Effective inhibition of acid and neutral ceramidases by novel B-13 and LCL-464 analogues

Article abstract of DOI:10.1016/j.bmc.2012.12.014

Induction of apoptosis mediated by the inhibition of ceramidases has been shown to enhance the efficacy of conventional chemotherapy in several cancer models. Among the inhibitors of ceramidases reported in the literature, B-13 is considered as a lead compound having good in vitro potency towards acid ceramidase. Furthermore, owing to the poor activity of B-13 on lysosoamal acid ceramidase in living cells, LCL-464 a modified derivative of B-13 containing a basic ω-amino group at the fatty acid was reported to have higher potency towards lysosomal acid ceramidase in living cells. In a search for more potent inhibitors of ceramidases, we have designed a series of compounds with structural modifications of B-13 and LCL-464. In this study, we show that the efficacy of B-13 in vitro as well as in intact cells can be enhanced by suitable modification of functional groups. Furthermore, a detailed SAR investigation on LCL-464 analogues revealed novel promising inhibitors of aCDase and nCDase. In cell culture studies using the breast cancer cell line MDA-MB-231, some of the newly developed compounds elevated endogenous ceramide levels and in parallel, also induced apoptotic cell death. In summary, this study shows that structural modification of the known ceramidase inhibitors B-13 and LCL-464 generates more potent ceramidase inhibitors that are active in intact cells and not only elevates the cellular ceramide levels, but also enhances cell death.

Full text of DOI:10.1016/j.bmc.2012.12.014

Bioorganic & Medicinal Chemistry 21 (2013) 874–882
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effective inhibition of acid and neutral ceramidases by novel B-13
and LCL-464 analogues
Krishna P. Bhabak a, Burkhard Kleuser b, Andrea Huwiler c, Christoph Arenz a,
⇑
a Humboldt Universität zu Berlin, Institute for Chemistry, Brook-Taylor-Str. 2, 12489 Berlin, Germany
b University of Potsdam, Institute of Nutritional Science, Dept. Nutritional Toxicology, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal (Bergholz-Rehbrücke), Germany
c Institute of Pharmacology, University of Bern, CH-3010 Bern, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Induction of apoptosis mediated by the inhibition of ceramidases has been shown to enhance the efficacy
of conventional chemotherapy in several cancer models. Among the inhibitors of ceramidases reported in
the literature, B-13 is considered as a lead compound having good in vitro potency towards acid
ceramidase. Furthermore, owing to the poor activity of B-13 on lysosoamal acid ceramidase in living cells,
Received 19 September 2012
Revised 7 December 2012
Accepted 13 December 2012
Available online 21 December 2012
LCL-464 a modified derivative of B-13 containing a basic
x-amino group at the fatty acid was reported to
have higher potency towards lysosomal acid ceramidase in living cells. In a search for more potent
inhibitors of ceramidases, we have designed a series of compounds with structural modifications of
B-13 and LCL-464. In this study, we show that the efficacy of B-13 in vitro as well as in intact cells can
be enhanced by suitable modification of functional groups. Furthermore, a detailed SAR investigation
on LCL-464 analogues revealed novel promising inhibitors of aCDase and nCDase. In cell culture studies
using the breast cancer cell line MDA-MB-231, some of the newly developed compounds elevated endog-
enous ceramide levels and in parallel, also induced apoptotic cell death. In summary, this study shows
that structural modification of the known ceramidase inhibitors B-13 and LCL-464 generates more potent
ceramidase inhibitors that are active in intact cells and not only elevates the cellular ceramide levels, but
also enhances cell death.
Keywords:
Sphingolipids
Ceramide
Ceramidase inhibitors
Structure–activity-relationship
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
(nCDase)8 and alkaline ceramidase (alkCDase).9–11 A deficiency of
aCDase leads to massive lysosomal accumulation of Cer, resulting
Sphingolipids (SLs) that act as major signaling molecules are
bioactive constituents of eukaryotic plasma membranes and play
important roles in many cellular processes such as cell recognition,
cell growth, cell differentiation, cell death.1 Among different SLs,
the central molecule is ceramide (Cer), which is produced either
by de novo biosynthesis, degradation of glycosphingolipids (GSLs)
or by degradation of sphingomyelin (SM), catalyzed by sphingomy-
elinases (SMases).2,3 The produced Cer undergoes further degrada-
tion to sphingosine (Sph) by various ceramidases (CDases).
Moreover, a further phosphorylation of Sph to sphingosine-1-
phosphate (S1P) via sphingosine kinases (SKs) can occur. Proper
equilibrium between Cer and S1P is crucial for a final cellular re-
sponse, cell homeostasis and normal cell development as they in-
duce contrasting cellular processes.4–6 The concentration of
cellular ceramide is highly dependent on the cleavage of produced
Cer by different ceramidases (CDases). Based on the pH-optima and
subcellular localizations, CDases can be classified into three major
sub-types such as acid ceramidase (aCDase),7 neutral ceramidase
in Farber disease.12,13 On the other hand it has been shown that
especially aCDase is up-regulated in prostate cancer14 and melano-
mas,15 leading to the hypothesis that aCDase could be a tumor
marker.14,15 Moreover, aCDase in tumor cells confers resistance
to chemo- and radiotherapy therefore the inhibition of this enzyme
has become a potential target for cancer therapy.16 Based on these
outcomes and indications, the inhibition of ceramidases has been
thought to be significantly important for the development of che-
motherapeutic agents in anti-cancer therapy.
Since last several years only few lead inhibitors of ceramidases
could be developed such as B-13, D-e-MAPP and NOE (Fig. 1).2
A
further improvement on the potency of inhibitors becomes compli-
cated as the exact molecular basis for the inhibition of ceramidases
by the existing inhibitors is still unknown. Several research groups
have attempted SAR studies for the development of new inhibitors
mainly by various modifications on the existing inhibitors such as
substituent, functional group, length of fatty acid, stereochemistry,
etc. For example, two decades ago, Hannun and co-workers have
reported that B-13 effectively inhibited the HL-60 cell growth in
a
1.5
dose-dependent manner with an IC50 of approximately
l
⇑
Corresponding author. Fax: +49 30 2093 6947.
E-mail address: christoph.arenz@chemie.hu-berlin.de (C. Arenz).
M.17 In that report, they have varied and optimized several
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.014

K. P. Bhabak et al. / Bioorg. Med. Chem. 21 (2013) 874–882
875
O
O
H
O
O2N
H
CH3
7
N
C13H27
OH
N
C13H27
HN
7
HO
CH3
OH
B-13
OH
D-e-MAPP
N-Oleoylethanolamine
(NOE)
H Cl
N
O
O
R
H
C13H27
OH
O2N
NH2
( )11
OH
O2N
N
( )11
OH
HN
HN
OH
OH
LCL-463
OH
LCL-464
LCL-204: R = NO2
LCL-385: R = H
Figure 1. Chemical structures of some literature known inhibitors of ceramidases.
structural factors on N-acyl-phenylaminoalcohol analogues such as
the length of fatty acid chain, importance of primary/secondary hy-
droxyl groups and stereochemistry of C1 and C2 atoms on the cell
growth. The same group subsequently developed D-e-MAPP, a
structurally modified B-13 analogue that selectively inhibits alka-
line ceramidase activity derived from HL-60 cell extracts with an
LCL-204 has been further studied in details by several research
groups. For example, it has been shown that the lysosomotropic
aCDase inhibitor LCL-204 induces apoptosis in prostate cancer
cells33 and enhances the cytotoxicity of the viral protein apoptin
in prostate cancer.34 This compound was also found to be active
in head and neck squamous cancer cells (HNSCC) in vitro and
in vivo in a xenograft model sensitizing cells to Fas-ligand induced
apoptosis.35 A more detailed biochemical investigation however
revealed LCL-204 and probably also LCL-385 are highly unspecific
and induce unwanted lysosomal permeabilization and degradation
of aCDase.25
IC50 of about 1–5 l
M.18 It should be noted that D-e-MAPP differs
from B-13 with respect to functional groups as well as the stereo-
chemistry of chiral centers. The inhibitory activity of D-e-MAPP
towards alkCDase was completely abolished upon the reversal of
stereochemistry of chiral centers (L-e-MAPP). This compound
rather acted as a competitive substrate analogue for alkCDase.
However, in a contradictory report after several years, it has been
shown that D-e-MAPP does not show any noticeable inhibition to-
wards alkCDase, while it shows poor activity towards aCDase in a
dose dependent manner in human melanoma and HaCaT cells.19
This study also showed B-13 to be a potent and selective inhibitor
of aCDase over alkCDase in HaCaT keratinocytes and human mela-
noma cells. The third compound N-oleoyl-ethanolamine (NOE) sig-
nificantly increased the cellular Cer level although it acted only as a
weak inhibitor of aCDase.20 In further studies however, two NOE
analogues have been reported that significantly inhibited aCDase
Based on these observations, a second generation basic B-13
analogue (LCL-464) was tested by Bielawska and co-workers.25
This compound has been shown to inhibit aCDase activity in cell
extracts (50% at ꢀ50
lM) with a lower potency than B-13 but
much higher than that of LCL-204. Furthermore, LCL-464 caused
an early inhibition of aCDase in cellular studies leading to de-
creased sphingosine levels and a specific increase in C14- and
C16-ceramide contents. Unlike LCL-204, the new derivative LCL-
464 neither induces lysosomal destabilization nor the degradation
of aCDase.25 These results show that the cellular inhibition of acid
ceramidase depends both on their in vitro activity and on their
ability to accumulate in cellular lysosomes. As both LCL-46425
and B-1317 have been shown previously to be moderate inhibitors
of acid ceramidase, the present study was focused on the design of
several B-13 and LCL-464 analogues with the intention to have
more potent inhibitors of ceramidases. In the first category, some
B-13 analogues have been developed with the modification of
important functional groups. In a second series, a number of LCL-
464 analogues have been designed with the basic structures of
D-e-MAPP and some recently published B-13 analogues.24 Inhibi-
tion potencies of new derivatives are studied towards both of acid
and neutral ceramidases in order to better interpret results from
cell culture experiments. Furthermore, apoptotic behavior as well
as the effect of these compounds on the endogenous ceramide level
has been studied to understand their pharmacokinetics of
inhibition.
in cellular studies (IC50 ꢀ15
l
M) and also at the same time exhib-
ited cytotoxicity to A549 cells (IC50 ꢀ40
l
M).21,22
Bielawska and co-workers have recently carried out several
very detailed and interesting studies with aCDase inhibitors (LCL-
analogues) containing basic amino functionalities that help the
molecules to accumulate in lysosomes (Fig. 1).23–25 A number of
similar compounds were also developed previously by Gatt and
co-workers as inhibitors of sphingolipid biosynthesis.26,27 Lyso-
somal accumulation is also observed for a group of functional
inhibitors, the tricyclic antidepressants such as imipramine and
desipramine.28 The major drawback is that the tricyclic com-
pounds are highly non-selective in their nature of inhibition. In
addition to inhibiting aCDase,29 they inhibit acid sphingomyeli-
nase30 and lysosomal phospholipases.31 These amine-based mole-
cules generally are in equilibrium between the protonated and
neutral forms depending on pH. Only the latter can permeate
through lysosomal membranes and enter the lysosomes. However,
in the acidic environment of the lysosome, compounds undergo
full protonation leading to an increased local concentration of sub-
stances in lysosome. In lysosome these compounds interfere with
the substrate (membrane) binding of lipid hydrolases, making
them highly susceptible to proteolytic degradation.32 Indeed,
spatial preferences for the LCL-analogues such as LCL-204 and
LCL-385 have been observed for the accumulation in acidic
compartment of cells. Among different sets of LCL-analogues,
2. Results and discussion
Recently we have developed a series of new aromatic ethanol-
amine-based inhibitors with the partial modifications of B-13, D-
e-MAPP as well as NOE structures and some of the new compounds
exhibited significantly higher potency than the parent inhibitors
towards acid and neutral ceramidases.36 An enhanced selectivity
towards aCDase over nCDase has been observed in those analogues
upon the substitution of alkyl chains at the aromatic ring. In this

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K. P. Bhabak et al. / Bioorg. Med. Chem. 21 (2013) 874–882
study, we also showed for the first time that the lead compound B-
13 shows significant inhibition towards nCDase along with its good
aCDase activity. Therefore, the elevation of endogenous ceramide
level by B-13 might be the result of both, the inhibition of aCDase
and nCDase. Furthermore, the potency of B-13 and some of the
amide-based inhibitors could be enhanced significantly upon the
conversion of amide group to the corresponding sulfonamide
counterparts.
labeled ceramide analogues to understand their relative hydrolytic
behavior towards ceramidases.39 Among the four different combi-
nations of NBD and NR-based substrates, Acyl-NBD-C12-Cer was
found to be superior candidate with respect to the ceramidase-
mediated hydrolysis in vitro and therefore this substrate was cho-
sen for the detailed inhibition studies in this report in the presence
of recombinant human aCDase and nCDase as enzyme sources.
Ceramidase-mediated hydrolysis of the amide bond in Acyl-
NBD-C12-Cer produces fluorescent NBD-C12-fatty acid, which was
separated from the uncleaved substrate using thin layer chromato-
graphic (TLC)-method and quantified by fluorescent imaging meth-
od. It should be mentioned that the absolute IC50/EC50 values in an
inhibition experiment are dependent on the substrate and enzyme
used and also on their respective concentrations, which suggested
us to consider literature-known inhibitors such as B-13, D-e-MAPP
and LCL-464 as references during the inhibition experiments.
As shown in Figure 3 and also reported very recently from our
group,36 B-13 has been found to be a moderate inhibitor of recom-
binant human aCDase in vitro. The newly designed B-13 analogues
such as KPB-49, KPB-67 and KPB-105 exhibited significantly higher
inhibition than that of B-13 towards aCDase at a concentration of
In the present study, we wanted to understand the effect of dif-
ferent functionalities in B-13 such as primary and secondary hy-
droxyl groups, p-nitro group etc. Accordingly, we have
synthesized three different modified analogues with the alteration
of one functional group at a time. To probe the importance of pos-
sible hydrogen bonding interactions in B-13 at the enzyme active
site, the secondary hydroxyl group was converted to the corre-
sponding ketone (KPB-49). As shown in Scheme 1A, KPB-49 could
be synthesized from B-13 by a single step selective oxidation of
the secondary hydroxyl group using fine powder of manganese
dioxide. In contrast, the primary hydroxyl group could also be
selectively oxidized to the corresponding carboxylic acid (KPB-
105) using sodium hypochloride (NaOCl) in the presence of TEMPO
as radical initiator. The electron withdrawing p-nitro group was
converted to the electron donating amino group by the reduction
mediated by zinc dust in concentrated acetic acid (Scheme 1A).
All the new derivatives were screened along with B-13 for their
inhibition potencies towards both of acid and neutral ceramidases.
In another set of compounds, a series of N,N-dimethylamino
substituted compounds has been synthesized following the meth-
od developed by Bielawska and co-workers with minor modifica-
tions.25 These compounds could be synthesized in three steps
starting from the corresponding aminols as shown in Scheme 1B.
Some of the aminols could be prepared from the corresponding
aldehydes using Henry reaction as reported previously.37,38 The
amino group of aminols with different substituents can be coupled
80 lM (Fig. 3). For example, KPB-49 exhibited almost 1.5 times
higher potency than B-13 towards inhibition of aCDase. Inhibition
was found to be even more pronounced towards nCDase upon this
modification as the inhibition potency of KPB-49 was almost dou-
ble of B-13 (Fig. 4).These results indicate that the in vitro inhibitory
activity of B-13 could be considerably improved upon the replace-
ment of a hydrogen-bond donor atom (secondary hydroxyl group)
with a hydrogen-bond acceptor (keto group). However, a similar
modification of secondary hydroxyl group in B-13 analogues lack-
ing primary hydroxyl group led to a weaker inhibition towards
ceramidases as shown recently.36 These observations suggest that
the presence of at least one hydroxyl group as hydrogen-bond do-
nor is possibly important for an effective inhibition towards
ceramidases. To understand the role of primary hydroxyl group
in B-13, compound KPB-105 was synthesized upon the selective
oxidation of primary hydroxyl group to the corresponding carbox-
ylate. A significant increase in inhibition (ꢀ1.75 times) was
observed towards aCDase upon this modification. However, the
scenario was opposite in case of nCDase as KPB-105 did not exhibit
any noticeable inhibition towards nCDase. Unlike KPB-49, com-
pound KPB-105 produced an enhanced selectivity towards aCDase
over nCDase. The selective inhibition of KPB-105 towards aCDase
may be highly influenced by the presence of pH-sensitive carbox-
ylate group. For example, during the inhibition of aCDase (pH
with
x-bromo-acyl chloride to form x-bromo-amides. Subse-
quently, the bromo group was reacted with N,N-dimethylamine
leading to the formation of different N,N-dimethylamino substi-
tuted compounds as shown in Figure 2. All the new compounds
were thoroughly characterized by NMR spectroscopic (1H and
13C) and ESI-MS spectrometric analyses. Optical rotation was mea-
sured for the stereochemically pure compounds.
The inhibitory potency of the newly synthesized inhibitors was
evaluated using NBD-ceramide analogue as a substrate.36 Very re-
cently we have reported a detailed comparative kinetic study with
possible combination of NBD and Nile Red (NR) substituted singly
O
O
O
NH2
(i)
O2N
H
Br
O2N
N
C13H27
OH
HN
C13H27
OH
( )11
HN
(i)
R1
R2
OH
R1
R2
OH
O
OH
Aminol
(1R,2R) B-13
(2R) KPB-49
A
ω−Bromo-Amide
(ii)
B
(ii)
(iii)
O
O
O
H2N
O2N
N
R1 = Substituents on
aromatic ring
HN
C13H27
OH
HN
C13H27
CO2H
( )11
HN
R1
R2
R2 = H or Me
OH
OH
OH
(2S,3R) KPB-105
(1R,2R) KPB-67
N,N-dimethylamino Analogues
Scheme 1. Synthetic route to different B-13 (A) and N,N-dimethylamino substituted compounds (B) as inhibitors of ceramidases. Reagents and conditions: (A) (i) MnO2,
CH2Cl2, 35 °C, 24 h; (ii) NaOCl, TEMPO, NaHCO3, (nBu)4NBr, CH2Cl2, H2O, rt, 30 min; (iii) Zn dust, concd AcOH, rt to 70 °C, 30 min, (B) (i) Br-C11H23-COCl, 50% NaOAc (aq), THF,
2 h, 0 °C to rt; (ii) Me2NH, 1.5 N NaOH, THF, reflux, 12 h.

K. P. Bhabak et al. / Bioorg. Med. Chem. 21 (2013) 874–882
877
O
O
O
Y
Y
N
N
( )11
( )11
X
HN
C13H27
HN
X
HN
R
OH
(+/-) KPB-20: X = N, Y = CH
DP-24c
OH
OH
KPB-99:
(+/-)
X = N; Y = CH
(+/-) KPB-102: X = CH; Y = N
KPB-93: R = Me
(+/-)
: X = CH, Y = N
(1S,2R)
KPB-94: R = H
(+/-)
(+/-) DP-24a: X = Y = CH
Figure 2. Chemical structures of already reported amide-based compounds (KPB-20, DP-24c and DP-24a) and the newly synthesized N,N-dimethylamino substituted
compounds as inhibitors of ceramidases.
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
***
***
***
***
***
***
***
**
***
***
***
***
***
***
***
**
***
***
4
ol
Contr
ol
Contr
PP
PP
102
A
A
B-13
KPB-K4P9B-67
B-13
KPB-K4P9B-67
DP-24a
KPB-94 KPB-K2P0B-99
DP-24c
KPB-
DP-24a
KPB-94 KPB-K2P0B-99
DP-24c
KPB-102
KPB-93
KPB-93
LCL-46
LCL-464
KPB-105
KPB-105
D-e-M
D-e-M
Figure 3. Plot for the relative potencies of various compounds for the inhibition of
Figure 4. Plot for the relative potencies of various compounds for the inhibition of
aCDase activity. The final concentrations of fluorescent substrate and inhibitors
nCDase activity. The final concentrations of fluorescent substrate and inhibitors
were 15 and 80
lM, respectively. Inhibitory data are shown as mean SD from
were 15 and 80 lM, respectively. Inhibitory data are shown as mean SD from
three independent experiments under standard assay condition. Statistical signif-
icances of the results have been compared to the control reaction; ⁄, 0.01 < p 6 0.05;
⁄⁄, 0.001 < p 6 0.01; ⁄⁄⁄, p 60.001. The control value represents the percentage
hydrolysis of the substrate by aCDase in the absence of any inhibitor.
three independent experiments under standard assay condition. Statistical signif-
icances of the results have been compared to the control reaction; ⁄, 0.01 < p 6 0.05;
⁄⁄, 0.001 < p 6 0.01; ⁄⁄⁄, p 60.001. The control value represents the percentage
hydrolysis of the substrate by nCDase in the absence of any inhibitor.
4.5), the likely active form is carboxylic acid (–COOH) whereas, the
corresponding carboxylate (COOꢁ) might be the most active spe-
cies at neutral pH during the inhibition of nCDase (pH 7.0). This
might be an interesting aspect for the design of selective inhibitors
modulated by pH of the cellular medium/environment. A third
modification with the conversion of electron withdrawing nitro
group to the basic amino group (KPB-67) also led to a slightly in-
creased inhibition towards aCDase while the effect was negligible
towards nCDase. This modification however made KPB-67 to be
more or at least equally potent as B-13 towards ceramidases. With
the presence of primary amino group in the aromatic ring, KPB-67
could be considered as an analogue of lysosomotropic inhibitor
LCL-464. While LCL-464 exhibited almost similar inhibition as B-
13, compound KPB-67 was found to be almost 1.5 times more po-
tent than LCL-464 towards aCDase.
As the lysosomotropic inhibitor LCL-464 exhibited moderate
inhibition towards both aCDase and nCDase in the in vitro assays,
we decided to screen the newly developed analogues with the var-
iation of substituents as shown in Figure 2. In addition to compar-
ing their inhibitory potencies to LCL-464, we also included the
corresponding amide counterparts, reported recently from our
group, such as KPB-20, DP-24c and DP-24a to understand the effect
of basic N,N-dimethylamino group towards their activities. While
D-e-MAPP has been reported to be a potent inhibitor of alkaline
ceramidase in HL-60 cells18 subsequent reports revealed this
compound to be relatively poor inhibitor of both aCDase and
nCDase in HaCaT keratinocytes.19 With these complications in its
activity in different experimental conditions and with different cel-
lular lysates as enzyme sources, we decided to screen the potency
of D-e-MAPP using recombinant human aCDase and nCDase as
sources of enzymes. Interestingly, in our present study, difference
in inhibition potency of D-e-MAPP towards recombinant human
aCDase and nCDase has been observed. For example, while
D-e-MAPP exhibited significant inhibition towards aCDase (60%
inhibition at 80 lM concentration, Fig. 3), it showed very weak
inhibitory activity for nCDase (8%). These results indicate the selec-
tivity of D-e-MAPP towards aCDase over nCDase. However, the
weak activity of D-e-MAPP towards nCDase could be dramatically
enhanced upon the incorporation of N,N-dimethylamino group. For
example, while D-e-MAPP exhibited very weak inhibition (8%) to-
wards nCDase, the corresponding N,N-dimethylamino derivative
(KPB-93) showed almost 2.5 times higher potency (63% inhibition),
which was even much higher than the already reported amino-
substituted compound LCL-464 (30%). These observations indicate
that N,N-dimethylamino derivatives of D-e-MAPP is more effective
inhibitors of ceramidases than the parent compound at least in the
in vitro inhibition assays.
As shown in our previous reports, more simplified amide-
substituted analogues of B-13 such as DP-24a, KPB-20 and
DP-24c also exhibited relatively higher inhibition potency towards

878
K. P. Bhabak et al. / Bioorg. Med. Chem. 21 (2013) 874–882
potency than B-13 over the entire range of concentrations studied
and the IC50 value of KPB-93 was found to be almost 2.5 times
lower than B-13 under the identical experimental condition.
To assess the pro-apoptotic potential of our compounds, we
have carried out apoptosis studies. In the present study, apoptotic
cell death in MDA-MB-231 breast cancer cells was investigated,
detecting the occurrence of DNA fragmentation by using an ELISA.
Cells were incubated with various inhibitors at two different con-
B-13 (IC50 = 120
KPB-93 (IC50 = 45
μ
M)
M)
100
80
60
40
20
0
μ
centrations (25 and 50 lM) to see their apoptotic behavior. As
shown in Figure 6, most of the newly synthesized inhibitors
showed significantly higher fragmentation (ꢀ150–400%) than the
control, indicating the occurrence of apoptosis. At 50 lM concen-
tration of inhibitors, at least double or even more fragmentation
was observed for most compounds. Induction of apoptosis by com-
pounds in the present study is probably the result of significant
inhibition of ceramidases and consequently an increase of cellular
ceramide levels. Among the modified B-13 analogues and newly
synthesized basic N,N-dimethylamino substituted compounds,
KPB-67 having a primary amino group in the phenyl ring of B-13
was found to be most potent, showing a three- to fourfold
0
50
100
150
200
M)
250
300
350
[Inhibitor] (
μ
Figure 5. Plot for the effect of concentrations of B-13 and KPB-93 for the inhibition
of nCDase activities. IC50 values represent the concentration of inhibitors required
for 50% inhibition of control activities. Inhibitory data are shown as mean SD from
three independent experiments under standard assay condition.
enhancement of DNA fragmentation, while
a more detailed
investigation revealed an EC50 of 59.02 M (Fig. 6). Obviously,
l
the presence of a polar primary amino group in the phenyl ring
of B-13 may have high significance for the development of a potent
chemotherapeutic agent.
both of aCDase and nCDase.36,38 To see the effect of N,N-dimethyl-
amino group on their potencies, we compared the activities of KPB-
94, KPB-99 and KPB-102 to the corresponding parent compounds.
As shown in Figures 3 and 4, while the activities of KPB-94 and DP-
24a were comparable, a much weaker inhibition was observed for
the N,N-dimethylamino derivatives (KPB-99 and KPB-102) of the
pyridyl-substituted compounds. This observation supports the
assumption that the incorporation of a weakly basic group apart
from the hydrophobic fatty acid part in B-13 and D-e-MAPP ana-
logues is a better choice for the development of more potent cer-
amidase inhibitors. However, this could be verified only after
screening the compounds in cellular as well as in animal model
experiments.
Interestingly, and in contrast to the apoptosis data, a cell viabil-
ity assay using the AlamarBlueÒ blue reagent showed only minor
and non-significant responses for most compounds (data not
shown). Although this result was unexpected, it may indicate that
our ceramidase inhibitors are specifically pro-apoptotic, rather
than being cytotoxic. To understand whether the addition of inhib-
itors indeed enhances endogenous ceramide levels in the cells, we
have determined the total ceramide levels in MDA-MB-231 cancer
cells upon treatment of cells with various inhibitors of interest.
Cells were incubated at 37 °C for 24 h with two different concen-
trations of inhibitors (25 and 50
quantified using mass spectrometry. As shown in Figure 7, at
50 M concentration, all the compounds with the exception of
lM) and the ceramide levels were
As KPB-93 was found to be one of the most active inhibitors
among the new N,N-dimethylamino-substituted compounds in
the present study, we further studied the dose-dependency of this
compound and compared to that with B-13 towards the inhibition
of nCDase. As shown in Figure 5, KPB-93 exhibited much higher
l
KPB-99 increased total ceramide content in cells to at least 150%
or more upon their incubation. B-13 showed a significant elevation
of total ceramide content (350%), which is in agreement with ear-
lier reports by Bielawska and co-workers.25 It should be noted that
450
***
25
50
μ
μ
M
M
400
350
300
250
200
150
100
50
***
***
***
1.00
***
**
*
*
***
***
**
**
0.75
**
**
0.50
***
0.25
0.00
1
10
100
0
concentration of KPB-67 (μM)
a
4
ol
ontr
05
-1
-24
DP
B-13
EC50 = 59.02 µM (R2 = 0,9816)
C
KPB-67
KPB-93 KPB-94 KPB-99
KPB-102
LCL-46
KPB
Figure 6. Left: Induction of apoptosis by different ceramidase inhibitors Data are expressed as % of control and are means S.D (n = 3). Right: Dose–response curve for KPB-
67. Data are expressed as optical density. MDA-MB-231 cells were treated for 24 h with either vehicle (DMSO) or 25 M (red) and 50 M (green) of the different compounds.
l
l
Thereafter, DNA fragmentation as a measure of apoptosis was determined by a cell death detection plus ELISA as described in the methods part. Statistical significances of the
results have been compared to the control reaction; ⁄, 0.01 < p 6 0.05; ⁄⁄, 0.001 < p 6 0.01; ⁄⁄⁄, p 60.001.

K. P. Bhabak et al. / Bioorg. Med. Chem. 21 (2013) 874–882
879
inhibitors including LCL-464 and other newly developed N,N-
dimethylamino derivatives tested in the present study. The eleva-
tion of total ceramide level by B-13 and KPB-67 is in agreement
with the results on their apoptotic behavior and this clearly indi-
cates that the apoptosis of MDA-MB-231 cells upon the adminis-
tration of inhibitors such as B-13 or KPB-67 is most probably due
to the inhibition of ceramidases leading to the accumulation of cel-
lular ceramide. Among the N,N-dimethylamino-substituted com-
pounds, KPB-102 showed reasonable elevation of ceramide in
comparison to other derivatives.
450
400
350
300
250
200
150
100
50
25
50
μ
μ
M
M
**
*
***
*
**
**
***
*
*
*
To understand the influence of inhibitors on different ceramides
such as C16-Cer, C18-Cer and C24-Cer, we have chosen the highly
active inhibitors (B-13 and KPB-67) for further detailed studies. As
shown in Figure 8, the effect of these compounds on different cera-
*
mides was analyzed at 25 and 50 lM concentrations. In general,
the elevation of C16-Cer was highest, while the elevation was
found to be least for C18-Cer. The increase of all ceramides
0
5
2
ol
Contr
67
0
0
24a
464
B-13
L-
LC
P-
was much lower for B-13 at lower concentration (25
lM) but it
D
KPB-
KPB-93 KPB-94 KPB-99
KPB-1
KPB-1
was found to be significantly higher at 50
lM concentration.
Figure 7. Effect of ceramidase inhibitors on the cellular ceramide content. A final
concentration of 25 (red) and 50 (green) of various inhibitors were
lM
lM
incubated with MDA-MB-231 breast cancer cells for 24 h at 37 °C for their effect
on cellular ceramide level. Data are shown as mean SD from three independent
experiments under standard assay condition. Statistical significances of the results
have been compared to the control reaction; ⁄, 0.01 < p 6 0.05; ⁄⁄, 0.001 < p 6 0.01;
⁄⁄⁄, p 60.001.
3. Conclusion
In summary, we report the design and synthesis of two series of
novel analogues of B-13 and LCL-464 having improved inhibition
potencies towards aCDase and nCDase in vitro and in intact cells.
This study suggests that the in vitro potency of B-13 could be en-
hanced further with a suitable modification of functional groups.
While B-13 did not show much selectivity, replacement of the pri-
mary hydroxyl group with a carboxylic acid led to an enhanced
selectivity towards aCDase over nCDase. Furthermore, the inhibi-
tory activity of D-e-MAPP and some of the simplified B-13 ana-
logues could be increased upon the incorporation of polar basic
N,N-dimethylamino group at the fatty acid chain. While the
replacement of p-nitro group in B-13 with a primary amino group
(KPB-67) had marginal effect on the in vitro inhibition of aCDase
and nCDase, this modification made the molecule to be highly ac-
tive at the cellular level leading to apoptosis with significant eleva-
tion of endogenous ceramide content, suggesting a more detailed
investigation of KPB-67 in future experimental therapies.
4. Experimental section
4.1. Materials and method
Chemicals were purchased from Sigma–Aldrich or Acros.
Acyl-C12-NBD-Cer was purchased from Avanti-Polar Lipids, Inc. Re-
combinant neutral ceramidase (nCDase) was obtained from R&D
systems, Inc. Solvents were freshly distilled whenever required
for the reaction. All the moisture sensitive reactions were carried
out under dry argon atmosphere. Thin layer chromatographic
(TLC) studies are performed on pre-coated silica gel 60 F254 on
aluminum sheets (Merck KGaA). 1H (500 or 300 MHz) and 13C
(125 or 75 MHz) NMR spectra were obtained on a Bruker Avance
III 500 MHz or Bruker Avance DPX 300 MHz NMR spectrometers.
Chemical shifts are cited with respect to Me4Si as internal stan-
dard. Mass spectral studies were carried out on a Hewlett-Packard
GCMS 5995-A mass spectrometer with ESI-MS or EI-MS modes.
Optical rotation of the stereochemically pure compounds was
determined using Perkin-Elmer 241 Polarimeter. The inhibition
data were plotted using Origin 6.1 or Graph Pad Prism 4 software
and the statistical significance was assessed using students T-test
with IBM SPSS software. Previously published compounds from
our group such as DP-24a/DP-24c38 and KPB-2036 were synthe-
sized following the literature procedure.
Figure 8. Effect of B-13 and KPB-67 on the elevation different ceramide levels in
MDA-MB-231 cells at two different concentrations. Data shown in the plot
represent average of three independent experiments under standard assay condi-
tion and the results are shown as mean SD values. Statistical significances of the
⁄
⁄⁄
,
results have been compared to the control reaction;
,
0.01 < p 6 0.05;
0.001 < p 6 0.01; ⁄⁄⁄, p 60.001.
the elevation was much higher at 50
lM concentration (350%) of
B-13 than at 25 M (150%). For highly polar B-13 analogues such
l
as KPB-67 and KPB-105, it has been observed that, as compared
to KPB-105, the primary amino substituted compound KPB-67
showed a significant increase in total ceramide level upon the
incubation at both the concentrations. For example, while KPB-
105 increased the ceramide level by ꢀ150%, the enhancement
was around 300–380% for KPB-67. KPB-67 was the compound that
exhibited highest increase in ceramide level among all the

880
K. P. Bhabak et al. / Bioorg. Med. Chem. 21 (2013) 874–882
4.1.1. Synthesis of N-((R)-3-hydroxy-1-(4-nitrophenyl)-1-
oxopropan-2-yl)tetradecanamide (KPB-49)
chloride (1.10 mmol) was added dropwise over 1 min. The reaction
mixture was allowed to attain room temperature and stirred at
room temperature for another 2 h and the completion of the reac-
tion was monitored by TLC method. The mixture was diluted with
ethyl acetate and extracted with water followed by brine solution.
The combined organic extract was dried over anhydrous sodium
sulfate, filtered and evaporated to dryness under reduced pressure
to afford crude product. This crude material was purified by silica
gel column chromatography using cyclohexane and ethyl acetate
mixture or ethyl acetate and methanol mixture as eluent.
To a stirred solution of B-13 (50.0 mg, 0.11 mmol) in dichloro-
methane was added fine powder of manganese dioxide (0.1 g,
1.14 mmol) and the reaction mixture was stirred at 35 °C for
24 h. The mixture was filtered through a pad of celite to remove
the unreacted MnO2. The solvent was evaporated to obtain the
crude product as white solid. The compound was purified by silica
gel column chromatography using cyclohexane and ethyl acetate
as eluent. The solvent was evaporated to afford the pure product
as yellowish amorphous solid. Yield: 15 mg (33%), mp: 78–80 °C.
Rf = 0.30 (100% Ethyl acetate). 1H NMR (CDCl3, 500 MHz, ppm):
d = 0.90 (t, J = 6.5 Hz, 3H), 1.27–1.32 (m, 20H), 1.64–1.70 (m, 2H),
2.29–2.35 (m, 2H), 3.94–4.02 (m, 2H), 5.61–5.64 (m, 1H), 6.75 (d,
J = 6.5 Hz, 1H), 8.22 (d, J = 9.0 Hz, 2H), 8.37 (d, J = 8.5 Hz, 2H). 13C
NMR (CDCl3, 125 MHz, ppm): 14.1, 22.7, 25.6, 29.2, 29.3, 29.5,
29.6, 31.9, 36.5, 57.2, 64.1, 124.1, 129.9, 138.9, 150.8, 174.0,
4.2.3. Preparation of LCL-464 analogues
To a solution of the product (0.76 mmol) in a mixture of THF
(8.0 mL) and 1.5 N NaOH solution (4.0 mL) (2:1), N,N-dimethyl-
amine (1.52 mmol) was added at room temperature and the reac-
tion mixture was refluxed overnight. The solution was diluted with
CH2Cl2 and extracted with water and brine solution. The combined
organic layer was dried over anhydrous sodium sulfate. The
solvent was evaporated under reduced pressure to afford the crude
product as yellowish oil. The product was purified by silica gel
column chromatography using cyclohexane, ethyl acetate and
methanol mixture in the presence of 1% NEt3.
196.1.
½
a 2D5
+7.1 (c 0.01 in MeOH). ESI-MS: m/z calcd for
ꢂ
C23H36N2O5: 419.2551 [MꢁH]ꢁ; observed: 419.2553.
4.1.2. Synthesis of N-((1R,2R)-1-(4-aminophenyl)-1,3-
dihydroxypropan-2-yl)tetradecanamide (KPB-67)
The compound was synthesized following the literature proce-
dure with minor modifications.24 To a stirred solution of B-13
(0.10 g, 0.23 mmol) in acetic acid (80%, 2.0 mL) was added zinc
dust (0.15 g, 2.29 mmol). After 10 min, the reaction mixture was
heated to 70 °C for 30 min. The unreacted zinc dust was removed
by filtration. The filtrate was diluted with ethyl acetate and ex-
tracted with saturated sodium bi-carbonate solution. The com-
bined organic layer was dried over anhydrous sodium sulfate.
The solvent was evaporated and the compound was purified by sil-
ica gel column chromatography using cyclohexane/ethyl acetate
and finally with ethyl acetate/methanol mixtures as eluent. The
solvent was evaporated to afford the pure product as light reddish
solid. Yield: 30 mg (34%), mp: 112–114 °C. Rf = 0.35 (100% ethyl
acetate). 1H NMR (CDCl3, 500 MHz, ppm): d = 0.90 (t, J = 7.0 Hz,
3H), 1.27 (br, 20H), 1.62 (m, 2H), 2.15 (m, 2H), 2.76 (m, 2H), 3.61
(dd, J1 = 5.5 Hz, J2 = 11.0 Hz, 2H), 3.70 (dd, J1 = 3.1 Hz,
J2 = 11.0 Hz, 1H), 4.10 (m, 1H), 5.65 (d, J = 4.3 Hz, 1H), 6.66 (d,
J = 8.3 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H). 13C NMR (CDCl3, 125 MHz,
ppm): 14.1, 22.7, 25.7, 29.2, 29.3, 29.5, 29.7, 31.9, 36.2, 36.8,
4.2.3.1. Synthesis of 12-(dimethylamino)-N-((1S,2R)-1-hydroxy-
1-phenylpropan-2-yl)dodecanamide (KPB-93).
Yield: (47%)
as light yellow amorphous solid. Mp: 52–54 °C. Rf = 0.2 (25% MeOH
in ethyl acetate with 2% NEt3). 1H NMR (CDCl3, 500 MHz, ppm):
d = 0.95 (d, J = 7.0 Hz, 3H), 1.24 (br, 14H), 1.42 (t, J = 6.5 Hz, 2H),
1.58 (t, J = 6.0 Hz, 2H), 2.16 (s, 6H), 2.23 (t, J = 7.5 Hz, 2H), 4.23 (t,
J = 6.5 Hz, 1H), 4.78 (s, 1H), 5.15 (br, 1H), 6.11 (br, 1H), 7.22 (t,
J = 7.0 Hz, 1H), 7.28–7.33 (m, 4H). 13C NMR (CDCl3, 125 MHz,
ppm): 14.0, 25.7, 27.2, 27.4, 29.1, 29.2, 29.3, 29.4, 36.8, 45.0,
50.8, 59.6, 75.9, 126.2, 127.2, 128.0, 141.6, 173.5. ½a D25
ꢂ
+12.9 (c
0.01 in MeOH). ESI-MS: m/z calcd for C23H40N2O2: 377.3163
[M+H]+; observed: 377.3163.
4.2.3.2. Synthesis of 12-(dimethylamino)-N-(2-hydroxy-2-phen-
ylethyl)dodecanamide (KPB-94).
Yield: (41%) as light yellow
amorphous solid. Mp: 60–62 °C. Rf = 0.25 (25% MeOH in ethyl ace-
tate with 2% NEt3). 1H NMR (CDCl3, 500 MHz, ppm): d = 1.24 (br,
14H), 1.39 (br, 2H), 1.55 (br, 2H), 2.11 (s, 6H), 2.17 (t, J = 7.5 Hz,
2H), 3.20–3.24 (m, 1H), 3.59–3.62 (m, 1H), 4.72 (d, J = 8.0 Hz,
1H), 5.44 (br, 1H), 6.41 (br, 1H), 7.21–7.33 (m, 5H). 13C NMR (CDCl3,
125 MHz, ppm): 25.7, 27.3, 27.4, 29.2, 29.3, 29.4, 29.5, 36.6, 45.1,
47.3, 59.7, 73.0, 125.8, 127.5, 128.3, 142.5, 174.3. ESI-MS: m/z calcd
for C22H38N2O2: 363.3006 [M+H]+; observed: 363.3002.
53.2, 64.9, 115.4, 127.2, 130.0, 145.1, 174.1. ½a D25
ꢂ
+5.7 (c 0.01 in
MeOH). ESI-MS: m/z calcd for C23H40N2O3: 393.3115 [M+H]+; ob-
served: 393.3112.
4.2. Synthesis of LCL-464 analogues
These compounds were synthesized following the literature
procedure with minor modifications.25
4.2.3.3. Synthesis of 12-(dimethylamino)-N-(2-hydroxy-2-(pyri-
din-4-yl)ethyl)dodecanamide (KPB-99).
Yield: (39%) as off-
white amorphous solid. Mp: 61–63 °C. Rf = 0.10 (25% MeOH in
ethyl acetate with 2% NEt3). 1H NMR (CDCl3, 500 MHz, ppm):
d = 1.22 (br, 14H), 1.38 (br, 2H), 1.55 (br, 2H), 2.11–2.21 (m,
10H), 3.19–3.26 (m, 1H), 3.62–3.67 (m, 1H), 4.76 (t, J = 7.5 Hz,
1H), 6.64 (br, 1H), 7.27 (d, J = 6.0 Hz, 2H), 8.42 (dd, J1 = 4.0 Hz,
J2 = 2.0 Hz, 2H). 13C NMR (CDCl3, 125 MHz, ppm): 25.6, 27.4,
29.1, 29.2, 29.3, 29.4, 29.5, 36.4, 45.2, 46.9, 59.7, 71.6, 121.1,
149.4, 152.0, 174.6. ESI-MS: m/z calcd for C21H37N3O2: 364.2959
[M+H]+; observed: 364.2961.
4.2.1. Preparation of 12-bromododecanoyl chloride
12-Bromododecanoic acid (0.30 g, 1.10 mmol) was dissolved in
dry cyclohexane (4.0 mL) by stirring at 45 °C for 5 min. To this
well-stirred solution, one drop of dry pyridine followed by oxalyl
chloride (0.14 mL, 1.65 mmol) were added over 1 min. The reaction
mixture was heated to 50 °C for 30 min. After stirring for an addi-
tional 30 min at room temperature, the reaction mixture was evap-
orated to dryness by purging dry argon gas into the reaction flask
and dried under the high vacuum. The freshly prepared 12-brom-
ododecanoyl chloride was dissolved in 3 mL anhydrous THF and
used directly to the next step without purification.
4.2.3.4. Synthesis of 12-(dimethylamino)-N-(2-hydroxy-2-(pyri-
din-3-yl)ethyl)dodecanamide (KPB-102).
Yield: (37%) as off-
white amorphous solid. Mp: 54–56 °C. Rf = 0.10 (25% MeOH in
ethyl acetate with 2% NEt3). 1H NMR (CDCl3, 500 MHz, ppm):
d = 1.27 (br, 14H), 1.44 (br, 2H), 1.61 (t, J = 6.5 Hz, 2H), 2.17–2.27
(m, 10H), 3.31–3.36 (m, 1H), 3.67–3.71 (m, 1H), 4.86 (t,
J = 4.5 Hz, 1H), 6.42 (br, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.74 (d,
4.2.2. Coupling of acyl chloride with amines
To a well-stirred ice-cooled solution of the corresponding amine
(1.00 mmol) in THF (5.0 mL) and 50% aqueous solution of sodium
acetate (5.0 mL), the freshly prepared 12-bromododecanoyl

K. P. Bhabak et al. / Bioorg. Med. Chem. 21 (2013) 874–882
881
J = 8.0 Hz, 1H), 8.48 (s, 1H), 8.55 (s, 1H). 13C NMR (CDCl3, 125 MHz,
ppm): 25.6, 27.3, 29.1, 29.2, 29.3, 29.4, 30.3, 36.5, 45.1, 47.4, 59.7,
71.2, 123.4, 133.8, 138.0, 147.7, 148.8, 174.8. ESI-MS: m/z calcd for
the volume of DMSO was maintained less than 5% (v/v). The mix-
ture was incubated for 2 h at 37 °C and the enzymatic reaction was
quenched by adding 200 lL of chloroform/methanol (2:1) mixture.
The mixture was vortexed thoroughly and centrifuged at
10,000 rpm for 2 min. to separate the organic layer. Fifty microli-
ters of the organic layer was transferred into new sets of vials
and the percentage hydrolysis was determined as mentioned for
the aCDase activities.
C
21H37N3O2: 364.2959 [M+H]+; observed: 364.2962.
4.2.3.5. Synthesis of (2S,3R)-3-hydroxy-3-(4-nitrophenyl)-2-(tet-
radecanamido)propanoic acid (KPB-105). To a solution of B-
13 (0.20 g, 0.47 mmol) in CH2Cl2 (1.0 mL) and 2,2,6,6-tetramethyl
piperidine-N-oxide (TEMPO, 1.0 mol %, 0.7 mg in CH2Cl2), 0.9 mL
of saturated solution of sodium bicarbonate with 5.3 mg of KBr
and 10.0 mg of tetrabutylammonium bromide hydrate were added.
The mixture was cooled to 0 °C with external ice-bath and 1.5 mL
of NaOCl (5% solution), 0.8 mL of saturated sodium bicarbonate
solution and 1.5 mL of brine solution was added. The reaction mix-
ture was stirred at 0 °C for 30 min. The solution was diluted with
CH2Cl2, extracted with aqueous solution and the combined organic
layer was dried over anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure and the crude product was
purified by silica gel column chromatography using cyclohexane/
ethyl acetate and ethyl acetate/methanol mixtures, respectively.
The solvent was evaporated to afford the pure product as yellowish
semi-solid. Yield: 76 mg (37%). Rf = 0.1 (25% MeOH in ethyl ace-
tate). 1H NMR (CD3OD, 500 MHz, ppm): d = 0.92 (t, J = 7.0 Hz, 3H),
1.05 (br, 2H), 1.31 (br, 20H), 2.14 (m, 2H), 4.74 (br, 1H), 5.49 (br,
1H), 7.68 (d, J = 8.6 Hz, 2H), 8.17 (d, J = 7.8 Hz, 2H). 13C NMR
(CD3OD, 125 MHz, ppm): 14.5, 23.8, 26.9, 30.2, 30.5, 30.6, 30.8,
4.2.3.8. Apoptosis assay (DNA fragmentation).
Apoptosis
was detected by measuring the appearance of cytoplasmic his-
tone-associated DNA fragments by photometric enzyme-linked
immunoassay (Cell Death Detection ELISA Plus, Roche Diagnostics).
MDA-MB-231 cells were seeded in 96 well plates for 24 h and then
treated with the indicated concentrations of the compounds in
Dulbecco’s modified Eagle medium (DMEM) for 24 h. The prepara-
tion of cell lysates and the assay procedures were performed
according to the manufacturer’s protocol.
4.2.3.9. Cell viability assay.
Cell vitality was detected by mea-
suring the fluorescence of the AlamarBlueÒ reagent (AlamarBlue
reagent, Invitrogen). MDA-MB-231 cells were seeded in 96 well
plates for 24 h and then treated with different concentrations of
the compounds in Dulbecco’s modified Eagle medium (DMEM)
for 48 or 72 h. AlamarBlue reagent was added and fluorescence
was monitored after 2 and 4 h, according to the manufacturer’s
protocol.
33.1, 37.2, 60.6, 74.2, 124.0, 128.5, 148.5, 151.5, 175.7. ½a D25
ꢂ
+9.1
(c 0.01 in MeOH). ESI-MS: m/z calcd for C23H36N2O6: 435.2501
[MꢁH]ꢁ; observed: 435.2493.
4.2.3.10. Determination of cellular ceramide content.
Cells
(MDA-MB-231) upon the administration of different ceramidase
inhibitors were collected, transferred into siliconized glass tubes
and dissolved in 1 mL of methanol. Different ceramides in the sam-
ples were then extracted by a modified Bligh & Dyer extraction
method.40 As an internal standard, 20 pmol of C17-ceramide in
methanol was added, followed by the addition of 1 mL each of
chloroform, aqua dest and methanol. After 2 min of intensive stir-
ring, 1 mL chloroform was added. Subsequently, the sample was
stirred again for 2 min and 1 mL of aqua dest was added. The mix-
ture was intensely vortexed and centrifuged for 5 min (800ꢃg). The
lower organic phase was transferred into a siliconized glas tube
and the aqueous phase was further extracted twice with 1 mL of
chloroform. Solvent in the combined organic phase was evaporated
using a Speed Vac SC201 ARC vacuum system (Thermo Fischer Sci-
entific, Dreieich, Germany) and the dried lipids were re-dissolved
4.2.3.6. Acid ceramidase (aCDase) assay.
The acid cerami-
dase assay was performed in sodium acetate buffer (200 mM, pH
4.5) with 200 mM sodium chloride and 0.1% Triton-X100. The stock
solutions of the fluorescent substrate and all the inhibitors were
prepared in DMSO. Human recombinant acid ceramidase (aCDase)
was used as the enzyme source for the cleavage of the fluorescent
substrate. The total volume of the assay mixture was 100
final concentration of the substrate and inhibitors were 15 and
80 M, respectively and the total protein concentration in the as-
say mixture was 60 g/mL. In all the assay mixtures the volume
lL. The
l
l
of DMSO was maintained less than 5% (v/v). The assay mixture
was incubated for 8 h at 37 °C. The enzymatic reaction was
quenched by adding 200 lL of chloroform/methanol (2:1) mixture.
The assay mixture was vortexed well and centrifuged to separate
the organic and aqueous layers. Fifty microliters of the organic
layer was transferred into new sets of vials and the total amount
was spotted on non-fluorescent silica gel TLC plates pre-coated
on aluminum sheets (mobile phase: cyclohexane/ethyl acetate/
acetic acid = 40:60:2). The fluorescent spots of the cleaved and
uncleaved substrate on the TLC plates were detected by Fluores-
cent Imaging System (Kodac Image Station 4000 MM PRO) using
430 and 550 nm as excitation and emission wavelengths for the
NBD-linked substrate. The percentage ceramidase activity and
the inhibition were determined by the quantification of fluorescent
intensity of the cleaved and uncleaved substrates.
in 200 lL of methanol by rigorous vortexing and sonicating on
ice. Sample analysis was performed using rapid resolution liquid
chromatography/mass spectrometry (LC–MS). The LC equipment
consisted of an Agilent 1200 Series binary pump, degasser and
autosampler (Agilent Technologies, Waldbronn, Germany). A quad-
rupole/time-of flight (QTOF) 6530 mass spectrometer equipped
with Jet-Stream Technology operating in the positive Electrospray
Ionization (ESI) mode was used for the detection (Agilent Technol-
ogies, Waldbronn, Germany). Highly pure nitrogen gas for the mass
spectrometer was produced by nitrogen generator (Parker Balston,
Maidstone, UK). Chromatographic separations were obtained using
a ZORBAX Eclipse XDB-C18 (C18, 4.6 ꢃ 50 mm, 1.8
size, 80 Å pore size, Agilent Technologies, Waldbronn, Germany).
The injection volume per sample was 10 L. An isocratic solvent
lm particle
4.2.3.7. Neutral ceramidase (nCDase) assay.
Human recom-
binant neutral ceramidase (nCDase) (R&D Systems GmbH) was
used as the enzyme source for the cleavage of the fluorescent sub-
strates. The neutral ceramidase assay was performed in sodium
acetate buffer (200 mM, pH 7.0) with 200 mM sodium chloride
and 0.1% Triton-X100. The stock solutions of the fluorescent sub-
strates were prepared in DMSO. The total volume of the assay mix-
l
system consisting of acetonitrile/2-propanol 3:2 with 1% formic
acid with a flow rate of 1 mL/min over 15 min was used. For mass
spectrometric measurements the following ion source conditions
and gas settings for positive LC–MS/MS were adjusted: sheat gas
temperature = 400 °C, sheat gas flow = 9 L/min, nebulizer
pressure = 30 psig, drying gas temperature = 350 °C, drying gas
flow = 8 L/min, capillary voltage = 2000 V, fragmentor volt-
age = 355 V, nozzle voltage = 2000 V. All ceramides gave the same
ture was 100
inhibitors were 15 and 80
of recombinant enzyme was 25 ng/mL. In all the assay mixtures
lL. The final concentration of the substrates and
l
M, respectively. The final concentration

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K. P. Bhabak et al. / Bioorg. Med. Chem. 21 (2013) 874–882
14. Liu, X.; Cheng, J. C.; Turner, L. S.; Elojeimy, S.; Beckham, T. H.; Bielawska, A.;
Keane, T. E.; Hannun, Y. A.; Norris, J. S. Expert Opin. Ther. Targets 2009, 13, 1449.
15. Musumarra, G.; Barresi, V.; Condorelli, D. F.; Scire, S. Biol. Chem. 2003, 384, 321.
16. Saad, A. F.; Meacham, W. D.; Bai, A.; Anelli, V.; Elojeimy, S.; Mahdy, A. E.;
Turner, L. S.; Cheng, J.; Bielawska, A.; Bielawski, J.; Keane, T. E.; Obeid, L. M.;
Hannun, Y. A.; Norris, J. S.; Liu, X. Cancer Biol. Ther. 2007, 6, 1455.
17. Bielawska, A.; Linardic, C. M.; Hannun, Y. A. J. Biol. Chem. 1992, 267, 18493.
18. Bielawska, A.; Greenberg, M. S.; Perry, D.; Jayadev, S.; Shayman, J. A.; McKay, C.;
Hannun, Y. A. J. Biol. Chem. 1996, 271, 12646.
fragment ion of m/z 264.27 at different retention times, depending
on their chain length. Quantification was performed using Mass
Hunter Software. Calibration curves of reference ceramides were
performed from 1 to 100 pmol and were constructed by linear fit-
ting, using the least squares linear regression calculation. The
resulting slope of the calibration curve was used to calculate the
concentration of the respective analytes in the unknown samples.
19. Raisova, M.; Goltz, G.; Bektas, M.; Bielawska, A.; Riebeling, C.; Hossini, A. M.;
Eberle, J.; Hannun, Y. A.; Orfanos, C. E.; Geilen, C. C. FEBS Lett. 2002, 516, 47.
20. Ueda, N.; Yamanaka, K.; Yamamoto, S. J. Biol. Chem. 2001, 276, 35552.
21. Grijalvo, S.; Bedia, C.; Triola, G.; Casas, J.; Llebaria, A.; Teixido, J.; Rabal, O.;
Levade, T.; Delgado, A.; Fabrias, G. Chem. Phys. Lipids 2006, 144, 69.
22. Bedia, C.; Canals, D.; Matabosch, X.; Harrak, Y.; Casas, J.; Llebaria, A.; Delgado,
A.; Fabrias, G. Chem. Phys. Lipids 2008, 156, 33.
23. Bielawska, A.; Bielawski, J.; Szulc, Z. M.; Mayroo, N.; Liu, X.; Bai, A.; Elojeimy, S.;
Rembiesa, B.; Pierce, J.; Norris, J. S.; Hannun, Y. A. Bioorg. Med. Chem. 2008, 16,
1032.
Acknowledgements
We thank H. Schulze and K. Sandhoff (Bonn) for a donation of
aCDase-transformed baculovirus. The authors are grateful for gen-
erous funding by the DFG (SPP 1267). K.P.B. thanks the Alexander
von Humboldt Foundation for a research fellowship.
24. Szulc, Z. M.; Mayroo, N.; Bai, A.; Bielawski, J.; Liu, X.; Norris, J. S.; Hannun, Y. A.;
Bielawska, A. Bioorg. Med. Chem. 2008, 16, 1015.
25. Bai, A.; Szulc, Z. M.; Bielawski, J.; Mayroo, N.; Liu, X.; Norris, J.; Hannun, Y. A.;
Bielawska, A. Bioorg. Med. Chem. 2009, 17, 1840.
26. Dagan, A.; Wang, C.; Fibach, E.; Gatt, S. Biochim. Biophys. Acta 2003, 1633, 161.
27. Labaied, M.; Dagan, A.; Dellinger, M.; Geze, M.; Egee, S.; Thomas, S. L.; Wang, C.;
Gatt, S.; Grellier, P. Malar. J. 2004, 3, 49.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.014.
28. Elojeimy, S.; Holman, D. H.; Liu, X.; El-Zawahry, A.; Villani, M.; Cheng, J. C.;
Mahdy, A.; Zeidan, Y.; Bielwaska, A.; Hannun, Y. A.; Norris, J. S. FEBS Lett. 2006,
580, 4751.
29. Zeidan, Y. H.; Pettus, B. J.; Elojeimy, S.; Taha, T.; Obeid, L. M.; Kawamori, T.;
Norris, J. S.; Hannun, Y. A. J. Biol. Chem. 2006, 281, 24695.
References and notes
1. Kolter, T.; Sandhoff, K. Angew. Chem., Int. Ed. 1999, 38, 1532.
2. Delgado, A.; Casas, J.; Llebaria, A.; Abad, J. L.; Fabrias, G. Biochim. Biophys. Acta
2006, 1758, 1957.
3. Canals, D.; Perry, D. M.; Jenkins, R. W.; Hannun, Y. A. Br. J. Pharmacol. 2011, 163,
694.
4. Pettus, B. J.; Chalfant, C. E.; Hannun, Y. A. Biochim. Biophys. Acta 2002, 1585, 114.
5. Maceyka, M.; Payne, S. G.; Milstien, S.; Spiegel, S. BBA-Mol. Cell. Biol. L 2002,
1585, 193.
6. Taha, T. A.; Mullen, T. D.; Obeid, L. M. Biochim. Biophys. Acta 2006, 1758, 2027.
7. Li, C. M.; Park, J. H.; He, X.; Levy, B.; Chen, F.; Arai, K.; Adler, D. A.; Disteche, C.
M.; Koch, J.; Sandhoff, K.; Schuchman, E. H. Genomics 1999, 62, 223.
8. Liu, B.; Hassler, D. F.; Smith, G. K.; Weaver, K.; Hannun, Y. A. J. Biol. Chem. 1998,
273, 34472.
9. El Bawab, S.; Bielawska, A.; Hannun, Y. A. J. Biol. Chem. 1999, 274, 27948.
10. El Bawab, S.; Roddy, P.; Qian, T.; Bielawska, A.; Lemasters, J. J.; Hannun, Y. A. J.
Biol. Chem. 2000, 275, 21508.
11. Tani, M.; Okino, N.; Mitsutake, S.; Ito, M. J. Biochem. 1999, 125, 746.
12. Koch, J.; Gartner, S.; Li, C. M.; Quintern, L. E.; Bernardo, K.; Levran, O.; Schnabel,
D.; Desnick, R. J.; Schuchman, E. H.; Sandhoff, K. J. Biol. Chem. 1996, 271, 33110.
13. Ehlert, K.; Frosch, M.; Fehse, N.; Zander, A.; Roth, J.; Vormoor, J. Pediatr.
Rheumatol. Online J. 2007, 5, 15.
30. Kornhuber, J.; Tripal, P.; Reichel, M.; Muhle, C.; Rhein, C.; Muehlbacher, M.;
Groemer, T. W.; Gulbins, E. Cell Physiol. Biochem. 2010, 26, 9.
31. Pappu, A.; Hostetler, K. Y. Biochem. Pharmacol. 1984, 33, 1639.
32. Kolter, T.; Sandhoff, K. Annu. Rev. Cell Dev. Biol. 2005, 21, 81.
33. Holman, D. H.; Turner, L. S.; El-Zawahry, A.; Elojeimy, S.; Liu, X.; Bielawski, J.;
Szulc, Z. M.; Norris, K.; Zeidan, Y. H.; Hannun, Y. A.; Bielawska, A.; Norris, J. S.
Cancer Chemother. Pharmacol. 2008, 61, 231.
34. Liu, X.; Elojeimy, S.; El-Zawahry, A. M.; Holman, D. H.; Bielawska, A.; Bielawski,
J.; Rubinchik, S.; Guo, G. W.; Dong, J. Y.; Keane, T.; Hannun, Y. A.; Tavassoli, M.;
Norris, J. S. Mol. Ther. 2006, 14, 637.
35. Elojeimy, S.; Liu, X.; McKillop, J. C.; El-Zawahry, A. M.; Holman, D. H.; Cheng, J.
Y.; Meacham, W. D.; Mahdy, A. E.; Saad, A. F.; Turner, L. S.; Cheng, J.; Day, T. A.;
Dong, J. Y.; Bielawska, A.; Hannun, Y. A.; Norris, J. S. Mol. Ther. 2007, 15, 1259.
36. Bhabak, K. P.; Arenz, C. Bioorg. Med. Chem. 2012, 20, 6162.
37. Kolter, T.; Van Echten-Deckert, G.; Sandhoff, K. Tetrahedron 1994, 50, 13425.
38. Proksch, D.; Klein, J. J.; Arenz, C. J. Lipids 2011, 2011, 971618.
39. Bhabak, K. P.; Proksch, D.; Redmer, S.; Arenz, C. Bioorg. Med. Chem. 2012, 20,
6154.
40. Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Phys. 1959, 37, 911.