Radical scavenging by N-aminoazaaromatics

Article abstract of DOI:10.1016/S0968-0896(00)00118-8

N-Aminoazaaromatics were found to react with nitric oxide in the presence of oxygen to afford deaminated products in high yields. The reaction proceeded almost instantaneously in various solvents including water, and one to two equivalent of NO was consumed depending upon the amount of oxygen coexisted, and 1 equivalent of N₂O was released in the reaction. In addition, N-aminoazoles were deaminated by potassium superoxide to give parent azoles in good yields. Two equivalents of superoxide was consumed, and about half equivalents of both nitrite and nitrate ion were released. The results demonstrated that N-aminoazoles have ability to protect the biological system against the oxidation promoted by radicals such as nitrogen oxides and superoxide. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00118-8

Bioorganic & Medicinal Chemistry 8 (2000) 1983±1989
Radical Scavenging by N-Aminoazaaromatics
Takashi Itoh, Michiko Miyazaki, Hiromi Maeta, Yuji Matsuya,
Kazuhiro Nagata and Akio Ohsawa*
School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Received 22 March 2000; accepted 28 April 2000
AbstractÐN-Aminoazaaromatics were found to react with nitric oxide in the presence of oxygen to a€ord deaminated products in
high yields. The reaction proceeded almost instantaneously in various solvents including water, and one to two equivalent of NO
was consumed depending upon the amount of oxygen coexisted, and 1 equivalent of N2O was released in the reaction. In addition,
N-aminoazoles were deaminated by potassium superoxide to give parent azoles in good yields. Two equivalents of superoxide was
consumed, and about half equivalents of both nitrite and nitrate ion were released. The results demonstrated that N-aminoazoles
have ability to protect the biological system against the oxidation promoted by radicals such as nitrogen oxides and superoxide.
# 2000 Elsevier Science Ltd. All rights reserved.
Introduction
yields.13 NO or higher nitrogen oxides was transformed
stoichiometrically to nitrous oxide N2O. In addition, N-
aminoazole derivatives were also found to quench super-
oxide anion, which is a precursor of peroxynitrite14, 15 and
other active oxygen species.16 This paper describes these
results.
Despite its simple structure, nitric oxide (NO) has been
found to play diverse roles in biological systems.1,2
Accompanied by the positive physiological roles, NO
causes extensive damage to the body in the cases that NO
is released at relatively higher concentration.3 In these cir-
cumstances, NO is activated by the reaction with various
active oxygens to a€ord more reactive nitrogen oxides, and
the ultimate reactive species included in the processes has
been an important target for toxicological studies.4 From
the viewpoint of environmental sciences, the conversion
of NO into species such as N2O and N2 has important
consequences.5,6
Results and Discussion
Reaction of N-aminoazaaromatics with nitric oxide in
the presence of various amounts of oxygen
N-Aminopyrazole (1)17 was allowed to react for 5 min
with NO under air in acetonitrile to give pyrazole in 100%
yield accompanied by 1 equivalent (equiv) of N2O, whose
quantitative formation was con®rmed by gas chromato-
graphy (GC). Other N-aminated compounds of indazoles
and imidazoles were deaminated in the same manner to
give the products (Scheme 1 and Fig. 1). The yield was
For the understanding of reactive species of nitrogen
oxides toward organic molecules, we have recently been
studying the reaction of nitric oxide with nitrogen
compounds in the presence and absence of oxygen,7 10
and found that oxygen plays two types of roles in this
system, that is, one in which oxygen is consumed according
to a stoichiometry, and the other where oxygen functions
in a catalytic manner.11,12 With these results in hand, we
next applied the reaction system to development of a
new methodology of nitric oxide quenching, and found
that N-aminoazaaromatics reacted rapidly with NO in
the presence of various amounts of oxygen to a€ord
corresponding deaminated products in quantitative
1
measured with H NMR using mesitylene or 1,4-diox-
ane as an internal standard.
As shown in Table 1, an equimolar amount of NO
a€orded about 50% yield of the product, and the starting
material was almost entirely consumed by 2 equiv of nitric
oxide in a very short reaction time. Although the reaction
time (5 min) was adopted to obtain the reproducible data,
the reaction completed instantaneously, which was indi-
cated by TLC. Unexpectedly, the same amount of NO is
consumed even when the substrate has a positive charge
(substrates 10±13).
*Corresponding author.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00118-8

1984
T. Itoh et al. / Bioorg. Med. Chem. 8 (2000) 1983±1989
Figure 1. N-Aminoazaaromatics used for the reaction.
Table 2. The relationship between the yield of the deaminated pro-
duct and the amounts of NO and O2 using N-aminopyrazole (1) as a
substrate
Entry
NO
(equiv)
O2
(equiv)
Time
(h)
Yield
(%)
Recovery
(%)
Scheme 1.
1
2
3
4
5
6
7
8
9
10
2
2
2
0.5
0.25
0.1
0.05
0.5
0.25
0.5
0.5
0.25
excess
1
1
0.5
4
1
13
1
4
13
9
96
93
49
46
100
73
67
77
80
65
Ð
Ð
46
52
Ð
26
31
20
20
33
Table 1. The reaction of N-aminoazaaromatics with NO in air
N-Aminoazaaromatic
NO
(equiv)
Reaction
time
Yield
(%)a
4
4/3
4/3
1
1
1
1
1
2
2
1
2
2
1
2
2
1
2
1
2
9 h
5 min
5 min
5 h
5 min
5 min
9 h
5 min
5 min
5 h
5 min
5h
5 min
65 (33)
100
72 (17)
57 (43)
97
92 (6)
55 (43)
90
90
53 (43)
100
2
4
1
6
10
11
12
One of the plausible mechanisms based on the stoichio-
metry in Table 2 is shown in Scheme 2. In the presence
of excess amount of O2, NO is transformed to NO2 (or
0.5 N2O4), and the reaction might proceed through the
electrophilic attack of N2O4 to N-amino group to give the
deaminated product, N2O, and nitrate ion (eqs (1) and
(2)). As decreasing the amount of O2, the active nitrogen
oxide mainly involved might be dinitrogen trioxide N2O3
(eq (3)), and N2O3 attacks amino group to a€ord the
product, N2O, and nitrite ion (eq (4)). Nitrite ion thus
obtained is known to dimerize to regenerate N2O3 (eq
(5)), thus the overall stoichiometry of the reaction is
thought to be as shown in eq (6). In this occasion, the
stoichiometrical amount of O2 should be 0.25 equiv.
Although the two types of the reaction processes men-
tioned above might consume over 0.25equiv of O2, there
is another pathway that involves a catalytic contribution
of O2 as shown in Entry 4 of Table 2, in which 0.05 equiv
of O2 a€orded 46% of the product. We have also found
this type of the O2 catalytic process in the reaction of
aromatic primary amines with NO,7 and presume that
N2O3 reacts with the substrate so as to give NO and
NO2, which regenerates N2O3 via bonding to NO (eqs (7)
and (8)).
13
51 (19)
92
aThe numbers in the parentheses show the recovery (%) of the starting
materials.
In order to determine the stoichiometry of oxygen, the
reaction using N-aminopyrazole (1) was carried out in
the presence of various amounts of NO and O2, and the
results are summarized in Table 2.
When 2 equiv of NO was applied, the reaction completed
by the use of 0.25 equiv of O2. In the case of the use of
0.05 equiv of O2, the reaction proceeded at nearly half
point in the presence of 4 equiv of NO (entry 4). Thus it
was suggested that the partial catalytic behavior of O2
be involved in the reaction. The use of equimolar
amount of NO resulted in 80% yield of the deaminated
product in the presence of 0.25 equiv of O2, and excess
O2 caused the slight decrease of the yield (entries 7±10).
Thus, there seems to exist at least three pathways that
consume di€erent amounts of NO and O2.

T. Itoh et al. / Bioorg. Med. Chem. 8 (2000) 1983±1989
1985
Table 4. a. The reaction of 2 with NO and O2 in various solvents
Solvent
Time
(h)
Yield
(%)
Recovery
(%)
CH2Cl2
DCEa
THF
EtOH
DMF
2
2
2
24
2
92
94
91
82
88
Ð
Ð
Ð
17
2
b. The reaction of 11 with NO and O2 in various solvents
Scheme 2.
Solvent
Time
(h)
Yield
(%)
Recovery
(%)
The same stoichiometry was observed when N-amino-
pyridinium perchlorate was adopted as a substrate using
various amounts of NO and O2 (Table 3). Consequently,
it was suggested that the two types of the substrates
undergo the deamination through the same reaction
process. Although it seems improbable for N-amino qua-
ternary salt to receive electrophilic attack of nitrosating
agent, one possible explanation is that N-amino quaternary
salts initialize the reaction by deprotonation of N-amino
group to form an imine 15, which undergoes the reaction
with N2O3 or N2O4 to give an N-nitroso derivative
(Scheme 3).
CH2Cl2
DCEa
THF
EtOH
DMF
2
2
2
2
2
98
98
36
Ð
64
Ð
Ð
36
90
18
aDCE, 1,2-dichloroethane.
ethanol. These data suggest that there can be di€erent
reaction paths in these two types of substrates. In ethanol,
nitrosating agents would react with the solvent to form
a nitrite ester, which could have di€erent reactivity
toward 2 and 11.
The above data indicated that the N-aminoazaaromatics
react with NO (in the presence of O2), N2O3, and N2O4
in di€erent manners, and eliminate these species as a
form of N2O. For studying the applicability of the
reaction, we investigated the solvent e€ect of the reaction,
and Table 4a and b show the data. While the reaction
proceeded smoothly in any type of solvents in the case
of a pyrazole derivative 2, a pyridinium salt 11 reacted
slowly in THF, and gave no deaminated product in
Table 5 represents the results of deamination reaction of
N-aminoazaaromatics in water. Except the substrates
insoluble in water (2 and 4), the reaction proceeded
smoothly to give the deaminated product.
Reaction of N-aminoazoles with potassium superoxide
Next, we investigated the reaction of N-aminoazaaro-
matics with superoxide. Since superoxide has been
known to be a precursor of other active oxygen species16
and peroxynitrite,14,15,18 the scavenging of this anion
radical is one of the most important issues in the biological
system.
Table 3. The relationship between the yield of the deaminated pro-
duct and the amounts of NO and O2 using N-aminopyridinium per-
chlorate (10) as a substrate
Entry
NO
(equiv)
O2
(equiv)
Time
(h)
Yield
(%)
Recovery
(%)
1
2
3
4
5
6
2
2
2
4/3
1
1
0.5
0.25
0.1
0.5
0.5
0.5
1
1
4
4
100
100
46
100
68
Ð
Ð
50
Ð
30
43
Table 5. Deamination of N-aminoazzaaromatics with NO (2 equiv)
and O2 (0.5 equiv) in water
Substrate
Reaction
time (h)
Yield
(%)
excess
9
55
1
2
3
4
3
2
2
1
1
3
1
1
1
96
49
95
63
100
83
69
95
5
10
11
12
14
76
Scheme 3.

1986
T. Itoh et al. / Bioorg. Med. Chem. 8 (2000) 1983±1989
When 1-amino-5-methyl-3-phenylpyrazole (2) was
allowed to react with potassium superoxide in acetonitrile,
the deaminated product was obtained in a good yield,
which was measured with 1H NMR using mesitylene as an
internal standard. Table 6 shows the results using various
amounts of superoxide and 18-Crown-6 (Scheme 4).
One of the plausible mechanisms is as follows (Scheme 6).
The reaction is commenced with abstraction of a proton
from N-amino group by superoxide to form an N-imino
anion 1621 and hydroperoxy radical 17. The obtained 17
is well known to react rapidly with superoxide to give
hydrogen peroxide and oxygen.22 Oxygen thus formed
might react with 16 to give an aminyl radical 18, which
further reacts with superoxide to a€ord a peroxy anion
19. The anion 19 is supposed to decompose to an N-
nitroso compound 20 and OH . The overall stoichiometry
of eq (10) to eq (14) is representated as eq (15), which
means that the same amount of HOO and HO was
formed by the reactions. The N-nitroso compound 20
derived from azaaromatics is thought to be unstable
under the conditions to decompose by nucleophilic
The data indicated that the presence of 18-Crown-6 was
crucial for the rapid progress of the reaction (entries 1±5),
and the reaction demanded 2 equiv of superoxide (entries
5, 8, and 9). Unexpectedly, the presence of O2 did not
a€ord any in¯uences upon the yields (entries 6, 8, and 5
versus 10, 11, and 12), which indicated that O2 did not
participate in rate determining step(s). The reaction also
proceeded by the use of electrogenerated superoxide.
For the understanding of the stoichiometrical relationship
of the reaction, the fate of the nitrogen atom of N-
amino group was investigated, and it was found that
nearly half equiv of nitrite and nitrate ion were released in
every case (Scheme 5). The amounts of nitrite19 and
nitrate20 were determined using reported methods.
Another possible nitrogen derivatives such as N2, N2O,
and NO were not detected in the mixture. Thus, the reac-
tion equation should be formulated as follows (eq (9)).
Scheme 5.
The other substrates were also applied to the reaction
system and the results are summarized in Table 7. Any
N-aminoazoles tested were found to be good substrates
for the reaction, and the stoichiometry of the reac-
tion was in accord with that of eq (9) in every case.
The N-aminated quaternary salts of azaaromatics,
however, resulted in low yields of deaminated products
accompanied by complex mixtures.
Table 7. The yield of deaminated product by the reaction of N-ami-
noazaaromatics with potassium superoxide (2 equiv) under Ar
Substrate
Reaction Time (h)
Yield (%)
1
2
3
4
7
8
9
10
1
1
1
1
4
1
2
2
82
91
80
84
75
97
91
13
Table 6. The relationship between the yield of the deaminated pro-
duct and the amounts of KO2 using 1-amino-5-methyl-3-phenylpyr-
azole (2) as a substrate
Entrya
KO2
(equiv)
18-Crown-6
(equiv)
Time
(h)
Yield
(%)
Recovery
(%)
1
2
3
4
5
6
7
8
9
2
2
2
2
2
1
1
1
1.5
0
0.5
2
2
2
1
1
1
1.5
25
25
0.1
0.2
1
0.5
1
17
3
53
76
67
86
91
55
61
63
81
20
4
28
8
1
41
35
31
12
10
11
12
1
1
2
1
1
2
0.5
17
1
54
62
92
44
32
1
aEntries 1±9 were the experiments under argon, and 10±12 were under O2.
Scheme 4.
Scheme 6.

T. Itoh et al. / Bioorg. Med. Chem. 8 (2000) 1983±1989
1987
attack of either OH or HOO to give nitrite or nitrate
ion, respectively. In fact, there are few reports concerning
stable N-nitrosoazoles other than indoles23 and carba-
zole.24 N-Nitrosocarbazole was synthesized according to
the reported method,24 and was subjected to the reaction
with OH or HOO . The results showed that denitrosa-
tion proceeded at similar rates with both nucleophiles.
Thus, in the reaction, these two nucleophilic attacks
seem to occur at about the same rate to a€ord two
anions in nearly the same yields. Another possible
mechanism for the formation of 1:1 of nitrite: nitrate is
hydration of N2O4.25 However, in the presence of equimo-
lar N-aminoazole and H2O, N2O4 was found to react
quite faster with N-aminoazole than H2O. Thus, we
concluded that N2O4 (NO2) was not released in the
reaction. In the case of N-aminated quaternary salts,
N-imine 16 is readily formed13 and would be more
stable than those of N-aminoazoles, thus the step shown
in eq (12) should be slow.
spectra were recorded on a JMS-SX102A instrument. The
nuclear magnetic resonance spectra (NMR) were measured
with JEOL GX400 and LA500 spectrometers using tet-
ramethylsilane as an internal standard. The abbreviations
used are as follows: s, singlet; d, doublet; dd, double
doublet; dt, double triplet; dq, double quartet; t, triplet;
q, quartet; quin, quintet; m, multiplet. High perfor-
mance liquid chromatography (HPLC) analysis was
carried out using a JASCO UV-1575 PU-1570 system.
Materials. Nitric oxide gas (99.9%) was purchased from
Takachiho Chemical Company Ltd, and was passed
through 10 M NaOH aqueous solution and a column of 4±
8 mesh soda lime to remove NOX impurities. All chemicals
except N-aminoazaaromatics were of analytical grade and
were used as received.
Synthesis of N-aminoazaaromatics
Method A. According to the reported method,17,29,30 an
azaaromatic was dissolved in aqueous NaOH. Hydro-
xylamine-O-sulfonic acid (HAS) in limited amounts was
added to the solution, which was kept at 50 ꢀC. Then
the mixture was allowed to stir at room temperature to
complete the reaction. The mixture was extracted with
CH2Cl2, and the organic layer was dried over MgSO4,
and evaporated o€ to leave the residue, which was
chromatographed on alumina (aluminium oxide 90 (70±
230 mesh, Merck)) or silica gel (Kieselgel 60 (70±230
mesh, Merck)) to give the product.
Conclusion
In this paper, we described the deamination of N-
aminoazaaromatics with various nitrogen oxides. It was
found that the substrates reacted rapidly with nitrogen
oxides to form N2O. The reaction proceeded in the pre-
sence of various amounts of O2, and the products were
only deaminated azaaromatics and N2O. In addition,
diverse N-aminoazaaromatics were found to be applicable
for the reaction, and a variety of solvents are available
to the reaction including water. This nitrosation±
deamination of N-amino group has an advantage over
that of C-amino compounds in that deamination pro-
duces a stable azaaromatics whereas C-amino deriva-
tives give carbocations or carbon radicals,7 which might
be toxic to the body. In addition, deamination of N-ami-
noazaaromatics with nitrogen oxides is superior to that of
general N-amino compounds because the latter gives N-
nitroso compounds, which may be carcinogenic,26 as ®nal
products. Thus the reaction system is thought to be of use
for the quenching nitrogen oxides. Moreover, there are
many azaaromatics that act as inhibitors of nitric oxide
synthase (NOS), some of which involve 7-nitroindazole27
and 1-phenylimidazole.28 Our reaction system can a€ord
these compounds by the deamination of corresponding N-
amino derivatives, and this indicates the reaction might
provide a system in which NOS inhibitor is released only
in the presence of enough NO.
Method B. O-Mesitylenesulfonylhydroxylamine (MSH)
was synthesized according to the reported method.31 The
activity of MSH, which included considerable amount of
water in synthetic process, was determined by iodide±
thiosulfate titration, and 2.5 mmol of MSH was dis-
solved in CH2Cl2. Water was separated o€, and the
organic solution was dried over MgSO4. An azaaromatic
(2.0 mmol) and NaH (2.5 mmol) were reacted in THF at
0 ꢀC for 30 min, then the MSH solution was added
dropwise. After the mixture was allowed to stand at
room temperature for several hours, methanol was
added to quench excess NaH, and the mixture was ®l-
tered. The ®ltrate was evaporated to dryness under
reduced pressure, and the residue was chromatographed
on alumina or silica gel to give the product. Some of the
N-aminoazaaromatics were dicult to separate from
the starting materials by simple chromatography. In
these cases, N-aminoazaaromatics were transformed to
imines by the treatment with acetone in the presence of a
catalytic amount of p-toluenesulfonate. After the separa-
tion of the imines and the starting material, the isolated
imines were hydrolyzed to give the N-amino compounds.
Among 14 N-aminoazaaromatics used for the
reaction,17,32 34 6, 8, 11, and 12 are new compounds.
Moreover, it was found that N-aminoazoles are good
scavenger of superoxide, which is a precursor of various
active oxygens and peroxynitrite. Therefore, they are
found to be e€ective toward most radicals originating
from the biological systems. The detailed reaction
mechanisms and the application to the biological system
is now under investigation.
1-Amino-7-nitroindazole (6). This was synthesized using
method B. Yellow powder from hexane±ethyl acetate: mp
157±158 ꢀC; 1H NMR (400 MHz, DMSO-d6) d 6.49 (2H,
bs), 7.26 (1H, dd, J=8.0Hz, 7.6 Hz), 7.95 (1H, d,
J=7.6Hz), 8.08 (1H, d, J=8.0 Hz), 8.21 (1H, s). Anal.
Experimental
Melting points were measured with a Buchi535 micro-
melting point apparatus and are uncorrected. The mass

1988
T. Itoh et al. / Bioorg. Med. Chem. 8 (2000) 1983±1989
calcd for C7H6N4O2: C, 47.19; H, 3.39; N, 31.45. Found:
C, 47.43; H, 2.81; N, 31.15.
11 in H2O, the analysis indicated that 90% of N2O was
present in the gas phase. This datum also suggested that
an equivalent of N2O was formed in the reaction.
1-Amino-3,5-diphenyl-1,2,4-triazole (8). This was syn-
thesized using method B. Colorless granules from etha-
nol: mp 193±194 ꢀC; 1H NMR (400 MHz, CDCl3) d
7.39±7.52 (6H, m), 8.12 (2H, dd, J=6.6Hz, 2.0 Hz), 8.25
(2H, dd, J=7.3Hz, 2.0 Hz); 13C NMR (100 MHz,
CDCl3) d 126.07, 127.06, 128.30, 128.47, 128.78, 129.20,
129.98, 130.65, 152.47, 158.31. Anal. calcd for C14H12
N4: C, 71.17; H, 5.12; N, 23.71. Found: C, 71.04; H,
4.97; N, 23.69.
General procedure for the reaction of N-aminoazaaro-
matics with potassium superoxide. KO2 and 18-Crown-6
was dissolved in CH3CN, and the solution was bubbled
with dry Ar gas for 20 min when necessary. Then the
¯ask was sealed, and a substrate in CH3CN was intro-
duced to the reaction mixture using a syringe. The
mixture was allowed to stir at room temperature for the
desired hours. Thereafter, the solvent was evaporated
o€ to leave the residue, whose analysis was performed
by 1H NMR using mesitylene or 1,4-dioxane as an
internal standard. In the cases that the products are
volatile, the reaction was carried out in CD3CN, and the
mixture was analyzed directly by NMR. Electro-
generated superoxide also a€orded a similar result. In the
experiment, N-aminopyrazole was dissolved in 40 mL of
0.1 M tetraethylammonium perchlorate solution of ace-
tonitrile and a stream of oxygen was bubbled into the
solution through a gas dispersion tube which was inser-
ted into the cathode chamber of a H cell containing
platinum electrode. The electroreduction of oxygen was
carried out with Nikko Keisoku potentiogalvanostat
NPGS-2501. The potential was set and maintained at
1.10 V versus SCE until the starting material was dis-
appeared.
1-Amino-4-phenylpyridinium perchlorate (11). This was
synthesized using meꢀthod A. Pale yellow powder from
ethanol: mp 120±121 C; 1H NMR (400 MHz, CD3OD)
d 7.60±7.62 (3H, m), 7.92±7.94 (2H, m), 8.29 (2H, d,
J=7.0 Hz), 8.73 (2H, d, J=7.0 Hz). Anal. calcd for
C11H11ClN2O4: C, 48.80; H, 4.10; N, 10.35. Found: C,
49.08; H, 3.88; N, 10.26.
1-Amino-3-phenylimidazolium perchlorate (12). This was
synthesized using method B. Colorless plates from eth-
anol: mp 132±133 ꢀC; H NMR (400 MHz, CDCl3) d
1
7.49 (5H, s), 7.53 (1H, d, J=1.5 Hz), 7.69 (1H, d,
J=1.5 Hz), 9.11 (1H, s). Anal. calcd for C9H10ClN3O4:
C, 41.63; H, 3.88; N, 16.18. Found: C, 41.76; H, 4.18; N,
15.97.
General procedure for the reaction of N-aminoazaaro-
matics with nitric oxide in the presence of oxygen. The
substrate (0.2 mmol) was placed in a two-necked ¯ask
equipped with a rubber septum and a three-way stop-
cock, one outlet of which was attached to an Ar balloon,
and another to a pump. The ¯ask was degassed in vacuo
and ®lled with Ar gas. A solvent (10 mL) was added and
the solution was bubbled with Ar gas for 20 min, then
the ¯ask was sealed. NO gas was passed through a column
of soda lime and a necessary amount was measured
using a Hamilton gas-tight syringe, and added to the
reaction vessel. Then, a certain amount of oxygen was
added and the reaction mixture was allowed to stir at
room temperature. When the reaction was completed,
Ar was bubbled to degass excess NO and O2, and the
solvent was evaporated o€ to leave the residue, whose
analysis was performed by 1H NMR using mesitylene or
1,4-dioxane as an internal standard. In the cases that the
products are volatile, the reaction was carried out in
deuterated solvents, and the mixture was analyzed
directly by NMR.
The analysis of HNO2 and HNO3. The amount of
nitrite ion was determined by Saltzman reagent
method.19 HNO3 was determined using the reported
method which included brucine as a reagent.20
Reaction of N-nitrosocarbazole. N-Nitrosocarbazole was
synthesized according to the reported method.24 It
(0.05 mmol) was dissolved in CH3CN (1 mL), and the
solution was bubbled with dry Ar gas for 15 min, then
the ¯ask was sealed. 30% H2O2 (0.5 mmol) was added to
the solution, and the mixture was allowed to react for 2 h
to give a quantitative yield of carbazole. The same result
was obtained when N-nitrosocarbazole (0.1 mmol) was
subjected to the reaction with KOH powder (2 mmol),
18-Crown-6 (2 mmol), and H2O (2 mmol) in CH3CN
(2 mL) for 2 h.
The experiment for excluding NO2 as a donor of nitrite
and nitrate ion. KOH (1.2 mmol) was suspended in
CH3CN, and the solution was bubbled with dry Ar gas
for 15 min. Then the ¯ask was sealed, and 1-amino-5-
methyl-3-phenylpyrazole (2) (0.6 mmol) in CH3CN
(1 mL) was introduced to the reaction mixture using a
syringe. Then NO2 (0.6 mmol) was added with a gas-tight
syringe, and the mixture was allowed to stir at room
temperature for 2 h. Thereafter, Ar was bubbled to
degass excess NO2, and the solvent was separated to two
parts, and one was evaporated o€ to leave the residue,
The analysis of gaseous products. The gaseous products
were analyzed by Shimadzu GC-14BPTF gas chroma-
tography with TCD detector using SHINCARBON T
(N2O) and Porapak Q (N2 and O2) columns. Since N2O
was found to partially dissolve in CH3CN (ca. 25%) or
H2O (ca. 10%) under the reaction conditions, the analyzed
data were corrected based on these results. When the
compound 2 (0.2 mmol) reacted with NO (0.4 mmol)
and O2 (0.1 mmol), the analysis of the gaseous part
showed that N2O was obtained in 70% (in CH3CN) or
85% (in H2O). Thus, it was concluded that N2O was
formed stoichiometrically. In the case of the compound
1
whose analysis was performed by H NMR. The result
showed that the deaminated product was obtained in
40% yield accompanied by the recovery of 60% of 2.
The other part was subjected to the analysis of nitrite
and nitrate ion, and it was shown that 0.3 mmol of
nitrate ion was formed accompanied by no nitrite ion.

T. Itoh et al. / Bioorg. Med. Chem. 8 (2000) 1983±1989
1989
These data indicated that NO2 reacted with 2 much
faster than with H2O, thus the formation of 1:1 of
nitrite and nitrate in the reaction of N-aminoazoles and
superoxide was not derived from hydrolysis of NO2.
14. Palmer, R. M. J.; Ashton, D. S.; Moncada, S. Nature
1998, 333, 664.
15. Moro, M. A.; Darley-Usmar, V. M.; Goodwin, D. A.;
Read, N. G.; Zamora-Pino, R.; Feelisch, M.; Radomski, M.
W.; Moncada, S. Proc. Natl. Acad. Sci. USA 1994, 91, 6702.
16. Afanas'ev, I. B. Superoxide Ion: Chemistry and Biological
Implication. CRC: Boca Raton, 1991. Vol. 2.
17. Ohsawa, A.; Arai, H.; Ohnishi, H.; Itoh, T.; Kaihoh, T.;
Okada, M.; Igeta, H. J. Org. Chem. 1985, 50, 5520.
18. Beckman, J. S. In Nitric Oxide. Principles and Actions;
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Acknowledgements
The authors are grateful to Professor Masaaki Hirobe
(the President of University of Shizuoka) for helpful
discussions and many insightful comments. This work
was supported in part by a Grant-in-Aid for Scienti®c
Research from the Ministry of Education, Science,
Sports and Culture of Japan.
20. Saito, G.; Sugimoto, K.; Hagino, K. Bunseki Kagaku
1971, 20, 542.
21. There are some papers which claim the reactions with
superoxide were initialized by deprotonation from N±H
bonds; see, (a) Crank, G.; Makin, M. I. H. Tetrahedron Lett.
1979, 20, 2169. (b) Stuehr, D. J.; Marletta, M. A. J. Org.
Chem. 1985, 50, 694.
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