Involvement of apoptosis and autophagy in the death of RPMI 8226 multiple myeloma cells by two enantiomeric sigma receptor ligands

Article abstract of DOI:10.1016/j.bmc.2013.11.033

Over-expression of σ receptors by many tumor cell lines makes ligands for these receptors attractive as potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential σ-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the σ₁ and σ₂ receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of σ receptors, both compounds inhibited cell proliferation with IC₅₀ values in the low μM range. No chiral differentiation between either the σ receptor binding affinity or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as the expression of bcl₂ family members bax, bad and bid. However, apoptosis appeared to be caspase independent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However, further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotective. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the lipid antioxidant α-tocopherol attenuated LPO. Interestingly, α-tocopherol reduced significantly both apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autophagy in RPMI 8226 cells.

Full text of DOI:10.1016/j.bmc.2013.11.033

Bioorganic & Medicinal Chemistry 22 (2014) 221–233
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Involvement of apoptosis and autophagy in the death of RPMI 8226
multiple myeloma cells by two enantiomeric sigma receptor ligands
a
b
b
b
a,
⇑
Katharina Korpis , Frauke Weber , Stefanie Brune , Bernhard Wünsch , Patrick J. Bednarski
a
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Greifswald, F.-L.-Jahn-Straße 17, 17487 Greifswald, Germany
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster Corrensstraße 48, 48149 Münster, Germany
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Over-expression of r receptors by many tumor cell lines makes ligands for these receptors attractive as
potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential
Received 17 September 2013
Revised 8 November 2013
Accepted 16 November 2013
Available online 25 November 2013
r-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds
showed high affinities for the
r
1
and
r
r
2
receptors. In the human multiple myeloma cell line RPMI
receptors, both compounds inhibited cell proliferation with
8226, a line expressing high levels of
IC50 values in the low lM range. No chiral differentiation between either the r receptor binding affinity
Keywords:
or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was
evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the
cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as
Sigma-receptor
RPMI 8226 cells
Apoptosis
Cancer
2
the expression of bcl family members bax, bad and bid. However, apoptosis appeared to be caspase inde-
Lipid peroxidation
Autophagy
pendent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-
II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However,
further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotec-
tive. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the
lipid antioxidant
a-tocopherol attenuated LPO. Interestingly, a-tocopherol reduced significantly both
apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating
LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autoph-
agy in RPMI 8226 cells.
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
Receptors were first described by Martin et al.1 and initially
r
2
receptor, has been cloned from many tissues but there is little
similarity to known receptors except for a 30% homology with
the yeast D /D isomerase. Since the discovery of the r receptors,
8 7
they have been implicated in many neurological disorders like
4
r
2
classified as a subtype of the opioid receptor. However, it is now
known that they are a unique class of receptors with two subtypes,
schizophrenia, alzheimer’s disease and depression, due to the fact
3
5–7
termed
r
1
(25 kDa) and
r
2
(21.5 kDa). The
r
1
receptor, but not the
that they modulate some neurotransmitter systems.
In the cen-
tral nervous system, both subtypes are involved in the modulation
2
+
+
8–11
of Ca and K channels.
r
1
Receptors are suggested in the mod-
receptors in the modulation of sphingo-
Interestingly, both subtypes are over-expressed in
many human tumor cells. These findings have led to the develop-
ment of ligands for both diagnostic imaging and cancer treat-
Abbreviations: AI, apoptosis inhibitor;
BODIPY 665/676 4-difluoro-3,5-bis(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-
diaza-s-indacene; DCFDA, 2 ,7 -dichlorofluorescein diacetate; DAPI, 4,6-diamino-
-phenylindole; DMSO, dimethylsulfoxide; DTG, ditolylguanidine; IC50, half max-
imal inhibitory drug concentration; K , ligand dissociation constant; K , inhibitory
B
max
,
maximal ligand binding sites;
ulation of ankyrin and r
lipid levels.
2
C
11
,
12,13
0
0
2
r
d
i
1
4,15
dissociation constant; LC3B, microtubule-associated protein light chain 3; LPO,
Lipid peroxidation; 3-MA, 3-methyladenine; MMP, mitochondrial membrane
potential; MTT, 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide;
PARP, poly(ADP-ribose) polymerase; PBS, Dulbeccos phosphate buffered saline; PI,
propidium iodide; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen
species; SEM, standard error of mean; SD, standard deviation; TBH, tertiary-
ment.
shown to be involved in programmed cell death (apoptosis),
whereas receptor agonists and receptor antagonists have
antiapoptotic and neuroprotective activity.
antagonists induce caspase-dependent apoptosis , whereas
1 2
r Receptor antagonists and r agonists have been
r
1
r
2
1
6
In particular,
r
r
1
1
7
1
6
butyl-hydroperoxide; z-VAD-FMK, z-Val-Ala-Asp(Ome)-CH
Corresponding author. Tel.: +49 3834 864883; fax: +49 3834 864802.
E-mail addresses: korpis@uni-greifswald.de (K. Korpis), fwebe_01@uni-muen-
2
F.
agonists appear to prevent caspase activation. On the other hand,
agonists have been shown to initiate a caspase-independent
pathway of programmed cell death and have been postulated to
⇑
r
2
ster.de (F. Weber), sbrun_02@uni-muenster.de (S. Brune), wuensch@uni-muen-
ster.de (B. Wünsch), bednarsk@uni-greifswald.de (P.J. Bednarski).
1
8–20
be potential anti-cancer drugs.
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.033

2
22
K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
The ethylenediamine substructure is a crucial pharmacophore
element for receptor ligands. Several studies indicate that the eth-
ylenediamine can also be incorporated into a piperazine ring, lead-
ing to 1,4-disubstituted piperazine derivatives with high
r
r
1
21,22
receptor affinity, (e.g., (1), Fig. 1).
piperazine derivatives with an aromatic residue at N-4 showed very
potent receptor affinity. This was further enhanced by developing
the p-methoxybenzyl derivative 2, which had a 3-fold higher affinity
Hydroxymethyl-substituted
r
1
2
1
for the
1
r receptor. We previously reported on bridged piperazine
6
,8-diazabicyclo[3.2.2]nonane derivatives as sigma ligands with
2
3
high
growth inhibition activities in various human cancer cell lines, with
IC50 values comparable to those of cisplatin in cell lines expressing
receptors. In that study, the antiproliferative activity correlated with
both the affinity and stereochemistry. Later, we synthesized con-
formationally restricted receptor ligands with a 7,9-diazabicy-
clo[4.2.2]decane scaffold and found that the affinities for and
receptors were lower for the four-membered bridge than their cor-
1
r affinity. In addition, these compounds displayed strong
r
r
1
r
1
r
1
r
2
24
responding three-carbon bridged piperazines. The cytotoxic activ-
ity against the same tumor cell lines was diminished but still
moderate with IC50 values in the low
PB28 (3) is a potent agonist and also contains an ethylenedi-
amine unit within a piperazine ring. Based on knowledge of the
structure–activity relationships of 1–3, compounds with
hydroxyethyl-containing side chain and bulky residues at both
N-atoms were synthesized, exemplified by enantiomers (S)-4 and
lM range.
r
2
25
Figure 1. Structural formulas of monocyclic piperazine derivatives.
a
3
3
[ H]-(+)-pentazocine and [ H]-DTG are in range with what others
2
7
have reported for various human cancer cell lines.
The and affinities of the enantiomeric piperazines (S)-4
and (R)-4 are summarized in Table 1. Both enantiomers show high
affinity towards the guinea pig brain receptor, but reduced
affinity towards the human receptor of the RPMI 8226 cells.
The affinity of both compounds in both assays is in the same
two-digit nanomolar range. Whereas comparison of the human
receptor affinities reveals only a low selectivity of 1.3, the
selectivity is considerably higher when the results with the
(
R)-4, with a biphenylmethyl moiety at N-1 and a cyclohexylmeth-
yl residue at N-4. The goal of the present study was to understand
the cellular mechanisms by which two enantiomeric receptor li-
gands cause cell death in the multiple myeloma cell line RPMI
226, a line that over-expresses
receptors.26 Our data indicates
r
1
r
2
r
r
1
r
1
8
r
r
2
that both apoptosis and autophagy are operative during cell death,
but that autophagy appears incomplete and does not protect dying
cells against apoptosis.
r
1 2
r /r
1 2
r /r
membrane preparations produced from guinea pig brain and rat li-
ver are taken into account. Since (S)-4 has a lower r2 affinity than
2
2
2
4
. Results
2
(R)-4, its selectivity factor against the r subtype is almost 4-fold
.1. Chemistry
higher than those of (R)-4.
.1.1. Synthesis of the hydroxyethylpiperazines (S)-4 and (R)-4
The synthesis of the (S)-configured hydroxyethyl piperazine (S)-
started with the proteinogenic amino acid (S)-aspartate, which
2.3. Cytotoxity in RPMI 8226 cells
Exposing RPMI 8226 cells to increasing concentrations of either
3, (S)-4 or (R)-4 for 72 h resulted in a dose-dependent decrease in
cell viability/number, as determined by the MTT assay. The po-
tency of the compounds is expressed in the IC50 value (Table 1)
measured by the MTT method, which shows (R)-4 to be somewhat
more potent than (S)-4, but this difference was not significant.
Notable is that the commercially available r2 agonist PB 28 (3),
was transformed into the HCl salt of the diester (S)-5 (Scheme 1).
The chloroacetamide (S)-7 was obtained in a three step sequence
without purification of the intermediates. (S)-5 was condensed with
4
the aldehyde 6 to an imine, which was reduced with NaBH to afford
a secondary amine. Acylation of the secondary amine with chloro-
acetyl chloride provided the chloroacetamide (S)-7 in 34% yield over
three reaction steps. The piperazinedione (S)-8 was formed by a tan-
with an IC50 value of 9.29
lM, is only slightly more potent than
dem reaction comprising of an S
S)-7 with cyclohexylmethylamine and subsequent intramolecular
aminolysis of the resulting amino ester. Finally, LiAlH reduction
of (S)-8 led to the hydroxyethyl substituted piperazine (S)-4 in
7% yield. The enantiomeric piperazine derivative (R)-4 was pre-
pared in the same manner starting with (R)-aspartate.
N
2 reaction of the chloroacetamide
(R)-4. The large difference between receptor K and cellular IC
i
50
(
values for 3, (S)-4 and (R)-4 may be a result of two mechanisms:
(1) the highly lipophile ligands become widely distributed within
the cells, lowering the effective concentration present at the recep-
tors, (2) nearly full receptor occupancy is required for cytotoxicity,
4
7
as has been reported for other
r ligands (see Discussion below). In
contrast to the RPMI 8226 cell line, no antiproliferative effects
were observed for (S)-4 and (R)-4 in the human HL60 leukemia cell
2
.2. r Receptor affinity
line at 20
Enantiomeric differences in
have previously been observed for various r
l
M, evidence for a selective action in the former cell line.
receptor affinity and cytotoxicity
ligands possessing rig-
The K
with the highly selective
system and the ligand DTG in the
ence of an excess of (+)-pentazocine (2 M) to block
mean K -value of 36.0 ± 0.3 nM and Bmax-value of 477 ± 7 fmol/mg
protein for [ H]-(+)-pentazocine and a mean K
and Bmax-value of 371 ± 25 fmol/mg protein for [ H]-DTG were cal-
d
- and Bmax-values were determined for RPMI 8226 cells
r
3
r
1
agonist [ H]-(+)-pentazocine in the
system in the pres-
receptors. A
2
8
r
1
r
1
/r
2
r
2
idly-bridged bicyclopiperazines. Thus, it came as some surprise
that the two enantiomers studied here showed no chiral discrimi-
nation with respect to receptor binding and cytotoxicity in human
RPMI 8226 cells. This lack of discrimination may be due to the
chiral center being located in a flexible piperazine ring that can
interconvert at both nitrogens to keep the hydroxyethyl group of
l
r
1
d
3
d
-value of 12 ± 3 nM
3
26
d
culated from three independent experiments. Our K values for

K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
223
Scheme 1. Synthetic route to (S)-4. Reagents and synthetic reaction conditions: (a) CH
rt, 45 min, (3) chloroacetyl chloride, CH Cl , NEt , rt, 2.5 h, 34% (over 3 steps); (c) cyclohexylmethylamine (C
6 h, 77%. (R)-4 was prepared in the same manner.
3
OH, (H
3
C)
3
SiCl, rt, 16 h, 100%; (b) (1) 6, CH
2
Cl
2
, NEt
3
, Na
2
SO
4
, rt, 16 h, (2) NaBH
4 2
, Et O,
2
2
3
6
H
11CH NH ), CH CN, NEt
2
2
3
3
, rt, 16 h, 68%; (d) LiAlH
4
, THF, reflux,
1
Table 1
i 2
Receptor affinities (K values) and cytotoxicity in RPMI 8226 cells (IC50 values) of the monocyclic lead compounds 1 and 2, the known r -agonist PB 28 (3) and our newly
r
developed enantiomers (S)-4 and (R)-4
Compd
K
i
± SEM (nM)
IC50 ± SD (lM)
r
1
Affinity
r
2
Affinity
1 2
r /r Selectivity animal/human
a,f
b,f
1
2
3
0.47 ± 0.04
161 ± 19
342/n.d.
5.8/n.d.
46.4/n.d.
20.8/1.3
4.5/1.3
n.d.
n.d.
9.29 ± 4.25
21.32 ± 6.46
13.04 ± 4.90
a,g
b,g
12 ± 1
70 ± 10
c,h
c,h
(PB 28)
13
0.28
a
d
d
b
e
(S)-4
R)-4
3.5 ± 0.5 , 34 ± 8
7.8 ± 2.4 , 29 ± 7
73 ± 45 , 43 ± 13
a
b
e
(
38 ± 1.0 , 39 ± 11
Values determined here are the averages of at least three independent determinations.
n.d.: not determined.
K values of compounds measured with receptor preparations from:
i
a
Guinea pig brain.
Rat liver.
MCF-7 cells.
RPMI 8226 cells.
RT-4 cells.
b
c
d
e
f
K
K
K
i
i
i
values are from Ref. 22.
values are from Ref. 21.
values are from Ref. 42.
g
h
either isomer in an equatorial position without dramatic changes
in the orientations of any of the three ring substituents, thus mak-
ing possible comparable poses within the
r receptor active site.
This structural similarity is visualized by superimposing a low en-
ergy conformation of the two enantiomers, as shown in Figure 2 (or
in the video of Supplementary Fig. 1).
2
.4. Induction of apoptosis
To determine whether the decrease in cell viability/number was
Figure 2. Superimposed structures of
a low energy conformer for (S)-4
caused by apoptosis, flow cytometric analysis of RPMI 8226cells trea-
ted with equi-toxic concentrations of (S)-4 (36 M) or (R)-4 (23 M)
were stained with PI + annexin-V-FITC. While no changes in the dis-
tribution of cells were observed after 6 h, significant increases in the
population of apoptotic (annexin-V positive) cells after 24 and 48 h
werenoted(Fig. 3A). Moreoverchromatincondensation,anotherhall-
(28.857 kcal/mol) and (R)-4 (28.880 kcal/mol). Structures and energies were
calculated with the MM3 force field and aligned by best least-squares fit,
implemented with the PCMODEL (ver 8.5) software (Serena Software, Bloomington,
IN).
l
l
mark of apoptosis, was visualized in fluorescence micrographs of
DAPI-stained cells (Fig. 3B).

2
24
K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
2
.5. Caspase-3 and caspase-8 activation
To determine if apoptosis was mediated by caspases, the activ-
2.6. Apoptosis is caspase-independent
To evaluate whether caspases are involved in drug induced
apoptosis, the effect of the general caspase irreversible inhibitor,
z-VAD-FMK, was examined by annexin-V/PI double staining of
ities of caspase-3, caspase-8 and caspase-9 were measured by a
flow cytometry method based on the Green Caspase Staining Kit
(
Promokine, Heidelberg, Germany). The results indicate that all
three caspases were activated in a time-dependent manner
Fig. 4A), suggesting that both the extrinsic and intrinsic pathways
cells treated with (S)-4 (36 lM). Preincubation with 100 lM z-
VAD-FMK 1 h before drug treatment did not prevent apoptosis
0
(
(Fig. 5). In addition, the apoptosis inhibitor 2,2 -methylene-
2
9,30
were activated. Caspase-8 activation was also demonstrated by
western blot analysis (Fig. 4B and C); activation of caspase-8
involves a proteolysis of the inactive procaspase (55 kDa) to
intermediate cleavage fragments (26–31 kDa) and an active
bis(1,3-cyclohexandione)
at 50 lM also failed to block
apoptosis, suggesting that apoptosis caused by (S)-4 and (R)-4 is
caspase-independent (Supplementary Fig. 2).
1
8 kDa fragment. Moreover, a 6 h drug treatment resulted in a
2.7. Sigma ligands induced lipid peroxidation
significant cleavage of the poly (ADP-ribose) polymerase-1
(
(
PARP-1), one of the main cleavage targets of caspase-3 in cells
Fig. 4B and C). However, caspase-9 expression in RPMI 8226 cells
To measure whether lipid peroxidation (LPO) might play a role
in cell death, treated and untreated cells were loaded with the fluo-
rescent dye BODIPY 665/676 C11. This lipophilic dye with a conju-
gated polyene system is vulnerable to oxidation by lipid peroxyl
radicals, resulting in a shift in fluorescence. As shown in Figure 6A,
a shift in fluorescence was observed by flow cytometry in cells trea-
was below the detection limits of the Western blotting assay (data
not shown).
To investigate further the mechanisms of drug induced apopto-
sis, expression of bcl-2 family proteins was studied. Western blot-
3
1
ting revealed that time-dependent exposure to (S)-4 (36
l
M)
ted with (R)-4 (23
remained for 24 h (Fig. 6B). Moreover, pretreatment with the lipid
antioxidant -tocopherol (200 M) almost completely inhibited
LPO (Fig. 6A and B). Treatment with the scavenger alone did not in-
duce LPO production. On the other hand, no significant reactive
lM). This shift in fluorescence began by 5 h and
decreased bax levels over 96 h, whereas the bcl levels increased
2
significantly by 72 h (Fig. 4B and C). However, significant decreases
in bid levels due to the cleavage to the truncated form were not ob-
served over 96 h (Fig. 4B and C).
a
l
Figure 3. Apoptotic effects of (S)-4 and (R)-4. (A) Analysis of apoptotic cells by annexin/PI double staining. Annexin-V positive/PI negative RPMI 8226 cells after treatment
with (S)-4 (36 M) or (R)-4 (23 M) for 6, 24 and 48 h. Apoptosis was evaluated by determing the percentage of annexin-V positive cells and expressed as mean ± SD of at
least three determinations, con = untreated cells, p <0.05, p <0.01. (B) Nuclear morphological changes in RPMI 8226 cells untreated (control) or treated with 36
M (R)-4 for 24 h and 48 h were observed after DAPI staining (magnification 40 ꢀ 1300).
l
l
⁄
⁄⁄
l
M (S)-4 or
23 l

K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
225
Figure 4. Effects of (S)-4 and (R)-4 on caspase activation and apoptosis regulatory proteins. (A) Activation of caspase-3,-8 and -9 after treatment of RPMI 8226 cells with
M (S)-4 and 23 M (R)-4 after 24 and 48 h and flow cytometry analysis. Bars indicate mean ± SD of at least three determinations. (B) Time dependent expression levels of
, PARP, bax, caspase-8 and bid were analyzed by western blotting. RPMI 8226 cells were treated with (S)-4 (36 M) for 6–96 h. Protein lysates were collected for western
blot analysis. GAPDH acts as a loading control. Con = controls without treatment, representative immunoblots from three independent experiments are shown. (C)
3
6
l
l
bcl
2
l
⁄
⁄⁄
⁄⁄
⁄
Quantitative data of western blot analysis are represented as the mean ± SD. p <0.001, p <0.01, p <0.05.
oxygen species (ROS) formation, measured with DCFDA, could be
detected by flow cytometry in treated cells (Fig. 6C).
2
.8. Effects on mitochondrial membrane potential
Mitochondria play an important role in apoptosis and autoph-
agy. The mitochondrial membrane potential (MMP,
m
D-W )
regulates the permeability of mitochondria, which serves a crucial
role in triggering apoptotic and autophagic pathways.32 Disruption
of the mitochondrial transmembrane potential is one of the earliest
intracellular events after inducing apoptosis. We examined the
m
effect of (R)-4 (23 lM) on D-W by JC-1 staining of the mitochon-
Figure 5. Analysis of apoptotic cells by annexin/PI double staining. RPMI 8226 cells
were treated with 36 M (S)-4 for 48 h with or without pretreatment of 100 M z-
VAD FMK for 1 h. Apoptosis was evaluated by determing the percentage of annexin-
V positive/PI negative cells and expressed as mean ± SD of at least three determi-
nations, con = controls without treatment.
dria. This cationic dye shows transmembrane potential-dependent
accumulation in mitochondria due to a fluorescence shift. In vital
cells JC-1 fluoresces either as green monomers in the cytosol
or as red aggregates in the mitochondria. In apoptotic cells with
a reduced membrane potential the dye is not able to aggregate and
l
l
3
3
only green fluorescence in the cytosol can be detected. Flow
cytometry studies revealed time-dependent depression in
, which was first detected after 1 h (Fig. 6D), becoming
a
2.9. LPO triggered apoptosis
D-W
m
most notable at 3 h and normalized by 24 h. This behavior
indicates that loss of MMP is an early event in the cytotoxicity of
To assess whether LPO might be involved in apoptosis, the ef-
fect of the antioxidant
a-tocopherol on the cytotoxicity of (S)-4
(R)-4.
and (R)-4 in RPMI 8226 cells was investigated. The annexin-V/PI

2
26
K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
Figure 6. Effects of (R)-4 on formation of LPO, ROS and mitochondrial membrane dissipation. (A) LPO-Detection: Representative flow cytometric histograms of control (black
line) and cells (orange line) treated for 24 h with 23 M (R)-4 and a) no preincubation with the antioxidant -tocopherol or b) with preincubation after staining with the
fluorescent dye BODIPY 665/676 C11. (B) Quantification of the mean fluorescence intensity of control (con) or drug treated RPMI 8226 cells with or without -tocopherol
Toco). The results indicate mean ± SD of at least three determinations. (C) ROS-Detection: Representative flow cytometric histograms of control (black line) and cells treated
with 23 M (R)-4 (green line) for indicated times. DCFDA was used for estimation of ROS. TBH (220 M, red line) served as a positive control, (D) MMP-Detection:
M (R)-4 (blue line) for indicated times after staining with JC-1.
l
a
a
(
l
l
Representative flow cytometric histograms of control (black line) and cells treated with 23
l
double staining revealed no change in annexin-positive cells after
LC3-II levels increased significantly. Another hallmark of autoph-
agy are changes in the levels of the polyubiquitin-binding p62 pro-
tein (p62 or sequestosome 1 (SQSTM1), which binds to LC3 and
attracts protein aggregates to the autophagosome for degrada-
2
4 h when (S)-4 was co-incubated with antioxidant compared to
controls treated with the drug alone, but by 48 h a statistical de-
crease in apoptotic cells was found (Fig. 7A). (R)-4 showed attenu-
ated effects (Fig. 7B). In addition, when the fraction of early and
late apoptotic cells (annexin and PI positive cells) at 48 h was con-
sidered, an intense protective effect of the LPO-scavenger was
found for both compounds (Fig. 7C).
3
5
tion. Consequently, the levels of p62 decrease during autoph-
3
4
agy. However, in the cases of both (S)-4 and (R)-4, increases in
p62 expression were observed in western blotting analysis
(Fig. 8A and B), which can be seen as evidence that autophagy is
incomplete.
2
.10. Sigma ligands induced incomplete autophagy
2
.11. LPO triggers autophagy
To determine if the ligands were involved in autophagic pro-
r
cesses, western blot analysis of the classical autophagosome mar-
ker, microtubule-associated protein light chain 3 (LC3) was done.
LC3 exists in three isoforms (LC3A, LC3B and LC3C); in this study
the LC3B form was assessed. During autophagy LC3 is cleaved to
its cytosolic form LC3-I. Lipidation leads to the LC3-II form, which
becomes associated with the autophagosomes. Conversion of LC3-I
Having established that the compounds induce LPO, it was of
interest to know if LPO triggered autophagy in RPMI 8226 cells.
Therefore, the effect of -tocopherol was investigated on the
expression levels of LC3 and p62. Pretreatment with -tocopherol
a
a
1 h before drug exposure blocked LC3-II expression completely, as
shown representatively for (R)-4 in Figure 8C and D. Likewise, p62
levels diminished (Fig. 8C and D).This strongly supports the idea
that LPO plays a key role in the induction of autophagy.
3
4
to LC3-II is therefore a marker for the autophagosome formation.
As shown representatively for (R)-4 in Figure 8A and B, by 48 h

K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
227
Figure 7. Effects of (S)-4 and (R)-4 after pretreatment with the antioxidant
a-tocopherol. Annexin-V positive/PI negative cells after treatment with (A) 36
l
M (S)-4 or (B)
2
3
lM (R)-4 for 24 and 48 h with or without 1 h preincubation with 200 lM a-tocopherol (Toco). (C) Annexin-V/PI positive cells, which represents the early and late
apoptotic fraction, after treatment with 36
mean ± SD of at least three determinations, p <0.001, p <0.01, p< 0.05.
l
M (S)-4 or 23
l
M (R)-4 for 48 h with or without 1 h preincubation with 200
lM
a
-tocopherol. Con = control, the results represent
⁄
⁄⁄
⁄⁄
⁄
2
.12. Effects of autophagy inhibitors
dual role in autophagy, on the one hand it is known to act as an
early phase autophagy inhibitor, blocking the activity of class III
phosphoinositide 3-kinase (PI3K), which together with beclin-1
plays important roles in preautophagosome formation.36 On the
other hand, it was found to induce autophagy after a prolonged
incubation in full medium under nutrient-rich conditions.37 In our
study a 48 h incubation of RPMI 8226 cells with 10 mM 3-MA alone
and in combination with (R)-4 increased the expression of LC3-II,
thus 3-MA appears to act as an autophagy inducer (Fig. 8C and D).
Wortmannin, a late phase autophagy inhibitor, blocks the fusion
PARP-cleavage fragments, an indication of apoptosis, were first
observed at 6 h drug treatment (Fig. 4B and C), but LC3B-II forma-
tion, an indicator of autophagy, was first noted at 24 h and levels
became significant at 48 h (Fig. 8A and B). Thus, it appears that
apoptosis precedes autophagy, and as a result autophagy cannot
be protecting cells from apoptosis. To confirm this, RPMI 8226 cells
were pretreated with autophagy inhibitors 3-methyladenine
(
3-MA) and wortmannin 1 h before drug treatment. 3-MA has a

2
28
K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
Figure 8. Effects on p62 and LC3B processing during autophagy, representative for (R)-4. (A) RPMI 8226 cells were treated with (R)-4 (23
collected for western blot analysis. GAPDH acts as a loading control. Con = controls without treatment, representative immunoblots are shown. (B) Quantitative western blot
data are represented as the means ± SD. (C) Effects of (R)-4 after preincubation with the antioxidant -tocopherol (Toco, 200 M) or autophagy inhibitors wortmannin (Wort,
M) and 3-MA (10 mM) for 48 h. (D) The relative densitiy of protein bands were quantified, normalized to that of GAPDH and expressed as percentage relative to the
lM) for 6–96 h. Protein lysates were
a
l
2
l
⁄
⁄⁄
⁄⁄⁄
expression level in control cells. All results are of at least three determinations, (B) and (D) show means ± SD, p <0.05, p <0.01, p <0.001.
of autophagosomes and lysosomes at the end of the autophagic pro-
observing typical morphological blebbing of the cells and nuclear
3
8
cess. Wortmannin (at 2
but together with (R)-4 significantly increased the levels of LC3-II
Fig. 8C and D). However, p62 levels remained unchanged, indicat-
ing incomplete autophagy. Wortmannin has low toxicity at 1 M in
lM) had no effect on LC3-II expression
fragmentation by DAPI staining. Blebbing of the cell membrane
and formation of apoptotic bodies follows the translocation of
the phospholipid phosphotidylserine (PS) from the inner face of
(
4
4
l
the plasma membrane to the cell surface. Flow cytometry with
annexin-V staining of PS in the outer membrane leaflet provided
additional evidence for apoptosis. The caspase family plays a cen-
tral role in apoptosis: induction via the intrinsic apoptotic pathway
results in the activation of initiator-caspase-9, while induction by
RPMI 8226 cells so this concentration was used in combination
experiments. In combined treatments of wortmannin with either
(R)-4 or (S)-4, no significant increases in annexin-V positive cells
were observed after 48 h compared to cells treated with the
r
ligand alone, providing further evidence that autophagy is
incomplete and does not protect against apoptosis (Supplementary
Fig. 3).
death receptors activate initiator-caspase-8, which in turn acti-
vates the effector-caspase-3 by proteolytic processing.4
5,46
Flow
cytometry with fluorogenic, irreversible binding caspase inhbitiors
was used to show the activation of caspases-3, -8 and -9 in cells
treated with (S)-4 or (R)-4 for 24 and 48 h. Thus, diverse evidence
points at apoptosis as a key mechanism of cell death for both
compounds.
Apoptosis can arise either via the extrinsic or intrinsic path-
way, but it is also possible that both ways are linked by cleavage
2
of the blc -protein bid. However, while western blot analysis
3
. Discussion
The use of
r receptor ligands as potential anticancer drugs has
6,39–41
been attracting attention in recent years.
ported on the antiproliferative activity of diverse
Depending on the structure of the ligand and the tumor cell line,
We have also re-
2
3,24,28
r
ligands.
showed cleavage products of PARP-1 and caspase-8, no evidence
for caspase-9 fragments or cleaved bid were observed. Moreover,
the broad spectrum caspase inhibitor z-VAD-FMK and the apop-
tosis inhibitor 2,2 -methylenebis(1,3-cyclohexandione) failed to
inhibit apoptosis, even at high concentrations. These findings
r
ligands are known to induce cell death by various mecha-
1
8,20,40,42
nisms.
In this study, we examined cell death caused by
receptor ligands with high and
receptor affinities. For these studies the multiple myeloma cell line
RPMI 8226 was used, which is known to express high levels of
two newly synthesized
r
r
1
r
2
0
r
2
6
suggest that (S)-4 and (R)-4 activate apoptosis in a caspase-inde-
receptors.
Both (S)-4 and (R)-4 have good affinity to both the
receptors in the nanomolar range but inhibit the growth of RPMI
226 cells with IC50 values in the low M range. The large differ-
ences between receptor affinity and cytotoxic potency have been
observed in the past for ligands and were explained by data indi-
cating that near complete occupancy of the receptor population
pendent manner. There is reported evidence that some
receptor ligands, typically agonists, induce apoptosis by a cas-
pase-independent pathway, and caspases play only a bystander
r
1 2
r and r
r
2
8
l
1
8,42
role.
The mitochondrion plays a central role in both apoptotic path-
ways. Disruption of accompanied by the release of apopto-
genic proteins is critical in caspase-dependent and-independent
r
D-W
m
r
is required for cell death.43 This together with the fact that the li-
gands are highly lipophilic and distribute strongly into cell mem-
branes are likely explanations for the differences between
receptor binding affinity and cytotoxic potency.
4
7
2
mechanisms. The pro- and anti-apoptotic bcl family members
act as key regulators of the mitochondrial membrane. An imbalance
in their levels disrupt the integrity of the outer mitochondrial mem-
4
8
brane. Treatment with (S)-4 resulted in a reduction in
m
DW after
It was hypothesized that inhibition of cell survival by (S)-4 and
1
–3 h. Western blot analysis of the anti-apoptotic protein
(R)-4 was caused by apoptosis. This idea was substantiated by

K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
229
bcl
2
revealed increasing levels, and the pro-apoptotic bax levels de-
Evidence is provided that both compounds initiate mitochondrial
disruptions as well as LPO generation, leading to the release of pro-
creased after a 72 h treatment with (S)-4. It is possible that both fac-
tors can antagonize each other in their function, leading to a steady
state modus. Although caspases play a pivotal role in the apoptotic
cell death, apoptogenic proteins exist that do not require caspase
activation. One of these proteins is the apoptosis-inducing factor
survival and pro-apoptotic bcl
2
-family members. A reduction of
DW-
m
and an imbalance in LPO generation results in the release of
AIF from mitochondria into cytosol. Consequently, cancer cells are
stimulated to undergo a caspase-independent apoptotic cell death
that is not protected from by autophagy. However, the exact path-
waystocelldeathremainelusiveandfurtherstudieswillberequired
(
m
AIF). A lessening of DW is accompanied by the translocation of
AIF to the nucleus, which then mediates caspase-independent
apoptosis with characterictic features like chromatin condensation
and DNA fragmentation.49 Preliminary western blotting data re-
vealed the presence of AIF in the cytosolic fraction in cells treated
with (S)-4 (data not shown).
to unravel the mode of action of these interesting new
r ligands.
5
5
. Materials and methods
.1. Materials
Cancer is often associated with an excessive generation of reac-
tive oxygen species (ROS), which can damage DNA, cellular pro-
50
teins and cell membrane lipids as well as directly affecting
RPMI 1640 medium was obtained from PAN-Biotech (Alden-
bach, Germany). Chloromethylsilane, (S)- and (R)-aspartate, chlo-
roacetylchloride, fetal bovine serum, penicillin/streptomycin, 3-
various signaling pathways.2
0,51,52
Therefore, we investigated the
ability of our ligands to induce both ROS and LPO. While ROS
r
could not be detected by the classical staining with the fluorescent
dye DCFDA, both enantiomers triggered LPO generation as an early
event, which can be completely suppressed by preincubation with
the antioxidant
apoptosis, the annexin-V/PI double staining was assessed after pre-
treatment with -tocopherol. Decreased apoptotic levels provided
evidence that LPO act upstream of the apoptosis cascade. Thus, LPO
may play a key role in the induction of cell death. Although cas-
pase-dependent apoptosis is the most common pathway of physi-
ological cell death, an imbalance of LPO generation might prompt
the cells to turn down a caspase-independent route.
(
(
(
4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium-bromide
0
MTT), 4 ,6-diamidino-2-phenylindole (DAPI), propidium iodide
0
0
PI) and protease inhibitor cocktail (PIC), 2 ,7 -dichlorofluorescein
-tocoph-
a-tocopherol. To investigate if LPO also triggers
diacetate (DCFDA), tertiary-butyl-hydroperoxide (TBH),
a
erol, wortmannin and 3-methyladenine (3-MA) were purchased
from Sigma–Aldrich (Taufkirchen, Germany). The guinea pig brains
and rat livers were obtained from Harlan-Winkelmann (Borchen,
a
3
Germany). [ H](+)-pentazocine (specific activity 22 Ci/mmol) was
3
obtained from Perkin Elmer (Boston, MA), [ H](+)-pentazocine
3
(
specific activity 29–50 Ci/mmol) and [ H]-ditolylguanidine (spe-
cific activity 50 Ci/mmol) were from ARC (St. Louis, MO). DMSO
for cell culture) was obtained from Roth (Karlsruhe, Germany).
The effects of the new
r ligands on autophagy were also studied.
(
Compared to apoptosis much less is known about the mechanisms
underlying autophagy. It is widely accepted to be a survival mech-
Accutase was obtained from PAA Laboratories GmbH (Cölbe, Ger-
many), 4-difluoro-3,5-bis(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-
diaza-s-indacene (BODIPY 665/676 C11) was purchased from Invit-
rogen (Eugene, OR). The annexin/PI Apoptosis Detection Kit was
obtained from Miltenyi Biotec (Teterow, Germany). The Green Cas-
pase-3, Caspase-8 and Caspase-9 Staining kit, the general caspase
inhibitor z-VAD-FMK and the Mitochondrial Membrane Potential
kit were purchased from Promokine (Heidelberg, Germany). The
anism during metabolic or therapeutic stress, leading to self-degra-
dation of the damaged organelles.5
3,54
On the other hand, evidence
is growing that autophagy, induced by anticancer drugs, can di-
rectly cause tumor cell killing.5
5–57
Moreover, regulation of autoph-
agy and apoptosis are intertwined at various levels.58 RPMI 8226
cells treated with either (S)-4 or (R)-4 underwent time-dependent
conversion of LC3-I to LC3-II, first noticeable at 24 h and significant
by 48 h. However, p62 levels did not decrease during the same time,
as might have been expected for classical autophagy, but rather in-
0
apoptosis inhibitor 2,2 -methylenebis(1,3-cyclohexandione) was
30
synthesized in our laboratories. Primary rabbit polyclonal anti-
2
human bcl , bax, bid, caspase-8, caspase-9 and a horseradish per-
creased. This would suggest that the
plete autophagy, as has been observed for geranylgeranoic acid
and neuregulin.
port of ligands inducing incomplete autophagy.
Preincubation of RPMI 8226 cells with the autophagy inhibitor
wortmannin actually led to increased LC3-II formation. Wortman-
nin, as a late autophagy inhibitor, blocks autophagy when auto-
phagosomes fuse with lysosomes. Therefore, if autophagy was
incomplete, further increases in LC3-II levels might be expected.
r ligands induce an incom-
oxidase-conjugated secondary antibody were obtained from Acris
Antibodies (Herford, Germany), primary rabbit monoclonal antihu-
man GAPDH, polyclonal PARP, LC3B, p62 and AIF were obtained
from Cell Signaling Technology (Danvers, USA). The chemilumines-
cence kit was purchased from Biorad (München, Germany). All
other not mentioned reagents for western blot analysis were
obtained from Roth (Karlsruhe, Germany). The water for all exper-
iments was supplied by a Milli-Q water purification system from
Millipore.
5
9,60
To the best of our knowledge, this is the first re-
r
Interestingly, pretreatment with the antioxidant
a-tocopherol
completely blocked the conversion of LC3-I to LC3-II, indicating
an interfering role of the antioxidant in autophagic mechanisms.
Autophagy constitutes a cell strategy to survive cellular insults
by removing damaged organelles.53 However, the known autoph-
agy inhibitor wortmannin had no effect on the apoptotic activity
5
.2. General procedures
Moisture sensitive reactions were conducted under dry nitro-
gen. THF was dried with sodium/benzophenone and was freshly
distilled before use. Thin layer chromatography: Silica gel 60
F254 plates (Merck). Flash chromatography (fc): Silica gel 60, 40–
of (S)-4 and (R)-4, suggesting that autophagy induced by these
r
ligands is not cytoprotective, perhaps because it is incomplete. This
is consistent with the observation that the initiation of apoptosis,
indicated by PARP cleavage at 6 h, substantially precedes the first
signs of autophagy, indicated by LC3-II formation at 24 h.
4
3
lm (Merck); parentheses include: diameter of the column,
eluent, R value. Melting point: Melting point apparatus SMP 3
f
1
13
(
Stuart Scientific), uncorrected. H NMR (400 MHz),
C NMR
(
100 MHz): Unity Mercury Plus AS 400 NMR spectrometer (Var-
4
. Conclusion
ian); in ppm related to tetramethylsilane; coupling constants are
given with 0.5 Hz resolution; the assignments of C and 1H NMR
signals were supported by 2D NMR techniques. MS: MAT GCQ
(Thermo-Finnigan): EI, MAT LCQ (Thermo Finnigan): ESI, Micro-
TOF-QII (Bruker Daltonics): APCI. IR: IR spectrophotometer 480Plus
13
To the best of our knowledge this is the first report of
r ligands
that do not induce cytoprotective autophagy, which could offer
(S)-4 and (R)-4 an advantage over other ligands in cancer therapy.
r

2
30
K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
FT-ATR-IR (Jasco) or FT/IR Prestige 21 (Shimadzu). Polarimetry:
Polarimeter 341 (Perkin Elmer), sample length 1 dm, k = 589 nm,
2.5 h at room temperature. The solvent was removed in vacuo
and diethyl ether (50 ml) was added to the resulting residue. The
suspension was filtered, washed and the filtrate was concentrated
in vacuo. Purification of the residue was performed by fc ( 3.5 cm,
+20 °C. HPLC (method ACN): Merck Hitachi Equipment; UV detec-
tor: L-7400; autosampler:L-7200; pump: L-7100; degasser: L-
7
2
5
614; column: LiChrospherÒ 60 RP-select B (5
50–4 mm cartridge; flow rate: 1.0 ml/min; injection volume:
.0 l; detection at k = 210 nm; solvents: A: demineralized water
l
m); LiCroCARTÒ
h = 17 cm,
Colorless oil, yield 683.6 mg (34% over 3 steps). C21
= 403.9. HPLC (method ACN): t = 20.8 min, purity 76.6%. Spe-
f
v = 30 ml, cyclohexane/ethyl acetate = 7/3, R = 0.27).
H22ClNO5,
l
M
r
R
2
D
0
1
with 0.05% (v/v) trifluoroacetic acid; B: acetonitrile with 0.05%
v/v) trifluoroacetic acid: gradient elution: 0.0 min: 90.0% of A,
cific rotation: ½
a
ꢂ
ꢃ29.4 (c 1.03; EtOAc). H NMR (CDCl
3
): d
), 2.79 (dd,
), 3.05 (dd, J = 17.3 / 6.7 Hz,
), 3.32 (dd, J = 17.1/6.5 Hz, 0.8H, CHCH
NR
(
[ppm] = 2.75 (dd, J = 17.3/6.7 Hz, 0.2H, CHCH CO CH
2 2 3
HR
1
1
0.0% of B; 4.0 min: 90.0% of A, 10.0% of B; 29.0 min: 0.0% of A,
00.0% of B; 31.0 min: 0.0% of A, 100.0% of B; 31.5 min: 90.0% of
2 2 3
J = 17.1/6.8 Hz, 0.8H, CHCH CO CH
0.2H, CHCH
NR
2
CO
2
CH
3
2
CO2-
), 3.66
), 4.10 (d,
HR
NR
NR
A, 10.0% of B; 40.0 min: 90.0% of A, 10.0% of B.
CH
s, 3 ꢀ 0.8H, OCH
2
J = 12.6 Hz, 0.8H, O = CCH Cl ), 4.16 (d, J = 11.6 Hz, 0.8H, O = CCH2-
3
), 3.56 (s, 3 ꢀ 0.2H, OCH
3
), 3.64 (s, 3 ꢀ 0.2H, OCH
3
HR
HR
(
3
), 3.70 (s, 3 ꢀ 0.8H, OCH
3
HR
5
.3. Syntheses of (S)-4 and (R)-4
HR
NR
Cl ), 4.28 (d, J = 15.6 Hz, 0.2H, NCH
2
Ar ), 4.48 (s, broad, 2 ꢀ 0.2H,
NR
HR
5
5
.3.1. Dimethyl (S)-2-aminobutanedioate hydrochloride ((S)-
ꢁHCl)
O@CCH
J = 16.7 Hz, 0.8H, NCH
4.95 (d, J = 15.5 Hz, 0.2H, NCH
2
Cl ), 4.55 (t, J = 6.6 Hz, 0.8H, CHCH
2
CO
2
CH
3
), 4.73 (d,
HR
HR
2
Ar ), 4.80 (d, J = 16.5 Hz, 0.8H, NCH
2
Ar ),
2
Ar ), 5.07 (t, J = 7.2 Hz, 0.2H, CHCH2-
NR
Under ice cooling, chlorotrimethylsilane (33.2 ml, 263.0 mmol)
NR
HR + NR
was added dropwise to a suspension of (S)-aspartate (10 g,
5.1 mmol, 98% ee) in methanol abs. (150 ml) and the reaction
CO
80:20. C NMR (CD
34.6 (1C, CHCH CO
Cl ), 47.3 (1C, NCH
2
CH
3
), 7.30–7.65 (m, 9H, Ar-H
). Ratio of rotamers is
1
3
HR
7
3
OD): d [ppm] = 34.4 (1C, CHCH
2
CO
2
CH
3
),
NR
HR
mixture was stirred for 16 h at room temperature. The solvent
and volatile byproducts were evaporated in vacuo. The residue
was suspended in methanol (1 ꢀ 30 ml) and the solvent was re-
moved in vacuo. Then diethyl ether was added (30 ml) and the sol-
vent was removed under reduced pressure. This procedure was
repeated three times. The product was dried in high-vacuum. Col-
2
2
CH
3
), 41.2 (1C, O@CCH
2
Cl ), 42.0 (1C,
NR
NR
HR
O@CCH
2
2
Ar ), 52.2 (1C, OCH
3
), 52.8 (1C,
Ar ),
), 127.1 (2C,
NR
HR
NR
HR
OCH
57.2 (1C, CHCH
3
), 53.0 (1C, OCH
3
), 53.1 (1C, OCH
), 57.5 (1C, CHCH CO CH
2 2 3
3
), 53.2 (1C, NCH
2
NR
HR
2
CO
2
CH
3
NR
HR
NR
NR
Ar-C ), 127.2 (2C, Ar-C ), 127.3 (2C, Ar-C ), 127.5 (1C, Ar-C ),
127.7 (1C, Ar-C ), 127.8 (2C, Ar-C )128.0 (2C, Ar-C ), 128.1
HR
HR
HR
NR
NR
HR
orless solid, mp 112–115 °C, y0ield 15 g (100%).
C
6
H
12ClNO4,
q
(2C, Ar-C ), 128.9 (2C, Ar-C ), 129.0 (2C, Ar-C ), 134.3 (1C, Ar-C -
2
1
HR
NR
NR
HR
M
r
= 197.6. Specific rotation: ½
CD OD): d [ppm] = 3.07 (d, J = 5.3 Hz, 2H, CHCH
s, 3H, OCH ), 3.84 (s, 3H, OCH ), 4.40 (t, J = 5.4 Hz, 1H, CHCH
). The signal for the protons of the NH
NMR (CD OD): d [ppm] = 35.0 (1C, CHCH
CHCH CO CH ), 53.0 (1C, CO CH ), 54.0 (1C, CO
CO CH
aꢂ
+10.3 (c 1.00; H
2
O). H NMR
), 135.1 (1C, Ar-C
q
), 136.1 (1C, Ar-C
), 141.3 (1C, Ar-C
q
q
), 140.4 (1C, Ar-C
), 167.2 (1C, C@O ), 167.9
q
),
D
NR
HR
HR
NR
(
(
CH
3
2
CO
2
CH ), 3.75
3
140.7 (1C, Ar-C
(1C, C@O ), 169.7 (1C, CO
(1C, CO
(M+Na, 100). IR (neat):
q
NR
HR
3
3
2
CO2-
13
2
CH
3
), 169.9 (1C, CO
2
CH
). MS (ESI): m/z [%] = 426
[cm ] = 2952 (C–H aliph.), 1732 (C@Oester),
3
), 170.6
+
NR
HR
3
3
-group is not seen.
C
2
CH
3
), 171.7 (1C, CO CH
2 3
ꢃ1
3
2
CO
2
CH
CH
3
), 50.4 (1C,
), 169.8 (1C,
m
2
2
3
2
3
2
3
1658 (C@Oamide), 796 (C–Cl), 756, 698 (arom. monosubst.).
2
3
), 171.2 (1C, CO
2
CH
3
). MS (EI): m/z [%] = 162 (M–Cl, 22).
ꢃ1
IR (neat):
m
[cm ] = 2864 (C–Haliph.), 1736 (C@Oester).
5.3.4. Dimethyl (R)-2-[N-(biphenyl-4-ylmethyl)-N-(2-
chloroacetyl)amino] butanedioate ((R)-7)
.
5
5
.3.2. Dimethyl (R)-2-aminobutanedioate hydrochloride ((R)-
ꢁHCl)
As described for (S)-5, (R)-aspartate (6.0 g, 45.1 mmol, 98% ee)
Compound (R)-5 HCl (1 g, 5.06 mmol) was dissolved in CH
2
Cl
2
abs. (15 ml) and triethylamine (0.74 ml, 5.06 mmol) and biphe-
nyl-4-carbaldehyde (6, 0.92 g, 5.06 mmol) were added slowly.
was reacted with chlorotrimethylsilane (19.9 ml, 157.8 mmol) in
Then Na
room temperature. For work-up, the suspension was filtered, the
precipitate was washed with CH Cl and the filtrate was concen-
trated in vacuo. Diethyl ether was added to the resulting residue
and the mixture was filtered again. Evaporation of the filtrate gave
the imine (760 mg, 2.34 mmol) as a colorless solid, which was dis-
solved in diethyl ether (15 ml) and reduced directly by addition of
2 4
SO was added and the mixture was stirred for 16 h at
abs. methanol (90 ml). Colorless solid, mp 105–110 °C, yield 8.9 g
2
D
0
(
1
99.9%). C
6
H
12ClNO4,
M
r
= 197.6. Specific rotation: ½
aꢂ
ꢃ13.8 (c
2
2
.00; H
2
O). MS (ESI): m/z [%] = 162 (M – Cl, 100).
5
.3.3. Dimethyl (S)-2-[N-(biphenyl-4-ylmethyl)-N-(2-
chloroacetyl)amino] butanedioate ((S)-7)
.
Compound (S)-5 HCl (1.0 g, 5.06 mmol) was dissolved in CH
abs. (10 ml) and triethylamine (0.74 ml, 5.06 mmol) and biphenyl-
-carbaldehyde (6, 0.92 g, 5.06 mmol) were added slowly. Then
Na SO was added and the mixture was stirred for 16 h at room
temperature. For work-up, the suspension was filtered, the precip-
itate was washed with CH Cl and the filtrate was concentrated in
2
Cl
2
4
NaBH (150.8 mg, 3.98 mmol) over 40 min under ice cooling. The
mixture was stirred at room temperature for another 5 min, before
the solvent was evaporated in vacuo. Water (15 ml) was added to
the residue and the aqueous layer was extracted with diethyl ether
4
2
4
(5 ꢀ 10 ml). The combined organic layers were dried (K
2 3
CO ), fil-
2
2
tered and the solvent was removed under reduced pressure. The
oily residue of the resulting amine (725.6 mg, 2.22 mmol) was dis-
vacuo. Diethyl ether was added to the resulting residue and the
mixture was filtered again. Evaporation of the filtrate gave the
imine (1.3 g, 4.01 mmol) as a colorless solid, which was dissolved
solved in CH
and chloroacetyl chloride (0.28 ml, 3.56 mmol) were added drop-
wise under N -atmosphere and ice cooling. The mixture was stir-
2 2
Cl abs. (10 ml). Triethylamine (0.25 ml, 1.78 mmol)
in diethyl ether (15 ml) and reduced directly by addition of NaBH
257.7 mg, 6.82 mmol) over 40 min under ice cooling. The mixture
4
2
(
red for 2.5 h at room temperature. The solvent was removed in
vacuo and diethyl ether (50 ml) was added to the resulting residue.
The suspension was filtered, washed and the filtrate was concen-
trated in vacuo. Purification of the residue was performed by fc (
was stirred at room temperature for another 5 min, before the sol-
vent was evaporated in vacuo. Water (15 ml) was added to the res-
idue and the aqueous layer was extracted with diethyl ether
(
5 ꢀ 10 ml). The combined organic layers were dried (K
2
CO
3
), fil-
3 cm, h = 19 cm,
= 0.26). Colorless oil, yield 360 mg (15% over 3 steps). C21
ClNO5, = 403.9. HPLC (method ACN): = 21.0 min, purity
+26.8 (c = 0.97; EtOAc). MS (EM,
23ClNO 404.1265 (M+H), found
v = 30 ml, cyclohexane/ethyl acetate = 4/1,
tered and the solvent was removed under reduced pressure. The
oily residue of the resulting amine (1.2 g, 3.68 mmol) was dissolved
in abs. CH
chloroacetyl chloride (0.47 ml, 5.88 mmol) were added dropwise
under N -atmosphere and ice cooling. The mixture was stirred for
R
f
H
22-
M
r
t
R
2
0
2
Cl
2
(10 ml). Triethylamine (0.41 ml, 2.94 mmol) and
93.9%. Specific rotation: ½ ꢂ
a
D
APCI): m/z = calcd for
C
21
H
5
2
404.1212.

K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
231
5
.3.5. Methyl (S)-2-[1-(biphenyl-4-ylmethyl)-4-
NCH), 2.56–2.60 (m, 1H, NCH
NCH), 2.92–3.02 (m, 1H, NCH , NCH), 3.46 (d, J = 13.2 Hz, 1H, NCH2-
Ar), 3.74–3.84 (m, 1H, CHCH CH OH), 3.84–3.96 (m, 1H, CHCH CH2-
OH), 4.16–(d, J = 12.8 Hz, 1H, NCH Ar), 7.32–7.45 (m, 5H, Ar-H),
7.53–7.59 (m, 4H, Ar-H). The signal for the proton of the OH group
2 2
, NCH), 2.83–2.90 (m, 1H, NCH ,
(
cyclohexylmethyl)-3,6-dioxopiperazin-2-yl]acetate ((S)-8)
Compound (S)-7 (195.7 mg, 0.48 mmol) was dissolved in dry
2
2
2
2
acetonitrile (molecular sieves 3 Å, 10 ml). Triethylamine (0.1 ml,
.58 mmol) and cyclohexylmethylamine (0.1 ml, 0.63 mmol) were
2
0
1
3
added slowly and the reaction mixture was stirred at room tem-
perature for 16 h. For workup, the solvent was evaporated in vacuo
and the residue was dissolved in ethyl acetate (10 ml). The organic
layer was washed with 0.5 M HCl (2 ꢀ 4 ml), 0.5 M NaOH
is not seen. C NMR (CDCl
32.0/34.9 (7C, NCH 11, CHCH
(1C, NCH or NCH), 56.8 (1C, NCH
NCH or NCH), 61.2 (1C, CHCH CH
127.1 (2C, Ar-C), 127.2 (2C, Ar-C), 127.3 (1C, Ar-C), 128.9 (2C, Ar-
C), 129.6 (2C, Ar-C), 137.5 (1C, Ar-C ), 140.2 (1C, Ar-C ), 141.0 (1C,
Ar-C ). MS (EM, APCI): m/z = calcd for C26 O 393.2906 (M+H),
found 393.2877. IR (neat): [cm ] = 3338 (O–H), 2920, 2848
3
): d [ppm] = 26.2/26.3/26.9/31.2/31.9/
CH OH), 49.4 (1C, NCH ), 52.1
), 57.8 (1C, NCH Ar), 58.1 (1C,
OH), 65.0 (1C, NCH
2
C
6
H
2
2
2
2
2
2
2
2
2
2 6
C H11),
(
1 ꢀ 4 ml) and brine (1 ꢀ 4 ml), dried (Na
2 4
SO ), filtered and con-
centrated in vacuo. The residue was purified by fc ( 2 cm,
h = 18 cm, = 10 ml, cyclohexane/ethyl acetate = 7/3, R = 0.11).
Colorless solid, mp 135–137 °C, yield 146.3 mg (68%).
q
q
v
f
q
37 2
H N
ꢃ1
m
C
27
H
32
N
2
O
4,
M
r
= 448.5. HPLC (method ACN): t
R
= 21.7 min, purity
(C–Haliph.), 819 (arom. disubst.), 751, 685 (arom. monosubst.).
20
9
8.5%. Specific rotation: ½ ꢂ
a
+46.3 (c 0.42; EtOAc). MS (EM, APCI):
449.2440 (M+H), found 449.2395.
D
m/z = calcd for C27
H
33
N
2
O
4
5.3.8. (R)-2-[1-(Biphenyl-4-ylmethyl)-4-
(
cyclohexylmethyl)piperazin-2-yl]ethanol ((R)-4)
5
.3.6. Methyl (R)-2-[1-(biphenyl-4-ylmethyl)-4-
As described for the synthesis of (S)-4, (R)-8 (100 mg,
(
cyclohexylmethyl)-3,6-dioxopiperazin-2-yl]acetate ((R)-8)
As described for the synthesis of (S)-8, (R)-7 (200 mg,
.50 mmol) was reacted with triethylamine (0.1 ml, 0.71 mmol)
0.22 mmol) was reacted with LiAlH
1.3 mmol) in THF abs. (10 ml). The crude product was purified by
( 2 cm, h = 16 cm, = 10 ml, cyclohexane/ethyl acetate = 3/7,
= 0.15). Colorless oil, yield 84.1 mg (96%).
= 392.6. HPLC (method ACN): t = 21.0 min, purity 99.6%. Spe-
ꢃ29.6 (c 0.37; EtOAc). MS (EM, APCI): m/
O 393.2906 (M+H), found 393.2910.
4
solution (1 M in THF, 1.3 ml,
0
f
R
M
c
v
and cyclohexylmethylamine (0.1 ml, 0.64 mmol) in dry acetonitrile
f
26 36 2 ,
C H N O
(
(
R
C
9
(
3
5
CO
(
molecular sieves 3 Å, 10 ml). The crude product was purified by fc
2.5 cm, h = 18 cm, = 10 ml, cyclohexane/ethyl acetate = 7.5/2.5,
= 0.05). Colorless solid, mp 134–137 °C, yield 164.4 mg (74%).
= 448.5. HPLC (method ACN): t = 21.8 min, purity
r
R
2
D
0
v
cific rotation: ½ ꢂ
a
f
37 2
z = calcd for C26H N
27
H
32
N
2
O
4,
M
r
R
2
D
0
1
8.0%. Specific rotation: ½
): d [ppm] = 0.93–1.01 (m, 2H, NCH
H, NCH 11), 1.58–1.74 (m, 6H, NCH
.1 Hz, 1H, CHCH CO CH ), 3.04 (dd, J = 17.2/3.7 Hz, 1H, CHCH2-
CH ), 3.18–3.31 (m, 2H, NCH 11), 3.61 (s, 3H, OCH ), 3.95
d, J = 17.2 Hz, 1H, O@CCH N), 4.15 (t, J = 4.3 Hz, 1H, CHCH
CH ), 4.28 (d, J = 15.1 Hz, 1H, NCH Ar), 4.35 (d, J = 17.2 Hz, 1H,
O@CCH N), 5.11 (d, J = 15.1 Hz, 1H, NCH Ar), 7.31 (d, J = 8.1 Hz,
H, Ar-H), 7.35 (t, J = 7.3 Hz, 1H, Ar-H), 7.42–7.26 (m, 2H, Ar-H),
.54–7.57 (m, 4H, Ar-H). 13C NMR (CDCl
): d [ppm] = 25.8 (1C,
11), 25.9 (1C, NCH 11), 26.4 (1C, NCH 11), 30.6
1C, NCH 11), 30.8 (1C, NCH 11), 35.2 (1C, CHCH CO CH ),
5.5 (1C, NCH 11), 47.1 (1C, NCH Ar), 50.9 (1C, O@CCH N),
2.1 (1C, OCH ), 52.8 (1C, NCH 11), 56.3 (1C, CHCH CO CH ),
27.2 (2C, Ar-C), 127.6 (1C, Ar-C), 127.8 (2C, Ar-C), 128.6 (2C, Ar-
), 140.6 (1C, Ar-C ), 141.17
), 164.7 (1C, C@O), 165.5 (1C, C@O), 170.3 (1C, CO CH ).
449.2440 (M+H), found
aꢂ
ꢃ43.1 (c 0.42; EtOAc). H NMR
11), 1.14–1.28 (m,
11), 2.83 (dd, J = 17.2/
5.4. Biology
CDCl
3
2 6
C H
2
C
6
H
C
2 6
H
5.4.1. Receptor binding studies
2
2
3
The affinities of the enantiomeric piperazines (S)-4 and (R)-4 to-
2
3
C
2 6
H
3
wards the
as previously described.
nates as well as homogenates of the human multiple myeloma cell
line RPMI 8226 were used as receptor material in the assays. In
both assays, tritium labeled [ H]-(+)-pentazocine was employed as
radioligand. In the assays, rat liver homogenates as well as
r receptors were determined in receptor binding studies
2
6,61,62
2
2
CO2-
Briefly, guinea pig brain homoge-
3
2
2
2
r
1
3
2
7
NCH
3
r
2
2
C
6
H
2
C
6
H
2
6
C H
homogenates of the human urinary bladder tumor cell line RT-4
(
2
C
6
H
2
C
6
H
2
2
3
(DSMZ, Braunschweig, Germany) served as receptor material. The
3
3
5
1
2
C
6
H
2
2
radioligand was [ H]-ditolylguanidine (DTG), which interacts with
3
C
2 6
H
2
2
3
2
both r receptor subtypes. In order to gain r selectivity (+)-pentaz-
ocine was added to mask the
1
r receptors.
C), 128.9 (2C, Ar-C), 134.7 (1C, Ar-C
1C, Ar-C
q
q
(
q
2
3
5.4.2. Cell culture
MS (EM, APCI): m/z = calcd for C27
H
33
N
2
O
4
The human multiple myeloma cell line RPMI 8226 was obtained
from the DSMZ (Braunschweig, Germany) and grown in RPMI 1640
medium supplemented with 10% fetal bovine serum, penicillin
ꢃ1
4
49.2469. IR (neat):
m
[cm ] = 2926, 2846 (C-H aliph.), 1728
(
C@Oester), 1650 (C@Oamide), 751, 692 (arom. monosubst.).
(
10,000 U/ml) and streptomycin (10 mg/ml) at 37 °C in a humidi-
5
(
.3.7. (S)-2-[1-(Biphenyl-4-ylmethyl)-4-
cyclohexylmethyl)piperazin-2-yl]ethanol ((S)-4)
Under N (S)-8 (220.5 mg, 0.5 mmol) was dissolved in THF
12 ml) and the mixture was cooled down to 0 °C. At this tempera-
solution (1 M in THF, 2.9 ml, 2.9 mmol) was added.
The reaction mixture was stirred at 0 °C for 10 min and then heated
to reflux for 16 h. Then water was added under ice cooling until H
fied incubator with 5% CO
harvesting.
2
atmosphere. Accutase was used for cell
2
(
5.4.3. In vitro cytotoxicity assay
Cell viability was assessed by the MTT assay. Cells were seeded
out in 96-well plates at a density of 10,000 cells/well in a volume of
ture a LiAlH
4
2
100 ll culture medium. Cells were then exposed to five serial dilu-
evolution was finished. The mixture was stirred at 0 °C for 10 min
and then heated to reflux for 30 min. After cooling to room temper-
ature the mixture was filtered and the solvent was removed in va-
cuo. The crude product was purified by fc ( 3 cm, h = 16 cm,
tions of substances, added to the medium from 1000-fold concen-
trated stock solutions in DMSO. For controls, cells were exposed to
0.1% DMSO alone. After a 72 h incubation, 20
DMSO) were added to each well followed by a 6 h incubation. Next,
100 l of 0.04 N HCl in isopropanol were added to each well to dis-
ll MTT (2.5 mg/ml in
v
= 10 ml, cyclohexane/ethyl acetate = 3/7, R
yield 196.3 mg (77%). C26 = 392.6. HPLC (method ACN):
= 17.4 min, purity 97.7%. Specific rotation: ½
f
= 0.15). Colorless oil,
l
36
H N
2
O
,
M
r
solve the MTT-formazan product, and the well optical density was
measured at k = 570 nm with a 2010 microplate reader (Anthos,
Salzburg, Austria). The relative cell viability was expressed as the
percentage of the treated versus control (T/C). Linear regression
analysis of the T/C versus log concentration allowed for an estima-
tion of the IC50 value; that is the concentration that reduced cell
viability by 50%.
20
t
R
a
ꢂ
+28.3 (c 0.88;
D
1
EtOAc). H NMR (CDCl
3
): d [ppm] = 0.77–0.96 (m, 2H, NCH
.07–1.28 (m, 3H, NCH 11), 1.40–1.57 (m, 1H, NCH
.62–1.81 (m, 5H, NCH 11, CHCH CH
11, CHCH CH OH), 2.04–2.07 (m, 1H, NCH
OH), 2.12 (d, J = 7.2 Hz, 2H, NCH 11), 2.31–2.45 (4H, NCH
2
C
6
H11),
1
1
NCH
CH
2 6
C H
2
C
6
H
11),
C
2 6
H
2
2
OH), 1.84–1.87 (m, 1H,
11, CHCH
2
2 6
C H
2
2
2
C
6
H
2
2
C
6
H
2
,

2
32
K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
5
.4.4. Fluorescence microscopy
with 1 ll MitoCapture (JC-1) reagent for 20 min at 37 °C according
To study changes in nuclear morphology, cells were treated for
4 and 48 h in six well plates with concentrations ca. 75% higher
to the instructions of the Mitochondrial Apoptosis Staining Kit
(Promokine, Heidelberg, Germany), the cells were washed, resus-
pended in 1 ml of incubation buffer and fluorescence was mea-
sured by flow cytometry. The FITC channel (kEx/Em = 488/530 nm)
was used for green monomers and the PI channel (kEx/Em = 488/
590 nm) for red aggregates.
2
than the respective IC50; that is, 36
l
M for (S)-4 and 23
R)-4. Afterwards, cells were stained with DAPI/methanol
2.5 mg/ml) at 37 °C for 15 min and visualized at a magnification
lM for
(
(
of 400 fold; cells were irradiated with light at kEx = 365 nm (BP-fil-
ter) and observed at kEm >397 nm (LP-filter).
5
.4.11. Western blot analysis
5
.4.5. Flow cytometry
Cells were lysed in lysis buffer (50 mM Tris ph 7.6, 100 mM
Flow cytometry analysis was done with a Macs Quant flow
3 4
NaCl, 100 mM NaF, 5 mM EDTA, 0.2 mM Na VO and 0.1% Triton
cytometer and Macs Quantify software (Miltenyi Biotec, Bergisch
Gladbach, Germany). A total of 104 cells were scored in all
determinations.
X-100) with protease inhibitor cocktail for 30 min on ice. After son-
ication for 5 min, the lysate was centrifuged at 18,000g for 20 min
at 4 °C. Protein concentrations of the supernatant were determined
with the Bradford assay. Equal amounts of protein (20 lg) were
5
.4.6. Apoptosis staining
Apoptosis was measured by using the protocol of the manufac-
loaded onto 12% SDS–Page gels, electrophoretically separated,
transferred to nitrocellulose membrane and blocked with 10%
non-fat milk in TBST buffer for 3 h. The membranes were then
incubated over night with the following primary antibodies: anti-
turer of the annexin-V-FITC Apoptosis Detection Kit (Miltenyi Bio-
5
tec, Telterow, Germany). After treating 7.5 ꢀ 10 cells with
substances (S)-4 (36
lM) and (R)-4 (23
l
M) for 24 or 48 h, the cells
2
bcl , anti-bax, anti-caspase-8, anti-caspase-9, anti-bid, anti-PARP,
were harvested and washed with binding buffer. The cell pellet
was resuspended in 100 l binding buffer and stained with10
annexin-V for 15 min in the dark. After another washing step cells
were resuspended, incubated with 5 l propidium iodide (PI) and
subsequently analyzed by flow cytometry using the FITC channel
anti-p62 and anti-LC3 followed by an 1 h incubation with second-
ary antibody conjugated with horseradish peroxidase at a 1:10,000
dilution. Protein bands were detected by enhanced chemilumines-
cence kit and digitalized by an INTAS Chemocham (Göttingen,
Germany) instrument. An equal loading was verified by using
anti-GAPDH. For stripping western blots, the blot was incubated
with stripping buffer for 30 min at 56 °C. Expression of the protein
bands was quantified using LabImage 1D software, normalized to
GAPDH and expressed as percentage relative to the expression le-
vel in control cells.
l
ll
l
k
k
Ex/Em = 488/530 nm for annexin-V positive cells and the PI channel
Ex/Em = 488/590 nm for late apoptotic and dead cells.
5
.4.7. Caspase activity assay
Apoptosis was induced in 1 ꢀ 10 RPMI 8226 cells with sub-
stances (S)-4 (36 M) and (R)-4 (23 M) for 24 or 48 h. Cells were
harvested, aliquoted in Eppendorf tubes and incubated with 1 l of
6
l
l
l
5
.4.12. Statistical analysis
caspase-3 (FITC-DEVC-FMK), caspase-8 (FITC-IETD-FMK) or cas-
pase-9 (FITC-LETD-FMK) inhibitors according to the instructions
in the Green Caspase Staining kit, which measures the irreversible
binding of selective, fluorogenic caspase inhibitors within cells by
flow cytometry. After 1 h incubation, cells were washed, resus-
The results of three independent experiments were expressed
as the means ± SD. Statistical significance was determined using
the analysis of variance (ANOVA) followed by a multiple compari-
son test with a Dunnetts test or Bonferroni test. (Graph Pad Prism
⁄
⁄⁄
⁄⁄⁄
software). p <0.05, p <0.01, p <0.001 was considered statisti-
pended in 500
ll wash buffer and kept on ice prior to analyzing
cally significant.
by flow cytometry in the FITC channel kEx/Em = 488/530 nm.
Acknowledgments
5
.4.8. Detection of reactive oxygen species
After incubating RPMI 8226 cells with either 23 lM (R)-4 or
00 lM TBH for times between 30 min, 5 h and 24 h, the cells were
0
We thank Heidi Lemmerhirt for the synthesis of 2,2 -methyl-
2
enebis(1,3-cyclohexandione). This work was supported in part by
the German Research Foundation [DFG grants BE 1287/3-1, WU
harvested and washed with Dulbeccos phosphate buffered saline
PBS, containing 0.2 g KCl, 0.1 g MgSO O, 1.55 g Na HPO ꢁ2H2-
ꢁ7H
O, 0.2 g KH PO , 8 g NaCl ad 1000 ml H O). The cell pellet was
resuspended in 1 ml PBS, stained with 2
cein diacetate (DCFDA) for 30 min at 37 °C and 5% CO
two times with PBS and resuspended in 500 l PBS. Fluorescently
(
4
2
2
4
1
76/6-1].
2
4
2
0
0
lM 2 ,7 -dichloro fluores-
Supplementary data
2
and washed
l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.11.033.
labeled cells were analyzed by flow cytometry with the FITC chan-
nel (kEx/Em = 488/530 nm).
References and notes
5
.4.9. Detection of lipid peroxidation
Cells were incubated with 23 M (R)-4 for times between
0 min, 5 and 48 h. Cells were harvested and washed with PBS
M 4-difluoro-3,5-bis(4-phe-
nyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene (BODIPY 665/
76 C11) in 1 ml PBS for 30 min. After washing twice, cells were
resuspended in 500 l PBS and the fluorescence intensity was sub-
sequently measured with the flow cytometer with the APC channel
Ex/Em = 620/675 nm.
l
1.
Martin, W. R.; Eades, C. G.; Thompson, J. A.; Huppler, R. E.; Gilbert, P. E. J.
Pharmacol. Exp. Ther. 1976, 197, 517.
3
and incubated in the presence of 2
l
2. Walker, J. M.; Bowen, W. D.; Walker, F. O.; Matsumoto, R. R.; Decosta, B.; Rice,
K. C. Pharmacol. Rev. 1990, 42, 355.
3.
Hellewell, S. B.; Bruce, A.; Feinstein, G.; Orringer, J.; Williams, W.; Bowen, W. D.
6
Eur. J. Pharmacol. (Mol. Pharmacol. Sect.) 1994, 268, 9.
4. Hanner, M.; Moebius, F. F.; Flandorfer, A.; Knaus, H. G.; Striessnig, J.; Kempner,
l
E.; Glossmann, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8072.
5
6
7
8
.
.
.
.
Volz, H. P.; Stoll, K. D. Pharmacopsychiatry 2004, 37, S214.
Maurice, T.; Su, T.-P. Pharmacol. Ther. 2009, 124, 195.
Hayashi, T.; Su, T. P. CNS Drugs 2004, 18, 269.
k
Monnet, F. P.; Morin-Surun, M. P.; Leger, J.; Combettes, L. J. Pharmacol. Exp. Ther.
5
.4.10. Detection of the mitochondrial membrane potential (D-
2003, 307, 705.
m
W )
9.
Aydar, E.; Palmer, C. P.; Klyachko, V. A.; Jackson, M. B. Neuron 2002, 34, 399.
RPMI 8226 cells were treated with (R)-4 (23
lM) for 1, 3 and
10. Bowen, W. D. Pharm. Acta Helv. 2000, 74, 211.
11. Zhang, H. L.; Cuevas, J. J. Neurophysiol. 2002, 87, 2867.
2
4 h, harvested and washed with incubation buffer. After labeling

K. Korpis et al. / Bioorg. Med. Chem. 22 (2014) 221–233
233
1
1
1
2. Hayashi, T.; Su, T. P. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 491.
3. Crawford, K. W.; Coop, A.; Bowen, W. D. Eur. J. Pharmacol. 2002, 443, 207.
4. Wheeler, K. T.; Wang, L. M.; Wallen, C. A.; Childers, S. R.; Cline, J. M.; Keng, P. C.;
Mach, R. H. Br. J. Cancer 2000, 82, 1223.
5. Vilner, B. J.; Decosta, B. R.; Bowen, W. D. J. Neurosci. 1995, 15, 117.
6. Yang, S.; Bhardwaj, A.; Cheng, J.; Alkayed, N. J.; Hurn, P. D.; Kirsch, J. R. Anesth.
Analg. 2007, 104, 1179.
7. Spruce, B. A.; Campbell, L. A.; McTavish, N.; Cooper, M. A.; Appleyard, M. V. L.;
O’Neill, M.; Howie, J.; Samson, J.; Watt, S.; Murray, K.; McLean, D.; Leslie, N. R.;
Safrany, S. T.; Ferguson, M. J.; Peters, J. A.; Prescott, A. R.; Box, G.; Hayes, A.;
Nutley, B.; Raynaud, F.; Downes, C. P.; Lambert, J. J.; Thompson, A. M.; Eccles, S.
Cancer Res. 2004, 64, 4875.
8. Crawford, K. W.; Bowen, W. D. Cancer Res. 2002, 62, 313.
9. Riganas, S.; Papanastasiou, I.; Foscolos, G. B.; Tsotinis, A.; Bourguignon, J.-J.;
Serin, G.; Mirjolet, J.-F.; Dimas, K.; Kourafalos, V. N.; Eleutheriades, A.; Moutsos,
V. I.; Khan, H.; Georgakopoulou, S.; Zaniou, A.; Prassa, M.; Theodoropoulou, M.;
Pondiki, S.; Vamvakides, A. Bioorg. Med. Chem. 2012, 20, 3323.
0. Ostenfeld, M. S.; Fehrenbacher, N.; Hoyer-Hansen, M.; Thomsen, C.; Farkas, T.;
Jaattela, M. Cancer Res. 2005, 65, 8975.
1. Bedurftig, S.; Wunsch, B. Bioorg. Med. Chem. 2004, 12, 3299.
2. Foster, A.; Wu, H. F.; Chen, W. B.; Williams, W.; Bowen, W. D.; Matsumoto, R.
R.; Coop, A. Bioorg. Med. Chem. Lett. 2003, 13, 749.
3. Holl, R.; Schepmann, D.; Froehlich, R.; Gruenert, R.; Bednarski, P. J.; Wuensch, B.
J. Med. Chem. 2009, 52, 2126.
4. Sunnam, S. K.; Schepmann, D.; Rack, E.; Froehlich, R.; Korpis, K.; Bednarski, P. J.;
Wuensch, B. Org. Biomol. Chem. 2010, 8, 5525.
5. Cassano, G.; Gasparre, G.; Contino, M.; Niso, M.; Berardi, F.; Perrone, R.;
Colabufo, N. A. Cell Calcium 2006, 40, 23.
36. Li, L.; Ishdorj, G.; Gibson, S. B. Free Radical Biol. Med. 2012, 53, 1399.
37. Wu, Y. T.; Tan, H. L.; Shui, G. H.; Bauvy, C.; Huang, Q.; Wenk, M. R.; Ong, C. N.;
Codogno, P.; Shen, H. M. J. Biol. Chem. 2010, 285, 10850.
38. Tassa, A.; Roux, M. P.; Attaix, D.; Bechet, D. M. Biochem. J. 2003, 376, 577.
39. Hornick, J. R.; Vangveravong, S.; Spitzer, D.; Abate, C.; Berardi, F.;
Goedegebuure, P.; Mach, R. H.; Hawkins, W. G. J. Exp. Clin. Cancer Res. 2012, 31.
40. Zeng, C.; Rothfuss, J.; Zhang, J.; Chu, W.; Vangveravong, S.; Tu, Z.; Pan, F.;
Chang, K. C.; Hotchkiss, R.; Mach, R. H. Br. J. Cancer 2012, 106, 693.
41. Megalizzi, V.; Le Mercier, M.; Decaestecker, C. Med. Res. Rev. 2012, 32, 410.
42. Azzariti, A.; Colabufo, N. A.; Berardi, F.; Porcelli, L.; Niso, M.; Simone, G. M.;
Perrone, R.; Paradiso, A. Mol. Cancer Ther. 2006, 1807, 5.
43. Rybczynska, A. A.; Dierckx, R. A.; Ishiwata, K.; Elsinga, P. H.; van Waarde, A. J.
Nucl. Med. 2008, 49, 2049.
44. van Engeland, M.; Nieland, L. J. W.; Ramaekers, F. C. S.; Schutte, B.;
Reutelingsperger, C. P. M. Cytometry 1998, 31, 1.
45. Elmore, S. Toxicol. Pathol. 2007, 35, 495.
46. Budihardjo, I.; Oliver, H.; Lutter, M.; Luo, X.; Wang, X. D. Annu. Rev. Cell Dev.
Biol. 1999, 15, 269.
47. van Gurp, M.; Festjens, N.; van Loo, G.; Saelens, X.; Vandenabeele, P. Biochem.
Biophys. Res. Commun. 2003, 304, 487.
48. Breckenridge, D. G.; Xue, D. Curr. Opin. Cell Biol. 2004, 16, 647.
49. Daugas, E.; Susin, S. A.; Zamzami, N.; Ferri, K. F.; Irinopoulou, T.; Larochette, N.;
Prevost, M. C.; Leber, B.; Andrews, D.; Penninger, J.; Kroemer, G. Faseb J. 2000,
14, 729.
50. Selvaraj, S.; Mohan, A.; Narayanan, S.; Sethuraman, S.; Krishnan, U. M. J. Med.
Chem. 2013, 56, 970.
51. Yen, Y. P.; Tsai, K. S.; Chen, Y. W.; Huang, C. F.; Yang, R. S.; Liu, S. H. Arch. Toxicol.
2012, 86, 923.
52. Fiers, W.; Beyaert, R.; Declercq, W.; Vandenabeele, P. Oncogene 1999, 18, 7719.
53. Burscha, W.; Karwan, A.; Mayer, M.; Dornetshuber, J.; Frohwein, U.; Schulte-
Hermann, R.; Fazi, B.; Di Sano, F.; Piredda, L.; Piacentini, M.; Petrovski, G.;
Fesus, L.; Gerner, C. Toxicology 2008, 254, 147.
54. Bhutia, S. K.; Mukhopadhyay, S.; Sinha, N.; Das, D. N.; Panda, P. K.; Patra, S. K.;
Maiti, T. K.; Mandal, M.; Dent, P.; Wang, X.-Y.; Das, S. K.; Sarkar, D.; Fisher, P. B.
Adv. Cancer Res. 2013, 118, 61.
1
1
1
1
1
2
2
2
2
2
2
2
6. Brune, S.; Schepmann, D.; Lehmkuhl, K.; Frehland, B.; Wuensch, B. Assay Drug
Dev. Technol. 2012, 10, 365.
2
2
7. Vilner, B. J.; John, C. S.; Bowen, W. D. Cancer Res. 1995, 55, 408.
8. Geiger, C.; Zelenka, C.; Weigl, M.; Froehlich, R.; Wibbeling, B.; Schepmann, D.;
Gruenert, R.; Bednarski, P. J.; Wuensch, B. J. Med. Chem. 2007, 50, 6144.
9. Tsuda, T.; Ohmori, Y.; Muramatsu, H.; Hosaka, Y.; Takiguchi, K.; Saitoh, F.; Kato,
K.; Nakayama, K.; Nakamura, N.; Nagata, S.; Mochizuki, H. Eur. J. Pharmacol.
2
2
001, 433, 37.
0. Yu, J.-J.; Wang, L.-M.; Liu, J.-Q.; Guo, F.-L.; Liu, Y.; Jiao, N. Green Chem. 2010, 12,
16.
55. Yoo, S. H.; Yoon, Y. G.; Lee, J. S.; Song, Y. S.; Oh, J. S.; Park, B. S.; Kwon, T. K.; Park,
C.; Choi, Y. H.; Yoo, Y. H. Int. J. Oncol. 2012, 41, 1443.
56. Garcia-Escudero, V.; Gargini, R. Autophagy 2008, 4, 923.
57. Liu, N.; Tai, S.; Ding, B. X.; Thor, R. K.; Bhuta, S.; Sun, Y.; Huang, J. T. Endocr. -
Relat. Cancer 2012, 19, 711.
3
3
2
1. Dominguez-Rebolledo, A. E.; Martinez-Pastor, F.; Fernandez-Santos, M. R.; del
Olmo, E.; Bisbal, A.; Ros-Santaella, J. L.; Garde, J. J. Reprod. Domest. Anim. 2010,
4
5, e360.
58. Inbal, B.; Bialik, S.; Sabanay, I.; Shani, G.; Kimchi, A. J. Cell Biol. 2002, 157, 455.
59. Okamoto, K.; Sakimoto, Y.; Imai, K.; Senoo, H.; Shidoji, Y. Biochem. J. 2011, 440,
63.
60. Schmukler, E.; Shai, B.; Ehrlich, M.; Pinkas-Kramarski, R. PloS ONE 2012, 7.
61. Schepmann, D.; Lehmkuhl, K.; Brune, S.; Wuensch, B. J. Pharm. Biomed. Anal.
2011, 55, 1136.
3
3
2. Suen, D.-F.; Norris, K. L.; Youle, R. J. Genes Dev. 2008, 22, 1577.
3. Smiley, S. T.; Reers, M.; Mottolahartshorn, C.; Lin, M.; Chen, A.; Smith, T. W.;
Steele, G. D.; Chen, L. B. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 3671.
4. Mizushima, N.; Yoshimori, T. Autophagy 2007, 3, 542.
5. Ichimura, Y.; Kirisako, T.; Takao, T.; Satomi, Y.; Shimonishi, Y.; Ishihara, N.;
Mizushima, N.; Tanida, I.; Kominami, E.; Ohsumi, M.; Noda, T.; Ohsumi, Y.
Nature 2000, 408, 488.
3
3
62. Meyer, C.; Neue, B.; Schepmann, D.; Yanagisawa, S.; Yamaguchi, J.;
Wuerthwein, E.-U.; Itami, K.; Wuensch, B. Bioorg. Med. Chem. 2013, 1844, 21.