Synthesis of tetraalkyl (pyrrolidine-2,2-diyl)bisphosphonates and 2,2- bis(diethoxyphosphoryl)-3,4-dihydro-2H-pyrrole 1-oxide; ESR study of derived nitroxides

Full text of DOI:10.1021/jo980092z

J . Org. Chem. 1998, 63, 9095-9099
9095
Notes
Syn th esis of Tetr a a lk yl
P yr r olid in e-2,2-d iyl)bisp h osp h on a tes a n d
Sch em e 1
(
2
,2-Bis(d ieth oxyp h osp h or yl)-3,4-d ih yd r o-2H-
p yr r ole 1-Oxid e; ESR Stu d y of Der ived
Nitr oxid es
Gilles Olive, Fran c¸ ois Le Moigne, Anne Mercier,
†
Antal Rockenbauer, and Paul Tordo*
group takes part in the stabilization of DEPMPO-OOH.
It was then interesting to investigate the influence of the
introduction of a second phosphorylated group on the
same carbon atom.
Laboratoire Structure et R e´ activit e´ des Esp e` ces
Paramagn e´ tiques, CNRS UMR 6517, Chimie, Biologie et
Radicaux Libres, Universit e´ s d’Aix-Marseille I et III, Centre
de St J e´ r oˆ me, Service 521, Avenue Escadrille
Tetraalkyl (pyrrolidine-2,2-diyl)bisphosphonates 5 were
Normandie-Niemen, 13397 Marseille Cedex 20, France
1,2,7-12
selected as precursors
of the targeted gem-bis-
phosphorylated nitrones. We describe hereafter the
synthesis of 5 and that of the bis(diethoxyphosphoryl)-
Received J anuary 20, 1998
3
,4-dihydro-2H-pyrrole 1-oxide 6 (Scheme 1). We will
In tr od u ction
also describe the spin trapping properties of 6 and the
ESR features of various nitroxides derived from 5.
Many investigations are currently undertaken to ad-
dress the role played by oxygen-centered radicals in
numerous pathological processes. We recently reported
the synthesis of a new spin trap, the 2-diethoxyphos-
phoryl-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide 1 (DEP-
MPO), which was shown to be particularly efficient in
trapping oxygen-centered radicals in biological milieu.1
DEPMPO is a phosphorylated analogue of the 2,2-
dimethyl-3,4-dihydro-2H-pyrrole 1-oxide 2 (DMPO), which
is the most popular spin trap in free radical biology.
Resu lts a n d Discu ssion
Syn th esis of Bisp h osp h on a tes 5. Aminomethylene
gem-bisphosphonates exhibit a number of useful biologi-
13-16
cal properties,
and different synthetic approaches for
-4
these compounds are now available. However, simple
and efficient syntheses of the alicyclic analogues are still
14,17,18
scarce.
Yokomatsu18 prepared the tetraethyl(piperidine-2,2-
•
-•
The kinetics of HO and O
2
addition on 1 or 2 are
diyl)bisphosphonate 7 via a Beckman rearrangement. We
first tried to extend his approach to the synthesis of 5
from cyclobutanone 8. However, following his procedure,
we obtained a crude reaction mixture containing at least
seven products, with only 10% of the desired bisphos-
phonates. Then, we decided to prepare the bisphospho-
nates 5 through esterification of the corresponding
almost identical. However, the DEPMPO superoxide
spin adduct (DEPMPO-OOH) 3 has a significantly larger
half-life (15 times at pH 7.4) than DMPO-OOH.4
To understand and, possibly, to optimize the influence
of the phosphoryl group on the half-life of superoxide spin
adducts5 we prepared a series of new phosphorylated
nitrones and investigated their spin trap properties. We
showed that if a methylene spacer is inserted between
the carbon adjacent to the nitrogen of the nitroxyl
,6
(
pyrrolidine-2,2-diyl)bisphosphonic acid 9. The com-
pound 9 was obtained from the pyrrolidin-2-one according
14
17
to the general method of Pl o¨ ger modified by Zilch. It
is soluble in basic aqueous medium only, and all our
5
function and the phosphorylated group, the spin trap-
ping properties of the resulting nitrone and the persis-
tence of the superoxide spin adduct are very similar to
those of DMPO. This result clearly suggests that the
electron-withdrawing effect exerted by the phosphoryl
(
7) Murashi, S.-I.; Shiota, T. Tetrahedron Lett. 1987, 28, 2383-2386.
(8) Murashi, S.-I.; Mitsui, H.; Shiota, T.; Tsuda, T.; Watanabe, S.
J . Org. Chem. 1990, 55, 1736-1744.
(
9) Ballistreri, F. P.; Chiacchio, U.; Rescifina, A.; Tomaselli, G. A.;
Toscano, R. M. Tetrahedron Lett. 1992, 48, 8677-8684.
(10) Marcantoni, E.; Petrini, M.; Polimanti, O. Tetrahedron Lett.
1995, 36, 3561-3562.
*
Tel: (33) 4.91.28.85.62. Fax: (33) 4.91.98.85.12. E-mail: paul@
srepir1.univ-mrs.fr.
†
Central Research Institute for Chemistry, P.O. Box 17, Budapest
(11) Zajac, W. W.; Walters, T. R.; Darcy, M. G. J . Org. Chem. 1988,
53, 5856-5860.
H-1525, Hungary. E-mail: rocky@cric.chemres.hu.
(
1) Fr e´ javille, C.; Karoui, H.; Tuccio, B.; Le Moigne, F.; Culcasi, M.;
Pietri, S.; Lauricella, R.; Tordo, P. J . Chem. Soc., Chem. Commun.
994, 1793-1794.
2) Fr e´ javille, C.; Karoui, H.; Tuccio, B.; Le Moigne, F.; Culcasi, M.;
Pietri, S.; Lauricella, R.; Tordo, P. J . Med. Chem. 1995, 38, 258-265.
3) Fr e´ javille, C.; Karoui, H.; Le Moigne, F.; Culcasi, M.; Pietri, S.;
Tordo, P. Brevet fran c¸ ais PV 9308906, 20 juillet 1993.
4) Tuccio, B.; Lauricella, R.; Fr e´ javille, C.; Bouteiller, J .-C.; Tordo,
P. J . Chem. Soc., Perkins Trans 2 1995, 295-298.
5) Roubaud, V.; Mercier, A.; Olive, G.; Le Moigne, F.; Tordo, P. J .
Chem. Soc., Perkin Trans. 2 1997, 9, 1827-1830.
6) Barbati, S.; Cl e´ ment, J . L.; Olive, G.; Roubaud, V.; Tuccio, B.;
(12) Barbati, S.; Clement, J . L.; Fr e´ javille, C.; Bouteiller, J . C.; Tordo,
P., to be published.
(13) Sietsema, W. K.; Ebetino, F. H.; Salvagno, A. M.; Bevan, J . A.
Drugs Exp. Clin. Res. 1989, XV, 389-396.
(14) Ploger, W.; Schmidt-Dunker, M.; Gloxhuber, C. U.S. Patent,
3,988,443, 1976.
(15) Fleisch, H. In Handbook of experimental pharmacology; Baker,
P. F., Ed.; Springer-Verlag: Berlin, 1988; Vol. 83, pp 440-466.
(16) Nugent, R. A.; Murphy, M.; Schlachter, S. T.; Dunn, C. J .;
Smith, R. J .; Staite, N. D.; Galinet, L. A.; Shields, S. K.; Aspar, D. G.;
Richard, K. A.; Rohloff, N. A. J . Med. Chem. 1993, 36, 134-139.
(17) Zilch, H.; Esswein, A.; Bauss, F. Offenlegunggchrift, DE 41 14
586 A 1, 1992.
1
(
(
(
(
(
Tordo, P. In Free Radicals in Biology and Environment; Minisci, F.,
Ed.; Kluwer Academic Publishers: Dordrecht/Boston/ London, 1997;
Vol. 27, pp 39-47.
(18) Yokomatsu, T.; Yoshida, Y.; Nakabayashi, N.; Shibuya, S. J .
Org. Chem. 1994, 59, 7562- 7564.
1
0.1021/jo980092z CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/01/1998

9
096 J . Org. Chem., Vol. 63, No. 24, 1998
Notes
Ta ble 1. Ca lcu la ted ESR P a r a m eter s of 6-OOH a n d 6-SG Sp in Ad d u cts Obta in ed by Sim u la tion w ith Ch em ica l
Exch a n ge (Tw o-Site Mod el)
adduct
source
H2O2 30%
aN, G
aHâ, G
aP1, G
aP2, G
aHγ, G
%
exchng time, ns
47.9
6
-OOH
-OOH
-SG
12.8
10.3
11.1
8.2
11.8
17.2
16.1
42.0
47.7
39.2
46.7
43.5
54.7
39.3
39.1
39.4
38.6
41.5
27.7
0.2(4)
0.2(4)
0.75(4)
0.75(4)
0.4
52
48
41
59
90
10
1
2.9
12.9
2.7
13.7
1.8
6
6
HX/XOD/buffer, pH 5.8
GSSG/ UV/buffer, pH 5.8
12.6
30.8
1
1
0.4
Sch em e 2
Sch em e 3
attempts to get the corresponding esters failed, even with
the use of ethylorthoformate.17
Finally, we succeeded in preparing 5 in reasonable
yield by modifying the Zilch synthesis (Scheme 2). POCl
2 mol) was added to a mixture of pyrrolidin-2-one (1 mol)
3
(
and trialkyl phosphite (2 mol) at -5 °C under nitrogen.
The reaction mixture was stirred for 5 h at room
temperature and then poured over a cold aqueous satu-
4
rated NH OH solution. After workup, the aminobis-
phosphonates 5a or 5b were obtained in about 50% yield.
Resulting from the coupling with two phosphorus
nuclei which exhibit the same chemical shift, the R and
F igu r e 1. (a) ESR spectra of the 6-OOH adduct (H O , 30%).
2
2
â carbons of 5 (C
â
-C
R
-O-P(O)-O-CR′-Câ′) were expected to
(b) Complex adduct obtained with 6 (50 mM) with a DTPA
(4.5 mM)/light/riboflavin system (0.1 M) in phosphate buffer
pH 6.0.
appear as second-order quintets (AA′X systems) in the
1
3
C NMR spectra. However, the two outer lines of the
2
quintet were not observed, thus indicating that J PP′
1
g
-
The 6-CH
3
and 6-CO
2
adducts were also generated
5 Hz.19 If we assume that J CP is negligible, then the
5
using a Fenton system, in the presence of DMSO or
sodium formate, respectively. (6-CH :a ) 14.6 G, aHâ
21.8 G, aP1 ) 45.5 G, aP2 ) 44.3 G, aHγ (4H) ) 0.5 G;
magnitude of the different coupling constants can be
3
N
2
measured directly on the observed virtual triplets:
J
CRP
)
4
2
4
3
(
+ J CRP′) ) 5.3 Hz, J CR′P (+ J CR′P′) ) 5.8 Hz, J CâP ) 5.5
-
6
-CO
2 N
: a ) 14.3 G, aHâ ) 20.3 G, aP1 ) 43.0 G, aP2 )
3
2
4
Hz, J Câ′P ) 7.2 Hz for 5a and J CRP (+ J CRP′) ) 6.2 Hz,
4
2.3 G, aHγ (4H) ) 0.4 G) Moderate line width distortions
2
CR′P (+ 4J CR′P′) ) 6.7 Hz, J Câ1P ) 6.2 Hz, J Câ2P ) 6.7
3
3
J
were observed in the ESR spectra of these spin adducts,
and very good calculated spectra were obtained consider-
ing slightly different couplings for the two diastereotopic
phosphorus nuclei.
The 6-OOH adduct was formed with three different
generating systems (Table 1). It was well characterized
Hz for 5b. No coupling was observed for Câ′1 and Câ′2.
The oxidation of 5a at 0 °C with dimethyldioxirane
(
DMD), prepared in situ from oxone and acetone in
2
0
biphasic conditions (Brik procedure), led to the gem-
bisphosphorylated nitrone 6 in 11% yield after purifica-
tion (Scheme 3). The use of DMD in an acetone solution
2
1
from the UV photolysis of 30% H
2 2
O in the presence of 6
instead of the in situ procedure did not lead to a
substantial increase of the yield.
ESR Stu d ies. Sp in Tr a p p in g. Spin trapping with
(Figure 1a). The use of the hypoxanthine/xanthine
oxidase (HX/XOD) system at pH 6.0 also led to 6-OOH
together with 6-OH, the later probably being formed
from the decomposition of the superoxide adduct. Work-
ing in the presence of superoxide dismutase (SOD) (85
6
5
1
using a Fenton system in aqueous phosphate buffer (pH
.8) led to the formation of the 6-OH spin adduct (a
3.5 G, aHâ ) 13.3 G, aP1 ) 44.1 G, aP2 ) 43.4 G, a2 Hγ
N
)
-1
units mL ) inhibited the formation of the signal at-
2
(4H) ) 0.5 G, determined from computer simulation ).
tributed to 6-OOH; when glutathione (100 mM)/glu-
-
1
tathione peroxidase (10 units mL ) was added to 6 in
the HX/XOD system, 6-OOH was reduced to 6-OH.
When superoxide was produced from a riboflavin/light/
DTPA system in phosphate buffer at pH 5.8, a complex
spectrum was obtained resulting from the superposition
of 6-OOH with other spin adducts (6-OH and carbon-
(19) Veps a¨ l a¨ inen, J . J .; Nupponen, H.; Pohjala, E.; Ahlgren, M.;
Vainiotalo J . Chem. Soc., Perkin Trans. 2 1992, 835-842.
(
20) Brik, M. E. Tetrahedron Lett. 1995, 36, 5519-5522.
(21) Adam, W.; Bialas, J .; Hadjiarapoglou, L. Chem. Ber. 1991, 124,
2
4
377.
22) Rockenbauer, A.; Korecz, L. Appl. Magn. Reson. 1996, 10, 29-
(
3.

Notes
J . Org. Chem., Vol. 63, No. 24, 1998 9097
Sch em e 4
centered radical adducts) and no “pure” ESR line of the
superoxide adduct could be found (Figure 1b) to measure
its kinetic of decay. At pH 7.0, 6-OOH was not identi-
fied. The kinetic of decay of the superoxide adduct,
generated in pyridine by the lumiflavin/light/DTPA
system, was estimated. With this superoxide generating
system, the persistence of 6-OOH was found to be
similar to that of DMPO-OOH but 4.8 times lower than
that of DEPMPO-OOH. UV photolysis of glutathione
disulfide in the presence of 6 in phosphate buffer pH 5.8
led to 6-SG. For 6-OOH (Figure 1a) and 6-SG, the
line width alternation was rather important and satisfac-
tory simulations could only be obtained taking into
account a chemical exchange between two forms of these
adducts (two-site model). The calculated ESR param-
eters are listed in Table 1.
The trapping of the superoxide with the bisphos-
phorylated nitrone 6 presented the same limitations as
those observed with DMPO. The persistence of 6-OOH
is low (not observed in phosphate buffer at pH 7.0), and
the decay of 6-OOH in pyridine is closer to that of
DMPO-OOH rather than to that of DEPMPO-OOH. On
the other hand, a fast decomposition of the superoxide
adduct into the hydroxyl adduct was observed. Work in
progress in our laboratory23 indicates that, in addition
F igu r e 2. ESR spectra at 25 °C: (a) radical 10a in methylene
a
chloride, (b) radical 10b in methylene chloride, (c) 10b in
a
a
2
4
benzene, (d) 11 in aqueous solution of NaOH (pH ) 13). (2,
to its strong electron-withdrawing effect, steric crowding
of the (EtO) P(O) plays a role in the stabilization of the
H
H
P
P
H
1
lines for which m
I
1
+ m
I
2
) 0; b, m
) 0).
I
1
+ m
I
2
) 0; (, m
I
+
2
H
P
P
m
I
2
) 0 and m
I
1
+ m
I
2
DEPMPO-OOH spin adduct. However, for 6, the im-
portant increase of intramolecular steric strain related
to the introduction of a second phosphorylated group can
induce the opposite, a destabilization of the spin adducts.
1
0a could be explained assuming a complex chemical
exchange between four sites. However, at 298 K, the
interconversion rate between the different sites is suf-
ficiently high to average the ESR parameters, and no
significant line broadening was observed (Figure 2a).
The ESR spectrum of radical 10b was recorded in
either methylene chloride or benzene. The lines corre-
Furthermore, we have shown that when only one (EtO)
O) is present, low-energy conformations with the phos-
phorus group in a pseudoaxial position are adopted by
the molecule. When a second (EtO) P(O) group is intro-
2
P-
(
2
duced in a gem-position, one group is forced to occupy a
high-energy pseudoequatorial position.
ESR F ea tu r es of Bisp h osp h or yla ted Nitr oxid es
Der ived fr om Am in obisp h osp h on a tes 5a a n d 5b
a n d Th eir Cor r esp on d in g Acid . The oxidation of 5a
and 5b with m-CPBA led to nitroxides 10a and 10b
H1
H2
P1
P2
sponding either to m
I
+ m
I
) 0 or m
I
+ m
I
) 0 are
broadened, the broadening being much stronger in
benzene (Figure 2c) than in methylene chloride (Figure
2b). The existence of a π complex between benzene and
a nitroxide has been previously reported26 and could
explain such a difference.The calculated ESR parameters
obtained by using a two-site chemical exchange model
are listed in Table 2. The complete analysis of the
chemical exchange observed for 10b will be published in
a forthcoming paper.
(
a
Scheme 4) that exhibited 27-line ESR spectra (a
(2H), a (2P)). The ESR spectrum of 10a was recorded
in different solvents. No significant changes were ob-
served in aprotic solvents (a ) 13.9-14.0 G, a ) 42.3-
2.8 G, a ) 17.8-18.1 G, g ) 2.0060) and even in water
changes are not very large (a ) 14.5 G, a ) 44.3 G, a
19.3 G, g ) 2.0058). This result could be accounted
N
(1N),
H
P
N
P
4
H
Oxidation of 9 with m-CPBA in an aqueous solution
of NaOH (pH 13) led to the ESR signal shown in Figure
2d, which was assigned to nitroxide 11 (Scheme 5). The
ESR parameters of 11 were obtained by computer
simulation: aN ) 16.1 G, aH ) 21.1 G, aP ) 40.6 G, g )
N
P
H
)
for by either the existence of a largely predominant
conformer or the presence of an equilibrium between
2
5
15,27
equivalent conformers. We have recently shown that
at low temperature (below 250 K) the ESR spectrum of
2.0054. Assuming that the pKa4 of 9 is lower than 11,
the nitroxide 11 is expected to be formed as a tetrasodium
salt.
(
23) Le Moigne, F.; Karoui, H.; Tordo, P., To be published.
(24) Katzhendler, J .; Ringel, I.; Karaman, R.; Zaher, H.; Breuer, E.
(26) Burnett, G. M.; Cameron, G. G.; Cameron, J . J . Chem. Soc.,
Faraday Trans. 1973, 69, 864- 870.
(27) Curry, J . D.; Nicholson, D. A. In Topics in phosphorus chemistry;
Wiley: New York, 1972; Vol. 7, p 37.
J . Chem. Soc., Perkin Trans. 2 1997, 341-349.
25) Rockenbauer, A.; Mercier, A.; Le Moigne, F.; Olive, G.; Tordo,
P. J . Phys. Chem. A 1997, 101, 7965-7970.
(

9
098 J . Org. Chem., Vol. 63, No. 24, 1998
Notes
solutions. Proton-decoupled 31P NMR spectra were recorded on
Ta ble 2. Ca lcu la ted ESR P a r a m eter s of Nitr oxid e 10b
in Ben zen e a n d Meth ylen e Ch lor id e a t 25 °C Obta in ed by
a Bruker AC 100 at 40.54 MHz, and the chemical shifts (δ) in
2
2,25
Sim u la tion w ith Ch em ica l Exch a n ge
Mod el)
(Tw o-Site
3 4
ppm were referenced to external 85% H PO . All J values are
given in hertz. IR spectra were recorded on a MATTSON 1000
series FTIR. ESR measurements were performed on a Brucker
ESP 300 spectrometer equipped with an X-band resonator (9.41
GHz). All ESR spectra were recorded at 100 kHz magnetic field
modulation. Solvents were purchased from SDS. Pyrrolidin-
methylene chloride
benzene
aN, G
aP1, G
aP2, G
aH1, G
aH2, G
other couplings, G
13.9, 14.3
44.4, 40.0
41.2, 45.3
18.8, 16.3
17.2, 19.9
0.3(2)
13.8, 13.8
43.6, 38.8
41.1, 44.9
18.5, 15.6
17.1, 19.9
0.4(2)
2
-one, m-CPBA (m-chloroperbenzoic acid) (57-86%), phosphorus
oxychloride, and triethyl phosphite were Aldrich reagents and
were used as purchased, as was phosphorous acid purchased
from Acros Chimica. Ammonia solution (ca. 32%) was a Prolabo
reagent and was used as purchased.
0
.4(2)
0.4(2)
g
2.0060
68, 32
11.1
2.0060
66, 34
38.5
P r ep a r a t ion of Tet r a a lk yl (P yr r olid in e-2,2-d iyl)b is-
p h osp h on a tes 5a a n d 5b). Phosphorus oxychloride (40 mL,
population, %
exchng time, ns
0
.44 mol) was added over 1.25 h at -5 °C to a mixture of
pyrrolidin-2-one (18.49 g, 0.22 mol) and trialkyl phosphite (0.42
mol). The reaction mixture was stirred for 5 h at room
temperature and then poured over a mixture of ice (300 g) and
ammonia 32% (300 mL). The aqueous layer was extracted with
methylene chloride (4 × 100 mL). The organic layer was
concentrated to obtain a yellow oil. This oil was dissolved in
Sch em e 5
1
00 mL of methylene chloride. Water was added (200 mL), and
then concentrated hydrochloric acid (37%) was added until pH
. The aqueous layer was separated and washed with methylene
1
chloride (4 × 50 mL). Sodium hydroxide and sodium carbonate
were added until pH 10 to the aqueous layer, which was
extracted with methylene chloride (4 × 50 mL). The organic
layer was dried over sodium sulfate and filtered. Removal of
the solvent afforded compound 5a or 5b.
5
a (33.4 g, 47%): IR (neat) 3480 (NH), 1243 (PdO), 1164 (P-
-
1 1
2 5 6 6
O-C H ) cm ; H NMR (C D , 400 MHz) δ 4.17 (8H, m), 2.88
(
2H, t, J ) 6.5), 2.42 (2H, n, J PH ) 17.7, J HaHb ) 7.2), 1.69 (2H,
In water, nitroxides 10a and 11 exhibit significantly
different nitrogen couplings and g factors (a ) 16.1 G,
g ) 2.0054 for the former and a ) 14.5 G, g ) 2.0058
q, J ) 6.8, J HH ) 7.2), 1.11 (6H, t, J ) 7.1), 1.10 (6H, t, J ) 7.1);
N
31
13
P NMR (CDCl
3 6 6
) δ 22.5; C NMR (C D , 100.61 MHz) δ 63.4
N
(
t, J CP ) 5.3), 62.7 (t, J CP ) 3.6), 62.8 (t, J CP ) 151.8), 47.7 (t,
for the latter). These changes result from the strong
donor effect exerted by the phosphonato groups, which
favors the mesomeric form 11b (Scheme 5). Although
J CP ) 4.0), 31.2 (t, J CP ) 3.0), 26.5 (t, J CP ) 3.1), 16.6 (t, J
)
CP
-
2
5.5), 16.5 (t, J CP ) 7.2); pK
a
) 3.5; bp 140 °C at 6 × 10 mmHg.
1
5
a gave a picrate: mp 128-130 °C; H NMR (CDCl
3
, 100 MHz)
δ 8.86 (2H, s), 7.99 (2H, s), 4.24 (8H, m), 3.64 (2H, t, J ) 6.9),
this increase in a
of the â-phosphorus coupling constant a
observed a decrease in a for nitroxide 11 as compared
to 10a (11, a ) 40.6 G; 10a , a ) 44.3 G). This could be
N
should be accompanied by an increase
2
.67-2.15 (4H, m), 1.36 (6H, t, J ) 7.1), 1.33 (6H, t, J ) 7.1);
P
, actually we
31
3 30 4 13 2
P NMR (CDCl ) δ 16.23. Anal. Calcd for C18H N O P : C,
P
3
7.75; H, 5.28; N, 9.79. Found: C, 37.24; H, 5.34; N, 9.28.
P
P
5b (37.2 g, 45%): IR (neat) 3471 (NH), 1246 (PdO), 989 (P-
-
1 1
reasonably attributed to a decrease in the hyperconju-
3 2 6 6
O-CH-(CH ) ) cm ; H NMR (C D , 400 MHz) δ 5.00 (1H, m),
4
.87 (1H, m), 2.95 (2H, t, J ) 6.5), 2.37 (2H, tt, J PH ) 17.7, J HH
gative proportionality constant value, B, which appears
2
) 7.3), 1.75 (2H, q, J ) 6.8, J HH ) 6.9), 1.31 (6H, d, J HH ) 6.2),
in the McConnell relationship (a
P
) B
P
cos θ; B
P
N
) BF ;
1
)
.28 (6H, d, J HH ) 6.3), 1.27 (6H, d, J HH ) 6.4), 1.22 (6H, d, J HH
F
N
is the spin density on nitrogen), rather than to
31
13
3 6 6
6.1); P NMR (CDCl ) δ 21.2; C NMR (C D , 100.61 MHz)
2
conformational changes.
2
1
δ 71.7 (t, J CP ) 6.2), 70.8 (t, J CP ) 6.7), 63.1 (t, J CP ) 151.1),
3
2
3
Although the dismutation was reported to be the main
disappearance process for â-hydrogen-bearing nitroxides,
we observed that nitroxides 10a , 10b, and 11 were
persistent for days regardless of the presence of two
â-hydrogens.
47.7 (t, J ) 4.6), 31.0 (t, J ) 3.4), 26.4 (t, J ) 3.3), 24.7
CP
CP
CP
3
3
(s), 24.4 (s), 24.0 (t, J CP ) 6.4), 23.8 (t, J CP ) 6.7). Anal. Calcd
for C16 : C, 48.12; H, 8.83; N, 3.51. Found: C, 48.21;
H
6 2
35NO P
H, 8.80; N, 3.51. mp ) -27 °C.
P r ep a r a tion of Cyclobu ta n on e Oxim e 8.2
8,29
An aqueous
solution (10 mL of water) of sodium carbonate (4.80 g, 45.3 mmol)
was added under stirring to an aqueous solution (11 mL of water)
of hydroxylamine hydrochloride (6.22 g, 89.5 mmol) and cyclobu-
tanone (5.15 g, 73.5 mmol), and the temperature was kept below
In addition to the previously reported 2,2-bis(diethoxy-
phosphoryl)-5,5-dimethylpyrrolidinoxyl,25 nitroxides 10a ,
1
0b, and 11 are, to our knowledge, the first reported
4
5 °C. The reaction mixture was stirred for 1 h. The aqueous
examples of gem-â-bisphosphorylated nitroxides. We will
now study the extension of this synthetic route to other
cyclic aminobisphosphonates by varying the nature of
both the phosphite and the lactam.
Further ESR studies of bisphosphonic acid-bearing
nitroxides are planned, including metal-complexation
properties and pH-correlated variations of the coupling
constants. The good persistence of nitroxide 11 opens
interesting perspectives for the use of this kind of
nitroxide as MRI contrast enhancing agents.
layer was extracted with diethyl ether (4 × 25 mL), and the
solvent was removed to dryness to give a white powder. The
powder was then dissolved in acetone (10 mL), dried over sodium
sulfate, filtered, and evaporated to dryness to give compound 8
1
(4.7 g, 75%) as white crystals:
3
H NMR (CDCl , 400 MHz) δ
8.88 (1H, s, -OH), 2.92 (2H, dt, J
1
) 8.3, J ) 1.0), 2.87 (2H, dt,
2
13
J
1
) 8.2, J
2
3
) 1.0), 1.97 (2H, q, J ) 8.1); C NMR (CDCl ) δ
2
8
1
59.86, 31.51, 30.61, 14.54; mp 87 °C (lit. 84-85 °C).
P r ep a r a tion of (P yr r olid in e-2,2-d iyl)bisp h osp h on ic a cid
9
.17 The pyrrolidin-2-one (17.36 g, 0.2 mol) and phosphorous
acid (32.9 g, 0.4 mol) were heated under nitrogen at 80 °C until
Exp er im en ta l Section
(
28) Iffland, D. C.; Criner, G. X.; Koral, M.; Lotspeich, F. J .;
Papanastassiou, Z. B.; White, S. M., J r. J . Am. Chem. Soc. 1953, 75,
1H and 13C NMR spectra were recorded on Bruker AC 100,
00, and 400 spectrometers, and the chemical shifts (δ) in ppm
were referenced to internal TMS or HOD (4.81 ppm) for D
4
044-4046.
2
(29) Hawkes, G. E.; Herwig, K.; Roberts, J . D. J . Org. Chem. 1974,
2
O
39, 1017-1028.

Notes
J . Org. Chem., Vol. 63, No. 24, 1998 9099
the acid was dissolved. The phosphorus oxychloride (39 mL, 0.4
mol) was carefully added over 1 h. The reaction mixture was
stirred for 8 h at 100 °C to give a yellow solid. Water (1 L) was
added and the mixture was heated for 1 h at 100 °C and then
filtered on a B u¨ chner. Crystallization of the filtrate afforded
compound 6 (8.0 g, 18%): IR (KBr) 2979 (νCHsp3), 2778 (νNH+),
tions indicated below correspond to initial conditions after
mixing of all reactants.
Sp in Tr a p p in g of th e Ra d ica l Hyd r oxyl (HO ). A classical
•
Fenton system was used. A solution of FeSO
mM) in phosphate buffer pH 5.8 was added to a solution of
nitrone 6 (100 mM) and H (2 mM). The spectrum was
recorded 40 s after the addition of FeSO The 6-CH and
adducts were obtained in the same manner but with
4
(2 mM)/DTPA (1
2
O
2
2
707 (P-OH), 2462 (P-OH), 1614 (δ NH+), 1435 (δCHsp3), 1241
4
.
3
-
1
1
-
2
(
PdO), 1119, 986 (P-O-H) cm
;
H NMR (D
2
O/NaOD, 200
6-CO
MHz) δ 3.42 (2H, t, J ) 7.02), 2.38 (2H, h, J HcHb ) 7.13, J PHc
)
the addition of dimethyl sulfoxide (DMSO) (100 mM) or sodium
formiate (50 mM) prior to the addition of ferrous sulfate.
P r od u ction of th e Su p er oxid e Ad d u ct; P h otolysis of
Hyd r ogen P er oxid e 30%. A solution of 6 (50 mM) in hydrogen
peroxide (30%) was illuminated by UV light for 15 s in the ESR
cavity, and the spectrum was recorded just after photolysis.
Hyp oxa n th in e/Xa n th in e Oxid a se System . A solution of
3
1
1
4.46), 2.06 (2H, q, J ) 7.04); P NMR (D
2
O/NaOD) δ 15.01;
1
3
C NMR (D
mp 281 °C (dec). Anal. Calcd for C
N, 6.06; P, 26.82. Found C, 20.65; H, 4.90; N, 5.64; P, 26.37.
,2-Bis(d ieth oxyp h osp h or yl)-3,4-d ih yd r o-2H-p yr r ole 1-
2
O/NaOD) δ 65.77 (t, J CP ) 118), 48.61, 31.31, 25.36;
4
6 2
H11NO P : C, 20.78; H, 4.80;
2
Oxid e 6. A mixture of 5 (1.6 g, 4.6 mmol), acetone (80 mL),
methylene chloride (60 mL), tetrabutylammoniumhydrogeno-
sulfate (81.4 mg, 0.24 mmol), 0.1 M Na HPO buffer (60 mL),
2 4
and 2 M aqueous sodium hydroxide (2 mL) was cooled to 0 °C.
A solution of oxone (9.5 g; 15.5 mmol) in water (90 mL) was
added over 1 h. Then, the organic layer was separated, and the
aqueous layer was extracted with methylene chloride (2 × 100
mL). The combined organic fractions were reconcentrated under
vacuum. Acetone (1 mL) was added to the residual material.
-
1
xanthine oxidase (0.4 U mL ) was added to an air-bubbled
solution of 10 (100 mM), DTPA (1 mM), and hypoxanthine (0.4
mM) in phosphate buffer pH 6.0. Air was bubbled into the
reaction mixture for 30 s, and the ESR spectra were recorded
40 s after the addition of xanthine oxidase.
F la vin /Ligh t/DTP A System s. A solution of 6 (50 mM),
DTPA (4.5 mM), and riboflavin (0.1 M) in phosphate buffer pH
6.0 was prepared. Air was bubbled into the reaction mixture
for 30 s. The superoxide production was initiated by irradiation
with the tungsten lamp. In pyridine, the riboflavin was replaced
by lumiflavin.
Preparative TLC on Silicagel (CH
2 2
Cl /anhydrous EtOH, 10/1 v/v;
extraction with MeOH) afforded nitrone 6 in 11% yield (0.154
1
g): H NMR (C
t, J ) 7.1), 2.11 (2H, m, J HH ) 2.7, J HP ) 2.9, J HH ) 7.3), 2.69
2H, m, J HH ) 7.2, J HP ) 17.2), 4.20 (4H, m, J ) 10.3), 4.37 (4H,
6 6
D , 400 MHz) δ 1.09 (6H, t, J ) 7.1), 1.11 (6H,
•
•
GS
Tr a p p in g. GS was produced by UV photolysis of a
(
solution containing glutathione disulfide (50 mM) and nitrone
6 (50 mM) in phosphate buffer pH 5.8. The ESR spectrum was
recorded after 15 s of UV photolysis.
1
3
3
m, J ) 10.3), 6.42 (1H, q, J HH ) 2.8, J HP ) 3.1); C NMR (CDCl ,
00 MHz) 16.4 (s), 27.7 (s), 27.8 (s), 63.9 (s), 64.5 (s), 136.5 (t);
P NMR (CDCl
1
3
1
3 7 2
, 40 MHz) 15.5; HRMS for C12H25NO P calcd
3
R
57.1106, found 357.1127; MS (m/z) 220, 203, 138, 94, 67, 28.
Ack n ow led gm en t. This work was supported by the
Centre National de la Recherche Scientifique (C.N.R.S.),
the Universities of Aix-Marseille I and III, and Grants
OTKA (Hungarian National Research Fund) T-015841
and Balaton (intergovernemental project of Hungary
and France) F-34/96.
f
(methylene chloride/ethanol 19/1) 0.26.
ESR Stu d y of Nitr oxid es 10a , 10b, a n d 11. Compounds
a , 5b, or 9 (0.03 mmol) were dissolved in 100 µL of solvent.
5
m-CPBA (7.2 mg, 0.03 mmol) was added, and the spectrum was
immediately recorded after nitrogen bubbling.
Sp in Tr a p p in g Exp er im en ts. Xanthine oxidase (XOD) and
bovine erythrocyte superoxide dismutase (SOD) were purchased
from Boerhringer Mannheim Biochemica Co. Glutathione per-
oxidase, diethylenetriaminepentaacetic acid (DTPA) and chelat-
ing iminodiacetic resin Chelex 100 were purchased from Sigma
Chemical Co. Phosphate buffers (0.1 M) were stirred in the
presence of Chelex (4 h, 4 g/100 mL) to remove traces of metal
impurities. UV photolysis was performed with an Oriel Xe-
Hg (1000 W) lamp. Illumination of flavins/DTPA solutions was
achieved with a tungsten filament 100 W lamp. The concentra-
Su p p or t in g In for m a t ion Ava ila b le: 1H NMR and 13C
NMR, with attributions, of compounds 5a , 5b, 6, 8, and 9 (2
pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
J O980092Z