Synthesis of Sydnonimine Derivatives as Potential Trypanocidal Agents

Article abstract of DOI:10.1002/jhet.5570400533

N-(p-Nitrophenoxy)carbonyl-3-morpholino-sydnonimine (NCMS) has been prepared from 3-morpholinosyd-nonimine hydrochloride. Using the Griess assay and the superoxide-mediated reduction of ferricytochrome c, the nitric oxide (NO.) and superoxide anion (O

Full text of DOI:10.1002/jhet.5570400533

943
Synthesis of Sydnonimine Derivatives as
Potential Trypanocidal Agents
Laurent Soulère, Frédéric Bringaud, and Pascal Hoffmann
Sep-Oct 2003
*
a,
a,†
b
a
Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique UMR CNRS 5068 – Université
Paul Sabatier, 118 route de Narbonne – 31062 Toulouse cedex 4, France
b
Laboratoire de Parasitologie Moléculaire – ESA CNRS 5016 – Université Victor Segalen, 146 rue Léo
Saignat - 33076 Bordeaux cedex, France
Received March 18, 2003
N-(p-Nitrophenoxy)carbonyl-3-morpholino-sydnonimine (NCMS) has been prepared from 3-morpholinosyd-
nonimine hydrochloride. Using the Griess assay and the superoxide-mediated reduction of ferricytochrome c,
-
the nitric oxide (NO•) and superoxide anion (O • ) - releasing properties in phosphate buffer pH 7.4 of this novel
2
peroxynitrite donor was studied and compared with the known 3-morpholino-sydnonimine (SIN-1). From
compound NCMS, a series of N-substituted sydnonimine derivatives were easily prepared that contain purine or
melaminophenyl groups which specify a recognition by a trypanosomal purine transporter. The ability of these
3
new sydnonimines to inhibit the uptake of [2 H]adenosine on Trypanosoma equiperdum was studied.
J. Heterocyclic Chem., 40, 943 (2003).
Introduction.
3-morpholino-sydnonimine 1 (NCMS), a new sydnon-
imine that spontaneously decomposes in aqueous solution
to yield both nitric oxide and superoxide anion radicals.
The nitric oxide and superoxide releasing properties of this
intermediate were investigated and compared with the
known SIN-1.
Several types of cells, such as endothelial neutrophils or
macrophages, produce significant amounts of both nitric
oxide radical (NO•) and superoxide radical (O • ) which
can recombine in a near diffusion-controlled reaction to
-
2
-
give peroxynitrite (ONOO ) [1], a potent oxidant which
decompose to generate highly reactive species capable of
nitrating tyrosine residues in proteins and oxidizing DNA
or lipids [2]. Decomposition of ONOO generates radicals
Results.
Synthesis of Sydnonimines.
-
SIN-1 was synthesized as previously described in a one-
pot procedure from N-aminomorpholine and sodium
formaldehyde bisulfite as starting material [12]. The imine
intermediate was treated with potassium cyanide to yield a
nitrile, which was then nitrosated to give the correspond-
ing nitrosohydrazine. Cyclisation of this intermediate
under acidic conditions gave SIN-1 with an overall yield
of more than 50 %. The p-nitrophenyl sydnonimine com-
pound, NCMS, was obtained by treatment of SIN-1 with
4-nitrophenyl chloroformate in pyridine. Reaction of
NCMS in refluxing acetonitrile with various alcohols gave
sydnonimines 4-6 (Scheme 1). For the synthesis of com-
pound 4, the alcohol precursor was obtained from 2-
chloro-4,6-diamino-1,3,5-triazine and 4-aminophenethyl
alcohol in refluxing aqueous sodium hydroxide. The alco-
hol used to obtain the adenine sydnonimine derivative 6
was synthesized in two steps from adenine, which was first
alkylated with 1,2-dibromoethane, and the resulting inter-
mediate was allowed to react with 2-(4-aminophenyl)-
ethanol. The ethyl- (molsidomine, 2) and tert-butyl- (3)
derivatives of SIN-1, which lack a P2-recognition moiety
were synthesized as reference compounds from NCMS in
refluxing ethanol and tert-butanol, respectively.
such as carbonate, nitrogen dioxide or hydroxyl radicals
that could account for its toxic properties [3], but the
mechanism by which it decomposes has been a subject of
controversy [4-6]. Sydnonimines are a class of compounds
that generate simultaneously both nitric oxide radical and
superoxide radical in neutral oxygenated aqueous media
[7-8]. As a consequence, these compounds can be used as
possible peroxynitrite donor and constitute an important
class of compounds for understanding the contribution and
the relevance of peroxynitrite in NO•-related biological
processes. Sydnonimines exert different pharmacological
actions [9], and also constitute an alternative to the organic
nitrates in the treatment of cardiovascular diseases whose
use is often limited by the development of nitrate tolerance
[10]. They release nitric oxide either spontaneously or
after enzymatic conversion and subsequent decarboxyla-
tion to SIN-1 (like molsidomine). In a preceding study
[11], we reported the synthesis and the biological evalua-
tion of a series of nitric oxide-releasing compounds (S-
nitrosothiol and organic nitrate compounds) that contained
melaminyl, adenine and adenosine moieties and exhibited
a high affinity for a trypanosomal purine transporter (P2
transporter). We now extent this study to the preparation
and the biological evaluation of a series of 3-morpholino-
sydnonimines designed to deliver specifically peroxyni-
trite into the parasite via this transporter so that it accumu-
lates and displays its cytotoxic effect. All these compounds
were prepared starting from N-(p-nitrophenoxy)carbonyl-
Formation of Nitric Oxide and Superoxide.
The rate of formation of superoxide was measured with
the commonly used spectrophotometric assay based on
the superoxide-mediated reduction of ferricytochrome c

944
Communication to the Editor
Vol. 40
Scheme 1. Synthesis of Sydnonimine derivatives. (a) 4-Nitrophenyl chloroformate, pyridine 25 °C, 16 h (70 %). (b) 2: Refluxing ethanol, 3.5 h (83 %); 3:
Refluxing tert-butanol, 18 h (70 %); 4: (2-4-[(4,6-Diamino-1,3,5-triazin-2-yl)amino]phenyl-1-ethanol), acetonitrile 82 °C, 4 h (26 %); 5: 2’,3’-O-
Isopropylidene-adenosine, acetonitrile 82 °C, 4 h (50 %); 6: 2-(4-[2-(6-Amino-9H-9-purinyl)ethyl]aminophenyl)-1-ethanol, acetonitrile 82 °C, 3 h (46 %).
[13]. Figure 1A depicts the difference spectral scans of
ferricytochrome c (5 µM) to ferrocytochrome c during
the decomposition of a solution of 3-morpholinosydnon-
imine (SIN-1, 500 µM) in non-deoxygenated phosphate
buffer (pH 7.4) at 25 °C, with a characteristic increase of
the absorbance at 550 nm. The reaction of superoxide
release followed a pseudo-first order kinetic with a half-
life time value of around 30 minutes. The spontaneous
hydrolysis of NCMS (50 µM) was studied spectrophoto-
metrically by monitoring the formation of the p-nitrophe-
noxide group at 420 nm. This reaction followed a typical
first order kinetic with a half-life time value of around 40
minutes (not shown). As for SIN-1, the generation of
-
O • from NCMS (500 µM) was measured by the reduc-
2
tion of cytochrome c. The same characteristic increase in
absorbance at 550 nm with a t value of 55 minutes was
1/2
observed, in addition of that at 420 nm due to the nitro-
phenoxide release (Figure 1B). The yields of nitric oxide
from SIN-1 and NCMS were indirectly quantified using
the Griess method [14] based on measurement of nitrite
ions, products of the oxidative metabolism of NO•. The
kinetics of NO• release in a mixture of DMSO and phos-
phate buffer pH 7.4 followed a first order for the two
compounds (17 mM) with t values of about 6 h for
1/2
SIN-1 (data not shown) and 14 h for NCMS (Figure 2).
The difference of NO•- liberation between the two com-
pounds can be explained by the further hydrolytic step
that is required for the formation of the NO•- generating
form NCMS.
Figure 1. Difference spectral scans of reduction of ferricytochrome c (50
-
µM) to ferrocytochrome c by O • generated from 500 µM SIN-1 (A) and
2
from 500 µM NCMS (B). Repeated scans were performed at 2-min (A) or
10-min (B) intervals in 50 mM phosphate buffer (pH 7.4) containing 20 %
DMSO and 1 mM EDTA

Sep-Oct 2003
Communication to the Editor
Discussion.
945
Early experiments have evidenced the mechanism of
NO•- release from sydnonimines and the role of oxygen in
initiating this reaction [7] (Figure 3): in a first step, SIN-1
decomposes spontaneously through ring opening under
neutral conditions to give SIN-1A. Then, oxygen present
in solution oxidises SIN-1A to give superoxide plus the
corresponding cation radical (SIN-1B), which then sponta-
+
neously releases nitric oxide, H and SIN-1C. Therefore,
sydnonimine derivatives may be considered as peroxyni-
trite donors as they release simultaneously stoichiometric
-
amounts of NO• and O • [18], thus mimicking the release
2
of these radicals by macrophages, neutrophils, or endothe-
lial cells, and the activity of iNOS [19]. For a therapeutic
purpose, a strategy based on peroxynitrite releasing mole-
cules could be exploited for the development of drugs with
trypanocidal activity. Because of their general mode of
Figure 2. Nitrite ions formation from NCMS (17 mM) in 50 mM phos-
phate buffer (pH 7.4) containing 1 mM EDTA and 90 % DMSO (25 °C).
Concentrations of nitrite ions were determined using the assay based on
Griess reaction [14].
Affinity for the P2 Purine Transporter.
Compounds 1-6 were tested on Trypanosoma equiper-
3
dum for their ability to inhibit the uptake of [2- H]adeno-
sine via the purine transporter P2 [15-16] (Table I).
Because the natural substrate adenosine enters through
both trypanosomal adenosine transporters P1 (K = 0.6
m
µM) and P2 (K = 0.7 µM), all experiments were carried
m
out in the presence of a saturating concentration of inosine
that is known to block transporter P1 [17]. Compared to
the K value of the natural substrate adenosine, the values
m
of inhibitory constants show that sydnonimines 4, 5 and 6,
structurally related to melaminyl, adenine and adenosine,
respectively, efficiently inhibit adenosine transport with K
i
ranging from 0.22 to 2.90 µM. In the same experimental
conditions, the uptake of cymelarsan, an arsenical drug
currently used in African trypanosomiasis treatment and
known to enter through the P2-transporter [15], is less effi-
cient. Compounds that lacked a P2 recognition group
including SIN-1 and NCMS did not inhibit the adenosine
transport with K values higher than 300 µM.
i
Table 1
Figure 3. Mechanism of nitric oxide radical and superoxide anion radical
release from sydnonimine SIN-1.
Inhibition of P2 adenosine transporter in Trypanosoma equiperdum by
Cymelarsan and Sydonimine Compounds
Compounds
K (µM)
i
action, these compounds may overcome the drawbacks of
current drugs such as resistance development. In the pre-
sent work, three compounds (4-6) related to 3-morpholino-
sydnonimine were designed in order to specifically target
parasites that possess a P2-nucloside transporter, so that
they generate transient and high NO• and superoxide lev-
els that might be responsible for the destruction of invad-
ing pathogens. These compounds that bear a P2-trans-
porter recognition motif such as a melaminyl, adenine or
adenosine group, were found to efficiently inhibit the
Adenosine
Cymelarsan
SIN-1
NCMS (1)
2
3
4
5
6
0.70 [a] ± 0.01
22.9 ± 0.2
> 300
> 300
> 300
> 300
0.22 ± 0.03
0.85 ± 0.10
2.90 ± 0.18
[ a] K value of adenosine for transporter P2.
m

946
Communication to the Editor
Vol. 40
c (50 µM), superoxide-generating compound (SIN-1 or NCMS,
500 µM), EDTA (1 mM) and DMSO (20 %). Spectra were
recorded between 400 and 600 nm at 2- or 10-minutes intervals.
uptake of adenosine with K values of the same order of
i
magnitude of the K value of the natural substrate adeno-
m
sine. The lethal dose (LD ) of compounds 3 (tert-butyl
100
Griess Assay.
ester) and 5 (adenosine derivative) in Trypanosoma
equiperdum were determined to be 25 and 50 µM within
48 h, respectively. In spite of a poor affinity for the trans-
Nitrite ions were quantified using the Griess method: 10 µL of
a solution of SIN-1 or NCMS (16.7 mM) in a mixture of DMSO
(90 %) and 50 mM phosphate buffer pH 7.4 (10 %) containing 1
mM EDTA were periodically taken and added to 890 µL of
water. 50 µL of solution A and 50 µL of solution B were then
successively added, and the absorbance at 540 nm was recorded
after 10 minutes (solution A: 1 g of sulfanilamide and 5 g of
phosphoric acid in 100 ml water; solution B: 0.1 g N-(1-naph-
thyl)-ethylenediamine dihydrochloride in 100 ml water). A
sodium nitrite solution (1 mM) in water was used as reference.
porter (K > 300 µM), LD
of NCMS was found to be
i
100
200 µM. The other compounds were less toxic for the par-
asites with LD values superior to 200 µM. In accor-
100
dance with previous works, which showed that nitric oxide
from NO•-donors inhibit the growth of several parasites
including Trypanosoma cruzi or Leishmania major [20],
we can hypothesized that the trypanocidal activity of the
active compounds is due to the production of nitric oxide
and superoxide with releasing rates compatible with the
parasitic cellular replication cycle. Compounds that bear a
P2-transporter recognition motif would probably require
enzymatic activation, like molsidomine (2) that is known
to be enzymatically converted in the liver to SIN-1 [21].
The new sydnonimine NCMS constitutes a key intermedi-
ate for the synthesis of these N-substituted compounds.
In contrast to most NO•-donor compounds, which require
either enzymatic bioactivation or the presence of cofactors
such as thiols (i.e. furoxans, organic nitrite and nitrate) or
metal ions (i.e. S-nitrosothiols), NCMS decomposes sponta-
neously to yield the active NO•-releasing agent 3-morpholi-
nosydnonimine, which then gives concomitantly nitric
oxide and superoxide radicals. Despite the increasing dis-
coveries relative to the biological role of peroxynitrite, com-
pounds able to spontaneously release this potent oxidant,
such as NCMS, are relatively limited. Moreover, this new
sydnonimine provides a starting point for the synthesis of
compounds with antimicrobial or other biological activities
that could specifically deliver nitric oxide and/or peroxyni-
trite with controlled release rates, for example compatible
with the rapid proliferation of trypanosomes.
N-[(4-Nitrophenoxy)carbonyl]-3-morpholinosydnon-imine (1,
NCMS).
To a solution of 3-morpholinosydnonimine hydrochloride (2 g,
9.7 mmole) in dry pyridine (60 ml) was added 4-nitrophenyl chlo-
roformate (2.9 g, 14.5 mmole). The reaction mixture was stirred at
room temperature for 16 h. Pyridine was then removed under
reduced pressure. The residue was triturated with hot
dichloromethane and filtered to give 1 (2.26 g, 70 %) as a white
-1 1
powder. ir (KBr): 1691, 1614, 1510, 1261 cm ; H nmr (dimethyl
sulfoxide-d ): δ 3.62 (t, J = 4.6 Hz, 4H), 3.84 (t, J = 4.6 Hz, 4H),
6
13
7.41 (d, J = 7 Hz, 2H), 8.27 (d, J = 7 Hz, 2H), 8.42 (s, 1H);
C
nmr (dimethyl sulfoxide-d ): δ 53.3, 64.7, 100.2, 122.5, 124.9,
6
+
143.8, 157.3, 168.4, 173.7; ms (DCI, NH ): m/z 336 [M+H] .
3
Anal. Calcd. for C H N O : C, 46.57; H, 3.91; N, 20.89.
13 13
5 6
Found: C, 46.60; H, 3.91; N, 20.67.
N-(Ethoxycarbonyl)-3-morpholinosydnonimine (2).
A solution of 1 (0.3 g, 0.89 mmole) in ethanol (15 ml) was
refluxed for 3.5 h. The solvent was evaporated and the residue
was purified by column chromatography using ethyl acetate as
eluent to yield 2 (0.18 g, 83 %) as a white powder; ir (KBr): 1653,
-1
1
1562, 1266 cm ; H nmr (CD OD): δ 1.27 (t, J = 7 Hz, 3H),
3
3.60 (t, J = 4.75 Hz, 4H), 3.94 (t, J = 4.75 Hz, 4H), 4.12 (q, J = 7
13
Hz, 2H), 8.03 (s, 1H); C nmr (CD OD): δ 14.9, 55.6, 62.3,
3
66.7, 100.8, 162.3, 175.4.
N-(tert-Butoxycarbonyl)-3-morpholinosydnonimine (3).
A solution of 1 (0.3 g, 0.89 mmole) in tert-butyl alcohol (15
ml) was refluxed for 18 h. The solvant was evaporated and the
residue was purified by chromatography using a mixture of
dichloromethane and ethyl acetate (8:2) as eluent to give 3 (0.17
EXPERIMENTAL
General.
-1 1
Nuclear magnetic resonance spectra were recorded on a
g, 70 %) as a white powder; ir (KBr): 1662, 1614, 1295 cm ; H
1
13
Brucker AC 250 at 250 MHz ( H) or 60 MHz ( C). Ultraviolet
spectra were recorded with a CARY 1E spectrophotometer
(VARIAN), and IR spectra on a Perkin-Elmer 1600 FTIR spec-
trophotometer. Silica gel 60 (70 – 230 mesh, Merck) was used for
column chromatography and silica plates (60F254, Merck) were
used for thin layer chromatography. SIN-1 hydrochloride was
synthesized from N-aminomorpholine as previously described
[12]. All chemicals were obtained from Aldrich, and used with-
out further purification. Elemental analyses were performed by
the Ecole Nationale Supérieure de Chimie de Toulouse, France.
nmr (deuteriochloroform): δ 1.47 (s, 9H), 3.48 (t, J = 4.7 Hz, 4H),
13
3.93 (t, J = 4.7 Hz, 4H), 7.66 (s, 1H); C nmr (deuteriochloro-
form): δ 28.2, 54.8, 65.5, 78.9, 96.7, 160.6, 173.7.
Anal. Calcd. for C H N O : C, 48.88; H, 6.71; N, 20.73.
11 18
4 4
Found: C, 49.24; H, 6.92; N, 19.89.
N-[(4-[(4,6-Diamino-1,3,5-triazin-2-yl)amino]phenethyl-
oxy)carbonyl]-3-morpholino-sydonimine (4).
Alcohol precursor of 4 (2-4-[(4,6-diamino-1,3,5-triazin-2-
yl)amino]phenyl-1-ethanol) was synthesized as follows: an aque-
ous 1 M sodium hydroxide solution (2 x 6.87 ml, 13.74 mmole)
was slowly added during a period of 3.5 h to a refluxing solution
of 2-chloro-4,6-diamino-1,3,5-triazin (2 g, 13.74 mmole) and
4-aminophenethyl alcohol (1.9 g, 13.7 mmole) in water (50 ml).
Cytochrome c Assay.
Measurements of superoxide formation was carried out in
50 mM phosphate buffer, pH 7.4 (20 °C) containing cytochrome

Sep-Oct 2003
Communication to the Editor
947
The mixture was then cooled (ice-bath), and the precipitate was
collected by filtration and dried to yield the alcohol; H nmr
the residue was purified by chromatography using a mixture of
ethyl acetate and methanol (7:3) as eluent to yield 6 (0.23 g, 46
1
-1 1
(dimethyl sulfoxide-d ): δ 2.65 (t, J = 7 Hz, 2H), 3.56 (m, 2H),
%) as a white powder; ir (KBr): 1633, 1512, 1263 cm
H nmr
6
;
6
4.60 (m, 1H), 6.29 (s, 2H), 7.05 (d, J = 8 Hz, 2H), 7.63 (d, J = 8
(dimethyl sulfoxide-d ): δ 2.68 (t, J = 7 Hz, 2H), 3.53 (t, J = 6 Hz,
4H), 3.60 (t, J = 6 Hz, 2H), 3.80 (t, J = 4.7 Hz, 4H), 4.17 (m, 2H),
4.30 (t, J = 5 Hz, 1H), 4.65 (t, J= 4.7 Hz, 1H), 6.98 (d, J = 8 Hz,
2H), 7.08 (d, J = 8 Hz, 2H), 7.11 (s, 2H), 8.02 (s, 1H), 8.00 (s,
13
Hz, 2H), 8.73 (s, 1H); C nmr (dimethyl sulfoxide-d ): δ 38.36,
6
62.39, 119.63, 128.50, 132.09, 138.39, 164.72, 166.00. A solu-
tion of this alcohol (0.125 g, 0.5 mmole) and 1 (0.185, 0.5
mmole) in acetonitrile (10 ml) was refluxed for 4 h. The reaction
mixture was filtered and the filtrate evaporated to dryness. The
residue was purified by column chromatography using a mixture
13
1H), 8.08 (s, 1H); C nmr (deuteriochloroform): δ 42.6, 48.0,
54.8, 53.5, 62.0, 64.9, 97.9, 118.6, 126.9, 128.6, 136.4, 140.7,
141.3, 149.6, 152.1, 155.7, 159.8, 171.6; ms (DCI, NH ): m/z
3
+
of ethyl acetate and methanol (9:1) as eluent to yield 5 (0.056 g,
495 [M+H] .
-1 1
26 %) as a white powder; ir (KBr): 1664, 1597, 1264 cm
H
Anal. Calcd. for C H N O •1.5H CO : C, 49.64; H, 5.07;
;
22 26 10
4
2
3
nmr (dimethyl sulfoxide-d ): δ 2.84 (t, J = 6.5 Hz, 2H), 3.56 (t, J
N, 25.17. Found: C, 49.51; H, 5.37; N, 25.22.
6
= 4.6 Hz, 4H), 3.84 (t, J = 4.6 Hz, 4H), 4.15 (t, J = 6.5 Hz, 2H),
6.25 (s, 4H), 7.09 (d, J = 8Hz, 2H), 7.66 (d, J = 8 Hz, 2H), 8.15 (s,
REFERENCES AND NOTES
13
1H), (s, 1H); C nmr (dimethyl sulfoxide-d ): δ 34.1, 53.4,
6
64.8, 65.2, 98.9, 119.6, 128.5, 130.8, 138.8, 160.1, 164.7, 167.0,
[*] Correspondence to: Dr. Pascal Hoffmann – Université Paul
Sabatier – LSPCMIB, Bat. 2R1 - 118 Route de Narbonne – 31062
Toulouse cedex 4, France; Email: hoffmann@cict.fr; Fax: (33) 5 61
55 60 11.
[†] Present address: Department of Chemical Biology, Max-
Planck Institut für Molekulare Physiologie, Dortmund, Germany.
[1] R. E. Huie and S. Padmaja, Free Radic. Res. Commun., 18,
195 (1993).
[2] J. T. Groves, Curr Opin. Chem. Biol., 3, 226 (1999).
[3] R. Radi, G. Peluffo, M. N. Alvarez, M. Naviliat and A.
Cayota, Free Radic. Biol. Med., 30, 463 (2001).
[4] A. U. Khan, D. Kovacic, A. Kolbanovskiy, M. Desai, K.
Frenkel and N. E. Geacintov, Proc. Natl. Acad. Sci. USA, 97, 2984
(2000).
+
173.5; ms (DCI, NH ): m/z 443 [M+H] .
3
Anal. Calcd. for C H N O .H CO : C, 45.24; H, 4.80; N,
18 22 10
4
2
3
27.77. Found: C, 45.34; H, 4.85; N, 27.95.
N-(2',3'-Isopropylidene-5’-carbonyl-adenosine)-3-morpholi-
nosydnonimine (5).
A solution of 2',3'-O-isopropylidene-adenosine (0.155 g, 0.5
mmole) and 1 (0.185, 0.5 mmole) in acetonitrile (10 ml) was
heated to reflux for 4 h. The solvent was evaporated to dryness
and the residue was purified by chromatography using a mixture
of dichloromethane and methanol (95:5) as eluent to give 6
(0.125 g, 50 %) as a white powder; ir (KBr): 1652, 1594, 1263
-1 1
[5] G. Merenyi, J. Lind, G. Czapski and S. Goldstein, Proc. Natl.
Acad. Sci. USA, 97, 8216 (2000).
cm
H nmr (deuteriochloroform): δ 1.37 (s, 3H), 1.62 (s, 3H),
;
3.49 (t, J = 4.7 Hz, 4H), 3.92 (t, J = 4.7 Hz, 4H), 421 (dd, J = 12
and 4 Hz, 1H), 4.40 (dd, J = 12 and 4 Hz, 1H), 4.59 (q, J = 4 Hz,
1H), 5.06 (dd, J = 6 and 2 Hz, 1H), 5.40 (dd, J= 6 and 2 Hz, 1H),
6.19 (s, 2H), 6.22 (d, J = 2 Hz, 1H), 7.72 (s, 1H), 8.23 (s, 1H),
[6] G. R. Martinez, P. Di Mascio, M. G. Bonini, O. Augusto, K.
Briviba, H. Sies, P. Maurer, U. Rothlisberger, S. Herold and W. H.
Koppenol, Proc. Natl. Acad. Sci. USA, 97, 10307 (2000).
[7] M. Feelisch, J. Ostrowski and E. Noack, J. Cardiovasc.
Pharmacol., 14, S13 (1989).
[8] H. Bohn and K. Schonafinger, J. Cardiovasc. Pharmacol., 14,
S6 (1989).
[9] P. G. Wang, M. Xian, X. Tang, X. Wu, Z. Wen, T. Cai and A.
J. Janczuk, Chem. Rev., 102, 1091 (2002).
13
8.33 (s, 1H); C nmr (deuteriochloroform): δ 25.3, 27.2, 54.6,
65.2, 65.4, 81.9, 84.5, 84.6, 90.9, 99.4, 114.3, 119.7, 139.5,
149.7, 153.2, 155.6, 160.8, 174.4; ms (DCI, NH ): m/z 504
3
+
[M+H] .
Anal. Calcd. for C H N O •1.5H CO : C, 43.29; H, 4.73;
20 25
9
7
2
3
[10] B. Hinz and H. Schroder, Pharm. Res., 16, 633 (1999).
[11] L. Soulere, P. Hoffmann, F. Bringaud and J. Perie, J. Bioorg.
Med. Chem. Lett., 10, 1347 (2000).
N, 21.13. Found: C, 43.84; H, 4.60; N, 20.88.
N-([(4-[2-(6-Amino-9H-9-purinyl)ethyl]aminophenethyl)oxy]-
carbonyl)-3-morpholino sydnonimine (6).
[12] K. Masuda, Y. Imashiro and T. Kaneko, Chem. Pharm. Bull.,
19, 72 (1970).
Alcohol precursor of 6 (2-(4-[2-(6-amino-9H-9-purinyl)ethyl]-
aminophenyl)-1-ethanol) was synthesized as follows: a solution
of 9-(2-bromoethyl)-9H-6-purinamine (2 g, 8.26 mmole), 4-
aminophenethyl alcohol (1.135 g, 8.26 mmole) and triethylamine
(0.835 g, 8.26 mmole) in methanol (45 ml) was refluxed for 24 h.
The solvent was evaporated to dryness and the residue was puri-
fied by chromatography using a mixture of ethyl acetate and
methanol (9:1) as eluent to yield the alcohol (0.79 g, 32 %) as a
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1
6
yellow powder; H nmr (dimethyl sulfoxide-d ): δ 2.56 (t, J = 7
Hz), 3.42-3.53 (m, 4H), 4.29 (t, J = 6 Hz), 4.54 (s, 1H), 5.74 (s,
1H), 6.53 (d, J = 8 Hz, 2H), 6.92 (d, J = 8 Hz, 2H), 7.24 (s, 2H),
13
6
8.08 (s, 1H), 8.18 (s, 1H); C nmr (dimethyl sulfoxide-d ): δ
38.2, 42.4, 42.5, 62.7, 112.0, 118.7, 126.6, 129.3, 141.0, 146.3,
149.5, 152.3, 155.8. A solution of this alcohol (0.3 g, 1 mmole)
and 1 (0.335 g, 1 mmole) in acetonitrile (15 ml) was refluxed for
3 h. The solvent was then removed under reduced pressure and
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