals in Biology and Medicine,’’ 2nd. ed., Oxford Uni-
versity Press, Oxford (1989).
Scheme 3.
2
3
a) E. G. Janzen and J. I.-P. Liu, J. Magn. Reson., 9, 510
(1973). b) E. G. Janzen and D. L. Haire, in ‘‘Advances in
Free Radical Chemistry,’’ JAI Press, Inc., (1990), p 253.
a) E. Finkelstein, G. M. Rosen, and E. J. Rauckman, Arch.
Biochem. Biophys., 200, 1 (1980). b) G. M. Rosen and E.
Finkelstein, Adv. Free Radical Biol. Med., 1, 345 (1985).
c) J. R. Harbour, V. Chow, and J. R. Bolton, Can. J. Chem.,
52, 3549 (1974). d) G. R. Buettner, Free Radical Biol. Med.,
3, 259 (1987). e) S. Pou, D. J. Hassett, E. B. Britigan, M. S.
Cohen, and G. M. Rosen, Anal. Biochem., 177, 1 (1989).
E. Finkelstein, G. M. Rosen, and E. J. Rauckman, J. Am.
Chem. Soc., 102, 4994 (1980).
4
5
Figure 2. ESR Spectra of spin adducts obtained by
the reaction with superoxide radicals (a) 3a-OOH
(pH 7.4); 3b-OOH (pH 7.4).
a) G. M. Rosen, E. Finkelstein, and E. J. Rauckman, Arch.
Biochem. Biophys., 215, 367 (1982). b) M. J. Turner, III and
G. M. Rosen, J. Med. Chem., 29, 2439 (1986).
6
a) D. L. Haire, J. W. Hilborn, and E. G. Janzen, Can. J.
Chem., 60, 1514 (1982). b) E. G. Janzen and Y.-K. Zhang,
J. Org. Chem., 60, 5441 (1995). c) V. Roubaud, A. Mercier,
G. Olive, F. L. Moigne, and P. Tordo, J. Chem. Soc., Perkin
Trans. 2, 1997, 1827. d) R. Sato, K. Ito, H. Igarashi, M.
Uejima, K. Nakanishi, and M. Takeishi, Chem. Lett.,
1998, 1059.
Table 1. Hyperfine splitting constants of pyrroline N-oxide 3
adduct (mT)
Entry
Spin adduct
AN
Ab-H
AP
1
2
3
4
3a-OH
3b-OH
3a-OOH
3b-OOH
1.42
1.34
1.24
1.22
2.18
2.00
1.21
1.18
3.64
3.95
4.17
4.61
7
a) H. Karoui, C. Nsanzumuhire, F. L. Moigne, and P. Tordo,
J. Org. Chem., 64, 1471 (1999). b) Y.-K. Xu, Z.-W. Chen, J.
Sun, K. Liu, W. Chen, W. Shi, H.-M. Wang, and Y. Liu, J.
Org. Chem., 67, 7624 (2002).
The kinetics of decay of the superoxide adducts were mea-
sured in phosphate buffer (pH 7.4). The superoxide was gener-
ated with a xanthine/xanthine oxidase system and was stopped
by adding SOD. The spin adduct decay was followed by mon-
itoring the decrease of an appropriate line of the spin adduct.
The ESR signals of 3a-OOH and 3b-OOH were observed for
40 min and 6 min, respectively. The decay of 3a-OOH produced
under these conditions was pure first-order and had a rate con-
stant of 1:1 ꢅ 10ꢁ3 sꢁ1, which corresponds to a half-life of
633 s. These half-life times are longer than that of DMPO
(t1=2 ¼ 50 s, at pH 7.0) and the same as that of DEPMPO
(t1=2 ¼ 780 s, at pH 7.0) under similar conditions.10
´
8
9
C. Frejaville, H. Karoui, B. Tuccio, F. L. Moigne, M.
Culcasi, S. Pietri, R. Lauricella, and P. Tordo, J. Med.
Chem., 38, 258 (1995).
Satisfactory elemental analyses were obtained for all new
compounds. 2-(methylphenylphosphoryl)-2-methyl-3,4-di-
hydro-2H-pyrroline N-oxide (3a); 1H NMR (CDCl3,
400 MHz) d ¼ 1:38{1:46 (m, 1H) 1.90 (d, J ¼ 12 Hz, 3H)
2.085 (d, J ¼ 18:4 Hz, 3H), 1.99–2.09 (m, 1H), 2.24-2.32
(m, 1H), 2.79–2.89 (m, 1H), 6.57 (dt, J ¼ 1:2 Hz, 2.4 Hz,
1H), 7.27–7.912 (m, 5H). 2-(ethoxyphenylphosphoryl)-2-
methyl-3,4-dihydro-2H-pyrroline N-oxide (3b); 1H NMR
(CDCl3, 400 MHz) d ¼ 1:27 (t, J ¼ 7:2 Hz, 3H), 1.31 (t,
J ¼ 7:2 Hz, 3H) 1.49 (d, J ¼ 15:2 Hz, 3H), 1.70 (d,
J ¼ 15:2 Hz, 3H), 1.85–2.10 (m, 2H), 2.05-2.55 (m, 1H),
2.80–3.13 (m, 1H) 3.92–4.18 (m, 2H) 6.61 (db,
J ¼ 2:4 Hz, 0.5H), 6.86 (db, J ¼ 3:2 Hz, 0.5H), 7.43–
7.908 (m, 5H).
To estimate the relative lipophilicity of the pyrroline N-ox-
ide 3, we determined the partition coefficient of 3 between 1-oc-
tanol and the water system (Table 2). The presence of a phenyl
group at the phosphorus enhanced the lipophilicity of 3. It was
Table 2. Partition of pyrroline N-oxide 3 between 1-octanol
and watera
´
10 B. Tuccio, R. Lauricella, C. Frejaville, J. C. Bouteiller, and
P. Tordo, J. Chem. Soc., Perkin Trans. 2, 1995, 295.
11 E. G. Janzen, J. L. Poyer, C. F. Schaefer, P. E. Downs, and
C. M. Dubose, J. Biochem. Biophys. Methods, 30, 239
(1995).
3a
3b
DEPMPO
0.06c
DMPO
0.11 (0.10d)
Pb
0.70
0.44
aConditions: 1-octanol 5 ml, H2O 5 ml, 0.15 mmol, at 36 ꢂC.
bP = [3 in 1-octanol]/[3 in H2O]. Ref. 8. Ref. 11.
c
d
Published on the web (Advance View) June 17, 2003;DOI 10.1246/cl.2003.604