Cytotoxic activity of new neodymium (III) complexes of bis-coumarins

Article abstract of DOI:10.1016/j.ejmech.2004.06.002

Complexes of neodymium (III) with bis-coumarins: 3,3′-benzylidene- bis(4-hydroxy-2H-1-benzopyran-2-one); bis(4-hydroxy-2-oxo-2H-chromen-3-yl)- piridin-2-yl-methane; bis(4-hydroxy-2-oxo-2H-chromen-3-yl)-piridin-4-yl-methane; bis(4-hydroxy-2-oxo-2H-chromen-

Full text of DOI:10.1016/j.ejmech.2004.06.002

European Journal of Medicinal Chemistry 39 (2004) 765–775
www.elsevier.com/locate/ejmech
Original article
Cytotoxic activity of new neodymium (III) complexes of bis-coumarins
Irena Kostova a,*, Ilia Manolov b, Georgi Momekov c
a Department of Chemistry, Faculty of Pharmacy, Medical University, 2 Dunav St., Sofia 1000, Bulgaria
b Department of Organic Chemistry, Faculty of Pharmacy, Medical University, 2 Dunav St., Sofia 1000, Bulgaria
c Department of Pharmacology and Toxicology, Faculty of Pharmacy, Medical University, 2 Dunav St., Sofia 1000, Bulgaria
Received 9 January 2004; received in revised form 28 May 2004; accepted 1 June 2004
Available online 23 July 2004
Abstract
Complexes of neodymium (III) with bis-coumarins: 3,3′-benzylidene-bis(4-hydroxy-2H-1-benzopyran-2-one); bis(4-hydroxy-2-oxo-2H-
chromen-3-yl)-piridin-2-yl-methane; bis(4-hydroxy-2-oxo-2H-chromen-3-yl)-piridin-4-yl-methane; bis(4-hydroxy-2-oxo-2H-chromen-3-
yl)-(1H-pyrazol-3-yl)-methane were synthesized by reaction of neodymium (III) salt and the ligands, in amounts equal to metal:ligand molar
ratio of 1:2. The complexes were prepared by adding an aqueous solution of neodymium (III) salt to an aqueous solution of the ligand
subsequently raising the pH of the mixture gradually to ca. 5.0 by adding dilute solution of sodium hydroxide. The neodymium (III) complexes
with bis-coumarins were characterized by different physicochemical methods—elemental analysis, IR-, 1H- and 13C-NMR-spectroscopies
and mass-spectral data. The spectral data of neodymium (III) complexes were interpreted on the basis of comparison with the spectra of the
free ligands. This analysis showed that in the Nd (III) complexes the ligands coordinated to the metal ion through both deprotonated hydroxyl
groups. On the basis of the m(C=O) red shift observed, participation of the carbonyl groups in the coordination to the metal ion was also
suggested. Cytotoxic screening by MTT assay was carried out. The complexes were tested on HL-60, HL-60/Dox and SKW-3 cell lines. The
overall results from the preliminary screening program revealed that all of the new Nd (III) complexes reach 50% inhibition of the malignant
cells proliferation and thus could be considered as biologically active. On the basis of the IC50 values obtained compounds Nd(L1)(OH).H2O
and Nd(L3)(OH).2H2O were found to exert superior activity in comparison to the remaining complexes.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Bis-coumarins; Neodymium (III) complexes; IR- and NMR-spectra; Cytotoxic activity
1. Introduction
mined cytostatic and cytotoxic nature of 8-nitro-7-
hydroxycoumarin using both human (including K-562 and
HL-60) and animal cell lines grown in vitro. Coumarin and
its 4-hydroxy and 7-hydroxy derivatives, as well as o-, m-
and p-coumaric acid were tested against P-815 and P-388
tumor cells in vitro. All compounds were more or less cyto-
toxic against tumor cells [3]. The effect of warfarin on tumor
cell growth was studied [4]. Warfarin inhibits metastasis of
Mtln3 rat mammary carcinoma without affecting primary
tumor growth. Seven known coumarins, showing significant
cytotoxic activities on P388 cell lines, were isolated from the
roots of Angelica gigas (Umbelliferae) [5]. Akman et al. [6]
had investigated synergistic cytotoxicity between menadione
and the related anticoagulant dicumarol, inhibited growth of
murine leukemia L1210 in liquid suspension culture. The
cytotoxicity of 22 natural and semisynthetic simple cou-
marins was evaluated in GLC4, a human small cell lung
Coumarin is used widely as a therapeutic agent and is
administered clinically in the treatment of certain lymphede-
mas and malignancies. 7-hydroxy- and 4-hydroxycoumarins
are naturally occurring substances with a variety of biologi-
cal activities, e.g. antitumoral action.
The antitumor activities of coumarin and its known me-
tabolite 7-hydroxy-coumarin were tested in several human
tumor cell lines by Steffen et al. [1]. Both compounds inhib-
ited cell proliferation of a gastric carcinoma cell line, a
colon-carcinoma cell line (Caco-2), a hepatoma-derived cell
line (Hep-G2) and a lymphoblastic cell line (CCRF cem).
Egan et al. [2] have synthesized, characterized and deter-
* Corresponding author.
E-mail address: irenakostova@yahoo.com (I. Kostova).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.06.002

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I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
carcinoma cell line, and in COLO 320, a human colorectal
cancer cell line, using the microculture tetrazolium (MTT)
assay [7].
some interest as a possibility to influence of resistant tumors.
The corresponding lanthanide salts are found to be of very
low or missing activity. So far we can conclude that the
structure of metal-ligand determines the antitumor spectrum
of the newly formed complexes. Those in vitro effects are not
so clearly expressed as it is in the case of cis-DDP (II).
Nevertheless their study is interesting in connection with
other cell lines and tumors in order to find out the differences
in their spectrum of activity.
Unfortunately, little is known about the complexing abil-
ity of neodymium (III) with coumarins. A survey of the
literature reveals that no work has been done on the reactions
of neodymium (III) with 3,3′-benzylidene-bis(4-hydroxy-
2H-1-benzopyran-2-one) and its derivatives. It was, there-
fore, considered worthwhile to study the complexation and in
the first place the objective of this study was to determine
whether the new complexes were active as cytotoxic agents.
In the present study, we perform investigation of the coor-
dination ability of 3,3′-benzylidene-bis(4-hydroxy-2H-1-
benzopyran-2-one); bis(4-hydroxy-2-oxo-2H-chromen-3-
A number of 4-hydroxycoumarin derivatives have been
studied as to their HIV integrase inhibitory potency [8]. The
main purpose was to simplify the large structure of the
compounds while maintaining their potency. It was found
that the minimum active pharmacophore consists of a cou-
marin dimer containing an aryl substituent on the central
linker, methylene. The addition of 4- and 7-hydroxy substitu-
ents in the coumarin rings improved the potency of the
compounds. Among the systems studied, the 3,3′-benzyli-
dene-bis(4-hydroxycoumarin) has been tested as a HIV inte-
grase inhibitor and has shown significant activity [8]. The
complexation ability of the 3,3′-benzylidene-bis(4-hydro-
xycoumarin) with lanthanides was not reported so far. It is
expected that the complexes with this ligand and with similar
ligands will retain or improve its biological activity as in the
case of other lanthanide complexes with hydroxycoumarin
derivatives.
The complexes of rare earth ions have aroused much
interest. Lanthanides are a subject of increasing interest in
bioinorganic and coordination chemistry [9,10].
Nowadays, a lot of studies report complexes of coumarin
derivatives with rare earth metals, which possess biological
activity. Thus, lanthanide complexes of 3-sulfo-4-hydroxy-
coumarin [11] and bis-(4-hydroxy-3-coumarinyl)-acetic acid
[12] have been synthesised and characterised. The complexes
have revealed good anticoagulant action.
yl)-piridin-2-yl-methane;
bis(4-hydroxy-2-oxo-2H-chro-
men-3-yl)-piridin-4-yl-methane; bis(4-hydroxy-2-oxo-2H-
chromen-3-yl)-(1H-pyrazol-3-yl)-methane in complexation
reaction with neodymium (III). The obtained Nd (III) com-
plexes with these coumarin ligands was characterized by
elemental analysis, physicochemical methods, mass-, NMR-
and IR-spectroscopy. The complicated vibrational spectra of
neodymium (III) complexes were interpreted on the basis of
comparison with the vibrational spectra of the free ligands.
The most sensitive to coordination modes of the ligands have
been assigned and discussed.
Lanthanium chloride manifests an antitumor activity. Fur-
thermore, literature data show that the coumarins have also
these properties. These previous data from literature are in
accordance with our investigations. They give our reason to
suppose that complexes of coumarins with lanthanides could
present interesting metalorganic compounds with antitumor
activity. As a result from our earlier work the cytotoxic
profile of some complexes of mendiaxon, warfarin, cou-
machlor and niffcoumar with lanthanides against P3HR1,
K-562 and THP-1 cell lines was proved [13–18]. The com-
plexes of cerium, lanthanum and neodymium with these
coumarin ligands induced approximately 30% reduction of
the survival of P3HR1 Burkitt lymphoma cells at concentra-
tions 100 and 400 µM. The cerium and lanthanum complexes
of mendiaxon and niffcoumar induce similar low cytotoxic
effect onAML derived THP-1 myeloleukemia cells. With the
relatively resistant CML derived erythroleukemic K-562 cell
line we obtained very interesting in vitro results. It is note-
worthy that the lanthanum and neodymium complexes with
niffcoumar exert more pronounced cytotoxic effects in com-
parison to cerium complex. They have a strong cell prolifera-
tion inhibiting effects (only about 30% of the cells survived).
This means that the resistant tumor cells may be inhibited
well with lanthanide complexes. This means also that the
spectrum of cytotoxicity of these complexes is different from
cis-DDP (II) and from Pt (II) complexes. These results are of
We observed that Nd (III) possess a cytotoxic activity and
literature data show that the coumarins have also these prop-
erties. That is why our synthesis of complexes of Nd (III) is
taken into consideration with cytotoxic screening and further
pharmacological study.
2. Chemistry
The compounds used for preparing the solutions were
Merck products, p.a. grade: Nd(NO3)3.6H2O. 3,3′-benzyli-
dene-bis(4-hydroxy-2H-1-benzopyran-2-one), bis(4-hydroxy-
2-oxo-2H-chromen-3-yl)-piridin-2-yl-methane, bis(4-hy-
droxy-2-oxo-2H-chromen-3-yl)-piridin-4-yl-methane and
bis(4-hydroxy-2-oxo-2H-chromen-3-yl)-(1H-pyrazol-3-yl)-
methane were used for the preparation of metal complexes as
ligands (Scheme 1). These ligands were synthesized by con-
densation of 4-hydroxycoumarin and aromatic or heterocy-
clic aldehyde in ethanol medium at reflux and stirring until
crystals appeared to us.
The complexes of neodymium (III) with 3,3′-benzyli-
dene-bis(4-hydroxy-2H-1-benzopyran-2-one) (H2L1); bis-
(4-hydroxy-2-oxo-2H-chromen-3-yl)-piridin-2-yl-methane
(H2L2); bis(4-hydroxy-2-oxo-2H-chromen-3-yl)-piridin-4-
yl-methane (H2L3); bis(4-hydroxy-2-oxo-2H-chromen-3-

I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
767
in this cell line. Cell lines were obtained from the Department
of Human and Animal Cell Cultures at the German Collec-
tion of Microorganisms and Cell Cultures (DSMZ). They
were grown as suspension-type cultures under standard
conditions—RPMI 1640 medium (Sigma), supplemented
with 10% heat inactivated fetal bovine serum (Sigma)
and
2
mM L-glutamine (Sigma), in controlled
environment—‘Heraeus’ incubator with humidified atmo-
sphere and 5% carbon dioxide, at 37 °C in cell culture flasks.
In order to maintain the cells in log-phase, cell suspension
was discarded 2–3 times per week and the remaining culture
was supplemented with fresh medium aliquots. The RPMI
medium for HL-60/Dox contained 0.2 µM doxorubicin.
3.2. Cytotoxicity determination
The cytotoxic activity of the investigated neodymium
complexes was assessed by the MTT (3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye-
reduction assay as described by Mosmann [19], with some
modifications [20]. The method is based on the ability of the
vital cells to metabolize the yellow tetrazolium dye MTT to
violet, water-insoluble formazan. After the dissolution of the
latter, using acidification and organic solvent addition its
concentration, which is proportional to dye number of viable
cells is determined spectrophotometrically. Briefly logarith-
mically growing cells were seeded into 96-well microplates
(100 µl/well at a density of 1 × 105 cells/ml) and exposed to
various concentrations of the investigated compounds for or
72 h. After the incubation with the test-compound, MTT-
solution (10 mg/ml in PBS) was added (10 µl/well). Plates
were further incubated for 4 h at 37 °C and the formazan
crystals formed were dissolved by adding 100 µl/well of 5%
formic acid in 2-propanol. Absorption was measured by an
ELISA reader (Uniscan–Titertek) at 540 nm, reference filter
690 nm. For each concentration at least eight wells were
used. 100 µl RPMI 1640 medium with 10 µl MTT stock and
100 µl 5% formic acid in 2-propanol was used as blank
solution.
Scheme 1. Structures of the ligands
yl)-(1H-pyrazol-3-yl)-methane (H2L4) were synthesized by
reaction of neodymium (III) salt and the ligand, in amounts
equal to metal:ligand molar ratio of 1:2. The complexes were
prepared by adding an aqueous solution of neodymium (III)
salt to an aqueous solution of the ligand subsequently raising
the pH of the mixture gradually to ca. 5.0 by adding dilute
solution of sodium hydroxide. The reaction mixtures were
stirred with an electromagnetic stirrer at 25 °C for 1 h. At the
moment of mixing of the solutions, precipitates were ob-
tained. The precipitates were filtered, washed several times
with water and dried in a desiccator to constant weight. The
complexes were insoluble in water, slightly soluble in metha-
nol and ethanol and soluble in DMSO.
3. Pharmacology
3.1. Human tumor cell lines and culture conditions
The antineoplastic activity of the tested compounds was
assessed on the lymphoid SKW-3 cell line (DSMZ No. ACC
53, cell type: human T-cell leukemia, origin: established
from the peripheral blood of a 61-year-old man with T-cell
chronic lymphocytic leukemia in 1977, doubling time of ca.
30–40 h, cytogenetics: human near diploid karyotype with
4% polyploidy and on the myeloid HL-60 (DSMZ No. ACC
3, cell type: human acute myeloid leukemia, origin: estab-
lished from the peripheral blood of a 35-year-old woman
with acute myeloid leukemia in 1976, doubling time of ca.
25 h, cytogenetics: human flat-moded hypotetraploid karyo-
type with hypodiploid sideline and 1.5% polyploidy); as well
as on the resistant variant HL-60/Dox, which is characterized
by the expression of the multi-drug resistance-associated
protein MRP-1, which conditions pleiotropic drug resistance
4. Results and discussion
4.1. Chemistry
The complexes were characterized by elemental analysis.
The metal ion was determined after mineralisation. The wa-
ter content in the complexes was determined by Karl Fisher
analysis. The formation of the complexes was confirmed by
IR-spectroscopy, 1H, 13C-NMR-spectroscopy and mass-
spectral data.
Table 1 shows the data of the elemental analysis of the
complexes serving as a basis for the determination of their
empirical formulae. The elemental analysis data of the Nd
(III) complexes obtained are in agreement with the presented
formulas.

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I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
Table 1
spectra of 3,3′-benzylidene-bis(4-hydroxy-2H-1-benzo-
pyran-2-one) (H2L1); bis(4-hydroxy-2-oxo-2H-chromen-3-
yl)-piridin-2-yl-methane (H2L2); bis(4-hydroxy-2-oxo-2H-
chromen-3-yl)-piridin-4-yl-methane (H2L3); bis(4-hydroxy-
2-oxo-2H-chromen-3-yl)-(1H-pyrazol-3-yl)-methane
(H2L4) and of the neodymium complexes with these ligands
are presented in Table 3.
Elemental analysis data for Nd (III) complexes with bis-coumarins
Experimental/calculated
Complex
%C
%H
%N
–
%H2O %Nd
Nd(L1)(OH).H2O
51.27
50.93
47.6
3.24
2.89
3.42
2.96
3.28
2.96
3.05
2.85
3.5
24.18
24.45
23.45
23.68
23.36
23.68
23.97
24.12
–
3.06
6.38
5.92
6.29
5.92
6.3
Nd(L2)(OH).2H2O
Nd(L3)(OH).2H2O
Nd(L4)(OH).2H2O
2.64
2.3
2.55
2.3
5.11
4.69
2–
47.37
47.44
47.37
44.24
44.22
4.2.1. IR-spectrum of the complex of 3,3′-benzylidene-
bis(4-hydroxy-2H-1-benzopyran-2-one) (H2L1)
6.03
2–
2–
L1
=
C25H14O6
;
L2
=
C24H13NO6
;
L3
=
C24H13NO6 ;
The bands appear in the IR spectrum of 3,3′-benzylidene-
bis(4-hydroxy-2H-1-benzopyran-2-one) (H2L1) at 3074,
3032; 1660, 1617; 1605, 1568; 1496, 1182, 1160, 1092,
1074 cm–1. The bands at 1660 and 1617 cm–1 can be attrib-
uted to the stretching vibrations of the carbonyl groups of the
lacton rings. Bands at 1605 and 1568 cm–1 can be related to
the stretching vibrations of the conjugated olefinic system.
The vibrations at 1496 cm–1 correspond to the aromatic
systems.
A broad band, characteristic of mOH of coordinated water
was observed in the range 3300–3400 cm–1 in the spectrum
of the complex. The weak bands observed at 3074 and
3032 cm–1 in the spectrum of the free ligand is missing in the
spectrum of the complex. A comparison of the infrared spec-
tra of the ligand and of the complex reveals the disappearance
of absorption bands observed in the free ligand at 3074,
3032 cm–1 and 1345, 1336 cm–1 associated with the stretch-
ing and deformation OH of the phenolic groups, indicating
the loss of phenolic protons on complexation, thus forming
2–
L4 = C22H12N2O6
.
The suggested formulas were further confirmed by mass-
spectral fragmentation analysis. As it is seen from Table 2,
the first peaks in the Nd (III) complexes spectra (although
with low intensity) correspond to the mass-weight of the
complex formation and the next ones to that of the ligands.
The results thus obtained are in agreement with metal:ligand
ratio 1:1 in the investigated complexes. The data of mass-
spectral fragmentation of the ligands and of the complexes
are presented in Table 2.
4.2. IR spectra of the complexes
The mode of bonding of the ligands to Nd (III) was
elucidated by recording the IR spectra of the complexes as
compared with this of the free ligands.
IR-spectra of the compounds were recorded on solid state
in Nujol in the range 3800–400 cm–1. The data of the IR
Table 2
Mass-spectral data of bis-coumarins and their Nd (III) complexes
Ligand
m/z
412
249
221
162
120
413
395
252
162
120
92
(%)
8
Complex
m/z
589
410
307
176
(%)
2
H2L1=C25H16O6
Nd(L1)(OH).H2O
100
17
20
37
7
1
68
100
H2L2=C24H15NO6
H2L3=C24H15NO6
H2L4=C22H14N2O6
Nd(L2)(OH).2H2O
Nd(L3)(OH).2H2O
Nd(L4)(OH).2H2O
607
573
552
411
307
176
604
573
552
410
307
176
600
579
562
410
307
176
2
2
4
7
1
30
28
38
0
1
45
100
4
413
252
250
162
120
92
18
50
62
74
86
0
5
6
4
44
100
1
402
241
240
162
120
92
16
100
72
74
98
3
1
1
48
100

I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
769
Table 3
Selected experimental IR frequencies of the ligands and their Nd (III) complexes (cm–1
)
Compound
mOH/H2O
m(C=O)
m(C=C)
m(Py)
m(Ar)
d(COH)
m(C–O)
1182m
1160m
1092s
H2L1=C25H16O6
3074m
3032m
1660s
1617s
1605s
1568s
–
–
1496m
1345m
1336m
772
750
1074m
1192w
1150w
1109m
1094w
1181m
1164m
1111s
Nd(L1)(OH).H2O
H2L2=C24H15NO6
Nd(L2)(OH).2H2O
H2L3=C24H15NO6
Nd(L3)(OH).2H2O
H2L4=C22H14N2O6
Nd(L4)(OH).2H2O
3391br
1625sh
1599s
1505s
1450m
1489m
1436m
1498m
1436 m
1496m
1460m
–
757
1620
1559
1505
1410
1622
1558
1506
1418
1620
1558
1520
1405
1622
1558
1516
1418
1620
1559
1507
1417
1622
1559
1505
1420
3122m
3060m
1696s
1635s
1608s
1539s
1350m
1332m
770
751
1039m
1212w
1150w
1109m
1078w
1181m
1155m
1107s
3400br
1652sh
1598s
1522s
–
759
3180m
3120m
1699s
1635s
1610s
1538s
1340m
1315m
770
750
1037m
1186w
1150w
1109m
1075w
1187m
1150m
1110s
3382br
1653sh
1600s
1520s
–
760
3139m
3070m
1669s
1635s
1610s
1539s
1360m
1300m
770
748
1044m
1195w
1145w
1108m
1054w
3375br
1653sh
1599s
1520s
–
758
a br-broad, s-strong, m-medium, sh-shoulder, w-weak.
metal–oxygen bonds which appear as bands in the far IR
region.
1620, 1559, 1505, 1410 cm–1 can be attributed to the
stretching vibrations of pyridine and they remain almost
the same in the complex.
The mC=O bands at 1660 and 1617 cm–1 exhibits a shift of
30–40 cm–1 to lower wavenumber values on complexation
which may be taken as evidence for the participation of the
C=O groups in coordination.
A broad band, characteristic of mOH of coordinated water
was observed in the range 3300–3400 cm–1 in the spectrum
of the complex. The weak bands observed at 3122 and
3060 cm–1 in the spectrum of the free ligand is missing in the
spectrum of the complex. A comparison of the infrared spec-
tra of the ligand and of the complex reveals the disappearance
of absorption bands observed in the free ligand at 3122,
3060 cm–1 and 1350, 1332 cm–1 associated with the stretch-
ing and deformation OH of the phenolic groups, indicating
the loss of phenolic protons on complexation, thus forming
metal–oxygen bonds which appear as bands in the far IR
region.
The C–C and C–O stretch and the C–O–C band are all
shifted in the complex. Similar frequency shifts are observed
for the other complexes and are attributed to complexation of
the positive ion with the carbonyl oxygen [21].
4.2.2. IR-spectrum of the complex of bis(4-hydroxy-2-oxo-
2H-chromen-3-yl)-piridin-2-yl-methane (H2L2)
The bands appear in the IR spectrum of bis(4-hydroxy-
2-oxo-2H-chromen-3-yl)-piridin-2-yl-methane (H2L2)
at 3122, 3060; 1696, 1635; 1608, 1539; 1489, 1181,
1164, 1111, 1039 cm–1. The bands at 1696 and 1635 cm–1
can be attributed to the stretching vibrations of the carbo-
nyl groups of the lacton rings. Bands at 1608 and
1539 cm–1 can be related to the stretching vibrations of
the conjugated olefinic system. The vibrations at
1489 cm–1 correspond to the aromatic systems. Bands at
The mC=O bands at 1696 and 1635 cm–1 exhibits a shift of
30–40 cm–1 to lower wavenumber values on complexation
which may be taken as evidence for the participation of the
C=O groups in coordination.
The C–C and C–O stretch and the C–O–C band are all
shifted in the complex. Similar frequency shifts are observed
for the other complexes and are attributed to complexation of
the positive ion with the carbonyl oxygen [21].

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I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
4.2.3. IR-spectrum of the complex of bis(4-hydroxy-2-oxo-
2H-chromen-3-yl)-piridin-4-yl-methane (H2L3)
3070 cm–1 and 1360, 1300 cm–1 associated with the stretch-
ing and deformation OH of the phenolic groups, indicating
the loss of phenolic protons on complexation, thus forming
metal–oxygen bonds which appear as bands in the far IR
region.
The bands appear in the IR spectrum of bis(4-hydroxy-2-
oxo-2H-chromen-3-yl)-piridin-4-yl-methane (H2L3) at
3180, 3120; 1699, 1635; 1610, 1538; 1498, 1181, 1155,
1107, 1037 m–1. The bands at 1699 and 1635 m–1 can be
attributed to the stretching vibrations of the carbonyl groups
of the lacton rings. Bands at 1610 and 1538 m–1 can be
related to the stretching vibrations of the conjugated olefinic
system. The vibrations at 1498 cm–1 correspond to the aro-
matic systems. Bands at 1620, 1558, 1520, 1405 cm–1 can be
attributed to the stretching vibrations of pyridine and they
remain almost the same in the complex.
A broad band, characteristic of mOH of coordinated water
was observed in the range 3300–3400 cm–1 in the spectrum
of the complex. The weak bands observed at 3180 and
3120 cm–1 in the spectrum of the free ligand is missing in the
spectrum of the complex. A comparison of the infrared spec-
tra of the ligand and of the complex reveals the disappearance
of absorption bands observed in the free ligand at 3180,
3120 cm–1 and 1340, 1315 cm–1 associated with the stretch-
ing and deformation OH of the phenolic groups, indicating
the loss of phenolic protons on complexation, thus forming
metal–oxygen bonds which appear as bands in the far IR
region.
The mC=O bands at 1669 and 1635 cm–1 exhibits a shift of
20–30 cm–1 to lower wavenumber values on complexation
which may be taken as evidence for the participation of the
C=O groups in coordination.
The C–C and C–O stretch and the C–O–C band are all
shifted in the complex. Similar frequency shifts are observed
for the other complexes and are attributed to complexation of
the positive ion with the carbonyl oxygen [21].
IR-spectra of the compounds were recorded on solid state
in Nujol in the range 700–220 cm–1. The spectrum of the
complex shows new bands, in comparison with this of the
free ligand, which have been assigned to the rocking, wag-
gling and metal–oxygen stretching vibrations.
4.3. 1H- and 13C-NMR spectra of the ligands and their Nd
(III) complexes
Metal ion coordination with ligand by means of oxygen
atoms of C=O groups and of the deprotonated hydroxyl
groups was shown owing to data of 1H- and 13C-NMR spec-
tra.
Proton spectra of the compounds recorded at 250 MHz in
DMSO-d6, confirmed the formation of the complex. The
typical chemical shifts of the 1H-NMR spectra in DMSO-d6
are presented in Table 4. As it is seen from Table 4, chemical
shifts to higher ppm were observed in the complexes and they
were attributed to coordination of ligands to Nd (III).
13C-NMR spectra of the ligands and of the complexes
were recorded at 62.9 MHz in DMSO-d6. The results of
The mC=O bands at 1699 and 1635 cm–1 exhibits a shift of
30–40 cm–1 to lower wavenumber values on complexation
which may be taken as evidence for the participation of the
C=O groups in coordination.
The C–C and C–O stretch and the C–O–C band are all
shifted in the complex. Similar frequency shifts are observed
for the other complexes and are attributed to complexation of
the positive ion with the carbonyl oxygen [21].
Table 4
1H NMR spectral shifts, d (ppm) of the ligands and their Nd (III) complexes
4.2.4. IR-spectrum of the complex of bis(4-hydroxy-2-oxo-
2H-chromen-3-yl)-(1H-pyrazol-3-yl)-methane (H2L4)
(250 MHz, DMSO-d6)
Compound
d (ppm)
a
a
a
H5–H8
H9
H2′–H6′
H2L1=C25H16O6
Nd(L1)(OH).H2O
H2L2=C24H15NO6
Nd(L2)(OH).2H2O
H2L3=C24H15NO6
Nd(L3)(OH).2H2O
H2L4=C22H14N2O6
Nd(L4)(OH).2H2O
7.11–7.39
7.47–7.60
7.24–7.58
7.20–7.75
7.22–7.58
7.20–7.80
7.23–7.55
7.18–7.80
6.37
6.64
6.54
6.3
7.56–7.92
7.88–8.16
7.80–8.64
8.35–8.81
7.80–8.68
8.40–8.80
7.83–8.15
8.02–8.48
The bands appear in the IR spectrum of bis(4-hydroxy-2-
oxo-2H-chromen-3-yl)-(1H-pyrazol-3-yl)-methane (H2L4)
at 3139, 3070; 1669, 1635; 1610, 1539; 1496, 1187, 1150,
1110, 1044 cm–1. The bands at 1669 and 1635 cm–1 can be
attributed to the stretching vibrations of the carbonyl groups
of the lacton rings. Bands at 1610 and 1539 cm–1 can be
related to the stretching vibrations of the conjugated olefinic
system. The vibrations at 1496 cm–1 correspond to the aro-
matic systems. Bands at 1620–1417 cm–1 can be attributed to
the stretching vibrations of pyrazol and they remain almost
the same in the complex.
6.46
6.72
6.36
5.89
A broad band, characteristic of mOH of coordinated water
was observed in the range 3300–3400 cm–1 in the spectrum
of the complex. The weak bands observed at 3139 and
3070 cm–1 in the spectrum of the free ligand is missing in the
spectrum of the complex. A comparison of the infrared spec-
tra of the ligand and of the complex reveals the disappearance
of absorption bands observed in the free ligand at 3139,

I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
771
Table 5
13C NMR spectral shifts, d (ppm) of the ligands and their Nd(III) complexes (62.9 MHz, DMSO-d6)
Atom d (ppm)
H2L1=C25H16O6 Nd(L1)(OH).H2O H2L2=C24H15NO6 Nd(L2)(OH).2H2O H2L3=C24H15NO6 Nd(L3)(OH).2H2O H2L4=C22H14N2O6 Nd(L4)(OH).2H2O
C-2 165.3
C-4 164.9
C-8a 152.2
C-1′ 139.9
C-7 131.9
C-3′ 128.1
C-5′ 128.1
C-4′ 126.7
C-6′ 125.6
C-2′ 125.6
C-5 123.9
C-6 123.8
C-4a 117.9
C-8 115.9
C-3 104.1
C-9 35.9
167.7
164.6
152.5
142.3
130.9
127.6
127.6
126.6
124.8
124.8
124.1
122.9
119.9
115.4
103.4
36.5
168.6
164.0
157.6
152.9
146.5
141.9
141.9
131.9
125.9
–
164.8
161.9
157.28
152.58
148.3
135.8
135.8
130.3
124.2
–
168.2
164.9
164.2
152.8
141.0
131.7
131.7
–
164.7
157.0
155.8
148.3
146.2
134.5
134.5
–
167.9
163.9
152.7
150.5
134.3
131.6
–
169.1
157.6
157.1
152.6
142.1
132.8
–
–
–
125.3
125.3
124.3
123.3
119.5
115.9
101.5
37.9
129.8
129.8
126.2
121.9
117.1
116.8
102.7
34.4
–
–
106.1
124.3
123.3
119.5
115.8
101.4
30.1
129.9
122.1
120.1
117.6
117.4
103.8
38.5
124.4
123.4
119.3
115.9
100.5
36.7
122.4
120.4
117.6
115.5
103.5
38.5
13C-NMR spectra of the compounds in d (ppm) are presented
in Table 5.
carbon atoms of the complex and they confirmed the ex-
pected coordination of the ligand through both deprotonated
hydroxyl and carbonyl oxygen atoms. The other carbon at-
oms were only slightly affected from the coordination of the
metal. Similar chemical shifts were observed for the other
ligands and their complexes (Table 5). On the basis of the
results thus obtained, it was suggested that the ligands act as
tetradentate ones in the Nd (III) complex formation.
The ligand 3,3′-benzylidene-bis(4-hydroxy-2H-1-benzo-
pyran-2-one) (H2L1) showed seven signals in the 13C-NMR
spectra resonating at d 131.91, 128.08, 126.70, 125.58,
123.92, 123.76 and 115.95 ppm for 13 methine carbons
(Table 5). In agreement with literature data, the peaks at d
131.91, 123.92, 123.76 and 115.95 ppm were related to C-7,
C-5, C-6 and C-8 (the atom numbering is in agreement with
the scheme in Table 4 carbons, respectively of the coumarin
moieties. The signals at d 128.08, 126.70 and 125.58 ppm
were assigned to C-3′ (and C-5′), C-4′ and C-2′ (and C-6′)
carbons of the phenyl ring. The chemical shifts at d 165.36,
164.87, 152.23, 139.94, 117.96 and 104.13 ppm are due to
the C-2, C-4, C-8a, C-1′, C-4a and C-3 quaternary carbons,
respectively. Due to electron transfer from the hydroxyl and
carbonyl oxygen atoms to Nd (III), a difference in chemical
shifts was observed for the neighboring C-4, C-3 and C-2
4.4. Pharmacology
The in vitro screening data for the investigated com-
pounds are presented in Tables 6–9 and Figs. 1–12. The
investigations carried out enabled the construction of dose–
response curves with consequent calculation of the IC50
values for all compounds under investigation. The data for
the cytotoxic efficacy of the tested compounds on HL-60
cells indicate that Nd(L1)(OH).H2O proved to be the most
Table 6
Spectrophotometric data from MTT assay concerning the cytotoxic activity of the investigated Nd complexes on HL-60 cells after 72 h incubation
Compound
MTT-formazan absorption at 580 nm
Untreated control
0.6047 0.0351
0.6047 0.0351
0.6047 0.0351
0.6047 0.0351
31.25 (µM)
62.5 (µM)
125 (µM)
250 (µM)
500 (µM)
Nd(L1)(OH).H2O
Nd(L2)(OH).2H2O
Nd(L3)(OH).2H2O
Nd(L4)(OH).2H2O
0.489 0.0576
0.592 0.0176
0.5893 0.0646
0.5913 0.0187
0.335 0.0137
0.511 0.0303
0.5995 0.0058
0.5325 0.0239
0.2658 0.0337
0.3228 0.0189
0.4863 0.0504
0.468 0.0422
0.233 0.0103
0.252 0.0187
0.2143 0.0172
0.3028 0.0170
0.1548 0.0076
0.2133 0.0047
0.1778 0.6387
0.22 0.0117
Table 7
Spectrophotometric data from MTT assay concerning the cytotoxic activity of the investigated Nd complexes on HL-60/Dox cells after 72 h incubation
Compound
MTT-formazan absorption at 580 nm
Untreated control
1.1204 0.0650
1.1204 0.0650
1.1204 0.0650
1.1204 0.0650
31.25 (µM)
62.5 (µM)
125 (µM)
250 (µM)
500 (µM)
Nd(L1)(OH).H2O
Nd(L2)(OH).2H2O
Nd(L3)(OH).2H2O
Nd(L4)(OH).2H2O
0.8293 0.0163
1.244 0.0360
1.2153 0.0277
1.1178 0.0641
0.4068 0.0415
1.3575 0.0699
1.2888 0.0716
0.9735 0.1152
0.2233 0.0125
0.8335 0.1546
1.229 0.2140
0.8013 0.1005
0.1755 0.0185
0.333 0.0614
0.6348 0.0311
0.8318 0.0349
0.1888 0.0199
0.2198 0.0269
0.1823 0.0222
0.3663 0.0627

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I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
Table 8
Spectrophotometric data from MTT assay concerning the cytotoxic activity of the investigated Nd complexes on SKW-3 cells after 72 h incubation
Compound
MTT-formazan absorption at 580 nm
Untreated control
0.2772 0.0114
0.2772 0.0114
0.2772 0.0114
0.2772 0.0114
31.25 (µM)
62.5 (µM)
125 (µM)
250 (µM)
500 (µM)
Nd(L1)(OH).H2O
Nd(L2)(OH).2H2O
Nd(L3)(OH).2H2O
Nd(L4)(OH).2H2O
0.2973 0.0220
0.3173 0.008
0.3045 0.0121
0.267 0.0102
0.1785 0.0214
0.2995 0.0244
0.2625 0.0443
0.244 0.0161
0.1455 0.0068
0.137 0.0244
0.1398 0.0121
0.1905 0.0094
0.1073 0.0119
0.1215 0.0019
0.1158 0.0102
0.0958 0.0213
0.082 0.0095
0.1103 0.0078
0.089 0.0116
0.0995 0.0099
Fig. 1. Cytotoxic activity of Nd(L1)(OH).H2O on HL-60 cells after 72 h
incubation as assessed by MTT assay. Each data point represents the arith-
metic mean of at least six independent experiments.
Fig. 3. Cytotoxic activity of Nd(L3)(OH).2H2O on HL-60 cells after 72 h
incubation as assessed by MTT assay. Each data point represents the arith-
metic mean of at least six independent experiments.
Fig. 2. Cytotoxic activity of Nd(L2)(OH).2H2O on HL-60 cells after 72 h
incubation as assessed by MTT assay. Each data point represents the arith-
metic mean of at least six independent experiments.
Fig. 4. Cytotoxic activity of Nd(L4)(OH).2H2O on HL-60 cells after 72 h
incubation as assessed by MTT assay. Each data point represents the arith-
metic mean of at least six independent experiments.
Nd(L1)(OH).H2O and Nd(L2)(OH).2H2O also caused the
most prominent maximal efficacy at the higher concentration
investigated (500 µM)—about 25% of the cells were viable.
The other complexes investigated caused less pronounced
proliferation inhibition with approximately 35% vital cells at
the highest concentration. The results obtained for the effects
on the resistant HL-60/Dox cells revealed that Nd(L1)
(OH).H2O and Nd(L2)(OH).2H2O were the most active com-
pounds with IC50 values 51.4 and 161.85 µM, respectively.
These data suggest that there is no cross resistance to both
complexes in HL-6/Dox and even collateral sensitivity to
Nd(L1)(OH).H2O was discovered, since its IC50 value in the
resistant sub-line is almost twice smaller than that in the
sensitive line HL-60. Interestingly we found that HL-60/Dox
was quite less sensitive to the other compounds under inves-
active compound with IC50 value 90.1 µM, whereas the other
compunds could be ranged according to the decrease in
potency as follows: Nd(L2)(OH).2H2O (IC50 = 161.64 µM)
> Nd(L3)(OH).2H2O (IC50 = 209.4 µM) > Nd(L4)
(OH).2H2O (IC50 = 255.24 µM). The most active compounds
Table 9
IC50 values of the investigated Nd complexes on HL-60, HL-60/Dox and
SKW-3 cells after 72 h incubation
Compound
IC50 value (µM)
HL-60
90.01
161.64
209.4
255.4
HL-60/Dox
SKW-3
131.59
123.1
Nd(L1)(OH).H2O
Nd(L2)(OH).2H2O
Nd(L3)(OH).2H2O
Nd(L4)(OH).2H2O
51.4
161.85
290.61
396.7
130.8
192.4

I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
773
Fig. 5
.
Cytotoxic activity of Nd(L1)(OH).H2O on HL-60/Dox cells after
Fig. 8. Cytotoxic activity of Nd(L4)(OH).2H2O on HL-60/Dox cells after
72 h incubation as assessed by MTT assay. Each data point represents the
arithmetic mean of at least six independent experiments.
72 h incubation as assessed by MTT assay. Each data point represents the
arithmetic mean of at least six independent experiments.
Fig. 9. Cytotoxic activity of Nd(L1)(OH).H2O on SKW-3 cells after 72 h
incubation as assessed by MTT assay. Each data point represents the arith-
metic mean of at least six independent experiments.
Fig. 6. Cytotoxic activity of Nd(L2)(OH).2H2O on HL-60/Dox cells after
72 h incubation as assessed by MTT assay. Each data point represents the
arithmetic mean of at least six independent experiments.
Fig. 10. Cytotoxic activity of Nd(L2)(OH).2H2O on SKW-3 cells after 72 h
incubation as assessed by MTT assay. Each data point represents the arith-
metic mean of at least six independent experiments.
Fig. 7. Cytotoxic activity of Nd(L3)(OH).2H2O on HL-60/Dox cells after
72 h incubation as assessed by MTT assay. Each data point represents the
arithmetic mean of at least six independent experiments.
whereas Nd(L4)(OH).2H2O caused less pronounced cell sur-
vival inhibition with almost 33% viable cells at the highest
concentration applied. The antineoplastic efficacy of the in-
dividual tested compounds on the lymphoid cell line SKW-3
did not differ to the extent, found for HL-60 and HL-60/Dox.
tigation Nd(L3)(OH).2H2O and Nd(L4)(OH).2H2O with in-
dices of resistance 1.4 and 1.55, respectively. The maximal
efficacy of Nd(L2)(OH).2H2O, Nd(L1)(OH).H2O and
Nd(L3)(OH).2H2O did not differ significantly with approxi-
mately 16, 17 and 20% vital cells at 500 µM, respectively,

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I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
(III) complexes confirmed the suggested coordination of the
ligands through both the hydroxyl and carbonyl oxygen at-
oms.
The overall results from the preliminary screening pro-
gram revealed that all of the novel Nd complexes reach 50%
inhibition of the malignant cells proliferation and thus
could be considered as biologically active. On the basis of the
IC50 values obtained compounds Nd(L1)(OH).H2O and
Nd(L3)(OH).2H2O were found to exert superior activity in
comparison to the remaining complexes. These findings
as well as the practical lack of cross-resistance to these
agents in HL-60/Dox give us reason to conclude that
Nd(L1)(OH).H2O and Nd(L3)(OH).2H2O should undergo
further thorough pharmacological and toxicological investi-
gations.
According to our expectations the complexes of neody-
mium (III) possess a cytotoxic activity and their in vitro
effects are clearly expressed. These results confirmed our
previous observations on the cytotoxicity of neodymium (III)
complexes.
Fig. 11. Cytotoxic activity of Nd(L3)(OH).2H2O on SKW-3 cells after 72 h
incubation as assessed by MTT assay. Each data point represents the arith-
metic mean of at least six independent experiments.
6. Experimental protocols
6.1. Chemistry
The carbon, hydrogen and nitrogen contents of the com-
pounds were determined by elemental analysis. The water
content was determined by Metrohn Herizall E55 Karl Fisher
titrator. IR spectra (Nujol) were recorded on a IR-
spectrometer FTIR-8101M Shimadzu (3800–400 cm–1) and
on a IR-spectrometer Perkin–Elmer GX Auto image system
Fig. 12. Cytotoxic activity of Nd(L4)(OH).2H2O on cells after 72 h incuba-
tion as assessed by MTT assay. Each data point represents the arithmetic
mean of at least six independent experiments.
1
(700–200 cm–1). H-NMR spectra were recorded at room
temperature on Brucker WP 250 (250 MHz) spectrometer in
DMSO-d6. Chemical shifts are given in ppm. 13C-NMR
spectra were recorded at ambient temperature on Brucker
250 WM (62.9 MHz) spectrometer in DMSO-d6. Chemical
shifts are given in ppm, downfield from TMS. Mass spectra
were recorded on a Jeol JMS D 300 double focusing mass
spectrometer coupled to a JMA 2000 data system. The com-
pounds were introduced by direct inlet probe, heated from
50 to 400 °C at a rate of 100 °C/min. The ionization current
was 300 mA, the accelerating voltage 3 kV and the chamber
temperature 150 °C.
On the basis of the IC50 values obtained Nd(L2)(OH).2H2O
was found to be the most active compound (IC50 123.1 µM),
whereas the IC50 values for Nd(L1)(OH).H2O and
Nd(L3)(OH).2H2O were approximately 131 µM and for
Nd(L4)(OH).2H2O IC50 value of 192.4 µM was calculated.
As the concentration–response curves indicate, however,
there were no large differences in the maximal efficacy
of Nd(L3)(OH).2H2O, Nd(L4)(OH).2H2O and Nd(L2)
(OH).2H2O with 32.1, 35.9 and 39.8% vital cells, respec-
tively, whereas Nd(L1)(OH).H2O caused more than 70%
inhibition of malignant cell proliferation at the highest con-
centration investigated.
6.2. General method of synthesis
Cytotoxicity determination by MTT assay shows that the
inorganic salt neodymium nitrate did not show any signifi-
cant activity [13–18] but the complexes with Nd (III) were
found to be cytotoxic.
The complexes of neodymium (III) with 3,3′-benzyli-
dene-bis(4-hydroxy-2H-1-benzopyran-2-one) (H2L1); bis-
(4-hydroxy-2-oxo-2H-chromen-3-yl)-piridin-2-yl-methane
(H2L2); bis(4-hydroxy-2-oxo-2H-chromen-3-yl)-piridin-4-
yl-methane (H2L3); bis(4-hydroxy-2-oxo-2H-chromen-3-
yl)-(1H-pyrazol-3-yl)-methane (H2L4) were synthesized by
reaction of neodymium (III) salt and the ligand, in amounts
equal to metal:ligand molar ratio of 1:2. The complexes were
prepared by adding an aqueous solution of neodymium (III)
5. Conclusions
The coordination ability of the ligands has been proved in
1
complexation reaction with neodymium (III) ion. H-, 13C
NMR- and IR-spectral analysis of the ligands and their Nd

I. Kostova et al. / European Journal of Medicinal Chemistry 39 (2004) 765–775
775
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the pH of the mixture gradually to ca. 5.0 by adding dilute
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