Nitroanilines are the reduction products of benzofuroxan system by oxyhemoglobin (HbO22+)

Article abstract of DOI:10.1016/S0014-827X(01)01139-9

Benzofuroxans are interesting compounds which display several biochemical and pharmacological properties. Recent studies from our laboratory demonstrate that they are reduced by ferrous salts at room temperature and that the principal reaction products are o-nitroanilines. This paper shows that simple benzofuroxan derivatives are also able to oxidise HbO₂²⁺ to methemoglobin (MetHb³⁺) (UV detection) and to form o-nitroanilines (HPLC detection). From a toxicological point of view this reaction is interesting, since it indicates that the blood is a site for metabolism of these compounds with consequent methemoglobinemia and formation of toxic compounds.

Full text of DOI:10.1016/S0014-827X(01)01139-9

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Il Farmaco 56 (2001) 799–802
Short Communication
Nitroanilines are the reduction products of benzofuroxan system
by oxyhemoglobin (HbO22+)
Claudio Medana, Sonja Visentin, Giorgio Grosa, Roberta Fruttero, Alberto Gasco *
Dipartimento di Scienza e Tecnologia del Farmaco, 6ia P. Giuria 9, 10125 Turin, Italy
Received 18 April 2001; accepted 23 May 2001
Abstract
Benzofuroxans are interesting compounds which display several biochemical and pharmacological properties. Recent studies
from our laboratory demonstrate that they are reduced by ferrous salts at room temperature and that the principal reaction
products are o-nitroanilines. This paper shows that simple benzofuroxan derivatives are also able to oxidise HbO22+ to
methemoglobin (MetHb3+) (UV detection) and to form o-nitroanilines (HPLC detection). From a toxicological point of view this
reaction is interesting, since it indicates that the blood is a site for metabolism of these compounds with consequent
methemoglobinemia and formation of toxic compounds. © 2001 Elsevier Science S.A. All rights reserved.
Keywords: Benzofuroxans; o-Nitroanilines; Hemoglobin
this heterocycle system as a potential NO donor [5] and
for designing new 1,4-dihydropyridines, able either to
1. Introduction
block or to activate voltage-dependent L-type calcium
Benzofuroxan (benzofurazan oxide) is an interesting
channels [6,7]. We also studied the reduction of com-
ring system whose chemistry has been recently reviewed
pounds 1–3 by ferrous salts at room temperature and
on various occasions [1–3]. NMR evidence shows that,
we found that the principal reaction products were
at room temperature, benzofuroxan derivatives in solu-
substituted o-nitroanilines [8]. In this paper, we address
tion exhibit rapid tautomeric equilibrium a = b (Fig.
our study to the interaction of benzofuroxans 1–5 with
1).
oxyhemoglobin and we show that the reaction is largely
Thermodynamic equilibrium concentrations are de-
comparable with the one with ferrous salts.
pendent on a number of factors: solvent, temperature,
the nature and position of the substituents at the ring.
Benzofuroxans display several biochemical and phar-
macological properties and a specific review was
devoted to this aspect [4]. There are many other possi-
ble applications of these compounds [1–3] and worthy
of note among them is the use of ‘potassium dini-
trobenzofuroxan’ and of the corresponding barium salt
as detonating agents. Recently, we were interested in
* Corresponding author.
E-mail address: alberto.gasco@pharm.unito.it (A. Gasco).
Fig. 1. Tautomeric equilibrium and studied compounds.
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01139-9

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C. Medana et al. / Il Farmaco 56 (2001) 799–802
Table 1
Yields % and reaction times with oxyhemoglobin or FeSO4
Compd. Yield (with HbO22+
)
Yield of
Yield (with Fe2+
)
Reaction time (with HbO22+
)
Reaction time (with Fe2+
)
MetHb3+
6
8995
3892
5494
Quantitative
3591
5492
6591
1592
9493
8192
6093
2691
7092
1091
6791
1092
7891
60 min
60 min
60 min
60 min
60 min
60 min
60 min
60 min
10 min
60 min
60 min
24 h
60 min
60 min
24 h
7a
7b
8b
9a
9b
10b
11
9293
9296
8895
9495
24 h
2. Experimental
agreement with literature data. Compound 11 is identi-
cal to the one obtained by the alternative synthesis
described below (Scheme 3).
2.1. General
2.3. 3-Amino-2-nitrobenzonitrile (10b)
Derivatives 1 [9], 2, 3 [10], 4, 5 [5], and 12 [11] were
synthesised according to literature. Melting points were
determined in a Bu¨chi 540 apparatus after introducing
the sample into the bath at a temperature 10 °C lower
than the melting point and are uncorrected. A heating
rate of 1 °C min−1 was used. The compounds were
routinely checked by infrared spectrophotometry (Shi-
madzu FT-IR 8101M), 1H and 13C NMR (Bruker
AC200) and mass spectrometry (Finnigan-Mat TSQ-
700). HPLC analyses were performed with a diode
array UV detector (Shimadzu LC10A). Column chro-
matography was performed on silica gel (Merck
Kieselgel 60, 230–400 mesh ASTM). Thin layer chro-
matography (TLC) was carried out on plates precoated
with Merck silica gel 60 F254, with a layer thickness of
0.25 mm. Petroleum ether 40–60 °C was used. Anhy-
drous MgSO4 was used as drying agent. Solvent re-
moval was achieved under reduced pressure at room
temperature (r.t.). Elemental analyses of the new com-
pounds were performed by REDOX (Cologno M.), and
the results are within 90.4% of the theoretical values.
The figures in brackets are standard errors (9SE).
Caution: Arylamines should be treated as potential
carcinogens and handled accordingly with protective
gloves in a well-ventilated fume hood.
M.p. 198 °C (ethyl acetate–petroleum ether). 1H
NMR (DMSO-d6): l 7.20–7.23 (m, 1H), 7.31–7.35 (m,
1H), 7.46–7.54 (m, 1H), 7.79 (br, 2H). 13C NMR
(DMSO-d6): l 106.55, 117.11 (CN), 124.61, 124.98,
126.47, 134.31, 147.03. EI MS; m/z: M+ 163 (100).
Anal. (C7H5N3O2): C, H, N.
2.4. Synthesis of 4-benzofurazanylcarboxylic acid
methyl ester (13)
A mixture of benzofurazan-4-carboxylic acid (0.6 g,
3.66 mmol), thionyl chloride (5 ml, 69 mmol) and a
catalytic amount of DMF was refluxed for 30 min and
then evaporated in vacuo. Anhydrous methanol (6 ml)
was added and the pure solid separated was collected
by filtration and washed with water. Quantitative yield,
1
m.p. 102 °C (ethanol). H NMR (DMSO-d6): l 4.30 (s,
3H), 7.76–7.80 (m, 1H), 8.29–8.41 (m, 2H). 13C NMR
(DMSO-d6): l 163.24 (COO), 149.61, 146.44, 137.16,
132.07, 121.67, 119.10, 52.98 (CH3). EI MS; m/z: M+
178 (45), [M−31]+ 147 (85). Anal. (C8H6N2O3): C, H,
N.
2.5. Synthesis of 4-benzofurazanylcarboxyamide (11)
2.2. Synthesis of deri6ati6es 7–11
To a stirred ice-water cooled solution of 13 (0.5 g, 2.3
mmol) in methanol (6 ml), an aqueous solution (32%)
of ammonia (9.5 ml) was added. The mixture was kept
under stirring at r.t. overnight. The solid obtained was
filtered off, washed with water and dried to give the
pure title product. Yield 53%, m.p. 184 °C (ethanol).
1H NMR (DMSO-d6): l 7.70–7.77 (m, 1H), 7.91–8.03
(br, 2H, NH2), 8.07–8.10 (m, 1H), 8.23–8.27 (m, 1H).
13C NMR (DMSO-d6): l 163.94 (CONH2), 149.38,
146.74, 133.73, 132.40, 123.32, 119.39. EI MS; m/z: M+
163 (100), [M−16]+ 147 (55). Anal. (C7H5N3O2): C, H,
N.
A previously published method [8] was used, partly
modified as follows: a solution of the appropriate ben-
zofuroxan (1 mmol) in DMSO was added at r.t. to a
stirred solution of FeSO4×7H2O (6 mmol) in water.
The progress of the reaction was followed by TLC, the
reaction mixture was poured into water, extracted with
ethyl acetate, and purified by flash chromatography
(eluent: petroleum ether–ethyl acetate 7:3). Yields and
reaction times are reported in Table 1. Analytical pro-
files of compounds 7a, 7b, 8b [8], 9a, 9b [12] are in

C. Medana et al. / Il Farmaco 56 (2001) 799–802
801
Table 2
Kinetic data
Purospher RP-18 column (250×4 mm; 5 mm particles)
at 40 °C, eluting (1 ml min−1) with 65% aqueous ace-
tonitrile containing 0.1% trifluoroacetic acid. Yields of
the reaction products are reported in Table 1.
Compd.
DA (min−1
)
1
2
3
4
5
0.092090.0039
0.10590.011
0.013790.0014
0.088490.0053
0.017190.0029
MetHb3+ formation in the reaction mixture was
determined spectrophotometrically at u=630 nm
(mMetHb=3630) and 577 nm (mHbO2=15 000) [13].
2.8. Oxyhemoglobin oxidation kinetic study
The rate of oxidation was determined using a spec-
trophotometric technique based on the conversion of
HbO22+ to MetHb3+. The formation of MetHb3+ was
followed by recording the absorbance increase (DA at
u=401 nm) using a Perkin–Elmer Lambda 5 spec-
trophotometer and a thermostated (37 °C) glass cu-
vette. The reaction was started by adding DMSO
solutions of the compound (final concentrations 50 mM)
to a 4 mM HbO22+ solution in 50 mM phosphate buffer
(pH 7.4). The increase of absorbance was recorded over
the first 3 min. The initial oxidation rates expressed as
DA min−1 (Table 2) were calculated from the slope of
the straight line portion of each curve. Rate value is the
average of at least three determinations.
2.6. Preparation of oxyhemoglobin
Pure oxyhemoglobin was prepared by adding an
excess of the reducing agent sodium dithionite
(Na2S2O4) to a solution of commercially available
bovine hemoglobin (H-2500 Sigma Chemical Co.), in
50 mM phosphate buffer (pH 7.4), at 4 °C and pro-
tected from light. After 5 min the resulting solution was
loaded onto a chromatographic column (Sephadex G-
25, Pharmacia, Uppsala, Sweden) and eluted with phos-
phate buffer. The purity of the oxyhemoglobin solution
was determined spectrophotometrically (umax=414 nm;
m=125 000).
Analogously pure methemoglobin and carboxy-
hemoglobin (HbCO) were prepared by treating solu-
tions of commercially available bovine hemoglobin with
excess of K3Fe(CN)6 and CO, and then purified by gel
chromatography.
3. Results and discussion
The reaction of benzofuroxans and HbO22+ was run
by treating in pH 7.4 phosphate buffer, at 37 °C, each
benzofuroxan derivative with purified oxyhemoglobin
in 1:2 molar ratio (benzofuroxan/heme). Analysis of the
reaction products showed that HbO22+ was transformed
into MetHb3+ and the benzofuroxans into the corre-
2.7. Study of the reaction with oxyhemoglobin
A solution of the appropriate benzofuroxan in
methanol was added to 1 ml of 50 mM phosphate
buffer (pH 7.4) containing freshly prepared HbO22+and
2-nitro-4-chloroaniline as internal standard (IS). The
final concentrations were 0.2 mM for benzofuroxans
and IS and 0.4 mM (heme) for HbO22+. After 1 h, the
mixture was diluted 1:1 with acetonitrile, centrifuged
(10 min, 9600g) and analysed by HPLC, using a Merck
sponding o-nitroanilines (Scheme 1). HbO22+
“
MetHb3+ transformation was monitored by UV–Vis
spectroscopy, while the formation of nitroanilines was
detected by HPLC using the appropriate nitroaniline as
standard. After 1 h the reactions were completed by
over 90% (Table 1). This means that the reduction of
Scheme 1.

802
C. Medana et al. / Il Farmaco 56 (2001) 799–802
NO release by NO donors (oxyhemoglobin assay)
[14,15]. In fact NO is able to transform HbO22+ into
MetHb3+ and nitrate according to equation
HbO22+ +NO“MetHb3+ +NO−3
Since benzofuroxans, in these conditions, are unable
to release NO [5], this is a good example as the above
reaction should be used carefully because potential NO
donors can directly promote HbO22+ “MetHb3+
transformation.
In conclusion, this paper suggests that blood is a
possible site for metabolism of benzofuroxan deriva-
tives with the formation of methemoglobin and toxic
o-nitroanilines.
Scheme 2.
Scheme 3.
benzofuroxan system 1 by HbO22+ involves two elec-
trons (Scheme 2) and parallels the reduction by ferrous
salts. The most relevant difference is that the former
process is quite faster and the yields are partly different.
In some cases with Fe2+ ions the reduction was not
completed even after 24 h.
Acknowledgements
The financial support from MURST, Rome, is grate-
fully acknowledged.
Literature data [5] show that both 2 and 3 exist in
tautomeric forms (2a/2b ca. 1:1, 3a/3b ca. 3:1, CHCl3,
−40 °C). Reduction by HbO22+ affords in the case of
2 a mixture of the nitroanilines 7a (38%) and 7b (54%).
After 24 h the reduction with ferrous salts also gives
similar results but the yields in anilines were reversed
(7a, 60%; 7b, 26%). Reduction of 3, both with HbO22+
and ferrous salts, produces only nitroaniline 8b. There-
fore, in both cases the thermodynamically favoured
4-methyl isomer is selectively less reactive than the
7-methyl one. This is probably due to steric hindrance
to electron transfer to N-3 of the benzofuroxan system
[8]. Also, compounds 4 and 5 exist in tautomeric forms
(4a/4b ca. 1:2, acetone −20 °C; 5a/5b ca. 5.7:1, ace-
tone, 0 °C) [5]. Reduction of 4 by HbO22+ parallels the
reduction of the corresponding methyl derivative 2
affording the expected mixture of nitroanilines 9a (35%)
and 9b (54%). Again, different yields are obtained in
the reaction with Fe2+. The reduction of 5 gives only
partly similar results to those obtained in the reduction
of the methyl analogue 3, since besides the sole ni-
troaniline 10b (65%) we isolated in low yield (15%) the
benzofurazan derivative 11, as a result of the combined
CN hydrolysis and N“O deoxygenation processes.
The structure of 11 was confirmed by direct synthesis
(Scheme 3). This same derivative is formed as the
principal product in the reaction by Fe2+ salts. Reduc-
tion of benzofuroxans by oxyhemoglobin does not seem
to involve auto-oxidation of the heme group with pro-
duction of intermediate O−2 , since the same results were
obtained using HbCO2+. Initial rates of these reduc-
tions, using an excess of benzofuroxan derivatives with
respect to HbO22+, were measured following the forma-
tion of MetHb3+ by UV spectroscopy. 5(6)-Substituted
benzofuroxans display initial rates similar to that of 1,
while 4(7)-tautomers were significantly slower. This
technique is frequently used to detect nitric oxide (NO)
under aerobic conditions and to measure initial rates of
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