Influence of methoxy groups on the antiproliferative effects of [Fe III(salophene-OMe)Cl] complexes

Article abstract of DOI:10.1016/j.ejmech.2010.08.037

We synthesized methoxy-substituted iron(III)-salophene complexes ([Fe ^III(OMe-salophene)Cl] with salophene = N,N′-bis(salicylidene)- 1,2-phenylenediamine) and analyzed their biological activity in MCF-7 and MDA-MB-231 breast cancer as well as i

Full text of DOI:10.1016/j.ejmech.2010.08.037

European Journal of Medicinal Chemistry 45 (2010) 5486e5492
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Influence of methoxy groups on the antiproliferative effects
of [FeIII(salophene-OMe)Cl] complexes
,b,
Annegret Hille a, Ronald Gust a
*
a Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Strasse 2 þ 4, D-14195 Berlin, Germany
b Institute of Pharmacy, Department of Pharmaceutical Chemistry, University of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
a r t i c l e i n f o
a b s t r a c t
We synthesized methoxy-substituted iron(III)-salophene complexes ([FeIII(OMe-salophene)Cl] with
salophene ¼ N,N0-bis(salicylidene)-1,2-phenylenediamine) and analyzed their biological activity in MCF-
7 and MDA-MB-231 breast cancer as well as in HT-29 colon carcinoma cells. The results obtained in
a time-dependent chemosensitivity test clearly demonstrated the correlation between the cytotoxicity of
the complexes and the position of methoxy substituents in the salicylidene moieties: 3-OCH3 (4) < 5-
OCH3 (8) < H (2) < 4-OCH3 (6) ¼ 6-OCH3 (10). Compounds 6 and 10 caused cytocidal effects already at
Article history:
Received 27 January 2010
Received in revised form
30 June 2010
Accepted 13 August 2010
Available online 24 August 2010
a concentration of 0.5 mM. Both lead compound 2 and complex 8 showed similar time response curves,
Keywords:
Iron-salophene complexes
MCF-7
MDA-MB-231
HT-29
Anticancer activity
however, with a 5-fold lower activity compared to 6 and 10, respectively. Referring to [FeIII(salophene)Cl]
(2), methoxy substitution was accompanied with the loss of tumor cell selectivity. Moreover, the free
ligands (1, 3, 5, 7, and 9) were inactive.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
stacked between two base pairs. They confirmed an external elec-
trostatic interaction between the negatively charged DNA double
The discovery of the inorganic complex cisplatin revolutionized
the treatment of testicular cancer and led to increased research of
organometallic complexes as antitumor agents [1]. Among them,
a variety of iron containing compounds has emerged due to their
outstanding biological properties. The group of Neuse reported
about antitumor activity of a series of ferrocene complexes against
Ehrlich ascites tumor in CF1 mice [2,3]. Moreover, according to the
findings of James et al. enantioselective ferrocenyl nucleoside
analogues turned out to be apoptosis-inducing compounds in Bur-
kitt-like lymphoma (BJAB) tumor cells [4]. Hillard et al. developed
ferrocenyl complexes combining antiestrogenicity and estrogen-
independent cytotoxicity in the same molecule in order to address
both estrogen receptor positive and negative breast cancer cells [5].
Furthermore, it was demonstrated that the DNA cleavage ability of
hydroxyl-substituted iron-salen complexes (salen ¼ N,N0-bis(sali-
cylidene)ethylenediamine) strongly depended on the position of
the hydroxyl groups in the salicylidene moiety [6]. The group of
Tumminello postulated a non-intercalative mode of action with
DNA of iron-salen complexes, although the flat molecules can be
helix analogously to porphyrazines and metaleporphyrazine
complexes [7]. This might induce apoptosis via mitochondrial
pathway as demonstrated for human HEK-293 cells [8].
In a previously published structureeactivity relationship study
we described the development of [N,N0-bis(salicylidene)-1,2-phe-
nylenediamine]iron(III) ([FeIII(salophene)Cl]) as antitumor agent
and the evaluation of the mode of action [9]. The same lead
structure was used by Lange et al. [10] and they demonstrated
a selective action against platinum-resistant ovarian cancer cells if
a 3-methoxy substituent is present at the salicylidene moiety. This
study strengthened our intention to increase the cytotoxicity of
[FeIII(salophene)Cl] complexes by introduction of OCH3 substitu-
ents (Scheme 1).
2. Results and discussion
2.1. Synthesis
The N,N0-bis(salicylidene)-1,2-phenylenediamine ligands were
synthesized as previously described [9]. The commercially available
1,2-phenylenediamine was reacted in ethanol with two equivalents
of the respective salicylaldehyde (Scheme 1). The precipitated
products were collected and characterized by 1H NMR spectroscopy
(Fig. 1).
* Corresponding author. Department of Pharmaceutical Chemistry, University of
Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria. Tel.: þ43 512 507 5245; fax: þ43
512 507 2940.
E-mail address: ronald.gust@uibk.ac.at (R. Gust).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.037

A. Hille, R. Gust / European Journal of Medicinal Chemistry 45 (2010) 5486e5492
5487
H
O
d
b
FeCl3
EtOH
2
d
b
N
N
d
b
d
b
N
O
N
O
d
b
- 2 HCl(aq)
- 2 H2O
c
OH
Fe
Cl
H2N
NH2
c
OH HO
c
c
c
a
a
a
a
a
1,3,5,7,9
2,4,6,8,10
a
H
b
c
H
d
H
compound
H
H
H
H
1
2
H
H
H
OMe
OMe
H
H
H
3
H
H
4
OMe
OMe
H
H
H
5
H
H
H
6
H
OMe
OMe
H
H
7
H
H
H
8
H
H
OMe
OMe
9
H
H
H
10
Scheme 1. Synthesis pathway of the salophene ligands and their iron(III) complexes.
As shown in Fig. 1, the position of the methoxy group affected
most pronounced effect was observed for the para-substituted
derivative 7.
The observed shifts might be the consequence of H-bridges of
different strength, due to the changed electron density at the
oxygen. It is well known, that the phenolic protons of the salophene
the OH signals (
d
¼ 12e14), while the resonance of the imine proton
He was nearly identical for all investigated ligands. Methoxy groups
located in ortho- or para-position (3 and 7) shifted the OH signal to
higher field compared to that of the meta derivatives 5 and 9. The
Fig. 1. 1H NMR spectra of the ligands 1, 3, 5, 7 and 9 in DMSO-d6. The resonance peaks in the area of 12e14 ppm belong to the phenolic proton.

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}

A. Hille, R. Gust / European Journal of Medicinal Chemistry 45 (2010) 5486e5492
5489
Fig. 2. Antiproliferative effects of cisplatin on MCF-7 and MDA-MB-231 breast cancer and HT-29 colon carcinoma cells.
comparable to very effective minor-groove binders CD-measure-
ments and melting curve analysis provided no evidence of inter-
calation into the DNA double helix [9].
Interestingly the most favoured ortho- and para-position led to
a reduction of activity.
This finding induced us to study the effects of þI/þM and eI/eM
substituents at the salicylidene moiety on the cytotoxicity of the
complexes. The results will be published in a forthcoming paper.
Exchanging the salophene by N,N0-bis(salicylidene)-1,2-cyclo-
hexanediamine (saldach) reduced the in vitro effects drastically. An
unequivocal mode of action cannot be deduced from these results,
but it seems to be very likely that cell death is caused by interfer-
ence with more than one intracellular target.
3. Materials and methods
Methoxy groups at the salicylidene moiety seem to modify the
pharmacological properties. It is obvious that the substitution in
meta-position to the hydroxyl group bound to iron led to a much
higher in vitro activity than in ortho- and para-position. Compared
to [FeIII(salophene)Cl] (2) this means an increase in activity at the
MCF-7 and HT-29 cell lines and a slight loss of activity against MDA-
MB-231 cells. It isverylikelythatmethoxysubstituents at 2 probably
influence the cell unspecific redox behaviour of the complex. This is
howeverspeculativeandhas tobeconfirmedin aforthcomingstudy.
An indication is given by Routier et al. who showed that the
para-substituted [bis(hydroxy)salen]iron(II) complex displayed
cell-damaging properties because the hydroxyl group built
a hydroquinone system cooperating with the redox-active metal to
facilitate spontaneous formation of free radicals [6]. In our
complexes such formation is impossible since the cleavage of the
O-methyl ether was not observed under cell culture conditions.
Nevertheless, the methoxy groups might influence the electron
density at the FeeO bond due to their electron donating effects.
3.1. General procedures
The following instrumentation was used: 1H NMR, Bruker ADX
400 spectrometer at 400 MHz (internal standard, TMS); EI-MS
spectra, CH-7AVarian (70 eV); IR spectra (KBr pellets), PerkineElmer
model 580 A; FT-Raman spectra were measured on a Bruker RFS 100
spectrometer (excitation with 1064 nm line of Nd:YAG laser; power,
100 mW, resolution 3 cmꢀ1). Elemental C, H, N analyses were carried
out with a PerkineElmer 240 B and 240 C elemental analyzer.
Analyses indicated by the symbols of the elements or functions were
within ꢀ0.4% of the theoretical values. Chemicals were obtained
from Sigma Aldrich (Germany) and used without further purifica-
tion. Compounds 1 and 2 were synthesized as recently published [9].
3.2. Synthesis of the ligands and their iron(III) complexes
Two equivalents of the respective salicylaldehyde (7.00 mmol)
in ethanol (20 mL) were added dropwise to a solution of one
Fig. 3. Antiproliferative effects of [FeIII(salophene)Cl] (2) on MCF-7 and MDA-MB-231 breast cancer and HT-29 colon carcinoma cells.

5490
A. Hille, R. Gust / European Journal of Medicinal Chemistry 45 (2010) 5486e5492
Fig. 4. Antiproliferative effects of the [FeIII(salophene-OMe)Cl] complexes 4, 6, 8, and 10 on the MCF-7 and MDA-MB-231 breast cancer and HT-29 colon carcinoma cells.
equivalent of 1,2-phenylenediamine in ethanol (10 mL). The
mixture was stirred under reflux for 1e2 h and then allowed to cool
down to room temperature. The product was collected, washed
with ethanol and dried (P2O5). The obtained ligand (0.6 mmol) was
dissolved in ethanol (10 mL) and heated to reflux in the presence
one equivalent of FeCl3$6H2O in ethanol (5 mL). After 1e2 h, the
mixture was cooled to room temperature, the solid was filtered off,
washed with ethanol and dried in vacuo.
a crystalline, black powder. IR (KBr):
n
[cmꢀ1] ¼ 1602 (w), 1579 (s),
1546 (s), 1446 (s), 1435 (s), 1313 (m), 1253 (s), 1199 (m),þ529 (w), 456
(m), 340 (s). MS (EI, 200 ꢁC): m/z (%) ¼ 465 (30) [M ], 430 (100)
[MþeCl]. Anal. C22H18N2O4FeCl (C, H, N).
3.2.3. Synthesis of 5
Compound 5 was obtained from 1,2-phenylenediamine and
4-methoxysalicylaldehyde. Yield: 0.96 g (2.55 mmol, 84%) of
a crystalline, yellow powder. 1H NMR (DMSO-d6):
d
¼ 13.52 (s,
3.2.1. Synthesis of 3
Compound 3 was obtained from 1,2-phenylenediamine and
3-methoxysalicylaldehyde. Yield: 1.31 g (3.48 mmol, 70%) of
2H, AreOH), 8.84 (s, 2H, He), 7.56e7.54 (d, 2H, Hd, 3J ¼ 8.64 Hz),
7.45e7.34 (m, 4H, Hg þ Hf, 3J ¼ 9.33 and 9.30 Hz, 4J ¼ 2.41 Hz),
6.56e6.53 (dd, 2H, Hc, 3J ¼ 8.62 Hz, 4J ¼ 2.37 Hz), 6.50e6.49
(d, 2H, Ha, 4J ¼ 2.31 Hz), 3.81 (s, 3H, eOCH3). IR (KBr):
a crystalline, dark orange powder. 1H NMR (DMSO-d6):
d
¼ 13.00 (s,
2H, AreOH), 8.93 (s, 2H, He), 7.48e7.40 (m, 4H, Hg þ Hf, 3J ¼ 9.36
and 9.33 Hz, 4J ¼ 2.67 and 2.29 Hz), 7.26e7.24 (dd, 2H, Hb,
3J ¼ 7.83 Hz, 4J ¼ 1.16 Hz), 7.14e7.12 (dd, 2H, Hd 3J ¼ 8.08
4J ¼ 1.09 Hz), 6.93e6.89 (m, 1H 3J ¼ 7.88 and 7.87 Hz), 3.81 (s,
n
[cmꢀ1] ¼ 3433 (br), 1616 (m), 1566 (w), 1464 (w), 1513 (m), 1295
(m), 1204 (s). MS (EI, 120 ꢁC): m/z (%) ¼ 376 (52) [Mþ]. Anal.
C22H20N2O4 (C, H, N).
3H, eOCH3). IR (KBr):
n
[cmꢀ1] ¼ 3427 (br), 1612 (s), 1573 (m), 1466
3.2.4. Synthesis of 6
(s), 1255 (s), 1205 (m). MS (EI, 90 ꢁC): m/z (%) ¼ 376 (74) [Mþ]. Anal.
Compound
6
was obtained from N,N0-bis(4-methox-
C22H20N2O4 (C, H, N).
ysalicylidene)-1,2-phenylenediamine (0.10 g, 0.32 mmol) and
FeCl3$6H2O (0.07 g, 0.27 mmol). Yield: 104.0 mg (0.22 mmol, 84%) of
3.2.2. Synthesis of 4
a crystalline, black powder. IR (KBr):
n
[cmꢀ1] ¼ 1608 (w), 1574 (s),
Compound
4
was obtained from N,N0-bis(3-methox-
1522 (w), 1374 (w), 1309 (s), 1266 (s), 1252 (s), 1208 (s), 520 (w), 504
(s), 480 (w), 439 (m), 338 (s), 332 (s). MS (EI, 275 ꢁC): m/z (%) ¼ 465
(70) [Mþ], 430 (100) [MþeCl]. Anal. C22H18N2O4FeCl (C, H, N).
ysalicylidene)-1,2-phenylenediamine (0.10 g, 0.27 mmol) and
FeCl3$6H2O (0.072 g, 0.27 mmol). Yield: 72.10 mg (0.16 mmol, 58%) of

A. Hille, R. Gust / European Journal of Medicinal Chemistry 45 (2010) 5486e5492
5491
Fig. 4. (continued).
3.2.5. Synthesis of 7
4J ¼ 3.66 Hz, 2H, Hb, 3J ¼ 8.38 Hz, 4J ¼ 1.69 Hz), 6.55e6.51 (m, 4H,
Compound 7 was obtained from 1,2-phenylenediamine and 5-
methoxysalicylaldehyde. Yield: 1.19 g (3.16 mmol, 82%) of a crys-
Hc þ Ha, 3J ¼ 9.04 and 9.07 Hz), 3.84 (s, 3H, eOCH3). IR (KBr):
n
[cmꢀ1] ¼ 3431 (br), 1614 (m), 1586 (s), 1469 (s), 1457 (s), 1358 (s),
1251 (s),1187 (s),1192 (s). MS (EI,150 ꢁC): m/z (%) ¼ 376 (100) [Mþ$].
Anal. C22H20N2O4 (C, H, N).
talline orange powder. 1H NMR (DMSO-d6):
d
¼ 12.27 (s, 2H,
AreOH), 8.89 (s, 2H, He), 7.44e7.37 (m, 4H, Hg þ Hf, 3J ¼ 9.34 and
9.35 Hz, 4J ¼ 2.63 and 2.64 Hz), 7.28e7.27 (d, 2H, Hd, 4J ¼ 3.09 Hz),
7.03e7.00 (dd, 2H, Hb, 3J ¼ 8.92 Hz, 4J ¼ 3.13 Hz), 6.89e6.86 (d, 2H,
3.2.8. Synthesis of 10
Ha, 3J ¼ 8.96 Hz). IR (KBr):
n
[cmꢀ1] ¼ 3419 (br), 1623 (m), 1571 (s),
Compound 10 was obtained from N,N0-bis(6-methoxysali-
cylidene)-1,2-phenylenediamine (0.13 g, 0.34 mmol) and FeCl3$6H2O
(0.097 g, 0.36 mmol). Yield: 97.00 mg (0.21 mmol, 60%) of a crystal-
1489 (s), 1270 (s), 1212 (m), 1160 (m). MS (EI, 150 ꢁC): m/z (%) ¼ 376
(63) [Mþ]. Anal. C22H20N2O4 (C, H, N).
line, black powder. IR (KBr):
n
[cmꢀ1] ¼ 1604 (w), 1572 (s), 1541 (s),
3.2.6. Synthesis of 8
1459 (m), 1431 (s), 1366 (m), 1258 (m),þ540 (w), 480 (m)þ, 322 (s). MS
(EI, 225 ꢁC): m/z (%) ¼ 465 (31) [M ], 430 (100) [M eCl]. Anal.
C22H18N2O4FeCl (C, H, N).
Compound
8
was obtained from N,N0-bis(5-methox-
ysalicylidene)-1,2-phenylenediamine (0.14 g, 0.37 mmol) and
FeCl3$6H2O (0.10 g, 0.38 mmol). Yield: 56.00 mg (0.12 mmol, 33%) of
a crystalline, black powder. IR (KBr):
n
[cmꢀ1] ¼ 1616 (s), 1536 (s), 1469
3.3. Biological methods
(s), 1362 (m), 1287 (m), 1223 (m), 636 (m), 476 (w), 321 (s). MS (EI,
250 ꢁC): m/z (%) ¼ 465 (21) [Mþ], 430 (100) [MþeCl]. Anal.
C22H18N2O4FeCl (C, H, N).
3.3.1. Cell culture conditions
The human MCF-7 and MDA-MB-231 breast cancer and HT-29
colon cancer cell lines were obtained from the American Type
Culture Collection (ATCC). Both cell lines were maintained as
3.2.7. Synthesis of 9
Compound 9 was obtained from 1,2-phenylenediamine and 6-
methoxysalicylaldehyde. Yield: 0.78 g (2.07 mmol, 63%) of a crys-
a monolayer culture in L-glutamine containing Dulbecco’s Modified
Eagle’s Medium (DMEM) with 4.5 g/L glucose (PAA Laboratories
GmbH, Austria), supplement with 10% fetal calf serum (FCS; Gibco,
Germany) in a humidified atmosphere (5% CO2) at 37 ꢁC. The cell
talline, light orange powder. 1H NMR (DMSO-d6):
d
¼ 13.78 (s, 2H,
AreOH), 9.08 (s, 2H, He), 7.41e7.31 (m, 6H, Hg þ Hf, 3J ¼ 9.42 Hz,

5492
A. Hille, R. Gust / European Journal of Medicinal Chemistry 45 (2010) 5486e5492
lines were passaged twice a week after previous treatment with
trypsin (0.05%)/ethylenediaminetetraacetic acid (0.02% EDTA;
Boehringer, Germany).
Acknowledgment
Dr. J. Spandl (Institute of Chemistry and Biochemistry-Inorganic
Chemistry, Freie Universität Berlin, Fabeckstr. 34/36, 14195 Berlin)
is gratefully acknowledged for Raman measurements. The financial
support of DFG-Deutsche Forschungsgemeinschaft (FOR 630) is
greatly appreciated.
3.3.2. In vitro chemosensitivity assay
The in vitro testing of the substances for antitumor activity in
adherent growing cell lines was carried out on exponentially
dividing human cancer cells according to a previously published
microtiter assay [18]. Exponential cell growth was ensured
during the whole time of incubation. Briefly, 100
mL of a cell
suspension was placed in each well of a 96-well microtiter plate
at 7200 cells/mL (MCF-7), at 3000 cells/mL (MDA-MB-231) and at
1600 cells/mL (HT-29) of culture medium and incubated at 37 ꢁC
in a humidified atmosphere (5% CO2) for 3 d (MCF-7) and for 2 d
(MDA-MB-231 and HT-29), respectively. Then the old medium
References
[1] B.K. Keppler, Metal Complexes in Cancer Chemotherapy. VCH, Weinheim,
1993.
[2] P. Köpf-Maier, H. Köpf, E.W. Neuse, J. Cancer Res. Clin. Oncol. 108 (1984)
336e340.
[3] E.W. Neuse, Macromolecules containing metal and metal-like elements. in:
A.S. Abd-El-Aziz, C.E. Carraher Jr., C.U. Pittman Jr., J.E. Sheats, M. Zeldin (Eds.),
Biomedical Applications, vol. 3. John Wiley & Sons, New York, USA, 2004, pp.
89e117 (Chapter 6).
[4] P. James, J. Neudörfl, M. Eissmann, P. Jesse, A. Prokop, H.-G. Schmalz, Org. Lett.
8 (2006) 2763e2766.
[5] E. Hillard, A. Vessières, L. Thouin, G. Jaouen, C. Amatore, Angew. Chem. 118
(2006) 291e296.
[6] S. Routier, H. Vezin, E. Lamour, J.-L. Bernier, J.-P. Catteau, C. Bailly, Nucl. Acids
Res. 27 (1999) 4160e4166.
[7] A. Silvestri, G. Barone, G. Ruisi, M.T. Lo Giudice, S. Tumminello, J. Inorg. Bio-
chem. 98 (2004) 589e594.
[8] G.A. Woldemariam, S.S. Mandal, J. Inorg. Biochem. 102 (2008)
740e747.
was exchanged by 200
mL of fresh medium together with an
adequate volume of a stock solution of metal complex to obtain
the desired test concentration. Cisplatin was dissolved in dime-
thylformamide (DMF) while dimethylsulfoxide (DMSO) was used
for all other compounds. Eight wells were used for each test
concentration and the control containing the corresponding
amount of DMF and DMSO, respectively. The medium was
removed after appropriate incubation time. Subsequently, the
cells were fixed with a solution of 1% (v/v) glutaric dialdehyde in
phosphate buffered saline (PBS) and stored under PBS at 4 ꢁC.
Cell biomass was determined by means of a crystal violet staining
technique as described earlier [16]. The effectiveness of the
[9] A. Hille, I. Ott, A. Kitanovic, I. Kitanovic, H. Alborzinia, E. Lederer, S. Wölfl,
N. Metzler-Nolte, S. Schäfer, W. Sheldrick, C. Bischof, U. Schatzschneider,
R. Gust, J. Biol. Inorg. Chem. 14 (2009) 711e725.
[10] T.S. Lange, K.K. Kim, R.K. Singh, R.M. Strongin, C.K. McCourt, L. Brard, PLoS One
3 (5) (2008) e2603.
complexes is expressed as corrected T/Ccorr [%] or s[%] values
according to the following equation:
[11] F. Voegtle, E. Goldschmitt, Chem. Ber. 109 (1976) 1e40.
[12] A. Belaïd, S. Landreau, S. Djebbar, O. Benali-Baïtich, M.A. Khan, G. Bouet,
Transition Met. Chem. 33 (2008) 511e516.
[13] Y.-M. Zhou, X.-R. Ye, F.-B. Xin, X.-Q. Xin, Transition Met. Chem. 24 (1999)
118e120.
Cytostatic effect : T=Ccorr½%ꢂ ¼ ½ðT ꢀ C0Þ=ðC ꢀ C0Þꢂ ꢃ 100
Cytocidal effect :
½%ꢂ ¼ ½ðT ꢀ C0Þ=C0ꢂ ꢃ 100
s
[14] K.P. Krause, O. Kayser, K. Mäder, R. Gust, R.H. Müller, Int. J. Pharm. 196 (2000)
169e172.
[15] T. Kapp, A. Dullin, R. Gust, Bioconjug. Chem. 21 (2010) 328e337.
[16] G. Bernhardt, H. Reile, H. Birnböck, T. Spruß, H. Schönenberger, J. Cancer Res.
Clin. Oncol. 118 (1992) 35e43.
[17] H. Reile, T. Spruß, G. Bernhardt, R. Müller, R. Gust, H. Schönenberger, Arch.
Pharm. (Weinheim, Ger.) 324 (1991) 405e409.
[18] H. Reile, H. Birnböck, G. Bernhardt, T. Spruß, H. Schönenberger, Anal. Biochem.
187 (1990) 262e267.
whereby T (test) and C (control) are the optical densities at 590 nm
of crystal violet extract of the cells in the wells (i.e. the chromatin-
bound crystal violet extracted with ethanol (70%) with C0 corre-
sponding to the density of the cell extract immediately before
treatment. For the automatic estimation of the optical density of
the crystal violet extract in the wells, a microplate autoreader
(Flashscan S 12; Analytik Jena, Germany) was used.