N-Metallocenoylsphingosines as targeted ceramidase inhibitors: Syntheses and antitumoral effects

Article abstract of DOI:10.1016/j.bioorg.2020.103703

Three N-metallocenoylsphingosines with variance in the central metal (Fe, Co, Ru), the charge (neutral or cationic), and the arene ligands (Cp₂, Cp*Ph) were synthesized from serine and metallocene carboxylic acids as substrate-analogous inhibit

Full text of DOI:10.1016/j.bioorg.2020.103703

BioorganicChemistry97(2020)103703
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
N-Metallocenoylsphingosines as targeted ceramidase inhibitors: Syntheses
and antitumoral effects
Matthias Rothemunda,1, Alexander Bära,1, Felix Klattb, Sascha Weidlerc, Leonhard Köhlera,
⁎
Carlo Unverzagtc, Claus-D. Kuhnb, Rainer Schoberta,
a Department of Chemistry, University Bayreuth, Universitaetsstrasse 30, 95440 Bayreuth, Germany
b Gene Regulation by Non-Coding RNA, Elite Network of Bavaria and University of Bayreuth, Universitaetsstr. 30, 95447 Bayreuth, Germany
c Bioorganic Chemistry, University of Bayreuth, Universitaetsstr. 30, 95447 Bayreuth, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three N-metallocenoylsphingosines with variance in the central metal (Fe, Co, Ru), the charge (neutral or ca-
tionic), and the arene ligands (Cp2, Cp*Ph) were synthesized from serine and metallocene carboxylic acids as
substrate-analogous inhibitors of human acid ceramidase (AC). Their inhibitory potential was examined using
the recombinant full length ASAH1 enzyme, expressed and secreted from High Five insect cells, and the fluor-
escent substrate Rbm14-12. All complexes inhibited AC, most strongly so ruthenium(II) complex 13a. Some
antitumoral effects of the complexes, such as the interference with the microtubular and F-actin cytoskeleton of
cancer cells, were correlated to their AC-inhibition, whereas others, e.g. their cytotoxicity and their induction of
caspase-3/-7 activity in cancer cells, were not. All complexes accumulated preferentially in the lysosomes of
cancer cells like their target AC, arrested the cells in G1 phase of the cell cycle, and displayed cytotoxicity with
mostly single-digit micromolar IC50 values while inducing cancer cell apoptosis.
Ceramidase inhibitors
Anticancer agents
Colocalization
Organometallic drugs
Sphingolipids
1. Introduction
ceramidases are known, called alkaline, neutral and acid ceramidase
according to their pH optimum [1,2]. Due to its role in Farber’s disease,
The sphingolipid rheostat is a delicately balanced network of
sphingosine derivatives and pertinent enzymes, regulating growth,
differentiation, motility, survival and apoptosis of endothelial and
cancer cells [1–4]. Key players of this rheostat are the cell death pro-
moting ceramides (1, cer) and sphingosine (2a, sph), as well as sphin-
gosine-1-phosphate (3, s1p) which promotes cell survival and pro-
liferation (Fig. 1) [5–8]. Increased levels of cer and sph are associated
with greater permeability of lysosomal and mitochondrial membranes,
and thus with an activation of the lysosomal and the intrinsic apoptotic
pathway [9–11]. Both metabolites were reported to impair cell cycle
progression. Imbalancing the rheostat led to Golgi fragmentation and
loss of cell-cell contacts, and eventually to anoikis and cell starvation
[12,13]. The anti-apoptotic s1p can be generated only by conversion of
cer to sph by ceramidases and a subsequent phosphorylation of sph by
sphingosine kinases (SK) [14,15]. In human cells three types of
a deficiency in lysosomal N-acylsphingosine-amidohydrolase activity,
acid ceramidase (AC) probably is the most thoroughly investigated and
most important ceramidase [16]. Ceramidases are often upregulated in
cancer cells, resulting in an increase of s1p and consequently in higher
survival rates and sustained cell proliferation [17–20]. Thus, the cer/
s1p rheostat was recognized as a promising target for chemotherapeutic
intervention in the treatment of cancer [6]. The inhibition of cer-
amidases is expected to lead to an accumulation of pro-apoptotic cer
and to a depletion of anti-apoptotic s1p.
Early ceramidase inhibitors were structurally modelled on the nat-
ural ceramides, yet showed low activities and poor selectivities in in
vitro assays [21–24]. Only a few derivatives showed a significant ac-
tivity, e.g. analogues of N-oleoylethanolamine (NOE) and the ceramide
analogue (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol
(D-e-MAPP), whose antitumoral and ceramidase inhibitory effects were
Abbreviations: AC, acid ceramidase; ASAH1, N-acylsphingosine amidohydrolase 1; cer, ceramide; cpm, cells per mL; DIPEA, diisopropylethylamide; DMF, di-
methylformamide; HBTU, (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate); JACoP, Just another colocalization plugin; LICQ, Li’s
Intensity Correlation Quotient; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NBT, nitroblue tetrazolium; PC, Pearson correlation coefficient;
P-gp1, p-glycoprotein 1; sph, sphingosine; SK, sphingosine kinase; s1p, sphingosine-1-phosphate; vbl, vinblastine; vpm, verapamil
⁎ Corresponding author.
E-mail address: Rainer.Schobert@uni-bayreuth.de (R. Schobert).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.103703
Received 30 October 2019; Received in revised form 22 February 2020; Accepted 24 February 2020
Availableonline25February2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

M. Rothemund, et al.
BioorganicChemistry97(2020)103703
Fig. 1. Ceramide/sphingosine-1-phosphate rheostat: sphingosine-1-phosphate (3, s1p), promoting cell survival and proliferation, can be synthesized via hydro-
lyzation of cell death promoting ceramides (1, cer) by ceramidase to give sphingosine (2a, sph) and its downstream phosphorylation by sphingosine kinases (SK).
studied in detail [21,25,26]. More potent inhibitors, structurally un-
related to ceramide, were later identified by Draper et al. (e.g. ceranib-
2) [27], Realini et al. (carmofur) [28] and others [21]. A major problem
with developing new ceramidase inhibitors is the low comparability of
literature data. Multiple methods for determining the efficacy of cer-
amidase inhibitors are in use, that differ in the conditions of the cellular
assays, the purity of the enzymes, and the nature of the reporter sub-
strates. More often than not, potential inhibitors were tested not on
purified enzymes but on crude cell extracts of unknown enzyme con-
centration.
Following our successful strategy of covalently combining natural
metabolites with bioactive metal complex fragments and metallocenes
in particular [29], we developed new sphingosine–metallocene con-
jugates and investigated their antitumoral modes of action, as well as
their AC-inhibitory activity on a purified human ceramidase enzyme.
2. Results and discussion
Scheme 2. Synthesis of organometallic ceramide analogues 11–13. Reagents
2.1. Chemistry
and conditions: (i) HBTU, DIPEA, DMF, 1 h, r.t.
A series of novel organometallic AC-inhibitor candidates was pre-
good yields.
To estimate the stability of the N-metallocenoylsphingosines 11a,
pared by N-acylation of sph (2a) and alkyne-labeled sph (2b) with
different metallocene carboxylic acids. The two sphingoid bases 2 were
synthesized from doubly protected serine ester 4 which was treated
with excess lithium dimethyl methylphosphonate to be converted to the
β-ketophosphonate 5 in excellent yield (Scheme 1). Its HWE reactions
with tetradecanal and tetradec-13-ynal using NEt3 as a base in the
presence of anhydrous LiCl afforded the corresponding 3-keto-
sphingosines 6a and 6b [30]. The diastereoselective reduction of the
enones was achieved using LiAlH(OtBu)3 in analogy to a literature
protocol [31]. Gratifyingly, only the desired anti-configured alcohols 7
were obtained in 75% and 77% yield, respectively. Their deprotection
with TFA afforded the free sphingoid bases 2 in total yields of 67% and
64% over four steps. The sphingoid bases 2 were then linked to car-
boxyferrocene (8), carboxycobaltocenium hexafluorophosphate (9) and
[(η5-Cp*)(η6-C6H5CO2H)Ru]PF6 (10) using HBTU as a coupling reagent
in the presence of excess DIPEA (Scheme 2). Purification by column
chromatography over silica gel (for 11 and 13), or precipitation with
NaBPh4 in MeOH (for 12) afforded the corresponding amides 11–13 in
12a and 13a under biological conditions, UV–vis spectra of their so-
lutions in a water–DMF (99:1) mixture were recorded over the course of
48 h (ESI Figure S1). They showed distinct spectra with a maximum at
λ = 240–260 nm (11a, 13a), or two absorption maxima in the same
range (12a). While the shape of the spectral curves remained largely
the same, their height (=absorbance) diminished over time, most ra-
pidly so for the cobalt complex 12a. This observation can be rationa-
lized by a decreasing concentration of the amphiphilic complexes due
to partial precipitation or micelle formation. However, all complexes
appear to be sufficiently long-lived to be taken up by cancer cells and to
reach their biological targets [32].
2.2. Inhibition of purified acid ceramidase
The N-metallocenoylsphingosines 11a, 12a, and 13a were tested for
their ability to inhibit human AC (ASAH1) in vitro. Using a fluorescence
based assay developed by Bedia et al. [16] we measured the IC50 values
for the inhibition of AC by complexes 11a, 12a and 13a (Fig. 2). In
contrast to the original work by Bedia et al. we expressed human acid
ceramidase recombinantly in High Five insect cells and purified the
enzyme by affinity chromatography and gel filtration, instead of using a
crude cell extract (cf Supporting Information). In this way we were able
to control the exact amount of enzyme used in our assays, rather than
relying on an approximation for its quantification. By a modified var-
iant of Bedia’s fluorescence assay, we measured a KM value of 14 µM for
the affinity of our purified enzyme for the Rbm14-12 substrate, com-
pared to 26 µM for the affinity of Bedia’s enzyme sample. Using this
measured KM value as a basis for the Rbm14-12 substrate concentra-
Scheme 1. Synthesis of sphingoid bases 2a and 2b. Reagents and conditions: (i)
n-BuLi, dimethyl methylphosphonate, THF, −78 °C, 2 h; (ii) tetradecanal or
tetradec-13-ynal, NEt3, LiCl, THF, r.t., 24 h; (iii) LiAlH(OtBu)3, EtOH, −78 °C,
1.5 h; (iv) TFA, CH2Cl2, r.t., 1 h.
tion, we found ruthenium complex 13a (23.5
AC inhibitor, while complexes 11a (142.6
0.5 µM) to be the best
7.6 µM) and 12a
2

M. Rothemund, et al.
BioorganicChemistry97(2020)103703
substrate of efflux transporter P-gp1 since each showed similar activ-
ities against the mdr Kb-V1Vbl cervix carcinoma cells, both in the pre-
sence and absence of the competitive P-gp1 inhibitor verapamil. In the
HCT116 wildtype cell line (HCT116wt) 12a was the most active com-
plex, whereas in the p53 knock-out mutant (HCT116−/−) Ru complex
13a was most active, again with low single-digit micromolar IC50 va-
lues. None of the complexes was as cytotoxic as ceranib-2.
2.5. Cell cycle progression
The effect of 11a, 12a, 13a, sph and ceranib-2 on the cell cycle of
518A2 melanoma cells was investigated by flow cytometry (Fig. 4).
Upon treatment for 24 h with varying concentrations, all complexes
arrested the melanoma cells in G1-phase. While ferrocene complex 11a
showed effects only at 10 µM, the cobaltocenium and ruthenium arene
complexes 12a and 13a already had a noticeable impact on the cell
cycle at a concentration of 2 µM. This is at variance with the results of
the MTT assays. A higher concentration of 11a was also necessary to
cause distinct effects in other biological assays, as discussed below.
Compounds 12a and 13a seem to arrest the melanoma cells during the
G1 phase without causing cell death over the course of 24 h. Sph had a
negligible effect on the cell cycle, increasing the cell population in G1-
phase only slightly. Ceranib-2 showed the greatest effect, arresting cells
at submicromolar concentrations in S- and G2-phase which hints at a
different mode of action. Neither sph nor ceranib-2 induced cell death
at these concentrations.
Fig. 2. Inhibition of AC (0.05 µg) after 30 min of incubation with ceranib-2,
11a, 12a or 13a at 37 °C at a final concentration range of 200–0.05 µM, or
10 µM–2 nM of carmofur. Means
SD from four independent values [16].
(98.1
14.0 µM) were approximately five to seven times less active.
The IC50 value of ca. 110 µM obtained for ceranib-2 under these assay
conditions is not reliable due to its limited solubility. In earlier in-
hibition assays with cellular ceramidase enzymes under less well-de-
fined conditions ceranib-2 was active with an IC50 = 28 µM [27]. For
the better soluble, known AC-inhibitor carmofur [33] we measured, as
expected, a superior activity with IC50 = 0.55
0.09 µM.
2.6. Reactive oxygen species (ROS)
2.3. Intracellular localization
Ceramides can have an indirect impact on the function of membrane
associated proteins. Moreover, ceramide accumulation is associated
with elevated ROS levels, due to deregulated mitochondrial functions
[11]. We monitored ROS levels in 518A2 melanoma cells treated with
complexes 11a, 12a and 13a using the nitroblue tetrazolium (NBT)
assay. In comparison to the solvent control all complexes caused an
increase in ROS levels after 24 h of incubation, accompanied by a re-
duced cell vitality. Complexes 11a and 13a doubled the ROS levels at
the highest concentration of 10 µM, which is less than their IC50 (24 h)
values. In contrast, the cobalt complex 12a nearly quadrupled the ROS
per vital cell at this concentration, which is, however, about twice its
IC50 (24 h) value (Fig. 5).
Acid ceramidases are predominantly found in lysosomes or nuclei of
mammalian cells [34–36]. To test if complexes 11a, 12a or 13a not
only inhibit AC but actually accumulate in these organelles we treated
518A2 melanoma cells with the alkyne-tagged N-metallocenoyl-
sphingosines 11b, 12b, and 13b (30 µM, 0.5 h). Due to the bioortho-
gonality of the acetylene group, these derivatives behave just like their
saturated a-analogues. We tracked their intracellular localization by
“clicking” them with a mixture of 3-azido-7-hydroxycoumarin, sodium
ascorbate and copper sulphate to form a fluorescent triazole which was
visualized by confocal fluorescence microscopy (Fig. 3) [37–41]. We
found all three complexes to colocalize with lysosomes. The neutral
ferrocenoyl complex 11b was also present in other cell compartments,
resulting in a lower Pearson correlation coefficient (PC = 1 for a
complete match; PC = 0.56 for 11b). Due to overlapping fluorescence
spectra of the triazole products and stained nuclei, their co-staining was
not possible. In contrast to 11b, the positively charged cobalt and ru-
thenium complexes 12b and 13b were found mainly in the lysosomes,
resulting in high values for PC and Li’s Intensity Correlation Coefficient
(12b: PC = 0.92, LICQ = 0.47; 13b: PC = 0.95, LICQ = 0.42).
2.7. Interference with cytoskeletons
The microtubules and F-actin components of the cytoskeleton of
518A2 melanoma cells, previously treated with complexes 11a, 12a, or
13a at concentrations close to their IC50 values, were visualized by
immunofluorescence staining and the results were documented in
fluorescence images (Fig. 6). While the microtubules were evenly dis-
tributed in control cells, they seemed to be focussed near cell nuclei and
to radiate toward peripheral cell compartments in cells treated with the
metal complexes. The microtubules of cells treated with ruthenium
complex 13a appeared to have lost the regular filamentous structure
visible in control cells. All complexes strongly influenced the organi-
zation of the actin cytoskeleton, causing stress fiber formation and
depolymerization of actin filaments throughout the cells.
2.4. Inhibition of cancer cell growth
Having shown that the N-metallocenoylsphingosines inhibit AC and
accumulate mainly in AC-rich lysosomes of cancer cells, we studied
them for other antitumoral effects and their correlation with AC-in-
hibition. The cytotoxicities of complexes 11a, 12a, 13a and their al-
kyne derivatives 11b, 12b, 13b, of sph, and of the known ceramidase
inhibitor ceranib-2 were determined in MTT-assays against nine human
cancer cell lines of four entities, including the HCT116 p53 knock out
mutant HCT116−/− and the P-gp1 overexpressing multi-drug resistant
(mdr) cell line KB-V1Vbl. The resulting IC50 values are listed in table 1.
All complexes were active with IC50 values in the low micromolar
range. Except for the HCT116 cells, ferrocene 11a was the most active
metal complex. None of the three a-type complexes appears to be a
As mentioned in the introduction, increased levels of ceramides are
frequently associated with Golgi fragmentation. Thus, we examined
518A2 melanoma cells treated with 11a, 12a or 13a for such effects by
staining their nuclei with DAPI (blue) and by staining α-N-acet-
ylgalactosamine residues in their Golgi apparatus with Alexa Fluor 647
HPA lectin antibody (Supporting Information, Figure S2). In compar-
ison to the control, treated cells indeed revealed a Golgi apparatus that
was more diffuse around the nucleus, hinting at its fragmentation.
3

M. Rothemund, et al.
BioorganicChemistry97(2020)103703
Fig. 3. Colocalization of alkyne-labeled
complexes 11b, 12b, 13b (30 µM,
0.5 h) and lysosomes (red) in 518A2
melanoma cells after “click” reaction
with 3-azido-7-hydroxycoumarin to
give triazoles (cyan). Yellow box: cell
that was analyzed for PC and LICQ va-
lues using ImageJ (JaCOPI) [42–44];
PC, Pearson Coefficient; LICQ, Li’s In-
tensity Correlation Quotient. Scales:
white: 50 µm; yellow 15 µm. (For in-
terpretation of the references to colour
in this figure legend, the reader is re-
ferred to the web version of this article.)
2.8. Induction of caspase activity
Caspase-3 and -7 are often activated upon deregulation of the cer/
s1p rheostat, especially by elevated levels of cer or sph. By means of
Apo-One® Homogeneous Caspase-3/-7 Assays (Promega) we ascer-
tained that all complexes 11a, 12a and 13a caused caspase-3/-7 acti-
vation in 518A2 melanoma cells, most strongly so the cobaltocenium
complex 12a, which was, however, still inferior to the established
apoptosis inducer staurosporine (Fig. 7)
3. Conclusions
The replacement of the fatty acyl residue in ceramides by metallo-
cenoyl residues led to metallodrugs 11–13 with properties distinctly
different from those of ceramides. Our main objective, to find new
substrate-like, targeted inhibitors of ceramidase was realized with ru-
thenium complex 13a. It inhibited purified human AC with an IC50 of
Fig. 4. Cell cycle analysis of 518A2 melanoma cells, treated with 11a (10 μM),
12a (2 μM), 13a (2 μM), sph (10 μM), cer2=ceranib-2 (200 nM) or the solvent
as control for 24 h.
Table 1
Means
SD of IC50 values [µM] of compounds 11–13, sph, and ceranib-2 in MTT assays with human cell linesa after 72 h of incubation as calculated from four
independent measurements.
Cell lines
b
+ vpm b
518A2
HT-29
HCT116wt
HCT116−/−
DLD-1
EaHy.926
U87
Hela
Kb-V1Vbl
Kb-V1Vbl
11a
11b
12a
12b
13a
13b
sph
5.0
3.6
5.8
5.7
7.6
5.7
11.9
0.1
0.5
0.2
0.8
0.1
0.9
1.4
0.3
0.2
0.5
0.3
6.6
0.7
11.9
0.2
0.5
7.3
0.2
4.7
0.2
3.3
0.7
0.1
0.5
1.0
1.0
4.2
0.3
0.2
7.5
0.4
0.9
4.4
0.5
0.5
4.1
10.0
2.4
0.1 11.8
13.0
10.0
17.4
12.8
20.9
16.6
0.6
6.5
0.3
0.5
0.3
0.6
4.5
5.7
7.6
4.9
4.3
0.2
6.3
3.2
2.8
0.3
0.4
1.0
0.8
2.2
1.9
7.3
5.7
10.3
11.4
5.6
0.8
20.4
31.2
27.4
31.7
9.5
0.7
12.0
24.5
32.9
44.2
4.7
0.6
6.3
8.9
0.4
0.3
0.7
0.1
6.6
8.4
1.0
1.3
0.4
3.0
0.4
1.7
2.8
10.6
27.3
6.4
0.2
0.5
0.4
9.4
5.2
7.6
0.2
27.8
12.7
0.3 16.9
0.7 15.6
0.5
27.4
21.8
0.3
0.9
21.5
19.1
0.7 28.8
1.5 31.4
0.4
0.6
0.9
0.7
1.8
0.4
ceranib-2 0.70
0.03 0.59
0.04 1.2
0.0
0.78
0.05 0.69
0.08 0.20
0.05 3.1
0.2
0.63
0.11 0.79
0.11 0.69
0.02
a
518A2 – melanoma, HT-29 – colon adenocarcinoma, HCT116wt – colon carcinoma (wildtype), HCT116−/− - colon carcinoma (p53 knock-out mutant), DLD-1 –
Dukes type C colorectal adenocarcinoma, Ea.Hy926 – endothelial hybrid, U87-MG – likely glioblastoma, Hela – cervix carcinoma, Kb-V1Vbl – cervix carcinoma.
b
Vbl – vinblastine, vpm – verapamil.
4

M. Rothemund, et al.
BioorganicChemistry97(2020)103703
Fig. 5. Left: Relative levels of reactive
oxygen species (ROS) in 518A2 mela-
noma cells, after 24 h incubation with
1, 2.5, 5 or 10 µM of 11a, 12a or 13a or
the solvent as control, calculated in re-
lation to the percentage of vital cells.
Right: Vitality of 518A2 melanoma cells
from analogously conducted MTT pro-
liferation assays. Means
three independent values.
SD from
ca. 23 µM, a value which, though inferior by a factor of 10 to that of
dedicated AC-inhibitors such as carmofur, is pharmacologically mean-
ingful. The distinctly weaker ceramidase inhibition by the neutral fer-
rocene conjugate 11a and the “cationic” cobaltocenium derivative 12a
indicates that the steric demand of the arene ligands might be more
decisive for their interference with the target enzyme than the complex
charge and the nature of the central metal. The complex charge, how-
ever, seems to be crucial for the intracellular accumulation: the cationic
cobalt and ruthenium complexes readily accumulated in lysosomes,
whereas the neutral ferrocene complex 11a was also found in the cy-
tosol. Thus, our data suggest that the next generation of AC-inhibitors of
this type should carry bulky ligands and a positive charge in order to
accumulate at sites rich in the target enzyme.
inhibitors 11a and 12a were on average slightly more antiproliferative
against our cancer cell lines than complex 13a. This indicates that
ceramidase inhibition is only one aspect among several antitumoral
factors. All three metallocenoylsphingosines 11–13 showed additional
antitumoral effects, whose intensities are also not stringently correlated
with their AC-inhibitory potential. For instance, the best AC-inhibitor
13a also had the most pronounced impact on the cytoskeleton of mel-
anoma cells, while the cobalt complex was the strongest inducer of
caspase-3/-7 activity and ROS production in these cells.
The results of this study warrant a broader screening of a library of
(cationic) metallocenoylsphingosines with variance in the arene ligands
to establish a structure–activity relation and to identify those secondary
effects that are most closely associated with AC-inhibition.
Concerning the antiproliferative activity, the less effective AC-
Fig. 6. Immunofluorescence staining of microtubules (red), actin filaments (green) and nuclei (blue) of 518A2 melanoma cells after 24 h of incubation with 11a
(10 µM), 12a (5 µM), 13a (5 µM), or the solvent as control. Scale: 50 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
5

M. Rothemund, et al.
BioorganicChemistry97(2020)103703
and the resulting mixture was stirred at room temperature for 18 h.
Then 1 M citric acid (150 mL) was added, the phases were separated
and the aqueous phase was extracted with EtOAc (2 × 100 mL). The
combined organic phases were washed with brine (300 mL), dried over
MgSO4, and concentrated in vacuo. The crude product was purified by
flash chromatography (silica gel, n-hexane/ethyl acetate 30:1) to leave
6b as a colorless oil. Yield: 3.38 g (6.66 mmol, 89%). [α]2D3= +43.4
(c = 1.1, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 6.95 (dt, J = 15.8,
7.0 Hz, 1H), 6.26 (d, J = 15.8 Hz, 1H), 4.53 (dt, J = 7.4, 4.0 Hz, 1H),
3.96 (dd, J = 10.4, 4.0 Hz, 1H), 3.82 (dd, J = 10.4, 4.0 Hz, 1H), 2.21
(q, J = 7.0 Hz, 2H), 2.17 (td, J = 7.1, 2.6 Hz, 2H), 1.92 (t, J = 2.6 Hz,
1H), 1.47–1.55 (m, 2H), 1.43 (m, 11H), 1.34–1.40 (m, 2H), 1.22–1.32
(m, 12H), 0.81–0.85 (m, 9H), −0.01 (m, 6H). 13C NMR (126 MHz,
CDCl3): δ = 196.4, 155.3, 149.2, 126.9, 79.6, 68.0, 63.6, 59.4, 32.6,
29.5, 29.4, 29.35, 29.2, 29.1, 28.7, 28.35, 28.3, 28.0, 25.7, 18.4, 18.2.
HRMS (ESI): m/z calculated for C29H53O4NSi + Na+ [M+Na+]:
530.3636. Found: 530.3627.
Fig. 7. Activation of caspase-3 and -7 after 4 h of incubation with 11a (10 µM),
12a (5 µM), 13a (5 µM); negative control: DMF; positive control: staurosporine
(st, 2 µM). Means
SD from three independent experiments.
4.1.3. tert-Butyl ((2S,E)-1-((tert-Butyldimethylsilyl)oxy)-3-hydroxyoctadec-
4-en-17-yn-2-yl)carbamate (7b)
4. Experimental
7b was prepared according to a literature procedure [31] from 6b
(1.33 g, 2.60 mmol, 1.0 equiv) and LiAlH(OtBu)3 (1.32 g, 5.20 mmol,
4.1. Chemistry
2.0 equiv) as
a colorless oil. Yield: 1.02 g (2.00 mmol, 77%).
Melting points are uncorrected; IR spectra were recorded on an FT-
IR spectrometer with ATR sampling unit; NMR spectra were run on a
500 MHz spectrometer; chemical shifts are given in ppm (δ) downfield
from tetramethylsilane as internal standard; Mass spectra: direct inlet,
EI, 70 eV; HRMS: UPLC/Orbitrap MS system in ESI mode;
Microanalyses: Elementar Unicube analyzer. Specific optical rotations
were measured at 589 nm (Na-D-line) and are given in deg cm3 g−1
dm−1. Absorption and fluorescence measurements were obtained using
a plate reader (Tecan Infinite F200). Cell cycle analysis was done using
a flow cytometer (Beckman Coulter Cytomics FC500). Fluorescence
images were obtained using an AxioCAM MRm (Zeiss, Axioplan2) or a
Leica TCS SP5 confocal microscope. Serine ester 4 [45], tetradecanal
[46], tetradec-13-ynal [47], 9 [48], 10 [49], and Rbm14-12 [50] were
prepared according to literature procedures. All tested compounds
were > 95% pure by elemental analysis and/or UPLC–HRMS.
[α]2D3= +16.6 (c = 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 5.75
(dtd, J = 15.3, 6.9, 0.9 Hz, 1H), 5.50 (dd, J = 15.3 Hz, 5.9, 2H), 5.24
(d, J = 8.2 Hz, 1H), 4.16–4.22 (m, 1H), 3.93 (dd, J = 10.2, 3.1 Hz,
1H), 3.75 (dd, J = 10.2, 2.4 Hz, 1H), 3.53–3.60 (m, 1H), 3.33 (d,
J = 8.24 Hz, 1H), 2.17 (dt, J = 7.2, 2.7 Hz, 2H), 2.03 – 2.07 (m, 2H),
1.93 (t, J = 2.7 Hz, 1H), 1.48–1.55 (m, 2H), 1.44 (s, 9H), 1.33–1.41 (m,
4H), 1.26 (br. s., 12H), 0.89 (s, 9H), 0.06 (d, J = 1.53 Hz, 6H). 13C
NMR (126 MHz, CDCl3): δ = 155.8, 133.0, 129.4, 84.8, 79.4, 74.66,
68.0, 63.5, 54.4, 32.3, 29.6, 29.55, 29.5, 29.2, 29.1, 28.7, 28.5, 28.4,
25.8, 18.4, 18.1. HRMS (ESI): m/z calculated for C29H55O4NSi + Na+
[M+Na+]: 532.3793. Found: 532.3784.
4.1.4. (2S,E)-2-Aminooctadec-4-en-17-yne-1,3-diol (2b)
TFA (20 mL) was added to a solution of 7b (1.89 g, 3.73 mmol, 1.0
equiv) in CH2Cl2 (20 mL) at 0 °C. The mixture was stirred at room
temperature for 1.5 h. Volatiles were removed in vacuo. To remove
excess TFA the residue was dissolved in MeOH (10 mL) and con-
centrated at reduced pressure. This was repeated twice. The remaining
residue was dissolved in 1 M NaOH (10 mL) and extracted with EtOAc
(3 × 10 mL). The combined organic phases were dried over MgSO4 and
concentrated in vacuo. The crude product was purified by flash chro-
matography (silica gel, CHCl3/MeOH/NH4OH 95:5:1) to give 2b as a
4.1.1. tert-Butyl (S)-(1-((tert-Butyldimethylsilyl)oxy)-4-(dimethoxyphosphoryl)
-3-oxobutan-2-yl)carbamate (5)
A solution of dimethyl methylphosphonate (5.34 mL, 49.5 mmol,
3.3 equiv) in THF (150 mL) at −78 °C was treated with n-BuLi (2.5 M in
hexane, 19.2 mL, 48.0 mmol, 3:2 equiv). After stirring for 30 min, a
solution of 4 (5.0 g, 15.0 mmol, 1.0 equiv) in THF (50 mL) was added
dropwise. The mixture was stirred for 1 h at −78 °C. Then sat. aq.
NH4Cl (200 mL) was added, the phases were separated, and the aqu-
eous phase was extracted with EtOAc (2 × 100 mL). The combined
organic phases were washed with brine (300 mL), dried over MgSO4,
and concentrated in vacuo. The crude product was purified by flash
chromatography (silica gel, n-hexane/ethyl acetate 1:1) to give 5 as a
colorless oil. Yield: 6.31 g (14.8 mmol, 99%). [α]2D3 = +62.3 (c = 1.0,
CHCl3). 1H NMR (500 MHz, CDCl3): δ = 5.53 (d, J = 7.6 Hz, 1H),
4.41–4.49 (m, 1H), 4.07 (dd, J = 10.4, 3.7 Hz, 1H), 3.84 (dd, J = 10.4,
4.0 Hz, 1H), 3.80 (d, J = 2.9 Hz, 3H), 3.78 (d, J = 2.9 Hz, 3H), 3.41
(dd, J = 22.0, 14.6 Hz, 1H), 3.14 (dd, J = 22.0, 14.6 Hz, 1H), 1.45 (s,
9H), 0.86 (s, 9H), 0.04 (s, 6H). 13C NMR (126 MHz, CDCl3): δ = 199.7,
155.2, 79.9, 63.0, 61.9, 60.4, 53.1, 38.5 (d, 1J(C,P) = 131.7 Hz), 28.3,
white solid. Yield: 1.04 g (3.50 mmol, 94%). mp = 69 °C. [α]D23
=
+8.9 (c = 1.0, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 5.76 (dtd,
J = 15.5, 6.8, 0.6 Hz, 1H), 5.47 (ddt, J = 15.5, 6.8, 1.2 Hz, 1H), 4.05
(t, J = 6.8 Hz, 1H), 3.66 (qd, J = 10.8, 5.3 Hz, 2H), 2.86 (q,
J = 5.3 Hz, 1H), 2.18 (td, J = 7.2, 2.7 Hz, 2H), 2.06 (q, J = 6.8 Hz,
2H), 1.94 (t, J = 2.7 Hz, 1H), 1.48–1.58 (m, 2H), 1.34–1.43 (m, 4H),
1.24–1.33 ppm (m, 12H). 13C NMR (126 MHz, CDCl3) δ = 134.7,
129.3, 84.8, 75.5, 68.0, 64.1, 56.1, 32.3, 29.55, 29.5, 29.4, 29.2, 29.1,
28.7, 28.6, 18.4. HRMS (ESI): m/z calculated for C18H34O2N [M+H+]:
296.2576. Found: 296.2584.
4.1.5. General procedure for the synthesis of complexes 11–13 from 2
To a solution of the respective organometallic carboxylic acid 8–10
(1.0 equiv) in dry dimethylformamide (0.05 M), diisopropylethylamine
(2.0 equiv), HBTU (1.1 equiv) and the corresponding sphingoid base 2
(1.1 equiv) were added at 0 °C. The mixture was stirred for 1 h while it
was allowed to reach room temperature. Volatiles were removed under
reduced pressure and the remainder was dissolved in CH2Cl2 (10 mL),
washed with 1 M NaOH (10 mL), 1 M aqueous HCl (10 mL), and water
(10 mL). The organic phase was dried (MgSO4) and evaporated to
dryness.
25.7,
18.2,
−5.6.
HRMS
(ESI):
m/z
calculated
for
C17H36O7NPSi + Na+ [M+Na+]: 448.1891. Found: 448.1890.
4.1.2. tert-Butyl (S,E)-(1-((tert-Butyldimethylsilyl)oxy)-3-oxooctadec-4-
en-17-yn-2-yl)carbamate (6b)
NEt3 (3.1 mL, 22.44 mmol, 3.0 equiv) was added to a solution of 5
(3.18 g, 7.46 mmol, 1.0 equiv), LiCl (0.95 g, 22.44 mmol, 3.0 equiv)
and tetradec-13-ynal (3.12 g, 15.0 mmol, 2.0 equiv) in THF (150 mL)
6

M. Rothemund, et al.
BioorganicChemistry97(2020)103703
4.1.6. N-((2S,3R,E)-1,3-Dihydroxyoctadec-4-en-2-yl)
dissolved in 3 mL MeOH followed by the addition of 45 mg of NaBPh4
(0.13 mmol, 1.0 equiv) in 2 mL MeOH. A yellow precipitate formed,
which was isolated by filtration, washed with MeOH (5 mL) and dried
in vacuo to leave a yellow solid. Yield: 59 mg (55%). mp = 97.5 °C.
[α]2D3 = +27.5 (c = 0.2, CHCl3). 1H NMR (500 MHz, acetone‑d6):
δ = 7.70–7.80 (m, 1H), 7.32–7.39 (m, 8H), 6.94 (t, J = 7.5 Hz, 8H),
6.77–6.82 (m, 6H), 6.34–6.38 (m, 1H), 6.30–6.33 (m, 1H), 5.81–5.84
(m, 5H), 5.80 (t, J = 2.1 Hz, 2H), 5.71–5.78 (m, 1H), 5.60 (dd,
J = 15.3, 6.1 Hz, 1H), 4.24–4.29 (m, 1H), 4.11–4.19 (m, 1H), 3.97 (t,
J = 5.3 Hz, 1H), 3.76–3.89 (m, 2H), 2.31 (t, J = 2.7 Hz, 1H), 2.16 (td,
ferrocenecarboxamide (11a)
11a was prepared according to the general procedure from 8
(50 mg, 0.22 mmol), DIPEA (74 µL, 0.43 mmol), HBTU (91 mg,
0.24 mmol) and 2a (71 mg, 0.24 mmol). The crude product was pur-
ified by flash chromatography (silica gel, n-hexane/acetone 2:1) to give
an orange solid. Yield: 86 mg (0.17 mmol, 77%). mp = 81 °C. [α]D23
=
+7.7 (c = 0.2, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 6.58 (d,
J = 7.6 Hz, 1H), 5.83 (dt, J = 15.5, 7.0 Hz, 1H), 5.57 (dd, J = 15.5,
6.4 Hz, 1H), 4.71 (d, J = 8.9 Hz, 2H), 4.39 – 4.37 (m, 1H), 4.35 (s, 2H),
4.20–4.23 (m, 5H), 4.01–4.06 (m, 1H), 3.96 (dd, J = 11.3, 4.0 Hz, 1H),
3.76 (dd, J = 11.3, 2.7 Hz, 1H), 3.56 (br. s., 2H), 2.06 (q, J = 7.0 Hz,
2H), 1.33–1.41 (m, 2H), 1.20–1.32 (m, 20H), 0.87 ppm (t, J = 6.9 Hz,
3H). 13C NMR (126 MHz, CDCl3): δ = 171.6, 134.2, 128.8, 75.3, 74.1,
70.7, 69.9, 68.4, 68.3, 62.4, 54.9, 32.3, 31.9, 29.7, 29.6, 29.6, 29.5,
29.3, 29.2, 29.2, 22.7, 14.1. MS (ESI + ): m/z 512,4 [M+H+].
Elemental analysis calculated (%) for C29H45FeNO3: C 68.09, H 8.87, N
2.74. Found: C 68.33, H 8.60, N 3.01.
J
= 7.0, 2.7 Hz, 2H), 2.01–2.09 (m, 2H), 1.45–1.53 (m, 2H),
1.22–1.44 ppm (m, 16H). 13C NMR (126 MHz, acetone‑d6): δ = 165.9,
165.5, 165.2, 164.8, 162.6, 137.5, 133.9, 131.9, 126.5, 122.8, 87.6,
87.4, 87.3, 85.6, 85.2, 73.5, 70.3, 61.9, 57.9, 33.4, 30.8, 30.7, 30.6,
30.5, 30.3, 30.1, 30.0, 29.9, 29.8, 19.1. MS (ESI+): m/z 510.3
[M−BPh4−]. MS (ESI−): m/z 319.1 [BPh4−]. Elemental analysis cal-
culated (%) for C53H61BCoNO3: C 76.71, H 7.41, N 1.67. Found: C
76.77, H 7.85, N 2.03.
4.1.7. N-((2S,3R,E)-1,3-Dihydroxyoctadec-4-en-17-yn-2-yl)
4.1.10. {η6-[N-((2S,3R,E)-1,3-dihydroxyoctadec-4-en-17-yn-2-yl)
benzamide)]}(η5-1,2,3,4,5-pentamethylcyclopentadienyl)-ruthenium (13a)
13a was prepared according to the general procedure from 10
(50 mg, 0.10 mmol), DIPEA (34 µL, 0.20 mmol), HBTU (42 mg,
0.11 mmol) and 2a (33 mg, 0.11 mmol). The crude product was pur-
ified by flash chromatography (silica gel, CH2Cl2/MeOH 95:5) to give a
ferrocenecarboxamide (11b)
11b was prepared according to the general procedure from 8
(50 mg, 0.22 mmol), DIPEA (74 µL, 0.43 mmol), HBTU (91 mg,
0.24 mmol) and 2b (71 mg, 0.24 mmol). The crude product was pur-
ified by flash chromatography (silica gel, n-hexane/acetone 2:1) to
leave an orange solid. Yield: 100 mg (0.20 mmol; 91%). mp = 67 °C.
[α]2D3 = +12.5 (c = 0.2, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 6.51
(d, J = 7.0 Hz, 1H), 5.86 (dt, J = 15.6, 7.0 Hz, 1H), 5.61 (dd, J = 15.6,
6.4 Hz, 1H), 4.70–4.73 (m, 2H), 4.42–4.47 (m, 1H), 4.36–4.39 (m, 2H),
4.24 (s, 5H), 4.01–4.08 (m, 2H), 3.78–3.84 (m, 1H), 2.78–2.83 (m, 2H),
2.19 (td, J = 7.1, 2.6 Hz, 2H), 2.10 (q, J = 7.0 Hz, 2H), 1.95 (t,
J = 2.6 Hz, 1H), 1.49–1.57 (m, 2H), 1.36–1.44 (m, 4H), 1.24–1.33 ppm
(m, 12H). 13C NMR (126 MHz, CDCl3): δ = 171.3, 134.4, 128.9, 74.7,
70.6, 69.8, 68.3, 68.2, 68.0, 62.6, 54.7, 32.3, 29.6, 29.5, 29.4, 29.2,
29.1, 28.7, 28.5, 18.4. MS (ESI+): m/z 508.4 [M+H+]. Elemental
analysis calculated (%) for C29H41FeNO3: C 68.63, H 8.14, N 2.76.
Found: C 68.89, H 8.46, N 2.88.
white solid. Yield: 70 mg (0.05 mmol, 90%). mp = 129 °C. [α]D23
=
+7.5 (c = 0.2, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 7.42 (d,
J = 7.9 Hz, 1H), 6.36–6.45 (m, 2H), 5.79–5.97 (m, 4H), 5.54 (dd,
J = 15.6, 6.1 Hz, 1H), 4.39–4.44 (m, 1H), 4.00–4.09 (m, 2H), 3.81 (d,
J = 8.2 Hz, 1H), 2.00–2.08 (m, 2H), 1.87–1.96 (m, 15H), 1.32–1.39 (m,
2H), 1.18–1.31 (m, 26H), 0.87 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz,
CDCl3): δ = 163.3, 134.0, 128.5, 97.8, 92.3, 88.3, 87.2, 87.1, 85.6,
85.4, 74.0, 61.7, 55.9, 32.3, 31.9, 29.7, 29.6, 29.6, 29.5, 29.5, 29.3,
29.3, 29.2, 29.1, 22.7, 14.1, 10.1. MS (ESI+): m/z 640.4 [M−PF6+].
MS (ESI−): m/z 144.9 [PF6−]. Elemental analysis calculated (%) for
C
35H56F6NO3PRu: C 53.56, H 7.19, N 1.78. Found: C 53.28, H 7.28, N
2.07.
4.1.8. N-((2S,3R,E)-1,3-Dihydroxyoctadec-4-en-2-yl)cobalto-
4.1.11. {η6-[N-((2S,3R,E)-1,3-Dihydroxyoctadec-4-en-17-yn-2-yl)
benzamide)]}(η5-1,2,3,4,5-pentamethylcyclopentadienyl)ruthenium (13b)
13b was prepared according to the general procedure from 10
(50 mg, 0.10 mmol), DIPEA (34 µL, 0.20 mmol), HBTU (42 mg,
0.11 mmol) and 2b (32 mg, 0.11 mmol). The crude product was pur-
ified by flash chromatography (silica gel, CH2Cl2/MeOH 98:2) to leave
ceniumcarboxamide Tetraphenylborate (12a)
12a was prepared according to the general procedure from 9
(100 mg, 0.26 mmol), DIPEA (90 µL, 0.53 mmol), HBTU (109 mg,
0.29 mmol) and 2a (110 mg, 0.29 mmol). The crude product was dis-
solved in 3 mL MeOH followed by the addition of 90 mg of NaBPh4
(0.26 mmol, 1.0 equiv) in 2 mL MeOH. A yellow precipitate formed,
which was isolated by filtration, washed with MeOH (5 mL) and dried
in vacuo to give a yellow solid. Yield: 184 mg (83%). mp = 109 °C.
[α]2D3 = +25.2 (c = 0.2, CHCl3). 1H NMR (500 MHz, acetone‑d6):
δ = 7.74 (d, J = 8.9 Hz, 1H), 7.35 (m, 8H), 6.94 (t, J = 7.5 Hz, 8H),
6.76–6.82 (m, 4H), 6.35–6.37 (m, 1H), 6.32 (m, 1H), 5.83 (s, 5H), 5.80
(t, J = 2.1 Hz, 2H), 5.74 (dt, J = 15.6, 7.0 Hz, 1H), 5.60 (dd, J = 15.6,
6.1 Hz, 1H), 4.24–4.30 (m, 1H), 4.11–4.18 (m, 1H), 3.98 (t, J = 5.5 Hz,
1H), 3.77–3.88 (m, 2H), 2.08–2.03 (m, 2H), 1.33–1.40 (m, 2H),
1.22–1.32 (m, 20H), 0.88 ppm (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz,
acetone‑d6): δ = 165.9, 165.5, 165.1, 164.7, 162.6, 137.4, 137.4,
133.9, 131.8, 126.4, 122.7, 95.5, 87.6, 87.4, 87.3, 85.6, 85.2, 73.5,
61.8, 57.9, 33.4, 33.0, 30.8, 30.7, 30.6, 30.4, 30.3, 30.1, 30.0, 29.8,
23.7, 14.8. MS (ESI+): m/z 514,3 [M - BPh4−]. MS (ESI−): m/z 319.1
[BPh4−]. Elemental analysis calculated (%) for C53H65BCoNO3: C
76.34, H 7.86, N 1.68. Found: C 76.44, H 7.54, N 2.08.
a white solid. Yield: 40 mg (0.05 mmol, 51%). mp = 118 °C. [α]D23
=
+6.5 (c = 0.2, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 7.35 (d,
J = 7.6 Hz, 1H), 6.41 (dd, J = 14.5, 6.0 Hz, 2H), 5.91–5.96 (m, 1H),
5.80–5.91 (m, 3H), 5.55 (dd, J = 15.4, 6.0 Hz, 1H), 4.44 (s, 1H),
4.01–4.10 (m, 2H), 3.80–3.88 (m, 1H), 3.40 (br. s, 1H), 3.12–3.24 (br.
m, 1H), 2.18 (td, J = 7.1, 2.6 Hz, 2H), 2.02–2.10 (m, 2H), 1.95 (s,
16H), 1.53 (quin, J = 7.3 Hz, 2H), 1.33–1.43 (m, 4H), 1.22–1.32 ppm
(m, 12H). 13C NMR (126 MHz, CDCl3): δ = 163.2, 134.1, 128.4, 97.8,
92.3, 88.2, 87.1, 85.6, 85.4, 84.8, 74.0, 68.0, 61.7, 55.8, 32.3, 29.6,
29.5, 29.2, 29.1, 28.7, 28.5, 18.4, 10.1. MS (ESI+): m/z 636.4
[M−PF6−]. MS (ESI−): m/z 144.9 [PF6−]. Elemental analysis calcu-
lated (%) for C35H52F6NO3PRu: C 53.84, H 6.71, N 1.79. Found: C
53.55, H 6.85, N 2.13.
4.2. Biological evaluation
4.2.1. Recombinant expression of ASAH1 in High Five insect cells
Recombinant full length ASAH1 was expressed and secreted from
High Five insect cells. The used vector containing the genetic in-
formation for ASAH1 was obtained from Genescript. The first 21 amino
acids were exchanged for the melittin signal sequence (MKFLVNVAL-
VFMVVYISYIYA) [51]. At the C-terminus a TEV site, followed by a His-
4.1.9. N-((2S,3R,E)-1,3-Dihydroxyoctadec-4-en-17-yn-2-yl)
Cobaltoceniumcarboxamide tetraphenylborate (12b)
12b was prepared according to the general procedure from 9
(50 mg, 0.13 mmol), DIPEA (45 µL, 0.26 mmol), HBTU (55 mg,
0.14 mmol) and 2b (42 mg, 0.14 mmol). The crude product was
7

M. Rothemund, et al.
BioorganicChemistry97(2020)103703
tag (ENLYFQGGGGHHHHHH) was added. 48 h after infection with the
respective baculovirus, the cells (1.2 mio cells/mL) were centrifuged
(4500 rpm, 4 °C, 5 min), and the supernatant containing the secreted
ASAH1 was dialysed against Ni2+-IMAC binding buffer (50 mM
and the plate was again incubated for 24 h. The cells were treated with
30 µM of 11b, 12b or 13b for 30 min at 37 °C. The old medium was
discarded and the cells were washed three times with 1 mL of PBS. After
fixation (4% formaldehyde in PBS) for 20 min and permeabilization
(0.5% Triton X-100, 1% BSA in PBS), 200 µL of click working solution
(2 mM CuSO4, 5 mM sodium ascorbate, 0.1 mM 3-azido-7-hydro-
xycoumarin, 1% BSA in PBS) were added into each well. The cells were
incubated at room temperature in the dark for 30 min before the so-
lution was discarded once more. The cells were washed twice with PBS
(1% BSA) before the coverslips were embedded in mowiol.
Fluorescence images were taken using a Leica TCS SP5 confocal mi-
croscope (pinhole at 1 Airy). Colocalization parameters were calculated
for one cell using the ImageJ plugin JaCOP [42–44].
NaH2PO4, 300 mM NaCl, pH 7.8, 4 × 1 L, 4 °C). After dialysis, Ni2+
-
IMAC was performed (washing buffer 50 mM NaH2PO4, 300 mM NaCl,
20 mM imidazol, pH 7.8; elution buffer 50 mM NaH2PO4, 300 mM
NaCl, 125 mM imidazole, pH 7.8). The protein was concentrated
(amicon stirred cell, NADIR/UP020P) and the protein was further
purified via gel filtration (flow 0.6 mL/min, Hiload 16/600 Superdex
200 pg, buffer 50 mM Tris/HCl, 200 mM NaCl, pH 7.6). The purity of
the protein was determined via SDS-page (cf. Supporting Information)
The protein concentration was determined at A280 using the extinction
coefficient ε = 79215 M−1 cm−1. From 320 mL of medium we ob-
tained 2.4 mg of pure enzyme.
4.2.6. IC50 determination using the MTT assay
The cytotoxicity of 11–13, sph and ceranib-2 was evaluated via
MTT based proliferation assays. Cells were seeded at 0.05 × 106 cpm or
0.1 × 106 cpm (DLD-1) into the wells of 96 well plates (100 µL/well)
and incubated for 24 h to establish confluency. Appropriate predilu-
tions in H2O made from fresh stock solutions (10 mM) in DMF or DMSO
(ceranib-2) or EtOH (sph) were added into the wells to reach final
concentrations ranging from 25 nM to 100 µM. After 72 h of incubation
at 37 °C the plates were centrifuged for 5 min (300 g, 4 °C) and the
medium was discarded by swiftly turning the plates onto fresh cell
tissue paper. 50 µL of an MTT solution (0.05% in PBS) was added into
each well and the plates were further incubated at 37 °C for 2 h. The
MTT solution was removed as before, and the cells and the formazan
were lysed by addition of 25 µL of SDS/DMSO (1%, 0.6% acetic acid).
For complete solution of the formazan the plates were incubated for at
least another hour at 37 °C before the absorptions of the formazan and
the background were measured at 570 nm, respectively at 630 nm. The
vitality of the cells treated with the solvent was set to 100% viable cells
for each concentration and the vitality of the cells inside the wells
treated with 11, 12, 13 was calculated accordingly. The IC50 values
4.2.2. Determination of KM of the fluorescence substrate Rbm14-12
The assay for the determination of the KM and IC50 values is based
on the fluorescence assay of Bedia et al. [16]. In 1.5 mL Eppendorf
reaction tubes 750 µL of reaction mix containing 645 µL NaOAc buffer
(25 mM, pH 4.5), 5 µL of Rbm14-12 predilutions in EtOH (final con-
centrations 0, 2.5, 5, 7.5, 10, 20, 40, 60 µM, 0.5% EtOH) were prepared.
The reactions were started with 250 µL of ASAH1 solution in NaOAc
buffer (final concentration 500 ng/mL). The reaction tubes were in-
cubated at 37 °C. After 0, 5, 10, 15, 20, 30, 40, and 50 min, 100 µL
samples were withdrawn from each tube and added to 50 µL of MeOH
in the wells of a 96 black well plate to stop the enzyme reaction. 100 µL
of a 2.5 mg/mL NaIO4 solution (100 mM glycine/NaOH, pH 10.6) were
added to each well and the plate was incubated at room temperature in
the dark for 2 h. For background measurements reaction tubes con-
taining blanks were treated analogously. The fluorescence of the re-
sulting umbelliferone was measured at λex = 340 and λem = 465 nm.
The KM value (cf. Supporting Information) was determined using
GraphPad Prism.
were finally calculated via GraphPad Prism. Means
SDs were cal-
4.2.3. Determination of ceramidase inhibition (IC50 values)
culated from four independent values.
In a 96 black well plate 75 µL of reaction mix, containing 64.5 µL
NaOAc buffer (25 mM, pH 4.5), 0.5 µL of fluorescence substrate
Rbm14-12 (final concentration 14 µM), and 10 µL of a predilution of
11a, 12a, 13a, carmofur, ceranib-2 or the solvent in NaOAc buffer
(final concentrations 200 µM to 50 nM, or 10 µM to 2 nM in the case of
carmofur) were prepared. The enzyme reaction was started by addition
of 25 µL of NaOAc containing 0.05 µg ASAH1, resp. 25 µL of pure
NaOAc for background fluorescence determination, into each well. The
reaction was stopped after 30 min (37 °C) by addition of 50 µL of MeOH
to each well. Then 100 µL of a 2.5 mg/mL NaIO4 solution (100 mM
glycine/NaOH, pH 10.6) were added into each well and the plate was
incubated at room temperature in the dark for 2 h. The fluorescence of
the product umbelliferone was measured at 340 nmex / 465 nmem. IC50
4.2.7. Cell cycle analysis
The effects of 11–13, sph, and ceranib-2 on the progression of the
cell cycle of 518A2 melanoma cells were analyzed via propidium iodide
(PI) staining and flow cytometry. Cells were seeded at 0.05 × 106 cpm
into the wells of 6-well plates (3 mL/well) and the plates were in-
cubated under standard cell culture conditions for 24 h. Dilutions of the
test complexes in H2O were added to the wells to reach final con-
centrations of 10 µM (11a, sph), 2 µM (12a, 13a), 200 nM (ceranib-2)
or equal amounts of the respective solvent. The cells were incubated for
another 24 h before the medium of each well (3 wells per concentra-
tion) was transferred into an ice cooled centrifugation tube, the cells
were washed with 1 mL of PBS which was transferred into the re-
spective tube as well. The cells were harvested via trypsination and
thorough washing of the wells with PBS, and were transferred into the
respective tube. The cells were centrifuged at 300 g (5 min, 4 °C) and
the supernatant was discarded. The resulting pellet was resuspended in
1 mL of ice cold EtOH (70%) and kept on ice for at least 1 h. For PI
staining the cells were centrifuged at 400 g (5 min, 23 °C) and the
supernatant was discarded. The cells were layered with 1 mL PBS and
incubated for 5 min at 23 °C. After centrifugation (5 min, 400 g, 4 °C)
the PBS was discarded and the pellet was resuspended with 200 µL of PI
staining buffer (50 µg/mL propidium iodide, 0.1% sodium citrate, 50
µg/mL RNAse I) and the cells were incubated for 30 min at 37 °C.
Thereafter, flow cytometry measurements were done with a Cytomics™
FC 500.
values (means
SD) were calculated from four independent values
using GraphPad Prism. We checked that the metal complexes do not
themselves interfere with the fluorescence intensity of umbelliferone
(cf. Figure S5).
4.2.4. Cell culture conditions
The cell lines used for the biological evaluation were cultivated in
Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and 1%
Antibiotic-Antimycotic. The cells were cultivated at 37 °C, 5% CO2 and
95% humidity.
4.2.5. Intracellular colocalization
518A2 cells were seeded at a density of 0.03 × 106 cells per mL
(cpm) onto glass cover slips (Ø 12 mm) inside the wells of a 24 well
plate (0.5 mL/well). After 24 h of incubation under cell culture con-
ditions the medium was exchanged for 500 µL of complete DMEM
containing 6 µL of Cell light Lysosomes-RFP, BacMam (Thermo Fisher)
4.2.8. (Immuno-)fluorescence staining
518A2 cells were seeded at 0.05 × 106 cpm analogously to the
preparation of the colocalization. The cells were incubated at standard
8

M. Rothemund, et al.
BioorganicChemistry97(2020)103703
cell culture conditions for 24 h and then treated with 11a (10 µM), 12a
(5 µM), 13a (5 µM) for another 24 h. The medium was removed and the
cells were washed thrice with PBS. After fixation with 3.7% for-
maldehyde in PBS for 20 min at room temperature, the cells were again
washed three times with PBS.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Microtubules. For staining of the microtubules the cells were in-
cubated for 30 min (room temperature) with blocking and permeabi-
lization buffer (1% BSA , 0.1% Triton X-100 in PBS). The buffer was
replaced with the primary mouse anti-tubulin antibody (1:500 in 1%
BSA in PBS) and the plates were incubated for 1 h at 37 °C. The buffer
was discarded and the cells were washed three times with PBS, before
the secondary goat anti-mouse phalloidin 488 antibody (1:500 in 1%
BSA in PBS) was added to each well. After another hour of incubation at
room temperature and in the dark, the buffer was discarded again and
the cells were washed twice with PBS before the coverslips were layered
with 500 µL of sterile water. The coverslips were carefully embedded
into mowiol mounting buffer (2.5% DABCO, 1 mg/mL DAPI) and stored
at 4 °C.
Acknowledgement
R.S. thanks the Deutsche Forschungsgemeinschaft, Germany, for a
grant (Scho 402/12-2).
Appendix A. Supplementary material
Experimental data of 2a, 6a, and 7a; NMR spectra of 2–13; UV-vis
stability studies of 2–8; Fluorescence staining of Golgi apparatus in
518A2 melanoma cells; SEC chromatogram and SDS-page of the ex-
pressed ASAH1; Michaelis Menten curve of Rbm14-12 at doi:xxxx.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.103703.
Actin filaments. Staining of the actin filaments was done using
Actin-stainTM 488 phalloidin (Cytoskeleton) and following the protocol
provided by the manufacturer. After staining the cells were washed and
the coverslips embedded as described above.
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for 30 min (room temperature). The cells were embedded as before.
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