Fluorination as tool to improve bioanalytical sensitivity and COX-2-selective antitumor activity of cobalt alkyne complexes

Article abstract of DOI:10.1039/c9dt03330k

The cobalt alkyne complex [(prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS) is an auspicious lead, which exhibits its anticancer activity mainly by inhibition of both cyclooxygenases (COX-1 and COX-2). Since COX-2 participates in carcinogenesis, a selective inhibition of that isoenzyme is aimed. To study if fluorination increases the COX-2/COX-1 inhibition ratio of the lead, substitution was respectively performed in the positions 3, 4, 5, and 6 of the aromatic moiety. The complexes 3/4/5F-Co-ASS and to a much lower extent also 6F-Co-ASS showed cytotoxic, antimetabolic, and apoptotic effects in COX-1/2-positive HT-29 and MDA-MB-231 cells and remarkably less activity in the COX-1/2-negative MCF-7 cell line. The metabolic activity in MCF-7 cells was even unaffected up to a concentration of 40 μM. With exception of 6F-Co-ASS, the complexes strongly reduced the PGE₂ synthesis in HT-29 cells and all complexes inhibited COX-2 more effectively than COX-1 in an assay at isolated enzymes. These findings point to an interference in the COX cascade as part of the mode of antitumor action. The limited cellular effects of 6F-Co-ASS are related to its poor uptake as determined by HR CS AAS/MAS. Moreover, the cellular uptake studies confirm fluorination as beneficial tool for bioanalytical labeling. The higher quantification of fluorine by HR CS MAS makes this method about 5-fold more sensitive than HR CS AAS measuring cobalt. As a further positive result, 3/4/5/6-Co-ASS demonstrated high selectivity to tumor cells due to lack of antimetabolic activity against the non-tumorigenic bone marrow stromal cell line HS-5.

Full text of DOI:10.1039/c9dt03330k

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Fluorination as tool to improve bioanalytical
sensitivity and COX-2-selective antitumor activity
of cobalt alkyne complexes†
Cite this: Dalton Trans., 2019, 48,
15856
Daniel Baecker,a Victoria Obermoser,a Elisabeth Anna Kirchner,a Andrea Hupfauf,a
a
Brigitte Kircher b,c and Ronald Gust
*
The cobalt alkyne complex [(prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS) is an aus-
picious lead, which exhibits its anticancer activity mainly by inhibition of both cyclooxygenases (COX-1
and COX-2). Since COX-2 participates in carcinogenesis, a selective inhibition of that isoenzyme is aimed.
To study if fluorination increases the COX-2/COX-1 inhibition ratio of the lead, substitution was respect-
ively performed in the positions 3, 4, 5, and 6 of the aromatic moiety. The complexes 3/4/5F-Co-ASS and
to a much lower extent also 6F-Co-ASS showed cytotoxic, antimetabolic, and apoptotic effects in
COX-1/2-positive HT-29 and MDA-MB-231 cells and remarkably less activity in the COX-1/2-negative
MCF-7 cell line. The metabolic activity in MCF-7 cells was even unaffected up to a concentration of 40 µM.
With exception of 6F-Co-ASS, the complexes strongly reduced the PGE2 synthesis in HT-29 cells and all
complexes inhibited COX-2 more effectively than COX-1 in an assay at isolated enzymes. These findings
point to an interference in the COX cascade as part of the mode of antitumor action. The limited cellular
effects of 6F-Co-ASS are related to its poor uptake as determined by HR CS AAS/MAS. Moreover, the cellular
uptake studies confirm fluorination as beneficial tool for bioanalytical labeling. The higher quantification of
fluorine by HR CS MAS makes this method about 5-fold more sensitive than HR CS AAS measuring cobalt. As
a further positive result, 3/4/5/6-Co-ASS demonstrated high selectivity to tumor cells due to lack of antimeta-
bolic activity against the non-tumorigenic bone marrow stromal cell line HS-5.
Received 15th August 2019,
Accepted 19th September 2019
DOI: 10.1039/c9dt03330k
rsc.li/dalton
discovered to exert therapeutic activities,4,5 especially as che-
motherapeutics due to antiproliferative and antimicrobial
Introduction
Cobalt is a trace element essential for various biological pro- properties.1,6,7
cesses. It is mainly present in our body in the form of cobala-
Considering the lower toxicity of cobalt compared to platinum
min (vitamin B12), which acts as a cofactor for several and the minor expenses for the design of new cobalt complexes,
enzymes.1 Vitamin B12 is important since it contributes to it was found to be very attractive to investigate cobalt containing
physiological functions, e.g. the nervous system and the organometallic compounds as alternatives to the antitumor
brain.2 Interestingly, the counterpart of vitamin B12, the so- agents Cisplatin, Carboplatin, and Oxaliplatin.8,9
called antivitamins B12 were attributed to exhibit the capa-
Due to the different coordination mode of the ligands in
bility as anticancer agents.3 Moreover, cobalt complexes were metal complexes (e.g. platinum complexes) and organometallic
compounds it is very likely that intrinsic and acquired resis-
tance of tumor cells against platinum complexes may be cir-
cumvented and side-effects caused by platinum-based drugs
aDepartment of Pharmaceutical Chemistry, Institute of Pharmacy, CMBI – Center for
Molecular Biosciences Innsbruck, University of Innsbruck, CCB – Center for
can be minimized.
Chemistry and Biomedicine, Innrain 80-82, 6020 Innsbruck, Austria.
Therefore, research was intensified to extend the potential
E-mail: ronald.gust@uibk.ac.at; Tel: +43-512-507-58200
of cobalt complexes as anticancer drugs.10–14 Rational drug
design also aims to induce a mode of action distinct from
platinum complexes by introduction of a biologically active
ligand.
bImmunobiology and Stem Cell Laboratory, Department of Internal Medicine V
(Hematology and Oncology), Innsbruck Medical University, Anichstraße 35, 6020
Innsbruck, Austria
cTyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck, Austria
†Electronic supplementary information (ESI) available: Synthesis and character-
ization of intermediate products; graphite furnace time–temperature programs;
tabular data of COX-1/2 inhibition; IR and 1H NMR spectra of the ligands and
the complexes. See DOI: 10.1039/c9dt03330k
Following this approach, the combination of dicobalthexa-
carbonyl with cyclooxygenase (COX) inhibiting non-steroidal
anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid
15856 | Dalton Trans., 2019, 48, 15856–15868
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(ASS) led to the development of the lead [(prop-2-ynyl)-2-acet- was studied against COX-1/2-positive colon (HT-29) and breast
oxybenzoate]dicobalthexacarbonyl (Co-ASS) by our group.15–18 cancer (MDA-MB-231) cell lines. The COX-1/2-negative MCF-7
Prior structure activity relationship (SAR) studies focused cell line was used as a reference.
on the dicobalthexacarbonyl moiety to optimize the pharma-
The characterization was completed by cellular uptake
cological profile of Co-ASS. Higher carbonyl clusters of studies, which covered the sequential analysis of both cobalt
cobalt or ruthenium did not enhance the cytotoxic effects. The and fluorine using high-resolution continuum-source atomic
use of iron cluster even entirely diminished the activity.19 respectively molecular absorption spectrometry (HR CS AAS/
Moreover, modifications using manganese, cobalt, moly- HR CS MAS).
bdenum, and rhodium containing [cyclopentadienyl]metal-
carbonyl cluster also failed to increase the growth inhibitory
potency. Therefore, the [alkyne]dicobalthexacarbonyl scaffold
Results and discussion
Synthesis
is the best choice to be combined with a bioactive NSAID
ligand.20
The synthesis (Scheme 1) started from the respectively fluo-
rine-substituted salicylic acids (SS). As educts, the 3F-, 4F-, and
5F-SS derivatives (3/4/5F-SS) were commercially available. The
6-fluorosalicylic acid (6F-SS) had to be synthesized by reaction
of 2,6-difluorobenzoic acid with powdered sodium hydroxide
under anhydrous conditions using dimethyl sulfoxide (DMSO)
as solvent. The selective exchange of one fluorine substituent
by a hydroxyl group yielded 6F-SS.36
Subsequent acetylation with acetic anhydride and triethyl-
amine (TEA) in tetrahydrofuran (THF) resulted in the acetyl-
salicylic acids (3/4/5/6F-ASS), which were further esterified
with propargyl alcohol to give propargyl-3/4/5/6F-ASS, respect-
ively. N,N′-Dicyclohexylcarbodiimide (DCC) as an activating
agent and 4-(dimethylamino)pyridine (DMAP) as catalyst were
applied in this reaction.36 Related propargyl fluorosalicylates
arose as by-products, which can be reacetylated without prior
separation from the ASS ester.
The final synthesis of the respective dicobalthexacarbonyl
complexes (3/4/5/6F-Co-ASS) was performed by reaction of the
propargyl-3/4/5/6F-ASS with dicobaltoctacarbonyl in absolute
THF under an argon atmosphere.37 All complexes were charac-
terized by NMR, FT-IR and HR MS analogously to prior
studies.15,19,20,23 The purity was determined by HPLC in each
case and was higher than 95% (except where noted).
The results of the above mentioned studies also assure that
inhibition of COX-1/2 is the plausible mode of action of Co-
ASS,21 while it might also take its effect otherwise, e.g. as a CO
releasing molecule (CORM). Due to the pronounced inter-
action of Co-ASS with the isoenzyme COX-1, which is generally
associated with limiting side-effects in the therapy applying
NSAIDs, a selectivity shift towards COX-2 is desirable. COX-2 is
the isoenzyme involved in the development of cancer and it
therefore represents a favorable target.22 An approach to shift
the selectivity towards COX-2 is the utilization of the larger
binding cave of COX-2 and the attachment at selective amino
acids as already performed in the design of COX-2 selective
NSAIDs. Therefore, the ASS moiety of Co-ASS was chlorinated
and indeed the complexes showed higher inhibitory potency
against COX-2.23
In continuation to this SAR study, we exchanged chlorine
by fluorine. Fluorination is a frequently used strategy in medi-
cinal chemistry.24–27 Fluorine influences the intrinsic potency,
the metabolic stability, and the binding affinity of potential
drugs to target proteins. It further increases the membrane
permeability of a drug due to altered conformation, lipophili-
city, and basicity.28,29
This concept was applied to Co-ASS resulting in the deriva-
tives 3F-Co-ASS, 4F-Co-ASS, 5F-Co-ASS, and 6F-Co-ASS. Beyond
the impact of fluorination on pharmacokinetic and physico-
chemical properties, fluorine can also be used with the objec-
tive of labeling.
Biological evaluation
COX-1/2 isoenzyme inhibition. The inhibition of the target
The most famous labeling using fluorine represents the enzymes COX-1/2 was investigated initially at the isolated pro-
utilization of the isotope F18, which is detectable by positron teins. ASS served as a reference and acts as NSAID by inhi-
emission tomography.30 However, this technique is limited by bition of both COX isoenzymes though exhibiting preferential
the relatively short half-time (109.8 min) of the nuclide. Owing COX-1 selectivity.38
to the advancement of high-resolution continuum-source
ASS showed at the concentration of 10 μM merely a slight
molecule absorption spectrometry (HR CS MAS), the analysis inhibition of COX-1 (28.8%) and no effect towards COX-2
of some non-metals is feasible,31 e.g. fluorine being quantified (0.6%). The inhibitory effect was enhanced at the concen-
by the molecular absorption of gallium mono-fluoride tration of 100 μM to 73.3% (COX-1) and 59.4% (COX-2).
(GaF).32,33 Analytical methods for the determination of fluo-
Fluorination of ASS slightly increased the inhibition (at
rine containing drugs in biological samples were elaborated by 100 μM) of COX-1 in case of 4F-ASS to 85.4%, while the activity
our group34,35 and herewith applied for the investigations of of 5F-ASS and 6F-ASS was reduced to 50.4% and 40.5%,
the fluorinated cobalt alkyne complexes.
respectively. Interestingly, 3F-ASS was completely inactive
The present study includes the synthesis of the fluorinated (1.6%). At COX-2 all compounds showed nearly the same
compounds and the evaluation of their potential to inhibit iso- inhibitory properties in the range of 43–51%. Transformation
lated COX isoenzymes and the PGE2 production in HT-29 cells. to the propargyl esters decreased the activity at COX-1
Their antiproliferative, antimetabolic and apoptotic activity (15–30%), while this structural modification had no influence
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Scheme 1 Synthesis route of the propargyl-3/4/5/6F-ASS and the related dicobalthexacarbonyls 3/4/5/6F-Co-ASS. Ar, argon; DCC, N,N’-dicyclo-
hexylcarbodiimide; DCM, dichloromethane; DMAP, 4-(dimethylamino)pyridine; DMSO, dimethyl sulfoxide; RT, room temperature; TEA, triethylamine;
THF, tetrahydrofuran.
on the effects at COX-2 (44–50%). Compared to propargyl-ASS tive up to a concentration of 100 µM (only ASS and propargyl-
(34.5%) the effects at COX-2 slightly increased upon fluorina- ASS are exemplarily shown in Table 1). Co-ASS reduced the cell
tion (see Fig. 1a and ESI, Table S3†).
mass of HT-29 (IC50 = 1.15 µM) more effectively than Cisplatin
As already observed for the lead Co-ASS, the inhibitory (IC50 = 3.52 µM). Against the breast cancer cell lines, Co-ASS
potency 10-fold increased upon coordination to dicobalthexa- had minor activity (MDA-MB-231: IC50 = 10.1 µM; MCF-7: IC50
carbonyl in case of propargyl-3/4/5/6F-ASS, too (Fig. 1b). At = 16.4 µM).
10 µM, Co-ASS inhibited COX-1 and COX-2 to the same extent
Fluorine substituents at position 3 (IC50 = 2.78 µM), 4 (IC50
(COX-1: 82.7%; COX-2: 78.5%). Fluorination of the lead dis- = 3.33 µM) or 5 (IC50 = 1.73 µM) of the ASS moiety of Co-ASS
tinctly influenced the inhibitory effect on the isoenzymes, only marginally influenced the effects at the HT-29 cell line,
independent of the position of the fluorine substituent at the while 6F-Co-ASS with an IC50 = 20.2 µM was 10-fold less active
aromatic ring. The inhibition of COX-1 was reduced to about than its isomers.
38–45%, while the inhibition of COX-2 remained nearly
unchanged (67–74%). Thus, fluorination affected a selectivity However, 5F-Co-ASS (IC50 = 5.33 µM) was more active than Co-
shift towards COX-2. ASS. The activity of 3F-Co-ASS (IC50 = 10.5 µM) and 4F-Co-ASS
The same trend was observed at the MDA-MB-231 cell line.
Antiproliferative effects in cancer cell lines. The antiproli- (IC50 = 9.23 µM) was identical to Co-ASS. The 6F isomer
ferative activity caused by the fluorinated cobalt alkyne com- (6F-Co-ASS) was the less potent derivative (IC50 = 33.9 µM)
plexes was evaluated in vitro using a crystal violet assay.23 As again.
an indication of the influence of the compounds on the
Interestingly, the antiproliferative effects towards MCF-7
growth of COX-1/2 expressing HT-29 and MDA-MB-231 cells as cells were equal for all complexes, independent of the presence
well as COX-negative MCF-7 cells, the cell biomass was quanti- and the position of the fluorine substituent. The IC50 values of
fied via chromatin staining after 72 h of incubation.
16.3–24.6 µM further document that the effects are minor in
The precursors and the cobalt alkyne complexes were cells lacking COX-1/2 (MCF-7).
included in this study. Cisplatin served as positive control
(Table 1).
Antimetabolic activity in cancer cell lines. To get a closer
look into the mechanism of cell death, the metabolic activity
Cisplatin as unselective antitumor agent reduced the of the cells was quantified upon treatment with the complexes.
growth independent of the cell lines used with IC50 The MTT assay is a colorimetric assay, that is based on the
3.5–4.0 µM. The ASS and propargyl-ASS derivatives were inac- ability of metabolically active cells to reduce 3-(4,5-dimethyl-
=
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Fig. 1 Inhibition of isolated COX-1 and COX-2 caused by (a) the
organic precursors [100 µM] and (b) the cobalt complexes [10 µM]. The
values are given as % COX inhibition calculated from the initial activity of
vehicle treated enzyme. The values represent the mean + SD of 3 inde-
pendent experiments.
Fig. 2 Antimetabolic activity of the cobalt alkyne complexes after 72 h
of incubation against (a) HT 29, (b) MDA-MB-231, and (c) MCF 7 cells.
The values represent the mean + SD of ≥4 independent experiments.
Table 1 Inhibition of cell-growth determined using a crystal violet cell
biomass assay. The IC50 values represent the mean SD of ≥3 indepen-
dent experiments
as most potent compound reduced the metabolic activity at
40 µM to 7%, while the lead diminished it to only 26%. 6F-Co-
ASS had no influence on the cells.
MDA-MB-231 cells were more sensitive to the complexes.
The inhibitory properties increased in the series 4F-Co-ASS <
3F-Co-ASS < Co-ASS < 5F-Co-ASS. 6F-Co-ASS was inactive (see
Fig. 2b). 5F-Co-ASS caused a reduction to 9% at 40 µM and
34% at 20 µM.
As already determined in the crystal violet assay, COX-1/
2-negative MCF-7 cells are not as sensitive as MDA-MB-231 or
HT-29 cells to the treatment with the complexes. The meta-
bolic activity remained unaffected (Fig. 2c). After exposure of
the cells to 40 µM of the complexes, which was the highest
concentration used within this study, the metabolic activity
was still 87% for 3F-Co-ASS, 86% for 4F-Co-ASS, 96% for
IC50 [µM]
Compound
HT-29
MDA-MB-231
MCF-7
ASS
≥100
≥100
≥100
≥100
≥100
≥100
Propargyl-ASS
3F-Co-ASS
4F-Co-ASS
5F-Co-ASS
6F-Co-ASS
Co-ASS
2.78 0.57
3.33 0.60
1.73 0.34
20.2 5.7
1.15 0.10
3.52 0.48
10.5 1.0
9.23 0.83
5.33 0.37
33.9 3.4
10.1 0.6
3.57 1.06
24.6 5.0
16.3 1.1
18.2 1.7
16.6 2.4
16.4 1.4
4.02 1.11
Cisplatin
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its 5F-Co-ASS and 95% for 6F-Co-ASS. Even the lead structure Co-
purple-colored formazan derivative, which is purple. The ASS did not influence the metabolic activity of MCF-7 cells.
absorbance measured at 570 nm correlates with the number of
metabolically active cells.39
Antimetabolic activity in human bone marrow stromal cells.
The human bone marrow stromal cell line HS-5 was used as
The complexes showed in this assay a concentration-depen- an example to evaluate the effects of the fluorinated cobalt
dent reduction of the metabolic activity in HT-29 cells alkyne complexes on the metabolic activity of a non-tumor cell
(Fig. 2a). 3F-Co-ASS and 4F-Co-ASS achieved a 50% reduction line. All complexes did not show antimetabolic activities
at 40 µM, 5F-Co-ASS and Co-ASS already at 20 µM. 5F-Co-ASS (Fig. 3) at the tested concentrations.
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Inhibition of PGE2 synthesis. The high sensitivity of HT-29
and MBA-MB-231 cells upon treatment with the complexes
compared to the MCF-7 cell line might be due to the presence
of COX-1/2. Therefore, the influence of the complexes on the
prostaglandin E2 (PGE2) level as parameter of cellular COX-1/2
inhibition was determined on the example of HT-29 cells.23
The isoenzymes COX-1 and COX-2 catalyze the double oxy-
genation of the substrate arachidonic acid to prostaglandin
G2 (PGG2) and the following reduction to prostaglandin
Fig. 3 Antimetabolic activity of the cobalt alkyne complexes after 72 h H2 (PGH2). The latter is then isomerized to the prostaglandin
of incubation against the non-tumorigenic human bone marrow stromal
HS-5 cells. The values represent the mean + SD of ≥4 independent
experiments.
E2 (PGE2) by tissue- and cell-specific isomerases. PGE2 is of
sufficient stability and released from the cells into the
medium. The concentration can then be quantified in an
enzyme immunoassay (EIA).43–45 The results of the PGE2
inhibitory assay are depicted in Fig. 5.
Therefore, as a first result of this study, it can be stated that
ASS as reference did not inhibit the cellular PGE2 synthesis
the complexes exhibit antimetabolic activity selectively towards
at a concentration of 10 µM (2.34%), whereas it considerably
COX-positive tumor cell lines. Normal, non-tumor cells grow
reduced it at 100 µM (25.4%).
unaffectedly.
As already determined in the assay at the isolated enzymes,
Induction of apoptosis. Activation of the cysteine-dependent
Co-ASS is the most potent complex inhibiting the PGE2 syn-
aspartate-directed proteases (caspases 3/7) is characteristic for
thesis to 57.5% at 10 µM. The fluorine containing complexes
cells initiating apoptosis.40–42 Therefore, the induction of cas-
are less active. The inhibitory potential decreased in the order
pases 3/7 was used to investigate the potency of the fluorinated
3F-Co-ASS (43.4%) > 4F-Co-ASS = 5F-Co-ASS (35.5%). Because
cobalt alkyne complexes to cause apoptotic cell death.
the complexes inhibited COX-2 in the enzyme assay to the
A distinct induction of caspases 3/7 was merely detected at
same extent, the limited cellular activity might be the conse-
a concentration of 40 µM (Fig. 4).
quence of the reduced COX-1 inhibition. As in the other cellu-
The complexes caused the greatest extent of apoptosis in
lar assays, 6F-Co-ASS was inactive.
HT-29 cells. The lead Co-ASS showed a 4.8-fold caspases 3/7
Cellular uptake using high-resolution continuum-source
induction. Fluorination at position 3, 4, or 5 reduced this
atomic (molecular) absorption spectrometry. The above dis-
effect to 3.5–3.8-fold. Complex 6F-Co-ASS was inactive.
cussed findings led to the question about the uptake of the
A similar pattern was determined in the MDA-MB-231 cell
complexes into the cells. This is especially necessary for 6F-Co-
line, although at a lower level. 3F-Co-ASS, 4F-Co-ASS, and
ASS, which was inactive in most of the cellular assays. Another
5F-Co-ASS induced the generation of the proteases only to
aspect is whether the intact complexes are accumulated during
about 2.0-fold. Again, 6F-Co-ASS did not cause apoptosis and
the time of incubation.
Co-ASS was the most active complex (3.3-fold).
None of the complexes stimulated caspases 3/7 production
in MCF-7 cells. In all cases only a 0.9–1.3-fold induction was
determined.
These results correspond well with the antimetabolic
activity in the respective cell lines. Comparison with the crystal
violet assay shows that cell mass reduction in MCF-7 cells
appears via interference in additional pathways.
Fig. 4 Induction of caspases 3/7 in HT-29, MDA-MB-231, and MCF-7
cells upon exposure with the cobalt alkyne complexes for 24 h. The
values represent the mean + SD of ≥4 independent experiments.
Fig. 5 Inhibition of the intracellular PGE2 synthesis in HT-29 cells upon
exposure with the cobalt alkyne complexes for 24 h. The values rep-
resent the mean + SD of 3 independent experiments.
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Therefore, uptake studies of the fluorinated cobalt alkyne
complexes into HT-29, MDA-MB-231, and MCF-7 were per-
formed using HR CS AAS. The cobalt content was quantified
as parameter. As a second feature, the determination of the
fluorine at the ASS moiety is achievable via HR CS MAS. The
comparison of the results allows statements about the stability
of the compounds upon cellular uptake.
With respect to metal complexes, the technique of atomic
absorption spectrometry (AAS) belongs, apart from inductively-
coupled plasma mass spectrometry (ICP MS), to the methods of
choice for the quantification of trace elements in biological
samples.46 Both analytical methods show similar sensitiveness,
but the great advantage of AAS over ICP MS are the lower expenses
of purchasing and maintaining the spectrometer.47 The simul-
taneous or sequential measurements further allow multi-element
analysis with AAS.47 Moreover, the development of HR CS MAS
broadened the applicability to non-metals such as fluorine.48 In
contrast, sensitive determination of fluorine employing commer-
cial argon plasma ICP MS is hardly feasible.49,50
For the quantification of fluorine containing drugs in biologi-
cal matrices, a HR CS MAS was developed. In a graphite furnace
atomic absorption spectrometer, it is feasible to detect fluorine
as GaF with a selective absorption band at 211.2480 nm.32
Based on these capabilities, the approach of sequentially
quantifying cobalt within the metal cluster by the means of
HR CS AAS as well as fluorine within the ligand using HR CS
MAS was followed. For comparison, the free propargyl-3/4/5/
6F-ASS ligands were studied with HR CS MAS, too.
Surprisingly, none of the propargyl esters was taken up into
MDA-MB-231 cells, which were used exemplarily to investigate
the uptake of the ligands in cancer cells. No GaF absorption
related to the fluorine of the ligands was measured in the cell
matrices (n = 3, data not shown) after incubation with propar-
gyl-3/4/5/6F-ASS.
Because it is well known that coordination of the alkyne
ligand to the cobalt cluster increases the lipophilicity, cellular
uptake might be enhanced, which is in agreement with
Tweedy’s chelation theory.51
Fig. 6 Cellular uptake into (a) HT-29, (b) MDA-MB-231, and (c) MCF-7
cells after 1, 2, 4, 6, and 24 h of incubation with 10 µM of 3F-Co-ASS ( ),
◆
4F-Co-ASS ( ), 5F-Co-ASS ( ), 6F-Co-ASS ( ), and Co-ASS ( ) quan-
tified based on cobalt. The values are given as pmol complex per µg
protein and represent the mean SE of 3 independent experiments.
Cellular uptake into HT-29 cells quantified by HR CS AAS
The profile of 4F-Co-ASS was comparable to the lead Co-ASS
(Fig. 6a). The cellular uptake is expressed as quantity of with about 2.0 pmol µg−1 after 1 h, 3.6 pmol µg−1 after 6 h,
complex [pmol] related to the mass of cellular protein [μg]. and about 5.0 pmol µg−1 after 24 h. 5F-Co-ASS showed an
3F-Co-ASS, 4F-Co-ASS, and 5F-Co-ASS showed a fast initial initial uptake of 3.4 pmol µg−1, which considerably raised
uptake of about 4.0 pmol µg−1 after 1 h of incubation, compar- until 4 h of incubation (5.9 pmol µg−1) and remained nearly
able to the lead Co-ASS (4.5 pmol µg−1). The maximum cobalt unchanged (6.5 pmol µg−1).
content was reached after 4 h of incubation (3F-Co-ASS: 6.5
pmol µg−1; Co-ASS: 6.4 pmol µg−1; 4F-Co-ASS: 5.8 pmol µg−1
The highest intracellular content was observed for 3F-Co-
ASS, which rose from 4.4 pmol µg−1 after 1 h to 9.0 pmol µg−1
;
5F-Co-ASS: 5.2 pmol µg−1). During the following 20 h the levels after 6 h. Its uptake was more than 2-fold higher than that of
slightly decreased.
Co-ASS or 4F-Co-ASS. Interestingly, 6F-Co-ASS had the same
The uptake of 6F-Co-ASS into HT-29 cells differed from that accumulation profile as in HT-29 cells and was the compound
of the other derivatives. After 1 h, an amount of 2.7 pmol µg−1 with the least intracellular cobalt content (0.60 pmol µg−1 after
was quantified and reached a maximum of 3.6 pmol µg−1 after 1 h, 2.5 pmol µg−1 after 6 h, and 3.1 pmol µg−1 after 24 h).
4 h, which maintained until the end of the test.
Cellular uptake into MCF-7 cells quantified by HR CS AAS
Cellular uptake into MDA-MB-231 cells by HR CS AAS (Fig. 6b). (Fig. 6c). The uptake kinetics of the complexes in MCF-7 cells are
In contrast to HT-29 cells, the uptake into MDA-MB-231 cells comparable to those in HT-29 cells, however, with a slower
differed dependent on the position of the fluorine at the ASS uptake rate during 6 h. 4F-Co-ASS and 5F-Co-ASS caused a
moiety.
maximum of 3.6 pmol µg−1 and 3.4 pmol µg−1 after 6 h, respect-
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ively. Slightly higher values were determined for 3F-Co-ASS (4.2 better probe than cobalt. The quantification is 3- to 6-fold
pmol µg−1) and Co-ASS (4.1 pmol µg−1). 6F-Co-ASS was again less higher using HR CS MAS. For instance, the intracellular fluo-
effective. It reached a maximum of only 1.3 pmol µg−1 after 4 h.
rine content caused by 3F-Co-ASS was 27.5 pmol µg−1, which
Cellular uptake into HT-29 cells quantified by HR CS MAS enhanced during 6 h to 55.4 pmol µg−1. After 24 h a decrease
(Fig. 7a). The cellular uptake can also be followed by fluorine to 41.1 pmol µg−1 was observed.
analysis. All complexes exhibited more or less the same
Cellular uptake into MCF-7 cells quantified HR CS MAS
accumulation profile in HT-29 cells using HR CS AAS (Fig. 6a) (Fig. 7c). A completely different profile was obtained in MCF-7
or HR CS MAS (Fig. 7a). Only 3F-Co-ASS slightly differed if fluo- cells. In contrast to the HR CS AAS analysis (see Fig. 6c) a con-
rine was analyzed and showed a higher uptake after 1 h (8.3 tinuous increase of the complex concentrations in cells during
pmol µg−1), which further increased to 13.2 pmol µg−1 (6 h). the incubation of 24 h was observed by HR CS MAS.
After 24 h, the intracellular content decreased and was nearly
The fluorine content rose in each case during the obser-
comparable regardless whether cobalt (4.5 pmol µg−1) or fluo- vation period of 24 h. The initial amounts (1 h) are 5.9 pmol
rine (7.1 pmol µg−1) was used as tracer.
µg−1 for 3F-Co-ASS, about 3.7 pmol µg−1 for 4F-Co-ASS, 3.4
Cellular uptake into MDA-MB-231 cells quantified HR CS MAS pmol µg−1 for 5F-Co-ASS, and 2.4 pmol µg−1 for 6F-Co-ASS,
(Fig. 7b). In MDA-MB-231 cells fluorine revealed to be a much which increased after 24 h to 32.6–34.0 pmol µg−1 for 3F-Co-
ASS, 4F-Co-ASS, and 5F-Co-ASS. The less potent 6F-Co-ASS
reached a value of only 19.8 pmol µg−1
.
The discrepancy between HR CS AAS and HR CS MAS in
case of MCF-7 cells needs further investigations. Thus far, the
findings of a disparate ratio between cobalt and fluorine in the
samples point to different physiological behavior of the com-
plexes in the cells. It might be possible that they degrade intra-
cellularly. Cobalt undergoes efflux (perhaps bound to pro-
teins), while the fluorine containing ligands accumulated in
the cells. Degradation in the medium prior to the transport
into the cells can be excluded, because the free ligand did not
enter the cells (see above).
6F-Co-ASS was the complex with the lowest activity in the
cellular assays (antiproliferative, antimetabolic, and apoptotic
properties; PGE2 inhibition in HT-29 cells) compared to the
other derivatives. This can be attributed to its minor uptake
into the cells based on HR CS ASS and HR CS MAS analyses.
The uptake studies further suggested that utilizing fluorine
(HR CS MAS) instead of cobalt (HR CS AAS) as probe seems to
be approximately 5-fold more sensitive. Fluorine analysis by
HR CS MAS seems generally feasible for the application to
future investigations not only of fluorinated metal complexes
but also principally for evaluating fluorinated compounds in
biological samples. Another great advantage of fluorination
represents the feature of introducing a permanent tracer into
the molecule structure without any further modification,
which would actually lead to new chemical entities.
Protein binding. In general, the binding to macromolecules,
especially to plasma proteins, is an essential criterion that
influences the biological efficacy of a drug.52,53 The protein
binding of the lead Co-ASS was previously evaluated and
suggested that sufficient plasma levels of free drug can be
achieved.54
It is of interest, if the fluorine substituent influences the
binding to proteins (mainly related to the proteins contained
in FCS) included in the medium used for the cell culture
experiments. On the one hand, proteins can prevent the cellu-
lar uptake, but on the other hand they can also be involved in
uptake mechanisms. However, it is supposed that the uptake
of a drug into cells is merely caused by the unbound
fraction.55
Fig. 7 Cellular uptake into (a) HT-29, (b) MDA-MB-231, and (c) MCF-7
cells after 1, 2, 4, 6, and 24 h of incubation with 10 µM of 3F-Co-ASS ( ),
◆
4F-Co-ASS ( ), 5F-Co-ASS ( ), 6F-Co-ASS ( ), and Co-ASS ( ) quan-
tified based on fluorine. The values are given as pmol complex per µg
protein and represent the mean SE of 3 independent experiments.
15862 | Dalton Trans., 2019, 48, 15856–15868
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Paper
case of measuring fluorine by HR CS MAS instead of quantify-
ing cobalt by HR CS AAS. Therefore, the technique of HR CS
MAS was demonstrated as a highly sensitive method for the
quantification of fluorinated cobalt complexes. This approach
can be applied in future for investigating other fluorinated
compounds in biological samples.
Experimental section
General materials and methods
Fig. 8 Protein binding after exposure of 10 µM of 3F-Co-ASS ( ),
◆
4F-Co-ASS ( ), 5F-Co-ASS ( ), 6F-Co-ASS ( ), and Co-ASS ( ) to the
All the applied educts, solvents, and other chemicals were
acquired from Alfa Aesar, Fluka, Sigma-Aldrich, or TCU
Chemicals. The solvents were obtained either in appropriate
purity or distilled before usage. Milli-Q water from a Milli-Q
Gradient A10 water purification system (Merck Millipore,
Billerica, USA) was deployed. Silica gel 60 (particle size
40–63 µm) was used for column chromatography and
Polygram® SIL G/UV254 polyester foils coated with a 0.2 mm
of silica gel with a fluorescence indicator (Macherey-Nagel,
Düren, Germany) were applied for thin layer chromatography
(TLC). Observation under UV light at 254 and/or 365 nm
served as detection. The melting points were determined using
cell culture medium for 1, 2, 4, 6, and 24 h. The values represent the
mean SE of 3 independent experiments.
It was previously reported that C–F⋯OvC interactions may
influence the binding, if a close contact of the fluorine to the
carbonyl moiety exists.56
Therefore, the cobalt alkyne complexes (10 µM) were incu-
bated with DMEM containing 10% FCS. The proteins were pre-
cipitated with ice-cold ethanol and the cobalt content in the
supernatant was quantified by HR CS AAS.
All complexes showed a fast binding (62–70%) to the pro-
teins in the medium, which slightly decreased over 24 h. The
binding is nearly independent of the presence and the posi-
tion of the fluorine substituent (Fig. 8). After 24 h, the protein
binding decreased to about 54–63%. As depicted in Fig. 8,
3F-Co-ASS showed the least binding after 6 h (52%). This
might be the consequence for the higher uptake into the cells,
based on the amount of free complex.
1
a microscope Kofler melting point apparatus. H nuclear mag-
netic resonance (1H NMR) spectroscopy was performed
employing either a Gemini 2000 NMR Spectrometer (200 MHz,
Varian, Paolo Alto, USA, now part of Agilent Technologies,
Santa Clara, USA) or a Bruker Avance 4 Neo spectrometer
(400 MHz) in deuterated DMSO or CDCl3. Tetramethylsilane
was used as internal standard. The chemical shifts are given in
parts per million (ppm) and the coupling constants in Hertz
(Hz). High resolution mass spectrometry (HR MS) was per-
formed on an Orbitrap Elite (Thermo Fisher Scientific,
Waltham, USA) via direct infusion and electrospray ionisation.
The samples were dissolved in MeOH and doped with a
Conclusions
[(Prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS) diluted NaCl solution to obtain better ionisation.
is a metal complex that displayed outstanding growth inhibi- Fragmentation experiments (HCD – higher energy collisional
tory potency against certain tumor cell lines. As one possible dissociation) were conducted to confirm the structure of the
mode of action, the unselective inhibition of both cyclooxygen- R-Co2(CO)6 complexes. Low levels of fragmentation energy
ase isoenzymes (COX-1 and COX-2) is considered. It was aimed (typically 5% normalized collision energy) resulted in the step-
to shift the selectivity towards COX-2 since this isoenzyme wise loss of six single CO entities, confirming the structure of
plays a substantial role in tumorigenesis.
the complexes. Infrared (IR) spectroscopy was carried out
The goal of generating COX-2-selective derivatives was employing an Alpha FT-IR Spektrometer (Bruker, Billerica,
pursued by fluorination of the aromatic moiety. Introducing of USA). HPLC investigations were performed using an instru-
fluorine effected a preferential inhibition of isolated COX-2 ment from Shimadzu (Duisburg, Germany) provided with the
(about 70% at 10 µM) compared to COX-1 (about 45%). autosampler SIL-20A HT, the pump LC20 AD, the column oven
Notably, the fluorinated cobalt alkyne complexes showed CT0-10AS VP, and the diode-array detector (DAD) SPD-M20A
hardly any apoptotic and antimetabolic property in the COX-1/ with a deuterium and tungsten lamp. The Eurospher 100-5
2-negative MCF-7 cell line. In contrast, the compounds C18 (250 × 4 mm) column (Knauer, Berlin, Germany) was uti-
induced apoptosis in the COX-1/2 expressing HT-29 and lized for separation purposes. The detection was performed
MDA-MB-231 cells, reduced the cell biomass in the low micro- using a DAD and the purity of the final products was
molar range and exhibited a concentration-dependent anti- greater than 95% (except where noted) at the exemplary wave-
metabolic activity. Hence, the results approved an interference length of 254 nm. An Enspire multimodal plate reader
with the COX cascade, which is presumed as mechanism of (PerkinElmer Life Sciences, Waltham, USA) was applied to
anticancer action. Moreover, the cellular uptake studies mani- measure the absorbance or the luminescence within the bio-
fested an about 5-fold higher bioanalytical sensitivity in the logical testing.
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Chemistry
and subsequently added to the initial solution, which was
then stirred at room temperature for 3–4 h. Within the first
General procedure for the synthesis of propargyl 2-acetoxy- minutes, both formation of carbon monoxide and change of
fluorobenzoates. 500 mg (2.52 mmol) of F-ASS was dissolved color to reddish-brown were observed. Addition of 1 g of silica
in absolute DCM and 1.1 eq. of DCC, 0.2 eq. of DMAP and 1.1 gel to the mixture finally caused the quenching of the reaction.
eq. of propargyl alcohol were added, which was then stirred at The THF was removed under reduced pressure using an evap-
room temperature for 1–3 h. DCU (N,N′-dicyclohexylurea) was orator. The purification of the product was performed by
formed as byproduct during the reaction, which was removed column chromatography. Silica gel was utilized as stationary
by filtration. The DCM was removed under reduced pressure phase and a mixture of diethyl ether/petroleum ether (2 : 1)
employing an evaporator and the product was purified by acted as mobile phase. The reddish-brown fractions were col-
column chromatography using a mixture of diethyl ether/pet- lected and used to obtain the fluorinated alkynyl cobalt
roleum ether (2 : 1) as mobile phase. Characterization was per- complex. Characterization was performed by FT IR and 1H
formed by IR and 1H NMR analyses (see ESI, Fig. S1–S8†).
Propargyl 2-acetoxy-3-fluorobenzoate (propargyl-3F-ASS). Yield:
NMR analysis (see ESI, Fig. S9–S16†).
[Propargyl 2-acetoxy-3-fluorobenzoate]dicobalthexacarbonyl
78% as colorless, crystalline powder; mp: 60 °C; HPLC: 95% (3F-Co-ASS). Yield: 100% as reddish-brown, crystalline powder;
1
purity; H NMR (400 MHz, CDCl3): δ 2.41 (s, 3H, CH3), 2.54 (t, mp: 87 °C (decomposition); HPLC: 95% purity; 1H NMR
4
4J = 2.4 Hz, 1H, CH), 4.89 (d, J = 2.4 Hz, 2H, CH2), 7.29 (ddd, (200 MHz, CDCl3): δ 7.85 (d, 1H, Ar–H), 7.38 (m, 2H, Ar–H),
3
4
3J = 8.0 Hz, J = 8.0 Hz, J(H–F) = 5.2 Hz, 1H, ArH-5), 7.38 (ddd, 6.11 (s, 1H, CH), 5.48 (s, 2H, CH ), 2.40 (s, 3H, CH ); IR (ν in
¯
2
3
3J(H–F) = 9.4 Hz, J = 8.0 Hz, J = 1.6 Hz, 1H, ArH-4), 7.82 (ddd, cm−1): 3091 w (Aryl-C–H), 2943 w (C–H), 2098 m, 2055 s, 2018
3
4
3J = 8.0 Hz, 4J = 1.6 Hz, J(H–F) = 1.6 Hz, 1H, ArH-6); IR (ν in s (CO), 1771 s (CvO), 1726 m (CvO), 1593 m (CvC), 1271 s,
5
¯
cm−1): 3265 m (uC–H), 3081 w (Aryl-C–H), 2949 w (C–H), 1193 s, 980 s, 719 s; HR MS (m/z) calculated for [M + Na]+:
1759 m (CvO), 1719 s (CvO), 1588 w (CvC), 1201 s, 1172 s, 544.8736, found: 544.8780.
1142 s, 1006 s, 907 s.
[Propargyl
2-acetoxy-4-fluorobenzoate]dicobalthexacarbonyl
Propargyl 2-acetoxy-4-fluorobenzoate (propargyl-4F-ASS). Yield: (4F-Co-ASS). Yield: 71% as reddish-brown, crystalline powder;
78% as colorless, highly viscous oil; HPLC: 95% purity; 1H mp: 102 °C (decomposition); HPLC: 95% purity; 1H NMR
NMR (400 MHz, CDCl3): δ 2.38 (s, 3H, CH3), 2.53 (t, 4J = 2.4 Hz, (200 MHz, CDCl3): δ 8.13 (s, 1H, Ar–H), 7.02 (s, 2H, Ar–H), 6.11
4
3
1H, CH), 4.87 (d, J = 4.87 Hz, 2H, CH2), 6.86 (dd, J(H–F) = 8.8 (s, 1H, CH), 5.46 (s, 2H, CH ), 2.36 (s, 3H, CH ); IR (ν in cm−1):
¯
2
3
4
3
3
Hz, J = 2.4 Hz, 1H, ArH-3), 7.04 (ddd, J(H–F) = 7.8 Hz, J = 8.8 3103 w (Aryl-C–H), 2931 w (C–H), 2098 m, 2053 s, 1999 s (CO),
4
3
4
Hz, J = 2.6 Hz, 1H, ArH-5), 8.10 (dd, J = 8.8 Hz, J(H–F) = 6.4 1777 s (CvO), 1727 m (CvO), 1618 s (CvC), 1275 s, 1187 s,
Hz, 1H, ArH-6); IR (ν in cm−1): 3282 w (uC–H), 3082 w (Aryl-C– 1102 s, 1026 s; HR MS (m/z) calculated for [M + Na]+: 544.8736,
¯
H), 2943 w (C–H), 1771 s (CvO), 1723 s (CvO), 1607 s (CvC), found: 544.8784.
1251 s, 1191 s, 1116 s, 1074 s.
[Propargyl
2-acetoxy-5-fluorobenzoate]dicobalthexacarbonyl
Propargyl 2-acetoxy-5-fluorobenzoate (propargyl-5F-ASS). Yield: (5F-Co-ASS). Yield: 73% as reddish-brown, crystalline powder;
92% as colorless, crystalline powder; mp: 69 °C; HPLC: 96% mp: 94 °C; HPLC: 95% purity; 1H NMR (200 MHz, CDCl3): δ
1
purity; H NMR (400 MHz, CDCl3): δ 2.36 (s, 3H, CH3), 2.54 (t, 7.77 (dd, 1H, Ar–H), 7.26 (s, 1H, Ar–H), 7.10 (dd, 1H, Ar–H),
4
4J = 2.4 Hz, 1H, CH), 4.88 (d, J = 2.4 Hz, 2H, CH2), 7.09 (dd, 6.12 (s, 1H, CH), 5.48 (s, 2H, CH ), 2.35 (s, 3H, CH ); IR (ν in
¯
2
3
3J = 8.8 Hz, J(H–F) = 4.8 Hz, 1H, ArH-3), 7.28 (ddd, J = 8.8 Hz, cm−1): 3123 w (Aryl-C–H), 2932 w (C–H), 2095 m, 2033 s, 2001
4
3
3J(H–F) = 7.4 Hz, J = 3.2 Hz, 1H, ArH-4), 7.75 (dd, J(H–F) = 8.4 s (CO), 1760 s (CvO), 1726 m (CvO), 1597 s (CvC), 1260 s,
4
3
Hz, 4J = 3.2 Hz, 1H, ArH-6); IR (ν in cm−1): 3281 m (uC–H), 1202 s, 1170 s, 1066 s, 966 s; HR MS (m/z) calculated for [M +
¯
3102 w (Aryl-C–H), 2941 w (C–H), 1763 s (CvO), 1725 s (CvO), Na]+: 544.8736, found: 544.8744.
1593 m (CvC), 1214 s, 1170 s, 1086 s, 680 s.
[Propargyl
2-acetoxy-6-fluorobenzoate]dicobalthexacarbonyl
Propargyl 2-acetoxy-6-fluorobenzoate (propargyl-6F-ASS). Yield: (6F-Co-ASS). Yield: 87% as reddish-brown, crystalline powder;
91% as colorless, highly viscous oil; HPLC: 95% purity; 1H mp: 85 °C (decomposition); HPLC: 93% purity; 1H NMR
NMR (400 MHz, CDCl3): δ 2.33 (s, 3H, CH3), 2.54 (t, 4J = 2.4 Hz, (200 MHz, CDCl3): δ 7.45 (s, 1H, Ar–H), 6.99 (m, 2H, Ar–H),
4
3
4
1H, CH), 4.90 (d, J = 2.4 Hz, CH2), 6.93 (ddd, J = 8.2 Hz, J = 6.10 (s, 1H, CH), 5.51 (s, 2H, CH ), 2.31 (s, 3H, CH ); IR (ν in
¯
2
3
1.2 Hz, J(H–F) = 1.2 Hz, 1H, ArH-3), 7.06 (ddd, 3J = 8.2 Hz, cm−1): 3103 w (Aryl-C–H), 2931 w (C–H), 2098 m, 2053 s,
5
3J(H–F) = 9.2 Hz, J = 1.2 Hz, 1H, ArH-5), 7.48 (ddd, J = 8.2 Hz, 1999 s (CO), 1777 s (CvO), 1727 s (CvO), 1618 s (CvC),
4
3
3J = 8.2 Hz, J(H–F) = 6.0 Hz, 1H, ArH-4); IR (ν in cm−1): 3285 w 1466 s, 1275 s, 1187 s, 1102 s, 1026 s; HR MS (m/z) calculated
4
¯
(uC–H), 3103 w (Aryl-C–H), 2941 w (C–H), 1770 m (CvO), for [M + Na]+: 544.8736, found: 544.8755.
1731 s (CvO), 1618 m (CvC), 1274 s, 1186 s, 1102 s, 1025 s.
Biology
General procedure for the synthesis of cobalt alkyne com-
plexes. 50 mg (0.21 mmol) of propargyl-F-ASS were dissolved
General cell culture methods. The human bone marrow
in absolute THF and transferred into a triple-necked flask, stromal cell line HS-5 was provided by Dr Karin Jöhrer-
armed with an argon connection, a condenser with a bubble Deym from the Tyrolean Cancer Research Institute (TKFI,
counter and a plug. 76 mg (0.22 mmol, 1.05 eq.) of dicobalt- Innsbruck, Austria). The colon carcinoma cell line HT-29 as
octacarbonyl were promptly weighed into a dried weighing dish well as the breast cancer cell lines MCF-7 (hormone-depen-
15864 | Dalton Trans., 2019, 48, 15856–15868
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dent) and MDA-MB-231 (hormone-independent) were pur- comprising 5 × 104 cells per well in 50 µL of medium (HT-29,
chased from the cell line service (CLS, Eppelheim, Germany).
MCF-7, MDA-MB-231) or 2 × 104 cells per well in 50 µL of
HS-5 cells were maintained in RPMI-1640 medium without medium (HS-5). The compounds were added in appropriate
phenol red (PAA Laboratories, Pasching, Austria), sup- concentrations 3 h (HS-5: 1 h) thereafter, followed by the incu-
plemented with L-glutamine (292 mg L−1), penicillin (100 U bation for another 72 h at 37 °C in a humidified atmosphere
mL−1), streptomycin (100 µg mL−1) and fetal bovine serum (5% CO2/95% air). The cultures were then evaluated for the
(FBS; 10%; all from Invitrogen Corporation, Gibco, Paisley, metabolic activity performing a modified 3-(4,5-dimethyl-
Scotland). HT-29, MCF-7, and MDA-MB-231 cells were cultivated thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
as monolayer cultures in Dulbecco’s Modified Eagle Medium (EZ4U kit, Biomedica, Vienna, Austria) following the protocol
(DMEM) high modified with glucose (4.5 mg L−1
) and of the manufacturer. The optical density of the respective
L-glutamine (584 mg L−1) but without phenol red (GE Healthcare culture medium was subtracted in order to exclude unspecific
BioSciences, Pasching, Austria), supplemented with fetal calf staining due to FCS containing medium. The metabolic
serum (FCS; 10%; Biochrom, Cambridge, UK). All cell lines were activity omitting the compound was set to 100%. The values
grown at 37 °C in a humidified atmosphere (5% CO2/95% air) represent the arithmetic mean
using 75 cm2 flasks and were serially passaged twice weekly. The experiments.
SD of ≥4 independent
cell lines were authenticated by typing short tandem repeats and
routinely monitored for mycoplasma contamination.
Determination of caspases 3/7 induction. Exponentially
growing cells were seeded in triplicates into 96-well plates
COX inhibition assay. The impact of the compounds (10 µM comprising 5 × 104 cells per well in 50 µL of medium.
and/or 100 µM) to inhibit both isolated ovine COX-1 and Appropriate concentrations of the compounds were added 3 h
human recombinant COX-2 was evaluated performing an thereafter, followed by the incubation for another 24 h at 37 °C
enzyme immunoassay (EIA) (COX Inhibitor Screening Assay, in a humidified atmosphere (5% CO2/95% air). The super-
Cayman Chemicals, Ann Arbor, USA) following the protocol of natant was put into white 96-well plates and the induction
the manufacturer. Thereby, the isoenzymes were accurately of apoptosis was determined by measurement of caspases 3/7
incubated with the respective compound for solely 10 min. employing a Caspase-3/7 Glo® Kit (Promega, Madison, USA)
The untreated control was set to 0% inhibition of COX activity. following the protocol of the manufacturer. The values were
The given values were calculated as the mean of duplicate normalized to the luminescence signal of solely medium. The
determinations and express the mean SD of three indepen- final results were calculated in respect of the caspases 3/7
dent experiments.
Determination of cell biomass with crystal violet staining. metic mean SD of ≥four independent experiments.
The testing of the complexes for their impact to reduce cell Determination of the inhibition of cellular PGE2 synthesis.
activity of the untreated cells. The values represent the arith-
biomass in vitro was evaluated by performing a crystal violet The COX-expressing HT-29 cells were seeded into 24-well
assay following a modified procedure as previously pub- plates comprising 5 × 104 cells per well in 500 µL of medium
lished.57 Exponentially growing cells were seeded in quadru- and incubated at 37 °C in a humidified atmosphere (5% CO2/
ples into 96-well plates (HT-29: 3 × 103 cells in 100 µL per well, 95% air). The compounds (10 µM) were added and incuba-
MCF-7 and MDA-MB-231: 2 × 103 cells in 100 µL per well). The tion was carried on for further 24 h. In order to evaluate the
plates were kept for 24 h at 37 °C in a humidified atmosphere COX-catalyzed enzymatic potential, the substrate arachidonic
(5% CO2/95% air) to allow adhesion of the cells prior to acid (Sigma-Aldrich, St Louis, USA) was added at a con-
adding of completed medium containing the vehicle DMSO, centration of 50 µM. The enzymatic turnover was stopped
the controls Cisplatin and Co-ASS and the fluorinated cobalt after 1 h of incubation by collecting the supernatant medium.
alkyne complexes at designated concentrations, respectively. The concentration of generated PGE2 in the supernatant was
The medium was removed after another 72 h of incubation quantified employing an EIA (Prostaglandin E2 Kit
–
and the cells were washed with PBS followed by fixation with a Monoclonal, Cayman Chemicals, Ann Arbor, USA) following the
solution of glutaraldehyde in PBS (1%, v/v). The cell biomass protocol of the manufacturer. The untreated control was set to
was determined by staining the chromatin of adherent cells 100% activity or rather 0% inhibition of PGE2 synthesis. The
with the dye crystal violet, following extraction of the stain capability of the compounds to inhibit cellular PGE2 synthesis
with ethanol (70%, v/v) and subsequent measurement of the was calculated as the mean of duplicate determinations and
absorbance at the wavelength of 590 nm. The cell biomass is expresses the arithmetic mean
expressed as the percentage of cell biomass of the vehicle experiments.
SD of three independent
treated control that was set to 100%. The IC50 values are given
Determination of cellular uptake. The cellular content of
as the mean SD of ≥three independent experiments. The both cobalt and fluorine, respectively, was quantified by
values were calculated with Prism 7.0 (GraphPad, San Diego, absorption spectrometry in order to evaluate the uptake of
USA) using nonlinear regression and decal logarithm of the the compounds into each HT-29, MCF-7, and MDA-MB-231
inhibitor versus variable slope equation, while the top con- cells. The quantification of cobalt was carried out performing
straint was set to 100%.
HR CS AAS according to a method previously described and
Determination of antimetabolic activity. Exponentially applied for chlorinated cobalt alkyne complexes (furnace
growing cells were seeded in triplicates into 96-well plates program is provided in the ESI, Table S1†).23 Fluorine was
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measured in the means of GaF employing HR CS MAS were pipetted into a 96-well plate and each 200 µL of Bradford
following a modified protocol (furnace program is provided reagent were added. All samples and standards were measured
in the ESI, Table S2†).35 The cobalt or fluorine contents in duplicate. After 5 min of incubation at room temperature,
were related to the protein contents determined by the method the absorbance was read at the wavelength of 595 nm employ-
of Bradford58 and the extent of cellular uptake was ing
expressed as pmol compound per µg protein. The values were Scientific, Waltham, USA).
a microplate reader (Multiskan GO, Thermo Fisher
calculated as the arithmetic mean SE of three independent
experiments.
Determination of the protein binding. The evaluation was
carried out following a protocol as reported before.59 The
For cellular uptake studies, HT-29, MCF-7, and cobalt alkyne complexes were applied at the concentration
MDA-MB-231 cells were seeded, respectively, in their exponen- (10 µM) equal to the cellular uptake studies and incubated
tially growing phase in 2 mL of cell culture medium into 6-well with the same cell culture medium as used within the cellular
tissue culture plates. After incubation for 24 h in a 5% CO2/ uptake studies. The samples were put in a thermomixer
95% air atmosphere, they should reach about 70% of con- (Eppendorf, Germany) running at 400 rpm and 37 °C. After the
fluency. Then, the medium was displaced by drug (10 µM, respective incubation times (1, 2, 4, 6, and 24 h), the amount
0.1% v/v DMSO) or vehicle (DMSO) containing medium and of each 150 µL was taken and added to 300 µL of ice-cold
incubated for 1, 2, 4, 6, and 24 h, respectively. Afterwards, the ethanol (96%). Notably, no precipitation of the complexes
medium was aspirated and the cells were washed with PBS fol- occurred upon diluting with ethanol. The dilutions were kept
lowed by the detaching using Accutase (GE Healthcare at −20 °C overnight to afford further protein precipitation.
BioSciences, Pasching, Austria) and the harvesting with PBS. After centrifugation at 4000 rpm and 4 °C for 10 min, 400 µL
The cell suspensions were centrifuged (8000 rcf, 3 min, RT) of the supernatant were removed. The unbound amount of the
and the supernatants were removed. The obtained cell pellets complexes was quantified using HR CS AAS in terms of cobalt
were washed once more with PBS by resuspending, centrifu- with two instrumental replicates each. The samples containing
ging, and removing of the supernatant and stored at −20 °C only the cell culture medium and the vehicle DMSO served as
until further analysis. The cell pellets were resuspended in blank. Their absorbances were subtracted from the values
Milli-Q water and lysed using a sonotrode (20 s, nine cycles, when using the complexes. The percental protein binding was
80–85% power) straight after thawing. The cell lysates were obtained indirectly by subtracting the unbound amount from
diluted appropriately with an aqueous solution of Triton-X-100 the initially set concentration (10 µM), which was assigned to
(final concentration: 0.1%) for the purpose of quantification 100%. The final outcomes represent the arithmetic mean SD
by absorption spectrometry.
of three independent conductions.
Absorption spectrometry measurements. The absorption
spectrometry measurements were performed with a contrAA
700 high-resolution continuum-source absorption spectro-
meter (Analytik Jena AG, Jena, Germany) based on the furnace
Abbreviations
technique. Samples of the respective compounds dissolved in ASS
DMSO were used as standards and calibration was performed Co-ASS
in a matrix matched manner (seven data points, R2 ≥ 0.99).
Acetylsalicylic acid
[(Prop-2-ynyl)-2-acetoxybenzoate]
dicobalthexacarbonyl
Cobalt was quantified at the wavelength of 240.7254 nm by CORM
direct injecting of the samples into PIN platform graphite COX
tubes applying a furnace program reported before (pyrolysis: CDCl3
1100 °C, atomization: 2300 °C, see ESI, Table S1†).23 In con- DAD
trast, fluorine was measured in terms of GaF at the wavelength DCC
of 211.2480 nm referring to a method described previously,35 DCM
while the pyrolysis (520 °C) and molecular forming (1400 °C) DCU
temperatures were adjusted with the present fluorinated DMAP
cobalt alkyne complexes (see ESI, Table S2†). For fluorine ana- DMEM
lysis, the same samples were spiked with 3 µM of the respect- DMSO
ive complex, injected into a standard graphite tube lined with EIA
a tantalum foil and measured. The mean integrated absor- FCS
bances of duplicate injections were used throughout the FT IR
CO releasing molecule
Cyclooxygenase
Deuterated chloroform
Diode array detector
N,N′-Dicyclohexylcarbodiimide
Dichloromethane
N,N′-Dicyclohexylurea
4-(Dimethylamino)pyridine
Dulbecco’s Modified Eagle Medium
Dimethyl sulfoxide
Enzyme immunoassay
Fetal calf serum
Fourier transform infrared spectroscopy
Gallium mono-fluoride
High performance liquid chromatography
experiments.
GaF
Quantification of the protein content. The protein content HPLC
of the cell lysates was determined according to Bradford’s HR
method.58 Briefly, the cell lysates were appropriately diluted AAS
with Milli-Q water. Solutions of bovine albumin diluted with HR
Milli-Q water (31.25–1000 µg mL−1) served as calibration stan- MAS
dards. Each 20 µL of the diluted samples or the standards HR MS
CS High-resolution
continuum-source
atomic
absorption spectrometry
CS High-resolution continuum-source molecular
absorption spectrometry
High-resolution mass spectrometry
15866 | Dalton Trans., 2019, 48, 15856–15868
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Dalton Transactions
Paper
ICP MS
MTT
Inductively coupled plasma mass spectrometry
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide
10 Q.-P. Qin, J.-L. Qin, T. Meng, W.-H. Lin, C.-H. Zhang,
Z. Z. Wei, J.-N. Chen, Y.-C. Liu, H. Liang and Z.-F. Chen,
Eur. J. Med. Chem., 2016, 124, 380–392.
mp
Melting point
11 V. Thamilarasan, N. Sengottuvelan, A. Sudha, P. Srinivasan
and G. Chakkaravarthi, J. Photochem. Photobiol., B, 2016,
162, 558–569.
12 B. Y. K. Law, Y. Q. Qu, S. W. F. Mok, H. Liu, W. Zeng,
Y. Han, F. Gordillo-Martinez, W.-K. Chan, K. M.-C. Wong
and V. K. W. Wong, Oncotarget, 2017, 8, 55003–55021.
13 D. O. Abe, A. Eskandari and K. Suntharalingam, Dalton
Trans., 2018, 47, 13761–13765.
14 S. Ambika, Y. Manojkumar, S. Arunachalam, B. Gowdhami,
K. K. Meenakshi Sundaram, R. V. Solomon,
P. Venuvanalingam, M. A. Akbarsha and M. Sundararaman,
Sci. Rep., 2019, 9, 2721.
15 I. Ott, K. Schmidt, B. Kircher, P. Schumacher, T. Wiglenda
and R. Gust, J. Med. Chem., 2005, 48, 622–629.
NMR
NSAID
PBS
Nuclear magnetic resonance
Non-steroidal anti-inflammatory drug
Phosphate buffered saline
Prostaglandin
PG
RPMI-1640 Rosewell Park Memorial Institute Medium
RT
SAR
SD
SE
SS
TEA
THF
Room temperature
Structure activity relationship
Standard deviation
Standard error
Salicylic acid
Triethylamine
Tetrahydrofurane
16 K. Schmidt, M. Jung, R. Keilitz, B. Schnurr and R. Gust,
Inorg. Chim. Acta, 2000, 306, 6–16.
17 I. Ott, B. Kircher and R. Gust, J. Inorg. Biochem., 2004, 98,
485–489.
Conflicts of interest
There are no conflicts to declare.
18 I. Ott and R. Gust, Arch. Pharm., 2007, 340, 117–126.
19 G. Rubner, K. Bensdorf, A. Wellner, B. Kircher,
S. Bergemann, I. Ott and R. Gust, J. Med. Chem., 2010, 53,
6889–6898.
Acknowledgements
20 G. Rubner, K. Bensdorf, A. Wellner, S. Bergemann, I. Ott
and R. Gust, Eur. J. Med. Chem., 2010, 45, 5157–5163.
21 I. Ott, B. Kircher, C. P. Bagowski, D. H. W. Vlecken,
E. B. Ott, J. Will, K. Bensdorf, W. S. Sheldrick and R. Gust,
Angew. Chem., Int. Ed., 2009, 48, 1160–1163.
22 N. Hashemi Goradel, M. Najafi, E. Salehi, B. Farhood and
K. Mortezaee, J. Cell. Physiol., 2019, 234, 5683–5699.
23 V. Obermoser, D. Baecker, C. Schuster, V. Braun, B. Kircher
and R. Gust, Dalton Trans., 2018, 47, 4341–4351.
24 I. Ojima, ChemBioChem, 2004, 5, 628–635.
Daniel Baecker wishes to give thanks for the financial support
of the research work granted by “Tiroler Wissenschaftsfonds
(TWF)” (GZ: UNI-0404/1982) and to Werner Pöttschacher
(Analytik Jena AG) for technical support. The Austrian
Research Promotion Agency FFG [West Austrian BioNMR
858017] is also kindly acknowledged.
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