Development of a new class of potential antiatherosclerosis agents: NO-donor antioxidants

Article abstract of DOI:10.1016/j.bmcl.2004.10.006

A new class of NO-donor phenol derivatives is described. The products were obtained by joining appropriate phenols with either nitrooxy or 3-phenylsulfonylfuroxan-4-yloxy moieties. All the compounds proved to inhibit the ferrous salt/ascorbate induced lip

Full text of DOI:10.1016/j.bmcl.2004.10.006

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5971–5974
Development of a new class of potential antiatherosclerosis
agents: NO-donor antioxidants
Clara Cena,a Donatella Boschi,a Gian Cesare Tron,b Kostantin Chegaev,a
Loretta Lazzarato,a Antonella Di Stilo,a Manuela Aragno,c
Roberta Frutteroa and Alberto Gascoa,*
aDipartimento di Scienza e Tecnologia del Farmaco, Via P. Giuria 9, I-10125 Torino, Italy
bDipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Via Bovio 6, I-28100 Novara, Italy
cDipartimento di Medicina e Oncologia Sperimentale, C.so Raffaello 30, I-10125 Torino, Italy
Received 18 June 2004; revised 1 September 2004; accepted 4 October 2004
Available online 22 October 2004
Abstract—A new class of NO-donor phenol derivatives is described. The products were obtained by joining appropriate phenols
with either nitrooxy or 3-phenylsulfonylfuroxan-4-yloxy moieties. All the compounds proved to inhibit the ferrous salt/ascorbate
induced lipidic peroxidation of membrane lipids of rat hepatocytes. They were also capable of dilating rat aorta strips precontracted
with phenylephrine.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
tures in which a number of selected antioxidants were
linked to NO-donor moieties through appropriate spac-
ers. Here we report the preliminary results of a study on
the capacity of inhibiting the ferrous salt/ascorbate in-
duced lipid peroxidation of membrane lipids of rat
hepatocytes and on the in vitro vasodilating properties
of a first series of such products. As NO-donor moieties,
we chose either the nitrooxy function present in simple
vasodilating nitric esters 1 (der.s 17, 20, 24) or the 3-phen-
ylsulfonylfuroxan-4-yloxy moiety present in 4-ethoxy-3-
phenylsulfonylfuroxan (CHF 2363) 2, a known NO-
dependent vasodilator4 (der.s 10, 11, 15). As antioxi-
dants, we considered phenols characterized by having
different O–H bond dissociation energy (BDE), which
is one of the factors that contribute to the antioxidant
capacity of the products: p-cresol (25, BDE,
86.2 0.6kcal/mol), 2,6-di-t-butyl-p-methylphenol (26,
BDE, 81.02 0.13kcal/mol) and 6-hydroxy-2,2,5,7,8-
pentamethylchroman, the phenol substructure present
in the vitamin E (27, BDE, 78.2 0.18kcal/mol).5
Atherosclerosis is the main cause of morbidity and mor-
tality in the western societies.1 This pathology is closely
correlated with endothelial dysfunction resulting from
an increase of plasma lipids, peroxidation of low-density
lipoproteins (LDLs) and an impairment of endothelial-
derived relaxing factor (EDRF, nitric oxide, NOÅ)-medi-
ated bioactions. Oxidative modifications of high levels
of LDLs following oxidative stress, lead to the form-
ation of foam cells, precursors of atherosclerotic plaques.
Experimental evidence supports the hypothesis that the
treatment of hypercholesterolemic animals and human
subjects with appropriate amounts of antioxidants can
reduce the LDL oxidation and the expression of athero-
sclerosis.1,2 In an atherosclerotic blood vessel, NOÅ bio-
actions are impaired by a number of reasons including a
possible decreased NOÅ production, an increased NOÅ
inactivation and a decrease in responsiveness of the tar-
get cells to NOÅ. By contrast, vasodilation to exogenous
sources of NOÅ seems to be preserved.3 On these bases
we have recently designed a large series of hybrid struc-
2. Chemistry
The preparation of the furoxan derivatives follows the
synthetic routes that are illustrated in Scheme 1.6 The
hydroxyl groups of the p-hydroxybenzaldehyde (3) and
of its 2,3-di-t-butyl derivative 4 were protected with
Keywords: Phenol antioxidants; Nitric oxide; NO donors; NO-donor
phenol antioxidants.
*
Corresponding author. Tel.: +39 011 6707670; fax: +39 011
6707286; e-mail: alberto.gasco@unito.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.006

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C. Cena et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5971–5974
R
OH
SO2Ph
N+
R
HO
HO
R
R
ONO2
N
O-
O
14
R = Alkyl
O
1
2 R = EtO
9 R = PhSO2
25 R = H
27
Br
16
26 R = t-Bu
vi
R
HO
i
HO
R
3 R = H
MEMO
H
OH
4 R = t-Bu
O
OH
O
12
O
O
O
vii, viii
21
i,ii
R
MEMO
R'O
R
vii
ONO2
5 R = H, R' = TBDMS
6 R = t-Bu, R' = MEM
OEt
O
OEt
OH
17
13
O
O
OH
iii, iv
iv
R
R
MEMO
R'O
R
MEMO
7 R = H, R' = TBDMS
8 R = t-Bu, R' = MEM
O
OH
O
OH
O
22
OH
14
v, vi
18
HO
R
v, vi
ii
ii, iii
SO2Ph
O-
HO
O
N
N+
SO2Ph
O-
O
10 R = H
O
O
11 R = t-Bu
OBoc
N+
MEMO
N
15
O
O
OTs
O
Scheme 1. Reagents and conditions: (i) 3, TBDMSCl, NaH, THF; 4,
MEMCl, di-iso-propyl ethylamine, C2H4Cl2, reflux; (ii) (EtO)2POCH2-
COOEt/t-BuOK, THF, À78°C; (iii) H2, Pd/C, EtOH; (iv) LiAlH4,
THF; (v) 9, NaH, THF; (vi) HCl/dioxane to obtain 10, TFA/CH2Cl2
to obtain 11 and 15; (vii) p-TSA, EtOH, reflux; (viii) MEMCl, NaH,
THF.
23
OTs
19
iv, v
iv, v
OH
t-butyldimethylsilyl chloride (TBDMSCl) and 2-meth-
oxyethoxymethyl chloride (MEMCl), respectively. The
resulting products were subjected to the modified Wittig
reaction in the presence of the phosphonacetic acid tri-
ethyl ester in basic medium to obtain the a,b-unsatu-
rated esters 5 and 6, respectively. Both the products
were transformed into the corresponding saturated
alcohols 7 and 8 by reduction first with H2, Pd/C and
then with LiAlH4. Selective nucleophilic displacement
of the 4-phenylsulfonyl group of the 3,4-bis(phenylsulfon-
yl)furoxan (9) by the intermediates 7 and 8 in dry THF
in the presence of NaH followed by acid deprotection of
the phenol functions afforded the final products 10, 11.
The starting material for the preparation of the furoxan
15 was the commercial acid 12. This product was treated
with ethanol in the presence of p-toluenesulfonic acid (p-
TSA) to obtain the corresponding ester that was MEM
protected on the hydroxy group to afford 13. This latter
compound was reduced with LiAlH4 in dry THF to the
corresponding alcohol 14 and then left to react with 9 in
basic medium to give, after deprotection of the phenol
function, the final product 15.
HO
O
ONO2
O
24
ONO2
20
Scheme 2. Reagents and conditions: (i) AgNO3, CH3CN, 60°C; (ii)
TsCl, TEA, CH2Cl2, rt; (iii) (t-Boc)2O, DMAP, CH2Cl2, rt; (iv)
Bu4NNO3, benzene, reflux; (v) TFA, CH2Cl2, rt; (vi) allyl bromide,
NaH, DMF, rt; (vii) a. 9-BBN, THF, rt, b. H2O2, NaOAc, 0°C.
18 was treated with p-toluenesulfonyl chloride (TsCl)
in the presence of triethylamine (TEA). The correspond-
ing tosylate, obtained in fair yield, was later protected
on the phenol group with di-t-butyl dicarbonate
(t-Boc)2O, in the presence of 4-N,N-dimethylaminopyr-
idine (DMAP) to give 19. This latter product was treated
in refluxing benzene with tetrabutylammonium nitrate
(Bu4NNO3) and then at room temperature with a
CH2Cl2 solution of trifluoroacetic acid to afford the final
compound 20. The MEM protected 6-hydroxychroman
structure 14 was used as a starting material for the prep-
aration of 24. It was transformed into 21 by reaction
with allyl bromide and NaH in N,N-dimethylformamide
(DMF). This latter product was left to react first with
9-borabicyclo[3.3.1]nonane (9-BBN) in THF and then
with hydrogen peroxide and sodium acetate to give the
Nitrooxy derivatives were synthesized according to the
pathway reported in Scheme 2.7 The simple nitrooxy
derivative 17 was obtained by the action of AgNO3 on
4-(3-bromopropyl)phenol (16) in acetonitrile at 60°C.
To prepare the final product 20, the alcoholic group of

C. Cena et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5971–5974
5973
propanol derivative 22. The corresponding tosylate 23,
obtained under the same conditions used to prepare
the tosylate of 18, was transformed into the final nitro-
oxyderivative 24 following procedures similar to those
used to transform 19 into 20.
The potential antioxidant activity of furoxan derivatives
is an interesting aspect of the furoxan chemistry that is
worthy of additional investigation.
All the hybrid products behaved as potent in vitro vaso-
dilators. They were capable of dilating in a dose depend-
ent manner rat aorta strips precontracted with
phenylephrine.9 One example is reported in Figure 2.
3. Results and discussion
All the products described in the present work were as-
sessed as inhibitors of ferrous salt/ascorbate induced
lipidic peroxidation of membrane lipids of rat hepato-
cytes.8 The progress of the peroxidation was followed
by visible spectroscopy detection of 2-thiobarbituric
acid reactive substances (TBARS), which are the final
metabolites of the autooxidation. All the products were
able to inhibit the lipid oxidation in a dose dependent
manner. One example is reported in Figure 1.
The vasodilating potencies (EC50) of all the compounds
are collected in Table 1. This action is cyclic GMP
(cGMP) dependent since a considerable decrease in the
potency was observed when the experiments were re-
peated in the presence of 1H-[1,2,4]oxadiazolo[4,3-a]qui-
noxalin-1-one (ODQ), a well known inhibitor of the
sGC.10 A comparison between the IC50 and EC50 values
shows that in this series of products the vasodilating ac-
tion prevails over the antioxidant one. Also the nitrooxy
derivatives proved to be dose dependent antioxidants
with a potency similar to that of the reference phenols
(Table 1). They too display in vitro sGC dependent
vasodilating activity but it is less potent than the corre-
sponding furoxan derivativesÕ activity (Table 1). Work is
in progress in order to obtain products with better bal-
anced antioxidant and vasodilating activities. All the
products reported in this preliminary paper could be
The potencies as antioxidants of these compounds (IC50)
are collected in Table 1 together with those of the parent
phenols. The hybrid products show potencies near to
those of the reference phenols, with the only partial
exception of the derivative 10. It is noteworthy that also
the NO-donor furoxan 2 displays an antioxidant action,
which is close to that of 25.
20
20
10
control
0.25µm
10
0.50µm
0
0.75µm
1.0µm
2.5µm
-10.0 -7.5
-5.0
-2.5
log conc (M)
0
0
10
20
time (min)
30
Figure 2. Concentration–response curves for vasodilating activity of
compound 15 in the absence (open circle) and in the presence (solid
circle) of ODQ.
Figure 1. Effect of 15 on time course of lipid peroxidation.
Table 1. Antioxidant and vasodilating activity of the reference NO-donors 1–2, of the hybrids 10, 11, 15, 17, 20, 24 and of the parent phenols 25–27
Compd
Antioxidant activity
Vasodilating activity
IC50 (95% CL), lMa
EC50 SE, lMa
41
+ODQ
EC50 SE, lM
b
c
1 (R = n-pr)
2
6
110 (98–122)
47 (45–48)
0.012 0.002
0.012 0.001
0.11 0.03
0.044 0.004
1.0 0.2
1.2 0.2
0.36 0.09
4.8 0.5
10
11
15
17
20
24
25
26
27
2.0 (1.9–2.0)
0.49 (0.48–0.50)
143 (133–153)
2.0 (1.9–2.1)
0.15 (0.15–0.16)
290 (260–324)
1.7 (1.6–1.9)
0.17 (0.16–0.17)
0.67 0.09
c
c
40
1.2 0.1
1
10
1
—
—
—
—
—
—
a Values are means of at least five experiments.
b Derivative 1 was inactive when tested at 1mM.
c The products 1, 17 and 20 induced, respectively, 4.6 0.6%, 38 9%, 15 5% of vasodilation when tested at 100lM.

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C. Cena et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5971–5974
m/z (EI): 309 (M+); 1H NMR (CDCl3): d 6.96 (s, 2H, Ph),
5.10 (s, 1H, OH), 4.47 (t, 2H, J = 6.5Hz, CH2O), 2.65 (t,
2H, J = 7.7Hz, CH2Ph), 2.06–2.00 (m, 2H, CH2CH2CH2O),
1.43 (s, 18H, t-Bu). Anal. (C17H27NO4) C, H, N. Com-
pound 24: oil; m/z (EI): 339 (M+); 1H NMR (CDCl3): d 4.57
(t, 2H, J = 6.5Hz, CH2ONO2), 4.19 (s, 1H, OH), 3.63–3.58
(m, 2H, OCH2CH2), 3.47 (1H, d, AB system, J = 9.8Hz,
CHaHbO), 3.41(1H, d, AB system, J = 9.8Hz, CHaHbO),
of value as potential antiatherosclerotic agents and they
are worthy of in vivo further investigations.
Acknowledgements
This study was supported by a MIUR grant.
2.61(m, 2H, 4-CH -chromane), 2.15 (s, 3H), 2.11 (s, 3H),
2
2.09 (s, 3H) (5,7,8-CH3-chromane), 2.03–1.92 (m, 3H) (3-
CHH-chromane and CH2CH2ONO2), 1.79–1.72 (m, 1H, 3-
CHH-chromane), 1.57 (s, 3H, 2-CH3-chromane). Anal.
(C17H25NO6) C, H, N.
References and notes
1. Eberhardt, M. K. Reactive Oxygen Metabolites; CRC:
BocaRaton, 2000.
2. Keaney, J. F.; Vita, J. A. Prog. Cardiovasc. Dis. 1995, 38,
129.
8. Microsomal membranes from male Wistar rats (200–250g)
were prepared by differential centrifugation (8000g,
20min; 120,000g, 1h) in
a HEPES/sucrose buffer
3. Dillon, G. A.; Vita, J. A. In Nitric Oxide and the
Cardiovascular System; Loscalzo, J., Vita, A. J., Eds.;
Humana: Totowa, 2000; pp 207–226.
4. Civelli, M.; Giossi, M.; Caruso, P.; Razzetti, R.; Bergam-
aschi, M.; Bongrani, S.; Gasco, A. Br. J. Pharmacol. 1996,
118, 923–928.
(10mM, 250mM, pH = 7.4) and stored at À80°C. Incu-
bation was performed at 37°C in a Tris–HCl/KCl buffer
(100mM, 150mM, pH = 7.4) containing microsomal
membranes (2mg prot/mL), ascorbic acid (100lM) and
DMSO solutions of the tested compounds. Addition of
DMSO alone (maximal amount 5%) did not change
significantly the extent of peroxidation in the control
experiments. Lipid peroxidation was initiated by adding
FeSO4 2.5lM. Aliquots were taken from the incubation
mixture at 5, 15 and 30min and treated with trichloro-
acetic acid (TCA) 10% p/v. Lipid peroxidation was
assessed by spectrophotometric (543nm) determination
of the TBARS consisting mainly of malondialdehyde
(MDA), and TBARS concentrations (expressed in nmol/
mg protein) were obtained by interpolation with a MDA
standard curve.11 The antioxidant activity of tested
compounds was evaluated as the % of inhibition of
TBARS production with respect to control samples, using
the plateau values obtained after 30min of incubation.
IC50 values were calculated by non linear regression
analysis.
5. Luccarini, M.; Pedrielli, P.; Pedulli, G. F.; Cabiddu, S.;
Fattuoni, C. J. Org. Chem. 1996, 61, 9259–9263.
6. Compound 10: mp 92–93°C (EtOH); m/z (EI): 376 (M+);
1H NMR (DMSO-d6): d 9.19 (s, 1H, OH), 8.05–8.03 (m,
2H), 7.93–7.88 (m, 1H), 7.78–7.73 (m, 2H) (PhSO2), 7.00–
6.98 (m, 2H), 6.70–6.67 (m, 2H) (PhOH), 4.34 (t, 2H,
J = 6.2Hz, CH2O), 2.57 (t, 2H, J = 7.5Hz, CH2Ph), 2.00
(m, 2H, CH2CH2CH2). Anal. (C19H20N2O8S) C, H, N.
Compound 11: mp 110–111°C (EtOH); m/z (EI): 488 (M+);
1H NMR (DMSO-d6): d 8.05–8.02 (m, 2H), 7.89–7.86 (m,
1H), 7.77–7.72 (m, 2H) (PhSO2), 6.86 (s, 2H, PhOH), 6.74
(s, 1H, OH), 4.33 (t, 2H, J = 5.9Hz, CH2O), 2.57 (t, 2H,
J = 7.2Hz, CH2Ph), 1.99 (m, 2H, CH2CH2CH2), 1.33 (s,
18H, t-Bu). Anal. (C25H32N2O6S) C, H, N. Compound 15:
mp 68–72°C dec (MeOH/H2O); m/z (EI): 460 (M+); 1H
NMR (CDCl3): d 8.00–7.98 (m, 2H), 7.72–7.68 (m, 1H),
7.53–7.48 (m, 2H) (PhSO2), 4.46 (1H, d, AB system,
J = 10.4Hz, CHaHbO), 4.40 (1H, d, AB system,
J = 10.4Hz, CHaHbO), 2.71(m, 2H, 4-CH 2-chromane),
1.94–1.87 (m, 1H), 1.28–1.22 (m, 1H) (3-CH2-chromane),
2.16 (s, 3H), 2.14 (s, 3H), 2.02 (s, 3H) (5,7,8-CH3-
chromane), 1.41 (s, 3H, 2-CH3-chromane). Anal.
(C22H24N2O7S) C, H, N.
9. The experiments were assessed according to the procedure
described in J. Med. Chem. 1997, 40, 463–469 with little
modification: the aortic strips were contracted with 1lM
L-phenylephrine; the effect of 1lM ODQ on relaxation
was evaluated in a separate series of experiments in which
it was added to the bath at least 5min before the
contraction; responses were recorded by isometric trans-
ducer connected to the MacLab System PowerLab.
10. Schrammel, A.; Behrends, S.; Schmidt, K.; Koesling, D.;
Mayer, B. Mol. Pharmacol. 1996, 50, 1–5.
7. Compound 17: oil; m/z (EI): 197 (M+); 1H NMR (CDCl3): d
7.05–7.02 (m, 2H, Ph), 6.80–6.78 (m, 2H, Ph), 5.92 (br, 1H,
OH), 4.42 (t, 2H, J = 6.5Hz, CH2O), 2.65 (t, 2H,
J = 8.0Hz, CH2Ph), 2.04–1.95 (m, 2H, CH2CH2CH2O).
Anal. (C9H11NO4) C, H, N. Compound 20: mp 78–79°C;
11. Mastrocola, R.; Aragno, M.; Betteto, S.; Brignardello, E.;
Catalano, M. G.; Danni, O.; Boccuzzi, G. Life Sci. 2003,
73, 289–299.