5488
A. Hille, R. Gust / European Journal of Medicinal Chemistry 45 (2010) 5486e5492
ligands were incorporated in H-bonds to the imine nitrogen
OeH/N]C (salicylic effect) [11].
relation to cisplatin. This drug is a well accepted reference for the
design of metal based drugs.
The resonance signals of the 1,2-phenylenediamine moiety
(Hf and Hg, see Fig. 1 and Scheme 1) appeared in the same region
irrespectively of OMe substituents located at the salicylidene,
indicating no significant influence on the aromatic ring.
Cisplatin showed at a concentration of 5 mM cytostatic activity
against MCF-7 and MDA-MB-231 cells (T/Ccorr ¼ 3%, respectively,
see Fig. 2). On the HT-29 cell line it was less active (T/Ccorr ¼ 35%).
The time response curves (5 mM) are characterized by a slow onset
The paramagnetism of the iron complexes made a character-
isation by NMR spectroscopy impossible. However, the coordina-
tion of the ligands to iron(III) could be verified by infra red and
Raman spectroscopy (Table 1). The broad signal in the region from
3300 to 2400 cmꢀ1 confirmed the involvement of the phenolic OH
group and the imine nitrogen in H-bridges. Upon coordination, the
of activity and a maximum of cytotoxicity at the end of the
experiment (150e200 h).
The lead structure of this study the [FeIII(salophene)Cl] (2) was
more active than cisplatin (compare Figs. 2 and 3). The highest
activity was determined at the MDA-MB-231 cell line. Already at
a concentration of 0.1
T/Ccorr ¼ 10%). To achieve the same efficacy, 0.5
at the MCF-7 and 1 M at the HT-29 cell line.
m
M cytostatic effects were determined (min.
band disappeared and the
n
(C]N) at about 1610 cmꢀ1 was shifted
mM were necessary
to lower frequencies. The Dn of z10 cmꢀ1 is in agreement with the
literature. Two further vibration bands at about 1570 and
1470 cmꢀ1 were influenced in the same way (Dn ¼ 30e40 cmꢀ1).
m
In contrast to cisplatin, 2 already reached its maximum inhibi-
tion of cell growth during an incubation time of 60 h followed by
a recuperation of the cells. Such “V shaped profile” was docu-
mented by Bernhardt et al. during the establishment of this “crystal
violet assay” [16]. As possible reasons for this behaviour they
proposed inactivation of the drugs in the culture medium, the
damage of only a subpopulation of cancer cells and the develop-
ment of secondary resistance. In accordance with this, we deter-
mined the influence of the metal complexes on tumor cells over
a time period up to 244 h and abstain from the determination of
IC50 values, which documented the effects at only one time point.
For high cytotoxicity it was necessary to coordinate the ligands
to iron. Both, FeCl3 and free ligands did not significantly influence
New bands arise in the region between 600 and 300 cmꢀ1
.
Vibration bands of the FeeN
(n
z
540 cmꢀ1), the FeeO
(n
z 470 cmꢀ1) and the FeeCl bond ( z 320 cmꢀ1) could be
n
assigned by Raman spectroscopy (see Table 1) [12,13].
2.2. Solubility and stability in aqueous solution
Satisfying solubility and stability in aqueous solution are
a prerequisite for successful in vitro studies. Testing at the limit of
solubility and decomposition prior to cellular uptake would lead to
inadequate results. Therefore, stock solutions of all complexes in
tumor cell growth within a concentration range of 1e5 mM (data
DMSO (0.01 M) were diluted with aqua bidest. to 100 mM and stored
not shown). The methoxy groups in the salicylidene rings led to
a loss of tumor cell selectivity. The effects at MCF-7, MDA-MB-231
and HT-29 cells are comparable. Furthermore, only in the case of 8
a marginal recuperation of the cells was observed at the lowest
at room temperature for 2 h. After filtration, the amount of iron was
quantified by graphite furnace atomic absorption spectroscopy
according to published procedures [14,15].
The solubility of the complexes depended on the position of the
concentration used (0.5
3-methoxy group reduced the antiproliferative effects.
[FeIII(salophene-3-OMe)Cl] (4) was inactive at 0.5 and 1
M but
reached the cytocidal area at 5 M, indicating a steep concentration
mM).
methoxy group: 2 (46.5
(97.6 M) < 10 (100.1 M). However, in each case it is guaranteed
that the complexes showed a higher solubility than the maximal
concentration of 5 M used in the in vitro tests.
mM) < 4 (70.9 mM) < 6 (93.1 mM) < 8
A
m
m
m
m
m
activity relation. The shift of the methoxy group from the 3- into the
4- (6) or the 6-position (10) enormously increased the growth
inhibitory effects at the cell lines. Compounds 6 and 10 caused
nearly identical curves as 4 but at 10 fold lower concentrations.
An exception regarding the concentration dependent response
represents complex 8. The activity of 8 was comparable to 4 at
Furthermore, the aqueous solutions were investigated for
stability using HPLC. All complexes caused only a single peak in the
HPLC chromatograms whose areas did not change during the
observation time of 48 h.
It should be noted that the maximal amount of DMSO in the test
solutions was 0.1%.
a concentration of 5
0.5 M.
mM, but inhibited cell growth also at 1 and
m
2.3. Biological activity
2.4. Discussion
The biological activity was determined in MCF-7 and MDA-MB-
231 breast cancer as well as in HT-29 colon carcinoma cell lines in
The graphs of the time response curves not only give informa-
tion about possible development of resistance but also suggest
about side effects observable in in vivo studies. Highly reactive
platinum(II) complexes such as aquasulfatoplatinum(II) derivatives
with very steep concentration activity curves were active in diverse
mouse models but showed toxic side effects such as loss of body
weight [17]. A reduction of the respective dose reduced both, the
antitumor activity and the side effects nearly independent on the
used tumor model. From the analysis of the graphs depicted in
Fig. 4, a comparable behaviour can be deduced for 4, 6, and 10.
The influence of methoxy groups on the cytotoxicity and the
selectivity of iron(III)-salophene complexes is pronounced. Espe-
cially the loss of tumor cell selectivity was unusual and indicated
a cell unspecific cellular interaction. Studies on the mode of action
of this complex type were part of the first paper of this series [9].
[FeIII(salophene)Cl] (2) was able to generate reactive oxygen species
(ROS) and induced apoptosis but showed only low DNA binding.
Although the iron(salophene) complexes possess a planar structure
Table 1
Selected Raman frequencies [cmꢀ1] of methoxy-substituted compounds 3e10.
Compound
n(C]N)
n(AreO)
n(FeeN)
n(FeeO)
n(FeeCl)
3
1612s
1573m
1467s
1546s
1435s
1566w
1464w
1522w
1374w
1571s
1489s
1535s
1463s
1586s
1469s
1541s
1431s
e
e
e
4
1602w
1616m
1608w
1623m
1616s
{529w}
{456m}
{340s}
5
e
e
e
6
{520w}
{504s}
e
{480w}
{439m}
e
{338s}
{332s}
e
7
8
{636m}
e
{476w}
e
{321m}
e
9
1614m
1604w
10
{540w}
{480m}
{322s}
Hille, Annegret
