Relationship between anticancer activity and stereochemistry of saldach ligands and their iron(III) complexes

Article abstract of DOI:10.1002/ardp.200900048

(R,R)-, (S,S)- and (R,S)-N,N′-bis(salicylidene)-1,2- diaminocyclohexane (saldach) and their iron(III) complexes were screened for anticancer activity against MCF-7 and MDA-MB 231 breast cancer as well as HT-29 colon carcinoma cells. Antiproliferative effects depended on the presence of the central atom iron but were independent on the configuration at the saldach ligand. While the free ligands were inactive, the iron(III) derivatives displayed anticancer activity within a concentration range of 1 to 5 μM irrespective of the used cell line. At 5 μM they were even more active than cis-platin. A mode of action comparable to cis-platin can be excluded because it is very likely that the DNA is not the primary target of [Fe^III (saldach)] complexes.

Full text of DOI:10.1002/ardp.200900048

Arch. Pharm. Chem. Life Sci. 2009, 342, 625–631
A. Hille and R. Gust
625
Full Paper
Relationship Between Anticancer Activity and Stereochemistry
of Saldach Ligands and their Iron(III) Complexes
Dedicated to Professor H. Schꢀnenberger on his 85. birthday
Annegret Hille and Ronald Gust
Institute of Pharmacy, Freie Universitꢁt Berlin, Berlin, Germany
(R,R)-, (S,S)- and (R,S)-N,N9-bis(salicylidene)-1,2-diaminocyclohexane (saldach) and their iron(III)
complexes were screened for anticancer activity against MCF-7 and MDA-MB 231 breast cancer as
well as HT-29 colon carcinoma cells. Antiproliferative effects depended on the presence of the
central atom iron but were independent on the configuration at the saldach ligand. While the
free ligands were inactive, the iron(III) derivatives displayed anticancer activity within a concen-
tration range of 1 to 5 lM irrespective of the used cell line. At 5 lM they were even more active
than cis-platin. A mode of action comparable to cis-platin can be excluded because it is very likely
that the DNA is not the primary target of [FeIII (saldach)] complexes.
Keywords: Anticancer activity / HT-29 / Iron saldach complexes / MCF-7 / MDA-MB 231 /
Received: March 10, 2009; Accepted: May 20, 2009
DOI 10.1002/ardp.200900048
promising carrier ligand, there is only few information
about non-platinum 1,2-diaminocyclohexane complexes
displaying anticancer activity [5–7]. Therefore, we modi-
fied DACH as well as its phenylenediamine congener in
such a way that they were suitable to obtain metal com-
plexes.
We already demonstrated that the [N,N9-bis(salicyli-
dene)-1,2-phenylenediamine]iron(II/III) complexes strong-
ly reduced cell proliferation more effectively than cis-
platin. This was ascribable to the presence of iron as the
central atom in combination with an intact phenylene-
diamine moiety. The reduction of the ligand resulting in
rac-N,N9-bis(salicylidene)-1,2-cyclohexanediamine strong-
lyreducedtheefficacyoftheironcomplexes.
The contribution of the DACH ligands chirality to the
anticancer activity of oxaliplatin prompted us to study
its influence on the cytotoxic properties of [FeIII(sal-
dach)Cl] complexes (see Fig. 1) [8]. We selected the oxida-
tion state +III because an earlier study demonstrated
nearly identical effects of iron(II) and iron(III) complexes.
Furthermore, we also investigated the cytotoxicity of the
free ligands, because Gao et al. recently identified related
Introduction
Transition metal compounds containing an 1,2-diamino-
cyclohexane (DACH) building block have been used as cat-
alysts for many years [1, 2]. The use in pharmaceutical
research increased with the observation of Kidani et al. in
the early 1980s that platinum complexes bearing DACH
as carrier ligand exhibited excellent antitumor activity
[3, 4]. Already at the beginning of the studies, a clear
dependence of the tumor-growth inhibiting effects on
the configuration of the DACH ligand was observed: The
meso form was less active than the racemic form and the
(R,R)-enantiomer was more active than its (S,S)-congener,
e.g. against the L1210 leukemia of the mouse. Optimiza-
tion of the leaving group led to [(R,R)-1,2-diamino-cyclo-
hexane]-oxalato-platinum(II) which is now established as
oxaliplatin in the systemic treatment of advanced color-
ectal carcinoma both, as a single agent and in combina-
tion regimens. Although the DACH ligand represents a
Correspondence: Ronald Gust, Institute of Pharmacy, Freie Universitꢁt
Berlin, Kꢀnigin-Luise-Strasse 2+4, D-14195 Berlin, Germany.
E-mail: rgust@zedat.fu-berlin.de
chiral
N,N9-bis-salicyl-1,2-diaminocyclohexane
com-
Fax: +49 30 8385-6906
pounds as effective anticancer agents in the down regula-
tion of Bcl-2-family gene expression in human breast can-
cer cells [9].
Abbreviations: 1,2-diaminocyclohexane (DACH); poly{ADP-ribose} poly-
merase (PARP); reactive oxygen species (ROS)
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

626
A. Hille and R. Gust
Arch. Pharm. Chem. Life Sci. 2009, 342, 625–631
Figure 1. Synthesized saldach ligands 1-4 and their [FeIII(saldach)Cl] complexes 5-8.
oxygen to iron indicated by the disappearance of the
broad O-H…N=C band in the IR spectra.
Results and discussion
Synthesis and structural characterization
Saldach ligands as well as the iron complexes (see Fig. 1) Biological activity
Biological properties were evaluated in relation to cis-
were synthesized as described previously [8, 10].
The formation of the Schiff bases was confirmed by the platin, which showed cytostatic activity against MCF-7
presence of a m(C=N) at about 1630 cm–1 in the IR spectra and MDA-MB 231 cells (T/Ccorr = 3%, respectively). On HT-29
cells it was less active (T/Ccorr = 35%) (see Fig. 2).
Free ligands as well as their complexes were adminis-
(see Experimental). Furthermore, the imine nitrogen is
part of an H-bond from the phenolic hydrogen (O-H…N=C;
salicylic effect) resulting in a broadening of the OH tered at equimolar concentrations. At 0.5, 1, and 5 lM all
stretching vibration in the region between 3500 to 2500 ligands were completely inactive (data not shown), while
1
cm–1 [11]. In the H-NMR spectra of 1 to 4, recorded in
the complexes showed concentration-dependent antipro-
DMSO-d6 and CDCl3, the imine proton appeared at d = liferative effects. Compared to cis-platin, the cytotoxicity
8.49 (1), 8.26 (2, 3), and 8.29 (4), while the OH resonance is was somewhat higher. The time response curve of cis-
located at d = 13.33 (1), 13.32 (2, 3), and 13.41 (4), respec- platin is characterized by a maximum effect at the end of
the test (after an incubation time of 150 to 220 h),
tively.
After coordination, NMR analysis is impossible due to whereas the highest antiproliferative effects of the iron
the paramagnetism of the iron complexes. In the IR spec- complexes were detected already after 48 to 72 h. There-
tra, the stretching vibration of the imine bond is shifted fore, it is necessary to evaluate the cytotoxicity in time-
by Dm = 18 cm–1 to lower frequencies. This shift is in
respond curves instead of concentration-respond curves
accordance with that observed for [N,N9-bis(salicylidene)- as realized for the calculation of IC50 values.
1,2-phenylenediamine]iron(II/III) and confirmed the coor-
dination of the imine via the nitrogen to iron. A further
chelate ring is built due to the binding of the phenolic
The effects depended on the used cell line. Cytostatic
or low cytocidal effects were caused at the highest con-
centration of 5 lM. Significantly reduced cell growth was
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 625–631
Saldach Ligands and their Iron(III) Complexes
627
Figure 2. Antiproliferative effects of cis-platin on the human MCF-7 and MDA-MB 231 breast cancer as well as the human HT-29
colon carcinoma cell lines.
caused at 1 lM only in the case of (R,R)-trans-[FeIII(sal- on the coordination to iron suffers from the cyclohexane
dach)Cl] complex 6 against the MDA-MB 231 cell line. ring: It changes between chair-chair or chair-boat confor-
These very steep concentration-activity curves indicated mations (see Fig. 4).
nonspecific cell toxic effects independent of the configu-
ration at the DACH ligand (see Fig. 3).
Configurations at the asymmetric C atoms determine
the orientation of the cyclohexane ring relative to the
Gao et al. investigated the influence of N,N9-bis-salicyl- iron plane (see Fig. 4).
1,2-diaminocyclohexane isomers on the growth of tumor
In the case of (R,R)- and (S,S)-configurated compounds a
cells, e.g. the MCF-7 breast cancer cells. The (R,R)-configu- coplanarity of the most likely chair conformation and
rated isomer (IC50 = 0.4 lM) was somewhat more active the chelate ring could be assumed. A change to the (R,S)-
than its (S,S)-enantiomer (IC50 = 0.6 lM) and both were configuration led to a nearly perpendicular orientation
remarkably more cytotoxic than the (R,S)-isomer. Fur- both, in the chair and the boat conformation.
thermore, the (R,R)-enantiomer displayed in SRB (growth
FA very complex mode of action was already discussed
inhibition assay using Sulforhodamine B staining tech- for [rac-N,N9-bis(salicylidene)-1,2-cyclohexanediamine]iro-
nique [12]) and colony-formation assays a significantly n(II/III). Generation of reactive oxygen species (ROS) or
greater cytotoxic activity toward MCF-7 cells than non- DNA interaction followed by induction of apoptosis indi-
tumorigenic MCF-10A breast cells and was an extremely cated by PARP cleavage experiments (PARP = an 116-kDa
efficient regulator of anti-apoptotic genes, Bcl-xL, Bcl-2, nuclear poly{ADP-ribose} polymerase, appears to be
and the cell cycle related gene, cyclin D1 [7].
involved in DNA repair and serves as a marker of cells
Exchange of the amine by an imine bond completely undergoing apoptosis [16]) would be a possible explana-
abolished the cytotoxic properties. The N,N9-bis(salicyli- tion for the cytotoxicity which is depended on the pres-
dene)-1,2-cyclohexanediamines 1–4 did not influence the ence of an iron center.
growth of MCF-7, MDA-MB 231, and HT-29 cells. This
The 3D structure of the [FeIII(saldach)Cl] complexes (see
effect might be the consequence of a reduced degree of Fig. 4) allows a DNA intercalation by the iron salene moi-
freedom which hinders the interaction with specific tar- ety. If this is realized, a clear difference in antiprolifera-
gets.
tive potency should be observed in the in-vitro assay (see
The imine bond and the salicyl effect caused a very Fig. 3). The racemic form and the separated (R,R)- and
rigid conformation with rotation possible only around (S,S)-enantiomers are plane structures and should be bet-
the N-C axis at the cyclohexane. The latter is prohibited ter attached to the DNA compared to the (R,S)-isomer. The
upon coordination to iron. The flexibility of the build cyclohexane ring, located perpendicular to the iron
five-membered iron ethylenediamine chelate is marginal salene, would hinder or even prevent the intercalation
and allows only an interconversion between a d- and a k- between nucleobases. From Fig. 3, however, it is obvious
form as it is well known from platinum(II) complexes. [3, that neither conformation nor configuration played a
13–15] A further dynamic process which is independent critical role for cell-growth inhibiting effects. Since DNA
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

628
A. Hille and R. Gust
Arch. Pharm. Chem. Life Sci. 2009, 342, 625–631
Figure 3. Time-
dependent analysis of
antiproliferative effects
of the [FeIII(saldach)Cl]
complexes 5, 6, 7 and 8
on the human MCF-7
and MDA-MB 231
breast cancer and the
HT-29 colon carcinoma
cell lines.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 625–631
Saldach Ligands and their Iron(III) Complexes
629
Figure 4. Conformations of the (R,R)-isomer 6 (above) and the (R,S)-isomer 8 (below); interconversion of the cyclohexane ring.
standard, TMS); EI-MS spectra, CH-7A Varian (70 eV; Varian Inc.,
Palo Alto, CA, USA); IR spectra (KBr pellets), Perkin-Elmer model
580 A (Perkin Elmer, USA). Elemental C, H, N analyses were car-
ried out with a Perkin-Elmer 240 B and 240 C elemental ana-
lyzer. Chemicals were obtained from Sigma Aldrich (Germany)
and were used as received.
is a chiral macromolecule and differentiates between
enantiomeric cytostatics, it is not the target of the com-
plexes 5–8 [17, 18].
Such considerations were subject of many SAR studies
on oxaliplatin and related compounds [3]. Although the
DNA is the accepted target of platinum complexes, it can
be excluded in the case of our iron salene complexes (see
also [8]). Cell-death promoting effects were unspecific,
depended on the presence of an iron centre and inde-
pendent of the conformation/configuration of the sal-
dach ligand.
Synthesis of ligands (method A)
Schiff bases were prepared involving reaction of salicylaldehyde
with the corresponding diamine (2:1 molar ratio) in ethanol.
The imines were filtered, washed with ethanol, dried over P2O5,
and used without further purifications.
It is well known that iron salene complexes are able to
generate reactive oxygen species (ROS) [8], able to damage
essential signal cascades in the cells. The generation of
ROS in MCF-7 was already verified for the racemic com-
plex 5. Therefore, in a further study, we will look for the
real intracellular target of iron salene complexes and try
to optimize the cytotoxic potency. If cell damage is
caused by redox reactions, it will be of interest to investi-
gate how substituents at the aromatic rings coordinated
to iron influence the redox behavior and the cytotoxicity.
First results with methoxy groups located at the salicyli-
dene moieties were promising [19].
Synthesis of ligands (method B)
Schiff bases were prepared according to a published procedure
with minor modifications [10]. The respective 1,2-diammonium-
cyclohexane mono-(+/–)-tartrate salt (4.60 mmol) and potassium
carbonate (9.95 mmol) were dissolved in a mixture of distilled
water (30 mL) and ethanol (10 mL). The mixture was heated with
salicylaldehyde (9.95 mmol) in ethanol (10 mL) under reflux for
2 h. Subsequently, the yellow slurry was cooled to 58C or lower
in an ice bad, filtered and dissolved in CH2Cl2 (30 mL). The
organic layer was washed twice with water (10 mL) and brine (20
mL), dried over Na2SO4, and the solvent was removed in vacuo. A
yellow solid was obtained after crystallization in the refrigera-
tor.
Synthesis of the complexes
Experimental
The complexes were prepared by reaction of an ethanolic solu-
tion of FeCl366 H2O with a boiling solution of the respective
Schiff base in ethanol (1:1 molar ratio) under reflux for 1 h. The
mixture was allowed to cool to room temperature, the precipi-
tated complexes were filtered off, washed with ethanol, and
dried over P2O5.
Chemistry
General procedures
The following instrumentation was used: H-NMR, Bruker ADX
400 spectrometer (Bruker Bioscience, USA) at 400 MHz (internal
1
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

630
A. Hille and R. Gust
Arch. Pharm. Chem. Life Sci. 2009, 342, 625–631
2H), 1.56–1.44 (m, 2H); MS (EI, 1008C) m/z: 322 (48%) [M+]. Anal.
calcd. for C20H22N2O2: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.39;
H, 6.85; N, 8.45.
rac-trans-N,N9-Bis(salicylidene)-1,2-cyclohexanediamine
1
Compound 1 was obtained from cis/trans-1,2-cyclohexanedi-
amine (3.00 g, 26.3 mmol) and salicylaldehyde (6.42 g, 52.5
mmol) (method A).
Iron(III) saldach complex 5
Yield: 4.76 g (14.76 mmol, 56%); crystalline yellow powder;
Compound 5 was obtained from rac-trans-N,N9-bis(salicylidene)-
1,2-cyclohexanediamine (100.00 mg, 0.31 mmol) and iron(III)
chloride hexahydrate (84.70 mg, 0.31 mmol).
20
m.p.: 114–1158C [20]; ½aꢀD = 0 (EtOH, c = 0.1), IR (KBr) m [cm–1]:
1
3433 br, 3011 w, 3068 w, 2993 m, 1630 s, 1501 m, 1280 s; H-
Yield: 90.00 mg (0.22 mmol, 70%); black solid; IR (KBr) m [cm–1]:
2924 m, 1612 s, 1543 m, 1312 m; MS (EI, 2758C) m/z: 411 (64%)
[M+], 376 (100%) [M+9–Cl], 56 (20) [Fe]. Anal. calcd. for C20H20N2O2-
FeCl: C, 58.35; H, 4.90; N, 6.80. Found: C, 58.52; H, 4.80; N, 6.82.
NMR (DMSO-d6) d: 13.33 (s, 2H, Ar-OH), 8.49 (s, 2H, NCH), 7.36–
7.32 (dd,3J = 7.7 Hz, 4J = 1.6 Hz, 2H, Hd), 7.29–7.25 (td,3J = 7.7 Hz, 4J
= 1.6 Hz, 2H, Hb), 6.85–6.80 (m, 3J = 7.5 Hz, 4J = 1.0 Hz, 4H, Ha + Hc),
3.42–3.36 (m, 2H), 1.90–1.79 (m, 4H), 1.63–1.61 (m, 2H), 1.48-1.43
(m, 2H); MS (EI, 758C) m/z: 322 (57%) [M+]. Anal. calcd. for
C20H22N2O2: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.52; H, 6.74; N,
8.53.
Iron(III) saldach complex 6
Compound 6 was obtained from (R,R)-N,N9-bis(salicylidene)-1,2-
cyclohexanediamine (103.00 mg, 0.32 mmol) and iron(III) chlor-
ide hexahydrate (86.00 mg, 0.32 mmol).
(R,R)-trans-N,N9-Bis(salicylidene)-1,2-
Yield: 101.00 mg (0.24 mmol, 77%); black solid; IR (KBr) m
[cm–1]: 2925 s, 1614 s, 1543 m, 1312 m; MS (EI, 2508C) m/z: 411
(64%) [M+], 376 (100%) [M+9–Cl], 56 (20) [Fe]. Anal. calcd. for
C20H20N2O2FeCl: C, 58.35; H, 4.90; N, 6.80. Found: C, 58.36; H,
5.19; N, 6.99.
cyclohexanediamine 2
Compound 2 was obtained from (R,R)-1,2-diammoniumcyclohex-
ane mono-(+)-tartrate salt (1.23 g, 4.65 mmol) and salicylalde-
hyde (1.21 g, 9.91 mmol) (method B).
20
Yield: 1.15 g (3.57 mmol, 77%); crystalline yellow solid; ½aꢀD
=
–444.0 (EtOH, c = 0.1); IR (KBr) m [cm–1]: 3453 br, 3052 w, 2935 m,
2856 m, 1630 s, 1497 m, 1278 s; 1H-NMR (CDCl3) d: 13.32 (s, 2H,
Ar-OH), 8.26 (s, 2H, NCH), 7.26–7.22 (td, 3J = 6.98 and 6.86 Hz, 4J =
1.12 and 1.21 Hz, 2H, Hb), 7.16–7.13 (dd, 3J = 7.70 and 7.60 Hz, 4J =
1.70 and 1.62 Hz, 2H, Hd), 6.89–6.87 (d, 3J = 8.27 Hz, 2H, Ha), 6.81–
6.77 (td, 3J = 6.63 and 7.52 Hz, 4J = 1.13 and 0.92 Hz, 2H, Hc), 3.35–
3.28 (m, 2H), 1.96–1.88 (m, 4H), 1.77–1.69 (m, 2H), 1.50–1.45 (m,
2H); MS (EI, 908C) m/z: 322 (60%) [M+]. Anal. calcd. for C20H22N2O2:
C, 74.51; H, 6.88; N, 8.69. Found: C, 74.69; H, 7.07; N, 8.78.
Iron(III) saldach complex 7
Compound 7 was obtained from (S,S)-N,N9-bis(salicylidene)-1,2-
cyclohexanediamine (157.00 mg, 0.49 mmol) and iron(III) chlor-
ide hexahydrate (130.80 mg, 0.49 mmol).
Yield: 79.00 mg (0.22 mmol, 70%); black solid; IR (KBr) m [cm–1]:
2936 m, 1612 s, 1544 m, 1312 m; MS (EI, 2758C) m/z: 411 (64%)
[M+], 376 (100) [M+9–Cl], 56 (20) [Fe]. Anal. calcd. for C20H20N2O2-
FeCl: C, 58.35; H, 4.90; N, 6.80. Found: C, 58.76; H, 5.38; N, 6.98.
(S,S)-trans-N,N9-Bis(salicylidene)-1,2-
cyclohexanediamine 3
Compound 3 was obtained from (S,S)-1,2-diaminocyclohexane
(0.22 g, 1.93 mmol) and salicylaldehyde (0.47 g, 3.86 mmol)
(method A).
Iron(III) saldach complex 8
Compound 8 was obtained from (R,S)-N,N9-bis(salicylidene)-1,2-
cyclohexanediamine (178.00 mg, 0.55 mmol) and iron(III) chlor-
ide hexahydrate (148.70 mg, 0.55 mmol).
Yield: 123.00 mg (0.30 mmol, 54%); black solid; IR (KBr) m
[cm–1]: 2928 w, 1612 s, 1543 m, 1301 m; MS (EI, 2758C) m/z: 411
(64) [M+], 376 (100) [M+9–Cl], 56 (20) [Fe]. Anal. calcd. for C20H20N2O2-
FeCl: C, 58.35; H, 4.90; N, 6.80. Found: C, 58.52; H, 4.80; N, 6.82.
20
Yield: 0.24 g (0.75 mmol, 39%); yellow solid; ½aꢀD = + 377.4
(EtOH, c = 0.1); IR (KBr) m [cm–1]: 3443 br, 3052 w, 2935 m, 2857 m,
1631 s, 1497 m, 1278 s. 1H-NMR (CDCl3) d: 13.32 (s, 2H, Ar-OH),
8.26 (s, 2H, NCH), 7.26–7.21 (td, 3J = 7.16 and 7.73 Hz, 4J = 1.47 Hz,
2H, Hb), 7.16–7.14 (dd, 3J = 7.64 Hz, 4J = 1.24 Hz, 2H, Hd), 6.90–6.87
(d, 3J = 8.23 H, 2H, Ha), 6.81–6.77 (td, 3J = 7.49 and 7.42 Hz, 2H, Hc),
3.38–3.26 (m, 2H), 1.96–1.82 (m, 4H), 1.77–1.72 (m, 2H), 1.54–1.43
(m, 2H); MS (EI, 908C) m/z: 322 (45%) [M+]. Anal. calcd. for
C20H22N2O2: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.69; H, 6.65; N,
8.45.
Biochemicals, chemicals, and materials
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 a
monolayer culture in L-glutamine containing Dulbecco’s Modi-
fied Eagle’s Medium (DMEM) with 4.5 g/L glucose (PAA Laborato-
ries GmbH, Austria), supplemented with 10% fetal calf serum
(FCS; Gibco, Germany) using 25 cm2 culture flasks in a humidi-
fied atmosphere (5% CO2) at 378C. The cell lines were passaged
twice a week after previous treatment with trypsin (0.05%)/ethyl-
enediaminetetraacetic acid (0.02% EDTA; Boehringer, Germany).
(R,S)-cis-N,N9-Bis(salicylidene)-1,2-cyclohexanediamine
4
Compound 4 was obtained from (R,S)-1,2-diaminocyclohexane
(0.96 g, 8.39 mmol) and salicylaldehyde (2.05 g, 16.80 mmol)
(method A).
Yield: 2.41 g (7.47 mmol, 89%); crystalline yellow solid; IR (KBr)
m [cm–1]: 3437 br, 3049 w, 2933 s, 2858 m, 1629 s, 1500 m, 1281 s;
1H-NMR (CDCl3) d: 13.41 (s, 2H, Ar-OH), 8.29 (s, 2H, NCH), 7.23–
7.21 (dd, 3J = 8.65 and 7.85 Hz,4J = 0.83 and 1.63 Hz, 2H, Hd), 7.19–
7.16 (td, 3J = 7.83 Hz, 4J = 1.68 and 1.42 Hz, 2H, Hb), 6.86–6.84 (d,
3J = 8.37 Hz, 2H, Ha), 6.80–6.77 (td, 3J = 7.06 and 7.22 Hz, 4J = 0.92
Hz, 2H, Hc), 3.56–3.54 (m, 2H), 1.95–1.80 (m, 4H), 1.73–1.68 (m,
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 [21, 22]. Exponential cell growth was ensured
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 625–631
Saldach Ligands and their Iron(III) Complexes
631
during the whole time of incubation. Briefly, 100 lL of a cell sus-
pension 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 378C
in a humidified atmosphere (5% CO2) for 3 d (MCF-7) and for 2 d
(MDA-MB 231 and HT-29), respectively. By removing the medium
and adding 200 lL of fresh medium containing an adequate vol-
ume of a stock solution of the metal complex, the desired test
concentration was obtained. Cis-platin was dissolved in DMF
while DMSO was used for all other compounds. Eight wells were
used for each test concentration and for the control, which con-
tained the corresponding amount of DMF and DMSO, respec-
tively. The medium was removed after reaching the appropriate
incubation time. Subsequently, the cells were fixed with a solu-
tion of 1% (v/v) glutaric dialdehyde in phosphate buffered saline
(PBS) and stored under PBS at 48C. Cell biomass was determined
by means of a crystal violet staining technique as described ear-
lier [21, 22]. The effectiveness of the complexes is expressed as
corrected T/Ccorr [%] or t [%] values according to the following
equations:
[3] M. Noji, K. Okamoto, Y. Kidani, J. Med. Chem. 1981, 24,
508–515.
[4] K. Okamoto, M. Noji, T. Tashiro, Y. Kidani, Chem. Pharm.
Bull. 1981, 29, 929–939.
[5] A. H. Braun, W. Achterrath, H. Wilke, U. Vanhoefer, A. Har-
strick, P. Preusser, Cancer 2004, 100, 1558–1577.
[6] J. J. Bonire, S. P. Fricker, J. Inorg. Biochem. 2001, 83, 217–
221.
[7] J. Gao, Y. Liu, Y. Zhou, L. M. Boxer, F. R. Woolley, ChemBio-
chem 2007, 8, 332–340.
[8] A. Hille, I. Ott, A. Kitanovic, I. Kitanovic, H. Alborzinia, E.
Lederer, S. Wꢀlfl, N. Metzler-Nolte, S. Schꢁfer, W. Shel-
drick, C. Bischof, U. Schatzschneider, R. Gust, J. Biol. Inorg.
Chem. 2009, 14, 711–725.
[9] J. Gao, Y. Liu, Y. Zhou, R. A. Zingaro, ChemMedChem 2007, 2,
1723–1729.
[10] J. Yang, Y. Yu, Q. Li, Y. Li, A. Cao, J. Polym. Sci. A 2005, 43,
373–384.
[11] F. Vꢀgtle, E. Goldschmitt, Chem. Ber. 1976, 109, 1–40.
cytostatic effect: T/Ccorr [%] = [(T – C0)/(C – C0)]6100
cytocidal effect: t [%] = [(T – C0)/C0]6100
(1)
(2)
[12] J. Gao, F. R. Woolley, R. A. Zingaro, J. Med. Chem. 2005, 48,
7192–7197.
[13] R. Gust, T. Burgemeister, A. Mannschreck, H. Schꢀnen-
berger, J. Med. Chem. 1990, 33, 2535–2544.
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 chroma-
tin-bound crystal violet extracted with ethanol (70%)) with C0
being the density of the cell extract directly 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.
[14] R. Gust, H. Schꢀnenberger, K. J. Range, U. Klement, Arch.
Pharm. 1993, 326, 967–976.
[15] R. Gust, H. Schꢀnenberger, Arch. Pharm. 1993, 326, 405–
413.
[16] F. J. Olivier, G. de la Rubia, V. Rolli, M. C. Ruiz-Ruiz, G.
Murcia, J. Mꢂnissier–de Murica, J. Biol. Chem. 1998, 273,
33533–33539.
The financial support of DFG – Deutsche Forschungsgemeinschaft (pro-
ject: FOR 630) is greatly appreciated.
[17] H. M. Er, T. W. Hambley, J. Inorg. Biochem. 2009, 103, 168–
173.
[18] Q. Yang, P. Yang, X. Qian, L. Tong, Bioorg. Med. Chem. Lett.
2008, 18, 6210–6213.
The authors have declared no conflict of interest.
[19] A. Hille, R. Gust, J. Inorg. Biochem., submitted.
[20] E. T. G. Cavalheiro, F. C. D. Lemos, Z. J. Schpector, E. R.
References
Dockal, Thermochim. Acta 2001, 370, 129–133.
[21] G. Bernhardt, H. Reile, H. Birnbꢀck, T. Spruss, H. Schꢀnen-
[1] Y. L. Bennani, S. Hanessian, Chem. Rev. 1997, 97, 3161–
berger, J. Cancer Res. Clin. Oncol. 1992, 118, 35–43.
3196.
[2] E. N. Jacobsen, W. Zhang, A. R. Muci, J. R. Ecker, L. Deng, J.
Am. Chem. Soc. 1991, 113, 7063–7064.
[22] H. Reile, H. Birnbꢀck, T. Spruss, H. Schꢀnenberger, Anal.
Biochem. 1990, 187, 262–267.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com