Synthesis and biological evaluation of Zeise’s salt derivatives with acetylsalicylic acid substructure

Article abstract of DOI:10.3390/ijms19061612

The development of novel biologically active organometallic compounds bearing an acetylsalicylic acid (ASA) substructure led to the synthesis of analogical Zeise-type salts that accordingly inhibit cyclooxygenase (COX) enzymes. In order to determine the i

Full text of DOI:10.3390/ijms19061612

International Journal of
Molecular Sciences
Article
Synthesis and Biological Evaluation of Zeise’s Salt
Derivatives with Acetylsalicylic Acid Substructure
Alexander Weninger 1, Daniel Baecker 1, Victoria Obermoser 1, Dorothea Egger 1, Klaus Wurst 2
and Ronald Gust 1,
*
1
Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular Biosciences
Innsbruck, University of Innsbruck, CCB—Centrum for Chemistry and Biomedicine, Innrain 80-82,
6020 Innsbruck, Austria; alexander.weninger@uibk.ac.at (A.W.); daniel.baecker@uibk.ac.at (D.B.);
victoria.obermoser@gmx.at (V.O.); dorothea.egger@gmx.at (D.E.)
Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, CCB—Centrum for
Chemistry and Biomedicine, Innrain 80-82, 6020 Innsbruck, Austria; klaus.wurst@uibk.ac.at
Correspondence: ronald.gust@uibk.ac.at; Tel.: +43-512-507-58200
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 April 2018; Accepted: 21 May 2018; Published: 30 May 2018
Abstract: The development of novel biologically active organometallic compounds bearing
an acetylsalicylic acid (ASA) substructure led to the synthesis of analogical Zeise-type salts that
accordingly inhibit cyclooxygenase (COX) enzymes. In order to determine the influence of the
length of the alkyl chain between the platinum(II) center and the ASA moiety, compounds with
varying methylene groups (n = 1–4) were synthesized and characterized. For the propene derivative
structural elucidation by X-ray crystallography was possible. Prior to evaluation of biological
activity, the complexes were investigated regarding their stability in different media, such as water,
physiological sodium chloride, and phosphate buffered saline. Therefore, an analytical method
based on capillary electrophoresis was established. All of the compounds were tested for their COX
inhibitory potential. In general, complexes with longer alkyl chains caused higher inhibition of COX
enzymes and the inhibitory potential towards COX enzymes was enhanced when compared to Zeise’s
salt. The growth inhibitory effects of the synthesized substances were investigated in vitro against
colon carcinoma (HT-29) and breast cancer (MCF-7) cells. The IC50 values of the new derivatives
ranged from 30 to 50 µM, whereas neither Zeise’s salt itself nor ASA showed any antiproliferative
activity at the used concentrations.
Keywords: anticancer; antiproliferative activity; capillary electrophoresis; COX inhibition; cyclooxygenase
enzyme; platinum chemistry; Zeise’s salt
1. Introduction
The most famous platinum complex in anticancer therapy by far is Cisplatin, which was first
synthesized by M. Peyrone in 1844 [
1]. However, it took more than 120 years until its antitumor effect
was discovered by B. Rosenberg [ ], and to date it is in therapeutic use for 40 years. During that time,
2
various derivatives of Cisplatin were synthesized that differ in the leaving groups and/or in the carrier
ligands to improve its pharmacological profile. Yet, only a few derivatives, such as Carboplatin and
Oxaliplatin, are approved for tumor therapy [3–5]. Meanwhile, the DNA was identified to be the
main target of platinum complexes where intrastrand cross-links between adjacent guanine bases
or adjacent guanine and adenine bases are formed, causing defective replication mechanisms and
ultimately leading to cell death [
that they are restricted by dose limiting side effects as well as frequent development of resistance.
Unfortunately, they show cross-resistance due to the same mode of action by targeting the DNA [ ].
4]. Cisplatin, Carboplatin, and Oxaliplatin (Figure 1) have in common
4,5
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www.mdpi.com/journal/ijms

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Figure 1. (a) Cisplatin; (b) Carboplatin; (c) Oxaliplatin; (d) Zeise’s salt; and (e) Co-ASS.
To overcome these disadvantages and to obtain alternative drugs for a second line therapy, novel
approaches are aimed at following the use of other metal centers or the design of bioorganometallic
compounds, which address other targets than the DNA [3,6–17].
A possible target is represented by both cyclooxygenase (COX) enzymes. The COX-1 isoform is
constitutively active in nearly all human tissues, whereas the isoenzyme COX-2 is basally expressed
only in the central nervous system, the kidney and in female reproduction organs [18]. Relevant
upregulation of COX-2 takes place during inflammation or carcinogenesis. Overexpression of COX-2
in various types of tumors, such as prostate, colon, or breast cancer, is also well known. Moreover,
the prognosis is in general poor for patients suffering breast and prostate carcinoma, which display
a high COX expression [19,20]. It was shown in animal models that the use of COX-inhibitors led
to the reduction of both growth and progression of these tumors. Such inhibitors are renowned as
nonsteroidal anti-inflammatory drugs (NSAIDs), of which acetylsalicylic acid (ASA) might be the most
famous representative.
A series of metal complexes bearing the ASA substructure was already investigated by our
group [12–15]. In this context, ASA derivatives of Zeise’s salt were examined as well [21]. Zeise’s salt,
potassium trichlorido[ethylene]platinate(II) (see Figure 1), was named after its discoverer, the Danish
pharmacist W. C. Zeise. This compound is considered as one of the first, if not the very first,
organometallic compound. In contrast to Cisplatin, Zeise’s salt was never under clinical investigation,
because of its high instability. However, we demonstrated that Zeise’s salt is pharmacologically active
as it inhibited the COX isoenzymes during an incubation time of 10 min [21]. Binding of Zeise’s salt via
a methylene spacer to the carboxylic group of ASA analogously to the design of Co-ASS (see Figure 1)
increased the stability, as well as the COX inhibitory potency. The resulting Pt-Propene-ASA complex
(
1a, see Scheme 1) represents not only a COX-1/2 inhibitor, but it also suppressed the growth of various
tumor cells [21].
Interestingly, if the degradation of Pt-Propene-ASA takes place, the Zeise moiety is not reduced
but a platinum mediated ester cleavage takes place.
These findings unequivocally showed that Zeise’s salt derivatives represent a promising class of
organometallic compounds to be optimized as antitumor agents with a mode of action different from
already established platinum complexes. However, it is necessary to further improve the stability in
aqueous solution in order to realize preclinical evaluations.
In this study, the alkyl spacer between ASA and the Zeise component was elongated in 1a
(–(CH2)n– with n = 1–4) in order to reduce the influence of the platinum on the ester bonding.
The homologous series of Pt-Propene-ASA (n = 1: 1a), Pt-Butene-ASA (n = 2: 2a), Pt-Pentene-ASA
(n = 3: 3a), and Pt-Hexene-ASA (n = 4: 4a) was investigated regarding the COX inhibitory potential and
the influence on the proliferation of the HT-29 colon carcinoma and the MCF-7 breast cancer cell lines.

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2. Results
2.1. Syntheses and Characterization
2.1.1. Syntheses
The syntheses of the target compounds 1a
–
4a are summarized in Scheme 1. The ligands
1–
4
were
obtained by conventional Steglich esterification of ASA with the respective alkenole [22]. In a first
step, ASA built with N,N0-dicyclohexylcarbodiimide (DCC) in the presence of catalytic amounts of
4-dimethylaminopyridine (DMAP) in dry dichloromethane an active N-acyl species that readily reacted
◦
at a low temperature (0 C
→
room temperature (rt)) in a second step with the respective alcohol.
with Zeise’s salt was possible in dry ethanol by stirring at
Complexation of the resulting esters 1–4
elevated temperature within 3 h in a slight excess of the ligand. All of the desired products were
obtained in overall good yields. It is notable that the final organometallic compounds are hygroscopic
and sensitive to light. Thus, they should be stored in a desiccator with protection from light.
The platinum compounds are easily soluble in polar solvents, such as water, alcohol, and acetone,
and they are insoluble in ether or hydrocarbons. In dimethyl sulfoxide (DMSO) rapid Cl−/DMSO
exchange took place, as confirmed by NMR spectroscopy.
Scheme 1. Syntheses of the ligands (top row) and of the final Zeise’s salt derivatives (bottom row).
All of the reactions were carried out under a protective atmosphere of argon. rt = room temperature.
2.1.2. X-ray Structure Analysis
The structure of the Zeise’s salt derivatives was determined by X-ray analysis on the example of
Pt-Propene-ASA (1a). Crystal data and refinement for 1a are submitted in the Supplementary Materials.
Selected bond lengths, distances, and angles are listed in Table 1. For the numbering of the atoms,
see Figure 2.
Crystals of 1a were obtained from a mixture of ethanol/diethyl ether. In the crystal, the potassium
ion is fixed by interactions to two carbonyl oxygens, the oxygen of the co-crystallized ether molecule
and the four chlorido ligands of two PtCl3 moieties (Figure 3, left). The olefin is perpendicularly
π
-bonded at the PtCl3 moiety in a square planar coordination sphere around the platinum (see Figure 2
and Figure 3, middle).

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Table 1. Selected bond lengths and bond angles in the crystal of 1a.
◦
Structural Element
Bond Angle/
Bond Length/Å
Pt(1)–C(1)
Pt(1)–C(2)
Pt(1)–Cl(3) (cis)
Pt(1)–Cl(2) (cis)
Pt(1)–Cl(1) (trans)
C(1)–C(2)
2.114(3)
2.155(3)
2.3030(7)
2.3033(7)
2.3244(7)
1.405(4)
1.484(4)
C(2)–C(3)
C(1)–C(2)–C(3)
C(1)–C(2)–H(2)
C(3)–C(2)–H(2)
Pt(1)–C(2)–C(1)
Pt(1)–C(2)–C(3)
Pt(1)–C(2)–H(2)
119.0(3)
120.0(2)
115.0(2)
69.2(2)
119.5(2)
104.6(2)
Figure 2. ORTEP drawing of complex 1a.
Figure 3. Left: K+ interactions in the crystal of 1a; middle: single molecule of 1a; right: conformational
change of the ASA moiety in 1a.
The distance from the midpoint of the C-C double bond of 1a to the platinum centre (2.015 Å) is
slightly smaller, as in Zeise’s salt (2.022 Å) [23]. The dπ-pπ* back bonding to the vinyl group is stronger
in 1a because of the electron drawing properties of the ASA-CH2- residue and slightly reduces the
length of the C-C bond (1.40 Å) when compared to that of Zeise’s salt (1.44 Å). Contrary to expectations,

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the Pt-Cl bond oriented trans to the ethylene is not substantially elongated (2.32 Å) compared to the
other ones (2.30 Å), which points to only a marginal trans effect.
Overall, the trichlorido[ethylene]platinate moiety of 1a is very similar to Zeise’s salt regarding
bond lengths and angles. The ASA moiety is oriented in the crystal above the square planar
coordination plane. Thereby, the carbonyl oxygen of the acetoxy moiety is at a distance of 3.97 Å above
the platinum (Figure 3, middle).
2.1.3. Molecular Characterization by NMR Spectroscopy
All of the synthesized ligands and the corresponding complexes were fully characterized by
1H and 13C NMR spectroscopy. The assignment of the signals was performed by [1H,1H]-COSY,
[1H,13C]-HSQC, and [1H,13C]-HMBC 2D NMR experiments (see Supplementary Materials).
The coordination of the ligands to platinum was confirmed by a significant upfield shift of the
vinyl protons. The alkene is bound to the platinum in a π-donor/π-acceptor bonding, thus reducing
the double bond character (to about 1.5 as determined in the X-ray structure). As a consequence,
the resonances of the vinyl protons and the carbons of the platinum complexes are shifted 0.87–1.06
and 49.83–55.87 ppm, respectively, to higher field (summarized in Table 2).
Table 2. Selected 1H and 13C chemical shifts of the vinyl group of the ligands
complexes 1a–4a 1.
1–4 and the corresponding
1H NMR
13C NMR
Compound
–CH=CH2
∆δ
–CH=CH2
∆δ
–CH=CH2
∆δ
–CH=CH2
∆δ
1
1a
6.06
5.00
5.42/5.28
4.34/4.31
133.43
77.56
118.65
65.10
1.08/0.97
1.06
55.87
51.08
49.68
49.73
53.55
2
2a
5.90
5.02
5.18/5.08
4.25
135.26
84.18
117.59
65.96
0.93/0.83
0.91/0.82
0.89/0.80
0.88
0.88
0.87
51.63
50.54
50.34
3
3a
5.88
5.00
5.08/4.99
4.17
138.59
88.91
115.59
65.05
4
4a
5.84
4.97
1
5.04/4.95
4.15
139.33
89.60
115.18
64.84
Spectra measured in Acetone-d6. Chemical shifts (δ) are given in ppm.
Because of the unsymmetrical substitution, three =C-H resonances appear in the 1H NMR spectra,
splitted by geminal and vicinal couplings. When compared to Zeise’s salt ( = 4.20), they are shifted to
lower field (e.g.,
1a) = 4.31, 4.34 (=CH2) and 5.00 (=CH–R)). The 2JPt-H remained nearly unchanged
δ
δ
(
(Zeise’s salt: 64 Hz; 1a: 73 Hz (=CH2) and 62 Hz (=CH–R), see Figure 4).
1
Interestingly, the H NMR spectra demonstrate a diastereotopic splitting of the –CH2–CH=
methylene protons for the complexes 1a
δ
–
4a. In the spectrum of 1a (see Figure 4), the resonances are at
= 4.90 (dd, 2J = 11.9 Hz, 3J = 5.5 Hz, 1H, –OC αHβ–), and 4.50 (dd, 2J = 11.9 Hz, 3J = 7.7 Hz, 1H,
β–). The 3J couplings of the –CH2– protons to –CH= reveal a conformational change of the
H
–OCHα
H
ASA-CH2-moiety. When compared to the dihedral angles found in the X-ray structure (60◦ and 180◦),
the coupling constants of 5.5 Hz and 7.7 Hz point to a reduction of the dihedral angle to 40–50◦ and
140–150◦, respectively. The 3JPt-H couplings of about 32 Hz confirm this change (see Figure 4).
While in the crystal of 1a, the spatial structure of the ligand is determined by interactions
to potassium, it can adopt an energetically more preferred conformation in solution. This can
include a reorientation of the carbonyl oxygen of the ester above the platinum (see Figure 3, right).
In this case, a Pt-O distance of about 2 Å can be realized, which is very similar to those of
platinum(IV) acetate complexes [24]. This reorientation facilitates the nucleophilic attack of a water
molecule at the carbonyl C-atom, because the transition state is stabilized by the Pt-O-C interaction.
When compared to a “normal” ester function the attack of a water molecule is eased and the ester is
cleaved platinum-mediated.

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Figure 4. Excerpt of the 1H NMR spectrum of 1a featuring the 2/3JPt–H couplings and the assignments
of the proton signals. The methylene protons show a diastereotopic splitting.
2.1.4. Characterization by Mass Spectrometry
Platinum has six naturally occurring isotopes (190Pt, 192Pt, 194Pt, 195Pt, 196Pt, 198Pt), of which five
are stable (not stable: 190Pt). Upon complexation characteristic mol peaks with the significant line
pattern representing these platinum isotopes confirmed the binding of the ethylene moiety to the
platinum. As an example, the mass spectrum of 1a is submitted in the Supplementary Materials.
2.2. Evaluation of Stability
Capillary electrophoresis (CE) was employed in order to determine the stability of the anionic
platinum(II) complexes in aqueous media. Therefore, a method was established that allowed the
detection of the respective complex and possible degradation products in a short period of time
(for further information on the setup see Section 4). Methanol was chosen as a non-coordinating
co-solvent for the sample solutions (final amount of methanol: 50%) in order to guarantee the solubility
of decomposition products. In contrast to HPLC analysis, it was possible to separate the platinum
complex (tm = 6.3 min) from the free ligand (tm = 4.4 min) using a 50 mM sodium tetraborate solution
◦
(pH = 9.3) as the background electrolyte (temperature: 25 C; UV-detection: 230 nm; capillary:
fused silica 64.5 cm
×
75 µm, effective length 56 cm; 20 kV). The possible degradation products ASA and
salicylic acid (SA) showed migration times of 7.0 min and 8.2 min, respectively. The reactions followed
pseudo-first order kinetics (R2 ≥ 0.99) under the experimental conditions that were implemented.
Complex 1a was stable in methanol for 48 h. Upon the addition of water (1:1 mixture), a fast
decomposition of 1a took place. As previously determined by HPLC analysis, the main degradation
was the cleavage of ASA from the [propenol]PtCl3 moiety. In a further step, ASA was deacetylated to
SA. Neither a release of the ligand
1 from the platinum nor oxidation reactions of Pt(II) were detected.
The half-live (τ1/2) of 1a was determined to be 35.7
the result published before [21].
±
0.6 min. This value is in good agreement with

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The butenol derivative 2a was much more stable with τ1/2 = 69.6
±
3.0 h, indicating that
an additional methylene group between ASA and the alkene-platinum reduced the influence of
the platinum on the ester cleavage. While in 1a the carbonyl group is arranged to the platinum in
a six-membered ring, the additional methylene group would enlarge the ring, for which reason other
conformational orientations might be energetically preferred. A platinum-mediated ester cleavage
played a subordinate role and the stability increased. Further elongation of the spacer between ASA and
the Zeise moiety did not further improve the stability (τ1/2 = 73.5
for 4a).
± 4.7 h for 3a and τ1/2 = 55.4 ± 4.0 h
Substitution reactions with chloride ions can take place under physiological conditions [25].
−
For instance, high Cl concentrations can lead to a substitution of the alkene ligand and the formation
of the tetrachloridoplatinate(II) complex. Therefore, the stability of 2a was examined in the presence
of 154 mM Cl− (extracellular Cl− concentration) in the 50% methanol/water mixture. Interestingly,
instead of the alkene release, the stability significantly increased (recovery of 80.3
complex 2a after 168 h). This finding points to the hindered positioning of the carbonyl oxygen above
the platinum in the presence of high chloride concentrations.
±
3.2% of the intact
The stability of 2a was further investigated in the presence of phosphate-buffered saline (PBS).
In PBS, the chloride concentration is comparable to that used in the above described experiment, but it
is additionally buffered with potassium phosphate to a physiological pH of 7.4. The same conditions
were adjusted in the methanol/water mixture.
To our great surprise, incubation of 2a with PBS resulted in a faster degradation (τ1/2 = 7.95
±
0.09 h)
as in methanol/water. However, no cleavage of the alkenol ester took place, but 2a was degraded to
Pt-Butene-SA. The release of the acetate results in an organometallic compound with lower mobility
and a characteristically changed UV-spectrum. This reaction happened most probably due to the
slightly basic conditions in PBS, allowing for alkaline-caused hydrolysis of the acetyl ester group.
Nevertheless, these results clearly demonstrated that residues at the ethylene ligand of Zeise’s
salt have an impact on the stability of the complex. Zeise’s salt itself is very unstable under aqueous
conditions [25] and it degrades in a redox reaction building acetaldehyde and Pt(0). This reaction was
not observed in case of the complexes 1a–4a. To determine the biological effects of Zeise’s salt only short
time incubations (up to 15 min) are possible, e.g., determination of COX the inhibition (see Section 2.3.1;
incubation time: 10 min). Other experiments, such as cytotoxicity assays (see Section 2.3.2; incubation
time 72 h), are only significant for the complexes 1a–4a.
2.3. Biological Evaluation
2.3.1. COX-1/2 Isoenzyme Inhibition
The impact of the compounds (10 µM) to inhibit the isoenzymes COX-1 and COX-2, respectively,
was investigated (Figure 5). The parent compound ASA displayed a COX-1 inhibition of 29.2% and was
inactive towards COX-2 (1.0%). In contrast, Zeise’s salt and its derivatives revealed greater inhibitory
properties. Zeise’s salt caused a strong inhibition of COX-1 (83.8%), while it showed just a marginal
reduction of COX-2 activity (11.3%). The inhibitory potency of the complexes 1a–4a depended on the
spacer between ASA and the Zeise moiety. The compounds 1a and 2a exhibited slightly less COX-1
inhibition when compared to Zeise’s salt (67.3% and 70.0%, respectively). 3a (93.8%) and 4a (96.6%)
nearly reached a 100% inhibition. The same trend was observed at COX-2, however, on a much
lower level: Zeise’s salt (11.3%) < 1a (17.4%) < 2a (28.7%) < 4a (33.0%). Only 3a did not fit this series.
It reduced COX-2 merely to the same amount (15.3%) as Zeise’s salt.

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Figure 5. Inhibition of isolated ovine COX-1 and COX-2. Values are given as % COX inhibition
calculated from initial activity of vehicle treated enzyme and represent the mean
of ≥2 independent experiments.
±
standard deviation
2.3.2. Antiproliferative Effects
The growth inhibitory effects of ASA, Zeise’s salt as well as the thereof derived complexes 1a–4a
were investigated in vitro against the HT-29 and the MCF-7 cell lines. Thereby, an already published
crystal violet assay [26] was employed, in which the cell biomass reduced by the substances was
determined as parameter. Cisplatin was applied as positive control. The resulting IC50 values are listed
in Table 3.
Table 3. Inhibition of the cell growth in colon carcinoma (HT-29) and breast cancer (MCF-7) cell lines
determined in a crystal violet cell biomass assay.
IC50/µM 1
Compound
HT-29
MCF-7
ASA
Zeise’s salt
≥50
≥50
≥50
≥50
1a
2a
3a
49.7 ± 1.8
31.4 ± 0.4
44.2 ± 3.4
41.4 ± 0.3
2.6 ± 0.1
≥50
30.1 ± 1.5
37.4 ± 2.5
43.7 ± 5.7
3.7 ± 0.3
4a
Cisplatin
1
IC50 values represent the concentration where 50% of the compounds maximum antiproliferative activity
compared to vehicle treated control was achieved. Results are given as mean
independent experiments.
±
standard error of mean of
≥
2
ASA and Zeise’s salt did not show antiproliferative activity towards both cell lines at the used
concentrations (IC50 50 M). Both of the cell lines are comparably sensitive to the Zeise’s salt
derivatives. The lowest effects showed 1a with a 50% inhibition at 50 M (IC50 50 M). Elongation
of the spacer by one methylene group (2a) increased the cytotoxicity to an IC50 value of about 31 M.
Further elongation slightly reduced the activity, but 3a (IC50 (HT-29) = 44.2 M; IC50 (MCF-7) = 37.4 M)
and 4a (IC50 (HT-29) = 41.4 M; IC50 (MCF-7) = 43.7 M) were even more active than ASA and Zeise’s
≥
µ
µ
≈
µ
µ
µ
µ
µ
µ
salt. However, all of the complexes were 10- to 20-fold less active than Cisplatin (IC50 (HT-29) = 2.6 µM
and IC50 (MCF-7) = 3.7 µM; see Table 3).
3. Discussion
NSAIDs are drugs with analgetic, antiphlogistic, anti-inflammatory, and anticoagulative
properties. All of them share the ability to inhibit cyclooxygenase enzymes.

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Out of this drug class ASA is the most prominent representative and is the subject of extensive
pharmacological investigations. During the last years, various epidemiological studies revealed that
ASA could be used for the prevention of tumor development, such as colon and breast cancer [27,28].
However, no information is available that ASA can be used for the treatment of established tumors.
Nevertheless, COX enzymes might be targets for the design of new anticancer drugs, because some
tumor entities grow under the influence of COX.
In our studies, ASA was inactive against the HT-29 colon cancer and the MCF-7 breast cancer
cell lines in vitro up to a concentration of 100 µM. In contrast, Zeise’s salt derivatives, which are more
potent inhibitors of COX-1/2, caused antiproliferative effects against these cell lines. Zeise’s salt itself
is not stable in cell culture medium and it degraded immediately after addition. The derivatives
1a–4a might also be not stable under these conditions, but as the experiments with 2a in PBS
demonstrated, at first, only a loss of the acetyl residue can be assumed preventing the essential
trichlorido[alkene]platinum(II) moiety. The high chloride concentration should decrease the cleavage
of ASA or SA from the trichlorido[alkenol]platinum(II) moiety.
However, degradation by ester cleavage might be the most critical point. Complex 1a having
the lowest stability under aqueous conditions showed the lowest activity against the used cell lines.
The derivatives 2a–4a, which were more stable, reduced the cell growth with IC50 values that were
between 30 and 40 µM. When compared to Cisplatin (IC50 values of about 3 µM), they are more than
10-fold less active.
The reason for this weaker cytotoxicity might be the difference of the intracellular targets. While
the main target of Cisplatin represents the DNA, Zeise’s salt derivatives can bind to isolated DNA [21],
but it seems that the intrastrand cross-links, which are necessary for high cytotoxicity, are not built in
the case of Zeise’s salt derivatives or were not as stable as found for Cisplatin.
As already published earlier, Cisplatin and Zeise’s salt showed nearly the same uptake kinetic
in tumor cells, while 2a was four- to five-fold higher accumulated. Therefore, partial inactivation by
ingredients of the cell culture medium can also be considered as a reason for their low cytotoxicity.
Although the X-ray structure revealed only a marginal elongation of the Pt–Cl bond trans to
the alkene, it is well accepted that the trans effect of the alkene moiety forces on the one hand a fast
exchange of the chlorido ligand trans to the olefin by stronger ligands, such as amines and thiols.
On the other hand strong σ-donors might have a weakening effect on the Pt-alkene bonding and can
cause olefin release. Incubation of 2a with an excess of L-cysteine yielded a yellow precipitate that was
not soluble in common solvents. We assume that Pt(Cys)2 was built, which is a well-known degradation
product of Cisplatin. Formation of amino acid-platinum compounds is likewise responsible for the
partial deactivation of Cisplatin upon injection into the patient’s blood stream [29,30].
In summary, we synthesized a series of Zeise’s salt derivatives with ASA substructure. We were
able to enhance the stability of the complexes on the one hand and the COX inhibitory potential on the
other. Interference in the arachidonic acid cascade as mode of action is assumed. However, further
investigations regarding the mode of action are necessary and the improvement of activity at the COX
enzymes is currently undertaken.
4. Materials and Methods
4.1. General Aspects
All of the chemicals were purchased either from Sigma-Aldrich, Alfa Aesar, Fluka, Euriso-Top,
or TCI Chemicals and were used as received without further purification. Solvents were purchased in
appropriate purity or were freshly distilled prior to utilization. Water was deionized using a Millipore
Milli-Q Gradient A10 Water Purification system (Merck Millipore, Billerica, MA, USA). Column
chromatography was performed using silica gel 60 (particle size 40–63 µm). Reactions were monitored
by thin layer chromatography (TLC) that was carried out on “TLC Silica gel 60 F254 aluminium sheets”
with a fluorescence indicator (Merck, Darmstadt, Germany). Detection was performed by observation

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under UV-light at 254 and/or 365 nm, respectively. Melting points (m.p.) were determined using
a Kofler hot bench (Wagner & Munz, Munich, Germany) and uncorrected values are given.
1H and 13C NMR spectroscopy was performed employing either a Bruker Avance 4 Neo
(1H resonance frequency: 400 MHz) or an Agilent Direct Drive 2 (1H resonance frequency:
500 MHz) spectrometer. For the correct assignment of the signals, [1H,1H]-COSY, [1H,13C]-HSQC and
[1H,13C]-HMBC 2D NMR experiments were carried out. Chemical shifts
δ
are given in parts per million
(ppm). Coupling constants J are given in Hertz (Hz). Chemical shifts of H and 13C experiments
1
were referenced using the center of the internal residual peak of the solvent multiplet, which was
related to tetramethylsilane (TMS) as
The subscripts and indicate the downfield shifted proton (subscript
proton (subscript β) of a diastereotopic methylene moiety.
δ
= 2.05 (1H NMR) and
δ
= 29.84 (13C NMR) for Acetone-d6 [31].
), respectively, upfield shifted
α
β
α
High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed using
an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The ligands were
analyzed in positive mode and the platinum complexes in negative mode. The peaks of greatest
intensity are presented.
Capillary electrophoresis experiments were carried out on a 3D-CE system (Agilent, Santa Clara,
CA, USA), which was equipped with an autosampler, a diode array detector (DAD) and a temperature
controlled column compartment. Agilent fused-silica capillaries (75
64.5 cm total length) were purchased from VWR (Vienna, Austria).
µm i.d.; 56 cm effective length,
The purity of all the compounds was above 95%, as determined by CE.
4.2. Capillary Electrophoresis
A 50 mM sodium tetraborate solution with a pH of 9.3 (adjusted with 1 M NaOH solution) was
used as the background electrolyte (BGE). Prior to the first utilization new capillaries were washed
with 1 M NaOH solution (45 min), water (45 min) and BGE (45 min). Samples were injected into the
◦
capillary via hydrodynamic mode (50 mbar for 2 s). The capillary was thermostated at 25 C, whereas
the autosampler was kept at 37 ◦C by an external water bath. The separation voltage was 20 kV and
the detection wavelength was set to 230 nm. The required run time was 10 min. The capillary was
flushed consecutively with 0.1 M NaOH solution (3 min), water (3 min), and BGE (5 min) before
each measurement. For the purpose of stability testing, the complexes were dissolved in methanol
and diluted 1:1 (v/v) with water, 1.8% aqueous NaCl solution, or double-concentrated PBS to a final
concentration of 1 mM. Every experiment was performed in triplicate. The half-lives are presented
as mean
±
standard deviation. All of the samples, buffers, and washing solutions were membrane
filtered (0.22 µm porosity) and degassed by ultrasonication prior to usage.
4.3. X-ray Crystallography
For single crystal structure analysis, a suitable crystal has been prepared under a polarization
microscope and immediately placed into a stream of cold nitrogen (173 K) inside a Bruker
D8 Quest diffractometer (Photon 100) that was equipped with an Incoatec Microfocus source
generator (multi layered optics monochromatized Mo-Kα radiation,
λ = 71.073 pm). Multi-scan
absorption corrections were applied with the program SADABS-2014/5. After structure solution
and parameter refinement with anisotropic displacement parameters for all of the atoms using the
SHELXS/L-13 [32,33] software suite, the space group P21/c was found to be correct. Hydrogen
atoms at C1 and C2 were found and refined with isotropic displacement parameters without any
restraints. Additional details of the crystal structure investigation may be obtained from the Cambridge
Crystallographic Data Centre (CCDC). The supplementary crystallographic data of 1a were deposited
as CCDC number 1831275 and these data are provided free of charge.

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4.4. General Procedure for the Synthesis of the Acetylsalicylic Acid Esters 1–4
Following the protocol of Steglich et al. [22], under a protective atmosphere of argon
1.00 equivalents of ASA (10.0 mmol), 1.10 equivalents of the respective alcohol (11.0 mmol) and
10.0 mol% DMAP were dissolved in 30 mL of anhydrous dichloromethane and were cooled with
an ice bath. Then, a solution containing 1.05 equivalents of DCC (10.5 mmol) in 10 mL of anhydrous
dichloromethane was added over a period of 5 min via syringe. The reaction mixture was stirred
for another 10 min at 0 ◦C, and then for 3.5 h at room temperature. After completion of the reaction
(TLC monitoring; eluent: petroleum ether/ethyl acetate = 5:1), the mixture was washed each three
times with aqueous 1 M HCl solution and saturated aqueous NaHCO3 solution and once with brine.
The organic phase was dried over anhydrous Na2SO4 and evaporated. Pure products were obtained
after column chromatography (petroleum ether/ethyl acetate = 5:1).
4.4.1. (Prop-2-en-1-yl)-2-acetoxybenzoate (Propene-ASA, 1)
Yield: 80% as colorless oil. HR-ESI-MS: calculated for C12H12O4Na [
1
+Na]+: 243.0628. Found:
1
3
4
m/z 243.0628. H NMR (400 MHz, Acetone-d6):
δ
= 8.02 (dd, J = 7.8 Hz, J = 1.7 Hz, 1H, ArH-6),
7.66 (ddd, 3J = 8.1 Hz, 3J = 7.5 Hz, 4J = 1.7 Hz, 1H, ArH-4), 7.40 (ddd, 3J = 7.7 Hz, 3J = 7.7 Hz, 4J = 1.2 Hz,
3
4
3
3
1H, ArH-5), 7.20 (dd, J = 8.1 Hz, J = 1.2 Hz, 1H, ArH-3), 6.06 (ddt, J = 17.3 Hz, J = 10.5 Hz,
3
2
4
3J = 5.7 Hz, 1H, –C
H=CH2), 5.42 (ddt, J = 17.2 Hz, J = J = 1.6 Hz, 1H, =CH2, trans), 5.28 (ddt,
3J = 10.5 Hz, 2J = 4J = 1.4 Hz, 1H, =CH2, ), 4.78 (dt, 3J = 5.7 Hz, 4J = 1.4 Hz, 4J = 1.4 Hz, 2H, –OCH2–),
cis
2.26 (s, 3H, –CH3); 13C NMR (101 MHz, Acetone-d6):
δ = 169.69 (–(C=O)–CH3), 164.76 (Ar-(C=O)),
151.72 (C2), 134.83 (C4), 133.43 (C20), 132.27 (C6), 126.86 (C5), 124.92 (C3), 124.59 (C1), 118.65 (C30),
66.23 (C10), 21.01 (–CH3).
4.4.2. (But-3-en-1-yl)-2-acetoxybenzoate (Butene-ASA, 2)
Yield: 78% as colorless oil. HR-ESI-MS: calculated for C13H14O4Na [
2
+Na]+: 257.0784. Found:
1
m/z 257.0785. H NMR (400 MHz, Acetone-d6):
δ
= 8.00 (dd, 3J = 7.8 Hz, 4J = 1.8 Hz, 1H, ArH-6), 7.65
(ddd, 3J = 8.1 Hz, 3J = 7.4 Hz, 4J = 1.7 Hz, 1H, ArH-4), 7.38 (ddd, 3J = 7.6 Hz, 3J = 7.6 Hz, 4J = 1.2 Hz, 1H,
ArH-5), 7.19 (dd, 3J = 8.1 Hz, 4J = 1.2 Hz, 1H, ArH-3), 5.90 (ddt, 3J = 17.1 Hz, 3J = 10.3 Hz, 3J = 6.8 Hz, 1H,
–C
4J = 1.3 Hz, 1H, =CH2, ), 4.32 (t, 3J = 6.7 Hz, 2H, –OCH2–), 2.51 (tdt, 3J = 6.7 Hz, 3J = 6.7 Hz, 4J = 1.4 Hz,
H
=CH2), 5.18 (ddt, 3J = 17.2 Hz, 2J = 4J = 1.7 Hz, 1H, =CH2, trans), 5.08 (ddt, 3J = 10.3 Hz, 2J = 2.3 Hz,
cis
2H, –CH2–), 2.28 (s, 3H, –CH3); 13C NMR (101 MHz, Acetone-d6):
δ = 169.65 (–(C=O)–CH3), 164.97
(Ar-(C
=O)), 151.73 (C2), 135.26 (C30), 134.71 (C4), 132.20 (C6), 126.79 (C5), 124.89 (C3), 124.67 (C1),
117.59 (C40), 64.76 (C10), 33.79 (C20), 21.02 (–CH3).
4.4.3. (Pent-4-en-1-yl)-2-acetoxybenzoate (Pentene-ASA, 3)
Yield: 60% as colorless oil. HR-ESI-MS: calculated for C14H16O4Na [
m/z 271.0920. H NMR (500 MHz, Acetone-d6):
3
+Na]+: 271.0941. Found:
1
δ
= 8.02 (dd, 3J = 7.8 Hz, 4J = 1.5 Hz, 1H, ArH-6), 7.65
(ddd, 3J = 8.1 Hz, 3J = 7.3 Hz, 4J = 1.6 Hz, 1H, ArH-4), 7.39 (ddd, 3J = 7.6 Hz, 3J = 7.6 Hz, 4J = 1.1 Hz, 1H,
ArH-5), 7.19 (d, 3J = 8.1 Hz, 1H, ArH-3), 5.88 (ddt, 3J = 17.0 Hz, 3J = 10.2 Hz, 3J = 6.7 Hz, 1H, –C
=CH2),
5.08 (ddt, 3J = 17.2 Hz, 2J = 4J = 1.6 Hz, 1H, =CH2, trans), 4.99 (ddt, 3J = 10.2 Hz, 2J = 2.2 Hz, 4J = 1.2 Hz,
1H, =CH2, ), 4.28 (t, 3J = 6.6 Hz, 2H, –OCH2–), 2.28 (s, 3H, –CH3), 2.24–2.18 (m, 2H, –C
2–CH=), 1.85
= 169.63 (–( =O)–CH3),
H
H
cis
(tt, 3J = 7.0 Hz, 3J = 7.0 Hz, 2H, –CH2–); 13C NMR (126 MHz, Acetone-d6):
δ
C
165.01 (Ar-(C
=O)), 151.67 (C2), 138.59 (C40), 134.65 (C4), 132.20 (C6), 126.79 (C5), 124.85 (C3), 124.69
(C1), 115.59 (C50), 65.08 (C10), 30.76 (C30), 28.63 (C20), 21.01 (–CH3).
4.4.4. (Hex-5-en-1-yl)-2-acetoxybenzoate (Hexene-ASA, 4)
Yield: 82% as colorless oil. HR-ESI-MS: calculated for C15H18O4Na [
4
+Na]+: 285.1097. Found:
1
3
4
m/z 285.1070. H NMR (500 MHz, Acetone-d6):
δ
= 8.00 (dd, J = 7.9 Hz, J = 1.7 Hz, 1H, ArH-6),
7.67–7.62 (m, 1H, ArH-4), 7.39 (dd, 3J = 7.6 Hz, 3J = 7.6 Hz, 1H, ArH-5), 7.19 (d, 3J = 7.9 Hz, 1H, ArH-3),

Int. J. Mol. Sci. 2018, 19, 1612
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5.84 (ddt, 3J = 17.0 Hz, 3J = 10.1 Hz, 3J = 6.7 Hz, 1H, –C
H
=CH2), 5.08–5.00 (m, 1H, =CH2, trans), 4.95
3
2
4
3
(ddt, J = 10.2 Hz, J = 2.1 Hz, J = 1.0 Hz, 1H, =CH2, ), 4.28 (t, J = 6.7 Hz, 2H, –OCH2–), 2.28
cis
3
3
3
3
(s, 3H, –CH3), 2.13 (dt, J = 7.8 Hz, J = 7.8 Hz, 2H, –C
2H, –OCH2–C
δ = 169.62 (–(
H2–CH=), 1.77 (tt, J = 6.7 Hz, J = 6.7 Hz,
H
C
2–), 1.54 (tt, 3J = 7.7 Hz, 3J = 7.7 Hz, 2H, –CH2–); 13C NMR (126 MHz, Acetone-d6):
=O)–CH3), 165.04 (Ar-(C0=O)), 151.65 (C2), 139.303 (C50), 134.63 (C4), 132.18 (C6), 126.78
0
0
0
(C5), 124.86 (C3), 124.75 (C1), 115.18 (C6 ), 65.54 (C1 ), 34.00 (C4 ), 28.88 (C2 ), 25.97 (C3 ), 21.02 (–CH3).
4.5. General Procedure for the Synthesis of the Zeise’s Salt Derivatives 1a–4a
In an argon atmosphere, 1.0 equivalents of Zeise’s salt (0.30 mmol) was dissolved in 8 mL of
degassed (three cycles freeze-pump-thaw), anhydrous ethanol under protection from light, and then
stirred at room temperature. Then, 1.2 equivalents of the respective ligand (0.36 mmol), which was
dissolved in approximately 2 mL of degassed and dry ethanol was added dropwise via syringe.
◦
After addition, the mixture was stirred at 48 C for 3 h. The mixture was then allowed to cool to
room temperature, upon which it was filtered and evaporated. Recrystallization from ethanol/diethyl
ether afforded pure solid products. The crystals that were obtained in case of 1a were suitable for
X-ray analysis.
4.5.1. Potassium {trichlorido[
η
2-(prop-2-en-1-yl)-2-acetoxybenzoate]platinate(II)} (Pt-Propene-ASA, 1a
)
◦
Yield: 95% as yellow crystalline needles. m.p.: 94 C (decomposition). HR-ESI-MS: calculated for
C12H12Cl3O4Pt [1a–K]−: 520.9432. Found: m/z 520.9431. H NMR (400 MHz, Acetone-d6):
δ = 8.10
1
(dd, 3J = 7.8 Hz, 4J = 1.7 Hz, 1H, ArH-6), 7.65 (ddd, 3J = 8.1 Hz, 3J = 7.4 Hz, 4J = 1.7 Hz, 1H, ArH-4),
7.39 (ddd, 3J = 7.6 Hz, 3J = 7.6 Hz, 4J = 1.2 Hz, 1H, ArH-5), 7.19 (dd, 3J = 8.1 Hz, 4J = 1.2 Hz, 1H, ArH-3),
2
3
3
3
3
5.00 (dddd, JPt-H = 62 Hz, J = 13.0 Hz, J = 7.7 Hz, J = 7.7 Hz, J = 5.5 Hz, 1H, –C
(dd, 3JPt-H = 32 Hz, 2J = 11.9 Hz, 3J = 5.5 Hz, 1H, –OC αHβ–), 4.50 (dd, 3JPt-H = 32 Hz, 2J = 11.9 Hz,
3J = 7.7 Hz, 1H, –OCHα β–), 4.34 (dd, 2JPt-H = 73 Hz, 3J = 12.8 Hz, 2J = 1.4 Hz, 1H, =CH2, trans), 4.31
(dd, 2JPt-H = 74 Hz, 3J = 7.9 Hz, 2J = 1.4 Hz, 1H, =CH2, ), 2.33 (s, 3H, –CH3); 13C NMR (101 MHz,
H=CH2), 4.90
H
H
cis
Acetone-d6): δ = 169.84 (–(C=O)–CH3), 164.98 (Ar-(C=O)), 151.64 (C2), 134.67 (C4), 132.57 (C6), 126.79
(C5), 124.81 (C3), 124.68 (C1), 77.56 (C20), 65.37 (C10), 65.10 (C30), 21.26 (–CH3).
4.5.2. Potassium {trichlorido[η2-(but-3-en-1-yl)-2-acetoxybenzoate]platinate(II)} (Pt-Butene-ASA, 2a)
Yield: 93% as yellow powder. m.p.: 118 ◦C (decomposition). HR-ESI-MS: calculated for
1
C13H14Cl3O4Pt [2a–K]−: 535.9590. Found: m/z 535.9581. H NMR (400 MHz, Acetone-d6): δ = 8.08
(dd, 3J = 7.8 Hz, 4J = 1.8 Hz, 1H, ArH-6), 7.64 (ddd, 3J = 7.7 Hz, 3J = 7.7 Hz, 4J = 1.8 Hz, 1H, ArH-4),
7.39 (ddd, 3J = 7.6 Hz, 3J = 7.6 Hz, 4J = 1.2 Hz, 1H, ArH-5), 7.18 (dd, 3J = 8.1 Hz, 4J = 1.2 Hz, 1H, ArH-3),
5.16–4.87 (m, 2JPt-H = 62 Hz, 1H, –C
H
=CH2), 4.70–4.59 (m, 2H, –OCH2–), 4.38–4.11 (m, 2JPt-H = 60 Hz,
αHβ–), 2.30 (s, 3H,
2H, =CH2), 2.57 (dddd, 2J = 14.1 Hz, 3J = 8.0 Hz, 3J = 6.0 Hz, 3J = 6.0 Hz, 1H, –C
–CH3), 2.10–1.99 (m, 1H, –CHα = 169.76 (–(
β–); 13C NMR (101 MHz, Acetone-d6):
165.11 (Ar-(
65.96 (C40), 64.86 (C10), 33.36 (C20), 21.15 (–CH3).
H
H
δ
C
=O)–CH3),
0
C
=O)), 151.68 (C2), 134.61 (C4), 132.43 (C6), 126.81 (C5), 124.81 (C3), 124.74 (C1), 84.18 (C3 ),
4.5.3. Potassium {trichlorido[η )
2-(pent-4-en-1-yl)-2-acetoxybenzoate]platinate(II)} (Pt-Pentene-ASA, 3a
Yield: 57% as yellow powder. m.p.: 96 ◦C (decomposition). HR-ESI-MS: calculated for
1
C14H16Cl3O4Pt [3a–K]−: 548.9734. Found: m/z 548.9771. H NMR (500 MHz, Acetone-d6): δ = 8.06
(dd, 3J = 7.9 Hz, 4J = 1.7 Hz, 1H, ArH-6), 7.64 (ddd, 3J = 8.1 Hz, 3J = 7.5 Hz, 4J = 1.8 Hz, 1H, ArH-4),
3
3
4
3
4
7.38 (ddd, J = 7.6 Hz, J = 7.6 Hz, J = 1.2 Hz, 1H, ArH-5), 7.18 (dd, J = 8.1 Hz, J = 1.2 Hz,
2
1H, ArH-3), 5.10–4.89 (m, JPt-H = 66 Hz, 1H, –C
H
=CH2), 4.35–4.33 (m, 2H, –OCH2–), 4.24–4.09
(m, 2JPt-H = 64 Hz, 2H, =CH2), 2.40–2.31 (m, 1H, 20-Hα), 2.30 (s, 3H, –CH3), 2.28–2.21 (m, 1H, 30-Hα),
2.15–2.07 (m, 1H, 20-Hβ), 1.70 (dddd, 2J = 13.2 Hz, 3J = 9.3 Hz, 3J = 6.2 Hz, 3J = 6.2 Hz, 1H, 30-Hβ);
13C NMR (126 MHz, Acetone-d6):
δ = 169.75 (–(C=O)–CH3), 165.08 (Ar-(C=O)), 151.67 (C2), 134.56

Int. J. Mol. Sci. 2018, 19, 1612
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(C4), 132.39 (C6), 126.77 (C5), 124.77 (C3), 88.91 (C40), 65.43 (C10), 65.05 (C50), 30.60 (C30), 29.04 (C20),
21.13 (–CH3); C1 not observed.
4.5.4. Potassium {trichlorido[η )
2-(hex-5-en-1-yl)-2-acetoxybenzoate]platinate(II)} (Pt-Hexene-ASA, 4a
Yield: 19% as yellow powder. m.p.: 100 ◦C (decomposition). HR-ESI-MS: calculated for
1
C15H18Cl3O4Pt [4a–K]−: 562.9891. Found: m/z 562.9928. H NMR (500 MHz, Acetone-d6): δ = 8.06
(dd, 3J = 7.9 Hz, 4J = 1.7 Hz, 1H, ArH-6), 7.63 (ddd, 3J = 7.7 Hz, 3J = 7.7 Hz, 4J = 1.7 Hz, 1H, ArH-4),
7.40 (ddd, 3J = 7.6 Hz, 3J = 7.6 Hz, 4J = 1.2 Hz, 1H, ArH-5), 7.18 (dd, 3J = 8.2 Hz, 4J = 1.2 Hz, 1H, ArH-3),
5.10–4.84 (m, 2JPt–H = 60 Hz, 1H, –C =CH2), 4.37–4.25 (m, 2H, –OCH2–), 4.25–4.04 (m, 2JPt-H = 59 Hz,
H
20H, =CH2), 2.29 (s, 3H, –CH3), 2.27–2.19 (m, 1H, 40-Hα), 2.03–1.97 (m, 1H, 20-Hα), 1.94–1.87 (m, 1H,
0
0
3 -Hα), 1.88–1.81 (m, 1H, 3 -Hβ), 1.81–1.71 (m, 1H, 2 -Hβ), 1.65 (ddt, 2J = 13.1 Hz, 3J = 9.0 Hz, 3J = 6.5 Hz,
1H, 40-Hβ); 13C NMR (126 MHz, Acetone-d6):
δ = 169.71 (–(C=O)–CH3), 165.13 (Ar-(C=O)), 151.62
(C2), 134.51 (C4), 132.44 (C6), 126.88 (C5), 124.82 (C1), 124.75 (C3), 89.60 (C50), 65.69 (C10), 64.84 (C60),
33.80 (C40), 29.15 (C20), 26.36 (C30), 21.12 (–CH3).
4.6. General Methods for the Cell Culture
The HT-29 colon carcinoma and the MCF-7 breast cancer cell lines were obtained from the cell line
service (CLS, Eppelheim, Germany). The HT-29 and the MCF-7 cells were cultivated as a monolayer
in Dulbecco’s Modified Eagle Medium (DMEM) containing glucose (4.5 g/L) and lacking phenol
red (GE Healthcare, Little Chalfont, UK), supplemented with fetal calf serum (FCS; 10%; Biochrom)
and L-glutamine (584 mg/L; GE Healthcare). The cells were cultured in a humidified atmosphere
◦
(5% CO2/95% air) at 37 C and were passaged regularly twice a week. The cell lines were characterized
by typing short tandem repeats and routinely monitored for mycoplasma infection.
4.6.1. COX-1/2 Isoenzyme Inhibition
The inhibition of both isolated isoenzymes, ovine COX-1 and ovine COX-2, respectively,
were investigated at a concentration of 10
µM of the respective substances while employing
an enzyme immunoassay (COX Inhibitor Screening Assay, Cayman Chemicals, Ann Arbor, MI, USA).
The conduction of the assay was performed following the manufacturer’s protocol. Thereby,
the isoenzymes were incubated with the particular compounds for exactly 10 min. The values express
the mean of duplicate determinations and they represent the mean
independent experiments. The untreated control was set to 0% inhibition.
±
standard deviation of
≥
2
4.6.2. Antiproliferative Effects
The effects of the newly synthesized substances on the cell biomass were determined in vitro
while employing a crystal violet assay. The testing was performed following a modified protocol, as
previously published [26]. Exponentially growing HT-29 and MCF-7 cells were seeded in 96-well tissue
culture plates with a density of 3
×
103 and 2
×
103 cells, respectively, in 100
µL of completed DMEM
per well in quadruples. The plates were kept in a humidified atmosphere (5% CO2/95% air) at 37 ◦C
for 24 h prior to the addition of completed medium containing the vehicle DMF, the positive control
Cisplatin, and the compounds to be tested, respectively. After 72 h of incubation, the medium was
removed, the cells were washed with PBS and fixed using a solution of glutaraldehyde in PBS (1%, v/v).
The cell biomass was examined by staining the chromatin of adherent cells with crystal violet, extraction
of the stain with ethanol (70%, v/v) and following measurement of absorbance (λ = 590 nm). The cell
viability is given as percentage of cell viability of the vehicle treated control, which was set to 100%.
The IC50 values represent the mean
they were calculated with Prism 7.0 (GraphPad, San Diego, CA, USA) using nonlinear regression and
decadal logarithm of the inhibitor versus variable slope equation. The top constraint was set to 100%.
±
standard error of mean of
≥
2 independent experiments and

Int. J. Mol. Sci. 2018, 19, 1612
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Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/6/
1612/s1.
Author Contributions: A.W. synthesized and characterized the compounds and tested their stability; D.B. and
D.E. determined cytotoxicity values; V.O. and D.B. evaluated the COX inhibition. K.W. recorded and processed the
X-ray crystal structure of 1a. A.W., K.W. and R.G. analyzed the X-ray crystal structure of 1a. A.W. and D.B. wrote
the article under the supervision of R.G. All authors have given approval to the final version of the manuscript.
Acknowledgments: The authors would like to thank Monika Cziferszky and Peter Enoh (both Department of
Pharmaceutical Chemistry, Institute of Pharmacy, University of Innsbruck) for recording of the mass spectra and
the group of Martin Tollinger (Institute of Organic Chemistry, University of Innsbruck) for recording the 500 MHz
NMR spectra. The publication of this article (APC) has been funded by the University of Innsbruck. We gratefully
acknowledge the support by the FFG (Projekt 858017—West-Austrian BioNMR).
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
ASA
acetylsalicylic acid
BGE
CCDC
CE
background electrolyte
Cambridge Crystallographic Data Centre
capillary electrophoresis
cyclooxygenase
COX
DAD
DCC
DMAP
DMF
DMSO
HR-ESI-MS
m.p.
diode array detector
0
N,N -dicyclohexylcarbodiimide
4-dimethylaminopyridine
dimethylformamide
dimethyl sulfoxide
high resolution electrospray ionization mass spectrometry
melting point
NMR
NSAID
PBS
nuclear magnetic resonance
nonsteroidal anti-inflammatory drug
phosphate-buffered saline
salicylic acid
SA
TLC
TMS
thin layer chromatography
tetramethylsilane
τ1/2
half-live
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