Design, synthesis, and biological characterization of potential antiatherogenic nitric oxide releasing tocopherol analogs

Article abstract of DOI:10.1016/j.bmc.2005.05.060

Synthesis and biological characterization of a series of α-tocopherol analogs with NO-releasing capacity are reported. The selected NO-donor moieties were nitrooxy and furoxan. All products were tested for their in vitro NO-releasing capacities, vasodilat

Full text of DOI:10.1016/j.bmc.2005.05.060

Bioorganic & Medicinal Chemistry 13 (2005) 5787–5796
Design, synthesis, and biological characterization of potential
antiatherogenic nitric oxide releasing tocopherol analogsq
a,d,
b,d,
´
´
Carlos Batthyany,
Gloria V. Lopez,
´
Fabiana Blanco,c,d Horacio Botti,b,d
´
Andres Trostchansky, Eduardo Migliaro,c,d Rafael Radi,b,d Mercedes Gonzalez,
b,d
a
Hugo Cerecettoa,* and Homero Rubbob,d,
*
a
´ ´
Departamento de Quımica Organica, Facultad de Ciencias-Facultad de Quımica, Universidad de la Republica, Montevideo, Uruguay
b
´
Departamento de Bioquımica, Universidad de la Republica, Montevideo, Uruguay
´
´
Departamento de Fisiologıa, Universidad de la Republica, Montevideo, Uruguay
´
c
´
Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay
´
d
´
Received 16 April 2005; revised 25 May 2005; accepted 26 May 2005
Available online 1 July 2005
Abstract—Synthesis and biological characterization of a series of a-tocopherol analogs with NO-releasing capacity are reported.
The selected NO-donor moieties were nitrooxy and furoxan. All products were tested for their in vitro NO-releasing capacities,
vasodilating properties, and antiplatelet activity. They were also capable of preventing LDL oxidation.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
idant trails,6 emphasizing the importance of developing
new drugs with improving antioxidant capacities in vivo.
Human LDL contains a number of antioxidants that
inhibit lipid oxidation, a-tocopherol (1, vitamin E,
Chart 1) being the most abundant (–6 a-tocopherol mol-
ecules per LDL particle). a-Tocopherol is selectively tar-
geted into LDL during its metabolism and as a way to
its delivery to all cells, due to the action of the a-tocoph-
erol-transfer protein.14,15 Besides, nitric oxide (ÅNO) is a
free radical species that has strong biological antioxi-
dant actions.16 In fact, it has been previously shown that
ÅNO inhibits LDL oxidation by scavenging of lipid
Atherosclerosis is a complex disease of diverse etiology,
where oxidation, increased deposition, and altered
metabolism of lipoproteins are key events associated
with lesion development.1,2 Lipid oxidation was first
proposed as an early step in atherogenesis, since modifi-
cation of low density lipoprotein (LDL) by cells or other
mechanisms could cause it to become cytotoxic.3–5 The
oxidation hypothesis suggests that antioxidant supple-
mentation might prevent or retard the development of
the atheroma plaque, improving patients prognosis.6
Although the oxidation hypothesis of atherosclerosis
has received considerable experimental support, recent
negative results from large scale supplementation studies
using antioxidant vitamins have questioned this idea.7–13
The oxidative hypothesis of atherosclerosis is not neces-
sarily disproved by the failure of these particular antiox-
Å
propagatory radicals.17–22 Moreover, NO is able to dif-
fuse into LDL and that makes it a potentially more
effective lipid antioxidant than a-tocopherol.23–29 There-
fore, hybrid molecules combining the vitamin E struc-
Å
Å
ture and NO releasing moieties, to target NO delivery
in vivo specifically into LDL, should be a possible ther-
apeutic strategy to protect LDL from oxidative modifi-
cations, contributing to the treatment of atherosclerosis.
Furoxans (1,2,5-oxadiazole N-oxide derivatives), organ-
ic nitrates, thionitrites, and nonoates, represent impor-
tant classes of NO donors.30,31 Organic nitrates
(RONO2) are the oldest class of NO donors that have
been clinically applied. Also, it is well known that furox-
Keywords: NO donor; Vitamin E; Antioxidant; LDL oxidation.
q
Part of this research is presented in the Uruguayan patent of
´ ´ ´
invention: Gonzalez, M., Lopez, G.V., Cerecetto, H., Batthyany, C,
´
Radi, R, Rubbo, H. UR Patent No. 28445, 2004: Analogos de
´
Tocoferol Dadores de Oxido Nıtrico.
´
*
Corresponding authors. Tel.: +598 2 5258516x216; fax: +598 2 525 07
49; (H.C.); tel.: +598 2 9249561; fax: +598 2 9249563 (H.R.); e-mail
addresses: hcerecet@fq.edu.uy; hrubbo@fmed.edu.uy
These authors contributed equally to this article.
Å
ans are able to release NO at physiological pH, in the
presence of thiol cofactors.32,33 Herein, we describe the
synthesis and preliminary biological characterization
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.060

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G. V. Lopez et al. / Bioorg. Med. Chem. 13 (2005) 5787–5796
5788
Chart 1. Chemical structure of a-tocopherol and TroloxÒ, and general structure of the designed compounds; tetramethylchroman system is linked to
the NO releasing moieties by adequate functionalities.
Å
of novel tocopherol analogs—ÅNO donors obtained by
coupling, through appropriate spacers, either furoxan
or nitrooxy groups with a-tocopherol or its analog,
trolox (Chart 1). These hybrid compounds release
ÅNO, due to the presence of a furoxan or nitrooxy sub-
structure, inhibiting platelet aggregation and exhibiting
vasorelaxation properties. Moreover, they effectively
protect LDL from oxidation, combining the tocopherol
substructure with affinity to LDL and antioxidant prop-
tyl chloride in the presence of pyridine to obtain the cor-
responding ester 3. This latter compound was treated
with AgNO3 in acetonitrile at 80 °C to afford the final
product 4 in a moderate yield. To prepare compound
6, the a-tocopherol derivative 5 was prepared as be-
fore.34 This latter compound was transformed into the
final nitrooxy derivative 6 following the same procedure
used to transform 3 into 4. The simple nitrooxy deriva-
tive, 8,35 was synthesized by direct dicyclohexylcarbodii-
mide (DCC) promoted esterification of 1 as described in
Scheme 1. The starting material for the preparation of
the nitrooxy derivatives 11 and 13 (Scheme 2) was the
acetate derivative of the commercial acid 2 (Trolox,
Chart 1). The final product 11 was obtained by treating
9 with 3-(nitrooxy)propanol (10) in presence of DCC.
To obtain the final product 13, compound 9 was first
transformed into the corresponding acyl chloride, which
was directly treated with 2-chloroethylamine to afford
the corresponding amide 12. This latter product was
treated with AgNO3 in acetonitrile at 80 °C to afford 13.
Å
erties of NO-donor.
2. Results and discussion
2.1. Chemistry
The preparation of the organic nitrate derivatives fol-
lows the synthetic routes that are illustrated in Schemes
1 and 2. Compounds 4, 6, and 8 were prepared from a-
tocopherol. This reactant was treated with 2-chloroace-
Scheme 1. Synthesis of a-tocopherol analog—nitrooxy derivatives 4, 6, and 8. Reagents and conditions: (a) 2-Chloroacetyl chloride, pyridine, toluene,
15 min; (b) AgNO3, CH3CN, 80 °C, 35 h; (c) acetic anhydride, pyridine, rt, 3 days; (d) HgO, Br2, dry CC14, rt, 3 h; (e) DCC, CH2C12, 40 °C, 24 h.

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G. V. Lopez et al. / Bioorg. Med. Chem. 13 (2005) 5787–5796
5789
Scheme 2. Synthesis of a-tocopherol analog—nitrooxy derivatives 11 and 13. Reagents and conditions: (a) Acetic anhydride, pyridine, rt, 2 h; (b)
DCC, CH2C12, reflux, 12 h; (c) (i) 9, SOC12, DMF, toluene, reflux, 90 min; (ii) 2-chloroethylamine hydrochloride, Et3N, CH2C12, rt; (d) AgNO3,
CH3CN, 80 °C, 35 h.
The preparation of furoxan derivatives follows the
synthetic routes that are illustrated in Schemes 3 and
4. a-Tocopherol (1) was treated with either 3,4-bis(phen-
ylsulfonyl)furoxan, 14, or 3-chloromethyl-4-phenylfuro-
xan, 17, yielding compounds 15, 16, and 18, respectively
(Scheme 3). In the case of the reaction of 1 with furoxan
14, the expected product (Fig. 1) was not obtained even
in mild conditions.
Scheme 3. Synthesis of a-tocopherol analog—furoxan derivative 18. Reagents and conditions: (a) K2CO3 (1.2 eq.), 18:crown:6, THF, rt, 24 h;
(b) K2CO3 (0.5 eq.), 18:crown:6, THF, reflux, 9 h.
Scheme 4. Synthesis of tocopherol analog—foroxan derivatives 22 and 24, and tocopherol analog—forazan derivative 21. Reagents and conditions:
(a) (i) 9, SOCl2, DMF, toluene, reflux, 90 min; (ii) 19, 20, or 23, Et3N, CH2C12, rt.

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G. V. Lopez et al. / Bioorg. Med. Chem. 13 (2005) 5787–5796
5790
Table 1. ÅNO releasing rate of the tested compounds
Compound
[ÅNO]releasea,b(kNO releasing rate 10ꢀ3 (minꢀ1))
c
4
6
—
c
—
c
8
—
c
—
Figure 1. Expected product of the reaction between furoxan 14 and
a-tocopherol, 1.
11
13
14
17
19
20
21
22
23
24
c
—
3.7 0.5
c
—
c
Indeed (6-hydroxy-2,7,8-trimethyl-2-(4,8,12-trimethyl-
tridecyl)chroman-5-yl)methyl benzenesulfinate, 15,36
was generated as the main product. a-Tocopheryl qui-
none, 16, was also isolated as the result of oxidation pro-
moted by furoxan 14. Compound 15 could be obtained
by the reaction between highest reactive 5a-methyl
group (Mills–Nixon effect)37 of the ortho-quinone met-
hide I with the nucleophile PhSO2ꢀ (benzenesulfonic an-
ion) resulted from furoxan 14 decomposition (Fig. 2).38
—
1.17 0.06
c
—
0.21 0.02 (7 1)
1.2 0.1
0.35 0.02 (13.3 0.7)
a Determined at 6 lM compound concentration in the presence of
30 lM of cysteine.
b All values are mean SEM (n = 5).
Å
c No NO liberation was observed at 6–100 lM compound concen-
The final products 21, 22, and 24 (Scheme 4) were pre-
pared by treatment of the appropriate alcohols 19 and
20, and amine 23, respectively, with the corresponding
acid chloride of trolox acetate 9, following a procedure
similar to those used to transform 9 into 12. Furazan
derivative 21 was included in the experiments to know
the relevance of the N-oxide moiety in the studied bio
activities.16
tration in the presence of 30–500 lM of cysteine.
lM minꢀ1, were linearly dependent on the concentra-
tion of the single compounds (Fig. 3). In contrast, furo-
xan derivative 18, furazan derivative 21, and nitrooxy
Å
derivatives were unable to release NO (Table 1) in the
conditions assayed. The potential anti-aggregatory ef-
fects of these compounds were studied on ADP-induced
platelet aggregation model.40 The anti-aggregatory
potency of furoxan derivatives correlated with their abil-
Å
2.2. NO releasing capacity
Å
The ability of nitrooxy derivatives 4,6, 8, 11, and 13,
furoxans 18, 22, and 24, and furazan 21 to release NO
ity to produce NO (data not shown).
Å
was determined at different concentrations in the pres-
ence of cysteine (5- to 20-fold molar excess) following
hemoglobin oxidation.39 Furoxan reactants 14, 17, 20,
2.3. Vasorelaxation properties
All the hybrid a-tocopherol analogs—ÅNO donor deriv-
atives had the ability to induce rat aortic rings vasore-
laxation (Figs. 4A–D). These resÅults indicate that
organic nitrate derivatives release NO. As observed
for platelet aggregation, the vasoactive properties of
Å
and 23 were included to compare their NO releasing
behavior with the new compounds. The new furoxan
Å
derivatives, 22 and 24, generate high levels of NO
(Table 1). Initial rates of NO donation, expressed as
Figure 2. Speculative proposal for the generation of derivatives 15 and 16.

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G. V. Lopez et al. / Bioorg. Med. Chem. 13 (2005) 5787–5796
5791
these novel compounds correlated with the extent of
ÅNO released. Thus, derivatives 22 and 24 showed the
greatest aortic vasodilating effects, whereas the parent
compounds (a-tocopherol acetate and trolox acetate 9)
were unable to produce vasodilatation in the same con-
ditions (Fig. 4A). Derivative 24 dose dependently
(EC50 = 1.0 0.1 lM) vasodilated aortic rings by a
ÅNO release-dependent mechanism (Fig. 4B). As oxyhe-
moglobin has been reported to scavenge ÅNO, decreasing
the biological activity of ÅNO and related com-
pounds,41,42 we examined the effect of the hemoprotein
on 24-elicited vasorelaxation. The structural motive
present in derivative 18 and, in particular, the substitu-
tions on furoxan moiety promote a partial lack of
vasodilating activity (Fig. 4A). The N-oxide relevance
in the observed vasodilating activity was confirmed with
the furazan analog 21 (Fig. 4D). In fact, at 10.0 lM
furoxan 24 was found to be about 10 times more potent
than the deoxygenated analog, furazan 21 in causing
vasorelaxation. On the other hand, the organic nitrates
were drastically less active than compound 24 in its
vasodilating properties (Fig. 4D; compare compound
Å
Figure 3. Correlation curves between initial NO formation rate (lM
ÅNO minꢀ1) and tested compound concentration (C, lM): (m) 22, (.)
24. The reaction was followed by detecting the increase of absorbance
(DA) at k = 401 nm over the first 10 min. The reaction was started by
adding the tested compounds (0–24 lM) to a 10 lM HbO2 and
cysteine (30–100 lM) solution in 50 mM phosphate buffer, pH 7.4,
37 °C.
Figure 4. (A) Percentage of vasodilating activity of a-tocopherol acetate (1 acetate), 9, 18, 21, 22, and 24. Tested compounds (10 lM) were added to
the tissue–organ bath system after contraction of the thoracic aorta ring with NA (1 lM). *p < 0.01 and **p < 0.05. (B) Percentage of vasodilating
activity of 24 at different concentrations (1–10 lM) in the absence of HbO2 and at 1 lM concentration in the presence of HbO2 (10 lM) added to the
bath 10 min before the tested compound; (C) percentage of vasodilating activity of 4, 6, 8, 11, and 13. Tested compounds (20 lM) were added as in
*
(A). p < 0.01 and **p < 0.05; (D) percentage of vasodilating capacity in rat aortic model of a-tocopherol analogs—furoxan 24 (.), furazan 21 (Ç),
and organic nitrate 13 (d) derivatives at different compound concentrations (1.0–20.0 lM). Tested compounds were added as previously.

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5792
Our observations emphasize the necessity of performing
further studies to analyze the LDL protective activity of
these compounds in vivo.
4. Experimental
4.1. Chemistry
Argon and nitrogen were purchased from AGA S.A.
(Montevideo, Uruguay). Other chemicals were pur-
chased from Sigma (St. Louis, MO, USA) or Aldrich
(Milwaukee, WI, USA) at the highest purity available.
Compounds 5,34 7,35 8,35 10,44 14,45 17,46 19,47 20,47
and 2348 were synthesized according to literature
methods. Elemental analyses were obtained from vacu-
um-dried samples (over phosphorous pentoxide at 3–
4 mmHg, 24 h at room temperature) and performed on
a Fisons EA 1108 CHNS-O analyzer and were within
0.4% of theoretical values. Infrared spectra were record-
ed on a Bomen, Hartman & Braun FTIR spectropho-
tometer, using potassium bromide tablets. 1H NMR
and 13C NMR spectra were recorded on a Bruker
DPX-400 instrument, with CDCl3 as solvent and tetra-
methylsilane as the internal reference. Electron impact
(EI) and electrospray (ES+) mass spectra were obtained
at 70 eV on a Shimadzu GC-MS QP 1100 EX or on a
Hewlett Packard 1100 MSD spectrometer, respectively.
TLC was carried out on AlugramÒ Sil G/UV254 or alu-
minum oxide on polyester plates. Column chromatogra-
phy (CC) was carried out on silica gel (Merck, 60–230
mesh) or aluminum oxide (Merck, 70–230 mesh). All
solvents were dried and distilled prior to use.
Figure 5. Compound 24 incubated with LDL inhibits oxygen uptake
during its ABAP-mediated oxidation. (a) Oxidation of LDL (8 lM) in
50 mM phosphate buffer by 5 mM ABAP was monitored using a Clark
type oxygen electrode at 37 °C. (b) Control trace. (c) Oxidation of
LDL previously incubated with 24 in 50 mM phosphate buffer.
24 to its analog 13), emphasizing the special behavior of
furoxan moiety in the NO-release capacity.
Å
In summary, the tocopherol derivatives belonging to ser-
ies a (see Chart 1) displayed low vasodilating activity.
However, derivative 6, series b, showed modest vasodi-
lating activity at 20 lM. On the other hand, derivatives
belonging to series c, 11, 22, 24, and in a less extent 13,
displayed a good vasodilating activity.
2.4. Antioxidant properties
4.2. 2,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)chro-
man-6-yl 2-chloroacetate (3)
To test the capacity of these compounds to protect LDL
from oxidative modifications, we studied the effect of
ÅNO donors on azo-compound (ABAP, 2,20-azobis(2-
amidopropane))-mediated LDL oxidationÅ.43 We select-
ed furoxan derivative, 24, due to its high NO-releasing
ability. As expected, compound 24 protected LDL from
azo compound-mediated oxidation. In fact, when LDL
was incubated with derivative 24, it decreased ABAP-
mediated oxygen consumption (Fig. 5).
To
a stirred solution of a-tocopherol (500 mg,
1.2 mmol) and pyridine (0.1 mL) in toluene (2.0 mL),
was added dropwise over 15 min a solution of 2-chloro-
acetyl chloride (0.1 mL) in toluene (2 mL). The reaction
mixture was filtered, and then the filtrate was washed
with brine. The organic layer was dried with sodium sul-
fate and the solvent evaporated in vacuo. The product
was purified by column chromatography (SiO2, hex-
1
ane:ethyl ether (9:1)). Yellow oil, yield 81%. H NMR:
d = 4.34 (s, 2H), 2.62 (t, J = 6.6 Hz, 2H), 2.12 (s, 3H),
2.05 (s, 3H), 2.01 (s, 3H), 1.88–1.74 (m, 2H), 1.61–1.48
(m, 3H), 1.47–1.35 (m, 4H), 1.36–1.21 (m, 11H), 1.19–
1.04 (m, 6H), 0.90–0.86 (m, 12H). 13C NMR:
d = 166.39, 150.19, 140.61, 126.84, 125.12, 123.70,
118.00, 75.61, 40.97, 40.74–12.19 (other methylic,
methylenic, and methynic carbons). IR: vmax = 2951,
2926, 2868, 1779, 1757, 1460, 1414, 1377, 1235, 1148,
1109, 1067 cmꢀ1. Anal. (C31H51ClO3) C, H.
3. Conclusion
Our results show that the new tocopherol analogs, 6, 11,
13, 22, and 24, represent a new class of ÅNO donors having
different capacity to release ÅNO. These tocopherol deriv-
atives exhibited vasorelaxation properties with deriva-
tives belonging to series c (Chart 1), 11, 22, 24, and in a
less extent 13 displayed greater vasodilating effects. This
Å
suggests that these compounds stimulate NO signaling
pathways in vascular tissue. Moreover, the observed
LDL-protective activity of derivative 24 suggest the po-
tential use of these compounds for prevention of athero-
sclerosis disease. These Ôsite-specificÕ observations are
significant in view of the fact that the protective effect of
4.3. 2,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)chro-
man-6-yl 2-nitrooxyacetate (4)
To a stirred solution of silver nitrate (340 mg, 2 mmol)
in acetonitrile (2.0 mL) protected from light, was added
dropwise compound 3 (100 mg, 0.2 mmol) in acetoni-
Å
typical NO donors or antioxidants decrease with time
and distances of the biological targets, that is, LDL.

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G. V. Lopez et al. / Bioorg. Med. Chem. 13 (2005) 5787–5796
5793
trile. The reaction mixture was allowed to stir for 24 h at
room temperature, and then for 35 h at 80 °C. The mix-
ture was concentrated under reduced pressure and the
residue was treated with water. The precipitated AgCl
was removed by filtration, and the filtrate was extracted
with ethyl ether. The combined organic layers were dried
with sodium sulfate and evaporated in vacuo. The prod-
uct was purified by column chromatography (SiO2, hex-
chromatography (SiO2, hexane/ethyl ether (1:1)). Color-
less oil, yield 28%. 1H NMR: d = 4.28 (br s, 1H), 4.14 (br
s, 2H), 4.06 (br s, 1H), 2.69–2.45 (m, 3H), 2.33 (s, 3H),
2.18 (s, 3H), 2.05 (s, 3H), 1.95 (s, 3H), 1.91–1.85 (m,
3H), 1.65 (s, 3H). 13C NMR: d = 174.14, 169.74,
149.86, 141.92, 127.72, 125.64, 123.31, 117.47, 77.86,
69.65, 61.13, 31.96, 23.02, 21.26, 20.85, 14.46, 13.24,
12.35, 12.11. IR (KBr): v = 2934, 1755, 1632, 1456,
1369, 1331, 1280, 1213, 1179, 1140, 1111 cmꢀ1. MS
(IE, 70 eV): m/z (%) = 395 (M+, 8), 353 (17), 307 (9),
247 (11), 231 (10), 205 (35), 164 (3), 135 (3), 107 (3),
43 (100). Anal. (C19H25NO8) C, H, N.
1
ane/ethyl ether (95:5)). Yellow oil, yield 42%. H NMR:
d = 5.20 (s, 2H), 2.61 (t, J = 6.7 Hz, 2H), 2.11 (s, 3H),
2.04 (s, 3H), 1.99 (s, 3H), 1.88–1.74 (m, 2H), 1.62–1.51
(m, 3H), 1.46–1.37 (m, 3H), 1.34–1.21 (m, 12H), 1.19–
1.06 (m, 6H), 0.90–0.86 (s, 12H). 13C NMR:
d = 164.98, 150.36, 140.10, 126.78, 125.09, 123.83,
118.08, 75.66, 67.29, 39.78–12.19 (other methylic,
methylenic, and methynic carbons). IR: vmax = 2951,
2928, 2868, 1778, 1659, 1458, 1412, 1377, 1288, 1186,
1109, 1070, 841 cmꢀ1. MS (IE, 70 eV): m/z (%) = 533
(M+, 1), 486 (37), 458 (9), 430 (100), 261 (7), 221 (28),
193 (7), 165 (42), 149 (8), 69 (6), 57 (11). Anal.
(C31H51NO6) C, H, N.
4.7. 2-(2-Chloroethylcarbamoyl)-2,5,7,8-tetramethylchro-
man-6-yl acetate (12)
To a stirred solution of trolox acetate 9 (150 mg,
0.5 mmol) in dry toluene (1.5 mL), thionyl chloride
(0.06 mL, 0.8 mmol), and a drop of dry DMF were add-
ed. The mixture was heated at reflux for 90 min and
cooled to room temperature. Solvent and thionyl chlo-
ride excess was evaporated in vacuo and the residue
was dissolved in dry dichloromethane (1.0 mL). Then,
solutions of 2-chloroethylamine hydrochloride (58 mg,
0.34 mmol) and triethylamine (0.3 mL) in dichloro-
methane were added dropwise. The reaction mixture
was stirred at room temperature until the disappear-
ance of the reactant. It was washed with 10% sodium
bicarbonate solution and then with water. The organic
layer was dried with sodium sulfate and the solvent was
evaporated in vacuo. The product was purified by
column chromatography (SiO2, hexane/ethyl ether
(7:3)). Yellow oil, yield 28%. 1H NMR: d = 6.92
(br s, 1H), 3.62–3.49 (m, 4H), 2.68–2.58 (m, 2H), 2.35
(s, 4H), 2.20 (s, 3H), 2.07 (s, 3H), 1.99 (s, 4H), 1.55
(s, 3H). 13C NMR: d = 174.80, 169.86, 148.36, 142.08,
127.80, 126.03, 122.94, 118.47, 79.00, 44.19, 41.13,
29.56, 27.75/26.71, 20.87, 20.67, 13.35, 12.49, 12.38.
MS (IE, 70 eV): m/z (%) = 355 (M++2, 3), 353 (M+,
8), 313 (9), 311 (27), 247 (11), 205 (71), 189 (7), 175
(3), 161 (3), 91 (8), 63 (9), 43 (100). Anal.
(C18H24ClNO4) C, H, N.
4.4. 5-Nitrooxymethyl-2,7,8-trimethyl-2-(4,8,12-trimeth-
yltridecyl)chroman-6-yl acetate (6)
The title compound was prepared from 5 (136 mg,
0.25 mmol) following the procedure used for the synthesis
1
of 4. Yellow oil, yield 28%. H NMR: d = 5.41 (s, 2H),
2.78 (t, J = 6.7 Hz, 2H), 2.36 (s, 3H), 2.15 (s, 3H), 2.04
(s, 3H), 1.87–1.75 (m, 2H), 1.61–1.51 (m, 3H), 1.46–1.21
(m, 15H), 1.18–1.03 (m, 6H), 0.90–0.86 (s, 12H). 13C
NMR: d = 170.05, 150.31, 142.07, 131.24, 129.19,
128.49, 119.26, 76.11, 68.03, 39.78–12.72 (other methylic,
methylenic, and methynic carbons). MS (IE, 70 eV): m/z
(%) = 533 (M+, 2), 445 (12), 71 (13), 57 (29), 43 (100).
Anal. (C31H51NO6) C, H, N.
4.5. 6-Acetoxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (9)
A mixture of TroloxÒ (500 mg, 2 mmol), pyridine (5.1
mL), and acetic anhydride (4.3 mL) was stirred for 2 h
at room temperature. The reaction mixture was diluted
with water (10.0 mL), and extracted several times with
ethyl ether. The combined organic layers were washed
with saturated copper sulfate solution and brine. It was
dried with sodium sulfate and the solvent evaporated in
vacuo to give 9 as a white solid, which was used without
further purification. The acetylation process was con-
firmed by 1H NMR.
4.8. 2,5,7,8-Tetramethyl-2-(2-nitrooxyethylcarbamoyl)-
chroman-6-yl acetate (13)
The title compound was prepared from 12 (50 mg,
0.14 mmol) following a similar procedure used for the
synthesis of 4. The reaction mixture was stirred for 24 h
at 80 °C. The product was purified by column chromatog-
raphy (SiO2, hexane/ethyl ether (7:3)). Yellow oil, yield
1
18%. H NMR: d = 7.20 (br s, 1H), 4.53–4.52 (m, 2H),
4.6. 3-Nitrooxypropyl 6-acetoxy-2,5,7,8-tetramethyl-
chroman-2-carboxylate (11)
4.43–4.39 (m, 1H), 4.35–4.30 (m, 1H), 2.64–2.50 (m,
2H), 2.44–2.40 (m, 1H), 2.33 (s, 3H), 2.17 (s, 3H), 2.05
(s, 3H), 1.95 (s, 3H), 1.96–1.85 (m, 1H), 1.64 (s, 3H).
13C NMR: d = 173.8, 164.2, 149.8, 141.1, 127.6, 125.5,
123.6, 117.4, 77.60, 70.61, 61.02, 30.66, 25.69, 21.12,
20.88, 13.28, 12.38, 12.13. IR: vmax = 2924, 2851, 1754,
1638, 1458, 1370, 1279, 1213, 1198, 1140, 1109. MS
(IE, 70 eV): m/z (%) = 381(M+ + 1, 20), 339 (41), 293
(27), 247 (31), 231 (27), 205 (100). Anal. (C18H24N2O7)
C, H, N.
To a stirred solution of 9 (100 mg, 0.34 mmol) in dry
dichloromethane (2.0 mL) was added DCC (71 mg,
0.34 mmol). The reaction mixture was allowed to stir
for 1 h in ice-water bath. Then, alcohol 12 (42 mg,
0.34 mmol) was added and allowed to stir at room tem-
perature. The reaction mixture was heated at reflux for
12 h, filtered, and then solvent evaporated under re-
duced pressure. The product was purified by column

´
G. V. Lopez et al. / Bioorg. Med. Chem. 13 (2005) 5787–5796
5794
4.9. 4-Phenyl-3-[2,5,7,8-tetramethyl-2-(4,8,12-trimethylt-
ridecyl)chroman-6-oxyl]methyl-l,2,5-oxadiazole N2-oxide
(18)
(ES+): m/z (%) = 597 (M+Å+23, 10), 575 (M+Å+l, 5), 513
(5). Anal. (C27H30N2O10S) C, H, N, S.
4.12. 6-Acetyloxy-2,5,7,8-tetramethyl-N-[2-(3-phen-
ylsulfonyl-N2-oxide-l,2,5-oxadiazole-4-oxyl)ethyl]chro-
man-2-carboxamide (24)
Catalytic amounts of 18-crown-6 were added to a stirred
solution of 1 (100 mg, 0.23 mmol) in dry THF (5.0 mL),
6 (58 mg, 0.28 mmol), and potassium carbonate (17 mg,
0.12 mmol). The mixture was heated at reflux until the
disappearance of the reactants. Then, the solvent was
evaporated in vacuo and the product was purified by
column chromatography (SiO2, hexane/ethyl ether
The title compound was prepared from 9 (100 mg,
0.34 mmol) and amine 25 (97 mg, 0.34 mmol) following
the procedure used for the synthesis of 21. The crude
product was purified by column chromatography (SiO2,
hexane/ethyl ether (2:8)). Colorless oil that crystallized
1
(8:2)). Yellow oil, yield 65%. H NMR: d = 7.78–7.76
1
(m, 2H), 7.58–7.51 (m, 3H), 4.81 (s, 2H), 2.58–2.56 (m,
2H), 2.14 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 1.99–1.88
(m, 2H), 1.87–1.34 (m, 15H), 1.30 (s, 3H), 1.18–1.00
(m, 6H), 0.92–0.87 (s, 12H). 13C NMR: d = 157.88,
149.04, 147.71, 131.49, 129.49, 128.43, 128.09, 126.80,
126.28, 123.76, 118.23, 112.94, 75.38, 62.52, 40.98–
12.24 (other methylic, methylenic, and methynic car-
at 4 °C, yield 88%. H NMR: d = 8.04 (d, J = 7.6 Hz,
2H), 7.75 (t, J = 7.6 Hz, 1H), 7.58 (t, J = 8.0 Hz, 2H),
6.95 (br s, 1H), 4.48 (br s, 2H), 3.78–3.73 (m, 2H), 2.67–
2.59 (m, 2H), 2.36 (s, 3H), 2.23 (s, 3H), 2.06 (s, 3H), 2.00
(br s, 5H), 1.57 (s, 3H). 13C NMR: d = 175.22, 169.92,
159.15, 148.32, 142.16, 138.40, 136.05, 130.07, 128.90,
127.98, 126.00, 123.18, 118.46, 110.80, 79.08, 70.64,
38.46, 29.70, 25.0/24.1, 20.90, 20.71, 13.40, 12.53(2C).
IR: vmax = 3434, 2932, 1754, 1676, 1213, 1171 cmꢀ1. MS
(ES+): m/z (%) = 560 (M+Å+l, 30), 465 (32), 463 (35), 283
(60). Anal. (C26H29N3O9S) C, H, N, S.
bons). IR: vmax = 2926, 1601, 1458, 1248, 1082 cmꢀ1
.
MS (ES+): m/z (%) = 560 (M+ÅꢀCH2(CH3)2, 5), 417
(7), 342 (10), 247 (2). Anal. (C38H56N2O4) C, H, N.
4.10. 3-(3-Phenylsulfonyl-l,2,5-oxadiazole-4-oxyl)propyl
6-acetyloxy-2,5,7,8-tetramethylchroman-2-carboxylate
(21)
Å
4.13. NO release evaluation
Å
The rate of NO release was determined by measuring
the oxidation of oxyhemoglobin (HbO2) to methemo-
globin (MetHb) at k = 401 nm, at 37 °C using a Shimad-
zu spectrophotometer.37 The reaction was started by
adding the tested compounds (6–100 lM in 50 mM
phosphate buffer) to a 10 lM HbO2 solution in 50 mM
phosphate buffer, pH 7.4, in the presence of 5- to 20-fold
molar excess of cysteine. 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 five
determinations. The molar extinction coefficient
The title compound was prepared from 9 (100 mg,
0.34 mmol) and alcohol 19 (69 mg, 0.27 mmol) following
the procedure used for the synthesis of 12. The product
was purified by column chromatography (SiO2 hexane/
ethyl ether (8:2)). Yellow oil, yield 7%. 1H NMR:
d = 8.07 (d, J = 8.6 Hz, 2H), 7.75 (t, J = 7.5 Hz, 1H),
7.62 (t, J = 7.7 Hz, 2H), 4.33–4.28 (m, 1H), 4.14–4.09
(m, 2H), 4.07 (br s, 1H), 2.66–2.61 (m, 1H) 2.49–2.43
(m, 2H), 2.3l (s, 3H), 2.16 (s, 3H), 2.03 (s+m, 5H), 1.90
(m, 4H), 1.64 (s, 3H). 13C NMR: d = 174.12, 169.95,
149.84 (two carbons), 149.17, 141.59, 138.39, 135.79,
130.04, 129.28, 127.72, 125.59, 123.30, 117.42, 70.09,
66.22, 60.82, 30.83–12.15 (other methylic, methylenic,
and methynic carbons). IR: vmax = 2934, 1752, 1574,
1213, 1198 cmꢀ1. MS (ES+): m/z (%) = 581 (M+Å+23,
40), 559 (M+Å+l, 30). Anal. (C27H30N2O9S) C, H, N, S.
De ¼ e401MetHb ꢀ e401HbO was determined by quantitative
2
oxidation of five different concentrations (1–10 lM) of
HbO2 in pH 7.4 phosphate buffer with a 20 lM solution
of NOC-7 (l-hydroxy-2-oxo-3-(N-3-methyl-aminopro-
pyl)-3-methyl-1-triazene). The slope (De) of the straight
line (r = 0.999) obtained plotting the increase of the
absorbance DA at k = 401 nm against the HbO2 concen-
trations was 57 2 mMꢀ1 cmꢀ1. The ability of these
compounds to inhibit platelet aggregation in vitro was
evaluated as previously described.38
4.11. 3-(3-Phenylsulfonyl-N2-oxide-l,2,5-oxadiazole-4-
oxyl)propyl 6-acetyloxy-2,5,7,8-tetramethyl-chroman-2-
carboxylate (22)
4.14. Vasorelaxation assays
The title compound was prepared from 9 (100 mg,
0.34 mmol) and alcohol 20 (102 mg, 0.34 mmol) follow-
ing the procedure used for the synthesis of 12. The prod-
uct was purified by column chromatography (SiO2
hexane/ethyl ether (8:2)). Yellow oil, yield 9%. 1H
NMR: d = 8.04 (d, J = 8.6 Hz, 2H), 7.75 (t, J = 7.5 Hz,
1H), 7.61 (t, J = 8.2 Hz, 2H), 4.42–4.36 (m, 1H), 4.17–
4.11 (m, 2H), 4.02 (br s, 1H), 2.62–2.46 (m, 3H), 2.29
(s, 3H) 2.16 (s, 3H), 2.02 (s+m, 5H), 1.88 (s, 4H), 1.65
(s, 3H). 13C NMR: d = 174.23, 169.90, 155.05, 149.89,
141.80, 138.56, 135.92, 130.01, 128.91, 127.70, 125.89,
123.26, 117.47, 110.84, 80.20, 67.68, 60.84, 30.87–12.43
(other methylic, methylenic, and methynic carbons).
IR: vmax = 2932, 1752, 1553, 1213, 1171 cmꢀ1. MS
Wistar rats (250–300 g) were anesthetized (40 mg kgꢀ1
pentobarbital i.p.), and descending thoracic aorta was
excised and cut in rings (4 mm in length) (experimental
´
procedures approved by Comision Honoraria de Exper-
´
´
imentacion Animal, Universidad de la Republica). Tis-
sues were mounted under 2 g of passive tension in a
Radnoti tissue–organ bath system containing 30 mL of
tyrode solution, maintained at 37 °C and gassed with
95% O2–5% CO2, pH 7.4. Aortic rings were allowed to
equilibrate for 1 h and a dose-dependent contraction
was obtained by their incubation in the presence of
1 lM noradrenaline (NA). Tested compounds were add-
ed after plateau, using a-tocopherol and Trolox as con-

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G. V. Lopez et al. / Bioorg. Med. Chem. 13 (2005) 5787–5796
5795
Lakka, T. A.; Rissanen, T.; Leskinen, L.; Tuomainen, T.
P.; Valkonen, V. P.; Ristonmaa, U.; Poulsen, H. E. J.
Intern. Med. 2000, 248, 377.
trols. Drug vehicle (DMSO) also served as control and
did not affect the experiments.The effect of 10 lM
HbO2 on relaxation was evaluated by its addition to
the bath at least 10 min before compound addition.
10. Stephens, N. G.; Parsons, A.; Schofield, P. M.; Kelly, F.;
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4.15. Antioxidant properties
Human LDL (8 lM) obtained by ultracentrifugation
from healthy plasma donors as before,49 was incubated
for 15 min at 37 °C in 50 mM potassium phosphate buff-
er, pH 7.4, in the presence of 2 mM tested compound,
added in DMSO (<1% final). Then, the free compound
was removed by size exclusion HPLC. The LDL fraction
with compound of interest was then incubated in the pres-
ence of 5 mM ABAP and LDL oxidation was monitored
by oxygen consumption as before.43 Controls were done
using the same experimental conditions, but without
LDL and compound, and just without the compound.
The presence of the compounds in LDL fraction was
checked by RP-HPLC with UV detection after extraction
of the compounds from LDL with methanol and using the
compound as the standard for all chromatographic
purposes.
19. Hogg, N.; Struck, A.; Goss, S. P. A.; Santanam, N.;
Joseph, J.; Parthasarathy, S.; Kalyanaraman, B. J. Lipid
Res. 1995, 36, 1756.
20. Struck, A. T.; Hogg, N.; Thomas, J. P.; Kalyanaraman, B.
FEBS Lett. 1995, 361, 291.
21. Rubbo, H.; Raddi, R.; Trujillo, M.; Telleri, R.; Kalyana-
ramen, B.; Barnes, S.; Kirk, M.; Freeman, B. A. J. Biol.
Chem. 1994, 269, 26066.
4.16. Data analysis
Data are expressed as mean SEM or 95% confidence
interval. Statistical comparisons were carried out with
Fisher, ANOVA, Dunnett, or Student tests.
22. Rubbo, H.; Parthasarathy, S.; Barnes, S.; Kirk, M.;
Kalyanaraman, B.; Freeman, B. A. Arch. Biochem. Bio-
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Acknowledgments
´
23. Denicola, A.; Batthyany, C.; Lissi, E.; Freeman, B. A.;
Rubbo, H.; Radi, R. J. Biol. Chem. 1995, 277, 932.
We gratefully acknowledge C.S.I.C. (Universidad de la
Repu´blica) and PEDECIBA for financial support. This
work was supported by grants from FOGARTY-NIH
´
24. Moller, M.; Botti, H.; Batthyany, C.; Rubbo, H.; Radi,
R.; Denicola, A. J. Biol.Chem. 2005, 280, 8850.
25. Rubbo, H.; Radi, R.; Anselmi, D.; Kirk, M.; Barnes, S.;
Butler, J.; Eiserich, J. P.; Freeman, B. A. J. Biol. Chem.
2000, 275, 10812.
´
to H.R. and R.R., Wellcome Trust to H.R., Fundacion
´
ÔManuel PerezÕ to C.B., Howard Hughes Medical Insti-
tute (HHMI, USA) to RR and the Guggenheim Foun-
dation to R.R. and H.R.. R.R. is a Howard Hughes
International Research Scholar. G.V.L., C.B., F.B.
and A.T. thank PEDECIBA for scholarship.
26. Trostchansky, A.; Batthyany, C.; Botti, H.; Radi, R.;
Denicola, A.; Rubbo, H. Arch. Biochem. Biophys. 2001,
395, 225.
27. Trostchansky, A.; Ferrer-Sueta, G.; Batthyany, C.; Botti,
H.; Batinic-Haberle, I.; Radi, R.; Rubbo, H. Free Radic.
Biol. Med. 2003, 35, 1293.
28. Sanguinetti, S. M.; Batthyany, C.; Trostchansky, A.;
Botti, H.; Lopez, G. I.; Wikinski, R. L.; Rubbo, H.;
Schreier, L. E. Arch. Biochem. Biophys. 2004, 423, 302.
29. Botti, H.; Batthyany, C.; Trostchansky, A.; Radi, R.;
Freeman, B. A.; Rubbo, H. Free Radic. Biol. Med. 2004,
36, 152.
30. Wang, P. G.; Xiaoping Tang, M. X.; Wu, X.; Cai, T.;
Janczuk, A. J. Chem. Rev. 2002, 102, 1091.
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