close

Вход

Забыли?

вход по аккаунту

?

New Lanthanum III Complex Synthesis Characterization and Cytotoxic Activity.

код для вставкиСкачать
598
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
Full Paper
New Lanthanum (III) Complex – Synthesis, Characterization,
and Cytotoxic Activity
Irena Kostova1, Vinod K. Rastogi2, Wolfgang Kiefer3, and Aleksandar Kostovski4
1
Department of Chemistry, Faculty of Pharmacy, Medical University, Sofia, Bulgaria
Department of Physics, CCS University, Meerut, India
3
Institut fr Physikalische Chemie, Universitt Wrzburg, Wrzburg, Germany
4
Laboratory of Experimental Chemotherapy, Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Medical University, Sofia, Bulgaria
2
The complex of lanthanum (III) was synthesized reacting the respective inorganic salt with 5aminoorotic acid in amounts equal to the metal : ligand molar ratio of 1 : 3. The complex was
prepared by adding an aqueous solution of lanthanum (III) nitrate to an aqueous solution of the
ligand, subsequently raising the pH of the mixture gradually to approx. 5.0 through addition of
a dilute solution of sodium hydroxide. The structure of the final complex was determined by
means of spectral data (IR, Raman, 1H-NMR) and elemental analysis. Significant differences in
the IR spectrum of the complex were observed as compared to the spectrum of the ligand. A
comparative analysis of the Raman spectrum of the complex with that of the free 5-aminoorotic
acid allowed a straightforward assignment of the vibrations of the ligand groups involved in
coordination. The ligand and the complex were tested for the cytotoxic activities on the chronic
myeloid leukemia derived K-562, overexpressing the BCR-ABL fusion protein and the non-Hodgkin lymphoma derived DOHH-2, characterized by a re-expression of the anti-apoptotic protein
bcl-2 cell lines. The results obtained indicate that the tested compounds exerted a considerable
cytotoxic activity upon the evaluated cell lines in a concentration-dependent matter, which enabled the construction of dose-response curves and the calculation of the corresponding IC50
values. The inorganic salt exerted a very weak cytotoxic effect on these cells, which is in contrast
to the lanthanum (III) complex.
Keywords: 5-Aminoorotic acid / Cytotoxicity / Lanthanum (III) complex / NMR / Raman /
Received: April 28, 2006; accepted: September 11, 2006
DOI 10.1002/ardp.200600077
Introduction
The coordination chemistry of orotic acid (2,6-dioxo1,2,3,6-tetrahydropyrimidine-4-carboxylic acid, vitamin
B13, H3L9) has been an area of great research activity, ranging from bioinorganic to pharmaceutical and material
chemistry. Orotic acid (vitamin B13, Fig. 1) and its salts
Correspondence: Dr. Irena Kostova, Department of Chemistry, Faculty
of Pharmacy, Medical University, 2 Dunav St., Sofia 1000, Bulgaria.
E-mail: irenakostova@yahoo.com
Fax: +359 2 987 9874
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
play an important role in biological systems as precursors of pyrimidine nucleosides [1] and are found in cells
and body fluids of many living organisms [2 – 4]. These
compounds are applied in medicine as biostimulators of
ionic exchange processes in organisms, and different
metal complexes of orotic acid were studied [5 – 9]. There
is also a great interest in orotic acid in relation to food
protection and nourishment research [10 – 12]. The crystal and molecular structure of orotic acid was determined by Takusawaga and Shimada [13]. Mutikainen [14]
studied the crystal structure of metal complexes of orotic
acid. Because of the importance of orotic acid and its
metal complexes in living systems, a reliable assignment
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
New Lanthanum (III) Complex with Cytotoxic Activity
599
Figure 1. The structure of orotic acid (H3L9, HOA).
of their vibrational spectra is a useful basis in the study
of their interactions with other chemical species present
in the biological milieu. The orotic acid molecule is
related to the molecules of uracil or thymine. Different
studies on these type of molecules were done by vibrational spectroscopy [15, 16] as well as normal coordinate
analysis (NCA) and ab initio calculations [16 – 27]. The
results of these studies may help in the assignment of the
spectra of orotic acid.
Metal ion complexes of orotic acid (Fig. 1) and its substituted derivatives continue to attract attention because
of its multidentate functionality and its pivotal role in
bioinorganic chemistry. It is an interesting multidentate
ligand capable of coordinating to metal ions through the
nitrogen atoms, the two carbonyl oxygens, and the carboxylate oxygens. Existing studies of its coordination
complexes demonstrate that it occurs as a di-anion coordinating often via the N1 atom and the carboxylic acid
group, so forming a five-membered chelate ring. Despite
its polydentate nature, only a few polymeric complexes
of orotic acid have been observed.
The multifunctionality of the hydroorotate, H2L9–, and
orotate, HL92– anions offer interesting possibilities in crystal engineering as a versatile ligand for supramolecular
assemblies. Metal-ion coordination may occur through
the two N atoms of the pyrimidine ring as well as the two
carbonyl oxygen atoms or the carboxylic group, which
results in a multi-faceted coordination chemistry. The
coordinated orotate anions exhibit a ligand surface with
double or triple hydrogen-bonding capabilities, depending on the metal coordination mode, and has, thus, a
potential to adopt several modes of interligand hydrogen
bonding to allow the formation of extended, selfassembled structures.
Thus, besides the biological relevance, the orotic acid
and its anions H2L9–, HL92–, and L93– are interesting multidentate ligands as they can coordinate through the two
pyrimidine nitrogen atoms, the two carbonyl oxygens,
and the oxygens from the carboxyl group. The equilibrium composition of the reactant mixture and thus the
solution pH are critical factors which determine the
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. The structure of the ligand 5-aminoorotic acid (H4L,
HAOA).
mode of coordination. Between pH 3 and 9, orotic acid
exists mainly as readily-coordinating monodeprotonated
H2L9– (the carboxylic group has pKa 2.07). The literature
lists many reports on the coordinating preferences of the
orotate moiety in metal complexes. It was found that in
solutions with neutral or slightly acidic pH, Cu(II), Zn(II),
Co(II), Mn(II), Fe(III), Cr(III), VO(II), Cd(II), Hg(II), and Ag(I)
are coordinated through the carboxylate group, while
Ni(II), Co(II), and Cu(II) are coordinated through the carboxylate end and the adjacent N1 [28 – 31]. Bidentate
binding through N1 and the carboxylate group was
observed by several crystal structure determinations
[32 – 36]. In the complexes [Co(HL9)(OH)(H2O)(NH3)]n and
[Ni(HL9)(OH)(H2O)2(NH3)]n the orotate anion bridges the
metal ions through the carboxylate and the N1 and O2
atoms, forming one-dimensional polymeric chains [37]. A
recent reinvestigation of nickel (II) orotate pentahydrate
[Ni(HL9)(H2O)4] N H2O by modern diffraction, spectroscopic
and theoretical methods revealed novel structural features [38].
Despite the interest in orotate metal complexes, the
coordination chemistry of the derivatives of orotic acid
has received rather scant attention. One of these derivatives is 5-aminoorotic acid (5-amino-2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid, H4L) which has a
relatively unknown coordination chemistry [6].
We have recently synthesized lanthanide complexes
with a number of biologically active ligands, and we
reported their significant cytotoxic acitivity in different
human cell lines [39 – 48]. These promising results
prompted us to search for new lanthanide complexes
with 5-aminoorotic acid (H4L, see Fig. 2). Thus, the aim of
this work was to synthesize and characterize a new
lanthanum (III) complex of 5-aminoorotic acid and to
determine the cytotoxic activity of the obtained complex
in the selected tumor cell lines. In the present study, the
following cell lines were exploited as in vitro tumor test
systems: the chronic myeloid leukemia derived K-562,
www.archpharm.com
600
I. Kostova et al.
overexpressing the BCR-ABL fusion protein and the nonHodgkin lymphoma derived DOHH-2, characterized by a
rexpression of the antiapoptotic protein bcl-2 cell lines.
Results and discussion
Chemistry
The new complex was characterized by elemental analysis. The metal ions were determined after mineralization. The water content in the complex was determined
by Karl-Fisher analysis. The nature of the complex was
confirmed by IR, Raman, and 1H-NMR-spectroscopy. The
data of the elemental analysis of the new lanthanum (III)
complex obtained serving as a basis for the determination of its empirical formula and the results of the KarlFisher analysis are presented below. Elemental analysis
of La(III) complex of 5-aminoorotic acid: (% calculated/
found): La(AOA)363 H2O: C: 25,60/25,14; H: 2,56/2,24; N:
17,92/17,48; H2O: 7,68/7,25; La: 19,77/20,06, where HAOA
= C5N3O4H5 and AOA= C5N3O4H4– .
IR spectra of the ligand and its lanthanum complex
The mode of bonding of the ligand to La(III) ions was elucidated by recording the IR spectrum of the complex as
compared with that of the free ligand. The monoanions
of orotic acid and its derivatives show a preference for
monodentate Ocarboxylate coordination as it was described
in the literature data [49 – 51].
Tentative assignments of selected IR bands of the complex are given in Table 1. The assignments have been
given by studying literature reports [50] and comparing
the spectra of the ligand and the metal complex. As a general remark, we must emphasize that some stretching
and deformation modes are coupled so that the proposed
assignments should be regarded as approximate descriptions of the vibrations.
In the m(OH)water region the spectrum of La(III) complex
shows one medium band at 3448 cm–1, attributed to the
presence of coordinated water [52]. This band overlaps
with the masym(NH2) band [51].
In the spectrum of La(III) complex the bands due to the
masym(NH2), msym(NH2), m(C2O), and m(C4O) vibrations appear
at 3448, 3339, 1718, and 1684 cm–1 [50]. The absence of
large systematic shifts of these bands in the spectrum of
the complex implies that there is no interaction between
the amino nitrogen or the carbonyl oxygens and the
lanthanum (III) ions. It was not possible to differentiate
clearly the spectroscopic behavior of the different carbonyl groups of the complex [51].
The masym(CO2) and msym(CO2) bands are at 1637 and
1424 cm–1 for the complex and thus the participation of
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
Table 1. Assignments of the more relevant absorption bands of
5-aminoorotic acid and its La(III) complex in the 3800 –
1300 cm–1 IR-region.
m (cm – 1)
HAOA
La(AOA)363 H2O
mOH
masymNH2
msymNH2
mNH
–
3457
3322
3196
mC2=0
mC4=0
masymCOO –
mC=C
1689
1667
–
1566
1604
1436
1457
–
1405
3448
3448
3339
3263
3171
1718
1684
1637
1556
–
1499
mC-N
msymCOO –
dNH
1424
1390
Figure 3. IR spectra of 5-aminoorotic acid and its lanthanum
(III) complex between 3500 – 400 cm–1.
the carboxylate group of H3L– in coordination was suggested.
The assignments of the more relevant absorption
bands of 5-aminoorotic acid and its complex in the
3500 – 1300 cm–1 IR-region are presented in Table 1 and
also on Fig. 3 and they are in agreement with the literature data for such kind of complexes. The IR spectrum of
the presented new lanthanum complex is similar to that
of other complexes of this type. Notwithstanding in these
studies it was not possible to differentiate clearly the
spectroscopic behavior of the different functional groups
present in these systems. Therefore, we have attempted
to advance in that direction using Raman spectroscopy.
www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
New Lanthanum (III) Complex with Cytotoxic Activity
601
Table 2. Experimental infrared and Raman bands (cm – 1) for 5aminoorotic acid and its La(III) complex in the 2000 – 200 cm – 1
region.
Band assignment (cm – 1)
d(HOH), m(C2=O2), m(C7=O3)
m(C7=O3), m(C4=O4)
m(C4=O4), m(C5=C6)
masymCOO –
m(C5=C6), m(C4=O4)
m(N1-C2), m(N1-C6), m(N3-C2)
m(N3-C2), m(N1-C6), msymCOOd(N3H3), m(C2=O2)
m(C7=O1), q(HOH)
m(Ura ring), d(N1H1), d(C=Oca)
Figure 4. Raman spectra of 5-aminoorotic acid and its lanthanum (III) complex between 1800 – 200 cm–1.
Raman spectra of the ligand and its lanthanum complex
The Raman spectra of the free 5-aminoorotic acid and of
the lanthanum complex, in the pertinent range, are
shown in Fig. 4. In the well-defined high-frequency field
present in the spectra, dramatic intensity changes are
observed in going from the acid to the complex (Table 2).
Spectral region 3500 – 2000 cm – 1
In this region, the O – H, N – H, and C – H stretches give
rise to intense IR bands (Fig. 3, Table 1). The assignment
of the O – H and N – H stretching bands is rather difficult.
These bands appear overlapped in the same spectral
region, and the involvement of these groups in hydrogen
bonds affects their wavenumbers and produces a relevant band broadening in the IR and Raman spectra.
Spectral region 1800 – 900 cm – 1
The bands that appear in this region are mainly due to inplane vibrations. Some mixed modes with ring and carboxylic contributions are observed in this region
(Table 2); a significant vibrational coupling between the
ring and carboxylic moieties is observed. The double
bond stretching vibrations m(C=O) and m(C=C) are the
internal coordinates that dominate in the modes with
fundamentals in the 1800 – 1600 cm–1 spectral range.
The infrared spectrum of the title complex shows a
broad and relatively strong band with a maximum at
1718 cm–1. The considerable width of this band is due to a
superposition of m(C=O) and m(C=C) stretching modes and
bending modes of water molecules, of very similar frequencies. Fortunately, in the Raman spectrum, bands
arising from d(H2O) vibrations are very weak, hence, the
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
m(Ura ring)
q(HOH), c(C2O2)
d(O3C7O1)
c(C7O3)
d(Ura ring), d(C2O2)
c(C7O3)
d(C4O4), d(Ura ring)
m(Ln-O1)
HAOA
La(AOA)363 H2O
IR
R
IR
R
1689s
1667w
1604s
–
1566w
1511w
1457m
1436m
1405m
1311m
1255m
1235m
923w
871w
826m
794w
–
499m
445m
–
1699m
1670w
1609vs
–
1560m
1492w
1440w
1420w
1341m
1300w
1247m
1718m –
1684s 1673m
–
1623vs
1637s
–
1556m 1542m
1499m 1494w
1424s,br 1420s
920w
–
780w
–
584w
483m
430w
–
941w
884br,w
825m
798m
594w
499m
441m
No data
1390m 1384s
1306m 1295s
1237w 1230s
933w
–
763w
–
581w
480m
425m
375w
m(C=O) and m(C=C) stretching vibrations can be clearly
observed. The Raman band of the highest frequency,
1673 cm–1, should be assigned to the m(C7=O3) stretching
vibrations in the carboxylate group, whereas the next
two strong bands at 1623 and 1542 cm–1 are mainly
caused by the m(C4=O4) and m(C5=C6) stretching vibrations.
The medium intensity infrared band at 1499 cm–1 and
Raman band at 1494 cm–1 arise mainly from carbonnitrogen stretching vibrations in the uracilate ring.
These frequencies are higher than the stretching vibrations of single C – N bonds [53]. This fact indicates that
upon deprotonation of the carboxylate group and coordination to La(III), p-electron density increases within the
uracilate ring. The strong band at 1424 cm–1 (IR) can be
also assigned to the C – N bonds and this vibration corresponds to the so-called msym(COO) vibration in the metalcarboxylate complex [53]. The medium band at 1390 cm–1
in the IR spectrum of the complex is assigned to the
N3H3 in-plane bending vibration coupled with the
C2=O2 stretching vibrations, which is in agreement with
the literature data [38]. The C7-O1 stretching vibrations
in the title complex are assigned at 1306 (IR) and 1295
(Raman) cm–1. These stretching vibrations are coupled
with water deformations [38].
Different stretches of the uracil ring contribute to the
bands in the 1600 – 900 cm–1 region. The assignment of
all observed IR and Raman bands in this spectral range is
www.archpharm.com
602
I. Kostova et al.
difficult, because of the presence of highly coupled
modes and combination bands that may overlap with
those due to fundamentals, and they interact with one
another, leading to distortions of the observed bands.
The medium bands at 1255 cm–1 in the IR spectrum
and at 1247 cm–1 in the Raman spectrum of the ligand
are assigned to the m(Ura ring), d(N1H1), and d(C=Oca) and
they are shifted in the spectrum of the complex to lower
wavenumbers. The strong Raman band at 1230 cm–1 in
the Raman spectrum of the complex can be assigned to
the stretching vibration of the uracilate ring. The weak
bands at 1150 cm–1 in the IR spectrum and at 1130 cm–1
in the Raman spectrum of the ligand are assigned to the
m(N3-C4), m(N3-C2), m(N1-C2), and mrg and they are also
shifted in the spectrum of the complex to lower wavenumbers (Figs. 3 and 4). The same shifts were observed
for the bands at 1083 cm–1 in the IR spectrum and at
1045 cm–1 in the Raman spectrum of the ligand which
are assigned to the dOH, mCrg-Cca, and mC-Oca (Figs. 3 and 4).
Spectral region below 900 cm – 1
In this spectral region, the normal modes appear to be
rather delocalized. Nevertheless, the IR bands of the
ligand at 923, 755, 719, and 445 cm–1 are mainly due to
vibrations of the uracil ring whereas the IR bands at 826,
794, and 499 cm–1 are mostly due to vibrations of the carboxylic group. The Raman band of the complex at
763 cm–1 is assigned to the in-plane d(C7OO) vibration of
the carboxylate group [38]. The corresponding infrared
band is observed at 825 cm–1. The out-of-plane vibration
of the carboxylate C7=O3 groups are observed as the medium intensity infrared band at 798 cm–1. They are not
observed in the Raman spectra. The bands of the C2=O2,
C4=O4, C-Oca, and Crg-Cca bonds contribute to the bands
observed in the range of 790 – 750 cm–1 (Figs. 3 and 4).
The spectra in the frequency region below 600 cm–1 are
particularly interesting, since they provide information
about the metal-ligand vibrations. The bands centered at
about 594 cm–1 arise from the uracilate ring deformation
coupled with d(C2O2) vibration [38]. The infrared band at
441 cm–1 is caused by the in-plane d(C4O4) vibration [38]
coupled with ring deformation, as shown in Table 2.
In the Raman spectrum of the complex, the new band
at 375 cm–1 can be assigned to the in-phase (symmetric)
stretching vibration of the La-O bond, which is in accordance with the literature data for similar coordination
compounds [38]. The Raman spectra are particularly useful in studying the metal-oxygen stretching vibrations,
since these vibrations give rise to medium intensity
bands in Raman, but are weak in the infrared spectra.
The detailed assignment of the remaining bands in the
vibrational spectra is shown in Table 2.
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
Table 3. 1H-NMR spectra of 5-aminoorotic acid and its La(III)
complex (250 MHz, DMSO-d6).
Nn – H
HAOA
La(AOA)363 H2O
N1 – H
N3 – H
C5NH2-2H
11.47
9.44
6.00
11.22
9.03
5.57
1
H-NMR spectra of the ligand and its lanthanum complex
Metal ion coordination with ligand by means of carboxyl
oxygens was shown owing to data of 1H-NMR spectra. 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 3.
The 1H-NMR spectrum of the ligand in DMSO-d6 shows
the expected three resonances due to the NH protons.
The carboxamido and imido protons give singlets at dH
11.47 and 9.44 and the amino protons give a broad signal
at dH 6.00 [51]. In the spectrum of the complex in the
same solvent, the amido and imido protons are observed.
Its spectrum shows a very broad peak at about dH 3.4,
which is due to the intermolecular exchange of protons
between the amino group and water (contained in the
solvent). The fact that we did not observe two sharp signals of the separated species or an averaged signal are
attributed to the intermediate rate of the exchange [54,
55].
The presence of the N(1)H and N(3)H proton resonances
in the spectrum of the complex (Table 3) clearly shows
that those nitrogen atoms are not engaged in complex
formation. So far, we can conclude that La(III) ions appear
to bind the 5-aminoorotic acid at the carboxylate group
as reported for most orotate complexes [54 – 57].
We should mention that, as it has been reported earlier
in the literature for similar complexes [51], the 1H-NMR
spectrum of the new La(III) complex in DMSO-d6 showed
the presence of one carboxamido, one imido, and two
amino protons. Moreover, the signals of the complex in
this solvent are very similar in the regions of the resonances due to NH and NH2 protons. Recently published
literature data are in accordance with our suggestions
[51, 54 – 57].
The metal-ligand binding mode of 5-aminoorotic acid
was recently explained by us through modeling of the
La(III)-5-aminoorotic acid structure [58]. It was suggested
that 5-aminoorotic acid binds to the La(III) ion through
both oxygen atoms of the carboxylic group from all three
ligands, and the central ion La(III) is six-coordinated. The
density-function calculations revealed that the mode of
binding was bidentate through the carboxylic oxygen
www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
New Lanthanum (III) Complex with Cytotoxic Activity
603
Table 4. Spectrophotometrical data from MTT assay concerning the cytotoxic activity of 5-aminoorotic acid, La(III) complex of 5-aminoorotic acid, and cisplatin on K-562 cells.
MTT-formazan absorption at 580 nm
Compound
Untreat. control 12.5 (lM)
25 (lM)
50 (lM)
100 (lM)
200 (lM)
HAOA
La(AOA)363 H2O
Cisplatin
0.616 l 0.080
0.475 l 0.039
0.814 l 0.045
0.291 l 0.013
0.422 l 0.009
0.572 l 0.050
0.286 l 0.009
0.401 l 0.020
0.230 l 0.021
0.293 l 0.055
0.370 l 0.019
0.183 l 0.025
0.272 l 0.065
0.203 l 0.017
0.090 l 0.030
0.290 l 0.024
0.460 l 0.029
0.726 l 0.026
Figure 6. Cytotoxic effects of HAOA on the chronic myeloid leukemia-derived K-562 cell line after 48 h exposure, as assessed
by the MTT-dye reduction assay. Each data point represents the
mean l sd (n F 6).
Figure 5. Structural formula of the lanthanum (III) complex of 5aminoorotic acid.
atoms. On the bases of our experimental spectral data
and our theoretical investigations [58] we are able to suggest the most probable structural formula of the complex
as presented in Fig. 5.
Pharmacology
The screening performed revealed that the ligand and its
La(III) complex exerted cytotoxic effects against the
chronic myeloid leukemia derived K-562, overexpressing
the BCR-ABL fusion protein and the non-Hodgkin lymphoma derived DOHH-2, characterized by a re-expression
of the anti-apoptotic protein bcl-2 cell lines in a concentration dependent matter, which enabled the construction of concentration response curves as depicted on
Figs. 6 – 11 and Tables 4 – 7. The corresponding La(III)
nitrate salt was found to be inactive in the investigated
concentration range (data not shown) [39 – 47].
An even more intriguing discrepancy between the
cytotoxic efficacy of the lanthanum (III) complex of 5aminoorotic acid and cisplatin was encountered in
DOHH-2 cells. Notwithstanding the practically equivalent relative potencies (in terms of IC50), the novel com-
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 7. Cytotoxic effects of HAOA on the non-Hodgkin lymphoma-derived DOHH-2 cell line after 48 h exposure, as
assessed by the MTT-dye reduction assay. Each data point
represents the mean l sd (n F 6).
plex compound caused almost total eradication of
DOHH-2 within the higher concentrations exploited,
whilst the efficacy of the referent drug cisplatin reached
a plateau with more than 30% viable cells even at the
highest concentration exploited (200 lM). This encounter could be generally ascribed to the well-established
high level of bcl-2 expression in DOHH-2 cells [59]. This
anti-apoptotic protein abolishes several of the cell-death
www.archpharm.com
604
I. Kostova et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
Figure 8. Cytotoxic effects of La(AOA)363 H2O on the chronic
myeloid leukemia-derived K-562 cell line after 48 h exposure, as
assessed by the MTT-dye reduction assay. Each data point
represents the mean l sd (n F 6).
Figure 10. Cytotoxic effects of cisplatin on the chronic myeloid
leukemia-derived K-562 cell line after 48 h exposure, as
assessed by the MTT-dye reduction assay. Each data point
represents the mean l sd (n F 6).
Figure 9. Cytotoxic effects of La(AOA)363 H2O on the nonHodgkin lymphoma-derived DOHH-2 cell line after 48 h exposure, as assessed by the MTT-dye reduction assay. Each data
point represents the mean l sd (n F 6).
Figure 11. Cytotoxic effects of cisplatin on the non-Hodgkin
lymphoma-derived DOHH-2 cell line after 48 h exposure, as
assessed by the MTT-dye reduction assay. Each data point
represents the mean l sd (n F 6).
Table 5. Relative potency of the investigated compounds in the
panel of human tumor cell line K-562, following 48 h treatment
cells.
Compound
IC50 value (mM)
HAOA
La(AOA)3 N 3 H2O
Cisplatin
11.81
182.18
36.68
signaling pathways, which are involved in the cytotoxic
mode of action of cisplatin, and conversely higher levels
of bcl-2 are well established to confer resistance to platinum-based drugs [60 – 62]. Thus, the established superior
inhibition of DOHH-2 proliferation by the novel compound implies that this class of tumor-inhibiting metal
coordination compounds bypasses the bcl-2 mechanisms
of cellular resistance.
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
As the results obtained clearly indicate the lanthanum (III) complex of 5-aminoorotic acid proved to be a
very active cytotoxic agent against the non-Hodgkin lymphoma-derived DOHH-2, characterized by a re-expression
of the anti-apoptotic protein bcl-2 cells. Taken together
the results from the cytotoxicity screening give us reason
to conclude that the La(III) complexes with 5-aminoorotic
acid, necessitates further more detailed pharmacological
evaluation.
Conclusion
The complex of lanthanum (III) with 5-aminoorotic acid
has been synthesized by a new method. It has been
proved that the La(III) ions are coordinated with 5-aminoorotic acid via the oxygen atoms from the carboxyl
group. In conclusion, the complex described above
demonstrates once more the versatility of the 5-aminoorwww.archpharm.com
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
New Lanthanum (III) Complex with Cytotoxic Activity
605
Table 6. Spectrophotometrical data from MTT assay concerning the cytotoxic activity of 5-aminoorotic acid, La(III) complex of 5-aminoorotic acid, and cisplatin on DOHH-2 cells.
Compound
Untreat. control 12.5 (lM)
25 (lM)
50 (lM)
100 (lM)
200 (lM)
HAOA
La(AOA)363 H2O
Cisplatin
0.903 l 0.057
0.251 l 0.022
1.264 l 0.073
0.508 l 0.027
0.125 l 0.006
0.450 l 0.051
0.405 l 0.009
0.122 l 0.013
0.446 l 0.040
0.348 l 0.057
0.108 l 0.011
0.462 l 0.044
0.303 l 0.006
0.004 l 0.006
0.397 l 0.063
0.61 l 0.064
0.149 l 0.008
0.488 l 0.048
Table 7. Relative potency of the investigated compounds in the
panel of human tumor cell line DOHH-2, following 48 h treatment
cells.
Compound
IC50 value (mM)
HAOA
La(AOA)363 H2O
Cisplatin
38.67
24.79
10.25
otate ligand, which adopts different coordination modes.
The different charge and coordination mode of the
ligand have a major effect on the supramolecular structures adopted by the complex. From previous results and
this work it is clear that the nature of 5-aminoorotic acid
makes its various anionic forms versatile ligands for use
with a variety of metals and for a variety of objectives/
advantages, including variable coordination modes,
high-nuclearity aggregate formation and/or linking of
aggregates into polymeric arrays. Thus, we believe that
H4L has a great potential as a generally useful new polyfunctional ligand in metal chemistry and it will prove
attractive to a variety of coordination chemists.
In our hands, the new La(III) complex under investigation exhibited in vitro cytotoxic effects in micromolar
concentrations against the chronic myeloid leukemia
derived K-562, overexpressing the BCR-ABL fusion protein
and the non-Hodgkin lymphoma derived DOHH-2, characterized by a re-expression of the anti-apoptotic protein
bcl-2 cell lines. According to our expectations, the La(III)
complex, as other by us investigated lanthanide (III) complexes, possesses a cytotoxic activity and its in vitro effects
are clearly expressed. These results confirmed our previous observations on the cytotoxicity of cerium (III),
lanthanum (III), and neodymium (III) complexes with
other biologically active ligands.
Experimental
Synthesis of the complex
The compounds used for preparing the solutions were Merck
products (Merck, Darmstadt, Germany), p.a. grade: La(NO3)36
6 H2O N 5-Aminoorotic acid (H4L, Fig. 2) was used for the prepara-
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tion of the metal complex as a ligand. The complex was synthesized by reaction of lanthanum (III) salt and the ligand, in
amounts equal to the metal : ligand molar ratio of 1 : 3. The
synthesis of the complex was made in different ratios (1 : 1, 1 : 2,
1 : 3) but in all the cases, the product composition was 1 : 3. The
complex was prepared by adding an aqueous solution of lanthanum (III) salt to an aqueous solution of the ligand, subsequently
raising the pH of the mixture gradually to approx. 5.0 by addition of a dilute solution of sodium hydroxide. The reaction mixture was stirred with an electromagnetic stirrer at 258C for one
hour. At the moment of mixing of the solutions, a precipitate
was obtained. The precipitate was filtered (pH of the filtrate was
5.0), washed several times with water, and dried in a desiccator
to constant weight. The complex was insoluble in water, methanol, and ethanol and well soluble in DMSO.
Analytical and spectroscopic methods
The carbon, hydrogen, and nitrogen contents of the compound
were determined by elemental analysis. The water content was
determined by Metrohn Herizall E55 Karl Fisher Titrator
(Metrohm, Herisau, Switzerland). The solid-state infrared spectra
of the ligand and its La(III) complex were recorded in KBr in the
4000 – 400 cm–1 frequency range by FT-IR 113V Bruker spectrometer (Bruker, Germany) and in Nujol by IR-spectrometer FTIR8101M Shimadzu (Shimadzu, Tokyo, Japan).
The Raman spectra of 5-aminoorotic acid and its lanthanum (III) complex were recorded with a Dilor Labram spectrometer (Horiba-Jobin-Yvone, Inc) using the 514.5 nm excitation
line from a Spectra Physics argon ion laser (Spectra Physics,
Mountain View, CA, USA). The Labram integrated system is
coupled trough an Olympus LMPlanFL 1006objective (Olympus)
to the optical microscope. The spectra were collected in the backscattering geometry with a resolution of 2 cm–1. The detection of
a Raman signal was carried out with a CCD camera (Photometric,
model 9000, Dongwoo Optron, Co., Ltd.). The laser power varied
from 100 to 250 mW and is indicated for each figure caption.
1
H-NMR spectra were recorded at room temperature on
Brucker 250 WM (250 MHz) spectrometer in DMSO- d6. Chemical
shifts are given in ppm, downfield from TMS.
Pharmacology
All of the procedures concerning cell culture maintenance, solution preparation and treatment were carried out in a laminar
flow cabinet (Heraeus). The stock solutions of the tested compounds (at 20 mM) were freshly prepared in DMSO, and thereafter consequently diluted in RPMI-1640 medium, in order to
achieve the desired final concentrations. At the final dilutions
obtained, the concentration of DMSO never exceeded 1%. For
the cell viability assessment MTT formazan absorption was
measured using Uniskan-Titertek ELISA reader at 580 nm (Flow
Laboratories, France).
www.archpharm.com
606
I. Kostova et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
Data processing was performed using Microsoft Excel and the
plots were generated using Microcal Origin, version 3.5.
Human tumor cell lines and culture conditions
The cytotoxic effects of the tested compounds were assessed on
the chronic myeloid leukemia derived K-562, overexpressing the
BCR-ABL fusion protein and the non-Hodgkin lymphoma derived
DOHH-2, characterized by a re-expression of the anti-apoptotic
protein bcl-2 cell lines. They were all grown as suspension-type
cultures in a controlled environment: RPMI 1640 medium
(Sigma), with 10% heat inactivated fetal bovine serum (Sigma)
and 2mM L-glutamine (Sigma), in a Heraeus incubator with
humidified atmosphere and 5% carbon dioxide, at 378C. In order
to maintain the cells in log phase, cell suspension was discarded
2 or 3 times per week and the cell culture was re-fed with fresh
RPMI-1640 aliquots.
,
Cytotoxicity determination
The cell viability was determined using the MTT-dye reduction
assay. Briefly, exponentially growing cells were seeded in 96well microplates (100 mL/well) at a density of 16105 cells per ml
and after 24 h incubation at 378C they were exposed to various
concentrations of the compounds for 48 h. After the incubation
with the tested compounds, MTT solution (10 mg/mL in PBS) was
added (10 mL/well). The plates were further incubated for 4 h at
378C and the formazan crystals formed were dissolved through
addition of 100 mL/well 5% solution of formic acid in 2-propanol
(Merck). The absorption of the samples was then measured using
an ELISA reader (Uniscan Titertec) at wavelength of 580 nm. The
blank solution consisted of 100 mL RPMI 1640 medium (Sigma),
10 mL MTT stock and 100 mL 5% formic acid in 2-propanol. The
survival fractions were calculated as percentage of the untreated
control using the formula:
SF% = Atest/Acontrol6100, where Atest is the average value for the
absorption at a given concentration and Acontrol is the average
absorption of the untreated control, respectively.
References
[1] P. L. Panzeter, D. P. Ringer, Biochem. J. 1993, 293, 775 –
779.
[2] M. Berthelot, G. Cornu, M. Daudon, M. Helbert, C. Laurence, Clin. Chem. 1987, 33, 2070 – 2073.
[3] P. J. Banditt, J. Chromatogr. B 1994, 660, 176 – 179.
[4] M. T. MacCann, M. M. Thompson, I. C. Gueron, M. Tuchman, Clin. Chem. 1995, 41, 739 – 743.
[5] A. Sarpotdar, G. J. Burr, J. Inorg. Nucl. Chem. 1979, 41,
549 – 553.
[6] D. Lalart, G. Dodin, J. E. Dubois, J. Chim. Phys. 1982, 79,
449 – 453.
[7] I. Bach, O. Kumberger, H. Schmidbaur, Chem. Ber. 1990,
123, 2267 – 2271.
[8] O. Kumberger, J. Riede, H. Schmidbaur, Chem. Ber. 1991,
124, 2739 – 2742.
[9] E. J. Baran, R. C. Mercader, F. Hueso-Urena, M. MorenoCarretero, et al., Polyhedron 1996, 15, 1717 – 1721.
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[10] E. Fernandez Garcia, J. U. McGregor, J. Dairy Sci. 1994, 77,
2934 – 2939.
[11] A. S. Akalin, S. Gone, Sci. Int. 1996, 51, 554 – 556.
[12] P. Ruasmadiedo, J. C. Badagancedo, E. Fernandez Garcia,
D. G. Dellano, C. G. de los Reyes Gavilan, J. Food Protect
1996, 59, 502 – 508.
[13] F. Takusawaga, A. Shimada, Bull. Chem. Soc. Jpn. 1973, 46,
2011 – 2019.
[14] I. Mutikainen, Ann Acad. Sci. Fenn 1988, 217, 1 – 39.
[15] R. Letellier, M. Ghomi, E. Taillandier, Eur. Biophys. J. 1987,
14, 243 – 252.
[16] W. B. Person, K. Szczepaniak in Vibrational Spectra and
Structure (Ed.: J. R. Durig), Elsevier, Amsterdam, 1993, pp.
239 – 325.
[17] H. Rostkowska, K. Szczepaniak, M. J. Nowak, J. Leszczynski, et al., J. Am. Chem. Soc. 1990, 112, 2147 – 2160.
[18] I. R. Gould, I. H. Hillier, J. Chem. Soc. Perkin Trans 1990, 2,
329 – 330.
[19] J. J. Leszczynski, Phys. Chem. 1992, 96, 1649 – 1653.
[20] G. J. Thomas Jr., M. Tsuboi in Advances in Biophysical Chemistry, (Ed.: C. A. Bush), JAI Press, Greenwich, CT, USA,
1993, pp. 1 – 70.
[21] J. Florian, V. Hrouda, Spectrochim. Acta 1993, 49A, 921 –
938.
[22] P. Lagant, G. Vergoten, R. Efremov, W. L. Peticolas, Spectrochim. Acta. 1994, 50A, 961 – 971.
[23] W. L. Peticolas, T. Rush, J. Comput. Chem. 1995, 16, 1261 –
1270.
[24] T. Rush, W. L. Peticolas, J. Phys. Chem. 1995, 99, 14647 –
14658.
[25] A. Aamouche, G. Berthier, C. Coulombeau, J. P. Flament,
et al., Chem. Phys. 1996, 204, 353 – 363.
[26] A. Aamouche, M. Ghomi, C. Coulombeau, H. Jobic, et al.,
J. Phys. Chem. 1996, 100, 5224 – 5234.
[27] A. Aamouche, M. Ghomi, C. Coulombeau, L. Grajcar, et
al., J. Phys. Chem. A. 1997, 101, 1808 – 1817.
[28] G. Maistralis, A. Koutsodimou, N. Katsaros, Transit. Met.
Chem. 2000, 25, 166 – 173.
[29] F. Nepveu, N. Gaultier, N. Korber, J. Jaud, P. Castan, J.
Chem. Soc. Dalton Trans. 1995, 24, 4005 – 4014.
[30] E. R. Tucci, B. E. Doody, N. C. Li, J. Phys. Chem. 1961, 65,
1570 – 1574.
[31] E. R. Tucci, C. H. Ke, N. C. Li, J. Inorg. Nucl. Chem. 1967, 29,
1657 – 1667.
[32] M. Sabat, D. Zglinska, B. Jezowska-Trzebiatowska, Acta
Crystallogr. B 1980, 36, 1187 – 1188.
[33] I. Mutikainen, P. Lumme, Acta Crystallogr. B 1980, 36,
2233 – 2237.
[34] T. Solin, K. Matsumoto, K. Fuwa, Bull. Chem. Soc. Jpn. 1981,
54, 3731 – 3734.
[35] T. S. Khodashova, M. A. Porai-Koshits, N. K. Davidenko, N.
N. Vlasova, Koord. Khim. 1984, 10, 262 – 268.
[36] A. Karipides, B. Thomas, Acta Crystallogr. C 1986, 42,
1705 – 1707.
www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2006, 339, 598 – 607
New Lanthanum (III) Complex with Cytotoxic Activity
[37] I. Mutikainen, Finn. Chem. Lett. 1985, 193 – 200.
[38] R. Wysokinski, B. Morzyl-Ociepa, T. Glowiak,
Michalska, J. Mol. Struct. 2002, 606, 241 – 251.
D.
[39] I. Kostova, I. Manolov, S. Konstantinov, M. Karaivanova,
Eur. J. Med. Chem. 1999, 34, 63 – 68.
[40] I. Kostova, I. Manolov, I. Nicolova, N. Danchev, Farmaco
2001, 56, 707 – 713.
[41] I. Kostova, I. Manolov, I. Nicolova, S. Konstantinov, M.
Karaivanova, Eur. J. Med. Chem. 2001, 36, 339 – 347.
607
[51] N. Lalioti, C. P. Raptopoulou, A. Terzis, A. Panagiotopoulos, et al., J. Chem. Soc., Dalton Trans. 1998, 1327 – 1333.
[52] L. S. Gelfand, F. J. Iaconianni, L. L. Pytlewski, A. N. Speca,
et al., J. Inorg. Nucl. Chem. 1980, 42, 377 – 385.
[53] K. Nakamoto, Infrared and Raman spectra of inorganic and
coordination compounds, Part B, 5th Ed, Wiley, New York,
1997.
[54] R. J. Abraham, P. Loftus in Proton and Carbon-13 NMR Spectroscopy, Heyden, London, 1978, pp. 23, 24, 165 – 168.
[43] I. Kostova, I. Manolov, M. Radulova, Acta Pharm. 2004, 54,
119 – 131.
[55] L. M. Jackman, S. Sternhel in Applications of Nuclear Magnetic Spectroscopy in Organic Chemistry, International Series of Monographs in Organic Chemistry, Pergamon,
Oxford, 2nd Ed., 1969, Vol. 5, pp. 53 – 60, 103, 104, 215 –
218, 359, 360, 380 – 384.
[44] I. Kostova, I. Manolov, G. Momekov, Eur. J. Med. Chem.
2004, 39, 765 – 775.
[56] S. Bekiroglu, O. Kristiansson, J. Chem. Soc., Dalton Trans.
2002, 1330 – 1335.
[45] I. Kostova, N. Trendafilova, G. Momekov, J. Inorg. Biochem.
2005, 99, 477 – 487.
[57] S. Lencioni, A. Pellerito, T. Fiore, A. M. Giuliani, et al.,
Appl. Organometal. Chem. 1999, 13, 145 – 157.
[46] I. Kostova, G. Momekov, M. Zaharieva, M. Karaivanova,
Eur. J. Med. Chem. 2005, 40, 542 – 551.
[58] I. Kostova, N. Peica, W. Kiefer, Chem. Phys. 2006, 327, 494 –
505.
[47] I. Kostova, R. Kostova, G. Momekov, N. Trendafilova, M.
Karaivanova, J. Tr. Elem. Med. Biol. 2005, 18, 219 – 226.
[59] H. G. Drexler, W. Dirks, R. A. F. MacLeod, H. Quentmeier,
et al., (Eds.) DSMZ Catalogue of Human and Animal Cell
Lines., 6th Ed., DSMZ GmbH, Braunschweig, 1997.
[42] I. Kostova, I. Manolov, M. Radulova, Acta Pharm. 2004, 54,
37 – 47.
[48] I. Kostova, N. Trendafilova, T. Mihailov, Chem. Phys. 2005,
314, 73 – 84.
[49] T. W. Hambley, R. I. Christopherson, E. S. Zvargulis, Inorg.
Chem. 1995, 34, 6550 – 6552.
[50] S. P. Perlepes, V. Lazaridou, B. Sankhla, J. M. Tsangaris,
Bull. Soc. Chim. Fr. 1990, 127, 597 – 608.
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[60] S. I. Akiyama, Z.-S. Chen, T. Sumizawa, T. Furukawa, AntiCancer Drug Des. 1999, 14, 143 – 151.
[61] T. Miyashita, J. C. Reed, Blood 1993, 81, 151 – 157.
[62] T. C. Fisher, A. E. Milner, C. D. Gregory, A. L. Jackman, et
al., Cancer Res. 1993, 53, 3321 – 3326.
www.archpharm.com
Документ
Категория
Без категории
Просмотров
2
Размер файла
718 Кб
Теги
complex, synthesis, activity, characterization, lanthanum, cytotoxic, iii, new
1/--страниц
Пожаловаться на содержимое документа