Synthesis of N-hydroxycinnamoyl amino acid ester analogues and their free radical scavenging and antioxidative activities

Article abstract of DOI:10.1007/s00044-011-9713-2

Hydroxycinnamic acids have a variety of biological activities, including antioxidant activity. To find more active antioxidants with hydroxycinnamoyl moiety, we synthesized a series of N-hydroxycinnamoyl amino acid esters and evaluated their antioxidative activities by 2,2- diphenyl-1-picryl hydrazyl (DPPH) radical scavenging and human red blood cells (RBCs) haemolysis methods. It was found that N-caffeoyl amides exhibited the highest DPPHscavenging activities, whereas N-feruloyl amides demonstrated the highest antihaemolysis activities among the three different hydroxycinnamamides (caffeoyl, feruloyl, and p-coumaroyl), and that hydroxycinnamoyl amides were more effective than their corresponding free acid and ester compounds in both DPPH and RBC haemolysis tests.

Full text of DOI:10.1007/s00044-011-9713-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1905–1911
DOI 10.1007/s00044-011-9713-2
ORIGINAL RESEARCH
Synthesis of N-hydroxycinnamoyl amino acid ester analogues
and their free radical scavenging and antioxidative activities
•
Qing-Yi Wei Hong Jiang Jian-Xun Zhang
•
•
•
Peng-Fei Guo Huan Wang
Received: 13 November 2010 / Accepted: 15 June 2011 / Published online: 30 June 2011
ꢀ Springer Science+Business Media, LLC 2011
Abstract Hydroxycinnamic acids have a variety of bio-
logical activities, including antioxidant activity. To find
more active antioxidants with hydroxycinnamoyl moiety,
we synthesized a series of N-hydroxycinnamoyl amino acid
esters and evaluated their antioxidative activities by 2,2-
diphenyl-1-picryl hydrazyl (DPPH) radical scavenging and
human red blood cells (RBCs) haemolysis methods. It was
found that N-caffeoyl amides exhibited the highest DPPH-
scavenging activities, whereas N-feruloyl amides demon-
strated the highest antihaemolysis activities among the
three different hydroxycinnamamides (caffeoyl, feruloyl,
and p-coumaroyl), and that hydroxycinnamoyl amides
were more effective than their corresponding free acid
and ester compounds in both DPPH and RBC haemolysis
tests.
mediated peroxidation of membrane lipids and oxidative
damage of DNA are associated with a variety of chronic
health problems, such as cancer, atherosclerosis, and age-
ing (Anderson et al., 2001; Lass and Sohal, 1998; Wei
et al., 2006a; Cheng et al., 2006). Therefore, inhibition of
free radical–induced oxidative damage by supplementation
of antioxidants has become an attractive therapeutic strat-
egy for reducing the risk of these diseases (Wei et al.,
2006b; Tsuda et al., 2004).
Phenolic acids, especially hydroxycinnamic acid
derivatives, are widely distributed in plants and found
in considerable amounts in propolis, fruits, vegetables,
and beverages of human diet (Scalbert and Williamson,
2000; Robbins, 2003). As one important kind of these
derivatives, hydroxycinnamamides are also widely
spread in plants (Tanguy et al., 1978; Tanguy, 1985).
The plant hydroxycinnamamides are known to have
antioxidant, HIV-1 integrase, antimicrobial, antiplatelet,
antituberculosis, and tyrosinase properties (Lee et al.,
2003, 2004; Tebayashi et al., 2000; Charvat et al.,
2006; Narasimhan et al., 2004; Lai et al., 2002; Yoya
et al., 2009; Kang et al., 2009). Because of the limi-
tation of availability of hydroxycinnamic acid deriva-
tives from nature, the knowledge to compare their
antioxidative activities is still limited. Therefore, we
synthesized a series of N-hydroxycinnamoyl amino acid
esters and conducted a structure–activity relationship
study of them in this study. In this study, three series of
N-hydroxycinnamoyl amino acid esters (p-coumaroyl-,
feruloyl- and caffeic and their structure were shown in
Scheme 1) were synthesized. Their efficiency as radical
scavengers was evaluated by their activity toward
DPPH-scavenging and their potency as antioxidants was
evaluated by antihaemolysis of human red blood cells
(RBCs).
Keywords Hydroxycinnamic acid Á Antioxidant Á
DPPH Á Haemolysis Á Amide Á Amino acid
Introduction
A large body of clinical and experimental evidences
accumulated in the past decade suggested that free radical–
Q.-Y. Wei
College of Light Industry and Food Sciences, South China
University of Technology, 510641 Guangzhou, China
H. Jiang (&) Á J.-X. Zhang Á P.-F. Guo Á H. Wang
Department of Chemistry, College of Science, Huazhong
Agricultural University, Wuhan 430070, Hubei, China
e-mail: jianghong0066@126.com
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Med Chem Res (2012) 21:1905–1911
O
Scheme 1 The tested
compounds structure
O
O
N
H
O
N
H
O
N
H
O
O
O
O
HO
HO
H3CO
HO
HO
c
b
a
Ph
Ph
Ph
O
O
O
N
H
O
N
H
O
N
H
O
O
O
O
HO
HO
H3CO
HO
HO
d
e
f
OH
OH
O
O
OH
N
O
HO
HO
H
N
O
O
H
O
O
H3CO
HO
O
HO
Vitamin C
g
h
O
O
O
O
O
O
HO
H3CO
HO
HO
HO
Ethyl caffeate (EC)
Ethyl felulate (EF)
Ethyl p-coumarate (EP)
O
O
O
OH
OH
OH
HO
HO
H3CO
HO
HO
Caffeic acid (CA)
Felulic acid (FA)
p-Coumaric acid (CoA)
Results and discussion
free radicals. This assay is based on a decolorization
technique by UV–vis spectroscopy at 517 nm in which the
radical is generated directly in a stable form before reaction
with putative antioxidants. DPPH method has been widely
used to assess radical-scavenging activity of phenolic
compounds (Goupy et al., 2003). On addition of
Scavenging effect on DPPH radicals
The DPPH method was carried out in a homogeneous
phase to measure the ability of antioxidants to intercept
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Med Chem Res (2012) 21:1905–1911
1907
hydroxycinnamic acids analogues to an ethanol solution of
DPPH, the absorption at 517 nm decreased immediately.
The concentrations giving 50% reduction in the absorbance
of DPPH solution (IC50) were shown in Table 1. Their
scavenging activity of DPPH radicals decreased in the
following order: d [ a [ caffeic acid (CA) [ Vitamin
C [ ethyl caffeate (EC) [ ferulic acid (FA) [ e [ b [
h [ ethyl ferulate (EP) [ c, f, g, ethyl p-coumarate (EP). It
can be seen from Table 1 that the activity of these com-
pounds depends significantly on the introduction of elec-
tron-donating groups (hydroxyl and methoxyl). Therefore,
the scavenging activity has the following sequence: CA
derivatives [ FA derivatives [ p-coumaric acid (CoA)
derivatives (as we can see from IC50 values, for amide:
a \ b \ c; d \ e \ f; for ester: EC \ EF \ EP; and for
free acid: CA \ FA \ CoA, respectively). The scavenging
activity for the same parent molecule has the following
order: amide [ ester [ free acid. It is also noticeable that
free CA and its amide derivative are more effective than
Vitamin C (a famous antioxidant, positive control) in
DPPH-scavenging activity.
RBC membranes to induce lipid peroxidation. Then, the
RBC membrane is quickly damaged, leading to haemoly-
sis. Antioxidants (ArOHs) present or added to RBCs react
with the chain-propagating peroxyl radicals to stop the
peroxidation and hence inhibit haemolysis.
Figure 1 shows the AAPH-induced RBC haemolysis in
an aerobic atmosphere. Addition of AAPH induced, after
an inhibition period, fast haemolysis. As the blank (1)
experiment shows, haemolysis does not occur at the
beginning of the reaction because the endogenous antiox-
idants, e.g., vitamin E and/or ubiquinol-10, present in
biomembranes defense the attack from radicals. The
inhibitory time of haemolysis was 108 min in the absence
of added antioxidants when the concentration of AAPH
was 50 mM.
Addition of hydroxycinnamic acid and its analogues
(ArOHs) to the 5% RBC suspension significantly increased
the inhibition time of the RBCs. The inhibition time pro-
duced by ArOHs was illustrated in Fig. 1. The inhibitory
times produced by 10-lM test compounds in decreasing
order were e [ b [ a [ d [ h [ EF [ EA [ c [ Vita-
min C, with the inhibition times, being 198, 193, 180, 160,
158, 150, 148, 138, and 133 min, respectively. It is clearly
seen that the activity of these compounds depends signifi-
cantly on the number of phenolic groups and methoxyl
group in the molecules. Interestingly, N-feruloyl amino
acid esters that possess a methoxyl groups demonstrated
better inhibitory activities towards RBCs haemolysis than
corresponding N-caffeoyl amides of corresponding amino
Inhibition of RBC haemolysis by hydroxycinnamic acid
derivatives
Although DPPH assay has been widely used to conveniently
test the free radical scavenging activity of phenolic com-
pounds, the method is only chemical relevance and the sys-
tem used is a homogenous solution. It has been recognized
that the antioxidant activity in homogenous solutions may
not parallel that in heterogeneous media (Zhou et al., 2005).
Human RBCs are heterogeneous media. Therefore, the
antioxidative effect of hydroxycinnamoyl amide and ester
analogues was investigated in RBC model to evaluate the
influence of microenvironment on the antioxidative activity.
Thermal decomposition of AAPH produces an initiating
radical that can attack the polyunsaturated lipids (LH) in
Table 1 Scavenging activity of antioxidants for DPPH radical
Compounds
IC50 (lM)
Compounds
IC50 (lM)
a
b
c
d
e
f
12.80
64.58
[200
1.45
Ethyl caffeate (EC)
Ethyl ferulate (EF)
Ethyl p-Coumarate (EP)
Caffeic acid (CA)
Ferulic acid (FA)
p-Coumaric acid (CoA)
Vitamin C
48.98
150.02
[200
18.25
50.13
[200
29.61
52.35
[200
[200
87.24
Fig. 1 Inhibition of AAPH-induced haemolysis of 5% human RBCs
in PBS (0.15 M, pH 7.4) in an aerobic atmosphere at 37ꢁC by
hydroxycinnamic derivatives (ArOH). [ArOH]0 = 10 lM and
[AAPH]0 = 50 mM. The numbers on the lines represent different
compounds: (1) native RBCs, (2) e, (3) b, (4) a, (5) d, (6) h, (7) EF,
(8) EA, (9) c, and (10) Vitamin C. Lines for compound g, f, CA, FA,
EP, and CoA are not shown for clarity. Data are expressed as
means SD
g
h
Note: Vitamin C was used for positive control. IC50 value was
determined to be the effective concentration at which DPPH radical
was scavenged by 50%. The IC50 value was obtained by interpolation
from linear regression analysis
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Med Chem Res (2012) 21:1905–1911
acid. Therefore, compound e, the most active one, is fol-
lowed by compound b.
it impossible to form quinone oxidation products, so they are
inefficient in quenching radicals in DPPH systems. So CA
and its derivatives are more effective than FA corresponding
derivatives in DPPH system. Accordingly, it is understand-
able for the DPPH-scavenging ability in the following order
d [ e [ f. Compound g, with two hydroxyl groups on dif-
ferent phenyl ring, cannot form intramolecular hydrogen
bond, so its antiradical and antihaemolysis activities were
lower than CA and FA analogues.
The antioxidant mechanism
It is proposed nowadays that the use of more than one marker
of oxidation is required to evaluate the efficacy of an anti-
oxidant. Hence, we evaluated the antioxidant effects of
hydroxycinnamic acid analogues to scavenge DPPH and
protection of human red blood cells (RBCs) from oxidative
haemolysis.
In the biomembrane systems, antioxidative activity
depends not only on the number of hydroxyl groups but
also on the solubility, hydrophobicity (or partition coeffi-
cient, log P) (Rice-Evans et al., 1996). It has been recog-
nized that peroxyl radicals produced by AAPH in the
aqueous phase attack phospholipids at the membrane sur-
face (Niki, 1990), suggesting that antioxidants need to be
located near the surface of the membrane to act, so the
lipophilicity of the antioxidants makes great effects in
inhibiting the lipid peroxidation. It is reasonable for the
highly active antihaemolysis of N-feruloyl amino acid
esters, especially for compounds e, for its high log P val-
ues. For the same reason, amide and ester have better
activity compared with their corresponding free acid.
In conclusion, our results show that the synthetic amide
analogues of hydroxycinnamic acid exhibit stronger antioxi-
dativeactivity than their corresponding freeacids and estersin
the both assay systems. The observation that the feruloyl
amides bearing ortho-methoxyl group vicinal to the hydroxyl
functionality exhibit markedly higher antihaemolytic activi-
ties gives us useful information for antioxidant drug design.
The structural feature responsible for the better DPPH free
radical scavenging activity of d, a, and CA is the ortho-
dihydroxyl functionality in the catechol ring; therefore, the
phenolic group plays a major role in the activity of
hydroxycinnamic acid analogues. The presence of the elec-
tron-donating hydroxyl group at the ortho-position makes
H-atom transfer to peroxyl radicals easier (Lucarini et al.,
2002), resulting in the formation of a phenoxy radical. It was
also known that the ortho-hydroxyl substitution on phenol
would make the oxidation intermediate, ortho-hydroxy-
phenoxyl radical, more stable because of the intramolecular
hydrogen bonding interaction as reported recently from both
experimental (Foti and Ruberto, 2001) and theoretical cal-
culations (Wright et al., 2001). In addition, ortho-OH phe-
noxyl radical and/or ortho-semiquinone radical anion shall
be easier to further oxidize to form the final product ortho-
quinone (Scheme 2). Although the ortho-methoxyl group
can also form intramolecular hydrogen bond with the phe-
nolic hydrogen, methoxylation of one hydroxyl group makes
O
O
O
O
C
OH LOO
OH
LOOH
HN
HN
H
O
O
C
R1
C
R1
OR2
OR2
O
O
O
HN
LOO
LOOH
C
O
O
HN
O
OH
C
R1
O
C
R1
OR2
OR2
Scheme 2 The proposed antioxidative mechanism of N-hydroxycinnamoyl amino acid ester analogues
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Med Chem Res (2012) 21:1905–1911
Experiment
1909
(C=C–H), 1,728 (C=O), 1,654 (O=C–N), 1,605 (C=C). MS
(m/z, %): 264.7 (M1, 75.0), 219 (76.5), 55 (100).
Materials and methods
Ethyl N-[3-(4-hydroxy-3-methoxyphenyl)-1-oxo-2-propen-
1-yl] glycinate (b)
CA, FA, and CoA were purchased from Sigma Chemical
(St. Louis, MO, USA) and DPPH (1,1-Diphenyl-2-picryl-
hydrazyl) and AAPH (2,2’-Azobis(2-amidinopropane
hydrochloride)) were purchased from Aldrich (St. Louis,
MO, USA). All other chemicals were of the highest quality
available.
Synthesized compounds were identified by IR, 1HNMR,
and MS analysis. Melting points were determined on a RY-
2 melting apparatus; IR spectra were recorded on a Avatar
330 infrared spectrophotometer (KBr pellet); 1H NMR data
were acquired on a Bruker AV 400-MHz operating at
400 MHz. CDCl3 or DMSO-d6 was used as solvent; mass
is performed on a Saturn 2000 mass spectrometer.
A Spectronic Genesys 8 UV/VIS spectrophotometer was
used in the DPPH and RBC haemolysis assays.
Compound b, pale yellow solid, m.p. 154–155ꢁC, 1H NMR
(400 MHz, CDCl3), 7.595 (d, 1H, J = 15.6, HC=C), 7.083
(d, 1H, J = 8.0, ArH), 7.012 (S, 1H, ArH), 6.924 (d, 1H,
J = 8.0, ArH), 6.329 (d, 1H, J = 15.6, C=CH), 6.118 (br s,
1H, NH), 5.877 (br s, 1H, OH), 4.270–4.252 (q, 2H,
J = 7.2, –OCH2), 4.181 (d, 2H, J = 5.2, –CH2), 3.9725 (s,
3H, OCH3), 1.328–1293 (t, 3H, J = 7.2, –CH3). IR (KBr)
t, cm-1: 3,503 (HO–), 3,365 (N–H), 3,046 (Ar–H), 3,027
(C=C–H), 1,728 (C=O), 1,654 (O=C–N), 1,605 (C=C). MS
(m/z, %): 278 (M1, 100.0).
Ethyl N-[3-(4-hydroxyphenyl)-1-oxo-2-propen-1-yl]
glycinate (c)
Synthetic procedure for N-Hydroxycinnamoyl amino
acid esters
Compound c, white solid, m.p. 169–170ꢁC, 1H NMR
(400 MHz, CDCl3), 7.609 (d, 1H, J = 15.6, HC=C), 7.410
(d, 2H, J = 8.4, ArH), 6.849 (d, 2H, J = 8.4, ArH), 6.329
(d, 1H, J = 15.6, C=CH), 6.118 (br s, 1H, NH),
4.270–4.252 (q, 2H, J = 7.2, –OCH2), 4.181 (d, 2H,
J = 5.2, CH2), 1.328–1.292 (t, 3H, J = 7.2, –CH3). IR
(KBr) t, cm-1: 3,503 (HO–), 3,365 (N–H), 3,046 (Ar–H),
3,027 (C=C–H), 1,728 (C=O), 1,654 (O=C–N), 1,605
(C=C), 1,210. MS (m/z, %): 249 (M1, 100.0).
N-hydroxycinnamoyl amino acid esters a–h were synthe-
sized from hydroxycinnamic acid and the corresponding
amino acid esters according to literature with some
modifications (Orlandi et al., 2001). Generally, to a solu-
tion of 3,4-dihydroxycinnamic acid (3.0 g, 16.5 mmol),
ethyl glycinate hydrochloride (2.2 g, 16.5 mmol) and
1-hydroxybenzotriazole (2.3 g, 17.0 mmol) in N,N-
dimethylformamide (50 ml) were added to triethylamine
(6.9 ml, 49.5 mmol) at 0ꢁC. After 10 min, 1-[3-(dimeth-
ylamino)propyl]-3-ethylcarbodiimidehydrochloride (3.2 g,
16.7 mmol) was added at 0ꢁC and the resultant reaction
mixture was stirred for 18 h at room temperature. The
mixture was poured into water (200 ml), extracted with
ethyl acetate (4 9 200 ml), washed with water
(2 9 100 ml) and brine (100 ml), and dried over MgSO4.
After removal of the solvent under reduced pressure, the
residual paste was purified by column chromatography
(silica gel, Hexane/EtOAc [1:1]) to give a–h.
Ethyl N-[3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]-
L-phenylalaninate (d)
Compound d, pale yellow solid, m.p. 167–168ꢁC, 1H NMR
(400 MHz, DMSO-d6): 9.430 (br s, 1H, OH), 9.208 (br s,
1H, OH), 8.414 (d, 1H, J = 7.6, NH), 7.288 (d, 1H,
J = 15.6, HC=C), 7.270–7.187 (m, 5H HC=C, ArH), 6.939
(S, 1H, ArH), 6.844 (d, 1H, J = 8.0, ArH), 6.748 (d, 1H,
J = 8.0, ArH), 6.402 (d, 1H, J = 15.6, C=CH),
4.580–4.524 (m, 1H, CH), 4.089–4.070 (q, 2H, J = 7.6,
CH2), 3.073–2.924 (m, 2H, CH2), 1.128–1.093 (t, 3H,
J = 7.6, CH2CH3). IR (KBr) t, cm-1: 3,503 (HO–), 3,365
(N–H), 3,046 (Ar–H), 3,027 (C=C–H), 1,728 (C=O), 1,654
(O=C–N), 1,605 (C=C). MS (m/z, %): 355 (M1, 81.2),
284.0 (28.3), 149.0 (65.7), 73 (100.0).
Ethyl N-[3-(3,4-dihydroxyphenyl)-1-oxo-2-propeny-yl]
glycinate (a)
Compound a, pale yellow solid, m.p. 170–171ꢁC, 1H NMR
(400 MHz, DMSO-d6), 9.415 (S, 1H, OH), 9.170 (S, 1H,
OH), 8.414 (br s, 1H, NH), 7.282 (d, 1H, J = 15.6, HC=C),
6.964 (S, 1H, ArH), 6.870 (d, 1H, J = 8.0, ArH), 6.757(d,
1H, J = 8.0, ArH), 6.419 (d, 1H, J = 15.6, C=CH), 4.113
(d, 2H, J = 6.8, CH2), 3.930–3.946 (q, 2H, J = 6.8,
–OCH2), 1.217–1.181 (t, 3H, J = 7.6, CH3). IR (KBr) t,
cm-1: 3,503 (HO–), 3,365 (N–H), 3,046 (Ar–H), 3,027
Ethyl N-[3-(4-hydroxy-3-methoxyphenyl)-1-oxo-2-propen-
1-yl]-L-phenylalaninate (e)
Compound e, pale yellow solid, m.p. 154–155ꢁC, 1H NMR
(400 MHz, DMSO-d6): 9.483 (S, 1H, OH), 8.411 (d, 1H,
NH), 7.314–7.273 (m, 6H, HC=C, ArH), 7.119 (S, 1H,
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Med Chem Res (2012) 21:1905–1911
Determination of partition coefficient (Log P)
ArH), 6.996 (d, 1H, J = 8.0, ArH), 6.795 (d, 1H, J = 8.0,
ArH), 6.521 (d, 1H, J = 15.6, C=CH), 4.600–4.554 (m,
1H, CH), 4.085–4.032 (q, 2H, J = 7.2, OCH2), 3.801 (S,
3H, OCH3), 3.065–2.929 (m, 2H, CH2), 1.135–1.099 (t,
3H, J = 7.2, CH3). IR (KBr) t, cm-1: 3,503 (HO–), 3,365
(N–H), 3,046 (Ar–H), 3,027 (C=C–H), 1,728 (C=O), 1,654
(O=C–N), 1,605 (C=C). MS (m/z, %): 369.0 (M1, 43.0),
295.0 (80.0), 221.0 (100.0), 73 (95.3).
The logarithm of the partition coefficient for n-octanol/
water was computed using CS ChemPropPro software, an
add-on program to ChemDraw Ultra (CambridgeSoft). The
log P values of our test compounds were as follows:
a (0.61), b (0.87), c (1), d (2.77), e (3.04), f (3.16), g (2.77),
h (2.25), VC (-3.36), EC (1.75), EF (2.02), EP (2.14), CA
(1.15), FA (1.42), and CoA (1.54).
Ethyl N-[3-(4-hydroxyphenyl)-1-oxo-2-propen-1-yl]-
L-phenylalaninate (f)
Assay of DPPH radical scavenging activity
Compound f, white solid, m.p. 129–130ꢁC, 1H NMR
(400 MHz, DMSO-d6): 9.879 (S, 1H, OH), 8.444 (d, 1H,
J = 7.6, NH), 7.395 (d, 2H, J = 8.4, ArH), 7.314 (d, 1H,
J = 15.6, CH=C), 7.269–7.210 (d, 5H, ArH), 6.794 (d, 2H,
J = 8.4, ArH), 6.478 (d, 1H, J = 15.6, C=CH), 4.568 (d,
1H, J = 4.8, CH), 4.081–4.028 (q, 2H, J = 7.2, OCH2),
3.075–2.927 (m, 2H, CH2), 1.130–1.094 (t, 3H, J = 7.2,
CH3). IR (KBr) t, cm-1: 3,503 (HO–), 3,365 (N–H), 3,046
(Ar–H), 3,027 (C=C–H), 1,728 (C=O), 1,654 (O=C–N),
1,605 (C=C). MS (m/z, %): 339.8 (M1, 32.0), 129.9
(100.0).
The DPPH free radical scavenging activity was determined
by the method in literature with some modifications
(Anselmi et al., 2004). Each sample at different concen-
tration in ethanol (0.5 ml) was mixed with 2.5 ml of eth-
anolic solution containing 0.1-mM DPPH. The mixture was
shaken vigorously, and then left to stand for 30 min in
the dark. The absorbance was measured at 517 nm. The
absorbance of the control was obtained by replacing
the sample with ethanol. The radical-scavenging activity of
the samples (antioxidants) was expressed in terms of IC50
(concentration in lM required for a 50% decrease in
absorbance of DPPH radical).
Methyl N-[3-(4-hydroxyphenyl)-1-oxo-2-propen-1-yl]-
L-tyrosinate (g)
Preparation of RBC
Compound g, white solid, m.p. 173–175ꢁC, 1H NMR
(400 MHz, DMSO-d6): 9.885 (S, 1H, OH), 9.249 (S, 1H,
OH), 8.388 (d, 1H, NH), 7.396 (d, 2H, J = 8.4, ArH),
7.309 (d, 1H, J = 15.6, HC=C), 7.025 (d, 2H, J = 8.4,
ArH), 6.797 (d, 2H, J = 8.4, ArH), 6.666 (d, 2H, J = 8.4,
ArH), 6.482 (d, 1H, J = 15.6, C=CH), 4.523–4.468 (q, 1H,
J = 8.0, CH), 3.602 (S, 3H, OCH3), 2.964–2.794 (m, 2H,
CH2). IR (KBr) t, cm-1: 3,503 (HO–), 3,365 (N–H), 3,046
(Ar–H), 3,027 (C=C–H), 1,728 (C=O), 1,654 (O=C–N),
1,605 (C=C). MS (m/z, %): 341.9 (M1, 100.0).
Human red blood cells were separated from heparinized
blood from a healthy volunteer. Erythrocytes were sepa-
rated from blood plasma by centrifugation (10 min at
2,000 rpm at 4ꢁC), and then the RBCs were washed three
times with phosphate-buffered saline (PBS) at pH 7.4.
During the last washing, the cells were centrifuged at
exactly 2,000 rpm for 10 min to obtain a constantly packed
cell volume.
Assay for haemolysis
The 5% suspension of RBCs in PBS (pH 7.4) was
incubated under air atmosphere at 37ꢁC for 5 min, and
haemolysis was initiated by introducing a PBS solution
of AAPH. This reaction mixture was incubated at 37ꢁC
with gentle shaking. The extent of haemolysis was
determined spectrophotometrically by measuring the
absorbance of haemolysate at 540 nm. Briefly, aliquots
of the reaction mixture were taken at appropriate time
intervals, diluted with 0.15-M NaCl, and centrifuged at
2,000 rpm for 10 min to separate the RBCs. The per-
centage haemolysis was determined by measuring the
absorbance of the supernatant at 540 nm and compared
with that of complete haemolysis by treating the same
RBC suspension with distilled water. In the case
Ethyl N-[3-(4-hydroxy-3-methoxyphenyl)-1-oxo-2-propen-
1-yl]-L-valinate (h)
Compound h, white solid, m.p. 79–80ꢁC, 1H NMR
(600 MHz, CDCl3), 7.573 (d, 1H, J = 15.6, HC=C), 7.068
(d, 1H, J = 7.8, ArH), 7.009 (S, 1H, ArH), 6.915 (d, 1H,
J = 7.8, ArH), 6.347 (d, 1H, J = 15.6, C=CH), 6.136 (br s,
1H, NH), 4.728–4.706 (m, 1H, CH), 4.239 (q, 2H, J = 6.0,
CH2 CH3), 3.927 (S, 3H, OCH3), 2.241–2.230 (m, 1H,
CH), 1.316–1.292 (t, 3H, J = 4.2, CH3), 0.995–0.950 (m,
6H, CH3). IR (KBr) t, cm-1: 3,503 (HO–), 3,365 (N–H),
3,046 (Ar–H), 3,027 (C=C–H), 1,728 (C=O), 1,654 (O=C–
N), 1,605 (C=C). MS (m/z, %): 322.0 (M1, 100.0), 177
(47.0).
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Med Chem Res (2012) 21:1905–1911
1911
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Acknowledgments This work was supported by the National Nat-
ural Science Foundation of China (Grant NO. 20702016).
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