Design and synthesis of novel NO-donor-ferulic acid hybrids as potential antiatherosclerotic drug candidates a

Article abstract of DOI:10.1002/ddr.20442

Novel NO-donor-ferulic acid hybrids were designed and synthesized through a symbiotic approach using ferulic acid and three different NO-donating groups, such as nitric ester, 4-hydroxyl-3-phenylfuroxan, and 4-hydroxymethyl-3- phenylsulfonylfuroxan. Antioxidant, nitric oxide release, and vasodilator properties studies showed that the target phenylsulfonylfuroxan 14, especially 14c, while keeping the antioxidant activity, showed more NO release activity and vasodilating activity than isosorbide dinitrate (ISDN). Thus, 14c may be considered a novel potent anti-atherosclerosis drug candidate.

Full text of DOI:10.1002/ddr.20442

DRUG DEVELOPMENT RESEARCH 72 : 405–415 (2011)
Research Article
Design and Synthesis of Novel NO-Donor-Ferulic Acid
Hybrids as Potential Antiatherosclerotic Drug Candidates
Ã
Nian-Guang Li,1,2 Rong Wang,1 Zhi-Hao Shi,3 Yu-Ping Tang,1 Bao-Quan Li,1
Zhen-Jiang Wang,1 Shu-Lin Song,1 Li-Hua Qian,1 Wei Li,2 Jian-Ping Yang,1 Li-Juan Yao,1
Ã
Jun-Zuan Xi,1 Jia Xu,1 Feng Feng,4 Da-Wei Qian,1 and Jin-Ao Duan1
1Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Chinese Medicine,
Nanjing, Jiangsu 210046, People’s Republic of China
2Department of Medicinal Chemistry, Nanjing University of Chinese Medicine, Nanjing, Jiangsu
210046, People’s Republic of China
3Department of Organic Chemistry, China Pharmaceutical University, Nanjing, Jiangsu 211198,
People’s Republic of China
4Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, Jiangsu
210009, People’s Republic of China
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Toxicology, Formulation Phases I-III
Technology,
Genomics,
Proteomics
Preclinical
Research
Postmarketing
Phase IV
Drug Delivery,
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
Novel NO-donor-ferulic acid hybrids were designed and synthesized through a symbiotic
approach using ferulic acid and three different NO-donating groups, such as nitric ester, 4-hydroxyl-3-
phenylfuroxan, and 4-hydroxymethyl-3-phenylsulfonylfuroxan. Antioxidant, nitric oxide release, and
vasodilator properties studies showed that the target phenylsulfonylfuroxan 14, especially 14c, while
keeping the antioxidant activity, showed more NO release activity and vasodilating activity than
isosorbide dinitrate (ISDN). Thus, 14c may be considered a novel potent anti-atherosclerosis drug
candidate. Drug Dev Res 72:405–415, 2011.
r 2011 Wiley-Liss, Inc.
Key words: atherosclerosis; nitric oxide donor; ferulic acid; nitric oxide release
INTRODUCTION
deaths worldwide, making it the leading cause of morbidity
and mortality [Hennekens, 1998]. Atherosclerosis is
Cardiovascular disease (CD), such as heart attack,
stroke, and hypertension, is to blame for about 30% of all
Abbreviations used: CD, cardiovascular disease; DCC,
dicyclohexylcarbodiimide; DEAD, diethyl azodicarboxylate;
DMAP, 4-N,N-dimethylaminopyridine; DPPH, 2,2-diphenyl-1-
Grant sponsor: Program for New Century Excellent Talents by
the Ministry of Education; Grant number: NCET-09-0163; Grant picrylhydrazyl; EDC, 1-ethyl-(3-dimethylaminopropyl)carbodii-
sponsor: Research Fund for the Doctoral Program of Higher Education mide; EDRF, endothelial-derived relaxing factor; FA, ferulic acid;
of China; Grant number: 20093237120012; Grant sponsor: Main
Training Fund of Nanjing University of Chinese Medicine; Grant
ISDN, isosorbide dinitrate; LDL, low-density lipoprotein; mp,
melting point; ROS, reactive oxygen species; TBA, 2-thiobarbi-
number: 10XPY02; Grant sponsor: National Natural Science Founda- turic acid; TBARS, 2-thiobarbituric acid reactive substances.
tion of China; Grant numbers: 30873235; 81001382; Grant sponsor:
Ã
Correspondence to: Yu-Ping Tang and Jin-Ao Duan, Jiangsu
Key Laboratory for TCM Formulae Research, Nanjing University of
Chinese Medicine, 138 Xianlin Road, 210046, Nanjing, Jiangsu,
People’s Republic of China. E-mail: yupingtang@njutcm.edu.cn
(Y.-P. Tang); dja@njutcm.edu.cn (J.-A. Duan)
Natural Science Foundation of Jiangsu Province of China; Grant
number: BK2008455; Grant sponsor: Construction Project for Jiangsu
Key Laboratory for High Technology Research of TCM Formulae;
Grant number: BM2010576; Grant sponsors: Construction Project for
Jiangsu Engineering Center of Innovative Drug from Blood-Condition-
ing TCM Formulae; Program for Outstanding Scientific and Techno-
logical Innovation Team of Jiangsu Higher Education (2009); Key
Research Project in Basic Science of Jiangsu College and University;
Grant numbers: 06KJA36022; 07KJA36024; 10KJA360039.
Received 18 February 2011; Accepted 19 March 2011
Published online in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/ddr.20442
ꢀc
2011 Wiley-Liss, Inc.

406
LI ET AL.
unquestionably the main underlying pathology of CD, their capacity of inhibiting the ferrous salt/ascorbate-
among which the major risk factors for the develop- induced lipid peroxidation of membrane lipids of rat
ment of atherosclerosis are increased levels of low- hepatocytes and on the in vitro vasodilating properties.
density lipoprotein (LDL), oxidative modification of As antioxidants, we considered ferulic acid (FA) 1
LDL, and an impairment of endothelial derived (Fig. 1), one of the most ubiquitous phenolic com-
relaxing factor (EDRF)-, nitric oxide (NO)-mediated pounds in nature, especially as rich as an ester form in
bioactions [Steinberg et al., 1989; Napoli et al., 2006]. rice bran pitch [Graf, 1992], which has been largely
The accumulation of LDL and reactive oxygen used as a food preservative because of its ability to
species (ROS) in the subendothelial space induces a inhibit the autoxidation of oils [Dziedzic and Hudson,
high degree of LDL oxidation. According to the 1984]. It has been reported that FA inhibited the
oxidative hypothesis of atherosclerosis, this is an early autoxidation of methyl linoleate and linoleic acid as
event in a complex process leading to the formation of well as the oxidation of liposomes induced by the
foam cells that constitute a fatty streak, a forerunner in water-soluble initiator 2,2-diphenyl-1-picrylhydrazyl
the development of mature atherosclerotic plaques (DPPH) [Son and Lewis, 2002; Medina et al., 2002].
[Keaney and Vita, 1995]. An increasing number of Moreover, FA has been shown to reinforce the
studies indicate that the administration of exogenous antioxidant capacity of lactoferrin by inhibiting the
antioxidants might decrease the impact of atherosclero- oxidation in liposomes and oil-in-water emulsions
sis in animals and humans through the regulation and [Medina et al., 2002]. In addition, FA increased the
protection of several aspects of endothelial function antioxidant activity of plasma and the resistance of
`
[Ogita and Liao, 2004; Cherubini et al., 2005; Pratico, LDLs to oxidation [Ohta et al., 1997; Laranjinha et al.,
2005]. Endothelium-dependent vascular relaxation is 1996; Andreasen et al., 2001]. As N-donor moieties, we
mediated predominantly by NO. In the vessels affected chose the nitroxy function present in simple vasodilating
by atherosclerosis, inactivation of NO is enhanced as a nitric esters 2 (in 8, 9), the 3-phenylfuroxan-4-yloxy
result of its reaction with the superoxide anion (Oꢁ2 ꢂ), substructure present in the 4-hydroxyl-3-phenylfuroxan
leading to the formation of peroxynitrite (ONOOꢁ). As 3 (in 12), and the 3-phenylsulfonylfuroxan-4-yloxy
with decreasing the bioavailability of protective NO, the substructure present in 4-hydroxymethyl-3-phenylsul-
peroxynitrite formed in this reaction is a precursor for fonylfuroxan 4 (in 14) (Fig. 1) as well. These reference
the highly reactive and very toxic hydroxyl radical compounds display extremely modulated in vitro NO-
(OHꢂ ). In addition, peroxynitrite is concerned to be of dependent vasodilator properties [Civelli et al., 1996].
high cytotoxicity in its own right and can mediate lipid
peroxidation reactions. Furthermore, a decrease in the
production of NO by endothelial cells cannot be
excluded, at least in advanced atherosclerotic disease
MATERIALS AND METHODS
Synthesis
[Ogita and Liao, 2004].
Reagents and solvents were purchased from
commercial sources and used without further purifica-
tion unless otherwise specified. Air- and moisture-
sensitive liquids and solutions were transferred via
syringe or stainless steel cannula. Reactions were
monitored by thin-layer chromatography (TLC) carried
out on 0.15–0.20-mm Yantai silica gel plates (RSGF
254) using ultraviolet (UV) light as the visualizing
agent. Chromatography was performed on Qingdao
silica gel (16–200 mesh) using petroleum ether (6–90)
and ethyl acetate as the eluanting solvent.
On this basis, some researchers have recently
proposed and synthesized a new class of NO-donor
hybrid drugs obtained by joining antioxidants, such as
p-cresol, vitamin E, and vitamin C with appropriate
NO-donor moieties as potential agents for the treat-
ment of CD involving atherosclerotic vascular changes
[Cena et al., 2004, 2008; Boschi et al., 2006]. In
pursuing some anti-atherosclerosis agents, we report
on the design and synthesis of another series of such
compounds, and the preliminary results of a study on
COOH
HXOH2C
Ph
HYO
SO2Ph
RONO2
2
4
3
N
N
N
N
1
HO
O
O
O
O
O
X: a = /
R : a = -(CH2)3-
b = -(CH2)4-
Y: a = (CH2)2O
b = m-Ph-O
c = p-Ph-O
d = m-Ph-CH2O
e = p-Ph-CH2O
b = (CH2)2CH(CH3)O
c = (CH2)4O
d = (CH2)2O(CH2)2O
c = -(CH2)5-
d = -(CH2)6-
e = -CH2CH=CHCH2-
Fig. 1. Ferulic acid and reference NO-donor compounds.
Drug Dev. Res.

NOVEL NO-DONOR-FERULIC ACID HYBRIDS
407
The melting points (mp) were measured on a 1961; Farrar, 1964; Kelley et al., 1977; Gasco et al.,
WRS-1B apparatus and were not corrected. 1H NMR 1991]. Direct esterification of FA with hydroxyl
spectra were obtained using
a Bruker AV-300 compound 3 or 4 was tried using many catalysts such
(300 MHz). Chemical shifts were recorded in ppm as dicyclohexylcarbodiimide (DCC)/4-N,N-dimethyla-
downfield from tetramethylsilane. J-values were given minopyridine (DMAP), 1-ethyl-(3-dimethylaminopro-
in Hertz (Hz). Abbreviations used were s (singlet), pyl)carbodiimide (EDC), BF3/Et2O, and diethyl
d (doublet), t (triplet), q (quartet), b (broad), and azodicarboxylate (DEAD)/Ph3P; unfortunately, no pro-
m (multiplet). ESI-MS spectra were recorded on a duct was obtained, probably because of the weak
Waters Synapt HDMS spectrometer.
acidity of the –COOH. Various protecting groups and
The synthetic routes of nine nitrate/FA hybrids conditions, such as Ac2O and ethyl chlorformate, were
(8a–8e and 9a–9d) are outlined in Figure 2. FA (1) then used to protect the phenolic hydroxyl group of FA;
was first treated with dibromoalkanes (5a–5d) bearing ethyl chlorformate was found to give optimum results
three to six carbons in the presence of Et3N and when used in conjunction with NaOH in water to
acetone at 501C to generate compounds 6a–6d as the afford 10 in 90% yields. Subsequently, 10 was
major products, and 7a–7d were also obtained as by esterified with 3 or 4 in the presence of DCC and
products no matter the stoichiometry between FA (1) DMAP to generate compounds 11a–11e and
and dibromoalkanes (5a–5d) were changed. In order to 13a–13d. The ethoxycarbonyl groups in 11 and 13
increase the diversity of target compounds, 6a–6d or were removed in ethanolamine solution, without the
7a–7d were further converted to the corresponding breakdown of other ester bonds, to produce target
nitrates 8a–8d or 9a–9d, respectively, with AgNO3 in compounds 12a–12e and 14a–14d.
THF/CH3CN. However, when FA (1) was treated with
(E)-1,4-dibromobut-2-ene (5e), only 6e was obtained,
and then 6e was further converted to the correspond-
ing nitrates 8e with AgNO3 in THF/CH3CN.
EXPERIMENTS
General Procedure for the Preparation of 6(a–e) and
7(a–d)
The other nine target compounds were synthe-
sized through modifying the carboxyl group of FA
with phenylfuroxan or phenylsulfonylfuroxan (Fig. 3).
A total of five phenyl-substituted furoxans (3) and
four phenylsulfonyl-substituted furoxans (4) were
synthesized as described previously [Kenney et al.,
To a stirring mixture of FA (5.0 g, 25.8 mmol) and
dibromo-alkane (100 mmol) in dry acetone (150 ml) at
room temperature was added Et3N (10 ml). The
reaction mixture was refluxed gently for 4–6 h. The
solution was cooled to room temperature and then
COORONO2
8(a-e)
COORBr
AgNO3, CH3CN
50 °C
HO
HO
6(a-e)
COOH
O
O
O
Et3N, acetone
+
BrRBr
5
1
HO
60 °C
COORBr
O
COORONO2
9(a-d)
AgNO3, CH3CN
50 °C
BrRO
O2NORO
7(a-d)
O
R : a = -(CH2)3-, b = -(CH2)4-, c = -(CH2)5-, d = -(CH2)6-, e = -CH2CH=CHCH2-
Fig. 2. Synthesis of nitric ester–ferulic acid hybrids.
Ph
Ph
NH2(CH2)2OH,
95% EtOH
COXOCH2
COXOCH2
N
O
N
O
N
N
11(a-e)
12(a-e)
EtO2CO
HO
COOH
COOH
O
O
N
O
O
O
O
ClCO2Et, 1 N NaOH
3
DCC, DMAP, CH2Cl2
SO2Ph
N
HO
SO2Ph
N
50 °C
EtO2CO
NH2(CH2)2OH,
95% EtOH
25 °C
COYO
COYO
14(a-d)
O
4
O
O
1
N
10
O
O
13(a-d)
O
EtO2CO
HO
X: a = /, b = m-Ph-O, c = p-Ph-O, d = m-Ph-CH2O, e = p-Ph-CH2O
Y: a = (CH2)2O, b = (CH2)2CH(CH3)O, c = (CH2)4O, d = (CH2)2O(CH2)2O
Fig. 3. Synthesis of 4-hydroxyl-3-phenylfuroxa–ferulic acid hybrids and 4-hydroxymethyl-3-phenylsulfonylfuroxa–ferulic acid hybrids.
Drug Dev. Res.

408
LI ET AL.
filtered. Removal of the solvent under vacuo followed (E)-3-bromopropyl-3-(4-(3-bromopropoxy)-3-methoxy-
by silica gel column chromatographic purification of phenyl)acrylate (7a)
the residue using 20% ethyl acetate in petroleum ether
afforded the compounds (yields 4.3–65.8%).
Yellow solid, 11% yield; mp 63.2–63.81C. 1H
NMR (CDCl3) d 2.22–2.30 (m, 2H, CH2), 2.34–2.43
(m, 2H, CH2), 3.52 (t, J 5 6.6 Hz, 2H, CH2Br), 3.62
(t, J 5 6.6 Hz, 2H, CH2Br), 3.89 (s, 3H, OCH3), 4.19
(t, J 5 6.0 Hz, 2H, ArOCH2), 4.35 (t, J 5 6.0 Hz, 2H,
COOCH2), 6.31 (d, J 5 15.9 Hz, 1H, C==CH), 6.90
(d, 1H, Ar–H), 7.05–7.11 (m, 2H, Ar–H), 7.64
(d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 457 [M1Na]1.
(E)-3-bromopropyl-3-(4-hydroxy-3-methoxyphenyl)
acrylate (6a)
Dark red oil, 56% yield. 1H NMR (CDCl3) d 2.26
(m, 2H, CH2), 3.52 (t, J 5 3.7 Hz, 2H, CH2Br), 3.94
(s, 3H, OCH3), 4.34 (t, J 5 3.7 Hz, 2H, COOCH2), 5.83
(s, 1H, OH), 6.30 (d, J 5 9.5 Hz, 1H, C==CH), 6.92
(d, 1H, Ar–H), 7.06–7.15 (m, 2H, Ar–H), 7.65
(d, J 5 9.5Hz, 1H, CH==C). ESI-MS: 353 [M1K]1.
(E)-4-bromobutyl-3-(4-(4-bromobutoxy)-3-methoxy-
phenyl)acrylate (7b)
Brown solid, 7% yield; mp 72.6–75.11C. 1H NMR
(CDCl3) d 1.85–1.93 (m, 2H, CH2), 1.99–2.13 (m, 6H,
CH2), 3.45–3.52 (m, 4H, CH2Br), 3.89 (s, 3H, OCH3),
4.08 (t, J 5 5.9 Hz, 2H, ArOCH2), 4.24 (t, J 5 6.2 Hz,
2H, COOCH2), 6.30 (d, J 5 15.9 Hz, 1H, C==CH), 6.85
(d, 1H, Ar–H), 7.05–7.10 (m, 2H, Ar–H), 7.62
(d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 485 [M1Na]1.
(E)-4-bromobutyl-3-(4-hydroxy-3-methoxyphenyl)
acrylate (6b)
White solid, 43% yield; mp 81.9–82.31C. 1H
NMR (CDCl3) d 1.87–2.07 (m, 4H, CH2), 3.47
(t, J 5 6.4 Hz, 2H, CH2Br), 3.93 (s, 3H, OCH3), 4.24
(t, J 5 6.4 Hz, 2H, COOCH2), 5.85 (s, 1H, OH), 6.28
(d, J 5 15.9 Hz, 1H, C==CH), 6.92 (d, 1H, Ar–H),
7.03–7.10 (m, 2H, Ar–H), 7.62 (d, J 5 15.9 Hz, 1H,
CH==C). ESI-MS: 351 [M1Na]1.
(E)-5-bromopentyl-3-(4-(5-bromopentyloxy)-3-methoxy-
phenyl)acrylate (7c)
White solid, 8% yield; mp 52.9–53.61C. 1 H NMR
(CDCl3) d 1.55–1.76 (m, 6H, CH2), 1.89–1.99 (m, 6H,
CH2), 3.42 (m, 4H, CH2Br), 3.89 (s, 3H, OCH3), 4.06
(t, J 5 6.4 Hz, 2H, ArOCH2), 4.21 (t, J 5 6.4 Hz, 2H,
COOCH2), 6.30 (d, J 5 15.9Hz, 1H, C==CH), 6.85
(d, 1H, Ar–H), 7.06–7.09 (t, 2H, Ar–H), 7.62
(d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 513 [M1Na]1.
(E)-5-bromopentyl-3-(4-hydroxy-3-methoxyphenyl)
acrylate (6c)
White solid, 66% yield; mp 66.3–66.61C. 1H
NMR (CDCl3) d 1.58 (m, 2H, CH2), 1.70 (m, 2H,
CH2), 1.90 (m, 2H, CH2), 3.44 (m, 2H, CH2Br), 3.92
(s, 3H, OCH3), 4.20 (m, 2H, COOCH2), 5.91 (s, 1H,
OH), 6.28 (d, J 5 15.9 Hz, 1H, C==CH), 6.91 (d, 1H,
Ar–H), 7.03–7.09 (m, 2H, Ar–H), 7.61 (d, J 5 15.9 Hz,
1H, CH==C). ESI-MS: 365 [M1Na]1.
(E)-6-bromohexyl-3-(4-(6-bromohexyloxy)-3-methoxy-
phenyl)acrylate (7d)
Yellow oil, 28% yield. 1H NMR (CDCl3) d
1.42–1.48 (m, 8H, CH2), 1.67–1.89 (m, 8H, CH2), 3.41
(m, 4H, CH2Br), 3.89 (s, 3H, OCH3), 4.05 (t, J 5 6.6 Hz,
2H, ArOCH2), 4.20 (t, J 5 6.6 Hz, 2H, COOCH2), 6.30
(d, J 5 15.9 Hz, 1H, C==CH), 6.85 (d, 1H, Ar–H),
7.06–7.09 (m, 2H, Ar–H), 7.62 (d, J 5 15.9 Hz, 1H,
CH==C). ESI-MS: 541 [M1Na]1.
(E)-6-bromohexyl-3-(4-hydroxy-3-methoxyphenyl)
acrylate (6d)
Yellow solid, 53% yield; mp 50.9–51.41C. 1H
NMR (CDCl3) d 1.39–1.45 (m, 4H, CH2), 1.65–1.70
(m, 2H, CH2), 1.85–1.93 (m, 2H, CH2), 3.41 (m, 2H,
CH2Br), 3.91 (s, 3H, OCH3), 4.19 (m, 2H, COOCH2),
5.94 (s, 1H, OH), 6.28 (d, J 5 15.9 Hz, 1H, C==CH),
6.91 (d, 1H, Ar–H), 7.03–7.08 (m, 2H, Ar–H), 7.61
(d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 379 [M1Na]1.
General Procedure for the Preparation of 8(a–e) and
9(a–d)
(E)-((E)-4-bromobut-2-enyl)-3-(4-hydroxy-3-methoxy-
phenyl)acrylate (6e)
To a stirring solution of 6 or 7 (0.7 mmol) in
CH3CN (20 ml) at room temperature was added
Pale solid, 4% yield; mp 69.7–72.81C. 1 H NMR AgNO3 (0.6 g, 2.5 mmol); then the reaction mixture
(CDCl3) d 3.92 (s, 3H, OCH3), 3.97 (d, J 5 6.9 Hz, 2H, was stirred at 501C in dark for 6–8 h. The solution was
CH2Br), 4.71 (d, J 5 5.3 Hz, 2H, COOCH2), 5.88 cooled to room temperature and then filtered. Removal
(s, 1H, OH), 5.92–6.05 (m, 2H, CH==CH), 6.30 of the solvent under vacuo followed by silica gel
(d, J 5 15.9 Hz, 1H, C==CH), 6.91 (d, 1H, Ar–H), column chromatographic purification of the residue
7.03–7.08 (m, 2H, Ar–H), 7.63 (d, J 5 15.9 Hz, 1H, using 30% ethyl acetate in petroleum ether afforded
CH==C). ESI-MS: 349 [M1Na]1.
the compounds (yields 17–96%).
Drug Dev. Res.

NOVEL NO-DONOR-FERULIC ACID HYBRIDS
409
(E)-3-(nitroxy)propyl-3-(4-hydroxy-3-methoxyphenyl) (E)-3-(nitroxy)propyl-3-(3-methoxy-4-(3-(nitroxy)
acrylate (8a)
The title compound was obtained in 30% as
propoxy)phenyl)acrylate (9a)
The title compound was obtained in 22% as
yellow oil starting from 6a. 1H NMR (CDCl3) d yellow solid starting from 7a; mp 57.9–58.21C. 1H
2.13–2.19 (m, 2H, CH2), 3.94 (s, 3H, OCH3), 4.33 NMR (CDCl3) d 2.15 (t, J 5 6.1 Hz, 2H, CH2), 2.27
(t, J 5 6.1 Hz, 2H, COOCH2), 4.59–4.64 (m, 2H, (t, J 5 6.1 Hz, 2H, CH2), 3.89 (s, 3H, OCH3), 4.15
CH2ONO2), 5.91 (br, 1H, OH), 6.28 (d, J 5 15.9 Hz, (t, J 5 6.0 Hz, 2H, ArOCH2), 4.32 (t, J 5 6.1 Hz, 2H,
1H, C==CH), 6.92 (d, 1H, Ar–H), 7.03–7.10 (m, 2H, COOCH2), 4.62 (t, J 5 6.1 Hz, 2H, CH2ONO2), 4.70
Ar–H), 7.65 (d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 320 (t, J 5 6.4 Hz, 2H, CH2ONO2), 6.30 (d, J 5 15.9 Hz,
[M1Na]1.
1H, C==CH), 6.87 (d, 1H, Ar–H), 7.06–7.10 (m, 2H,
Ar–H), 7.64 (d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 423
[M1Na]1.
(E)-4-(nitroxy)butyl-3-(4-hydroxy-3-methoxyphenyl)
acrylate (8b)
The title compound was obtained in 92% as white (E)-4-(nitroxy)butyl-3-(3-methoxy-4-(4-(nitroxy)
solid starting from 6b; mp 58.1–62.71C. 1H NMR butoxy)phenyl)acrylate (9b)
(CDCl3) d 1.81–1.90 (m, 4H, CH2), 3.93 (s, 3H,
The title compound was obtained in 96% as white
OCH3), 4.24 (t, J 5 5.9 Hz, 2H, COOCH2), 4.52 solid starting from 7b; mp 58.1–58.61C. 1 H NMR
(t, J 5 6.2 Hz, 2H, CH2ONO2), 5.90 (s, 1H, OH), (CDCl3) d 1.82–1.86 (m, 4H, CH2), 1.95–1.99 (m, 4H,
6.28 (d, J 5 15.9 Hz, 1H, C==CH), 6.92 (d, 1H, Ar–H), CH2), 3.89 (s, 3H, OCH3), 4.09 (t, J 5 5.9 Hz, 2H,
7.03–7.10 (m, 2H, Ar–H), 7.62 (d, J 5 15.9 Hz, 1H, ArOCH2), 4.25 (t, J 5 5.9 Hz, 2H, COOCH2),
CH==C). ESI-MS: 334 [M1Na]1.
4.50–4.59 (m, 4H, CH2ONO2), 6.30 (d, J 5 15.9 Hz,
1H, C==CH), 6.85 (d, 1H, Ar–H), 7.05–7.10 (m, 2H,
Ar–H), 7.62 (d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 451
[M1Na]1.
(E)-5-(nitroxy)pentyl-3-(4-hydroxy-3-methoxyphenyl)
acrylate (8c)
The title compound was obtained in 83% as
yellow solid starting from 6c; mp 51.3–53.71C. 1H (E)-5-(nitroxy)pentyl-3-(3-methoxy-4-(5-(nitroxy)
NMR (CDCl3) d 1.49–1.63 (m, 2H, CH2), 1.71–1.84 pentyloxy)phenyl)acrylate (9c)
(m, 4H, CH2), 3.93 (s, 3H, OCH3), 4.21 (t, J 5 6.4 Hz,
The title compound was obtained in 86% as yellow
2H, COOCH2), 4.47 (t, J 5 6.5 Hz, 2H, CH2ONO2), solid starting from 7c; mp 75.51C. 1 H NMR (CDCl3) d
5.91 (s, 1H, OH), 6.28 (d, J 5 15.9Hz, 1H, C==CH), 1.52–1.67 (m, 4H, CH2), 1.71–1.93 (m, 8H, CH2), 3.90
6.92 (d, 1H, Ar–H), 7.03–7.09 (m, 2H, Ar–H), 7.61 (s, 3H, OCH3), 4.06 (t, J 5 6.4 Hz, 2H, ArOCH2), 4.22
(d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 348 [M1Na]1. (t, J 5 6.4 Hz, 2H, COOCH2), 4.48 (m, 4H, CH2ONO2),
6.30 (d, J 5 15.9 Hz, 1H, C==CH), 6.85 (d, 1H, Ar–H),
(E)-6-(nitroxy)hexyl-3-(4-hydroxy-3-methoxyphenyl)
acrylate (8d)
7.07 (m, 2H, Ar–H), 7.62 (d, J 5 15.9 Hz, 1H, CH==C).
ESI-MS: 479 [M1Na]1.
The title compound was obtained in 89% as white
solid starting from 6d; mp 64.1–66.61C. 1 H NMR (E)-6-(nitroxy)hexyl-3-(3-methoxy-4-(6-(nitroxy)
(CDCl3) d 1.45–1.49 (m, 4H, CH2), 1.70–1.78 (m, 4H, hexyloxy)phenyl)acrylate (9d)
CH2), 3.92 (s, 3H, OCH3), 4.20 (t, J 5 6.5 Hz, 2H,
The title compound was obtained in 17% as
COOCH2), 4.46 (t, J 5 6.6 Hz, 2H, CH2ONO2), 5.90 yellow solid starting from 7d; mp 55.8–56.81C. 1H
(s, 1H, OH), 6.28 (d, J 5 15.9 Hz, 1H, C==CH), 6.91 NMR (CDCl3) d 1.45–1.52 (m, 8 H, CH2), 1.70–1.89
(d, 1H, Ar–H), 7.04–7.09 (m, 2H, Ar–H), 7.61 (m, 8H, CH2), 3.89 (s, 3H, OCH3), 4.05 (t, J 5 6.6 Hz,
(d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 362 [M1Na]1. 2H, ArOCH2), 4.20 (t, J 5 6.6 Hz, 2H, COOCH2), 4.46
(t, J 5 6.6 Hz, 4H, CH2ONO2), 6.30 (d, J 5 15.9 Hz, 1H,
(E)-((E)-4-(nitroxy)but-2-enyl)-3-(4-hydroxy-3-methoxy-
phenyl)acrylate (8e)
C==CH), 6.85 (d, 1H, Ar–H), 7.07 (m, 2H, Ar–H), 7.62
(d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 507 [M1Na]1.
The title compound was obtained in 46% as yellow
solid starting from 6e; mp 71.1–71.91C. 1 H NMR (E)-3-(4-(ethoxycarbonyloxy)-3-methoxyphenyl)
(CDCl3) d 3.93 (s, 3H, OCH3), 4.74 (d, J 5 5.1 Hz, 2H, acrylic acid (10)
COOCH2), 4.94 (d, J 5 6.2 Hz, 2H, CH2ONO2), 5.92
To a stirring solution of FA (11.6 g, 60mmol)
(s, 1H, OH), 6.03–6.12 (m, 2H, CH==CH), 6.30 dissolved in 75ml NaOH (1mol/L) at room temperature
(d, J 5 15.9 Hz, 1H, C==CH), 6.92 (d, 1H, Ar–H), was added ethyl chlorformate (7.1 ml, 75 mmol); then
7.03–7.10 (m, 2 H, Ar–H), 7.64 (d, J 5 15.9 Hz, 1H, the reaction mixture was stirred at 501C for 4 h. The
CH==C). ESI-MS: 332 [M1Na]1.
solution was cooled to room temperature and then
Drug Dev. Res.

410
LI ET AL.
poured into cold water, the solid obtained was filtered to 6.76–7.82 (m, 12H, Ar–H), 7.81 (d, J 5 15.9 Hz, 1H,
give the target compound 10 in 90% yield as white solid CH==C). ESI-MS: 555 [M1Na]1.
that was used in the next step without purication; mp
177.4–178.41C. 1H NMR (CDCl3) d 1.40 (t, J 5 7.1 Hz,
3H, CH3), 3.91 (s, 3H, OCH3), 4.35 (q, J 5 7.1 Hz, 2H,
OCH2), 6.48 (d, J 5 15.9 Hz, 1H, C==CH), 7.15–7.19
(m, 3H, Ar–H), 7.82 (d, J 5 15.9 Hz, 1H, CH==C). ESI-
MS: 289 [M1Na]1.
(E)-3-((4-phenyl-1,2,5-oxadiazol-3-yl)methoxy)
benzyl-3-(4-(ethoxycarbonyloxy)-3-methoxyphenyl)
acrylate (11d)
The title compound was obtained in 55% yield as
yellow oil starting from 10 and 3d. 1H NMR (CDCl3) d
1.38 (t, J 5 7.1 Hz, 3H, CH3), 3.88 (s, 3H, OCH3), 4.33
(q, J 5 7.1 Hz, 2H, OCH2), 5.15 (s, 2H, CH2), 5.22
(s, 2H, Ar–CH2), 6.45 (d, J 5 15.9 Hz, 1H, C==CH),
6.90–7.92 (m, 12H, Ar–H), 7.71 (d, J 5 15.9 Hz, 1H,
CH==C). ESI-MS: 569 [M1Na]1.
General Procedure for the Preparation of 11(a–e)
and 13(a–d)
To a stirring solution of 10 (2.1 mmol), DCC
(433 mg, 2.1 mmol), and DMAP (25.5 mg, 0.21 mmol)
in dry CH2Cl2 (40 ml) at room temperature was added
3 or 4 (2.1 mmol); then the reaction mixture was stirred
at room temperature for 4–10 h, at last the reaction
mixture was filtered. Removal of the solvent under
vacuo followed by silica gel column chromatographic
purification of the residue using 20% ethyl acetate in
petroleum ether afforded the compounds (yields
36–83%).
(E)-4-((4-phenyl-1,2,5-oxadiazol-3-yl)methoxy)
benzyl-3-(4-(ethoxycarbonyloxy)-3-methoxyphenyl)
acrylate (11e)
The title compound was obtained in 55% yield as
yellow solid starting from 10 and 3e; mp 50.2–53.11C.
1H NMR (CDCl3) d 1.38 (t, J 5 7.1 Hz, 3H, CH3), 3.87
(s, 3H, OCH3), 4.32 (q, J 5 7.1 Hz, 2H, OCH2), 5.11
(s, 2H, CH2), 5.19 (s, 2H, Ar–CH2), 6.41 (d, J 5
15.9 Hz, 1H, C==CH), 6.99–7.89 (m, 12H, Ar–H),
7.68 (d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 569
[M1Na]1.
(E)-(4-phenyl-1,2,5-oxadiazol-3-yl)methyl-3-(4-(ethoxy-
carbonyloxy)-3-methoxyphenyl)acrylate (11a)
The title compound was obtained in 83% yield
as yellow solid starting from 10 and 3a; mp
130.2–131.01C. 1H NMR (CDCl3) d 1.39 (t, J 5
7.1 Hz, 3H, CH3), 3.88 (s, 3H, OCH3), 4.32 (q,
J 5 7.1 Hz, 2H, OCH2), 5.28 (s, 2H, CH2), 6.38
(d, J 5 15.9 Hz, 1H, C==CH), 7.09–7.18 (m, 3H,
Ar–H), 7.53–7.58 (m, 3H, Ar–H), 7.66 (d, J 5
15.9 Hz, 1H, CH==C), 7.73 (m, 2H, Ar–H). ESI-MS:
463 [M1Na]1.
(E)-2-(4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yloxy)
ethyl-3-(4-(ethoxycarbonyloxy)-3-methoxyphenyl)
acrylate (13a)
The title compound was obtained in 38% yield as
yellow oil starting from 10 and 4a. 1H NMR (CDCl3) d
1.39 (t, J 5 7.1 Hz, 3H, CH3), 3.90 (s, 3H, OCH3), 4.33
(q, J 5 7.1 Hz, 2H, OCH2), 4.62 (m, 2H, COOCH2),
4.71 (m, 2H, CH2), 6.41 (d, J 5 15.9 Hz, 1H, C==CH),
7.13–8.07 (m, 8H, Ar–H), 7.71 (d, J 5 15.9 Hz, 1H,
CH==C). ESI-MS: 557 [M1Na]1.
(E)-3-((4-phenyl-1,2,5-oxadiazol-3-yl)methoxy)
phenyl-3-(4-(ethoxycarbonyloxy)-3-methoxyphenyl)
acrylate (11b)
The title compound was obtained in 60% yield as
yellow solid starting from 10 and 3b; mp 109.2–111.21C.
1H NMR (CDCl3) d 1.40 (t, J 5 7.1 Hz, 3H, CH3), 3.91
(s, 3H, OCH3), 4.33 (q, J 5 7.1 Hz, 2H, OCH2), 5.11
(s, 2H, CH2), 6.45 (d, J 5 15.9 Hz, 1H, C==CH),
6.84–7.82 (m, 12H, Ar–H), 7.80 (d, J 5 15.9 Hz, 1H,
CH==C). ESI-MS: 555 [M1Na]1.
(E)-4-(4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yloxy)
butan-2-yl-3-(4-(ethoxycarbonyloxy)-3-methoxy-
phenyl)acrylate (13b)
The title compound was obtained in 36% yield as
white oil starting from 10 and 4b. 1H NMR (CDCl3) d
1.40 (m, 6H, CH3), 2.20 (m, 2H, CH2), 3.89 (s, 3H,
OCH3), 4.30 (q, J 5 7.1 Hz, 2H, OCH2), 4.50
(t, J 5 6.2 Hz, 2H, CH2O), 5.24 (m, 1H, COOCH),
6.36 (d, J 5 15.9 Hz, 1H, C==CH), 7.02–8.09 (m, 8H,
Ar–H), 7.61 (d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 585
[M1Na]1.
(E)-4-((4-phenyl-1,2,5-oxadiazol-3-yl)methoxy)
phenyl-3-(4-(ethoxycarbonyloxy)-3-methoxyphenyl)
acrylate (11c)
The title compound was obtained in 67% yield as (E)-4-(4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yloxy]
yellow solid starting from 10 and 3c; mp 92.7–93.61C. butyl3-(4-(ethoxycarbonyloxy)-3-methoxyphenyl)
1H NMR (CDCl3) d 1.40 (t, J 5 7.1Hz, 3 H, CH3), 3.91 acrylate (13c)
(s, 3H, OCH3), 4.33 (q, J 5 7.1 Hz, 2H, OCH2), 5.08
The title compound was obtained in 38% yield as
(s, 2 H, CH2), 6.57 (d, J 5 15.9 Hz, 1H, C==CH), white solid starting from 10 and 4c; mp 107.5–108.01C.
Drug Dev. Res.

NOVEL NO-DONOR-FERULIC ACID HYBRIDS
411
1H NMR (CDCl3) d 1.39 (t, J 5 4.2 Hz, 3H, CH3), 1.91 (E)-4-((4-phenyl-1,2,5-oxadiazol-3-yl)methoxy)
(m, 2H, CH2), 2.03 (m, 2H, CH2), 3.89 (s, 3H, OCH3), phenyl-3-(4-hydroxy-3-methoxyphenyl)acrylate (12c)
4.30 (m, 4H, OCH2), 4.50 (t, J 5 3.8 Hz, 2 H, CH2O), The title compound was obtained in 79% yield as
6.42 (d, J 5 9.6 Hz, 1H, C==CH), 7.11–7.16 (m, 3H, yellow solid starting from 11c; mp 123.2–126.71C. 1H
Ar–H), 7.60–7.76 (m, 3H, Ar–H), 7.66 (d, J 5 9.6 Hz, NMR (CDCl3) d 3.95 (s, 3H, OCH3), 5.10 (s, 2H, CH2),
1H, CH==C), 8.05–8.07 (m, 2H, Ar–H). ESI-MS: 585 5.93 (s, 1H, OH), 6.47 (d, J 5 15.9 Hz, 1H, C==CH),
[M1Na]1.
6.94–7.82 (m, 12H, Ar–H), 7.78 (d, J 5 15.9 Hz, 1H,
CH==C). ESI-MS: 483 [M1Na]1.
(E)-2-(2-(4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yloxy)
ethoxy)ethyl3-(4-(ethoxycarbonyloxy)-3-methoxy-
phenyl)acrylate 13d)
The title compound was obtained in 81% yield as
white oil starting from 10 and 4d. 1H NMR (CDCl3) d
1.41 (m, 3H, CH3), 3.86 (s, 3H, OCH3), 3.88–4.61 (m,
10H, OCH2), 6.39 (d, J 5 15.9 Hz, 1H, C==CH),
7.11–7.15 (m, 3H, Ar–H), 7.56–7.63 (m, 3H, Ar–H),
7.69 (d, J 5 15.9 Hz, 1H, CH==C) 8.05–8.08 (m, 2 H,
Ar–H). ESI-MS: 601 [M1Na]1.
(E)-3-((4-phenyl-1,2,5-oxadiazol-3-yl)methoxy)
benzyl-3-(4-hydroxy-3-methoxyphenyl)acrylate (12d)
The title compound was obtained in 73% yield as
white solid starting from 11d; mp 118.8–120.41C. 1H
NMR (CDCl3) d 3.92 (s, 3H, OCH3), 5.13 (s, 2H,
CH2), 5.17 (s, 2H, COOCH2), 5.91 (s, 1H, OH), 6.35
(d, J 5 15.9 Hz, 1H, C==CH), 6.90–7.87 (m, 12H,
Ar–H), 7.67 (d, J 5 15.9 Hz, 1H, CH==C). ESI-MS:
497 [M1Na]1.
General Procedure for the Preparation of 12(a–e)
and 14(a–d)
(E)-4-((4-phenyl-1,2,5-oxadiazol-3-yl)methoxy)
benzyl-3-(4-hydroxy-3-methoxyphenyl)acrylate (12e)
The title compound was obtained in 91% yield as
yellow oil starting from 11e. 1H NMR (CDCl3) d 3.91
(s, 3H, OCH3), 5.11 (s, 2H, CH2), 5.18 (s, 2H,
COOCH2), 5.92 (b, 1H, OH), 6.31 (d, J 5 15.9 Hz,
1H, C==CH), 6.89–7.83 (m, 12H, Ar–H), 7.64
(d, J 5 15.9 Hz, 1H, CH==C). ESI-MS: 497 [M1Na]1.
To a stirring solution of 11 or 13 (1.0 mmol) in
95% EtOH (20 ml) was added ethanolamine (0.26 ml,
4.0 mmol) at room temperature, after 30 min, 2 ml HCl
(2 mol/L) was added to the reaction mixture; then the
EtOH was removed under vacuo. The residue was
diluted with water and extracted with EtOAc. The
obtained organic layer was dried, filtered, and evapo-
rated under vacuo. The crude product was purified by
silica gel column chromatography using 30% ethyl
acetate in petroleum ether afforded the compounds
(yields 64–98%).
(E)-2-(4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yloxy)
ethyl-3-(4-hydroxy-3-methoxyphenyl)acrylate (14a)
The title compound was obtained in 82% yield as
red solid starting from 13a; mp 139.2–141.11C. 1H
NMR (CDCl3) d 3.94 (s, 3H, OCH3), 4.61 (t, J 5 2.4 Hz,
2H, CH2O), 4.71 (t, J 5 2.4 Hz, 2H, COOCH2), 5.88
(s, 1H, OH), 6.31 (d, J 5 15.9 Hz, 1H, C==CH),
7.05–7.12 (m, 3H, Ar–H), 7.63 (d, J 5 15.9 Hz, 1H,
CH==C), 7.54–7.73 (m, 3H, Ar–H), 8.06 (d, J 5 7.3 Hz,
2H, Ar–H). ESI-MS: 485 [M1Na]1.
(E)-(4-phenyl-1,2,5-oxadiazol-3-yl)methyl 3-(4-
hydroxy-3-methoxyphenyl) acrylate (12a)
The title compound was obtained in 89% yield as
yellow solid starting from 11a; mp 142.7–145.31C. 1H
NMR (CDCl3) d 3.92 (s, 3H, OCH3), 5.26 (s, 2H, CH2),
5.91 (s, 1H, OH), 6.27 (d, J 5 15.9 Hz, 1H, C==CH),
6.91 (d, 1H, Ar–H), 7.00–7.08 (m, 2H, Ar–H), 7.52–7.57
(m, 3H, Ar–H), 7.63 (d, J 5 15.9 Hz, 1H, CH==C),
7.73–7.77 (m, 2H, Ar–H). ESI-MS: 391 [M1Na]1.
(E)-4-(4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yloxy)
butan-2-yl-3-(4-hydroxy-3-methoxyphenyl)acrylate
(14b)
The title compound was obtained in 98% yield as
yellow solid starting from 13b; mp 70.1–73.21C. 1H
NMR (CDCl3) d 1.40 (d, J 5 6.2 Hz, 3H, CH3), 2.22
(E)-3-((4-phenyl-1,2,5-oxadiazol-3-yl)methoxy)
phenyl-3-(4-hydroxy-3-methoxyphenyl)acrylate (12b)
The title compound was obtained in 64% yield as (m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.53 (t, J 5 6.2 Hz,
white solid starting from 11b; mp 57.3–59.11C. 1H 2H, CH2O), 5.26 (q, J 5 6.2 Hz, 1H, COOCH), 5.90 (b,
NMR (CDCl3) d 3.95 (s, 3H, OCH3), 5.10 (s, 2H, CH2), 1H, OH), 6.29 (d, J 5 15.9 Hz, 1H, C==CH), 6.90–7.08
5.92 (s, 1H, OH), 6.45 (d, J 5 15.9 Hz, 1H, C==CH), (m, 3H, Ar–H), 7.61 (d, J 5 15.9 Hz, 1H, CH==C),
6.84–7.86 (m, 12H, Ar–H), 7.80 (d, J 5 15.9 Hz, 1H, 7.60–7.77 (m, 3H, Ar–H), 8.08 (m, 2H, Ar–H). ESI-
CH==C). ESI-MS: 483 [M1Na]1.
MS: 513 [M1Na]1.
Drug Dev. Res.

412
LI ET AL.
(E)-4-(4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yloxy)
DPPH radical was expressed as a percentage:
butyl-3-(4-hydroxy-3-methoxyphenyl)acrylate (14c)
The title compound was obtained in 92% yield as
light brown solid starting from 13c; mp 143.3–146.91C.
1H NMR (CDCl3) d 1.88–2.05 (m, 4H, 2CH2),
3.91 (s, 3H, OCH3), 4.30 (t, J 5 6.2 Hz, 2H, CH2O),
4.50 (t, J 5 6.2 Hz, 2H, COOCH2), 5.88 (b, 1H,
OH), 6.29 (d, J 5 15.9 Hz, 1H, C==CH), 6.91–7.09
(m, 3H, Ar–H), 7.62 (d, J 5 15.9 Hz, 1H, CH==C),
7.62–7.77 (m, 3H, Ar–H), 8.06 (m, 2H, Ar–H). ESI-MS:
513 [M1Na]1.
Scavenged DPPH ð%Þ ¼ ð1 ꢁ Atest=AcontrolÞ ꢃ 100;
ð1Þ
where Atest is the absorbance of a sample at a given
concentration after 10 min reaction time and Acontrol is
the absorbance recorded for 10 ml ethanol. The EC50
value is defined as the concentration of sample that
causes 50% loss of the DPPH radical.
All the final compounds were assessed as inhibitors
of ferrous salt/ascorbate induced peroxidation of mem-
brane lipids of rat hepatocytes. FA was used for
comparison. The 2-thiobarbituric acid (TBA) assay was
used to follow the progress of the autooxidation. The
procedure involves the detection of the final metabolites
of the lipid autooxidation, namely 2-thiobarbituric acid
reactive substances (TBARS) by visible spectroscopy
[Buege and Aust, 1978]. Female Wister rats weighing
250720 g were starved overnight before cervical disloca-
tion. Liver microsomes were prepared by tissue homo-
genization with ice-cold 0.25 M sucrose–0.01 M Tris
buffer, pH 7.4, with 1 mM EDTA (STE buffer), in a
motor-driven glass homogenizer. Microsomal fractions
were isolated by the removal of nuclear faction at 8,000 g
for 20 min and removal of mitochondrial fraction at
18,000 g for 10 min. The microsomal fraction was
sedimented in a CP100MX ultracentrifuge at 105,000 g
for 60 min, washed two times with 0.15 M KCl at
105,000 g for 30 min. The membranes, suspended in
0.1 M potassium phosphate buffer, pH 7.5, were stored in
a deep freezer maintained at ꢁ201C. Rat liver micro-
somes were incubated at 371C in a Tris-HCl/KCl buffer
(100 mM, 150 mM, pH 7.4), containing microsomal
membranes (2 mg mlꢁ1), ascorbic acid (100 mM), and
either ethanol solutions of the tested compounds or
ethanol alone. Lipid peroxidation was initiated by the
addition of FeSO4 (2.5 mm). Aliquots were taken from the
incubation mixture at 5, 15, and 30 min and were treated
with trichloroacetic acid (TCA, 10% w/v) and 2-thiobar-
bituric acid (TBA, 2% w/v). Lipid peroxidation was
assessed by spectrophotometric (l5532 nm) determina-
tion [25] of the TBARS consisting mainly of malondial-
dehyde (MDA), and TBARS concentrations (expressed in
nmol mgꢁ1 protein) were obtained by interpolation with
an MDA standard curve. The antioxidant activity of tested
compounds was evaluated the percentage inhibition of
TBARS production with respect to control samples
treated with ethanol alone. IC50 values were calculated
by nonlinear regression analysis.
(E)-2-(2-(4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yloxy)
ethoxy)ethyl-3-(4-hydroxy-3-methoxyphenyl)
acrylate (14d)
The title compound was obtained in 87% yield as
white solid starting from 13d; mp 115.5–115.81C. 1H
NMR (CDCl3) d 3.86 (t, J 5 4.7 Hz, 2H, OCH2), 3.91
(s, 3H, OCH3), 3.93 (t, J 5 3.5 Hz, 2H, CH2O), 4.39
(t, J 5 4.7 Hz, 2H, CH2O), 4.59 (t, J 5 3.5 Hz, 2H,
COOCH2), 5.89 (b, 1H, OH), 6.29 (d, J 5 15.9 Hz, 1H,
C==CH), 6.88–7.09 (m, 3H, Ar–H), 7.63 (d, J 5 15.9 Hz,
1H, CH==C), 7.51–7.74 (m, 3H, Ar–H), 8.06 (m, 2H,
Ar–H). ESI-MS: 529 [M1Na]1.
BIOLOGICAL SCREENING
Antioxidant Assays by Evaluation of DPPH Radical
Scavenging Activity and Inhibition of Lipid
Peroxidation in Rat Liver Homogenate
DPPH assay measures the hydrogen-donating
ability of antioxidants to convert the stable DPPH free
radical into 1,1-diphenyl-2-picrylhydrazine [Brand-
Williams et al., 1995]. The reaction is accompanied
by a change in color from deep-violet to light-yellow
and is monitored spectrophotometrically. FA serves as
reference antioxidants in this assay. DPPH radical
scavenging activity was determined using the method
of Wang et al. [Wang et al., 2008] with minor
modifications. The solution of the sample (10 ml) in
ethanol was added to 90 ml of a 0.1 mM DPPH radical
in ethanol in a 96-well plate. The sample solution refers
to the tested compounds and the reference antioxidants
at various concentrations, as well as ethanol as a
control. The solutions of the tested compounds had
concentrations ranging from 3 mg/ml to 1,000 mg/ml,
whereas the concentrations of the reference com-
pounds solutions varied from 0.1 mg/ml to 1,000 mg/ml.
The reaction leading to the scavenging of DPPH
radical was complete within 10 min at 251C. The
absorbance of the mixture was then measured at
NO Release Assay In Vitro
The ability of the target compounds to release
517 nm using a microplate reader. The reduction of NO was indirectly evaluated through their capacity to
Drug Dev. Res.

NOVEL NO-DONOR-FERULIC ACID HYBRIDS
413
produce nitrite in physiological pH 7.4 buffered water
RESULTS AND DISCUSSION
Antioxidant Activity
and kept at 371C for 2 and 4 h in the presence of a
strong excess of cysteine (1:50). Nitrite was the most
important product of the oxidation of NO in aerobic
aqueous solution. Detection of nitrite can be used to
infer the previous presence of NO. It was detected by
Griess reaction, according to the procedure previously
described [Sorba et al., 1997]. Isosorbide dinitrate
(ISDN) was chosen as a positive control. A solution of
the appropriate 80 ml furoxan (0.01 mol/L) in DMSO
was added to 8 ml phosphate buffer (pH 7.4) contain-
ing 5 mmol/L L-cysteine. The final concentration of
drug was 10ꢁ4 mol/L. After 2 h at 371C, 100 ml of the
reaction mixture was treated with 100 ml of the Griess
reagent [sulfanilamide (4 g), N-naphthylethylenedia-
mine 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 550 nm; 6.25–200 mmol/ml sodium nitrite
standard solutions were used for the calibration curve.
Detection of nitrite can be used to infer the previous
presence of NO.
Table 1 summarizes the results obtained in the
DPPH and lipid peroxidation assay. The dual nitrates 9,
where both the hydroxyl groups were occupied by
nitrates, were screened as side-products in the reaction
and had no antioxidant activity. While the mono-
nitrates 8, phenylfuroxan nitrates 12 and phenylsulfo-
nylfuroxan nitrates 14 had a higher antioxidant activity
than the dual nitrates 9, suggesting the FA phenolic
hydroxyl group was required for free radical scavenging
activity. In the series of mono-nitrates 8, the saturated
nitrates (8a–8d) showed a higher antioxidant activity
than the unsaturated one (8e). Furthermore, the result
showed that the antioxidant activity decreased as the
number of atoms in the nitric esters increased, such as
8c (IC50 69.3 mM for DPPH, 67.2 mM for lipid
peroxidation) and 8d (IC50 94.1 mM for DPPH,
99.7 mM for lipid peroxidation). In the series of
phenylfuroxan nitrates 12, no substituent between FA
and phenylfuroxan 12a (IC50 98.3 mM for DPPH,
104.4 mM for lipid peroxidation) showed a much higher
antioxidant activity than analogues with benzene ring
substituent (12b, 12c, 12e), with 12d as an exception
(IC50 85.6 mM for DPPH, 94.8 mM for lipid peroxida-
tion). For the series of phenylsulfonylfuroxan nitrates
14, the butyl ether derivatives 14b (IC50 83.4 mM for
DPPH, 89.7 mM for lipid peroxidation) and 14c (IC50
66.9 mM for DPPH, 72.1 mM for lipid peroxidation) had
a much higher antioxidant activity than the diethyl
ether derivatives 14a (IC50 154.3 mM for DPPH,
165.4 mM for lipid peroxidation) and 14d (IC50
124.7 mM for DPPH, 130.4 mM for lipid peroxidation).
Vasodilator Activity
The vasodilating activity of the target compounds
was determined through an in vitro vascular relaxation
assay (organ bath) using PGF2a-precontracted porcine
pulmonary arteries. ISDN was chosen as positive
controls. Pulmonary arteries were cut into rings
(2 mm long, 1.5–2-mm diameter) [Klinger and Mu¨ller,
1974]. The rings were suspended between two
L-shaped platinum hooks and mounted in a 10-ml
organ bath filled with modified a Krebs–Henseleit
solution. The solution was kept at 371C and aerated
with 95% O2/5% CO2. Preparations were connected to
an isometric force transducer (BL-410, Chengdu
Technology and Market, China) for continuous mea-
surement of changes in tension. A resting tension of 20
mN was maintained throughout the experiment. After
an equilibration period of 60 min, contractions were
induced at intervals of 45 min. The ring segments were
initially made to contract with KCl (45 mM) and
subsequently with PGF2a (2–3 ꢃ 3 mM) until the
contractions became constant. Endothelial integrity
Nitric Oxide Release Assay In Vitro
Comparison of the data reported in Table 2
indicates that the mono-nitrates 8 generated the
TABLE 1. In Vitro Antioxidant Activity of NO-Donor-Ferulic Acid
Hybrids (IC50 in lM) in DPPH and Lipid Peroxidation Assays
Lipid
peroxidation
(mM)
Lipid
DPPH
(mM)
DPPH peroxidation
(mM)
Compd.
Compd.
(mM)
was assessed by the bradykinin (10 nM)-induced 8a
31.3
54.9
69.3
39.1
56.9
67.2
12a
12b
12c
12d
12e
14a
14b
14c
14d
98.3
165.6
196.5
85.6
256.4
154.3
83.4
104.4
174.7
203.2
94.8
175.4
165.4
89.7
8b
relaxation of PGF2a-precontracted vessels. In mechani-
cally endotheliumdenuded arterial rings, pretreated
with 0.2 mM L-NAME, the relaxation induced by
8c
8d
8e
94.1
99.7
258.3
41,000
41,000
41,000
41,000
16.2
260.4
41,000
41,000
41,000
41,000
17.6
bradykinin was less than 10%. The relaxation response
9a
to test compounds was studied after the second or third 9b
9c
9d
FA
66.9
124.7
72.1
130.4
PGF2a-induced contraction had stabilized, by con-
structing a cumulative concentration-response curve.
Results are expressed as EC507SE (mM).
Drug Dev. Res.

414
LI ET AL.
TABLE 2. NO Generation Properties and Vasodilating Potency of the Target Compounds
NOꢁ2 (mM)
NOꢁ2 (mM)
Compd.
2 h
4 h
EC50 (mM)
Compd.
2 h
4 h
EC50 (mM)
8a
8b
8c
8d
8e
9a
9b
9c
9d
ISDN
1.6370.24
1.0070.25
1.0570.39
1.1070.30
1.7970.38
9.9571.25
11.2771.24
10.4570.76
10.5270.45
12.1471.17
2.6170.48
1.3470.28
1.2470.26
0.9670.20
2.2770.23
8.1370.83
8.3770.83
9.8370.60
9.5270.52
14.2370.94
13.4971.09
16.8571.56
14.9671.17
14.3871.27
12.6771.43
0.726770.0088
0.523170.0059
0.641870.0068
0.589770.0082
0.112370.0067
12a
12b
12c
12d
12e
14a
14b
14c
14d
FA
10.7070.78
4.1170.37
3.7170.42
6.5870.65
3.9270.31
22.3271.14
22.0671.33
26.0071.32
27.5371.17
—
12.2270.46
3.4470.25
2.1070.17
5.1770.29
3.1670.24
35.1971.21
34.8671.40
36.7971.95
38.8871.64
—
0.714770.0054
1.643870.0091
1.834270.0094
1.625370.0089
1.721470.0076
0.175470.0091
0.114370.0058
0.092870.0046
0.103970.0077
—
minimum amount of NO, and the introduction of vasodilating potency. The EC50 values followed the
different substituents between FA and nitrate does not sequence 841249414, and the most active com-
modify the extent of the NO release ability induced by pounds belonged to the phenylsulfonyl series 14. 14b
the thiol cofactor, such as 8a and 8e, the nitrites (EC50 0.1143 mM) and 14d (EC50 0.1039 mM) showed
detected were 1.63 mM and 1.79 mM, respectively, after a roughly similar vasodilating activity to those of ISDN
2 h at 371C when treated with 100 ml Griess reagent. (EC50 0.1123 mM). Importantly, Derivative 14c, which
The di-nitrates 9 showed a stronger NO release ability showed the most potent antioxidant activity (72.1 mM
when compared with the mono-nitrates 8, and were as for anti-lipid peroxidation), was more potent than
potent as ISDN taken as reference, however, the NO ISDN in in vitro vascular relaxation assay (EC50 values
release time of di-nitrates 9 was much shorter, such as were 0.0928 mM and 0.1123 mM, respectively).
9a, the nitrites detected were 9.95 mM after 2 h at
Previous study has shown that FA exhibited the
371C, but they were reduced into 8.13 mM after 4 h at antioxidant activity of plasma and the resistance of
371C. The phenyl-substituted furoxans 12 showed a LDLs to oxidation [Ohta et al., 1997; Laranjinha et al.,
slightly stronger NO release ability and were 3–10 1996; Andreasen et al., 2001]. In the present study, we
times stronger than mono-nitrates 8; most of these found that esterification of FA with phenylsulfonyl
series compounds showed a much shorter NO release group through different side chains could afford some
time with 12a as an exception, and the nitrites of 12a derivatives with high antioxidant, NO release, and
detected were added from 10.70 mM after 2 h into vasodilating activity; one of the most important
12.22 mM after 4 h. In the phenylsulfonyl series 14 all examples was 14c, which could potentially be used for
the compounds displayed good NO donating proper- therapeutic intervention of atherosclerosis in the clinic.
ties, and the nitrites detected were about two times
more than that of the positive control ISDN after 2 h at
371C, such as 14a, the nitrites detected after 2 h were
22.32 mM, while the nitrites detected after 2 h from
CONCLUSIONS
In summary, we have designed and synthesized
18 novel ferulic acid hybrid compounds with three
different NO-donating groups (e.g., nitric ester, 4-
hydroxyl-3-phenylfuroxan, and 4-hydroxymethyl-3-
phenylsulfonylfuroxan), via alkyl or benzyl spacer. The
target phenylsulfonylfuroxan compounds 14, in parti-
cular while maintaining antioxidant activity, showed
more NO release activity and vasodilating activity
compared with ISDN. Compound 14c moderately
relaxed the porcine pulmonary arteries in in vitro
vasorelaxation experiments (organ bath), aided by the
NO donor part of the molecule. The results suggest
that these NO-donor-ferulic acid hybrids, especially
ISDN were only 12.14 mM. Furthermore, the substi-
tuents between FA and the phenylsulfonyl nitrate
showed that the NO release activity increased with the
side chain became much longer, such as 14a and 14d,
with the ether group increased from ethyl ether into
diethyl ether, the nitrites detected after 2 h increased
from 22.32 mM into 27.53 mM. In the homologues 14b
and 14c, the nitrites detected after 2 h increased from
22.06 mM into 26.00 mM with the side chain between
FA and the phenylsulfonyl nitrate changed from
isobutyl group into butyl group.
Vasodilating Activity
Analysis of EC50 values reported in Table 2 compound 14c, may be considered to be a novel more
indicates that the compounds covered a wide range of potent drug candidate for anti-atherosclerosis.
Drug Dev. Res.

NOVEL NO-DONOR-FERULIC ACID HYBRIDS
415
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