Water soluble furoxan derivatives as NO prodrugs

Article abstract of DOI:10.1021/jm960379t

The synthesis, characterization, NO donor properties, and in vitro vasodilating activity of a series of water soluble furoxans (5-14a,b) are described. All of the compounds released NO when treated with a large excess of cysteine under physiological condi

Full text of DOI:10.1021/jm960379t

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J . Med. Chem. 1997, 40, 463-469
463
Wa ter Solu ble F u r oxa n Der iva tives a s NO P r od r u gs
Giovanni Sorba, Claudio Medana, Roberta Fruttero, Clara Cena, Antonella Di Stilo, Ubaldina Galli, and
Alberto Gasco*
Dipartimento di Scienza e Tecnologia del Farmaco, Universita` degli Studi di Torino, via P. Giuria 9, 10125 Torino, Italy
Received May 28, 1996X
The synthesis, characterization, NO donor properties, and in vitro vasodilating activity of a
series of water soluble furoxans (5-14a ,b) are described. All of the compounds released NO
when treated with a large excess of cysteine under physiological conditions (pH 7.4; 37 °C).
The amount of NO produced after 1 h of incubation was evaluated by detecting nitrites, via
the Griess reaction. Derivatives 5b, 7b, and 14b were able to release nitric oxide also in the
absence of the thiol cofactor. The initial rates of NO release were determined at different
concentrations, using a spectrophotometric technique based on the NO-induced oxidation of
oxyhemoglobin (HbO2) to methemoglobin (MetHb). The initial rates of NO release were linearly
dependent on the concentrations of the single compounds. The vasodilating potency (EC50) of
all the derivatives was assessed on rat aortic strips precontracted with noradrenaline.
Correlation between potency and initial NO release rate is discussed.
In tr od u ction
Most of the furoxans we have studied so far are poorly
water soluble. This limits the study of their NO donor
capability under physiological conditions. This article
describes the preparation of a series of water soluble
furoxans and a study of their NO donor properties as
well as of their in vitro vasodilating activity. The
correlation between the initial rates of the NO release
and the vasodilating potencies of the compounds is also
analyzed.
The discovery that nitric oxide is an important physi-
ological mediator in cardiovascular, immune, central,
and peripheral nervous systems has given rise to a great
interest in NO prodrugs, those derivatives able to
release this messenger under physiological conditions.1
The classical employment of NO donors is in the
treatment of cardiovascular diseases, but a variety of
new therapeutic possibilities are emerging.2,3
Recently it has been shown that a few derivatives of
1,2,5-oxadiazole 2-oxide (furoxan, furazan oxide) are
able to increase the level of cytosolic cGMP in human
platelets4 and to activate rat liver guanylate cyclase in
the presence of thiols.5 This behavior is explained by
the finding that furoxans can release nitric oxide under
the action of thiol cofactors.5 With furoxancarboxamides
as models, nitroso thiols were isolated in release.5
Generally speaking the overall reaction of NO donation
by furoxans appears to be complex. Some mechanisms
have been hypothesized.5,6 They take into account not
only the intermediacy of nitroso thiols but also direct
release of nitroxyl anion (NO-) and NO•. The involve-
ment of the ring 3-position has been pointed out.6
Anyway the subject still needs a specific study. Re-
cently we synthesized and tested for their antiaggre-
gatory and vasodilating properties many new deriva-
tives of the furoxan system.7 CHF 2206 (1)8 and CHF
2363 (2)9 merited further investigation.
Ch em istr y
A classical strategy to obtain water soluble derivatives
of a ring system is the direct attachment to it of basic
side chains containing amine functions.12 We used this
approach to achieve our aim of preparing water soluble
furoxans. In a recent paper we have shown that
phenylsulfonyl-substituted furoxans are flexible inter-
mediates for the synthesis of new functionalized furoxan
derivatives.13 All of the compounds described in the
present work have been synthesized from such starting
materials, according to the pathways shown in Scheme
1.
Dimethylaminoalkoxy-substituted phenylfuroxans 5a ,
6a , and 5b, 6b were obtained by action of the appropri-
ate dimethylamino alcohol either on 4-(phenylsulfonyl)-
3-phenylfuroxan (4a ) or on its 4-phenyl isomer 4b. The
reaction was run in tetrahydrofuran (THF) in the
presence of sodium hydroxide at room temperature. The
(aminoalkyl)thio compounds 7a , 8a , and 7b, 8b were
prepared by a similar procedure. The [2-(dimethylami-
no)ethyl]sulfonyl-substituted phenylfuroxan isomers 9a,b
were easily synthesized by the action of potassium
permanganate on acetic acid solutions of 8a and 8b,
respectively. We obtained the phenylsulfonyl-substi-
tuted furoxans 11-13 from 10 under conditions similar
to those employed for the synthesis of the phenyl
analogues. With the amino alcohols we isolated only
the 3-phenylsulfonyl isomers (11-13), but with 2-(di-
methylamino)ethanethiol as a reagent we obtained the
two possible isomers 14a and 14b in the ratio ∼1:4. It
is noteworthy that the major product in the reaction
with the thiol is that formed by substitution of the
O
HOCH2
N
NH2
–
SO2
+
CH3CH2O
SO2
+
+
N
N
N
N
N
O
–
–
O
O
O
O
O
3
1
2
Simultaneously, researchers at the Cassella Institute
undertook a wide study on furoxancarboxamides from
which CAS 1609 (3) was selected as a candidate for
further preclinical work.10 The furoxan system is also
a flexible tool in the design of hybrid drugs with mixed
R1-antagonist properties and NO dependent vasodilating
activity.11
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
S0022-2623(96)00379-2 CCC: $14.00 © 1997 American Chemical Society

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464 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Sorba et al.
Sch em e 1
3-sulfonyl group, the one which is retained in the amino
alcohol reaction.
2.2 × 10-2 M (22 °C); the comparable solubility of the
analogue 3-(phenylsulfonyl)-4-ethoxyfuroxan (2) is 5.7
× 10-5 M.
All of the proposed structures were established by 1H
and 13C NMR spectroscopy. The structures of the
isomer pairs 5-9a ,b, in which a phenyl group is directly
linked to the furoxan ring, were confirmed on the basis
of the well-known shielding effect exerted by the N+-
O- moiety both on the C′-1 and, to a lesser extent, on
the C′-4 of the 3-phenyl ring compared with the corre-
sponding carbons of the 4-phenyl isomers.14 This same
effect influences also the chemical shifts of hydrogens
present in side chains.15 Indeed, the protons of the
aminoalkyl group in the 4-phenyl isomers display small
but significant upfield shifts with respect to the corre-
sponding resonances in the 3-phenyl isomers. In a
similar manner the structure was assigned to the isomer
pair 14a and 14b. A more complex situation arises from
the 3-phenylsulfonyl derivatives 11-13, for which only
one isomer is available. In this case we based our
structural assignments on the close resemblance of their
spectra to those of 3-(phenylsulfonyl)-4-ethoxyfuroxan.
In this last compound the connectivity relative to C-4
and C-3 in the furoxan ring was assigned by using 1D
(INEPT) and 2D (HETCOR) polarization transfer tech-
niques.13
Exten ts a n d Ra tes of Nitr ic Oxid e Relea se. In
order to evidence spontaneous release of nitric oxide,
each compound was dissolved in pH 7.4 buffered water
and kept at 37 °C for 1 and 24 h. The amount of NO
released was evaluated by detecting nitrites, which are
the oxidative products of nitric oxide, by the Griess
reaction. The extent of the thiol-induced NO generation
was similarly determined after 1 h of incubation in the
presence of a large excess of cysteine (1:50). The results
-
expressed as percent NO2 (mol/mol) are summarized
in Table 1.
The initial rates of NO release in the presence of
cysteine (5-fold molar excess) and, if necessary, in its
absence were determined at different concentrations in
the range 10-3-10-6 M. In this study a spectrophoto-
metric technique, based on the NO-induced oxidation
of oxyhemoglobin (HbO2) to methemoglobin (MetHb),
was employed.16 With the furoxan derivatives unable
to release spontaneously NO, no oxidation occurred in
the absence of cysteine. The reaction was followed by
detecting the increase of absorbance (∆A) at λ ) 401
nm. The initial rates of nitric oxide donation, expressed
as ∆A min-1, were linearly dependent on the concentra-
tions of the single compounds. Correlation coefficients
(r) of the regression lines ranged from 0.98 to 0.99.
Typical examples are reported in Figure 1. From these
All of the derivatives we synthesized, isolated as
oxalates, are freely water soluble in the concentration
range we used for studying NO release (10-3-10-6 M).
The increase of solubility obtained grafting a basic chain
to the furoxan ring is relevant. For example derivative
12‚H2C2O4 is water soluble at concentration of about
data the concentrations able to give ∆A ) 0.1 min-1
1min
0.1
(C
) were evaluated for each member of the series. A

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Water Soluble Furoxan Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 465
Ta ble 1. Vasodilating Potency and NO Generation Properties of Water Soluble Furoxansd
1min
0.1
compd
EC50 (µM)
EC50 (µM) + HbO2 10 µM
C
(M)
% NO2- (mol/mol)
% NO2- (mol/mol) L-Cys 50X
5a
5b
7.35 ( 2.16
1.23 ( 0.23
68.0 ( 8.2
8.64 × 10-3 a
0
3.9 ( 0.2
2.22 ( 0.64
5.00 × 10-4
6.3 ( 0.2
62.9 ( 3.1
(97 ( 2)c
6a
6b
7a
7b
14.3 ( 3.8
1.03 ( 0.15
15.2 ( 1.4
3.48 ( 0.65
124.4 ( 17.7
3.63 ( 0.42
78.0 ( 9.5
15.0 ( 1.7
-
0
0
0
3.9 ( 0.2
91.1 ( 0.9
2.8 ( 0.1
7.19 × 10-4
-
1.01 × 10-3 a
4.5 ( 0.2
37.9 ( 0.5
(56 ( 1)c
8a
8b
9a
9b
48.1 ( 3.7
1.55 ( 0.02
0.101 ( 0.012
0.0826 ( 0.0075
123.8 ( 12.1
11.5 ( 1.8
0.43 ( 0.02
1.01 ( 0.14
-
0
0
0
0
2.9 ( 0.2
35.0 ( 2.1
8.8 ( 0.3
7.07 × 10-4
1.59 × 10-3 a
1.82 × 10-4
36.3 ( 1.2
(33 ( 1)c
11
12
13
14a
14b
0.013 ( 0.003
0.046 ( 0.009
0.048 ( 0.010
0.13 ( 0.03
0.35 ( 0.05
0.33 ( 0.06
1.64 × 10-5
2.04 × 10-5
1.53 × 10-5
6.30 × 10-5
6.07 × 10-5 b
0
0
0
0
19.7 ( 0.6
58.9 ( 0.4
20.2 ( 0.6
27.9 ( 0.4
58.5 ( 1.5
0.0041 ( 0.0009
0.030 ( 0.0040
0.0520 ( 0.0066
0.0557 ( 0.0066
95.3 ( 2.3
(100 ( 1)c
1.5 ( 0.6
0
SNP
GTN
0.0215 ( 0.0026
0.0224 ( 0.0039
0.0481 ( 0.0071
-
2.05 × 10-4
58 ( 2
45 ( 1
-
a
b
-
d
Values extrapolated. 3.63 × 10-5 in the absence of L-cysteine. c % NO2 (mol/mol) after 24 h. Tested as oxalates.
smooth muscle preparations, it could be matter of
further investigations.
Discu ssion
All of the investigated furoxans released nitric oxide
when treated under physiological conditions with an
excess of cysteine (1:50). The extent of release covers a
wide range (3-91%). Derivatives 5b, 7b, and 14b are
able to donate NO also in the absence of the thiol
cofactor. After 1 h the yield of NO spontaneously
released by 14b was 95%, while in the case of 5b and
7b only 6% and 5% were released, respectively. After
24 h the yields increase to 100%, 97%, and 56%,
respectively. Also derivative 9b over 24 h behaves as a
spontaneous NO donor (yield 33%). With the interest-
ing exception of the derivative 14b, the presence of
cysteine increases the extent of NO donation. In all of
these compounds, the basic chain at the 3-position,
ionizable under physiological conditions, is essential for
non-thiol-mediated release. Indeed their analogues 5a ,
7a , 9a , and 14a , with the same chain at the 4-position
do not spontaneously release NO, not even over 24 h.
Also the suppression of the basic function on the lateral
chain cancels spontaneous donation, as observed in
4-phenylfuroxans 3-EtX-substituted (X ) S, O, SO2) and
in 4-(phenylsulfonyl)-3-ethylthiofuroxan.17
Comparison of the data reported in Table 1 indicates
that, in the phenylfuroxan series, 4-phenyl isomers are
markedly better NO donors than the corresponding
3-phenyl isomers. This is in line with our previous
findings on other phenylfuroxan derivatives.18 Phenyl-
furoxans bearing (aminoalkyl)oxy moieties at the 3-po-
sition release an amount of NO larger than the corre-
sponding thio and sulfonyl analogues. In the couple of
homologues 5b and 6b, both the members are good NO
donors but 5b only is able of spontaneous release. The
introduction of two methyl groups on the amino function
present on the lateral chain of 7b to give 8b does not
modify the extent of the release induced by thiol cofactor
but removes the spontaneous donation. In the 3-phen-
ylsulfonyl series all of the compounds display good NO-
donating properties. The best NO donor is the deriva-
tive 12 in which either the increase of the length of the
lateral chain or the elimination of the methyl groups
F igu r e 1. Examples of correlation curves between initial NO
formation rate (∆A min-1) and drug concentration (C): (9) 12,
(0) 14b, (b) 5b, (O) 7b.
variation in the optical density ∆A ) 0.1 was chosen in
order to have both sufficiently precise experimental data
1min
0.1
and the most part of C
values intrapolated on the
lines. Using derivative 14b as a source of NO, a molar
extinction coefficient ∆ꢀ ) ꢀ401MetHb - ꢀ401HbO2 ) 38.8 (
0.1 mM-1 cm-1 was evaluated for the oxidation reaction.
On the basis of this value, in line with previous results
reported in literature,16 ∆A ) 0.1 min-1 corresponds to
2.6 µmol L-1 of NO developed in 1 min.
Va sod ila tin g Activities. Vasodilating effects of all
of the derivatives were assessed on endothelium-
denuded strips of rat aorta precontracted with nor-
adrenaline. The compounds were able to bring about a
concentration-dependent relaxation of the strips. Con-
centration-response curves were also evaluated in the
presence of HbO2 (10 µM), a well-known NO scavenger.
EC50 values are reported in Table 1. With all of the
compounds, at the maximum concentrations tested, only
a partial recovery (60-85%) of the noradrenergic tone
occurred, after 1 h of washing the aortic preparations
with fresh Krebs solution. This effect seems to be
concentration dependent. Indeed a detailed study per-
formed with the pair of isomers 9a ,b shows that it
decreases with the concentration of the compounds and
disappears at concentration below 1 × 10-7 M. In
addition it is not specific to noradrenaline since we
obtained similar results working with K+ precontracted
rat aorta strips. Whether this effect is common to other

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466 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Sorba et al.
Sch em e 2
present on the amino nitrogen, as well as the substitu-
tion of sulfur for oxygen at the 4-position to afford 13,
11, and 14a , respectively, decreases the extent of the
release. The sole member of the 4-phenylsulfonyl series
at our disposal, the 4-(phenylsulfonyl)-3-((2-(dimethyl-
amino)ethyl)thio)furoxan (14b) shows strong NO donor
character both in the presence and in the absence of
cysteine. The behavior of this compound is worthy of
comment since NO-releasing capabilities of all of the
previous studied furoxans were highly dependent on the
presence of thiol cofactors. One could speculate that
14b undergoes attack at the 3-position by hydroxyl
anion with consequent nucleophilic displacement of
2-(dimethylamino)ethanethiol (17), through the inter-
mediacy of the adduct 15 (Scheme 2). Thus thiol 17
could be the ultimate responsible for NO release.
Although other data do not seem to support this
hypothesis (e.g., the behavior of 5b and 9b), we incu-
bated, under physiological conditions, 14b (10-4 M) with
N-ethylmaleimide (NEM), a well known agent able to
trap thiols.19 No change in the extent of nitrite ion was
detectable. In order to exclude the influence in the
release by traces of contaminating heavy metals, we
repeated the experiment also in the presence of chela-
tors (EDTA, 0.1 mM). Again no change in the amount
Substitution of the phenyl ring for phenylsulfonyl
moiety in 14b to give 8b suppresses the spontaneous
release, perhaps because the beneficial effect of an
electron-withdrawing function in the 4-position is lost.
In this compound NO release requires, as in most
furoxans, the attack at the 3-position by thiolate anion,
a stronger nucleophile than OH-, to give an adduct
which is a reasonable intermediate on the route of thiol-
mediated NO donation.5,6 The finding that the sup-
pression of the two methyl groups present on the amino
function of 8b affords the weak spontaneous NO donor
7b suggests that charged lateral chain plays fine roles
in NO donation. The substitution in 8b either of the
((dimethylamino)ethyl)oxy chain or of the ((dimethyl-
amino)ethyl)sulfonyl chain for the ((dimethylamino)-
ethyl)thio one affords 5b and 9b, respectively. These
compounds give rise to slow spontaneous release of
different extent. This suggests that the introduction in
such models at the 4-position of the phenylsulfonyl
group should afford potent spontaneous donors. The
lack of spontaneous release moving from 5b to 6b could
be partially due to the “damping effect” of the methylene
group on the electron-withdrawing influence of the
charged dimethylamino function. In conclusion these
data show that the first step on the route of NO
production, under physiological conditions, by furoxans,
without the assistance of thiol cofactors, could be the
result of attack at the 3-position by OH- group, a
consequence of a complex balance of electronic and steric
factors exerted by the substituents at the ring. On the
basis of these considerations, studies are in progress to
obtain new classes of spontaneously NO-releasing furox-
ans.
-
of NO2 was observed. A more attracting explanation
of the behavior of 14b is that 15 does not evolve to the
substitution product 16 but gives the intermediate 18
since thiolate anion could be, in this system, a poor
leaving group. Some formally correct mechanisms able
to justify NO formation from 18 can be envisaged. One
apparently straightforward implies the loss of nitroxyl
anion (NO-) (see Scheme 2) which, in turn, should react
-
either with molecular oxygen to yield NO and then NO2
Also the initial rates of NO release vary widely in the
-
or dimerize to N2O2 with subsequent generation of
1min
0.1
N2O. If this is the case, the extent of NO2- production
(96%) indicates that, under the experimental conditions
used, the former pathway have to be strongly predomi-
nant otherwise other schemes of NO generation must
operate or be paramount. Anyway, these results, as
other previous findings, indicate that the overall mech-
anism of NO donation by furoxans is quite complex and
that the matter is in need of specific experimental
studies.
presence of cysteine (5-fold molar excess). The C
values are spread over the range 1.6 × 10-5 to 8.6 ×
10-3 M. 4-Phenyl derivatives are faster release agents
than 3-phenyl derivatives. In the former series the
1min
0.1
C
values follow the sequence 7b > 6b = 8b > 5b >
9b. In the latter series, the sequence is 5a > 9a ; the
other terms display a release rate too low to be mea-
sured. Phenylsulfonyl-substituted furoxans are fast-
releasing agents, particularly when aminoalkoxy moi-

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Water Soluble Furoxan Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 467
lating potencies of furoxans are principally dependent
on initial rates of NO release. Selected members of the
series described in this work are under investigation in
order to evaluate their activity in vivo and to clarify if
there is some correlation with the corresponding in vitro
behavior.
Exp er im en ta l Section
Gen er a l. All of the compounds were characterized as
oxalates which melted with decomposition. Melting points
were determined on a Bu¨chi 530 apparatus after introducing
the sample into the bath at a temperature 10 °C lower than
the melting point. A heating rate of 3 °C min-1 was used. The
compounds were routinely checked by infrared spectropho-
tometry (Perkin-Elmer Model 781, Shimadzu FT-IR 8101M)
and HPLC (Shimadzu LC10A). 1H and 13C nuclear magnetic
resonance spectra were recorded in dimethyl sulfoxide-d6
(DMSO-d6) at 200 and 50 MHz, respectively, with a Brucker
AC-200 spectrometer. The 13C-NMR signal of the oxalate
anion is normally observed in the range 165-163 ppm. The
1H-NMR signals of exchangeable protons are very broad and
appear in the range 6-11 ppm. Column chromatography was
performed on silica gel (Merck Kieselgel 60, 230-400 mesh
ASTM) with the indicated solvent system. Thin layer chro-
matography (TLC) was carried out on 5 × 20 cm plates
precoated with Merck silica gel 60 F254, with a layer thickness
of 0.25 mm. Anhydrous MgSO4 was used as drying agent.
Solvent removal was achieved under reduced pressure at room
temperature. Elemental analyses of the new compounds were
performed by REDOX (Cologno M.), and the results are within
(0.4% of the theoretical values. Intermediates 4a ,13 4b,13 and
1021 were synthesized according to literature.
F igu r e 2. Concentration-response curves for vasodilating
activity of compound 12 in the absence (b) and in the presence
(O) of 10-5 M HbO2.
eties are present at the 4-position. The couple of
isomers 14a and 14b show similar donation rates. The
spontaneous release by 14b is slightly faster than that
induced by cysteine. Under the experimental conditions
used for our study, no clear correlation exists between
extent and rate of NO release.
Analysis of EC50 values reported in Table 1 indicates
that the compounds cover a wide range of vasodilating
potency. The EC50s follows the sequence 8a > 7a = 6a
> 5a > 7b > 8b = 5b = 6b > 9a > 9b > 14b = 14a >
13 > 11 > 12. The most active compounds belong to
the phenylsulfonyl series. Derivative 12 is about 5
times as potent as glyceryl trinitrate (GTN) and sodium
nitroprusside (SNP) taken as references. 11 and 13
display potencies comparable to those of references. The
two isomers 14a and 14b show the same potency
roughly similar to those of GTN and SNP. In the
phenylfuroxan series, 4-phenyl isomers are always more
potent than the corresponding 3-phenyl ones. 3-((2-
(Dimethylamino)ethyl)sulfonyl)-4-phenylfuroxan (9b) is
the most active member of this group.
The figures in brackets for the regression lines are standard
errors ((SEM).
4-((2-(Dim eth yla m in o)eth yl)oxy)-3-p h en ylfu r oxa n (5a )
Oxa la te. To a stirred solution of 4a (1 g, 3.3 mmol) in THF
(20 mL) were added 2-(dimethylamino)ethanol (0.67 mL, 6.6
mmol) and then 0.79 g of 50% w/w water NaOH solution (9.9
mmol). NaOH solution was added portionwise while the
temperature was maintained at 25 °C. The reaction mixture
was stirred at room temperature for 6 h and then evaporated
in vacuo. The residue was treated with water and extracted
with CH2Cl2. The combined organic layers were dried and
concentrated in vacuo, and the oily residue was filtered on a
short silica gel column (CH2Cl2:MeOH, 95:5) to give the title
product as an oily free base (0.77 g, 94%). The base was
immediately transformed into the corresponding oxalate: mp
In the presence of 10 µM HbO2, the concentration-
response curves were shifted to the right in a parallel
manner. A typical example is reported in Figure 2. The
increase of EC50 values is in keeping with involvement
of nitric oxide in the vasorelaxant action.
When the log EC50 (M) values are plotted against log
1min
0.1
C
(M) values, the quite satisfactory linear regres-
sion equation (1) is obtained and is able to explain 77%
of the variance in the data. The significance of the
equation was improved by omitting derivative 9a , which
is the sole outlier; in fact its residual (observed minus
estimated value) surpasses double the value of s. At
the moment we have not been able to explain this partly
deviant behavior. The clean equation (2) is able to
explain 90% of the variance of the data.
1
157-158 °C dec (MeOH/H2O); H NMR (DMSO-d6) δ 2.81 (s,
6H, NCH3), 3.58 (t, 2H, NCH2), 4.82 (t, 2H, OCH2), 8.1-7.6
(m, 5H, C6H5); 13C NMR (DMSO-d6) δ 43.1 (NCH3), 54.9
(NCH2), 65.9 (OCH2), 107.8 (C3), 161.9 (C4), 121.9 (PhC1),
126.5/129.1 (PhC2/C3), 130.9 (PhC4). Anal. (C12H15N3O3‚
H2C2O4) C, H, N.
4-((3-(Dim eth ylam in o)pr opyl)oxy)-3-ph en ylfu r oxan (6a)
Oxa la te. Prepared as 5a starting from 4a (1 g, 3.3 mmol)
and 3-(dimethylamino)propanol (0.77 mL, 9.9 mmol) with
stirring at room temperature for 4 h: column eluent, CH2Cl2:
MeOH, 95:5; 0.78 g, 90%; mp of the oxalate 146-147 °C dec
(i-PrOH/MeOH); 1H NMR (DMSO-d6) δ 2.29 (m, 2H, CCH2)
2.79 (s, 6H, NCH3), 3.25 (t, 2H, NCH2), 4.55 (t, 2H, OCH2),
8.1-7.4 (m, 5H, C6H5); 13C NMR (DMSO-d6) δ 23.7 (CCH2),
42.5 (NCH3), 53.8 (NCH2), 68.4 (OCH2), 107.9 (C3), 162.3 (C4),
122.1 (PhC1), 126.5/129.4 (PhC2/C3), 131.0 (PhC4). Anal.
(C13H17N3O3‚H2C2O4) C, H, N.
1min
0.1
log EC50 ) 0.755 ((0.129) log C
+ 1.42 ((0.88)
(1)
n ) 12
r ) 0.879
s ) 0.44
F ) 33.95
1min
0.1
log EC50 ) 0.782 ((0.083) log C
+ 1.50 ((0.56)
(2)
3-((2-(Dim eth yla m in o)eth yl)oxy)-4-p h en ylfu r oxa n (5b)
Oxa la te. Prepared as 5a starting from 4b (1 g, 3.3 mmol)
and 2-(dimethylamino)ethanol (1 mL, 9.9 mmol) with stirring
at room temperature for 30 min: column eluent, EtOAc:
acetone, 1:1; 0.56 g, 68%; mp of the oxalate 143-144 °C dec
n ) 11
r ) 0.952
s ) 0.30
F ) 74.16
In conclusion this work shows that (1) it is possible
to modulate over a wide range the NO-donating proper-
ties of furoxan derivatives; (2) furoxan system, if ap-
propriately substituted, can behave under physiological
conditions as NO donor without the assistance of thiol
cofactors (spontaneous release); (3) the in vitro vasodi-
1
(MeOH); H NMR (DMSO-d6) δ 2.60 (s, 6H, NCH3), 3.10 (m,
2H, NCH2), 3.28 (m, 2H, OCH2), 7.9-7.6 (m, 5H, C6H5); 13C
NMR (DMSO-d6) δ 43.1 (NCH3), 55.9 (NCH2), 67.3 (OCH2),
131.2 (C3), 151.1 (C4), 125.2 (PhC1), 126.8/129.4 (PhC2/C3),
131.7 (PhC4). Anal. (C12H15N3O3‚H2C2O4) C, H, N.

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468 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Sorba et al.
3-((3-Dim eth ylam in o)pr opyl)oxy)-4-ph en ylfu r oxan (6b)
Oxa la te. Prepared as 5a starting from 4b (1 g, 3.3 mmol)
and 3-(dimethylamino)propanol (1.16 mL, 9.9 mmol) with
stirring at room temperature for 30 min: eluent, CH2Cl2:
MeOH, 95:5; 0.45 g, 52%; mp of the oxalate 134-135 °C dec
(DMSO-d6) δ 42.9 (NCH3), 49.7/50.5 (SO2CH2/NCH2), 113.4
(C3), 156.9 (C4), 120.6 (PhC1), 129.8/128.9 (PhC2/C3), 131.5
(PhC4). 9b: stirring at room temperature for 4 h (0.85 g, 75%);
mp of the oxalate 159-160 °C dec (MeOH/H2O); 1H NMR
(DMSO-d6) δ 2.38 (s, 6H, NCH3), 3.97 (t, 2H, SO2CH2), 3.06
(t, 2H, NCH2), 7.7-7.6 (m, 5H, C6H5); 13C NMR (DMSO-d6) δ
43.6 (NCH3), 50.3 (SO2CH2/NCH2, coincident signals), 117.4
(C3), 155.4 (C4), 124.8 (PhC1), 129.8/128.5 (PhC2/C3), 131.5
(PhC4). Anal. (C12H15N3O4S‚H2C2O4) C, H, N.
1
(MeOH); H NMR (DMSO-d6) δ 2.15 (m, 2H, CCH2) 2.71 (s,
6H, NCH3), 3.12 (t, 2H, NCH2), 4.52 (t, 2H, OCH2), 7.9-7.6
(m, 5H, C6H5); 13C NMR (DMSO-d6) δ 24.7 (CCH2), 42.6
(NCH3), 53.7 (NCH2), 70.3 (OCH2), 131.2 (C3), 151.2 (C4), 125.3
(PhC1), 128.7/129.5 (PhC2/C3), 131.7 (PhC4). Anal. (C13H17
-
3-(P h en ylsu lfon yl)-4-((2-(d im et h yla m in o)et h yl)oxy)-
fu r oxa n (12) Oxa la te. To a stirred solution of 10 (1 g, 2.7
mmol) in THF (25 mL) was first added 2-(dimethylamino)-
ethanol (0.55 mL, 5.5 mmol) and then, keeping the tempera-
ture at 25 °C with water cooling, 0.65 g of 50% w/w water
NaOH solution (8.1 mmol). The reaction mixture was kept
under stirring for 30 min at room temperature. The solvent
was removed in vacuo, and the residue was treated with water
and extracted with CH2Cl2. The combined organic layers were
dried and concentrated in vacuo. The concentrated solution
was treated with carbon and filtered on a Celite bed and then
evaporated in vacuo to give the title compound as a white solid
(0.51 g, 60%) which was immediately transformed into the
N3O3‚H2C2O4) C, H, N.
4-((2-Am in oeth yl)th io)-3-p h en ylfu r oxa n (7a ) Oxa la te.
To a stirred solution of 4a (1 g, 3.3 mmol) in THF (30 mL)
kept under nitrogen were added first 2-aminoethanethiol
hydrochloride (0.75 g, 6.6 mmol) and then 1.06 g of 50% w/w
water NaOH solution (13.2 mmol), NaOH solution was added
portionwise while the temperature was maintained at 25 °C.
The reaction mixture was stirred under nitrogen at room
temperature for 3 h. In vacuo solvent removal afforded a
residue which was treated with water and extracted with CH2-
Cl2. The combined organic layers were dried and evaporated
in vacuo, and the obtained oily free base 7a (0.70 g, 90%) was
transformed into the corresponding oxalate: mp 186-187 °C
1
corresponding oxalate: mp 158-159 °C dec (MeOH/H2O); H
1
dec (MeOH/H2O); H NMR (DMSO-d6) δ 3.18 (t, 2H, SCH2),
NMR (DMSO-d6) δ 2.83 (s, 6H, NCH3), 3.52 (t, 2H, NCH2),
4.78 (t, 2H, OCH2), 8.1-7.7 (m, 5H, C6H5); 13C NMR (DMSO-
d6) δ 43.2 (NCH3), 54.8 (NCH2), 66.8 (OCH2), 110.8 (C3), 158.6
(C4), 137.0 (PhC1), 130.1/128.5 (PhC2/C3), 136.3 (PhC4). Anal.
(C12H15N3O5S‚H2C2O4) C, H, N.
3.43 (t, 2H, NCH2), 7.9-7.6 (m, 5H, C6H5); 13C NMR (DMSO-
d6) δ 28.3 (SCH2), 37.8 (NCH2), 114.5 (C3), 154.1 (C4), 121.9
(PhC1), 129.3/127.7 (PhC2/C3), 131.1 (PhC4). Anal. (C10H11
-
N3O2S‚H2C2O4) C, H, N.
3-((2-Am in oeth yl)th io)-4-p h en ylfu r oxa n (7b) Oxa la te.
Prepared as 7a starting from 4b and 2-aminoethanethiol
hydrochloride with stirring at room temperature for 30 min:
column eluent at first CH2Cl2 and then CH2Cl2:MeOH, 95:5;
(0.31 g, 40%); mp of the oxalate 193-194 °C dec (MeOH/H2O);
1H NMR (DMSO-d6) δ 2.97 (m, 2H, SCH2), 3.20 (m, 2H, NCH2),
7.9-7.6 (m, 5H, C6H5); 13C NMR (DMSO-d6) δ 28.6 (SCH2),
39.2 (NCH2), 111.1 (C3), 157.9 (C4), 125.9 (PhC1), 129.2/128.0
(PhC2/C3), 131.5 (PhC4). Anal. (C10H11N3O2S‚H2C2O4) C, H,
N.
3-(P h en ylsu lfon yl)-4-((3-d im eth yla m in o)p r op yl)oxy)-
fu r oxa n (13) Oxa la te. Prepared as 12 starting from 10 and
3-(dimethylamino)propanol with stirring at room temperature
for 1 h (0.71 g, 80%): mp of the oxalate 141-142 °C dec
1
(MeOH, i-PrOH); H NMR (DMSO-d6) δ 2.20 (m, 2H, CCH2),
2.77 (s, 6H, NCH3), 3.13 (t, 2H, NCH2), 4.49 (t, 2H, OCH2),
8.1-7.7 (m, 5H, C6H5); 13C NMR (DMSO-d6) δ 23.3 (CCH2),
42.4 (NCH3), 53.5 (NCH2), 68.9 (OCH2), 110.6 (C3), 158.8 (C4),
137.1 (PhC1), 130.1/128.6 (PhC2/C3), 136.2 (PhC4). Anal.
(C13H17N3O5S‚H2C2O4) C, H, N.
4-((2-(Dim eth ylam in o)eth yl)th io)-3-ph en ylfu r oxan (8a)
Oxa la te. Prepared as 7a starting from 4a and 2-(dimethyl-
amino)ethanethiol hydrochloride with stirring at room tem-
perature for 1 h: column eluent CH2Cl2:MeOH, 95:5 (0.79 g,
80%); mp of the oxalate 166-167 °C dec (MeOH/H2O); mp of
the hydrochloride 201 °C dec;21 1H NMR (DMSO-d6) δ 2.69 (s,
6H, NCH3), 3.29 (t, 2H, SCH2), 3.58 (t, 2H, NCH2), 7.8-7.6
(m, 5H, C6H5); 13C NMR (DMSO-d6) δ 42.8 (NCH3), 25.7
(SCH2), 55.1 (NCH2), 114.5 (C3), 154.2 (C4), 121.9 (PhC1),
129.3/127.7 (PhC2/C3), 131.1 (PhC4). Anal. (C12H15N3O2S‚
H2C2O4) C, H, N.
3-(P h en ylsu lfon yl)-4-((2-a m in o)eth yloxy)fu r oxa n (11)
Oxa la te. Prepared as 12 starting from 10 and 2-aminoethanol
with stirring at 15-20 °C for 30 min. The concentrated
solution was saturated with NaCl and extracted with CH2Cl2.
The free base was purified by flash chromatography (EtOAc:
MeOH, 95:5) (0.43 g, 55%): mp of the oxalate 183-184 °C dec
1
(MeOH/H2O); H NMR (DMSO-d6) δ 3.30 (t, 2H, NCH2), 4.65
(t, 2H, OCH2), 8.1-7.7 (m, 5H, C6H5); 13C NMR (DMSO-d6) δ
37.7 (NCH2), 68.4 (OCH2), 111.0 (C3), 159.0 (C4), 137.0 (PhC1),
130.1/128.8 (PhC2/C3), 136.3 (PhC4). Anal. (C10H11N3O5S‚
H2C2O4) C, H, N.
3-((2-(Dim eth ya m in o)eth yl)th io)-4-p h en ylfu r oxa n (8b)
Oxa la te. Prepared as 7a starting from 4b and 2-(dimethyl-
amino)ethanethiol hydrochloride with stirring at room tem-
perature for 40 min: column eluent CH2Cl2:MeOH, 95:5; (0.67
g, 77%); mp of the oxalate 169-170 °C dec (MeOH); 1H NMR
(DMSO-d6) δ 2.56 (s, 6H, NCH3), 3.07 (m, 2H, SCH2), 3.26 (m,
2H, NCH2), 7.9-7.6 (m, 5H, C6H5); 13C NMR (DMSO-d6) δ 42.8
(NCH3), 26.3 (SCH2), 56.4 (NCH2), 111.2 (C3), 158.0 (C4), 126.0
(PhC1), 129.2/128.1 (PhC2/C3), 131.5 (PhC4). Anal. (C12H15N3-
O2S‚H2C2O4) C, H, N.
P r ep a r a t ion of 3-(P h en ylsu lfon yl)-4-((2-(d im et h yl-
a m in o)eth yl)th io)fu r oxa n (14a ) Oxa la te a n d th e 4-P h en -
ylsu lfon yl Isom er (14b) Oxa la te. To a stirred solution of
10 (2 g, 5.5 mmol) in THF (25 mL) kept under nitrogen was
added first 2-(dimethylamino)ethanethiol hydrochloride (1.54
g, 11 mmol) and then, portionwise, 1.75 g of 50% w/w water
NaOH solution (22 mmol). The temperature during the
addition was kept at 25-30 °C. The reaction mixture was
stirred at room temperature for 1 h. The solvent was removed
in vacuo, and the water solution of the residue was extracted
with CH2Cl2. The combined organic layers were dried and
evaporated in vacuo to afford a mixture of the two title isomers.
The mixture was resolved by flash chromatography (EtOAc:
MeOH, 95:5); first eluted 14b (0.82 g, 45%), second eluted 14a
(0.22 g, 12%). Both the isomers were transformed into the
corresponding oxalates. 14a ‚H2C2O4: mp 143-144 °C dec
(MeOH/H2O); 1H NMR (DMSO-d6) δ 2.72 (s, 6H, NCH3), 3.55
(m, 2H, NCH2), 3.32 (m, 2H, SCH2), 8.1-7.6 (m, 5H, C6H5);
13C NMR (DMSO-d6) δ 42.6 (NCH3), 54.7 (NCH2), 24.8 (SCH2),
117.3 (C3), 152.8 (C4), 136.4/136.5 (PhC1/PhC4), 130.2/128.5
(PhC2/C3). 14b‚H2C2O4: mp 144-145 °C dec (MeOH/H2O);
1H NMR (DMSO-d6) δ 2.59 (s, 6H, NCH3), 3.23 (t, 2H, NCH2),
2.95 (t, 2H, SCH2), 8.1-7.8 (m, 5H, C6H5); 13C NMR (DMSO-
d6) δ 42.5 (NCH3), 56.1 (NCH2), 26.0 (SCH2), 108.9 (C3), 159.7
(C4), 136.0/136.4 (PhC1/PhC4), 130.3/129.3 (PhC2/C3). Anal.
(C12H11N3O4S‚H2C2O4) C, H, N.
P r ep a r a tion of 4-((2-(Dim eth yla m in o)eth yl)su lfon yl)-
3-p h en ylfu r oxa n (9a ) Oxa la te a n d th e 4-P h en yl Isom er
(9b) Oxa la te. To a stirred and water-cooled solution of the
appropriate thio derivative 8a or 8b (1 g, 3.8 mmol) in acetic
acid (10 mL) potassium permanganate (1.2 g, 7.6 mmol) was
added portionwise. The mixture was stirred at room temper-
ature for 6 h. Acetic acid was removed at room temperature
in vacuo. The residue was treated with water, and the excess
potassium permanganate and the formed manganese dioxide
were destroyed by sodium sulfite. The water solution was
basified with sodium carbonate and extracted with EtOAc. The
combined organic layers were dried and concentrated in vacuo
to give an oil which was purified by flash chromatography
(CH2Cl2:MeOH, 95:5) to give the title products as free bases.
9a (0.66 g, 58%): mp of the oxalate 150-151 °C dec (MeOH/
1
H2O); H NMR (DMSO-d6) δ 2.57 (s, 6H, NCH3), 4.13 (t, 2H,
SO2CH2), 3.29 (t, 2H, NCH2), 7.8-7.6 (m, 5H, C6H5); 13C NMR
Qu a n tita tive Nitr ite Detection . A solution of the ap-

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Water Soluble Furoxan Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 469
propriate furoxan (20 µL) in distilled water was added to 2
mL of 50 mM phosphate buffer (pH 7.4) containing 5 mM
L-cysteine or in its absence. The final concentration of drug
was 10-4 M. After 1 h at 37 °C, 1 mL of the reaction mixture
was treated with 250 µL of the Griess reagent [sulfanilamide
(4 g), N-naphthylethylenediamine dihydrochloride (0.2 g), and
85% phosphoric acid (10 mL) in distilled water (final volume:
100 mL)]. After 10 min at room temperature, the absorbance
was measured at 540 nm; 10-80 nmol/mL sodium nitrite
standard solutions were used for the calibration curve. The
eluted with phosphate buffer. Two Pasteur pipets were used
as columns for 1 mL of this solution.
The purity of the oxyhemoglobin solution was determined
spectrophotometrically (600-360 nm) (λmax ) 576 nm, 541 nm;
ꢀ ) 14 600, 13 800) by dissolving 10 µL of the eluted solution
into 3 mL of buffer (concentrated oxyHb (µM) ) A × 2290).
The solutions were freshly prepared daily.
Ack n ow led gm en t. This work was supported by
grants from CNR and MURST Studi e Ricerche Final-
izzate 40%, Roma.
-
yield in nitrite was expressed as percent NO2 (mol/mol) (
SEM.
NO Relea se Kin etic Stu d y. The rate of NO release was
determined using a spectrophotometric technique16 based on
the oxidation of oxyhemoglobin (HbO2) to methemoglobin
(MetHb) according to the equation
Refer en ces
(1) Kerwin, J . F., J r.; Lancaster, J . R., J r.; Feldman, P. L. Nitric
oxide: A new Paradigm for Second Messengers. J . Med. Chem.
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(2) Lefer, A. M.; Lefer, D. J . Therapeutic Role of Nitric Oxide Donors
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HbO22+ + NO f MetHb3+ + NO3
-
(3) (a) Po¨rsti, I.; Paakkari, I. Nitric Oxide-based Possibilities for
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The formation of MetHb was followed by recording the
absorbance increase (∆A) at λ ) 401 nm using a Perkin-Elmer
λ5 spectrophotometer and a thermostated (37 °C) cuvette.
The reaction was started by adding drug water solutions of
at least three different concentrations (final concentration 10-5
to 7.5 × 10-4 M) to a 4 µM HbO2 solution in 50 mM phosphate
buffer (pH 7.4) containing a 5-fold molar excess of cysteine as
to the furoxan solute. The increase of the absorbance (∆A)
was recorded over the first 3 min. The initial rates were
calculated from the slope of the straight line portion of each
curve. Every NO-releasing rate is the average of at least three
determinations. The molar extinction coefficient ∆ꢀ ) ꢀ401MetHb
A.; Bosia, A.; Pescarmona, G. Characterization of
a New
Compound, S35b, as a Guanylate Cyclase Activator in Human
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(5) Feelisch, M.; Scho¨nafinger, K.; Noack, E. Thiol-Mediated Gen-
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Furoxans. Biochem. Pharmacol. 1992, 44, 1149-1157.
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Gasco, A. Furoxans as nitric oxide donors. 4-Phenyl-3-
furoxancarbonitrile: Thiol-mediated NO release and biological
evaluation. J . Med. Chem. 1994, 37, 4412-4416.
(7) Gasco, A.; Fruttero, R.; Sorba, G. NO-Donors: an emerging class
of compounds in medicinal chemistry. Il Farmaco 1996, 51, 617-
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(8) Civelli, M.; Caruso, P.; Giossi, M.; Bergamaschi, M.; Razzetti,
R.; Bongrani, S.; Gasco, A. CHF 2206, a New Potent Vasodilating
and Antiaggregating Drug as Potential Nitric Oxide Donor. Eur.
J . Pharmacol. 1994, 255, 17-24.
(9) Civelli, M.; Giossi, M.; Caruso, P.; Razzetti, R.; Bergamaschi,
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System as a Useful Tool for Balancing “Hybrids” with Mixed
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(13) Sorba, G.; Ermondi, G.; Fruttero, R.; Galli, U.; Gasco, A.
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- ꢀ401HbO was determined by quantitative oxidation of five
2
different concentrations (2-6 µM) of HbO2 in pH 7.4 phosphate
buffer with a 7.5 × 10-5 M water solution of 14b. The slope
(∆ꢀ) of the straight line (r ) 0.999) obtained plotting the
increase of the absorbance ∆A at λ ) 401 nm against the HbO2
concentrations was 38.8 ( 0.1 mM-1 cm-1. Using K3Fe(CN)6
as oxidant, a similar value of 39.9 ( 1.4 mM-1 cm-1 was
obtained.
Va soa ct ivit y Det er m in a t ion s. Thoracic aortas were
isolated from male Wistar rats weighing 180-200 g. The
vessels were helically cut, the endothelium was removed, and
two strips were obtained from each aorta. The tissues were
mounted under 1g tension in organ baths containing 30 mL
of Krebs-Heinseleit solution (NaCl, 137; KCl, 2.68; MgCl2, 0.50;
CaCl2, 5.44; NaH2PO4, 0.54; NaHCO3, 8.93; glucose, 8.30;
ascorbic acid, 0.10 mM) at 37 °C and gassed with 95% O2-5%
CO2 (pH 7.4). The aortic strips were allowed to equilibrate
for 1 h and then were contracted with noradrenaline (1 µM),
which causes a submaximal response. During this first
contraction the absence of intact endothelium was verified by
adding acetylcholine (1 µM), which was found not to induce
relaxation. The preparations were then extensively washed
with Krebs-Heinseleit solution, and a second contraction was
evoked by noradrenaline (1 µM). When the response to the
agonist plateaued, cumulative concentrations of the vasodi-
lating agent were added. When maximal vasodilation was
obtained, aortic strips were washed repeatedly and a third
contraction was induced by noradrenaline (1 µM) so as to verify
the reversibility of vasodilation. The effects of oxyhemoglobin
on relaxation were evaluated in a separate series of experi-
ments by exposing aortic strips, precontracted with 1 µM
noradrenaline, to oxyhemoglobin 10 µM for at least 10 min
before addition of vasodilating agent.
(14) Calvino, R.; Fruttero, R.; Gasco, A.; Mortarini, V.; Aime, S.
Unsymmetrically Substituted Furoxans. Part 7. A 13C NMR
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(15) Gasco, A.; Boulton, A. J . Furoxans and Benzofuroxans. Adv.
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(16) Noack, E.; Kubitzek, D.; Kojda, G. Spectrophotometric Deter-
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(17) Medana, C.; Gasco, A. Unpublished results.
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Fruttero, R.; Civelli, M.; Gasco, A. A New Class of Furoxan
Derivatives as NO-donors: Mechanism of Action and Biological
Activity. Br. J . Pharmacol. 1995, 114, 816-820.
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In The Chemistry of the Thiol Group; Patai, S., Ed.; J ohn Wiley
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(20) Farrar, W. W. The 3,4-Bisarenesulfonylfuroxans. J . Chem. Soc.
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Chem. Abstr. 1994, 120, 245116.
P r ep a r a tion of Oxyh em oglobin . Bovine hemoglobin
type 1 (H-2500 Sigma Chemical Co.) contains a mixture of
oxyhemoglobin and the oxidized derivative methemoglobin.
Pure oxyhemoglobin was prepared by adding a solution of
commercially available hemoglobin in 50 mM phosphate saline
buffer, pH 7.4, to an excess of the reducing agent sodium
dithionite (Na2S2O4) at 4 °C and was then protected from the
light. Hemoglobin (64.5 mg) was gently dissolved into 1 mL
of distilled water; 53.6 mg of sodium dithionite was then added
to the solution which was loaded onto a chromatographic
column (Sephadex G-25, Pharmacia, Uppsala, Sweden) and
J M960379T