A one-step synthesis of 2-(2-pyridyl)-3H-indol-3-one N-oxide: Is it an efficient spin trap for hydroxyl radical?

Full text of DOI:10.1021/jo0006122

4
460
J . Org. Chem. 2000, 65, 4460-4463
A On e-Step Syn th esis of
Sch em e 1
2
-(2-P yr id yl)-3H-in d ol-3-on e N-Oxid e: Is It
a n Efficien t Sp in Tr a p for Hyd r oxyl
Ra d ica l?
Gerald M. Rosen,*, Pei Tsai, Eugene D. Barth,
†,‡
†
§
|
|
|
Gilbert Dorey, Patrick Casara, Michael Spedding, and
Howard J . Halpern§
Department of Pharmaceutical Sciences, University of
Maryland School of Pharmacy, Baltimore, Maryland 21201,
Medical Biotechnology Center, University of Maryland
Biotechnology Institute, Baltimore, Maryland 21201,
Department of Radiation and Cellular Oncology, University
of Chicago, Illinois 60637, and Institute de Recherche
Servier, Croissy sur Seine 7829, France
Sch em e 2
grosen@umaryland.edu
Received April 20, 2000
The field of free radicals in biology has its origins in a
series of publications in the late 1960s in which the
•
-
secretion of superoxide (O
2
) during the enzymic cycling
1
7
of xanthine oxidase was first described. Soon thereafter,
a publication caught our fancy in which 2-(2-pyridyl)-
3H-indol-3-one N-oxide 4 was reported to spin trap HO .
•
a Cu/Zn-containing enzyme was found to disproportionate
2
this free radical into O
2
and H
2
O
2
. This enzyme, which
The corresponding spin trapped adduct, 2-hydroxy-2-(2-
pyridyl)-3H-indol-3-on-1-oxyl (11), exhibited remarkable
stability when compared to the shorter lifetime of 3-hy-
became known as superoxide dismutase (SOD), has
played a pivotal role in defining the ubiquitous nature
•
-
•- 3
7
of O
2
and other free radicals generated from O
2
.
droxy-5,5-dimethyl-1-pyrrolin-1-oxyl (13). However, en-
In the intervening years, a variety of methods have
been developed to detect free radicals in biological milieu.
Of those, spin trapping/EPR spectroscopy is singular in
its ability to characterize specific free radicals, generated
thusiasm for such robustness must be tempered by the
fact that 4, by lacking a hydrogen atom at the R-carbon,
has lost one of the strengths of spin trapping, additional
hyperfine splittings that can aid in the characterization
of the parent free radical.8 Despite this, there are a
number of experimental paradigms that would greatly
benefit from readily available sources of 2-aryl-3H-indol-
3-one N-oxides.
4
in situ, and identified in animal models in real time.
•
Based on our earlier success at identifying HO in
5
irradiated leg tumors of mice, we have become particu-
larly interested in syntheses of newer spin traps that
•
would allow the in vivo in situ detection of HO under
There have been a number of synthetic approaches to
other experimental paradigms. During the course of our
investigations, we have studied the specificity of 3-sub-
stituted 5,5-dimethyl-1-pyrroline N-oxides and a number
2
3
2
-aryl-3H-indol-3-one N-oxides, including 2-(2-pyridyl)-
H-indol-3-one N-oxide (see, for instance, Schemes 1 and
). However, multistep pathways, especially those in
•
6
of imidazoline N-oxides toward HO . Recently, however,
which intermediates are exposed to sunlight to obtain
the desired product, have often resulted in poor yields of
*
To whom correspondence should be addressed at the University
of Maryland School of Pharmacy. Tel: 410-706-0514. Fax: 410-706-
184.
the coveted nitrone.9
8
We thought, based on the earlier work of Castro and
†
10a
10b
University of Maryland School of Pharmacy.
University of Maryland Biotechnology Institute.
University of Chicago.
Stephens
and Sonogashira et al., that it might be
‡
§
|
possible to adapt these methods to the synthesis of the
title compounds. To our surprise, we were able to
synthesize a family of 2-aryl-3H-indol-3-one N-oxide in
a one-step reaction in excellent yields. Herein, we de-
scribed our preparative design.
Institute de Recherche Servier.
(1) McCord, J . M.; Fridovich, I. J . Biol. Chem. 1968, 243, 5753.
(2) McCord, J . M.; Fridovich, I. J . Biol. Chem. 1969, 244, 6049.
(3) (a) Fridovich, I. Science, 1978, 201, 875. (b) Fridovich, I. In
Superoxide Dismutase; Oberley, L. W., Ed.; CRC Press: Boca Raton,
FL, 1982; Vol. I, p 79. (c) Fridovich, I. Annu. Rev. Biochem. 1995, 64,
9
7. (d) Fridovich, I. J . Biol. Chem. 1997, 272, 18515.
4) (a) J anzen, E. G. Acc. Chem. Res. 1971, 4, 31. (b) Pou, S.;
Halpern, H. J .; Tsai, P.; Rosen, G. M. Acc. Chem. Res. 1999, 32, 2,
55.
5) Halpern, H. J .; Yu, C.; Barth, E.; Peric, M.; Rosen, G. M. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 796.
6) (a) Rosen, G. M.; Turner, M. J ., III. J . Med. Chem. 1988, 31,
28. (b) Arya, P.; Stephens, J . C.; Griller, D.; Pou, S.; Ramos, C. L.;
(
(7) Nepveu, F.; Souchard, J .-P.; Rolland, Y.; Dorey, G.; Spedding,
M. Biochem. Biophys. Res. Commun. 1998, 242, 272.
1
(8) (a) J anzen, E. G.; Liu, J . I.-P. J . Magn. Reson. 1973, 9, 510. (b)
J anzen, E. G.; Evans, C. A.; Liu, J . I.-P. J . Magn. Reson. 1973, 9, 513.
(c) Buettner, G. R. Free Radical Biol. Med. 1987, 3, 259.
(9) (a) Ruggli, P.; Cuenin, H. Helv. Chim. Acta 1944, 27, 649. (b)
Kr o¨ hnke, F.; Vogt, I. Chem. Ber. 1952, 85, 376. (c) Bond, C. C.; Hooper,
M. J . Chem. Soc. C 1969, 2453. (d) Bond, C. C.; Hooper, M. Synthesis
1974, 443. (e) Bristow, T. H. C.; Foster, H. E.; Hooper, M., J . Chem.
Soc., Chem. Commun. 1974, 677.
(
(
4
Pou, W. S.; Rosen, G. M. J . Org. Chem. 1992, 57, 2297. (c) Kirilyuk, I.
A.; Grigor’ev, I. A.; Volodarskii, L. B. Bull. Acad. Sci. USSR 1992, 40,
1
871. (d) Haseloff, R. F.; Kirilyuk, I. A.; Dikalov, S. I.; Khramstov, V.
V.; Utepbergenov, D. I.; Blasig, I. E.; Grigor’ev, I. A. Free Radical Res.
997, 26, 159. (e) Tsai, P.; Pou, S.; Straus, R.; Rosen, G. M. J . Chem.
Soc., Perkin Trans. 2 1999, 1759.
(10) (a) Castro; C. E.; Stephens, R. D. J . Org. Chem. 1963, 28, 2163.
(b) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahderon Lett. 1975,
4470.
1
1
0.1021/jo0006122 CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/10/2000

Notes
J . Org. Chem., Vol. 65, No. 14, 2000 4461
Sch em e 3
Our initial approach was to prepare 10 and then,
following literature methods,11 cyclize it to 4 (Scheme 3).
In the experiments described by Sonogashira et al.,1 the
reactions were run in ethyl acetate with dimethylamine
added as a catalyst to accelerate the reaction. As the reac-
tants were readily soluble in triethylamine, we felt this
amine might serve as both the solvent and promoter of
the reaction. We, therefore, combined 8 and 9 in triethyl-
0b
3 2
amine and added catalytic amounts of Pd(PPh ) Cl and
CuI at room temperature. By following the reaction by
TLC, we found that after about 6-8 h a second more
polar product appeared, which with time began to be the
dominant product. The reaction mixture was allowed to
stir for several days to completion, as judged by TLC and
filtered. Ethyl acetate was then added to the remaining
solid, and again the mixture was filtered. After evapora-
tion, in vacuo, the residual oil was passed through a
flash-chromatographic column containing silica gel and
eluted with pentane/ethyl acetate mixtures, resulting in
the isolation of 2-(2-pyridyl)-3H-indol-3-one N-oxide 4 in
good yields. Using a similar procedure, other analogues
F igu r e 1. (A) EPR spectrum of 4 (3 mM in H
.5%) in the presence of H (100 µM). The spectrum was
recorded 3 min after mixing the reagents. Receiver gain was
2
O and DMF,
3
2 2
O
4
2+
1
.25 × 10 . (B) Same as (A) except Fe (100 µM) was added,
3
and the receiver gain was 2.5 × 10 . (C) EPR spectrum of 4
(3 mM in 50 mM phosphate buffer, pH 7.4 and DMF, 3.5%) in
3
the presence of H
2
O
2
(1 mM). Receiver gain was 2.5 × 10 .
of 4 (see, Scheme 1), such as R
were prepared in reasonable yields. Attempts to general-
ize this reaction to aliphatic alkynes, where R ) H and
1
) H and R ) phenyl,
has been reported for the oxidation of N-hydroxy-2-
phenylindole with either lead tetraacetate or p-nitro-
perbenzoic acid, leading to 2-hydroxy-2-phenyl-3H-indol-
1
R ) methyl, resulted in poor yields of the desired nitrone.
3
-on-1-oxyl.13
In general, the complete reaction from 8 and 9 to 4
can be accomplished by stirring the mixture at room
temperature for 3-4 days. The availability of the catalyst
and reagents, the ease of the procedure, and the gentle-
ness of experimental conditions suggest that this one-
step preparative scheme to 2-aryl-3H-indol-3-one N-
oxides will become widely used as these compounds
have been shown to exhibit significant pharmacological
activity.7
Next, we decided to estimate the efficiency of 4 to spin
•
trap HO as compared to other nitrones, whose reactions
with this free radical are well documented: 5,5-dimethyl-
1
-pyrroline N-oxide (12),1 5-(diethoxyphosphoryl)-5-
4a
1
4b
methyl-1-pyrroline N-oxide (14), and R-(4-pyridyl 1-ox-
ide)-N-tert-butylnitrone (16) and EtOH.1 For these
experiments, we used the metal ion-catalyzed Haber-
Weiss reaction in the presence of a continued flux of O
at 6 µM/min, as a source of HO .
4c
•
-
2
,
•
6e,15
To our surprise, we
2 2
When nitrone 4 was incubated with H O (100 µM),
we obtained a small but discernible EPR spectrum
corresponding to the nitroxide, 2-hydroxy-2-(2-pyridyl)-
obtained no EPR spectrum corresponding to nitroxide 11,
whereas under identical conditions, nitrones 12, 14, and
•
1
6 with EtOH readily spin trapped HO (Figure 2). In
3
H-indol-3-on-1-oxyl (Figure 1A). With the addition of
the case of the spin trapping system of 16 and EtOH,
2
+
•
•
Fe , generating HO , the EPR spectrum became consid-
erably large (Figure 1B). Of interest was the finding that
HO reacts with EtOH. This leads to R-hydroxyethyl
radical that is spin trapped by nitrone 16, yielding
at higher concentrations of H
2
O
2
(1 mM) in the absence
of exogenous Fe2 we observed the same EPR spectrum
whose intensity was identical to that found when Fe2
was present (compare Figure 1B,C). In the case of data
depicted in Figure 1C, we suggest a direct oxidation of
nitrone 4 resulted in the formation of 2-hydroxy-2-(2-
pyridyl)-3H-indol-3-on-1-oxyl (11).11 A similar reaction
+
(12) Hiremath, S. P.; Hooper, M. Adv. Heterocycl. Chem. 1978, 22,
+
123.
(13) Russell, G. A.; Myers, C. L.; Bruni, P.; Neugebauer, F. A.;
Blankespoor, R. J . Am. Chem. Soc. 1970, 92, 2762.
(14) (a) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J . J . Am. Chem.
Soc. 1980, 102, 4994. (b) Frejaville, C.; Karoui, H.; Tuccio, B.; Le
Moigne, F.; Culcasi, M., Pietri, S.; Lauicella, R.; Tordo, P. J . Med.
Chem. 1995, 38, 258. (c) Pou, S.; Ramos, C. L.; Gladwell, T.; Renks,
E.; Centra, M.; Young, D.; Cohen, M. S.; Rosen, G. M. Anal. Biochem.
1
994, 217, 76.
(
11) Hooper, M.; Patterson, D. A.; Wibberley, D. G. J . Pharm.
(15) (a) Haber, F.; Weiss, J . Proc. R. Soc. London A 1934, 147, 332.
Pharmacol. 1965, 17, 734.
(b) Dunford, H. B. Free Radical Biol. Med. 1987, 3, 405.

4
462 J . Org. Chem., Vol. 65, No. 14, 2000
Notes
Sch em e 4
Exp er im en ta l Section
Gen er a l Meth od s. Hypoxanthine and xanthine oxidase were
obtained from Sigma Chemical Co. (St. Louis, MO). R-(4-Pyridyl
1
-oxide)-N-tert-butylnitrone (16) was purchased from Aldrich
Chemical Co. (Milwaukee, WI). Superoxide dismutase (SOD) was
purchased from Boehringer Mannheim (Indianapolis, IN). 5,5-
Dimethyl-1-pyrroline N-oxide (12) was synthesized as described
in the literature16 and purified by Kugelrohr distillation. 5-
(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (14) was
obtained from OXIS International (Portalnd, OR). IR spectra
1
3
were recorded on an FT-IR spectrometer in CHCl solution. H
NMR spectra were obtained using a GE QE-300/Tecmake NMR
spectrometer. Melting points were taken on a Thomas-Hoover
capillary melting point apparatus and are corrected. The prepa-
ration of 2-(2-pyridyl)-3H-indol-3-one N-oxide (4) is illustrative
of the general syntheses of 2-substituted 3H-indol-3-one N-
oxides.
F igu r e 2. (A) EPR spectrum following the addition of xan-
thine oxidase to hypoxanthine (400 µM), in the presence of 4
Syn th esis of 2-(2-P yr id yl)-3H-in d ol-3-on e N-Oxid e (4).
1-Iodo-2-nitrobenzene (2.41 g, 9.67 mmol, Aldrich Chemical Co.,
Milwaukee, WI) was dissolved in freshly distilled triethylamine
(50 mL) to which 2-ethynylpyridine (1 g, 9.69 mmol, Aldrich
Chemical Co.) was added. The reaction was stirred at ambient
(10 mM, 5% DMF in 50 mM phosphate buffer, pH 7.4, 1 mM
2
+
DTPA) and Fe (400 µM). Superoxide was generated at 6 µM/
min. EPR spectra were recorded 2 min after commencing the
4
reaction. Receiver gain was 1.25 × 10 . (B) Same as (A) except
2
temperature under N for 30 min at which point dichlorobis-
nitrone 12 (10 mM) was substituted for 4. (C) Same as (A)
except nitrone 14 (10 mM) was used instead of 4. (D) Same as
(triphenylphosphine)palladium (0.2 g, 0.29 mmol, Aldrich Chemi-
cal Co.) and copper(I)iodide (0.185 g, 0.97 mmol, Aldrich
Chemical Co.) were added. The mixture was stirred at room
temperature for 3-4 days. The reaction mixture was filtered,
the remaining solid was washed with ethyl acetate, and the
combined solutions were evaporated to dryness, leaving an oil.
The residual material was passed through a chromatographic
column containing silica gel (Aldrich, mesh 230-400), eluting
first with pentane/ethyl acetate (2:1), which removed a small
amount of starting 2-ethynylpyridine. Changing to pentane/ethyl
acetate (1:1), a second product was isolated to which ether (25
mL) was added. After the ether solution was refluxed for 30 min,
the remaining solid was collected, yielding 2-(2-pyridyl)-3H-
indol-3-one N-oxide 4 as a yellow solid (1.58 g, 70%). If desired,
this product can be further purified by recrystallization from
(
A) except nitrone 16 (10 mM) and EtOH (68 mM) were used
3
instead of 4 and the receiver gain was 1.25 × 10 .
nitroxide 17 (Scheme 4).14c Using γ-radiation as a source
•
of HO resulted in poor yields of nitroxide 11. In fact, by
comparing intensities of the corresponding EPR spectra,
we determined nitrone 16 and EtOH was 500 times more
•
sensitive at reporting HO than was nitrone 4 (data not
shown).
We then explored the ability of nitrone 4 to spin trap
•
-
enzymatically secreted O
2
. To our delight, we were
unable to detect an EPR spectrum (data not shown) when
9a
ethanol: mp 180-182 °C; IR (CHCl
3
) 1731, 1714 (CdO), 1180
•
-
-1
nitrone 4 was incubated with the model O
2
generating
(N-O) cm ; NMR (CDCl ) δ 7.30-7.40 (1, m), 7.50-7.90 (5, m),
3
system consisting of xanthine and xanthine oxidase,
where under identical experimental conditions pyrroline
8.49 (2, d, J ) 0.026 Hz), 8.89 (1, s).
In a similar fashion, 2-phenyl-3H-indol-3-one N-oxide was
obtained as a red solid in 65% yield, mp 184-186 °C, from
ethanol.9c In contrast, poor yields of about 10-15% for 2-methyl-
•
- 6
N-oxides and some imidazoline N-oxides spins trap O
2
.
In conclusion, data presented herein demonstrate that
3
H-indol-3-one N-oxide9e suggest that the general applicability
•
the efficiency of nitrone 4 to spin trap HO is limited.
of this synthetic approach is limited to 2-aryl-3H-indol-3-one
N-oxides.
However, the inability of 2-(2-pyridyl)-3H-indol-3-one
•
-
N-oxide to spin trap O
2
points to the utility of this
•
nitrone to characterize HO under specific experimental
designs.
(16) Bonnett, R.; Brown, R. F. C.; Clark, V. M.; Sutherland, I. O.;
Todd, A. J . Chem. Soc. 1959, 2094.

Notes
EP R Sp ectr a l Mea su r em en ts. Spin-trapped adducts, de-
J . Org. Chem., Vol. 65, No. 14, 2000 4463
hypoxanthine (400 µM), in the presence of nitrone 4 (10 mM,
5% DMF in 50 mM phosphate buffer, pH 7.4, 1 mM DTPA) and
•
•-
rived from the reaction of HO and O
recorded using an EPR spectrometer (Varian Associates E-9) at
5 °C. Reaction mixtures were transferred to a flat quartz cell,
2
with the nitrones, were
2
+
Fe (400 µM). Superoxide was generated at 6 µM/min. Other
spin traps were used, and data are presented in Figure 2: 5,5-
dimethyl-1-pyrroline N-oxide (12) (10 mM); 5-(diethoxyphos-
phoryl)-5-methyl-1-pyrroline N-oxide (14) (10 mM); R-(4-pyridyl
1-oxide)-N-tert-butylnitrone (16) (10 mM); and EtOH (68 mM).
In each of the experiments, the reaction mixture was transferred
to a flat quartz cell and fitted into the cavity of the EPR
spectrometer. Spectra were recorded 3 min after commencing
the reaction.
2
fitted into the cavity of the EPR spectrometer, and spectra were
recorded at room temperature. Instrumentation settings were
as follows: microwave power, 20 mW; modulation frequency, 100
kHz; modulation amplitude, 1.0 G; response time, 1 s; and sweep,
1
2.5 G/min. Receiver gain is indicated in each figure legend.
Su p er oxid e Gen er a tion fr om Hyp oxa n th in e/Xa n th in e
•
-
Oxid a se. Production of O
2
was determined by mixing hypox-
anthine (400 µM), ferricytochrome c (80 µM), and sufficient
Hydroxyl radical was produced from irradiating an aqueous
solution of sodium phosphate, 50 mM, pH 7.4 and spin trapped
using either nitrone 4 (50 mM) or nitrone 16 (50 mM) and EtOH
xanthine oxidase in sodium phosphate buffer (50 mM) containing
•
-
DTPA (1 mM), at pH 7.4. The rate of O
2
generation was
•
60
estimated by measuring the SOD-inhibitable reduction of fer-
2
(150 mM). The source of HO was from Co of H O in a
1
8
ricytochrome c (80 µM) at 550 nm using an extinction coefficient
Gammacell irradiator at a dose of 3000 Gy/h for 1 h. This yields
-
1
-1 17
•
of 21 mM cm
.
∼1 mM of HO .
Sp in Tr a p p in g of Hyd r oxyl Ra d ica l. The Fenton reaction
•
2+
was used as a source of HO by mixing H
µM), and nitrone 4 (3 mM in H O and DMF, 3.5%) in either H
or phosphate buffer at pH 7.4.
2
O
2
(100 µM), Fe (100
Ack n ow led gm en t. This research was supported in
part by grants from the National Institutes of Health,
CA-69538 and RR 12257.
2
2
O
The metal ion-catalyzed Haber-Weiss reaction was used as
•
an alternative source of HO . Xanthine oxidase was added to
J O0006122
(
17) Kuthan, H.; Ullrich, V.; Estabrook, R. W. Biochem. J . 1982,
(18) Halpern, H. J .; Pou, S.; Yu, C.; Barth, E.; Rosen, G. M. J . Am.
Chem. Soc. 1993, 115, 218.
2
03, 551.