Synthesis, computational study and cytotoxic activity of new 4-hydroxycoumarin derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.05.005

Six new 4-hydroxycoumarin derivatives have been synthesized. They were characterized by UV-vis, IR, ¹H NMR, ¹³C NMR, mass spectral data, elemental analysis, TLC and melting point determination. The new 4-hydroxycoumarin derivatives a

Full text of DOI:10.1016/j.ejmech.2007.05.005

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European Journal of Medicinal Chemistry 43 (2008) 694e706
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, computational study and cytotoxic activity of
new 4-hydroxycoumarin derivatives
Stancho Stanchev a,c, Georgi Momekov b, Frank Jensen c, Ilia Manolov a,
*
a Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Sofia, 2 Dunav St., BG-1000 Sofia, Bulgaria
b Department of Pharmacology and Toxicology, Faculty of Pharmacy, Medical University of Sofia, 2 Dunav St., BG-1000 Sofia, Bulgaria
c Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Received 19 January 2007; received in revised form 8 May 2007; accepted 10 May 2007
Available online 27 May 2007
Abstract
Six new 4-hydroxycoumarin derivatives have been synthesized. They were characterized by UVevis, IR, 1H NMR, 13C NMR, mass spectral
data, elemental analysis, TLC and melting point determination. The new 4-hydroxycoumarin derivatives are studied by computational methods e
DFT (B3LYP) and force field methods (MM2 and OPLS), in order to optimize their geometry and calculate quantum-chemical properties and
conformational analysis. Five new 4-hydroxycoumarin derivatives were tested for cytotoxic activity in two tumor cell lines e HL-60 and EJ. The
obtained results are compared with the utilized anticancer drug melphalan. Two of these compounds e ethyl 2-[(3,4-dihydroxyphenyl)-
(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl]-3-oxobutanoate (SS-16) and ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(3-nitrophenyl)methyl]-
3-oxobutanoate (SS-21) show comparatively good cytotoxic properties. Their activity is weaker than melphalan. SS-16 seems to be more active
than SS-21.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 4-Hydroxycoumarins; Knoevenagel reaction; Michael reaction; DFT calculations; Cytotoxic activity; MTT
1. Introduction
MCF-7 and RP-1788. In studies on the antiproliferative actions
of coumarin compounds, we discovered that dicoumarol (a cou-
marin anticoagulant, 3,30-methylenebis[4-hydroxycoumarin])
inhibits the first cleavage of Strongylocentrotus purpuratus
(sea urchin) embryos in a concentration-dependent manner
with 50% inhibition occurring at a concentration of 10 mM
[2]. It was found that dicoumarol binds to bovine brain tubulin
with a K(d ) of 22 mM and that 0.1 mM dicoumarol strongly sta-
bilizes the growing and shortening dynamics at the plus ends of
the microtubules in vitro. Dicoumarol and taxol in combination
gave results in a synergistic inhibition of cell division of sea
urchin embryos. These two compounds can be used in combina-
tion, in order to reduce the high toxicity of taxol. There are data
about cytotoxic action of some coumarin derivatives and their
metal complexes established by brine shrimp bioassay [3]. Some
substituted benzopyranobenzothiazinones are synthesized by
the interaction of 4-hydroxycoumarin and 2-aminothiophenol.
Coumarins are highly active biological substances. They
have spasmolytic, antioxidant, anticoagulant, antibacterial, an-
tiviral and antifungal activities. There are a lot of data about an-
tineoplastic action of coumarin derivatives. They are acting at
different stages of cancer formation. Some of them have cyto-
static properties and the others have cytotoxic activity. It has
been found that coumarin and 7-hydroxycoumarin (7-HC) have
antitumor activity [1]. 7-Hydroxycoumarin is the active metab-
olite of coumarin. Both coumarin and 7-HC were found to be
growth-inhibitory (cytostatic) for the following human malig-
nant cell lines: A549, ACHN, Caki-2, Dakiki, HS-Sultan, H727,
HCT-15, HL-60, K562, LNCaP, PC-3, Du 145 COLO-232,
* Corresponding author. Fax: þ359 2 9879874.
E-mail address: imanolov@gmx.net (I. Manolov).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.05.005

S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
695
These compounds express estrogenic activity on MCF-7 breast
carcinoma cells [4]. Their structure can be expressed as follows:
CH3
H3C
O
R
HN
R
S
O
O
O
O
R1
2-methyl-2-[4-methylhex-3-enyl]-2H,5H-pyrano[3,2-c]chromen-5-one
Also such tri- or tetracyclic 4-hydroxycoumarin derivatives
were obtained by asymmetric Michael reaction [7]. This
reaction was performed as interaction of 4-hydroxycoumarin
and 4-substituted-2-oxo-3-butenoate esters in the presence
of (S )-tBu-BOXeCu(OTf)2 (tBu-bisoxazolineseCu(OTf)2
complexes) as catalyst in Et2O. The first product of this
reaction is 3-substituted 4-hydroxycoumarin, which contains
4-substituted-2-oxo-3-butanoate fragment in the third place.
There is equilibrium between this 3-substitued 4-hydroxycou-
marin and its cyclic form in which one more pyran ring is
formed. The interaction of 6-substituted 4-hydroxycoumarins
and 1-(3-oxo-1,4-benzoxazin-6-yl)-3-allylpropen-1-ones was
implemented according to the Michael reaction [8]. The product
is substituted 4-hydroxycoumarin in the third place, containing
propyonyl-2H-[1,4]-benzoxazin-3(4H )-ones. The reaction was
carried out with pyridine as a basic agent and also as a medium.
The Michael addition to benzylydenecyclohexanones and 4-hy-
droxycoumarins was carried out in order to obtain new deriva-
tives with better anticoagulant activity [9]. The products of
the reaction are different 4-hydroxy-3-[(2-oxo)-cyclohexyl]-
benzylcoumarins. The reaction was accomplished in dioxane
medium with piperidine as a basic agent. Enantioselective syn-
thesis of warfarin was performed via Michael reaction [10]. This
reaction was carried out via interaction of 4-hydroxycoumarin
The most active compound is when R ¼ H and R1 ¼ OH.
The results are compared with 17b-estradiol. Their structural
properties and structureeactivity relationship were also stud-
ied by molecular modeling. These compounds may contribute
for building a new chemical library, used for treatment of dis-
eases, which are estrogenic-dependent.
The third position in 4-hydroxycoumarin ring is highly ac-
tivated, because of the influence of hydroxyl group with elec-
tron-donating properties and electron-withdrawing effects of
carbonyl oxygen atom at the second place. There is a conjuga-
tion of p-electrons from the double bond and lone p-electron
pairs from oxygen atom. These factors make the third position
in the coumarin ring very convenient for Michael addition to
the compounds with activated double bond (Michael
acceptors). The electronic and molecular properties of some
substituted di(4-hydroxycoumarins) were calculated by the hy-
brid DFT method e B3LYP [5]. Different basis sets were
tested in the course of the calculations: 6-31G*, 6-31 þ G**
and 6-311G*. Electron density distribution, molecular electro-
static potential, hardness, electrophilicity index and reactive
sites of the compounds are also calculated and discussed.
The possible places of electrophylic attack and hydrogen
bond formation were predicted. The different tautomeric
forms of 4-hydroxycoumarin have been established [6].
O
O
O
OH
H
H
O
O
III
OH
O
O
I
II
The nature of these three forms was elucidated by using
different spectral methods. There are also some reactions
with the compounds with activated double bond, which
are giving very interesting polycyclic structures [6]. For ex-
ample, when 4-hydroxycoumarin is reacting with farnesal
the product is:
and optically active amines in the presence of p-toluenesulfonic
acid. The different enamines were the product of that reaction.
After that two reactions were performed. The first reaction is be-
tween the obtained enamine with benzalacetone in the presence
of p-toluenesulfonic acid in methanol media at room tempera-
ture, which gives very low yield of (S ) warfarin and (R)

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S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
warfarin. Another reaction is in the presence of LDA and Lewis acids (TMSCl) in THF media at ꢀ78 ꢁC which gives the high yield of
optically active warfarin. The reaction of acylation at the third place of 4-hydroxycoumarin has been performed with acetic acid and
phosphoroxychloride [11]. The product is 3-acetylcoumarin, which is used in further reaction of aldol condensation with aromatic
aldehydes for obtaining antibacterial 4-hydroxycoumarins. Long 3-acyl derivatives of 4-hydroxycoumarin were obtained [12] via
acylation with undec-10-enoylchloride. The reaction was performed in nitrogen atmosphere in sonochemical reactor with dry pyr-
idine as a solvent and piperidine as a catalyst. The reaction mixture is sonicated at 21 kHz for 1.5 h at 38 ꢁC.
2. Chemistry
Differently substituted aromatic aldehydes are used for the synthesis of arylydene-b-ketoesters via Knoevenagel reaction with
ethylacetoacetate in the presence of piperidine as a basic agent and a glacial acetic acid. These arylydene-b-ketoesters may be
presented as:
O
CH3
O
O
R
H3C
where R ¼ m-NO2 (ethyl 2-(3-nitrobenzylidene)-3-oxobutanoate) (SS-19), R ¼ p-NO2 (ethyl 2-(4-nitrobenzylidene)-3-oxobuta-
noate) (SS-1), R ¼ p-OH (ethyl 2-(4-hydroxybenzylidene)-3-oxobutanoate) (SS-4), R ¼ m,p-diOH (ethyl 2-(3,4-dihydroxybenzy-
lidene)-3-oxobutanoate) (SS-8), R ¼ p-COOH (ethyl 2-(4-carboxybenzylidene)-3-oxobutanoate) (SS-6).
O
O
O
CH3
O
CH3
piperidine, CH3COOH
-H2O
O
O
+
O
H3C
CH3
R
R
3-Acetyl-8-methoxycoumarin (SS-5) is synthesized via Knoevenagel reaction of 2-hydroxy-3-methoxybenzaldehyde and
ethylacetoacetate with above-mentioned conditions.
O
O
CH3
O
reesterification
H3C
O
CH3COOH, piperidine
-H2O
+
O
OH
O
OH
CH3
CH3
O
O
O
CH3
CH3
O
CH3
O
O
O
CH3

S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
697
This process of reesterification is carried out spontaneously.
The second step of the reaction is a condensation of the obtained arylydene-b-ketoesters with 4-hydroxycoumarin through Michael
reaction, by using sodium methoxide or piperidine as a basic agent. This reaction can be expressed as follows:
H3C
O
H3C
O
O
O
O
O
OH
O
HO
CH3
Δ, piperidine or
CH3O-Na+
+
O
H3C
O
O
R
R
where R ¼ m-NO2 ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(3-nitrophenyl)methyl]-3-oxobutanoate (SS-21), R ¼ p-NO2 ethyl
2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-nitrophenyl)methyl]-3-oxobutanoate (SS-3), R ¼ p-OH ethyl 2-[(4-hydroxy-2-oxo-2H-
chromen-3-yl)(4-hydroxyphenyl)methyl]-3-oxobutanoate (SS-14), R ¼ m,p-diOH ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(3,4-
dihydroxyphenyl)methyl]-3-oxobutanoate (SS-16), R ¼ p-COOH ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-carboxyphenyl)-
methyl]-3-oxobutanoate (SS-17).
Compound SS-5 interacts with 4-hydroxycoumarin (according to Michael reaction), giving polycyclic compound e oxabicy-
clononane (chromanocoumarin) derivative (SS-20). The reaction scheme can be written as follows:
O
H3C
O
OH
O
O
CH3
NaOH, Δ
+
O
O
CH3
O
O
O
O
O
CH3
It can be proposed that the real product of reaction is:
O
O
H3C
H3C
O
O
O
which is isomer of SS-20.
The probable mechanism is expressed in Scheme 1 and for the isomeric product, the reaction mechanism is as follows
(Scheme 2).
All of the synthesized compounds are characterized by 1H NMR, 13C NMR, UVevis, IR, mass spectrometry and also by TLC,
elemental analysis and melting point determination.

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S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
CH3
CH3
O
O
O
O
OH
O
CH3
OH
O
O
OH
OH
O
H2O
NaOH
+
O
O
O
O
OH
O
CH3
O
O
O
O
O
H3C
H3C
H3C
O
O
H3C
H3C
OH
rotation
HO
OH
O
OH
O
-CO2
O
-H2O
CH3
O
O
O
H3C
O
O
H3C
O
O
Scheme 1. Probable mechanism of the interaction of 3-acetyl-8-methoxycoumarin with 4-hydroxycoumarin.
CH3
CH3
O
O
O
OH
O
O
CH3
OH
O
O
OH
O
H2O
OH
+
O
NaOH
O
O
O
OH
O
CH3
O
O
O
O
H3C
H3C
O
H3C
CH3
OH
H3C
O
OH
O
O
CH3
O
OH
CH3
HO
-CO2
OH
O
OH
O
O
O
O
H3C
O
O
O
O
H3C
-H2O
H3C
O
O
O
Scheme 2. Alternative mechanism of the interaction of 3-acetyl-8-methoxycoumarin with 4-hydroxycoumarin.

S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
699
3. Computational studies
methyl]-3-oxobutanoate
(SS-21),
4-[1-(4-hydroxy-2-oxo-
2H-chromen-3-yl)-2-(ethoxycarbonyl)-3-oxobutyl]benzoic acid
(SS-17), ethyl 2-[(3,4-dihydroxyphenyl)(4-hydroxy-2-oxo-
2H-chromen-3-yl)methyl]-3-oxobutanoate (SS-16) and 7-ace-
tonyl-11-methoxy-6-oxo-6H,7H-(1)-benzopyrano[4,3b]-(1)-
benzopyran (SS-20). These compounds are synthesized by two
steps: Knoevenagel reaction of aromatic aldehyde and
ethylacetoacetate as the first step. The second step is Michael
reaction of the obtained arylydene-b-ketoester with 4-hydrox-
The purpose of this investigation is to study the silico con-
formational behavior of the newly synthesized 4-hydroxycou-
marin compounds and determine physicochemical parameters
like dipole moment, Log P, HOMO and LUMO energies, mo-
lecular volume and charges, which will be useful for future
QSAR analysis.
4. Pharmacology
1
ycoumarin. They are characterized by spectral methods e H
NMR, 13C NMR, UVevis, IR, mass spectrometry and also by
TLC, elemental analysis and melting point determination.
These five 4-hydroxycoumarin derivatives were tested for
cytotoxic activity on urinary bladder carcinoma-derived EJ cells
and promyelocyte leukemia-derived HL-60 cells using MTT
test e [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] dye reduction assay as described by Mosmann [13]
with some modifications [14]. Absorption of the samples was
measured by an ELISA reader at 580 nm. Survival fraction
was calculated as percentage of the untreated control. The ex-
perimental data were processed and were fitted to sigmoidal
concentrationeresponse curves via non-linear regression.
The cytotoxic activity of the newly synthesized coumarin
derivatives was evaluated in vitro against HL-60 and EJ cell-
lines, after 72 h of continuous exposure. The cell viability
was assessed using MTT-dye reduction assay and the corre-
sponding IC50 values were calculated as the concentrations
of tested compounds causing 50% decrease of cell survival.
The clinically utilized antineoplastic drug melphalan e (2-
amino-3-[4-bis(2-chloroethyl)amino]phenylpropanoic acid), was
exploited as positive control.
5.2. Computational studies
Computationalstudiesofthesenewcompoundsweremadeby
DFT (B3LYP) and force field methods (MM2 and OPLS2005).
Different quantum-chemical parameters were calculated by
B3LYP/6-31G** methods such as ESP charges, Log P, energy
of HOMO and LUMO orbitals, dipole moment and molecular
volume. These descriptors can be used in future QSAR analysis.
These data have significance for the biological activity e for in-
teraction with the receptor/enzyme, penetration through the cell
membrane, chemical properties of the molecule during drug
metabolism. The generation of the conformations was made by
using force field (MM2 and OPLS2005) and Monte Carlo
simulation method. DFT (B3LYP/6-31G*) methods are used
for calculating their single point energy. All of the studied com-
pounds have many conformations in the bioactive energy range
0e15 kJ/mol, which means that entropy factor may be important
for the interaction with protein receptor. Many conformers are
stabilized by intramolecular hydrogen bonding between
hydroxyl group from 4-hydroxycoumarin fragment and acetyl
group from side chain. Cluster analysis of one SS-3 as an exam-
ple was made in order to generate representative conformations.
Nine representative conformations are found and more signifi-
cant difference is in the ethyl-2-acetyl-propionate chain.
5. Results and discussion
5.1. Chemistry
Six 4-hydroxycoumarin derivatives are synthesized e ethyl
2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hydroxyphenyl)-
methyl]-3-oxobutanoate (SS-14), ethyl 2-[(4-hydroxy-2-oxo-
2H-chromen-3-yl)(4-nitrophenyl)methyl]-3-oxobutanoate (SS-3),
ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(3-nitrophenyl)-
Table 1 contains atomic charges, calculated by the ESP proce-
dure, which might participate in hydrogen bonding with the
Table 1
ESP charges for more important atoms
R
q1(O)
ꢀ0.45
q2(O)
ꢀ0.51
q3(OH)
q4(O)
ꢀ0.45
q5(O)
ꢀ0.44
q6(O)
ꢀ0.5
q8(C)
ꢀ0.05
q9(C)
ꢀ0.56
q(NO2)
q(OH)
q(COOH)
3-NO2-(SS-21)
O ꢀ0.51;
N 0.61;
O1 ꢀ0.39;
O2 ꢀ0.38
e
e
e
H 0.41
3,4-diOH-(SS-16)
4-COOH-(SS-17)
ꢀ0.44
ꢀ0.41
ꢀ0.51
ꢀ0.54
O ꢀ0.52;
ꢀ0.46
ꢀ0.48
ꢀ0.43
ꢀ0.39
ꢀ0.51
ꢀ0.48
0.20
0.21
ꢀ0.5
O1 ꢀ0.55;
O2 ꢀ0.58;
H1 0.43;
H2 0.44
e
e
H 0.41
O ꢀ0.48;
ꢀ0.30
e
C 0.49;
O1 ꢀ0.519;
O2 ꢀ0.522;
H 0.42
H 0.42
4-NO2-(SS-3)
4-OH-(SS-14)
ꢀ0.43
ꢀ0.45
ꢀ0.52
ꢀ0.53
O ꢀ0.52;
ꢀ0.54
ꢀ0.47
ꢀ0.37
ꢀ0.42
ꢀ0.54
ꢀ0.56
0.25
0.48
ꢀ0.57
ꢀ0.75
N 0.62;
O1 ꢀ0.390;
O2 ꢀ0.386
e
e
e
H 0.43
O ꢀ0.55;
O ꢀ0.56;
e
H 0.42
H 0.41

700
S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
enzyme and/or receptor. The Log P is a measurement for hydro-
phobicity and is important for evaluating the distribution of the
compound between lipid/water phases and for penetration
through a cell membrane, metabolism of the drug molecule, hy-
drophobic interactions with proteins and toxicity. The dipole mo-
ment is a measure of the polarity of the molecule. This is useful
also for determining the penetration through cell membrane (sol-
ubilityinlipidbilayer)andforthe speed ofexcretion. TheHOMO
and LUMO energies are important for evaluating the reduction
and oxidation potentials of the compound. Information about
these orbitals may be significant for drug metabolism, because
manydrugsaremetabolizedbyoxidationandreductionreactions.
The molecular volume may provide further information, regard-
ing the interaction between drug molecule and receptor.
Conformational analysis is useful for searching the proba-
ble bioactive conformation. Usually, the bioactive conforma-
tion is in the range between 0 kJ/mol and 15 kJ/mol of the
global minimum. A conformational analysis can also be used
for predicting the Boltzmann distribution and entropy of
interaction between conformation family and receptor.
The B3LYP/6-31G** method was used for calculating the
above molecular properties and a conformational search was
performed with the aid of MM2 and OPLS 2005 force field
(for SS-20). Monte Carlo simulation method at 20 000 itera-
tions is used in order to generate well minimized conforma-
tions. Refined estimates of relative energies were done by
using B3LYP/6-31G* method. This hybrid DFT method
(B3LYP) was chosen due to its high accuracy and reasonable
computational cost for systems of the present size (4-hydrox-
ycoumarin derivatives have up to 60 atoms).
The optimized geometries of the compounds are shown in
Fig. 1. The geometry of 4-[1-(4-hydroxy-2-oxo-2H-chromen-
3-yl)-2-(ethoxycarbonyl)-3-oxobutyl]benzoic acid (SS-17) is
taken for example.
The atomic charges derived from the electrostatic potential
for some of the more significant atoms are presented in Table
1, for which the labeling is indicated in the structure below.
The results for quantum-chemical parameters are presented
in Table 2.
The geometry of the compound SS-20 was optimized and
the charges are calculated in the same way as above-men-
tioned compounds (Fig. 2)
This chromanocoumarin derivative has the values of quan-
tum-chemical parameters as follows:
Fig. 1. Optimized geometry of SS-17 obtained by B3LYP/6-31G** method.
Log P ¼ ꢀ0.18, m ¼ 4.48 D,
E
(HOMO) ¼ ꢀ6.29 eV,
3
˚
E (LUMO) ¼ ꢀ1.87 eV, molecular volume ¼ 331.3 A .
It can be inferred that these compounds possess hydropho-
bic properties, from their Log P values. Hydrophobicity con-
tributes for good penetration through cell membrane and for
resorption. The compound SS-20 is an exception of that order,
as it has hydrophilic properties. All the compounds are polar
molecules, as judged from the dipole moments. These com-
pounds are relatively small molecules, according to their
molecular volume data and they have several negative ESP
charges and mobile hydrogen atoms for hydrogen bond forma-
tion and van der Waals interaction with protein fragments. The
energies of HOMO orbitals are between ꢀ6.00 eV and
ꢀ6.53 eV, depending on the nature of the substituent (elec-
tron-withdrawing or electron-donating), for 3,4-dihydroxy
these values are between ꢀ5.00 eV and ꢀ6.00 eV. The same
relationship is visible for LUMO orbitals. Their energy is in-
creasing of electron-donating substituents (between ꢀ1.29 eV
and ꢀ2.63 eV).
The results from the conformational analysis are presented
as a graphical relationship between the relative energy of the
conformers (in kJ/mol) and the number of conformers. From
these charts it can be seen that how many conformations exist
in the energy range 0e15 kJ/mol, i.e. the conformations,
R
Table 2
Main quantum-chemical properties
6
H3C
3
O
OH
7
R
Log P
(GhoseeCrippen) [D] [eV]
m
E (HOMO) E (LUMO) Molecular
[eV]
3
˚
volume [A ]
O
5
8
1 3-NO2-
2.82
5.65 ꢀ6.47
6.99 ꢀ5.90
2.70 ꢀ6.36
4.90 ꢀ6.44
4.92 ꢀ6.07
ꢀ2.63
ꢀ1.56
ꢀ1.75
ꢀ2.41
ꢀ1.59
401.2
392.1
406.0
399.3
385.3
9
2 3,4-diOH- 2.01
3 4-COOH- 2.34
O
O
1
H3C
O
2
4 4-NO2-
5 4-OH-
2.82
2.4
4

S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
Conformations/Energy relationship
701
50
40
30
20
10
0
0
10
20
30
Conformation number
40
50
60
70
Fig. 4. Graphical relationship between number of conformations and their en-
ergy for compound SS-21.
Among the five coumarin derivatives only compounds
SS-16 and SS-21 displayed significant cytotoxic activity
causing 50% inhibition of cell viability within the investigated
concentration range, whereas the remaining analogues exerted
only marginal effects. On the basis of the IC50 values obtained
SS-16 was found to be more active than SS-21 in both cell lines.
All of the tested coumarin analogues are less active than
melphalan, which is utilized as neoplastic drug (see Table 3).
On the basis of the established micromolar cytotoxicity of
SS-16 in both the cells this compound could be considered
as lead for further development of cytotoxic coumarin
derivatives.
Fig. 2. Optimized geometry and ESP charges of SS-20 obtained by B3LYP/6-
31G** method.
which are within an acceptable energy for binding to the re-
ceptor or enzyme.
These charts are presented as Figs. 3e8.
It is seen that all of the compounds have many low-energy
conformations and the number of conformations is connected
with the entropy factor for the interaction with a protein
receptor. Some of the bioactive conformations are stabilized
by intramolecular hydrogen bonds between OH group from
4-hydroxycoumarin fragment and acetyl group (carbonyl
oxygen atom) from the side chain.
6. Conclusion
Six 4-hydroxycoumarin derivatives are synthesized e ethyl
2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hydroxyphenyl)-
methyl]-3-oxobutanoate (SS-14), ethyl 2-[(4-hydroxy-2-oxo-
2H-chromen-3-yl)(4-nitrophenyl)methyl]-3-oxobutanoate (SS-3),
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), ethyl 2-[(3,4-dihydroxyphenyl)(4-hydroxy-2-oxo-
2H-chromen-3-yl)methyl]-3-oxobutanoate (SS-16) and 7-ace-
tonyl-11-methoxy-6-oxo-6H,7H-(1)-benzopyrano[4,3b]-(1)-
benzopyran (SS-20). These compounds are synthesized by
two steps: Knoevenagel reaction of aromatic aldehyde and
ethylacetoacetate as the first step. The second step is
Michael reaction of the obtained arylydene-b-ketoester with
4-hydroxycoumarin. They are characterized by spectral
Based on a cluster analysis, we have selected some repre-
sentative structures of the compounds and Fig. 9 shows the
overlaid representative structures of compound SS-3. From
49 found conformations, after cluster analysis 9 representative
structures were found in cluster level 41. The main differences
are in the position of ethyl-2-acetyl-propionate chain, where
the bigger part of the rotatable bonds is located.
5.3. Pharmacology
These compounds are tested for cytotoxic activity with
MTT-dye reduction assay on urinary bladder carcinoma cells
(EJ-60) and leukemia-derived HL-60 cells. The data about
new compounds are compared with the clinically utilized an-
tineoplastic drug melphalan e (2-amino-3-[4-bis(2-chloroe-
thyl)amino] phenylpropanoic acid).
Conformations/Energy relationship
Conformations/Energy relationship
50
60
45
40
35
30
25
20
15
10
5
50
40
30
20
10
0
0
0
50
100
150
200
0
50
100
Conformation number
150
200
-10
Conformation number
Fig. 3. Graphical relationship between number of conformations and their
energy for compound SS-16.
Fig. 5. Graphical relationship between number of conformations and their en-
ergy for compound SS-17.

702
S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
Conformations/Energy relationship Conformations/Energy relationship
35
30
25
20
15
10
5
45
40
35
30
25
20
15
10
5
0
0
0
10
20
30
40
Conformation number
50
60
5
10
Conformation number
15
20
25
0
Fig. 6. Graphical relationship between number of conformations and their
energy for compound SS-3.
Fig. 8. Graphical relationship between number of conformations and their
energy for compound SS-20.
methods e 1H NMR, 13C NMR, UVevis, IR, mass spectrom-
etry and also by TLC, elemental analysis and melting point
determination.
less active than melphalan. The compound (SS-16) expresses
the best cytotoxic activity, according to the EJ-60 and
HL-60 cell lines. The compound (SS-21) also possesses signif-
icant cytotoxic activity. Oxabicyclononane derivative (SS-20)
is in hand to be tested for cytotoxic activity.
Computational studies of these new compounds were made
by DFT (B3LYP) and force field methods (MM2 and
OPLS2005). Different quantum-chemical parameters were
calculated by B3LYP/6-31G** methods such as ESP charges,
Log P, energy of HOMO and LUMO orbitals, dipole moment
and molecular volume. These descriptors can be used in future
QSAR analysis. These data have significance for the biologi-
cal activity e for interaction with the receptor/enzyme, pene-
tration through the cell membrane, chemical properties of the
molecule during drug metabolism. The generation of the con-
formations was made by using force field (MM2 and
OPLS2005) and Monte Carlo simulation method. DFT
(B3LYP/6-31G*) methods are used for calculating their single
point energy. All of the studied compounds have many confor-
mations in the bioactive energy range 0e15 kJ/mol, which
means that entropy factor may be important for the interaction
with protein receptor. Many conformers are stabilized by intra-
molecular hydrogen bonding between hydroxyl group from 4-
hydroxycoumarin fragment and acetyl group from side chain.
Cluster analysis of one representative compound was made in
order to generate representative conformations. Nine represen-
tative conformations are found and more significant difference
is in the ethyl-2-acetyl-propionate chain.
7. Experimental protocols
7.1. Synthesis of 4-hydroxycoumarin derivatives
7.1.1. Materials and methods
All starting materials were purchased from Merck, Sigmae
Aldrich and Fluka. They are used without further purification.
Melting points are measured in open capillary tubes on a Buchi
¨
535 melting point apparatus. The IR spectra were recorded at
Shimadzu FTIR 8101M spectrometer in nujol and frequencies
are expressed in cmꢀ1. The 1H NMR spectra were recorded in
These compounds are tested for cytotoxic activity with
MTT-dye reduction assay on urinary bladder carcinoma cells
(EJ-60) and leukemia-derived HL-60 cells. The data about
new compounds are compared with the clinically utilized an-
tineoplastic drug melphalan e (2-amino-3-[4-bis(2-chloroe-
thyl)amino] phenylpropanoic acid). All the compounds are
Conformations/Energy relationship
60
50
40
30
20
10
0
0
20
40
60
80
100
120
-10
Conformation number
Fig. 7. Graphical relationship between number of conformations and their
energy for compound SS-14.
Fig. 9. Overlaid representative conformations of the compound SS-3.

S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
703
Table 3
Cytotoxic activity of the newly synthesized compounds and melphalan in a
panel of tumor cell lines after 72 h exposure (MTT-dye reduction assay)
67 MHz): d ¼ 30, 110, 135, 140, 160, 190; EIMS: m/z
(%) ¼ 234 (100, 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.
Compound
IC50 (mM)a
HL-60b
EJc
SS-14
SS-16
SS-17
SS-21
SS-3
>200
35.1 ꢂ 2.4
>200
>200
115.8 ꢂ 7.9
>200
90.8 ꢂ 8.1
>200
172.5 ꢂ 7.3
>200
Melphalan
a
11.4 ꢂ 3.2
22.1 ꢂ 2.7
7.1.2.3. 3-Acetyl-8-methoxy-2H-chromen-2-one (SS-5). Yel-
low crystals, m.p. 171e172 ꢁC. The substance crystallizes
from ether and purified after recrystallization from ethanol.
Yield: 28%; UVevis: lmax ¼ 220, 254, 316 nm; FTIR (nujol):
1728.4, 1689.8, 1601.1, 1465, 1207.6, 1093.8, 765.8 cmꢀ1; 1H
NMR (acetone, 200 MHz): d ¼ 2.7 (s, 3H, aliphatic), 4.0 (s,
3H, aliphatic), 7.4 (m, 3H, aromatic), 8.4 (s, 1H, aliphatic);
13C NMR (acetone, 67 MHz): d ¼ 30, 60, 115, 125, 130, 150,
205; TLC: Rf ¼ 0.51 (hexane/acetone ¼ 2:1). Anal.: C12H10O5
(218) (C,H) (calcd/found): %C 66.05/66.04, %H 4.62/4.38.
Arithmetic mean ꢂ sd of eight independent experiments.
b
c
Human acute promyelocyte leukemia.
Human urinary bladder carcinoma.
Brucker 250 MHz in DMSO-d6 or acetone using TMS as an
internal standard (chemical shifts are reported in ppm units, cou-
pling constants (J ) in Hz). Abbreviations used are as follows:
s e singlet, d e doublet, dd e double doublet, t e triplet, m e
multiplet, br e broad.
Mass spectral analysis was performed by electron ioniza-
tion on mass spectrometer HewlettePackard 5973 at 70 eV.
7.1.2.4.
4-[2-(Ethoxycarbonyl)-3-oxobut-1-en-1-yl]benzoic
7.1.2. General procedure for the preparation
of arylydene-b-ketoesters
acid (SS-6). Yellow crystals, m.p. 148e150 ꢁC. The substance
crystallizes from water and purified after recrystallization from
ethanol. Yield: 57%; UVevis: lmax ¼ 204, 292 nm; FTIR (nu-
Aromatic aldehyde and ethylacetoacetate in equimolar
quantities are mixed in round-bottomed flask. Piperidine
(0.03 mol) and glacial acetic acid (0.04 mol) are also added
to the reaction mixture. The latter is stirred at room tempera-
ture for 90 min. After that 20 ml of ether and/or 150 ml dis-
tilled water are added to the reaction mixture and crystals
with different colors are formed. These crystals are filtered
and washed. Then they are dried at room temperature and re-
crystallized in appropriate solvents e mainly alcohols (etha-
nol, propanol, 2-propanol) and water (for SS-8).
jol): 3300e2400, 1736.1, 1689.8, 1608.8, 1460.3, 848 cmꢀ1
;
1H NMR (DMSO, 250 MHz): 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/chloro-
form/acetone/methanol ¼ 5:3:2:1). Anal.: C14H14O5 (262)
(C,H) (calcd/found): %C 64.12/64.44, %H 5.38/5.26.
7.1.2.1. Ethyl 2-(4-nitrobenzylidene)-3-oxobutanoate (SS-1).
White crystals, m.p. 160e161 ꢁC. The substance crystallizes
from ether and purified after recrystallization from isopropyl
alcohol. Yield: 66%; UVevis: lmax ¼ 204, 270 nm; FTIR
7.1.2.5. Ethyl 2-(3,4-dihydroxybenzylidene)-3-oxobutanoate
(SS-8) [19]. Brown-yellow crystals, m.p. 147.8e151 ꢁC. The
substance crystallizes from water and purified after recrystalli-
zation from water. Yield: 19%; UVevis: lmax ¼ 206, 252,
344 nm; FTIR (nujol): 3540, 1714.9, 1643.6, 1603, 1464.1,
(nujol): 1732.3, 1711.1, 1608.8, 1529.7, 1464.1, 844.9 cmꢀ1
;
1H NMR (acetone, 200 MHz): d ¼ 0.9 (t, 3H, aliphatic), 1.3
(s, 3H, aliphatic), 2.8 (q, 2H, aliphatic), 6.1 (s, 1H, aliphatic),
7.5 (d, 2H, aromatic), 8.2 (d, 2H, aromatic); 13C NMR
(acetone, 67 MHz): d ¼ 15, 30, 45, 125, 130, 145, 165, 175,
200; TLC: Rf ¼ 0.75 (hexane/chloroform/acetone/methanol ¼
5:3:2:1). Anal.: C13H13NO5 (263) (C,H) ¼ (calcd/found): %C
59.31/58.28, %H 4.98/5.92, %N 5,32/3,52.
1
1197 cmꢀ1; H NMR (DMSO, 250 MHz): d ¼ 1.0 (t, 3H, ali-
phatic), 2.3 (s, 3H, aliphatic), 4.2 (q, 2H, aliphatic), 6.9 (d,
2H, aromatic), 7.0 (s, 1H, aromatic), 7.6 (s, 1H, aliphatic), 8.4
(s, 2H, hydroxyl); EIMS: m/z (%) ¼ 250 (100, Mþ), 249 (40),
235 (20), 233 (29.6), 222 (17.4), 205 (31), 189 (76.5), 177
(10.4), 176 (31.3), 163 (42.6), 161 (73.9), 147 (4.3), 134
(19.1), 117 (9.6), 103 (5.2), 89 (15.7), 88 (11.3), 77 (11.3), 69
(4.3), 62 (8.7), 51 (8.7); TLC: Rf ¼ 0.4 (hexane/chloroform/ace-
tone/methanol ¼ 5:3:2:1). Anal.: C13H14O5 (250) (C,H) (calcd/
found): %C 62.39/62.31, %H 5.64/5.64.
7.1.2.2. Ethyl 2-(4-hydroxybenzylidene)-3-oxobutanoate (SS-4)
[15e18]. Yellow crystals, m.p. 141e143 ꢁC. The substance
crystallizes from ether and purified after recrystallization
from isopropyl alcohol. Yield: 51%; UVevis: lmax ¼ 206,
224, 286 nm; FTIR (nujol): 3325.7, 1732.3, 1641.6, 1597.3,
1
1462.2, 1205.7, 819.8 cmꢀ1; H NMR (acetone, 200 MHz):
d ¼ 1.3 (t, 3H, aliphatic), 2.3 (s, 3H, aliphatic), 4.3 (q, 2H, al-
iphatic), 6.9 (dd, 2H, aromatic), 7.4 (dd, 2H, aromatic), 7.6 (s,
1H, aliphatic), 10.5 (s, 1H, hydroxyl); 13C NMR (acetone,
7.1.2.6. Ethyl 2-(3-nitrobenzylidene)-3-oxobutanoate (SS-19).
White crystals, m.p. 100e103 ꢁC. The substance crystallizes
from water and purified after recrystallization from isopropyl

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S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
alcohol. Yield: 17%. UVevis: lmax ¼ 210, 266 nm; FTIR
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, hy-
droxyl); 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/chloroform/ace-
tone/methanol). Anal.: C22H20O7 (396) (C,H) (calcd/found):
%C 66.66/66.36, %H 5.09/5.13.
1
(nujol): 1728.4, 1660.9, 1628.1, 1529.7, 780, 735 cmꢀ1; H
NMR (DMSO, 250 MHz): d ¼ 0.9 (t, 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 (29.6), 63 (9.6), 51 (13), 45 (2.6); TLC: Rf ¼ 0.5 (hex-
ane/acetone ¼ 2:1). Anal.: C13H13NO5 (263) (C,H) (calcd/
found): %C 59.31/59.54, %H 4.98/H 5.13.
7.1.3.3. Ethyl 2-[(3,4-dihydroxyphenyl)(4-hydroxy-2-oxo-2H-
chromen-3-yl)methyl]-3-oxobutanoate
(SS-16). Tiled-red
crystals, m.p. 243.4e247 ꢁC. Purified after recrystallization
from isopropyl alcohol. Yield: 5%; UVevis: lmax ¼ 208,
280 nm; FTIR (nujol): 3451, 1732.3, 1662.8, 1608.8, 1460.3,
7.1.3. General procedure for the preparation of
condensation products with 4-hydroxycoumarin
Arylydene-b-ketoester, obtained in previous reaction, and
4-hydroxycoumarin are mixed in equimolar quantities in
25e30 ml methanol (used as a solvent). Sodium methoxide
(0.003 mol) as a basic agent is also added to the reagents.
The reaction mixture is boiled and stirred for 60 h under re-
flux. The reaction is controlled by TLC (hexane:acetone ¼ 2:1
or hexane:acetone:chloroform:methanol ¼ 5:3:2:1). When the
quantities of reagents are depleted the heating was stopped.
The residue from the reaction mixture was filtered off and
washed with hot water, in order to remove the 4-hydroxicou-
marin, which was not reacted. After that the residue is dried
at room temperature and recrystallized in appropriate solvent
(methanol, ethanol or 2-propanol).
1180.6, 1109.2, 825.6, 756.2 cmꢀ1 1H NMR (DMSO,
;
250 MHz): d ¼ 1.2 (t, 3H, aliphatic), 2.1 (s, 3H, aliphatic), 4.1
(q, 2H, aliphatic), 4.4 (m, 1H, aliphatic), 4.6 (m, 1H, aliphatic),
6.8 (m, 1H, aromatic), 7.1 (m, 1H, aromatic), 7.2 (m, 2H, aro-
matic), 7.4 (m, 1H, aromatic), 7.8 (m, 1H, aromatic), 7.9 (s, 1H,
aromatic), 8.1 (s, 3H, hydroxyl); EIMS: Mþ is probably very
unstable and it goes to a fragmentation spontaneously. m/z
(%) ¼ 396 (0.09), 374 (0.9), 348 (0.5), 331 (0.9), 317 (5.3),
282 (8.8), 281 (12.3), 265 (9.6), 241 (1.8), 228 (26.3), 213
(0.9), 200 (48.2), 189 (1.8), 171 (8.8), 162 (93), 144 (7), 134
(5.3), 120 (100), 110 (58.8), 92 (74.6), 81 (8.8), 64 (35), 51
(11.4), 45 (3.5); TLC: Rf ¼ 0.12 (hexane/chloroform/acetone/
methanol ¼ 5:3:2:1). Anal.: C22H20O8 (412) (C,H) (calcd/
found): %C 64.07/64.44, %H 4.89/4.52.
7.1.3.1. Ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-nitro-
phenyl)methyl]-3-oxobutanoate (SS-3). White crystals, m.p.
250e254 ꢁC. Purified after recrystallization from ethanol.
Yield: 33%; UVevis: lmax ¼ 206, 272 nm; FTIR (nujol):
7.1.3.4. 4-[1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-2-(ethoxy-
carbonyl)-3-oxobutyl]benzoic acid (SS-17). White crystals,
m.p. 150e155 ꢁC. Purified after recrystallization from metha-
nol. Yield: 28%; UVevis: lmax ¼ 208, 282, 308 nm; FTIR
(nujol): 3442, 3300e2400, 1732.3, 1693.7, 1612.7, 1462.4,
3362.3, 1732.3, 1651.3, 1616.5, 1601.1, 833.3, 765.1 cmꢀ1
;
1H NMR (DMSO, 250 MHz): d ¼ 1.0 (t, 3H, aliphatic), 2.0
(s, 3H, aliphatic), 3.9 (q, 2H, aliphatic), 4.2 (m, 1H, aliphatic),
5.0 (m, 1H, aliphatic), 7.2 (m, 3H, aromatic), 7.5 (m, 2H,
aromatic), 7.8 (m, 1H, aromatic), 7.9 (m, 2H, aromatic), 10
(s, 1H, hydroxyl); EIMS: m/z (%) ¼ 426 (0.8, Mþ), 380
(0.8), 368 (0.4), 343 (0.8), 327 (5.3), 317 (3.5), 302 (1.8),
284 (0.9), 274 (0.8), 256 (7), 242 (2.6), 230 (6.1), 213
(27.2), 202 (4.4), 187 (4.4), 176 (5.3), 163 (10), 162 (80.7),
149 (7.9), 134 (1.8), 121 (48.2), 120 (100), 105 (4.4), 92
(57), 77 (7.9), 63 (18.4), 51 (6.1), 46 (1.8); TLC:
Rf ¼ 0.48 (hexane/acetone ¼ 2:1). Anal.: C22H19NO8 (426)
(C,H) (calcd/found): %C 62.12/62.02, %H 4.5/4.38, %N
3.29/3.21.
1109, 846.3, 756.2 cmꢀ1 1H NMR (DMSO, 250 MHz):
;
d ¼ 1.0 (t, 3H, aliphatic), 2.1 (s, 3H, aliphatic), 3,9 (q, 2H, al-
iphatic), 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, hy-
droxyl), 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.
7.1.3.2. Ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(4-hy-
droxyphenyl)methyl]-3-oxobutanoate (SS-14). White crystals,
m.p. 195e197 ꢁC. Purified after recrystallization from ethanol.
Yield: 21%; UVevis: lmax ¼ 214, 280, 308 nm; FTIR (nujol):
7.1.3.5. 7-acetonyl-11-methoxy-6-oxo-6H,7H-(1)-benzopyra-
no[4,3b]-(1)-benzopyran (SS-20). Beige crystals, m.p. 149e
1
1
3391.3, 1699.5, 1622.3, 1599.2, 1464.1, 821, 760 cmꢀ1; H
152 ꢁC. Yield: 7%; H NMR (DMSO, 250 MHz): d ¼ 1.9 (s,
NMR (DMSO, 250 MHz): 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,
3H, aliphatic), 3.3 (m, 2H, aliphatic), 3.9 (s, 3H, aliphatic),
5.0 (s, 1H, aliphatic), 6.9 (m, 3H, aromatic), 7.3 (m, 2H, aro-
matic), 7.6 (m, 1H, aromatic), 7.8 (m, 1H, aromatic); EIMS:

S. Stanchev et al. / European Journal of Medicinal Chemistry 43 (2008) 694e706
705
m/z (%) ¼ 336 (100, Mþ), 321 (77.2), 305 (38.6), 293 (17.5),
279 (21), 265 (3.5), 238 (3.5), 227 (4.4), 213 (53.5), 201 (7.9),
188 (2.6), 175 (24.6), 160 (9.6), 152 (9.6), 132 (7.9), 121
(15.8), 103 (2.6), 77 (6.1), 65 (5.3), 51 (2.6); TLC: Rf ¼ 0.76
(hexane/ether ¼ 1:2).
bromide] dye reduction assay. Exponentially growing cells
were seeded in 96-well microplates (100 ml/well at a density
of 3.5 ꢃ 105 cell/ml for the adherent and 1 ꢃ 105 cells/ml for
the suspension cell lines) and allowed to grow for 24 h prior
to the exposure to the studied compounds. Stock solutions of
the tested compounds were freshly prepared in DMSO and
then diluted with growth medium. At the final dilutions the
solvent concentration never exceeded 0.5%. Cells were ex-
posed to the tested agents for 72 h, whereby for each concen-
tration a set of eight separate wells were used. After the
exposure period MTT solution (10 mg/ml in PBS) aliquots
were added to each well. The plates were further incubated
for 4 h at 37 ꢁC and the formazan crystals formed were dis-
solved by adding 110 ml of 5% HCOOH in 2-propanol. Ab-
sorption of the samples was measured by an ELISA reader
(Uniscan Titertec) at 580 nm. Survival fraction was calculated
as percentage of the untreated control. The experimental data
were processed by means of GraphPad Prism software and
were fitted to sigmoidal concentrationeresponse curves via
non-linear regression.
7.1.3.6. Ethyl 2-[(4-hydroxy-2-oxo-2H-chromen-3-yl)(3-nitro-
phenyl)methyl]-3-oxobutanoate (SS-21). White crystals, m.p.
135e140 ꢁC. Purified after recrystallization from ethanol.
Yield: 37%; UVevis: lmax ¼ 210 nm; FTIR (nujol): 3335,
1
1732.3, 1674.4, 1620.4, 1529.7, 1068.7, 763, 736 cmꢀ1; H
NMR (DMSO, 250 MHz): d ¼ 1.0 (t, 3H, aliphatic), 1.9 (s,
3H, aliphatic), 3.9 (q, 2H, aliphatic), 4.4 (m, 1H, aliphatic),
4.6 (m, 1H, aliphatic), 7.2 (m, 1H, aromatic), 7.4 (m, 1H, ar-
omatic), 7.5 (m, 1H, aromatic), 7.7 (m, 3H, aromatic), 7.9 (m,
2H, aromatic), 9.8 (s, 1H, hydroxyl); EIMS: 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).
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