Novel antioxidant agents deriving from molecular combination of Vitamin C and NO-donor moieties

Article abstract of DOI:10.1016/j.bmc.2008.03.014

In this paper, we describe a new class of products in which NO-donor moieties are linked to either the 3-OH (4a-f) or 2-OH group (7a-c) of ascorbic acid (ASA). Log Ps and pK_as of these products were experimentally evaluated. All the compounds were tested for their antioxidant activity on lipidic peroxidation induced by Fe³⁺-ADP/NADPH in lipids of microsomal membranes of rat hepatocytes. Only 3-O series displays antioxidant activity and it seems to be principally dependent on the lipophilicity. Both series trigger in vitro NO-dependent vasodilator properties.

Full text of DOI:10.1016/j.bmc.2008.03.014

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5199–5206
Novel antioxidant agents deriving from molecular combination
of Vitamin C and NO-donor moieties
Clara Cena, Konstantin Chegaev, Silvia Balbo, Loretta Lazzarato, Barbara Rolando,
Marta Giorgis, Elisabetta Marini, Roberta Fruttero and Alberto Gasco*
`
Dipartimento di Scienza e Tecnologia del Farmaco, Universita degli Studi di Torino, Via Pietro Giuria 9, I-10125 Torino, Italy
Received 8 November 2007; revised 26 February 2008; accepted 4 March 2008
Available online 6 March 2008
Abstract—In this paper, we describe a new class of products in which NO-donor moieties are linked to either the 3-OH (4a–f) or 2-
OH group (7a–c) of ascorbic acid (ASA). Log Ps and pKas of these products were experimentally evaluated. All the compounds were
tested for their antioxidant activity on lipidic peroxidation induced by Fe3+-ADP/NADPH in lipids of microsomal membranes of rat
hepatocytes. Only 3-O series displays antioxidant activity and it seems to be principally dependent on the lipophilicity. Both series
trigger in vitro NO-dependent vasodilator properties.
ꢀ 2008 Elsevier Ltd. All rights reserved.
HO
HO
HO
HO
HO
HO
1. Introduction
O
O
O
O
O
O
Today there is an interest in multi-target drugs, namely
in products which are able to modulate, directly or
through metabolites, more than one physiological tar-
get. These compounds could represent an alternative
to the use of cocktails of single target drugs in the treat-
ment of complex diseases by combining therapeutic
mechanisms. Usually, multi-target drugs are obtained
by joining two drugs, or a crucial part of them, through
metabolically stable or cleavable linkers.1 A particular
family of these products is represented by appropriate
drugs linked to nitric oxide (NO) donor moieties.2
NO-donor antioxidants belong to this family.3 These
products combine NO-dependent pharmacological
properties with radical scavenger activities. They are
potentially interesting in the treatment of some forms
of cardiovascular disease associated with endothelial
dysfunction, such as atherosclerosis, hypercholesterol-
emia, and hypertension. In these pathologies, an in-
creased concentration of reactive oxygen species
(ROS), a decreased ability to produce NO by endothelial
cells, a decreased sensitivity of the vessel to the actions
of NO, and a NO destruction are heavily implicated.4
L-Ascorbic acid (ASA, 1) (Fig. 1) is a very important,
HO OH
O
OH
HO
O
H3C
CH3
1
1a
1b
Figure 1. Ascorbic acid (ASA), 3-O methyl, and 2-O methyl ASA
derivatives.
highly hydrophilic radical scavenger. It is able to effi-
ciently trap a number of radicals, including anion super-
oxide, peroxyl and hydroxyl radicals, and to restore the
antioxidant properties of Vitamin E, a highly lipophilic
radical scavenger, by reducing the a-tocopheroxyl radi-
cal (a-TOÅ) to a-tocopherol.5 ASA seems to play a role
in preventing endothelial dysfunction. The mechanism
of such an action is largely unknown. Several hypothe-
ses have been formulated including an ascorbate-in-
duced decrease in low density lipoproteins (LDL)
oxidation, a scavenging of intracellular superoxide, a
potentiated release of NO from circulating or tissue S-
nitrosothiols, a direct reduction of nitrite to NO, an acti-
vation of either endothelial NO synthase (e-NOS) or
smooth muscle guanylate cyclase.6 ASA decomposes
upon exposure to heat, UV light, metal ions, and other
oxidants. A number of pro-drugs and ASA derivatives
have been developed in order to increase the stability
of these compounds,7 including 3-O- and 2-O-alkylasc-
orbic acids, as for example the methyl derivatives 1a
Keywords: Multitarget drugs; NO-donor antioxidants; NO-donor
ascorbic acid derivatives.
*
Corresponding author. Tel.: +39 0116707670; fax: +39
0116707286; e-mail: alberto.gasco@unito.it
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.014

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C. Cena et al. / Bioorg. Med. Chem. 16 (2008) 5199–5206
R
R'
ONO2
H3C
R= OC2H5, R'= SO2Ph
R= CH2OH, R'= CONH2
R= OCH3, R'= Ph
i
ONO2
ONO2
n= 0, 1
N+
HO
HO
N
(
)
n
ONO2
O
O
2
3c
Figure 2. Reference NO-donor compounds.
HO
O
O
ii
and 1b (Fig. 1).8 A number of these latter products dis-
played inhibitory effects on some lipid peroxidation
models and were capable of alleviating myocardial le-
sions induced by ischemia reperfusion.
R
O H
1
+
HO
O
OH
R
ONO
ONO
2
2
3a R =
3b R =
3c R =
4a R =
4b R =
4c R =
ONO
ONO
2
As a development of our work in the field of NO-donor
antioxidants,9 here we describe a new class of products
in which alkyl chains containing NO-donor nitrooxy
and furoxan moieties are linked to either the 3-OH
(4a–f) or 2-OH group (7a–c) of ASA. The NO-donor
moieties we used in our approach were nitrooxy-substi-
tuted alkyl moieties which are present in the simple ni-
tric esters as well as the 3-phenylsulfonylfuroxan-4-
yloxy substructure present in 4-ethoxy-3-phenylsulfo-
nylfuroxan, the 3-carbamoylfuroxan-4-ylmethyl sub-
2
2
2
ONO
ONO
2
ONO
ONO
2
H N
H N
2
2
O
O
3d R =
3e R =
3f R =
4d R =
4e R =
4f R =
+
+
N
N
N
N
O
O
O
O
O
O
+
+
N
N
N
N
O
O
O
O
O
O
O
O
S
S
structure
present
in
the
4-hydroxymethyl-3-
O
O
+
+
N
N
N
N
O
furoxancarboxamide and the 3-phenylfuroxan-4-yloxy
substructure present in the 4-methoxy-3-phenylfuroxan
(Fig. 2).9 These reference compounds display extremely
modulated in vitro NO-dependent vasodilator proper-
ties. Synthesis of the NO-donor derivatives of Vitamin
C, determination of their structures through pKa and
NMR analysis, as well as the study of their lipophilic–
hydrophilic balance are reported. Antioxidant proper-
ties, assessed in the thiobarbituric acid reactive sub-
stances (TBARS) assay and the ability to relax rat
aorta strips precontracted with phenylephrine of all
the products are also discussed.
O
O
O
H N
2
Br
O
iii
4d
1
+
+
N
N
O
O
5
Scheme 1. Reagents and conditions: (i) I2, AgNO3, CH3CN, rt, then
reflux; (ii) PPh3, DIAD, THF dry, ꢀ78 ꢁC, then heat up to rt; (iii)
NaHCO3, DMSO dry, 50 ꢁC.
HO
O
2. Synthesis
O
O
O
O
i, ii
O
3a, 3b, 3e
HO
+
All NO-donor alcohols (3a–f) were previously described
in the literature. The synthetic procedure for 3c was
modified with respect to the literature one: the desired
product was obtained by treating 5-hexen-1-ol (2) with
I2 and AgNO3 (Scheme 1). The 3-O substituted com-
pounds 4a–f were prepared by the reactions of corre-
sponding alcohols with ASA under the Mitsunobu
conditions (Scheme 1), namely by treating the adduct
of PPh3 and diisopropyl azodicarboxylate (DIAD) in
THF solution with ASA, followed by the addition of
the appropriate alcohol, resulting in the regioselective
formation of the 3-O substituted compounds.10 The
products were obtained in a fairly good yields (35 –
43%), with the exception of products 4d (17%) and 4f
(17%). In the case of 4d, the yield could be slightly im-
proved (30%) by treating 1 directly with 4-bromometh-
ylfuroxan derivative (5) in DMSO solution, in the
presence of NaHCO3 (Scheme 1).
HO
O
O
OH
R
O
ONO
7a R =
7b R =
7c R =
6
2
ONO
2
O
+
N
N
O
O
Scheme 2. Reagents and conditions: (i) PPh3, DIAD, ꢀ15 ꢁC, then rt;
(ii) MeOH, HCl 4 M, rt.
ment with HCl in methanol followed by purification
gave rise to the title compounds (Scheme 2).
3. Results and discussion
3.1. Ionization constant and NMR studies
To synthesize the final 2-O substituted compounds 7a–c,
5,6-O-isopropyliden-3-O-(methoxymethyl) ascorbic acid
(6) was treated with the appropriate alcohol under the
same Mitsunobu conditions used to prepare the 3-O
analogues. Removal of the protective groups by treat-
Dissociation constants (pKa) of all the compounds de-
scribed in the present work (4a–f, 7a–c), together with
those of ASA, 1a and 1b taken as references, are col-

C. Cena et al. / Bioorg. Med. Chem. 16 (2008) 5199–5206
5201
Table 1.
ASA
Compound Antioxidant action
Vasodilation
Ionization and lipophilicity
log D1.0 SD (CLOGP) log D7.4 SD
IC50 (95%CL) (lM) EC50 SEM (lM)a (+1 lM ODQ) pKa SD
b
c
—
—
—
4.08 0.01 (3-OH) ꢀ1.65 0.06 (ꢀ1.76)
10.85 0.03 (2-OH)
1a
4a
4b
4c
4d
4e
4f
>1 mM
—
7.54 0.01
7.55 0.01
7.60 0.02
7.64 0.01
7.07 0.01
7.60 0.03
7.62 0.02
3.23 0.02
3.19 0.01
3.56 0.01
3.44 0.01
ꢀ1.41 0.04 (ꢀ1.74)
ꢀ0.48 0.05 (ꢀ0.94)
0.86 0.05 (0.40)
0.78 0.01 (0.21)
ꢀ0.99 0.04 (ꢀ2.96)
1.53 0.03 (0.74)
1.18 0.01 (0.99)
ꢀ1.44 0.04 (ꢀ1.46)
ꢀ0.26 0.02 (ꢀ0.66)
0.84 0.03 (0.68)
1.71 0.04 (1.02)
ꢀ1.61 0.05
ꢀ0.55 0.04
0.76 0.02
0.66 0.02
ꢀ1.44 0.03
1.13 0.05
0.98 0.01
454 (414–497)
80 (77–83)
28 (26–32)
55 9 (>100)
2.1 0.7 (>100)
0.72 0.13 (>100)
6.0 0.8 (>100)
3.8 0.4 (80.9 11.7)
0.034 0.004 (7.0 0.5)
—
>1 mM
17 (16–18)
80 (76–83)
c
1b
7a
7b
7c
Inactive at 1 mM
Inactive at 1 mM
>1 mM
—
c
d
—
—
ꢀ1.93 0.03
ꢀ1.35 0.04
7.1 0.8 (>100)
32 2 (>100)
392 (373–414)
a EC50 values are means of at least five experiments.
b Maximal inhibition of lipid peroxidation in the range 10–50 lM: 77% at 50 lM (see discussion).
c Log D could not be measured by shake-flask method (log D < ꢀ2).
d EC50 could not be calculated because at the maximal concentration tested (100 lM), 50% of tissue relaxation was not achieved.
lected in Table 1. The values were determined by poten-
tiometric titration with GlpKa apparatus (Sirius Analyt-
ical Instruments Ltd.). Most of the compounds showed
good aqueous solubility and titrations were performed
in water. Products 4e and 4f required titrations in the
presence of methanol as a cosolvent and pKa values were
obtained by extrapolation to zero content of the cosol-
vent. The pKa measurements showed that between the
two enol hydroxyl groups of the ASA’s lactone ring,
the 3-OH is significantly more acidic than 2-OH (Table
1). This difference is partly retained in 3-OCH3 and 2-
OCH3 derivatives. The pKa values for the other com-
pounds of the table are consistent with the assigned
structures. In fact, 3-O substituted compounds have
pKa near that of the 3-OCH3 model, while 2-O substi-
tuted compounds are near that of 2-OCH3. This means
that the NO-donor moieties linked to the lateral 3-O and
2-O alkyl chains of 4a–f and 7a–c, scantly influence the
pKa values of these compounds. The only exception is
the amide derivative 4d, in which the electron-withdraw-
ing furoxan substructure is joined to the 3-O position
through a methylene bridge. The assigned structures
were also confirmed by 13C NMR spectra. Recently,
13C NMR spectra of a series of 3-O and 2-O alkyl deriv-
atives of 5,6-O-isopropylidene-L-ASA have been criti-
cally discussed.11 In the 3-O alkyl derivatives C-3
carbons display characteristic upfield chemical shifts
with respect to C-3 carbons in 2-O alkyl substituted
compounds, while the C-2 carbon chemical shifts are
scarcely influenced regardless of the site of alkylation.
The same situation occurs for the O-alkyl substituted
compounds described in the present work (see Section
5).
lated log D1.0 represent the logarithms of the partition
coefficients of these species (log PN). The measured val-
ues are in a good agreement with the calculated values
using CLOGP algorithm,12 with the only partial excep-
tion of 4d. At pH 7.4 all the products are ionized for
about 40%, and consequently their log D7.4 are lower
than their log D1.0 of about 0.2 U. A partly different sit-
uation occurs for the 2-O substituted compounds.
According to their pKa values, they are largely present
in the unionized form at pH 1 and in the ionized form
(I) at pH 7.4, and consequently log D1.0 ffi log PN and
log D7.4 ffi log PI. Generally speaking, all the NO-donor
derivatives of ASA described in the present work are
definitely more lipophilic than ascorbic acid.
3.3. Antioxidant properties
All the final compounds were assessed as inhibitors of
Fe3+-ADP/NADPH-induced peroxidation of membrane
lipids of rat hepatocytes. ASA and its 3-OCH3 and 2-
OCH3 derivatives were also considered for comparison.
The 2-thiobarbituric acid (TBA) assay was used to fol-
low the progress of the autooxidation. The procedure in-
volves the detection of the final metabolites of the lipid
autooxidation, namely 2-thiobarbituric acid reactive
substances (TBARS) by visible spectroscopy.13 A num-
ber of products under study were able to inhibit, in a
concentration-dependent manner, the progress of the
reaction (see an example in Fig. 3). The behavior of
ASA is worthy of a special comment. The complex role
of this product in different lipid peroxidation systems
resulting either in antioxidant or pro-oxidant actions
has been extensively reported.14 Under the conditions
we set up to carry out our study, ASA was able to inhi-
bit lipidic peroxidation in a concentration-dependent
manner in the range from 10 to 50 lM, showing 77%
maximal inhibition. By contrast, at higher concentra-
tions it showed a pro-oxidant effect. 1a and 1b deriva-
tives were inactive when tested at 1 mM concentration.
This is in agreement with the known fact that monoalky-
lation of the 3-O or 2-O position of ASA notably re-
duces the electron-donating activity of the resulting
3.2. Lipophilicity studies
Distribution coefficients (log D) between n-octanol and
water were measured by shake flask technique at room
temperature at pH 1 and pH 7.4. The results are re-
ported in Table 1. The 3-O substituted compounds,
according to their pKa values, at pH 1 are present essen-
tially as neutral forms (N), and consequently the tabu-

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C. Cena et al. / Bioorg. Med. Chem. 16 (2008) 5199–5206
mechanism of this action involves their conversion into
25
20
15
10
5
NO in vascular smooth muscle cells with consequent
activation of the soluble guanylate cyclase (sGC). In
the case of nitrates this conversion prevalently follows
an enzymatic pathway,15a while for furoxans it should
occur under the action of intracellular thiols.15b The
vasodilator activities of the new compounds described
in the present work were evaluated on denuded rat aorta
strips pre-contracted with phenylephrine. All the prod-
ucts were capable of relaxing the contracted tissue in a
concentration-dependent manner. The vasodilator
potencies, expressed as EC50, are reported in Table 1.
In the 3-O substituted nitric esters series, the most active
product is the dinitrooxy compound 4c, followed by the
nitrooxyhexyl derivative 4b and its analogue 4a with a
shorter alkyl chain. In the furoxan family, the phenylsul-
fonyl substituted furoxan 4f is a very potent vasodilator,
followed by 4e and then by 4d, which bear 3-phenylfuro-
xan-4-yloxy and 3-carbamoylfuroxan-4-yl substruc-
tures, respectively. Certainly, the very high NO-donor
capacity of the 4-phenylsulfonylfuroxan-3-yloxy sub-
structure present in 4f,15b probably combined with its
high lipophilicity, plays paramount roles in the high
vasodilator potency of the product. In the 2-O substi-
tuted series, the mononitrooxyhexyl derivative 7b was
the most potent vasodilator, followed by the phenylf-
uroxan derivative 7c and by the mononitrooxypropyl
derivative 7a. The members of the 3-O series resulted
to be more potent than their isomers of the 2-O series.
When the vasodilator experiments were repeated in the
presence of 1 lM ODQ (1H-[1,2,4]oxadiazolo[4,3-a]qui-
noxalin-1-one), a well-known inhibitor of sGC, a de-
crease in the potencies was observed, which is in
keeping with NO-activation of the sGC.
control
5μM
10μM
20μM
30μM
40μM
50μM
0
0
10
20
30
time (min)
Figure 3. Effect of compound 4e on time course of lipid peroxidation.
products.14c The potencies of the NO-donor ASA deriv-
atives expressed as IC50, namely the molar concentra-
tion able to reduce autooxidation by 50%, are
collected in Table 1. Analysis of the data shows that 2-
O substituted compounds do not display any antioxi-
dant activity, with the exception of the feebly active phe-
nylfuroxan derivative (7c), which is the most lipophilic
member of the group. This behavior probably largely
depends on the low lipophilicity of the products, due
to their almost complete ionization under physiological
pH conditions used to carry out the antioxidant experi-
ments. Actually, it is known that 2-O-alkyl ascorbic
acids require long lipophilic alkyl moieties to inhibit li-
pid peroxidation.8a The antioxidant behavior of 3-O
substituted compounds (4a–f) is quite different from that
of the 2-O isomers. The products are more lipophilic ow-
ing to their higher pKa values. At pH 7.4, they are ion-
ized for about 40% with the only exception of 4d which
is ionized for 68%. The most active product of the or-
ganic nitrate series is the dinitrooxy derivative 4c fol-
lowed by mononitrooxy derivatives 4b and 4a. Their
activities parallel their lipophilicities. In the furoxan ser-
ies the two active products are the lipophilic compounds
4e and 4f, while the hydrophilic amide derivative 4d is
inactive. The important role that the lipophilicity seems
to exert on the antioxidant activity in this class of prod-
ucts is in accordance with the finding that the antioxi-
dant potency of 3-O-alkyl ascorbic acids, assessed on
lipid peroxidation induced in rat microsomes by Fe2+
and Fe3+-ADP, displays a parabolic dependence on
lipophilicity.14c Actually we calculated that the optimal
CLOGP of this latter products ranges from 3 to 4. Con-
sequently, their log D should range from 2.60 to 3.70,
assuming for these products the same pKa as the parent
3-OCH3 derivative and neglecting the partition of the
ionized forms. The low lipophilicity of the 3-O NO-do-
nor ascorbic acid derivatives described in the present
work indicates that they should be positioned on the
ascending branch of parabolic dependence. In the condi-
tions set up to evaluate antioxidant properties of NO-
donor ASA derivatives, none of the reference NO-donor
substructures (Fig. 2) displayed lipid peroxidation inhi-
bition at 500 lM concentration.
4. Conclusions
A new class of 2-O-alkyl and 3-O-alkyl ascorbic acids
bearing on the alkyl chains NO-donor nitrooxy and
furoxan moieties was synthesized. The antioxidant prop-
erties of these compounds were evaluated on lipid perox-
idation induced in rat microsomes by Fe3+-ADP/
NADPH using TBARS detection and taking as refer-
ences ASA and its O-methyl derivatives 1a and 1b. Un-
like the 2-O series the 3-O one displays antioxidant
activities, which seem to be principally dependent on
the higher lipophilicity of the products belonging to this
class. All the products were able to relax rat aorta strips
precontracted with phenylephrine in a dose-dependent
manner. The vasodilator action was decreased by the
presence of ODQ, and this is in keeping with a NO acti-
vation of the sGC. Selected members of 3-O series are
worthy of additional in vivo study since they are poten-
tially useful in the treatment of some forms of the car-
diovascular disease associated with endothelial
dysfunction.
3.4. Vasodilator activity
5. Experimental
1H and 13C NMR spectra were recorded on a Bruker
Avance 300 at 300 and 75 MHz, respectively, using
It is known that organic nitrates and furoxan derivatives
display vasodilator activities. The generally accepted

C. Cena et al. / Bioorg. Med. Chem. 16 (2008) 5199–5206
5203
SiMe4 as the internal standard. Low resolution mass
spectra were recorded with a Finnigan-Mat TSQ-700.
Melting points were determined with a capillary appara-
CH2Cl2/MeOH) and further by HPLC (RP-18, eluent
1/1 MeOH/H2O, 39 mL/min, 100 mg/injection) to give
the title compound as a pale yellow oil, 35%. 1H
NMR (CD3OD): d 2.16–2.24 (m, 2H, CH2CH2CH2),
3.67 (d, 2H, J = 6.9 Hz, CH(OH)CH2(OH)), 3.86 (t,
1H, J = 6.6 Hz, CH(OH)CH2(OH)), 4.61–4.82 (m, 4H,
CH2CH2CH2), 4.82 (d, 1H, J = 1.6 Hz, CHOC=O);
13C NMR (CD3OD): d 28.5, 63.4, 68.8, 70.5, 71.2,
76.6, 121.0, 151.2, 172.9. MS: m/z 280 (M+H)+. Anal.
(C9H13NO9 Æ 0.75 H2O) C, H, N: C calcd 36.93, found
36.82, H calcd 4.99, found 4.79, N calcd 4.79, found
4.51.
tus (Buchi 540). Flash column chromatography was per-
¨
formed on silica gel (Merck Kieselgel 60, 230-400 mesh
ASTM); PE stands for 40–60 petroleum ether. The pro-
gress of the reactions was followed by thin layer chro-
matography (TLC) on 5 · 20 cm plates with a layer
thickness of 0.2 mm. Anhydrous magnesium sulfate
was used as the drying agent for the organic phases. Or-
ganic solvents were removed under vacuum at 30 ꢁC.
Preparative HPLC was performed on a LiChrospherꢂ
C18 column (250 · 25 mm, 10 lm) (Merck Darmstadt,
Germany) with a Varian ProStar mod-210 with Varian
UV detector mod-325. Elemental analyses (C, H, N)
were performed by REDOX (Monza). Compounds
1a,16 1b,16 3a,17 3b,17 3e,18 3f,19 520, and 68a were synthe-
sized according to the literature. The compound 3d was
a gift from Sanofi-Aventis Deutschland GmbH.
5.2.2. 5-(1,2-Dihydroxyethyl)-3-hydroxy-4-(6-nitrooxy-
hexyloxy)-5H-furan-2-one (4b). The resulting oil was
purified by flash chromatography (eluent 95/5 CH2Cl2/
MeOH) to give the title compound as a pale yellow oil,
1
43%. H NMR (CD3OD): d 1.47–1.49 (m, 4H), 1.72–
1.80 (m, 4H) (CH2(CH2)4CH2), 3.65 (d, 2H, J = 6.9 Hz,
CH(OH)CH2(OH)), 3.84 (t, 1H, J = 6.6 Hz, CH(OH)-
CH2(OH)), 4.47–4.54 (m, 4H, CH2(CH2)4CH2), 4.77 (d,
1H, J = 1.5 Hz, CHOC@O); 13C NMR (CD3OD): d
26.2, 26.4, 27.8, 30.6, 63.5, 70.6, 72.5, 74.6, 76.7, 120.5,
152.2, 173.2. MS m/z 322 (M+H)+. Anal. (C12H19
NO9 Æ 0.25 H2O) C, H, N: C calcd 44.24, found 43.97, H
calcd 6.03, found 5.89, N calcd 4.30, found 4.27.
5.1. 6-Hydroxyhexane-1,2-diyl dinitrate (3c)
To a stirred solution of 2 (2.0 mL, 17 mmol) and AgNO3
(9.0 g, 53 mmol) in CH3CN, I2 (4.3 g, 17 mmol) was
added in one portion. The reaction was stirred at room
temperature (rt) until all I2 dissolved, then it was heated
at reflux for 5 h. The precipitate was filtered off and the
reaction mixture was concentrated and diluted with
EtOAc (100 mL). The organic phase was washed twice
with H2O (100 mL), brine, dried and evaporated. The
resulting dark yellow oil was purified by flash chromatog-
raphy (eluent 7/3 PE/EtOAc) to give the title compound as
a yellow oil, 55%. 1H NMR (CDCl3): d 1.51–1.86 (m, 7H,
HOCH2(CH2)3), 3.67 (t, 2H, J = 5.9 Hz, HOCH2), 4.48
(dd, 1H), 4.76 (dd, 1H) (AMX like, CH2ONO2), 5.28–
5.35 (m, 1H, CHONO2); 13C NMR (CD3OD): d 21.4,
29.1, 31.9, 62.2, 71.3, 79.2. MS: m/z 225 (M+H)+.
5.2.3. 4-(5,6-Bisnitrooxyhexyloxy)-5-(1,2-dihydroxyeth-
yl)-3-hydroxy-5H-furan-2-one (4c). The resulting oil
was purified by flash chromatography (eluent 95/5
CH2Cl2/MeOH) to give the title compound as a pale yel-
1
low oil, 37%. H NMR (CD3OD): d 1.57–1.63 (m, 2H),
1.79–1.85 (m, 4H) ((O2NO)CH(CH2)3CH2O), 3.66 (d,
2H, J = 7.0 Hz, CH(OH)CH2(OH)), 3.82–3.85 (m, 1H,
CH(OH)CH2(OH)), 4.52–4.63 (m, 3H, (O2NO)CHH-
CH(ONO2)(CH2)3CH2O), 4.79 (d, 1H, J = 1.5 Hz,
CHOC=O), 4.90–4.94 (m, 1H, (O2NO)CHH (over-
lapped with OH signal)), 5.41–5.44 (m, 1H, CH(O-
NO2)); 13C NMR (CD3OD): d 22.2, 29.7, 30.4, 63.4,
70.6, 72.1, 72.9, 76.7, 81.1, 120.6, 151.9, 173.1. MS m/z
383 (M+H)+. Anal. (C12H18N2O12 Æ 1.25 H2O) C, H,
N:: C calcd 35.61, found 36.21, H calcd 5.10, found
4.88, N calcd 6.92, found 6.31.
1
Lit. data:21 pale yellow oil. H NMR (500 MHz, Ace-
tone-d6): d 1.51–1.58 (m, 4H), 1.80–1.88 (m, 2H), 3.50
(t, 1H), 3.55 (d, 2H), 4.72 (dd, 1H), 5.00 (dd, 1H),
5.47–5.53 (m, 1H).
5.2. General procedure for the preparation of products
4a–f
5.2.4. 4-[2-(1,2-Dihydroxyethyl)-4-hydroxy-5-oxo-2,5-
dihydrofuran-3-yloxymethyl]-furoxan-3-carboxylic acid
amide (4d). The resulting oil was purified by flash chroma-
tography (eluent 95/5 CH2Cl2/MeOH) to give the title
compound as a white solid, mp 186–187 ꢁC (MeOH),
To a stirred solution of PPh3 (1.2 eq.) in THF dry under
positive N2 pressure, DIAD (1.2 eq.) was added drop-
wise at ꢀ78 ꢁC. After 10 min, the precipitate of Mitsun-
obu betaine was formed. Stirring was continued for
10 min, then a solution of ASA (1.0 eq.) in DMF dry
was added to the above mixture at ꢀ78 ꢁC. The cooling
bath was removed and a solution of the corresponding
alcohol (1.0 eq.) in THF dry was added. The mixture
was allowed to reach rt and was stirred overnight. The
reaction mixture was concentrated, diluted with EtOAc
and the organic phase was washed with H2O, brine,
dried, and evaporated. The resulting oil was purified
by flash chromatography with eluents indicated.
1
17%. H NMR (DMSO-d6 + CD3OD): d 3.63 (d, 2H,
J = 6.3 Hz, CH(OH)CH2(OH)), 3.86 (t, 1H, J = 6.9 Hz,
CH(OH)CH2(OH)), 4.87 (s, 1H, CHOC@O), 5.77, 5.87
(dd, 2H, J = 14.3 Hz, CH2O); 13C NMR (DMSO-d6):
61.5, 63.4, 68.4, 74.4, 110.1, 120.4, 149.1, 155.0, 155.6,
169.9. MS m/z 318 (M+H)+. Anal. (C10H11N3O9 Æ 0.5
H2O) C, H, N: C calcd 36.82, found 36.74, H calcd 3.71,
found 3.49, N calcd 12.88, found 13.25.
5.2.5. 5-(1,2-Dihydroxyethyl)-3-hydroxy-4-[3-(3-phenylf-
uroxan-4-yloxy)propoxy]-5H-furan-2-one
(4e).
The
5.2.1. 5-(1,2-Dihydroxyethyl)-3-hydroxy-4-(3-nitrooxy-
propoxy)-5H-furan-2-one (4a). The resulting oil was
purified first by flash chromatography (eluent 95/5
resulting oil was purified by flash chromatography (elu-
ent gradient from 9/1 to 7/3 CH2Cl2/EtOAc then 95/5
CH2Cl2/MeOH) to give the title compound as a color-

5204
C. Cena et al. / Bioorg. Med. Chem. 16 (2008) 5199–5206
less oil which turns into a white foam during desiccation,
39%. 1H NMR (CD3OD): d 2.34–2.42 (m, 2H,
CH2CH2CH2), 3.63 (d, 2H, J = 6.0 Hz, CH(OH)-
CH2(OH)), 3.85 (t, 1H, J = 7.2 Hz, CH(OH)CH2(OH)),
4.65–4.78 (m, 5H, CH2CH2CH2 + CHOC@O), 7.49–
7.57 (m, 3H), 8.11–8.13 (m, 2H) (C6H5); 13C NMR
(CD3OD): d 30.3, 63.5, 68.7, 69.1, 70.6, 76.7, 108.8,
121.0, 123.9, 127.4, 130.0, 131.6, 151.4, 163.8, 172.9.
MS m/z 395 (M+H)+. Anal. (C17H18N2O9 Æ 0.75 H2O)
C, H, N: C calcd 50.06, found 50.14, H calcd 4.82, found
4.58, N calcd 6.87, found 6.57.
(ii) To a stirred solution of the protected 2-O substituted
ASA derivatives (0.80–1.0 g) in MeOH (10 mL), 4 M
HCl (2 mL) was added and the reaction was stirred at
rt until complete (TLC control). The reaction mixture
was concentrated, diluted with H2O and extracted with
EtOAc. The organic phase was washed with brine, dried
and evaporated. The title products were purified as
indicated.
5.4.1. 5-(1,2-Dihydroxyethyl)-4-hydroxy-3-(3-nitrooxy-
propoxy)-5H-furan-2-one (7a). (i) The intermediate pro-
tected compound was purified by flash chromatography
(eluent 98/2 CH2Cl2/ EtOAc), 76%. (ii) The resulting oil
was purified by flash chromatography (eluent EtOAc)
and lyophilized to give the title compound as colorless
oil, 81%. 1H NMR (CD3OD): d 2.04–2.13 (m, 2H,
CH2CH2CH2), 3.67 (d, 2H, J = 6.9 Hz, CH(OH)-
CH2(OH)), 3.92 (t, 1H, J = 6.3 Hz, CH(OH)CH2(OH)),
4.04–4.12 (m, 2H), 4.67 (t, 2H, J = 6.6 Hz) (CH2CH2
CH2), 4.86 (s, 1H, CHOC@O (overlapped with OH sig-
nal)); 13C NMR (CD3OD): d 28.3, 63.4, 69.1, 70.5, 71.4,
76.9, 121.8, 161.7, 172.8. MS: m/z 280 (M+H)+. Anal.
(C9H13NO9 Æ 0.75 H2O) C, H, N: C calcd 36.93, found
36.50, H calcd 4.99, found 4.51, N calcd 4.78, found
4.67.
5.2.6. 4-[3-(3-Phenylsulfonylfuroxan-4-yloxy)propoxy]-5-
(1,2-dihydroxyethyl)-3-hydroxy-5H-furan-2-one (4f). The
resulting oil was purified by flash chromatography
(eluent gradient from 98/2 to 95/5 CH2Cl2/MeOH) to
give the title compound as a white solid, mp 144–
147 ꢁC (dec.), 17%. 1H NMR (CD3OD): d 2.26–2.34
(m, 2H, CH2CH2CH2), 3.64 (d, 2H, J = 6.9 Hz,
CH(OH)CH2(OH)), 3.82–3.87 (m, 1H, CH(OH)-
CH2(OH)), 4.58 (t, 2H), 4.68 (t, 2H) (CH2CH2CH2),
4.81 (d, 1H, J = 1.3 Hz CHOC@O), 7.67–7.72 (m,
2H), 7.80–7.85 (m, 1H), 8.04–8.06 (m, 2H) (C6H5);
13C NMR (CD3OD): d 30.1, 63.4, 68.7, 69.0, 70.6,
76.7, 111.8, 121.0, 129.6, 131.0, 137.0, 139.5, 151.4,
160.4, 172.9. MS m/z 459 (M+H)+. Anal.
(C17H18N2O11S Æ 0.5 H2O) C, H, N: C calcd 43.68,
found 43.91, H calcd 4.10, found 3.89, N calcd 5.99,
found 5.78.
5.4.2. 5-(1,2-Dihydroxyethyl)-4-hydroxy-3-(6-nitrooxy-
hexyloxy)-5H-furan-2-one (7b). (i) The intermediate pro-
tected compound was purified by flash chromatography
(eluent 99/1 CH2Cl2/ EtOAc), 87%. (ii) The resulting oil
was dissolved in H2O and water was washed twice with
Et2O and then extracted with EtOAc. The organic solvent
was removed to give colorless oil which was lyophilized to
give the title product asan extremely hydroscopic pale yel-
low solid, 45%. 1H NMR (CD3OD): d 1.40–1.53 (m, 4H),
1.76–1.78 (m, 4H) (CH2(CH2)4CH2), 3.67 (d, 2H,
J = 6.9 Hz, CH(OH)CH2(OH)), 3.89–4.05 (m, 3H), 4.49
(t, 2H, J = 6.6 Hz) (CH(OH)CH2(OH), CH2(CH2)4CH2),
4.84 (d, 1H, J = 1.8 Hz, CHOC@O (overlapped with OH
signal)); 13C NMR (CD3OD): d 26.3, 26.5, 27.4, 29.5,
63.5, 70.5, 72.9, 74.7, 76.8, 122.1, 161.2, 173.0. MS m/z
322 (M+H)+. Anal. (C12H19NO9 Æ 0.5 H2O) C, H, N: C
calcd 43.64, found 43.83, H calcd 6.10, found 6.02, N
calcd 4.24, found 4.32.
5.3. Alternative procedure for the preparation of 4d
To a stirred solution of ASA (0.20 g, 1.1 mmol) in
DMSO dry (10 mL) under positive N2 pressure, NaH-
CO3 (0.14 g, 1.7 mmol) was added at rt and the reaction
mixture was stirred for 1 h. Then 5 (0.22 g, 1 mmol) was
added and the reaction mixture was heated at 50 ꢁC
overnight. The next day the reaction mixture was
poured into H2O (30 mL) and extracted with EtOAc
(3 · 20 mL). Organic phases were combined, washed
with brine, dried and evaporated. The resulting solid
was crystallized twice from MeOH to give the title com-
pound as a white solid, 30%.
5.4. General procedure for the preparation of products
7a–c
5.4.3. 5-(1,2-Dihydroxyethyl)-4-hydroxy-3-[3-(3-phenylf-
uroxan-4-yloxy)propoxy]-5H-furan-2-one (7c). (i) The
intermediate protected compound was purified by flash
chromatography (eluent 98/2 CH2Cl2/EtOAc), 80%. (ii)
The resulting oil was dissolved in H2O and the water
was washed twice with CH2Cl2 and then extracted with
EtOAc. The organic solvent was removed to give a col-
orless oil which was lyophilized to give the title com-
(i) To a stirred solution of PPh3 (1.2 eq.) in THF dry un-
der positive N2 pressure, DIAD (1.2 eq.) was added
dropwise at ꢀ15 ꢁC. After 15 min, a precipitate of Mits-
unobu betaine was formed. Stirring was continued for
10 min, then 6 (1.1 eq.) was added, followed by an addi-
tion of the corresponding alcohol (1.0 eq.) (solid or in
THF dry solution). The mixture was stirred at ꢀ15 ꢁC
for 30 min, then the cooling bath was removed, the reac-
tion was allowed to reach rt and was stirred for addi-
tional 2 h. Then the reaction mixture was
concentrated, diluted with EtOAc and the organic phase
washed with H2O, NaHCO3 sat. sol., brine, dried, and
evaporated. The resulting oil was purified by flash chro-
matography with the eluents indicated. Intermediate
protected products were immediately used for the depro-
tection reaction.
1
pound as a white solid, mp 153–156 ꢁC (dec.), 52%. H
NMR (CD3OD): d 2.24–2.32 (m, 2H, CH2CH2CH2),
3.67 (d, 2H, J = 6.9 Hz, CH(OH)CH2(OH)), 3.91 (t,
1H, J = 7.2 Hz, CH(OH)CH2(OH)), 4.22 (t, 2H,
J = 6.0 Hz), 4.69 (t, 2H, J = 6.0 Hz) (CH2CH2CH2),
4.84 (s, 1H, CHOC@O), 7.48–7.54 (m, 3H), 8.10–8.13
(m, 2H) (C6H5); 13C NMR (CD3OD): d 30.1, 63.6,
68.9, 69.3, 70.5, 76.9, 108.9, 122.0, 123.9, 127.0, 129.6,
131.6, 161.6, 163.8, 172.8. MS m/z 395 (M+H)+. Anal.

C. Cena et al. / Bioorg. Med. Chem. 16 (2008) 5199–5206
5205
(C17H18N2O9) C, H, N: C calcd 51.78, found 51.55, H
calcd 4.60, found 4.62, N calcd 7.10, found 7.18.
peroxidation was assessed by spectrophotometric
(543 nm) determination of the TBARS consisting
mainly of malondialdehyde (MDA), and TBARS con-
centrations (expressed in nmol/mg protein) were ob-
tained by interpolation with a MDA standard curve.
The antioxidant activity of tested compounds was eval-
uated as the % of inhibition of TBARS production with
respect to control samples, using the plateau values ob-
tained after 30 min of incubation. IC50 values were cal-
culated by nonlinear regression analysis of at least five
experiments.
5.5. Ionization and lipophilicity studies
Potentiometric titrations were performed using the
GLpKa apparatus (Sirius Analytical Instruments Ltd.,
Forrest Row, East Sussex, UK). Ionization constants
were determined by at least four separate titrations for
each compound: different aqueous solutions (ionic
strength adjusted to 0.15 M with KCl) of the com-
pounds (20 mL, about 1 mM) were initially acidified to
pH 1.8 with 0.5 N HCl; the solutions were then titrated
with standardized 0.5 N KOH to pH 10.5. The low
aqueous solubility of 4e and 4f required titrations in
the presence of methanol as a cosolvent: at least five dif-
ferent hydro-organic solutions (ionic strength adjusted
to 0.15 M with KCl) of the compounds (20 mL, about
1 mM in 15–50 wt% methanol) were initially acidified
to pH 1.8 with 0.5 N HCl; the solutions were then ti-
trated with standardized 0.5 N KOH to pH 10.5. The
initial estimates of the psKa values (the apparent ioniza-
tion constants in the water–methanol mixtures) were ob-
tained and aqueous pKa values were determined by
extrapolation to zero content of the cosolvent according
to the Yasuda-Shedlovsky procedure.22 All the titrations
were performed under argon at 25.0 0.1 ꢁC.
5.7. Vasodilator activity
Thoracic aortas were isolated from male Wistar rats
weighing 180–200 g. As few animals aspossible were used.
The purposes and the protocols of our studies have been
approved by the Ministero della Salute, Rome, Italy.
The endothelium was removed and the vessels were heli-
cally cut: three strips were obtained from each aorta.
The tissues were mounted under 1.0 g tension in organ
baths containing 30 ml of Krebs-bicarbonate buffer with
the following composition (mM): NaCl 111.2, KCl 5.0,
CaCl2 2.5, MgSO4 1.2, KH2PO4 1.0, NaHCO3 12.0, glu-
cose 11.1, maintained at 37 ꢁC and gassed with 95% O2–
5% CO2 (pH 7.4). The aortic strips were allowed to equil-
ibrate for 120 min and then contracted with 1 lM L-phen-
ylephrine. When the response to the agonist reached a
plateau, cumulative concentrations of the vasodilating
agent were added. Results are expressed as EC50 SEM
(lM). The effects of 1 lM ODQ on relaxation were eval-
uated in separate series of experiments in which it was
added to the organ bath 5 minutes before the contraction.
Responses were recorded by an isometric transducer con-
nected to the MacLab System PowerLabꢂ. The addition
of the drug vehicle, DMSO, had no appreciable effect on
contraction level.
The apparent partition coefficients (log DpH) were mea-
sured by the shake-flask procedure at pH 1.0 and 7.4
(HCl and phosphate buffer solutions with ionic strength
adjusted to 0.15 M with KCl, respectively). n-Octanol
was added to the buffers and the two phases were mutu-
ally saturated by shaking for 4 h. The compounds were
solubilized in the buffered aqueous phase at a concentra-
tion of about 0.1 mM and an appropriate amount of n-
octanol was added. The two phases were shaken for
about 20 min, by which time the partitioning equilib-
rium of solutes was reached, and then centrifuged
(10,000 rpm, 10 min). The concentration of the solutes
in the aqueous phase was measured by UV spectropho-
tometer (UV-2501PC, Shimadzu) at kmax. For each
compound at least seven log D values at different pHs
were measured.
Acknowledgment
This work was supported by a MIUR Grant (COFIN
2005).
5.6. Antioxidant activity
References and notes
Microsomal membranes from male Wistar rats (180–
200 g) were prepared by differential centrifugation
(8000g, 20 min; 120,000g, 1 h) in a HEPES/Sucrose buf-
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in a Tris–HCl/KCl buffer (100 mM/150 mM, pH 7.4),
recentrifugation (120,000g, 1 h), and were stored at
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