Cytotoxicity and acid ceramidase inhibitory activity of 2-substituted aminoethanol amides

Article abstract of DOI:10.1016/j.chemphyslip.2008.07.012

The acid ceramidase (AC) inhibitory activity of octanoylamides, p-tert-butylbenzamides and pivaloylamides of several 2-substituted aminoethanols is reported. All the aminoethanol amides bearing a hexadecyl substituent (C16), as well as (S)-N-(1-(hexadecylthio)-3-hydroxypropan-2-yl)pivaloylamide (SC16-tb) were inhibitory in cell lysates overexpressing AC, while all other compounds were not inhibitors. Kinetic experiments with (R,E)-N-(1-hydroxyoctadec-3-en-2-yl)pivaloylamide (E-tb) and SC16-tb showed that inhibition was competitive, with K_i values of 34 and 94.0 μM, respectively. None of the compounds inhibited neutral ceramidase. Compounds E-tb and E-c7 (the octanoylamide of the unsaturated base E), which elicited a dose-response inhibition with IC₅₀ values around 15 μM, were the only AC inhibitors in intact cells. Both compounds were toxic to A549 cells with LD₅₀ values nearly 40 μM. Flow cytometry studies with E-tb evidenced that this compound induced a concentration-dependent cell cycle arrest at G(1) and a 20-25% apoptosis/late apoptosis/necrosis after a 24-h incubation at 50 μM. In agreement with its activity as acidic ceramidase inhibitor, this effect was accompanied with an increase in the amounts of C14, C16 and C18 ceramides (LC-MS analyses), which suggested that these lipids may be responsible for the cytotoxic activity of E-tb.

Full text of DOI:10.1016/j.chemphyslip.2008.07.012

Chemistry and Physics of Lipids 156 (2008) 33–40
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Cytotoxicity and acid ceramidase inhibitory activity of 2-substituted
aminoethanol amides
Carmen Bediaa,1, Daniel Canalsa,1, Xavier Mataboscha, Youssef Harraka, Josefina Casasa,
Amadeu Llebariaa, Antonio Delgadoa,b, Gemma Fabriása,∗
a Research Unit on BioActive Molecules (RUBAM), Departament de Química Biomèdica, Institut de Química Avanc¸ ada de Catalunya IQAC-C.S.I.C.,
Jordi Girona 18-26, E-08034 Barcelona, Spain
b Universitat de Barcelona, Facultat de Farmàcia, Unitat de Química Farmacèutica (Unitat Associada al CSIC), Avgda. Juan XXIII, s/n, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The acid ceramidase (AC) inhibitory activity of octanoylamides, p-tert-butylbenzamides and pivaloy-
lamides of several 2-substituted aminoethanols is reported. All the aminoethanol amides bearing a
hexadecyl substituent (C16), as well as (S)-N-(1-(hexadecylthio)-3-hydroxypropan-2-yl)pivaloylamide
(SC16-tb) were inhibitory in cell lysates overexpressing AC, while all other compounds were not inhibitors.
Kinetic experiments with (R,E)-N-(1-hydroxyoctadec-3-en-2-yl)pivaloylamide (E-tb) and SC16-tb showed
that inhibition was competitive, with Ki values of 34 and 94.0 ␮M, respectively. None of the compounds
inhibited neutral ceramidase. Compounds E-tb and E-c7 (the octanoylamide of the unsaturated base E),
which elicited a dose–response inhibition with IC50 values around 15 ␮M, were the only AC inhibitors
in intact cells. Both compounds were toxic to A549 cells with LD50 values nearly 40 ␮M. Flow cytometry
studies with E-tb evidenced that this compound induced a concentration-dependent cell cycle arrest at
G(1) and a 20–25% apoptosis/late apoptosis/necrosis after a 24-h incubation at 50 ␮M. In agreement with
its activity as acidic ceramidase inhibitor, this effect was accompanied with an increase in the amounts of
C14, C16 and C18 ceramides (LC–MS analyses), which suggested that these lipids may be responsible for
the cytotoxic activity of E-tb.
Received 29 April 2008
Received in revised form 9 July 2008
Accepted 23 July 2008
Available online 8 August 2008
Keywords:
Sphingolipids
Ceramide
Farber disease
Cancer
Apoptosis
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
tions in the cell depending on the local pH of its subcellular
location.
Acid ceramidase (AC), a key regulatory enzyme involved in sph-
ingolipid metabolism, catalyzes the degradation of ceramide into
sphingosine and free fatty acids within the lysosomes. Mutations
in the AC gene ASAH1) result in Farber disease or lipogranulo-
matosis, a fatal human genetic disorder (Sugita et al., 1972; Koch
et al., 1996). Importantly, AC not only hydrolyzes ceramide into
sphingosine, but can also synthesize ceramide from sphingosine
and free fatty acids in vitro and in situ (Okino et al., 2003). This
enzymatic activity occurs at a distinct pH from the hydrolysis
reaction, suggesting that the enzyme may have different func-
The involvement of AC in cell death in both physiological and
pathological conditions has been extensively documented (Park
and Schuchman, 2006). Thus, AC is required for TNF␣-induced
PGE2 production (Zeidan et al., 2006), it may prevent insulin resis-
tant type II diabetes promoted by free fatty acids (Chavez et al.,
2005), and its inactivation provokes death in alveolar macrophages
through a decrease in ERK and Akt activities (Monick et al., 2004).
Furthermore, AC has been recently identified as a novel factor
required for early embryo survival (Eliyahu et al., 2007).
Different studies have shown that AC is aberrantly expressed
in several human diseased cells, such as in Alzheimer’s disease in
brain (Huang et al., 2004) and a number of cancers (Seelan et al.,
2000; Liu et al., 2006a,b; Saad et al., 2007), and that cells over-
expressing AC are more resistant to pharmacological induction of
programmed cell death. For instance, overexpression of AC pro-
tects from caspase-dependent TNF␣ (Strelow et al., 2000) and Fas
(Elojeimy et al., 2007) induced cell death, as well as from caspase-
independent death (Thon et al., 2005, 2006). Human glyoma cells
with functional p53 are resistant to gamma-radiation in part by
induction of AC expression and are sensitized to gamma-radiation
Abbreviations: AC, acid ceramidase; BSA, bovine serum albumin; EDTA,
sodium ethylendiaminetetracetate; FACS, fluorescence-activated cell sorting; FBS,
fetal bovine serum; HRMS, high-resolution mass spectrometry; IR, infrared;
MTT, 3-(4.5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide; NMR, nuclear
magnetic resonance; PBS, phosphate-buffered saline; TOF, time-of-flight; UPLC,
ultra-performance liquid chromatography.
∗
Corresponding author. Tel.: +34 93 4006115; fax: +34 93 2045904.
E-mail address: gfdqob@iiqab.csic.es (G. Fabriás).
1
Both authors contributed equally to this work.
0009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2008.07.012

34
C. Bedia et al. / Chemistry and Physics of Lipids 156 (2008) 33–40
by AC inhibitors (Hara et al., 2004). The use of AC inhibitors as
pharmacological tools has strengthened the notion that AC may
be a useful cancer drug target (Liu et al., 2008). For example, the
anti-proliferative and apoptotic responses induced by anandamide
(Mimeault et al., 2003a,b) or androgen ablation (Eto et al., 2003)
in human prostatic cancer cell lines LNCaP, DU145, and PC3 are
potentiated by the AC inhibitor, N-oleoylethanolamine. It has also
been shown that ceramide-mediated tumor-induced dendritic cell
apoptosis (Kanto et al., 2001) is enhanced by AC inhibitors. Using a
bioinformatic approach, AC was found to be among the most impor-
tant genes for melanoma therapy and diagnosis (Musumarra et al.,
2003).
In agreement with these experimental proofs, AC inhibitors
have been successfully used to slow the growth of cancer cells,
alone or in combination with other established anticancer treat-
ments. The AC inhibitor B13 prevents tumor growth of aggressive
human colon cancer cell lines metastatic to the liver (Selzner et
al., 2001), induces apoptosis in human melanoma cells (Raisova et
al., 2002) and sensitizes prostate tumors to the effects of radiation
(Samsel et al., 2004). Compound LCL204, a lysosomotropic analog
of B13 that increases degradation of AC in a cathepsin-dependent
manner, induces apoptosis in prostate cancer cells (Holman et
al., 2008). A similar effect has been reported for another lysoso-
motropic agent, desipramine (Elojeimy et al., 2006). The cytotoxic
activity of the AC inhibitor AD2646 towards Jurkat leukemic cells
has been demonstrated (Granot et al., 2006). Pharmacological inhi-
bition or small interfering RNA targeting AC sensitizes hepatoma
cells to chemotherapy (Morales et al., 2006), HNSCC cell lines to
Fas-induced apoptosis (Elojeimy et al., 2007), and a number of neu-
roectodermal tumor cell lines to fenretinide cytotoxicity in hypoxia
(Batra et al., 2004). Finally, the anti-tumor agent apoptin activates
apoptosis in several human cancers with accumulation of ceramide
through down-regulation of AC at the protein level (Liu et al.,
2006a,b).
Fig. 1. Compounds investigated in this study.
(ppm) relative to the singlet at 7.24 ppm of CDCl3 for 1H and in
ppm relative to the center line of a triplet at 77.0 ppm of CDCl3 for
13C. ESI/HRMS spectra were recorded on a Waters LCT Premier Mass
spectrometer.
2.2. Acylation of Z-NH2 and E-NH2
A solution of the starting amine (Z)-NH2 or (E)-NH2 (0.1 mmol)
in 5 ml of a 1:1 mixture of tetrahydrofuran and 50% aq. NaOAc was
cooled to 0 ◦C and stirred vigorously at this temperature for 15 min.
The corresponding acyl chloride (0.19 mmol) was next added drop-
wise over the reaction mixture, which was allowed to warm to 25 ◦C
and stirred for additional 20 h. The mixture was extracted with
EtOAc (3× 10 ml) and the combined organic phases were washed
with 1N NaOH and brine, dried over MgSO4, filtered and evapo-
rated. The resulting solid was purified by flash chromatography to
give the final amide.
2.2.1. (R,Z)-4-tert-Butyl-N-(1-hydroxyoctadec-3-en-2-yl)
benzamide (Z-tbph)
In a previous article we reported on the modest activity of
several aminoethanol 2-oxooctanamides and oleamides as AC
inhibitors (Grijalvo et al., 2006), which turned out to exhibit
low toxicity in A549 human lung adenocarcinoma cells (the low-
est LD50/24 h were around 80 ␮M for the 2-oxooctanamide of
base SC16 and the 2-oxooctanamide and oleoylamide of base
SAr).
In connection with our ongoing projects aimed at the discovery
of novel AC inhibitors by the screening of combinatorial libraries
(Bedia et al., 2005; Grijalvo et al., 2006) in this article we describe
the synthesis and biological activities of several amides of dif-
ferently C2-substitued aminoethanols. Substituents include two
C16-unsaturated chains (coded as Z- and E-) and their saturated
counterpart (C16-), a saturated thioether (SC16-) and two aromatic-
containing chains (SAr- and OAr-). The amino group was acylated
with octanoyl (-c7), p-tert-butylphenyl (-tbph) and tert-butyl (-tb)
moieties. Along the paper, compounds are abbreviated with a code
resulting from the combination of the side chain and acyl identifiers
(Fig. 1).
39 mg (88% from Z-NH2); 1H NMR (400 MHz): 7.68 (AAꢀ, 2H,
J = 8.0 Hz; 7.41 (BBꢀ, 2H, J = 8.0 Hz), 6.32 (b, 1H, amide), 5.65
(m, 1H), 5.41 (m, 1H), 4.97 (m, 1H), 3.73–3.71 (m, 2H), 2.15
(m, 2H), 1.33 (s, 9H), 1.25 (m, 24H), 0.85 (t, 3H); 13C NMR
(100 MHz): 168.1, 155.3, 135.7, 131.1, 130.0, 126.8, 125.5, 125.2,
66.7, 50.4, 34.9, 31.9, 31.1, 29.6–29.3, 28.0, 22.7, 14.1.; [␣]D +125.2
(c = 0.5, CHCl3); HRMS: calculated for C29H49NO2: 443.3763; found:
444.3836.
2.2.2. (R,E)-4-tert-Butyl-N-(1-hydroxyoctadec-3-en-2-yl)
benzamide (E-tbph)
42 mg (84% from E-NH2); 1H NMR (500 MHz): 7.71 (AAꢀ, 2H,
J = 8.0 Hz), 7.41 (BBꢀ, 2H, J = 8.0 Hz), 6.44 (b, 1H, amide), 5.73 (m,
1H), 5.47 (dd, 1H, J = 15.5, Jꢀ = 6.0), 4.68 (m, 1H), 3.76–3.71 (m,
2H), 2.03 (m, 2H), 1.31 (s, 9H), 1.23 (m, 24H), 0.86 (t, 3H); 13C
NMR (125 MHz): 167.7, 155.2, 134.4, 131.3, 126.8, 126.0, 125.5,
65.9, 53.8, 34.9, 32.4, 31.8, 31.1, 29.6–29.0, 22.6, 14.2; [␣]D +28.1
(c = 0.5, CHCl3); HRMS: calculated for C29H49NO2: 443.3763; found:
444.3838.
2. Experimental procedures
2.2.3. (R,E)-N-(1-Hydroxyoctadec-3-en-2-yl)octanamide (E-c7)
38 mg (92% from E-NH2); 1H NMR (300 MHz): 5.83 (b, 1H.
amide), 5.65 (m, 1H), 5.37 (dd, 1H, J = 16.5, Jꢀ = 6.3), 4.47 (m, 1H),
3.68–3.56 (m, 2H), 2.20 (t, 2H, J = Jꢀ = 7.2), 2.05 (m, 2H), 1.61 (m, 2H),
1.29–1.22 (m, 32H), 0.87 (t, 3H), 0.85 (t, 3H); 13C NMR (75 MHz):
173.8, 134.2, 126.0, 91.8, 66.0, 53.5, 36.8, 32.4, 31.8, 31.6, 29.6–28.9,
25.7, 22.6, 14.1, 14.0; [␣]D −6.6 (c = 0.5, CHCl3); HRMS: calculated
for C26H51NO2: 409.3920; found: 410.4009.
2.1. Chemistry
2.1.1. General
Solvents were distilled prior to use and dried by standard meth-
ods. Melting points are uncorrected. FT-IR spectra are reported in
cm−1 1H and 13C NMR spectra were obtained in CDCl3 solutions
.
at 500, 400 or 300 MHz (for 1H) and 125, 75 or 50 MHz (for 13C).
Chemical shifts were reported in delta ı units, parts per million

C. Bedia et al. / Chemistry and Physics of Lipids 156 (2008) 33–40
35
2.3. Catalytic hydrogenation of Z and E amides
Cells treated with 0.1% ethanol were used as controls (Villorbina et
al., 2006).
A solution of the starting amide (0.15 mmol) in 5 ml of CH3OH
was stirred under H2 (1 atm) in the presence of 5 mg of 10% Pd/C
for 24 h. The reaction mixture was filtered through a pad of Celite®
and evaporated in vacuo to give the crude amide as oil. Purification
by flash chromatography (10% hexanes/EtOAc) afforded the final
compound.
2.4.4. Apoptosis
After trypsinization, cells were stained with Alexa Fluor 488
Annexin V/propidium iodide staining kit (Molecular Probes, Inc.,
OR), and apoptosis was evaluated by FACS (Villorbina et al., 2006).
Liquid chromatography–mass spectrometry: A549 cells
(5 × 105 cells/ml) were incubated with E-tb (50 ␮M in 1 ml of
medium in six-well plates) for 24 h. Next, cells were collected
by brief trypsinization, washed in PBS and transferred to glass
vials. Sphingolipid extracts, fortified with internal standards, were
prepared as described (Merrill et al., 2005) and analysed by liquid
chromatography–mass spectrometry using a Waters Aquity UPLC
system connected to a Waters LCT Premier orthogonal accelerated
time-of-flight mass spectrometer (Waters, Millford, MA), oper-
ated in positive electrospray ionization mode in the conditions
previously reported (Munoz-Olaya et al., 2008).
2.3.1. (R)-N-(1-Hydroxyoctadecan-2-yl)pivalamide (C16-tb)
47 mg (white solid, 84% from Z-tb); 1H NMR (400 MHz): 5.68 (d,
1H, J = 8.0); 4.12 (d, 2H, J = 8.0); 4.02 (t, 1H, J = Jꢀ = 8.0); 1.46 (m, 2H),
1.23 (m, 28H), 1.18 (s, 3H); 1.16 (s, 6H); 0.84 (t, 3H, J = Jꢀ = 7.5); 13C
NMR (100 MHz): 178.5, 65.7, 48.4, 38.8, 31.9, 29.6–29.3 (12 C), 27.3
(3C), 25.7, 22.7, 14.1; [␣]D + 23.2 (c = 1.0, CHCl3); HRMS: calculated
for C23H47NO2: 369.3607; found: 370.3680.
2.3.2. (R)-4-tert-Butyl-N-(1-hydroxyoctadecan-2-yl)benzamide
(C16-tbph)
59 mg (89% from Z-tbph); 1H NMR (400 MHz): 7.70 (AAꢀ, 2H,
J = 8.0 Hz), 7.42 (BBꢀ, 2H, J = 8.0 Hz), 6.22 (b, 1H, amide), 4.12 (m,
1H), 3.76 (m, 1H), 3.66 (m, 1H), 1.58 (m, 2H), 1.31 (s, 9H), 1.23 (m,
28H), 0.87 (t, 3H); 13C NMR (100 MHz): 168.2, 155.2, 131.4, 126.8,
125.5, 66.1, 52.4, 34.9, 31.3, 31.1, 29.6–29.3, 26.1, 22.6, 14.1; [␣]D
+14.1 (c = 0.5, CHCl3); HRMS: calculated for C29H51NO2: 445.3920;
found: 446.4018.
2.4.5. Inhibition of ceramidases
The effect of the compounds on AC activity was deter-
mined in 96-well plates as previously reported (Bedia et al.,
2007) using (1ꢀS,2ꢀR)-N-[1-hydroxymethyl-2-hydroxy-4-(2-oxo-
2H-chromen-7-yloxy)butyl]hexadecanoylamide as substrate. The
experiments in vitro were performed using AC10X cell lysates.
Cell pellets were resuspended in 0.25 M sucrose (1 ml/106 cells).
The cell suspension was frozen by immersing the tube in liq-
uid nitrogen and then it was thawed at 37 ◦C (water bath). After
gentle mixing, the freeze–thaw cycle was repeated twice and
the lysate was used as the protein source in the assay, which
was performed in 96-well plates as reported (Bedia et al., 2007)
using 24 ␮l of lysate, 75 ␮l of buffer (20 mM AcOH/AcONa pH
4.5) and 1 ␮l of substrate (1.6 mM in ethanol, final concentra-
tion of 16 ␮M) with or without inhibitor. After 3 h at 37 ◦C, the
reactions were stopped with methanol (50 ␮l/well) and 100 mM
glycine–NaOH buffer pH 10.6 (25 ␮l/well). To each well was then
sequentially added freshly prepared solutions of NaIO4 (25 ␮l,
10 mg/ml) and bovine serum albumin (25 ␮l, 2 mg/ml) in 0.1 M
phosphate buffer, pH 8. After 16 h in the dark, the fluorescence
released was measured at excitation and emission wavelengths of
355 and 460 nm, respectively. For enzyme activity determination,
a calibration line prepared with umbelliferone in a concentration
range of 0–3.2 ␮M was used to correlate the experimental flu-
orescence values with the amount of product released in each
sample.
2.3.3. (R)-N-(1-Hydroxyoctadecan-2-yl)octanamide (C16-c7)
55 mg (90% from E-c7); 1H NMR (400 MHz): 5.59 (b, 1H, amide),
3.90 (m, 1H), 3.67 (m, 1H), 3.54 (m, 1H), 2.18 (m, 2H), 1.64–1.22 (m,
40H), 0.85 (t, 2× 3H); 13C NMR (100 MHz): 174.3, 66.2, 52.0, 36.9,
31.9, 31.7, 31.2, 29.6, 28.9, 26.1, 25.8, 22.6, 22.5, 14.1, 14.0; [␣]D +19.6
(c = 0.5, CHCl3); HRMS: calculated for C26H53NO2: 411.4076; found:
412.4164.
2.4. Biological protocols
2.4.1. Cell biology
Human alveolar epithelial A549 cells (American Type Culture
Collection) and MOHNSEN cells overexpressing AC (AC10X) were
grown in HAM F12 and DMEM, respectively, both supplemented
with glutamine (2 mmol/l), penicillin (100 U/ml), streptomycin
(100/zg/ml) and heat-inactivated fetal calf serum (10%). Both cell
lines were kept at 37 ◦C in 5% CO2/95% air.
2.4.2. Cytotoxicity
Inhibition of the neutral ceramidase activity was determined in
microsomes following our previously reported procedure (Grijalvo
et al., 2006), using 25 mM Tris–HCl buffer pH 7.4, 0.1 mg of micro-
somal protein, 3 nmol each of test compound and fluorogenic
substrate and a 1 h incubation period. Reaction was stopped with
methanol and further processed as above.
The cytotoxicity of all compounds shown in Fig. 1 and that of the
AC inhibitors N-oleoylethanolamine and B13 was investigated. At
the time of the experiments, cells were seeded at a density of 105
cells per well in 96-well plates. Media were renewed after 24 h and
compounds were added to give final concentrations of 10–400 ␮M.
Twenty-four hours later, the number of viable cells was quantified
by measuring the formazan produced from MTT (Villorbina et al.,
2006). All compounds were administered in ethanol and control
experiments were performed with ethanol (0.1%).
3. Results
3.1. Synthesis of compounds
2.4.3. Cell cycle
A549 cells (5 × 105 cells/ml) were incubated with E-tb (6, 12
and 25 ␮M in 1 ml of medium in six-well plates) for 24 h. Next,
cells were collected by brief trypsinization, washed with PBS,
10 mM EDTA at 4 ◦C, resuspended in PBS, 70% ethanol at 4 ◦C,
106 cells/ml and stored at −20 ◦C. On the day of the analysis,
cells were washed with PBS and treated with DNase-free RNase
(0.05 mg/ml) at 37 ◦C for 1 h. Cells were then stained with propid-
ium iodide for 15 min and analyzed with a FACS flow cytometer.
Compounds Z-NH2, E-NH2 (Scheme 1), Z-tb, E-tb and Z-c7 (Fig. 1)
were obtained as described (Mormeneo et al., 2007). Amides con-
taining SC16, SAr, and OAr moieties were obtained as reported
(Grijalvo et al., 2008). The remaining amides were obtained as
outlined in Scheme 1 by acylation of the free amine with the cor-
responding acyl chloride under Schotten-Bauman conditions (THF,
50% aq. NaOAc) followed by catalytic hydrogenation with 10% Pd/C
at atmospheric pressure at room temperature.

36
C. Bedia et al. / Chemistry and Physics of Lipids 156 (2008) 33–40
Scheme 1. Synthesis of amides: (a) acyl chloride, THF/50% aq. NaOAc; (b) H2, 10% Pd/C, 1 atm.
3.2. Inhibition of ceramidases
ture (Table 2). AC inhibition by E-tb and E-c7 in intact cells
was dose-dependent with IC50 values of 13.5 and 15.7 ␮M,
Activity on AC was screened both with cell lysates (in vitro) and
in intact cells using the previously reported fluorogenic procedure
(Bedia et al., 2007) at equimolar concentrations of substrate and
test compound. Transduced Farber cells (MOHNSEN) overexpress-
ing AC (AC10X) were also used as enzyme source. All Z, E, and, to a
lesser extent, C16-derived amides reduced the production of fluo-
rescence from the fluorogenic ceramidase substrate in vitro. Among
the amide moieties, pivaloylamides (tb) were the most active ones
and only pivaloylamide SC16-tb was active among the ether and
thioether amides (Tables 1 and 2).
The type of inhibition was determined with E-tb and SC16-tb as
representative members. Incubation of AC10X cell lysates with dif-
ferent inhibitor doses at varied substrate concentrations revealed
that both compounds were competitive AC inhibitors towards the
substrate with Ki values of 33.9 ␮M (E-tb) and 94.0 ␮M (SC16-tb)
(Fig. 2).
None of the compounds inhibited the neutral ceramidase,
as concluded in experiments carried out in vitro with rat liver
microsomes, which showed that all compounds, at equimolar con-
centrations with respect to the substrate (40 ␮M), were inactive at
inhibiting the production of fluorescence at neutral pH (data not
shown). In the same experiments, sphingosine (40 ␮M), which was
used as a positive control (Usta et al., 2001), produced a 48–62%
inhibition.
respectively.
3.3. Cytotoxicity
Screening for cytotoxicity in A549 human lung adenocarci-
noma cells using the MTT test evidenced that cell viability was
reduced by some molecules (Table 3). The most toxic compounds
(LD50 values below 50 ␮M) were SAr-tbph, pivaloylamides Z-tb, E-
tb and SC16-tb, and octanamides Z-c7 and E-c7. Less toxic molecules
included OAr-tbph and, with lower potency, OAr-c7 and SC16-
tbph. The remaining compounds were not toxic at concentrations
up to 400 ␮M. A good relationship between AC inhibition in situ
(AC10X cells) and cytotoxicity in A549 cells was only observed
for pivaloylamide E-tb and octanamide E-c7 (Fig. 3). These two
amides were not cytotoxic at concentrations up to 150 ␮M in
AC10X cells. In the same experimental conditions, the IC50 values
for N-oleoylethanolamine and B13 were 212 and 81 ␮M, respec-
tively.
Flow cytometry analysis revealed that compound E-tb induced
a
concentration-dependent cell cycle arrest at G(1) (Fig. 4).
Table 2
Activity of 2-substituted 2-aminoethanols as AC inhibitors in intact cells
Only pivaloylamides E-tb and C16-tb, the octanoylamide E-
c7 and the p-tert-butylbenzamide C16-tbph caused a significant
decrease of fluorescence from the substrate in AC10X cell cul-
tb
c7
tbph
S/I = 1a
IC50b, ␮M
S/I = 1a
IC50b, ␮M
S/Ia = 1
Z
E
77 17
60 4.0*
78 1.1*
107 8.5
105 12
99 8.9
nd
13.5
nd
nd
nd
nd
85 26
62 20*
109 16
107 15
104 11
117 19
nd
15.7
nd
nd
nd
nd
101 24
102 18
72 4.7*
102 11
103 19
101 17
C16
SC16
SAr
OAr
Table 1
Activity of 2-substituted 2-aminoethanols as AC inhibitors in vitro
tb
c7
66.5 21#
tbph
The experiments were carried out as described in Section 2, using intact AC10X
cells and the fluorogenic substrate (Bedia et al., 2007). Twenty-four hours before
the experiment, cells were seeded in 96-well plates. The medium was removed and
100 ␮l of the same medium containing 16 ␮M of both substrate and test compound
(substrate only in controls) were added and incubations proceeded for 4 h. After this
time, samples were processed as reported (Bedia et al., 2007).
§
Z
E
34.3 8.2*
25.2 5.0*
76.1 15#
64.7 17
82.5 12
97.2 8.2
66.4 14
§
66.4 15
38.3 17*
74.3 20#
85.5 6.9
87.1 17
C16
SC16
SAr
OAr
71.9 25#
112.5 11
91.0 9.2
88.6 1.1
§
84.8 9.1
a
Data correspond to the % of control at equimolar concentrations of substrate and
test compound (16 ␮M) and are expressed as the mean S.D. of three experiments
with triplicates. Results are normalized to the number of cells, which was similar
in all cases. The asterisk indicates statistical significance with respect to controls at
p ≤ 0.05 (unpaired two-tail t-test).
The experiments were conducted with AC10X cell lysates using a fluorogenic sub-
strate (Bedia et al., 2007) as described in Section 2. Data correspond to the % of
control at equimolar concentrations of substrate and test compound (40 ␮M) and
are expressed as the mean S.D. of two or three experiments with three repli-
cates. Superscript symbols indicate statistical significance with respect to controls
b
In the IC50 determinations, data was obtained in one experiment with triplicates
§
(*p ≤ 0.0005; #p ≤ 0.005, p ꢀ 0.05 unpaired two-tail t-test).
and was analysed with the four parameter logistic equation. nd, not determined.

C. Bedia et al. / Chemistry and Physics of Lipids 156 (2008) 33–40
37
Fig. 3. Correlation between AC inhibition in AC10X intact cells and cytotoxicity in
A549 cells at 24 h (LD50).
Table 3
Cytotoxicity of compounds over A549 cells
tb
41
39
400
50
c7
21
40
400
400
400
187
tbph
Z
E
400
400
400
172
43
C16
SC16
SAr
OAr
400
400
78
A549 cells were seeded at 105 cells per well in 96-well plates. Media were renewed
after 24 h and compounds were added in ethanol solution (0.1% final concentration)
to give final concentrations of 10–400 ␮M. Twenty-four hours later, the number of
viable cells was determined with the MTT test. Data correspond to the LD50 values
(␮M) obtained by regression analysis of the dose–response curves obtained in a
single experiment with triplicates.
3.4. Effect of E-tb on ceramide and sphingosine production
The effect of E-tb on ceramide production was determined
by UPLC/TOF. The results are summarized in Fig. 6. N-
Tetracosanoylamide and, to a lesser extent, the N-palmitoylamide
were the most abundant ceramide species. Treatment with
Fig. 2. Type of inhibition of AC by SC16-tb (A) and E-tb (B). Inhibitory effect
was investigated in AC10X cell lysates with different amounts of inhibitor at var-
ied substrate concentrations. Line equations: (A) y = 1.8438x + 173; R2 = 0.98; (B)
y = 2.6016x + 88,167; R2 = 0.96. Ki: (A) 94 ␮M; (B) 34 ␮M. Data correspond to the
mean S.D. of three replicates.
E-tb caused
a time-course increase of N-acylated ceramides
containing N-tetradecanoyl, N-hexadecanoyl and N-octadecanoyl
moieties. N-Tetradecanoylceramide levels raised up to three-
fold after 12 h with respect to levels at 30 min, whereas the
C16 and C18 amides increased around twofold. Conversely,
ceramides with N-acyl moieties of eicosanoic, docosanoic and
tetracosanoic acids did not increase after E-tb treatment up
to 12 h. Amounts of the respective dihydroceramide species
remained low and unchanged throughout the monitored period
(data not shown). Finally, levels of sphingosine were not
This compound increased the number of dead cells at con-
centrations around the LD50 (50 ␮M) (Fig. 5). In addition
to necrosis (ca. 30%), E-tb produced a 13–15% of propidium
iodide (+)/annexin V (+) cells (necrosis/late apoptosis) and a
7–10% of propidium iodide (−)/annexin V (+) cells (apoptosis)
(Fig. 5).
Fig. 4. Effect of E-tb on cell cycle. 5 × 105 cells/ml were treated with the compound at 0 (controls) 6, 12 and 25 ␮M for 24 h. Cells were stained with propidium iodide/RNase
A solution for 30 min and then analyzed by flow cytometry using a FACScan flow cytometer. Cell number (arbitrary units) was plotted against DNA content (fluorescence). (A)
Representative cell cycle profiles obtained after 24 h treatment with 25 ␮M E-tb. (B) Histogram representing the mean percentage of cells in each cell cycle phase after 24 h
treatment with different concentrations of E-tb. Data are derived from one experiment with duplicates that was repeated twice with similar results. The standard deviation
values were always lower than the 8% of the mean.

38
C. Bedia et al. / Chemistry and Physics of Lipids 156 (2008) 33–40
Fig. 5. Apoptosis induction. A549 cells were treated with 50 ␮M of E-tb for 24 h and evaluated by annexin V and PI double staining. FITC and PI fluorescence intensities were
recorded through 520–530 and 575 nm filters, respectively. (A) Lower left quadrant shows viable cells; lower right quadrant shows viable cells in early stages of apoptosis;
upper right quadrant shows cells in later stage of apoptosis; upper left quadrant shows dead cells. Shown is a representative experiment of two with incubations carried out
in duplicate. (B) The stacked bars represent the mean S.D. of cells found in each quadrant after the treatment. The standard deviation values were always lower than the
11% of the mean.
affected by treatment with E-tb throughout the period investi-
gated.
field, several AC inhibitors have been identified within a second
series de 2-substituted aminoethanol amides. Using lysates from
AC10X and the fluorogenic substrate previously reported (Bedia
et al., 2007), three amides derived from two unsaturated sph-
ingoid bases differing at the double bond stereochemistry have
been identified as AC inhibitors, the corresponding pivaloylamides
being the most potent ones. Double bond reduction in the above
compounds led to less potent inhibitors, which stresses the impor-
3.5. Metabolism of E-tb
The same extracts used to quantify total ceramides were ana-
lyzed for the presence of E-tb metabolites. As shown in Fig. 7, the
1-phosphocholine derivatives was the main metabolite identified,
reaching a 20% of the total at 12 h, which corresponded to around
740 pmol. Other metabolites included the O-glucosylated deriva-
tive, the N-deacylated product and the N-palmitoyl, N-stearoyl
and N-lignoceroyl amides. Although the levels of these metabo-
lites increased over the time of incubation, their percentages never
reached the 0.5% of the total. The amounts of the glucosylated tert-
butylamide, and the palmitoyl and tetracosanoylamides reached
8-10 pmol at 12 h, whereas only 1 pmol of the stearoylamide was
detected at the same time point.
4. Discussion
Due to its ability to regulate the ceramide/sphingosine balance,
AC has become an important target in cancer therapy (Park and
Schuchman, 2006). Continuing with our ongoing research in this
Fig. 6. Effect of E-tb on ceramide production. A549 cells were treated with 50 ␮M of
E-tb for the specified times. Then, cells were trypsinized and lipids were extracted,
methanolysed and the different ceramide species were quantitated by UPLC/TOF.
Results are referred to samples from cells treated with vehicle for 24 h (the same
ceramide profiles was observed in extracts from cells treated with vehicle for
30 min). N-dodecylsphingosine (500 pmol/sample) was added for quantification of
ceramides. Amounts are corrected for the protein concentration of each sample.
Symbols correspond to: (ꢀ), C22; (᭹), C14; (ꢁ), C24; (ꢂ), C20; (ꢁ), C18; (ꢃ), C16;
(ꢂ), so. The amounts of each species at 12 h (mean pmol S.D., n = 3) are given next
to the symbols.
Fig. 7. Metabolization of E-tb in A549 cells. After treatment with 50 ␮M of E-tb for
the specified times, cells were scrapped off and lipids were extracted, methanol-
ysed and the different metabolites of E-tb were investigated and quantitated by
UPLC/TOF. Results are referred to samples from cells treated with E-tb for 30 min.
N-dodecylsphingosine (500 pmol/sample) was added for quantification. Amounts
are corrected for the protein concentration of each sample. Only the folds increase
over the time is shown. Symbols correspond to: (ꢀ), E-tb; (᭹), E-tbGlc; (ꢁ), E-C16;
(ꢂ), E-NH2; (ꢁ), E-C18; (ꢃ), E-tbPC; (ꢂ), E-C24. The metabolite structures are given
below. aActual amounts of amine E-NH2 could not be calculated due to the absence
of the suitable internal standard in the samples.

C. Bedia et al. / Chemistry and Physics of Lipids 156 (2008) 33–40
39
tance of the sphingoid-like base unsaturation for AC inhibition. In
order to explore whether these unsaturations could be replaced
with an electron rich element without loss of inhibitory activity,
the effect of amides derived from sphingoid-like bases SC16, SAr
and OAr was determined. Results showed that only the pivaloy-
lamide of SC16 was effective, although its inhibitory activity was
only slightly higher than that of C16-tb, and significantly lower than
those of the unsaturated base amides. All the other amides of the
ether/thioether bases (SAr and OAr) were inactive. In the light of the
results obtained with pivaloylamides, compounds E-tb and SC16-tb
were investigated further. Kinetic analysis revealed that AC inhibi-
tion by these compounds is competitive towards the substrate, with
Ki values of 34 and 94 ␮M, respectively.
acteristic C3 stereogenic centre present in sphingolipids. This is
a valuable improvement from a synthetic point of view, since it
greatly eases the preparation, and entail an important advantage
over the previously reported AC inhibitors (Selzner et al., 2001;
Raisova et al., 2002; Dagan et al., 2003; Samsel et al., 2004; Bedia
et al., 2005; Grijalvo et al., 2006). Finally, Tsuboi et al. (2005)
reported that AC hydrolyzes also N-acylethanolamines and that N-
acylethanolamine acid amidase has a low but measurable ceramide
hydrolyzing activity. However, since hydrolysis of the fluorogenic
substrate used here by the MOHNSEN cells is negligible (Bedia et
al., 2007), should the N-acylethanolamine acid amidase be present
in the MOHNSEN fibroblasts, its contribution to hydrolysis by the
AC10X cells does not account for the inhibitory effects found. How-
ever, the fact that some of the compounds reported here inhibit
other amidases cannot be disregarded at this point and further
experiments are necessary to clarify this issue.
Some of these inhibitors elicited cytotoxicity in A549 cells with
higher potencies than those of N-oleoylethanolamine and B13. On
the other hand, only a limited number of compounds inhibited AC in
intact AC10X cells. Significant AC inhibitory activity occurred only
with pivaloylamides E-tb and C16-tb, octanoylamide E-c7 and the
p-tert-butylamide C16-tbph. A good correlation between AC inhi-
bition in intact AC10X cells and cytotoxicity in A549 cells was only
found for E-tb and E-c7, which had similar AC inhibitory potencies
(IC50 around 15 ␮M) and comparable cytotoxicities (LD50 around
40 ␮M). Conversely, saturated base amides C16-tb and C16-tbph
were less active AC inhibitors and were not cytotoxic. Flow cytom-
etry analysis revealed that E-tb was apoptotic to A549 cells. In
contrast, neither this compound nor their octanoylamide analogs
E-c7 were cytotoxic in MOHNSEN cells overexpressing AC. This
result suggested that the apoptotic activity of E-tb over A549
cells might be caused, at least in part, by a buildup of ceramide
occurring by AC inhibition. Such a buildup would be counterbal-
anced by AC overexpression, with a resulting lack of toxicity of
the inhibitors in the AC10X cell model. In order to obtain support
for this assumption, ceramides were analysed by UPLC-MS (Fig. 6).
These analyses evidenced that A549 cells treated with E-tb exhib-
ited a time-course increase of C14, C16 and C18 ceramides with
respect to controls, but the longer chain ceramides (C20 to C24)
remained unaffected. This may indicate the inhibition of a cerami-
dase selectively involved in the hydrolysis of short-chain ceramides.
Alternatively, AC may hydrolyze all ceramides, but this would be
followed by selective reacylation of the released sphingosine to the
long chain ceramides. Unexpectedly, as found with the D-e-MAPP
N-alkyl analog LCL284 (Bielawska et al., 2007), sphingosine levels
did not change significantly throughout the time of treatment with
E-tb. These overall findings may be explained by assuming that
the sphingosine decrease resulting from inhibition of short-chain
ceramides hydrolysis is compensated by an increased of long chain
ceramides deacylation, the pool of which is replenished through
biosynthesis de novo or sphingomyelin hydrolysis.
The role of the N-pivaloyl group in the observed inhibition is
so far unknown. A pivaloylamide unit has also been reported in
a SMase inhibitor, whose N-octanoyl analog is not active (Taguchi
et al., 2003a,b). One attractive explanation is that pivaloylamides
bind to the enzyme active site, but they are not hydrolyzed due
to the steric hindrance of the bulky tert-butyl group, which ham-
pers the attack of the hydrolytic nucleophilic cysteine. The fact
that both E-tb and SC16-tb are competitive inhibitors with respect
to the substrate and that very low amounts of the free base and
reacylated amides are detected in the UPLC/TOF analyses of E-tb
treated A549 cells support this notion. Adamantyl ceramides, also
having a bulky N-acyl moiety, have been reported to display selec-
tive cytotoxicity on drug-resistant breast tumor cells compared to
normal breast epithelial cells. The authors suggest that an effect
on ceramidase may contribute to the cytotoxicity elicited by these
compounds (Crawford et al., 2003). A very interesting feature of
the AC inhibitors reported in this article is the absence of the char-
Acknowledgements
We thank Dr. S. Grijalvo and Dr. D. Mormeneo for the previously
reported compounds used in this study, Mrs. Eva Dalmau for excel-
lent technical assistance and Prof. T. Levade and Dr. J.A. Medin for
providing the AC10X cell line. This work was funded by the Span-
ish Council for Scientific Research (CSIC, Grant 200580F0211 and
predoctoral fellowship to X.M.), Generalitat de Catalunya (Grant
2005SGR05-01063), Fundació La Marató de TV3 (Projects 040730
and 040731), and the Ministerio de Educación y Ciencia (Grant
CTQ2005-00175/BQU, Juan de la Cierva contract to Y.H. and a pre-
doctoral fellowship to C.B.).
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