Amide and ester derivatives of chlorido[4-carboxy-1,2-disalicylideneaminobenzene]iron(iii) as necroptosis and ferroptosis inducers

Article abstract of DOI:10.1039/d0dt00168f

In continuation of the structure-activity study about 4-substituted chlorido[N,N'-disalicylidene-1,2-phenylenediamine]iron(iii) complexes as necroptosis and ferroptosis inducers, we introduced a 4-COOH group at the 1,2-phenylenediamine moiety of the lead

Full text of DOI:10.1039/d0dt00168f

View Article Online
View Journal
Dalton
Transactions
An international journal of inorganic chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: D. Bäcker, B. Ma,
J. Sagasser, L. Schultz, C. Hörschläger, M. Weinreich, L. Steiner, B. Kircher and R. Gust, Dalton Trans.,
2020, DOI: 10.1039/D0DT00168F.
Volume 46
Number 10
14 March 2017
Pages 3073-3412
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Dalton
Transactions
An international journal of inorganic chemistry
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
rsc.li/dalton
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
ISSN 0306-0012
COMMUNICATION
Douglas W. Stephen et al.
shall the Royal Society of Chemistry be held responsible for any errors
N-Heterocyclic carbene stabilized parent sulfenyl, selenenyl,
and tellurenyl cations (XH+, X = S, Se, Te)
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
rsc.li/dalton

Page 1 of 12
Dalton Transactions
Please do not adjust margins
View Article Online
DOI: 10.1039/D0DT00168F
ARTICLE
Amide and ester derivatives of chlorido[4-carboxy-1,2-disalicylide-
neaminobenzene]iron(III) as necroptosis and ferroptosis inducers
a
a
Daniel Baecker,‡ Benjamin N. Ma,‡ Jessica Sagasser,a Lukas Schultz,a Carina Hörschläger,a Maria
Received 00th January 20xx,
Accepted 00th January 20xx
Weinreich,a Lucy Steiner, b,c Brigitte Kircherb,c and Ronald Gust*a
DOI: 10.1039/x0xx00000x
In continuation of the structure-activity study about 4-substituted chlorido[N,N’-disalicylidene-1,2-phenylenediamine]-
iron(III) complexes as necroptosis and ferroptosis inducers, we introduced a 4-COOH group at the 1,2-phenylenediamine
moiety of the lead ([Fe(III)salopheneCl]) and derived the resulting complex 15 to the respective ethyl, propyl, or butyl amides
(16-18) and esters (19-21). The compounds 16-21 exerted concentration-dependent antiproliferative and antimetabolic
effects against HL-60 cells. The esters were more active than the analogous amides. Elongation of the alkyl chain enhanced
the activity of the amides, while that of the esters decreased. The complexes 16-21 induced necroptosis and/or ferroptosis
but not apoptosis. Studies on protein binding and uptake into HL-60 cells indicated that the complexes mainly accumulated
by passive transport. The high binding tendency of all complexes to apo-Transferrin, however, points to participation of a
carrier-mediated transport into the cells, too.
approach in tumor therapy.7 Ferrostatin (Fer-1) was already
identified as ferroptosis inhibitor and can be used to verify this effect.
Chlorido[N,N’-disalicylidene-1,2-phenylenediamine]iron(III)
([Fe(III)salopheneCl]) emerged as promising lead structure
representing an iron(III) complex with antileukemic properties. It was
feasible to overcome multiple drug resistance in leukemia cells
resistant to the cytostatics Vincristine and Daunorubicine8 and
showed also cytotoxicity against Cisplatin-resistant ovarian cancer
cells.9
In many cases, the approved analogous Carboplatin and
Oxaliplatin possess cross-resistance to Cisplatin, since they cause
antitumor activity by the same mode of action, the binding to the
DNA. The usage in a second line therapy is therefore impossible. The
strong but unselective reactivity against other biologically relevant
biomolecules is the reason for unwanted and less tolerable side
effects. Nevertheless, the great success of platinum drugs as
antitumor agents encouraged medicinal chemists to search for other
metal complexes in cancer research.
A reduction of toxicity leading to less adverse events as well as
overcoming of resistances by a mode of action different from that of
Cisplatin are hoped to be achieved by the use of endogenous and/or
biologically essential metals such as iron.10 Iron complexes were
already investigated as antitumor agents inducing cell death e.g. via
the induction of oxidative stress (ferroptosis or necroptosis).11,12
Introduction
Cell death either appears accidentally and uncontrolled or it
occurs following regulated mechanisms.1 In 1972, apoptosis was
described for the first time as pathway to cause programmed killing
of cells.2 The finding that apoptosis participates in physiological and
pathological processes enabled new approaches for therapeutic
applications, e.g. in the treatment of cancer.3 During the past years,
further non-apoptotic modalities, e.g. necroptosis (2005) and
ferroptosis (2012) were discovered.1
Necroptosis is a form of regulated necrotic cell death, mediated
by certain kinases, for example the receptor interacting protein
kinases 1 and 3 (RIPK-1/RIPK-3).4 It can be utilized to induce specific
annihilation of tumor cells and represents a promising therapeutic
approach for bypassing acquired or intrinsic apoptosis-resistance.5
Abrogation of antiproliferative effects by the specific necroptosis
inhibitor Necrostatin-1 (Nec-1), which suppresses phosphorylation of
RIP1 and interaction with RIP3, points to participation of this
pathway in the mode of action.
Ferroptosis is characterized by the iron-dependent generation of
reactive oxygen species (ROS) as part of lipid peroxidation.6 Specific
induction of ferroptosis in malignant cells is a new and hopeful
a. Department of Pharmaceutical Chemistry, Institute of Pharmacy, CMBI – Center for
Molecular Biosciences Innsbruck, University of Innsbruck, CCB – Center for Chemistry
and Biomedicine, Innrain 80-82, 6020 Innsbruck, Austria
b. Immunobiology and Stem Cell Laboratory, Department of Internal Medicine V
(Hematology and Oncology), Innsbruck Medical University, Anichstraße 35, 6020
Innsbruck, Austria
c. Tyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck, Austria
* E-mail: ronald.gust@uibk.ac.at; Tel: +43-512-507-58200
‡ These authors contributed equally to the publication.
Please do not adjust margins

Dalton Transactions
Please do not adjust margins
Page 2 of 12
ARTICLE
Journal Name
View Article Online
DOI: 10.1039/D0DT00168F
Scheme 1. Synthesis pathway of the amide precursors 2-4 and the ester precursors 5-7, the respective ligands 8-14, and the corresponding iron(III) salophene complexes 15-21.
In a previously published structure-activity relationship (SAR)
Results and discussion
study, we demonstrated the relevance of substituents in position 4
Synthesis
of the 1,2-phenylenediamine moiety at [Fe(III)salopheneCl] for the
The synthesis of the compounds covered three main steps
comprising the preparation of the precursors 2-7, the formation of
the ligands 8-14, and the coordination to iron(III) yielding the
complexes 15-21 (Scheme 1).
contribution of necroptosis or ferroptosis to the mode of action in
leukemic cell lines.12
To increase the structural flexibility, we introduced a 4-COOH
group (complex 15) and studied the influence of amidation and
esterification (ethyl, propyl, or butyl amides (16-18) and esters (19-
21)) on the induction of necroptosis and ferroptosis. Furthermore,
such derivation opens the possibility to bind the complexes to
biomolecules (peptides or proteins) or synthetic polymers such as
polyethylene glycols or dendrimers used for targeted drug delivery
systems.13,14
The antiproliferative and antimetabolic activity of 15-21 against
the acute myeloid leukemia cell line HL-60 was examined and the
contribution of apoptosis, necroptosis, and ferroptosis to the mode
of action was studied.
To obtain the amides 2-4, 3,4-dinitrobenzoic acid was activated
using thionyl chloride, followed by reaction with the respective
primary amine (R-NH2, R= ethyl, propyl, butyl) under basic
conditions, and reduction of the resulting 3,4-dinitrobenzamides
with hydrogen employing palladium on carbon as catalyst.
The analogous esters 5-7 were prepared by reaction of 3,4-
diaminobenzoic acid (1) with an excess of the respective alcohol (R-
OH, R= ethyl, propyl, butyl) under acidic conditions (sulfuric acid).
Condensation of the 1,2-phenylenediamines 1-7 with two
equivalents of salicylaldehyde yielded the salophenes 8-14. Finally,
complexation with iron(III) chloride according to published
procedures15 gave the complexes 15-21.
Additionally, the binding behaviour of the complexes to the iron
specific carrier protein apo-Transferrin (apo-Tf) and the uptake into
HL-60 cells were quantified to get more insight into the mode of
action of these iron(III) salophene complexes.
The precursors 2-7 and the ligands 8-14 were characterized by
1H-NMR and additionally by FT IR spectroscopy (Figures S3-S15) in
case of the salophenes.
The intramolecular H-bridge from the OH to the imine nitrogen
(salicylic effect) causes a broadening of the OH stretching vibration
in the IR-spectra of the ligands and results in a slightly visible OH
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 12
Dalton Transactions
Please do not adjust margins
Journal Name
ARTICLE
signal in the range 3400-2400 cm-1. The C=N vibration is located at even a complete inhibition was determined (0.3V%iewaAnrtdicle0O.n1li%ne,
1614-1609 cm-1.
respectively).
DOI: 10.1039/D0DT00168F
Due to their paramagnetism, characterization of the iron(III)
The ester derivatives 19-21 were more effective than the
salophene complexes 15-21 with 1H-NMR spectroscopy was not respective amide analogues (16-18). A correlation between activity
feasible. Therefore, they were characterized by FT IR spectroscopy and length of the alkyl chain also exists, but with opposed tendency
and high-resolution mass spectrometry (HR MS).
(Figure 1). The esters were even active at 1 µM and the cytotoxicity
In the IR-spectra, the broad stretching vibration of the ArO-H decreased in the order ethyl (1 µM: 43%; 3 µM: 18%) < propyl (1 µM:
disappeared, indicating the coordination of the phenolate to iron(III). 53%; 3 µM: 20%) < butyl (1 µM: 72%; 3 µM: 51%). The maximum of
Additionally, the Ar-O vibration changed to 1297-1314 cm-1. The activity was reached at 2 µM (19), 3 µM (20), and 10 µM (21) with a
stretching vibration of the salicylideneamino group was shifted upon proliferation rate of about 20% each.
coordination to 1604-1601 cm-1 and confirmed the building of an
Fe(III)N2O2 coordination sphere.
Cell death induction. The participation of early apoptosis as well as
The crystal structure of iron(III) salophene complexes documents late apoptosis/necrosis in the antiproliferative activity were
a distorted quadrangle pyramidal geometry as determined for examined by staining HL-60 cells with Annexin V conjugated to the
[Fe(III)salopheneCl]. In accordance with the literature16 the two green fluorescent dye fluorescein isothiocyanate (FITC) in
nitrogen and the two oxygen atoms of the ligand form the bottom of combination with propidium iodide (PI, red fluorescence) and
a pyramid, while the chlorido ligand builds its top. Because of the analysis by flow cytometry after 24 h of incubation with 3 µM of the
Fe(III)-Cl bond, the metal ion is positioned out of the N2O2 plane and complexes 15-21 (Figure 2).
represents the centre of a distorted tetraeder.
The 4-COOH derivative 15 (1.0-fold) as well as its amide
The purity was ascertained by HPLC employing a previously
published method,17 using a Chromolith®HighResolution RP-18e
column and an eluent of acetonitrile/25mM phosphate buffer
pH 3 (25/75). In case of 15 and 19 the usage of trifluoroacetic acid
(TFA) 0.1% and MeCN (78/22) as mobile phase allows an optimized
separation. All complexes showed purity higher than 98% (Figures
S16-S22).
Biological evaluation
Figure 2. Cell death induction by the compounds 15-21 (3 µM) against HL-60 cells. Cell
death induction in the absence of the complexes was set at 1. Asterisks represent
statistical significance (p < 0.05 vs. controls).
Antiproliferative activity. The antiproliferative effects were
examined using the acute myeloid leukemia cell line HL-60. Cells
were treated with increasing concentrations of the complexes 15-21
(0.5-20 µM) and the incorporation of [3H]-thymidine into DNA during
the S-phase of mitosis was assayed after an incubation time of 72 h.
The resulting radioactivity in the DNA serves as parameter for the
extent of mitotic cell division.18 The proliferation upon treatment
with the complexes 15-21 is depicted in Figure 1.
derivatives 16 (1.1-fold) and 17 (1.0-fold) did not induce early
apoptosis. The propyl amide 18 gave an almost negligible effect (1.5-
fold). Generally, apoptosis is indicated, if the staining is higher than
at least 3-fold intensified compared to an untreated control.
Slightly detectable apoptosis-involved cell death was measured
for the ester derivatives 19 (1.8-fold), 20 (2.2-fold), and 21 (2.1-fold).
However, these effects are too weak to consider apoptosis as cell
death-inducing mechanism.
Complex 15 bearing a 4-COOH group at the 1,2-phenylene-
diamine moiety did not influence the proliferation of HL-60 cells up
to the highest tested concentration of 20 µM (Figure 1).
Marginal late apoptosis/necrosis was monitored for 17 (2.5-fold)
and 18 (2.4-fold), while 15 (1.1-fold) and 16 (1.3-fold) did not show
noteworthy effects. The esters 19 (3.4-fold), 20 (2.7-fold), and 21
(1.8-fold) were slightly more active. The observed trend regarding
the length of the alkyl chain follows that of the proliferation assay.
Increasing the incubation time to 48 h did not change the late
apoptosis/necrosis rate of 15 and 16. In case of 17-21, a 4-5-fold
induction of cell death was found, indicating that necrosis might be
involved in the mode of action. In this experiment, the effects were
independent of the length of the alkyl chain (Figure 2).
Figure 1. Antiproliferative activity of the compounds 15-21 against HL-60 cells. The
proliferation in the absence of the complexes was set at 100%. Asterisks represent
statistical significance (p < 0.05 vs. controls).
Antimetabolic activity. The influence of the complexes 15-21
(concentration: 0.5-20 µM for 72 h) on the metabolic activity of
HL-60 cells was evaluated with a modified MTT assay. Such a cell
viability test principally relies on the capability of metabolically
competent cells to reduce tetrazolium salts (like the representative
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Amidation increased the antiproliferative potency of the
complexes. The ethyl derivative 16 was inactive at 3 µM (91%), but
reduced the proliferation at 10 µM to 26%. The propyl (17) and butyl
(18) amides showed distinctly higher activity and diminished the
thymidine uptake at 3 µM to 69% and 31%, respectively. At 10 µM
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Dalton Transactions
Please do not adjust margins
Page 4 of 12
ARTICLE
Journal Name
for instance) to a coloured formazan derivative, followed by
colorimetric analysis.19
The amide 18 exhibited low antimetabolic activity at the used
View Article Online
concentrations. The reduced activity at 5 µMD(tOoI:7100%.10)3w9a/Ds0aDbrTo0g0a16te8dF
A metabolic activity of 80-100% indicates no influence of the test partially by Nec-1 (79%) and completely by Fer-1, already at 1 µM
compounds. Complex 15 did not reduce the viability of the cells at all (Figure 4a). Administration of both inhibitors did not enhance the
used concentrations as well as 16-18 at 0.5-3 µM. The amides (16- effect of Fer-1 (Figure 4c).
18) decreased the activity at 10-20 µM to about 20%. The effect was
independent of the length of the N-alkyl chain (Figure 3).
These outcomes suggest
contribution of ferroptosis to the antitumor action. The other amide
a marginally more considerable
In contrast, the effects of the esters 19-21 follow the expected derivatives 16 and 17 exerted a comparable behaviour (Figures S24-
SAR (see antiproliferative activity). The most active derivative 19 S25, S28).
reduced the metabolic turnover to 38% at 1 µM and at higher
Complex 15, bearing a free 4-COOH group, showed slight
concentrations to the maximum of about 20%. Elongation of the alkyl antimetabolic properties at 5 µM, which were also revoked by the
residue of the ester led to complexes with less pronounced influence inhibitors Nec-1 and Fer-1, respectively. However, a preferred mode
on the cells. To reach the maximum effect (20-30%) concentrations of action with participation of necroptosis and ferroptosis cannot
of 10 µM or higher were needed.
unequivocally be deduced (Figures S23, S28-S29).
a
b
Figure 3. Antimetabolic activity of the compounds 15-21 against HL-60 cells. The
metabolic activity in the absence of the complexes was set at 100%. Asterisks represent
statistical significance (p < 0.05 vs. controls).
The cell death caused by the complexes was also investigated in
the presence of Fer-1 or/and Nec-1 to determine the participation of
necroptosis as well as ferroptosis in the mode of action. Fer-1 is a
radical-trapping antioxidant, which protects cells from lipid oxidation
by cytotoxic radicals.20 Nec-1 acts as inhibitor of the RIPK-121 and
preserves cells from RIP-mediated necroptosis.
c
According to an already published procedure, Fer-1 was used at
the concentrations of 1 µM and 3 µM, Nec-1 at 20 µM.12 Their
influence is most effectively studied up to a drug concentration of
5 µM. Therefore, the cells were treated for 72 h with the complexes
15-21 (0.1-5 µM) and in addition concomitantly with the inhibitors.
The data of 18 (most active amide derivative) and 19 (most active
ester derivative) are depicted in Figure 4. The results of the other
complexes are given in Figures S23-S27.
Complex 19 exhibited a concentration-dependent potency. At
1 µM, the metabolic activity was 25%. This effect can completely be
abrogated by Nec-1 (20 µM) and Fer-1 (3 µM). Lower concentrations
of Fer-1 (1 µM) did not influence the inhibitory potential of 19. At
higher concentrations of 19, Nec-1 and Fer-1 were not as effective.
At 5 µM, Nec-1 increased the activity to 50%, while Fer-1 (3 µM) led
only to 12% metabolically competent cells (Figure 4b).
This finding indicates that necroptosis and ferroptosis are not
independent of each other. Via both pathways the viability can be
restored at low drug concentrations. At higher concentrations,
contribution of necroptosis to the mode of action is dominant. A
similar profile was observed for the ester derivatives 20 and 21
(Figures S26-S27, S29).
Simultaneous administration of Fer-1 and Nec-1 with 19 did not
rise the capacity of Nec-1 (Figure 4c). At a drug concentration of
1 µM as well as 3 µM, nearly the same effects were discovered as
observed with Nec-1 alone.
Figure 4. Influence of the inhibitors Nec-1 and Fer-1, respectively, on the
antimetabolic activity of complex 18 (a), 19 (b), and (c) combination of 18 as well
as 19 with both inhibitors.
The antimetabolic activity of the complexes 15-21 was also
investigated using fibroblasts, which served as representatives for
non-cancerous cells. The effects (Figure S30) were comparable to
those observed against HL-60 cells. This fact indicates the necessity
of a targeted drug delivery into cancer cells, e.g. accomplished by the
connection of the complexes to dendrimers as suggested above.
Binding behaviour to proteins. It is basically considered that merely
free, unbound drug molecules are able to cross passively cell
membranes, unless a specific transport mechanism drives the
cellular uptake.22 The binding behaviour of drugs to proteins is hence
an essential criterion influencing both pharmacokinetic and
pharmacodynamic properties and should be examined to facilitate a
rational drug design.23
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 12
Dalton Transactions
Please do not adjust margins
Journal Name
ARTICLE
Human serum albumin is the major drug binding protein due to having an initial binding of about 58%, the derivative 19 finally
View Article Online
its widespread occurrence in the plasma.24 The general interaction of attained a high degree of 79%. The propyl derivative 20 revealed a
DOI: 10.1039/D0DT00168F
metal complexes with this protein was already reported before25 and constant behaviour (about 64%) over 300 min.
the binding to it was even utilized to accumulate drugs in cancer
cells.26
In summary, there is a clear relation of the binding profile of the
ester derivatives 19-21 to apo-Tf (ethyl > propyl > butyl). The amide
The uptake of iron is achieved by receptor-mediated endocytosis derivatives showed an attachment to the protein comparable to the
after iron is bound to its specific carrier protein apo-Tf.27 This process esters, but distinctly lower antiproliferative effects, which
was already suggested for the uptake of similar iron(III) salophene documents that apo-Tf does not influence the cytotoxic properties.
complexes into HL-60 cells.12
The binding of the complexes 15-21 to apo-Tf (Figure 5) as well Binding to the proteins in the cell culture medium. For the
as to the proteins (predominant albumin in FBS) contained in the interpretation of the in vitro results, it is of interest to know the
applied cell culture medium (Figure 6) was investigated. The amount of free unbound complexes. The attachment to proteins in
evaluation was performed indirectly by quantifying the unbound cell culture medium (30-55% after 60 min) was remarkably lower
amount in the supernatant using high-resolution continuum source compared to apo-Tf. The lead [Fe(III)salopheneCl] showed the lowest
atomic absorption spectrometry (HR CS AAS) after protein (20-30%), complex 15 with a polar 4-COOH the highest values. The
precipitation with ice-cold ethanol.
curve of 15 increased from initially 45% to 75% after 300 min
(Figure 6). Amidation changed the profile (Figure 6a). At the
Binding to apo-Transferrin. All amides 16-18 showed apo-Tf binding beginning, a relatively low association of about 20% was observed
ranging from 65% to 80%, which was reached with exception of 16 and reached the maximum after 180 min (17: 63%; 18: 43%) or 300
already after 10 min of incubation and maintained over the min (16: 75%).
observation time of 300 min (Figure 5a). The kinetic of 16 showed a
The results of the esters 19-21 (Figure 6b) were comparable to
relatively low binding (41%) at the beginning, which increased during that of the amides, however, with a faster initial onset. The maxi-
120 min to the maximum of 81%. The behaviour of the references mum effects were already obtained after 30 min and remained
[Fe(III)salopheneCl] and 15 were comparable after 10 min (68% and constant up to 300 min.
75%, respectively) and maintained for 300 min (65% and 78%).
a
a
b
b
Figure 6. Binding of 15  and the respective ethyl  , propyl , and butyl 
Figure 5. Binding of 15  and the respective ethyl , propyl , and butyl 
amides (a) and esters (b) to the proteins contained in the cell culture medium
amides (a) and esters (b) to apo-Tf , compared to [Fe(III)salopheneCl] .
compared to [Fe(III)salopheneCl] .
The initial association of the ester derivatives 19-21 to apo-Tf was
From these results it is obvious that the ethyl and propyl
very fast and the maximum was already reached after 10 min, stayed
derivatives bound to a higher extent to proteins in the medium than
constant for 300 min, and ranged between 50% and 80% (Figure 5b).
the butyl analogue. The ester 19-21 showed a lower binding
Complex 21 showed the lowest values (53% after 300 min). Though
compared to 15 but higher than the reference [Fe(III)salopheneCl].
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Dalton Transactions
Please do not adjust margins
Page 6 of 12
ARTICLE
Journal Name
The strong binding of 15 to the proteins present in the medium is the
consequence of ionic interactions, because esterification increased protein-bound complexes can reach the cellular environment by a
the amount of free and pharmacologically available drug molecules. passive transport. The binding to proteins in the medium correlates
The results further suggest that it is very likely that only free, non-
View Article Online
DOI: 10.1039/D0DT00168F
The presence of unbound or free drugs in the medium is an
essential prerequisite for the accumulation in cultured cells.
Therefore, the uptake into HL-60 cells was investigated.
with the poorer uptake of the complexes. An active transport, e.g.
mediated by the transferrin receptor as proposed for iron(III)
salophene complexes before12 cannot be excluded, but has to be
investigated more detailed in a forthcoming SAR study.
Cellular uptake. HL-60 cells were incubated with the iron(III)
salophene complexes (3 µM) up to 6 h and the cellular iron
concentration (related to the protein content) was quantified by HR
CS AAS (Figure 7) and compared with the lead [Fe(III)salopheneCl].
The latter displayed an uptake of 1.60 pmol µg-1 after 1 h and the
maximum of 2.84 pmol µg-1 after 2 h. This level marginally dropped
to 2.47 pmol µg-1 after 6 h.
The 4-COOH group strongly reduced the enrichment in the cells.
Only 0.63 pmol µg-1 were measured for 15 during the incubation
time of 1 h and 6 h, respectively. Derivatization to the amide 16 did
not change the behaviour of the complex (Figure 7a). The cellular
level was comparable to that of 15 with 0.44 pmol µg-1 (1 h) and
0.32 pmol µg-1 (6 h). Increasing the length of the N-alkyl chain from
ethyl (16) to propyl (17) enhanced the iron content in cells to
1.87 pmol µg-1 after 1 h, which then reduced to 1.20 pmol µg-1
(2-6 h). Complex 18 exhibited an accumulation profile identical to
that of the lead with the maximum of 4.02 pmol µg-1 after 2 h and a
decrease to 3.49 pmol µg-1 after 6 h.
The ester derivatives 19-21 (Figure 7b), which displayed a more
or less similar binding to apo-Tf and to proteins in the medium, acted
differently regarding the cellular uptake. All complexes caused a
higher cellular iron content than 15 and reached a maximum of about
2.5 pmol µg-1 after 2-4 h comparable to [Fe(III)salopheneCl]. This
level kept constant until the end of the experiment (6 h).
While the propyl ester 20 exhibited a very fast uptake
(2.13 pmol µg-1 after
1 h), the kinetics of 19 (ethyl ester:
0.73 pmol µg-1 after 1 h) and 21 (butyl ester: 1.38 pmol µg-1 after 1 h)
were somewhat slower. Nevertheless, the accumulation was
completed after 4 h.
In contrast to the amide derivatives 16-18, the uptake of the
esters 19-21 did not depend on the alkyl chain. All complexes were
taken up to the same amount. This fact correlates on the one hand
with the extent of free drug molecules, but also with their similar
antiproliferative and antimetabolic activity. Ester cleavage in the cell
culture medium leading to 15 cannot be excluded, but it seems to be
very unlikely, because the esters were higher accumulated than 15.
Nevertheless, the stability of the compounds was investigated on the
example of complex 19.
a
Stability in cell culture medium. Esterases and amidases occur
ubiquitously intracellular or in the serum.28-30 Therefore, 19 was
dissolved in the cell culture medium containing fetal bovine serum
a
b
15
19
b
Figure 7. Cellular uptake of 15  and the respective ethyl , propyl , and butyl 
amides (a) and esters (b) into HL-60 cells, compared to [Fe(III)salopheneCl] .
19
These findings showed that the restricted uptake of the 4-COOH
derivate is not the consequence of a negative charge under
physiological conditions. The enhanced uptake owing to the
elongation of the N-alkyl chain is in agreement with the increased
antiproliferative and antimetabolic activity. This demonstrates the
relevance of the lipophilicity of the complexes on the cell penetrating
properties and hence also on the biological activity.
Figure 8. Chromatograms of an equimolar mixture containing 15 and 19 (a) and of the
stability study of 19 after 18 h of incubation with FBS-containing cell culture medium
(b).
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 12
Dalton Transactions
Please do not adjust margins
Journal Name
ARTICLE
(FBS, 10%) as applied in the biological investigations within the (Merck Millipore, Billerica, USA) was applied. Column
View Article Online
DOI: 10.1039/D0DT00168F
chromatography: Silica gel 60 (particle size 40-63 μm). Thin layer
present study.
The complex was incubated for 0, 6, and 18 h, respectively, at chromatography: Polygram® SIL G/UV254 polyester foils coated with
37 °C with FBS-containing medium. At each time point, the enzyme 0.2 mm of silica gel with a fluorescence indicator (Macherey-Nagel,
activity was stopped by protein precipitation with ice-cold ethanol Düren, Germany); detection: under UV light at 254 and/or 365 nm.
(96%) and centrifugation. Analysis of the supernatant for either 19 or 1H nuclear magnetic resonance (1H-NMR) spectroscopy: Gemini
the 4-carboxy derivative 15 was performed by HPLC. The 2000 NMR Spectrometer (200 MHz, Varian, Paolo Alto, USA, now
optimization of the previously published procedure17 as described part of Agilent Technologies, Santa Clara, USA) or Bruker Avance 4
above ensures a baseline separation of both complexes (19 (tret
=
Neo Spectrometer (400 MHz); solvent: deuterated DMSO with
8.65 min); 15 (tret= 3.02 min), Figure 8a). Furthermore, the high tetramethylsilane as internal standard; the chemical shifts are given
solubility of 15 in ethanol guarantees that the possible degradation in parts per million (ppm) and the coupling constants in Hertz (Hz).
of 19 to 15 can be identified by the HPLC method.
Infrared (IR) spectroscopy: Alpha FT IR Spectrometer (Bruker,
The chromatograms obtained at each time point display only the Billerica, USA). High-resolution mass spectrometry (HR MS): Orbitrap
peak related to complex 19 (Figure 8b, Figures S31-S34) and indicate Elite (Thermo Fisher Scientific, Waltham, USA) via direct infusion and
that no enzymatic hydrolysis occurred. Thus, stability of the electrospray ionization (ESI). HPLC analyses: Shimadzu (Duisburg,
complexes during the cellular accumulation phase can be assumed.
Germany) provided with the autosampler SIL-20A HT, the pump LC20
AD, the column oven CT0-10AS VP, and the diode-array detector
(DAD) SPD-M20A with a deuterium and tungsten lamp; column:
Chromolith® HighResolution RP-18e (100×4.6 mm, Merck) column;
the purity of the final products was higher than 98% at the exemplary
wavelength of 310 nm. Measurements of absorbance within the
biological testing were performed with an EnSpire multimodal plate
reader (PerkinElmer Life Sciences, Waltham, USA).
Conclusions
Chlorido[N,N’-disalicylidene-1,2-phenylenediamine]iron(III)
complexes were developed to exert antitumor activity against
leukemia cells by the induction of cell death via necroptosis and
ferroptosis. With the purpose to extend the SAR study of 4-
substituted iron(III) salophene complexes, a 4-COOH group was
introduced and the resulting complex (15) was derived to ethyl,
propyl, or butyl amides (16-18) and esters (19-21), respectively.
Chemistry
General procedure for the synthesis of the 4-substituted N,N’-
disalicylidene-1,2-phenylenediamine ligands. 1 eq. of the respective
1,2-phenylenediamine (1-7) was dissolved under heating in EtOH
(giving a 0.1 M solution). Two eq. of salicylaldehyde were added and
the reaction mixture was refluxed for 3 h. The solvent was removed
in vacuo to obtain the respective ligands 8-14 after crystallization
overnight.31 The ligands were characterized by 1H-NMR and FT IR
spectroscopy and were readily applied for the synthesis of the
respective iron(III) salophene complexes.
The complexes 16-21 induced
a concentration-dependent
inhibition of proliferation and reduction of metabolic activity of HL-
60 cells. The effects of the amides 16-18, which were lower than that
of the esters, were improved by elongation of the N-alkyl chain. The
esters 19-21 revealed an inverse SAR.
The complexes 16-21 did not induce apoptosis, but necroptosis
and ferroptosis. In particular, the amides 16-18 rather caused
necroptosis than ferroptosis especially at higher concentrations. The
participation of ferroptosis in the mode of action was slightly
enhanced for the esters 19-21. The stability of the complexes against
hydrolysing enzymes such as esterase present in the cell culture
medium was confirmed based on the example of 19.
The uptake of the esters correlated with the non-protein-bound
fraction and in case of the amides with the length of the N-alkyl
chain. This finding points to a more pronounced contribution of a
passive accumulation. Nevertheless, because of the high binding
tendency of all complexes to apo-Tf, a transferrin receptor-mediated
uptake is also possible. This, however, needs to be confirmed in an
extended upcoming study.
The inactivity of complex 15 is a consequence of the low cellular
uptake, which is not caused by an anionic repulsion, since the ethyl
amide derivative 16 showed the same uptake kinetics.
3,4-Disalicylideneaminobenzoic acid (8). From 3,4-diaminobenzoic
acid (5 mmol, 0.61 g) and salicylaldehyde (10.2 mmol, 1.24 g). Orange
crystalline powder, yield 1.14 g (3.15 mmol, 63%). FT IR (ATR):

max/cm-1= 3200-2500 (b) COOH/Ar-OH, 1680 (s) C=O, 1613 (s) C=N,
1557 (s), 1263 (m) Ar-O, 752 Ar-H (s) oop. HR MS as C21H17N2O4+ m/z
calculated: 361.1183, found: 361.1272.
N-Ethyl-3,4-disalicylideneaminobenzamide (9). From N-ethyl-3,4-
diaminobenzamide (3.35 mmol, 0.60 g) and salicylaldehyde (6.7
mmol, 0.82 g). Orange crystalline powder, yield 0.992 g (2.56 mmol,
76%). FT IR (ATR): max/cm-1= 3300-2300 (b) ArOH, 3287 (m) –N=C-
OH, 3058 (w) aryl CH, 2977 (w), 2932 (w), 2872 (w) aliphatic CH, 1649
(m) N=C-O, 1609 (m) C=N, 1545 (s), 1278 (m) Ar-O, 755 Ar-H (s) oop.
1H-NMR (DMSO-d6): δ= 12.80 (s, 2H, Ar-OH), 9.01 (s, 1H,
azomethine), 8.99 (s, 1H, azomethine), 8.59 (t, 1H, CONH), 7.91 (s,
1H, Ar-H), 7.88 (s, 1H, Ar-H), 7.74-7.68 (m, 2H, Ar-H), 7.56-7.40 (m,
3H, Ar-H), 7.03-6.96 (m, 4H, Ar-H), 1.22-1.14 (t, 3H, CH3).
N-Propyl-3,4-disalicylideneaminobenzamide (10). From N-propyl-
3,4-diaminobenzamide (2.87 mmol, 0.56 g) and salicylaldehyde (5.74
mmol, 0.70 g). Orange crystalline powder, yield 0.841 g (2.56 mmol,
76%). FT IR (ATR) max/cm-1: 3300-2300 (b) ArOH, 3390 (m) –N=C-OH,
3064 (w) Ar-H, 2963 (w), 2926 (w), 2867 (w) aliphatic CH, 1650 (m)
Experimental section
General material and methods
All educts, solvents, or applied chemicals were purchased from
Sigma-Aldrich, TCI Chemicals, Fluka, or Alfa Aesar. The solvents were
obtained either in appropriate purity or were purified by distillation
prior to use. Water from a Gradient A10 water purification system
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Dalton Transactions
Please do not adjust margins
Page 8 of 12
ARTICLE
Journal Name
N=C-O, 1613 (m) C=N, 1539 (m), 1274 (m) Ar-O, 749 (s) Ar-H oop. 1H- complex (15-21) was formed immediately. To complete the reaction,
View Article Online
DOI: 10.1039/D0DT00168F
NMR (DMSO-d6): δ= 12.80 (s, 2H, Ar-OH), 9.01 (s, 1H, azomethine), the mixture was refluxed for 30 min. The solvent was evaporated to
8.98 (s, 1H, azomethine), 8.60-8.55 (t, 1H, CONH), 7.91 (s, 1H, Ar-H), obtain the dark-brown to black amorphous powder. The complexes
7.86 (s, 1H, Ar-H), 7.75-7.68 (m, 2H, ArH), 7.56-7.40 (m, 3H, Ar-H), were characterized with FT IR spectroscopy and HR MS in the positive
7.03-6.96 (m, 4H, Ar-H), 1.63-1.48 (qt, 2H, CH2), 0.96-0.88 (t, 3H, mode. The purity of the respective complexes was determined with
CH3).
a previous described HPLC method.17 The purity of 15 was
N-Butyl-3,4-disalicylideneaminobenzamide (11). From N-butyl-3,4- reconfirmed with a slightly optimized HPLC method, using a mobile
diaminobenzamide (1.21 mmol, 0.25 g) and salicylaldehyde (2.41 phase of TFA 0.1% and MeCN (78/22), while other conditions were
mmol, 0.30 g). Orange crystalline powder, yield 0.275 g (0.662 mmol, kept equal.
55%). FT IR (ATR): max/cm-1= 3300-2300 (b) ArOH, 3393 (w) -N=C-OH,
Chlorido[4-carboxy-1,2-disalicylideneaminobenzene]iron(III)
3062 (w) Ar-H, 2957 (w), 2927 (w), 2856 (w) aliphatic CH, 1651 (m) (15). From 8 (0.5 mmol, 181 mg) and anhydrous iron(III)
N=C-O, 1612 (m) C=N, 1535 (m), 1273 (m) Ar-O, 748 (s) Ar-H oop. 1H-
NMR (DMSO-d6): δ= 12.73 (s, 2H, Ar-OH), 9.01 (s, 1 H, azomethine),
8.98 (s, 1H, azomethine), 8.55 (t, 1H, CONH), 7.90 (s, 1H, Ar-H), 7.86
(s, 1H, Ar-H), 7.74-7.68 (m, 2H, Ar-H), 7.55-7.40 (m, 3H, Ar-H), 7.02-
6.96 (m, 4H, Ar-H), 1.58-1.26 (m, 4H, (CH2)2), 0.96-0.88 (t, 3H, CH3).
Ethyl-3,4-disalicylideneaminobenzoate (12). From ethyl-3,4-diamino-
benzoate (5 mmol, 0.901 g) and salicylaldehyde (10 mmol, 1.22 g).
Orange crystalline powder, yield 1.42 g (3.65 mmol, 73%). FT IR
(ATR): max/cm-1=3600-2400 (b) ArOH, 3054 (w) Ar-H, 2977 (w), 2937
(w), 2904 (w) aliphatic CH, 1716 (s) C=O, 1610 (s) C=N, 1558 (m), 1277
chloride (0.5 mmol, 82 mg). Dark-brown amorphous powder,
yield: 171 mg (0.380 mmol, 76%), purity: 99.98%. FT IR (ATR):

max/cm-1= 3500-2500 (b) COOH, 1681 (s) C=O, 1602 (s) C=N,
1574 (s), 1524 (s), 1288 (m) Ar-O, 757 (s) Ar-H oop, HR MS as
+
C21H14FeN2O4 , m/z calculated: 414.0298, found: 414.0374.
Chlorido[4-ethylaminocarbonyl-1,2-disalicylideneaminobenze-
ne]iron(III) (16). From 9 (0.5 mmol, 192 mg) and anhydrous
iron(III) chloride (0.5 mmol, 80 mg). Dark-brown amorphous
powder, yield: 51.0 mg (0.107 mmol, 21%), purity: 99.60%. FT IR
(ATR): max/cm-1= 3291 (w) N=C-OH, 3052 (w) Ar-H, 2973 (w),
2928 (w), 2869 (w) aliphatic CH, 1636 (m) N=C-O; 1601 (m) C=N,
1580 (s), 1533 (s), 1311 (m) C-O, 1193 (m), 1149 (m), 916 (w),
811 (w), 756 (s) Ar-H oop, 605 (m), 547 (m). HR MS as
1
(m) Ar-O, 1220 (m) C-O, 753 (s) Ar-H oop. H-NMR (DMSO-d6): δ=
12.66 (m, 2H, Ar-OH), 9.02 (s, 1H, azomethine), 8.98 (s, 1H,
azomethine), 7.98 (s, 1H, Ar-H), 7.95 (d, 1H, Ar-H), 7.75-7.70 (m, 2H,
Ar-H), 7.58-7.53 (d, 1H, Ar-H), 7.49-7.39 (m, 2H, Ar-H), 7.02-6.94 (m,
4H, Ar-H), 4.42-4.31 (q, 2H, CH2), 1.38-1.33 (t, 3H, CH3).
Propyl-3,4-disalicylideneaminobenzoate (13). From propyl-3,4-
diaminobenzoate (5 mmol, 0.973 g) and salicylaldehyde (10.1 mmol,
+
C23H19FeN3O3 , m/z calculated: 441.0770, found: 441.0853.
Chlorido[4-propylaminocarbonyl-1,2-disalicylideneaminobenze-
ne]iron(III) (17). From 10 (0.5 mmol, 199 mg) and anhydrous
iron(III) chloride (0.5 mmol, 85 mg). Dark-brown amorphous
powder, yield: 200 mg (0.408 mmol, 82%), purity: 99.47%; FT IR
1.23 g). Orange crystalline powder, yield 1.39 g (3.45 mmol, 69%). (ATR): max/cm-1= 3395 (w) N=C-OH, 3056 (w) Ar-H, 2962 (w),
FT IR (ATR): max/cm-1=2600-2400 (b) ArOH, 3056 (w) Ar-H, 2958 (w),
2938 (w), 2878(w) aliphatic CH, 1713 (s) C=O, 1613 (s) C=N, 1559 (m),
1277 (m) Ar-O, 1222 (m) C-O, 751 (s) Ar-H oop. 1H-NMR (DMSO-d6):
δ= 12.71 (m, 2H, Ar-OH), 9.02 (s, 1H, azomethine), 8.98 (s, 1H,
azomethine), 7.98-7.97 (d, 1H, Ar-H), 7.95-7.94 (d, 1H, Ar-H), 7.75-
7.70 (m, 2H, Ar-H), 7.58-7.54 (d, 1H, Ar-H), 7.49-7.39 (m, 2H, Ar-H),
7.03-6.95 (m, 4H, Ar-H), 4.32-4.25 (t, 2H, CH2), 1.82-1.71 (s6, 2H,
CH2), 1.03-0.96 (t, 3H, CH3).
2931 (w), 2872 (w) aliphatic CH, 1642 (m) N=C-O, 1601 (m) C=N,
1533 (s), 1378 (m), 1314 (m) Ar-O, 1193 (m), 1149 (s), 830 (m),
754 (s) Ar-H oop, 608 (m), 550 (m). HR MS as C24H21FeN3O3 , m/z
+
calculated: 455.0927, found: 455.1012.
Chlorido[4-butylaminocarbonyl-1,2-disalicylideneaminobenze-
ne]iron(III) (18). From 11 (0.5 mmol, 207 mg) and anhydrous
iron(III) chloride (0.5 mmol, 83 mg). Dark-brown amorphous
powder, yield: 100 mg (0.198 mmol, 40 %), purity: 99.38%; FT IR
(ATR): max/cm-1= 3378 (w) N=C-OH, 3056 (w) Ar-H; 2953 (w),
2871 (w) aliphatic CH, 1640 (m) N=C-O, 1602 (w) C=N, 1531 (s),
1378 (s), 1314 (m) Ar-O, 1192 (m), 1149 (m), 915 (w), 834 (w),
Butyl-3,4-disalicylideneaminobenzoate (14). From butyl-3,4-diami-
nobenzoate (5 mmol, 2.09 g) and salicylaldehyde (10 mmol, 1.23 g).
Orange crystalline powder, yield 1.37 g (2.70 mmol, 54%). FT IR
(ATR): max/cm-1=2600-2400 (b) ArOH, 3058 (w) Ar-H, 2956 (w), 2928
(w), 2870 (w) aliphatic CH, 1711 (s) C=O, 1614 (s) C=N, 1559 (m), 1278
+
754 (s) Ar-H oop, 605 (m), 551 (m). HR MS as C25H23FeN3O3 , m/z
calculated: 469.1083, found: 469.1170.
Chlorido[4-ethoxycarbonyl-1,2-disalicylideneaminobenzene]-
iron(III) (19). From 12 (0.5 mmol, 192 mg) and anhydrous
iron(III) chloride (0.5 mmol, 86 g). Dark-brown amorphous
powder, yield: 172 mg (0.36 mmol, 72%), purity: 100%. FT IR
(ATR): max/cm-1= 2977 (w), 2925 (w) aliphatic CH, 1707 (s) C=O,
1604 (s) C=N, 1573 (s), 1529 (s), 1370 (s), 1299 (s) Ar-O, 760 (s)
1
(m) Ar-O, 1220 (m) C-O, 753 (s) Ar-H oop. H-NMR (DMSO-d6): δ =
12.71 (m, 2H, Ar-OH), 9.02 (s, 1H, azomethine), 8.98 (s, 1H,
azomethine), 7.98-7.94 (m, 2H, Ar-H), 7.76-7.71 (m, 2H, Ar-H), 7.58-
7.54 (d, 1H, Ar-H), 7.440 (m, 2H, Ar-H), 6.99-6.96 (m, 4H, Ar-H), 4.36-
4.30 (t, 2H, CH2), 1.77-1.70 (tt, 2H, CH2), 1.50-1.39 (qt, 2H, CH2), 0.99-
0.92 (t, 3H, CH3).
+
Ar-H oop, 604 (m), 549 (m). HR MS as C23H18FeN2O4 , m/z
calculated: 442.0611, found: 442.0635.
Chlorido[4-propoxycarbonyl-1,2-disalicylideneaminobenzene]-
iron(III) (20). From 13 (0.5 mmol, 203 mg) and anhydrous
iron(III) chloride (0.5 mmol, 83 mg). Dark-brown amorphous
powder, yield: 169 mg (0.34 mmol, 69%), purity: 100%. FT IR
(ATR): max/cm-1= 2963 (w), 2933 (w), 2897 (w) aliphatic CH,
General procedure for the synthesis of the substituted chlorido[4-
carboxy-1,2-disalicylideneaminobenzene]iron(III) complexes. 1 eq.
of the respective ligand (8-14) was dissolved in MeCN under heating
to give a 0.1 M solution. An equimolar and freshly prepared solution
of anhydrous iron(III) chloride in MeCN was added and the respective
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 9 of 12
Dalton Transactions
Please do not adjust margins
Journal Name
ARTICLE
given as mean + SE of 4 independent experimeVnietws.ArAticslteeOrnisliknes
DOI: 10.1039/D0DT00168F
represent statistical significance (p < 0.05 vs. controls).
1711 (s) C=O, 1602 (s) C=N, 1577 (s), 1531 (s), 1297 (m) Ar-O,
750 (s) Ar-H oop, 603 (m), 549 (m). HR MS as C24H20FeN2O4 , m/z
+
calculated: 456.0767, found: 456.0794.
Determination of the antimetabolic activity. The antimetabolic
activity of the compounds was investigated in vitro using a modified
MTT assay (EZ4U kit, Biomedica, Vienna, Austria) according to the
manufacturer’s protocol. The cells were seeded into U-bottomed 96-
well plates in triplicates (5 × 104 cells in 50 µL per well). After
incubation for 1 h at 37 °C, the compounds were added reaching the
appropriate final concentrations. Depending on the experimental
approach, concomitant administration of Fer-1 and/or Nec-1 with
the compounds was conducted. After 72 h, the cells were examined
regarding their metabolic activity. To exclude the unspecific staining
generated by FBS-containing medium, the optical density of the cell
culture medium was subtracted. The final data are given as mean +
SE of 4 independent experiments. Asterisks represent statistical
significance (p < 0.05 vs. controls).
Chlorido[4-butoxycarbonyl-1,2-disalicylideneaminobenzene]-
iron(III) (21). From 14 (0.5 mmol, 210 mg) and anhydrous
iron(III) chloride (0.5 mmol, 84 mg). Dark-brown amorphous
powder, yield: 184 mg (0.36 mmol, 73%), purity: 99.57%. FT IR
(ATR): max/cm-1= 2960 (w), 2930 (w), 2897 (w) aliphatic CH,
1713 (s) C=O, 1603 (s) C=N, 1575 (s), 1531 (s), 1298 (m) Ar-O,
+
760 Ar-H oop, 605 (m), 551 (m). HR MS as C25H22FeN2O4 , m/z
calculated: 470.0924, found: 470.0956.
Biology
General cell culture methods. The acute myeloid leukemia cell line
HL-60 was purchased from the DSMZ, German Collection of
Microorganisms and Cell Cultures, Braunschweig, Germany and
cultured in RPMI 1640 without phenol red (BioWhittaker, Lonza,
Walkersville, USA), supplemented with fetal bovine serum (10%, FBS,
Biowest, Nuaillé, France), L-glutamine (2 mM), penicillin (100 U mL-1)
and streptomycin (100 µg mL-1; all from Invitrogen Coorporation
Gibco Paisley, Scotland). The HL-60 cells were cultivated in a
humidified atmosphere (37 °C, 5% CO2/95% air) and fed twice a
week. HL-60 cells were routinely monitored for mycoplasma
contamination (VenorGeM mycoplasma detection kit, Minerva) and
the authentication was confirmed by typing short tandem repeats.
Ferrostatin-1 (Fer-1), Necrostatin-1 (Nec-1) and human apo-
Transferrin (apo-Tf) were acquired from Sigma-Aldrich (Vienna,
Austria). The complexes were dissolved in DMSO and diluted with
cell culture medium to reach the test concentration.
Atomic absorption spectrometry measurements. A contrAA 700
high-resolution continuum source atomic absorption spectrometer
(Analytik Jena AG) was utilized for quantifying the complexes in
biological matrices based on the absorption of iron (λ =
248.3270 nm). A volume of 10 µL of the samples was injected
straight into coated standard graphite tubes (PIN-platform, Analytik
Jena AG), which were impregnated with a Zirconium AAS standard
solution before the first usage. The graphite furnace program as
reported previously was applied, which is characterized by a pyrolysis
and an atomization temperature of 1400 °C and 2250 °C,
respectively.12
Determination of the protein binding. The experiments were
performed according to methods described earlier.12,32 The binding
of the iron(III) salophene complexes (3 µM) to human apo-Tf
(4 mg mL-1) in PBS (pH 7.4) or to proteins of the medium used for all
cell culture experiments was examined. The samples were incubated
in a thermomixer (Eppendorf, Germany) at 400 rpm and 37 °C. After
the particular incubation periods (0, 10, 30, 60, 120, 180, 300 min),
an aliquot of 150 µL each was taken and diluted with ice-cold ethanol
(96%) in order to precipitate the proteins. Notably, the dark-coloured
complexes did not precipitate upon addition of ethanol. Thereafter,
the samples were stored at -20 °C overnight to enable continuing
protein precipitation, followed by centrifugation (4000 rpm, 10 min,
4 °C). An amount of 200 µL of the supernatant was removed and kept
at -20 °C until further analysis.
The extent of unbound complex was detected by quantification
of the iron content in the supernatant using HR CS AAS. Samples
containing no iron(III) salophene complex but only the solvent DMSO
served as blanks. Their absorbances were attributed to trace iron in
the set-up and they were subtracted from the values obtained when
investigating the complexes. The mean integrated absorbance of two
injections was used throughout the experiments. Pure samples of the
complexes served as standards and the calibrations were conducted
matching the matrix of the experiments when evaluating the binding
behaviour to the proteins. The percentage of binding to the proteins
was obtained by subtracting the quantified unbound extent from the
entire initial amount (3 µM), which was set at 100%. The presented
Determination of apoptosis/necrosis. HL-60 cells (5 × 104 cells in
50 µL per well) were incubated in duplicates in 96-well plates for 24
or 48 h with 3 µM of each complex. The potency of the compounds
to induce apoptosis/necrosis was determined by staining the cells
with Annexin V conjugated to the dye fluorescein isothiocyanate
(green fluorescent) in combination with propidium iodide (red
fluorescence), followed by analysis using flow cytometry (FACSCanto
II, Becton Dickinson, San José, USA). The final data are given as mean
+ SE of 4 independent experiments. Asterisks represent statistical
significance (p < 0.05 vs. controls).
Determination of the antiproliferative activity. The antiproliferative
activity of the complexes was evaluated in vitro using a [3H]-
thymidine incorporation assay. Exponentially growing cells were
seeded into U-bottomed 96-well plates with a density of 5 × 104 cells
in 50 µL per well. After incubation for 1 h, the compounds were
added to achieve the tested concentrations, respectively. For the last
12-16 h of incubation, each well was exposed to [3H]-thymidine
(2 Ci mmol-1, Hartmann Analytic, Braunschweig, Germany). Cells
were harvested using a semiautomated device and the [3H]-
thymidine uptake expressed in counts per minute (cpm) was
measured in a scintillation counter (Microbeta Trilux, Perkin Elmer,
Waltham, USA). The proliferation in the absence of the complexes
was set at 100% and the compound’s activity was calculated as
percentage of the control (without compound). The final data are
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Dalton Transactions
Please do not adjust margins
Page 10 of 12
ARTICLE
Journal Name
data express the mean ± SD of 2 independent conductions with two Statistical analysis. The Wilcoxon rank sum test was uVsieewd AtorticalenOalnyliznee
DOI: 10.1039/D0DT00168F
instrumental replicates, respectively.
the significance of differences between proliferation, metabolic
activity, and cell death induction in the absence and the presence of
a variable concentration of the investigated complexes (NCSS
software, Kaysville, UT, USA).
Cellular uptake studies. The studies were conducted following a
previously described procedure.12 HL-60 leukemic cells were seeded
in their exponentially growing phase into 6-well tissue culture plates
(1 × 106 cells in 1400 µL of medium per well). The cell suspension was
incubated for 2 h (37 °C, 5% CO2/95% air atmosphere) before adding
600 µL of medium containing the complex or vehicle (DMSO) leading
to a volume of 2000 µL and a concentration of 3 µM in each well. The
plates were incubated for 1, 2, 4, and 6 h, respectively. After the set
time points, the content of each well was transferred into a tube
followed by centrifugation (8000 rcf, 3 min, RT). The supernatant was
removed gently and the cell pellets were washed twice by
resuspending with PBS and centrifuging. The thus collected pellets
were kept at -20 °C until further analysis.
The cell pellets were resuspended in MilliQ water directly after
thawing and lysed by sonication (20 s, nine cycles, 80-85% power).
The lysates were quantified employing HR CS AAS based on
determining iron. Samples of the respective compounds dissolved in
DMSO were utilized as standards and calibration was conducted in a
matrix matched manner. The mean integrated absorbance of
triplicate injections was applied throughout the experiments. The
samples containing the lysates of cells that were incubated merely
with medium and the vehicle DMSO served as blank.
Abbreviations
apo-Tf
apo-Transferrin
DMSO
Dimethyl sulfoxide
eq.
Equivalent
ESI
EtOH
Electrospray ionisation
Ethanol
FBS
Fetal bovine serum
[Fe(III)salopheneCl]
Chlorido[N,N’-disalicylidene-1,2-
phenylenediamine]iron(III)
Ferrostatin-1
Fourier transform infrared
Acute myeloid leukemic cell line
High performance liquid chromatography
High-resolution continuum source atomic
absorption spectrometry
High-resolution mass spectrometry
Acetonitrile
3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
Necrostatin-1
Nuclear magnetic resonance
Phosphate buffered saline
Receptor interacting protein kinase 1/3
Reactive oxygen species
Rosewell Park Memorial Institute Medium
Room temperature
Fer-1
FT IR
HL-60
HPLC
HR CS AAS
HR MS
MeCN
MTT
Because the uptake is given as the amount of compound (pmol)
referred to the cellular protein mass (μg), protein quantification of
each sample was implemented following the protocol of Bradford.33
The final values of the cellular uptake were calculated as the mean ±
SD of 2-3 independent experiments.
Nec-1
NMR
PBS
RIPK-1/3
ROS
RPMI 1640
Quantification of protein content. In brief, dilutions of albumin
bovine fraction V prepared with MilliQ water (31.25-1000 μg mL-1) RT
SAR
SE
SD
TEA
TFA
Structure-activity relationship
Standard error
Standard deviation
Triethylamine
Trifluoroacetic acid
served as standards for calibration. 200 μL of Bradford reagent were
added to each 20 μL of the lysates (see above) and the calibration
standards in 96-well plates, respectively. After incubation for 5 min
at RT, the absorbance (λ= 595 nm) was measured with a microplate
reader (Thermo Scientific Multiskan GO). All the samples were
measured in duplicate.
Conflicts of interest
There are no conflicts to declare.
Stability study. For the stability study of 19 against enzymatic
hydrolysis, the complex was dissolved in DMSO (10 mM) and diluted
with FBS-containing medium (10%) to obtain a solution of 300 µM.
This solution was incubated for 0, 6, and 18 h at 37 °C. Inactivation of
the enzymes in an aliquot of the sample (333 µL) was achieved at
each time point by enzyme precipitation with ice-cold ethanol (96%,
667 µL). Following centrifugation (4000 rpm, 10 min, 4 °C) yielded a
supernatant that was analyzed by HPLC. The concentration of the
samples was sufficient (100 µM in case of no hydrolysis, blank,
medium without FBS) to quantify the complex and/or the respective
hydrolysis product. The HPLC method17 applied for the purity testing
of the complexes was slightly optimized with respect to the mobile
phase (TFA 0.1% and MeCN (78/22)) to warrant a baseline separation
of 19 and the possible hydrolysis product 15. For the comparison of
chromatograms, complex 15, 19, respectively, an equimolar mixture
of them, and medium itself were subject of the HPLC analysis.
Acknowledgements
The Austrian Research Promotion Agency FFG [West Austrian
BioNMR858017] is kindly acknowledged.
References
1
2
3
D. Tang, R. Kang, T. V. Berghe, P. Vandenabeele and G. Kroemer,
Cell Res, 2019, 29, 347-364.
J. F. Kerr, A. H. Wyllie and A. R. Currie, Br J Cancer, 1972, 26, 239-
257.
B. Favaloro, N. Allocati, V. Graziano, C. Di Ilio and V. De Laurenzi,
Aging, 2012, 4, 330-349.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 11 of 12
Dalton Transactions
Please do not adjust margins
Journal Name
ARTICLE
4
5
6
B. Shan, H. Pan, A. Najafov and J. Yuan, Genes Dev 2018, 32, 327-
View Article Online
DOI: 10.1039/D0DT00168F
340.
Y. Gong, Z. Fan, G. Luo, C. Yang, Q. Huang, K. Fan, H. Cheng, K. Jin,
Q. Ni, X. Yu and C. Liu, Mol Cancer, 2019, 18, 100, 1-17.
S. J. Dixon, K. M. Lemberg, M. R. Lamprecht, R. Skouta, E. M.
Zaitsev, C. E. Gleason, D. N. Patel, A. J. Bauer, A. M. Cantley, W.
S. Yang, B. Morrison and B. R. Stockwell, Cell, 2012, 149, 1060-
1072.
7
8
9
Y. Mou, J. Wang, J. Wu, D. He, C. Zhang, C. Duan and B. Li, J
Hematol Oncol, 2019, 12:34, 1-16.
S. Y. Lee, A. Hille, I. Kitanovic, P. Jesse, G. Henze, S. Wölfl, R. Gust
and A. Prokop, Leuk Res, 2011, 35, 387-393.
T. S. Lange, K. K. Kim, R. K. Singh, R. M. Strongin, C. K. McCourt
and L. Brard, PLoS One, 2008, 3, e2303.
10 C. P. Tan, Y. Y. Lu, L. N. Ji and Z. W. Mao, Metallomics, 2014, 6,
978-995.
11 T. Xu, W. Ding, X. Ji, X. Ao, Y. Liu, W. Yu and J. Wang, J Cell Mol
Med, 2019, 23, 4900-4912.
12 J. Sagasser, B. N. Ma, D. Baecker, S. Salcher, M. Hermann, J.
Lamprecht, S. Angerer, P. Obexer, B. Kircher and R. Gust, J Med
Chem, 2019, 62, 8053-8061.
13 T. Kapp, A. Dullin and R. Gust, Bioconjug Chem, 2010, 21, 328-
337.
14 D. Smilowicz and N. Metzler-Nolte, Dalton Trans, 2018, 21,
15465-15476.
15 A. Hille, I. Ott, A. Kitanovic, I. Kitanovic, H. Alborzinia, E. Lederer,
S. Wölfl, N. Metzler-Nolte, S. Schäfer, W. S. Sheldrick, C. Bischof,
U. Schatzschneider and R. Gust, J Biol Inorg Chem, 2009, 14, 711-
725.
16 Y. X. Zhou, X. F. Zheng, D. Han, H. Y. Zhang, X. Q. Shen, C. Y. Niu,
P. K. Chen, H. W. Hou and Y. Zhu, Synth React Inorg M, 2006, 36,
693-699.
17 S. El Deeb, B. N. Ma, D. Baecker and R. Gust, Inorg Chim Acta,
2019, 487, 76-80.
18 E. Vega-Avila and M. K. Pugsley, Proc West Pharmacol Soc, 2011,
54, 10-14.
19 P. Kumar, A. Nagarajan and P. D. Uchil, Cold Spring Harb Protoc,
2018, 2018, pdb.prot095505, 1-4.
20 O. Zilka, R. Shah, B. Li, J. P. Friedmann Angeli, M. Griesser, M.
Conrad and D. A. Pratt, ACS Cent Sci, 2017, 3, 232-243.
21 C. Zhuang and F. Chen, J Med Chem, 2019, 63, 1490-1510.
22 K. Vuignier, J. Schappler, J. L. Veuthey, P. A. Carrupt and S. Martel,
Anal Bioanal Chem, 2010, 398, 53-66.
23 G. L. Trainor, Exp Opin Drug Discov, 2007, 2, 51-64.
24 T. Bohnert and L.-S. Gan, J Pharm Sci, 2013, 102, 2953-2994.
25 T. Topală, A. Bodoki, L. Oprean and R. Oprean, Clujul Med, 2014,
87, 215-219.
26 E. Frei, Diabetol Metab Syndr, 2011, 3, 11, 1-4.
27 E. Gammella, P. Buratti, G. Cairo and S. Recalcati, Metallomics,
2017, 9, 1367-1375.
28 M. F. Montenegro, M. T. Moral-Naranjo, E. Munoz-Delgado, F. J.
Campoy and C. J. Vidal, FEBS J, 2009, 276, 2074-2083.
29 T. Uete, M. Asahara and H. Tsuchikura, Clin Chem, 1970, 16, 322-
330.
30 M. P. Hazenberg and H. de Visser, Eur J Clin Chem Clin Biochem,
1992, 30, 141-144.
31 E. Fischer and A. Speier, Ber Dtsch Chem Ges, 1895, 28, 3252-
3258.
32 D. Baecker, V. Obermoser, E. A. Kirchner, A. Hupfauf, B. Kircher
and R. Gust, Dalton Trans, 2019, 48, 15856-15868.
33 M. M. Bradford, Anal Biochem, 1976, 72, 248-254.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins

Dalton Transactions
Page 12 of 12
View Article Online
DOI: 10.1039/D0DT00168F
161x156mm (150 x 150 DPI)