Facile synthesis and NO-generating property of 4H-[1, 2, 5]oxadiazolo[3, 4-d]pyrimidine-5, 7-dione 1-oxides

Article abstract of DOI:10.1021/jo980732y

4H-[1, 2, 5]Oxadiazolo[3, 4-d]pyrimidine-5, 7-dione 1-oxides (2) are conveniently prepared in high yields by the oxidative intramolecular cyclization of 6-amino-5-mtro-lH-pyrimidine-2, 4-diones (1) employing iodosylbenzene diacetate as an oxidant in the presence of lithium hydride. The generation of nitric oxide (NO) and NO-related species from 2 occurs in the presence of thiols such as AT-acetylcysteamine, cysteine, and glutathione under physiological conditions. The evidence for the NO generation derives from mechanistic interpretations for the reaction of 2 with thiols and other chemical observations.

Full text of DOI:10.1021/jo980732y

J . Org. Chem. 1998, 63, 6947-6951
6947
F a cile Syn th esis a n d NO-Gen er a tin g P r op er ty of
4H-[1,2,5]Oxa d ia zolo[3,4-d ]p yr im id in e-5,7-d ion e 1-Oxid es
Magoichi Sako,* Souichi Oda, Seiji Ohara, Kosaku Hirota, and Yoshifumi Maki
Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi, Gifu 502-8585, J apan
Received April 20, 1998
4H-[1,2,5]Oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-oxides (2) are conveniently prepared in high yields
by the oxidative intramolecular cyclization of 6-amino-5-nitro-1H-pyrimidine-2,4-diones (1) employ-
ing iodosylbenzene diacetate as an oxidant in the presence of lithium hydride. The generation of
nitric oxide (NO) and NO-related species from 2 occurs in the presence of thiols such as
N-acetylcysteamine, cysteine, and glutathione under physiological conditions. The evidence for
the NO generation derives from mechanistic interpretations for the reaction of 2 with thiols and
other chemical observations.
Nitric oxide (NO) generates in vivo in the process of
the oxygenative conversion of L-arginine to L-citrulline,
catalyzed by a heme-containing NO synthase,1 and has
been recognized to have a number of important physi-
ological roles in systems as varied as the transmission
of nerve impulses,2 regulation of blood flow,3 synergistic
inhibition of platelet aggregation,4 and nonspecific im-
mune response to bacterial infection.5 NO has limited
solubility (1.9 mM/L at 25 °C) in aqueous solutions6 and
is highly sensitive to autoxidation (-d[O2]/dt ) k1 [NO]2-
[O2] with k1 ) 2.9 × 106 M-2 s-1 at 22 °C)7 to convert
into the nitrite ion (NO2-) together with a slight amount
of the nitrate ion (NO3-). In view of physiological
significance and transient physicochemical property of
NO, much attention has been paid to the compounds that
serve as a NO donor with well-controlled releasing
property under physiological conditions and have the
potential pharmacological effects.8
that 4,6-dimethyl-4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-
5,7-dione 1-oxide (2a )10 causes relaxation of vascular
smooth muscles and inhibition of platelet aggregation in
guinea pig.11 This fact implies the release of NO from
2a in the biological systems and has directed our atten-
tion to the chemistry of this heterocycle having a struc-
turally unique fused ring system. In this paper, we
describe a new and facile synthetic method for 4,6-
unsubstituted or 4- and/or 6-substituted 4H-[1,2,5]oxa-
diazolo[3,4-d]pyrimidine-5,7-dione 1-oxides 2 and dem-
onstrate the generation of NO and NO-related species
from 2 under physiological conditions in order to sub-
stantiate our assumption on the biological activity of 2a .
During the course of our investigations on syntheses,
chemical reactivities, and biological activities of novel
nitrogen-containing heterocyclic N-oxides,9 we have found
(1) Butler, A. R.; Williams, D. L. H. Chem Soc. Rev. 1993, 233-
241. Anbar, M. Experientia 1995, 51, 545-550. Kerwin, J . F., J r.;
Lancaster, J . R., J r.; Feldman, P. L. J . Med. Chem. 1995, 38, 4343-
4362. Williams, R. J . P. Chem. Soc. Rev. 1996, 77-83. Wiseman, H.;
Halliwell, B. Biochem. J . 1996, 313, 17-29.
(2) Wang, T.; Xie, Z.; Lu, B. Nature 1995, 374, 262-266.
(3) Huang, P. L.; Huang, Z.; Mashimo, H.; Bloch, K. D.; Moskowitz,
M. A.; Bevan, J . A.; Fishman, M. C. Nature 1995, 377, 239-242;
Stamler, J . S. Nature 1996, 380, 108-111; Perutz, M. F. Nature 1996,
380, 205-206; J ia, L.; Bonaventura, C.; Bonaventura, J .; Stamler, J .
S. Nature 1996, 380, 221-226; Hoshino, M.; Maeda, M.; Konishi, R.;
Seki, H.; Ford, P. C. J . Am. Chem. Soc. 1996, 118, 5702-5707.
(4) Katzenschlager, R.; Weiss, K.; Rogatti, W.; Peskar, B. A.;
Sinzinger, H. Prostaglandins Leukot. Essent. Fatty Acids 1992, 45,
207-210.
Resu lts a n d Discu ssion
Syn th esis. [1,2,5]Oxadiazolo[3,4-d]pyrimidine 1-ox-
ides (furazano[3,4-d]pyrimidine 1-oxides; pyrimidofurox-
ans) have been used as versatile intermediates for the
preparation of biologically interesting fused pyrimidines
such as 8-substituted 9H-purines,10 4-aminopteridine 5,8-
dioxides,12 and 1H-benzo[g]pteridine-2,4-dione 5-oxides.13
There have been two primary methods for the construc-
tion of this fused ring system: (a) the thermal decom-
(5) Groves, J . T.; Marla, S. S. J . Am. Chem. Soc. 1995, 117, 9578-
9579. Suzuki, T.; Yamaoka, R.; Nishi, M.; Ide, H.; Makino, K. J . Am.
Chem. Soc. 1996, 118, 2515-2516. Fukuto, J . M.; Ignarro, L. J . Acc.
Chem. Res. 1997, 30, 149-152.
(6) Shaw, A. W.; Vosper, A. J . J . Chem. Soc., Faraday Trans. 1 1977,
73, 1239-1244. NO is much more soluble in apolar solvents such as
n-hexane, with a concentration at saturation of 0.13 M.
(7) Goldstein, S.; Czapski, G. J . Am. Chem. Soc. 1995, 117, 12078-
12084.
(9) Maki, Y.; Sako, M. J . Synth. Org. Chem. J pn. 1994, 52, 149-
160.
(10) Yoneda, F.; Tachibana, T.; Tanoue, J .; Yano, T.; Sakuma, Y.
Heterocycles 1981, 15, 341-344.
(11) Unpublished results. During the course of our investigation,
7-morpholino-[1,2,5]oxadiazolo[3,4-d]pyrimidine 1-oxide has been docu-
mented to relax potassium-depolarized guinea pig pulmonary arteries,
which implies
a NO-mediated mechanism, without any chemical
(8) Wink, D. A.; Darbyshire, J . F.; Nims, R. W.; Saavedra, J . E.;
Ford, P. C. Chem. Res. Toxicol. 1993, 6, 23-27. Ignarro, L. J .; Fukuto,
J . M.; Griscavage, J . M.; Rogers, N. E.; Byrns, R. E. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 8103-8107. Lefer, A. M.; Lefer, D. J . Drugs Future
1994, 19, 665-672. Saavedra, J . E.; Billiar, T. R.; Williams, D. L.; Kim,
Y.-M.; Watkins, S. C.; Keefer, L. K. J . Med. Chem. 1997, 40, 1947-
1954.
evidence for the NO generation (cf. Brendel, J .; Schoenafinger, K.;
Bohn, H. Eur. Pat. Appl. EP 573829, 1993; Chem. Abstr. 1994, 120,
217724v).
(12) Binder, D.; Noe, C. R.; Prager, B. C.; Turnowsky, F. Arzneim.-
Forsch. 1983, 33, 803-805.
(13) Yoneda, F.; Sakuma, Y.; Matsumoto, S. Heterocycles 1975, 3,
113-116.
S0022-3263(98)00732-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/05/1998

6948 J . Org. Chem., Vol. 63, No. 20, 1998
Sako et al.
position of 6-azido-5-nitropyrimidines, which are pre-
pared by treatment of 6-chloro-5-nitropyrimidines with
sodium azide or by the nitrosation of 6-hydrazino-5-
nitropyrimidines, with accompanying loss of nitrogen;14
(b) the nitrosative or nitrative cyclization of 6-hydroxy-
lamino-1H-pyrimidine-2,4-diones (cf. pyrimidine-2,4,6-
trione 4-oximes 8)15 in acidic media. These synthetic
methods are not so effective (29-65% yield) to prepare
the desired 4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-
dione 1-oxides 2 because of concurrent occurrence of
undesirable side reactions during the intramolecular
cyclization, i.e., tetrazole-ring formation without loss of
nitrogen in the former case and dehydrative dimerization
of the starting pyrimidine-2,4,6-trione 4-oximes leading
to the corresponding 1H,9H-pyrimido[5,4-g]pteridine-
2,4,6,8-tetrones in the latter case.
F igu r e 1.
succinimides in place of IBD. Their efficiency, however,
was lower than that of the IBD oxidation assisted with
LiH.
Taking the chemical reactivities of these oxidants into
consideration, a most plausible reaction sequence for the
oxidative formation of the oxadiazolopyrimidinedione
1-oxides 2 from the 6-amino-5-nitropyrimidinediones 1
is depicted in Figure 1 for the case of 1a with IBD.
The reaction is initiated by activation of the C6-amino
group in 1a with IBD after treatment with LiH to form
a N-halogenated intermediate. The transient intermedi-
ate undergoes a neighboring group participation by the
C5-nitro group assisted by LiH to afford the ultimate
product 2a , accompanied with the release of iodobenzene
and lithium acetate.18 Characteristics of this synthetic
method are (a) the use of 6-amino-5-nitro-1H-pyrimidine-
2,4-diones 1, which can be prepared from readily avail-
able 6-amino-1H-pyrimidine-2,4-diones in high yields, as
the starting material, (b) the use of IBD which is a
commercially available mild oxidant, and (c) the reaction
proceeding smoothly even under mild conditions without
accompanying undesired side reactions. The present
synthetic method was applicable to the syntheses of other
oxadiazolopyrimidines, e.g., [1,2,5]oxadiazolo[3,4-d]pyri-
midine-5,7-diamine 1-oxide (3)14 and 5-amino-6H-[1,2,5]-
oxadiazolo[3,4-d]pyrimidin-7-one 1-oxide (4).14
Gen er ation of NO an d NO-Related Species. Chem-
ical reactivities of the [1,2,5]oxadiazolo[3,4-d]pyrimidine
1-oxides (cf. 2) have been investigated on the behavior
toward primary amines,10,13,15b,19 alcohols,20 and active
methylene compounds12 under thermal conditions. The
most electrophilic site of these heterocyclic N-oxides has
been consequently demonstrated to be significantly de-
pendent on the nature of substituents on the pyrimidine
ring and, in the case of the oxadiazolopyrimidinedione
1-oxides 2, to be the 3a-position of the fused ring system.21
The chemical reactivities of 2a to the primary alkyl- or
arylamines were successfully utilized for the syntheses
of 8-substituted 1,3-dimethyl-3,9-dihydropurine-2,6-di-
ones (theophyllines)10 and 1,3-dimethyl-1H-benzo[g]-
pteridine-2,4-dione 5-oxides13 as described above. The
chemical behavior of this compound 2a toward other
Our recent work has developed a facile method for the
preparation of 4,6-dimethyl-4H-[1,2,5]oxadiazolo[3,4-d]-
pyrimidine-5,7-dione, the deoxygenated compound of 2a ,
involving an oxidative intramolecular cyclization of
6-amino-1,3-dimethyl-5-nitroso-1H-pyrimidine-2,4-di-
one with iodosylbenzene diacetate (IBD) after treatment
with lithium hydride (LiH).16 From a mechanistic point
of view, this method appears to be applicable to the
synthesis of 2 by using appropriate 6-amino-5-nitro-1H-
pyrimidine-2,4-diones 1 as starting materials.17 Along
this line, the IBD oxidation of 6-amino-1,3-dimethyl-5-
nitro-1H-pyrimidine-2,4-dione (1a ) was examined.
To a solution of 1a pretreated with two equimolar
amounts of LiH in DMF for 0.5 h, slightly excess IBD
was added. Warming the mixture at 50 °C with stirring
caused smooth consumption of 1a to give the correspond-
ing 4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-ox-
ide 2a almost quantitatively after 1 h. The structure of
2a was confirmed both by its microanalytical results and
spectral data and by independent synthesis starting from
1,3-dimethylpyrimidine-2,4,6-trione 4-oxime (8a ) as pre-
viously reported.15 Analogous results were obtained in
the IBD oxidations of 6-amino-1(or 3)-methyl-5-nitro-1H-
pyrimidine-2,4-dione (1b or 1c) and 1,3-unsubstituted
6-amino-5-nitro-1H-pyrimidine-2,4-dione (1d ) leading to
the corresponding oxadiazolopyrimidinedione 1-oxides
2b-d . The pretreatment with LiH was required for the
smooth conversion of 1a to 2a in this synthetic method,
i.e., the IBD oxidation of 1a without the LiH treatment
under similar conditions caused a significant delay in the
formation of 2a (the yield of 2a : 28% after 1 h; 61% after
3 h). The formation of 2a was also observed by employ-
ment of sodium hydride in place of LiH or N-halogeno-
(14) (a) Temple, C., J r.; Kussner, C. L.; Montgomery, J . A. J . Org.
Chem. 1968, 33, 2086-2089. (b) Nutiu, R.; Boulton, A. J . J . Chem.
Soc., Perkin Trans. 1 1976, 1327-1331. (c) Dang, V. T.; Stadlbauer,
W. Molecules 1996, 1, 201-206.
(15) (a) Yoneda, F.; Sakuma, Y.; Ueno, M. J . Heterocycl. Chem. 1973,
10, 415. (b) Yoneda, F.; Sakuma, Y. J . Heterocycl. Chem. 1973, 10, 993-
996. The 6-hydroxyimino-1H-pyrimidine-2,4-diones possess exclusively,
so far as can be judged by those spectral data, an oxime form (cf. 8) in
a solution.14b
(18) Analogous reaction sequence has been proposed for the oxida-
tive cyclization of o-aminonitrobenzenes leading to benzo[1,2,5]-
oxadiazoles. Cf. Dyall, L. K.; Kemp, J . E. Aust. J . Chem. 1973, 26,
1969-1976.
(19) Tennant, G.; Yacomeni, C. W. J . Chem. Soc., Chem. Commun.
1982, 60-62. Tennant, G.; Wallece, G. M. J . Chem. Soc., Chem.
Commun. 1982, 267-268.
(20) Remennikov, G. Y.; Pirozhenko, V. V.; Dyachenko, I. A. Khim.
Geterosikl. Soedin. 1992, 101-106.
(16) Sako, M.; Oda, S.; Hirota, K.; Beardsley, G. P. Synthesis 1997,
1255-1257.
(21) Previously, Yoneda et al. have demonstrated that the initial
attack of anilines occurs at the N1 position of the oxadiazolopyrim-
idinedione ring after isomerization on the basis of the structural
consideration of products, 1,3-dimethyl-1H-benzo[g]pteridine-2,4-dione
5-oxides.13 Our recent results of experiments using 15N-labeled com-
pound of 2a as a starting material have indicated that the formation
of the benzopteridinedione 5-oxides also can be reasonably explained
by the initial attack of the anilines to the 3a position of this fused
ring. These results will be published elsewhere.
(17) In fact, benzo[1,2,5]oxadiazoles are prepared by means of the
oxidative cyclization of appropriate o-aminonitrobenzenes with IBD
or alkaline hypochloride. Cf. Boulton, A. J .; Ghosh, P. B. In Advances
in Heterocyclic Chemistry, Vol. 10; Katritzky, A. R., Boulton, A. J ., Eds.
Academic Press: New York, 1969; pp 1-41. Gasco, A.; Boulton, A. J .
In Advances in Heterocyclic Chemistry, Vol. 29; Katritzky, A. R.,
Boulton, A. J ., Eds.; Academic Press: New York, 1981; pp 251-340.

4H-[1,2,5]Oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-Oxides
J . Org. Chem., Vol. 63, No. 20, 1998 6949
Ta ble 1. p H Dep en d en cy in th e Rea ction of th e N-Oxid e
2a w ith N-Acetylcystea m in ea
nucleophiles such as thiols and hydroxide ion, however,
is hitherto unknown.
The N-oxide 2a , being very stable in organic solvents
such as acetonitrile, was fairly unstable in aqueous
solution to undergo gradually an alkaline hydrolysis to
give 4-methylaminofurazan-3-carboxylic acid methyla-
mide 2-oxide (5), e.g., the 15% conversion of 2a leading
to 5 was observed after heating in a pH 7.5 phosphate
buffer solution at 37 °C for 5 h. In sharp contrast, 2a
was very unstable in the pH 7.5 buffer solution contain-
ing two equimolar amounts of N-acetylcysteamine to
convert into 5-(2-acetylaminoethyl)thio-1,3-dimethylpy-
rimidine-2,4,6-trione (6a ) (64%), 5,6-bis[(2-acetylamino-
ethyl)thio]-1,3-dimethyl-1H-pyrimidine-2,4-dione (7a) (3%),
and 1,3-dimethylpyrimidine-2,4,6-trione 4-oxime (8a )
(8%), together with the formation of bis(2-acetylamino-
remaining
2a (%)
pH
6 (%)
8 (%)
5.91
6.45
6.98
7.55
8.04
73
50
25
17
10
21
41
63
64
70
3
5
7
8
9
a
Reaction conditions: 2a (0.025 mmol) in buffer-MeCN (1:1)
(1.5 mL) containing N-acetylcysteamine (2 equiv), at 37 °C for 1
h.
of thiols in pH 7.5 buffer solution was most effective for
the generation of the NO-related species. Analogous
results were obtained by the use of cysteine and glu-
tathione in place of N-acetylcysteamine and by the use
of 2b-d in place of 2a as shown in Table 2, although
their efficiency in the generation of NO and NO-related
species was lower than that in the case of 2a and
N-acetylcysteamine. Thus, a substituent in the N4-
position of the N-oxides 2 seems to enhance their NO-
generating capacity.
On the basis of the above and precedent facts on the
chemical reactivities of 2, reasonable mechanisms for the
present reactions resulting in the NO generation are
outlined as shown in Figure 2 for the case of 2a . The
attack of thiols to both of the 3a and 7a positions in 2a
and the subsequent ring opening produce the key inter-
mediary adduct A. Further attack of another thiol to the
C-5 nitroso group of A releases thionitrite, a NO precur-
sor, together with the formation of 6a , 7a , and 8a .24
It should be noted that no formation of the NO or NO-
related species was observed in reactions of the deoxy-
genated compound of 2a and 6-amino-1,3-dimethyl-5-
nitroso(or nitro)-1H-pyrimidine-2,4-dione with N-acetyl-
cysteamine under similar conditions.
ethyl) disulfide. No formation of detectable amounts of
5 was observed in the reaction with N-acetylcysteamine.22
The structures of 6a and 7a were assigned on the basis
of these microanalytical results and spectral data, and
that of 8a was confirmed by spectral comparison with
the authentic compound prepared by the reaction of
6-chloro-1,3-dimethyl-1H-pyrimidine-2,4-dione with hy-
droxylamine.15 The formation of these products led us
to expect the generation of NO or S-nitroso-N-acetylcys-
teamine during the reaction. Thionitrites, however, have
been demonstrated to be unstable to undergo homolytic
cleavage of the S-N bond even at ambient temperature
and physiological pH to give the corresponding disulfides,
accompanied with the generation of NO.23 Furthermore,
the generated NO is very sensitive to air oxidation to
convert to NO2- as described above. Therefore, the total
amounts of NO and NO-related species generated during
the reaction of 2a with N-acetylcysteamine were evalu-
ated by the detection of NO2-, which was formed after
autoxidation of the reaction mixture, using the Griess
reagent (see Experimental Section). For example, the
Con clu sion
The IBD oxidation of 6-amino-5-nitro-1H-pyrimidine-
2,4-diones 1 in the presence of LiH afforded the corre-
sponding 4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione
1-oxides 2 in high yields. The present synthetic method
is widely applicable for the preparation of other 4H-[1,2,5]-
oxadiazolo[3,4-d]pyrimidine 1-oxides. The generation of
NO and NO-related species from 2a ,b in the presence of
thiols such as N-acetylcysteamine under physiological
conditions was strongly supported on the basis of the
structural elucidation of the reaction products and other
chemical evidence. The present results point out that
the 4-substituted 4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-
5,7-dione 1-oxides, e.g., 2a ,b, behave as useful NO
donors8 by the action of thiol cofactors.
-
evaluated NO2 was 28% yield in the 25 µM scale
experiment of 2a . In this reaction, the consumption of
2a and the generation of NO and NO-related species were
significantly dependent on pH in the media and the
concentration of the thiol employed as shown in Tables
-
1 and 2. No formation of NO2 was observed in the
reaction which was carried out in the absence of N-
acetylcysteamine, even on exposure of the reaction
mixture to air. The reaction using two equimolar amounts
Exp er im en ta l Section
Melting points are uncorrected. 1H NMR and 13C NMR
spectra were recorded at 400 and 75 MHz, respectively, by
using DMSO-d6 (unless otherwise noted) as a solvent and TMS
(22) Previously, Hecht et al. have documented that the deoxygen-
ation of heterocyclic N-oxides proceeds under analogous conditions,
accompanied with the generation of a hydroxyl radical. However, no
formation of detectable amounts of the deoxygenated product of 2a
was observed in this reaction, indicating no generation of hydroxyl
radicals under these reaction conditions.
(23) Oae, S.; Shinhama, K. Org. Prep. Proc. Int. 1983, 15, 167-198.
Williams, D. L. H. Chem. Soc. Rev. 1985, 14, 171-196. Roy, B.;
d′Hardemare, A. du M.; Fontecave, M. J . Org. Chem. 1994, 59, 7019-
7026. Williams, D. L. H. J . Chem. Soc., Chem. Commun. 1996, 1085-
1091. Beloso, P. H.; Williams, D. L. H. J . Chem. Soc., Chem. Commun.
1997, 89-90.
(24) Analogous reaction sequence has been proposed for the genera-
tion of NO from [1,2,5]oxadiazoles in the presence of thiol cofactors.
Cf. Medana, C.; Ermondi, G.; Fruttero, R.; Stilo, A. D.; Ferretti, C.;
Gasco, A. J . Med. Chem. 1994, 37, 4412-4416. In sharp contrast to
their results, the product 5 was very stable under the conditions
employed even in the presence of N-acetylcysteamine. See: Sorba, G.;
Medana, C.; Fruttero, R.; Cena, C.; Stilo, A. D.; Galli, U.; Gasco, A. J .
Med. Chem. 1997, 40, 463-469 and references therein.

6950 J . Org. Chem., Vol. 63, No. 20, 1998
Sako et al.
Ta ble 2. Gen er a tion of NO a n d NO-Rela ted Sp ecies in th e Rea ction of th e N-Oxid es 2 w ith Th iolsa
generated NO and NO-related species
N-oxides
thiols
after 1 h (%)
after 2 h (%)
0 (86)
after 3 h (%)
2a
2a
2a
2a
2a
2a
2b
2c
2d
without
0 (92)b
23 (69)
28 (23)
25 (7)
4 (91)
2 (86)
13 (66)
7 (90)
7 (89)
N-acetylcysteamine (1 equiv)
N-acetylcysteamine (2 equiv)
N-acetylcysteamine (3 equiv)
cysteine (2 equiv)
21 (6)
22 (trace)
3 (87)
21 (ND)
22 (ND)
glutathione (2 equiv)
4 (84)
N-acetylcysteamine (2 equiv)
N-acetylcysteamine (2 equiv)
N-acetylcysteamine (2 equiv)
a
Reaction conditions: a solution of 2 (0.025 mmol) and an appropriate thiol in 0.1 M phosphate buffer (pH 7.5)-MeCN (1:1) (1.5 mL)
was stirred at 37 °C in test tube equipped with balloon containing argon atmosphere. At hourly intervals, TLC analyses were done to
determine the consumption of 2 and then quantitative analyses were carried to evaluate nitrite ion generated in these reactions after air
b
treatment of the mixture. Values in parentheses are the yields of 2 that remained in these reactions.
residue with chloroform, the resulting precipitate was collected
by filtration and washed with chloroform to isolate the
corresponding 4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione
1-oxides (2) in almost pure state.
4,6-Dim et h yl-4H -[1,2,5]oxa d ia zolo[3,4-d ]p yr im id in e-
5,7-d ion e 1-oxid e (2a ): 95%; mp 243 °C (lit.10,14b,15 mp 245
°C dec]; mass m/z (rel intensity) 198 (M+, 100), 182 (M+ - O,
5), 168 (M+ - NO, 36), 149 (14), 83, 81; IR (KBr) 1740, 1689,
1640 cm-1; UV (MeOH, ꢀ) λmax 312 (2.1 × 103), 268 (1.7 × 104)
1
nm; H NMR δ 3.49 (3H, s), 3.57 (3H, s); 13C NMR δ 151.9,
151.8, 149.6, 101.2, 29.4, 28.1. This product was identical in
every respect with the authentic compound prepared indepen-
dently by the reaction of 6-chloro-1,3-dimethyl-1H-pyrimidine-
2,4-dione with hydroxylamine and subsequent nitrosation.10,14b,15
F igu r e 2.
4-Meth yl-4H-[1,2,5]oxa d ia zolo[3,4-d ]p yr im id in e-5,7-d i-
on e 1-oxid e (2b): 72%; mp 203 °C; mass m/z (rel intensity)
184 (M+, 100), 168 (M+ - O, 8), 154 (M+ - NO, 44), 149 (9) 81
(69); IR (KBr) 3160, 1741, 1688, 1642 cm-1; UV (MeOH, ꢀ) λmax
as an internal standard. Mass spectra were determined at
an ionizing voltage of 70 eV. Elemental analyses were
performed by the microanalytical laboratory of our university.
For thin-layer chromatographic (TLC) analyses, Merck pre-
coated TLC plates (Merck No. 5715; silica gel 60-F254) were
used. Column chromatography was performed on silica gel
(Merck No. 7734-5B; silica gel 60). Anhydrous solvents were
distilled and stored over activated 4 Å sieves before use.
Unless otherwise noted, materials obtained from commercial
suppliers were used without further purification. Starting
materials, 6-amino-1,3-dimethyl-5-nitro-1H-pyrimidine-2,4-di-
one (1a ),25 6-amino-1(or 3)-methyl-5-nitro-1H-pyrimidine-2,4-
dione (1b or 1c),26 6-amino-5-nitro-1H-pyrimidine-2,4-dione
(1d ),27 5-nitropyrimidine-2,4,6-triamine,28 and 2,6-diamino-5-
nitro-3H-pyrimidin-4-one,28 were prepared by the nitration of
the corresponding 6-aminopyrimidines (Aldrich or Tokyo
Kasei; 95-98% purity) in 88-99% yields.
1
311 (1.5 × 103), 266 (1.4 × 104) nm; H NMR δ 3.41 (3H, s),
12.1 (1H, br s); 13C NMR δ 153.2, 151.9, 149.5, 101.6, 28.3.
Anal. Calcd for C5H4N4O4: C, 32.62; H, 2.19; N, 30.43.
Found: C, 32.60; H, 2.27; N, 30.35.
6-Meth yl-4H-[1,2,5]oxa d ia zolo[3,4-d ]p yr im id in e-5,7-d i-
on e 1-oxid e (2c): 85%; mp 212 °C (lit.10,14b,15a mp 210-211
°C); mass m/z (rel intensity) 184 (M+, 100), 168 (M+ - O, 26),
154 (M+ - NO, 36); IR (KBr) 3108, 1738, 1680, 1639 cm-1
;
UV (MeOH, ꢀ) λmax 302 (1.5 × 103), 266 (1.2 × 104) nm; 1H
NMR δ 3.23 (3H, s), 12.8 (1H, br s); 13C NMR δ 152.4, 150.8,
149.7, 100.8, 27.4.
4H-[1,2,5]Oxa d ia zolo[3,4-d ]p yr im id in e-5,7-d ion e 1-ox-
id e (2d ): 97%; mp 257 °C (lit.14a mp 260 °C dec); mass m/z
(rel intensity) 170 (M+, 100), 154 (M+ - O, 13), 140 (M+
-
P r ep a r a tion of 4H-[1,2,5]oxa d ia zolo[3,4-d ]p yr im id in e-
5,7-d ion e 1-Oxid es (2) Usin g Iod osylben zen e Dia ceta te;
Gen er a l P r oced u r e. To a stirred suspension of the ap-
propriate 6-amino-5-nitropyrimidinedione (1) (1.0 mmol) in dry
DMF (3.0 mL) was added LiH (Aldrich, 95% purity) [17.7 mg
(2.1 mmol) for 2a ; 26.0 mg (3.1 mmol) for 2b-d ] in one portion.
After stirring for 0.5 h at ambient temperature, iodosylbenzene
diacetate (Aldrich, 98% purity) (483.1 mg, 1.5 mmol) was added
to the mixture and the solution was heated with stirring at
50 °C for 1 h. The mixture was decolorized gradually from
pale yellow during the reaction. After ensuring complete
consumption of the starting 6-amino-5-nitropyrimidinedione
1 by TLC analysis [developing solvent: chloroform-MeOH-
acetic acid (40/8/1)], the reaction mixture was diluted with
water (50 mL), neutralized with dilute hydrochloride, and
extracted with ethyl acetate (3 × 50 mL). The extract was
washed with brine (50 mL), dried over anhydrous magnesium
sulfate, and evaporated to dryness. After trituration of the
NO, 70), 127 (2) 111 (3); IR (KBr) 3305, 3190, 3100, 1743 (sh),
1714, 1643 cm-1; UV (MeOH, ꢀ) λmax 302 (2.0 × 103), 264 (1.6
× 104) nm; 1H NMR δ 11.7 (1H, br), 12.3 (1H, br); 13C NMR δ
152.5, 152.3, 149.8, 101.2.
In an analogous manner, 5-amino[1,2,5]oxadiazolo[3,4-d]-
pyrimidine 1-oxides 3 and 4 were prepared by using appropri-
ate 2,6-diamino-5-nitropyrimidines as starting materials.
[1,2,5]Oxa d ia zolo[3,4-d ]p yr im id in e-5,7-d ia m in e 1-ox-
id e (3): 57%; mp >300 °C (lit.14a mp >264 °C); mass m/z (rel
intensity) 168 (M+, 100), 152 (M+ - O, 13), 138 (M+ - NO, 7),
122 (19); IR (KBr) 3428, 3163, 1640 cm-1; UV (MeOH, ꢀ) λmax
1
371 (2.6 × 103), 290 (1.3 × 104), 230 (1.4 × 104) nm; H NMR
δ 8.58 (1H, br), 7.67 (1H, br), 7.15 (1H, br), 7.07 (1H, br); 13C
NMR δ 164.5, 162,2, 152.3, 98.7.
5-Am in o-6H -[1,2,5]oxa d ia zolo[3,4-d ]p yr im id in -7-on e
1-oxid e (4): 71%; mp >300 °C (lit.14a mp >264 °C); mass m/z
(rel intensity) 169 (M+, 100), 153 (M+ - O, 8), 139 (M+ - NO,
33), 123 (33); IR (KBr) 3367, 1725, 1631 cm-1; UV (MeOH, ꢀ)
1
λmax 329 (2.0 × 103), 275 (1.0 × 104) 227 (2.0 × 104) nm; H
NMR δ 11.3 (1H, br), 7.5 (1H, br), 6.7 (1H, br); 13C NMR δ
160.2, 155.2, 152,7, 101.1.
(25) Taylor, E. C.; McKillop, A. J . Org. Chem. 1965, 30, 3153-3155.
(26) Pfleiderer, W.; Hutzenlaub, W. Chem. Ber. 1973, 106, 3149-
3174.
In the cases of 2b, 3, and 4, small amounts of the corre-
sponding starting materials were recovered under the condi-
tions described above, respectively.
(27) Bitterli, P.; Erlenmeyer, H. Helv. Chim. Acta 1951, 34, 835-
840.
(28) Carbon, J . A. J . Org. Chem. 1961, 26, 455-461.

4H-[1,2,5]Oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-Oxides
J . Org. Chem., Vol. 63, No. 20, 1998 6951
The oxidation of 1a (12.5 mg, 0.063 mmol) with IBD (30.6
mg, 0.093 mmol), which was carried out without the LiH
treatment or after treatment with sodium hydride in place of
LiH, was followed by TLC densitometry [chloroform-MeOH
(20/1)] at hourly intervals. The yields of 2a in these reactions
were as follows: 28% after 1 h, 51% after 2 h, and 61% after
3 h in the former case; 89% after 1 h in the latter case,
accompanied with recovery of the starting 1a .
Oxid a tion of th e 6-Am in o-5-n itr op yr im id in ed ion e 1d
w ith N-Ha logen osu ccin im id es. Addition of N-bromosuc-
cinimide (Aldrich; 95% purity) (11.6 mg, 0.062 mmol) to the
solution of 1d (86.0 mg, 0.05 mmol) in dry DMF (1.0 mL)
containing LiH (1.3 mg, 0.15 mmol) and subsequent treatment
of the mixture at 50 °C for 1 h with stirring afforded 2d in
19% yield (by TLC densitometry) after 1 h, with recovery of
1d . Employment of N-iodosuccinimide (NIS) or N-chlorosuc-
cinimide (NCS) in place of NBS in this reaction did not effect
increase in the formation of 1d , i.e., the yields of 2d were 5%
in the case of NIS and 7% in the case of NCS.
Alk a lin e Hyd r olysis of 2a . A solution of 2a (198 mg, 1.0
mmol) in acetonitrile (12 mL) containing 1 N NaOH (5 mL)
was stirred at ambient temperature for 0.5 h. After neutral-
ization with diluted hydrochloride followed by concentration
under reduced pressure, the resulting precipitate was collected
by suction and was recrystallized from acetone to give 4-meth-
ylaminofurazan-3-carboxylic acid methylamide 2-oxide (5) (126
mg, 73%): mp 279 °C; mass m/z (rel intensity) 172 (M+, 100),
155 (M+ - OH, 67), 142 (M+ - NO, 87), 126 (8) 83 (19); IR
(KBr) 3236, 1749, 1647 cm-1; UV (MeOH) λmax 287, 250 nm;
1H NMR δ 3.01 (3H, s), 3.49 (3H, s), 11.02 (1H, br s), 11.34
(1H, br s). Anal. Calcd for C5H8N4O3: C, 34.89; H, 4.68; N,
32.55. Found: C, 34.91; H, 4.62; N, 32.45. The reaction was
carried out in the acetonitrile solution containing 0.1 M pH
7.0 (or pH 7.5) phosphate buffer and was followed by TLC
densitometry. The analyses of the reaction mixtures showed
the formation of 5 in 8% (at pH 7.0) and 15% (at pH 7.5) yields,
respectively, with recovery of 2a after stirring at 37 °C for 5
h.
Rea ction s of 2a a n d 2b w ith N-Acetylcystea m in e. A
solution of 2a (99.1 mg, 0.5 mmol) in 0.1 M K2HPO4-KH2PO4
buffer (pH 7.5)-acetonitrile (1/1) (10.0 mL) containing N-
acetylcysteamine (Aldrich, 95% purity) (112.0 µL, 1.0 mmol)
was stirred at 37 °C under argon for 1 h. After removal of
the solvent under reduced pressure, the resulting residue was
subjected to column chromatography and was eluted with
chloroform-MeOH (10/1 to 4/1) to separate 5-(2-acetylamino-
ethyl)thio-1,3-dimethylpyrimidine-2,4,6-trione (6a ) [99.6 mg
(64% as a potassium salt); mp >300 °C; mass m/z (rel intensity)
213 (M+ - AcNH2 - 1, 16), 156 (M+ - AcNHCH2CH2S + 1,
3.39 (3H, s), 3.40 (2H, q), 3.48 (2H, q), 3.73 (3H, s), 6.42 (1H,
br t), 6.82 (1H, br t). Anal. Calcd for C14H22N4O4S2: C, 44.90;
H, 5.92; N, 14.96. Found: C, 44.74; H, 5.92; N, 14.79], 1,3-
dimethylpyrimidine-2,4,6-trione 4-oxime (8a ) (6.4 mg, 8%), and
bis(2-acetylaminoethyl) disulfide (34.3 mg, 27%), together with
recovered 2a (16.8 mg, 17%) and N-acetylcysteamine (40.9 mg,
34%). The product 8a was identical in every respect with the
authentic compound prepared independently by the reaction
of 6-chloro-1,3-dimethyl-1H-pyrimidine-2,4-dione with hydrox-
ylamine.
Under analogous conditions, the reaction of 2b (18.4 mg,
0.1 mmol) with N-acetylcysteamine (21.3 µL, 0.2 mmol) was
carried out to give 5-(2-acetylaminoethyl)thio-1-methylpyri-
midine-2,4,6-trione (6b) [20.9 mg (70% as a potassium salt);
mp >300 °C; mass m/z (rel intensity) 259 (M+ - 1, 1), 200
(M+ - AcNH2, 2), 142 (M+ - AcNHCH2CH2S + 1, 95), 118
(47), 86 (100); IR (KBr) 3424, 1686, 1637, 1562 cm-1; UV
(MeOH, ꢀ) λmax 259 (1.9 × 104) nm; 1H NMR δ 1.81 (3H, s),
2.32 (2H, t), 3.03 (3H, s), 3.08 (2H, q), 8.56 (1H, br t), 9.53
(1H, br s); 13C NMR δ 168.5, 165.2, 164.5, 152.0, 76.4, 37.9,
35.6, 26.8, 22.7] and 5,6-bis[(2-acetylaminoethyl)thio-1-methyl-
1H-pyrimidine-2,4-dione (7b) [4.1 mg (11%); mass m/z (rel
intensity) 360 (M+, 7), 301 (M+ - AcNH2, 8), 86 (100); IR (KBr)
3308, 1676, 1645 cm-1; UV (MeOH) λmax 302 nm; 1H NMR
(CDCl3) δ 2.01 (3H, s), 2.02 (3H, s), 2.98 (2H, t), 3.21 (2H, t),
3.41 (2H, q), 3.50 (2H, q), 3.69 (3H, s), 6.37 (1H, br t), 6.69
(1H, br t), 8.89 (1H, br). Anal. Calcd for C14H22N4O4S2: m/z
360.0938. Found: m/z 360.0932], accompanying with bis(2-
acetylaminoethyl) disulfide.
Qu a n tita tive An a lyses of Nitr ite Ion . The appropriate
oxadiazolopyrimidinedione 2 (0.025 mmol) was added to a
mixed solution of 0.1 M phosphate buffer (pH 5.91-8.04) and
acetonitrile (1:1) (1.5 mL) containing the appropriate thiol (N-
acetylcysteamine, cysteine, or glutathione) or without, with
stirring. After continuation of stirring at 37 °C for 1 h in a
test tube equipped with an argon balloon followed by air
treatment for 10 min, 10 µL of the reaction mixture was diluted
with 1.0 mL of water and was treated with 100 µL of the Griess
reagent [sulfanilamide (4 g), N-naphthylenediamine dihydro-
chloride (0.2 g), and 85% phosphoric acid (10 mL) in distilled
water (final volume: 100 mL)] for 10 min at room temperature.
After dilution of the solution with water (3.1 mL), the absor-
bance was measured at 540 nm; 2-10 nmol/mL sodium nitrite
standard solutions were used for the calibration curve. The
results estimated were shown in Table 2.
Ack n ow led gm en t. This work was supported by a
Grant-in-Aid for Scientific Research (C) (No. 08672564)
from the Ministry of Education, Science, Sports and
Culture in J apan.
8), 86 (18), 44 (100); IR (KBr) 3388, 1676, 1652, 1562 cm-1
;
UV (MeOH, ꢀ) λmax 260 (1.7 × 104) nm; H NMR δ 1.81 (3H,
s), 2.34 (2H, t), 3.08 (2H, q), 3.10 (6H, s), 8.48 (1H, br t); 13C
NMR δ 168.7, 163.9, 152.6 (2C), 77.0, 38.1, 35.6, 27.7 (2C),
22.7. Anal. Calcd for C10H14N3O4SK: C, 38.57; H, 4.53; N,
13.49. Found: C, 38.44; H, 4.49; N, 13.65], 5,6-bis[(2-acetyl-
aminoethyl)thio]-1,3-dimethyl-1H-pyrimidine-2,4-dione (7a )
[5.8 mg (3%); mp 140-141 °C; mass m/z (rel intensity) 374
(M+, 7), 315 (M+ - AcNH2, 8), 86 (100); IR (KBr) 3281, 1699,
1
Su p p or tin g In for m a tion Ava ila ble: Spectral data and
spectra for compounds prepared (12 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.
1
1642 cm-1; UV (MeOH, ꢀ) λmax 300 (1.5 × 104) nm; H NMR
(CDCl3) δ 2.00 (3H, s), 2.02 (3H, s), 2.96 (2H, t), 3.18 (2H, t),
J O980732Y