Investigation of the antioxidant properties of some new 4-hydroxycoumarin derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.07.007

The aim of this investigation was to measure the activity of four 4-hydroxycoumarin derivatives - three of them were described before and one was newly synthesized. The substances were ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hydroxyphenyl)methyl]-3-oxobutanoate (SS-14), 4-[1-(4-hydroxy-2-oxo-2H-chromen-3-yl)-2-(ethoxycarbonyl)-3-oxobutyl]benzoic acid (SS-17), ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(3-nitrophenyl)methyl]-3-oxobutanoate (SS-21) and ethyl 2-[(3,4,5-trimethoxyphenyl)(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl]-3-oxobutanoate (T-2). The synthesis of T-2 consists of two steps. First step was Knoevenagel reaction between 3,4,5-trimethoxybenzaldehyde and ethylacetoacetate. Ethyl 2-(3,4,5-trimethoxy)-phenylmethyleneacetoacetate was the product. Second step was Michael addition reaction between the latter product and 4-hydroxycoumarin. All the compounds were tested in vitro for antioxidant activity in hypochlorous system. The assay was based on the luminol-dependent chemiluminescence of free radicals, which decreased in the presence of 4-hydroxycoumarin derivative. Compound SS-14 (ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hydroxyphenyl)methyl]-3-oxobutanoate) expresses the best scavenger activity at the highest concentration (10^-4 mol/L).

Full text of DOI:10.1016/j.ejmech.2008.07.007

European Journal of Medicinal Chemistry 44 (2009) 3077–3082
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Investigation of the antioxidant properties
of some new 4-hydroxycoumarin derivatives
,
b
b
a
a
S. Stanchev a , V. Hadjimitova , T. Traykov , T. Boyanov , I. Manolov
*
a Department of Organic Chemistry, Faculty of Pharmacy, Medical University – Sofia, 2 Dunav st., 1000 Sofia, Bulgaria
b Department of Physics and Biophysics, Medical Faculty, Medical University – Sofia, 2 Zdrave st., 1431 Sofia, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this investigation was to measure the activity of four 4-hydroxycoumarin derivatives – three of
them were described before and one was newly synthesized. The substances were ethyl 2-[(4-hydroxy-2-
oxo-2H-chromen-3-yl)(4-hydroxyphenyl)methyl]-3-oxobutanoate (SS-14), 4-[1-(4-hydroxy-2-oxo-2
H-chromen-3-yl)-2-(ethoxycarbonyl)-3-oxobutyl]benzoic acid (SS-17), ethyl 2-[(4-hydroxy-2-oxo-2H-
chromen-3-yl)(3-nitrophenyl)methyl]-3-oxobutanoate (SS-21) and ethyl 2-[(3,4,5-trimethoxyphenyl)
(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl]-3-oxobutanoate (T-2). The synthesis of T-2 consists of two
steps. First step was Knoevenagel reaction between 3,4,5-trimethoxybenzaldehyde and ethylacetoacetate.
Ethyl 2-(3,4,5-trimethoxy)-phenylmethyleneacetoacetate was the product. Second step was Michael
addition reaction between the latter product and 4-hydroxycoumarin. All the compounds were tested in
vitro for antioxidant activity in hypochlorous system. The assay was based on the luminol-dependent
chemiluminescence of free radicals, which decreased in the presence of 4-hydroxycoumarin derivative.
Compound SS-14 (ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hydroxyphenyl)methyl]-3-oxobuta-
noate) expresses the best scavenger activity at the highest concentration (10ꢀ4 mol/L).
Received 20 March 2008
Received in revised form 7 July 2008
Accepted 8 July 2008
Available online 16 July 2008
Keywords:
4-Hydroxycoumarins
Synthesis
Chemiluminescence
Hypochlorous
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
substituents on the third place in the coumarin ring. The principle of
the method is that the solution of DPPH in ethanol is intensively
4-Hydroxycoumarins are very important class of biologically
active substances in nature and in medicine. They exhibit mainly
anticoagulant activity and there are some drugs which are widely
used as anticoagulants – Warfarin and Acenocoumarol. They
exhibit cytotoxic and spasmolytic activities and also activity against
HIV virus [1–5]. The mechanism of anti-HIV activity is probably
blocking of HIV-1 protease and integrase [3–5].
Three of the tested 4-hydroxycoumarin derivatives in the
present study – ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(3-
nitrophenyl)methyl]-3-oxobutanoate (SS-21), 4-[1-(4-hydroxy-2-
oxo-2H-chromen-3-yl)-2-(ethoxycarbonyl)-3-oxobutyl]benzoic acid
(SS-17) and ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hydrox-
yphenyl)methyl]-3-oxobutanoate (SS-14) – were synthesized and
investigated for cytotoxic activity in two tumor cell lines – HL-60 and
EJ [6]. The results are compared with the utilized anticancer drug
Melphalan. SS-21 shows comparatively good cytotoxic properties. Its
activity is weaker than Melphalan.
colored and has an absorption maximum at 517 nm. In the presence
of scavenger substance the solution becomes colorless. The results
show that 4-hydroxycoumarins with electron-donating groups on
the third position and on the seventh position in the coumarin ring
have the best scavenger activity.
Structure–activity relationship of some coumarin antioxidants
as protective agents against linoleic acid hydroperoxide (LOOH)
toxicity in cultivated human umbilical vein endothelial cells was
explored [8]. Coumarin, 4-hydroxycoumarin, 7-hydroxycoumarin,
esculetin (6,7-dihydroxycoumarin), scopoletin (7-hydroxy-6-
methoxycoumarin) and 8-acetyl-6-hydroxy-7-methoxycoumarin
were used in the assay. There are two types of treatment – one is
pretreatment, when after treatment with antioxidant the medium
is changed with Earle solution (containing LOOH) and consequently
the cells incubated for 3 h. The second type is concurrent treat-
ment, when the cells are treated for 3 h with Earle solution con-
taining both LOOH and antioxidant. Among the coumarins
examined, esculetin has the best protective activity against LOOH-
induced toxicity in both the treatments – pretreatment and
concurrent treatment. Esculetin is known as a strong lipoxygenase
inhibitor. The presence of o-phenolic hydroxyl groups can be
important for the protection against LOOH-induced toxicity.
The antioxidative activity of some coumarins is investigated by
measuring their protective action toward linoleic acid peroxidation
Differently substituted 4-hydroxycoumarins are tested for free
radical scavenger activity in the system of 1,1-diphenyl-2-picrylhy-
drazyl (DPPH) stable radical [7]. Investigated 4-hydroxycoumarins
are substituted with electron-donating and electron-withdrawing
* Corresponding author.
E-mail address: stancho_stanchev@yahoo.com (S. Stanchev).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.07.007

3078
S. Stanchev et al. / European Journal of Medicinal Chemistry 44 (2009) 3077–3082
R1
O
2. Results and discussion
R
2.1. Chemistry
Compound T-2 was synthesized in a two-step synthetic scheme.
R2
O
First step was a Knoevenagel reaction between 3,4,5-trimethox-
ybenzaldehyde and ethylacetoacetate. The product was ethyl 2-
(3,4,5-trimethoxy)-phenylmethyleneacetoacetate (Fig. 2).
The second step was Michael reaction between 4-hydrox-
ycoumarin and ethyl-2-(3,4,5-trimethoxy)-phenylmethyleneaceto
R = R2 = H, R1 = - OH 4- hydroxycoumarin
R = R1 = H, R2 = - OH 7- hydroxycoumarin
R = H, R1 = -CH3, R2 = - OH 7-hydroxy-4-methylcoumarin
R = H, R1 = - CH3, R2 = -NH2 7-amino-4-methylcoumarin
acetate. Arylmethylene-b-ketoester fragment was added to the
third position in the coumarin ring, because of the lowest electronic
density on that position. The reason for this was the electron-
R = H, R1 = -CF3, R2 = -NH2 7-amino-4-trifluoromethylcoumarin
R = - COOH, R1 = R2 = - H coumarin-3-carboxilyc acid
withdrawing effects of carbonyl and hydroxyl groups and the p,
conjugation between lone pair electrons of the hydroxyl oxygen
atom and -electrons of the C]C bond (Fig. 3).
The other investigated compounds have chemical structures as
shown in Fig. 4:
p-
p
O
O
O
O
CH3
8-methoxypsoralen
Ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(3-nitrophenyl)
methyl]-3-oxobutanoate (SS-21) [6],
Fig. 1. Chemical structure of the investigated compounds.
4-[1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-2-(ethoxycarbonyl)-
3-oxobutyl] benzoic acid (SS-17) [6],
Ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hydroxyphenyl)
methyl]-3-oxobutanoate (SS-14) [6].
in micelles of sodium dodecylsulfate in buffer solution (pH ¼ 7.4)
[9]. Results are expressed as relative antioxidant efficiency (RAE),
defined as the ratio of the antioxidant efficiency (AE) of the tested
These compounds were synthesized in the same way as T-2 [6]
(Fig. 5 and Fig. 6).
compound to that of
a-tocopherol. The coumarins tested are 4-
First step was Knoevenagel reaction between differently
substituted aromatic aldehydes and ethylacetoacetate at room
temperature in the presence of piperidine. The products of this
type of reaction were substituted in the aromatic ring aryl-
hydroxycoumarin, 7-hydroxycoumarin (umbelliferone), scopoletin,
5,7-dihydroxy-4-methylcoumarin, 6,7-dihydroxy-4-methylcoumarin,
7,8-dihydroxycoumarin (daphnetine) and 4-methyldaphnetine. The
best RAE was established for daphnetine (7,8-dihydroxycoumarin).
4-Methyldaphnetine and 6,7-dihydroxy-4-methylcoumarin also dis-
played a very good relative antioxidant efficiency. The reason for that
is probably the presence of o-phenolic (catechol) hydroxyl groups. The
activity of such compounds can be connected with the different
capabilities to transfer hydrogen atom to LOOꢁ. Hydrophilicity also
methylene-
The second step was Michael reaction between the aryl-
methylene- -ketoesters and 4-hydroxycoumarin [6] (Fig. 5).
b-ketoesters.
b
The products were 4-hydroxycoumarin derivatives, substituted
on the third place with ethyl 2-acetyl-3-phenylbutanoate fragment.
played
a role in penetration through sodium dodecylsulfate
micelles and antioxidative activity.
The chemiluminescence characteristics of seven different
coumarins were studied in detail in peroxyoxalate–hydrogen
peroxide system [10]. Their structure is shown in Fig. 1.
The fluorescence and chemiluminescence spectra were
compared and all used coumarins were found to be good fluo-
rescers. The intensity and kinetic parameters of the chemilumi-
nescent systems were evaluated from computer fitting of the
resulting intensity–time plots. Among different coumarins, 7-
amino-4-trifluoromethylcoumarin revealed the most promising
characteristics as an efficient blue fluorescence emitter.
The aim of the present investigation was to test some new 4-
hydroxycoumarin derivatives for antioxidative (scavenger) activity
in model system, which generates free radicals.
2.2. Chemiluminescent antioxidant assay
The measurements of the luminol-dependent CL in the presence
of OClꢀ showed that SS-14 has significant antioxidant effect at
concentration 10ꢀ4 M. In Fig. 7 are shown typical kinetic curves for
such systems, for control (phosphate buffer solution) and for SS-14.
The corresponding CL-SI values are presented in Fig. 8.
There is a very good linearity between the concentration and
chemiluminescence’s scavenger index (Fig. 9) (R2 ¼ 0.95–1.00), so
the decreasing CL-SI is proportional to the increasing concentration
of the tested 4-hydroxycoumarin derivative. The most significant
standard deviation was measured for SS-21 at concentration
5 ꢂ10ꢀ5 mol/L and also for T-2 at concentration 10ꢀ5 mol/L.
O
O
CH3
O
O
O
H3C
H3C
piperidine, g. CH3COOH
-H2O
O
H3C
+
O
O
O
O
CH3
O
CH3
CH3
O
O
CH3
CH3
CH3
Fig. 2. Knoevenagel interaction between 3,4,5-trimethoxybenzaldehyde and ethylacetoacetate.

S. Stanchev et al. / European Journal of Medicinal Chemistry 44 (2009) 3077–3082
3079
H3C
H3C
O
O
O
O
CH3
H3C
HO
O
O
O
piperidine, t0
HO
O
H3C
+
O
CH3
O
O
O
CH3
O
O
CH3
CH3
O
O
CH3
Fig. 3. Michael addition reaction between ethyl 2-(3,4,5-trimethoxy)-phenylmethyleneacetoacetate and 4-hydroxycoumarin.
It can be seen from Fig. 8 that a strong scavenger activity for SS-
14, SS-17 and T-2 is found at 10ꢀ4 mol/L.
The worst radical capturing activity was found for SS-21. At the
highest tested concentration (10ꢀ4 mol/L) CL-SI had the value only
7% of the control (control sample has 100%). At the lowest
concentrations there was lack of antioxidant effects.
From all of the tested compounds, SS-14 showed the strongest
scavenger activity at the concentration 10ꢀ4 mol/L, followed by T-2
and SS-17.
Superoxide and H2O2 are weak reactive species, but the latter
dismutates spontaneously, because it is the substrate of myelo-
peroxidase (MPO). Hypochloric acid can be formed as a product,
which reacts rapidly with biological macromolecules. Finally
hypochloric acid can interact with biogenic amines and form
product chloramines, which are weak, but long lasting oxidants.
The stationary concentrations of superoxide and hydrogen
peroxide are very small, because of the spontaneous dismutation
of H2O2. The basic oxidizing agent in phagocytes is HOCl. The
action of the phagocytic HOCl concerns both pathogenic micro-
organism and the human body, because it causes oxidative
damage in the surrounding tissues. Substances which can scav-
enge HOCl could decrease the effect of the action of the phago-
cytes [11].
The chemical structure–scavenger activity relationship can be
made. The antioxidant activity in general of 4-hydroxycoumarins
can be explained with the presence of enolic hydroxyl group at the
fourth position in coumarin ring. The hydroxyl group, which is also
present at p-position in the aromatic ring in SS-14, has high elec-
tron-releasing properties (positive mesomeric effect is higher than
negative inductive effect) and it activates aromatic ring. The same is
valid for methoxy groups at positions 3, 4 and 5 in aromatic ring in
a molecule of T-2. These groups have a bit higher electron-releasing
activity than hydroxyl group and also activate the aromatic ring.
Hydroxyl and methoxy groups have a good capability to catch free
radicals by themselves. Carboxyl and nitro groups are electron-
withdrawing substituents, they deactivate aromatic ring and have
no capability to bind the free radicals. The better scavenger activity
of SS-17 is probably due to the lower electron-withdrawing effect of
carboxyl group, than that of nitro group. So, that could be the
probable explanation of the good antioxidative activity of SS-14 and
T-2, and the worst activity of SS-21.
The basic part of the phagocyte bactericidal effect is due to the
ROS (reactive oxygen species) produced by them. The phagocytosis
signal activates one cascade of biochemical reactions, which gives
hypochloric acid as product. The latter is an extremely strong
oxidative agent, which oxidizes biogenic amines, thioles and
proteins. The reaction in which superoxide anions are formed from
oxygen at the beginning and they cause a subsequent cascade of
processes in which other ROS are obtained is called respiratory
burst. It can be seen in Fig. 10.
3. Conclusions
The four tested 4-hydroxycoumarin derivatives, 2-[(4- hydroxy-
2-oxo-2H-chromen-3-yl)(4-hydroxyphenyl)methyl]-3-oxobutanoate
(SS-14) [6], 4-[1-(4-hydroxy-2-oxo-2H-chromen-3-yl)-2-(ethox-
ycarbonyl)-3-oxobutyl]benzoic acid (SS-17) [6], ethyl 2-[(4-hydroxy-
2-oxo-2H-chromen-3-yl)(3-nitrophenyl)methyl]-3-oxobutanoate
(SS-21) [6] and ethyl 2-[(3,4,5-trimethoxy-phenyl)(4-hydroxy-2-
oxo-2H-chromen-3-yl)methyl]-3-oxobutanoate (T-2), were tested
for antioxidant activity in a model system of hypochlorous anion. SS-
14 has the best hypochlorous scavenging activity at 10ꢀ4 mol/L. The
reason for higher scavenger activity of this compound is the presence
of electron-donating group as substituent in the aromatic ring in
the side chain.
T-2 was a brand new synthesized compound. It was synthesized
by a two-step method. The first step was Knoevenagel reaction
between 3,4,5-trimethoxybenzaldehyde and ethylacetoacetate.
The product of this reaction was ethyl 2-(3,4,5-trimethoxy)-phe-
nylmethyleneacetoacetate. That product was reacted with 4-
hydroxycoumarin according to Michael addition reaction. The
arylmethylene-
b
-ketoester added on the third position in coumarin
OH
HO
O
O
N+
O-
H3C
O
H3C
O
H3C
O
O
O
HO
OH
O
OH
O
O
O
O
O
O
O
O
H3C
O
H3C
SS-14
H3C
SS - 17
SS-21
Fig. 4. Chemical structures of the investigated compounds.

3080
S. Stanchev et al. / European Journal of Medicinal Chemistry 44 (2009) 3077–3082
O
1400
1200
1000
800
600
400
200
0
____
control
------- SS -14 (10-4 mol/L.)
O
O
CH3
O
O
H3C
O
25°C, piperidine
-H2O
+
O
R
R
CH3
CH3
Fig. 5. Knoevenagel reaction between substituted benzaldehydes and ethylacetoacetate.
0
2
4
Time (sec)
6
8
-200
ring. The product was characterized by UV–vis, 1H NMR and mass
spectrometry.
Fig. 7. Chemiluminescence kinetic curves of SS-14 and control.
67 MHz):
d
¼ 30, 110, 135, 140, 160, 190; EIMS: m/z (%) ¼ 234 (100,
4. Experimental part
Mþ), 233 (57), 220 (10), 219 (69), 217 (17), 205 (15), 191 (25.4), 189
(38.25), 187 (11.3), 175 (8.7), 163 (11.3), 161 (11.3), 160 (28.7), 151
(28.7), 147 (68.7), 146 (11.3), 145 (37.4), 131 (7), 123 (30.4), 120 (9.6),
119 (20), 118 (19.1), 115 (2.6), 107 (6), 91 (20), 89 (19.1), 77 (7), 65
(11.3), 63 (12.1), 53 (5), 45 (0.9); TLC: Rf ¼ 0.39 (hexane/
acetone ¼ 2:1); Anal.: C13H14O4 (234), (C, H) ¼ (calcd/found): %C,
66.66/66.50; %H, 6.02/5.94.
4.1. Chemical part
All starting materials were purchased from Merck (Germany),
Sigma–Aldrich (St. Louis, USA) and Fluka (Switzerland). They were
used without further purification. Melting points were measured in
open capillary tubes on a Bu¨chi 535 melting point apparatus.
1H NMR spectra were recorded in Brucker 250 MHz in DMSO-d6
or acetone using TMS as an internal standard (chemical shifts are
reported in ppm units). Abbreviations were as follows: s – singlet,
d – doublet, dd – double doublet, t – triplet, m – multiplet, br –
broad.
4.1.1.2. 4-[2-(Ethoxycarbonyl)-3-oxobut-1-en-1-yl]benzoic acid (SS-
6). Yellow crystals, m.p. 148–150 ꢃC. The substance crystallizes
from water. Purified after recrystallization from ethanol. Yield: 57%;
UV–vis: lmax ¼ 204, 292 nm; FTIR (nujol): band 3300–2400, 1736.1,
1689.8, 1608.8, 1460.3, 848 cmꢀ1 1H NMR (DMSO, 250 MHz):
;
Mass-spectral analysis was performed by electron ionization on
mass spectrometer Hewlett–Packard 5973 at 70 eV.
d
¼ 1.0 (t, 3H) (aliphatic), 2.4 (s, 3H) (aliphatic), 4.2 (q, 2H)
(aliphatic), 7.4 (s, 1H) (aliphatic), 7.6 (s, 1H) (aromatic), 7.8 (s, 1H)
(aromatic), 8.0 (d, 2H) (aromatic), 13.23 (s, 1H) (carboxyl group);
EIMS: m/z (%) ¼ 262 (64, Mþ), 261 (16.7), 247 (18.4), 233 (8), 218
(18.4), 217 (100), 191 (10), 189 (14.9), 179 (9.6), 175 (26.3), 173 (27.2),
171 (11.4), 155 (14.9), 151 (16.7), 147 (7.9), 131 (9.6), 129 (17.5), 115
(7.9), 103 (18.4), 101 (14.9), 91 (5.3), 77 (11.4), 75 (8.7), 63 (3.5), 51
(2.6), 45 (1.8); TLC: Rf ¼ 0.33 (hexane/chloroform/acetone/meth-
anol ¼ 5:3:2:1); Anal.: C14 H14 O5 (262), (C, H) (calcd/found): %C,
64.12/64.44; %H, 5.38/5.26.
4.1.1. General procedure for the preparation of arylmethylene-
b-ketoesters
Aromatic aldehyde and ethylacetoacetate in equimolar quanti-
ties were mixed in round-bottomed flask. Piperidine (0.03 mol) and
glacial acetic acid (0.04 mol) were also added to the reaction
mixture. The latter was stirred at room temperature for 90 min.
After 20 ml ether and/or 150 ml distilled water was added to the
reaction mixture, crystals with different colors were formed. These
crystals were filtered off and washed. Then they were dried at room
temperature and were recrystallized from appropriate solvents –
mainly alcohols (ethanol, 1-propanol or 2-propanol).
4.1.1.3. Ethyl
2-(3-nitrophenylmethylene)-3-oxobutanoate
(SS-
19). White crystals, m.p.100–103 ꢃC. The substance crystallizes
from water. Purified after recrystallization from 2-propanol. Yield:
17%; UV–vis: lmax ¼ 210, 266 nm; FTIR (nujol): 1728.4, 1660.9,
4.1.1.1. Ethyl 2(4-hydroxyphenylmethylene)-3-
oxobutanoatee (SS-4).
1628.1,1529.7, 780, 735 cmꢀ1; 1H NMR (DMSO, 250 MHz):
d
¼ 0.9 (t,
Yellow crystals, m.p. 141–143 ꢃC. The substance crystallizes from
ether. Purified after recrystallization from 2-propanol. Yield: 51%;
UV–vis: lmax ¼ 206, 224, 286 nm; FTIR (nujol): 3325.7, 1732.3,
3H) (aliphatic), 1.6 (s, 3H) (aliphatic), 4.8 (q, 2H) (aliphatic), 7.4 (s,
1H) (aliphatic), 7.6 (t, 1H) (aromatic), 7.9 (d, 1H) (aromatic), 8.1 (d,
1H) (aromatic), 8.3 (s, 1H) (aromatic); EIMS: m/z (%) ¼ 263 (65.2,
Mþ), 262 (20), 248 (99.1), 246 (100), 234 (19.1), 220 (32.1), 218
(51.8), 216 (15.7), 202 (35.7), 200 (24.3), 192 (13), 180 (18.3), 176
(66.09), 174 (27), 160 (10.4), 152 (21.7), 146 (13), 130 (20.9), 129
(36.5), 120 (17.4), 115 (19.1), 102 (35.7), 101 (47.8), 89 (13.9), 75
;
1641.6, 1597.3, 1462.2, 1205.7, 819.8 cmꢀ1 1H NMR (acetone,
200 MHz):
d
¼ 1.3 (t, 3H) (aliphatic), 2.3 (s, 3H) (aliphatic), 4.3 (q,
2H) (aliphatic), 6.9 (dd, 2H) (aromatic), 7.4 (dd, 2H) (aromatic), 7.6
(s, 1H) (aliphatic), 10.5 (s, 1H) (hydroxyl); 13C NMR (acetone,
R
O
HO
O
CH3
H3C
t°, piperidine or CH3O-Na+
O
O
OH
O
+
O
O
R
CH3
O
O
O
H3C
Fig. 6. Michael addition of arylmethylene-b-ketoester to 4-hydroxycoumarin.

S. Stanchev et al. / European Journal of Medicinal Chemistry 44 (2009) 3077–3082
3081
Fig. 8. Relationship between CL-SI and the concentration of the tested compound as
bar graph.
(29.6), 63 (9.6), 51 (13), 45 (2.6); TLC: Rf ¼ 0.5 (hexane/
acetone ¼ 2:1); Anal.: C13H13NO5 (263), (C, H) (calcd/found): %C,
59.31/59.54; %H, 4.98/5.13.
The investigated compounds were 2-[(4-hydroxy-2-oxo-2H-
chromen-3-yl)(4-hydroxyphenyl)-methyl]-3-oxobutanoate (SS-14)
[6], 4-[1-(4-hydroxy-2-oxo-2H-chromen-3-yl)-2-(ethoxy-carbonyl)-
3-oxobutyl]benzoic acid (SS-17) [6], ethyl 2-[(4-hydroxy-2-oxo-2H-
chromen-3-yl)(3-nitrophenyl)methyl]-3-oxobutanoate (SS-21) [6].
Their characteristics and synthetic procedure are presented below.
Fig. 10. The metabolic pathway of oxygen explosion [11].
1622.3, 1599.2, 1464.1, 821, 760 cmꢀ1
;
1H NMR (DMSO, 250 MHz):
4.1.2. General procedure for the preparation of condensation
products with 4-hydroxycoumarin
d
¼ 1.0 (t, 3H) (aliphatic), 2.0 (s, 3H) (aliphatic), 4.1 (q, 2H)
(aliphatic), 4.4 (m, 1H) (aliphatic), 4.6 (m, 1H) (aliphatic), 6.9 (m,
2H) (aromatic), 7.3 (m, 1H) (aromatic), 7.4 (m, 1H) (aromatic), 7.6
(m, 2H) (aromatic), 7.9 (m, 1H) (aromatic), 8.1 (m, 1H) (hydroxyl),
8.6 (s, 1H) (hydroxyl); EIMS: m/z (%) ¼ 396 (0.09, Mþ), 364 (0.09),
350 (0.09), 321 (0.09), 307 (0.9), 279 (0.4), 266 (56.1), 265 (100), 249
(31.6), 237 (10.5), 221 (2.6), 210 (2.6), 181 (1.8), 165 (1.8), 153 (2.6),
146 (7), 130 (7), 121 (19.3), 118 (17.5), 102 (4.4), 92 (15.8), 85 (12.3),
76 (2.6), 63 (10.5), 53 (2.6), 46 (0.09); TLC: Rf ¼ 0.48 (hexane/chlo-
roform/acetone/methanol= 5:3:2:1); Anal.: C22 H20 O7 (396), (C, H)
(calcd/found): %C, 66.66/66.36; %H, 5.09/5.13.
Arylmethylene-b-ketoester, obtained in previous reaction, and
4-hydroxycoumarin were mixed in equimolar quantities in 25–
30 ml methanol (used as a solvent). Sodium methoxide (0.003 mol)
as a basic agent was also added to the reagents. The reaction
mixture was boiled and stirred for 60 h under reflux. The reaction
was controlled by TLC (hexane/acetone ¼ 2:1 or hexane/acetone/
chloroform/methanol ¼ 5:3:2:1). When the quantities of reagents
depleted the heating was stopped. The residue from the reaction
mixture was filtered off and was washed with hot water, in order to
remove the unreacted 4-hydroxycoumarin. After that the residue
was dried at room temperature and recrystallized from appropriate
solvents (methanol, ethanol or 2-propanol).
4.1.2.2. 4-[1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-2-(ethoxycarbonyl)-
3-oxobutyl]benzoic acid (SS-17) [6]. White crystals, m.p. 150–155 ꢃC.
Purified after recrystallization from methanol. Yield: 28%; UV–vis:
lmax ¼ 208, 282, 308 nm; FTIR (nujol): 3442, band 3300–2400, 1732.3,
4.1.2.1. Ethyl
2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hydrox-
1693.7, 1612.7, 1462.4, 1109, 846.3, 756.2 cmꢀ1 1H NMR (DMSO,
;
yphenyl)methyl]-3-oxobutanoate (SS-14) [6]. White crystals, m.p.
195–197 ꢃC. Purified after recrystallization from ethanol. Yield:
21%; UV–vis: lmax ¼ 214, 280, 308 nm; FTIR (nujol): 3391.3, 1699.5,
250 MHz):
d
¼ 1.0 (t, 3H) (aliphatic), 2.1 (s, 3H) (aliphatic), 3.9 (q, 2H)
(aliphatic), 4.2 (m, 1H) (aliphatic), 4.6 (m, 1H) (aliphatic), 7.2 (m, 1H)
(aromatic), 7.4 (m, 1H) (aromatic), 7.5 (m, 2H) (aromatic), 7.6 (m, 2H)
(aromatic), 7.8 (m, 1H) (aromatic), 8.0 (m, 1H) (hydroxyl), 12.83 (s, 1H)
(carboxyl); EIMS: m/z (%) ¼ 424 (1.3, Mþ), 392 (0.4), 378 (17.5), 360
(1.8), 335 (48.2), 317 (19.3), 307 (9.6), 294 (44.7), 293 (34.2), 265 (6.1),
257 (50.9), 250 (22.8), 249 (100), 239 (5.3), 229 (1.8), 215 (12.3), 205
(1.8), 187 (2.6), 173 (2.6), 165 (4.4), 146 (2.6), 130 (6.1), 120 (19.3), 102
(6.1), 92 (30.7), 75 (6.1), 64 (8.8), 51 (2.6); TLC: Rf ¼ 0.62 (hexane/
chloroform/glacial acetic acid ¼ 10:10:4); Anal.: C23H20O8 (424) (C, H)
(calcd/found): %C, 65.09/65.07; %H, 4.75/4.90.
120
R2 = 1
100
80
60
40
20
0
R2 = 0.971
R2 = 0.9804
4.1.2.3. Ethyl
2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(3-nitro-
Linear (SS-14)
Linear (T-2)
Linear (SS-17)
Linear (SS-21)
phenyl)methyl]-3-oxobutanoate (SS-21) [6]. White crystals, m.p.
135–140 ꢃC. Purified after recrystallization from ethanol. Yield:
37%; UV–vis: lmax ¼ 210 nm; FTIR (nujol): 3335, 1732.3, 1674.4,
R2 = 0.9451
1620.4, 1529.7, 1068.7, 763, 736 cmꢀ1 1H NMR (DMSO, 250 MHz):
;
d
¼ 1.0 (t, 3H) (aliphatic), 1.9 (s, 3H) (aliphatic), 3.9 (q, 2H)
0
20
40
Drug concentrations (µmol/L)
60
80
100
120
(aliphatic), 4.4 (m,1H) (aliphatic), 4.6 (m,1H) (aliphatic), 7.2 (m,1H)
(aromatic), 7.4 (m, 1H) (aromatic), 7.5 (m, 1H) (aromatic), 7.7 (m,
3H) (aromatic), 7.9 (m, 2H) (aromatic), 9.8 (s, 1H) (hydroxyl); EIMS:
Fig. 9. Linear relationship between concentrations of the tested compounds and CL-SI.

3082
S. Stanchev et al. / European Journal of Medicinal Chemistry 44 (2009) 3077–3082
m/z (%) ¼ 425 (4.4, Mþ), 382 (4.4), 361 (12.3), 336 (38.6), 320 (1.8),
308 (2.6), 294 (58.8), 278 (100), 266 (8.8), 257 (14.9), 249 (48.2),
248 (91.2), 239 (1.8), 220 (8.8), 205 (1.8), 176 (3.5), 165 (10.5), 139
(5.3), 130 (15.8), 120 (71.9), 101 (13.2), 92 (68.4), 85 (18.4), 75 (14.9),
64 (17.5), 51 (6); TLC: Rf ¼ 0.34 (hexane/acetone ¼ 2:1).
vis: lmax ¼ 206, 234 nm; 1H NMR (DMSO, 250 MHz):
¼ 1.0 (t, 3H),
d
2.0 (s, 3H), 3.5 (m, 3H), 3.8 (m, 6H), 4.2 (q, 2H), 7.2 (m, 4H), 7.6 (m,
2H),11.2 (s, 1H); EIMS: m/z (%) ¼ 470 (0.4), 450 (0.4), 420 (44.7), 406
(0.4), 392 (0.4), 374 (17.4), 359 (0.8), 347 (28), 331 (4.5), 318 (1.5),
301 (100), 286 (6.1), 260 (26.5), 245 (9.8), 232 (8.3), 210 (1.5), 199
(4.5), 181 (5.3), 168 (8.3), 153 (5.3), 135 (5.3), 115 (6.8), 98 (9.8), 77
(6.1), 65 (2.3), 53 (4.5).
Ethyl 2-[(3,4,5-trimethoxyphenyl)(4-hydroxy-2-oxo-2H-chro-
men-3-yl)methyl]-3-oxobutanoate (T-2) is a brand new compound.
Its synthetic procedure can be presented as follows.
4.1.2.4. Synthetic procedure for T-2
4.2. Investigation of the antioxidant properties
4.1.2.4.1. Knoevenagel
reaction
between
3,4,5-trimethox-
ybenzaldehyde and ethylacetoacetate. About 1.96 g (0.01 mol) of
3,4,5-trimethoxybenzaldehyde, 5.2 g (0.04 mol) of ethyl-
acetoacetate, 1.7 g (0.02 mol) of piperidine, and 1.2 g (0.02 mol) of
The luminol-dependent chemiluminescence (CL) was used for
registration of ROS (reactive oxygen species) with LKB 1251
luminometer (Bioorbit, Turku, Finland) set at 310 K and connected
with an AT-type computer. Data collection was performed by
MultiUse program, version 1.08 (Bioorbit, Turku, Finland).
glacial acetic acid were consequently poured into
a round-
bottomed flask. The reaction mixture was stirred with electro-
magnetic stirrer at room temperature for 2 h. 20 ml of ether was
added at the end of the reaction time. The reaction mixture was left
to stay for 2 days at room temperature and white-yellow crystals
were formed. After that new quantities of 5–10 ml of ether was
added and the crystal residue was filtered off in vacuo. These
crystals were washed, dried at room temperature and recrystallized
from appropriate solvents.
Luminol-dependent CL in a system of NaOCl generated ClOꢀ.
The sample contained the following substances in 1 ml PBS:
0.1 mM luminol, 0.06 mM NaOCl and the tested drug at concen-
trations between 1 and 100 mM, or a buffer for the controls. The
chemiluminescence was registered after the addition of NaOCl
using the ‘‘flash assay’’ option of the MultiUse program, every
50 ms. The ratio of CL in the presence and in the absence of the drug
was termed CL scavenging index (CL-SI).
4.1.2.4.1.1. Ethyl-2-(3,4,5-trimethoxy)-
phenylmethyleneacetoacetate
White crystals. The product was recrystallized from ethanol. M.p.
159–160.5 ꢃC.; UV–vis lmax ¼ 206, 236, 320 nm; Anal. C16H20O6
(308) (C, H) ¼ (calcd/found): %C, 62.33/61.8; %H, 6.54/6.64.
4.1.2.4.2. Michael condensation between ethyl-2-(3,4,5-trime-
thoxy)-phenylmethyleneacetoacetate and 4-hydroxycoumarin. About
1.99g (0.0064 mol) of ethyl-2-(3,4,5-trimethoxy)phenylmethy
leneacetoacetate, 2.09 g (0.0257 mol) of 4-hydroxycoumarin and 20
ml dioxane were poured and mixed in a round-bottomed flask. After
that 1–2 ml of piperidine was added. The reaction mixture was
stirred under reflux for 30 h. Then 20 ml of water was added to the
reaction mixture and the stirring under reflux was continued for 2 h.
After that the heating was stopped for 48 h. The reaction mixture
was stirred under reflux for additional 30 h, according to TLC control.
At the end of the reaction time the mixture was left at room
temperature and white crystal residue was formed.
References
[1] Y. Yamada, N. Nakatani, H. Fuwa, Agric. Biol. Chem. 51 (1987) 1711–1713.
[2] Y. Jacquot, L. Bermont, H. Giorgi, B. Refouvelet, G. Adessi, E. Daubrosse,
A. Xicluna, Eur. J. Med. Chem. 36 (2001) 127–136.
[3] S. Kirkiacharian, D.T. Thuy, S. Sicsic, R. Bakhchinian, R. Kurkijan, T. Tonnaire, Il
Farmaco 57 (2002) 703–708.
[4] A. Mazumder, S. Wang, N. Neamati, M. Nicklaus, S. Sunder, J. Chen,
G.W.A. Milne, W.G. Rice, T.R. Burke, Y. Pommler, J. Med. Chem. 39 (1996)
2472–2481.
[5] P.J. Tummino, D. Ferguson, L. Hupe, D. Hupe, Biochem. Biophys. Res. Commun.
200 (1994) 1658–1664.
[6] S. Stanchev, G. Momekov, F. Jensen, I. Manolov, Eur. J. Med. Chem. 43 (2008)
694–706.
[7] S. Kirkiachiarian, R. Bakhchinian, H. Chidiak, M. Mazmanian, C. Planche, Ann.
Pharm. Fr. 57 (1999) 251–254.
[8] T. Kaneko, N. Baba, M. Matsuo, Cytotechnology 35 (2001) 43–55.
[9] M. Foti, M. Piatelli, M.T. Barata, G. Ruberto, J. Agric. Food Chem. 44 (1996)
497–501.
[10] M.J. Chaichi, A.R. Karami, A. Shockravi, M. Shamsipur, Spectrochim Acta A 59
(2003) 1145–1150.
[11] V. Hadjimitova, T. Traykov, M. Mileva, S. Ribarov, Z. Naturforsch. 57c (2002)
1066–1071.
4.1.2.4.2.1. Ethyl 2-[(3,4,5-trimethoxyphenyl)(4-hydroxy-2-oxo-2H-
chromen-3-yl)methyl]-3-oxobutanoate (T-2)
White crystals. The substance was recrystallized from ethanol.
Yield: 32%, m.p. 155–157 ꢃC, Rf ¼ 0.38 (hexane/acetone ¼ 2:1); UV–