Novel meriolin derivatives as rapid apoptosis inducers

Article abstract of DOI:10.1016/j.bmc.2019.06.029

3-(Hetero)aryl substituted 7-azaindoles possessing multikinase inhibitor activity are readily accessed in a one-pot Masuda borylation-Suzuki coupling sequence. Several promising derivatives were identified as apoptosis inducers and, emphasizing the multik

Full text of DOI:10.1016/j.bmc.2019.06.029

Accepted Manuscript
Novel meriolin derivatives as rapid apoptosis inducers
Daniel Drießen, Fabian Stuhldreier, Annika Frank, Holger Stark, Sebastian
Wesselborg, Björn Stork, Thomas J.J. Müller
PII:
S0968-0896(19)30526-7
https://doi.org/10.1016/j.bmc.2019.06.029
BMC 14966
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
29 March 2019
28 May 2019
18 June 2019
Please cite this article as: Drießen, D., Stuhldreier, F., Frank, A., Stark, H., Wesselborg, S., Stork, B., Müller, T.J.J.,
Novel meriolin derivatives as rapid apoptosis inducers, Bioorganic & Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.bmc.2019.06.029
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Graphical Abstract
Novel meriolin derivatives as rapid
apoptosis inducers
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Daniel Drießen,a Fabian Stuhldreier,b Annika Frank,c Holger Stark,c Sebastian Wesselborg,b Björn Stork,b and
Thomas J. J. Müller*a
a Institut für Organische Chemie und Makromolekulare Chemie, Heinrich-Heine-Universität Düsseldorf,
Universitätsstraße 1, D-40225 Düsseldorf, Germany. E-mail: ThomasJJ.Mueller@hhu.de; Fax: +49 (0)211
8114324; Tel: +49 (0)2118112298
b Institut für Molekulare Medizin I, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, D-40225
Düsseldorf, Germany
c Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf,
Universitätsstraße 1, D-40225 Düsseldorf, Germany

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Novel meriolin derivatives as rapid apoptosis inducers
Daniel Drießen,a Fabian Stuhldreier,b Annika Frank,c Holger Stark,c Sebastian Wesselborg,b Björn Stork,b
and Thomas J. J. Müller*a
a Institut für Organische Chemie und Makromolekulare Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, D-40225 Düsseldorf, Germany
b Institut für Molekulare Medizin I, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, D-40225 Düsseldorf, Germany
c Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße1, D-40225 Düsseldorf, Germany
This work is dedicated to Prof. Dr. Peter Proksch on the occasion of his 65th birthday.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
3-(Hetero)aryl substituted 7-azaindoles possessing multikinase inhibitor activity are readily
accessed in a one-pot Masuda borylation-Suzuki coupling sequence. Several promising
derivatives were identified as apoptosis inducers and, emphasizing the multikinase inhibition
potential, as sphingosine kinase 2 inhibitors. Our measurements provide additional insights into
the structure-activity-relationship of meriolin derivatives, suggesting derivatives bearing a
pyridine moiety with amino groups in 2-position as most active anticancer compounds and thus
as highly promising candidates for future in vivo studies.
Available online
Keywords:
Synthetic methods
One-pot reaction
Masuda borylation-Suzuki coupling
Meriolin
2009 Elsevier Ltd. All rights reserved.
Apoptosis
Sphingosine kinase 2
1. Introduction
Further studies also indicated a high potential of meriolins in the
treatment of neurodegenerative diseases, bipolar disorders,
A variety of marine compounds consists of indole or 7-
azaindole core elements1 and quite some have revealed
interesting biological and pharmacological activity. Meridianins
originate from the ascidian Aplidium meridianum, while variolins
were first isolated from the antarctic sponge Kirkpatrickia
variolosa.2,3 In comparison to meridianins and variolins (Fig. 1)
meriolins can be considered to be truncated hybrid derivatives
combining structural motifs of both classes.4 Quite early 3-
(hetero)aryl substituted 7-azaindoles have therefore become a
lead structure for the development of kinase inhibitors.
strokes, cancer, and chronic inflammations.6
Multikinase inhibitors are well-known inducers of apoptotic
cell death.8-10 For application in therapeutic context initiating
apoptosis is much more favorable than induction of necrosis as
apoptosis does not harm the host by causing inflammatory
reactions.11 The major effectors of the highly regulated apoptotic
signaling network are cysteinyl-aspartate specific proteases
(caspases) including the most potent executioner of apoptosis,
caspase-3.11 Besides caspases several interconnected kinase-
driven signaling pathways are involved in the regulation of
apoptosis. Since many of these signaling cascades negatively
regulate apoptosis in cancer cells, inhibition of kinases is a
reasonable approach for anticancer therapy.12 Particularly in
treatment of acute leukemias targeting kinases seems to be a
valid strategy as demonstrated by the recent approval of the
multitargeted kinase inhibitor midostaurin for acute myeloid
leukemia.13 Furthermore, also lymphomas are reported to be most
vulnerable to multikinase inhibition.14 Interestingly, multiple
kinases which were reported to be inhibited by meriolin
derivatives5 are heavily linked to downregulation of apoptosis in
cancer cells, for instance GSK3 or CDK7.15-17 Summing up,
meriolin derivatives appear to be promising candidates for
induction of apoptosis in leukemia and lymphoma cells.
N
N
N
NH2
NH2
NH2
OH
N
N
N
N
N
N
N
N
H
H
H2N
Variolin B
Meridianin A
Meriolin 1
Figure 1. Natural products meridianin A and variolin B with
high bioactivity and meriolin 1 as a structural hybrid.
Indeed, meriolins have been reported to strongly inhibit
various protein kinases.5,6 This finding correlates excellently with
the ability of 7-azaindoles to bind to the hinge region of kinases.
In some cases, also ligands with kinase domains of signaling
cascade proteins (e.g. PDK1) were successfully cocrystallized.7

The sphingosine kinases 1 and 2 (SK1, SK2) are important
regulators of the sphingolipid metabolism.18 By fine-tuning the
levels of sphingosine-1-phosphate (through phosphorylation of
sphingosine) the enzymes are involved in cell proliferation and
apoptosis and hence connected to cancerous diseases. Recent
research even reported on the connection between SK2 inhibition
and induction of caspase-3 cleavage in TRAIL resistant cancer
cells.19
screened with respect to their apoptosis inducing potential and to
establish first structure-activity relationships (SAR). In addition,
the inhibition of sphingosine kinases 1 and 2 (SK1, SK2) was
assessed by initial screening studies.
2. Results and Discussion
2.1. Masuda borylation-Suzuki coupling sequence
Based upon our experience in applying the MBSA sequence to
the synthesis of marine natural products and analogues, we first
established, starting from N-protected 3-iodo-7-azaindole 1 and
equistoichiometric amounts of (hetero)aryl halides 2, an
optimized protocol for efficiently preparing meriolin derivatives
3 (Scheme 3, Figure 2).26,27,28
As a consequence, rational design of meriolins as kinase
inhibitors by a reliable, modular methodology is highly desirable.
First syntheses of meriolins reported longest linear sequences of
four steps, however, resulting in low overall yields.20,21 Based
upon the availability and accessibility of (hetero)aryl halides, the
retrosynthetic analysis of meriolins suggests Suzuki-Miyaura-
coupling22 as a reliable, versatile terminal key step for the
formation of the desired compounds (Scheme 1).
3.0 mol % Pd(PPh3)4
I
(hetero)aryl
1.7 equivs HBpin, 10 equivs NEt3
1,4-dioxane, 80 °C, 4 h
2
then: 1.0 equiv (hetero)aryl halide
N
N
N
N
N
2.5 equivs Cs2CO3, MeOH, 100 °C, 18 h
then: 2.5 equivs NaOH, 4 h
NH2
R
H
N
1
3
(15 examples, 40-96%)
Hal
N
NH2
+
Suzuki
coupling
Masuda
borylation
Scheme 3. MBSA one-pot synthesis of meriolin derivatives 3.
N
Bpin
I
The N-protected 7-azaindoles  were prepared in quantitative
yields using a one-pot protocol of iodination followed by
subsequent protection in dimethylformamide under basic
conditions.28 A change from N-Boc protected 7-azaindoles to a
tosyl protection group was chosen to provide a more stable
reagent with respect to the reaction conditions. In comparison to
our initial protocol by raising the amount of pinacol borane
dehydrohalogenation of the starting material can be efficiently
suppressed. Also increasing the excess of triethylamine in the
Masuda borylation step turned out to be favorable.23 With a
N
PG
N
PG
N
N
N
N
H
PG = protecting group
Hal = I, Br, Cl
Scheme 1. Retrosynthetic analysis of meriolin 1.
Pinacolyl boronates are stable to moisture, light, can be stored
at room temperature and can be obtained by Masuda borylation
upon reacting an (hetero)aryl halide in the presence of pinacol
borane, triethylamine and a palladium catalyst.23 In comparison
to the related Miyaura borylation24 with B2pin2 the Masuda
reaction represents a more atom-economical approach. Most
favorably, both catalytic processes, borylation and
(hetero)arylation can be concatenated in a one-pot fashion, i.e. in
reaction time of
4 h in 1,4-dioxane conversion to the
corresponding pinacolyl ester is essentially quantitative,
according to its yields, if isolated. Excessive pinacol borane is
efficiently scavenged by the addition of methanol after
completion of the Masuda borylation. The addition of cesium
carbonate and (hetero)aryl halide is sufficient to facilitate the
Suzuki coupling. In comparison to Boc-protected (7-aza)indoles
the tosyl protecting group remains intact during the Suzuki
arylation step. The concurrent deprotection by the alcoholic
carbonate solution can be suppressed.30 The catalytic system
obviously remains stable to give a full conversion to the
protected meriolin derivatives. Finally, by addition of sodium
hydroxide to the reaction mixture deprotection of the tosyl group
gives rise to the isolation of meriolin derivatives 3 in good to
excellent yields. In essence, a single and simple catalyst system
sequentially catalyzes both borylation and (hetero)arylation, and
finally, the tosyl group can be selectively cleaved by addition of
sodium hydroxide. It is noteworthy to mention that all
compounds can be uneventfully isolated by a single simple flash
chromatography.
the sense of
a
one-pot Masuda borylation-Suzuki
(hetero)arylation (MBSA) sequence.
First examples of MBSA were reported on the synthesis of
biaryls,25 but it was not before 2011 when we came up with a
general MBSA sequence employing heterocyclic halides as
substrates, which were successfully applied to concise total
syntheses of several marine alkaloids26-28 and kinase inhibitors
(Scheme 2).7
I
(hetero)aryl
[Pd0], HBpin
N
N
then: (hetero)aryl-Hal, base
then: NaOH
N
PG
N
H
Scheme 2. Masuda borylation-Suzuki coupling (MBSA)
sequence in a one-pot fashion.
Besides the highly practical nature of one-pot processes,
where all transformations are conducted in a consecutive or
sequential fashion, the MBSA synthesis of bi(hetero)aryls
starting from two different (hetero)aryl halides can be considered
as a sequentially Pd-catalyzed process.29 Most characteristically,
the initial catalyst also will catalyze the subsequent reaction(s)
without further addition of catalyst loading.
Herein we report an optimized MBSA sequence for the
synthesis of novel meriolin derivatives that have previously been
identified to be potent inhibitors of PDK1.7 Various 3-pyrimidyl
and 3-pyridyl substituted 7-azaindoles as key pharmacophores, in
particular with amino substituents on the azine moieties, were

NH2
HN
Measuring activation of caspase-3 by using two concentrations
per selected compound with regard to their cytotoxicity profile,
we showed that all six compounds distinctly induce apoptosis
within 3-4 h (Fig. 3). Of note, compound 3f caused even higher
activation of caspase-3 in Ramos cells than the multikinase
inhibitor staurosporine (STS; used as positive control), although
STS was applied in a considerably higher concentration (2.5 µM
vs. 0.1 µM). To further verify rapid induction of apoptosis, we
detected cleavage of the caspase-3 substrate PARP1 via
immunoblotting (Fig. 3). Again, every compound of the
mentioned subset induced apoptosis within a few hours, in line
with the results of the caspase-3 activity assay.
N
N
N
N
NH2
NH2
N
N
N
N
H
N
H
N
N
N
N
H
H
3a
3c
3d
(81%)
3b
(75%)
NH2
(77%)
(91%)
N
H2N
N
N
N
N
N
OMe
NH2
SMe
N
N
N
N
H
N
H
N
N
N
N
H
H
a
3f
(67%)
3h
3e
3g
(83%)
(40%)
(47%)
MeO
H2N
H
N
N
N
N
N
N
NH2
NH2
NH2
N
N
N
H
N
N
N
H
N
N
N
N
H
H
3i
(62%)
3k
(83%)
3j
3l
(75%)
(53%)
N
N
NH2
NH2
N
N
N
N
H
N
N
N
N
b
c
c
3m
3n
3o
(93%)
(82%)
(96%)
Figure 3. Selected meriolin derivatives 3 exhibit pronounced
cytotoxicity on human lymphoma and leukemia cell lines. (A)
Ramos (Burkitt‘s lymphoma) and (B) Jurkat J16 (acute T-cell
leukemia) cells were incubated with escalating concentrations of
meriolin derivatives 3 for 24 or 72 h. After incubation, cell
viability was determined by resazurin reduction assay as
described in methods. Relative viability of DMSO (0.1% v/v)-
treated control cells was set to 100%. Data points shown are the
mean of three independent experiments, error bars = SD. IC50
values (IC50 = half maximal inhibitory concentration) were
calculated via nonlinear regression curve fitting using Prism 6
(GraphPad Software).
Figure 2. Scope of MBSA one-pot synthesis of meriolin
derivatives 3 (isolated yields are given in parentheses; color code
for halides 2: red = chloride, blue = bromide, green = iodide).
aSecond regioisomer was formed. DME/water was used in the
b
c
Suzuki coupling step. No deprotection after Suzuki coupling
step.
2.2. Biological activity
The class of meriolins is known to act highly cytotoxic in
cancer cells.31 In order to characterize their cytotoxicity profile
we measured cell viability of leukemia and lymphoma cells after
treatment with several novel meriolin derivatives 3 using the
resazurin reduction assay. Out of 13 tested meriolins 3, 6
exhibited pronounced cytotoxicity against the tested cancer cell
lines with IC50 values ranging from 0.06 to 6 µM (Fig. 3, Table
Table 1. Cytotoxicity of meriolin derivatives 3 towards leukemia
and lymphoma cells as determined by resazurin reduction assay.
IC50 expressed as mean from three independent experiments.
Bracketed values indicate 95% confidence intervals.
compound
Ramos
24h
Ramos
72h
Jurkat J16 Jurkat J16
24h
72h
1). The remaining meriolins 3 possessed low cytotoxicity (IC50
>
10 µM) or were inactive in the tested concentration range up to
30 µM (data not shown). Meriolin derivative 3f demonstrated the
highest potency in both cell lines, as reflected by its remarkably
low IC50 values. Compared to meriolin 1, compound 3f showed
up to 50-fold increased cytotoxicity. The high quantity of tested
meriolin derivatives provides insight into their structure-activity
relationship. The amino group in 2-position of the azine moiety
seems to be crucial for the cytotoxic efficacy since the vast
majority of tested meriolin derivative lacking this group failed to
potently induce cytotoxicity (3e, 3g, 3h, 3m). In addition,
3a
4.80 µM
[4.28-5.37]
0.97 µM
[0.91-1.05]
0.30 µM
[0.26-0.34]
0.11 µM
[0.10-0.11]
6.04 µM
[5.15-7.08]
0.37 µM
[0.34-0.40]
3.73 µM
[2.63-5.28]
0.60 µM
[0.50-0.71]
0.18 µM
[0.16-0.20]
0.07 µM
[0.06-0.08]
3.90 µM
[2.67-5.72]
0.24 µM
[0.21-0.27]
4.53 µM
[4.00-5.13]
0.87 µM
[0.81-0.93]
0.24 µM
[0.21-0.27]
0.11 µM
[0.10-0.11]
5.56 µM
[4.93-6.26]
0.43 µM
[0.37-0.49]
2.74 µM
[2.42-3.11]
0.48 µM
[0.44-0.51]
0.11 µM
[0.10-0.13]
0.06 µM
[0.06-0.07]
3.08 µM
[2.51-3.78]
0.21 µM
[0.19-0.23]
3c
3d
3f
3i
meriolin derivatives carrying
a pyridine substituent are
apparently more cytotoxic than derivatives with a pyrimidine
substituent (see 3a/j vs. 3c/f). Comparing the cytotoxicity of
compounds 3a and 3j, a second amino group at the azine in meta-
orientation seems to further increase the compound’s effect.
In order to determine whether the observed cytotoxicity of
compounds 3a, 3c, 3d, 3f, 3i and 3j is related to induction of
apoptosis we performed fluorimetric caspase-3 activity assays.
3j

Figure 4. A subset of meriolin derivatives 3 induces apoptosis in rapid kinetics. (A) Ramos or (B) Jurkat J16 cells were treated with the
indicated meriolin derivatives 3 for up to 8 h. After a lysis step, the pro-fluorescent caspase-3 substrate Ac-DEVD-AMC was added.
Increase in fluorescence of AMC was measured at 37 °C for at least 60 min using a microplate spectrophotometer and reflects caspase-3
activity. The multikinase inhibitor staurosporine (STS, 2.5 µM) served as positive control for caspase-3 activation. Curves for STS are
reused in corresponding diagrams. Ramos cells were treated with (C) 3a, (D) 3c, (E) 3d, (F) 3f, (G) 3i, or (H) 3j in defined
concentrations (selected on the basis of initial cytotoxicity testings) for 2-8 h. Subsequently, cells were harvested, lysed and deployed
for immunoblotting against the caspase-3 substrate PARP1. Expression of GAPDH was used as protein loading control. Co-treatment
with the broad-spectrum caspase inhibitor Q-VD-OPh (QVD, 10 µM) was conducted in order to confirm caspase dependency of PARP1
cleavage. The uncleaved form of PARP (p116) is indicated by filled arrowheads, the cleaved form (p85) by open arrowheads. STS (2.5
µM) served as positive control.
In order to improve multikinase inhibitor abilities of the
compounds biological activities, selected compounds were
evaluated by screening against sphingosine kinase 1 and 2. The
selected compounds 3a, 3c and 3f showed clear preference for
SK2 in a screening at 10 µM. Compound 3c was the least active
compound with 10% ± 5% and 39% ± 5% inhibition of SK1 and
SK2, respectively. Compounds 3a and 3f showed pronounced
inhibition of SK2 with 72% ± 3% and 87% ± 7%, respectively,

while SK1 inhibition was only moderate (10% ± 4% and 18% ±
1%, respectively).
mg) and cesium carbonate (823 mg, 2.50 mmol) the mixture was
stirred in a preheated oil bath at 100 °C for 18 h. After the Suzuki
coupling was completed the mixture was cooled to room temp
(water bath). Sodium hydroxide (100 mg, 2.50 mmol) was added
and the reaction was stirred at 100 °C for 4 h. Then, after cooling
to room temp (water bath), the solvents were removed in vacuo
and the residue was absorbed onto Celite®. After purification by
chromatography on silica gel (dichloromethane/methanol/
aqueous ammonia, see Supporting Information) and after drying
in vacuo at 80 °C for 18 h compound 3f (151 mg, 67%) was
obtained as an orange solid. 1H-NMR (DMSO-d6, 300 MHz):  =
5.33 (bs, 4 H), 6.05 (s, 2 H), 7.15 (dd, J = 4.7, 7.9 Hz, 1 H), 7.74
(d, J = 2.5 Hz, 1 H), 8.14-8.44 (m, 2 H), 11.88 (s, 1 H); 13C-NMR
(DMSO-d6, 75 MHz):  = 93.1, 113.6, 115.9, 117.3, 124.0, 127.7,
142.9, 144.3, 149.0, 159.2; EI MS (m/z (%)): 226 (14) [M+H+],
All tested compounds showed a marked inhibition of SK2 and
weak action on SK1. It has to be noted that the compounds
showed fluorescence quenching that may influence the assay
results. Having the similar assay conditions for both isoenzymes,
the selectivities on SK2 for 3a and 3f are clearly based on
biological activity and not on physicochemical properties. The
SK2 is known as a target involved in cell proliferation and
apoptosis. As recently reported inhibition of SK2 led to TRAIL-
dependent caspase-3 cleavage and was able to overcome TRAIL
resistance in non-small cell lung cancer cells.19 Collectively, the
presented data support the pharmacological activity observed in
cell viability assays and serve as additional argument for the
proof of concept of the presented compounds on multi-kinase
inhibition.
+
+
225 (100) [M+], 198 (21) [C11H10N4 ], 170 (7) [C10H8N3 ], 155
+
+
(4) [C10H7N2 ], 118 (5) [C7H6N2 ]; Anal. calcd. for C12H11N4
(225.25): C 63.99, H 4.92, N 31.09; Found: C 63.89, H 4.88, N
30.80. (For full experimental and analytical details, see
Supporting Information)
3. Conclusions
The MBSA sequence is a concise and reliable methodology
for easily accessing a broad spectrum of novel active agents in a
Cell lines and reagents: Adult lymphoblastic leukemia T
cells (Jurkat J16, no. ACC-282) and Burkitt's lymphoma B cells
(Ramos, no. ACC-603) were obtained from the German
Collection of Microorganisms and Cell Cultures (DSMZ,
Germany). Both cell lines were routinely cultured in RPMI 1640
media supplemented with 10% FCS, 120 IU/ml penicillin, and
120 µg/ml streptomycin. Resazurin sodium salt and Q-VD-OPh
hydrate (QVD) were purchased from Sigma Aldrich Life Science
(Darmstadt, Germany), staurosporine (STS) from LC
Laboratories (Woburn, MA, USA) and Ac-DEVD-AMC from
Biomol (Hamburg, Germany). The antibodies used for
immunoblotting were mouse anti-PARP1 (Enzo Life Sciences,
New York, NY, USA; #BML-SA250) and mouse anti-GAPDH
(Abcam, Cambridge, United Kingdom; #ab8245).
one-pot fashion. Moreover,
a modular diversity oriented
synthesis can be readily achieved by simply varying the halides.
A particular advantage is the sequentially Pd-catalyzed process,
which is highly catalyst economical. This methodological tool is
currently applied in various concise total syntheses of natural
products and bioactive compounds. Here, we used the
methodology for generating several new inducers of apoptotic
cell death in cancer cells. In particular, 3f showed great a great
potential as a valuable lead compound for future anticancer
studies.
4. Experimental Section
Synthesis of 1a:28 7-Azaindole (3.54 g, 30.0 mmol) and
potassium hydroxide (4.96 g, 75.0 mmol) were dissolved in DMF
(55.0 mL). A solution of iodine (7.63 g, 30.3 mmol) in DMF
(55.0 mL) was added dropwise to the solution at room temp and
the resulting mixture was stirred for 30 min. After addition of a
second portion of potassium hydroxide (4.96 g, 75.0 mmol) the
mixture was stirred at room temp for 10 min before a solution of
p-toluene sulfonyl chloride (12.14 g, 63.0 mmol) in DMF (55.0
mL) was added dropwise. The resulting mixture was stirred at
room temp for 3 h. After complete conversion (monitored by
TLC) the reaction mixture was poured onto ice water (200 mL)
and stored in the refrigerator overnight. The resulting yellow
precipitate was filtrated, washed with ice water (50.0 mL) and
dried in vacuo to give the desired product 1a (11.74 g, 98%) as a
light yellow solid (for full analytical details, see the Supporting
Information).
Resazurin reduction assay: Cell viability was evaluated
using the resazurin reduction assay. In viable cells the non-
fluorescent dye resazurin gets reduced to highly fluorescent
resorufin.32 Therefore resorufin fluorescence can serve as a
measure for metabolic activity. For viability testings, Ramos and
Jurkat J16 cells were seeded in a density of 0.5 x 106 cells/mL
and treated with the compounds of interest for the indicated time.
Resazurin (40 µM) was added 3 h prior to end of incubation time.
Finally, the fluorescence of resorufin (excitation: 535 nm,
emission: 590 nm) was measured using
a
microplate
spectrophotometer. Viability of control cells (0.1% DMSO) was
set to 100%.
Immunoblotting: Immunoblotting was performed according
to standard protocol as described earlier.33 Signals were detected
using IRDye800- or IRDye680- conjugated secondary antibodies
and an infrared imaging system (LI-COR Biosciences, Lincoln,
NE, USA).
General procedure MBSA (Compound 3f): The 7-azaindole
1a (1.00 mmol, 398 mg) and tetrakis(triphenyl-
phosphane)palladium(0) (35.0 mg, 0.03 mmol) were placed in a
dry screw-cap vessel with a magnetic stir bar. After evacuating
and refilling with argon for three times dry 1,4-dioxane (5.0 mL)
was added and the resulting mixture was degassed with argon for
10 min. Dry triethylamine (1.40 mL, 10.0 mmol) and 4,4,5,5-
tetramethyl-1,3,2-dioxaborolane (0.25 mL, 1.70 mmol) were
successively added. Then the reaction mixture was stirred in a
preheated oil bath at 80 °C for 4 h. The mixture was cooled to
room temp (water bath). Dry methanol (5.0 mL) was added and
the mixture was stirred at room temp for 10 min. After the
addition of 2,6-diamino-4-bromopyridine (2f) (1.00 mmol, 129
Fluorimetric caspase-3 activity assay: Activation of
caspase-3 was detected as described previously.33 Briefly, cells
plated in 96-well microtiter plates were lysed in caspase assay
buffer (20 mM HEPES, 84 mM KCl, 10 mM MgCl2, 200 μM
EDTA, 200 μM EGTA, 0.5% NP-40, 1 µg/mL leupeptin, 1
μg/mL pepstatin,
5 μg/mL aprotinin) after the indicated
incubation time. Subsequently, reaction buffer containing the
pro-fluorescent caspase-3 substrate Ac-DEVD-AMC was added.
In the final step, increase in AMC fluorescence, indicating
caspase-3 activity, was measured over a period of at least 60 min.
Sphingosine kinase inhibition assays: The inhibition of the
sphingosine kinase was measured as reported previously18a with

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minor changes. Briefly, the compounds of interest (10 µM) were
incubated with sphingosine kinase (SK) 1 or 2 (0.5 U/mL or 0.1
U/mL final concentration, respectively) for 15 min. Then 5 µM
sphingosine and ATP (final concentration, assay volume of 10
µL) were added and incubated for 30 or 60 min (SK1 and 2,
respectively) at room temperature. Afterwards the detection
reagent (BellBrook Labs) was added and incubated for 60 min in
the dark. The buffer contained 50 mM HEPES (pH 7.5), 4 mM
MgCl2, 100 µM sodium orthovanadate, 1 mM dithiothreitol, 0.01
% Brij 35 and 2 mM egtazic acid. Assays were performed in
triplicates in two independent experiments (3a and 3c at SK1
with n=1). Relative fluorescence intensity of the compounds was
calculated relative to specific fluorescence intensity (total
fluorescence intensity minus blank was set to 100%). Inhibition
values are derived by calculating 100 minus relative fluorescence
intensity. Values are reported as global means of all replicates
with standard error of mean.
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Acknowledgements
The authors cordially thank the GRK 2158 (Deutsche
Forschungsgemeinschaft) for financial support.
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Supporting Information:
Supporting Information to this article can be found online at
https://doi.org/j.bmc.
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